Patent Application
20150368195
The invention relates to a method for treating and/or preventing an inflammation disease comprising administering indoline compounds. Particularly, the method uses 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines or 1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles to treat and/or prevent inflammation diseases.
Cytokines are soluble proteinaceous substances produced by a wide variety of haemopoietic and non-haemopoietic cell types, and are critical to the functioning of both innate and adaptive immune responses. Apart from their role in the development and functioning of the immune system, and their aberrant modes of secretion in a variety of immunological, inflammatory and infectious diseases, cytokines are also involved in several developmental processes during human embryogenesis. Thus, cytokines often act locally, but can also have effects on the whole body. For example, cytokines are able to interact directly with the evolving biology of an injury, trauma, or disease. Compounds having cytokine mediating activity have application in rheumatoid arthritis and inflammation.
Inflammatory events play a central role in the pathology of disease conditions and this process is mediated by cytokines, a system of polypeptides that enable one cell to signal to initiate events in another cell that initiate inflammatory sequelae. Normally, the system acts as part of a defensive reaction against infectious agents, harmful environmental agents, or malignantly transformed cells. But when inflammation exceeds the requirements of its defensive role, it can initiate adverse clinical effects, such as arthritis, septic shock, inflammatory bowel disease, and a range of other human disease conditions.
As one example, fibrosis of organs occurs in such a manner that extracellular matrix is excessively accumulated in the organs through invasion or injury of organs due to some cause. Excessive deposition of extra cellular matrix (ECM) components such as fibronectin (FN) and type I collagen by organ fibroblasts is defined as fibrosis. Organ fibrosis is the final common pathway for many diseases that result in end-stage organ failure. Uncontrollable wound-healing responses, including acute and chronic inflammation, angiogenesis, activation of resident cells, and ECM remodeling, are thought to be involved in the pathogenesis of fibrosis. However, effective therapy for organ fibrosis is still unavailable
As another example, rheumatoid arthritis (RA) is a systemic chronic autoimmune disease that results in destructive arthropathy. The complex interactions between the synovial and immune system cells result in synoviocyte proliferation, release of inflammatory cytokines/chemokines that recruit immune cells into the affected joints and activate infiltrated cells, and expression of degradative enzymes, resulting in progressive joint damage. Thus, these two cell types are key effector cells in RA and provide targets for pathological investigation and drug development. Classic drugs used for treating RA fall into three categories: nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, and disease-modifying anti-rheumatic drugs (DMARDs). However, some adverse effects of these drugs remain major concerns. Recently developed therapies that targeted cytokines have a major impact on the disease course of RA, however, its usage may be difficult because of increased risks of infection and nonresponse rates. Therefore, novel treatments that target critical intracellular molecules in synovial inflammation are required.
Histone deacetylases (HDACs) are categorized into four categories: class I (HDAC1, 2, 3, and 8); class IIa (HDAC4, 5, 7, and 9) and class IIb (HDAC6 and 10); class III (SIRT1-7); and class IV (HDAC11). These are involved in the post-translational modifications of core histone and nonhistone proteins. Recent proteomic analyses have shown that a substantial number of key signal transduction components and transcription factors that regulate immune responses and inflammation are HDAC substrates (Choo Q Y, Ho P C, Lin H S. Histone deacetylase inhibitors: new hope for rheumatoid arthritis? Curr Pharm Des 2008; 14: 803-820; Shakespear M R, Halili M A, Irvine K M, Fairlie D P, Sweet M J. Histone deacetylases as regulators of inflammation and immunity. Trends Immunol 2011; 32: 335-343). Thus, HDAC inhibitors have been examined as possible anti-inflammatory agents. However, there are very few HDAC inhibitors that have been sufficiently developed to undergo clinical trials for RA treatment.
ITF2357 (givinostat) ameliorated joint inflammation and prevented cartilage and bone destruction in an animal model (Joosten L A, Leoni F, Meghji S, Mascagni P. Inhibition of HDAC activity by ITF2357 ameliorates joint inflammation and prevents cartilage and bone destruction in experimental arthritis. Mol Med 2011; 17: 391-396). However, a phase II safety and efficacy clinical trial of ITF2357 that evaluated patients with active systemic onset of juvenile idiopathic arthritis, but not those with RA, suggested that HDAC inhibitors still require considerable development for use as RA therapeutics.
Therefore, there is still a need to develop an anti-inflammatory candidate; particularly, a candidate against RA and fibrosis.
The invention provides a method for inhibiting cytokine release from a cell, comprising administering an effective amount of the compound having formula (I) or a pharmaceutically acceptable salt, prodrug or solvate thereof to a subject:
wherein is a single bond or a double bond;
R1 is SO2Ra, wherein Ra is a aryl unsubstituted or substituted by 1 to 3 substituent selected from the group consisting of: —OC1-10alkyl, halogen, —NO2, —NH2, —OH, —C1-6alkyl, —C2-10alkenyl, —C2-10alkynyl, —C3-10cycloalkyl, —C5-10cycloalkenyl, 6 to 10 membered aryl or 6 to 10 membered heteroaryl;
R2, R5 and R6 are each independently H, —OC1-10alkyl, halogen, —NO2, —NH2, —OH, —C1-10alkyl, —C2-10alkenyl or —C2-10alkynyl; and
R4 is H, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, aryl, 5 to 14 membered heteroaryl, C3-10cycloalkyl, C5-10cycloalkenyl, C5-14heterocycloC1-10alkyl, C5-14heterocyclo C2-10alkenyl, halo, cyano, nitro, ORb, SRb, S(O)Rb, CH═CH—C(O)NRcRd, NHC(O)—CH═CH—C(O)Rb, NHC(O)—CH═CH—C(O)NRcRd, SO2NRcRd, OC(O)Rb, C(O)NRcRd, NRcRd, NHC(O)Rb, NHC(O)NRcRd, or NHC(S)Rc, in which each of Rb, Rc, and Rd, independently, is H, hydroxy, C1-10alkoxy, C6-10aryloxy, C5-14heteroaryloxy, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C6-10aryl, C5-14heteroaryl, C3-10cycloalkyl, C5-10cycloalkenyl, C3-14heterocycloC1-6alkyl, or C5-14heterocycloC2-10alkenyl.
the invention provides a method for inhibiting HDACs 1, 2, 3, and 8 in a cell or a subject, comprising administering an effective amount of the compound having formula (I) or a pharmaceutically acceptable salt, prodrug or solvate thereof to the cell or subject.
