WO1997013750A1 - Azetidinones and their therapeutic use as cytokine inhibitors - Google Patents

Abstract

A compound of formula (I) wherein: R1 is C¿1-6? alkyl-R?5; R2¿ is hydrogen, C¿1-6? alkyl or C1-6 alkyl-R?5¿, wherein R?1 and R2¿ may be the same or different; R?3 is COR7¿ or S(O)¿0-2?R?8; R4 is COR9¿ or SO¿2R?10.

Description

AZETIDINONES AND THEIR THERAPEUTIC USE AS CYTOKINE INHIBITORS

Field of the Invention

This invention relates to novel /S-lactam compounds and their use in treating cytokine-mediated diseases. Background of the Invention

Interleukin-1 (IL-l) and tumour necrosis factor (TNF) are two of the most studied proinflammatory cytokines. These cytokines and others (e.g. IL-6 and IL-8) may play important roles in mediating many chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease and psoriasis.

IL-l has multiple effects on cells involved in inflammation and wound healing. Subcutaneous injection of IL-l leads to argination of neutrophils and maximal extravascular infiltration of the polymorphonuclear leukocytes (PMN) . In vitro studies reveal IL-l to be a

"chemotactic attractant for PMN, to activate PMN to metabolize glucose more rapidly to reduce nitroblue tetrazolium and to release their lysozomal enzymes. Endothelial cells are stimulated to proliferate by IL-l to produce thro boxane, to become more adhesive and to release procoagulant activity. IL-l also enhances collagen type IV production by epidermal cells, induces osteoblast proliferation and alkaline phosphatase production and stimulates osteoclasts to resorb bone. Even macrophages have been reported to be chemotactically attracted to IL-l to produce prostaglandins in response to IL-l and to exhibit a more prolonged and active tumoricidal state. IL-l is also a potent bone resorptive agent capable upon infusion into mice of causing hypercalcemia and increase in bone resorptive surface as revealed by histomorpho etry (Sabatini et al . , PNAS 85:5235-5239, (1988)) . TNF is a cytokine which is produced initially as a cell-associated 28kDa precursor. It is released as an active, 17kDa form (Jue et al . , Biochemistry 29:8371-8377 (1990)), which can mediate a large number of deleterious effects in vivo . When administered to animals or humans it causes inflammation, fever, cardiovascular effects, haemorrhage, coagulation and acute phase responses, similar to those seen during acute infections and shock states. Chronic administration can also cause cachexia and anorexia. Accumulation of excessive TNF can be lethal.

There is considerable evidence from animal model studies that blocking the effects of TNF with specific antibodies can be beneficial in acute infections, shock states, graft versus host reactions and autoimmune disease. TNF is also an autocrine growth factor for some myelomas and lymphomas and can act to inhibit normal haematopoiesis in patients with these tumours. Preventing the production or action of TNF is, therefore, predicted to be a potent therapeutic strategy for many inflammatory, infectious, immunological or malignant diseases. These include, but are not restricted to, septic shock, haemodynamic shock and sepsis syndrome (Mathison et al . (1988) J. Clin. Invest. 81:1925-1937; Miethke et al . (1992) J. Exp. Med. 175:91-98), post- ischaemic reperfusion injury, malaria (Grau et al . , (1989) Immunol. Rev. 112:49-70); mycobacterial infection (Barnes et al . (1992) Infect. Imm. 60:1441-6), meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, rheumatoid arthritis, multiple sclerosis, radiation damage, toxicity following administration of immunosuppressive monoclonal antibodies such as OKT3 or CAMPATH-1 and hyperoxic alveolar injury.

Accordingly, disease states in which the cytokine inhibitors may be useful as therapeutic agents include, but are not limited to, infectious diseases where active infection exists at any body site, such as meningitis and salpingitis; complications of infections including toxic shock, disseminated intravascular coagulation, and/or adult respiratory distress syndrome; acute or chronic inflammation due to antigen, antibody, and/or complement deposition; inflammatory conditions including arthritis, cholangitis, colitis, encephalitis, endocarditis, glomerulonephritis, hepatitis, myocarditis, pericarditis, reperfusion injury and vasculitis. Immune-based diseases which may be responsive to cytokine inhibitors inlude but are not limited to conditions involving T-cells and/or macrophages such as acute and delayed hypersensitivity, graft rejection, and graft-versus-host-disease; auto-immune diseases including Type 1 diabetes mellitus and multiple sclerosis. Cytokine inhibitors may also be useful in the treatment of bone and cartilage resorption as well as diseases resulting in excessive deposition of extracellular matrix. Such diseases include periodontal diseases, interstitial pulmonary fibrosis, cirrhosis, systemic sclerosis, and keloid formation. Cytokine inhibitors may also be useful in treatment of certain tumors which produce

^* IL-l as an autocrine growth factor and in preventing the cachexia associated with certain tumors. These and other relevant disease states or conditions associated with TNF and other relevant factors are described in PCT/GB96/01862, the content of which is incorporated herein by reference. Summary of the Invention The invention encompasses compounds of formula (I)

wherein:

R¹ is C,.₆alkyl-R⁵ R is hydrogen, C .₆ alkyl or C₁.₆ alkyl-R⁵, wherein R¹ and R² may be the same or different;

R³ is COR⁷ or S(O)₀.₂R⁸;

R* is COR⁹ or S0₂R¹⁰;

R⁵ is C0₂H, tetrazolyl, NHS0₂CF₃ or C0₂R⁶;

R is C,^ alkyl, C,.₆ alkylaryl or a group that may be removed in vivo (for example by enzymic attack) to provide a carboxylic acid (i.e. an ester prodrug) ; examples of such groups include C₂.₆ alkenyl, C ₆ alkoxy-C^ alkyl, C .₆ alkyl or C,_.6 alkylOCO_jC,^ alkyl;

R⁷ is OC,.₆ alkyl, OC^ alkyl-aryl, aryl, C,_₆ alkyl aryl, ^*C ₆ alkyl, HC^ alkyl, NHC,_₆ alkyl-aryl, NHaryl or U (C ₆ alkyl)₂;

R⁸ is aryl, C,_₆ alkyl, C^ alkyl-aryl, NHC,^ alkyl, NHC,.₆ alkyl-aryl, NHaryl or N(C₁.₆alkyl)₂;

7 8 wherein when R and R is C,_₆ alkyl or aryl, these groups may each be optionally substituted with OH, NH₂, C0₂H, COR⁹, S0₂R¹°, tetrazolyl, NHS0₂CF₃, NHCOR⁹, NHS0₂R¹⁰, NHCOC,.₆ alkyl or NHCOaryl;

R⁹ is C₁.₆ alkyl, aryl, C₁.₆ alkyl-aryl, CF₃, OC,^ alkyl, OC,_₆ alkyl-aryl, NHC^ alkyl, NHaryl, HC,^ alkyl-aryl or NfC,^ alkyl)₂; and

R¹⁰ is CF_j, C,.₆ alkyl-aryl, C,.₆ alkyl-aryl, NHC₁.₆ alkyl, NHaryl, N(C₁.₆ alkyl)₂ or NHC,.₆ alkyl-aryl; and salts, solvates and hydrates thereof. R 7, R 8, R 9 and R 10 may also each be N(C_1-6 alkyl)C,^ alkyl-aryl. It will be readily appreicated that combinations of substituents and/or variables are allowed only if they result in stable compounds. Description of the Invention

It will be appreciated that the compounds according to the invention can contain one or more asymmetrically substituted carbon atom. The presence of one or more of these asymmetric centres in a compound of formula (I) can give rise to stereoisomers, and in each case the invention is to be understood to extend to all such stereoisomers, including enantio ers and diastereomers, and mixtures including racemic mixtures thereof.

In the formulae herein, the - line is used at a potential asymmetric centre to represent the possibility of

R- and S- configurations, the < line and the line to represent a unique configuration at an asymmetric centre. As used in this specification, alone or in combination, the term "C,^ alkyl" refers to a straight or branched chain alkyl moiety having from one to six carbon atoms, including for example, methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl and the like.

