Heterocyclic compounds as phosphodiesterase inhibitors

FIELD

Also described herein is the process for the preparation of the above said novel compounds of formula (I), their analogs, stereoisomers, diastereomers, polymorphs, hydrates, solvates, pharmaceutically acceptable salts, pharmaceutical compositions, metabolites, prodrugs and intermediates useful in the preparation of such compounds.

The compounds described herein are phosphodiesterase type 4 (PDE4) inhibitors. More particularly, they down regulate or inhibit the production of tumor necrosis factor (TNF-α) which are mediated by PDE4 enzymes and therefore are useful in the treatment of a variety of allergic and inflammatory diseases including asthma, COPD, chronic bronchitis, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, vernal conjunctivitis, psoriasis, rheumatoid arthritis, ulcerative colitis, Crohn's disease, uveitis, NASH and lupus.

BACKGROUND

Airway inflammation characterize's a number of severe lung diseases including asthma and chronic obstructive pulmonary disease (COPD). Events leading to airway obstruction include edema of airway walls, infiltration of inflammatory cells into the lungs, production of various inflammatory mediators and increased mucous production. The airways of asthmatic patients are infiltrated by inflammatory leukocytes, of which the eosinophil is the most prominent component. The magnitude of asthmatic reactions is correlated with the number of eosinophils present in the lungs. The accumulation of eosinophils is found dramatically in the lungs of asthmatic patients, which are capable of lysing and activating cells and destroying tissues. Upon activation eosinophils release inflammatory cytokines such as IL-1, IL-1β, IL-3, IL-6, IL-8, IL-12, TNF-α and inflammatory mediators such as PAF (Platelet-Activating Factor), LTD4 (Leukotriene-D4) and relative oxygen species that can produce edema, bronchoconstriction. TNF-α is involved in the pathogenesis of a number of autoimmune and inflammatory diseases (Bartolome Celli,Chest,2006, vol 129 no. 1, 5-6). Consequently, manipulation of the cytokine signaling or biosynthetic pathways associated with these proteins may provide therapeutic benefit in disease states mentioned above. It has been well demonstrated that TNF-α production in pro-inflammatory cells becomes attenuated by an elevation of intracellular cyclic adenosine 3′,5′-monophosphate (cAMP); this second messenger is regulated by the phosphodiesterase (PDE) family of enzymes. The phosphodiesterase enzymes play an integral role in cell signaling mechanisms by hydrolyzing cAMP and cGMP to their inactive 5′ forms. Inhibition of PDE enzymes thus results in an elevation of cAMP and/or cGMP levels and alters intracellular responses to extra cellular signals by affecting the processes mediated by cyclic nucleotides. Since eosinophils are believed to be a critical proinflammatory target for asthma, identification of the expression of PDE4 gene family in eosinophils led to the PDE4 as potential therapeutic target for asthma [Rogers, D. F., et al.,Trends Pharmacol. Sci.,1998, 19, 160-164; Barnes, P. J., Trends Pharmacol. Sci., 1998, 19, 415-423, L. Pages, et. al.,Expert Opin. Ther. Patents2009, 19, 1501-1519].

Phosphodiesterase type 4 (PDE4) is cAMP-specific and Ca2+independent enzyme and hydrolyses cAMP in mast cells, basophils, eosinophils, monocytes and lymphocytes. The association between cAMP elevation in inflammatory cells with airway smooth muscle relaxation and inhibition of mediator release has led to widespread interest in the design of PDE4 inhibitors [Trophy, T. J.,Am. J. Respir. Crit. Care Med.,1998, 157, 351-370; P. J. Barnes,Eur Respir Rev2005, 14: 94, 2-11; Wolfgang Bäumer et al.,Inflammation&Allergy—Drug Targets,2006, 6, 17-26; Joseph P. Grande et al.,Exp Biol Med2007, 232, 38-51]. Excessive or unregulated TNF-α production has been implicated in mediating or exacerbating a number of undesirable physiological conditions such as diseases including osteoarthritis and other arthritic conditions; septic shock, endotoxic shock and respiratory distress syndrome. Since TNF-α also participates in the onset and progress of autoimmune diseases, PDE4 inhibitors may find utility as therapeutic agents for rheumatoid arthritis, multiple sclerosis and Crohn's disease. [Nature Medicine,1995, 1, 211-214 and ibid., 244-248]. TNF-α is also reported to be a factor of insulin-resistant diabetes because it declines the phosphorylating mechanism of insulin receptors of muscle and fat cells [J. Clin. Invest.,1994, 94, 1543-1549].