In one embodiment, the inhibition of cytokine release is associated with an inflammatory disease, particularly, a chronic inflammation disease. The inflammatory disease include, but not limited to, arthritis, synovitis, vasculitis, conditions associated with inflammation of the bowel, atherosclerosis, multiple sclerosis, Alzheimer's disease, vascular dementia, pulmonary inflammatory diseases, fibrotic diseases, inflammatory diseases of the skin, systemic inflammatory response syndrome, sepsis, inflammatory and/or an autoimmune disorder (for example, autoimmune conditions of the liver, and/or the complications thereof. Preferably, the arthritis is osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthropathies like ankylosing spondylitis, reactive arthritis (Reiter's syndrome), psoriatic arthritis, enteropathic arthritis associated with inflammatory bowel disease, Whipple disease and Behcet disease, septic arthritis, gout (also known as gouty arthritis, crystal synovitis, metabolic arthritis), pseudogout (calcium pyrophosphate deposition disease) or Still's disease. Preferably, the fibrosis is pulmonary fibrosis, liver fibrosis or renal fibrosis.
(TRAP) stained, and the numbers of TRAP-positive multinuclear cells were counted (
The invention is, at least in part, based on the discovery that 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines and 1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles has great potential as a novel agent to be used in the treatment of inflammation-associated diseases, particularly, inflammatory arthritis and fibrosis.
The invention surprisingly found that the compounds of the invention show 2˜10-fold increases in activity compared to SAHA to suppress cytokine production. The compounds of the invention also caused markedly reduction in acute inflammation. Taken together, these results indicate that 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines and 1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles HDAC inhibitors exhibit a potent anti-inflammatory activities. The also invention surprisingly found that the compounds of the invention are >10 times potent than the marketed HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on HDACs inhibition in rheumatoid arthritis. The compounds of the invention also have a longer half-life, higher systemic exposure and oral bioavailability than SAHA.
As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising,” “comprises” and “comprised” are not intended to exclude other additives, components, integers or steps.
As used herein, except where the context requires otherwise, the method steps disclosed are not intended to be limiting nor are they intended to indicate that each step is essential to the method or that each step must occur in the order disclosed.
As used herein, the term “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In specific embodiments, the subject is a human. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human.
As used herein, the terms “treat,” “treating” and “treatment” refer to the eradication or amelioration of a disease or disorder, or of one or more symptoms associated with the disease or disorder. In certain embodiments, the terms refer to minimizing the spread or worsening of the disease or disorder resulting from the administration of one or more prophylactic or therapeutic agents to a subject with such a disease or disorder. In some embodiments, the terms refer to the administration of a compound or dosage form provided herein, with or without one or more additional active agent(s), after the diagnosis or onset of symptoms of the particular disease.
As used herein, the terms “prevent,” “preventing” and “prevention” refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or an antibody or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to patients at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In this regard, the term “prevention” may be interchangeably used with the term “prophylactic treatment.
As used herein, the terms “co-administration” and “in combination with” include the administration of two or more therapeutic agents simultaneously, concurrently or sequentially within no specific time limits unless otherwise indicated. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms.
As used herein, the term “effective amount” is the quantity of compound in which a beneficial outcome is achieved when the compound is administered to a subject or alternatively, the quantity of compound that possess a desired activity in-vivo or in-vitro. In the case of inflammatory disorders and immune disorders, a beneficial clinical outcome includes reduction in the extent or severity of the symptoms associated with the disease or disorder and/or an increase in the longevity and/or quality of life of the subject compared with the absence of the treatment. The precise amount of compound administered to a subject will depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of inflammatory disorder. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
As used herein, the term “aryl” means a monocyclic or polycyclic-aromatic ring or ring radical comprising carbon and hydrogen atoms. Examples of suitable aryl groups include, but are not limited to, phenyl, tolyl, anthacenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. An aryl group can be unsubstituted or substituted with one or more substituents (including without limitation alkyl (preferably, lower alkyl or alkyl substituted with one or more halo), hydroxy, alkoxy (preferably, lower alkoxy), alkylthio, cyano, halo, amino, and nitro. In certain embodiments, the aryl group is a monocyclic ring, wherein the ring comprises 6 carbon atoms.
As used herein, the term “alkyl” means a saturated straight chain or branched non-cyclic hydrocarbon typically having from 1 to 6 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl and n-hexyl; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl etc, and the like. Alkyl groups included in compounds of this invention may be optionally substituted with one or more substituents, such as amino, alkylamino, alkoxy, alkylthio, oxo, halo, acyl, nitro, hydroxyl, cyano, aryl, alkylaryl, aryloxy, arylthio, arylamino, carbocyclyl, carbocyclyloxy, carbocyclylthio, carbocyclylamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylthio, and the like. In addition, any carbon in the alkyl segment may be substituted with oxygen, sulfur, or nitrogen.
The term “alkoxy,” as used herein, refers to an alkyl group which is linked to another moiety though an oxygen atom. Alkoxy groups can be substituted or unsubstituted.
As used herein, the term “alkenyl” means a straight chain or branched, hydrocarbon radical typically having from 2 to 10 carbon atoms and having at least one carbon-carbon double bond. Representative straight chain and branched alkenyls include vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl and the like. Alkenyl groups can be substituted or unsubstituted.
As used herein, the term “alkynyl” means a straight chain or branched, hydrocarbon radical typically having from 2 to 10 carbon atoms and having at lease one carbon-carbon triple bond. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl, -1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 9-decynyl and the like. Alkynyl groups can be substituted or unsubstituted.
As used herein, the term “cycloalkyl” means a saturated, mono- or polycyclic alkyl radical typically having from 3 to 10 carbon atoms. Representative cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantly, decahydronaphthyl, octahydropentalene, bicycle[1.1.1]pentanyl, and the like. Cycloalkyl groups can be substituted or unsubstituted.
As used herein, the term “cycloalkenyl” means a cyclic non-aromatic alkenyl radical having at least one carbon-carbon double bond in the cyclic system and typically having from 5 to 10 carbon atoms. Representative cycloalkenyls include cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl, cyclooctatrienyl, cyclooctatetraenyl, cyclononenyl, cyclononadienyl, cyclodecenyl, cyclodecadienyl and the like. Cycloalkenyl groups can be substituted or unsubstituted.
