EA046372B1 - 2-FLUORINATED BALL ACIDS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES - Google Patents

Description

The present invention relates to new compounds that are used in the treatment of neurodegenerative disorders. In particular, the invention relates to bile acid derivatives, pharmaceutical compositions containing them, methods for their preparation and the use of these compounds in the treatment or prevention of neurodegenerative disorders.

Neurodegenerative diseases are a group of disorders of the central nervous system and include Parkinson's disease, mild cognitive impairment, dementia (including Alzheimer's disease, vascular dementia and dementia with Lewy bodies), Huntington's disease and amyotrophic lateral sclerosis (motor neuron disease). The incidence of neurodegenerative diseases increases with age, and such conditions are therefore a growing problem in societies where the average age of the population is increasing. There is currently no cure for any of these diseases, although there are some medications that improve the symptoms of Parkinson's disease and some types of cognitive impairment and dementia.

Symptoms of Parkinson's disease are resting tremor, bradykinesia, and rigidity, and these symptoms are caused by neurodegeneration and loss of dopaminergic neurons. There is ample evidence indicating that there is a strong link between mitochondrial dysfunction and Parkinson's disease. A mild deficiency of NADH dehydrogenase (complex I) activity in the mitochondrial electron transport chain has been found in tissues from patients with Parkinson's disease, and a number of proteins associated with familial Parkinson's disease are either mitochondrial proteins or associated with mitochondria.

Alzheimer's disease results in progressive cognitive impairment and is characterized by the presence of extracellular neuritic plaques and intracellular neurofibrillary tangles. Mitochondrial dysfunction is thought to lead to the deposition of β-amyloid proteins, which are a major component of neuritic plaques, and to the formation of neurofibrillary tangles.

Huntington's disease is an inherited, progressive neurodegenerative disorder characterized by movement disorders, personality changes, and cognitive decline. The pathology of Huntington's disease suggests an association with mitochondrial dysfunction.

Amyotrophic lateral sclerosis is also thought to be associated with mitochondrial dysfunction. This disease affects the motor neurons of the central nervous system, resulting in muscle weakness, wasting, and death within 2 to 3 years of diagnosis.

Attempts have been made to discover compounds that can treat neurodegenerative disorders, and several compounds have been developed that target mitochondria. For example, bile acids such as UDCA (ursodeoxycholic acid) are known to have beneficial effects on mitochondrial dysfunction in tissues of some Parkinson's disease patients, particularly in tissue from Parkinson's disease patients with a mutant parkin gene (Mortiboys et al ., Ursocholanic acid rescues mitochondrial function in common forms of familial Parkinson's disease, Brain, 136(10), 3038-3050 (2013)) and patients with Parkinson's disease with the LRRK2 ^G2019S mutation (Mortiboys et al., Neurology, 85, 846- 852 (2015)). In addition, bile acids such as UDCA are known to have beneficial effects on fibroblasts from patients suffering from both sporadic Alzheimer's disease and familial Alzheimer's disease due to mutations in the PSEN1 (presenilin) gene (Bell et al., Ursodeoxycholic Acid Improves Mitochondrial Function and Redistributes Drp1 in Fibroblasts from Patients with either Sporadic or Familial Alzheimer's Disease. Journal of Molecular Biology, pii: S0022-2836(18)30987-2. 2018).

WO 2014/036379, WO 2015/061421 and WO 2016/145216 report that bile acids can be used in the treatment of neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis. WO 2015/061421 relates to deuterated bile acids and WO 2016/145216 to fluorinated bile acids, especially bile acids fluorinated at the 3- and/or 7-positions.

The inventors of the present invention, in this state of affairs, have discovered that certain fluorinated bile acids have excellent mitochondrial repair properties and are particularly effective in the treatment of neurodegenerative disorders.

The present invention provides a compound of general formula (I):

where one of R ¹ and R ² represents F, and the other of R ¹ and R ² represents H or F;

Y represents a bond or a ^-^alkylene, C _2-20 alkenylene or C _2-20 alkynylene linker group;

R ³ represents C(O)OR ¹² , C(O)NR ¹² R ¹³ , S(O)2R ¹² , OS(O)2R ¹² , S(O)2OR ¹² , OS(O)2OR ¹² ,

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S(O)2NR ¹² R ¹³ , C(O)NR ¹² S(O)2R ¹³ , NHC(O)NR ¹² S(O)2R ¹³ , OP(O)(OR ¹² )2, C(O)NR ¹² [CH(R ¹⁵ )]nR ¹⁶ or C(O)NR ^{1 2} C(O)CH ₂ NR|CH(R' ⁵ )]nR1 ⁶ ;

each R ¹² independently represents H or C _1-6 alkyl, optionally substituted with one or more substituents selected from halogen, OR ¹⁰ , NR ¹⁰ R ¹¹ , R ¹⁶ and aryl;

R ¹⁰ and R ¹¹ are each independently H or C^alkyl;

R ¹³ represents H, C _1-6 alkyl, optionally substituted with one or more substituents selected from halogen and aryl; or a 3-8 membered carbocyclic ring or heterocyclic ring, wherein said carbocyclic or heterocyclic ring is optionally substituted with one or more substituents selected from =O and R ¹⁶ ; or phenyl or a 5-6 membered heteroaryl ring, wherein said phenyl or heteroaryl ring is optionally substituted with an R ¹⁶ substituent; or when R ³ represents C(O)NR ¹² R ¹³ or S(O)2NR ¹² R ¹³ , R ¹² and R ¹³ together with the nitrogen atom to which they are attached form a 3-8 membered heterocyclic ring, which may contains one or more additional heteroatoms selected from N, O and S; and optionally substituted with one or more substituents selected from CH2C(O)OH, C(O)OH, C^alkyl, C(O)OC _1-6 -alkyl, S(O) ₂ OH, =O and =N-OH; and optionally fused with a phenyl group that is unsubstituted or substituted with one or more substituents selected from halogen and nitro;

n is 1, 2 or 3;

each R ¹⁵ independently represents H or C^alkyl, optionally substituted with one or more substituents selected from halogen, phenyl and 5- or 6-membered heteroaryl; 3-8 membered cycloalkyl group; or an R ¹⁴ group, where R ¹⁴ represents a side chain of an amino acid; or when n is 2 or 3, two R ¹⁵ groups, together with the carbon atoms to which they are attached, and possibly a carbon atom present between them, can combine to form -(CH2)p, so that the [CH(R ¹⁵ ) group ]n represents a 3-8 membered carbocyclic ring;

p is equal to 1, 2, 3, 4, 5 or 6;

R ¹⁶ is selected from C(O)OH, S(O) ₂ OH, the group S(O) ₂ (C _1-( ^km), OS(O) ₂ OH and P(O)(OH) ₂ ;

or a pharmaceutically acceptable salt thereof or an isotopic modification thereof.

Compounds of general formula (I) are used for the treatment of neurodegenerative disorders of the central nervous system, including Parkinson's disease, dementia and amyotrophic lateral sclerosis.

Detailed Description of the Invention

As used herein, unless the context otherwise requires due to express language or intended meaning, the word contains or variants such as includes or containing are used in an inclusive sense, that is, to indicate the presence of the claimed features, but not to exclude the presence or adding additional features in various embodiments of the invention.

All publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and separately identified for inclusion herein by reference as if set forth in their entirety.

As used herein, the term C-x/alkyl refers to a linear or branched, fully saturated hydrocarbon group having from 1 to 20 carbon atoms. The term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Other alkyl groups, for example C _1-12 alkyl, C _1-10 alkyl, C _1-8 alkyl, C _1-6 alkyl, C _1-5 alkyl, C _1-4 alkyl, C _1-3 alkyl or C _{1- 2} alkyl are as defined above, but contain a different number of carbon atoms.

The term C _2-20 alkenyl refers to a straight or branched hydrocarbon group having from 2 to 20 carbon atoms and at least one carbon-carbon double bond. Examples of alkenyl groups include -CH=CH2, -CH=CH(CH3), -CH2CH=CH2, -CH=CHCH3, -CH2CH2CH=CH2, -CH ₂ CH=CH(CH ₃ )- and -CH ₂ CH=CH (CH ₂ CH ₃ ). Other alkenyl groups, for example C _2-12 alkenyl, C _2-10 alkenyl, C _2-8 alkenyl, C _2-6 alkenyl, C _2-5 alkenyl, C _2-4 alkenyl or C _2-3 alkenyl, are those as defined above, but contain a different number of carbon atoms.

The term C _2-20 alkynyl refers to a straight or branched hydrocarbon group having from 2 to 20 carbon atoms and at least one carbon-carbon triple bond. Examples of alkynyl groups include -CbC'H. -C.'H ₂ CbC.'H. -CbC-C.'H ₃ . -CH ₂ CH ₂ C^CH, -CH ₂ CbCCH ₃ and -CH ₂ CbCCH ₂ CH ₃ . Other alkynyl groups, such as ^-^alkynyl, ^-^alkynyl, ^^alkynyl, ^^alkynyl, ^^alkynyl, ^^alkynyl or ^^alkynyl, are as defined above but contain a different number of carbon atoms.

The term alkylene refers to a straight or branched, fully saturated hydrocarbon chain. Suitable alkylene is C-^ alkylene, C _1-12 alkylene, C-^ alkylene, C _1-8 alkylene, C _1-6 alkylene, C _1-5 alkylene, C ^ alkylene, C _1-3 alkylene or C _{- 2} alkylene. Examples of alkylene groups include -CH2-, -CH2CH2-, -CH(CH ₃ )-CH2-, -CH2CH(CH ₃ )-, -CH2CH2CH2-, -CH2CH(CH2CH ₃ )- and -CH ₂ CH(CH ₂ CH ₃ )CH ₂ -.

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The term alkenylene refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond. Suitably, alkenylene is ^-^alkenylene, ^-^alkenylene, ^-^alkenylene, C.'2-halkenylene, ^^alkenylene, ^-alkenylene, ^^alkenylene or ^^alkenylene. Examples of alkenylene groups include -CH=CH-, -CH=C(CH ₃ )-, -CH2CH=CH-, -CH=CHCH2-, -CH2CH2CH=CH-, -CH2CH=C(CH3)- and -CH2CH= C(CH2CH3)-.

The term alkynylene refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. Accordingly, alkenylene is ^-^al^nylene, ^-^al^nylene, ^-^al^nylene, ^^alkynylene, ^^alkynylene, C'2-Alkynylene. C ₂ - ₄ alkynylene or C ₂ - ₃ alkynylene. Examples of alkynylene groups include -C^C-, -CH ₂ C^C-, -C^C-CH ₂ -, -CH;CH;C C-, -CH;C CCH ₂ - and -CH;CC-CH ;CH;-.

The terms aryl and aromatic refer to a cyclic group with aromatic character having from 6 to 14 ring carbon atoms (unless otherwise noted, for example 6 to 10 ring carbon atoms) and containing up to three rings. If an aryl group contains more than one ring, not all rings need to be aromatic in character. Examples include phenyl, naphthyl and anthracenyl, as well as partially saturated systems such as tetrahydronaphthyl (eg 1,2,3,4tetrahydronaphthyl), indanyl and indenyl.

The terms heteroaryl and heteroaromatic refer to a cyclic group with aromatic character having from 5 to 14 ring atoms (unless otherwise specified, for example from 5 to 10 ring atoms), containing at least one heteroatom selected from N, O and S, and containing up to three rings. If a heteroaryl group contains more than one ring, not all rings need to be aromatic in character. Examples include pyridine, pyrimidine, pyrrole, thiophene, furan, thiazole, oxazole, fused systems such as indole, benzimidazole and benzothiophene; and partially saturated systems such as indoline and dihydrobenzofuran.

The terms carbocyclic and carbocyclyl refer to a non-aromatic hydrocarbon ring system containing from 3 to 10 ring carbon atoms (unless otherwise noted), which may be a fused or bridged ring system and which optionally contains one or more carbon-carbon double bonds. Examples include cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl; and cycloalkenyl groups such as cyclohexenyl and cycloheptenyl; and bridging groups such as adamantyl. More preferably, the carbocyclyl group is a monocyclic, fully saturated (cycloalkyl) ring.

The terms heterocyclic and heterocyclyl refer to a non-aromatic ring system containing from 3 to 10 ring carbon atoms (unless otherwise noted) and at least one heteroatom selected from N, O and S, and which may be a fused or bridged ring system, and which may be fully saturated or may contain one or more carbon-carbon or carbon-nitrogen double bonds. Examples include piperidinyl, morpholinyl, thiomorpholinyl, thiozolidinyl, tetrahydrothiophenyl and tetrahydrothiopyranyl. More preferably, the heterocyclyl group is a fully saturated monocyclic ring.

The term halogen refers to fluorine, chlorine, bromine or iodine and to fluorine, chlorine, bromine or iodine groups.

The term '^-(haloalkyl) refers to a linear or branched alkyl group as defined above, having from 1 to 6 carbon atoms and substituted by one or more halogen atoms, up to perhalogen substitution. Examples include trifluoromethyl, chloroethyl and 1,1difluoroethyl. Other haloalkyl groups, such as Cl- ₅ haloalkyl, Cl- ₄ haloalkyl, Cyhaloalkyl or C^haloalkyl, are as defined above but contain a different number of carbon atoms.

The term amino acid side chain refers to the side chain of a naturally occurring amino acid, which may be a D-amino acid or an L-amino acid, but more suitably a D-amino acid. Examples of naturally occurring amino acids include glycine, proline, cysteine, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

The term side chain refers to the -YR ³ moiety. In UDCA, -YR ³ is -CH2CH2C(O)OH, and references to a modified side chain refer to -YR ³ moieties other than this.

References to the =O substituent refer to an oxygen atom bound by a double bond to an adjacent atom, which is typically a carbon or sulfur atom and which may be a ring atom. Examples of moieties containing a =O substituent include -C(O)-, -S(O)- and -S(O) ₂ -.

Suitable pharmaceutically acceptable salts of the compounds of general formula (I) include base addition salts such as sodium, potassium, calcium, aluminum, zinc, magnesium and other metal salts, as well as choline, diethanolamine, ethanolamine, ethyldiamine, meglumine and other good ones.

- 3 046372 known base addition salts, which are presented in Paulekuhn et al., J. Med. Chem. 2007, 50, 6665-6672 (incorporated herein by reference) and/or known to those skilled in the art.

The term isotopic modification refers to isotopically labeled compounds that are identical to the compounds represented by formula (I) except 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 more commonly of all those found in nature, or in which the proportion of an atom having an atomic mass or mass number rarely found in nature has been increased (the latter method being called isotopic enrichment). Examples of isotopes that may be included in the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine, such as ² H (deuterium), ³ H, 11 C, ¹³ C, ¹⁴ C, ¹⁸ F, ¹²³ I or ¹²⁵ I (eg ³ H, ⁿ C, ¹⁴ C, ¹⁸ F, ¹²³ I or ¹²⁵ I), which may be naturally occurring or non-naturally occurring isotopes.

In some cases, a compound of general formula (I) may be a compound of formula (I'):

O') where one of R ¹ and R ² represents F and the other of R ¹ and R ² represents H or F;

Y represents a bond or a C _1-20 α-alkylene, ^-^ alkenylene or ^-^ alkynylene linker group;

R ³ represents C(O)OR ¹² , C(O)NR ¹² R ¹³ , S(O)2R ¹² , OS(O)2R ¹² , S(O)2OR ¹² , OS(O)2OR ¹² , S( O)2NR ^I2 R ¹³ , C(O)NR ^I2 S(O)2R ¹³ , NHC(O)NR ^I2 S(O)2R ¹³ , OP(O)(OR ¹² )2, C(O)NR ^I2 [ CH(R ^I5 )]„R ¹⁶ or C(O)NR ¹² C(O)CH2NR ¹² [CH(R ¹⁵ )]nR ¹⁶ ;

each R ¹² independently represents H or ^alkyl, optionally substituted with one or more halogen or aryl groups;

R ¹³ represents H, Ci-balkyl, optionally substituted with one or more halogen or aryl groups, or a 5- or 6-membered carbocyclic ring, optionally substituted with R ¹⁶ ; or

R ¹² and R ¹³ together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocyclic ring, optionally containing an additional nitrogen atom, optionally substituted by one or more substituents selected from the groups CH ₂ C(O)OH , C(O)OH, S(O) ₂ OH, =O and =N-OH, and optionally fused with a phenyl group that is unsubstituted or substituted with one or more substituents selected from halogen and nitro;

each R ¹⁵ independently represents H or C _1- ^Kna, optionally substituted with one or more halogen or aryl groups, or an R ¹⁴ group, where R ¹⁴ represents an amino acid side chain;

n is 1, 2 or 3;

R ¹⁶ is selected from C(O)OH, S(O)2OH, OS(O)2OH and P(O)(OHb);

or a pharmaceutically acceptable salt or isotopic modification thereof.

The compound of general formula (I) may be a compound of general formula (IA), (IB), (IC) or (ID):

where R ¹ , R ² , Y and R ³ are as defined above for the general formula (I).

Some particularly suitable compounds of the invention are those of general formula (IA).

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Other suitable compounds of the invention are those of general formula (IB).

Other suitable compounds of the invention are those of general formula (IC).

Other suitable compounds of the invention are those of general formula (ID).

In certain suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ¹ and R ² are both F.

In other suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ¹ is F and R ² is H.

In other suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ¹ is H and R ² is F.

Compounds in which R ¹ and R ² are both F are particularly suitable.

In compounds of general formulas (I), (IA), (IB), (IC) and (ID), Y respectively represents a bond, either a C _1-15 alkylene linker or a C _2-15 alkenylene linker. Preferably, Y is a bond, either a C _1-12 , C _1-10 , C _1-8 , C _1-6 , C _1-4 , C _1-3 or C _1-2 alkylene linker, or a C _2-12 , C _2-10 , C _2-8 , C _2-6 , C _2-4 , C _2-3 or C ₂ alkenylene linker, and is unsubstituted or substituted with an OH group.

In certain suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), Y represents a bond, either a C _1-3 alkylene or a C _2-3 alkenylene linker group. Accordingly, Y represents C _1-3 alkylene or C _2-3 alkenylene.

Preferably, Y represents a bond or a C _1-3 alkylene linker group. More preferably, Y represents a C _1-3 alkylene linker group.

Examples of particularly suitable Y linkers include a -CH2-, -CH2CH2-, -CH(OH)-CH2-, -CH=CH- or -CH=C(CH ₃ )- bond, especially a -CH ₂ - bond. -CH ₂ CH ₂ -, -CH=CH- or -CH=C(CH ₃ )-, especially -CH2-, -CH2CH2-, -CH=CH- or -CH=C(CH3)-, and especially - CH2CH2-.

As discussed above, in compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ³ is C(O)OR ¹² , C(O)NR ¹² R ¹³ , S(O )2R ¹² , OS(O)2R ¹² , S(O)2OR ¹² , OS(O)2OR ¹² , S(O)2NR ¹² R ¹³ , C(O)NR ¹² S(O)2R ¹³ , NHC(O )NR ¹² S(O)2R ¹³ , OP(O)(OR ¹² )2, C(O)NR ¹² [CH(R ¹⁵ )]nR ¹⁶ or C(O)NR ¹² C(O)CH2NR ¹² [CH (R ¹⁵ )] _n R ¹⁶ .

In some cases, in compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ³ is preferably C(O)OR ¹² , OS(O);R. OS(O)OR, S(O)2NR ¹² R ¹³ , C(O)NR ¹² S(O)2R ¹³ , NR ¹² C(O)NR ¹² S(O)2R ¹³ or C(O)NR ¹² [ CH(R ¹⁵ )]nR ¹⁶ ; where R ¹² , R ¹³ , R ¹⁵ , n and R ¹⁶ are as defined above.

In this case, more suitable compounds are those in which R ³ is C(O)OR ¹² , C(O)NR ¹² CH(R ¹⁴ )C(O)OH or C(O)NR ¹² CH(R ¹⁵ ) CH(R ¹⁵ )S(O)2OH, where R ¹² and R ¹⁴ are as defined above, and R ¹⁵ represents H or C^lkyl, optionally substituted with one or more halogen or aryl group.

In other more suitable compounds, R ³ is C(O)OR ¹² , (C(O)N(R ¹² )(R ¹³ ) or C(O)NR ¹² [CH(R ¹⁵ )] _n R ¹⁶ ; where R ¹² , R ¹³ , R ¹⁵ , R ¹⁶ and n are as defined above.

In compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ¹² respectively represents H, C _1-6 alkyl, which may be unsubstituted or substituted as described above, preferably H , benzyl or C _1-4 alkyl, optionally substituted with R ¹⁶ or N(R ¹⁰ )(R ¹¹ ), especially H, or methyl, or ethyl, optionally substituted with R ¹⁶ or N(R ¹⁰ )(R ¹¹ ).

In some compounds in which R ³ is C(O)NR ¹² S(O) ₂ R ¹³ , NHC(O)NR ¹² S(O)2R ¹³ , C(O)NR ¹² [CH(R ¹⁵ )] nR ¹⁶ or C(O)NR ¹² C(O)CH2NR ¹² [CH(R ¹⁵ )]nR ¹⁶ , R ¹² is preferably H, methyl or ethyl, especially H or methyl.

In compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ¹³ , when present, is suitably a 5- or 6-membered carbocyclyl or heterocyclyl, optionally substituted with R ¹⁶ or = O, where =O substituents may be attached to the C or S ring atom; or phenyl, optionally substituted with R ¹⁶ .

Alternatively, in certain suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ is C(O)NR ¹² R ¹³ or S(O) ₂ NR ¹² R ¹³ , R ¹² and R ¹³ together with the nitrogen atom to which they are attached may form a 5- or 6-membered heterocyclic ring, optionally substituted with one or more substituents selected from R ¹⁶ and =O, and optionally containing one or more than one additional heteroatom selected from O, N and S.

In compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ¹⁶ when present is preferably C(O)Oh, S(O) ₂ OH, S(O ) ₂ (C _1-6 alkyl) or OS(O) ₂ 0h, especially C(O)OH or S(O)2OH.

When R ¹⁶ represents C(O)OH, S(O) ₂ OH, OS(O) ₂ OH or P(O)(OH) ₂ , a compound of general formula (I), (IA), (IB), ( IC) or (ID) may be in salt form. Suitable salts are as discussed above, but metal salts, such as sodium and potassium salts, especially sodium salts, are particularly suitable.

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In certain suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ³ is C(O)OR ¹² , C(O)NR ¹² R ¹³ or C(O) NR ¹² [CH(R ¹⁵ )]nR ¹⁶ , where:

when R ³ represents C(O)OR ¹² , each R ¹² independently represents H or C^alkyl, optionally substituted with one or more substituents selected from halogen, OR ¹⁰ , NR ¹⁰ R ¹¹ , R ¹⁶ and aryl;

R ¹⁰ and R ¹¹ are each independently H or C^alkyl; or when R ³ is C(O)NR ¹² R ¹³ :

each R ¹² is H or C^alkyl, optionally substituted with one or more substituents selected from halogen, OR ¹⁰ and NR10R ¹¹ ;

R ¹⁰ and R ¹¹ are each independently H or C^alkyl; And

R ¹³ is 1,1-tetrahydrothiopyran dioxide or 1,1-tetrahydrothiophene dioxide; or

R ¹² and R ¹³ , together with the nitrogen atom to which they are attached, form a 5- or 6-membered ring containing an SO ₂ group or a substituted C(O)OH; or when R ³ is C(O)NR ¹² [CH(R ¹⁵ )] _n R ¹⁶ , R ¹² is H or methyl, R ¹⁵ is H, and either

R ¹⁶ represents C(O)OH, and n is 1; or

R ¹⁶ represents S(O) ₂ OH, S^^Q-^Kra) or OS(O) ₂ OH; and n is 2 or 3.

or a pharmaceutically acceptable salt or isotopic modification thereof.

In certain particularly suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ³ is C(O)OR ¹²

Accordingly, in these compounds, R ¹² is H, or methyl or ethyl, optionally substituted with R ¹⁶ , and preferably R ¹² is H, CH2R ¹⁶ or -CH2CH2R ¹⁶ . where R ¹⁶ is as defined above, but in particular represents S(O) ₂ OH. More preferably, R ¹² is H.

Particularly suitable compounds of this kind are those in which R ³ represents C(O)OH, and their salts, as discussed above, for example metal salts such as sodium and potassium salts, especially sodium salts.

In compounds of general formulas (I), (IA), (IB), (IC) and (ID), where R ³ represents C(O)OR ¹² and R ¹² represents H or C^alkyl (for example methyl or ethyl) substituted with R ¹⁶ , where R ¹⁶ represents C(O)OH, S(O) ₂ OH, OS(O) ₂ OH or P(O)(OH) ₂ , the compound may be in the form of a salt. Suitable salts are as discussed above, but metal salts, such as sodium and potassium salts, especially sodium salts, are particularly suitable.

In other particularly suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ³ is C(O)NR ¹² [CH(R ¹⁵ )] _n R ¹⁶ and salts thereof, as discussed above, for example metal salts such as sodium and potassium salts, especially sodium salts.