It has been demonstrated that increasing cAMP levels within these cells results in suppression of cell activation, which in turn inhibits the production and release of pro-inflammatory cytokines such as TNF-α. Since eosinophils are believed to be a critical pro-inflammatory target for asthma, identification of the expression of PDE4 gene family in eosinophils led to the PDE4 as potential therapeutic target for asthma. The IL-6, tumor necrosis factor TNF-α, E-selectin, and nitric oxide (NO) production have been reported to be involved in the pathogenesis of LPS-induced uveitis, PDE4 inhibitors are known to suppress cutaneous inflammation and LPS-induced TNF-α expression including IL-6 inhibition, forming a viable strategy for treatment of uveitis (Investigative Ophthalmology and Visual Science,2004, 45, 2497-2502).

Interest in the drugs capable of selective inhibition of PDE4 has taken much attention due to several factors: (a) tissue distribution of PDE-4 strongly suggested that the pathologies related to the central nervous and immune systems could be treated through the selective PDE4 inhibitors (b) increase in intracellular cAMP concentration, the obvious biochemical consequence of PDE-4 inhibition, has been well characterized in immuno-competent cells where it acts as a deactivating signal.

Four human cDNA isoforms of PDE-4 (PDE4-A, B, C and D) were identified. mRNA for all these four isoforms was expressed in the human lungs. PDE4-A, B, C and D were expressed in eosinophils. Of these gene families, PDE-4 characterized as the cAMP-specific gene family has been shown to predominate in proinflammatory human lymphoid and myeloid lineage cells.

Objective

The application of several PDE4 inhibitors is limited due to their undesirable side effect profile, which include nausea, emesis and gastric acid secretion due to action on PDE4 in parietal cells in the gut. [Barnette et al., T. J.,J. Pharmacol. Exp. Ther.,1995, 273, 1396-1402]. One of the earliest PDE4 inhibitors, Rolipram, was withdrawn from clinical development because of its severe unacceptable side effect profile. [Zeller E. et al.,Pharmacopsychiatry,1984, 17, 188-190]. The cause of the severe side effects of several PDE4 inhibitor molecules in human clinical trials has recently become apparent. [Jacobitz, et al.,J., Mol. Pharmacol.,1996, 50, 891-899]. The human recombinant PDE4 exists in four isoforms A, B, C, and D [Muller, T et al.,Trends in Pharmacol. Sciences,1996, 17, 294-298] accordingly compounds displaying more PDE4D isoenzyme selectivity over the A, B or C are found to have fewer side effects than Rolipram [Hughes, B et al.,Br. J. Pharmacol.1996, 118, 1183-1191]. Therefore, selective inhibitors of PDE4 isozymes would have therapeutic effects in inflammatory and respiratory diseases and fewer undesirable side effects.

Although researchers all over the world are working in this direction to achieve the desired selective PDE4 isozyme inhibition, so far success is limited. Among the various compounds, which showed clinically proven PDE 4 inhibition, Oglemilast, Apremilast and ELB-353 have reached advanced stage of human clinical trials.

Described herein is the use of therapeutically effective amount of the compounds of the general formula (I) or a pharmaceutically acceptable salt thereof as a medicament for therapeutic treatment of PDE4 and TNF-α mediated diseases such as asthma, COPD and chronic inflammatory diseases, specific autoimmune diseases, sepsis-induced organ injury, in the subjects like human beings and animals.

SUMMARY

Described are compounds of the general formula (I),

wherein: X and Y independently represents O, S or NR6;

R4and R5can be combined together to form a substituted or unsubstituted 5 to 7 membered ring, having 0-3 heteroatoms selected from O, N and S;

R2represents substituted or unsubstituted groups selected from alkyl, haloalkyl, cycloalkyl and the like;

A or B represents —CR6, NR6, ═N—, —O— or —S—; when one of A or B represents —CR6, then the other represents —NR6—, —O— or —S—;

when X is NR6, then R2and R6can be combined together to form a substituted or unsubstituted 5 to 7 membered ring having 0-3 heteroatoms selected from O, N and S;

‘’ represents a double bond or a single bond;

DETAILED DESCRIPTION

Described are compounds of the general formula (I),

wherein X and Y independently represents O, S or NR6;

R4and R5can be combined together to form a substituted or unsubstituted 5 to 7 membered ring, having 0-3 heteroatoms selected from O, N and S;

when X is NR6, then R2and R6can be combined together to form a substituted or unsubstituted 5 to 7 membered ring having 0-3 heteroatoms selected from O, N and S;

‘’ represents a double bond or a single bond.