The term "C₂.₆ alkenyl" refers to a straight or branched chain alkyl moiety having two to six carbon atoms and having in addition one double bond, of either E or Z stereochemistry where applicable. This term would include for example, vinyl, 1-propenyl, 1- and 2-butenyl, 2-methyl- 2-propenyl etc.

The term "aryl" means an optionally substituted phenyl or naphthyl group with the substituent(s) being selected, for example, from halogen, trifluoromethyl, C₁.₆alkyl, alkoxy, phenyl and the like. The term "halogen" means fluorine, chlorine, bromine or iodine.

The term "C .₆ alkoxy" refers to a straight or branched chain alkyl moiety having from one to six carbon atoms and one oxygen atom, including for example, methoxy, ethoxy , propoxy, ethoxymethyl, hexyloxy and the like. Salts of compounds of formula (I) include phar aceutically-acceptable salts, for example acid addition salts derived from inorganic or organic acids, such as hydrochlorides, hydrobromides, hydroiodides, p- toluenesulphonates, phosphates, sulphates, perchlorates, acetates, trifluoroacetates, propionates, citrates, malonates, succinates, lactates, oxalates, tartrates and benzoates.

Salts may also be formed with bases. Such salts include salts derived from inorganic or organic bases, for example alkali metal salts such as magnesium or calcium salts, and organic amine salts such as morpholine, piperidine, dimethylamine or diethylamine salts.

Particularly preferred compounds according to the invention include: trans (±)-l-Acetyl-4-benzoyl-3-[2-(ethoxycarbonyl)ethyl]-2- azetidinone

'cis (±) -l-Acetyl-4-benzoyl-3-[2-(ethoxycarbonyl)ethyl]-2- azetidinone cis (±)-l-Acetyl-4-benzoyl-3-[ (ethoxycarbonyl)methyl]-2- azetidinone cis (±)-l-Acetyl-4-benzoyl-3-[ (benzyloxycarbonyl)methyl]-2- azetidinone trans (±)-l-Acetyl-4-benzoyl-3-[ (ethoxycarbonyl) ethyl]-2- azetidinone cis (±) -l-Acetyl-4-benzoyl-3-[2-(benzyloxycarbonyl)ethyl]- 2-azetidinone cis (±)-l-Acetyl-4-benzoyl-3-[3-(ethoxycarbonyl)propyl]-2- azetidinone and others exemplified herein.

Compounds of the general formula (I) may be prepared by any suitable method known in the art and/or by the following processes. It will be appreciated that where a particular stereoisomer of formula (I) is required, the synthetic processes described herein may be used with the appropriate homochiral starting material and/or isomers may be resolved from mixtures using conventional separation techniques (e.g. hplc) .

In the description and formulae below, the groups R , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are as defined above, except where otherwise indicated. It will be appreciated that functional groups, such as amino, hydroxyl or carboxyl groups, present in the various compounds described below, and which it is desired to retain, may need to be in protected form before any reaction is initiated. In such instances, removal of the protecting group may be the final step in a particular reaction. Suitable protecting groups for such functionality will be apparent to those skilled in the art. For specific details see "Protective Groups in Organic Synthesis", Wiley Interscience, T W Greene, PGM Wutts.

A compound of general formula (I) may be prepared by acylation of a compound of general formula (II)

with an active derivative of an acid of formula (III) , i.e.

9 9 3 . . .

R COOH or R SO H. Active derivatives of acids of formula (III) include for example acid anhydrides or acid halides, such as acid chlorides. The coupling reaction may be performed using standard conditions for acylation reactions of this type. Thus, for example the reaction may be achieved in a solvent, for example an inert organic solvent such as an ether, e.g. a cyclic ether such as tetrahydrofuran, an amide e.g. a substituted amide such as dimethylformamide, or a halogenated hydrocarbon such as dichloromethane at a low temperature e.g. -20 C to ambient temperature, such as 0°C, in the presence of a base, e.g. an organic base such as an amine, e.g. triethylamine or 4-dimethylaminopyridine.

Acid derivatives of general formula (III) may be commercially available, or readily prepared by methods known by those skilled in the art.

It will be appreciated by those skilled in the art that the above transformations can only be carried out when suitable protecting groups are incorporated in the groups R¹, R² and R³. It will be appreciated that where a particular stereoisomer of formula (I) is required, this may be obtained by conventional resolution techniques such as high performance liquid chromatography. Where desired, however, appropriate homochiral starting materials may be used in the coupling reaction to yield a particular stereoisomer of formula (I) . This is exemplified below.

Intermediates of general formula (II) may be prepared ^*by deprotection of intermediates of general formula (IV)

The deprotection reaction may be performed using standard conditions for reactions of this type. Thus, for example, the reaction may be achieved in a solvent, for example an inert organic solvent such as an ether, e.g. a cyclic ether such as tetrahydrofuran, or any alkyl nitrile, such as acetonitrile, optionally in the presence of a cosolvent such as water, in the presence of eerie ammonium nitrate at a low temperature such as -20°C to ambient temperature, preferably 0°C. Intermediates of general formula (IV) may be prepared by [2+2] cycloaddition reactions of imines of general structure (V)

and acid chlorides of general structure (VI)

Cycloaddition reactions of this type may be performed in a solvent, for example an inert organic solvent such as a halogenated hydrocarbon for example dichloromethane, at a temperature ranging from ambient temperature to the boiling point of the solvent, preferably the boiling point of the solvent, in the presence of a base, e.g. an organic base such as an amine, e.g. triethylamine or a cyclic amine such as N-methylmorpholine. Imines of general formula (V) may be either commercially available or readily prepared from commercially available starting materials using the procedure of W. Hagmann et al . , J. Med. Chem. (1993) 36, 771. Acid chlorides of general formula (VI) may be commercially available or readily prepared by methods known to those skilled in the art, from commercially available starting materials.

It will be appreciated that where appropriate chirality can be incorporated into molecules of general formula (I) by the use of appropriately resolved commercially available starting materials, or by standard methods known to those in the art (e.g. resolution of acids or amines via diastereomeric salt formation; chiral induction using chiral catalysts etc) . As a further extension to the present invention, intermediates of general formula (IV) with cis stereochemistry may be converted into the same intermediates of general formula (IV) with trans stereochemistry. Isomerisation reactions of this type may be performed in a solvent, such as an inert organic solvent, for example a hydrocarbon such as toluene, at a temperature ranging from ambient temperature to the boiling point of the solvent, preferably 50°C, in the presence of a base, such as an organic base, for example 1,5-diazabicyclo[4.3.0]non- 5-ene.

Compounds of formula (I) may also be prepared by interconversion of other compounds of formula (I) . Thus, for example, a compound of formula (I) wherein R contains a C0₂H group may be prepared by hydrogenation (using palladium on carbon in a suitable solvent, such as an alcohol, e.g. ethanol) of a compound of formula (I) wherein R contains a CO_∑R group in which R is a C,^ alkylaryl group (e.g. benzyl) . Intermediates of formula (IV) in which R is an ester or an amide may also be prepared from corresponding acids (R = COOH) using standard conditions known to those skilled in the art. These acids may be prepared from corresponding aldehydes (R == CHO) using any appropriate oxidation procedure known to those skilled in the art. The aldehydes may be prepared by [2+2] cycloaddition reactions of the known bisimine [p-CH₃OPh)-N=CH-]₂ and acid chlorides of formula (V) followed by acidic hydrolysis of the intermediate imine. Cycloadditions of this type may be performed as previously described for reactions of acid chlorides of formula (VI) with imines of formula (V) . Any mixtures of final products or intermediates obtained can be separated on the basis of the physico¬ chemical differences of the constituents, in known manner, into the pure final products or intermediates, for example by chromatography, distillation, fractional crystallization, or by formation of a salt if appropriate or possible under the circumstances.