In such compounds, R ¹² is preferably H, methyl or methyl substituted with R ¹⁶ , for example -CH2C(O)OH. In some compounds of this type, R ¹² represents H; in other compounds of this type, R ¹² represents methyl; in other compounds of this kind, R ¹² is -CH2R ¹⁶ In this case, R ¹⁵ is correspondingly H, and n is correspondingly 1, so that R ³ is C(O)N(CH2R ¹⁶ ) ₂ and the two groups are R ¹⁶ may be the same or different, but are preferably the same and represent, for example, C(O)OH.

When R ³ is C(O)NR ¹² [CH(R ¹⁵ )]nR ¹⁶ , it may be a C(O)NR ¹² CH(R ¹⁴ )C(O)OH group or a C(O)NR ¹² [ group CH(R ¹⁵ )]nR ¹⁶ where R ¹² represents H or C^alkyl, preferably H or C1-3alkyl, especially H or methyl; n is 2 or 3; each R ¹⁵ is H or C^alkyl, optionally substituted with one or more halogen or aryl groups, preferably H or C1-6alkyl and more preferably H; and R ¹⁶ is S(O) ₂ OH, S(O) ₂ (C _1-6 alkyl) or OS(O) ₂ OH, especially S(O) ₂ OH, S(O) ₂ (methyl) or OS (O) _2OH , especially S(O)2OH.

When R ³ is C(O)NR ¹² CH(R ¹⁴ )C(O)OH, R ¹⁴ is in particular a side chain of an amino acid selected from glycine, alanine, valine, leucine or isoleucine, i.e. R ¹⁴ represents H, CH ₃ , CH(CH ₃ ) ₂ or CH(CH ₃ )(C ₂ H ₅ ). Preferably, R ¹⁴ is H. Particularly suitable R ¹² moieties are as defined above. When R ¹² is H and R ¹⁴ is H, R ³ is a glycine conjugate; and when R ¹² is methyl and R ¹⁴ is H, R ³ is an N-methylglycine conjugate. Preferably, R ¹² is H and R ³ is a glycine conjugate.

When R ³ is C(O)NR ¹² [CH(R ¹⁵ )]nR ¹⁶ , it may be a C(O)NR ¹² CH(R ¹⁵ )CH(R ¹⁵ )S(O)2OH group, where R ¹⁴ represents a side chain of an amino acid, and R ¹⁵ represents H or C^alkyl, optionally substituted with one or more halogen or aryl groups. The compound may be in the form of a salt, as discussed above, for example metal salts such as sodium and potassium salts, especially sodium salts.

When R ³ is C(O)NR ¹² CH(R ¹⁵ )CH(R ¹⁵ )S(O) ₂ OH, each R ¹⁵ is respectively H or C^alkyl. Preferably, both R ¹⁵ moieties are H. Especially

- 6 046372 suitable R ¹² moieties are as defined above, but in particularly suitable compounds R ¹² is H or methyl. When R ¹² is H and both R ¹⁵ moieties are H, R ³ is a taurine conjugate. When R ¹² is methyl and both R ¹⁵ moieties are H, R ³ is an N-methyltaurine conjugate.

In certain suitable compounds in which R ³ is C(O)NR ¹² [CH(R ¹⁵ )]nR ¹⁶ , each R ¹⁵ independently represents H or C _1-4 alkyl, optionally substituted as above. Preferably R ¹⁵ is H or unsubstituted C^alkyl, more preferably H, methyl or ethyl and especially H.

Alternatively, in compounds where R ³ is C(O)NR ¹² [CH(R ¹⁵ )]nR ¹⁶ , and n is 2 or 3, the two R ¹⁵ groups may combine with the carbon atoms to which they are attached, and possibly with the intervening carbon atom, if present, to form a carbocyclic ring as above. Preferably, the two R ¹⁵ groups are on adjacent carbon atoms. The carbocyclic ring thus formed is a 5-7 membered ring, for example a 6-membered ring.

In other particularly suitable compounds of general formulas (I), (IA), (IB), (IC) and (ID), R ³ is C(O)NR ¹² R ¹³ .

In some suitable compounds of this type, R ¹² is H or C _1-z ^km, optionally substituted with one or more substituents, for example one substituent selected from R ¹⁶ and NR ¹⁰ R ¹¹ , where R ¹⁶ , R ¹⁰ and R ¹¹ are as defined above. R ¹⁰ and R ¹¹ may be the same or different and are preferably selected from H and C-alkyl, especially C-alkyl. More suitable R ¹⁶ groups are as defined above.

In particularly suitable compounds in which R ³ is C(O)NR ¹² R ¹³ , R ¹² is H or C^alkyl substituted with one R ¹⁶ substituent. More suitable R ¹⁶ groups are as defined above, and the compound may be present in the form of a salt as discussed above, for example a metal salt such as a sodium or potassium salt, especially a sodium salt.

When R ³ represents C(O)NR ¹² R ¹³ , R ¹³ suitably represents phenyl or a 5-7 membered cycloalkyl or heterocyclyl group, any of which may optionally be substituted by one R ¹⁶ substituent, and wherein cycloalkyl and heterocyclyl groups may be substituted one or more than one =O substituent.

More preferably, the cycloalkyl group is unsubstituted cyclopentyl, cyclohexyl or cycloheptyl.

More preferably, the heterocyclyl group is a 5- or 6-membered sulfur-containing group such as tetrahydrothiophene and tetrahydrothiopyran, or oxides thereof such as 1,1-dioxotetrahydrothiophene and 1,1-dioxotetrahydropyran.

In other suitable compounds wherein R ³ is C(O)NR ¹² R ¹³ , each R ¹² is H or C _1-6 alkyl, optionally substituted with one or more substituents selected from halogen, OR ¹⁰ and NR ¹⁰ R ¹¹ ;

R ¹⁰ and R ¹¹ are each independently H or C^alkyl; And

R ¹³ is 1,1-tetrahydrothiopyran dioxide or 1,1-tetrahydrothiophene dioxide or

R ¹² and R ¹³ , together with the nitrogen atom to which they are attached, form a 5- or 6-membered ring containing an SO ₂ group or a substituted C(O)OH.

In these compounds, R ¹² is preferably H and R ¹³ is a 1,1-tetrahydrothiopyran dioxide ring. Alternatively, R ¹² and R ¹³ , together with the nitrogen atom to which they are attached, form a 6-membered ring containing an SO2 moiety, especially a thiomorpholine dioxide ring, or a piperidine substituted with C(O)OH.

Particularly preferred R ³ groups include C(O)OH, C(O)NHCH2C(O)OH, C(O)N(CH3)CH2C(O)OH, C(O)NHCH2CH2S(O)2OH and C(O) N(CH3)CH2CH2S(O)2OH, especially C(O)OH, C(O)NHCH2C(O)OH, C(O)NHCH2CH2S(O)2OH and C(O)N(CH3)CH2CH2S(O)2OH .

- 7 046372

In one embodiment, the compound of formula (I) is selected from the group consisting of 23-fluorochenodeoxycholic acid (compound 1);

23-fluoro-33,7a-dihydroxy-53-cholanic acid (compound 2);

2a-fluoro-33,7a-dihydroxy-53-cholanic acid (compound 3);

2a-fluoro-33,73-dihydroxy-53-cholanic acid (compound 4);

2a-fluoro-3a,7a-dihydroxy-53-cholanic acid (compound 5);

2a-fluoro-3a,73-dihydroxy-53-cholanic acid (compound 6);

2,2-difluoro-33,73-dihydroxy-53-cholanic acid (compound 7);

2,2-difluoro-Za,7a-dihydroxy-53-cholanic acid (compound 8);

2,2-difluoro-Za,73-dihydroxy-53-cholanic acid (compound 9);

7U-(2,2-difluoro-33,73-dihydroxy-53-cholan-24-amide)-ethylsulfonic acid (compound 10);

7U-(2,2-difluoro-33,73-dihydroxy-53-cholan-24-amide)-propionic acid (compound I);

7U-(methyl),7U-(2,2-difluoro-33,73-dihydroxy-53-cholan-24-amide)-acetic acid (compound 12);

7U-(2,2-difluoro-33,73-dihydroxy-53-cholan-24-amide)-from/?ans-2cyclohexanecarboxylic acid (compound^);

1-(2,2-difluoro-33,73-dihydroxy-53-cholan-24-oil)-piperidine-3-carboxylic acid (compound);

3-(2,2-difluoro-33,73-dihydroxy-53-cholan-24-amide)-4-thiazolidine-carboxylic acid (compound^);

7U-(2,2-difluoro-33,73-dihydroxy-53-cholan-24-oil)-morpholine (compound 16);

- 8 046372

7/-(2,2-difluoro-3p,73-dihydroxy-5p-cholan-24-amide)-methylcarboxylic acid (compound 17)

7/-(carboxymethyl)-7/-(2,2-difluoro-33,73-dihydroxy-5p-cholan-24-oyl)-2-aminoacetic acid (compound 18);

7/-(methyl)-7/-(2,2-difluoro-33,7p-dihydroxy-53-cholan-24-amide)-ethylsulfonic acid (compound 19);

3-(2,2-difluoro-3p,7p-dihydroxy-5p-cholan-24-oil)-amino-propanesulfonic acid (compound 20);

7/-(2,2-difluoro-3p,73-dihydroxy-5p-cholan-24-amide)-methanesulfonic acid (compound 21);

7/-(2,2-difluoro-3p,73-dihydroxy-5p-cholan-24-oil)-2-aminoethylsulfuric acid (compound 22);

O-(2,2-difluoro-33,7p-dihydroxy-5p-cholan-24-oil)-2-hydroxyethylsulfonic acid (compound 23);

7/-(2,2-difluoro-3p,73-dihydroxy-5P-cholan-24-oil)-aniline-2-sulfonic acid (compound 24);

7/-(cyclohexyl)-7/-(2,2-difluoro-3p,73-dihydroxy-53-cholan-24-oyl)-3-aminopropanesulfonic acid (compound 25);

7/-(cyclohexyl)-7/-(2,2-difluoro-3p,73-dihydroxy-53-cholan-24-oyl)-2-aminoethanesulfonic acid (compound 26);

7/-(2,2-difluoro-3p,73-dihydroxy-5p-cholan-24-oil)-2-aminoethylmethylsulfone (compound 27);

7/-(ethyl)-7/-(2,2-difluoro-3p,7p-dihydroxy-5p-cholan-24-oil)-3-aminotetrahydrothiophene dioxide (compound 28);

7/-(2-(diisopropylamino)ethyl)-7/-(2,2-difluoro-3p,73-dihydroxy-5P-cholan-24oyl)-3-amino-tetrahydrothiophene dioxide a (compound 29);

7/-(2,2-difluoro-3p,73-dihydroxy-53-cholan-24-oil)-thiomorpholine dioxide (compound 30);

7/-(2,2-difluoro-3 β,73-dihydroxy-5P-cholan-24-oil)-1,1-dioxidotetrahydro-2Hthiopyran-3-ylamine (compound 31); and pharmaceutically acceptable salts thereof (where appropriate), especially metal salts such as sodium and potassium salts, especially sodium salts or (for compound 18) disodium salt.

Methods for preparing compounds of general formula (I) are described below. These methods constitute a further aspect of the invention.

Compounds of general formulas (IB) and (IC) in which R ¹ represents F and R ³ represents C(O)OR ^12a , where R ^12a represents Q-balkyl, optionally substituted by one or more (im)halogen or aryl group, can be obtained from compounds of general formula (II):

n (II), where Y and R ³ are as defined for the general formula (I); R ^12a represents C1 ₆ alkyl, optionally substituted with one or more halogen or aryl groups; and R ²¹ represents an acid-sensitive OH protecting group;

- 9 046372 by treatment with an acid, for example hydrochloric acid, as described in General Method L below.

Suitable acid-sensitive protecting groups for R ²¹ include alkyl ethers, for example methoxymethyl.

Compounds of general formula (II) can be formed as a mixture of isomers of general formulas (IIB) and (IIC):

12a (IIB) n

(PS) where Y and R ³ are as defined for the general formula (I), and R ^12a and R ²¹ are as defined for the general formula (II);

by reducing the compound of general formula (III):

where Y and R ³ are as defined for the general formula (I), and R ^12a and R ²¹ are as defined for the general formula (II).

Removal of the protecting groups R ²¹ from compounds of general formulas (IIB) and (IIC), as described above, results in compounds of general formulas (IB) and (IC), respectively.

Suitable reducing agents for the reduction of compounds of general formula (III) include hydrides, for example sodium borohydride. The reduction can be carried out in an organic solvent such as tetrahydrofuran at a temperature of from about 15 to 25°C, preferably at room temperature.

Compounds of general formula (III) can be prepared from compounds of general formula (IV):

where Y and R ³ are as defined for the general formula (I); R ^12a and R ²¹ are as defined for the general formula (II); and R ²² represents an OH protecting group;

by fluorination using an agent such as Selectfluor®, (1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) which has the structure:

bf;

Ν» ^g

When using Selectfluor®, the reaction is usually carried out at a temperature of 15 to 25°C, usually at room temperature, in a polar organic solvent such as acetonitrile.

Suitable R ²² protecting groups include the silyl protecting groups Si(R ²³ )3, where each R ²³ is independently C1-6 alkyl or phenyl.

Examples of R ²² groups include trimethylsilyl (TMS), triethylsilyl (TES), triphenylsilyl (TPS), triisopropylsilyl (TIPS), texyldimethylsilyl (TDS), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS or TBS), di-tert-butylmethylsilyl ( DTBMS), diethylisopropylsilyl (DEIPS) and dimethylisopropylsilyl (DMIPS), in particular TMS, TES, TIPS, TBDMS and TBDPS.

- 10 046372

Compounds of general formula (IV) can be prepared from compounds of general formula (V):

(V) where Y and R ³ are as defined for the general formula (I); R ^12a and R ²¹ are as defined for the general formula (II); by reacting with a compound of general formula (VI):

(R ²³ ) ₃ Si-OR ²⁴ (VI) wherein each R ²³ is independently as defined above and R ²⁴ represents a leaving group such as trifluoromethanesulfonate (triflate), toluenesulfonyl (tosyl) or methanesulfonyl (mesyl).

The reaction is usually carried out under alkaline conditions, for example in the presence of a weak base such as triethylamine, at a temperature of from about 15 to 25°C, preferably at room temperature.

Compounds of general formula (V) can be prepared from chenodeoxycholic acid by esterification of a carboxylic acid by reaction with an R ^12a OH alcohol, for example as described in General Method A below, followed by protection of the 7-OH group by reaction with a compound of general formula (VII ):

R ²¹ -X (VII) where R ²¹ is as defined for general formula (II), and X represents a leaving group, usually a halogen, for example chlorine; for example, as described in General Method K below. This may be followed by oxidation of the 3-OH group as described in General Method M below.

Compounds of general formula (I) in which R ² represents F and Y represents C(O)OR ^12a , wherein R ^12a is as defined above, can be prepared as follows.

Compounds of general formulas (IA) and (IC) in which R ² represents F and R ³ represents C(O)OR ^12a can be prepared by reducing compounds of general formula (XIIa):

,V4°

OR ^12a

Η (XIIa) where Y is as defined for the general formula (I), and R ^12a is as defined for the general formula (II).

Accordingly, reduction is carried out using a hydride, for example sodium borohydride, in the presence of cerium(III) chloride. The reaction is usually carried out at a temperature of from about 15 to 25°C, preferably at room temperature.

The resulting product is a mixture of compounds of the general formula (IA) and the general formula (IC), which can be separated by conventional methods, for example by chromatography.

The compound of general formula (XIIa) can also be used to prepare compounds of general formulas (IB) and (ID) in which R ² represents F and R ³ represents C(O)OR ^12a In this case, the compound of general formula (XIIa ) can be reacted with a carboxylic acid of general formula (XIII):

R ²⁵ -C(O)OH (XIII) where R ²⁵ represents C _1-6 αlkyl or benzyl, but more preferably benzyl;

- 11 046372 in the presence of triphenylphosphine and diethyl azodicarboxylate (DEAD) using the Mitsunobu reaction to obtain a compound of general formula (XIV):

(XIV) where Y is as defined for the general formula (I); R ^12a is as defined for the general formula (II), and R ²⁵ is as defined for the general formula (XIII).

A compound of general formula (XIV) can be hydrolyzed using a weak base such as potassium carbonate to give a compound of general formula (XIIb):

,UCH°

OR ^12a n

(XL) where Y is as defined for the general formula (I), and R ^12a is as defined for the general formula (II).

The compound of general formula (XIIb) can then be reduced to obtain a mixture of compounds of general formula (IB) and general formula (ID) using the conditions described above for reducing the compound of general formula (XIIa).

A compound of general formula (XIIa) can be prepared from a compound of general formula (XIV):

,w

OR ^12a t t n

(xiv) where Y is as defined for the general formula (I), and R ^12a is as defined for the general formula (II); through interaction with the hydrogen fluoride-pyridine complex (HF·pyridine).

Accordingly, the reaction is carried out in an anhydrous organic solvent such as dichloromethane (DCM).

A compound of general formula (XIV) can be prepared by epoxidizing a compound of general formula (XV):

where Y is as defined for the general formula (I), and R ^12a is as defined for the general formula (II).

A suitable oxidizing agent is meta-chloroperbenzoic acid (mCPBA), and the reaction can be carried out in an organic solvent such as DCM and at a temperature of about 15 to 25°C, preferably at room temperature.

- 12 046372

A compound of general formula (XV) can be prepared from a compound of general formula (XVI):

where Y is as defined for the general formula (I), and R ^12a is as defined for the general formula (II); through an elimination reaction.

Trifluoromethanesulfonic acid anhydride (triflate anhydride) is an example of a suitably activated leaving group that can be used in combination with a base such as dimethylaminopyridine (DMAP). The reaction can be carried out at a temperature of from about 5 to 20°C, preferably from 10 to 15°C.

Compounds of general formula (XVI) can be prepared from 7-keto-lithocholic acid by esterification as described in General Method A below. 7-Keto-lithocholic acid is commercially available.

Compounds of general formula (IA) in which both R ¹ and R ² are fluorine and R ³ is C(O)OR ^12a , wherein R ^12a is as defined for general formula (II), can be prepared from compounds of general formula (XXI):

where Y is as defined for the general formula (I), and R ^12a and R ²¹ are as defined for the general formula (II);

by reacting with an acid, for example hydrochloric acid, as described in General Method L below.

Compounds of general formula (IA) can be converted into compounds of general formulas (IB) and (ID) using the following procedure.

A compound of general formula (IA) can be oxidized to give a compound of general formula (XXII):

where Y is as defined for the general formula (I), and R ^12a and R ²¹ are as defined for the general formula (II).

Suitable oxidizing agents for this procedure include Dess-Martin periodinane and the reaction conditions for this procedure are as described below in General Method M.

The diketone of general formula (XXII) can then be reduced to obtain a mixture of compounds of general formulas (IB) and (ID). Suitable reducing agents for this procedure include a hydride, for example sodium borohydride, in the presence of cerium(III) chloride, as described in General Method B below. Compounds of general formulas (IB) and (ID) can be separated by conventional methods, for example by chromatographic methods.

- 13 046372

Compounds of general formula (XXI) can be prepared by fluorination of compounds of general formula (XXIII):

where Y is as defined for the general formula (I), and R ^12a and R ²¹ are as defined for the general formula (II).

Suitable fluorinating agents for this interaction include N,Ndiethylaminosulfur trifluoride (DAST). The reaction with DAST can occur in an organic solvent, such as dichloromethane, at a temperature of about 15 to 25°C, usually at room temperature.

Compounds of general formula (XXIII) can be prepared by oxidation of compounds of general formula (XXIV):

where Y is as defined for the general formula (I), and R ^12a and R ²¹ are as defined for the general formula (II).

Suitable oxidizing agents include Dess-Martin periodinane, as described in General Method M below.

Compounds of general formula (XXIV) can be prepared by reacting a compound of general formula (XXV):

where Y is as defined for the general formula (I); R ^12a and R ²¹ are as defined for the general formula (II); and R ²⁶ is a base-sensitive protecting group;

with a compound of general formula (VII) as described above, followed by reaction with a base to remove the protecting group R ²⁶ .

Examples of the R ²⁶ protecting groups include acyl groups R ²⁵ C(O)-, where R ²⁵ is as defined above for the general formula (XIII).

The reaction of a compound of general formula (XXV) with a compound of general formula (VII) can be carried out using the general method K below.

The protecting group R ²⁶ can be removed with a base such as an alkoxide, for example sodium or potassium alkoxide, usually sodium alkoxide, for example sodium methoxide or sodium ethoxide. Deprotection is suitably carried out in an alcohol solvent such as methanol or ethanol at a temperature of about 15 to 25°C, usually at room temperature.

A compound of general formula (XXV) can be prepared from a compound of general formula (XXVI):

- 14 046372 where Y is as defined for the general formula (I); R ^12a and R ²¹ are as defined for the general formula (II); by epoxidation to obtain a compound of general formula (XXVIa):

(XXVIa) where Y is as defined for general formula (I); R ^12a and R ²¹ are as defined for the general formula (II);

followed by ring opening by reaction with a compound of general formula (XXVII):

R ²⁶ -OH (XXVII) where R ²⁶ is as defined above for the general formula (XXV).

A suitable oxidizing agent is meta-chloroperbenzoic acid (m-CPBA), and the reaction can be carried out in an organic solvent such as DCM and at a temperature of about 15 to 25°C, preferably at room temperature. As a result of the interaction, an inseparable mixture of Δ2β,3β-epoxide (XXVIa) and Δ3β,4β-epoxide is formed. When the mixture is treated with a compound of general formula (XXVII), a compound of general formula (XXVIa) reacts to form the desired product.

In the ring opening reaction where the protecting group R ²⁶ is an acyl group R ²⁵ C(O)-, the compound of formula (XXVII) is a compound of general formula (XIII) as defined above. The reaction of a compound of general formula (XXVIa) with a compound of general formula (XIII) can occur at elevated temperature, for example from about 40 to 60°C, usually at about 50°C.

A compound of general formula (XXVI) can be prepared from a compound of general formula (XXVIII):

(XXVIII) where Y is as defined for general formula (I); R ^12a and R ²¹ are as defined for the general formula (II); through an elimination reaction.

Trifluoromethanesulfonic acid anhydride (triflate anhydride) is an example of a suitably activated leaving group that can be used in combination with a base such as lutidine. The reaction can be carried out at a temperature of from about 5 to 20°C, preferably from 10 to 15°C.

A compound of general formula (XXVIII) can be prepared from a compound of general formula

divided for general formula (II); and R ²⁶ is as defined for the general formula (XXV); by reacting with a compound of general formula (XXVII) as defined above. Accordingly, the compound of general formula (XXVII) is a carboxylic acid of general formula (XIII), such that R ²⁶ represents an acyl group of formula R ²⁵ C(O)-.

The reaction can occur in an alcohol solvent such as methanol or ethanol at a temperature of about 15 to 25°C, usually at room temperature.

- 15 046372

A compound of general formula (XXIX) can be prepared from a compound of general formula (XXX):

n (XXX) where Y is as defined for the general formula (I); R ^12a is as defined for the general formula (II); and R ²⁶ is as defined for the general formula (XXV);

by reacting with a compound of general formula (VII). Suitable reaction conditions are as described in General Method K below.

A compound of general formula (XXX) can be prepared from a compound of general formula (XXXI):

(XXXI) where R ^12a is as defined for the general formula (II);

by reacting with a compound of general formula (XXVII) or, more often, with a compound of general formula (XXXII):

(R ²⁶ ) ₂ O (XXXIII) where R ²⁶ is as defined for the general formula (XXV).

When R ²⁶ represents the acyl group of R ²⁵ , the reagent of general formula (XXXIII) is a carboxylic acid anhydride.

The compound of general formula (XXXI) is a UDCA ester, which can be prepared from UDCA by reaction with the alcohol R ^12a OH, for example, as described in General Method A.

Compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents C(O)OR ^12a , where R ^12a represents ^^alkyl, possibly substituted by one ) or more than one halogen or aryl group, can be converted into other compounds of general formulas (I), (IA), (IB), (IC) and (ID).

Compounds of general formulas (I), (IA), (IB), (IC) and (ID) in which R ³ is C(O)OH can be prepared by hydrolysis of an equivalent compound in which R ³ is C (O)OR ^12a Hydrolysis may be acid or alkaline hydrolysis. Alkaline hydrolysis is often more suitable and can be carried out, for example, using an alkali metal hydroxide such as lithium, sodium or potassium hydroxide, more commonly lithium hydroxide. Alkaline hydrolysis is described in General Method C below.

Compounds of general formula (I) in which R ³ is C(O)NR ¹² R ¹³ can be prepared from a carboxylic acid by reaction with an amine of the formula H-NR ¹² R ¹³ , where R ¹² and R ¹³ are as follows: as defined above for general formula (I); in a suitable solvent when heated. Accordingly, the reaction is carried out in the presence of a coupling agent and under alkaline conditions, for example in the presence of an amine such as diisopropylethylamine (DIPEA) or triethylamine (TEA), and in an organic solvent such as DMF, as described in General Method Q below.

Suitable coupling agents include known peptide coupling agents such as O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(benzotriazol-1-yl)X,X tetrafluoroborate ,I,I-tetramethyluronium (TBTU), O-(7-αzabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(7-azabenzotriazol-1-yl) tetrafluoroborate -N,N,N',N'-tetramethyluronium (TATU), (benzotriazol-1-yloxy)-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), and triazoles such as 1-hydroxy-7-azabenzotriazole (HOAt) or hydroxybenzotriazole (HOBt); and chloroformates such as isobutyl chloroformate.

