Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • 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
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C53/00—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen
  • C07C53/02—Formic acid
  • C07C53/06—Salts thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C53/00—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen
  • C07C53/15—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen containing halogen
  • C07C53/16—Halogenated acetic acids
  • C07C53/18—Halogenated acetic acids containing fluorine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
  • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
  • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
  • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
  • C07D235/08—Radicals containing only hydrogen and carbon atoms
• • 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

Definitions

• the present invention relates to novel compounds which have activity as inhibitors of the epithelial sodium channel (ENaC).
• ENaC epithelial sodium channel
• the invention also relates to the use of these compounds in treating diseases and conditions modulated by ENaC, particularly respiratory diseases and conditions, methods of preparing the compounds and pharmaceutical compositions containing them.
• 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% solids under which conditions the mucus is cleared by mucociliary action.
• the hydration of the airway mucosa is regulated by the coordinated activity of a number of ion channels and transporters.
• the balance of anion (Cl / HCOT) secretion mediated via the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the Calcium Activated Chloride Conductance (CaCC; TMEM16A, also known as And) and Na + absorption through the epithelial Na + channel (ENaC) determine the hydration status of the airway mucosa.
• CFTR Cystic Fibrosis Transmembrane Conductance Regulator
• CaCC Calcium Activated Chloride Conductance
• ENaC epithelial Na + channel
• 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 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.
• ENaC is expressed in renal, colonic, corneal, sweat duct and respiratory epithelia where it forms a low conductance channel ( ⁇ 4 pS) with a selectivity for Na + over K + of approximately 10-fold (Kellenberger 2002).
• Loss and gain of function mutations in the channel can cause human disease including pseudohypoaldosteronism type 1 (PHA1), a salt wasting disease (Chang et al, 1996), and Liddles’s syndrome, a disease associated with salt retention and hypertension (Botero-Velez et al, 1994).
• PHA1 pseudohypoaldosteronism type 1
• PHA1 salt wasting disease
• Liddles’s syndrome a disease associated with salt retention and hypertension
• ENaC is expressed in the cortical collecting duct of the kidney epithelium and block of the channel here can lead to a systemic accumulation of K + .
• an inhaled ENaC blocker avoids renal exposure following absorption from the lung. This could be achieved through either a high lung retention of ENaC blocker therefore enabling only a low dose to be administered or through the design of a compound that will be rapidly transformed to an inactive metabolite before it reaches the kidney.
• ENaC blockers have also been implicated in the hydration of skin and the surface of the eye (Frateschi et al, 2010; Thelin et al, 2012).
• WO 201 1/1 13894 relates to compounds which are said to be of use for treating inflammatory or obstructive diseases of the airways or for promoting mucosal hydration.
• the compounds are of the formula: where A is N or CR 4a and R 2 is haloalkyl. None of the compounds exemplified in this document contain a benzimidazole moiety.
• WO 2011/079087 relates to compounds of the formula:
• WO 2015/007516, WO 2015/007517 and WO 2015/007519 all relate to compounds of the formula:
• WO 2016/113168, WO 2016/113167 and WO 2016/113169 relate to compounds of the formula:
• WO 2016/113170 relates to compounds of the formula: r
• WO 2017/028926 relates to ENaC inhibiting compounds of the formula:
• WO 2017/028927 relates to ENaC inhibiting compounds of the formula:
• WO 2017/221008 also relates to compounds comprising a pyrazine group with a single amino substituent at the 3-position, with most of these compounds also having a substituent at the pyrazine 6-position.
• the present inventors have surprisingly discovered that compounds with alternative structures to the 6-halo-3, 5-diamino pyrazine or 6-substituted-3-aminopyrazine also have ENaC blocking activity and may have beneficial properties compared with the known compounds, particularly in relation to the ADME (Absorption, Excretion, Distribution and Metabolism) properties.
• our application PCT/GB2017/053499 relates to pyrrolopyrazine compounds having ENaC blocking activity.
• X is an anion
• R 1 is:
• L 1 is:
• each of Z ⁇ Z 2 and Z 3 is independently CM2 alkylene, C2-12 alkenylene, C2-12 alkynylene any of which is optionally substituted by one or more substituents selected from halo, OH, C(0)NR 15 R 16 , C(0)OR 15 and NR 15 R 16 ;
• each R 15 and R 16 is independently H or Ci-e alkyl or R 15 and R 16 together with the nitrogen atom to which they are attached may form a 5- or 6- membered heterocyclic ring optionally containing one or more further heteroatoms selected from N, O and S;
• each of Q 1 , Q 2 and Q 3 is independently carbocyclyl, heterocyclyl, aryl or heteroaryl any of which is optionally substituted with one or more substituents selected from halo, OH, C 1-4 alkyl, C 1.4 haloalkyl, C(0)NR 15 R 16 , C(0)OR 15 and NR 15 R 16 , and, for cycloalkyl and heterocyclyl groups, oxo, wherein R 15 and R 16 are as defined above; n is 1 to 6;
• each R 7 and R 8 is independently selected from H or C1.12 alkyl optionally substituted with one or more halo or OH groups, or
• R 7 and an R 8 or two R 8 groups when attached to a nitrogen atom they may, together with the nitrogen atom combine to form a 5- or 6-membered heterocyclic ring optionally comprising one or more further heteroatoms selected from N, O and S;
• R 9 is H or C1-6 alkyl
• each R 12 and R 13 is independently H, Ci-e alkyl, C2-6 alkenyl, C2-6 alkynyl, C 3-8 cycloalkyl or C 3-8 heterocyclyl, any of which is optionally substituted by one or more substituents selected from halo, OR 7 , C(0)OR 7 , -N(R 7 )R 8 and C(0)N(R 7 )R 8 and, in the case of cycloalkyl or heterocyclyl groups, oxo; wherein
• R 7 , R 8 and R 9 are as defined above; each of R 2 and R 3 is independently CMO alkyl, wherein one or more -CH2- groups is optionally replaced by -0-, -S- or -NR 7 - provided that adjacent -CH2- groups are not so replaced and which is optionally substituted with one or more substituents selected from halo, OH, SH, N(R 7 )R 8 , aryl, heteroaryl, cycloalkyl, heterocyclyl, -C(0)0R 7 , -C(0)N(R 7 )R 8 , OR 7 and -N(R 7 )R 8 , wherein R 7 and R 8 are as defined above;
• R 4 is H, halo, cyano, Ci- 6 alkyl, C(0)0R 16 or C(0)N(R 16 )R 17 ;
• alkyl groups are optionally substituted with one or more substituents selected from halo, -OR 7 and -N(R 7 )R 8 , wherein R 7 and R 8 are as defined above;
• each R 16 and R 17 is independently H or Ci-e alkyl or R 16 and R 17 together with the nitrogen atom to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing one or more further heteroatoms selected from O, N and S.
• the compounds of general formula (I) have ENaC blocking activity and, furthermore, are expected to have one or both of the following advantageous properties.
• 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 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.
• the anion X can have any negative charge and will be balanced by the appropriate number of cations.
• a compound of general formula (I) in which X is an anion having a single negative charge will have a 1 : 1 ratio of catiomanion whereas if the anion X has a charge of -2, the ratio of catiomanion in the compound of general formula (I) will be 2: 1.
• the anion X is suitably a pharmacologically acceptable anion, although other anions may also be useful, particularly in synthetic precursors to the compounds of general formula (I).
• Suitable anions, X include halide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate, fumarate, citrate, tartrate, oxalate, succinate, mandelate, methane sulfonate and p-toluene sulfonate.
• An additional anion X or an anion with additional negative charge, e.g. a charge of -2, will be required if the R 1 substituent contains a moiety R 10 which is cationic such that the charge in the compound of general formula (I) is balanced.
• references to salts of the compounds of formula (I) may refer to salts of an additional basic nitrogen atom, for example a nitrogen atom to which R 7 and R 8 moieties are attached. Counter ions for such salts are as defined for X .
• R 1 , R 2 or R 3 comprises a carboxyl group C(0)OH
• salts may be formed.
• Suitable counter ions for such salts include sodium, potassium, lithium calcium, aluminium, zinc, magnesium and other metal ions as well as choline, diethanolamine, ethanolamine, ethyl diamine, megulmine and other well-known basic addition salts as summarised in Paulekuhn et at., (2007) J. Med. Chem. 50: 6665-6672 and/or known to those skilled in the art.
• R 2 or R 3 may comprise an anionic group, for example C(0)0 , which may act as counter ion to the N + moiety in the benzimidazolium ring.
• Ci-e alkyl refers to a straight or branched fully saturated hydrocarbon group having from 1 to 6 carbon atoms.
• the term encompasses methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl.
• Other alkyl groups for example C1 -12 alkyl and Ci -4 alkyl are as defined above but contain different numbers of carbon atoms.
• C2-6 alkenyl refers to a straight or branched hydrocarbon group having from 2 to 6 carbon atoms and at least one carbon-carbon double bond. Examples include ethenyl, prop-1 -enyl, hex-2-enyl etc. Other alkenyl groups, for example C1 -12 alkenyl are as defined above except that they contain the specified number (e.g. 1 to 12) carbon atoms.
• C2-6 alkynyl refers to a straight or branched hydrocarbon group having from 2 to 6 carbon atoms and at least one carbon-carbon triple bond. Examples include ethynyl, prop-1 -ynyl, hex-2-ynyl etc. Other alkynyl groups, for example C2-12 alkynyl are as defined above except that they contain the specified number (e.g. 2 to 12) carbon atoms.
• C1 -6 alkylene refers to a straight or branched fully saturated hydrocarbon chain having from 1 to 6 carbon atoms.
• alkylene groups include -CH2-, -CH2CH2-, CH(CH 3 )-CH 2 -, CH 2 CH(CH 3 )-, -CH2CH2CH2-, -CH 2 CH(CH 2 CH 3 )- and CH2CH(CH2CH 3 )CH2-.
• Other alkylene groups, for example C1 -12 alkylene are as defined above except that they contain the specified number (e.g. 1 to 12) carbon atoms.
• C2-6 alkenylene refers to a straight or branched hydrocarbon chain containing from 2 to 6 carbon atoms and at least one carbon-carbon double bond.
• Other alkenylene groups, for example C 2 -i 2 alkenylene, are as defined above except that they contain the specified number (e.g. 2 to 12) carbon atoms.
• C 2-6 alkynylene refers to a straight or branched hydrocarbon chain containing from 2 to 6 carbon atoms and at least one carbon-carbon triple bond.
• alkenylene groups include -CoC-, -CH 2 CoC-, -CoC-CH 2 -, CH 2 CH 2 CoC-, CH 2 CoCCH 2 - and -CH 2 CHoC-CH 2 CH 2 -.)-.
• Other alkynylene groups for example C 2 -i 2 alkynylene, are as defined above except that they contain the specified number (e.g. 2 to 12) carbon atoms.
• carbocyclic and “carbocyclyl” refer to a non-aromatic hydrocarbon ring system containing from 3 to 10 ring carbon atoms, unless otherwise indicated, and optionally one or more double bond.
• the carbocyclic group may be a single ring or may contain two or three rings which may be fused or bridged. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl and cyclohexenyl.
• heterocyclic and“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.