The compounds of formula (I) or a pharmaceutically- acceptable salt thereof can be used in the manufacture of a medicament for the prophylactic or therapeutic treatment of any disease state in a human, or other mammal, which is exacerbated or caused by excessive or unregulated cytokine production by such mammal's cell, such as but not limited ^* to monocytes and/or macrophages.

Compounds of formula (I) are capable of inhibiting proinfla matory cytokines, such as IL-l, IL-6, IL-8 and TNF and are therefore of use in therapy. IL-l, IL-8 and TNF affect a wide variety of cells and tissues and these cytokines, as well as other leukocyte-derived cytokines, are important and critical inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these pro-inflammatory cytokines is of benefit in controlling, reducing and alleviating many of these disease states. Accordingly, the present invention provides a method of treating a cytokine-mediated disease whch comprises administering an effective cytokine-interferring amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In particular, compounds of formula (I) or pharmaceutically-acceptable salts thereof are of use in the prophylaxis or therapy of any disease state in a human, or other mammal, which is exacerbated by or caused by excessive or unregulated IL-l, IL-8 or TNF producton by such mammal's cell, such as, but not limited to, monocytes and/or macrophages. Accordingly, in another aspect, this invention relates to a method of inhibition of the production of IL-l in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of formula

(I) or a pharmaceutically acceptable salt thereof. There are many disease states in which excessive or unregulated IL-l production is implicated in exacerbating and/or causing the disease. These include rheumatoid arthritis, osteoarthritis, endotoxemia and/or toxic shock syndrome, other acute or chronic inflammatory disease states such as the inflammatory reaction induced by endotoxin or inflammatory bowel disease, tuberculosis, atherosclerosis, muscle degeneration, multiple sclerosis,

'cachexia, bone resorption, psoriatic arthritis, Reiter's syndrome, gout, traumatic arthritis, rubella arthritis and acute synovitis. Recent evidence also links IL-l activity to diabetes, pancreatic 0-cells and Alzheimer's disease.

In a further aspect, this invention relates to a method of inhibiting the production of TNF in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Excessive or unregulated TNF production has been implicated in mediating or exacerbating a number of diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, gram-negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoisosis, bone resorption diseases, such as osteoporosis, reperfusion injury, graft versus host reaction, allograft rejections, fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS) , AIDS, ARC (AIDS-related complex), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis and pyresis.

Compounds of formula (I) are also useful in the treatment of viral infections, where such viruses are sensitive to upregulation by TNF or will elicit TNF production in vivo . The viruses contemplated for treatment herein are those that produce TNF as a result of infection, or those which are sensitive to inhibition, such as by decreased replication, directly or indirecly, by the TNF inhibiting-compounds of formula (I) . Such viruses include, but are not limited to HIV-1, HIV-2 and HIV-3, Cytomegalovirus (CMV) , Influenza adenovirus and the Herpes group of viruses, such as but not limited to, Herpes Zoster and Herpes Simplex. Accordingly, in a further aspect, this

^* invention relates to a method of treating a mammal afflicted with a human immunodeficiency virus (HIV) which comprises administering to such mammal an effective TNF inhibiting amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Compounds of formula (I) may also be used in association with the veterinary treatment of mammals, other than in humans, in need of inhibition of TNF production. TNF-mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted above, but in particular viral infections. Examples of such viruses include, but are not limited to, the lentivirus infections such as equine infectious anaemia virus, caprine arthritis virus, visna virus, or the aedi virus, or the retroviruses, such as feline immunodeficiency virus (FIV) , bovine immunodeficiency virus, or canine immunodeficiency virus. The compounds of formula (I) may also be used topically in the treatment of prophylaxis of topical disease states mediated by or exacerbated by excessive cytokine production, such as by IL-l or TNF respectively, such as inflamed joints, eczema, psoriasis and other inflammatory skin conditions such as sunburn; inflammatory eye conditions including conjuctivitis; pyresis, pain and other conditions associated with inflammation.

Compounds of formula (I) have also been shown to inhibit the production of IL-8. Accordingly, in a further aspect, this invention relates to a method of inhibiting the production of IL-8 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of formula (I) or a pharmaceutically- acceptable salt thereof.

There are many disease states in which excessive or unregulated IL-8 production is implicated in exacerbating and/or causing the disease. These diseases are characterized by massive neutrophil infiltration such as, psoriasis, inflammatory bowel disease, asthma, cardiac and renal reperfusion injury, adult respiratory distress syndrome, thrombosis and glo erulonephritis. All of these diseases are associated with increased IL-8 production which is responsible for the chemotaxis of neutrophils into the inflammatory site. In contrast to other inflammatory ctyokines (IL-l, TNF, and IL-6) , IL-8 has the unique property of promoting neutrophil chemotaxis and activation. Therefore, the inhibition of IL-8 production would lead to a direct reduction in the neutophil infiltration.

The compounds of formula (I) are administered in an amount sufficient to inhibit a cytokine, in particular IL- 1, IL-8 or TNF, such that its production is regulated down to normal levels, or in some case to subnormal levels, so as to ameliorate or prevent the disease stnte. Abnormal levels of IL-l, IL-8 or TNF, for instance in the context of the present invention, constitute: (i) levels of free (not cell bound) IL-l, IL-8 or TNF greater than or equal to 1 picogram per ml; (ii) any cell associated IL-l, IL-8 or TNF; or (iii) the presence of IL-l, IL-8 or TNF mRNA above basal levels in cells or tissues in which IL-l, IL-8 or TNF, respectively, is produced.

The discovery that the compounds of formula (I) are inhibitors of cytokines, specifically IL-l, IL-8 and TNF is based upon the effects of the compounds of formula (I) on the production of the IL-l, IL-8 and TNF in in vitro assays, e.g. as in Example A.

As used herein, the term "inhibiting the production of

IL-l (IL-8 or TNF)" refers to: a) a decrease of excessive in vivo levels of the cytokine (IL-l, IL-8 or TNF) in a human to normal or sub-normal levels by inhibition of the in vivo release of the cytokine by all cells, including but not limited to monocytes or macrophages; b) a down regulation, at the genomic level, of excessive in vivo levels of the cytokine (IL-l,

IL-8 or TNF) in a human to normal to sub-normal levels; c) a down regulation, by inhibition of the direct synthesis of the cytokine (IL-l, IL-8 or TNF) as a post translational event; or d) a down regulation, at the translational level, of excessive in vivo levels of the cytokine (IL- 1, IL-8 or TNF) in a human to normal or sub¬ normal levels. As used herein, the term "TNF-mediated disease or disease state" refers to any and all disease states in which TNF plays a role, either by production of TNF itself, or by TNF causing another monokine to be released, such as but not limited to IL-l, IL-6 or IL-8. A disease state in which, for instance, IL-l is a major component, and whose production or action, is exacerbated or secreted in response to TNF, would therefore be considered a disease state mediated by TNF.

As used herein, the term "cytokine" refers to any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions between cells in the immune, inflammatory or hematopoietic response. A cytokine includes, but is not limited to, onokines and lymphokines, regardless of which cells produce them. For instance, a onokine is generally referred to as being produced and secreted by a mononuclear cell, such as a macrophage and/or monocyte. Many other cells however also produce monokines, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epideral keratinocytes and B-lymphocytes. Lymphokines are generally referred to as being produced by lymphocyte cells. Examples of cytokines include, but are not limited to, Interleukin-1 (IL-l), Interleukin-6 (IL-6) , Interleukin-8 (IL-8) , Tumor Necrosis Factor-alpha (TNF-α) and Tumor Necrosis Factor-_/3 (TNF-/?) .