Amines of the formula H-NR ¹² R ¹³ are known and readily available or can be prepared by methods known to those skilled in the art.

Compounds of general formula (I) in which R ⁷ is C(O)NR ¹² R ¹³ or OS(O) ₂ OR ¹² can also be prepared by methods similar to those described by Festa et al., J. Med. Chem., 2014, 57, 8477-8495 (incorporated herein by reference).

- 16 046372

Compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents C(O)NR ¹² [CH(R ¹⁵ )] _n R ¹⁶ , can be prepared from compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents C(O)OH, by reacting with a compound of general formula (XL):

HNR ¹² [CH(R ¹⁵ )] _n R ¹⁶ (XL) where R ¹² , R ¹⁵ , n and R ¹⁶ are as defined above; in the presence of a coupling agent and under alkaline conditions, for example in the presence of an amine such as diisopropylethylamine (DIPEA) or triethylamine (TEA), and in an organic solvent such as DMF, as described in General Method Q below. Suitable coupling agents are as described above.

For example, compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents C(O)NR ¹² CH(R ¹⁴ )C(O)OH, can be prepared from compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents C(O)OH, by reaction with an amino acid of general formula (XLI):

R ^{14 p,} ' ² L ^0H o (XY) where R ¹² and R ¹⁴ are as defined above; in the presence of a coupling agent and under alkaline conditions, for example in the presence of an amine such as diisopropylethylamine (DIPEA) or triethylamine (TEA), and in an organic solvent such as DMF, as described in General Method Q below. Suitable coupling agents are as described above, with HATU being particularly suitable.

Accordingly, in the compound of general formula (XLI), R ¹² represents H or methyl, and R ¹⁴ represents H.

Likewise, compounds of general formulas (I), (IA), (IB), (IC) and (ID) in which R ³ is C(O)NR ¹² CH(R ¹⁵ )CH(R ¹⁵ )S(O ) ₂ OH, can be prepared from compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents C(O)OH, by reacting with a compound of general formula ( XLII):

R ¹⁵

(XLII) wherein R ¹² and R ¹⁵ are as defined above;

in the presence of a coupling agent and under alkaline conditions, for example in the presence of an amine such as diisopropylethylamine (DIPEA) or triethylamine (TEA), and in an organic solvent such as DMF, as described in General Method Q below. Suitable coupling agents are as described above, with HATU and isobutyl chlorophosphate being particularly suitable.

Accordingly, in the compound of general formula (XLII), R ¹² represents H or methyl, and each R ¹⁵ represents H.

Amino acids of the general formula (XLI) and taurine and its derivatives of the general formula (XLII) are widely known and are readily available or can be synthesized by methods known in the art.

Compounds of general formulas (I), (IA), (IB), (IC) and (ID) in which R ³ is C(O)OH can be converted into compounds in which R ³ is C(O )NR ¹² S(O) ₂ R ¹³ where R ¹³ is as defined above by reacting with a compound of the formula:

NHR ¹² S(O) ₂ R ¹³ where R ¹² and R ¹³ are as defined above, in the presence of a coupling agent and under alkaline conditions, for example in the presence of an amine such as diisopropylethylamine (DIPEA) or triethylamine (TEA), and in an organic solvent such as DMF, as described in General Method Q below. Suitable coupling agents are as described above, with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDCI) being particularly suitable.

Compounds of the general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents NHC(O)NR ¹² S(O) ₂ R ¹³ , can be prepared from compounds of the general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents C(O)OH, by the method shown in Scheme 1:

- 17 046372

Scheme 1 o

I diphenylphosphoryl azide/triethylamine

II heating

III R ¹³ SO ₂ NHR ¹²

Compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents S(O)2OR ¹² , can be synthesized from compounds of general formulas (I), (IA) , (IB), (IC) and (ID), in which R ³ represents C(O)OH. The compound where R ³ is C(O)OH may first be reacted with a C _1-6 alkanoyl or benzoyl chloride, or a C _1-6 alkanoic anhydride to protect any OH groups. The protected compound can then be reacted with a reducing agent such as a hydride, preferably lithium aluminum hydride or sodium borohydride, to reduce the carboxyl group to OH. The alcohol group can be replaced with a halogen, such as bromine or iodine, for example using the triphenylphosphine/imidazole/halogen method described by Classon et al., J. Org. Chem., 1988, 53, 6126-6130 (incorporated herein by reference). The halogenated compound can then be reacted with sodium sulfite in an alcohol solvent to produce the SO ₃ ^- Na ⁺ -substituted compound.

Compounds of the general formulas (I), (IA), (IB), (IC) and (ID) in which R ³ represents OS(O)2OR ¹² can be prepared by protecting the OH groups of the compounds of the general formulas (I) , (IA), (IB), (IC) or (ID), in which R ³ represents C(O)OR ¹² , using any suitable protecting group; reducing the carboxylic acid or ester to produce an alcohol and reacting it with a chlorosulfonic acid in the presence of a base such as triethylamine to produce a protected triethylamine salt of the compound wherein R ³ is OS(O)2OR ¹² . Protecting groups can be removed using alkaline hydrolysis.

Reaction of an alcohol with a sulfonyl chloride produces a compound of general formula (I), (IA), (IB), (IC) or (ID) in which R ³ is OS(O) ₂ R ¹² .

Compounds of general formulas (I), (IA), (IB), (IC) and (ID), in which R ³ represents S(O) ₂ R ¹² , can be prepared from alcohol by reaction with Lawesson's reagent followed by oxidation the resulting product.

Surprisingly, it has been shown that the compounds of the present invention are capable of restoring mitochondrial function and can penetrate the blood-brain barrier. Therefore, they are used in the treatment of neurodegenerative disorders, including Parkinson's disease, mild cognitive impairment, dementia (including Alzheimer's disease, vascular dementia and dementia with Lewy bodies), Huntington's disease and amyotrophic lateral sclerosis (motor neuron disease).

The compounds of the present invention are surprisingly active compared to bile acids with slightly different substitutions on the A and B rings.

In a further aspect of the invention there is provided a compound of general formula (I) for use in medicine.

A compound of general formula (I) is also provided for use in the treatment or prevention of a neurodegenerative disorder.

The invention also provides the use of a compound of general formula (I) in the preparation of an agent for the treatment or prevention of a neurodegenerative disorder.

The invention also provides a method for treating or preventing a neurodegenerative disorder, which method comprises administering to a patient in need of such treatment an effective amount of a compound of general formula (I).

Examples of neurodegenerative disorders include Parkinson's disease, mild cognitive impairment, dementia (including Alzheimer's disease, vascular dementia, and dementia with Lewy bodies), Huntington's disease, amyotrophic lateral sclerosis (motor neuron disease), progressive supranuclear palsy, and Wilson's disease. Disorders that are particularly suitable for

- 18 046372 treatments with compounds of the present invention include Parkinson's disease, mild cognitive impairment, dementia (including Alzheimer's disease, vascular dementia and dementia with Lewy bodies), Huntington's disease and amyotrophic lateral sclerosis, and especially Parkinson's disease, mild cognitive impairment and dementia ( including Alzheimer's disease, vascular dementia and dementia with Lewy bodies).

Compounds of general formula (IA) and (IB) are particularly effective in the treatment or prevention of Parkinson's disease, especially compounds of general formula (IA) and (ID) in which both R ¹ and R ² are F.

Examples of particularly suitable compounds for use in the treatment of Parkinson's disease include the compound of general formula (IA), which is 2,2-difluoro-3b,7b-dihydroxy-5vcholanic acid (compound 7) and the compound of general formula (ID), which is 2,2difluoro-3a,7b-dihydroxy-5b-cholanic acid (compound 9). 2,2-Difluoro-3b,7b-dihydroxy-5vcholanic acid (compound 7) is particularly suitable.

The compounds of general formula (IB) are particularly effective in the treatment or prevention of dementia, such as Alzheimer's disease. This is especially true for a compound of general formula (IB) in which both R ¹ and R ² are F.

Accordingly, when the neurodegenerative disorder is dementia, especially Alzheimer's disease, the compound of general formula (IB) is 2,2-difluoro-3a,7adihydroxy-5b-cholanic acid (compound 8).

The compounds of general formula (I) are usually administered as part of a pharmaceutical composition.

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 or carrier.

The composition may be formulated for administration by any route, for example parenteral, including intravenous, intramuscular, subcutaneous or intradermal; or for oral, rectal, nasal, topical (including eye drops, topical pulmonary, buccal and sublingual) or vaginal administration.

Preferably, the composition is formulated for parenteral administration or for local administration to the lungs (by inhalation).

The composition can be prepared by combining the above-defined active agent with a carrier. Typically, preparations are prepared by uniformly and thoroughly combining the active agent with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product. The invention includes methods for preparing a pharmaceutical composition, comprising combining or combining a compound of general formula (I) with a pharmaceutically acceptable carrier or excipient.

The oral preparations of the present invention may be presented in the form of discrete units such as capsules, sachets or tablets, each containing a predetermined amount of the active agent; in the form of powder or granules; in the form of a solution or suspension of the active agent in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-oil emulsion liquid or a water-in-oil liquid emulsion; or in the form of a bolus and so on.

In some cases, the compositions may be formulated for delayed, sustained or controlled release of a compound of general formula (I).

For compositions for oral administration (for example, tablets and capsules), the term acceptable carrier includes carriers such as conventional excipients, for example, binders, for example syrup, gum acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose , hydroxypropyl methylcellulose, 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 metal stearates, glycerol stearate, stearic acid, silicone fluid, talc waxes, oils and colloidal silicon dioxide. Flavorings such as peppermint, wintergreen, cherry flavoring and the like may also be used. It may be desirable to add a coloring agent to make the dosage form easily identifiable. Tablets can also be coated by methods well known in the art.

The tablet may be prepared by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active agent into particulate form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant. Molded tablets can be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally film-coated or scored and may be formulated to provide delayed

- 19 046372 or controlled release of the active agent.

Other preparations suitable for oral administration include lozenges containing the active agent in a flavored base, usually sucrose and gum acacia or tragacanth; lozenges containing the active agent on an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes containing the active agent in a suitable liquid carrier.

For topical application to the skin, the compounds of general formula (I) may be formulated as a cream, ointment, jelly, solution or suspension, etc. The cream or ointment formulations that may be used for the medicament are conventional formulations well known in the art, for example as described in standard pharmaceutical textbooks such as the British Pharmacopoeia.

Local administration to the lungs can be achieved using an aerosol formulation. Aerosol formulations typically contain the active ingredient suspended or dissolved in a suitable aerosol propellant such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFC propellants include trichloromonofluoromethane (Propellant 11), dichlorotetrafluoromethane (Propellant 114) and dichlorodifluoromethane (Propellant 12). Suitable HFC propellants include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227). The propellant typically makes up 40%-99.5%, for example 40%-90%, by weight of the total inhalation composition. The formulation may contain excipients, including cosolvents (eg ethanol) and surfactants (eg lecithin, sorbitan trioleate and the like). Other possible excipients include polyethylene glycol, polyvinylpyrrolidone, glycerin and the like. Aerosol formulations are packaged in canisters and the appropriate dose is delivered using a dispenser (such as those supplied by Bespak, Valois or 3M, or alternatively Aptar, Coster or Vari).

Local administration to the lungs can also be achieved using a non-pressurized drug such as an aqueous solution or suspension. They can be administered via a nebulizer, such as a nebulizer that may be hand-held, portable, or for home or hospital use (ie, non-portable). The formulation may contain excipients such as water, buffers, tonicity adjusting agents, pH adjusting agents, surfactants and co-solvents. Suspension liquid and aerosol preparations (both pressurized and non-pressurized) typically contain the compound of the invention in finely divided form, for example with a D ₅₀ of 0.5-10 µm, for example about 1-5 µm. The particle size distribution can be represented using the values of D ₁₀ , D ₅₀ and D ₉₀ . The median value of the particle size distribution, D50, is defined as the particle size in microns that bisects the distribution. The measurement obtained by laser diffraction is more accurately described as a volume distribution, and therefore the D ₅₀ value obtained by this method is more properly called the Dv ₅₀ value (median for volume distribution). The Dv values used here refer to the particle size distribution measured using laser diffraction. Similarly, the values of _D10 and _D90 used in relation to laser diffraction are taken to indicate the values of _Dv10 and _Dv90 and refer to the particle size at which 10% of the distribution lies below the _D10 value and 90% of the distribution lies below the _D90 value respectively.

Local administration to the lungs can also be achieved using the drug in dry powder form. A dry powder formulation will contain the compound of the invention in finely divided form, typically having a mass median diameter (MMAD) of 1-10 µm or a D ₅₀ of 0.5-10 µm, for example about 1-5 µm. Powders of the compound of the invention in finely divided form can be obtained through a micronization process or a similar grinding process. Micronization can be carried out using a jet mill such as those manufactured by Hosokawa Alpine. The resulting particle distribution can be measured using laser diffraction (eg Malvern's Mastersizer 2000S analyzer). The formulation will typically contain a topically acceptable diluent, for example lactose, glucose or mannitol (preferably lactose), usually of a relatively large particle size, for example with a mass median diameter (MMAD) of 50 µm or more, for example 100 µm or more or D ₅₀ 40 -150 microns. As used herein, the term lactose refers to the lactose-containing component, including α-lactose monohydrate, β-lactose monohydrate, α-lactose anhydrous, β-lactose anhydrous and amorphous lactose. Lactose components can be processed by micronization, sieving, grinding, pressing, agglomeration or spray drying. Also included are commercially available lactose in various forms, such as the products Lactohale® (inhalation grade lactose; DFE Pharma), InhaLac®70 (sifted dry powder inhaler lactose; Meggle), Pharmatose® (DFE Pharma) and Respitose® (sifted lactose inhalation grade; DFE Pharma). In one embodiment, the lactose component is selected from the group consisting of α-lactose monohydrate, α-lactose anhydrous, and amorphous lactose. Preferably, the lactose is α-lactose monohydrate.

Dry powder preparations may also contain other excipients. Thus, in one

- 20 046372 embodiment of the dry powder preparation according to the present disclosure contains magnesium or calcium stearate. Such preparations may have high chemical and/or physical stability, especially when such preparations also contain lactose.

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

In one embodiment, the compound of general formula (I) is provided in the form of a micronized dry powder preparation, for example containing lactose of a suitable grade.

Thus, as an aspect of the invention, there is provided a pharmaceutical composition containing a compound of general formula (I) in particulate form in combination with particulate lactose, which composition optionally contains magnesium stearate.

In one embodiment, the compound of general formula (I) is provided as a micronized dry powder preparation containing appropriate grade lactose and magnesium stearate, which is filled into a device such as DISCUS. Accordingly, such a device is a multi-dose device, for example, the drug is filled into blisters for use in a multi-dose unit dose device such as DISCUS.

In another embodiment, the compound of general formula (I) is provided as a micronized dry powder preparation, eg containing a suitable grade of lactose, which is filled into hard capsules for use in a single-dose device such as an AEROLISER.

In another embodiment, the compound of general formula (I) is provided as a micronized dry powder preparation containing appropriate grade lactose and magnesium stearate, which is filled into hard capsules for use in a single-dose device such as an AEROLISER.

In another embodiment, the compound of general formula (I) is provided in the form of a fine powder for use in an inhalation dosage form, where the powder is in the form of fine particles with a D50 of 0.5-10 μm, for example about 1-5 μm, which were obtained using a grinding process other than jet mill micronization, such as spray drying, spray freezing, microfluidization, high pressure homogenization, supercritical fluid crystallization, ultrasonic crystallization, or combinations of these methods, or other suitable particle formation methods known in the art, which are used to produce small particles with an aerodynamic particle size of 0.5-10 microns. The resulting particle distribution can be measured using laser diffraction (eg Malvern's Mastersizer 2000S analyzer). The particles may contain the compound either alone or in combination with other suitable excipients which may aid in processing. The resulting fine particles may form a 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 present invention can also be administered rectally, for example in the form of suppositories or enemas, which include aqueous or oily solutions, as well as suspensions, emulsions and foams. Such compositions are prepared in accordance with standard methods well known to those skilled in the art. For example, suppositories can be prepared by mixing the active ingredient with a conventional suppository base such as cocoa butter or other glycerides. In this case, the drug is mixed with a suitable non-irritating excipient that is solid at normal temperatures but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such substances are cocoa butter and polyethylene glycols.

Preparations for parenteral administration usually must be sterile.

A medical practitioner or other qualified person will be able to determine the appropriate dosage for a compound of general formula (I) and, therefore, the amount of a compound of the invention to be included in any particular pharmaceutical preparation (whether in unit dosage form or not).

The compounds of general formula (I) can be used in combination with one or more other active agents that are useful in the treatment or prevention of neurodegenerative disorders.

Therefore, in an additional aspect of the invention there is provided a product containing a compound of general formula (I) and an additional agent useful in the treatment or prevention of a neurodegenerative disorder as a combination preparation for simultaneous, sequential or

- 21 046372 separate use in the treatment or prevention of a neurodegenerative disorder, as described above.

The invention will now be described in more detail with reference to the following examples and drawings in which:

In fig. 1 (FIGS. 1A, 1B and 1C) shows data for fibroblast cell lines from controls and 6 patients with sporadic Parkinson's disease (sPD). Controls are compared to untreated sPD fibroblasts, sPD fibroblasts incubated with UDCA, and sPD fibroblasts incubated with compound 7; fig. 1A: basal oxygen consumption; fig. 1B: maximum respiratory rate; fig. 1C: ATP-linked respiration.

In fig. Figure 2 shows the rate of extracellular acidification in fibroblast cell lines from a control group and from 6 patients with sporadic Parkinson's disease (sPD). Control fibroblasts are compared with untreated sPD fibroblasts, sPD fibroblasts incubated with UDCA, and sPD fibroblasts incubated with compound 7.

In fig. Figure 3 shows the activity of mitochondrial respiratory chain complex I in a homogenate from an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced mouse that was either not treated or was treated with compound 7 at a dose of 4 mg/kg or 12 mg/kg.

In fig. 4 shows a comparison of mitochondrial respiratory chain complex I activity in a homogenate of mouse brain tissue from the left striatum from a mouse treated with vehicle, vehicle plus 12 mg of Compound 7, MPTP, or MPTP plus 1 mg, 4 mg, or 12 mg of Compound 7.

Abbreviations.________________________________________________________________

AcOH acetic acid Vos otreot-butyloxycarbonyl 'VpON /7?/7<l7?-butanol BzOH benzoic acid Count calculated fromSRVA l/<l7?<7-chloroperbenzoic acid With day DAST diethylaminosulfur trifluoride DCM dichloromethane DEAD diethyl azodicarboxylate DIPA diisopropyl alcohol DIPEA ΛζΑ-diisopropylethylamine DMF ΛζΑ-dimethylformamide EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Equiv. equivalents EtOAc ethyl acetate EtOH ethanol Et _3N triethylamine h watch HATU (1 - [bis(dimethylamino)methylene]-1 Η-1,2,3 -

- 22 046372

triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate HOBt hydroxybenzotriazole HPLC high performance liquid chromatography MeOH methanol mole moths PTO methoxymethyl NaOMe sodium methoxide RE petroleum ether RRyZ triphenylphosphine R.M. reaction mixture K.t. room temperature Saturation saturated S.M. starting material _Tf2O trifluoromethanesulfonic acid anhydride (triflate anhydride) THF tetrahydrofuran TLC thin layer chromatography UDCA ursodeoxycholic acid

General methods.

General Method A for protecting a 24-carboxylic acid as a methyl ester.

The Pellicari method was used (ACS Med. Chem. Lett. 2012, 3, 273-277). Free bile acid (25.0 g; 64 mmol; 1 eq.) was dissolved in HPLC grade methanol (20 volumes) before addition of p-toluenesulfonic acid (0.1 eq.) and sonication at 30°C for 2 h. Once the reaction was complete, as determined by TLC analysis, the solvent was removed in vacuo before dissolving the residue in EtOAc (16 volumes), washing the organic phase with sat. NaHCO ₃ (twice), water and brine. The organic phase was then dried (Na ₂ SO ₄ ) and concentrated to give the methyl ester.

General Method B for 3-keto/7-keto reduction using NaBH ₄ /CeCl ₃ .

Using Cerny's conditions (Steroids 2012, 77, 1233-1241). To a solution of ketone (1 eq.) and CeCl ₃ (1.2 eq.) in MeOH (approximately 50 volumes) and EtOAc (2 ml) was added NaBH ₄ (1.1 eq.) over 5 minutes. The solution was stirred for 30 minutes, after which additional NaBH ₄ (1 eq.) was added and stirred for another 30 minutes to complete the reaction. The reaction was stopped by adding ice-cold 2 M HCl and the aqueous phase was washed with EtOAc (twice). The combined organic phases were washed with sat. NaHCO ₃ and water, dried (Na ₂ SO ₄ ) and concentrated, then further purified by column chromatography to obtain both aOH and β-OH epimers.

General Method C for saponification of methyl ester using LiOH to MeOH.

To a solution of methyl ester (43 mg; 0.11 mmol; 1 eq.) in MeOH (approximately 70 volumes) was added 2 M LiOH (10 eq.), and the solution was allowed to stir until reaction was complete at room temperature. The solvent was removed in vacuo and the crude residue was acidified with 2 M HCl before extraction with EtOAc (twice). The combined organic phases were washed with water and brine, dried (Na ₂ SO ₄ ) and concentrated to give the free acid.

General Method D for saponification of methyl ester using LiOH in THF.

Methyl ether (1.0 eq.) was dissolved in THF (approximately 20 volumes) and LiOH (2 M in H2O; 10 eq.) was added. After stirring at room temperature until the reaction was complete, the reaction mixture was reduced in vacuo, the resulting residue was acidified with 2 M HCl, and the aqueous phase was extracted with EtOAc (twice). The combined organic phases were then washed with water and brine before drying over Na ₂ SO ₄ and reduction in vacuo to give the free acid.

General Method E for acetate/benzoate methanolysis.

Acetate/benzoate protected bile acid (3.1 g; 6.12 mmol; 1 eq.) was dissolved in anhydrous MeOH (approximately 10 volumes) before adding 25% NaOMe in MeOH (approximately

- 23046372 volumes), and the reaction mixture was stirred at room temperature. Once the reaction was complete, the mixture was acidified to pH 4-5 with 2 M HCl and diluted with H ₂ O. The aqueous phase was extracted with DCM (twice), the combined organic phases were washed with NaHCO ₃ , dried (Na ₂ SO ₄ ) and concentrated to give removed protection. Used without further purification.

General method F for ester saponification using NaOH.

Ether (1.0 eq), NaOH (12.95 eq), and MeOH (HPLC grade; 8.35 mL/mol) were added to a round bottom flask. The reaction mixture was stirred at room temperature overnight. After completion of the reaction, as shown by TLC analysis, the solvent was removed under reduced pressure. The residue was diluted with water, acidified with aqueous HCl and extracted with EtOAc (three times). The combined organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give the desired compound (purification by flash chromatography if necessary).

General Method G for the reduction of ketone derivatives using NaBH ₄ .

Ketone (1.0 eq), sodium borohydride (2.4 eq), and anhydrous THF (approximately 40 volumes) were added to a round bottom flask. The reaction mixture was stirred at room temperature overnight, quenched with H ₂ O and extracted with ethyl acetate (three times). The combined organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude product was purified by flash chromatography and by HPLC where necessary.

General Method K for protecting secondary alcohol as MOM ester.

To a solution of the starting secondary alcohol (1 eq.) in anhydrous DCM (approximately 17 volumes) were added DIPEA (3 eq.) and MOM-Cl (5 eq.) at 0°C. The reaction mixture was warmed to room temperature before being left to stir overnight. Once stirring was complete, the reaction mixture was quenched with water (approximately 3.4 mL/mmol) and methanol (approximately 3.4 mL/mmol) before separating the layers, extracting the aqueous layer with EtOAc (four times), and washing the combined organic layers with brine (twice). . The organic phase was then dried (Na2SO4) and concentrated in vacuo to give the crude material. The crude material was purified by flash chromatography (HPLC if necessary) to obtain the desired compound.

General method L for eliminating the MOM group using HCl.

MOM-protected substance (1.5 g; 1.76 mmol; 1 eq.) was dissolved in MeOH (approximately 30 volumes) and 2 M HCl (approximately 5.7 ml/mmol), then the mixture was heated to 70°C for 5 hours. The reaction mixture was cooled and concentrated in vacuo, subjected to azeotropic distillation to dryness (MeOHx3, CHCl ₃ x1) to obtain the desired substance.

General Method M for secondary alcohol oxidation using DessaMartin periodinane.

To a solution of the starting secondary alcohol (1.0 eq.) in dichloromethane at 0°C, Dess-Martin periodinane (approximately 1.2 eq.) was added in portions over 10 min. After warming to room temperature for 18 hours, the reaction was determined to be complete by TLC analysis and the reaction mixture was quenched by adding sat. Na ₂ S ₂ O ₃ solution and sat. NaHCO ₃ solution. The aqueous phase was separated and extracted with dichloromethane (three times) and the combined organic fractions were washed with sat. NaHCO ₃ solution, water and brine, dried over MgSO ₄ , filtered and concentrated in vacuo to give the crude desired product. The crude product was purified by flash chromatography (HPLC if necessary) to obtain the desired compound.

