WO2009060206A1

WO2009060206A1 - 3,4,6,7-tetrahydro-1h-pyrrolo[3,4-d]pyrimidine-2,5-diones and their therapeutic use

Info

Publication number: WO2009060206A1
Application number: PCT/GB2008/003755
Authority: WO
Inventors: Janusz Kulagowski; Christine Edwards
Original assignee: Argenta Discovery Limited
Priority date: 2007-11-07
Filing date: 2008-11-07
Publication date: 2009-05-14

Abstract

Compounds in which two substituted 3,4,6,7-tetrahydro-1 H-pyrrolo[3,4-d]pyrimidine-2,5- dione molecules are covalently linked via a linker radical which has contains a betaine or zwitterionic motif are inhibitors of human neutrophil elastase activity, for the treatment of respiratory diseases, especially by inhalation.

Description

3,4,6,7-TETRAHYDRO-i H-PYRROLO[3,4-D]PYRIMIDINE-2,5-DIONES AND THEIR

THERAPEUTIC USE

Field of the Invention This invention relates to substituted 3,4,6,7-tetrahydro-1 H-pyrrolo[3,4-d]pyrimidine-2,5- diones which are inhibitors of human neutrophil elastase activity, 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, 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)₁ 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 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-mediated inflammation, as supported by the increased number of neutrophils observed in bronchoalveolar leakage (BAL) fluids of COPD patients (Thompson, A. B.; Daughton, D.; etal. 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 (α_rAT), 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 ai, 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, CrC₆-alkyl, C₂-C₆-alkenyl, C₂-C₆-alkynyl, hydroxy or CrC₆-alkoxy or C₂-C₆-alkenyloxy,, wherein C₁-C₆- 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 CrC₄-alkoxy; R and R⁴ each independently represent a radical of formula -[X]_m-[Alk¹]_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 C₁-C₃ 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)C", -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^Bi 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)-(Ci -C₆ alkyl)-, and the other is hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, HO- (C₁-C₆ alkyl)-, HOCK=O)-(C₁-C₈ alkyl)- or R^AR^BN-(C_rC₆ alkyl)- wherein R^A and R^B are independently hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, or HOCt=O)-(C₁ -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. Such compounds are thus neutral, despite the presence of both a positively charged nitrogen and a negatively charged oxygen. The compounds are thus examples of betaines, which are 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 related to those of formula M-L-M¹ as discussed above which are inhibitors of HNE, and which are characterised by the presence in the molecule of a zwitterionic or betaine motif. Such 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, since the zwitterionic or betaine motif modulates the residence time in the lung before systemic absorbtion of the compound.

Detailed Description of the Invention In one embodiment, the invention provides a compound formula (IA) or (IB):

wherein

A is aryl or heteroaryl;

D is oxygen or sulphur; R¹, R², R³ and R⁵ are independently each hydrogen, halogen, nitro, cyano, C_rC₆-alkyl,

C₂-C₆-alkenyl, C₂-C₆-alkynyl, hydroxy or C_rC₆-alkoxy or C₂-C₆-alkenyloxy,, wherein C_rC₆- alkyl and GrCβ-alkoxy can be further substituted with one to three identical or different radicals selected from the group consisting of halogen, hydroxy and CrC₄-alkoxy;

R⁴ is hydrogen or an optional substituents; k, m, p and q are independently 0 or 1 ;

AIk¹ and AIk² each independently represent an optionally substituted Ci-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;

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

Q represents a divalent radical which contains an anionic-cationic pair selected from (i) a negatively charged nitrogen and positively charged nitrogen, (ii) a negatively charged oxygen and a positively charged nitrogen or (iii) a negatively charged nitrogen and a positively charged sulfur or (iv) a negatively charged oxygen and a positively charged 55

6 sulfur, PROVIDED THAT Q is not a radical -N⁺(R^P)(R^Q)- wherein one of R^p and R^Q is HOC(=O)-(Ci-C₆ alkyl)-, and the other is hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, HO- (C₁-C₆ alkyl)-, HOC(=O)-(d-C₆ alkyl)- or R^AR^BN-(C_rC₆ alkyl)- wherein R^A and R⁵ are independently hydrogen, C_rC₆ alkyl, or C₃-C₆ cycloalkyl, or HOC^O)-(C₁ -C₆ alkyl)-.

Compounds (IA) and (IB) are of course dinners or heterodimers of the form M-L-M¹ where the head groups M and M¹ are the same or different and L is a divalent linker radical -[X]m-[Alk¹]p-Q-[Alk²]_q-[X¹]k-- Hereafter, the term "dimer" will be used to include both homo and heterodimers compounds (IA( and (IB), linked by such a linker radical.

Compounds of the invention thereof may be prepared in the form of salts, particularly pharmaceutically acceptable salts, N-oxides, hydrates and solvates thereof. Any claim to a compound herein, or reference to "compounds of the invention", compounds with which the invention is concerned", compounds of formula (I), and the like includes salts, N- oxides, 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 and cystic fibrosis.

Hence other aspects of the invention are (i) a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier or excipient; and (ii) 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.

Terminology

As used herein, the term "(C_a-C_b)alkyP wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms. Thus when a is 1 and b is 6, for example, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term "(C_a-C_b)alkenyl" wherein a and b are integers refers to a straight or branched chain alkenyl moiety having from a to b carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. Thus when a is 2 and b is 6, for example, the term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2- methyl-2-propenyl.

As used herein the term "C₃-C_b alkynyl" wherein a and b are integers refers to straight chain or branched chain hydrocarbon groups having from a to b carbon atoms and having in addition one triple bond. Thus when a is 1 and b is 6, for example, the term includes for example, ethynyl (-C=CH), 1 -propynyl, 1 - and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.

As used herein the term "divalent (C_a-C_b)alkylene radical" wherein a and b are integers refers to a saturated hydrocarbon chain having from a to b carbon atoms and two unsatisfied valences.

As used herein the term "divalent (C_a-C_b)alkenylene radical" wherein a and b are integers refers to a divalent hydrocarbon chain having from 2 to 6 carbon atoms, and at least one double bond.

As used herein the unqualified term "carbocyclic" refers to a mono-, bi- or tricyclic radical having up to 16 ring atoms, all of which are carbon, and includes aryl and cycloalkyl.

As used herein the unqualified term "cycloalkyl" refers to a monocyclic saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the unqualified term "aryl" refers to a mono-, bi- or tri-cyclic carbocyclic aromatic radical, and includes radicals having two monocyclic carbocyclic aromatic rings which are directly linked by a covalent bond. Illustrative of such radicals are phenyl, biphenyl and napthyl.

As used herein the unqualified term "heteroaryl" refers to a mono-, bi- or tri-cyclic aromatic radical containing one or more heteroatoms selected from S, N and O, and includes radicals having two such monocyclic rings, or one such monocyclic ring and one monocyclic aryl ring, which are directly linked by a covalent bond. Illustrative of such radicals are thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl. As used herein the unqualified term "heterocyclyl" or "heterocyclic" or "heterocycloalkyl" includes "heteroaryl" as defined above, and in its non-aromatic meaning relates to a mono-, bi- ortri-cyclic non-aromatic radical containing one or more heteroatoms selected from S, N and O, and to groups consisting of a monocyclic non-aromatic radical containing one or more such heteroatoms which is covalently linked to another such radical or to a monocyclic carbocyclic radical. Illustrative of such radicals are pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido groups.

Unless otherwise specified in the context in which it occurs, the term "substituted" as applied to any moiety herein means substituted with up to four compatible substituents, each of which independently may be, for example, (Ci-C₆)alkyl, cycloalkyl, (C_rC₆)alkoxy, hydroxy, hydroxy(CrC₆)alkyl, mercapto, mercapto(CrC₆)alkyl, (CrC₆)alkylthio, phenyl, monocyclic heteroaryl having 5 or 6 ring atoms, halo (including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, -COOH, -COOR^A, -COR^A, -SO₂R^A, -CONH₂, -SO₂NH₂, -C0NHR^A, -SO₂NHR^A, -CONR^AR^B, -SO₂NR^AR^B, -NH₂, -NHR^A, -NR^AR^B, -OCONH₂, -OCONHR^A ,

-OCONR^AR^B, -NHCOR^A, -NHCOOR^A, -NR^BCOOR^A, -NHSO₂OR^A, -NR⁸SO₂OH,

-NR^BS0₂OR^A,-NHCONH₂, -NR^ACONH₂, -NHCONHR^B,-NR^ACONHR^B, -NHCONR^AR^B or

-NR^AC0NR^AR^B wherein R^A and R^B are independently a (Ci-C₆)alkyl, (C₃-C₆) cycloalkyl , phenyl or monocyclic heteroaryl having 5 or 6 ring atoms, or R^A and R^B when attached to the same nitrogen atom form a cyclic amino ring, such as piperidinyl, morpholinyl or piperazinyl. An "optional substituent" may be one of the foregoing substituent groups.

As used herein the term "salt" includes base addition, acid addition and quaternary salts. Compounds of the invention which are acidic can form salts, including pharmaceutically acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine and the like. Those compounds (I) which are basic can form salts, including pharmaceutically acceptable salts with inorganic acids, e.g. with hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the 2008/003755

like, and with organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p-toluenesulphonic, benzoic, benzenesunfonic, glutamic, lactic, and mandelic acids and the like. Those compounds (I) which have a basic nitrogen can also form quaternary ammonium salts with a pharmaceutically acceptable counter-ion such as chloride, bromide, acaetate, formate, p-toluenesulfonate, succinate, hemi-succinate, naphthalene-bis sulfonate, methanesuifonate, xinafoate, and the like. For a review on salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

Compounds of the invention which contain one or more actual or potential chiral centres, because of the presence of asymmetric carbon atoms, can exist as a number of diastereoisomers with R or S stereochemistry at each chiral centre. The invention includes all such diastereoisomers and mixtures thereof.

Individual compounds of the invention may exist in several polymorphic forms and may be obtained in different crystal habits. The compounds may also be administered in the form of prodrugs thereof.

So-called 'pro-drugs' of the compounds of formula (I) are also within the scope of the invention. Thus certain derivatives of the compounds which may be active in their own right or may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as

'prodrugs'. Further information on the use of prodrugs may be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and V.J. Stella) and

Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (ed. E. B. Roche,

American Pharmaceutical Association; CS. Larsen and J. østergaard, Design and application of prodrugs, In Textbook of Drug Design and Discovery, 3^rd Edition, 2002,

Taylor and Francis ).

Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985). Such examples could be a prodrug of a carboxyl group (such as -CO-O-CH₂-O-COtBu as used in the pivampicillin prodrug of ampicillin), an amide (-CO-NH-CH₂-NAIk₂) or an amidine ( -C(=N-O-CH₃)-NH₂).

Also included within the scope of the invention are metabolites of compounds of formula (I) that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites include

(i) where the contains a methyl group, an hydroxymethyl derivative thereof (-CH₃ -> - CH₂OH):

(ii) where the compound contains an alkoxy group, an hydroxy derivative thereof (-OR -> -OH);

(iii) where the compound contains a carboxylic acid ester group, a carboxylic acid derivative thereof (-C00R -> -COOH);

(iv) where the compound contains a tertiary amino group, a secondary amino derivative thereof (-NR¹R² -> -NHR¹ or -NHR²);

(v) where the compound contains a secondary amino group, a primary derivative thereof (-NHR¹ -> -NH₂);

(vi) where the compound contains a phenyl moiety, a phenol derivative thereof (-Ph - > -PhOH); and

(vii) where the compound contains an amide group, a carboxylic acid derivative thereof (-CONH₂ -> COOH).