As used herein, the term "cytokine-interfering" -"cytokine-suppresive amount" refers to an effective compound of formula (I) which will cause a decrease in the in vivo levels of the cytokine to normal or sub-normal levels, when given to a patient for the prophylaxis or treatment of a disease state which is exacerbated by, or caused by, excessive or unregulated cytokine production. As used herein, the cytokine referred to in the phrase "inhibition of a cytokine, for use in the treatment of a HIV-infected human" is a cytokine which is implicated in (a) the initiation and/or maintenance of T cell activation and/or (b) any cytokine-mediated disease associated problem such as cachexia or muscle degeneration. As TNF-/3 (also known as lymphotoxin) has close structural homology with TNF-α (also known as cachectin) and since each induces similar biological responses and binds to the same cellular receptor, both TNF-α and TNF-/3 are inhibited by the compounds of the present invention and thus are herein referred to collectively as "TNF" unless specifically indicated otherwise. In order to use a compound of formula (I) or a pharmaceutically-acceptable salt thereof in therapy, it will normally be formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice. This invention, therefore, also relates to a pharmaceutical composition comprising an effective, non- toxic amount of a compound of formula (I) and a pharmaceutically-acceptable carrier or diluent.

The pharmaceutical compostition containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group ^■consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the US Patents 4,256,108;4,166,452; and 4,265,874, to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally- occurring phosphatide, for example lecithin, or - condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain a l i pha t i c a l c oho l s , f or examp l e heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified, for example sweetining, flavouing and colouring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occuring gums, for example gum acacia or gum

- tragacanth, naturally-occurring phosphatides, for example soya bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, for example gycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents. 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 in 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 compounds of formula (I) may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions, etc containing the compounds of Formula (I) are employed. (For the purposes of this application, topical application shall include mouth washes and gargles. )

Dosage levels of the order of from about 0.05 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above indicated conditions (about 2.5 mg to about 7 gms per patient per day) . For example, inflammation may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day (about 0.5 mg to about

3.5 gms per patient per day). The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may vary from about 5 to about 95% of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. The following non-limiting Examples are intended to illustrate the preparation of compounds of Formula (I) , and as such are not intended to limit the invention as set forth in the claims appended, thereto.

In the Examples, the following abbreviations are used: RT Room temperature

DMAP 4-Dimethylaminopyridine

DBN 1,5-Diazabicyclo[4.3.0]non-5-ene

Intermediate 1 ( ± ) - c i s - 4 - B e n z o y l - 3 - [ ( ethoxycarbonyl ) methyl ] -l- ( 4- methoxyphenyl)-2-azetidinone

A solution of N-(4-methoxyphenyl)iminoacetophenone (1.2 g) in dichloromethane was treated with triethylamine (0.84 ml) at RT. 3-Ethoxycarbonylpropanoyl chloride (823 mg) in 1,2-dichloroethane (2 ml) was added and the resultant mixture was stirred at RT for 16 h, and at reflux for 7 h. 2M Hydrochloric acid (10 ml) was added, the organic phase was removed and washed with saturated sodium bicarbonate (10 ml) and brine (10 ml), dried (Na₂S0₄) and concentrated in vacuo to give a dark brown oil. Purification by flash chromatography [eluting with hexane:ethyl acetate (2:1)] provided the title compound as a pale yellow oil (135 mg) .

TLC R_f = 0.2 (1% MeOH - CH₂C1₂) Similarly prepared were:

Intermediate 2 ( ± ) - c i s - 4 - B e n z o y l - 3 - [ 2 -

(ethoxycarbonyl)ethyl]-l-( -methoxyphenyl)- 2-azetidinone From N-(4-methoxyphenyl)i inoacetophenone (1.2 g) and ethyl glutaryl chloride (0.94 ml) as an off-white solid (215 mg) .

TLC R_f = 0.25 (1%-MeOH - CH₂C1₂) .

Intermediate 3 ( ± ) - σ i s - 4 - B e n z o y l - 3 -

[ (benzyloxycarbonyl)methyl]-l- (4- methoxyphenyl) -2-azetidinone

From N-( 4 -methoxyphenyl) iminoacetophenone (1.0 g) and benzylsuccinyl chloride (0.95 g) as a white solid (1.8 g) .

TLC R_f = 0.35 (1% EtOAc - CH₂C1₂) .

Intermediate 4 ( ± ) - c i s - 4 - B e n z o y l - 3 - [ 2 - <benzyloxycarbonyl) ethyl] -l- {4- methoxyphenyl)-2-azetidinone

From N-(4-methoxyphenyl)iminoacetophenone (1.0 g) and benzyl glutaryl chloride (1.0 g) as a yellow solid (0.9 g) .

TLC R_f = 0.12 (1% EtOAc - CH₂C1₂) .

Intermediate 5 (±) -cis-4-Benzoyl-3-[3-(ethoxycarbonyl) propyl]-l-( -methoxyphenyl)-2-azetidinone From N-(4-methoxyphenyl)iminoacetophenone (3.1 g) and 5- (ethoxycarbonyl)pentanoyl chloride (2.9 g) as a yellow solid (2.2 g) .

TLC R_f = 0.30 (5% EtOAc - CH₂C1₂) .

Intermediate 6 ( ± ) - σ i s - 4 - B e n z o y l - 3 -

[(ethoxycarbonyl) ethyl]-2-azetidinone A solution of Intermediate 1 (135 mg) in acetonitile (20 ml) was cooled to -20 C. A solution of eerie ammonium nitrate (406 mg) in water (10 ml) was added dropwise and the resulting mixture stirred at -20 C for 30 minutes, and at 0°C for 30 minutes. Saturated sodium bicarbonate solution (30 ml) was added and the mixture was extracted with ethyl acetate (3 x 100 ml) . The combined organic fractions were washed with brine (50 ml) , dried (MgS0₄) and concentrated in vacuo to yield a yellow oil. Purification by flash chromatography (eluting with 2% ethanol- dichloro ethane) gave the title compound as a buff-coloured powder (50 mg) .

TLC R_f = 0.2 [hexane - EtOAc (1:1)]

Similarly prepared were:

Intermediate 7 ( ± ) - c i s - 4 - B e n z o y l - 3 - [ 2 -

(ethoxycarbonyl)ethyl]-2-azetidinone From Intermediate 2 (1.91 g) as a pale yellow solid (369 mg) .

TLC R_f = 0.2 [hexane:EtOAc (1:1)].

intermediate 8 ( ± ) - c i s - 4 - B e n z o y l - 3 -

[ (benzyloxycarbonyl)methyl]-2-azetidinone From Intermediate 3 (730 mg) as a pale yellow gum (244 mg) .

TLC R_f = 0.4 [EtOAc-.hexane (3:1)].

Intermediate 9 ( ± ) - c i s - 4 - B e n z o y l - 3 - [ 2 -

(benzyloxycarbonyl)ethyl]-2-azetidinone

From Intermediate 4 (350 mg) as an off-white solid (150 mg) .

TLC R_f = 0.18 (5% EtOAc - CH₂C1₂) . Intermediate 10 ( ± ) - ci s - 4 - B e n z oy l - 3 - [ 3 -

(ethoxycarbonyl) ropyl]-2-azetidinone From Intermediate 5 (700 mg) as a white solid (410 mg) .

TLC R_f = 0.1 (5% EtOAc - CH₂C1-) .

intermediate ll (±) -cis-4-Benzoyl-3-carboxymethyl-l-

(4-methoxyphenyl) -2-azetidinone A solution of Intermediate 3 (350 mg) in tetrahydrofuran (5 ml) was hydrogenated at RT using hydrogen in the presence of 5% palladium-on-carbon (20 mg) . The reaction mixture was stirred for 16h, filtered through Celite and the filtrate concentrated in vacuo to give a white solid (190 mg) .

TLC R_f = 0.05 (5% EtOAc - CH₂C1₂ - 3 drops AcOH)

Intermediate 12 (± ) -trans-4-Bβnzoyl-3- [2-

( ethoxycarbonyl) ethyl] - l- ( 4 - methoxyphenyl)-2-azetidinone

A mixture of Intermediate 2 (1.01 g) , DBN (10 ml) and toluene (200 ml) was heated at reflux for 6h, and then cooled to RT. The reaction mixture was washed with 2N hydrochloric acid (2 x 30 ml) and brine (50 ml) , dried (MgS0₄) and concentrated in vacuo to give the product as a yellow oil (1 g) .

