Synthesis and antitumor activity of alkylated bile acids and oxazolines

Accepted Manuscript

Srđan Bjedov, Dimitar Jakimov, Mihalj Poša, Olivera R. Klisurić, Marija Sakač

PII:

S0040-4020(17)31095-5

10.1016/j.tet.2017.10.058

TET 29061

DOI:

Reference:

To appear in: Tetrahedron

Received Date: 13 July 2017

Revised Date: 16 October 2017

Accepted Date: 20 October 2017

Please cite this article as: Bjedov Srđ, Jakimov D, Poša M, Klisurić OR, Sakač M, Synthesis

and antitumor activity of alkylated bile acids and oxazolines, Tetrahedron (2017), doi: 10.1016/

j.tet.2017.10.058.

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ACCEPTED MANUSCRIPT

Synthesis and antitumor activity of alkylated bile acids and oxazolines

Srđan Bjedov ^a,* Dimitar Jakimov ^b, Mihalj Poša ^c, Olivera R. Klisurić ^d, Marija Sakač ^a

^aDepartment of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University

of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia

^bOncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Put Dr Goldmana 4, 21204

Sremska Kamenica, Serbia

^cDepartment of Pharmacy, Faculty of Medicine, University of Novi Sad, Hajduk Veljka 3, 21000 Novi Sad,

Serbia

^dDepartment of Physics, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 4, 21000

Novi Sad, Serbia

Corresponding author: Srđan Bjedov

e-mail: srdjan.bjedov@dh.uns.ac.rs

Key words:

•

Bile acid

Stereoselective alkylation

Grignard reaction

Antitumor activity

Oxazoline

Abstract

Bile acid derivatives with a free carboxylic group or an oxazoline ring in the side chain and with different

lengths of alkyl chains on steroid skeleton were synthetized and their antitumor activity against six

human cancer cell lines was investigated. Methyl, ethyl, butyl or octyl chains were introduced

stereoselectively by Grignard reaction at C-7 of acid and oxazoline, and at C-12 of oxazoline. Carbonyl

group at C-12 of acid compound gave addition product only with methyl Grignard reagent, and complex

mixture of products with other used reagents. Due to enolization, the C-3 carbonyl group did not

participate in the Grignard reaction. Steric reasons are a main cause of this chemical behavior.

Compounds with a butyl chain at the C-7 position showed very good antitumor activity with IC₅₀<5 µM.

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1. Introduction

Perception of bile acids (BAs) as merely lipid emulsifying and solubilizing molecules was fundamentally

changed after discovery of their hormonal roles. BAs are natural agonists for a number of nuclear and

transmembrane receptors involved in a variety of metabolic functions, ranging from BA biosynthesis to

energy homeostasis.¹These findings considerably broadened therapeutic potential of BA and their

derivatives.

Relation of hydrophobic/hydrophilic area of BA is important for their biological properties. Hydrophobic

BAs, such as secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA) which lack one hydroxyl

group relative to corresponding primary BAs, can induce apoptosis and have been implicated as tumor

promoters.^{2, 3}BAs with oxo groups have lower membranolytic activity then their hydroxyl analogues.^{4, 5}

Also, obeticholic acid, semisynthetic BA, with ethyl group connected to steroid C-6 is a strong farnesoid

X receptor (FXR) agonist, and in the final phases of clinical studies for treatment of some liver diseases.^6,

7

In our previous study we have showed that introduction of an ethylidene group at the C-7 position on

the steroid skeleton greatly influenced antitumor activity of these compounds.⁸Perception of BA steroid

skeleton as a pharmacophore, and findings that introduction of hydrophobic groups on BA steroid

skeleton can significantly influence biological activity prompted us to investigate the most common way

to change hydrophobicity-introduction of alkyl groups. This work should give insights into specific

chemical behavior of bile acid steroid skeleton in alkylation reactions with Grignard reagents and in vitro

antitumor activity of alkyl derivatives.

2. Results and discussion

2.1. Chemistry

In order to investigate the possibilities of bile acid alkylation we decided to employ Grignard reactions as

they are simple, cheap and fast. Also we decided to try Grignard reactions on bile acid derivatives with

free carboxylic groups, as well as with protected carboxylic groups in form of oxazoline. For the starting

compound we used cheap and commercially available cholic acid (CA). By known procedures we

oxidized CA in corresponding 7-oxo (1), 12-oxo (2) and 3-oxo (3) derivatives.^9-117,12-Diformyloxy-3-oxo

compound 4 was obtained by known method.¹²

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Scheme 1. Synthesis of oxazoline derivatives

Protection of carboxylic group in the form of oxazoline can resolve low solubility of bile salt in ether

solvents and potential problems of carboxylic carbon participation in Grignard reaction. The synthesis of

bile oxazoline 5 is described in our previous publication.⁸By similar manner we have synthesized 12-oxo

6 and 3-oxo 7 oxazoline in good yields (Scheme 1.). The sequence included formylation (8), condensation

of carboxylic group with 2-amino-2-methyl-1-propanol (9, 10) using EEDQ as coupling agent followed by

cyclization to oxazoline (11, 12) ring by treatment with thionyl chloride and deformylation (6 and 7).

Bile acid derivatives, 7-oxo bile acid 1 and 7-oxo bile oxazoline 5, were subjected to Grignard reaction

with methyl, ethyl, butyl and octylmagnesium halides. Reaction condition, products and yields of

reactions are shown in Scheme 2.

Eqvivalents

of R²MgX

34

Reaction

time [min]

Yield

[%]

Substrate

Reagent

Product

MeMgI

EtMgBr

BuMgCl

OctMgBr

30

90

30

13

14

15

16

R²=Me

R²=Et

R²=Bu

R²=Oct

83

88

77

83

24

25

30

1

135

19

MeMgI

EtMgBr

BuMgCl

OctMgBr

20

15

60

17

18

19

20

R²=Me

63

74

72

56

45

23

28

R²=Et

R²=Bu

R²=Oct

5

Scheme 2. 7-Oxo derivatives in Grignard reaction

Both oxo derivatives, 1 and 5, gave corresponding alkyl adducts in good yields. However, adducts with

free carboxyl group are formed in better yields then oxazolines with similar reaction conditions.

Reaction times were slightly shorter for 5 then for 1 probably due to better solubility of oxazolines in

THF. Reactions with Grignard reagent were carried out in dry THF on temperature of –20 °C. When

higher temperature was applied oxazoline ring was partially hydrolyzed into amides 21, 22 and 23 (Fig.

1.), though alkylation of C-7 carbonyl group still occurs.

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NH

O

CH₂OH

OH

H

R

OH

HO

H

21 R=Me

22 R=Bu

23 R=Oct

Fig. 1. Alkylated amides formed by partial oxazoline hydrolysis

Yields of alkylated amides at –20 °C were less than 8%, while at reflux yields of byproduct were

increased up to 26%. Alkylation reactions were stereoselective, and only one epimer was detected.

Determination of configuration in steroid skeleton is always challenging, especially in the case when

newly introduced substituent is alkyl group. NOE NMR experiments usually give ambiguous results due

to considerable overlap of signals in NMR spectra, also bile acid derivatives with long hydrocarbon

chains are hard to crystallize. Fortunately, we succeeded to crystallize carboxylic derivate with methyl

group 13, and determined configuration at C-7 by X-ray crystallography experiment. Structure of 13

shown in Fig 2, shows that methyl group at C-7 is β orientated. This configuration was expected

considering considerable steric hindrance of α side of steroid by A ring, and β methyl group has

thermodynamically favorable equatorial orientation.

If a nucleophile approaches from the α side of the steroidal skeleton the maximal overlap of HOMO

orbitals of nucleophile and LUMO orbitals of carbonyl group would result in 105° to 107°angle of attack

(Bürgi-Dunitz angle α_BD, Nu-C(carbonyl)-O(carbonyl)). This would make nucleophilic carbon of Grignard

reagent in syn-axial orientation with C-4, C-9 and C-12 hydrogens. That means that there is no variance

in Flippin-Lodge angle (α_FL) in α-azimuth plane, and that nucleophile attack from α side is excluded.

While in attack from β side at α_BD≈ 107° angle Flippin-Lodge angle (β-azimuth plane) shows maximal

variance, i. e. attack is not sterically inhibited (Appendix A – Supplementary data).

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Fig. 2. ORTEP drawing of the molecular structure of compound 13 with labeled non-H atoms.

Displacement ellipsoids are shown at 30% probability, and H atoms are drawn as spheres of arbitrary

radii.

Example of 13 gave enough evidence that is safe to conclude, with considerable degree of certainty, that

other 7-alkyl derivatives have also β-orientated alkyl groups.

Somewhat different results were obtained in the reaction of 12-oxo BA 2 or 12-oxo oxazoline 6 with

Grignard reagents. Reaction condition, products and yields of these reactions are shown in Scheme 3.

Eqvivalents

of R²MgX

Reaction

conditions

90 min, -20

°C

5 h, 0 °C or

RT

Yield

[%]

Substrate

Reagent

Product

MeMgI

EtMgBr

BuMgCl

20

24

CA

R²=Me

R²=H

27

/

2

5 h, 0 °C or

RT

R²=H

10

MeMgI

EtMgBr

21

22

30 min, 0 °C

2 h -20 °C →

3 h 0 °C

2 h -20 °C →

3 h 0 °C

25

26

27

28

27

29

R²=Me

R²=Et

R²=H

53

48

17

49

19

67

R²=Bu

R²=H

BuMgCl

51

52

6

OctMgBr

3 h 0 °C

R²=Oct

Scheme 3. 12-Oxo derivatives in Grignard reaction

Alkylation of 12-oxo BA was only successful with MeMgI, and 24 was isolated in moderate yield.

