WO2018103583A1

WO2018103583A1 - N-hydroxy-benzene-sulfonamide derivatives and their uses thereof

Info

Publication number: WO2018103583A1
Application number: PCT/CN2017/113857
Authority: WO
Inventors: Shijun Zhang; Jingde WU; Xiaoxiao SUN; Xiufang LIU; Xiangye LI
Original assignee: Zibo Anxuan Pharmaceutical Science And Technology Co., Ltd.
Priority date: 2016-12-05
Filing date: 2017-11-30
Publication date: 2018-06-14

Abstract

Inhibitors with anti-inflammatory agents are provided, as are methods of using the analogs to inhibit inflammation and prevent or treat diseases and conditions associated with inflammation, such as multiple sclerosis and autoinflammatory diseases.

Description

N-hydroxy-benzene-sulfonamide derivatives and their uses thereof

BACKGROUND OF THE INVENTION Field of the Invention

The invention generally relates to compounds that modulate immune responses and methods of their use to inhibit inflammatory responses involved in human diseases. In particular, the invention provides small molecule inhibitors of the NLRP3 inflammasome activation, and methods of using the analogs to prevent or treat NRLP3 inflammasome associated diseases and disorders such as multiple sclerosis, arthritis, gout, and autoimmune and autoinflammatory diseases.

Background of the Invention

Inflammatory diseases are one of the major health problems worldwide. Non-steroid antiinflammatory drugs (NSAIDs) are the most prescribed drugs for treatment of inflammatory diseases. The NSAIDs provide the patients with only symptomatic relief; however, they do not modify the pathogenesis of inflammation. Furthermore, prolonged use of NSAIDs may cause severe side effects particularly on gastric mucosa. Therefore, searching for new drug candidates in the treatment of chronic inflammation has great importance.

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which is composed of NLRP3, the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) , and caspase-1, is one of the essential components of innate immunity and has been extensively studied. NLRP3 inflammasome is believed to be critically involved in the maturation and production of interleukin (IL) -1β and IL-18. Emerging evidence have indicated a critical role for the NLRP3 inflammasome and IL-1β in the pathogenesis of many human diseases including multiple sclerosis (MS) , arthritis, gout, Alzheimer’s disease, atherosclerosis, metabolic syndrome, and age-related macular degeneration. Therefore, development of novel small molecular inhibitors that selectively target this protein platform may provide new opportunities to disease intervention for therapeutic benefits in the clinic.

The immunopathology of MS is characterized by the infiltration of myelin-reactive T cells into the central nervous system (CNS) and induction of demyelination which disrupts the communication of the nervous system. Although the exact etiology and pathogenesis of MS remain unknown, emerging evidence supports a critical role for NLRP3 inflammasome and IL-1β. The involvement of inflammasome in MS progression was first revealed by the observation that absence of the inflammasome products caspase-1, IL-1β and IL-18 rendered mice resistance to experimental autoimmune encephalomyelitis (EAE) , a mouse model that mimics human MS. Additionally, an increased nlrp3 expression was observed in the spinal cord of EAE mice, and NLRP3 deficiency substantially delayed onset and reduced severity of EAE symptoms, decreased neuroinflammation, demyelination and oligodendrocyte loss progression. Clinical studies showed that expression of caspase-1, IL-1β, and IL-18 was elevated in MS plaques and peripheral mononuclear cells of MS patients. Intriguingly, the effectiveness of IFN-β, a drug that has been used for more than 15 years as the first-line treatment for MS, is NLRP3 inflammasome dependent. Given the critical role of the NLRP3 inflammasome in the pathogenesis of this devastating disease, development of novel small molecular inhibitors with defined MOA will not only lead to a better understanding of the NLRP3 inflammasome in MS and potentially other inflammatory diseases, but also may provide new opportunities to disease intervention for therapeutic benefits in the clinic.

