Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5β-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile

Abstract

Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J. Med. Chem., 2018, 61, 6759–6778), we identified N-phenyl-3α,7α,12α-trihydroxy-5β-cholan-24-amide as an inhibitor of C. difficile strain R20291 with an IC₅₀ of 1.8 μM. Studies of bile salts on spore germination have shown that chenodeoxycholate, ursodeoxycholate and lithocholate are more potent inhibitors of germination compared to cholate. Given this, we created amide analogs of chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids using amines identified from our previous studies. We found that chenodeoxy- and deoxycholate derivatives were active with potencies equivalent to those for cholanamides. This indicates that only 2 out of the 3 hydroxyl groups are needed for activity and that the alpha stereochemistry at position 7 is required for inhibition of spore germination.

Graphical Abstract

INTRODUCTION

Clostridioides (Clostridium difficile) is a common Gram-positive, spore-forming bacterium that can cause severe and even deadly infections in the colon.^1–2 Once the infection has been established in the gut, C. difficile produces toxins including the enterotoxin and cytotoxin, TcdA and TcdB respectively, resulting in diarrhea and severe damage to the intestinal lining of the gut.³ A 2019 CDC report indicated that in 2017, over 223,900 Americans contracted C. difficile resulting in 12,800 deaths and $1 billion in healthcare costs.⁴ People 65 years or older are more susceptible and make up 80% of C. difficile infections deaths.² Immunocompromised individuals and patients using broad-spectrum antibiotics are also at risk from C. difficile. The CDC has listed C. difficile as one of the top five urgent threats to public health in its 2019 report.⁴

C. difficile is spread by an oral-fecal route of transmission.² The vegetative bacteria themselves are unable to accomplish this transition since they are susceptible to the relatively high oxygen content of air and the highly acidic conditions of the stomach. Instead, C. difficile is spread by spores that are heat-resistant, aerobically stable, acid- and radiation-resistant, and most importantly are not affected by antiseptic cleaners.^2,5–6 Spores are dormant and are viable for months on surfaces. Outbreaks are most common in hospitals and healthcare facilities as the spore is not killed with standard cleaning procedures.⁷ Given the role of spores in the transmission of C. difficile, we hypothesized that eradicating spores or preventing their germination could prevent the development of C. difficile infections.

Spore germination is regulated in vivo by bile acids, glycine, and other nutrients present within the gut.^5–6,8–11 Several studies have shown that taurocholate, cholate, and deoxycholate activate germination, albeit with different potencies.^10–14 In contrast, the bile acid chenodeoxycholate inhibits spore germination.^13–14

Taurocholate produced by the liver is quickly reabsorbed after entering the distal ileum via enterohepatic circulation.^10–14 The taurocholate remaining in the gut is metabolized by the intestinal microbiome to generate cholic acid and taurine.¹⁵ Cholic acid is further metabolized to chenodeoxycholate. Thus, under normal conditions, the concentration of taurocholate in the intestines is low and the concentration of chenodeoxycholate is high, resulting in inhibition of spore germination.^{6,8–9,11,13} Upon treatment with antibiotics, the gut microbial population is depleted and altered, leading to an increase in the concentration of taurocholate and a decrease in chenodeoxycholate levels, thus promoting germination.^6,8–9 The connection between the gut microbiome, bile salt composition, and antibiotic use explains why patients on antibiotics have an increased risk of acquiring C. difficile.^2,7

Given the role of bile salts in germination, it is not surprising that these agents have been examined as inhibitors of germination. Studies have found that the natural bile salt, chenodeoxycholate (Fig. 1) inhibits germination with a K_i of 378 μM.¹⁴ Other natural compounds such as ursodeoxycholic acid and lithocholate have a K_i of 213 μM and 104 μM respectively.¹⁴ Ursodeoxycholic acid has been used to treat a single patient with C. difficile ileal pouchitis and has been reported to reduce C. difficile recurrence in humans.^16–17 However, another study has suggested that ursodeoxycholic acid may not be useful in preventing C. difficile infections in humans.¹⁸ Analogs of ursodeoxycholate have also been prepared and these inhibited germination with K_i’s in the 5–100 μM range.¹⁹

Figure 1.

Structures of bile acids: cholic acid (1), chenodeoxycholic acid (2), deoxycholic acid (3), lithocholic acid (4), and ursodeoxycholic acid (5)

Analogs of cholic acid have also been explored as anti-germinants. CamSA (6 in Fig. 2) is a potent inhibitor of spore germination (K_i 50 μM) that has been shown to actively stop C. difficile germination in both the hamster and mouse models and hence prevent infection.^10,20–23 Unfortunately, CamSA showed no inhibitory or prophylactic activity against the hypervirulent R20291 strain. The inability of CamSA to work on a hypervirulent strain, coupled with an increased prevalence of these strains in clinical settings, necessitated continued exploration of cholic acid analogs. Recently, we examined a range of aliphatic and aromatic N-phenylcholan-24-amides for their ability to inhibit spore germination.²⁴ We identified four lead compounds that displayed IC₅₀ values in the 1.8–16 μM (7-10 in Fig. 2) range. To improve potency of these agents, we hypothesized that since cholic acid itself is not a natural spore germination inhibitor whereas other bile salt acids are, changing the steroid portion of these molecules should enhance potency.¹¹ In this paper, we discuss the synthesis of a series of amide analogs (11-26) of chenodeoxy-, deoxy-, litho-, and ursodeoxycholic acids and present their biological activity against the R20291 strain. Since the primary difference between these acids is the presence or stereochemistry of the hydroxyl groups at the 7- or 12-position of the steroid, this study will also yield information on the necessity of the hydroxyl groups in inhibition.

Figure 2.

N-(Aryl)-3α,7α,12α-trihydroxy-5β-cholan-24-amides (7-10) identified as potent inhibitors of the epidemic strain of C. difficile R202091.

RESULTS

Synthesis of the Bile Salt Analogues.