Structural features of compounds of the invention

In the compounds of the invention of formula (I), in any compatible combination: The atom D may be O or S, but O is currently preferred.

The ring A is aryl or heteroaryl and may be any of those rings listed above as B2008/003755

11 examples of aryl or heteroaryl, especially phenyl and monocyclic heteroaryl having 5 or 6 ring atoms. Specific examples include pyridyl, such as 2- and 3- pyridyl, or pyrimidinyl such as pyrimidin-2-yl, but presently it is preferred that A be phenyl.

R¹ and R² may be selected from any of the substituent types for which they are defined in relation to formula (I), including hydrogen, halogen, nitro, cyano, C_rC₃- alkyl, C₂-C₃-alkenyl, C₂-C₃-alkynyl, hydroxyl or C_rC₃-alkoxy or C₂-C₃-alkenyloxy. Specific examples of such substitutuents include hydrogen, fluoro, chloro, bromo, cyano, methyl, methoxy and -C=CH. For example, -AR¹R² may be 4- cyanophenyl or 4-ethynylphenyl.

R³ and R⁵ too may be selected from any of the substituent types for which they are defined in relation to formula (I), but in one currently preferred type of compound of the invention R⁵ is hydrogen and R³ is 3-trifluoromethyl, 3-chloro or

3-bromo.

R⁴ as an optional substituents may vary widely, For example, R⁴ may be selected from Ci-C₆-alkyl, formyl, aminocarbonyl, mono- or di-CrC₄-alkylaminocarbonyl, C₃-C_a-cycloalkylcarbonyl, C_rC₆-alkylcarbonyl, CrCe-alkoxycarbonyl, N-(C₁-C₄- alkylsulfonyl)-aminocarbonyl, N-(Ci-C₄-alkylsulfonyl)-N-(C_rC₄-alkyl)- aminocarbonyl, heteroaryl, heterocycloalkyl, heteroarylcarbonyl or heterocycloalkylcarbonyl; wherein CrC₆-alkyl, mono- and di-C_rC₄- alkylaminocarbonyl, CrCβ-alkylcarbonyl, CrCβ-alkoxycarbonyl, heteroaryl and heterocycloalkyl can be substituted with one to three identical or different radicals selected from the group consisting of aryl, heteroaryl, hydroxyl, CrC₄-alkoxy, hydroxycarbonyl, CrCβ-alkoxycarbonyl, aminocarbonyl, mono and di-CrC₄- alkylaminocarbonyl, amino, mono- and di-Ci-C₄-alkylamino, CrC₄- alkylcarbonylamino, cyano, N-(mono- and di-Ci-C₄-alkylamino-C_rC₄-alkyl)- aminocarbonyl, N-(C₁-C₄-alkoxy-C₁-C₄-alkyl)-aminocarbonyl and halogen.

In particular subclasses of compounds of the invention, R⁴ is hydrogen.

The radical Q is defined as a divalent radical which contains an anionic-cationic pair selected from (i) a negatively charged nitrogen and positively charged nitrogen, (ii) a negatively charged oxygen and a positively charged nitrogen or (iii) a negatively charged nitrogen and a positively charged sulfur or (iv) a negatively charged oxygen and a positively charged sulfur, PROVIDED THAT Q is not a radical -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)- (C₁-C₆ alkyl)- or R^AR^BN-(C_rC₆ alkyl)- wherein R^A and R⁵ are independently hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, or HOC(=O)-(CrC₆ alkyl)-. The proviso in the above definition of Q has the effect of excluding compounds referred to in International patent application no. PCT/GB2007/001638 having a that particular betaine motif. There are many examples of the functional groups comprised in the types (i) to (iv) ion pairs above, and many ways of arranging the pairs structurally. Examples of particular cases of ion pairings and structural types include cases 1 -9 below.

Radical Q. Case 1 : In this case, Q represents a divalent radical of formula:

-S⁺(A-Q¹-(B)_S-Z¹)-, -(R^B)N⁺(A-Q¹-(B)_S-Z¹)- or X NΛ⁺ Tλ -WA-Q¹ -(B)₃-Z¹), x ^y wherein s is 0 or 1 ; ring L represents a mono- or bicyclic ring or ring system having 3 to 6 ring atoms in the case of a monocyclic ring and up to 10 ring atoms in the case of a bicyclic ring system, A and B each independently represent an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic link wherein R^A is hydrogen C₁-C₃ alkyl or aryl(Cr C₃ alkyl)-; Q¹ is an anionic divalent radical selected from (1) to (11):

R^B is hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl or aryl(C_rC₃ alkyl)-; and Z¹ is hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members

Radical Q, Case 2:

In this case, Q represents a divalent radical of formula:

wherein ring T represents a mono- or bicyclic ring or ring system having 3 to 6 ring atoms in the case of a monocyclic ring and up to 10 ring atoms in the case of a bicyclic ring system, and Q¹ is an anionic divalent radical selected from those of formulae (1) to (11) as defined in Case 1.

Radical Q, Case 3: In this case, Q represents a divalent radical of formula:

-S⁺(A-Zⁱ)-, -N+(A-Z¹)(R^B)- or N+ -HA-Z¹) 03755

14 wherein ring L is as defined in case 1 , A represents an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic link wherein R^A is hydrogen, C_rC₃ alkyl or aryl(CrC₃ alkyl)-; R^B is hydrogen, C₁-C₆ alkyl, 3PyI(C₁-C₃ alkyl)-, or C₃-C₆ cycloalkyl; and Z¹ is hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members, and wherein radical -A-Z¹ is substituted by an anionic group selected from those of formulae (12) to (20):

or by an anionic group selected from formulae (1) to (11) as defined in case 1 but wherein one unsatisfied valency is satisfied by a group -A-Z¹.

Radical Q, Case 3A:

In this case, Q represents a divalent radical of formula:

N⁺

wherein ring L represents a mono- or bicyclic ring or ring system having 3 to 6 ring atoms in the case of a monocyclic ring and up to 10 ring atoms in the case of a bicyclic ring system, and S¹ represents an anionic group selected from those of formulae (12) to (20):as defined in claim 5 or S¹ represents an anionic divalent radical selected from formulae (1 ) to (11 ) as defined in relation to case 1 , but wherein one unsatisfied valency is satisfied by hydrogen or a C₁-C₃ alkyl group. B2008/003755

15

Radical Q, Case 4:

In this case, Q represents a divalent radical-N(E-A-Q¹-B-Q²-(D)_s-Z¹)- or -(R^B)C(E-A-Q¹-B-Q²-(D)_S-Z¹)- wherein E is a bond, -C(=O)-, or-S(=0)₂-; s is 0 or 1 ; A, B and D independently represent an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted Ci-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic link wherein R^A is hydrogen, d-C₃ alkyl or aryl(d-C₃ alkyl)-; Z¹ is hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; one of Q¹ and Q₂ is an anionic divalent radical selected from those of formulae (1) to (11) as defined in case 1 , while the other of Q¹ and Q² is a divalent cationic radical selected from those of formulae (21) to (27):

wherein ring K is a 4- to 8-membered saturated ring which may contain 1 or 2 additional heteroatoms N, O or S; ring J is a 5- or 6-membered heteroaromatic rung; R¹ and R" are each hydrogen, or an optional substituent, or R' and R" taken together represent a divalent C₁-C₆ alkylene or C₂-C₆ akenylene radical in which up to three carbon atoms may be replaced by N O or S atoms.

Radical Q, Case 4A

In this case, Q represents a divalent radical a divalent cationic radical selected from those of formulae (20A) (21 A), (24A) and (25A):

wherein wherein ring K is a 4- to 8-membered saturated ring which may contain 1 or 2 additional heteroatoms N, O or S; ring J is a 5- or 6-membered heteroaromatic rung; R' is hydrogen or an optional substituents, and R'" is a group S¹ as defined in case 3A above or R'" is a group -A-Z¹ wherein A and Z¹ are as defined in case 3 above and -A-Z¹ is substituted by S¹.

Radical Q, Case 5:

In this case Q represents a divalent radical of formula -N(E-A-Q¹ -B-Z¹)- or -(R^B)C(E-A-Q¹-B-Z¹)-, wherein E is a bond, -C(=O)-, or-S(=0)₂-; A and B independently represent an optionally substituted divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic radical, or an optionally substituted CrC₆ alkylene, or C₂-Ce alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic link wherein R^A is hydrogen, CrC₃ alkyl or aryl(CrC₃ alkyl)-; Z¹ is hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; radical -B-Z¹ is substituted by Q²; Q¹ is selected from divalent anionic radicals (1 ) to (11 ) as defined in case 1 and Q² is selected from cationic radicals (12) to (20) as defined in case 3, OR Q¹ is selected from divalent cationic radicals (21 ) to (27) as defined in case 4 and Q² is selected from anionic groups of formulae (12) to (20) as defined in case 3 or anionic groups selected from formulae (1) to (11) as defined in case 1 but wherein one unsatisfied valency is satisfied by a group -A-Z¹.

In this Case 5, Q may represent, for example, a divalent radical of formula (II):

wherein Q₃ is an anionic radical O-C(=O)-(C_rC₃ alkyl)- or O-S(=O)₂-(Ci-C₃ alkyl)-. In formula (II) Q₃ may be an anionic radical O-C(=O)-CH₂- or ^"O-S(=O)₂-CH₂-. Radical Q. Case 6:

In this case, Q represents -(R^B)C(A-Z¹)- wherein A represents an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted Ci-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic link wherein R^A is hydrogen or CrC₃ alkyl; R^B is hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl; Z¹ is hydrogen, trifluoromethyl or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; and wherein radical -A-Z¹ is substituted by a group selected from:

(28) (29) wherein R¹ and R" are each hydrogen, C₁-C₃ alkyl or aryl(CrC₃ alkyl)-, or R' and R" taken together with the nitrogen to which they are attached form a 3- to 8-membered heterocyclic ring, and wherein R'" represents hydrogen or one or more optional substituents.

Radical Q, Case 7: In this case, Q represents a divalent radical of formula

-(Z²-(B¹)_r-Q²-A¹)C(A-Q¹-(B)_s-Z¹)- wherein r and s are independently 0 or 1 ; A, A¹ , B and B¹ each independently represent an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic link wherein R^A is hydrogen, C₁-C₃ alkyl or aryl(CrC₃ alkyl)-; Z¹ and Z² independently represent hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; one of Q¹ and Q² is an anionic divalent radical selected from those of formulae (1) to (11) as defined in Case 1 while the other of Q¹ and Q² is a cationic divalent radical selected from those of formulae (21) to (27) as defined in case 4. Radical Q. case 8:

In this case, Q represents a divalent radical of formula -(Z²-A¹)C(A-Q¹ -(B)₃-Z¹)- wherein r and s are independently 0 or 1 ; A, A¹ , and B each independently represent an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted Ci-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic link wherein R^A is hydrogen, C₁-Ce alkyl or aryl(Cr C₃ alkyl)-; Z¹ and Z² independently represent hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; radical Z²-A¹- is substituted by Q²; and radical -B-Z¹ is substituted by Q²; Q¹ is selected from divalent anionic radicals (1) to (11) as defined in case 1 and Q² is selected from cationic groups (12) to (20) as defined in case 3, OR Q¹ is selected from divalent cationic radicals (21) to (27) as defined in case 4 and Q² is selected from anionic groups of formulae (12) to (20) as defined in case 3 or groups of formulae (1 ) to (11 ) as defined in case 1 but wherein one unsatisfied valency is satisfied by a group -A-Z¹.