Reactions of 2 with EtMgBr and BuMgCl were carried out at three temperatures: at –20 °C after 2 h the

reaction did not occur; at 0 °C or room temperature (RT) after 5 h complex mixture of products was

obtained with CA as main product (10% with BuMgCl). In reaction with EtMgBr reaction mixture was not

purified, ad CA as main product was detected by TLC check.

Oxazoline 6 with MeMgI at 0 °C after 30 minutes gave only adduct 25 but with EtMgBr and BuMgCl, at –

20 °C and then at 0 °C, beside alkylated products 26 i.e 28, gave also reduced product 27. Reduced

product 27, was created as a result of the reduction of the keto group with Grignard reagent, and it is

related to steric hindrance of C-12 carbonyl group.¹²Reaction of 6 with OctMgBr was carried out at 0 °C

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for 3 h, and only octyl product 29 was isolated. These findings show that higher reaction temperatures

(0 °C) disfavors reduction reaction, and only addition of alkyl group occurs.

Configuration at C-12 of 24 was determined by ROESY NMR experiment, and a strong cross-peak

between methyl groups on C-12 and C-13 indicate β orientation of C-12 methyl group. Same

stereochemistries at C-12 presumably have all C-12 alkyl derivatives, as Flippin-Lodge angle in α-azimuth

plane tends to zero value (Appendix B - Supplementary data).

The difference in the chemical behavior between 7-oxo and 12-oxo BAs is attributed to a lower steric

accessibility of C-12 carbonyl carbon. In this way it could be also explained different reactivity of these

groups in Grignard reaction. The Grignard reagent reacted relatively quickly with the C-7 carbonyl group

of 1, to give the corresponding addition product, while in the case of 2 only methyl adducts were

isolated in poor yield, and ethyl- and butyl- reagents gave complex mixture of products. Approach of the

alkylmagnesium halide reagent to C-12 carbonyl of 2 is hampered by C-18 methyl group, therefore

reaction occurs only at the higher temperatures (RT), and at higher temperatures side reactions also

occur. Weakly reactive carboxylic carbon C-24 undergoes addition reactions on room temperature,

which with the combination of C-12 adducts and reduction of C-12 carbonyl group leads to complex

mixture of products. Smaller value of Flippin-Lodger angle in β-azimuth plane of C-12 carbonyl makes

successful collision with Grignard reagent molecules only with molecules that approach carbonyl group

in angle no greater than α_FL-2 angle. Therefore, increasing the temperature increases the number of

collisions and this number tends to number of collisions seen at C-7 carbonyl group (attack from β side)

(Appendix B).

In the next phase Grignard reactions of acid 3 with MeMgI (THF, 1 h RT; benzene, 3 h reflux), EtMgBr

(THF, 100 min 0 °C) and BuMgCl (THF, 4 h RT) were investigated. The reactions have not occurred, and

only a starting compound was isolated. This result was unexpected given that the carbonyl group at C-3

position is the least hindered in comparison to the C-7 and C-12 carbonyl groups. Explanation of this

result is the formation of a nucleophilic enolate anion caused by abstraction of α-protons by Grignard

reagent. This is confirmed by reaction of 3 with EtMgBr which is quenched by addition of D₂O, to afford

compound 3D (Scheme 4) (Appendix C - Supplementary data)

Scheme 4. (a) EtMgBr, THF, −20 °C, 2.5 h; (b) i) D₂O; ii) AcOH.

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Also, addition reaction did not occur in the case of oxazoline analogue 7 and EtMgBr or BuMgCl (–20 °C,

RT or reflux), and only partial hydrolysis of oxazoline ring to amide 30 occurred in a 8% yield.

2.2. Antitumor activity

Antiproliferative activities of synthesized derivatives (13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 26, 28, 29)

were evaluated in vitro against six tumor cell lines (estrogen receptor positive breast adenocarcinoma

MCF-7, estrogen receptor negative breast adenocarcinoma MDA-MB-231, human prostate cancer PC3,

cervix carcinoma HeLa, colon adenocarcinoma HT-29, adenocarcinomic human alveolar basal epithelial

cells A549) and one normal cell line (fetal fibroblasts MRC-5). Cytotoxicity was evaluated by using

standard MTT assay,¹⁴after exposure of cells to the tested compounds for 72 h. The commercial

nonselective antitumor agent doxorubicin (DOX) was used as a positive control in this assay.

Three compounds showed antiproliferative activity with the IC₅₀values lower than 10 µM. 7-Butyl-acid

derivative 15 showed activity against MCF-7 with IC₅₀value of 9.00 µM, and against MDA-MB-231 with

IC₅₀value of 3.07 µM. 7-Butyl-oxazoline 19 showed activity in low micromolar range (IC₅₀4.43 µM)

against HeLa cell line. Against MDA-MB-231 cell line compound 20 showed antiproliferative activity of

IC₅₀9.70 µM. It is important to highlight that none of the tested compounds were active against fetal

fibroblast normal cell line MRC-5, unlike Doxorubicine which is very toxic towards this cell line. Data of In

vitro antiproliferative activities of other synthetized compounds could be found in Table 1. in the

supplementary data.

3. Conclusion

We have explored alkylation of bile acid derivatives at the positions C-3, C-7 and C-12 by using Grignard

reaction. Alkylation was done on the derivatives with either free carboxylic group or oxazoline group in

side chain Alkylation of substrates with free the carboxylic group gave slightly better yields, but

considering the additional synthetic steps for the protection of the carboxylic group, direct alkylation is

definitively method of choice. Oxazoline group was beneficial for the synthesis of C-12 alkyl derivatives

since all alkyl products were obtained in the reactions with corresponding reagents, while only 12-

methyl derivative was successfully synthesized from the derivative with free carboxylic group (alkylation

with ethyl and butyl reagents gave a substantial amount of by-products due to side reactions such as

alkylation of C-24 and reduction of C-12 carbonyl group). Alkylation at C-12 is also stereoselective with

only β-alkyl products isolated. Substrates with the C-3 carbonyl group failed to react with Grignard

reagents due to formation of unreactive enol anion. Selected synthesized derivatives were evaluated for

their in vitro antiproliferative activity against tumor cell lines and one normal cell line. The most potent

activities were recorded in the cultures of MDA-MB-231 cells (7-butyl compounds 15 and 19 with IC₅₀

3.07 µM and 4.43 µM, respectively) and MCF-7 cells (7-butyl compound 15 with IC₅₀9.00 µM). None of

the synthesized compounds were active toward normal MRC-5 cells.

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4. Experimental section

¹H and ¹³C NMR spectra were recorded on a Bruker AC 250 apparatus (¹H 250 MHz and ¹³C 62.5 MHz)

1

Bruker Avance III HD 400 (400 MHz H, 101 MHz ¹³C) Avance III HD 500 (500 MHz H, 125 MHz ¹³C)

apparatus using tetramethylsilane as the internal standard. IR spectra were recorded on a Nexus 670 FT-

IR spectrometer. HRMS spectra (TOF) were recorded on a 6210 Time-of-Flight LC/MS Agilent

Technologies (ESI+) instrument. Melting points were determined using a Nagema Boeitus melting point

apparatus and the results are uncorrected. The colorimetric MTT assay was carried out following the

reported procedure.¹⁴Flash chromatography was performed on silica gel 60 (0.04–0.063 mm, Merck).

Grignard’s reagents (EtMgBr, 2 M solution in Et₂O; BuMgCl, 2 M solution in Et₂O; OctMgBr, 2 M solution

in Et₂O) were purchased from Sigma-Aldrich, solutions were dried with anhydrous Na₂SO₄.

Tetrahydrofuran (THF) was dried and distilled from sodium metal using benzophenone as an indicator.

4.1 3α,7α-Diformyloxy-12-oxo-5β-cholanoic acid (8)

Oxo BA 2 (0.487 g, 1.20 mmol) was dissolved in formic acid (3.5 mL) with addition of HClO₄(0.05 mL).

Solution was heated at 50–55 °C, after 1 h solution was cooled to room temperature and acetic

anhydride (1 mL) was added until appearance of large amount of bubbles (decomposition of acetic

formic anhydride formed after acetic anhydride reacted with all water from formic acid ), and then

poured in cold water (100 mL). White precipitate was filtered, washed with water to neutral pH and

dried on air. Compound 8 was obtained in yield of 0.530 g (96%). Rf (CHCl₃/acetone 7:1 with glacial

acetic acid 0.05 mL/10 mL of eluent) 0.80.

IR (cm^-1): 2941, 2874, 1717, 1182.

¹H NMR (400 MHz, DMSO-

4.60 (m, 1H, H-3), 5.00 (s, 1H, H-7), 8.17 and 8.22 (2s, 2×1H, 2CHO), 11.94 (s, 1H, COOH).

d6) δ = 0.76 (d, J=6 Hz, 3H, H-21), 0.99 (s, 3H, H-18) and 1.03 (s, 3H, H-19),

¹³C NMR (101 MHz, DMSO-

d6) δ = 11.6 (C-18), 19.0 (C-21), 22.1 (C-19), 23.5, 26.9, 27.3, 30.7, 31.4, 31.5,

34.5, 34.7, 35.4, 35.5, 37.1, 37.6, 37.8, 40.1, 46.5, 53.1, 56.9, 70.6 (C-7), 73.4 (C-3), 162.1 (2CHO), 175.2

(C-24), 213.5 (C-12).