The synthesis of the bile acid analogs (11-26) is outlined in Scheme 1. Chenodeoxycholic (2), deoxycholic (3), lithocholic (4), and ursodeoxycholic (5) acids were pre-activated with HBTU/NMM in anhydrous DMF or THF or a combination of both at room temperature and subsequently treated in situ with the appropriate aniline analog which produced the desired compounds (11–26) in moderate to good (49–88%) yields.^24,25 The isolation and purification of the products (11–26) were carried out by treating the concentrated post-reaction mixture with ice cold 2% aqueous HCl followed by sonication and filtration. Wherever purity of the compound was found not appropriate, the products were further purified by column chromatography with silica gel and eluted from a mixture of CH₂Cl₂ and MeOH.

Scheme 1.

Chemical synthesis of N-(Aryl)-3α,7α-dihydroxy-5β-chloan-24-amides (11-14), N-(Aryl)-3α,12α-dihydroxy-5β-chloan-24-amides (15-18), N-(Aryl)-3α-hydroxy-5β-chloan-24-amides (19-22), and N-(Aryl)-3α,7β-dihydroxy-5β-cholan-24-amides (23-26).

Antigerminant Activity of Compounds.

All compounds were analyzed as inhibitors of spore germination using a standard optical density assay, which measures germination as a decrease in absorbance at 580 nm.²⁴ A two-step process was taken for the analysis of the biological activity of the compounds. Compounds were first analyzed for their ability to inhibit spore germination of C. difficile R20291 at a single concentration of 125 μM. Compounds that were able to slow spore germination >40% compared to untreated samples were then reanalyzed at different concentrations to determine their IC₅₀ values. The anti-germinant activity of the compounds are shown in Table 1.

Table 1.

C. difficile spore germination inhibitory activities of N-(Aryl)-3α,7α-dihydroxy-5β- chloan-24-amides (11-14), N-(Aryl)-3α,12α-dihydroxy-5β-chloan-24-amides (15-18), N-(Aryl)-3α-hydroxy-5βchloan-24-amides (19-22), and N-(Aryl)-3α,7β-dihydroxy-5β-chloan-24-amides (23-26)

Base	Compounda	R1	R2	R3	R4	R5	R6	% Germination (125 μM)b	IC50 (μM)c
Chenodeoxycholate	11	OH	H	H	H	H	H	14 ± 0.6	4 ± 0.5
	12	OH	H	H	OCH3	H	H	31 ± 2	6 ± 1
	13	OH	H	H	H	F	H	100	NDd
	14	OH	H	H	CH3	H	F	66 ± 6	NDd
Deoxycholate	15	H	H	OH	H	H	H	3 ± 0.2	1.3 ± 0.1
	16	H	H	OH	OCH3	H	H	0.4 ± 3	2.4 ± 0.3
	17	H	H	OH	H	F	H	59 ± 5	NDd
	18	H	H	OH	CH3	H	F	65 ± 0.9	NDd
Lithocholate	19	H	H	H	H	H	H	100	NDd
	20	H	H	H	OCH3	H	H	100	NDd
	21	H	H	H	H	F	H	100	NDd
	22	H	H	H	CH3	H	F	100	NDd
Ursodeoxycholate	23	H	OH	H	H	H	H	57 ± 0.8	NDd
	24	H	OH	H	OCH3	H	H	100	NDd
	25	H	OH	H	H	F	H	100	NDd
	26	H	OH	H	CH3	H	F	100	NDd

^a.Number corresponding to scheme 1.

^b.Percent (%) germination of each compound was reported with standard deviations and tested at a final concentration of 125 μM. A 96-well plate was prepared by adding individual CamSA analogs to separate wells in triplicate along with 6mM taurocholate and 12mM glycine. Upon the addition of spores, the OD₅₈₀ was measured once every minute for 2 hours and normalized using the OD₅₈₀ obtained at time zero [relative OD₅₈₀ = OD₅₈₀(t)/OD₅₈₀(t₀)]. 100% indicates no inhibition at 125 μM.

^c.C. difficile spores were incubated with various concentrations of analogs along with 6 mM taurocholate and 12 mM glycine. The IC₅₀ was calculated by plotting the extent of germination versus the logarithm of the concentration of the analog.

^d.ND IC₅₀ was not determined.

The most biologically active compounds came from the chenodeoxy- and deoxycholan-24-amide series (11-18), where several compounds (11, 12, 15, 16) showed single micromolar IC₅₀ values (IC₅₀ 1–6 μM). Compounds 14, 17, and 18 inhibited germination, but with less potency (34–40% inhibition at 125 μM) compared to the simple aniline and o-methoxyaniline derivatives in the series. The most active compound in this series, 15, is approximately the same potency as the lead compound 7 (1.3 vs 1.8 μM). The deoxycholate derivative 16 is the next most potent compound (2.4 μM) and is 4-fold more potent than the cholate derivative, 8. The remaining active compounds 12 and 16 are within 1.5–2 fold of the potency of the cholate derivatives 7 and 8. Our results indicate that for the aniline and o-methoxyaniline amide derivatives, cholate, chenodeoxycholate and deoxycholate give potent compounds. These results suggest that neither the 7α-OH nor 12α-OH are required for the activity of these agents.

However, removal of both the 7- and 12-hydroxyl groups to give the N-phenyllithocholan-24-amide (19-22) series yielded inactive agents. Although the N-phenyllithocholan-24-amides are extremely hydrophobic, they were soluble to at least 125 μM and thus solubility was not the reason for their poor activity. Given the fact that the chenodeoxy- and dexoycholanamides contain two hydroxyl groups and were active, while the lithocholanamides only contain a single hydroxyl group (3α-OH) and were inactive, our data indicates that at least two hydroxyl groups are needed for activity.

To determine whether the hydroxyl groups form a specific interaction with their unknown target, we examined the stereospecificity of the 7-OH group on germination inhibition. Compounds 23-26 are ursodeoxycholanamides that possess a 3α-OH and 7β-OH functionality but lacked the 12α-OH. When compared to compounds 11-14, the ursodeoxycholanamides are inactive with marginal active seen for 23 (43% inhibition at 125 μM) only. This indicates that the hydroxyl groups must be in the α-configuration for their interaction with their target. This result also indicates that activity is due to a specific interaction with a target and not a non-specific detergent-like disruption of the spore.