Radical Q, Case 9;

In this case, Q represents a divalent radical selected from:

wherein R' is hydrogen, Ci-C₃ alkyl or aryl(C_rC₃ alkyl)-.

Specific examples of anionic and cationic radicals, and pairings thereof, which may be present in the radical Q include those present in the compounds of the Examples herein.

Dimers of the invention are those of formula M-L-M¹, wherein L is the divalent linker radical-[X]_m-[Alk¹ ]_p-[Q]_n-[Alk²]_q-[X¹ ]_k- and M and M¹ are each independently a radical of formula (IA) or (IB) above. Particular dimers of the invention include those of formula (X):

wherein m, p, q, k, X, X¹ , AIk¹ and AIk² are as defined and discussed above, and Q is as defined in any of cases 1 -9 above. In such compounds, m and k may each be 0, p and q may each be 1 , and AIk¹ and AIk² may each be -(CH₂W-

Specific examples of dimeric compounds of the invention include those of the Examples herein.

As mentioned above, and as is commonly expected in medicinal chemistry, the compounds of the invention may be administered in appropriate cases as prodrugs. One important class of prodrugs relevant to the present compounds is the class of esters of compounds (I) (including dimers thereof as discussed herein) which have a carboxylate, sulfonate or phosphonate group in the radical Q. Ester prodrugs are well known, and include C₁-C₆ alkyl esters.

Utilities

The therapeutic utility of the compounds of the invention 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 present invention is also concerned with pharmaceutical formulations comprising, as an active ingredient, a compound of the invention. Other compounds may be combined with compounds of this invention for the prevention and treatment of inflammatory diseases of the lung. Thus the present invention is also concerned with pharmaceutical compositions for preventing and treating inflammatory diseases of the lung comprising a therapeutically effective amount of a compound of the invention and one or more other therapeutic agents. T/GB2008/003755

20

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, and will generally be detrmined by clinical trial as required in the pharmaceutical art. 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 B2008/003755

21 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 1μg 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 non-steroidal antiinflammatory 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 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).

Methods of Synthesis

The compounds of the present invention can be prepared according to the procedures of the following general schemes, using appropriate materials, and are further exemplified by the following specific examples. Moreover, by using the procedures described with the disclosure contained herein, one of ordinary skill in the art can readily prepare additional compounds of the present invention claimed herein. The compounds illustrated in the examples are not, however, to be construed as forming the only genus that is considered as the invention. The examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds.

The compounds of the invention may be isolated in the form of their pharmaceutically acceptable salts, such as those described previously herein above. The free acid or base form corresponding to isolated salts can be generated by treatment with a suitable base or acid such as NaOH, potassium carbonate, acetic acid and hydrochloric acid and extraction of the liberated free acid or base into an organic solvent followed by evaporation. The free form isolated in this manner can be further converted into another pharmaceutically acceptable salt by dissolution in an organic solvent followed by addition of the appropriate acid or base and subsequent evaporation, precipitation, or crystallisation.

It may be necessary to protect reactive functional groups (e.g. hydroxyl, amino, thio or carboxy) in intermediates used in the preparation of compounds of the invention to avoid their unwanted participation in a reaction leading to the formation of the compounds. Conventional protecting groups, for example those described by T. W. Greene and P. G. M. Wuts in "Protective groups in organic chemistry" John Wiley and Sons, 1999, may be used.

Compounds of the invention may be prepared according to the routes illustrated in Schemes 1 and 2 where the linker component used in the dimerisation step contains a betaine or protected betaine, or a group (or groups) which can serve as a precursor to the desired betaine.

Step i) is optional R' = H if step i) is omitted

H₂N-linker-NH₂ NaHCO₃, CH₃CN

L^inker ∞ntains a betaine o^r group(s) which can be converted to the desired

betaine

Scheme 1

Br e.g. CO₂H be N

Bifunctional linker

brominatioπ bromination

R¹NH₂, NaHCO₃ R¹NH₂, NaHCO₃ CH₃CN CH₃CN

Scheme 2

In general synthetic Schemes 1 and 2, the Linker part may be one which already contains the ionic groups forming the desired betaine ion pair. However, as indicated, in some cases it may be more convenient if the linker part contains one of the ionic groups of the desired betaine, and also a reactive group onto which a radical containing the other ionic group of the desired betaine is subsequently be introduced. In other cases, the linker part may contain neither of the ionic groups of the desired betaine, but has reactive sites onto which radicals containing the ionic groups of the desired betaine may subsequently be introduced.

The following Examples illustrate 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 prepacked polypropylene column containing unbonded activated silica with irregular particles with average size of 50 μm and nominal 60.A 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. '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.

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 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 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/mm %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/mm %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

LC-MS method 5

Finnigan AQA 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

MS ionisation method - Electrospray (positive ion)

Abbreviations used in the experimental section:

DCE = 1 ,1-dichloroethane DCM = dichloromethane DMAP = Λ/,Λ/-dimethylaminopyridine DMF = Λ/,Λ/-dimethylformamide

HATU = 0-(7-Azabenzotriazole-1-yl)-/V,/V,/V,A/'-tetramethyluronium hexafluorophosphate HPLC = high performance liquid chromatography mCPBA = mefa-chloroperbenzoic acid

NBS = Λ/-bromosuccinimide

RT = room temperature

Rt = retention time

THF = tetrahydrofuran

Intermediates 1 and 2

Intermediates 1 and 2 were prepared according to WO06082412.

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+Hf

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

To a solution of Intermediate 4 (0.4 g, 0.787 mmol) in acetonitrile (10 ml) were added DlPEA (408 μl, 2.35 mmol) and /V-(3-aminopropyl)-1 ,3-propanediamine (50 mg, 0.392 mmol). The reaction mixture was stirred at 4O⁰C 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 7

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 /V,Λ/-bis(3- aminqpropyl)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 8

To a solution of 5-methylpyrazine-2-carboxylic acid (750 mg, 5.40 mmol) in fert-butanol (25 ml) was added di-tert-butyl dicarbonate (1.4 g, 6.5 mmol) and the reaction mixture was allowed to stir at 55⁰C for 15 min. DMAP (197 mg, 1.6 mmol) was added and stirring was continued at 55⁰C for 17 h. The solvent was evaporated and the crude product was purified using an Isolute® Si Il cartridge and 2% EtOAc in cyclohexane as eluent. Yield: 900 mg (86%)

1 H NMR (400 MHz, CDCI₃) δ = 1.42 (s, 9H), 2.65 (s, 3H), 8.55 (s, 1 H)₁ 9.1 (s, 1 H) ppm

Intermediate 9

Intermediate 8 (900 mg, 4.6 mmol) was dissolved in DCE (50 ml) and NBS (900 mg, 5.1 mmol) and benzoyl peroxide (111 mg, 0.46 mmol) were added. The reaction mixture was illuminated with a 140W lamp for 24 h. The mixture was diluted with DCM (200 ml) and the solution was washed with sat. NaHCO₃(aq) (100 ml), water (50 ml), dried (Na₂SO₄) and evaporated. The crude material was purified using an Isolute® Si Il cartridge, eluting with 20% EtOAc in cyclohexane to give the product as a colourless oil. Yield: 800 mg, (64%) 1 H NMR (400 MHz, CDCI₃) δ = 1.65 (s, 9H), 4.6 (s, 2H), 8.8 (s, 1 H), 9.15 (s, 1 H) ppm

Intermediate 10

tert-Butyl 4-bromomethylbenzoate (500 mg, 1.84 mmol) and Intermediate 7 (272 mg, 0.30 mmol) were dissolved in acetonitrile (5 ml). The solution was heated under N₂ at 9O⁰C for

17 h. The solvent was removed and the residue was triturated with Et₂O to give a pale yellow solid.

Yield: 300 mg (85%)

LC-MS (Method 2): Rt = 2.95/2.99 min, m/z = 1098 [M]⁺

The following intermediates were prepared in an analogous manner from Intermediate 7 and an alkylating agent:

Intermediate 14

4-Fluorobenzenesulphonamide (1.75 g, 10 mmol) was suspended in toluene (25 ml) and bromoacetyl bromide (3.03 g, 15 mmol) was added. The mixture was stirred at 110⁰C for 3 h and then left at RT for 17 h. The product was filtered from the cooled mixture and obtained as a white crystalline solid. Yield: 1.1 g (37%) 1 H NMR (400 MHz, CDCI₃) δ = 3.85 (s, 2H), 7.25 (m, 2H), 8.1 (m, 2H), 8.8 (br s, 1 H), ppm

The following intermediates were prepared using a similar method:

Intermediate 17

Intermediate 6 (400 mg, 0.448 mmol), tert-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 4O⁰C for 24 h. The crude product was purified on a CombiFlash® companion using 0-15% MeOH in DCM as eluent.

Yield: 200 mg (44%)

LC-MS (Method 2): Rt = 2.84 min, m/z = 1008 [M+H]⁺

The following intermediates were prepared in an analogous manner from Intermediate 6 and an alkylating agent:

Intermediate 21

Intermediate 17 (200 mg, 0.199 mmol) and benzyl bromide (170 mg, 0.992 mmol) were dissolved in acetonitrile (5 ml) and the reaction mixture was stirred at RT for 3 days. A further amount of benzyl bromide (170 mg, 0.992 mmol) in acetonitrile (1 ml) was added and, after 24 h, the solvent was evaporated. The residue was taken up into a minimum amount of DCM and the product was precipitated by the addition of Et₂O.

Yield: 200 mg (86%)

LC-MS (Method 1): Rt = 2.98 min, m/z = 1098 [M]⁺

Intermediate 22

Intermediate 17 (150 mg, 0.15 mmol), 4-chlorobenzyl bromide (205 mg, 1.00 mmol) and sodium hydrogen carbonate (84 mg, 1.00 mmol) in acetonitrile (10 ml) were stirred at

8O⁰C for 5 h. The reaction was treated with water (20 ml) and the volatiles were evaporated. The product was extracted into EtOAc (20 ml) and the organic layer was washed with water (10 ml), dried (Na₂SO₄) and evaporated. The oily residue was treated with Et₂O and the pale yellow solid which formed was collected by filtration.

Yield: 110 mg (60%)

LC-MS (Method 2): Rt = 3.79 min, m/z = 1132 [M]⁺

Intermediate 23

Intermediate 17 (150 mg, 0.148 mmol) and 4-bromomethylbenzene sulfonamide (249 mg, 0.992 mmol) were dissolved in acetonitrile (10 ml). To the clear solution, NaHCO₃

(100 mg, 1.19 mmol) was added and the reaction mixture was stirred at 50 ⁰C for 48 h.

The solvent was evaporated in vacuo. The residue was poured into a beaker containing

200 ml of water; trituration gave a white solid which was used without purification for the next step.

