WO2010034996A1

WO2010034996A1 - Crystalline acid addition salts and their use as enzyme inhibitors

Info

Publication number: WO2010034996A1
Application number: PCT/GB2009/002278
Authority: WO
Inventors: Christine Edwards; Janusz Kulagowski; Harry Finch
Original assignee: Argenta Discovery 2009 Limited
Priority date: 2008-09-23
Filing date: 2009-09-23
Publication date: 2010-04-01
Also published as: GB0817429D0

Abstract

A crystalline acid addition salt of the compound of formula (I), said salt being selected from the hydrogen sulfate, p-toluene sulfonate, naphthalene-2-sulfonate, fumarate, and ethanesulfonate salts thereof. The said salts are inhibitors of human neutrophil elastase (HNE), and thus of use in the treatment of inflammatory conditions, including those of the respiratory tract, especially when administered by inhalation for pulmonary delivey.

Description

CRYSTALLINE ACID ADDITION SALTS AND THEIR USE AS ENZYME INHIBITORS

Field of the Invention

This invention relates to crystalline salts of a compound ("Compound (I)") which is an inhibitor of human neutrophil elastase (HNE), and thus of use in the 5 treatment of inflammatory conditions, including those of the respiratory tract. Also disclosed herein is a method of synthesis of the class of compounds of which Compound (I) is a member. Background to the Invention

Compound (I) with which the invention is concerned has the formula:

and is disclosed as the formate salt in Example 18 of WO 2007/129060. Compound (I) is an inhibitor of HNE and has been found to be particularly suitable for pulmonary delivery by inhalation.

However, as for most pharmaceutical products, it is desirable that compound

15 (I) should be presented in a stable crystalline form. Detailed Description of the Invention

Accordingly, the present invention provides a crystalline acid addition salt of compound (I) above, said salt being selected from the hydrogen sulfate, p-toluene sulfonate, naphthalene-2-sulfonate, fumarate, ethanesulfonate salts thereof 0 As more fully elaborated below, the crystallinity of the salts of the invention have been confirmed by X Ray Powder Diffraction (XRPD), and they have been shown to be acceptably non-hygroscopic by Dynamic Vapour Sorption (DVS) studies.

The therapeutic utility of the salts of the invention is pertinent to any disease 5 that is known to be at least partially mediated by the action of human neutrophil elastase. For example, they may be used 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 salt of the invention. Other compounds may be combined with salts 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 salt of the invention and one or more other therapeutic agents.

Suitable therapeutic agents for a combination therapy with compounds of the invention include: (1) a corticosteroid, for example fluticasone or budesonide; (2) a β2-adrenoreceptor agonist, for example salmeterol or formeterol; (3) a leukotriene modulator, for example montelukast or pranlukast; (4) anticholinergic agents, for example selective muscarinic-3 (M3) receptor antagonists such as tiotropium bromide; (5) phosphodiesterase-IV (PDE-IV) inhibitors, for example roflumilast or cilomilast; (6) an antitussive agent, such as codeine or dextramorphan; (7) a non- steroidal anti-inflammatory agent (NSAID), for example ibuprofen or ketoprofen; (8) a mucolytic, for example N acetyl cysteine or fudostein; (9) a expectorant/mucokinetic modulator, for example ambroxol, hypertonic solutions (e.g. saline or mannitol) or surfactant; (10) a peptide mucolytic, for example recombinant human deoxyribonoclease I (dornase-alfa and rhDNase) or helicidin; and (11) antibiotics, for example azithromycin, tobramycin and aztreonam.

The magnitude of prophylactic or therapeutic dose of a salt 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 determined 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 salt 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 salt of the invention, additional active ingredient(s), and pharmaceutically acceptable excipients.

Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a salt of the present invention. In therapeutic use, the active salt 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. 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 the salt. Preferably, a unit dose comprises the active compound in an amount of 1 μg to 10 mg.

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

For delivery by inhalation, the active compound is preferably in the form of microparticles. They may be prepared by a variety of techniques, including spray- drying, freeze-drying and micron isation.

By way of example, a composition of the invention may be prepared as a suspension for delivery from a nebuliser or as an aerosol in a liquid propellant, for example for use in a pressurised metered dose inhaler (PMDI). Propellants suitable for use in a PMDI are known to the skilled person, and include CFC-12, HFA-134a, HFA-227, HCFC-22 (CCI2F2) and HFA-152 (CH4F2 and isobutane).

