STEROIDAL COMPOUNDS

FIELD OF THE INVENTION

The present invention relates to steroid-based compounds useful in the treatment of inflammatory conditions.

BACKGROUND OF THE INVENTION

WO 2004/101595 discloses a class of estratriene derivatives which suppress inflammation.

The present invention provides a novel class of 6-benzooxadiazolylalkoxyimino estratriene derivatives which have surprisingly different selectivities of action when compared to the estratriene derivatives exemplified in WO 2004/101595.

The core structure of the estratriene derivatives showing positions C2, C3, C6, C13 and C17 of the molecule is as follows:

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a compound of Formula I, or pharmaceutically acceptable salts, derivatives or prodrugs thereof.

wherein:

Ri is selected from the group consisting of H, OR ₆ , halo, NH ₂ , NO ₂ , OC(O)R ₆ , and -CH-N-O-R ₇ ;

R ₂ is selected from the group consisting of OCi _-3 alkylC(O)OR ₆ , OCi _-3 alkylOR ₆ , OC(O)R ₆ , OR ₆ , S(O) _1n R ₈ , halo and H;

R ₃ is H;

R ₄ Is H;

R ₅ is selected from the group consisting of H, OR ₆ , C(O)R ₆ , OCi _-3 alkylOR ₆ , OC(O)R ₆ , C _1-3 alkylOS(O) _m R ₈ and S(O) ₀₁ R ₈ ;

R ₆ is selected from the group consisting of H, lower alkyl, aryl, alkylaryl and S(O) _1n R ₈ ;

R ₇ is selected from the group consisting of H, lower alkenyl, aryl and alkylaryl;

R ₈ is selected from the group consisting of H ₅ lower alkyl and NRgR ₁₀ ;

Rg and R ₁₀ are each independently selected from H and lower alkyl; and

m is an integer selected from O ₃ 1, 2 or 3.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier, diluent or excipient.

In a third aspect, the present invention provides a method of treating a disease or condition comprising an inflammatory component in a subject comprising the administration of a compound according to the first aspect or a pharmaceutical composition according to the second aspect to the subject.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the effect of CP92 (a compound according to the present invention) on an LPS induced model of lung inflammation. The upper panel shows the effect of CP92 on BALF protein levels. The lower panel shows the effect of CP92 on BAL cell numbers.

Figure 2 shows the effect of CP92 on a zymosan challenge model of peritonitis. The upper panel shows the effect of CP92 on peritoneal lavage cell numbers. The lower panel shows the effect of CP92 on peritoneal lavage protein content.

Figure 3, in the upper panel shows the effect of CP92 on the proliferation of cells in response to the proliferative stimulus of either thrombin (human airway smooth muscle) or basic fibroblast growth factor (bFGF) (human lung parenchymal fibroblasts, pFb). The upper panel shows the effect of CP92 on the stimulus by bFGF. The lower panel shows the effect of CP92 on the stimulus by thrombin.

Figure 4, shows the effect of CP92 and analogues of CP92 (CP-I -66, CP-1-68, CP-I -73) against a control in a Prostaglandin E ₂ assay.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a compound of Formula I, or pharmaceutically acceptable salts, derivatives or prodrugs thereof.

wherein:

Ri is selected from the group consisting of H, OR ₆ , halo, NH ₂ , NO ₂ , OC(O)R ₆ , and -CH=N-O-R ₇ , preferably H, hydroxy or lower alkoxy;

R ₂ is selected from the group consisting of OC ₁ - _S aIlCyIC(O)OR ₆ , OC _1-3 alkylOR ₆ , OC(O)R ₆ , OR ₆ , S(O) _1n R ₈ , halo and H, preferably halo, hydroxy, lower alkoxy, OC _1-3 alkylC(O)OR ₆ or OS(O) _m R ₈ , preferably OS(O) _m R ₈ is OS(O) ₂ NH ₂ ;

R ₃ is H;

R ₄ Is H;

R ₅ is selected from the group consisting of H, OR ₆ , C(O)R ₆ , OC _]-3 alkylOR ₆ , OC(O)R ₆ , and S(O) ₀₁ R ₈ , preferably H, lower alkoxy or S(O) ₀₁ R ₈ , preferably S(O)JR ₈ is S(O) ₂ NH ₂ ;

R ₆ is selected from the group consisting of H, lower alkyl preferably C1. ₃ a.kyl, aryl preferably phenyl, alkyϊaryl preferably benzyl, and S(O) _1n R ₈ preferably S(O) ₂ NH ₂ ;

R ₇ is selected from the group consisting of H, lower alkenyl preferably C _2-4 alkenyl, aryl preferably phenyl and alkylaryl preferably benzyl;

R ₈ is selected from the group consisting of H, lower alkyl preferably Ci _-3 alkyl and NR9R10;

R ₉ and Rio are each independently selected from H and lower alkyl, preferably both R ₉ and Rio are H; and

m is an integer selected from 0, 1, 2 or 3, preferably m is 2.

In a preferred embodiment of the invention, R ₂ is OH.

Preferably, the compound of formula I is:

In another preferred embodiment, R ₂ is OS(O) ₂ NH ₂ . A preferred compound of formula I wherein R ₂ is OS(O) ₂ NH ₂ is:

Advantageously, compounds of formula I having a OS(O) _m R ₈ group, preferably a OS(O) ₂ NH ₂ group, in the C3 position may demonstrate improved bioavailability.

In still another preferred embodiment, R ₂ is lower alkoxyl, preferably Ci _-3 alkoxyl. A preferred compound of formula I wherein R ₂ is lower alkoxyl is:

In yet another preferred embodiment, R ₂ is OC _1-3 alkylC(O)OR ₆ , preferably OC _1-3 alkylC(O)Oloweralkyl. Preferred compounds of formula I wherein R ₂ is OCi _-3 alkylC(O)OR ₆ are:

In still a further preferred embodiment of the first aspect, the present invention provides compounds of formula I wherein R ₅ is H. Preferred compounds of formula I wherein R ₅ is H are:

In another preferred embodiment R ₅ is S(O) _1n R ₈ , preferably S(O) ₂ NH ₂ . Preferred compounds of formula I wherein R ₅ is S(O) ₀₁ R ₈ are:

and

The compound of Formula I is shown with a particular conformation around the C=N double bond of the 6-benzooxadiazolylalkoxyimino group which is considered to be the

"E" conformation. Compounds having the "Z" conformation are also contemplated by the present invention. The "E" conformation is preferred.

