Sulfisoxazole compounds for the treatment of inflammasome-mediated pulmonary diseases

Abstract

Disclosed herein are methods, compounds, and compositions for treating or preventing an inflammasome-mediated disease or condition, including an inflammasome-mediated pulmonary disease or condition.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 045,253, filed June 29, 2020, the contents of which are hereby incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The technology of the present disclosure generally relates to methods, compounds, and compositions for treating or preventing an inflammasome-mediated disease or condition, including an inflammasome-mediated pulmonary disease or condition. BACKGROUND

[0004] Inflammation is an adaptive response to noxious stimuli. Innate immunity includes a system of germline-encoded receptors that survey intracellular and extracellular compartments for signs of infection and recognize highly conserved microbial motifs or pathogen-associated molecular patterns (PAMPs). These pattern recognition receptors (PRRs) are expressed by host cells of defense, such as macrophages, monocytes, dendritic cells, and epithelial cells. Membrane-bound Toll-like receptors (TLRs) and C-type lectins are PRRs that survey the extracellular environment and endosomal compartments for PAMPs, while the cytoplasm is scanned by intracellular nucleic acid sensors, such as interferon-inducible proteins (also known as AIM2) and retinoic acid-inducible gene-like helicases. Activation of these receptors elicits proinflammatory cytokine production and type I interferon-dependent antiviral responses through the transcription factor NF-κΒ.

[0005] Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a class of intracellular PRRs that recognize PAMPs and host-derived signals, DAMPs (danger-associated molecular patterns). NLRs are composed of a conserved central domain that mediates nucleotide binding and oligomerization, a COOH-terminal leucine- rich domain (LRR) that senses NLR agonists and has self-inhibitory properties in their absence, and an NH2-terminal region that is required for protein-protein interactions. The human NLR gene family consists of 22 members, which are divided into four subfamilies based on their NH2-terminal domains: NLRA, NLRB, NLRC, and NLRP. Activation of certain NLRs (NLRP1 (NACHT, LRR and PYD domain containing 1), NLRP3 (NACHT, LRR and PYD domain containing 3), and NLRC4 (NLR family CARD domain containing 4)) leads to the assembly of inflammasomes. Inflammasomes are intracellular multimeric protein complexes that regulate the maturation and release of IL-1 family proinflammatory cytokines (e.g., IL-1 β and IL-18) in response to pathogenic and endogenous danger signals. Increasing evidence suggests that inflammasomes play a key role in the pathogenesis of acute and chronic respiratory diseases. Thus, there is a need to develop improved technologies to treat such conditions associated with inflammasome activity. SUMMARY

[0006] In one aspect, the present disclosure provides a compound having the structure of Formula I,

[0007]

[0008] a tautomer thereof and / or a pharmaceutically acceptable salt thereof;

[0009] wherein

[0010] A is absent, or is selected from the group consisting of C(O)N(R 3 ), phenylene, oxazolylene, thiazolylene, piperidylene, and

[0011]

[0012] L is absent or C 1-10 alkylene;

[0013] X is H, CHO, COOH, C(O)NR 4 R 5 , COOR 6 , NH2, or NHR;

[0014] R is 2-chloropyrimidin-4-yl;

[0015] R 1 and R2 independently substituted or unsubstituted C 1-6 alkyl, or R 1 and R 2 one is H and the other is cyclohexyl-NH-C(O), or R 1 and R 2 together are C 4-6 alkylene and form a 5-, 6-, or 7-membered ring with the nitrogen to which they are attached, which ring is optionally substituted with phenyl;

[0016] R 3 and R 4 are independently selected from H or C 1-6 alkyl;

[0017] R 5 is selected from H, PEG, or C 1-6 alkyl; and

[0018] R 6 is selected from substituted or unsubstituted C 1-10 alkyl, C 2-10 alkenyl, or C 7-14 aralkyl.

[0019] In some embodiments, A is absent. In some embodiments, A is C(O)N(R 3 ). In some such embodiments, N is attached to L or X. In other such embodiments, C is attached to L or X. In some embodiments, R 3 is C 1-6 alkyl, and in some embodiments, R 3 is H or methyl. In some embodiments, A is phenylene, oxazolylene, thiazolylene, piperidinylene, or

[0020]

[0021] In some embodiments, A is phenylene, oxazolylene, thiazolylene, or piperidinylene.

[0022] In some embodiments, L is absent. In some embodiments, L is C 1-10 alkylene.

[0023] In some embodiments, X is H. In some embodiments, X is COOH. In some embodiments, X is C(O)NR 4 R 5 . In some embodiments, X is COOR 6 . In some embodiments, X is NH2or NHR.

[0024] In some embodiments, R 1 and R2 each of R 1-6 alkyl optionally substituted with one or more F, OH, CF3, C 3-7 cycloalkyl, or SO2-alkyl. In some embodiments, R 1 and R 2 together are C 4-6 alkylene, and form a 5-, 6-, or 7-membered ring with the nitrogen to which they are attached. In some embodiments, one of R 1 and R 2 is H and the other is cyclohexyl-NH-C(O).

[0025] In some embodiments, A is absent, or is selected from the group consisting of C(O)N(R 3 ), phenylene, oxazolylene, thiazolylene, and piperidinylene; L is absent or is C 1-10 alkylene; X is H, COOH, or NH2; R 1 and R 2 are independently substituted or unsubstituted C 1-6 alkyl; and R 3 is selected from H or C 1-6 alkyl.

[0026] In some embodiments, A is absent and L is C 3-10 alkylene. In some embodiments, A is phenylene, oxazolylene, thiazolylene, or piperidinylene, and L is absent or is C 1-5 alkylene.

[0027] In some embodiments, the compound, tautomer thereof, and / or pharmaceutically acceptable salt thereof is selected from the group consisting of:

[0028]

[0029]

[0030] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising the compound, tautomer thereof, and / or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0031] In one aspect, the present disclosure relates to a method for treating or preventing an inflammasome-mediated disease or condition in a mammalian subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of the present technology, a tautomer thereof, and / or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier.

[0032] In some embodiments, the inflammasome-mediated disease or condition is associated with NLRP1 inflammasome activation and / or NLRP3 inflammasome activation.

[0033] In some embodiments, the inflammasome-mediated disease or condition is an inflammasome-mediated pulmonary disease or condition.

[0034] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0035]

[0036] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0037]

[0038] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0039]

[0040] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0041]

[0042] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0043]

[0044] In some embodiments, the inflammasome-mediated pulmonary disease or condition is caused by a pathogen selected from the group consisting of: pandemic influenza; Streptococcus pneumoniae; Pseudomonas aeruginosa; Mycobacterium tuberculosis; respiratory syndrome caused by rhinovirus, flavivirus, dengue virus, Zika virus, or West Nile virus; idiopathic pulmonary fibrosis (IPF); chronic obstructive pulmonary disease (COPD); acute exacerbation of chronic obstructive pulmonary disease (AECOPD); asthma; acute respiratory distress syndrome (ARDS); COVID-19; Middle East respiratory syndrome (MERS); severe acute respiratory syndrome (SARS); silicosis; and asbestosis.

[0045] In some embodiments, the inflammasome-mediated lung disease or condition is associated with an inhaled irritant. In some embodiments, the inhaled irritant comprises a gas, a mist, a smoke, or a dust. In some embodiments, the inhaled irritant is selected from the group consisting of silica, asbestos, smoke, cigarette smoke, and nanoparticles.

[0046] In some embodiments, the administering step is selected from the group consisting of intranasal administration, intramuscular administration, subcutaneous administration, administration by inhalation, and oral administration.

[0047] In some embodiments, treating or preventing an inflammasome-mediated disease or condition comprises reducing the level of one or more inflammatory cytokines in the subject as compared to an untreated control subject.

[0048] In some embodiments, the one or more inflammatory cytokines is selected from the group consisting of IL-1β, IL-18, IL-1α, IL-6, IL-33, TNF-α, CCL2, IFN-γ, IL-10, IL12p70, MCP-1, HMGB1, and any combination thereof. In some embodiments, the one or more inflammatory cytokines is IL-1β.

[0049] In some embodiments, treating or preventing an inflammasome-mediated lung disease or condition comprises reducing the level of cellular infiltrates in the lung of the subject as compared to an untreated control subject, wherein the cellular infiltrates comprise alveolar macrophages, neutrophils, inflammatory Ly6C + one or more of macrophages and dendritic cells.

[0050] In some embodiments, treating or preventing an inflammasome-mediated disease or condition comprises reducing apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) speck formation in the subject as compared to an untreated control subject.

[0051] In one aspect, the disclosure relates to a use of a composition in the manufacture of a medicament for treating or preventing an inflammasome-mediated disease or condition, wherein the composition comprises a therapeutically effective amount of a compound of the present technology, a tautomer thereof, and / or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier.

[0052] In some embodiments, the inflammasome-mediated disease or condition is associated with NLRP1 inflammasome activation and / or NLRP3 inflammasome activation.

[0053] In some embodiments, the inflammasome-mediated disease or condition is an inflammasome-mediated lung disease or condition.

[0054] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0055]

[0056] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0057]

[0058] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0059]

[0060] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0061]

[0062] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0063]

[0064] In some embodiments, the inflammasome-mediated pulmonary disease or condition is caused by a pathogen selected from the group consisting of: pandemic influenza; Streptococcus pneumoniae; Pseudomonas aeruginosa; Mycobacterium tuberculosis; respiratory syndrome caused by rhinovirus, flavivirus, dengue virus, Zika virus, or West Nile virus; idiopathic pulmonary fibrosis (IPF); chronic obstructive pulmonary disease (COPD); acute exacerbation of chronic obstructive pulmonary disease (AECOPD); asthma; acute respiratory distress syndrome (ARDS); COVID-19; Middle East respiratory syndrome (MERS); severe acute respiratory syndrome (SARS); silicosis; and asbestosis.

[0065] In some embodiments, the inflammasome-mediated pulmonary disease or condition is associated with an inhaled irritant. In some embodiments, the inhaled irritant comprises a gas, a mist, a smoke, or a dust. In some embodiments, the inhaled irritant is selected from the group consisting of: silicon dioxide, asbestos, smoke, cigarette smoke, a nanoparticle.

[0066] In some embodiments, the administering step is selected from the group consisting of: intranasal administration, intramuscular administration, subcutaneous administration, administration by inhalation, and oral administration.

[0067] In some embodiments, treating or preventing an inflammasome-mediated disease or condition comprises reducing the level of one or more inflammatory cytokines in the subject compared to an untreated control subject.

[0068] In some embodiments, the one or more inflammatory cytokines are selected from the group consisting of IL-1b, IL-18, IL-1a, IL-6, IL-33, TNF-a, CCL2, IFN-g, IL-10, IL12p70, MCP-1, HMGB1, and any combination thereof. In some embodiments, the one or more inflammatory cytokines is IL-1b.

[0069] In some embodiments, treating or preventing an inflammasome-mediated pulmonary disease or condition comprises reducing the level of cellular infiltrates in the lung of the subject compared to an untreated control subject, wherein the cellular infiltrates comprise alveolar macrophages, neutrophils, inflammatory Ly6C + one or more of macrophages and dendritic cells.

[0070] In some embodiments, treating or preventing an inflammasome-mediated disease or condition comprises reducing the level of apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) speck formation in the subject compared to an untreated control subject.

[0071] In one aspect, the present disclosure relates to a compound of the present technology, a tautomer thereof, and / or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier, for use in treating or preventing an inflammasome-mediated disease or condition in a subject in need thereof.

[0072] In some embodiments, the inflammasome-mediated disease or condition is associated with NLRP1 inflammasome activation and / or NLRP3 inflammasome activation.

[0073] In some embodiments, the inflammasome-mediated disease or condition is an inflammasome-mediated pulmonary disease or condition.

[0074] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprises:

[0075]

[0076] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof, or the pharmaceutical composition comprises:

[0077]

[0078] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0079]

[0080] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0081]

[0082] In some embodiments, the compound, tautomer, and / or pharmaceutically acceptable salt thereof or the pharmaceutical composition comprises:

[0083]

[0084] In some embodiments, the inflammasome-mediated pulmonary disease or condition is caused by a pathogen selected from the group consisting of: pandemic influenza; Streptococcus pneumoniae; Pseudomonas aeruginosa; Mycobacterium tuberculosis; respiratory syndromes caused by rhinovirus, flavivirus, dengue virus, Zika virus, or West Nile virus; idiopathic pulmonary fibrosis (IPF); chronic obstructive pulmonary disease (COPD); acute exacerbation of chronic obstructive pulmonary disease (AECOPD); asthma; acute respiratory distress syndrome (ARDS); COVID-19; Middle East respiratory syndrome (MERS); severe acute respiratory syndrome (SARS); silicosis; and asbestosis.

[0085] In some embodiments, the inflammasome-mediated pulmonary disease or condition is associated with an inhaled irritant. In some embodiments, the inhaled irritant comprises a gas, a mist, a smoke, or a dust. In some embodiments, the inhaled irritant is selected from the group consisting of: silicon dioxide, asbestos, smoke, cigarette smoke, a nanoparticle.

[0086] In some embodiments, the administering step is selected from the group consisting of: intranasal administration, intramuscular administration, subcutaneous administration, administration by inhalation, and oral administration.

[0087] In some embodiments, treating or preventing an inflammasome-mediated disease or condition comprises reducing the level of one or more inflammatory cytokines in the subject compared to an untreated control subject.

[0088] In some embodiments, the one or more inflammatory cytokines are selected from the group consisting of IL-1b, IL-18, IL-1a, IL-6, IL-33, TNF-a, CCL2, IFN-g, IL-10, IL12p70, MCP-1, HMGB1, and any combination thereof. In some embodiments, the one or more inflammatory cytokines is IL-1b.

[0089] In some embodiments, treating or preventing an inflammasome-mediated pulmonary disease or condition comprises reducing the level of cellular infiltrates in the lung of the subject as compared to an untreated control subject, wherein the cellular infiltrates comprise alveolar macrophages, neutrophils, inflammatory Ly6C + one or more of macrophages and dendritic cells.

[0090] In some embodiments, treating or preventing an inflammasome-mediated disease or condition comprises reducing apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) speck formation in the subject as compared to an untreated control subject. BRIEF DESCRIPTION OF DRAWINGS

[0091] Figure 1 is a graph showing the dose response of propenecid analogs on inflammasome activity inhibition as assessed by IL-1b ELISA secreted in macrophages pre-activated with lipopolysaccharide (LPS; 100 ng / mL). Macrophages were stimulated with NLRP3 activator silica (250 pg / mL). From left to right: the first bar for each compound is non-silica activated; the second bar is silica activated but without compound; the following bars are the compound at 300 mM, 150 mM, 30 mM, and 3 mM. Prob = propenecid; MCC = MCC950, a specific small molecule inhibitor of NLRP3 inflammasome. From left to right, the compounds listed on the x-axis are as follows: Prob, BT004, BT005, BT006, BT007, BT008, BT009, BT010, BT011, BT026, BT027, BT028, BT029, BT030, BT031, BT032, BT033, BT034, BT035, BT041, BT043, BT052, BT053, BT054, BT055, BT056, BT057, BT058, MCC.

[0092] Figure 2Figure 1 is a graph showing the dose response of propenecid analogs BT135, BT136, BT137, BT138, BT139, and BT140 on inflammasome activity inhibition, as assessed by IL-1 beta ELISA secreted in macrophages pre-activated by lipopolysaccharide (LPS; 100 ng / mL). Macrophages were stimulated with NLRP3 activator Nigericin (6 mM). From left to right, the first bar for each compound (cluster) is non-silica activated, the second bar is activated but without compound, the following bars are 300 mM, 150 mM, 30 mM, and 3 mM of the compound. Prob / D = propenecid dissolved in DMSO; Prob / P = propenecid dissolved in PBS. Experiments were performed in triplicate.

[0093] Figure 3A and 3B Figure 2 is a graph showing the dose response of propenecid analogs BT032, BT132, BT133, and BT134 on inflammasome activity inhibition, as assessed by IL-1 beta ELISA secreted in macrophages pre-activated by lipopolysaccharide (LPS; 100 ng / mL). Macrophages were stimulated with NLRP3 activator Nigericin (6 mM) ( Figure 3A ) or silica (250 pg / mL) ( Figure 3B ). From left to right, the first bar for each compound (cluster) is non-Nigericin or non-silica activated, the second bar is activated but without compound, the following bars are 300 mM, 150 mM, 30 mM, and 3 mM of the compound. Experiments were performed in triplicate.

[0094] Figures 4A-4E Figure 3 is a graph showing the mean percent change in body weight ( 5 pfu) intranasally infected C57BL / 6 mice groups (n = 5-14 per group) and survival curves ( Figure 4A and 4D ) and survival curves ( Figure 4B , 4C and 4E). Mice were treated intranasally with 40 mg / kg of a propenecid analog of the present technology (BT032, BT133, or BT135) or PBS at day 1 or day 3 post-infection, and once every 48 hours thereafter.

[0095] Figures 5A-5E Figure 4 is a graph showing the administration of a propenecid analog of the present technology (e.g., BT032) reduces excessive inflammation in the airways during HKx31 infection. Groups of C57BL / 6 mice were intranasally infected with a high dose of HKx31 (10 5pfu; n = 8 for PBS, IAV alone, and IAV with BT032 treatment; n = 4 for BT032 alone (no infection). Mice were treated intranasally with 20 mg / kg of the present technology probenecid analog (BT032) or PBS on day 3 post-infection. Total numbers of white blood cells in the BAL were determined by live cell counts Figure 5A ), and total CD11c + I-A b低 macrophages Figure 5B ), Ly6G + neutrophils Figure 5C ), Ly6C + inflammatory macrophages Figure 5D ), and CD11c + I-A b低 dendritic cells Figure 5E ).

[0096] Figure 5F are graphs showing macrophage infiltration and pro-inflammatory chemokine concentrations in the airways of mice treated with 10 5 PFU HKx31 IAV or no treatment as indicated (n = 8 mice per group). Mice were treated with 20 mg / kg BT032 on day 3 post-infection and euthanized 24 hours post-treatment. Bronchoalveolar lavage (BAL) fluid (BALF) was obtained from mice and the number of macrophages (CD11c+, I-Ablow) and MCP-1 concentrations were determined by cytokine bead analysis, as determined by flow cytometry. BALF concentrations of the chemokine MCP-1 were determined by cytokine bead array.

[0097] Figure 6A and 6B are graphs showing inflammasome activity determined by measuring secreted IL-1β concentrations in mouse immortalized BMDMs pre-activated with lipopolysaccharide (LPS; 100 ng / mL) for 3 hours. Macrophages were stimulated with the NLRP3 activator Nigericin (3 μΜ) Figure 6A ) or the NLRP1 agonist L18-MDP (100 μg / mL) Figure 6B ) with or without treatment with BT032 (3.9-350 μΜ). Secreted IL-1β concentrations were measured by ELISA and expressed as the mean ± SEM of the pooled results of 3 independent experiments performed in triplicate, where activity was normalized to the percentage of activity associated with DMSO-treated control cells and non-stimulation Figure 6A ) or to IL-1β concentrations Figure 6B ) and shown as curves of Log[M] BT032 versus normalized response (variable slope).