DISCUSSION

We have previously shown that cholonamides are potent inhibitors of spore germination with the most potent compound, 7, displaying an IC₅₀ of 1.8 μM.²⁴ Cholic acid, the base bile acid used in these analogs, is an activator of germination whereas chenodeoxycholate, ursodeoxycholate and lithocholate are potent natural bile salt inhibitors.^{10–11,13–14} Thus, amide-based analogs derived from chenodeoxycholate, ursodeoxycholate and lithocholate should be more potent inhibitors of spore germination. Given this, we synthesized and evaluated the inhibition of a series of bile salt amides.

Our results indicate that one of the two hydroxyl groups at the 7 or 12 position is expendable, but removal of both group leads to inactive compounds. Alteration of the stereochemistry at the 7-position also eliminated activity suggesting a specific interaction between the target and the anti-germinant. These results are distinctly different from the natural bile acids in which lithocholate is the most potent inhibitor followed by ursodeoxycholic acid.^11,13 Both our work as well as the previous research on the effects of bile acids on germination were conducted in a ribotype 027 C. difficile strain. Studies on amide-based bile salt anti-germinants have also been conducted against the C. difficile 630 strain.²⁶ Compound 7, the simple phenyl cholanamide, gave an IC₅₀ of 270 μM against 630 while the chenodeoxycholic version (11) was inactive up to the maximum tested concentration.²⁶ The chenodeoxycholic analog of CamSA had an IC₅₀ of 6.5 mM, which is approximately 100-fold less potent than the cholate derivative (6, IC₅₀=58 μM).²⁶ However, other analogs examined in the study gave greater potency for the chenodeoxycholate versus the cholate derivative. Thus, no definitive conclusions can be reached regarding a strict preference for the steroid nucleus used in bile salt inhibitors for the 630 strain. Comparison of data across strains is also problematic since it is known that there are substantial differences in the effects of amide-based anti-germinants between the 630 and R20291 strains. For example, CamSA is active against the 630 strain, but inactive in the R20291 strain.²⁴ Compound 7 inhibits the 630 strain with an IC₅₀ of 270 μM, but inhibits the R20291 strain with an IC₅₀ of 1.8 μM.²⁶

The observation that one of the hydroxyl groups can be removed, but not more than one, is interesting. It is believed that germinants bind to the CspC protein to regulate germination.^{6,8,11,27–30} While the crystal structure of CspC has been solved, there is no structural or biophysical information on the binding of any bile salt to the protein.³⁰ Thus, the bile acid binding site and protein-ligand interactions are unknown. An examination of cholate and chenodeoxycholate binding sites in other lipid and bile salt binding proteins (PDB codes: 2QO5, 1TW4, 3EM0, 2FT9, 5L8O, 2QO6, 2QO4, 4QE6, 6HL1, 1OSV, 1OT7, 7CFN) reveals that often only 2 of the 3 hydroxyl groups interact with the protein suggesting that interaction with all of the hydroxyl groups is not required for activity. In about half of the cases listed above, a bridging water molecule is part of the interaction between the ligand and the protein. The presence of the water molecule provides flexibility in the interaction between the protein and the hydroxyl groups, all of which occur on the alpha face the steroid. In the other structures listed above, there is an extensive network of interactions with specific hydroxyl groups. Loss of a specific hydroxyl group should result in a change in affinity; however, this not always seen. In the case of the FXR receptor, it has been noted that 3-deoxy-chenodeoxycholate binds well and activates even though the 3-hydroxyl group forms critical interactions with the receptor.³¹ Structural analysis of this interaction reveals plasticity in the receptor allowing binding and activation.³¹

Despite this analysis, it should be noted that our spore germination assay utilizes the intact spore and thus does not directly measure binding to a target protein. The variances observed in our assay could also be the result of differences in compound transport through the spore.⁸ Ultimately, the rationale for the structure-activity data observed here will have to await structural data on the binding of anti-germinates to their target(s).

EXPERIMENTAL SECTION

General Comments.

Chenodeoxycholic acid (3α,7α-Dihydroxy-5β-cholan-24-oic acid), deoxycholic acid (3α,12α-Dihydroxy-5β-cholan-24-oic acid), lithocholic acid (3α-Hydroxy-5β-cholan-24-oic acid), and ursodeoxycholic acid (3α,7β-Dihydroxy-5β-cholan-24-oic acid) were purchased from Pfaltz and Bauer, and Chem-Impex International. Silica gel for column chromatography was purchased from Sorbent technologies. All reagents and solvents were purchased from Sigma-Aldrich, Acros Organics, TCI Chemicals or Chem-Impex International and were used without further purification. Thin layer chromatography (TLC) were performed on pre-coated (0.25 mm) silica gel plate (Sorbtech, 60 F-254), and visualization was done either by UV (254 nm), or iodine chamber. Column chromatographic purifications of compounds were performed on silica-gel (Sorbtech, 60–230 mesh, 0.063–0.20mm). ¹H and ¹³C NMR spectra were recorded on a Varian VNMRS 600 MHz or Bruker 400 MHz spectrometer by dissolving the compounds in deuterated solvents as methanol-d₄ (CD₃OD) or dimethyl sulfoxide-d₆ (DMSO-d₆) containing TMS as an internal standard. All the peaks in ¹H NMR spectra were referenced with TMS whereas in the ¹³C NMR spectra, the peaks were either reference with TMS or to the solvents used as CD₃OD (49.00), and DMSO-d₆ (39.52). The chemical shifts are expressed in ppm (δ) whereas coupling constants (J) are listed in hertz (Hz) and the multiplicities are recorded by following abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad signal). The purities of the testing compounds were determined by ¹H NMR. Melting points were determined using Mel-temp II apparatus by Laboratory Device, in open capillaries and are uncorrected.

N-(Phenyl)-3α,7α-dihydroxy-5β-cholan-24-amide (11).

