Synthesis of novel aryl brassinosteroids through alkene cross-metathesis and preliminary biological study

Article abstract of DOI:10.1016/j.steroids.2017.08.010

A series of phenyl analogues of brassinosteroids was prepared via alkene cross-metathesis using commercially available styrenes and 24-nor-5α-chola-2,22-dien-6-one. All derivatives were successfully docked into the active site of BRI1 using AutoDock Vina. Plant growth promoting activity was measured using the pea inhibition biotest and Arabidopsis root sensitivity assay and then was compared with naturally occuring brassinosteroids. Differences in the production of plant hormone ethylene were also observed in etiolated pea seedlings after treatment with the new and also five known brassinosteroid phenyl analogues. Antiproliferative activity was also studied using normal human fibroblast and human cancer cell lines.

Full text of DOI:10.1016/j.steroids.2017.08.010

Steroidsxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of novel aryl brassinosteroids through alkene cross-metathesis and
preliminary biological study
Petra Korinkovaa,1, Vaclav Bazgierb,1, Jana Oklestkovaa, Lucie Rarovac, Miroslav Strnada,
Miroslav Kvasnicaa,⁎
a
Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University & Institute of Experimental Botany
ASCR, Šlechtitelů 27, 783 71 Olomouc, Czech Republic
b
Regional Centre of Advanced Technologies and Material, Department of Physical Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, 771 46 Olomouc,
Czech Republic
c
Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University,
Šlechtitelů 27, 783 71 Olomouc, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of phenyl analogues of brassinosteroids was prepared via alkene cross-metathesis using commercially
available styrenes and 24-nor-5α-chola-2,22-dien-6-one. All derivatives were successfully docked into the active
site of BRI1 using AutoDock Vina. Plant growth promoting activity was measured using the pea inhibition biotest
and Arabidopsis root sensitivity assay and then was compared with naturally occuring brassinosteroids.
Differences in the production of plant hormone ethylene were also observed in etiolated pea seedlings after
treatment with the new and also five known brassinosteroid phenyl analogues. Antiproliferative activity was also
studied using normal human fibroblast and human cancer cell lines.
Brassinosteroids
Cross-metathesis
Organic synthesis
Molecular docking
BRI1 receptor kinase
Plant bioassays
1. Introduction
independent X-ray diffraction structural determinations by two groups
[9,10]. The structure of the ligand-binding domain resembles a superhelix of
Brassinosteroids (BRs, Fig. 1) represent a large group of plant steroids
with more than 70 structurally and functionally related compounds [1]. BRs
have been found at low concentrations throughout the plant kingdom,
widely distributed in higher and lower plants, and have been detected in
various plant parts such as pollen, seeds, leaves, stems, roots, and flowers.
They are essential for many aspects of plant growth and development, such
as cell division, elongation and differentiation, pollen tube growth, seed
germination, regulation of gene expression, enzyme activation and photo-
synthesis [2–5]. At the molecular level, BRs change the gene expression and
the metabolism of nucleic acids and proteins. BRs have structures similar to
those of animal steroid hormone. Unlike animals, plants perceive steroids at
cell membrane, using the membrane-integral receptor kinase brassinos-
teroid insensitive 1 (BRI1) [6–8]. The encoded protein, BRI1, belongs to a
large family of plant LRR (leucine-rich repeat) receptor-like kinases, char-
acterized by an extracellular LRR domain, a single-pass transmembrane
segment and a cytoplasmic kinase domain. BRI1 has been established as an
authentic brassinosteroid receptor by genetic and biochemical investigations
[9]. Crystal structures of BRI1 in both free (PDB ID: 3RIZ, 3RGX), and
brassinolide-bound (PDB ID: 3RJ0, 3BRZ), forms are available, following
25 twisted LRRs. A 70-amino acid island domain between LRRs 21 and 22
folds back into the interior of the superhelix, creating a surface pocket
where the brassinosteroids bind. These recently published structures of
Arabidopsis thaliana BRI1 enable the rational design of brassinosteroid-like
antagonists and agonists. Recent studies [11,12] have indicated that mole-
cular docking is a powerful tool to predict how effective incorporation of
different functional groups into brassinosteroid skeleton is and to design
new types of BRs with biological activities comparable to natural BRs [11].
The aim of our study is related to the synthesis of new aryl analo-
gues of BRs by alkene cross-metathesis and to study of their biological
properties. Alkene cross-metathesis was chosen for preparation of all
aryl analogues as an efficient method for construction of the new side
chains using different commercially available substituted styrenes. The
biological activities of newly prepared derivatives were evaluated using
plant bioassays (pea inhibition biotest and Arabidopsis root sensitivity
bioassay) and Calcein AM cytotoxicity assay. All derivative structures
were subjected to docking studies using AutoDock Vina [13] in order to
analyze the results with theoretical studies.
⁎
Corresponding author at: Laboratory of Growth Regulators, Institute of Experimental Botany ASCR & Palacký University, Šlechtitelů 27, 78371 Olomouc, Czech Republic.
E-mail address: kvasnica@ueb.cas.cz (M. Kvasnica).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.steroids.2017.08.010
Received 14 June 2017; Received in revised form 18 July 2017; Accepted 9 August 2017
0039-128X/©2017ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:Korinkova,P.,Steroids(2017),http://dx.doi.org/10.1016/j.steroids.2017.08.010

P. Korinkova et al.
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(2.48 mmol) in dichloroethane (5 mL). The reaction mixture was he-
ated at 80 °C for 5 h. Then, another portion of H-G catalyst (19 mg;
0.03 mmol) was added and the reaction mixture was heated at 80 °C for
additional 5 h. Then, the solvent was evaporated and crude solid was
purified by column chromatography on silica gel (mobile phase – 3%
ethyl acetate in cyclohexane, Rf of products 0.18–0.25). In some cases,
stated in each experiment, HPLC had to be used due to very close re-
tention time of product and starting material (mobile phase – 0.5%
ethyl acetate in cyclohexane).
2.1.2. (22E)-23-phenyl-24-nor-5α-chola-2,22-dien-6-one (8a)
The general procedure with styrene afforded 120 mg (81%) of the
title compound 8 as a colorless oil. 1H NMR (CDCl3) δ 0.72, 0.74 (both
s, 3H, CH3), 1.14 (d, 3H, J = 6.4 Hz, CH3), 1.69–1.80 (m, 2H),
1.96–2.04 (m, 4H), 2.07 (dt, 1H, J = 12.6, J′ = 3.3 Hz), 2.23–2.31 (m,
2H), 2.34–2.37, (m, 2H), 5.57 (m, 1H, H-3), 5.69 (m, 1H, H-2), 6.06
(dd, 1H, J = 15.9, J′ = 8.9 Hz, H-22), 6.30 (d, 1H, J = 15.9 Hz, H-23),
7.19 (m, 1H, Ar-H), 7.27–7.35 (m, 4H, 4×Ar-H). 13C NMR δ 12.19 (C-
18), 13.50 (C-19), 20.38 (C-21), 21.10, 21.70, 23.92, 28.21, 37.68,
39.35, 39.37, 40.04, 40.39, 42.92, 46.96, 53.40, 53.83, 55.83, 56.75,
124.49 (C-3), 124.95 (C-2), 125.92 (2×C), 126.72, 127.42 (C-23),
128.45 (2×C), 136.94 (C-22), 137.98, 211.98 (C-6). Spectral data in
agreement with literature [11].
Fig. 1. Structures of most common natural brassinosteroids; castasterone (1), brassinolide
(2), 24-epicastasterone (3), 24-epibrassinolide (4), 28-homocastasterone (5), 28-homo-
brassinolide (6).
2.1.3. (22E)-23-(2-fluorophenyl)-24-nor-5α-chola-2,22-dien-6-one (8b)
The general procedure with o-fluorostyrene afforded 84 mg (65%)
of the title compound 9a as a colorless oil: IR ν (cm−1) 2930, 1702,
1655, 1593, 1560, 965. 1H NMR (CDCl3) δ 0.73, 0.75 (both s, 3H, CH3);
1.15 (d, 3H, J = 6.7 Hz, CH3); 1.72–1.81 (m, 2H); 1.97–2.06 (m, 4H);
2.08 (dt, 1H, J = 12.6, J′ = 3.5 Hz); 2.24–2.33 (m, 2H); 2.34–2.38 (m,
2H); 5.58 (m, 1H, H-3); 5.70 (m, 1H, H-2); 6.14 (dd, 1H, J = 15.9,
J′ = 8.6 Hz, H-22); 6.47 (d, 1H, J = 15.9 Hz, H-23); 7.01 (ddd,
J = 10.9, J′ = 7.8, J″ = 0.9 Hz, Ar-H); 7.07 (td, 1H, J = 7.8,
J′ = 1.2 Hz, Ar-H); 7.16 (m, 1H, Ar-H); 7.41 (td, 1H, J = 7.8,
J′ = 1.8 Hz, Ar-H). 13C NMR δ 12.19 (C-18), 13.48 (C-19), 20.27 (C-
21), 21.08, 21.70, 23.91, 28.17, 37.65, 39.32, 39.35, 40.01, 40.76,
42.92, 46.93, 53.37, 53.80, 55.68, 56.71, 115.56 (d, J = 22.8 Hz),
119.79 (d, J = 3.6 Hz), 123.91 (d, J = 3.6 Hz), 124.48 (C-3), 124.94
(C-2), 125.62 (d, J = 13.2 Hz), 126.92 (d, J = 3.6 Hz), 127.86 (d,
J = 8.4 Hz), 139.50 (d, J = 3.6 Hz), 159.94 (d, J = 248.3 Hz, C-F),
211.90 (C-6). 19F NMR {1H} δ −118.83 (s, 1F). HRMS: (API+) cal-
culated for C29H38FO ([M+H]+) 421.2907, Found 421.2910.
2. Experimental
2.1. General methods
The melting points were determined on a Stuart SMP30 instrument
(Bibby Scientific Ltd., UK). Elemental analyses were performed using an
EA 1108 elemental analyzer (Fison Instruments); the values (C, H, N)
agreed with the calculated values within acceptable limits. The infrared
spectra were recorded on a Thermo Scientific Nicolet spectrometer iZ10
using the ATR technique. The wave numbers are given in cm−1. The
NMR spectra were taken on a JEOL JNM-ECA 500 (JEOL, Tokyo, Japan;
1H, 500 MHz; 13C, 125 MHz) spectrometer equipped with a 5 mm
JEOL Royal probe. 1H NMR and 13C NMR chemical shifts (δ) were
calibrated using tetramethylsilane (TMS, 1H δ = 0 ppm) or solvents:
CDCl3 (1H δ = 7.26 ppm, 13C δ = 77.00 ppm) or DMSO-d6 (1H
δ = 2.46 ppm, 13C δ = 40.00 ppm). Chemical shifts are given in ppm
(δ-scale), coupling constants (J) in Hz. All values were obtained by first-
order analysis. For API HRMS analysis, the samples were dissolved in
chloroform (or chloroform: methanol; 1:1; v/v, in case of hydroxylated
compounds) to a concentration 10 µg.mL−1. The ASAP (Atmospheric
Solids Analysis Probe) was dipped into the sample solution, placed into
the ion source and analysed in fullscan mode. The source of the Synapt
G2-Si Mass Spectrometer (Waters, Manchester, UK) was operated in
positive ionisation mode (ASAP+), if not stated otherwise, at source
temperature of 120 °C. The Corona needle current was kept at 5 µA and
the collision energy at value 4. The probe temperature was ramped up
from 50 °C to 600 °C in 3 min. Data were acquired from 50 to 1000 Da
with 1.0 s scan time in High Resolution Mode. The data were processed
using the Masslynx 4.1 software (Waters). Mass accuracy of 1 ppm or
less was achieved with the described instrumentation for all com-
pounds. Merck silica gel Kieselgel 60 (230–400 mesh) was used for
column chromatography. The HPLC system consisted of a Waters semi-
preparative HPLC system including quaternary pump, liquid handler,
UV–VIS and ELSD detectors. The semi preparative column was filled
with silica gel. Reagents and solvents were purchased from
Sigma–Aldrich and were not purified.