HRMS (TOF) m/z: calculated for C₂₆H₃₈O₇[M+Na]⁺485.2510, found 485.2514.

4.2 2-(3α,7α-Diformyloxy-12-oxo-5β-cholan-24-amido)-2-methyl-1-propanol

(9)

To the suspension of compound 8 (0.530 g, 1.15 mmol) in EtOAc (13 mL) were added: triethylamine (0.3

mL), 2-amino-2-methyl-1-propanol (0.2 mL, 2.1 mmol), H₂O (1 mL) and EEDQ (0.42 g, 1.7 mmol).

Reaction mixture was refluxed for 4 h. After cooling to room temperature, the reaction mixture was

washed successively with 3 M HCl (1×4 mL), H₂O (1×4 mL), 10% NaHCO₃(2×10 mL) and then with water

to neutrality (3×5 mL). The organic layer was dried and evaporated in vacuum to give an oily residue

which was purified by flash column chromatography (CH₂Cl₂/acetone 8:1). Pure 9 was obtained as

colorless oil in yield of 0.366 g (60%). Rf (CHCl₃/acetone 7:1) 0.61.

IR (cm^-1): 3393, 2936, 2873, 2249, 1717, 1643, 1184.

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¹Н NMR (250 MHz, CDCl₃) δ = 0.86 (d,

J=6 Hz, 3H, H-21), 1.04 and 1.06 (2s, 2×3H, H-18 i H-19), 1.29 (s,

6H, 2xCH₃amide), 3.57 (s, 2H, CH₂amide), 4.72 (m, 1H, H-3), 5.15 (s, 1H, H-7), 5.56 (s, 1H, NH), 8.01 (s,

1H, CHO), 8.04 (s, 1H, CHO).

¹³C NМR (62.5 MHz, CDCl₃) δ = 11.5, 18.7, 22.1, 23.7, 24.8, 25.9, 26.5, 27.3, 31.1, 33.5, 34.1, 34.4, 34.7,

35.4, 35.5, 37.5, 37.7, 37.7, 45.0, 49.00, 52.74, 56.2, 57.0, 70.4, 70.9, 72.5, 160.3 (CHO), 160.6 (CHO),

174.5 (C-24), 213.6 (C-12).

HRMS (TOF) m/z: calculated for C₃₀H₄₇O₇[M+H]⁺534.34253, found 534.34165.

4.3 2-(3α,7α-Diformyloxy-12-oxo-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (11)

Freshly distilled SOCl₂(0.06 mL, 0.83 mmol), was added dropwise to the stirred ice-cooled solution of

compound 9 (0.092 g, 0.17 mmol) in THF (0.5 mL) and the reaction mixture was stirred further for 1 h. A

saturated NaHCO₃solution (5 mL) was added with vigorous stirring. Reaction mixture was extracted with

EtOAc (4×5 mL). The organic extracts were combined, dried and evaporated to dryness. Crude product

was purified by flash chromatography (EtOAc /CH₂Cl₂2:1). Pure 11 was obtained as oil in yield of 0.086 g

(94%). Rf (CHCl₃/acetone 7:1) 0.80.

IR (cm^-1): 2964, 2940, 2873, 1665, 1464, 1448

¹Н NMR (250 MHz, CDCl₃) δ = 0.74 (s, 3H, H-18), 0.86 (d,

J=6.0 Hz, 3H, H-21), 1.20 (s, 3H, H-19), 1.25 (s,

6H, 2xCH₃on C-4'), 3.88 (s, 2H, H-5’), 4.81 (m, 1H, H-3), 5.27 (s, 1H, H-12), 7.98 (s, 1H, CHO on C-3), 8.10

(s, 1H, CHO on C-12).

¹³C NМR (62.5 MHz, CDCl₃) δ = 12.4 (C-18), 17.6 (C-21), 22.6 (C-19), 24.0, 25.1, 25.9, 26.3, 27.5, 28.3

(CH3), 28.4 (CH3), 31.9, 32.9, 33.5, 34.6, 34.8, 36.5, 41.8, 44.9, 45.6, 46.3, 49.0, 66.8 (C-4’), 72.5 (C-3),

74.6 (C-12), 78.9 (C-5'), 160.3 and 160.4 (2xCHO), 166.2 (C-2’), 210.5 (C-7).

HRMS (TOF) m/z: calculated for C₃₀H₄₅NO₆516.33196, found 516.33113.

4.4 2-(3α,7α-Dihydroxy-12-oxo-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (6)

Solution of 11 (0.322 g, 0.63 mmol) and KOH (0.28 g, 4.99 mmol) in methanol (6 mL) was refluxed for 20

min., whereupon saturated solution of NH₄Cl in H₂O (40 mL) was added to neutrality. Solution was

extracted with EtOAc (4×8 mL). The organic extracts were combined, dried and evaporated to dryness

and purified by flash chromatography (CH₂Cl₂/acetone 1:1). Compound 6 was obtained as a small white

needles crystal (mp 193 °C after recrystallization from acetone) in yield of 0.273 g (90%). Rf

(EtOAc/acetone 1:1) 0.34.

IR (cm^-1): 3386, 2965, 2930, 2870, 1701, 1664.

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¹Н NMR (400 MHz, DMSO-

d6) δ = 0.78 (d, J=5.5 Hz, 3H, H-21), 0.94 (s, 3H, H-19), 0.96 (s, 3H, H-18), 1.15

(s, 6H, 2xCH₃on C-4'), 3.18 (m, 1H, H-3), 3.72 (s, 1H, H-7), 3.84 (s, 2H, H-5’), 4.33–4.36 (more signals, 2H,

OH on C-3 i C-7).

¹³C NМR (101 MHz, DMSO-

d6) δ = 11.6 (C-18), 19.1 (C-21), 22.5 (C-19), 23.7, 25.2, 27.6, 28.7 i 28.8

(2xCH₃on C-4'), 30.8, 31.9, 35.1, 35.5, 35.6, 35.9, 37.1, 37.9, 41.4, 46.6 (C-17), 53.9, 56.8, 66.5 (C-7), 67.2

(C-4'), 70.4 (C-3), 78.4 (C-5'), 165.1 (C-2'), 214.42 (C-12).

HRMS (TOF) m/z: calculated for C₂₈H₄₅NO₄[M+H]⁺460.34214, found 460.34070.

4.5 2-(7α,12α-Diformyloxy-3-oxo-5β-cholan-24-amido)-2-methyl-1-propanol

(10)

To the suspension of compound 4 (0.799 g, 1.73 mmol) in EtOAc (17 mL) were added: triethylamine (0.4

mL), 2-amino-2-methyl-1-propanol (0.3 mL, 3.1 mmol) and EEDQ (0.74 g, 2.99 mmol). Reaction mixture

was refluxed for 3 h and stirred at room temperature for 12 h. The reaction mixture was washed

successively with 3 M HCl (1×4 mL), H₂O (1×4 mL), 10% NaHCO₃(2×10 mL) and then with water to

neutrality (3×5 mL). The organic layer was dried and evaporated in vacuum to give an oily residue which

was purified by flash column chromatography (CH₂Cl₂/acetone 5:1). Pure 10 was obtained as colorless

oil in yield of 0.703 g (76%). Rf (CH₂Cl₂/acetone 5:1) 0.48.

IR (cm^-1): 3391, 2963, 2919, 1715, 1261, 1095, 1021.

¹H NMR (400 MHz, CDCl₃) δ = 0.81 (s, 3H, CH₃), 0.87 (d,

J=6.5 Hz, 3H, H-21), 1.05 (s, 3H, CH₃), 1.29 (s, 6H,

2xCH₃amide), 3.58 (s, 2H amide), 4.84 (bs, 1H, OH amide), 5.17 (d,

8.10 (s, 1H, CHO), 8.17 (s, 1H, CHO).

J=2.5 Hz, 1H, H-12), 5.51 (s, 1H, H-7),

¹³C NMR (101 MHz, CDCl3) δ = 12.2, 17.6, 21.6, 22.8, 24.8, 24.8, 25.9, 27.2, 29.5, 31.1, 31.4, 33.9, 34.4,

34.7, 36.2, 36.5, 37.8, 42.2, 42.9, 44.6, 45.1, 47.3, 56.2, 70.6, 70.9, 75.2, 160.3, 160.4, 174.3 (C-24), 211.6

(C-3).

HRMS (TOF) m/z: calculated for C₃₀H₄₇NO₇[M+Na]⁺556.32447, found 556.32315.

4.6 2-(7α,12α-Diformyloxy-3-oxo-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (12)

Freshly distilled SOCl₂(0.1 mL, 1.37 mmol), was added dropwise to the stirred ice-cooled solution of

compound 10 (0.168 g, 0.31 mmol) in THF (3 mL) and the reaction mixture was stirred further for 1 h. A

saturated NaHCO₃solution (10 mL) was added with vigorous stirring. Reaction mixture was extracted

with EtOAc (4×10 mL). The organic extracts were combined, dried and evaporated to dryness. Crude

product was purified by flash chromatography (CH₂Cl₂/acetone 8:1). Pure 12 was obtained as colorless

oil in yield of 0.112 g (69%). Rf (CH₂Cl₃/acetone 5:1) 0.53.

IR (cm^-1): 3416, 2961, 2927, 2872, 1722, 1415, 1667, 1176.

