WO2021014168A1

WO2021014168A1 - Solid forms of 4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]-n-(1,1-dimethylprop-2-ynyl)pyridine-2-carboxamide

Info

Publication number: WO2021014168A1
Application number: PCT/GB2020/051780
Authority: WO
Inventors: Janet MAAS; Emma Sharp
Original assignee: Enterprise Therapeutics Limited
Priority date: 2019-07-25
Filing date: 2020-07-24
Publication date: 2021-01-28
Also published as: GB201910665D0

Abstract

The invention relates to novel crystalline forms of 4-[[2-(5-Chloro-2-hydroxy- phenyl)acetyl]amino]-N-(1,1-dimethylprop-2-ynyl)pyridine-2-carboxamide (Compound 1). In particular, the invention relates to the Form 2 and Form 6 crystalline polymorphs.

Description

SOLID FORMS OF 4-[[2-(5-CHLORO-2-HYDROXY-PHENYL)ACETYL]AMINO]-N-(1 ,1-DIMETHYLPROP-2-YNYL) PYRIDINE-2-CARBOXAMIDE

Field of the invention

The present invention relates to a novel crystalline form, of a compound which has activity 5 as a positive modulator of the calcium-activated chloride channel (CaCC), TMEM16A. The invention also relates to methods of preparing the novel crystalline form and to compositions containing it as well as to its use in treating diseases and conditions in which TMEM16A plays a role, particularly respiratory diseases and conditions.

10 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 (such as bacteria, viruses and fungal spores) to enter the airways. 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 15 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.

20 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% w/v 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 25 (Cl- / HCO₃-) secretion mediated via the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the Calcium Activated Chloride Conductance (CaCC; TMEM16A) 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.

30

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 the ability of the airway to secrete fluid, the % solids can be increased to 15% which is 35 believed to contribute towards the plugging of small airways and failure of mucus clearance. Strategies to increase the hydration of the airway mucus include either the stimulation of anion secretion and thereby fluid secretion or the inhibition of Na⁺ absorption. To this end, stimulating the activity of TMEM16A channels will increase anion secretion and therefore increase fluid accumulation in the airway mucosa, hydrate mucus and enhance mucus clearance mechanisms.

TMEM16A, also referred to as Anoctamin-1 (And), is the molecular identity of calcium- activated chloride channels (Caputo et al, 2008; Yang et al, 2008). TMEM16A channels open in response to elevation of intracellular calcium levels and allow the bidirectional flux of chloride, bicarbonate and other anions across the cell membrane. Functionally TMEM16A channels have been proposed to modulate transepithelial ion transport, gastrointestinal peristalsis, nociception and cell migration/proliferation (Pedemonte & Galietta, 2014).

TMEM16A channels are expressed by the epithelial cells of different organs including the lungs, liver, kidney, pancreas and salivary glands. In the airway epithelium TMEM16A is expressed at high levels in mucus producing goblet cells, ciliated cells and in submucosal glands. Physiologically TMEM16A is activated by stimuli which mobilise intracellular calcium, particularly purinergic agonists (ATP, UTP), which are released by the respiratory epithelium in response to cyclical shear stress caused by breathing and other mechanical stimuli such as cough. In addition to increasing anion secretion leading to enhanced hydration of the airways, activation of TMEM16A plays an important role in bicarbonate secretion. Bicarbonate secretion is reported to be an important regulator of mucus properties and in controlling airway lumen pH and hence the activity of native antimicrobials such as defensins (Pezzulo et al, 2012).

Indirect modulation of TMEM16A, via elevation of intracellular calcium, has been clinically explored eg. denufosol (Kunzelmann & Mall, 2003). Although encouraging initial results were observed in small patient cohorts this approach did not deliver clinical benefit in larger patient cohorts (Accurso et al 201 1 ; Kellerman et al 2008). This lack of clinical effect was ascribed to only a transient elevation in anion secretion, the result of a short half-life of denufosol on the surface of the epithelium and receptor/pathway desensitisation, and unwanted effects of elevating intracellular calcium such as increased release of mucus from goblet cells (Moss, 2013). Compounds which act directly upon TMEM16A to enhance channel opening at low levels of calcium elevation are expected to durably enhance anion secretion and mucociliary clearance in patients and improve innate defence. As TMEM16A activity is independent of CFTR function, TMEM16A positive modulators have the potential to deliver clinical benefit to all CF patients and non-CF respiratory diseases characterised by mucus congestion including chronic bronchitis and severe asthma.

TMEM16A modulation has been implicated as a therapy for dry mouth (xerostomia), resultant from salivary gland dysfunction in Sjorgen’s syndrome and radiation therapy, dry eye, cholestasis and gastrointestinal motility disorders.

The present inventors have developed novel compounds and novel forms of these compounds which are positive modulators of TMEM16A and which are therefore of use in the treatment of diseases and conditions in which TMEM16A plays a role, particularly respiratory diseases and conditions. These compounds were described for the first time in our earlier application WO 2019/145726, the contents of which are hereby incorporated by reference in their entirety. In particular, WO 2019/145726 discloses 4-[[2-(5-Chloro-2- hydroxy-phenyl)acetyl]amino]-N-(1 , 1 -dimethylprop-2-ynyl)pyridine-2-carboxamide (Compound 1), which has the following structural formula:

Summary of the invention

The inventors have now developed novel forms of Compound 1.

In a first aspect of the invention there is provided Compound 1 in the form of its Form 2 Form B) anhydrous solid crystalline polymorph.

In a second aspect of the invention there is provided Compound 1 in the form of its Form 6 (Form F) anhydrous crystalline polymorph.

Brief description of the figures

Figure 1 is an XRPD diffractogram of solid crystalline polymorphic Form 1 , also known as Form A, of Compound 1.

Figure 2 shows overlaid thermogravimetric analysis (TGA; upper trace) and differential scanning calorimetry (DSC; lower trace) thermograms of solid crystalline polymorphic Form 1 Compound 1. There is a single endotherm: integral 70 J/g; onset 174.8 °C, peak 177.9 °C.

Figure 3 are XRPD diffractograms from the solubility assessment of Example 2:

Trace 1 : Form 1 (reference)

Traces 2-7: Samples consistent with Form 1 ;

Trace 8: sample cooled in ethyl acetate;

Trace 9: sample cooled in isopropyl alcohol;

Trace 10: Sample cooled in acetone;

T race 1 1 : sample cooled in tetrahydrofuran;

Trace 12: sample cooled in dichloromethane;

Trace 13: sample matured in tert- butyl methyl ether.

Figure 4 is the XRPD diffractogram, measured in reflectance mode, of the material obtained from 5% aqueous ACN in Example 2 (upper trace) compared with Form 1 (lower trace). This sample showed subtle differences from Compound 1 , Form 1 when analysed on an XRPD instrument in transmission mode’ but is consistent with Form 1 when analysed on a different instrument in reflectance mode.

Figure 5 is the XRPD diffractogram measured in reflectance mode, of the material obtained from acetone/n-heptane in Example 3 (upper trace) compared with Form 1 (lower trace). This sample showed subtle differences from Compound 1 Form 1 , when analysed on an XRPD instrument in transmission mode, but is consistent with Form 1 when analysed on a different instrument in reflectance mode.

Figure 6 is the XRPD diffractogram for the Form 2, also known as Form B, polymorph of Compound 1.

Figure 7 shows overlaid TGA (upper) and DSC (lower) thermograms of the Form 2 (Form B) polymorph of Compound 1. TGA shows a weight loss of 1.4% at low temperature. DSC lobe 1 : integral -31 J/g, peak 57.3 °C; lobe 2: -4 J/g; onset 139.7 °C, peak 142.9 °C; Lobe 3: integral 19 J/g, onset 150.3 °C, peak 155.6 °C; Lobe 4: integral -79 J/g, onset 173.8 °C, peak 175.9 °C.

Figure 8 is the XRPD diffractogram for the static stability test for the Form 2 (Form B) polymorph of Compound 1 showing that Form 2 is stable after 7 days at 40°C and 75% relative humidity: Trace 1 : initial sample Trace 2: after 7 days at 40°C and 75% relative humidity.

Figure 9 is the XRPD diffractogram for the Form 3, also known as Form C, polymorph of Compound 1.

Figure 10 shows overlaid TGA (upper trace) and DSC (lower trace) thermograms of the Form 3 polymorph of Compound 1. TGA shows weight loss of 10.0% at temperature corresponding to Lobe 1 of the DSC. In the DSC, Lobe 1 : integral -98 J/g, onset 88.4 °C, peak 96.7 °C; Lobe 2: integral -5 J/g, onset 124.7 °C, peak 129.8 °C; Lobe 3: integral -78 J/g, onset 176.4 °C, peak 177.7 °C.

Figure 11 is the XRPD diffractogram for the static stability test for the Form 3 polymorph of Compound 1 in which the XRPD diffractograms of the initial sample and the sample after 7 days at 40°C and 75% relative humidity are compared with the XRPD diffractograms of Forms 1 and 2.

Trace 1 : Form 3 sample (initial)

Trace 2: sample after 7 days at 40°C and 75% relative humidity

Trace 3: Form 2

Trace 4: Form 1

Figure 12 is the XRPD diffractogram for the Form 4, also known as Form D, polymorph of Compound 1.

Figure 13 shows overlaid TGA (upper trace) and DSC (lower trace) thermograms of the Form 4 polymorph of Compound 1. DSC, Lobe 1 : integral 10 J/g, onset 117.0 °C, peak 127.3 °C; Lobe 2: integral 6 J/g onset 137.6 °C, peak 146.0 °C; Lobe 3: integral -76 J/g, onset 174.1 °C, peak 176.3 °C.

