WO2020260558A1 - 3-modified iso-/isoallo-lithocholic acid derivatives or their homo-analogs for preventing and treating clostridioides difficile-associated diseases - Google Patents

Abstract

The present invention relates to isolithocholic acid (3β-hydroxy-5β-cholan-24-oic acid) and isoallolithocholic acid (3β-hydroxy-5α-cholan-24-oic acid) together with the respective 22-homo-analogs or the deuterated analogs, which are modified in 3-position, for preventing or treating Clostridioides difficile-associated disease in a mammalian subject.

Description

3-Modified iso-/isoallo- lithocholic acid derivatives or their homo- analogs for preventing and treating Clostridioides difficile-a ssociated diseases

The present invention relates to isolithocholic acid (3p-hydroxy^-cholan-24-oic acid; iso- LCA) and isoallolithocholic acid (3p-hydroxy-5a-cholan-24-oic acid) together with the respective 22-homo-analogs or the deuterated analogs, which are modified in 3-position, for preventing or treating Clostridioides difficile- associated disease in a mammalian subject.

Introduction

Clostridioides difficile (formerly Clostridium difficile, abbreviated C. difficile or C. diff.) is an anaerobic, gram-positive spore-forming rod-shaped bacterium. Sporulation is important to the capacity of this organism to cause disease, as the spores (endospores) persist on environmental surfaces, and are resistant to a number of disinfectants and antibiotics, thereby facilitating transmission. Change of environmental conditions trigger the germination of the spores and the bacteria can proliferate. Members of the genus Clostridium are: C. perfringens, C. tetani, C. botulinium, C. sordellii and C. difficile. Clostridia are associated with diverse human diseases including tetanus, gas gangrene, botulism and pseudomembranous colitis and can be a causative agent in food poisoning.

C. diff. causes Clostridioides difficile-associated diseases (CDAD) or Clostridioides difficile infection (CDI). Over the past decade, the number of CDI has significantly increased with hyper-virulent and drug resistant strains now becoming endemic. CDI is primarily of concern in the hospital setting and is of particular concern amongst elderly patients where mortality rates are particularly high. Of particular concern is the emergence of new endemic strains. A particularly pertinent example is the hyper-virulent BI/NAP1/027 (also known as ribotype 027) strain, which shows increased toxin A and B production as well as the production of additional novel binary toxins.

CDI is a serious issue in the Western World with estimates of up to 700.000 cases of CDI per year in the US alone. The US Center for Disease Control and Prevention report that CDI is responsible for 14.000 deaths per annum in the US and has designated C. diff. as one of three pathogens that poses an immediate public health threat and requires urgent and aggressive action.

C. diff. is a commensal enteric bacterium, the levels of which are kept in check by the normal gut flora. Disruption of indigenous bacterial flora in the intestinal tract by antimicrobial therapy (or, occasionally, by chemotherapy) is a critical element in the pathogenesis of infection. With the understanding that this infection is a complication of antimicrobial therapy, an important therapeutic intervention is discontinuation of the offending drug when possible. Exposure to C. diff. may lead to asymptomatic colonization or infection. Infection is associated with a wide spectrum of clinical manifestations from mild diarrhea through to death.

The current standard of care CDI treatments are the broad spectrum antibiotics, vancomycin and metronidazole. While effective at reducing levels of C. diff., these antibiotics also cause significant collateral damage to the gut flora because of their broad spectrum activity and leave patients vulnerable to disease recurrence, the primary clinical issue. Each additional episode of the disease is associated with greater disease severity and higher mortality rates.

It has been reported that approximately 25% of CDI patients suffer a second episode of the infection, and the risk of further recurrence rises to 65%. Recurrent disease is associated with an increased burden on the healthcare system. Although clindamycin is the major antibiotic associated with CDAD, the disease is now associated with nearly all antibiotics including members of the fluoroquinolone, cephalosporin, macrolide, b-lactam and many others classes.

Current treatment options for CDI

Antibiotic therapy

The Infectious Diseases Society of America (IDSA) currently recommends Metronidazole as the therapeutic agent of choice for mild CDI and Vancomycin for severe CDI.

Newer antibiotics, which are already approved (Fidaxomicin approved by the FDA in May 2011 , DIFICID^®, formerly referred to as OPT-80) or which are in development (Ridinilazole), are aiming to be more effective against C. diff., but trying to spare the healthy gut microbiome. Additionally, these antibiotics are limited to the intestine with no systemic exposure due to low oral bioavailability.

Antitoxins

Bile acid sequestrants like Cholestyramine (Questran) binds toxins A and B of C. diff., but the clinical experience of different investigators has shown marked variation in results. Cholestyramine binds vancomycin and should not be used concurrently with vancomycin therapy.

Actoxumab and Bezlotoxumab are fully human monoclonal antibodies which binds toxins A and B of C. diff., respectively. These antibodies are designed for the prevention of recurrence of CDI but due to the mechanism of action, reduce only the symptoms of the disease but do not eradicate the cause of the disease the CDI. Bezlotoxumab (Zinplava) was approved in October 2016 by the U.S. FDA.

Vaccination

Vaccines based on the neutralization of bacterial toxins have already proven efficacy as illustrated by the decreased prevalence of disease caused by Corynebacterium diphteriae or Clostridium tetani in countries where vaccination programs include these two toxoid vaccines. Currently, two vaccine candidates against CDI are being clinically tested, Sanofi- Pasteur’s toxoid ACAM-CDIFF™ composed of a mixture of formalin-inactivated toxin A and B and Intercell’s recombinant fusion protein containing a part of the receptor-binding domain of toxins A and B as an anti-CDI vaccine candidate.

Gut Microbiome Modulation

Probiotics are not recommended as a single agent for the treatment of active CDI owing to limited data supporting their benefit and a potential risk for septicemia.

However, as it becomes more and more obvious that disruption of the healthy bowel flora is in general the basis for relapsing CDI, restoration of the normal colonic bacterial flora seems to be optimal for the prevention of disease recurrence. This could be e.g. achieved by fecal microbiota transplantation (FMT) which reported clinical cure rates for recurrent CDI with more than 90%.

Prior Art

Bile acids and Clostridioides difficile

Bile acids as germination drivers of C. difficile

Germination of spores of C. diff. within the gastrointestinal tract of a host is critical to initiate C. cf/Tf.-associated diseases since only the vegetative form produces toxin. In general, bacterial spores germinate in a specific environment in the host, often in response to the binding of one and or more small molecules. In case of C. diff. it was first shown in vitro that different conjugates as well as unconjugated primary bile acids such as cholate, taurocholate and glycocholate are able to stimulate germination (J.A. Sorg & A.L. Sonenshein, J. Bacteriol. 2008; 180:2505).

Later experiments in mice proved in vivo that bile acids are related to the germination and disease initiation. Treatment of mice with cholestyramine, a bile salt binding resin, severely decreased the germination capacity of C. diff. spores. On the other hand, treatment of mice with antibiotics stimulated the germination capability in vivo. It was further shown in mice that this effect of antibiotics in the animal model was related to a higher proportion of primary to secondary bile acids in the stool of antibiotic treated mice (J.L. Giel et al. , PlosOne 2010;5:e8740).

These new findings directly lead to the idea that bile acids or bile acids derivatives could be therapeutically used to block spore germination in vivo and thereby the initiation of CDI disease. It was shown that all bile acids lacking a 12a-hydroxyl group on the bile acid scaffold could be in principal used as competitive inhibitors of spore germination (competitive to all bile acids in the host with a 12a-hydroxyl moiety that drives germination).

Further structure-activity-relationship work done on the bile acid scaffold by the Sorg group exemplified that an ester moiety compared to a free carboxylic acid would be an even more preferred structure since this was associated with significant lower inhibitor constants ( ).

Furthermore, it was postulated that effective inhibitors need to resist uptake by the colonic epithelium and to resist 7-dehydroxylation by the colonic gut flora. Therefore the acetylation of i.e. the 7-OH position of the bile acid scaffold was proposed to be preferred (J.A. Sorg & A.L. Sonenshein, J. Bacteriol. 2008; 180:2505 as well as W02010/062369 and WO2015/076788). Claim 9 of WO2010/062369 mentions LCA derivatives with free carboxylic acid moiety (Ri is CO2H), however for the closest analog towards the present invention, i.e. 5p-cholanic acid 3a-ol acetate (see Table 3) no effect on C. diff. spore germination was shown. In WO2010/062369 only 3a-0 bile acids are described, while the racemat is claimed. Noteworthy, in all described bile acids the oxygen in 3-position is in a- orientation. WO2015/076788 is restricted to muricholic acid-based compounds (containing a 6-hydroxy moiety in the steroidal core).

R. Thanissery et al. (Anaerobe 2017;45:86) describe the relationship of germination, growth and toxicity in several C.Diff. strains in the presence of secondary bile acids including iso- LCA. However, they do not mention derivatives of /so-LCA, in particular modifications at the 3-O-position.

US2008/0026077 describes a method for the delivery of a therapeutic to epithelial cells through the use of a bile acid, e.g. /so-LCA, conjugated to a peptide via the carboxylic acid moiety of the bile acid.

For LCA several liabilities in the literature are reported: for example oral administration of LCA results in elevation of alanine transaminase (ALT) indicating hepatocellular injury (A.F. Hofmann, Drug Metab. Rev. 2004;36:703; B.L. Woolbright et al., Toxicol. Lett. 2014;228:56). In addition, LCA is described as Vitamin D agonist (M. Ishizawa et al., J. Lipid Res. 2008;49:763; R. Adachi et al., J. Lipid Res. 2005;46:46), however higher doses can lead to hypercalcemia followed by polyuria. We showed that this Vitamin D agonism is also more pronounced for LCA analogs compared to the matched-pair /so-LCA analogs (see comparison at Example 202).

Bile acids as inhibitors of C. difficile growth

Until 2015 the therapeutic application of bile acid derivatives for treatment of C. diff.- infections was only seen in their inhibitory potential on the germination step which usually takes place in the small intestine of a host. However a recent series of papers showed in different mouse models that secondary bile acids are even capable to prevent the outgrowth of C. diff. vegetative cells in the large intestine - an effect that was fully independent of the before reported effects of bile acids on spore germination (C.G. Buffie et al. , Nature 2015;517:205, M.J. Koenigsknecht et al., Infect. Immun. 2015;83:934, C.M. Theriot & V.B. Young, Annu. Rev. Microbiol. 2015;69:445).

It could be shown in murine models of CDI that there is a direct correlation between the large intestinal amount of secondary bile acids (LCA and deoxycholic acid (DCA)) and the severity of CDI. It was demonstrated that a healthy microbiota that is able to generate enough secondary bile acids through the process of bile acid metabolism in the gastrointestinal tract protects the host against the outgrowth of C. diff. in the large intestine even if the host is challenged with vegetative C. diff. bacteria.

Once the natural bile acid metabolism is interrupted by antibiotic therapy, no or not enough secondary bile acids are generated which makes the host vulnerable for a C. diff. disease.

Of special importance for the protection of the host seem to be the secondary bile acid producing bacteria strains, e.g. Clostridium scindens, which carry the enzyme 7a- dehydroxylase. The reconstitution of the microbiome after antibiotic challenge with the single bacterial strain Clostridium scindens was e.g. sufficient to protect against CDI in a mouse model (C.G. Buffie et al. Nature 2015;517:205).

These findings in murine models were further confirmed by human data: A patient with recurrent C. diff. infections was treated with UDCA and remained infection-free for over 10 months (A.R. Weingarden et al., J. Clin. Gastroenterol. 2016;50:624). Also systematic bile acid profiling in the stool of patients with first time CDI, patients with recurrent CDI and healthy controls mirrors the profiles seen in the mouse models. Secondary bile acids in stool were significantly depleted in the most severe cases of CDI whereas primary bile acids in stool were elevated in recurrent CDI (J.R. Allegretti et al., Aliment. Pharmacol. Ther. 2016;43: 1142).

The impact of various secondary bile acids (including isolithocholic acid) on different C. diff. strains was investigated in vitro by R. Thanissery et al. in Anaerobe 2017;45:86. The study illustrates how C. diff. strains can have different responses when exposed to secondary bile acids in vitro. Many secondary bile acids are able to inhibit TCA mediated spore germination and outgrowth and toxin activity in a dose dependent manner, but the level of inhibition and resistance varied across all strains and ribotypes. Bile acid sensitivity and in vivo virulence of C. diff. clinical isolates using LCA and DCA in in vitro investigations was described by B.B. Lewis et al. in Anaerobe 2016;41 :23.

Finally, also the enormous clinical success of fecal microbiota transplantation (FMT) for last line treatment of patients with several rounds of recurrent CDI could be attributed to reintroduction of bacterial strains with 7a-dehydroxylase activity. Literature mentioning 3-substituted iso- LCA-based bile acids

The synthesis of 3-methyl ether of iso- LCA is mentioned in J. Am. Chem. Soc. 1952;74:5472 while the quantitative determination via mass spectroscopy was described in Biomed. Mass Spectrom. 1980; 11-12:515.

In W02004/037275 and J. Lipid Res. 2005;46:2325 the following compounds

are described as intermediate in the preparation of bile acid conjugates and their use in liposomes for targeted drug delivery into liver cells.

The synthesis of acetamido analog

was described in Nippon Kagaku Zasshi 1959;80: 1310.

WO2017/035501 describes e.g. D-glutamic acid, L-glutamic acid or D-aspartic acid esters of iso- LCA as sialyltransferase inhibitors for treating cancer, inflammatory and immune diseases.

WO2017/125929 describes treatments for modulating gut microbiota, wherein the bile acid is represented by W-X-G, herein G represents the bile acid, X is a bonding member selected from heteroatom, direct C-C or C=C bond and W represents one or two fatty acid radicals. Preferred bile acid derivative is aramchol:

ES2296463 described procedures for the preparation and application of new amide derivatives of bile acids functionalized at C-3 as e.g. tensioactive modifiers in cosmetics with the following representative structure:

W02014/160480 and WO2018/075699 describes methods of preparation and use of neuroactive steroids (e.g. as NMDA receptor modulators). An example is the following structure

There is additional literature, which mention 3p-hydroxy or 3p-amino LCA analogs, however no treatment of C. cf/Tf.-associated diseases is mentioned.

