WO2009060158A1 - 4- (4-cyanophenyl) -1- (3-trifluoromethylphenyl) -3,4, 6, 7-tetrahydro-1h-pyrrolo [3, 4- d] pyrimidine-2, 5-dione derivatives and their use as human neutrophil elastase inhibitors - Google Patents

Abstract

Thirty six specific compounds having human neutrophil elastase inhibitory activity are disclosed, one of which has the structural formula (1). The compounds are useful inter alia for the treatment of inflammatory respiratory disease, and may be administered by inhalation.

Description

- (4-CYANOPH ENYL) -1- (3-TRIFLUOROMETHYLPHENYL) -3,4 , 6, 7-TETRAHYDRO-1H

-PYRROLO [3 , 4-D] PYRI M I Dl N E-2 , 5-DIONE DERIVATIVES AND THEIR USE

AS HUMAN NEUTROPHIL ELASTASE INHIBITORS

Field of the Invention

This invention relates to some specific substituted 3,4,6,7-tetrahydro-1 H- pyrrolo[3,4-d]pyrimidine-2,5-diones which are inhibitors of human neutrophil

5 elastase, and their use in therapy. Background to the invention

Human neutrophil elastase (HNE) 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, io 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

15 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

20 inhibit the inflammatory response and the development of emphysema (Wright, J. L. et al. Am. J. Respir. Cut. Care Med. 2002, 166, 954-960; Churg, A. etal. 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

25 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 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

30 injury following acute myocardial infarction.

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-

35 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 (CH-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 develop emphysema that increases in severity over time (Laurrell, C. B.; Erikkson, S Scand. J. CHn. 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 sequentially in a step-wise manner 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. et al., Bioorg. Med. Chem. Letts. 1999, 7, 2569-2575). Also, multiple binding interactions (either sequential or parallel) 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.

Our co-pending International Patent Application No. PCT/GB2007/001638 relates, inter alia, to homodimeric or heterodimeric compounds of formula M-L-M¹ wherein L is a divalent linker radical and M and M¹ are each independently a radical of formula (A') or (B'):

wherein

A is aryl or heteroaryl; D is oxygen or sulphur;

R¹, R², R³ and R⁵ are independently each hydrogen, halogen, nitro, cyano,

C|-C₆-alkyl, C₂-C₆-alkenyl, C₂-C₆-alkynyl, hydroxy or CrC₆-alkoxy or C₂-Ce- alkenyloxy, wherein CrC₆-alkyl and CrC₆-alkoxy can be further substituted with one to three identical or different radicals selected from the group consisting of halogen, hydroxy and Ci-C₄-alkoxy;

R and R⁴ each independently represent a radical of formula -[X]₀₁-[AIk¹ ]_p- [Q]n-[Alk²]_q-[X¹]_k-Z wherein k, m, n, p and q are independently 0 or 1 ;

AIk¹ and AIk² each independently represent an optionally substituted C₁-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-) or amino (-NR^A-) link wherein R^A is hydrogen or CrC₃ alkyl;

Q represents (i) -O-, -S-, -S(=O)-, -S(=O)₂-, -S⁺(R^A)-, -N(R^A)-, -N⁺(R^A)(R^B)-,

-C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR^A-, -NR^AC(=O)-, -S(O₂)NR^A-₍

-NR^AS(O₂),-NR^AC(=O)NR^B-, -NR^AC(=NR^A)NR^B-, -C(=NR^D)NR^E-, -NR^EC(=NR^D)-, wherein R^A, R^B, R^D and R^E are independently hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, or R^A and R^B' or R^D and R^E taken together with the nitrogen to which they are attached form a monocyclic heterocyclic ring of 5 to 7 ring atoms which my contain a further heteroatom selected from N, O and S, or (ii) an optionally substituted divalent mono- or bicyclic carbocyclic or heterocyclic radical having 3- 6 ring members;

X represents -(C=O)-, -S(O₂)-, -C(=O)O-, -(C=O)NR^A-, or -S(O₂)NR^A-, wherein R^A is hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl;

X^I represents -O-, -S-, or -NH; and

Z is hydrogen or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members. In some embodiments of the compounds (A') and (B') of International

Patent Application No. PCT/GB2007/001638, the linker radical L is a divalent straight chain, saturated or unsaturated hydrocarbon radical having from 2 to 12 carbon atoms in the said chain, and wherein one or more carbons is replaced by -N⁺(R^P)(R^Q)-, wherein one of R^p and R^Q is HOC(=O)-(C_rC₆ alkyl)-, and the other is hydrogen, CrC₆ alkyl, or C₃-C₆ cycloalkyl, HO-(CrC₆ alkyl)-, HOC(=O)-(d-C₆ alkyl)- or R^AR^BN-(C_rC₆ alkyl)- wherein R^A and R⁵ are independently hydrogen,

CrC₆ alkyl, or C₃-C₆ cycloalkyl, or HOC(=O)-(Ci-C₆ alkyl)-. In such compounds, given the presence of the positively charged nitrogen of the radical -N⁺(R^P)(R^Q)-, the carboxyl group in R^p and R^Q is in the negatively charged carboxylate species form. The compound is thus neutral, despite the presence of both a positively charged nitrogen and a negatively charged oxygen. The compound is thuis an example of a betaine, betaines being chemical compounds with both a positively charged cationic functional group such as an ammonium ion or phosphonium ion (an "onium ion") which bears no hydrogen atom, and a negatively charged functional group such as a carboxylate group which may not be adjacent to the cationic site. Betaines are examples of the class of compounds known as zwitterions which are electrically neutral but carry formal positive and negative charges on different atoms which may be adjacent or non-adjacent. Brief description of the invention

This invention relates to compounds within the scope of PCT/GB2007/001638 but specifically disclosed therein. Like the compounds of PCT/GB2007/001638, the present compounds are useful in the treatment of diseases or conditions in which HNE activity plays a part. The compounds are particularly useful in the case of topical pulmonary application by inhalation. Detailed description of the invention

The present invention provides compounds selected from the group consisting of formulae (1) to (36) as set out in claim 1 herein, and salts, hydrates and solvates thereof. In compounds (9), (12), (13), (25), (27) and (30), 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, mesylate, 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 other compounds of the invention include acid addition salts such as a hydrochloride, hydrobromide, phosphate, sulfate, acetate, diacetate, fumarate, maleate, tartrate, citrate, oxalate, methanesulfonate or p-toluenesulfonate. Compounds of the invention above and salts thereof may be prepared in the form of hydrates, and solvates thereof. 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 have asymmetric carbon atoms, and thus can exist as mixtures of isomeric forms or in single stereisomeric form. Preferably, the compounds of the invention are purely or predominantly in the form shown in the Examples herein, i.e:

(i) Compounds having any of formulae 1-18, or 31-33, are purely or predominantly in the form wherein the stereochemical orientation of the bond between the 4-cyanophenyl group and the fused pyrimidine ring is as shown in formula (X):

(ii) Compounds having any of formulae 19-25, 29, 30 or 34-36 are purely or predominantly in the form wherein the stereochemical orientation of the bond between the left hand side 4-cyanophenyl group and fused pyrimidine ring is as shown in formula (X) above, and the stereochemical orientation of the bond between the right hand side 4-cyanophenyl group and fused pyrimidine ring is as shown in formula (Z):

Compounds having any of formulae 26-28, are purely or predominantly in the form wherein the stereochemical orientation of the bond between the left hand side 4-cyanophenyl group and fused pyrimidine ring is as shown in formula (V), and the stereochemical orientation of the bond between the right hand side 4-cyanophenyl group and fused pyrimidine ring is as shown in formula (W):

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 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 nontoxic 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 micron isation.

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 non-steroidal 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 (domase-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 propel lant-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 of compounds of the invention:

General Experimental Details:

All solvents and commercial reagents were used as received. Where products were purified using an Isolute® SPE Si Il cartridge, 'Isolute SPE Si cartridge' refers to a pre-packed polypropylene column containing unbonded activated silica with irregular particles with average size of 50 μm and nominal 6θA porosity. Where an Isolute® SCX-2 cartridge was used, 'Isolute® SCX-2 cartridge' refers to a pre-packed polypropylene column containing a non end- capped propylsulphonic acid functionalised silica strong cation exchange sorbent. 'Isolute® Al-N cartridge' refers to a pre-packed polypropylene column containing neutral alumina with average particle size 50-200 μm and 120 A pore diameter. 'CombiFlash® companion' refers to an automated flash silica chromatography system which uses pre-packed polypropylene (RediSep®) columns containing silica with average particle size 35-70 μm (230-400 mesh). 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 with a flow rate typically 17 ml/min and gradient of 1%/min increasing in B. UV detection at 254 nm. Compounds were obtained as the formate salt where stated. HPLC system 2:

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; B: acetonitrile with a flow rate typically 17 ml/min and gradient of 1%/min increasing in B. UV detection at 254 nm.

HPLC system 3:

Phenyl hexyl column (250 x 21.20 mm Luna column with 10 μm particle size), eluting with a gradient of A: water; B: acetonitrile at a flow rate of 20 ml/min with UV detection at 254 nm. After HPLC purification, fractions containing product were combined and freeze-dried, unless otherwise stated, to give the product as a white or off-white solid. In some cases, where HPLC system 1 or 3 was used and where the compound contained a basic centre, the product was obtained as the formate salt. 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 C 18-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) Abbreviations used in the experimental section: DCM = dichloromethane

DIPEA = Diisopropylethylamine

DMA = Λ/,Λ/-dimethylacetamide

DMF = Λ/,/V-dimethylformamide

HATU = 0-(7-Azabenzotriazole-1-yl)-Λ/,Λ/,Λ/',Λ/'-tetramethy]uronium hexafluorophosphate

HBTU = <>Benzotriazole-/V,Λ/,/V₎ΛMetramethyluronium hexafluorophosphate

HPLC = high performance liquid chromatography

IMS = industrial methylated spirits

RT = room temperature

Rt = retention time

TFA = trifluoroacetic acid

THF = tetrahydrof uran

Intermediates 1 and 2

Intermediates 1 and 2 were prepared according to WO06/082412. Intermediate 3

Intermediate 1 (40.0 g, 0.010 mol) was dissolved in a mixture of MeOH (100 ml) and toluene (200 ml) and a 2M solution of (trimethylsilyl)diazomethane (100 ml, 0.2 mol) in hexanes was added dropwise. After stirring at RT for 30 min, the reaction mixture was evaporated to dryness. The product was a cream solid. Yield: 40.4 g (95%) LC-MS (Method 1): Rt = 3.68, m/z = 416 [M+H]⁺ Intermediate 4

A solution of Intermediate 3 (35.0 g, 0.09 mol) in chloroform (150 ml) was treated with bromine (14.87 g, 0.09 mmol) in chloroform (50 ml). The reaction mixture was stirred at RT for 30 min and then evaporated to give an orange foam. The product was purified by crystallization from EtOAc and pentane to give the bromo ester as a cream solid. Yield: 38.04 g (91%)

LC-MS (Method 2): Rt = 3.66 min, m/z = 494/496 [M+H]⁺ Intermediate 5

Intermediate 5 was prepared from Intermediate 2 using a similar procedure to that used in the synthesis of Intermediate 4.

LC-MS (Method 2): Rt = 3.82 min, m/z = 508/510 [M+H]⁺

Intermediate 6

Example 7 (684 mg, 1.50 mmol) was dissolved in pyridine (15 ml) under an atmosphere of N₂. Methanesulphonyl chloride (188 mg, 1.65 mmol) was added and the reaction was stirred at RT for 1 h after which time the pyridine was evaporated. The residue was dissolved in EtOAc (100 ml) and washed with 10% aqueous citric acid (50 ml), sat. aqueous sodium hydrogen carbonate (50 ml) and brine (50 ml). The organic solution was dried (MgSO₄) and evaporated to give a pink foam.

Yield: 620 mg (59%)

LC-MS (Method 1): Rt = 3.21 min, m/z = 535 [M+Hf

Intermediate 7

Intermediate 7 was prepared from Example 8 by a similar procedure to that used for Intermediate 6. Yield: 240 mg (73%)

LC-MS (Method 2): Rt = 3.20 min, m/z = 549 [M+H]⁺ Intermediate 8

A mixture of piperidin-4-yl-carbamic acid tert-butyl ester (470 mg, 2.35 mmol), 2- bromopyridine (370 mg, 2.35 mmol), 2-dicyclohexylphosphino-2-(Λ/,Λ/- dimethylamino)biphenyl (111 mg, 0.28 mmol) and bis(dibenzylineneacetone)palladium (129 mg, 0.14 mmol) in toluene (8 ml) was heated at 140°C in the microwave for 30 min. The mixture was diluted with water (150 ml) and washed with EtOAc (3 x 100 ml). The combined organic extracts were dried (MgSO₄) and evaporated. The product was chromatographed using a CombiFlash® companion eluting with 0-5% MeOH in DCM, giving 340 mg of Boc- protected material of -70% purity. This material was dissolved in DCM (5 ml) and treated with TFA (3 ml). After 1 h the volatiles were evaporated and the residue was dissolved in MeOH. The solution was loaded onto an Isolute® SCX-2 cartridge which had been conditioned with MeOH. After flushing with a further portion of MeOH, Intermediate 8 was eluted from the cartridge with 2M NH₃ in MeOH. Evaporation gave an orange oil which was purified by chromatography using a CombiFlash® companion system and eluting with 0-40% MeOH in DCM. Yield: 125 mg (30%)