It has been well established that CD4⁺ T lymphocytes, IL-17A-producing T-helper cells (i.e., Th17 cells) in particular, play a causative role in autoimmune damage to the CNS in MS. There are high levels of IL-17 in both plaques and cerebrospinal fluid of patients with MS. IL-17 production by CNS-infiltrating T cells is correlated with blood–brain barrier disruption in MS patients. IL-17 deficiency or neutralization resulted in delayed disease onset, decreased severity scores and histological changes with early recovery of EAE. Our recent study discovered that inflammatory CD11b⁺Gr-1⁺ myeloid cells, which are excessively expanded several immune pathologies, including EAE, have a high capacity to drive a Th17 response through IL-1β production and contribute to the disease pathogenesis. Therefore, inhibition of NLRP3 inflammasome activation in CD11b⁺Gr-1⁺ myeloid cells will potentially reduce their activity in promoting a pathogenic Th17 response in MS, thereby leading to alleviation of the disease.

SUMMARY OF THE INVENTION

A family of N-hydroxy-benzene-sulfonamide analogs have been designed and the resulting compounds inhibit the formation and activity of the NLRP3 inflammasome and the production of IL-1β, thus acting as anti-inflammatory agents. Accordingly, the present disclosure provides novel N-hydroxy-benzene-sulfonamide analogs as anti-inflammatory agents, and methods of their use to inhibit inflammation, e.g. by inhibiting NRLP3 inflammasome formation and/or activity and the production of IL-1β. In some aspects, the agents and methods are used for the treatment of MS, e.g. in order to prevent neurodegenration associated with inflammation. In other aspects, the inhibitors are used to prevent or treat other NRLP3 inflammasome-mediated diseases such as gout and various auto-inflammatory diseases. The inhibitors are advantageously non-toxic when administered in vivo.

Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

It is an object of this invention to provide a compound of Formula I:

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

W is carbonyl or CH₂ or CH₂OH;

X is NH, O, or S; and

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl In one aspect, the compound is

wherein

R4 is halogen, amino, nitro or cyano;

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl

In another aspect, the compound is

The invention also provides compositions comprising each of these compounds, and variants thereof as described herein, combined with a physiologically acceptable carrier.

The invention also provides methods of preventing or treating NRLP3 inflammasome-associated inflammation in a subject in need thereof, comprising a step of administering to said subject a therapeutically effective amount of the compound of Formula I:

wherein

R4 is halogen, amino, nitro or cyano;

W is carbonyl or CH₂ or CH₂OH;

X is NH, O, or S; and

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

In some aspects of the methods, the compound is Formula II.

wherein

R4 is halogen, amino, nitro or cyano;

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

In other aspects of the methods, the compound is

In exemplary methods, the NRLP3 inflammasome-associated inflammation is selected from the group consisting of adverse cardiac remodeling after acute myocardial infarction (AMI) ; peritonitis, and an autoinflammatory condition.

The invention also provides methods of preventing or treating heart failure in a subject who has had multiple sclerosis (MS) , comprising a step of administering to said subject a therapeutically effective amount of the compound of Formula I:

wherein

R4 is halogen, amino, nitro or cyano;

W is carbonyl or CH₂ or CH₂OH;

X is NH, O, or S; and

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

In exemplary methods, the compound is

wherein

R4 is halogen, amino, nitro or cyano;

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

In additional exemplary methods, the compound is

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A. JC-171 treatment (100 mg/kg) inhibits disease progression of experimental autoimmune encephalomyelitis (EAE)

Fig. 1B. JC-171 treatment selectively blocks IL-1β production in EAE mice

Fig. 1C. JC-171 treatment reduced pathogenic Th17 response in EAE mice

Fig. 1D. JC-171 therapy at disease onset inhibits EAE and Th17 response

Fig. 1E. Treatment with JC-171 blocks MSU-induced IL-1β production

Figure 1F. JC-171 blocks NLRP3 inflammasome activation through interrupting NLRP3 and ASC interaction. Bone marrow derived macrophages (BMDCs) from C57BL/6 mice were pre-treated with JC-171 (400 μm) for 1.5 h followed by stimulation with LPS (100 ng/mL) and ATP (10 mM) to activate NLRP3 inflammasome. Culture media was collected 24 hours later for ELISA assay of the indicated cytokines (A) . Cells were also collected after LPS/ATP stimulation for immunoprecipitation (IP) assays using anti-NLRP3 antibodies (B) . Immunocimplexes were subjected to immunoblotting with anti-ASC antibodies. Total cell lysates were also used as input controls. Serum levels of IL-1β and TNF-α from C57BL/6 mice pretreated with JC-171 (100 mg/kg) or vehicle control as measured by ELISA 2.5 h after i.p. LPS injection (20 mg/kg) (C) . *, p < 0.05. NS, not significant.