2.1.4. (22E)-23-(3-fluorophenyl)-24-nor-5α-chola-2,22-dien-6-one (8c)
The general procedure with m-fluorostyrene afforded 94 mg (73%)
of the title compound 10a as a colorless oil: IR ν (cm−1) 2933, 1705,
1656, 1593, 1560, 966. 1H NMR (CDCl3) δ 0.73, 0.74 (both s, 3H, CH3);
1.14 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.80 (m, 2H); 1.97–2.04 (m, 4H);
2.07 (dt, 1H, J = 12.5, J′ = 3.2 Hz); 2.23–2.31 (m, 2H); 2.34–2.38 (m,
2H); 5.58 (m, 1H, H-3); 5.70 (m, 1H, H-2); 6.08 (dd, 1H, J = 15.7,
J′ = 8.7 Hz, H-22); 6.28 (d, 1H, J = 15.7 Hz, H-23); 6.88 (td, 1H,
J = 8.3, J′ = 2.6 Hz, Ar-H); 7.03 (m, 1H, Ar-H); 7.08 (b d, 1H,
J = 8.3 Hz, Ar-H); 7.24 (td, 1H, J = 7.8, J′ = 6.1 Hz, Ar-H). 13C NMR δ
12.16 (C-18), 13.46 (C-19), 20.22 (C-21), 21.06, 21.68, 23.87, 28.16,
37.62, 39.30, 39.32, 39.98, 40.30, 42.90, 46.90, 53.33, 53.77, 55.67,
56.66, 112.25 (d, J = 21.6 Hz), 113.44 (d, J = 21.6 Hz), 121.81 (d,
J = 2.4 Hz), 124.45 (C-3), 124.92 (C-2), 126.46 (d, J = 2.4 Hz),
129.79 (d, J = 8.4 Hz), 138.28, 140.35 (d, J = 7.2 Hz), 163.08 (d,
J = 244.7 Hz, C-F), 211.82 (C-6). 19F NMR {1H} δ −113.78 (s, 1F).
HRMS: (API+) calculated for C29H38FO ([M+H]+) 421.2907, Found
421.2910.
2.1.5. (22E)-23-(4-fluorophenyl)-24-nor-5α-chola-2,22-dien-6-one (8d)
The general procedure with p-fluorostyrene afforded 96 mg (75%)
of the title compound 8 as a colorless oil. 1H NMR (CDCl3) δ 0.72, 0.73
(both s, 3H, CH3), 1.13 (d, 3H, J = 6.7 Hz, CH3), 1.71–1.80 (m, 2H),
2.1.1. General procedure for cross metathesis
Hoveyda-Grubbs 2nd generation catalyst (19 mg; 0.03 mmol) was
added to a solution of dien 7 (100 mg; 0.31 mmol) and styrene
2

P. Korinkova et al.
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1.96–2.04 (m, 4H), 2.08 (dt, 1H, J = 12.5, J′ = 3.2 Hz), 2.22–2.31 (m,
2H), 2.33–2.37, (m, 2H), 5.57 (m, 1H, H-3), 5.69 (m, 1H, H-2), 5.97
(dd, 1H, J = 15.7, J′ = 8.7 Hz, H-22), 6.26 (d, 1H, J = 15.7 Hz, H-23),
6.97 (m, 2H, 2×Ar-H), 7.27 (m, 2H, 2×Ar-H). 13C NMR δ 12.17 (C-18),
13.48 (C-19), 20.35 (C-21), 21.08, 21.69, 23.88, 28.21, 37.65, 39.32,
39.34, 40.01, 40.33, 42.89, 46.92, 53.36, 53.80, 55.79, 56.71, 115.25
(d, J = 20.4 Hz, 2×C), 124.47 (C-3), 124.93 (C-2), 126.25 (C-23),
127.28 (d, J = 8.4 Hz, 2×C), 134.07 (d, J = 3.6 Hz), 136.65 (d,
J = 2.4 Hz, C-22), 161.79 (d, J = 244.7 Hz, C-F), 211.93 (C-6).
Spectral data in agreement with literature [11].
(C-19), 20.28 (C-21), 21.10, 21.70, 23.96, 28.21, 37.66, 39.34, 39.36,
40.03, 40.47, 42.98, 46.95, 53.39, 53.83, 55.66, 56.72, 123.26, 124.50
(C-3), 124.96 (C-2), 126.50, 126.81, 127.33, 128.06, 132.77, 137.80,
139.93, 211.97 (C-6). HRMS: (API+) calculated for C29H3879BrO ([M
+H]+) 481.2106, Found 481.2107.
2.1.10. (22E)-23-(3-bromophenyl)-24-nor-5α-chola-2,22-dien-6-one (8i)
The general procedure with m-bromostyrene afforded 97 mg (66%)
of the title compound 14a as a colorless oil: IR ν (cm−1) 2944, 1700,
1655, 1588, 1560, 964. 1H NMR (CDCl3) δ 0.72, 0.73 (both s, 3H, CH3);
1.13 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.80 (m, 2H); 1.96–2.04 (m, 4H);
2.06 (dt, 1H, J = 12.5, J′ = 3.4 Hz); 2.22–2.31 (m, 2H); 2.34–2.37, (m,
2H); 5.58 (m, 1H, H-3); 5.69 (m, 1H, H-2); 6.06 (dd, 1H, J = 15.7,
J′ = 8.7 Hz, H-22); 6.23 (d, 1H, J = 15.7 Hz, H-23); 7.14 (t, 1H,
J = 7.8 Hz, Ar-H); 7.22 (m, 1H, Ar-H); 7.30 (m, 1H, Ar-H); 7.47 (t, 1H,
J = 1.7 Hz, Ar-H). 13C NMR δ 12.19 (C-18), 13.50 (C-19), 20.25 (C-21),
21.09, 21.70, 23.91, 28.20, 37.66, 39.34, 39.36, 40.03, 40.41, 42.95,
46.94, 53.37, 53.82, 55.69, 56.71, 122.69, 124.48 (C-3), 124.69,
124.96 (C-2), 126.15, 128.70, 129.56, 129.95, 138.55, 140.17, 211.92
(C-6). HRMS: (API+) calculated for C29H3879BrO ([M+H]+) 481.2106,
Found 481.2108.
2.1.6. (22E)-23-(2-chlorophenyl)-24-nor-5α-chola-2,22-dien-6-one (8e)
The general procedure with o-chlorostyrene afforded 86 mg (64%)
of the title compound 11a as a colorless oil: IR ν (cm−1) 2941, 1701,
1655, 1593, 1563, 964. 1H NMR (CDCl3) δ 0.73, 0.75 (both s, 3H, CH3);
1.16 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.81 (m, 2H); 1.96–2.04 (m, 4H);
2.08 (dt, 1H, J = 12.5, J′ = 3.2 Hz); 2.22–2.33 (m, 2H); 2.34–2.38 (m,
2H); 5.58 (m, 1H, H-3); 5.70 (m, 1H, H-2); 6.04 (dd, 1H, J = 15.6,
J′ = 8.9 Hz, H-22); 6.69 (d, 1H, J = 15.9 Hz, H-23); 7.13 (m, 1H,
ΣJ = 16.8 Hz, Ar-H); 7.19 (m, 1H, ΣJ = 15.9 Hz, Ar-H); 7.33 (dd, 1H,
J = 7.9, J′ = 1.2 Hz, Ar-H); 7.47 (dd, 1H, J = 7.9, J′ = 1.5 Hz, Ar-H).
13C NMR δ 12.19 (C-18), 13.46 (C-19), 20.26 (C-21), 21.07, 21.68,
23.91, 28.17, 37.62, 39.30, 39.32, 39.98, 40.51, 42.93, 46.91, 53.34,
53.77, 55.63, 56.68, 123.80, 124.46 (C-3), 124.91 (C-2), 126.53,
126.64, 127.73, 129.52, 132.58, 136.00, 139.72, 211.87 (C-6). HRMS:
2.1.11. (22E)-23-(4-bromophenyl)-24-nor-5α-chola-2,22-dien-6-one (8j)
The general procedure with p-bromostyrene afforded 107 mg (73%)
of the title compound 8 as a colorless oil. 1H NMR (CDCl3) δ 0.72, 0.73
(both s, 3H, CH3), 1.13 (d, 3H, J = 6.7 Hz, CH3), 1.70–1.79 (m, 2H),
1.96–2.04 (m, 4H), 2.07 (dt, 1H, J = 12.5, J′ = 3.3 Hz), 2.22–2.30 (m,
2H), 2.33–2.37, (m, 2H), 5.57 (m, 1H, H-3), 5.69 (m, 1H, H-2), 6.05
(dd, 1H, J = 15.7, J′ = 8.7 Hz, H-22), 6.24 (d, 1H, J = 15.7 Hz, H-23),
7.18 (m, 2H, 2×Ar-H), 7.39 (m, 2H, 2×Ar-H). 13C NMR δ 12.19 (C-18),
13.49 (C-19), 20.24 (C21), 21.08, 21.70, 23.90, 28.19, 37.65, 39.32,
39.35, 40.02, 40.40, 42.92, 46.93, 53.36, 53.81, 55.71, 56.70, 120.30,
124.47 (C-3), 124.95 (C-2), 126.33 (C-23), 127.47 (2×C), 131.48
(2×C), 136.89, 137.77 (C-22), 211.92 (C-6). Spectral data in agree-
ment with literature [11].
(API+) calculated for
437.2614.
C ) 437.2611, Found
29H38ClO ([M+H]+
2.1.7. (22E)-23-(3-chlorophenyl)-24-nor-5α-chola-2,22-dien-6-one (8f)
The general procedure with m-chlorostyrene afforded 94 mg (70%)
of the title compound 12a as a colorless oil: IR ν (cm−1) 2944, 1701,
1654, 1593, 1562, 964. 1H NMR (CDCl3) δ 0.72, 0.73 (both s, 3H, CH3);
1.13 (d, 3H, J = 6.7 Hz, CH3); 1.70–1.80 (m, 2H); 1.96–2.05 (m, 4H);
2.07 (dt, 1H, J = 12.5, J′ = 3.2 Hz); 2.22–2.31 (m, 2H); 2.33–2.37 (m,
2H); 5.58 (m, 1H, H-3); 5.69 (m, 1H, H-2); 6.07 (dd, 1H, J = 15.9,
J′ = 8.9 Hz, H-22); 6.25 (d, 1H, J = 15.9 Hz, H-23); 7.14–7.22 (m, 3H,
3×Ar-H); 7.31 (t, 1H, J = 1.7 Hz, Ar-H). 13C NMR δ 12.18 (C-18),
13.48 (C-19), 20.24 (C-21), 21.08, 21.69, 23.89, 28.19, 37.64, 39.31,
39.33, 40.01, 40.39, 42.92, 46.92, 53.34, 53.79, 55.66, 56.68, 124.23,
124.47 (C-3), 124.94 (C-2), 126.22, 126.63, 127.74, 129.63, 134.36,
138.47, 139.85, 211.92 (C-6). HRMS: (API+) calculated for C29H38ClO
([M+H]+) 437.2611, Found 437.2615.