1H NMR (400 MHz, CDCI₃): δ = 1.38 (m, 2H), 1.91 (m, 2H), 2.91 (m, 3H), 4.24 (m, 2H), 6.58 (dd, 1 H), 6.67 (d, 1H), 7.45 (m, 1H), 8.17 (m, 1 H) ppm. Intermediate 9

Intermediate 8 (125 mg, 0.70 mmol), 3-tø/t-butoxycarbonylaminopropionic acid (150 mg, 0.77 mmol) and DIPEA (225 mg, 1.75 mmol) were dissolved in DMF (10 ml) and HATU (320 mg, 0.84 mmol) was added. The solution was allowed to stand at RT for 1 h and was then diluted with water (200 ml). The product was extracted with EtOAc (3 x 150 ml) and the combined organics were dried (MgSO₄) and evaporated. Chromatography using a CombiFlash® companion and eluting with 0-20% MeOH in DCM gave, after evaporation of the solvent and trituration with Et₂O, 165 mg of a white solid. The solid was dissolved in DCM (3 ml) and the solution was treated with TFA (3 ml). After 30 min the volatiles were evaporated and the residue was dissolved in MeOH. The solution was loaded onto an Isolute® SCX-2 cartridge which had been conditioned with MeOH. After flushing with a further portion of MeOH, Intermediate 9 was eluted from the cartridge with 2M NH₃ in MeOH. Evaporation gave a solid which was triturated with Et₂O.

Yield: 55 mg (32%)

1H NMR (400 MHz, CDCI₃): δ = 1.39-1.63 (m, 2H), 2.02 (m, 2H), 2.31 (m, 2H), 3.04 (m, 4H), 4.06 (br s, 1 H), 4.16 (m, 2H), 6.60 (dd, 1H), 6.67 (d, 1H), 7.15 (br s, 1H), 7.46 (m, 1H), 8.18 (br d, 1H) ppm. Intermediate 10

A mixture of 2-[2-(2-chloroethoxy)ethoxy]ethanol (5.5 g, 32.5 mmol) and potassium phthalimide (5.58 g, 30.2 mmol) in DMF (5 ml) was heated at 110⁰C for 5 h. After cooling the mixture was filtered, the filtrate was diluted with Et₂O, and the mixture was chromatographed using a CombiFlash® companion, eluting with 0-7% MeOH in DCM. The product was obtained as a colourless oil. Yield: 2.37 g (25%)

1 H NMR (400 MHz, CDCI₃): δ = 3.55 (m, 2H), 3.62 (m, 2H), 3.66 (m, 4H), 3.77 (m,

2H), 3.92 (m, 2H), 7.73 (m, 2H), 7.85 (m, 2H) ppm.

Intermediate 11

To a solution of Intermediate 10 (1.0 g, 2.86 mmol) in pyridine (30 ml) was added methanesulphonyl chloride (360 mg, 3.15 mmol). After 100 min the pyridine was evaporated. The residue was dissolved in EtOAc (200 ml) and the solution was washed with 10% aqueous citric acid (100 ml), brine (2 x 100 ml), dried (MgSO₄) and evaporated. Yield: 780 mg (76%)

LC-MS (Method 1): Rt = 2.93 min, m/z = 358 [M+H]⁺ Intermediate 12

Phenol (186 mg, 1.98 mmol) and Intermediate 11 (780 mg, 2.18 mmol) were dissolved in DMF (10 ml). The solution was stirred under N₂ whilst 60% sodium hydride in mineral oil (88 mg, 2.18 mmol) was added, and stirring was continued at RT overnight. The mixture was diluted with water (150 ml) and washed with EtOAc (3 x 100 ml). The combined organic extracts were washed with water (100 ml) and brine (100 ml), dried (MgSO₄) and evaporated. The product was purified using a CombiFlash® companion eluting with 0-100% EtOAc in Et₂O. The product was obtained as a clear oil. Yield: 105 mg (15%) 1H NMR (400 MHz, CDCI₃) δ = 3.68 (s, 4H), 3.73-3.81 (m, 4H), 3.91 (m, 2H), 4.04 (m, 2H), 6.91 (m, 3H), 7.26 (m, 2H), 7.69 (m, 2H), 7.83 (m, 2H) ppm. Intermediate 13

A solution of Intermediate 12 (100 mg, 0.28 mmol) and hydrazine hydrate (1 ml) in EtOH (10 ml) was heated at reflux for 1 h. The solution was evaporated and dissolved in MeOH. The solution was loaded onto an Isolute® SCX-2 cartridge which had been conditioned with MeOH. After flushing with a further portion of MeOH, Intermediate 13 was eluted from the cartridge with 2M NH₃ in MeOH. Evaporation gave the amine as a clear oil. Yield: 60 mg (95%)

1H NMR (400 MHz, CDCI₃): δ = 2.87 (t, 2H), 3.52 (t, 2H), 3.66 (m, 2H), 3.74 (m, 2H), 3.87 (m, 2H), 4.14 (m, 2H), 6.93 (m, 3H), 7.28 (m, 2H) ppm. Intermediate 14

To a solution of bis-(2-aminoethyl)carbamic acid te/f-butyl ester (J. Med. Chem., 2006, 49(13), 3872-3887) (5.22 g, 25.71 mmol) in acetonitrile (250 ml) was added sodium hydrogen carbonate (1.73 g, 20.60 mmol). The mixture was warmed to 6O⁰C and a solution of Intermediate 4 (5.22 g, 10.27 mmol) in DMF (25 ml) was added dropwise over 2 h whilst maintaining the temperature at 60⁰C. Evaporation gave a residue to which was added water (100 ml). The product was extracted with EtOAc (3 x 100 ml) and the combined extracts were dried (MgSO₄) and evaporated. Chromatography using a CombiFlash® companion and eluting with 0-20% MeOH in DCM gave, on evaporation of the pure fractions, a residue which was triturated with Et₂O to give a cream solid. Yield: 3.37 g (56%) LC-MS (Method 1): Rt = 2.41 min, m/z = 585 [M+H]⁺ Intermediate 15

