Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

• the present invention relates to novel compounds which have activity as positive modulators of the calcium-activated chloride channel (CaCC), TMEM16A.
• the invention also relates to methods of preparing the compounds and pharmaceutical compositions containing them as well as to the use of these compounds in treating diseases and conditions in which TMEM16A plays a role, particularly respiratory diseases and conditions.
• the hydration of the mucus gel is critical to enable mucus clearance (Boucher 2007; Matsui et al, 1998).
• the mucus gel 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 % solids of the mucus gel is increased as the hydration is reduced and mucus clearance is reduced (Boucher, 2007).
• 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 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 anions 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 (Ano1), 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).
• 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.
• WO2019/145726 relates to 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.
• the present inventors have developed further compounds which have excellent activity as positive modulators of TMEM16A.
• at least some of the compounds have one or more advantages compared with the compounds exemplified in WO2019/145726.
• Some of the compounds of the invention have increased metabolic stability, as represented by their low clearance rates from human microsomes and hepatocytes. This makes them particularly suitable for oral administration since lower first pass metabolism, leads to increased oral bioavailability and a lower dose for a given pharmacological effect
• Other compounds of the present invention have metabolic stability which is decreased in comparison with the compounds exemplified in WO2019/145726 to an extent which makes them particularly suitable for administration by inhalation.
• the decreased in vivo half-life in blood ensures that compounds administered directly to the lungs will have a reduced chance of interacting with TMEM16A at other locations in the body, and potential side effects will therefore be minimised.
• the compounds of the invention also have adequate water solubility as represented by log D values, leading also to modest lipophilicity which is an advantage when preparing a pharmaceutical formulation.
• R 1 is selected from H, ethynyl, CN, methyl, fluoromethyl, difluoromethyl, trifluoromethyl, chlorodifluoromethyl, dichlorofluoromethyl and hydroxymethyl;
• R 2 is selected from methyl and CH2OH
• R 3 is selected from H and methyl
• R 2 and R 3 together with the carbon atom to which they are attached form a 3- to 10- membered carbocyclic or oxygen-containing heterocyclic ring system either of which is optionally substituted, in addition to the R 1 group, with one or more substituents selected from C1 -4 alkyl, C1 -4 haloalkyl, halo and OH; or
• R 1 , R 2 and R 3 together with the carbon atom to which they are attached combine to form a 5- to 8-membered bridged carbocyclic or heterocyclic ring system optionally substituted with one or more substituents selected from OH, halo, C1 -4 alkyl and C1 -4 haloalkyl;
• R 4 is H or halo
• R 5 is selected from H, halo, CN and C1 -4 alkyl optionally substituted with one or more substituents selected from halo and OH;
• X 1 is CR 6 or N
• R 6 is selected from H, halo, CN and C1 -4 alkyl optionally substituted with one or more substituents selected from halo and OH;
• X 2 is CR 7 or N
• R 7 is H, halo, CN; a 3- to 7-membered carbocyclic or heterocyclic ring system optionally substituted with one or more substituents selected from halo and OH; or Ci alkyl optionally substituted with one or more substituents selected from halo, OH and a 3- to 7-membered carbocyclic or heterocyclic ring system optionally substituted with one or more substituents selected from halo and OH;
• X 3 is CR 8 or N
• R 8 is selected from H, halo, CN and C1 -4 alkyl optionally substituted with one or more substituents selected from halo and OH.
• the compounds of general formula (I) are positive modulators of TMEM16A, they are useful for treating diseases and conditions in which modulation of TMEM 16A plays a role, especially respiratory diseases and conditions.
• 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.
• 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.
• Salts and solvates (such as hydrates) of the compounds of general formula (I) are suitably pharmaceutically acceptable.
• suitable pharmaceutically acceptable salts are well known to those of skill in the art and are described, for example by Gupta et al (2016).
• Some particularly suitable salts of the compounds of general formula (I) include basic addition salts such as sodium, potassium, calcium, aluminium, zinc, magnesium and other metal salts as well as choline, diethanolamine, ethanolamine, ethyl diamine and meglumine salts.
• acid addition salts may be formed, for example hydrochloride, mesylate, hydrobromide, sulphate, and fumarate salts. Salts of synthetic intermediates need not be pharmaceutically acceptable.
• C1-4 alkyl refers to a straight or branched fully saturated hydrocarbon group having from 1 to 4 carbon atoms.
• the term encompasses methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl and t-butyl.
• Other alkyl groups for example C1-6 alkyl are as defined above but contain the stated numbers of carbon atoms.
• the term “3- to 10-membered carbocyclic” refers to a non-aromatic hydrocarbon ring system containing from 3 to 10 ring carbon atoms.
• the carbocyclic ring system may be a single ring or may contain two rings which may be fused or in a spiro arrangement or bridged, where carbon atoms in a bridge are included in the number of ring carbon atoms. Examples include cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, as well as spiro systems and bridged systems such as bicyclo[1.1.1]pentyl. Carbocyclic ring systems may contain other numbers of ring atoms as specified, for example 5 to 8 ring atoms or 3 to 7 ring atoms.
• cycloalkyl refers to a fully saturated carbocyclic ring system as defined above.
• examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, as well as bridged cycloalkyl systems such as bicyclo[1.1.1]pentyl.
• the terms“3- to 10-membered heterocyclic” and “3- to 10-membered heterocyclyl” refer to a non-aromatic ring system containing 3 to 10 ring atoms including at least one heteroatom selected from N, O and S.
• a heterocyclic ring system may contain one or more carbon-carbon double bonds but preferably is fully saturated.
• the heterocyclic ring system may be a single ring or may contain two or three rings which may be fused or in a spiro arrangement or bridged, where bridge atoms are included in the number of ring atoms.
• An oxygen-containing heterocyclic ring system contains at least one oxygen as a ring atom and optionally one or two further heteroatoms selected from O, N and S.
• heterocyclic ring systems examples include oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and 2-oxaspiro[3.3] heptan-6-yl.
• Heterocyclic ring systems may contain other numbers of ring atoms as specified, for example 5 to 8 ring atoms or 3 to 7 ring atoms.
• halogen refers to fluorine, chlorine, bromine or iodine and the term“halo” to fluoro, chloro, bromo or iodo groups.
• halide refers to fluoride, chloride, bromide or iodide.
• C1 -4 haloalkyl refers to a C1 -4 alkyl group as defined above in which one or more of the hydrogen atoms is replaced by a halo group. Any number of hydrogen atoms may be replaced, up to perhalo substitution. Examples include trifluoromethyl, chloroethyl and 1 , 1-difluoroethyl.
• a fluoroalkyl group is a haloalkyl group in which halo is fluoro.
• Other haloalkyl groups, for example C1 -3 haloalkyl, are as defined above but contain the stated number of carbon atoms.
• isotopic variant refers to isotopically-labelled compounds which are identical to those recited in formula (I) but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature, or in which the proportion of an atom having an atomic mass or mass number found less commonly in nature has been increased (the latter concept being referred to as“isotopic enrichment”).
• isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as 2H (deuterium), 3H, 1 1 C, 13C, 14C, 18F, 1231 or 1251 (e.g. 3H, 1 1 C, 14C, 18F, 1231 or 1251), which may be naturally occurring or non-naturally occurring isotopes.
• some or all methyl groups are replaced by CD3.
• one of, two of or all of R 1 , R 2 and R 3 may be CD3.
• R 1 is H, methyl, difluoromethyl trifluoromethyl, ethynyl or CN, especially methyl, difluoromethyl trifluoromethyl, ethynyl or CN.
• R 2 is methyl. In other compounds of the invention, R 2 is CH2OH;
• R 3 is methyl
• R 2 is methyl and R 3 is methyl.
• R 1 is not H and is suitably CN, ethynyl or methyl optionally substituted with one or more substituents selected from fluoro and OH.
• R 1 is methyl, difluoromethyl trifluoromethyl, ethynyl or CN.
