Design, synthesis and biological activities of new brassinosteroid analogues with a phenyl group in the side chain

Article abstract of DOI:10.1039/c6ob01479h

We have prepared and studied a series of new brassinosteroid derivatives with a p-substituted phenyl group in the side chain. To obtain the best comparison between molecular docking and biological activities both types of brassinosteroids were synthesized; 6-ketones, 10 examples, and B-lactones, 8 examples. The phenyl group was introduced into the steroid skeleton by Horner-Wadsworth-Emmons. The docking studies were carried out using AutoDock Vina 1.05. Plant biological activities were established using different brassinosteroid bioassays in comparison with natural brassinosteroids. Differences in the production of the plant hormone ethylene were also observed in etiolated pea seedlings after treatment with new brassinosteroids. The most active compounds were lactone 8f and 6-oxo derivatives 8c and 9c, their biological activities were comparable or even better than naturally occurring brassinolide. Finally the cytotoxicity of the new derivatives was studied using human normal and cancer cell lines.

Full text of DOI:10.1039/c6ob01479h

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Russinova and M. Strnad, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB01479H.
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DOI: 10.1039/C6OB01479H
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ARTICLE
Design, synthesis and biological activities of new brassinosteroid
analogues with phenyl group in the side chain
00th January 20xx,
Accepted 00th January 20xx
M. Kvasnica,a,† J. Oklestkova,a V. Bazgier,a,b L. Rárová,c P. Kořínková,a J. Mikulík,a M. Budesinsky,d T.
Béres,c K. Berka,b,e Q. Lu,f,g E. Russinova,f,g M. Strnada
DOI: 10.1039/x0xx00000x
www.rsc.org/
We have prepared and studied a series of new brassinosteroid derivatives with p-substituted phenyl group in
the side chain. To have the best comparison between molecular docking and biological activities both types
of brassinosteroid were synthesized; 6-ketones, 10 examples, and B-lactones, 8 examples. The phenyl group
was introduced into steroid skeleton by Horner–Wadsworth–Emmons. The docking studies were carried out
using AutoDock Vina 1.05. Plant biological activities were established using different brassinosteroid
bioassays in comparison with natural brassinosteroids. Differences in the production of plant hormone
ethylene were also observed in etiolated pea seedlings after treatment with new brassinosteroids. The most
active compounds were lactone 8f and 6-oxoderivatives 8c and 9c, their biological activities were
comparable or even better than naturally occurring brassinolide. Finally cytotoxicity of new derivatives was
studied using human normal and cancer cell lines.
mammalian nuclear steroid receptor, BRs are perceived at the cell
surface by the transmembrane receptor complex formed by the
receptor kinase BRI1 and its co-receptor BAK1.16-19 The BRI1
Introduction
receptor has a binding site for BRs located in the extracellular
ectodomain. There, the nonpolar side of BRs fit into a highly
nonpolar cavity of the receptor cleft and hydroxyl groups of BRs are
exposed to the solvent or towards interaction with BAK1 or
SERK1.18,20 This structural knowledge, formed the basis of the idea
of the brassinosteroid side chain modifications using a nonpolar
group such as the phenyl group. Moreover, some analogues
containing cykloalkyl subtituents at C-24 (replacing the isopropyl
group of brassinolide 1) exhibited significant activity in rice lamina
inclination biotest, where the compound with the cyclohexyl group,
showed the lowest activity, whereas the cyclopentyl analogue was
comparably active as brassinolide in this bioassay.21,22
Brassinosteroids (BRs, Fig. 1) are a class of plant steroid hormone,
that are now known to be 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 photosynthesis.1-3 They are
also involved in defense against a wide range of biotic and abiotic
stresses, such as water, temperature, oxidative stresses and high
salinity.4,5 Moreover, recent studies have shown that natural BRs
have potential application to medicine due to antiviral,6,7
immunomodulatory and neuroprotective activity,8,9 and anti-
proliferative effects in animal cells in vitro.10-15 In contrast to the
The aim of this study was to synthesize new brassinosteroid
derivatives with a p-substituted phenyl group in the side chain and
study their biological properties. The phenyl group was chosen
owing to its successful molecular docking into the active site of BRI1
using AutoDock Vina. Some compounds showed marked interaction
with the BRI1 receptor. The biological activities of newly prepared
derivatives were confirmed using by plant bioassay (pea inhibition
biotest, Arabidopsis root and hypocotyl sensitivity bioassay and
BES-1 dephosphorylation assay). Their cytotoxic activities were
studied using human normal and cancer cell lines.
a.Laboratory of Growth Regulators, Centre of the Region Haná for
Biotechnological and Agricultural Research, Institute of Experimental
Botany ASCR & Palacký University, Šlechtitelů 27, 78371 Olomouc, Czech
Republic.
b.Department of Physical Chemistry, Faculty of Science, Palacký University, tř. 17.