Intermediate 14 (128 mg, 0.22 mmol) was dissolved in DCM (10 ml) and a solution of potassium carbonate (303 mg, 2.20 mmol) in water (10 ml) was added. With vigorous stirring, a solution of 4-(pyrrolidine-1-sulfonyl)benzenesulphonyl chloride (341 mg, 1.10 mmol) in DCM (5 ml) was added dropwise over 10 min. The organic layer was separated and the aqueous solution was washed with DCM (2 x 50 ml). The organics were combined, dried (MgSO₄) and evaporated. The product was purified using a CombiFlash® companion eluting with 0-10% MeOH in DCM, followed by trituration with Et₂O. Yield: 120 mg (64%)

LC-MS (Method 1): Rt = 3.67/3.81 min, m/z = 858 [M+Hf Intermediate 16

o Intermediate 16 was prepared from Intermediate 14 and methanesulphonyl chloride using a similar procedure to that used in the synthesis of Intermediate 15. Yield: 48%

LC-MS (Method 1): Rt = 3.39 min, m/z = 663 [M+Hf Intermediate 17

A mixture of Intermediate 5 (1.00 g, 1.97 mmol), glycine terf-butyl ester hydrochloride (330 mg, 1.97 mmol) and potassium carbonate (410 mg, 2.95 mmol) in acetonitrile (20 ml) was heated at 160°C for 20 min in the microwave. The mixture was filtered and evaporated. The residue was taken up into EtOAc 20 (150 ml) and the solution was washed with 10% aqueous citric acid (100 ml) and water (50 mi), dried (Na₂SO₄) and evaporated to give the pure product. Yield: quantitative LC-MS (Method 2): Rt = 3.59/3.76 min, m/z = 457 [M+H-tBu]⁺ Intermediates 18 and 19

18 19

Borane dimethyl sulphide complex (2.5 ml, 25.0 mmol) was added to a solution of 3,3'-thtodipropionitrile (1.0 g, 7.14 mmol) and the reaction mixture was allowed to stand at RT. After 1 h, the solid which had formed was dissolved in THF (5 ml) and the reaction was left for a further 1h. The reaction was treated cautiously with water (20 ml) followed by cone. HCI (20 ml), and heated at reflux for 1 h. After cooling to RT, the mixture was adjusted to pH14 by the addition of 10N NaOH. The products were extracted into EtOAc (2 x 200 ml) and the combined extracts were dried (MgSO₄) and evaporated to give a clear oil. The mixture of Intermediates 18 and 19 was used without further purification. Intermediate 20

To a solution of Intermediate 4 (0.4 g, 0.787 mmol) in acetonitrile (10 ml) were added DIPEA (408 μl, 2.35 mmol) and Λ/-(3-aminopropyl)-1 ,3-propanediamine (50 mg, 0.392 mmol). The reaction mixture was stirred at 40⁰C for 3 h and the solvent was evaporated. The crude mixture was separated using a CombiFlash® companion eluting with 0-15% MeOH in DCM. The desired product was obtained as a cream solid. Yield: 204 mg (59%)

LC-MS (Method 2): Rt = 2.68/2.75 min, m/z = 894 [M+H]⁺ Intermediate 21

A mixture of Intermediate 20 (200 mg, 0.224 mmol), tert-butyl bromoacetate (1.31 g, 6.72 mmol) and DIPEA (1.17 ml, 6.72 mmol) in acetonitrile (50 ml) was heated at 40°C for 5 days. The solvent was evaporated and the resulting residue was dissolved in EtOAc (100 ml). Upon concentration of the solution, a solid precipitated. The solid was filtered and purified using a CombiFlash® companion system, eluting with 0-15% MeOH in DCM. Yield: 100 mg (37%)

LC-MS (Method 1): Rt = 2.99 min, m/z = 1122 [M]⁺ Intermediate 22

Intermediate 22 was prepared according to WO2006/082412. Intermediate 23

Intermediate 22 (1.65 g, 1.70 mmol) was dissolved in chloroform (50 ml) and bromine (681 mg, 4.26 mmol) was added dropwise with stirring. A further quantity of bromine (681 mg, 4.26 mmol) was added and, after 15 min, the volatiles were evaporated. The residue was chromatographed on an Isolute® SI Il cartridge (20 g) eluting with 50-100% EtOAc in pentane and then 5-10% MeOH in EtOAc. Evaporation gave a colourless gum. Yield: 1.17 g (61%) LC-MS (Method 2): Rt = 3.40 min, m/z = 1128 [M+H]⁺ Intermediate 24

Intermediate 23 (774 mg, 0.66 mmol) was dissolved in acetonitrile (40 ml) and 2M ethylamine in THF (8 ml, 16 mmol) was added. The solution was stirred at RT for 3 days after which time the mixture was filtered and evaporated. The product was purified by HPLC (system 1). The pure fractions were combined and approximately one fifth of the solution of the resulting formate salt was freeze- dried. The remainder was converted to the free base; the solution was loaded onto an Isolute® SCX-2 cartridge which had been conditioned with MeOH. The cartridge was flushed with MeOH and the amine was eluted with 2M NH₃ in MeOH. Both the formate salt and free base were a white solid. Yield: 92 mg (14%) formate salt 335 mg (53%) free base

LC-MS (Method 3): Rt = 9.10 min, m/z = 964.34 [M+H]⁺ Intermediate 25

Intermediate 24 (285 mg, 0.296 mmol) and te/t-butyl bromoacetate (58 mg,

2.96 mmol) were dissolved in acetonitrile (5 ml) and the solution was heated at

8O⁰C for 17 h. The solvent was evaporated and the residue was triturated with

Et₂O to give a colourless glassy solid.

Yield: 266 mg (78%)

LC-MS (Method 2): Rt = 3.06 min, m/z = 1078 [M]⁺ Intermediate 26

Intermediate 5 (108.6 g, 0.217 mol) was dissolved in THF (1500 ml) and triethylamine (240 ml, 1.74 mol) was added, followed by a solution of Λ/,Λ/-bis(3- aminopropyl)methylamine (23.55 g, 0.162 mol) in THF (100 ml) dropwise over 30 min. The reaction was stirred at RT for 17 h and the volume was reduced to 300-

400 ml. Water (3000 ml) was added and the mixture was stirred vigorously for 90 min. The supernatant was decanted off and a further amount of water (3000 ml) was added. Stirring was continued for 30 min. The water was decanted off and the remaining solid was dissolved in EtOAc (1500 ml). The solution was dried

(Na₂SO₄), evaporated to a small volume, and poured into Et₂O with vigorous stirring. The white precipitate was filtered off and washed with Et₂O and dried in vacuo.

