Accepted Manuscript Synthesis and biological activity of novel deoxycholic acid derivatives Irina I. Popadyuk, Andrey V. Markov, Oksana V. Salomatina, Evgeniya B. Logashenko, Andrey V. Shernyukov, Marina A. Zenkova, Nariman F. Salakhutdinov PII: DOI: Reference:

S0968-0896(15)00412-5 http://dx.doi.org/10.1016/j.bmc.2015.05.012 BMC 12308

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

12 February 2015 27 April 2015 4 May 2015

Please cite this article as: Popadyuk, I.I., Markov, A.V., Salomatina, O.V., Logashenko, E.B., Shernyukov, A.V., Zenkova, M.A., Salakhutdinov, N.F., Synthesis and biological activity of novel deoxycholic acid derivatives, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.012

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Synthesis and biological activity of novel deoxycholic acid derivatives Irina I. Popadyuka+, Andrey V. Markovb+, Oksana V. Salomatinaa, Evgeniya B. Logashenkob∗, Andrey V. Shernyukova, Marina A. Zenkovab, Nariman F. Salakhutdinova, c a

N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch Russian Academy of Sciences, 9, Lavrent’ev ave., Novosibirsk, 630090, Russian Federation b Institute of Chemical Biology and Fundamental Medicine, Siberian Branch Russian Academy of Sciences, 8, Lavrent’ev ave., Novosibirsk, 630090, Russian Federation c Novosibirsk State University, 2, Pirogova Str., Novosibirsk, 630090, Russian Federation [+] These authors contributed equally to this work.

A B S TR A C T We report the synthesis and biological activity of new semi-synthetic derivatives of naturally occurring deoxycholic acid (DCA) bearing 2-cyano-3-oxo-1-ene, 3-oxo-1(2)-ene or 3-oxo-4(5)-ene moieties in ring A and 12-oxo or 12-oxo-9(11)-ene moieties in ring C. Bioassays using murine macrophage-like cells and tumour cells show that the presence of the 9(11) double bond associated with the increased polarity of ring A or with isoxazole ring joined to ring A, improves the ability of the compounds to inhibit cancer cell growth. Keywords: Bile acids Deoxycholic acid derivatives Cytotoxicity Nitric oxide Biological activity

1. Introduction

Bile acids (BAs) are steroidal molecules synthesised from cholesterol in the liver of mammalian. BAs circulate in the human body from the liver to the bile, which is followed by entry into the small intestine, absorption into the distal part of the ileum, transport back to the liver in the portal venous blood and re-secretion into bile; this process is named enterohepatic circulation.1,2,3 All BAs consist of two units, that is, a rigid steroid nucleus containing one to three hydroxyl groups and a short aliphatic side chain with a carboxyl group at the end. The structures of these compounds have a number of functional groups defining a wide range of possible synthetic transformations.4,5 BAs are widespread in nature; they possess a high enantiomeric purity and a broad spectrum of native biological activities (anti-inflammatory, antiviral, anticancer, immunostimulatory).1,5,6,7 Originally, BAs were known for their functions in the regulation of lipid, glucose and cholesterol homeostasis as well as solubilisation and transport of lipids and fat-soluble vitamins.5,6,8 However, recent investigations have found that BAs can also act like hormones by binding to nuclear (FXR, TGR5, PXR, CAR, VDR)6,7,9,10,11,12 and membrane13 receptors. Also, BAs play an important role as promoters of colon and esophageal cancers.7 BAs are cytotoxic at abnormally high concentrations, either intra-cellularly or extra-cellularly8 and can cause necrosis or apoptosis.14 All of these facts make BAs interesting and perspective starting materials for organic synthesis in order to obtain new derivatives with new biological properties. Earlier, it was discovered that the introduction of a 2-cyano-3-oxo-1-ene moiety into pentacyclic triterpene molecules (betulin,15 betulinic acid,16,17 oleanolic,18 ursolic18 and glicyrrhetinic acid19,20,21) enhances anti-inflammatory and anticancer activities compared to the corresponding natural precursors. The most active semi-synthetic derivatives bardoxolone methyl (methyl 2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-28-oate)22 and Soloxolone methyl (methyl 2-cyano-3,12dioxo-18βH-olean-9(11),1(2)-dien-30-oate)21,23 were obtained by direct A- and C-ring modification of oleanolic and glycyrrhetinic acids, respectively. These compounds contain a 2-cyano-3-oxo-1(2)-ene moiety in the A ring and a 12-oxo11(9)-ene moiety in the C ring. It was interesting to investigate the effect of such modification on biological activity in case of other scaffold. BAs and pentacyclic triterpenes are biogenetic relatives, as both are products of squalene bio-cyclisation. Compared with pentacyclic triterpenoids, BAs contain four fused rings, which mean a smaller molecular weight that is beneficial in terms of membrane permeability. Furthermore, BA derivatives may afford better bioavailability than triterpenes, owing to the fact that BAs are metabolic products. Among BAs only deoxycholic acid does not contain hydroxyl groups in ring B that allow us to realize direct modifications affecting only A- and C- rings. This work aimed at revealing the structure–activity relationships (SARs) of new semi-synthetic derivatives of deoxycholic acid (DCA). Taking inspiration from the results previously mentioned on triterpenes, a series of compounds based on DCA was synthesised using the following strategies: building up 3-oxo-1(2)-en, 3-oxo-4(5)-en and 2-cyano-3∗ Corresponding author. Tel.: +7-383-363-5161; fax: +7-383-363-5153; e-mail: [email protected]

oxo-1(2)-en moieties in the A ring of DCA, creating 12-oxo-9(11)-en and 12-oxo moieties in ring C of DCA (Fig. 1). These derivatives 1–6 and their intermediates were synthesised and then screened in vitro for their ability to inhibit NO production and for their cytotoxic activities.

Figure 1. Semi-synthetic derivatives of DCA (1–6).

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2. Results and discussion 2.1. Chemical synthesis To improve the anti-inflammatory and anticancer activity of DCA, novel derivatives of DCA were synthesised by combining the chemical modifications in rings A and C (Fig.1). The key intermediates in the synthesis were 3,12-dioxo derivatives of DCA 8 and 13 (Scheme 1). The methyl ester of DCA 7 (DCA-Me) was chosen as a starting material. It was obtained in quantitative yield by esterification of DCA with diazomethane in MeOH (Scheme 1) analogously to the known procedure. 24

Scheme 1. Synthesis of DCA key intermediates (8, 13); reagents and conditions: (a) Jones reagent, acetone, RT; (b) Ac2 O, pyridine, RT; (c) SeO2, AcOH, reflux; (d) SeO2, AcOH, MW; (e) KOH, MeOH, RT.

Diketone 8 was synthesised from DCA-Me through a Jones oxidation with a yield of 94%.25 The sequence of reactions to obtain key intermediate 13 is shown in Scheme 1. The 3-hydroxy group was protected by Ac2O at room temperature in pyridine26 (compound 9); reaction course was monitored by TLC to avoid interaction of the 12-hydroxy group with Ac2O, followed by Jones oxidation gave the carbonyl group at C-12 (10). Further formation of 9(11)-double bond in the C ring of the steroid skeleton was attempted by bromination–dehydrobromination, according to previously reported data.27 α,βUnsaturated carbonyl compounds can also be prepared through the reaction of carbonyl compounds with SeO2 in acetic acid.28,29,30 Interaction of 10 with a six-fold excess of SeO2 in glacial acetic acid for 24 h (reflux) gives 11 with a yield of 75%. Subsequent deprotection of the acetate group by KOH in methanol freed the 3-hydroxy group (compound 12), and Jones oxidation gives the second intermediate 13 with a total yield of 45% after five steps from 7. The main disadvantage of the key synthetic step (oxidation of 10 with SeO2) was the long duration of the reaction, as full conversion of 10 to 11 was achieved after 24 h. Moreover, in the case of partial conversion of 10 to 11, the isolation of 11 from the mixture was very laborious. The application of microwave techniques has proven to be useful in organic synthesis.31 In order to optimise the conditions for the conversion of 10 to 11, we conducted a series of microwave-assisted oxidation experiments of 10 with SeO2 in acetic acid (Table 1). Data show that microwave irradiation alone has no effect on the reaction rate and, when using a six-fold excess of SeO2 at 120°C (b.p. AcOH 118°C), the conversion of 10 to 11 was equivalent to one, when under reflux at atmospheric pressure. Experiments performed at various temperatures revealed that full conversion was achieved at 180°C. In order to optimise the amount of SeO2 used, experiments with varying amounts of SeO2 were carried out. It turned out that a two-fold excess was not enough to complete the reaction and three-fold excess of SeO2 resulted in a conversion of 60%. It should be noted that a three-fold excess of SeO2 under reflux conditions at atmospheric pressure resulted in complete conversion after 48 h. Significantly better results (full conversion) were obtained when the amount of 10 was increased four times to 400 mg (in 2 mL of AcOH) with a three-fold excess of SeO2 (Table 1, line 9). Thus, the microwave-assisted synthesis conditions allowed us to carry out the experiments at 180°C, reduce the reaction time from 24 h to 30 min and reduce the amount of SeO2 used from a six-fold excess to three-fold.