10

ACCEPTED MANUSCRIPT

¹H NMR (400 MHz, CDCl₃) δ = 0.79 (s, 3H, H-18), 0.88 (d,

J

=6 Hz, 3H, H-21), 1.04 (s, 3H, H-19), 1.25 (s, 6H,

2xCH₃on C-4'), 3.01 (t,

J

=15 Hz, H-4α) 1H, 3.89 (s, 2H, H-5'), 5.15 (d, J=2 Hz, 1H, H-7), 5.31 (s, 1H, H-12),

8.09 (s, 1H, CHO), 8.15 (s, 1H, CHO).

¹³C NMR (101 MHz, CDCl₃) δ = 12.1 (C-18), 17.6 (C-21), 21.6 (C-19), 22.8, 24.9, 25.9, 27.2, 28.4 (CH₃on C-

4'), 28.4 (CH3 on C-4'), 29.5, 31.1, 31.8, 34.4, 34.8, 36.2, 36.5, 37.7, 42.2, 42.9, 44.6 (C-4), 45.1, 47.0, 66.8

(C-4'), 70.6 (C-7), 75. (C-12), 78.9 (C-5'), 160.3 (CHO), 160.4 (CHO), 166.3 (C-2'), 211.6 (C-3).

HRMS (TOF) m/z: calculated for C₃₀H₄₅NO₆[M+H]⁺516.33196, found 516.33071.

4.7 2-(7α,12α-Dihydroxy-3-oxo-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (7)

Solution of 12 (0.602 g, 1.17 mmol) and KOH (0.60 g, 10.69 mmol) in methanol (15 mL) was refluxed for

20 min., whereupon saturated solution of NH₄Cl in H₂O (60 mL) was added to neutrality. Solution was

extracted with EtOAc (5×10 mL). The organic extracts were combined, dried and evaporated to dryness

and purified by flash chromatography (CH₂Cl₂/acetone 1:1). Compound 7 was obtained as a white

needle crystal (mp 148 °C after recrystallization from acetone) in yield of 0.416 g (77%). Rf

(CHCl₃/acetone 2:1) 0.54.

IR (cm^-1): 3428, 2941, 2868, 1708, 1663.

¹H NMR (400 MHz, CDCl₃) δ = 0.73 (s, 3H, CH₃), 0.99 (s, 3H, CH₃), 1.01 (d,

2xCH₃on C-4'), 3.90 (d, =3, 1H, H-7), 3.92 (s, 2H, H-5'), 4.00 (s, 1H, H-12).

J=3 Hz, 3H, H-21), 1.26 (2s, 6H,

J

¹³C NMR (101 MHz, CDCl₃) δ = 12.4, 17.7, 21.5, 23.3, 24.4, 26.8, 27.8, 28.2, 28.4, 28.4, 31.3, 34.2, 34.9,

35.6, 36.8, 36.8, 39.3, 41.9, 43.4, 45.5, 46.2, 46.6, 66.7 (C-4'), 67.9 (C-7), 73.1 (C-12), 79.0 (C-5'), 167.4 (C-

2'), 213.5 (C-3).

HRMS (TOF) m/z: calculated for C₂₈H₄₅NO₄[M+H]⁺460.34214, found 460.34053.

General procedure for Grignard reaction

Appropriate Grignard reagent was added dropwise over 3 min to a cooled (–20 °C) solution of

corresponding compound in dry THF and stirred at same temperature (unless stated otherwise) for the

reaction time. Reaction was quenched by addition of saturated aqueous solution of NH₄Cl, and then

glacial acetic acid. After extraction with EtOAc, organic phases were combined, dried and evaporated in

vacuum. The crude reaction mixture was purified by flash column chromatography.

4.8 3α,7α,12α-Trihydroxy-7β-methyl-5β-cholanic acid (13)

MeMgI in Et₂O (2 M, 7.4 mL, 14.8 mmol); 1 (0.173 g, 0.43 mmol) in THF (12.5 mL); reaction time 30 min;

NH₄Cl (10 mL); CH₃COOH (3 mL); EtOAc (5x10 mL); flash chromatography: CH₂Cl₂/MeOH 9:1 with

addition of glacial acetic acid 0.05 mL/10 mL of eluent. Compound 13 was obtained as transparent cube

crystals (mp. 224 °C, after crystallization from acetone, lit.¹⁵mp. 221–223 °C) in yield of 0.154 g (83%). Rf

(same solvent system as for flash chromatography) 0.28.

11

ACCEPTED MANUSCRIPT

¹H NMR (400 MHz, DMSO-

d

6) δ = 0.62 (s, 3H, H-18), 0.78 (s, 3H, H-19), 0.94 (d,

J

=6 Hz, 3H, H-21), 1.10 (s,

=3.5 Hz, 1H, OH

3H, CH₃on C-7), 3.22 (m, 1H, H-3), 3.56 (s, 1H, OH on C-7), 3.76 (s, 1H, H-12), 4.09 (d,

on C-12), 4.31 (d, =4 Hz, 1H, OH on C-3), 11.92 (bs, 1H, COOH).

J

¹³C NMR (101 MHz, DMSO-

d6) δ = 13.2 (C-18), 17.5 (C-21), 23.2 (C-19), 27.4, 28.0, 28.9, 29.5, 30.7, 31.2,

31.3, 33.7 (CH₃on C-7), 34.5, 35.4, 36.1, 38.6, 42.5, 42.7, 43.6, 45.0, 45.1, 47.5, 70.8 (C-3), 70.9 (C-12),

71.4 (C-7), 175.6 (C-24).

Structure Determination and Refinement for 13

A suitable single crystal of compound 13 was mounted on a glass ﬁber and crystallographic data were

collected using an Oxford Diffraction Gemini S diffractometer with a CCD area detector (λ_MoKα= 71073 Å,

monochromator: graphite) at 293 K. CrysAlisPro and CrysAlis RED software packages (Oxford Diffraction,

2009, Abingdon) were used for data collection and data integration. Analysis of the integrated data did

not reveal any decay. Final cell parameters were determined by a global reﬁnement of 1,942 reﬂections

(2.836 <

θ < 28.903°). Collected data were corrected for absorption effects by using a Multi-scan

absorption correction.¹⁵Structure solution and reﬁnement were carried out with the programs SHELXT

and SHELXL-2014/6 respectively.¹⁶ORTEP-3 for Windows¹⁷was employed for molecular graphics and

WinGx software was used to prepare material for publication.¹⁸

2

Full-matrix least-squares reﬁnement was carried out by minimizing (F_o

–

F_c²). All nonhydrogen atoms

were reﬁned anisotropically and reﬁnement was carried out without geometric or ADP restraints.

Hydrogen atoms attached to carbon atoms were placed in geometrically idealized positions and reﬁned

as riding on their parent atoms, with C–H = 0.98–1.00 Å with U_iso(H) = 1.2U_eq(C) for methylene and

methyne groups, and U_iso(H) = 1.5 U_eq(C) for methyl groups. At the final stage of the refinement, H

atoms attached to O atoms were identified on difference electron density maps and isotropically

refined. Crystal data and experimental details of the structure determination are listed in Table 1.

Table 1 Crystal data and refinement parameters for compound 13

Compound 13

Parameters

Chemical formula

M_r

C₂₅H₄₂O₅

422.58

Crystal system

Space group

Temperature

Unit cell dimensions

a, b, c (Å)

Orthorhombic

P2₁2₁2₁

293 K

a = 9.0862 (6) Å

b = 15.8629 (12) Å

c = 16.8304 (11) Å

2425.8 (3) Å³

4

Volume

Z

Absorption coefficient

Crystal size

0.08 mm^-1

0.57 × 0.31 × 0.29 mm³

12

ACCEPTED MANUSCRIPT

Absorption correction

Multi-scan

CrysAlis PRO 1.171.38.41 (Rigaku Oxford Diffraction, 2015)

Empirical absorption correction using spherical harmonics,

implemented in SCALE3 ABSPACK scaling algorithm.

T_min, T_max

0.975, 1.000

5979

Reflections collected

Independent reflection

Observed [I > 2s(I)] reflections

Data/restrains/parameters

Goodness-of-fit on F²

Final R [ F²> 2σ(F²)]

R indices (all data)

3817 [R_int= 0.02]

3425

3817/0/291

1.061

R1 = 0.0442, wR2 = 0.0950

R1 = 0.0502, wR2 = 0.0984

0.21, -0.22

ρ_max, ρ

_min(e Å^-3)

Crystallographic data for compound 13 are deposited in the Cambridge Crystallographic Data Centre as

supplementary material number CCDC 1561497. The CCDC files 1561497. contain the supplementary

crystallographic data. Copies of this information may be obtained free of charge from the Director,

CCDC, 12 Union Road, Cambridge, CB21FZ, UK (email: deposit@ccdc.cam.ac.uk or

http://www.ccdc.cam.ac.uk).

4.9 3α,7α,12α-Trihydroxy-7β-ethyl-5β-cholanic acid (14)

EtMgBr in Et₂O (3 M, 3 mL, 9 mmol); 1 (0.154 g, 0.38 mmol) in THF (6.4 mL); reaction time 90 min; NH₄Cl

(10 mL); CH₃COOH (3 mL); EtOAc (5x10 mL); flash chromatography: CH₂Cl₂/MeOH 9:1 with addition of

glacial acetic acid 0.05 mL/10 mL of eluent. Compound 14 (0.146 g; 88%) was obtained in the form of oil.

Rf (same solvent system as for flash chromatography) 0.31.

IR (cm^-1): 3401, 2930, 1713, 1377, 1097, 1034.

