Chloride-hydrochloride salt of a benzodiazolium enac inhibitor

Abstract

The invention relates to 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6- (4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3- diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride. The invention further relates to a method for preparing this salt, to pharmaceutical compositions containing it and to its 5 use in methods for treating a respiratory disease or condition.

Description

[0001] COMPOUNDS AND PHARMACEUTICAL COMPOSITIONSThe present invention relates to a compound which has activity as an inhibitor of theepithelial sodium channel (ENaC), in particular to a particular salt of the compound, topharmaceutical compositions comprising this salt and to the use of such compositions forthe treatment of diseases and conditions modulated by ENaC, particularly respiratory diseases and conditions, methods of preparing the compounds and pharmaceutical compositions containing them. Background of the Invention Humans can inhale up to 12,000 L of air each day and with it comes the potential for airborne pathogens (bacteria, viruses, fungal spores). To protect against these airborne pathogens, the lung has evolved innate defence mechanisms to minimise the potential for infection and colonisation of the airways. One such mechanism is the mucus clearance system, whereby secreted mucus is propelled up and out of the airways by the coordinated beating of cilia together with cough clearance. This ongoing ‘cleansing’ of the lung constantly removes inhaled particles and microbes thereby reducing the risk of infection. In recent years it has become clear that the hydration of the mucus gel is critical to enable mucus clearance (Boucher 2007; Matsui et al, 1998). In a normal, healthy airway, the mucus gel is typically 97% water and 3% solids under which conditions the mucus is cleared by mucociliary action. The hydration of the airway mucosa is regulated by the coordinated activity of a number of ion channels and transporters. The balance of anion(Cl- / HCO3-) secretion mediated via the Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR) and the Calcium Activated Chloride Conductance (CaCC; TMEM16A, also known as Ano1) and Na+absorption through the epithelial Na+channel (ENaC) determine the hydration status of the airway mucosa. As ions are transported across the epithelium, water is osmotically obliged to follow and thus fluid is either secreted or absorbed. In respiratory diseases such as chronic bronchitis and cystic fibrosis, the % solids of the mucus gel is increased as the hydration is reduced and mucus clearance is reduced (Boucher, 2007). In cystic fibrosis, where loss of function mutations in CFTR attenuates ability of the airway to secrete fluid, the % solids can be increased to 15% which is believed to contribute towards the plugging of small airways and failure of mucus clearance.Furthermore, in cystic fibrosis an increase in ENaC activity has been reported by severalgroups (Knowles et al, 1983; Middleton et al, 1993) and this increase in ENaC functionhas been shown to correlate with disease severity (Fajac et al, 2004; Leal et al, 2008). Strategies to increase the hydration of the airway mucus include either the stimulation of anion and thereby fluid secretion or the inhibition of Na+absorption. To this end, blockingthe activity of ENaC will inhibit Na+ absorption and therefore increase fluid accumulationin the airway mucosa, hydrate mucus and enhance mucus clearance mechanisms.ENaC is expressed in renal, colonic, corneal, sweat duct and respiratory epithelia whereit forms a low conductance channel (~4 pS) with a selectivity for Na+over K+of approximately 10-fold (Kellenberger 2002). Loss and gain of function mutations in the channel can cause human disease including pseudohypoaldosteronism type 1 (PHA1), a salt wasting disease (Chang et al, 1996), and Liddles’s syndrome, a disease associated with salt retention and hypertension (Botero-Velez et al, 1994). Of particular note to lung physiology is the observation that patients with PHA1 loss-of-function mutations in ENaC have an enhanced rate of airway mucociliary clearance (MCC) compared with the normal healthy population, typically 3-4 fold faster (Kerem et al, 1999). Furthermore, the upper airways of these patients appear to be ‘wet’ and have extra-hydration compared to normal.These observations further support the salient role that ENaC plays in the human airwayin the regulation of hydration and the therapeutic benefit that blocking ENaC in the airwaycould deliver in terms of enhancing MCC and innate defence. Amiloride, a small compound blocker of ENaC, has been demonstrated to increase MCC in both healthy controls and also patients with cystic fibrosis (CF), further supporting thephysiological significance of this mechanism (App et al, 1990). However, the compounddid not achieve clinical endpoints in CF patients (Bowler et al, 1995; Graham et al, 1993;Knowles et al, 1990; Pons et al, 2000).Further ENaC blockers were therefore developed and these include:QBW276 believed to be the compound disclosed in Example 1.0 of WO 2012 / 035158,which has the structure: AZD5634, disclosed in Example 2 of WO 2015 / 140527 and having the structure: BI 1265162, disclosed in WO 2017 / 028927 (Example 2.04), which has the structure: .However, none of these compounds showed any clinical benefit in studies in patients withcystic fibrosis.Our earlier application WO 2018 / 096325 relates to ENaC inhibiting compounds containinga pyrrolopyrazine moiety. Example 50 describes the preparation of 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetic acid trifluoroacetate, which is said to have the structure: , although subsequent analysis has shown that the salt is actually the trifluoroacetate di(trifluoroacetic acid). The 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-(4- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium cation has been found to be a potent ENaC inhibitorwhich persists for several hours in lung tissue in pharmacologically active amounts andleads to a significant increase in mucociliary clearance in a sheep model compared withanimals dosed with water. The compound is intended for administration to the lungs forthe treatment of conditions such as cystic fibrosis, chronic obstructive pulmonary disease,chronic bronchitis, bronchiectasis, severe asthma and primary ciliary dyskinesia.Trifluoroacetic acid salts are not established salts for inhalation and so the trifluoroacetatedi(trifluoroacetic acid) of this cation may not be tolerated when administered to the lungs.The inventors aimed to provide another form of the 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium cation with merits oradvantages, for example by having suitable physical characteristics when in solid form and having superior tolerability in the context of a medicine intended for administration to the lungs. Summary of the InventionIn a first aspect of the present invention there is provided a compound which is 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-(4-{bis[(2S,3R,4R,5R)- 2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride having the formula:including all tautomeric and resonance forms thereof.Further aspects of the invention derive from the fact that this salt has proved difficult tosynthesise and it has been necessary to develop a suitable process for its production. Thisparticular salt form has the merit that it is expected to be well tolerated as a pharmaceuticalproduct for administration to the lungs. In addition, this salt form can be stored and handledwith relative ease compared with other forms of this cation. Detailed Description of the Invention As used herein, the 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-(4- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium cation is sometimes referred to as the “Compound Ication”.All tautomeric and resonance forms of the Compound I cation are included within the scopeof the invention. As would be well understood by a person of skill in the art, the CompoundI cation may exist in alternative resonance forms as follows: where R represents: in which * represents the point of attachment to the remainder of the molecule.Both of these resonance forms are included within the scope of the invention.In the present specification, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth. In the present specification, references to “pharmaceutical use” refer to use for administration to a human or an animal, in particular a human or a mammal, for example a domesticated or livestock mammal, for the treatment or prophylaxis of a disease or medical condition. The term “pharmaceutical composition” refers to a composition which is suitable for pharmaceutical use and “pharmaceutically acceptable” refers to an agent which is suitable for use in a pharmaceutical composition. Other similar terms should be construed accordingly. In the present specification, the compound of the invention or substances consisting of or consisting essentially of the compound of the invention may be stored in dry conditions and under an inert atmosphere. Storage in dry conditions refers to storage in a vessel fromwhich water has been removed and which is sealed to minimise the entry of water andstorage under an inert atmosphere refers to storage in a vessel in which air has been replaced by an inert gas such as nitrogen or argon, especially nitrogen. References to a dry inert atmosphere refer to an inert atmosphere from which water is removed and into which the entry of water is minimised. In the present specification, “wet ethanol” refers to ethanol having a water content of fromabout 0.2 to 0.9% w / w, suitably from about 0.4% to about 0.7% w / w. Similarly, “wetisopropanol” refers to isopropanol having a water content of from about 0.2 to 0.9% w / w,suitably from about 0.4% to about 0.7% w / w.In the present specification a “unit dosage form” is a medication formulated and packaged in the correct amount to be taken as a single dose. If a unit dosage form is intended foronce daily administration, one unit dosage form is administered per day, and when a unitdosage form is for twice daily administration, two unit dosage forms are administered perday. As noted above, WO 2018 / 096325 describes in Example 50 the preparation of a 2-[({3- amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)- 2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetic acid trifluoroacetate salt, which is said to have thestructure: ,although subsequent further analysis of the product of Example 50 showed that it was, infact 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis [(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetate di(trifluoroacetic acid). This is referred to as theCompound I cation trifluoroacetate ditrifluoroacetic acid salt in the discussion below. In the course of developing alternative forms of the Compound I cation, the inventorsprepared a chloride dihydrochloride salt using a salt exchange process. Surprisingly,however, attempts to purify this salt led to the production of a chloride hydrochloride salt.The Compound I cation chloride dihydrochloride salt has relatively poor chemical stabilityeven when stored under inert conditions.The chloride hydrochloride salt unexpectedly proved to have markedly improved stabilitycompared with the chloride dihydrochloride salt.The invention therefore provides in a first aspect a compound which is 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride having the formula: Including all tautomeric and resonance forms thereof(sometimes referred to herein as the “Compound I cation chloride hydrochloride salt”). The Compound I cation chloride hydrochloride salt has substantially improved chemical stability compared with the Compound I cation chloride dihydrochloride salt, can beprepared as a free-flowing powder, and can be stored as such provided that it is kept in asealed container in dry conditions and under an inert atmosphere, for example undernitrogen.Suitably, the Compound I cation chloride hydrochloride salt as a solid is in amorphousform, i.e. it is an amorphous solid. There is also provided a free-flowing powder comprising, consisting essentially of or consisting of the Compound I cation chloride hydrochloride salt. The free-flowing powder is suitably stored in a sealed container in dry conditions and under an inert atmosphere, suitably a nitrogen atmosphere.Suitably, the powder comprises the Compound I cation chloride hydrochloride salt inamorphous form.The powder may comprise impurities in addition to the Compound I cation chloridehydrochloride salt and it is desirable that the content of these impurities is minimised.Therefore, the powder may be free from water or, optionally, in addition to the CompoundI cation chloride hydrochloride salt, the powder may also comprise water in an amount of 0-5.0% ww, or 0-4.0% w / w, more suitably 0-2.5% w / w.The powder may be free from aluminium chloride but, alternatively may optionallycomprise ammonium chloride in an amount of 0-0.15% w / w, more suitably 0-0.05% w / w.The powder may be free from acetamide but, alternatively, may optionally compriseacetamide in an amount of 0-0.15% w / w, more suitably 0-0.075% w / w.The powder may be free from trifluoroacetic acid but, alternatively, may optionally comprisetrifluoroacetic acid in an amount of 0-0.2% w / w, more suitably 0-0.1% w / w.The powder may be free from acetonitrile but, alternatively, may optionally compriseacetonitrile in an amount of 0-0.5% w / w, more suitably 0-0.01% w / w.The powder may be free from ethanol but, alternatively, may optionally comprise ethanolin an amount of 0-1.0% w / w, more suitably in an amount of 0-0.5% w / w.The powder may be free from DMSO but, alternatively, may optionally comprise DMSO inan amount of 0-0.5% w / w, more suitably 0-0.05% w / w.Suitably, when analysed by HPLC, the powder comprises total impurities in an amount of ≤ 3% area, more suitably ≤ 1.5% area. Suitably, the powder comprises no single impurity in an amount of ≥ 0.5% area. The Compound I cation chloride hydrochloride salt may be produced from the