3

Table 1. Effect of temperature, ratio and concentration of reagents in the microwave-assisted oxidation of 10 with SeO2 in glacial AcOH. №

Compound 10, mg

Molar ratio 10:SeO2

T, °C

t, h

Ratio 10:11 NMR 1H data

1

100

1:6

120

0.5

1:0

2

100

1:6

150

0.5

1:1

3

100

1:6

180

0.5

0:1

4

100

1:2

180

0.5

3:2

5

100

1:2

180

1

1:1

6

100

1:2

180

2

1:1

7

100

1:3

180

0.5

1:2

8

50

1:3

180

0.5

2:3

9

400

1:3

180

0.5

0:1

Volume of AcOH used in each experiment was 2 mL.

According to our efforts to synthesise compounds containing the 12-oxo-9(11)-ene fragment and multiple bonds in different positions of ring A, we have synthesised compounds 2 and 3. For introduction into the steroid framework 4(5)double bond, we used a previously published procedure for steroidal compounds.32,33 It included obtaining a 4-bromine derivative followed by dehydrobromination. Compound 3 was obtained by reacting 13 with bromine in acetic acid, followed by heating in DMF with lithium carbonate and lithium bromide. The reaction mixture contained desirable product 3 and side product 2 in the ratio 4:1 (1H NMR data). Compound 3 was obtained by flash column chromatography in 60% yield. Another common method of producing α, β-unsaturated carbonyl compounds is through the interaction with PhSeCl and subsequent oxidation of the selenium-containing intermediate.34 Thus, reaction of 13 with PhSeCl in AcOEt, followed by decomposition of the Se-intermediate with H2O2/THF gave compound 2, containing a 3-oxo-1(2)-en moiety, and trace amounts of 3 and also 3-oxo-1,4-diene derivative. Both of these reactions to introduce a double bond in the alpha position of the carbonyl group proceed via the enol, and we can assume the factor that determines the direction of the reaction is the nature of the solvent. Compounds 5 and 6 were prepared in a similar way to 2 and 3, based on compound 8. For the formation of compound 5, the conversion of 8 achieved 50%, and when the reaction time was increased, the conversion was not changed. Increasing the amount of PhSeCl resulted in the formation of a complex mixture of products. Bromination and dehydrobromination of 8 led to the formation 5 and 6 in a 1:4 ratio. Under these conditions, a complex mixture of products can be formed; however, according to NMR data we did not observe formation of any products other than 5 and 6. The compounds were obtained by flash column chromatography with yields of 16 and 63%, respectively. Among the different ways of administration of the CN-group, we chose a method that included formation and subsequent cleavage of the isoxazole ring, as this method is often used in the chemistry of natural compounds.15-21 Initially, we conducted a condensation reaction of compound 13 with methyl formate in the presence of sodium methoxide in benzene, which resulted in the formation of 2- (14a) and 4-hydroxymethylene (14b) derivatives in a 10:1 ratio (1 H NMR data). Further transformation was performed with a mixture of compounds 14a and 14b. Isoxazole derivatives, 15a and 15b, were prepared by reacting the hydroxymethylene derivatives 14a and 14b with NH2OH*HCl. Cleavage of the isoxazole ring was carried out with sodium methoxide in methanol. The isoxazole ring in compound 15a was opened with formation of the CN-group (16), whereas the isoxazole ring in compound 15b was not cleaved under these conditions. In this step, compounds 16 and 15b were separated by column chromatography on silica gel. Further interaction between 16 and 2,3-dichloro-5,6-dicyanquinone (DDQ) in benzene under reflux results in the formation of desirable product 1 containing 1(2) double bond (total yield 15% after four steps from 13). Simultaneously, we modified DCA to obtain a compound containing the 2-cyano-1(2)-en-3-one fragment in ring A and 12-oxo group in ring C. As the carbonyl group in rings A and C of compound 8 differ in reactivity, we decided to use the same synthetic scheme as for 1. Compound 8 reacted with methyl formate in benzene in the presence of sodium methoxide and gave 2- (17a) and 4-hydroxymethylene (17b) derivatives in a 2:1 ratio (1H NMR data). The ratio of 17a to 17b became 8:1 when sodium methoxide was replaced with NaH (1H NMR data). Further transformations were performed with a mixture of compounds 17a and 17b. Subsequent condensation with NH2OH*HCl led to the formation of compounds 18a and 18b. At this stage, it was necessary to control the temperature and reactant ratio, as high temperatures and excess NH2OH*HCl may cause condensation of the 12-oxo group with NH2OH. The reaction of compound 18a with sodium methoxide in MeOH led to the opening of the isoxazole ring, giving 19, which was proven by 1H and13C NMR data. As for 15b, the isoxazole ring in compound 18b was not cleaved under these conditions. The desirable product, compound 4, was obtained by reaction of 19 with DDQ in benzene (total yield was 35% after four steps from 8). Thus, in the present study, a series of new compounds based on the available animal metabolite—DCA—was synthesised. The scheme proposed the introduction of a 2-cyano-1(2)-en-3-ones fragment in ring A of DCA steroidal framework, which has been successfully implemented. All target compounds and intermediates were characterised by 1 H and 13C NMR and high-resolution mass spectroscopy (HRMS). Configurations for 14a, 14b, 17a, 17b were assigned using NMR data. Chemical shifts of OH-groups of all these compounds are in the range of 14.2-15.5 ppm in the 1H NMR

4

spectra showing strong H-bonds, and leaving the only one possible orientation of double bond in respect to carbonyl group at C-3 (Z-orientation). This (Z) orientation is in a good agreement with 1H-1H NOESY data (for 14a NOE between (H-26)(H-1,1’) and (H-26)-(H-11); for 17a NOE between (H-26)-(H1,1’) and (H-26)-(H-11e); for 14b and 17b NOE between (H-26)-(H-5), (H-26)-(H-6e), (H-26)-(H-7a)).

Scheme 2. Modification of DCA ring A. Reagents and conditions: (a) PhSeCl, AcOEt, RT; H2O2, THF, 1h, RT; (b) Br2, AcOH, 1.5 h, RT; LiBr, Li2 CO3, DMF, 2h, reflux; (c) HCOOMe, NaOMe, benzene, 4h, RT; (d) NH2OH· HCl, MeOH-H2 O, reflux; (e) NaOMe, MeOH-Et2 O (0°C to RT); f) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), benzene, reflux.

2.2. Biological activity

5

Here, we screened compounds 1–6 and all intermediates leading to them (see Figure 1, Scheme 2) for their cytotoxicity with respect to different cancer cells and for their ability to inhibit NO production. We also compared the biological activity of the new compounds with that of Soloxolone methyl (SM; as a positive control), which is a semi-synthetic derivative of glycyrrhetinic acid that, as we showed earlier, exhibits high antiproliferative and pro-apoptotic activities.23 Owing to the specificity of the metabolism of BAs for the investigation of novel derivatives of DCA, human duodenal cancer cells (HuTu-80), human hepatocellular liver carcinoma cells (HepG2) and murine hepatoma cells (MH-22a) were used. The cytotoxic activity of each compound was determined using the MTT assay, a colorimetric test of cell viability during treatment with each compound. Human epidermoid cancer cells KB-3-1, which are not related to enterohepatic and non-cancer cells J774, were chosen as a control. The IC50 values (concentration of a compound allowing survival of 50% of the cells in a population) of the synthesised DCA derivatives with respect to the tested cells are listed in Table 2.