Remaining challenges

In summary, secondary bile acids have a direct impact on vegetative C. diff. cells by growth inhibition, so that they could be used theoretically to treat and/or prevent CDI or recurrent CDI as a kind of supplement for too low colonic secondary bile acid exposure due to antibiotic disturbance of the microbiota. However, several issues are attached to this:

1) A first disadvantage of a direct C. diff. therapy with either natural LCA or DCA are the toxic properties of high LCA/DCA concentrations on colon and liver tissue. E.g. high systemic LCA exposure is related i.e. to liver diseases (B.L. Woolbright et al. in Toxicol. Lett. 2014;228:56.) whereas DCA is known for his proliferative effect on colon tissue and is related to the occurrence of colon tumors (Y.H. Ha et al. in J. Korean Soc. Coloproctol. 2010;26:254). Therefore, systematic and intestinal exposure to these secondary bile acids should be rather limited.

2) A second limitation is the pharmacokinetics of secondary bile acids i.e. with limited colonic exposure due to high absorption especially by the ileal ASBT transporter. This makes it difficult to achieve sufficient exposure in colon and is directly linked to the first disadvantage - the unwanted systemic exposure of both compounds. Based on this, we started testing of isolithocholic acid (5p-cholanic acid-3p-ol) or isoallolithocholic acid (5a-cholanic acid-3p-ol) derivatives, which were modified in 3-position of the bile acid scaffold and we surprisingly identified the claimed compounds as being preferred over the LCA-analogs (with 3a-orientation of the heteroatom) in animal models of C. difficile. A head-to-head comparison of an iso- LCA-analog compared to the LCA-matched pair confirmed a better efficacy expressed as a higher surviving rate in the acute mouse model (Example 1 vs. C1 ; Figure 1 vs. Figure 3).

Summary of the invention

The present invention relates to a compound according to Formula (I)

or a pharmaceutically acceptable salt, co-crystal or solvate thereof for use in the prophylaxis or treatment of Clostridioides difficile associated disease, wherein R, R', X, Y, y and the dotted line are defined as in claim 1.

The present invention further relates to a method of preventing or treating Clostridioides difficile- associated disease in a mammalian subject, comprising administering to a mammalian subject having or is at risk of developing C. difficile- associated disease an effective amount of a compound of Formula (I)

or a pharmaceutically acceptable salt, co-crystal or solvate thereof,

wherein each R, R', X, Y, y and the dotted line are defined as in claim 1.

The present invention also encompasses a pharmaceutical composition comprising a compound according to Formula (I) and a pharmaceutically acceptable carrier or excipient.

Detailed description of the invention

More precisely, the present invention relates to a compound according to Formula (I) or a pharmaceutically acceptable salt, co-crystal or solvate thereof for use in the prophylaxis or treatment of Clostridioides difficile associated disease, wherein:

R' is selected from H, CrC₆-alkyl, C₂-C₆-alkenyl and C₂-C₆-alkynyl,

wherein alkyl, alkenyl and alkynyl is unsubstituted or substituted with 1 to 7 substituents independently selected from the group consisting of CN, halogen, azide, oxo, OR¹⁰, O- C₂-C₆-alkylene-OR¹⁰, 0-C₃-io-cycloalkyl, 0-C₃-io-heterocycloalkyl, -(CH₂-CH₂-0)_n- CH2CH2R¹², -0-(CH₂-CH2-0)n-CH2CH₂R¹², Co-Cs-alkylene-R¹⁰, CO2R¹⁰, CONR¹⁰R¹¹ , CONR¹⁰SO₂R¹⁰, COR¹⁰, SO_XR^{1 0}, SO₃H , SO₂NR¹⁰R¹¹ , NR¹⁰COR¹¹ , N R¹⁰SO₂R^{1 1} , N R¹⁰- CO-NR¹⁰R¹¹ , NR¹⁰-SO₂-NR¹⁰R¹¹ , NR¹⁰R¹¹ and N(R¹⁰R¹⁰R¹¹)⁺;

R is selected from H, Ci-Cio-alkyl, C₂-Cio-alkenyl, C₂-Cio-alkynyl, Co-Cio-alkylene-C₃-io- cycloalkyl and Co-Cio-alkylene-C₃-io-heterocycloalkyl,

wherein alkyl, alkenyl, alkynyl, alkylene, cycloalkyl and heterocycloalkyl is unsubstituted or substituted with 1 to 7 substituents independently selected from the group consisting of CN, halogen, azide, oxo, OR¹⁰, 0-C₂-C₆-alkylene-OR¹⁰, O-C_3-10- cycloalkyl, 0-C₃-io-heterocycloalkyl, -(CH₂-CH2-0)n-CH2CH₂R¹², -0-(CH₂-CH₂-0)_n- CH2CH2R¹², -(CH2-CH₂-0)n-CH₂-(C=0)-R¹², -0-(CH2-CH₂-0)n-CH₂-(C=0)R¹², C₀-C₈- alkylene-R¹⁰, CO₂R¹⁰, CONR¹⁰R¹¹ , CONR¹⁰SO₂R¹⁰, COR¹⁰, SO_xR¹⁰, S0₃H, SO₂NR¹⁰R¹¹ , NR¹⁰COR^{1 1} , NR¹⁰SO₂R^{1 1} , NR¹⁰-CO-NR¹⁰R^{1 1} , NR¹⁰-SO₂-NR¹⁰R^{1 1} , NR¹⁰R¹¹ and N(R¹⁰R¹⁰R¹¹)⁺;

X is selected from O, NH, NCi-₆-alkyl, N-halo-Ci-₆-alkyl, N-CO-Ci-₆-alkyl and N-CO-halo-Ci- ₆-alkyl;

Y is selected from a bond, C=0 and (C=0)-NH;

y = 1 or 2;

the dotted line represents an optional double bond;

R¹⁰ and R¹¹ is independently selected from H, Ci-₆-alkyl, halo-Ci-₆-alkyl, -(CH₂-CH₂-0)_m- CH₂CH₂R¹², Co-C₈-alkylene-C₃-io-cycloalkyl and Co-Cs-alkylene-Cs-io-heterocycloalkyl, wherein

alkyl, alkylene, cycloalkyl and heterocycloalkyl are unsubstituted or substituted with 1 to 6 substituents independently selected from the group consisting of halogen, CN, OH, oxo, Ci-₃-alkyl, halo-Ci-₃-alkyl, O-Ci-₃-alkyl, O-halo-Ci-₃-alkyl, S0₂-Ci-₃-alkyl, NR¹¹¹ R¹¹², CO₂R¹¹¹ and CONR¹¹¹ R¹¹²; or wherein R¹⁰ and R¹¹ when taken together with the atom(s) to which they are attached complete a 3- to 8-membered ring containing carbon atoms and optionally containing 1 to 4 heteroatoms selected from O, S and N, wherein the ring is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of fluoro, OH, oxo, Ci- 3-alkyl and halo-Ci-3-alkyl;

R¹² is independently selected from halogen, OH, O-Ci-₆-alkyl, azide, NH2, NH-(CH₂-CH₂-0)_m- Ci-6-alkyl, N((CH₂-CH₂-0)m-C₁-6-alkyl)2, NH-CO-Ci-e-alkyl, NCi-e-alkyl-CO-Ci-e-alkyl, NH-CO- CH₂0-(CH₂-CH₂-0)m-H, NH-C0-CH₂0-(CH₂-CH₂-0)m-C_1-6-alkyl and NH-(CH₂-CH₂-0)_m-C_1-6- alkyl;

R¹¹¹ and R¹¹² is independently selected from H, Ci-4-alkyl, halo-Ci-4-alkyl, Co-C₄-alkylene-C₃- 10-cycloalkyl and Co-C4-alkylene-C3-io-heterocycloalkyl, wherein

alkyl, alkylene, cycloalkyl and heterocycloalkyl are unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of halogen, CN, OH, oxo, Ci-3-alkyl, halo-Ci-3-alkyl, O-Ci-3-alkyl, O-halo-Ci-3-alkyl, S02-Ci-3-alkyl, NH2, NHCi-3-alkyl, N(Ci.₃-alkyl)₂, C0₂H, C0₂-C_1-3-alkyl, CONH₂, CONHC^-alkyl and CON(Ci-3-alkyl)₂;

or wherein R¹¹¹ and R¹¹² when taken together with the nitrogen to which they are attached complete a 3- to 8-membered ring containing carbon atoms and optionally containing 1 to 3 heteroatoms selected from O, S or N, wherein the ring is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of fluoro, OH, oxo, C1-3- alkyl and halo-Ci-3-alkyl;

m is independently selected from 0 to 15;

n is independently selected from 0 to 30;

x is selected from 0 to 2; and

one or more hydrogen(s) in Formula (I) or in the residues may be replaced by deuterium(s); with the proviso, that when R' is hydrogen or deuterium, y is 1 and the dotted line is not present then -X-Y-R is not OH.

In a preferred embodiment in combination with any of the above and below embodiments, the compound for use is represented by Formula (II)

In a more preferred embodiment in combination with any of the above and below embodiments, the compound for use is represented by Formula (III)

In a preferred embodiment in combination with any of the above and below embodiments, y is 1.

In a preferred embodiment in combination with any of the above and below embodiments, R' is H.

In a preferred embodiment in combination with any of the above and below embodiments, X is O.

In a further preferred embodiment in combination with any of the above and below embodiments,

R' is H;

Y-X is selected from O, (C=0)-0 and NH-(C=0)-0;

R is selected from CrC4-alkyl and C2-C4-alkenyl,

wherein alkyl and alkenyl is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, OR¹⁰, NR¹⁰R^{1 1} , -0-(CH₂- CH₂-0)n-CH2CH₂R¹², -0-(CH₂-CH₂-0)n-CH₂-(C=0)R¹² and C0₂R¹⁰;

R¹⁰ and R¹¹ is independently selected from H, CrC4-alkyl and fluoro-Ci-C4-alkyl;

R¹² is selected from OH, 0-Ci-₄-alkyl, NH₂, NHCi-₆-alkyl, N(Ci.₆-alkyl)₂, NH-CO-C^-alkyl and NCi-e-alkyl-CO-Ci-e-alkyl; and

n is selected from 0 to 10.

In an even more preferred embodiment in combination with any of the above and below embodiments,

R' is H;

Y-X is selected from O, (C=0)-0 and NH-(C=0)-0;

R is CrC4-alkyl,

wherein alkyl is substituted with -0-(CH₂-CH₂-0)_n-CH₂CH₂R¹² or -0-(CH₂-CH₂-0)_n- CH₂-(C=0)R¹²; R¹² is selected from OH, O-C^-alkyl, NH₂, NHCi-e-alkyl, I Ci-e-alkyOa, NH-CO-Ci-e-alkyl and NCi-₆-alkyl-CO-Ci-₆-alkyl; and

n is selected from 0 to 10.

In an even more preferred embodiment in combination with any of the above and below embodiments,

R' is H;

p = 1 to 5.

In an even more preferred embodiment in combination with any of the above and below embodiments,

R' is H;

p = 2 to 5.

In a similar more preferred embodiment in combination with any of the above and below embodiments,

R' is H;

X-Y-R is OEt;

y = 1 or 2;

and the dotted line represents an optional double bond. In an alternatively preferred embodiment in combination with any of the above and below embodiments, one or more hydrogen(s) in Formula (I) or in the residues is/are replaced by deuterium(s).

In a further preferred embodiment in combination with any of the above and below embodiments, one or more hydrogen(s) in R or R' is/are replaced by deuterium(s).

In an even further preferred embodiment in combination with any of the above and below embodiments, R is D.

In a similar even further preferred embodiment in combination with any of the above and below embodiments, X-Y-R is OD2CD₃.

In an alternatively preferred embodiment in combination with any of the above and below embodiments,

R' is selected from CrC₆-alkyl, C2-C6-alkenyl and C2-C6-alkynyl,

wherein alkyl, alkenyl and alkynyl is unsubstituted or substituted with 1 to 7 substituents independently selected from the group consisting of CN, halogen, azide, oxo, OR¹⁰, O- C2-C6-alkylene-OR¹⁰, 0-C₃-io-cycloalkyl, 0-C₃-io-heterocycloalkyl, -(CH₂-CH₂-0)_n- CH2CH2R¹², -0-(CH₂-CH2-0)n-CH2CH₂R¹², C₀-C₈-alkylene-R¹⁰, CO2R¹⁰, CONR¹⁰R^{1 1} , CONR¹⁰SO₂R¹⁰, COR¹⁰, SO_XR¹⁰, SO₃H, SO₂NR¹⁰R^{1 1} , N R¹⁰COR^{1 1} , N R¹⁰SO₂R¹¹ , NR¹⁰- CO-NR¹⁰R¹¹ , NR¹⁰-SO₂-NR¹⁰R¹¹ , NR¹⁰R¹¹ and N(R¹⁰R¹⁰R¹¹)⁺; and

X is O.

In a more alternatively preferred embodiment in combination with any of the above and below embodiments,

R' is CrC4-alkyl,

wherein alkyl is unsubstituted or substituted with -0-(CH2-CH2-0)_n-CH2CH2R¹²;

R is selected from H and CrC4-alkyl,

wherein alkyl is unsubstituted or substituted with -0-(CH2-CH2-0)_n-CH2CH2R¹²;

Y is selected from a bond or C=0;

R¹² is independently selected from OH or OMe; and

n is independently selected from 0 to 5.

In an even more preferred embodiment in combination with any of the above and below embodiments, the compound for use is selected from

or a pharmaceutically acceptable salt, co-crystal or solvate thereof.

In a most preferred embodiment in combination with any of the above and below embodiments, the compound for use is selected from

or a pharmaceutically acceptable salt, co-crystal or solvate thereof.

In an even most preferred embodiment in combination with any of the above and below embodiments, the compound for use is selected from or a pharmaceutically acceptable salt, co-crystal or solvate thereof.

In an upmost preferred embodiment in combination with any of the above and below embodiments, the compound for use is

or a pharmaceutically acceptable salt, co-crystal or solvate thereof.

Also provided is a pharmaceutical composition comprising the compound of the invention and a pharmaceutically acceptable carrier or excipient.

