WO2009037413A1

WO2009037413A1 - Dimers of 5- [ (4-cyanophenyl) sulfinyl] -6-methyl-2-oxo-1- [3- (trifluoromethyl)phenyl] -1,2-dihydropyridine-3-carboxamide as inhibitors of human neutrophil elastase for treating respiratory diseases

Info

Publication number: WO2009037413A1
Application number: PCT/GB2007/003537
Authority: WO
Inventors: Harry Finch; Nicholas Charles Ray; Christine Edwards
Original assignee: Argenta Discovery Limited
Priority date: 2007-09-19
Filing date: 2007-09-19
Publication date: 2009-03-26

Abstract

Six specific compounds having human neutrophil elastase inhibitory activity are disclosed, one of which has the structural formula wherein A- is a pharmaceutically acceptable anion. The compounds are useful inter alia for the treatment of inflammatory respiratory disease, and may be administered by inhalation.

Description

DIMERS OF 5- [ (4-CYANOPHENYL) SULFINYL] -6-METHYL-Z-OXO-I- [3-

(TRIFLUOROMETHYL)PHENYL] -I ^-DIHYDROPYRIDINE-S-CARBOXAMIDE

AS INHIBITORS OF HUMAN NEUTROPHIL ELASTASE

FOR TREATING RESPIRATORY DISEASES

Field of the Invention

This invention relates to multimeric heterocyclic compounds which are inhibitors of human neutrophil elastase (HNE), and their use in therapy, for example the treatment of chronic obstructive pulmonary disease (COPD) and other conditions where HNE is implicated. Background to the invention

Human neutrophil elastase is a 32 kDa serine proteinase found in the azurophilic granules of neutrophils. It has a role in the degradation of a wide range of extracellular matrix proteins, including fibronectin, laminin, proteoglycans, Type III and Type IV collagens as well as elastin (Bieth, G. In Regulation of Matrix accumulation, Mecham, R. P. (Eds), Academic Press, NY, USA 1986, 217-306).

HNE has long been considered to play an important role in homeostasis through repair and disposal of damaged tissues via degradation of the tissue structural proteins. It is also relevant in the defence against bacterial invasion by means of degradation of the bacterial body. In addition to its effects on matrix tissues, HNE has been implicated in the upregulation of IL-8 gene expression and also induces IL-

8 release from the epithelial cells of the lung. In animal models of Chronic

Obstructive Pulmonary Disease induced by tobacco smoke exposure both small molecule inhibitors and protein inhibitors of HNE inhibit the inflammatory response and the development of emphysema (Wright, J. L. et al. Am. J. Respir. Crit. Care

Med. 2002, 166, 954-960; Churg, A. et al. Am. J. Respir. Crit Care Med. 2003, 168,

199-207). Thus, HNE may play a role both in matrix destruction and in amplifying inflammatory responses in chronic respiratory diseases where neutrophil influx is a characteristic feature. Indeed, HNE is believed to play a role in several pulmonary diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis

(CF), acute respiratory distress syndrome (ARDS), pulmonary emphysema, pneumonia, severe asthma, sarcoidosis, bronchiectasis and lung fibrosis. It is also implicated in several cardiovascular diseases in which tissue remodelling is involved, for example, in heart failure and the generation of ischaemic tissue Injury following acute myocardial infarction. Elevated HNE levels are also correlated with the severity of inflammation in inflammatory bowel disease (Silberer H et al, Clin

Lab. 2005;51(3-4):117-26) and may play a role in impaired mucosal repair in patients with ulcerative colitis. COPD is an umbrella term encompassing three different pathological conditions, all of which contribute to limitation of airflow: chronic bronchitis, emphysema and small-airway disease. Generally all three will exist to varying extents in patients presenting with COPD, and all three may be due to neutrophil- mediated inflammation, as supported by the increased number of neutrophils observed in bronchoalveolar leakage (BAL) fluids of COPD patients (Thompson, A. B.; Daughton, D.; et al. Am. Rev. Respir. DIs. 1989, 140, 1527-1537). The major pathogenic determinant in COPD has long been considered to be the protease-anti- protease balance (also known as the 'elastase:anti-elastase hypothesis'), in which an imbalance of HNE and endogenous antiproteases such as α1 -antitrypsin (O₁-AT), Secretory leukocyte protease inhibitor (SLPI) and pre-elafin leads to the various inflammatory disorders of COPD. Individuals that have a genetic deficiency of the protease inhibitor α1 -antitrypsin (α1-AT) develop emphysema that increases in severity over time (Laurrell, C. B.; Erikkson, S Scand. J. Clin. Invest. 1963 15, 132- 140). An excess of HNE is therefore destructive, leading to the breakdown of pulmonary morphology with loss of elasticity and destruction of alveolar attachments of airways in the lung (emphysema) whilst simultaneously increasing microvascular permeability and mucus hypersecretion (chronic bronchitis).