• R 2 and R 3 together with the carbon atom to which they are attached form a 3- to 10-membered carbocyclic or oxygen-containing heterocyclic ring system optionally substituted as described above.
• More suitable substituents for such rings include fluoro, chloro, methyl, ethyl, trifluoromethyl and OH.
• ring systems formed by R 2 and R 3 and the carbon atom to which they are attached are unsubstituted (except for the R 1 group) or substituted in addition to the R 1 group with one or more, for example one or two, substituents selected from methyl and fluoro.
• Ring systems formed by R 2 and R 3 and the carbon atom to which they are attached include C3-6 cycloalkyl rings, particularly cyclopropyl, cyclobutyl and cyclopentyl, any of which may be unsubstituted or substituted as described above.
• R 2 and R 3 and the carbon atom to which they are attached include 3- to 8-membered oxygen-containing heterocyclic ring systems, which may be monocyclic ring systems such as tetrahydropyranyl, tetrahydrofuranyl and oxetanyl or alternatively may be fused or spiro-linked ring systems such as oxaspiro[3.3] heptan-6-yl. These ring systems may be unsubstituted or substituted as described above.
• R 1 , R 2 and R 3 together with the carbon atom to which they are attached combine to form a 5- to 8-membered bridged carbocyclic or heterocyclic ring system.
• the carbon to which R 1 , R 2 and R 3 are attached is a bridgehead atom.
• Examples of such ring systems include bicyclo[1.1.1]pentyl.
• the ring system formed by R 1 , R 2 and R 3 together with the carbon atom to which they are attached may be unsubstituted or substituted as described above but is more suitably unsubstituted.
• R 4 is H.
• R 5 is H or halo, especially H, fluoro or chloro. More suitably, R 5 is H.
• X 1 is N.
• X 1 is CR 6 .
• R 6 is suitably H or halo, for example H, fluoro or chloro. More suitably, R 6 is H.
• X 2 may be CR 7 or N. More suitably, X 2 is CR 7 and, in this case R 7 is suitably H, C 1-3 alkyl optionally substituted with OH, C 1-3 haloalkyl optionally substituted with OH, methyl substituted with a 3- to 6- membered carbocyclic or heterocyclic ring system; ora 3- to 6- membered carbocyclic or heterocyclic ring system.
• R 7 is H, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkyl substituted with OH, C 1-3 haloalkyl substituted with OH, a 3- to 6- membered carbocyclic or heterocyclic ring system or CH 2 -R 11 , where R 1 1 is a nitrogen-containing 3- to 6-membered heterocyclic ring linked to the CH 2 moiety via a ring nitrogen atom.
• R 7 is C 1-3 alkyl substituted with OH or C 1-3 haloalkyl substituted with OH, it may take the form -C(CH 3 )(OH)-R 12 , wherein R 1 2 is C 1-2 alkyl or C 1-2 haloalkyl.
• R 7 is H, methyl, ethyl, isopropyl, trifluoromethyl, morpholinylmethyl, tetrahydrofuryl or 1 -hydroxy- 1 -trifluoromethyl(ethyl), especially H, methyl, ethyl, isopropyl and trifluoromethyl.
• R 7 is H.
• X 3 is CR 8 or N.
• X 3 is CR 8 .
• Rs is suitably H, methyl or trifluoromethyl, especially H or trifluoromethyl and more especially H.
• R 8 is N.
• X 1 is CR 6 and X 2 is CR 7 , such that the compound is of general formula (la):
• X 1 is CR 6
• X 2 is CR 7
• X 3 is N
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined for general formula (I).
• X 1 is CR 6
• X 2 is CR 7
• X 3 is CR 8 , such that the compound is of general formula (lc):
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are as defined for general formula (I).
• R 5 , R 6 and R 7 is H.
• the compounds of the invention may be prepared by the methods set out below, which are novel and form a further aspect of the invention.
• a compound of general formula (I) may be prepared by reacting a compound of general formula (II):
• R 1 , R 2 , R 3 and R 4 are as defined for general formula (I);
• the reaction is conducted in the presence of a coupling reagent and under basic conditions, for example in the presence of an amine such as diisopropylethylamine (DIPEA) and in an organic solvent such as DMF.
• DIPEA diisopropylethylamine
• Suitable coupling reagents include known peptide coupling agents such as O- (Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU), O- (Benzotriazol-1-yl)- N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU), 0-(7- Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), 0-(7- Azabenzotriazol-1-yl)- N,N,N’,N’-tetramethyluronium tetrafluoroborate (TATU), (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (Benzotriazol-l-yloxy)tripyrrol
• the coupling reagent may be propylphosphonic anhydride (T3P®).
• T3P propylphosphonic anhydride
• the reaction may be conducted under basic conditions, for example in the presence of an amine such as diisopropylethylamine (DIPEA) or triethylamine (TEA) and in an organic solvent such as 1 ,4-dioxane.
• DIPEA diisopropylethylamine
• TEA triethylamine
• a compound of general formula (II) may be prepared by reacting a compound of general formula (IV):
• R 1 , R 2 and R 3 are as defined for general formula (I);
• reaction is suitably carried out in the presence of a coupling reagent as described above, for example TBTU and in the presence of base such as triethylamine.
• a coupling reagent as described above, for example TBTU and in the presence of base such as triethylamine.
• a compound of general formula (I) may be prepared by deprotecting a protected compound of general formula (Iz):
• R 1 , R 2 , R 3 , R 4 R 5 , X 1 , X 2 and X 3 are as defined for general formula (I) and R 15 is an amine protecting group, for example a cyclic ether such as tetrahydropyran-2-yl or a benzyl or substituted benzyl group such as 4-methoxybenzyl.
• R 15 is an amine protecting group, for example a cyclic ether such as tetrahydropyran-2-yl or a benzyl or substituted benzyl group such as 4-methoxybenzyl.
• Deprotection may be achieved by treatment with an acid, for example trifluoroacetic acid.
• the protected compound of general formula (Iz) may be prepared by reacting a compound of general formula (II) with a protected compound of general formula (lllz):
• R 5 , X 1 , X 2 and X 3 are as defined for general formula (I) and R 15 is as defined for general formula (Iz).
• the reaction may be conducted in the presence of a coupling agent as described above for the reaction between the compounds of general formulae (II) and (III).
• the crude product may be used in the subsequent deprotection step without further purification.
• Some compounds of general formula (lllz) may be prepared from a compound of general formula (III) by known methods depending on the nature of the protecting group.
• a compound of general formula (III) may be reacted with 3,4-dihydro-2H-pyran in the presence of pyridinium p- toluenesulfonate.
• the group R 15 is benzyl or a benzyl derivative
• the compound of general formula (III) may be reacted with chloromethyl benzene or a substituted derivative thereof such as 1-chloromethyl-4-methoxybenzene. This reaction usually leads to a mixture of the required protected compound of general formula (lllz) as a mixture with the estes of the compound of formula (lllz).
• the ester can be converted to the compound of general formula (lllz) by hydrolysis using an aqueous base such as lithium or sodium hydroxide.
• an aqueous base such as lithium or sodium hydroxide.
• R 4 R 5 , X 1 , X 2 and X 3 are as defined for general formula (I) and R 15 is as defined for general formula (Iz).
• reaction is carried out in the presence of a coupling agent under similar conditions to those described above for the reaction of the compound of general formula (II) with the compound of general formula (III).
• a compound of general formula (XI I z) may be prepared by hydrolysis of a compound of general formula (Xlllz):
• R 4 R 5 , X 1 , X 2 and X 3 are as defined for general formula (I)
• R 15 is as defined for general formula (Iz)
• R 16 is Ci-e alkyl or benzyl, for example methyl.
• the hydrolysis is conducted under basic conditions, for example in the presence of lithium, sodium or potassium hydroxide and in a mixed solvent comprising an alcohol such as methanol, water and an organic solvent such as tetrahydrofuran.
• a mixed solvent comprising an alcohol such as methanol, water and an organic solvent such as tetrahydrofuran.
• the reaction may be carried out at a temperature of about 15 to 25 °C, typically at room temperature.