As used herein, the term “heterocycle” or “heterocyclyl” means a monocyclic or polycyclic heterocyclic ring (typically having 3- to 14-members) which is either a saturated ring or a unsaturated non-aromatic ring. A 3-membered heterocycle can contain up to 3 heteroatoms, and a 4- to 14-membered heterocycle can contain from 1 to about 8 heteroatoms. Each heteroatom is independently selected from nitrogen, which can be quaternized; oxygen; and sulfur, including sulfoxide and sulfone. The heterocycle may be attached via any heteroatom or carbon atom. Representative heterocycles include morpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrindinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like. A heteroatom may be substituted with a protecting group known to those of ordinary skill in the art, for example, the hydrogen on a nitrogen may be substituted with a tert-butoxycarbonyl group. Furthermore, the heterocyclyl may be optionally substituted with one or more substituents (including without limitation a halogen atom, an alkyl radical, or aryl radical). Only stable isomers of such substituted heterocyclic groups are contemplated in this definition. Heterocyclyl groups can be substituted or unsubstituted.
As used herein, the term “heteroaryl” means a monocyclic or polycyclic heteroaromatic ring (or radical thereof) comprising carbon atom ring members and one or more heteroatom ring members (such as, for example, oxygen, sulfur or nitrogen). Typically, the heteroaryl has from 5 to about 14 ring members in which at least 1 ring member is a heteroatom selected from oxygen, sulfur and nitrogen. In another embodiment, the heteroaryl is a 5 or 6 membered ring and may contain from 1 to about 4 heteroatoms. In another embodiment, the heteroaryl has a 7 to 14 ring members and may contain from 1 to about 7 heteroatoms. Representative heteroaryls include pyridyl, furyl, thienyl, pyrrolyl, oxazolyl, imidazolyl, indolizinyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, pyridinyl, thiadiazolyl, pyrazinyl, quinolyl, isoquinolyl, indazolyl, benzoxazolyl, benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl, isothiazolyl, tetrazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinozalinyl, purinyl, pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl or benzo(b)thienyl and the like. These heteroaryl groups may be optionally substituted with one or more substituents.
In one aspect, the invention provides a method for inhibiting cytokine release from a cell or a subject, comprising administering an effective amount of the compound having formula (I) or a pharmaceutically acceptable salt, prodrug or solvate thereof to the cell or the subject:
wherein is a single bond or a double bond;
R1 is SO2Ra, wherein Ra is aryl unsubstituted or substituted by 1 to 3 substituent selected from the group consisting of: —OC1-10alkyl, halogen, —NO2, —NH2, —OH, —C1-6alkyl, —C2-10alkenyl, —C2-10alkynyl, —C3-10cycloalkyl, —C5-10cycloalkenyl, 6 to 10 membered aryl or 6 to 10 membered heteroaryl;
R2, R5 and R6 are each independently H, —OC1-10alkyl, halogen, —NO2, —NH2, —OH, —C1-10alkyl, —C2-10alkenyl or —C2-10alkynyl; and
R4 is H, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, aryl, 5 to 14 membered heteroaryl, C3-10cycloalkyl, C5-10cycloalkenyl, C3-14heterocycloC1-10alkyl, C5-14heterocyclo C2-10alkenyl, halo, cyano, nitro, ORb, SRb, S(O)Rb, CH═CH—C(O)NRcRd, NHC(O)—CH═CH—C(O)Rb, NHC(O)—CH═CH—C(O)NRcRd, SO2NRcRd, OC(O)Rb, C(O)NRcRd, NRcRd, NHC(O)Rb, NHC(O)NRcRd, or NHC(S)Rc, in which each of Rb, Rc, and Rd, independently, is H, hydroxy, C1-10alkoxy, C6-10aryloxy, C5-14heteroaryloxy, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C6-10aryl, C5-14heteroaryl, C3-10cycloalkyl, C5-10cycloalkenyl, C3-14heterocycloC1-6alkyl, or C5-14heterocycloC2-10alkenyl.
In one embodiment, aryl is 6 to 10 membered aryl; C1-10alkyl is C1-4alkyl or C1-6alkyl; C2-10alkenyl is C2-6alkenyl; or C2-10alkynyl is C2-6alkynyl.
In one embodiment, Ra is 6 to 10 membered aryl unsubstituted or substituted by 1 to 3 substituent selected from the group consisting of: —OC1-6alkyl, halogen, —NO2, —NH2, or —OH. Preferably, Ra is phenyl. Preferably, Ra is phenyl substituted by one to three, same or different, —OCH3, halogen, NO2 or NH2.
In one embodiment, R4 is CH═CH—C(O)NRcRd, NHC(O)—CH═CH—C(O)Rb, NHC(O)—CH═CH—C(O)NRcRd, NHC(O)Rb, NHC(O)NRcRd, or NHC(S)Rc.
In one embodiment, R2, R5 and R6 are each independently H, halogen, —NO2, —NH2, or —OH.
In one embodiment, R4 is CH═CH—C(O)NRcRd, and Ra is a 6 to 10 membered aryl unsubstituted or substituted by 1 to 3 substituent selected from the group consisting of: —OC1-6alkyl, halogen, —NO2, —NH2, or —OH. Preferably, R4 is CH═CH—C(O)NRcRd, and Ra is phenyl or naphthyl.
In one embodiment, when is a double bond, R4 is CH═CH—C(O)NRcRd, and Ra is a 6 to 10 membered aryl unsubstituted or substituted by 1 to 3 substituent selected from the group consisting of: —OC1-6alkyl, halogen, —NO2, —NH2, or —OH. Preferably, R4 is CH═CH—C(O)NRcRd, and Ra is phenyl or naphthyl.
In one embodiment, when is a single bond, R4 is CH═CH—C(O)NRcRd, and Ra is a 6 to 10 membered aryl unsubstituted or substituted by 1 to 3 substituent selected from the group consisting of: —OC1-6alkyl, halogen, —NO2, —NH2, or OH. Preferably, Ra is phenyl or naphthyl unsubstituted or substituted by 1 to 3 substituent selected from the group consisting of: —OC1-6alkyl, halogen, —NO2, —NH2, or OH. More preferably, Ra is phenyl or naphthyl unsubstituted or substituted by 1 to 2 substituent selected from the group consisting of: —OCH3, halogen, —NO2, —NH2, or OH.
In one embodiment, the compound is one of the following compounds:
In another embodiment, the invention provides a method for inhibiting HDACs 1, 2, 3, and 8 in a cell or a subject, comprising administering an effective amount of the compound having formula (I) or a pharmaceutically acceptable salt, prodrug or solvate thereof to the cell or subject.