General N method for hydrogenation/hydrogenolysis using a catalyst

Benzyl-protected bile acid (1.0 eq), catalyst [Pd/C or PtO ₂ ] (10 mol%) and solvent [MeOH, EtOH, etc.] were added to a round bottom flask. The reaction mixture was degassed with hydrogen gas and then stirred under H2 at atmospheric pressure or elevated pressure for 16-72 hours. The catalyst was filtered through celite and the filtrate was concentrated and purified by flash chromatography.

General O method for producing enol silyl ether from ketone derivatives

DIPA (12.6 eq) and THF (1.25 mL/mmol) were added to the round bottom flask. The solution was cooled to -78°C, then n-butyllithium (2.5 M in hexanes; 12 eq.) was added dropwise and stirred at -78°C for 30 minutes. TMSCl (10 eq.) was added and stirred for 20 minutes. A solution of the ketone derivative (1 eq) in THF (6.5 ml/mmol) was then added dropwise over 10 min and stirred at this temperature for 45 min, followed by the addition of triethylamine (18 eq) and stirred for 1 h The reaction mixture was warmed to -20°C, quenched with saturated NaHCO3 solution and warmed to room temperature for 2 hours. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (three times). The combined organic layer was washed with saturated NaHCO3 solution, water, brine, dried over Na ₂ SO ₄ , filtered and concentrated to obtain the desired enol silyl ether intermediate, which was used for subsequent reaction without any purification.

General Method P for electrophilic fluorination of enol silyl ether using

- 24 046372

Selectfluor (bis-(tetrafluoroborate) 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane) in DMF.

Enol silyl ether derivative (1.0 eq), DMF (2 mL/mmol), and a solution of Selectfluor (1.5 eq) in DMF (3 mL/mmol) were added to a round bottom flask at 0°C. The reaction mixture was stirred at room temperature overnight, quenched with H2O and extracted with ethyl acetate (four times). The combined organic layer was washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude product was purified by flash chromatography and HPLC where necessary.

General Q method for the formation of conjugates.

Fluorinated bile acid (1 eq.) was dissolved in anhydrous DMF (12 volumes) with stirring under argon. HATU (1 eq.), DIPEA (3.0 eq.), and amino acid (1.1 eq.) were added and the reaction mixture was stirred at room temperature for 16 hours. Once the reaction was complete, the reaction mixture was dry loaded directly onto silica and purified by C18 column chromatography (gradient elution MeOH in H2O, 0100%) to give the conjugate as either the DIPEA salt or the free acid. The DIPEA salts were then treated with a sodium ion exchange resin column to obtain the desired sodium salt of the compounds as a residue.

Example 1. Synthesis of 2b-fluoro compounds.

A. Methyl 3a,7a-dihydroxy-5b-cholanoate (1A.1)

1A.1

The Pellicari method was used (ACS Med. Chem. Lett. 2012, 3, 273-277). CDCA (chenodeoxycholic acid) (25.0 g; 64 mmol; 1 eq.) was dissolved in HPLC grade methanol (500 ml) before adding para-toluenesulfonic acid (1.21 g; 6.4 mmol; 0.1 eq.). ) and sonication at 30°C for 2 hours. Once complete, as determined by TLC analysis, the solvent was removed under vacuum before dissolving the residue in EtOAc (400 ml), washing the organic phase with sat. NaHCO3 (2x150 ml), water (250 ml) and brine (250 ml). The organic phase was then dried (Na ₂ SO ₄ ) and concentrated to give the title compound as a white/pale yellow solid (26.0 g; quantitative) (General Method A).

^1H NMR (400 MHz, CDCl ₃ ): δ 3.84 (1H, q, J=2.4 Hz), 3.66 (3H, s), 3.44 (1H, tt, J=10.9, 4.5 Hz) , 2.34 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.28-2.15 (2H, m), 2.12-0.97 (26H, m), 0.93 (3H, d, J=6 ,2 Hz), 0.90 (3H, s), 0.65 (3H, s) ppm.

MCHP (low resolution mass spectrometry) (ESI+ (electrospray ionization)) m/z: 429.1 [M+Na]+.

B. Methyl 7a-hydroxy-3-oxo-5b-cholanoate (1B.1)

B.1

To a solution of methyl 3a,7a-dihydroxy-5b-cholanoate (10.0 g; 24.6 mmol; 1 eq) in water (25 ml) and tert-butanol (100 ml) was added KBr (5.9 g; 0 mmol; 2 eq.), KHCO3 (24.6 g; 246 mmol; 10 eq.) and TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl) (5.0 g; 32.0 mmol; 1.3 eq.). The solution was cooled to 0°C before adding approximately 11% NaClO solution (54.2 ml; 73.2 mmol; 3.0 eq.) in portions over 6 hours. The reaction was stopped by slowly adding sodium thiosulfate solution (300 ml; 1.2 M; 350 mmol). The aqueous phase was extracted with EtOAc (2x300 ml), the extracts were combined and washed with brine (300 ml) and water (300 ml) before drying (Na ₂ SO ₄ ) and removing the solvent under vacuum. The resulting bright red thick oily crude (15 g) was purified using flash chromatography (PE/EtOAc, 80:20 to 65:35) to give a white solid (6.5 g; 16.0 mmol; 66% ).

1H NMR (400 MHz, CDCl ₃ ): δ 3.93 (1H, br s), 3.67 (3H, s), 3.40 (1H, t, J=14.4 Hz), 2.46-1.10 (30H, m), 1.01 (3H, s), 0.95 (3H, d, J=6.6 Hz), 0.71 (3H, s) ppm.

MSNR (IER+) m/z: 422.1 [M+NH ₄ ] ⁺ , 427.1 [M+Na] ⁺ .

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C. Methyl 7a-methoxymethoxyl-3-oxo-5b-cholanoate (1C.1)

1B.1

1C.1

To a solution of methyl 7a-hydroxy-3-oxo-5b-cholanoate (3.0 g; 7.41 mmol; 1 eq.) in anhydrous DCM (50 ml) was added DIPEA (3.83 ml; 22.2 mmol; 3 equiv.) and MOM-Cl (2.82 ml; 37.1 mmol; 5 equiv.) at 0°C. The reaction mixture was warmed to room temperature before being left to stir overnight. Once the reaction was complete, the reaction mixture was quenched with water (25 mL) and methanol (25 mL) before separating the layers, and extracting the aqueous layer with EtOAc (4 x 75 mL), and washing the combined organic layers with brine (2 x 150 mL). The organic phase was then dried (Na ₂ SO ₄ ) and concentrated in vacuo to give 3.8 g of crude material, which was purified by flash chromatography (PE/EtOAc, 75:25) to give a white solid (3.10 g, 6.9 mmol; 93%).

^1H NMR (400 MHz, CDCl ₃ ): δ 4.68 (1H, d, J=6.8 Hz), 4.55 (1H, d, J=6.8 Hz), 3.72-3.62 (4H, m), 3.43 -3.28 (4H, m), 2.49-1.05 (27H, m), 1.03 (3H, s), 0.94 (3H, d, J=6.4 Hz), 0.69 (3H, s) ppm.

MSNR (IER+) m/z: 449.3 [M+H]+, 471.1 [M+Na]+.

D. Methyl 7a-methoxymethoxyl-3-trimethylsilyloxy-5b-chol-2-enoate (1D.1) and methyl 7a-methoxymethoxyl-3-trimethylsilyloxy-5b-chol-3-enoate (ID.2)

The method of Barlow et al. was followed. (Eur. J. Med. Chem. 2011, 46, 1545-1554). Et ₃ N (0.62 mL; .46 mmol; 2 eq.) and trimethylsilyl triflate (0.44 ml; 2.45 mmol; 1.1 eq.). The reaction mixture was left to stir for 1 h before diluting with additional DCM (150 ml) and quenching with sat. NaHCO ₃ (100 ml). The layers were separated and the aqueous phase was extracted with additional DCM (3x100 ml), the extracts were combined and washed with brine (150 ml), dried (Na ₂ SO ₄ ) and concentrated to give a colorless oil (1.2 g) which contained methyl-7a -methoxymethoxyl-3-trimethylsilyloxy-5b-chol-2-enoate (IA.1) and methyl 7a-methoxymethoxyl3-trimethylsilyloxy-5b-chol-3-enoate (1A.2) in a ratio of approximately 1:1. This crude material was used in subsequent steps without further purification.

^1H NMR (400 MHz, CDCh): δ 4.80-4.44 (3H, m), 3.67 (3H, s), 3.66-3.56 (1H, m), 3.40 (1.5H, s), 3.36 (1.5H , s), 2.51-1.08 (31H, m), 0.99-0.95 (3H, m), 0.93 (3H, d, J=6.2 Hz), 0.66 (1.5H, br. s), 0.65 (1 ,5H, br. s), 0.21-0.15 (9H, m) ppm

E. Methyl 2b-fluoro-7a-methoxymethoxyl-3-oxo-5b-cholanoate (1E.1) and methyl 4b-fluoro-7amethoxymethoxyl-3-oxo-5 β-cholanoate (1E.2)

The method of Fujimoto et al. was followed. (Bioorg. Med. Chem. Lett. 2011, 21, 6409-6413). To a solution of 7α-methoxymethoxy-3-trimethylsilyloxy-5v-chol-2-enoate and methyl 7a-methoxymethoxy-3-trimethylsilyloxy-5v-chol-3-enoate (1.10 g; 2.2 mmol; 1 eq.) in anhydrous Selectfluor® was added to acetonitrile

- 26 046372 (1.20 g; 3.3 mmol; 1.5 eq.), leaving the reaction mixture to stir at room temperature for 4 hours. The solvent was removed under vacuum before dilution with EtOAc (100 ml) and water (100 ml). The layers were separated before extracting the aqueous layer with additional EtOAc (2x100 ml). The combined organic layers were then washed with brine (150 ml), dried (Na ₂ SO ₄ ) and concentrated to give 1.05 g of a pale yellow crude solid. The crude material was purified using flash chromatography (PE/EtOAc, 80:20) to give methyl 2bfluoro-7a-methoxymethoxyl-3-oxo-5b-cholanoate as a white solid (377 mg; 0.81 mmol; 36% in two steps) and methyl 4b-fluoro-7a-methoxymethoxyl-3-oxo-5b-cholanoate as a white solid (321 mg; 0.69 mmol; 31% in two steps).

1E.1: 1H NMR (400 MHz, CDCl ₃ ): δ 5.05 (1H, ddd, J=49.4, 13.5, 5.6 Hz), 4.67 (1H, d, J=6.8 Hz) , 4.54 (1H, d, J=6.8 Hz), 3.67 (4H, s), 3.48 (1H, t, J=13.9 Hz), 3.37 (3H, s), 2.52 (1H, dt, J =12.7, 6.1 Hz), 2.42-1.12 (27H, m), 1.08 (3H, s), 0.95 (3H, d, J=6.6 Hz), 0.69 (3H, s) ppm .

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -195.19 (ddt, J=49.2, 9.6, 6.2 Hz) ppm.

MSNR (ESI+) m/z: 484.2 [M+NH ₄ ] ⁺ , 489.1 [M+Na] ⁺ .

1E.2: ^1H NMR (400 MHz, CDCl ₃ ): δ 5.78 (1H, dd, J=46.8, 11.7 Hz), 4.77 (1H, d, J=6.8 Hz), 4.59 ( 1H, d, J=6.8 Hz), 3.78 (1H, q, J=2.7 Hz), 3.67 (3H, s), 3.40 (3H, s), 2.52 (1H, td, J=14, 4, 4.9 Hz), 2.42 1.11 (24H, m), 1.07 (3H, s), 0.94 (3H, d, J=6.4 Hz), 0.70 (3H, s) ppm

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -200.67 (ddd, J=46.8, 12.1, 6.9 Hz) ppm.

MSNR (IER ⁺ ) m/z: 484.2 (M+NH ₄ ) ⁺ , 489.1 (M+Na) ⁺ .

F. Methyl 2b-fluoro-3a-hydroxy-7a-methoxymethoxyl-5b-cholanoate (1F.1) and methyl 2b-fluoro-3bhydroxy-7a-methoxymethoxyl-5 β-cholanoate (1F.2)

To a solution of methyl 2b-fluoro-7a-methoxymethoxyl-3-oxo-5b-cholanoate (260 mg; 0.56 mmol; 1 eq.) in anhydrous tetrahydrofuran (20 ml) was added sodium borohydride (64 mg; 1.70 mmol ; 3 eq.), and the reaction mixture was left to stir overnight at room temperature. Once the reaction was complete, as determined by TLC analysis, the reaction mixture was diluted with EtOAc (150 mL) and quenched with water (100 mL), separating the layers before extracting the aqueous layer with additional EtOAc (2x100 mL). The combined organic layers were then washed with water (150 ml) and brine (150 ml) before drying (Na ₂ SO ₄ ) and concentration in vacuo to give 306 mg of a pale crude oil. The crude material was purified by flash chromatography (PE/EtOAc, 65:35) to give methyl 2b-fluoro-3a-hydroxy-7a-methoxymethoxyl-5b-cholanoate as a white solid (144 mg; 0.307 mmol; 55%) and methyl 2b-fluoro-3b-hydroxy-7amethoxymethoxyl-5b-cholanoate as a colorless gum (88 mg; 0.19 mmol; 34%).

1F.1: 1H NMR (400 MHz, CDCl ₃ ): δ 4.68 (1H, d, J=6.8 Hz), 4.54 (1H, d, J=7.1 Hz), 4.40 (1H, dddd, J =52.3, 12.0, 8.6, 4.4 Hz), 3.66 (3H, s), 3.62-3.47 (3H, m), 3.37 (3H, s), 2.48-1.01 (27H, m) , 0.99 (3H, s), 0.92 (3H, d, J=6.4 Hz), 0.64 (3H, s) ppm.

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -187.47 (ddq, J=52.3, 12.7, 7.1 Hz) ppm.

MSNR (IER ⁺ ) m/z: 486.1 [M+NH ₄ ] ⁺ , 491.2 [M+Na] ⁺ .

1F.2: 1H NMR (400 MHz, CDCl ₃ ): δ 4.72-4.51 (3H, m), 4.20-4.09 (1H, m), 3.66 (3H, s), 3.59 (1H, d, J=2, 4 Hz), 3.38 (3H, s), 2.45 (1H, ddd, J=15.2, 12.4, 2.2 Hz), 2.35 (1H, ddd, J=15.0, 10.3, 5 ,1 Hz), 2.22 (1H, ddd, J=15.6, 9.5, 6.5 Hz), 2.06-1.05 (25H, m), 1.02 (3H, s), 0.93 (3H, d, J =6.6 Hz), 0.65 (3H, s) ppm.

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -187.31 (dquin, J=47.2, 7.5 Hz) ppm.

MSNR (IER ⁺ ) m/z: 486.0 [M+NH ₄ ] ⁺ , 491.1 [M+Na] ⁺ .

G. 2b-Fluorochenodeoxycholic acid (compound 1)

1F.1 Connection 1 (70%)

Using General Method L followed by General Method C, methyl 2b-fluoro-3a-hydroxy-7a-methoxymethoxyl-5b-cholanoate (1F.1; 118 mg; 0.25 mmol; 1 eq.) was deprotected to give 2b-fluorochenodeoxycholic acid (compound 1) as a pale yellow solid (72 mg; 0.18 mmol; 70%).

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Compound 1: 1H NMR (400 MHz, CD ₃ OD): δ 4.32 (1H, dddd, J=52.5, 12.5, 8.6, 4.0 Hz), 3.78 (1H, q, J=2 ,3 Hz), 3.44 (1H, tdd, J=12.0, 8.7, 5.0 Hz), 2.43 (1H, q, J=13.2 Hz), 2.33 (1H, ddd, J=15 ,5, 11.0, 5.0 Hz), 2.25-2.11 (2H, m), 2.04 (1H, dt, J=12.4, 2.9 Hz), 1.99-1.04 (22H, m), 1.00 (3H, s), 0.97 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CD3OD): δ -186.77 (ddq, J=52.2, 11.9, 7.8 Hz) ppm.

MSNR (IER+) m/z: 393.1 [M+HH ₂ O] ⁺ , 821.2 [2M+H] ⁺ , 843.3 [2M+Na] ⁺ .

H. 2b-Fluoro-3b,7a-dihydroxy-5b-cholanic acid (compound 2)

Using General Method L followed by General Method C, methyl 2b-fluoro-3b-hydroxy-7a-methoxymethoxyl-5b-cholanoate (1F.2; 25 mg; 0.053 mmol; 1 eq.) was deprotected to give 2b- fluoro-3b,7a-dihydroxy-5b-cholanic acid (compound 2) as a gummy solid (21 mg; 0.05 mmol; 96%).

Compound 2: ^1H NMR (400 MHz, _acetone4O6 ): δ 10.42 (1H, br. s), 4.58 (1H, dddd, J=47.7, 12.1, 4.4, 2.8 Hz), 4.12-4.00 (1H, m), 3.80 (1H, q, J=2.4 Hz), 3.59 (1H, br. s), 3.29 (1H, br. s), 2.61 (1H, ddd, J=15 ,3, 12.8, 2.3 Hz), 2.34 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.21 (1H, ddd, J=15.5, 9.6 , 6.4 Hz), 2.03-1.03 (31H, m), 1.01 (3H, s), 0.97 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm.

¹⁹ F NMR (376 MHz, acetone-O ₆ ): δ -186.90 (dqum, J=47.6, 7.6 Hz) ppm.

MSNR (IER ⁺ ) m/z: 393.4 [M+H-H2O] ⁺ , 373.4 [M+H-H2O-HF] ⁺ , 355.5 [M+H-2H2O-HF] ⁺ .

Example 2. Synthesis of 2a-fluoro compounds.

A. Methyl 7-oxo-5v-chol-2-enoate (2A.1) and methyl 7-oxo-5v-chol-3-enoate (2A.2)

66% _2A1 2A.2

Methyl 3a-hydroxyl-7-oxo-5b-cholanoate (60 g; 148 mmol; 1.0 eq.; synthesized from 7-ketolithocholic acid using Method A) and DMAP (30 g; 122 mmol; 2.0 eq.) dissolved in DCM (500 ml) and cooled to 0°C on ice. Triflate anhydride (26.1 mL; 156 mmol; 1.05 eq.) was then added over 15 min. The reaction mixture was stirred at 0°C for 2 hours, although there was no reaction. The reaction mixture was then slowly heated to 10-12°C and the reaction was monitored by TLC. After confirmation of completion of the reaction after 2 hours, the reaction mixture was quenched with 2 M HCl (500 ml) and stirred at room temperature for 10 minutes. The layers were separated and the aqueous layer was extracted with brine (500 ml), dried (Na ₂ SO ₄ ) and concentrated to give 78 g of a brown tarry solid. The crude material was purified by flash chromatography (petroleum ether/EtOAc, 95:5 to 90:10) to give a mixture of alkenes methyl 7-oxo-5B-chol-2-enoate and methyl 7-oxo-5B-chol- 3-enoate as a colorless gum (37.5 g; 97 mmol; 66%).

2A.1 and 2A.2: 1H NMR (400 MHz, CDCl ₃ ): δ 5.67-5.30 (2H, m), 3.65 (3H, s), 2.89-2.79 (1H, m), 2.58-2.48 (1H, m), 2.44-1.26 (21H, m), 1.23 (2H, s), 1.21 (1H, s), 0.91-0.88 (3H, m), 0.65 (3H, m) ppm.

MSNR (IER ⁺ ) m/z: 387.2 [M+H] ⁺ , 404.2 [M+NH ₄ ] ⁺ .

B. Methyl 2v,3v-epoxy-7-oxo-5v-cholanoate (2B.1) and methyl 3v,4v-epoxy-7-oxo-5v-cholanoate (2B.2)

2A.1 +AZ,4-isomer 2A2 2B.1 (-11%) 2B.2 (-22%)

A mixture of methyl 7-oxo-5v-chol-2-enoate and methyl 7-oxo-5v-chol-3-enoate alkenes (20 g; 51.8 mmol; 1 eq.) was dissolved in DCM (200 ml) at room temperature before adding mCPBA (19.1 g; 77.7 mmol; 1.5 eq.). The completion of the reaction was confirmed after 1.5 hours, and during the reaction the mixture turned from a solution to a suspension. The reaction mixture was quenched with sat. aq. Na ₂ S ₂ O ₃ (150 ml) and left to stir for 30 minutes. Additional DCM (200 ml) and H ₂ O (150 ml) were added to facilitate solvation. The layers were separated and the aqueous layer was extra

- 28 046372 were treated with additional DCM (200 ml), then the combined organic layers were washed with sat. aq. NaHCO ₃ (200 ml) and dried (Na ₂ SO ₄ ) and concentrated to obtain 20.5 g of a pale yellow tarry solid. The crude material was purified by flash chromatography (petroleum ether/EtOAc, 92.5:7.5 to 92:8 to 80:10 to 88:12 to 80:20) to obtain pure Δ3β,4β-epoxide (2.00 g) together with Δ2β,3β-epoxide 80% purity (1.85 g) and a significant amount of mixed fractions (8.5 g). The fraction mixture was repurified (petroleum ether/EtOAc, 93:7 to 92:8 to 91:9 to 80:10 to 88:12 to 85:15 to 80:20) to obtain pure Δ3β,4β-epoxide (0. 8 g) together with 80% pure Δ3β,4β-epoxide (2.15 g) and 60% pure Δ2,3-epoxide (1.30 g). A total of methyl 2b,3b-epoxy-7-oxo-5b-cholanoate was isolated as a white crystalline solid (approximately 2.3 g; 5.8 mmol; 11%) along with methyl 3b,4b-epoxy -7-oxo-5b-cholanoate as a white solid (approximately 4.5 g; 11.3 mmol; 22%).

2B.1: ^1H NMR (400 MHz, CDCl ₃ ): δ 3.63 (3H, s), 3.13-3.09 (1H, m), 2.98 (1H, dd, J=5.3, 4.4 Hz), 2.78 (1H, dd, J=12.3, 4.3 Hz), 2.39-2.11 (5H, m), 1.91 (6H, m), 1.61-1.17 (10H, m), 1.13 (3H, m), 1.09-0.92 (2H, m), 0.89 (3H, d, J=6.5 Hz), 0.63 (3H, s) ppm.

MSNR (IER) m/z: 403.1 [M+H] ⁺ , 425.2 [M+Na] ⁺ , 403.1 [M+H-MeCN] ⁺ .

2B.2: 1H NMR (400 MHz, CDCl3): δ 3.64 (3H, s), 3.16-3.13 (1H, m), 2.89 (1H, dd, J=12.6, 7.0 Hz), 2.82 ( 1H, d, J=3.8 Hz), 2.42-1.17 (24H, m), 1.13 (3H, s), 0.89 (3H, d, J=6.5 Hz), 0.64 (3H, s) m. d.

MSNR (IER) m/z: 403.2 [M+H] ⁺ , 425.2 [M+Na] ⁺ .

C. Methyl 2a-fluoro-3b-hydroxy-7-oxo-5b-cholanoate (2C.1) \ O \ O ^Z HF pyr. (70%) F/,, ¹

0°C-K.t.

2B.1 2C.1 (80%)

A solution of methyl 2b,3b-epoxy-7-oxo-5b-cholanoate (830 mg; 2.06 mmol; 1 eq.) in anhydrous DCM (25 ml) was cooled to 0°C before adding 70% HF-pyridine complex ( 830 µl) and left to warm to room temperature. After confirmation of completion of the reaction after 2 days, the reaction mixture was again cooled to 0°C and carefully quenched by dropwise addition of saturated NaHCO ₃ (20 ml). The layers were separated and the aqueous layer was extracted with additional DCM (20 ml); the combined organic phases were washed with 2 M HCl and brine (30 ml each), dried (Na2SO4) and concentrated to give 840 mg of a white foamy solid. The crude material was purified by flash chromatography (PE/EtOAc, 70:30) to obtain methyl 2fluoro-3b-hydroxy-7-oxo-5b-cholanoate as a gummy solid (700 mg; 1.66 mmol; 80%) .

1H NMR (400 MHz, CDCl3): δ 4.53 (1H, dq, J=47.0, 2.6 Hz), 4.04-3.96 (1H, m), 3.65 (3H, s), 2.87 (1H, dd, J=12.7, 6.1 Hz), 2.42-1.25 (25H, m), 1.22 (3H, s), 1.20-1.00 (3H, m), 0.90 (3H, d, J=6.4 Hz) , 0.64 (3H, s) ppm

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -184.60 (tt, J=48.6, 8.7 Hz) ppm.

MSNR (IER) m/z: 423.1 [M+H] ⁺ , 445.1 [M+Na] ⁺ , 845.5 [2M+H] ⁺ .