¹Н NMR (400 MHz, DMSO-d6) δ = 0.64 (s, 3H, H-18), 0.76 (s, 3H, H-19), 0.79 (t,

chain), 0.94 (d, =6 Hz, 3H, H-21), 3.22 (m, 1H, H-3), 3.41 (s, 1H, OH on H-7), 3.75 (s, 1H, H-12), 4.10 (s,

1H, OH on C-12), 4.33 (s, 1H, OH on C-3),

J=7 Hz, 3H, CH₃ethyl

J

¹³C NMR (101 MHz, DMSO-d6) δ = 10.0 (CH₃ethyl chain), 13.2 (C-18), 17.5 (C-21), 21.5, 23.0 (C-19), 26.2,

27.8, 28.8, 29.7, 30.6, 31.3, 31.5, 34.2, 35.5, 36.1, 37.0, 38.9, 39.0, 42.0, 43.0, 44.9, 47.7, 70.8, 70.9, 73.8

(C-7), 175.6 (C-24).

HRMS (TOF) m/z: calculated for C₂₆H₄₃O₅[M+K]⁺475.28203, found 475.28151.

13

ACCEPTED MANUSCRIPT

4.10 3α,7α,12α-Trihydroxy-7β-butyl-5β-cholanic acid (15)

BuMgCI in Et₂O (2 M, 3.4 mL, 6.8 mmol); 1 (0.111 g, 0.27 mmol) in THF (4.25 mL); reaction time 30 min;

NH₄Cl (10 mL); CH₃COOH (3 mL); EtOAc (5x10 mL); flash chromatography: CH₂Cl₂/MeOH 9:1 with

addition of glacial acetic acid 0.05 mL/10 mL of eluent. Compound 15 was obtained in the form of oil in

yield of 0.080 g (94% calculated in regard to reacted 1), and regenerated 1, 0.037 g. Rf (same solvent

system as for flash chromatography) 0.33.

IR (cm^-1): 3401, 2932, 2871, 1709, 1379, 1078, 1033, 736.

¹Н NMR (400 MHz, CDCl₃) δ = 0.75 (s, 3H, H-18), 0.85 (s, 3H, H-19), 0.92 (t,

1.03 (d, =6 Hz, 3H, H-21), 3.54 (m, 1H, H-3), 3.95 (s, 1H, H-12).

J=7 Hz, 3H, CH₃butyl chain),

J

¹³C NMR (101 MHz, CDCl₃) δ = 13.0 (C-18), 14.2 (CH₃butyl chain), 17.3 (C-21), 22.4, 23.3, 26.4, 27.4, 27.8,

28.8, 28.9, 29.7, 30.7, 31.1, 34.0, 35.5, 38.0, 39.3, 40.5, 41.6, 43.1, 44.4, 45.4, 48.0, 72.1 (C-3), 72.6 (C-

12), 75.4, 77.2, 178.3 (C-24).

HRMS (TOF) m/z: calculated for C₂₈H₄₈O₅[M+K]⁺503.31333, found 503.31222.

4.11 3α,7α,12α-Trihydroxy-7β-octyl-5β-cholanic acid (16)

OctMgBr in Et₂O (2 M, 11.5 mL, 23 mmol); 1 (0.311 g, 0.76 mmol) in THF (15 mL); reaction time 135 min;

NH₄Cl (10 mL); CH₃COOH (3 mL); EtOAc (5x10 mL); flash chromatography: CH₂Cl₂/MeOH 9:1 with

addition of glacial acetic acid 0.05 mL/10 mL of eluent. Compound 16 was obtained in the form of oil in

yield of 0.330 g (83%). Rf (same solvent system as for flash chromatography) 0.33.

IR (cm^-1): 3400, 2925, 2855, 1711, 1377, 1262, 1081, 1032.

¹Н NMR (400 MHz, DMSO-

chain), 0.93 (d, =6 Hz, 3H, H-21), 3.22 (m, 1H, H-3), 3.41 (s, 1H, OH on C-7), 3.74 (s, 1H, H-12), 4.11 (s,

1H, OH on C-12), 4.33 (s, 1H, OH on C-3).

d6) δ = 0.62 (s, 3H, H-18), 0.75 (s, 3H, H-19), 0.85 (t, J=7 Hz, 3H, CH₃octyl

J

¹³C NMR (101 MHz, DMSO-

d6) δ = 13.1 (C-18), 14.4 (CH₃octyl chain), 17.5 (C-21), 22.6 (C-19), 23.0, 25.1,

26.2, 27.8, 28.9, 29.1, 29.4, 29.7, 30.3, 30.6, 31.2, 31.3, 31.7, 34.2, 35.5, 36.1, 38.9, 40.5, 42.1, 43.1, 44.9,

45.0, 47.6 (C-13), 70.9 (C-3), 70.9 (C-12), 73.6 (C-7), 175.4 (C-24).

HRMS (TOF) m/z: calculated for C₃₂H₅₆O₅[M+CHO]⁻565.41098, found 565.40997.

4.12 2-(3α,7α,12α-Trihydroxy-7β-methyl-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (17), 2-(3α,7α,12α-trihydroxy-7β-methyl-5β-cholano -24-

amido)-2-methyl-1-propanol (21)

MeMgI in Et₂O (2 M, 6 mL, 12 mmol); 5 (0.289 g, 0.63 mmol) in THF (13 mL); reaction time 20 min; NH₄Cl

(10 mL); EtOAc (5x10 mL); flash chromatography: CH₂Cl₂/MeOH 9:1. Compound 17 was obtained as a

white needle crystal (mp. 105 °C crystalized from acetone, lit.¹⁹mp. 103–104.5 °C) in the yield of 0.193 g

(63%), and 21 in the form of oil in yield of 0.019 g (6%). Rf (CH₂Cl₂/MeOH 92:8) for 17 0.20, and for 21

0.10.

14

ACCEPTED MANUSCRIPT

Spectroscopic data for compound 17:

¹H NMR (400 MHz, DMSO-

6) δ = 0.61 (s, 3H, H-18), 0.78 (s, 3H, H-19), 0.95 (d, J=6 Hz, 3H, H-21), 1.09 (s,

d

3H, CH₃on C-7), 1.14 (s, 6H, 2xCH₃on C-4'), 3.21 (m, 1H, H-3), 3.57 (s, 1H, OH on C-7), 3.76 (s, 1H, H-12),

3.83 (s, 2H, H-5'), 4.10 (d, =3.1 Hz, 1H, OH on C-12), 4.33 (d, =3.9 Hz, 1H, OH on C-3).

J

¹³C NMR (101 MHz, DMSO-

d6) δ = 13.1 (C-18), 17.6 (C-21), 23.1 (C-19), 24.9, 27.4, 28.1, 28.7 and 28.8

(2xCH₃on C-4'), 28.9, 29.5, 30.7, 32.3, 33.7 (CH₃on C-7), 34.4, 35.3, 36.1, 38.6, 42.4, 42.7, 43.6, 45.0,

45.0, 47.5 (C-13), 67.1 (C-4'), 70.8, 70.9, 71.4 (C-7), 78.4 (C-5'), 165.3 (C-2').

Spectroscopic data for compound 21:

IR (cm^-1): 3369, 2930, 1649, 1549.

¹H NMR (400 MHz, CDCl₃) δ= 0.70 (s, 3H, H-18), 0.86 (s, 3H, H-19), 1.01 (d,

J = 6 Hz, 3H, H-21), 1.22 (s, 3H,

CH₃on C-7), 1.29 (s, 6H, 2xCH₃on amide), 3.45 (m, 1H, H-3), 3.57 (s, 2H, CH₂on amide), 3.94 (s, 1H, H-

12), 6.15 (s, 1H, NH).

¹³C NMR (101 MHz, CDCl₃) δ= 12.8 (C-18), 17.6 (C-21), 22.5 (C-19), 24.6 and 24.5 (2xCH₃on amide), 27.5,

27.8, 28.6, 28.8, 30.2, 31.6, 33.1, 33.3 (CH₃on C-7), 34.3, 35.0, 35.6, 38.3, 41.9, 42.5, 43.2, 44.5, 44.7,

47.6, 55.9, 70.7 (C-7), 71.8 (CH₂on amide), 72.6 (C-3), 73.3 (C-12), 175.2 (C-24).

HRMS (TOF) m/z: calculated for C₂₉H₅₁NO₅[M+H]⁺494.3840, found 494.3843.

4.13 2-(3α,7α,12α-Trihydroxy-7β-ethyl-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (18)

EtMgBr in Et₂O (3 M, 3 mL, 9 mmol); 5 (0.091 g, 0.2 mmol) in THF (2 mL); reaction time 15 min; NH₄Cl (8

mL); EtOAc (5x10 mL); flash chromatography: acetone/EtOAc 1:3. Compound 18 was obtained as an oil

in the yield of 0.072 g (74%). Rf (CH₂Cl₂/MeOH 9:1) 0.29.

IR (cm^-1): 3391, 2964, 2935, 2873, 1724, 1663, 1462, 1366, 1271, 1159, 1086, 1038, 996, 933, 912, 735.

¹Н NMR (400 MHz, CDCl₃) δ = 0.73 (s, 3H, H-18), 0.84 (s, 3H, H-19), 0.87 (t,

J

=7 Hz, 3H, CH₃ethyl chain),

1.03 (d,

12).