2.1.12. (22E)-23-(2-methylphenyl)-24-nor-5α-chola-2,22-dien-6-one (8k)
The general procedure with o-methylstyrene afforded 83 mg (65%)
of the title compound 15a as a colorless oil: IR ν (cm−1) 2932, 1701,
1653, 1593, 1563, 964. 1H NMR (CDCl3) δ 0.72, 0.74 (both s, 3H, CH3);
1.14 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.80 (m, 2H); 1.96–2.04 (m, 4H);
2.07 (dt, 1H, J = 12.6, J′ = 3.6 Hz); 2.22–2.31 (m, 2H); 2.32 (s, 3H,
Ar-CH3); 2.33–2.37 (m, 2H); 5.57 (m, 1H, H-3); 5.69 (m, 1H,
J = 15.6 Hz, H-2); 5.89 (dd, 1H, J = 15.6, J′ = 8.9 Hz, H-22); 6.49 (d,
1H, J = 15.6 Hz, H-23); 7.10–7.15 (m, 3H, 3×Ar-H); 7.37 (d, 1H,
J = 7.3 Hz, Ar-H). 13C NMR δ 12.20 (C-18), 13.48 (C-19), 19.58 (Ar-
CH3), 20.53 (C-21), 21.08, 21.69, 23.92, 28.30, 37.65, 39.31, 39.34,
40.01, 40.72, 42.87, 46.93, 53.35, 53.78, 55.71, 56.73, 124.48 (C-3),
124.93 (C-2), 125.28, 125.45, 125.92, 126.67, 130.06, 134.94, 137.15,
138.43, 211.98 (C-6). HRMS: (API+) calculated for C30H41O ([M
+H]+) 417.3157, Found 417.3160.
2.1.8. (22E)-23-(4-chlorophenyl)-24-nor-5α-chola-2,22-dien-6-one (8g)
The general procedure with p-chlorostyrene afforded 100 mg (75%)
of the title compound 8 as a colorless oil. 1H NMR (CDCl3) δ 0.72, 0.73
(both s, 3H, CH3), 1.13 (d, 3H, J = 6.7 Hz, CH3), 1.70–1.80 (m, 2H),
1.96–2.04 (m, 4H), 2.07 (dt, 1H, J = 12.5, J′ = 3.4 Hz), 2.22–2.30 (m,
2H), 2.34–2.37, (m, 2H), 5.57 (m, 1H, H-3), 5.69 (m, 1H, H-2), 6.03
(dd, 1H, J = 15.9, J′ = 8.9 Hz, H-22), 6.27 (d, 1H, J = 15.9 Hz, H-23),
7.24 (s, 4H, 4×Ar-H). 13C NMR δ 12.19 (C-18), 13.50 (C-19), 20.27 (C-
21), 21.09, 21.70, 23.91, 28.21, 37.66, 39.33, 39.36, 40.04, 40.39,
42.93, 46.94, 53.37, 53.82, 55.75, 56.72, 124.49 (C-3), 124.96 (C-2),
126.30 (C-23), 127.12 (2×C), 128.55 (2×C), 132.22, 136.45, 137.64
(C-22), 211.96 (C-6). Spectral data in agreement with literature [11].
2.1.13. (22E)-23-(3-methylphenyl)-24-nor-5α-chola-2,22-dien-6-one (8l)
The general procedure with m-methylstyrene afforded 88 mg (69%)
of the title compound 16a as a colorless oil: IR ν (cm−1) 2933, 1700,
1653, 1593, 1562, 964. 1H NMR (CDCl3) δ 0.71, 0.73 (both s, 3H, CH3);
1.12 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.79 (m, 2H); 1.95–2.04 (m, 4H);
2.06 (dt, 1H, J = 12.6, J′ = 3.5 Hz); 2.20–2.30 (m, 2H); 2.32 (s, 3H,
Ar-CH3); 2.33–2.37 (m, 2H); 5.57 (m, 1H, H-3); 5.68 (m, 1H, H-2); 6.04
(dd, 1H, J = 15.9, J′ = 8.9 Hz, H-22); 6.27 (d, 1H, J = 15.9 Hz, H-23);
7.00 (d, v, J = 7.3 Hz, Ar-H); 7.11–7.19 (m, 3H, 3×Ar-H). 13C NMR δ
12.19 (C-18), 13.49 (C-19), 20.40 (C-21), 21.09, 21.40 (Ar-CH3), 21.70,
23.90, 28.20, 37.66, 39.32, 39.35, 40.03, 40.40, 42.88, 46.94, 53.37,
53.80, 55.82, 56.73, 123.09, 124.49 (C-3), 124.94 (C-2), 126.60,
127.45, 127.51, 128.36, 136.73, 137.89, 137.95, 211.99 (C-6). HRMS:
(API+) calculated for C30H41O ([M+H]+) 417.3157, Found 417.3161.
2.1.9. (22E)-23-(2-bromophenyl)-24-nor-5α-chola-2,22-dien-6-one (8h)
The general procedure with o-bromostyrene afforded 87 mg (59%)
of the title compound 13a as a colorless oil: IR ν (cm−1) 2944, 1699,
1654, 1593, 1564, 964. 1H NMR (CDCl3) δ 0.72, 0.75 (both s, 3H, CH3);
1.15 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.81 (m, 2H); 1.96–2.04 (m, 4H);
2.08 (dt, 1H, J = 12.5, J′ = 3.1 Hz); 2.22–2.38 (m, 4H); 5.57 (m, 1H,
H-3); 5.69 (m, 1H, H-2); 5.99 (dd, 1H, J = 15.6, J′ = 8.9 Hz, H-22);
6.63 (d, 1H, J = 15.6 Hz, H-23); 7.05 (m, 1H, ΣJ = 16.8 Hz, Ar-H);
7.23 (m, 1H, ΣJ = 16.2 Hz, Ar-H); 7.45 (dd, 1H, J = 7.6, J′ = 1.5 Hz,
Ar-H); 7.52 (m, 1H, ΣJ = 9.2 Hz, Ar-H). 13C NMR δ 12.24 (C-18), 13.51
3

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Steroidsxxx(xxxx)xxx–xxx
2.1.14. (22E)-23-(4-methylphenyl)-24-nor-5α-chola-2,22-dien-6-one
(8m)
2), 125.38, 125.40 (q, J = 3.6 Hz, 2×C), 126.04 (2×C), 126.35,
128.56 (q, J = 32.4 Hz), 139.66, 211.91 (C-6). 19F NMR {1H} δ -62.25
(s, 1F). HRMS: (API+) calculated for C30H38F3O ([M+H]+) 471.2875,
Found 471.2879.
The general procedure with p-methylstyrene afforded 91 mg (71%)
of the title compound 8 as a colorless oil. 1H NMR (CDCl3) δ 1H NMR
(CDCl3) δ 0.72, 0.73 (both s, 3H, CH3), 1.12 (d, 3H, J = 6.7 Hz, CH3),
1.70–1.79 (m, 2H), 1.96–2.04 (m, 4H), 2.07 (dt, 1H, J = 12.5,
J′ = 3.4 Hz), 2.22–2.30 (m, 2H), 2.32 (s, 3H, CH3), 2.33–2.37, (m, 2H),
5.57 (m, 1H, H-3), 5.69 (m, 1H, H-2), 6.00 (dd, 1H, J = 15.9,
J′ = 8.9 Hz, H-22), 6.27 (d, 1H, J = 15.9 Hz, H-23), 7.09 (d, 2H,
J = 7.9 Hz, 2×Ar-H), 7.22 (d, 2H, J = 7.9 Hz, 2×Ar-H). 13C NMR δ
12.19 (C-18), 13.49 (C-19), 20.42 (C-21), 21.09 (CH2, Ar-CH3), 21.70,
23.90, 28.19, 37.68, 39.34, 39.36, 40.04, 40.34, 42.88, 46.95, 53.39,
53.81, 55.89, 56.75, 124.50 (C-3), 124.95 (C-2), 125.80 (2×C), 127.21
(C-23), 129.13 (2×C), 135.18, 135.94 (C-22), 136.40, 212.00 (C-6).
Spectral data in agreement with literature [11].
2.1.18. (22E)-23-(2,4-dimethylphenyl)-24-nor-5α-chola-2,22-dien-6-one
(8q)
The general procedure with 2,4-dimethylstyrene and purification on
HPLC afforded 81 mg (61%) of the title compound 20a as a colorless
oil: IR ν (cm−1) 2935, 1701, 1652, 1593, 1560, 964. 1H NMR (CDCl3) δ
0.72, 0.74 (both s, 3H, CH3); 1.13 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.80
(m, 2H); 1.96–2.04 (m, 4H); 2.07 (dt, 1H, J = 12.5, J′ = 3.4 Hz);
2.22–2.37 (m, 4H); 2.28 (s, 6 H, 2×Ar-CH3); 5.57 (m, 1H, H-3); 5.69
(m, 1H, H-2); 5.85 (dd, 1H, J = 15.6, J′ = 8.9 Hz, H-22); 6.45 (d, 1H,
J = 15.6 Hz, H-23); 6.94 (s, 1H, Ar-H); 6.95 (d, 1H, J = 8.3 Hz, Ar-H);
7.27 (d, 1H, J = 8.3 Hz, Ar-H). 13C NMR δ 12.20 (C-18), 13.48 (C-19),
19.76 (Ar-CH3), 20.57 (C-21), 20.97 (Ar-CH3), 21.08, 21.70, 23.92,
28.28, 37.66, 39.32, 39.35, 40.02, 40.68, 42.85, 46.93, 53.37, 53.79,
55.78, 56.75, 124.48 (C-3), 124.93 (C-2), 125.09, 125.38, 126.64,
130.84, 134.29, 134.77, 136.28, 137.58, 211.98 (C-6). HRMS: (API+)
calculated for C31H43O ([M+H]+) 431.3314, Found 431.3318.
2.1.15. (22E)-23-(2-trifluoromethylphenyl)-24-nor-5α-chola-2,22-dien-6-
one (8n)
The general procedure with o-trifluoromethylstyrene afforded
82 mg (57%) of the title compound 17a as a colorless oil: IR ν (cm−1
)
2942, 1701, 1653, 1593, 1560, 1128, 964. 1H NMR (CDCl3) δ 0.73,
0.76 (both s, 3H, CH3); 1.15 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.81 (m,
2H); 1.96–2.04 (m, 4H); 2.07 (dt, 1H, J = 12.6, J′ = 3.6 Hz); 2.23–2.31
(m, 2H); 2.31–2.38 (m, 2H); 5.58 (m, 1H, H-3); 5.69 (m, 1H, H-2); 6.00
(dd, 1H, J = 15.6, J′ = 8.9 Hz, H-22); 6.68 (qd, 1H, J = 15.6,
J′ = 2.1 Hz, H-23); 7.29 (t, 1H, J = 7.7 Hz, Ar-H); 7.46 (t, 1H,
J = 7.7 Hz, Ar-H); 7.55 (d, 1H, J = 7.7 Hz, Ar-H); 7.60 (d, 1H,
J = 7.7 Hz, Ar-H). 13C NMR δ 12.19 (C-18), 13.48 (C-19), 20.17 (C-21),
21.08, 21.70, 23.94, 28.02, 37.63, 39.32 (2×C), 40.00, 40.59, 42.95,
46.92, 53.37, 53.80, 55.68, 56.69, 123.72, 124.38 (q, J = 273.5 Hz),
124.47 (C-3), 124.93 (C-2), 125.58 (q, J = 6.0 Hz), 126.44, 126.92 (q,
J = 30.0 Hz), 127.28, 131.65, 137.26, 141.19, 211.90 (C-6). 19F NMR
{1H} δ -59.56 (s, 1F). HRMS: (API+) calculated for C30H38F3O ([M
+H]+) 471.2875, Found 471.2876.