TLC R_f = 0.23 (1% MeOH - CH₂C1₂)

Similarly prepared were:

Intermediate 13 ( ± ) - trans - 4 - B en z oy l - 3

[ (ethoxycarbonyl) ethyl] -l- ( methoxyphenyl)-2-azetidinone

From Intermediate 1 (99 mg) as a pale yellow oil (14 mg) TLC R_f = 0.20 [hexane:EtOAc (2:1)].

intermediate 14 ( ± ) - trans- 4 - B e n z oy l - 3 -

[ (benzyloxycarbonyl)methyl]-l-(4- methoxyphenyl)-2-azetidinone

From Intermediate 3 (500 mg) as a yellow gum (59 mg) .

TLC R_f = 0.51 [EtOAc:hexane (1:1)].

intermediate 15 ( ± ) - t ra n s - 4 - B e n z o y 1 - 3 - [ 3 -

(ethoxycarbonyl) ropyl] -1- { - methoxyphenyl)-2-azetidinone

From Intermediate 5 (1.5 g) as yellow gum (1.4 g) .

TLC R_f = 0.27 (5% EtOAc - CH₂C1₂) .

Intermediate 16 ( ± ) - trans- 4 - B e n z oy l - 3 -

[ ( ethoxycarbony 1 ) ethy 1 ] - 2 - azetidinone. Using the method described for the preparation of Intermediate 6, the title compound was prepared from Intermediate 13 (37 mg) as a white solid (65 mg) .

Mp 84-85°C.

intermediate 17 ( ± ) - tra n s - 4 - B e n z oy 1 - 3 - [ 2 -

(ethoxycarbonyl)ethyl]-2-azetidinone Using the method described for the preparation of Intermediate 6 the title compound was prepared from intermediate 12 (1.0 g) as a pale yellow oil (333 mg) .

TLC R_f = 0.21 [hexane:EtOAc (1:1)].

intermediate 18 ( ± ) - c i s - 4 - C a rb om e t h o xy - 3 - [ ( ethoxycarbonyl) methyl] -l- ( - methoxyphenyl)-2-azetidinone

Prepared from methyl [ (4-methoxyphenyl) imino]acetate (966 mg) and ethylsuccinyl chloride (0.88 ml) using the procedure described for Intermediate 1, as a pale yellow oil (531 mg) .

TLC R_f = 0.2 [hexane:EtOAc (2:1)].

Similarly prepared were:

Intermediate 19 (±)-cis-3-[ (Benzyloxycarbonyl)- methyl]-4-carbomethoxy-l-(4- methoxyphenyl)-2-azetidinone

From methyl [ (4-methoxyphenyl) imino]acetate (0.9 g) and benzylsuccinyl chloride (1.07 g) as a white solid (400 mg) .

TLC R_f = 0.32 (1% EtOAc - CH₂Cl₂) .

Intermediate 20 (+)-cis-4-carbomethoxy-3-[2-

(ethoxycarbonyl)ethyl]-1-(4- methoxyphenyl)-2-azetidinone From methyl [ (4-methoxyphenyl) imino] acetate (8.8 g) and benzylglutaryl chloride (5 ml) as a pale yellow oil (9.5 g).

TLC R_f = 0.30 [EtOAc:hexane (1:2)]

Intermediate 21 (±)-cis-3-[ (Ethoxycarbonyl)methyl]-4- formyl-i-(4-methoxyphenyl)-2- azetidinone

A solution of ethylsuccinyl chloride (11.1 ml) in dichloromethane (240 ml) was added dropwise over 5h to a solution of , N' -1 , 2-ethanediylidenebis [ 4- methoxyphenyl]benzenamine (16.8 g) and triethylamine (13.23 ml) in dichloromethane (240 ml) at reflux under nitrogen. The mixture was stirred at reflux for a further 3h then cooled to RT. 5N HCl (100 ml) was added and the mixture was vigorously stirred at RT for 2h. The layers were separated and the organic phase was washed with 2N HCl (80 ml), water (80 ml) and brine (100 ml), dried (MgSO and concentrated in vacuo to give a dark brown oil. Purification by flash chromatography using 1% ethyl acetate in dichloromethane gave the title compound as a light brown solid (8.8 g) .

TLC R_f = 0.25 (1% EtOAc - CH₂C1₂) .

Intermediate 22 (±)-cis-4-Carbomethoxy-3- [ (ethoxycarbonyl) ethyl]-2- azetidinone

Prepared from Intermediate 18 (503 mg) using the procedure described for Intermediate 6, as a pale yellow oil (197 mg) .

TLC R_f = 0.27 [EtOAc:hexane (1:1)].

Similarly prepared were:

Intermediate 23 (±) -cis-4-Carbomethoxy-3- t (benzyloxycarbonyl)methyl]-2- azetidinone

From Intermediate 19 (0.51 g) as a colourless oil (197 mg) .

TLC R_f = 0.30 [EtOAc:hexane (1:1)].

Intermediate 24 (±)-cis-4-Carbomethoxy-3-[2-

(ethoxycarbonyl)ethyl]-2-azetidinone

From Intermediate 3 (2.2 g) as a yellow gum (0.3 g) .

TLC R_f = 0.15 (10% EtOAc - CH-.C1,)

Intermediate 25 (+)-cis-3-[ (Ethoxycarbonyl)methyl]-l-

⁽4-methoxyphenyl)-2-oxo-4-azetidine- carboxylic acid

A solution of sodium chlorite (503 mg) and sodium phosphate (470 mg) in water (5.5 ml) was added to a suspension of Intermediate 21 (162 mg) in 2-methyl-2-butene (0.30 ml) and tert-butanol (14 ml). The solution was stirred at RT for 4h and then concentrated in vacuo . The residue was partitioned between ethyl acetate (20 ml) and 2N HCl (20 ml) . The mixture was separated and the aqueous phase was extracted with ethyl acetate (2 x 20 ml) . The combined organic extracts were washed with brine (20 ml) , dried (MgS0₄) and concentrated in vacuo to give a yellow gum. The residue was azeotroped twice with dichloromethane to give the title compound as a buff coloured solid (178 mg) .

NMR (200MHz, CDC1₃) d 1.25 (3H, t) , 2.7 - 3.0 (2H, m) , 3.8 (3H, s), 4.2 (2H, q) , 4.8 (IH, d) , 6.9 (2H, d) , 7.3 (2H, d) , 8.5 (IH, br.s).

Intermediate 26 (±)-cis-4-(Benzylaminocarbonyl)-3-

[(ethoxycarbonyl)methyl]-1-(4- methoxyphenyl)-2-azetidinone A solution of Intermediate 25 (340 mg) , EEDQ (301 mg) and benzylamine (0.15 ml) in acetonitrile (50 ml) was heated at reflux for 6h. The cooled reaction mixture was washed with 2N HCl (2 x 25 ml) and brine (30 ml), dried (MgSO and concentrated in vacuo to give a brown solid. Purification by flash chromatography eluting with ethyl acetate:hexane (2:3) gave the title compound as a white solid (160 mg) .

TLC R_f = 0.21 [EtOAc - hexane (2:3)].

Similarly prepared were:

Intermediate 27 (±)-cis-4-(Benzyloxycarbonyl)-3-

[ (ethoxycarbonyl)methyl]-l-(4 methoxyphenyl)-2-azetidinone From Intermediate 25 (340 mg) and benzyl alcohol (0.14 ml) as a yellow oil (202 mg) .

TLC R_f = 0.30 [EtOAc - hexane (1:3)]. Intermediate 28 (±)-cis-4-(Benzylaminocarbonyl)-3- t (ethoxycarbonyl)methyl]-2- azetidinone

Prepared as a yellow oil (107 mg) from Intermediate 26 (155 mg) using the procedure described for Intermediate 22.