• the heterocyclic group may be a single ring or may contain two or three rings which may be fused or bridged. Examples include tetrahydrofuranyl, tetrahydroypranyl, pyrrolidine, piperidinyl, morpholinyl, piperazinyl and thiomorpholinyl.
• aryl and“aromatic” in the context of the present specification refer to a ring system with aromatic character having from 5 to 14 ring carbon atoms and containing up to three rings. Where an aryl group contains more than one ring, not all rings must be fully aromatic in character. Examples of aromatic moieties are benzene, naphthalene, fluorene, indane and indene.
• heteroaryl and“heteroaromatic” in the context of the specification refer to a ring system with aromatic character having from 5 to 14 ring atoms, at least one of which is a heteroatom selected from N, O and S, and containing up to three rings. Where a heteroaryl group contains more than one ring, not all rings must be fully aromatic in character. Examples of heteroaryl groups include pyridine, pyrimidine, indole, benzofuran, benzimidazole and indolene.
• halogen refers to fluorine, chlorine, bromine or iodine
• halo to fluoro, chloro, bromo or iodo groups
• halide to fluoride, chloride, bromide or iodide
• Ci-e haloalkyl refers to a Ci-e 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. Other haloalkyl groups, for example C1 -12 haloalkyl are as defined above except that they contain the specified number (e.g. 1 to 12) 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.
• the R 1 substituent is suitably at the 5- or the 6-position and thus the compound of general formula (I) can be a compound of general formula (IA):
• R 1 , R 2 , R 3 , R 4 and X are as defined for general formula (I); or a compound of general formula (IB):
• R 1 , R 2 , R 3 , R 4 and X are as defined for general formula (I).
• R 1 is:
• R 1 is H, halo, C(0)0H, C(0)0(Ci- 6 alkyl), Ci-e alkyl, Ci- 6 haloalkyl, Ci-e alkoxy or Ci-e haloalkoxy.
• R 1 groups include H, chloro, C(0)0H, methyl, trifluoromethyl, methoxy and trifluoromethoxy.
• R 1 is -L 1 R 10 .
• U is:
• L 1 is:
• the cyclic groups Q 1 , Q 2 and Q 3 are independently selected from 5- and 6-membered aryl and heteroaryl groups and 4 to 8- membered carbocyclyl and heterocyclyl groups.
• Q 1 , Q 2 and Q 3 are selected from phenyl, 5- and 6-membered heteroaryl groups and 4- to 7-membered and heterocyclyl groups, still more suitably phenyl, 5- and 6-membered nitrogen-containing heteroaryl and 4- to 7-membered nitrogen-containing heterocyclyl groups.
• heteroaryl Q 1 , Q 2 and Q 3 groups examples include pyridyl, pyrimidinyl, pyrazolyl, imidazolyl and oxazolyl groups, with 5-membered rings such as imidazolyl and oxazolyl and especially pyrazolyl being particularly suitable.
• Q 1 , Q 2 or Q 3 is pyrazolyl, it may have the following regiochemistry:
• heterocyclyl Q 1 , Q 2 and Q 3 groups include azetidinyl, piperidinyl, piperazinyl, and aziridinyl, with 6-membered rings such as piperazinyl and piperidinyl being more suitable.
• Piperidinyl is a particularly suitable heterocyclyl group, especially 1 ,4-piperidinyl.
• Q 1 , Q 2 and Q 3 groups include cyclohexyl and tetrohydropyran groups, either or which may be substituted with one or more substituents selected from OH, or NR 15 R 16 , especially OH, NH 2 or NHCH 3 .
• the Q moiety may be a nitrogen containing heterocyclyl ring in which C(O) is linked to the nitrogen atom.
• Q 1 is suitably a 5- or 6-membered heterocyclyl ring which is linked to the -C(O) moiety via a ring nitrogen atom.
• Q 1 is a 5- or 6-membered nitrogen-containing heterocyclyl ring such as piperidin-1-yl or pyrrolidine-1 -yl, more suitably piperidin-1 yl.
• Q 1 is piperidin-1-yl the remainder of the molecule is linked to the 4-position of the piperidine ring.
• Q 1 is pyrrolidine-1 -yl
• the remainder of the molecule may be linked to the 3-position of the pyrrolidine ring.
• Q 2 is suitably a 5- or 6-membered heterocyclyl ring which is linked to the -C(O) moiety via a ring nitrogen atom.
• Q 2 is piperidin-1-yl or pyrrolidine-1 -yl, more suitably piperidin-1 yl.
• Q 1 is piperidin-1 -yl
• the remainder of the molecule is linked to the 4-position of the piperidine ring.
• Q 1 is pyrrolidine-1 -yl
• the remainder of the molecule may be linked to the 3-position of the pyrrolidine ring.
• the Q moiety may be a heterocyclyl group which is linked to the C(O) moiety via a ring carbon atom.
• heterocyclyl groups include 5- and 6-membered rings, suitably nitrogen-containing rings such as piperidinyl or pyrrolidinyl.
• Q 1 is a piperidine ring it is a piperidin-4-yl group such that the piperidine 4-position is linked to the C(O) moiety.
• the piperidine nitrogen atom is linked to the remainder of the molecule.
• U moieties of this type include C(0)Q 1 Z 1 , in which Q 1 may be a piperidine ring in which the 4-position is linked to C(O) and the 1 -position is linked to Z 1 .
• the Q moiety is suitably a heterocyclyl ring, e.g. a 5- or 6-membered nitrogen-containing heterocyclyl ring, which is linked to the -C(0)N(R 7 )- moiety via a ring carbon atom.
• Q 1 is suitably a 5- or 6-membered heterocyclyl ring which is linked to the -C(0)N(R 7 )- moiety via a ring carbon atom, with the remainder of the molecule being linked to a ring nitrogen atom.
• Q 1 is piperidin-4-yl or pyrrolidinyl.
• the U comprises a -Q 1 Q 2 - or -Q 2 Q 3 - moiety
• this may be, for example:
• -Q 1 Q 2 - and -Q 2 Q 3 - moieties include biphenyl, suitably a 1 , 1’-biphenyl- 4-yl moiety.
• This type of -Q 1 Q 2 - or -Q 2 Q 3 - moiety is suitably linked at each side to a Z moiety, for example as in -C(0)N(R 7 )Z 1 Q 1 Q 2 Z 2 -
• each of Z 1 , Z 2 and Z 3 is independently Ci-i2 alkylene, optionally substituted by one or more halo or OH groups.
• R 1 is L 1 R 10 and U comprises a Z 1 , and optionally a Z 2 and optionally a Z 3 moiety.
• Z 1 and, where present, Z 2 and Z 3 suitably comprise Ci-e alkylene, C 2 -s alkenylene, C 2 -s alkynylene any of which is optionally substituted by one or more halo, OH, C(0)NR 15 R 16 , C(0)OR 15 or NR 15 R 16 ; wherein R 15 and R 16 are as defined above.
• the groups Z 1 and, where present, Z 2 and Z 3 comprise C alkylene optionally substituted by one or more halo, OH, C(0)NR 15 R 16 , C(0)OR 15 or NR 15 R 16 ; wherein R 15 and R 16 are as defined above.
• the groups Z 1 and, where present, Z 2 and Z 3 comprise Ci -4 alkylene which is unsubstituted or substituted by one or more halo, OH, C(0)NR 15 R 16 , C(0)OR 15 or NR 15 R 16 , wherein each R 15 and R 16 is H.
• Z 1 and where present, Z 2 and Z 3 comprise Ci -4 alkylene which is unsubstituted or substituted with one or more OH, halo, C(0)NH 2 or C(0)OH.
• Z 1 and, where present, Z 2 and Z 3 are unsubstituted Ci -4 alkylene.
• Z 1 is Ci -4 alkylene substituted with one or more halo, C(0)NH 2 or C(0)OH.
• Z ⁇ Z 2 or Z 3 may be directly linked to a cyclic group via a ring nitrogen atom. This may occur, for example, in compounds where R 1 is L 1 R 10 and U is:
• the Z 1 or Z 2 or Z 3 group is also linked to R 10 .
• the Z 1 or Z 2 or Z 3 group may be CM 2 alkylene substituted with one or more OH group, suitably by a plurality of OH groups, for example 2-1 1 OH groups.
• the number of OH groups will be one less than the number of carbon atoms in the alkylene group;
• Z 1 is a Ci- 3 alkylene group substituted with 2-7 OH groups, for example 5-7 OH groups.
• Z 1 or Z 2 or Z 3 groups of this type include -CH 2 [CH(OH)] n -, where n is suitably 3-7. Most suitably, Z 1 or Z 2 or Z 3 where appropriate is -CH 2 -CH(OH)-CH(OH)- CH(OH)-CH(OH)-CH(OH)-.
• R 10 is suitably H such that the Z 1 R 10 , Z 2 R 10 or Z 3 R 10 moiety is -CH 2 [CH(OH)] n -H, where n is suitably 3-7, for example -CH 2 -[CH(OH)] 4 -CH 2 OH.
• R 10 is -L 1 R 10
• suitable R 10 groups include H, -N(R 7 )R 8 , -
• R 10 when R 10 is H, U is -OZ 1 , where Z 1 is as defined above but is suitably Ci-e alkylene optionally substituted as described above. More suitably in these compounds, Z 1 is Ci -4 alkylene such that the group -OZ 1 R 10 is -0(Ci- 4 alkyl), for example methoxy, ethyoxy isopropoxy or f-butyloxy. In one embodiment (e.g. as used in Compounds 1 , 7, 10-16, 61 , 64 and 67-73), R 1 is methoxy.
• R 10 when R 10 is H, U is -Q 1 -, -Q 1 Q 2 - or -C(0)N(R 7 )Q 1 -, where the Q 1 group or, for -Q 1 Q 2 -, the Q 2 group, is a nitrogen-containing heterocyclyl group which is linked to the R 10 group via a ring nitrogen atom.
• Q 1 R 10 or Q 2 R 10 groups include:
• R 1 is -Q 1 -H.
• R 1 is -Q 1 Q 2 -H.
• R 1 is -C(0)N(R 7 )Q 1 -H.
• the cylic group when R 10 is H, and which contain a cyclic group Q 1 , Q 2 or Q 3 linked to Z 1 or Z 2 or Z 3 , the cylic group may be a nitrogen containing heterocyclyl group linked to Z 1 or Z 2 or Z 3 via a ring nitrogen atom. This may occur, for example, in compounds where R 1 is L 1 R 10 and U is:
• Particularly suitable compounds of this type are those in which R 10 is H and U is -Q 1 Z 1 -, - Q 1 Q 2 Z 1 -, -C(0)N(R 7 )Q 1 Z 1 -, or -C(0)Q 1 Z 1 -; or, still more suitably, -Q 1 Z 1 -, -Q 1 Q 2 Z 1 - or - C(0)N(R 7 )Q 1 Z 1 -.
• the Z 1 , Z 2 or Z 3 moiety is suitably a CM2 alkylene group substituted by a plurality of OH groups, for example 2 to 1 1 OH groups.
• the number of OH groups will be one less than the number of carbon atoms in the alkylene group;
• Z 1 is a Ci-s alkylene group substituted with 2-7 OH groups, for example 5-7 OH groups.
