7-DIFLUORINATED STEROIDS FOR USE IN THE TREATMENT OF NEUROLOGICAL OR MITOCHONDRIAL DISEASES

Abstract

Compounds of formula (I): , wherein the linkage , R1, R2, R3, R4a and R4b are as defined herein, are capable of restoring damaged mitochondria and are useful for the treatment of neurodegenerative and neuromuscular diseases and disorders.

Description

[0001] 7-DIFLUORINATED STEROIDS FOR USE IN THE TREATMENT OF NEUROLOGICAL OR MITOCHONDRIAL DISEASES

[0002] The present invention relates to novel compounds which are of use in the treatment of neurodegenerative disorders and other conditions in which mitochondrial dysfunction is implicated and / or conditions in which modulating mitochondrial function is useful. In particular, the invention relates to bile acid derivatives, to pharmaceutical compositions containing them, process for preparing them and to the use of the compounds in the treatment or prevention of neurodegenerative and neuromuscular disorders and mitochondrial disease.

[0003] Background of the Invention

[0004] Neurodegenerative diseases are a group of disorders of the central nervous system (CNS) and include dementia (including Alzheimer’s disease, vascular dementia, frontotemporal dementia (FTD) and dementia with Lewy bodies), Parkinson’s disease, mild cognitive impairment, Huntington’s disease, amyotrophic lateral sclerosis (motor neurone disease), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and Wilson’s disease. The incidence of neurodegenerative disease increases with age and therefore such conditions are 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 available which alleviate the symptoms of Parkinson’s disease, some types of cognitive impairment and dementia.

[0005] Alzheimer’s disease leads to progressive cognitive impairment and is characterised by the presence of extracellular neuritic plaques and intracellular neurofibrillary tangles. It is thought that mitochondrial dysfunction leads to the deposition of the p-amyloid proteins which are the major component of the neuritic plaques and to the formation of the neurofibrillary tangles. Alzheimer’s disease may be either sporadic or familial, with the most common cause of familial Alzheimer’s disease being mutations in the PSEN1 gene, which encodes presenilin-1 (Kelleher and Shen, 2017) as well as the PSEN2 (presenilin-2) and APP (amyloid precursor protein) genes (Petit et al (2022). Presenilin-1 and 2 are associated with the mitochondrial membrane causing disease related pathology. Familial Alzheimer’s disease is associated with early onset, in which symptoms may manifest in patients as young as 24 years of age. Sporadic Alzheimer’s disease typically manifests much later, generally in patients over 65 years of age.

[0006] The 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 a large body of evidence which suggests that there is a strong association between mitochondrial dysfunction and Parkinson’s disease. A mild deficiency of mitochondrial electron transport chain NADH dehydrogenase (complex I) activity has been found in the tissues of Parkinson’s disease patients and a number of the proteins that are linked to the familial form of Parkinson’s disease are either mitochondrial proteins or are associated with mitochondria.

[0007] Huntington’s disease is an inherited progressive neurodegenerative disease and is characterised by motor impairment, personality changes and cognitive decline. The pathology of Huntington’s disease provides evidence for a link with mitochondrial dysfunction.

[0008] Amyotrophic lateral sclerosis is also thought to be linked to mitochondrial dysfunction. This disease targets motor neurons in the CNS resulting in muscle weakness, atrophy and, death within 2-3 years of diagnosis. Phase 3 clinical trials have been conducted on TLIDCA for ALS (Albanese et al, 2022).

[0009] Attempts have been made to find compounds which are capable of treating neurodegenerative disorders and several compounds have been developed which target mitochondria. For example, it is known that bile acids such as LIDCA (ursodeoxycholic acid) exert a beneficial effect on mitochondrial dysfunction in tissue from certain patients suffering from Parkinson’s disease, in particular in tissue from parkin mutant Parkinson’s disease patients (Mortiboys, et al 2013) and LRRK2G2019Smutant Parkinson’s disease patients (Mortiboys et al 2015). Furthermore it is known that bile acids such as LIDCA exert a beneficial effect on fibroblasts from patients suffering from both sporadic Alzheimer’s Disease and familial Alzheimer’s Disease due to PSEN1 mutations (Bell et a / 2018). Furthermore, additional studies have shown that LIDCA is beneficial to cells from sporadic Parkinson’s patients (Carling et al 2020). Furthermore, an innovative Phase 2a clinical trial testing LIDCA in Parkinson’s patients has shown some positive effects of LIDCA treatment in this small patient group (Payne et al, 2023).

[0010] WO 2014 / 036379, WO 2015 / 061421 and WO 2016 / 145216 teach that bile acids may be of use 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 discloses fluorinated bile acids particularly bile acids fluorinated at the 3- and / or 7-positions, for example 7-fluoro, 3,7-difluoro and 3, 3,7,7- tetrafluoro analogues of ursodeoxycholic acid. WO 2020 / 128514 relates to 2-fluorinated bile having mitochondrial rescue properties and WO 2023 / 233164 relates to further bile acid derivatives which also have mitochondrial rescue properties. If a compound is to be used for the treatment of a neurodegenerative disorder it is, of course, necessary for it to enter the plasma, cross the blood brain barrier and remain in the CNS in sufficient amounts for activity.

[0011] In order for an orally administered drug to enter the CNS, it must first enter the plasma and this has proved problematic in the case of bile acids, even when the intrinsic activity of the compounds is relatively high. When taken orally, bile acids are transported from the gut to the liver where they are conjugated, especially with taurine or glycine residues, to form bile salts. The bile salts are then exported to the gall bladder and enter the enterohepatic circulation (Dawson, 2009). The result of the bile acids / salt entering the enterohepatic circulation is that only a small proportion of the administered compound enters the blood stream and is therefore available to cross the blood brain barrier.

[0012] The blood brain barrier is formed by a monolayer of endothelial cells joined at tight junctions and other elements including a basement membrane, glial cells, pericytes and neurons. The function of the blood brain barrier is to prevent harmful substances from entering the brain, while allowing access to oxygen and nutrients such as glucose.

[0013] The ability of a drug molecule to achieve an effective concentration in the CNS depends on a number of factors, which are discussed in Banks, 2009. Most drugs cross the CNS by transmembrane diffusion and this is favoured by low molecular weight and lipophilicity. The drug must be sufficiently lipid soluble to pass into the cell membrane but not so lipophilic that it remains in the blood brain barrier rather than passing through it or is taken up by peripheral tissues. Drugs can, of course, diffuse across the blood brain barrier in both directions and if the drug is to achieve an effective concentration in the brain, it is also preferable that the rate of passage from the plasma into the brain (influx) is higher than the rate of passage from the brain into the plasma (efflux) (Dolghih, 2013; Doan, 2002. Efflux may be enhanced by binding of the drug to P-glycoprotein (also known as MDR1), which is an active efflux transporter and brain penetration of a drug can be predicted by the efflux ratio, which is represented by the following equation:

[0014] Efflux Ratio = ^app (B~A)Papp (A-B)

[0015] Where Papp<B-A) represents the permeability coefficient in the apical to basolateral (A-B) direction, representing transport of the compound from the plasma to the CNS, and Papp<A-B) represents the permeability coefficient in the basolateral to apical (B-A) direction, representing efflux from the CNS. This is discussed in greater detail in Biological Example 3 below. For effective CNS concentration, the efflux ratio should preferably be less than 2.5 (Doan, 2002), which is not the case with bile acids such as ursodeoxycholic acid (LIDCA). In some cases, however, if a compound is particularly effective at passing through the blood brain barrier, it may achieve a pharmaceutically effective concentration in the CNS even if the efflux ratio is higher than the preferred value. However, more suitable compounds have lower efflux ratios.

[0016] Another relevant factor is the degree of protein binding, since this can prevent a drug from diffusing across the blood brain barrier (Banks, 2009). The problem of protein binding is particularly acute with bile acids, many of which are bound in plasma at high levels.

[0017] Neuromuscular disorders include conditions such as muscular dystrophies, myopathies, neuromuscular junction diseases, motor neurone diseases, peripheral nerve diseases, mitochondrial diseases and ion channel diseases. Many of these diseases arise because of mitochondrial defects (Canto-Santos, 2020) and therefore can be treated by compounds which are capable of rescuing dysfunctional mitochondria. When a bile acid derivative is intended for use in the treatment of neuromuscular diseases, the ability to cross the blood brain barrier is not so important as for compounds intended for the treatment of neurodegenerative diseases. However, it is still necessary to ensure that the bile acid derivative does not become trapped in the enterohepatic circulation and that the degree of binding to plasma proteins is not sufficiently high to prevent the compound from crossing the mitochondrial membrane.

[0018] Mitochondrial diseases include Barth syndrome, chronic progressive external opthalomplegia (CPEO), Kearns-Sayre syndrome (KSS), Leigh syndrome, 3-methylglutaconic aciduria, deafness, encephalopathy, and Leigh-like syndrome (MEGDEL syndrome), myoclonic epilepsy and ragged-red fibers syndrome (MERRF syndrome), mitochondrial encephalomyopathy, lactic acidosis and stroke-like syndrome (MELAS), mitochondrial neurogastrointestinal encephalopathy (MNGIE) and Senger syndrome. These diseases can also be treated by compounds which are capable of rescuing dysfunctional mitochondria.

[0019] It is therefore desirable to develop bile acid derivatives which are able to rescue dysfunctional mitochondria, which are not recirculated in the enterohepatic circulation, which do not bind in large quantities to plasma proteins and which cross the blood brain barrier and are retained in the CNS in an effective concentration.

[0020] Summary of the Invention

[0021] In a first aspect of the present invention there is provided a compound of formula (I): wherein: each is independently a single or a double bond when connected to R1is a single bond, R1is selected from H, fluoro, chloro, OH, NH2, N3,

[0022] C1-4 alkyl, O(Ci-4 alkyl), NH(CI-4alkyl), C(O)OH, C(O)NHR1aand NHC(O)OR1 b; wherein when R1is C1.4 alkyl or O(Ci-4 alkyl), it is optionally substituted with one or more substituents selected from fluoro, chloro, OH, NH2, N3, O(Ci-4 alkyl) and NH(CI-4 alkyl); wherein each of R1aand R1 bis independently H or C1.4 alkyl optionally substituted with halo; when connected to R1is a double bond, R1is selected from CHR8and O; wherein R8is H or CH3;

[0023] R2is selected from C(O)OR5, C1.6 alkyl, C2-6 alkenyl and C(O)N(R5a)(R5b); wherein R5is H, C1.4 alkyl or benzyl; each of R5aand R5bis H or C1.4 alkyl or R5aand R5btogether with the nitrogen atom to which they are attached form a piperidine, pyrrolidine, piperazine or morpholine ring; and wherein alkyl and alkenyl groups of R2are optionally substituted with one or more substituents selected from OH, NH2, N3, fluoro and chloro; when connected to R3is a single bond, R3is OH; when connected to R3is a double bond, R3is O; each of R4aand R4bis independently H, F, Cl, C1.2 alkyl or C1.2 haloalkyl; or a salt or solvate thereof.

[0024] Compounds of formula (I) include all isotopic variants but, in particular, isotopic variants in which: in an alkyl group R1, one or more hydrogen atoms are present as2H (deuterium) or3H (tritium), especially deuterium; and / or when connected to R3is a single bond, the hydrogen atom at the 3-position of the bile acid skeleton is replaced by2H (deuterium) or3H (tritium), especially deuterium. In a second aspect of the invention, there is provided a compound of formula (IZ): wherein:

[0025] R1Zis selected from H, fluoro, chloro, OH, C1.4 alkyl and O(Ci-4 alkyl), wherein when R1Zis Ci- 4 alkyl it is optionally substituted with one or more substituents selected from fluoro, chloro, OH and O(Ci-4alkyl);

[0026] R5is H, Ci-6 alkyl or benzyl; or a salt or solvate thereof.

[0027] Compounds of formula (IZ) include all isotopic variants thereof.

[0028] Detailed Description of the Invention

[0029] In the present specification, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

[0030] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

[0031] In the present specification, references to “pharmaceutical use” refer to use for administration to a human or an animal, in particular a human or a mammal, for example a domesticated or livestock mammal, for the treatment or prophylaxis of a disease or medical condition. The term “pharmaceutical composition” refers to a composition which is suitable for pharmaceutical use and “pharmaceutically acceptable” refers to an agent which is suitable for use in a pharmaceutical composition. Other similar terms should be construed accordingly. In the present application, the term “Ci-e” alkyl refers to a straight or branched fully saturated hydrocarbon group having from 1 to 6 carbon atoms. The term encompasses methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, s-butyl and t-butyl. Other alkyl groups, for example C1.4 alkyl, C1.3 alkyl, or C1.2 alkyl are as defined above but contain different numbers of carbon atoms.

[0032] The term “Ci-e alkylene” refers to a straight or branched fully saturated hydrocarbon chain having from one to 6 carbon atoms. The term encompasses -CH2-, -CH2CH2-, -CH(CH3)-CH2- , -CH2CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH2CH3)- and -CH2CH(CH2CH3)CH2-. Other alkylene groups, for example C1.5 alkylene, C1.4 alkylene, C1.3 alkylene, or C1.2 alkylene are as defined above but contain different numbers of carbon atoms.

[0033] The term “C2-6 alkenyl” refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms and containing one or more carbon-carbon double bonds. Examples include -CH=CH2, -CH2CH=CH2, -CH=CH-CH3, -CH=C(CH3)2, -CH2CH=CH-CH3, and CH=CH-CH2CH3. Other alkenyl groups, for example C2-4 alkenyl and C2-3 alkyl are as defined above but contain different numbers of carbon atoms.

[0034] The term “protected OH group” refers to a hydroxyl protected by any known protecting group. Examples of protected OH groups of this type include R18C(O)O, where R18is Ci-e alkyl or benzyl, especially methyl. Silyl ether protecting groups may also be used and OH can also be protected as an ether, for example a Ci-e alkyl, benzyl or p-methoxybenzyl ether. Other suitable protecting groups for OH are well known to those of skill in the art (see e.g. Wuts, PGM and Greene, TW (2006) “Greene’s Protective Groups in Organic Synthesis”, 4thEdition, John Wiley & Sons, Inc., Hoboken, NJ, USA).

[0035] Salts of the compounds of formula (I) may be basic addition salts.

[0036] Any salts intended to be administered to a patient will be pharmaceutically acceptable but other salts may also be used during the synthesis of a pharmaceutically acceptable final product. Pharmaceutically acceptable salts are known to those of skill in the art and are summarised in Gupta et al, Molecules, 23, 1719 (2018).

[0037] Pharmaceutically acceptable basic addition salts include sodium, potassium, calcium, aluminium, zinc, magnesium and other metal salts as well as choline, amine salts including triethylamine, / V, / V-diisopropylethylamine (DI PEA), diethanolamine, ethanolamine, ethyl diamine, meglumine and other well-known basic addition salts. The compounds of formula (I) include all stereoisomers. In the compounds of the invention, the stereochemistry of the bile acid ring system is fixed and therefore the term “stereoisomers” as used herein refers only to stereoisomers of the R1and / or the R3substituents in the compounds of formula (I) and not to stereoisomers of the bile acid ring system.

[0038] The compounds of formula (I) include all isotopic variants. The term “isotopic variant” refers to isotopically-labelled compounds which are identical to those recited in formula (I) but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature, or in which the proportion of an atom having an atomic mass or mass number found less commonly in nature has been increased (the latter concept being referred to as “isotopic enrichment”). Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as2H (deuterium),3H,11C,13C,14C,18F,123l or125l (e.g.3H,11C,14C,18F,123l or125l), which may be naturally occurring or non-naturally occurring isotopes.

[0039] As noted above, the invention provides a compound of formula (I) as defined above or a salt, solvate and / or isotopic variant thereof.

[0040] The compounds of formula (I) have significant advantages when compared to the prior art compounds such as ursodeoxycholic acid and the fluorinated derivatives described in WO 2016 / 145216.

[0041] Firstly, the compounds of formula (I) have a side chain which is one carbon atom shorter than that of ursodeoxycholic acid and the ursodeoxycholic acid derivatives described in WO 2016 / 145216. Surprisingly, it appears that this prevents recognition of the compounds by the enzymes which control conjugation of bile acids in the liver. As a result, bile salts of the compounds of formula (I) are not formed in the liver and the compounds of formula (I) do not enter the enterohepatic circulation but pass to the bloodstream. This means that if a compound of formula (I) and the ursodeoxycholic acid derivatives described in WO 2016 / 145216 are administered at the same dose, a larger amount of the compound of formula (I) passes to the bloodstream. This is the case for both parenteral and oral administration and is demonstrated in the assay described in Biological Example 4, where a number of compounds of formula (I) are compared with Comparative Compound A, a compound falling within the claim scope of WO 2016 / 145216. Secondly, the 7,7-difluoro substitution pattern of the compounds of formula (I) leads to an unexpectedly low efflux ratio. Particularly preferred compounds of formula (I) retain acceptable permeability but the efflux ratio is significantly decreased compared to LIDCA. For example, as shown in Biological Example 3 below, Compound 1 of Example 1 has an efflux ratio of 1.2, whereas LIDCA has an efflux ratio of 3.6. Because of this, the achievable concentration in the CNS is significantly greater for compounds of formula (I) than for LIDCA.

[0042] Thirdly, the compounds of formula (I) bind to proteins in lower amounts than LIDCA or the compounds of WO 2016 / 145216. Thus for example, 89% of Compound 1 of Example 1 binds to plasma proteins but 99% of LIDCA is protein-bound in plasma. This means that the unbound concentration in the blood is significantly higher, for the compounds of the invention than for LIDCA meaning that penetration across the blood brain barrier is higher.

[0043] The above factors all lead to an unexpected increase in oral bioavailability of the compounds of formula (I) compared to either LIDCA or the compounds of WO 2016 / 145216. Biological Example 1 describes a dose response assay for mitochondrial membrane potential (MMP) and mitochondrial morphology as represented by the percentage of long mitochondria. In these assays, Compound 1 , which is a compound of formula (I) was compared with Comparative Compound A, which has the same difluoro substitution at the 7-positon of the steroid ring system but has a side chain one carbon atom longer. The results showed that Compound 1 achieved a higher maximum MMP value than Comparative Compound A and a similar maximum for long mitochondria. However, there is likely to be a more significant difference in the in vivo activities of Compound 1 and Comparative Compound A than might have been predicted from their in vitro activity because levels in the CNS will be significantly higher for a compound of formula (I) than for compounds with a longer side chain.

[0044] In some suitable compounds of formula (I), connected to R1is a single bond.

[0045] In some compounds of this type, R1is suitably selected from H, fluoro, chloro, OH, NH2, N3, C1.4 alkyl, O(Ci-4alkyl), NH(CI-4alkyl) and C(O)OH.

[0046] In some suitable compounds of this type, R1is H.

[0047] In other suitable compounds of this type, R1is C1.4 alkyl which is unsubstituted or is substituted as described above. The C1.4 alkyl group may be unsubstituted and, for example may be methyl or ethyl. Alternatively, the C1.4 alkyl group may be substituted with one or more substituents selected from fluoro, chloro, OH, NH2, N3, and O(Ci-4 alkyl), especially fluoro, chloro, OH, NH2 and methoxy. In particularly suitable compounds of this type, R1is methyl, ethyl, or methyl substituted with one or more substitutents selected from OH, methoxy, NH2 or F. For example, R1is methyl, ethyl, CH2OH, CH2F, CHF2, CH2OCH3 or CH2NH2. In some cases, the compound is an isotopic variant in which one or more of the hydrogen atoms of a substituted or unsubstituted alkyl group R1is replaced by2H (deuterium, D) or3H (tritium, T). For example, R1may be CD3.

[0048] In still other suitable compounds of this type, R1is fluoro, chloro or azido (N3), especially fluoro or chloro.

[0049] In still other suitable compounds of this type, R1is OH, NH2, O(Ci-4 alkyl) or NH(CI-4 alkyl), suitably OH, methoxy, ethoxy, NH2, methyl amino or dimethylamino. In some still more suitable compounds, R1is OH or methoxy and in other still more suitable compounds, R1is NH2.

[0050] In still other suitable compounds of this type, R1is C(O)OH.

[0051] In still other suitable compounds in which connected to R1is a single bond, R1is C(O)NHR1aor NHC(O)OR1 b. Suitably, R1aand R1 bare each independently selected from H and methyl.

[0052] In some suitable compounds of formula (I), connected to R1is a double bond.

[0053] In some suitable compounds of this type, R1is CHR8, wherein R8is as defined above but is still more suitably H.

[0054] In other suitable compounds of the type, R1is O.

[0055] In some suitable compounds of formula (I) R2is C(O)OR5.

[0056] As noted above, R5may be H, Ci-e alkyl or benzyl.

[0057] In particularly suitable compounds of formula (I) and formula (IZ), R5is H such that R2in formula (I) is C(O)OH and C(O)OR5in formula (IZ) is C(O)OH.

[0058] In other compounds of formula (I) and formula (IZ), R5is a group R6, where R6is Ci-e alkyl or benzyl, and more suitably C1.4 alkyl, for example methyl. In other suitable compounds of formula (I), R2is C1.4 alkyl or C2-4 alkenyl wherein said alkyl and alkenyl groups are optionally substituted with one or more substituents selected from OH, NH2, N3, fluoro and chloro.

[0059] More suitably in these compounds, R2is methyl, ethyl or ethenyl, any of which is optionally substituted with one or more substituents selected from OH, NH2, N3, fluoro and chloro, for example unsubstituted ethyl, unsubstituted ethenyl or methyl substituted with one or more substituents selected from OH, NH2, N3, fluoro and chloro.

[0060] Still more suitably in these compounds, R2is unsubstituted ethyl, unsubstituted ethenyl, CH2OH, CH2NH2, CH2N3, CH2F or CHF2.

[0061] In still other suitable compounds, R2is C(O)NR5aR5b, where R5aand R5bare as defined above. More suitably, R5aand R5bare each independently H, methyl or ethyl, for example H or methyl. In some still more suitable compounds, R5ais H and R5bis methyl.

[0062] In alternative compounds where R2is C(O)NR5aR5b, R5aand R5btogether with the nitrogen atom to which they are attached form a piperidine, pyrrolidine, piperazine or morpholine ring, more suitably a piperidine, piperazine or morpholine ring, for example a morpholine ring.

[0063] In some suitable compounds of the invention, each of R4aand R4bis independently H, F, Cl, methyl or methyl substituted with one or more fluoro substituents. More suitably, one of R4aand R4bis H and the other of R4aand R4bis H, F, Cl, methyl or methyl substituted with one or more fluoro substituents.

[0064] In some suitable compounds, both R4aand R4bare H.

[0065] In some suitable compounds R4ais H and R4bis F, Cl, methyl or trifluoromethyl, more suitably F or Cl and especially F.

[0066] In other suitable compounds R4bis H and R4ais F, Cl, methyl or trifluoromethyl, more suitably F or Cl and especially F.

[0067] In some suitable compounds of the invention, connected to R3is a double bond, and R3is O. More suitably, however, connected to R3is a single bond, and R3is OH.

[0068] In particularly suitable compounds, connected to R3is a single bond, and R3is a-OH such that the compound of formula (I) is a compound of formula (IY):

[0069] In compounds of formula (IY), R1, R2, R4aand R4bare as described above for compounds of formula (I).

[0070] Particularly suitable compounds of the invention include: 7,7-difluoro 24-norlithocholic acid (Compound 1);

[0071] Methyl 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate (Compound 1 Me ester) 3a-hydroxy-7,7-difluoro-5p-24-nor-22-methyl-cholanic acid (Compound 2);

[0072] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-ethyl-cholanic acid (Compound 3);

[0073] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-fluoro-cholanic acid (Compound 4);

[0074] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxy-cholanic acid (Compound 5);

[0075] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-chloro-cholanic acid (Compound 6);

[0076] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-(methyl-d3)-cholanic acid (Compound 7);

[0077] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-methoxy-cholanic acid (Compound 8);

[0078] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-keto-cholanic acid (Compound 9);

[0079] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-azido-cholanic acid (Compound 10);

[0080] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-amine-cholanic acid (Compound 11);

[0081] 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-ol (Compound 12);

[0082] 3a-hydroxy-7,7-difluoro-5p-24-nor-chol-22-ene (Compound 13);

[0083] 3a-hydroxy-7,7-difluoro-5p-24-nor-cholane (Compound 14);

[0084] 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-azide (Compound 15);

[0085] 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-amine (Compound 16);

[0086] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholanic acid (Compound 17);

[0087] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-fluoromethyl-cholanic acid (Compound 18);

[0088] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-di-fluoromethyl-cholanic acid (Compound 19);

[0089] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-methylene-cholanic acid (Compound 20); 3a-Hydroxy-7,7-difluoro-5p-24-nor-22-methoxymethyl-cholanic acid (Compound 21); 3a-hydroxy-7,7-difluoro-5p-24-nor-22-aminomethyl-cholanic acid (Compound 22);

[0090] 3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-ol (Compound 23); 3-keto-7,7-difluoro-5p-24-nor-cholan-23,23-dicarboxylic acid (Compound 24);

[0091] 3-keto-7,7-difluoro-5p-24-nor-cholanic acid (Compound 25); 3p-Hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 26); 2p-fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 27);

[0092] 2p-fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 28);

[0093] 4p-fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 29);

[0094] 4p-fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 30);

[0095] 4a-chloro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 31); [2H]-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oic acid (Compound 32);

[0096] A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oyl)-(morpholine) (Compound 33); and A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oyl)-(methylamine) (Compound 34); and salts and solvates of any one thereof.

[0097] Preparation of Compounds of the Invention

[0098] Compounds of formula (I) in which connected to R1is a single bond, R2is C(O)OR5, where R5is H, R3is a-OH and R4aand R4bare both H. Compound 1 may be prepared from a protected compound of formula (II): wherein R1is as defined for formula (I), R6is Ci-e alkyl or benzyl and R7is a protected OH group; by hydrolysis, particularly base hydrolysis, for example by treatment with a strong base such as sodium or potassium hydroxide in an alcoholic solvent such as methanol, ethanol or isopropanol.

[0099] Compounds of formula (II) are new and form an aspect of the invention.

[0100] Suitably, R7is R18C(O)O, where R18is Ci-e alkyl or benzyl, especially methyl. These protecting groups can easily be removed by hydrolysis at the same time as the R6group. A compound of formula (II) may be prepared by fluorination of a compound of formula (III): wherein R1is as defined for formula (I) and R6and R7are as defined for formula (II).

[0101] The fluorination may be carried out using a fluorinating agent such as diethylaminosulfur trifluoride (DAST), a DAST alternative, for example morpholino-DAST, SF4 or Deoxo-Fluor® (bis(2-methoxyethyl)aminosulfur trifluoride solution).

[0102] Suitably, fluorination using DAST or a DAST derivative is conducted at elevated temperature, for example at about 30 °C to 70 °C, for example about 40 °C to 60 °C and typically at about 50 °C. Fluorination with SF4 is generally conducted at low temperature, typically about -78°C.

[0103] A compound of formula (III) may be prepared by oxidation of a compound of formula (IV): wherein R1is as defined for formula (I) and R6and R7are as defined for formula (II).

[0104] One suitable method is a Dess-Martin periodinane (1 , 1 ,1 -triacetoxy-1 ,1 -dihydro-1 , 2- benziodoxol) oxidation, which may be carried out in a chlorinated solvent such as chloroform or dichloromethane at a temperature of about 15 to 25 °C, suitably at room temperature.

[0105] An alternative oxidation method is oxidation using a hypochlorite, for example sodium hypochlorite, under acidic conditions, for example provided by acetic acid. The reaction may be carried out in an aqueous solvent and at a temperature of 0 to 15 °C, more usually at about O to 10 °C. Other oxidation methods include a Jones reaction using sodium dichromate or, more usually, chromic trioxide in dilute sulfuric acid. This process is known to be reliable for the clean conversion of bile acid hydroxyl groups to the corresponding keto derivatives (Bortolini et al, J. Org. Chem., 2002, 67, 5802). Alternatively oxidation may be carried out using TEMPO ((2,2,6,6-Tetramethyl-piperidin-1-yl)oxy) or a derivative thereof.

[0106] A compound of formula (IV) may be prepared by protecting an ester of formula (V): wherein R1is as defined for formula (I) and R6is as defined for formula (II).

[0107] For example, when the protected OH group R7of the compound of formula (IV), is R18C(O)O, the compound of formula (V) may be reacted with a carboxylic acid of formula R18C(O)OH, or its acid chloride or anhydride.

[0108] The reaction may be conducted in an organic solvent, for example a solvent such as tetra hydrofuran, at the reflux temperature of the solvent.

[0109] An ester of formula (V) may be prepared from a carboxylic acid of formula (VI): wherein R1is as defined for formula (I); by reaction with an alcohol of formula (VII):

[0110] R6-OH (VII) where R6is as defined for formula (II). The reaction may be conducted at about 15 °C to 25 °C, for example at room temperature. Compounds of formulae (VI) and (VII) are known and are commercially available or may be prepared by methods known to those of skill in the art.

[0111] In compounds of formulae (II), (III), (IV), (V) and (VI) above, it is preferred that R1is H and therefore this method is particularly suitable for preparing compounds of formula (I) in which R1is H, for example Compound 1 , which has the formula:

[0112] Compound 1 ;

[0113] In this case, the starting material of formula (VI) is nor-ursodeoxycholic acid.

[0114] Compounds of formula (I) in which R1is other than hydrogen may be prepared from compounds of formula (II) in which R1is hydrogen.

[0115] A compound of formula (II) in which R1is hydrogen and R7is R18C(O)O, where R18is Ci-e alkyl or benzyl, may be converted to a compound of formula (la), which is a compound of formula (I) in which R2is C(O)OR5and R5is a group R6: wherein R6is as defined for formula (II); by hydrolysis with a base such as an alkali metal alkoxide in an alcoholic solvent, for example with sodium or potassium methoxide in methanol. The reaction is suitably conducted at about 15 °C to 25 °C, for example at room temperature.

[0116] The hydrolysis is selective for the removal of the R18C(O) protecting group, while leaving the ester on the side chain intact.

[0117] In some cases, transesterification may occur for example, hydrolysis of a compound of formula (II) in which R6is other than methyl with an alkali metal methoxide would result in the production of a compound of formula (la) in which R6is methyl.

[0118] A compound of formula (la) may be reacted with a compound of formula (XI) comprising an ether protecting group:

[0119] R12-O-X2-R10(XI) wherein R10is halo, especially chloro or bromo, X2is Ci-e alkylene and R12is Ci-e alkyl; to give a protected compound of formula (XII): wherein R6is as defined for formula (II) and R12and X2are as defined for formula (XI).

[0120] Suitably, the reaction is carried out under an inert atmosphere such as nitrogen, in mildly basic conditions, for example using N,N-diisopropylethylamine (DIPEA).

[0121] The compound of formula (XII) may be reacted with a compound of formula (XIII):

[0122] R1a-R13(XIII) where R1ais C1.4 alkyl optionally substituted as defined above for R1and R13is halo, for example chloro, bromo or iodo, especially iodo to give a compound of formula (XIV): wherein R6is as defined for formula (II) and R12and X2are as defined for formula (XI) and R1ais C1.4 alkyl optionally substituted as defined above for R1.

[0123] The reaction is suitably carried out under strongly basic conditions, for example using lithium diisopropylamide (LDA) in the presence of hexamethylphosphoric triamide (HMPT), hexamethylphosphoramide (HMPA), tris(N,N-tetramethylene)phosphonic acid triamide (tripyrrolidinophosphoric acid triamide; TPPA). The reaction is suitably conducted under an inert atmosphere, for example nitrogen.

[0124] The protecting group R12-O-X2may be removed by reaction of the compound of formula (XIV) with an acid, for example hydrochloric acid to give a compound of formula (lb): wherein R6is as defined for formula (II) and R1ais C1.4 alkyl.

[0125] Compounds of formula (lb) are compounds of formula (I) in which R1is C1.4 alkyl and R2is C(O)OR5, where R5is C1.6 alkyl or benzyl.