[0098] Figure 6C This is a graph illustrating the effects of BT032 treatment on macrophages for NLRP3 (from left to right: nigrain, monosodium urate (MSU), and silica) and NLRP1 (L18-MDP)-induced inflammasome activation, non-canonical inflammasome activity (LPS(B4)), and AIM2 (poly dA:dT) and NLRC4 (flagellate)-mediated inflammasome activation. MCC950 is an NLRP3-specific inhibitor; MSU = monosodium urate; LPS = lipopolysaccharide; BT32 = BT032. Secreted IL-1β concentrations were measured by ELISA and are expressed as the mean ± SEM of the pooled results from three independent experiments performed in triplicate, where activity was normalized to the percentage of activity associated with DMSO-treated control cells (“drug-free”) and unstimulated (“NS”) cells.

[0099] Figure 7 This demonstrates that BT032 (20, 100, and 350 μM) inhibits the activity of 5 x 10 primary human bronchial epithelial cells (5 x 10⁻⁶ cells) obtained from consenting volunteers and treated with NLRP1 viral mimic dsRNA poly I:C (1.0 μg / ml). 4 A graph illustrating the effect of attack on the virus mimicry transfected into cells with lipidamine. The NLRP3 selective inhibitor MCC950 (MCC: 5 μM) was used as a negative control. Secreted IL-1β concentrations were determined from the culture supernatant by ELISA, and results are shown as the mean ± SEM of two independent experiments performed in triplicate.

[0100] Figure 8A This is a schematic diagram of an experiment using a mouse model of silicosis in the lungs.

[0101] Figures 8B-8E This is a graph illustrating the reduction of excessive airway inflammation following silica challenge by administration of probenecid analogs (e.g., BT132) of the current technology. C57BL / 6 mice (n=5 per group) were intranasally treated for 24 hours with PBS (50 μl), silica (1 mg in 50 μl PBS), or silica (1 mg) in combination with a total volume of 50 μl of BT132 (40 mg / kg) for 24 hours. Airway leukocytes in bronchoalveolar lavage (BAL) fluid (BALF) were determined by viable cell counting. Figure 8B Ly6G + Neutrophils ( Figure 8C ) and total CD11c + IA b低 macrophages Figure 8D The concentration of IL-1β in BALF was determined by ELISA. Figure 8E). Data are expressed as mean ± SEM of 5 mice per group, significant differences compared to mice challenged with silica alone, *p<0.05, ***p<0.001, one-way ANOVA.

[0102] Figure 9A is a schematic representation showing an inflammasome multiprotein complex (adapted from Review InvivoGen, Inflammasomes, available at www.invivogen.com / review-inflammasome (2021)). In this illustrative inflammasome, the inflammasome complex contains a Nod-like receptor (NLR), an adaptor apoptosis-associated speck-like (ASC) protein and caspase-1. As shown in this illustrative example of an inflammasome multiprotein complex, the NLR portion contains a pyrin domain (PYD) and a nucleotide-binding and oligomerization domain (NACHT); the caspase-1 portion contains a caspase recruitment domain (CARD) and p20 and plO subunits; the ASC portion contains a PYD and a CARD.

[0103] Figures 9B-9D is a picture showing the ability of the probenecid analog of the present technology (e.g. BT032) to inhibit inflammasome complex formation after NLRP3 activation. NLRP3-deficient immortalized BMDMs recombinant for ASC-Cerulean (pseudocolor RED) and NLRP3-Flag were either left unstimulated ( Figure 9B ), or stimulated with the NLRP3 agonist Nigericin (3 mM) for 90 min ( Figure 9C ). Prior to Nigericin challenge, ASC-Cerulean macrophages were also pre-treated with BT032 (350 mM) for 60 min ( Figure 9D ). Macrophages were fixed with 4% paraformaldehyde and imaged for inflammasome speck formation, as identified by intense punctate staining in the cytoplasm of the cells. Nuclei were stained with DAPI (4',6'-diamidino-2-phenylindole; blue). Representative images shown are maximum intensity projections of 3D deconvoluted z-stacks using ImageJ.

[0104] Figure 9E is a graph showing the comparison of the number of ASC specks detected per field (6-7 fields per sample) with the total number of cells / field determined by staining the nuclei. Data are expressed as percentage of ASC specks per field for each treatment group, as shown in Figures 9B-9D .

[0105] Figure 10A is a schematic representation of the mouse IP LPS challenge model used for NLRP3 inflammation experiments.

[0106] Figure 10B Figure 24 is a graph showing IL-1 b levels (pg / mL) in mouse serum following intraperitoneal (IP) administration of PBS or LPS (10 mg / kg), with or without administration of BT032 (100 mg / kg) or BT132 (160 mg / kg) one hour prior to LPS administration.

[0107] Figure 10C Figure 25 is a graph showing TNF a levels (pg / mL) in mouse serum following IP administration of PBS or LPS (10 mg / kg), with or without administration of BT032 (100 mg / kg) or BT132 (160 mg / kg) one hour prior to LPS administration.

[0108] Figure 10D Figure 24 is a graph showing IL-1 b levels (pg / mL) in mouse serum following intraperitoneal (IP) administration of PBS or LPS (10 mg / kg), with or without administration of BT032 (100 mg / kg) or BT132 (160 mg / kg) one hour prior to LPS administration.

[0109] Figure 10E Figure 25 is a graph showing TNF a levels (pg / mL) in mouse serum following IP administration of PBS or LPS (10 mg / kg), with or without administration of BT032 (100 mg / kg) or BT132 (160 mg / kg) one hour prior to LPS administration.

[0110] Figure 11 Figure 24 is a graph showing IL-1 b levels (pg / mL) in mouse serum following intraperitoneal (IP) administration of PBS or LPS (10 mg / kg), with or without administration of BT032 (100 mg / kg) or BT132 (160 mg / kg) one hour prior to LPS administration. DETAILED DESCRIPTION

[0111] I. DEFINITIONS

[0112] The following terms are used herein, the definitions of which are intended to be illustrative only.

[0113] Generally, "substituted" refers to an organic radical (e.g., alkyl) as defined below, in which one or more bonds from a hydrogen atom contained therein are replaced by a bond to a non-hydrogen or non-carbon atom. Substituted radicals also include radicals in which one or more bonds to a carbon atom or a hydrogen atom are replaced by one or more bonds to a heteroatom, including double or triple bonds. Thus, unless otherwise specified, a substituted radical is substituted with one or more substituents. In some embodiments, a substituted radical is substituted with 1, 2, 3, 4, 5, or 6 substituents. Those skilled in the art will appreciate that substituted radicals of the present technology are chemically stable radicals that allow for isolation of the compounds in which they occur. Examples of substituents include: halogen (i.e., F, CI, Br, and I); hydroxyl; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy; carbonyl (oxy); carboxylate; ester; polyurethane; oxime; hydroxylamine; alkoxyamine; aralkoxyamine; thiol; sulfide; sulfoxide; sulfone; sulfonyl; sulfonamide; amine; N-oxide; azide; amide; urea; amidine; guanidine; nitro; nitrile (i.e., CN); and the like.

[0114] Alkyl includes straight-chain and branched-chain alkyl groups having (unless otherwise specified) 1 to 12 carbon atoms and typically 1 to 10 carbons or in some embodiments 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups can be substituted or unsubstituted. Examples of straight-chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched-chain alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, t-butyl, neopentyl, isoamyl, and 2,2-dimethylpropyl. Representative substituted alkyl groups can be substituted one or more times with substituents such as those listed above, and include, but are not limited to, haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like. In some embodiments, alkyl groups are substituted with 1, 2, or 3 substituents.

[0115] Alkenyl groups contain straight-chain and branched-chain alkyl groups as defined above except that there is at least one double bond between two carbon atoms. Alkenyl groups can be substituted or unsubstituted. Alkenyl groups have 2 to 12 carbon atoms and typically 2 to 10 carbons or in some embodiments 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, alkenyl groups have one, two, or three carbon-carbon double bonds. Examples include, but are not limited to, ethenyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, and the like. Representative substituted alkenyl groups can be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted with substituents such as those listed above.

[0116] Aryl is a monocyclic aromatic hydrocarbon radical containing 6 to 10 carbon atoms. Aryl groups can be substituted or unsubstituted. In some embodiments, aryl groups contain 6 to 10 carbon atoms. Representative aryl groups include, but are not limited to, phenyl and naphthyl. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted with substituents such as those listed above.

[0117] Heteroalkyl and heteroalkenyl are alkyl (as defined herein) and alkenyl (as defined herein), respectively, that contain 1 to 6 heteroatoms selected from N, O, and S. It is understood that each heteroatom present is bonded to at least one carbon atom within the heteroalkyl or heteroalkenyl. In some embodiments, the heteroalkyl or heteroalkenyl contains 1, 2, or 3 heteroatoms. Heteroalkyl and heteroalkenyl groups can be substituted or unsubstituted. Examples of heteroalkyl groups include, but are not limited to, CH3CH2OCH2, CH3NHCH2, CH3CH2N(CH3)CH2, CH3CH2SCH2, CH3CH2OCH2CH2OCH2CH2. Examples of heteroalkenyl groups include, but are not limited to, CH2=CHN(CH3)CH2, and CH2=CHSCH2. Representative substituted heteroalkyl or heteroalkenyl groups can be mono-substituted or substituted more than once (e.g., 1, 2, or 3 times) with substituents such as those listed above, and include, but are not limited to, haloheteroalkyl (e.g., trifluoromethyloxyethyl), carboxyalkylaminoalkyl, methacrylate, and the like. 2＝ CHOCH2, CH2=CHN(CH3)CH2, and CH2=CHSCH2. Representative substituted heteroalkyl or heteroalkenyl groups can be mono-substituted or substituted more than once (e.g., 1, 2, or 3 times) with substituents such as those listed above, and include, but are not limited to, haloheteroalkyl (e.g., trifluoromethyloxyethyl), carboxyalkylaminoalkyl, methacrylate, and the like.

[0118] Cycloalkyl groups include monocycloalkyl, bicycloalkyl, or tricycloalkyl groups having 3 to 12 carbon atoms in the ring (or in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms). Cycloalkyl groups can be substituted or unsubstituted. Exemplary monocycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, cycloalkyl groups have 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms is in the range of 3 to 5, 3 to 6, or 3 to 7. Bicyclo and tricyclo ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decahydronaphthyl, and the like. Substituted cycloalkyl groups can be substituted one or more times with non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings that are substituted with linear or branched alkyl groups as defined above. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4-, 2,5-, or 2,6-disubstituted cyclohexyl groups, which can be substituted with substituents such as those listed above.

[0119] Cycloalkylalkyl is an alkyl group as defined above, wherein a hydrogen bond or carbon bond of the alkyl group is replaced with a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups can be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups can be substituted at both the alkyl, cycloalkyl, or both alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted with substituents such as those listed above.

[0120] Groups described herein that have two or more points of attachment within the compounds of the technology (i.e., di-, tri-, or polyvalent) are named using the suffix “ene.” For example, a divalent alkyl group is an alkylene group, a divalent cycloalkyl group is a cycloalkylene group, a divalent heteroalkyl group is a heteroalkylene group, a divalent alkenyl group is an alkenylene group, and the like. Substituted groups that have a single point of attachment to the compounds of the technology are not referred to using “ene” nomenclature. Thus, for example, chloroethyl is not referred to herein as chloroethylene.

[0121] The term "administering" a molecule to a subject means delivering the molecule to the subject or cell. "Administering" includes prophylactic administration of a composition (i.e., before a disease and / or one or more symptoms of a disease is detectable) and / or therapeutic administration of a composition (i.e., after a disease and / or one or more symptoms of a disease is detectable). The methods of the present technology include administering one or more compounds. If more than one compound is to be administered, the compounds can be administered together at essentially the same time, and / or at different times in any order. Likewise, the compounds of the present technology can be administered prior to, concurrently with, and / or after other types of drugs or treatment procedures (e.g., surgery).

[0122] The use of the term "include," "includes" or "including," "comprise," "comprises" or "comprising" to describe or define the elements of a compound, composition or method, is by way of disclosure of examples, and not by way of limitation. In other words, the use of "include," "includes" or "including," "comprise," "comprises" or "comprising" should not be interpreted as an intention to limit the examples to the recited elements, but to the contrary, an intention to encompass all elements of the compound, composition or method, as well as additional elements, without material effect on the nature of the recited examples. Likewise, the use of "consisting" or "consisting of" should be interpreted as an intention to encompass only the elements recited in the example, and not additional elements.

[0123] The term "conjugate" and grammatical equivalents, when referring to the conjugation of a molecule of interest and a polymer, means covalently linking the molecule of interest to the polymer. The linkage can be direct. Alternatively, the linkage can be indirect through a linker or moiety. Methods of conjugating to polymers are known in the art, including methods of conjugating to polypeptides to produce fusion proteins (Pasut, Polymers 6: 160-178 (2014); Medscape, Nanomedicine 5(6): 915-935 (2010)). In some embodiments, the conjugate includes probenecid conjugated to a PEG polymer.

[0124] As used herein, the term “effective amount” or “therapeutically effective amount” or “pharmaceutically effective amount” refers to an amount that is sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount that results in complete or partial amelioration of an inflammasome-mediated pulmonary disease or a symptom associated with an inflammasome-mediated pulmonary disease in a subject in need thereof. In the context of therapeutic or prophylactic applications, the amount of composition administered to a subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, multiple therapeutic compositions or compounds are administered. In the methods described herein, the therapeutic compound can be administered to a subject having one or more signs or symptoms of an inflammasome-mediated pulmonary disease (e.g., elevated lung concentrations of inflammatory cytokines such as IL-1β or IL-18).

[0125] Pharmaceutically acceptable salts of the compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and are not biologically undesirable (e.g., the salt does not have undue toxicity, allergenicity, or irritancy, and is bioavailable). When the compounds of the present technology have basic groups, such as, for example, amino groups, pharmaceutically acceptable salts can be formed with inorganic acids such as hydrochloric acid, hydroboronic acid, nitric acid, sulfuric acid, and phosphoric acid, with organic acids such as alginic acid, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid, or with acidic amino acids such as aspartic acid and glutamic acid. When the compounds of the present technology have acidic groups, such as, for example, carboxylic acid groups, they can form salts with metals such as alkali and alkaline earth metals (e.g., Na + , Li + , K + , Ca 2+ , Mg 2+ , Zn 2+ ), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine), or basic amino acids (e.g., arginine, lysine, and ornithine). Such salts can be prepared in situ during the isolation and purification of the compounds, or by separately reacting the purified compound in free base or free acid form, respectively, with a suitable acid or base, and isolating the salt thus formed.

[0126] A "polymer" is a substance having a molecular structure consisting primarily or entirely of a large number of similar units bonded together. Polymers can exist naturally (e.g., cellulose, polypeptides, nucleotide sequences, etc.) or artificially (e.g., plastics, resins, etc.). Polymers can be used as carriers of drugs conjugated thereto (i.e., polymer carriers) and can enhance the solubility of the conjugated drugs, improve their pharmacokinetic characteristics, protect the drugs from degradation, release the drugs under certain conditions (such as changes in pH) or in the presence of enzymes (such as esterases, lipases, or proteases). In addition, targeting moieties or solubilizers can also be introduced into the conjugates to improve their therapeutic index (Medscape, Nanomedicine 5(6): 915-935 (2010)). Polymers (including polymer carriers) can also be used to limit the distribution of drugs conjugated thereto, for example, by preventing the conjugated drugs from entering specific body compartments (e.g., from the lumen of the gastrointestinal tract to underlying tissues). Polymers (including polymer carriers) are pharmaceutically acceptable and can be natural polymers and / or synthetic linear polymers, and include polyethylene glycol (PEG), dextran, periodate-oxidized dextran, polysialic acid (PSA), hyaluronic acid (HA), dextrin, hydroxyethyl starch (HES), poly(2- ethyl 2-oxazoline) (PEOZ), polyglutamic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(D,L-lactide-co-glycolide) (PLA / PLGA), poly(hydroxyalkyl methacrylamide), polyglycerol, 25 polyamidoamine (PAMAM), polyethylenimine (PEI), and polypeptides.

[0127] As used herein, "inflammasome activation" means the formation of an inflammasome through the association of a pattern recognition receptor such as NLRP3 with an apoptosis-associated speck-like protein containing CARD (ASC) and procaspase-1 due to a stimulatory factor such as a pathogen component, and caspase-1 activation. Caspase-1 cleaves the proinflammatory cytokines IL-1 β and IL-18 into their active forms and mediates a form of inflammatory cell death known as pyroptosis. Other intracellular pattern recognition receptors (PRRs) such as NLR family members NLRPl and NLRC4, non-NLR PRRs such as absent in melanoma 2 (AIM2) and interferon-gamma-inducible protein 16 (IFI16) are also capable of forming inflammasomes. The sulfonsulfone analogs of the present technology can inhibit inflammasome activation, thereby inhibiting the production of activated caspase-1. As a result, the sulfonsulfone analogs of the present technology can inhibit the release of one or more inflammatory cytokines such as IL-1 β, IL-18, IL-1 α, IL-6, IL-33, TNF-α, CCL2, IFN-γ, IL-10, IL12p70, MCP-1, and HMGB1, reduce lung cell infiltration, reduce ASC speck formation, and provide protection against inflammasome-mediated lung disease.

[0128] As used herein, "inflammasome-mediated disease or condition" refers to a disease or condition associated with NLRP3 and / or NLRPl inflammasome activation or activity. In some embodiments, the inflammasome-mediated disease or condition is an inflammasome-mediated lung disease or condition.

[0129] As used herein, "inflammasome-mediated lung disease or condition" refers to acute or chronic respiratory inflammation-related diseases or conditions caused by pathogens, including but not limited to pandemic influenza (e.g., influenza A), Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis and other bacterial infections, acute respiratory distress syndrome (ARDS), and diseases caused by novel and emerging respiratory viruses (i.e., Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which causes COVID-19, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) which causes MERS). This includes respiratory syndromes caused by SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), respiratory syndromes caused by rhinovirus, flavivirus, dengue virus, Zika virus, and / or Nile virus, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), acute exacerbation of COPD (AECOPD), asthma, and inflammasome-mediated lung diseases or symptoms induced by environmental exposure to irritants, including but not limited to silica (e.g., silicosis), asbestos (e.g., asbestosis), smoke, cigarette smoke, and nanoparticles (e.g., titanium dioxide). As demonstrated by the experimental examples presented herein, probenecid analogues of this technology (e.g., BT032, BT132) are effective as inhibitors of inflammasome activation and are effective in methods for the prevention or treatment of inflammasome-mediated lung diseases or symptoms. Therefore, since probenecid analogs of this technology are effective in such methods, those skilled in the art will understand that probenecid analogs of this technology (e.g., BT032, BT132) are effective in methods for treating any inflammasome-mediated lung disease or condition, and are not limited to the illustrative diseases / pathogens that cause the inflammasome-mediated lung diseases or conditions listed herein.

[0130] As used herein, “inhibition of inflammasome activation” means complete or partial inhibition of inflammasome activation by stimulating factors. In other words, “inhibition of inflammasome activation” means that, compared with an untreated control, the compounds of this technique reduce the amount of activated caspase-1 produced or the amount of released inflammatory cytokines (such as IL-1β, IL-18, IL-1α, IL-6, IL-33, TNF-α, MCP-1, and HMGB1) or the formation of ASC spots.

[0131] Those skilled in the art will appreciate that the compounds of the technology can exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism, and / or stereoisomerism. Since the structural formulas within the specification and claims can represent only one of the possible tautomeric, conformational isomer, stereoisomer, or geometric isomer forms, it is understood that the technology encompasses any tautomeric, conformational isomer, stereoisomer, and / or geometric isomer form of the compounds described herein having one or more of the utilities described herein, as well as mixtures of these various different forms.