2.1.19. (22E)-23-(2,5-dimethylphenyl)-24-nor-5α-chola-2,22-dien-6-one
(8r)
The general procedure with 2,5-dimethylstyrene and purification on
HPLC afforded 77 mg (58%) of the title compound 21a as a colorless
oil: IR ν (cm−1) 2931, 1699, 1653, 1593, 1560, 963. 1H NMR (CDCl3) δ
0.72, 0.74 (both s, 3H, CH3); 1.13 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.80
(m, 2H); 1.96–2.04 (m, 4H); 2.08 (dt, 1H, J = 12.5, J′ = 3.3 Hz);
2.22–2.31 (m, 2H); 2.27, 2.30 (both s, 3H, Ar-CH3); 2.32–2.37 (m, 2H);
5.57 (m, 1H, H-3); 5.69 (m, 1H, H-2); 5.88 (dd, 1H, J = 15.6,
J′ = 8.9 Hz, H-22); 6.46 (d, 1H, J = 15.6 Hz, H-23); 6.92 (dd, 1H,
J = 7.6, J′ = 1.2 Hz, Ar-H); 7.00 (d, 1H, J = 7.6 Hz, Ar-H); 7.18 (b s,
1H, Ar-H). 13C NMR δ 12.20 (C-18), 13.48 (C-19), 19.36 (Ar-CH3),
20.57 (C-21), 21.00 (Ar-CH3), 21.09, 21.70, 23.92, 28.31, 37.66, 39.33,
39.37, 40.02, 40.73, 42.87, 46.93, 53.37, 53.80, 55.74, 56.77, 124.48
(C-3), 124.94 (C-2), 125.37, 126.07, 127.43, 130.01, 131.89, 135.23,
2.1.16. (22E)-23-(3-trifluoromethylphenyl)-24-nor-5α-chola-2,22-dien-6-
one (8o)
The general procedure with m-trifluoromethylstyrene afforded
136.88, 138.12, 211.95 (C-6). HRMS: (API+) calculated for C31H43
O
95 mg (66%) of the title compound 18 as a colorless oil: IR ν (cm−1
)
([M+H]+) 431.3314, Found 431.3318.
2940, 1701, 1655, 1593, 1561, 1124, 964. 1H NMR (CDCl3) δ 0.72,
0.74 (both s, 3H, CH3); 1.14 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.80 (m,
2H); 1.97–2.05 (m, 4H); 2.07 (dt, 1H, J = 12.6, J′ = 3.3 Hz); 2.22–2.32
(m, 2H); 2.34–2.37 (m, 2H); 5.58 (m, 1H, H-3); 5.69 (m, 1H, H-2); 6.14
(dd, 1H, J = 15.7, J′ = 8.7 Hz, H-22); 6.34 (d, 1H, J = 15.7 Hz, H-23);
7.37–7.44 (m, 2H, 2×Ar-H); 7.48 (d, 1H, J = 7.3 Hz, Ar-H), 7.56 (b s,
1H, Ar-H). 13C NMR δ 12.19 (C-18), 13.50 (C-19), 20.24 (C-21), 21.09,
21.70, 23.91, 28.23, 37.66, 39.36, 39.37, 40.03, 40.48, 42.96, 46.93,
53.37, 53.82, 55.64, 56.71, 122.52 (q, J = 3.6 Hz), 123.26 (q,
J = 3.6 Hz), 124.19 (q, J = 272.3 Hz), 124.48 (C-3), 124.96 (C-2),
126.27, 128.85, 129.14, 130.81 (q, J = 32.4 Hz), 138.71, 138.90,
211.92 (C-6). 19F NMR {1H} δ -62.59 (s, 1F). HRMS: (API+) calculated
for C30H38F3O ([M+H]+) 471.2875, Found 471.2879.
2.1.20. (22E)-23-(4-t-butylphenyl)-24-nor-5α-chola-2,22-dien-6-one (8s)
The general procedure with p-t-butylstyrene and purification on
HPLC afforded 84 mg (60%) of the title compound 22a as a colorless
oil: IR ν (cm−1) 2939, 1702, 1651, 1593, 1564, 1372, 963. 1H NMR
(CDCl3) δ 0.73, 0.74 (both s, 3H, CH3); 1.13 (d, 3H, J = 6.7 Hz, CH3);
1.32 (s, 9 H, t-Bu); 1.71–1.80 (m, 2H); 1.97–2.05 (m, 4H); 2.08 (dt, 1H,
J = 12.7, J′ = 3.4 Hz); 2.23–2.32 (m, 2H); 2.35–2.38 (m, 2H); 5.59 (m,
1H, H-3); 5.70 (m, 1H, H-2); 6.03 (dd, 1H, J = 15.7, J′ = 8.7 Hz, H-
22); 6.30 (d, 1H, J = 15.7 Hz, H-23); 7.27–7.29 (m, 2H, 2×Ar-H);
7.32–7.34 (m, 2H, 2×Ar-H). 13C NMR δ 12.18 (C-18), 13.48 (C-19),
20.46 (C-21), 21.08, 21.70, 23.90, 28.17, 31.28 (3×C, t-Bu), 37.66,
39.32, 39.35, 40.02, 40.39, 42.86, 46.94, 53.38, 53.80, 55.84, 56.74,
124.48 (C-3), 124.94 (C-2), 125.35 (2×C), 125.60 (2×C), 127.09,
135.17, 136.17, 149.72, 211.97 (C-6). HRMS: (API+) calculated for
2.1.17. (22E)-23-(4-trifluoromethylphenyl)-24-nor-5α-chola-2,22-dien-6-
one (8p)
C
33H47O ([M+H]+) 459.3627, Found 459.3629.
The general procedure with p-trifluoromethylstyrene afforded
102 mg (71%) of the title compound 19a as a colorless oil: IR ν (cm−1
)
2.1.21. (22E)-23-(4-phenylphenyl)-24-nor-5α-chola-2,22-dien-6-one (8t)
The general procedure with p-phenylstyrene afforded 47 mg (32%)
of the title compound 23a as a colorless oil: IR ν (cm−1) 2949, 1700,
1654, 1593, 1565, 1465, 960. 1H NMR (CDCl3) δ 0.72, 0.75 (both s, 3H,
CH3); 1.15 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.80 (m, 2H); 1.96–2.04 (m,
4H); 2.08 (dt, 1H, J = 12.5, J′ = 3.1 Hz); 2.22–2.31 (m, 2H); 2.33–2.37
(m, 2H); 5.57 (m, 1H, H-3); 5.69 (m, 1H, H-2); 6.11 (dd, 1H, J = 15.7,
J′ = 8.7 Hz, H-22); 6.34 (d, 1H, J = 15.7 Hz, H-23); 7.33 (m, 1H, Ar-
H); 7.39–7.44 (m, 4H, 4×Ar-H); 7.52–7.54 (m, 2H, 2×Ar-H);
7.57–7.60 (m, 2H, 2×Ar-H). 13C NMR δ 12.20 (C-18), 13.50 (C-19),
2940, 1702, 1653, 1593, 1562, 1127, 963. 1H NMR (CDCl3) δ 0.72,
0.74 (both s, 3H, CH3); 1.15 (d, 3H, J = 6.7 Hz, CH3); 1.71–1.81 (m,
2H); 1.96–2.05 (m, 4H); 2.07 (dt, 1H, J = 12.5, J′ = 3.4 Hz); 2.21–2.31
(m, 2H); 2.34–2.37 (m, 2H); 5.58 (m, 1H, H-3); 5.69 (m, 1H, H-2); 6.17
(dd, 1H, J = 15.7, J′ = 8.7 Hz, H-22); 6.34 (d, 1H, J = 15.7 Hz, H-23);
7.41 (d, 2H, J = 8.4 Hz, 2×Ar-H); 7.53 (d, 2H, J = 8.4 Hz, 2×Ar-H).
13C NMR δ 12.21 (C-18), 13.51 (C-19), 20.19 (C-21), 21.10, 21.70,
23.92, 28.19, 37.67, 39.34, 39.37, 40.04, 40.49, 42.99, 46.94, 53.38,
53.83, 55.85, 56.70, 124.28 (q, J = 271.5 Hz), 124.48 (C-3), 124.97 (C-
4

P. Korinkova et al.
Steroidsxxx(xxxx)xxx–xxx
20.39 (C-21), 21.10, 21.70, 23.92, 28.21, 37.68, 39.34, 39.37, 40.04,
40.47, 42.93, 46.95, 53.39, 53.82, 55.84, 56.74, 124.50 (C-3), 124.96
(C-2), 126.32 (2×C), 126.86 (2×C), 126.98, 127.12, 127.16 (2×C),
128.73 (2×C), 137.04, 137.15, 139.51, 140.83, 211.98 (C-6). HRMS:
(API+) calculated for C35H43O ([M+H]+) 479.3314, Found 479.3317.
2.2.2. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(3-fluorophenyl)-24-nor-
5α-cholan-6-one (10c)
The general procedure for dihydroxylation of 8c (60 mg;
0.14 mmol) afforded 59 mg (85%) of the title compound 10c as a white
solid: m. p. 278–279 °C (EtOH); IR ν (cm−1) 3334 vb, 2939, 1710,
1615, 1591, 1461, 763. 1H NMR (DMSO-d6) δ 0.34 (H-18), 0.59 (H-19)
(both s, 3H, CH3); 0.86 (d, 3H, J = 6.4 Hz, H-21, CH3); 1.80 (m, 1H);
1.86 (m, 1H); 1.98 (dd, 1H, J = 12.8, J′ = 4.4 Hz); 2.07 (t, 1H,
J = 12.8 Hz); 2.57 (dd, 1H, J = 12.0, J′ = 3.0 Hz); 3.43–3.48 (m, 2H,
H-2, H-3); 3.74 (m, 1H, H-22); 4.18 (d, 1H, J = 2.4 Hz, OH); 4.32 (d,
1H, J = 6.1 Hz, OH); 4.39 (dd, 1H, J = 7.9, J′ = 3.4 Hz, H-23); 4.57
(d, 1H, J = 4.3 Hz, OH); 5.24 (d, 1H, J = 4.0 Hz, OH); 7.04–7.12 (m,
3H); 7.35 (m, 1H). 13C NMR δ 11.38 (C-18), 12.53 (C-19), 13.35 (C-21),
20.78, 23.28, 26.81, 27.25 36.48, 37.01, 39.08, 40.09, 41.83, 42.05,
45.94, 50.28, 51.88, 52.85, 55.94, 67.09 (C-2), 67.48 (C-3), 74.56 (C-
23), 76.17 (C-22), 113.46 (d, J = 21.6 Hz), 113.88 (d, J = 21.6 Hz),
123.05, 129.96 (d, J = 8.4 Hz), 146.53 (d, J = 7.2 Hz), 162.04 (d,
J = 242.3 Hz, C-F), 211.54 (C-6). 19F NMR {1H} δ -113.41 (s, 1F).