Figure 14 is the XRPD diffractogram for the static stability test for the Form 4 polymorph of Compound 1 in which the XRPD diffractograms of the initial sample and the sample after 7 days at 40°C and 75% relative humidity are compared with the XRPD diffractograms of Forms 1 and 2.

Trace 1 : Form 4 sample (initial)

Trace 2: sample after 7 days at 40°C and 75% relative humidity

Trace 3: Form 2

Trace 4: Form 1 Figure 15 is the XRPD diffractogram for the Form 5, also known as Form E, polymorph of Compound 1.

Figure 16 shows overlaid TGA (upper trace) and DSC (lower trace) thermograms of the Form 5 polymorph of Compound 1. TGA shows weight loss of 10.3% at temperature corresponding to Lobe 1 of the DSC. In the DSC, Lobe 1 : integral -71 J/g, onset 96.1 °C peak 108.0 °C; Lobe 2: integral -83 J/g, onset 175.9 °C, peak 177.5 °C.

Figure 17 is the XRPD diffractogram for the static stability test for the Form 5 polymorph of Compound 1 in which the XRPD diffractograms of the initial sample and the sample after 7 days at 40°C and 75% relative humidity are compared with the XRPD diffractograms of Forms 1 and 2.

Trace 1 : Form 5 sample (initial)

Trace 2: sample after 7 days at 40°C and 75% relative humidity

Trace 3: Form 2

Trace 4: Form 1.

Figure 18 is the XRPD diffractogram for the Form 6, also known as Form F, polymorph of Compound 1.

Figure 19 shows overlaid TGA (upper trace) and DSC (lower trace) thermograms of the Form 6 (Form F) polymorph of Compound 1. In the DSC plot, Lobe 1 : integral 6 J/g, onset 135.7 °C, peak 143.0 °C; Lobe 2, integral 88 J/g, onset 177.5 °C, peak 178.6 °C.

Figure 20 is the XRPD diffractogram for the static stability test for the Form 6 (Form F) polymorph of Compound 1 measured in reflectance mode and showing that after 7 days at 40°C and 75% relative humidity, the sample remained unchanged.

Trace 1 : Form 6 sample (initial)

Trace 2: sample after 7 days at 40°C and 75% relative humidity

Trace 3: Form 1.

Figure 21 is the XRPD diffractogram for the Form 7, also known as Form G, polymorph of Compound 1.

Figure 22 shows overlaid TGA (upper trace) and DSC (lower trace) thermograms of the Form 7 polymorph of Compound 1. The TGA plot has a weight loss of 18.5% at a temperature consistent with Lobe 1 of the DSC plot. In the DSC plot, Lobe 1 : integral - 116 J/g, onset 90.7 °C, peak 97.4 °C; Lobe 2: integral 70 J/g, onset 176.4 °C, peak 177.7 °C.

Figure 23 is the XRPD diffractogram for the static stability test for the Form 7 (Form G) polymorph of Compound 1 in which the XRPD diffractograms of the initial sample and the sample after 7 days at 40°C and 75% relative humidity are compared with the XRPD diffractogram of Form 1.

Trace 1 : Form 7 sample (initial)

Trace 2: sample after 7 days at 40°C and 75% relative humidity

Trace 3: Form !

Figure 24 is the XRPD diffractogram for the Form 8, also known as Form H, polymorph of Compound 1.

Figure 25 shows overlaid TGA (upper trace) and DSC (lower trace) thermograms of the Form 6 (Form F) polymorph of Compound 1. The TGA plot shows a weight loss of 13.1 % consistent with Lobe 1 of the DSC plot. In the DSC, Lobe 1 : integral -85 J/g, onset 85.4 °C, peak 97.8 °C; Lobe 2: integral -81 J/g, onset 176.8 °C, peak 178.0 °C.

Figure 26 is the XRPD diffractogram for the static stability test for the Form 6 (Form F) polymorph of Compound 1 in which the XRPD diffractograms of the initial sample and the sample after 7 days at 40°C and 75% relative humidity are compared with the XRPD diffractograms of Forms 1 and 2.

Trace 1 : Form 6 (Form F) sample (initial)

Trace 2 sample after 7 days at 40°C and 75% relative humidity

Trace 3: Form 1 (Form A)

Trace 4: Form 2 (Form B).

Figure 27 is the XRPD diffractogram of the re-synthesised sample of Form B (ethyl acetate) from Example 5. It is consistent with the XRPD of Figure 6.

Figure 28 is the TGA plot of the re-synthesised sample of Form B (ethyl acetate) from Example 5. Heating from 30 to 500 °C at 10 °C per minute. There are weight loss events as follows: -1.1190%: onset 57.95 °C, endset 72.68 °C; -2.0921 %, onset 98.26 °C, endset 112.95 °C; -5.6770%, onset 172.22 °C, endset 203.93 °C; -53.8619%, onset 288.51 °C, endset 403.32 °C. Figure 29 is the TGA plot of the re-synthesised sample of Form B (water) from Example 5. Heating from 30 to 500 °C at 10 °C per minute. There are weight loss events as follows: -1.2601 , onset 59.65 °C, endset 73.12 °C; -4.6919, onset 174.87 °C, endset 203.68 °C; - 54.3281 %, onset 310.46 °C, endset 394.10 °C.

Detailed description 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 literature and patent documents referred to herein are incorporated by reference to the fullest extent possible.

Where the specification refers to a volume of solvent per unit mass of Compound 1 , this refers to mg of Compound 1 and mL of solvent such that 50 mg of Compound 1 in 20 volumes of solvent is a sample of 1 mL volume; 30 mg of Compound 1 in 60 volumes of solvent is a sample of 1.8 mL volume and 30 mg of Compound 1 in 70 volumes of solvent is sample 2.1 mL volume.

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.

Compound 1 disclosed herein is 4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]-N-(1 , 1- dimethylprop-2-ynyl)pyridine-2-carboxamide. which has the structure shown above. This compound is exemplified in our earlier application WO 2019/145726and the method exemplified in that document results in the production of Compound 1 as the Form 1 crystalline polymorph, also known as the Form A crystalline polymorph.

The present inventors have found a number of different crystalline forms of Compound 1 , namely forms 1 to 8 (also known as A to H). Polymorphic Forms 1 , 2 and 6 (A, B and F) are particularly useful as these were all unchanged after 7 days at 40°C and 75% relative humidity and the present invention relate to the novel Forms 2 (B) and 6 (F) polymorphs.

Therefore, in one aspect of the invention, there is provided Compound 1 in the form of its Form 2 (Form B) anhydrous solid crystalline polymorph (the Form 2 polymorph), for example as characterised by an XRPD diffractogram substantially as shown in Figure 6.

The XRPD diffractogram of the Form 2 polymorph has major peaks at 14.6 and 22.5 (± 0.2 degrees, 2-theta values).

Suitably, major peaks at 14.6 and 22.5 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six or all seven) at positions selected from 7.2, 9.2,

18.2, 20.4, 23.9, 24.7 and 26.0 (± 0.2 degrees, 2-theta values) are observable in the XRPD diffractogram of the Form 2 polymorph.

More suitably, major peaks at 14.6 and 22.5 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven or all eight) at positions selected from

7.2, 9.2, 18.2, 20.4, 23.9, 24.7, 26.0 and 29.1 (± 0.2 degrees, 2-theta values) are observable in the XRPD diffractogram of the Form 2 polymorph.

More suitably, major peaks at 14.6 and 22.5 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven, eight, nine, ten, eleven, twelve or all thirteen) of the peaks at positions 7.2, 9.2, 1 1.6, 12.7, 15.6, 18.2, 20.4, 23.9, 24.7, 26.0, 27.4, 27.8 and 29.1 (± 0.2 degrees, 2-theta values) are observable in the XRPD diffractogram of the Form 2 polymorph of Compound 1.

Still more suitably, major peaks at 14.6 and 22.5 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or all eighteen) of the peaks at positions 7.2,

9.2, 1 1.6, 12.7, 13.0, 15.6, 18.2, 18.8, 20.4, 20.9, 23.2, 23.9, 24.7, 26.0, 27.4, 27.8, 28.6 and 29.1 (± 0.2 degrees, 2-theta values) are observable.

Suitably, peaks at 9.9 and 10.4 (± 0.2 degrees, 2-theta values), which are characteristic of the Form 1 polymorph of Compound 1 , are not observable in the XRPD diffractogram of the Form 2 polymorph of Compound 1. The XRPD peak positions and intensities in the diffractogram pattern of the Form 2 polymorph are as set out below.

XRPD analysis may be carried out using the instruments set out below in the examples.

Suitably, the Form 2 polymorph is substantially free from other forms of Compound 1 , such that, for example, in a sample of Compound 1 , at least 97%, 98%, 99%, 99.5%, 99.6% 99.7%, 99.8% or 99.9% by weight of Compound 1 is present as the Form 2 polymorph. The Form 2 (Form B) polymorph may be prepared by crystallisation from a suitable solvent, especially from ethyl acetate. Therefore, in a further aspect of the invention, there is provided a process for the preparation of the Form 2 crystalline polymorph of Compound 1 , the process comprising crystallising Compound 1 from ethyl acetate.

Suitably, the process comprises the steps of:

(i). dissolving Compound 1 in ethyl acetate at a temperature of about 40 to 60 °C, for example 45 to 55 °C, and typically 50°C;

(ii). Cooling the solution obtained in step (i) to -5 to 15°C, suitably 0 to 10°C and typically about 5°C;

(iii) allowing crystallisation to take place; and

(iv). isolating the crystals obtained in step (iii).