Multimeric ligands consist of multiple binding domains which are tethered together through a suitable scaffold. Hence individual binding domains are linked together into a single molecule, increasing the probability that the multimer will bind simultaneously with multiple active sites resulting in high-affinity interactions (Handl, H. L. et al. Expert OpIn. Ther. Targets 2004, 8, 565-586; Han, Y. F. ef al., Bioorg. Med. Chem. 1999, 7, 2569-2575). Also, multiple binding interactions with relatively high off-rates can combine to yield an overall low off-rate for the multimeric ligand. Thus, a molecule consisting of a suitable linker and ligands may be expected to show advantage over the monomeric ligands alone in terms of potency and/or duration of action. Multimeric compounds are unlikely to be orally bioavailable (as predicted by Lipinski's "Rule of 5") which may be advantageous where an inhaled route of administration to the lungs is targeted, since even after inhaled administration, a large proportion of drug is likely to enter the Gl tract. Thus such compounds may be expected to show reduced systemic exposure after inhalation administration and hence an improved toxicity profile over orally administered therapies. Compounds (herein described as "monomers") which are described as inhibitors of human neutrophil elastase are disclosed in WO2004/043924, WO2005/021509, WO2005/021512, WO2005/026123, WO2005/026124, WO 2006/098683 and WO 2006/098684. Summary of the invention

Our copending intenational Patent Application No. PCT/GB2007/000919 is concerned, inter alia, with compounds of formula (IA) or (IB), which are inhibitors of human neutrophil elastase (HNE), and useful, for example in the treatment of chronic obstructive pulmonary disease (COPD) and other conditions where HNE is implicated:

KER

wherein X represents -C= or -N=;

LINKER is a divalent linker radical;

R₁ is a group of formula Z-[AIk¹ ]_m-[X]_p-[Alk²]_n- wherein: m, n and p are independently 0 or 1;

Z is hydrogen or an optionally substituted monocyclic carbocyclic or heterocyclic group having 3 to 7 ring atoms; AIk¹ and AIk² are each independently an optionally substituted divalent C₁-C₆ alkylene, C₂-C₆ alkenylene or C₂-C₆ alkynylene radical, which may optionally be interrupted by -O-, -S-, -S(=O)-, -S(=O)₂- or -NR₅- wherein R₅ is hydrogen, C₁-C₃ alkyl, or cyclopropyl; and X is -O-, -S-, -S(=O)-, -S(=O)₂- or -NR₅- wherein R₅ is hydrogen, C₁-C₃ alkyl, or cyclopropyl;

R₂ represents hydrogen or C₁-C₆ alkyl; or in the case of formula (IA), R₁ and R₂ taken together with the carbon atoms to which they are attached form a 5-, 6- or 7-membered carbocyclic or heterocyclic ring fused to the ring containing X and N, said fused ring being optionally substituted by one or more optional substituents, or one or more optionally substituted C₁-C₃ alkyl, C₂-C₃ alkenyl, or C₂-C₃ alkynyl groups; or in the case of formula (IB), R₂ is linked with a carbon or nitrogen atom in the LINKER radical to form a 5-, 6- or 7-membered carbocyclic or heterocyclic ring fused to the ring containing X;

R₃ represents hydrogen, or 1 or 2 optional substituents, or 1 or 2 optionally substituted C₁-C₃ alkyl, C₂-C₃ alkenyl, or C₂-C₃ alkynyl;

R₄ represents a radical of formula -[AIk]₃-Q wherein a is 0 or 1 ; AIk represents an optionally substituted divalent C₁-C₄ alkylene radical, which may terminate in or be interrupted by -O-, -S-, -S(=O)-, -S(=O)₂- or -NR₅- wherein R₅ is hydrogen, C₁-C₃ alkyl, or cyclopropyl;

Q is hydrogen, optionally substituted monocyclic carbocyclic or heterocyclic having from 3 to 6 ring atoms; or R₄-NH- represents an optionally substituted monocyclic heterocyclic ring having 5 or 6 ring atoms and linked to the carbonyl via a ring nitrogen.

The present invention relates to HNE inhibitory compounds falling within the scope of Formula (IA) of PCT/GB2007/000919, but not specifically disclosed therein. Description of the invention According to the present invention, there is provided a compound selected from the group consisting of those of formula (1) to (5) in the following Table, or a pharmaceutically acceptable salt thereof, wherein A^' is a pharmaceutically acceptable anion.

In compounds (1), (4) and (5), A^" is a pharmaceutically acceptable anion, which may be selected, for example, from the group comprising chloride, bromide, sulfate, methanesulfonate, benzenesulfonate, toluenesulfonate (tosylate), napadisylate (naphthalene-1 ,5-disulfonate or naphthalene-1 -(sulfonic acid)-5- sulfonate), edisylate (ethane-1 ,2-disulfonate or ethane-1 -(sulfonic acid)-2-sulfonate), isethionate (2-hydroxyethylsulfonate), phosphate, acetate, citrate, lactate, tartrate, maleate, malate, fumarate, xinafoate, p-acetamidobenzoate and succinate; wherein the number of any quaternary ammonium species balances the anion A^" such that compound has no net charge. The invention includes all permissible ratios of cationic ammonium species to anion A^', for example hemi-napadisylate and napadisylate.