[0126] Suitably, the reaction is conducted at a temperature of about 30 °C to 50 °C.

[0127] If a compound of formula (I) in which R2is C(O)OH is required, the protecting group R6may be removed by reaction of the compound of formula (lb) with a strong base, such as a sodium or potassium hydroxide, in an alcoholic such as methanol to give a product of formula (I) in which R2is C(O)OH and R1is C1.4 alkyl. Suitably, the reaction is conducted at a temperature of about 50 °C to 70 °C.

[0128] A compound of formula (XII) may also be converted to compound of formula (XVI): wherein R6is as defined for formula (II), R12and X2are as defined for formula (XI) and R1 bis fluoro or chloro by reaction with a fluorinating agent such as N-fluorobenzenesulfonimide (NFSI) or a chlorinating agent such as N-chlorosuccinimide. The reaction is suitably carried out under strongly basic conditions, for example using LDA in the presence of an agent such as hexamethylphosphorous triamide (HMPT) hexamethylphosphoramide (HMPA) or tripyrrolidinophosphoric acid triamide (TPPA). Such agents enhance the rate and selectivity of lithiation reactions. The reaction is suitably carried out under an inert atmosphere such as nitrogen.

[0129] Removal of the protecting groups to give a compound of formula (I) may be carried out in two steps. Firstly, the compound of formula (XVI) may be reacted with an acid, for example hydrochloric acid to give a compound of formula (Ic): wherein R6is as defined for formula (II) and R1 bis as defined for formula (XVI).

[0130] Compounds of formula (Ic) are compounds of formula (I) in which R1is fluoro or chloro and R2is C(O)OR5, where R5is Ci-e alkyl or benzyl.

[0131] Subsequently the protecting group R6may be removed to give a product of formula (I) in which R1is fluoro or chloro and R2is C(O)OH by reaction of the compound of formula (XVII) with a strong base, such, for example sodium hydroxide, in an alcoholic such as methanol. Suitably, the reaction is conducted at a temperature of about 35 °C to 55 °C.

[0132] Compounds of formula (XII) may also be converted to compounds of formula (I) in which R1is OH or alkoxy as shown in Scheme 1 below. In Scheme 1 , the products of steps (ii) are compounds of formula (I) in which R2is C(O)OR5, where R5is a group R6and is Ci-e alkyl or benzyl, and the products of steps (iii) are compounds of formula (I) in which R2is C(O)OR5, where R5is H. In these compounds of formula (I), R1is either OH or O(Ci-4) alkyl optionally substituted as described above for formula (I). Scheme 1

[0133] Conditions:

[0134] (i) The compound of formula (XII) is oxidised, for example by treating with a base such as1 M lithium diisopropylamide (LDA) 1 M in THF / hexanes, Davis reagent ((3-phenyl-2- (phenylsulfonyl)-1 ,2-oxaziridine or 2-(benzenesulfonyl)-3-phenyloxaziridine) and tris (N,N- tetramethlene)phosphonic acid triamide in tetra hydrofuran (THF) to give an OH substituted compound of formula (XXa) wherein R6is as defined for formula (II) and R12and X2are as defined for formula (XI). (ii) Treatment of the compound of formula (XXa) or the compound of formula (XXb) with an acid such as HCI, suitably in an alcoholic solvent such as methanol and at a temperature of about 45 °C to give a compound of formula (I) in which R1is OH and R2is C(O)OR5, where R5is a group R6, which is Ci-e alkyl or benzyl or a compound of formula (I) in which R1is O(Ci.

[0135] 4 alkyl) optionally substituted as described above and R2is C(O)OR5, where R5is a group R6, which is Ci-6 alkyl or benzyl.

[0136] (iii) Hydrolysis of a compound of formula (I) in which R1is OH or 0(Ci-4 alkyl) optionally substituted as described above and R2is C(O)OR5, where R5is a group R6, which is Ci-e alkyl or benzyl with a strong base such as sodium hydroxide, suitably in an alcoholic solvent such as methanol and at a temperature of about 60 °C to give a compound of formula (I) in which R2is C(O)OH and R1is OH or R2is C(O)OH and R1is O(Ci-4 alkyl) optionally substituted as described above.

[0137] (iv) Treatment of the compound of formula (XXa) with a compound of formula R15-R16, where R15is C1.4 alkyl and R16is halide, especially chloride, bromide or iodide, but particularly iodide to give a compound of formula (XXb) in which R1is a group OR15, where R15is C1.4 alkyl, optionally substituted as described above for formula (I) and R6, X2and R12are as defined for formula (XX). The reaction may be conducted in a solvent such as THF under an inert atmosphere such as nitrogen an in the presence of a strong base such as NaHMDS. Compounds of formula (XII) may also be converted to compounds of formula (I) in which R1is

[0138] C1.4 alkyl substituted with fluoro, chloro, OH or O(Ci-4 alkyl) as shown in Scheme 2 below.

[0139] Scheme 2 Conditions: (i) Treatment of the compound of formula (XII) with a base such as lithium diisopropylamide (LDA) and a formic acid ester of formula:

[0140] HC(O)OR6wherein R6is as defined for formula (II) in a suitable solvent such as THF and in the presence of an activating agent such as TPPA, suitably under an inert atmosphere such as nitrogen; to yield a compound of formula (XXV) wherein R6, R12and X2are as defined for formula (XII).

[0141] (ii) Reduction of the compound of formula (XXV), for example with a hydride reducing agent such as sodium borohydride in a suitable solvent such as THF and suitably in an inert atmosphere such as nitrogen, to give a compound of formula (XXVIa) wherein R6is as defined for formula (II) and R12and X2are as defined for formula (XI).

[0142] (iii) Treatment of the compound of formula (XXVIa) with an acid such as TFA suitably in a solvent such as dichloromethane or HCI, suitably in an alcoholic solvent such as methanol and at a temperature of about 45 °C to give a compound of formula (I) wherein R6is as defined for formula (XII) and R1CH2OH and R2is C(O)OR5, where R5is C1.6 alkyl or benzyl; or treatment of the compound of formula (XXVIb) to give a compound of formula (I) in which R1is methyl in which one or more hydrogen atoms are replaced by F or Cl and R2is C(O)OR5, where R5is C1.6 alkyl or benzyl.

[0143] (iv) Treatment of the product of step (iii) with sodium hydroxide in MeOH at 60 °C to give a compound of formula (I) in which R1is CH2OH and R2is C(O)OH or a compound of formula (I) in which R1is methyl in which one or more hydrogen atoms are replaced by F or Cl and R2is C(O)OH.

[0144] (v) T reatment of the compound of formula (XXVIa) with a fluorinating agent such as DAST in dichloromethane or a chlorinating agent such as carbon tetrachloride / triphenylphosphine to give a compound of formula (XXVIb) wherein R6, R12and X2are as defined for formula (XII) and R1is methyl in which one or more hydrogen atoms are replaced by F or Cl.

[0145] In Scheme 2, the products of steps (iii) are compounds of formula (I) in which R2is C(O)OR5, where R5is a group R6and is C1.6 alkyl or benzyl, and the products of steps (iv) are compounds of formula (I) in which R2is C(O)OR5, where R5is H. In these compounds of formula (I), either R1is CH2OH or methyl substituted with one or more F or Cl (e.g. CH2F, CH2CI, CHF2 or CF3). Compounds of formula (XXa) of Scheme 1 may also be converted into compounds of formula (XXc) in which R1is N3, (XXd) in which R1is NH2 and (XXe) in which connected to R1is a double bond and R1is O. This is shown in Scheme 3. Scheme 3

[0146] Conditions:

[0147] (i) Treatment of the compound of formula (XXa) to replace OH with a leaving group, followed by replacement of the leaving group with azide. For example, the compound of formula (XXa) may be treated with methanesulfonyl chloride in the presence of a base such as triethylamine. Suitably, the reaction is carried out in a solvent such as dichloromethane under an inert atmosphere such as nitrogen and at reduced temperature, for example about -5 °C to 5 °C, typically about 0 °C. The product is then treated with an azide, typically sodium azide, in a solvent such as DMF in order to obtain compound (XXc), in which R1is N3, R6is as defined for formula (II) and R12and X2are as defined for formula (XI).

[0148] (ii) Treatment of the compound of formula (XXc) with a reducing agent such as triphenylphosphine in the presence of water to give a compound of formula (XXd) in which R1is NH2, R6is as defined for formula (II) and R12and X2are as defined for formula (XI). Typically, the reaction is conducted in a solvent such as THF and at an elevated temperature, suitably the reflux temperature of the solvent.

[0149] (iii) Oxidation of the compound of formula (XXa) with a suitable oxidising agent, for example Dess Martin periodinane to give a compound of formula (XXe) in which connected to R1is a double bond, R1is O R6is as defined for formula (II) and R12and X2are as defined for formula (XI).

[0150] Compounds of formulae (XXc), (XXd) and (XXe) can be converted to compounds of formula (I) using the same method described above for compounds of formulae (XXa) and (XXb), i.e. by employing the procedures described for steps (ii) and (iii) of Scheme 1 . Compounds of formula (XXVIa) of Scheme 2 may also be converted into compounds of formula (XXVc), in which connected to R1is a double bond and R1is CHR8, where R8is H or CH3, compounds of formula (XXVId), (XXVIe) and (XXVIf), in which connected to R1is a single bond and R1is CH2OR20, where R20is C1.4 alkyl, N3 or NH2, or compound of formula (XXVIg) in which connected to R1is a single bond, R1is CH2OH and R2is CH2OH. These reactions are shown in Scheme 4.

[0151] Conditions:

[0152] (i) Treatment of the compound of formula (XXVIa) to replace OH with a leaving group, for example by reaction with methanesulfonyl chloride, followed by reaction with rubidium fluoride. For example, the compound of formula (XXVIa) may be treated with methanesulfonyl chloride in the presence of a base such as triethylamine. Suitably, the reaction is carried out in a solvent such as dichloromethane under an inert atmosphere such as nitrogen and at reduced temperature, for example about -5 °C to 5 °C, typically about 0 °C. Conversion of the mesylate intermediate to an alkene may be achieved by treating a solution of the mesylate in a solvent such as DMF with Rubidium fluoride, suitably at elevated temperature, for example about 40 °C to 60 °C. The product is a compound of formula (XXVIc) in which R6is as defined for formula (II) and R12and X2are as defined for formula (XI), connected to R1is a double bond and R1is CHR8.

[0153] (ii) Treatment of the compound of formula (XXVIa) with a compound R20-R21, where R20is Ci-4 alkyl and R21is a halide, for example chloride, bromide or iodide, but especially iodide. Suitably, the reaction is carried out in a solvent such as THF and in the presence of a base such as NaHMDS. The product is a compound of formula (XXVId), in which R6is as defined for formula (II) and R12and X2are as defined for formula (XI), connected to R1is a single bond and R1is CH2OR20, where R20is C1.4 alkyl.

[0154] (iii) Treatment of a compound of formula (XXVIa) to replace OH with a leaving group, for example by reaction with methanesulfonyl chloride, followed by reaction of the intermediate with an azide, for example sodium azide. For example, the compound of formula (XXVIa) may be treated with methanesulfonyl chloride in the presence of a base such as triethylamine. Suitably, the reaction is carried out in a solvent such as dichloromethane under an inert atmosphere such as nitrogen and at reduced temperature, for example about -5 °C to 5 °C, typically about 0 °C. Reaction of the mesylate intermediate with the azide may be carried out in a solvent such as DMF. The product is a compound of formula (XXVIe) in which R6is as defined for formula (II) and R12and X2are as defined for formula (XI) and R1is CH2N3.

[0155] (iv) Treatment of the compound of formula (XXVIe) with a reducing agent such as triphenylphospine in the presence of water to give a compound of formula (XXVIf) in which R1is CH2NH2. Typically, the reaction is conducted in a solvent such as THF and at an elevated temperature, suitably the reflux temperature of the solvent.

[0156] (v) Reduction of compound (XXVIa), for example with a hydride reducing agent such as lithium borohydride gives rise to a product of formula (XXVIg) in which R6is as defined for formula (II) and R12and X2are as defined for formula (XI), R1is CH2OH and R2is CH2OH.

[0157] Compounds of formulae (XXVIc), (XXVId), (XXVIe) and (XXVIf) can be converted to compounds of formula (I) by the same methods as used for the compounds of formulae (XXVIa) and (XXVIb), i.e. by employing the procedures described for steps (iii) and (iv) of Scheme 2. To convert a compound of formula (XXVIg) to a compound of formula (I), it may be treated with an acid according to step (iii) of Scheme 2. As shown in Scheme 5, Compounds of formula (I) in which connected to R3is a single bond and R3is a-OH can be converted to compounds of formula (I) in which connected to R3is a double bond and R3is O by oxidation. Appropriate oxidising agents vary depending on the nature of R1and R2but suitable oxidising agents include Jones reagent (chromium trioxide in aqueous sulfuric acid) and Dess Martin periodinane. When Jones reagent is used, the reaction is suitably conducted in a solvent such as acetone but when Dess Martin periodinane is used, a more suitable solvent is dichloromethane. In some cases, oxidation of the R3group may also result in oxidation of the R1and / or R2groups. For example, CH2OH groups may be converted to C(O)OH as shown in Example 24 below. Other suitable oxidising agents are as described above for the oxidation of a compound of formula (IV) to give a compound of formula (III).

[0158] Compounds of formula (I) in which connected to R3is a double bond and R3is O can be reduced to give of compounds formula (I) in which connected to R3is a single bond and R3is P-OH, for example using a reducing agent such as potassium tri-sec-butylborohydride (K-selectride®) in a solvent such as tetrahydrofuran. Again, this is illustrated in Scheme 5.

[0159] Compounds of formula (I) in which connected to R3is a double bond, R3is O and R4aand R4bare both H can be converted into a mixture of isomers of compounds of formula (I) in which connected to R3is a double bond, R3is O and R4aand R4bare either H or fluoro, chloro, Ci-2 alkyl or C1.2 haloalkyl. These compounds of formula (I) can then be reduced to give compounds of formula (I) in which connected to R3is a single bond and R3is a-OH or p- OH. This is shown in Scheme 6 and illustrated in Example 27 below. Scheme 6

[0160] Conditions:

[0161] (i) Conversion of the ketone to the silyl enol ether followed by reaction with an electrophilic agent. Fluorination can be achieved by reaction with an electrophilic fluorinating regent such as 1-(chloromethyl)-4-fluoro-1 ,4-diazabicyclo[2.2.2]octane-1 ,4-diium ditetrafluoroborate (sold under the trade mark Selectfluor®). When R4aor R4bis Cl, the agent may be a chlorinating agent such as N-chlorosuccinimide or carbon tetrachloride / triphenylphosphine. For the preparation of compounds in which R4aor R4bis C1.2 alkyl or C1.2 haloalkyl, the reagent may be an alkyl or haloalkyl iodide or trimethane sulfonate. Preparation of the silyl enol ether may be achieved by treatment with trimethylsilyl trifluoromethanesulfonate under basic conditions, for example in the presence of a triethylamine, in a solvent such as dichloromethane and under an inert atmosphere, such as nitrogen. Reactions with an electrophilic reagent, e.g. fluorinations with Selectfluor® are suitably carried out under an inert atmosphere such as nitrogen and in a solvent such as acetonitrile. The product is a mixture of isomers of formula (I) in which one of R4aand R4bis F, Cl, C1.2 alkyl or C1.2 haloalkyl and the other of R4aand R4bis H. The isomers are suitably separated before the reduction step (ii), for example using column chromatography as described in Example 27. Di-substituted compounds may be prepared from the monosubstituted products by repeating step (i).

[0162] (ii) Reduction of the separate isomers isolated after step (i), for example using a hydride reducing agent such as sodium borohydride. Such reductions are suitably carried out under an inert atmosphere such as nitrogen, in a solvent such as THF and at reduced temperature, for example about -25 °C to -15 °C. The product is a mixture of isomers 3a-0H and 3P-OH isomers, which can be separated as described in Example 27.

[0163] Compounds of formula (I) in which R2is C(O)N(R5a)(R5b) can be prepared from compounds of formula (I) in which R2is C(O)OH by reaction with an amine of formula (XXVII)

[0164] H-N(R5a)(R5b) (XXVII) wherein R5aand R5bare as defined for formula (I).

[0165] In some cases, the compound of formula (I) may be reacted with ethyl chloroformate under basic conditions, for example in the presence of triethylamine, to form an acid anhydride, which may then be reacted with the compound of formula (XXVII). Suitably, both steps of the reaction are conducted at reduced temperature for example -5 °C to 5 °C, typically about 0 °C.

[0166] In reaction with the anhydride, the compound of formula (XXVII) may be in aqueous solution under basic conditions, for example provided by sodium bicarbonate.

[0167] Alternatively, the reaction may be carried out under basic conditions, for example using triethylamine and in the presence of a coupling agent.

[0168] Suitable coupling reagents include known peptide coupling agents such as O-(benzotriazol-1- yl)- / V, / V, / V’, / V’-tetramethyluronium hexafluorophosphate (HBTLI), O-(benzotriazol-l-yl)- / V, / V, / V’, / V’-tetramethyluronium tetrafluoroborate (TBTLI), O-(7-azabenzotriazol-1-yl)-A / ,A / ,A / ’,A / ’- tetramethyluronium hexafluorophosphate (HATLI), O-(7-azabenzotriazol-1-yl)- N,N,N’,N’- tetramethyluronium tetrafluoroborate (TATLI), (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.

[0169] Compounds of formulae (II), (XII), (XIV), (XVI), (XX), (XXV), (XXVI) and (XXVIg) are new and form a further aspect of the invention:

[0170] (XIV) (XVI)

[0171] Wherein:

[0172] R1is as defined for formula (I);

[0173] R1ais C1.4 alkyl optionally substituted as defined above for R1;

[0174] R1 bis fluoro or chloro;

[0175] R1cis OH, O(Ci-4 alkyl), N3NH2or =0;

[0176] R1dis CH2OH, methyl in which one or more H is replaced by Cl or F, CH2N3, CH2NH2 or =CHR8;

[0177] R6is C1.6 alkyl or benzyl;

[0178] R7is a protected OH group, for example R18C(O)O, where R18is C1.6 alkyl or benzyl;

[0179] R8is H or CH3;

[0180] R12is Ci-6 alkyl; and

[0181] X2is Ci-6 alkylene.

[0182] Therapeutic Methods

[0183] The compounds of the invention are able to restore mitochondrial function. Surprisingly, it has been shown that they do not enter the enterohepatic recirculation, can cross the blood brain barrier in relatively high levels and bind to plasma proteins in lower levels than compounds such as LIDCA, meaning that they have excellent bioavailability in comparison with known bile acid derivatives. They are therefore of use in the treatment or prevention of neurodegenerative disorders including Parkinson’s disease, mild cognitive impairment, dementia (including Alzheimer’s disease, vascular dementia, dementia with Lewy bodies and frontotemporal dementia (FTD)), Huntington’s disease, amyotrophic lateral sclerosis (motor neurone disease), progressive supranuclear palsy and Wilson’s disease.

[0184] In the discussion below, references to compounds of formula (I) for use in medicine, the use of compounds of formula (I) in the preparation of a medicament, methods of treatment employing compounds of formula (I) and pharmaceutical compositions comprising compounds of formula (I) apply equally to the pharmaceutically acceptable salts and solvates of compounds of formula (I).

[0185] The compounds of formula (I) are also useful in treating or preventing conditions in which modulating mitochondrial function is advantageous, particularly neurodegenerative disorders such as Parkinson’s disease, mild cognitive impairment, dementia (including Alzheimer’s disease, vascular dementia, dementia with Lewy bodies and FTD), Huntington’s disease, amyotrophic lateral sclerosis (motor neurone disease), multiple system atrophy, progressive supranuclear palsy and Wilson’s disease.

[0186] Other diseases and conditions in which modulating mitochondrial function is advantageous include neuromuscular diseases such as muscular dystrophies, myopathies, neuromuscular junction (NMJ) diseases, motor neuron diseases, peripheral nerve diseases, mitochondrial diseases and ion channel diseases.

[0187] In a further aspect of the invention, there is provided a compound of formula (I) for use in medicine.

[0188] There is also provided:

[0189] • A compound of formula (I) for use in the treatment of a neurodegenerative disorder;

[0190] • A compound of formula (I) for use in the prevention of a neurodegenerative disorder;

[0191] • A compound of formula (I) for use in the treatment of a neuromuscular disorder;

[0192] • A compound of formula (I) for use in the prevention of a neuromuscular disorder.

[0193] The invention also provides:

[0194] • The use of a compound of formula (I) in the manufacture of a medicament for the treatment of a neurodegenerative disorder;

[0195] • The use of a compound of formula (I) in the manufacture of a medicament for the prevention of a neurodegenerative disorder; • The use of a compound of formula (I) in the manufacture of a medicament for the treatment of a neuromuscular disorder;

[0196] • The use of a compound of formula (I) in the manufacture of a medicament for the prevention of a neuromuscular disorder.

[0197] The invention further provides:

[0198] • A method for the treatment of a neurodegenerative disorder, the method comprising administering to a patient in need of such treatment an effective amount of a compound of formula (I);

[0199] • A method for the prevention of a neurodegenerative disorder, the method comprising administering to a patient in need of such treatment an effective amount of a compound of formula (I);

[0200] • A method for the treatment of a neuromuscular disorder, the method comprising administering to a patient in need of such treatment an effective amount of a compound of formula (I);

[0201] • A method for the prevention of a neuromuscular disorder, the method comprising administering to a patient in need of such treatment an effective amount of a compound of formula (I).

[0202] Examples of neurodegenerative disorders which can be treated with the compounds of formula (I) include Parkinson’s disease, mild cognitive impairment, dementia (including Alzheimer’s disease, vascular dementia, dementia with Lewy bodies and FTD), Huntington’s disease, amyotrophic lateral sclerosis (motor neurone disease), multiple system atrophy (MSA), progressive supranuclear palsy and Wilson’s disease. Disorders which are particularly suitable for treatment with the compounds of the present invention include Parkinson’s disease, mild cognitive impairment, dementia (including Alzheimer’s disease, vascular dementia, dementia with Lewy bodies and FTD), Huntington’s disease and amyotrophic lateral sclerosis and especially Parkinson’s disease, mild cognitive impairment and dementia (including Alzheimer’s disease, vascular dementia, dementia with Lewy bodies and FTD).

[0203] It has been shown that the compounds of the invention are particularly suitable for the treatment of dementia, for example Alzheimer’s disease, vascular dementia, dementia with Lewy bodies and FTD and especially Alzheimer’s disease.

[0204] Examples of neuromuscular disorders which can be treated with the compounds of formula (I) include muscular dystrophies, myopathies, NMJ diseases, motor neuron diseases, peripheral nerve diseases, mitochondrial diseases and ion channel diseases. Examples of muscular dystrophies include Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDM D), Fasiosca-pulohume-ra1 muscular dystrophy (FSHD), limb-girdle muscular dystrophy (LGMD), Oculopha-ryngeal MD (OPMD) and distal muscular dystrophy (DD).

[0205] Examples of myopathies include congenital myopathies, endocrine myopathies, inflammatory myopathies, metabolic myopathies, myofibrillar myopathies and scapula-peroneal myopathies.

[0206] Examples of NMJ diseases include congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS) and myasthenia gravis (MG).

[0207] Examples of motor neuron diseases include amyotrophic lateral sclerosis (ALS, also included in the list of neurodegenerative disorders above since it arises from degeneration of nerve cells in the brain and spinal cord), spinal-bulbar muscular atrophy (SBMA) and spinal muscular atrophy (SMA).

[0208] Examples of peripheral nerve diseases include Charcot-Marie-Tooth disease (CMT) and giant axonal neuropathy (GAN).

[0209] Examples of mitochondrial diseases include mitochondrial myopathies and Friedreich’s ataxia as well as Barth syndrome, chronic progressive external opthalomplegia (CPEO), Kearns- Sayre syndrome (KSS), Leigh syndrome, 3-methylglutaconic aciduria, deafness, encephalopathy, and Leigh-like syndrome (MEGDEL syndrome), myoclonic epilepsy and ragged-red fibers syndrome (MERRF syndrome), mitochondrial encephalomyopathy, lactic acidosis and stroke-like syndrome (MELAS), mitochondrial neurogastrointestinal encephalopathy (MNGIE) and Senger syndrome.

[0210] Examples of ion channel diseases include Andersen-Tawil syndrome, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, myotonia congenita, paramyotonia congenita and potassium-aggravated myotonia.

[0211] Pharmaceutical Compositions

[0212] The compounds of formula (I) will generally be 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 formula (I) or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable excipient or carrier.

[0213] The composition may be formulated for administration by any route, for example parenteral, including intravenous, intramuscular, subcutaneous or intradermal; or oral, rectal, nasal, topical (including transdermal, eye drops, topical administration to the lung, buccal and sublingual) or vaginal administration.

[0214] More suitably, the composition is formulated for parenteral administration or for oral administration.

[0215] In some embodiments, the composition is formulated for oral administration.

[0216] In other embodiments, the composition is formulated for parenteral administration, especially intravenous administration.

[0217] The composition may be prepared by bringing into association the above defined active agent with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of formula (I) in conjunction or association with a pharmaceutically acceptable excipient or carrier.

[0218] Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion; or as a bolus etc.

[0219] In some cases, the compositions may be formulated for delayed, slow or controlled release of the compound of formula (I).

[0220] For compositions for oral administration (e.g. tablets and capsules), the term “acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate, stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of Wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

[0221] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.

[0222] Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

[0223] For topical application to the skin, compounds of formula (I) may be made up into a cream, ointment, jelly, solution or suspension etc. Cream or ointment formulations that may be used for the drug are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia.

[0224] Topical administration to the lung may be achieved by use of an aerosol formulation. Aerosol formulations typically comprise the active ingredient suspended or dissolved in a suitable aerosol propellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFC propellants include trichloromonofluoromethane (propellant 11), dichlorotetrafluoromethane (propellant 114), and dichlorodifluoromethane (propellant 12). Suitable HFC propellants include tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227). The propellant typically comprises 40%-99.5% e.g. 40%-90% by weight of the total inhalation composition. The formulation may comprise excipients including co-solvents (e.g. ethanol) and surfactants (e.g. lecithin, sorbitan trioleate and the like). Other possible excipients include polyethylene glycol, polyvinylpyrrolidone, glycerine and the like. Aerosol formulations are packaged in canisters and a suitable dose is delivered by means of a metering valve (e.g. as supplied by Bespak, Valois or 3M or alternatively by Aptar, Coster or Vari). Topical administration to the lung may also be achieved by use of a non-pressurised formulation such as an aqueous solution or suspension. These may be administered by means of a nebuliser e.g. one that can be hand-held and portable or for home or hospital use (ie nonportable). The formulation may comprise excipients such as water, buffers, tonicity adjusting agents, pH adjusting agents, surfactants and co-solvents. Suspension liquid and aerosol formulations (whether pressurised or unpressurised) will typically contain the compound of the invention in finely divided form, for example with a D50 of 0.5-10 pm e.g. around 1-5 pm. Particle size distributions may be represented using D10, D50 and D90 values. The D50 median value of particle size distributions is defined as the particle size in microns that divides the distribution in half. The measurement derived from laser diffraction is more accurately described as a volume distribution, and consequently the D50 value obtained using this procedure is more meaningfully referred to as a Dvso value (median for a volume distribution). As used herein Dv values refer to particle size distributions measured using laser diffraction. Similarly, D10 and D90 values, used in the context of laser diffraction, are taken to mean Dv and Dvgo values and refer to the particle size whereby 10% of the distribution lies below the D10 value, and 90% of the distribution lies below the D90 value, respectively.

[0225] Topical administration to the lung may also be achieved by use of a dry-powder formulation. A dry powder formulation will contain the compound of the disclosure in finely divided form, typically with a mass mean diameter (MMAD) of 1-10 pm or a D50 of 0.5-10 pm e.g. around 1- 5 pm. Powders of the compound of the invention in finely divided form may be prepared by a micronization process or similar size reduction process. Micronization may be performed using a jet mill such as those manufactured by Hosokawa Alpine. The resultant particle size distribution may be measured using laser diffraction (e.g. with a Malvern Mastersizer 2000S instrument). The formulation will typically contain a topically acceptable diluent such as lactose, glucose or mannitol (preferably lactose), usually of comparatively large particle size e.g. a mass mean diameter (MMAD) of 50 pm or more, e.g. 100 pm or more or a D50 of 40-150 pm. As used herein, the term “lactose” refers to a lactose-containing component, including a- lactose monohydrate, p-lactose monohydrate, a-lactose anhydrous, p-lactose anhydrous and amorphous lactose. Lactose components may be processed by micronization, sieving, milling, compression, agglomeration or spray drying. Commercially available forms of lactose in various forms are also encompassed, for example Lactohale® (inhalation grade lactose; DFE Pharma), lnhaLac®70 (sieved lactose for dry powder inhaler; Meggle), Pharmatose® (DFE Pharma) and Respitose® (sieved inhalation grade lactose; DFE Pharma) products. In one embodiment, the lactose component is selected from the group consisting of a-lactose monohydrate, a-lactose anhydrous and amorphous lactose. Preferably, the lactose is a- lactose monohydrate. Dry powder formulations may also contain other excipients. Thus in one embodiment a dry powder formulation according the present disclosure comprises magnesium or calcium stearate. Such formulations may have superior chemical and / or physical stability especially when such formulations also contain lactose.

[0226] A dry powder formulation is typically delivered using a dry powder inhaler (DPI) bevice. Example dry powder delivery systems include SPINHALER®, DISKHALER®, TURBOHALER®, DISKUS®, SKYEHALER®, ACCUHALER® and CLICKHALER®. Further examples of dry powder delivery systems include ECLIPSE, NEXT, ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER, BREEZHALER / NEOHALER, MONODOSE, FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN, ELPENHALER, MIATHALER, TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIEL dry powder inhaler, MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN, PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

[0227] In one embodiment a compound of formula (I) is provided as a micronized dry powder formulation, for example comprising lactose of a suitable grade.

[0228] Thus, as an aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula (I) in particulate form in combination with particulate lactose, said composition optionally comprising magnesium stearate.

[0229] In one embodiment a compound of formula (I) is provided as a micronized dry powder formulation, comprising lactose of a suitable grade and magnesium stearate, filled into a device such as DISKUS. Suitably, such a device is a multidose device, for example the formulation is filled into blisters for use in a multi-unit dose device such as DISKUS.

[0230] In another embodiment a compound of formula (I) is provided as a micronized dry powder formulation, for example comprising lactose of a suitable grade, filled into hard shell capsules for use in a single dose device such as AEROLISER.

[0231] In another embodiment a compound of formula (I) is provided as a micronized dry powder formulation, comprising lactose of a suitable grade and magnesium stearate, filled into hard shell capsules for use in a single dose device such as AEROLISER.

[0232] In another embodiment a compound of formula (I) is provided as a fine powder for use in an inhalation dosage form wherein the powder is in fine particles with a D50 of 0.5-10 pm e.g. around 1-5 pm, that have been produced by a size reduction process other than jet mill micronisation e.g. spray drying, spray freezing, microfluidisation, high pressure homogenisation, super critical fluid crystallisation, ultrasonic crystallisation or combinations of these methods thereof, or other suitable particle formation methods known in the art that are used to produce fine particles with an aerodynamic particle size of 0.5-10 pm. The resultant particle size distribution may be measured using laser diffraction (e.g. with a Malvern Mastersizer 2000S instrument). The particles may either comprise the compound alone or in combination with suitable other excipients that may aid the processing. The resultant fine particles may form the final formulation for delivery to humans or may optionally be further formulated with other suitable excipients to facilitate delivery in an acceptable dosage form.