In the context of the present invention“Ci-C₆-alkyl” means a saturated alkyl chain having 1 to 6 carbon atoms which may be straight chained or branched. Examples thereof include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert- butyl, n- pentyl, isopentyl, n-hexyl and isohexyl. Similarly,“Ci-C4-alkyl” means a saturated alkyl chain having 1 to 4 carbon atoms which may be straight chained or branched. Examples thereof include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tert- butyl. Same applies to“Ci-Cio-alkyl” which means a saturated alkyl chain having 1 to 10 carbon atoms which may be straight chained or branched.

The term "halo-Ci-C₆-alkyl" means that one or more hydrogen atoms in the alkyl chain are replaced by a halogen. A preferred example thereof is CH2F, CHF2 and CF₃.

“Co-Cs-alkylene” means that the respective group is divalent and connects the attached residue with the remaining part of the molecule. Moreover, in the context of the present invention, “Co-alkylene” is meant to represent a bond, whereas Ci-alkylene means a methylene linker, C2-alkylene means an ethylene linker or a methyl-substituted methylene linker and so on.

A C2-Cio-alkenyl means a straight chained or branched alkyl chain having 2 to 10 carbon atoms and at least one carbon to carbon double bond. Examples thereof include ethenyl, propenyl and decenyl. Consequently, a C2-C6-alkenyl means a straight chained or branched alkyl chain having 2 to 6 carbon atoms and at least one carbon to carbon double bond.

Similarily, a C2-Cio-alkynyl means a straight chained or branched alkyl chain having 2 to 10 carbon atoms and at least one carbon to carbon triple bond. Examples include ethynyl, propynyl and decynyl. Consequently, a C2-C6-alkynyl means a straight chained or branched alkyl chain having 2 to 6 carbon atoms and at least one carbon to carbon triple bond.

A C3-io-cycloalkyl group means a saturated or partially unsaturated mono-, bi-, spiro- or multicyclic ring system comprising 3 to 10 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octanyl, spiro[3.3]heptyl, bicyclo[2.2.1]heptyl, adamantyl and pentacyclo[4.2.0.0²'⁵.0³'⁸.0⁴'⁷]octyl. Consequently, a 3- to 6-membered cycloalkyl group means a saturated or partially unsaturated mono-, bi- or spirocyclic ring system comprising 3 to 6 carbon atoms whereas a 5- to 8-membered cycloalkyl group means a saturated or partially unsaturated mono-, bi-, or spirocyclic ring system comprising 5 to 8 carbon atoms.

A C3-io-heterocycloalkyl group means a saturated or partially unsaturated 3- to 10-membered carbon mono-, bi-, spiro- or multicyclic ring wherein 1 , 2, 3 or 4 carbon atoms are replaced by 1 , 2, 3 or 4 heteroatoms, respectively, wherein the heteroatoms are independently selected from N, O, S, SO and SO2 if not indicated otherwise. Examples thereof include epoxidyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl tetrahydropyranyl, 1 ,4-dioxanyl, morpholinyl, 4-quinuclidinyl, 1 ,4-dihydropyridinyl and 6- azabicyclo[3.2.1]octanyl. The heterocycloalkyl group can be connected with the remaining part of the molecule via a carbon, nitrogen (e.g. in morpholine or piperidine) or sulfur atom. An example for a S-linked heterocycloalkyl is the cyclic sulfonimidamide

Halogen is selected from fluorine, chlorine, bromine and iodine, more preferably fluorine or chlorine and most preferably fluorine.

Any formula or structure given herein, is also intended to represent unlabelled forms as well as isotopically labelled forms of the compounds. Isotopically labelled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as, but not limited to ²H (deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹ P, ³²P, ³⁵S, ³⁶CI and ¹²⁵l. Various isotopically labelled compounds of the present disclosure, for example those into which radioactive isotopes such as ³H, ¹³C and ¹⁴C are incorporated. Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays or in radioactive treatment of patients. Isotopically labelled compounds of this disclosure and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

The disclosure also includes“deuterated analogs” of compounds of Formula (I) to Formula (III) in which from 1 to z hydrogens attached to a carbon atom is/are replaced by deuterium, in which z is the number of hydrogens in the molecule. Such compounds may exhibit increased resistance to metabolism and thus be useful for increasing the half-life of any compound of Formula (I) to Formula (III) when administered to a mammal, e.g. a human. See, for example, Foster in Trends Pharmacol. Sci. 1984:5;524. Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens have been replaced by deuterium.

Deuterium labelled or substituted therapeutic compounds of the disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and/or an improvement in therapeutic index.

The concentration of such a heavier isotope, specifically deuterium, may be defined by an isotopic enrichment factor. In the compounds of this disclosure any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is designated specifically as“H” or“hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition. Accordingly, in the compounds of this disclosure any atom specifically designated as a deuterium (D) is meant to represent deuterium (e.g. Example 1 -5 or 1 -9).

Furthermore, the compounds of the present invention are partly subject to tautomerism. For example, if a heteroaromatic group containing a nitrogen atom in the ring is substituted with a hydroxy group on the carbon atom adjacent to the nitrogen atom, the following tautomerism can appear:

It will be appreciated by the skilled person that when lists of alternative substituents include members which, because of their valency requirements or other reasons, cannot be used to substitute a particular group, the list is intended to be read with the knowledge of the skilled person to include only those members of the list which are suitable for substituting the particular group.

Where tautomerism, like e.g. keto-enol tautomerism, of compounds of the present invention or their prodrugs may occur, the individual forms, like e.g. the keto and enol form, are each within the scope of the invention as well as their mixtures in any ratio. Same applies for stereoisomers, like e.g. enantiomers, cis/trans- isomers, atropisomers, conformers and the like.

If desired, isomers can be separated by methods well known in the art, e.g. by liquid chromatography.

The compounds of the present invention can be in the form of a pharmaceutically acceptable salt or a solvate. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids. In case the compounds of the present invention contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts. Thus, the compounds of the present invention which contain acidic groups can be present on these groups and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. The compounds of the present invention which contain one or more basic groups, i.e. groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art. If the compounds of the present invention simultaneously contain acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). The respective salts can be obtained by customary methods which are known to the person skilled in the art like, for example, by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the compounds of the present invention which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

Further, the compounds of the present invention may be present in the form of solvates, such as those which include as solvate water, or pharmaceutically acceptable solvates, such as alcohols, in particular ethanol.

Moreover, the compounds of the present invention may be present in the form of co-crystals. Co-crystals consist of two or more components that form a unique crystalline structure having unique properties. More preferred, co-crystals are solids that are crystalline single phase materials composed of two or more different molecular or ionic compounds generally in a stoichiometric ratio which are neither solvates nor simple salts.

Furthermore, the present invention provides pharmaceutical compositions comprising at least one compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof as active ingredient together with a pharmaceutically acceptable carrier.

"Pharmaceutical composition" means one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing at least one compound of the present invention and a pharmaceutically acceptable carrier.

The compounds described by Formula (I) to Formula (III) are useful for preventing or treating diseases associated with C. difficile. Exposure to C. diff. may lead i.e. in elderly and immune- compromised people to colonization and infection. Once colonized, C. diff. produces toxins which results in a range of clinical signs and symptoms, from inflammation of the mucosal epithelium, diarrhea and cramping in mild cases to the development of pseudomembranous colitis and death in severe cases. Pseudomembranous colitis and C. diff. colitis represent the more severe clinical pictures.

The compositions are suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation) or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy. Experimental Section

The compounds of the present invention can be prepared by a combination of methods known in the art including the procedures described in this section.

Abbreviations

Bn benzyl

Cbz carbobenzoxy-(benzyloxycarbonyl)

EA ethyl acetate

FCC flash column chromatography on silica gel

h hour(s)

PE petroleum ether

sat. saturated (aqueous)

Isolithocholic acid (CAS: 1534-35-6) is commercially available (e.g. Steraloids; catalogue ID: C1475-000) or can be prepared from LCA by Mitsunobu reaction using 4-nitrobenzoic acid, triphenylphosphine and diethyl diazodicarboxylate and subsequent saponification with aqueous KOH (P. Miro et al.; Chem. Commun. 2016;52:713). Isoallolithocholic acid (CAS: 2276-93-9) is commercially available (e.g. Alfa Chemistry; order number: ACM2276939 or Steraloids; catalog ID: C0700-000).

In an alternative approach, the same procedure can be applied starting with LCA methyl ester:

Step 1 : (3S.5 8 9S, 10S, 13R 14S, 17 )-17-(( )-5-Methoxy-5-oxopentan-2-yl)-10, 13- dimethylhexadecahvdro-1 /-/-cvclopentaralphenanthren-3-yl 4-nitrobenzoate (P1a)

To a solution of lithocholic acid methyl ester (19.5 g, 50 mmol), 4-nitrobenzoic acid (8.4 g, 50 mmol) and PPhb (13.1 g, 50 mmol) in tetrahydrofuran (500 mL) was added diisopropyl azodicarboxylate (75 ml_, 75 mmol) under N₂ at 0°C and then the mixture was stirred at rt overnight. The mixture was filtered and the solid was dried under reduced pressure to afford the compound P1 a as a white solid.

Step 2: Isolithocholic acid (P1)

To a solution of compound P1a (17.6 g, 33 mmol) in tetrahydrofuran (300 ml_) was added 1 N NaOH (50 ml_) and then the mixture was stirred at rt for 1 h, quenched by addition of 1 N HCI (60 ml_) and extracted with EA (3 x 300 ml_). The combined organic layer was concentrated and purified by FCC (EA:PE = 3: 1) to give compound P1 as a white solid. ¹ H- NMR (500 MHz, DMSO-d₆): d 1 1.95 (br s, 1 H), 4.17 (br s, 1 H), 3.87 (s, 1 H), 2.27-2.21 (m, 1 H), 2.13-2.04 (m, 1 H), 1.93-1.62 (m, 6H), 1.54-1.48 (m, 1 H), 1.40-0.98 (m, 19H), 0.89 (s, 3H), 0.87 (d, J = 6.5 Hz, 3H), 0.61 (s, 3H). MS: 375.2 (M-H)-.

Preparative Example P1 -1

Using a similar procedure as described for Example 1 , the following compound was prepared:

# educt structure analytical data

¹H-NMR (500 MHz, DMSO-de) d:

3-d1-lsolithocholic acid (P2)

To a solution of 3-oxo-5p-cholanoic acid (5.00 g, 13.4 mmol) in CD₃OD (30 ml_) was added NaBD₄ (730 mg, 17.4 mmol) at 0°C. The mixture was stirred at this temperature for 3 h, quenched with sat. NH₄CI (100 ml_) and extracted with EA (3 x 200 ml_). The combined organic layer was concentrated and purified by FCC (EA:PE = 3: 1) to afford compound P2 as a white solid (beside the 3a-hydroxy isomer). ¹H-NMR (500 MHz, DMSO-d6) d: 1 1.93 (s, 1 H), 4.13 (s, 1 H), 2.25-2.18 (m, 1 H), 2.14-2.06 (m, 1 H), 1.93-1.62 (m, 6H), 1.55-0.96 (m, 20H), 0.89 (s, 3H), 0.87 (d, J = 6.5 Hz, 3H), 0.61 (s, 3H). MS: 376.3 (M-H)-. (R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-Ethoxy-10, 13-dimethylhexadecahvdro-1 H-cvclo- pentaralphenanthren-17-yl)pentanoic acid (1)

To a solution of compound P1 (200 g, 0.5 mmol) in dry THF (50 ml_) was added NaH (80 mg, 1.0 mmol) and ethyl iodide (70 mg, 0.5 mmol). The mixture was reflux at 60°C overnight, quenched with 5% HCI and extracted with EA (3 x 50 ml_), The combined organic layer was concentrated and purified by FCC (MeOH/DCM = 1:15) to give compound 1 as a white solid. ¹H-NMR (500 MHz, CDC ): d 3.60 (s, 1H), 3.46-3.39 (m, 2H), 2.40-2.36 (m, 1H), 2.29-2.26 (m, 1H), 1.97-1.95 (m, 1H), 1.86-1.79 (m, 4H), 1.62-1.02 (m, 24H), 1.06-0.92 (m, 6H), 0.65 (s, 3H). MS: 403.3 (M-H)-.

Example 1-1 to 1-12

Using a similar procedure as described for Example 1, the following compounds were prepared:

# conditions structure analytical data

Ή-NMR (500 MHz, DMSO-de):

8 eq. methyl iodide, d 11.94 (brs, 1H), 4.43 (s, 1H), l_^ 8 eq. NaH; stirred at 3.16 (s, 3H), 2.26-0.98 (m,

70°C overnight 28H), 0.88-0.86 (m, 6H), 0.61

(s, 3H).

¹H-NMR (500 MHz, DMSO-de): d 11.91 (brs, 1H), 3.20 (s, 3H), isoallolithocholic acid, 3.10-3.03 (m, 1H), 2.26-2.19 3.3 eq. methyl iodide, (m, 1 H), 2.13-2.07 (m, 1H), 1-2 6.7 eq. NaH; stirred at 1.94-1.88 (m, 1H), 1.83-0.81

60°C overnight (m, 27H), 0.74 (s, 3H), 0.64-

0.58 (m, 4H). MS: 389.2 (M-H)

¹H-NMR (500 MHz, DMSO-de):

8 eq.1-fluoro-2- d 11.92 (s, 1 H), 4.49 (dt, J = iodoethane, 8 eq. NaH; 48.0, 4.0 Hz, 2H), 3.60-3.52 (m, 1-3 stirred at 70°C 3H), 2.26-0.97 (m, 28H), 0.89- overnight 0.86 (m, 6H), 0.62 (s, 3H). MS:

421.3 (M-H)-

¹H-NMR (500 MHz, DMSO-de): d 11.94 (brs, 1H), 5.92-5.80

8 eq.3-iodoprop-1-ene, (m, 1 H), 5.23 (dd, J = 17.3, 1.8 8 eq. NaH; stirred at Hz, 1 H), 5.09 (J = 10.5, 1.0 Hz, 70°C overnight 1 H), 3.89-3.87 (m, 2H), 3.59 (s,

1 H), 2.25-0.98 (m, 28H), 0.89-

0.86 (m, 6H), 0.61 (s, 3H). # conditions structure analytical data

Ή-NMR (400 MHz CDCI ) d

¹H-NMR (500 MHz, DMSO-de)

8 eq. NaH, 8 eq. d: 11.95 (s, 1H), 3.56-3.48 (m,

5H), 3.43-3.42 (m, 4H), 3.24 (s,

1-6 ^Br^" stirred 3H), 2.26-0.98 (m, 28H), 0.88- at 70°C overnight 0.86 (m, 6H), 0.61 (s, 3H). MS:

477.3 (M-H)-.