Listopadu 12, 77146 Olomouc, Czech Republic.
c. Department of Chemical Biology and Genetics, Centre of the Region Haná for
Biotechnological and Agricultural Research, Palacký University, Šlechtitelů 27,
78371 Olomouc, Czech Republic.
d.Institute of Organic Chemistry and Biochemistry, ASCR, Flemingovo n. 2, 16610
Prague 6, Czech Republic.
e. Regional Centre of Advanced Technologies and Materials, Department of Physical
Chemistry Palacky University in Olomouc, 17. listopadu 1131, Olomouc CZ779 00,
Czech Republic.
f. Department of Plant Systems Biology, VIB, 9052 Gent, Belgium.
g.Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052
Gent, Belgium.
† Corresponding author: kvasnica@ueb.cas.cz
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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isolated. Dihydroxylation of the double bond on the A-ring is stereo
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controlled by A-ring conformation and the presence of a methyl
DOI: 10.1039/C6OB01479H
group on C10. The attack of reagent is always from the bottom side
of molecule and thus only 2α,3α-diol was detected.
As brassinosteroids with lactone in the B-ring are known to show
higher biological activity than corresponding 6-ketones, we decided
to prepare them as well. The direct Baeyer-Villiger oxidation of
tetrahydroxy-ketones with freshly prepared trifluoroperoxyacetic
acid led to both isomers in a ratio approx. 10-15:1 (with natural
isomer favoured). However, these mixtures were inseparable even
by HPLC (Scheme 1).
Scheme 1: Baeyer-Villiger oxidation of B-ring showing formation of
natural and unnatural isomeric lactones.
Fig. 1: Structures of most common natural brassinosteroids;
castasterone (1), brassinolide (2), 24-epicastasterone (3), 24-
epibrassinolide (4), 28-homocastasterone (5), 28-homobrassinolide
(6).
For this reason, we had to prepare tetraacetates 8d-17d first and
carry out the oxidation reaction on these. This “detour” allowed us
to prepare and easily separate the required lactones 8e-11e, 13e,
14e, 16e, and 17e in good yields (79-88 %). Unfortunately, two aryl-
ketones 12d and 15d were unstable during the reaction and only a
mixture of products was obtained in both cases. The instability of
iodophenyl derivative 12d, was predictable owing to the presence
of easily oxidizable iodine. Due to the presence of the activating
group, the methoxyphenyl derivative 15d is more nucleophilic on
the benzene ring than other derivatives and thus also undergoes
electrophilic aromatic substitution reaction (substitution with
trifluoroacetate). These two reactions also failed using 3-
chloroperoxybenzoic acid.
The last reaction step was basic hydrolysis of tetraacetoxy-lactones
to corresponding tetrahydroxy-lactones 8f-11f, 13f, 14f, 16f, and
17f in almost quantitative yields. The reaction steps can be seen in
Scheme 2. All compounds were characterized by NMR, IR and MS
techniques. Compounds used for biological testing were also
characterized for purity by elemental analysis and melting point.
Results and discussion
Chemistry
For the preparation of desired phenyl analogues of
brassinosteroids, we set out from the known aldehyde 7 ((20S)-6,6-
ethylenedioxy-5α-pregn-2-en-20-carbaldehyde) prepared according
to
a
published procedure.23 With this aldehyde Horner–
Wadsworth–Emmons (HWE) reaction was then carried out with
different commercially available p-substituted benzylphosphonates.
Based on the character of the BRI1 non-polar part of the cavity for
side chain, we used these substituents in para position: fluorine,
chlorine, bromine, iodine, nitro, methyl, methoxy, nitrile, and
isopropyl. Benzyl triphenylphosphonium chloride was used for
preparation of non-substituted aryl analogues.