Suitable pharmaceutically acceptable salts of compounds (2) and (3), and dual ammonium/pharmaceutically acceptable salts of compounds (1), (4) and (5) include acid addition salts such as a hydrochloride, hydrobromide, phosphate, sulfate, acetate, diacetate, fumarate, maleate, tartrate, citrate, oxalate, methanesulfonate or p-toluenesulfonate.

Compounds (1)-(5) above and salts thereof may be prepared or recovered in the form of hydrates and solvates. Any reference herein, including the claims herein, to "compounds with which the invention is concerned" or "compounds of the invention" or "the present compounds", and the like, includes reference to salts hydrates, and solvates of such compounds.

Compounds of the invention may be useful in the treatment or prevention of diseases in which HNE is implicated, for example chronic obstructive pulmonary disease (COPD), chronic bronchitis, lung fibrosis, pneumonia, acute respiratory distress syndrome (ARDS), pulmonary emphysema, smoking-induced emphysema or cystic fibrosis, asthma, rhinitis, psoriasis, dermatitis, (atopic and non-atopic),

Crohn's disease, ulcerative colitis, and irritable bowel disease.

Another aspect of the invention is a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier or excipient.

Preferred compositions are those adapted for pulmonary administration by inhalation.

Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for the treatment or prevention of a disease or condition in which HNE is implicated. Thus, compounds of the invention may be used in a method of therapy, for the treatment of a patient suffering from a condition or disease as defined above.

The therapeutic utility of the present compounds is pertinent to any disease that is known to be at least partially mediated by the action of human neutrophil elastase. For example, the present compounds may be beneficial in the treatment of chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), acute respiratory distress syndrome (ARDS), pulmonary emphysema, pneumonia and lung fibrosis.

The weight ratio of the first and second active ingredients may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used.

The magnitude of prophylactic or therapeutic dose of a compound of the invention will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound and its route of administration. It will also vary according to the age, weight and response of the individual patient. In general, the daily dose range will lie within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per kg, and • most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases. Another aspect of the present invention provides pharmaceutical compositions which comprise a compound of the invention and a pharmaceutically acceptable carrier. The term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the invention, additional active ingredient(s), and pharmaceutically acceptable excipients.

The pharmaceutical compositions of the present invention comprise a compound of the invention as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.

Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a compound of the present invention. In therapeutic use, the active compound may be administered by any convenient, suitable or effective route. Suitable routes of administration are known to those skilled in the art, and include oral, intravenous, rectal, parenteral, topical, ocular, nasal, buccal and pulmonary. Delivery by inhalation is preferred.

Compositions suitable for administration by inhalation are known, and may include carriers and/or diluents that are known for use in such compositions. The composition may contain 0.01-99% by weight of active compound. Preferably, a unit dose comprises the active compound in an amount of 1 μg to 10 mg.

The most suitable dosage level may be determined by any suitable method known to one skilled in the art. It will be understood, however, that the specific amount for any particular patient will depend upon a variety of factors, including the activity of the specific compound that is used, the age, body weight, diet, general health and sex of the patient, time of administration, the route of administration, the rate of excretion, the use of any other drugs, and the severity of the disease undergoing treatment. For delivery by inhalation, the active compound is preferably in the form of microparticles. They may be prepared by a variety of techniques, including spray- drying, freeze-drying and micronisation.

By way of example, a composition of the invention may be prepared as a suspension for delivery from a nebuliser or as an aerosol in a liquid propellant, for example for use in a pressurised metered dose inhaler (PMDI). Propellants suitable for use in a PMDI are known to the skilled person, and include CFC-12, HFA-134a,

HFA-227, HCFC-22 (CCI2F2) and HFA-152 (CH4F2 and isobutane).

In a preferred embodiment of the invention, a composition of the invention is in dry powder form, for delivery using a dry powder inhaler (DPI). Many types of DPI are known.

Microparticles for delivery by administration may be formulated with excipients that aid delivery and release. For example, in a dry powder formulation, microparticles may be formulated with large carrier particles that aid flow from the DPI into the lung. Suitable carrier particles are known, and include lactose particles; they may have a mass median aerodynamic diameter of greater than 90 μm. In the case of an aerosol-based formulation, a preferred composition is: Compound of the invention 24 mg / canister

Lecithin, NF Liq. Cone. 1.2 mg / canister

Trichlorofluoromethane, NF 4.025 g / canister Dichlorodifluoromethane, NF 12.15 g / canister.