In a preferred embodiment of the invention, a composition of the invention is in dry powder form, for delivery using a dry powder inhaler (DPI). Many types of DPI are known. Microparticles for delivery by administration may be formulated with excipients that aid delivery and release. For example, in a dry powder formulation, microparticles may be formulated with large carrier particles that aid flow from the DPI into the lung. Suitable carrier particles are known, and include lactose particles; they may have a mass median aerodynamic diameter of greater than 90 μm As mentioned, salts 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 salt of the invention. When a salt 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 salt of the invention. As stated, salts 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)

In the synthesis of compound (I) for the formation of the present salts, a general synthetic route has been devised which differs from the route described in WO2007/129060 for the preparation of compound (I) and other members of the class to which it belongs. The process enables the preparation of, for example, compounds of formula (E):

wherein

X is =C- or =N-; n and m are independently 1 , 2, or 3;

R⁵ is hydrogen or C₁-C₃ alkyl;

R², R³ and R⁴ are independently each hydrogen, halogen, nitro, cyano, Q-C₆- alkyl, C₂-C₆-alkenyl, C₂-C₆-alkynyl, hydroxy or CrC₆-alkoxy or C₂-C₆-alkenyloxy, wherein CrC₆-alkyl and Ci-C₆-alkoxy can be further substituted with one to three identical or different radicals selected from the group consisting of halogen, hydroxy and C_rC₄-alkoxy.

The process involves (i) bromination of compound of formula (A) to form a compound of formula (B):

wherein R¹ is C₁-C₃ alkyl, and R is -NH₂ or -OH, (ii) if R in compound B is a group -OH, conversion of that group to -NH₂ to form the amide (B) wherein R is -NH₂; (iii) dehydration of the compound (B) wherein R is -NH₂ to form the compound of formula (B')

(iv) reaction of compound (B¹) with a diamine of formula (C) in the presence of a base to form the desired compound (E):

(C)

A variation of the process involves carrying out step (i) as described above, then (ii) reaction of compound (B) with a diamine of formula (C) wherein R⁵ is (a) hydrogen or (b) or an amino protecting group which can subsequently be deprotected and generate R5 as hydrogen or (c) C_rC₃ alkyl, the said reaction with (C) being carried out in the presence of a base to form the substituted diamine (D):

then (iii) if R in compound D is -OH or -OR', conversion to -NH₂ to form the diamide (D) wherein R is -NH₂, and (iv) dehydration of the diamide (D) wherein R is -NH₂ to convert the amide groups to nitrile groups, thereby formingthe desired compound (E).

A further variation of the process involves carrying out step (i) as described above except that R is a group -OR' wherein R' is C₁-C₃ alkyl, then (ii) reaction of compound (B) wherein R is the said group -OR' with a diamine of formula (C) as described above to form the substituted diamine (D) above but wherein R is the said group -OR'; (iii) then conversion of the said group -OR' to form the diamide (D) wherein R is -NH₂, and (iv) dehydration of the diamide (D) wherein R is -NH₂ to convert the amide groups to nitrile groups, thereby formingthe desired compound (E).

Where a compound (D) having the specific stereochemistry shown in the formula for compound (I) is required, resolution of the monomeric intermediates (A) or (B) or (B¹) may be carried out, and the correct enantiomer selected for the subsequent stages of the method. In that connection, resolution of the monomer (A) or (B) wherein R is -OH is preferred.

Further details of the above process and process variant are given below in connection with Schemes 1-4 and the Examples herein.

The process and process variant described above may be used, for example, to prepare compounds of formula (E) wherein R³ and R⁴ are each hydrogen and R² is 3-trifluoromethyl, 3-chloro or 3-bromo. Synthesis of Compound (I)

The routes shown in Schemes 1 to 4 describe alternative routes for the synthesis of amine (6) through various intermediates. The Biginelli reaction between an arylaldehyde (X = C or N) and an alkyl or aryl acetoacetate, forming compounds (1 ) and (7), may be carried out in the presence of a catalyst such as polyphosphoric acid or chlorotrimethylsilane. Formation of a diastereomeric mixture of salts between (1) and a chiral base, typically an organic amine, may allow crystallization of the (R)-enantiomer (2) preferentially, to permit isolation of (2) after treatment with acid. For the amide formation, reactions may be accomplished using standard conditions, for example, conversion of the acid into the corresponding acid chloride followed by reaction with ammonia or reaction of the acid with ammonia and an amide coupling reagent. The acid chloride may be formed using various reagents including thionyl chloride and oxalyl chloride, and the coupling reagent may be selected from reagents commonly used for that purpose such as HATU, DCC, EDC, Bop and PyBop etc. The bromination steps may be achieved using, for example, bromine or NBS. The dehydration reactions may be effected using standard dehydrating conditions such as POCI₃ or TFAA. The dimerisation reaction with a triamine may benefit from the use of a suitable base e.g. triethylamine, NaHCO₃, DIPEA etc and the triamine may be used in a protected form i.e. the secondary amine may be protected by e.g. tert-butyloxycarbonyl, benzyloxycarbonyl, etc. with the protecting group being removed at a later stage. The secondary amine (6) may be purified by chromatographic means or by the crystallization of a suitable salt such as a 4-toluenesulphonic acid salt. All of the reactions may be performed in various solvents that must be compatible with the reagents used, and may be carried out at various suitable temperatures, typically 0-80°C.

Scheme 1 shows one of the routes which may be used to prepare amine (6).