• Z 1 groups of this type include -CH2[CH(OH)] n -, where n is suitably 3- 7. Most suitably, n is 5 and in this case, Z 1 is -CH2[CH(OH)]4-CH(OH)- such that the group zi R i°, Z 2 R IO or Z 3 R IO j s a moiety -CH 2 [CH(OH)] 4 -CH 2 OH.
• Examples of compounds where the group Z 1 R 10 is a moiety -CH 2 [CH(0H)] 4 -CH 2 0H include those in which R 1 is:
• R 1 is -Q 1 Z 1 -H.
• R 1 is -Q 1 Q 2 Z 1 -H.
• R 1 is C(0)N(R 7 )Q 1 Z 1 -H.
• R 10 is H
• R 10 is attached to a ring nitrogen atom of a moiety Q 1 , Q 2 or Q 3 or the U group.
• Z 1 , Z 2 and Z 3 may be -(CH2) n - where n is 1 to 6 or -0(CH 2 ) m -, where m is 1 to 5.
• Q 1 or, for -Q 1 Q 2 -, Q 2 is a carbocyclyl or heterocyclyl group and is linked to R 10 via a ring carbon atom; or C(0)N(R 7 )Q 1 , where Q 1 is a is a carbocyclyl or heterocyclyl group and is linked to R 10 via a ring nitrogen atom.
• R 7 is suitably Ci-e alkyl, still more suitably Ci -4 alkyl, for example t- butyl.
• R 1 is -Q 1 C(0)OR 7 , where Q 1 is piperidin-4-yl and R 7 is f-butyl.
• R 1 is -Q 1 Q 2 C(0)0R 7 , where Q 1 is pyrazol-4-yl, Q 2 is piperidin-4-yl and R 7 is f-butyl.
• R 1 is C(0)NHQ 1 C(0)0R 7 , where Q 1 is piperidin-1-yl and R 7 is
• each of R 7 and R 8 is independently either H or Ci-e alkyl optionally substituted with one or more OH groups. In some cases, both R 7 and R 8 are H. In the case where either or both of R 7 and R 8 is Ci-e alkyl, it may be substituted with a plurality of OH groups, for example 2-7 OH groups. Typically, the number of OH groups will be one less than the number of carbon atoms in the alkyl group. More suitably in compounds of this type, one or preferably both, of R 7 and R 8 may be - CH 2 [CH(OH)] m CH 2 OH, where m is suitably 2-6. Most suitably, m is 4 and in this case, the R 7 and/or R 8 group is a moiety -CH 2 [CH(OH)]4-CH 2 OH.
• R 10 is -N ⁇ CH 2 [CH(OH)]4-CH 2 OH ⁇ 2 .
• U is -Z 1 - and R 10 is NH 2
• U is -OZ 1 - and R 10 is NH 2 .
• U is -C(0)N(R 7 )Z 1 - and R 10 is NH 2 .
• U is -C(0)Q 1 and and R 10 is NH 2 .
• U is -C(0)N(R 7 )Z 1 Q 1 and R 10 is NH 2 .
• U is -OZ 1 - and R 10 is -N ⁇ CH 2 [CH(OH)]4-CH 2 OH ⁇ 2.
• U is -Z 1 - and R 10 is - N ⁇ CH 2 [CH(OH)] 4 -CH 2 OH ⁇ 2 .
• U is -C(0)N(R 7 )Z 1 - and R 10 is -N ⁇ CH 2 [CH(OH)] 4 -CH 2 OH ⁇ 2 ..
• U is -C(0)Q 1 and R 10 is -N ⁇ CH 2 [CH(OH)] 4 -CH 2 OH ⁇ 2 .
• U is -C(0)N(R 7 )Z 1 Q 1 and R 10 is -N ⁇ CH 2 [CH(OH)] 4 -CH 2 OH ⁇ 2 .
• U is -C(0)N(R 7 )Q 1 and R 10 is -N ⁇ CH 2 [CH(OH)] 4 -CH 2 OH ⁇ 2 .
• U is -C(0)N(R 7 )Z 1 Q 1 Q 2 Z 2 - and R 10 is -N ⁇ CH 2 [CH(OH)] 4 -CH 2 OH ⁇ 2 .
• each of R 7 and R 9 is suitably H or Ci- 4 alkyl, particularly H or methyl and especially H; and each R 8 is independently either H or Ci-s alkyl optionally substituted with one or more OH groups.
• R 8 groups is a
• Ci-s alkyl group it may be substituted with a plurality of OH groups, for example 2-7 OH groups. Typically, the number of OH groups will be one less than the number of carbon atoms in the alkyl group. More suitably in compounds of this type, one or preferably both, R 8 groups may be -CH 2 [CH(OH)] m CH 2 OH, where m is suitably 2-6. Most suitably, m is 4 and in this case, one or preferably both the R 8 groups is a moiety -CH 2 [CH(OH)] 4 -CH 2 OH.
• each of R 7 and R 8 is suitably H or Ci- 6 alkyl. More suitably, R 7 is H and R 8 is Ci- 6 alkyl, still more suitably Ci- 4 alkyl, for example f-butyl.
• R 1 is -OR 12 -S0 2 R 12 , -C(0)OR 12 , -
• Suitable groups Q 1 and Q 2 are as set out above.
• Suitable R 12 and R 13 groups include H and Ci-e alkyl optionally substituted with one or more substituents selected from halo and OR 7 .
• each of R 2 and R 3 is independently CM O alkyl in which one or more -CH2- groups is optionally replaced by -0-, -S- or -NR 7 - and which is optionally substituted as defined above. There may, for example, be no such substituents or a single substituent.
• each of R 2 and R 3 is independently CM O alkyl in which one or more -CH2- groups is optionally replaced by -O- or -S- and which is optionally substituted as defined above.
• R 2 and R 3 groups include -(CH2) S CH3 or (CH 2 CH 2 0) t -H, either of which is optionally substituted as defined above; and wherein s is 0-9, more suitably 0-6 and still more suitably 0-3; and t is 1-3, especially 2 or 3.
• Particularly suitable compounds of general formula (I) are those in which R 2 and R 3 are the same or different and are both unsubstituted Ci -4 alkyl, for example methyl or ethyl. In some such compounds R 2 and R 3 are the same and are both methyl or both ethyl. In other such compounds, one of R 2 and R 3 is methyl and the other of R 2 and R 3 is ethyl.
• Suitable substituents for R 2 and R 3 include OH, SH, halo, N(R 7 )R 8 , C(0)OR 7 , C(0)N(R 7 )R 8 , phenyl or pyridyl, where R 7 and R 8 are as defined above.
• R 2 Particularly suitable substituents for R 2 include OH, SH, phenyl or pyridyl, particularly OH, phenyl, pyridyl, C(0)0-Ci- 6 alkyl, C(0)OH, C(0)NH 2 or C(0)N(R 7 )R 8 , where each of R 7 and R 8 is C1.3 alkyl or R 7 and R 8 together with the nitrogen atom to which they are attached form a pyrrolidine, piperidine, piperazine or morpholine ring.
• At least one of R 2 and R 3 is -(CH2) S CH3, wherein s is as defined above, and is optionally substituted with a single substituent as defined above.
• At least one of R 2 and R 3 is methyl, ethyl, benzyl, pyridylmethyl, -CH 2 OH, -CH 2 NH 2 , -CH 2 CH 2 OH or CH 2 CH 2 NH 2 .
• At least one of R 2 and R 3 is -CH2CH2OCH2CH2OH or -CH2CH2OCH2CH2OCH2CH2OH
• R 4 is H, halo, cyano or C1-3 alkyl. In more suitable compounds of general formula (I), R 4 is H, chloro, bromo, cyano or methyl. In particularly suitable compounds of general formula (I), R 4 is H or methyl. In other compounds of general formula (I), R 4 is H.
• R 4 is methyl
• Some particularly suitable compounds of the present invention include those having a cation selected from:
• Compounds of general formula (I) may be prepared by reacting a compound of general formula (II) or a salt or activated derivative thereof:
• R 1 , R 2 and R 3 are as defined for general formula (I);
• the reaction is carried out under basic conditions in the presence of a coupling reagent, which may generate an activated acid as an intermediate.
• the basic conditions may be supplied by a non-nucleophilic base such as N,N-diisopropylethylamine (DIPEA) or trimethylamine.
• DIPEA N,N-diisopropylethylamine
• Suitable coupling reagents include 0-(7-Azabenzotriazol-1-yl)- N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), O-(Benzotriazol-l-yl)- L/,L/,L/',L/'-tetramethyluronium tetrafluoroborate (TBTU), A/,/ ⁇ /,/ ⁇ /',/ ⁇ /-tetramethyl-0-(1/-/- benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) or a combination of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) with 1-hydroxy-7-azabenzotriazole (HOAt) or hydroxybenzotriazole (HOBt).
• HATU 0-(7-Azabenzotriazol-1-yl)- N,N,N’,N’-t
• carbonyldiimidazole may be used as a coupling agent.
• imidazole or a salt thereof e.g. imidazole hydrochloride
• imidazole hydrochloride e.g. imidazole hydrochloride
• the reaction may be conducted at a temperature of about 10 to 50°C, more usually at 15 to 30°C, or room temperature and in an organic solvent such as A/,/ ⁇ /-dimethylformamide.
• a compound of general formula (I) may be prepared by reacting a compound of general formula (III) as defined above with an activated derivative of a compound of general formula (II), which is a compound of general formula
• R 4 and R 5 are as defined for general formula (I).
• reaction is suitably conducted at a temperature of about 10 to 50°C, more usually at 15 to 30°C, or room temperature and in an organic solvent such as A/,/ ⁇ /-dimethylformamide.
• the activated compounds of general formula (IVa) are prepared by reacting a compound of general formula (II) as defined above or a salt thereof with carbonyl diimidazole (CDI).
• a compound of general formula (II) as defined above or a salt thereof with carbonyl diimidazole (CDI).
• CDI carbonyl diimidazole
• the reaction takes place in an organic solvent such as A/,/ ⁇ /-dimethylformamide and at a temperature of from about 10 to 30°C, more usually 15 to 25°C or room temperature.
• the activated compounds of general formula (IVb) are prepared by reacting a compound of general formula (II) as defined above, or a salt thereof, with carbonyl diimidazole (CDI) under basic conditions.
• a compound of general formula (II) as defined above, or a salt thereof with carbonyl diimidazole (CDI) under basic conditions.
• the reaction takes place in an organic solvent such as A/,/ ⁇ /-dimethylformamide and at a temperature of from about 10 to 30°C, more usually 15 to 25°C or room temperature.
• the compounds of general formulae (III) may comprise a protected R 1 , R 2 and/or R 3 substituent and/or the compounds of general formulae (II) and (IVa) or (IVb) may comprise a protected R 4 substituent.
• R 4 is as defined for general formula (I) and R 21 is Ci-e alkyl or benzyl.
• the hydrolysis is base hydrolysis such that the compound of general formula (V) is reacted with a base, suitably a strong aqueous base such as lithium hydroxide, potassium hydroxide or sodium hydroxide.
• a base suitably a strong aqueous base such as lithium hydroxide, potassium hydroxide or sodium hydroxide.