NMR (200MHz, CDC1₃) d.

Intermediate 29 (±)-cis-4-(Benzyloxycarbonyl)-3- [ (ethoxycarbonyl)methyl]-2- azetidinonβ

Prepared as a yellow oil (83 mg) from Intermediate 27 (202 mg) using the procedure described for Intermediate 22.

NMR (200MHz, CDC1₃) d 1.2 (3H, t ), 2.4 - 2.8 (2H, m) , 4.1 (2H, q), 4.4 (IH, d) , 5.1 - 5.3 (2H, dd) , 6.5 (IH, s) , 7.4 (5H, s).

Intermediate 30 ( ± ) -cis- 4 -( Car borne thoxy- 3 -[ 2 - (benzylo y carbonyl) ethyl] -l- (4- methoxyphenyl)-2-azetidinone

Prepared as a white solid (0.63 g) from methyl [ (4- methoxyphenyl)imino] acetate (0.81 g) and benzylsuccinyl chloride (1.0 g) using the procedure described for Intermediate 1.

TLC R_f = 0.1 (1% EtOAc - CH₂C1₂) .

Intermediate 31 (±) -cis-4-( Carbornethoxy-3 -[ 2-

(benzyloxycarbonyl) -e hyl] -2 - azetidinone

From Intermediate 30 (1.1 g) using the procedure for Intermediate 6, as a yellow oil (0.59 g) .

TLC R_f 0.22 (MeOH: EtOAc: CH₂C1₂ 1:10: 89) Similarly prepared were:

Intermediate 32 ( ± ) - trans- 4 -Benzoyl-3 - [ 3 -

(ethoxycarbonyl) ropyl]-2-azetidinone

From Intermediate 15 (1.35 g) as a white crystalline solid (0.6 g) .

Intermediate 33 ( ± ) - t rans - 4 - B e n z o y l - 3 - [benzyloxycarbony1 ) ethyl ] -2 - azetidinone

From Intermediate 14 (2.7 g) as an orange oil (0.65 g) .

Intermediate 34 ( ± ) - trans- 3 -[ 4 -B enz oy1 - 1 - ( 4 - methoxyphenyl) -2-oxoazetidin-3-yl]- propanoic acid

A solution of lithium hydroxide monohydrate (220 mg) in water (25 ml) was added to a solution of Intermediate 2 (798 mg) in THF (25 ml) at room temperature. The resultant dark orange solution was stirred for 3 hours and then acidified to pH 1 with 2M hydrochloric acid. The mixture was extracted with ethyl acetate (3 x 50 ml) and the combined organic extracts were washed with brine (50 ml) . Drying (MgS0₄) and concentration in vacuo provided the title compound (720 mg) as a yellow solid.

Intermediate 35 (±) -trans-3- [4-Benzoyl-l-[2-(t- butyloxy-carboxyl)ethyl]-i-(4- methoxyphenyl)-2-azetidinone

A solution of Intermediate 34 (204 mg) , isobutene (about 3 ml) and concentrated sulphuric acid (2 drops) in dichloromethane (10 ml) was sealed into a pressure tube at -78°C and then stored at room temperature for 3 days. The solution was diluted with dichloromethane (30 ml) and then washed with aqueous saturated sodium hydrogen carbonate solution (15 ml) and brine (20 ml) . Drying (MgSO and concentration in vacuo provided a yellow oil which was applied to a silica column, and eluted with 2% ethyl acetate in dichloromethane. The title compound was obtained as a yellow oil (134 mg) .

TLC R_f 0.35 (2% EtOAc - CH₂CH₂)

Intermediate 36 (±)-trans-4-Benzoyl-3-[2-(t-butyloxy- carbonyl)ethyl]-2-azetidinone

From Intermediate 35 (134 mg) as a yellow oil (99 mg) using the procedure described for Intermediate 6.

TLC R_f 0.1 (3% EtOAc - CH₂C1₂)

Intermediate 37 (±)-cis-4-[ (N-Benzyl-N-methylamino)- carbonyl]-3-[ (ethoxycarbonyl)methyl]-1- ( -methoxyphenyl)-2-azetidinone

Triethylamine (25 μl) was added a suspension of Intermediate 25 (151 mg) in dichlormethane (5 ml) at room temperature under nitrogen. The resultant solution was treated with BOP reagent (217 mg) and then, after stirring for 5 minutes, with N-benzyl methylamine (70 μl) . The resultant solution was stirred for 2h and then diluted with dichloromethane (30 ml) . The solution was washed with ₂M hydrochloric acid (3 x 8 ml), saturated aqueous sodium hydrogen carbonate solution (10 ml) and brine (₁5 ml₎. Drying (MgSO and concentration in vacuo provided a yellow oil which was applied to a silica column, and eluted with ethyl acetate-hexane (1:1). The title compound was obtained as a pale yellow oil (146 mg) .

TLC R_f 0.25 (EtOAc - hexane 1:1) Intermediate 38 (±) -cis-4-[ (N-Benzyl-N-methylamino)- carbonyl]-3-[ (ethoxycarbonyl)methyl]-2- azetidinone

From Intermediate 37 (146 mg) as a pale yellow oil (97 mg) using the procedure for Intermediate 6.

TLC R_f 0.1 (70% EtOAc - hexane)

Example 1 (±) -cis-i-Acetyl-4-benzoyl-3-

[ (ethoxycarbonyl) methyl] -2- azetidinone A mixture of Intermediate 16 (181 mg) , triethylamine (0.14 ml), DMAP (10 mg) and acetic anhydride (78 ml) in dichloromethane (10 ml) was stirred at RT for 3h. The reaction mixture was washed successively with 2N hydrochloric acid (3 x 20 ml) , saturated sodium "bicarbonate (25 ml) and brine (25 ml) , dried (MgSO and concentrated in vacuo to give an orange oil. Purification by flash chromatography (eluting with 1% methanol- dichloromethane) provided the title compound as a colourless oil (165 mg) .

TLC R_f = 0.2 (1% MeOH - CH₂C1₂) .

¹H NMR (200MHz, CDCl₃, Ref., TMS) d 1.0 (3H, t) , 2.5 (3H, s) , 2.7 (2H, d) , 3.65 - 4.0 (2H, m) , 4.18 - 4.25 (IH, m) , 5.7 (IH, d) , 7.5 - 7.7 (3H, m) , 8.0 (2H, m) .

Similarly prepared were:

Example 2 (±) -cis-1-Acety1-4-benzoy1-3-[2-

(ethoxycarbonyl)ethyl]-2-azetidinone

From Intermediate 7 (350 mg) as a colourless oil (310 mg) .

TLC R_f = 0.26 (1% methanol - CH₂C1₂) ¹H NMR (200MHz, CDCl₃, Ref., TMS) d 1.2 (3H, t) , 1.65 - 1.90 (2H, m) , 2.52 (2H, t) , 2.55 (3H, s) , 3.9 (IH, m) , 4.15 (2H, q) , 5.6 (IH, d) , 7.5 - 7.8 (3H, m) , 8.0 (2H, m) .

Example 3 ( ± ) - cis - l -Ac e ty 1 - 4 -ben z oy 1 - 3 -

[ ( benzy loxycarbonyl ) ethyl ] -2 - azetidinone From Intermediate 8 (240 mg) as a yellow solid (271 mg) .

TLC R_f = 0.62 [hexane:EtOAc (1:1)].

¹H NMR (200MHz, CDC1₃, Ref., TMS) d 2.5 (3H, s) , 2.6 (IH, dd) , 2.8 (IH, dd) 4.2 (IH, m) , 4.75 (2H, q) , 5.7 (IH, d) , 7.5 - 7.8 (3H, m) , 8.0 (2H, m) .

Example 4 (±) -trans-l-Acetyl-4-benzoyl-3-[2-

( ethoxycarbonyl) ethyl] -2-azetidinone From Intermediate 17 (824 mg) as a colourless oil (803 mg) .