As used herein, the term "alkyl" either used alone or in compound terms such as NH(alkyl) or N(alkyl) ₂ , refers to monovalent straight chain or branched hydrocarbon groups. The term "lower alkyl" refers to monovalent straight chain or branched Ci _-6 alkyl, preferably Ci- ₃ alkyl. For example, suitable alkyl groups include, but are not limited to methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 2-, 3- or 4-methylpentyl, 2-ethylbutyl, n-hexyl or 2-, 3-, 4- or 5-methylpentyl.

As used herein, the term "alkenyl" refers to straight chain or branched hydrocarbon groups having one or more double bonds between carbon atoms. The term "lower alkenyl" refers to straight chain or branched C _2-6 alkenyl, preferably C _2-4 alkenyl. Suitable alkenyl groups include, but are not limited to ethenyl, propenyl, isopropenyl, butenyl, pentenyl and hexenyl.

As used herein, the term "alkynyl" refers to straight chain or branched hydrocarbon groups having one or more triple bonds between carbon atoms. The term "lower alkynyl" refers to straight chain or branched C ₂ - ₆ alkynyl, preferably C _2-4 alkynyl. Suitable alkynyl groups include, but are not limited to ethynyl, propynyl, isopropynyl, butynyl, pentynyl and hexynyl.

As used herein, the term "alkoxy" refers to straight chain or branched chain hydrocarbon groups having an oxygen atom linkage between carbon atoms in the chain. The term "lower alkoxyl" refers to straight chain or branched C _1-6 alkoxyl, preferably C _1-3 alkoxyl.

As used herein, the term "amino" refers to a group -NRR where each R may be independently selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl and alkylaryl. Suitable amino groups include, but are not limited to, NH ₂ , NHloweralkyl, N(loweralkyl) ₂ , and NHaryl.

As used herein, the term "aryl" refers to any of mono-, bi- or polycyclic (including conjugated and fused) hydrocarbon ring systems preferably having 6 to 10 carbon atoms

and containing an aromatic residue. Suitable aryl groups include, but are not limited to, phenyl.

As used herein, the term "alkylaryl" refers to any of mono-, bi- or polycyclic (including conjugated and fused) hydrocarbon ring systems preferably having 6 to 10 carbon atoms and containing an aromatic residue together with an alkyl linkage. Suitable alkylaryl groups include, but are not limited to, benzyl (i.e. -CH ₂ phenyl).

As used herein, the term "halo" or "halogen" refers to fluorine (fluoro), chlorine (chloro), bromine (bromo) or iodine (iodo).

As used herein, the term "sulfoxide", either alone or in a compound word, refers to -S(O)R where R is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl and alkylaryl.

As used herein, the term "sulfonyl", either alone or in a compound word, refers to -S(O) ₂ R, where R is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl and alkylaryl.

As used herein, the term "sulfonamide" or "sulfonamyl" or "sulfonamido", either alone or in a compound word, refers to a group S(O) ₂ NRR, where each R is independently selected from R hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl and alkylaryl.

As used herein, the term "sulfate" refers to a group -OS(O) ₂ OR where R is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl and alkylaryl.

As used herein, the term "sulfonate" refers to a group -SO ₃ R where R is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl and alkylaryl.

As used herein, the term "thio" refers to a group -SR where R is selected from hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl and alkylaryl.

Each alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group and alkylaryl group may be optionally substituted with one or more substituents selected from the group consisting of Ci-C ₃ alkyl, C ₆ aryl, alkylaryl, OH, OCi-C ₃ alkyl, halo, CN, NO ₂ , CO ₂ H, CO ₂ C,.C ₃ alkyl, CONH ₂ , CONH(C _1- C ₃ alkyl), CON(C _1- C ₃ alkyl) ₂ , trifluoromethyl, NH ₂ , NH(Ci.C ₃ alkyl) and N(C].C ₃ alkyl) ₂ . For example, an optionally substituted alkyl group

may be 2-hydroxyethyl, trifluoromethyl, or difluoromethyl. Each aryl may optionally be fused with a dioxolane ring. Any of the above substituents may additionally be substituted by optional substituents.

Optional substituents also include suitable nitrogen protecting groups (see "Protective Groups in Organic Synthesis" Theodora Greene and Peter Wuts, third edition, Wiley Interscience, 1999).

The salts of the compound of formula I are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts.

The term "pharmaceutically acceptable derivative" may include any pharmaceutically acceptable salt, hydrate or prodrug, or any other compound which upon administration to a subject, is capable of providing (directly or indirectly) a compound of formula I or a pharmaceutically active metabolite or residue thereof.

Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, zinc, ammonium, alkylammonium such as salts formed from triethylamine, alkoxyammonium such as those formed with ethanolamine and salts formed from ethylenediamine, choline or amino acids such as arginine, lysine or histidine. General information on types of pharmaceutically acceptable salts and their formation is known to those skilled in the art and is as described

in general texts such as "Handbook of Pharmaceutical salts" P.H.Stahl, C.G.Wermuth, 1 ^st edition, 2002, Wiley- VCH.

Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

This invention also encompasses pharmaceutical compositions containing prodrugs of compounds of formula I. Compounds of formula I having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs.

Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (eg, two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy and carboxylic acid groups of compounds of formula

I. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvlin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of formula I through the carbonyl carbon prodrug sidechain. Prodrugs also include phosphate derivatives of compounds of formula I

(such as acids, salts of acids, or esters) joined through a phosphorus-oxygen bond to a free hydroxyl of compounds of formula I.

It will also be recognised that the compounds of formula I may possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form. The invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres eg., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.

The compounds of the present invention may be administered by any suitable means, for example, parenterally, such as by subcutaneous, intravenous, intramuscular, or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions). The compounds of the present invention may also be administered intranasally or via inhalation, for example by atomiser, aerosol or nebulizer means. In a preferred embodiment, the compounds of the present invention are administered by infusion, even more preferably by IV infusion. In still another preferred embodiment, the compounds of the present invention are administered to a subject by IV infusion as an infusion formulation wherein the infusion formulation comprises: a compound of general formula I or pharmaceutically acceptable salts, derivatives or prodrugs thereof; solutol; and ethanol.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of the first aspect and a pharmaceutically acceptable carrier, diluent or excipient.