Despite standard use of sodium hydride as a base in the HWE
reaction, we observed isomerization of the methyl group on C-20
(see supporting information for detailed reaction conditions and
analysis). Using this base along with steric hindrance of the reaction
site for bulky aryl group, the reaction time increased and this led to
enolization of aldehyde. The aryl group of phosphonates used, is
crucial for this epimerization as it was not observed when smaller
stabilizing groups were used (e.g. COOR, CN).24 Using n-butyllithium
instead, solved the problem and only desired aryl diens 8a-17a with
22E configuration were obtained. In almost all cases, the reaction
gave products in good yields (80-90 %). Only compound 12a was
prepared in lower yield (65 %) due to the presence of reactive
iodine.
<Scheme 2>
Scheme 2: Synthesis of brassinosteroid phenyl analogues: a)
benzyltriphenylphosphonium bromide or diethyl arylphosphonates,
n-BuLi/THF; b) 5% HCl/THF; c) OsO4, CH3SO2NH2, K3[Fe(CN)6],
K3CO2,hydroquinidine
4-chlorobenzoate/t-BuOH,
H2O;
d)
Ac2O/pyridine; e) trifluoroperoxyacetic acid/ CH2Cl2; f) NaOH/THF,
H2O.
Molecular docking
Further hydrolysis of ketal group gave aryl dienons 8b-17b which
were subjected to dihydroxylation in almost quantitative yield.
Simultaneous Sharpless dihydroxylation of both double bonds was
used to minimize formation of unnatural configuration of 22 and 23
hydroxy groups. As a chiral ligand, we used hydroquinidine 4-
chlorobenzoate. The reaction rate was increased by addition of
methansulfonamide.25 Without methansulfonamide, the reaction
took more than 48 hours. Such reaction conditions allowed us to
isolate only the desired 22R,23R-isomers 8c-17c in good yields (75-
83 %). The correct configuration was determined according to a
published alternative preparation of 8c.26 The unnatural isomers
(22S,23S) were formed only in trace amounts and therefore not
Molecular docking is a useful tool for understanding of the pose and
energetics of a protein-ligand complex. The binding site of BRI1 is
located on the surface of the receptor ectodomain as a nonpolar
cleft lined by nonpolar aromatic and aliphatic residues (I540, I563,
W564, Y599, Y642, M657, F681, I682, I706), whereas hydroxyl
groups form the cleft ridge (Y597, Y599, Y642, S647). Brassinolide
fits into the cleft via its nonpolar side and displays its hydroxyl
groups towards the solvent and protein partners (Fig. 2).
Since there is a space left in the cleft around the brassinolide chain,
we were running molecular docking of BRs derivatives with a phenyl
ring on the tail replacing 1',2'-dimethylpropyl moiety of
brassinolide. Molecular docking predicted similar or better binding
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energies than for brassinolide for compounds 8c, 8f, 9c, 9f, 10c, 10f,
ARTICLE
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and 14f (Fig. 3). This implies that derivatives with a phenyl ring on Tab. 1: IC50 (mol/L) values obtained from the pea inhibition biotest.
DOI: 10.1039/C6OB01479H
IC50 (mol.L1-)
the tail or phenyl ring with small groups such as fluorine, chlorine or
Compound
methyl should be accommodated within the BRI1 cleft at least as
easily as brassinolide itself and hence they were the best candidates
for showing similar binding experimentally. On the other hand,
derivatives with larger groups did not fit well into the cleft and the
docked pose often revealed the tail out of the cleft completely (for
the pose of all compounds see sup. inf.). In all docking cases,
lactones showed better binding energies than 6-ketones due to
better fit to cavity, close to Y599. The best compound to emerge
from molecular docking was compound 8f.
Brassinolide
8c
2.2.10-5± 2.10-6
2.5.10-6± 3.10-7
1.8.10-6± 5.10-8
2.0.10-6± 3.10-7
2.6.10-6± 8.10-8
1.8.10-5± 3.10-6
2.3.10-5± 2.10-6
2.7.10-4± 2.10-5
1.7.10-6± 3.10-7
2.1.10-2± 4.10-3
4.0.10-2± 5.10-4
no inhibition
8f
9c
9f
10c
10f
11c
11f
12c
13c
13f
14c
14f
1.8.10-4± 7.10-6
1.4.10-6± 2.10-7
no inhibition
15c
16c
16f
2.1.10-4± 5.10-6
no inhibition
Fig. 2: 3D view of brassinolide with amino acid around active site.