• R 4 is Ci-e alkyl
• R 21 is Ci-e alkyl or benzyl
• R 18 is a leaving group, for example halo such as chloro
• reaction is carried out at elevated temperature such as 50°C.
• R 18 and R 21 are as defined above for general formula (XIX), R 20 is halo, for example bromo and R 26 is an amine protecting group, for example butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc) or benzyloxycarbonyl (Cbz);
• each R 27 is independently C alkyl
• R 18 and R 21 are as defined above for general formula (XIX) and R 20 is as defined for general formula (XX) with a suitable protecting group using standard methods.
• R 21 is as defined above for general formula (XIX) and R 20 is as defined for general formula (XX) by reaction of a compound of general formula (XXIa) with a suitable halogenating agent.
• a suitable halogenating agent for example, when R 18 is chloro, the halogenating agent may be phosphorous oxychloride as described for the synthesis of Intermediate 2 below.
• R 21 is as defined above for general formula (XIX) and X 2 is as defined for general formula (XX) by oxidation, for example with metachloroperbenzoic acid (mCPBA) as described for the preparation of Intermediate 1 below.
• mCPBA metachloroperbenzoic acid
• compounds of general formula (V) may be converted to other compounds of general formula (V).
• compounds of general formula (V) in which R 4 is halo may be prepared from compounds of general formula (V) in which R 4 is H by reaction with a halogenating agent such as /V-chlorosuccinimide, /V-bromosuccinimide or N- iodosuccinimide.
• Compounds of general formula (V) in which R 4 is halo may be converted into compounds of general formula (V) in which R 4 is alkyl or cyano.
• Compounds in which R 4 is alkyl may be prepared from compounds in which R 4 is halo by reaction with an organometallic reagent, for example dialkyl zinc.
• Compounds of general formula (V) in which R 4 is cyano may be prepared from compounds of general formula (V) in which R 4 is halo by reaction with potassium ferrocyanide. For this reaction, the nitrogen at the 5-position of the pyrrazolopyrazine ring should be protected, for example with a trimethylsilylethoxymethyl protecting group.
• R 4 is as defined for general formula (I) and R 22 is a protecting group, for example Ci-e alkyl, or benzyl optionally substituted with OH, halo, Ci-e alkyl, Ci-e alkoxy, C haloalkyl or Ci-e haloalkoxy.
• Deprotection may be achieved by heating under acidic conditions, typically also with microwave irradiation as described for the synthesis of Intermediate 1 1.
• a compound of general formula (XXIII) may be prepared from a compound of general formula (XXIV):
• R 4 is as defined for general formula (I) and R 22 is as defined for general formula (XXIII);
• reaction by reaction with 2-cyanoacetic acid under basic conditions as described for the preparation of Intermediate 10.
• the reaction is carried out at a temperature of 1 10-160°C, more usually 130-150°C, with microwave irradiation.
• a compound of general formula (XXIV) may be prepared from a compound of general formula (XXV):
• R 4 is as defined for general formula (I) and R 22 is as defined for general formula (XXIII);
• R 1 , R 2 and R 3 are as defined for general formula (I) and X is as defined for general formula (I) and which may be the same or different from the X of the product of general formula (III);
• R 23 is O(Ci-e) alkyl optionally substituted with aryl
• R 24 is H
• the compound of general formula (III) may be prepared by deprotection of a compound of general formula (Vila):
• R 2 and R 3 are as defined for general formula (I) and X is as defined for general formula (I) and may be the same or different from the X of the product of general formula (III); R 23 and R 24 are as defined for general formula (VII); and R 1a is a protected group R 1 , for example when R 1 is a carboxylic acid, R 1a may be a Ci-e alkyl or benzyl carboxylic ester.
• the removal of the protecting group C(0)R 23 may be achieved by reaction with an acid. This is appropriate for alkyloxycarbonyl protecting groups, for example when R 23 is‘butyloxy. Acid deprotection is also appropriate for use with compounds of general formula (Vila) when the protecting group R 1a is acid sensitive as in the case of carboxylic esters.
• Reaction with an acid may result in a change in the anion X .
• the compound of formula (III) will usually be present in the form of its acid addition salt.
• Fmoc i.e. when R 23 is fluorenylmethyloxy
• a base for example piperidine or morpholine.
• R 23 is benzyloxy or fluoren-9- ylmethyloxy and R 1a comprises another protecting group, for example‘butyloxycarbonyl (Boc), such that the two protecting groups are stable under different conditions. Removal of the protecting group R 23 will then result in production of a protected compound of general formula (Ilia):
• R 2 , R 3 are described for general formula (I)
• R 1a is as defined for general formula (Vila)
• X is as defined for general formula (I) and which may be the same or different from the X of the product of general formula (Vila)
• Examples of cyclic N(R 24 )C(0)R 23 groups include 1 ,3-dioxo-2,3-dihydro-1 /-/-isoindol-2-yl.
• Examples of individual R 23 groups include methoxy, ethoxy, n propoxy, 'propoxy, "butyloxy, s butyloxy,‘butyloxy, benzyloxy, fluorenylmethyloxy and phenyl optionally substituted with C(0)0H.
• the compound of general formula (III) or (Ilia) may be synthesised by reacting the compound of general formula (VII) or (Vila) with hydrazine hydrate.
• this reaction is carried out in an alcoholic solvent such as methanol and at elevated temperature, for example about 60-90°C, typically about 75°C as described for the synthesis of Intermediate 14 below.
• R 1 and R 3 are as defined for general formula (I); R 1a is as defined for general formula (Vila) and R 23 and R 24 are as defined for general formula (VII);
• X 1 is as defined above for general formula (IX) and R 2a is a protected R 2 group.
• R 2a may be protected using standard protecting groups, for example silyl protecting groups such as trimethylsilyl (TMS),‘butyldimethylsilyl (TBDMS) etc.
• the compound of general formula (VIII) is suitably one in which R 23 is -0(Ci- 6 ) alkyl optionally substituted with aryl, or R 23 is aryl optionally substituted with C(0)0H; and R 24 is H; because, in this case, the silyl and carbonyloxy protecting groups can be removed using an acid such as hydrogen chloride solution.
• Some compounds of general formulae (VII) and (VIII) may be synthesised from other compounds of general formulae (VII) and (VIII).
• a compound of general formula (VIII) in which R 1 is C(0)OR 5 , where R 5 is other than H may be converted to a compound of general formula (VIII) in which R 1 is C(0)OH by hydrolysis, for example with a base, suitably an alkali metal hydroxide such as lithium hydroxide.
• the compound in which R 1 is C(0)OH may then be converted to a compound in which R 1 is C(0)OR 12 , where R 12 is other than H by reaction with a compound of general formula (X):
• R 12 is as defined for general formula (I) except that it is not H and R 25 is Ci -4 alkyl.
• R 1 and R 3 are as defined in general formula (I). This is exemplified by the synthesis of Intermediate 18 from Intermediate 17.
• the compound of general formula (XI) may be re-protected to obtain a new compound of general formula (VIII), for example by reaction with a compound of general formula (XII):
• R 23 is as defined above for general formula (VII).
• the reaction may be conducted in the presence of a base such as trimethylamine in a polar organic solvent, for example dichloromethane and at a temperature of from 10 to 30°C, more usually 15 to 25°C, typically at room temperature.
• a base such as trimethylamine
• a polar organic solvent for example dichloromethane
• An example of this type of process is the synthesis of Intermediate 19 from Intermediate 18.
• a compound of general formula (VII) or (VIII) in which R 1 is halo, particularly bromo may be reacted with an alkyne to give a compound of general formula (VII) in which R 1 is -L 1 R 10 , where U comprises an alkynylene group.
• the reaction may be catalysed with a copper (I) salt, for example copper (I) iodide. This is illustrated in the synthesis of Intermediate 72.
• a compound of general formula (VII) or (VIII) in which R 1 is halo, particularly bromo, may also be reacted with a compound of general formula (XX): where R 1a is as defined above for R 1 except that it is not halo and X 2 is an organoborane group, for example 4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl.
• XX is an organoborane group, for example 4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl.
• R 1 or R 3 for compounds of general formula (VIII)
• R 1 or R 3 comprises a -C(0)OR 12 or -C(0)OR 7 group in which R 12 or R 7 is other than H; or a -C(0)N(R 7 )R 8 group
• base hydrolysis may be used, for example using a base such as lithium hydroxide, sodium hydroxide or potassium hydroxide.
• the hydrolysis may be acid hydrolysis using an acid such as hydrochloric acid. This is particularly suitable when the R 12 or R 7 group is an alkyl group such as tert-butyl.
• R 10 is N(H)R 7 or N(R 7 )R 8 where R 7 is CH 2 -R 7a and R 8 is CH 2 -R 8a and each R 7a and R 8a is independently selected from H or CM I alkyl optionally substituted with one or more halo or OH groups or protected OH groups.
• the conversion can be achieved by reductive amination using a reducing agent such as a hydride, for example sodium cyanoborohydride, with an aldehyde or acetal as shown below: H
• R 7 is -ChhR 73 , where R 7a is H or CM I alkyl optionally substituted with one or more halo or OH groups;
• R 19 is C1 -12 alkyl
• A is a fragment of a compound of general formula (VII) or (VIII) not including R 1 as follows:
• a cyclic hemiacetal can be used in place of the aldehyde or acetal.
• the scheme below shows an example where a 6-membered hemiacetal is used to give a compound in which R 1 is U-NHR 7 where R 7 is (0H 2 ) 4 0H 2 0H:
• a and L 1 are as defined above. If a large excess of the aldehyde, acetal or cyclic hemiacetal is used, both of the amine hydrogen atoms will be replaced. In some cases, it may be possible to react successively with different aldehydes, acetals or hemicacetals to yield compounds in which R 7 and R 8 are different.
• R 7a and R 8a are CM I alkyl optionally substituted with one or more halo or OH or protected OH groups; and most suitably each R 7 and R 8 is CH 2 [CH(0H)] 4 CH 2 0H, wherein OH groups are optionally protected, for example as acetals, such as benzylidene acetals.
• Examples of this type of reaction include the conversion of Intermediate 83 to Intermediate 84 and the conversion of Intermediate 87 to Intermediate 88.
• R 10 may be converted to compounds in which R 10 is H by hydrolysis, for example acid hydrolysis using hydrochloric acid in a solvent such as dioxane.
• R 10 is H; by reductive amination with an aldehyde, acetal or cyclic hemiacetal equivalent compound using a method similar to that described above for the conversion of compounds of general formulae (VII) and (VIII) in which R 1 is U NH2 to compounds of general formulae (VII) and (VIII) in which R 1 is L 1 N(R 7 )R 8 where R 7 is CH2-R 7a and R 8 is CH2-R 8a and each each R 7a and R 8a is independently selected from H or CM I alkyl optionally substituted with one or more halo or OH groups.
• Z 1 , Z 2 or Z 3 is CH2-C1.1 1 alkyl optionally substituted with one or more halo or OH groups; and most suitably is CH 2 [CH(0H)] 4 CH 2 0H, wherein OH groups are optionally protected, for example as acetals, such as benzylidene acetals.