D. Methyl 2a-fluoro-3a-benzoyloxy-7-oxo-5b-cholanoate (2D.1)

2C.1 2D.1

To a solution of methyl 2a-fluoro-3b-hydroxy-7-oxo-5b-cholanoate (1.05 g; 2.5 mmol; 1 eq.), PPh ₃ (980 mg; 3.7 mmol; 1.5 eq. .) and benzoic acid (450 mg; 3.7 mmol; 1.5 eq.) in anhydrous THF (25 ml) was added DEAD (650 μl; 3.7 mmol; 1.5 eq.). The solution was left to stir at 30°C over the weekend, after which ¹⁹ F NMR analysis of the crude material showed approximately 40% conversion to the desired benzoate. Additional amounts of PPh ₃ , BzOH and DEAD (1.5 eq. each) were added and the reaction mixture was left to stir overnight, after which the conversion was approximately 60%. Additional amounts of PPh ₃ , benzoic acid and DEAD (0.5 eq. each) were added, stirred overnight and 80% conversion was achieved. More PPh ₃ , benzoic acid and DEAD (0.5 eq each) were added and stirred again overnight, although no further interaction was observed. The solvent was removed in vacuo and the crude bright yellow material was separated by flash chromatography (PE/EtOAc, 98:2 to 95:5 to 85:15 to 70:30 to 0:100) to give 285 mg of methyl-2a -fluoro-3a-benzoyloxy-7-oxo-5b-cholanoate (approximately 90% purity) together with 1.28 g of additional mixed fractions.

1H NMR (400 MHz, CDCl ₃ ): δ 8.04 (2H, dd, J=7.8, 1.2 Hz), 7.56 (1H, tt, J=7.6, 1.2 Hz), 7.44 (2H , t, J=7.8

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Hz), 5.12-4.79 (2H, m), 3.67 (3H, s), 2.92 (1H, dd, J=12.6, 5.9 Hz), 2.53-1.29 (21H, m), 1.26 (3H, s), 1.24-1.04 (4H, m), 0.93 (3H, d, J=6.4 Hz), 0.67 (3H, s) ppm.

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -199.45 (tdd, J=49.9, 28.6, 8.7 Hz) ppm.

MCHP (IER+) m/z: 527.2 [M+H]+, 544.1 [M+NH ₄ ]+, 549.1 [M+Na]+.

E. Methyl 2a-fluoro-3a-hydroxy-7-oxo-5b-cholanoate (2E.1)

The method of Zhao et al. (Eur. J. Org. Chem., 2005, 2005, 4414-4427). A mixture of methyl 2a-fluoro3a-benzoyloxy-7-oxo-5b-cholanoate (400 mg; 0.76 mmol; 1 eq.) and potassium carbonate (20 mg; 0.15 mmol; 0.2 eq.) was suspended in anhydrous MeOH (20 ml) and left to stir for 16 hours at room temperature. After 16 h, the reaction mixture turned into a colorless solution, and completion of the reaction was determined by TLC analysis. The solvent was removed in vacuo and the crude residue was partitioned between EtOAc/H ₂ O (5 ml each) and the aqueous layer was extracted with additional EtOAc (2x5 ml). The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated to give 320 mg of pale gum. The crude material was purified by flash chromatography (PE/acetone, 70:30) to give methyl 2a-fluoro-3a-hydroxy-7-oxo-5b-cholanoate (275 mg; 0.65 mmol; 86%) as a gummy solid matter.

¹ H NMR (400 MHz, CDCl ₃ ): δ 4.71 (1H, d, J=52.0 Hz), 3.62 (3H, s), 3.59-3.45 (1H, m), 2.84 (1H, dd, J= 12.5, 6.0 Hz), 2.43 (1H, d, J=8.3 Hz), 2.39-2.26 (3H, m), 2.24-2.08 (2H, m), 2.01-1.20 (18H, m) , 1.18 (3H, s), 1.14-1.02 (3H, m), 0.88 (3H, d, J=6.5 Hz), 0.61 (3H, s) ppm

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -202.32 (tdd, J=51.2, 29.5, 8.7 Hz) ppm.

MSNR (IER+) m/z: 423.4 [M+H]+.

F. Methyl 2a-fluoro-3b,7a-dihydroxy-5b-cholanoate (2F.1) and methyl 2a-fluoro-3b,7b-dihydroxy-5b cholanoate (2F.2)

Using General Method B, methyl 2a-fluoro-3b-hydroxy-7-oxo-5vcholanoate (300 mg; 0.71 mmol; 1 eq.) was reduced. The crude material was purified by flash chromatography (PE/EtOAc, 65:35 to 55:45) to give methyl 2a-fluoro-3b,7a-dihydroxy-5b cholanoate (140 mg; 0.33 mmol; 46%) and methyl 2a-fluoro-3b,7b-dihydroxy-5b-cholanoate (107 mg; 0.25 mmol; 35%).

2F.1: 1H NMR (400 MHz, CDCl ₃ ): δ 4.55 (1H, dq, J=47.4, 2.8 Hz), 4.00 (1H, dq, J=7.2, 3.4 Hz) , 3.86 (1H, q, J=2.6 Hz), 3.66 (3H, s), 2.72 (1H, tt, J=14.3, 2.4 Hz), 2.35 (1H, ddd, J=15, 5, 11.0, 5.0 Hz), 2.22 (1H, ddd, J=15.7, 9.4, 6.5 Hz), 2.11-1.06 (27H, m), 0.97 (3H, s), 0.92 (3H, d, J=6.5 Hz), 0.66 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl3): δ -184.70 (tt, J=48.6, 8.7 Hz) ppm.

MSNR (ESI+) m/z: 447.3 [M+Na]+.

2F.2: 1H NMR (400 MHz, CDCl ₃ ): δ 4.50 (1H, dq, J=47.3, 2.8 Hz), 3.95 (1H, dq, J=7.2, 3.4 Hz) , 3.63 (3H, s), 3.55 (1H, td, J=9.7, 5.1 Hz), 2.32 (1H, ddd, J=15.4, 10.2, 5.0 Hz), 2.19 ( 1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.111.02 (27H, m), 0.96 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -184.43 (tt, J=47.7, 8.7 Hz) ppm.

MSNR (ESI+) m/z: 447.2 [M+Na]+.

G. Methyl 2a-fluoro-3a,7a-dihydroxy-5b-cholanoate (2G.1) and methyl 2a-fluoro-3a,7b-dihydroxy-5v-cholanoate (2G.2)

Using General Method B, methyl 2a-fluoro-3a-hydroxy-7-oxo-5b-cholanoate was reduced

- 30 046372 (270 mg; 0.64 mmol; 1 eq.). The crude material was purified by flash chromatography (PE/acetone, 75:25) to obtain 33 mg of pure 7a-OH analogue along with 140 mg of a mixture of both 7a-OH and 7e-OH epimers. The mixture was repurified by flash chromatography (PE/EtOAc, 60:40 to 50:50) to provide additional pure methyl 2a-fluoro-3a,7a-dihydroxy-5b-cholanoate (total 74 mg; 0.17 mmol ; 27%) and pure methyl 2a-fluoro-3a,7b-dihydroxy-5b-cholanoate (45 mg; 0.11 mmol; 17%), both as gummy solids.

2G.1: ^1H NMR (400 MHz, CDCl ₃ ): δ 4.67 (1H, d, J=52.1 Hz), 3.78 (1H, q, J=2.4 Hz), 3.59 (3H, s) , 3.37 (1H, dddd, J=28.5, 12.0, 4.4, 2.5 Hz), 2.46 (1H, q, J=13.0 Hz), 2.37-2.23 (2H, m), 2.20-2.10 (1H, m), 2.04-0.88 (27H, m), 0.88-0.83 (6H, m), 0.59 (3H, s) ppm

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -202.71 (tdd, J=52.0, 27.7, 8.7 Hz) ppm.

MSNR (IER+) m/z: 407.4 [M+HH ₂ O] ⁺ , 387.3 [M+HH ₂ O-HF] ⁺ .

2G.2: 1H NMR (400 MHz, CDCl ₃ ): δ 4.74 (1H, d, J=52.1 Hz), 3.66 (3H, s), 3.63-3.35 (2H, m), 2.43-2.29 (2H , m), 2.28-2.14 (1H, m), 2.08-1.00 (29H, m), 0.96 (3H, s), 0.92 (3H, d, J=6.2 Hz), 0.67 (3H, s) m .d.

¹⁹ F NMR (CDCl ₃ , 376 MHz): δ -202.49 (tdd, J=52.0, 29.5, 8.7 Hz) ppm.

MSNR (IER ⁺ ) m/z 407.4 [M+H-H2O] ⁺ , 387.3 [M+H-H2O-HF] ⁺ .

H. 2a-Fluoro-3b,7a-dihydroxy-5b-cholanic acid (compound 3)

2F.1 Compound 3 (94%)

Using the general method C, methyl 2a-fluoro-3b,7a-dihydroxy-5b-cholanoate (105 mg; 0.25 mmol; 1 eq.) was hydrolyzed to give 2a-fluoro-3b,7a-dihydroxy-5b-cholanic acid (compound 3) (96 mg; 0.23 mmol; 94%) as a pale solid.

1H NMR (400 MHz, acetone^): δ 10.51 (1H, br. s), 4.58 (1H, dq, J=48.0, 2.7 Hz), 3.96 (1H, dq, J=7.2, 3 ,4 Hz), 3.91 (1H, q, J=2.8 Hz), 2.89 (1H, tt, J=14.2, 2.6 Hz), 2.43 (1H, ddd, J=15.5, 10 ,7, 5.3 Hz), 2.30 (1H, ddd, J=15.0, 9.4, 6.7 Hz), 2.20-2.06 (4H, m), 2.01-1.13 (21H, m), 1.11 -0.97 (6H, m), 0.78 (3H, s) ppm

¹⁹ F NMR (376 MHz, acetone^): δ - 184.37 (tt, J=49.4, 8.7 Hz) ppm.

MSNR (PER ^- ) m/z: 409.1 [MH] ^- , 819.5 [MH] ^- .

I. 2a-Fluoro-3b,7b-dihydroxy-5b-cholanic acid (compound 4)

2F.2 Compound 4 (93%)

Using the general method C, methyl 2a-fluoro-3b,7b-dihydroxy-5b-cholanoate (90 mg; 0.21 mmol; 1 eq.) was hydrolyzed to give 2a-fluoro-3b,7b-dihydroxy-5b-cholanic acid (compound 4) (80 mg; 0.19 mmol; 93%) as a colorless solid.

1H NMR (400 MHz, acetone^): δ 10.43 (br. s), 4.58 (1H, dq, J=48.0, 3.1 Hz), 3.99 (1H, dq, J=7.1, 3.3 Hz ), 3.57 (1H, tdd, J=10.2, 5.0, 1.0 Hz), 2.42 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.29 (1H, ddd, J=15.8, 9.2, 6.8 Hz), 2.22-2.06 (4H, m), 2.04-1.14 (23H, m), 1.07 (3H, s), 1.05 (3H, d, J =6.6 Hz), 0.79 (3H, s) ppm.

¹⁹ F NMR (376 MHz, acetone^): δ -184.29 (tt, J=49.4, 8.7 Hz) ppm.

MSNR (PER ^- ) m/z: 409.1 [MH] ^- , 819.5 [2M-H] ^- .

J. 2a-Fluoro-3a,7a-dihydroxy-5b-cholanic acid (compound 5)

2G.1 Connection 5 (93%)

Using General Method C, methyl 2a-fluoro-3a,7a-dihydroxy-5b-cholanoate (74 mg; 0.17 mmol; 1 eq.) was hydrolyzed to give 2a-fluoro-3a,7a-dihydroxy-5b-cholanic acid (compound 5) (65 mg; 0.16 mmol; 93%) as a colorless solid.

1H NMR (400 MHz, acetone^): δ 4.65 (dq, J=52.3, 1.7 Hz), 3.81 (1H, q, J=2.8 Hz), 3.40 (1H, dddd, J=29 ,7, 12.0, 3.9, 2.1 Hz), 2.69 (1H, q, J=12.6 Hz), 2.39-2.16 (3H, m), 2.02-1.01 (25H, m), 0.98 -0.91 (6H, m), 0.69 (3H, s) ppm

¹⁹ F NMR (376 MHz, acetone^): δ -200.79 (tdd, J=51.2, 29.5, 8.7 Hz) ppm.

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MSNR (PER ^- ) m/z: 841.4 [2M+H]+, 393.4 [M+H-H2O] ⁺ , 375.4 [M+H-H2O-HF] ⁺ , 373.4 [M +H-2H ₂ O] ⁺ .

K. 2a-Fluoro-3a,7b-dihydroxy-5b-cholanic acid (compound 6)

Using General Method C, methyl 2a-fluoro-3a,7b-dihydroxy-5b-cholanoate (44 mg; 0.10 mmol; 1 eq.) was hydrolyzed to give 2a-fluoro-3a,7b-dihydroxy-5b-cholanic acid (compound 6) (40 mg; 0.97 mmol; 97%) as a colorless solid.

¹ H NMR (400 MHz, acetone^): δ 4.67 (1H, dq, J=52.1, 1.7 Hz), 3.61-3.42 (2H, m), 2.39-2.15 (3H, m), 2.02- 1.06 (29H, m), 0.99 -0.93 (6H, m), 0.70 (3H, s) ppm.

¹⁹ F NMR (376 MHz, acetone^): δ -200.58 (tdd, J=50.7, 30.3, 6.9 Hz) ppm.

MSNR (PER ^- ) m/z: 393.4 [M+H-H2O] ⁺ , 375.4 [M+H-H2O-HF] ⁺ , 373.4 [M+H-2H2O] ⁺ .

Example 3. Synthesis of 2,2-difluorinated analogues.

A. Methyl 3a,7b-dihydroxy-5b-cholanoate (3A.1)

UDCA FOR.1 (quantitatively)

General Method A protected with UDCA (100 g; 250 mmol; 1 eq.) to give methyl 3a,7b-dihydroxy-5b-cholanoate as a white solid (103 g; 250 mmol; quantified).

1H NMR (400 MHz, CDCl ₃ ): δ 3.66 (3H, s), 3.73-3.63 (2H, m), 3.58 (2H, td, J=10.4, 5.3 Hz), 2.35 (1H, ddd, J =15.3, 10.1, 4.8 Hz), 2.21 (1H, ddd, J=15.6, 9.6, 6.4 Hz), 1.99 (1H, dt, J=12.3, 2 ,8 Hz), 1.95-0.97 (26H, m), 0.94 (3H, s), 0.92 (3H, d, J=6.4 Hz), 0.67 (3H, s) ppm.

MSNR (IER+) m/z: 389.5 [M+HH ₂ O] ⁺ , 371.5 [M+H-2H2O] ⁺ .

The data are consistent with the literature (except for the melting point); see J. Ren, Y. Wang, J. Wang, J. Lin, K. Wei, R. Huang, Steroids 2013, 78, 53-58.

B. Methyl-3a-acetoxy-7b-hydroxy-5b-cholanoate (3B.1)

ZA.1 3B.1 (76%)

Methyl 3a,7b-dihydroxy-5b-cholanoate (30.0 g; 73.8 mmol; 1 eq.), acetic anhydride (35 ml; 369 mmol; 1 eq.) and NaHCO ₃ (37.2 g; 443 mmol; 6 eq.) was taken into THF (600 ml) and the reaction mixture was heated to 85°C overnight. The reaction mixture was cooled, filtered, and the supernatant concentrated in vacuo to give a crude residue. This was placed in EtOAc and brine (300 ml each), the layers were then separated and the aqueous layer was extracted with additional EtOAc (2x200 ml). The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated to obtain 37 g of clear resin/liquid. The crude material was purified by flash chromatography (petroleum ether/EtOAc, 85:15 to 80:20 to 70:30) to give methyl 3a-acetoxy-7b-hydroxy-5vholanoate as a gummy solid (25.3 g; 56.4 mmol; 76%).

^1H NMR (400 MHz, CDCl ₃ ): δ 4.64 (1H, tt, J=10.5, 5.5 Hz), 3.64 (3H, s), 3.55 (1H, ddd, J=11.5, 8 ,7, 5.1 Hz), 2.33 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.20 (1H, ddd, J=15.6, 9.6, 6.4 Hz), 2.00 (3H, s), 1.97-0.98 (24H, m), 0.93 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

MSHP (ESI+) m/z: 471.5 [M+Na] ⁺ , 371.4 [M+HH ₂ O-HOAc] ⁺ .

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C. Methyl-3 a-acetoxy-7b-methoxymethoxyl-5b-cholanoate (3C.1)

Using the general method, K methyl 3a-acetoxy-7b-hydroxy-5c-cholanoate (72 g, 160.5 mmol) was protected as MOM ester. The crude material was purified by flash chromatography (petroleum ether/EtOAc, 85:15 to 80:20 to 70:30 to 60:40) to give methyl 3aacetoxy-7b-methoxymethoxyl-5b-cholanoate as a gummy solid (62 g; 126 mmol; 79%).

¹ H NMR (400 MHz, CDCl ₃ ): δ 4.70-4.61 (1H, m), 4.60 (2H, s), 3.64 (3H, s), 3.39-3.25 (4H, m), 2.33 (ddd, J= 15.5, 11.0, 5.0 Hz), 2.19 (1H, ddd, J=15.5, 9.6, 6.4 Hz), 2.00 (3H, s), 1.90-0.98 (25H, m ), 0.94 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

MSHP (ESI+) m/z: 515.5 [M+Na]+, 371.5 [M+H-HOCH ₂ OCH ₃ -HOAc] ⁺ .

D. Methyl 3a-hydroxy-7b-methoxymethoxyl-5β-cholanoate (3D.1)

3C.1 3D.1 (quantitative)

Using General Method E, methyl 3a-acetoxy-7b-methoxymethoxyl-5b-cholanoate (82 g; 166 mmol; 1 eq.) was hydrolyzed to give methyl 3a-hydroxy-7b-methoxymethoxyl-5b-cholanoate as pale yellow resin (75 g; 166 mmol; quantitative yield).

^1H NMR (400 MHz, CDCl ₃ ): δ 4.61 (2H, s), 3.65 (3H, s), 3.56 (1H, tt, J=10.5, 5.0 Hz), 3.41-3.25 (4H, m), 2.34 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.20 (1H, ddd, J=15.5, 9.6, 6.4 Hz), 2.02-1.90 (1H, m), 1.89-1.72 (6H, m), 1.70-0.97 (19H, m), 0.94 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.66 (3H, s ) m.d.

MSHP (ESI ⁺ ) m/z: 371.5 [M+H-HOCH ₂ OCH ₃ -H ₂ O] ⁺ .

E. Methyl 7v-methoxymethoxyl-5v-chol-2-enoate (3E.1) and methyl 7v-methoxymethoxyl-5v-chol-3-enoate (3E.2)

3E.1 + 3E.2 (89%)

Methyl 3a-hydroxy-7b-methoxymethoxyl-5b-cholanoate (75 g; 166 mmol; 1 eq.) was dissolved in DCM (650 ml) and cooled to 5°C on ice before adding lutidine (58 ml; less than 500 mmol; 3 eq.) and Tf ₂ O (31 ml; 183 mmol; 1.1 eq.). The reaction mixture was heated to 8-10°C for 1 hour, but the reaction was not complete, additional lutidine (25 ml) and Tf ₂ O (15 ml) were added, and the reaction mass was further heated to 12-14°C for another 1.5 hours. Completion of the reaction was determined by TLC analysis. The reaction mixture was dry loaded onto silica and purified by flash chromatography (petroleum ether/EtOAc, 98:2 to 97:3 to 95:5) to give an inseparable mixture of methyl-7 β-methoxymethoxyl-5 in-chol -2-enoate and methyl 7b-methoxymethoxyl-5b-chol-3-enoate as a pale yellow gum (64.1 g; 148 mmol; 89%).

3E.1/3E.2: 1H NMR (400 MHz, CDCl ₃ ): δ 5.74-5.34 (2H, m), 4.68-4.62 (2H, m), 3.66 (3H, s), 3.37 (3H, s) , 3.13 (1H, td, J=10.2, 5.0 Hz), 2.32 (1H, ddd, J=15.5, 10.3, 5.0 Hz), 2.26-2.16 (1H, m), 2.15-1.00 (27H, m), 0.98 (2H, s), 0.92 (2H, d, J=6.4 Hz), 0.86 (2H, d, J=6.6 Hz), 0.68 (3H, s) m.d.

- 33 046372

F. Methyl 2a-acetoxy-3b-hydroxy-7b-methoxymethoxyl-5b-cholanoate (3F.1) and methyl-3b,4b-epoxy7b-methoxymethoxyl-5c-cholanoate (3F.2)

A mixture of methyl 7β-methoxymethoxyl-5β-chol-2-enoate and methyl 7β-methoxymethoxyl-5β-chol-3-enoate (63.0 g; 146 mmol; 1 eq.) together with mCPBA (54.0 g ; 1.5 eq.) was dissolved in DCM and stirred for 1 hour at room temperature. The reaction mass was quenched with sat. aq. Na ₂ S ₂ O ₃ (250 ml) and stirred for 20 minutes at room temperature. The layers were separated and the aqueous layer was extracted with DCM (300 ml). The combined organic phases were washed with sat. aq. NaHCO ₃ (300 ml), dried (Na ₂ SO ₄ ) and concentrated to obtain 72 g of a pale yellow resin containing an inseparable mixture of Δ2β,3β- and Δ3β,4β-epoxides (assumed quantitative yield). This mixture was then dissolved in AcOH (600 ml) and heated to 50°C for 16 hours. The reaction mixture was concentrated in vacuo, then subjected to azeotropic distillation (EtOAcx3; DCMx1), before the crude material was purified by flash chromatography (petroleum ether /EA, 85:15 to 80:20 to 70:30 to 60:40 to 50:50) to give methyl 2a-acetoxy-3b-hydroxy-7b-methoxymethoxyl-5b-cholanoate as a gummy solid (11 .1 g; 21.9 mmol; 15% - 2 stages) and methyl 3v,4v-epoxy-7v-methoxymethoxyl-5v-cholanoate as a resinous solid (40.5 g; 90 mmol; 62% - 2 stages ).

3F.1: 1H NMR (400 MHz, CDCl ₃ ): δ 4.74 (1H, q, J=3.9 Hz), 4.62 (2H, s), 3.79 (1H, q, J=3.7 Hz), 3.64 (3H, s), 3.36-3.31 (4H, m), 2.33 (1JH, ddd, J=15.5, 11.0, 5.0 Hz), 2.20 (1H, ddd, J=15.5, 9.6, 6.4 Hz), 2.03 (3H, s), 2.01-0.99 (29H, m), 0.98 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.65 (3H , s) ppm

MSNR (ESI+) m/z: 531.6 [M+Na] ⁺ , 387.4 [M+H-HOCH ₂ OCH ₃ -HOCH ₂ OCH ₃ ] ⁺ .

3F.2: 1H NMR (400 MHz, CDCl ₃ ): δ 4.64 (2H, s), 3.64 (3H, s), 3.35 (3H, s), 3.19 (1H, br. s), 3.10 (1H, td , J=10.8, 4.5 Hz), 2.88 (1H, d, J=3.7 Hz), 2.32 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.19 (1H , ddd, J=15.5, 9.6, 6.4 Hz), 2.12-2.04 (1H, m), 2.03-0.95 (25H, m), 0.90 (3H, d, J=6.4 Hz) , 0.87 (3H, s), 0.65 (3H, s) ppm

MSHP (ESI ⁺ ) m/z: 417.4 [M, partial -MOM elimination] ⁺ , 387.4 [M+H-HOCH ₂ OCH ₃ ] ⁺ .

G. Methyl 2a-acetoxy-3b,7b-dimethoxymethoxyl-5b-cholanoate (3G.1)

Using the general method, K methyl 2a-acetoxy-3b-hydroxy-7b-methoxymethoxy-5vholanoate (11.0 g; 21.6 mmol; 1 eq.) was protected as the MOM derivative to give methyl-2a-acetoxy-3b,7b- dimethoxymethoxyl-5b-cholanoate as a pale yellow oil/resin (13.0 g; quantitative).

1H NMR (400 MHz, CDCl ₃ ): δ 4.83 (1H, q, J=3.1 Hz), 4.62 (2H, s), 4.62 (2H, s), 3.67 (1H, q, J=2.9 Hz), 3.63 (3H, s), 3.33 (3H, s), 3.33 (3H, s), 3.32-3.30 (1H, m), 2.32 (1H, ddd, J=15.5, 11.0, 5 ,0 Hz), 2.19 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.01 (3H, s), 1.99-1.25 (25H, m), 0.95 (3H, s), 0.89 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

MCHP (IER ⁺ ) m/z: 575.6 [M+Na] ⁺ , 491.6 [M-HOCH2OCH3] ⁺ , 429.5 [M-2HOCH2-OCH3] ⁺ .

H. Methyl 2a-hydroxy-3b,7b-dimethoxymethoxyl-5b-cholanoate (3H.1)

3G.1 3H.1 (85%)

- 34 046372

Using General Method E, methyl 2a-acetoxy-3b,7b-dimethoxymethoxyl-5b-cholanoate (13.0 g; 21.6 mmol; 1 eq.) was methanolysed to give methyl 2a-hydroxy-3b,7b-dimethoxymethoxyl -5b-cholanoate in the form of a pale yellow resin (9.5 g; 18.3 mmol; 85%).