Compounds of the invention may be used in combination with other drugs that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which present compounds are useful. Such other drugs may be administered, by a route and in an amount commonly used therefore, contemporaneously or sequentially with a compound of the invention. When a compound of the invention is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of the invention is preferred. Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of the invention. Suitable therapeutic agents for a combination therapy with compounds of the invention include: (1) a corticosteroid, for example fluticasone or budesonide; (2) a β2-adrenoreceptor agonist, for example salmeterol or formeterol; (3) a leukotriene modulator, for example montelukast or pranlukast; (4) anticholinergic agents, for example selective muscarinic-3 (M3) receptor antagonists such as tiotropium bromide; (5) phosphodiesterase-IV (PDE-IV) inhibitors, for example roflumilast or cilomilast; (6) an antitussive agent, such as codeine or dextramorphan; (7) a nonsteroidal anti-inflammatory agent (NSAID), for example ibuprofen or ketoprofen; (8) a mucolytic, for example N acetyl cysteine or fudostein; (9) a expectorant/mucokinetic modulator, for example ambroxol, hypertonic solutions (e.g. saline or mannitol) or surfactant; (10) a peptide mucolytic, for example recombinant human deoxyribonoclease I (dornase-alfa and rhDNase) or helicidin; and (11) antibiotics, for example azithromycin, tobramycin and aztreonam.

The agents of the invention may be administered in inhaled form. Aerosol generation can be carried out using, for example, pressure-driven jet atomizers or ultrasonic atomizers, preferably using propellant-driven metered aerosols or propellant-free administration of micronized active compounds from, for example, inhalation capsules or other "dry powder" delivery systems.

The active compounds may be dosed as described depending on the inhaler system used. In addition to the active compounds, the administration forms may additionally contain excipients, such as, for example, propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, fillers (e.g. lactose in the case of powder inhalers) or, if appropriate, further active compounds. For the purposes of inhalation, a large number of systems are available with which aerosols of optimum particle size can be generated and administered, using an inhalation technique which is appropriate for the patient. In addition to the use of adaptors (spacers, expanders) and pear-shaped containers (e.g. Nebulator®, Volumatic®), and automatic devices emitting a puffer spray (Autohaler®), for metered aerosols, in particular in the case of powder inhalers, a number of technical solutions are available (e.g. Diskhaler®, Rotadisk®, Turbohaler® or the inhalers for example as described EP-A-0505321).

The following Examples illustrate the preparation and properties of compounds of the invention. Preparative HPLC conditions:

HPLC system 1 :

C18-reverse-phase end-capped column (250 x 21.2 mm Gemini column with

5 μm particle size), eluting with a gradient of A: water + 0.1% formic acid; B: acetonitrile + 0.1 % formic acid at a flow rate of 17 ml/min and gradient of 1%/min increasing in B. UV detection at 254 nm. After HPLC purification, pure fractions were combined and freeze-dried. Compounds were obtained as the formate salt where stated.

LC/MS Systems The Liquid Chromatography Mass Spectroscopy (LC-MS) systems used:

LC-MS method 1

Waters Platform LC with a C18-reverse-phase column (30 x 4.6 mm

Phenomenex Luna 3 μm particle size), elution with A: water + 0.1% formic acid; B: acetonitrile + 0.1% formic acid. Gradient:

Gradient - Time flow ml/min %A %B

0.00 2.0 95 5

0.50 2.0 95 5

4.50 2.0 5 95

5.50 2.0 5 95

6.00 2.0 95 5

Detection - MS, ELS, UV (100 μl split to MS with in-line UV detector) MS ionisation method - Electrospray (positive and negative ion) LC-MS method 2

Waters Micromass ZMD with a C18-reverse-phase column (30 x 4.6 mm Phenomenex Luna 3 μm particle size), elution with A: water + 0.1% formic acid; B: acetonitrile + 0.1% formic acid. Gradient:

Gradient - Time flow ml/min %A %B

0.00 2.0 95 5

0.50 2.0 95 5

4.50 2.0 5 95

5.50 2.0 5 95

6.00 2.0 95 5 Detection - MS, ELS, UV (100 μl split to MS with in-line UV detector) MS ionisation method - Electrospray (positive and negative ion) LC-MS method 3

Micromass Platform LCT with a C18-reverse-phase column (100 x 3.0 mm Higgins Clipeus with 5 μm particle size), elution with A: water + 0.1% formic acid; B: acetonitrile + 0.1% formic acid. Gradient:

Gradient - Time flow ml/min %A %B

0.00 1.0 95 5

1.00 1.0 95 5

15.00 1.0 5 95

20.00 1.0 5 95

22.00 1.0 95 5

25.00 1.0 95 5

Detection - MS, ELS, UV (100 μl split to MS with in-line UV detector) MS ionisation method - Electrospray (positive ion) LC-MS method 4 Waters Micromass ZQ2000 with a C18-reverse-phase column (100 x 3.0 mm

Higgins Clipeus with 5 μm particle size), elution with A: water + 0.1% formic acid; B: acetonitrile + 0.1 % formic acid. Gradient:

Gradient - Time flow ml/min %A %B

0.00 1.0 95 5

1.00 1.0 95 5

15.00 1.0 5 95

20.00 1.0 5 95

22.00 1.0 95 5

25.00 1.0 95 5

Detection - MS, ELS, UV (100 μl split to MS with in-line UV detector) MS ionisation method - Electrospray (positive ion) NMR Spectrometers

NMR's were run on either a Varian Unity Inova 400 MHz spectrometer or a

Bruker Avance DRX 400 MHz Spectrometer.