TLC R_f = 0.16 [hexane: EtOAc (3:1)].

¹H NMR (200MHz, CDC1₃, Ref., TMS) d 1.2 (3H, t) , 2.2 - 2.4 (2H, ) , 2.5 (3H, s) , 2.55 - 2.60 (2H, ) , 3.25 (IH, ) , 4.10 (2H, q) , 5.2 (IH, d) , 7.5 - 7.75 (3H, m) , 8.0 (2H, m) .

Example 5 ( ± ) - trans- 1- Acetyl- 4 -benzoyl-3-

( (ethoxycarbonyl) ethyl] -2- azetidinone

From Intermediate 13 (50 mg) as a colourless oil (45 mg) .

TLC R_f = 0.20 [hexane:EtOAc (5:2)].

¹H NMR (200MHz, CDC1₃, Ref., TMS) d 1.15 (3H, t) , 2.45 (3H, s) , 2.9 (2H, m) , 3.5 (IH, m) , 4.1 (2H, q) , 5.35 (IH, d) , 7.4 - 7.7 (3H, m) , 8.0 (2H, m) . Example 6 (±) -cis-i- Acetyl -4 -benzoy 1-3- [₂-

(benzyloxycarbonyl) ethyl] -2 - azetidinone

From Intermediate 9 (240 mg) as a yellow oil (270 mg) .

TLC R_f = 0.26 [EtOAc:hexane (1:2)].

¹H NMR (200MHz, CDCl₃, Ref., TMS) d 1.8 (2H, m) , 2.5 (3H, s) , 2.55 (2H, m) , 3.9 (IH, ) , 5.1 (2H, s) , 5.5 (IH, d) , 7.4 - 7.7 (3H, m) , 8.0 (2H, m) .

Exam^ple 7 ( + ) -cis-l-Acety 1-4 -benzoy 1-3 - [3-

( ethoxycarbonyl) propyl ] - 2 - azetidinone From Intermediate 10 (335 mg) as a yellow oil (190 mg) .

TLC R_f = 0.15 (5% EtOAc - CH₂C1₂) .

¹H NMR (200MHZ, CDCl_j, Ref., TMS) d 1.1 (3H, t) , 1.5 - 1.9 (4H, m) , 2.2 (2H, m) , 2.5 (3H, s) , 3.7 (IH, m) 4.05 (2H, q) , 5.6 (IH, d) , 7.5 - 7.7 (3H, m) , 8.0 (2H, m) .

Example 8 (±) - cis- 1 -Ace ty 1- -benzoy 1-3- (2- carboxyethyl) -2-azetidinone

Using the procedure described for the preparation of Intermediate 11, the title compound was prepared from Example 6 (118 mg) as a white solid (87 mg) .

TLC R_f = 0.05 (1% EtOAc - CH₂Cl₂ + 3 drops AcOH) .

¹H NMR (200MHz, CDC1₃, Ref., TMS) d 1.8 (2H, ) , 2.5 (3H, s) , 2.55 (2H, ) 3.8 (IH, m) , 5.6 (IH, d) , 7.5- 7.7 (3H, m) , 8.0 (2H, m) . Example 9 (±) -cis-l-Acetyl-4-carbomethoxy-3-

[ (ethoxycarbonyl) methyl] - 2 - azetidinone

Using the procedure described for Example 1, the title compound was prepared from Intermediate 22 (184 mg) , as a colourless oil (184 mg) .

TLC R_f = 0.26 [EtOAc - hexane (1:2)].

NMR (200MHz, CDC1₃) d 1.2 (3H, t) , 2.4 (3H, s) , 2.6 - 2.9 (2H, ) , 3.8 (3H, s) , 4.0 (lH,m) , 4.2 (2H, q) , 4.7 (IH, d) .

Similarly prepared were:

Example 10 (±)-cis-l-Acetyl-4-carbomethoxy-3-

[ (benzyloxycarbonyl) ethyl]-2- azetidinone

From Intermediate 23 (200 mg) as a colourless oil (223 mg) .

TLC R_f = 0.30 [EtOAc - hexane (1:2)].

NMR (200MHz, CDC1₃) d 2.4 (3H, s) , 2.7 - 3.0 (2H, m) , 3.6 (3H, s) , 4.1 (IH, ) , 4.7 (IH, d) , 5.2 (2H, d) , 7.4 (5H, s) .

Example 11 (±) -cis-l-Acetyl-4-carbomethoxy-3-[2-

(ethoxycarbonyl)ethyl]-2-azetidinone

From Intermediate 24 (0.6 g) as a yellow gum (320 mg) .

TLC R_f = 0.16 [EtOAc:hexane (1:2)]

NMR (200MHz, CDCl₃) dl.3 (3H, t) , 1.95 (2H, m) , 2.4 (3H, s) , 2.5 (2H, t) , 3.65 (IH, m) , 3.8 (3H, s) , 4.2 (2H, q) , 4.6 (lH,d). Example 12 (±)-cis-l-Acetyl-4 (benzyloxycarbonyl)

-3-[ (ethoxycarbonyl)methyl]-2- azetidinone.

From Intermediate 29 (75 mg) as a colourless oil (71 mg) .

TLC R_f = 0.24 [EtOAc - hexane (1:2)].

NMR (200MHz, CDCl₃) d 1.2 (3H, t) , 2.4 (3H, s) , 2.5 - 2.9 (2H, m) , 3.9 - 4.1 (3H, m) , 4.7 (IH, d) , 5.2 (2H, d) , 7.4 (5H, s ) .

Example 13 (±)-cis-l-Acetyl-4-

(benzyla inocarbonyl) -3- [ (ethoxycarbonyl)methyl]-2- azetidinone

From Intermediate 28 (107 mg) as a white solid (78 mg) .

^*TLC R_f = 0.18 [EtOAc:hexane (2:3)].

NMR (200MHZ, CDC1₃) d 1.2 (3H, t) , 2.4 (3H, s) , 2.9 (2H, d) , 3.8 (2H, q) , 3.9 - 4.1 (IH, m) , 4.4 (2H, d) , 4.55 (IH, d) , 6.8 (IH, m) , 7.3 (5H, m) .

Example 14 ( ± ) - c i s - 1 - Ac e t y l - 3 - [ 2 - (benzyloxycarbonyl) ethyl]-4- methoxycarbony 1-2 -azetidinone

From Intermediate 31 (0.54 g) as a straw-coloured oil (0.6 g) .

Example 15 (±) - trans-1-Acety l-4-benzoyl-3-

[ (ethoxycarbonyl) ropyl] -2- azetidinone

From Intermediate 31 (0.38 g) as a colourless oil (0.3 g) . 97/13750

37

Example 16 ( ± ) - trans- 1 -Ace ty 1 - -benzoyl-3-

[ (benzy loxycarbonyl ) methyl ] -2- azetidinone

From Intermediate 33 (0.35 g) , as a pale yellow oil (0.17 g) .

TLC R 0.29 (1% EtOAc - CH₂Cl₂)

Exam^ple 17 (±) -trans-l-Acetyl-4-benzoyl-3-[2- (t- butyloxycarbonyl) ethyl]-2-azetidinone

From Intermediate 36 (99 mg) as a pale yellow oil (92 mg) .

TLC R_f 0.33 (30% EtOAc - hexane)

Example 18 (±)-cis-l-Acetyl-4-[N-benzyl-N-methyl)- carboxamide] -3- [ (ethoxycarbonyl) methyl ] -2-azetidone

From Intermediate 38 (97 mg) as a colourless oil (74 mg) .

TLC R_f 0.3 (50% EtOAc - hexane)

Exam^ple 19 (±) -cis- 3- [l-Acety 1-4 -me thoxy car bony 1-

2-oxoazetidin-3-yl]propanoic acid

From Example 14 (0.45 g) using the procedure described for Intermediate 11, as a pale straw-coloured gum (0.4 g) .