¹H-NMR (500 MHz, DMSO-de)

8 eq. NaH, 8 eq. d: 11.94 (brs, 1H), 3.57-3.49

(m, 9H), 3.44-3.41 (m, 4H),

; stirred 3.24 (s, 3H), 2.26-0.97 (m, at 70°C overnight 28H), 0.88-0.86 (m, 6H), 0.61

(s, 3H). MS: 521.1 (M-H) .

¹H-NMR (500 MHz DMSO-d )

¹

¹H-NMR (500 MHz, DMSO-de) d: 11.96 (brs, 1H), 6.05 (dd, J

J = ,

0.83 (s, 3H), 0.69 (s, 3H).401.3

(M-H)-.

¹H-NMR (500 MHz, DMSO-de)

8 eq. NaH, 8 eq. d: 11.94 (s, 1H), 3.56 (s, 1H),

1 -bromo-2-methoxy 3.44-3.41 (m, 4H), 3.25 (s, 3H),-11 ethane; stirred at 70°C 2.26-0.98 (m, 28H), 0.88-0.86 overnight (s, 6H), 0.61 (s, 3H).433.1 (M-

H)-.

8 eq. NaH, 8 eq. methyl

4-bromobutanoate;

-12 stirred at 70°C

overnight

(R)-4-((3S.5R.8R.9S.10S.13R.14S.17R)-10.13-Dimethyl-3-propoxyhexadecahvdro-1 H- cyclopentaralphenanthren-17-yl)pentanoic acid (2)

To a solution of compound 1 -4 (100 g, 0.10 mmol) in THF (10 ml_) was added 10%Pd on charcoal (10 mg) and then stirred at rt overnight under a H2 atmosphere. The mixture was filtered, concentrated and then purified by FCC (EA:PE = 1 :5) to give compound 2 as a white solid. ¹ H-NMR (500 MHz, DMSO-d₆) d: 11.93 (s, 1 H), 3.52 (s, 1 H), 3.27-3.24 (m, 2H), 2.26- 0.98 (m, 30H), 0.88-0.84 (m, 9H), 0.61 (s, 3H).

Example 3

(4R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-10, 13-Dimethyl-3-(oxiran-2-ylmethoxy)hexadeca- hydro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (3)

To a solution of compound 1 -4 (200 mg, 0.20 mmol) in DCM (50 ml_) was added m-CPBA (344 mg, 0.20 mmol). The mixture was stirred at rt for 2 h, filtered and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 : 15) to give compound 3 as a white solid.

Example 4

(4R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-(2,3-Dihvdroxypropoxy)-10, 13-dimethylhexa- decahvdro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (4)

To a solution of compound 3 (100 mg, 0.10 mmol) in THF (20 ml_) was added 1 N NaOH (2 ml_). The mixture was stirred at 40°C for 8 h, quenched with 5% HCI and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 :15) to give compound 4 as a white solid. ¹ H-NMR (500 MHz, DMSO-d₆) d: 11.95 (s, 1 H), 4.52 (br s, 1 H), 4.41 (br s, 1 H), 3.53-3.50 (m, 2H), 3.40-3.19 (m, 4H), 2.26-0.97 (m, 28H), 0.88-0.86 (m, 6H), 0.61 (s, 3H). MS: 449.2 (M-H) .

(R)-4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-3-((Ethylcarbamoyl)oxy)-10, 13-dimethylhexadeca- hydro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (5)

To a solution of compound P1 (200 mg, 0.50 mmol) in toluene (50 ml_) was added isocyanatoethane (100 mg, 1.00 mmol) and EΐbN (102 mg, 1.00 mmol). The mixture was stirred at 100°C overnight, cooled to rt, quenched with 5% HCI and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 :15) to give compound 5 as a white solid. ¹ H-NMR (500 MHz, DMSO-d₆) d: 11.95 (s, 1 H), 6.94 (t, J = 5.3 Hz, 1 H), 4.79 (s, 1 H), 2.99-2.95 (m, 2H), 2.25-0.97 (m, 31 H), 0.91 (s, 3H), 0.87 (d, J = 7.0 Hz, 3H), 0.62 (s, 3H). MS: 446.1 (M-H)-.

(R)-4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-3-((3-Carboxypropanoyl)oxy)-10, 13-dimethylhexa- decahvdro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (6)

To a solution of compound P1 (200 g, 0.50 mmol) in toluene (50 ml_) was added succinic anhydride (100 mg, 1.00 mmol) and EΐbN (102 mg, 1.00 mmol). The mixture was stirred at 100°C overnight, cooled to rt, quenched with 5% HCI and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 :15) to afford compound 6 as a white solid. ¹ H-NMR (500 MHz, DMSO-d₆) d: 12.07 (br s, 2H), 4.95 (s, 1 H), 2.48-2.46 (m, 4H), 2.26-0.97 (m, 28H), 0.92 (s, 3H), 0.87 (d, J = 6.0 Hz, 3H), 0.62 (s, 3H). MS: 499.3 (M+Na)⁺. (R)-4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-3-(2-Methoxyacetoxy)-10, 13-dimethylhexadeca- hydro-1 H-cyclopentaralohenanthren-17-yl)pentanoic acid (7)

To a solution of compound P1 (200 mg, 0.50 mmol) in DCM (50 ml_) was added 2- methoxyacetyl chloride (108 mg, 1.00 mmol) and EΐbN (102 mg, 1.00 mmol). The mixture was stirred at rt overnight, quenched with 5% HCI and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 : 15) to give compound 7 as a white solid. ¹ H-NMR (500 MHz, DMSO-d₆) d: 1 1.95 (s, 1 H), 5.05 (s, 1 H), 4.02 (s, 2H), 3.31 (s, 3H), 2.26-0.97 (m, 28H), 0.92 (s, 3H), 0.87 (d, J = 7.0 Hz, 3H), 0.62 (s, 3H). MS: 471.4 (M+Na)⁺.

Step 1 : Benzyl (R)-4-((3R.5R.8R.9S.10S.13R.14S.17R)-3-hvdroxy-10.13-dimethylhexa- decahydro-1 H-cyclopentaralphenanthren-17-yl)pentanoate (8a)

Compound 8a was prepared as described in W02017101789 (K₂CO₃ in DMF at 85°C for 2 h).

Step 2: Benzyl (R)-4-((3S.5R.8R.9S.10S.13R.14S.17R)-3-(2-(2-(2-methoxy- ethoxy)ethoxy)acetoxy)-10, 13-dimethylhexadecahvdro-1 H-cyclopentaralohenanthren-17- vDoentanoate

Compound 8a (1.76 g), 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (1.0 ml_) and 2-diphenyl- phosphinopyridine (1.6 g) were combined in dry THF (20 ml_) and cooled in an ice bath under a flow of argon. Diisopropylazodicarboxylate (1.4 ml_) was diluted in dry THF (2 ml_) and added dropwise to the mixture. The mixture was allowed to reach rt, stirred over the weekend and then partitioned between 4N HCI and EA. The organic phase was washed with 4N HCI, water and brine, dried over Na2SC>4, concentrated and purified by FCC (cyclohexane/EA) to afford compound 8b as a colorless solid.

Step 3: (R)-4-((3S.5R.8R.9S.10S.13R.14S.17R)-3-(2-(2-(2-Methoxy- ethoxy)ethoxy)acetoxy)-10, 13-dimethylhexadecahvdro-1 H-cyclopentafalphenanthren-l 7- vDoentanoic acid (8)

Palladium on activated charcoal (10%, 90 mg) was suspended in a deoxygenated solution of the compound 8b (2.3 g) in MeOH (25 ml_). The flask was flushed with argon, then with hydrogen. After 14 h at rt under an atmospheric pressure the mixture was filtered through a pad of celite and concentrated. FCC (cyclohexane/EA = 5: 1 + 0.05% of formic acid) afforded compound 8 as a colorless solid. ¹ H-NMR (300 MHz, CDCh) d: 9.85 (br s, 1 H), 5.15 (s, 1 H), 4.10 (s, 2H), 3.73-3.62 (m, 6H), 3.56-3.52 (m, 2H), 3.36 (s, 3H), 2.41-2.16 (m, 2H), 1.99-0.97 (m, 26H), 0.93 (s, 3H), 0.89 (d, J = 7.0 Hz, 3H), 0.63 (s, 3H). ¹³C-NMR (75 MHz, CDCh) d: 180.00, 170.11 , 71.90, 71.62, 70.87, 70.65, 70.53, 68.85, 59.01 , 56.52, 55.96, 42.76, 40.14, 39.90, 37.35, 35.63, 35.31 , 34.85, 31.00, 30.77, 30.71 , 30.59, 28.15, 26.44, 26.11 , 25.02, 24.17, 23.85, 21.07, 18.25, 12.08. MS: 559.9 (M+Na)⁺. (Preparative) Example 8-1 to 8-5

Using a similar procedure as described for Example 8, the following compounds were prepared:

# conditions structure analytical data

1H-NMR (300 MHz, CDCh) d: 8.92 (br s,

1 H), 5.17 (s, 1 H), 4.13

30H), 0.89 (s, 3H), 0.88-0.81 (m, 6H), 0.58 # conditions structure analytical data

(s, 3H). MS: 557.0 (M+Na)⁺.

3H). MS: 545.8 (M+Na)⁺.

Example 9

Step 1 : Benzyl (R)-4-((3R.5R.8R.9S.10S.13R.14S.17R)-10.13-dimethyl-3-((methyl- sulfonyl)oxy)hexadecahvdro-1 H-cyclopentaralphenanthren-17-yl)pentanoate (9a)

To a solution of benzyl lithocholate (1.00 g, 2.00 mmol) in DCM (100 ml_) was added MsCI (228 mg, 2.00 mmol) and EίbN (808 mg, 8.00 mmol) and then the mixture was stirred at rt overnight, quenched with H2O (100 ml_) and extracted with EA (3 x 100 ml_). The combined organic layer was concentrated and purified by FCC (EA:PE = 1 :5) to give compound 9a as a white solid.

Step 2: Benzyl (R)-4-((3S.5R.8R.9S.10S.13R,14S.17R)-3-(2-(2-hvdroxyethoxy)ethoxy)-

10, 13-dimethylhexadecahvdro-1 H-cyclopentaralphenanthren-17-yl)pentanoate (9b)

To a solution of compound 9a (500 mg, 0.90 mmol) in 2,2’-oxydiethanol (10 ml_) was added pyridine (0.5 ml_) and then the mixture was stirred at 100°C for 4 h, cooled, quenched with water (100 ml_) and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (EA:PE = 1 :5) to give compound 9b as a white solid.

Step 3: (R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-(2-(2-Hvdroxyethoxy)ethoxy)-10, 13-di- methylhexadecahydro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (9)

To a solution of compound 9b (100 mg, 0.10 mmol) in THF (10 ml_) was added Pd/C (10 mg) and then the mixture was stirred at rt overnight under a H2 atmosphere, filtered, concentrated and purified by FCC (EA:PE = 1 :5) to give compound 9 as a white solid. ¹ H-NMR (500 MHz, DMSO-de): d 12.01 (br s, 1 H), 4.56 (br s, 1 H), 3.51-3.41 (m, 9H), 2.26-0.97 (m, 28H), 0.88- 0.86 (m, 6H), 0.61 (s, 3H). MS: 463.3 (M-H)-.

Example 9-1 to 9-2

Using a similar procedure as described for Example 9, the following compounds were prepared:

# reagent structure analytical data

¹

(R)-4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-3-(3-Carboxypropoxy)-10, 13-dimethylhexadeca- hydro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (10)

To a solution of compound 1 -12 (100 mg, 0.20 mmol) in THF (10 ml_) was added 1 N NaOH (0.5 ml_) and then the mixture was stirred at rt for 4 h, quenched with 1 N HCI (1 ml_) and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 : 15) to give compound 10 as a white solid. ¹ H-NMR (500 MHz, CDsOD) d: 3.54 (t, J = 6.5 Hz, 2H), 3.31-3.28 (m, 1 H), 2.39 (t, J = 7.3 Hz, 2H), 2.36-2.33 (m, 1 H), 2.25-2.18 (m, 1 H), 2.07-2.01 (m, 1 H), 1.98-0.99 (m, 27H), 0.99-0.97 (m, 6H), 0.71 (s, 3H). MS: 461.1 (M-H)-.

Step 1 : Benzyl (R)-4-((3S.5R.8R.9S.10S.13R.14S.17R)-3-hvdroxy-10.13-dimethylhexa- decahydro-1 H-cyclopentaralphenanthren-17-yl)pentanoate (11 a)

To a solution of compound P1 (400 g, 1.06 mmol), benzyl alcohol (172 mg, 1.60 mmol) and DMAP (26 mg, 0.21 mmol) in DCM (20 ml_) was added DCC (240 mg, 1.17 mmol) at rt. The mixture was stirred at rt for 6 h and filtered. The filtrate was concentrated and purified by FCC (PE/EA = 3: 1) to give compound 11 a as a white solid.

Step 2: 1-Benzyl 4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-17-((R)-5-(benzyloxy)-5-oxopentan-

2-yl)-10, 13-dimethylhexadecahvdro-1 H-cyclopentaralohenanthren-3-yl)

((benzyloxy)carbonyl)-L-aspartate (11 b)

To a solution of compound 11 a (300 g, 0.64 mmol), (S)-4-(benzyloxy)-3-(((benzyl- oxy)carbonyl)amino)-4-oxobutanoic acid (276 mg, 0.77 mmol) and DMAP (16 mg, 0.13 mmol) in DCM (20 ml_) was added DCC (171 mg, 0.83 mmol) at rt. The mixture was stirred at rt overnight and filtered. The filtrate was concentrated and purified by FCC (PE/EA = 4: 1) to give compound 11 b as a yellow oil.