The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Pharmaceutical formulations include those for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The compounds of the invention, together with a conventional adjuvant, carrier or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids as solutions, suspensions, emulsions, elixirs or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.

In a preferred embodiment, the pharmaceutical formulations of the present invention are administered by infusion.

The pharmaceutical compositions for the administration of the compounds of this invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The compositions of the present invention may contain other therapeutic agents as described below, and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned 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-butane diol. 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 oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds which are usually applied in the treatment of inflammatory conditions. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Suitable agents for use in combination with the compounds of the present invention include, for example, the glucocorticoids.

When other therapeutic agents are employed in combination with the compounds of the present invention they may be used for example in amounts as noted in the Physician Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

In a third aspect, the present invention provides a method of treating a condition or disease comprising an inflammatory component in a subject comprising the administration of an effective amount of a compound according to the first aspect or a pharmaceutical composition according to the second aspect to the subject.

Representative conditions or diseases comprising an inflammatory component include, but are not limited to, (1) inflammatory or allergic diseases such as systemic anaphylaxis or hypersensitivity responses, drug allergies, insect sting allergies; inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, ileitis and enteritis; vaginitis; psoriasis and inflammatory dermatoses such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis; spondyloarthropathies; scleroderma; respiratory allergic diseases such as asthma, allergic rhinitis, hypersensitivity lung diseases, and the like, (2) autoimmune diseases, such as arthritis (rheumatoid and psoriatic), osteoarthritis,

multiple sclerosis, systemic lupus erythematosus, diabetes mellitus, glomerulonephritis, and the like, (3) graft rejection (including allograft rejection and graft-v-host disease), and (4) other diseases in which undesired inflammatory responses are to be inhibited (e.g., atherosclerosis, myositis, neurological conditions such as stroke and closed-head injuries, neurodegenerative diseases, Alzheimer's disease, encephalitis, meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonary disease, sinusitis and Behcet's syndrome).

In a preferred embodiment, the condition or disease affects the respiratory system of the subject such as acute respiratory distress syndrome or cystic fibrosis, preferably acute cystic fibrosis. Even more preferably the condition or disease is a respiratory allergic disease such as asthma, allergic rhinitis, hypersensitivity lung diseases, and the like, preferably asthma.

In a preferred embodiment a compound according to the first aspect or a pharmaceutical composition according to the second aspect of the invention is administered to a subject with said respiratory conditions.

Conditions and diseases comprising an inflammatory component which are arranged in accordance with the system of the body which they affect are listed in Table 1 below.

Table 1

In a further embodiment, the inflammatory disease is selected from the group consisting of chronic inflammatory disease and acute inflammatory disease.

In another embodiment, the disease or condition is associated with hypersensitivity.

Preferably, the hypersensitivity is selected from the group consisting of Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and delayed type hypersensitivity.

More preferably, the delayed type hypersensitivity is selected from the group consisting of contact dermatitis and drug eruption.

More preferably, the T lymphocyte-mediated hypersensitivity is selected from the group consisting of helper T lymphocyte mediated hypersensitivity and cytotoxic T lymphocyte mediated hypersensitivity.

Yet more preferably, the helper T lymphocyte-mediated hypersensitivity is selected from the group consisting of T _h1 lymphocyte mediated hypersensitivity and T _h2 lymphocyte mediated hypersensitivity.

In another embodiment, the disease or condition is associated with autoimmune disease.

Preferably, the autoimmune disease is selected from the group consisting of cardiovascular disease, rheumatoid disease, glandular disease, gastrointestinal disease, cutaneous disease, hepatic disease, neurological disease, muscular disease, nephric disease, disease related to reproduction, connective tissue disease and systemic disease.

More preferably, the cardiovascular disease is selected from the group consisting of occlusive disease, atherosclerosis, myocardial infarction, thrombosis, Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome, anti-factor VIII autoimmune disease, necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis, antiphospholipid syndrome, antibody induced heart failure, thrombocytopenic purpura, autoimmune hemolytic anemia, cardiac autoimmunity in Chagas' disease and anti-helper T lymphocyte autoimmunity.

More preferably, the rheumatoid disease is selected from the group consisting of rheumatoid arthritis and ankylosing spondylitis.

More preferably, the glandular disease is selected from the group consisting of pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome.

More preferably, the gastrointestinal disease is selected from the group consisting of colitis, ileitis, Crohn's disease, chronic inflammatory intestinal disease and celiac disease.

More preferably, the cutaneous disease is selected from the group consisting of autoimmune bullous skin disease, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

More preferably, the hepatic disease is selected from the group consisting of autoimmune hepatitis and primary biliary cirrhosis.

More preferably, the neurological disease is selected from the group consisting of neurodegenerative disease, multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-Eaton myasthenic syndrome, paraneoplastic neurological disease, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de Ia Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia, arthrogryposis multiplex, optic neuritis and stiff-man syndrome.

More preferably, the muscular disease is selected from the group consisting of autoimmune myositis, primary Sjogren's syndrome and smooth muscle autoimmune disease.

More preferably, the nephric disease is autoimmune interstitial nephritis.

More preferably, the disease related to reproduction is repeated fetal loss.

More preferably, the connective tissue disease is selected from the group consisting of autoimmune ear disease and autoimmune disease of the inner ear.

More preferably, the systemic disease is selected from the group consisting of systemic lupus erythematosus and systemic sclerosis.

In a further embodiment, the disease or condition is associated with an infectious disease.

Preferably, the infectious disease is selected from the group consisting of chronic infectious disease, subacute infectious disease, acute infectious disease, viral disease, bacterial disease, protozoan disease, parasitic disease, fungal disease, mycoplasma disease and prion disease.

In a further embodiment, the disease or condition is associated with a disease associated with transplantation of a graft.

Preferably, the disease is selected from the group consisting of graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.

Preferably, the graft is selected from the group consisting of a syngeneic graft, an allograft and a xenograft.

Preferably, the graft is selected from the group consisting of a cellular graft, a tissue graft, an organ graft and an appendage graft.