In step (i), Compound 1 used in Step (i) is suitably the Form 1 (Form A) polymorph. The amount of ethyl acetate used in step (i) to dissolve Compound 1 is suitably at least 20 volumes, for example 20 to 60 volumes, per unit mass of Compound 1. Step (i) may also include the removal of solid Compound 1 , for example by filtering, to obtain a clear solution.

In step (ii), cooling suitably takes place at a rate of about 0.01 to 0.5°C/min, more suitably 0.05 to 0.2 °C/min, for example 0.1 °C/min.

In step (iii), crystallisation may be achieved by stirring the cooled solution from step (ii). Stirring may be prolonged, for example overnight.

In step (iv), the crystals are suitably isolated by filtration.

In an alternative procedure, step (ii) may be replaced by a process of controlled evaporation of the solvent to yield a super saturated solution and achieve crystal nucleation.

Isostructural pseudopolymorphs of Form 2 (Form B) have been found in which ethyl acetate present in the lattice is replaced by isopropanol or water. These pseudopolymorphs are designated From B (isopropanol) and Form B (water).

Form B (isopropanol) may be prepared as described in the Examples below by keeping Form 3 (Form C) at 43% relative humidity for 12 days and then increasing the relative humidity to 60% for a further 6 days. The Form 2 (Form B) prepared by this method contains 2.7% w/w isopropanol.

Form B (water) may be prepared by storing Form B (ethyl acetate) at 95% relative humidity and 15 to 25 °C for 20 to 30 days, suitably about 25 days. Under these conditions, ethyl acetate (2.9% w/w) is replaced by water.

In another aspect of the invention, there is provided Compound 1 in the form of its Form 6 (Form F) anhydrous solid crystalline polymorph (the Form 6 polymorph), for example as characterised by an XRPD diffractogram substantially as shown in Figure 18.

The XRPD diffractogram of the Form 6 polymorph has major peaks at 15.5 and 25.3 (± 0.2 degrees, 2-theta values).

Suitably, major peaks at 15.5 and 25.3 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven or all eight) at positions selected from 7.2,

9.3, 15.5, 16.5, 17.0, 18.9, 24.7, 25.3, 27.4 and 28.1 (± 0.2 degrees, 2-theta values) are observable in the XRPD diffractogram.

More suitably, major peaks at 15.5 and 25.3 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven, eight, nine, ten, eleven, twelve or all thirteen) of the peaks at positions 7.2, 9.3, 12.0, 15.5, 16.5, 17.0, 18.9, 21.6, 23.0, 24.3, 24.7, 25.3, 27.4, 28.1 and 30.6 (± 0.2 degrees, 2-theta values) are observable in the XRPD diffractogram of the Form 6 polymorph of Compound 1.

Still more suitably, major peaks at 15.5 and 25.3 (± 0.2 degrees, 2-theta values) and at least major peaks (for example three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or all eighteen) of the peaks at positions 7.2,

8.4, 9.3, 12.0, 15.5, 16.5, 17.0, 18.9, 21.6, 22.3, 22.6, 23.0, 24.3, 24.7, 25.3, 26.9, 27.4, 28.1 , 30.6 and 31.2 (± 0.2 degrees, 2-theta values) are observable in the XRPD diffractogram of the Form 6 polymorph of Compound 1.

Suitably, peaks at 9.9 and 10.4 (± 0.2 degrees, 2-theta values), which are characteristic of the Form 1 polymorph of Compound 1 , are not observable in the XRPD diffractogram of the Form 6 polymorph of Compound 1. The XRPD peak positions and intensities in the Form 6 (Form F) diffractogram pattern are as set out below.

Suitably, the Form 6 polymorph is substantially free from other forms of Compound 1 , such that, for example, in a sample of Compound 1 , at least 97%, 98%, 99%, 99.5%, 99.6% 99.7%, 99.8% or 99.9% by weight of Compound 1 may be present as the Form 6 polymorph.

The Form 6 polymorph may be prepared by crystallisation from a suitable solvent, especially from dichloromethane. Therefore, in a further aspect of the invention, there is provided a process for the preparation of the Form 6 crystalline polymorph of Compound 1 , the process comprising crystallising Compound 1 from dichloromethane.

Suitably, the process comprises the steps of:

(i). dissolving Compound 1 in dichloromethane at a temperature of about 40 to 60 °C, for example 45 to 55 °C, and typically 50°C;

(iii) allowing crystallisation to take place; and

(iv). isolating the crystals obtained in step (iii).

In step (i), Compound 1 used in Step (i) is suitably Form 1. The amount of dichloromethane used in step (i) to dissolve Compound 1 is suitably at least 40 volumes, for example 40 to 60 volumes, per unit mass of Compound 1. Step (i) may also include the removal of solid Compound 1 , for example by filtering, to obtain a clear solution. This step is particularly useful when the Compound 1 used in Step (i) is Form 1 as it is not highly soluble in dichloromethane.

In step (iv), the crystals are suitably isolated by filtration. In an alternative procedure, step (ii) may be replaced by a process of controlled evaporation of the solvent to yield a super saturated solution and achieve crystal nucleation.

Compound 1 is a modulator of TMEM16A and therefore, in a further aspect of the invention, there is provided Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above for use in the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A.

There is also provided Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in the manufacture of a medicament for the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A.

There is also provided a method for the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A, the method comprising administering to a patient in need of such treatment an effective amount of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above.

The diseases and conditions affected by modulation of TMEM16A include respiratory diseases and conditions, dry mouth (xerostomia), intestinal hypermobility, cholestasis and ocular conditions.

There is also provided:

• Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above for use in the treatment or prophylaxis of respiratory diseases and conditions.

• Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above for use in the treatment or prophylaxis of dry mouth (xerostomia).

• Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above for use in the treatment or prophylaxis of intestinal hypermobility.

• Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above for use in the treatment or prophylaxis of cholestasis.

• Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above for use in the treatment or prophylaxis of ocular conditions.

The invention also provides:

• The use of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in the manufacture of a medicament for the treatment or prophylaxis of respiratory diseases and conditions.

• The use of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in the manufacture of a medicament for the treatment or prophylaxis of dry mouth (xerostomia).

• The use of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in the manufacture of a medicament for the treatment or prophylaxis of intestinal hypermobility.

• The use of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in the manufacture of a medicament for the treatment or prophylaxis of cholestasis.

• The use of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in the manufacture of a medicament for the treatment or prophylaxis of ocular conditions.

There is further provided:

• A method for the treatment or prophylaxis of respiratory diseases and conditions, the method comprising administering to a patient in need of such treatment an effective amount of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above.

• A method for the treatment or prophylaxis of dry mouth (xerostomia), the method comprising administering to a patient in need of such treatment an effective amount of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above.

• A method for the treatment or prophylaxis of intestinal hypermobility, the method comprising administering to a patient in need of such treatment an effective amount of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above.

• A method for the treatment or prophylaxis of cholestasis, the method comprising administering to a patient in need of such treatment an effective amount of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above.

• A method for the treatment or prophylaxis of ocular conditions, the method comprising administering to a patient in need of such treatment an effective amount of Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above.

Respiratory diseases and conditions which may be treated or prevented by Compound 1 in the form of its Form 2 or Form 6 polymorph include cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, including non-cystic fibrosis bronchiectasis, asthma and primary ciliary dyskinesia.

Dry mouth (xerostomia) which may be treated or prevented by Compound 1 in the form of its Form 2 or Form 6 polymorph may result from Sjorgens syndrome, radiotherapy treatment and xerogenic drugs.

Compound 1 will generally be administered as part of a pharmaceutical composition and therefore the invention further provides a pharmaceutical composition comprising Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above together with a pharmaceutically acceptable excipient.

The pharmaceutical composition may be formulated for oral, rectal, nasal, , topical (including topical administration to the lung, dermal, transdermal, eye drops, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration and may be prepared by any methods well known in the art of pharmacy. Compositions for oral administration or topical administration to the lung are particularly suitable.

The composition may be prepared by bringing into association the above defined active agent with the excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in conjunction or association with a pharmaceutically acceptable carrier or vehicle.

Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion; or as a bolus etc.

For compositions for oral administration (e.g. tablets and capsules), the term“acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate, stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

For topical application to the skin, Compound 1 in the form of its Form 2 or Form 6 polymorph may be made up into a cream, ointment, jelly, solution or suspension etc. Cream or ointment formulations that may be used for the drug are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia.

Topical administration to the lung may be achieved by use of an aerosol formulation. Aerosol formulations typically comprise the active ingredient suspended or dissolved in a suitable aerosol propellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFC propellants include trichloromonofluoromethane (propellant 1 1), dichlorotetrafluoromethane (propellant 1 14), and dichlorodifluoromethane (propellant 12). Suitable HFC propellants include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227). The propellant typically comprises 40%-99.5% e.g. 40%-90% by weight of the total inhalation composition. The formulation may comprise excipients including co solvents (e.g. ethanol) and surfactants (e.g. lecithin, sorbitan trioleate and the like). Other possible excipients include polyethylene glycol, polyvinylpyrrolidone, glycerine and the like. Aerosol formulations are packaged in canisters and a suitable dose is delivered by means of a metering valve (e.g. as supplied by Bespak, Valois or 3M or alternatively by Aptar, Coster or Vari).