A or B represents —CR6, NR6, ═N—, —O— or —S—; when one of A or B represents —CR6, then the other represents —NR6, ═N—, —O— or —S—;

The term “alkyl” refers to straight or branched aliphatic hydrocarbon groups having the specified number of carbon atoms, which are attached to the rest of the molecule by a single atom. Preferred alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl and the like.

The term “alkenyl” refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond, which may be straight or branched chain having about 2 to 10 carbon atoms, which may be optionally substituted by one or more substituents. Preferred alkenyl groups include, without limitation, ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like.

The term “alkynyl” refers to straight or branched hydrocarbyl radicals having at least one carbon-carbon triple bond in the range of 2-12 carbons. Preferred alkynyl groups include, without limitation, ethynyl, propynyl, butynyl and the like.

The term “aryl” refers to aromatic radicals having 6 to 14 carbon atoms, which may be optionally substituted by one or more substituents. Preferred aryl groups include, without limitation, phenyl, naphthyl, indanyl, biphenyl and the like or substituted or unsubstituted arylene groups such as phenylene, biphenylene, naphthylene, anthracenylene, phenanthrylene, indanylene and the like.

The term “aralkyl” refers to an aryl group directly bonded to an alkyl group, which may be optionally substituted by one or more substituents. Preferred aralkyl groups include, without limitation, —CH2C6H5, —C2H4C6H5and the like.

The term “aralkenyl” refers to an aromatic ring radical directly bonded to an alkenyl group. The aryl radical may be attached to the main structure at any carbon from the alkenyl group. Examples of such aralkenyl groups include but are not limited to, phenylethenyl and phenylpropenyl.

The term “aralkynyl” refers to an aromatic ring radical directly bonded to an alkynyl group. The aryl radical may be attached to the main structure at any carbon from the alkynyl group. Examples of such aralkynyl groups include but are not limited to, phenylethynyl and phenylpropynyl.

The term “alkanoyl” represents a group of the formula —C(O)alkyl. Preferred alkanoyl groups include, without limitation, acetyl, propanoyl, butanoyl and the like.

The term “aroyl” denotes an aryl-CO— group, wherein aryl is as defined above. Examples of such aroyl groups include but are not limited to, benzoyl, naphthoyl and the like.

The term “aralkanoyl” refers to aralkyl-C(O)— group. Examples of such aralkanoyl groups include but are not limited to, phenylacetyl, phenylpropanoyl, naphthylacetyl, naphthylpropanoyl and the like.

The term “heteroaryl” refers to an aromatic heterocyclic ring radical as defined above. The heteroaryl ring radical may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure.

The term “heteroarylalkyl” refers to a heteroaryl ring radical as defined above, directly bonded to an alkyl group. The heteroarylalkyl radical may be attached to the main structure at any carbon atom from an alkyl group.

The term “cycloalkyl” refers to non-aromatic mono or polycyclic ring systems of about 3 to 12 carbon atoms. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctanyl and the like; preferred polycyclic rings include, without limitation, perhydronaphthyl, adamantyl and norbornyl groups, bridged cyclic groups or spirobicyclic groups e.g. spiro[4.4]-non-2-yl and the like.

The term “cycloalkenyl” refers to a non-aromatic cyclic ring radical containing about 3 to 8 carbons with at least one carbon-carbon double bond, which may be optionally substituted. Preferred cycloalkenyl groups include, without limitation, cyclopropenyl, cyclopentenyl and the like.

The term “cycloalkynyl” refers to a non-aromatic cyclic ring radical containing about 3 to 8 carbon atoms with at least one carbon-carbon triple bond, which may be optionally substituted. Preferred cycloalkynyl groups include, without limitation, cyclopropynyl, cyclopentynyl and the like.

The term “alkylthio” refers to an alkyl group attached via a sulfur linkage to the rest of the molecule, which may be optionally substituted. Preferred alkylthio groups include, without limitation, —SCH3, —SC2H5and the like.

The term “alkoxy” refers to an alkyl group attached via an oxygen linkage to the rest of the molecule. Preferred alkoxy groups include, without limitation, —OCH3, —OC2H5and the like.