[0233] The compound of the invention may also be administered rectally, for example in the form of suppositories or enemas, which include aqueous or oily solutions as well as suspensions and emulsions and foams. Such compositions are prepared following standard procedures, well known by those skilled in the art. For example, suppositories can be prepared by mixing the active ingredient with a conventional suppository base such as cocoa butter or other glycerides. In this case, the drug is mixed with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0234] Parenteral formulations will generally be sterile.

[0235] The medical practitioner, or other skilled person, will be able to determine a suitable dosage for the compound of formula (I), and hence the amount of the compound of the invention that should be included in any particular pharmaceutical formulation (whether in unit dosage form or otherwise).

[0236] Compounds of formula (I) may be used in combination with one or more other active agents which are useful in the treatment or prophylaxis of neurodegenerative disorders or neuromuscular disorders, for example the neurodegenerative and neuromuscular disorders described above.

[0237] Therefore, in a further aspect of the invention, there is provided a pharmaceutical composition as described above further comprising an additional active agent useful in the treatment or prophylaxis of neurodegenerative disorders or neuromuscular disorders, for example the neurodegenerative and neuromuscular disorders described above. There is also provided a product comprising a compound of formula (I) and an additional active agent useful in the treatment or prevention of a neurodegenerative or neuromuscular disorder as a combined preparation for simultaneous, sequential or separate use in the treatment or prevention of a neurodegenerative disorder or neuromuscular disorder, for example the neurodegenerative and neuromuscular disorders as described above.

[0238] Figures and Examples

[0239] The invention will now be further described with reference to the following examples and to the drawings in which:

[0240] FIGURE 1 is a plot showing blood concentrations of Compound 1 following IV Administration to male C57 mice at 1.00 (mg / kg) where reference numbers 19, 20 and 21 each refer to an individual mouse.

[0241] FIGURE 2 is a plot showing blood concentrations of Comparative Compound A following IV Administration to male C57 mice at 1.00 (mg / kg) where reference numbers 13, 14 and 15 each refer to an individual mouse.

[0242] FIGURE 3 is a plot showing blood concentrations of Compound 1 following oral administration to male C57 mice at 3.00 (mg / kg) where reference numbers 22, 23 and 24 each refer to an individual mouse.

[0243] FIGURE 4 is a plot showing blood concentrations of Comparative Compound A following oral administration to male C57 mice at 3.00 (mg / kg) where reference numbers 16, 17 and 18 each refer to an individual mouse.

[0244] Abbreviations

[0245] General Experimental Procedures

[0246] Proton (1H), carbon (13C) and fluorine (19F) NMR-spectra were recorded on a Bruker AV400 spectrometer at 298K unless otherwise stated. Chemical shifts (5) are quoted in parts per million (ppm) and coupling constants ( ) in Hertz (Hz). Abbreviations indicating multiplicity are: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, hept = heptet, m = multiplet, br = broad, app. apparent. NMR spectra were referenced using residual solvent peaks set to 5 7.26 and 77.16 ppm (CDC ), or 5 3.31 and 49.00 ppm (CD3OD), or 5 2.50 and 39.52 ppm (de- DMSO), or 5 2.05 and 29.82 ppm (acetone-de). Low resolution ES mass spectra were recorded on a WATERS ZMD single quadrupole system or an Agilent 1260 Infinity II, iQ MSD single quadrupole system. High resolution mass spectra were recorded on the Bruker Apex III FT-ICR-MS. Optical rotations were recorded on an OPTICAL ACTIVITY POLAAR 2001 polarimeter at 589 nm. All reactions were monitored by thin layer chromatography (TLC) using 0.2 mm silica gel (Merck Kieselgel 60 F254) precoated aluminium or glass plates, using UV light, cerium ammonium molybdate stain, p-anisaldehyde, ninhydrin or potassium permanganate staining solution to visualize. Flash column chromatography was performed on a Biotage® I solera automated flash system with Biotage® Star Columns. Preparative HPLC chromatography was performed on an Agilent 1260 II LC system with preparative binary pump, using a multiple wavelength detector and a Phenomenex Prodigy ODS-3 100A, 5p, 250 x 21.2 mm column. Solvents for reactions and chromatography were analytical grade and were used as supplied unless otherwise stated.

[0247] Comparative Example A - Synthesis of 7,7-difluoro lithocholic acid (Comparative Compound A) i. Synthesis of methyl 3a,7a-dihydroxy-5p-cholan-24-oate

[0248] To a round-bottom flask were added CDCA (1 g, 2.55 mmol, 1 eq), MeOH (20 mL) and p- toluenesulfonic acid monohydrate (50 mg, 0.26 mmol, 0.1 eq). The reaction mixture was stirred until the solution was homogeneous, and then sonicated at 30 °C for 2 h. The reaction mixture was concentrated, the residue was dissolved in chloroform (30 mL), washed with saturated NaHCCh (2 x 10 mL), water (10 mL) and 10% NaCI (10 mL), dried over Na2SC>4, filtered and concentrated to afford the methyl ester quantitatively as gummy solid (95%), and used for next step without any purification.

[0249] 1H NMR (300 MHz, CDCh) 6 3.79-3.90 (m, 1 H, H7), 3.66 (s, 3H, OCH3), 3.37-3.55 (m, 1 H, H3), 0.92 (d, J = 6.4 Hz, 3H, H21), 0.90 (s, 3H, H19), 0.66 (s, 3H, H18);13C NMR (75 MHz, CDCh) 6 174.7 (C24), 71.9, 68.5, 55.8, 51.4, 50.4, 42.6, 41.5, 39.9, 39.6, 39.4, 35.33, 35.31 , 35.0, 34.6, 32.8, 30.97, 30.95, 30.6, 28.1 , 23.7, 22.7, 20.5, 18.2, 11.7. ii. Synthesis of methyl 3a-hydroxy-7-keto-5p-cholan-24-oate

[0250] To a round-bottom flask were added methyl 3a,7a-dihydroxy-5p-cholan-24-oate from step i (3.59 g, 8.83 mmol, 1 eq), sodium bromide (0.057 eq), TBAB (3.30 eq) and a solution of MeOH / MeCOOH / FW / AcOEt (v / v:3 / 1 / 0.25 / 6.5, 8.43 mL / mmol) and the reaction mixture was stirred till the solution was homogeneous. Sodium hypochlorite solution (11-14%, 1.1 eq) was added at 0 °C until the hypochlorite test (peroxide test paper) was positive. The reaction mixture was stirred at room temperature for 6 h. After TLC indicated the completion of the reaction, the reaction mixture was quenched with sodium bisulfite solution (3.3%) until the hypochlorite test was negative. Water was added and stirred for 15 min at room temperature. The mixture was extracted with ethyl acetate (x3) and the combined organic phase washed with aqueous sodium bisulfite solution (3.3%), water and dried over anhydrous sodium sulphate, filtered and concentrated. The crude was purified by flash chromatography to afford the desired compound (acetone / petroleum ether 12:88-80:20) to afford (2.15 g, 60%) as a gummy solid.

[0251] 1H NMR (300 MHz, CDCh) 6 3.65 (s, 3H, OCH3), 3.52-3.63 (m, 1 H, H3 ), 2.85 (dd, J = 12.6, 6.3 Hz, 1 H, H6P), 2.28-2.44 (m, 2H), 2.12-2.27 (m, 2H), 1.19 (s, 3H, H19), 0.91 (d, J = 6.2 Hz, 3H, H21), 0.64 (s, 3H, H18);13C NMR (75 MHz, CDCh) 6 212.0 (C7), 174.6 (C24), 70.8 (C3), 54.7, 51.4, 49.5, 48.9, 46.1 , 45.4, 42.7, 42.6, 38.9, 37.4, 35.2, 35.1 , 34.1 , 31.0, 31.0, 29.8, 28.2, 24.8, 23.0, 21.6, 18.3, 12.0. iii. Synthesis of methyl 3a-benzoyloxy-7-keto-5p-cholan-24-oate

[0252] To a round-bottom flask were added methyl 3a-hydroxy-7-keto-5p-cholan-24-oate from step ii (5 g, 12.4 mmol, 1.0 eq), toluene (50 mL), pyridine (1.6 mL) and BzCI (2.3 mL, 1.5 eq). The reaction mixture was stirred at ambient overnight. Upon completion indicated by TLC analysis, the reaction was quenched with H2O and extracted with ethyl acetate. The organic layer was washed with aq. HCI (2.0 M), H2O, 10% NaCI, and then dried over Na2SO4, filtered and concentrated. The crude was purified by flash chromatography (acetone / petroleum ether 8:92 - 20:80) to afford product (86%) as white solid. The crystals were obtained from recrystallisation in acetone / petroleum ether.

[0253] [Q]D -14.0 (c 0.7, CHC , 20 °C); m.p. 136-138 °C (acetone / petroleum ether); Rf 0.22 (acetone / petroleum ether 10:90); IR (neat) 2940 (m), 2869 (m), 1736 (m), 1710 (s), 1445 (w), 1380 (w), 1274 (s), 1173 (w), 1112 (m), 756 (w), 711 (m) cm’1;1H NMR (400 MHz, CDCI3) 6 8.00 (dd, J = 8.4, 1 .3 Hz, 2H, HAr(o)), 7.53 (tt, J = 7.3, 1.8 Hz, 1 H, HAr(p)), 7.41 (t, J = 7.9 Hz, 2H, HAr(m)), 4.94 (tt, J = 11.4, 4.6 Hz, 1 H, H3P), 3.66 (s, 3H, OCH3), 2.89 (dd, J = 12.4, 5.9 Hz, 1 H, H6P), 2.42 (t, J = 11.3 Hz, 1 H, H8P), 2.30-2.38 (m, 1 H), 2.15-2.28 (m, 2H), 1.24 (s, 3H, H19), 0.92 (d, J= 6.3 Hz, 3H, H21), 0.67 (s, 3H, H18);13C NMR (101 MHz, CDCI3) 6211.8 (C7), 174.6 (C24), 165.9 (PhCO),132.8 (CAr(p)), 130.5 (CArCO), 129.5 (CAr(o)), 128.2 (CAr(m)), 73.4 (C3), 54.7 (CH), 51.4 (OCH3), 49.5 (C8), 48.8 (CH), 45.9 (CH), 45.2 (C6), 42.8 (CH), 42.6 (C), 38.9 (CH2), 35.17 (C), 35.16 (CH), 33.8 (CH2), 33.2(CH2), 31.0 (CH2), 30.9 (CH2), 28.2 (CH2), 26.1 (CH2), 24.7 (CH2), 23.0 (C19), 21.7 (CH2), 18.3 (C21), 12.0(C18); MS (ESI+) m / z 526.2 ([M+NH4]+, 100%), 531.2 ([M+Na]+, 17%); HRMS(ESH-) for C32H44NaO5[M+Na]+calcd 531.3081 , found 531.3072 (1.7 ppm error). iv. Synthesis of methyl 3a-benzoyloxy-7,7-difluoro-5p-cholan-24-oate

[0254] To a round-bottom flask were added methyl 3a-benzoyloxy-7-keto-5p-cholan-24-oate from step iii (1.36 g) and DAST (15 mL). The reaction mixture was heated at 80 °C overnight. Upon completion monitored by TLC analysis, the reaction mixture was quenched with sat. aq. NaHCO3solution then extracted with ethyl acetate. The combined organic layer was washed with 10% NaCI, dried over Na2SO4, filtered, and concentrated. The crude was purified by flash chromatography (acetone / petroleum ether 4:96-8:92-20:80) and HPLC (acetone / hexane 5:95) to afford the desired compound.

[0255] [a]D+ 30.4 (c 1.1 , CHCh, 20 °C); m.p. 140-144 °C (acetone / hexane); Rf 0.29 (acetone / petroleum ether 10:90); IR (neat) 2940 (m), 2868 (w), 1740 (s), 1711 (s), 1455 (w), 1321 (w), 1275 (s), 1176 (m), 1141 (m), 1121 (s), 1065 (s), 1026 (m), 977 (m), 760 (s), 718 (s) cm’1;1H NMR (400 MHz, CDCh) 6 8.02 (dd, J = 8.5, 1.5 Hz, 2H, HAr(o)), 7.51 (tt, J = 7.5, 1.4 Hz, 1 H, HAr(p)), 7.40 (t, J = 7.5 Hz, 2H, HAr(m)), 4.87 (tt, J = 11.0, 4.5 Hz, 1 H, H3 ), 3.64 (s, 3H, OCH3), 2.27-2.40 (m, 1 H), 0.97 (s, 3H, H19), 0.92 (d, J = 6.5 Hz, 3H, H21), 0.66 (s, 3H, H18);13C NMR (101 MHz, CDCh) 174.6 (C24), 166.0 (PhCO), 132.7 (CAr(p)), 130.7 (CArCO), 129.5 (CAr(o)), 128.2 (CAr(m)), 124.6 (t, J= 244.5 Hz, C7), 74.0 (C3), 55.0 (CH), 51.4 (OCH3), 48.6 (d, J = 3.5 Hz, CH), 43.1 (C), 41.8 (dd, J = 22.9, 19.3 Hz, C8), 41.1 (d, J = 10.6 Hz, CH), 39.4 (CH2), 37.1 (d, J = 9.0 Hz, CH), 36.1 (t, J = 23.7 Hz, C6), 35.3 (CH), 34.40 (CH2), 34.36 (C), 33.1 (d, J = 4.4 Hz), 31.01 (CH2), 30.98 (CH2), 28.3 (CH2), 26.5 (CH2), 25.3 (d, J = 3.9 Hz, CH2), 22.6 (C19), 20.9 (CH2), 18.3 (C21), 11.8 (C18);19F NMR (376 MHz, CDCh) 6 -84.7 (br. d, J = 241.0 Hz, F7), -101.4 (dddd, J = 241.0, 38.1 , 25.1 , 14.7 Hz, F7);19F {1H} NMR (376 MHz, CDCh) -84.7 (d, J = 241.0 Hz, F7), -101.4 (d, J = 241.0 Hz, F7); MS(ESI+) m / z 553.3 ([M+Na]+, 100%); HRMS (ESI+) for C32H44F2NaO4[M+Na]+calcd 553.3100, found 553.3101 (- 0.2 ppm error). v. Synthesis of 7,7-difluoro lithocholic acid (Comparative Compound A)

[0256] To a round-bottom flask were added methyl 3a-benzoyloxy-7,7-difluoro-5p-cholan-24-oate from step iv (40 mg), NaOH (200 mg) and MeOH (2 mL). The reaction mixture was stirred at rt overnight. Upon the completion indicated by TLC analysis, the solvent was firstly removed and then acidified with aq. 6 M HCI solution. The solution was further extracted with ethyl acetate. The combined organic layer was washed with 10% NaCI, dried over Na2SO4, filtered and concentrated. NMR analysis of the crude mixture indicated a quantitative conversion to the desired product. The crude was purified by flash chromatography (DCM / MeOH 97:3, addition of 0.5% formic acid) to afford Comparative Compound A as white solid, recrystallised from neat MeOH.

[0257] IR (neat) 3361 (br. w), 2952 (s), 2927 (s), 2869 (s), 1708 (s),1470 (m), 1455 (m), 1379 (m), 1072 (s), 978 (m), 916 (m), 732 (s) cm-1;1H NMR (400 MHz, CDCh) 6 3.47 (tt, J = 11.0, 5.3 Hz, 1 H.H30), 2.29-2.45 (m, 1 H), 0.98 (s, 3H, H19), 0.97 (d, J = 6.9 Hz, 3H, H21), 0.71(s, 3H, H18);13C NMR (101 MHz, CDCh) 6 178.2 (C24), 126.0 (dd, J = 244.5, 243.2 Hz, C7), 72.1 (C3), 56.6, 50.2 (d, J = 3.9 Hz), 44.4, 43.3 (dd, J = 23.3, 20.0 Hz), 42.7 (d, J = 10.6 Hz), 41.0, 38.6 (d, J = 9.0 Hz), 38.2 (d, J = 4.0 Hz), 37.5 (t, J = 23.5 Hz), 36.8, 35.9, 35.5, 32.4, 32.1 , 31 .0, 29.4, 26.7 (d, J = 3.9 Hz), 23.2, 22.1 , 19.0, 12.4;19F NMR (376 MHz, CDCh) 84.7 (br. d, J = 241.0 Hz, F70), -102.0 (dddd, J = 241.0, 38.6, 25.1 , 14.7 Hz, F7a); MS (ESI+) m / z 825.4 ([2M+H]+, 46%); HRMS (ESI+) for C24H42F2NO3 [M+NH4]+calcd 430.3127, found 430.3124 (0.7 ppm error).

[0258] Example 1 - Synthesis of 7,7-difluoro 24-norlithocholic acid (Compound 1)

[0259] Compound 1 was synthesised as shown in Scheme 7

[0260] Scheme 7 To a suspension of nor-UDCA (40 g, 105.7 mmol) in MeOH (HPLC grade, 300 mL) was charged cone, sulfuric acid (-200 pL). The reaction was stirred at ambient temperature for 3 days, then quenched with triethylamine and concentrated to dryness. The residue was dispersed in ethyl acetate (200 mL) and water (40 mL), stirred for 1 h then filtered, washed with water and dried. The filtrate was separated, the organic phase washed with water (40 mL), concentrated and combined with the isolated solid to afford nor-UDCA methyl ester as a white solid (41.57 g, quant).

[0261] 1H NMR (400 MHz, CDCI3) 6 3.64 (s, 3H), 3.60-3.51 (m, 2H), 2.41 (dd J = 14.4, 3.2 Hz, 1 H), 2.03-1.94 (m, 2H), 1.94-1.72 (m, 5H), 1.68-1.53 (m, 4H), 1.53-1.38 (m, 5H), 1.38-0.99 (m, 7H), 0.96 (d, J = 6.3 Hz, 3H), 0.92 (s, 3H), 0.69 (s, 3H). ii. Methyl 3a-acetylhydroxy-7p-hydroxy-5p-24-nor-cholan-23-oate

[0262] To a solution of nor-UDCA methyl ester (24.13 g, 61.5 mmol) in THF (482 mL) at reflux was charged NaHCCh (30.1 g, 369 mmol), followed by the dropwise addition of acetic anhydride (29.1 mL, 307 mmol). The reaction mixture was stirred at reflux for 20 h and then cooled to ambient temperature, diluted with EtOAc (300 mL) and added slowly into a stirred solution of sat. NaHCOs. The organic phase was dried over MgSCU, concentrated in vacuo and purified by column chromatography (SiC>2, heptane in ethyl acetate 0-50%) to afford the title compound (20.5 g, 76%).

[0263] 1H NMR (400 MHz, CD3OD) 5 4.68-4.59 (m, 1 H), 3.65 (s, 3H), 3.50-3.42 (m, 1 H), 2.46 (dd, J = 14.5, 3.4 Hz, 1 H), 2.07-1.98 (m, 2H), 2.00 (s, 3H), 1.95-1.79 (m, 5H), 1.74-1.64 (m, 3H), 1.59-1.05 (m, 13H), 1.07-0.68 (m, 6H), 0.75 (s, 3H). iii. Methyl 3a-acetylhydroxy-7-keto-5p-24-nor-cholan-23-oate

[0264] To a solution of methyl 3a-acetylhydroxy-7 -hydroxy-5 -24-nor-cholan-23-oate from step ii (20 g, 46.0 mmol) in DCM (200 mL) was charged DMP (23.4 g, 55.2 mmol) portion-wise maintaining the temperature at < 30 °C. The reaction mixture was stirred at ambient temperature for 17 h then filtered and washed with DCM. The solution was stirred with sat. NaHCOs (150 mL) for 1 h before charging 5% sodium thiosulfate (140 mL). The phases were separated and the organic phase was washed with water (2 x 200 mL) and 10% NaCI (200 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiCh, acetone / DCM) to afford the title compound (18 g, 90%).

[0265] 1H NMR (400 MHz, CDCh) 6 4.74-4.63 (m, 1 H), 3.66 (s, 3H), 2.88-2.82 (m, 1 H), 2.46-2.36 (m, 2H), 2.26-2.15 (m, 1 H), 2.05-1.68 (m, 10H), 1.99 (s, 3H), 1.59-1.40 (m, 4H), 1.40-1.26 (m, 3H), 1.26-1.10 (m, 3H), 1.20 (s, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.69 (s, 3H).

[0266] Iv(a). Methyl 3a-acetylhydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate

[0267] To methyl 3a-acetylhydroxy-7-keto-5p-24-nor-cholan-23-oate from step iii (17.8 g, 41.1 mmol) was charged DAST (50 g). The reaction mixture was heated to 50 °C for 2 weeks, then diluted with DCM and quenched by slowly charging into 5% NaHCCh and stirring overnight. The phases were separated and the aqueous phase was extracted with DCM. The combined extracts were washed with 5% NaHCOs and concentrated in vacuo. The resulting residue was purified by column chromatography (SiC>2, acetone / heptane), to afford the title compound (3.34 g, 18%).

[0268] 1H NMR (400 MHz, CDCh) 6 4.69-4.58 (m, 1 H), 3.66 (s, 3H), 2.44 (dd, J = 14.4, 3.2 Hz, 1 H), 2.25-2.06 (m, 1 H), 2.06-1.97 (m, 3H), 1.97-1.89 (m, 1 H), 1.89-1.58 (m, 10H), 1.57-1.44 (m, 2H), 1.44-1.24 (m, 4H), 1.24-1.07 (m, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.96 (s, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -84.5 (d, J = 240.8 Hz), -101.3 (d, J = 240.8 Hz).

[0269] Iv(b) Methyl 3a-acetylhydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate (alternative synthesis)

[0270] Methyl 3a-acetylhydroxy-7-keto-5p-24-nor-cholan-23-oate (15 g, 0.034 mol, 1 equiv.) and water (1.87 g, 0.1 mol, 3 equiv.) were placed in a 300 mL autoclave made of Hastelloy nickel alloy. The reaction vessel was cooled down by liquid nitrogen, and SF4 (37.5 g, 0.34 mol, 10 equiv.) was condensed into a reaction vessel. The cooling bath was removed, and the mixture was allowed to warm up to room temperature and stirred at this temperature for 24h. Then gaseous products were vented off into a trap with an aqueous solution of NaOH (1 M). The reaction vessel was again cooled and another portion of SF4 (37.5 g, 0.34 mol, 10 equiv.) was condensed into autoclave. The cooling bath was removed, and the mixture was allowed to warm up to room temperature and stirred at this temperature for additional 24h. Then gaseous products were vented off into a trap with an aqueous solution of NaOH (1 M) and residue was poured onto ice (200 g), extracted with DCM (200 ml), and an organic extract was washed with saturated aq. NaHCCh (150 mL). The organic layer was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to afford crude product. Crude product was purified by recrystallization from 220 ml of isopropyl alcohol to gave 11.6 g, 74% yield of material as slightly yellow solid. v. 7,7-difluoro 24-norlithocholic acid (Compound 1)

[0271] Methyl 3a-acetylhydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate from step iv (0.2 g, 0.44 mmol) was suspended in IPA (2 mL), 1.0 M NaOH (1.45 mL) was charged and the mixture was heated at 40 °C for 18 h. The reaction mixture was concentrated and charged into 2.0 M H2SO4 (0.73 mL). the resulting solid was collected by filtration and purified by column chromatography (SiO2, acetone / heptane) to afford the final compound as an off white solid (167 mg, 95%). pKa4.1 ;1H NMR (400 MHz, CD3OD) 5 3.52-3.43 (m, 1 H), 2.46-2.40 (m, 1 H), 2.30-2.12 (m, 1 H), 2.06 (dt, J = 12.6, 3.2 Hz, 1 H), 2.01-1.48 (m, 14H), 1.45-1.16 (m, 6H), 1.10 (td, J = 14.1 , 2.9 Hz, 1 H), 1 .03 (d, J = 6.2 Hz, 3H), 0.99 (s, 3H), 0.75 (s, 3H);13C NMR (100 MHz, CD3OD) 5 177.4, 125.8 (t, J = 243.6 Hz), 71.9, 56.5, 50.1 ( = 4.2 Hz), 44.3, 43.1 (dd, J = 22.9, 19.3 Hz), 42.5 (br. d, J = 10.7 Hz), 42.5, 40.8, 38.5 (d, J = 9.0 Hz), 38.1 (d, J = 4.2 Hz), 37.3 (t, J = 23.0 Hz), 35.8, 35.4 (d, J = 1 .2 Hz), 34.9, 30.9, 29.4 (d, J = 1 .5 Hz), 26.5 (d, J = 4.3 Hz), 23.0, 22.0, 20.0, 12.2;19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 241.0 Hz), -102.4 (d, J = 241.0 Hz); MS (ESI’) m / z 397.3 [M-H]’.

[0272] Example 2 - Synthesis of 3a-hydroxy-7,7-difluoro-5 -24-nor-22-methyl-cholanic acid (Compound 2)

[0273] Compound 2 was synthesised from methyl 3a-acetylhydroxy-7,7-difluoro-5p-24-nor-cholan- 23-oate, i.e the product of Example 1 , step iv, by the method shown in Scheme 8. Scheme 8 i. Methyl 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate (Compound 1 Me ester)

[0274] The product of this step is the starting material for Compound 25 of Example 25 (3-keto).

[0275] To a suspension of methyl 3a-acetylhydroxy-7,7-difluoro-5 -24-nor-cholan-23-oate (5.17 g, 11.4 mmol) in MeOH (100 mL) was charged 25 wt% NaOMe in MeOH to pH 12. The reaction mixture was stirred ay ambient temperature for 18 h then quenched with Amberlyst 15 H+resin, filtered and concentrated in vacuo to afford the title compound (4.59 g, quant.).

[0276] 1H NMR (400 MHz, CDCh) 6 3.66 (s, 3H), 3.59-3.49 (m, 1 H), 2.44 (dd, J = 14.4, 3.1 Hz, 1 H), 2.24-2.05 (m, 1 H), 2.05-1.97 (m, 2H), 1.97-1.63 (m, 10H), 1.61-1.41 (m, 3H), 1.41-1.12 (m, 6H), 1.07 (td, J = 14.2, 3.2 Hz, 1 H), 0.99 (d, J = 6.4 Hz, 3H), 0.95 (s, 3H), 0.70 (s, 3H).

[0277] 11. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate

[0278] The product of this step is the starting material for Compound 3 of Example 3, Compound 4 of Example 4, Compound 5 of Example 5, Compound 6 of Example 6, Compound 12 of Example

[0279] 12, Compound 17 of Example 17.

[0280] To a solution of methyl 3a-hydroxy-7,7-difluoro-5 -24-nor-cholan-23-oate from step i (4.69 g, 11.4 mmol) in DCM (20 mL) was charged DI PEA (6.34 mL, 36.4 mmol) dropwise, followed by MOMCI (1.64 mL, 21 .6 mmol), which was washed in with additional DCM (30 mL). The reaction mixture was stirred under nitrogen at ambient temperature for 4 days then quenched by the drop-wise addition of water (50 mL). After stirring for 1 h the phases were separated. The aqueous phase was extracted with DCM (2 x 15 mL) and the combined extracts were washed with 0.5 M HCI (3 x 15 mL, till pH < 5), 5% NaHCCh (25 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 2-10% ethyl acetate in heptane) to afford the title compound as an off white solid (4.46, 86%).

[0281] 1H NMR (400 MHz, CDCh) 6 4.67 (s, 2H), 3.66 (s, 3H), 3.49-3.39 (m, 1 H), 3.36 (s, 3H), 2.44 (dd, J = 14.4, 3.1 Hz, 1 H), 2.24-2.05 (m, 1 H), 2.05-1.97 (m, 2H), 1.96-1.88 (m, 1 H), 1.88-1.67 (m, 9H), 1.59-1.43 (m, 3H), 1.40-1.23 (m, 4H), 1.22-1.10 (m, 2H), 1.05 (td, J = 14.2, 3.2 Hz, 1 H), 0.99 (d, J = 6.4 Hz, 3H), 0.95 (s, 3H), 0.70 (s, 3H). iii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-methyl-cholanic acid (Compound 2)

[0282] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate from step ii (0.2 g, 0.44 mmol) in THF (8 mL) was charged HMPT (40 pL, 0.22 mmol). The reaction mixture was cooled to -78 °C under nitrogen then LDA (1.0 M in THF / hexanes, 0.66 mL, 0.66 mmol) was charged. After stirring at -78 °C for 30 min Mel (41 pL, 0.66 mmol) in THF (1 mL) was charged and the solution was allowed to warm to ambient temperature whist stirring under nitrogen. After 18 h the reaction was quenched by the addition of 1 M HCI (10 mL), stirred for 30 min then the phases were separated and the aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined extracts were washed with 5% NaHCOs (20 mL), 10% NaCI (20 mL) and concentrated in vacuo. The residue was dissolved in MeOH (14 mL), 2 M HCI (1.1 mL, 2.2 mmol) was charged and the reaction mixture was heated to 40 °C for 18 h, then 50% NaOH (0.24 mL, 4.4 mmol) was charged and the solution was heated at 40 °C for 5 days. The solution was concentrated in vacuo, water (15 mL) was charged the aqueous solution was extracted with TBME (15 mL). The aqueous phase was acidified with 2 M HCI, concentrated in vacuo and purified by column chromatography (SiC>2, 0-10% acetone in toluene) to afford the title compound as a single diastereomer an off white solid (88 mg, 48%). pKa4.9;1H NMR (400 MHz, CD3OD) 5 3.52-3.43 (m, 1 H), 2.58 (ddd, J = 14.1 , 7.0, 3.0 Hz, 1 H), 2.30-2.12 (m, 1 H), 2.12-2.06 (m, 1 H), 2.06-1.99 (m, 1 H), 1.92-1.49 (m, 12H), 1.44-1.17 (m, 6H), 1.10 (td, J = 14.0, 2.9 Hz, 1 H), 0.99 (s, 3H), 0.97 (d, J = 7.1 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.76 (s, 3H);13C NMR (100 MHz, CD3OD) 5 180.3, 125.9 (t, J = 241.6 Hz), 71.9, 53.9, 50.2 (d, J = 4.2 Hz), 44.2, 43.2 (dd, J = 23.4, 19.7 Hz), 43.1 , 42.6 (br. d, J = 9.8 Hz), 40.9, 38.5, 38.5 (d, J = 8.2 Hz), 38.3 (dd, J = 32.5, 4.2 Hz), 37.4 (t, J = 23.3 Hz), 35.8, 35.4, 30.9, 29.0 (d, J = 1.7 Hz), 26.4 (d, J = 4.3 Hz), 23.0, 22.0, 14.9, 12.2, 8.9;19F {1H} NMR (376 MHz, CD3OD) -85.2 (d, J = 241.0 Hz), -102.5 (d, J = 241.0 Hz); MS (ESI’) m / z 411.3 [M-H]’.

[0283] Example 3 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-ethyl-cholanic acid (Compound 3)

[0284] Compound 3 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor- cholan-23-oate, i.e. the product of Example 2, step ii according to Scheme 9.

[0285] Scheme 9 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-ethyl-cholan-23-oate

[0286] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate (0.5 g, 1.10 mmol) and HMPT (95 pL, 0.52 mmol) in THF (15 mL) at -78 °C under nitrogen was charged LDA (1.0 M in THF / hexanes, 1.64 mL, 1.64 mmol). After stirring for 30 min ethyl iodide (132 pL, 1.64 mmol) in THF (1 mL) was charged. The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 18 h. The mixture was partitioned between 1 M HCI (30 mL) and ethyl acetate (30 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined extracts were washed with 5% NaHCO3(30 mL) and 10% NaCI (30 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 0-10% ethyl acetate in heptane) to afford the title compound as a single diastereomer (396 mg, 74%).