[0132] Unless specifically indicated, stereoisomers (also known as optical isomers) of a compound encompass all chiral, diastereomeric, and racemic forms of structures. Thus, the compounds disclosed herein include enriched or resolved optical isomers at any or all asymmetric atoms, as can be evident from the depiction of the structures. Both racemic and diastereomeric mixtures, as well as individual optical isomers, can be isolated or synthesized to be substantially free of their enantiomeric or diastereomeric counterpart, and are within the scope of the technology.

[0133] “Tautomer” refers to isomeric forms of a compound that are in equilibrium with each other and involve the migration of a proton and a change in at least one bond order (e.g., between single and double bonds). The existence and concentration of the isomeric forms will depend on the environment in which the compound is placed, and can vary, for example, depending on whether the compound is a solid or in an organic or aqueous solution, the temperature, and whether an acid or base is present. For example, in aqueous solution, imines can be in equilibrium with enamines, which are referred to as tautomers of each other, as shown below:

[0134]

[0135] Similarly, those skilled in the art will be familiar with other tautomeric forms, such as, for example, keto / enol tautomers, keto / phenol tautomers, and the like. Since the structural formulas are representative of the compounds, it is understood that all chemical formulas of the compounds described herein represent all tautomeric forms of the compounds and are within the scope of the technology.

[0136] As used herein, “treating,” “treat,” “treated,” “treatment” encompass treatment of a disease or disorder or condition described herein (e.g., inflammasome-mediated lung disease or an inflammasome-mediated lung condition) in a subject, such as a human, and include: (i) inhibiting the disease or disorder, i.e., arresting its development; (ii) relieving the disease or disorder, i.e., causing the condition to regress; (iii) slowing the progression of the condition; and / or (iv) inhibiting, relieving, or slowing the progression of one or more symptoms of the disease or disorder. Symptoms can be assessed by methods known in the art.

[0137] As used herein, "prevention" or "preventing" of a disorder or condition refers to a compound that reduces the occurrence of the disorder or condition in a treated sample relative to a control sample, or delays the onset of one or more symptoms of the disorder or condition relative to a control sample, in a statistical sample.

[0138] It is also to be understood that the various methods described to treat or prevent medical diseases and conditions are intended to mean "substantial", which includes complete treatment or prevention but also treatment or prevention less than complete and where some biologically or medically relevant result is achieved.

[0139] As used herein, the term "subject," "individual," or "patient" can be a single organism, a vertebrate, a mammal, or a human. "Mammal" includes humans, non-human primates, murines (e.g., mice, rats, hamsters, gerbils), ovines, bovines, ruminants, leporines, porcines, caprines, equines, canines, felines, avians, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0140] A subject "in need" of treatment according to the methods and / or compositions of the present technology includes a subject "suffering" from an inflammasome-mediated pulmonary disease or condition (i.e., a subject experiencing and / or exhibiting one or more clinical and / or subclinical symptoms of an inflammasome-mediated pulmonary disease or condition), as well as a subject "at risk" of developing an inflammasome-mediated pulmonary disease or condition. A subject "in need" of treatment includes animal models of an inflammasome-mediated pulmonary disease or condition. A subject "at risk" of developing an inflammasome-mediated pulmonary disease or condition refers to a subject who is not currently exhibiting symptoms of an inflammasome-mediated pulmonary disease or condition and who is predisposed to express one or more symptoms of the disease or condition. Such predisposition can be based on family history, genetic factors, environmental factors such as exposure to harmful compounds present in the environment, and the like. It is not intended to limit the present technology to any particular signs or symptoms. Thus, it is intended for the present technology to encompass subjects experiencing any range of disease or condition, from subclinical symptoms to full-blown inflammasome-mediated pulmonary disease, wherein the subject exhibits at least one sign (e.g., a sign and a symptom) associated with an inflammasome-mediated pulmonary disease or condition.

[0141] II. OVERVIEW

[0142] Activation of certain NLRs (NLRP1, NLRP3, and NLRC4) leads to the assembly of inflammasomes, which are large macromolecular signaling complexes that control the proteolytic activation of IL-1 family proinflammatory cytokines (e.g., IL-1 β and IL-18) in response to any array of stimuli, such as pathogens (e.g., viral or bacterial infection), environmental irritants, and endogenous danger signals. Increasing evidence suggests that inflammasomes play a key role in the pathogenesis of acute and chronic respiratory diseases. For example, inflammasome activation can be involved in acute lung inflammation following viral infection and during the progression of several chronic lung diseases, including idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), acute exacerbation of COPD (AECOPD), and asthma.

[0143] In one aspect, the technology provides methods, compounds, and compositions for treating, preventing, or ameliorating inflammasome-mediated diseases. In some embodiments, the inflammasome-mediated disease is an inflammasome-mediated lung disease. In some embodiments, the inflammasome-mediated lung disease includes acute or chronic respiratory inflammation-related diseases or diseases caused by pathogens, including but not limited to pandemic influenza (e.g., influenza A), Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and other bacterial infections, acute respiratory distress syndrome (ARDS), respiratory syndromes caused by novel emerging respiratory viruses (i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19, Middle East respiratory syndrome coronavirus (MERS-CoV) causing MERS, severe acute respiratory syndrome coronavirus (SARS-CoV) causing SARS), respiratory syndromes caused by rhinovirus, flavivirus, dengue virus, Zika virus, and / or Nile virus, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), acute exacerbation of COPD (AECOPD), asthma, and inflammasome-mediated lung diseases triggered by environmental exposure to irritants, such as but not limited to silica (e.g., silicosis), asbestos (e.g., asbestosis), smoke, cigarette smoke, and nanoparticles (such as titanium dioxide).

[0144] As demonstrated by the experimental examples set forth herein, the propenidazole analogs of the present technology (e.g., BT032, BT132, BT135, BT136, BT137, and BT159) are effective as inhibitors of inflammasome activation and are effective in methods for preventing or treating inflammasome-mediated diseases or conditions. The experimental examples also demonstrate that the propenidazole analogs of the present technology are effective in methods for preventing or treating inflammasome-mediated pulmonary diseases. Accordingly, because the propenidazole analogs of the present technology are effective in such methods, one of ordinary skill in the art will understand that the propenidazole analogs of the present technology (e.g., BT032, BT132, BT135, BT136, BT137, and BT159) are effective in methods for treating any inflammasome-mediated pulmonary disease and are not limited to the illustrative diseases / pathogens listed herein that cause inflammasome-mediated pulmonary diseases.

[0145] In some embodiments, treating or preventing and inflammasome-mediated pulmonary disease comprises reducing the production of pro-inflammatory cytokines, cellular infiltration in the lung, formation of ASC specks, and / or reducing the excessive inflammation that often accompanies inflammasome-mediated pulmonary diseases. In some embodiments, the pro-inflammatory cytokines comprise IL-1β, IL-18, IL-1α, IL-6, IL-33, TNF-α, MCP-1, and HMGB1.

[0146] III. Compounds of the Present Technology

[0147] The present technology provides compositions for treating inflammasome-mediated diseases. In some embodiments, the present technology provides compositions for treating inflammasome-mediated pulmonary diseases and conditions.

[0148] In some embodiments, the present technology discloses propenidazole analogs defined by Formula I:

[0149]

[0150] tautomers thereof and / or pharmaceutically acceptable salts thereof;

[0151] wherein

[0152] A is absent or selected from the group consisting of C(O)N(R 3 ), phenylene, oxazolylene, thiazolylene, piperidinylene, and

[0153]

[0154] L is absent or C 1-10 alkylene;

[0155] X is H, CHO, COOH, C(O)NR4 R 5 , COOR 6 , NH2or NHR;

[0156] R is 2-chloropyrimidin-4-yl;

[0157] R 1 and R 2 are independently substituted or unsubstituted C 1-6 alkyl, or R 1 and R 2 are H and the other is cyclohexyl-NH-C(O), or R 1 and R 2 together are C 4-6 alkylene and form a 5-, 6-, or 7-membered ring with the nitrogen to which they are attached, which is optionally substituted with phenyl;

[0158] R 3 and R 4 are independently selected from H or C 1-6 alkyl;

[0159] R 5 is selected from H, PEG, or C 1-6 alkyl; and

[0160] R 6 is selected from substituted or unsubstituted C 1-10 alkyl, C 2-10 alkenyl, or C 7-14 aralkyl.

[0161] In some embodiments of the compound of Formula I, A can be absent. In some embodiments, A can be C(O)N(R 3 ), wherein R 3 is H or C 1-6 alkyl. In some such embodiments, R 3 may be H or methyl. In some embodiments, A can be phenylene, oxazolylene, thiazolylene, piperidylene, or

[0162] For example, A can be phenylene.

[0163] In some embodiments of the compound of Formula I, L can be absent. In some embodiments, L can be C 1-10 alkylene, for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 alkylene or a range between and including any two of the foregoing L values.

[0164] In some embodiments of the compound of Formula I, X can be H. In some embodiments, X can be COOH. In some embodiments, X can be COOR 6 , wherein R 6 may be as defined herein. For example, R 6 may be substituted or unsubstituted C 1-10 alkyl, or C 1-6 alkyl, e.g., substituted or unsubstituted methyl or ethyl. R 6 may be substituted or unsubstituted C 2-10 alkenyl, or C 2-6 alkenyl, e.g., substituted or unsubstituted alkenyl. R 6 may be substituted or unsubstituted C 7-14 aralkyl, or substituted or unsubstituted C 7-10 aralkyl, e.g., substituted or unsubstituted benzyl or phenethyl. In some embodiments, X can be NH2or X can be NHR, wherein R is R is 2-chloroimidazol-4-yl. In some embodiments, X can be C(O)NR 4 R 5 , wherein R 4 and R 5 may be as defined herein. For example, R 4 may be H, or R 4 may be C 1-6 alkyl. In some embodiments, R 5 may be H. In some embodiments, R 5 may be C 1-6 alkyl. In some embodiments, R 5 may be polyethylene glycol (PEG).

[0165] The PEG can have any suitable geometry (linear, branched, multi-armed) and any suitable average molecular weight. In some embodiments, the PEG is a linear PEG. In some embodiments, the PEG can have an average molecular weight in the range of about 100 Da to about 40 kDa. (Unless otherwise specified, “average molecular weight” refers to weight average molecular weight.) In some embodiments, the average molecular weight of the polymer is about 100 Da, 200 Da, 300 Da, 400 Da, 500 Da, 550 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1 kDa, 1.5 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 7.5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, or any range between and including any two of these values. For example, the average molecular weight of the PEG can be in the range of about 500 Da to about 2 or to about 3 kDa.

[0166] In some embodiments, the PEG can be functionalized at one or more of its terminal ends with an amine (NH2) and / or an aldehyde (CHO) group, and include linear monoamines and monoaldehydes, linear diamines and dialdehydes, multi-armed amines and multi-armed aldehydes, branched mono-, di- and multi-armed amines and aldehydes, and multi-armed forked amines and aldehydes. The PEG can terminate in a hydroxyl or C 1-6 ether, e.g., methyl or ethyl ether. In some embodiments, the PEG can be functionalized at one end with an amine and at the other end with a hydroxyl or C 1-6 ether group. In some such embodiments, the PEG is a linear PEG.

[0167] In some embodiments of the compound of Formula I, R 1 and each of R 2 may independently be C 1-6 alkyl. The latter can optionally be substituted, e.g., with one or more (e.g., 1, 2, or 3) F, OH, CF3, C 3-7 cycloalkyl, or SO2-alkyl. In some embodiments, R 1 and R 2 together can be C 4-6 alkylene, and form a 5-, 6-, or 7-membered ring with the nitrogen to which they are attached. Thus, R 1 and R 2 together can be C 4-6 alkylene and form a pyrrolidine, piperidine, or azepane, each of which can optionally be substituted with a phenyl group. In some embodiments, one of R 1 and R 2 is H, and the other is cyclohexyl-NH-C(O).

[0168] In some embodiments of the compound of Formula I, A is absent, or is selected from the group consisting of C(O)N(R 3 ), phenylene, oxazolylene, thiazolylene, and piperidinylene, L is absent or is C 1-10 alkylene; X is H, COOH, or NH2; R 1 and R 2 are independently substituted or unsubstituted C 1-6 alkyl; and R 3 is selected from H or C 1-6 alkyl. In some embodiments, A can be absent, and L can be C 3-10 alkylene. In some embodiments, A can be phenylene, oxazolylene, thiazolylene, or piperidinylene, and L is absent or is C 1-5 alkylene. In some embodiments, X can be COOH or NH2.

[0169] As will be appreciated by those skilled in the art, the disclosure of any compound herein, including the compounds of Formula I and other probenecid analogs, also discloses tautomers thereof and / or pharmaceutically acceptable salts thereof. In some embodiments, the present technology discloses a probenecid analog defined by Formula Ia (BT004):

[0170]

[0171] In some embodiments, the present technology discloses a probenecid analog defined by Formula Ib (BT005):

[0172]

[0173] In some embodiments, the present technology discloses a probenecid analog defined by Formula Ic (BT006):

[0174]

[0175] In some embodiments, the present technology discloses a probenecid analog defined by Formula Id (BT007):

[0176]

[0177] In some embodiments, the present technology discloses a probenecid analog defined by Formula Ie (BT008):

[0178]

[0179] In some embodiments, the present technology discloses a probenecid analog defined by Formula If (BT009):

[0180]

[0181] In some embodiments, the present technology discloses a probenecid analog defined by Formula Ig (BT010):

[0182]

[0183] In some embodiments, the present technology discloses a probenecid analog defined by Formula Ih (BT011):

[0184]

[0185] In some embodiments, the present technology discloses a probenecid analog defined by Formula Ii (BT026):

[0186]

[0187] In some embodiments, the present technology discloses a probenecid analog defined by Formula Ij (BT027):

[0188]

[0189] In some embodiments, the present technology discloses a probenecid analogue (BT028) defined by formula Ik:

[0190]

[0191] In some embodiments, the present technology discloses a probenecid analogue (BT029) defined by formula Il:

[0192]

[0193] In some embodiments, the present technology discloses a probenecid analogue (BT030) defined by formula Im:

[0194]

[0195] In some embodiments, the present technology discloses a probenecid analogue (BT031) defined by formula In:

[0196]

[0197] In some embodiments, the present technology discloses a probenecid analogue (BT032) defined by formula Io:

[0198]

[0199] In some embodiments, the present technology discloses a probenecid analogue (BT033) defined by formula Ip:

[0200]

[0201] In some embodiments, the present technology discloses a probenecid analogue (BT034) defined by formula Iq:

[0202]

[0203] In some embodiments, the present technology discloses a probenecid analogue (BT041) defined by formula Is:

[0204]

[0205] In some embodiments, the present technology discloses a probenecid analogue (BT043) defined by formula It:

[0206]

[0207] In some embodiments, the present technology discloses a probenecid analog (BT052, also known as BT159) defined by Formula Iu:

[0208]

[0209] In some embodiments, the present technology discloses a probenecid analog (BT053) defined by Formula Iv:

[0210]

[0211] In some embodiments, the present technology discloses a probenecid analog (BT054) defined by Formula Iw:

[0212]

[0213] In some embodiments, the present technology discloses a probenecid analog (BT055) defined by Formula Ix:

[0214]

[0215] In some embodiments, the present technology discloses a probenecid analog (BT056) defined by Formula Iy:

[0216]

[0217] In some embodiments, the present technology discloses a probenecid analog (BT057) defined by Formula Iz:

[0218]

[0219] In some embodiments, the present technology discloses a probenecid analog (BT058) defined by Formula Iaa:

[0220]

[0221] In some embodiments, the present technology discloses a probenecid analog (BT132) defined by Formula Iab:

[0222]

[0223] In some embodiments, the present technology discloses a probenecid analog (BT133) defined by Formula Iac:

[0224]

[0225] In some embodiments, the present technology discloses a probenecid analog (BT134) defined by Formula Iad:

[0226]

[0227] In some embodiments, the present technology discloses a probenecid analog (BT135) defined by Formula Iae:

[0228]

[0229] In some embodiments, the present technology discloses a probenecid analog (BT136) defined by Formula Iaf:

[0230]

[0231] In some embodiments, the present technology discloses a probenecid analog (BT137) defined by Formula Iag:

[0232]

[0233] In some embodiments, the present technology discloses a probenecid analog (BT138) defined by Formula Iah:

[0234]

[0235] In some embodiments, the present technology discloses a probenecid analog (BT139) defined by Formula Iai:

[0236]

[0237] In some embodiments, the present technology discloses a probenecid analog (BT140) defined by Formula Iaj:

[0238]

[0239] In another aspect, the present disclosure provides a compound of Formula II:

[0240]

[0241] a tautomer thereof, and / or a pharmaceutically acceptable salt thereof;

[0242] wherein

[0243] A is absent, or is selected from the group consisting of C(O)N(R 3 ), phenylene, oxazolylene, thiazolylene, piperidinylene, and

[0244]

[0245] L 2 is absent or is C 1-12 alkylene or C 1-12 heteroalkylene, or a peptide comprising 2-10 amino acid residues;

[0246] X is H, CHO, COOH, C(O)NR 4 R 5 , COOR 6 , NH2, or NHR;

[0247] R is 2-chloropyrimidin-4-yl;

[0248] R 1 and R 2 are independently substituted or unsubstituted C 1-6 alkyl, or R 1 and R 2 are H, and the other is cyclohexyl-NH-C(O), or R 1 and R 2 together are C 4-6 alkylene and form a 5-, 6-, or 7-membered ring with the nitrogen to which they are attached, which ring is optionally substituted with phenyl;

[0249] R 3 and R 4 are independently selected from H or C 1-6 alkyl;

[0250] R 5 is selected from H, a polymeric carrier, or C 1-6 alkyl, wherein the polymeric carrier is a pharmaceutically acceptable polymer; and

[0251] R 6 is selected from substituted or unsubstituted C 1-10 alkyl, C 2-10 alkenyl, or C 7-14 aralkyl.

[0252] In the present aspect, propenecid (or an analog thereof, e.g., as defined in Formula II) is linked to a polymeric carrier via a linker L 2 In some embodiments, the linker can act as a spacer to pull the propenecid analog and the polymer apart to avoid, for example, interference with binding ability. The linker includes one or more atoms, e.g., one or more atoms selected from C, N, or O. In some such embodiments, the linker can further include one or more H atoms, e.g., NH, N(CH3), or CH2.

[0253] In some embodiments, the linker is a biodegradable linker. In some embodiments, the biodegradable linker includes an oligopeptide having 2 to 10 amino acid residues. The residues can be selected from naturally occurring amino acids.