The “pharmaceutically acceptable salt” is a salt formed from an acid and a basic group of one of the compounds of any one of formulas (I) mentioned herein. Illustrative salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. The “pharmaceutically acceptable salt” also refers to a salt prepared from a compound of any one of formulas (I) having an acidic functional group and a pharmaceutically acceptable inorganic or organic base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris-(2-hydroxyethyl)-amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like. The pharmaceutically acceptable salt also refers to a salt prepared from a compound of any one of formulas (I) having a basic functional group, such as an amino functional group, and a pharmaceutically acceptable inorganic or organic acid. Suitable acids include, but are not limited to, hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide, hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
As used herein, the term “pharmaceutically acceptable solvate,” is a solvate formed from the association of one or more solvent molecules to one or more molecules of a compound of any one of formulas (I). The term solvate includes hydrates (e.g., hemi-hydrate, mono-hydrate, dihydrate, trihydrate, tetrahydrate, and the like).
The compounds of this invention may be prepared by methods generally disclosed in U.S. patent application Ser. No. 12/912,260.
In one embodiment, the inhibition of cytokine release is associated with an inflammatory disease, particularly, a chronic inflammation disease. The inflammatory disease include, but not limited to, arthritis (such as rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis), synovitis, vasculitis, conditions associated with inflammation of the bowel (such as Crohn's disease, ulcerative colitis, inflammatory bowel disease and irritable bowel syndrome), atherosclerosis, multiple sclerosis, Alzheimer's disease, vascular dementia, pulmonary inflammatory diseases (such as asthma, chronic obstructive pulmonary disease and acute respiratory distress syndrome), fibrotic diseases (including idiopathic pulmonary fibrosis, cardiac fibrosis and systemic sclerosis (scleroderma)), inflammatory diseases of the skin (such as contact dermatitis, atopic dermatitis and psoriasis), systemic inflammatory response syndrome, sepsis, inflammatory and/or an autoimmune disorder (for example, autoimmune conditions of the liver (such as autoimmune hepatitis, primary biliary cirrhosis, alcoholic liver disease, sclerosing cholangitis, and autoimmune cholangitis), and/or the complications thereof. Preferably, the arthritis is osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthropathies like ankylosing spondylitis, reactive arthritis (Reiter's syndrome), psoriatic arthritis, enteropathic arthritis associated with inflammatory bowel disease, Whipple disease and Behcet disease, septic arthritis, gout (also known as gouty arthritis, crystal synovitis, metabolic arthritis), pseudogout (calcium pyrophosphate deposition disease) or Still's disease. Preferably, the fibrosis is pulmonary fibrosis, liver fibrosis or renal fibrosis.
In general, compounds of the invention will be administered in therapeutically effective amounts by any of the usual modes known in the art, either singly or in combination with at least one other compound of this invention and/or at least one other conventional therapeutic agent for the disease being treated. A therapeutically effective amount may vary widely depending on the disease, its severity, the age and relative health of the animal being treated, the potency of the compound(s), and other factors. As anti-inflammatory agents, therapeutically effective amounts of compounds of this invention may range from 25-250 mg/Kg body weight/day, such as from 25 mg/Kg/day; for example, 25 mg/Kg/day. A person of ordinary skill in the art will be conventionally able, and without undue experimentation, having regard to that skill and to this disclosure, to determine a therapeutically effective amount of a compound for the treatment of inflammatory diseases such as arthritis and fibrosis.
In general, the compounds disclosed herein will be administered as pharmaceutical compositions by one of the following routes: oral, topical, systemic (e.g. transdermal, intranasal, or by suppository), or parenteral (e.g. intramuscular, subcutaneous, or intravenous injection). Compositions may take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions; and comprise at least one compound of this invention in combination with at least one pharmaceutically acceptable excipient. Suitable excipients are well known to persons of ordinary skill in the art, and they, and the methods of formulating the compositions, may be found in such standard references as Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, Pa. Suitable liquid carriers, especially for injectable solutions, include water, aqueous saline solution, aqueous dextrose solution, and glycols.
In particular, the compound is administered in any particular dosage form. For example, the compound can be administered, orally, for example, as tablets, troches, lozenges, aqueous or oily suspension, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
Tablets contain the compound in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch or alginic acid; binding agents, for example, maize starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate or stearic acid or tale. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. Formulations for oral use may also be presented as hard gelatin capsules wherein the compound is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
Aqueous suspensions or oily suspensions may be formulated with excipients suitable for the manufacture of aqueous or oily suspensions.
The compounds of the invention can also be administered by injection or infusion, either subcutaneously or intravenously, or intramuscularly, or intrasternally, or intranasally, or by infusion techniques in the form of sterile injectable or oleaginous solution or suspension. The compound may be in the form of a sterile injectable aqueous or oleaginous solution or suspensions. These solution or suspensions may be formulated according to the known art using suitable solvent or dispersing of wetting agents and suspending agents that have been described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oils may be conventionally employed including synthetic mono- or diglycerides. In addition fatty acids such as oleic acid find use in the preparation of injectables. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided dosages may be administered daily or the dosage may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
It is especially advantageous to formulate the compounds in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each containing a therapeutically effective quantity of the compound and at least one pharmaceutical excipient. A drug product will comprise a dosage unit form within a container that is labelled or accompanied by a label indicating the intended method of treatment, such as the treatment of an inflammatory disease such as arthritis and fibrosis.
Scheme 1 describes the synthesis of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines beginning with commercially available indole-carboxylate 16. Treatment of 16 with sodium cyanoborohydride in the presence of acetic acid yielded indoline 17. The resulting product was reacted with various benzenesulfonyl chlorides to provide 18a-h. The ester functionalities were reduced by LAH followed by oxidation with PDC, yielding the corresponding aldehydes, 19a-h. Subsequently, the resulting products were subjected to the Wittig reaction with methyl (triphenylphosphoranylidene)acetate followed by conversion into acrylic acids (20a-h) by treatment with lithium hydroxide. The corresponding acrylic acids were reacted with NH2OTHP to afford protected hydroxamates followed by TFA deprotection, yielding compounds 7-15.
General procedures for preparation of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines (7-15).
To a solution of 16 (0.30 g, 1.71 mmol) in AcOH (2 mL) was added sodium cyanoborohydride (0.16 g, 2.57 mmol) at 0° C., and allowed to stir at room temperature for 2 h. The reaction was quenched with water at 0° C., concentrated NaOH was added up to pH 10. The aqueous layer was extracted with CH2Cl2 (15 mL×3). The combined organic layer was dried over anhydrous MgSO4 and purified by chromatography over silica gel to afford 17 as a yellow solid (92% yield; 1:2 EtOAc/n-hexane): 1H NMR (500 MHz, CDCl3) δ 3.06 (t, J=8.5 Hz, 2H), 3.65 (t, J=8.5 Hz, 2H), 3.84 (s, 3H), 6.54 (dd, J=8.6, 4.7 Hz, 1H), 7.75-7.76 (m, 2H).