J=6 Hz, 3H, H-21), 1.26 (s, 6H, 2xCH₃on C-4’), 3.47 (m, 1H, H-3), 3.90 (s, 2H, H-5’), 3.94 (s, 1H, H-

¹³C NMR (101 MHz, CDCl₃) δ = 9.5 (CH₃ethyl chain), 12.9 (C-18), 17.5 (C-21), 22.4 (C-19), 25.0, 26.4, 27.7,

28.4 and 28.4 (2xCH₃on C-4’), 28.8, 29.7, 30.2, 32.0, 34.1, 35.3, 35.7, 36.9, 38.6, 38.7, 39.9, 41.6, 42.7,

45.1, 47.9, 66.7 (C-4'), 71.7 (C-3), 72.4 (C-12), 75.5 (C-7), 78.9 (C-5'), 166.8 (C-2').

HRMS (TOF) m/z: calculated for C₃₀H₅₁NO₄[M+H]⁺490.38909, found 490.38929.

15

ACCEPTED MANUSCRIPT

4.14 2-(3α,7α,12α-Trihydroxy-7β-butyl-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (19), 2-(3α,7α,12α-trihydroxy-7β-butyl-5β-cholano-24-

amido)-2-methyl-1-propanol (22)

Procedure A: BuMgCl in Et₂O (2 M, 75 mL, 0.15 mol); 5 (3.0 g, 6.52 mmol) in THF (50 mL); reaction time

60 min; NH₄Cl (100 mL); EtOAc (5x30 mL); flash chromatography: acetone/EtOAc 3:1, then CH₂Cl₂/MeOH

9:1. Compound 19 was obtained as a colorless oil in the yield of 2.41 g (79% in regard to reacted 5) and

compound 22 (0.263 g, 8% in regard to reacted 5), and regenerated 5, 0.290 g. Rf (EtOAc/acetone 1:3)

for 19 0.41, for 22 0.35.

Procedure B: BuMgCl in Et₂O (2 M, 0.2 mL, 0.4 mmol); 5 (0.064 g, 0.14 mmol) in THF (1.6 mL); heated to

reflux 2 h; NH₄Cl (10 mL); EtOAc (5x10 mL); flash chromatography same as in procedure A. Compound 19

was obtained as an oil in the yield of 0.019 g (33% in regard to reacted 5) and compound 22 (0.015 g,

20% in regard to reacted 5), and regenerated 5, 0.012 g.

Spectroscopic data for compound 19:

IR (cm^-1): 3370, 2957, 2930, 2868, 1663, 1463, 1366, 1081, 908, 734.

¹Н NMR (500 MHz, DMSO-

chain), 0.94 (d, =6 Hz, 3H, H-21), 1.14 (s, 6H, 2xCH₃on C-4'), 3.21 (m, 1H, H-3), 3.40 (s, 1H, OH on C-7),

=3 Hz, 1H, C-12), 3.83 (s, 2H, H-5'), 4.09 (d, =4 Hz, 1H, OH on C-12), 4.32 (d, =4 Hz, 1H, OH on

d6) δ = 0.62 (s, 3H, H-19), 0.75 (s, 3H, H-18), 0.83 (t, J=7 Hz, 3H, CH₃butyl

J

3.74 (d,

C-3).

J

¹³C NMR (125 MHz, DMSO-

d6) δ = 12.7 (C-18), 14.1 (CH₃butyl chain), 17.1 (C-21), 22.6 (C-19), 23.0, 24.4,

25.7, 27.0, 27.4, 28.2 and 28.3 (2xCH₃on C-4'), 28.4, 29.2, 30.2, 31.8, 33.7 (C-10), 34.8, 35.6, 38.4, 40.1,

41.6, 42.6, 44.3, 44.4, 47.2 (C-13), 66.6 (C-4'), 70.4 (C-3), 70.4 (C-12), 73.1 (C-7), 77.9 (C-5'), 164.8 (C-2').

HRMS (TOF) m/z: calculated for C₃₂H₅₅NO₄[M+H]⁺518.42039, found 518.41963.

Spectroscopic data for compound 22:

IR (cm^-1): 3369, 2932, 2869, 1651, 1552, 1456, 1378, 1266, 1181, 1102, 1074, 1035, 994, 738, 703.

¹Н NMR (250 MHz, CDCl₃) δ = 0.69 and 0.81 (2s, 2x3H, H-18 and H-19), 0.87 (t,

chain), 0.99 (d, =5 Hz, 3H, H-21), 1.27 (s, 6H, 2xCH₃amide), 3.42 (bs, 1H, OH on C-7), 3.53 (s, 2H, CH₂

J=6 Hz, 3H, CH₃butyl

J

amide), 3.88 (s, 1H, H-12), 4.00 (bs, 1H, OH on C-12), 5.45 (bs, 1H, OH on C-3), 6.44 (s, 1H, NH).

¹³C NMR (62.5 MHz, CDCl3) δ = 12.8 (CH₃), 14.1 (CH₃), 17.5 (CH₃), 22.4 (CH₃), 23.2, 24.3 (CH₃amide), 24.4

(CH₃amide), 26.2, 27.3, 27.6, 28.7, 29.6, 30.1, 31.5, 33.0, 34.0, 35.0, 35.6, 38.5, 39.5, 40.3, 41.6, 42.7,

44.4, 44.4, 47.7, 55.6 (qC amide), 70.5 (CH₂amide), 71.6, 72.5, 75.3 (C-7), 175.1 (C-24).

HRMS (TOF) m/z: calculated for C₃₂H₅₇NO₅[M+Na]⁺558.41290, found 558.41193.

16

ACCEPTED MANUSCRIPT

4.15 2-(3α,7α,12α-Trihydroxy-7β-octyl-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (20), 2-(3α,7α,12α-trihydroxy-7β-octyl-5β-cholano-24-

amido)-2-methyl-1-propanol (23)

Procedure A: OctMgBr in Et₂O (2 M, 32 mL, 64 mmol); 5 (1.031 g, 2.24 mmol) in THF (17 mL); reaction

time 1 h; NH₄Cl (50 mL); EtOAc (5x20 mL); flash chromatography: acetone/EtOAc 2:3, then CH₂Cl₂/MeOH

93:7. Compound 20 was obtained as an oil in the yield of 0.720 g (69% in regard to reacted 5) and

regenerated 5, 0.196 g. Rf (CH₂Cl₂/MeOH 9:1) for 20 0.28.

Procedure B: OctMgBr in Et₂O (2 M, 0.2 mL, 0.4 mmol); 5 (0.069 g, 0.15 mmol) in THF (1.7 mL); heated to

reflux 2 h; NH₄Cl (20 mL); EtOAc (5x10 mL); flash chromatography same as in procedure A. Compound 20

was obtained as an colorless oil in the yield of 0.016 g (18% in regard to reacted 5) and compound 23

(0.022 g, 26% in regard to reacted 5), and regenerated 5, 0.012 g. Rf (CH₂Cl₂/MeOH 9:1) for 23 0.40.

Spectroscopic data for compound 20:

IR (cm^-1): 3370, 2925, 2855, 1726, 1653, 1553, 1464, 1378, 1263, 1174, 1082, 1037, 941, 911, 731, 610.

¹Н NMR (500 MHz, DMSO-

chain), 0.95 (d, =6 Hz, 3H, H-21), 1.14 (s, 6H, 2xCH₃on C-4'), 3.21 (m, 1H, H-3), 3.41 (s, 1H, OH on C-7),

3.75 (s, 1H, H-12), 3.83 (s, 2H, H-5'), 4.10 (d, =3.5 Hz, 1H, OH on C-12), 4.33 (d, =3.3 Hz, 1H, OH on C-3).

d6) δ = 0.62 (s, 3H, H-18), 0.75 (s, 3H, H-19), 0.86 (t, J=7 Hz, 3H, CH₃octyl

J

¹³C NMR (125 MHz, DMSO-

d6) δ = 13.1 (C-18), 14.4 (CH₃octyl chain), 17.6 (C-21), 22.6, 23.1 (C-19), 24.8,

25.1, 26.2, 27.1, 27.9, 28.7, 28.8, 28.9, 29.1, 29.4, 29.7, 30.2, 30.6, 31.7, 32.3, 34.2, 35.3, 36.1, 38.9, 40.5,

42.1, 43.0, 44.9, 45.0, 47.7 (C-13), 67.1 (C-4'), 70.8 (C-3), 70.9 (C-12), 73.6 (C-7), 78.4 (C-5'), 165.3 (C-2').

HRMS (TOF) m/z: calculated for C₃₆H₆₃NO₄[M+H]⁺574.48299, found 574.48370.

Spectroscopic data for compound 23:

IR (cm^-1): 3369, 2927, 2857, 1648, 1550, 1465, 1378, 1073, 1035, 758.

¹Н NMR (250 MHz, CDCl₃) δ = 0.62 (s, 3H, H-18), 0.75 (s, 3H, H-19), 0.86 (t,

0.94 (d, =6 Hz, 3H, H-21), 1.16 (s, 6H, 2xCH₃amide), 3.22 (m, 1H, H-3), 3.37 (d,

3.40 (s, 1H, OH on C-7), 3.74 (s, 1H, H-12), 4.07 (d, =4 Hz, 1H, OH on C-12), 4.33 (d,

3), 4.91 (t, =6 Hz, 1H, OH amide), 7.23 (s, 1H, NH).