[0254] In some embodiments, the linker includes substituted or unsubstituted C1-C zalkylene, cycloalkylene, cycloalkyl, heteroalkylene, alkenyl, or heteroalkenyl, where z can be any integer from 1 to 12, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. For example, the linker can include a C1-Czfluoroalkyl, where one or more of the hydrogen atoms are fluorine atoms, such as 1, 2, or 3 or more fluorines. In some embodiments, L 2 is a heteroalkylene containing one or two NH groups, including but not limited to (C1-C 10 alkylene)-NH (e.g., CH2CH2NH, CH2CH2CH2NH, CH2CH2CH2CH2NH, CH2CH(CH3)CH(CH3)CH2NH), (C n alkylene)NH(C p alkylene), where n, p are independently integers from 1-10, but n+p does not exceed 10 (e.g., CH2CH2CH2NH CH2CH2), NH-(C1-C 10 alkylene)NH (e.g., NH(CH2)5NH, NH(CH2)6NH, NH(CH2)8NH), or NH(C n alkylene)NH(C p alkylene), where n and p are integers as previously defined (e.g., NHCH2CH2CH2NH CH2CH2, NH(CH2)6NHCH2). In some embodiments, L 2 is a heteroalkylene containing one or two oxygen atoms, including but not limited to (C1-C 10 alkylene)-O (e.g., CH2CH2O, CH2CH2CH2O, CH2CH2CH2CH2O, CH2CH(CH3)CH(CH3)CH2O), (C n alkylene)O(C p alkylene), where n, p are independently integers from 1-10, but n+p does not exceed 10 (e.g., CH2CH2CH2OCH2CH2), O-(C1-C 10 alkylene)O (e.g., O(CH2)5O, O(CH2)6O, O(CH2)8O), or O(C n alkylene)O(C p alkylene), where n and p are integers as previously defined (e.g., OCH2CH2CH2O CH2CH2, O(CH2)6OCH2). In some embodiments, L 2 is a heteroalkylene containing O and NH groups, including but not limited to NH-(C1-C 10 alkylene)O (e.g., NH(CH2)5O, NH(CH2)6O, NH(CH2)8O), or NH(C n alkylene)O(Cp alkylene), wherein n and p are integers as previously defined (e.g., NHCH2CH2OCH2CH2, O(CH2)6NHCH2).

[0255] In some embodiments, the polymeric carrier is selected from the group consisting of PEG, dextran, periodate-oxidized dextran, polysialic acid (PSA), hyaluronic acid (HA), dextrin, hydroxyethyl starch (HES), poly(2- ethyl 2-oxazoline) (PEOZ), polyglutamic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(D,L-lactide-co-glycolide) (PLA / PLGA), poly(hydroxyalkyl methacrylamide), polyglycerol, 25 polyamidoamine (PAMAM), polyethylenimine (PEI), and a polypeptide (i.e., comprising a- amino acid residues). The polymer can have any suitable weight average molecular weight, e.g., from about 100 Da to about 40 kDa. In some embodiments, the average molecular weight of the polymer is about 100 Da, 200 Da, 300 Da, 400 Da, 500 Da, 550 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1 kDa, 1.5 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 7.5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, or any range between and including any two of these values. In some embodiments, the polymeric carrier is PEG and can have a structure disclosed herein above.

[0256] Further, the variables A, X, R, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 may also have any of the values disclosed herein, e.g., any of the values disclosed with respect to Formula I, as all such embodiments are also intended for use with Formula II.

[0257] Propenecid-polymer conjugates can be prepared using standard techniques known in the art. In some embodiments, a bifunctional linker containing at least two functional groups containing a heteroatom selected from N, O, and S, one of which is protected, can be conjugated using standard ester, thioester, and amide bond formation techniques. For example, a diaminoalkylene linker in which one amino group is protected by a polyurethane protecting group (e.g., Boc, Cbz, etc.) can be coupled to propenecid in the presence of a coupling agent (e.g., DCC, EDC / HOBt, etc.). Alternatively, a reactive ester, mixed anhydride, or acid halide derivative of propenecid can be prepared and reacted with the mono-protected diamine. (See, e.g., Bodanszky, M. and Bodanszky, A., The Practice of Peptide Synthesis, Springer- Verlag, New York, 1984.) The protecting group can be removed and the free amine reacted with an aldehyde derivative of the polymer under reductive conditions to provide the conjugate. Similarly, a linker having a protected aldehyde (e.g., 1,1-dimethoxy) and an amine can be coupled to propenecid, deprotected to form an aldehyde, and subjected to reductive amination with an amino-containing polymer to form the conjugate. Variations of these schemes using a, co-carboxyamines, a, co-amino alcohols, a, co-carboxy alcohols, a, co-amino thiols, etc. to link propenecid and a polymer will be readily appreciated by those skilled in the art.

[0258] IV. Uses of compositions of the present technology

[0259] The present technology provides methods for treating, preventing, or ameliorating an inflammasome-mediated pulmonary disease or condition in a mammalian subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of one or more probenecid analogs. In some embodiments, the inflammasome-mediated pulmonary disease or condition is a pathogen or caused by a pathogen selected from, but not limited to, one or more of the following: pandemic influenza (e.g., influenza A), Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, acute respiratory distress syndrome (ARDS), COVID-19, MERS, SARS, rhinovirus, flavivirus, dengue virus, Zika virus, and / or West Nile virus-induced respiratory syndrome, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), acute exacerbation of COPD (AECOPD), asthma, and silicosis. In some embodiments, the probenecid analog is a compound of Formula I and pharmaceutically acceptable salts thereof, including a compound of any one of Formulas la-la j. In some embodiments, the probenecid analog is a compound of Formula Io (BT032), lab (BT132), lac (BT133), lae (BT135), laf (BT136), or lag (BT137). In further embodiments, the probenecid analogs of the present technology reduce the production of pro-inflammatory cytokines and / or reduce cellular infiltration in the lung. In some embodiments, the probenecid analogs of the present technology reduce the secretion of IL-1b.

[0260] As demonstrated by the experimental examples set forth herein, the probenecid analogs of the present technology (e.g., BT032, BT132) are effective as inhibitors of inflammasome activation and are effective in methods for preventing or treating inflammasome-mediated pulmonary diseases or conditions. Accordingly, because the probenecid analogs of the present technology are effective in such methods, one of ordinary skill in the art will appreciate that the probenecid analogs of the present technology (e.g., BT032, BT132) are effective in methods for treating any inflammasome-mediated pulmonary disease or condition and are not limited to the illustrative diseases / pathogens causing the inflammasome-mediated pulmonary diseases or conditions listed herein.

[0261] V. Combination therapy

[0262] In some embodiments, the probenecid analogs of the present technology can be combined with one or more additional therapeutic agents for preventing, ameliorating, or treating an inflammasome-mediated pulmonary disease or condition.

[0263] In one embodiment, an additional therapeutic agent is administered to a subject in combination with a probenecid analog of the present technology (e.g., a compound of Formula I, including but not limited to Formula lo (BT032), Iab (BT132), Iac (BT133), Iae (BT135), Iaf (BT136), or Iag (BT137)) such that a synergistic therapeutic effect is produced.

[0264] In some embodiments, a probenecid analog of the present technology (e.g., a compound of Formula I, including but not limited to Formula lo (BT032), Iab (BT132), Iac (BT133), Iae (BT135), Iaf (BT136), or Iag (BT137)) is combined with one or more compounds for use in treating or preventing an inflammasome-mediated pulmonary disease or condition or a disease or condition caused by a pathogen including but not limited to pandemic influenza (e.g., influenza A), Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, acute respiratory distress syndrome (ARDS), COVID-19, MERS, SARS, rhinovirus, flavivirus, dengue virus, Zika virus, and / or West Nile virus-induced respiratory syndrome, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), acute exacerbation of COPD (AECOPD), asthma, and silicosis.

[0265] As demonstrated by the experimental examples set forth herein, the probenecid analogs of the present technology (e.g., BT032, BT132) are effective as inhibitors of inflammasome activation and are effective in methods for preventing or treating inflammasome-mediated pulmonary diseases or conditions. Accordingly, because the probenecid analogs of the present technology are effective in such methods, one of ordinary skill in the art will understand that the probenecid analogs of the present technology (e.g., BT032, BT132) are effective in methods for treating any inflammasome-mediated pulmonary disease or condition and are not limited to the illustrative diseases / pathogens that cause the inflammasome-mediated pulmonary diseases or conditions listed herein.

[0266] The multiple therapeutic agents can be administered in any order or even simultaneously. If administered simultaneously, the multiple therapeutic agents can be provided in a single, unified form or can be provided in multiple forms (as mere examples, as a single formulation or as two separate formulations). One of the therapeutic agents can be administered multiple times, or both therapeutic agents can be administered multiple times. If not administered simultaneously, the time between multiple administrations can vary from more than zero weeks to less than four weeks. Moreover, the combination methods, compositions, and formulations are not limited to the use of only two agents.

[0267] In some embodiments, the methods of the present technology can further comprise administering one or more antibiotics and / or anti-inflammatory agents. Examples of antibiotics / anti-inflammatory agents used alone or in combination in the methods of the present technology include, but are not limited to, tetracyclines, macrolide antibiotics (e.g., azithromycin), fluoroquinolones, ceftazidime, ciprofloxacin, levofloxacin, gentamicin, cefepime, aztreonam, carbapenems, ticarcillin, ureidopenicillins, isoniazid, rifampin, ethambutol, pyrazinamide, streptomycin, corticosteroids (e.g., hydrocortisone, cortisone, esamethasone, flurohydrocortisone, betamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, dexamethasone), non-steroidal drugs (e.g., COX inhibitors, LOX inhibitors, p38 kinase inhibitors), immunosuppressants (e.g., cyclosporine) and cytokine synthesis inhibitors, minocycline and doxycycline, and diuretics or any combination thereof.

[0268] VI. Mode of Administration

[0269] Any method known to those of skill can be employed for contacting a cell, organ, or tissue with a compound of the present technology. Suitable methods include in vitro, ex vivo, or in vivo methods.

[0270] In vitro methods typically involve a cultured sample. For example, cells can be placed in a reservoir (e.g., a tissue culture plate) and incubated with a compound under appropriate conditions suitable for obtaining the desired result. Suitable incubation conditions can be readily determined by one of skill in the art.

[0271] Ex vivo methods typically involve a cell, organ, or tissue removed from a mammal, such as a human. The cell, organ, or tissue can be, for example, incubated with a compound under appropriate conditions. The contacted cell, organ, or tissue is typically returned to the donor, placed in a recipient, or stored for future use. Thus, the compound is typically in a pharmaceutically acceptable carrier.

[0272] In vivo methods typically involve administering a compound of the present technology to a mammal, such as a human. When used in vivo for therapy, a compound of the present technology is administered to a mammal in an amount effective to obtain the desired result or to treat the mammal. The effective amount is determined during pre-clinical and clinical trials by methods well known to physicians and clinicians. The dosage and dosing regimen will depend on the degree of disease or condition in the subject, the nature of the compound of the present technology used, e.g., its therapeutic index, the subject, and the subject’s history.

[0273] A compound of the present technology, such as an effective amount of a compound of the present technology, useful in the methods of the present application, in a pharmaceutical composition or medicament, can be administered to a mammal in need thereof by any of a variety of known methods for administering pharmaceutical compositions or medicaments. A compound of the present technology can be administered systemically or locally.

[0274] The compounds of the present technology described herein can be incorporated into pharmaceutical compositions to be administered to a subject, alone or in combination, for the treatment or prevention of a disorder described herein. Such compositions typically comprise an active agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0275] In some embodiments, the pharmaceutical compositions of the present disclosure contain a pharmaceutically acceptable carrier and / or excipient suitable for oral administration of the compound or mixture in the form of a tablet, capsule, or pill, or parenteral, intravenous, intradermal, intramuscular, intracutaneous, subcutaneous, or transdermal administration.

[0276] Pharmaceutical compositions are typically formulated to be compatible with the intended route of administration. Administration of the pharmaceutical compositions of the present disclosure can be accomplished through any means known to those of skill in the art. Routes of administration include, but are not limited to, parenteral, intranasal / respiratory (e.g., inhalation), intravenous, intramuscular, intradermal, intraperitoneal, intratracheal, intracutaneous, subcutaneous, oral, transdermal (topical), sublingual, ocular, vaginal, rectal, and transmucosal administration. Systemic routes include oral and parenteral. Several types of devices are commonly used for inhalation administration. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhalers (DPI), spacer / holding chambers in conjunction with MDI, and nebulizers.

[0277] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the present disclosure to be formulated in tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by a subject to be treated. Oral pharmaceutical formulations can be obtained as solid excipient, which after addition of the suitable auxiliaries (if desired) are processed to give tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally, the oral formulations can also be formulated in saline or buffers in order to mimic the natural conditions in the body. The compounds of the present disclosure can also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository

[0278] Orally administrable pharmaceutical preparations include -push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The -push fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Microspheres that are formulated for oral administration can also be used. Such microspheres have been defined in detail in the art. All orally administrable formulations should be in a dose suitable for such administration.

[0279] For buccal administration, the compositions can take the form of tablets or troches formulated in the conventional manner.

[0280] For inhalation administration, the compounds used according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0281] In some embodiments, the compounds for use according to the present disclosure can be formulated for intrabronchial administration, e.g., in some embodiments, the compounds for use according to the present disclosure can be administered during bronchoscopic or microendoscopic surgery that delivers the compound to the alveolar space (i.e., microdosing in the lung).

[0282] When systemic delivery is desired, the compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules, or in multi-dose containers, with an added preservative. These compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0283] Pharmaceutical formulations intended for parenteral administration comprise an aqueous solution of the active compound in a water-soluble form. Alternatively, suspensions of the active compound can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or mediators include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of high-concentration solutions.

[0284] Alternatively, the active compound may be in powder form so that it can be prepared together with a suitable medium (e.g., sterile, pyrogen-free water) before use.

[0285] These compounds can also be formulated into rectal or vaginal compositions (such as suppositories or retention enemas) containing, for example, a conventional suppository base (such as cocoa butter or other glycerides).

[0286] In some embodiments, application is local and / or onto the luminal surface of the tissue to be treated. “Local” application of the composition means bringing the composition into contact with the skin. “Luminal surface” refers to the internal open space or cavity of a tubular organ, such as the central space inside an artery or vein through which blood flows; the interior of the gastrointestinal tract; the route of the bronchi in the lungs; the interior of the renal tubules and collecting ducts; and the route of the female reproductive tract, beginning with a single route through the vagina, splitting into two cavities within the uterus, both of which continue through the fallopian tubes.

[0287] In some embodiments, the compounds of this technology are applied topically and / or onto the luminal surface of the target tissue. This helps to reduce the potential systemic toxicity of the compounds.

[0288] Other delivery systems can include time-release, delayed release, or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increase convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. These include polymer-based systems such as poly(lexpolactide-co-glycolide), copolyoxalates, polycaprolactones, poly- esteramides, polyorthoesters, polyhydrosbutanoic acid, and polyanhydrides. Microencapsula- tion of drugs in these delivery systems is described in, for example, U.S. Patent No. 5,075,109. Delivery systems also include non-polymer systems that are biode-gradable, including lipids such as steroids and cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosion systems wherein the agent of the present disclosure is contained in a matrix that slowly dissolves, such as those described in U.S. Patent Nos. 4,452,775; 4,675,189; and 5,736,152, and (b) diffusion systems wherein the active component permeates at a controlled rate from a polymer, such as those described in U.S. Patent Nos. 3,854,480; 5,133,974; and 5,407,686. Additionally, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

[0289] Experimental Examples

[0290] The present technology is further illustrated by the following examples, which should not be construed as in any way limiting.

[0291] Example 1: Synthesis of Probenecid analogs.

[0292] Illustrative examples of the general synthesis of the probenecid analogs of the present technology are shown in Schemes 1-14.

[0293] Scheme 1 - Synthesis of BT132 (also referred to as BT032-1)

[0294]

[0295] General procedure for Suzuki biaryl coupling reaction. To a solution of boronate 1 (Scheme 1 ; 1.5 mmol, 1.0 equiv) in aqueous dioxane (5.0 mL) was added 4-(4-bromophenyl)butanoic acid (1.65 mmol, 1.1 equiv), potassium phosphate (4.5 mmol, 3.0 equiv) and palladium acetate ((Pd(OAc)2), 0.075 mmol, 0.5 equiv). The reaction mixture was stirred at 100 °C for 18 h. The reaction mixture was cooled to room temperature, diluted with water (10.0 mL), acidified with 1 M aqueous HC1 and extracted with ethyl acetate (10.0 mL x 2). The organic phase was washed with brine (20.0 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by chromatography. 4-(4'-(N,N-dipropylsulfamoyl)-[1,1 '-biphenyl]-4-yl)butanoic acid 3, also referred to herein as BT032, was obtained as a white solid (0.8 mmol, 76% yield). MS: m / z = 404 (M+H) + .

[0296] General procedure for amide coupling reaction and PEG amide formation. To a solution of acid 3 (Scheme 1 ; 1.0 mmol, 1.0 equiv) and polyethylene glycol amine ((PEG 550 -NH2), 1.2 mmol, 1.2 equiv) in dichloromethane (3.0 mL) was added BOP (benzotriazol-1 -yloxytris(dimethylamino)phosphonium hexafluorophosphate, 1.2 mmol, 1.2 equiv) and triethylamine (2.0 mmol, 2 equiv). The reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was poured into water and extracted with ethyl acetate (10.0 mL x 2). The combined organic layers were washed successively with 1 M aqueous HC1 (10 mL), saturated aqueous sodium bicarbonate (10 mL), brine (10.0 mL x 2), dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by combi-flash chromatography eluting with 5-10% MeOH in CH2CI2. PEG 550 amide BT032-1 was obtained as a white solid (55 mg, 0.26 mmol, 26% yield). MS (electrospray ionization (ESI) = 936 (average MW).

[0297] Scheme 2 - Synthesis of BT133 (also referred to as BT032-2)

[0298]

[0299] PEG 1000Amide BT032-2 was prepared from compound 3 (prepared as in Scheme 1) by adjusting the general procedure of Scheme 1 using NH2-PEG 1000 PEG amidation was performed. Reaction details are shown in the table below. BT032-2 was obtained as a white solid (100 mg) in 49% yield. MS (ESI) = 1386 (average MW).

[0300]

[0301] Scheme 3 - Synthesis of BT134 (also referred to as BT032-3)

[0302]

[0303] PEG 2000 Amide BT032-3 was prepared from compound 3 (prepared as in Scheme 1) by adjusting the general procedure of Scheme 1 using NH2-PEG 2000 PEG amidation was performed. Reaction details are shown in the table below. BT032-3 was obtained as a white solid (800 mg) in 67% yield. MS (ESI) = 2386 (average MW).

[0304]

[0305] Scheme 4 - Synthesis of BT135 (also referred to as BT0135) and BT138 (also referred to as BT0138)

[0306]

[0307] Preparation of BT0135. To a solution of fluoride 3 (its preparation method is described in the following literature: Kayumov, M., Advanced Synthesis & Catalysis 362 (4): 776-781 (2020)) (Scheme 4; 1.0 mmol, 1.0 equiv) and piperidine 4 (1.1 equiv) in dimethyl sulfoxide (5 mL) was added excess cesium carbonate (4.0 equiv). The reaction mixture was stirred at 90 °C for 16 hours. The reaction mixture was poured into water (5 mL), acidified with 1 M aqueous HC1 and extracted with ethyl acetate (10.0 mL x 2). The organic phase was washed with brine (20.0 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by chromatography. 4-(1-(4-(N,N-dipropylsulfamoyl)-phenyl)piperidin-4-yl)butanoic acid BT0135 was obtained as a white solid (0.53 mmol, 53% yield). MS: m / z = 411 (M+H) + .

[0308] PEG 2000Amide BT0138 was prepared by adapting the general procedure of Scheme 1 using NH2-PEG as shown in Scheme 4 2000 PEG amidation was performed. Reaction details are shown in the table below. BT0138 was obtained as a white solid in 48% yield (1.40 g). MS (ESI) = 2408 (average MW).

[0309]

[0310] Scheme 5 - Synthesis of BT136 (also referred to as BT0136) and BT139 (also referred to as BT0139)

[0311]

[0312] Compound 3 of Scheme 5 was prepared by adapting the general procedure of Suzuki coupling in Scheme 1 using boronate 1 and dibromothiazole 2. Reaction details are shown in the table below.