Step 3: (R)-4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-3-(((S)-3-Amino-3-carboxypropanoyl)oxy)- 10, 13-dimethylhexadecahvdro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (11)

To a solution of compound 11 b (200 mg, 0.25 mmol) in MeOH (10 ml_) and THF (10 ml_) was added Pd/C (40 mg) at rt. Then the mixture was stirred at 50°C overnight under 50 psi of H2 and filtered. The filtrate was concentrated and washed with EA (10 ml_) to give compound 11 as a white solid. ¹ H-NMR (400 MHz, DMSO-de) d: 4.98 (s, 1 H), 3.76-3.72 (m, 1 H), 2.82-0.86 (m, 36H), 0.62 (s, 3H). MS: 492.1 (M+H)⁺.

Example 11 -1 to 11 -4

Using a similar procedure as described for Example 11 , the following compounds were prepared:

# reagent structure analytical data

o H-NMR (500 MHz, DMSO-de)

,

. , . . - .

, , , ,

0.62 (s, 3H). MS: 623.1 (M-H) . Example 12

Step 1 : Benzyl (R)-4-((3S.5R.8R.9S.10S.13R.14S.17R)-3-((3-bromopropanoyl)oxy)-10.13- dimethylhexadecahydro-1 H-cyclopentaralphenanthren-17-yl)pentanoate (12a)

To a solution of compound 11a (500 mg, 1.00 mmol) in DCM (10 ml_) was added BrCH CH COCI (155 mg, 1.00 mmol) and K₂CO₃ (324 mg, 3.00 mmol) and then the mixture was stirred at rt overnight, quenched with H O (100 ml_) and extracted with DCM (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (EA:PE = 1 : 10) to give compound 12a as a white solid.

Step 2: Benzyl (R)-4-((3S,5R.8R.9S.10S.13R.14S, 17R)-3-((3- (dimethylamino)propanoyl)oxy)-10, 13-dimethylhexadecahydro-1 H- cvclopentaralphenanthren-17-yl)pentanoate (12b)

To a solution of compound 12a (250 g, 0.50 mmol) in THF (10 ml_) was added 2N NHMe in THF (1 ml_) and then the mixture was stirred at rt overnight, quenched with H O (100 ml_) and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (EA:PE = 1 :5) to give compound 12b as a white solid.

Step 3: (R)-4-((3S,5R,8R,9S, 10S,13R,14S, 17R)-3-((3-(Dimethylammonio)propanoyl)oxy)-

10, 13-dimethylhexadecahvdro-1 H-cyclopentafalphenanthren-l 7-yl)pentanoate (12)

To a solution of compound 12b (200 mg, 0.40 mmol) in THF (10 ml_) was added Pd/C (20 mg) and then the mixture was stirred at rt overnight under a H atmosphere, filtered, concentrated and purified by FCC (EA:PE = 1 :5) to give compound 12 as a white solid. ¹ H- NMR (500 MHz, CD₃OD) d: 5.12 (br s, 1 H), 3.00 (t, J = 8.8 Hz, 2H), 2.68 (t, J = 8.8 Hz, 2H), 2.55 (s, 6H), 2.28-1.10 (m, 28H), 0.99 (s, 3H), 0.95 (d, J = 8.0 Hz, 3H), 0.70 (s, 3H). MS: 474.1 (M-H)-. Example 12-1 to 12-2

Using a similar procedure as described for Example 12, the following compounds were prepared:

# reagent structure analytical data

¹H-NMR (500 MHz CD OD) d:

Example 13

(R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-10, 13-Dimethyl-3-((3-(trimethylammonio)propan- oyl)oxy)hexadecahydro-1 H-cvcIopentafalphenanthren-l 7-yl)pentanoate (13)

To a solution of compound 12 (90 mg, 0.20 mmol) in THF (10 ml_) was added Mel (0.1 ml_) and then the mixture was stirred at rt overnight, quenched with H2O (100 ml_) and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 :15) to give compound 13 as a white solid. ¹ H-NMR (500 MHz, DMSO-d₆) d: 1 1.97 (s, 1 H), 5.04 (s, 1 H), 3.56 (t, J = 10.0 Hz, 2H), 3.07 (s, 9H), 2.95 (t, J = 10.0 Hz, 2H), 2.23-1.02 (m, 27H), 0.94 (s, 3H), 0.87 (d, J = 8.0 Hz, 3H), 0.62 (s, 3H). MS: 490.4 (M+H)⁺.

Example 13-1 to 13-2

Using a similar procedure as described for Example 13, the following compounds were prepared:

# reagent structure analytical data

¹H-NMR (500 MHz DMSO-d ) d: 1 1 95

22 ,

# reagent structure analytical data

Ή-NMR (500 MHz DMSO-d ) d: 12 13

Example 14

(R)-4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-3-(2-(2-(2-Acetamidoethoxy)ethoxy)acetoxy)- 10, 13-dimethylhexadecahvdro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (14)

To a solution of compound P8-3 (170 g) in dry DMF (5 ml_) was added piperidine (22.5 pl_) and the mixture was stirred for 75 min at rt. Then AC2O (100 mI_) was added and the mixture was stirred overnight, diluted with 0.1 N HCI (50 ml_) and extracted with EA (3 x 50 ml_). The combined organic layer was dried over Na2SC>4, filtered, concentrated and purified by FCC (cyclohexane: isopropanol = 10:3 + 0.8% formic acid) to give compound 14 as a brittle mass. ¹ H-NMR (300 MHz, CD₃OD) d: 5.17 (br s, 1 H), 4.16 (s, 2H), 3.73-3.70 (m, 2H), 3.67-3.63 (m, 2H), 3.55 (t, J = 5.5 Hz, 2H), 3.35 (t, J = 5.4 Hz, 2H), 2.38-2.14 (m, 2H), 2.08-1.04 (m, 27H), 1.95 (s, 3H), 0.99 (s, 3H), 0.95 (d, J = 6.4 Hz, 3H), 0.70 (s, 3H). ¹³C-NMR (75 MHz, CD₃OD) d: 178.15, 173.40, 171.89, 73.21 , 72.00, 71.28, 70.57, 69.55, 57.85, 57.50, 43.96, 41.50, 41.17, 40.50, 38.84, 37.08, 36.74, 36.03, 32.35, 32.03, 31.86, 31.74, 29.27, 27.65, 27.31 , 26.01 , 25.29, 24.41 , 22.57, 22.35, 18.82, 12.55.

Example 15

Step 1 : (3S.5R.8R.9S, 10S.13R.14S.17R)-17-((R)-5-(Benzyloxy)-5-oxopentan-2-yl)-10.13- dimethylhexadecahvdro-1 H-cvclopentaralphenanthren-3-yl 10-oxo-2,5,8, 14, 17-pentaoxa-

1 1-azanonadecan-19-oate (15a)

To a solution of the benzyl ester of compound P8-3 (530 g) in dry DMF (5 ml_) was added piperidine (70 pl_) and the mixture was stirred for 45 min. Then 2-[2-(2-methoxy- ethoxy)ethoxy]acetic acid (120 mI_) and HATU ( in dry 190 mg) in dry DMF (2 ml_) was added and the mixture was stirred overnight, diluted with 0.1 N HCI (50 ml_) and extracted with EA (3 x 50 ml_). The combined organic layer was dried over Na₂SC>₄, filtered, concentrated and purified by FCC (cyclohexane: isopropanol = 5: 1) to give compound 15a as a colorless mass.

Step 2: (R)-4-((3S,5R,8R,9S, 10S,13R,14S, 17R)-10, 13-Dimethyl-3-((10-oxo-2,5,8, 14, 17- pentaoxa-1 1-azanonadecan-19-oyl)oxy)hexadecahydro-1 H-cyclopentaralphenanthren-17- yl)pentanoic acid (15)

To a solution of compound 15a (285 mg) in MeOH (15 ml_) was added 10%-Pd/C (75 mg) and then the mixture was stirred at rt overnight under a H₂ atmosphere, filtered over celite and concentrated to give compound 15 as a white solid. ¹ H-NMR (300 MHz, CD₃OD) d: 5.16 (br s, 1 H), 4.15 (s, 2H), 3.99 (s, 2H), 3.73-3.54 (m, 14H), 3.46-3.43 (m, 2H), 3.37 (s, 3H), 2.38-1.28 (m, 29H), 0.99 (s, 3H), 0.95 (d, J = 6.4 Hz, 3H), 0.71 (s, 3H). ¹³C-NMR (75 MHz, CD₃OD) d: 178.10, 172.85, 171.82, 73.05, 72.96, 72.02, 71.96, 71.40, 71.34, 71.26, 70.49, 69.58, 59.17, 57.85, 57.50, 42.96, 41.51 , 51.18, 39.81 , 38.83, 37.07, 36.74, 36.03, 32.36, 32.06, 31.88, 31.76, 30.83, 29.27, 27.66, 27.34, 26.02, 25.31 , 24.46, 22.27, 18.86, 12.60. MS: 682.7 (M+H)⁺, 704.5 (M+Na)⁺.

Example 16

Step 1 : Benzyl (R)-4-((3S,5R,8R,9S, 10S,13R,14S, 17R)-10, 13-dimethyl-3-(((4-nitrophen- oxy)carbonyl)oxy)hexadecahydro-1 H-cyclopentaralphenanthren-17-yl)pentanoate (16a)

To a solution of compound 11 a (500 mg, 1.10 mmol) in DCM (100 ml_) was added 4-nitro- phenylchloroformate (442 mg, 2.20 mmol) and EtsN (340 mg, 3.30 mmol) and then the mixture was stirred at rt overnight, washed with sat. NaHCCh (100 ml_) and H₂O (100 ml_), dried over Na₂SC>₄, concentrated and purified by FCC (EA:PE = 1 :7) to give compound 16a as a white solid.

Step 2: terf-Butyl 1-(((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-17-((R)-5-(benzyloxy)-5-oxopentan-

2-yl)-10, 13-dimethylhexadecahydro-1 H-cyclopentaralphenanthren-3-yl)oxy)-1 -oxo-5, 8, 11- trioxa-2-azatridecan- 13-oate (16b) To a solution of compound 16a (200 g, 0.30 mmol) in DCM (100 ml_) was added tert- butyl 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetate (160 mg, 0.60 mmol), TEA (101 mg, 1.00 mmol) and DMAP (40 mg, 0.30 mmol) and then the mixture was stirred at reflux overnight, cooled to rt, washed with sat. NaHCCh (100 ml_) and H2O (100 ml_), dried over Na2SC>4, concentrated and purified by FCC (EA:PE = 1 :5) to give compound 16b as a white solid.

Step 3: 1 -(((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-17-((R)-5-(Benzyloxy)-5-oxopentan-2-yl)-

10, 13-dimethylhexadecahydro-1 H-cyclopentaralphenanthren-3-yl)oxy)-1 -oxo-5,8, 11 -trioxa-

2-azatridecan-13-oic acid

To a solution of compound 16b (210 mg, 0.20 mmol) in DCM (50 ml_) was added TFA (5 ml_) and then the mixture was stirred at rt overnight, concentrated and purified by FCC (EA:PE = 3: 1) to give compound 16c as a white solid.

Step 4: 1-(((3S,5R,8R,9S, 10S, 13R,14S, 17R)-17-((R)-4-Carboxybutan-2-yl)-10, 13-dimethyl- hexadecahydro-1 H-cyclopentaralphenanthren-3-yl)oxy)-1 -oxo-5,8, 11 -trioxa-2-azatridecan-

13-oic acid (16)

To a solution of compound 16c (150 mg, 0.20 mmol) in THF (10 ml_) was added Pd/C (30 mg) and then the mixture was stirred at rt overnight under a H2 atmosphere, filtered, concentrated and purified by FCC (EA:PE = 7: 1) to give compound 16 as a white solid. ¹ H- NMR (500 MHz, CD₃OD) d: 4.90 (br s, 1 H), 4.13 (s, 2H), 3.72-3.61 (m, 8H), 3.53 (t, J = 5.5 Hz, 2H), 3.28 (t, J = 5.5 Hz, 2H), 2.37-2.30 (m, 1 H), 2.24-2.16 (m, 1 H), 2.07-1.06 (m, 27H), 0.98 (s, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.70 (s, 3H). MS: 608.1 (M-H)-.

Example 16-1

Using a similar procedure as described for Example 16, the following compound was prepared:

# reagent structure analytical data

# reagent structure analytical data

3H), 0.85 (d, J = 6.5 Hz, 3H), 0.60 (s, 3H). MS: 564.1 (M-H) .

Example 17

Step 1 : 4-(((3S.5R.8R.9S.10S.13R.14S.17R)-17-((R)-5-(Benzyloxy)-5-oxopentan-2-yl)-

10, 13-dimethylhexadecahvdrc-1 H-cvclopentaralphenanthren-3-yl)oxy)-4-oxobutanoic acid

(17a)

A mixture cf ccmpcund 11 a (1.00 g, 2.10 mmcl), dihydrcfuran-2,5-dicne (650 g, 6.50 mmcl) and DMAP (550 mg, 4.50 mmcl) in pyridine (30 ml_) was refluxed fcr 16 h, ccncentrated and purified by prep-HPLC tc give ccmpcund 17a as a brown oil.

Step 2: Benzyl (R)-4-((3S.5R.8R.9S.10S,13R.14S.17R)-3-((4-((1-hvdroxy-2-methylpropan-

2-yl)amino)-4-oxobutanoyl)oxy)-10, 13-dimethylhexadecahydro-1 H- cyclopentaralphenanthren-17-yl)pentanoate (17b)

To a solution of compound 17a (200 mg, 0.35 mmol), 2-amino-2-methylpropan-1-ol (38 mg, 0.42 mmol) and HATU (201 mg, 0.53 mmol) in DCM (20 ml_) was added TEA (107 mg, 1.10 mmol). The mixture was stirred at rt for 16 h, concentrated and purified by FCC (DCM:EA = 5:1) to give compound 17b as a brown oil.