To a round bottom flask, chenodeoxycholic acid 2 (393 mg, 1.0 mmol) and HBTU (476 mg, 1.25 mmol) were placed and dissolved in anhydrous DMF (5.0 mL). NMM (0.14 mL) was added into above reaction flask and it was sealed with a rubber septum and purged with argon and the resulting solution was stirred for 30 min at room temperature. A solution of aniline (112 mg, 1.20 mmol) in 2 mL of DMF was added to the reaction followed by 0.14 mL of NMM and the reaction was stirred for 30 min and then left at room temperature for overnight. The DMF was removed from the solution using a vacuum rotary evaporator using hot bath, dry further on high vacuum pump, and the residue so obtained, was treated with 150 mL of 2% ice cold HCl and sonicated twice for 10 min each time. A white precipitate formed during the sonication step. The flask was removed from the sonicator bath and allowed the precipitate to settled down on the bottom of flask, the liquid layer (water soluble material) was decanted, and the procedure was repeated two additional times with 100 mL of 2% HCl. The solid was collected by filtration, washed with cold water and dried under high vacuum to give a white solid which was subjected to purification by column chromatography over silica gel when eluted from a mixture of CH₂Cl₂-MeOH (95:5; 90:10; 85:15; 80:20 and 50:50) to give 383 mg (82% yield) of a white solid. mp: 104–106°C; TLC R_f. 0.53 (CH₂Cl₂:CH₃OH, 9:1); ¹H NMR (DMSO-d₆, 400 MHz): δ 9.82 (s, 1H), 7.57 (d, 2H, J = 8.0 Hz), 7.27 (t, 2H, J = 7.6 Hz), 7.00 (t, 1H, J = 7.2 Hz), 4.29 (d, 1H, J = 4.4 Hz), 4.10 (d, 1H, J = 3.2 Hz), 3.63 (s, 1H), 3.19–3.16 (m, 1H), 2.37–2.29 (m, 1H), 2.25–2.15 (m, 2H), 1.92 (d, 1H, J = 11.6 Hz), 1.88–1.68 (m, 6H), 1.48–1.17 (m, 14H), 1.14–0.84 (m, 8H), 0.61 (s, 3H); ¹H NMR (CD₃,OD, 400 MHz): δ 7.53 (d, 2H, J = 8.0 Hz), 7.28 (t, 2H, J = 7.6 Hz), 7.06 (t, 1H, J = 7.2 Hz), 3.79 (s, 1H), 3.39–3.33 (m, 1H), 2.46–2.38 (m, 1H), 2.31–2.22 (m, 2H), 2.02–1.82 (m, 6H), 1.74 (m, 1H), 1.66–1.53 (m, 2H), 1.50–1.40 (m, 5H), 1.36–1.27 (m, 5H), 1.23–1.17 (m, 3H), 1.14–0.92 (m, 7H), 0.69 (s, 3H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 171.61, 139.37, 128.57, 122.82, 118.97, 70.30, 66.13, 55.54, 50.00, 41.90, 41.39, 39.39, 35.28, 35.05, 34.81, 34.71, 33.32, 32.26, 31.33, 30.53, 27.77, 23.13, 22.68, 20.23, 18.35, 11.64; ¹³C NMR (CD₃,OD, 100 MHz): δ 175.16, 139.98, 129.77, 125.09, 121.30, 72.87, 69.05, 57.37, 51.57, 43.72, 43.20, 41.09, 40.80, 40.50, 36.99, 36.59, 36.24, 35.94, 35.02, 34.08, 33.20, 31.39, 29.31, 24.66, 23.43, 21.82, 19.02, 12.23.

N-(2’-Methoxyphenyl)-3α,7α-dihydroxy-5β-cholan-24-amide (12).

This compound was prepared from chenodeoxycholic acid 2 and 2-methoxyaniline in 81% yield. mp: 85–87°C; TLC R_f. 0.54 (CH₂Cl₂:CH₃OH, 9:1); ¹H NMR (DMSO-d₆, 400 MHz): δ 8.98 (s, 1H), 7.91 (d, 1H, J = 7.2 Hz), 7.02 (m, 2H), 6.87 (t, 1H, J = 7.2 Hz), 4.30 (d, 1H, J = 4.0 Hz), 4.09 (s, 1H), 3.81 (s, 3H), 3.63 (s, 1H), 3.19 (br, 1H), 2.40–2.15 (m, 3H), 1.92 (d, 1H, J = 10.8 Hz), 1.82–1.68 (m, 6H), 1.48–1.10 (m, 14H), 1.04–0.83 (m, 8H), 0.61 (s, 3H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 171.73, 149.52, 127.46, 124.03, 121.94, 120.10, 111.03, 70.31, 66.13, 55.57, 49.99, 41.90, 41.40, 39.40, 35.29, 35.06, 34.81, 34.70, 33.05, 32.26, 31.40, 30.53, 27.77, 23.14, 22.67, 20.24, 18.35, 11.63.

N-(3’-Fluorophenyl)-3α,7α-dihydroxy-5β-cholan-24-amide (13).

This compound was prepared from chenodeoxycholic acid 2 and 3-fluoroaniline in 65% yield. mp: 106–108 (softening), 189–192^oC; TLC R_f. 0.51 (CH₂Cl₂:CH₃OH, 9:1); ¹H NMR (DMSO-d₆, 400 MHz): δ 10.06 (s, 1H), 7.59 (d, 1H, J = 12.0 Hz), 7.34–7.26 (m, 2H), 6.83 (t, 1H, J = 7.6 Hz), 3.63 (s, 1H), 3.19 (m, 1H), 2.38–2.31 (m, 1H), 2.26–2.15 (m, 2H), 1.92 (d, 1H, J = 10.8 Hz), 1.83–1.68 (m, 6H), 1.48–1.37 (m, 7H), 1.32–1.18 (m, 7H), 1.14–0.84 (m, 8H), 0.61 (s, 3H); ¹H NMR (CD₃OD, 400 MHz): δ 7.51 (dt, 1H, J = 11.2 and 2.0 Hz), 7.31–7.22 (m, 2H), 6.78 (td, 1H, J = 9.6 and 1.6 Hz), 3.79 (s, 1H), 3.38–3.34 (m, 1H), 2.44–2.39 (m, 1H), 2.32–2.22 (m, 2H), 2.03–1.82 (m, 6H), 1.75 (m, 1H), 1.66–1.59 (m, 2H), 1.54–1.48 (m, 5H), 1.45–1.27 (m, 5H), 1.24–1.17 (m, 3H), 1.02–0.92 (m, 7H), 0.70 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 175.23, 165.45, 163.04, 141.87, 141.76, 131.13, 131.03, 116.29, 116.26, 111.32, 111.10, 108.08, 107.82, 72.81, 69.00, 57.27, 51.51, 43.66, 43.12, 41.02, 40.72, 40.42, 36.93, 36.52, 36.18, 35.87, 34.96, 34.01, 33.01, 31.32, 29.27, 24.61, 23.39, 21.76, 18.96, 12.18.