• R 1 and R 3 are as defined for general formula (I);
• R 23 is as defined above for general formula (VII).
• the reaction suitably takes place in the presence of a base, typically a non-nucleophilic base, for example an amine such as A/,/ ⁇ /-diisopropylethylamine (DIPEA) or triethylamine and a peptide coupling agent, for example HATU, TBTU, HBTU or a combination of EDC with HOAt or HOBt.
• a base typically a non-nucleophilic base, for example an amine such as A/,/ ⁇ /-diisopropylethylamine (DIPEA) or triethylamine and a peptide coupling agent, for example HATU, TBTU, HBTU or a combination of EDC with HOAt or HOBt.
• DIPEA A/,/ ⁇ /-diisopropylethylamine
• a peptide coupling agent for example HATU, TBTU, HBTU or a combination of EDC with HOAt
• R 1 and R 3 are as defined for general formula (I);
• catalytic hydrogenation suitably using a palladium catalyst.
• the hydrogenation is suitably carried out at 1 atmosphere pressure and at a temperature of about 10 to 30°C, usually 15 to 25°C, for example at room temperature.
• R 1 is as defined for general formula (I) and X 2 is a leaving group, particularly a halo group such as chloro or fluoro;
• R 3 is as defined for general formula (I).
• the reaction is suitably carried out under pressure, at a temperature of about 30-70°C, more usually about 40-60°C, typically about 50°C and in an organic solvent such as tetrahydrofuran.
• R 1 , R 3 and R 4 are as defined for general formula (I);
• compounds of general formula (I) in which R 3 comprises a -C(0)0R 7 group in which R 7 is other than H or a -C(0)N(R 7 )R 8 group can be converted to compounds in which R 3 comprises a -C(0)0H or C(0)0 group by hydrolysis.
• the hydrolysis may be base hydrolysis, for example using a base such as lithium hydroxide, sodium hydroxide or potassium hydroxide.
• acid hydrolysis may be employed, for example using hydrochloric acid.
• R 1 is L 1 R 10 where R 10 is -N(R 7 )-C(0)0R 8
• R 1 is L 1 R 10 where R 10 is -NHR 7 by hydrolysis, for example acid hydrolysis using hydrochloric acid in a solvent such as dioxane.
• R 7 is H such that in the product compound of general formula (I) R 10 is NH 2 .
• R 1 is LR 10
• R 10 is NH2
• R 10 is N(R 7 )R 8
• R 7 is Chh-R 73 and R 8 is CH2-R 8a and one of R 7a and R 8a is CM I alkyl optionally substituted with one or more halo or OH groups
• the other of R 7a and R 8a is H or CM I alkyl optionally substituted with one or more halo or OH groups.
• the compounds of general formula (I) in which R 1 is LR 10 , and U comprises a moiety Q 1 , Q 2 or Q 3 linked to R 10 via a ring nitrogen atom and in which R 10 is H can be converted to compounds of general formula (I) in which Q 1 , Q 2 or Q 3 is linked to a Z 1 , Z 2 or Z 3 moiety via a ring nitrogen atom, wherein Z ⁇ Z 2 or Z 3 is CH2-C1.1 1 alkyl optionally substituted with one or more halo or OH groups; and R 10 is H.
• R 1 is C(0)0H
• R 1 is - C(0)NR 12 R 13 , wherein R 12 and R 13 are as defined above, or in which R 1 is -L 1 R 10 , wherein U is -C(0)N(R 7 )Z 1 -, -C(0)N(R 7 )Q 1 -, -C(0)N(R 7 )Z 1 Q 1 -, -C(0)N(R 7 )Z 1 Q 1 Z 2 -, -C(0)N(R 7 )Q 1 Z 1 -, -C(0)N(R 7 )Q 1 Q 2 -, -C(0)N(R 7 )Q 1 Q 2 Z 1 -, -C(0)N(R 7 )Z 1 Q 1 Q 2 Z 2 -, -C(0)N(R 7 )Z (CH 2 CH 2 0) n Z 2 - -C(0)N(R 7 )Z (CH 2 0) n Z 2 -,
• U is -C(0)Q 1 -, -C(0)Q 1 Z 1 -, -C(0)Q 1 Q 2 -, -C(0)Q 1 Q 2 Z 1 -,
• the reaction is carried out under basic conditions in the presence of a coupling reagent.
• the basic conditions may be supplied by a non-nucleophilic base such as N,N- diisopropylethylamine (DIPEA) or trimethylamine.
• DIPEA N,N- diisopropylethylamine
• Suitable coupling reagents include O- (7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), O- (Benzotriazol-1-yl)-/ ⁇ /,/ ⁇ /,/ ⁇ /',/ ⁇ /'-tetramethyluronium tetrafluoroborate (TBTU), N,N,N',N'- tetramethyl-0-(1 /-/-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with 1-hydroxy-7- azabenzotriazole (HOAt) or hydroxybenzotriazole (HOBt).
• HATU O- (7-Azabenzotriazol-1-yl)-N,N,N’,N’-t
• U is -C(0)N(R 7 )Z 1 - where R 7 is H and Z 1 is as defined above; R 10 is -N(R 7 )-C(0)0R 8 ; R 7 is H and R 8 is as defined above.
• U is -C(0)N(R 7 )Q 1 - where R 7 is H and Q 1 is heterocyclyl linked to R 10 via a ring nitrogen atom; R 10 is -C(0)0R 7 where R 7 is as defined above.
• U is -C(0)Q 1 - where Q 1 is heterocyclyl linked to C(O) via a ring nitrogen atom and to R 10 via a ring carbon atom; R 10 is -N(R 7 )-C(0)0R 8 ; R 7 is H and R 8 is as defined above.
• U is -C(0)N(R 7 )Z 1 Q 1 - where R 7 is H, Z 1 is as defined above, Q 1 is heterocyclyl linked to R 10 via a ring carbon atom; R 10 is -N(R 7 )-C(0)0R 8 ; R 7 is H and R 8 is as defined above.
• U is C(0)N(R 7 )Z 1 where R 7 is H or methyl and Z 1 is as defined above;
• R 10 is N(R 7 )R 8 , where R 7 and R 8 are both C 1-12 alkyl substituted with one or more OH groups.
• U is C(0)N(R 7 )Q 1 Z 1 where R 7 is H, Q 1 is linked to - C(0)N(R 7 )- via a ring carbon atom and to Z 1 via a ring nitrogen, Z 1 is C 1-12 alkylene substituted with one or more OH; R 10 is H.
• U is C(0)Q ⁇ where Q 1 is linked to C(O) via a ring nitrogen atom;
• R 10 is N(R 7 )R 8 , where R 7 and R 8 are both C 1-12 alkyl substituted with one or more OH groups.
• U is -C(0)N(R 7 )Z 1 Q 1 - where R 7 is H; Z 1 is as defined above, Q 1 is linked to R 10 via a ring carbon atom, R 10 is N(R 7 )R 8 , where R 7 and R 8 are both C 1-12 alkyl substituted with one or more OH groups.
• U is -C(0)Q 1 Z 1 - where Q 1 is linked to the C(O) moiety via a ring nitrogen atom and Z 1 as defined above;
• R 10 is N(R 7 )R 8 , where R 7 and R 8 are both C 1-12 alkyl substituted with one or more OH groups.
• U is -C(0)N(R 7 )Q 1 - where R 7 is H, Q 1 is linked to - C(0)N(R 7 )- via a ring carbon atom and to R 10 via a ring nitrogen atom; R 10 is N(R 7 )R 8 , where R 7 and R 8 are both C 1-12 alkyl substituted with one or more OH groups.
• U is -C(0)N(R 7 )Z 1 0(CH 2 CH 2 0) n Z 2 , where R 7 is H, Z ⁇ n and Z 2 are as defined above; R 10 is N(R 7 )R 8 , where R 7 and R 8 are both C 1-12 alkyl substituted with one or more OH groups.
• U is -C(0)N(R 7 )Z 1 Q 1 Q 2 Z 2 -, where R 7 is H, Z 1 , Q 1 , Q 2 and Z 2 are as defined above; R 10 is N(R 7 )R 8 , where R 7 and R 8 are both C 1-12 alkyl substituted with one or more OH groups.
• R 7 and R 8 are as defined for general formula (I);
• U is as defined for general formula (I) and is linked to H via an amine of a Z 1 moiety or a ring nitrogen atom of a Q 1 moiety; may be prepared from a protected compound of general formula (XXXI):
• R 7 and R 8 are as defined for general formula (I)
• U is as defined for general formula (XXX)
• R 30 is an amine protecting group such as fluorenylmethyloxycarbonyl (Fmoc), butyloxycarbonyl (Boc) or benzyloxycarbonyl (Cbz).
• Deprotection may be by hydrogenation for compounds where R 30 is Cbz or by reaction with an acid such as hydrochloric or hydrobromic acid in the case where R 30 is a protecting group such as Boc or Cbz or with a weak base such as morpholine or piperidine when R 30 is a protecting group such as Fmoc.
• R 7 and/or R 8 is a group -CH 2 [CH(0H)] 4 CH 2 0H, this may be protected in the compound of general formula (XXXI), for example as a benzylidene acetal.
• An amine of general formula (XXXI) in which R 7 and/or R 8 is a group -CH2[CH(0H)]4CH20H, protected as a benzylidene acetal may be reacted with a compound of general formula (VII) or (VIII).
• the protecting group R 23 is an acid labile group such as Boc
• the protecting group R 23 and the benzylidene acetal protecting groups can be removed simultaneously using an acid.
• a two step deprotection process may be used wherein the R 23 group is removed by hydrogenation and the benzylidene acetal is subsequently removed by treatment with an acid.
• the protecting group R 23 is a group such as Fmoc it may be removed by treatment with a base as described above. Treatment with an acid will then be required to remove the benzylidene acetal protection.
• the compounds of general formula (I) are ENaC blockers and are therefore useful in the treatment or prevention of respiratory diseases and conditions.
• a compound of general formula (I) for use in medicine is provided.
• the compound of general formula (I) is for use in the treatment or prophylaxis of a disease or condition mediated by ENaC.
• the invention further 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 skin conditions and 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 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.
• cystic fibrosis chronic obstructive pulmonary disease (COPD)
• COPD chronic obstructive pulmonary disease
• chronic bronchitis chronic bronchitis
• emphysema emphysema
• bronchiectasis including non-cystic fibrosis bronchiectasis
• asthma and primary ciliary dyskinesia include cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, including non-
• Skin conditions which may be treated by the compounds of the present invention include psoriasis, atopic dermatitis and ichthyosis.
• Ocular conditions which may be treated by the compounds of the present invention included dry eye disease.
• Compounds of the present invention have good ENaC blocking activity. They are particularly suitable for treating respiratory diseases because they have a prolonged retention time in the lungs.
• the patient to be treated is suitably a mammal and more suitably a human.
• the compounds of general formula (I) may be administered in a pharmaceutical composition and therefore in a further aspect of the invention there is provided a pharmaceutical composition comprising a compound of general formula (I) and a pharmaceutically acceptable excipient.
• Other pharmacologically active materials may also be present, as considered appropriate or advisable for the disease or condition being treated or prevented.
• each of the carriers must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
• formulations include those suitable 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.