Topical administration to the lung may also be achieved by use of a non-pressurised formulation such as an aqueous solution or suspension. These may be administered by means of a nebuliser e.g. one that can be hand-held and portable or for home or hospital use (ie non-portable). The formulation may comprise excipients such as water, buffers, tonicity adjusting agents, pH adjusting agents, surfactants and co-solvents. Suspension liquid and aerosol formulations (whether pressurised or unpressurised) will typically contain the compound of the invention in finely divided form, for example with a D₅₀ of 0.5- 10 mm e.g. around 1-5 mm. Particle size distributions may be represented using D_{1 0}, D₅₀ and D₉₀ values. The D₅₀ median value of particle size distributions is defined as the particle size in microns that divides the distribution in half. The measurement derived from laser diffraction is more accurately described as a volume distribution, and consequently the D₅₀ value obtained using this procedure is more meaningfully referred to as a Dv₅₀ value (median for a volume distribution). As used herein Dv values refer to particle size distributions measured using laser diffraction. Similarly, D_{1 0} and D₉₀ values, used in the context of laser diffraction, are taken to mean Dv₁₀ and Dv₉₀ values and refer to the particle size whereby 10% of the distribution lies below the D_{1 0} value, and 90% of the distribution lies below the D₉₀ value, respectively.

Topical administration to the lung may also be achieved by use of a dry-powder formulation. A dry powder formulation will contain the compound of the disclosure in finely divided form, typically with a mass mean diameter (MMAD) of 1-10 mm or a D₅₀ of 0.5-10 mm e.g. around 1-5 mm. Powders of the compound of the invention in finely divided form may be prepared by a micronization process or similar size reduction process. Micronization may be performed using a jet mill such as those manufactured by Hosokawa Alpine. The resultant particle size distribution may be measured using laser diffraction (e.g. with a Malvern Mastersizer 2000S instrument). The formulation will typically contain a topically acceptable diluent such as lactose, glucose or mannitol (preferably lactose), usually of comparatively large particle size e.g. a mass mean diameter (MMAD) of 50 mm or more, e.g. 100 mm or more or a D₅₀ of 40-150 mm. As used herein, the term“lactose” refers to a lactose-containing component, including a-lactose monohydrate, b-lactose monohydrate, a-lactose anhydrous, b-lactose anhydrous and amorphous lactose. Lactose components may be processed by micronization, sieving, milling, compression, agglomeration or spray drying. Commercially available forms of lactose in various forms are also encompassed, for example Lactohale^® (inhalation grade lactose; DFE Pharma), lnhaLac^®70 (sieved lactose for dry powder inhaler; Meggle), Pharmatose^® (DFE Pharma) and Respitose^® (sieved inhalation grade lactose; DFE Pharma) products. In one embodiment, the lactose component is selected from the group consisting of a-lactose monohydrate, a-lactose anhydrous and amorphous lactose. Preferably, the lactose is a- lactose monohydrate.

Dry powder formulations may also contain other excipients. Thus in one embodiment a dry powder formulation according the present disclosure comprises magnesium or calcium stearate. Such formulations may have superior chemical and/or physical stability especially when such formulations also contain lactose.

A dry powder formulation is typically delivered using a dry powder inhaler (DPI) device. Example dry powder delivery systems include SPINHALER®, DISKHALER®, TURBOHALER®, DISKUS®, SKYEHALER®, ACCUHALER® and CLICKHALER®. Further examples of dry powder delivery systems include ECLIPSE, NEXT, ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER, BREEZHALER/NEOHALER, MONODOSE, FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN, ELPENHALER, MIATHALER, TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIEL dry powder inhaler, MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN, PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

In one embodiment Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above is provided as a micronized dry powder formulation, for example comprising lactose of a suitable grade.

Thus, as an aspect of the invention there is provided a pharmaceutical composition comprising Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in particulate form in combination with particulate lactose, said composition optionally comprising magnesium stearate.

In one embodiment Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above is provided as a micronized dry powder formulation, comprising lactose of a suitable grade and magnesium stearate, filled into a device such as DISKUS. Suitably, such a device is a multidose device, for example the formulation is filled into blisters for use in a multi-unit dose device such as DISKUS.

In another embodiment Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above is provided as a micronized dry powder formulation, for example comprising lactose of a suitable grade, filled into hard shell capsules for use in a single dose device such as AEROLISER.

In another embodiment Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above is provided as a micronized dry powder formulation, comprising lactose of a suitable grade and magnesium stearate, filled into hard shell capsules for use in a single dose device such as AEROLISER.

In another embodiment Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above is provided as a fine powder for use in an inhalation dosage form wherein the powder is in fine particles with a D₅₀ of 0.5-10 mm e.g. around 1-5 mm, that have been produced by a size reduction process other than jet mill micronisation e.g. spray drying, spray freezing, microfluidisation, high pressure homogenisation, super critical fluid crystallisation, ultrasonic crystallisation or combinations of these methods thereof, or other suitable particle formation methods known in the art that are used to produce fine particles with an aerodynamic particle size of 0.5-10 mm. The resultant particle size distribution may be measured using laser diffraction (e.g. with a Malvern Mastersizer 2000S instrument). The particles may either comprise the compound alone or in combination with suitable other excipients that may aid the processing. The resultant fine particles may form the final formulation for delivery to humans or may optionally be further formulated with other suitable excipients to facilitate delivery in an acceptable dosage form.

Compound 1 in the form of its Form 2 or Form 6 polymorph may also be administered rectally, for example in the form of suppositories or enemas, which include aqueous or oily solutions as well as suspensions and emulsions and foams. Such compositions are prepared following standard procedures, well known by those skilled in the art. For example, suppositories can be prepared by mixing the active ingredient with a conventional suppository base such as cocoa butter or other glycerides. In this case, the drug is mixed with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

Generally, for compositions intended to be administered topically to the eye in the form of eye drops or eye ointments, the total amount of Compound 1 in the form of its Form 2 or Form 6 polymorph will be about 0.0001 to less than 4.0% (w/w).

Preferably, for topical ocular administration, the compositions comprising Compound 1 in the form of its Form 2 or Form 6 polymorph will be formulated as solutions, suspensions, emulsions and other dosage forms. Aqueous solutions are generally preferred, based on ease of formulation, as well as a patient's ability to administer such compositions easily by means of instilling one to two drops of the solutions in the affected eyes. However, the compositions may also be suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions. Suspensions may be preferred for compounds that are sparingly soluble in water.

An alternative for administration to the eye is intravitreal injection of a solution or suspension of Compound 1 in the form of its Form 2 or Form 6 polymorph. In addition, Compound 1 in the form of its Form 2 or Form 6 polymorph may also be introduced by means of ocular implants or inserts.

The compositions comprising Compound 1 in the form of its Form 2 or Form 6 polymorph may also include various other ingredients, including, but not limited to, tonicity agents, buffers, surfactants, stabilizing polymer, preservatives, co-solvents and viscosity building agents. Suitable pharmaceutical compositions include Compound 1 in the form of its Form 2 or Form 6 polymorph formulated with a tonicity agent and a buffer. The pharmaceutical compositions of Compound 1 in the form of its Form 2 or Form 6 polymorph may further optionally include a surfactant and/or a palliative agent and/or a stabilizing polymer.

Various tonicity agents may be employed to adjust the tonicity of the composition, preferably to that of natural tears for ophthalmic compositions. For example, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, simple sugars such as dextrose, fructose, galactose, and/or simply polyols such as the sugar alcohols mannitol, sorbitol, xylitol, lactitol, isomaltitol, maltitol, and hydrogenated starch hydrolysates may be added to the composition to approximate physiological tonicity. Such an amount of tonicity agent will vary, depending on the particular agent to be added. In general, however, the compositions will have a tonicity agent in an amount sufficient to cause the final composition to have an ophthalmically acceptable osmolality (generally about 150-450 mOsm, preferably 250-350 mOsm and most preferably at approximately 290 mOsm). In general, the tonicity agents of the invention will be present in the range of 2 to 4% w/w. Preferred tonicity agents of the invention include the simple sugars or the sugar alcohols, such as D-mannitol.

An appropriate buffer system (e.g. sodium phosphate, sodium acetate, sodium citrate, sodium borate or boric acid) may be added to the compositions to prevent pH drift under storage conditions. The particular concentration will vary, depending on the agent employed. Preferably however, the buffer will be chosen to maintain a target pH within the range of pH 5 to 8, and more preferably to a target pH of pH 5 to 7.

Surfactants may optionally be employed to deliver higher concentrations of Compound 1 in the form of its Form 2 or Form 6 polymorph. The surfactants function to solubilise the compound and stabilise colloid dispersion, such as micellar solution, microemulsion, emulsion and suspension. Examples of surfactants which may optionally be used include polysorbate, poloxamer, polyosyl 40 stearate, polyoxyl castor oil, tyloxapol, Triton, and sorbitan monolaurate. Preferred surfactants to be employed in the invention have a hydrophile/lipophile/balance "HLB" in the range of 12.4 to 13.2 and are acceptable for ophthalmic use, such as TritonX114 and tyloxapol.

Additional agents that may be added to the ophthalmic compositions of Compound 1 in the form of its Form 2 or Form 6 polymorph are demulcents which function as a stabilising polymer. The stabilizing polymer should be an ionic/charged example with precedence for topical ocular use, more specifically, a polymer that carries negative charge on its surface that can exhibit a zeta-potential of (-)10-50 mV for physical stability and capable of making a dispersion in water (i.e. water soluble). A preferred stabilising polymer of the invention would be polyelectrolyte, or polyelectrolytes if more than one, from the family of cross- linked polyacrylates, such as carbomers and Pemulen(R), specifically Carbomer 974p (polyacrylic acid), at 0.1-0.5% w/w.

Other compounds may also be added to the ophthalmic compositions of Compound 1 in the form of its Form 2 or Form 6 polymorph to increase the viscosity of the carrier. Examples of viscosity enhancing agents include, but are not limited to: polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family; vinyl polymers; and acrylic acid polymers.