More preferably, the cellular graft is selected from the group consisting of a stem cell graft, a progenitor cell graft, a hematopoietic cell graft, an embryonic cell graft and a nerve cell graft.

More preferably, the tissue graft is selected from the group consisting of a skin graft, a bone graft, a nerve graft, an intestine graft, a corneal graft, a cartilage graft, a cardiac tissue graft, a cardiac valve graft, a dental graft, a hair follicle graft and a muscle graft.

More preferably, the organ graft is selected from the group consisting of a kidney graft, a heart graft, a skin graft, a liver graft, a pancreatic graft, a lung graft and an intestine graft.

More preferably, the appendage graft is selected from the group consisting of an arm graft, a leg graft, a hand graft, a foot graft, a finger graft, a toe graft and a sexual organ graft.

In a further embodiment, the disease or condition is associated with an allergic disease.

Preferably, the allergic disease is selected from the group consisting of asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.

In another embodiment, the disease or condition is associated with a neurodegenerative disease.

In a further embodiment, the disease or condition is associated with a cardiovascular disease.

In a further embodiment, the disease or condition is associated with a gastrointestinal disease.

In a further embodiment, the disease or condition is associated with a tumor.

Preferably, the tumor is selected from the group consisting of a malignant tumor, a benign tumor, a solid tumor, a metastatic tumor and a non-solid tumor.

In a further embodiment, the disease or condition is associated with septic shock.

In a further embodiment, the disease or condition is associated with anaphylactic shock.

In a further embodiment, the disease or condition is associated with toxic shock syndrome.

In a further embodiment, the disease or condition is associated with cachexia.

In a further embodiment, the disease or condition is associated with necrosis.

In a further embodiment, the disease or condition is associated with gangrene.

In a further embodiment, the disease or condition is associated with a prosthetic implant.

Preferably, the prosthetic implant is selected from the group consisting of a breast implant, a silicone implant, a dental implant, a penile implant, a cardiac implant, an artificial joint, a

bone fracture repair device, a bone replacement implant, a drug delivery implant, a catheter, a pacemaker and a respirator tube.

In a further embodiment, the disease or condition is associated with menstruation.

In a further embodiment, the disease or condition is associated with an ulcer.

Preferably, the ulcer is selected from the group consisting of a skin ulcer, a bed sore, a gastric ulcer, a peptic ulcer, a buccal ulcer, a nasopharyngeal ulcer, an esophageal ulcer, a duodenal ulcer and a gastrointestinal ulcer.

In a further embodiment, the disease or condition is associated with an injury.

Preferably, the injury is selected from the group consisting of an abrasion, a bruise, a cut, a puncture wound, a laceration, an impact wound, a concussion, a contusion, a thermal burn, frostbite, a chemical burn, a sunburn, a desiccation, a radiation burn, a radioactivity burn, smoke inhalation, a torn muscle, a pulled muscle, a torn tendon, a pulled tendon, a pulled ligament, a torn ligament, a hyperextension, a torn cartilage, a bone fracture, a pinched nerve and a gunshot wound.

In a further embodiment, the disease or condition is a musculoskeletal inflammation.

Preferably, the musculoskeletal inflammation is selected from the group consisting of a muscle inflammation, myositis, a tendon inflammation, tendinitis, a ligament inflammation, a cartilage inflammation, a joint inflammation, a synovial inflammation, carpal tunnel syndrome and a bone inflammation.

In a further embodiment, the disease or condition is selected from the group consisting of an idiopathic inflammation and an inflammation of unknown etiology.

Preferably, the disease or condition comprising an inflammatory component is selected from the group consisting of arthritis, nephritis, asthma, bronchitis, chronic granulomatous disease, and psoriasis. More preferably, the disease or condition is asthma.

In addition to primates, such as humans, a variety of other mammals can be treated according to the method of the present invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated. However, the method can also be practiced in other species, such as avian species (e.g., chickens). Preferably, the subject is a human.

The term "effective amount" means the amount of the subject composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

The terms "administration of and or "administering a" compound should be understood to mean providing a compound of the invention to the individual in need of treatment.

In the treatment or prevention of inflammatory conditions, an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5 mg/kg per day, 0.5 to 5 mg/kg per day or 5 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of

administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples.

COMPOUND SYNTHESIS

Experimental procedure for the synthesis of 6-[((Benzo[c][l,2,5]oxadiazol-5- yl)methoxy)imino]-2-methoxyestradiol (CP92)

(Benzo[c][l,2,5]oxadiazol-5-yl)methoxyamine hydrochloride (0.096 g, 0.475 mmol) was added to a solution of 2-methoxy-6-oxoestradiol (see below) (0.104 g, 0.327 mmol) in methanol (8 ml). Poly(4-vinylpyridine) (25% cross-linked) (0.668 g) was added. The suspension was stirred at room temperature under nitrogen for 24h and concentrated to dryness. Tetrahydrofuran (8 ml) followed by PS-isocyanate (1.46 mmol/g) (0.195 g) were added. The suspension was stirred at room temperature under nitrogen for 2Oh and filtered through Celite™. The filtrate was concentrated to a yellow oil. This was purified by column chromatography (silica), eluting with 1:2 hexane/ethyl acetate to give 6-[((benzo[c][l,2,5]oxadiazol-5-yl)methoxy)imino]-2-methoxye stradiol (CP9-2) as a foamy yellow solid which was washed with hexane (0.139 g, 92%).

Rf- 0.61 (8:2 ethyl acetate/hexane).

¹ H nmr (CDCl ₃ ): δ 0.79 (s, 3H); 1.22-1.68 (m, 6H); 1.76-1.84 (m, IH); 1.98-2.02 (m, 2H); 2.12-2.24 (m, 2H); 2.28-2.36 (m, IH); 3.18 (dd, J 18.1, 4.4 Hz, IH); 3.77 (t, J 8.8 Hz, IH); 3.92 (s, 3H); 5.27 (s, 2H); 5.46 (brs, IH); 6.80 (s, IH); 7.44 (dd, J9.3, 1.2 Hz, IH); 7.49 (s, IH); 7.78 (t, J 1.0 Hz, IH); 7.81 (dd, J 9.3, 1.0 Hz, IH).