Chiral base

Biginelli reaction

Amide Dehydration formation

Scheme 1 Scheme 2 shows an alternative synthesis for compound (3). The (R)- enantiomer (8) may be separated from the racemic mixture of Biginelli products (7) by a chromatographic method which employs a chiral stationary phase. The ester in (8) (CO₂R⁶) may be selectively hydrolyzed using an alkali metal hydroxide or other standard method e.g. potassium trimethylsilanoate. Diester (7) may also be converted directly to the chiral acid (2) by enantioselective enzymatic hydrolysis

Ester Direct

Enzymatic hydrolysis amidation hydrolysis

Amide formation

Scheme 2

Scheme 3 shows how compound (4) may be converted into (6) utilizing the reactions shown in Scheme 1 in a different order.

Scheme 3

Scheme 4 describes an alternative order of reactions for the synthesis of intermediate (9) from diester (8). Bromination H₂NH4τ^Λ-HirNH₂ base, solvent

Scheme 4

The routes in Schemes 1 to 4 represent methods for the formation of amine (6), howeverthe processes described maybe carried out in an alternative order and those skilled in the art will readily understand that known variations of the conditions and processes of the preparative procedures can be used.

Salts of amine (6) may be formed by reaction with an acidic molecule. The salt may precipitate or crystallize from the reaction medium.

The following Examples illustrate the invention. General Methods

All solvents and commercial reagents were used as received. Where products were purified using an Isolute® SPE Si Il cartridge, 'Isolute SPE Si cartridge' refers to a pre-packed polypropylene column containing unbonded activated silica with irregular particles with average size of 50 μm and nominal 60A porosity. Melting points were measured using a Bϋchi B-540 apparatus. Analytical LC-MS Systems LC-MS method 1

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)

LC-MS method 2 Waters Micromass ZQ2000 with a C18-reverse-phase column (100 * 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 3.

Waters Micromass ZMD with a C18-reverse-phase column (30 * 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) Chiral HPLC method

Analytical chiral HPLC (250 x 4.6 mm ChiralPak IA column with 5 μm particle size) eluting with 30% THF in n-heptane and with a flow rate of 1 ml/min. UV detection at 254 nm. Differential scanning calorimetrv (DSC)

DSC measurements were performed on a Mettler Toledo DSC823e equipped with a Mettler Toledo TS0801 RO sample robot and automated sample carousel. Samples were prepared in 40 μl aluminium pans, the sample lids were automatically pierced by the robot and the analysis undertaken between 30 and 250⁰C at 10°C/min. Typically, 1-3 mg were used for analysis and the experiment was performed under dry nitrogen purged at 50 mlmin^"1. The instrument was calibrated for energy and temperature using certified indium. X-Rav Powder Diffractometers (XRPD) D5000 High resolution diffractometer The data was collected using a Siemens D5000 diffractometer using Cu Ka radiation (4OkV, 40mA), θ-θ goniometer, divergence of V20 and receiving slits, a graphite secondary monochromatorand a scintillation counter. The instrument was performance checked using a certified Corundum standard (NIST 1976). The software used for data collection was Diffrac Plus XRD Commander v2.3.1 and the data were analysed and presented using Diffrac Plus EVA v 11.0.0.3. Samples were run under ambient conditions as flat plate specimens using powder as received. The sample was gently packed into a cavity cut into polished, zero- background (510) silicon wafer. The sample was rotated in its own plane during analysis. The data was collected between 2 to 42 2Θ, using a step size of 0.05 °2Θ and a collection time of 4 s.step^"1. C2 Low resolution diffractometer

The data was collected using a Bruker AXS C2 GADDS diffractometer using Cu Ka radiation (40 kV, 40 mA), automated XYZ stage, laser video microscope for auto-sample positioning and a HiStar 2-dimensional area detector. X-ray optics consists of a single Gδbel multilayer mirror coupled with a pinhole collimator of 0.3 mm. The beam divergence, i.e. the effective size of the X-ray beam on the sample, was approximately 4 mm. A θ-θ continuous scan mode was employed with a sample - detector distance of 20 cm which gives an effective 2Θ range of 3.2° - 29.7°. Typically the sample would be exposed to the X-ray beam for 120 seconds. The software used for data collection was GADDS for WNT 4.1.16 and the data were analysed and presented using Diffrac Pius EVA v 9.0.0.2 or v 13.0.0.2. Samples were run under ambient conditions as flat plate specimens using powder as received without grinding. Approximately 1-2 mg of the sample was lightly pressed on a polished, zero-background (510) silicon wafer to obtain a flat surface. Single crystal X-ray

Single crystal x-ray analysis was performed on a Bruker-Nonius FR591 rotating anode system fitted with a Bruker-Nonius Roper CCD camera using X-rays at 0.71073 angstroms from MoK using a graphite monochromator. Data was collected at a temperature of 120K. Data collection was using COLLECT (Hooft, R.W.W., 1998), Cell refinement by DENZO (Otwinowski & Minor, 1997) & COLLECT (Hooft, R.W.W., 1998). Structure solution and refinement by SHELX (Sheldrick, 2008). Dynamic Vapour Sorption (DVS)