Yield: 60 mg (34%)

LC-MS (Method 2): Rt = 3.37 min, m/z = 1177 [M]⁺

The following intermediate was prepared using a similar method:

Intermediates 25

Intermediate 19 (160 mg, 0.151 mmol) and tert-butyl bromoacetate (600 mg, 3.09 mmol) were dissolved in acetonitrile (15 ml). To the clear solution, NaHCO₃(500 mg, 5.95 mmol) was added and the reaction mixture was stirred at 40°C for 7 days. The volatiles were removed in vacuo. The reaction mixture was poured into water (100 ml) and extracted with ethyl acetate (2 x 100 ml). The organic layer was dried (Na₂SO₄) and the solvent was evaporated to afford the product as white solid. Yield = 150 mg LC-MS (Method 1): Rt = 3.12 min, m/z = 1174 [M]⁺

The following intermediates were prepared using a similar method: For intermediate 27 the alkylating agent was methyl bromide rather than tert-butyl bromoacetate.

Intermediate 28

Intermediate 6 (150 mg, 0.168 mmol), 1 -(tert-butoxycarbonyl^-piperidinecarboxylic acid

(46 mg, 0.202 mmol), DIPEA (87 mg, 0.672 mmol) and HATU (77 mg, 0.202 mmol) were dissolved in DMF (4 ml) and the solution was allowed to stand at RT for 3 days. The solvent was evaporated and the residue was dissolved in EtOAc (20 ml). The solution was washed with sat. NaHCO₃(aq) (20 ml), dried (Na₂SO₄) and evaporated to give a glassy solid.

Yield: 177 mg (95%)

LC-MS (Method 2): Rt = 3.77 min, m/z = 1105 [M+H]⁺

Intermediate 29

Intermediate 28 (177 mg, 0.160 mmol) was dissolved in a mixture of DCM (16 ml) and TFA (4 ml). After 1 h the volatiles were evaporated and the residue was dissolved in MeOH (10 ml). The solution was loaded onto an Isolute® SCX-2 cartridge (5 g) which had been conditioned with MeOH. After flushing briefly with MeOH, the product was eluted with 2M methanolic NH₃. Evaporation gave a pale cream glassy solid. Yield: quantitative LC-MS (Method 1 ): Rt = 2.69/2.74 min, m/z = 1005 [M+H]⁺ Intermediate 30

A solution of Intermediate 29 (203 mg, 0.202 mmol), tert-butyl bromoacetate (39 mg, 0.202 mmol) and DIPEA (52 mg, 0.404 mmol) in THF (4 ml) was heated in a sealed tube at 50⁰C. After 4 h the solution was allowed to stand at RT for 17 h and then a further equivalent of tert-butyl bromoacetate was added. Heating at 5O⁰C was resumed. After 4 h the solvent was evaporated and the residue was dissolved in MeOH (10 ml). The solution was loaded onto an Isolute® SCX-2 cartridge (5 g) which had been conditioned with MeOH. The cartridge was flushed with MeOH and then the product was eluted with 2M methanolic NH₃. Evaporation gave an amber glassy solid. Yield: 221 mg (98%) LC-MS (Method 1 ): Rt = 2.91 min, m/z = 1119 [M+H]⁺

Intermediate 31

Intermediate 30 (216 mg, 0.193 mmol) was dissolved in a 30% solution of bromomethane in acetonitrile (10 ml). Sodium hydrogen carbonate (16 mg, 0.193 mmol) and water (17 mg, 0.965 mmol) were added, and the reaction mixture was stirred at RT for 24 h. The mixture was filtered and evaporated to give a glassy solid.

Yield: quantitative

LC-MS (Method 1): Rt = 2.89/2.92 min, m/z = 1133 [M]⁺ 03755

40

Intermediate 32

To a solution of diethylenetriamine (0.41 g, 3.94 mmol) and triethylamine (1.59 g, 15.75 mmol) in THF (10 ml) was added a solution of Intermediate 4 (2.0 g, 3.94 mmol) in THF (30 ml). The reaction mixture was stirred at RT for 19 h and the solvent was evaporated. The residue was partitioned between water and EtOAc. The organic layer was separated, washed with water and brine, dried (Na₂SO₄) and evaporated. The crude product was purified on an isolute Si Il cartridge (25 g) eluting with DCM, 20% EtOAc in DCM and then 10% MeOH in DCM. The desired product was obtained as a cream solid. Yield: 1.0 g (59%) LC-MS (Method 4): Rt = 7.82 min. m/z = 866 [M+H]⁺

Intermediate 33

A mixture of isonicotinic (1.5 g, 12.2 mmol) and 1 ,3-propanesultone (9.0 g, 73.8 mmol) in anhydrous DMF (30 mi) was stirred at 6O⁰C for 24 h. After cooling to RT, the solid was filtered and washed with DMF. The damp solid was suspended in Et₂O, filtered, washed with ether and dried under vacuum to give the required product as a white solid. Yield: 2.94 g (98%) LC-MS (Method 1 ): Rt = 0.40 min. m/z = 246 [M+H]⁺

Intermediate 34

Intermediate 34 was prepared from nicotinic acid using a similar procedure to that described for Intermediate 33. Yield: quantitative LC-MS (Method 1): Rt = 0.41 min. m/z = 246 [M+H]⁺ Intermediate 35

tert-Butyl-4-bromobutyrate (1.0 g, 4.5 mmol) was added to a stirred suspension of isonicotinic acid (0.25 g, 2.0 mmol) in DMF (2 ml) under argon and heated at 80⁰C for 18 h. The cooled mixture was diluted with Et₂O and the supernatant liquid decanted away from the resultant gummy solid. This was triturated using Et₂O then dissolved in acetonitrile/water and freeze dried to give an off-white solid.

Yield: 0.6 g (86%)

LC-MS (Method 5): Rt = 0.44, m/z = 210 [M+H-'Buf

Intermediate 36

Intermediate 6 (100 mg, 0.11 mmol), Intermediate 35 (60 mg, 0.17 mmol), DIPEA (60μl, 0.34 mmol) and HATU (70 mg, 0.18 mmol) were dissolved in DMF (0.5 ml) and the solution stirred at RT for 20 h. The solvent was evaporated and the residue purified using an Isolute® Al-N cartridge eluted with a 0-60% MeOH in DCM gradient to give the desired product. Yield: 31 mg (23%)

LC-MS (Method 5): Rt = 3.36 min, m/z = 1085 [M+H-'Buf Intermediate 37

Intermediate 37 was prepared from Intermediates 32 and 35 using a procedure analogous to that employed in the synthesis of Intermediate 36.

Yield: 41 mg (31%)

LC-MS (Method 5): Rt = 3.36 min, m/z = 1057 [M+H-'Bu]⁺

Intermediate 38

A solution of Intermediate 6 (0.25 g, 0.28 mmol) in acetonitrile (20 ml) was treated with sodium hydrogen carbonate (0.05 g, 0.60 mmol) and bromomethylcyclopropane (0.20 g, 1.5 mmol). The reaction mixture was stirred at 50⁰C for 7 days, then filtered and evaporated to dryness. The crude product was dissolved in MeOH (5 ml) and loaded onto an Isolute® SXC-2 cartridge (25g) which had been conditioned with MeOH. The cartridge was flushed with MeOH then the product was eluted with 2M methanolic NH₃. Evaporation gave a glassy solid which was triturated with Et₂O. Yield: 0.19 g (71%) LC-MS (Method 1): Rt = 2.72 min, m/z = 948 [M+H]⁺ Intermediate 39

To a solution of Intermediate 38 (0.15 g, 0.158 mmol) in acetonitrile (10 ml) was added sodium hydrogen carbonate (0.013 g, 0.158 mmol), DIPEA (0.102 g, 0.79 mmol) and allyl 4-bromomethylbenzoate (WO9741098) (0.06 g, 0.238 mmol). The reaction mixture was stirred under nitrogen for 3 days at 55⁰C and then filtered, evaporated to dryness and triturated with Et₂O. The crude product was purified using a Combif lash® companion (12 g cartridge), eluting with 0-20% MeOH in DCM. The product was triturated with Et₂O to give an orange solid. Yield: 0.1O g (52%) LC-MS (Method 2): Rt = 2.91 min, m/z = 1122 [M]⁺

Intermediate 40

^"SO₃H To a solution of 4-(bromomethyl)benzenesulphonyl chloride (1.078 g, 4.00 mmol) in 1 ,4- dioxane (30 ml) was added aqueous HCI (10%, 30 ml) and the reaction mixture was allowed to stir at 4O⁰C for 4 h. The solvent was removed and the residue was purified by crystallization from chloroform to give a white solid. Yield: 1.17 g (48%) 1 H NMR (400 MHz, DMSO-Cf₆) δ = 7.61 (d, 2H), 7.40 (d, 2H), 4.76 (s, 1 H) ppm.

Intermediate 41

To a solution of ethyl 1-aza-bicyclo[2.2.2]octane-4-carboxylate (PCT2005104745) (9.610 g, 52.44 mmol) in THF (480 ml) at -78⁰C was added borane-THF complex (73.41 ml,

73.41 mmol) and the resulting solution was stirred at this temperature for 3.5 h. The reaction was quenched with slow addition of H₂O (50 ml), warmed to RT, and allowed to stir for 45 min. The solution was diluted with EtOAc (100 ml), washed with brine (15 ml), extracted with EtOAc (3 x 50 ml), dried (Na₂SO₄) and evaporated. The crude product was purified on a CombiFlash® companion using 0-50% EtOAc in cyclohexane as eluent to give a bright yellow solid.

Yield: 7.236 g (70%)

1 H NMR (400 MHz, CDCI₃) δ = 4.15 (q, 2H), 3.07 (t, 4H)₁ 1.96 (t, 4H), 1.26 (t, 3H) ppm.

Intermediate 42

A solution of Intermediate 41 (1.00 g, 5.07 mmol) in THF (45 ml) was treated with 1 N LiOH (7.61 ml, 7.61 mmol) and the resulting mixture was allowed to stir at RT for 18 h. The reaction was neutralised with 1 N HCI (7.6 ml), washed with EtOAc (2 x 20 ml), dried (Na₂SO₄) and evaporated. The white residue was suspended in a 1 :4 mixture of Et₂O/cyclohexane and filtered off to give the acid as a white solid. Yield: 696 mg (81%)

1 H NMR (400 MHz, CDCI₃) δ = 3.11 (t, 4H), 1.98 (t, 4H), ppm.

Intermediate 43

Intermediate 6 (200 mg, 0.224 mmol), Intermediate 42 (76 mg, 0.448 mmol), HATU (213 mg, 0.560 mmol) and DIPEA (0.160 ml, 0.896 mmol) in DMF (3.2 ml) were stirred at RT for 4 h. The solvent was removed and the residue was dissolved in EtOAc (10 ml), washed with a 10% citric acid solution (10 ml), dried (Na₂SO₄) and evaporated. Using HPLC (system 1 ), the pure betaine was obtained as a white solid. Yield: 152 mg (65%)

LC-MS (method 1 ): Rt = 3.63 min, m/z = 1045.32 [M+H]⁺ Intermediate 44

To a solution of Intermediate 43 (100 mg, 0.096 mmol) in anhydrous acetone (2 ml) at 0⁰C was added dropwise a 1.25 M HCI solution in MeOH (0.540 ml, 0.672 mmol) and the solution was allowed to stir at this temperature for 1 h. The reaction mixture was quenched by addition of sat. NaHCO₃(aq) (10 ml) at O⁰C, warmed to RT, diluted with EtOAc (15 ml), washed with sat. NaHCO₃(aq) (20 ml) dried (Na₂SO₄) and evaporated. The oily residue was dissolved in the minimum amount of MeOH, treated with Et₂O and the pale yellow solid which formed was collected by filtration. Yield: quantitative LC-MS (method 2): Rt = 2.72 min, m/z = 1031.19 [M+H]⁺

Intermediate 45

Intermediate 45 was prepared from Intermediate 4 using a method similar to that used in the preparation of Intermediate 7. Yield: (68%) LC-MS (method 1): Rt = 2.77 min, m/z = 880 [M+H]⁺ Intermediate 46

Intermediate 46 was prepared from intermediate 45 and tert-butyl bromoacetate using a similar procedure to that described for the synthesis of Intermediate 10.