Table 2. IC50 values for of the intermediates and target compounds 1–6 Compound

ICHuTu80 50

HepG2

IC50

ICMH-22 50

ICKB 50

ICJ774 50

ICNO 50

1

18.3±0.4

19.6±0.5

31.1±1.4

>100

>100

14.3±0.9

2

43.3±5.8

>100

23.2±0.4

>100

>100

11.4±1.5

3

55.7±3.6

>100

51.5±1.4

>100

>100

12.2±1.9

4

94.2±0.9

>100

51.5±0.1

>100

>100

11.9±0.1

5

37.2±4.8

>100

43.1±2.5

>100

>100

8.7±0.3

6

39.9±4.2

>100

33.8±1.6

>100

63.4±5.2

10.3±1.9

8

56.3±8.2

>100

>100

>100

>100

10.5±0.1

13

26.8±2.7

>100

>100

>100

>100

10.1±0.6

14a

10.5±0.5

16.9±0.8

38.2±0.3

>100

>100

9.3±0.3

14b

13.2±0.6

68.1±8.1

>100

21.9±0.6

>100

9.4±0.3

15a

13.0±0.2

20.3±1.6

21.8±1.1

21.9±1.4

>100

8.4±0.6

17a

6.5±0.5

8.1±0.2

9.8±0.3

4.9±0.5

20.9±1.6

7.9±0.8

17b

18.9±1.7

>100

70.7±1.1

>100

>100

10.1±1.3

18a

38.2±3.3

38.9±1.6

30.1±2.9

>100

>100

24.9±0.3

18b

19.5±0.6

21.5±0.7

24.8±0.8

>100

>100

37.7±5.2

DCA*

82.9±1.9

>100

51.8±0.6

>100

>100

50.7±6.5

SM**

0.7±0.2

2.4±0.4

2.5±0.2

0.3±0.1

3.3±0.1

0.1±0.1 HepG2

The results are presented as the mean ± SEM of three independent experiments. The ICHuTu80 , IC50 , 50 MH-22 J774 IC50 , ICKB 50 and IC 50 values were defined as the compound concentration providing 50% of HuTu80, HepG2, MH-22, KB-3-1 and J774 cells survival, respectively, as measured by the MTT assay (see Section 4); incubation time: 24 h. The IC୒୓ 50 was defined as the compound concentration providing a 50% decrease in NO production by LPS-stimulated J774 cells in comparison with the control (not compounds added). The grey highlighting indicates the most active compounds. *DCA: deoxycholic acid, **SM: Soloxolone methyl.

As can be seen from the data presented in Table 2, all tested compounds (except 4) exhibit toxicity against the HuTu-80 cell line. It is not surprising, because many BAs absorb through intestinal epithelial cells. These DCA derivatives were found to be more active than their precursor. The most active compounds were 14a, 14b, 15a and 17a (ICHuTu80 values 50 10.46, 13.23, 13.00 and 6.46, respectively). Among target compounds 1–6, only compounds 1 displayed moderate cytotoxicity for duodenal cells (ICHuTu80 = 18.34±0.43), whereas all others displayed low (2, 3, 5, 6) or very low 50 cytotoxicity (4). We compared the activity of compounds against the duodenal cell line with that against the HepG2 liver cell line; less than half of the compounds tested were cytotoxic against HepG2, despite the fact that DCA is absorbed into the bloodstream by the liver and then secreted in the bile composition. The IC50 values for HepG2 cells turned out to be somewhat higher than that for HuTu-80 cells, except for compound 14b, which was almost inactive with respect to HepG2 cells. The precursor compound DCA did not exhibit any toxicity against human cancer liver cells. It is interesting to note that, at the same time, almost all compounds were more or less cytotoxic against murine hepatoma cells, MH-22a, which is probably owing to different metabolisms of enterohepatic circulation of bile salts between humans and rodents. With regard to normal cell lines, J774, only two compounds showed some toxicity (Table 2), that is, compounds 6 and 17a. So, among the tested derivatives, compound 17a was toxic for all used cell lines, including normal and non-cancer cells, and the values of IC50 differed slightly. The toxicity of target compounds 1–6 with respect to cancer cells was much

6

lower compared to that of the intermediate derivatives. As the minimal value corresponds to greater toxicity, compounds 14a, 14b and 15a can be identified as the most toxic to cancer cells. The criterion used to determine the effectiveness of the compounds was the Selectivity Index (SI). The SI was defined as the ratio of the cytotoxicity of a compound with respect to normal cells (ICJ774 50 ) versus cancer cells (Table 3). Taking into account that a higher SI corresponds to greater overall anticancer activity, we can identified the leading compounds as 14a, 14b and 15a (SI values for HuTu-80 cells 9.6, 7.6 and 7.6, respectively); the SI for 1 was 5.5. Table 3. SI values for of the intermediates and target compounds Compound

SIHuTu80

SIHe pG2

SI MH-22

SI KB

1

5.5

5.1

3.2

-

2

2.3

-

4.3

-

3

1.6

-

1.7

-

4

1.06

-

1.9

-

5

2.7

-

2.3

-

6

1.5

-

1.9

-

8

1.7

-

-

-

13

3.7

-

-

-

14a

9.6

5.8

2.6

-

14b

7.6

1.4

-

4.5

15a

7.6

4.9

4.5

4.6

17a

3.2

2.6

2.1

4.2

17b

5.3

-

1.4

-

18a

2.6

2.5

3.3

-

18b

5.1

4.6

4.0

-

DCA*

1.2

-

1.9

-

SM**

4.7

1.4

1.3

11

୎଻଻ସ

HuTu80 The SI was the ratio of IC50 (cytotoxicity on normal J774 cells) to the IC50 , HepG2 MH-22 IC50 , IC50 and ICKB 50 (cytotoxicity on HuTu-80, HepG2, MH-22a and KB-3-1 cells, respectively). The grey highlighting indicates the most effective compounds. *DCA: deoxycholic acid, **SM: Soloxolone methyl.

To understand the flexibility of the action of novel DCA derivatives, we analysed their cytotoxicity against nonenterohepatic KB-3-1 cells (Table 2). Only derivatives 14b, 15a and 17a decreased the viability of KB-3-1 cells, with almost the same value of SI (4.5, 4.6 and 4.2, respectively). So, comparison of the cytotoxicity of the compounds with the SI values highlighted the lead compounds - intermediates 14a, 14b and 15a, but not designed derivatives 1–6. The inorganic free radical NO, synthesised by a family of enzymes, termed NO synthases (NOS), acts through a hostdefence mechanism, damaging pathogenic DNA35 and as a regulatory molecule with homeostatic activities.36 However, excessive production of NO, owing to reactions with superoxides in biological systems, gives rise to various diseases such as inflammation, carcinogenesis and atherosclerosis.37,38 NO also plays an important role in angiogenesis, invasion and metastasis progression. 39,40 Therefore, inhibition of NO production could facilitate anticancer therapy. The inhibitory activities (ICNO 50 ) of DCA derivatives on NO production, induced by LPS in J774 macrophage-like cells, are shown in Table 2. Also, in this experiment, SM was used as the positive control. Preliminary screening showed that almost all of the new derivatives of DCA exhibit inhibitory activities against LPSinduced NO production in J774 cells at the level of 10 µM, except compounds 18a and 18b (24.9 and 37.7 µM, respectively). The inhibitory activity of SM was two orders higher (0.11 µM). A comparison of the structures of the synthesised compounds with pronounced biological activity makes it possible to identify some structure/biological activity relationships for synthetic analogues of DCA: (i) The 9(11)-double bond generally increased the cytotoxicity of the compounds (compare 13 and 8 or 1 and 4) or expanded the pattern of biological action (compare 15a and 18a or 14b and 17b); the absence of the 9(11)-double bond significantly increases toxicity against both malignant and non-malignant cells (compare 17a and 14a). (ii) The increasing of polarity of ring A by introduction of a 2- or 4-hydroxymethyl group increased the cytotoxicity of the compounds and expanded the pattern of biological action (compare 14a or 14b and 13). Notably, the location of the

7

hydroxymethyl group affects the activity of the compound; 2-hydroxymethylene is more preferable than the 4hydroxymethylene group (compare 14a and 14b). (iii) Introduction of an isoxazole ring joined with ring A increased the cytotoxicity of the compounds and expanded the pattern of biological action (compare 18a or 18b and 8). Notably, the position of ring fusion affects the activity of the compound; [3,4] fusion is more preferable than [2,3] (compare 18a and 18b). All newly synthesised compounds display more pronounced biological activity than the initial parent compound DCA.