The term “aryloxy” refers to an aryl group attached via an oxygen linkage to the rest of the molecule. Preferred aryloxy groups include, without limitation, —O-phenyl, —O-biphenyl and the like.

The term “alkylamino” refers to an alkyl group as defined above attached via an amino linkage to the rest of the molecule. Preferred alkylamino groups include, without limitation, —NHCH3, —N(CH3)2and the like.

The term “alkylsulfonyl” refers to straight or branched hydrocarbyl radicals group attached via a —SO2— linkage to the rest of the molecule. Preferred alkylsulfonyl groups include without limitation methylsulfonyl, ethylsulfonyl, n-propylsulfonyl or isopropylsulfonyl.

The term “arylsulfonyl” refers to an aryl group attached via a —SO2— linkage to the rest of the molecule. Preferred arylsulfonyl groups include without limitation phenylsulfonyl or naphthylsulfonyl.

The term “arylamino” refers to an aryl group attached via a amino linkage to the rest of the molecule. Preferred arylamino groups include without limitation phenylamino or naphthylamino.

The term “ring” refers to substituted or unsubstituted monocyclic or polycyclic, saturated or partially saturated or aromatic containing 0 to 4 heteroatoms selected from O, S and N.

The term “metabolite”, as used herein, refers to a derivative of a compound of formula (I) which is formed when the compound is metabolized.

The term “prodrug”, as employed herein, denotes a compound that is a drug precursor which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of formula (I).

The term “derivative” refers to a chemical compound or molecule made from a parent compound by one or more chemical reactions such as, by oxidation, hydrogenation, alkylation, esterification, halogenation and the like.

Pharmaceutically acceptable solvates may be hydrates or comprising of other solvents of crystallization such as alcohols.

Compounds disclosed herein may exist as single stereoisomers, racemates and or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the subject matter described.

The phrase “pharmaceutically acceptable” refers to compounds or compositions that are physiologically tolerable and do not typically produce allergic or similar untoward reactions, including but are not limited to, gastric upset or dizziness when administered to mammals.

The active compounds disclosed can also be prepared in any solid or liquid physical form, for example the compound can be in a crystalline form, in amorphous form and have any particle size. Furthermore, the compound particles may be micronized or nanoized, or may be agglomerated, particulate granules; powders, oils, oily suspensions or any other form of solid or liquid physical forms.

Described herein are also pharmaceutical compositions, containing one or more of the compounds of the formula (I), as defined above, their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, metabolites, prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates in combination with the usual pharmaceutically employed carriers, diluents and the like, useful for the treatment of inflammatory diseases.

The pharmaceutical composition may be in the forms normally employed, such as tablets, capsules, powders, syrups, solutions, suspensions and the like, may contain flavorants, sweeteners etc. in suitable solid or liquid carriers or diluents, or in suitable sterile media to form injectable solutions or suspensions. The compositions may be prepared by processes known in the art. Suitable pharmaceutically acceptable carriers include solid fillers or diluents and sterile aqueous or organic solutions. The active compound will be present in such pharmaceutical compositions in the amounts sufficient to provide the desired dosage in the range as described above. Suitable routes of administration include systemic, such as orally or by parenteral administration such as subcutaneous, intramuscular, intravenous and intradermal routes. Thus for oral administration, the compounds can be combined with a suitable solid or liquid carrier or diluent to form capsules, tablets, powders, syrups, solutions, suspensions and the like. The pharmaceutical compositions, may, if desired, contain additional components such as flavorants, sweeteners, excipients and the like. For parenteral administration, the compounds can be combined with a sterile aqueous or organic media to form injectable solutions or suspensions. For example, solutions in sesame or peanut oil, aqueous propylene glycol and the like can be used, as well as aqueous solutions of water-soluble pharmaceutically-acceptable acid addition salts or alkali or alkaline earth metal salts of the compounds. The injectable solutions prepared in this manner can then be, administered intravenously, intraperitoneally, subcutaneously or intramuscularly.

The compounds of the formula (I) are effective in inhibiting or lowering levels of TNF-α, a important mediator in cellular and molecular process leading to inflammatory and allergic disorders.

Described herein are compounds of formula (I), which is effective in the treatment of allergic and inflammatory diseases or disorder or conditions which may be associated with an undesirable inflammatory immune response or associated with the increase in secretion of TNF-α and/or PDE4 which comprises administering to a subject a therapeutically effective amount of a compound of formula (I).