DVS analysis was performed on a Surface Measurement Systems (SMS) DVS-lntrinsic moisture sorption analyser. The instrument was controlled by SMS Analysis Suite software (DVS-lntrinsic Control v1.0.0.30). Analysis of the data was performed using Microsoft Excel 2007 together DVS Standard Analysis Suite (v6.0.0.7). Sample temperature was maintained at 25⁰C and the sample humidity was obtained by mixing streams of wet and dry nitrogen at a total flow rate of 200mlmin^"1. The relative humidity was measured using a calibrated Rotronic probe (dynamic range 1-100% relative humidity (RH)) located close to the sample. The weight change of the sample as a function of %RH was constantly monitored by the microbalance (accuracy ± 0.005mg). 20 mg of sample was then placed in a tared stainless steel mesh basket under ambient conditions. The sample was loaded and unloaded at 40% RH and 25°C (typical room conditions) and the sample subjected to a graduated DVS regime over 2 cycles using the parameters shown in Table 1. A DVS isotherm was calculated from this date.

Table 1 : Method parameters for DVS experiment Abbreviations used in the experimental section: DCM = dichloromethane DMF = Λ/,Λ/-dimethylformamide

HPLC = high performance liquid chromatography RT = room temperature Rt = retention time THF = tetrahydrofuran Intermediate 1

A 5 litre glass reactor fitted with an overhead stirrer, thermometer and dropping funnel was charged with 3-trifluoroaniline (200 g, 155 ml, 1.24 mol). Stirring was commenced and glacial acetic acid (600 ml) was added to give a clear solution. The reactor content was diluted with water (1 I) to give a cloudy mixture (minor exotherm to 30°C). Sodium cyanate (100 g, 85% pure, containing residual sodium carbonate) was dissolved in water (1 I) and added dropwise to the reaction mixture over 1 h. During the addition the temperature rose to 34-35°C. Stirring was continued at room temperature for 2Oh. The solids were collected by filtration, washed with water (2 x 500 ml) and dried under vacuum at 50-55⁰C to constant weight. The product was isolated as an off-white to pale yellow solid. Yield: 219 g (87%) LC-MS (Method 3): Rt = 2.74 min, m/z = 205.13 [M+Hf Intermediate 2

Intermediate 2 was prepared by a modified method according toSynthesis (2007) 3, 417-427. Chloro trimethylsilane (19 ml, 150 mmol) was added to a stirred solution of 4-carboxybenzaldehyde (6.80 g, 44 mmol), Intermediate 1 (8.60 g, 40 mmol) and methyl acetoacetate (4.80 ml, 44 mmol) in dry DMF (50 ml) at RT. It was stirred for 48 h before volatile components were removed in vacuo and the resultant DMF solution poured into water (250 ml). Ethyl acetate (2 x 150 ml) extracts were washed with water and saturated brine then dried over magnesium sulfate, filtered and solvent removed in vacuo to give a yellow solid. Diethyl ether (50 ml) was added and the product isolated by filtration as a whte solid. Yield: 14.8 g (85%)

LC-MS (Method 1): Rt = 3.62 min, m/z = 435.2 [M+Hf Chiral HPLC: Rt = 20 and 30 min Intermediate 3

Intermediate 2 (5.90 g, 13.6 mmol) and (+)-cinchonine (4.00 g, 13.6 mmol) were dissolved in hot ethanol (45 ml). The solution was allowed to cool to RT overnight and the crystalline solid filtered off. This salt was suspended in 1 N HCI (50 ml) and extracted into ethyl acetate (3 x 50 ml). Combined extracts were dried (MgSO₄), filtered and evaporated to give a white foam. Yield: 2.63 g (44.5%) (89% of theoretical) LC-MS (Method 1): Rt = 3.61 min, m/z = 435.3 [M+Hf Chiral HPLC: Rt = 30 min Intermediate 4

Intermediate 4 was prepared according to the method of Intermediate 2 from chloro trimethylsilane (19 ml, 150 mmol), methyl 4-formylbenzoate (5.75 g,

35 mmol), Intermediate A (8.75 g, 40 mmol) and methyl acetoacetate (3.90 ml,

36 mmol) in dry DMF (50 ml) at RT. Yield: 11.O g (60%)

LC-MS (Method 1): Rt = 4.07 min, m/z = 449.3 [vl+H]⁺ Intermediate 5

Thionyl chloride (5.0 ml, 68.5 mmol) was added to a stirred suspension of Intermediate 2 (10.0 g, 23.0 mmol) in dry DCM (100 ml) at RT followed by DMF (6 drops). Solvent was removed in vacuo after 24 h to obtain an orange foam/solid which was then added gradually to ammonia in methanol (2M, 150 ml) with stirring at RT. The solution was purged with a stream of air to remove excess ammonia and then solvent removed in vacuo to give a residue that was dissolved in ethyl acetate (300 ml). It was transferred to a separating funnel, washed with water and saturated brine then dried over magnesium sulfate, filtered and solvent removed in vacuo to give a yellow semi-solid. This was triturated using diethyl ether and small amount of DCM to enable the product to be isolated by filtration as a pale yellow solid. Yield: 9.4 g (94%) LC-MS (Method 1): Rt = 3.38 min, m/z = 434.3 [M+H]⁺ Intermediate 6