17c
17f
2.0.10-3± 6.10-5
2.1.10-4± 5.10-6
The IC50 values obtained from the pea inhibition biotest are
summarized in Table 1. The most active lactones were 8f, 9f, 11f
and 14f (IC50 2.56.10-6 - 1.4.10-6mol.L-1) whereas brassinolide (BL),
the most active natural BRs used as a positive control, was about
ten time less active (IC50 2.2.10-5mol.L-1). 6-oxoderivatives 8c and 9c
were also active in this bioassay. Their IC50 values were comparable
with active lactones (Table 1). Compounds 15c, 13f and 16f showed
no inhibition of etiolated pea plants. Dose response curves for the
most active BRs derivatives are shown in Fig. 4.
Fig. 3: Pose of 8f within BRI1 binding site. Structure of brassinolide
and BRI1 binding site are shown in white, structure of 8f is shown in
blue.
Biology
The response of plant tissues to applied BRs varies with BR
concentration. In most cases, low concentration induces elongation
and curvature as a result of cell division and cell elongation. Most
BR bioassays are based on this effect.27 However, there are other
ways of regulating growth by BRs. For example, BRs inhibit the
growth of etiolated pea seedlings at high concentration and this is
probably caused by increased ethylene production. Ethylene effects
alteration of the normal planes of cell growth. Radial swelling or
abnormal radial expansion of stem, such as that seen in the
response of etiolated pea seedlings to ethylene application, results
from inhibited elongation, increased radial expansion and probably
also it accounts for leaf epinasty.28 These effects are known as the
“triple response” of etiolated seedlings to ethylene.29
Fig. 4: Effect of selected brassinosteroid derivatives on the
inhibition of etiolated pea seedlings. Error bars represent s.d.
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DOI: 10.1039/C6OB01479H
Fig. 5: 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.
Levels of ethylene were measured in cultivation vessels during the
incubation of etiolated pea plants after treatment with different BR
derivatives (Fig. 5). The highest concentration of ethylene (235 and
224 nL. L-1) was determined after 24 h treatment by 8c and 14f
compared to 206 nL.L-1 for BL treatment. While level of this gaseous
plant hormone produced by untreated control pea plants, was
found to be significantly lower (about 80 nL.L-1, Fig.5). Arteca et al30
also observed stimulated ethylene production in etiolated mung
bean segments 4 hours after treatment with 1 μM BL and this
increased production became greater over the following 20 h.
The most potent compounds (8c, 8f and 9c) were tested in
Fig. 6: A. Immunoblot analysis of BES1 in Arabidopsis thaliana (Col-
0) seedlings showing dephosphorylation of BES1 after BRs
treatment. B. Graph shows percentage of dephosphorylated BES1
relative to the total BES1 detected in Arabidopsis. The data are the
average of two biological repeats. Error bars indicate s.e.m. pBES1,
phosphorylated BES1.
Arabidopsis
brassinosteroid
sensitivity
and
BES-1
dephosphorylation bioassays. The potency of these compounds
was as follows: BL8f>9c>8c in Arabidopsis sensitivity bioassays.
The effects of tested compounds on the Arabidopsis roots and
hypocotyls, are shown in supplementary information (Fig. S3 and
Fig. S4). Compound 8f significantly increased dephosphorylation of
BES1 (Fig. 6), which is an important transcription factor in the BR
signalling pathway. Taken together, these results confirm that the
biological activity of compound 8f is comparable with natural BR-
brassinolide.