Topical ophthalmic products are typically packaged in multidose form. Preservatives are thus required to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben, phenylethyl alcohol, edentate disodium, sorbic acid, polyquaternium-1 , or other agents known to those skilled in the art. Such preservatives are typically employed at a level of from 0.001 to 1.0% w/v. Unit dose compositions of Compound 1 in the form of its Form 2 or Form 6 polymorph will be sterile, but typically unpreserved. Such compositions, therefore, generally will not contain preservatives.

Parenteral formulations will generally be sterile.

The medical practitioner, or other skilled person, will be able to determine a suitable dosage for Compound 1 in the form of its Form 2 or Form 6 polymorph and hence the amount of the compound of the invention that should be included in any particular pharmaceutical formulation (whether in unit dosage form or otherwise).

Compound 1 in the form of its Form 2 or Form 6 polymorph may be used in combination with one or more other active agents which are useful in the treatment or prophylaxis of respiratory diseases and conditions.

An additional active agent of this type may be included in the pharmaceutical composition described above but alternatively it may be administered separately, either at the same time as Compound 1 in the form of its Form 2 or Form 6 polymorph or at an earlier or later time.

Therefore, in a further aspect of the present invention there is provided a product comprising Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above and an additional agent useful in the treatment or prevention of respiratory conditions as a combined preparation for simultaneous, sequential or separate use in the treatment of a disease or condition affected by modulation of TMEM16A and especially a respiratory disease or condition, for example one of the diseases and conditions mentioned above.

There is also provided Compound 1 in the form of its Form 2 or Form 6 polymorph as defined above in combination with an additional agent useful in the treatment or prevention of respiratory conditions as a combined preparation for simultaneous, sequential or separate use in the treatment of a disease or condition affected by modulation of TMEM16A and especially a respiratory disease or condition, for example one of the diseases and conditions mentioned above.

Suitable additional active agents which may be included in a pharmaceutical composition or a combined preparation with Compound 1 in the form of its Form 2 or Form 6 polymorph include:

Suitable additional active agents which may be included in a pharmaceutical composition or a combined preparation with Compound 1 in the form of its Form A polymorph or Form B polymorph, especially its Form B(l) pseudopolymorph, as defined above or in amorphous form include:

b2 adrenoreceptor agonists such as metaproterenol, isoproterenol, isoprenaline, albuterol, salbutamol, formoterol, salmeterol, indacaterol, terbutaline, orciprenaline, bitolterol mesylate, pirbuterol, olodaterol, vilanterol and abediterol;

antihistamines, for example histamine Hi receptor antagonists such as loratadine, cetirizine, desloratadine, levocetirizine, fexofenadine, astemizole, azelastine and chlorpheniramine or H₄ receptor antagonists;

dornase alpha;

corticosteroids such as prednisone, prednisolone, flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate mometasone furoate and fluticasone furoate;

Leukotriene antagonists such as montelukast and zafirlukast;

anticholinergic compounds, particularly muscarinic antagonists such as ipratropium, tiotropium, glycopyrrolate, aclidinium and umeclidinium;

CFTR repair therapies (e.g. CFTR potentiators, correctors or amplifiers) such as Ivacaftor, QBW251 , Bamacaftor (VX659), Elexacaftor (VX445), VX561/CPT-656, VX152, VX440, GLP2737, GLP2222, GLP2451 , PTI438, PTI801 , PTI808, FDL-169 and FDL-176 and CFTR correctors such as Lumacaftor and Tezacaftor or combinations thereof (for example a combination of Ivacaftor, Tezacaftor and Elexacaftor);

ENaC modulators, particularly ENaC inhibitors;

Antibiotics;

Antivirals such as ribavirin and neuraminidase inhibitors such as zanamivir;

Antifungals such as PUR1900;

Airway hydrating agents (osmoloytes) such as hypertonic saline and mannitol (Bronchitol®); and

Mucolytic agents such as N-acetyl cysteine. When the additional active agent is an ENaC modulator, it may be an ENaC inhibitor such as amiloride, VX-371 , AZD5634, QBW276, SPX-101 , BI443651 , BI265162 and ETD001. Other suitable ENaC blockers are disclosed in our applications WO 2017/221008, WO 2018/096325, WO2019/077340 and WO 2019/220147 and any of the example compounds of those applications may be used in combination with the compounds of general formula (I). Particularly suitable compounds for use in combination with the compounds of general formula (I) include compounds having a cation selected from: 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) ethyl]-6-(4-{bis[(2S,3R,4R,5R)-

2.3.4.5.6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1 ,3-diethyl-H-1,3- benzodiazol-3-ium;

2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-{[2-(4-

{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidin-1-yl)ethyl]carbamoyl}- 1 ,3-diethyl-1 H-1 ,3-benzodiazol-3-ium;

2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-[4-({bis[(2S,3R,4R,5R)-

2.3.4.5.6-pentahydroxyhexyl]amino}methyl)piperidine-1-carbonyl]-1 ,3-diethyl-1H-1 ,3- benzodiazol-3-ium;

2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3R)-3- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-1 ,3- diethyl-1 H-1 ,3-benzodiazol-3-ium;

2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3S)-3- {bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-1 ,3- diethyl-1 H-1 ,3-benzodiazol-3-ium;

2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1 ,3-diethyl-6-{[(1r,4r)-4-

{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1 ,3- benzodiazol-3-ium;

2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1 ,3-diethyl-6-{[(1s,4s)-4-

and a suitable anion, for example halide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate, fumarate, citrate, tartrate, oxalate, succinate, mandelate, methane sulfonate or p-toluene sulfonate.

The invention will now be described in greater detail with reference to the Examples. INSTRUMENTATION AND GENERAL CONDITIONS

For Example 1 :

The starting materials and intermediates and Compound 1 may be isolated and purified using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation, and chromatography. Unless otherwise stated, all starting materials are obtained from commercial suppliers and used without further purification. Salts may be prepared from compounds by known salt-forming procedures.

MS

Mass spectra were run on LC-MS systems using electrospray ionization. These were run using either a Waters Acquity uPLC system with Waters PDA and ELS detectors or Shimadzu LCMS-2010EV systems. [M+H]+ refers to mono-isotopic molecular weights.

NMR

NMR spectra were recorded on a Bruker Avance III HD 500 MHz or a Bruker Avance III HD 250 MHz using the solvent as internal deuterium lock. Spectra were recorded at room temperature unless otherwise stated and were referenced using the solvent peak.

HPLC

The analytical HPLC conditions are as follows:

Method A

Column: Phenomenex Kinetix-XB C18 2.1 x 100 mm, 1.7mm

Column Temp 40 °C

Eluents: A: H20 0.1 % formic acid, B: acetonitrile, 0.1% formic acid

Flow Rate: 0.6 mL/min

Gradient: 0-5.3mins 5-100%B, 5.3-5.8mins 100%B, 5.8-5.82mins 100-5%B,

5.82-7.00mins 5%B

Method E

Column: Kinetex Core-Shell C18 2.1 x 50mm 5mm

Column Temp 40 °C

Eluents: A: H20+0.1 % formic acid, B: acetonitrile+ 0.1% formic acid

Flow Rate: 1.2 mL/min

Gradient: 0-1.20mins 5-100%B, 1.20-1.30mins 100%B, 1.30-1.31 mins 100-

5%B For Examples 2 to 4

X-ray Powder Diffraction (XRPD)

Bruker AXS D8 Advance

XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Ka radiation (40 kV, 40 mA) and a q-2 q goniometer fitted with a Ge monochromator. The incident beam passes through a 2.0 mm divergence slit followed by a 0.2 mm antiscatter slit and knife edge. The diffracted beam passes through an 8.0 mm receiving slit with 2.5° Soller slits followed by the Lynxeye Detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA respectively.

Samples were run under ambient conditions as flat plate specimens using powder as received. The sample was prepared on a polished, zero-background (510) silicon wafer by gently pressing onto the flat surface or packed into a cut cavity. The sample was rotated in its own plane.

The details of the standard data collection method are:

• · Angular range: 2 to 42° 2q

• · Step size: 0.05° 2q

• · Collection time: 0.5 s/step (total collection time: 6.40 min)

PANalytical Empyrean

XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Ka radiation (45 kV, 40 mA) in transmission geometry. A 0.5° slit, 4 mm mask and 0.04 rad Soller slits with a focusing mirror were used on the incident beam. A PIXcel3D detector, placed on the diffracted beam, was fitted with a receiving slit and 0.04 rad Soller slits. The software used for data collection was X’Pert Data Collector using X’Pert Operator Interface. The data were analysed and presented using Diffrac Plus EVA or HighScore Plus.

Samples were prepared and analysed in either a metal or Millipore 96 well-plate in transmission mode. X-ray transparent film was used between the metal sheets on the metal well-plate and powders (approximately 1 - 2 mg) were used as received. The Millipore plate was used to isolate and analyse solids from suspensions by adding a small amount of suspension directly to the plate before filtration under a light vacuum.

The scan mode for the metal plate used the gonio scan axis, whereas a 2q scan was utilised for the Millipore plate. The details of the standard screening data collection method are:

• · Angular range: 2.5 to 32.0° 2q

• · Step size: 0.0130° 2q

• · Collection time: 12.75 s/step (total collection time of 2.07 min)

Nuclear Magnetic Resonance (NMR)

1 H NMR spectra were collected on a Bruker 400 MHz instrument equipped with an auto sampler and controlled by a DRX400 console. Samples were prepared in DMSO-d6 solvent, unless otherwise stated. Automated experiments were acquired using ICON-NMR configuration within Topspin software, using standard Bruker-loaded experiments (1 H). Off-line analysis was performed using ACD Spectrus Processor.