^1H NMR (400 MHz, CDCl ₃ ): δ 4.65 (1H, d, J=6.8 Hz), 4.62 (1H, d, J=6.8 Hz), 4.60 (1H, d, J=6, 8 Hz), 4.57 (1H, d, J=6.8 Hz), 3.65-3.59 (4H, m), 3.43-3.36 (1H, m), 3.34 (3H, s), 3.33-3.31 (1H, m ), 3.31 (3H, s), 2.96 (1H, br. s), 2.29 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.16 (1H, ddd, J=15, 6, 9.4, 6.5 Hz), 1.97-1.23 (21H, m), 1.16-0.96 (4H, m), 0.92 (3H, s), 0.86 (3H, d, J=6.4 Hz) , 0.61 (3H, s) ppm

MSNR (ESI+) m/z: 533.7 [M+Na]+, 399.5 [M-HOCH ₂ OCH ₃ -H ₂ O-OMe] ⁺ , 387.4 [M+H-2HOCH2OCH3].

I. Methyl 2-oxo-3b,7b-dimethoxymethoxyl-5b-cholanoate (3I.1)

Using the general method, M methyl 2a-hydroxy-3b,7b-dimethoxymethoxyl-5b-cholanoate (9.2 g; 18.0 mmol; 1 eq.) was oxidized, then purified by flash chromatography (petroleum ether/EtOAc, from 80:20 to 70:30 to 65:35) to give methyl 2-oxo-3b,7b-dimethoxymethoxyl-5vcholanoate as a pale tarry solid (8.5 g; 16.7 mmol; 93%).

¹ H NMR (400 MHz, CDCl3): δ 4.62 (2H, d, J=7.0 Hz), 4.58 (2H, d, J=7.0 Hz), 3.76 (1H, t, J=2.6 Hz), 3.63 (3H, s), 3.34 (3H, s), 3.32 (3H, s), 3.31-3.23 (1H, m), 2.64 (1H, d, J=12.8 Hz), 2.38-2.03 (5H, m), 2.01-1.25 (14H, m), 1.21-1.10 (2H, m), 1.09 (3H, s), 0.87 (3H, d, J=6.4 Hz), 0.63 (3H, s ) m.d.

MSNR (ESI ⁺ ) m/z: 531.6 [M+Na] ⁺ , 477.6 [M-OMe] ⁺ , 415.5 [M-HOCH ₂ OCH ₃ -OMe] ⁺ .

J. Methyl 2,2-difluoro-3c,7c-dimethoxymethoxyl-5c-cholanoate (3J.1) and methyl 2-fluoro-3c,7c-dimethoxymethoxyl-5 β-chol-1-enoate (3J.2)

31.1 3J.1 (21%) 3J.2 (12%)

Methyl 2-oxo-3b,7b-dimethoxymethoxyl-5b-cholanoate (8.0 g; 15.7 mmol; 1 eq.) was dissolved in DCM (40 ml) before adding DAST (1004 ml; 78.6 mmol; 5 eq.), and the reaction mixture was stirred at room temperature for 5 hours. The mixture was then diluted with DCM (100 ml) before adding dropwise to ice-cold sat. aq. NaHCO ₃ solution (150 ml), then stirred for 20 minutes. The layers were separated, then the aqueous layer was extracted with DCM (100 ml), the combined organic layers were then dried (Na ₂ SO ₄ ) and concentrated to give 7.5 g of a pale brown gum/oil. The crude material was purified by flash chromatography (petroleum ether/EtOAc, 90:10 to 85:15 to 80:20) to give methyl 2,2-difluoro-3b,7b-dimethoxymethoxyl-5b-cholanoate (1.75 g ; 3.3 mmol; 21%) together with methyl 2-fluoro-3b,7b-dimethoxymethoxyl-5b-chol-1-enoate (970 mg; 1.9 mmol; 12%), both as gummy solids.

3J.1: 1H NMR (400 MHz, CDCl ₃ ): δ 4.72 (1H, d, J=6.6 Hz), 4.66 (1H, d, J=6.6 Hz), 4.63 (2H, s), 3.81 (1H, br. s), 3.66 (3H, s), 3.38 (3H, s), 3.36 (3H, s), 3.31-3.21 (1H, s), 2.34 (1H, ddd, J=15.6 , 10.5, 5.0 Hz), 2.21 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.12 (1H, br. s), 2.05-1.08 (25H, m) , 1.04 (3H, s), 0.92 (3H, d, J=6.4 Hz), 0.67 (3H, s)

m.d.

¹⁹ F NMR (376 MHz, CDCl3): δ -99.89 (d, J=259.0 Hz), -102.89 (ddt, J=250.6, 40.7, 5.0 Hz) ppm.

MSNR (ESI ⁺ ) m/z: 553.5 [M+Na] ⁺ , 437.5 [M-HOCH ₂ OCH ₃ -OCH ₃ ] ⁺ .

3J.2: 1H NMR (400 MHz, CDCl ₃ ): δ 5.39 (1H, d, J=17.7 Hz), 4.71 (1H, d, J=6.8 Hz), 4.69 (1H, d, J =6.8 Hz), 4.64 (2H, s), 4.12-4.08 (1H, m), 3.66 (3H, s), 3.40 (3H, s), 3.36 (3H, s), 3.27-3.17 (1H, m), 2.34 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.21 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.12-1.14 (24H, m), 1.12 (3H, s), 1.10-0.96 (2H, m), 0.91 (3H, d, J=6.4 Hz), 0.68 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -115.57 (dt, J=17.0, 8.5 Hz) ppm.

MCHP (IER ⁺ ) m/z: 448.6 [M+H-HOCH2OCH3] ⁺ , 387.4 [M+H-2HOCH2OCH3] ⁺ .

- 35 046372

K. Methyl 2,2-difluoro-3v,7v-dihydroxy-5v-cholanoate (3K.1)

Using General Method L, methyl 2,2-difluoro-3 β,7 β-dimethoxymethoxyl-5b-cholanoate (1.5 g; 1.76 mmol; 1 eq.) was deprotected to give methyl-2,2- difluoro-3b,7b-dihydroxy-5vcholanoate as a gummy solid (1.3 g; quantitative yield).

^1H NMR (400 MHz, CDCl ₃ ): δ 3.89 (1H, t, J=5.7 Hz), 3.67 (3H, s), 3.54 (1H, ddd, J=11.6, 9.2, 5 ,1 Hz), 2.36 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.22 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.17 -1.07 (27H, m), 1.05 (3H, s), 0.93 (3H, d, J=6.4 Hz), 0.69 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -100.05 (d, J=251.4 Hz), -105.16 (ddt, J=252.1, 40.5, 7.2 Hz) ppm.

MCHP (IER+) m/z: 425.5 [M+HH ₂ O] ⁺ , 405.5 [M+HH ₂ O-HF] ⁺ .

L. Methyl 2,2-difluoro-3,7-dioxo-5v-cholanoate (3L.1) plus hydrate (3L.2)

(91%)

Using the general method, M methyl 2,2-difluoro-3b,7b-dihydroxy-5b-cholanoate (1.0 g; 2.26 mmol; 1 eq.) was oxidized to give a mixture of methyl 2,2-difluoro-3 ,7-dioxo-5v-cholanoate and hydrate (900 mg; 2.05 mmol; 91% total yield).

3L.1 1H NMR (400 MHz, CDCl ₃ ): δ 3.67 (3H, s), 2.93 (1H, ddd, J=13.2, 5.5, 0.8 Hz), 2.77-2.63 (2H, m ), 2.52-1.38 (23H, m), 1.37 (3H, s), 1.35-0.96 (6H, m), 0.94 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm .

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -104.37 (ddd, J=263.6, 39.9, 12.1 Hz), -111.15 (dq, J=263.6, 5.0 Hz) m .d.

MSNR (IER ⁺ ) m/z: 439.5 [M+H] ⁺ .

3L.2 MSNR (IER ⁺ ) m/z: 457.5 [M+H] ⁺ .

M. Methyl 2,2-difluoro-3a,7a-dihydroxy-5b-cholanoate (3M.1) plus methyl 2,2-difluoro-3a,7bdihydroxy-5b-cholanoate (3M.2)

Using General Method B, a mixture of methyl 2,2-difluoro-3,7-dioxo-5vcholanoate and hydrate (750 mg; 1.71 mmol; 1 eq.) was reduced. The crude material was purified by flash chromatography (petroleum ether/EtOAc, 70:30 to 60:40 to 50:50) to give methyl 2,2-difluoro

3a,7a-dihydroxy-5b-cholanoate (300 mg; 0.68 mmol; 40%) and methyl 2,2-difluoro-3a,7b-dihydroxy-5v-cholanoate (26 mg; 0.06 mmol; 4%).

3M.1: 1H NMR (400 MHz, CDCl ₃ ): δ 3.86 (1H, q, J=2.7 Hz), 3.67 (3H, s), 3.65-3.56 (1H, m), 2.54 (1H, q , J=13.2 Hz), 2.47-2.31 (2H, m), 2.28-2.17 (1H, m), 2.05-1.04 (19H, m), 1.00 (3H, s), 0.94 (3H, d, J =6.5 Hz), 0.67 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -101.58 (dquin, J=235.8, 5.2 Hz), -119.95 (dddd, J=235.8, 39.9, 20.8, 10, 4 Hz) ppm

MSNR (IER ⁺ ) m/z: 425.5 [M+HH ₂ O] ⁺ .

3M.2: 1H NMR (400 MHz, CDCl ₃ ): δ 3.73 (1H, ddt, J=19.7, 10.9, 5.4 Hz), 3.67 (3H, s), 3.57 (1H, ddd, J=11.3, 9.5, 5.1 Hz), 2.43-2.30 (2H, m), 2.22 (2H, ddd, J=15.7, 9.4, 6.6 Hz), 2.04-1.05 (31H , m), 1.02 (3H, s), 0.93 (3H, d, J=6.4 Hz), 0.68 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -101.31 (dquin, J=237.6, 5.2 Hz), -119.06 (dddd, J=237.6, 38.2, 19.0, 10, 2 Hz) ppm

MCHP (IER ⁺ ) m/z: 425.5 [M+HH ₂ O] ⁺ .

- 36 046372

N. 2,2-Difluoro-3v,7v-dihydroxy-5v-cholanic acid (compound 7)

When using the general method C methyl 2,2-difluoro-3b,7b-dihydroxy-5b-cholanoate (60 mg;

0.14 mmol; 1 eq.) was hydrolyzed to give 2,2-difluoro-3b,7b-dihydroxy-5b-cholanic acid (compound 7) as a pale solid (50 mg; 0.12 mmol; 83%).

^1H NMR (400 MHz, CDCh): δ 3.89 (1H, br. s), 3.55 (1H, ddd, J=11.4, 9.2, 5.2 Hz), 2.39 (1H, ddd, J= 15.6, 10.5, 5.0 Hz), 2.26 (1H, ddd, J=15.8, 9.4, 6.5 Hz), 2.18-1.07 (26H, m), 1.05 (3H, s ), 0.94 (3H, d, J=6.5 Hz), 0.69 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -100.00 (d, J=253.2 Hz), -105.12 (ddt, J=251.4, 41.6, 8.0 Hz) ppm.

MCHP (IER+) m/z: 411.5 [M+H-H2O] ⁺ , 391.5 [M+H-H2O-HF] ⁺ .

O. 2,2-Difluoro-3a,7a-dihydroxy-5b-cholanic acid (compound 8)

3M.1 Compound 8 (96%)

Using the general method C, methyl 2,2-difluoro-3a,7a-dihydroxy-5b-cholanoate (75 mg; 0.17 mmol; 1 eq.) was hydrolyzed to give 2,2-difluoro-3a,7a-dihydroxy- 5b-cholanic acid (compound 8) as a white solid (70 mg; 0.16 mmol; 96%).

1H NMR (400 MHz, CDCh): δ 33.86 (1H, q, J=2.0 Hz), 3.62 (1H, ddt, J=19.7, 10.9, 5.4 Hz), 2.54 (1H, q, J=13.0 Hz), 2.44-2.35 (2H, m), 2.25 (1H, ddd, J=15.9, 9.6, 6.5 Hz), 2.02-1.02 (25H, m), 0.99 (3H, s), 0.94 (3H, d, J=6.4 Hz), 0.67 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl3): δ - 119.67 (d, J=235.8 Hz), -119.67 (dddd, J=235.8, 38.2, 19.1, 10.4 Hz) m. d.

MCHP (IER ⁺ ) m/z: 411.5 [M+H-H2O] ⁺ , 393.4 [M+H-2H2O] ⁺ .

P. 2,2-Difluoro-3,7-dioxo-5b-cholanic acid (reference compound E) plus hydrate (3P.1)

93%

Using the general method C, methyl 2,2-difluoro-3,7-dioxo-5v-cholanoate and hydrate (60 mg; 0.14 mmol; 1 eq.) was hydrolyzed to give 2,2-difluoro-3,7- dioxo-5b-cholanic acid (compound E) and hydrate as a white solid as a mixture of ketone/hydrate/acetal adducts (55 mg; 0.13 mmol; 93%).

1H NMR (400 MHz, CD3CN): δ 2.93 (1H, dd, J=13.1, 5.9 Hz), 2.90-2.84 (1H, m), 2.70-1.97 (9H, m), 1.930.93 (19H, m), 0.91 (2H, d, J=6.4 Hz), 0.89 (1H, d, J=6.4 Hz), 0.67 (2H, s), 0.64 (1H, s) ppm.

¹⁹ F NMR (376 MHz, CD ₃ CN): δ -103.96 (1F, ddd, J=261.6, 38.1, 15.0 Hz), -108.59 (0.2F, ddd, J=246.2, 39.0, 11.3 Hz), -110.91 (1F, dq, J=263.6, 5.2 Hz), -113.71 - -112.69 (0.1F, dq, J=246.2, 5.2 Hz ), -116.15 (0.1F, dq, J=246.2, 5.2 Hz), -117.09 (0.2F, dq, J=246.2, 5.2 Hz) ppm.

MCHP (IER ⁺ ) m/z: ketone: 425.5 [M+H] ⁺ ; hydrate: 443.5 [M+H] ⁺ ; hemiacetal: 457.7 [M+H] ⁺ , 439.5 [M+H-H2O] ⁺ ; acetal: 443.5 [M+H] ⁺ , 439.5 [M+H-MeOH] ⁺ .

- 37 046372

Q. 2,2-Difluoro-3a,7b-dihydroxy-5b-cholanic acid (compound 9)

Using the general method C, methyl 2,2-difluoro-3a,7b-dihydroxy-5b-cholanoate (25 mg; 0.06 mmol; 1 eq.) was hydrolyzed to give 2,2-difluoro-3a,7b-dihydroxy- 5b-cholanic acid (compound 9) as a gummy solid (20 mg; 0.05 mmol; 78%).

1H NMR (400 MHz, CD3OD): δ 3.69 (1H, ddt, J=21.0, 11.0, 5.0 Hz), 3.44 (1H, ddd, J=11.5, 9.8, 5, 0 Hz), 2.38-2.26 (2H, m), 2.25-2.14 (2H, m), 2.08-1.06 (32H, m), 1.03 (3H, s), 0.96 (3H, d, J=6.5 Hz ), 0.72 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CD3OD): δ -101.64 (dquin, J=239.3, 5.0 Hz), -120.18 (dddd, J=239.3, 38.2, 20.8, 10.4 Hz) ppm

MSNR (ESI+) m/z: 411.4 [M+H-H2O] ⁺ , 393.3 [M+H-2H2O] ⁺ .

Example 4. Synthesis of conjugates of compound 9.

A. Sodium salt of No. (2,2-Difluoro-3v,7v-dihydroxy-5v-cholan-24-amide)ethylsulfonic acid (compound 10)

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (7.4 mg, 0.017 mmol) was conjugated to give sodium salt N(2,2-difluoro-3b,7b-dihydroxy-5b -cholan24-amide)ethylsulfonic acid as a clear residue (7.4 mg; 77%).

1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.61-3.56 (2H, m), 3.40 (1H, ddd, J=14.6, 9.7, 5.2 Hz), 2.95 (2H, app.t., J=6.9 Hz), 2.24 (1H, ddd, J=15.0, 10.6, 5.2 Hz), 2.12-1.07 (23H, m), 1.05 (3H , s), 0.97 (3H, d, J=6.5 Hz), 0.71 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -101.00 (d, J=251.4 Hz), -105.55 (ddt, J=250.3, 40.3, 7.8 Hz) ppm.

MCHP (MER ^- ): [M-Na] ^- : counted 534.2706; discovered 534.2709.

B. Sodium salt of ^(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-amide)propionic acid (compound 11)

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give sodium salt N(2,2-difluoro-3b,7b-dihydroxy-5b -cholan24-amide)propionic acid in the form of a transparent residue (17.66 mg; 73%).

1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.45 -3.35 (3H, m), 2.40-2.30 (2H, m), 2.23 (1H, m), 2.121.06 (23H, m ), 1.05 (3H, s), 0.96 (3H, d, J=6.4 Hz), 0.71 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.62 (d, J=250.4 Hz), -105.55 (ddt, J=250.4, 40.8, 7.6 Hz) ppm.

MCBP (High Resolution Mass Spectrometry) (ESI ⁺ ): [M+Na] ⁺ : calculated 522.3002; discovered 522.3007.

C. No. (Methyl), No. (2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-amide)acetic acid (compound 12)

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (25.36 mg, 0.059 mmol) was conjugated to give Na(methyl),Na(2,2-difluoro-3b,7b- dihydroxy-5b-cholan-24amide)acetic acid as a clear residue (13.3 mg; 45%).

1H NMR (400 MHz, MeOD) δ 3.96 (2H, br. d, J=18.1 Hz), 3.78 (1H, br. s), 3.40 (1H, m), 3.08 (1.5H, s), 2.94 (1.5H, s), 2.48 (1H, m), 2.41-1.06 (23H, m), 1.05 (1.5H, s), 1.05 (1.5H, s), 1.00 (1.5H, d , J=6.5 Hz), 0.96

- 38 046372 (1.5H, d, J=6.5 Hz), 0.73 (1.5H, s), 0.71 (1.5H, s) ppm; ¹⁹ F NMR (376 MHz, MeOD) δ -100.62 (d, J=250.7 Hz), 105.55 (ddt, J=250.0, 40.2, 7.2 Hz) ppm.

MSHR (ESI ⁺ ): [M+Na] ⁺ : calculated 522.3002; discovered 522.2998.

D. Sodium salt of ^(2,2-difluoro-3b,7b-dihydroxy-5b-cholan-24-amide)-trans-2-cyclohexanecarboxylic acid (compound 13)

Η

Η

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give sodium salt N(2,2-difluoro-3b,7b-dihydroxy-5b -cholan24-amide)-trans-2-cyclohexanecarboxylic acid as a clear residue (18.18 mg; 68%).

1H NMR (400 MHz, MeOD) δ 3.89-3.72 (2H, m), 3.40 (1H, m), 2.28-1.06 (33H, m), 1.05 (3H, s), 0.96 (3H, d, J=6 .5 Hz), 0.70 (3H, s) ppm.

¹⁹ F { ¹ H} NMR (376 MHz, MeOD) δ -100.61 (d, J=249.4 Hz), -105.55 (d, J=250.2, 4 Hz) ppm.

MSVR (iER ⁺ ): [M+H] ⁺ : counted 554.3652; discovered 554.3656.

E. Sodium 1-(2,2-Difluoro-3v,7v-dihydroxy-5v-cholan-24-oil)piperidine-3-carboxylate (compound 14)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give 1-(2,2-difluoro-3b,7b-dihydroxy-5b- sodium cholane-24oyl)piperidine-3-carboxylate as residue (21.88 mg; 83%).

1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.41 (1H, ddd, J=14.8, 9.9, 5.2 Hz), 2.57-1.07 (33H, m), 1.05 (3H, s), 0.99 (3H, d, J=6.5 Hz), 0.72 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.63 (d, J=250.3 Hz), -105.55 (ddt, J=249.8, 40.9, 7.8 Hz) ppm.

MSVR (IER ⁺ ): [M+H] ⁺ : counted 540.3495; discovered 540.3507.

F. Sodium 3-(2,2-Difluoro-3b,7b-dihydroxy-5b-cholan-24-amide)-4-thiazolidine carboxylate (compound 15)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give 3-(2,2-difluoro-3b,7b-dihydroxy-5b- cholane-24-amide)-4thiazolidine-sodium carboxylate as a residue (21.41 mg; 81%).

1H NMR (400 MHz, MeOD) δ 4.90-4.78 (0.4H, m), 4.77 (0.6H, d, J=9.8 Hz), 4.71 (0.4H, d, J=8.4 Hz), 4.60 (0.4H, d, J=8.4 Hz), 4.59 (0.6H, m), 4.48 (0.6H, d, J=9.8 Hz), 3.78 (1H, br. s), 3.46-3.15 ( 3H, m), 2.58-1.07 (24H, m), 1.04 (3H, br. s), 0.99 (1.2H, d, J=6.5 Hz), 0.96 (1.8H, d, J=6.5 Hz), 0.73 (1.2H, s), 0.71 (1.8H, s) ppm

¹⁹ F NMR (376 MHz, MeOD) δ -100.63 (d, J=249.1 Hz), -105.52 (ddt, J=250.1, 41.4, 7.8 Hz) ppm.

MSVR (IER+): [M+H]+: counted 544.2903; discovered 544.2904.

G. №(2,2-Difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)morpholine (compound 16)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give N(2,2-difluoro-3b,7b-dihydroxy-5b-cholan -24oil)morpholine as a clear residue (9.83 mg; 65%).

- 39 046372

1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.72-3.59 (4H, m), 3.59-3.50 (4H, m), 3.40 (1H, ddd, J=14.8, 9, 7, 5.2 Hz), 2.42 (1H, m), 2.30 (1H, m), 2.13-1.06 (22H, m), 1.05 (3H, s), 0.99 (3H, d, J=6.5 Hz ), 0.72 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -101.64 (d, J=250.3 Hz), -105.55 (ddt, J=249.6, 40.6, 7.8 Hz) ppm.

MSVR (IER+): [M+H]+: counted 498.3389; 498.3394 discovered.

H. Sodium salt of No(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-amide)methylcarboxylic acid (compound 17)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give sodium salt N(2,2-difluoro-3b,7b-dihydroxy-5b -cholan24-amide)methylcarboxylic acid as a clear residue (18.12 mg; 77%).

1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.75 (2H, s), 3.40 (1H, ddd, J=14.8, 9.7, 5.2 Hz), 2.30 (1H , m), 2.13-1.06 (23H, m), 1.05 (3H, s), 0.98 (3H, d, J=6.4 Hz), 0.72 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -101.61 (d, J=249.7 Hz), -105.55 (ddt, J=249.6, 40.6, 7.8 Hz) ppm.

MSVR (IER ⁺ ): [M+H] ⁺ : counted 486.3026; 486.3029 discovered.

I. Disodium salt of N(carboxymethyl)-^(2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-oyl)-2aminoacetic acid (compound 18)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give Na(carboxymethyl)-N-(2,2-difluoro-3b) disodium salt ,7vdihydroxy-5v-cholan-24-oil)-2-aminoacetic acid as a clear residue (14.81 mg; 59%).

1H NMR (400 MHz, MeOD) δ 4.00 (4H, d, J=19.4 Hz), 3.78 (1H, br. s), 3.40 (1H, ddd, J=14.8, 9.7, 5, 2 Hz), 2.42 (1H, m), 2.23 (1H, m), 2.14-1.07 (22H, m), 1.05 (3H, s), 0.96 (3H, d, J=6.4 Hz), 0.71 ( 3H, s) ppm

¹⁹ F NMR (376 MHz, MeOD) δ -100.65 (d, J=250.7 Hz), -105.55 (ddt, J=249.6, 40.6, 7.8 Hz) ppm.

MSVR (IER ⁺ ): [M+H] ⁺ : counted 544.3080; 544.3081 discovered.

J. Sodium salt of N(methyl)-^(2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-amide)ethylsulfonic acid (compound 19)

Using the general method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg; 0.047 mmol) was conjugated to give \-(L1ethyl')-\-(2,2-di) sodium salt (|)tor-3b,7b-dihydroxy5b-cholan-24-amide)ethylsulfonic acid as a clear residue (9.49 mg; 36%).

1H NMR (400 MHz, MeOD) δ 3.84-3.68 (3H, m), 3.40-3.36 (1H, m), 3.11 (1.5H, s) 3.08-2.99 (2H, m), 2.92 (1.5H, m), 2.57-1.07 (23H, m), 1.05 (3H, s), 0.99 (1.5H, d, J=6.5 Hz), 0.98 (1.5H, d, J=6.5 Hz) , 0.72 (3H, s)

m.d.

¹⁹ F NMR (376 MHz, MeOD) δ -100.64 (d, J=249.4 Hz), -105.54 (ddt, J=250.3, 40.3, 7.8 Hz) ppm.

MSHR (IER ⁺ ): [M+Na] ⁺ : calculated 572.2828; discovered 572.2830.

K. Sodium 3-(2,2-Difluoro-3v,7v-dihydroxy-5v-cholan-24-oyl)aminopropanesulfonate (compound 20)

Connection 7

Using the general method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholane was conjugated

- 40 046372 acid (25.0 mg; 0.058 mmol) to obtain sodium 3-(2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24oyl)aminopropanesulfonate in the form of a transparent residue (25.44 mg; 76% ).