HRMS: (API+) calculated for C29H42FO5 ([M+H]+) 489.3016, Found
489.3017. Anal. Calcd for C29H41FO5: C, 71.28; H, 8.46. Found: C,
71.26; H, 8.49%.
2.1.22. (22E)-23-(2-isopropoxyphenyl)-24-nor-5α-chola-2,22-dien-6-one
(9)
The general procedure with different bis-ortho substituted styrene or
without styrene afforded 20 mg (14%) of the title compound 9 as a
colorless oil: IR ν (cm−1) 2940, 1701, 1653, 1591, 1561, 963. 1H NMR
(CDCl3) δ 0.72, 0.74 (both s, 3H, CH3); 1.14 (d, 3H, J = 6.7 Hz, CH3);
1.34 (d, 3H, J = 6.1 Hz, CH(CH3)2); 1.35 (d, 3H, J = 6.1 Hz, CH
(CH3)2); 1.71–1.81 (m, 2H); 1.96–2.03 (m, 4H); 2.08 (dt, 1H, J = 12.5,
J′ = 3.2 Hz); 2.22–2.31 (m, 2H); 2.33–2.37 (m, 2H); 4.49 (septet, 1H,
J = 6.1 Hz, OCH(CH3)2); 5.58 (m, 1H, H-3); 5.69 (m, 1H, H-2); 6.03
(dd, 1H, J = 15.9, J′ = 8.9 Hz, H-22); 6.62 (d, 1H, J = 15.9 Hz, H-23);
6.85–6.90 (m, 2H); 7.13 (m, 1H); 7.40 (dd, 1H, J = 7.6, J′ = 1.5 Hz).
13C NMR δ 12.21 (C-18), 13.50 (C-19), 20.51 (C-21), 21.10, 21.70,
22.25 (2×C, CH(CH3)2), 23.94, 28.21, 37.69, 39.34, 39.37, 40.05,
40.70, 42.89, 46.97, 53.41, 53.82, 55.94, 56.77, 71.04 (OCH(CH3)2),
114.70, 120.71, 122.32, 124.51 (C-3), 124.94 (C-2), 126.31, 127.49,
128.43, 137.13, 154.79, 212.05 (C-6). HRMS: (API+) calculated for
2.2.3. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(2-chlorophenyl)-24-nor-
5α-cholan-6-one (10e)
C
32H45O2 ([M+H]+) 461.3420, Found 461.3423.
The general procedure for dihydroxylation of 8e (60 mg;
0.14 mmol) afforded 54 mg (78%) of the title compound 10e as a white
solid: m. p. 290–292 °C (EtOH); IR ν (cm−1) 3307 vb, 2936, 1693,
1595, 1572, 1463, 760. 1H NMR (DMSO-d6) δ 0.38 (H-18), 0.60 (H-19)
(both s, 3H, CH3); 0.93 (d, 3H, J = 6.7 Hz, H-21, CH3); 1.79 (m, 1H);
1.89 (m, 1H); 1.98 (dd, 1H, J = 13.4, J′ = 4.7 Hz); 2.07 (t, 1H,
J = 12.3 Hz); 2.58 (dd, 1H, J = 12.3, J′ = 3.4 Hz); 3.45 (m, 1H, H-2);
3.58 (bd, 1H, J = 8.0 Hz, H-3); 3.74 (m, 1H, H-22); 4.18 (bd, 1H,
J = 2.4 Hz, OH); 4.31 (d, 1H, J = 6.1 Hz, OH); 4.58 (bd, 1H,
J = 3.4 Hz, H-23); 4.79 (d, 1H, J = 7.3 Hz, OH); 5.28 (bs, 1H, OH);
7.26 (m, 1H); 7.34 (td, 1H, J = 7.5, J′ = 1.2 Hz); 7.39 (dd, 1H, J = 7.9,
J′ = 1.2 Hz); 7.44 (dd, 1H, J = 7.8, J′ = 1.7 Hz). 13C NMR δ 11.37 (C-
18), 13.36 (C-19), 13.39 (C-21), 20.76, 23.33, 26.81, 27.40, 37.01,
37.36, 39.04, 40.10, 41.82, 42.08, 45.93, 50.26, 52.13, 52.84, 55.93,
67.09 (C-2), 67.47 (C-3), 71.00 (C-23), 75.66 (C-22), 127.19, 128.65,
128.92, 129.13, 131.92, 141.13, 211.54 (C-6). HRMS: (API+) calcu-
lated for C29H42ClO5 ([M+H]+) 505.2721, Found 505.2720. Anal.
Calcd for C29H41ClO5: C, 68.96; H, 8.18. Found: C, 68.93; H, 8.19%.
2.2. General procedure for dihydroxylation of dienes
To a solution of diene (0.2 mmol); hydroquinidine 4-chlorobenzoate
(23 mg; 0.049 mmol); methansulfonamide (33 mg; 0.35 mmol); po-
tassium carbonate (140 mg; 1.01 mmol); and potassium ferricyanide
(350 mg; 1.06 mmol) in the mixture of t-butanol and water (15 mL;
1:1 v/v) was added 0.1 mL of osmium tetroxide in t-butanol (1 g/
20 mL; 0.02 mmol). Reaction mixture was stirred at room temperature
for 24 h. A saturated solution of sodium sulfite (3 mL) was then added.
After an additional 30 min of stirring, the reaction mixture was diluted
with ethyl acetate (30 mL) and extracted with water (2 × 20 mL). The
combined organic fractions were dried over anhydrous magnesium
sulfate and evaporated under reduced pressure. Column chromato-
graphy on silica gel (mobile phase – MeOH/CHCl3 – 1/16) gave the
desired product.
Compounds 10a (m. p. 269–271 °C, EtOH), 10d (m. p. 276–278 °C,
EtOH), 10g (m. p. 251–253 °C, EtOH), 10j (m. p. 248–249 °C, i-PrOH),
and 10m (m. p. 271–273 °C, EtOH) were prepared according to general
procedure which is also described in literature [11]. All data for these
compounds are in agreement with same literature.
2.2.4. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(3-chlorophenyl)-24-nor-
5α-cholan-6-one (10f)
The general procedure for dihydroxylation of 8f (60 mg;
0.14 mmol) afforded 56 mg (80%) of the title compound 10f as a white
solid: m. p. 269–270 °C (EtOH); IR ν (cm−1) 3257 vb, 2936, 1709,
1600, 1576, 1461, 781, 699. 1H NMR (DMSO-d6) δ 0.35 (H-18), 0.60
(H-19) (both s, 3H, CH3); 0.86 (d, 3H, J = 6.7 Hz, H-21, CH3); 1.79 (m,
1H); 1.87 (m, 1H); 1.96 (dd, 1H, J = 13.4, J′ = 4.6 Hz); 2.07 (t, 1H,
J = 12.3 Hz); 2.58 (dd, 1H, J = 12.3, J′ = 3.2 Hz); 3.43–3.46 (m, 2H,
H-2, H-3); 3.74 (m, 1H, H-22); 4.18 (bd, 1H, J = 2.1 Hz, OH); 4.33 (d,
1H, J = 5.8 Hz, OH); 4.38 (bd, 1H, J = 8.3 Hz, H-23); 4.59 (d, 1H,
J = 3.4 Hz, OH); 5.26 (bs, 1H, OH); 7.23 (m, 1H); 7.29–7.31 (m, 2H);
7.35 (t, 1H, J = 7.6 Hz). 13C NMR δ 11.44 (C-18), 12.58 (C-19), 13.39
(C-21), 20.81, 23.33, 26.85, 27.31, 36.59, 37.06, 39.12, 40.11, 41.88,
42.09, 45.98, 50.33, 51.93, 52.90, 55.98, 67.13 (C-2), 67.52 (C-3),
74.56 (C-23), 76.16 (C-22), 125.73, 126.77, 127.12, 130.01, 132.75,
146.12, 211.64 (C-6). HRMS: (API+) calculated for C29H42ClO5 ([M
+H]+) 505.2721, Found 505.2716. Anal. Calcd for C29H41ClO5: C,
68.96; H, 8.18. Found: C, 68.95; H, 8.23%.
2.2.1. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(2-fluorophenyl)-24-nor-
5α-cholan-6-one (10b)
The general procedure for dihydroxylation of 8b (60 mg;
0.14 mmol) afforded 54 mg (77%) of the title compound 10b as a white
solid: m. p. 288–290 °C (EtOH); IR ν (cm−1) 3348 vb, 2941, 1710,
1620, 1587, 1492, 757. 1H NMR (DMSO-d6) δ 0.34 (H-18), 0.60 (H-19)
(both s, 3H, CH3); 0.86 (d, 3H, J = 6.4 Hz, H-21, CH3); 1.80 (m, 1H);
1.87 (m, 1H); 1.98 (m, 1H); 2.07 (t, 1H, J = 12.6 Hz); 2.58 (d, 1H,
J = 12.4 Hz); 3.44 (m, 1H, H-2); 3.58 (m, 1H, H-3); 3.74 (m, 1H, H-22);
4.18 (br s, 1H, OH); 4.31 (d, 1H, J = 5.8 Hz, OH); 4.62 (d, 1H,
J = 1.8 Hz, OH); 4.66 (d, 1H, J = 8.3 Hz, H-23); 5.25 (br s, 1H, OH);
7.12 (m, 1H); 7.18 (m, 1H); 7.29 (m, 1H); 7.38 (m, 1H). 13C NMR δ
11.32 (C-18), 12.51 (C-19), 13.30 (C-21), 20.73, 23.27, 26.73, 27.21
36.77, 36.98, 39.06, 40.10, 41.79, 42.00, 45.94, 50.25, 51.89, 52.86,
55.93, 67.08 (C-2), 67.45 (C-3), 68.48 (C-23), 75.65 (C-22), 114.92 (d,
J = 22.8 Hz), 124.31 (d, J = 3.6 Hz), 128.82 (d, J = 3.6 Hz), 128.87,
130.01 (d, J = 14.2 Hz), 159.61 (d, J = 243.5 Hz, C-F), 211.39 (C-6).
19F NMR {1H} δ −118.49 (s, 1F). HRMS: (API+) calculated for
2.2.5. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(2-bromophenyl)-24-nor-
5α-cholan-6-one (10h)
C
C
29H42FO5 ([M+H]+) 489.3016, Found 489.3017. Anal. Calcd for
29H41FO5: C, 71.28; H, 8.46. Found: C, 71.30; H, 8.45%.