To a solution of 17 (0.28 g, 1.58 mmol) in pyridine (2 mL) was added 4-methoxybenzenesulfonyl chloride (0.32 g, 1.58 mmol) and heated to reflux for 6 h. The reaction mixture was purified by chromatography over silica gel to afford 18c as a white solid (85% yield; 1:1 EtOAc/n-hexane): 1H NMR (300 MHz, CDCl3) δ 2.96 (t, J=8.7 Hz, 2H), 3.80 (s, 3H), 3.85 (s, 3H), 3.93 (t, J=8.7 Hz, 2H), 6.89 (d, J=8.7 Hz, 2H), 7.62 (d, J=8.4 Hz, 1H), 7.20-7.76 (m, 3H), 7.88 (d, J=8.4 Hz, 1H).
To a solution of 18c (0.45 g, 1.26 mmol) in THF (10 mL), LAH (0.10 g, 2.52 mmol) was added at 0° C. The reaction was warmed to room temperature and stirred for 2 h. The reaction was quenched with water followed by extraction with CH2Cl2 (15 mL×3). The combined organic layer was dried over anhydrous MgSO4 and purified by silica gel chromatography (1:1; EtOAc/n-hexane) to afford a brown solid. A solution of the resulting solid in CH2Cl2 (10 mL), PDC (0.63 g, 1.66 mmol) and MS (0.63 g) was stirred at room temperature for 1 h. The reaction was filtered through celite and the filtrate was purified by chromatography over silica gel to afford 19c (62% yield; 1:1 EtOAc/n-hexane): 1H NMR (300 MHz, CDCl3) δ 3.04 (t, J=8.4 Hz, 2H), 3.83 (s, 3H), 3.98 (t, J=8.4 Hz, 2H), 6.93 (d, J=9.0 Hz, 2H), 7.61 (s, 1H), 7.70-7.72 (m, 2H), 7.78 (d, J=9.0 Hz, 2H), 9.84 (s, 1H).
To a solution of 19c (0.20 g, 0.66 mmol) in CH2Cl2 (15 mL), methyl (triphenylphosphoranylidene)acetate (0.27 g, 0.80 mmol) was added and allowed to stir at room temperature for 6 h. The reaction mixture was quenched with water and extracted with CH2Cl2 (25 mL×3). The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to give a yellow residue. To a solution of the crude adduct in dioxane (15 mL), 1M LiOH(aq) (3.4 mL) was added and stirred at 40° C. for 6 h. The reaction was acidified by concentrated HCl to give the precipitate which was recrystallized in MeOH to afford 20c as a white solid (87%; overall 46% yield from 17): 1H NMR (500 MHz, CD3OD) δ 2.91 (t, J=8.5 Hz, 2H), 3.92 (t, J=8.5 Hz, 2H), 6.33 (d, J=15.9 Hz, 1H), 7.00 (d, J=8.9 Hz, 2H), 7.38 (s, 1H), 7.40 (d, J=8.4 Hz, 1H), 7.55-7.58 (m, 2H), 7.74-7.76 (m, 2H).
To a solution of 21c (0.2 g, 0.56 mmol), PyBOP (0.31 g, 0.59 mmol), triethylamine (0.19 ml, 1.34 mmol) in DMF (2 mL), NH2OTHP (0.08 g, 0.67 mmol) was added and stirred at room temperature. After being stirred for 2 h, the reaction was quenched with water, followed by extraction with EtOAc (15 mL×3). The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography (30:1:1%; CH2Cl2/CH3OH/NH3(aq) to give a white solid, which was treated with TFA (1.8 mL, 24.2 mmol) in the presence of CH3OH (33 mL) and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure to give a white residue, which was recrystallized by CH3OH to afford 9 as a white solid (96% yield): mp: 158-160° C.; 1H NMR (500 MHz, CD3OD) δ 2.91 (t, J=8.5 Hz, 2H), 3.81 (s, 3H), 3.92 (t, J=8.5 Hz, 2H), 6.32 (d, J=15.5 Hz, 1H), 7.00 (d, J=9.0 Hz, 2H), 7.32 (s, 1H), 7.37 (d, J=8.0 Hz, 1H), 7.47 (d, J=15.5 Hz, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.74 (d, J=9.0 Hz, 2H); MS (EI) m/z: 373 (M+, 3.33%), 98 (100%); HRMS (EI) for C18H18N2O5S (M+) calcd 374.0936. Found 374.0939.
The title compound was obtained in 97% overall yield from compound 20a in a manner similar to that described for the preparation of 9: mp 128-130° C.; 1H NMR (500 MHz, CD3OD) δ 2.91 (t, J=8.5 Hz, 2H), 3.95 (t, J=8.5 Hz, 2H), 6.32 (d, J=15.5 Hz, 1H), 7.32 (s, 1H), 7.38 (d, J=8.5 Hz, 1H), 7.46 (d, J=15.5 Hz, 1H), 7.51 (dd, J=7.5, 8.0 Hz, 2H), 7.58 (d, J=8.5 Hz, 1H), 7.61 (dd, J=1.0, 8.0 Hz, 1H), 7.81 (d, J=7.5 Hz, 2H); MS (EI) m/z: 344 (M+, 3.21%), 170 (100%); HRMS (EI) for C17H16N2O4S (M+) calcd 344.0831. Found 344.0829.
The title compound was obtained in 95% overall yield from compound 20b in a manner similar to that described for the preparation of 9: mp: 156-157° C.; 1H NMR (300 MHz, CD3OD) δ 2.82 (t, J=8.5 Hz, 2H), 3.65 (s, 3H), 3.82 (t, J=8.5 Hz, 2H), 6.18 (d, J=15.5 Hz, 1H), 6.97-7.00 (m, 1H), 7.14-7.15 (m, 2H), 7.23-7.28 (m, 3H), 7.42 (d, J=15.5 Hz, 1H), 7.49 (d, J=8.5 Hz, 1H); MS (EI) m/z: 413 (M++K). Anal. Calcd for C18H18N2O5S.1.5 H2O: C, 53.86; H, 5.27; N, 6.98. Found: C, 53.73; H, 5.12; N, 6.70.
The title compound was obtained in 68% overall yield from compound 20d in a manner similar to that described for the preparation of 9: mp: 192-193° C.; 1H NMR (500 MHz, CD3OD) δ 2.90 (t, J=8.5 Hz, 2H), 3.72 (s, 3H), 3.85 (s, 3H), 3.93 (t, J=8.5 Hz, 2H), 6.35 (d, J=15.5 Hz, 1H), 7.06 (d, J=8.5 Hz, 1H), 7.19 (d, J=1.5 Hz, 1H), 7.36 (s, 1H), 7.42 (d, J=8.5 Hz, 1H), 7.45 (d, J=8.5 Hz, 1H), 7.48 (d, J=15.5 Hz, 1H), 7.61 (d, J=8.5 Hz, 1H); MS (EI) m/z: 389 (M+−15, 55%), 170 (100%); HRMS (EI) for C19H20N2O6S (M+) calcd 402.1042. Found 404.1042.