Differential Scanning Calorimetry (DSC)

DSC data were collected on a TA Instruments Q2000 equipped with a 50 position auto sampler. Typically, 0.5 - 3 mg of each sample, in a pin-holed aluminium pan, was heated at 10 °C/min from 25 °C to 250 °C. A purge of dry nitrogen at 50 ml/min was maintained over the sample.

The instrument control software was Advantage for Q Series and Thermal Advantage and the data were analysed using Universal Analysis.

Thermo-Gravimetric Analysis (TGA)

TGA data were collected on a TA Instruments Discovery TGA, equipped with a 25 position auto-sampler. Typically, 5 - 10 mg of each sample was loaded onto a pre-tared aluminium DSC pan and heated at 10 °C/min from ambient temperature to 350 °C. A nitrogen purge at 25 ml/min was maintained over the sample.

The instrument control software was TRIOS and the data were analysed using Universal Analysis.

Chemical Purity Determination by HPLC

Purity analysis was performed on an Agilent HP1100 series system equipped with a diode array detector and using ChemStation software. The full method details are provided below:

Abbreviations

br broad

d doublet

dd doublet of doublets

DCM dichloromethane

DIPEA diisopropylethylamine

DMF N,N-dimethylformamide

EtOAc ethyl acetate

HATU 2-(7-aza-1 H-benzotriazole-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate

m multiplet

MeCN acetonitrile

min minute(s)

mL millilitre(s)

MS mass spectrometry

m/z mas to charge ratio

NMR nuclear magnetic resonance

Rt retention time

s singlet

sat saturated

t triplet TBTU N , N , N’, N’-tetramethyl-0-(benzotriazole-1 -yl)uranium tetrafluoroborate

TEA triethylamine

Example 1 - Preparation of 4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]-N-(1,1- dimethylprop-2-ynyl)pyridine-2-carboxamide (Compound 1) - Method of

PCT/GB2019/050209

Step 1 : Methyl 4-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]pyridine-2-carboxylate

2-(5-Chloro-2-methoxy-phenyl)acetic acid (24.19 g, 120.59 mmol) was suspended in thionyl chloride (103.68 mL, 1427.48 mmol) and heated at 70 °C for 1.5 hours. After cooling to room temperature the mixture was concentrated in vacuo. The residue was then dissolved in DCM (135 mL) and re-concentrated. The resulting brown viscous oil was dissolved in DCM (135 mL) and added dropwise to a cooled (ice bath) suspension of methyl 4-aminopyridine-2-carboxylate (17.9 g, 1 17.65 mmol) and DIPEA (30.82 mL, 176.47 mmol) in DCM (225 mL). The reaction mixture was allowed to warm to room temperature and stirred overnight. The resulting mixture was diluted with water (180 mL) and stirred for 10 mins. The organic portion was separated, dried over anhydrous Na2SC>4, filtered and concentrated in vacuo. The residue was purified by chromatography on silica eluting with EtOAc to afford the titled compound as an orange glassy solid.

1 H NMR (500 MHz, DMSO-d6) d 10.72 (s, 1 H), 8.54 (d, J = 5.5 Hz, 1 H), 8.30 (d, J = 1.9 Hz, 1 H), 7.77 (dd, J = 5.5, 2.2 Hz, 1 H), 7.32 - 7.29 (m, 2H), 7.02 - 6.99 (m, 1 H), 3.86 (s, 3H), 3.75 (s, 3H), 3.71 (s, 2H).

LC-MS (Method E): Rt 1.04 mins; MS m/z 335.0/337.0 = [M+H]+ (98% @ 215nm) Step 2: 4-[[2-(5-Chloro-2-methoxy-phenyl)acetyl]amino]pyridine-2-carboxylic acid

To a solution of methyl 4-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]pyridine-2- carboxylate (step 1) (95%, 38.09 g, 108.1 mmol) in THF (200 mL) was added a 2M aqueous solution of lithium hydroxide hydrate (162.15 mL, 324.29 mmol) and the resulting mixture was stirred at room temperature for 1 hour. The volatile organics were removed in vacuo and the aqueous residue cooled (ice-bath) and treated with the gradual addition of 3M aqueous HCI (150 mL). The resulting suspension was filtered, washed with water (3 x 200 mL), diethyl ether (2 x 250 mL), dried under suction and then further dried in a high vacuum oven at 40 °C to afford the titled compound as a beige solid.

1 H NMR (500 MHz, DMSO-d6) d 10.78 (s, 1 H), 8.53 (d, J = 5.6 Hz, 1 H), 8.27 (d, J = 2.0 Hz, 1 H), 7.81 (dd, J = 5.6, 2.2 Hz, 1 H), 7.34 - 7.28 (m, 2H), 7.04 - 6.98 (m, 1 H), 3.75 (s, 3H), 3.72 (s, 2H).

LC-MS (Method E): Rt 0.88 mins; MS m/z 320.9/ 323.0 = [M+H]+ (97% @ 215nm)

Step 3: 4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxylic acid

1 M BBr3 in DCM (308.67 mL, 308.67 mmol) was added slowly over 1 hour to a suspension of 4-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]pyridine-2-carboxylic acid (step 2) (25.0 g, 77.17 mmol, 99%) in DCM (500 mL) at 0 - 5°C under an inert atmosphere of N2. The mixture was allowed to warm to room temperature and stirred for a further hour. The reaction mixture was concentrated in vacuo and the residue was suspended in EtOAc (500 mL) and water (500 mL) at 0°C. The resulting mixture was allowed to warm to room temperature and the aqueous layer adjusted to pH 4 by portion-wise addition of sat. aq. NaHC0₃ (350 mL). The precipitate was collected by filtration, washed with water (2 x100 mL), EtOAc (2 x100 mL), diethyl ether (2 x 150 mL) and dried in a high vacuum oven at

40 °C to afford the titled compound as a beige solid.

1 H NMR (500 MHz, DMSO-d6) d 10.72 (s, 1 H), 9.82 (br. s, 1 H), 8.53 (d, J = 5.5 Hz, 1 H), 8.27 (d, J = 2.0 Hz, 1 H), 7.80 (dd, J = 5.6, 2.2 Hz, 1 H), 7.22 (d, J = 2.7 Hz, 1 H), 7.13 (dd, J = 8.6, 2.7 Hz, 1 H), 6.80 (d, J = 8.6 Hz, 1 H), 3.67 (s, 2H).

LC-MS (Method E): Rt 0.86 mins; MS m/z 306.9/308.9 = [M+H]+ (97% @ 215nm)

Step 4

To a solution of 4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]pyridine-2-carboxylic acid (step 3) (20 g, 63.25 mmol, 97%), 2-methylbut-3-yn-2-amine (7.99 mL, 75.9 mmol) and DIPEA (16.57 mL, 94.88 mmol) in DMF (315 mL) was added HATU (28.86 g, 75.9 mmol) and the mixture stirred at room temperature for 1 hour. The resulting mixture was concentrated in vacuo and the residue was dissolved in EtOAc and washed sequentially with 1 M HCI, 1 M NaOH and brine. The acid washing was re-extracted with EtOAc and the combined organic extracts were washed with 1 M NaOH, brine, dried over Na₂S0₄ and concentrated in vacuo to afford a yellow foam. The foam was absorbed onto silica and purifed by chromatography eluting with 0-100% EtOAc in heptanes. The isolated material was suspended in MeCN (50 mL) and heated to reflux until all solids had dissolved. The resulting solution was allowed to cool to room temperature and stand for 8 hours to yield crystals. The crystals were filtered, washed with ice cooled MeCN and dried in a vacuum oven overnight to afford crop 1 of the titled compound. The filtrate from was combined with the impure fractions from chromatography and repurified by chromatography on silica eluting with 0-100% EtOAc in heptanes. The isolated material was combined with crop 1 and recrystallised again by heating at reflux in MeCN. The solution was allowed to cool and stand at room temperature to afford crystals which were filtered, washed with ice- cooled MeCN and dried in a vacuum oven to afford the titled compound as a crystalline solid.1 H NMR (500 MHz, DMSO-d6) d 10.71 (br s, 1 H), 9.82 (br s, 1 H), 8.46 (d, J = 5.5 Hz, 1 H), 8.31 (s, 1 H), 8.19 (d, J = 1.9 Hz, 1 H), 7.84 (dd, J = 5.5, 2.2 Hz, 1 H), 7.22 (d, J = 2.7 Hz, 1 H), 7.12 (dd, J = 8.6, 2.7 Hz, 1 H), 6.81 (d, J = 8.6 Hz, 1 H), 3.68 (s, 2H), 3.20 (s, 1 H), 1.64 (s, 6H).

LC-MS (Method A): Rt 3.07 mins; MS m/z 372.1/374.1 = [M+H]+ (99% @ 215 nm)

The product was crystalline and was designated the Form 1 (or Form A) crystalline polymorph. The XRPD diffractogram and TGA and DSC thermograms for this polymorph are shown in Figures 1 and 2 and the XRPD peaks are listed in Table 1.

Table 1 - XRPD Peaks for Form 1 polymorph of Compound 1

TGA (Figure 2) and ¹H NMR analysis showed that Form 1 is an anhydrous non-solvated form of Compound 1 , although this batch contained a very small amount of residual acetonitrile. On heating, there are no events seen in the DSC data before the sharp endotherm with an onset temperature of 175 °C, likely to be a melting event (see Figure 2). There was no evidence of conversion to other any forms on heating, suggesting that Form 1 is a stable form.