Abbreviations used in the experimental section: AIBN = 2,2'-azobis(2-methylpropionitrile)

Al-N = neutral alumina

DBU = 1 ,8-diazabicyclo[5.4.0]undec-7-ene

DCE = 1 ,1-dichloroethane

DCM = dichloromethane DME = 1 ,2-dimethoxyethane

DMF = Λ/,Λ/-dimethylformamide

DIPEA = di-isopropylethylamine

RT = room temperature

HATU = O-(7-Azabenzotriazol-1-yl)-Λ/,Λ/,Λ/',/V- tetramethyluroniumhexafluorophosphate

IMS = industrial methylated spirit

NBS = Λ/-bromosuccinimide

NIS = Λ/-iodosuccinimide

TFA = trifluoroacetic acid THF = tetrahydrofuran

Rt = retention time

SCX = strong cation exchange

Intermediate 1

Bromine (0.25 ml, 4.5 mmol) was added to a stirred solution of 6-methyl-2- oxo-1 -(3-trifluoromethylphenyl)-1 ,2-dihydropyridine-3-carboxylic acid (WO04043924)

(1.0 g, 3.3 mmol) in acetic acid. After 2 h a further quantity of bromine (0.25 ml, 4.5 mmol) was added and stirring was continued for 2 h. After standing at RT for 18 h, the mixture was diluted with half sat. aqueous sodium thiosulphate (50 ml) and extracted with DCM (2 x 20 ml). The combined organics were washed with water (20 ml) and dried (Na₂SO₄) before evaporation of the solvent. The desired product was obtained as a pale yellow solid.

Yield: 1.24 g (100%) LC-MS (Method 1 ): Rt 3.49 min, m/z 376/378 [MH⁺]

Intermediate 2

2(4.5 g, 11.97 mmol) was heated at 100^aC in 5M aqueous NaOH (400 ml) for

2 h. The solution was allowed to cool and filtered. After diluting with water (200 ml), the solution was acidified by the addition of cone, aqueous HCI. The solution was extracted with EtOAc (3 x 200 ml) and the organic extracts were combined, washed with water (200 ml) and brine (100 ml), dried (Na₂SO₄) and evaporated. The residue was triturated with Et₂O and the brown solid was filtered and dried. Yield: 475 mg (13%) LC-MS (Method 1): Rt 3.01 min, m/z 314 [MH⁺]

Intermediate 3

A solution of 2(470 mg, 1.50 mmol), 2-bromoethyl acetate (1.00 g, 5.99 mmol) and DBU (913 mg, 6.01 mmol) in DMF (3 ml) was heated under argon at 80^QC for 1.5 h. On cooling the reaction mixture was diluted with EtOAc (50 ml) and 1 M aqueous HCI (50 ml). The organic layer was separated, washed with water_, (40 ml) and brine (40 ml), dried (Na₂SO₄) and evaporated. The crude product was chromatographed on an Isolute™ Si Il cartridge (20 g) eluting with 50-100% EtOAc in pentane. Yield: 457 mg (63%)

LC-MS (Method 1): Rt 3.22 min, m/z 486 [MH⁺]

Intermediate 4

Intermediate 3 (457 mg, 0.924 mmol) was dissolved in MeOH (15 ml) and 2M aqueous NaOH (15 ml) was added. The solution was allowed to stand for 4 h and then filtered through celite. The filtrate was acidified by the addition of cone. aqueous HCI and the product was extracted into EtOAc (100 ml). The organic extract was washed with water (70 ml) and brine (50 ml), dried (Na₂SO₄) and evaporated to give a brown oil which solidified on standing.

Yield: 309 mg (92%)

LC-MS (Method 1): Rt 2.83 min, m/z 358 [MH⁺]

Intermediate 5

NIS (11.7 g, 0.05 mol) was added portion-wise to a stirred solution of 6- methyl-2-oxo-1 -(3-trifluoromethylphenyl)-1 ,2-dihydropyridine-3-carboxylic acid

(WO04043924) (10.3 g, 0.035 mol) in a mixture of TFA (30 ml) and DCM (30 ml).

Stirring was continued for 1.5 h the reaction mixture was partitioned between water

(200 ml) and DCM (200 ml). The organic phase was separated and the aqueous was extracted further with DCM (2 x 100 ml). The organic extracts were combined, washed with sat. aqueous sodium thiosulphate (100 ml), dried (Na₂SO₄) and concentrated to give a beige solid.

Yield: 13.6 g (93%)

LC-MS (Method 2): Rt 3.46 min, m/z 424 [MH⁺] Intermediate 6

Intermediate 5 (4.00 g, 9.46 mmol) was suspended in a mixture of toluene (40 ml) and MeOH (40 ml) and (trimethylsilyl)diazomethane (10 ml, 2M in hexane) was added over 10 min. After nitrogen evolution had ceased a further portion of (trimethylsilyl)diazomethane (2 ml) was added. The solvents were evaporated and the residue was triturated with Et₂O to give a pale cream solid.