The general procedure for dihydroxylation of 8h (60 mg;
5

P. Korinkova et al.
Steroidsxxx(xxxx)xxx–xxx
0.13 mmol) afforded 55 mg (81%) of the title compound 10h as a white
solid: m. p. 288–289 °C (EtOH); IR ν (cm−1) 3313 vb, 2944, 1699,
1593, 1564, 1471, 786, 682. 1H NMR (DMSO-d6) δ 0.39 (H-18), 0.60
(H-19) (both s, 3H, CH3); 0.95 (d, 3H, J = 6.7 Hz, H-21, CH3); 1.78(m,
1H); 1.89 (m, 1H); 1.98 (dd, 1H, J = 13.1, J′ = 4.6 Hz); 2.07 (t, 1H,
J = 12.3 Hz); 2.58 (dd, 1H, J = 12.3, J′ = 3.2 Hz); 3.46 (m, 1H, H-2);
3.58 (bd, 1H, J = 7.0 Hz, H-3); 3.74 (m, 1H, H-22); 4.20 (d, 1H,
J = 1.8 Hz, OH); 4.34 (d, 1H, J = 5.8 Hz, OH); 4.58 (m, 1H, H-23);
4.73 (d, 1H, J = 7.0 Hz, OH); 5.31 (bs, 1H, OH); 7.18 (m, 1H); 7.38 (m,
1H); 7.42 (dd, 1H, J = 7.9, J′ = 1.8 Hz); 7.55 (dd, 1H J = 7.9,
J′ = 1.2 Hz). 13C NMR δ 11.45 (C-18), 13.40 (C-19), 13.69 (C-21),
20.81, 23.40, 26.86, 27.47, 37.07, 37.60, 39.10, 40.10, 41.89, 42.15,
45.98, 50.34, 52.22, 52.89, 55.97, 67.13 (C-2), 67.52 (C-3), 73.47 (C-
23), 75.65 (C-22), 122.72, 127.76, 129.08, 129.46, 132.26, 142.83,
2.28 (s, 3H, CH3); 2.58 (dd, 1H, J = 12.1, J′ = 3.2 Hz); 3.44–3.49 (m,
2H, H-2, H-3); 3.74 (m, 1H, H-22); 4.18 (d, 1H, J = 2.1 Hz, OH);
4.30–4.33 (m, 2H, H-23, OH); 4.46 (d, 1H, J = 4.0 Hz, OH); 5.03 (d,
1H, J = 3.4 Hz, OH); 7.03–7.05 (m, 2H); 7.07 (s, 1H); 7.19 (t, 1H,
J = 7.5 Hz). 13C NMR δ 11.45 (C-18), 12.51 (C-19), 13.37 (C-21), 18.59
(Ar-CH3), 20.80, 23.32, 26.83, 27.23, 36.35, 37.04, 39.12, 40.10,
41.86, 42.08, 45.97, 50.31, 51.91, 52.88, 55.97, 67.11 (C-2), 67.50 (C-
3), 75.15 (C-23), 76.21 (C-22), 124.15, 127.67, 127.83, 127.94, 136.99,
143.20, 211.62 (C-6). HRMS: (API+) calculated for C30H45O5 ([M
+H]+) 485.3267, Found 485.3269. Anal. Calcd for C30H44O5: C, 74.34;
H, 9.15. Found: C, 74.31; H, 9.16%.
2.2.9. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(2-trifluoromethylphenyl)-24-
nor-5α-cholan-6-one (10n)
211.68 (C-6). HRMS: (API+) calculated for C29H4279BrO5 ([M+H]+
)
The general procedure for dihydroxylation of 8n (60 mg;
0.13 mmol) afforded 50 mg (73%) of the title compound 10n as a white
solid: m. p. 268–270 °C (EtOH); IR ν (cm−1) 3418 vb, 2939, 1701,
1613, 1589, 1315, 1114, 772. 1H NMR (DMSO-d6) δ 0.38 (H-18), 0.60
(H-19) (both s, 3H, CH3); 0.87 (d, 3H, J = 6.4 Hz, H-21, CH3); 1.82 (m,
1H); 1.88 (m, 1H); 1.99 (dd, 1H, J = 13.1, J′ = 4.6 Hz); 2.07 (t, 1H,
J = 12.6 Hz); 2.58 (dd, 1H, J = 12.2, J′ = 3.1 Hz); 3.45 (m, 1H, H-2);
3.66 (m, 1H, H-3); 3.74 (bs, 1H, H-22); 4.18 (d, 1H, J = 2.1 Hz, OH);
4.31 (d, 1H, J = 6.1 Hz, OH); 4.63 (d, 1H, J = 4.3 Hz, H-23); 4.70 (d,
1H, J = 4.6 Hz, OH); 5.32 (bs, 1H, OH); 7.46 (m, 1H); 7.65–7.70 (m,
3H). 13C NMR δ 11.31 (C-18), 12.83 (C-19), 13.35 (C-21), 20.75, 23.34,
26.81, 27.38, 37.00, 37.77, 39.04, 40.10, 41.82, 42.11, 45.91, 50.28,
52.32, 52.84, 55.91, 67.09 (C-2), 67.47 (C-3), 69.76 (C-23), 75.19 (C-
22), 124.49 (q, J = 274.7 Hz, CF3), 126.26 (q, J = 28.8 Hz), 125.21 (q,
J = 5.0 Hz), 127.59, 129.38, 132.39, 142.56, 211.54 (C-6). 19F NMR
{1H} δ -55.95 (s, 1F). HRMS: (API+) calculated for C30H42F3O5 ([M
+H]+) 539.2984, Found 539.2986. Anal. Calcd for C30H41F3O5: C,
66.89; H, 7.67. Found: C, 66.88; H, 7.69%.
549.2216, Found 549.2211. Anal. Calcd for C29H41BrO5: C, 63.38; H,
7.52. Found: C, 63.35; H, 7.56%.
2.2.6. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(3-bromophenyl)-24-nor-
5α-cholan-6-one (10i)
The general procedure for dihydroxylation of 8i (60 mg; 0.13 mmol)
afforded 54 mg (79%) of the title compound 10i as a white solid: m. p.
264–265 °C (EtOH); IR ν (cm−1) 3242 vb, 2935, 1709, 1596, 1572,
1460, 782, 700. 1H NMR (DMSO-d6) δ 0.35 (H-18), 0.60 (H-19) (both s,
3H, CH3); 0.86 (d, 3H, J = 6.7 Hz, H-21, CH3); 1.79 (m, 1H); 1.87 (m,
1H); 1.98 (dd, 1H, J = 13.1, J′ = 4.8 Hz); 2.07 (t, 1H, J = 12.3 Hz);
2.58 (dd, 1H, J = 12.3, J′ = 3.2 Hz); 3.42–3.46 (m, 2H, H-2, H-3); 3.74
(m, 1H, H-22); 4.19 (bs, 1H, OH); 4.33 (bd, 1H, J = 5.5 Hz, OH); 4.37
(bd, 1H, J = 8.3 Hz, H-23); 4.59 (bs, 1H, OH); 5.26 (bs, 1H, OH);
7.26–7.30 (m, 2H); 7.43 (m, 1H); 7.45 (m, 1H). 13C NMR δ 11.44 (C-
18), 12.58 (C-19), 13.38 (C-21), 20.80, 23.32, 26.84, 27.30, 36.60,
37.05, 39.11, 40.10, 41.87, 42.09, 45.97, 50.32, 51.93, 52.89, 55.97,
67.12 (C-2), 67.51 (C-3), 74.52 (C-23), 76.15 (C-22), 121.39, 126.10,
129.69, 130.00, 130.33, 146.38, 211.62 (C-6). HRMS: (API+) calcu-
lated for C29H4279BrO5 ([M+H]+) 549.2216, Found 549.2211. Anal.
Calcd for C29H41BrO5: C, 63.38; H, 7.52. Found: C, 63.32; H, 7.59%.
2.2.10. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(3-trifluoromethylphenyl)-
24-nor-5α-cholan-6-one (10o)
The general procedure for dihydroxylation of 8o (60 mg;
0.13 mmol) afforded 53 mg (77%) of the title compound 10o as a white
solid: m. p. 243–245 °C (EtOH); IR ν (cm−1) 3257 vb, 2934, 1712,
1618, 1457, 1329, 1113, 706. 1H NMR (DMSO-d6) δ 0.32 (H-18), 0.59
(H-19) (both s, 3H, CH3); 0.86 (d, 3H, J = 6.4 Hz, H-21, CH3); 1.79 (m,
1H); 1.86 (m, 1H); 1.97 (dd, 1H, J = 13.1, J′ = 4.6 Hz); 2.07 (t, 1H,
J = 12.6 Hz); 2.57 (dd, 1H, J = 12.2, J′ = 3.3 Hz); 3.42–3.49 (m, 2H,
H-2, H-3); 3.74 (bs, 1H, H-22); 4.19 (d, 1H, J = 1.8 Hz, OH); 4.33 (d,
1H, J = 5.5 Hz, OH); 4.50 (d, 1H, J = 7.9 Hz, OH); 4.65 (bs, 1H, H-23);
5.35 (bs, 1H, OH); 7.54–7.62 (m, 4H). 13C NMR δ 11.30 (C-18), 12.59
(C-19), 13.36 (C-21), 20.78, 23.30, 26.83, 27.22, 36.54, 37.01, 39.08,
40.10, 41.84, 42.09, 45.94, 50.31, 51.93, 52.86, 55.94, 67.11 (C-2),
67.50 (C-3), 74.52 (C-23), 76.23 (C-22), 123.38 (q, J = 4.0 Hz), 123.84
(q, J = 4.0 Hz), 124.37 (q, J = 272.3 Hz, CF3), 128.77 (q,
J = 31.8 Hz), 129.14, 131.12, 145.01, 211.59 (C-6). 19F NMR {1H} δ
2.2.7. (22R,
nor-5α-cholan-6-one (10k)
23R)-2α,3α,22,23-tetrahydroxy-23-(2-methylphenyl)-24-
The general procedure for dihydroxylation of 8k (60 mg;
0.14 mmol) afforded 53 mg (76%) of the title compound 10k as a white
solid: m. p. 269–271 °C (EtOH); IR ν (cm−1) 3300 vb, 2942, 1706,
1631, 1604, 1461, 981, 759. 1H NMR (DMSO-d6) δ 0.29 (H-18), 0.55
(H-19) (both s, 3H, CH3); 0.81 (d, 3H, J = 6.4 Hz, H-21, CH3); 1.76 (m,
1H); 1.83 (m, 1H); 1.97 (dd, 1H, J = 13.1, J′ = 4.9 Hz); 2.02 (t, 1H,
J = 12.3 Hz); 2.26 (s, 3H, CH3); 2.58 (dd, 1H, J = 12.3, J′ = 3.2 Hz);
3.40 (m, 1H, H-2); 3.59 (dd, 1H, J = 8.1, J′ = 2.6 Hz, H-3); 3.69 (m,
1H, H-22); 4.13 (d, 1H, J = 2.1 Hz, OH); 4.27 (d, 1H, J = 5.8 Hz, OH);
4.44 (d, 1H, J = 4.0 Hz, OH); 4.52 (dd, 1H, J = 8.3, J′ = 1.8 Hz, H-
23); 4.94 (d, 1H, J = 3.4 Hz, OH); 7.06–7.08 (m, 2H); 7.10 (m, 1H);
7.20 (d, 1H, J = 7.3 Hz). 13C NMR δ 11.42 (C-18), 13.25 (C-19), 13.37
(C-21), 19.32 (Ar-CH3), 20.79, 23.35, 26.83, 27.35, 36.79, 37.04,
39.07, 40.10, 41.86, 42.09, 45.96, 50.31, 52.21, 52.87, 55.95, 67.11 (C-
2), 67.50 (C-3), 71.41 (C-23), 75.50 (C-22), 125.74, 126.82, 127.38,
130.15, 135.06, 141.42, 211.61 (C-6). HRMS: (API+) calculated for
-60.97 (s, 1F). HRMS: (API+) calculated for C30H42F3O5 ([M+H]+
)
539.2984, Found 539.2985. Anal. Calcd for C30H41F3O5: C, 66.89; H,
7.67. Found: C, 66.85; H, 7.71%.
2.2.11. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-trifluoromethylphenyl)-
24-nor-5α-cholan-6-one (10p)
C
C
30H45O5 ([M+H]+) 485.3267, Found 485.3268. Anal. Calcd for
30H44O5: C, 74.34; H, 9.15. Found: C, 74.29; H, 9.19%.