[0313]

[0314]

[0315] Preparation of BT0136. To an ice-cold solution of alcohol 6 (Scheme 5; 1.0 mmol, 1.0 eq) in acetonitrile (1.5 mL) was added excess of freshly prepared Jones reagent (2.67 molar; 0.5 mL). The reaction mixture was stirred at 0-5 °C for 2 h. The reaction mixture was poured into ice water (5 mL) and extracted with ethyl acetate (10.0 mL x 2). The combined organic layers were washed with brine (10.0 mL x 2), dried over Na2S04, filtered and concentrated under reduced pressure to get a residue. The residue was purified by chromatography. 4-(2-(4-(N,N-dipropylaminosulfonyl)phenyl)thiazol-4-yl)butanoic acid BT0136 was obtained as a white solid (0.3 mmol, 31% yield). MS: m / z = 411 (M+H) + .

[0316] PEG 2000 Amide BT0139 was prepared by adapting the general procedure of Scheme 1 using NH2-PEG as shown in Scheme 5 2000 PEG amidation was performed. Reaction details are shown in the table below. BT0139 was obtained as a white solid in 28% yield (160 mg). MS (ESI) = 2408 (average MW).

[0317]

[0318] Scheme 6 - Synthesis of BT137 (also referred to as BT0137) and BT140 (also referred to as BT0140)

[0319]

[0320] Acid BT137 was prepared by adjusting the general procedure of Scheme 1, Suzuki coupling using aryl bromide and boronate ester as shown in Scheme 6. Reaction details are listed in the table below. BT0137 was obtained as a white solid in 89% yield. MS: m / z = 445 (M+H) + .

[0321] PEG 2000 Amide BT0140 was prepared by adjusting the general procedure of Scheme 1, PEG amidation using NH2-PEG 2000 as shown in Scheme 6. Reaction details are listed in the table below. BT0140 (160 mg) was obtained as a white solid in 33% yield. MS (ESI) = 2442 (average MW).

[0322]

[0323] Scheme 7 - Synthesis of BT004

[0324]

[0325] To a solution of acid probenecid 1.1 (Cayman Chemical Company, 1180 East Ellsworth Rd., Ann Arbor, MI, 48108; 1 mmol, 1.0 eq) and n-hexylamine (1.2 eq) in dichloromethane (3.0 mL) was added benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP, 1.2 eq) and diisopropylethylamine (2.0 mmol, 2 eq). The reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was poured into water and extracted with ethyl acetate (10 mL x 2). The combined organic layers were washed successively with 1 M aqueous hydrochloric acid (HC1; 10 mL), saturated aqueous sodium bicarbonate (NaHC03; 10 mL), brine (10 mL x 2), dried over sodium sulfate (Na2S04), filtered and concentrated under reduced pressure to give a residue. The residue was purified by chromatography. 4-(N,N-dipropylaminosulfonyl)-N-hexylbenzamide BT004 (0.89 mmol, 89% yield) was obtained as a white solid. MS: m / z = 370 (M+H) + .

[0326] Scheme 8 - Synthesis of BT030

[0327]

[0328] To a cold solution of alcohol 2.1 (Intonation Research Laboratories, A-1B, Chilka Nagar Main Rd, Industrial Development Area, Nacharam, Secunderabad, Telangana 500076, India; 1 mmol, 1.0 eq) in dichloromethane (DCM; 5 mL) was added triethylamine (2 eq) followed by methanesulfonyl chloride (MsCl, 1.2 eq). The reaction mixture was stirred at 5 °C for 3 h. The reaction mixture was poured into water and extracted with ethyl acetate (10 mL x 2). The combined organic layers were washed successively with 1 M aqueous hydrochloric acid (HC1; 10 mL), saturated aqueous sodium bicarbonate (NaHC03; 10 mL), brine (10 mL x 2), dried over sodium sulfate (sodium sulfate), filtered, and concentrated under reduced pressure to give the corresponding methanesulfonate. The crude sulfonate was dissolved in dimethylformamide (5 mL) and the solution was cooled. Sodium azide (NaN3; 2 eq) was then added and the reaction mixture was stirred at 60 °C for 6 h. The reaction mixture was poured into water and extracted with ethyl acetate (10 mL x 2). The combined organic layers were washed successively with 1 M aqueous hydrochloric acid (HC1; 10 mL), saturated aqueous sodium bicarbonate (NaHC03; 10 mL), brine (10 mL x 2), dried over sodium sulfate (sodium sulfate), filtered, and concentrated under reduced pressure to give the corresponding azide 2.2. Compound 2.2 was used directly for the next reaction. The azide 2.2 was dissolved in ethanol (10 mL) and 10% palladium on carbon (Pd / C; 0.3 eq). The reaction mixture was stirred at ambient temperature and pressure under an atmosphere of hydrogen gas for 12 h. The reaction mixture was filtered to remove the Pd / C catalyst, the solvent was removed under reduced pressure to give a residue, which was purified by chromatography. 4-(4-aminobutyl)-N,N-dipropylbenzenesulfonamide BT030 was obtained as a white solid (0.45 mmol, 45% yield). MS: m / z = 389 (M-H).

[0329] Scheme 9 - Synthesis of BT031

[0330]

[0331] The same reaction conditions used for the preparation of BT030 were applied to the synthesis of BT031 starting from alcohol 3.1. 3-(4-aminobutyl)-N,N-dipropylbenzenesulfonamide BT030 was obtained as a white solid. MS: m / z = 389 (M-H).

[0332] Scheme 10 - Synthesis of BT053

[0333]

[0334] (6-(methylamino)hexyl)carbamic acid tert-butyl ester (4.2). To a solution of (6- hydroxyhexyl)carbamic acid tert-butyl ester 4.1 (4.0 g, 18.4 mmol) in DCM (100 mL) was added a solution of diiodomethane (9.37 g, 22.0 mmol) at 0 °C and the resulting mixture was stirred for 2 hours. The reaction was then quenched with a saturated solution of sodium bisulfite (100 mL) and a saturated solution of NaHC03(100 mL). The phases were separated and the aqueous phase was extracted with DCM (100 mL). The organic phases were combined and dried over MgS04, filtered and concentrated under reduced pressure to give the crude aldehyde. Freshly prepared EtOH (80 mL) was stirred at room temperature. After 2 minutes, a solution of MeNH2 2M in MeOH (73 mL) was added and the mixture was stirred vigorously. After 3 hours of stirring, the resulting mixture was cooled to 0 °C and NaBH4(731 mg, 19.3 mmol) was added. The reaction was stirred for 30 minutes and the resulting mixture was quenched with a saturated solution of NaHC03, the organic material was extracted with DCM (3 x 300 mL), dried over sodium sulfate and concentrated under reduced pressure to give the crude 4.2 (4.2 g, 99%). LC-MS: RT = 1.09 min; MS calculated for 230.35; mass found: [M-Boc+H]: 175.0. 1 H NMR (500 MHz, CDC13) δ 4.51 (s, 1H), 3.10 (d, J = 6.0 Hz, 2H), 2.64 - 2.52 (m, 2H), 2.43 (s, 3H), 1.54 - 1.46 (m, 4H), 1.44 (s, 9H), 1.33 (dd, J = 6.7, 2.9 Hz, 4H).

[0335] N-(6-((2-chloropyrimidin-4-yl)amino)hexyl)-4-(N,N-dipropylaminosulfonyl)-N- methylbenzamide (BT053). Triethylamine (10.3 mL, 73.6 mmol) was added to a solution of 4-(N,N-dipropylaminosulfonyl)benzoic acid 1.1 (5.25 g, 18.4 mmol) dissolved in DMF (46.0 mL). HATU (7.07 g, 18.4 mmol) was then added to the mixture at 0 °C and stirred for 5 minutes. A solution of tert-butyl (6-(methylamino)hexyl)carbamate 4.2 (4.24 g, 18.4 mmol) was added dropwise to the mixture at 0 °C over 15 minutes and the reaction was allowed to warm slowly to room temperature and stirred for 18 hours. EtOAc (50 mL) and water (50 mL) were then added and the organic layer was washed with water (2 x 50 mL), washed with a saturated solution of NaHC03(1 x 50 mL), washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude Boc-protected amine (8.0 g, 93%) which was used without purification in the next step. LC-MS: RT = 1.86 min; MS calculated: 497.69; mass found: [M-Boc+H]: 398.3. Trifluoroacetic acid (11.4 mL, 148 mmol) was added to a solution of the crude in DCM (36.9 mL). The reaction was stirred at room temperature for 5 hours. LC-MS was performed after the reaction and starting material was still present. Trifluoroacetic acid (2.0 mL) was added and the reaction was stirred for 18 hours. The mixture was washed with a saturated solution of NaHC03(2 x 20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude amine (7.65 g) which was used without purification in the next step. LC-MS: RT = 1.34 min; MS calculated: 397.58; mass found: [M+H]: 398.3. 2,4-Dichloropyrimidine (3.64 g, 23.9 mmol) was added to a solution of the crude and triethylamine (20.6 mL, 147 mmol) dissolved in DCM (46.0 mL). The reaction was stirred at 0 °C for 3 hours (LCMS was performed after the reaction and it was not complete), Et3N (4.0 mL) was added and the resulting mixture was stirred at room temperature for 18 hours. Water (40 mL) was then added and extracted with EtOAc (2 x 40 mL), washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was purified by flash column chromatography (220 g of silica, EtOAc / hexanes, 15 to 100%) to give compound BT053 (5.09 g, 54%) as a yellow oil. LC-MS: RT = 1.73 min; purity: 98.9%; MS calculated: 510.09; mass found: [M+H]: 510.3. 1H NMR (500 MHz, CDC13) δ 7.99 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 7.9 Hz, 2H), 6.26 (d, J = 6.0 Hz, 1H), 5.31 (s, 1H), 3.56 (t, J = 6.9 Hz, 1H), 3.22 - 3.13 (m, 2H), 3.09 (s, 6H), 2.96 (s, 3H), 2.89 (d, J = 11.7 Hz, 2H), 1.84 - 1.46 (m, 12H), 0.88 (t, J = 7.4 Hz, 6H).

[0336] Scheme 11 - Synthesis of BT054

[0337]

[0338] 4-(8-azidooctyl)-N,N-dipropylbenzenesulfonamide (5.2). Methanesulfonyl chloride (1.53 mL, 19.6 mmol) was added to a solution of 4-(8-hydroxyoctyl)-N,N-dipropylbenzenesulfonamide 5.1 (4.84 g, 13.1 mmol) and triethylamine (3.67 mL, 26.2 mmol) dissolved in DCM (43.7 mL) at 0 °C. The reaction was stirred at 0 °C for 30 min and at room temperature for 30 min. Water (30 mL) was then added and extracted with EtOAc (2 x 30 mL), washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude mesylate as a yellow oil. LC-MS: RT = 2.08 min; MS calcd for: 447.65; mass found: [M+H]]: 448.4. 1H NMR (400 MHz, CDC13) δ 7.71 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 4.24 (t, J = 6.5 Hz, 2H), 3.12 - 3.03 (m, 4H), 3.00 (s, 3H), 2.44 (t, J = 7.0 Hz, 2H), 1.84 - 1.75 (m, 2H), 1.69 - 1.59 (m, 2H), 1.55 - 1.43 (m, 8H), 0.86 (t, J = 7.4 Hz, 6H). Sodium azide (1.71 mL, 26.2 mmol) was added to a solution of the crude mesylate (13.1 mmol) dissolved in DMF (65.5 mL). The reaction was stirred at room temperature for 18 hours. EtOAc (30 mL) and water (30 mL) were then added and the organic phase was washed with water (3 x 30 mL), brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was purified by flash column chromatography (100 g silica, 1 to 20% EtOAc / hexanes) to give azide 5.2 as a colorless oil (3.65 g, 71% over 2 steps). LC-MS: RT = 2.32 min; MS calculated for: 394.67; mass found: [M-N2]: 367.4. 1 H NMR (400 MHz, CDC13) δ 7.71 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 4.24 (t, J = 6.5 Hz, 2H), 3.12 - 3.03 (m, 4H), 3.00 (s, 3H), 2.44 (t, J = 7.0 Hz, 2H), 1.84 - 1.75 (m, 2H), 1.69 - 1.59 (m, 2H), 1.55 - 1.43 (m, 8H), 0.86 (t, J = 7.4 Hz, 6H). Sodium azide (1.71 mL, 26.2 mmol) was added to a solution of the crude mesylate (13.1 mmol) dissolved in DMF (65.5 mL). The reaction was stirred at room temperature for 18 hours. EtOAc (30 mL) and water (30 mL) were then added and the organic phase was washed with water (3 x 30 mL), brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was purified by flash column chromatography (100 g silica, 1 to 20% EtOAc / hexanes) to give azide 5.2 as a colorless oil (3.65 g, 71% over 2 steps). LC-MS: RT = 2.32 min; MS calculated for: 394.67; mass found: [M-N2]: 367.4.

[0339] 4-(8-Aminooctyl)-N,N-dipropylbenzenesulfonamide (5.3). 10% by weight palladium on activated carbon (150 mg) was added to a solution of 4-(8-azidooctyl)-N,N-dipropylbenzenesulfonamide 5.2 (1.45 g, 3.67 mmol) dissolved in EtOAc (36.9 mL) and MeOH (3.69 mL). After the resulting mixture was flushed (x 3) and a balloon was added with hydrogen gas, the reaction was stirred at room temperature under a hydrogen atmosphere for 2 hours. The mixture was filtered through celite (MeOH for washing) and concentrated under reduced pressure to give the crude amine 5.3 as a yellow oil (1.23 g, 91%). LC-MS: RT = 1.57 min; MS calculated for: 368.58; mass found: [M+H]: 369.3.

[0340] 4-(8-aminooctyl)-N,N-dipropylbenzenesulfonamide hydrochloride (BT054 HC1 salt). Di-tert-butyl dicarbonate (1.53 mL, 6.67 mmol) was added to a solution of crude 5.3 (1.23 g, 3.34 mmol) dissolved in DCM (16.7 mL). The reaction was stirred at room temperature for 30 minutes. The resulting mixture was concentrated under reduced pressure and purified by flash column chromatography (100 g silica, 5 to 35% EtOAc / hexane) to give the Boc-protected amine as a yellow oil (1.45 g, 93%). LC-MS: RT = 2.24 min; MS calculated for: 468.69; mass found: [M-Boc+H]: 369.4. Hydrochloric acid (3.09 mL, 12.4 mmol) (4 M solution in dioxane) was added to a solution of the Boc-protected amine (1.45 g, 3.09 mmol). The reaction was stirred at room temperature for 1 hour. The resulting mixture was concentrated in a flask flushed with air to give BT054 HC1 salt as a white solid (1.20 g, 96%). LC-MS: RT = 1.64 min; purity: 95.5%; MS calculated for: 405.04; mass found: [M-Cl]: 369.2. 1 H NMR (500 MHz, CDC13) δ 8.28 (s, 3H), 7.69 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 3.08 - 3.03 (m, 4H), 2.95 (m, 2H), 2.64 (t, J = 7.7 Hz, 2H), 1.80 - 1.71 (m, 2H), 1.64 - 1.59 (m, 2H), 1.54 (dq, J = 14.9, 7.5 Hz, 4H), 1.42 - 1.35 (m, 2H), 1.30 (s, 6H), 0.86 (t, J = 7.4 Hz, 6H).

[0341] Scheme 12 - Synthesis of BT055

[0342]

[0343] 2-(4-(N,N-dipropylaminosulfonyl)phenyl)oxazole-4-carboxylic acid ethyl ester (6.3). Oxazole-4-carboxylic acid ethyl ester 6.1 (4.0 g, 28 mmol) was placed in a sealed tube (250 mL) with DBU (8.47 mL, 57 mmol), Pd(OAc)2(318.2 mg, 1.4 mmol) and Cy-John-Phos ligand (994 mg, 2.8 mmol). A solution of bromide 6.2 (9.0 g, 28 mmol) in dry dioxane (80 mL) was added and the resulting mixture was purged with nitrogen for 10 minutes. The mixture was stirred at 110 °C for 18 hours. After filtration through celite and concentration in vacuo, the crude product was purified by flash column chromatography (330 g silica, 5-35% EtOAc / hexanes) to give 6.3 as a white powder (6.70 g, 62%). LC-MS: RT = 1.86 min; MS calcd for: 380.46; mass found: [M+H]]: 381.2. 1 H NMR (400 MHz, CDC13) δ 8.32 (s, 1H), 8.25 - 8.22 (m, 2H), 7.93 - 7.88 (m, 2H), 4.44 (q, J = 7.1 Hz, 2H), 3.14 - 3.07 (m, 4H), 1.57 - 1.49 (m, 4H), 1.42 (t, J = 7.1 Hz, 3H), 0.87 (t, J = 7.4 Hz, 6H).

[0344] 4-(4-(bromomethyl)oxazol-2-yl)-N,N-dipropylbenzenesulfonamide (6.4). Lithium aluminium hydride (10.1 mL, 20.1 mmol, 2.0 M in THF) was added dropwise over 10 minutes to a solution of ethyl 2-(4-(N,N-dipropylaminosulfonyl)phenyl)oxazole-4- carboxylate 6.3 (3.83 g, 10.1 mmol) dissolved in THF (101 mL) at -40 °C (using dry ice and acetonitrile bath). The reaction was stirred at -40 °C for 2 hours. A solution of saturated Rochelle salt (30 mL) was then added to the reaction mixture at -40 °C, anhydrous sodium sulfate was added and the mixture was stirred vigorously at room temperature. The mixture was filtered and concentrated under reduced pressure to give the crude alcohol as a yellow solid (3.17 g, 93%). LC-MS: RT = 1.56 min; MS calculated for 338.42; mass found: [M+H]: 339.3. At 0 °C, phosphorus tribromide (1.78 mL, 18.7 mmol) was added dropwise to a solution of the crude alcohol (3.17 g, 9.37 mmol) dissolved in DCM (62.4 mL). The reaction was stirred at this temperature for 1 hour. The mixture was stirred at room temperature for 4 hours. Water (50 mL) was then added and extracted with EtOAc (2 x 50 mL), washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified with silica gel pad (30% EtOAc / hexane) to give 6.4 as a yellow powder (2.68 g, 71%). LC-MS: RT = 1.91 min; MS calculated for 401.32; mass found: [M+H]: 403.0.

[0345] ((2-(4-(N,N-dipropylaminosulfonyl)phenyl)oxazol-4-yl)methyl)triphenylphosphonium bromide (6.5). Triphenylphosphine (1.47 g, 5.61 mmol) was added to a solution of 6.4 (2.25 g, 5.61 mmol) dissolved in THF (22.4 mL). The reaction was refluxed for 18 hours. The resulting mixture was then concentrated under reduced pressure to give crude 6.5 as a light yellow solid (3.73 g, 100%). The material was used in the next step without purification. LC-MS: RT = 1.80 min; MS calculated for 663.60; mass found: [M-Br]: 583.4. 1H NMR (500 MHz, CDC13) δ 8.48 (d, J = 4.4 Hz, 1H), 7.90 (ddt, J = 9.4, 3.6, 1.3 Hz, 8H), 7.84 - 7.76 (m, 5H), 7.69 - 7.64 (m, 6H), 5.60 (d, J = 14.0 Hz, 2H), 3.13 - 3.04 (m, 4H), 1.60 - 1.50 (m, 4H), 0.87 (t, J = 7.4 Hz, 6H).