Figure 1G. Therapeutic JC-171 treatment attenuates EAE severity and Th17 response in EAE mice. C57BL/6 mice (n = 5) were immunized with MOG peptide (200 μg) emulsified in CFA on day 0. JC-171 treatment (10 mg/kg) were started when the clinical scores of individual mice have reached 1 (flaccid tail) and continued every other days. EAE development in mice was followed, and clinical scores were recorded (A) . Twenty-eight days after immunization, spinal cords were dissected from mice and subjected to Luxol fast blue/periodic acid–Schiff staining for analysis of demyelination (B) . The frequency of IL-17A⁺ CD4⁺ Th17 cells in the spleen and spinal cord was determined by flow cytometry (C) . Bar=50 μm

DETAILED DESCRIPTION

Analogs that inhibit the NLRP3 inflammasome activity are provided, as are methods of their use to treat various NLRP3-inflammasome related diseases and disorers. The analogs have the generic structures of Formula I:

wherein

R4 is halogen, amino, nitro or cyano;

W is carbonyl or CH₂ or CH₂OH;

X is NH, O, or S; and

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

In one aspect, the compound is

wherein

R4 is halogen, amino, nitro or cyano;

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

In another aspect, the compound is

In the Examples section below and the accompanying figure legends, Formula III 5-chloro-N- (4- (N-hydroxysulfamoyl) phenethyl) -2-methoxybenzamide is also referred to as JC-171.

The analogs disclosed herein are used to treat any disorder or condition associated with (e.g. caused by or related to or which exacerbates) unwanted NLRP3 inflammasome activation and/or consequences of such activation, e.g. unwanted production of pro-inflammatory cytokines pro-IL-1β and pro-IL-18. Such diseases/conditions may be caused by so-called sterile inflammation (e.g. various inflammatory diseases, second wave inflammation after heart attack, stroke or other ischemic or traumatic injury) , or by inflammation that is caused by an infection (e.g. by an infectious organism such as a bacterium or virus) . Such diseases and conditions result from a wide array of stimuli. For example, numerous microbes including various bacteria, viruses, fungi, and protozoan parasites can activate the NLRP3 inflammasome, e.g., the bacterial toxin nigericin has also been reported to induce the activation of NLRP3 by causing potassium efflux in a pannexin-1-dependent manner. In addition to microbial activators, endogenous “danger” signals such as ATP, monosodium urate (MSU) activate the NLRP3 inflammasome, as do various other types of cellular damage resulting e.g. from metabolic stress, ischemia and trauma. The NLRP3 inflammasome is implicated in metabolic disorders and sterile inflammatory responses including multiple sclerosis, arthritis, type II diabetes mellitus, gout and ischemia. A number of endogenous and exogenous crystalline molecules activate the NLRP3 inflammasome, e.g. uric acid crystals and calcium pyrophosphate dihydrate, the causative agents of gout and pseudogout respectively. Silica and asbestos particles, which cause the fibrotic lung disorders silicosis and asbestosis respectively, also activate the NLRP3 inflammasome. Release of ATP from necrotic cells is a danger signal that activates the innate or sterile inflammatory immune response. Inhibiting NLRP3 inflammasome activation has beneficial effects in preventing the damage mediated by the sterile inflammatory response in diseases such as renal-, cardiac-, and cerebral-ischemia. In addition, necrosis-induced sterile inflammation in trauma and secondary to infections and sepsis are modulated by the inhibitors of the NLRP3 pathway described herein. The NLRP3 inflammasome can also be activated by molecules associated with stress or danger, including crystalline and particulate substances.