1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.40 (1H, ddd, J=14.6, 9.7, 5.2 Hz), 3.33-3.25 (2H, m), 2.86 -2.78 (2H, m), 2.24 (1H, m), 2.17-1.07 (25H, m), 1.05 (3H, s), 0.97 (3H, d, J=6.5 Hz), 0.72 (3H, s ) m.d.

¹⁹ F NMR (376 MHz, MeOD) δ -100.59 (d, J=250.1 Hz), -105.55 (ddt, J=249.4, 40.8, 7.5 Hz) ppm.

MSVR (IER+): [M+Na] ⁺ : calculated 594.2647; discovered 594.2648.

L. Sodium salt of No(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-amide)methanesulfonic acid (compound 21)

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (25.0 mg, 0.058 mmol) was conjugated to give sodium salt N(2,2-difluoro-3b,7b-dihydroxy-5b -cholan24-amide)methanesulfonic acid as a clear residue (27.63 mg; 87%).

1H NMR (400 MHz, MeOD) δ 4.32 (1H, d, J=17.8 Hz), 4.28 (1H, d, J=17.8 Hz), 3.78 (1H, br. s), 3.41 (1H, ddd, J=14.6, 9.8, 5.1 Hz), 2.33 (1H, m), 2.25-1.07 (23H, m), 1.05 (3H, s), 0.98 (3H, d, J=6 .4 Hz), 0.72 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.61 (d, J=250.8 Hz), -105.55 (ddt, J=249.4, 40.8, 7.5 Hz) ppm.

MSHR (ESI ⁺ ): [M-2H+D+2Na] ⁺ : calculated 567.2397; discovered 567.2387.

M. №(2,2-Difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)-2-aminoethyl sulfate sodium (compound 22)

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (30.0 mg, 0.070 mmol) was conjugated to give N(2,2-difluoro-3b,7b-dihydroxy-5b-cholan Sodium -24-oil)aminoethyl sulfate.

1H NMR (400 MHz, MeOD) δ 4.04 (2H, arr. t, J=5.5 Hz), 3.78 (1H, br. s), 3.44 (2H, arr. t, J=5.5 Hz), 3.41 (1H, m), 2.26 (1H, ddd, J=15.5, 10.5, 5.2 Hz), 2.18-1.06 (23H, m), 1.05 (3H, s), 0.98 (3H, d , J=6.5 Hz), 0.71 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.59 (d, J=250.0 Hz), -105.48 (ddt, J=249.4, 40.8, 7.5 Hz) ppm.

MSVR (IER ⁺ ): [M+Na] ⁺ : calculated 574.2621; discovered 574.2620.

N. O-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-hydroxyethylsulfonic acid sodium salt (compound 23)

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give sodium salt O-(2,2-difluoro-3β,7β-dihydroxy- 5β-cholan24-oil)-2-hydroxyethylsulfonic acid as a clear residue (15.06 mg; 58%).

1H NMR (400 MHz, MeOD) δ 4.42 (2H, arr. t, J=7.0 Hz), 3.78 (1H, br. s), 3.41 (1H, ddd, J=14.8, 9.8, 5.0 Hz), 3.12 (2H, arr. t, J=7.2 Hz), 2.39 (1H, m), 2.26 (1H, m), 2.14-1.06 (22H, m), 1.05 (3H, s ), 0.95 (3H, d, J=6.7 Hz), 0.71 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.65 (d, J=250.5 Hz), -105.48 (ddt, J=249.8, 40.9, 7.6 Hz) ppm.

MSHR (ESI ⁺ ): [M+Na] ⁺ : calculated 581.2331; discovered 581.2332.

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O. Sodium salt of (2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-oyl)aniline-2-sulfonic acid (compound 24)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give sodium salt N(2,2-difluoro-3b,7b-dihydroxy-5b -cholan24-oil)aniline-2-sulfonic acid as a white residue (24.04 mg; 85%).

1H NMR (400 MHz, MeOD) δ 8.30 (1H, d, J=8.2 Hz), 7.86 (1H, dd, J=7.9, 1.5 Hz), 7.40 (1H, m), 7.12 ( 1H, m), 3.79 (1H, br. s), 3.41 (1H, ddd, J=14.8, 9.8, 5.0 Hz), 2.50 (1H, m), 2.34 (1H, m), 2.15-1.08 (22H, m), 1.05 (3H, s), 1.02 (3H, d, J=6.3 Hz), 0.73 (3H, s) ppm

¹⁹ F NMR (376 MHz, MeOD) δ -100.62 (d, J=251.2 Hz), -105.49 (ddt, J=249.3, 40.6, 7.2 Hz) ppm.

MSHR (ESI ⁺ ): [M-H+2Na] ⁺ : calculated 628.2491; discovered 628.2494.

P. No. (Cyclohexyl)-No. (2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-oil)-3-aminopropanesulfonate sodium (compound 25)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give N(cyclohexyl)-X-(2,2-difluoro-3b,7b Sodium -dihydroxy-5vholan-24-oil)-3-aminopropanesulfonate as a clear residue (27.97 mg; 92%).

1H NMR (400 MHz, MeOD) δ 4.17 (0.4H, m), 3.78 (1H, br. s), 3.62 (0.6H, m), 3.45-3.33 (3H, m), 2.872.75 (2H, m ), 2.41 (1H, m), 2.30 (1H, m), 2.14-1.07 (34H, m), 1.05 (3H, br. s), 1.02-0.97 (3H, m), 0.73 (1.8H, s) , 0.72 (1.2H, s) ppm

¹⁹ F NMR (376 MHz, MeOD) δ -100.60 (d, J=249.8 Hz), -105.48 (ddt, J=250.1, 41.0, 7.6 Hz) ppm.

MSVR (IER ⁺ ): [M+Na] ⁺ : calculated 654.3610; discovered 654.3606.

Q. Sodium (Cyclohexyl)^-(2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-oil)-2-aminoethanesulfonate (compound 26)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give N(cyclohexyl)-X-(2,2-difluoro-3b,7b Sodium -dihydroxy-5vholan-24-oil)-2-aminoethanesulfonate as a clear residue (28.41 mg; 95%).

1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.73-3.56 (3H, m), 3.40 (1H, ddd, J=14.7, 9.7, 5.0 Hz), 3.09 -2.96 (2H, m), 2.44 (1H, m), 2.31 (1H, m), 2.15-1.07 (32H, m), 1.05 (3H, br. s), 1.02-0.97 (3H, m), 0.73 (1.8H, s), 0.72 (1.2H, s) ppm

¹⁹ F NMR (376 MHz, MeOD) δ -100.57 (d, J=251.0 Hz), -105.49 (ddt, J=250.3, 40.9, 7.0 Hz) ppm.

MCBP (IER+): [M-H+2Na] ⁺ : calculated 662.3273; discovered 662.3285.

R. №(2,2-Difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)-2-aminoethylmethylsulfone (compound 27)

Using the general method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give X-(2,2-difluoro-3b,7b-dihydroxy-5b— holan-24-oil)2aminoethylmethylsulfone as a clear residue (22.49 mg; 90%).

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1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.62 (2H, arr. t, J=6.7 Hz), 3.40 (1H, ddd, J=14.7, 9.6, 4.9 Hz), 3.29 (2H, arr. t, J=6.5 Hz), 2.99 (3H, s), 2.26 (1H, ddd, J=15.3, 10.4, 5.3 Hz) , 2.17-1.06 (23H, m), 1.05 (3H, s), 0.97 (3H, d, J=6.4 Hz), 0.71 (3H, s) ppm

¹⁹ F NMR (376 MHz, MeOD) δ -100.60 (d, J=249.7 Hz), -105.49 (ddt, J=249.9, 41.2, 7.1 Hz) ppm.

MCBP (IER+): [M+Na] ⁺ : counted 556.2879; discovered 556.2873.

S. ^(Ethyl)A-(2,2-difluoro-3b,7b-dihydroxy-5b-cholan-24-oil)-3-aminotetrahydrothiophene dioxide (compound 28)

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give Na(ethyl)-Na(2,2-difluoro-3b,7b- dihydroxy-5b-cholan-24oyl)-3-aminotetrahydrothiophene dioxide as a clear residue (20.71 mg; 77%).

1H NMR (400 MHz, MeOD) δ 4.56 (1H, m), 3.78 (1H, br. s), 3.51 (4H, m), 3.33-3.21 (2H, m), 3.08 (1H, m), 2.63- 1.06 (29H, m), 1.05 (3H, s), 0.99 (3H, d, J=6.5 Hz), 0.72 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.59 (d, J=250.4 Hz), -105.48 (ddt, J=250.4, 41.5, 7.4 Hz) ppm.

MSVR (IER ⁺ ): [M+H] ⁺ : counted 574.3372; 574.3370 discovered.

T. №(2-(Diisopropylamino)ethyl)-№(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)-3aminotetrahydrothiophene dioxide (compound 29)

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give N(2-(diisopropylamino)ethyl)-N(2,2-difluoro -3в,7вdihydroxy-5в-cholan-24-oil)-3-aminotetrahydrothiophene dioxide as a residue (23.90 mg; 76%).

1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.50-0.88 (m), 0.72 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.56 (d, J=250.5 Hz), -105.49 (m) ppm.

MSVR (IER ⁺ ): [M+H] ⁺ : counted 673.4420; discovered 673.4430.

U. №(2,2-Difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)thiomorpholine dioxide (compound 30)

O\O

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give Na(2,2-difluoro-3b,7b-dihydroxy-5b-cholan -24oil)thiomorpholine dioxide as a clear residue (22.2 mg; 87%).

1H NMR (400 MHz, MeOD) δ 4.09-3.93 (4H, m), 3.78 (1H, br. s), 3.40 (1H, ddd, J=14.6, 9.6, 5.1 Hz), 3.23 -3.04 (4H, m), 2.52 (1H, m), 2.38 (1H, m), 2.14-1.07 (22H, m), 1.05 (3H, s), 0.99 (3H, d, J=6.6 Hz ), 0.73 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.63 (d, J=249.3 Hz), -105.54 (ddt, J=250.6, 41.6, 7.5 Hz) ppm.

MSVR (IER ⁺ ): [M+H] ⁺ : counted 546.3059; discovered 546.3065.

V. N-(2,2-Difluoro-3β,7β-dihydroxy-5β-cholan-24-oil)-1,1-dioxidotetrahydro-2H-thiopyran-3-ylamine (compound 31)

Connection 7

Using General Method Q, 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to give Na(2,2-difluoro-3b,7b-dihydroxy-5b-cholan -24-oil)-1,1dioxidotetrahydro-2H-thiopyran-3-ylamine as a residue (22.44 mg; 88%).

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1H NMR (400 MHz, MeOD) δ 4.26 (1H, tt, J=11.1, 3.6 Hz), 3.78 (1H, br. s), 3.40 (1H, ddd, J=14.5, 10.0, 5.1 Hz), 3.27 (1H, m), 3.08-2.91 (3H, m), 2.30-1.06 (26H, m), 1.05 (3H, s), 0.97 (3H, d, J=6.5 Hz ), 0.71 (3H, s) ppm.

¹⁹ F NMR (376 MHz, MeOD) δ -100.63 (d, J=250.3 Hz), -105.49 (ddt, J=250.7, 41.5, 7.9 Hz) ppm.

MSVR (IER ⁺ ): [M+H] ⁺ : counted 560.3216; discovered 560.3217.

Comparative example 5. Synthesis of 2-fluoroalkene compounds.

A. Methyl 2-fluoro-3b,7b-dihydroxy-5b-chol-1-enoate (4A.1)

3J-2 4A1 (quantitatively)

Using General Method L, methyl 2-fluoro-3b,7b-dimethoxymethoxyl 5b-chol-1-enoate from Example 4J (900 mg; 1.76 mmol; 1 eq.) was deprotected to give methyl 2-fluoro-3b ,7β-dihydroxy5β-chol-1-enoate as a white resinous solid (750 mg; quantitative yield).

1H NMR (400 MHz, CDCl ₃ ): δ 5.34 (1H, d, J=17.6 Hz), 4.20 (1H, ddd, J=7.7, 4.7, 1.3 Hz), 3.67 (3H , s), 3.50 (1H, ddd, J=11.2, 9.6, 4.8 Hz), 2.35 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.22 ( 1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.12 (1H, td, J=14.0, 5.3 Hz), 2.04-1.21 (23H, m), 1.12 (3H , d, J=0.7 Hz), 0.92 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -117.65 (dt, J=17.0, 8.5 Hz) ppm.

MSNR (IER ⁺ ) m/z: 405.5 [M+HH ₂ O] ⁺ , 387.5 [M+H-2H2O] ⁺ .

B. Methyl 2-fluoro-3,7-di-oxo-5v-chol-1-enoate (4B.1)

4A.1 4B.1 (67%)

Using the general method, M methyl 2-fluoro-3b,7b-dihydroxy-5b-chol-1-enoate (600 mg;

1.42 mmol; 1 eq.) oxidized, then purified by flash chromatography (petroleum ether/EtOAc, 80:20 to 70:30 to 60/40) to give methyl 2-fluoro-3,7-di-oxo-5v-chol -1-enoate as a gummy solid (400 mg; 0.96 mmol; 67%).

1H NMR (400 MHz, CDCl ₃ ): δ 6.34 (1H, d, J=14.7 Hz), 3.66 (3H, s), 2.89 (1H, dd, J=12.8, 5.9 Hz), 2.61 (1H, dtd, J=13.5, 5.8, 2.0 Hz), 2.55-2.11 (7H, m), 2.07 (1H, dd, J=13.2, 2.0 Hz), 2.04 -1.71 (5H, m), 1.51 (3H, s), 1.49-0.94 (8H, m), 0.92 3H, (d, J=6.4 Hz), 0.70 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -131.67 (dd, J=15.6, 3.5 Hz), ppm.

MSNR (IER+) m/z: 419.5 [M+H] ⁺ , 460.5 [M+H+MeCN] ⁺ .

C. Methyl 2-fluoro-3a,7a-dihydroxy-5b-chol-1-enoate (4C.1)

4B.1 4C.1 (25%)

Using General Method B, methyl 2-fluoro-3b,7b-dihydroxy-5b-chol-1-enoate (400 mg; 0.96 mmol; 1 eq.) was reduced, then purified by flash chromatography (petroleum ether/EtOAc , 70:30 to 60:40 to 50:50) to give methyl 2-fluoro-3a,7a-dihydroxy-5v-chol-1-enoate as a gummy solid (99 mg; 0.22 mmol; 25 %).

1H NMR (400 MHz, CDCl ₃ ): δ 5.25 (1H, d, J=18.2 Hz), 4.35 (1H, t, J=7.9 Hz), 3.85 (1H, q, J=l,7 Hz), 3.67 (3H, s), 3.65 (1H, d, J=l,8 Hz), 2.53-2.32 (2H, m), 2.29-2.17 (1H, m), 2.04-1.07 (29H, m) , 1.04 (3H, d, J=0.9 Hz), 0.93 (3H, d, J=6.4 Hz), 0.68 (3H, s) ppm.

¹⁹ F NMR (376 MHz, CDCl ₃ ): δ -125.36 (dd, J=19.1, 6.9 Hz) ppm.

MSNR (IER ⁺ ) m/z: 405.5 [M+HH ₂ O] ⁺ , 387.5 [M+H-2H2O] ⁺ .

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D. 2-Fluoro-3b,7b-dihydroxy-5b-chol-1-enoic acid (reference compound F)

Using General Method C, methyl 2-fluoro-3b,7b-dihydroxy-5b-chol-1-enoate from Step A (60 mg; 0.14 mmol; 1 eq.) was hydrolyzed to give 2-fluoro-3b,7b -dihydroxy-5b-chol-1-enoic acid (compound F) as a white solid (40 mg; 0.10 mmol; 70%).

^1H NMR (400 MHz, CD3OD): δ 5.32 (1H, d, J=17.7 Hz), 4.10 (1H, ddd, J=8.0, 4.5, 1.0 Hz), 3.38 (1H, td , J=10.5, 4.8 Hz), 2.40-1.15 (29H, m), 1.13 (3H, s), 0.95 (3H, d, J=6.5 Hz), 0.73 (3H, s) m .d.

¹⁹ F NMR (376 MHz, CD3OD): δ -117.71 (dt, J=15.6, 7.6 Hz) ppm.

MSNR (IER+) m/z: 391.5 [M+HH ₂ O] ⁺ , 373.5 [M+H-2H2O] ⁺ .

E. 2-Fluoro-3,7-dioxo-5v-chol-1-enoic acid (reference compound G)

Using General Method C, methyl 2-fluoro-3,7-di-oxo-5v-chol-1-enoate from Step B (50 mg; 0.12 mmol; 1 eq.) was hydrolyzed to give 2-fluoro-3 ,7-dioxo-5b-chol-1-enoic acid (compound G) as a white solid (45 mg; 0.11 mmol; 93%).

^1H NMR (400 MHz, CDCh): δ 6.34 (1H, d, J=14.7 Hz), 2.89 (1H, dd, J=13.0, 6.1 Hz), 2.62 (1H, dtd, J =13.5, 5.8, 2.0 Hz), 2.55-1.73 (13H, m), 1.51 (3H, s), 1.49-0.95 (9H, m), 0.93 (3H, d, J=6, 4 Hz), 0.70 (3H, s)

m.d.

¹⁹ F NMR (376 MHz, CDCI3): δ -131.63 (dd, J=13.9, 3.5 Hz) ppm.

MSNR (IER ⁺ ) m/z: 405.4 [M+H] ⁺ , 446.5 [M+H+MeCN] ⁺ .

F. 2-Fluoro-3a,7a-dihydroxy-5b-chol-1-enoic acid (reference compound H)

Using General Method C, methyl 2-fluoro-3a,7a-dihydroxy-5b-chol-1-enoate from Step C (50 mg; 0.11 mmol; 1 eq.) was hydrolyzed to give 2-fluoro-3a,7a -dihydroxy-5b-chol-1-enoic acid (Compound H) as a pale solid (45 mg; 0.11 mmol; 97%).

^1H NMR (400 MHz, acetone^): δ 5.16 (1H, d, J=18.6 Hz), 4.23 (1H, ddd, J=9.0, 7.0, 2.5 Hz), 4.07 ( 1H, br. s), 3.82 (1H, q, J = 2.7 Hz), 3.32 (1H, br. s), 2.54 (1H, td, J = 13.7, 10.0 Hz), 2.34 ( 1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.21 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.02-1.06 (25H, m) , 1.05 (3H, d, J=l,0 Hz), 0.96 (3H, d, J=6.6 Hz), 0.71 (3H, s) ppm.

¹⁹ F NMR (376 MHz, acetone-06): δ -123.32 (dd, J=19.1, 6.9 Hz) ppm.

MSNR (ESI ⁺ ) m/z: 373.5 [M+H-2H2O] ⁺ .

Biological examples

Abbreviations.________________________________________________________________________________

BDNF brain-derived neurotrophic factor SuZ cyanine 3 DAPI 4',6-diamide ino-2-phenylindole DAT dopamine transporter antibody dcAMP dibutyrylcyclic adenosine monophosphate

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DMEM Dulbecco's modified Eagle's medium EDTA ethylenediaminetetraacetic acid FGFb fibroblast growth factor, basic form FGF8 fibroblast growth factor 8 GDNF glial cell-derived neurotrophic factor HBSS Hanks balanced salt solution HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid iNPC induced neural progenitor cell MEM minimum required environment MMP transmembrane potential of mitochondria MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine NEEA addition of non-essential amino acids for cell cultures PBS phosphate buffered saline solution BP Parkinson's disease (sPD is a sporadic form of Parkinson's disease TGF-b3 transforming growth factor beta 3 TMRM Tetramethylrhodamine methyl ester perchlorate Tuj antibody to beta-III tubulin

Example 6.

A. Primary fibroblast culture, iNPC generation and culture.

Fibroblasts were cultured in DMEM (Invitrogen) and regularly subcultured every 3-5 days using trypsin to dissociate them. Induced neural progenitor cells (iNPCs) were generated as previously described (Meyer et al., Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS Proc Natl Acad Sci USA 2014). iNPCs were maintained in DMEM/Ham F12 (Invitrogen) supplemented with N2, B27 (Invitrogen) and FGFb (Peprotech) in fibronectin-coated (Millipore) Petri dishes and subcultured regularly every 2-3 days using accutase to separate them.

B. Differentiation of iNPC dopaminergic neurons.

Briefly, iNPCs were plated in a 6-well plate and cultured for 2 days in DMEM/F-12 with Glutamax™ supplemented with 1% NEAA, 2% B27 (Gibco) and 2.5 μM DAPT (N-[N-( 3,5difluorophenacetyl)^-alanyl]^-phenylglycine-tert-butyl ether). On day 3, DAPT was removed and 1 μM protein agonist Smoothened (SAG) and FGF8 (75 ng/ml) were added to the medium for an additional 10 days. At this stage, neurons are reseeded. SAG and FGF8 were then removed and replaced with BDNF (30 ng/ml), GDNF (30 ng/ml), TGF-b3 (2 mM), and dcAMP (2 mM; Sigma) for 15 days.

C. Immunofluorescence staining and ELISA (enzyme-linked immunosorbent assay)

Cells were seeded in 96-well plates and fixed using 4% paraformaldehyde for 30 min. After washing with PBS, cells were permeabilized using 0.1% Triton™ X-100 for 10 min and blocked using 5% goat serum for 1 h. Cells were incubated with primary antibodies to tyrosine hydroxylase (St John's Laboratory); DAT (Abcam); Tuj (Millipore); Tom20 (BD Biosciences); activated caspase 3 (Cell Signaling); alpha-synuclein (Cell Signaling); phosphorylated alpha-synuclein (Millipore) at 4°C for 16 h. Cells were washed using PBS-Tween® and incubated with 1 μM Alexa Fluor™-conjugated secondary antibodies 488 and 568 (Invitrogen) and Hoescht (Sigma) before imaging. Imaging was performed using the Opera Phenix™ multiparameter imaging system (Perkin Elmer).

Dopamine ELISA is performed using a dopamine ELISA kit (Labor Diagnostika Nord GmbH & Co. KG) according to the manufacturer's instructions. Dopamine release is achieved by incubating cells at 37°C using three different conditions simultaneously for each line. The medium is removed from all wells, then the first well is incubated with HBSS with Ca ²⁺ and Mg ²⁺ (Gibco, Life Technologies) for 30 min, the second well is incubated with HBSS with Ca ²⁺ and Mg ²⁺ for

- 46 046372 min and then add 56 mM KCl (Fisher chemical) for another 15 min, and the third well is incubated with HBSS without Ca ²⁺ and Mg ²⁺ (Gibco, Life Technologies), but with 2 mM EDTA for 15 min and then add 56 mM KCl for another 15 min. The medium is immediately collected in an Eppendorf tube and cells are collected using Accutase®, centrifuged at 400 g for 4 min and resuspended in 10 µl PBS. Both the medium and the pellet were supplemented with 1 mM EDTA and 4 mM sodium metabisulfite (Sigma) to preserve dopamine.

Protocol for MMP.

Fibroblasts were cultured and seeded into a black μClear® Griener 384-well plate at 10,000 cells per well in 50 μl of media. The plates are left in an incubator overnight to allow the fibroblasts to adhere to the surface of the plate. The next morning, the glucose-based medium is replaced with 25 μl of galactose-based medium. Compounds were then added to the plates using an ECHO® 550 Liquid Dispensing Station. Wells were dispensed to provide an 8-point range of compound concentrations from 0.06 nM to 300 nM. After dispensing, another 25 μl of galactose-based medium is added to the wells and then left in the incubator for 24 hours. After 24 hours, the medium is removed from the wells and replaced with 25 μl of phenol-free minimum essential medium with 100 nM TMRM (Sigma) and 10 μM Hoechst dye (Sigma). The plate is returned to the incubator for another hour, after which the staining medium is removed and replaced with 25 μl of phenol-free MEM. The plate is then imaged using an IN Cell Multiparameter Microscope (GE Healthcare) with 10 fields of view per well in 2 channels, Cy3 excitation 542 nm, emission 604-64 nm; and DAPI excitation 350 nm, emission 450-55 nm at 37°C with CO ₂ . After imaging, the plate is discarded and the images are processed using the INCell developer Toolkit (GE Healthcare).

Protocol for ATP (adenosine triphosphate).

The protocol for ATP is generally as described in Mortiboys et al., Mitochondrial function and morphology are impaired in parkin-mutant fibroblasts, Ann Neurol. 2008 Nov; 64(5):555-65. Briefly, fibroblasts were cultured and seeded in white 384-well plates at 5000 cells per well in 50 μl of medium. The plates are left in an incubator overnight to allow the fibroblasts to adhere to the surface of the plate. The next morning, the glucose-based medium is replaced with 25 μl of galactose-based medium. Compounds are added to plates using an ECHO 550 liquid dispensing station. Dispense into wells to provide an 8-point range of compound concentrations from 0.06 nM to 300 nM. After dispensing, another 25 μl of galactose-based medium is added to the wells and then left in the incubator for 24 hours. After incubation, the medium is removed from the plate and the wells are washed twice with sterile PBS. The wells are filled with 25 μl of sterile PBS followed by 12.5 μl of lysis solution from the ATPlite™ Luminescence ATP Assay System (Perkin Elmer), including 16 cell-free wells for use as blanks. The plate is then placed on a rotary shaker for 5 min at 700 rpm. After vortexing, add 12.5 µl of ATP substrate solution (Perkin Elmer) to each well and vortex for another 5 min. The tablet is then placed in the dark for 10 min before reading. Using a PHERAStar® tablet reader, the luminescence intensity is recorded. After ATP analysis, the plates are immediately analyzed for DNA (deoxyribonucleic acid) content using the CyQUANT® assay.