MS ESI +ve: M ⁺ +H, 464.5.

MS ESI -ve: M ⁺ -H, 462.5

m.p. 149-15O ⁰ C.

Experimental procedure for the synthesis of 6-(Benzo[c][l,2,5]oxadiazol-5- yl)methoxyamine hydrochloride

(Benzo[c][l,2,5]oxadiazol-5-yl)methoxyphthalimide

5-(Bromomethyl)benzo[c][l,2,5]oxadiazole (0.441 g, 2.07 mmol) was added to a solution of N-hydroxyphthalimide (0.336 g, 2.06 mmol) in dry tetrahydrofuran (15 ml). N ₅ N-

Diisopropylethylamine (0.519 g, 4.02 mmol) was added and the solution was stirred at reflux under nitrogen for 23h and cooled to room temperature. The suspension was concentrated in vacuo to a pale beige solid. This was diluted with water and filtered. The residue was washed with plenty of water and dried under vacuum to give (benzo[c][l,2,5]oxadiazol-5-yl)methoxyphthalimide (0.571 g, 94%).

Rf- 0.50 (100% dichloromethane)

¹ H nmr (CDCl ₃ ): 55.30 (s, 2H); 7.75-7.78 (m, 3H); 7.83-7.85 (m, 2H); 7.91 (d, J 1.46 Hz, IH); 7.92 (d, J 9.8 Hz, IH).

Benzo[c][l,2,5]oxadiazol-5-yl)methoxyamine hydrochloride

Concentrated hydrochloric acid (2 ml) was added to a suspension of (benzo[c][l,2,5]oxadiazol-5-yl)methoxyphthalimide (0.571 g, 1.94 mmol) in ethanol (15 ml). The suspension was stirred at reflux for 18h and allowed to cool down slightly before water was added. The aqueous phase was washed with chloroform (x3), then concentrated to give (benzo[c][l,2,5]oxadiazol-5-yl)methoxyamine hydrochloride as an off-white solid (0.390 g, 99%).

¹ H nmr (d6-DMSO): δ 5.15 (s, 2H); 7.61 (dd, J 9.3, 1.4 Hz, IH); 8.09 (t, J 1.0 Hz, IH); 8.11 (d, J 9.3, LO Hz ₅ IH).

MS ESI +ve: M ⁺ +H, 166.1.

Experimental procedure for the synthesis of 2-Methoxy~6-oxoestradiol

The synthesis of this compound has previously been described in WO 2004/101595, the disclosure of which is hereby incorporated by reference.

Experimental procedure for the synthesis of 3-0-(Ethoxycarbonylmethyl)~2- methoxy6-([l,2,5]oxadiazolebenzyloxy)iminoestradiol (Compound AA-1-66)

Anhydrous potassium carbonate (0.312 g, 2.26 mmol) was added to a solution of

2-methoxy-6-([l,2,5]oxadiazolebenzyloxy) iminoestradiol (0.150 g, 0.323 mmol) in acetonitrile (5 ml). Ethyl bromoacetate (0.054 g, 0.323 mmol) was added, and the suspension was stirred at reflux for 4h and cooled to room temperature. Water (20 ml) was added and the mixture was extracted with ethyl acetate (x3). The combined organic extracts were washed with water, brine, dried over sodium sulphate, filtered and concentrated. The crude product was purified by column chromatography (silica) eluting with 1: 2 hexane /ethyl acetate to give AA- 1-66 (0.145 g, 82%).

Rf- 0.50 (1 :3 hexane /ethyl acetate)

Experimental procedure for the synthesis of 3-0-(Ethoxycarbonylmethyl)-2-methoxy~ 6-([l,2,5]oxadiazolebenzyIoxy) 17-sulphamoyliminoestradiol (Compound AA-1-69)

A solution of chlorosulfonyl isocyanate (0.108 g, 0.76 mmol) in anhydrous acetonitrile (ImI) was cooled in an ice bath under nitrogen. Formic acid (0.040 g, 0.86 mmol) was added. The mixture was stirred at room temperature for 2h. N,N-Dimethylacetamide (0.05 ml) was added and the mixture was stirred at room temperature for Ih then chilled in an ice bath. A solution of 3-O-(Ethoxycarbonylmethyl)-2-methoxy-6-

([l,2,5]oxadiazolebenzyloxy) iminoestradiol (AA-I -66) (0.050 g, 0.091 mmol) in N ₅ N- dimethylacetamide (5 ml) was added. The mixture was stirred at room temperature under nitrogen overnight. Water (10 ml) was added. This was extracted with ethyl acetate (x3). The combined organic extracts were washed with brine, dried over magnesium sulphate, filtered and concentrated. The crude product was purified by column chromatography (silica) eluting with with 1:1 hexane /ethyl acetate to give AA-1-69 (0.042 g, 74%).

Rf- 0.56 (1:1 hexane /ethyl acetate)

Experimental procedure for the synthesis of 2-methoxy-6-([l,2,5] oxadiazolebenzyloxy)iminoestradiol- 3,17-bis- sulphamate (Compound AA-1-68)

A solution of chlorosulfonyl isocyanate (0.368 g, 2.6 mmol) in anhydrous acetonitrile (ImI) was cooled in an ice bath under nitrogen. Formic acid (0.133 g, 2.9 mmol) was added. The mixture was stirred at room temperature for 1.5h. N,N-Dimethylacetamide (0.05 ml) was added and the mixture was stirred at room temperature for Ih then chilled in an ice bath. A solution of 2-methoxy-6-([l,2,5]oxadiazolebenzyloxy)iminoestradiol (CP92) (0.150 g, 0.323 mmol) in N,N-dimethylacetamide (5 ml) was added. The mixture was stirred at room temperature under nitrogen overnight. Water (20 ml) was added. This was extracted with ethyl acetate (x3). The combined organic extracts were washed with brine, dried over magnesium sulphate, filtered and concentrated. The crude product was purified by column chromatography (silica) eluting with 1 :2 hexane/ethyl acetate to give AA-1-68 (0.139 g, 70 %).

Rf- 0.54 (1 :2 hexane /ethyl acetate).