Yield: 86.0 g (88%)

Further purification was achieved by crystallization from either n-butanol or n- propanol.

LC-MS (Method 4): Rt = 7.88 min, m/z = 908.44 [M+H]⁺

Intermediate 27

Intermediate 1 (500 mg, 1.25 mmol) was dissolved in chloroform (20 ml) and bromine (64 ml, 1.25 mmol) was added with stirring. A further 4 drops of bromine were added to the decolourised solution and the colour of the excess bromine remained. The volatiles were evaporated and the product was obtained, after chromatography on an Isolute® Si Il cartridge (10 g) eluting with 10-40% EtOAc in pentane, as a white solid.

Yield: 217 mg (36%)

LC-MS (Method 2): Rt = 3.28 min, m/z = 480/482 [M+H]⁺

Intermediates 28 and 29

Intermediate 20 (160 mg, 0.19 mmol), 4-bromomethylbenzoic acid (43 mg, 0.20 mmol) and DIPEA (49 mg, 0.38 mmol) were dissolved in DMF (2 ml) and HATU (87 mg, 0.23 mmol) was added. After 20 h the solution was dripped into water (100 ml) and the white precipitate which formed was filtered, dissolved in EtOAc and evaporated. The residue was triturated with Et₂O and then chromatographed using a CombiFlash® companion system, eluting with 0-10% MeOH in DCM to give a mixture of Intermediates 29 and 30. Yield: 100 mg (1 :2 mixture of 28 and 29)

LC-MS (Method 2): Rt = 3.72 min, m/z = 1090/1092 [M+H]⁺ and Rt = 3.61 min, m/z = 1146 [M+H]⁺ Intermediate 30

To a solution of Intermediate 20 (0.22 g, 0.246 mmol) in acetonitrile (10 ml) were added DIPEA (640 μl, 3.69 mmol) and 4-bromobutyric acid tert-butyl ester (550 mg, 2.0 mmol). The reaction was stirred at RT for 7 days in the presence of molecular sieves (4 A). The reaction mixture was diluted by adding acetonitrile (20 ml) and filtered to remove insoluble material. The volatiles were evaporated in vacuo and the residue was poured into water (100 ml) and extracted with EtOAc (2 x 25 ml). The combined organic extracts was dried (Na₂SO₄) and the solvent was evaporated to obtain the crude product. The crude mixture was separated using a CombiFlash® companion eluting with 0-15% MeOH in DCM.

The desired product was obtained as a glassy liquid.

Yield: 130 mg (51%)

LC-MS (Method 2): Rt = 2.92 min, m/z = 1036 [M+H]⁺

Intermediate 31

Intermediate 30 (130 mg, 0.125 mmol) was dissolved in a 30% solution of bromomethane in acetonitrile (5 ml) and sodium hydrogen carbonate (11 mg,

0.125 mmol) was added. The reaction was stirred at RT for 24 h and then the volatiles were evaporated giving a pale yellow gummy solid.

Yield: quantitative

LC-MS (Method 1): Rt = 2.92 min, m/z = 1050 [M]⁺

Example 1

Intermediate 4 (508 mg, 1.00 mmol), 3-cyclohexylpropylamine (130 mg, 0.92 mmol) and sodium hydrogen carbonate (772 mg, 9.19 mmol) in acetonitrile were heated at reflux for 4 h. The reaction mixture was filtered, evaporated and partitioned between DCM (50 ml) and 1N HCI (50 ml). The organic layer was separated and washed with 1N NaOH (50 ml), dried (MgSO₄) and evaporated. The crude was purified using a CombiFlash® companion, eluting with 0-5% MeOH in DCM. Trituration with Et₂O gave several batches, the purest of which was obtained as a white solid. Yield: 56 mg (11%)

LC-MS (Method 3): Rt = 13.28 min, m/z = 523.26 [M+H]⁺

The following compounds were prepared in a similar manner:

57 7.74 531.24

6 4 and ethanolamine 60 7.67 443.10

* Required further purification using HPLC system 1 Example 9

Intermediate 6 (200 mg, 0.37 mmol) was heated at reflux in pyridine (10 ml) for

1 h. The solvent was evaporated and the product was obtained as the formate salt after HPLC (system 1) purification. The product was an off-white solid.

Yield: 120 mg (53%)

LC-MS (Method 3): Rt = 6.39 min, m/z = 518.24 [M]⁺

Example 10

Intermediate 6 (200 mg, 0.37 mmol) and 3-cyclohexylpropylamine (J. Med. Chem., 2002, 45(25), 5492-5505) (150 mg, 0.97 mmol) were dissolved in THF (10 ml) and the solution was heated at reflux for 17 h. The solution was loaded onto and Isolute® SCX-2 cartridge (25 g) which had been conditioned with MeOH. After flushing with a further amount of MeOH, the product was eluted with 2M NH₃ in MeOH. Evaporation gave the crude product which was purified using a CombiFlash® companion system, eluting with 0-10% MeOH in DCM. The product was obtained as a pale pink solid. Yield: 36 mg (16%) LC-MS (Method 2): Rt = 2.68 min, m/z = 594 [M+H]⁺ Example 11

Example 11 was prepared from Intermediate 6 and methyl-(3- phenoxypropyl)amine using a procedure similar to that described for the synthesis of Example 10, however further purification using HPLC (system 1) was required.

Yield: 30 mg (13%)

LC-MS (Method 3): Rt = 7.68 min, m/z = 604.32 [M+H]⁺

Example 12

Intermediate 7 (240 mg, 0.43 mmol) was dissolved in THF (10 ml) and 4- phenylquinuclidine (153 mg, 0.88 mmol) was added. The reaction mixture was heated at reflux for 17 h. After cooling, the solution was loaded onto an Isolute® Al-N cartridge (10 g) and the cartridge was flushed with DCM. The product was eluted with 2, 5, 10 and then 20% MeOH in DCM. After evaporation of the purest fractions, the solid was triturated with Et₂O and then further purified by HPLC (system 1). The product was obtained a white solid. Yield: 100 mg (32%) LC-MS (Method 3): Rt = 7.77 min, m/z = 640.36 [M]⁺ Example 13

Example 10 (28 mg, 0.047 mmol) was dissolved in a 30% solution of bromomethane in acetonitrile (1 ml). The solution was heated in a sealed tube for

20 h. The solvent was evaporated and the residue was triturated with Et₂O to give a cream solid.