Examples of particular auto-inflammatory diseases which may be prevented or treated by the agents described herein include but are not limited to:

i) Joint, bone and muscle diseases such as rheumatoid arthritis, psoriatic arthritis, osteoarthritis, ankylosing spondylitis, erosive osteoarthritis of the hand, recurrent multifocal osteomyelitis, traumatic knee injury; relapsing polychondritis, etc;

ii) Hereditary systemic autoinflammatory diseases such as familial Mediterranean fever (FMF) , cryopyrin-associated periodic syndrome (CAPS) ; Muckle-Wells Syndrome, TNF receptor-associated periodic syndrome (TRAPS) , hyper-IgD syndrome (HIDS) , periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) , deficiency of interleukin-1 (IL-1) receptor antagonist (DIRA) , etc; iii) Systemic inflammatory diseases such as systemic juvenile idiopathic arthritis, adult-onset Still’s disease, Schnitzler syndrome,

disease, PFAPA (Periodic Fever, Apthous Sstomatitis, Pharyngitis, Adenitis) , SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome, macrophage activation syndrome, etc; and

iv) Common inflammatory diseases such as gout, Type 1 diabetes, Type 2 diabetes, metabolic syndrome, insulin resistance, stroke, heart attack, myocarditis, cardiac toxicity due to drug or radiation, ischemic heart disease, cardiomyopathy on a familial or genetic basis, heart failure, cardiac arrest and anoxic brain injury, acute and chronic lung injury due to infection, ischemia, toxin, trauma; dry eye syndrome, pustular psoriasis; neutrophilic dermatoses; acute or chronic hepatitis due a virus, toxin, ischemia or drug; acute or chronic renal injury due to ischemia, hypertension, diabetes, toxin or drugs; sepsis, septic shock; etc.

In one aspect, the compounds are used to treat Multiple sclerosis (MS) . MS refers to all types of MS including relapse-remitting, secondary progressive, and primary progressive MS.

The present invention provides compositions comprising the compounds described herein, and/or pharmaceutically acceptable salts of the compounds. The compositions are generally for use in preventing or treating inflammation, e.g. inflammation caused by formation and activity of NLRP3 inflammasome. The compositions include one or more substantially purified compounds as described herein, and a pharmacologically suitable (compatible) carrier. The preparation of such compositions is known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated.

Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. In addition, the compositions may contain other agents with different but complementary activities, e.g. other anti-inflammatory agents, analgesics, blood thinners, antihistamines, etc. If it is desired to administer an oral form of the compositions, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The compositions of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of compound in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99%. Still other suitable formulations for use in the present invention can be found, for example in Remington's Pharmaceutical Sciences, Philadelphia, Pa., 19th ed. (1995) .

As used herein, "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention. These: salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-. beta. -hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts, " J. Pharm. Sci., 66, 1-19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like.

Precursors (generally inactive precursors) of the compounds which are metabolized after administration to yield the compounds/active agents described herein in an active form are also encompassed.

The therapeutic agents described herein are used alone or in combination with other suitable agents, e.g. other agents that prevent or treat inflammation (for example, by another mechanism) , including but not limited to: IL-1R antagonists such as anakinra; monoclonal antibodies against interleukin 1β such as canakinumab (Ilaris) ; various interleukin 1 binding proteins such as rilonacept; and the like. Accordingly, the compositions provided herein may include one or more of these additional agents.

The compositions (preparations) of the present disclosure may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to: by injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, and the like) , by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosal linings, and the like) , by inhalation, orally, intranasally, by ingestion of a food or probiotic product containing the antimicrobial peptide, topically (e.g. on areas such as eyes, skin, in ears or on inflamed areas) , as eye drops, via sprays, incorporated into dressings or bandages (e.g. lyophilized forms may be included directly in the dressing) , etc. Generally, the mode of administration is by injection so as to effect systemic distribution of the agent, or locally by direct application, via an appropriate means, at or near a site of inflammation or a site where inflammation is likely to occur.