Compound I cation trifluoroacetate ditrifluoroacetic acid salt described in Example 50 of WO2018 / 096325. In an attempt to produce a suitable salt for use in an inhaled formulation, the inventors attempted to use a conventional salt exchange process. This was notsuccessful, but the inventors did eventually succeed in developing a novel salt exchangeprocess, discussed in greater detail below, which gave rise to a solid product comprisingthe Compound I cation chloride dihydrochloride salt.The Compound I cation chloride dihydrochloride salt proved to have poor chemicalstability, even when stored in dry conditions under an inert atmosphere. It also containedimpurities which it is desirable to minimise before formation of a pharmaceuticalcomposition. For example, when the solvent used in the salt exchange process is a mixtureof acetonitrile and ethanol, the Compound I cation chloride dihydrochloride salt may alsocontain small amounts of ammonium chloride and acetamide. These impurities should bepresent in the lowest possible amounts in product intended for use in a pharmaceutical composition.Therefore, the inventors designed a purification step in which the solid product of the saltexchange step comprising the Compound I cation chloride dihydrochloride salt is dissolvedin a solvent to form a solution, which is then added to an anti-solvent to precipitate apurified product; and isolating the purified product as a solid.Although this step was originally designed to remove impurities such as ammoniumchloride and acetamide from the Compound I cation chloride dihydrochloride salt,surprisingly, it also resulted in the removal of a hydrochloride moiety, leading to theproduction of the Compound I cation chloride hydrochloride salt. The Compound I cationchloride hydrochloride salt, with appropriate selection of preparative conditions, could beisolated as a solid in a physical form which was easier to handle and proved to be morestable than the chloride dihydrochloride salt. Accordingly, the Compound I cation chloridehydrochloride salt appears to be more suitable for use as a component of an aqueouspharmaceutical composition than other forms of the Compound I cation.In a further aspect of the invention, there is provided a process for the preparation of 2- [({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)- 2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride, the process comprising:(I) precipitating 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3- diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride (Compound I cation chloride hydrochloride salt) from a solution comprising 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2- yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino} piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride dihydrochloride (Compound I cation chloride dihydrochloride salt) in a solvent by addition of the solution to an antisolvent for 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3- diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride to form a solid precipitate; and(II) optionally isolating the solid precipitate.The process for preparing the Compound I cation chloride hydrochloride salt is suitablycarried out under an inert atmosphere such as nitrogen. Suitably, it is carried out at a temperature of about 15 °C to 40 °C, suitably at 15 °C to 25 °C, or room temperature.The solution comprising Compound I cation chloride dihydrochloride salt may be preparedby dissolving a solid comprising Compound I cation chloride dihydrochloride salt in a suitable solvent. Suitable solvents for forming the solution include solvents comprising a dipolar aproticsolvent, water or mixtures thereof.A dipolar aprotic solvent refers to a solvent having a strong dipole and with no potential for donation of hydrogen to form hydrogen bonds. Examples of such solvents include dimethylsulfoxide (DMSO), sulfolane, N-methyl pyrrolidone (NMP), dimethyl acetamide (DMAc)and dimethyl formamide (DMF). Therefore, the suitable solvent of step (I) of the process may comprise DMSO, sulfolane, NMP, DMAc, DMF, water or mixtures thereof. DMSO is less toxic than other dipolar aprotic solvents and therefore, suitably, the solvent used in step (I) to form the solution may comprise DMSO, water or mixtures thereof. Surprisingly, it has been found that the addition of a C1-4 alcohol greatly improves the quality of the product. In particular, precipitation from a solution comprisingdimethylsulfoxide and / or water in admixture with a C1-4 alcohol such as ethanol or isopropanol gives rise to a product which is a filterable solid, On the other hand, the inventors found that precipitation from solution in DMSO and / or water without the presence of an alcohol may yield a product which is a gummy solid and which cannot be filtered.Therefore, more suitable solvents for use in preparing the solution of step (I) furthercomprise a C1-4 alcohol such as ethanol or isopropanol.Particularly suitable solvents comprise mixtures of DMSO and / or water with a C1-4 alcoholsuch as ethanol or isopropanol.Suitably, in the solvent of step (I), the volume ratio of water and / or DMSO to C1-4 alcoholis about 2:1 to 1:2, typically about 1:1 to 1:1.5. The water and / or DMSO component mayconsist of only water or only DMSO but more suitably is a mixture with volume ratio (water: DMSO) of between 1:2 and 1:20 e.g. 1:5 to 1:15 e.g. around 1:10. When a solvent comprising a mixture of DMSO and / or water with an alcohol is used as a solvent, the solidcomprising Compound 1 cation chloride dihydrochloride salt is suitably dissolved in 3 to 8volumes of solvent, more suitably in 4 to 7 volumes of solvent, for example about 6 volumes of solvent. A particularly suitable solvent for forming the solution in step (I) comprises a mixture of ethanol, DMSO and water. The ethanol used in the solvent mixture may be wet ethanol as defined above. More suitably, the solvent comprises 3 to 4 volumes, for example 3.5 volumes wet ethanol, 2 to 3 volumes, for example 2.5 volumes DMSO and 0.2 to 0.3 volumes, for example 0.25 volumes water per mass of solid comprising Compound I cation chloride dihydrochloride salt.As noted above, the precipitation of the Compound I cation chloride hydrochloride salt fromsolution is achieved by adding the solution comprising Compound I cation chloridedihydrochloride salt to an anti-solvent. The product obtained in this way is a filterable solid.Although the Compound I cation chloride hydrochloride salt can also be obtained by addingthe anti-solvent to the solution comprising Compound I cation chloride dihydrochloride salt,this leads to a product which is in the form of a gum and is difficult to filter and dry.In order to achieve precipitation of the Compound I cation chloride hydrochloride salt as afilterable solid, the solution comprising Compound I cation chloride dihydrochloride salt is suitably added dropwise to the anti-solvent, more suitably with stirring to ensure thorough mixing. Suitable anti-solvents for this step include C1-4 alcohols such as ethanol or isopropanol, especially ethanol. Again, wet ethanol or isopropanol may be used, especially wet ethanol. More suitably, when the anti-solvent comprises a C1-4alcohol such as ethanol or isopropanol (e.g. wet ethanol or wet isopropanol), the same alcohol is used as an anti- solvent.A large excess of anti-solvent is suitably used in the precipitation step, for example, 7 to11 volumes of anti-solvent per volume of solution comprising the Compound I cationchloride dihydrochloride salt. More suitably, 8 to 10 volumes, for example about 9 volumesof anti-solvent per volume solution comprising Compound I cation chloride dihydrochloridesalt is used.In step (II), isolation of the solid precipitate may for example comprise filtration, which issuitably carried out under an inert atmosphere, such as a nitrogen atmosphere. When thefiltered solid material is dried (after optional washing), a free-flowing amorphous powdercan be produced. Since the amorphous form of the Compound I cation chloride hydrochloride salt can be obtained as a free-flowing powder, it is easier to handle than the chloride dihydrochloridesalt and is suitable for use in preparing an aqueous pharmaceutical composition. It ischemically stable if stored in dry conditions under an inert atmosphere such as nitrogen and, although it is hygroscopic, it does not take up significant amounts of water and retainsits free-flowing form when stored in dry conditions under nitrogen.As noted above, the starting material for the process is a solution comprising Compound Ication chloride dihydrochloride, which may be prepared by dissolving a solid comprising Compound I cation chloride dihydrochloride in a suitable solvent. Attempts to form a Compound I cation salt containing chloride ions and hydrochloride moieties using aconventional acid base extraction salt exchange process were not possible because of thehigh solubility in water and low solubility in organic solvents of the Compound I cationtrifluoroacetate. Ion exchange was also unsuccessful because the Compound I cation was insufficiently stable in the presence of the ion exchange resin. However, after some experimentation, the inventors were able to develop a salt exchangeprocess which gave rise to a product comprising the Compound I cation chloridedihydrochloride salt.The process therefore includes obtaining a solid comprising the Compound I cationchloride dihydrochloride salt by a salt exchange step comprising: treating 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis [(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl- 1H-1,3-benzodiazol-3-ium trifluoroacetate di(trifluoroacetic acid) (Compound I cation trifluoroacetate di(trifluoroacetic acid) salt) with excess hydrochloric acid in a solventselected from acetonitrile, C1-4 alcohols and mixtures thereof to produce a product mixturecomprising a precipitate of 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3- diethyl-1H-1,3-benzodiazol-3-ium chloride dihydrochloride (Compound I cation chloridedihydrochloride salt) and isolating a solid product comprising said chloride dihydrochloridesalt. Suitably, the salt exchange step is carried out under an inert atmosphere, for example a nitrogen atmosphere. The salt exchange step comprises treating the Compound I cation trifluoroacetate di(trifluoroacetic acid) salt with excess hydrochloric acid in a suitable solvent selected from acetonitrile and C1-4 alcohols such as methanol or ethanol, or mixtures of such solvents. For example, a mixture of acetonitrile and ethanol may be used. Suitable mixtures e.g. comprise acetonitrile:ethanol in a ratio of from about 3:1 to 5:1, typically about 4:1. In some cases, in the salt exchange step, a solution of hydrogen chloride in a suitable solvent is added to a solution of the trifluoroacetate di(trifluoroacetic acid) salt of theCompound I cation, typically in the same solvent, to give a product mixture comprising theCompound I cation chloride dihydrochloride salt.Alternatively, a solution of hydrogen chloride in a suitable solvent may be added to thesolid Compound I cation trifluoroacetate di(trifluoroacetic acid) salt. Typically in this method, the hydrogen chloride solution is added rapidly to the solid Compound I cation trifluoroacetate di(trifluoroacetic acid) salt. The mixture may be stirred until salt exchange has occurred, for example for about 18 to 24 hours. Suitably, the salt exchange is carried out under an inert atmosphere such as nitrogen. The salt exchange may take place at room temperature, i.e. about 15°C to 25°C.The salt exchange step concludes by isolating the product comprising the chloridedihydrochloride salt as a solid. The process directly provides a product mixture comprisinga precipitate of the Compound I cation chloride dihydrochloride salt, which can be filteredoff from the solution. However, the yield and or purity of the Compound I cation chloridedihydrochloride salt may be maximised by mixing the product mixture with an anti-solventto achieve further precipitation. Therefore, the isolation may further comprise mixing theproduct mixture with a suitable anti-solvent, for example an ether such as a di(C1-4 alkyl)ether such as tert butyl methyl ether (TBME).The use of the anti-solvent has the further advantage that it effectively washes the productand so may lead to the removal of some of the trifluoroacetic acid produced in the saltexchange process.Mixing with an anti-solvent may be carried out under an inert atmosphere, typically undernitrogen and can be achieved either by the addition of the anti-solvent to the solution or by the addition of the solution to the anti-solvent. Suitably, the precipitated solid is filtered and it may then be dried. Drying may be carried out in vacuo, suitably at elevated temperature, for example at about 28°C to 35°C, typically about 30°C and for a time of about 18 to 30 hours, typically about 24 hours.The process for preparing Compound I cation chloride hydrochloride salt may furthercomprise washing the solid comprising Compound I cation chloride dihydrochloride beforecarrying out step (I) of the process and / or washing the Compound I cation chloridehydrochloride salt after step (II) of the process.Washing the solid comprising Compound I cation chloride dihydrochloride before carryingout step (I) of the process may comprise washing the solid comprising Compound I cationchloride dihydrochloride salt obtained from the salt exchange step. The washing may comprise forming a slurry of the solid comprising Compound I cationchloride dihydrochloride salt in a suitable liquid, stirring the slurry and filtering the washedproduct. Similarly, washing the Compound I cation chloride hydrochloride salt obtained from step (II) of the process may comprise forming a slurry of the Compound I cation chloride hydrochloride salt in a suitable liquid, stirring the slurry and filtering the washed product. Suitably, as with the remainder of the process, washing steps are carried out under an inert atmosphere, for example under a nitrogen atmosphere.Suitable washing liquids, e.g. liquids for the formation of a slurry are