[0346] 4-(4-(5-(benzyloxy)pent-1-en-1-yl)oxazol-2-yl)-N,N-dipropylbenzenesulfonamide (6.7). At 0 °C, Dess-Martin periodinane (2.91 g, 6.73 mmol) was added to a solution of 4-(benzyloxy)butan-1-ol 6.6 (987 μL, 5.61 mmol) dissolved in DCM (28.1 mL). The reaction was stirred at room temperature for 1 h 30 min. Then a saturated solution of NaHC03(50 mL) and a saturated solution of Na2S203(50 mL) were added and the resulting mixture was stirred for 30 min, extracted with DCM (2 x 50 mL), washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude aldehyde as a yellow oil. The crude was used in the next reaction without purification. At -40 °C under argon, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.28 mL, 8.42 mmol) was added to a solution of the crude aldehyde and phosphonium salt 6.5 (3.72 g, 5.61 mmol) dissolved in DMF (28.1 mL). The reaction was stirred at this temperature for 1 h then at room temperature for 1 h 30 min. Then water (50 mL) and EtOAc (50 mL) were added, washed with water (2 x 50 mL), washed with brine (x 2), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was purified by flash column chromatography (silica 100 g, 10 to 100% EtOAc / hexane) to give 6.7 (1.78 g, 66%) as a yellow oil. LC-MS: RT = 2.25 min; MS calcd: 482.63; mass found: [M+H]: 483.4. 1H NMR (500 MHz, CDC13) δ 8.20 - 8.13 (m, 2H), 7.91 - 7.83 (m, 2H), 7.74 (s) and 7.57 (s) (1H, E / Z isomers), 7.37 - 7.31 (m, 5H), 6.56 (dt, J = 15.6, 7.0 Hz, 0.5H), 6.27 (ddd, J = 13.5, 5.3, 3.9 Hz, 1H), 5.83 (dt, J = 11.5, 7.4 Hz, 0.5H), 4.52 (s, 2H), 3.55 (dt, J = 11.4, 6.4 Hz, 2H), 3.14 - 3.08 (m, 4H), 2.60 (qd, J = 7.5, 1.6 Hz, 1H), 2.34 (q, J = 6.9 Hz, 1H), 1.89 - 1.77 (m, 2H), 1.60 - 1.50 (m, 4H), 0.87 (td, J = 7.4, 2.8 Hz, 6H).

[0347] 4-(4-(5-hydroxypentyl)oxazol-2-yl)-N,N-dipropylbenzenesulfonamide (6.8). Palladium hydroxide (20 wt. % Pd on wet carbon, 178 mg) was added to a solution of 6.7 (1.78 g, 3.69 mmol) dissolved in MeOH (3.35 mL) and EtOAc (33.5 mL). After rinsing (x 3) the resulting mixture and adding hydrogen gas with a balloon, the reaction was stirred at room temperature under a hydrogen atmosphere for 1 hour. The mixture was filtered through celite (EtOAc was used for washing) and concentrated under reduced pressure to give the crude alcohol 6.8 (1.11 g, 76%) as a white solid. LC-MS: RT = 1.75 min; MS calcd: 394.53; mass found: [M+H]: 395.3. 1 H NMR (500 MHz, CDC13) δ 8.20 - 8.13 (m, 2H), 7.91 - 7.83 (m, 2H), 7.74 (s) and 7.57 (s) (1H, E / Z isomers), 7.37 - 7.31 (m, 5H), 6.56 (dt, J = 15.6, 7.0 Hz, 0.5H), 6.27 (ddd, J = 13.5, 5.3, 3.9 Hz, 1H), 5.83 (dt, J = 11.5, 7.4 Hz, 0.5H), 4.52 (s, 2H), 3.55 (dt, J = 11.4, 6.4 Hz, 2H), 3.14 - 3.08 (m, 4H), 2.60 (qd, J = 7.5, 1.6 Hz, 1H), 2.34 (q, J = 6.9 Hz, 1H), 1.89 - 1.77 (m, 2H), 1.60 - 1.50 (m, 4H), 0.87 (td, J = 7.4, 2.8 Hz, 6H).

[0348] 4-(4-(5-azidopentyl)oxazol-2-yl)-N,N-dipropylbenzenesulfonamide (6.9). Methanesulfonyl chloride (885 μL, 11.4 mmol) was added to a solution of 6.8 (3.00 g, 7.60 mmol) and triethylamine (2.13 mL, 15.2 mmol) dissolved in DCM (38.0 mL) at 0 °C. The reaction was stirred at this temperature for 10 minutes and at room temperature for 50 minutes. Water (50 mL) was then added, extracted with EtOAc (2 x 50 mL), washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude mesylate as a yellow oil. The crude was used in the next step without purification. LC-MS: RT = 1.90 min; MS calcd for: 472.62; mass found: [M+H]: 473.3. Sodium azide (993 μL, 15.2 mmol) was added to a solution of the crude mesylate (7.60 mmol) dissolved in DMF (38.0 mL). The reaction was stirred at room temperature for 18 hours. EtOAc (50 mL) and water (50 mL) were then added, washed with water (3 x 50 mL), washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was purified by flash column chromatography (silica 100 g, 5 to 100% EtOAc / hexanes) to give 6.9 as a colourless oil (2.61 g, 82% over 2 steps). LC-MS: RT = 2.12 min; MS calcd for: 419.54; mass found: [M+H]: 420.3. 1 H NMR (500 MHz, CDC13) δ 8.14 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.49 (s, 1H), 3.29 (t, J = 6.9 Hz, 2H), 3.14 - 3.06 (m, 4H), 2.62 (t, J = 7.3 Hz, 2H), 1.78 - 1.44 (m, 10H), 0.87 (t, J = 7.4 Hz, 6H).

[0349] 4-(4-(5-aminopentyl)oxazol-2-yl)-N,N-dipropylbenzenesulfonamide hydrochloride (BT055 HC1 salt). Palladium (10 wt% on activated carbon, 261 mg) was added to a solution of 6.9 (2.61 g, 6.22 mmol) dissolved in MeOH (5.66 mL) and EtOAc (56.6 mL). After rinsing (x 3) the resulting mixture and adding hydrogen gas with a balloon, the reaction was stirred at room temperature under a hydrogen atmosphere for 1 hour. The mixture was filtered through celite (EtOAc used for washing) and concentrated under reduced pressure to give the crude amine. Hydrochloric acid solution (4 M in dioxane, 3 equivalents) was then added and the reaction stirred at room temperature for 30 minutes. The resulting mixture was concentrated under reduced pressure, stirred in MTBE and filtered to give BT055 HC1 salt as a white solid (2.33 g, 87%). LC-MS: RT = 1.48 min; purity: 97.02%; MS calculated: 430.00; mass found: [M-Cl]: 394.3. 1 H NMR (400 MHz, CDC13) δ 8.35 (s, 3H), 8.28 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.6 Hz, 2H), 7.65 (s, 1H), 3.10 (dd, J = 8.6, 6.7 Hz, 4H), 3.01 (m, 2H), 2.70 (t, J = 7.5 Hz, 2H), 1.81 (m, 4H), 1.54 (m, 6H), 0.86 (t, J = 7.4 Hz, 6H).

[0350] Scheme 13 - Synthesis of BT056

[0351]

[0352] 4-bromo-N,N-dipropylbenzenesulfonamide (7.2). Dipropylamine (31.9 mL, 230 mmol) was added to a solution of 4-bromobenzenesulfonyl chloride 7.1 (20.0 g, 76.7 mmol) dissolved in THF (153 mL) at 0 °C. The reaction was stirred at room temperature for 1 hour. A saturated solution of NH4CI (50 mL) was then added, extracted with EtOAc (2 x 50 mL), washed with brine, dried over sodium sulfate and concentrated under reduced pressure to give compound 7.2 as a white solid (24.6 g, 100% yield). LC-MS: RT = 1.95 min; MS calculated: 320.25; mass found: [M+H]: 320.1. 1 H NMR (400 MHz, CDC13) δ 8.35 (s, 3H), 8.28 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.6 Hz, 2H), 7.65 (s, 1H), 3.10 (dd, J = 8.6, 6.7 Hz, 4H), 3.01 (m, 2H), 2.70 (t, J = 7.5 Hz, 2H), 1.81 (m, 4H), 1.54 (m, 6H), 0.86 (t, J = 7.4 Hz, 6H).

[0353] 4-(8-hydroxyoct-1 -yn-1 -yl)-N,N-dipropylbenzenesulfonamide (7.4). Tetra(triphenylphosphine)palladium(0) (5.17 g, 4.39 mmol) was added to a solution of oct-7-yn-1 -ol 7.3 (4.52 g, 35.1 mmol), 4-bromo-N,N-dipropylbenzenesulfonamide 7.2 (5.62 g, 17.6 mmol) and Cul (3.34 g, 17.6 mmol) dissolved in diisopropylethylamine (88.0 mL). The reaction was stirred at 150 °C for 2 hours. EtOAc (50 mL) and a saturated solution of NH4CI (50 mL) were then added, extracted with EtOAc (2 x 50 mL), washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash column chromatography (220 g, EtOAc / hexanes, 5 to 100%) to give compound 7.4 as a yellow oil (4.80 g, 75%). LC-MS: RT = 1.91 min; MS calculated: 365.53; mass found: [M+H]: 366.3. 1 H NMR (400 MHz, CDCI3) δ 7.71 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 3.66 (t, J = 6.6 Hz, 2H), 3.11 - 3.02 (m, 4H), 2.43 (t, J = 7.0 Hz, 2H), 1.73 - 1.36 (m, 12H), 0.86 (t, J = 7.4 Hz, 6H).

[0354] 8-(4-(N,N-dipropylaminosulfonyl)phenyl)octanoic acid (BT056). 10 wt% Palladium on activated charcoal (1.11 g, 13.1 mmol) was added to a solution of 4-(8-hydroxyoct-1 -yn-1 -yl)-N,N-dipropylbenzenesulfonamide 7.4 dissolved in MeOH (87.5 mL). After rinsing (x 3) the resulting mixture and purging with a balloon of hydrogen gas, the reaction was stirred at room temperature under an atmosphere of hydrogen gas for 2 hours. The mixture was filtered (MeOH used for washing) and concentrated under reduced pressure to give the crude alcohol as a colourless oil (4.32 g, 89%). LC-MS: RT = 1.98 min; MS calculated: 369.56; mass found: [M+H]: 370.3. H NMR (400 MHz, CDCI3) δ 7.71 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 3.66 (t, J = 6.6 Hz, 2H), 3.11 - 3.02 (m, 4H), 2.43 (t, J = 7.0 Hz, 2H), 1.73 - 1.36 (m, 12H), 0.86 (t, J = 7.4 Hz, 6H). 1H NMR (400 MHz, CDC13) δ 7.69 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 3.63 (t, J = 6.6 Hz, 2H), 3.10 - 2.99 (m, 4H), 2.71 - 2.58 (m, 2H), 1.72 - 1.44 (m, 10H), 1.32 (m, 6H), 0.86 (t, J = 7.4 Hz, 6H). To a solution of the crude alcohol (974 mg, 2.64 mmol) in DCM (7 mL) containing Tempo (8.40 mg, 52.7 μmol) was added saturated aqueous NaHC03(7 mL) containing KBr (47.5 mg, 395 μmol) and TBAC (75.5 mg, 264 μmol). The resulting mixture was then cooled to 0 °C and a solution of NaOCl (6 mL, 8.57 mmol), saturated NaHC03(3 mL), and brine (3 mL) was added dropwise over 15 min. LC-MS showed the corresponding carboxylic acid and still some starting material alcohol remained. Another portion of NaOCl (6 mL) mixed with saturated NaHC03(3 mL) and brine (3 mL) was added over 5 min and the reaction was stirred for 15 min. No more alcohol was observed by LC-MS. The reaction was diluted with water (10 mL) and DCM (10 mL) and the phases were separated. The organic material (DCM) was extracted with water (2 x 10 mL). The combined aqueous phases were treated with 10% HC1 at room temperature to a pH of about 4. The organic material was then extracted from the resulting acidic aqueous phase with EtOAc (3 x 10 mL). It was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude was purified by flash column chromatography (50 g silica, EtOAc / hexanes, 10 to 100%) to give compound BT056 as a white solid (795 mg, 79%). LC-MS: RT = 1.93 min; purity: 97%; MS calcd: 383.55; mass found: [M-H]: 382.5. 1 H NMR (500 MHz, CDC13) δ 7.70 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 3.12 - 3.01 (m, 4H), 2.71 - 2.59 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.63 (s, 3H), 1.55 (dq, J = 14.9, 7.4 Hz, 4H), 1.34 (m, 3H), 0.87 (t, J = 7.4 Hz, 6H).

[0355] Scheme 14 - Synthesis of BT057

[0356]

[0357] 5-(2-(4-(N,N-dipropylsulfamoyl)phenyl)oxazol-4-yl)pentanoic acid (BT057). Tetrapropylammonium perruthenate (111 mg, 317 pmol) was added to a solution of 6.8 (1.25 g, 3.17 mmol), 4-methylmorpholine N-oxide (3.44 g, 28.5 mmol) and H2O (85.5 pL, 4.75 mmol) dissolved in MeCN (12.7 mL). The reaction was stirred at room temperature for 18 hours. The reaction was quenched with isopropanol (15 mL) and concentrated under reduced pressure. The crude product was purified by flash column chromatography (50 g silica, 0 to 30% MeOH / DCM) to give BT057 as a white solid. LC-MS: RT = 1.73 min; purity: 96.4%; MS calcd: 408.51 ; mass found: [M+H]: 409.0. 1 H NMR (500 MHz, CDC13) δ 8.13 (d, J = 8.7 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 7.50 (s, 1H), 3.15 - 3.07 (m, 4H), 2.64 (t, J = 6.7 Hz, 2H), 2.43 (t, J = 7.0 Hz, 2H), 1.80 - 1.71 (m, 4H), 1.60 - 1.47 (m, 4H), 0.87 (t, J = 7.4 Hz, 6H).

[0358] The following compounds were prepared using the procedure given above or by adapting the procedure above by using appropriate starting materials.

[0359] As shown in the table, each compound prepared gave the expected molecular ion or would give the expected molecular ion.

[0360] Compound Name Molecular Weight m / z [M+H] + ]] BT032 403.53 404 BT137 444.54 445 BT136 410.55 411 BT135 410.57 411 BT163 389.51 390 BT160 369.52 370 BT004 368.21 370 BT005 298.14 299.92 BT006 312.15 313.96 BT007 285.10 286.89 BT008 301.10 302.92 BT009 249.31 250.85 BT010 283.09 284.90 BT011 345.41 346.91 BT026 397.58 BT027 452.61 454.13 BT028 412.54 413.31 BT029 398.52 399.24 BT030 388.57 BT031 388.57 385.99* BT032 403.53 404 BT033 403.53 405.10 BT034 467.58 468.39 BT041 383.55 384.3 BT043 411.61 412.6 BT052 496.07 496.4 BT053 510.09 510.3 BT054 368.58 369.2 BT055 393.53 394.3 BT056 383.55 382.5* BT057 408.51 409.0 BT058 352.41 351.4*

[0361] Example 2: Inhibition of inflammasome activation by compounds of the present technology.

[0362] This example demonstrates the efficacy of compounds of the present technology to inhibit NLRP3 inflammasome activation in vitro, and that compounds of the present technology exhibit dose-responsive inhibition of inflammasome activation assessed by IL-1 beta secretion.

[0363] Mouse macrophages were stimulated in vitro. Immortalized wild-type C57BL / 6 bone marrow-derived macrophages (iBMDM) were grown in DMEM supplemented with 10% heat-inactivated FBS and 2 mM glutamine. iBMDM were seeded in 96-well plates 24 hours prior to incubation with lipopolysaccharide (LPS; 100 ng / mL) for 3 hours. Cells were then incubated with control probenecid compounds (probenecid dissolved in DMSO (Prob / D), probenecid dissolved in PBS (Prob / P)) or one of the probenecid analogs of the present technology (BT032, BT132, BT133, BT134, BT135, BT136, BT137, BT138, BT139, or BT140) at a concentration of 300 µM, 150 µM, 30 µM, or 3 µM for 1 hour prior to stimulation with NLRP3 inflammasome activators silica (250 µg / mL) or Nigericin (6 µM). Six hours later, cell supernatants were collected and IL-1β levels were quantified by ELISA. These experiments were performed in triplicate three times.

[0364] Results. As shown in Figure 2 and Figure 3A and 3B Treatment with probenecid analogs of the present technology reduced IL-1β secretion in a dose-dependent manner in response to different NLRP3 stimuli (i.e., silica and Nigericin). Overall, probenecid analogs BT032, BT132, BT133, and BT136 were more effective than control probenecid compounds.

[0365] Thus, these results demonstrate that probenecid analogs of the present technology are useful in methods of inhibiting NLRP3 inflammasome activation and treating or preventing inflammasome-mediated pulmonary diseases or conditions.

[0366] Example 3: Probenecid analogs of the present technology for prevention and treatment of inflammasome-mediated lung disease - pandemic influenza model. Figure 4A

[0367] This example demonstrates the ability of probenecid analogs of the present technology to reduce inflammasome-mediated disease or condition and excessive inflammation in vivo during a high-dose influenza A virus challenge in mice.

[0368] Mice were infected with influenza virus. Six to eight-week-old C57BL / 6 male and female mice were maintained in a pathogen-free facility. The influenza A virus strain used in this study was the HKx31 (H3N2) pandemic strain. Other strains, such as A / PR / 8 / 34 (H1N1), can also be investigated. Virus was grown in 10-day embryonated chicken eggs by standard procedures and titrated on Madin-Darby canine kidney (MDCK) cells.

[0369] For viral infection studies, five to fourteen male and female C57BL / 6 mice were randomized into treatment groups (BT032, BT133, BT135, or PBS). Mice were lightly anesthetized and intranasally infected with 10 5 plaque forming units (pfu) of HKx31 (H3N2) in 50-μL PBS. One day after infection on day 0, mice were treated with 40 mg / kg of a probenecid analog of the technology (BT032, BT133, BT135) in 50 μL PBS via the intranasal route one day after infection (day 1) and every 48 hours thereafter (e.g., day 1, day 3, day 5 after infection) or three days after infection (day 3) and every 48 hours thereafter (e.g., day 3, day 5, day 7 after infection). Control mice were treated with PBS alone. Mice were weighed daily and assessed for visual signs of clinical illness, including inactivity, roughened fur, labored breathing, and hunched behavior. Animals that lost >20% of their original body weight or exhibited severe clinical signs of illness were euthanized.

[0370] To analyze the effect of a probenecid analog of the technology on lung cell infiltration, groups of C57BL / 6 mice were intranasally infected with a high dose of HKx31 (105pfu; n=8 for PBS, IAV alone, and IAV with BT032 treatment; n=4 for BT032 alone (no infection)) on day 0 and treated once with 40 mg / kg of a probenecid analog of the technology (BT032) or PBS on day 3 after infection, 24 hours later, mice were euthanized. Immediately after euthanasia, bronchoalveolar lavage (BAL) fluid was obtained by lavaging the lungs three times with 1 mL of PBS.

[0371] Leukocytes were recovered and characterized from mice. For flow cytometry analysis, BAL cells were treated with red blood cell lysis buffer (Sigma Aldrich) and cell number and viability were assessed by trypan blue exclusion using a hemocytometer. BAL cells were incubated with Fc blocker and then stained with fluorescent dye-conjugated Ly6C, Ly6G, CD11c, and I-A b monoclonal antibodies (BD Biosciences, USA). Neutrophils (Ly6G + ), airway macrophages (CD11c + I-A b高 ), inflammatory macrophages (Ly6G - Ly6C +), as described in Tate et al., Scientific Reports 6, 27912 (2016). Live cells (propidium iodide negative) were analyzed using flow cytometry. Total cell counts were calculated from live cell counts by trypan blue exclusion.