Also described herein is the method of treating an inflammatory condition or immune disorder in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a compound of formula (I). Examples of inflammatory or immune disorders are selected from asthma, COPD (chronic obstructive pulmonary disease), atopic dermatitis, allergic rhinitis, allergic conjunctivitis, psoriasis, rheumatoid arthritis, septic shock, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid spondylitis, osteoarthritis, uveitis eosinophilic granuloma, cystic fibrosis, chronic bronchitis, Lupus and Nonalcoholic steatohepatitis (NASH).

Described herein are compounds of formula (I), which are effective in the treatment and/or prophylaxis, the inflammatory and/or allergic diseases preferably COPD (Chronic obstructive pulmonary disease), asthma, rheumatoid arthritis, allergic rhinitis or uveitis, which comprises administering to a subject a therapeutically effective amount of a compound according to formula (I).

Described herein are compounds of formula (I), which are effective in treating diseases mediated by PDE4, which comprises administering to a subject a therapeutically effective amount of a compound according to formula (I).

A method of treating inflammatory diseases mediated by PDE4 enzyme comprising administering an effective amount of a compound of formula (I), to the mammal in need thereof.

A method of treatment of allergic or inflammatory diseases mediated by PDE4 comprising asthma, COPD, chronic bronchitis, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, vernal conjunctivitis, psoriasis, rheumatoid arthritis, ulcerative colitis, Crohn's disease, uveitis, NASH and lupus administering an effective amount of a compound of formula (I).

A method of treating inflammatory or immunological diseases by lowering plasma concentrations of anyone or a combination or all of TNF-α, IL-1β and IL-6 comprising administering an effective amount of a compound of formula (I), to the mammal in need thereof.

A method of treating immunological diseases, those mediated by cytokines selected from TNF-α, IL-1β, IL-6 and IL-12 comprising administering an effective amount of a compound of formula (I), to the mammal in need thereof.

A method of reducing inflammation in an inflamed organ or tissue by delivering a required amount of compound of formula (I).

The compounds of the formula (I) can also be administered as a pharmaceutical composition in a pharmaceutically acceptable carrier, preferably formulated for oral administration.

The compounds described herein may also exhibit polymorphism. This invention further includes different polymorphs of the compounds. The term polymorph refers to a particular crystalline state of a substance, having particular physical properties such as X-ray diffraction, IR spectra, melting point and the like.

This invention, in addition to the above listed compounds, is intended to encompass the use of analogs of such compounds. In this context, analogs are molecules having substantial biological similarities regardless of structural similarities.

The term “therapeutically effective amount” or “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.

The term “subject” as used herein is meant to include all mammals and in particular humans, in need of treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound of formula (I) chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can readily be determined by one of ordinary skill in the art.

Described below is a process for the preparation of compounds of formula (I) as shown in the scheme 1.

The compound of formula (1a) is reacted with a suitably substituted aryl halide (preferably bromide or iodide) in suitable solvents such as acetonitrile, DMF, DMSO, tetrahydrofuran, dioxane, halogenated solvents such as 1,2-dichloroethane, in presence of a suitable non-nucleophilic bases which include KF, Na2CO3, Cs2CO3at reflux temperatures and reaction time ranging from 1 hour to 48 hours followed by the isolation of the required product (1b) by the usual procedures. The compound of formula (1b) is cyclised, employing metal catalysts (palladium acetate in DMF, DMA or glacial acetic acid, nickel catalyst in pyridine or DMF, tetrakistriphenylphosphinepalladium in DMF and the like), preferably palladium acetate in DMF, to give the compound of formula (1c).

The compound of formula (1c) is oxidized to the acid through known literature procedures (such as sodium chlorite or potassium permanganate and the like). The acid intermediate is then converted to the corresponding amide. The amide on dehydration using dehydrating agents such as thionyl chloride or trifluoroactic anhydride (TFAA) or by direct dehydration of the oxime derivative of compound of formula (1c) using dehydrating agent such as thionyl chloride, TFAA and the like gives cyano compound. The cyano compound on reacting with P4S10or Lawesson's reagent gives compound of formula (1e).

Alternatively, the compound of formula (1d) is reacted with metal thiocyanate such as potassium thiocyanate in solvents such as acetonitrile, DMF, DMSO, tetrahydrofuran, dioxane, halogenated solvents such as 1,2-dichloroethane, ethers such as 1,2-dimethoxyethane, water, alkyl or haloalkyl sulfonic acids like methane sulfonic acid, trifluoromethane sulfonic acid and the like or a mixture thereof at temperatures ranging from 0° C. to reflux temperatures and reaction time ranging from 1 hour to 48 hours followed by the isolation of the required product (1e) by the usual procedures.