N-(2’-Methyl-5’-fluorophenyl)-3α,7α-dihydroxy-5β-cholan-24-amide (14).

This compound was prepared from chenodeoxycholic acid 2 and 2-methyl-5-fluoroaniline. The product was further purified by column chromatography over silica gel when eluted from a mixture of CH₂Cl₂-MeOH (95:5, 90:10, 80:20, and 70:30) to yield white solid in 69% yield. mp: 104–106°C; TLC R_f. 0.49 (CH₂Cl₂:CH₃OH, 9:1); ¹H NMR (CD₃OD, 400 MHz): δ 7.22–7.17 (m, 2H), 6.83 (td, 1H, J = 8.4 and 2.4 Hz), 3.79 (s, 1H), 3.38–3.31 (m, 1H), 2.47–2.43 (m, 1H), 2.36–2.32 (m, 1H), 2.27 (d, 1H, J = 11.6 Hz), 2.22 (s, 3H), 2.03–1.82 (m, 6H), 1.76–1.74 (m, 1H), 1.66–1.59 (m, 2H), 1.54–1.49 (m, 5H), 1.45–1.26 (m, 5H), 1.24–1.18 (m, 2H), 1.13–1.10 (m, 1H), 1.02 (d, 3H, J = 6.4 Hz), 0.98–0.94 (m 4H), 0.68 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 175.46, 163.45, 161.05, 138.30, 138.20, 132.51, 132.42, 128.95, 128.92, 113.42, 113.28, 113.21, 113.04, 72.81, 68.99, 57.32, 51.52, 43.68, 43.13, 41.04, 40.73, 40.43, 36.92, 36.54, 36.19, 35.89, 34.34, 34.02, 33.21, 31.33, 29.31, 24.62, 23.42, 21.78, 18.97, 17.46, 12.23.

N-(Phenyl)-3α,12α-dihydroxy-5β-cholan-24-amide (15).

This compound was prepared from deoxycholic acid 3 and aniline. The product was further purified by column chromatography over silica gel when eluted from a mixture of CH₂Cl₂-MeOH (95:5, 90:10, 80:20, and 70:30) to yield white solid in 70% yield. mp: 102–104 (softening), 185–87^oC; TLC R_f. 0.41 (CH₂Cl₂:CH₃OH, 95:5); ¹H NMR (CD₃OD, 400 MHz): δ 7.52 (d, 2H, J = 8.0 Hz), 7.28 (t, 2H, J = 7.6 Hz), 7.06 (t, 1H, J = 7.6 Hz), 3.96 (s, 1H), 3.56–3.47 (m, 1H), 2.43–2.39 (m, 1H), 2.32–2.26 (m, 1H), 1.93–1.75 (m, 7H), 1.65–1.58 (m, 3H), 1.54–1.37 (m, 9H), 1.32–1.25 (m, 2H), 1.18–1.05 (m, 5H), 1.01–0.92 (m, 4H), 0.71 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 175.28, 140.01, 129.79, 125.11, 121.30, 74.10, 72.57, 49.34, 48.15, 47.60, 43.66, 37.49, 37.24, 36.95, 36.47, 35.34, 35.02, 34.86, 33.21, 31.11, 29.96, 28.72, 28.44, 27.51, 24.92, 23.75, 17.77, 13.25.

N-(2’-Methoxyphenyl)-3α,12α-dihydroxy-5β-cholan-24-amide (16).

This compound was prepared from deoxycholic acid 3 and 2-methoxyaniline. The product was further purified by column chromatography over silica gel when eluted from a mixture of CH₂Cl₂-MeOH (95:5, 90:10, 80:20, and 70:30) to yield white solid in 63% yield. mp: 75–78°C; TLC R_f. 0.34 (CH₂Cl₂:CH₃OH, 95:5); ¹H NMR (CD₃OD, 400 MHz): δ 7.88 (dd, 1H, J = 8.0 and 1.2 Hz), 7.09 (td, 1H, J = 8.0 and 1.2 Hz), 6.99 (d, 1H, J = 7.6 Hz), 6.90 (td, 1H, J = 8.0 and 1.2 Hz), 3.97 (s, 1H), 3.87 (s, 3H), 3.56–3.48 (m, 1H), 2.52–2.45 (m, 1H), 2.39–2.31 (m, 1H), 1.93–1.75 (m, 7H), 1.65–1.59 (m, 3H), 1.54–1.37 (m, 9H), 1.33–1.25 (m, 2H), 1.85–1.05 (m, 5H), 1.02–0.93 (m, 4H), 0.72 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 175.33, 151.76, 128.20, 126.25, 123.79, 121.45, 111.84, 74.11, 72.57, 56.24, 49.34, 48.23, 47.62, 43.66, 37.49, 37.24, 36.90, 36.47, 35.34, 34.89, 34.86, 33.20, 31.11, 29.94, 28.72, 28.44, 27.51, 24.92, 23.74, 17.74, 13.25.

N-(3’-Fluorophenyl)-3α,12α-dihydroxy-5β-cholan-24-amide (17).