Yield: (65%)

LC-MS (method 2): Rt = 2.86 min, m/z = 994 [M]⁺

Intermediate 47

Intermediate 47 was prepared from Intermediate 45 using a similar procedure to that described for the synthesis of Intermediate 10, except that the reaction was carried out at

RT.

Yield: (77%)

LC-MS (method 2): Rt = 2.95 min, m/z = 1070 [M]⁺

Intermediate 48

A solution of Intermediate 2 (3.00 g, 6.99 mmol) in DMF (60 ml) was cooled to -10⁰C under nitrogen. Sodium hydride (60%, 280 mg, 6.99 mmol) was added portion wise and, after 15 min, benzyl bromide (1.79 g, 10.49 mmol) was added. The reaction mixture was allowed to warm from -10⁰C to O⁰C over 1 h before the addition of sat. NH₄CI(aq) (40 ml). The mixture was extracted with EtOAc and the extract was washed with water and brine, dried (Na₂SO₄) and evaporated. Purification was achieved using an Isolute® Si Il cartridge (20 g) eluting with a stepwise gradient of 0-10-20-30% EtOAc in pentane. The product was obtained as a white foam. Yield: 2.99 g (82%) LC-MS (Method 2): Rt = 4.22 min, m/z = 520 [M+H]⁺

Intermediate 49

Intermediate 49 was prepared from Intermediate 48 using a method similar to that used in the preparation of Intermediate 4.

Yield: quantitative

LC-MS (Method 2): Rt = 4.31 min, m/z = 598/600 [M+H]⁺

Intermediate 50

Intermediate 50 was prepared from Intermediate 49 according to a method similar to that described for Intermediate 7.

Yield: (56%)

LC-MS (Method 2): Rt = 3.29 min, m/z = 1088 [M+H]⁺ Intermediate 51

Intermediate 51 was prepared from Intermediate 50 and tert-butyl bromoacetate according to a method similar to that described for Intermediate 10.

Yield: (93%)

LC-MS (Method 1): Rt = 3.38 min, m/z = 1202 [M]⁺

lntermediate 52

Intermediate 52 was prepared from Intermediate 50 and 4-allyl 4-bromomethyl-benzoate (WO9741098) according to a method similar to that described for Intermediate 10. Yield: (94%) LC-MS (Method 1): Rt = 3.42 min, m/z = 1262 [M]⁺

Intermediate 53

To a solution of Intermediate 5 (4.26 g, 8.38 mmol) and bis(3-aminopropyl)benzylamine (0.926 g, 4.19 mmol) (J. Med. Chem., 2006, 49, 1291-1312) in THF (70 ml) was added triethylamine (3.39 g, 33.5 mmol) at RT with stirring. The reaction mixture was stirred at 45⁰C under argon for 20 h. After cooling to RT, the solvent was evaporated and the residue partitioned between water and ethyl acetate. The organic layer was separated, washed with water, dried (Na₂SO₄) and evaporated to give a yellow glass. The crude mixture was separated using a CombiFlash® companion eluting with 0-10% MeOH in DCM. The desired product was obtained as a cream solid. Yield: 3.12 g (75%) LC-MS (Method 4): Rt = 8.35 min. m/z = 984 [M+H]⁺

Intermediate 54

Intermediate 53 (120 mg, 0.122 mmol) and allyl 4-bromomethylbenzoate (WO9741098) (404 mg, 1.59 mmol) were dissolved in acetonitrile (10 ml) and NaHCO₃ (10 mg, 0.122 mmol) was added. The reaction mixture was heated at 80⁰C for 24 h. The reaction mixture was filtered and evaporated, and the product was purified using a Combiflash® companion (12 g cartridge) eluting with 0-20% MeOH in DCM. The solid product was triturated with Et₂O to give a cream solid. Yield: 240 g (79%) LC-MS (Method 2): Rt = 3.03 min, m/z = 1158 [M]⁺

Intermediate 5 (5.0 g, 9.84 mmol) and ethylenediamine (5.9 g, 98.4 mmol) were dissolved in THF and the solution was allowed to stand at RT for 17 h. The solvent was evaporated and the residue was partitioned between EtOAc and sat. NaHCO₃(aq). The organic layer was separated and the aqueous was extracted further. The combined organics were washed with water and brine, dried (Na₂SO₄) and evaporated to give a pale yellow foam. Yield: 4.26 g (98%) LC-MS (Method 1): Rt = 2.02/2.15 min, m/z = 442 [M+H]⁺

Intermediate 56

A solution of Intermediate 55 (1.5 g, 3.40 mmol), pyridine-3,5-dicarboxylic acid (284 mg, 1.70 mmol), DIPEA (3 ml) and HATU (1.55 g, 4.08 mmol) in DMF (40 ml) was allowed to stand at RT. After 1 h the DMF was evaporated and the residue was partitioned between

EtOAc and sat. NaHCO₃(aq). The organic layer was separated and the aqueous was extracted further. The combined organics were washed with water and brine, dried

(Na₂SO₄) and evaporated. The crude material was purified using an Isolute® Si Il cartridge (25 g) eluting with a stepwise gradient of 0-10-15% MeOH in EtOAc. The product was obtained as a pale pink solid.

Yield: 711 mg (41%)

LC-MS (Method 1): Rt = 3.25 min, m/z = 1014 [M+H]⁺

A portion (100 mg) of the above material was further purified using HPLC (system 2) Recovery: 60 mg (60%)

LC-MS (Method 4): Rt = 9.38 min, m/z = 1014.49 [M+H]⁺

Intermediate 57

Intermediate 6 (10.47 g, 11.73 mmol) was dissolved in acetonitrile (500 ml) and tert-butyl bromoacetate (10.0 g, 44.84 mmol) and DIPEA (9.08 mg, 70.38 mmol) were added. The reaction mixture was stirred at RT for 14 days and then filtered. Evaporation of the solvent gave crude product which was purified using a Combiflash® companion (120 g cartridge) eluting with 0-10% MeOH in DCM. The solid product was triturated with Et₂O to give a white solid. Yield: 8.79 g (72%) LC-MS (Method 2): Rt = 2.93 min, m/z = 1036 [M+H]⁺

Intermediate 58

Intermediate 57 (130 mg, 0.125 mmol) was dissolved in a 30% solution of bromomethane in acetonitrile (5 ml). Three drops of water and NaHCO₃ (11 mg, 0.125 mmol) were added and the reaction was stirred at RT for 24 h. The reaction mixture was filtered and the volatiles were evaporated to give a yellow gummy solid.

Yield: quantitative

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

Intermediate 59

Pyridine-2,5-dicarboxylic acid (1.00 g, 5.99 mmol) was suspended in allyl alcohol (10 ml) and cone. H₂SO₄ (0.4 ml) was added. The reaction was heated at 85⁰C for 24 h. 4A Molecular sieves were then introduced into the reaction vessel and heating was continued for a further 24 h. The mixture was allowed to cool and sat. NaHCO₃(aq) (50 ml) and DCM (80 ml) were added. The organic layer was separated, dried (hydrophobic frit) and evaporated. The product was isolated as a yellow oil after purification on an Isolute® Si Il cartridge (10 g) eluting with DCM. Yield: 908 mg (61%) LC-MS (Method 2): Rt = 3.28 min, m/z = 248 [M+H]⁺

Intermediate 60

Intermediate 59 (908 mg, 3.68 mmol) was dissolved in 1 ,4-dioxane (50 ml) and the solution was cooled in ice. To the mixture was added 1 N NaOH(aq) (0.4 ml) and a small amount of additional 1 ,4-dioxane was added to aid stirring. After 1 h the reaction mixture was treated with 1 N HCI(aq) (100 ml) and the product was extracted into EtOAc. The organic extracts were washed with water and brine, dried (Na₂SO₄) and evaporated. The product was purified on an Isolute® Si Il cartridge (10 g) eluting with a stepwise gradient of 0-5-10-15% MeOH in EtOAc. The product was a white solid. Yield: 200 mg (26%) LC-MS (Method 2): Rt = 2.52 min, m/z = 208 [M+H]⁺

Intermediate 61

Intermediate 61 was prepared from Intermediate 6 and Intermediate 60 using a method similar to that used in the synthesis of Intermediate 36.

Yield: (52%)

LC-MS (Method 1): Rt = 3.67 min, m/z = 1083 [M+H]⁺

Intermediate 62

Intermediate 61 (130 mg, 0.12 mmol) was dissolved in a 20% solution of bromomethane

(10 ml) and the solution was stirred at 5O⁰C for 7 days. The volatiles were evaporated.

The product was obtained as a glassy solid.

Yield: quantitative

LC-MS (Method 2): Rt = 2.83 min, m/z = 1097 [M]⁺

Intermediate 63

Intermediate 63 was prepared from Intermediate 5 and N-(2-aminoethyl)-1 ,3- propanediamine using a method similar to that used in the preparation of Intermediate 7.

The product was purified on an lsolute® Si Il cartridge eiuting with DCM then 5% MeOH in DCM and isolated as a white solid.

Yield: (56%) LC-MS (Method 2): Rt = 2.75 min, m/z = 880 [M+H]⁺

Intermediate 64

Intermediate 64 was prepared from Intermediate 4 and 2-[5-(2-aminoethyl)pyridin-3- yljethylamine using a method similar to that used in the preparation of Intermediate 7. The product was purified on an isolute® Si Il cartridge eluting with 0-15% MeOH in DCM. Yield: (80%) LC-MS (Method 1): Rt = 2.92 min, m/z = 928 [M+Hf

Intermediate 65

Intermediate 5 (6.00 g, 11.81 mmol), glycine tert-butyl ester hydrochloride (2.37 g, 14.17 mmol) and triethylamine (6.5 ml) in THF (100 ml) were heated at 60⁰C for 17 h. After cooling, the mixture was filtered and the filtrate evaporated. The crude mixture was separated using a CombiFlash® companion (80 g cartridge) eluting with 50-100% EtOAc in pentane. The desired product was obtained as an orange glass. Yield: 2.99 g (49%) LC-MS (Method 2): Rt = 3.62 min, m/z = 513 [M+H]⁺

Intermediate 66

Intermediate 65 (2.99 g, 5.84 mmol) was dissolved in a mixture of TFA (40 ml) and DCM (120 ml). After 5.5 h the volatiles were evaporated and the residue was triturated with Et₂O to give a cream solid. Yield: 2.33 g (87%) LC-MS (Method 1): Rt = 2.95 min, m/z = 457 [M+H]⁺

A solution of Intermediate 66 (510 mg, 1.19 mmol), diethylenetriamine (57 mg, 0.559 mmol), DIPEA (1 ml) and HATU (543 g, 1.43 mmol) in DMF (10 ml) was allowed to stand at RT. After 2 h the DMF was evaporated and the residue was partitioned between

EtOAc and sat. NaHCO₃(aq). The organic layer was separated, washed with water and brine, dried (Na₂SO₄) and evaporated. The crude material was purified using an Isolute®

Si Il cartridge (10 g) eluting with 20 then 40% MeOH in EtOAc to give the product as a white solid.