The compound of formula (1e) is then converted to the compounds of formula (IC) by reacting the thioamide compound of formula (1e) with the optionally substituted bromo pyurvates.

The compound of formula (1c) is oxidized to the acid through known literature procedures (such as sodium chlorite or potassium permanganate and the like). The acid is then coupled with the optionally substituted amino acid featuring a hydroxyl group under appropriate basic conditions (EDC, or triethylamine or HBTU (2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), HOBt, diisopropylamine or triethylamine and the like) reported in the literature. The amide derivative (10 is then cyclized to oxazoline through literature known methods (DAST, Deoxo-flour, triphenyl phosphine, (CBrCl2)2and the like). The oxazoline is then subjected to dehydrogenation, (methods include MnO2, DBU:CCl4:Pyridine, DDQ (2,3-Dichloro-5,6-Dicyano benzoquinone), chloroanil and the like) to give the oxazole compound of formula (IA).

The compound of formula (1c) is oxidized to the acid through known literature procedures (such as sodium chlorite or potassium permanganate and the like). The acid is then coupled with the optionally substituted diamino compound under appropriate basic conditions (EDC, or triethylamine or HBTU, HOBt, diisopropylamine or triethylamine and the like) reported in the literature, followed by cyclization under acidic conditions gives (IB).

The examples given below are provided by the way of illustration only and therefore should not be construed to limit the scope of the invention.

REPRESENTATIVE EXPERIMENTAL PROCEDURES

Synthesis of ethyl 6-(2-(4-methoxydibenzo[b,d]furan-1-yl)thiazol-4-yl)picolinate

Step 1: Preparation of 4-methoxydibenzo[b,d]furan

To a solution of 4-hydroxydibenzo[b,d]furan (2 g, 0.0109 mol) in DMF (5 mL) potassium carbonate (3.2 g, 0.0232 mol) and methyl iodide (1.35 mL, 0.0217 mol) was added. The reaction slurry was stirred at room temperature for 3 h. Subsequently the reaction mixture was poured into the cold water (100 mL) and extracted with hexane (3×100 mL). The organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was evaporated at reduced pressure to, give the desired product in 66% yield, Rf0.74 (Ethyl acetate:Hexane (3:7); MS m/z: 199.1 (M+1).

Step 2: Preparation of 4-methoxydibenzo[b,d]furan-1-carbothioamide

Step 3: Preparation of ethyl 6-(2-(4-methoxydibenzo[b,d]furan-1-yl)thiazol-4-yl)picolinate

Synthesis of 6-(2-(4-methoxydibenzo[b,d]furan-1-yl)thiazol-4-yl)picolinic acid

The following compounds were, prepared according to the procedure outlined above.

Synthesis of ethyl 2-(4-(difluoromethoxy)-8-nitrodibenzo[b,d]furan-1-yl)-1,3-oxazole-4-carboxylate

Step 1: Synthesis of 4-(difluoromethoxy)-3-hydroxy benzaldehyde

To a stirred solution of 3,4-dihydroxybenzaldehyde (25 g, 0.181 mol) in DMF (75 mL), potassium carbonate (70 g, 0.507 mol) was added. It was refluxed to 75-80° C. Chlorodifluoro methane gas was purged into the reaction mixture until 95% of the starting material was consumed. The reaction mixture was poured into the crushed ice and kept aside. After sometime the aqueous layer was extracted with ethyl acetate (3×500 mL). The organics were dried over sodium sulphate and filtered. The filtrate was evaporated under reduced pressure to give the crude product. The product was purified by column chromatography by using gradient (0-1) % of ethyl acetate in hexane. The solid obtained was washed with hexane and dried. Yield—25.3%. Rf—0.41 (Ethyl acetate:Hexane (3:7).