[0372] Macrophage infiltration and pro-inflammatory chemokine concentrations from mice. C57BL / 6 mice (n = 8 per group) were infected intranasally with 10 5 PFU of HKx31 IAV. On day 3 post-infection, mice were treated with 20 mg / kg of BT032 or not treated and euthanized 24 hours later. Bronchoalveolar lavage (BAL) fluid (BALF) was obtained from mice and the number of macrophages (CD11c+, I-Ablow) and MCP-1 concentrations were determined by cytokine bead analysis, as determined by flow cytometry.

[0373] Results. Intranasal treatment of mice with the probenecid analogs of the present technology reduced disease clinical signs, including weight loss Figure 4B and 4D , and prolonged survival of mice post-infection Figure 4B , 4C and 4E). Specifically, BT032 prolonged survival of mice from 5 days post-infection to 7-9 days Figures 4A-4E , 4C and 4E).

[0374] These results demonstrate that early treatment of mice with the probenecid analogs of the present technology (at day 1 post-infection with HKx31 and every 48 hours thereafter) is effective in methods for treating or preventing NLRP3-mediated lung disease or condition and does not result in adverse effects Figures 4C-4E .

[0375] These results also demonstrate that treatment of mice with the probenecid analogs of the present technology starting at day 3 post-infection (determined to be the peak of disease following HKx31 (H3N2) challenge; Tate et al. (2016)) and every 48 hours thereafter (e.g., day 5, day 7) results in reduced disease clinical signs, including weight loss and prolonged survival Figures 5A-5F . Thus, these results demonstrate that the probenecid analogs of the present technology are effective in methods for treating NLRP3-mediated lung disease or condition, for example, in the late stages of severe and highly virulent influenza A infection.

[0376] As shown in Figure 5A , the probenecid analog treatment groups exhibited reduced biomarkers of excessive lung inflammation and cellular infiltration, including reduced total number of cellular infiltrates in the airways Figure 5B , as well as alveolar macrophages (Figure 5C neutrophils ( Figure 5D ), inflammatory Ly6C + macrophages ( Figure 5E ), dendritic cells ( Figure 5F ), and chemokine MCP-1 ( Example 4: Probenecid analogs of the present technology for prevention and treatment of inflammasome-mediated lung disease. ).

[0377] Accordingly, these results demonstrate that the probenecid analogs of the present technology are effective in methods for preventing and treating inflammasome-mediated lung disease or conditions.

[0378] Animal Model

[0379] This example will demonstrate the ability of the probenecid analogs of the present technology to reduce inflammasome-mediated disease or conditions and excessive inflammation in vivo during a high dose influenza A challenge in a subject.

[0380] Human Subject

[0381] Animal models suitable for use in this example include, but are not limited to, animal influenza models, such as those described herein. Those of skill in the art will appreciate that the following description is illustrative and can be applied to other animal models as appropriate.

[0382] Mice are infected with influenza virus. Six to eight-week-old C57BL / 6 male and female mice will be maintained in a pathogen-free facility. Influenza A virus strains used in this study include A / PR / 8 / 34 (H1N1) and HKx31 (H3N2). Virus is grown in 10-day embryonated chicken eggs by standard procedures and titrated on Madin-Darby canine kidney (MDCK) cells.

[0383] For virus infection studies, eight male and female C57BL / 6 mice are randomized into treatment groups. Mice are lightly anesthetized and intranasally infected with 50-μL PBS containing 10 5 pfu of HKx31 (H3N2) or 50 pfu PR8 (H1N1). Following infection, mice are treated with 40 mg / kg probenecid analogs in 50 μL PBS by intranasal route on day 3 post-infection and every 48 hours thereafter (e.g., on days 5, 7, 9, 11, etc., post-infection). Control mice are treated with PBS alone or 40 mg / kg of a probenecid analog control compound. Uninfected mice treated with probenecid analogs of the present technology are included for comparison. Mice are weighed daily and assessed for visual signs of clinical illness, including inactivity, roughened fur, labored breathing, and hunched behavior. Animals that lose >20% of original body weight or exhibit severe clinical signs of illness are euthanized.

[0384] Bronchoalveolar lavage (BAL) fluid was obtained immediately after euthanasia by rinsing the lungs three times with 1 mL PBS. The lungs were then removed and immediately frozen in liquid nitrogen. The titer of infectious virus in lung homogenates was determined by standard plaque assay on MDCK cells.

[0385] Proinflammatory cytokines in BAL fluid were quantified. To detect cytokines, BAL fluid was collected and stored at -80 °C. IL-1 β was quantified by ELISA. Levels of IL-6, CCL2, IFN-γ, IL-10, IL12p70, and TNF-α proteins were determined by Cytometric Bead Array and Mouse Inflammation Kit (Becton Dickinson).

[0386] Leukocytes were recovered and characterized from mice. For flow cytometry analysis, BAL cells were treated with red blood cell lysis buffer (Sigma Aldrich) and cell number and viability were assessed by trypan blue exclusion using a hemocytometer. BAL cells were incubated with Fc blocker and then stained with fluorescent dye-conjugated Ly6C, Ly6G, CD11c, and I-A b Monoclonal antibodies (BD Biosciences, USA) were used for staining. Neutrophils (Ly6G+), airway macrophages (CD11c+I-Abhigh), and inflammatory macrophages (Ly6G- Ly6C+) were quantified by flow cytometry as described in Tate et al., Sci. Rep. 6, 27912 (2016). Live cells (propidium iodide negative) were analyzed using a flow cytometer. Total cell counts were calculated from live cell counts by trypan blue exclusion.

[0387] Results. It is expected that intranasal treatment of mice with the propenecid analogs of the present technology will reduce disease clinical signs including weight loss and prolong survival of mice after infection. It is also expected that these results will show that treatment of uninfected mice with the propenecid analogs of the present technology does not result in weight loss or any clinical signs of disease.

[0388] Treatment of mice with a sulfisoxazole analog of the technology beginning on day 3 post-infection (determined to be the peak of disease following H3N2 challenge; Tate et al. (2016)) and every 48 hours thereafter (e.g., days 7, 9, and 11) is expected to result in reduced clinical signs of disease, including weight loss, and prolonged survival. Similarly, treatment of mice with a sulfisoxazole analog of the technology beginning on day 7 (time of onset of severe disease following PR8 H1N1 challenge; Tate et al. (2016)) and every 48 hours thereafter is expected to result in reduced weight loss, and improved survival and recovery. Furthermore, treatment groups are expected to exhibit reduced biomarkers of excessive lung inflammation and cellular infiltration, including reduced total number of cellular infiltrates in the airways, such as alveolar macrophages, neutrophils, inflammatory Ly6C + macrophages and dendritic cells (DCs), and reduced IL-1β production levels and excessive inflammation in the airways. Thus, these results will demonstrate that a sulfisoxazole analog of the technology is effective in methods for treating an inflammasome-mediated lung disease or condition, for example, in the late stage of a severe and high virulence influenza A infection.

[0389] Early treatment of mice with a sulfisoxazole analog of the technology is also expected to be effective in methods for treating or preventing an inflammasome-mediated lung disease or condition and not result in adverse effects. Treatment with a sulfisoxazole analog of the technology on day 1 (HKx31) or day 5 (PR8) post-infection and every 48 hours thereafter is expected to result in reduced clinical signs of disease, including weight loss, and prolonged survival. Collectively, these results will demonstrate that a sulfisoxazole analog of the technology is effective in methods for treating or preventing an inflammasome-mediated lung disease or condition at any stage of clinical presentation, without adverse effects or disease potentiation.

[0390] Example 5: Inhibition of NLRP1 and NLRP3 inflammasome by probenecid analogs of the present technology.

[0391] Human subjects diagnosed with or suspected of having an inflammasome-mediated lung disease or condition or related disorder and who are currently exhibiting one or more symptoms and / or pathologies of an inflammasome-mediated lung disease or condition or related disorder are recruited using selection criteria known and accepted in the art.

[0392] Preventive and therapeutic methods: A sulfisoxazole analog of the technology is administered to a subject at a dose and frequency commensurate with the stage and severity of the disease. In some embodiments, the compound is administered once per day, week, or month. In some embodiments, the compound is administered multiple times per day, week, or month.

[0393] To demonstrate prophylactic and therapeutic methods in humans, the sulfipsopyraide analogs of the present technology are administered to a subject prior to or after the symptoms and / or pathological development of an inflammasome-mediated pulmonary disease or condition or related disorder, and the reversal of symptoms / pathology or the attenuation of expected symptoms / pathology is assessed using methods known in the art.

[0394] Results: It is expected that the sulfipsopyraide analogs of the present technology will induce the reversal of symptoms and / or pathology of inflammasome-mediated pulmonary diseases or conditions and related disorders in human subjects. These results will demonstrate that the compounds of the present technology are useful and effective for the prevention and treatment of such disorders.

[0395] Figure 6A

[0396] This example demonstrates the efficacy of the compounds of the present technology to inhibit NLRP1 and NLRP3 inflammasome activation in vitro, and the compounds of the present technology exhibit dose-responsive inhibition of inflammasome activation assessed by IL-1 beta secretion.

[0397] IC50 curves. Immortalized BMDM grown in DMEM / 10% FCS, 2 mM glutamine under 5% CO2 were seeded at 4 x 105cells in 96-well format 20 hours before challenge with 100 ng / ml LPS E. coli 055:B5 for 3 hours. Macrophages were treated with drugs (3.9-350 mM) or vehicle (DMSO) for 60 minutes in serum-free medium, then challenged with Nigericin (3 mM) for 120 minutes. Secreted IL-1 beta in the cultured supernatant was determined by ELISA according to the manufacturer's instructions. 4

[0398] In vitro stimulation of human macrophages. Immortalized BMDM grown in DMEM / 10% FCS, 2 mM glutamine under 5% CO2 were seeded at 4 x 105cells in 96-well format 20 hours before challenge with 100 ng / ml LPS E. coli 055:B5 for 3 hours (differences in that LPS (B4) challenge was done with Pam3Cys; 100 ng / ml). Macrophages were treated with drugs (3.9-350 mM) or vehicle (DMSO) for 60 minutes in serum-free medium, then challenged with Nigericin (3 mM) for 120 minutes. Secreted IL-1 beta in the cultured supernatant was determined by ELISA according to the manufacturer's instructions. 4 ​Cells were seeded in 96-well format. Prior to challenge with NLR agonists, macrophages were treated with vehicle (DMSO), BT032 (20, 100 mM) or MCC950 (5 mM) for 60 minutes in serum-free media; Nigericin (3 mM; 120 minutes), monosodium urate crystals; MSU (250 pg / ml; 6 hours), silica MSU (250 pg / ml; 6 hours), L18-MDP (100 pg / ml; 16 hours), LPS serotype 0111:B4 (2 pg; 16 hours), poly dA:dT (1 pg; 6 hours) and flagellin (200 pg; 6 hours) were all complexed with Lipofectamine 2000. Secreted IL-1 b in the supernatant from culture was determined by ELISA according to the manufacturer’s instructions.

[0399] Results. As shown in Figure 6C and 6B , the half maximal inhibitory concentration (IC 50 ) of BT032 for inhibition of NLRP3- and NLRP1 -mediated inflammasome activity was 30 mM, respectively. As shown in Example 6: Inhibition of NLRP1 activation in human bronchial epithelial cells by probenecid analogs of the present technology. , treatment of macrophages with BT032 specifically inhibited NLRP3 (Nigericin, monosodium urate; MSU and silica) and NLRP1 (L18-MDP) induced inflammasome activation in a dose-dependent manner. However, BT032 had limited effect on non-canonical inflammasome activity (LPS (B4)) and had no effect on AIM2 (poly dA:dT) and NLRC4 (flagellin) mediated inflammasome activation. Importantly, while the specific NLRP3 inhibitor MCC950 inhibited Nigericin, MSU and silica inflammasome activation, unlike BT032, it had no effect on NLRP1 activity. These findings demonstrate that the probenecid analogs of the present technology (e.g., BT032) are specific NLRP1 and NLRP3 inhibitors.

[0400] Accordingly, these results demonstrate that the probenecid analogs of the present technology (e.g., BT032) are useful in methods of inhibiting NLRP3 and NLRP1 inflammasome activation and treating or preventing inflammasome-mediated diseases or conditions.

[0401] Figure 7

[0402] Epithelial cell NLRP1 is activated by double-stranded (ds) RNA (e.g., rhinovirus, coronavirus, flavivirus, dengue virus, Zika virus, West Nile virus infection, etc.). This example demonstrates the efficacy of the compounds of the present technology in inhibiting NLRP1 inflammasome activation in primary bronchial epithelial cells isolated from human subjects.

[0403] Methods. Primary bronchial epithelial cells (PBECs) were obtained from bronchial brushings and cultured on collagen-coated flasks in submerged conditions supplemented with bronchial epithelial growth medium. PBECs were seeded at 2.5 x 10 5 / ml cells in collagen-coated 96-well plates overnight. PBECs were treated with BT032 (20, 100, 350 mM), MCC950 (MCC; 5 mM), or vehicle (DMSO) for 60 minutes in serum-free medium, followed by challenge with high-molecular-weight poly(I:C) complexed with Lipofectamine 2000 for 10 hours. Secreted IL-1 b in the culture supernatant was determined by ELISA according to the manufacturer’s instructions.

[0404] Results. As shown in Example 7: BT132 is effective in a mouse model of pulmonary silicosis. Figure 6, treatment with the probenecid analog BT032 inhibited poly I:C NLRP1 activation in primary human bronchial epithelial cells, as evidenced by a dose-dependent decrease in IL-1 b secretion.

[0405] Accordingly, these results demonstrate that probenecid analogs of the present technology, such as BT032, are useful in methods of inhibiting inflammasome activation in human bronchial epithelial cells and treating or preventing inflammasome-mediated pulmonary diseases or conditions. These results further demonstrate that probenecid analogs of the present technology, such as BT032, are useful in methods of treating viral infections associated with dsRNA-activated NLRP1, such as respiratory viral infections caused by, but not limited to, rhinovirus, coronavirus, flavivirus, dengue virus, Zika virus, and West Nile virus.

[0406] In summary, Example 5 and Example 6 demonstrate that BT032 is capable of dual inhibition of NLRP3 and NLRP1 inflammasomes. Accordingly, these results demonstrate that probenecid analogs of the present technology, such as BT032, inhibit inflammation mediated by macrophages and epithelial cells and are useful in methods of preventing or treating inflammasome-mediated pulmonary diseases or conditions, such as AECOPD.

[0407] Figures 8B-8E

[0408] This example demonstrates the efficacy of compounds of the present technology in reducing inflammation associated with silica inhalation in a mouse model of pulmonary silicosis.

[0409] Methods. Groups of 5 C57B1 / 6 mice (6-8 weeks old) were anesthetized and treated intranasally with 50 μΐ PBS, 1 mg silica / PBS, or 1 mg silica / BT132 (40 mg / kg). Mice were euthanized 24 hours after challenge and BAL was obtained from euthanized mice by lavaging the lungs three times with 1 ml PBS. For flow cytometry analysis, BAL cells were treated with red blood cell lysis buffer and cell number and viability were assessed by trypan blue exclusion using a hemocytometer. BAL cells were incubated with Fc blocker (2.4G2) and then stained with fluorescent dye-conjugated Ly6C, Ly6G, CD11c, and I-Ab monoclonal antibodies (MHC-II). Neutrophils (Ly6G+) and airway macrophages (CD11c+I-Ablow) were quantified by flow cytometry. Live cells (propidium iodide negative) were analyzed using a flow cytometer and FlowJo software. Total cell counts were calculated from live cell counts by trypan blue exclusion. BAL IL-1 β concentration was determined by ELISA according to the manufacturer's instructions.

[0410] Results. As shown in Figure 1, while silica induced a strong inflammatory response, as evidenced by an increase in cells in the mouse BAL fluid (BALF), constituting increased neutrophils and macrophages, mice treated concurrently with BT132 showed significantly reduced lung leukocyte influx and IL-1 β concentration, indicating a reduced lung inflammatory burden. Example 8: BT032 inhibits Nigeria nystin-induced ASC speck formation.

[0411] Thus, these results demonstrate that the probenecid analogs of the present technology, such as BT132, are useful in methods of reducing levels of lung cell infiltration (immune cell infiltration) and cytokine levels and treating or preventing inflammasome-mediated lung diseases or conditions, including inflammasome-mediated lung diseases or conditions associated with inhaled irritants.

[0412] Figures 9B-9E

[0413] This example demonstrates the efficacy of compounds of the present technology in inhibiting ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) speck formation.

[0414] Methods. NLRP3-deficient immortalized macrophages stably expressing ASC-cerulean and NLRP3 were seeded in 8-well Ibidi chambered slides (2 x 105cells / well) 24 hours prior to stimulation. Cells were treated with 10 μΜ BT132 or vehicle (DMSO) for 1 hour prior to stimulation with 100 μg / ml LPS, 100 μg / ml silica, or 100 μg / ml silica + 10 μΜ BT132. Cells were imaged every 5 minutes for 1 hour using a Nikon Ti-Eclipse inverted fluorescent microscope with a 20x objective lens and a Hamamatsu ORCA-Flash 4.0 camera. Images were analyzed using NIS-Elements software. 5 ​Macrophages were treated in serum-free medium with BT032 (350 μM) or mordant (DMSO) for 60 min, followed by challenge with nigericin (3 μM; 90 min). Ten minutes prior to harvest, cells were treated with Hoechst 33342, washed, fixed in 4% paraformaldehyde, and stored in PBS. Six random fields were imaged using a 40 z-plane at 60x magnification. Images were z-stacked deconvolutioned using overlapping scans processed with ImageJ. ASC-blue spots were counted for each field, and the percentage of spots for each field was counted and expressed as the percentage of total Hoechst-positive cells.

[0415] Result. Figure 9B As shown, although ASCs exhibit diffuse activity in the cytoplasm of unstimulated cells ( Figure 9E However, when stimulated with nigrain, ASC staining appeared strong and punctate, indicating activation of inflammasomes in approximately 20% of the cells. Figure 9D However, when macrophages were pretreated with BT032, they showed a significant reduction in ASC spots in the cells. Example 9: Lipopolysaccharide (LPS) model of NLRP3 inflammation. and 9E This indicates that BT032 inhibits the formation of oligomeric inflammasomes. Importantly, since ASC-blue macrophages do not require stimulation to activate, this result also suggests that BT032 specifically inhibits the formation of oligomeric inflammasome complexes rather than stimulation (i.e., NF-κB activation and upregulation of IL-1β and NLRP3).

[0416] Therefore, these results indicate that probenecid analogs (such as BT032) of this technology can be used in methods for inhibiting inflammasome formation and for treating or preventing inflammasome-mediated diseases or symptoms.

[0417] Figure 10A

[0418] This example demonstrates the efficacy of the compounds of this technology in inhibiting NLRP3 inflammasome activation following acute intraperitoneal (ip) attack with LPS.

[0419] Internal LPS attack. For example... Figure 10B As shown, female C57BL / 6 mice (6-8 weeks old) were intraperitoneally injected with PBS or the drug (i.e., BT132 or BT032) one hour before intraperitoneal (ip) injection of 10 mg / kg LPS E. coli O55:B5 (Sigma-Aldrich) or PBS. Two hours later, the mice were sacrificed, and serum and peritoneal fluid IL-1β and TNF-α levels were measured by ELISA.