The amount of a compound that is administered varies depending on several factors, including the disease or condition being treated, the stage of the disease, the overall health of the subject, the subject’s age, gender and weight, etc. In general, the amount is in the range of from about 0.01 to about 100 mg/kg of body weight, and usually is in the range of from about 1 to about 20 mg/kg of body weight. The subjects (patients) that are treated as described herein are generally mammals, e.g. humans, but veterinary applications of this technology are also encompassed, e.g. for companion pets such as cats and dogs.

The compounds of the disclosure are utilized to prevent and/or to treat conditions and/or diseases associated with (e.g. caused by) NRLP3 inflammasome activity (i.e. to treat NRLP3 inflammasome-associated inflammation) . By “prevent” we mean that the compounds are administered prophylactically to a subject who is likely to develop the disease or condition, but before symptoms or indications of disease develop, or early in development. For example, subjects who have experienced MS may be treated as described herein in order to prevent subsequent adverse cardiac remodeling during the “second wave” of inflammation. Alternatively, or in addition, the compounds may be administered in order to treat conditions/diseases that have already developed (e.g. when symptoms are already being exhibited, or are observable or measurable) . In this case, administration of the compounds ameliorates and may reverse the symptoms, or at least arrest the disease (e.g. prevent further disease development or progress) . Those of skill in the art will recognize that while a goal of prevention or treatment may be to completely prevent or alleviate disease symptoms, much benefit can also accrue if symptoms not fully eradicated but are lessened, decreased or their onset is slowed, even though a full-blown cure is not effected.

Methods of treating NRLP3 inflammasome-related diseases are provided. Such methods may include a step of identifying a subject in need of such treatment (e.g. a subject with one or more symptoms of an NRLP3 inflammasome-related disorder, or a subject who is likely to develop such a disorder) . For example, patients who have had a MS may be treated, as patients for whom there is reason to suspect the relapse of MS is likely to occur. The same is true for other conditions that are treated by the agents disclosed herein, i.e. a subject suitable for undergoing treatment may have one or more readily observable symptoms, or early symptoms, or a predisposition to development of the disease (e.g. genetically, due to life style, due to exposure to a substance that is known to cause inflammation, etc. ) that is being treated.

As indicated above, the present invention inter alia provides the specified compounds for use in a method of: method of preventing or treating NRLP3 inflammasome-associated inflammation, including neuroinflammations associated with MS, etc, as well as acute inflammation, or acute inflammatory response, which may occur in variety of illness in which an injury induces inflammation. Further, the present invention may provide the specified compound as an active therapeutic ingredient in the specified method. Further, the present invention may provide the specified compound for use in a method of treatment of the human or animal body by therapy, the method comprising the specified method.

Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms "a" , "an" , and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely, " "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

EXAMPLES

The synthesis of the exemplary compounds is described in Scheme 1

^aReagents and conditions: a) EDC, HOBt, Et₃N, methylene chloride; b) Chlorosulonic acid, methylene chloride; c) NH₂OH, N-methylmorpholine, MeOH.

EXAMPLE 1. Preparation of 5-chloro-2-methoxy-N-phenethylbenzamide (3) . 5-Chloro-2-methoxybenzoic acid 1 (1 mmol) and trimethylamine (Et₃N, 2 mmol) were dissolved in dimethyl formaldehyde (DMF, 25 mL) and cooled to 0 ℃. N- (3-dimethylaminopropyl) -N’-ethylcarbodiimide hydrochloride (EDC, 1.5 mmol) was added and after 30 min, hydroxybenzotriazole (HOBt, 1.5 mmol) was added. After 1 h, 2-phenylethanamine 2 (1 mmol) was added to the mixture and the reaction was allowed to warm to room temperature for overnight. The solution was concentrated under reduced pressure and H₂O was added. The solution was extracted with ethyl acetate (EtOAc) and combined organic phase was concentrated and purified by column chromatography (EtOAc/Hexane 20/80) to give compound 3 as a viscous oil (yield: 82%) . ¹H NMR (400 MHz, DMSO-d6) δ 8.22 (br. s., 1H) , 7.66 (br. s., 1H) , 7.50 (dd, J = 3.00, 8.80 Hz, 1H) , 7.32 (d, J = 7.22 Hz, 2H) , 7.28 (d, J = 8.03 Hz, 2H) , 7.20 -7.24 (m, 1H) , 7.16 (d, J = 9.46 Hz, 1H) , 3.81 (s, 3H) , 3.54 (q, J = 7.06 Hz, 2H) , 2.85 (t, J = 6.20 Hz, 2H) ; ¹³C NMR (100 MHz, DMSO-d6) δ 163.4, 155.7, 139.4, 131.5, 129.6, 128.7, 128.3, 126.1, 124.7, 124.3, 114.1, 56.2, 40.7, 34.9.