Yield: 280 mg (48%)

LC-MS (Method 1): Rt = 2.60/2.63 min, m/z = 980 [M+H]⁺

Intermediate 10 (300 mg, 0.26 mmol) was treated with a mixture of DCM (20 ml) and TFA

(5 ml). The solution was stirred at RT for 3 h before evaporation of the volatile materials.

The residue was triturated with sat. NaHCO₃(aq), water and then EtOAc. Using HPLC

(system 2), the pure product was obtained as a white solid.

Yield: 100 mg (37%) LC-MS (Method 3): Rt = 8.20 min, m/z = 1042.21 [M+H]⁺

The following examples were prepared using similar methodology:

Λ/-(2-Bromoacetyl)methanesulphonamicie (290 mg, 1.34 mmol) (Helvetica Chimica Acta, 2005, 88(3), 588-603) and Intermediate 7 (272 mg, 0.30 mmol) were dissolved in acetonitrile (10 ml). Sodium hydrogen carbonate (500 mg, 5.95 mmol) was added and the reaction was heated at 8O⁰C for 7 h. After filtering, the solvent was evaporated to give a pale yellow solid which was purified using HPLC (system 2). Yield: 100 mg (32%)

LC-MS (Method 3): Rt = 8.95 min, m/z = 1043.12 [M+H]

The following examples were prepared in a similar manner from Intermediate 7 and an alkylating agent:

Example 17

To a solution of Intermediate 7 (471 mg, 0.520 mmol) and 2-bromo-Λ/-hydroxyacetamide (400 mg, 2.50 mmol) in acetonitrile (30 ml) was added DIPEA (677 μl, 3.90 mmol). After stirring at RT for 2 h, the solvent was evaporated to give a pale yellow oil which was purified using HPLC (system 1). The formate salt obtained was dissolved in EtOAc (20 ml) and the solution was washed with sat. NaHCO₃(aq) (20 ml), dried (Na₂SO₄) and evaporated. The residue was dissolved in acetonitrile/water and freeze-dried. Further purification was achieved using HPLC (system 2). Yield 100 mg (18%) LC-MS (Method 4): Rt = 7.72 min, m/z = 981.32 [M+H]⁺

Example 18

A solution of Intermediate 6 (200 mg, 0.224 mmol), isonicotinic acid Λ/-oxide (34 mg, 0.246 mmol), DIPEA (154 μl, 0.896 mmol), and HATU (93 mg, 0.246 mmol) in DMF (5 ml) was allowed to stand at RT for 2 h before evaporation of the solvent. The residue was partitioned between EtOAc (50 ml) and sat. NaHCO₃(aq) (50 ml), with a small amount of MeOH added to aid solubility. The organic layer was separated, dried (Na₂SO₄) and evaporated. The resulting pale yellow solid was purified by HPLC (system 2) and the pure product was obtained as a white solid. Yield: 159 mg (70%) LC-MS (Method 4): Rt = 9.29 min, m/z = 1015.29 [M+H]⁺ Example 19

Intermediate 31 (219 mg, 0.193 mmol) was treated with a mixture of DCM (18 ml) and TFA (8 ml). After 17 h the volatiles were evaporated and the residue was dissolved in acetonitrile/water. The solution was basified by the addition of solid sodium hydrogen carbonate and then filtered. Purification using HPLC (system 2) gave a pale cream solid. Yield: 133 mg (64%) LC-MS (Method 4): Rt = 8.62 min, m/z = 1077.37 [M+H]⁺

Example 20

A solution of Intermediate 7 (100 mg, 0.110 mmol) in DCM (1 ml) was cooled in an ice bath and a solution of 70-75% mCPBA (50 mg, 0.20-0.22 mmol) in DCM (1 ml) was added. After stirring at O⁰C for 3 h, the mixture was diluted with DCM (4 ml) and the organic solution was washed with sat. NaHCO₃(aq) (4 ml). On the sides of the reaction vessel an oily residue had been deposited and this was dissolved in acetonitrile (8 ml) and added to the DCM solution. The solvents were evaporated and the product was purified using HPLC (system 1). The formate salt was obtained as a white solid. Yield: 40 mg (39%) LC-MS (Method 3): Rt = 8.09 min, m/z = 924.31 [M+H]⁺ Example 21

Intermediate 6 (300 mg, 0.336 mmol), Intermediate 33 (164 mg, 0.672 mmol), HATU (320 mg, 0.840 mmol) and DIPEA (0.240 ml, 1.344 mmol) were dissolved in DMF (5 ml) and the solution was allowed to stand at RT for 4 h. The solvent was evaporated and the residue was dissolved in EtOAc (15ml). The solution was washed with a 10% citric acid solution (10 ml), dried (Na₂SO₄) and evaporated. The residue was then dissolved in EtOAc (10 ml), washed with sat. NaHCO₃(aq) (2 x 10 ml), dried (Na₂SO₄) and evaporated to give a pale yellow solid. Yield : 162 mg (43%) LC-MS (method 4): Rt = 8.68 min, m/z = 1121.40 [M+Hf

To a suspension of Intermediate 32 (626 mg, 0,724 mmol) and 33 (355 mg, 1.45 mmol) in anhydrous DMF (8 ml) was added DIPEA (390 mg, 3.03 mmol) followed by HATU (688 mg, 1.81 mmol) at RT with stirring. The solution was stirred under nitrogen at RT for 20 h and then concentrated to half its volume under reduced pressure. The residue was added dropwise to sat. NaHCO₃(aq) (100 ml) with rapid stirring. The solid was collected by filtration, washed with water (60 ml) and dried under vacuum over P₂O₅ at 45°C to give a beige solid. Yield: 540 mg (68%) LCMS (Method 4): 95% purity, Rt = 8.78 min. m/z = 1093.31 [M+Hf The above product (200 mg) was crystallised from MeOH (3.5 ml) to give a white crystalline solid. Yield: 125 mg (63%) LCMS (Method 4): 97.5% purity, Rt = 8.76 min. m/z = 1093.31 [M+H]⁺

Example 23

Example 23 was prepared from Intermediates 32 and 34 using a procedure similarto that described for Example 22.

Yield: 290 mg (76%)

LCMS (Method 4): 94.5% purity, Rt = 8.71 min. m/z = 1093.47 [M+Hf

The above product (60 mg) was purified using HPLC (system 2).

Recovery: 46.9 mg, (78%) LCMS (Method 4): >99% purity, Rt = 8.70 min. m/z = 1093.47 [M+H]⁺

Example 24

Example 24 was prepared from Intermediates 6 and 34 using a similar procedure to that employed in the synthesis of Example 22. Yield: 308 mg (82%) LCMS (Method 4): 95% purity, Rt = 8.73 min. m/z = 1121.38 [M+H]⁺ The above product (250 mg) was purified using HPLC (system 2).

Recovery: 185 mg, (60%)

LCMS (Method 4): 99% purity, Rt = 8.73 min. m/z = 1121.38 [M+H]⁺

Example 25

Intermediate 6 (100 mg, 0.11 mmol), 3,4-bis-bromomethylbenzoic acid (J. Org. Chem.,

1995, 60{11 ), 3307-3310) ( 34 mg, 0.11 mmol), DIPEA (60 μl, 0.34 mmol) and acetonitrile

(3 ml) were sealed in a reaction tube under argon and stirred at RT for 18 h. The reaction mixture was then heated at 40⁰C for a further 18 h. Water (2 ml) was added and the product was isolated using HPLC (system 2). The pure product was obtained as a white solid.

Yield: 25 mg (22%)

LC-MS (Method 4): Rt = 8.06 min, m/z = 1040.31 [M+H]⁺

Example 26

Intermediate 36 (31 mg, 25 μmol) was treated with a mixture of DCM (1.5 ml) and TFA

(0.5 ml). The solution was stirred at RT for 18 h before evaporation of the volatile materials. The residue was purified using HPLC (system 2) and the pure product was obtained as a white solid.

Yield: 22 mg (81%)

LC-MS (Method 4): Rt = 8.66 min, m/z = 1085.54 [M+H]⁺

Example 27 was prepared from Intermediate 37 using a procedure analogous to that used for Example 26.

Yield: 24 mg (66%)

LC-MS (Method 4): Rt = 8.63 min, m/z = 1057.45 [MH-H]⁺

Example 28

Intermediate 39 (100 mg, 0.0833 mmol) was dissolved in THF (2 ml) and morpholine (72 mg, 0.83 mmol) and tetrakis(triphenyiphosphine)palladium(0) (10 mg) were added. The reaction mixture was stirred for 1 h at 2O⁰C before filtering and evaporating to dryness. The crude product was triturated with a 1 :1 mixture of Et₂O and EtOAc. The product was dissolved in MeOH (2 ml) and loaded into an Isolute® SXC-2 cartridge (10 g) which had been conditioned with MeOH. The cartridge was flushed with 2M methanolic NH₃. This ammonia solution was evaporated to dryness, re-dissolved in MeOH and washed with 1 M HCI before drying (MgSO₄) and evaporation of the solvent. The product was obtained as a white solid. Yield: 2 mg (2.2%) LC-MS (Method 3): Rt = 8.24 min, m/z = 1082.22 [M+Hf Example 29

Intermediate 7 (500 mg, 0.550 mmol), Intermediate 40 (552 mg, 2.200 mmol) and DIPEA

(0.670 ml, 3.850 mmol) were dissolved in acetonitrile (11 ml). The solution was heated at

5O⁰C for 2 days and the solvent was removed. The residue was dissolved in EtOAc (10 ml), washed with a 10% citric acid solution (10 ml), dried (Na₂SO₄) and evaporated.

Using HPLC (system 1 ), the pure sulphonic acid was obtained as a white solid. The acid was then dissolved in acetonitrile (10 ml) and stirred for 12 h with sodium hydrogen carbonate (100 mg). The solid was filtered off and thoroughly rinsed with acetonitrile and

MeOH. The filtrate was concentrated and the residue was triturated with Et₂O to give a white solid.

Yield : 119 mg (21%)

LC-MS (Method 4): Rt = 8.82 min, m/z = 1078.21 [M+H]⁺

Example 30

Intermediate 44 (99.0 mg, 0.096 mmol) and terf-butyl bromoacetate (37 mg, 0.192 mmol) were dissolved in DMF (5 ml) and the solution was stirred at 6O⁰C for 4 h. The solvent was removed. The residue was dissolved in the minimum of MeOH and precipitated with Et₂O. The pale yellow solid was filtered, rinsed with Et₂O and then treated with a 4M solution of HCI in dioxane (3 ml). The solution was stirred at RT for 12 h and the solvent was removed. Using HPLC (system 1), the pure betaine was obtained as a white solid. Yield: 50 mg (48%) LC-MS (Method 4): Rt = 8.66 min, m/z = 1089.26 [M+H]⁺ Example 31

A solution of intermediate 7 (100 mg, 0.11 mmol) and 1 ,3-propanesultone (268 mg, 2.20 mmol) in DMF (2.5 ml) was heated at 120°C for 30 min under microwave irradiation. The solvent was evaporated and the crude product was dissolved in MeOH and loaded onto an Isolute® SCX-2 cartridge (5 g) which had been conditioned with MeOH. The product was eluted with MeOH and the use of HPLC (system 1) gave a creamy solid. Yield: 33.5 mg (29%) LC-MS (Method 4): Rt = 8.62 min, m/z = 1030.32 [M+H]⁺

Example 32 was prepared from Intermediate 46 using a similar procedure to that described for the synthesis of Example 1.