[0420] Results. Overall, the results indicate that the propenecid analogs of the present technology are effective in methods for reducing serum and peritoneal fluid IL-1 beta production Figure 10C and 10D ). Moreover, the results indicate that the effect is NLPR3 specific, as serum and peritoneal fluid TNF-alpha levels were not affected ​ and 10E ).

[0421] Accordingly, these results indicate that the propenecid analogs of the present technology (e.g., BT032 and BT132) are useful in methods of inhibiting inflammasome activation and treating or preventing inflammasome-mediated diseases or conditions.

[0422] Example 10: Inhibition of NLRP1 inflammasome by propenecid analogs of the present technology.

[0423] This example demonstrates the efficacy of compounds of the present technology to inhibit NLRP1 inflammasome activation in vitro, and that compounds of the present technology exhibit dose-responsive inhibition of inflammasome activation assessed by IL-1 beta secretion.

[0424] Human macrophages were stimulated in vitro. Immortalized BMDM grown in DMEM / 10% FCS, 2 mM glutamine at 5% CO2 were seeded at 4 x 10 4 cells in 96-well format for 20 hours. Prior to challenge with the NLRP1 agonist L18-MDP (100 pg / mL; 16 hours), macrophages were treated with BT032 (20, 100, 350 mM), BT135 (20, 100, 350 mM), BT136 (20, 100, 350 mM), BT137 (20, 100, 350 mM), or BT159 (also known as BT052) (20, 100, 350 mM) in serum-free media for 60 minutes (NS = non-stimulated cells). Secreted IL-1 beta in the cultured supernatants was determined by ELISA according to the manufacturer’s instructions and expressed as percent of maximal activation of untreated macrophages (“no drug”) in Figure 11 . Results from three independent experiments were pooled in triplicate where activity was normalized to percent activity relative to DMSO-treated control cells (“no drug”) and non-stimulated (NS) cells.

[0425] Results. As shown in Figure 11 , treatment of macrophages with BT032, BT135, BT136, and BT159 (also referred to herein as BT052) inhibited NLRP1 (L18-MDP)-induced inflammasome activation in a dose-dependent manner. These results indicate that the propenecid analogs of the present technology (e.g., BT032, BT135, BT136, BT159) are NLRP1 inhibitors.

[0426] Accordingly, these results demonstrate that the propenecid analogs of the present technology, such as BT032, BT135, BT136, BT159, can be used in methods of inhibiting NLRP1 inflammasome activation and treating or preventing inflammasome-mediated diseases or conditions.

[0427] Example 11: Human microdosing.

[0428] Human microdosing has been used to safely investigate the pharmacodynamic effects of experimental drugs prior to full phase 1 studies (Lewis, 2009). Up to 100 pg of drug can be evaluated for local dosing by IV administration as well as by direct instillation into the lung.

[0429] Methods. At time 0, microdoses (100 pg, 30 pg, 3 pg) of BT032 are delivered into single alveoli using a bronchial catheter with alveolar lavage: i) healthy human lung; ii) healthy human lung challenged with LPS (50 pg) by inhalation; and iii) lung of a diseased human (COPD, acute respiratory distress syndrome (ARDS)). At 1 hour, 4 hours, and 8 hours, lavage is removed from the alveoli and immune cells (e.g., macrophages, neutrophils, total cells) and cytokines (e.g., IL-1B, IL-6, IL-18, TNF-a, etc.) are evaluated. Immune cell quantification is determined by flow cytometry in conjunction with cell-specific markers. Cytokine levels are determined using standard ELISA assays.

[0430] Results. BT032 is not expected to induce an inflammatory response when microdosed into healthy human lung, as assessed by immune cell infiltration and cytokine levels. LPS challenge in healthy human lung is expected to result in significant infiltration of total cells, neutrophils, and macrophages, as well as significant elevation of cytokine levels. Administration of BT032 to LPS-challenged lung is expected to result in dose-dependent inhibition of immune cell infiltration and cytokine induction. Lavage from COPD and ARDS patients is expected to exhibit elevated levels of both immune cell infiltration and cytokines. Instillation of microdoses of BT032 is expected to result in dose-dependent reduction of these inflammatory markers.

[0431] Accordingly, these results will demonstrate that the compounds of the present technology are useful and effective for treating inflammasome-mediated diseases or conditions of the human lung.

[0432] Example 12: Propenecid analogs of the present technology for prevention and treatment of inflammasome-mediated lung disease AECOPD model.

[0433] This example will demonstrate the ability of the propene sulfonate analogs of the present technology to reduce inflammasome-mediated disease or condition and excessive inflammation in vivo during an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in a subject.

[0434] Animal model

[0435] Animal models suitable for use in this example include, but are not limited to, animal models of AECOPD such as the model described by Chow et al. (2017 Dec 20), Animal Models of Chronic Obstructive Pulmonary Disease, COPD - An Update in Pathogenesis and Clinical Management, Cormac McCarthy, IntechOpen, DOI: 10.5772 / intechopen.70262, available from www.intechopen.com / books / copd-an-update-in-pathogenesis-and-clinical-management / animal-models-of-chronic-obstructive-pulmonary-disease). Those skilled in the art will appreciate that the following description is illustrative and can be applied to other animal models as appropriate.

[0436] Quantification of pro-inflammatory cytokines in BAL fluid. To detect cytokines, BAL fluid was collected and stored at -80 °C. IL-1 b was quantified by ELISA. Levels of IL-6, CCL2, IFN-g, IL-10, IL12p70, MCP-1, and TNF-a proteins were determined by Cytometric Bead Array and Mouse Inflammation Kit (BD Biosciences).

[0437] Recovery and characterization of leukocytes from mice. For flow cytometry analysis, BAL cells were treated with red blood cell lysis buffer (Sigma-Aldrich) and cell number and viability were assessed by trypan blue exclusion using a hemocytometer. BAL cells were incubated with Fc blocker and then stained with fluorescent dye-conjugated Ly6C, Ly6G, CD11c, and I-A bMonoclonal antibodies (BD Biosciences, USA) were used for staining. Neutrophils (Ly6G+), airway macrophages (CD11c+I-Abhigh), inflammatory macrophages (Ly6G-Ly6C+) were quantified by flow cytometry as described in Tate et al., Sci. Rep. 6, 27912 (2016). Live cells (propidium iodide negative) were analyzed using a flow cytometer. Total cell counts were calculated based on live cell counts by trypan blue exclusion.

[0438] Results. It is expected that intranasal treatment of mice with the propenecid analogs of the present technology will reduce clinical signs of disease and extend survival of mice. In addition, it is expected that the treatment groups will exhibit reduced biomarkers of excessive lung inflammation and cellular infiltration, including reduced total number of cellular infiltrates in the airways, such as alveolar macrophages, neutrophils, inflammatory Ly6C + macrophages and dendritic cells (DCs), and reduced levels of IL-1 beta production and excessive inflammation in the airways.

[0439] Accordingly, these results will demonstrate that the propenecid analogs of the present technology are effective in methods for treating inflammasome-mediated lung disease, for example, in AECOPD.

[0440] Human subject

[0441] Human subjects diagnosed with or suspected of having an inflammasome-mediated lung disease or condition or related disorder and who are currently exhibiting one or more symptoms and / or pathologies of an inflammasome-mediated lung disease or condition or related disorder, such as AECOPD, are recruited using selection criteria known and accepted in the art.

[0442] Preventative and therapeutic methods: The propenecid analogs of the present technology are administered to a subject at a dose and frequency commensurate with the stage and severity of the disease. In some embodiments, the compound is administered once per day, week, or month. In some embodiments, the compound is administered multiple times per day, week, or month. In some embodiments, the propenecid analogs of the present technology are formulated for intrapulmonary administration.

[0443] To demonstrate preventative and therapeutic methods in humans, the propenecid analogs of the present technology are administered to a subject prior to or after the development of symptoms and / or pathologies of an inflammasome-mediated lung disease or condition, for example, AECOPD, or related disorder, and the reversal of symptoms / pathologies or the attenuation of expected symptoms / pathologies is assessed using methods known in the art.

[0444] Results: It is expected that the propenecid analogs of the present technology will induce the reversal of symptoms and / or pathology of inflammasome-mediated lung disease or condition (e.g., AECOPD) and related disorders in human subjects. These results will demonstrate that the compounds of the present technology are useful and effective for the prevention and treatment of such disorders.

[0445] equivalents

[0446] The present technology is not to be limited to the particular embodiments described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be apparent to those of ordinary skill in the art without departing from the spirit and scope of the invention. Accordingly, it is to be understood that this present technology is not to be limited to the specific methods and devices disclosed, and that modifications and other devices are intended to be included within the scope of the present technology. Such modifications and variations are intended to fall within the scope of the appended claims and their equivalents. It should be understood that the present technology is not limited to a particular method, reagent, compound, composition or biological system, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0447] Each publication and patent mentioned in this specification is incorporated herein by reference in its entirety for all purposes. Various modifications and variations to the described methods and systems of the present technology, which are intended to fall within the scope of the present technology, will be apparent to those skilled in the art from consideration of the specification and practice of the present technology disclosed herein. Although the present technology has been described and illustrated with respect to particular embodiments, it is not to be inappropriately limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the present technology which are apparent to the skilled person in the art or relevant fields are intended to be within the scope of the appended claims.

Claims

1. A compound having the structure of formula I, I Its tautomers and / or its pharmaceutically acceptable salts; in A is a phenylene oxide; L is C 3-10 Alkylene; X is COOH, C(O)NR 4 R 5 COOR 6 Or NHR; R is 2-chloropyrimidin-4-yl; R 1 and R 2 Independently for unreplaced C 1-6 Alkyl, or R 1 and R 2 One of them is H, and the other is cyclohexyl-NH-C(O); R 3 and R 4 Independently selected from H or C 1-6 alkyl; R 5 Selected from H, PEG or C 1-6 Alkyl; and R 6 Selected from unreplaced C 1-10 alkyl.

2. The compound according to claim 1, wherein R 3 It is H or methyl.

3. The compound according to any one of claims 1 to 2, wherein X is COOH, C(O)NR 4 R 5 Or NHR.

4. The compound according to any one of claims 1 to 2, wherein X is COOH.

5. The compound according to any one of claims 1 to 2, wherein X is COOR 6 .

6. The compound according to any one of claims 1 to 2, wherein X is C(O)NR 4 R 5 .

7. The compound according to any one of claims 1 to 2, wherein X is NHR.

8. The compound according to any one of claims 1 to 2, wherein R 1 and R 2 One of them is H, and the other is cyclohexyl-NH-C(O).

9. The compound according to claim 1, wherein... A is a phenylene oxide. X is COOH; R 1 and R 2 Independently for unreplaced C 1-6 Alkyl; and R 3 Selected from H or C 1-6 alkyl.

10. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is 。 11. A pharmaceutical composition comprising the compound of claim 10, its tautomers and / or a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

12. Use of the compound, its tautomer, and / or pharmaceutically acceptable salt, or the pharmaceutical composition according to any one of claims 1 to 2 or 10, in the preparation of a medicament for treating or preventing inflammasome-mediated diseases or symptoms in mammalian subjects in need.

13. The use according to claim 12, wherein the inflammasome-mediated disease or symptom is associated with NLRP1 inflammasome activation and / or NLRP3 inflammasome activation.

14. The use according to claim 12, wherein the inflammasome-mediated disease or symptom is an inflammasome-mediated lung disease or symptom.

15. The use according to claim 12, wherein the compound, its tautomers, and / or pharmaceutically acceptable salts, or the pharmaceutical composition comprises: 。 16. The use according to claim 14, wherein the inflammasome-mediated lung disease or symptom is caused by a pathogen selected from the group consisting of: pandemic influenza; Streptococcus pneumoniae; Pseudomonas aeruginosa; Mycobacterium tuberculosis; respiratory syndrome caused by rhinovirus, flavivirus, dengue virus, Zika virus, or West Nile virus; idiopathic pulmonary fibrosis (IPF); chronic obstructive pulmonary disease (COPD); acute exacerbation of chronic obstructive pulmonary disease (AECOPD); asthma; acute respiratory distress syndrome (ARDS); COVID-19; Middle East respiratory syndrome (MERS); severe acute respiratory syndrome (SARS); silicosis; and asbestosis.

17. The use according to claim 14, wherein the inflammasome-mediated lung disease or symptom is related to inhaled irritants.

18. The use according to claim 17, wherein the inhaled irritant comprises gas, mist, smoke, or dust.

19. The use according to claim 17, wherein the inhaled irritant is selected from the group consisting of: silica, asbestos, smoke, cigarette smoke, and nanoparticles.

20. The use according to claim 12, wherein the drug is administered from the group consisting of: intranasal administration, intramuscular administration, subcutaneous administration, inhalation administration, and oral administration.

21. The use according to claim 12, wherein treating or preventing inflammasome-mediated diseases or conditions comprises reducing the levels of one or more inflammatory cytokines in the subject compared to an untreated control subject.

22. The use according to claim 21, wherein the one or more inflammatory cytokines are selected from the group consisting of: IL-1β, IL-18, IL-1α, IL-6, IL-33, TNF-α, CCL2, IFN-γ, IL-10, IL12p70, MCP-1, HMGB1, and any combination thereof.

23. The use according to claim 22, wherein one or more inflammatory cytokines are IL-1β.

24. The use according to claim 14, wherein treating or preventing inflammasome-mediated lung disease or symptoms comprises reducing the level of cellular infiltration in the lungs of the subject compared to an untreated control subject, wherein said cellular infiltration comprises alveolar macrophages, neutrophils, inflammatory Ly6C cells, etc. + One or more of macrophages and dendritic cells.

25. The use according to claim 12, wherein treating or preventing inflammasome-mediated diseases or conditions comprises reducing apoptosis-associated speckle-like proteins in said subjects containing caspase activation and recruitment domain (ASC) speckle formation, compared to untreated control subjects.

26. Use of a composition in the preparation of a medicament for treating or preventing inflammasome-mediated diseases or symptoms, wherein the composition comprises a therapeutically effective amount of the compound of claim 1, its tautomers and / or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 11.

27. The use according to claim 26, wherein the inflammasome-mediated disease or symptom is associated with NLRP1 inflammasome activation and / or NLRP3 inflammasome activation.

28. The use according to claim 26, wherein the inflammasome-mediated disease or symptom is an inflammasome-mediated lung disease or symptom.

29. The use according to claim 27, wherein the compound, its tautomers, and / or pharmaceutically acceptable salts, or the pharmaceutical composition comprises: 。 30. The use according to claim 28, wherein the inflammasome-mediated lung disease or symptom is caused by a pathogen selected from the group consisting of: pandemic influenza; Streptococcus pneumoniae; Pseudomonas aeruginosa; Mycobacterium tuberculosis; respiratory syndrome caused by rhinovirus, flavivirus, dengue virus, Zika virus, or West Nile virus; idiopathic pulmonary fibrosis (IPF); chronic obstructive pulmonary disease (COPD); acute exacerbation of chronic obstructive pulmonary disease (AECOPD); asthma; acute respiratory distress syndrome (ARDS); COVID-19; Middle East respiratory syndrome (MERS); severe acute respiratory syndrome (SARS); silicosis; and asbestosis.

31. The use according to claim 28, wherein the inflammasome-mediated lung disease or symptom is related to inhaled irritants.

32. The use according to claim 31, wherein the inhaled irritant comprises gas, mist, smoke, or dust.

33. The use according to claim 31, wherein the inhaled irritant is selected from the group consisting of: silica, asbestos, smoke, cigarette smoke, and nanoparticles.

34. The use according to claim 26, wherein the drug is administered from the group consisting of: intranasal administration, intramuscular administration, subcutaneous administration, inhalation administration, and oral administration.

35. The use according to claim 26, wherein treating or preventing inflammasome-mediated diseases or conditions comprises reducing the levels of one or more inflammatory cytokines in the subject compared to an untreated control subject.

36. The use according to claim 35, wherein the one or more inflammatory cytokines are selected from the group consisting of: IL-1β, IL-18, IL-1α, IL-6, IL-33, TNF-α, CCL2, IFN-γ, IL-10, IL12p70, MCP-1, HMGB1, and any combination thereof.

37. The use according to claim 36, wherein one or more inflammatory cytokines are IL-1β.

38. The use according to claim 28, wherein treating or preventing inflammasome-mediated lung disease or symptoms comprises reducing the level of cellular infiltration in the lungs of the subject compared to an untreated control subject, wherein said cellular infiltration comprises alveolar macrophages, neutrophils, and inflammatory Ly6C cells. + One or more of macrophages and dendritic cells.

39. The use according to claim 26, wherein treating or preventing inflammasome-mediated diseases or conditions comprises reducing apoptosis-associated speckle-like proteins in said subjects containing caspase activation and recruitment domain (ASC) speckle formation, compared to untreated control subjects.

40. A compound, its tautomer and / or pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 1 to 2 or any one of claim 10, for the treatment or prevention of inflammasome-mediated diseases or symptoms in subjects in need.

41. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 40, wherein the inflammasome-mediated disease or symptom is associated with NLRP1 inflammasome activation and / or NLRP3 inflammasome activation.

42. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 40, wherein the inflammasome-mediated disease or symptom is an inflammasome-mediated lung disease or symptom.

43. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 40, wherein the compound, its tautomer, and / or pharmaceutically acceptable salt, or the pharmaceutical composition comprises: 。 44. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 42, wherein the inflammasome-mediated lung disease or symptom is caused by a pathogen selected from the group consisting of: pandemic influenza; Streptococcus pneumoniae; Pseudomonas aeruginosa; Mycobacterium tuberculosis; respiratory syndrome caused by rhinovirus, flavivirus, dengue virus, Zika virus, or West Nile virus; idiopathic pulmonary fibrosis (IPF); chronic obstructive pulmonary disease (COPD); acute exacerbation of chronic obstructive pulmonary disease (AECOPD); asthma; acute respiratory distress syndrome (ARDS); COVID-19; Middle East respiratory syndrome (MERS); severe acute respiratory syndrome (SARS); silicosis; and asbestosis.

45. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 42, wherein the inflammasome-mediated lung disease or symptom is related to an inhaled irritant.

46. ​​The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 45, wherein the inhaled irritant comprises a gas, mist, smoke, or dust.

47. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 45, wherein the inhaled irritant is selected from the group consisting of: silica, asbestos, smoke, cigarette smoke, and nanoparticles.

48. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 40, wherein the administration step is selected from the group consisting of: intranasal administration, intramuscular administration, subcutaneous administration, inhalation administration, and oral administration.

49. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 40, wherein treating or preventing inflammasome-mediated diseases or conditions comprises reducing the levels of one or more inflammatory cytokines in the subject compared to an untreated control subject.

50. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 49, wherein the one or more inflammatory cytokines are selected from the group consisting of: IL-1β, IL-18, IL-1α, IL-6, IL-33, TNF-α, CCL2, IFN-γ, IL-10, IL12p70, MCP-1, HMGB1, and any combination thereof.

51. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 50, wherein one or more inflammatory cytokines are IL-1β.

52. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 42, wherein treating or preventing inflammasome-mediated lung disease or symptoms comprises reducing the level of cellular infiltration in the lungs of the subject compared to an untreated control subject, wherein said cellular infiltration comprises alveolar macrophages, neutrophils, inflammatory Ly6C cells, etc. + One or more of macrophages and dendritic cells.

53. The compound, tautomer, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 40, wherein treating or preventing inflammasome-mediated diseases or conditions comprises reducing apoptosis-associated speckle-like proteins containing caspase activation and recruitment domain (ASC) speckle formation in said subjects compared to untreated control subjects.

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