[0287] 1H NMR (400 MHz, CDCh) 6 4.67 (s, 2H), 3.67 (s, 3H), 3.50-3.40 (m, 1 H), 3.62 (s, 3H), 2.35 (dt, J = 11.5, 2.4 Hz, 1 H), 2.25-2.05 (m, 1 H), 1.96 (dt, J = 12.5, 3.4 Hz, 1 H), 1.91-1.67 (m, 9H), 1.67-1.44 (m, 4H), 1.42-1.22 (m, 5H), 1.17 (td, J = 12.9, 3.9 Hz, 1 H), 1.05 (td, J = 14.3, 3.2 Hz, 1 H), 0.95 (s, 3H), 0.90-0.84 (m, 6H), 0.69 (s, 3H). ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-ethyl-cholanic acid (Compound 3)

[0288] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-ethyl-cholan-23-oate from step i (198 mg, 0.41 mmol) was dissolved in MeOH (15 mL), 2 M HCI (1.0 mL, 2.0 mmol) was charged and the reaction mixture was heated to 40 °C for 18 h, then 50% NaOH (0.2 mL, 4.1 mmol) was charged and the solution was heated at 60 °C for 7 days. The solution was concentrated in vacuo, water (15 mL) was charged the aqueous solution was extracted with TBME (15 mL). The aqueous phase was acidified with 2 M HCI, concentrated in vacuo and purified by column chromatography (SiC>2, acetone / toluene) to afford the title compound as one diastereomer as an off white solid (142 mg, 81%). pKa4.9;1H NMR (400 MHz, CD3OD) 5 3.53-3.43 (m, 1 H), 2.32 (br. d t, J = 11.5, 2.3 Hz, 1 H), 2.30-2.12 (m, 1 H), 2.02 (br. dt, J = 12.6, 3.0 Hz, 1 H), 1.98-1.90 (m, 1 H), 1.89-1.48 (m, 13H), 1.48-1.17 (m, 7H), 1.14 (td, J = 14.1 , 2.9 Hz, 1 H), 0.98 (s, 3H), 0.92-0.89 (m, 6H), 0.74 (s, 3H);13C NMR (100 MHz, CD3OD) 5 179.5, 125.9 (t, J = 242.0 Hz), 71.9, 54.2, 51.7, 50.2 (d, J = 4.0 Hz), 44.2, 43.2 (dd, J = 23.0, 19.4 Hz), 42.5 (br. d, J = 10.6 Hz), 40.9, 39.4, 38.5 (d, J = 8.9 Hz), 38.1 (d, = 4.1 Hz), 37.3 (t, = 23.0 Hz), 35.8, 35.4 (d, J = 1.1 Hz), 30.9, 29.3, 26.4 (d, J = 4.2 Hz), 23.0, 22.0, 17.9, 15.3, 13.7, 12.2;19F {1H} NMR (376 MHz, CD3OD) 5 -85.1 (d, J = 240.8 Hz), -102.4 (d, J = 240.8 Hz); MS (ESI’) m / z 425.3 [M-H]’.

[0289] Example 4 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-fluoro-cholanic acid (Compound 4)

[0290] Compound 4 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor- cholan-23-oate, i.e. the product of Example 2, step ii according to Scheme 10.

[0291] Scheme 10 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-fluoro-cholan-23-oate co-

[0292] MOMO''

[0293] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate (0.3 g, 0.66 mmol) and tripyrrolidinophosphoric acid triamide (TPPA, 75 pL, 0.33 mmol) in THF (4.5 mL) at -78 °C under nitrogen was charged LDA (1.0 M in THF / hexanes, 0.99 mL, 0.99 mmol). After stirring for 30 min N-fluorobenzenesulfonimide (NFSI, 0.3 g, 0.99 mmol) in THF (1 mL) was charged. The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 18 h. The mixture was diluted with ethyl acetate (30 mL) and quenched with 5% NaHCOs (10 mL). The phases were separated, and the aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined extracts were washed with 10% NaCI (30 mL), dried over MgSCU and concentrated in vacuo. The crude residue was obtained as mixture of fluoro- diastereomers (89:11), used without further purification (370 mg).

[0294] 1H NMR (400 MHz, CDCh) 6 4.95 (dd, J = 48.9, 1.8 Hz, 1 H, CHF major), 4.83 (dd, J = 47.6, 3.3 Hz, 1 H, CHF minor), 4.65 (s, 2H), 3.78 (s, 3H), 3.48-3.37 (m, 1 H), 3.34 (s, 3H), 2.25-2.05 (m, 1 H), 2.05-1.62 (m, 13H), 1.62-1.22 (m, 8H), 0.97-0.91 (m, 6H), 0.69 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -84.4 (dd, J = 240.6, 8.5 Hz), -101.2 (dd, J= 240.6, 3.0 Hz), -208.7 (major), -191.0 (minor). ii. Methyl 3a-hydroxy-7,7-difluoro-5p-24-nor-22-fluoro-cholan-23-oate

[0295] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-fluoro-cholan-23-oate from step i (370 mg) was dissolved in MeOH (26 mL), 2 M HCI (1.0 mL, 2.0 mmol) was charged and the reaction mixture was heated to 40 °C for 18 h. After completion, the mixture was concentrated in vacuo, redissolve in water (10 mL) and extracted with EtOAc (3 x 20 mL). The combined extracts were washed with and 10% NaCI (30 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, acetone / heptane) to afford the title compound as a mixture of fluoro-diastereomers (96:4) (51 mg, 18%).

[0296] 1H NMR (400 MHz, CDCh) 6 4.97 (dd, J = 48.9, 1.8 Hz, 1 H, CHF major), 4.90 (dd, J = 47.7, 3.4 Hz, 1 H, CHF minor), 3.79 (s, 3H), 3.62-3.45 (m, 1 H), 2.26-2.03 (m, 1 H), 2.06-1.64 (m, 8H), 1.59-1.16 (m, 13H), 0.99-0.94 (m, 6H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -84.4 (d, J = 240.6 Hz), -101.2 (d, J = 240.6 Hz), -208.7 (major), -191.0 (minor). iii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-fluoro-cholanic acid (Compound 4)

[0297] Methyl 3a-hydroxy-7,7-difluoro-5p-24-nor-22-fluoro-cholan-23-oate from step ii (51 mg, 0.12 mmol) was dissolved in MeOH (5 mL), 50% NaOH (0.14 mL, 1.78 mmol) was charged and the solution heated at 45 °C for 18 h. The solution was concentrated in vacuo. The crude residue was diluted with water (15 mL), acidified with 2 M HCI and extracted with EtOAc (3 x 30 mL). The combined extracts were washed with and 10% NaCI (30 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by reverse phase column chromatography (C18, MeOH / FW) to afford the title compound as one diastereomer (26 mg, 52%). pKa3.2;1H NMR (400 MHz, CD3OD) 5 5.00-4.73 (m, 1 H, CHF), 3.61-3.43 (m, 1 H), 2.36-2.12 (m, 1 H), 2.11-1.85 (m, 6H), 1.82-1.09 (m, 15H), 1.05-1.00 (m, 6H), 0.79 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 240.9 Hz), -102.5 (d, J = 240.9 Hz), -203.8; MS (ESI’) m / z 415.2 [M-H]-.

[0298] Example 5 - Synthesis of 3a-hvdroxy-7,7-difluoro-5P-24-nor-22-hydroxy-cholanic acid (Compound 5)

[0299] Compound 5 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor- cholan-23-oate, i.e. the product of Example 2, step ii according to Scheme 11.

[0300] Scheme 11 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxy-cholan-23-oate

[0301] The product of this step is the starting material for Compound 8 of Example 8, Compound 9 of Example 9, Compound 10 of Example 10 and Compound 11 of Example 11.

[0302] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate (969 mg, 2.12 mmol) and tripyrrolidinophosphoric acid triamide (TPPA, 0.24 mL, 1.06 mmol) in THF (40 mL) at -78 °C under nitrogen was charged LDA (1.0 M in THF / hexanes, 10.6 mL, 10.6 mmol). After stirring for 30 min Davis reagent (2.77 g, 10.6 mmol) in THF (15 mL) was charged. The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 18 h. The mixture was partitioned between 1 M HCI (50 mL) and ethyl acetate (50 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (3 x 50 mL). The combined extracts were washed with 5% NaHCCh (50 mL) and 10% NaCI (50 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 5-10% acetone in heptane) to afford the title compound (0.50 g, 50%) as a mixture of diastereomers (89:11).

[0303] 1H NMR (400 MHz, CDCh) 6 4.67 (s, 2H), 4.26-4.23 (m, 1 H major), 4.21-4.18 (m, 1 H minor), 3.80 (s, 3H minor), 3.78 (s, 3H major), 3.49-3.39 (m, 1 H), 3.36 (s, 3H), 2.60 (d, J = 5.3 Hz, 1 H), 2.23-2.05 (m, 1 H), 2.04-1.66 (m, 13H), 1.63-1.16 (m, 7H), 1.05 (td, J = 14.2, 3.1 Hz, 1 H), 0.94 (s, 3H), 0.83 (d, J = 6.7 Hz, 3H), 0.70 (s, 3H major), 0.68 (s, 3H minor);19F {1H} NMR (376 MHz, CDCh) 6 -84.4 (d, J = 240.2 Hz), -101.2 (d, J = 240.2 Hz). ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxy-cholanic acid (Compound 5)

[0304] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxy-cholan-23-oate from step i (50 mg, 0.11 mmol) was dissolved in MeOH (5 mL), 2 M HCI (0.26 mL, 0.53 mmol) was charged and the reaction mixture was heated to 40 °C for 18 h, then to 50 °C for 4 h. After this time additional 2 M HCI (0.13 mL, 0.26 mmol) was charged and the reaction mixture was heated at 50 °C for a further 20 h. After completion, 50% NaOH (0.13 mL, 2.12 mmol) was charged and the solution heated at 50 °C for 24 h. The solution was concentrated in vacuo. The crude residue was diluted with water (10 mL), acidified with 2 M HCI and extracted with EtOAc (3 x 10 mL). The combined extracts were washed with 10% NaCI (10 mL), dried over MgSCU and concentrated in vacuo The residue was purified by column chromatography (SiCh, 0-10% 7:2:1 (ethyl acetate: MeOH:water) in ethyl acetate) to afford the title compound (27.2 mg, 60%) as a mixture of diastereomers (88:12). pKa3.9;1H NMR (400 MHz, CD3OD) 5 4.20 (d, J = 2.0 Hz, 1 H major), 4.15 (d, J = 3.0 Hz, 1 H minor), 3.52-3.42 (m, 1 H), 2.30-2.12 (m, 1 H), 2.08-1.49 (m, 15H), 1.48-1.19 (m, 5H), 1.10 (td, J = 14.1 , 2.9 Hz, 1 H), 1.02 (d, J = 6.8 Hz, 3H minor), 0.99 (s, 3H), 0.92 (d, J = 6.8 Hz, 3H major), 0.75 (s, 3H major), 0.74 (s, 3H minor);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 240.9 Hz), -102.4 (d, J = 240.9 Hz); MS (ESI’) m / z 413.3 [M-H]’.

[0305] Example 6 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-chloro-cholanic acid (Compound 6)

[0306] Compound 6 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor- cholan-23-oate, i.e. the product of Example 2, step ii according to Scheme 12.

[0307] Scheme 12 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-chloro-cholan-23-oate

[0308] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate (250 mg, 0.55 mmol) and tripyrrolidinophosphoric acid triamide (TPPA, 63 pL, 0.27 mmol) in THF (8 mL) at -78 °C under nitrogen was charged LDA (1.0 M in THF / hexanes, 1.10 mL, 1.10 mmol). After stirring for 30 min N-chlorosuccinimide (150 mg, 1.10 mmol) in THF (2 mL) was charged. The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 18 h. Then the reaction was cooled to -78 °C and additional LDA (1.0 M in THF / hexanes, 0.55 mL, 0.55 mmol) was charged. After stirring for 40 minutes additional N-chlorosuccinimide (73 mg, 0.55 mmol). The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 18 h. The mixture was partitioned between 1 M HCI (30 mL) and ethyl acetate (30 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (3 x 30 mL). The combined extracts were washed with 5% NaHCOs (30 mL) and 10% NaCI (30 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 5-10% acetone in heptane) to afford the title compound (39 mg, 14%) as a 1 :1 mixture of diastereomers and (49 mg, 18%) as a 92:8 mixture of diastereomers.

[0309] 1H NMR (400 MHz, CDCh) 6 4.66 (s, 2H), 4.54 (d, J = 2.2 Hz, 1 H major), 4.49 (d, J = 3.2 Hz, 1 H minor), 3.78 (s, 3H major), 3.77 (s, 3H minor), 3.49-3.39 (m, 1 H), 3.36 (s, 3H), 2.25-2.06 (m, 2H), 2.05-1.66 (m, 10H), 1.65-1.46 (m, 4H), 1.45-1.12 (m, 5H), 1.06 (td, J = 14.2, 3.1 Hz, 1 H), 0.78 (d, J = 6.5 Hz, 3H), 0.95 (s, 3H), 0.72 (s, 3H major), 0.69 (s, 3H minor). ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-chloro-cholanic acid (Compound 6)

[0310] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-chloro-cholan-23-oate from step i (49 mg, 0.10 mmol) was dissolved in MeOH (5 mL), 2M HCI (0.25 mL, 0.5 mmol) was charged and the solution heated at 40 °C for 18 h, then heated at 50 °C and additional 2M HCI charged (0.13 mL, 0.25 mmol). After stirring at 50 °C for 22 h 50% NaOH (0.1 mL, 2 mmol) was charged and the heating continued for 22 h. The solution was concentrated in vacuo and the crude residue was diluted with water (10 mL), acidified with 2 M HCI and extracted with EtOAc (4 x 15 mL). The combined extracts were concentrated in vacuo. The residue was purified by reverse phase column chromatography (C18, MeOH / H2O) to afford the title compound (42 mg, 97%) as a 96:4 mixture of diastereomers. pKa3.1 ;1H NMR (400 MHz, CD3OD) 5 4.54 (d, J = 1.8 Hz, 1 H major), 4.49 (d, J = 3.1 Hz, 1 H minor), 3.52-3.42 (m, 1 H), 2.34-2.12 (m, 2H), 2.08-1.96 (m, 1 H), 1.96-1.17 (m, 18H), 1.10 (td, J = 14.0, 2.8 Hz, 1 H), 1.01 (d, J = 6.6 Hz, 3H), 0.99 (s, 3H), 0.77 (s, 3H);13C NMR (100 MHz, CD3OD) 5 125.9 (t, J = 242.0 Hz), 71.9, 68.1 , 53.7, 50.3 (d, J = 4.0 Hz), 44.0, 43.2 (dd, J = 23.0, 19.4 Hz), 42.6 (br. d, J = 9.9 Hz), 41.1 , 40.8, 38.5 (d, J = 8.9 Hz), 38.1 (d, J = 4.1 Hz), 37.3 (t, J = 23.0 Hz), 35.8, 35.4 (d, J = 1.1 Hz), 30.9, 28.6, 26.3 (d, J = 4.3 Hz), 23.0, 22.0, 14.6, 12.5;19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 240.8 Hz), -102.5 (d, J = 240.8 Hz); MS (ESL) m / z 431.2 [M-H]’.

[0311] Example 7 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-(methyl-d3)-cholanic acid (Compound 7)

[0312] Compound 7 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor- cholan-23-oate, i.e. the product of Example 2, step ii according to Scheme 13. Scheme 13 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-(methyl-d3)-cholan-23-

[0313] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate (100 mg, 0.22 mmol) and tripyrrolidinophosphoric acid triamide (TPPA, 25 pL, 0.11 mmol) in THF (4 mL) at -78 °C under nitrogen was charged LDA (1 .0 M in THF / hexanes, 0.33 mL, 0.33 mmol). After stirring for 30 min iodomethane-ds (20.4 pg, 0.33 mmol) in THF (0.5 mL) was charged. The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 18 h. Then the reaction was cooled to -78 °C and additional LDA (1.0 M in THF / hexanes, 0.33 mL, 0.33 mmol) was charged. After stirring for 30 minutes additional iodomethane-d3 (20.4 pg, 0.33 mmol) in THF (0.5 mL) was charged. The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 2.5 h. The mixture was partitioned between 1 M HCI (15 mL) and ethyl acetate (15 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (3 x 15 mL). The combined extracts were washed with 5% NaHCOs (15 mL) and 10% NaCI (15 mL), dried over MgSCU and concentrated in vacuo to afford the title compound as a crude mixture (150 mg). The residue was continued on without purification. ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-(methyl-d3)-cholanic acid (Compound 7)

[0314] Methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22-(methyl-d3)-cholan-23-oate from step i (150 mg) was dissolved in MeOH (7 mL), 2M HCI (0.55 mL, 1.1 mmol) was charged and the solution heated at 45 °C for 18 h, then heated at 60 °C and additional 2M HCI charged (0.25 mL, 0.50 mmol). After stirring at 60 °C for 22 h the solution was concentrated in vacuo and the crude residue was diluted with ethyl acetate (15 mL) and washed with 5% NaHCCh (15 mL). The aqueous phase was extracted with ethyl acetate (2 x 15 mL). The combined extracts were dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 5-15% ethyl acetate in heptane) to afford the intermediate as a 85:15 mixture with methyl 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate (70 mg). The intermediate was dissolved in MeOH (5 mL), 50% NaOH (86 pL, 1.63 mmol) was charged and the solution heated at 40 °C for 18 h, then heated at 65 °C and additional 50% NaOH charged (86 pL, 1.63 mmol). After stirring at 50 °C for 22 h 50% NaOH (0.1 mL, 2 mmol) was charged and the heating continued for 44 h. The7 solution was concentrated in vacuo and the crude residue was diluted with water (5 mL), acidified with 2 M HCI and extracted with EtOAc (4 x 10 mL). The combined extracts were concentrated in vacuo. The residue was purified by column chromatography (SiO2, 5-10% acetone in water) to afford the title compound as a single diastereomer (26 mg, 39%). pKa4.9;1H NMR (400 MHz, CD3OD) 5 3.53-3.43 (m, 1 H), 2.58 (br. s, 1 H), 2.31-1.98 (m, 3H), 1.93-1.49 (m, 11 H), 1.46-1.17 (m, 6H), 1.10 (td, J = 14.4, 2.9 Hz, 1 H), 0.98 (s, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.75 (s, 3H);13C NMR (100 MHz, CD3OD) 5 180.4, 125.9 (t, J = 241.8 Hz), 71.9,

[0315] 53.9, 50.2 (d, J = 3.9 Hz), 44.2, 43.2 (dd, J = 23.0, 19.4 Hz), 42.9, 42.6 (br. d, J = 10.4 Hz),

[0316] 40.9, 38.5 (d, J = 8.4 Hz), 38.5, 38.1 (d, J = 4.2 Hz), 37.3 (t, J = 23.5 Hz), 35.8, 35.4 (d, J = 1.1 Hz), 30.9, 29.0, 28.6, 26.4 (d, J = 4.2 Hz), 23.0, 22.0, 14.9, 12.2;19F {1H} NMR (376 MHz, CD3OD) 5 -85.1 (d, = 240.7 Hz), -102.4 (d, J = 240.7 Hz); MS (ESI’) m / z 414.3 [M-H]’.

[0317] Example 8 - Synthesis of 3a-hydroxy-7,7-difluoro-58-24-nor-22-methoxy-cholanic acid (Compound 8)

[0318] Compound 8 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22- hydroxy-cholan-23-oate, i.e. the product of Example 5, step i according to Scheme 14.

[0319] Scheme 14 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-methoxy-cholan-23-oate

[0320] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxy-cholan-23- oate (200 mg, 0.47 mmol) and methyl iodide (90 pL, 0.71 mmol) in THF (8 mL) at -20 °C under nitrogen was charged NaHMDS (1.0 M in THF, 0.71 mL, 0.71 mmol) drop-wise. The reaction mixture was stirred at -20 °C for 2 h then warmed to 0 °C and stirred for 18 h. The mixture was quenched by the addition of 5% NaHCCh (10 mL). The mixture was extracted with ethyl acetate (2 x 20 mL). The combined extracts were washed with 10% NaCI (20 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiCh, ethyl acetate / heptane) to afford the title compound as a mixture of diastereomers (80:20, 86 mg, 38%).

[0321] 1H NMR (400 MHz, CDCh) 6 4.67 (s, 2H major), 4.66 (s, 2H minor), 3.79 (d, J = 2.4 Hz, 1 H major), 3.77 (d, J = 3.4 Hz, 1 H minor), 3.75 (s, 3H minor), 3.75 (s, 3H major), 3.49-3.37 (m, 1 H), 3.39 (s, 3H), 3.36 (s, 3H major), 3.36 (s, 3H minor), 2.25-2.05 (m, 1 H), 2.01-1.67 (m, 12H), 1.63-1.16 (m, 8H), 1.05 (td, J = 14.3, 3.2 Hz, 1 H), 0.98 (d, J = 6.8 Hz, 3H minor), 0.95 (s, 3H), 0.90 (d, J = 6.9 Hz, 3H major), 0.69 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -84.3 (d, J = 239.9 Hz), -101.1 (d, J = 239.9 Hz minor), -101.2 (d, J = 239.9 Hz major). ii. Methyl 3a-hydroxy-7,7-difluoro-5p-24-nor-22-methoxy-cholan-23-oate

[0322] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-methoxy-cholan-23- oate from step i (86 mg, 0.18 mmol) in MeOH (5 mL) was charged 2M HCI (0.43 mL, 0.86 mmol). After stirring at 30 °C for 3 days the solution was concentrated in vacuo and the crude residue was diluted with ethyl acetate (10 mL) and washed with 5% NaHCCh (10 mL). The aqueous phase was extracted with ethyl acetate (2 x 10 mL). The combined extracts were washed with 10% NaCI, dried over MgSCU and concentrated in vacuo to afford the title compound as a mixture of diastereomers (70 mg, 85:15) which was continued on without any further purification. iii. 3a-Hydroxy-7,7-difluoro-5p-24-nor-22-methoxy-cholanic acid (Compound 8)

[0323] Methyl 3a-hydroxy-7,7-difluoro-5p-24-nor-22-methoxy-cholan-23-oate from step ii (70 mg, 0.16 mmol) was dissolved in MeOH (5 mL), 50% NaOH (95 L, 2.37 mmol) was charged and the solution heated at 45 °C for 19 h. The solution was concentrated in vacuo and the crude residue was diluted with water (5 mL), acidified with 2 M HCI and extracted with EtOAc (2 x 10 mL). The combined extracts were concentrated in vacuo. The residue was purified by reverse phase column chromatography (C18, MeOH / FW) to afford the title compound as a single diastereomer (22 mg, 32%). pKa3.4;1H NMR (400 MHz, CD3OD) 5 3.64 (br. s, 1 H), 3.52-3.42 (m, 1 H), 3.35 (br. s, 3H), 2.31-2.11 (m, 1 H), 2.06-1.45 (m, 15H), 1.45-1.15 (m, 5H), 1.10 (td, J = 14.2, 3.0 Hz, 1 H), 0.98 (s, 3H), 0.94 (d, J = 6.6 Hz, 3H), 0.73 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 240.8 Hz), -102.5 (d, J = 240.8 Hz); MS (ESI’) m / z 427.2 [M-H]’.

[0324] Example 9 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-keto-cholanic acid (Compound 9)

[0325] Compound 9 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22- hydroxy-cholan-23-oate, i.e. the product of Example 5, step i, according to Scheme 15.

[0326] Scheme 15 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-keto-cholan-23-oate

[0327] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxy-cholan-23- oate (100 mg, 0.21 mmol) in DCM (2 mL) was charged DMP (106 mg, 0.25 mmol). The reaction was stirred at ambient temperature overnight and then quenched with Na2S20s. The phases were separated and the aqueous phase was extracted with DCM. The combined extracts were washed with 10% NaCI, dried over MgSC and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the title compound (74 mg, 75%).

[0328] 1H NMR (400 MHz, CDCh) 6 4.67 (s, 2H), 3.86 (s, 3H), 3.50-3.39 (m, 1 H), 3.36 (s, 3H), 3.26 (dq, J = 10.2, 6.9 Hz, 1 H), 2.24-2.05 (m, 1 H), 1.98-1.92 (m, 1 H), 1.90-1.66 (m, 10H), 1.60-1.46 (m, 2H), 1.42-1.24 (m, 5H), 1.23-1.12 (m, 1 H), 1.19, (d, J = 6.9 Hz, 3H), 1.06 (td, J = 14.3, 3.3 Hz, 1 H), 0.95 (s, 3H), 0.73 (s, 3H). ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-keto-cholanic acid (Compound 9)

[0329] Methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22-keto-cholan-23-oate from step i (74 mg, 0.16 mmol) was dissolved in MeOH (2.6 mL). 2 M HCI (0.22 mL, 0.44 mmol) was charged and the reaction mixture was heated to 45 °C for 18 h. After this time additional 2 M HCI (0.20 mL, 0.40 mmol) was charged and the reaction mixture was heated at 45 °C for a further 21 h, then heated to 60 °C 5 h. After completion, 5 M NaOH (to pH 14) was charged and the solution was stirred at 30 °C for 5 days. The solution was concentrated in vacuo. The crude residue was diluted with water (10 mL), acidified with 2 M HCI and extracted with EtOAc (3 x 10 mL). The combined extracts were washed with 10% NaCI (10 mL), dried over MgSCU and concentrated in vacuo. Purification by preparative HPLC (C18, MeCN / H2O / 0.1% formic acid) afforded the title compound (7.2 mg).

[0330] (Compound 9): pKa1.4;1H NMR (400 MHz, CDCh) 6 3.59-3.48 (m, 1 H), 3.46-3.35 (m, 1 H), 2.25-2.05 (m, 1 H), 2.02-0.98 (m, 20H), 1.16, (d, J = 6.9 Hz, 3H), 0.93 (s, 3H), 0.67 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -84.7 (d, J = 240.6 Hz), -101.1 (d, J = 240.6 Hz); MS (ESI’) m / z 411.2 [M-H]-.

[0331] Example 10 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-azido-cholanic acid (Compound 10)

[0332] Compound 10 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22- hydroxy-cholan-23-oate, i.e. the product of Example 5, step i, according to Scheme 16. Scheme 16 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-azido-cholan-23-oate

[0333] The product of this step is the starting material for Compound 11 of Example 11.

[0334] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxy-cholan-23-oate (50 mg, 0.11 mmol) was dissolved in DCM (2.5 mL), cooled to 0 °C and then MsCI (12 pL, 0.16 mmol) and Et3N (74 pL, 0.53 mmol) were charged. The reaction mixture was allowed to warm to ambient temperature, stirred for 18 h then additional MsCI (6 pL) and Et3N (37 pL) was charged. Upon completion, indicated by TLC analysis, water (5 mL) was added and extracted with DCM (2 x 10 mL). The combined organic layer was washed with 5% NaHCO3solution (aq., 2 x 10 mL) and 10% NaCI (10 mL), dried over Na2SO4, filtered and concentrated to afford the mesylate intermediate. The residue was dissolved in DMF (2.5 mL) and charged into a flask containing NaN3(40 mg, 0.62 mmol). The reaction mixture was stirred at ambient temperature for 3 days, then heated to 50 °C for a further 2 days. After this time the reaction was cooled to ambient temperature, diluted with ethyl acetate and washed with 5% NaHCO3. The aqueous phase was extracted with ethyl acetate and the combined extracts were washed with 10% NaCI and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the title compound as a 73:27 mix of diastereomers (52 mg). This was continued on without any further purification. ii. 3a-Hydroxy-7,7-difluoro-5p-24-nor-22-azido-cholanic acid (Compounds 10a and

[0335] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-azido-cholan-23-oate from step i (33 mg, 0.06 mmol) was dissolved in MeOH (5 mL). 2 M HCI (0.15 mL, 0.30 mmol) was charged and the reaction mixture was heated to 40 °C for 18 h. After completion, 2 M NaOH (to pH 14) was charged and the solution was stirred at 40 °C for 21 h. The solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 2 M HCI and extracted with EtOAc (x3). The combined extracts were washed with 10% NaCI (10 mL), dried over MgSCU and concentrated in vacuo. Purification by preparative HPLC (C18, MeCN / H2O / 0.1% formic acid) afforded the title compound major diastereomer (9 mg) and minor diastereomer (5 mg). Major diastereomer (Compound 10a): pKa3.5;1H NMR (400 MHz, acetone-de) 54.36 (br. d J = 2.4 Hz, 1 H), 3.51-3.42 (m, 1H), 2.31-2.10 (m, 2H), 2.08-1.22 (m, 19H), 1.10 (td, J= 14.2, 3.2 Hz, 1 H), 1.00 (s, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.76 (s, 3H);19F {1H} NMR (376 MHz, acetonede) 5 -84.2 (d, J = 240.0 Hz), -101.5 (d, J = 240.0 Hz); MS (ESI’) m / z 438.1 [M-H]’.

[0336] Minor diastereomer (Compound 10b): pKa3.5;1H NMR (400 MHz, acetone-de) 64.32 (br. d J = 2.9 Hz, 1 H), 3.51-3.42 (m, 1H), 2.31-2.12 (m, 1 H), 2.08-1.19 (m, 19H), 1.10 (td, J= 14.2, 3.2 Hz, 1 H), 1.08 (d, J = 6.8 Hz, 3H), 0.99 (s, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, acetone- de) 5 -84.2 (d, J = 239.0 Hz), -101.5 (d, J = 239.0 Hz); MS (ESI’) m / z 438.3 [M-H]’).

[0337] Example 11 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-amine-cholanic acid (Compound 11)

[0338] Compound 11 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22- azido-cholan-23-oate, i.e. the product of Example 10, step i, according to Scheme 17. i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-amine-cholan-23-oate

[0339] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-azido-cholan-23-oate (35 mg, 0.07 mmol) was dissolved in THF (1 mL), PPhs (51 mg, 0.20 mmol) and H2O (10.5 pL, 0.59 mmol) were charged. The reaction mixture was heated at reflux for 2.5 h then concentrated in vacuo. The residue was purified by column chromatography (SiC>2, DCM / MeOH with 0.1 % NEts) to afford the title compound as a 90:10 mix of diastereomers (22 mg). This was continued on without any further purification. ii. 3a-Hydroxy-7,7-difluoro-5p-24-nor-22-amine-cholanic acid (Compound 11)

[0340] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-amine-cholan-23-oate from step i (22 mg, 0.05 mmol) was dissolved in MeOH (3 mL). 2 M HCI (0.10 mL, 0.20 mmol) was charged and the reaction mixture was heated to 40 °C for 22 h. After completion, 2 M NaOH (to pH 14) was charged and the solution was stirred at 40 °C for 18 h. The solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 2 M HCI and extracted with EtOAc (x3). The aqueous phase was saturated with NaCI and extracted with THF. The combined extracts were dried over MgSCU and concentrated in vacuo. Purification by preparative HPLC (C18, MeCN / H2O / 0.1% formic acid) afforded the title compound as one diastereomer (1.3 mg). pKa2.4 (acidic); pKa8.8 (basic);1H NMR (400 MHz, CD3OD) 5 3.56 (br. s 1 H), 3.51-3.42 (m, 1 H), 2.30-2.10 (m, 2H), 2.06-1.99 (m, 1 H), 1.92-1.20 (m, 18H), 1.15-1.02 (m, 4H), 0.98 (s, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -84.2 (d, J = 240.0 Hz), -101.5 (d, J = 240.0 Hz); MS (ESI+) m / z 414.2 [M+H]+.

[0341] Example 12 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-cholan-23-ol (Compound 12)

[0342] Compound 12 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor- cholan-23-oate, i.e. the product of Example 2, step ii according to Scheme 18.

[0343] Scheme 18 i. 3a-Methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-ol

[0344] The product of this step is the starting material for Compound 13 of Example 13 and Compound 15 of Example 15.

[0345] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate (300 mg, 0.66 mmol) in dry THF (5 mL) was charged dry MeOH (186 pL, 4.6 mmol). The solution was cooled to 0 °C then LiBH4 (0.1 g, 4.6 mmol) was charged portion-wise. The reaction was stirred between 0 - 5 °C for 19 h and then quenched by the addition of 5% NH4CI (10 mL) and allowed to warm to ambient temperature. The mixture was extracted with EtOAc and the organic phase was washed with 5% NH4CI, 10% NaHCOs and 10% NaCI, dried over MgSCU and concentrated in vacuo to afford the title compound as a white solid (quant.). The material was continued on without any purification.