The title compound was obtained in 95% overall yield from compound 20e in a manner similar to that described for the preparation of 9: mp: 129-131° C.; 1H NMR (500 MHz, CD3OD) δ 2.93 (t, J=8.5 Hz, 2H), 3.95 (t, J=8.5 Hz, 2H), 6.80 (d, J=15.5 Hz, 1H), 7.25 (dd, J=8.5, 9.0 Hz, 2H), 7.33 (s, 1H), 7.38 (d, J=8.5 Hz, 1H), 7.40 (d, J=15.5 Hz, 1H), 7.56 (d, J=8.5 Hz, 1H), 7.87 (dd, J=5.5, 8.5 Hz, 2H); MS (EI) m/z: 362 (M+, 25%), 132 (100%); HRMS (EI) for C17H15FN2O4S (M+) calcd 362.0737. Found 362.0739.
The title compound was obtained in 94% overall yield from compound 20f in a manner similar to that described for the preparation of 9: mp: 159-160° C.; 1H NMR (300 MHz, CD3OD) δ 2.93 (t, J=8.5 Hz, 2H), 3.96 (t, J=8.5 Hz, 2H), 6.33 (d, J=15.5 Hz, 1H), 7.34 (s, 1H), 7.39 (d, J=8.5 Hz, 1H), 7.47 (d, J=15.5 Hz, 1H), 7.53 (d, J=8.5 Hz, 2H), 7.57 (d, J=8.0 Hz, 1H), 7.80 (dd, J=8.5 Hz, 1H); MS (EI) m/z: 378 (M+); Anal. Calcd for C17H15ClN2O4S.0.5 H2O: C, 52.65; H, 4.16; N, 7.22. Found: C, 52.45; H, 4.26; N, 7.01.
The title compound was obtained in 85% overall yield from compound 20g in a manner similar to that described for the preparation of 9: mp 163-165° C.; 1H NMR (500 MHz, CD3OD) δ 2.95 (t, J=8.5 Hz, 2H), 4.02 (t, J=8.5 Hz, 2H), 6.33 (d, J=16.0 Hz, 1H), 7.34 (s, 1H), 7.40 (d, J=8.0 Hz, 1H), 7.46 (d, J=16.0 Hz, 1H), 7.59 (d, J=8.0 Hz, 1H), 8.07 (d, J=9.0 Hz, 2H), 8.34 (d, J=9.0 Hz, 2H); MS (EI) m/z: 389 (M+); HRMS (EI) for C17H15N3O6S (M+) calcd, 389.0682. Found, 389.0680. Anal. Calcd for C17H15N3O6S.0.5 H2O: C, 51.25; H, 4.05; N, 10.55. Found: C, 51.28; H, 4.27; N, 10.73.
A mixture of 13 (0.3 g, 0.77 mmol), iron powder (0.13 g, 2.31 mmol) and ammonium chloride (0.08 g, 1.54 mmol) in isopropyl alcohol (8 mL) and water (1.5 mL) was heated to reflux for 4 h. The reaction was quenched with water and extracted with CH2Cl2 (25 mL×3). The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The reaction mixture was purified by chromatography over silica gel to afford 14 (47% yield; 10:1:1%; CH2Cl2/CH3OH/NH3(aq)): mp: 97-99° C.; 1H NMR (500 MHz, CD3OD) δ 2.90 (t, J=8.5 Hz, 2H), 3.87 (t, J=8.5 Hz, 2H), 6.49 (d, J=16.0 Hz, 1H), 6.58 (dd, J=2.0, 9.0 Hz, 2H), 7.34 (s, 1H), 7.37 (d, J=8.5 Hz, 1H), 7.45 (d, J=16.0 Hz, 1H), 7.45 (d, J=9.0 Hz, 2H), 7.53 (d, J=8.5 Hz, 1H); MS (EI) m/z: 341 (M+−18, 71%), 156 (100%); HRMS (EI) for C17H17N3O4S (M+) calcd 359.0940. Found 359.0940.
The title compound was obtained in 47% overall yield from compound 20h in a manner similar to that described for the preparation of 9: mp: 142-143° C.; 1H NMR (300 MHz, CD3OD) δ 2.85 (s, 6H), 2.92 (t, J=8.7 Hz, 2H), 4.04 (t, J=8.7 Hz, 2H), 6.32 (d, J=15.9 Hz, 1H), 7.23 (d, J=7.5 Hz, 1H), 7.35 (d, J=8.4 Hz, 1H), 7.44-7.60 (m, 4H), 8.23 (dd, J=1.5, 7.5 Hz, 1H), 8.35 (d, J=8.7 Hz, 1H), 8.58 (d, J=8.7 Hz, 1H). MS (EI) m/z: 435 (M+−2). Anal. Calcd for C23H23N3O4S.C2H5OH: C, 62.09; H, 6.04; N, 8.69. Found: C, 62.14; H, 5.96; N, 8.22.
The title compound was obtained in 52% overall yield from compound 17 in a manner similar to that described for the preparation of 20c: 1H NMR (500 MHz, CD3OD) δ 2.92 (t, J=8.5 Hz, 2H), 3.96 (t, J=8.5 Hz, 2H), 6.33 (d, J=16.0 Hz, 1H), 7.38 (s, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.52 (d, J=7.7 Hz, 1H), 7.55 (d, J=16.0 Hz, 1H), 7.59-7.64 (m, 3H), 7.82 (d, J=7.7 Hz, 2H).
The title compound was obtained in 45% overall yield from compound 17 in a manner similar to that described for the preparation of 20c: 1H NMR (300 MHz, CD3Cl3) δ 2.96 (t, J=8.4 Hz, 2H), 3.77 (s, 3H), 3.96 (t, J=8.7 Hz, 2H), 6.29 (d, J=16.0 Hz, 1H), 7.08-7.13 (m, 1H), 7.28-7.41 (m, 5H), 7.60 (d, J=16.0 Hz, 1H), 7.65 (s, 1H).