Step 3: (R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-((4-((1-Hvdroxy-2-methylpropan-2- yl)amino)-4-oxobutanoyl)oxy)-10, 13-dimethylhexadecahydro-1 H- cyclopentaralphenanthren-17-yl)pentanoic acid (17)

A mixture of compound 17b (200 mg, 0.31 mmol) and Pd/C (60 mg) in MeOH (10 ml_) was hydrogenated for 16 h at rt under one atmosphere of H2. The catalyst was filtered off and the filtrate was concentrated and purified by FCC (DCM:EA = 5:1) to give compound 17 as a white solid. ¹ H-NMR (400 MHz, DMSO-d₆) d: 11.97 (br s, 1 H), 7.32 (s, 1 H), 4.94 (s, 1 H), 4.78 (br s, 1 H), 3.35 (s, 2H), 2.43 (t, J = 6.4 Hz, 2H), 2.33 (t, J = 6.4 Hz, 2H), 2.26-0.98 (m, 34H), 0.93 (s, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.62 (s, 3H). MS: 548.2 (M+H)⁺.

Example 17-1 to 17-2

Using a similar procedure as described for Example 17, the following compounds were prepared:

# reagent structure analytical data

1H-NMR (400 MHz CD₃OD) d:

,

. .

Ή-NMR (400 MHz, DMSO-de) d: 1 1 .95 (br s 1 H) 7.91 (t J = 5.8

. , , . , .

652.2 (M+H)⁺.

Example 18

Step 1 : 1-Benzyl 4-((3S.5R.8R.9S.10S.13R.14S.17R)-17-((R)-5-(benzyloxy)-5-oxopentan- 2-yl)-10, 13-dimethylhexadecahvdro-1 H-cyclopentaralohenanthren-3-yl) ( tert - butoxycarbonyl)-L-aspartate (18a)

To a solution of compound 11 a (1.00 g, 2.15 mmol), (S)-4-(benzyloxy)-3-((te/f-butoxy- carbonyl)amino)-4-oxobutanoic acid (833 mg, 2.58 mmol) and DMAP (52 mg, 0.43 mmol) in DCM (50 ml_) was added DCC (531 mg, 2.58 mmol) at rt. The mixture was stirred at rt overnight, filtered, concentrated and purified by FCC (PE/EA = 10: 1) to give compound 18a as a yellow oil.

Step 2: 1-Benzyl 4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-17-((R)-5-(benzyloxy)-5-oxopentan- 2-yl)-10, 13-dimethylhexadecahvdro-1 H-cyclopentaralphenanthren-3-yl) L-aspartate (18b) To a solution of compound 18a (600 g, 0.78 mmol) in DCM (10 ml_) was added TFA (5 ml_) at rt. The mixture was stirred at rt for 2 h, diluted with water (20 ml_), basified to pH = 10 with sat. Na2CC>3. The organic layer was separated, dried over Na2SC>4, filtered and concentrated to give compound 18b as a yellow oil.

Step 3: 1-Benzyl 4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-17-((R)-5-(benzyloxy)-5-oxopentan- 2-yl)-10, 13-dimethylhexadecahydro-1 H-cyclopenta[alphenanthren-3-yl) acetyl-L-aspartate

(18c)

To a solution of compound 18b (150 mg, 0.22 mmol) and TEA (44 mg, 0.44 mmol) in DCM (10 ml_) was added AcCI (21 mg, 0.26 mmol) under cooling with an ice-bath. The mixture was stirred at rt overnight, concentrated and purified by FCC (PE/EA = 2:1) to give compound 18c as a yellow oil.

Step 4: (R)-4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-3-(((S)-3-Acetamido-3-carboxypropan- oyl)oxy)-10, 13-dimethylhexadecahydro-1 H-cyclopenta[alphenanthren-17-yl)pentanoic acid

(18)

To a solution of compound 18c (120 mg, 0.17 mmol) in MeOH (20 ml_) and THF (20 ml_) was added Pd/C (30 mg) at rt. The mixture was stirred at 40°C overnight under 50 psi of H2, filtered, concentrated and washed with EA (2 ml_) to give compound 18 as a white solid. ¹ H- NMR (400 MHz, DMSO-d₆) d: 8.08 (d, J = 7.6 Hz, 1 H), 4.95 (s, 1 H), 4.50-4.46 (m, 1 H), 2.72 (dd, J = 5.8, 15.8 Hz, 1 H), 2.57-2.50 (m, 1 H), 2.25-0.86 (m, 37H), 0.62 (s, 3H). MS: 534.2 (M+H)⁺.

Step 1 : 1-Benzyl 4-((3S,5R,8R,9S, 10S, 13R, 14S, 17R)-17-((R)-5-(benzyloxy)-5-oxopentan- 2-yl)-10, 13-dimethylhexadecahvdro-1 H-cyclopentaralphenanthren-3-yl) N,N-dimethyl-L- aspartate (19a) To a solution of compound 18b (350 g, 0.52 mmol), HCHO (0.78 ml_, 37% in H20, 10 mmol) and AcOH (0.5 ml_) in MeOH (15 ml_) was added NaBHsCN (262 mg, 4.16 mmol) at rt. The mixture was stirred at rt overnight, concentrated and then the residue was diluted with EA (30 ml_) and water (10 ml_). The aq. phase was basified to pH 9 with 1 N NaOH and extracted with EA (30 ml_) twice. The combined organic layer was concentrated and purified by FCC (PE/EA = 4: 1) to give compound 19a as a yellow oil.

Step 2: (R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-(((S)-3-carboxy-3-(dimethyl- amino)propanoyl)oxy)-10, 13-dimethylhexadecahvdro-1 H-cyclopentaralphenanthren-17- vDpentanoic acid (19)

To a solution of compound 19a (200 mg, 0.29 mmol) in MeOH (20 ml_) and THF (20 ml_) was added Pd/C (50 mg) at rt. The mixture was stirred at 40°C overnight under 50 psi of H2. The mixture was filtered, concentrated and washed with EA (5 ml_) to give compound 19 as a white solid. ¹ H-NMR (400 MHz, DMSO-d₆) d: 4.95 (s, 1 H), 3.52 (t, J = 7.4 Hz, 1 H), 2.68 (dd, J = 8.0, 15.6 Hz, 1 H), 2.54-2.50 (m, 1 H), 2.33 (s, 6H), 2.27-0.86 (m, 34H), 0.62 (s, 3H). MS: 520.2 (M+H)⁺.

Example 20

Step 1 : (R)-1 -Diazo-5-((3S,5R.8R.9S.10S, 13R, 14S, 17R)-3-ethoxy-10, 13-dimethylhexa- decahydro-1 H-cyclopentafalphenanthren-l 7-yl)hexan-2-one (20a)

To a solution of compound 1 (2.00 g, 5.00 mmol) in DCM (30 ml_) was added oxalyl chloride (1.90 g, 15.0 mmol) at 0°C and the mixture was stirred at rt for 16 h, concentrated and diluted with DCM (15 ml_) and Et₂0 (30 ml_). Then TMSCHN2 (2.30 g, 20.0 mmol) was added. The mixture was stirred at rt for 4 h, washed with H2O (30 ml_) and concentrated. The residue was filtrated through silica gel (DCM as eluent) and concentrated to give compound 20a as a yellow solid. Step 2: (R)-5-((3S,5R.8R.9S.10S.13R.14S.17 R)-3- Ethoxy- 10.13-dimethylhexadecahvdro- 1 H-cyclopentaralphenanthren-17-yl)hexanoic acid (20)

A mixture of compound 20a (2.50 g, crude), AgNCh (850 mg, 5.00 mmol) in THFihhO = 1 : 1 (30 ml_) was stirred at rt for 4 h. The THF was removed under reduced pressure and the aq. layer was extracted with DCM (100 ml_), dried over Na2SC>4, concentrated and purified by FCC (DCM) to give compound 20 as a white solid. ¹ H-NMR (400 MHz, CDC ) d: 3.56-3.48 (m, 2H), 3.28-3.22 (m, 1 H), 2.38-2.24 (m, 2H), 1.97-0.95 (m, 31 H), 0.93-0.91 (m, 6H), 0.61 (s, 3H). MS: 417.1 (M-H)-.

Example 21

Step 1 : Benzyl (R)-4-((3S,5R,8R,9S, 10S,13R,14S, 17R)-3-amino-10, 13-dimethylhexadeca- hydro-1 H-cyclopentaralphenanthren-17-yl)pentanoate hydrochloride (21a)

Lithocholic acid benzyl ester(10.1 g) and the first batch of triphenyl phosphine (5.25 g) were dissolved in dry THF (150 ml_). A gentle flow of argon was applied to the reaction flask and the first batch of diisopropylazo dicarboxylate (4.7 ml_) was added via a syringe. After 10 min of stirring, phosphoric acid diphenylester azide (4.6 ml_) was added. After 23 h of stirring at rt, the second batches of triphenyl phosphine (2.56 g) and diisopropylazo dicarboxylate (2.3 ml_) were added and stirring was continued for 5 h. The third batch of triphenyl phosphine (5.25 g) was then added to the mixture together with water (15 ml_) and stirring at rt was continued for another 4.5 days. The mixture was evaporated to approx. 40 ml_ and EtOAc (100 ml_) were added. When all solids were dissolved, Et2<D (100 ml_) was added and then 4N HCI (20 ml_). The mixture was intensely stirred for 15 min. The precipitate was isolated by centrifugation and washed with a mixture of EtOAc and Et₂0 (1 +1) twice, then twice with water. Finally, the residue was dried to afford intermediate 21 a as a white residue.

Step 2: Benzyl (R)-4-((3S.5R.8R.9S, 10S.13R.14S.17R)-3-(2-(2-(2-methoxyethoxy)eth- oxy)acetamido)-10, 13-dimethylhexadecahydro-1 H-cyclopentaralphenanthren-17- vDoentanoate (21 b)

2-[2-(2-Methoxyethoxy)ethoxy]acetic acid (100 mI_) and HATU (280 mg) were combined in CH2CI2 (15 ml_) and the mixture was stirred for 2 min. Then intermediate 21 a (225 mg) in CH2CI2 (20ml_) and then A/,/\/-diisopropylethylamine (75 mI_) was added. After stirring for 2 d, the second batch of 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (100 mI_) and HATU (280 mg) in CH2CI2 (15 ml_) was added to the reaction mixture. After stirring for another 2 d the mixture was extracted by 1 N HCI (2 x), water and brine and dried over Na2SC>4, concentrated and purified by FCC (cyclohexane: EtOAc) to give compound 21 b.

Step 3: (R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-(2-(2-(2-Methoxyethoxy)ethoxy)acet- amido)-10, 13-dimethylhexadecahvdro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid

Compound 21 b (198 mg) was as dissolved in MeOH (20 ml_) and argonized. Palladium (10% on carbon, 85 mg) was added and the reaction flask was flushed with hydrogen. After stirring at rt for 2 h, the mixture was filtered through a pad of celite and concentrated to dryness to afford compound 21 as an amorphous mass. ¹ H-NMR (300 MHz, CDC ) d: 8.95 (br s, 1 H), 7.08 (d, J = 7.7 Hz, 1 H), 4.20 (br s, 1 H), 4.00 (s, 2H), 3.75-3.63 (m, 6H), 3.55-3.52 (m, 2H), 3.36 (s, 3H), 2.41-2.16 (m, 2H), 2.07-0.84 (m, 26H), 0.95 (s, 3H), 0.90 (d, J = 6.2 Hz, 3H), 0.63 (s, 3H). ¹³C-NMR (75 MHz, CDCb) d: 178.96, 169.31 , 71.86, 71.02, 70.54, 70.34, 70.24, 58.98, 56.39, 55.91 , 44.95, 42.69, 40.04, 39.76, 37.78, 35.57, 35.27, 34.98, 31.19, 30.97, 30.79, 30.41 , 28.09, 26.66, 26.12, 24.73, 24.10, 24.00, 20.98, 18.19, 12.00. MS: 537.1 (M+H)⁺, 558.7 (M+Na)⁺.

Example 21 -1 to 21 -3

Using a similar procedure as described for Example 21 , following compounds were prepared:

# reagent structure analytical data

Example 22

Step 1 : Benzyl (R)-4-((3S.5R.8R.9S.10S.13R.14S.17R)-3-acetamido-10.13-dimethylhexa- decahydro-1 H-cyclopentaralphenanthren-17-yl)pentanoate (22a)

Intermediate 21 a (336 g) was dissolved with pyridine (5 ml_) and AC₂O (100 pl_) was added. The mixture was stirred for 90 min, then additional AC₂O (100 mI_) was added and the mixture was stirred for additional 1 h, diluted with water (2 ml_), stirred overnight and partitioned between diluted aq. HCI and EA. The precipitate appearing was isolated by filtration, washed with water and recrystallized from methanol to obtain compound 22a as colorless solid.

Step 2: (R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-Acetamido-10, 13-dimethylhexadeca- hydro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (22)

Compound 22a (185 mg) was as dissolved in MeOH (20 ml_) and argonized. Palladium (10% on carbon, 105 mg) was added and the reaction flask was flushed with hydrogen. After stirring at rt for 2 h, the mixture was filtered through a pad of celite and concentrated to dryness to afford compound 22 as an amorphous mass. ¹ H-NMR (300 MHz, CDCI₃/CD₃OD) d: 4.06 (br s, 1 H), 2.33-2.08 (m, 2H), 2.00-1.57 (m, 29H), 0.90 (s, 3H), 0.85 (d, J = 6.4 Hz, 3H), 0.58 (s, 3H). MS: 418.8 (M+H)⁺.

Example 100

1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (100)

To a solution of 3-oxo^-cholanoic acid (300 mg, 0.77 mmol) in THF (30 ml_) was added 1 N EtMgBr (3 ml_, 3.0 mmol) under argon at -78°C and the mixture was stirred at -78°C for 2 h, quenched by 5% HCI and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 :9) to afford compound 100 as a white solid (beside the other isomer). ¹ H-NMR (500 MHz, CDC ) d: 2.43-2.37 (m, 1 H), 2.29-2.22 (m, 1 H), 1.97-0.95 (m, 29H), 0.93-0.91 (m, 6H), 0.88 (t, J = 7.5 Hz, 3H), 0.64 (s, 3H). MS: 387.1 (M-OH , 427.0 (M+Na)⁺.