EXAMPLE 2. Preparation of 4- (2- [ (5-chloro-2-methoxyphenyl) formamido] ethyl) benzene-1-sulfonyl chloride (4) . Compound 3 (1.73 mmol) was dissolved in methylene chloride (2 mL) . To this, excess chlorosulfonic acid (1 mL) was added, and the solution stirred at 70 ℃ for 2 h. The reaction was cooled to room temperature, and then poured over crushed ice. The product was extracted into methylene chloride, and then concentrated under reduced pressure. The product was purified by column chromatography (EtOAc/Hexanes: 20/80 to 50/50) yielding compound 4 as a white solid (yield: 65%) . ¹H NMR (400 MHz, DMSO-d6) δ 8.20 (br. s., 1H) , 7.66 (s, 1H) , 7.56 (d, J = 8.28 Hz, 2H) , 7.50 (dd, J = 2.76, 8.78 Hz, 1H) , 7.22 (d, J = 8.53 Hz, 2H) , 7.16 (d, J = 9.04 Hz, 1H) , 3.80 (s, 3H) , 3.51 (q, J = 7.30 Hz, 2H) , 2.84 (t, J = 6.80 Hz, 2H) ; ¹³C NMR (100 MHz, DMSO-d6) δ 163.5, 155.7, 146.1, 139.8, 131.5, 129.5, 128.0, 125.6, 124.7, 124.3, 114.2, 56.2, 40.6, 34.6.

EXAMPLE 3. Preparation of 5-chloro-N- (2- [4- (hydroxysulfamoyl) phenyl] ethyl) -2-methoxybenzamide (JC-171) . Hydroxylamine (6.44 mmol) and N-methylmorpholine (0.2 mL) were dissolved in MeOH (2 mL) . To this, compound 4 (1.29 mmol) was added, and the solution was stirred for 4 h. H₂O was added, and the product was extracted into methylene chloride and concentrated. The product was purified by column chromatography (dichloromethane/methanol: 94/6) to give compound JC-171 as a white solid (yield: 56%) . ¹H NMR (400 MHz, DMSO-d6) δ 9.56 (d, J = 3.26 Hz, 1H) , 9.52 (d, J = 3.26 Hz, 1H) , 8.27 (t, J = 5.40 Hz, 1H) , 7.78 (d, J = 8.28 Hz, 2H) , 7.64 (d, J = 2.76 Hz, 1H) , 7.48 -7.53 (m, 3H) , 7.15 (d, J = 8.78 Hz, 1H) , 3.81 (s, 3H) , 3.55 (q, J = 6.70 Hz, 2H) , 2.94 (t, J = 6.90 Hz, 2H) ; ¹³C NMR (100 MHz, DMSO-d6) δ 163.6, 155.7, 145.2, 135.3, 131.5, 129.5, 129.2, 128.2, 124.8, 124.3, 114.2, 56.2, 40.2, 34.7.

EXAMPLE 4. Cell viability assays. J774A. 1 cells were plated into a 96-well plate (1 × 10⁵ cells/well) for 24 h in growth medium. Cells were then treated with different concentrations of JC-171 for 0.5 h. After treatment, 3- (4.5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT, 0.5%solution) was added. After incubation for 4 h, DMSO (100 μL) was added for 5 min. Absorbance at 570 nm was then immediately recorded using a FlexStation 3 plate reader.

EXAMPLE 5. Cell death rescue assays. J774A. 1 cells were plated into a 96-well plate (1 × 10⁵ cells/well) for 24 h in growth medium. Cells were primed with Escherichia coli 0111: B4 LPS (Sigma-Aldrich) (final concentration: 1 μg/mL) for 4.5 h. Next, JC-171 was added for 30 min. ATP (5 mM) was added at the same time when JC-171 was added to induce NLRP3 inflammasome formation, and cells were incubated another 30 min. Cell viability was measured by MTT assay as described above.