Yield: (65%)

LC-MS (Method 4): Rt = 8.62 min, m/z = 938.29 [M+H]⁺ Example 33

Example 33 was prepared from Intermediate 47 using a similar procedure to that described for the synthesis of Example 1.

Yield: (60%)

LC-MS (Method 4): Rt = 7.97 min, m/z = 1014.35 [M+H]⁺

Example 34

Intermediate 51 (216 mg, 0.168 mmol) was dissolved in a 20% solution of TFA in DCM (20 ml) and the solution was allowed to stand at RT for 24 h. The volatiles were evaporated and the residue was dissolved in acetonitrile (10 ml). An excess of sodium hydrogen carbonate was added and the mixture was filtered. After evaporation of the solvent, the product was purified by HPLC (system 2) and obtained as a white solid. Yield: 110 mg (57%)

LC-MS (Method 4): Rt = 11.81 min, m/z = 1146.24 [M+H]⁺ Example 35

Intermediate 52 (226 mg, 0.168 mmol), morpholine (29 mg, 0.336 mmol) and tetrakis(triphenylphosphine)palladiurn(0) (19 mg, 0.0168 mmol) were dissolved in DCM (5 ml). After standing at RT for 24 h, the solution was diluted with DCM, washed with 1 N

HCI(aq), sat. NaHCO₃(aq), water and brine, dried (Na₂SO₄), and evaporated. The residue was purified using HPLC (system 2) to give a white solid.

Yield: 92 mg (45%)

LC-MS (Method 4): Rt = 10.29 min, m/z = 1222.28 [M+H]⁺

Example 36

Intermediate 54 (510 mg, 0.44 mmol) and morpholine (77 mg, 0.88 mmol) were dissolved in a mixture of DCM (10 ml) and THF (3 ml). Tetrakis(triphenylphosphine)-palladium(0) (51 mg, 0.044 mmol) was added and the reaction was stirred at RT for 5 h. The mixture was filtered, washed with DCM and dried. The mixture was partially purified using HPLC (system 2) giving pure fractions and fractions which contained a mixture of the desired product and starting material. The latter were combined and loaded on an Isolute® SCX- 2 cartridge (20 g) which had been conditioned with MeOH. The cartridge was flushed with MeOH and the product was eluted with 2M methanolic NH₃. The solvent was evaporated to give a white solid. Yield: 98 mg (20%)

The pure fractions gave further amounts of the desired product, also as a white solid. Yield: 70 mg (14%) LC-MS (Method 4): Rt = 8.48 min, m/z = 1118.23 [M+H]⁺

Example 37

A solution of Intermediate 56 (350 mg, 0.346 mmol) and 1 ,3-propanesultone (343 mg, 2.81 mmol) in DMF (4 ml) was heated at 60⁰C for 1.5 h then at 100⁰C for 10 min. The solution was allowed to cool and then poured into water (40 ml) with stirring. The pale cream solid which precipitated was filtered and dried. The solid was dissolved in DMF (4 ml) and a further portion of 1 ,3-propanesultone (200 mg, 1.64 mmol) was added. The solution was heated at 8O⁰C for 5 h. After cooling, the solution was poured into water 940 ml) with rapid stirring. The precipitated solid was filtered and dried in vacuo. A portion of the solid (250 mg) was purified using HPLC (system 2). Yield: 36 mg LC-MS (Method 4): Rt = 8.71 min, m/z = 1136.49 [M+H]⁺

Example 38

Example 38 was prepared from intermediate 58 by a method similar to that described for the preparation of Example 34.

Yield: (55%)

LC-MS (Method 4): Rt = 7.79 min, m/z = 994.39 [M+Hf Example 39

Intermediate 4 (788 mg, 1.60 mmol), tris(3-aminopropyl)amine (300 mg, 1.60 mmol) and sodium hydrogen carbonate (670 mg, 7.98 mmol) in acetonitrile (20 ml) were stirred at RT for 4 h. Di-tert-Butyl dicarbonate (1.04 g, 4.79 mmol) was added and stirring was continued for a further 1 h. The solvent was evaporated and the residue was partitioned between EtOAc and sat. NaHCO₃(aq). The organic layer was separated and washed with water and brine, dried (Na₂SO₄) and evaporated. The crude material (600 mg) was dissolved in acetonitrile (5 ml). tert-Butyl bromoacetate (1.2 g, 6.15 mmol) was added and the resulting solution was heated at 6O⁰C for 17 h. The solvent was evaporated and the residue was triturated with 1 :1 Et₂O/pentane. The resulting pale yellow solid was dissolved in 20% TFA in DCM (25 ml) and the solution was allowed to stand at RT for 17 h. The volatiles were evaporated and the residue was dissolved in acetonitrile. An excess of sodium hydrogen carbonate was added and the mixture was filtered and evaporated. The product was purified using HPLC (system 2) to give the desired product as a white solid.

Yield: 50 mg (6%) LC-MS (Method 4): Rt = 7.33 min, m/z = 1009.26 [M+H]⁺

Example 40

Intermediate 62 (141 mg, 0.12 mmol) and tetrakis(triphenylphosphine)palladium(0) (7 mg, 0.0061 mmol) were dissolved in THF (4 ml) and sodium toluenesulphinate (22 mg, 0.12 mmol) was added. The mixture was sonicated until the mixture was homogeneous and then left at RT for 48 h. Further portions of tetrakis(triphenylphosphine)palladium(0) and sodium toluenesulphinate were added and, after sonication, the reaction mixture was stirred at RT for 2 weeks. A further two portions of the reagents were added and, after 24 h, the reaction was complete. The mixture was filtered and evaporated, and the product was purified using HPLC (system 2). The product was obtained as a white solid. Yield: 63 mg (50%) LC-MS (Method 4): Rt = 8.81 min, m/z = 1057.64 [M+Hf

Example 41

Intermediate 63 (480 mg, 0.546 mmol), Intermediate 33 (147 mg, 0.601 mmol), HATU (228 mg, 0.601 mmol) and DIPEA (4.7 ml) were dissolved in DMF (10 ml) and the solution was allowed to stand at RT for 2 h. The mixture was partitioned between EtOAc and sat NaHCO₃(aq). The organic layer was separated, washed with brine, dried (Na₂SO₄) and evaporated. The crude was purified on an Isolute® Si Il cartridge (10 g) eluting with 5-14% MeOH in EtOAc to give a pale yellow solid. Yield: 406 mg (67%) LC-MS (Method 4): Rt = 8.85 min, m/z = 1107.38 [M+H]⁺

Intermediate 64 (96 mg, 0.104 mmol) and 1 ,3-propanesultone (63 mg, 0.518 mmol) were dissolved in DMF (3 ml) and the solution was heated at 8O⁰C for 17 h. A further portion of 1 ,3-propanesultone (200 mg, 1.64 mmol) was added and heating was continued for 4 h. The DMF was evaporated and the crude was purified using HPLC (system 2) and the desired product was thereby obtained as a white solid. Yield: 45 mg (41%) LC-MS (Method 4): Rt = 8.73 min, m/z = 1050.38 [M+H]⁺

Example 43

Example 43 was prepared from Intermediates 67 and 33 using a method similar to that used in the synthesis of Example 22. Purification was achieved using HPLC (system 2) and the product was obtained as a white solid.

Yield: (30%)

LC-MS (Method 4): Rt = 8.39 min, m/z = 1207.58 [M+H]⁺

Biological Assays Compounds of the invention were tested for their HNE inhibitory activity.

Fluorescent peptide substrate

Assays were performed in 96-well plates at a total assay volume of 100μl. 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 10OμM. 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 50μl 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. All compounds of the Examples had activities in the range 1-100 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 (175-22Og) 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 2.7ml/kg) when the predose was given at less than

30 minutes prior to HNE administration and dosed either intratracheally (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

0.5ml/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 (600units/ml) or sterile saline was administered by transoral tracheal instillation at a volume of 100μl 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 (0.5ml to 1 ml sodium pentobarbitone) one h post HNE challenge. The trachea was exposed and a small incision made between two tracheal rings allowing a cannula (10gauge, O.D. 2-10mm, Portex Ltd.) to be inserted approximately 2cm 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 4ml aliquots of heparinised (1 Ounits/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 10Oμl 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 100μl 0.1% CETAB/PBS was included on the plate as a blank. The OD of the contents of each well was measured at 415nm using a spectramax 250 (Molecular devices).

A standard curve was constructed by measuring the OD (at 415nm) 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.

Comounds of the Examples 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 11 showed a statistically significant reduction in haemorrhage of 72% relative to control when administered at 30μg/kg i.t, 3 hs prior to HNE.

Claims

1. A compound of formula (IA) or (IB):

AX^{) l} _N [X]_m-[Alkn

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-C₆-alkenyloxy,, wherein C₁-C₆- alkyl and C_rC₆-alkoxy can be further substituted with one to three identical or different radicals selected from the group consisting of halogen, hydroxy and CrC₄-alkoxy;

R⁴ is hydrogen, CrCe-alkyl, formyl, aminocarbonyl, mono- or di-C_rC₄- alkylaminocarbonyl, C₃-C₈-cycloalkylcarbonyl, CrCe-alkylcarbonyl, C_rC₆-alkoxycarbonyl, N-(Ci-C₄-alkylsulfonyl)-aminocarbonyl, N-(C₁ -C₄-alkylsulfonyl)-N-(C_rC₄-alkyl)- aminocarbonyl, heteroaryl, heterocycloalkyl, heteroarylcarbonyl or heterocycloalkylcarbonyl; wherein C_rC₆-alkyl, mono- and di-CrC^alkylaminocarbonyl, CrCe-alkylcarbonyl, C_rC₆-alkoxycarbonyl, heteroaryl and heterocycloalkyl can be substituted with one to three identical or different radicals selected from the group consisting of aryl, heteroaryl, hydroxyl, C_rC₄-alkoxy, hydroxycarbonyl, C₁-C₆- alkoxycarbonyl, aminocarbonyl, mono and di-CrC^alkylaminocarbonyl, amino, mono- and di-C_rC₄-alkylamino, C_rC₄-alkylcarbonylamino, cyano, N-(mono- and di-C_rC₄- alkylamino-C₁-C₄-alkyl)-aminocarbonyl_> N-(CrC4-alkoxy-C₁-C₄-alkyl)-anninocarbonylancl halogen. or an optional substituents; k, m, p and q are independently 0 or 1 ; AIk¹ and AIk² each independently represent an optionally substituted C_rC₆ 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;

X represents -(C=O)-, -S(O₂)-, -C(=O)O-, -C=O)NR^A-, or -S(O₂)NR^A-, wherein R^A is hydrogen, Ci-C₆ alkyl, or C₃-C₆ cycloalkyl; X¹ represents -O-, -S-, or -NH; and

Q represents a divalent radical which contains an anionic-cationic pair selected from (i) a negatively charged nitrogen and positively charged nitrogen, (ii) a negatively charged oxygen and a positively charged nitrogen or (iii) a negatively charged nitrogen and a positively charged sulfur or (iv) a negatively charged oxygen and a positively charged sulfur, PROVIDED THAT Q is not a radical -N⁺(R^P)(R^Q)- wherein one of R^p and R^Q is HOC^O)-(C₁ -C₆ alkyl)-, and the other is hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, HO- (C₁-C₆ alkyl)-, HOCf=O)-(C₁-C₆ alkyl)- or R^AR^BN-(C_rC₆ alkyl)- wherein R^A and R⁵ are independently hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl, or HOC(SO)-(C₁ -C₆ alkyl)-.