Step 2: Synthesis of 3-(2-bromo-4-nitrophenoxy)-4-(difluoromethoxy)benzaldehyde

To a solution of 4-(difluoromethoxy)-3-hydroxy benzaldehyde (8.6 g, 0.046 mol) in dimethylsulphoxide (15 mL) was added potassium fluoride (5.36 g, 0.092 mol) at room temperature. Then it was refluxed to 100-120° C. To this 2-bromo-1-fluoro-4-nitrobenzene (20.3 g, 0.093 mol) in dimethylsulphoxide (10 mL) was added dropwise at 100-120° C. The heating was continued for 5 hours. The reaction mixture was poured into the crushed ice and stirred for 1 hour. Then the aqueous layer was decanted. The solid obtained was dissolved in ethyl acetate (500 mL). The organic layer was washed with water (3×200 mL). The organics were dried over sodium sulphate and filtered. The solvents were removed in vacuum to give the crude product. The product was purified by column chromatography by using gradient (0-2) % of ethyl acetate in hexane. The solid obtained was washed with hexane and dried. Yield—66%.1H-NMR (DMSO-d6) δ: 7.06 (d, 1H), 7.23-7.41-7.59 (t, 1H), 7.67 (d, 1H), 7.82 (d, 1H), 7.96-7.98 (m, 1H), 8.20-8.23 (m, 1H), 8.61 (d, 1H), 9.97 (s, 1H); HPLC (purity): 99.5%; Mass calculated for C14H8BrF2NO5-388.1, observed—388.1; Rf—0.47 (Ethyl acetate:Hexane (3:7).

Step 3: Synthesis of 4-(difluoromethoxy)-8-nitrodibenzo[b,d]furan-1-carbaldehyde

To a mixture of 3-(2-bromo-4-nitrophenoxy)-4-(difluoromethoxy)benzaldehyde (11.5 g, 0.030 mol) and sodium acetate (3.7 g, 0.045 mol) in dimethylformamide (50 mL) palladium(II) acetate (0.68 g, 0.003 mol) was added in 4 different lots with 1 hour duration at 120-125° C. After the completion of the addition, it was refluxed for 16 hours. The reaction mixture was filtered through hyflo, washed with dimethylformamide (10 mL). The filtrate was poured into the cold water. The solid separated out was filtered. The solid was washed with hexane and dried. Yield—73.3%. HPLC (purity): 84.5%; Rf—0.5 (Ethyl acetate:Hexane (3:7)

Step 4: Synthesis of 4-(difluoromethoxy)-8-nitrodibenzo[b,d]furan-1-carboxylic acid

Step 6: Synthesis of ethyl 2-(4-(difluoromethoxy)-8-nitrodibenzo[b,d]furan-1-yl)-4,5-dihydrooxazole-4-carboxylate

Step 7: Synthesis of ethyl 2-(4-(difluoromethoxy)-8-nitrodibenzo[b,d]furan-1-yl)-1,3-oxazole-4-carboxylate

A mixture of methanol (100 mL) and ethyl acetate (100 mL) was added to the ethyl 2-(4-(difluoromethoxy)-8-nitrodibenzo[b,d]furan-1-yl)1,3-oxazole-4-carboxylate (0.8 g, 1.91 mmol) from step 7 of example 47. To the reaction mixture, palladium-carbon (0.30 g) was added. The reaction mixture was transferred to the hydrogenation flask and hydrogenation was performed at room temperature (40 psi-hydrogen) for 2 hours. The reaction mixture was filtered through celite. The filtrate was evaporated at reduced pressure to give the desired product. Yield—87.5%. Rf—0.5 (Ethyl acetate:Hexane (1:1)

Synthesis of 2-(4-(difluoromethoxy)-8-(methylsulfonamido) dibenzo[b,d]furan-1-yl)-1,3-oxazole-4-carboxylic acid

Synthesis of 2-(4-(difluoromethoxy)-8-(methylsulfoamido)dibenzo[b,d]furan-4-yl)-N-(4-hydroxycyclohexyl)-1,3-oxazole-4-carboxamide

The following compounds were prepared according to the procedure outlined above:

This assay determines the effect of test compounds on the production of TNF-α in human Peripheral Blood Mononuclear Cells (PBMC). Compounds were tested for their ability to inhibit the activity of TNF α in human PBMC. PBMC were isolated from blood (of healthy volunteers) using BD Vacutainer CPT™ (Cell preparation tube, BD Bio Science) and suspended in RPMI (Rosewell park memorial institute) medium (Physiol. Res.2003, 52, 593-598). The test compounds were pre-incubated with PBMC (0.5 million/incubation well) for 15 minutes at 37° C. and then stimulated with Lipopolysaccharide (Escherichia coli: B4; 1 μg/mL) for 18 h at 37° C. in 5% CO2. The levels of TNF-α in the cell culture medium were estimated using enzyme-linked immunosorbent assay performed in a 96 well format as per the procedure of the manufacturer (Cayman Chemical, Ann Arbor, USA). Representative results of TNF-α inhibition are shown in the Table I.