3. Conclusion We have synthesised DCA derivatives with inhibitory effects on the overproduction of NO with in vitro antiproliferative activity with respect to tumour cells. Based on the obtained data, we conclude that, for the design of DCA derivatives, we need to consider that the presence of the 9(11) double bond with the increased polarity of ring A or with the isoxazole ring joined to ring A, increases the ability of the compounds to inhibit cancer cell growth. One important criterion for a therapeutic drug for cancer is to have minimal or no side effects to normal body cells of patients undergoing chemotherapy. One way to achieve this is by employing lower doses of drugs. This invariably implies that the drug should not only have a high potent activity at lower concentrations, but should also exhibit a high degree of selectivity. The present in vitro studies demonstrate the ability of synthetic compound 14a to have high selective toxicity at lower concentrations on human duodenal cancer cells (SI = 9.6).

4. Experimental 4.1. Synthesis 4. 1. 1. G ener al ex p er i m ent al pr oc edu r es Spectral and analytical investigations were carried out at Collective Chemical Service Center of Siberian Branch of the Russian Academy of Sciences. Melting points were determined on a METTLER TOLEDO FP900 thermosystem and are uncorrected. The element composition of the products was determined from high-resolution mass spectra recorded on a DFS (double focusing sector) Thermo Electron Corporation instrument. Optical rotations were measured with a PolAAr 3005 polarimeter. Microwave-assisted reactions were carried out in a microwave reactor Monowave 300 (AntonPaar). 1 H and 13C NMR spectra were measured on Bruker spectrometers: AM-400 (operating frequency 400.13 MHz for 1H and 100.61 MHz for 13C) and DRX-500 (500.13 MHz for 1H and 125.76 MHz for 13C) using CDCl3 solutions of the substances. The chemical shifts were recorded in δ (ppm) using the δ 7.24 of CHCl3 (1H NMR) and δ 76.90 (13C NMR) as internal standards. Chemical shift measurements are given in ppm and the coupling constants (J) in hertz (Hz). The structure of the compounds was determined by NMR using the standard one-dimensional and two-dimensional procedures (1H-1H COSY, 1H-13C HMBC/HSQC, 13C-1H HETCOR/COLOC). The purity of the final compounds and intermediates for biological testing was confirmed to be ≥95%, as determined by HPLC analysis. HPLC analyses were carried out on a MilichromA-02, using a ProntoSIL 120-5-C18 AQ column (BISCHOFF, 2.0×75 mm column, grain size 5.0 µm). Mobile phase: Millipore purified water with 0.1% trifluoroacetic acid at a flow rate of 150 µ L/min at 35°C and UV detection at 210, 220, 240, 260 and 280 nm. A typical run time was 25 min with a linear gradient of 0 to 100% methanol. Flash column chromatography was performed with silica gel (Merck, 60-200 mesh) and neutral alumina (Chemapol, 40-250 mesh). 4. 1. 2. Rea g ent s DCA and selenium (IV) oxide were purchased from abcr GmbH & Co. KG, phenylselenyl chloride was purchased from ACROS organics, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and sodium hydride 57–63% in oil dispersion were purchased from Alfa Aesar, Ac2O, hydroxylamine hydrochloride was purchased from Reachem, lithium carbonate was purchased from Vekton and lithium bromide was purchased from Panreac Quimica S.A.U. Methyl formate, sodium methoxide and solution of diazomethane in diethyl ether were prepared according to the previously reported methods. All solvents used in the reactions were previously purified and dried according to the previously reported procedures. 4. 1. 3. M et hyl 3 α , 1 2 α - d i hyd r ox y - 5 β -ch ol an - 24- oat e (7, DC A-M e) To a stirring solution of DCA (20 g, 51 mmol) in MeOH (100 mL) at 0°C, a solution of CH2N2 in Et2 O was added portion-wise until the reaction mixture became yellow (excess of diazomethane). After complete decomposition of diazomethane (reaction mixture became colourless with a transparent solution), the solvent was removed on a rotary evaporator. Compound 7 (20.7 g, quantitative yield) was obtained as a white solid. This material was used for the next reaction without further purification. An analytically pure sample was obtained by re-crystallisation from CH3OH. M.p. 58.6ºC [decomposition, lit. 58.0–60.0ºC (CH3OH)].41 HRMS: m/z calcd for C25H42O4: 406.3078; found: 406.3084. 1H NMR (CDCl3): δ = 3.92 (dd, 1H, J1~J2~3, H-12), 3.60 (s, 3H, CH3-25), 3.53 (dddd, 1H, J3a, 2a= J3a, 4a=11.1, J3a, 2e= J3a, 2 2 4e=4.6, H-3a(β)), 2.31 (ddd, 1H, J=15.8, J23, 22=9.8, J23, 22′ =5.3, H-23), 2.17 (ddd, 1H, J=15.8, J23′, 22′ =9.2, J23′, 22=6.8, H23’), 2.09 (br.s, 2H, OH-3, OH-12), 1.90-1.44 (m: 13H, [1.80]-H-16, [1.78]-H-6, [1.76]-H-9, [1.74]-H-22, 1.74 (ddd, 1H, 2 J=J4a, 5=13.0, J4a, 3a =11.1, H-4a(α)), 1.67 (ddd, 1H, 2 J=15.5, J1e, 2a=J1e, 2e=3.6, H-1e(α)), 1.66 (ddd, 1H, J17, 20=J17, 16a=J17, 2 16e=9.6, H-17), 1.60 (dm, 1H, J=12.6, H-2e(β)), [1.53]-H-15, [1.51]-H-14, [1.46]-H-4, [1.45]-H-11, [1.45]-H-11), 1.441.18 (m: 8H, 1.35 (dddd, 1H, 2J=J2a, 3a= J2a, 1a=12.6, J2a, 1e=3.6, H-2a(α)), [1.35]-H-7, [1.35]-H-8, [1.33]-H-5, [1.32]-H-20,