2. A compound as claimed in claim 1 wherein R⁴ is hydrogen.

3. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical of formula:

-S⁺(A-Q¹-(B)_S-Z¹)-, -(R^B)N⁺(A-Q¹-(B) -Z¹)- or

wherein s is O or 1 ; ring L represents a mono- or bicyclic ring or ring system having 3 to 6 ring atoms in the case of a monocyclic ring and up to 10 ring atoms in the case of a bicyclic ring system, A and B each independently represent an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene or C₂-C₆ alkenylene radical which may optionally contain an ether (-0-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic link wherein R^A is hydrogen C₁ -C₃ alkyl or aryl^ - C₃ alkyl)-; Q¹ is an anionic divalent radical selected from (1) to (11):

R^B is hydrogen, C₁-C₆ aikyl, C₃-C₆ cycloalkyl or aryl(C_rC₃ alkyl)-; and Z¹ is hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members

4. A compound as claimed in claim 3 wherein Q represents a divalent radical of formula:

wherein ring T represents a mono- or bicyclic ring or ring system having 3 to 6 ring atoms in the case of a monocyclic ring and up to 10 ring atoms in the case of a bicyclic ring system, and Q¹ is an anionic divalent radical selected from those of formulae (1 ) to (11 ) as defined in claim 3.

5. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical of formula: -S⁺(A-Z¹)-, -N⁺(A-Z¹ )(R^B)- or

wherein ring L is as defined in claim 3, A represents an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted CrC₆ alkylene or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic link wherein R^A is hydrogen, CrC₃ alkyl or aryl(CrC₃ alkyl)-; R^B is hydrogen, Ci-C₆ alkyl, aryl(C_rC₃ alkyl)-, or C₃-C₆ cycloalkyl; and Z¹ is hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members, and wherein radical -A-Z¹ is substituted by an anionic group selected from those of formulae (12) to (20):

or by an anionic divalent radical selected from formulae (1 ) to (11 ) as defined in claim 3 but wherein one unsatisfied valency is satisfied by a group -A-Z¹.

6. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical of formula:

wherein ring L represents a mono- or bicyclic ring or ring system having 3 to 6 ring atoms in the case of a monocyclic ring and up to 10 ring atoms in the case of a bicyclic ring system, and S¹ represents an anionic group selected from those of formulae (12) to (20):as defined in claim 5 or S¹ represents an anionic divalent radical selected from formulae (1 ) to (11 ) as defined in claim 3 but wherein one unsatisfied valency is satisfied by hydrogen or a C₁-C₃ alkyl group.

7. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical -N(E-A-Q¹-B-Q²-(D)_S-Z¹)- or -(R^B)C(E-A-Q¹-B-Q²-(D)_S-Z¹)- wherein E is a bond, -C(=O)-, or -S(=O)₂-; s is 0 or 1 ; A, B and D independently represent an optionally substituted divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene, or C₂-Ce alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic link wherein R^A is hydrogen, C₁-C₃ alkyl or aryl(Cr C₃ alkyl)-; Z¹ is hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; one of Q¹ and Q₂ is an anionic divalent radical selected from those of formulae (1) to (11) as defined in claim 3, while the other of Q¹ and Q² is a divalent cationic radical selected from those of formulae (20) to (27):

wherein ring K is a 4- to 8-membered saturated ring which may contain 1 or 2 additional heteroatoms N, O or S; ring J is a 5- or 6-membered heteroaromatic rung; R' and R" are each hydrogen, or an optional substituent, or R¹ and R" taken together represent a divalent C₁-C₆ alkylene or C₂-C₆ akenylene radical in which up to three carbon atoms may be replaced by N O or S atoms.

8. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical a divalent cationic radical selected from those of formulae (20A) (21 A), (24A) and (25A):

wherein wherein ring K is a 4- to 8-membered saturated ring which may contain 1 or 2 additional heteroatoms N, O or S; ring J is a 5- or 6-membered heteroaromatic rung; R' is hydrogen or an optional substituents, and R'" is a group S¹ as defined in claim 6 or R'" is a group -A-Z¹ wherein A and Z¹ are as defined in claim 5 and -A-Z¹ is substituted by S¹.

9. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical of formula -N(E-A-Q¹-B-Z¹)- or -(R^B)C(E-A-Q¹-B-Z¹)-, wherein E is a bond, -C(=O)-, or -S(=O)₂-; A and B independently represent an optionally substituted divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic link wherein R^A is hydrogen, C₁-C₃ alkyl or aryl(CrC₃ alkyl)-; Z¹ is hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; radical -B-Z¹ is substituted by Q²; Q¹ is selected from divalent anionic radicals (1 ) to (11 ) as defined in claim 3 and Q² is selected from cationic radicals (12) to (20) as defined in claim 5, OR Q¹ is selected from divalent cationic radicals (21) to (27) as defined in claim 7 and Q² is selected from anionic groups of formulae (12) to (20) as defined in claim 5 or anionic groups selected from formulae (1 ) to (11 ) as defined in claim 3 but wherein one unsatisfied valency is satisfied by a group - A-Z¹.

10. A compound as claimed in claim 9 wherein Q represents a divalent radical of formula (II):

wherein Q₃ is an anionic radical O-C(=O)-(C_rC₃ alkyl)- or O-S(^)₂-(C₁-C₃ alkyl)-. B2008/003755

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11. A compound as claimed in claim 10 wherein wherein Q₃ is an anionic radical O- C(=O)-CH₂- or O-S(=O)₂-CH₂-

12. A compound as claimed in claim 1 or claim 2 wherein Q represents -(R^B)C(A-Z¹)- wherein A represents an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (S-), amino (- NR^A-) or divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic link wherein R^A is hydrogen or C_rC₃ alkyl; R^B is hydrogen, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl; Z¹ is hydrogen, trifluoromethyl or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; and wherein radical -A-Z¹ is substituted by a group selected from:

(28) (29) wherein R' and R" are each hydrogen, C₁-C₃ alkyl or aryl(C_rC₃ alkyl)-, or R¹ and R" taken together with the nitrogen to which they are attached form a 3- to 8-membered heterocyclic ring, and wherein R'" represents hydrogen or one or more optional substituents.

13. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical of formula -(Z²-(B¹)_r-Q²-A¹)C(A-Q¹ -(B)₃-Z¹)- wherein r and s are independently 0 or 1 ; A, A¹ , B and B¹ each independently represent an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic link wherein R^A is hydrogen, C₁-C₃ alkyl or aryl(C_rC₃ alkyl)-; Z¹ and Z² independently represent hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; one of Q¹ and Q² is an anionic divalent radical selected from those of formulae (1) to (11) as defined in claim 3 while the other of Q¹ and Q² is a cationic divalent radical selected from those of formulae (21) to (27) as defined in claim 7.

14. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical of formula -(Z²-A¹)C(A-Q¹ -(B)₃-Z¹)- wherein r and s are independently 0 or 1 ; A, A¹, and B each independently represent an optionally substituted divalent monocyclic 3- to 6- membered carbocyclic or heterocyclic radical, or an optionally substituted C₁-C₆ alkylene, or C₂-C₆ alkenylene radical which may optionally contain an ether (-O-), thioether (-S-), amino (-NR^A-) or divalent monocyclic 3- to 6-membered carbocyclic or heterocyclic link wherein R^A is hydrogen, C₁-C₃ alkyl or aryl(C_rC₃ alkyl)-; Z¹ and Z² independently represent hydrogen, trifluoromethyl, or an optionally substituted mono- or bicyclic carbocyclic or heterocyclic radical having 3-6 ring members; radical Z²-A¹- is substituted by Q²; and radical -B-Z¹ is substituted by Q²; Q¹ is selected from divalent anionic radicals (1) to (11) as defined in claim 3 and Q² is selected from cationic groups (12) to (20) as defined in claim 5, OR Q¹ is selected from divalent cationic radicals (21 ) to (27) as defined in claim 7 and Q² is selected from anionic groups of formulae (12) to (20) as defined in claim 5 or groups of formulae (1 ) to (11 ) as defined in claim 3 but wherein one unsatisfied valency is satisfied by a group -A-Z¹.

15. A compound as claimed in claim 1 or claim 2 wherein Q represents a divalent radical selected from:

wherein R' is hydrogen, C₁-C₃ alkyl or aryl(CrC₃ alkyl)-

16. A compound as claimed in any of claims 1 to 15 wherein R¹, R² and R³ are independently each hydrogen, halogen, nitro, cyano, C_rC₃-alkyl, C₂-C₃-alkenyl, C₂-C₃- alkynyl, hydroxy or Ci-C₃-alkoxy or C₂-C₃-alkenyloxy.

17. A compound as claimed in any of claims 1 to 15 wherein R¹, R² and R³ are independently each hydrogen, fluoro, chloro, bromo, cyano, methyl, methoxy or -C≡CH..

18. A compound as claimed in any of the preceding claims wherein A is phenyl, pyridyl, or pyrimidinyl.

19. A compound as claimed in any of the preceding claims wherein one of R¹ and R² is methyl, -C≡CH or cyano.

20. A compound as claimed in any of claims 1 to 17 wherein -AR¹R² is 4- cyanophenyl or 4-ethynylphenyl.

21. A compound as claimed in any of the preceding claims wherein D is O.

22. A compound as claimed in any of the preceding claims wherein R⁵ is hydrogen and R³ is 3-trifluoromethyl, 3-chloro or 3-bromo.

23. A compound as claimed in claim 1 of formula (X):

wherein m, p, q, k, X, X¹, AIk¹ and AIk² are as defined in claim 1 , and Q is as defined in any of claims 1 to 13.

24. A compound as claimed in any of the preceding claims wherein m and k are each 0, p and q are each 1 , and AIk¹ and AIk² are each -(CH₂)₂-5-.

25. A compound as claimed in claim 24 wherein m and k are each 0, p and q are each 1 ; AIk¹ and AIk² are each -(CH₂W; and Q is a divalent radical of formula (II) as defined in claim 10 or claim 11.

26. An ester of any compound claimed in any of the preceding claims which has a carboxylate, sulfonate or phosphonate group in the radical Q.

27. An ester as claimed in claim 26 which is a C₁-C₆ alkyl ester.

28. A compound as claimed in any of the preceding claims in pharmaceutically acceptable salt form.

29. A pharmaceutical composition comprising a compound as claimed in any of claims 1 to 28 and a pharmaceutically acceptable carrier or excipient.

30. A pharmaceutical composition as claimed in claim 29 which is adapted for administration by the pulmonary route.

31. Use of a compound as claimed in any of claims 1 to 28, for the manufacture of a medicament for use in the treatment of prevention of a disease or condition in which HNE is implicated.

32. A method of treatment of a disease or condition in which HNE is implicated, comprising administering to a subject suffering such disease an effective amount of a compound as claimed in any of claims 1 to 28.

33. Use according to claim 31 , or a method of treatment according to claim 32, 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.

34. Use according to claim 31 or a method of treatment according to claim 32, wherein the disease or condition is asthma, rhinitis, psoriasis, dermatitis, (atopic and non- atopic), Crohn's disease, ulcerative colitis, or irritable bowel disease.