EXAMPLE 6. In vitro NLRP3 activation assays. J774A. 1 cells were primed and activated with LPS/ATP as described above. JC-171 was added at the time when ATP was added for 30 min. The supernatants were collected and levels of IL-1β were measured with a mouse IL-1β Enzyme-linked Immunosorbent Assay (ELISA) kit following the manufacturer’s instructions. BMDMs were similarly treated as described with J774. A1 cell line followed by ELISA analysis of IL-β production. To assess effect of JC-171 on induction of cytokine TNF-α or IL-6, BMDMs were pretreated with JC-171 (400 μM) for 2 h and then stimulated LPS (100 ng/mL) for 5.5 h, followed by ATP (5 mM) treatment for 30 min.

EXAMPLE 7. Western blotting assays. J774A. 1 cells were seeded on 6-well plates (2 × 10⁶ cells/well) in RPMI 1640 medium for 24 h. Cells were then primed and activated with LPS/ATP and treated with JC-171 as described above. After treatment, cells were collected and wash with ice-cold PBS twice. Cells pellets were lysed by sonication in a radioimmunoprecipitation (RIPA) buffer solution. Protein samples were quantified by the Bradford method. Equal amounts of protein (20.0 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) . Proteins were probed with primary antibodies overnight at 4 ℃: anti-NLRP3 (1: 800, abcam, USA) , anti-IL1β (1: 500, EMD Millipore, CA, USA) , anti-caspase-1 (P20) (1: 1000, Cell Signaling Technology, MA, USA) , anti-caspase-1 (P10) (1: 200, Santa Cruz Biotechnology, Santa Cruz, USA) and incubated with a 1: 1000 dilution of horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (Cell Signaling Technology, USA) . After washing three times in TBS-Tween 20 for 10 min, the proteins were visualized by employing chemiluminescent reagent (Thermo Fischer Scientific, Waltham, MA) . The blots were also probed with antibodies against α-tubulin to ensure equal loading of proteins.

EXAMPLE 8. Immunoprecipitation and immunoblotting. BMDM cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in modified lysis buffer (50 mM Tris-Cl (pH 7.4) , 1%Nonidet P-40, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) , 1 mM phenylmethylsulfonyl fluoride (PMSF) , 1 μg/ml each of aprotinin and leupeptin, and 1 mM Na₃VO₄) . 1 mg protein of cell extracts was incubated with 2 μg of anti-NLRP3 antibodies for 2 h at 4 ℃, followed by incubation with 40 μl of Protein A/G Plus-Sepharose beads (Santa Cruz Biotechnology, Inc. ) overnight at 4 ℃. The beads were washed with lysis buffer, and immune complexes were eluted by boiling in 2 × sodium dodecyl sulfate (SDS) Laemmli loading buffer for 5 min prior to immunoblotting analyses with indicated antibodies. For immunoblotting, the immune complexes were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were immunoblotted with antibodies for NLRP3 or ASC, followed by HRP-conjugated secondary antibodies. Specific bands were visualized using Pierce^TM ECL Western Blotting Substrate from ThermoFisher Scientific (Waltham, MA) . Band intensities of immunoprecipitated ASC or NLRP3 panel were quantified using Image Studio LITE (LI-COR biotechnology) software. The ratios of ASC/NLRP3 association were calculated with PBS control group set as 1.

EXAMPLE 9. LPS challenge in vivo and JC-171 treatment. C57BL/6 mice were injected intraperitoneally (i.p. ) with 20 mg/kg LPS (Sigma-Aldrich) or PBS one hour after JC-171 (100 mg/kg) or vehicle treatment. 2.5 h after LPS injection, serum levels of IL-1β and TNF-α were measured by ELISA.