• a compound of general formula (Xlllz) may be prepared by reacting a compound of general formula (lllz) as defined above with a compound of general formula (XIV):
• R 4 is as defined for general formula (I) and R 16 is as defined for general formula (Xlllz).
• reaction is carried out in the presence of a coupling reagent as described above.
• a coupling reagent as described above.
• Compounds of general formula (XIV) are known and are commercially available or may be prepared by known methods, for example by esterification of carboxylic acids of general formula (V) above.
• a compound of general formula (XI Iz) in which X 1 is CR 6 and X 2 is CR 7 is designated a compound of general formula (Xllaz):
• a compound of general formula (Xllaz) is particularly useful for preparing compounds of general formula (I) in which one of R 5 and R 6 is H and the other of R 5 and R 6 is halo and compounds in which R 7 is other than H.
• a compound of general formula (Xllaz) may be prepared by reacting a compound of general formula (lllz) in which X 1 is CR 6 and X 2 is CR 7 (i.e. a compound of general formula (lllaz)):
• a compound of general formula (lllaz) may be prepared by carbonylation of a compound of general formula (XXI):
• X 3 , R 5 , R 6 and R 7 are as defined for general formula (I); R 15 is as defined for general formula (Iz); and R 20 is halo, for example bromo.
• Carbonylation can be achieved by reaction with the alkali metal salt of a malonic acid monoester, such as methyl potassium malonate, in the presence of a Pd catalyst such as [Pd(allyl)CI)]2, a phosphine ligand such as 2, 2'-bis(diphenylphosphino)-1 ,1 '-binaphthyl (BINAP) and an organic base, such as DMAP, followed by hydrolysis of the resulting ester with aqueous base, for example an alkali metal hydroxide such as lithium hydroxide.
• a malonic acid monoester such as methyl potassium malonate
• Pd catalyst such as [Pd(allyl)CI)]2
• a phosphine ligand such as 2, 2'-bis(dip
• the reaction is carried out under an inert atmosphere such as nitrogen and at elevated temperature, typically 120° to 160°C.
• An alternative method for preparing a compound of general formula (lllaz) is by reacting a compound of general formula (XXI) as defined above with the alkali metal salt of cyanoacetic acid, such as potassium 2-cyanoacetate, in the presence of a phosphine ligand such as SPhos and a palladium catalyst such as [Pd(allyl)CI)]2 dichloride, followed by hydrolysis of the resulting nitrile with aqueous base, for example an alkali metal hydroxide such as sodium hydroxide.
• the reaction is carried out under an inert atmosphere such as nitrogen and at elevated temperature, typically 120° to 160°C.
• a compound of general formula (XXI) may be prepared by protecting a compound of general formula (XXII):
• R 11 is a nitrogen-containing 3- to 6-membered heterocyclic ring linked to the H atom via a ring nitrogen atom.
• the reaction may be carried out under reducing conditions, for example in the presence of a hydride such as sodium triacetoxyborohydride in an organic solvent such as tetrahydrofuran and at a temperature of about 15 to 25 °C, typically at room temperature and in the presence of an acid such as acetic acid.
• a hydride such as sodium triacetoxyborohydride
• organic solvent such as tetrahydrofuran
• a compound of general formula (XXX) may be prepared by protecting a compound of general formula (XXXII):
• each R 25 is independently C 1-6 alkyl and R 1 2 is C 1-2 alkyl or C 1-2 haloalkyl.
• the reaction may be conducted in the presence of a fluoride ion source such as tetrabutylammonium fluoride (TBAF).
• a fluoride ion source such as tetrabutylammonium fluoride (TBAF).
• TBAF tetrabutylammonium fluoride
• a compound of general formula (XXXV) may be prepared by oxidation of a compound of general formula (XXXVII): (XXXVII)
• Suitable oxidising agents include Dess-Martin periodinane.
• the oxidation is suitably conducted in an organic solvent such as dichloromethane.
• a compound of general formula (XXXVII) may be prepared by reacting a compound of general formula (XXX) as defined above with a methyl Grignard reagent.
• the OH group of a compound of general formula (XXI) in which R 7 is -C(CH3)(OH)-R 12 will generally be protected, for example as a benzyl ether, before carbonylation to yield a compound of general formula (lllaz).
• the protecting group can be retained in the compounds of general formulae (Xllaz) and (laz) and removed at the same time as the protecting group R 15 when the compound of general formula (laz) is converted to a compound of general formula (la).
• a compound of general formula (lllaz) is a protected compound of general formula (III) in which X 1 is CR 6 and X 2 is CR 7 and so it can be reacted with a compound of general formula (II) as described above to give a compound of general formula (laz):
• R 1 , R 2 , R 3 , R 4 R 5 , R 6 and R 7 are as defined for general formula (I) and R 15 is as defined for general formula (Iz); which can be deprotected to give a compound of general formula (la).
• a further alternative method for preparing a compound of general formula (I) is by reacting a compound of general formula (IV) as defined above with a compound of general formula (XII):
• R 4 R 5 , X 1 , X 2 and X 3 are as defined for general formula (I).
• reaction is carried out in the presence of a coupling agent under similar conditions to those described above for the reaction of the compound of general formula (II) with the compound of general formula (III).
• the compound of general formula (XII) may be prepared by deprotecting a compound of general formula (Xllz) as described above by reaction with an acid such as trifluoroacetic acid.
• R 1 , R 2 , R 3 , R 4 R 5 and R 6 are as defined for general formula (I); R 15 is as defined for general formula (Iz); and R 21 is halo, for example bromo or iodo;
• the protecting group R 15 is benzyl or a derivative such as paramethoxybenzyl, it will be removed by the hydrogenation process. Otherwise, an additional deprotection step may be required, for example treatment with an acid such as TFA.
• the borolane used in the first step may be 2-isopropenyl-4,4,5,5-tetramethyl-1 ,3,2-dioxaborolane, while for compounds in which R 7 is tetrahydrofuran-2-yl, 2-(2,3-dihydrofuran-4-yl)-4, 4,5,5- tetramethyl-1 ,3,2-dioxaborolane may be used.
• Other diborolanes may be selected for the introduction of alternative R 7 groups.
• a compound of general formula (XXVaz) may be prepared by reacting a compound of general formula (II) as defined above with a compound of general formula (XXVI).
• R 5 and R 6 are as defined for general formula (I); R 15 is as defined for general formula (Iz); and R 21 is as defined for general formula (XXVaz).
• the reaction may take place in the presence of a coupling reagent under the conditions described above for the reaction of the compound of general formula (II) with the compound of general formula (III).
• a compound of general formula (XXVI) may be prepared from a compound of general formula (XXVII): (XXVII)
• a compound of general formula (XXVII) may be prepared by halogenating a compound of general formula (XXVIII):
• the compounds of general formula (I) are positive modulators of TMEM 16A and therefore, in a further aspect of the invention, there is provided a compound of general formula (I) as defined above for use in medicine, particularly in the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A.
• a compound of general formula (I) in the manufacture of a medicament for the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A.
• a method for the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A comprising administering to a patient in need of such treatment an effective amount of a compound of general formula
• TMEM16A The diseases and conditions affected by modulation of TMEM16A include respiratory diseases and conditions, dry mouth (xerostomia), intestinal hypermobility, cholestasis and ocular conditions.
• a compound of general formula (I) for use in the treatment or prophylaxis of intestinal hypermobility • A compound of general formula (I) for use in the treatment or prophylaxis of intestinal hypermobility.
• a compound of general formula (I) for use in the treatment or prophylaxis of ocular conditions • A compound of general formula (I) for use in the treatment or prophylaxis of ocular conditions.
• the invention also provides:
• a method for the treatment or prophylaxis of respiratory diseases and conditions comprising administering to a patient in need of such treatment an effective amount of a compound of general formula (I).
• a method for the treatment or prophylaxis of intestinal hypermobility comprising administering to a patient in need of such treatment an effective amount of a compound of general formula (I).