Yield: 3.21 g (78%)

LC-MS (Method 2): Rt 3.45 min, m/z 438 [MH⁺]

Intermediate 7

Intermediate 6 (3.76 g, 8.60 mmol) and 2,4-dimethoxybenzylthiol (Synth. Commun., 1998, 28, 3219) (1.90 g, 10.33 mmol) were dissolved in DME (75 ml) and the solution was degassed with argon. Copper (I) iodide (83 mg, 0.44 mmol) and potassium carbonate (2.37 g, 17.17 mmol) were added and the reaction mixture was heated at 90^QC under an atmosphere of argon. After 17 h the mixture was allowed to cool and then filtered through celite. Evaporation gave a black-green residue which was chromatographed on an Isolute™ Si Il cartridge (50 g) eluting with 0-40% EtOAc in pentane. The impure fractions were combined and re-purified, and the pure fractions from both columns were evaporated to give a yellow foam.

Yield: 2.25 g (53%)

LC-MS (Method 2): Rt 4.05 min, m/z 494 [MH⁺] Intermediate 8

A solution of Intermediate 7 (2.25 g, 4.56 mmol) in DCE (90 ml) was treated with TFA (7 ml) and the solution was heated at reflux for 17 h under argon. The cooled reaction mixture was treated with a 1 :1 mixture of pentane and Et₂O (100 ml) and a white solid was removed by filtration. The filtrate was evaporated and triturated with a mixture of pentane, DCM, Et₂O and EtOAc to afford a yellow solid.

Yield: 650 mg (42%) LC-MS (Method 2): Rt 3.27 min, m/z 344 [MH⁺]

Intermediate 9

Intermediate 8 (320 mg, 0.933 mmol) and 6-chloronicotinitrile (129 mg, 0.93 mmol) were dissolved in 1 ,4-dioxane (15 ml) and caesium carbonate (304 mg, 0.93 mmol) was added. The reaction mixture was stirred at 40^QC under argon. After 2 h the mixture was filtered and the filtrate evaporated. The residue was purified on an Isolute™ Si Il cartridge (10 g) eluting with 30 and 40% EtOAc in pentane. The product was obtained as a pale yellow solid. Yield: 310 mg (75%) LC-MS (Method 2): Rt 3.46 min, m/z 446 [MH⁺] Intermediate 10

Intermediate 9 (400 mg, 0.674 mmol) was dissolved in 1 ,4-dioxane (10 ml) and 1 M aqueous NaOH (6 ml) was added. After stirring for 1.5 h, the reaction mixture was poured into 1 M aqueous HCI (70 ml). The product was extracted with

EtOAc (150 ml) which was washed with water (100 ml) and brine (50 ml), dried

(Na₂SO₄) and evaporated to give a beige solid.

Yield: 290 mg (100%) LC-MS (Method 2): Rt 3.55 min, m/z 432 [MH⁺]

The material contained about 20% of the corresponding amide. LC-MS (Method 2): Rt 3.03 min, m/z 450 [MH⁺]

Intermediate 11

Intermediate 10 (290 mg, 0.67 mmol) was suspended in acetic acid (3.5 ml) and 30% aqueous hydrogen peroxide (0.9 ml) was added. The mixture was heated at 50^sC for 2 h, after which time the solid had dissolved, and then diluted with water (20 ml) and filtered. After a few minutes a white solid had precipitated from the solution and was filtered off. The filtrate was purified using HPLC system 1 giving Intermediate 11 after freeze-drying of the HPLC fractions. The white solid which had previously been obtained was combined with the material obtained from HPLC.

Yield: 76 mg (25%) LC-MS (Method 1 ): Rt 3.30 min, m/z 448 [MH⁺] Intermediate 12

Intermediate 12 was obtained during the synthesis of Intermediatel 1. Yield: 24 mg (8%)

LC-MS (Method 1): Rt 2.74/2.80 min, m/z 466 [MH⁺]

Intermediate 13

A solution of Intermediate 8 (846 mg, 2.47 mmol) and 4-iodobenzonitrile (565 mg, 2.47 mmol) in DME (20 ml) was degassed with argon. Copper (I) iodide (23 mg, 0.12 mmol) and potassium carbonate (681 mg, 4.93 mmol) were added and the reaction mixture was heated at 80^QC under argon. After 2 h a further amount of 4- iodobenzonitrile (450 mg, 1.97 mmol) was added and heating was continued for a further 1 h. The mixture was allowed to cool and filtered. Evaporation gave a black- green oil which was purified on an Isolute™ Si Il cartridge (25 g) eluting with 0-30% EtOAc in pentane. The desired product was obtained as a yellow gum which crystallised on standing.