A solution of bromine (830 μl, 16.19 mmol) in chloroform (30 ml) was added dropwise to a stirred mixture of Intermediate 5 (7.00 g, 16.15 mmol) in chloroform (70 ml) during 1 hour at RT. After 2.5 h the mixture was transferred to a separating funnel, washed with aqueous sodium bicarbonate and saturated brine then dried over magnesium sulfate, filtered and solvent removed in vacuo. The residue was triturated using 50% ethyl acetate - diethyl ether (100ml) and filtered off to isolate the product as a pale yellow solid. Yield: 5.6O g (67%)

LC-MS (Method 1): Rt = 3.51 min, m/z = 512.3/514.4 [M+Hj Intermediate 7

A solution of N-(3-aminopropyl)-1 ,3-propanediamine (1.54 ml, 10.8 mmol) in dry THF (5 ml) was added to a mixture of Intermediate 6 (5.53 g, 10.8 mmol) and triethylamine (6.0 ml, 43.1 mmol) in dry THF (65 ml) at RT. This was stirred for 18 h before the solvent was removed in vacuo. The residue was slurried in aqueous sodium bicarbonate, filtered, washed with water and sucked dry to give a pale yellow solid. This was slurried in 40% ethyl acetate/diethyl ether and again sucked dry to give the product as a cream powder. Yield: 4.4 g (87%) LC-MS (Method 2): Rt = 6.65 min, m/z = 930.49 [M+Hj Intermediate 8

Thionyl chloride (1.60 ml, 21.93 mmol) was added to a stirred suspension of Intermediate 3 (2.54 g, 5.84 mmol) in dry DCM (25 ml) at RT followed by DMF (6 drops). Solvent was removed in vacuo after 24 h to obtain an orange foam/solid which was then added gradually to ammonia in methanol (2 M, 50 ml) with stirring at RT. The solution was purged with a stream of air to remove excess ammonia and then solvent removed in vacuo to give a residue that was dissolved in ethyl acetate (100 ml). It was transferred to a separating funnel, washed with water and saturated brine then dried over magnesium sulfate, filtered and solvent removed in vacuo to give a yellow semi-solid. This was triturated using diethyl ether and the product isolated by filtration as a pale yellow solid. Yield: 2.23 g (88%) LC-MS (Method 3): Rt = 3.11 min, m/z = 434.18 [M+H]⁺ Intermediate 9

A solution of bromine (264 μl, 5.15 mmol) in chloroform (10 ml) was added dropwise to a stirred mixture of Intermediate 8 (2.23 g, 5.14 mmol) in chloroform (20 ml) during 30 mins at RT. After 3 h the mixture was transferred to a separating funnel, washed with aqueous sodium bicarbonate and saturated brine then dried over magnesium sulfate, filtered and solvent removed in vacuo. The residue was triturated using 20% ethyl acetate - diethyl ether (25 ml) and filtered off to isolate the product as a pale yellow solid. Yield: 1.6O g (61%) LC-MS (Method 1): Rt = 3.51 min, m/z = 512.2/514.2 [M+Hj Intermediate 10

Phosphorus oxychloride (2.76 ml, 29.61 mmol) and DMF (10 drops) were added to a stirred mixture of Intermediate 9 (1.52 g, 2.96 mmol) in dry DCM (30 ml) at RT. After stirring overnight, the mixture was transferred to a separating funnel, washed with aqueous sodium bicarbonate, water and saturated brine then dried over magnesium sulfate, filtered and solvent removed in vacuo. Purification using an Isolute® SPE Si cartridge eluted with a 0 - 4% MeOH in DCM gradient afforded the product as a foam. Yield: 0.94 g (64%)

LC-MS (Method 1): Rt = 4.07 min, m/z = 494.2/496.2 [M+Hj Intermediate 11

Method A: (R.ffl-lsomer (11a)

Example 1 (265.2 g, 0.25 mol) was suspended in ethyl acetate (2 I). The suspension was shaken with 30% sodium carbonate solution until all the solid had dissolved. The layers were separated, and the organic layer was washed with water, then with brine and finally dried (Na₂SO₄). The solution was evaporated to dryness yielding a colourless foam.