The title compound was obtained in 52% overall yield from compound 17 in a manner similar to that described for the preparation of 20c: 1H NMR (500 MHz, CD3OD) δ 2.89 (t, J=8.4 Hz, 2H), 3.69 (s, 3H), 3.84 (s, 3H), 3.93 (t, J=8.4 Hz, 2H), 6.33 (d, J=15.9 Hz, 1H), 7.03 (d, J=8.5 Hz, 1H), 7.18 (d, J=1.8 Hz, 1H), 7.39 (s, 1H), 7.43 (dd, J=8.5, 1.8 Hz, 2H), 7.56 (d, J=15.9 Hz, 1H), 7.61 (d, J=8.5 Hz, 1H).
The title compound was obtained in 50% overall yield from compound 17 in a manner similar to that described for the preparation of 20c: 1H NMR (500 MHz, CD3OD) δ 2.94 (t, J=8.4 Hz, 2H), 3.96 (t, J=8.4 Hz, 2H), 6.33 (d, J=15.9 Hz, 1H), 7.24-7.27 (m, 1H), 7.40 (s, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.55-7.60 (m, 2H), 7.87-7.90 (m, 2H).
The title compound was obtained in 46% overall yield from compound 17 in a manner similar to that described for the preparation of 20c: 1H NMR (300 MHz, CD3OD) δ 2.91 (t, J=8.7 Hz, 2H), 3.95 (t, J=8.4 Hz, 2H), 6.39 (d, J=16.2 Hz, 1H), 7.27-7.37 (m, 3H), 7.50-7.57 (m, 3H), 7.77-7.82 (m, 2H).
The title compound was obtained in 31% overall yield from compound 17 in a manner similar to that described for the preparation of 20c: 1H NMR (500 MHz, CD3OD) δ 2.97 (t, J=8.5 Hz, 2H), 3.99 (t, J=8.5 Hz, 2H), 6.27 (d, J=16.0 Hz, 1H), 7.32 (s, 1H), 7.37 (d, J=8.6 Hz, 1H), 7.53-7.60 (m, 2H), 8.01 (d, J=8.8 Hz, 2H), 8.31 (d, J=8.8 Hz, 2H).
The title compound was obtained in 45% overall yield from compound 17 in a manner similar to that described for the preparation of 20c: 1H NMR (500 MHz, CD3OD) δ 2.84 (s, 6H), 2.92 (t, J=8.5 Hz, 2H), 4.04 (t, J=8.5 Hz, 2H), 6.35 (d, J=16.0 Hz, 1H), 7.22 (d, J=7.5 Hz, 1H), 7.38 (d, J=3.0 Hz, 2H), 7.44-7.48 (m, 2H), 7.54-7.58 (m, 2H), 8.23 (dd, J=7.5, 1.0 Hz, 1H), 8.30 (d, J=9.0 Hz, 1H), 8.57 (d, J=8.5 Hz, 1H).
The non-conjugated primary antibodies used were specific for iNOS was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA); COX-2 and β-actin were purchased from Epitomics Inc. (Burlingame, Calif., USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibodies were obtained from Jackson ImmunoResearch Inc. (Cambridgeshire, UK).
Mouse macrophage cell line RAW264.7 was obtained from the Bioresource Collection and Research Center. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories Inc.) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen™ Life Technologies, Carlsbad, Calif., USA), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Biological Industries, Kibbutz Beit Haemek, Israel) at 37° C. in a humidified atmosphere of 5% CO2 in air.
WI-38 cells, a normal human embryonic lung fibroblast cell line, were obtained from American Type Culture Collection (Manassas, Va.). Cells were grown in MEM nutrient mixture, containing 10% FCS, 2 mM L-glutamine, 0.1 mM NEAA, 1 mM sodium pyruvate, 50 U/ml penicillin G, and 100 μg/ml streptomycin, in a humidified 37° C. incubator with 5% CO2 Cells were used between passages 18 and 30 for all experiments. After reaching confluence, cells were seeded onto 6-cm dishes for immunoblotting.
The HeLa nuclear extract HDAC activity was measured by using the HDAC Fluorescent Activity Assay Kit (BioVision, CA) according to manufacturer's instructions. Briefly, the HDAC fluorometric substrate and assay buffer were added to HeLa nuclear extracts in a 96-well format and incubated at 37° C. for 30 min. The reaction was stopped by adding lysine developer, and the mixture was incubated for another 30 min at 37° C. Additional negative controls included incubation without the nuclear extract, without the substrate, or without both. TSA at 1 μM served as the positive control. A fluorescence plate reader with excitation at 355 nm and emission at 460 nm was used to quantify HDAC activity.
RAW 264.7 cells (1×106) were plated and pretreated with the indicated concentrations of compound 9 for 1 h, and subjected to stimulation with LPS (25 ng/mL) for 24 h, and then 100 μL of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride) was mixed with 100 μL of the cell supernatant and the optical density at 550 nm was measured. Nitrite concentration was determined using a dilution of sodium nitrite as a standard.
To investigate the effect of compound 9 and SAHA on PGE2 levels in LPS stimulated cells, RAW 264.7 cells (1×106) were treated in the presence or absence of test compounds for 1 h, and then stimulated with LPS (25 ng/mL) for 24 h at 37° C. The concentrations of PGE2 in the supernatants of RAW 264.7 cell cultures were determined using an EIA kit (R&D Systems, Minneapolis, Minn., USA).
To determine the effect of compound 9 and SAHA on the production of cytokines IL-6 and TNF-α from LPS-stimulated cells, RAW 264.7 cells (1×106) were plated and pretreated in the presence or absence of compound 9 and SAHA for 1 h, and then stimulated with LPS (25 ng/mL) for 24 h at 37° C. Supernatants were collected and the concentrations of cytokines IL-6 and TNF-α were measured by ELISA kit.
Animal experiments were approved by the Institutional Animal Care and Use Committee of National Taiwan University College of Medicine (IACUC number: 20120226). Animals were divided into four groups (n=5). 0.5% (w/v) suspension of carrageenan in normal saline was administered to male Wistar rats (7-weeks) by intradermal injection into the base of the right hind paw. One hour prior to carrageenan injection, rats were oral administration of vehicle (1% carboxymethyl cellulose and 0.5% Tween 80) or a fine suspension of Compound 9 (25 mg/kg), SAHA (200 mg/kg) in vehicle. A positive control group was included in which rats were pretreated with 5 mg/kg Indomethacin. Three hours after carrageenan administration, the thickness and volume of the right hind paw were measured by digital caliper and digital plethysmometer (Diagnostic & Research Instruments CO., Ltd, Taipei, Taiwan), respectively.