Yield: 24 mg (74%)

LC-MS (Method 3): Rt = 8.34 min, m/z = 608.43 [M]⁺

Example 14

Intermediate 15 (120 mg, 0.140 mmol) was dissolved in a 1:1 mixture of DCM and TFA (2 ml). After 30 min the volatiles were evaporated and the residue was triturated with Et₂O. The solid was dissolved in MeOH (20 ml) and loaded onto an Isolute® SCX-2 cartridge which had been conditioned with MeOH. After flushing with a further portion of MeOH, the product was eluted with 2M NH₃ in MeOH. The solvent was removed and trituration with Et₂O gave a white solid. Yield: 84 mg (79%) LC-MS (Method 3): Rt = 7.57 min, m/z = 758.11 [M+H]⁺ Example 15

Example 15 was prepared from Intermediate 16 using a method analogous to that used in the synthesis of Example 14.

Yield: 67%

LC-MS (Method 3): Rt = 6.25 min, m/z = 563.16 [M+Hf

Example 16

Intermediate 17 (600 mg, 1.17 mmol) was dissolved in DCM (9 ml) and treated with TFA (3 ml). The solution was stirred at RT for 17 h before evaporation of the volatiles. The residue was triturated with Et₂O to give a beige solid and purification was achieved using HPLC (system 1).

Yield: 57 mg (11%)

LC-MS (Method 3): Rt = 8.17 min, m/z = 457.14 [MH-H]⁺ Example 17

Example 16 (172 mg, 0.377 mmol), W-methylmorpholine (166 μl, 1.51 mmol) and HATU (358 mg, 0.944 mmol) were dissolved in DMF (2 ml) and 2-[methyl-(3- phenoxypropyl)amino]ethanol (Forschungszentrum Rossendorf e.V., [Bericht] FZR, 1997, (FZR-165), 1-5) (79 mg, 0.377 mmol) was added. The reaction mixture was stirred at RT for 17 h and then diluted with EtOAc (50 ml). The solution was washed with sat. aqueous sodium hydrogen carbonate (50 ml), 10% aqueous citric acid (50 ml) and brine (50 ml), dried (Na₂SO₄) and evaporated. The product was purified using a CombiFlash® companion eluting with 1-100% EtOAc in pentane followed by HPLC (system 1). Yield: 10 mg (4%)

LC-MS (Method 3): Rt = 7.80/8.22 min, m/z = 648.29 [M+H]⁺ Examples 18 and 19

Intermediate 4 (1.24 g, 2.50 mmol), sodium hydrogen carbonate (2.1 g,

25.00 mmol) and a mixture of Intermediates 18 and 19 (296 mg) in acetonitrile (50 ml) was heated at reflux for 2 h. The reaction mixture was evaporated and the resultant residue was dissolved in EtOAc (100 ml). The solution was washed with

1N HCI (100 ml) and brine (100 ml), dried (MgSO₄), and evaporated.

Chromatography using a CombiFlash® companion and eluting with 0-10% MeOH in DCM, followed by HPLC (system 1) gave the two products.

Example 18 yield: 110 mg

LC-MS (Method 3): Rt = 9.84 min, m/z = 526.16 [M+Hf

Example 19 yield: 6 mg

LC-MS (Method 3): Rt = 11.17 min, m/z = 911.35 [M+Hf

Example 20

Intermediate 21 (100 mg, 0.089 mmol) was dissolved in a mixture of DCM (5 ml) and TFA (5 ml). The solution was stirred at RT for 4V∑ h and the volatiles were evaporated. The residue was dissolved in DCM and Et₂O was added to precipitate the product. After filtering, the solid was purified by HPLC (system 1).

Yield: 33 mg (37%)

LC-MS (Method 3): Rt = 9.20 min, m/z = 1010.31 [M+H]⁺

Example 21

Intermediate 20 (400 mg, 0.448 mmol) was dissolved in acetonitrile (10 ml) and 1- bromobutane (614 mg, 4.48 mmol) and DIPEA (1.2 ml, 6.72 mmol) were added. The reaction was stirred at RT for 4 days before the solvent was evaporated. The residue was triturated with water (15 ml), the solid was filtered and washed with Et₂O. The crude material was chromatographed using a CombiFlash® companion, eluting with 0-15% MeOH in DCM to give a cream foam. Yield: 265 mg (62%)

LC-MS (Method 2): Rt = 2.79 min, m/z = 950 [M+Hf Example 22

Example 21 (260 mg, 0.274 mmol) and terf-butyl bromoacetate (534 mg, 2.74 mmol) were dissolved in acetonitrile (10 ml) and the reaction mixture was stirred at RT for 4 days. A further quantity of tert-butyl bromoacetate (534 mg, 2.74 mmol) was added and stirring was continued for 2 days. DIPEA (953 μl, 5.48 mmol) was then added. After 17 h the solvent was evaporated and the residue was dissolved in EtOAc (50 ml). The organic solution was dried (Na₂SO₄) and evaporated. Trituration with Et₂O gave 221 mg of an off-white powder which was treated with a 1 :1 mixture of DCM and TFA (10 ml). After stirring for 24 h at RT, the volatiles were evaporated. The addition of Et₂O followed by sonication gave a soild which was filtered off. The crude material was purified using HPLC (system 2). The pure fractions were combined and freeze-dried to give a fluffy white solid which was dissolved in EtOAc (50 ml). The solution was washed with sat. aqueous sodium hydrogen carbonate (50 ml), dried (Na₂SO₄) and evaporated. The residue was dissolved in acetonitrile/water and freeze-dried to give a white solid. Yield: 80 mg (29%)

LC-MS (Method 3): Rt = 9.17 min, m/z = 1008.15 [M+H]⁺ Example 23

Intermediate 4 (500 mg, 0.984 mmol), Λ/-(2-aminoethyl)-1,2-ethanediamine (51 mg, 0.492 mmol) and DIPEA (513 mg, 2.95 mmol) were dissolved in acetonitrile (10 ml) and the solution was heated at 40⁰C for 17 h. The solvent was evaporated and the residue was chromatographed on a CombiFlash® companion using 0-15% MeOH in DCM as eluent. 100 mg of this material was purified using HPLC (system 1) and the product was obtained as the formate salt. Yield: 80 mg (9%)