[0346] 1H NMR (400 MHz, CDCI3) 6 4.67 (s, 2H), 3.76-3.59 (m, 2H), 3.50-3.40 (m, 1 H), 3.36 (s, 3H), 2.24-2.06 (m, 1 H), 2.06-1.98 (m, 1 H), 1.95-1.66 (m, 9H), 1 .68-1.42 (m, 9H), 1 .38-1 .02-1 .01 (m, 4H), 0.96 (d, J = 6.7 Hz, 3H), 0.95 (s, 3H), 0.68 (s, 3H). ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-ol (Compound 12)

[0347] To a solution of 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-cholan-23-ol from step i (53 mg, 0.12 mmol) in MeOH (2 mL) was charged 2M HCI (0.3 mL). The solution stirred at ambient temperature for 24 h, then heated to 45 °C for 2 days. The solution was concentrated in vacuo extracted with ethyl acetate (not soluble), the aqueous phase was saturated with solid NaCI and then extracted with THF. The organic phase was concentrated in vacuo and purified by reverse phase column chromatography (C18, MeOH / FW) to afford the title compound (9.6 mg, 21 %). pKa13.0;1H NMR (400 MHz, CD3OD) 5; 3.67-3.43 (m, 3H), 2.30-2.11 (m, 1 H), 2.10-2.04 (m, 1 H), 1.97-1.45 (m, 15H), 1.44-1.05 (m, 7H), 0.98 (d, J = 6.5 Hz, 3H), 0.98 (s, 3H), 0.73 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 240.7 Hz), -102.5 (d, J = 240.7 Hz); MS (ESI+) m / z 407.2 [M+Na]+.

[0348] Example 13 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-chol-22-ene (Compound 13)

[0349] Compound 13 was synthesised from 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-cholan-23- ol, i.e. the product of Example 12, step i, according to Scheme 19.

[0350] Scheme 19 i. 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholanal

[0351] To 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-cholan-23-ol (250 mg, 0.60 mmol) in DCM (5 mL) was charged DMP (512 mg, 1.21 mmol) in two portions. The reaction mixture was stirred at ambient temperature for 4 h then quenched with 5% NaHCCh (aq) (30 mL). After stirring for 30 min the mixture was filtered and the filter cake washed with DCM. The filtrate was separated and the organic phase was concentrated in vacuo to afford the crude title compound which was continued on without any purification (210 mg, 82%). ii. 3a-Methoxymethoxy-7,7-difluoro-5p-24-nor-chol-22-ene

[0352] The product of this step is the starting material for Compound 14 of Example 14.

[0353] To 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-cholanal from step i (210 mg, 0.49 mmol) in anhydrous THF (5 mL) at 0 °C was charged KO‘Bu (221 mg, 1.97 mmol) in anhydrous THF (5 mL), followed by PhsPMeBr (703 mg, 1 .97 mmol). The reaction mixture was allowed to warm to ambient temperature then stirred for 20 h and quenched with sat. NH4CI (aq) (10 mL). After stirring for 10 min phases were separated and the organic phase was concentrated in vacuo. The crude residue was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the title compound (130 mg, 63%).

[0354] 1H NMR (400 MHz, CDCI3) 6 5.83-5.71 (m, 1 H), 5.02-4.96 (m, 2H), 4.67 (s, 2H), 3.49-3.40 (m, 1 H), 3.36 (s, 3H), 2.24-1 .01 (m, 24H), 0.95 (s, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.68 (s, 3H);19F {1H} NMR (376 MHz, CDCI3) 6 -84.3 (d, = 240.1 Hz), -101.1 (d, = 240.1 Hz). ii. 3a-Hydroxy-7,7-difluoro-5p-24-nor-chol-22-ene (Compound 13)

[0355] To 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-chol-22-ene from step ii (60 mg, 0.14 mmol) in

[0356] MeOH (5 mL) was charged 2 M HCI (353 pL, 7.06 mmol). The reaction mixture stirred at 40 °C for 20 h, then cooled to ambient temperature, neutralised with 5% NaHCOs and TBME (20 mL) was charged. The phases were separated and the organic phase was dried over MgSC and concentrated in vacuo. The crude residue was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the title compound (9 mg, 17%).

[0357] 1H NMR (400 MHz, CDCI3) 6 5.83-5.71 (m, 1 H), 5.02-4.96 (m, 2H), 3.59-3.50 (m, 1 H), 2.24- 1.01 (m, 24H), 0.95 (s, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.68 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -84.3 (d, J = 240.1 Hz), -101.1 (d, J = 240.1 Hz).

[0358] Example 14 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-cholane (Compound 14)

[0359] Compound 14 was synthesised from 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-chol-22-ene, i.e. the product of Example 13, step ii, according to Scheme 20.

[0360] Scheme 20 i. 3a-Methoxymethoxy-7,7-difluoro-5 -24-nor-cholane

[0361] 3a-Methoxymethoxy-7,7-difluoro-5p-24-nor-chol-22-ene (65 mg, 0.15 mmol) in ethyl acetate (2 mL) was charged to a suspension of 10% Pd / C in ethyl acetate (3 mL) under nitrogen. The reaction vessel was purged with nitrogen (x3) and then purged with H2 (x3), left under an H2 atmosphere and stirred at ambient temperature for 18 h. The suspension was filtered, washed with ethyl acetate and the filtrate was concentrated in vacuo to afford the title compound (40 mg, 61%) which was continued on without purification. ii. 3a-Hydroxy-7,7-difluoro-5p-24-nor-cholane (Compound 14) To 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholane from step i (40 mg, 0.09 mmol) in MeOH (3 mL) was charged 2 M HCI (234 pL, 0.47 mmol). The reaction mixture stirred at 40 °C for 20 h, then additional 2 M HCI (234 pL, 0.47 mmol) was charged and the reaction mixture was stirred at 40 °C for 24 h, followed by heating to 50 °C for 24 h. After this time the reaction was cooled to ambient temperature, neutralised with 5% NaHCCh and TBME (20 mL) was charged. The phases were separated and the organic phase was dried over MgSCU and concentrated in vacuo to afford the title compound (40 mg, Quant.).

[0362] 1H NMR (400 MHz, CDCh) 6 3.59-3.50 (m, 1 H), 2.24-2.06 (m, 1 H), 2.05-1.97 (m, 1 H), 1.92- 0.99 (m, 24H), 0.95 (s, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.86 (t, 7.0 Hz, 3H), 0.66 (s, 3H);13C NMR (100 MHz, CDCh) 6 124.8 (t, J = 243.2 Hz), 71.5, 55.5, 48.8 (d, J = 3.9 Hz), 43.2, 42.0 (dd, J = 22.9, 19.2 Hz), 41.4 (d, J = 10.6 Hz), 39.7, 38.5, 37.5 (d, 4.3 Hz), 37.2 (d, J = 9.0 Hz), 36.4 (t, J = 23.2 Hz), 35.7, 34.9 (d, 1.4 Hz), 34.4 (d, J = 1.5 Hz), 30.4, 28.6 (d, J = 1.9 Hz), 25.5 (d, J = 4.1 Hz), 22.8, 21.1 , 19.4, 18.9, 14.7, 11.9;19F {1H} NMR (376 MHz, CDCh) 6 -84.3 (d, = 240.1 Hz), -101.2 (d, J = 240.1 Hz).

[0363] Example 15 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-cholan-23-azide (Compound 15

[0364] Compound 15 was synthesised from 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-ol i.e. the product of Example 12, step i, according to Scheme 21.

[0365] Scheme 21 i. 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-cholan-23-azide

[0366] The starting material 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-ol (500 mg, 1.21 mmol) was dissolved in DCM (7.5 mL) and Et3N (0.84 mL, 6.03 mmol) then cooled to 0 °C. MsCI (107 pL, 1.82 mmol) was then charged, the reaction mixture was allowed to warm to ambient temperature and stirred for 18. Upon completion, indicated by TLC analysis, water (20 mL) was added and extracted with DCM (2 x 10 mL). The combined organic layer was washed with 5% NaHCOs solution (20 mL), 10% NaCI (20 mL), then dried over MgSCU, filtered and concentrated to afford the mesylate intermediate. The residue (100 mg, 0.2 mmol) was dissolved in DMF (5 mL) and charged into a flask containing NaNs (43 mg, 0.66 mmol). The reaction mixture was stirred at 50 °C for 3 days. After this time the reaction was cooled to ambient temperature, diluted with TBME and washed with 5% NaHCCh. The aqueous phase was back extracted with TBME and the combined extracts were washed with 10% NaCI and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the title compound (72 mg, 79%). This was continued on without any further purification. ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-azide (Compound 15)

[0367] To a solution of 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-azide from step i (35 mg, 0.077 mmol) in MeOH (5 mL) was charged 2M HCI (0.2 mL). After stirring at 40 °C for 3 days the solution was concentrated in vacuo to afford the title compound (29 mg, 92%). pKa14.3;1H NMR (400 MHz, acetone-d6) 3.54-3.37 (m, 2H), 3.31-3.24 (m, 1 H), 2.26-1.99 (m, 4H), 1.94-1.04 (m, 20H), 0.98 (d, J = 5.8 Hz, 3H), 0.97 (s, 3H), 0.70 (s, 3H).19F {1H} NMR (376 MHz, acetone-d6) 5 -84.1 (d, J = 240.1 Hz), -101.4 (d, J = 240.1 Hz); MS (ESI+) m / z 432.2 [M+Na]+.

[0368] Example 16 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-cholan-23-amine (Compound 16}

[0369] Compound 16 was synthesised from 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-cholan-23- azide i.e. the product of Example 15, step i, according to Scheme 22.

[0370] Scheme 22 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-cholan-23-amine

[0371] 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-azide (35 mg, 0.08 mmol) was dissolved in THF (1 mL), PPhs (60 mg, 0.23 mmol) and H2O (12.4 pL, 0.69 mmol) were charged. The reaction mixture was stirred at reflux for 18 h then concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 7:2:1 (ethyl acetate: MeOH: H2O) in ethyl acetate) to afford the title compound (18 mg, 55%). This was continued on without any further purification. ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-amine (Compound 16)

[0372] To a solution of 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-amine from step i (18 mg, 0.042 mmol) in MeOH (2 mL) was charged 2M HCI (0.2 mL). After stirring at 40 °C for 2 days the solution was concentrated in vacuo to afford the title compound (16 mg, quant.). pKa14.3 (acidic); pKa10.2 (basic);1H NMR (400 MHz, CD3OD) 3.53-3.44 (m, 1 H), 3.03-2.97 (m, 1 H), 2.90-2.84 (m, 1 H), 2.30-2.04 (m, 2H), 1.97-1.07 (m, 22H), 1.03 (d, J = 6.5 Hz, 3H), 0.98 (s, 3H), 0.74 (s, 3H).19F {1H} NMR (376 MHz, CD3OD) 5 -86.5 (d, J = 240.7 Hz), -103.7 (d, J = 240.7 Hz); MS (ESI+) m / z 384.2 [M+H]+.

[0373] Example 17 - Synthesis of 3a-hydroxv-7,7-difluoro-5P-24-nor-22-hvdroxvmethyl- cholanic acid (Compound 17)

[0374] Compound 17 was synthesised from methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor- cholan-23-oate, i.e. the product of Example 2, step ii according to Scheme 23. Scheme 23 i. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-formyl-cholan-23-oate

[0375] To a solution of methyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-cholan-23-oate (500 mg, 1.1 mmol) and tripyrrolidinophosphoric acid triamide (TPPA, 0.13 mL, 0.05 mmol) in THF (20 mL) at -78 °C under nitrogen was charged LDA (1.0 M in THF / hexanes, 5.48 mL, 5.48 mmol). After stirring for 30 min ethyl formate (0.88 mL, 11.0 mmol) was charged dropwise. The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 18 h. The mixture was partitioned between 1 M HCI (50 mL) and ethyl acetate (50 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (3 x 50 mL). The combined extracts were washed with 5% NaHCCh (50 mL) and 10% NaCI (50 mL), dried over MgSCU and concentrated in vacuo to afford the title compound as a crude mixture (0.87g). The residue was continued on without purification. ii. Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-

[0376] 23-oate

[0377] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-formyl-cholan-23-oate from step i (0.3 g, 0.62 mmol) was dissolved in MeOH (12 mL) and cooled to 0 °C under nitrogen. Sodium borohydride (47 mg, 1.2 mmol) was charged in one portion then the reaction was stirred at 0 °C for 5 minutes and then allowed to warm to ambient temperature and stirred for 18 h. The reaction was quenched by the drop wise addition of 1 M HCI to pH 6. The mixture was concentrated in vacuo, diluted with ethyl acetate (15 mL) and washed with NaHCCh (to pH 8). The aqueous phase was extracted with ethyl acetate (15 mL) and the combined extracts were washed with 10% NaCI, dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 10-40% ethyl acetate in heptane) to afford the crude intermediate as a 6:4 mixture of diastereomers (256 mg, 85%), which was continued on without any further purification. iii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholanic acid (Compound 17)

[0378] Methyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-oate from step ii (100 mg, 0.21 mmol) was dissolved in MeOH (3.5 mL). 2 M HCI (0.5 mL, 1.0 mmol) was charged and the reaction mixture was heated to 45 °C for 19 h. After this time additional 2 M HCI (0.20 mL, 0.40 mmol) was charged and the reaction mixture was heated at 45 °C for a further 7.5 h. After completion, 50% NaOH (to pH 14) was charged and the solution was stirred at 45 °C for 3 days. The solution was concentrated in vacuo. The crude residue was diluted with water (10 mL), acidified with 2 M HCI and extracted with EtOAc (3 x 10 mL). The combined extracts were washed with 10% NaCI (10 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (7:2:1 (ethyl acetate: MeOH: H2O) in ethyl acetate) to afford the title compound as a single diastereomer (19 mg, 22%) and as a 54:46 diastereomeric mixture (58 mg, 66%), pKa4.6;1H NMR (400 MHz, CD3OD) 5 3.80 (dd, J = 10.8, 9.6 Hz, 1 H), 3.56 (dd, J = 10.8, 3.9 Hz, 1 H), 3.52-3.42 (m, 1 H), 2.44 (dt, 9.6, 3.9 Hz, 1 H), 2.30-2.11 (m, 2H), 2.05-1.98 (m, 1 H), 1.95-1.45 (m, 13H), 1.44-1.15 (m, 5H), 1.09 (td, J = 14.2, 3.1 Hz, 1 H), 0.98 (s, 3H), 0.93 (d, J = 7.0 Hz, 3H), 0.74 (s, 3H);13C NMR (100 MHz, CD3OD) 5 183.3, 125.9 (t, J = 244.1 Hz), 71.9, 60.5, 54.3, 53.3, 50.3 (d, J = 3.8 Hz), 44.3, 43.2 (dd, J = 23.0, 19.4 Hz), 42.6 (br. d, J = 10.7 Hz), 40.9, 38.5 (d, J = 8.8 Hz), 38.1 (d, J = 4.0 Hz), 37.9, 37.4 (t, J = 23.3 Hz), 35.8, 35.4 (br. s), 30.9, 29.0, 26.5 (d, J = 3.9 Hz), 23.0, 22.0, 16.6, 12.0;19F {1H} NMR (376 MHz, CD3OD) 5 -85.1 (d, J = 240.8 Hz), -102.4 (d, J = 240.8 Hz); MS (ESI’) m / z 427.3 [M-H]’. Example 18 - Synthesis of 3a-hvdroxy-7,7-difluoro-50-24-nor-22-fluoromethyl-cholanic acid (Compounds 18a and 18b)

[0379] Compound 18 was synthesised from 7,7-difluoro 24-norlithocholic acid (Compound 1), according to Scheme 24.

[0380] Scheme 24 i. t-Butyl 3a, 7 -dihydroxy-50-24-nor-cholan-23-oate

[0381] To a suspension of 7,7-difluoro 24-norlithocholic acid (4.31 g, 11.2 mmol) in in dry THF (47 mL) at 0 °C was charged TFAA (10.11 mL, 40.2 mmol) dropwise, maintaining the temperature at < 25 °C. The reaction mixture was warmed to ambient temperature and stirred for 2 h then tBuOH (35.2 mL, 370 mmol) was added dropwise maintaining the temperature at < 25 °C After stirring at ambient temperature for 20 h cone, ammonia (15 mL) was charged dropwise (maintaining temperature < 15 °C). After 3 days the reaction was quenched by the addition of 5% NaHCOs (50 mL). Ethyl acetate (150 mL) and 10% NaCI (25 mL) was charged. The phases were separated and the aqueous phase was extracted with ethyl acetate (x3). The combined extracts were washed with 10% NaCI and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 0-100% acetone in toluene) to afford the title compound (3.30 g, 65%).1H NMR (400 MHz, CDCh) 6 3.59-3.49 (m, 1 H), 2.38-2.29 (m, 1 H), 2.24-2.06 (m, 1 H), 2.00 (dt, J = 12.4, 3.5 Hz, 1 H), 1.95-1.63 (m, 11 H), 1.63-1.41 (m, 3H), 1.44 (s, 9H), 1.41-1.12 (m, 6H), 1.07 (td, J = 14.4, 3.2 Hz, 1 H), 0.99 (d, J = 6.0 Hz, 3H), 0.95 (s, 3H), 0.70 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -83.4 (d, J = 240.8 Hz), -101.2 (d, J = 240.8 Hz). ii. t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate

[0382] To a solution of t-Butyl 3a, 7 -dihydroxy-5p-24-nor-cholan-23-oate from step i (3.30 g, 7.49 mmol) in DCM (33 mL) was charged DIPEA (3.20 mL, 24.0 mmol) dropwise, followed by MOMCI (1.05 mL, 14.2 mmol). The reaction mixture was stirred under nitrogen at ambient temperature for 4 h then quenched by the drop-wise addition of water (33 mL). After stirring for 18 h the phases were separated. The aqueous phase was extracted with DCM (2 x 15 mL) and the combined extracts were washed with 0.5 M HCI (3 x 15 mL, till pH < 5), 5% NaHCCh (15 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiCh, 0-40% ethyl acetate in heptane) to afford the title compound (3.10 g, 83%).

[0383] 1H NMR (400 MHz, CDCh) 6 4.66 (s, 2H), 3.48-3.39 (m, 1 H), 3.36 (s, 3H), 2.38-2.29 (m, 1 H), 2.24-2.06 (m, 1 H), 1.99 (dt, J = 12.5, 3.4 Hz, 1 H), 1.93-1.66 (m, 8H), 1.58-1.41 (m, 3H), 1.43 (s, 9H), 1.38-1.21 (m, 8H), 1.20-1.10 (m, 1 H), 1.04 (td, J = 14.4, 3.4 Hz, 1 H), 0.98 (d, J = 6.3 Hz, 3H), 0.93 (s, 3H), 0.69 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -84.3 (d, J = 240.4 Hz), -101.1 (d, J = 240.4 Hz). iii t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-formyl-cholan-23-oate

[0384] The product of this step is the starting material for Compound 19 of Example 19.

[0385] To a solution of f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-cholan-23-oate from step ii (3.08 g, 6.18 mmol) and tripyrrolidinophosphoric acid triamide (TPPA, 0.71 mL, 3.09 mmol) in THF (20 mL) at -78 °C under nitrogen was charged LDA (1.0 M in THF / hexanes, 30.8 mL, 30.8 mmol). After stirring for 30 min ethyl formate (4.97 mL, 61 .8 mmol) was charged dropwise. The reaction mixture was allowed to warm to ambient temperature then stirred under nitrogen for 18 h. The mixture was partitioned between 1 M HCI (60 mL) and ethyl acetate (50 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (2 x 40 mL). The combined extracts were washed with 5% NaHCCh (30 mL) and 10% NaCI (30 mL), dried over MgSCU and concentrated in vacuo to afford the title compound as a crude mixture (Quant.). The residue was continued on without purification. iv. t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan- 23-oate

[0386] The product of this step is the starting material for Compound 20 of Example 20, Compound 21 of Example 21 , Compound 22 of Example 22 and Compound 23 of Example 23.

[0387] The f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-formyl-cholan-23-oate from step iii (3.25 g, 6.18 mmol) was dissolved in MeOH (28 mL) and cooled to 0 °C under nitrogen. Sodium borohydride (0.7 g, 18.5 mmol) was charged in one portion then the reaction was stirred at 0 °C for 5 minutes and then allowed to warm to ambient temperature and stirred for 18 h. The reaction was quenched by the drop wise addition of 2 M HCI to pH < 5. Ethyl acetate (70 mL) was charged, followed by water (10 mL). The phases were separated and the organic phase was washed with 5% NaHCCh (to pH 8), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, 0-40% ethyl acetate in heptane) to afford the title compound as a crude 43:57 mixture of diastereomers (2.87 g, 88%), which was continued on without any further purification. v. t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-fluoromethyl-cholan-23-

[0388] The f-butyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22-hydroxymethyl-cholan-23-oate from step iv (200 mg, 0.38 mmol) was dissolved in DCM (3 mL) and DAST (75 pL, 0.57 mmol) was charged. The reaction was stirred at ambient temperature for 4 h and then diluted with DCM and transferred into rapidly stirring solution of 5% NaHCO3and stirred for 30 min. The phases were separated and the aqueous phase was extracted with DCM. The combined extracts were washed with 10% NaCI, dried over MgSC and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the title compound as a crude inseparable mixture (17 mg), which was continued on without any further purification. vi. 3a-Hydroxy-7,7-difluoro-5p-24-nor-22-fluoromethyl-cholanic acid (Compound 18)

[0389] To a solution of f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-fluoromethyl-cholan-23- oate from step v (75 mg, 0.14 mmol) in DCM (5 mL) at 0 °C was charged TFA (0.2 mL). The reaction was allowed to warm to ambient temperature and stirred for 18 h. The reaction mixture was concentrated in vacuo, co-evaporated with water, then dissolved in MeOH and stirred with Na2CC>3 (150 mg) for 3 h. The mixture was filtered and concentrated, acidified with 2 M HCI and then extracted into ethyl acetate and concentrated in vacuo to afford the title compound as a 7:3 mixture of diastereomers (36 mg). Purification by preparative HPLC (C18, MeCN / H2O / 0.1 % formic acid) afforded the title compound major diastereomer (4.9 mg) and minor diastereomer (2 mg).

[0390] Major diastereomer (Compound 18a): pKa4.8;1H NMR (400 MHz, (CDs^CO) 5 4.79 (ddd, J = 46.9 9.3, 7.9 Hz, 1 H), 4.66 (ddd, J = 46.9, 9.3, 4.3 Hz, 1 H), 4.57-4.41 (m, 1 H), 2.13-1.18 (m, 22H), 1.10 (td, J = 14.2, 3.0 Hz, 1 H), 1.00 (s, 3H), 0.99 (d, J = 9.0 Hz, 3H), 0.76 (s, 3H)19F {1H} NMR (376 MHz, (CD3)2CO) 5 -84.2 (d, J = 240.1 Hz), -101.5 (d, J = 240.1 Hz), -224.3; MS (ESP) m / z 429.2 [M-H]’.

[0391] Minor diastereomer (Compound 18b): pKa4.8;1H NMR (400 MHz, (CDs^CO) 5 4.73 (ddd, J = 47.0, 8.9, 7.8 Hz, 1 H), 4.48 (ddd, J = 47.0, 8.9, 6.7 Hz, 1 H), 4.57-4.41 (m, 1 H), 2.30-1.17 (m, 22H), 1.10 (td, J = 14.2, 3.0 Hz, 1 H), 1.00 (d, J = 7.0 Hz, 3H), 0.99 (s, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, (CD3)2CO) 5 -84.1 (d, J = 240.1 Hz), -101.6 (d, J = 240.1 Hz), -222.5; MS (ESP) m / z 429.2 [M-H]'. Example 19 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-di-fluoromethyl- cholanic acid (Compound 19)

[0392] Compound 19 was synthesised from f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22- formyl-cholan-23-oate i.e. the product of Example 18, step iii, according to Scheme 25. i. t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-di-fluoromethyl-cholan- 23-oate t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-formyl-cholan-23-oate (150 mg, 0.29 mmol) was dissolved in DCM (2.3 mL) and DAST (380 pL, 2.85 mmol) was charged. The reaction mixture was stirred at ambient temperature for 18 h and then diluted with DCM and transferred in to rapidly stirring solution of 5% NaHCCh and stirred for 30 min. The phases were separated and the aqueous phase was extracted with DCM. The combined extracts were washed with 10% NaCI, dried over MgSC and concentrated in vacuo. The residue was purified by column chromatography (SiCh, 10-20% acetone in heptane) to afford the title compound as a crude inseparable mixture (134 mg), which was continued on without any further purification. ii. t-Butyl 3a-hydroxy-7,7-difluoro-5p-24-nor-22-di-fluoromethyl-cholan-23-oate

[0393] To a solution of f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-di-fluoromethyl-cholan- 23-oate from step i (134 mg, 0.24 mmol) in DCM (3 mL) at 0 °C was charged TFA (56 pL, 0.73 mmol). The reaction mixture was stirred at ambient temperature for 6 days then concentrated in vacuo and co-evaporated with water. The residue was dissolved in MeOH and stirred with 5% NaHCOs for 2 h. The mixture was extracted with ethyl acetate (x2). The combined extracts were washed with 10% NaCI and concentrated in vacuo. The crude residue was purified by column chromatography (SiCh, ethyl acetate / heptane / 0.1% AcOH) to afford the title compound as a mixture of diastereomers (24 mg). Purification by preparative HPLC (C18, MeCN / H2O / 0.1 % formic acid) afforded the title compound major diastereomer (2.6 mg) and minor diastereomer (1 .3 mg).

[0394] Major diastereomer:1H NMR (400 MHz, CDCh) 6 6.03 (ddd, J = 57.2, 55.6, 7.4 Hz, 1 H), 3.63- 3.54 (m, 1 H), 2.88-2.78 (m, 1 H), 2.28-1.67 (m, 9H), 1.65-1.09 (m, 13H), 1.52 (s, 9H), 1.06 (d, = 6.8 Hz, 3H), 0.98 (s, 3H), 0.73 (s, 3H)19F {1H} NMR (376 MHz, CDCh) 6 -84.4 (d, = 241.0 Hz), -101.1 (d, J = 241.0 Hz), -120.3 (d, J = 285.5 Hz), -125.5 (d, J = 285.5 Hz).

[0395] Minor diastereomer:1H NMR (400 MHz, CDCh) 6 6.06 (ddd, J = 56.9, 55.3, 7.0 Hz, 1 H), 3.61- 3.49 (m, 1 H), 2.87-2.76 (m, 1 H), 2.25-1.62 (m, 11 H), 1.62-1.02 (m, 11 H), 1.47 (s, 9H), 1.14 (d, J = 7.0 Hz, 3H), 0.96 (s, 3H), 0.69 (s, 3H)19F {1H} NMR (376 MHz, CDCh) 6 -84.4 (d, = 241.3 Hz), -101.1 (d, J = 241.3 Hz), -116.2 (d, J = 288.7 Hz), -122.7 (d, J = 288.7 Hz). iii. 3a-Hydroxy-7,7-difluoro-5 -24-nor-22-di-fluoromethyl-cholanoic acid

[0396] (Compounds 19a and 19b)

[0397] To t- Butyl 3a-hydroxy-7,7-difluoro-5p-24-nor-22-di-fluoromethyl-cholan-23-oate from step ii (2.1 mg major diastereomer, 0.9 mg minor diastereomer, reacted on separately) was charged 4 M HCI in dioxane (0.1 mL). The reaction mixture was stirred for 2 days until complete by TLC then concentrated in vacuo to afford the title compound major diastereomer (1 mg) and minor diastereomer (1.3 mg).

[0398] Major diastereomer (Compound 19a): pKa4.5;1H NMR (400 MHz, CD3OD) 5 6.09 (td, J = 56.3, 7.3 Hz, 1 H), 3.52-3.41 (m, 1 H), 2.90-2.77 (m, 1 H), 2.30-1.04 (m 24H), 1.07 (d, J = 6.8 Hz, 3H), 0.98 (s, 3H), 0.75 (s, 3H)19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 240.9 Hz), -102.5 (d, J = 240.9 Hz), -121.6 (d, J = 285.3 Hz), -126.6 (d, J = 285.3 Hz); MS (ESI’) m / z 447.2 [M-H]'.

[0399] Minor diastereomer (Compound 19b): pKa4.5; MS (ESI ) m / z 447.2 [M-H]'.

[0400] Example 20 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-methylene-cholanic acid (Compound 20)

[0401] Compound 20 was synthesised from f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22- hydroxymethyl-cholan-23-oate i.e. the product of Example 18, step iv, according to Scheme 26.

[0402] Scheme 26 i. t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-methylenemethyl-cholan- 23-oate t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-oate (200 mg, 0.38 mmol) was dissolved in DCM (10 mL) and cooled to 0 °C under nitrogen. Triethylamine (0.263 mL, 1.89 mmol) and methanesulfonyl chloride (0.044 mL, 0.57 mmol) were charged then the reaction was stirred at 0 °C for 5 minutes and then allowed to warm to ambient temperature and stirred for 18 h. The reaction was monitored by TLC, further triethylamine (0.130 mL) and methanesulfonyl chloride (0.022 mL) was added and stirred at room temperature for a further 18 h. Upon the completion indicated by TLC analysis, water (5 mL) was added and extracted with DCM (2 x 10 mL). The combined organic layer was washed with 5% NaHCOs solution (aq., 2 x 10 mL) and 10% NaCI (10 mL), dried over Na2SO4, filtered and concentrated to afford the mesylate intermediate. The residue was dissolved in DMF (5 mL), RbF (0.099 g, 0.95 mmol) was charged and the reaction mixture heated to 50 °C for 4 days. The mixture was diluted with ethyl acetate (5 mL) and quenched with 5% NaHCCh (5 mL). The phases were separated, and the aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined extracts were washed with 10% NaCI (30 mL), dried over MgSCU and concentrated in vacuo. The resulting residue was purified by column chromatography (SiC>2, ethyl acetate / heptane), to afford the title compound (20 mg, 10%) which was continued on without any further purification. ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-methylene-cholanic acid (Compound 20)

[0403] To a solution of f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-methylenemethyl- cholan-23-oate from step i (82 mg, 0.16 mmol) in DCM (3 mL) at 0 °C was charged TFA (0.24 mL, 1.4 mmol). The reaction mixture was stirred at ambient temperature for 18 h then concentrated in vacuo and co-evaporated with water. The residue was dissolved in MeOH and stirred with Na2COs (150 mg) for 2 h, filtered and concentrated in vacuo. The crude residue was purified by column chromatography (SiC>2, ethyl acetate / heptane / 0.1% AcOH) to afford the title compound as a colourless solid (20 mg, 34%). pKa3.9;1H NMR (400 MHz, CD3OD) 66.15 (d, J = 1.3 Hz, 1 H), 5.58 (d, J = 1.0 Hz, 1 H), 3.52- 3.44 (m, 1 H), 2.72-2.64 (m, 1 H), 2.30-2.11 (m, 1 H), 2.11-2.04 (m, 1 H), 1.91-1.06 (m, 19H), 1.15 (d, J = 6.8 Hz, 3H), 0.99 (s, 3H), 0.76 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) -85.2 (d, J = 241.1 Hz), -102.4 (d, J = 241 .1 Hz); MS (ESP) m / z 409.2 ([M-H]’.

[0404] Example 21 - Synthesis of 3a-hydroxy-7,7-difluoro-5P-24-nor-22-methoxymethyl- cholanic acid (Compounds 21a and 21 b)

[0405] Compound 21 was synthesised from f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22- hydroxymethyl-cholan-23-oate i.e. the product of Example 18, step iv, according to Scheme 27.