• a method for the treatment or prophylaxis of cholestasis comprising administering to a patient in need of such treatment an effective amount of a compound of general formula (I).
• a method for the treatment or prophylaxis of ocular conditions comprising administering to a patient in need of such treatment an effective amount of a compound of general formula (I).
• Respiratory diseases and conditions which may be treated or prevented by the compounds of general formula (I) include cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, including non-cystic fibrosis bronchiectasis, asthma and primary ciliary dyskinesia.
• COPD chronic obstructive pulmonary disease
• COPD chronic bronchitis
• emphysema bronchiectasis
• non-cystic fibrosis bronchiectasis asthma and primary ciliary dyskinesia.
• Dry mouth which may be treated or prevented by the compounds of general formula (I) may result from Sjorgens syndrome, radiotherapy treatment and xerogenic drugs.
• Intestinal hypermobility which may be treated or prevented by the compounds of general formula (I) may be associated with gastric dyspepsia, gastroparesis, chronic constipation and irritable bowel syndrome.
• Ocular conditions which may be treated or prevented by the compounds of by the compounds of general formula (I) include dry eye disease.
• the compounds of the present invention will generally be administered as part of a pharmaceutical composition and therefore the invention further provides a pharmaceutical composition comprising a compound of general formula (I) together with a pharmaceutically acceptable excipient.
• the pharmaceutical composition may be formulated for oral, rectal, nasal, bronchial (inhaled), topical (including 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.
• the composition may be prepared by bringing into association the above defined active agent with the excipient.
• 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 a compound of general formula (I) 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.
• 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
• 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.
• compositions 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.
• compounds of general formula (I) 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.
• Aerosol formulations typically comprise the active ingredient suspended or dissolved in a suitable aerosol propellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC).
• a suitable aerosol propellant such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC).
• 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.
• the formulation may comprise excipients including co solvents (e.g. ethanol) and surfactants (e.g. lecithin, sorbitan trioleate and the like).
• 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.
• a non-pressurised formulation such as an aqueous solution or suspension.
• 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 pm e.g. around 1-5 pm. Particle size distributions may be represented using D10, 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 Dvso value (median for a volume distribution).
• Dv values refer to particle size distributions measured using laser diffraction.
• D10 and D90 values used in the context of laser diffraction, are taken to mean Dvio and Dvgo values and refer to the particle size whereby 10% of the distribution lies below the D10 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 pm or a D50 of 0.5-10 pm e.g. around 1-5 pm.
• 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 pm or more, e.g. 100 pm or more or a D50 of 40-150 pm.
• a topically acceptable diluent such as lactose, glucose or mannitol (preferably lactose)
• MMAD mass mean diameter
• 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.
• 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.
• the lactose component is selected from the group consisting of a-lactose monohydrate, a-lactose anhydrous and amorphous lactose.
• the lactose is a- lactose monohydrate.
• Dry powder formulations may also contain other excipients.
• 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.
• DPI dry powder inhaler
• Example dry powder delivery systems include SPINHALER®, DISKHALER®, TURBOHALER®, DISKUS®, SKYEHALER®, ACCUHALER® and CLICKHALER®.
• 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.
• a compound of general formula (I) is provided as a micronized dry powder formulation, for example comprising lactose of a suitable grade.
• composition comprising a compound of general formula (I) in particulate form in combination with particulate lactose, said composition optionally comprising magnesium stearate.
• a compound of general formula (I) is provided as a micronized dry powder formulation, comprising lactose of a suitable grade and magnesium stearate, filled into a device such as DISKUS.
• a device such as DISKUS.
• 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.
• a compound of general formula (I) 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.
• a compound of general formula (I) 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.
• a compound of general formula (I) 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 pm e.g. around 1-5 pm, that have been produced by a size reduction process other than jet mill micronisation e.g.
• 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.
• the compound of the invention 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.
• suppositories can be prepared by mixing the active ingredient with a conventional suppository base such as cocoa butter or other glycerides.
• 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.
• suitable non irritating excipient 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.
• compositions intended to be administered topically to the eye in the form of eye drops or eye ointments the total amount of the compound of general formula (I) will be about 0.0001 to less than 4.0% (w/w).
• compositions administered according to general formula (I) 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.
• 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 the compound of general formula (I).
• the compound of general formula (I) may also be introduced by means of ocular implants or inserts.
• compositions administered according to general formula (I) 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 of general formula (I) include a compound of the invention formulated with a tonicity agent and a buffer.
• the pharmaceutical compositions of general formula (I) may further optionally include a surfactant and/or a palliative agent and/or a stabilizing polymer.
• tonicity agents may be employed to adjust the tonicity of the composition, preferably to that of natural tears for ophthalmic compositions.
• 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.
• 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).
• ophthalmically acceptable osmolality generally about 150-450 mOsm, preferably 250-350 mOsm and most preferably at approximately 290 mOsm.
• 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
• the particular concentration will vary, depending on the agent employed.
• 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 of general formula (I).
• 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 TritonX1 14 and tyloxapol.
• Additional agents that may be added to the ophthalmic compositions of compounds of general formula (I) 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.
• 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 general formula (I) 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 the compound of general formula (I) 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).
• Compounds of general formula (I) 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 the compound of general formula (I) or at an earlier or later time.
• a product comprising a compound of general formula (I) 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.
• a compound of general formula (I) 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 the compounds of general formula (I), (lx), (IA), (IB), (IC), (ID) or (IE) 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 HU 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
• 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
• Lumacaftor and Tezacaftor or combinations thereof for example a combination of Ivacaftor, Tezacaftor and Elexacaftor
• ENaC modulators particularly ENaC inhibitors
• Antivirals such as ribavirin and neuraminidase inhibitors such as zanamivir;
• Antifungals such as PUR1900;
• Airway hydrating agents such as hypertonic saline and mannitol (Bronchitol®); and
• Mucolytic agents such as. N-acetyl cysteine.
• the additional active agent when it is an ENaC modulator, it may be an ENaC inhibitor such as amiloride, VX-371 , AZD5634, QBW276, SPX-101 , BI443651 , BI1265162 and ETD001.
• ENaC blockers are disclosed in our applications WO 2017/221008, WO 2018/096325, WO 2019/077340 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-5/-/-pyrrolo[2,3-b]pyrazin-2-yl ⁇ formamido) ethyl]-6-(4- ⁇ bis[(2S,3 ,4 ,5 )-
• a suitable anion for example halide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate, fumarate, citrate, tartrate, oxalate, succinate, mandelate, methane sulfonate or p-toluene sulfonate.
• FIGURE 1 is an example trace from a whole-cell patch clamp (Qpatch) TMEM16A potentiator assay as used in Biological Example Hand illustrates the methodology used in the assay.
• 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 spectra were recorded on a Bruker Avance III HD 500 MHz with a 5mm Broad Band Inverse probe, a Bruker Avance III HD 250 MHz, a 400MHz Avance III HD Nanobay fitted with a 5mm Broad Band Observed SmartProbe using the solvent as internal deuterium lock. Spectra were recorded at room temperature unless otherwise stated and were referenced using the solvent peak.
• the various starting materials, intermediates, and compounds of the preferred embodiments may be isolated and purified, where appropriate, 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.
• the title compound was prepared from 4-amino-N-[1-(trifluoromethyl)cyclopropyl]pyridine- 2-carboxamide (Intermediate AD) and 2-(1 H-indol-6-yl)acetic acid analogously to Example 1.
• Step _ T N-(1 , 1 -Dimethylprop-2-ynyl)-4-[[2-(1 -tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxamide
• Step 2 N-(1,1-Dimethylprop-2-ynyl)-4-[[2-(1H-indazol-6-yl)acetyl]amino]pyridine-2- carboxamide
• step 1 A cooled (0 °C) solution of N-(1,1-dimethylprop-2-ynyl)-4-[[2-(1-tetrahydropyran-2- ylindazol-6-yl)acetyl]amino]pyridine-2-carboxamide (step 1) (83%, 950 mg, 1.77 mmol) in DCM (12 ml_) was treated with TFA (1.74 ml_, 22.79 mmol). The mixture was allowed to warm to room temperature and stirred for 3 h. Additional TFA (0.87 ml_, 11.40 mmol) was added and the reaction allowed to continue for 3 h. The resulting mixture was concentrated in vacuo.