solvents in which theimpurities are soluble but the Compound I cation chloride dihydrochloride and CompoundI cation chloride hydrochloride salts are sparingly soluble or insoluble. Such liquids includeC1-4alcohols, such as ethanol and isopropanol, but especially ethanol, suitably in anamount of about 8 to 25 volumes, more suitably 8 to 20 volumes or 8 to 12 volumes, forexample about 10 volumes with respect to the mass of the solid comprising Compound I cation chloride dihydrochloride or Compound I cation chloride hydrochloride. In somecases, the liquid may also contain water in an amount of about 0.5 to 7% v / v. Most suitably,the liquid used for the slurry is ethanol, especially wet ethanol, in an amount of 8 to 12volumes, especially about 10 volumes with respect to the mass of the salt of the Compound I cation. A washing step after the preliminary salt exchange step is useful for the reduction ofimpurities such as ammonium chloride and acetamide from the solid comprisingCompound I cation chloride dihydrochloride. These impurities may be formed as by- products of the preliminary salt exchange step.A washing step carried out after (II) of the process is useful for reducing the amount ofDMSO in the Compound I cation chloride hydrochloride salt product of step (II). It may alsoreduce further the amounts of residual ammonium chloride, acetamide and other impuritiesin the product.Suitably, washing steps are employed both after the preliminary salt exchange step andafter step (II). After the washing steps, the product filtered from the slurry may be dried. Drying may beconducted in vacuum oven, suitably at a temperature of ≤ 30°C.It is particularly advantageous to include a step of drying the Compound I cation chloride hydrochloride salt obtained from a washing step carried out after step (II). There is also provided a process for the preparation of 2-[({3-amino-5H-pyrrolo[2,3- b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloridehydrochloride, the process comprising:A. treating 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl- 1H-1,3-benzodiazol-3-ium trifluoroacetate di(trifluoroacetic acid) (Compound I cation trifluoroacetate di(trifluoroacetic acid) salt) with excess hydrochloric acid in a solvent selected from acetonitrile, C1-4alcohols and mixtures thereof in a salt exchange step toproduce a product mixture comprising a precipitate of 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl] amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloridedihydrochloride (Compound I cation chloride dihydrochloride salt) and isolating a solidproduct comprising said chloride dihydrochloride salt;B. optionally washing the solid product of step (a) by forming a slurry of the productin a C1-4 alcohol and filtering and drying the washed product;C. converting the solid comprising Compound I cation chloride dihydrochloride salt ofstep A. or optional step B. to 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumchloride hydrochloride (Compound I cation chloride hydrochloride salt) by a processcomprising: (I) precipitating Compound I cation chloride hydrochloride salt from a solution of Compound I cation chloride dihydrochloride salt in a solvent by addition of the solution to an antisolvent for Compound I cation chloride hydrochloride salt to form a solid precipitate; and(II) isolating the solid precipitate;D. optionally washing the isolated solid precipitate of step C. by forming a slurry of theproduct in a C1-4 alcohol and filtering the washed product;E. drying the product of step C. or optional step D.Suitable reaction conditions, solvents, anti-solvents and liquids for forming slurries in theprocess are as set out above.In step E., the product may be dried until it contains ≤ 5.0% w / w water, or 4.0% w / w waterand preferably ≤ 2.0% w / w water, more preferably ≤ 1.5% w / w water. Drying may beconducted in a vacuum oven, suitably at a temperature of ≤ 30°C.In a further aspect of the invention there is provided 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin- 2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochlorideobtainable by or obtained by the process described above.This product preferably comprises the lowest possible levels of impurities such as water,ammonium chloride, acetamide, trifluoroacetic acid, acetonitrile, ethanol and DMSO.The product may therefore comprise impurities selected from one or more, e.g. all of:water, suitably in an amount of ≤ 5.0% ww, suitably ≤ 4.0% w / w, more suitably ≤ 2.5% w / w;ammonium chloride, suitably in an amount of ≤ 0.15% w / w, more suitably ≤ 0.05% w / w;acetamide, suitably in an amount of ≤ 0.15% w / w, more suitably ≤ 0.075% w / w;trifluoroacetic acid, suitably in an amount of ≤ 0.2% w / w, more suitably ≤ 0.1% w / w ;acetonitrile, suitably in an amount of ≤ 0.5% w / w, more suitably ≤ 0.01% w / w;ethanol, suitably in an amount of ≤ 1.0% w / w, more suitably in an amount of ≤ 0.5% w / w; andDMSO, suitably in amount of ≤ 0.5% w / w, more suitably ≤ 0.05% w / w.Although the Compound I cation chloride hydrochloride salt is more stable than theCompound I cation chloride dihydrochloride salt, it is still preferably stored in dry conditionsunder an inert atmosphere, especially under nitrogen. The Compound I cation chloride hydrochloride salt is hygroscopic and contact with moisture should be avoided.Therefore, further aspects of the invention provide a sealed container containing theCompound I cation chloride hydrochloride salt or a free-flowing powder comprising theCompound I cation chloride hydrochloride salt; or the Compound I cation chloridehydrochloride salt obtainable by or obtained by the process described above under a dryinert atmosphere, especially under a dry nitrogen atmosphere.The invention also provides the Compound I cation chloride hydrochloride salt or a free-flowing powder comprising the Compound I cation chloride hydrochloride salt; or theCompound I cation chloride hydrochloride salt obtainable by or obtained by the processdescribed above in a sealed container under a dry inert atmosphere, for example a nitrogen atmosphere.The Compound I cation chloride hydrochloride salt has the advantage that it is stable whenkept under a nitrogen atmosphere and is suitable for use in a pharmaceutical composition, especially a composition intended for topical administration to the lungs. Therefore, the invention also provides a pharmaceutical composition comprising a solutionof the Compound I cation chloride hydrochloride salt or a free-flowing powder comprisingthe Compound I cation chloride hydrochloride salt or the Compound I cation chloridehydrochloride salt obtainable by or obtained by the process described above in an aqueoussolvent. Suitably, the pharmaceutical composition is formulated for topical administration to the lungs, for example using a nebuliser.Suitably, the pharmaceutical composition has a pH of 5 to 7, especially a pH of 5.7 to 6.3.Suitably, it comprises a buffer, for example a citrate buffer.The pharmaceutical composition may be stored in a sealed container under an inertatmosphere such as nitrogen.The Compound I cation chloride hydrochloride salt and the pharmaceutical compositioncomprising it are useful for the treatment or prevention of respiratory diseases and conditions, especially cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, asthma and primary ciliary dyskinesia. The active species in pharmaceutical compositions is the Compound I cation and doses as described herein are calculated in μg or μg / kg of the Compound I cation rather than of the Compound I cation chloride hydrochloride salt.The Compound I cation has proved to have a number of advantages in the clinic comparedwith ENaC inhibitors which are known from the prior art. In a randomised trial with healthysubjects, administration of the Compound I cation led to no serious adverse events, evenat the highest doses administered. In particular, there were no clinically significant changesin ECGs, vital signs, clinical lab assessments or spirometry and there was nohyperkalaemia in any of the subjects (Danahay 2025).The Compound I cation also has an excellent pharmacokinetic profile. In particular, theCompound I cation has been shown to have very low membrane permeability, whichmeans that following inhalation it persists in the airway lumen / lung tissue for considerablylonger than other ENaC inhibitors, including VX-371, BI 1265162 and AZD5634 and willtherefore have a longer duration of action than these compounds. Furthermore, the lowpermeability led to relatively low mean plasma concentrations of Compound I cation.Plasma concentrations peaked at 1-2 hours post inhaled dose with a prolonged absorptionphase. The low plasma concentrations indicate that side effects arising from ENaCinhibition outside the lungs, for example in the kidneys, may be lower than for ENaCinhibitor compounds having higher permeability and that the Compound I cation maytherefore have a larger therapeutic window than these other ENaC inhibitors.Compound I cation was tested in a sheep mucociliary clearance (MCC) model in order todetermine the lowest dose required to stimulate 99mTc-SC clearance by ≥ 20% at 4 hoursafter inhaled drug administration. The predicted clinical dose for Compound I cation(suitably administered as the chloride hydrochloride) calculated from the sheep MCCmodel was found to be considerably lower than the highest dose administered in the studyon healthy subjects, demonstrating that the Compound I cation has a good safety profileand the potential to be used in a twice daily dosing regimen or even in a once daily dosingregimen.The ENaC Inhibitors VX-371, BI 1265162, AZD5634 and QBW276 were also tested in thesheep MCC model and it was found that, although the human in vitro IC50 was higher forthe Compound 1 cation than for these other ENaC inhibitors, the predicted effective clinicaldose of the Compound I cation as the chloride hydrochloride salt from the sheep MCCmodel was significantly lower than the predicted effective doses for BI 1265162, AZD5634and QBW276 and similar to that for VX-371 (Danahay, 2025). It is highly probable that thereduced dose also arises from the particularly low permeability of the Compound I cationcompared with the other ENaC inhibitors.The pharmaceutical composition may be provided as a unit dosage form. In one embodiment, the unit dosage form is intended for twice daily dosing and comprises from100 μg to 5400 μg of the Compound I cation, which is suitably included in the unit dosageform as the chloride hydrochloride salt or free-flowing powder comprising the Compound Ication chloride hydrochloride salt or the Compound I cation chloride hydrochloride saltobtainable by or obtained by the process described above. More suitably, the unit dosageform comprises 150 μg to 5400 μg, 200 μg to 4500 μg or 250 μg to 3000 μg of theCompound I cation (included in the unit dosage form as the Compound I cation chloridehydrochloride salt or free-flowing powder comprising the Compound I cation chloridehydrochloride salt or the Compound I cation chloride hydrochloride salt obtainable by orobtained by the process described above). In an alternative embodiment, the unit dosage form is intended for once daily dosing andcomprises from 150 μg to 10800 μg of the Compound I cation (included in the unit dosageform as the Compound I cation chloride hydrochloride salt or free-flowing powdercomprising the Compound I cation chloride hydrochloride salt or the Compound I cationchloride hydrochloride salt obtainable by or obtained by the process described above).More suitably, the unit dosage form comprises 200 μg to 10800 μg, 300 μg to 9000 μg or500 μg to 6000 μg of the Compound I cation (included in the unit dosage form as theCompound I cation chloride hydrochloride salt or free-flowing powder comprising theCompound I cation chloride hydrochloride salt or the Compound I cation chloridehydrochloride salt obtainable by or obtained by the process described above).In a further aspect of the invention there is provided the Compound I cation, suitably in theform of the chloride hydrochloride salt or a free-flowing powder comprising the CompoundI cation chloride hydrochloride salt or the Compound I cation chloride hydrochloride saltobtainable by or obtained by the process described above, or a pharmaceuticalcomposition as described above, for use in the treatment of a respiratory disease orcondition.There is also provided the use of the Compound I cation, suitably in the form of the chloridehydrochloride salt or a free-flowing powder comprising the Compound I cation chloridehydrochloride salt or the Compound I cation chloride hydrochloride salt obtainable by orobtained by the process described above, or a pharmaceutical composition as describedabove, in the manufacture of a medicament for the treatment of a respiratory disease orcondition. There is also provided a method for the treatment of a respiratory disease or condition, the method comprising administering to a patient in need of such treatment an effectiveamount of the Compound I cation, suitably in the form of the chloride hydrochloride salt ora free-flowing powder comprising the Compound I cation chloride hydrochloride salt or theCompound I cation chloride hydrochloride salt obtainable by or obtained by the processdescribed above, or a pharmaceutical composition as described above.Suitably, the respiratory disease or condition is selected from cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, asthma and primary ciliary dyskinesia.Suitably, the Compound I cation is administered to a patient in an amount of 150 μg to10800 μg per day, for example 200 μg to 10800 μg, 300 μg to 9000 μg or 500 μg to 6000μg per day.Alternatively, a dose may be calculated according to the weight of the patient and the Compound I cation may be administered to a patient in an amount of 2.5 to 180 μg / kg perday, for example 3.3 to 180 μg / kg per day, 50 to 150 μg / kg per day or 8.0 to 100 μg / kg perday.In some cases, administration of the Compound I cation (as the chloride hydrochloridesalt) will be once daily. In other cases, administration will be twice daily. The Compound Ication (as the chloride hydrochloride salt) may be administered in the form of apharmaceutical composition as described above comprising the Compound I cationchloride hydrochloride salt or a free-flowing powder comprising the Compound I cationchloride hydrochloride salt or the Compound I cation chloride hydrochloride salt obtainableby or obtained by the process described above. The composition may therefore be formulated for once daily or twice daily dosing. Brief Description of the DrawingsFigure 1 is a series of plots showing the effects of single inhaled acute doses (a) or repeatdoses (BiD for 3.5 days) (b) of the Compound I cation on the whole-lung clearance of99mTc-SC particles in conscious, healthy sheep (mean data ± SEM; n=2-5 sheep per group) as described in Example 7.Figure 2 shows the effect of a single acute dose (a) or once daily repeat doses (b) of theCompound I cation or comparator compounds amiloride or ET001976 on blood K+levels([K+]blood) administered by intra-tracheal instillation to rats as described in Example 6.Figure 3 is a series of plots showing (a) plasma levels of the Compound I cation followingeither intravenous (IV) or intratracheal (IT) administration (0.3 mg / kg) to rats as described in Example 6; (b) absolute levels of Compound I cation measured in bronchoalveolar lavage (BAL) fluid or lung tissue of rats following IT administration (0.3 mg / kg) as described in Example 6.Figure 4 shows (a) plasma levels of Compound I cation after inhaled dosing to sheep(12.2 µg / kg, mean data ± SEM; n=3 sheep); (b) effects of a single inhaled dose ofCompound I cation on99mTc-sulfur colloid clearance in sheep, assessed at either 4-6h, 8- 10h or 16-18h after drug administration (mean data ± SEM; n=3 sheep). Groups were compared for differences in99mTc clearance from vehicle control at 60 and 120min usingone-way ANOVA with post-hoc Dunnett’s test, *P=0.0008, **P=0.0001 ***P<0.0001 asdescribed in Example 7Figure 5 shows levels of Compound I cation, VX-371 and BI 1265162 measured inbronchoalveolar lavage (BAL) fluid (a) or lung tissue of rats (b) following IT administration (0.3 mg / kg) (mean data ± SEM; n=3-8 rats per group) as described in Example 9c.Figure 6 shows plasma concentrations of Compound I cation after administration of doses of 0.06 mg, 0.18 mg, 0.6 mg, 1.8 mg, 3.6 mg, 7.2 mg and 10.8 mg by inhalation to a small group of healthy human subjects.Figure 7 is a comparison of the mean plasma concentrations of Compound I cation andthe legacy compound BI 1265162 after administration by inhalation to healthy volunteers at a dose of 0.6 mg (A) shows plasma concentrations from 0 to 1440 minutes, while (B) shows the period of 0 to 180 minutes in greater detail. EXAMPLES The invention will now be described in greater detail with reference to the examples. Abbreviations BEGM Bronchial Epithelial Growth mediumDMEM Dulbecco’s Modified Eagle mediumDMF N,N-dimethyl formamideDMSO DimethylsulfoxideEtOH EthanolHBE Human bronchial epithelial cellsHPLC High performance liquid chromatographyIPA isopropanolMeCN Acetonitrilemin Minute(s)PBS Phosphate buffered salineRT Room temperatureTBME Tertiary butyl methyl etherTFA Trifluoroacetic acidExample 1 – 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetate di(trifluoroacetic acid); saltexchange of trifluoroacetate counter ion to chlorideThe material from Example 50 of WO 2018 / 096325 was lyophilised and the lyophilised material was used as the starting material in the procedure set out below.In WO 2018 / 096325, the material from Example 50 is described as the Compound I aniontrifluoroacetic acid trifluoroacetate salt. However, this product has subsequently been re-analysed and has been shown to be Compound I cation trifluoroacetate di(trifluoroaceticacid). To a solution of purified 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6- (4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3- diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetate di(trifluoroacetic acid) in 1:4 volume to volume (v / v) ethanol / acetonitrile (2 vol) is added, under nitrogen, rapidly, a 0.25 M solution of hydrochloric acid in 1:4 v / v ethanol / acetonitrile (19.5 vol). After stirring for 18 hours the reaction mixture is added to TBME (32.3 vol) with stirring. Further TBME (3 x 5 vol) is added (total 47.3 vol) with 5 minutes stirring time between each addition. The ensuing slurry is stirred at 23-27°C for a minimum of 4 hours. The ensuing precipitate is collected by filtration, washed with TBME (4 x 5.0 vol) and dried under reduced pressure at 30°C for 24 hours. The isolated product is re-suspended in TBME (15 vol), stirred for a further 18 hours at 45°C, and the product is collected by filtration, washed with TBME (3 x 5.0 vol) and dried under reduced pressure at room temperature to provide 2-[({3-amino-5H- pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumchloride dihydrochloride (isolated intermediate) as a yellow to orange amorphous solid.Using these process steps, starting from 239.77 g of Compound I cation trifluoroacetate di(trifluoroacetic acid), 169.01 g of Compound I cation chloride dihydrochloride salt was obtained (88%); MS: m / z = 818.4 [M+]. HPLC Method An Agilent 1260 HPLC with a binary pump was used. Column Waters xSelect CSH C18150 x 4.6 mm, 3.5 μmInjection volume 5 μLWavelength 262 nmMobile Phase A H2O / MeCN / TFA (95 / 5 / 0.05, v / v / v)Mobile Phase B H2O / MeCN / TFA (5 / 95 / 0.05, v / v / v)Method Time (min) %A %B0.0 100 0 2.0 100 0 17.0 85 15 32.0 0 100 35.0 0 100 35.1 100 0 40.0 100 0 Flow rate 1 mL / minColumn Temperature 40°CRun Time 40 minWas Vial Sample diluentAnalysis of the product showed the following for 3 batches: Purity by HPLC 98.1-98.5% areaTFA content by HPLC 0.68-0.93 % w / wChloride content by agentometric titration 10.5-13.5 % w / wResidual solvents by GC-HS Acetonitrile4796-10469 ppm TBME 1549-3385 ppm DMF <16-<153 ppm Ethanol 5672-8722 ppm Residual NH4Cl by 1H NMR 0.75-6.46% w / wExample 2 – Development of method for conversion of Compound I cation chloridedihydrochloride salt to Compound I cation chloride hydrochloride salt andpurification The product of Example 1 contains impurities, particularly ammonium chloride andacetamide. The initial aim of the experiments described below was to find a suitableprocess for the removal of these impurities. The methods tested were a slurry method anda precipitation method. Surprisingly, it was found that the precipitation method, in additionto the removal of impurities, produced a product which is the Compound I cation chloride hydrochloride salt rather than the chloride dihydrochloride salt which is the product of Example 1. Slurry MethodIn the slurry experiments S2 to S8, test material was charged to a flask under nitrogen, aliquid in which the test material is insoluble was added and the mixture stirred at the temperature specified to form a slurry with a good vortex. After the specified amount oftime, the slurry was filtered and dried and the ratio of Compound I cation : ammoniumchloride was calculated in both the solid and the filtrate. The % weight of ammonium chloride in the solid was also calculated. The results are set out in Table 1. The following slurry experiments were carried out. ^Experiment S1: analysis of the product obtained from Example 1.^ Experiment S2: the product from Example 1 was mixed with 10 volumes of ethanolto form a slurry and stirred for 0.5 hours at room temperature. ^Experiment S3: the product of Experiment S2 was mixed with 10 volumes ofethanol to form a slurry and stirred for 4 hours at room temperature. ^Experiment S4: the product from Example 1 was mixed with 10 volumes ofisopropanol to form a slurry and stirred for 2 hours at room temperature. ^Experiment S5: the product from Example 1 was mixed with 20 volumes of ethanolto form a slurry and stirred for 16 hours at room temperature. ^Experiment S6: the product from Example 1 was mixed with 10 volumes of a 20:1mixture of ethanol:water to form a slurry and stirred for 16 hours at room temperature. ^Experiment S7: the product from Example 1 was mixed with 10 volumes of ethanolto form a slurry and stirred for 16 hours at room temperature. ^Experiment S8: the product from Example 1 was mixed with 10 volumes of ethanolto form a slurry and stirred for 16 hours at 40°C. The products obtained from Example 1 and Experiments S2, S3, S4, S5, S7 and S8 were free-flowing powders but the product of Experiment S6 was a glass.Table 1 – Slurry Experiments for preparation and purification of Compound I cationchloride hydrochloride saltExp Slurry mix MixingMixing Compound I % wt Compound I time temp cation:NH4Cl NH4Cl in cation:NH4Cl (h) molar ratio in solid molar ratio in solid filtrate S1 1:0.9 4.9S2 10 vol EtOH 0.5 RT 1:0.33 1.9 1:21.86Exp Slurry mix MixingMixing Compound I % wt Compound I time temp cation:NH4Cl NH4Cl in cation:NH4Cl (h) molar ratio in solid molar ratio in solid filtrate S3 Product of 4 RT 1:0.30 1.7 1:1.80S2 in 10 vol EtOH S4 10 vol IPA 2 RT 1:0.9 4.9 N / AS5 20 vol EtOH 16 RT 1:0.28 1.6 1:23.20S6 10 vol 20:1 16 RT 1:0.19 1.1 1:34.96EtOH:water S7 10 vol EtOH 16 RT 1:0.32 1.8S8 10 vol EtOH 16 40°C 1:0.34 1.9The analysis conducted in Experiment S1 showed that in the product obtained fromExample 1, the molar ratio of product to ammonium chloride was 1:0.9, i.e. ammoniumchloride was present in an amount of 4.9% w / w. The initial slurry using ethanol (Experiment S2) resulted in a significant drop in the ammonium chloride content to 1.9% w / w (1:0.33molar ratio). However, a second slurry of the product of Experiment S2 (Experiment S3)had little effect, with only a small further reduction in ammonium chloride content to 1.7%w / w (1:0.30 molar ratio). As shown in Experiment S6, the addition of a small amount of water to the slurry led to lower levels of ammonium chloride in the product but the solid obtained from the slurry step was a glass rather than a free-flowing powder. This was confirmed in additional experiments using 10 volumes of a liquid comprising ethanol or IPA and water at ratios of 10:1 or 15:1 and ethanol:water in ratios of 100:1 or 200:1 (not shown in table). As shown in Experiment S4, IPA was a less suitable choice than ethanol for the slurry. Increasing the temperature to 40°C did not lead to any significant improvement, with the level of ammonium chloride in the final product being almost identical to that of the room temperature experiments (see Experiments S7 and S8). The results of the slurry experiment suggest that the most suitable liquid for the slurry is ethanol. The addition of water to the slurry further reduces the amount of ammonium chloride in the product, but also leads to a product which is in a form which is difficult to isolate by filtration from the slurry. Precipitation Method A precipitation approach was also assessed. As the Compound I cation chloride dihydrochloride salt has limited solubility in organic solvents but good solubility in water and DMSO, initial dissolution was performed in either water or DMSO with precipitation initiated by addition of ethanol to the water or DMSO solution. In these initial experiments solution in 2 volumes of water followed by precipitation with 20 volumes of ethanol led to a reduction in the molar ratio of the Compound I cation to ammonium chloride of from 1:0.9 (4.9 % w / w ammonium chloride) to 1:0.14 (0.80% w / w ammonium chloride), whiledissolution in 3 volumes of DMSO followed by precipitation with 20 volumes of ethanol ledto a reduction in the molar ratio to 1:0.045 (0.3% w / w ammonium chloride). However, the product obtained was a gum or a mixture of a gum and a solid. Therefore, additional Experiments were carried out in which a solution of the Compound Ication chloride dihydrochloride salt in a mixture of DMSO and ethanol (3 vol, 1:1) wasadded into ethanol (54 vol) or IPA (54 vol) instead of adding the anti-solvent to the solution.A series of precipitation experiments is described below. This series of experimentscompared the products obtained by changing the order of addition and the effects of furtherprocedures carried out on the precipitated product. The results are shown in Table 2.^ Experiment P1: analysis of the product obtained from Example 1.^ Experiment P2: 36 volumes of ethanol was added to a solution of Compound Ication chloride dihydrochloride salt in 2 volumes of water + 2 volumes of ethanol. ^Experiment P3: a solution of Compound I cation chloride dihydrochloride salt in 3volumes of DMSO + 3 volumes of ethanol was added to 54 volumes of ethanol.^ Experiment P4: slurry of the product of Experiment P3 in 10 volumes of ethanolusing the Slurry Method set out above for Experiment S2.^ Experiment P5: a solution of Compound I cation chloride dihydrochloride salt in 3volumes of DMSO + 3 volumes of isopropanol was added to 54 volumes of isopropanol. ^Experiment P6 a solution of the product of Experiment P5 in 3 volumes of DMSO+ 3 volumes of isopropanol was added to 54 volumes of isopropanol. ^Experiment P7: slurry of the product of Experiment P5 in 10 volumes of isopropanolusing the Slurry Method set out above for Experiment S4Table 2 – Precipitation Experiments for further purification of Compound I cation chloride dihydrochloride saltE N T -PExp Conditions Compound I3 % wt NH4Cl in % wt Compound I 693 cation:NH4Cl solid DMSO in cation:NH4Cl molar P C molar ratio in solid ratio in filtrate T solid P1 1:0.9P2 Addition of 36 vol EtOH to Compound I cation chloridedihydrochloride salt dissolved in 2 vol water + 2 vol EtOHP3 Compound I cation chloride dihydrochloride salt 1:0.0225 0.13 6.2 1:8dissolved in 3 vol DMSO + 3 vol EtOH and then addedto 54 vol EtOH30 P4 Slurry of the product of P3 in 10 vol EtOH NH4Cl belowNH4Cl below 0.10 detection limit detection limit P5 Compound I cation chloride dihydrochloride salt 1:0.135 0.77 6.9 Only NH4Cldissolved in 3 vol DMSO + 3 vol IPA and then added to significant 54 vol IPA P6 Product of P5 dissolved in 3 vol DMSO + 3 vol IPA addedNH4Cl below NH4Cl belowNot run 1:18to 54 vol IPA detection limit detection limit P7 Slurry of the product of P6 in 10 vol IPA NH4Cl belowNH4Cl below 1.32 detection limit detection limit