Western blot analyses were performed as described previously (Chen B C, Chang Y S, Kang J C, Hsu M J, Sheu J R, Chen T L, et al. Peptidoglycan induces nuclear factor-kappaB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages. J Biol Chem 2004; 279:20889-97). Briefly, WI-38 lung fibroblasts were cultured in 6-cm dishes. After reaching confluence, cells were pretreated with specific inhibitors (E028 and G009) as indicated for 30 min, and then treated with the vehicle (H2O) or 10 ng/ml TGF-β for 2 h (CTGF assay) or 24 h (collagen I assay). Whole-cell lysates (30 rig) were subjected to 12% (CTGF) or 8% (collagen I) SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane which was then incubated in TBST buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.02% Tween 20; pH 7.4) containing 5% BSA. Proteins were visualized by specific primary antibodies and then incubated with HRP-conjugated secondary antibodies. The immunoreactivity was detected using enhanced chemiluminescence (ECL) following the manufacturer's instructions. Quantitative data were obtained using a computing densitometer with scientific imaging systems (Kodak, Rochester, N.Y.).
Results are expressed as the mean±SEM for the indicated number of separate experiments. Means were checked for statistical difference using 1-test and P-values<0.05 were considered significant.
Using kits that contained different recombinant HDAC isoforms, we evaluated the ability of MPT0G009 to inhibit HDAC-mediated deacetylation of lysine residues on the substrates that were provided. As shown in Table 1, MPT0G009 demonstrated potent inhibitory activity for class I HDACs 1, 2, 3, and 8 and for class IIb HDAC6 but not for class Ha HDAC4, with IC50 values of 4.62, 5.16, 1.91, 22.48, 8.43, and >104 nM, respectively.
aData represent the mean ± SEM from three replicate experiments.
The effect of the synthesized 5-(N-hydroxyacrylamide)-1-benzenesulfonylindolines 7-15, 5-(N-hydroxyacrylamide)-1-benzenesulfonylindole 6, and reference compound 1 on inflammatory factors was summarized in Table 2. Lipopolysacharide (LPS) stimulated RAW 264.7 macrophages were treated with test compounds at the indicated concentrations and the IC50 value of the compounds for inhibiting inflammatory factors nitric oxide (NO), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) releasing were measured by ELISA kit (Table 2). Compound 6 possessing an indole nucleus exhibited comparable activity to 1. With the exception of 14, the conversion of central skeleton from indole to indoline led to overall improvement of activity decreasing the induction of inflammatory factors. The 4′-amino substitution of 14 caused a dramatic loss of potency, which is correlated with the result of HeLa nuclear HDAC enzyme inhibition. The replacement of N,N-dimethylnaphthalene (15) led to slight decrease of activity; however, it is still better than 6 and 1. Among all indoline analogues, 9 having a 4′-OMe group exhibited the most potent inhibiting the inflammatory factors secretion. 9 reduced the expression of NO, IL-6, PGE2, and TNF-α with IC50 values of 1.07, 0.01, 0.52, and 0.52 μM, respectively. (Table 2)
[a]Data represent mean ± SEM from three independent experiments.
Furthermore, the anti-inflammatory effects of MPT0G009 was evaluated. Supernatants from cultures of RAW264.7 cells (
MPT0G009 and SAHA inhibited PGE2 production by both cell types, NO production by RAW264.7 cells, and IL-6 production by RA-FLS in a concentration-dependent manner; MPT0G009 was more effective than SAHA. Because synoviocyte proliferation plays a pivotal role in RA pathogenesis, we assessed the effects of MPT0G009 and SAHA at the above mentioned concentrations on the proliferation of HIG-82 synoviocytes (
To investigate the effects of MPT0G009 and SAHA on cell cycle progression, cellular DNA contents were determined by flow cytometry. As shown in
To determine whether the inhibitory effect of compound 9 and SAHA (1) on inflammatory factors NO and PGE2 were related to the modulation of iNOS and COX-2 expression, Western blot analysis was performed. As shown in
To investigate the effects of compound 9 and SAHA on inflammation models, rats were initially oral treated with compound 9 (25 mg/kg), SAHA (200 mg/kg), and Indomethacin (5 mg/kg) for 1 h and then subjected to carrageenan-induced acute inflammatory hind paw edema. The results showed that compound 9 and SAHA significantly inhibited hind paw edema (
Because histone H3 is a target of HDACs, we examined whether a MPT0G009- or SAHA-induced decrease in HDAC activity resulted in changes in histone acetylation in HIG-82 synoviocytes and RA-FLS. Western blots of lysates of HIG-82 synoviocytes that were treated with 3 μM MPT0G009 (
Bone destruction is one characteristic of RA pathogenesis, resulting in joint dysfunction. Differentiation of mouse macrophages osteoclast-like cells can be induced in the presence of M-CSF and RANKL, which has been used as a model to investigate osteoclast differentiation. To evaluate the effect of MPT0G009 on osteoclast formation, RAW264.7 macrophages were incubated for 30 min with or without 5 nM MPT0G009 or 50 nM SAHA (
We also assessed the effect of MTPOG009 on the DNA-binding activity of NF-kB and NFATc1, two pivotal transcriptional factors involved in RANKL-induced pathways for promoting osteoclast differentiation. When RAW264.7 cells that had been transiently transfected with reporter plasmids were treated with 5 nM MPT0G009 for 30 min before and during 24-h stimulation with RANKL, MPT0G009 inhibited RANKL-induced NF-kB (
Next, we examined whether MPT0G009 inhibited cytokine release and osteoclast formation by inhibiting HDAC activity. As shown in
Next, we evaluated the in vivo anti-arthritic effects of MPT0G009 in a rat AIA model. As shown in
(
The pharmokinetic parameters of MPT0G009 in rats after single-dose intravenous (i.v.) and oral administration are summarized in Table 2. After i.v. administration, the half-life of MPT0G009 was 6.74 h, and systemic exposure and clearance were 665 ngh/mL and 5.12 L/h/kg, respectively. After oral administration, MPT0G009 showed Tmax=3.43 h, T1/2=9.53 h, and bioavailability (F)=13.0% (Table 3). Table 4 shows the maximum tolerated dose data for MPT0G009 in CD-1 mice with a daily×7 schedule. No significant adverse effects were observed within three weeks in a study of mice when MPT0G009 was administrated at a dosage of up to 1000 mg/kg/day.
To determine whether the inhibitory effects of MPT0E028 and MPT0G009 on pro-fibrogenic mediators (TGF-β, thrombin, and ET-1) were related to the modulation of CTGF and Collagen I production, Western blot analysis was performed. First, WI-38 lung fibroblasts were incubated with different concentrations of MPT0E028 (0.01, 0.03, 0.1, 0.3, or 1 μM) (