1 H NMR (400 MHz, D₆-DMSO) δ = 1.54 (m, 4H), 2.53 (m, 4H), 3.17 (m, 4H), 3.86 (m, 4H), 5.39 (d, 2H), 7.60-7.88 (m, 16H), 8.17 (d, 2H) ppm Method B: Mixture of (Rf?)-, (S,S)- and (RS)-isomers (1 :1 :2 ratio) (11b)

Intermediate 7 (4.25 g, 4.57 mmol) was dissolved in dry DMF (25 ml) with stirring at RT then cooled to 0-5⁰C and phosphorus oxychloride (2.15 ml, 23.06 mmol) added. Sodium bicarbonate solution was added after 1 hour and the resultant solid filtered off, washed with water and dried to give the product as a cream solid. Purification using an Isolute® SPE Si cartridge elutedwith a 0 - 20% MeOH in DCM gradient and freeze-drying the residue from relevant fractions afforded the product as a pale yellow powder. Yield: 1.11 g (27%) LC-MS (Method 2): Rt = 7.91 min, m/z = 894.41 [M+H]⁺

Method B could be used to prepare homochiral (R,R)-isomer 11a if Intermediate 7 was prepared from Intermediate 9 rather than Intermediate 6. Example 1

A solution of Intermediate 10 (207 g, 0.41 mol) in THF (4.0 I) was added dropwise over 55 min to a stirred solution of dipropylenetriamine (53.5 g, 0.41 mol) and triethylamine (225.2 ml, 1.63 mol) in THF (0.5 1), contained in a jacketed glass reactor maintained at 25°C. A white precipitate was observed to form during the course of the addition. After stirring for 22h, the reaction mixture was reduced under educed pressure to approx.2 litres in volume, and partitioned between water (2.01) and EtOAc (2 x 2.01). The combined organic layers were washed with water (2.01) and then brine (2.01), dried (MgSO₄) and evaporated to leave crude Intermediate 9a as a pale yellow foam (177.6 g), which was used in the next step without further purification.

A solution of p-toluene sulfonic acid monohydrate (37.7 g, 0.20 mol) in THF (120 ml) was added to a stirred solution of the crude Intermediate 9a. The mixture was stirred for 3 days, after which the solid was collected, washed with THF and dried, initially under suction and then under vacuum at 45°C, to afford Example 1 as a white solid. Yield: 113.5 g (52%) m.p. = 216-218°C LC-MS (Method 2): Rt = 7.84 min, m/z = 894.25 [M+H]⁺ amine; Rt = 3.91 min, m/z =

171.01 [M-H]^" tosylate

1H NMR (400 MHz, d₆-DMSO) δ = 1.59 (m, 4H)₁ 2.24 (s, 3H), 2.66 (m, 4H), 3.09-

3.30 (m, 4H), 3.79 (m, 4H), 5.40 (s, 2H), 7.06 (m, 2H), 7.43 (m, 2H), 7.63-7.90 (m,

16H), 8.07 (br s, 2H), 8.20 (d, 2H) Hygroscopicity

Desorption (change in mass (%) - ref @ 25°C and 80% RH) = 1.25

DSC analysis - Trace shown in Fig 1 - Single melting endotherm onsetting at about 21O⁰C. (Integral -50.11 mJ; normalized -36.31 Jg^"1; Onset 210.76⁰C; Peak

221.94⁰C; Heating Rate 10.00°C min^"1). XRPD on D5000 (High resolution) diffractometer - Trace of Lin (counts) vs.2-

Theta-Scale) shown in Fig. 2.

The eight major peaks (defined as those having the highest relative intensities) of the XRPD diffraction pattern characterising the crystalline sulphonate salt of Example 2 are, in degrees 2Θ: between 6.45 and 6.55; between 19.5 and 19.6; between 20.5 and 20.6; between 18.8 and 18.9; between 20.9 and 21.0; between 18.25 and 18.35; between 18.6 and 18.7; and between 12.9 and 13.0. In

Fig 2, those peaks are at (degrees 2Θ) 6.49, 19.56, 20.54, 18.88, 20.93, 18.31 ,

18.65, and 12.96.

Examples 2 - 5 (general procedure) To a solution/suspension of the acid (1.1 equiv.) in solvent (0.5 ml) was added a solution of Intermediate 11 a (100 mg, 0.11 mmol) in the same solvent (1 ml) with stirring. The mixtures were stirred at RT overnight. The salt was filtered, washed with a small amount of cold solvent and dried in vacuo.

Example 2a

Intermediate 11a (52.6 g, 0.06 mol) was dissolved in THF (400 ml) and a solution of cone, sulfuric acid (3.36 ml) in THF (100 ml) was added dropwise with stirring. Seed crystals (prepared using the general method described above for example 2) were added and the mixture was allowed to stir at room temperature.

Salt formation and crystallisation initiated after 4 h and appeared to be complete after 18 h. (Without the use of a seed, crystallisation can take several days). After a total of 36 h the solid was filtered off, washed with THF and dried in vacuo at 4O⁰C.

Yield: 43.0 g (72%)

Example 4a

Intermediate 11a (5.0 g, 5.60 mmol) was dissolved in methanol (40 ml) and a solution of fumaric acid (0.72 g, 6.16 mmol) in methanol (10 ml) was added. The solution was stirred at RT for 24 h and the solid formed was filtered, washed methanol and dried in vacuo at 45⁰C.