Example 100-1

Using a similar procedure as described for Example 100, the following compound was prepared:

# conditions structure analytical data

(R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-Ethoxy-3, 10,13-trimethylhexadecahvdro-1 H- cyclopentaralphenanthren-17-yl)pentanoic acid (101)

To a solution of compound 100-1 (100 g, 0.26 mmol) in THF (50 ml_) was added NaH (40 mg, 1.0 mmol) and Etl (70 mg, 0.50 mmol) and then the mixture was stirred at 60°C overnight, cooled, quenched with 5% HCI and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 : 15) to give compound 101 as a white solid. ¹ H-NMR (500 MHz, DMSO-de) d: 1 1.94 (s, 1 H), 2.26-2.19 (m, 1 H), 2.13-2.06 (m, 1 H), 1.93-0.97 (m, 34H), 0.88 (s, 3H), 0.87 (d, J = 6.5 Hz, 3H), 0.61 (s, 3H). MS: 417.3 (M-H)-.

(R)-4-((3S,5R,8R.9S.10S, 13R, 14S, 17R)-3-Acetoxy-3, 10,13-trimethylhexadecahvdro-1 H- cyclopentaralphenanthren-17-yl)pentanoic acid (102)

To a solution of compound 100-1 (100 mg, 0.26 mmol) in EΐbN (10 ml_) was added AC2O (100 mg, 1.00 mmol) and DMAP (120 mg, 1.00 mmol) and then the mixture was stirred at 50°C overnight, quenched with 5% HCI and extracted with EA (3 x 50 ml_). The combined organic layer was concentrated and purified by FCC (MeOH:DCM = 1 : 15) to compound 102 as a white solid. ¹ H-NMR (500 MHz, DMSO-d₆) d: 11.97 (s, 1 H), 2.25-2.19 (m, 1 H), 2.12-2.03 (m, 1 H), 1.95-0.97 (m, 32H), 0.91 (s, 3H), 0.87 (d, J = 6.5 Hz, 3H), 0.61 (s, 3H). MS: 431.1 (M- H) .

Example 103

(R)-4-((3S.5R.8R.9S.10S.13R.14S.17R)-3-Hvdroxy-3-(3-(2-methoxyethoxy)prop-1-vn-1-yl)- 10, 13-dimethylhexadecahvdro-1 H-cyclopentaralphenanthren-17-yl)pentanoic acid (103)

To the solution of 3-(2-methoxyethoxy)prop-1-yne (762 mg, 6.67 mmol) in THF (7 ml_) was cooled at -78°C. The BuLi was added at -78°C for 30 min under l\h and the minxture was stirred at -78°C for 30 min and then 3-oxo^-cholanoic acid (1.00 g, 2.67 mmol) was added. The mixture was stirred at -78°C -rt for 2 h, poured into water, adjusted to pH = 6 with 1 N HCI and then extracted with EA (3 x). The combined organic layer was dried over Na2SC>4, filtered, concentrated and purified by FCC (PE:EA =2: 1 to 1 : 1 ) to give compound 103 as a white solid (beside the other isomer). ¹ H-NMR (400 MHz, CDCh) d: 4.26 (s, 2H), 3.69-3.67 (m, 2H), 3.59-3.56 (m, 2H), 3.39 (s, 3H), 2.42-0.91 (m, 34H), 0.64 (s, 3H). MS: 487.1 (M-H) Example 104

(R)-4-((3R,5R,8R.9S.10S, 13R, 14S, 17R)-3-Hvdroxy-3-(3-(2-methoxyethoxy)propyl)-10, 13- dimethylhexadecahvdro-1 H-cvclopentaralphenanthren-17-yl)pentanoic acid (104)

Compound 103 (150 mg, 307 pmol) was dissolved in THF and Pd/C-20% (16 mg, 30 mg) was added. The mixture was stirred at rt under H2 overnight. The Pd/C solid was filtered off, the filtrate was concentrated and purified by FCC (PE: EA = 2: 1 to 1 : 1 ) to give compound 104 as a colourless oil. ¹ H-NMR (400 MHz, CDCh) d: 3.61-3.58 (m, 2H), 3.56-3.54 (m, 2H), 3.50 (t, J = 6.4 Hz, 2H), 3.39 (s, 3H), 2.39-0.82 (m, 38H), 0.63 (s, 3H). MS: 491.1 (M-H) . Example 105

Step 1 : Benzyl (R)-4-((3S.5R.8R.9S.10S.13R.14S, 17R)-3-hvdroxy-3.10.13-trimethylhexa- decahydro-1 H-cyclopentafalphenanthren-l 7-yl)pentanoate (105a)

To a solution of phenylmethanol (110 mg, 1.0 mmol) and compound 100-1 (200 mg, 0.51 mmol) in DCM (10 ml_) was added DCC (177 mg, 0.80 mmol) and DMAP (52 mg, 0.40 mmol) and then the mixture was stirred at rt for 12 h, diluted with water (100 ml_) and extracted with DCM (3 x 100 ml_). The combined organic layer was washed with brine (50 ml_), concentrated and purified by FCC (PE:EA = 6: 1) to give compound 105a as a white solid.

Step 2: Benzyl (R)-4-((3S.5R.8R.9S, 10S.13R.14S.17R)-3-(2-(2-(2-methoxyethoxy)eth- oxy)acetoxy)-3, 10, 13-trimethylhexadecahydro-1 H-cyclopentaralphenanthren-17- vDpentanoate (105b

To a solution of 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (178 mg, 1.0 mmol) and compound 105a (190 mg, 395 pmol) in DCM (10 ml_) was added DCC (177 mg, 0.80 mmol) and DMAP (52 mg, 0.40 mmol) and then the mixture was stirred at rt for 12 h, diluted with water (100 ml_) and extracted with DCM (3 x 100 ml_). The combined organic layer was washed with brine (50 ml_), concentrated and purified by FCC (PE:EA = 6: 1) to give compound 105b as a white solid.

Step 3: (R)-4-((3S.5R,8R.9S.10S.13R.14S.17R)-3-(2-(2-(2-Methoxy- ethoxy)ethoxy)acetoxy)-3, 10,13-trimethylhexadecahvdro-1 H-cyclopentafalphenanthren-l 7- vDpentanoic acid (105)

To a solution of compound 105b (150 mg, 0.20 mmol) in THF (10 ml_) was added Pd/C (30 mg) and then stirred at rt under H2 overnight. The mixture was filtered, condensed and purified by FCC (EA:PE = 7: 1) to give compound 105 as a white solid. ¹ H-NMR (500 MHz, DMSO-de) d: 1 1.93 (s, 1 H), 3.99 (s, 2H), 3.57 (t, J = 7.8 Hz, 2H), 3.53-3.51 (m, 4H), 3.44- 3.42 (m, 2H), 3.24 (s, 3H), 2.26-2.19 (m, 1 H), 2.13-2.06 (m, 1 H), 1.94-0.98 (m, 29H), 0.91 (s, 3H), 0.87 (d, J = 6.5 Hz, 3H), 0.61 (s, 3H). MS: 549.0 (M-H) .

Example 200

Determination of minimal inhibitory concentrations (MICs) of bile acid derivatives on Clostridioides difficile ( Clostridium difficile), human strain R20291 , ribotype RT027, reference DSMZ (DSM-27147)

All bacterial culturing steps and MIC experiments were performed under anoxic conditions (95% N2_, 5% H2) and 37°C in an incubator model 2002 that was placed in a Type B vinyl anaerobic chamber, both from Coy Laboratories Products. The strains were maintained as frozen stock cultures in Brain-Heart Infusion broth supplemented with 5% (w/v) yeast extract and 1 % (w/v) L-cysteine (BHIS) containing 40% (v/v) glycerine (Carl Roth GmbH, Cat. #3783.1) at -80°C. Brain-heart infusion broth (Sigma Aldrich, Cat. #53286) with the addition of with 5 g/L yeast extract (Carl Roth GmbH, Cat. #2363.5) was prepared according to the manufacturer’s instructions. After heat sterilization, 1 g L-cysteine (Sigma Aldrich, Cat. #C7352) was added that was dissolved in 10 mL of distilled water (H₂0_dd) and filter sterilized. Medium was placed over night into the anaerobic chamber for degasing. For the MIC-assay cryo-preserved strains were streaked out on BBL™ Columbia CNA Agar with 5% Sheep Blood (BD™, Cat. #221352) and grown for 2-3 d. These plates were kept at rt in the anaerobic chamber for up to two weeks for starting fresh liquid cultures. Several colonies were picked for inoculating 5 mL of BHIS in 50 mL tubes (Sarstedt, Cat. #62.547.254) and bacteria were grown overnight. Hundred microliters of this culture were used for inoculating 10 mL of fresh BHIS in a 50 mL tube and bacteria were grown to an optical density at 600 nm (OD600) of 0.6-0.8. The inhibitory action of the bile acid derivatives on C. diff. strains were performed in a 96-well format. For this, a 1 :1 dilution series in the range of 2 mM to 8 mM was prepared for each compound in 100 pL BHIS containing 10% (v/v) DMSO (Sigma Aldrich, Cat. #D8418) per well. The bacterial culture was diluted to an OD600 of 0.1 in 15 mL BHIS and 100 pL were transferred into each well of the dilution series, resulting in compound concentrations ranging from 1 mM to 4 mM and a final DMSO concentration of 5% (v/v). As a control, bacteria were grown in BHIS with 5% (v/v) DMSO. After 16 h of incubation, bacterial growth was monitored by measuring Oϋboo in a Varioskan microplate reader (ThermoFisher Scientific).

Results: The MIC for the RT027 ribotype is summarized below in Table 1 : Typical examples of the invention desirably have a MIC lower than 25 mM (Group A), from about 25 mM to 250 mM (Group B) and above 250 mM (Group C). Table 1

Example 201

Determination of minimal inhibitory concentrations (MICs) of bile acid derivatives on Clostridioides difficile(Clostridium difficile), mouse strain VPI 10463 (ATCC 43255)

Concentrations (0.015-250 mM) of test compounds are prepared by serial two-fold dilutions in pre-reduced brain heart infusion (BHI) broth. To each well containing test article, approximately 5 x 105 CFU of bacteria are added and incubated for 48 hours in an anaerobic chamber at 37°C. Following incubation, the MIC of each test article is determined by presence/absence of bacterial growth in each well.

Results: The MIC for the mouse strain VPI 10463 is summarized below in Table 2: Typical examples of the invention desirably have a MIC lower than 25 mM (Group A), from about 25 mM to 250 mM (Group B) and above 250 mM (Group C).

Table 2

Example 202

One-hybrid reporter assay for the vitamin D receptor

The vitamin D receptor (VDR; NR1 I1) reporter assay was performed by transient co transfection of HEK293 cells with pCMV-BD (Stratagene #211342) containing the GAL4 DNA-binding domain fused with the ligand binding domain of VDR (Genbank accession no. NP_000376, aa 88-427), pFR-Luc reporter and pRL-CMV reporter (Promega #E2261) using PEI solution (Sigma Aldrich cat# 40872-7) in a 96-well plate. Cells were incubated for 4-6 hours, and then cultured in MEM supplemented with 8.7% FCS, Glutamax, NEAA, sodium pyruvate and Pen/Strep in the presence of test compounds for 16-20 hours. Cells were incubated for 4 to 6 hours in 30 pL/well transfection mix in OPTIMEM and then cultured for further 16 to 20 hours after addition of 120 pL MEM supplemented with 8.7% FCS, Glutamax, NEAA, sodium pyruvate and Pen/Strep in the presence of test compounds. Medium was removed and cells were lysed with diluted (1x) passive lysis buffer (Promega). Firefly luciferase buffer was then added and firefly luciferase luminescence was read on BMG LUMIstar OMEGA luminescence plate reader. One second later, renilla luciferase buffer was added and renilla luciferase luminescence was read to evaluate cell viability and to be able to normalize for well to well differences in transfection efficiency.

Materials Company Cat. No.

HEK293 cells DSMZ ACC305

MEM Sigma-Aldrich M2279

FCS Sigma-Aldrich F7542

Glutamax Invitrogen 35050038

Pen/Strep Sigma Aldrich P4333

Sodium pyruvate Sigma Aldrich S8636

Non-essential amino acids (NEAA) Sigma Aldrich M7145

PEI Sigma Aldrich 40.872-7

Passive lysis buffer (5x) Promega E1941

D-Luciferine PJK 260150

Coelentrazine PJK 260350

Results: The AC₅₀ for Example 1 , 1 -2, 1 -6, 1 -7, 1-8, 1 -10, 2, 4, 6, 8, 8-1 , 8-2, P8-3, 8-5, 9- 2, 11, 11 -1, 11 -2, 11 -3, 11 -4, 12, 12-1, 12-2, 13, 13-1, 13-2, 14, 15, 16, 16-1, 17, 17-1, 17-2, 18, 19, 20, 100, 100-1, 103, 104, 105 were measured to be inactive in this assay. For comparison, the AC50 for LCA was measured to be 19 to 29 mM.

Matched pair comparison of selected examples towards the a-0-anomer (LCA analogs) in Table 3 show, that the claimed structures are usually more potent in the MIC (measured according Example 200) and exhibit lower vitamin D agonism (measured according Example 202):

Table 3

E

O

E

O

E

E

Example 203

Efficacy evaluation in a murine model of Clostridioides difficile-associated disease: Acute model

The efficacy of compounds in suppressing C. diff. infection was assessed in C57BL/6 female mice. Mice were made vulnerable to C. diff. infection by administration of a cocktail of antibiotics (1% glucose, kanamycin (0.5 mg/ml_), gentamicin (44 pg/mL), colistin (1062.5 U/mL), metronidazole (269 pg/mL), ciprofloxacin (156 pg/mL), ampicillin (100 pg/mL) and vancomycin (56 pg/mL)) in drinking water for a period of 9 days. 3 days prior to C. diff. infection, mice received a single dose of clindamycin (10 mg/kg) in a volume of 0.5 ml_ by oral gavage. After this antibiotic pre-treatment, mice received a challenge of approximately 4.5 Iog10 viable spores of strain VP1 10463 (ATCC-43255) administered by oral gavage. Test compounds and placebo were administered via oral gavage bid from day 0 to day 4 (study 1). Gavage medium was aqueous, PBS-buffered 0.5% hydroxypropyl methylcellulose (HPMC) suspension. Efficacy of test articles was assessed by enumeration of survival of test animals over 6 days following C. diff. challenge and by comparison of mortality, disease severity scores and assessment of body weight against placebo treatment.