8

[1.28]-H-22, [1.20]-H-16, [1.20]-H-6), 1.08 (dddd, 1H, J7a, 6a=14.0, 2J=13.7, J7a, 8=12.8, J7a, 6e=4.0, H-7a(α)), [1.00]-H-15, 0.90 (ddd, 1H, 2J=15.5, J1a, 2a =12.6, J1a, 2e=3.4, H-1a(β)), 0.90 (d, 3H, J21, 20 = 6.4, CH3 -21), 0.84 (s, 3H, CH3-19), 0.61 (s, 3H, CH3-18). 13C NMR (CDCl3): δ = 174.59 (s, C-24), 72.90 (d, C-12), 71.43 (d, C-3), 51.30 (q, C-25), 47.95 (d, C-14), 46.98 (d, C-17), 46.28 (s, C-13), 41.90 (d, C-5), 36.14 (t, C-4), 35.81 (d, C-8), 35.10 (t, C-1), 35.07 (d, C-20), 33.92 (s, C10), 33.35 (d, C-9), 30.96 (t, C-23), 30.71 (t, C-22), 30.12 (t, C-2), 28.40 (t, C-11), 27.34 (t, C-16), 26.97 (t, C-6), 25.95 (t, C-7), 23.52 (t, C-15), 22.92 (q, C-19), 17.03 (q, C-21), 12.50 (q, C-18). 4. 1. 4. M et hyl 3 α - ac et oxy - 12 α - h ydr ox y- 5 β - ch ol an- 24 - oa t e (9) Compound 7 (9.66 g, 23.8 mmol) was dissolved in pyridine (100 mL) and Ac2 O (6 mL, 0.06 mmol) was added dropwise with vigorous stirring; the reaction mixture was stirred for 4 h (the reaction course was monitored by TLC, CHCl3: AcOEt = 20: 3) and saturated aqueous NaCl (80 mL) was added. The reaction mixture was diluted with AcOEt (30 mL) and aqueous HCl (5%) was added until the pH reached 4–5. The organic layer was separated; the aqueous layer was extracted with AcOEt. The combined organic layer was washed with 5% aqueous HCl, saturated aqueous NaHCO3, brine, dried over anhydrous MgSO4 and evaporated to dryness. The crude product was purified by flash column chromatography (silica gel, 0–50% AcOEt in CH2Cl 2) to yield compound 9 (7.67 g, 72%) as an amorphous solid. An analytically pure sample was obtained by re-crystallisation from CH3OH. M.p. 111.1–113.0ºC [lit. 104.2–107.7ºC (AcOEt)].42 HRMS: m/z calcd for C27H44O5: 448.3183; found: 448.3176. 1H NMR (CDCl3): δ = 4.68 (dddd, 1H, J3a, 2a= J3a, 4a=11.4, J3a, 2e= J3a, 2 4e=4.7, H-3a(β)), 3.96 (br.t., 1H, J1~J2~3, H-12), 3.64 (s, 3H, CH3-25), 2.34 (ddd, 1H, J=15.8, J23, 22=9.8, J23, 22′=5.3, H2 23), 2.21 (ddd, 1H, J=15.8, J23′, 22′=9.2, J23′, 22=6.8, H-23′), 1.99 (s, 3H, CH3-27), 1.90-1.70 (m: 6H, [1.84]-H-16, [1.83]H-4, [1.82]-H-6, [1.80]-H-9, [1.79]-H-22, 1.74 (ddd, 1H, 2J=14.3, J1e, 2a=J1e, 2e=3.4, H-1e(α))), 1.70-1.18 (m: 15H, [1.67]H-17, [1.67]-H-2, [1.60]-H-15, [1.67]-H-17, [1.54]-H-14, [1.53]-H-4, [1.67]-H-17, [1.49]-H-11, [1.49]-H-11, [1.45]-H-5, [1.43]-H-2, [1.41]-H-8, [1.40]-H-7, [1.39]-H-20, [1.34]-H-22, [1.25]-H-16, [1.24]-H-6), 1.14-0.96 (m: 3H, [1.09]-H-7, [1.06]-H-15, 1.01 (ddd, 1H, 2 J=J1a, 2a=14.3, J1a, 2e=3.5, H-1a(β)), 0.95 (d, 3H, J21, 20 = 6.4, CH3 -21), 0.89 (s, 3H, CH3-19), 0.65 (s, 3H, CH3 -18).13C NMR (CDCl3): δ = 174.52 (s, C-24), 170.54 (s, C-26), 74.14 (d, C-3), 73.01 (d, C-12), 51.35 (q, C-25), 48.19 (d, C-14), 47.27 (d, C-17), 46.40 (s, C-13), 41.75 (d, C-5), 35.88 (d, C-8), 34.95 (d, C-20), 34.77 (t, C-1), 34.01 (s, C-10), 33.55 (d, C-9), 32.06 (t, C-4), 30.95 (t, C-23), 30.08 (t, C-22), 28.60 (t, C-11), 27.31 (t, C-16), 26.84 (t, C6), 26.39 (t, C-2), 25.90 (t, C-7), 23.84 (t, C-15), 22.99 (q, C-19), 21.31 (q, C-27), 17.22 (q, C-21), 12.62 (q, C-18). 4. 1. 5. M et hyl 3 α - ac et oxy - 12- ox o- 5 β - ch ol an- 24- oa t e (10 ) Jones reagent (4.5 mL), prepared from Na2Cr2O7*2H2O in aqueous H2SO4, was added drop-wise to a solution of compound 9 (3.51 g, 7.83 mmol) in acetone (100 mL) at 0°C over a period of 30 min until the brown colour persisted. The mixture was stirred for a further 1.5 h at room temperature (the reaction course was monitored by TLC) and EtOH (50 mL) was added. The resulting mixture was concentrated under reduced pressure (v ~ 50 mL) and diluted with H2 O (300 mL). The solid was filtered and dried. The crude product was purified by flash column chromatography (silica gel, 0–50% AcOEt in CH2Cl 2) to yield 10 (3.12 g, 89%) as a white solid. An analytically pure sample was obtained by recrystallisation from CH3OH. M.p. 143.7–148.4ºC [lit. 142.2–144.4ºC (AcOEt/hexane)].42 HRMS: m/z calcd for C27H42O5: 446.3027; found: 446.3022. 1H NMR (CDCl3): δ = 4.61 (dddd, 1H, J3a, 2a= J3a, 4a=11.4, J3a, 2e= J3a, 4e=4.6, H-3), 3.58 (s, 3H, CH3 -25), 2.54 (dd, 1H, J11a, 9=12.8, 2J=12.2, H-11a(β)), 2.31 (ddd, 1H, 2J=15.8, J23, 22 =9.8, J23, 22′=5.3, H-23), 2.18 (ddd, 1H, 2 J=15.8, J23′, 22′=9.2, J23′, 22=6.8, H-23’), 1.96 (dd, 1H, 2J=12.2, J11e, 9=4.4, H-11e(α)), 1.95 (ddd, 1H, J17, 20=J17, 16a=J17, 2 16e =9.7, H-17), 1.93 (s, 3H, CH3-27), 1.90-1.68 (m: 5H, [1.86]-H-16, 1.81 (dddd, 1H, J=J6a, 7a=14.0, J6a, 7e=J6a, 5=4.5, H6a(β)), 1.78 (ddd, 1H, J9, 11a=12.8, J9, 8=11.5, J9, 11e=4.4, H-9), [1.74]-H-22, [1.73]-H-8), 1.68-1.57 (m: 4H, [1.64]-H-15, [1.63]-H-4, [1.62]-H-2, [1.61]-H-1), 1.52-1.42 (m: 3H, [1.48]-H-4, [1.46]-H-5, [1.44]-H-7), 1.39-1.14 (m: 7H, [1.33]-H-2, [1.30]-H-22, [1.29]-H-14, [1.26]-H-15, [1.25]-H-16, [1.23]-H-6, [1.20]-H-20), 1.07-0.96 (m: 2H, [1.02]-H-7, [1.01]-H-1), 0.94 (s, 3H, CH3-19), 0.94(s, 3H, CH3-18), 0.77 (d, 3H, J21, 20 = 6.6, CH3-21).13 C NMR (CDCl 3): δ = 214.35 (s, C-12), 174.34 (s, C-24), 170.32 (s, C-26), 73.45 (d, C-3), 58.39 (d, C-14), 57.25 (s, C-13), 51.19 (q, C-25), 46.21 (d, C-17), 43.78 (d, C-9), 41.10 (d, C-5), 37.83 (t, C-11), 35.40 (d, C-8), 35.36 (d, C-20), 35.10 (s, C-10), 34.69 (t, C-1), 31.88 (t, C-4), 31.03 (t, C-23), 30.26 (t, C-22), 27.27 (t, C-16), 26.69 (t, C-6), 26.10 (t, C-2), 25.76 (t, C-7), 24.08 (t, C-15), 22.50 (q, C19), 21.13 (q, C-27), 18.33 (q, C-21), 11.43 (q, C-18). 4. 1. 6. M et hyl 3 α - ac et oxy - 12- ox o- 5 β - ch ol- 9(1 1)- en- 2 4- o at e (1 1 ) General procedure for the oxidation of methyl 3α-acetoxy-12-oxo-5β-cholan-24-oate 10 with selenium (IV) oxide. 1) SeO2 (4.51 g, 40.6 mmol, 6 equiv.) was added to a solution of compound 10 (3.02 g, 6.77 mmol) in glacial AcOH (150 mL). After heating under reflux for 24 h, the reaction mixture was cooled to room temperature and diluted with AcOEt; the selenium was removed by filtration. The filtrate was washed with saturated aqueous NaHCO3 until the pH reached 7–8 and then it was extracted with AcOEt. H2O2 (3 mL, 30%) was added to a stirring organic layer. After stirring, the colour of the organic phase changed from dark brown to pale yellow. The organic phase was washed with brine, dried over anhydrous MgSO4 and evaporated to dryness. The crude product was purified by flash column chromatography (silica gel, chloroform) to yield compound 11 (2.26 g, 75%) as a pale yellow solid. An analytically pure sample was obtained by re-crystallisation from hexane/Et2O mixture. M.p. 148.8–149.4ºC [lit. 141–142ºC (AcOEt/hexane)]42 HRMS: m/z calcd for C27H40O5: 444.2870; found: 444.2869. 1 H NMR (CDCl3): δ = 5.66 (d, 1H, J11, 8 = 2.3, H-11), 4.68, (dddd, 1H, J3a, 2a = J3a, 4a = 11.3, J3a, 2e = J3a, 4e = 4.5, H-3), 3.61 (s, 3H, CH3 -25), 2.45-2.15 (m: 3H, 2.35 (ddd, 1H, 2J = 15.8, J23, 22 = 9.8, J23, 22′ = 5.3, H-23), [2.33]-H-8, 2.23 (ddd, 1H, 2J=15.8, J23′, 22′ = 9.2, J23′, 22 = 6.8, H-23′)), 2.11-1.97 (m: 2H, [2.04]-H1, [2.04]-H-6), 1.95 (s, 3H, CH3-27), 1.92-1.17 (m: 18H, [1.91]-H-16, [1.82]-H-22, [1.82]-H-17, [1.75]-H-2, [1.75]-H-15, [1.71]-H-7, [1.68]-H-14, 1.64 (d, 1H, J5a, 4a = 13.0, H-5), [1.53]-H-4, [1.52]-H-2, [1.41]-H-16, [1.40]-H-20, [1.36]-H-6, [1.35]-H-22, [1.34]-H-15, [1.32]-H-4, [1.29]-H-1, [1.20]-H-7), 1.15 (s, 3H, CH3-19), 0.97 (d, 3H, J21, 20 = 6.6, CH3-21), 0.86 (s, 3H, CH3-18). 13C NMR (CDCl3): δ = 204.90 (s, C-12), 174.50 (s, C-24), 170.42 (s, C-26), 163.81 (s, C-9), 123.52