Immediately after ATP analysis, DNA content is assessed using the CyQUANT® NF Cell Proliferation Assay Kit (ThermoFisher). CyQUANT® buffer is prepared immediately prior to analysis and consists of 1 µl CyQUANT® dye per 1 ml x1 HBSS solution. Add 12.5 µl of CyQUANT® buffer to each well. The plate is left in the incubator for 1 hour, then read on a PHERAStar® plate reader with excitation at 497 nm and emission at 520 nm.

Quantitative determination of ATP for each well was carried out using the following formula:

_X7 .____ _{__ d} _ ATPlite analysis value corrected for blank samples at level a. 1H/ —------------------------------------------------ --------------------------------CyQUANT assay value corrected for blank samples

Analysis of data for primary screening studies.

After assays were performed in triplicate for each line and compound, the data were entered into Graph pad Prism 7 software, where a dose-response curve was generated using the default [Agonist] versus effect equation (three parameters ).

_π Upper value-Lower value y = Upper value + x x----------------------------EC ₅₀ +x

From these EC ₅₀ values, the minimum effect and maximum effect were taken and used to calculate the geometric mean between the 5 different lines evaluated.

Results obtained for compounds that showed an ambiguous result in the [Agonist] equation depending on the effect (three parameters) were excluded from the geometric mean calculations due to the large scatter that was introduced by their inclusion.

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Analysis using Seahorse technology.

Fibroblasts are seeded into 24-well Seahorse plates at a rate of 50,000 cells per well. The cells are left for 2 days to attach to the surface. The medium is replaced with Seahorse DMEM with glucose and sodium pyruvate and left to equilibrate at 37 degrees under normal conditions, CO ₂ for 1 hour. The plate is placed in the Seahorse analyzer and the program of mixing (2 minutes), waiting (3 minutes) and measuring is started (3 minutes). After three measurements of basal respiration and ECAR (extracellular acidification rate), 0.5 μM oligomycin is administered, followed by three more measurements; then 0.5 μM of the protonophore oxidative phosphorylation uncoupler FCCP (carbonyl cyanide-4-trifluoromethoxyphenylhydrazone) is injected and three measurements are taken, and finally 1 μM rotenone is injected and three measurements are taken. Once all measurements are completed, cells are stained with 10 μM Hoescht dye and imaged using InCell to count cells per well for normalization. This classic mitochondrial stress test experimental protocol allows us to calculate basal mitochondrial respiration, ATP-linked respiration (the amount that is associated with ATP production), maximum and reserve tidal volume, non-mitochondrial respiration rate, and extracellular acidification rate, which is a proxy for glycolysis.

Analysis of complex I.

Mouse brains were homogenized ex vivo in a buffer of 250 mM sucrose, 20 mM HEPES, 3 mM EDTA, pH 7.5 at 4°C. Homogenization was performed using a Dounce homogenizer for cortical samples and by repeated passage through a 0.5 mm syringe for isolated striatum. Samples were then incubated with 30 μL detergent from the Complex I Colorimetric Assay Kit (AbCam) on ice for 20 min. Samples are then centrifuged at 13,000 rpm for 30 min. Triplicate samples for each condition were blocked using the blocking buffer from the kit on a plate from the Colorimetric Complex I Assay Kit (AbCam) for 3 h. The samples were then washed 3 times using the wash buffer from the kit before adding assay buffer from the assay kit. containing NADH (reduced nicotinamide adenine dinucleotide) and a colorimetric dye. The assay plate is read on a plate reader in the kinetic analysis program, reading 450 nm at 30 second intervals for 50 minutes.

D. Measurements of mitochondrial function and morphology in induced neurons

Cells are seeded in 96-well plates; For direct imaging, cells are incubated for one hour at 37°C with 80 nM tetramethylrhodamine (TMRM), 1 μM LysoTracker® Green fluorophore (Invitrogen), and 1 μM Hoechst dye solution (Sigma) before imaging using Opera Phenix™. Cellular ATP measurements are performed using an ATPlite kit (Perkin Elmer) according to the manufacturer's instructions. The production of reactive oxygen species by mitochondria was assessed using mitochondrial NpFR2 (probe; a kind gift from Dr. Liz New, University of Sydney, Australia) at a concentration of 20 μM and a solution of Hoechst dye at a concentration of 1 μM for 30 min at 37°C, then the dyes were removed and the cells visualized using Opera Phenix™. Images obtained from direct imaging experiments were analyzed using Harmony® (Perkin Elmer software). We have developed protocols for segmentation of the nucleus, cell walls and processes, mitochondria, lysosomes, and autophagosomes. We analyzed only z-projection images collected from z-sections.

Results.

Fibroblasts.

Mitochondrial transmembrane potential was measured in fibroblasts from 6 (Table 1) or 3 (Table 2) patients with sporadic Parkinson's disease treated with compounds of the invention or reference compounds. The results are shown in table. 1 and 2, where the lower value represents the maximum response at the lowest dose of compound (0.06 nM) and the upper value represents the maximum response at the highest dose of compound (300 nM).

Table 1

Data on the transmembrane potential of mitochondria from fibroblasts of 6 patients with sporadic PD

Compound E F G N 2 7 9 Lower value (% of response to vehicle treatment) 104.9 110 103.4 99.83 107.3 100.4 109.8 Upper value (% of response to vehicle treatment) 111.8 108.1 123.3 109.1 112 114 112.4 EC ₅₀ (nM) 23.29 0.6738 282.1 4,581 20.82 4,066 2,186

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table 2

Data on the transmembrane potential of mitochondria from fibroblasts of 3 patients with sporadic PD

Compound 14 17 19 20 22 27 thirty 31 Lower value (% of response to vehicle treatment) 94.3 88.9 95.9 99.8 99.3 102.6 98.4 99.6 Upper value (% of response to vehicle treatment) 166.8 152.3 143.1 147.1 145.8 158.9 155.6 151.1 EC ₅₀ (nM) 18.9 127.6 104.4 17.7 45.4 171.1 21.01 5.5

Note: In patients with sPD, the average reduction in MMP compared to controls is 18%; therefore, increasing MMP levels in vehicle-treated sPD patients to 118% would restore MMP to control levels.

Cellular ATP levels were measured in fibroblasts from 6 (Table 3) or 3 (Table 4) patients with sporadic Parkinson's disease treated with compounds of the invention or comparator compounds. The results are shown in table. 3 and 4, where the lower value represents the maximum response at the lowest dose of compound (0.06 nM) and the upper value represents the maximum response at the highest dose of compound (300 nM).

Table 3

Data on cellular ATP levels from fibroblasts from 6 patients with sporadic PD

Compound E F G N 2 7 9 Lower value (% of response to vehicle treatment) 81.59 99.91 83.3 87.64 79.9 112 99.04 Upper value (% of response to vehicle treatment) 135.5 111.9 92.72 120.8 112.6 171.8 102.9 EC ₅₀ (nM) 18.79 1,912 58.35 0.1247 1.078 0.4293 21.32

Table 4

Data on cellular ATP levels from fibroblasts from 3 patients with sporadic PD

Compound 14 17 19 20 22 27 thirty 31 Lower value (% of response to vehicle treatment) 107 100.7 98.8 102.4 95.9 101.4 96.7 108.1 Upper value (% of response to vehicle treatment) 126.9 132.2 122.3 121.7 115.9 113.8 115.3 133 EC ₅₀ (nM) 1.3 17.2 8.9 34.7 6.2 16.6 4.2 13.3

Note: in fibroblasts with sPD, the average decrease in cellular ATP levels compared to the control group is 24%. Therefore, 124% of the level in vehicle-treated sPD fibroblasts is an increase over control ATP levels.

The MMP and ATP assays described above, together with toxicity measurements, involve an initial screening of compounds in primary patient fibroblasts. When considering which compound is most active in primary screens, all information is taken into account, including EC50 values indicating activity and %maximum responses for both assays; Based on the combined activity, biological experts make decisions on each compound.

Oxygen consumption data obtained by Seahorse technology analysis for 6 sporadic PD patient fibroblast lines and 6 control lines are shown in FIG. 1A, 1B and 1C, from which it can be seen that fibroblasts in sPD demonstrate a significant decrease in basal mitochondrial respiration by 42% (***p less than 0.005), maximum respiration by 48% (*p less than 0.05) and ATP-bound breathing by 18% (*p less than 0.05). No improvement was observed with UDCA treatment, but treatment with compound 7 significantly improved all three mitochondrial parameters. It should be noted that both compounds are dosed at EC90 concentrations that are 100 nM for UDCA and 50 nM for compound 7. Thus, it appears that compound 7 provides a greater increase in mitochondrial function, as measured by oxygen consumption, and provides this effect at a lower concentration.

The rate of extracellular acidification in 6 fibroblast samples with sporadic PD and 6 control samples.

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Fibroblasts in sPD have a significant reduction in ECAR (an indirect measure of glycolysis) by 44% compared to controls (***p less than 0.005). As shown in FIG. 2, no changes are observed when treated with UDCA, but there are significant improvements when treated with compound 7.

The above data show protective effects of the compounds on mitochondria in primary fibroblasts from patients with sPD, but the cell type that is primarily damaged in PD is the dopaminergic neuron. The data below shows results obtained on dopaminergic neurons obtained from three patients with sPD; these cultures are approximately 96% dopaminergic neurons, and this methodology is currently the only protocol for creating such a pure dopaminergic culture from patient cells (method developed by Mortiboys, University of Sheffield); therefore, this patient-derived model most accurately represents the neurons affected in PD.

In table Figure 5 below shows the results of measurements of mitochondrial function and neuronal morphology in induced neurons from sPD patients compared to controls in the absence of treatment or treatment with either UDCA or Compound 7.

Table 5

Induced neurons

Parameter % of the level in induced neurons from the control group Patients with sPD not receiving treatment Patients with sPD treated with UDCA Patients with sPD treated with compound 7 ATP normalization (% of control) 50 82 101 ECso ATP (nM) 5 0.8 Normalization of MMR (% of control) 52 73 100 ECso MWD (nM) 7.3 0.52 ROS (reactive oxygen species) (normalization) (% of control) 115 110 103 Morphology of neurons (elongation; % of control) 48 64 78 Levels of activated caspase 3 162 143 100 Dopamine levels 0 No effect Approximately 5% increase in dopamine release

Data in table. 5 very clearly show that compound 7 provides a protective effect on both mitochondrial parameters in dopaminergic neurons in sPD in addition to improving neuronal morphology and reducing apoptosis levels (as measured by activated caspase 3 levels). Apoptosis is the main mechanism of cell death of dopaminergic neurons in culture and of dopaminergic neurons in patients with PD.

In vivo data obtained in mice with compound 7.

In fig. Figure 3 shows that mitochondrial respiratory chain complex I activity is significantly increased in a dose-dependent manner in whole brain tissue homogenates of wild-type mice following administration of Compound 7 at 4 mg/kg and 12 mg/kg. At a dose of 4 mg/kg the increase is approximately 380%, and at a dose of 12 mg/kg there is an increase in complex I activity of approximately 800% compared to the untreated control (*p less than 0.05).

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Claims (30)

In fig. Figure 4 shows the results of the activity of complex 1 in a homogenate of mouse brain tissue from the left region of the striatum from mice that were injected with: Carrier. Carrier plus 12 mg Compound 7 MPTP MPTP plus 1 mg compound 7 MPTP plus 4 mg compound 7 MPTP plus 12 mg compound 7 Treatment with compound 7 alone caused an increase in complex I activity of approximately 30% compared to the untreated control (*p less than 0.05). Treatment with MPTP caused a 50% reduction in complex I activity compared to untreated control (**p less than 0.01), but co-treatment with 1 mg of compound 7 and MPTP prevented the MPTP-induced loss of complex I activity and maintained complex I at normal levels (* *p less than 0.01 compared to MPTP alone), increasing doses of compound 7 concomitantly with MPTP appears to prevent any loss of complex I activity by MPTP. Activity of compounds in fibroblasts from patients with Alzheimer's disease. Fibroblasts from patients with both sAD (sporadic form of Alzheimer's disease) and familial form of AD (PSEN1 mutants) were tested using the same primary screening assay to determine total cellular ATP levels. Fibroblasts from patients with sAD and PSEN1 showed a 21% decrease compared to controls. The data presented in table. 6 below represent the average increase in ATP levels after 24 hours of treatment with 100 nM compounds. Since cells experience an average 21% decrease in ATP levels, an increase of 21% restores control levels, anything above 21% increases control levels. Table 6 Compound UDCA 2 7 8% increase compared to vehicle treatment 12% 65% 24% 65% The data clearly show that treatment with compounds 2, 7 and 8 results in a more favorable restoration of cellular ATP levels than treatment with UDCA. Additionally, compounds 2 and 8 are particularly effective, dramatically increasing ATP levels. CLAIM 1. Compound of general formula (I) where one of R ¹ and R ² represents F, and the other of R ¹ and R ² represents H or F; Y represents a bond or a C _1-20 alkylene linker group; R ³ is C(O)OR ¹² , C(O)NR ¹² R ¹³ , S(O)2OR ¹² or S(O)2NR ¹² R ¹³ , C(O)NR ¹² [CH(R ¹⁵ )]nR ¹⁶ ; each R ¹² is independently H or C _|-6 cLikhj, optionally substituted with one or more substituents selected from OR ¹⁰ , NR10R ¹¹ and R ¹⁶ ; R ¹⁰ and R ¹¹ are each independently H or C1. ₆ okil; R ¹³ represents H, C1-^Knn or a 3-8 membered carbocyclic ring or heterocyclic ring having at least one heteroatom selected from N, O and S, wherein said carbocyclic or heterocyclic ring is optionally substituted by one or more a substituent selected from =O and R ¹⁶ ; or phenyl or a 5-6 membered heteroaryl ring containing at least one heteroatom selected from N, O and S, wherein said phenyl or said heteroaryl ring is optionally substituted with an R ¹⁶ substituent; or when R ³ represents C(O)NR ¹² R ¹³ or S(O)2NR ¹² R ¹³ , R ¹² and R ¹³ together with the nitrogen atom to which they are attached form a 3-8 membered heterocyclic ring, which may contains one or more additional heteroatoms selected from N, O and S; and optionally substituted with one or more substituents selected from CH2C(O)OH, C(O)OH, C _1-6 alkyl, S(O) ₂ OH and =O; n is 1, 2 or 3; each R ¹⁵ independently represents H or C1-^Knn and may represent an R ¹⁴ group, where R ¹⁴ represents a side chain of an amino acid selected from glycine, alanine, valine, leucine or isoleucine; or - 51 046372 when n is 2 or 3, two R ¹⁵ groups, together with the carbon atoms to which they are attached, and possibly the intervening carbon atom, if present, can combine to form -(CH2)p-, so that the [CH(R ¹⁵ )]n group is a 3-8 membered carbocyclic ring; p is equal to 1, 2, 3, 4, 5 or 6; R ¹⁶ is selected from C(O)OH, S(O^OH, S(0)2(C1^™), OS(O)2OH and P(O)(OH)2; or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, which is a compound of the general formula (IA)-(IC) or (ID) where R ¹ , R ² , Y and R ³ are as defined above for the general formula (I). 3. A compound according to claim 1 or 2, wherein ^R1 and R2 are both F. 4. A compound according to any one of claims 1 to 3, where Y represents a bond or C ₁ . ₃ alkylene linker group. 5. A compound according to any one of claims 1 to 4, where R ³ represents C(O)OR ¹² , C(O)NR ¹² CH(R ¹⁴ )C(O)OH or C(O)NR ¹² CH(R ¹⁵ )CH(R ¹⁵ )S(O)2OH, where R ¹² and R ¹⁴ are as defined in claim 1, and R ¹⁵ represents H or C ₁ . ₆ alkyl; or R ³ is C(O)OR ¹² , C(O)N(R ¹² )(R ¹³ ) or C(O)NR ¹² [CH(R ¹⁵ )]nR ¹⁶ ; where R ¹² , R ¹³ , R ¹⁵ , R ¹⁶ and n are as defined in paragraph 1. 6. A compound according to any one of claims 1 to 5, where R ³ represents C(O)OR ¹² , or a pharmaceutically acceptable salt thereof. 7. A compound according to claim 6, wherein R ¹² is H, CH2R ¹⁶ or -CH2CH2R ¹⁶ , wherein R ¹⁶ is as defined above. 8. A compound according to any one of claims 1 to 5, where R ³ represents C(O)NR ¹² [CH(R ¹⁵ )] _n R ¹⁶ , or a pharmaceutically acceptable salt thereof. 9. A compound according to claim 8, wherein R ¹² is H, methyl or methyl substituted with R ¹⁶ . 10. A compound according to claim 8 or 9, where R ³ is C(O)NR ¹² CH(R ¹⁴ )C(O)OH or C(O)NR ¹² CH(R ¹⁵ )CH(R ¹⁵ )S( O) ₂ OH, where R ¹⁴ represents a side chain of an amino acid selected from glycine, alanine, valine, leucine or isoleucine, and R ¹⁵ represents H or Ci-balkyl; or R ³ is C(O)NR ¹² CH(R ¹⁴ )C(O)OH, where R ¹² is H or methyl and R ¹⁴ is H; or R ³ is C(O)NR ¹² CH(R ¹⁵ )CH(R ¹⁵ )S(O) ₂ OH, where R ¹² is H or methyl and both R ¹⁵ moieties are H. 11. A compound according to claim 8 or 9, wherein each R ¹⁵ is independently H or ^alkyl or n is 2 or 3, and the two R ¹⁵ groups are combined with the carbon atoms to which they are attached, and optionally with the one in between carbon atom, if present, to form a 5-7 membered carbocyclic ring. 12. A compound according to any one of claims 1 to 5, where R ³ represents C(O)NR ¹² R ¹³ , or a pharmaceutically acceptable salt thereof. 13. A compound according to claim 12, where R ¹² represents H or ^^alkyl substituted with one R ¹⁶ substituent, where R ¹⁶ is as defined in claim 1, and/or where R ¹³ represents phenyl or 5- a 7-membered cycloalkyl or heterocyclyl group, each of which is optionally substituted with one R ¹⁶ substituent, and wherein the cycloalkyl and heterocyclyl groups are substituted with one or more =O substituents. - 52 046372 14. The connection according to claim 1, selected from 2b-fluorochenodeoxycholic acid (compound 1); 2b-fluoro-3b,7a-dihydroxy-5b-cholanic acid (compound 2); 2a-fluoro-3b,7a-dihydroxy-5b-cholanic acid (compound 3); 2a-fluoro-3b,7b-dihydroxy-5b-cholanic acid (compound 4); 2a-fluoro-3a,7a-dihydroxy-5b-cholanic acid (compound 5); 2a-fluoro-3a,7b-dihydroxy-5b-cholanic acid (compound 6); 2,2-difluoro-3в,7в-dihydroxy-5в-cholanic acid (compound 7); 2,2-difluoro-3a,7a-dihydroxy-5b-cholanic acid (compound 8); 2,2-difluoro-3a,7b-dihydroxy-5b-cholanic acid (compound 9); H-(2,2-di(|)tor-3в,7в-dihydroxy-5в-cholan-24-amide)ethylsul(|)onic acid (compound 10); N(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-amide)propionic acid (compound 11); Na(methyl)-N-(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-amide)acetic acid (compound 12); Na(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-amide)-trans-2-cyclohexanecarboxylic acid (compound 13); 1-(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)piperidine-3-carboxylic acid (compound 14); 3-(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-amide)-4-thiazolidine-carboxylic acid (compound 15); Na(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-oil)morpholine (compound 16); N(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-amide)methylcarboxylic acid (compound 17); Na(carboxymethyl)-N-(2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-oyl)-2-aminoacetic acid (compound 18); N(methyl)-N-(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-amide)ethylsulfonic acid (compound 19); 3-(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-oyl)aminopropanesulfonic acid (compound 20); N(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-amide)methanesulfonic acid (compound 21); N(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)-2-aminoethylsulfuric acid (compound 22); O-(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)-2-hydroxyethylsulfonic acid (compound 23); N(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)aniline-2-sulfonic acid (compound 24); N(cyclohexyl)-N-(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-oil)-3-aminopropanesulfonic acid (compound 25); N(cyclohexyl)-H-(2,2-difluoro-3в,7в-dihydroxy-5в-cholan-24-oil)-2-aminoethanesulfonic acid (compound 26); H-(2.2-difluoro-3v,7v-dihydroxy-5v-cholan-24-oil)-2-aminoethylmethylsulfone (compound 27); N(ethyl)-H-(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-oil)-3-aminotetrahydrothiophene dioxide (compound 28); N(2-(diisopropylamino)ethyl)-N-(2,2-difluoro-3В,7В-dihydroxy-5В-cholan-24-oil)-3-aminotetrahydrothio(ene-dioxide (compound 29); N(2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-oil)thiomorpholine dioxide (compound 30); N(2,2-difluoro-3v,7v-dihydroxy-5v-cholan-24-oil)-1,1-dioxidotetrahydro-2H-thiopyran-3-ylamine (compound 31) and their pharmaceutically acceptable salts, especially metal salts, such as sodium and potassium salts, especially sodium salts or (for compound 18) disodium salt. 15. Use of a compound according to any one of claims 1 to 14 in the treatment or prevention of a neurodegenerative disorder. 16. Use of a compound according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder. 17. A method of treating or preventing a neurodegenerative disorder, comprising administering to a patient in need of such treatment an effective amount of a compound according to any one of claims 1 to 14. 18. Use according to claim 15 or 16, wherein the neurodegenerative disorder is Parkinson's disease, mild cognitive impairment, dementia (including Alzheimer's disease, vascular dementia and dementia with Lewy bodies), Huntington's disease, amyotrophic lateral sclerosis (motor neuron disease), progressive supranuclear palsy or Wilson's disease. 19. Use according to claim 18, wherein the neurodegenerative disorder is Parkinson's disease, and the compound is selected from compounds of general formula (IA) and (ID) as defined in claim 2. 20. Use according to claim 19, wherein the compound is selected from 2,2-difluoro-3b,7v-dihydroxy-5v-cholanic acid (compound 7) and difluoro-3a,7v-dihydroxy-5v-cholanic acid (compound 9). - 53 046372 21. Use according to claim 18, wherein the neurodegenerative disorder is Alzheimer's disease and the compound is selected from compounds of general formula (IB) as defined in claim 2. 22. Use according to claim 21, wherein the compound is 2,2-difluoro-3a,7a-dihydroxy-5vcholanic acid (compound 8). 23. The method of claim 17, wherein the neurodegenerative disorder is Parkinson's disease, mild cognitive impairment, dementia (including Alzheimer's disease, vascular dementia and dementia with Lewy bodies), Huntington's disease, amyotrophic lateral sclerosis (motor neuron disease), progressive supranuclear palsy or Wilson's disease. 24. The method of claim 23, wherein the neurodegenerative disorder is Parkinson's disease and the compound is selected from compounds of the general formula (IA) and (ID) as defined in claim 2. 25. The method according to claim 24, where the compound is selected from 2,2-difluoro-3v,7v-dihydroxy-5v-cholanic acid (compound 7) and difluoro-3a,7v-dihydroxy-5v-cholanic acid (compound 9). 26. The method of claim 23, wherein the neurodegenerative disorder is Alzheimer's disease and the compound is selected from compounds of general formula (IB) as defined in claim 2. 27. The method according to claim 26, where the compound is 2,2-difluoro-3a,7a-dihydroxy-5vcholanic acid (compound 8). 28. A pharmaceutical composition containing a compound according to any one of claims 1 to 14 and a pharmaceutically acceptable excipient or carrier. 29. A method for preparing a compound according to any one of claims 1 to 14, which is a compound of general formula (IB) or (IC) as defined in claim 2, in which R ¹ represents F and R ³ represents C(O )OR ^12a , where R ^12a represents C _1-6 αlkyl, optionally substituted with one or more halogen or aryl groups, wherein the method includes acid treatment of a compound of general formula (II) "H OR ^12a (P), where Y and R ³ are as defined in claim 1; R ^12a is as defined above; and R ²¹ represents an acid-sensitive OH protecting group. 30. A method for preparing a compound according to any one of claims 1 to 14, which is a compound of general formula (IA) as defined in claim 2, in which R ¹ and R ² are both F and R ³ is C (O)OR ^12a , where R ^12a represents C _1- ^km, optionally substituted with one or more halogen or aryl groups, wherein the method involves treating a compound of general formula (XXI) with an acid "IIIH OR ^12a (XXI), where Y is as defined in claim 1; R ^12a is as defined above; and R ²¹ represents an acid-sensitive OH protecting group. -