[0002] Surprisingly, Experiments P2 to P7 yielded the Compound I cation chloride hydrochloride salt rather than the chloride dihydrochloride salt which was used to form the solutions for Experiments P2, P3 and P5. The process of Experiment P2, in which an anti-solvent (ethanol) was added to a solutionof Compound I cation salt resulted in a product which was an unfilterable gum. However,reversing the order of addition as in Experiment P3 such that the solution of Compound Ication salt was added to the anti-solvent greatly improved the quality of the precipitate forDMSO alcohol mixtures. The products obtained from Experiments P3, P5 and P6 werefilterable solids, which could be easily separated from the anti-solvent, and washed anddried to give a free-flowing powder. The products obtained from the slurries of P4 and P7were also filterable solids, which could be easily separated from the anti-solvent, and washed and dried to give a free-flowing powder. Precipitation of the Compound I cation chloride dihydrochloride salt from solution inDMSO / EtOH by addition of the solution to ethanol is able to lower the ammonium chloridecontent to less than 0.5% w / w on the first pass, although significant levels of DMSO remained in the product (Experiment P3). However, the DMSO content can be reduced using a further ethanol slurry (Experiment P4). DMSO / IPA solutions could also be used (see Experiment P5) but were less effective than DMSO / ethanol and two precipitations were required to reduce the ammonium chloride to a level not detectable by NMR (see Experiment P6). An IPA slurry could also be used tocontrol the level of DMSO in the product (Experiment P7) although it was not as effectiveas an ethanol slurry.Example 3 – Optimised method for purification and conversion of Compound Ication chloride dihydrochloride salt to Compound I cation chloride hydrochloridesalt The method was carried out using a batch of 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2- yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino} piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride dihydrochloride (Compound I cation chloride dihydrochloride salt) prepared according to the method ofExample 1. The ammonium chloride content of the starting material was 0.7% w / w, which was lower than for other batches produced by the same method, where the ammoniumchloride content varied from about 1.0 to 6.3% w / w. The water content was 2.1% w / w.(i) Compound I cation chloride dihydrochloride salt was suspended in ethanol (10 vol)under nitrogen and the slurry was stirred for 2 hours at 15–25°C. The solids were filteredunder nitrogen, washed with ethanol (2 x 2 vol) and dried under nitrogen.(ii) The solids from (i) were added to a stirred mixture of ethanol containing ~0.5% w / wwater (3.5 vol), DMSO (2.5 vol) and water (0.25 vol) at 15–25°C under nitrogen. Thesolution obtained was clarified (glass microfibre filter) and added, dropwise, overapproximately 45 minutes, to a stirred solution of ethanol containing ~0.5% w / w water (54vol) at 15–25°C. The mixture was stirred for a further 1 hour and the solids (Compound Ication chloride hydrochloride salt) collected by filtration under nitrogen, washed withethanol containing ~0.5% w / w water (5 x 5 vol) and dried at 15–25°C under nitrogen.(iii) The solids from (ii) were suspended in ethanol containing ~0.5% w / w water (10 vol)and stirred under nitrogen at 15–25°C for 1 hour. The solids were filtered under nitrogenand washed with ethanol containing ~0.5% w / w water (2 x 2 vol). The solids were driedovernight on the filter under a nitrogen stream. The dried solids were passed through a500 μm screen, under nitrogen, to yield Compound I cation chloride hydrochloride salt asa yellow to orange amorphous solid. If necessary to control residual ethanol to sufficiently low levels, Compound I cationchloride hydrochloride salt can be left in a covered tray open to the atmosphere for asuitable period of time (residual ethanol is replaced by water in this procedure), followed by drying at up to 30°C under reduced pressure for 24 hours. The dried solids are passed through a 500 μm screen, under nitrogen, to yield the product. Using these 3 process steps, starting from 170 g of Compound I cation chloridedihydrochloride salt, 113.1 g of Compound I cation chloride hydrochloride salt wasobtained (69%).Example 4 – Stability TestingStability testing was carried out on the Compound I cation chloride hydrochloride andcompound I cation chloride dihydrochloride salts. The test substances were stored indouble polyethylene bags within a heat-sealed aluminium / polyethylene bag in an HDPEdrum at either 25°C / 60% relative humidity (RH) or 5°C / ambient relative humidity for aperiod of up to 24 months for the Compound I cation chloride hydrochloride salt. TheCompound I chloride dihydrochloride salt was stored for only 6 months. The results of thestability tests are set out in Tables 3 to 6.Table 3 – 25°C / 60% RH Stability Data for Compound I cation chloridedihydrochloride salt Initial 1 month 3 months 6 monthsAppearance Orange As initial As initial As initialsolid Assaya (by HPLC) 95.2 % w / w 91.8 % w / w 89.7 % w / w 84.4 % w / wTotal related 3.54% 5.72% 9.59% 15.18% substances (by HPLC)aon an anhydrous solvent free basisTable 4 – 5°C / ambient RH Stability Data for Compound I cation chloridedihydrochloride salt Initial 1 month 3 months 6 monthsAppearance Orange As initial As initial As initialsolid Assaya (by HPLC) 95.2% w / w 94.4% w / w 93.6% w / w 94.4% w / wTotal related 3.54% 4.07% 4.41% 4.52% substances (by HPLC)aon an anhydrous solvent free basisTable 5 – 25°C / 60% RH Stability Data for Compound I cation chloride hydrochloridesalt Initial 1 month 3 months 6 months 24 monthsAppearance Yellow As initial As initial As initial As initialsolid Assaya (by HPLC) 98.5 %97.7% 96.9% w / w 97.9% 98.6% w / w w / w w / w w / wInitial 1 month 3 months 6 months 24 monthsTotal related 1.55% 1.61% 1.59% 1.65% 1.67% substances (by HPLC)aon an anhydrous solvent free basisTable 6 – 5°C / ambient RH Stability Data for Compound I cation chloridehydrochloride salt (Batch #1)Initial 1 month 3 months 6 monthsAppearance Yellow solid As initial As initial As initialAssaya (by HPLC) 98.5 % w / w 97.4% w / w 97.4% w / w 96.9% w / wTotal related 1.55% 1.58% 1.60% 1.70% substances (by HPLC) 9months 12 months 18 months 24 monthsAppearance As initial As initial As initial As initialAssaya (by HPLC) 97.6% w / w 97.9% w / w 98.2% w / w 99.0% w / wTotal related 1.62% 1.62% 1.68% 1.60% substances (by HPLC)aon an anhydrous solvent free basisTable 7 – 5°C / ambient RH Stability Data for Compound I cation chloridehydrochloride salt (Batch #2)Initial 9 months 12 months 18 monthsAppearance Yellow solid As initial As initial As initialAssaya (by HPLC) 98.5 % w / w 97.6% w / w 97.9% w / w 98.2% w / wTotal related substances 1.55% 1.62% 1.62% 1.68% (by HPLC) Initial 24 months 36 Months 48 MonthsAppearance Yellow solid As initial As initial As initialAssaya (by HPLC) 98.5 % w / w 99.0% w / w 97.9% w / w 97.2% w / wTotal related substances1.55% 1.60% 1.61% 1.82%(by HPLC) Further stability tests of the for the Compound I cation chloride hydrochloride salt were carried out at -80°C and -20°C, although the results are not shown. As shown in Table 3, the stability results for the Compound I cation chloride dihydrochloride salt at 25°C / 60% RH show significant degradation with an increase of > 6% in impurities detected by HPLC after 3 months and > 11% after 6 months, with a concomitant reduction in assay value. The results after storage at 5°C are shown in Table 4 and reveal a much slower rate of degradation, as evidenced by an increase in total impurity content by HPLC of ~ 1% after 3 and 6 months. No significant changes in appearance, chloride content or physical form by XRPD were detected at 5°C after 6 months. The results for the Compound I cation chloride hydrochloride salt suggest that it has notably improved stability over the Compound I cation chloride dihydrochloride salt, as no significant increase in total related substances has been detected after 12 months storageat -80°C or 24 months storage at -20°C or 48 months storage at 5°C (see Tables 6 and 7)or 6 months at 25°C / 60% RH (see Table 5). The only detectable significant trend afterstorage of the compound I cation chloride hydrochloride salt is a small increase in water content at all conditions (highest after storage at 25°C / 60%RH). As shown in Table 5, a 24-month sample was tested after storage at 25°C / 60% RH. The results further support the good stability of this salt of the Compound I cation with no significant evidence of degradation being observed in the stability parameters tested.The assays show that the Compound I cation chloride hydrochloride salt has excellentstability. When stored at 5°C and ambient relative humidity, it has been shown to be stable for 4 years and even when stored under more extreme conditions (25°C / 60% RH) it is stable for up to 2 years. This indicates that the Compound I cation chloride hydrochloride salt has excellent handling properties which make it suitable for use as a pharmaceutical since it can be stored for at least 2 years without refrigeration and for up to 4 years at 5°C.When required, it can be made up into a suitable formulation for administration to a patient.BIOLOGICAL EXAMPLESAs described below, the Compound I cation chloride hydrochloride salt was used in someof the biological examples, while in others, the Compound I trifluoroacetate di(trifluoroacetic acid) salt was used. However, the active species in both of these salts is the Compound I cation.Example 5 – Measurement of ENaC Inhibitory Activitya. Cell cultureHuman bronchial epithelial (HBE) cells Cryopreserved HBE cells were thawed and seeded into 2 plastic T-75 flasks and grown in 20 mL bronchial epithelial cell growth medium (BEGM; Lonza, UK) supplemented with bovine pituitary extract (52 µg / mL), hydrocortisone (0.5 µg / mL), human recombinant epidermal growth factor (0.5 ng / mL), epinephrine (0.5 µg / mL), transferrin (10 µg / mL), insulin (5 µg / mL), retinoic acid (0.1 µg / mL), triiodothyronine (6.5 µg / mL), gentamycin (50 µg / mL), and amphotericin B (50 ng / mL). Medium was changed 6-8h after seeding the cells and then every 48 hours until cells were 90% confluent. Cells were then passaged and seeded (8.25x104cells / insert) on polycarbonate Snapwell inserts (Costar®) in differentiation media containing 50% DMEM in BEGM with the same supplements as above but without triiodothyronine and a final retinoic acid concentration of 50 nM (all-trans retinoic acid; Sigma-Aldrich, UK). Cells were maintained submerged for the first 7 days in culture, after which time they were exposed to an apical air interface for the remainder of the culture period. During the period at air-liquid interface, cells were fedwith a DMEM:F12 (Life Technologies, UK) media containing 2% Ultroser™ G (v / v; PallBioSepra, France). At all stages of culture, cells were maintained at 37°C in 5% CO2 in an air incubator. From day 7 after establishing the air-liquid interface, cells were washed 3x each week with warmed PBS. PBS (0.5 mL) was added to the apical surface and incubated for 20-30 min before aspirating. Cells were used between days 7 and 21 after establishment of the apical-air interface. Cells were always fed the day before use in the Ussing chambers. Sheep tracheal epithelial cells Sheep epithelial cells were harvested from tracheas of euthanized ewes as previouslydescribed (Fulcher et al, 2013). P0 cells were expanded in PneumaCultTM Ex Plus medium(Stemcell Technologies, cat#05041) supplemented with gentamycin (50µg / mL, ThermoFisher Scientific) and amphotericin B (2.5 µg / mL, ThermoFisher Scientific) before seeding on Snapwell inserts (Corning®, cat# 3801) at a density of 2x105cells in ALI media. ALI media was prepared by mixing 1:1 DMEM (ThermoFisher Scientific, cat#11995-040) and LHC Basal Medium (ThermoFisher Scientific, cat#12677-027) and supplemented with (final concentrations): insulin (5µg / mL, FeF Chemicals), hydrocortisone (0.072µg / mL), transferrin (10µg / mL), triiodothyronine (0.0067µg / mL), epinephrine (0.6µg / mL, Corning), phosphoethanolamine (70µg / mL), ethanolamine (30µg / mL), bovine serum albumin (0.5mg / mL), penicillin / streptomycin (100U / 100µg / mL, ThermoFisher Scientific), epidermal growth factor (0.5ng / mL), retinoic acid (5x10-8M), ferrous sulfate (1.5x10-6M), magnesium chloride (6x10-4M), calcium chloride (1.1x10-4M), zinc sulfate (3µM), silicone (0.5µM), selenium (30x10-9M), manganese (1X10-9M), molybdenum (1x10-9M), vanadium (5x10-9M), nickel sulfate (1x10-9M), and tin (0.5x10-9M). All additives were purchased from Sigma- Aldrich unless otherwise specified. The cells were maintained in submerged culture until confluency upon which air-liquid interface was initiated. The media was replaced every other day and the apical surfaces were washed using DPBS (Corning, cat#21-030-CV). The cells were allowed to differentiate for 2-3 weeks before the experiments were carried out.b. Short circuit current assaysAt the stated time in culture, the ion transport properties of HBE cells or sheep tracheal epithelial cells were examined using the short circuit current technique. Cells on Snapwell inserts were mounted in Vertical Diffusion Chambers (Costar) and were bathed with continuously gassed Ringer solution (5% CO2 in O2; pH 7.4) maintained at 37°C containing (in mM): 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose. The solution osmolarity was always between 280 and 300 mosmol kg H2O-1for all physiological salt solutions used. Cells were voltage clamped to 0 mV (model EVC4000; WPI) and the short-circuit current (ISC) was measured. Data were recorded using aPowerLab workstation (ADInstruments, UK). direct ENaC blocker test compounds(including Compound I cation in the form of the trifluoroacetate di(trifluoroacetic acid) salt)were added to the apical chamber from a 1000-fold stock solution (prepared in DMSO) to achieve a cumulative concentration response in terms of the inhibition of the basal ISC. IC50 values were calculated assuming that the cumulative current inhibition achieved with thetest compound reflected the total ENaC-mediated ISC. ResultsCompound I cation inhibited ENaC function in cultured HBE cells with an IC50 value of 57.5nM and in cultured sheep tracheal epithelial cells with an IC50 value of 30.5 nM.Example 6 – Rat hyperkalaemia and pharmacokineticsThe test compound used in Study 1 and Study 2 was Compound I cation in the form ofthe trifluoroacetate di(trifluoroacetic acid)..Male Sprague-Dawley rats (295-340 g) (Charles River Laboratories, Harlow, UK) were used for these studies. All studies were performed in accordance with the guidelines of the United Kingdom Home Office on the operation of the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 (the Act). Animals were acclimatized for a minimum of 7 days before study, maintained on a 12h light-dark cycle at a temperature of 21 ± 2°Cand humidity of 55 ± 5%. Animals were allowed food (Teklad™ 2014C, pelleted diet) andwater ad libitum.a. Study 1Rats were dosed with vehicle (5% D-glucose in sterile water) or test compound by intra-tracheal instillation (IT) as previously described (Coote et al, 2015). Samples of blood fromthe tail vein were collected throughout the studies and changes in blood potassium levelswere measured as previously described (Coote et al, 2015) although for some studies aRadiometer ABL80 CO-OX Blood Gas Analyser was used. A 2-way analysis of variance(ANOVA; corrected for repeat measures) with a post-hoc Dunnett’s test was used to test for significant differences in absolute blood potassium levels between vehicle and test compound treated groups across the duration of the studies (Graph Pad Prism). Significance was assumed when P<0.05. ResultsThe effects of a single dose or once daily repeat doses of Compound I cationtrifluoroacetate di(trifluoroacetic acid) solution on blood K+levels ([K+]blood) administeredby intra-tracheal instillation to rats (mean data ± SEM; n=6 rats per group) was measuredand is shown respectively in Figures 2a and 2b. Groups were compared for differences in[K+]blood using two-way ANOVA with post-hoc Dunnett’s test, *P<0.01. ET001976 is apositive control ENaC blocker disclosed in WO 2014 / 099673 as compound II-d and which has the structure: . A single dose of Compound I cation (1,416 μg / kg, i.t.) stimulated a rise in blood potassium levels in rats at 6h after dosing which had returned to normal levels by 24h (Figure 2a). A lower dose of 425 μg / kg (i.t.) was without significant effect. Once daily dosing of Compound I cation trifluoroacetate di(trifluoroacetic acid) (142-425 μg / kg, i.t.) for 7 days, was without effect on blood potassium, whereas, in contrast, dosing with ET001976 led to a marked increase in blood potassium levels (Figure 2b).b. Study 2In separate studies, jugular vein cannulated rats, were similarly dosed with testcompounds by either intra-venous (IV) or intratracheal (IT) routes at a dose of 0.3 mg / kg.Serial blood samples were collected into K2EDTA blood tubes and plasma was stored at - 20°C until analysis by LC-MS / MS. Groups of animals were also euthanized at 1, 3 and 6h after IT. dosing, and bronchoalveolar lavage (BAL) was performed (3x 4 mL normal saline) to recover compound from the airway lumen. BAL fluid was snap frozen together with the lungs and stored at -20°C until analysis of test compounds by LC-MS / MS. Results Figure 3a shows the plasma levels of Compound I cation following either intravenous or intratracheal administration to rats at a dose of 0.3 mg / kg. Following IV dosing, there was a low volume of distribution (0.43 ± 0.13 L / kg [mean ± SD, n=3]) with a half-life for plasma clearance of 1.9 ± 0.5h (mean ± SD, n=3). The half-life for plasma clearance after IT dosing was increased to 5.9 ± 1.3h (mean ± SD, n=3), which is consistent with absorption out of the lung being rate limiting for compound clearance. Absolute levels of Compound I cation measured in BAL fluid or lung tissue of rats is shownin Figure 3b. Analysis of Compound I cation in BAL fluid after IT dosing showed levelsequating to approximately 50% of the administered dose remaining at 1h that was reduced to approximately 13% by 6h.Example 7 – Sheep Mucociliary Clearance (MCC)In these experiments, the results presented in Figures 1a, 1b were obtained with thetrifluoroacetate di(trifluoroacetic acid) salt and the results in Figure 4a were obtained usingthe Compound I cation in the form of the chloride hydrochloride salt. The 8-10 hour dataand the 16-18 hour data presented in Figure 4b were also obtained using the chloridehydrochloride salt but the 4-6 hour data is simply transcribed from Figure 1a (9.2 μ / kg dose) and therefore was obtained using the trifluoroacetate di(trifluoroacetic acid) salt.Whole lung MCC was measured in conscious sheep as previously described (Danahay etal, 2020). Test compounds were prepared as solutions and aerosolized for inhalation usinga jet nebulizer. Compound I cation and BI 1265162 (powder) were dissolved in sterile waterwhilst ethanol was used for all other compounds. Doses are shown as the nominal dose (total dose loaded into nebulizer) as the free-base equivalent. Time-matched, vehicle- specific controls were used throughout. Aerosolized technetium labeled sulfur colloid (99mTc-SC; 20 mCi) was administered to the sheep at various times after compound / vehicle administration and serial scintigraphic images were then obtained over a 2h period. A one- way ANOVA with post-hoc Dunnett’s test was used to test for significant differencesbetween vehicle and test groups at the 60- and 120-minute time-points after 99mTc-SCinhalation (Graph Pad Prism). Significance was assumed when P<0.05. Results Figures 1a and 1b illustrate the effects of both single, acute doses of Compound I cation (Figure 1a) or repeat doses (BiD for 3.5 days) (Figure 1b) on the whole-lung clearance of99mTc-SC particles in conscious, healthy sheep (mean data ± SEM; n=2-5 sheep pergroup).. Compound I cation or vehicle was administered 4h before starting themeasurement of MCC (i.e.4h before99mTc-SC administration). Groups were compared for differences in99mTc-SC clearance from vehicle control at 60 and 120min after99mTc-SC administration using one-way ANOVA with post-hoc Dunnett’s test, *P<0.05, **P<0.0001. A single dose of Compound I cation enhanced both the rate and magnitude of MCC whenassessed 4h after inhaled dosing to sheep (Figure 1a). Doses of ≥ 9.2 μg / kg resulted inthe clearance of approximately 20% of the99mTc-SC compared with approximately 10% in the time-matched vehicle control. On repeat dosing of Compound I cation twice daily for 3.5 days, the dose required to achieve full efficacy at 4h after the 7thand final dose was reduced to 2.1 μg / kg (Figure 1b).Figure 4a illustrates the plasma levels of Compound I cation after inhaled dosing to sheep(12.2 µg / kg, mean data ± SEM; n=3 sheep). Figure 4b shows the effects of a single inhaleddose of Compound I cation on99mTc-sulfur colloid clearance in sheep, assessed at either 4-6h, 8-10h or 16-18h after drug administration (9.2 to 13 µg / gk) mean data ± SEM; n=3 sheep). Groups were compared for differences in99mTc clearance from vehicle control at 60 and 120min using one-way ANOVA with post-hoc Dunnett’s test, *P=0.0008, **P=0.0001 ***P<0.0001.In sheep, inhaled Compound I cation displayed a similar pharmacokinetic profile in plasmato that observed in the rat (see Figure 4a for sheep and Figure 3a for rat). This is consistentwith a slow absorption of compound out of the lungs (half-life of 9.2 ± 8.7h; mean ± SD, n=3). This pharmacokinetic profile translated through to an extended duration of action ofCompound I cation in the sheep MCC model, where a single inhaled dose of 9.2-13 μg / kgresulted in accelerated MCC out to 16h after dosing (Figure 4b).As discussed above in Example 6, however, the pharmacokinetic studies in rats showedlow propensity to increase in blood potassium levels following IT administration ofCompound I cation. These data, together with the sheep MCC data suggested the potentialfor a meaningful safety window for Compound I cation chloride hydrochloride salt.Example 8 – Calculation of Predicted Nominal Dose in HumansIn the sheep MCC model, the lowest dose of the Compound I cation required to stimulate99mTc-SC clearance by ≥20%, at 4h after drug administration was 9.2 μg / kg.The minimally efficacious dose (MED) inducing ≥20% clearance, was then converted intothe sheep lung dose and subsequently the equivalent human lung dose (Table 8) using the following steps.1. Nominal sheep dose (total dose loaded into nebuliser) is multiplied by 0.5 toaccount for the 50% delivery efficiency of the sheep nebuliser system and provide a lung deposited dose (in µg / kg).2 The sheep lung dose (in µg / kg). is converted to a human lung dose by multiplyingby 60kg, as estimate of the average weight of a person with CF.3. From the equivalent human lung dose, an estimate of the required clinical dose toachieve this delivery was calculated based on the Pari eFlow® rapid nebuliser deviceused to administer the Compound I cation solution in the clinical study described byRussell et al, 2022. The efficiency of the Pari eFlow® device was assumed to be 50%(Pham et al, 2018).4. A ratio of the clinically used dose to the predicted, minimally required dose wascalculated for the Compound I cation chloride hydrochloride salt.Table 8 Nominal Sheep lung Equivalent Predicted Maximum sheep dose deposited human lung nominal dose actual clinical inducing ≥ dose [µg / kg] deposited dose using clinical dose 20% [µg] device [µg] administered clearance [µg] [µg / kg] 9.2 4.6 276 552 4,650Based on the safety profile of the Compound I cation chloride hydrochloride salt where adose of 4.65 mg BiD was well tolerated in a 14-day multiple ascending dose healthyvolunteer study (Russell et al, 2022), the data indicate that efficacious doses of up toapproximately 8-fold above the MED to provide a 4h duration of action can be achieved (Table 8).The data obtained in Biological Examples 5 to 8 suggest that the Compound I cationchloride hydrochloride salt is a potent ENaC blocker with a long duration of action in vivofollowing inhaled delivery. The MCC data for the Compound I cation (~10 µg / kg; see Example 7 and Figures 1 and 4b) illustrates sustained activity that would support a twice daily dosing regimen