LC-MS (Method 3): Rt = 8.00 min, m/z = 866.11 [M+Hf Example 24

Intermediate 20 (400 mg, 0.448 mmol), te/t-butyl bromoacetate (262 mg, 1.34 mmol) and DIPEA (311 μl, 1.79 mmol) were dissolved in acetonitrile (50 ml) and the solution was stirred at 40°C for 24 h. The crude product was purified on a CombiFlash® companion using 0-15% MeOH in DCM as eluent. The tert-butyl ester was dissolved in DCM (5 ml) and TFA (5 ml) was added. After 4 h the volatiles were evaporated. The residue was triturated with Et₂O and the white solid obtained was purified by HPLC (system 3). Yield: 39 mg (9%)

LC-MS (Method 3): Rt = 8.73 min, m/z = 952.19 [M+H]⁺ Example 25

Example 19 (30 mg, 0.033 mmol) was dissolved in a 30% solution of bromomethane in acetonitrile (2 ml) and the solution was heated in the microwave at 60⁰C for 40 min. The volatiles were evaporated and the residue was triturated with Et₂O, then Et₂O/EtOAc (1:1, 2 ml). Purification using HPLC (system 1) gave an off-white solid. Yield: 6 mg (18%)

LC-MS (Method 3): Rt = 8.73 min, m/z = 952.19 [M+H]⁺ Example 26

Example 26 was prepared from Intermediate 23 and 0.5M NH₃ in dioxane using an analogous method to that used for Intermediate 24. Yield: 6% formate salt 15% free base LC-MS (Method 3): Rt = 8.17 min, m/z = 908.36 [M+H]⁺ Example 27

Example 26 (47 mg, 0.0518 mmol) was dissolved in acetonitrile (3 ml) and a 30% solution of bromomethane in acetonitrile (3 ml) was added. The solution was heated at 80°C in a sealed tube for 17 h. After cooling, the off-white solid which had precipitated was filtered off and purified by HPLC (system 2).

Yield: 18 mg (35%)

LC-MS (Method 3): Rt = 8.20 min, m/z = 922.38 [M]⁺

Example 28

Intermediate 25 (260 mg, 0.225 mmol) was dissolved in DCM (32 ml) and TFA (8 ml) was added. After 1 h the volatiles were evaporated. The residue was dissolved in DCM (8 ml) and the solution was washed with sat. aqueous sodium hydrogen carbonate (5 ml). The organic layer was separated and evaporated, and the crude material was purified by HPLC (system 2). The inner-salt was obtained as a white solid. Yield: 101 mg (44%) LC-MS (Method 3): Rt = 10.25 min, m/z = 1022.46 [M+H]⁺ Example 29

Intermediate 20 (100 mg, 0.11 mmol) was dissolved in DCM (20 ml) and succinic anhydride (33 mg, 0.33 mmol) and pyridine (80 mg, 1.0 mmol) were added. The reaction was stirred at RT for 17 h before evaporation of the volatile materials. Trituration with water yielded a white solid which was filtered and washed with water. Purification was achieved using HPLC (system 1). Yield: 40 mg (37%)

LC-MS (Method 4): Rt = 9.59 min, m/z = 994.33 [M+H]⁺ Example 30

Intermediate 26 (500 mg, 0.551 mg) and te/t-butyl bromoacetate (422 mg,

2.75 mmol) were dissolved in acetonitrile (10 ml) and the solution was stirred at

RT for 5 h. The solvent was evaporated and the crude material was purified by

HPLC (system 2).

Yield: 40 mg (7%)

LC-MS (Method 3): Rt = 8.23 min, m/z = 980.15 [M]⁺

Example 31

A solution of 4-aminomethyl-Λ/-methylbenzamide (454 mg, 2.77 mmol) was dissolved in THF (20 ml) and triethylamine (247 mg, 2.44 mmol) was added. With stirring at RT, Intermediate 4 (300 mg, 1.00 mmol) was added over 4 h and then stirring was continued for 3 days. The mixture was concentrated, water (30 ml) was added and the product was extracted with EtOAc (3 x 30 ml). The combined organics were dried (MgSO₄) and evaporated to give a pale yellow oil which was chromatographed on silica eluting with 0-5% MeOH in EtOAc. The product was isolated as a white solid. Yield: 235 mg (71%) LC-MS (Method 4): Rt = 8.50 min, m/z = 546 [M+Hf Example 32

Example 32 was prepared from Intermediate 4 and 4-aminomethyl-Λ/- methylbenzenesulfonamide using a method similar to that used in the synthesis of Example 31. Yield: 92%

LC-MS (Method 4): Rt = 9.27 min, m/z = 582 [M+H]⁺ Example 33

Intermediate 27 (1.3 g, 2.7 mmol) was dissolved in anhydrous THF (20 ml) and 4- aminoacetanilide (1.01 g, 6.75 mmol) was added. After the addition of triethylamine (0.75 ml), the reaction mixture was stirred at RT for 6 h. Evaporation of the solvent gave a light brown foam which was triturated with hexane to give 900 mg an orange-brown solid. The solid and HBTU (800 mg, 2.11 mmol) were dissolved in anhydrous DMA (25 ml). DIPEA (0.27 ml) was added and the reaction mixture was stirred at RT for 3.5 h. The reaction was treated with water (100 ml) and the pale brown solid which precipitated was filtered off, washed with water and dried. The crude product was chromatographed on silica gel eluting with EtOAc and pure material was obtained as an orange crystalline solid. Yield: 120 mg (8%)

LC-MS (Method 4): Rt = 8.88 min, m/z = 532.06 [M+H]⁺ Example 34

Intermediate 20 (450 mg, 0.504 mmol) and 4-(bromomethyl)pyridine (152 mg, 0.601 mmol) were dissolved in DCM (50 ml) and a solution of DIPEA (200 mg, 1.55 mmol) in DCM (10 ml) was added dropwise over 1 h. The reaction mixture was allowed to stand for 24 h at RT after which time silica was added and the solvent was evaporated. The solid was loaded onto a RediSep® cartridge and eluted with 0-10% MeOH in DCM using a CombiFlash® companion. The product was dissolved in MeOH and loaded onto an SCX-2 cartridge which had been conditioned with MeOH. After flushing briefly with MeOH, the product was eluted with 2M NH₃ in MeOH. After evaporation of the solvent, the residue was triturated with Et₂O to give a cream solid. Yield: 50 mg (10%)