This compound was prepared from deoxycholic acid 3 and 3-fluoroaniline. The product was further purified by column chromatography over silica gel when eluted from a mixture of CH₂Cl₂-MeOH (95:5, 90:10, 80:20, and 70:30) to yield white solid in 61% yield. mp: 194–196^oC; TLC R_f. 0.50 (CH₂Cl₂:CH₃OH, 95:5); ¹H NMR (CD₃OD, 400 MHz): δ 7.51 (dt, 1H, J = 11.6 and 1.2 Hz), 7.30–7.22 (m, 2H), 6.81–6.76 (m, 1H), 3.96 (s, 1H), 3.55–3.49 (m, 1H), 2.47–2.39 (m, 1H), 2.32–2.25 (m, 1H), 1.92–1.75 (m, 7H), 1.65–1.58 (m, 3H), 1.54–1.37 (m, 9H), 1.32–1.28 (m, 2H), 1.25–1.05 (m, 5H), 1.02–0.93 (m, 4H), 0.71 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 175.34, 165.47, 163.06, 141.89, 141.78, 131.13, 131.04, 116.32, 116.29, 111.32, 111.11, 108.09, 107.83, 74.03, 72.51, 49.29, 48.07, 47.54, 43.60, 37.43, 37.18, 36.87, 36.41, 35.29, 34.97, 34.80, 33.01, 31.05, 29.90, 28.65, 28.38, 27.45, 24.85, 23.69, 17.70, 13.18.

N-(2’-Methyl-5’-Fluorophenyl)-3α,12α-dihydroxy-5β-cholan-24-amide (18).

This compound was prepared from deoxycholic acid 3 and 2-methyl-5-fluoroaniline. The product was further purified by column chromatography over silica gel when eluted from a mixture of CH₂Cl_2-MeOH (95:5, 90:10, 80:20, and 70:30) to yield white solid in 57% yield. mp: 91–93°C; TLC R_f. 0.47 (CH₂Cl₂:CH₃OH, 95:5); ¹H NMR (CD₃OD, 400 MHz): δ 7.21–7.18 (m, 2H), 6.84 (td, 1H, J = 8.4 and 2.4 Hz), 3.97 (s, 1H), 3.55–3.49 (m, 1H), 2.51–2.44 (m, 1H), 2.39–2.31 (m, 1H), 2.21 (s, 3H), 1.93–1.85 (m, 5H), 1.81–1.75 (m, 2H), 1.65–1.59 (m, 3H), 1.54–1.37 (m, 9H), 1.33–1.25 (m, 2H), 1.18–1.07 (m, 5H), 1.02–0.95 (m, 4H), 0.71 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 175.62, 163.51, 161.11, 138.35, 138.25, 132.55, 132.47, 129.10, 129.07, 113.50, 113.38, 113.29, 113.14, 74.08, 72.55, 49.33, 48.16, 47.60, 43.64, 37.47, 37.22, 36.90, 36.46, 35.33, 34.84, 34.37, 33.24, 31.08, 29.94, 28.73, 28.43, 27.49, 24.90, 23.75, 17.73, 17.46, 13.25.

N-(Phenyl)-3α-hydroxy-5β-cholan-24-amide (19).

This compound was prepared from lithocholic acid 4 and aniline in 88% yield. mp: 198–200°C; ¹H NMR (DMSO-d₆, 400 MHz): δ 9.83 (s, 1H), 7.57 (d, 2H, J = 8.0 Hz), 7.27 (t, 2H, J = 8.0 Hz), 7.00 (t, 1H, J = 7.6 Hz), 4.48 (b, 1H), 2.35–2.30 (m, 1H), 2.24–2.17 (m, 1H), 1.94 (d, 1H, J = 8.4 Hz), 1.89–1.75 (m, 3H), 1.70–1.63 (m, 2H), 1.60–1.49 (m, 2H), 1.36–1.03 (m, 17H), 0.93–0.87 (m, 7H), 0.62 (s, 3H); ¹H NMR (CD₃OD, 400 MHz): δ 7.52 (d, 2H, J = 7.2 Hz), 7.28 (t, 2H, J = 7.2 Hz), 7.07 (t, 1H, J = 6.8 Hz), 3.53 (br, 1H), 2.42–2.39 (m, 1H), 2.31–2.27 (m, 1H), 2.02 (d, 1H, J = 10.8 Hz), 1.89–1.71 (m, 5H), 1.61 (br s, 2H), 1.44–1.10 (m, 17H), 1.01–0.94 (m, 7H), 0.69 (s, 3H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 171.59, 139.37, 128.57, 122.82, 118.98, 69.83, 56.07, 55.55, 42.25, 41.50, 36.27, 35.36, 35.12, 34.95, 34.18, 33.33, 31.29, 30.35, 27.70, 26.86, 26.13, 23.82, 23.24, 20.38, 18.31, 11.86; ¹³C NMR (CD₃OD, 100 MHz): δ 175.17, 139.97, 129.75, 125.09, 121.29, 72.43, 57.95, 55.48, 43.94, 43.56, 41.90, 41.56, 37.25, 37.20, 36.93, 36.50, 35.69, 35.01, 33.17, 31.21, 29.29, 28.37, 27.66, 25.28, 23.94, 21.96, 18.94, 12.50.

N-(2’-Methoxyphenyl)-3α-hydroxy-5β-cholan-24-amide (20).

This compound was prepared from lithocholic acid 4 and 2-methoxyaniline in 79% yield. mp: 193–195°C; ¹H NMR (DMSO-d₆, 600 MHz): δ 9.02 (s, 1H), 7.91 (s, 1H), 7.02 (br s, 2H), 6.88 (s, 1H), 4.46 (s, 1H), 3.81 (s, 3H), 2.40 (s, 1H), 2.30 (s, 1H), 1.94 (d, 1H, J = 5.4 Hz), 1.81–1.76 (m, 3H), 1.68 (d, 1H, J = 12.6 Hz), 1.62–1.49 (m, 3H), 1.35–1.04 (m, 17H), 0.92–0.87 (2 br peaks, 7H), 0.62 (s, 3H); ¹³C NMR (DMSO-d₆, 150 MHz): δ 171.79, 149.60, 127.46, 124.13, 122.06, 120.15, 111.06, 69.88, 56.12, 55.61, 42.30, 41.53, 39.99, 39.71, 36.32, 35.40, 35.16, 35.02, 34.23, 33.06, 31.41, 30.40, 27.77, 26.91, 26.20, 23.89, 23.30, 20.43, 18.37, 11.91.

N-(3’-fluorophenyl)-3α-hydroxy-5β-cholan-24-amide (21).