• step 1 80%, 1.76 g, 3.57 mmol
• MeOH(6 ml_) and water (6 ml_) was added UOH.H2O (180 mg, 4.28 mmol) and the mixture stirred at room temperature for 2 h.
• Additional UOH.H2O 45 mg, 1.07 mmol was added and the mixture stirred at room temperature for a further hour.
• the resulting mixture was acidified to pH 5 using 1 M HCI then diluted with brine (15 ml_) and EtOAc (15 ml_) whereupon a precipitate formed which was collected by vacuum filtration.
• the phases of the filtrate were separated and the aqueous was extracted with EtOAc (15 ml_) then the combined organics were concentrated in vacuo.
• the resulting residue was triturated in ether (10 ml_) and filtered and the solids combined with those from the previous filtration.
• the aqueous phase was cooled to 2-8°C and allowed to sit for 3 days.
• the resulting suspension was filtered and the solids combined with those from the previous filtrations.
• the combined solids were dried in a vacuum oven to afford the title compound as a colourless solid.
• Step 3 N-(1 -Ethynylcyclopentyl)-4-[[2-(1 H-indazol-6-yl)acetyl]amino]pyridine-2- carboxamide
• step 2 To a solution of a mixture of 4-[[2-(1-tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-(2-tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid (step 2) (99%, 150 mg, 0.39 mmol) and 1- ethynylcyclopentanamine hydrochloride (68 mg, 0.47 mmol) in DMF (2 ml_) was added DIPEA (136 pl_, 0.78 mmol) followed by HATU (163 mg, 0.43 mmol) and the mixture stirred at room temperature for 3 h.
• DIPEA 136 pl_, 0.78 mmol
• Example 1 The compounds of the following tabulated Examples (Table 1) were prepared analogously to Example 4 step 3 from a mixture of 4-[[2-(1-tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-(2-tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid (Example 4 step 2) and the appropriate commercially available amine.
• Table 1 The compounds of the following tabulated Examples (Table 1) were prepared analogously to Example 4 step 3 from a mixture of 4-[[2-(1-tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-(2-tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid (Example
• the resulting mixture was partitioned between DCM (5 ml_) and water (5 ml_) and the organic portion was separated by filtration through a hydrophobic PTFE fritted tube.
• the filtrate was concentrated in vacuo and the residue was dissolved in TFA (50% in DCM) (2.0 ml_, 0.16 mmol).
• the resulting mixture was agitated for 4 h and then diluted with DCM (10 ml_). Saturated aq. NaHCC>3 (10 ml_) was slowly added and the free-based mixture separated via filtration through a hydrophobic PTFE fritted tube.
• Example 2 The compounds of the following tabulated Examples (Table 2) were prepared analogously to Example 5 from a mixture of 4-[[2-(1-tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-(2-tetrahydropyran-2-ylindazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid (Example 4 step 2) and the appropriate commercially available amine.
• Step 1 A mixture of 6-bromo-5-fluoro-1-[(4-methoxyphenyl)methyl] indazole and 6-bromo- 5-fluoro-2-[(4-methoxyphenyl)methyl]indazole
• Step 2 A mixture of 2-[5-fluoro-1-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid and 2-[5-fluoro-2-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid
• step 1 To a solution of a mixture of 6-bromo-5-fluoro-1-[(4-methoxyphenyl)methyl]indazole and 6-bromo-5-fluoro-2-[(4-methoxyphenyl)methyl]indazole (step 1) (1200 g, 3.58 mmol) and potassium 3-ethoxy-3-oxo-propanoate (914 mg, 5.37 mmol) in mesitylene (30 mLI) was added DMAP (44 mg, 0.36 mmol) and the resulting mixture was sparged with nitrogen for 5 mins and stirred at room temperature.
• Step 3 A mixture of methyl 4-[[2-[5-fluoro-1-[(4-methoxyphenyl)methyl]indazol-6- yl]acetyl]amino]pyridine-2-carboxylate and methyl 4-[[2-[5-fluoro-2-[(4- methoxyphenyl)methyl]indazol-6-yl]acetyl]amino] pyridine-2-carboxylate
• step 2 To a solution of a mixture of 2-[5-fluoro-1-[(4-methoxyphenyl) methyl]indazol-6-yl]acetic acid and 2-[5-fluoro-2-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid (step 2) (975 mg, 3.1 mmol) in 1 ,4-dioxane (25 ml_) was added methyl 4-aminopyridine-2-carboxylate (472 mg, 3.1 mmol), DIPEA (1.08 ml_, 6.2 mmol) followed by a 50% solution of T3P® in EtOAc (1.02 ml_, 3.41 mmol) and the reaction mixture was stirred at room temperature under an inert atmosphere for 16 h.
• Step 4 A mixture of 4-[[2-[5-fluoro-1-[(4-methoxyphenyl) methyl]indazol-6- yl]acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-[5-fluoro-2-[(4- methoxyphenyl)methyl]indazol-6-yl]acetyl]amino]pyridine-2-carboxylic acid
• step 3 To a solution of a mixture of methyl 4-[[2-[5-fluoro-1-[(4-methoxyphenyl)methyl]indazol-6- yl]acetyl]amino]pyridine-2-carboxylate and methyl 4-[[2-[5-fluoro-2-[(4- methoxyphenyl)methyl]indazol-6-yl]acetyl]amino] pyridine-2-carboxylate (step 3) (830 mg, 1.85 mmol) in THF (20 ml_) was added 1 M LiOH(4.63 ml_, 4.63 mmol) and the reaction mixture was stirred at room temperature for 3 h.
• Step 5 A/-te/f-Butyl-4-[[2-(5-fluoro-1 H-indazol-6-yl)acetyl]amino]pyridine-2-carboxamide
• step 4) To a solution of a mixture of 4-[[2-[5-fluoro-1-[(4-methoxyphenyl) methyl]indazol-6- yl]acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-[5-fluoro-2-[(4- methoxyphenyl)methyl]indazol-6-yl]acetyl]amino]pyridine-2-carboxylic acid (step 4) (200 g, 0.46 mmol) in DMF (5 ml_) was added HATU (193 mg, 0.51 mmol) followed by DIPEA (161 pl_, 0.92 mmol).
• the title compound was prepared from a mixture of 4-[[2-[5-fluoro-1-[(4- methoxyphenyl)methyl]indazol-6-yl]acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-[5- fluoro-2-[(4-methoxyphenyl)methyl]indazol-6-yl]acetyl]amino] pyridine-2-carboxylic acid (Example 6, step 4) and l-(trifluoromethyl) cyclopropanamine hydrochloride analogously to Example 6 step 5.
• the title compound was prepared from a mixture of 4-[[2-[5-fluoro-1-[(4- methoxyphenyl)methyl]indazol-6-yl]acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-[5- fluoro-2-[(4-methoxyphenyl)methyl]indazol-6-yl]acetyl]amino] pyridine-2-carboxylic acid (Example 6, step 4) and 2,2-difluorocyclopentanamine hydrochloride analogously to Example 6 step 5.
• Steps 1-4 4-[[2-[4-Chloro-1-[(4-methoxyphenyl)methyl]indazol-6-yl]acetyl]amino]pyridine- 2-carboxylic acid
• the title compound was prepared analogously to a mixture of 4-[[2-[5-fluoro-1-[(4- methoxyphenyl)methyl]indazol-6-yl]acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-[5- fluoro-2-[(4-methoxyphenyl)methyl]indazol-6-yl]acetyl]amino]pyridine-2-carboxylic acid (Example 6 steps 1-4) by replacing 6-bromo-5-fluoro-1 H-indazole (step 1) with 6-bromo- 4-chloro-1 H-indazole.