Yield: 3.67 g (65%)

Physical and spectroscopic data for Examples 2-5 Example 2

1 H NMR (400 MHz, d₆-DMSO) δ = 1.59 (m, 4H), 2.66 (m, 4H), 3.09-3.30 (m,

4H), 3.79 (m, 4H), 5.40 (s, 2H), 7.63-7.90 (m, 16H), 8.03 (br s, 2H), 8.20 (d, 2H). DSC - Trace shown in Fig. 3 - Single melting endotherm onsetting at about

21O⁰C. (Integral -51.89 mJ; normalized -39.31 Jg^"1; Onset 210.74⁰C; Peak 221.50⁰C; Heating Rate 10.00°C min^'1).

XRPD on D5000 (High resolution) diffractometer- Trace of Lin (counts) vs.2-

Theta-Scale) shown in Fig.4.

The eight major peaks (defined as those having the highest relative intensities) of the XRPD diffraction pattern characterising the crystalline sulphonate salt of Example 2 are, in degrees 2Θ: between 19.4 and 19.5; between 21.5 and

21.6; between 17.65 and 14.75; between 8.1 and 8.2; between 22.1 and 22.2; between 20.9 and 21.0; between 23.15 and 23.25; and between 20.4 and 20.5. In

Fig 4, those peaks are at (degrees 2Θ) 19.47, 21.56, 14.07, 8.15, 22.16, 20.97,

23.20, and 20.45. Single crystal X-ray crystallography - The salt was determined as the hydrogen sulfate of 11a by single crystal x-ray crystallography with a space group

P2i monoclinic, R factor of 0.1276, GOF 1.090 and Flack 0.18(+ 16 sd). All hydrogen atoms were placed in idealised positions and refined using a riding model.

The hydrogen of the hydrogen sulfate was located and fully refined. Example 3

1 H NMR (400 MHz, d₆-DMSO) δ = 1.63 (m, 4H), 2.71 (m, 4H), 3.15-3.31 (m,

4H), 3.82 (m, 4H), 5.45 (s, 2H), 7.52 (m, 2H), 7.67-7.99 (m, 20H), 8.08 (br s, 2H)₁

8.13 (s, 1 H), 8.24 (d, 2H). DSC - Trace shown in Fig.5 - Single melting endotherm onsetting at about

205⁰C. (Integral -25.84 mJ; normalized -28.71 Jg^"1; Onset 205.58⁰C; Peak

211.54°C; Heating Rate 10.00⁰C min^"1).

XRPD on C2 (Low resolution) diffractometer - Trace of Lin (counts) vs. 2-

Theta-Scale) shown in Fig. 6. The eight major peaks (defined as those having the highest relative intensities) of the XRPD diffraction pattern characterising the crystalline naphthalene-2-sulfonate salt of Example 3 are, in degrees 2Θ: between 5.6 and 5.7; between 18.3 and 18.4; between 20.6 and 20.7; between 19.1 and 19.2; and between 16.1 and 16.2; between 17.5 and 17.8; between 19.4 and 19.5; and between 22.47 and 22.57. In Fig 6, those peaks are at (degrees 2Θ) 5.68, 18.37,

20.67, 19.04, 16.06, 17.58, 19.44, and 22.52.

Example 4

1 H NMR (400 MHz, d₆-DMSO) δ = 1.59 (m, 4H), 2.56 (m, 4H), 3.11-3.30 (m,

4H), 3.81 (m, 4H), 5.44 (s, 2H), 6.43 (s, 2H), 7.67-7.94 (m, 16H), 8.21 (d, 2H). DSC - Trace shown in Fig. 7 - single melting endotherm onset at about

144⁰C. (Integral -31.95 mJ; normalized -18.47 Jg^"1; Onset 144.38°C; Peak

161.82°C; Heating Rate 10.00°C min^"1).

XRPD on C2 (Low resolution) diffractometer - Trace of Lin (counts) vs. 2-

Theta-Scale) shown in Fig. 8. The eight major peaks (defined as those having the highest relative intensities) of the XRPD diffraction pattern characterising the crystalline fumarate salt of Example 4 are, in degrees 2Θ: between 18.7 and 18.8; between 22.2 and

22.3; between 13.45 and 13.55; between 24.7 and 24.8; between 17.2 and 17.3; between 21.65 and 21.75; between 26.1 and 26.2; and between 26.85 and 26.95. In Fig 8, those peaks are at (degrees 2Θ) 18.78, 22.29, 13.49, 24.74, 17.23, 21.69,

26.16, and 26.91.

Example 5

1 H NMR (400 MHz, d₆-DMSO) δ = 0.99 (t, 3H), 1.60 (m, 4H), 2.09 (q, 2H),

2.67 (m, 4H), 3.09-3.30 (m, 4H), 3.79 (m, 4H), 5.40 (s, 2H), 7.63-7.90 (m, 16H), 8.08 (br s, 2H), 8.20 (d, 2H). DSC - Trace shown in Fig. 9 - Single melting endotherm onsetting at about 195⁰C. (Integral -24.07 mJ; normalized -20.93 Jg^"1; Onset 195.12⁰C; Peak 205.72⁰C; Heating Rate 10.00⁰C min^"1.).