J

=7 Hz, 3H, CH₃octyl chain),

=6 Hz, 2H, CH₂amide),

=4 Hz, 1H, OH on C-

J

¹³C NMR (62.5 MHz, CDCl₃) δ = 13.2 (C-18), 14.4 (CH₃octyl chain), 17.8 (C-21), 22.6, 23.1 (C-19), 24.2

(2xCH₃amide), 25.1, 26.2, 27.9, 28.9, 29.1, 29.4, 29.7, 30.2, 30.6, 31.7, 32.2, 33.7, 34.2, 35.5, 36.1, 38.9,

40.5, 42.1, 43.0, 45.0, 47.6, 54.6, 55.4, 68.2 (CH₂amide), 70.8 (C-3), 70.9 (C-12), 73.6 (C-7), 173.4 (C-24).

HRMS (TOF) m/z: calculated for C₃₆H₆₅NO₅[M+H]⁺592.49355, found 592.49248.

17

ACCEPTED MANUSCRIPT

4.16 3α,7α,12α-Trihydroxy-12β-methyl-5β-cholanic acid (24)

MeMgI in Et₂O (2 M, 5 mL, 10 mmol); 2 (0.197 g, 0.49 mmol) in THF (12.5 mL); reaction time 90 min;

NH₄Cl (30 mL); EtOAc (5x10 mL); flash chromatography: CH₂Cl₂/MeOH 9:1 with addition of glacial acetic

acid 0.05 mL/10 mL of eluent). Compound 24 was obtained as a colorless oil in yield of 0.056 g (27%). Rf

(CH₂Cl₂/MeOH 9:1) 0.44.

IR (cm^-1): 3401, 2926, 1707, 1454.

¹HNMR (400 MHz, DMSO-

d

6) δ = 0.67 (s, 3H, H-18), 0.80 (s, 3H, H-19), 0.89 (d,

J

=7 Hz, 3H, H-21), 1.13 (s,

=3 Hz, 1H, OH

3H, CH₃on C-12), 3.18 (m, 1H, H-3), 3.62 (s, 1H, H-7), 3.86 (s, 1H, OH on C-12), 4.00 (d,

on C-7), 4.35 (s, 1H, OH on C-3).

J

¹³C NMR (101 MHz, DMSO-

d6) δ = 12.7 (C-18), 21.1, 21.8 (C-21), 23.1, 23.2, 27.5 (CH₃on C-12), 28.3,

28.8, 30.9 (C-6), 32.3 (C-20), 33.3, 34.9, 35.4, 35.7, 36.8 (C-11), 39.0 (C-8), 40.0, 42.0 (C-5), 42.5 (C-14),

47.6 (C-17), 49.5 (C-13), 66.7 (C-7), 70.9 (C-3), 73.0 (C-12), 175.3 (C-24).

HRMS (TOF) m/z: calculated for C₂₅H₄₂O₅[M+Na]⁺445.2924, found 445.2921.

4.17 2-(3α,7α,12α-Trihydroxy-12β-methyl-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (25)

MeMgI in Et₂O (2 M, 7 mL, 14 mmol); 6 (0.303 g, 0.66 mmol) in THF (13 mL); reaction time 30 min; NH₄Cl

(20 mL); EtOAc (5x7 mL); flash chromatography: CH₂Cl₂/MeOH 92:8. Compound 25 was obtained as a

colorless oil in the yield of 0.166 g (53%). Rf (CH₂Cl₂/MeOH 92:8) 0.43.

IR (cm^-1): 3391, 2969, 2932, 1662, 1463.

¹H NMR (400 MHz, DMSO-

9H, 2xCH₃on C-4' and CH₃on C-12), 3.18 (m, 1H, H-3), 3.62 (s, 1H, H-7), 3.82–3.86 (more signals, 3H, H-

d6) δ = 0.65 (s, 3H, H-18), 0.80 (s, 3H, H-19), 0.91 (d, J=7 Hz, 3H, H-21), 1.14 (s,

5' and OH on C-12), 3.96 (d,

J=3 Hz, 1H, OH on C-7), 4.32 (d,

J=4 Hz, 1H, OH on C-3).

¹³C NMR (101 MHz, DMSO-

d6) δ = 12.7 (C-18), 21.2, 21.8, 23.1, 23.3, 24.2, 26.4, 27.6, 28.3, 28.8 (2xCH₃

on C-4'), 29.8, 30.9, 31.8, 34.9, 35.4, 35.7, 36.7, 39.0, 42.0, 42.5, 47.5, 49.5, 66.7 (C-7), 67.1 (C-4'), 70.9

(C-3), 73.0 (C-12), 78.4 (C-5'), 164.9 (C-2').

HRMS (TOF) m/z: calculated for C₂₉H₄₉NO₄[M+Na]⁺498.3554, found 498.3550.

4.18 2-(3α,7α,12α-Trihydroxy-12β-ethyl-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (26), 2-(3α,7α,12α-trihydroxy-24-nor-5β-cholano-23-yl)-

4,4-dimethyloxazoline (27)

EtMgBr in Et₂O (3 M, 2.2 mL, 6 mmol); 6 (0.122 g, 0.27 mmol) in THF (4 mL); reaction time 2 h; NH₄Cl (10

mL); EtOAc (5x5 mL); flash chromatography: EtOAc/acetone 3:2, EtOAc/acetone 3:1, CH₂Cl₂/MeOH

18

ACCEPTED MANUSCRIPT

98:2). Compound 26 was obtained as an colorless oil in the yield of 0.062 g (48%), and compound 27 in

the yield of 0.021 g (17%, mp 104 °C recrystallised from acetone as a white needle crystals, lit.²⁰mp. 104

°C). Rf (CH₂Cl₂/MeOH 9:1) of 26 0.30, and of 27 0.21.

Spectroscopic data for compound 26:

IR (cm^-1): 3392, 2961, 2931, 2871, 1663, 1463, 1366, 1275, 1115, 1079, 1004, 940, 910, 737, 611.

¹Н NMR (400 MHz, DMSO-

chain), 0.89 (d, =7 Hz, 3H, H-21), 1.14 (s, 3H, CH₃on C-4’), 1.15 (s, 3H, CH₃on C-4’), 3.20 (m, 1H, H-3),

3.63 (s, 1H, H-7), 3.69 (s, 1H, OH on C-12), 3.80 (d, =8 Hz, 1H, H-5’), 3.85 (d, =8 Hz, 1H, H-5’), 3.96 (d,

=4 Hz, 1H, OH on C-3).

d6) δ = 0.66 (s, 3H, H-18), 0.80 (s, 3H, H-19), 0.84 (t, J=7 Hz, 3H, CH₃ethyl

J

=3 Hz, 1H, OH on C-7), 4.33 (d, J

¹³C NMR (101 MHz, DMSO-

d6) δ = 8.1 (CH₃ethyl chain), 13.1 (C-18), 21.3, 21.6 (C-21), 23.1 (C-19), 23.4,

26.4, 27.9, 28.8 (2xCH₃on C-4’), 29.6, 30.3, 30.7, 30.8, 32.3, 35.2, 35.4, 35.7, 39.1, 40.0, 42.0, 42.7, 47.1,

50.3, 66.9 (C-7), 67.1 (C-4’), 70.9 (C-3), 75.0 (C-12), 78.4 (C-5’), 164.9 (C-2’).

HRMS (TOF) m/z: calculated for C₃₀H₅₁NO₄[M+H]⁺490.38909, found 490.38809.

Spectroscopic data for compound 27:

IR (cm^-1): 3370, 2931, 2867, 1727, 1662, 1463, 1366, 1276, 1195, 1119, 1078, 1046, 981, 950, 914, 754.

¹Н NMR (400 MHz, DMSO-

21), 1.14 (s, 6H, 2xCH₃on C-4’), 3.18 (m, 1H, H-3), 3.61 (s, 1H, H-7), 3.78 (s, 1H, H-12), 3.83 (s, 2H, H-5’),

4.02 (d, =3 Hz, 1H, OH on C-7), 4.12 (d, =3 Hz, 1H, OH on C-12), 4.34 (d, =4 Hz, 1H, OH on C-3).

d6) δ = 0.58 (s, 3H, CH₃, H-18), 0.81 (s, 3H, CH₃, H-19), 0.94 (d, J=6 Hz, 3H, H-

J

¹³C NMR (101 MHz, DMSO-

d6) δ = 12.7 (C-18), 17.5 (C-21), 23.1 (C-19), 23.3, 24.9, 26.7, 27.8, 28.7 and

28.8 (2xCH₃on C-4'), 29.0, 30.8, 32.3, 34.8, 35.3, 35.4, 35.8, 39.9, 40.0, 41.8 (C-17), 42.0, 46.3 (C-13),

46.5, 66.7 (C-7), 67.1 (C-4’), 70.9 (C-3), 71.5 (C-12), 78.4 (C-5’), 165.4 (C-2').

HRMS (TOF) m/z: calculated for C₂₈H₄₇NO₄[M+H]⁺462.35779, found 462.35760.

4.19 2-(3α,7α,12α-Trihydroxy-12β-butyl-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (28)

BuMgCl in Et₂O (2 M, 6.4 mL, 12.8 mmol); 6 (0.115 g, 0.25 mmol) in THF (4.25 mL); reaction time 2 h, –20

°C, 3 h 0 °C; NH₄Cl (20 mL); EtOAc (5x10 mL); flash chromatography: acetone/EtOAc 1:2. Compound 28

was obtained as an oil in the yield of 0.063 g (49%) and compound 27 (0.022 g, 19%). Rf (CH₂Cl₂/MeOH

9:1) 0.62.

Spectroscopic data for compound 28:

IR (cm^-1): 3391, 2957, 2931, 2870, 1663, 1462, 1365, 1267.