• Step _ 5 4-[[2-(4-Chloro-1 H-indazol-6-yl)acetyl]amino]-N-[1 -(trifluoromethyl) cyclopropyl]pyridine-2-carboxamide
• Step 1 4-[[2-(4-Chloro-1 H-indazol-6-yl)acetyl]amino]pyridine-2-carboxylic acid
• Step 2 4-[[2-(4-Chloro-1 H-indazol-6-yl)acetyl]amino]-N-(1-ethynylcyclopentyl) pyridine-2- carboxamide
• the title compound was prepared from 4-[[2-(4-chloro-1 H-indazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid (step 1) and 1-ethynylcyclopentanamine hydrochloride analogously to Example 6 step 5.
• Step 1 A mixture of 6-bromo-4-fluoro-1-[(4- ethoxyphenyl) ethyl]indazole and 6-bromo- 4-fluoro-2-[(4-methoxyphenyl) methyl]indazole
• Step 2 A mixture of 2-[4-fluoro-1-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid and 2-[4-fluoro-2-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid
• the title compound was prepared from a mixture of 6-bromo-4-fluoro-1-[(4- methoxyphenyl)methyl]indazole and 6-bromo-4-fluoro-2-[(4-methoxy phenyl)methyl]indazole (step 1), and potassium 3-ethoxy-3-oxo-propanoate analogously to Example 6 step 2.
• Step 3 A/-te/f-Butyl-4-[[2-(4-fluoro-1 H-indazol-6-yl)acetyl]amino]pyridine-2-carboxamide
• step 2 To a solution of a mixture of 2-[4-fluoro-1-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid and 2-[4-fluoro-2-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid (step 2)(87%, 1 14 mg, 0.32 mmol), 4-amino-A/-te/f-butyl-pyridine-2-carboxamide (Intermediate AB) (61 mg, 0.32 mmol) and DIPEA (110 mI_, 0.63 mmol) in 1 ,4-dioxane (5 ml_) was added a 50% solution of T3P® in EtOAc (103 mI_, 0.35 mmol
• Step 1 6-Bromo-4-chloro-1-[(4-methoxyphenyl)methyl]indazole
• the title compound was prepared from 6-bromo-4-chloro-1-[(4- methoxyphenyl)methyl]indazole (step 1) and potassium 3-ethoxy-3-oxo-propanoate analogously to Example 6 step 2.
• Step _ 3 A/-te/f-Butyl-4-[[2-[4-chloro-1-[(4-methoxyphenyl)methyl]indazol-6- yl]acetyl]amino]pyridine-2-carboxamide
• Step 4 A/-te/f-Butyl-4-[[2-(4-chloro-1 H-indazol-6-yl)acetyl]amino]pyridine-2-carboxamide
• Step 1 6-Bromo-1-[(4-methoxyphenyl) ethyl]-3-(trifluoro ethyl)indazole
• step 1 A mixture of 6-bromo-1-[(4-methoxyphenyl) ethyl]-3-(trifluoro ethyl)indazole (step 1) (250 g, 0.65 mmol), potassium 2-cyanoacetate (120 mg, 0.97 mmol), diallyldipalladium dichloride (10 mg, 0.03 mmol) and SPhos (16 mg, 0.04 mmol) in toluene (5 ml_) was degassed with nitrogen for 5 mins. The resulting mixture was heated using microwave radiation at 140°C for 1.5 h.
• Step 3 A/-te/f-Butyl-4-[[2-[3-(trifluoromethyl)-1 H-indazol-6-yl]acetyl]amino] pyridine-2- carboxamide
• Example 8 The title compound was prepared from 2-[1-[(4-methoxyphenyl)methyl]-3- (trifluoromethyl)indazol-6-yl]acetic acid (Example 8, step 2) and 4-amino-N-[1- (trifluoromethyl)cyclopropyl]pyridine-2-carboxamide (Intermediate AD) analogously to Example 7 step 3.
• Step 1 2-[3-lodo-1-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid
• step 1 A mixture of 2-[3-iodo-1-[(4-methoxyphenyl)methyl]indazol-6-yl]acetic acid (step 1) (84%, 428 mg, 0.85 mmol) and 4-amino-A/-te/f-butyl-pyridine-2-carboxamide (Intermediate AB) (181.09 mg, 0.94 mmol) in 1 ,4-dioxane (8.5 ml_) was treated with TEA (0.30 ml_, 1.7 mmol) followed by a 50% solution of T3P® in EtOAc (1.01 ml_, 1.7 mmol) and the reaction mixture was stirred at room temperature for 18 h.
• Step 3 A/-te/f-Butyl-4-[[2-[3-isopropenyl-1-[(4-methoxyphenyl)methyl]indazol-6- yl]acetyl]amino]pyridine-2-carboxamide
• step 2 To a mixture of A/-te/f-butyl-4-[[2-[3-iodo-1-[(4-methoxyphenyl)methyl]indazol-6- yl]acetyl]amino]pyridine-2-carboxamide (step 2) (158 g, 0.26 mmol), Pd(OAc)2 (12 mg, 0.05 mmol), P(Cy)3 (30 mg, 0.1 1 mmol) and tripotassium phosphate (225 mg, 1.06 mmol) under a nitrogen atmosphere was added toluene (2.7 ml_) followed by 2-isopropenyl- 4,4,5,5-tetramethyl-1 ,3,2-dioxaborolane (0.10 ml_, 0.53 mmol) and the reaction mixture was heated at 100 °C for 4 h. After cooling to room temperature, the resulting mixture was purified by chromatography on silica eluting with 0-100% EtOAc in heptan
• A/-te/f-Butyl-4-[[2-[3-isopropenyl-1-[(4-methoxyphenyl)methyl] indazol-6-yl]acetyl] amino]pyridine-2-carboxamide(step 3) (98%, 113 mg, 0.22 mmol) and 10 % Pd-C (23 mg, 0.02 mmol) were dissolved in EtOH (2.2 ml_), placed under hydrogen and stirred for 4 h. The resulting mixture was filtered through Kieselguhr and concentrated in vacuo. The residue was and treated with DCE (2.16 ml_) followed by TFA (0.017 ml_, 0.22 mmol) and the mixture was heated at 70 °C overnight.
• Step 1 4-[[2-[3-lodo-1-[(4-methoxyphenyl)methyl]indazol-6-yl]acetyl]amino]-N-[1-
• Example 9 step 1 The title compound was prepared from 2-[3-iodo-1-[(4-methoxyphenyl)methyl]indazol-6- yl]acetic acid (Example 9 step 1) and 4-amino-N-[1-(trifluoromethyl)cyclopropyl]pyridine- 2-carboxamide (Intermediate AD) analogously to Example 9 step 2.
• Step _ 2 ⁇ 4-[[2-[3-(2,3-Dihydrofuran-4-yl)-1-[(4-methoxyphenyl)methyl]indazol-6- yl]acetyl]amino]-N-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide
• the title compound was prepared from 4-[[2-[3-iodo-1-[(4-methoxyphenyl)methyl]indazol- 6-yl]acetyl]amino]-N-[1-(trifluoromethyl)cyclo propyl]pyridine-2-carboxamide (step 1) and 2-(2,3-dihydrofuran-4-yl)-4,4,5,5-tetramethyl-1 ,3,2-dioxaborolane analogously to Example 9 step 3.
• Step _ 3 4-[[2-(3-Tetrahydrofuran-2-yl-1 H-indazol-6-yl)acetyl]amino]-N-[1-
• Step 1 6-bromo-1-[(4-methoxyphenyl)methyl]indazole-3-carbaldehyde
• step 1 6-bromo-1-[(4-methoxyphenyl)methyl]indazole-3-carbaldehyde (step 1)(600 mg, 1.74 mmol) and AcOH (0.15 mL, 2.61 mmol) in THF (10 mL) was added morpholine (0.30 ml_, 3.48 mmol) and sodium triacetoxyborohydride (442 mg, 2.09 mmol) and the reaction mixture was stirred at room temperature for 16 h. The resulting mixture was diluted with EtOAc (80 ml_) and saturated sodium bicarbonate solution (80 ml_) and the phases were separated.