to provide 24h enhancement of MCC.It has been assumed by those working in the field that the translation of efficacious andlong-acting doses from a healthy sheep airway to the environment of a CF lung may bechallenging, as in the CF situation, factors such as excessive mucus production,increased plasma protein leak and non-homogeneous particle deposition may all contribute to establishing and maintaining a therapeutically relevant dose. In view of this,it has been assumed that it would therefore be desirable to administer therapies forcystic fibrosis at doses higher than the ‘sheep-predicted’ minimally efficacious dose (MED).However, results from a small clinical study show that after administration of theCompound I cation chloride hydrochloride salt by inhalation to both healthy volunteersand CF patients, plasma concentrations of the Compound I cation peak quite slowly andare then maintained at a high level for several hours. In the study conducted on healthyvolunteers, doses of 0.06 mg, 0.18 mg, 0.6 mg, 1.8 mg, 3.6 mg, 7.2 mg and 10.8 mg were administered to subjects and the plasma concentration of the Compound I cationwas measured over a 6 hour period. The results are shown in Figure 6. As can be seenfrom Figure 6, the pattern of concentration in the plasma was consistent for all doses of Compound I cation. For all doses, plasma concentration peaked at about 1 to 2 hours after administration and then declined only slowly. Similar results were found in CFpatients. This is an indication that the behaviour of Compound I cation in humans,whether healthy volunteers or CF patients, is similar to that in the sheep MCC studies inthat Compound I cation passes slowly from the lungs to the plasma and is thereforeretained in the lungs of both groups for a period of several hours after administration. This indicates that Compound I cation chloride hydrochloride salt is likely to be suitable for once or twice daily dosing and that a low dose may be sufficient to maintain the required concentration of Compound I cation in the lungs. Compound I cation has been well tolerated in Phase 1 single and multiple ascendingdose studies at doses up to and including 4.65 mg BiD (Russell et al, 2019), a dose thatis approximately 8x the predicted MED (Table 8). The observation that on repeat dosingof Compound I cation in sheep, there is a dose sparing effect (Figure 1b), suggests that the window between the predicted MED and the upper limit of dosing may be furtherexpanded and indicates that it will be possible to administer the Compound I cationchloride hydrochloride salt in doses that will be both safe and clinically effective.Example 9 – Comparisons with Other ENaC InhibitorsThe Compound I cation chloride hydrochloride salt was compared with several other ENaCinhibitors in a number of assays. The comparator compounds used were VX-371, BI1265216, AZD5634 and QBW276. The structures of these compounds were deducedbased on either external company disclosures or from analyses of the patent literature asshown in the Table 8 below. In these experiments, the Compound I cation trifluoroacetatedi(trifluoroacetic acid) salt was used to prepare a test solution. As noted above, however,the active species in the test solution is the Compound I cation and so the salt form used in the experiments is immaterial.Table 9 – Disclosure of Comparator CompoundsPatent Compound Example document VX-371 WO2013003386 Formula IaBI 1265162 WO2017028927 Example 2.04AZD5634 WO2015140527 Example 2QBW276 WO 2012035158 Example 1.0The structures are shown below together with the structure of the Compound I cation. a. ENaC Inhibitor ActivityThe IC50 values were measured using the methods described in Example 5 above. The invitro potency of the ENaC blockers used for the present studies is shown in Table 10. MeanIC50 values (± standard error of the mean) were generated from concentration-response studies using human bronchial or sheep tracheal epithelial cell cultures.Table 10 – Human and Sheep IC50 Values for ENaC blockersCompound Human IC50 Sheep IC50Compound I cation (in the form ofthe trifluoroacetate di(trifluoroacetic57.5 ± 19.0 nM (n=31) 30.5 ± 9.2 nM (n=3)acid salt) VX-371 14.7 ± 3.7 nM (n=6) 7.6 ± 0.6 nM (n=3)BI 1265162 6.3 ± 2.2 nM (n=5) 7.7 ± 2.6 nM (n=6)AZD5634 11.4 ± 6.3 nM (n=18) 10.1 ± 7.4 nM (n=3)QBW276 27.8 ± 5.9 nM (n=4) NDb. Permeability assaysSelected compounds were assessed for permeability by Cyprotex (UK) using both a cell-based Caco2 epithelial cell assay and a cell-free parallel artificial membrane permeabilityassay (PAMPA). Compound I cation was used in the form of the trifluoroacetatedi(trifluoroacetic acid salt. Results The permeability of Compound I cation could not be determined as levels were below the limit of quantification in both the PAMPA and Caco2 assays (Table 11). As such, Compound I cation is considered to be of extremely low permeability. Levels of VX-371, BI1265162 and AZD5634 were detected in all of the assays (with the exception of BI 1265162 in the PAMPA assay) consistent with each of these compounds showing a greater membrane / epithelial permeability than Compound I cation. The data are consistent with an approximate permeability order of: VX-371 = AZD5634 > BI 1265162 > ETD001.Table 11 – Data from permeability assaysCompound CACO2 PAMPA (pH 7.4)Papp (A→B) Papp (B→A)Compound I cation <0.02 <0.009 <0.1VX-371 1.72 15.2 0.269BI 1265162 0.228 0.196 <0.1AZD5634 2.25 15.8 0.847Apparent permeability (Papp) is calculated from compound transport / diffusion from the apical (A) to basolateral (B) compartments (A→B) and also B→A. Units are 106cm.s. Parallel artificial membrane permeability assay (PAMPA) data are generated at pH7.4 and data expressed in log cm.s.c. Lung PKVX-371 and BI 1265162 were dosed to rats by IT instillation (0.3 mg / kg) using identical methodologies and protocols to Compound I cation as described in Example 6b. VX-371 and BI 1265162 were detected in BAL fluid and lung tissue (Figure 5). Levels of BI 1265162at 1h after dosing, were similar to those observed with Compound I cation and declined byapproximately 5.5x by the 6h assessment, in contrast to Compound I cation which reducedby 3.0x over this time. Levels of VX-371 in BAL fluid were substantially lower than thoseof Compound I cation and BI 1265162 at 1h and declined by 9.7x by 6h. Lung tissue levelsof VX-371 were substantially higher than observed for either of the other 2 compounds. Lumen permeability of the 3 compounds is in the order of: VX-371 >> BI 1265162 > Compound I cation (see above). Confirmation that the lumen permeability of BI 1265162 is greater than that of CompoundI cation was obtained by comparisons of two independent studies of healthy humanvolunteers in which the compounds were administered by inhalation and plasmaconcentrations were then measured over several hours. The comparative results areshown in Figure 7, where Figure 7A shows plasma concentrations over 1440 minutes andFigure 7B shows plasma concentrations over 180 minutes. It can be seen that peakplasma concentration of BI 1265162 was reached rapidly and then declined until 720 minutes after administration. In contrast, peak plasma concentration of Compound 1 cationwas not achieved until around 60 to 120 minutes after administration. These datademonstrate that compound 1 cation is more slowly absorbed through the lung into theplasma following inhalation than BI 1265162.The data obtained in these experiments show that while Compound I cation has a higherIC50 value in both human bronchial and sheep tracheal epithelial cell cultures than theother ENaC inhibitors tested, its membrane permeability is significantly lower than that forthe other ENaC blocking compounds. As a result of this, it is retained in the lung lumen atpharmacologically active levels for a considerably longer period of time than the other compounds tested. This opens the possibility that pharmaceutical compositions comprising solutions of the Compound I cation chloride hydrochloride salt could be used in a twice daily, or even in a once daily dosing regimen. References App EM, King M, Helfesrieder R, Köhler D and Matthys H. Acute and long-term amiloride inhalation in cystic fibrosis lung disease. A rational approach to cystic fibrosis therapy. Am Rev Respir Dis., 1990, 141(3):605-12.Botero-Velez M, Curtis JJ and Warnock DG. Brief report: Liddle’s syndrome Revisited – a disorder of sodium reabsorption in the distal tubule. N Engl J Med., 1994, 330(3):178- 181. Boucher RC. Evidence for airway surface dehydration as the initiating event in CF airway disease. J Intern Med., 2007, 261(1):5-16. Bowler IM, Kelman B, Worthington D, Littlewood JM, Watson A, Conway SP, Smye SW, James SL and Sheldon TA. Nebulised amiloride in respiratory exacerbations of cystic fibrosis: a randomised controlled trial. Arch Dis Child., 1995, 73(5):427-30. Chang SS, Grunder S, Hanukoglu A, Rösler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC and Lifton RP. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet., 1996, 12(3):248-53. Coote KJ, Paisley D, Czarnecki S. et al. NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia. Br J Pharmacol.2015; 172(11): 2814-26. Danahay HL, Lilley S, Fox R, Charlton H, Sabater J, Button B, McCarthy C, Collingwood SP, Gosling M. TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis. Am J Respir Crit Care Med.2020; 201(8):946-954. Danahay H, McCarthy C, Schofield T, Fox R, Charlton H, Lilley S, Sabater J, Salathe M, Baumlin N, Collingwood S, Gosling M. ETD001: A novel inhaled ENaC blocker with an extended duration of action in vivo. Journal of Cystic Fibrosis 2025, 24:72–78 Fajac I, Hubert D, Guillemot D, Honoré I, Bienvenu T, Volt’r F, Dal’'Ava-Santucci J and Dusser DJ. Nasal airway ion transport is linked to the cystic fibrosis phenotype in adult patients. Thorax, 2004, 59(11):971-6.Fulcher ML, Randell SH, Human nasal and tracheo-bronchial respiratory epithelial cellculture. Methods Mol Biol.2013; 945: 109-21. Graham A, Hasani A, Alton EW, Martin GP, Marriott C, Hodson ME, Clarke SW and Geddes DM. No added benefit from nebulized amiloride in patients with cystic fibrosis. Eur Respir J., 1993, 6(9):1243-8. Kellenberger S and Schild L. Epithelial sodium channel / degenerin family of ion channels: a variety of functions for a shared structure. Physiol Rev., 200282(3):735-67. Kerem E, Bistritzer T, Hanukoglu A, Hofmann T, Zhou Z, Bennett W, MacLaughlin E, Barker P, Nash M, Quittell L, Boucher R and Knowles MR. Pulmonary epithelial sodium- channel dysfunction and excess airway liquid in pseudohypoaldosteronism. N Engl J Med., 1999, 341(3):156-62. Knowles MR, Stutts MJ, Spock A, Fischer N, Gatzy JT and Boucher RC. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science, 1983, 221(4615):1067-70. Knowles MR, Church NL, Waltner WE, Yankaskas JR, Gilligan P, King M, Edwards LJ, Helms RW and Boucher RC. A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. N Engl J Med., 1990, 322(17):1189-94. Leal T, Fajac I, Wallace HL, Lebecque P, Lebacq J, Hubert D, Dall’Ava J, Dusser D, Ganesan AP, Knoop C, Cumps J, Wallemacq P and Southern KW. Airway ion transport impacts on disease presentation and severity in cystic fibrosis. Clin Biochem., 2008, 41(10-11):764-72. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW and Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell, 1998, 95(7):1005-15. Middleton PG, Geddes DM and Alton EW. Effect of amiloride and saline on nasal mucociliary clearance and potential difference in cystic fibrosis and normal subjects. Thorax, 1993, 48(8):812-6. Pham S, Ferguson GT, Kerwin E, Goodin T, Wheeler A, Bauer A. In Vitro Characterization of the eFlow Closed System Nebulizer with Glycopyrrolate Inhalation Solution. J Aerosol Med Pulm Drug Deliv.2018; 31(3): 162-169 Pons G, Marchand MC, d’Athis P, Sauvage E, Foucard C, Chaumet-Riffaud P, Sautegeau A, Navarro J and Lenoir G. French multicenter randomized double-blind placebo-controlled trial on nebulized amiloride in cystic fibrosis patients. The Amiloride- AFLM Collaborative Study Group. Pediatr Pulmonol., 2000, 30(1):25-31. Russell P, Woodward K, Charlwood J, White R, Wilkes D, Danahay H, Morris D.ETD001: A long-acting inhaled ENaC blocker, is well tolerated in humans. EuropeanRespiratory Journal 202260: 4338.