LC-MS (Method 4): Rt = 7.84 min, m/z = 985.36 [M+Hf Example 35

A mixture of Intermediates 28 and 29 (25 mg) and 33% trimethylamine in EtOH (1 ml) were sealed in a reaction tube and left at RT for 17 h. The reaction was then heated at 50°C for 48 h. After evaporation of the solvent, the product was purified by HPLC (system 1). The pure fractions were combined and evaporated, and the solid was triturated with Et₂O to give an off-white solid. Yield: 5 mg

LC-MS (Method 4): Rt = 7.97 min, m/z = 1069.42 [M]⁺ Example 36

Intermediate 31 was converted into Example 36 using a method analogous to that used to convert Intermediate 25 into Example 28, but using EtOAc rather than DCM for the extraction. Yield: 55% LC-MS (Method 4): Rt = 7.79 min, m/z = 994.39 [M+H]⁺ Biological Results

The compounds of Examples 1 to 36 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 100 μM. The final concentration of DMSO was 1% in the assay buffer (0.05M Tris.HCl, 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 30m ins stopped by adding 50μl soybean trypsin inhibitor (Sigma T-9003) at a final concentration of 50 μg/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 15 μg/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 25000 nM to 1 nM. The results are means of two independent experiments, each performed in duplicate.

The compounds of Examples 1 to 36 were shown to have IC₅₀ values for HNE in the above assay of less than 100 nM. HNE induced lung haemorrhage in the rat Instillation of human neutrophil elastase (HNE) into rat lung causes acute lung damage. The extent of this injury can be assessed by measuring lung haemorrhage.

Male Sprague Dawley rats (175-220 g) were obtained from Harlan UK Ltd., full barrier-bred and certified free from specified micro-organisms on receipt. Animals were weighed and randomly assigned to treatment groups (7-12 animals per group).

The vehicle used was 1% DMSO/Saline. Inhibitors were dissolved in 1% DMSO before the addition of 0.9% saline.

Animals in each study used to determine the efficacy of the elastase inhibitors delivered locally to the lung by a variety of routes. Rats were anaesthetised with the inhaled anaesthetic lsoflurane (4%) when the dose was given from 30 minutes to 6h prior to human neutrophil elastase (HNE) administration or terminally anaesthetised with hypnorm:hypnovel:water (1.5:1 :2 at 2.7 ml/kg) when the predose was given at less than 30 minutes prior to HNE administration and dosed either intratracheal^ (i.t.) by transoral administration using a Penn Century microsprayer or intranasally (i.n.) by dropping the fluid on to the nares. Animals either received vehicle or compound at a dose volume of 0.5 ml/kg.

Animals that had been allowed to recover after dosing were terminally anaesthetised with hypnorm:hypnovel:water (1.5:1:2 at 2.7ml/kg). Once sufficiently anaesthetised, HNE (600 units/ml) or sterile saline was administered by transoral tracheal instillation at a volume of 100 μl using a Penn Century microsprayer. Animals were kept warm in a temperature controlled box and given top up doses of anaesthetic as required to ensure continuous anaesthesia until termination.

Animals were sacrificed (0.5 ml to 1 ml sodium pentobarbitone) one hour post HNE challenge. The trachea was exposed and a small incision made between two tracheal rings allowing a cannula (10 gauge, O.D. 2-10 mm, Portex Ltd.) to be inserted approximately 2 cm into the trachea towards the lung. This was secured into place with a cotton ligature. The lungs were then lavaged (BAL) three times with fresh 4 ml aliquots of heparinised (10 units/ml) phosphate buffered saline (PBS). The resultant BALF was kept on ice until it was centrifuged.

The BALF was centrifuged at 1000 r.p.m. for 10 minutes in a centrifuge cooled to between 4 and 10⁰C. The supernatant was discarded and the cell pellet resuspended in 1 ml 0.1% CETAB/PBS to lyse the cells. Cell lysates were frozen until spectrophotometric analysis for blood content could be made. Standards were prepared by making solutions of whole rat blood in 0.1% CETAB/PBS.

Once defrosted 100 μl of each lysed cell suspension was placed into a separate well of a 96 well flat bottomed plate. All samples were tested in duplicate and 100 μl 0.1% CETAB/PBS was included on the plate as a blank. The OD of the contents of each well was measured at 415 nm using a spectramax 250 (Molecular devices).

A standard curve was constructed by measuring the OD (at 415 nm) of different concentrations of blood in 0.1% CETAB/PBS (30, 10, 7, 3, 1 , 0.3, 0.1 μl/ml).

The amount of blood in each experimental sample was calculated by comparison to the standard curve. Data were then analysed as below: 1 ) The mean OD for duplicates was calculated 2) The value for the blank was subtracted from the value for all other samples

3) Data were assessed to evaluate the normality of distribution.

The compounds of Examples 2, 20, 22, 23, 24, 25, 27, 30 and 36 were tested in the above assay and were shown to be effective in reducing the quantity of blood haemorrhaged relative to control. For example, the compound of Example 25 showed a statistically significant reduction in haemorrhage of 56% relative to control when administered at 100 μg/kg i.t, 6 hours prior to HNE.

Claims

Claims:

1. A compound selected from the group consisting of those of formulae (1) to (36) wherein A- is a pharmaceutically acceptable anion, and salts, hydrates, solvates and N-oxides thereof:

2. A compound as claimed in claim 1 having any of formulae 1-18, or 31-33, which compound is predominantly in the form wherein the stereochemical orientation of the bond between the 4-cyanophenyl group and the fused pyrimidine ring is as shown in formula (X):

3. A compound as claimed in claim 1 having any of formulae 19-25, 29, 30 or 34-36 which compound is predominantly in the form wherein the stereochemical orientation of the bond between the left hand side 4-cyanophenyl group and fused pyrimidine ring is as shown in formula (X) in claim 2, and the stereochemical orientation of the bond between the right hand side 4-cyanophenyl group and fused pyrimidine ring is as shown in formula (Z):

4. A compound as claimed in claim 1 having any of formulae 26-28, which compound is predominantly in the form wherein the stereochemical orientation of the bond between the left hand side 4-cyanophenyl group and fused pyrimidine ring is as shown in formula (V), and the stereochemical orientation of the bond between the right hand side 4-cyanophenyl group and fused pyrimidine ring is as shown in formula (W):

5. A pharmaceutical composition comprising a compound as claimed in any of claims 1 to 4 and a pharmaceutically acceptable carrier or excipient.

6. A pharmaceutical composition as claimed in claim 5 which is adapted for delivery to the lungs by inhalation.

7. Use of a compound as claimed in any of claims 1 to 4, for the manufacture of a medicament for use in the treatment of prevention of a disease or condition in which HNE is implicated.

8. Use according to claim 7, 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.

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