• compositions for oral, nasal, bronchial or topical administration.
• the composition may be prepared by bringing into association the above defined active agent with the carrier.
• 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.
• a compound of formula (I) is administered topically to the lung, eye or skin.
• a pharmaceutical composition comprising a compound of the general formula (I) optionally in combination with one or more topically acceptable diluents or carriers.
• 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. 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 p .
• 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 TritonX114 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.
• Other compounds may also be added to the ophthalmic compositions of the compound of general formula (I) to increase the viscosity of the carrier.
• 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.
• 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 mediated by ENaC 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 mediated by ENaC and especially a respiratory disease or condition, for example one of the diseases and conditions mentioned above.
• additional active agents which may be included in a pharmaceutical composition or a combined preparation with the compounds of general formula (I) include:
• b2 adrenoreceptor agonists such as metaproterenol, isoproterenol, isoprenaline, albuterol, salbutamol, formoterol, salmeterol, indacaterol, terbutaline, orciprenaline, bitolterol mesylate and pirbuterol;
• antihistamines for example histamine Hi receptor antagonists such as loratadine, cetirizine, desloratadine, levocetirizine, fexofenadine, astemizole, azelastine and chlorpheniramine or HU receptor antagonists;
• 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
• CFTR repair therapies e.g. CFTR potentiators such as Ivacaftor and CFTR correctors such as Lumacaftor and Tezacaftor;
• TMEM16A modulators particularly TMEM16A potentiators
• System A stationary phase: Kinetex Core-Shell C18 2.1 x 50 mm, 5 pm, 40 °C; detection UV 215 nm-ELS-MS; mobile phase: A, water + 0.1 % formic acid; B, MeCN + 0.1 % formic acid; gradient (A:B ratio, time): 95:5-0:100, 1.20 min; 100:0, 0.10 min; 100:0-5:95, 0.01 min; flowrate: 1.2 ml/min;
• System B stationary phase: Phenomenex Gemini-NX C18 2.0 x 100 mm, 3 pm, 40 °C; detection UV 215 nm-ELS-MS; mobile phase: A, 2 mM ammonium bicarbonate pH 10; B, MeCN; gradient (A:B ratio, time): 95:5-0: 100, 5.5 min; 0:100, 0.4 min; 0: 100-95:5, 0.02 min; flowrate: 0.6 ml/min;
• System D stationary phase: Waters CSH C18 2.1 x 100 mm, 1.7 pm, 40 °C; detection UV 215 nm-ELS-MS; mobile phase: A, 5 mM ammonium acetate pH 7; B, MeCN; gradient (A:B ratio, time): 95:5-0: 100, 5.30 min; 100:0, 0.50 min; 100:0-5:95, 0.02 min; 5:95, 1.18 min; flowrate: 0.6 ml/min;
• System E stationary phase: Waters CSH C18 2.1 x 100 mm, 1.7 pm, 40 °C; detection UV 215 nm-ELS-MS; mobile phase: A, water + 0.1 % formic acid; B, MeCN + 0.1 % formic acid; gradient; gradient (A:B ratio, time): 95:5-0:100, 1.10 min; 100:0, 0.25 min; 100:0-5:95, 0.05 min; 5:95, 0.1 min; flowrate: 0.9 ml/min;
• System F stationary phase: Phenomenex Gemini-NX C18 2 x 50mm 3 pm, 40 °C; detection UV 215 nm-ELS-MS; mobile phase: A, 2mM ammonium bicarbonate pH 10; B, MeCN; gradient (A:B ratio, time): 99: 1-0:100, 1.80 min; 100:0, 0.20 min; 0: 100-99: 1 , 0.01 min; flowrate 1 mL/min
• Phosphorus oxychloride (1.61 ml, 17.3 mmol) was added dropwise over 5 min to a cooled (0 °C) mixture of 2-amino-5-bromo-3-(methoxycarbonyl)pyrazin-1-ium-1-olate, Intermediate 1 (2.15 g, 8.66 mmol) in DMF (20 ml). The resultant mixture was allowed to warm to RT then stirred at RT for 1 h. Additional phosphorus oxychloride (0.80 ml, 8.6 mmol) was added then the reaction was left to stir at RT for 20 min. The resultant mixture was added dropwise to water (150 ml) over 10 min.
• reaction mixture was concentrated in vacuo then the crude material was purified by flash column chromatography on a silica column (100 g). The column was eluted with EtOAc: heptane, increasing the gradient linearly from 0: 100 to 34:76 over 8.5 CV. The desired fractions were combined then evaporated to afford the product as a pale yellow solid (1.55 g, 89%).
• Aqueous HCI solution (4.0 M, 20 ml, 80 mmol) was added then the reaction mixture was stirred at 60 °C for 1.5 h.
• the reaction mixture was allowed to cool to RT then added dropwise over 5 min to saturated aqueous NaHCC> 3 solution (120 ml).
• the resultant mixture was extracted with EtOAc (150 ml).
• the phases were separated then aqueous KF solution (1 M, 100 ml, 100 mmol) was added to the organic layer.
• the mixture was stirred vigorously for 0.5 h then filtered through a Celite® pad (filter material). The pad was rinsed with EtOAc (30 ml) then the combined filtrates were transferred to a separating funnel.
• Aqueous LiOH solution (1.0 M, 360 mI, 0.36 mmol) was added to a mixture of methyl 6- amino-3-methyl-1/-/-pyrazolo[3,4-£>]pyrazine-5-carboxylate, Intermediate 5 (80%, 56 mg, 0.22 mmol) in MeOH (1 ml). The resulting mixture was stirred at 50 °C for 6 h then stirred at RT for 16 h. Additional LiOH solution (1.0 M, 30 mI, 0.030 mmol) was added then the reaction was heated at 50 °C for 4 h. Additional LiOH solution (1.0 M, 50 mI, 0.050 mmol) was added then the reaction was heated at 50 °C for 2 h.
• Aqueous LiOH solution (1.0 M, 2.7 ml, 2.7 mmol) was added to a mixture of methyl 6- amino-3-methyl-1/-/-pyrazolo[3,4-6]pyrazine-5-carboxylate, Intermediate 5 (80%, 310 mg, 1.20 mmol) in MeOH (2.5 ml). The resultant mixture was stirred at 50 °C for 1.5 h then concentrated in vacuo to afford a brown/orange solid (335 mg). A sample (165 mg) of the residue thus obtained was treated with CH2CI2 (4 ml) and TFA (0.5 ml). The resultant mixture was stirred at RT for 20 min then concentrated in vacuo. The residue thus obtained was suspended in water (4 ml) with sonication.
• CDI (95 mg, 0.58 mmol) was added to a solution of 6-amino-3-methyl-1/-/-pyrazolo[3,4- b]pyrazine-5-carboxylic acid; trifluoroacetic acid, Intermediate 7 (91 %, 149 mg, 0.441 mmol) in DMF (2 ml). The resulting mixture was stirred at RT for 0.5 h. CH2CI2 (4 ml) was added then the reaction was left to stand for 20 min. The solid was collected by filtration, rinsed with CH2CI2, then dried in vacuo to afford the product as a yellow solid (74 mg, 63%).
• CDI (104 mg, 0.640 mmol) was added to a solution of 6-amino-1 /-/-pyrazolo[3,4- b]pyrazine-5-carboxylic acid; trifluoroacetic acid, Intermediate 11 (125 mg, 0.426 mmol) in DMF (2 ml). The reaction mixture was left to stir at RT for 3 h. Additional CDI (69 mg, 0.43 mmol) was added then the reaction was stirred at RT for a further 1 h. The reaction mixture was concentrated to ⁇ 1 ml under a stream of nitrogen then diluted with CH2CI2 (2 ml).
• lodoethane (715 pi, 8.95 mmol) was added to a suspension of 2-[(1-ethyl-6-methoxy-1 /-/- 1 ,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1 /-/-isoindole-1 , 3-dione, Intermediate 13 (3.00 g, 8.95 mmol) in MeCN (20 ml). The mixture was heated at 80 °C for 4 h. lodoethane (715 mI, 8.95 mmol) was added and mixture was stirred at 80 °C for 16 h.
• lodoethane (715 mI, 8.95 mmol) was added and mixture was stirred at 80 °C for a further 24 h the allowed to cool to RT. The mixture was concentrated in vacuo to approximately one third of the original volume. The precipitate was collected by filtration then washed with MeCN to afford a grey solid (2.6 g). The filtrate was concentrated to afford a dark grey solid. The two batches of solid thus obtained were combined and suspended in MeCN (20 ml) lodoethane (715 mI, 8.95 mmol) was added then the reaction mixture was stirred at 80 °C for 18 h then at 100 °C for 4 h.
• the reaction mixture was split into two equal portions in pressure tubes lodoethane (300 mI, 3.75 mmol) was added to both reaction mixtures then the pressure tubes were sealed and left to heat at 100 °C for 16 h.
• the reaction mixtures were allowed to cool to RT then combined.
• the resultant mixture was concentrated in vacuo to ⁇ 5 ml then filtered.
• the collected solid was washed with the minimum of MeCN (0.5 ml) to yield the product as a grey solid (2.37 g).
• the filtrate was concentrated under reduced pressure to afford a dark brown solid, which was triturated with EtOAc ( ⁇ 10 ml) and filtered. The filtrate was left to stand for 16 h then it was filtered again.
• the aqueous phase was extracted with EtOAc (2 x 150 ml), then the combined organic phases were washed with water (4 x 100 ml) and brine (50 ml) then dried over Na 2 S0 4 , filtered and evaporated to afford the crude intermediate as a black oil (18 g).
• the oil thus obtained was dissolved in acetic acid (80 ml) and stirred at 70 °C for 1 h. The reaction was allowed to cool to RT then evaporated to afford a brown solid.
• the solid was suspended in EtOAc (200 ml) then filtered and was washed with EtOAc, then dried under vacuum to afford a pale pink solid (6.5 g). The solid thus obtained was suspended in EtOAc (200 ml).
• the filtrate was transferred to a separating funnel then extracted with saturated aqueous NaHCC>3 solution (3 x 100 ml), water (100 ml) and brine (50 ml) then dried over Na 2 S0 4 , filtered and evaporated to a yellow solid which was suspended in the minimum volume of EtOAc:heptane (1 :4) and filtered then dried under vacuum to afford a third batch of the product as a white solid (1.77 g).
• the filtrate from the first filtration was transferred to a separating funnel then extracted with saturated aqueous NaHCCh solution (3 x 100 ml), water (100 ml) and brine (50 ml) then dried over Na 2 S0 4 , filtered and evaporated to a dark brown solid.
• Aqueous LiOH solution (2.0 M, 16 ml, 32 mmol) was added to a suspension of methyl 2- ( ⁇ [(te/f-butoxy)carbonyl]ami no ⁇ methyl)- 1 -ethyl- 1 H- 1 , 3-benzodiazole-6-carboxylate, Intermediate 16 (6.91 g, 20.7 mmol) in THF (100 ml).
• the reaction mixture was stirred at 50 °C for 16 h then allowed to cool to RT.
• the reaction mixture was concentrated in vacuo then the resulting solid was suspended in water (50 ml).
• Aqueous HCI solution (2 M) was added dropwise until pH 4 was reached.