Experimental procedure for the synthesis of 17-sulphamoyl-6-

([l,2,5]oxadiazolebenzyloxy)imino-2-methoxy-3-0-methylest radiol

(Compound AA-1-73)

A solution of chlorosulfonyl isocyanate (0.197 g, 1.39 mmol) in anhydrous acetonitrile (1 ml) was cooled in an ice bath under nitrogen. Formic acid (0.071 g, 1.55 mmol) was added. The mixture was stirred at room temperature for 2h. N,N-Dimethylacetamide (0.05 ml) was added and the mixture was stirred at room temperature for Ih then chilled in an ice bath. A solution of 6-([l,2,5]oxadiazolebenzyloxy)imino-2,3-dimethoxyestratriene (0.08Og, 0.168 mmol) in N,N-dimethylacetamide (10 ml) was added. The mixture was stirred at room temperature under nitrogen overnight. Water (20 ml) was added. This was extracted with ethyl acetate (x3). The combined organic extracts were washed with brine, dried over magnesium sulphate, filtered and concentrated. The crude product was purified by column chromatography (silica) eluting with l :lhexane /ethyl acetate to give AA-1-73 (0.084 g, 90%).

Rf- 0.59 (1 :2 hexane/ethyl acetate)

BIOLOGICAL ACTIVITY

1. LPS Mouse Model of acute lung injury

CP92 demonstrates anti-inflammatory activity when dosed in the LPS Mouse model of acute lung injury.

The model used is very similar to the one described by Bozinovski, S., Jones, J.E., Vlahos, R., Hamilton, J.A. and Anderson, G.P. (2002) "Granulocyte/macrophage-colony- stimulating factor (GM-CSF) regulates lung innate immunity to lipopolysaccharide through Akt/Erk activation of NFkappa B and AP-I in vivo" J Biol Chem, 277, 42808-14.

Intratracheal administration of Lipopolysaccharide (LPS) to mice results in injury to the lung and a resulting increase in total recoverable bronchoalveolar lavage (BAL) total cells mainly due to the infiltration of neutrophils to the site of injury. The LPS injury also results in increased expression of genes involved in the inflammatory response including ICAM, GM-CSF and COX-2. The effect of CP92 on BAL cell number, total BAL protein and inflammatory gene expression was analysed. In this experiment female Balb/c mice (17-21 g) were given a single intraperitoneal (i.p.) injection of vehicle or CP92 (150, 50, 15 and 5 mg/kg in peanut oil DMSO) Two hours after the mice were anaesthetised with methoxyflurane and a droplet of LPS (1 μg in saline) or sterile saline was applied to the nose. As the animals breathe through their nose, the LPS or saline is inhaled and gets access to the lung tissue. Twenty four hours post-LPS administration, mice were euthanized with a mixture of 200 μl ketamine and 10 μl xylazine i.p., tracheotomised and bronchoalveolar lavage (BAL) performed for the assessment of cell number, cell type and protein content. The lungs were removed and weighed as an indicator of any adverse effects of the drug treatment. Total RNA was isolated from the lung tissues for analysis of gene expression (mRNA) using RT-PCR.

As shown in Figure 1 , CP92 caused dose-dependent decreases in total BAL cell numbers and protein content.

2. Pulmonary Lipopolysaccharide-Induced Neutrophilia SD rat assay

Test substance CP92 was evaluated for possible protective activity in a rat model of lipopolysaccharide-induced pulmonary neutrophilia.

Female Sprague-Dawley rats weighing 180 + 2Og (provided by BioLasco Taiwan under Charles River Laboratories Technology Licensee) were used. Space allocation for animals was 45 x 23 x 21 cm for 7 rats. The animals were housed in animal cages. All animals were maintained in a hygienic environment under controlled temperature (22 ⁰ C - 24 ⁰ C) and humidity (60 - 80%) with 12 hours light/dark cycles for at least one week prior to use. The rats were fasted overnight prior to use.

Test substance CP92 at doses of 15, 50 and 150mg/kg and vehicle (90% peanut oil (Sigma, USA)A 0% DMSO (Merck, Germany), 4mL/kg) were each administered intraperitoneally (IP) to test animals 2 hours after intratracheal challenge with ~50μg/kg of E. coli lipopolysaccharide (LPS, Sigma USA, serotype O111:B4, in sterile saline, lOμg in 300μL per rat). Dexamethasone 21 -acetate (Sigma, USA) as a positive control was given orally 2 hours before or intraperitoneally 2 hours after LPS.

The formulations are summarised as follows. The dosing volume of 4mL/kg was used.

(a) This is based on visual observation : S = soluble, I = insoluble (or a suspension), ppt = precipitation

(b) Y = formula is kept in tube or vial with brown colour, or covered with aluminium foil. N = no protection from light. (c) RT = prepared fresh and stored under 15 ⁰ C ~ 3O ⁰ C.

4 ⁰ C = prepared fresh and stored in the refrigerator or kept on ice.

Rats were anaesthetised intravenously (IV) by pentobarbital (60mg/kg, IP), twenty-four hours after LPS challenge, 2mL of phosphate buffered saline (PBS) was administered twice through a tracheal cannula after which about a total of 3.5mL of bronchoalveolar lavage fluid (BALF) was harvested for total and differential cell counts as well as determination of protein content. BALF return volume for each animal was measured (accurate to the nearest 10 microlitres) and reported. Total cell number/mL and % neutrophils as well as protein content in BALF were then determined and one-way ANOVA followed by Dunnett's test was used to analyse difference between the vehicle control and treated groups. Significant difference was considered at P<0.05.

In addition, the blood samples from group 1 to group 5 were collected into lithium heparinized tubes at 1, 2 and 4 hours after test compounds administration. The plasma samples were isolated (~100μL) and stored at -8O ⁰ C.

CP92 at 150mg/kg caused significant decreases in both total white cell count and neutrophil count in bronchoalveolar lavage fluid relative to the vehicle control (Table 1-1).

In addition, CP92 at 50 and 150 mg/kg caused a significant decrease in protein content of bronchoalveolar lavage fluid (BALF) relative to the vehicle control (Table 2-1).

Dexamethasone at 1 mg/kg, given orally 2 hours before or intraperitoneally 2 hours after LPS challenge, was associated with significant decreases in both total white cell count and neutrophil count as well as in protein content of BALF in rats.