[0406] Scheme 27 i. t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-methoxymethyl-cholan-

[0407] 23-oate

[0408] To a solution of f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan- 23-oate (240 mg, 0.45 mmol) and methyl iodide (283 pL, 4.54 mmol) in THF (12 mL) at -20 °C under nitrogen was charged NaHMDS (1.0 M in THF, 4.08 mL, 4.08 mmol) drop-wise. The reaction mixture was allowed to warm to ambient temperature and stirred for 18 h. After this time the reaction was incomplete by1H NMR, NaH (170 mg, 4.24 mmol) followed by Mel (140 pL, 2.27 mmol) was charged and the mixture was stirred at ambient temperature for 1 .5 h. The reaction was quenched by the addition of 5% NaHCCh (20 mL) and extracted with ethyl acetate (2 x 20 mL). The combined extracts were concentrated in vacuo and the residue was purified by column chromatography (SiCh, ethyl acetate / heptane) to afford the title compound as a mixture of diastereomers (1 :1 , 90mg, 37%).

[0409] 1H NMR (400 MHz, CDCh) 5 4.67 (s, 2H), 3.67-3.59 (m, 1 H), 3.49-3.42 (m, 1 H), 3.42-3.34 (m, 1 H), 3.36 (s, 3H), 3.33 (s, 3H, diastereomer 1), 3.32 (s, 3H, diastereomer 2), 2.70 (td, J = 7.2, 2.7 Hz, diastereomer 1), 2.60 (dt, J = 9.2, 3.2 Hz, diastereomer 2), 2.25-1.66 (m, 12H), 1.60- 0.99 (m, 10H), 1.48 (s, 9H, diastereomer 1), 1.45 (s, 9H, diastereomer 2), 0.95 (s, 3H, diastereomer 1), 0.95 (s, 3H, diastereomer 2), 0.94 (d, J = 7.Q Hz, 3H, diastereomer 1), 0.95 (d, J = 7.0 Hz, 3H, diastereomer 2), 0.69 (s, 3H, diastereomer 1), 0.68 (s, 3H, diastereomer 2);19F {1H} NMR (376 MHz, CDCh) 6 -84.3 (d, J = 240.3 Hz, diastereomer 1), -84.3 (d, J = 240.1 , diastereomer 2), -100.9 (d, J = 240.1 Hz, diastereomer 2), -101.1 (d, J= 240.3 Hz diastereomer 1). ii. 3a-Hydroxy-7,7-difluoro-5p-24-nor-22-methoxymethyl-cholanic acid (Compound 21)

[0410] To a solution of f-butyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22-methoxymethyl-cholan- 23-oate from step i (90 mg, 0.17 mmol) in DCM (10 mL) at 0 °C was charged TFA (0.27 mL). The reaction was allowed to warm to ambient temperature and stirred for 17 h. The reaction mixture was concentrated in vacuo and co-evaporated with water. The residue was dissolved in MeOH and stirred with Na2CC (150 mg) for 3 h. The mixture was filtered and concentrated, acidified with 2 M HCI and then extracted into ethyl acetate and concentrated in vacuo to afford the title compound as a crude mixture. The crude residue was purified by preparative HPLC (C18, MeCN / H2O / 0.1% formic acid) afforded the title compound major diastereomer (10.8 mg) and minor diastereomer (9.3 mg).

[0411] Major diastereomer (Compound 21a): pKa4.5;1H NMR (400 MHz, CD3OD) 5 3.70 (t, J = 9.3 Hz, 1 H), 3.50 (dd, J = 9.3, 3.4 Hz, 1 H), 3.54-3.45 (m, 1 H), 3.34 (s, 3H), 2.74-2.66 (m, 1 H), 2.32- 2.14 (m, 1 H), 2.07-1.20 (m, 20H), 1.12 (td, J = 14.1 , 3.1 Hz, 1 H), 1.00 (s, 3H), 0.98 (d, J = 7.0 Hz, 3H), 0.76 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 6 -85.2 (d, J = 240.9 Hz), -102.5 (d, J = 240.9 Hz); MS (ESI’) m / z 441.2 [M-H]’.

[0412] Minor diastereomer (Compound 21b): pKa4.5;1H NMR (400 MHz, CD3OD) 53.67 (dd, J= 9.4, 8.0 Hz, 1H), 3.52-3.43 (m, 1H), 3.41 (dd, J = 9.4, 6.5 Hz, 1H), 3.32 (s, 3H), 2.80-2.74 (m, 1H), 2.30-2.01 (m, 3H), 1.89-1.15 (m, 18H), 1.10 (td, J = 13.9, 3.6 Hz, 1 H), 1.00-0.96 (m, 6H), 0.73 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 240.9 Hz), -102.5 (d, J = 240.9 Hz); MS (ES ) m / z 441.3 [M-H]’.

[0413] Example 22 - Synthesis of 3a-hvdroxv-7,7-difluoro-5P-24-nor-22-aminomethyl-cholanic acid (Compounds 22a and 22b)

[0414] Compound 22 was synthesised from f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22- hydroxymethyl-cholan-23-oate i.e. the product of Example 18, step iv, according to Scheme 28.

[0415] Scheme 28 i. t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-azidomethyl-cholan-23- oate t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-oate (200 mg, 0.38 mmol) was dissolved in DCM (10 mL) and cooled to 0 °C under nitrogen. Triethylamine (0.263 mL, 1.89 mmol) and methanesulfonyl chloride (0.044 mL, 0.57 mmol) were charged then the reaction was stirred at 0 °C for 5 minutes and then allowed to warm to ambient temperature and stirred for 18 h. The reaction was monitored by TLC, further triethylamine (0.130 mL) and methanesulfonyl chloride (0.022 mL) was added and stirred at room temperature for a further 18 h. Upon the completion indicated by TLC analysis, water (5 mL) was added and extracted with DCM (2 x 10 mL). The combined organic layer was washed with 5% NaHCOs solution (aq., 2 x 10 mL) and 10% NaCI (10 mL), dried over Na2SO4, filtered and concentrated to afford the mesylate intermediate. A portion of the residue (114 mg, 0.17 mmol) was dissolved in DMF (5 mL) and transferred into a flask containing NaNs (30 mg, 0.46 mmol) and the reaction mixture heated to 50 °C for 4 days. After this time the reaction was cooled to ambient temperature, diluted with ethyl acetate and washed with 5% NaHCOs. The aqueous phase was extracted with ethyl acetate and the combined extracts were washed with 10% NaCI and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the title compound as a 1 :1 mix of diastereomers contaminated with the eliminated product f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor- 22-methylene-cholan-23-oate (44 mg total mass). This was continued on without any further purification. ii. t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-aminomethyl-cholan-23- oate t-Butyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22-azidomethyl-cholan-23-oate from step i (44 mg, 0.08 mmol) was dissolved in THF (1 mL) and PPhs (62.5 mg, 0.24 mmol) and H2O (12.8 pL, 0.71 mmol) were charged. The reaction mixture heated to reflux for 18 h then concentrated in vacuo. The residue was purified by column chromatography (SiC>2, DCM / MeOH with 0.1 % NEts) to afford the title compound as a 6:4 mix of diastereomers (22 mg). This was continued on without any further purification. iii. 3a-Hydroxy-7,7-difluoro-5p-24-nor-22-aminomethyl-cholanic acid (Compound 22)

[0416] To a solution of f-butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-aminomethyl-cholan- 23-oate from step ii (22 mg, 0.04 mmol) in DCM (3 mL) was charged TFA (0.1 mL). The reaction mixture was stirred at ambient temperature for 20 h then concentrated in vacuo and co-evaporated with water. The residue was dissolved in MeOH and stirred with Na2COs (100 mg) for 2 h, filtered and concentrated in vacuo. The crude residue was purified by preparative HPLC (C18, MeCN / FW, 0.1 % formic acid) to afford the f-butyl ester intermediates major diastereomer (3.9 mg), minor diastereomer (2.6 mg). The f-butyl esters were reacted on separately, dissolve in 4 M HCI in dioxane (0.1 mL) and stirred at ambient temperature for 2 days until complete by TLC. After this time they were concentrated in vacuo to afford the major diastereomer (3.5 mg) and minor diastereomer (2.3 mg).

[0417] Major diastereomer (Compound 22a): pKa3.8 (acidic), 8.7 (basic);1H NMR (400 MHz, CD3OD) 5 3.52-3.43 (m, 1 H), 3.35-3.28 (m, 1 H), 2.93 (dd, J = 12.8, 4.3 Hz, 1 H), 2.84-2.78 (m, 1 H), 2.32-1.99 (m, 3H), 1.92-1.16 (m, 18H), 1.11 (td, J = 14.1 , 2.9 Hz, 1 H), 1.01 (d, J = 6.8 Hz, 3H), 0.98 (s, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) -85.2 (d, J = 240.9 Hz), -102.5 (d, J = 240.9 Hz); MS (ESI+) m / z 428.2 ([M+H]+.

[0418] Minor diastereomer (Compound 22b): pKa3.8 (acidic), 8.7 (basic);1H NMR (400 MHz, CD3OD) 5 3.52-3.44 (m, 1 H), 3.18 (t, J = 12.3 Hz, 1 H), 3.00 (dd, J = 12.8, 2.4 Hz, 1 H), 2.82-2.74 (m, 1 H), 2.33-2.13 (m, 2H), 2.06-1.19 (m, 19H), 1.11 (td, J = 14.0, 3.2 Hz, 1 H), 0.99 (s, 3H), 0.93 (d, J = 6.9 Hz, 3H), 0.77 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) -85.2 (d, J = 240.7 Hz), - 102.5 (d, J = 240.7 Hz); MS (ESI+) m / z 428.2 ([M+H]+.

[0419] Example 23 - Synthesis of 3a-hvdroxy-7,7-difluoro-5P-24-nor-22-hvdroxymethyl- cholan-23-ol (Compound 23)

[0420] Compound 23 was synthesised from t-butyl 3a-methoxymethoxy-7,7-difluoro-5 -24-nor-22- hydroxymethyl-cholan-23-oate i.e. the product of Example 18, step iv, according to Scheme 29. i. 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-ol t-Butyl 3a-methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-oate (350 mg, 0.66 mmol) was dissolved in anhydrous THF (5 mL), then MeOH (0.16 mL) was added and the resulting solution cooled to 5 °C under nitrogen UBH4 (87 mg, 3.97 mmol) was charged portion-wise then stirred at ambient temperature for 18 h. The reaction was then quenched by the drop-wise addition of 5% NH4CI (10 mL), extracted with EtOAc (2 x 10 mL) and the combined organic phase was washed with 5% NH4CI (10 mL), brine (10 mL), dried over Na2SC>4 and concentrated in vacuo. The resulting residue was purified by column chromatography (SiC>2, ethyl acetate / heptane), to afford the title compound (160 mg, 53%) which was continued on without any further purification. ii. 3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-ol (Compound 23)

[0421] The product of this step is the starting material for Compound 24 of Example 24.

[0422] 3a-Methoxymethoxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-ol from step i (160 mg, 0.35 mmol) was dissolved in MeOH (5 mL), 2 M HCI (0.8 mL, 5 vol) was charged and the reaction mixture was heated to 45 °C for 18 h. After cooling to ambient, the mixture was diluted with ethyl acetate (10 mL) and quenched with 5% NaHCOs (10 mL). The phases were separated and the organic was concentrated in vacuo and purified by column chromatography (SiO2, ethyl acetate / heptane) to afford the title compound as an off white solid (11 mg, 8%). pKa 10.1 ;1H NMR (400 MHz, CD3OD) 6 3.75 (dd, J = 10.7, 5.6 Hz, 1 H), 3.69 (dd, J = 11.0, 3.9 Hz, 1 H), 3.52-3.43 (m, 2H), 3.37-3.32 (m, 1 H), 2.29-2.12 (m, 1 H), 2.04 (dt, J = 12.3, 3.2 Hz, 1 H), 1.99-1.06 (m, 21 H), 0.98 (s, 3H), 0.86 (d, J = 7.0 Hz, 3H), 0.73 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) -85.1 (d, J = 241.2 Hz), -102.5 (d, J = 241.2 Hz); MS (ESI’) m / z 395.2 ([M- H2O-H]-.

[0423] Example 24 - Synthesis of 3-keto-7,7-difluoro-5P-24-nor-cholan-23,23-dicarboxylic acid (Compound 24)

[0424] Compound 24 was synthesised from 3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl- cholan-23-ol i.e. the product of Example 23, step ii, according to Scheme 30. i. 3-keto-7,7-difluoro-5p-24-nor-cholan-23,23-dicarboxylic acid (Compound 24)

[0425] To a suspension of 3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-ol (50 mg, 0.12 mmol) in acetone (10 mL) was charged Jones reagent (2 M in H2SO4, 140 pL, 0.28 mmol). The reaction was stirred at ambient temperature for 5 h, then quenched with I PA (10 mL). The solution was diluted with ethyl acetate (5 mL) and washed with 10 % NaCI (9 mL) dried over MgSCU and concentrated in vacuo. The residue was passed through a silica plug (SiC>2, acetone / chloroform), then purified by preparative HPLC (C18, MeCN / H2O / 0.1% formic acid) afforded the title compound (4 mg, 8%). pKa3.1 ;1H NMR (400 MHz, acetone-d6) 5 3.53 (d, J = 3.4 Hz, 1 H), 2.72 (t, J = 14.3 Hz, 1 H), 2.51-2.42 (m, 1 H), 2.34-2.18 (m, 2H), 2.11-1.75 (m, 9H), 1.67-1.29 (m, 9H), 1.19 (d, J = 6.8 Hz, 3H), 1.12 (s, 3H), 0.81 (s, 3H);19F {1H} NMR (376 MHz, acetone-d6) 5 -84.2 (d, = 242.5 Hz), -101 .7 (d, J = 242.5 Hz); MS (ESI’) m / z 439.2 [M-H]’. is of 3-keto-7,7-difluoro-; -24-nor-cholanic acid

[0426] Compound 25 was synthesised from methyl 3a-hydroxy-7,7-difluoro-5 -24-nor-cholan-23- oate i.e. the product of Example 2, step i, according to Scheme 31. i. Methyl 3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate

[0427] The product of this step is the starting material for Compound 26 of Example 26, Compounds 27, 28, 29 and Compound 30 of Example 27 and Compound 31 of Example 28 and Compound 32 of Example 29.

[0428] To a solution of starting material methyl 3a-hydroxy-7,7-difluoro-5 -24-nor-cholan-23-oate (1.4 g, 3.39 mmol) in DCM (20 mL) was charged DMP (2.16 g, 5.09 mmol) portion-wise maintaining the temperature at < 30 °C. The reaction mixture was stirred at ambient temperature for 18 h then filtered and washed with DCM. The organic was washed with Na2S20s (100 mL), 5% NaHCOs (100 mL), 10% NaCI (100 mL), dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, ethyl acetate / toluene) to afford the title compound (1.35 g, 97%).

[0429] 1H NMR (400 MHz, CDCh) 6 3.66 (s, 3H), 2.72 (t, J = 14.8 Hz, 1 H), 2.44 (dd, J = 14.4, 3.1 Hz, 1 H), 2.35-1.75 (m, 14H), 1.58-1.14 (m, 8H), 1.06 (s, 3H), 0.99 (d, J = 6.3 Hz, 3H), 0.74 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -84.4 (d, J = 244.4 Hz), -101.4 (d, J = 244.4 Hz)). ii. 3-keto-7,7-difluoro-5p-24-nor-cholanic acid (Compound 25) Methyl 3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate from step i (50 mg, 0.12 mmol) was dissolved in I PA (2 mL). 1 M NaOH (0.24 mL, 0.24 mmol) was charged and the reaction mixture was heated to 45 °C for 18 h. Upon the completion indicated by TLC analysis, the solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 1 M HCI and extracted with EtOAc (x3). The combined extracts were washed with 10% NaCI (10 mL) dried over Na2SC>4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, acetone / toluene) to afford the title compound (44 mg, 92%). pKa4.0;1H NMR (400 MHz, CDCh) 62.70 (t, J = 14.7 Hz, 1 H), 2.47 (dd, J = 15.1 , 3.2 Hz, 1 H), 2.33-1.15 (m, 22H), 1.04-1.02 (m, 6H), 0.73 (s, 3H;19F {1H} NMR (376 MHz, CDCh) 6 -84.4 (d, J = 244.0 Hz), -101.4 (d, J = 244.0 Hz) MS (ESI’) m / z 395.2 [M-H]’.

[0430] Example 26 - Synthesis of 3P-hydroxy-7,7-difluoro-5P-24-nor-cholanic acid (Compound 26)

[0431] Compound 26 was synthesised from methyl 3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate i.e. the product of Example 25, step i, according to Scheme 32.

[0432] Scheme 32 i. Methyl 3p-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate

[0433] To a solution of methyl 3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate (100 mg, 0.24 mmol) in anhydrous THF (2 mL) at -78 °C, under nitrogen was charged K-selectride® (1 M in THF, 0.48 mL, 0.48 mmol) dropwise. The reaction mixture was allowed to warm to ambient temperature and stirred for 1.5 h. The reaction was quenched by the addition of 2 M HCI (1 mL) dropwise, stirred for 1 h and then ethyl acetate was charged. Additional HCI was charged to aid solubility, then the phases were separated and the aqueous phase was re-extracted with ethyl acetate. The combined extracts were washed with 5% NaHCCh, then 10% NaCI, dried over MgSCU and concentrated in vacuo. The crude residue was purified by column chromatography (SiO2, MeCN / toluene) to afford the title compound as a white solid (46 mg, 46%).1H NMR (400 MHz, CDCI3) 6 4.09-4.06 (m, 1 H), 3.65 (s, 3H), 2.43 (dd, J = 14.3, 3.1 Hz, 1 H), 2.27-2.09 (m, 1 H), 2.05-1.08 (m, 22H), 0.98 (d, J = 6.3 Hz, 3H), 0.98 (s, 3H), 0.70 (s, 3H);19F {1H} NMR (376 MHz, CDCI3) 6 -83.8 (d, J = 239.9 Hz), -101.1 (d, J = 239.9 Hz). ii. 3p-Hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 26)

[0434] To a solution of methyl 3p-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate from step i (46 mg, 0.11 mmol) in IPA (2 mL) and MeOH (0.5 mL) was charged 1 M NaOH (1 mL, 1.0 mmol). The reaction mixture was stirred at ambient temperature for 18 h. Upon the completion indicated by TLC analysis, the solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 1 M HCI and extracted with EtOAc (x3). The combined extracts were washed with 10% NaCI (10 mL) dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, acetone / DCM) to afford the title compound (22 mg, 50%). pKa4.0;1H NMR (400 MHz, CD3OD) 5 4.01-3.98 (m, 1 H), 2.46-2.40 (m, 1 H), 2.33-2.13 (m, 1 H), 2.09-1.14 (m, 22H), 1.06 (d, J = 6.2 Hz, 3H), 1.01 (s, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -84.6 (d, J = 240.6 Hz), -102.3 (d, J = 240.6 Hz).

[0435] Example 27 - Synthesis of 2P-fluoro-3a-hydroxy-7,7-difluoro-5P-24-nor-cholanic acid (Compound 27), 2P-fluoro-3P-hydroxy-7,7-difluoro-5P-24-nor-cholanic acid (Compound 28), 4P-fluoro-3a-hydroxy-7,7-difluoro-5P-24-nor-cholanic acid (Compound 29) and 4P- fluoro-3P-hydroxy-7,7-difluoro-5P-24-nor-cholanic acid (Compound 30)

[0436] Compounds 27, 28, 29 and 20 were synthesised from methyl 3-keto-7,7-difluoro-5p-24-nor- cholan-23-oate, i.e. the product of Example 25, step i according to Scheme 33.

[0437] i. Methyl 2p-fluoro-3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate and methyl 4p- fluoro-3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate

[0438] Methyl 3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate (410 mg, 1.0 mmol) was dissolved in anhydrous DCM (6 mL) under nitrogen and cooled to 0 °C. Triethylamine (0.28 mL, 2.0 mmol) was charged followed by TMSOTf (0.2 mL, 1.1 mmol) maintaining the temperature ca. 0 °C. The reaction mixture was stirred for 1 h at 0 °C then gradually warmed to ambient temperature. After 2 h at ambient the reaction was monitored by TLC analysis, further triethylamine (0.1 mL) was charged followed by TMSOTf (0.07 mL) and stirred for 2 h until complete by TLC. The reaction was diluted with DCM (5 mL) and quenched by the addition of 5% NaHCOs (aq) (15 mL) extracted with DCM (3 * 10 mL) and the combined organic phase was washed with 10% NaCI (10 mL), dried over Na2SO4 and concentrated in vacuo to afford the crude intermediate which was continued through without any purification. The intermediate was dissolved in anhydrous MeCN (7 mL) under nitrogen, transferred to a flask containing Selectfluor (709 mg, 2.0 mmol) and stirred for 18 h at ambient. The reaction was diluted with ethyl acetate, quenched by the addition of 5% NaHCOs (aq) (30 mL) and stirred for 1 h. The phases were separated and the aqueous back extracted with ethyl acetate. The combined extracts were washed with 10% NaCI (10 mL) dried over Na2SC>4 and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the 2-fluoro compound (85 mg, 20%) and 4-fluoro compound (260 mg, 61 %).

[0439] 2-Fluoro:1H NMR (400 MHz, CDCh) 64.95 (ddd, J= 48.7, 13.5, 6.0 Hz, 1 H), 3.67 (s, 3H), 2.83 (t, J = 14.3 Hz, 1 H), 2.54-1.56 (m, 15H), 1.48-1.15 (m, 6H), 1.12 (s, 3H), 1.01 (d, J = 7.1 Hz, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 5 -85.0 (d, J = 246.1 Hz), -101.4 (d, J = 246.1 Hz), -195.1 (s).

[0440] 4-Fluoro:1H NMR (400 MHz, CDCh) 6 5.13 (dd, = 47.2, 11.7 Hz, 1 H), 3.67 (s, 3H), 2.47-1.55 (m, 15H), 1.53-1.15 (m, 7H), 1.11 (s, 3H), 1.00 (d, J = 7.1 Hz, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 5 -85.5 (d, J = 248.2 Hz), -103.0 (d, J = 248.2 Hz), -200.5 (s). ii. a. Methyl 2p-fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate and methyl 2p-fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate

[0441] Methyl 2p-fluoro-3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate from step i (85 mg, 0.19 mmol) was dissolved in anhydrous THF (6 mL) under nitrogen and cooled to -20 °C. NaBH4 (7.3 mg, 0.19 mmol) was charged and stirred at -20 °C for 18 h. The reaction was warmed to ambient temperature, diluted with ethyl acetate and quenched by the addition of 2M HCI (aq) (10 mL). The phases were separated and the aqueous back extracted with ethyl acetate (10 mL). The combined extracts were washed with 5% NaHCCh (10 mL), 10% NaCI (10 mL), dried over Na2SC>4 and concentrated in vacuo to afford the crude material as a mixture of isomers (~7:3 ratio; 3cc3 ). The mixture was combined with other crude batches for purification by column chromatography (SiCh, acetone / heptane) to afford the 3a-hydroxy compound (50 mg, 29%) and 3p-hydroxy compound (32 mg, 19%).

[0442] 3a-hydroxy-2 -fluoro:1H NMR (400 MHz, CDCh) 5 4.46-4.24 (m, 1 H), 3.67 (s, 3H), 3.70-3.59 (m, 1 H), 2.44 (dd, J = 14.3, 3.4 Hz, 1 H), 2.26-1.14 (m, 21 H), 1.03 (s, 3H), 0.99 (d, J = 6.2 Hz, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 5 -85.1 (d, J = 241.7 Hz), -101.3 (d, J =

[0443] 241.7 Hz), -187.7 (s).

[0444] 3 -hydroxy-2 -fluoro:1H NMR (400 MHz, CDCh) 5 4.70-4.41 (m, 1 H), 4.20-4.14 (m, 1 H), 3.66 (s, 3H), 2.43 (dd, J = 14.6, 3.3 Hz, 1 H), 2.23-1.11 (m, 21 H), 1.05 (s, 3H), 0.99 (d, J = 6.3 Hz, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 5 -84.7 (d, J = 241.8 Hz), -101.6 (d, J =

[0445] 241.8 Hz), -187.9 (s). ii. b. Methyl 4p-fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate and methyl

[0446] 4p-fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate

[0447] Methyl 4p-fluoro-3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate from step i (260 mg, 0.59 mmol) was dissolved in anhydrous THF (18 mL) under nitrogen and cooled to -20 °C. NaBH4 (22 mg, 0.59 mmol) was charged and stirred at -20 °C for 18 h. The reaction was warmed to ambient temperature, diluted with ethyl acetate and quenched by the addition of 2M HCI (aq) (20 mL). The phases were separated and the aqueous back extracted with ethyl acetate (20 mL). The combined extracts were washed with 5% NaHCCh (20 mL), 10% NaCI (20 mL), dried over Na2SC>4 and concentrated in vacuo to afford the crude material as a mixture of isomers (~7:3 ratio; 3cc3p). The crude mixture was purified by column chromatography (SiC>2, ethyl acetate / heptane) to afford the 3a-hydroxy compound (120 mg, 47%) and 3p-hydroxy compound (68 mg, 27%).

[0448] 3a-hydroxy-4p-fluoro:1H NMR (400 MHz, CDCh) 64.57 (ddd, J = 50.8, 19.9, 8.2 Hz, 1 H), 3.67 (s, 3H), 3.68-3.57 (m, 1 H), 2.47-2.32 (m, 3H), 2.06-1.10 (m, 19H), 1.02 (s, 3H), 0.99 (d, J = 6.2 Hz, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -85.5 (d, J = 244.0 Hz), -102.9 (d, J = 244.0 Hz), -193.7 (s).

[0449] 3 -hydroxy-4 -fluoro:1H NMR (400 MHz, CDCh) 64.73 (ddd, J= 46.3, 11.4, 3.6 Hz, 1 H), 4.22- 4.16 (m, 1 H), 3.66 (s, 3H), 2.44 (dd, J = 14.4, 3.1 , 1 H), 2.36-1.08 (m, 21 H), 1.04 (s, 3H), 0.98 (d, J = 6.1 Hz, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -85.2 (d, J = 244.2 Hz), - 103.1 (d, J = 244.2 Hz), -194.2 (s). iii. a. 2p-Fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholanoic acid (Compound 27)

[0450] Methyl 2p-fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate from step ii. a (50 mg, 0.12 mmol) was dissolved in MeOH (5 mL). 2 M LiOH (1 mL) was charged and the reaction mixture was stirred at 40 °C for 3 days. Upon the completion indicated by TLC analysis, the solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 1 M HCI and extracted with EtOAc (x3). The combined extracts were washed with 10% NaCI (10 mL) dried over MgSC and concentrated in vacuo to afford the title compound (30 mg, 62%). pKa4.0;1H NMR (400 MHz, CD3OD) 4.37-4.18 (m, 1 H), 3.60-3.48 (m, 1 H), 2.43 (d, J = 12.1 Hz, 1 H), 2.27-1.19 (m, 21 H), 1.05 (s, 3H), 1.03 (d, J = 6.4 Hz, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 6 -81.7 (d, J = 242.3 Hz), -98.3 (d, J = 242.3 Hz), -183.1 (s); MS (ESI’) m / z 415.2 [M-H]-. iii. b. 2p-Fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholanoic acid (Compound 28)

[0451] Methyl 2p-fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate from step ii. a (32 mg, 0.07 mmol) was dissolved in MeOH (5 mL). 2 M LiOH (1 mL) was charged and the reaction mixture was stirred at 40 °C for 3 days. Upon the completion indicated by TLC analysis, the solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 1 M HCI and extracted with EtOAc (x3). The combined extracts were washed with 10% NaCI (10 mL) dried over MgSO4 and concentrated in vacuo to afford the title compound (13 mg, 42%). pKa4.0;1H NMR (400 MHz, CDCI3) 4.54 (dddd, J = 47.3, 12.5, 4.8, 3.0, 1 H), 4.21-4.15 (m, 1 H), 2.49 (dd, J = 14.8, 3.2 Hz, 1 H), 2.25-1.15 (m, 21 H), 1.06-1.04 (m, 6H), 0.72 (s, 3H);19F {1H} NMR (376 MHz, CDCI3) 6 -84.7 (d, = 242.0 Hz), -101.6 (d, = 242.0 Hz), -187.9 (s); MS (ESL) m / z 415.2 [M-H]’. iii. c. 4p-Fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholanoic acid (Compound 29)

[0452] Methyl 4 -fluoro-3a-hydroxy-7,7-difluoro-5 -24-nor-cholan-23-oate from step ii. b (120 mg, 0.28 mmol) was dissolved in MeOH (10 mL). 2 M LiOH (2 mL) was charged and the reaction mixture was stirred at 40 °C for 3 days. Upon the completion indicated by TLC analysis, the solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 1 M HCI and extracted with EtOAc (x3). The combined extracts were washed with 10% NaCI (10 mL) dried over MgSO4 and concentrated in vacuo to afford the crude compound. The residue was purified by column chromatography (SiCh, ethyl acetate / toluene + 0.1 % acetic acid) to afford the title compound (37 mg, 32%). pKa4.0;1H NMR (400 MHz, CDCh) 4.57 (ddd, J = 50.5, 19.8, 9.7 Hz, 1 H), 3.67-3.56 (m, 1 H), 2.49 (dd, J = 14.9, 2.9 Hz, 1 H), 2.10-1.14 (m, 21 H), 1.04 (d, J = Q.2 Hz, 3H), 1.02 (s, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -85.5 (d, J = 245.0 Hz), -102.9 (d, J = 245.0 Hz), - 193.7 (s); MS (ESI’) m / z 415.2 [M-H]’. iii. d. 4p-Fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholanoic acid (Compound 30)

[0453] Methyl 4p-fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate from step ii. b (68 mg, 0.16 mmol) was dissolved in MeOH (5 mL). 2 M LiOH (1 mL) was charged and the reaction mixture was stirred at 40 °C for 3 days. Upon the completion indicated by TLC analysis, the solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 1 M HCI and extracted with EtOAc (x3). The combined extracts were washed with 10% NaCI (10 mL) dried over MgSCU and concentrated in vacuo to afford the crude compound (64 mg, quant.). The residue (30 mg) was purified by preparative HPLC (C18, MeCN / H2O / 0.1% FA) to afford the title compound (13 mg). pKa4.0;1H NMR (400 MHz, CDCh) 4.73 (ddd, = 45.7, 11.7, 2.9 Hz, 1 H), 4.22-4.15 (m, 1 H), 2.44-2.27 (m, 2H), 2.11-1.12 (m, 20H), 1.04 (s, 3H), 1.02 (d, J = 5.2 Hz, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -85.2 (d, = 244.1 Hz), -103.1 (d, = 244.1 Hz), -194.1 (s); MS (ESP) m / z 415.2 [M-H]’.

[0454] Example 28 - Synthesis of 4a-chloro-3a-hydroxy-7,7-difluoro-58-24-nor-cholanic acid (Compound 31)

[0455] Compound 31 was synthesised from methyl 3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate, i.e. the product of Example 25, step i using the method outlined in Scheme 33 but using N- chlorosuccinimide as opposed to the fluorinating reagent Selectfluor. Methyl 4-chloro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate (82 mg, 0.18 mmol) was dissolved in MeOH (0.5 mL). 2 M LiOH (91 pL, 1.03 mmol) was charged and the reaction mixture was stirred at 40 °C until complete by TLC analysis. Upon completion the solution was concentrated in vacuo. The crude residue was diluted with water, acidified with 2 M HCI and extracted with EtOAc (x3). The combined extracts were washed with 5% NaHCCU (5 mL) dried over MgSCU and concentrated in vacuo to afford the crude material. The crude mixture was purified by column chromatography (SiC>2, ethyl acetate / heptane / 0.1 % AcOH) to afford the title compound (5.0 mg, 6%). pKa4.0;1H NMR (400 MHz, CD3OD) 3.9 (dd, J = 11.7, 9.6 Hz, 1 H), 3.4 (td, J = 9.6, 5.0 Hz, 1 H), 2.5 (dd, J = 13.5, 3.3 Hz, 1 H), 2.33 (dd, J = 11.4, 2.1 Hz, 1 H), 2.08 - 1.00 (m, 20H), 0.96 (s, 3H), 0.92 (d, J = 6.24 Hz, 3H), 0.64 (s, 3H).19F {1H} NMR (376 MHz, CD3OD) 5 -86.4 (d, J = 244.8 Hz), -106.3 (d, J = 244.8 Hz); MS (ESI’) m / z 431 .2 [M-H]’.