• the resultant suspension was filtered then the solid was washed with the minimum of water and MeCN then dried under vacuum to afford the product as a white solid (6.05 g, 90%).
• reaction mixture was allowed to cool to RT then 1 , 1-di-te/f-butoxy-A/,A/-dimethylmethanamine (6.77 ml, 28.3 mmol) was added dropwise over 15 min. The resultant mixture was heated at 100 °C for 45 min. The reaction mixture was cooled to 50 °C then 1 , 1-di-te/f-butoxy-A/,A/-dimethylmethanamine (3.38 ml, 14.2 mmol) was added dropwise over 5 min. The resultant mixture was heated at 100°C for 0.5 h then allowed to cool to RT. The reaction mixture was partitioned between EtOAc (50 ml) and water (50 ml).
• Reaction 1 a suspension of tert- butyl 2-( ⁇ [(benzyloxy)carbonyl]amino ⁇ methyl)-1 -ethyl-1 /-/-1 , 3- benzodiazole-6-carboxylate, Intermediate 20 (800 mg, 1.95 mmol) and iodoethane (629 pi, 7.82 mmol) in MeCN (10 ml) was heated under microwave irradiation for 2 h at 120 °C. The reaction was retreated with iodoethane (629 mI, 7.82 mmol) then the reaction mixture was heated under microwave irradiation for a further 2 h at 120 °C.
• Reaction 2 a suspension of tert- butyl 2-( ⁇ [(benzyloxy)carbonyl]amino ⁇ methyl)-1 -ethyl-1 /-/-1 , 3- benzodiazole-6-carboxylate, Intermediate 20 (800 mg, 1.95 mmol) and iodoethane (629 mI, 7.82 mmol) in MeCN (10 ml) was heated under microwave irradiation for 1 h 45 min at 120 °C. The reaction was retreated with iodoethane (629 mI, 7.82 mmol) then the reaction mixture was heated under microwave irradiation for a further 1.5 h at 120 °C.
• Reaction 3 a suspension of tert- butyl 2-( ⁇ [(benzyloxy)carbonyl]amino ⁇ methyl)-1 -ethyl-1 H-1 , 3- benzodiazole-6-carboxylate, Intermediate 20 (700 mg, 1.71 mmol) and iodoethane (591 mI, 6.84 mmol) in MeCN (10 ml) was heated under microwave irradiation for 1.5 h at 120 °C. The reaction was retreated with iodoethane (629 mI, 7.82 mmol) then the reaction mixture was heated under microwave irradiation for a further 1.5 h at 120 °C. The three reactions were combined and concentrated in vacuo.
• the crude material was purified by flash column chromatography on C18 (60 g).
• the column was eluted with MeCN:water + 0.1 % formic acid using the following gradient (%MeCN, column volumes): 5%, 2 CV; 5- 31 %, 5 CV; 31 %, 4 CV; 31-59%, 6 CV; 59-100%, 3 CV; 100% 1 CV.
• the desired fractions were combined and concentrated in vacuo to afford the product as a white foam (2.13 g, 67%).
• the reaction was recharged with 4,6-O-benzylidene-D-glucopyranose (23.3 g, 86.9 mmol) then left to stir at 60 °C for a further 6 h.
• the reaction was allowed to cool to RT then added to saturated aqueous NaHCC> 3 solution (200 ml) and EtOAc (200 ml).
• the resultant mixture was filtered through a Celite® pad then the filtrate was transferred to a separating funnel.
• the phases were separated then the organic phase was washed with brine:water (1 : 1 , 2 c 200 ml), brine (100 ml), dried over Na 2 S0 4 , filtered and concentrated in vacuo.
• the resultant suspension was stirred at RT for 15 min then filtered.
• the filtrate was concentrated in vacuo until solid was observed.
• the resultant suspension was stirred at RT for 15 min then filtered.
• the filtrate was purified by flash column chromatography on C18 (400 g).
• the column was eluted with MeCN:H 2 0 + 0.1 % formic acid using the following gradient (% MeCN, column volumes): 10%, 1 CVs; 10-25%, 6 CVs; 25%, 2 CVs; 25-50%, 1 CV; 50-100%, 1 CVs; 100%, 2 CVs.
• the desired fractions were combined and concentrated in vacuo to remove the majority of the solvent.
• the residual solution thus obtained was lyophilised to afford a pale-yellow solid (6.35g).
• the aqueous phase was extracted with EtOAc (50 ml) then the combined organic phases were washed with water (4 x 50 ml) and brine (50 ml), then dried over Na 2 S0 4 , filtered and evaporated to a black solid (4 g).
• the solid was dissolved in the minimum of CH 2 CI 2 /MeOH then evaporated onto silica (9 g).
• the crude material was purified by flash column chromatography on a silica column (120 g). The column was eluted with ChhCh MeOH, increasing the gradient linearly from 100:0 to 95:5 over 10 column volumes. The desired fractions were combined and evaporated to a black solid (2.9 g).
• the solid thus obtained was dissolved in EtOAc (100 ml) and extracted with saturated aqueous sodium bicarbonate solution (3 x 50 ml) and water (50 ml) then dried over Na 2 S0 4 , filtered and evaporated to a black soli d (2.5 g).
• the solid was dissolved in the minimum of ChhCh/MeOH then evaporated onto silica (10 g).
• the material was further purified by flash column chromatography on a silica column (120 g). The column was eluted with EtOAc: heptane, increasing the gradient linearly from 0:100 to 100:0 over 10 column volumes. The desired fractions were combined and evaporated to afford the product as a pink solid (1.78 g, 62%).
• lodomethane (497 pi, 7.98 mmol) was added to a suspension of tert- butyl A/-[(1 -ethyl-6- fluoro-1 /-/-1 ,3-benzodiazol-2-yl)methyl]carbamate, Intermediate 26 (780 mg, 2.66 mmol) in MeCN (12 ml) in a pressure tube. The tube was sealed then heated at 75 °C for 4 h. The reaction mixture was allowed to cool to RT then concentrated in vacuo to afford the product as a pale yellow solid (1.16 g, 99%).
• the solid was filtered, washed with water and dried under vacuum.
• the solid thus obtained was triturated in MeCN then filtered and dried under vacuum to afford the intermediate as a pale pink solid (5.4 g).
• the solid thus obtained was added portion-wise to acetic acid (30 ml).
• the resulting suspension was heated at 100 °C for 45 min then allowed to cool to RT over 16 h.
• the resulting suspension was filtered and washed with EtOAc then dried under vacuum to afford the product as a pale pink solid (585 mg).
• the solid was suspended in MeCN (5 ml) then MeCN:water (1 : 1 , 1 ml) was added.
• the resulting suspension was filtered then the solid was dried under vacuum to afford the product as a pale pink solid (430 mg, 13%).
• lodomethane (590 pi, 9.47 mmol) was added to a suspension of 2-[(1-ethyl-6-methoxy- 1 H-1 ,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1 /-/-isoindole-1 ,3-dione, Intermediate 13
• lodomethane (381 mI, 6.12 mmol) was added to a suspension of tert- butyl A/- ⁇ [1-ethyl-6- (trifluoromethyl)-1 /-/-1 ,3-benzodiazol-2-yl]methyl ⁇ carbamate, Intermediate 34 (700 mg, 2.04 mmol) in MeCN (10 ml) in a pressure tube. The tube was sealed and heated at 75 °C for 8 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo to afford the product as a pale yellow solid (1.01 g, >99%).
• the aqueous phase was extracted with EtOAc (2 x 50 ml) then the combined organic phases were washed with water (4 x 50 ml) and brine (50 ml) then dried over Na 2 S0 4 , filtered and evaporated to a black oil.
• the oil thus obtained was dissolved in acetic acid (10 ml) and the resulting solution was heated at 70 °C for 1.5 h.
• the reaction solution was concentrated in vacuo then the residue was partitioned between EtOAc (50 ml) and saturated aqueous NaHCOs solution (50 ml).
• lodomethane (208 mI, 3.34 mmol) was added to a suspension of tert- butyl L/- ⁇ [1 -ethyl-6- (trifluoromethoxy)-1 /-/-1 ,3-benzodiazol-2-yl]methyl ⁇ carbamate, Intermediate 38 (92%, 400 mg, 1.02 mmol) in MeCN (5 ml) in a pressure tube. The tube was sealed then heated at 75 °C for 5 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo to yield the product as a dark green solid (552 mg, >99%).
• HATU (8.53 g, 22.3 mmol) and DIPEA (7.1 ml, 41 mmol) were added to a solution of N- (te/f-butoxycarbonyl)glycine (3.93 g, 22.4 mmol) in DMF (40 ml).
• DMF 40 ml
• the resulting solution was stirred at RT for 0.5 h then a solution of 1-/ ⁇ /-ethyl-5-methoxybenzene-1 , 2-diamine (3.39 g, 20.4 mmol) in THF (20 ml) was added.
• the reaction was left to stir at RT for 80 min then the reaction mixture was diluted with EtOAc (200 ml) and water (150 ml).
• the phases were separated and the organic phase was washed with water (2 x 150 ml) and brine (150 ml), then dried over Na 2 S0 4 and concentrated in vacuo to afford the crude product as a dark red oil.
• the crude material was purified by flash column chromatography on a silica column (340 g). The column was eluted with CHaC MeOH, increasing the gradient linearly from 100:0 to 92:8 over 10 column volumes. The desired fractions were combined and evaporated to a viscous dark red oil which solidified on standing to yield the product as a dark red solid (5.02 g, 77%).
• Methyl bromoacetate (395 mI, 4.18 mmol) was added to a suspension of tert- butyl N-[ ⁇ 1- ethyl-6-methoxy-1 /-/-1 ,3-benzodiazol-2-yl)methyl]carbamate, Intermediate 45 (96%, 500 mg, 1.39 mmol) in MeCN (4 ml) in a pressure tube. The tube was sealed and the resulting mixture was stirred at 75 °C for 4 h then allowed to cool to RT. The resultant suspension was filtered then the solid was washed with cold MeCN and dried under vacuum to afford the product as a white solid (454 mg, 71 %).
• the crude material was purified by flash column chromatography on a silica column (50 g).
• the column was eluted with CHaCL MeOH, using the following gradient (%MeOH, column volumes): 0%, 1 CV; 0-9%, 8 CV; 9%, 3 CV; 9-14%, 2 CV, 14%, 1 CV; 14-20%, 2 CV.
• the desired fractions were combined and evaporated to afford the product as a pale magenta foam (638 mg, 84%).
• the column was eluted with ChhCh MeOH, increasing the gradient linearly from 100:0 to 90: 10 over 10 column volumes.
• the desired fractions were combined and evaporated to yield a viscous dark red oil (615 mg).
• the material was further purified by flash column chromatography on C18 (30 g).
• the column was eluted with MeCN:water + 0.1 % formic acid using the following gradient (%MeCN, column volumes): 10%, 2 CV; 10 - 36%; 36%, 2 CV; 36 - 50%, 4 CV; 50 - 100%, 3 CV; 100%, 3 CV.
• the desired fractions were combined and evaporated to yield the product as a viscous dark red oil (278 mg, 43%).