• EtOAc 80 ml_
• saturated sodium bicarbonate solution 80 ml_
• Step 3 2-[1-[(4-Methoxyphenyl)methyl]-3-(morpholin-4-ium-4-ylmethyl)indazol-6- yl]acetate
• the title compound was prepared from 4-[[6-bromo-1-[(4-methoxyphenyl)methyl]indazol- 3-yl]methyl]morpholine (step 2) and potassium 3-ethoxy-3-oxo-propanoate analogously to Example 6 step 2.
• Step 4 Methyl 4-[[2-[1 -[(4-methoxyphenyl)methyl]-3-(morpholinomethyl)indazol-6- yl]acetyl]amino]pyridine-2-carboxylate
• Step 5 4-[[2-[1-[(4-Methoxyphenyl)methyl]-3-(morpholin-4-ium-4-ylmethyl)indazol-6- yl]acetyl]amino]pyridine-2-carboxylate
• Step 6 4-[[2-[3-(Morpholinomethyl)-1 H-indazol-6-yl]acetyl]amino]-N-(2,2,2-trifluoro-1 , 1- dimethyl-ethyl)pyridine-2-carboxamide
• the title compound was prepared from 4-[[2-[1-[(4-methoxyphenyl)methyl]-3-(morpholin- 4-ium-4-ylmethyl)indazol-6-yl]acetyl] amino]pyridine-2-carboxylate (step 5) and 1 , 1 , 1- trifluoro-2-methyl-propan-2-amine hydrochloride analogously to Example 6 step 5.
• Step 1 4-[[2-(1 H-lndazol-6-yl)acetyl]amino]pyridine-2-carboxylic acid
• Example 4 step 2 A mixture of 4-[[2-(1-tetrahydropyran-2-ylindazol-6-yl)acetyl]amino]pyridine-2-carboxylic acid and 4-[[2-(2-tetrahydropyran-2-ylindazol-6-yl)acetyl]amino]pyridine-2-carboxylic acid (Example 4 step 2) (50%, 1.9 g, 2.5 mmol) in DCM (100 ml_) was treated with TFA (1.91 ml_, 24.97 mmol) and the reaction mixture was stirred at room temperature overnight. The resulting mixture was concentrated in vacuo and the crude residue which was suspended in DCM (50 ml_). The suspension was sonicated for 10 mins, filtered, washed with DCM (20 ml_) and dried to afford the title compound as a beige solid.
• Step 2 4-[[2-(1 H-lndazol-6-yl)acetyl]amino]-N-(3-methyloxetan-3-yl)pyridine-2- carboxamide
• HATU (83 mg, 0.22 mmol) was added to a mixture of 4-[[2-(1 H-indazol-6- yl)acetyl]amino]pyridine-2-carboxylic acid (step 1) (40%, 125 mg, 0.17 mmol), 3- methyloxetan-3-amine hydrochloride (42 mg, 0.34 mmol) and DIPEA (88 pl_, 0.51 mmol) in DMF (1 ml_) and the reaction mixture was stirred at room temperature overnight . The resulting mixture was diluted with EtOAc (10 ml_) and water (10 ml_) and the phases were separated.
• Example 11 step 1 The title compound was prepared from 4-[[2-(1 H-indazol-6-yl)acetyl]amino]pyridine-2- carboxylic acid (Example 11 step 1) and 2-oxaspiro[3.3]heptan-6-amine hydrochloride analogously to Example 1 1 step 2.
• Example 1 1 step 1 The title compound was prepared from 4-[[2-(1 H-indazol-6-yl)acetyl]amino]pyridine-2- carboxylic acid (Example 1 1 step 1) and 4-(trifluoromethyl)tetrahydropyran-4-amine hydrochloride analogously to Example 1 1 step 2.
• Example 11 step 1 The title compound was prepared from 4-[[2-(1 H-indazol-6-yl)acetyl]amino]pyridine-2- carboxylic acid (Example 11 step 1) and 3-(trifluoromethyl)oxetan-3-amine hydrochloride analogously to Example 1 1 step 2.
• Example 11 step 1 The title compound was prepared from 4-[[2-(1 H-indazol-6-yl)acetyl]amino]pyridine-2- carboxylic acid (Example 11 step 1) and 1-(difluoromethyl)cyclopropanamine hydrochloride analogously to Example 1 1 step 2.
• Example 11 step 1 The title compound was prepared from 4-[[2-(1 H-indazol-6-yl)acetyl]amino]pyridine-2- carboxylic acid (Example 11 step 1) and 3-fluorobicyclo[1.1.1]pentan-1 -amine hydrochloride analogously to Example 11 step 2.
• Step 1 6-Bromo-1 -[(4-methoxyphenyl)methyl]indazole-3-carbaldehyde
• step 1 6-Bromo-1-[(4-methoxyphenyl)methyl]indazole-3-carbaldehyde (step 1) (73%, 3.06 g, 6.48 mmol) in THF (64.76 mL) was cooled to -78 °C and treated dropwise with 3M bromo(methyl)magnesium in diethyl ether (2.37 mL, 7.12 mmol). The mixture was allowed to warm to room temperature and stirred for 4 hours. The reaction was quenched with saturated ammonium chloride (50 mL) and the volatile solvents were removed in vacuo. The resulting suspension was diluted with EtOAc (50 mL) and the phases separated.
• EtOAc 50 mL
• step 2 A mixture of 1-[6-Bromo-1-[(4- ethoxyphenyl) ethyl]indazol-3-yl]ethanol (step 2) (87%, 2.45 g, 5.9 mmol) in DCM (28 ml_) at 0 °C was treated with Dess-Martin Periodinane (3.0 g, 7.08 mmol). The mixture was allowed to warm to room temperature and stirred for 18 hours. Saturated aqueous sodium sulfite (20 ml_) was added and the phases were separated.
• Step 4 2-[6-Bromo-1-[(4-methoxyphenyl)methyl]indazol-3-yl]-1 ,1 , 1-trifluoro-propan-2-ol
• step 3 1-[6-Bromo-1-[(4-methoxyphenyl)methyl]indazol-3-yl]ethenone (step 3) (500 mg, 1.39 mmol) in THF (10 ml_) at -78 °C was treated with trimethyl(trifluoromethyl)silane (41 1 pl_, 2.78 mmol) followed by 1 M TBAF in THF (14 mI_, 0.014 mmol) and the stirred for 30 mins. The mixture was allowed to warm to room temperature and stirred for 2 hours. 1 M TBAF in THF (2.78 ml_, 2.78 mmol) was added and stirring continued for a further 2 hours.
• Step 5 3-(1-Benzyloxy-2,2,2-trifluoro-1-methyl-ethyl)-6-bromo-1-[(4-methoxyphenyl) methyl]indazole
• step 4 2-[6-Bromo-1-[(4-methoxyphenyl)methyl]indazol-3-yl]-1 ,1 , 1-trifluoro-propan-2-ol (step 4)(658 mg, 1.53 mmol) in DMF (15.3 ml_) was treated with NaH 60% dispersion in mineral oil (123 mg, 3.07 mmol) and bromomethylbenzene (0.36 ml_, 3.07 mmol) and stirred for 18 hours. The reaction was quenched with brine (20 ml_) and the mixture was extracted with EtOAc (3 x 20 ml_). The combined organic extracts were dried over Na2SC>4 and concentrated in vacuo. Purification by chromatography on silica eluting with 0-35% EtOAc in heptane afforded the title compound as a colourless oil.
• Step 6 2-[3-(1-Benzyloxy-2,2,2-trifluoro-1-methyl-ethyl)-1-[(4-methoxyphenyl)methyl] indazol-6-yl]acetic acid
• step 5 (632 mg, 1.22 mmol), potassium 3-ethoxy-3-oxo-propanoate (414 mg, 2.43 mmol), DMAP (15 mg, 0.12 mmol), BINAP (76 mg, 0.12 mmol) and diallyldipalladium dichloride (22 mg, 0.06 mmol) were added to a sealed tube and placed under a nitrogen atmosphere. Toluene (6.1 ml_) was added, the mixture was sparged with nitrogen for 10 minutes and then stirred at 140 °C for 20 hours. The resulting mixture was purified by chromatography on silica eluting with 0-100% EtOAc in heptane to afford the ester intermediate.