This compound was prepared from lithocholic acid 4 and 3-fluoroaniline in 82% yield. mp: 222–224°C; ¹H NMR (DMSO-d₆, 600 MHz): δ 10.08 (s, 1H), 7.60 (d, 1H, J = 12.0 Hz), 7.33–7.26 (m, 2H), 6.84 (t, 1H, J = 8.4 Hz), 4.46 (s, 1H), 3.34 (br, 1H), 2.36–2.32 (m, 1H), 2.24–2.19 (m, 1H), 1.93 (d, 1H, J = 9.6 Hz), 1.86–1.76 (m, 3H), 1.67 (d, 1H, J = 13.8 Hz), 1.64–1.58 (m, 1H), 1.54–1.49 (m, 2H), 1.361.23 (m, 9H), 1.18–1.08 (m, 5H), 1.03 (br, 3H), 0.92–0.87 (m, 7H), 0.62 (s, 3H); ¹H NMR (CD₃OD, 400 MHz): δ 7.52 (dt, 1H, J = 9.2 and 2.0 Hz), 7.31–7.22 (m, 2H), 6.79 (td, 1H, J = 7.2 and 1.2 Hz), 3.57–3.50 (m, 1H), 2.46–2.38 (m, 1H), 2.31–2.24 (m, 1H), 2.02 (d, 1H, J = 11.6 Hz), 1.97–1.72 (m, 5H), 1.63–1.60 (m, 2H), 1.48–1.05 (m, 17H), 1.01–0.94 (m, 7H), 0.70 (s, 3H); ¹³C NMR (DMSO-d₆, 150 MHz): δ 172.04, 162.94, 161.34, 141.16, 141.08, 130.30, 130.24, 114.65, 109.38, 109.24, 105.79, 105.62, 69.87, 56.09, 55.55, 42.28, 41.52, 39.97, 36.30, 35.39, 35.15, 34.98, 34.22, 33.37, 31.18, 30.39, 27.75, 26.90, 26.18, 23.87, 23.28, 20.42, 18.32, 11.89.

N-(2’-Methyl-5’-fluorophenyl)-3α-hydroxy-5β-cholan-24-amide (22).

This compound was prepared from lithocholic acid 4 and 2-methyl-5-fluoroaniline in 59% yield. mp: 182–184°C; ¹H NMR (DMSO-d₆, 400 MHz): δ 9.22 (s, 1H), 7.36 (d, 1H, J = 10.0 Hz), 7.20 (s, 1H), 6.88 (s, 1H), 4.42 (s, 1H), 2.44–2.23 (2 br, 2H), 2.17 (s, 3H), 1.94 (d, 1H, J = 7.2 Hz), 1.88–1.47 (m, 7H), 1.42–0.99 (m, 17H), 0.94 and 0.87 (2 br, 7H), 0.62 (s, 3H); ¹H NMR (CD₃OD, 400 MHz): δ 7.22–7.18 (m, 2H), 6.85 (td, 1H, J = 8.4 and 2.4 Hz), 3.57–3.50 (m, 1H), 2.50–2.43 (m, 1H), 2.38–2.31 (m, 1H), 2.21 (s, 3H), 2.04 (d, 1H, J = 11.6 Hz), 1.94–1.87 (m, 3H), 1.83–1.72 (m, 2H), 1.63–1.61 (m, 2H), 1.52–1.40 (m, 7H), 1.37–1.25 (m, 5H), 1.22–1.07 (m, 5H), 1.03–0.93 (m, 7H), 0.71 (s, 3H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 171.82, 161.24, 158.86, 137.82, 131.72, 131.27, 131.19, 126.33, 111.06, 110.89, 110.65, 69.84, 56.09, 55.57, 42.27, 41.51, 36.28, 35.37, 35.13, 34.93, 34.19, 32.81, 31.38, 30.37, 27.72, 26.87, 26.15, 23.82, 23.24, 20.39, 18.30, 17.11, 11.85.

N-(Phenyl)-3α,7β-dihydroxy-5β-cholan-24-amide (23).

This compound was prepared from ursodeoxycholic acid 5 and aniline in 65% yield. mp: 196–198°C; TLC R_f. 0.58 (CH₂Cl₂:CH₃OH, 9:1); ¹H NMR (CD₃OD, 400 MHz): δ 7.53 (d, 2H, J = 7.6 Hz), 7.28 (t, 2H, J = 7.6 Hz), 7.07 (t, 1H, J = 7.2 Hz), 3.48 (m, 2H), 2.46–2.38 (m, 1H), 2.31–2.24 (m, 1H), 2.05 (d, 1H, J = 12.0 Hz), 1.91–1.79 (m, 5H), 1.63–1.37 (m, 10H), 1.34–1.12 (m, 7H), 1.06–0.96 (m, 7H), 0.72 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 175.21, 140.00, 129.79, 125.12, 121.34, 72.17, 72.00, 57.57, 56.61, 44.85, 44.57, 44.09, 41.63, 40.78, 38.67, 38.07, 36.95, 36.15, 35.22, 35.09, 33.28, 31.09, 29.73, 28.00, 23.98, 22.44, 19.15, 12.70.

N-(2’-Methoxyphenyl)-3α,7β-dihydroxy-5β-cholan-24-amide (24).

This compound was prepared from ursodeoxycholic acid 5 and 2-methoxyaniline. The product was purified by column chromatography over silica gel when eluted from a mixture of CH₂Cl₂-MeOH (95:5, 90:10, 80:20, 70:30 and 50:50) to yield white solid in 74% yield. mp: 87–89°C; TLC R_f. 0.50 (CH₂Cl₂:CH₃OH, 9:1); ¹H NMR (CD₃OD, 400 MHz): δ 7.88 (d, 1H, J = 8.0 Hz), 7.09 (t, 1H, J = 7.6 Hz), 6.99 (d, 1H, J = 8.0 Hz), 6.90 (t, H, J = 7.6 Hz), 3.87 (s, 3H), 3.50–3.45 (m, 2H), 2.52–2.44 (m, 1H), 2.38–2.31 (m, 1H), 2.05 (d, 1H, J = 12.4 Hz), 1.93–1.79 (m, 5H), 1.64–1.10 (m, 17H), 1.07–0.96 (s, 7H), 0.72 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 174.98, 151.43, 128.24, 126.03, 123.44, 121.43, 111.74, 72.07, 71.87, 57.44, 56.53, 56.23, 44.76, 44.46, 43.98, 41.55, 40.67, 38.59, 38.02, 36.81, 36.09, 35.14, 34.95, 33.17, 31.04, 29.68, 27.93, 23.98, 22.38, 19.14, 12.72.