Yield: 400 mg (36%)

LC-MS (Method 1): Rt 3.79 min, m/z 445 [MH⁺]

Intermediate 14

Intermediate 14 was prepared from Intermediate 13 using a procedure similar to that used in the synthesis of Intermediate 10. Yield: (100%) LC-MS (Method 1): Rt 3.78 min, m/z 431 [MH⁺]

Intermediate 15

Intermediate 15 was prepared from Intermediate 14 by a similar method to that used to prepare Intermediate 11. Yield: (25%) LC-MS (Method 1): Rt 3.35 min, m/z 447 [MH⁺]

Intermediate 16

4-Cyanobenzene-1-sulfonyl chloride (4.0 g, 19.8 mmol) was added to a solution of 3,3'-diamino-/V-methyldipropylamine (1.66 ml, 9.9 mmol) and triethylamine (3.0 ml, 21.6 mmol) in DMF (15 ml) at RT under nitrogen then stirred for 5 h. The mixture was poured into water (250 ml) and extracted with EtOAc (3 x 100 ml). The extracts were washed with water (2 x 100 ml) and brine (50 ml) before the organic phase was isolated, dried (MgSO₄), filtered and concentrated. The crude product was triturated using cyclohexane, filtered and dried to afford the desired compound as a white solid. Yield: 2.69 g (57%)

LC-MS (Method 2): Rt 2.31 min, m/z 476 [MH⁺] Intermediate 17

Lithium aluminium hydride (1M in THF; 11 ml, 11 mmol) was added dropwise to a solution of Intermediate 16 (2.20 g, 4.62 mmol) in THF (30 ml) at -78⁹C under nitrogen then stirred for 1 h before the cooling bath was removed and the mixture allowed to warm to RT. The reaction was quenched using wet sodium sulphate and left to stand overnight. It was filtered through celite using THF and concentrated in vacuo, then loaded onto an Isolute™ SCX-2 cartridge which was flushed with MeOH. The product was then eluted using 2M NH₃ in MeOH to give Intermediate 17 as a pale yellow glass.

Yield: 0.50 g (22%)

LC-MS (Method 2): Rt 0.24 min, m/z 484 [MH⁺]

Illustrative Preparative Example A

2,2'-Diamino-Λ/-methyldiethylamine (197 mg, 1.68 mmol), 6-methyl-2-oxo-1-

(3-trifluoromethylphenyl)-1 ,2-dihydropyridine-3-carboxylic acid (WO04043924) (1.00 g, 3.367 mmol), and DIPEA (3 ml) were dissolved in DMF (50 ml) and HATU (1.14 mg, 3.70 mmol) was added. The solution was allowed to stand at RT for 3 h and the

DMF was evaporated. The residue was dissolved in EtOAc (150 ml) and the solution was washed with sat. aqueous NaHCO₃ (100 ml), water (100 ml) and brine

(50 ml). After drying (Na₂SO₄) the crude product was purified on an Isolute™ Si Il cartridge (20 g) eluting with 0-20% MeOH in EtOAc to give a pale yellow solid. A small amount of material was purified further using HPLC system 1 yielding Example

A as the free base.

Yield: 950 mg, (84%)

LC-MS (Method 3): Rt 8.16 min, m/z 676.19 [MH⁺] Illustrative Preparative Example B

Example B was prepared by a procedure similar to that of Example A from Intermediate 5 and 2,2'-diamino-Λ/-methyldiethylamine. Purification was accomplished on a RediSep™ silica cartridge eluting with 0-10% MeOH in DCM.

Yield: (79%)

LC-MS (Method 3): Rt 9.26 min, m/z 928.06 [MH⁺]

Illustrative Preparative Example C

Example C was prepared by a procedure similar to that of Example A from Intermediate 4 and 3,3'-diamino-Λ/-methyldipropylamine. Purification was achieved using HPLC system 1 giving the free base. Yield: (13%) LC-MS (Method 3): Rt 7.57 min, m/z 824.41 [MH⁺]

Illustrative Preparative Example D

Example D was prepared by a procedure similar to that of Example A from Intermediate 15 and 3,3'-diamino-Λ/-methylpropylamine. Purification was achieved using HPLC system 1 giving the formate salt.

Yield: (27%) LC-MS (Method 3): Rt 9.16 min, m/z 1002.37 [MH⁺]

Illustrative Preparative Example E

Example C (13 mg, 0.016 mmol) was dissolved in acetonitrile (2 ml) and iodomethane (1 ml) was added. The solution was heated at 100^sC for 20 min in the microwave. The volatiles were evaporated and the product was purified on an Isolute™ Al-N cartridge (2 g) eluting with DCM then 1% MeOH in DCM. After evaporation the pure product was dissolved in acetonitrile/water and freeze-dried to give a cream solid.

Yield: 7 mg (46%)

LC-MS (Method 3): Rt 7.50 min, m/z 838.48 [M⁺]

Illustrative Preparative Example F

Example B (200 mg, 0.215 mmol) was dissolved in acetonitrile (15 ml) and a 30% solution of bromomethane in acetonitrile (7 ml) was added. The solution was heated for 42 h at 80^QC in a steal reaction vessel. A further portion of bromomethane solution (7 ml) was added and heating was continued for 18 h. The volatiles were evaporated and the product was purified on an Isolute™ Al-N cartridge (10 g) eluting with 0-5% MeOH in DCM. Trituration with EtOAc gave a pale yellow solid.