PDE4 (Phosphodiesterase type IV) enzymes convert cyclic AMP (cAMP) into AMP. The assay is performed to determine the effect of test compounds on the inhibition of purified human PDE4B enzyme.

The assay involves the detection of the Tritiated AMP (product) using SPA beads known as yittrium silicate. The linear AMP bind preferentially to SPA yittrium silicate beads compared to cyclic nucleotides in the presence of zinc sulphate. The binding of the radiolabelled product to the bead brings the isotope into close proximity to allow radiation from the tritium to excite the scintillant within the bead to emit light. The unbound radiolabel is not close enough to allow this energy transfer and the light emitted due to the binding of tritiated AMP is detected as cpm (counts per minute).

PDE4B activity was inhibited by the compounds according to the invention in a modified SPA (scintillation proximity assay) test, supplied by GE Healthcare Life Sciences (see procedural instructions “Phosphodiesterase [3H]-cAMP SPA enzyme assay, code TRKQ 7090”), carried out in 96-well microtitre plates. The test volume is 100 μL and contains 50 mM Tris buffer (pH 7.4), 8.3 mM Mg2+, in the presence of inhibitor or test compound, and containing PDE4B2 enzyme (sufficient to ensure that 10-20% of the cAMP is converted, under the said experimental conditions). The final concentration of DMSO in the assays (1% v/v) does not substantially affect the activity of the PDEs investigated. After a pre-incubation of 5 minutes at 37° C., the reaction is started by adding the substrate (cAMP; 0.5 μM cAMP, including about 50,000 cpm of [3H]-cAMP) and the assays are incubated for a further 10 minutes; after that, they are stopped by adding SPA beads containing 18 mM ZnSO4(50 μl). After the beads have been sedimented (>30 minutes), the microtitre plates are analyzed in a Microplate luminescence detection device (TopCount* NXT; PerkinElmer Life Sciences). Where, the signal in the absence of enzyme is defined as the background. 100% activity was defined as the signal detected in the presence of enzyme and DMSO with the background subtracted. The corresponding IC50values of the compounds for the inhibition of PDE4B2 activity are determined from the concentration-effect curves by means of non-linear regression fit of the standard 4-parameter/multiple binding sites equation from a eight- to ten-point titration. Representative results of PDE4B inhibition are shown in the Table II at 100 nM. (Catherine B. et al.Anal. Biochem.,1999, 275(2), pp. 148-155, David M. E.Biochem. Pharmacol.,1999, 57, pp. 965-973)

The LPS induced sepsis model in mice was performed as described by Les sekut et al (J Lab Clin Med 1994; 124, 813-20). Female Swiss albino mice were selected and the body weights were equivalent within each group. The mice were fasted for 20 h with free access to water. The mice were dosed orally with the test compound suspended in vehicle containing 0.5% Tween 80 in 0.25% Carboxy-methylcellulose sodium salt. The control mice were administered the vehicle alone. After 30 minutes of oral dosing, mice were injected with 500 μg of Lipopolysaccharide (Escherichia coli, LPS: B4 from Sigma) in phosphate buffer saline solution into the intraperitoneal cavity of the mice. After 90 minutes of LPS administration mice were bled via retro-orbital sinus puncture. Blood samples were stored overnight at 4° C. Serum samples were collected by centrifuging the samples at 4000 rpm for 15 minutes at 4° C. Immediately the serum samples were analysed for TNF-α levels using commercially available mouse TNF-α ELISA kit (Amersham Biosciences) and assay was performed by the manufacturer instruction. Representative results of TNF-α inhibition are shown in the Table III.

LPS induced neutrophilia in Sprague Dawley rats was performed using the protocol described in PulmPharmacol&Ther17,133-140, 2004. Male Sprague. Dawley rats were acclimatized to laboratory conditions five to seven days prior to the start of the experiment. They were randomly distributed to various groups based on body weight. Except normal group all the animals were exposed to LPS 100 μg/ml for 40 minutes. The rats were dosed with the test compound suspended in the vehicle containing 0.25% carboxymethylcellulose before half an hour of LPS exposure. BAL was performed 6 h after LPS exposure, total cell count and DLC was done and compared with control and the standard drug. Percentage Inhibition for neutrophilia was calculated and is shown in Table IV.