Claims

CLAIMS1. A compound which is 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1- carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride having the formula:including all tautomeric and resonance forms thereof.

2. A compound according to claim 1 which is an amorphous solid.

3. A free-flowing powder comprising, consisting essentially of or consisting of acompound according to claim 1 or claim 2.

4. A free-flowing powder according to claim 3 which is free from one or moreimpurities selected from water, ammonium chloride, acetamide, trifluoroacetic acid, acetonitrile, ethanol and DMSO.

5. A free-flowing powder according to claim 3 optionally comprising:water in an amount of 0-5.0% ww; and / or ammonium chloride in an amount of 0-0.15% w / w; and / or acetamide in an amount of 0-0.15% w / w; and / or trifluoroacetic acid in an amount of 0-0.2% w / w; and / or acetonitrile in an amount of 0-0.5% w / w; and / or ethanol in an amount of 0-1% w / w; and / or DMSO in an amount of 0-0.5% w / w.

6. A process for the preparation of 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino} piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride, theprocess comprising: (I) precipitating 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3- diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride (Compound I cation chloridehydrochloride salt) from a solution comprising 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino} piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride dihydrochloride (Compound I cation chloride dihydrochloride salt) in a solvent by addition of the solution to an antisolvent for 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-(4- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3- diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride to form a solid precipitate; and(II) optionally isolating the solid precipitate.

7. A process according to claim 6 carried out under an inert atmosphere, for examplea nitrogen atmosphere.

8. A process according to claim 6 or claim 7, the solution comprising Compound Ication chloride dihydrochloride salt is prepared by dissolving a solid comprising CompoundI cation chloride dihydrochloride salt in a suitable solvent, wherein the solvent comprisesDMSO, sulfolane, NMP, DMAc, DMF, water or mixtures thereof.

9. A process according to claim 8, wherein the solvent comprises DMSO, water ormixtures thereof and a C1-4alcohol wherein the volume ratio of water and / or DMSO to C1-4 alcohol is about 2:1 to 1:2.

10. A process according to any one of claims 6 to 9 wherein, in step I, the solidcomprising Compound 1 cation chloride dihydrochloride salt is dissolved in 3 to 8 volumesof a solvent comprising a mixture of ethanol, DMSO and water.

11. A process according to claim 10, wherein the solvent in step (I) comprises 3 to 4volumes wet ethanol, 2 to 3 volumes DMSO and 0.2 to 0.3 volumes water per mass of solid comprising Compound I cation chloride dihydrochloride salt.

12. A process according to any one of claims 6 to 11 wherein the solution comprisingCompound I cation chloride dihydrochloride salt is added dropwise to the anti-solvent.

13. A process according to any one of claims 6 to 12 wherein the anti-solvent is a C1-4alcohol.

14. A process according to claim 13 wherein the anti-solvent is ethanol or isopropanol.

15. A process according to any one of claims 6 to 14 wherein the anti-solvent is usedin an amount of 7 to 11 volumes of anti-solvent per volume of solution of the Compound I cation chloride dihydrochloride salt.

16. A process according to any one of claims 8 to 15 wherein a solid comprisingCompound I cation chloride dihydrochloride salt is prepared by a salt exchange stepcomprising: treating 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis [(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl- 1H-1,3-benzodiazol-3-ium trifluoroacetate di(trifluoroacetic acid) (Compound I cation trifluoroacetate di(trifluoroacetic acid) salt) with excess hydrochloric acid in a solvent selected from acetonitrile, C1-4alcohols and mixtures thereof to produce a product mixture comprising a precipitate of 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]- 6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride dihydrochloride (Compound I cation chloridedihydrochloride salt) and isolating a solid product comprising said chloride dihydrochloridesalt.

17. A process according to claim 16 wherein the salt exchange step is carried out underan inert atmosphere, for example a nitrogen atmosphere.

18. A process according to claim 16 or claim 17 wherein, in the salt exchange step:the solvent comprises a mixture of acetonitrile and ethanol, suitably in a ratio of from about 3:1 to 5:1; and / or a solution of hydrogen chloride in the solvent is added to a solution of the Compound I cation trifluoroacetate di(trifluoroacetic acid) salt in the solvent to give a product mixturecomprising the Compound I cation chloride dihydrochloride salt; or a solution of hydrogenchloride in a suitable solvent is added to the solid Compound I cation trifluoroacetatedi(trifluoroacetic acid) salt; and / orisolating the Compound I cation chloride dihydrochloride salt as a solid comprises mixingthe product mixture with an anti-solvent which is a di(C1-4 alkyl) ether.

19. A process according to any one of claims 6 to 18 further including one or morewashing steps, wherein the washing steps comprise:washing the Compound I cation chloride dihydrochloride salt isolated as a solid beforecarrying out step (I); and / orwashing the Compound I cation chloride hydrochloride salt product isolated as a solidprecipitate in step (II).

20. A process according to claim 19 wherein the washing steps comprise forming aslurry of the solid Compound I cation chloride dihydrochloride salt or Compound I cationchloride hydrochloride salt, in a C1-4 alcohol, stirring the slurry and filtering the washedproduct.

21. A process according to claim 19 or claim 20, further including the step of drying thewashed solid Compound I cation chloride hydrochloride salt product of step (II).

22. A process for the preparation of 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride, the process comprising:A. treating 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl- 1H-1,3-benzodiazol-3-ium trifluoroacetate di(trifluoroacetic acid) (Compound I cation trifluoroacetate di(trifluoroacetic acid) salt) with excess hydrochloric acid in a solvent selected from acetonitrile, C1-4 alcohols and mixtures thereof in a salt exchange step to produce a product mixture comprising a precipitate of 2-[({3-amino-5H-pyrrolo[2,3- b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl] amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloridedihydrochloride (Compound I cation chloride dihydrochloride salt) and isolating a solidproduct comprising said chloride dihydrochloride salt;B. optionally washing the solid product of step (a) by forming a slurry of the productin a C1-4 alcohol and filtering and drying the washed product;C. converting the Compound I cation chloride dihydrochloride salt of step A. oroptional step B. to 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-(4- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3- diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride (Compound I cation chloride hydrochloride salt) by a process comprising: ((I) precipitating Compound I cation chloride hydrochloride salt from a solution of Compound I cation chloride dihydrochloride salt in a solvent by addition of the solution to an antisolvent for Compound I cation chloride hydrochloride salt to form a solid precipitate; and (II) isolating the solid precipitate;D. optionally washing the isolated solid product of step C. by forming a slurry of theproduct in a C1-4alcohol and filtering the washed product;E. drying the product of step C. or optional step D.

23. A compound which is 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride hydrochloride (Compound Ication chloride hydrochloride salt) obtained by or obtainable by a process according to anyone of claims 6 to 22.

24. A compound according to claim 1 or claim 2, a free-flowing powder according toany one of claims 3 to 5 or a compound according to claim 23 in a sealed container in dry conditions and under an inert atmosphere, for example a nitrogen atmosphere.

25. A pharmaceutical composition comprising a solution of a compound according toclaim 1 or claim 2 or a free-flowing powder according to any one of claims 3 to 5 or acompound according to claim 23 in an aqueous solvent.

26. A pharmaceutical composition according to claim 25 which is formulated for topicaladministration to the lungs.

27. A pharmaceutical composition according to claim 25 or claim 26 which comprisesa buffer and has a pH of 5 to 7.

28. A pharmaceutical composition according to claim 27 which comprises a citratebuffer.

29. A pharmaceutical composition according to any one of claims 25 to 28 in a sealedcontainer under an inert atmosphere such as nitrogen.

30. A pharmaceutical composition according to any one of claims 25 to 29 formulatedas a unit dosage form for twice daily dosing and comprising from 100 μg to 5400 μg of the Compound I cation; or formulated as a unit dosage form for once daily dosing and comprising from 150 μg to10800 μg of the Compound I cation.

31. A compound according to claim 1 or claim 2, or a free-flowing powder according toany one of claims 3 to 5, or a compound according to claim 23, or a pharmaceuticalcomposition according to any one of claims 25 to 30 for use in the treatment of a respiratorydisease or condition.

32. A compound, free-flowing powder or pharmaceutical composition for use accordingto claim 31 wherein the respiratory disease or condition is selected from cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, asthma and primary ciliary dyskinesia.

33. A compound, free-flowing powder or pharmaceutical composition for use accordingto claim 31 or claim 32, wherein the Compound I cation is administered to a patient in an amount of 150 μg to 10800 μg per day.

34. A compound, free-flowing powder or pharmaceutical composition for use accordingto claim 31 or claim 32, wherein the Compound I cation is administered to a patient in an amount of 2.5 to 180 μg / kg per day.

35. A compound, free-flowing powder or pharmaceutical composition for useaccording to any one of claims 31 to 34, wherein the compound or pharmaceutical composition is formulated for once daily or twice daily dosing.

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