9

(d, C-11), 73.61 (d, C-3), 53.34 (d, C-14), 52.89 (s, C-13), 51.28 (q, C-25), 47.11 (d, C-17), 41.66 (d, C-5), 39.78 (s, C10), 37.66 (d, C-8), 35.12 (d, C-20), 34.88 (t, C-1), 33.85 (t, C-4), 31.31 (t, C-23), 30.47 (t, C-22), 29.58 (q, C-19), 27.21 (t, C-16), 26.36 (t, C-7), 27.49 (t, C-2), 26.02 (t, C-6), 24.04 (t, C-15), 21.18 (q, C-27), 19.30 (q, C-21), 10.55 (q, C-18). 2) SeO2 (0.75 g, 6.75 mmol, 3 equiv.) was added to a solution of compound 10 (1.0 g, 2.24 mmol) in glacial AcOH (50 mL). After heating under reflux for 48 h, the reaction mixture was treated as described above. Conversion was monitored by 1H NMR. General procedure for the oxidation of methyl 3α-acetoxy-12-oxo-5β-cholan-24-oate 10 with selenium (IV) oxide under microwave irradiation. 3) In a vial (10 mL), 2 mL of glacial AcOH was placed and the quantity of selenium (IV) oxide and compound 10 were calculated. Reactions were carried out in the microwave reactor under the established conditions (time, temperature). After termination if the reaction, the reaction mixture was cooled to room temperature, diluted with AcOEt and washed with saturated aqueous NaHCO3 until the pH reached 7–8, after which it was extracted with AcOEt. The combined organic phase was stirred with H2O2 (3 mL, 30%) until the colour of the organic phase became pale orange; then it was washed with brine and dried over anhydrous MgSO4. The solvent was removed on a rotary evaporator. The ratio of starting compound 10 to product 11 was detected by 1H NMR. The results of the reaction and the ratio of reactants are shown in Table 1. 4) SeO2 (2.30 g, 20.7 mmol, 3 equiv.) was added to a solution of compound 10 (3.08 g, 7.0 mmol) in glacial AcOH (15 mL). Reaction was carried out in the microwave reactor in a vial (30 mL) at 180°C for 30 minutes. Then reaction mixture was treated as described above. The crude product was purified by flash column chromatography (silica gel, chloroform) to yield compound 11 (2.34 g, 76%) as a pale yellow solid. 4. 1. 7. M et hyl 3 α - hy d rox y- 1 2 -ox o- 5 β - ch ol- 9 (11 )- en - 24- o at e (1 2) A solution of KOH (4 g, 71 mmol) in MeOH (30 mL) was added to a solution of compound 11 (3.0 g, 6.8 mmol) in MeOH (20 mL) at room temperature. The reaction mixture was stirred for 1.5 h (reaction course was monitored by TLC CHCl3:AcOEt = 20:3). The solvent was removed on a rotary evaporator; the solid residue was dissolved in AcOEt (30 mL), and HCl (5%) was added until the pH reached 3–4. After extraction with AcOEt, the combined organic phase was dried over anhydrous MgSO4. The solvent was removed to give compound 12 (2.6 g, 95%) as an amorphous solid. This material was used for next reaction without further purification. An analytically pure sample was obtained by recrystallisation from CH3OH. M.p. 118ºC [decomposition; lit. 115–116ºC (petroleum ether)].27 HRMS: m/z calcd for C25 H38O4: 402.2765; found: 402.2757. 1H NMR (CDCl3): δ = 5.67 (d, 1H, J11, 8 = 2.3, H-11), 3.63 (s, 3H, CH3-25), 3.61 (dddd, 1H, J3a, 2a=J3a, 4a=11.0, J3a, 2e=J3a, 4e=4.3, H-3), 2.40-2.30 (m: 2H, 2.37 (ddd, 1H, 2J=15.8, J23, 22=9.8, J23, 22′=5.3, H23), 2.33 (dddd, 1H, J8, 7a=12.4, J8, 14=9.7, J8, 7e=5.9, J8, 11=2.3, H-8)), 2.24 (ddd, 1H, 2J=15.8, J23′, 22′=9.2, J23′, 22=6.8, H23′), 2.08-1.98 (m: 2H, 2.04 (dddd, 1H, 2J=J6a, 7a=14.2, J6a, 7e=J6a, 5=4.4, H-6a(β)), 2.03 (dm, 1H, 2 J=14.2, H-1e(β)), 1.92 (m, H-16), 1.86-1.64 (m: 6H, 1.83 (ddd, 1H, J17, 20=J17, 16a=J17, 16e=9.7, H-17), [1.82]-H-22, [1.78]-H-2, [1.75]–H-15, [1.71]-H-7, 1.69 (ddd, 1H, J14, 15a=11.7, J14, 8=9.7, J14, 15e=7.3, H-14)), 1.59 (dm, 1H, J5, 4a =12.3), 1.52 (dm, 1H, 2J=12.3, H-4e(β)), 1.48-1.19 (m: 9H, [1.43]-H-2, [1.41]-H-16, [1.40]-H-20, [1.37]-H-6, [1.34]-H-15, [1.33]-H-22, [1.26]-H-1, [1.25]-H-4, [1.24]-H-7), 1.15 (s, 3H, CH3-19), 0.98 (d, 3H, J21, 20 = 6.6, CH3-21), 0.87 (s, 3H, CH3-18). 13C NMR (CDCl3): δ = 205.04 (s, C-12), 174.61 (s, C-24), 164.29 (s, C-9), 123.49 (d, C-11), 71.37 (d, C-3), 53.34 (d, C-14), 52.89 (s, C-13), 51.33 (q, C-25), 47.12 (d, C-17), 41.87 (d, C-5), 39.82 (s, C-10), 37.94 (t, C-4), 37.75 (d, C-8), 35.16 (d, C-20), 35.26 (t, C-1), 31.64 (t, C-2), 31.36 (t, C-23), 30.50 (t, C-22), 29.67 (q, C-19), 27.25 (t, C-16), 26.46 (t, C-7), 26.22 (t, C-6), 24.08 (t, C-15), 19.34 (q, C-21), 10.62 (q, C-18). 4. 1. 8. M et hyl 3, 12- di ox o- 5 β - c h ol - 9 (11 )- en - 24- o at e (1 3) Compound 12 (2.6 g, 6.4 mmol) was oxidised by Jones reagent (3.2 mL) in acetone (200 mL), in a similar way as described for the synthesis of 10, to give 13 (2.5 g, 96%). An analytically pure sample was obtained by re-crystallisation from CH3OH. M.p. 126.9ºC [decomposition; lit. 129.5–130.5ºC (Et 2O/petr. ether)].30 HRMS: m/z calcd for C25H36O4: 400.2608; found: 400.2604. 1H NMR (CDCl3): δ = 5.78 (d, 1H, J11, 8=2.3, H-11), 3.61 (s, 3H, CH3-25), 2.48 (ddd, 1H, 2 J=J2a, 1a =14.5, J2a, 1e=5.8, H-2a), 2.40 (dddd, 1H, J8, 7a=12.7, J8, 7=9.7, J8e 7e=4.8, J8, 11=2.3, H-8), 2.34 (ddd, 1H, 2J=15.8, J23, 22=9.8, J23, 22′=5.3, H-23), 2.27 (ddd, 1H, 2J=14.5, J1e, 2a=5.8, J1e, 2e=2.2, H-1e), 2.24 (dd, 1H, 2J=J4a, 5=15.7, H-4a), 2.23 (ddd, 1H, 2J=15.8, J23′, 22′=9.2, J23′, 22 = 6.8, H-23′), 2.17 (dddd, 1H, 2J=14.5, J2e, 1a=4.5, J2e, 1e=J2e, 4e=2.2, H-2e), 2.07 (dddd, 1H, 2 J=J6a, 7a=14.1, J6a, 7e =J6a, 5=4.2, H-6a), 2.1-1.88 (m: 3H, [1.97]-H-5; [1.95]-H-4; [1.92]-H-16), 1.88-1.67 (m: 5H, [1.83]-H-17; [1.81]-H-22; [1.79]-H-7, [1.77]-H-15; [1.72]-H-14), 1.61 (ddd, 1H, 2 J=J1a, 2a=14.5, J1a, 2e=4.5, H-1a), 1.5-1.15 (m: 6H, [1.42]-H-16; [1.41]-H-20; [1.36]-H-6; [1.33]-H-15; [1.31]-H-22, [1.25]-H-7), 1.23 (s, 3H, CH3-19), 0.96 (d, 3H, J21, 20 =6.6, CH3-21), 0.90 (s, 3H, CH3-18). 13C NMR (CDCl3): δ = 211.28 (s, C-3), 204.50 (s, C-12), 174.38 (s, C-24), 162.46 (s, C-9), 123.20 (d, C-11), 53.35 (d, C-14), 53.08 (s, C-13), 51.23 (q, C-25), 47.13 (d, C-17), 44.20 (d, C-5), 43.49 (t, C-4), 39.87 (s, C-10), 37.77 (t, C-2), 37.47 (d, C-8), 36.81 (t, C-1), 35.08 (d, C-20), 31.27 (t, C-23), 30.43 (t, C-22), 28.99 (q, C-19), 27.16 (t, C-16), 25.93 (t, C-7), 25.77 (t, C-6), 24.00 (t, C-15), 19.26 (q, C-21), 10.46 (q, C-18). 4. 1. 9. M et hyl 3, 12- di ox o- 5 β - c h ol a n- 24- o at e (8 ) Methyl deoxycholate 7 (2.0 g, 5.0 mmol) in acetone (130 mL) was oxidised by Jones reagent (6 mL), in a similar way as described in a synthesis of 10, to give 8 (1.9 g, 94%) as a white solid after purification by flash column chromatography [silica gel, CH2Cl2 with gradient AcOEt (0–50%)]. An analytically pure sample was obtained by re-crystallisation from methanol. M.p. 126.8–129.2ºC [lit. 133.7–135.9ºC (CH2 Cl2/hexane)].41 HRMS: m/z calcd for C25H38O4: 402.2765; found: 402.2760. 1H NMR (CDCl3): δ = 3.62 (s, 3H, CH3 -25), 2.57 (dd, 1H, 2J=J11a, 9a=12.5, H-11a), 2.54 (dd, 1H, 2 J=15.0, J4a, 2 2 5a=13.3, H-4a), 2.35 (ddd, 1H, J=15.8, J23, 22 =9.8, J23, 22′=5.3, H-23), 2.30 (ddd, 1H, J=J2a, 1a=14.7, J2a, 1e=5.2, H-2a), 2.22