Test substance (CP92) was administered intraperitoneally (IP) to groups of 7 SD female rats 2 hours after challenge with E. CoIi lipopolysaccharide (LPS, 10μg/300μL/rat, IT). Dexamethasone, as a positive control was given orally 2 hours before or intraperitoneally 2 hours after LPS challenge. 24 hours after LPS challenge, bronchoalveolar lavage fluid of each test animal was taken for measurement of total cell and differential cell counts. One way ANOVA followed by Dunnett's test was used for the comparison between the vehicle control and test substance-treated groups. Significant difference was considered at P<0.05 and was denoted as * vs. vehicle control.

Test substance (CP92) was administered intraperitoneally (IP) to groups of 7 SD female rats 2 hours after challenge with E. CoIi lipopolysaccharide (LPS, 10μg/300μL/rat, IT). Dexamethasone, as a positive control, was given orally 2 hours before or intraperitoneally 2 hours after LPS challenge. 24 hours after LPS challenge, bronchoalveolar lavage fluid of each test animal was taken for measurement of protein contents of BALF and the return volume of BALF was also recorded. One-way ANOVA followed by Dunnett's test was used for the comparison between the vehicle control and test substance-treated groups. Significant difference was considered at P<0.05 and was denoted as * vs. vehicle control.

(Marley, S.B., et. al, 1994; Mohler, K.M, et. al, 1994; and Birrell, M.A., et. al, 2004).

3. Zymosan challenge as a model of peritonitis

The peritonitis model is conducted in C57BL/6 mice. Drag is administered subcutaneously in a vehicle of 10% DMSO 90 % peanut oil in a volume of 100 mL 2 hours before administration of 1 mg zymosan (50mg/kg, ip) into the peritoneal cavity in a volume of 100 mL. After a period of 4 hours, the mice are killed by inhalational anaesthetic, methoxyfluorane and the peritoneal cavity is lavaged using 1 mL of phosphate buffered saline. The number of viable cells and the protein content of the recovered lavage

fluid are measured by haemocytometer counting of propidium iodide excluding cells and Bradford protein assay, respectively.

The model is conducted according to the methods described by

Robert Harmon, Jamie D. Croxtall, Stephen J. Getting, Fiorentina Roviezzo, U Simon Yona, Mark J. Paul-Clark, Felicity N. E. Gavins, Mauro Perretti, John F. Morris, Julia C. Buckingham, and Roderick J. Flower. "Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse" The FASEB Journal express article (2002) 17:253-5..

In these experiments, CP92 administered at a dose of 50 mg/kg, sc significantly attenuated the increase in cell recruitment to the peritoneal cavity but had no detectable effect on protein levels (see Figure 2).

4. Effects of CP92 on the proliferation of cells in response to the proliferative stimulus of either thrombin (human airway smooth muscle) or basic fibroblast growth factor (bFGF)(human lung parenchymal fibroblasts, pFb)

We investigated the effects of CP92 on the proliferation of cells in response to the proliferative stimulus of either thrombin (human airway smooth muscle) or basic fibroblast growth factor (bFGF)(human lung parenchymal fibroblasts, pFb). Cells were seeded onto 6-well plates at a density of 1.5 x 10 cells cm ² , made quiescent by removal of serum- containing media for 24 hours and then stimulated for 48 h with either thrombin (0.3U/ml) or bFGF (30OpM). The test compounds were pre-incubated with HASM or pFb cells for 30 minutes before the addition of thrombin. At the end of the 48 hour incubation period, cells were detached from the culture plate by trypsin (0.5% w/v in PBS containing 1 mM EDTA), incubated for 5 minutes at ambient temperature in PBS containing 0.5% (w/v) trypan blue, washed twice (2% FCS in PBS), isolated by centrifugation (12,000 x g, 5 min) and resuspended in 200 μl 2% FCS in PBS for counting in a haemocytometer chamber.

CP92 shows inhibitory activity on proliferation of HASM cells over the concentration range (300 nM to 3 μM) (Figure 3). The inhibitory activity of CP92 was similar on pFb

stimulated with basic Fibroblast Growth Factor (bFGF) as the mitogen (Figure 3). In contrast, there was no detectable inhibition of the mitogenic response of the breast tumour cell line, MCF7 when stimulated to proliferate with 5% FCS (control proliferation 133 ± 1% unstimulated; CP92 1 μM, 128 ± 4%).

Methods

Human airway smooth muscle (HASM) cells were cultured from macroscopically normal bronchi (0.5-2 cm diameter) obtained from lung resection or heart-lung transplant specimens provided by the Alfred Hospital (Melbourne) according to methods published in detail previously (Fernandes et al., 1999).

Approximately 0.1 g of smooth muscle was stripped from the wall of the bronchus for each cell culture. Dissected tissue was immersed in Dulbecco's modified Eagle's medium (DMEM) (Flow Laboratories, Scotland), supplemented with 100 U/mL penicillin G (CSL, Australia) and 100 μg/mL streptomycin (CSL, Australia). The tissue was rinsed in phosphate buffered saline (PBS; Oxoid, England) and the airway smooth muscle was chopped into 2 mm ³ pieces and digested for 2 hours in DMEM containing elastase (0.5 mg/mL: Worthington Biochemical, USA) followed by a 12 hour incubation in DMEM containing collagenase (1 mg/mL) (Worthington Biochemical, USA), at 37°C with agitation.

The resulting cell suspension was centrifuged and washed three times in phosphate buffered saline (PBS). Following the last centrifugation step, the cells were resuspended in 25 mL of DMEM supplemented with L-glutamine (2 mM: Sigma, USA), penicillin G (100 U/mL), streptomycin (100 μg/mL), amphotericin B (2 μg/mL: Wellcome, UK) and heat- inactivated FCS (10% v/v: CSL, Australia) and seeded into 25 cm ² culture flasks. The primary isolates were incubated for 7 to 14 days to reach confluence.

Cells were harvested weekly by 10 min exposure to trypsin (0.5%: CSL, Australia) and EDTA (1 mM in PBS: BDH, Australia) and passaged at a 1 :3 ratio into 75 cm ² flasks.