Form 1 was stored at 40 °C/75 %RH, in an open vial. After 2 weeks, the residue was analysed by XRPD and no change in form was detected. Therefore, Form 1 is stable to exposure to elevated temperature and humidity for up to 2 weeks and no evidence of a hydrated form was observed.

Form 1 was used as the input material in the focussed polymorph screening experiments, which are described below.

Example 2 - Solubility Assessment

Compound 1 (Form 1 , approximately 30 mg) was weighed into 2 ml glass vials and was treated with aliquots of solvent. The samples were stirred at 50 °C, 400 rpm, for approximately 10 minutes between addition of aliquots of solvent. Solvent was added until a clear solution was formed or a maximum of 60 volumes (1.8 ml) of solvent was reached.

Samples that were solutions (or close to dissolution, sample in DCM) were cooled to 5 °C at 0.1 °C/min and were stirred overnight. After cooling, the samples that contained solids were filtered using PTFE frits, dried under suction for approximately 30 minutes and were analysed by XRPD.

Samples that were suspensions in 60 volumes of solvent were matured (shaken in cycles of 4 hours at 50 °C/4 hours at RT). After 5 days, samples were filtered using PTFE frits, dried under suction for approximately 30 minutes and were analysed by XRPD.

The solubility results for Compound 1 Form 1 are shown in Table 2.

Table 2 - Solubility results for Compound 1 Form 1

X; material dissolved at 50 °C; x: material did not dissolve; 0: partial dissolution

Results show that the sample was soluble in ethyl acetate, acetone, ethanol, THF and in the acetonitrile/water mixtures (the solubility was higher in ACN/water 80/20 than in 95/5 v/v). A lower solubility was observed in I PA and acetonitrile and partial dissolution occurred in DCM. n-heptane, water and TBME were suitable anti-solvents, with no observable dissolution of Compound 1.

Samples that were solutions were cooled and slurries were matured. Observations and XRPD results of the solids obtained by cooling or maturation are shown in Table 3 and XRPD data is given in Figure 3.

Table 3 - Observations and XRPD results for the solids from the solubility assessment with Compound 1

XRPD results showed that all samples were crystalline. Six of the solids recovered were consistent with Form 1. The remaining six solids were six new forms, named Form 2 to Form 7. A sample of each of these forms was characterised and the results are given in Example 4.

The sample obtained from 5% aqueous ACN had subtle differences from Form 1 in the initial XRPD diffractograms (when analysed in transmission mode). This sample was re analysed on a different XRPD instrument (in reflectance mode). Results (Figure 4) show that this sample was consistent with Form 1 , so the subtle differences seen in the initial analysis are likely to be caused by preferred orientation.

Example 3 - Anti-Solvent Screen

Stock solutions of Compound 1 were made up in the minimum volume of solvent at 50 °C. The solutions were filtered using 0.45 mm PTFE filters to remove crystalline seeds and were split into equal volumes to give approximately 30 mg Compound 1/vial.

Aliquots of anti-solvent (n-heptane/water/TBME) were added. The samples were stirred at 50 °C, 400 rmm, for approximately 15 minutes between addition of aliquots of anti-solvent. Anti-solvent was added until a suspension or oil was formed or a maximum of 70 volumes (2.1 ml) of anti-solvent was added.

Samples were stirred for approximately 1 hour at 50 °C (after the last addition of anti solvent), samples that contained solids were filtered using PTFE frits, dried under suction for approximately 30 minutes and were analysed by XRPD.

Samples that were solutions were cooled to 5 °C at 0.1 °C/min and were stirred overnight. Solids that formed on cooling were filtered, dried under suction for approximately 30 minutes and were analysed by XRPD. Samples that remained solutions on cooling were left at ambient conditions with loosened lids to evaporate to dryness. The resulting solids were analysed by XRPD.

The solvents selected for the stock solutions were based on the results of the solubility assessment of Example 2. Acetone and ethanol were the solvents in which Compound 1 was most soluble. Although Compound 1 was freely soluble in the acetonitrile/water mixtures, these were not used in order to avoid miscibility issues between the water in the stock solution and the n-heptane and TBME that were used as anti-solvents. The third solvent selected was THF as this solubilised Compound 1 reasonably well. Anti-solvents were selected to be miscible with the solvent used in the stock solutions, with n-heptane, water and TBME being used as anti-solvents. The results are shown in Table 4. Table 4 - Observations and XRPD results from the anti-solvent screen with Compound 1

XRPD results of the solids showed that the majority were crystalline and consistent with Form 1.

Three samples were not consistent with Form 1. Samples from THF/n-heptane and THF/TBME were Form 5, or Form 5 with extra peaks, respectively. Form 5 was originally isolated by cooling a solution in THF, so it is logical that this form should be isolated from experiments with THF as the solvent. The final sample (from acetone/TBME) was another new form, named Form 8. This form was characterised and results are given in Example 4. The sample obtained from acetone/n-heptane had subtle differences from Form 1 in the initial XRPD diffractograms (when analysed in transmission mode). This sample was re analysed on a different XRPD instrument (in reflectance mode). Results (Figure 5) show that this sample was consistent with Form 1 , so the subtle differences seen in the initial analysis are likely to be caused by preferred orientation.

Example 4 - Characterisation of New Forms of Compound 1

One sample of each of the new forms (Forms 2, 3, 4, 5, 5 + extra peaks, 6, 7 and 8) was analysed by ¹H NMR, TGA, DSC and stored at 40 °C/75 %RH. Results are summarised in Table 5 with Form 1 for comparison.

Table 5 - Summary of Characterisation of Forms of Compound 1

*degradation begins at -175 °C, **immediately followed by exothermic degradation, ***some Form 1 may be present in this sample,

Forms 2, 4 and 6 are non-solvated polymorphs of Compound 1 , although the solids contain small amounts of residual solvent. The XRPD diffractograms of Forms 2, 4 and 6 are shown in Figures 6, 12 and 18 respectively. The Form 2 sample contains approximately 1 wt% ethyl acetate. On heating the Form 2 sample, the solvent is lost at low temperatures, resulting in a small weight loss in the TGA data (Figure 7), which is consistent with a low temperature broad endotherm in the DSC data (Figure 7). No low temperature weight losses/broad endotherms were seen in the Form 4 (Figure 13) and 6 (Figure 19) samples as the amount of residual solvent is too small to be detected by TGA/DSC.

On heating Forms 2, 4 and 6 to temperatures in excess of 130 °C, broad exotherms are seen in the DSC data (figures 7, 13 and 19). This is suggestive of an exothermic solid- state transition, caused by the conversion of Forms 2, 4 or 6 to a more stable form. Further heating results in a sharp endotherm in the DSC data, at a similar temperature to the melt of Form 1. It is likely that Forms 2, 4 and 6 are converted to Form 1 on heating.

Forms 2 and 6 were stable to exposure to 40 °C/75 %RH for 7 days (Figures 8 and 20) but Form 4 converted to a mixture of Form 1 and Form 2 after being exposed to these conditions for 7 days (Figure 14). Initial analysis of the XRPD diffractogram of Form 6 after 7 days at 40 °C/75 %RH, measured on an XRPD instrument in transmission mode, looked as though additional peaks were present compared to the Form 6 reference. However, when the static stability residue is compared to the Form 6 reference obtained on an instrument in reflectance mode (Figure 20) it can be seen that these peaks were present in the original Form 6 sample. Therefore, it appears that the sample did not change after exposure to elevated temperature and humidity.

Forms 3, 5, 7 and 8 are solvated forms of Compound 1 and their XRPD diffractograms are shown in Figures 9, 16, 21 and 24 respectively. Forms 3 and 7 are mono I PA and TBME solvates respectively. Form 5 contains a significant amount of THF but is not a stoichiometric solvate. When made from THF/TBME additional peaks were seen in the XRPD diffractogram (Form 5 + extra peaks), which may be caused by the replacement of some of the THF with TBME. Form 8 is a mixed acetone/TBME solvate with a non- stoichiometric amount of solvent.

The four solvated forms have similar thermal behaviour with low temperature weight losses in the TGA data, which are consistent with the amount of solvent detected by 1 H NMR. Broad endotherms are seen in the DSC thermograms at similar temperatures to the weight losses in the TGA, which are consistent with the loss of solvent (Figures 10, 17, 22 and 25).

Further heating results in a sharp endotherm being detected in the DSC data, likely to be a melt endotherm, with an onset temperature similar to that of the melt of Form 1. These results suggest that the solvated forms may convert to Form 1 on heating.

Storage of the solvated forms in open vials at 40 °C/75 %RH results in conversion to Form 1 , or to a mixture of Form 1 and Form 2 (Figures 11 , 18, 23 and 26). Therefore, the solvated forms are not stable to exposure to elevated temperature and humidity for 7-14 days and are not suitable for further develomment.

Example 5 - Further Characterisation of Form 2 (Form B) Form B (ethyl acetate)

Compound 1 (500 mg, 1.0 wt.) was dissolved in ethyl acetate (2.5 ml, 5.0 vol) at 75°C, cooled to sub ambient temperature and allowed to stand undisturbed. (Note: this batch was cooled in the freezer but standing in the fridge also generated Form B). The product was isolated by filtration and dried under a steady stream of nitrogen to give Form B (369 mg, 73% yield not corr.).

Melt onset by DSC 174 °C, -61.5 J / g. The product contained ethyl acetate (2.9 % w/w) and heptane (0.24 % w/w). The XRPD and DSC plots are shown in Figures 27 and 28 respectively and it can be seen that these are consistent with Form B (Form 2) obtained previously.