Results study 1 (using compound Example 1 , dosed with 100 mg/kg daily dose via gavage bid). Results study 2 (using compound Example 8, dosed with 100 mg/kg daily dose via gavage bid). Results from study 3 for Example C1 are presented additionally to show the deleterious effect of compounds with a significant Vitamin D agonistic activity in a mouse in vivo model.

Table 4 Survival

Table 5 Body weight

Table 6 Clinical signs

Score

Normal: 0

Lethargic: 1

Lethargic + Hunched:

Lethargic + Hunched + Wet tail/abdomen:

Lethargic + Hunched + Wet tail/abdomen + Hypothermic:

The benefit of compound Example 1 could be demonstrated in an acute mouse model of C. diff. infection. Whereas in the vehicle group 70% of the animals died, only 40% of the animals in the treatment group died.

The benefit of compound Example 8 could be demonstrated in an acute mouse model of C. diff. infection. Whereas in the vehicle group 50% of the animals died, 90% of the animals in the treatment group survived with improved clinical signs and body weight.

Oral administration of Example C1 led to a dramatic weight loss and death within 5 days of all animals due to Vitamin D agonism.

Recurrence model mouse

The efficacy of compounds in suppressing recurrent C. diff. infection was assessed in C57BL/6 female mice. Mice were made vulnerable to C. diff. infection by administration of a cocktail of antibiotics (1 % glucose, kanamycin (0.5 mg/mL), gentamicin (44 pg/mL), colistin (1062.5 U/mL), metronidazole (269 pg/mL), ciprofloxacin (156 pg/mL), ampicillin (100 pg/mL) and vancomycin (56 pg/mL)) in drinking water for a period of 9 days. 3 days prior to C. diff. infection, mice received a single dose of clindamycin (10 mg/kg) in a volume of 0.5 mL by oral gavage. After this antibiotic pre-treatment, mice received a challenge of approximately 4.5 Iog10 viable spores of strain VPI 10463 (ATCC-43255) administered by oral gavage (day 0). After that mice received 50 mg/kg vancomycin from day 0 until day 4. Test articles (bile acid) and placebo were administered via oral gavage bid from day 5 through day 11. Efficacy of test articles was assessed by enumeration of survival of test animals over 15 days following C. diff. challenge and by comparison of mortality, disease severity scores and assessment of body weight against placebo treatment.

Results studies 4 and 5 (using compound Example 1 and Example 8) dosed with 100 mg/kg daily dose via gavage bid: Table 8 Survival

Table 9 Body weight

Table 10 Clinical signs

The benefit of compound Example 1 and Example 8 could be demonstrated in a recurrence mouse model of Clostridioides difficile infection. Whereas in the vehicle group 70 to 80% of the animals died, only 40% of the animals in the treatment groups died with improved clinical signs and body weights.

Summary

Lithocholic acid (LCA) is claimed in WO2010/062369 (Claim 9) to be useful in preventing C. cf/Tf.-associated diseases but no data for this assumption was presented. Even more, for closest analog in regard to our invention, 3-acetyl-LCA (Table 3, line 9 in WO2010/062369) the underlying mechanism (i.e. inhibition of germination) was reported to be not present. On the other hand, for LCA several liabilities in the literature are reported: for example oral administration of LCA results in elevation of alanine transaminase (ALT) indicating hepatocellular injury. Another potential liability of LCA is increased Vitamin D agonism leading to hypercalcemia followed by polyuria. We confirmed this Vitamin D agonism for LCA analogs and showed, that iso- LCA analogs are devoid of this Vitamin D agonism or have at least a lower AC50 or efficacy compared to the LCA-matched pair (Example 202).

As additional beneficial effects for the compounds of the present invention we found, that in a head-to-head comparison of iso- LCA analogs towards the LCA matched pairs we showed that in most cases the iso- LCA analogs exhibit a lower minimal inhibitory concentration (MIC) on growth of C. diff. (measured according Example 200 and shown at the end of Example 202).

These beneficial effects prolonged the survival of /so-LCA analogs in the acute mouse model of Clostridioides difficile infection (Example 1 vs. C1 ; Figure 1 vs. Figure 3).

Claims

What is claimed:

1. A compound according to Formula (I)

or a pharmaceutically acceptable salt, co-crystal or solvate thereof for use in the prophylaxis or treatment of Clostridioides difficile associated disease, wherein:

R' is selected from H, CrC₆-alkyl, C₂-C₆-alkenyl and C₂-C₆-alkynyl,

wherein alkyl, alkenyl and alkynyl is unsubstituted or substituted with 1 to 7 substituents independently selected from the group consisting of CN, halogen, azide, oxo, OR¹⁰, O- C₂-C₆-alkylene-OR¹⁰, 0-C₃-io-cycloalkyl, 0-C₃-io-heterocycloalkyl, -(CH₂-CH₂-0)_n- CH2CH2R¹², -0-(CH₂-CH2-0)n-CH2CH₂R¹², C₀-C₈-alkylene-R¹⁰, CO2R¹⁰, CONR¹⁰R¹¹ , CONR¹⁰SO₂R¹⁰, COR¹⁰, SO_XR^{1 0}, SO₃H , SO₂NR¹⁰R¹¹ , NR¹⁰COR¹¹ , N R¹⁰SO₂R^{1 1} , N R¹⁰- CO-NR¹⁰R¹¹ , NR¹⁰-SO₂-NR¹⁰R¹¹ , NR¹⁰R¹¹ and N(R¹⁰R¹⁰R¹¹)⁺;

R is selected from H, Ci-Cio-alkyl, C₂-Cio-alkenyl, C₂-Cio-alkynyl, Co-Cio-alkylene-C₃-io- cycloalkyl and Co-Cio-alkylene-C₃-io-heterocycloalkyl,

wherein alkyl, alkenyl, alkynyl, alkylene, cycloalkyl and heterocycloalkyl is unsubstituted or substituted with 1 to 7 substituents independently selected from the group consisting of CN, halogen, azide, oxo, OR¹⁰, 0-C₂-C₆-alkylene-OR¹⁰, O-C_3-10- cycloalkyl, 0-C₃-io-heterocycloalkyl, -(CH₂-CH2-0)n-CH2CH₂R¹², -0-(CH₂-CH₂-0)_n- CH2CH2R¹², -(CH2-CH₂-0)n-CH₂-(C=0)-R¹², -0-(CH2-CH₂-0)n-CH₂-(C=0)R¹², C₀-C₈- alkylene-R¹⁰, CO₂R¹⁰, CONR¹⁰R¹¹ , CONR¹⁰SO₂R¹⁰, COR¹⁰, SO_xR¹⁰, S0₃H, SO₂NR¹⁰R¹¹ , NR¹⁰COR^{1 1} , NR¹⁰SO₂R^{1 1} , NR¹⁰-CO-NR¹⁰R^{1 1} , NR¹⁰-SO₂-NR¹⁰R^{1 1} , NR¹⁰R¹¹ and N(R¹⁰R¹⁰R¹¹)⁺;

X is selected from O, NH, NCi-₆-alkyl, N-halo-Ci-₆-alkyl, N-CO-Ci-₆-alkyl and N-CO-halo-Ci- ₆-alkyl;

Y is selected from a bond, C=0 and (C=0)-NH;

y = 1 or 2;

the dotted line represents an optional double bond;

R¹⁰ and R¹¹ is independently selected from H, Ci-₆-alkyl, halo-Ci-₆-alkyl, -(CH₂-CH₂-0)_m- CH₂CH₂R¹², Co-C₈-alkylene-C₃-io-cycloalkyl and Co-C₈-alkylene-C₃-io-heterocycloalkyl, wherein alkyl, alkylene, cycloalkyl and heterocycloalkyl are unsubstituted or substituted with 1 to 6 substituents independently selected from the group consisting of halogen, CN, OH, oxo, Ci-3-alkyl, halo-Ci-3-alkyl, O-Ci-3-alkyl, O-halo-Ci-3-alkyl, S0₂-Ci-₃-alkyl, NR¹¹¹ R¹¹², CO2R^{1 1 1} and CONR¹¹¹ R¹¹²;

or wherein R¹⁰ and R¹¹ when taken together with the atom(s) to which they are attached complete a 3- to 8-membered ring containing carbon atoms and optionally containing 1 to 4 heteroatoms selected from O, S and N, wherein the ring is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of fluoro, OH, oxo, Ci- 3-alkyl and halo-Ci-3-alkyl;

R¹² is independently selected from halogen, OH, O-Ci-₆-alkyl, azide, NH₂, NH-(CH₂-CH₂-0)_m- Ci-₆-alkyl, N((CH₂-CH₂-0)_m-Ci-₆-alkyl)₂, NH-CO-Ci-e-alkyl, NCi-e-alkyl-CO-Ci-e-alkyl, NH-CO- CH₂0-(CH₂-CH₂-0)_m-H, NH-C0-CH₂0-(CH₂-CH₂-0)_m-Ci-₆-alkyl and NH-(CH₂-CH₂-0)_m-C_1-6- alkyl;

R¹¹¹ and R¹¹² is independently selected from H, Ci-4-alkyl, halo-Ci-4-alkyl, Co-C₄-alkylene-C₃- io-cycloalkyl and Co-C4-alkylene-C3-io-heterocycloalkyl, wherein

alkyl, alkylene, cycloalkyl and heterocycloalkyl are unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of halogen, CN, OH, oxo, Ci-3-alkyl, halo-Ci-3-alkyl, O-Ci-3-alkyl, O-halo-Ci-3-alkyl, S0₂-Ci-₃-alkyl, NH₂, NHCi-3-alkyl, N(Ci-₃-alkyl)₂, C0₂H, C0₂-Ci.₃-alkyl, CONH₂, CONHC^-alkyl and CON(Ci-3-alkyl)₂;

or wherein R¹¹¹ and R¹¹² when taken together with the nitrogen to which they are attached complete a 3- to 8-membered ring containing carbon atoms and optionally containing 1 to 3 heteroatoms selected from O, S or N, wherein the ring is unsubstituted or substituted with 1 to 4 substituents independently selected from the group consisting of fluoro, OH, oxo, C_h¬ alky! and halo-Ci-3-alkyl;

m is independently selected from 0 to 15;

n is independently selected from 0 to 30;

x is selected from 0 to 2; and

one or more hydrogen(s) in Formula (I) or in the residues may be replaced by deuterium(s); with the proviso, that when R' is hydrogen or deuterium, y is 1 and the dotted line is not present then -X-Y-R is not OH.

2. The compound for use according to claim 1 , which is represented by Formula (II)

3. The compound for use according to claim 1 or 2, which is represented by Formula (III)

4. The compound for use according to any one of claims 1 to 3, wherein y is 1.

5. The compound for use according to any one of claims 1 to 4, wherein R' is H.

6. The compound for use according to any one of claims 1 to 5, wherein X is O.

7. The compound for use according to any one of claims 1 to 6, wherein

R' is H;

Y-X is selected from O, (C=0)-0 and NH-(C=0)-0;

R is selected from CrC4-alkyl and C2-C4-alkenyl,

wherein alkyl and alkenyl is unsubstituted or substituted with 1 to 3 substituents independently selected from the group consisting of halogen, OR¹⁰, NR¹⁰R^{1 1} , -0-(CH₂- CH₂-0)n-CH2CH₂R¹², -0-(CH₂-CH₂-0)n-CH₂-(C=0)R¹² and C0₂R¹⁰;

R¹⁰ and R¹¹ is independently selected from H, CrC4-alkyl and fluoro-Ci-C4-alkyl;

R¹² is selected from OH, 0-Ci-₄-alkyl, NH₂, NHCi-₆-alkyl, N(Ci.₆-alkyl)₂, NH-CO-C^-alkyl and NCi-e-alkyl-CO-Ci-e-alkyl; and

n is selected from 0 to 10.

8. The compound for use according to any one of claims 1 to 7, wherein

R' is H; p = 1 to 5.

9. The compound for use according to claim 8, wherein

R' is H;

X-Y-R is OEt;

y = 1 or 2;

and the dotted line represents an optional double bond.

10. The compound for use according to any one of claims 1 to 4, wherein

R¹ is selected from CrC₆-alkyl, C2-C6-alkenyl and C2-C6-alkynyl,

wherein alkyl, alkenyl and alkynyl is unsubstituted or substituted with 1 to 7 substituents independently selected from the group consisting of CN, halogen, azide, oxo, OR¹⁰, O- C2-C6-alkylene-OR¹⁰, 0-C₃-io-cycloalkyl, 0-C₃-io-heterocycloalkyl, -(CH₂-CH₂-0)_n- CH2CH2R¹², -0-(CH₂-CH2-0)n-CH2CH₂R¹², C₀-C₈-alkylene-R¹⁰, CO2R¹⁰, CONR¹⁰R^{1 1} , CONR¹⁰SO₂R¹⁰, COR¹⁰, SO_XR¹⁰, SO₃H, SO₂NR¹⁰R^{1 1} , N R¹⁰COR^{1 1} , N R¹⁰SO₂R¹¹ , NR¹⁰- CO-NR¹⁰R¹¹ , NR¹⁰-SO₂-NR¹⁰R¹¹ , NR¹⁰R¹¹ and N(R¹⁰R¹⁰R¹¹)⁺; and

X is O.

1 1. The compound for use according to claim 10, wherein

R¹ is CrC4-alkyl,

wherein alkyl is unsubstituted or substituted with -0-(CH2-CH2-0)_n-CH2CH2R¹²;

R is selected from H and CrC4-alkyl,

wherein alkyl is unsubstituted or substituted with -0-(CH2-CH2-0)_n-CH2CH2R¹²; Y is selected from a bond or C=0;

R¹² is independently selected from OH or OMe; and

n is independently selected from 0 to 5.

12. The compound for use according to any one of claims 1 to 11 , which is selected from

or a pharmaceutically acceptable salt, co-crystal or solvate thereof.

13. The compound for use according to claim 12, which is selected from

or a pharmaceutically acceptable salt, co-crystal or solvate thereof.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier or excipient.