TLC R_f 0.08 (MeOH: EtOAc :CH₂C1₂, 1:10:89)

Similarly prepared were:

Exam^ple 20 (±)-cis-2-[l-Acetyl-4-methoxycarbonyl-

2-oxoazetidin-3-yl]ethanoic acid From Example 10 (0.43 g) as a white solid (0.3 g) .

TLC R_f 0.06 (MeOH:EtOAc:CH₂C1₂, 1:10:89)

Example 21 (±) -cis-2-[l-Acetyl-4-benzoyl-2- oxoazetidin-3-yl]ethanoic acid

From Example 3 (0.6 g) as a white crystalline solid (0.45 g) .

Exam^ple 22 (±)-trans-2-[1-Acety1-4-benzoy1-2-oxo- azetidone-3-yl]ethanoic acid

From Example 16 (0.13 g) as a white powder (20 mg) .

Example A

Measurement of Cytokine Release

The compounds of the invention inhibit the release of IL-13 as indicated in the following in vitro test using THP-1 cells. The effect on the release of other cytokines can also be measured in this assay.

800 ml of THP-1 cells (1 x 10° cells) in RPMI 1640 medium (containing 2mM glutamine and 20 mM 2- mercaptoethanol) are placed into 24 well culture plates. The compound to be tested is added in a volume of 200 ml. After 3 hours at 37°C in 5% C0₂/95% air, 10 ml of lipopolysaccharide (5 mg/ml) is added and the incubation continued for a further 18 hours. Appropriate controls (with and without stimulus, solvent) are also included. The media are harvested by centrifugation at 1000 g for 10 minutes and stored at -20°C until required. The viability of the cells are measured using the MTT assay as previously described (J.A. Plumb et al, Cancer Res., 49, 4435, (1983)) . The media are analysed for the presence of IL-13, TNFα, IL-6, and IL-8 using commercially available ELISA kits (R & D Systems) . Results are expressed as IC₅₀ values (concentration of compound which inhibits to 50% the release of cytokine being measured) .

Claims

1. A compound of formula (I):

wherein:

R¹ is C_1-6 alkyl-R⁵;

R² is hydrogen, C_1-6 alkyl or C_1-6 alkyl-R⁵, wherein R¹ and R² may be the same or different;

R³ is COR⁷ or S(O)_0-2R⁸;

R⁴ is COR⁹ or SO₂R¹⁰ ;

is CO₂H , tetrazolyl , NHSO₂CF₃ or CO₂R⁶;

R⁶ is C_1-6 alkyl, C_1-6 alkylaryl or a group that may be removed in vivo (for example by enzymic attack) to provide a carboxylic acid (i.e. an ester prodrug);

R⁷ is OC_1-6 alkyl, OC_1-6 alkyl-aryl, aryl, C_1-6 alkyl-aryl, C_1-6 alkyl, NHC_1-6 alkyl, NHC_1-6 alkyl-aryl, NHaryl or N(C_1-6 alkyl)₂;

R⁸ is aryl, C_1-6 alkyl, C_1-6 alkyl-aryl, NHC_1-6 alkyl, NHC_1-6 alkyl-aryl, NHaryl or N(C_1-6alkyl)₂; wherein when R⁷ and R⁸ is C_1-6 alkyl or aryl, these groups may each be optionally substituted with OH, NH₂, CO₂H, COR⁹, SO₂R⁹, tetrazolyl, NHSO₂CF₃, NHCOR⁹ , NHSO₂R¹⁰, NHCOC_1-6 alkyl or NHCOaryl;

R⁹ is C_1-6 alkyl, aryl, C_1-6 alkyl-aryl, CF₃, OC_1-6 alkyl, OC_1-6 alkyl-aryl, NHC_1-6 alkyl, NHaryl, NHC_1-6 alkyl-aryl or N(C_1-6 alkyl)₂; and

R¹⁰ is CF₃, C_1-6 alkyl-aryl, C_1-6 alkyl-aryl, NHC_1-6 alkyl, NHaryl, N(C_1-6 alkyl)₂ or NHC_1-6 alkyl-aryl; and salts, solvates and hydrates thereof.

2. A compound of claim 1, wherein R³ is S(O)_0-2R⁸.

3. A compound of claim 1, wherein R³ is COR⁷ and R⁷ is

OC_1-6 alkyl, OC_1-6 alkyl-aryl, NHC_1-6 alkyl, NHC_1-6 alkyl-aryl, NHaryl or N(C_1-6 alkyl)₂.

4. A compound of claim 1, wherein R³ is COR⁷ and R⁷ is aryl, C_1-6 alkyl-aryl or C_1-6 alkyl.

5. A compound of claim 1, selected from

cis-1-acetyl-4-benzoyl-3-[(ethoxycarbonyl)methyl]-2- azetidinone,

cis-1-acetyl-4-benzoyl-3-[2-(ethoxycarbonyl)ethyl]-2- azetidinone,

cis-1-acetyl-4-benzoyl-3-[(benzyloxycarbonyl)methyl]- 2-azetidinone,

trans-1-acetyl-4-benzoyl-3-[2-(ethoxycarbonyl)ethyl]- 2-azetidinone,

t rans-1-acetyl-4-benzoyl-3-[(ethoxycarbonyl)methyl]-2- azetidinone,

cis-1-acetyl-4-benzoyl-3-[2-(benzyloxycarbonyl)ethyl]- 2-azetidinone,

cis-1-acetyl-4-benzoyl-3-[3-(ethoxycarbonyl)propyl]-2- azetidinone,

cis-1-acetyl-4-benzoyl-3-(2-carboxyethyl)-2-azetidinone.

6. A compound of claim 1, selected from

cis-1-acetyl-4-carbomethoxy-3-[ (ethoxycarbonyl)-methyl]-2-azetidinone,

cis-1-acetyl-4-carbomethoxy-3-[(benzyloxycarbonyl)-methyl]-2-azetidinone,

cis-1-acetyl-4-carbomethoxy-3-[2-(ethoxycarbonyl)-ethyl]-2-azetidinone,

cis-1-acetyl-(benzyloxycarbonyl)-3-[(ethoxycarbonyl)-methyl]-2-azetidinone, and

cis-1-acetyl-4-(benzylaminocarbonyl)-3- [(ethoxycarbonyl)methyl]-2-azetidinone.

7. A compound of any preceding claim, in the form of a single enantiomer or diastereomer, or a mixture of such isomers.

8. A pharmaceutical composition for use in therapy, comprising a compound of any preceding claim and a pharmaceutically-acceptable diluent or carrier.

9. Use of a compound of any of claims 1 to 7, for the manufacture of a medicament for use in treatment or prevention of a condition mediated by TNF or IL-1.

10. The use of claim 9, wherein the condition is selected from inflammation, wounds, osteoblast proliferation, bone resorption and cancer.

11. The use of claim 9, wherein the condition is selected from inflammatory and autoimmune diseases.

12. The use of claim 9, wherein the condition is selected from joint inflammation, arthritis, rheumatoid arthritis, rheumatoid spondylitis and osteoarthritis, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, acute respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, asthma, bone resorption diseases, reperfusion injury, graft vs host reaction, allograft rejection, malaria, myalgias, HIV, AIDS, ARC, cachexia, Crohn's disease, ulcerative colitis, pyresis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes mellitus, psoriasis, Bechet's diseases disease, anaphy lactoid purpura nephritis, chronic glomerulonephritis, inflammatory bowel disease and leukaemia.

13. The use of claim 9, wherein the condition is asthma.

14. The use of claim 9, wherein the condition is acute respiratory distress syndrome, pulmonary inflammatory disease or pulmonary sarcoidosis.

15. The use of claim 9, wherein the condition is joint inflammation.

16. The use of claim 9, wherein the condition is tardive dyskinesia.

17. The use of claim 9, wherein the condition is a yeast or fungal infection.

18. The use of claim 9, for gastroprotection.