Yield: (55%)

LC-MS (Method 3): Rt 9.36 min, m/z 942.02 [M⁺] Example 1

Example 1 was prepared from the compound of Illustrative Preparative Example D using a procedure similar to that described for the preparation of Illustrative Preparative Example E.

Yield: 7 g (34%)

LC-MS (Method 4): Rt 8.69 min, m/z 1016.42 [M⁺]

Example 2

Example 2 was prepared from the compound of Illustrative Preparative Example D using a procedure similar to that described for the preparation of Illustrative Preparative Example F. Yield: 38 mg (57%) LC-MS (Method 3): Rt 8.89 min, m/z 1016.35 [M⁺]

Example 3

Example 3 was prepared from Intermediates 15 and 17 using a procedure similar to that described for the preparation of Illustrative Preparative Example A. Yield: 179 mg (50%) LC-MS (Method 3): Rt 9.50 min, m/z 1340.42 [MH⁺]

Example 4

Example 4 was prepared from Intermediate 15 and 2,2'-diamono-N- methyldiethylamine using a procedure similar to that described for the preparation of Illustrative Preparative Example A.

Yield: 106 mg (28%)

LC-MS (Method 3): Rt 9,84 min, m/z 974.31 [MH⁺]

Example 5

Example 5 was prepared from the compound of Example 3 using a procedure similar to that described for the preparation of Illustrative Preparative Example F.

Yield: 45 mg (78%)

LC-MS (Method 4): Rt 9.28 min, m/z 1354.56 [M⁺]

Example 6

Example 6 was prepared from Example 4 using a procedure similar to that described for the preparation of Illustrative Preparative Example F. Yield: 17 mg (29%) LC-MS (Method 3): Rt 9.09 min, m/z 988.17 [M⁺]

Biological Results

The compounds of Examples 1 to 6 were tested for their inhibitory activity towards

HNE.

Fluorescent peptide substrate Assays were performed in 96-well plates at a total assay volume of 100 μl.

The final concentration of the enzyme (human leukocyte elastase, Sigma E8140) was 0.00036 units/well. A peptide substrate (MeO-Suc-Ala-Ala-Pro-ValAMC, Calbiochem #324745) was used, at the final concentration of 100μM. The final concentration of DMSO was 1% in the assay buffer (0.05M Tris.HCI, pH 7.5, 0.1 M NaCI; 0.1 M CaCI2; 0.0005% brij-35).

The enzymatic reaction was started by adding the enzyme. The enzymatic reaction was performed at RT and after 30mins stopped by adding 50 μl soybean trypsin inhibitor (Sigma T-9003) at a final concentration of 50μg/well. Fluorescence was read on the FLEXstation (Molecular Devices) using 380 nm excitation and 460 nm emission filters. The potency of the compounds was determined from a concentration series of 10 concentrations in range from 1000 nM to 0.051 nM. The results are means of two independent experiments, each performed in duplicate. Using Fluorescently labelled elastin

Assays were performed in 96-well plate at a total assay volume of 100 μl. The final concentration of the enzyme (human leukocyte elastase, Sigma E8140) was 0.002 units/well. Fluorescently labelled, solubilised elastin from bovine neck ligament (Molecular Probes, E-12056) was used at the final concentration of 15μg/ml. The final concentration of DMSO was 2.5% in the assay buffer (0.1 M Tris- HCL, pH8.0, containing 0.2mM sodium azide). The enzymatic reaction was started by adding the enzyme. The enzymatic reaction was performed at RT and read after 120 minutes. Fluorescence was read on the FLEXstation (Molecular Devices) using 485 nm excitation and 530 nm emission filters. The potency of the compounds was determined from a concentration series of 10 concentrations in range from 2500OnM to 1nM. The results are means of two independent experiments, each performed in duplicate.

The compounds of Examples 1 to 6 were shown to have IC₅₀ values for HNE in the above assay of less than 50OnM.

Claims

Claims:

1. A compound selected from the group consisting of those of formulae (1) to (5)

or a pharmaceutically acceptable salt thereof, wherein A^" is a pharmaceutically acceptable anion.

2. A compound as claimed in claim 1 wherein A^" is Br^" or l\

3. A pharmaceutical composition comprising a compound as claimed in claim 1 or claim 2 and a pharmaceutically acceptable carrier or excipient.

4. A pharmaceutical composition as claimed in claim 3 which is adapted for delivery to the lungs by inhalation.

5. Use of a compound as claimed in claim 1 or claim 2, for the manufacture of a medicament for use in the treatment of prevention of a disease or condition in which HNE is implicated.

6. Use according to claim 5, wherein the disease or condition is chronic obstructive pulmonary disease (COPD), chronic bronchitis, lung fibrosis, pneumonia, acute respiratory distress syndrome (ARDS), pulmonary emphysema, smoking- induced emphysema or cystic fibrosis.

7. Use according to claim 5, wherein the disease or condition is asthma, rhinitis, psoriasis, dermatitis, (atopic and non-atopic), Crohn's disease, ulcerative colitis, and irritable bowel disease.