Human lung parenchymal fibroblast cell culture

Human lung parenchymal fibroblast cell culture (pFb) were cultured from macroscopically normal parenchymal tissue of patients without fibrotic disease obtained from lung resection or heart-lung transplant specimens provided by the Alfred Hospital (Melbourne). Subpleural biopsy tissue measuring 5 x 5 x 5 mm was extensively rinsed in sterile PBS, chopped into 1 mm ³ fragments and adhered to the bottom of 1 well of a 6 well plastic culture dish in a minimum volume of medium (~ 200 ml), after approximately 4-6 h to allow tissue adherence the wells were flooded with 2 ml DMEM. Cultures were grown in DMEM (containing 10 % v v ^"1 FCS, 100 U ml ^"1 Penicillin-G, 100 μg ml ^'1 , 2 μg ml ^"1 amphotericin B, 1% v v ^"1 non-essential amino acids, and 1 % v v ^"1 Sodium Pyruvate) until monolayer confluence was reached.

In vitro fibroblast proliferation assay

Human pFb were grown to confluence and serum-deprived for 24 h to synchronise cells in Go/Gi-phase of the cell-cycle prior to stimulation with thrombin (0.3u/ml), or bFGF, (300 pM). A growth supplement containing factors essential for cell mitogenesis including insulin, transferrin, and selenium (1% v v ^"1 Monomed A, JRH Biosciences, USA) was added (in experiments using bFGF as the mitogen) and after 48 h cells were detached from the culture plate by the addition of trypsin, washed twice (2% FCS in PBS), isolated by centrifugation (12,000 x g, 5 min) and resuspended in 300 μl (2% FCS in PBS, containing 0.5% w v ^"1 trypan blue) before counting in a haemocytometer.

It will be understood that such assays could be readily utilized to determine the ability of disclosed compounds to inhibit Thrombin or bFGF-induced proliferation of human airway smooth muscle cells. There was no evidence of cytotoxicity at concentrations at or below 10 μM as evidenced by lack of cell detachment from the culture plate and lack of staining of cells with trypan blue (fewer than 5%). Without wishing to be bound to any particular proposed mode of action, these results suggest that the compounds of the application act via the modulation of intracellular signalling pathways.

5. Prostaglandin E ₂ Assay

The U937 monocytic cell line (available from the ATCC, USA) is commonly used to evaluate anti-inflammatory agents. In the present experiment, U937 cells maintained in culture in a medium comprising RPMI containing (5% FCS v/v; supplemented with 100U/ml penicillin, and 100 mg/ml streptomycin) were treated with phorbol myristate acetate (10 nM) then immediately dispersed into a 24 well cell culture plate at a concentration of 2 x 10 ^s cells per ml in a volume of ImI per well. After 24 hours by which time the cells had adhered to the plastic substrate, they were treated with one of several analogues of CP92 (CP-I -66, CP-1-68, CP-1-73) or with the vehicle of 0.1% DMSO. After a further 24 hour incubation period the supernatant was harvested and the content of prostaglandin E ₂ was measured by a radioimmunoassay, as described by Vlahos, R., & Stewart, A.G. (1999). ("Interleukin-lα and Tumour necrosis factor-α inhibit airway smooth muscle DNA synthesis by a glucocorticoid-sensitive induction of cyclo-oxygenase- 2. British Journal of Pharmacology, 126, 1315-1324). The CP92 analogues were found to decrease the concentration of Prostaglandin E ₂ in the supernatant, consistent with these compounds having an anti-inflammatory effect. (See Figure 4)

6. Comparison of a compound according to the present invention with a compound according to the prior art

A comparison of CP92 (a compound according to the present invention) with the compound CP2117, first disclosed in WO 2004/101595, shows a surprisingly different selectivity of action between the two compounds.

CP2117

The pIC ₅₀ values for the airway smooth muscle assay are CP92, 6.46; CP2117, 7.70 and for fibroblasts CP92, 6.81 ; CP2117, 6.93. In complete contrast, the in vivo threshold dose in mice for reduction in BAL cell values is 5 mg/kg for CP92 and 50 mg/kg for CP2117.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present

embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

REFERENCES

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2. "Handbook of Pharmaceutical salts" P.H.Stahl, C.G.Wermuth, 1 ^st edition, 2002, Wiley-VCH.

3. Physician Desk Reference (PDR).

4. Bozinovski, S., Jones, J.E., Vlahos, R., Hamilton, J.A. and Anderson, G.P. "Granulocyte/macrophage-colony-stimulating factor (GM-CSF) regulates lung innate immunity to lipopolysaccharide through Akt/Erk activation of NFkappa B and AP-I in vivo" J Biol Chem, (2002) 277, 42808-14.

5. Robert Harmon, Jamie D. Croxtall, Stephen J. Getting, Fiorentina Roviezzo, U Simon Yona, Mark J. Paul-Clark, Felicity N. E. Gavins, Mauro Perretti, John F. Morris, Julia C. Buckingham, and Roderick J. Flower. "Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse" The FASEB Journal express article (2002) 17(2):253-5..

6. Fernandes, D. J., Guida, E., Kalafatis, V., Harris, T., Wilson, J. & Stewart, A.G. (1999). Glucocorticoids inhibit proliferation, cyclin Dl expression and retinoblastoma protein phosphorylation, but not mitogen-activated protein kinase activity in human cultured airway smooth muscle. American Journal of Respiratory Cell Molecular Biology, 21 :77-88.

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8. Mohler, K.M, Sleath, P.R., Fitzner, JIN ₅ Cerretti, D.P., Alderson, M., Kerwar, S.S., Torrance, D. S., Otten-Evans, C, Greenstreet, T., Weerawarna, K., Kronheim, S. R., Petersen, M., Gerhart, M., Kozlosky, C.J., March, CJ. and Black, R.A., "Protection

against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing" Nature (1994) 370: 218 - 220.

9. Birrell, M.A., Patel, H.J., McCluskie, K. ₅ Wong, S., Leonard, T., Yacoub, M.H. and Belvisi, M.G. "PPAR-gamma agonists as therapy for diseases involving airway neutrophilia" Eur. Respir. J. (2004) 24(1): 18 - 23.

10. Vlahos, R., & Stewart, A. G. "Interleukin-lα and Tumour necrosis factor-α inhibit airway smooth muscle DNA synthesis by a glucocorticoid-sensitive induction of cyclo-oxygenase-2. British Journal of Pharmacology, (1999) 126, 1315-1324.