The XRPD peak table is set out below:

Form B (ispropanol)

Form 3 (Form C; 36 mg, 10.3 % w/w isopropanol) was maintained at 43 % RH for several days to determine if replacement of isopropanol by water took place. After the 1 h, 2 h, 24 h, 4 d, 6 d and 12 d time points, the phase was isostructural with input Form C; at t = 6 d sample contained (6.6 % w/w isopropanol) and after t = 12 d, exchange had stalled and the sample plateaued at 6.9 % w/w isopropanol. This suggested that Form C was a non- stoichiometric solvate, able to vary its solvent occupancy without a significant structural reorganisation of the crystal lattice; as was the case when this form was maintained at 95 % RH and changed into Form A. After 12 days at 43 % RH, water activity was increased to 60 % RH and was maintained at this level for + 6 days, after this time isopropanol content had fallen to 2.7 % w/w and the phase had changed into isostructural Form B (2.7 % w/w isopropanol), a pseudopolymorph of Form B (ethyl acetate) and also a pseudopolymorph of Form B (water).

The XRPD spectrum was consistent with the XRPD spectrum of Form B (ethyl acetate) obtained previously and the peak table is set out below.

The DSC plot shows two events and the TGA plot (Figure 28) shows two weight changes (-1.1% w/w and -2.1 % w/w) consistent with isopropanol content of about 2.7% w/w. This demonstrates that phase change into Form B (isopropanol) was observed and not exchange of solvent by water.

Form B (water)

Compound 1 , Form B (50 mg, 1.0 wt) was maintained at 95% relative humidity at 18 to 23 °C for 25 days. Ethyl acetate (2.9% w/w) was supplanted by water.

The TGA plot is shown as Figure 29.

Example 6 - Further Characterisation of Form 6 (Form F)

Form A (441.3 mg, 1.0 wt.) was dissolved in acetone (2 ml, 2.3 vol), aided by gentle heating. To the vessel containing the solution, was charged dichloromethane (8.8 ml, 20 vol) and the resultant solution was stored under sub-ambient conditions over 20 h, to allow for the product to crystallise. The product was isolated by filtration, pulled free of surplus solvents and dried under a steady stream of nitrogen to afford the title compound (359 mg, 72% yield not corr.).

Melt onset by DSC was 171 °C, -74.3 J / g.

The XRPD, TGA and DSC were consistent with those previously obtained.

Claims

1. A compound which is 4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]-N-(1 , 1- dimethylprop-2-ynyl)pyridine-2-carboxamide (Compound 1), having the following structural formula:

in the form of its Form 2 anhydrous solid crystalline polymorph, for example a form having an XRPD diffractogram substantially as shown in Figure 6 or its Form 6 anhydrous solid crystalline polymorph, for example a form having an XRPD diffractogram substantially as shown in Figure 18.

2. A compound according to claim 1 in the form of its Form 2 crystalline polymorph characterised by an XRPD diffractogram comprising major peaks at 14.6 and 22.5 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six or all seven) at positions selected from 7.2, 9.2, 18.2, 20.4, 23.9, 24.7 and 26.0 (± 0.2 degrees, 2-theta values).

3. A compound according to claim 2 characterised by an XRPD diffractogram comprising major peaks at 14.6 and 22.5 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven, eight, nine, ten, eleven, twelve or all thirteen) of the peaks at positions 7.2, 9.2, 1 1.6, 12.7, 15.6, 18.2, 20.4, 23.9, 24.7, 26.0, 27.4, 27.8 and 29.1 (± 0.2 degrees, 2-theta values).

4. A compound according to claim 2 or claim 3 characterised by an XRPD diffractogram comprising major peaks at 14.6 and 22.5 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or all eighteen) of the peaks at positions 7.2, 9.2, 1 1.6, 12.7, 13.0, 15.6, 18.2, 18.8, 20.4, 20.9, 23.2, 23.9, 24.7, 26.0, 27.4, 27.8, 28.6 and 29.1 (± 0.2 degrees, 2-theta values).

5. A compound according to any one of claims 1 to 4 wherein the Form 2 crystalline polymorph is substantially free of other forms of Compound 1 such that at least 97% by weight of Compound 1 is present as the Form 2 polymorph.

6. A process for the preparation of the Form 2 crystalline polymorph of Compound 1 according to any one of claims 1 to 5 comprising crystallising Compound 1 from ethyl acetate.

7. A process according to claim 6 comprising the steps of

(iii) allowing crystallisation to take place; and

(iv). isolating the crystals obtained in step (iii).

8. A compound according to claim 1 in the form of its Form 6 crystalline polymorph characterised by an XRPD diffractogram comprising major peaks at 15.5 and 25.3 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven or all eight) at positions selected from 7.2, 9.3, 15.5, 16.5, 17.0, 18.9, 24.7, 25.3, 27.4 and 28.1 (± 0.2 degrees, 2-theta values).

9. A compound according to claim 8 characterised by an XRPD diffractogram comprising major peaks at 15.5 and 25.3 (± 0.2 degrees, 2-theta values) and at least three peaks (for example three, four, five, six, seven, eight, nine, ten, eleven, twelve or all thirteen) of the peaks at positions 7.2, 9.3, 12.0, 15.5, 16.5, 17.0, 18.9, 21.6, 23.0, 24.3, 24.7, 25.3, 27.4, 28.1 and 30.6 (± 0.2 degrees, 2-theta values).

10. A compound according to claim 8 or claim 9 characterised by an XRPD diffractogram comprising major peaks at 15.5 and 25.3 (± 0.2 degrees, 2-theta values) and at least major peaks (for example three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or all eighteen) of the peaks at positions 7.2, 8.4, 9.3, 12.0, 15.5, 16.5, 17.0, 18.9, 21.6, 22.3, 22.6, 23.0, 24.3, 24.7, 25.3, 26.9, 27.4, 28.1 , 30.6 and 31.2 (± 0.2 degrees, 2-theta values).

1 1. A compound according to any one of claims 1 or 8 to 10 wherein the Form 6 crystalline polymorph is substantially free of other forms of Compound 1 such that at least 97% by weight of Compound 1 is present as the Form 6 polymorph.

12. A process for the preparation of the Form 6 crystalline polymorph of Compound 1 according to any one of claims 1 or 8 to 11 comprising crystallising Compound 1 from dichloromethane.

13. A process according to claim 6 comprising the steps of

(iii) allowing crystallisation to take place; and

(iv). isolating the crystals obtained in step (iii).

14. A compound according to any one of claims 1 to 5 or 8 to 11 for use in medicine.

15. A compound for use according to claim 14 in the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A.

16. Use of a compound according to any one of claims 1 to 5 or 8 to 1 1 in the manufacture of a medicament for the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A.

17. A method for the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A, the method comprising administering to a patient in need of such treatment an effective amount of a compound according to any one of claims 1 to 5 or 8 to 1 1.

18. A compound for use, a use or a method according to any one of claims 15 to 17, wherein the disease or condition affected by modulation of TMEM16A is selected from respiratory diseases and conditions, dry mouth (xerostomia), intestinal hypermobility, cholestasis and ocular conditions.

19. A compound for use, a use or a method according to claim 18, wherein the respiratory diseases and conditions are selected from cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, including non-cystic fibrosis bronchiectasis, asthma and primary ciliary dyskinesia.

20. A compound for use, a use or a method according to claim 18, wherein the dry mouth (xerostomia) results from Sjorgens syndrome, radiotherapy treatment or xerogenic drugs.

21. A compound for use, a use or a method according to claim 18, wherein the intestinal hypermobility is associated with gastric dyspepsia, gastroparesis, chronic constipation or irritable bowel syndrome.

22. A compound for use, a use or a method according to claim 18, wherein the ocular disease is dry eye disease.

23. A compound for use, a use or a method according to claim 18 wherein the compound is used in combination with an additional agent useful in the treatment or prevention of respiratory conditions as a combined preparation for simultaneous, sequential or separate use in the treatment of a disease or condition affected by modulation of TMEM16A.

24. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 or 8 to 11 together with a pharmaceutically acceptable excipient.

25. A pharmaceutical composition according to claim 24 formulated for oral, rectal, nasal, topical (including topical administration to the lung, dermal, transdermal, eye drops, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.

26. A pharmaceutical composition according to claim 24 or claim 25 further including an additional active agent useful in the treatment or prevention of respiratory conditions.

27. a product comprising a compound according to any one of claims 1 to 5 or 8 to 11 and an additional agent useful in the treatment or prevention of respiratory conditions as a combined preparation for simultaneous, sequential or separate use in the treatment of a disease or condition affected by modulation of TMEM16A.

28. A compound for use, a use or a method according to claim 23, a pharmaceutical composition according to claim 26 or a product according to claim 27, wherein the additional agent useful in the treatment or prevention of respiratory conditions is selected from:

dornase alpha;

Leukotriene antagonists such as montelukast and zafirlukast;

Antibiotics;

Antivirals such as ribavirin and neuraminidase inhibitors such as zanamivir;

Antifungals such as PUR1900;

Mucolytic agents such as N-acetyl cysteine;

ENaC modulators, particularly ENaC inhibitors such as:

amiloride, VX-371 , AZD5634, QBW276, SPX-101 , BI443651 , BI1265162, ETD001 and compounds having a cation selected from:

2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) ethyl]-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;

2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido) methyl]-6-{[2-(4-

29. A process for the preparation of a pharmaceutical composition according to claim 24 or 25 comprising bringing a compound according to any one of claims 1 to 5 or 8 to 11 into association with the excipient.