10

(ddd, 1H, 2J=15.8, J23′, 22′=9.2, J23′, 22 =6.8, H-23′), 2.13 (dddd, 1H, 2J=14.7, J2e, 1a=4.3, J2e, 1e =3.2, J2e, 4e =2.3, H-2e), 2.06 (dd, 1H, 2 J=12.5, J11e, 9a=4.2, H-11e), 2.30-1.96 (m: 2H, [2.01]–H-17; 1.99 (ddd, 1H, 2J=15.0, J4e, 5a=4.5, J4e, 2e =2.3, H-4e)), 1.96-1.75 (m: 7H, [1.91]–H-16; [1.89]–H-6′; [1.88]–H-9; [1.87]–H-5; 1.86 (ddd, 1H, 2J=14.5, J1e, 2a=5.2, J1e, 2e =3.2, H-1e); [1.86]–H-8; 1.80 (dddd, 1H, 2J=13.5, J22, 23=9.8, J22, 23′=6.8, J22, 20 =2.7, H-22)), 1.72 (m, 1H, H-15), 1.57 (dm, 1H, 2J=13.6, H-7e), 1.45-1.18 (m: 7H, 1.41 (ddd, 1H, J1a, 2a = 14.7, 2J=14.5, J1a, 2e=4.3, H-1a); [1.39]–H-14; [1.35]–H-22′; [1.30]–H-15′; [1.32]–H-16′; [1.29]–H-6; [1.26]–H-20), 1.10 (m, 1H, H-7a), 1.07 (s, 3H, CH3-19), 1.02 (s, 3H, CH3-18), 0.82 (d, 3H, J21, 13 20=6.6, CH3 -21). C NMR (CDCl3): δ = 213.84 (s, C-12), 211.78 (s, C-3), 174.41 (s, C-24), 51.29 (q, C-25), 58.38 (d, C14), 57.42 (s, C-13), 46.39 (d, C-17), 44.13 (d, C-9), 43.56 (d, C-5), 41.97 (t, C-4), 38.21 (t, C-11), 36.75 (t, C-2), 36.65 (t, C-1), 35.45 (s, C-10), 35.45 (d, C-20), 35.30 (d, C-8), 31.14 (t, C-23), 30.35 (t, C-22), 27.32 (t, C-16), 26.45 (t, C-6), 25.32 (t, C-7), 24.15 (t, C-15), 21.98 (q, C-19), 18.44 (q, C-21), 11.57 (q, C-18). 4. 1. 1 0. Met h yl 3, 1 2-di o xo- 5 β - c hol - 1(2 ), 9(1 1 )-di en- 2 4- o at e (2 ) PhSeCl (0.07 g, 0.30 mmol) was added to a stirring solution of 13 (0.11 g, 0.28 mmol) in AcOEt (3 mL), at room temperature, until the colour of the solution changed from red to pale yellow. Then, the reaction mixture was diluted with AcOEt (10 mL), washed with saturated aqueous NaHCO3 and the aqueous layer was removed. A mixture THF–H2O2 (1 mL, 10: 1, v/v) was added to the organic phase and the resulting mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure (v ~ 10 mL), diluted with AcOEt, washed with brine and dried over anhydrous MgSO4. The solvent was removed on a rotary evaporator. Compound 2 (0.08 g, 75 %) was obtained by flash column chromatography [silica gel, CH2Cl2 with gradient AcOEt (0–30%)]. An analytically pure sample was 23 obtained by re-crystallisation from methanol. M.p. 139.1–142.5ºC. [α]D +154º (c 0.11, CHCl3). HRMS: m/z calcd for C25 H34O4: 398.2452; found: 398.2447. 1H NMR (CDCl3): δ = 6.77 (d, 1H, J1, 2=10.1, H-1), 5.99 (dd, 1H, J2, 1=10.1, J2, 2 4e=1.0, H-2), 5.60 (d, 1H, J11, 8 =2.3, H-11), 3.63 (s, CH3-25), 2.42-2.32 (m: 3H, [2.38]–H-8; 2.37 (dd, J=16.1, J4a, 5=14.6, 2 H-4a); 2.36 (ddd, 1H, J=15.8, J23, 22=9.8, J23, 22′=5.3, H-23)), 2.26 (dddd, 1H, J5, 4a = 14.6, J5, 6a=4.4, J5, 4e=3.0, J5, 6e=2.2, H5), 2.24 (ddd, 1H, 2 J=15.8, J23′, 22′=9.2, J23′, 22=6.8, H-23’), 2.11 (dddd, 2J=J6a, 7a =14.1, J6a, 7e=J6a, 5=4.4, H-6a), 2.09 (ddd, 1H, 2 J=16.1, J4e, 5 =3.0, J4e, 2=1.0, H-4e), 1.95 (m, 1H, H-16), 1.92-1.78 (m: 4H, [1.88]–H-7e; [1.84]–H-17; [1.82]–H-15; [1.81]–H-22), 1.66 (ddd, 1H, J14, 15a=11.6, J14, 8 =9.5, J14, 15e=7.5, H-14), 1.51-1.44 (m: 2H, 1.48 (dddd, 2J=14.1, J6e, 7a =4.4, J6e, 7e=J6e, 5=2.2, H-6e); [1.45]–H-16), 1.44-1.28 (m: 3H, [1.40]–H-15; [1.38]–H-20; [1.33]–H-22), 1.42 (s, 3H, CH3-19), 1.24 (m, 1H, H-7a), 0.96 (d, 1H, J21, 20=6.6, H-21), 0.93 (s, 3H, CH3-18). 13 C NMR (CDCl3): δ = 204.09 (s, C-12), 199.62 (s, C-3), 163.12 (s, C-9), 156.21 (d, C-1), 128.24 (d, C-2), 124.79 (d, C-11), 174.50 (s, C-24), 53.06 (s, C-13), 52.78 (d, C14), 51.33 (q, C-25), 46.93 (d, C-17), 42.76 (s, C-10), 41.51 (d, C-5), 40.10 (t, C-4), 37.31 (d, C-8), 35.20 (d, C-20), 31.28 (t, C-23), 30.47 (t, C-22), 27.46 (t, C-16), 27.41 (t, C-7), 26.90 (q, C-19), 25.55 (t, C-6), 24.03 (t, C-15), 19.19 (q, C-21), 10.74 (q, C-18). 4. 1. 1 1. Met h yl 3, 1 2-di o xo- 5 β - c hol - 4(5 ), 9(1 1 )-di en- 2 4- o at e (3 ) A solution of Br2 (0.24 mL, 4.68 mmol) in AcOH (4 mL) was added drop-wise to a stirring solution of compound 13 (1.7 g, 4.25 mmol) in AcOH (25 mL). The reaction mixture was stirred at room temperature for 1 h and then it was diluted with H2O and extracted with AcOEt. The combined organic layers were washed with saturated aqueous NaHCO3, brine and dried over anhydrous MgSO4. The solvent was removed to give 2.0 g of brown amorphous solid. This material was used for the next reaction without further purification. HRMS: m/z calcd for C25H35 O4Br: 478.1713; found: 478.1715. Li2CO3 (1 g, 13.5 mmol) and LiBr (0.5 g, 5.7 mmol) were added to a solution of bromoderivative (2.0g, 4.2 mmol) from the previous step in DMF (40 mL). The resulting mixture was refluxed for 2.5 h and then concentrated on a rotary evaporator (v ~ 10 mL). The concentrated solution was diluted with AcOEt, washed with brine and dried over anhydrous MgSO4. The solvent was removed to give a brown amorphous solid. According to 1H NMR results, the reaction mixture contained compounds 2 and 3 in a ratio of 1:4. Compound 3 (1.05 g, 60%) was obtained by flash column chromatography [silica gel, CH2Cl2 with gradient AcOEt (0–30%)] as a white solid. An analytically pure sample was obtained by re23 crystallisation from methanol. M.p. 118.5–-119.5ºC. [α ]D +116º (c 0.12, CHCl3). HRMS: m/z calcd for C25H34O4 : 398.2452; found: 398.2449. 