EXAMPLE 10. EAE induction and JC-171 treatment. EAE was induced as we previously described. ³² Briefly, mice were immunized subcutaneously with 200 μg Myelin oligodendrocyte glycoprotein (MOG) _35–55 peptide (ProSpec, East Brunswick, NJ) emulsified in Complete Freund's Adjuvant (CFA) on day 0 followed by injection of 200 ng pertussis toxin (Sigma-Aldrich, St. Louis, MO) i. p. on

days

0 and 2. After the first immunization, the severity of EAE was monitored and graded daily in a blinded fashion on a scale of 0–5 as we previously described. For prophylactic treatment, JC-171 was administered to mice (100 mg/kg, i. p. ) on

days

0, 1 and 2; and every other days thereafter. Therapeutic treatment with JC-171 (10 mg/kg, i. p. ) or MCC950 (Sigma-Aldrich, 10 mg/kg, i. p. ) was initiated when the clinical scores of individual mice have reached 1 (flaccid tail) , and given every other days. Upon termination of the experiment, mice were transcardially perfused with ice-cold PBS followed by 4%paraformaldehyde. Spinal cords with vertebrae were removed. Paraffin-embedded sagittal sections of cervicothoracic spinal cord were stained with Luxol fast blue/periodic acid–Schiff to determine demyelination.

EXAMPLE 11. Analyses of immune response in EAE mice. Mononuclear cells (MNCs) were isolated from spinal cords of EAE mice using percoll gradient centrifugation after perfusion with PBS as we previously described. ³² MNCs were stimulated for 5 h in the presence of phorbol 12-myristate 13-acetate (10 nM; Sigma-Aldrich) , ionomycin (1 μM; EMD Biosciences) with the addition of brefeldin A (5 μg/ml, Biolegend) during the last 3 hours of culture. After surface staining with anti-CD4 mAbs for 30 min at 4℃, cells were fixed, permeabilized, and stained with anti–IL-17A mAbs for 30 min at 4℃. For analysis of Th17 cells in lymphoid organs, cells were stimulated with MOG_35–55 (1 μg/ml) overnight at 37℃, 5%CO2 first and stained with the mAbs as decribed above. Data were acquired using BD FACSCalibur and analyzed using FlowJo software (Tree Star, Ashland, OR) . Cytokine levels in the serum were assayed using the ELISA kits according to the manufacturer’s instructions.

Claims

A compound of Formula I:

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

W is carbonyl or CH₂ or CH₂OH;

X is NH, O, or S; and

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl
The compound of claim 1, wherein said compound is

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl
The compound of claim 1, wherein said compound is
A composition comprising

A compound of Formula I

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

W is carbonyl or CH₂ or CH₂OH;

X is NH, O, or S; and

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl
The compound of claim 4, wherein said compound is

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl
The compound of claim 4, wherein said compound is
A method of preventing or treating NRLP3 inflammasome-associated inflammation in a subject in need thereof, comprising a step of administering to said subject a therapeutically effective amount of the compound of Formula I:

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

W is carbonyl or CH₂ or CH₂OH;

X is NH, O, or S; and

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl
The compound of claim 7, wherein said compound is

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl
The compound of claim 7, wherein said compound is
The method of claim 7, wherein said NRLP3 inflammasome-associated inflammation is selected from the group consisting of multiple sclerosis; arthritis, gout, and an autoinflammatory condition.
A method of preventing or treating heart failure in a subject who has had multiple sclerosis, comprising a step of administering to said subject a therapeutically effective amount of the compound of Formula I:

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

W is carbonyl or CH₂ or CH₂OH;

X is NH, O, or S; and

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl
The compound of claim 11, wherein said compound is

wherein

R1 is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkyl or is unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted C1-C8 alkoxyl;

R4 is halogen, amino, nitro or cyano;

R2, R3 and R5 may be the same or different and are independently selected from H, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkoxyl, C1-C8 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted carbonyl, halogen, hydroxyl, amino, nitro and cyano;

Y is unbranched, branched, C1-C4 alkyl and may be present or absent;

Z is C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl, or NHR wherein R is selected from C1-C5 unbranched, branched, saturated, unsaturated, cyclic or acyclic, substituted or unsubstituted alkyl
The compound of claim 11, wherein said compound is