The general procedure for dihydroxylation of 8p (60 mg;
0.13 mmol) afforded 54 mg (78%) of the title compound 10p as a white
solid: m. p. 265–266 °C (EtOH); IR ν (cm−1) 3224 vb, 2937, 1710,
1621, 1461, 1331, 1121, 838. 1H NMR (DMSO-d6) δ 0.33 (H-18), 0.59
(H-19) (both s, 3H, CH3); 0.86 (d, 3H, J = 6.4 Hz, H-21, CH3); 1.80 (m,
1H); 1.86 (m, 1H); 1.98 (dd, 1H, J = 13.1, J′ = 4.6 Hz); 2.07 (t, 1H,
J = 12.6 Hz); 2.58 (dd, 1H, J = 12.2, J′ = 3.1 Hz); 3.44 (m, 1H, H-2);
3.50 (d, 1H, J = 7.6 Hz, H-3); 3.74 (bs, 1H, H-22); 4.19 (bs, 1H, OH);
4.33 (d, 1H, J = 4.6 Hz, OH); 4.47 (d, 1H, J = 8.3 Hz, H-23); 4.66 (bs,
1H, OH); 5.34 (bs, 1H, OH); 7.50 (d, 2H, J = 8.1 Hz); 7.68 (d, 2H,
2.2.8. (22R,
nor-5α-cholan-6-one (10l)
23R)-2α,3α,22,23-tetrahydroxy-23-(3-methylphenyl)-24-
The general procedure for dihydroxylation of 8l (60 mg; 0.14 mmol)
afforded 55 mg (79%) of the title compound 10l as a white solid: m. p.
276–278 °C (EtOH); IR ν (cm−1) 3315 vb, 2938, 1708, 1611, 1593,
1460, 988, 707. 1H NMR (DMSO-d6) δ 0.32 (H-18), 0.60 (H-19) (both s,
3H, CH3); 0.85 (d, 3H, J = 6.4 Hz, H-21, CH3); 1.80 (m, 1H); 1.87 (m,
1H); 1.98 (dd, 1H, J = 13.2, J′ = 4.9 Hz); 2.07 (t, 1H, J = 12.6 Hz);
6

P. Korinkova et al.
Steroidsxxx(xxxx)xxx–xxx
J = 8.1 Hz). 13C NMR δ 11.41 (C-18), 12.57 (C-19), 13.36 (C-21),
20.80, 23.29, 26.83, 27.26, 36.61, 37.03, 39.11, 40.10, 41.85, 42.07,
45.96, 50.31, 51.92, 52.87, 55.95, 67.11 (C-2), 67.50 (C-3), 74.68 (C-
23), 76.10 (C-22), 124.40 (q, J = 271.9 Hz, CF3), 124.96 (q, 2×C,
J = 3.6 Hz), 127.70 (q, J = 31.2 Hz), 127.80 (2×C), 148.29, 211.59
(C-6). 19F NMR {1H} δ -60.59 (s, 1F). HRMS: (API+) calculated for
2), 67.47 (C-3), 74.95 (C-23), 76.05 (C-22), 124.72 (2×C), 126.75
(2×C), 140.23, 149.41, 211.53 (C-6). HRMS: (API+) calculated for
C
C
33H51O5 ([M+H]+) 527.3736, Found 527.3734. Anal. Calcd for
33H50O5: C, 75.25; H, 9.57. Found: C, 75.22; H, 9.61%.
2.2.15. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-phenylphenyl)-24-
nor-5α-cholan-6-one (10t)
C
C
30H42F3O5 ([M+H]+) 539.2984, Found 539.2986. Anal. Calcd for
30H41F3O5: C, 66.89; H, 7.67. Found: C, 66.86; H, 7.70%.
The general procedure for dihydroxylation of 8t (40 mg;
0.08 mmol) afforded 33 mg (72%) of the title compound 10t as a white
solid: m. p. 251–253 °C (EtOH); IR ν (cm−1) 3328 vb, 2955, 1705,
1643, 1600, 1580, 1465, 967, 763. 1H NMR (DMSO-d6) δ 0.34 (H-18),
0.59 (H-19) (both s, 3H, CH3); 0.89 (d, 3H, J = 6.4 Hz, H-21, CH3);
1.78–1.89 (m, 2H); 1.97 (dd, 1H, J = 13.1, J′ = 4.6 Hz); 2.07 (t, 1H,
J = 12.6 Hz); 2.58 (dd, 1H, J = 12.1, J′ = 3.0 Hz); 3.44 (m, 1H, H-2);
3.54 (d, 1H, J = 8.6 Hz, H-3); 3.74 (bs, 1H, H-22); 4.19 (bs, 1H, OH);
4.33 (bs, 1H, OH); 4.41 (d, 1H, J = 8.9 Hz, H-23); 4.55 (bs, 1H, OH);
5.15 (bs, 1H, OH); 7.33–7.37 (m, 3H); 7.45 (t, 2H, J = 7.6 Hz); 7.64 (d,
2H, J = 8.3 Hz); 7.68 (t, 2H, J = 8.3 Hz). 13C NMR δ 11.49 (C-18),
12.52 (C-19), 13.35 (C-21), 20.79, 23.29, 26.81, 27.27, 36.52, 37.02,
39.12, 40.09, 41.83, 42.05, 45.95, 50.28, 51.92, 52.86, 55.95, 67.10 (C-
2), 67.48 (C-3), 74.87 (C-23), 76.17 (C-22), 126.26 (2×C), 126.51
(2×C), 127.38, 127.67 (2×C), 128.96 (2×C), 138.74, 139.80, 142.58,
2.2.12. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(2,4-dimethylphenyl)-
24-nor-5α-cholan-6-one (10q)
The general procedure for dihydroxylation of 8q (60 mg;
0.14 mmol) afforded 52 mg (75%) of the title compound 10q as a white
solid: m. p. 253–255 °C (EtOH); IR ν (cm−1) 3350 vb, 2943, 1693,
1617, 1502, 1458, 987, 817. 1H NMR (DMSO-d6) δ 0.35 (H-18), 0.60
(H-19) (both s, 3H, CH3); 0.85 (d, 3H, J = 6.4 Hz, H-21, CH3);
1.78–1.88 (m, 2H); 1.98 (dd, 1H, J = 13.1, J′ = 4.6 Hz); 2.07 (t, 1H,
J = 12.5 Hz); 2.22 (s, 3H, Ar-CH3); 2.26 (s, 3H, Ar-CH3); 2.58 (dd, 1H,
J = 12.1, J′ = 3.0 Hz); 3.44 (m, 1H, H-2); 3.63 (dd, 1H, J = 8.3 Hz, H-
3); 3.74 (m, 1H, H-22); 4.19 (bs, 1H, OH); 4.33 (bs, 1H, OH); 4.46 (bs,
1H, OH); 4.52 (d, 1H, J = 8.3 Hz, H-23); 4.89 (bs, 1H, OH); 6.93 (s,
1H); 6.96 (d, 1H, J = 7.9 Hz); 7.12 (d, 1H, J = 7.9 Hz). 13C NMR δ
11.50 (C-18), 13.13 (C-19), 13.38 (C-21), 19.25 (Ar-CH3), 20.65 (Ar-
CH3), 20.80, 23.36, 26.84, 27.36, 36.79, 37.05, 39.09, 40.10, 41.87,
42.09, 45.98, 50.31, 52.17, 52.88, 55.97, 67.12 (C-2), 67.51 (C-3),
71.26 (C-23), 75.44 (C-22), 126.41, 127.40, 130.84, 134.94, 135.62,
138.30, 211.64 (C-6). HRMS: (API+) calculated for C31H47O5 ([M
+H]+) 499.3423, Found 499.3426. Anal. Calcd for C31H46O5: C, 74.66;
H, 9.30. Found: C, 74.63; H, 9.32%.
211.57 (C-6). HRMS: (API+) calculated for C35H47O5 ([M+H]+
)
547.3423, Found 547.3421. Anal. Calcd for C35H46O5: C, 76.89; H,
8.48. Found: C, 76.85; H, 8.53%.
3. Molecular docking
Docking was performed prediction of conformation and energy
ranking between BRI1 receptor (PDBID: 3RGZ) and. Docking studies
was carried out using AutoDock Vina 1.05 [13]. All 3D structures of
BRI1 ligands were prepared with Marvin 5.10.3 [10], software which
can be used for drawing, displaying and characterization of chemical
structure, substructures and reactions. Ligands were prepared as deri-
vatives of natural ligand brassinolide (BLD). Polar hydrogens were
added to all ligands and proteins with the AutoDock Tools (ADT) [14]
program prior to docking with Autodock Vina program. Grid box with
size of 40 Å were centered on active site of protein. Exhaustiveness
parameter was set to 20 (default 8). After docking we compared docked
ligand with brassinolide crystal-like poses and the best crystal-like
poses of each ligand were analyzed.
2.2.13. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(2,5-dimethylphenyl)-
24-nor-5α-cholan-6-one (10r)
The general procedure for dihydroxylation of 8r (60 mg;
0.14 mmol) afforded 49 mg (71%) of the title compound 10r as a white
solid: m. p. 281–283 °C (EtOH); IR ν (cm−1) 3332 vb, 2933, 1707,
1616, 1506, 1461, 985. 1H NMR (DMSO-d6) δ 0.36 (H-18), 0.60 (H-19)
(both s, 3H, CH3); 0.86 (d, 3H, J = 6.4 Hz, H-21, CH3); 1.82 (m, 1H);
1.87 (m, 1H); 1.98 (dd, 1H, J = 13.1, J′ = 4.6 Hz); 2.07 (t, 1H,
J = 12.6 Hz); 2.23 (s, 3H, Ar-CH3); 2.24 (s, 3H, Ar-CH3); 2.58 (dd, 1H,
J = 12.0, J′ = 3.1 Hz); 3.45 (m, 1H, H-2); 3.61 (bd, 1H, J = 7.9 Hz, H-
3); 3.74 (bs, 1H, H-22); 4.20 (bs, 1H, OH); 4.34 (d, 1H, J = 5.2 Hz,
OH); 4.47 (bs, 1H, OH); 4.52 (d, 1H, J = 7.9 Hz, H-23); 4.94 (bs, 1H,
OH); 6.92 (dd, 1H, J = 7.6, J′ = 1.2 Hz); 6.99 (d, 1H, J = 7.6 Hz); 7.06
(bs, 1H). 13C NMR δ 11.47 (C-18), 13.29 (C-19), 13.40 (C-21), 18.92
(Ar-CH3), 20.81, 20.85 (Ar-CH3), 23.42, 26.86, 27.32, 36.91, 37.08,
39.10, 40.11, 41.88, 42.13, 45.98, 50.34, 52.25, 52.91, 55.98, 67.14 (C-
2), 67.52 (C-3), 71.45 (C-23), 75.53 (C-22), 127.46, 127.99, 130.08,
131.91, 134.34, 141.26, 211.68 (C-6). HRMS: (API+) calculated for
4. The pea inhibition biotest
Pea seedlings (Pisum arvense L. sort Arvica) germinating for 2 days
were selected for uniformity from a large population and then trans-
ferred into pots containing perlite and 1/10 diluted Hoagland solution
(half concentration, pH 5.7) After 24 h in a dark cultivation room
(24 °C, humidity 75%) the seedlings were treated with different
amounts of tested compounds in 5 µl fractionated lanolin. The sub-
stances were applied as microdrops to the scar left after the removal of
bract. The control plants were treated with lanolin alone. At least seven
plants were used for each experiment and the assays were repeated at
least three times. The inhibition of etiolated pea stems were measured
after 4 days and the difference in length between treated and control
plants provided a measure of activity. For each treatment, 8 seedlings
were analyzed in two biological replicates. The mean values were
subjected to the statistical analysis using the Student’s t test.