[0456] Example 29 - Synthesis of r2Hl-3a-hvdroxy-7,7-difluoro-5 -24-nor-cholan-23-oic acid (Compound 32)

[0457] Compound 32 was synthesised from methyl 3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate i.e. the product of Example 25, step i, according to Scheme 34.

[0458] Scheme 34 i. Methyl 3-[2H]-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate

[0459] To a solution of methyl 3-keto-7,7-difluoro-5p-24-nor-cholan-23-oate (100 mg, 0.24 mmol) in anhydrous THF (2 mL) and MeOD (2 mL) at 0 °C, under nitrogen was charged NaBD4 (0.48 mmol, 2 eq). The reaction mixture was allowed to warm to ambient temperature and stirred for 1 h. The reaction was quenched by the addition of 2 M HCI (2 mL) dropwise, stirred for 1 h and then ethyl acetate was charged. The phases were separated and the aqueous phase was re-extracted with ethyl acetate. The combined extracts were washed with 5% NaHCOs, then 10% NaCI, dried over MgSCU and concentrated in vacuo. The crude residue was purified by column chromatography (SiO2, MeCN / toluene) to afford the title compound as a white solid (74 mg, 73%).

[0460] 1H NMR (400 MHz, CDCI3) 6 3.59 (s, 3H), 2.37 (dd, J = 14.4, 3.1 Hz, 1 H), 2.16-2.00 (m, 1 H), 1.93-0.96 (m, 22H), 0.92 (d, J = 6.3 Hz, 3H), 0.88 (s, 3H), 0.64 (s, 3H);19F {1H} NMR (376 MHz, CDCI3) 6 -85.14 (d, J = 240.9 Hz), -102.4 (d, J = 240.9 Hz). ii. 3-[2H]-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oic acid (Compound 32)

[0461] To a solution of Methyl 3-[2H]-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate from step i (74 mg, 0.18 mmol) in IPA (2 mL) was charged 1 M NaOH (1 mL). The reaction mixture was stirred at ambient temperature for 4 d. Upon the completion indicated by TLC analysis, the IPA was removed in vacuo. The resulting slurry was acidified with 2 M HCI and extracted with EtOAc. The combined extracts were washed with 10% NaCI (2 x 10 mL) dried over MgSCU and concentrated in vacuo. The residue was purified by column chromatography (SiC>2, acetone / DCM) to afford the title compound (19 mg, 27%). pKa4.0;1H NMR (400 MHz, CD3OD) 5 4.01-3.98 (m, 1 H), 2.46-2.40 (m, 1 H), 2.33-2.13 (m, 1 H), 2.09-1.14 (m, 22H), 1.06 (d, J = 6.2 Hz, 3H), 1.01 (s, 3H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -84.6 (d, J = 240.6 Hz), -102.3 (d, J = 240.6 Hz).

[0462] General Procedure for the formation of 7,7-difluoro 24-norlithocholic acid conjugates

[0463] Compound 1 (1 equiv.) was dissolved in THF (25 vol) and cooled to 0 °C. Ethyl chloroformate (1.2 eq) was added followed by triethylamine (1.2 eq) and the reaction stirred for 1 h at 0 °C. After complete conversion of the starting material by TLC a solution of amine (1.5 eq) and NaHCOs (1 .5 eq) in H2O (25 vol) is added in one potion then stirred 0 °C for 2 h or until complete by TLC. Upon completion the mixture is concentrated under reduced pressure, diluted with H2O and acidified to pH<2 with 2M HCI (aq.). The suspension was extracted (ethyl acetate or THF; x3); the organic layers were combined and washed with 10% NaCI, dried over magnesium sulphate and concentrated to afford a crude residue. The crude material was purified via column chromatography. Example 30 - Synthesis of Af-(3a-hvdroxy-7,7-difluoro-5g-24-nor-cholan-23-oyl)- (morpholine) (Compound 33)

[0464] Using the general procedure 7,7-difluoro 24-norlithocholic acid (60 mg, 0.12 mmol) was conjugated and purified by column chromatography (SiC>2, 7:2:1 (ethyl acetate: MeOH: H2O) in ethyl acetate) to yield the title compound (16.2 mg, 29%). pKa14.3;1H NMR (400 MHz, CDCh) 6 3.67-3.59 (m, 6H), 3.58-3.51 (m, 1 H), 3.49-3.45 (m, 2H), 2.39-2.34 (m, 1 H), 2.16-1.02 (m, 23H), 0.98 (d, J = 5.9 Hz, 3H), 0.94 (s, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 6 -84.4 (d, J = 240.5 Hz), -101.2 (d, J = 240.5 Hz).

[0465] Example 31 - Synthesis of / V-(3a-hydroxy-7,7-difluoro-5B-24-nor-cholan-23-oyl)- (methylamine) (Compound 34)

[0466] Using the general procedure 7,7-difluoro 24-norlithocholic acid (60 mg, 0.12 mmol) was conjugated and purified by column chromatography (SiC>2, 7:2:1 (ethyl acetate: MeOH: H2O) in ethyl acetate) to yield the title compound (28.3 mg, 57%). pKa12.5;1H NMR (400 MHz, CDCh) 6 5.42 (d, J = 3.6 Hz, 1 H), 3.58-3.50 (m, 1 H), 2.80 (d, J = 4.9 Hz, 3H), 2.40-2.35 (m, 1 H), 2.23-1.03 (m, 23H), 0.97 (d, J = 6.5 Hz, 3H), 0.95 (s, 3H), 0.71 (s, 3H);19F {1H} NMR (376 MHz, CDCh) 5 -84.4 (d, J = 240.1 Hz), -101.2 (d, J = 240.1 Hz).

[0467] Comparative Example 32 - Preparation of Comparative Compounds B to G

[0468] The General Procedure for the formation of conjugates was used for the synthesis of the following compounds. A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-amide)-ethylsulfonic acid (Comparative Compound B)

[0469] Using the general procedure 7,7-difluoro 24-norlithocholic acid (35 mg, 0.09 mmol) was conjugated to yield Comparative Compound B as a white powder (15.6 mg, 34%).

[0470] 1H NMR (400 MHz, DMSO-cfe) 6 7.66 (t, J = 5.5 Hz, 1 H), 6.82 (br. s, 1 H), 3.28-3.18 (m, 2H), 3.10 (qd, J = 7.3, 4.8 Hz, 1H), 2.58-2.52 (m, 2H), 2.20-2.06 (m, 1 H), 1.95 (d, J = 12.3 Hz, 1H), 1.87-1.20 (m, 15H), 1.20-0.96 (m, 7H), 0.91 (s, 3H), 0.87 (d, J = 6.4 Hz, 3H), 0.66 (s, 3H);19F {1H} NMR (376 MHz, DMSO-cfe) 6 -82.6 (d, J = 237.6 Hz), -99.9 (d, J = 237.6 Hz).

[0471] A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-amide)-propylsulfonic acid (Comparative Compound C)

[0472] Using the general procedure 7,7-difluoro 24-norlithocholic acid (35 mg, 0.09 mmol) was conjugated to yield Comparative Compound C as a white powder (29.1 mg, 62%).

[0473] 1H NMR (400 MHz, CD3OD) 5 3.53-3.42 (m, 1 H), 3.41-3.33 (m, 2H), 3.24 (t, J = 6.7 Hz, 1H), 2.94-2.80 (m, 3H), 2.50-2.37 (m, 1 H), 2.29-2.11 (m, 1 H), 2.11-1.06 (m, 22H), 1.01-0.96 (m, 6H), 0.76 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.1 (d, J = 241.0 Hz), -102.4 (d, J = 241.0 Hz).

[0474] A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-amide)-((S)-hexanoic acid (Comparative Compound D)

[0475] Using the general procedure 7,7-difluoro 24-norlithocholic acid (35 mg, 0.09 mmol) was conjugated to yield Comparative Compound D as a white powder (19.3 mg, 42%).1H NMR (400 MHz, CD3OD) 5 4.38 (dd, J = 9.0, 4.9 Hz, 1 H), 4.14-4.05 (m, 1 H), 3.55-3.42 (m, 1 H), 2.43-2.21 (m, 1 H), 2.31-2.11 (m, 1 H), 2.10-2.03 (m, 1 H), 2.04-1.49 (m, 10H), 1.47-1.09 (m, 15H), 1.05 (d, J = 5.6 Hz, 3H), 1.02 (s, 3H), 1.00-0.93 (m, 4H), 0.79 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (dd, J = 240.6, 12.6 Hz), -102.4 (dd, J = 240.6, 15.1 Hz).

[0476] A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oyl)-((1,1)-dioxidothiomorpholine) (Comparative Compound E)

[0477] Using the general procedure 7,7-difluoro 24-norlithocholic acid (35 mg, 0.09 mmol) was conjugated to yield Comparative Compound E as a white powder (7.4 mg, 16%).

[0478] 1H NMR (400 MHz, CD3OD) 5 4.09-3.98 (m, 4H), 3.53-3.43 (m, 1 H), 3.18 (br. d, J = 21.0 Hz, 4H), 2.58 (dd, J = 15.2, 3.2 Hz, 1 H), 2.38-1.51 (m, 15H), 1.51-1.09 (m, 8H), 1.02-0.97 (m, 6H), 0.81 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 241.0 Hz), -102.4 (d, J = 240.9 Hz).

[0479] A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-amide)-((S)-2-cyclopropylacetic acid) (Comparative Compound F)

[0480] Using the general procedure 7,7-difluoro 24-norlithocholic acid (35 mg, 0.09 mmol) was conjugated to yield Comparative Compound F as a white powder (4.5 mg, 10%).

[0481] 1H NMR (400 MHz, CD3OD) 5 3.71-3.66 (m, 1 H), 3.52-3.42 (m, 1 H), 2.38-2.05 (m, 3H), 1.93- 1.24 (m, 15H), 1.16-1.02 (m, 3H), 0.97 (d, J = 5.8 Hz, 3H), 0.95 (s, 3H), 0.90-0.74 (m, 4H), 0.71 (s, 3H), 0.66-0.39 (m, 3H), 0.38-0.24 (m, 1 H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (dd, J = 241.0, 7.0 Hz), -102.4 (dd, J = 241.0, 7.0 Hz). N-(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oyl)-(2-aminoethyl hydrogen sulfate) (Comparative Compound G)

[0482] Using the general procedure 7,7-difluoro 24-norlithocholic acid (35 mg, 0.09 mmol) was conjugated to yield Comparative Compound G as a white powder (18.2 mg, 39%).

[0483] 1H NMR (400 MHz, CD3OD) 5 3.51-3.43 (m, 1 H), 3.47 (t, J = 5.5 Hz, 2H), 3.38 (t, J = 5.6 Hz, 2H), 2.38 (dd, J = 13.2, 3.2 Hz, 1 H), 2.31-2.09 (m, 1 H), 2.09-2.03 (m, 1 H), 1.97-1.47 (m, 9H), 1.44-1.02 (m, 12H), 1.02-0.93 (m, 6H), 0.75 (s, 3H);19F {1H} NMR (376 MHz, CD3OD) 5 -85.2 (d, J = 241.0 Hz), -102.4 (d, J = 241.0 Hz).

[0484] Biological Examples

[0485] Biological Example 1 - Biological Activity of Compounds in Alzheimer’s Disease Cells

[0486] Cell Methods

[0487] Cell lines used were obtained from the Coriell cell repository (4 individual PSEN1 mutant patient lines, PSEN1A, PSEN1 B and PSEN1 D and PSEN1 E), and all show reductions in mitochondrial membrane potential (MMP; 25% reduction), increased percentage of long mitochondria (5% change) and a more fused mitochondrial network. Characterisation of these lines is described in Bell et al, 2018. Details of the mutations are provided below.

[0488] When confluent, cells were plated into 1536 well plates, at a density of 200 cells per 5pl of media per well. Cells were drugged 24 hours after plating, using the Echo® 550 Liquid Handling System (Labcyte). This is an automated system, which uses acoustic droplet ejection, by which sound waves are used to move very small volumes of liquid without any physical contact. This increases precision and accuracy, whilst also decreasing the chance of contamination (Q. Guo et al., 2021). For the primary screen, cells were drugged with each compound at 2 concentrations (100nm and 1 pM) in triplicate technical repeats, repeated twice on one 1536 well plate for two different cell lines.

[0489] To carry out the assay, cells were incubated for one hour with a working solution of 80nM tetramethylrhodamine, methyl ester (TM RM; Invitrogen), 1 pM MitoTracker Green (Invitrogen) and 10pM Hoechst (Sigma) in phenol red free minimum essential media (MEM; Gibco by ThermoFisher Scientific). Immediately before imaging, wells were washed with 5pl MEM. Cells were imaged using the Opera Phenix® (Revvity). A total of 5 fields of view, approximately 20 cells per well, were imaged per well for 1536 well plates, using the using the Cy3 channel (excitation 645 / 30; emission 705 / 72), AlexaFluor 488 (excitation 488 / emission 525) and the DAPI channel (excitation 350 / 50; emission 455 / 50). Exposure time was optimised for each plate.

[0490] Images were analysed using Harmony® software (Revvity), to obtain a range of mitochondrial parameters. Images were segmented to show the nuclei, cells, and individual mitochondria. As a quality control measure, any well with a nuclei count of less than 12 was excluded. As a functional parameter, mitochondrial membrane potential, based on the intensity of TMRM staining and normalised to cell area, was assessed. Furthermore, morphological parameters were calculated including the percentage of long mitochondria.

[0491] For dose response data, a 9-point dose response was carried out, including the following concentrations: 1nm, 3nm, 10nm, 30nm, 100nm, 300nm, 1 pM, 3pM and 10pM. Each condition was repeated in four technical repeats. The assay and analysis were carried out as previously described using the Opera Phenix® and Harmony® analysis software. EC50 values were calculated using GraphPad prism software using nonlinear regression analysis.

[0492] Compound 1 and Comparative Compound A were tested in cell lines PSEN1A, PSEN1B, PSEN1 D and PSEN1 E; Comparative Compounds B to G were tested in cell lines PSEN1A and PSEN1 B; and the remaining compounds were tested in cell lines PSEN1A, PSEN1 D and PSEN1E.

[0493] Results

[0494] The results from the primary screen and the dose response are set out in Table 1 below. In Table 1, mean values in the primary screen over the cell lines tested are provided for each compound for both MMP and Long Mitochondria data. The columns headed “Activity” represent the EC50 in the dose response assays for MMP and Long Mitochondria. In Table 1 , the activity codes, AA, A1 and NA are used and the meanings for the activity codes are as follows:

[0495] AA Consistent activity 2 or 3 cell lines

[0496] A1 Active in one cell line or active in multiple cell lines but at only 1 or 2 concentrations NA Not active

[0497] Table 1 - Primary Screen and Dose Response in MMP and Long Mitochondria Assays

[0498] In the primary screen, % MMP is an indication of mitochondrial function. If mitochondrial function is lower than control levels, the mitochondria will function less efficiently. A substantial reduction in MMP may mean that the mitochondria are sufficiently dysfunctional to cause cell damage. Since the cell lines used in the assay show a reduction in MMP of 25%, an increase in MMP of 25% will restore the MMP in the PSEN1 mutant patient cell lines to that of controls.

[0499] The % long mitochondria is a measurement of mitochondrial morphology. Healthy mitochondria vary in size and shape depending on their environment and a mixture of different morphologies is found in healthy cells. The increase in the percentage of long mitochondria in the PSEN1 mutant patient lines indicates that the balance of different mitochondrial morphologies has been upset. Since the cell lines used show a 5% increase in long mitochondria, a reduction of 5% in % long mitochondria in will restore the patient phenotype to that of controls.

[0500] The results of the primary screen indicate that treatment with Comparative Compound A or with many of the Compounds of the invention elevates MMP to levels approaching those in controls, indicating that mitochondrial function is significantly improved. Treatment with Compound 1 is particularly effective. Treatment with Comparative Compounds B to G does not elevate MMP to such levels and so these compounds do not significantly improve mitochondrial function.

[0501] In the primary screen, Compound A and the compounds of the invention appear to restore the balance of mitochondrial morphology, whereas Comparative Compounds B to G do not, restore the balance mitochondrial morphology to any great extent.

[0502] In the concentration response assay, the results for MMP show that Comparative Compound A and a number of the compounds of the invention gave a response of AA. Compounds 1 , 3, 5, 8, 9b, 12 and 19a were all particularly active in the concentration response assay and were consistently active in 2 or 3 of the cell lines tested.

[0503] The dose response assay results for long mitochondria show that Compound A and the majority of the compounds of the invention have an activity rating of AA. The remaining compounds of the invention have an activity rating of A1 , indicating that either they are active in only one of the Alzheimer’s disease cell lines tested or that they are active in multiple cell lines but at only one or two concentrations.

[0504] It is important to consider both the MMP and the long mitochondria data, since some compounds which do not appear to be active in the MMP screen may be highly active in the long mitochondria screen. This is an indication that these compounds will be effective in restoring mitochondrial morphology, which, in turn, is an indication that they are likely to have a positive effect on mitochondrial membrane potential but over a different timescale or a different dose to that tested.

[0505] It is worth noting, however, that Comparative Compounds B to G are not active in the primary screen either for MMP or for long mitochondria. This is an indication that the nature of the R2substituent has considerable influence on the activity of the compounds.

[0506] Biological Example 2 - Biological Activity in Parkinson’s Disease

[0507] Parkinson’s Disease Cell Methods

[0508] Fibroblasts from sporadic PD patients were grown as described in Carling et al, 2020.

[0509] ATP Protocol

[0510] The ATP protocol is generally as described in Mortiboys et al 2008. Briefly, fibroblasts were cultured as and plated into white 384 well plates at a concentration of 5000 cells per well in 50 pl of media volume. The plates are left overnight in an incubator to allow the fibroblasts to adhere to the plate surface. The following morning the Glucose based medium is replaced with 25pl of Galactose based medium. The plates were then spiked with the compounds using a ECHO 550 liquid handling system. The wells were dosed to provide an 8-point concentration range of 1-10000nM of compound. After dosing the wells are topped up with a further 25pl of Galactose based medium and then left in an incubator for 24 hours. Following this incubation, the medium is removed from the plate and the wells are washed twice with sterile PBS. The wells are filled with 25pl of Sterile PBS followed by 12.5pl of Lysis solution from the ATPIite™ Luminescence ATP detection assay system (Perkin Elmer), including 16 cell free wells to use as blank controls. The plate is then placed on a rotary shaker for 5 mins at 700 rpm. Following the shaking 12.5pl of ATP substrate solution (Perkin Elmer) is added to each well and a further 5 min of shaking. The plate is then placed in darkness for 10 minutes prior to reading. Using a PHERAStar® plate reader, luminescence intensity is recorded. The Results are shown in Table 3.

[0511] Table 3 - Dose Response in Parkinson’s Disease Fibroblasts

[0512] The data in sporadic Parkinson’s Disease patient fibroblasts suggest Compound 1 to have an excellent profile increasing mitochondrial function in these cells with high potency. This effect is similar to other reported bile acid derivatives, for example the compounds described in WO2020 / 128514 and PCT / GB2023 / 051450, performing better than UDCA in the same cells and assays.

[0513] Biological Example 3 - MDCK-MDR1 Permeability Assay

[0514] A. Assay 1

[0515] Compounds of the invention, along with comparative Compound A and UDCA were tested in an MDCK-MDR1 permeability assay to investigate CNS permeability and efflux ratio.

[0516] MKCK-MDR1 cells are derived from Madin Darby canine kidney (MDCK) cells and have been transfected with the MDR1 gene, which encodes the efflux protein, P-glycoprotein (P-gp).

[0517] MDR1-MDCK cells were seeded into 24 well Transwell™ plates and cultured for 3 days to form a confluent monolayer . The test compound was prepared at a concentration of 10 pM in Hanks’ Balanced Salt Solution containing 25 mM HEPES and loaded into the donor compartments of the Transwell™ plates bearing the cell monolayers (pH 7.4 for both donor and receiver compartments). Lucifer Yellow was added to the apical buffer in all wells to assess integrity of the cell monolayer. Duplicate wells were prepared and incubated at 37°C in a CO2 incubator. Samples were removed at times 0 and 60 minutes and the test compound analysed by LC-MS / MS. Concentrations of Lucifer Yellow in the samples were measured using a fluorescence plate reader. The apparent permeability (Papp) values of the test compound were determined both in the apical to basolateral (A-B) direction, representing transport of the compound from the plasma to the CNS, and in the basolateral to apical (B-A) direction, representing efflux from the CNS. The permeability coefficient Pappin each direction may be calculated from the following equation. where is the rate of permeation of the drug across the cells;

[0518] Co is the concentration of the test compound at time zero and A is the area of the cell monolayer; and

[0519] A is the area of the cell monolayer.

[0520] The efflux ratio was calculated from using the permeability coefficients in each direction:

[0521] Efflux Ratio

[0522] Percent recovery data was also reported.

[0523] The assay was conducted using two replicates for each of the A-B and B-A measurements and the % recovery of the compounds was also calculated in order to ensure the validity of the results.

[0524] The results for Compound 1 , Comparative Compound A and LIDCA are presented in Table 4.

[0525] Table 4

[0526] As noted above, for an effective concentration of the compound in the CNS to be achieved, the efflux ratio should preferably be less than 2.5 (Doan, 2002), The results indicate that while Compound 1 , Compound 2 and Comparative Compound A all have an acceptable efflux ratio, the efflux ratio for LIDCA is too high for it to be present in a pharmaceutically effective concentration in the CNS.

[0527] B. Assay 2

[0528] A further assay was carried out. The results are not directly comparable with Assay 1 because a different assay protocol was used.

[0529] Cell Culture

[0530] MDR1-MDCK I cells were seeded onto polycarbonate membrane (PC) in 96-well Corning insert systems at 4.44 x 105cells / mL and cultured for 4-7 days for confluent cell monolayer formation.

[0531] Experimental Procedures

[0532] The transport buffer in the study was HBSS with 10.0 mM HEPES at pH 7.40±0.05. Test compounds were tested at 2.00 pM bi-directionally in duplicate. Digoxin was tested at 10.0 pM bi-directionally in duplicate, while nadolol and metoprolol were tested at 2.00 pM in A to B direction in duplicate. Final DMSO concentration was adjusted to less than 1 %. The plate was incubated for 1.5 hours in CO2 incubator at 37±1°C, with 5% CO2 at saturated humidity without shaking. And all samples after mixed with acetonitrile containing internal standard were centrifuged at 3220 x g for 10 min. For all samples, 150 pL supernatant solution was diluted with 150 pL ultra-pure water for LC-MS / MS analysis. In addition, the efflux ratio of each compound was also determined. Test and reference compounds were quantified by LC- MS / MS analysis based on the peak area ratio of analyte / IS.

[0533] After transport assay, Lucifer yellow rejection assay was applied to determine the cell monolayer integrity. Buffers are removed from both apical and basolateral chambers, followed by the addition of 75 pL of 100 pM lucifer yellow in transport buffer and 250 pL transport buffer in apical and basolateral chambers, respectively. The plate was incubated for 30 minutes at 37°C with 5% CO2 and 95% relative humidity without shaking. After 30 minutes incubation, 20 pL of lucifer yellow samples were taken from the apical sides, followed by the addition of 60 pL of Transport Buffer, and then 80 pL of lucifer yellow samples were taken from the basolateral sides. The relative fluorescence unit (RFU) of lucifer yellow was measured at 425 / 528 nm (excitation / emission) with a microplate reader.

[0534] Data Analysis

[0535] The apparent permeability coefficient Papp (cm / s) was calculated using the equation:

[0536] — is the cumulative concentration of compound in the receiver chamber as a function of dtrtime (pM / s);

[0537] Vris the solution volume in the receiver chamber (0.075 mL on the apical side, 0.25 mL on the basolateral side);

[0538] A is the surface area for the transport, i.e. 0.143 cm2for the area of the monolayer;

[0539] Co is the initial concentration in the donor chamber (pM).

[0540] The efflux ratio was calculated using the equation:

[0541] Efflux Ratio

[0542] % recovery was calculated using the equation:

[0543] [( Vrx Cr)+ (Vdx Cd)]

[0544] % Solution Recovery = 100 x - — - — -

[0545] (Vdx Co) where:

[0546] Vrand Co are defined as above;

[0547] Vd is the volume in the donor chambers (0.075 mL on the apical side, 0.25 mL on the basolateral side);

[0548] Cd and Crare the final concentrations of transport compound in donor and receiver chambers, respectively.

[0549] In this assay, compounds of the invention were compared with nor LIDCA, which has the structure:

[0550] This is similar to Compound 1 of the invention but has a 7P-OH substituent rather than the 7,7- difluoro substitution pattern seen in the compounds of the invention. The mean efflux ratio was determined for each test compound, where efflux ratio is defined as: and the results are shown below in Table 5.

[0551] In this assay, the most suitable compounds have an efflux ratio of less than 4.0 Table 5

[0552] Although Compounds 4, 5, 6 and 17 have high efflux ratios, the data set out in Biological Example 5 demonstrate that they are able to achieve high concentrations in plasma in comparison with Comparative Compound A.

[0553] Biological Example 4 - Phamacokinetic Data (Plasma)

[0554] The test compositions were prepared by dissolving the test compounds in an amount of 20% w / v in a solvent comprising DMSO and HP-p-CD in a volume ratio of 5 / 95.

[0555] The test compositions were administered to male C57 mice. In a first leg, each test composition was administered intravenously in an amount of 1.00 mg / kg to three mice and blood sampling was carried out at 5 minutes (0.0833 hours), 0.25 hours, 1 hour, 2 hours, 4 hours, 7 hours and 24 hours after administration for Compound 1 and Compound A and 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours after administration for the other test compounds. In a second leg, each test composition was administered orally in an amount of 3.00 mg / kg to three mice and blood sampling was carried out at 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 7 hours and 24 hours after administration for Compound 1 and Compound A and 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours after administration for the other test compounds. The concentration of each test compound was determined at each sampling timepoint. The three mice used for IV administration of Comparative Compound A were given reference numbers 13, 14 and 15 and the three mice used for oral administration of Comparative Compound A were given reference numbers 16, 17 and 18. The three mice used for IV administration of Compound 1 were given reference numbers 19, 20 and 21 and the three mice used for oral administration of Compound 1 were given reference numbers 22, 24 and 24.

[0556] Tables 6 and 7 shows the results for all compounds after IV administration and Figures 1 and 2 each illustrate the results for three individual mice treated with Compound 1 (Figure 1) or

[0557] Comparative Compound A (Figure 2).

[0558] Tables 8 and 9 show the results for all compounds following oral administration and Figures 3 and 4 each illustrate the results for three individual mice treated respectively with Compound 1 (Figure 3) and Comparative Compound A (Figure 4).

[0559] Results for Test Compounds administered by IV bolus (0.2 mq / mL; nominal dose 1.0 mq / kq)

[0560] Table 6 - Mean Plasma Concentration

[0561] Concentrations of Compound A and Compound 1 measured at 7 hours, not 8 hours.

[0562] BLQ = below limit of quantification

[0563] Table 7 - Half-life and AUC

[0564] Results for Test Compounds administered per Oral (0.3 mq / mL; nominal dose 3.0 mq / kq)

[0565] Table 8 - Mean Plasma Concentration

[0566] Concentrations of Compound A and Compound 1 measured at 7 hours, not 8 hours.

[0567] ND = not done

[0568] Table 9 - Half-life and AUC

[0569] ND = not done

[0570] It can be seen from Figures 1 to 4 and Tables 6 and 8 that at all timepoints sampled, the plasma concentration of Compounds 1 , 3, 4, 5, 6, 7 and 17 is higher than that of Comparative Compound A when administered intravenously and at most timepoints sampled, the plasma concentration for these compounds is also higher than that of Comparative Compound A following oral administration.

[0571] Following IV administration of Comparative Compound A to Mice 13, 14 and 15, half-life was measured at 0.176 hours and AllCo-mf was 207 ng.h / mL. Clearance was measured at 80.6 mL / min / kg.

[0572] In contrast, following IV administration of Compound 1 , half-life was measured at 6.30 ± 0.588 hours and AUCo-mfwas 23397 ± 1464 ng.hr / ml_. Clearance was measured at 0.714 ± 0.0464 mL / min / kg. Half-life and AllCo-mf were also considerably higher for all the other example compounds than for Comparative Compound A.

[0573] Following PO administration of Compound A at 3.00 mg / kg to Mice 16, 17 and 18, a Cmax of 497 ng / mL was measured at 0.250 h, Ciast 7 71 ng / mL, and AllCo-t of 1140 ng.hr / mL. Due to the irregularity of the PO profiles, ti / 2 and associated parameters could not be determined for Compound A.

[0574] Following PO administration of Compound 1 at 3.00 mg / kg a Cmax of 7543 ± 522 ng / mL was measured at 1.00 h, Ciast 609 ± 72.3 ng / mL, half-life 7.53 ± 0.337 and AUC-inf of 57753 ± 3998 ng.hr / mL. Bioavailability calculated using AUCo-mfwas measured at 82.3 ± 5.70 %.

[0575] Due to animal 17's concentrations being significantly lower than the other animals in the PO dose group and the consistency of the other animals profiles and data, animal 17 has been excluded from averages and PK calculations.

[0576] Compounds 3, 4, 5, 6, 7 and 17 also demonstrated significantly longer half-life and greater AUCo-infthan Compound A when administered by IV bolus or orally.

[0577] These results clearly demonstrate the improved plasma half-life and bioavailability of Compound 1 and the other example compounds compared with Comparative Compound A. It is believed that this improvement arises because the shorter side chain of the compounds of the invention prevents recognition of the compounds by the enzymes which control conjugation of bile acids in the liver, leading to an inability of compounds of the invention to form salts or conjugates, which means that the compound passes to the blood rather than entering the enterohepatic circulation. The significance of this is that compounds can only pass from the plasma through the blood brain barrier into the CNS, if they reach the blood. Compounds such as Comparative Compound A which are trapped to a significant extent in the enterohepatic circulation attain only low concentrations in the plasma and are therefore cannot cross the blood brain barrier in significant amounts.

[0578] Biological Example 5 - Pharmacokinetic data (CNS)

[0579] Preparation of Test Compositions

[0580] For each compound, test compositions 1 to 3 were prepared by the compound in amounts of 0.3 mg / mL, 1 mg / mL or 3 mg / mL in a solvent comprising: a) DMSO and b) HP-p-CD in water (20% w / v) in a volume ratio a / b of 5 / 95.

[0581] The test compositions were administered orally to three groups of six male C57 mice at doses of 3.0 (G01), 10.0 (G02) and 30.0 (G03) mg / kg. For each group, levels of the compound were measured in plasma, brain and cerebrospinal fluid in three mice at 0.25 hours and in the other three mice at 0.5 hours.

[0582] The results for Compounds 1 , 2 and are shown in Tables 10 and 11 below. For comparison, results for LIDCA and nor LIDCA are provided in Tables 12 and 13.

[0583] Table 10 - Results for Compound 1

[0584] Table 11 - Results for Compound 2

[0585] Table 12 - Results for nor UDCA

[0586] Table 13 - Results for UDCA

[0587] Comparing Tables 12 and 13 shows that shortening side chain from UDCA to nor UDCA leads to a significant increase in plasma concentrations. It seems likely that this is because UDCA is trapped in the enterohepatic circulation whereas nor UDCA is not.