• the intermediate was dissolved in a 1 : 1 mixture of MeOH and 2 M LiOH (12 ml_) and stirred for 18 hours.
• the volatile solvents were removed in vacuo and the aqueous mixture was acidified to pH 1 with 3 M HCI.
• the aqueous suspension was extracted with EtOAc (3 x 50 ml_) and the combined organic extracts were dried over Na2S04 and concentrated in vacuo to afford the title compound as a pale yellow gum.
• Step 7 4-[[2-[3-(2, 2, 2trifluoro-1 -hydroxy-1 -methyl-ethyl)-1 H-indazol-6-yl]acetyl]amino]-N- [1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide
• Step 1 4-[[2-(1 H-lndol-6-yl)acetyl]amino]pyridine-2-carboxylic acid
• Step 2 4-[[2-(1H-lndol-6-yl)acetyl]amino]-N-(2, 2, 2-trifluoro-1,1 -dimethyl-ethyl) pyridine-2- carboxamide
• step 1 4-[[2-(1H-lndol-6-yl)acetyl]amino]pyridine-2-carboxylic acid (step 1) (79%, 100 mg, 0.27 mmol) and 1,1,1-trifluoro-2-methyl-propan-2-amine hydrochloride (48 mg, 0.29 mmol) in DMF (3 ml_) were treated with DIPEA (0.094 ml_, 0.54 mmol) and HATU (112 mg, 0.29 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The resulting mixture was diluted with EtOAc (10 ml_) and washed with saturated aqueous sodium hydrogen carbonate (2 x 10 ml_). The organic layer was dried over Na2SC>4 and concentrated in vacuo. Purification by preparative HPLC (acidic pH, early elution method) afforded the title compound as an off-white solid.
• the title compound was prepared from 4-[[2-(1H-indol-6-yl)acetyl]amino]pyridine-2- carboxylic acid (Example 13, step 1) and 3,3-difluoro-1-methyl-cyclobutanamine hydrochloride analogously to Example 13 step 2.
• the title compound was prepared from 4-[[2-(1 H-indol-6-yl)acetyl]amino]pyridine-2- carboxylic acid (Example 13, step 1) and 4-aminotetrahydropyran-4-carbonitrile hydrochloride analogously to Example 13 step 2.
• the resulting mixture was diluted with DCM (10 ml_) and washed with water and brine. The organic portion was separated, dried over Na2SC>4 and concentrated in vacuo.
• the crude mixture was dissolved in THF (10 ml_) and treated with 1M LiOH (10 ml_). After stirring at room temperature for 1 h, the pH was adjusted to pH 5 with 1 M HCI (2.5 mL). The mixture was diluted with water (30 ml_) and extracted with EtOAc (30 L). The organic extracts were dried over Na2SC>4 and concentrated in vacuo to afford the title compound as a yellow viscous oil.
• Example 14 Automated Whole-Cell Patch Clamp Assay To Detect TMEM16A Activity In Recombinant Cells Cell culture and preparation
• Fisher rat thyroid (FRT) cells stably expressing human TMEM16A (TMEM16Aabc variant; Dr Luis Galietta, Insituto Giannina, Italy) were cultured in T-75 flasks in Hams F-12 media with Coon’s modification (Sigma) supplemented with 10% (v/v) foetal bovine serum, penicillin-streptomycin (10,000 U/mL/10000 pg/mL), G-418 (750pg/mL), L-glutamine (2 mM) and sodium bicarbonate solution (7.5% v/v).
• FRT-TMEM16A cells were whole-cell patch clamped using an automated planar patch clamp system (Qpatch, Sophion). Briefly, once high resistance (GOhm) seals were established between the cells and the planar recording array the patch was ruptured using suction pulses to establish the whole-cell recording configuration of the patch clamp technique.
• the assay employed the following solutions (all reagents Sigma):
• Intracellular solution N-methyl-D-glucamine 130, CaCL 18.2, MgCL 1 , HEPES 10, EGTA 10, BAPTA 20, Mg-ATP 2, pH 7.25, 325mOsm with sucrose.
• Extracellular solution N-methyl-D-glucamine 130, CaCl2 2, MgCL 1 , HEPES 10, pH 7.3, 320 mOsm with sucrose.
• the intracellular solution buffers intracellular calcium at levels required to give -20% activation of the maximal TMEM16A mediated current (EC20 for calcium ions).
• Cells were voltage clamped at a holding potential of -70mV and a combined voltage step (to +70 mV)/ramp (-90 mv to +90 mV) was applied at 0.05 Hz.
• solubilised in 100% (v/v) DMSO and subsequently diluted into extracellular solution were applied to generate a cumulative concentration response curve. Each concentration of test compound was incubated for 5 minutes before addition of the next concentration.
• Peak TMEM16A current at +70mV was plotted as a function of time over the assay period.
• Baseline current (IBL) was measured after a period of stabilisation. The increase in current for each compound addition was determined by taking the peak current during the incubation period and subtracting the current from the previous recording period and then expressing this as a percentage of the baseline current (% potentiation). For test compound concentration 1 in Figure 1 this is:
• Compounds 1 , 1.1 , 13, 13.1 and 13.2 are indoles and may therefore be compared with Compound B. It can be seen that Compounds 1 , 2.2, 13.1 and 13.2 have lower ECso values than Compound B and Compounds 1.1 , 13.1 and 13.2 have improved maximum potentiation of TMEM16A.
• the remaining example compounds are indazoles and may therefore be compared with Compound A. All of the indazole example compounds have significantly lower ECso than Compound A and all also have similar or improved maximum potentiation of TMEM16A, particularly at the 370nM concentration.
• Microsomes (human) were obtained from Bioreclamation.
• Test and reference control compounds (raloxifene, diclofenac, terfenadine, propranolol, dextromethorphan and metoprolol) were dissolved to create a 100 mM stock (final concentrations; 91.5% Acetonitrile: 8.5% DMSO). Final test compound concentration in incubation was 1 mM ( ⁇ 0.1 % DMSO).
• the assay buffer is prepared from Potassium phosphate solutions 1 and 2 by combination to form a pH 7.42 solution at 37 °C.
• Solution 1 17.4 g potassium phosphate dibasic anhydrous (K2HPO4, 0.1 M) dissolved in 1 L deionised water.
• Solution 2 13.6 g potassium phosphate monobasic anhydrous (KH2PO4, 0.1 M) dissolved in 1 L deionised water. pH7.4 with 2 mM magnesium chloride.
• NADPH (10 mM) is prepared in deionised water.
• Microsomes (all species) were removed from the -80 °C thawed at 37 °C. Microsomes were diluted in assay buffer to achieve a final protein concentration of 0.5 mg/ml_ and 1 mM NADPH.
• the target value for Clint for such compounds in the microsomal stability assay is less than 30 pL/min/mg, preferably less than 20 pL/min/mg and particularly preferably less than 10 pL/min/mg.
• Compounds with a clearance rate in this range are particularly suitable for oral administration since they have a lower propensity for metabolism leading to reduced first pass clearance leading to higher systemic exposure and increased oral bioavailability also resulting in lower dose for a given pharmacological effect.
• the target value for Clint for such compounds in the microsomal stability assay is greater than 35 pL/min/mg, preferably greater than 40 pL/min/mg and particularly preferably greater than 50 pL/min/mg.
• the high microsomal clearance rate corresponding to low in vivo half-life after absorption into the systemic circulation, ensures that compounds administered directly to the lungs will have a reduced chance of interacting with TMEM16A at other locations in the body, and potential side effects will therefore be minimised.
• TMEM16 confers receptor-activated calcium-dependent chloride conductance. Nature, 455(7217):1210 - 1215.

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