¹H NMR (400 MHz, DMSO-

d6) δ = 0.65 (s, 3H, H-18), 0.80 (s, 3H, H-19), 0.87–0.91 (more signals, 6H, CH₃

butyl chain and H-21), 1.14 and 1.15 (2s, 6H, 2xCH₃on C-4'), 3.18 (s, 1H, H-3), 3.62 (s, 1H, H-7), 3.69 (s,

19

ACCEPTED MANUSCRIPT

1H, OH on C-12), 3.79–3.86 (more signals, 2H, H-5'), 3.95 (d, J=3 Hz, 1H, OH on C-7), 4.31 (d, J=4 Hz, 1H,

OH on C-3).

¹³C NMR (101 MHz, DMSO-

d6) δ= 13.1 (C-18), 14.7 (CH₃butyl chain), 21.7, 21.7 (C-19), 23.1, 23.5, 23.9,

25.5, 26.5, 28.0, 28.7 and 28.9 (2xCH₃on C-4'), 29.8, 30.8, 31.2, 32.4, 35.2, 35.4, 35.7, 38.3, 39.1, 42.0,

42.6, 47.0, 50.4, 55.4 (C-13), 66.9 (C-4'), 67.1 (C-7), 70.9 (C-3), 75.1 (C-12), 78.4 (C-5'), 165.0 (C-2').

HRMS (TOF) m/z: calculated for C₃₂H₅₅NO₄[M+H]⁺518.42039, found 518.41949.

4.20 2-(3α,7α,12α-Trihydroxy-12β-octyl-24-nor-5β-cholano-23-yl)-4,4-

dimethyloxazoline (29)

OctMgBr in Et₂O (2 M, 6 mL, 12 mmol); 6 (0.104 g, 0.23 mmol) in THF (6 mL); reaction time 3 h; NH₄Cl

(20 mL); EtOAc (5x10 mL); flash chromatography: CH₂Cl₂/MeOH 92:8. Compound 29 was obtained as a

small white crystals (mp. 96 °C after crystallization from acetone/MeOH) in the yield of 0.057 g (67%). Rf

(CH₂Cl₂/MeOH 9:1) 0.42.

IR (cm^-1): 3391, 2927, 2856, 1652, 1549, 1463, 1377.

¹H NMR (400 MHz, DMSO-

octyl chain and H-21), 1.14 and 1.15 (2s, 6H, 2xCH₃on C-4'), 3.18 (m, 1H, H-3), 3.62 (s, 1H, H-7), 3.69 (s,

d6) δ = 0.65 (s, 3H, H-18), 0.79 (s, 3H, H-19), 0.85–0.91 (more signals, 6H, CH₃

1H, OH on C-12), 3.80-3.85 (more signals, 2H, H-5' ), 3.95 (d,

OH on C-3).

J=3 Hz, 1H, OH on C-7), 4.32 (d, J=4 Hz, 1H,

¹³C NMR (101 MHz, DMSO-

d6) δ = 13.1 (C-18), 14.4 (CH₃octyl chain), 21.6, 21.7 (C-21), 22.5, 23.1 (C-19),

23.3, 23.5, 26.5, 28.0, 28.7 and 28.9 (2xCH₃on C-4'), 29.3, 29.7, 29.8, 30.8, 31.2, 31.7, 32.4, 35.2, 35.4,

35.7, 38.6, 39.1, 40.2, 42.0, 42.6, 47.0, 50.4 (C-13), 66.9 (C-4'), 67.1 (C-7), 70.9 (C-3), 75.1 (C-12), 78.4 (C-

5'), 164.9 (C-2').

HRMS (TOF) m/z: calculated for C₃₆H₆₃NO₄[M+H]⁺574.48299, found 574.48158.

4.21 7α,12α-Dihydroxy-3-oxo[4-²H₁]-5β-cholanic acid (3D)

EtMgBr in Et₂O (3 M, 2 mL, 6 mmol); 3 (0.114 g, 0.28 mmol) in THF (3 mL); reaction time 90 min; reaction

was quenched with addition of D₂O (0.5 mL), mixed for 10 min and then AcOH (3 mL) was added; EtOAc

(5x10 mL); flash chromatography: CH₂Cl₂/MeOH 93:7 with addition of glacial acetic acid 0.05 mL/10 mL

of eluent). Compound 3D (0.108 g; 95%) was obtained as a white needle crystal (mp. 158 °C

recrystallized from H₂O/MeOH). Rf (CH₂Cl₂/MeOH with addition of glacial acetic acid 0.05 mL/10 mL of

eluent) 0.58.

IR (cm^-1): 3435, 2938, 2865, 1707, 1268.

¹H NMR (400 MHz, DMSO-

1H, H-7), 3.83 (s, 1H, H-12), 4.19 (s, 1H, OH), 4.32 (s, 1H, OH).

d6) δ = 0.63 (s, 3H, H-18), 0.92-0.94 (more signals, 6H, H-19 and H-21), 3.69 (s,

20

ACCEPTED MANUSCRIPT

¹³C NMR (101 MHz, DMSO-

d6) δ = 12.8 (C-18), 17.4 (C-21), 21.9 (C-19), 23.2, 27.0, 27.7, 29.1, 31.3, 31.3,

34.3, 34.9, 35.5, 36.7, 36.9, 41.8, 43.2, 45.5 (m, C-D), 46.3, 46.6, 55.4, 66.8 (C-7), 71.4 (C-12), 175.4 (C-

24), 212.4 (C-3).

HRMS (TOF) m/z: calculated for C₂₄H₃₇DO₅[M+K]⁺446.24136, found 446.24155.

4.22 2-(7α,12α-Trihydroxy-3-oxo-5β-cholano-24-amido)-2-methyl-1-propanol

(30)

BuMgCl in Et₂O (2 M, 2 mL, 4 mmol); 7 (0.071 g, 0.15 mmol) in THF (2 mL); after 2 h one more portion of

BuMgCl was added (2 M, 2 mL, 4 mmol) and mixed for 2 h, and then reaction temperature was heated

to the room temperature and mixed for 18 h more; NH₄Cl (10 mL); EtOAc (4x10 mL); flash

chromatography: EtOAc/toluene/MeOH 7:2:1). Compound 30 was obtained as an oil in the yield of

0.006 g (8%). Rf (same solvent system as for flash chromatography) 0.2.

IR (cm^-1): 3401, 2925, 2865, 1705, 1652, 1548.

¹H NMR (400 MHz, DMSO-

d6) δ = 0.62 (s, 3H, H-18), 0.92-0.94 (more signals, 6H, H-19 i H-21), 1.16 (s,

6H, 2×CH₃amide), 3.69 (s, 1H, H-7), 3.82 (s, 2H, H-12), 4.16 (d,

4.91 (s, 1H, OH amide), 7.24 (s, 1H, NH).

J=9 Hz, 1H, OH), 4.33 (d, J=3 Hz, 1H, OH),

¹³C NMR (101 MHz, DMSO-

d6) δ = 12.8 (C-18), 17.7 (C-21), 21.9, 23.2, 24.2 (2×CH₃amide), 27.0, 27.7,

29.2, 32.2, 33.6, 34.3, 34.9, 35.6, 36.6, 36.9, 41.8, 43.2, 45.8, 46.3, 46.7, 54.6, 66.8, 68.2, 71.4, 173.4 (C-

24), 212.3 (C-3).

HRMS (TOF) m/z: calculated for C₂₈H₄₇NO₅[M+H]⁺478.3527, found 478.3536.

Acknowledgement

This work is supported by the Ministry of Education, Science and Technological Development of Serbia

(Project No 172021).

References

1. Copple BL, Li T. Pharmacol. Res. 2016; 104:9.

2. Reddy BS, Watanabe K, Weisburger JH, Wynder EL. Cancer Res. 1977; 37:3238.

3. Hori T, Matsumoto K, Sakaitani Y, Sato M, Morotomi M. Cancer Lett. 1998; 124:79.

4. Poša M, Kuhajda K. Steroids 2010; 75:424.

5. Poša M, Rašeta M, Kuhajda K. Hem. Ind. 2011; 65:115.

6. Pellicciari R, Fiorucci S, Camaioni E, Clerici C, Costantino G, Maloney PR, Morelli A, Parks DJ,

Willson TM. J. Med. Chem. 2002; 45:3569.

7. https://clinicaltrials.gov/ct2/results?term=obeticholic+acid

8. Bjedov S, Jakimov D, Pilipović A, Poša M, Sakač M. Steroids 2017; 120:19.

9. Tsemg K, Klein PD. Steroids 1977; 29:635.

21

ACCEPTED MANUSCRIPT

10. Fieser LF, Rajagopalan S. J. Am. Chem. Soc. 1949; 71:3935.

11. Bertolasi V, Bortolini O, Fantin G, Fogagnolo M, Perrone D. Steroids 2007; 72:756.

12. Kramer W, Schneider S. J. Lipid Res. 1989; 30:1281.

13. Kuroki S, Une M, Mosbach EH. J. Lipid Res. 1985; 26:1205.

14. Whitmore FC, George RS. J. Am. Chem. Soc. 1942; 64:1239.

15. Blessing RH. Acta. Crystallogr. A. 1995; 51:33.

16. Sheldrick GM. Acta. Crystallogr. A. 2015; 71:3.

17. Farrugia LJ. J. Appl. Crystallogr. 1997; 30:565.

18. Farrugia LJ. J. Appl. Crystallogr. 1999; 32:837.

19. Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D,

Boyd MR. Cancer Res. 1988; 48:4827.

20. Cohen BI, May PS, McSherry CK, Mosbach EH. Steroids 1982; 40:701.

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