XRPD on D5000 (High resolution) diffractometer- Trace of Lin (counts) vs.2- Theta-Scale) shown in Fig. 10.

The eight major peaks (defined as those having the highest relative intensities) of the XRPD diffraction pattern characterising the crystalline ethane sulfonate salt of Example 5 are, in degrees 2Θ: between 19.55 and 19.65; between 7.05 and 7.15; between 18.1 and 18.2; between 14.15 and 14.25; between 23.1 and 23.2; between 18.3 and 18.4; between 22.67 and 22.77; and between 25.33 and 25.43. In Fig 10, those peaks are at (degrees 20) 19.59, 7.09, 18.14, 14.19, 23.04, 18.35, 22.72, and 25.38. 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 100 μl. The final concentration of the enzyme (human leukocyte elastase, Sigma E8140) was 0.00036 units/well. A peptide substrate (MeO-Suc-Ala-Ala-Pro-ValAMC, Calbiochem #324745) was used, at the final concentration of 100 μM. The final concentration of DMSO was 1% in the assay buffer (0.05M Tris.HCI, pH 7.5, 0.1 M NaCI; 0.1 M CaCI₂; 0.0005% brij-35).

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

Compound (I) and its salts had activities in the range 1-10 nM.

Claims

1. A crystalline acid addition salt of the compound of formula (I),

said salt being selected from the hydrogen sulfate, p-toluene sulfonate, naphthalene-2-sulfonate, fumarate, and ethanesulfonate salts thereof.

2. A crystalline form of the p-toluene sulfonate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern with peak intensities, in degrees 2Θ, between 6.45 and 6.55; between 19.5 and 19.6; between 20.5 and 20.6; between 18.8 and 18.9; between 20.9 and 21.0; between 18.25 and 18.35; between 18.6 and 18.7; and between 12.9 and 13.0.

3. A crystalline form of the p-toluene sulfonate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern corresponding to that shown in Fig 2.

4 A crystalline form of the p-toluene sulfonate salt as claimed in claim 2 or claim 3 having a melting point of 216-218⁰C.

5. A crystalline form of the hydrogen sulfate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern with peak intensities, in degrees 2Θ, between 19.4 and 19.5; between 21.5 and 21.6; between 17.65 and 14.75; between 8.1 and 8.2; between 22.1 and 22.2; between 20.9 and 21.0; between 23.15 and 23.25; and between 20.4 and 20.5.

6. A crystalline form of the hydrogen sulfate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern corresponding to that shown in Fig 4.

7 A crystalline form of hydrogen sulfate salt as claimed in claim 5 or claim 6 having a melting point of 216-218⁰C.

8. A crystalline form of the naphthalene-2-sulfonate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern with peak intensities, in degrees 20, between 5.6 and 5.7; between 18.3 and 18.4; between

20.6 and 20.7; between 19.1 and 19.2; and between 16.1 and 16.2; between 17.5 and 17.8; between 19.4 and 19.5; and between 22.47 and 22.57.. 9. A crystalline form of the naphthalene-2-sulfonate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern corresponding to that shown in Fig 6.

10 A crystalline form of the naphthalene-2-sulfonate salt as claimed in claim 8 or claim 9 having a melting point of 206-208°C. 11. A crystalline form of the fumarate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern with peak intensities, in degrees 2Θ, between 18.7 and 18.8; between 22.2 and 22.3; between 13.45 and

13.55; between 24.7 and 24.8; between 17.2 and 17.3; between 21.65 and 21.75; between 26.1 and 26.2; and between 26.85 and 26.95. 12. A crystalline form of the fumarate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern corresponding to that shown in Fig 8.

13 A crystalline form of the fumarate salt as claimed in claim 11 or claim 12 having a melting point of 176-178°C. 14. A crystalline form of the ethanesulfonate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern with peak intensities, in degrees 2Θ, between 19.55 and 19.65; between 7.05 and 7.15; between 18.1 and

18.2; between 14.15 and 14.25; between 23.1 and 23.2; between 18.3 and 18.4; between 22.67 and 22.77; and between 25.33 and 25.43. 15. A crystalline form of the ethanesulfonate salt as claimed in claim 1 that is characterized by having an X-ray powder diffraction pattern corresponding to that shown in Fig 10.

16 A crystalline form of the ethanesulfonate salt as claimed in claim 14 or claim

15 having a melting point of 200-202⁰C. 17. A pharmaceutical composition comprising a salt as claimed in any of the preceding claims, together with one or more pharmaceutically acceptable carriers and/or excipients.

18. A pharmaceutical composition as claimed in claim 17, which isadapted for pulmonary administration by inhalation.