1H NMR (CDCl3): δ = 5.76 (d, 1H, J4, 2a=1.9, H-4), 5.73 (d, 1H, J11, 8 =2.1, H-11), 3.62 (s, 3H, CH3 -25); 2.66-4.43 (m: 3H, 2.60 (dddd, 1H, 2 J=14.8, J6a, 7a=14.2, J6a, 7e=4.8, J6a, 4=1.9, H-6a); 2.53 (dddd, 1H, J8, 7a=12.7, J8, 14=9.7, J7e, 8=4.8, J8, 11=2.3, H-8); [2.48]–H-2), 2.38 (ddd, 1H, 2J=14.8, J6e, 7a=3.6, J6e, 7e=2.9, H-6e), 2.36 (ddd, 1H, 2 J=15.8, J23, 22=9.8, J23, 22’=5.3, H-23), 2.24 (ddd, 1H, 2J=15.8, J23’, 22’=9.2, J23’, 22=6.8, H-23’), 2.15-1.99 (m: 2H, [2.11]–H1; 2.06 (dddd, 1H, 2J=12.7, J7e, 6a=J7e, 8=4.8, J7e, 6e=2.9, H-7e)), 1.96 (m, 1H, H-16), 1.88-1.73 (m: 3H, [1.84]–H-17; [1.83]– H-15; [1.83]–H-22), 1.68 (ddd, 1H, J14, 15a=11.5, J14, 8=9.7, J14, 15e=7.5, H-14), 1.53-1.25 (m: 4H, [1.46]–H-16; [1.46]–H16; [1.42]–H-20; 1.31 (ddd, 2J=13.4, J22’, 23’=9.2, J22’, 23=5.3, H-22’)), 1.43 (s, 3H, CH3-19), 1.16 (dddd, 1H, J6a, 7a=14.2, 2 J=J7a, 8=12.7, J7a, 6e=3.6, H-7a), 0.97 (d, 3H, J21, 20=6.6, CH3-21), 0.95 (s, 3H, CH3-18). 13C NMR (CDCl3): δ = 204.43 (s, C-12), 197.92 (s, C-3), 174.43 (s, C-24), 166.48 (d, C-5), 165.37 (s, C-9), 124.98 (d, C-4), 122.01 (d, C-11), 52.55 (d, C14), 52.76 (s, C-13), 51.29 (q, C-25), 47.00 (d, C-17), 41.34 (s, C-10), 38.62 (d, C-8), 35.18 (d, C-20), 33.80 (t, C-7), 33.80 (t, C-2), 33.19 (t, C-1), 32.10 (t, C-6), 31.27 (t, C-23), 30.47 (t, C-22), 27.38 (t, C-16), 26.25 (q, C-19), 24.00 (t, C15), 19.22 (q, C-21), 10.52 (q, C-18). 4. 1. 1 2. Met h yl 3, 1 2-di o xo- 5 β - c hol - 1(2 )- en - 24- o at e (5 ) PhSeCl (0.29 g, 1.5 mmol) and 8 (0.5 g, 1.24 mmol) were reacted in AcOEt (10 mL) for 24 h, in a similar way as described in a synthesis of 2. According to 1H NMR, the conversion of 8 was 50%. 4. 1. 1 3. M et h yl 3, 12- di o xo- 5 β -c hol - 1(2 )- en- 2 4- o at e (5 ) and m et hy l 3, 12 - di ox o- 5 β - c h ol- 4 (5)- en24 - oat e (6) A solution of Br2 (0.14 mL, 2.74 mmol) in AcOH (4 mL) was added drop-wise to a stirring solution of compound 8 (1.0 g, 2.49 mmol) in AcOH (20 mL). The reaction mixture was stirred at room temperature for 1 h and then it was diluted

11

with H2O and extracted with AcOEt. The combined organic layers were washed with saturated aqueous NaHCO3, brine and dried over anhydrous MgSO4. The solvent was removed to give 1.02 g of a brown amorphous solid. This material was used for the next reaction without further purification. HRMS: m/z calcd for C25H37 O4Br: 480.1870; found: 480.1875. Li2CO3 (0.63 g, 8.5 mmol) and LiBr (0.31 g, 3.6 mol) were added to a solution of bromoderivative from the previous step in DMF (30 mL). The resulting mixture was refluxed for 2.5 h and then concentrated on a rotary evaporator (v ~ 10 mL) and diluted with AcOEt. The organic phase was washed with brine and dried over anhydrous MgSO4. The solvent was removed to give a brown amorphous solid. According to 1 H NMR results, the reaction mixture contained compounds 5 and 6 in a ratio of 1:4. Compound 6 (0.54 g, 63 %) and compound 5 (0.14g, 16%) were obtained by flash column chromatography [silica gel, n-hexane with gradient AcOEt (10–25%)] as white solids. Analytically pure samples were 32 obtained by re-crystallisation from methanol. M.p. (compound 5) 128.2ºC (decomposition). [α ]D +204º (c 0.10, CHCl3). HRMS: m/z calcd for C25H36O4: 400.2608; found: 400.2602. 1H NMR (CDCl3): δ = 6.62 (d, 1h, J1, 2=10.2, H-1), 5.89,( d, 1H, J2, 1=10.2, H-2), 3.63 (s, 3H, CH3 -25), 2.72 (dd, 1H, J11a, 9=12.8, 2J=12.2, H-11a(β)), 2.63 (m, 1H, H-4), 2.36 (ddd, 1H, 2 J=15.8, J23, 22=9.8, J23, 22′=5.3, H-23), 2.22 (ddd, 1H, 2J=15.8, J23′, 22′=9.2, J23′, 22=6.8, H-23’), 2.15-1.84 (m: 6H, [2.11]-H-5, [2.10]-H-4, 2.05 (dd, 1H, 2J=12.2, J11e, 9=4.2, H-11e(α)), [1.99]-H-17, [1.92]-H-16, [1.90]-H-6a(β)), 1.84-1.65 (m: 4H, [1.80]-H-22, [1.78]-H-8, [1.76]-H-9, [1.73]-H-15), 1.60 (dddd, 1H, 2J=13.4, J=J=J