• the column was eluted with MeCN:water + 0.1 % formic acid using the following gradient (%MeCN, column volumes): 10%, 2 CV; 10 - 14%, 2 CV; 14 - 18%, 1 CV; 18 - 27%, 2 CV; 27 - 31 %, 0.5 CV; 31 - 60%, 0.5 CV; 60 - 100%, 1 CV; 100%, 1 CV.
• the desired fractions were combined and evaporated to yield the product as a yellow amorphous solid (1.72 g, 47%).
• Benzyl bromide (88 pi, 0.74 mmol) was added to a suspension of 2-[(6-chloro-1 -ethyl- 1 /-/- 1 ,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1 /-/-isoindole-1 , 3-dione, Intermediate 20 (0.25 g, 0.74 mmol) in MeCN (5 ml) in a pressure tube. The tube was sealed then the reaction was stirred at 80 °C for 16 h then allowed to cool to RT. Benzyl bromide (88 mI, 0.74 mmol) was added then the reaction was stirred at 80 °C for a further 6 h.
• Benzyl bromide (1.29 ml, 10.8 mmol) was added to a mixture of tert- butyl L/-(1 /-/-1 ,3- benzodiazol-2-ylmethyl)carbamate (85%, 3.15 g, 10.8 mmol) and K 2 CO 3 (2.25 g, 16.3 mmol) in DMF (25 ml). The resulting mixture was stirred at RT for 16 h. The reaction mixture was partitioned between water (150 ml) and EtOAc (150 ml). The phases were separated then the aqueous phase was extracted with EtOAc (5 x 150 ml). The combined organics were dried over Na 2 S0 4 then concentrated in vacuo to approximately 50 ml. The resultant slurry was recrystallized from the minimum volume of refluxing EtOAc to afford the product as white solid (2.22 g, 61 %).
• the column was eluted with MeCN:water + 0.1 % formic acid using the following gradient (%MeCN, column volumes): 10-45%, 12 CV; 45%, 2 CV; 45-53%, 3 CV; 53-100%, 3 CV; 100%, 1 CV.
• the desired fractions were combined and evaporated to afford the product as a pale orange foam (209 mg, 53%).
• Morpholine (3.46 ml, 40.0 mmol) was added to a solution of 6-(2 - ⁇ [ ⁇ tert- butoxy)carbonyl]amino ⁇ ethoxy)-1 ,3-diethyl-2-( ⁇ [(9/-/-fluoren-9- ylmethoxy)carbonyl]amino ⁇ methyl)-1 /-/-1 ,3-benzodiazol-3-ium iodide, Intermediate 64 (95%, 3.00 g, 4.00 mmol) in THF (50 ml). The reaction mixture was stirred at RT for 25 min then diluted with diethyl ether (150 ml). The resulting mixture was agitated then the supernatant was decanted off. The residual gum was washed further with ether (2 x 60 ml) then dried under vacuum. The residue was dissolved in THF then concentrated in vacuo afford the product as a pale orange solid (1.99 g, 83%).
• the filter pad was rinsed with EtOAc then the combined filtrate was extracted with water (2 x 150 ml) and brine (150 ml) then dried over Na 2 S0 4 and concentrated in vacuo to afford the product as a viscous yellow/orange oil (4.69 g, 93%).
• the crude material was purified by flash column chromatography on a silica column (340 g).
• the column was eluted with CH 2 CI 2 :MeOH, using the following gradient (%MeOH, column volumes): 0%, 1 CV; 0-3.3%, 7 CV; 3.3%, 1 CV; 3.3-4.5%, 2 CV.
• the desired fractions were combined and evaporated to afford the product as a pale orange solid (4.73 g, 53%).
• the column was eluted with CH 2 CI 2 :MeOH, using the following gradient (%MeOH, column volumes): 0%, 1 CV; 0-3.4%, 7 CV; 3.4-4.3%, 2 CV, 4.3-6.0%, 2 CV.
• the desired fractions were combined and evaporated to afford the product as a pale orange foam (1.03 g, 61 %).
• Morpholine (1.10 ml, 12.7 mmol) was added to a solution of 6-(3 - ⁇ [ ⁇ tert- butoxy)carbonyl]amino ⁇ propoxy)-1 ,3-diethyl-2-( ⁇ [(9/-/-fluoren-9- ylmethoxy)carbonyl]amino ⁇ methyl)-1 /-/-1 ,3-benzodiazol-3-ium iodide, Intermediate 69 (90%, 1.03 g, 1.27 mmol) in THF (10 ml). The reaction mixture was stirred at RT for 2 h then diluted with diethyl ether (40 ml). The mixture was agitated then the supernatant was decanted off. The procedure was repeated with further diethyl ether washes (2 x 20 ml). The residue thus obtained was dried under vacuum to afford the product as a pale orange foam (700 mg, 99%).
• the resulting precipitate was collected by filtration then washed with water and dried under vacuum to afford the intermediate as a light grey solid.
• the solid thus obtained was added portionwise to acetic acid (150 ml).
• the resulting suspension was heated at 100 °C for 2.5 h then allowed to cool to RT.
• the reaction mixture was concentrated in vacuo then the residue was partitioned between EtOAc (300 ml) and water (300 ml).
• the resulting precipitate was collected by filtration and washed with EtOAc (200 ml) and water (200 ml) then dried under vacuum to afford the product as a pink solid (17.9 g, 76%).
• the reaction was recharged with palladium on carbon (10 wt%, 278 mg) and stirred at RT under a hydrogen atmosphere for a further 24 h.
• the reaction was re-charged with palladium on carbon (10 wt%, 278 g) and stirred at RT under a hydrogen atmosphere for a further 24 h.
• the reaction mixture was filtered through a Celite pad.
• the Celite pad was rinsed with EtOH (100 ml), MeOH (100 ml), EtOAc (100 ml), and DMF (5 ml).
• the combined filtrate was concentrated in vacuo then the crude material was purified by flash column chromatography on a silica column (25 g).
• lodoethane (1.04 ml, 13.0 mmol) was added to a solution of tert- butyl A/-(3- ⁇ 2-[(1 ,3-dioxo- 2, 3-dihydro-1 /-/-isoindol-2-yl)methyl]-1 -ethyl-1 /-/-1 ,3-benzodiazol-6-yl ⁇ propyl)carbamate, Intermediate 73 (86%, 1.20 g, 2.23 mmol) in MeCN (18 ml) in a pressure tube. The tube was sealed and heated at 1 10 °C for 4 h.
• the column was eluted with EtOAc: heptane, using the following gradient (%EtOAc, column volumes): 0%, 1 CV; 0-50%, 7 CV; 50-79%, 3 CV; 79%, 2 CV; 79-92%, 2 CV, 100%, 2 CV.
• the desired fractions were combined and concentrated in vacuo to afford the product as a white solid (670 mg, 50%).
• reaction was recharged with palladium on carbon (10 wt%, 140 mg) and stirred at RT under a hydrogen atmosphere for a further 48 h.
• the reaction mixture was filtered through Celite pad then concentrated in vacuo to afford the product as a colourless oil (635 mg, 94%).
• CS 2 CO 3 (848 mg, 2.60 mmol) was added then the resulting suspension was de-gassed by bubbling a stream of nitrogen through the reaction mixture for 10 min.
• XPhos-Pd-G2 (61 mg, 0.078 mmol) was added then the nitrogen bubbling was continued for a further 5 min.
• the tube was sealed then heated at 100 °C for 16 h.
• the reaction mixture was concentrated in vacuo then partitioned between EtOAc (100 ml) and water (100 ml). The phases were separated then the organic phase was washed with water (50 ml) and brine (2 x 50 ml), then dried over MgSCL, filtered and evaporated.
• the crude material was purified by flash column chromatography on a silica column (50 g).
• the column was eluted with EtOAc: heptane, increasing the gradient linearly from 0: 100 to 100:0 over 10 column volumes.
• the desired fractions were combined and evaporated to yield the product as a light yellow foam (226 mg, 31 %).
• lodoethane (159 mI, 1.98 mmol) was added to a solution of tert- butyl 4-(4- ⁇ 2-[(1 ,3-dioxo- 2, 3-dihydro-1 /-/-isoindol-2-yl)methyl]-1 -ethyl-1 /-/-1 ,3-benzodiazol-6-yl ⁇ -1 /-/-pyrazol-1- yl)piperidine-1-carboxylate, Intermediate 80 (220 mg, 0.397 mmol) in MeCN (5 ml). The reaction mixture was heated under microwave irradiation for 2 h at 120 °C.
• Morpholine (4.77 ml, 55.1 mmol) was added to a stirred mixture of 5-(2- ⁇ bis[(2S,3 )-2,3- dihydroxy-3-[(4 ,5 )-5-hydroxy-2-phenyl-1 ,3-dioxan-4-yl]propyl]amino ⁇ ethoxy)-1 ,3- diethyl-2-[( ⁇ [(9/-/-fluoren-9-yl)methoxy]carbonyl ⁇ amino)methyl]-1 /-/-1 ,3-benzodiazol-3-ium chloride, Intermediate 84 (84% 6.42 g, 5.26 mmol) in THF (60 ml). The resulting mixture was stirred at RT for 4 h.
• Morpholine (659 pi, 7.62 mmol) was added to a mixture of 5-(3- ⁇ bis[(2S,3 )-2,3-dihydroxy- 3-[(4 ,5 )-5-hydroxy-2-phenyl-1 ,3-dioxan-4-yl]propyl]amino ⁇ propoxy)-1 ,3-diethyl-2- ( ⁇ [(9/-/-fluoren-9-ylmethoxy)carbonyl]amino ⁇ methyl)-1 /-/-1 ,3-benzodiazol-3-ium chloride hydrochloride, Intermediate 88 (60%, 1.32 g, 0.762 mmol) in THF (10 ml). The mixture was stirred at RT for 2.5 h.
• Step 1 A suspension of hydrazine hydrate (1.27 ml, 1.31 mmol) and tert- butyl 4- ⁇ 2-[(1 ,3- dioxo-2,3-dihydro-1 /-/-isoindol-2-yl)methyl]-1 -ethyl-1 /-/-1 ,3-benzodiazol-6-yl ⁇ piperidine-1- carboxylate
• Intermediate 77 (2.56 g, 5.24 mmol) in MeOH (30 ml) was heated under reflux for 2.5 h then allowed to cool to RT. The resultant suspension was left to stand at RT for 16 h then filtered.
• Step 2 A solution of (2,5-dioxopyrrolidin-1-yl) 9/-/-fluoren-9-ylmethyl carbonate (1.16 g, 3.43 mmol) in MeCN (20 ml) was added dropwise over 10 min to a mixture of the intermediate from Step 1 and NaHCC>3 (576 mg, 6.86 mmol) in MeCN (40 ml) and water (30 ml). The reaction mixture was stirred at RT for 21 h then partitioned between EtOAc (100 ml) and water (100 ml).
• the column was eluted with ChhCh MeOH, using the following gradient (%MeOH, column volumes): 0%, 1 CV; 0-2.3%, 5 CV; 2.3%, 2 CV; 2.3-10%, 7 CV, 10%, 7 CV.
• the desired fractions were combined and evaporated to afford the product as a light pink foam (1.60 g, 75%).

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