N-(3’-Fluorophenyl)-3α,7β-dihydroxy-5β-cholan-24-amide (25).

This compound was prepared from ursodeoxycholic acid 5 and 3-fluoroaniline in 72% yield. mp: 120–122 (softening), 201–203°C; TLC R_f. 0.55 (CH₂Cl₂:CH₃OH, 9:1); ¹H NMR (CD₃OD, 600 MHz): δ 7.53 (d, 1H, J = 11.4 Hz), 7.30–7.23 (m, 2H), 6.79 (t, 1H, J = 7.2 Hz), 3.51–3.44 (m, 2H), 2.45–2.40 (m, 1H), 2.30–2.51 (m, 1H), 2.04 (d, 1H, J =12.0 Hz), 1.92–1.80 (m, 4H), 1.62–1.38 (m, 11H), 1.36–1.10 (m, 7H), 1.05–0.96 (m, 7H), 0.71 (s, 3H); ¹³C NMR (CD₃OD, 150 MHz): δ 175.23, 165.05, 163.44, 141.85, 131.12, 131.06, 116.31, 111.31, 111.17, 108.08, 107.90, 72.10, 71.91, 57.46, 56.48, 44.77, 44.46, 43.99, 41.55, 40.69, 38.60, 38.00, 36.89, 36.08, 35.15, 35.07, 33.09, 31.03, 29.69, 27.94, 23.96, 22.38, 19.12, 12.68.

N-(2’-Methyl-5’-fluorophenyl)-3α,7β-dihydroxy-5β-cholan-24-amide (26).

This compound was prepared from ursodeoxycholic acid 5 and 2-methyl-5-fluoroaniline. The product was purified by column chromatography over silica gel when eluted from a mixture of CH₂Cl₂-MeOH (95:5, 90:10, 80:20, 70:30 and 50:50) to yield white solid in 49% yield. mp: 93–95 (softening), 101–103^oC; TLC R_f. 0.52 (CH₂Cl₂:CH₃OH, 9:1); ¹H NMR (CD₃OD, 400 MHz): δ 7.22–7.18 (m, 2H), 6.85 (td, 1H, J = 8.4 and 2.8 Hz), 3.50–4.46 (m, 2H), 2.51–2.44 (m, 1H), 2.39–2.31 (m, 1H), 2.21 (s, 3H), 2.06 (d, 1H, J = 12.4 Hz), 1.93–1.90 (m, 3H), 1.87–1.79 (m, 2H), 1.63–1.57 (m, 3H), 1.54–1.42 (m, 7H), 1.41–1.10 (m, 7H), 1.06–0.94 (m, 7H), 0.71 (s, 3H); ¹³C NMR (CD₃OD, 100 MHz): δ 175.48, 163.45, 161.05, 138.30, 138.20, 132.52, 132.43, 128.97, 113.46, 113.31, 113.25, 113.07, 72.09, 71.91, 57.50, 56.55, 44.80, 44.47, 44.00, 41.58, 40.70, 38.60, 37.99, 36.88, 36.08, 35.16, 34.39, 33.28, 31.02, 29.72, 27.95, 23.95, 22.39, 19.09, 17.46, 12.68.

Bacterial Strains and Spore Preparation.

C. difficile R20291 was the kind gift of Prof. Nigel Minton (University of Nottingham). C. difficile cells were streaked onto BHIS (Brain heart infusion supplemented with 20 mg/ml yeast extract, 0.1% L-cysteine, and 0.05% sodium taurocholate) agar to yield single colonies. Single C. difficile colonies were grown in BHIS (Brain heart infusion supplemented with 5 mg/mL yeast extract) broth overnight and spread onto BHIS agar to obtain bacterial lawns. The plates were incubated for 7 days at 37°C in an anaerobic environment (10% CO₂, 10% H₂, and 80% N₂). The resulting bacterial lawns were collected by flooding the plates with ice-cold deionized water. The spores were pelleted and washed three times by centrifugation at 8,800 x g for five minutes. To remove any contaminating vegetative cells, the spores were purified through a 20% to 50% HistoDenz gradient at 18,200 x g for 30 minutes. The resulting spore pellet was washed five times with water, resuspended in a 0.05% sodium thioglycolate solution, and stored at 4°C.

C. difficile Spore Germination Assays.

Purified C. difficile spores were pelleted and washed with deionized water three times by centrifugation at 9,400 x g to remove the storage buffer. The spores were heat activated at 68°C for 30 minutes, then washed an additional three times to remove any spores that auto germinated. The spores were diluted to an optical density of 580 nm (OD₅₈₀) to 1.0 with a 100 mM sodium phosphate buffer, pH 6.0, containing 5 mg/ml sodium bicarbonate. To test for antagonists of spore germination, a 96-well plate was prepared by adding compounds to a final concentration of 125 μM into separate wells in triplicate along with 6mM taurocholate and 12mM glycine. Upon the addition of spores, the OD₅₈₀ was measured once every minute for 2 hours and normalized using the OD₅₈₀ obtained at time zero [relative OD₅₈₀ = OD₅₈₀(t)/OD₅₈₀(t₀)]. Selected compounds were further tested for germination inhibition at increasing concentrations to determine the concentration that reduces spore germination by 50% (IC₅₀).

Ernesto Abel-Santos and Steven Firestine are founders and officers of Able Therapeutics, LLC. Able Therapeutics is a virtual biotech company dedicated to targeting spore germination as prophylactic approaches to infectious diseases. Able Therapeutics has exclusive licensing rights to all IP generated from the Abel-Santos and Firestine labs, including all compounds tested in this manuscript. Able Therapeutics has not generated any income and none of the authors have received compensation from Able Therapeutics.