C
C
31H47O5 ([M+H]+) 499.3423, Found 499.3425. Anal. Calcd for
31H46O5: C, 74.66; H, 9.30. Found: C, 74.61; H, 9.33%.
2.2.14. (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-t-butylphenyl)-24-
nor-5α-cholan-6-one (10s)
The general procedure for dihydroxylation of 8s (60 mg;
0.13 mmol) afforded 53 mg (77%) of the title compound 10s as a white
solid: m. p. 255–256 °C (EtOH); IR ν (cm−1) 3234 vb, 2938, 1709,
1616, 1512, 1461, 1084, 991, 831. 1H NMR (DMSO-d6) δ 0.32 (H-18),
0.59 (H-19) (both s, 3H, CH3); 0.85 (d, 3H, J = 6.4 Hz, H-21, CH3);
1.26 (s, 3H, t-Bu); 1.77–1.88 (m, 2H); 1.98 (dd, 1H, J = 13.1,
J′ = 4.6 Hz); 2.07 (t, 1H, J = 12.6 Hz); 2.58 (dd, 1H, J = 12.3,
J′ = 3.4 Hz); 3.44 (m, 1H, H-2); 3.51 (d, 1H, J = 8.3 Hz, H-3); 3.74 (m,
1H, H-22); 4.20 (vb s, 2H, 2×OH); 4.32 (d, 1H, J = 8.3 Hz, H-23); 4.42
(vb s, 1H, OH); 4.99 (vb s, 1H, OH); 7.18 (d, 2H, J = 8.5 Hz); 7.33 (d,
2H, J = 8.5 Hz). 13C NMR δ 11.44 (C-18), 12.48 (C-19), 13.33 (C-21),
20.77, 23.29, 26.81, 27.13, 31.19 (3×C, t-Bu), 34.21, 36.33, 37.00,
39.10, 40.10, 41.81, 42.03, 45.94, 50.27, 51.95, 52.84, 55.92, 67.08 (C-
5. Determination of ethylene production
For measurement of ethylene production, pea seedlings (8 plants/
tested amount of substance) were placed in a 0.5 L glass container for
24 h in the dark. One milliliter of headspace gas from the chamber was
withdrawn for each measurement and injected into a gas chromato-
graph (Agilent Technologies, GC System, USA) equipped with a flame
7

P. Korinkova et al.
Steroidsxxx(xxxx)xxx–xxx
and helium was used as carrier gas. The area under the resultant peak
(y-axis) versus sensitivity (x-axis; nL.mL−1) was representing a quanti-
tative measure of ethylene concentration. The measurements were done
in triplicates and data were statistically analyzed using the Student’s t
test.
Table 1
Isolated yields of products in alkene cross-metathesis.
Entry
a
Ar
Yield (%)
81
Entry
l
Ar
Yield (%)
69
6. Arabidopsis brassinosteroid sensitivity assay
b
c
65
73
m
n
71
57
Arabidopsis thaliana L. (Heyhn.) (Columbia ecotype, Col-0; referred
to Arabidopsis) seedlings were stratified for 2 d at 4 °C and germinated
on vertical half-strength Murashige and Skoog (1% w/v sucrose) agar
plates with different concentrations of 24-epiBL and BR derivatives at
22 °C in a 16 h/8 h light-dark cycle for 5 d. Roots were then straigh-
tened on solid media plates, scanned with an Epson high-resolution
scanner and the entire root length measured with ImageJ (http://
rsbweb.nih.gov/ij/). For each treatment, more than 15 seedlings were
analyzed in two biological repeats. P values were calculated with a two-
tailed Student t-test using Excel software.
d
e
75
64
o
p
66
71
7. Cytotoxicity
f
70
q
61
Calcein AM cytotoxicity assay was performed exactly as described
earlier [11].
g
h
75
59
r
s
58
60
8. Results and discussion
8.1. Chemistry
For the preparation of above mentioned compounds we started with
the known [15] 24-nor-5α-chola-2,22-dien-6-one (7). With this com-
pound cross-metathesis were carried out using twenty commercially
available styrenes substituted with fluorine, chlorine, bromine, tri-
fluoromethyl, alkyls and phenyl group. The reaction was catalyzed by
Hoveyda-Grubbs second generation catalyst in refluxing di-
chloroethane. This catalyst has proven to be efficient for different
steroid side chain cross-metatheses [15–18]. Due to the fact that com-
pound 7 and used styrenes belongs to the group of type I olefins (non-,
meta- and para- substituted styrenes) and type II olefins (ortho- sub-
stituted styrenes) if Hoveyda-Grubbs second generation catalyst is used
[19], the reactions proceed in two steps. Firstly, the homodimerization
of styrene with strong release of ethylene takes place followed by the
secondary cross-metathesis of steroid and in-situ formed stilbene. This is
the main reason of long reaction time. In most cases, the cross-me-
tathesis gave the corresponding 23-aryl products in good isolated yields
60–80% (see Scheme 1). Lower yield is typical for ortho-substituted
styrenes as their homodimers are sparingly consumable during sec-
ondary cross-metathesis. In the case of 4-phenylstyrene the low yield
(32%) is caused by formation of poorly soluble 4,4′-diphenylstilbene
i
66
t
32
j
73
65
u
v
na
na
k
na – compound not isolated.
ionic detector (FID) and a capillary column (HP-AL/S stationary phase,
15 lm, i.d. = 0.535). The chromatographic analytical parameters were
as follows: column temperature: 150 °C; detector temperature: 220 °C;
Scheme 1. Preparation of aryldienones and aryl-tetraols. For Ar see
Table 1.
Scheme 2. Formation of isopropoxy derivative 9 if bis-ortho sub-
stituted or no styrenes are used in the reaction.
8

P. Korinkova et al.
Steroidsxxx(xxxx)xxx–xxx
Table 2
IC50 (mol/L) values obtained from the pea inhibition biotest.
Compounds 10a, 10d, 10g, 10j, and 10m are presented for
comparison; the results were published [11].
Compound
IC50 (mol/L)
24-epibrassinolide
10f
1.66 × 10−5
1.80 × 10−5
2.15 × 10−5
2.30 × 10−5
2.52 × 10−6
2.0 × 10−6
1.8 × 10−5
2.7 × 10−4
1.8 × 10−4
10b
10n
10a
10d
10g
10j
10m
Fig. 3. Effect of selected brassinosteroid derivatives on ethylene production (nL.mL−1) in
etiolated pea seedlings determined by GC-FID 24 h after ventilation. Error bars represent
s.d.
during homodimerization step. No desired products were observed if
bis-ortho substituted styrenes were used. In these cases we were able to
isolate only very small amount of 2-isopropoxyphenyl derivative 9 as a
product of reaction between steroid olefin and Hoveyda-Grubbs catalyst
(see Scheme 2). This was probably caused by steric hindrance near
steroidal terminal double bond. All products 8a-t were obtained as trans
double bond isomers as indicated by NMR analysis.
Next, Sharpless simultaneous dihydroxylation of both double bonds
was used to minimize formation of unnatural configuration of 22 and
23 hydroxy groups. Hydroquinidine 4-chlorobenzoate was used as
chiral ligand. The reaction rate was increased by addition of methan-
sulfonamide [20]. Such reaction conditions led only to the desired
22R,23R-isomers 10a-t in very good yields (75–83%).
applied BRs stimulate ethylene production by stabilizing the ACS pro-
tein. When low levels of ethylene are applied to etiolated pea seedlings,
the characteristic triple response can be observed: inhibition of stem
elongation, radial swelling of the stem and the absence of a normal
geotropic response [22]. Production of ethylene was measured in cul-
tivation vessels during the incubation of etiolated pea plants after
treatment of different BR derivatives (SFig. 2). The high concentrations
of ethylene (196, 142 and 165 nL.L−1) were determined after treatment
of 10f, 10b and 10n and compared to 229 nL.L−1 for 24-epiBL treat-
ment. While level of this gaseous plant hormone produced by untreated
control pea plants, was found to be significantly lower (about
60 nL.L−1, Fig. 3).
BRs derivatives were further assessed for biological activity based
on their inhibitory effect on Arabidopsis root growth [23]. The effects
of compounds 10f, 10b and 10n on the Arabidopsis roots are shown in
Fig. 4. Results of all prepared analogues are summarized in SFig. 3.
Molecular docking into BRI1 receptor shows that several com-
pounds binds with very high binding energy. In some cases the binding
energy was even better than for naturally occurring brassinolide
(−10.6 kcal/mol) [11] (see SI for molecular docking of all new com-
pounds). The high binding affinity of compounds 10f (−11.0 kcal/
mol), 10b (−10.7 kcal/mol), and 10n (−11.9 kcal/mol) also proves
the good biological activities of these analogues (Fig. 5).
All compounds were characterized by NMR, IR and MS techniques
together with elemental analysis for all tetraols. Compounds 10a, 10d,
10g, 10j, and 10m were also prepared by different synthetic strategy
[11] (see Table 1).
8.2. Biological activity and docking
Biological activity of new BRs derivatives was monitored by pea
inhibition biotest. This test is based on that BRs inhibit the growth of
etiolated seedlings at high concentration and the inhibition is probably
caused by ethylene production which is mediated by BRs. Dose re-
sponse curves for all prepared BRs derivatives are shown in SFig. 1. The
IC50 values obtained from the pea inhibition biotest are summarized in
Table 2. The most active BRs derivatives were 10f, 10b, and 10n (IC50
1.8 × 10−5–2.3 × 10−5 mol.L−1
) compared to 24-epibrassinolide
Relationship between biological activity and substitution pattern in
the phenyl group shows that no substitution or substitution with one
small group (fluorine, chlorine) led to promising compounds with high
(IC50 1.6 × 10−5 mol.L−1), used as a positive control (Fig. 2).
The crosstalk of BRs and ethylene regulates various aspects of plant
growth and development. Hansen et al. [21] showed that exogenously
Fig. 4. Effect of selected brassinosteroid derivatives on the inhibition of Arabidopsis root
lenght. 5 days old Arabidopsis thaliana seedlings (Columbia ecotype, Col-0) were treated
by DMSO/24epiBL/BR analogues. For each treatment more than 25 seedlings were
analyzed in two biological repeats.
Fig. 2. Effect of selected brassinosteroid derivatives on the inhibition of etiolated pea
seedlings. Error bars represent s.d.
9

P. Korinkova et al.
Steroidsxxx(xxxx)xxx–xxx
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.steroids.2017.08.010.
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This work was supported by grant GJ15-08202Y of the Grant
Agency of the Czech Republic and by the by project of the Ministry of
Education, Youth and Sports CR NPUI LO1204. This work was also
supported by student project IGA_PrF_2017_013 and IGA_PrF_2017_028
of the Palacky University in Olomouc.
10