[0588] Comparing the results presented in Table 10 with those in Table 12 shows that Compound 1 achieves significantly higher brain / plasma and CSF / plasma ratios than nor UDCA. These two compounds are similar in structure except that in nor UDCA, the bile acid skeleton has OH at the 7-position, whereas in Compound 1 , the bile acid skeleton has a 7,7-difluoro substitution pattern. This is confirmation that, as predicted by the efflux ratio determined in Biological Example 3, the 7,7-difluoro substitution has a significant effect on the ability of the molecule to achieve an effective concentration in the CNS. The plasma concentration achieved for nor UDCA is, in any case, lower than that achieved for Compound 1 and this, combined with the less favourable efflux ratio for nor UDCA leads to significantly lower concentrations of nor UDCA in the brain and CSF than can be achieved for Compound 1. As shown in Table 11 , Compound 2 achieves slightly lower brain and CSF concentrations than Compound 1 , although the levels are still significantly higher than those for nor UDCA. The brain / plasma and CSF / plasma ratios achieved by Compound 2 are similar to those achieved by Compound 1.

[0589] The results presented in Tables 10 and 11 show that, although the levels of Compounds 1 and 2 in the plasma were considerably higher than the levels in the brain and the CSF, both compounds were detected in tissue homogenates from the brain and the CSF at 15 minutes and 30 minutes after oral dosing for all three doses given. The amounts detected in the brain homogenates were sufficient for pharmacological activity.

[0590] In summary, Biological Examples 1 and 2 demonstrate that the compounds of the invention are active in cells from both Alzheimer’s disease and Parkinson’s disease patients. The compounds of the invention and Comparative Compound A are active in one or both MMP and long mitochondria in cells from Alzheimer’s disease patients.

[0591] Biological Example 3 shows that LIDCA has a high efflux ratio and consequently would not be expected to remain in the CNS in sufficient quantities for it to be active. This is not the case for either Comparative Compound A or Compound 1.

[0592] However, although Comparative Compound A has a similar efflux ratio to Compound 1, Biological Example 4 demonstrates that it becomes trapped in the enterohepatic circulation and is therefore not present in the plasma in sufficient levels for an effective amount of the compound to be available to enter the CNS. On the other hand, the bioavailability of Compounds 1 , 3 to 7 and 17 is significantly greater than that of Comparative Compound A and they enter in the plasma in sufficient concentrations for them to be available to cross the blood brain barrier. The results for LIDCA and nor LIDCA support the theory that the longer side chain present in LIDCA and Compound A results in entrapment of the compounds in the enterohepatic circulation, whereas this is avoided by nor LIDCA and Compounds 1 and 2, all of which have a shorter side chain.

[0593] Biological Example 5 demonstrates that Compounds 1 and 2 do indeed cross the blood brain barrier and can both be found in both brain tissue and in CSF 15 minutes and 30 minutes after oral dosing in mice. This is not the case for nor LIDCA even though the plasma concentrations achieved are comparable to those for Compounds 1 and 2. The inventors speculate that this is because nor LIDCA lacks the 7,7-difluoro substitution pattern present in the compounds of the invention.

[0594] References

[0595] Albanese, A; Ludolph, A C; McDermott, C J; Corcia, P; Van Damme, P; Van den Berg, L H; Hardiman, O; Rinaldi, G; Vanacore, N; Dickie, B; TUDCA-ALS Study Group, “Tauroursodeoxycholic acid in patients with amyotrophic lateral sclerosis: The TUDCA-ALS trial protocol”, Frontiers in Neurology, 13:1009113, (2022). doi: 10.3389 / fneur.2022.1009113 D’Amore, C.; Di Leva, F. S.; Sepe, V.; Renga, B.; Del Gaudio, C.; D’Auria, M. V.; Zampella, A.; Fiorucci, F.; Limongelli, V. “Design, Synthesis, and Biological Evaluation of Potent Dual Agonists of Nuclear and Membrane Bile Acid Receptors”, J. Med. Chem. 57, 937-954 (2014)

[0596] Arber C, Villegas-Llerena C, Toombs J, Pocock JM, Ryan NS, Fox NC, Zetterberg H, Hardy J, Wray S. “Amyloid precursor protein processing in human neurons with an allelic series of the PSEN1 intron 4 deletion mutation and total presenilin-1 knockout”. Brain Commun. (2019); 1(1), fcz024. doi: 10.1093 / braincomms / fcz024. Epub 2019 Oct 14. PMID: 32395715; PMCID: PMC7212081.

[0597] Arber C, Lovejoy C, Harris L, Willumsen N, Alatza A, Casey JM, Lines G, Kerins C, Mueller AK, Zetterberg H, Hardy J, Ryan NS, Fox NC, Lashley T, Wray S. “Familial Alzheimer's Disease Mutations in PSEN1 Lead to Premature Human Stem Cell Neurogenesis”. Cell Rep. (2021); 34(2), 108615. doi: 10.1016 / j.celrep.2020.108615. PMID: 33440141 ; PMCID: PMC7809623.

[0598] W.A. Banks “Characteristics of compounds that cross the blood-brain barrier”, BMC Neurology, 9(Suppl l):S3, (2009); doi : 10.1186 / 1471 -2377-9-S1 -S3.

[0599] S. M. Bell, K. Barnes, H. Clemmens, A. R. Al-Rafiah, E. A. Al-ofi, V. Leech, O. Bandmann, P.

[0600] J. Shaw, D. J. Blackburn, L. Ferraiuolo, and H. Mortiboys, “Ursodeoxycholic Acid Improves Mitochondrial Function and Redistributes Drp1 in Fibroblasts from Patients with either Sporadic or Familial Alzheimer's Disease.” Journal of Molecular Biology, 430(21), 3942-3953 (2018). DOI: 10.1016 / j.jmb.2018.08.019

[0601] Ben-Gedalya T, Moll L, Bejerano-Sagie M, Frere S, Cabral WA, Friedmann-Morvinski D, Slutsky I, Burstyn-Cohen T, Marini JC, Cohen E. “Alzheimer's disease-causing proline substitutions lead to presenilin 1 aggregation and malfunction”. EMBO J. (2015); 34(22), 2820-2839. doi: 10.15252 / embj.201592042. Epub 2015 Oct 5. PMID: 26438723; PMCID: PMC4682640.

[0602] Campion D, Flaman JM, Brice A, Hannequin D, Dubois B, Martin C, Moreau V, Charbonnier F, Didierjean O, Tardieu S, et al. “Mutations of the presenilin I gene in families with early- onset Alzheimer's disease”, Hum Mol Genet. (1995); 4(12), 2373-2377. doi: 10.1093 / hmg / 4.12.2373. PMID: 8634712. Campion D, Dumanchin C, Hannequin D, Dubois B, Belliard S, Puel M, Thomas-Anterion C, Michon A, Martin C, Charbonnier F, Raux G, Camuzat A, Penet C, Mesnage V, Martinez M, Clerget-Darpoux F, Brice A, Frebourg T. “Early-onset autosomal dominant Alzheimer disease: prevalence, genetic heterogeneity, and mutation spectrum”, Am J Hum Genet. (1999); 65(3), 664-670. doi: 10.1086 / 302553. PMID: 10441572; PMCID: PMC1377972.

[0603] Canto-Santos J, Grau-Junyent, J M, Garrabou G; “The Impact of Mitochondrial Deficiencies in Neuromuscular Diseases”, Antioxidants, 9, 964-992 (2020).

[0604] DOI: 10.3390 / antiox9100964

[0605] Carling PJ, Mortiboys H, Green C, Mihaylov S, Sandor C, Schwartzentruber A, Taylor R, Wei W, Hastings C, Wong S, Lo C, Evetts S, Clemmens H, Wyles M, Willcox S, Payne T, Hughes R, Ferraiuolo L, Webber C, Hide W, Wade-Martins R, Talbot K, Hu MT, Bandmann O. “Deep phenotyping of peripheral tissue facilitates mechanistic disease stratification in sporadic Parkinson's disease”. Prog Neurobiol. ,187, 101772 (2020). doi: 10.1016 / j.pneurobio.2020.101772.

[0606] P. A. Dawson, T. Lan, A. Rao; “Bile acid transporters”, Journal of Lipid Research, 50, 2340- 2357 (2009).

[0607] De Jonghe C, Cruts M, Rogaeva EA, Tysoe C, Singleton A, Vanderstichele H, Meschino W, Dermaut B, Vanderhoeven I, Backhovens H, Vanmechelen E, Morris CM, Hardy J, Rubinsztein DC, St George-Hyslop PH, Van Broeckhoven C. “Aberrant splicing in the presenilin-1 intron 4 mutation causes presenile Alzheimer's disease by increased Abeta42 secretion”. Hum Mol Genet. (1999); 8(8), 1529-1540. doi: 10.1093 / hmg / 8.8.1529. PMID: 10401002.

[0608] K. M. Doan, J.E. Humphreys, L.O. Webster, S. A Wring, L.J Shampine, C.J Serabjit-Singh, K.K. Adkinson, JW. Polli “Passive Permeability and P-Glycoprotein-Mediated Efflux Differentiate Central Nervous System (CNS) and Non-CNS Marketed Drugs”, Journal of Pharmacology and Experimental Therapeutics, 303(3), 1029-1037 (2002);

[0609] DOI : https: / / doi.Org / 10.1124 / jpet.102.039255 E. Dolghih, M.P. Jacobson, “Predicting Efflux Ratios and Blood-Brain Barrier Penetration from Chemical Structure: Combining Passive Permeability with Active Efflux by P-Glycoprotein”, ACS Chem. Neurosci., 4, 361-367 (2013); dx.doi.org / 10.1021 / cn3001922.

[0610] Dumanchin C, Tournier I, Martin C, Didic M, Belliard S, Carlander B, Rouhart F, Duyckaerts C, Pellissier JF, Latouche JB, Hannequin D, Frebourg T, Tosi M, Campion D. “Biological effects of four PSEN1 gene mutations causing Alzheimer disease with spastic paraparesis and cotton wool plaques”. Hum Mutat. (2006); 27(10), 1063. doi: 10.1002 / humu.9458. PMID: 16941492.

[0611] Gliebus G, Rosso A, Lippa CF. “Progranulin and beta-amyloid distribution: a case report of the brain from preclinical PS-1 mutation carrier”. Am J Alzheimers Dis Other Demen. (2009 Dec-2010 Jan); 24(6), 456-460. doi: 10.1177 / 1533317509346209. Epub 2009 Sep 23. PMID: 19776335; PMCID: PMC10846111.

[0612] Guo, Q; Su X; Zhang, X; Shao, M; Yu, H; Li, D. (2021), “A review on acoustic droplet ejection technology and system”, Soft Matter. The Royal Society of Chemistry, 17(11), 3010-3021. doi: 10.1039 / D0SM02193H

[0613] Hook V, Podvin S, Mosier C, Boyarko B, Seyffert L, Stringer H, Rissman RA. “Emerging evidence for dysregulated proteome cargoes of tau-propagating extracellular vesicles driven by familial mutations of tau and presenilin”. Extracell Vesicles Circ Nucl Acids. (2023); 4(4), 588-598. doi: 10.20517 / evcna.2023.44. Epub 2023 Nov 21. PMID: 38125374; PMCID: PMC10732590.

[0614] S. D. Mhatre, J. Iyer, S. Puukila, A. M. Paul, C.G.T. Tahimic, L. Rubinstein, M. Lowe, J.S. Alwood, M. B. Sowa, S. Bhattacharya, R.K. Globus, and A.E. Ronca “Neuro-consequences of the spaceflight environment” Neuroscience and Biobehavioural Reviews, 132, 908-935 (2022)

[0615] H. Mortiboys, K.J. Thomas, W.J. H. Koopman, S. Klaffke, P. Abou-Sleiman, S. Olpin, N. W. Wood, P.H.G.M. Willems, J.A.M. Smeitink, M.R. Cookson, and O. Bandmann, “Mitochondrial function and morphology are impaired in parkin-mutant fibroblasts”, Ann Neurol. Nov; 64(5):555-65 (2008) H. Mortiboys, J. Aasly, and O. Bandmann, “Ursocholanic acid rescues mitochondrial function in common forms of familial Parkinson’s disease”, Brain, 136(10), 3038-3050 (2013)

[0616] H. Mortiboys, R. Furmston, G. Bronstad, J. Aasly, C. Elliott, and O. Bandmann, “UDCA exerts beneficial effect on mitochondrial dysfunction in LRRK2G2019S carriers and in vivo” Neurology, 85, 846-852 (2015)

[0617] Jacquemont ML, Campion D, Hahn V, Tallaksen C, Frebourg T, Brice A, Durr A. “Spastic paraparesis and atypical dementia caused by PSEN1 mutation (P264L), responsible for Alzheimer's disease”. J Med Genet. (2002); 39(2): E2. doi: 10.1136 / jmg.39.2. e2. PMID: 11836371 ; PMCID: PMC1735046.

[0618] Kelleher and Shen, Presenilin-1 mutations and Alzheimer’s disease, Proc. Natl. Acad. Sci.

[0619] USA, 114(4), 629-631 (2017); doi / 10.1073 / pnas.1619574114

[0620] Nelson O, Tu H, Lei T, Bentahir M, de Strooper B, Bezprozvanny I. “Familial Alzheimer disease-linked mutations specifically disrupt Ca2+ leak function of presenilin 1”. J Clin Invest. (2007); 117(5), 1230-1239. doi: 10.1172 / JCI30447. Epub 2007 Apr 12. PMID: 17431506; PMCID: PMC1847535.

[0621] Payne, T; Sassani, M; Buckley, E; Moll, S; Anton, A; Appleby, M; Maru, S; Taylor, R; McNeill, A; Hoggard, N; Mazza, C; Wilkinson, I D; Jenkins, T; Foltynie, T; Bandmann, O, “Ursodeoxycholic acid as a novel disease-modifying treatment for Parkinson’s disease: protocol for a two-centre, randomised, double-blind, placebo-controlled trial, The 'UP' study”, BMJ Open 2020;10:e038911. doi: 10.1136 / bmjopen-2020-038911

[0622] Petit D, Gutierrez Fernandez S, Zoltowska K M, Enzlein T, Ryan N S, O’Connor A, Szaruga M, Hill E, Vandenberghe R, Fox N C, Chavez-Gutierrez L, “A profiles generated by Alzheimer’s disease causing PSEN1 variants determine the pathogenicity of the mutation and predict age at disease onset”, Nature Molecular Psychiatry, 27, 2821-2832, (2022)

[0623] Podvin S, Jones A, Liu Q, Aulston B, Mosier C, Ames J, Winston C, Lietz CB, Jiang Z, O'Donoghue AJ, Ikezu T, Rissman RA, Yuan SH, Hook V. “Mutant Presenilin 1 Dysregulates Exosomal Proteome Cargo Produced by Human-Induced Pluripotent Stem Cell Neurons”. ACS Omega. (2021); 6(20), 13033-13056. doi: 10.1021 / acsomega.1c00660. PMID: 34056454; PMCID: PMC8158845. Raux G, Guyant-Marechal L, Martin C, Bou J, Penet C, Brice A, Hannequin D, Frebourg T, Campion D. “Molecular diagnosis of autosomal dominant early onset Alzheimer's disease: an update.” J Med Genet. (2005); 42(10), 793-795. doi: 10.1136 / jmg.2005.033456. Epub 2005 Jul 20. PMID: 16033913; PMCID: PMC1735922.

[0624] Schwartzentruber A, Boschian C, Lopes FM, Myszczynska MA, New EJ, Beyrath J, Smeitink J, Ferraiuolo L, Mortiboys H. “Oxidative switch drives mitophagy defects in dopaminergic parkin mutant patient neurons” Sci Rep, 10(1), 15485 (2020 Sep 23). doi: 10.1038 / s41598- 020-72345-4

[0625] Tysoe C, Whittaker J, Xuereb J, Cairns NJ, Cruts M, Van Broeckhoven C, Wilcock G, Rubinsztein DC. “A presenilin-1 truncating mutation is present in two cases with autopsy- confirmed early-onset Alzheimer disease”. Am J Hum Genet. (1998); 62(1), 70-76. doi: 10.1086 / 301672. PMID: 9443865; PMCID: PMC1376799.

[0626] W. A. da Silveira, H. Fazelinia, S. Brin Rosenthal, E. C. Laiakis, M. S. Kim, C. Meydan, Y. Kidane, K. S. Rathi, S. M. Smith, B. Stear, Y. Ying, Y. Zhang, J. Foox, S. Zanello, B. Crucian,

[0627] D. Wang, A. Nugent, H. A. Costa, S. R. Zwart, S. Schrepfer, R.A. L. Elworth, N. Sapoval, T. Treangen, M. MacKay, N. S. Gokhale, S. M. Horner, L. N. Singh, D. C. Wallace, J. S. Willey,

[0628] J. C. Schisler, R. Meller, J. T. McDonald, K. M. Fisch, G. Hardiman, D. Taylor, C. E. Mason, S. V. Costes, and A. Beheshti, “Comprehensive Multi-omics Analysis Reveals Mitochondrial Stress as a Central Biological Hub for Spaceflight Impact”, Cell 183, 1185-1201, (2020)

[0629] Vanova T, Sedmik J, Raska J, Amruz Cerna K, Taus P, Pospisilova V, Nezvedova M, Fedorova V, Kadakova S, Klimova H, Capandova M, Orviska P, Fojtik P, Bartova S, Plevova

[0630] K, Spacil Z, Hribkova H, Bohaciakova D. “Cerebral organoids derived from patients with Alzheimer's disease with PSEN1 / 2 mutations have defective tissue patterning and altered development”. Cell Rep. (2023);42(11), 113310. doi: 10.1016 / j.celrep.2023.113310. Epub 2023 Oct 20. PMID: 37864790.

[0631] E. Wood, K. H. Hall, and W. Tate “Role of mitochondria, oxidative stress and the response to antioxidants in myalgic encephalomyelitis / chronic fatigue syndrome: A possible approach to SARS-CoV-2 long-haulers’?” Chronic Diseases and Translational Medicine, 7(1), 14-26 (2021) Yang J, Zhao H, Ma Y, Shi G, Song J, Tang Y, Li S, Li T, Liu N, Tang F, Gu J, Zhang L, Zhang Z, Zhang X, Jin Y, Le W. “Early pathogenic event of Alzheimer's disease documented in iPSCs from patients with PSEN1 mutations”. Oncotarget. (2017); 8(5), 7900-7913. doi: 10.18632 / oncotarget.13776. PMID: 27926491 ; PMCID: PMC5352369.

Claims

CLAIMS1 . A compound of formula (I):wherein: each is independently a single or a double bond when connected to R1is a single bond, R1is selected from H, fluoro, chloro, OH, NH2, N3,C1-4 alkyl, O(Ci-4 alkyl), NH(CI-4alkyl), C(O)OH, C(O)NHR1aand NHC(O)OR1 b; wherein when R1is C1.4 alkyl or O(Ci-4 alkyl), it is optionally substituted with one or more substituents selected from fluoro, chloro, OH, NH2, N3, O(Ci-4 alkyl) and NH(CI-4 alkyl); wherein each of R1aand R1 bis independently H or C1.4 alkyl optionally substituted with halo; when connected to R1is a double bond, R1is selected from CHR8and O; wherein R8is H or CH3;R2is selected from C(O)OR5, C1.6 alkyl, C2-6 alkenyl and C(O)N(R5a)(R5b); wherein R5is H, C1.4 alkyl or benzyl; each of R5aand R5bis H or C1.4 alkyl or R5aand R5btogether with the nitrogen atom to which they are attached form a piperidine, pyrrolidine, piperazine or morpholine ring; and wherein alkyl and alkenyl groups of R2are optionally substituted with one or more substituents selected from OH, NH2, N3, fluoro and chloro; when connected to R3is a single bond, R3is OH; when connected to R3is a double bond, R3is O; each of R4aand R4bis independently H, F, Cl, C1.2 alkyl or C1.2 haloalkyl; or a salt or solvate thereof.

2. A compound or a salt or solvate thereof according to claim 1 which is a compound of formula (IZ):wherein:R1Zis selected from H, fluoro, chloro, OH, C1.4 alkyl and O(Ci-4 alkyl), wherein when R1Zis Ci- 4 alkyl it is optionally substituted with one or more substituents selected from fluoro, chloro, OH and O(Ci-4alkyl);R5is H, Ci-6 alkyl or benzyl; or a salt or solvate thereof.

3. A compound or a salt or solvate thereof according to claim 1 which is an isotopic variant in which: in an alkyl group R1, one or more hydrogen atoms are present as2H (deuterium) or3H (tritium), especially deuterium; and / or when connected to R3is a single bond, the hydrogen atom at the 3-position of the bile acid skeleton is replaced by2H (deuterium) or3H (tritium), especially deuterium.

4. A compound or a salt or solvate thereof according to claim 1 or claim 3 wherein connected to R1is a single bond.

5. A compound or a salt or solvate thereof according to claim 4 wherein R1is selected from H, fluoro, chloro, OH, NH2, N3, C1.4 alkyl, O(Ci-4 alkyl), NH(CI-4 alkyl) and C(O)OH.

6. A compound or a salt or solvate thereof according to claim 5 wherein R1is H.

7. A compound or a salt or solvate thereof according to claim 4 wherein R1is C1.4 alkyl which is unsubstituted or substituted with one or more substituents selected from fluoro, chloro, OH, NH2, N3, and O(Ci-4 alkyl), especially fluoro, chloro, OH, NH2 and methoxy.

8. A compound or a salt or solvate thereof according to claim 7 wherein R1is methyl, ethyl, CH2OH, CH2F, CHF2, CH2OCH3, CH2NH2or CD3.

9. A compound or a salt or solvate thereof according to claim 4 wherein:R1is fluoro, chloro or azido (N3); orR1is OH, NH2, O(CI-4 alkyl) or NH(CI-4alkyl); orR1is C(O)OH; orR1is C(O)NHR1aor NHC(O)OR1 bwherein R1aand R1 bare each independently selected from H and methyl.

10. A compound or a salt or solvate thereof according to claim 1 or claim 3 wherein connected to R1is a double bond.

11. A compound or a salt or solvate thereof according to claim 10 wherein:R1is CHR8, wherein R8is H; orR1is O.

12. A compound or a salt or solvate thereof according to any one of claims 1 or 3 to 11 , wherein R2is C(O)OR5, wherein R5is as defined in claim 1.

13. A compound or a salt or solvate thereof according to claim 2 or claim 12, wherein R5is H.

14. A compound or a salt or solvate thereof according to any one of claims 1 or 3 to 11 , wherein R2is Ci-4alkyl or C2-4 alkenyl wherein said alkyl and alkenyl groups are optionally substituted with one or more substituents selected from OH, NH2, N3, fluoro and chloro.

15. A compound or a salt or solvate thereof according to claim 14 wherein R2is unsubstituted ethyl, unsubstituted ethenyl, CH2OH, CH2NH2, CH2N3, CH2F or CHF2.

16. A compound or a salt or solvate thereof according to any one of claims 1 to 3 or 11 , wherein R2is C(O)NR5aR5b, where R5aand R5bare as defined in claim 1.

17. A compound or a salt or solvate thereof according to any one of claims 1 or 3 to 16 wherein each of R4aand R4bis independently H, F, Cl, methyl or methyl substituted with one or more fluoro substituents.

18. A compound or a salt or solvate thereof according to claim 17 wherein: both R4aand R4bare H; orR4ais H and R4bis F; orR4bis H and R4ais F.

19. A compound or a salt or solvate thereof according to any one of claims 1 or 3 to 18 wherein connected to R3is a double bond, and R3is O.

20. A compound or a salt or solvate thereof according to any one of claims 1 or 3 to 18 wherein connected to R3is a single bond, and R3is OH.

21. A compound or a salt or solvate thereof according to any one of claims 1 to 18 which is a compound of formula (IY):wherein in compounds of formula (IY), R1, R2, R4aand R4bare as defined in claim 1.

22. A compound according to claim 1 selected from: 7,7-difluoro 24-norlithocholic acid (Compound 1);Methyl 3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oate (Compound 1 Me ester) 3a-hydroxy-7,7-difluoro-5p-24-nor-22-methyl-cholanic acid (Compound 2);3a-hydroxy-7,7-difluoro-5p-24-nor-22-ethyl-cholanic acid (Compound 3);3a-hydroxy-7,7-difluoro-5p-24-nor-22-fluoro-cholanic acid (Compound 4);3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxy-cholanic acid (Compound 5);3a-hydroxy-7,7-difluoro-5p-24-nor-22-chloro-cholanic acid (Compound 6);3a-hydroxy-7,7-difluoro-5p-24-nor-22-(methyl-d3)-cholanic acid (Compound 7);3a-hydroxy-7,7-difluoro-5p-24-nor-22-methoxy-cholanic acid (Compound 8);3a-hydroxy-7,7-difluoro-5p-24-nor-22-keto-cholanic acid (Compound 9);3a-hydroxy-7,7-difluoro-5p-24-nor-22-azido-cholanic acid (Compound 10);3a-hydroxy-7,7-difluoro-5p-24-nor-22-amine-cholanic acid (Compound 11);3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-ol (Compound 12);3a-hydroxy-7,7-difluoro-5p-24-nor-chol-22-ene (Compound 13);3a-hydroxy-7,7-difluoro-5p-24-nor-cholane (Compound 14);3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-azide (Compound 15);3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-amine (Compound 16);3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholanic acid (Compound 17); 3a-hydroxy-7,7-difluoro-5p-24-nor-22-fluoromethyl-cholanic acid (Compound 18); 3a-hydroxy-7,7-difluoro-5p-24-nor-22-di-fluoromethyl-cholanic acid (Compound 19); t-Butyl 3a-hydroxy-7,7-difluoro-5p-24-nor-22-di-fluoromethyl-cholan-23-oate (Compound 19 t- butyl ester)3a-hydroxy-7,7-difluoro-5p-24-nor-22-methylene-cholanic acid (Compound 20); 3a-Hydroxy-7,7-difluoro-5p-24-nor-22-methoxymethyl-cholanic acid (Compound 21); 3a-hydroxy-7,7-difluoro-5p-24-nor-22-aminomethyl-cholanic acid (Compound 22);3a-hydroxy-7,7-difluoro-5p-24-nor-22-hydroxymethyl-cholan-23-ol (Compound 23); 3-keto-7,7-difluoro-5p-24-nor-cholan-23,23-dicarboxylic acid (Compound 24);3-keto-7,7-difluoro-5p-24-nor-cholanic acid (Compound 25); 3p-Hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 26); 2p-fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 27);2p-fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 28);4p-fluoro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 29);4p-fluoro-3p-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 30);4a-chloro-3a-hydroxy-7,7-difluoro-5p-24-nor-cholanic acid (Compound 31); [2H]-3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oic acid (Compound 32);A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oyl)-(morpholine) (Compound 33); and A / -(3a-hydroxy-7,7-difluoro-5p-24-nor-cholan-23-oyl)-(methylamine) (Compound 34); or a salt or solvate of any one thereof.

23. A process for the preparation of a compound according to claim 1 or a salt or solvate thereof, the process comprising:A. for a compound of formula (I) in whichconnected to R1is a single bond, R2is C(O)OR5, where R5is H, R3is a-OH and R4aand R4bare both H: hydrolysis of a protected compound of formula (II):wherein R1is as defined for formula (I), R6is Ci-e alkyl or benzyl and R7is a protected OH group;B. for a compound of formula (la), which is a compound of formula (I) in which R2is C(O)OR5and R5is a group R6:wherein R6is as defined for formula (II); hydrolysis of a compound of formula (II) with an alkali metal alkoxide in an alcoholic solvent;C. for a compound of formula (lb), which is a compound of formula (I) in which R1is C1.4 alkyl and R2is C(O)OR5, where R5is C1.6 alkyl or benzyl:wherein R6is as defined for formula (II) and R1ais C1.4 alkyl; reaction of a compound of a compound of formula (XIV):wherein X2is C1.6 alkylene, R12is C1.6 alkyl, R6is as defined for formula (II) and R1ais C1.4 alkyl optionally substituted as defined in claim 1 for R1; with an acid to remove protecting group R12-O-X2;D. for a compound of formula (Ic):wherein R6is as defined for formula (II) and R1 bis R1 bis fluoro or chloro; reaction of a compound of formula (XVI):wherein X2is Ci-e alkylene, R12is Ci-e alkyl, R6is as defined for formula (II) and R1 bis fluoro or chloro by reaction with a fluorinating agent or a chlorinating agent;E. for a compound of formula (I) in which R1is OH or alkoxy i. reaction of a compound of formula (XX):wherein X2is Ci-e alkylene, R12is Ci-e alkyl, R6is as defined for formula (II) and R1cis OH, O(Ci-4 alkyl), N3NH2or =O; with an acid to give a compound of formula (I) in which R2is C(O)OR6, wherein R6is as defined for formula (II); and optionally ii. hydrolysis of the product of step i. to give a compound of formula (I) in which R2is C(O)OH;F. For a compound of formula (I) in which R1is CH2OH, methyl in which one or more H is replaced by Cl or F, CH2N3, CH2NH2 or =CHR8; reaction of a compound of formula (XXVI):wherein X2is Ci-e alkylene, R12is Ci-e alkyl and R6is as defined for formula (II) and R1disCH2OH, methyl in which one or more H is replaced by Cl or F, CH2N3, CH2NH2 or =CHR8; orG. for a compound of formula (I) in which R2is C(O)N(R5a)(R5b); reacting a compound of formula (I) in which R2is C(O)OH by reaction with an amine of formula (XXVII)H-N(R5a)(R5b) (XXVII) wherein R5aand R5bare as defined in claim 1.

24. A compound of (II), (XII), (XIV), (XVI), (XX), (XXV), (XXVI) or (XXVIg):(XIV) (XVI)wherein:R1is as defined for formula (I);R1ais C1.4 alkyl optionally substituted as defined above for R1;R1 bis fluoro or chloro;R1cis OH, O(Ci-4 alkyl), N3NH2or =0;R1dis CH2OH, methyl in which one or more H is replaced by Cl or F, CH2N3, CH2NH2 or =CHR8;R6is C1.6 alkyl or benzyl;R7is a protected OH group, for example R18C(O)O, where R18is C1.6 alkyl or benzyl;R8is H or CH3;R12is Ci-6 alkyl; andX2is Ci-6 alkylene.

25. A compound or salt or solvate thereof according to any one of claims 1 to 22 for use in medicine.

26. A compound or salt or solvate thereof according to any one of claims 1 to 22 for use in the treatment or prevention of a neurodegenerative disorder, a neuromuscular disorder or a mitochondrial disease.

27. The use of compound or salt or solvate thereof according to any one of claims 1 to 22in the manufacture of a medicament for the treatment or prevention of a neurodegenerative disorder, a neuromuscular disorder or a mitochondrial disease.

28. A method for the treatment or prevention of a neurodegenerative disorder, a neuromuscular disorder or a mitochondrial disease, the method comprising administering to a patient in need of such treatment an effective amount of a compound or salt or solvate thereof according to any one of claims 1 to 22.

29. A compound for use, a use or a method wherein: the neurodegenerative disorder is selected from Parkinson’s disease, mild cognitive impairment, dementia (including Alzheimer’s disease, vascular dementia, dementia with Lewy bodies and FTD), Huntington’s disease, amyotrophic lateral sclerosis (motor neurone disease), multiple system atrophy (MSA), progressive supranuclear palsy and Wilson’s disease;30. A pharmaceutical composition comprising a compound or salt or solvate thereof according to any one of claims 1 to 22 and a pharmaceutically acceptable excipient or carrier.

31. A composition according to claim 30 which is formulated for parenteral administration or for oral administration.

32. A phamaceutical composition according to claim 31 or claim 32 further comprising an additional active agent useful in the treatment or prophylaxis of neurodegenerative disorders or neuromuscular disorders.

33. A product comprising a compound of formula (I) and an additional active agent useful in the treatment or prevention of a neurodegenerative or neuromuscular disorder as a combined preparation for simultaneous, sequential or separate use in the treatment or prevention of a neurodegenerative disorder or neuromuscular disorder.

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