The antiproliferative activity of prepared brassinosteroid derivatives
was tested using several models of normal and cancer cell lines. We
compared the in vitro cytotoxic activity of selected analogues
against human foreskin fibroblasts (BJ) and cancer cell lines of
various histopathological origins, including T-lymphoblastic
leukemia CEM, breast carcinoma (MCF7) and cervical carcinoma
(HeLa). Cells were exposed to six 3-fold dilutions of each drug for
72 h prior to determination of cell survival. The IC50 (concentration
leading to 50% inhibition of viability) values obtained from Calcein
AM cytotoxicity assay are presented in supplementary information
(Table S2). Most tested BRs analogues had no detectable cytotoxic
activity, even when tested in concentrations of up to 50 µM. Only
compounds 10f, 11f and 13f showed moderate cytotoxic activity
against CEM and Hela cell lines (IC50 around 35 µM). No BRs
derivative mediated loss of viability was observed in the BJ
fibroblasts. 24-Epibrassinolide was used as a control. It is a natural
brassinosteroid with modest cytotoxicity against CEM cells (IC50 44
µM).15
Experimental
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
1
chemical shifts (δ) were calibrated using tetramethylsilane (TMS, H
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 minutes. 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 compounds. Merck silica gel Kieselgel 60 (230−400 mesh) was
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used for column chromatography. The HPLC system consisted of a (CH2), 33.34 (CH), 35.90 (C), 39.68 (CH2), 40.45 (CH), 41.20 (CH2),
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Waters semi-preparative HPLC system including quaternary pump, 41.24 (CH ), 42.63 (C), 48.08 (CH), 53.50 (CH), 55.92 (CH), 56.00
DOI: 10.1039/C6OB01479H
2
liquid handler, UV-VIS and ELSD detectors. The semi preparative (CH), 64.07 (CH2), 65.55 (CH2), 109.99 (C), 115.22 (d, J = 21.6 Hz,
column was filled with silica gel. Reagents and solvents were 2×CH), 124.77 (CH), 125.69 (CH), 126.04 (CH), 127.25 (d, J = 7.2 Hz,
purchased from Sigma–Aldrich and were not purified. For 2×CH), 134.18 (d, J = 3.6 Hz, C), 137.01 (d, J = 2.4 Hz, CH), 161.77 (d,
experimental procedures and data for compounds of series b, d, e, J = 244.7 Hz, C). 19F NMR {1H} δ -116.36 (s, 1F). HRMS: (API+)
and f see supplementary information.
calculated for C31H42FO2 ([M+H]+) 465.3169, Found 465.3170.
(22E)-6,6-Ethylenedioxy-23-phenyl-24-nor-5α-chola-2,22-diene
(8a)
(22E)-6,6-Ethylenedioxy-23-(4-chlorophenyl)-24-nor-5α-chola-
2,22-diene (10a)
The general procedure for WHE reaction with diethyl 4-
chlorophenylphosphonate and chromatography on silica
(Et2O/cyclohexane - 1/19) yielded 225 mg (87 %) of the title
To a suspension of benzyl triphenylphosphonium chloride (0.81
mmol) in dried THF (10 mL) was added n-BuLi (1.6 M solution in n-
hexane, 0.81 mmol) at 0 °C and stirred for 1 h. To the resultant red
solution was added a solution of aldehyde 7 (200 mg, 0.54 mmol) in
THF (10 mL) and stirred for 4 h at 25 °C. The reaction mixture was
quenched by water and extracted with Et2O (2 × 10 mL). The
combined organic fractions were washed with brine and dried over
anhydrous magnesium sulfate. Evaporation of the volatiles under
reduced pressure followed by column chromatography on silica gel
(Et2O/cyclohexane - 1/19) gave 211 mg (88 %) of the title
compound 8a as a colorless oil: IR ν (cm-1) 2933, 1655, 1598. 1H
NMR (CDCl3) δ 0.74, 0.89 (both s, 3H, CH3), 1.11 (d, 3H, J = 6.7 Hz,
CH3), 1.68-1.74 (m, 2H), 1.78 (m, 2H), 1.93-2.03 (m, 3H), 2.09 (m,
1H), 2.26 (m, 1H, ΣJ = 37.6 Hz), 3.78 (m, 1H, ΣJ = 24.1 Hz, OCH), 3.88-
3.99 (m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H-2), 6.06 (dd,
1H, J = 15.7, J´ = 8.7 Hz, H-22), 6.29 (d, 1H, J = 15.7 Hz, H-23), 7.17
(m, 1H, Ar-H), 7.25-7.34 (m, 4H, 4×Ar-H). 13C NMR δ 12.25 (CH3),
13.60 (CH3), 20.41 (CH3), 20.87 (CH2), 21.43 (CH2), 24.15 (CH2), 28.36
(CH2), 33.35 (CH), 35.91 (C), 39.68 (CH2), 40.49 (CH), 41.20 (CH2),
41.24 (CH2), 42.65 (C), 48.08 (CH), 53.50 (CH), 55.93 (CH), 56.00
(CH), 64.08 (CH2), 65.56 (CH2), 110.02 (C), 124.80 (CH), 125.70 (CH),
125.90 (2×CH), 126.64 (CH), 127.19 (CH), 128.43 (2×CH), 137.30
(CH), 138.08 (C). HRMS: (API+) calculated for C31H43O2 ([M+H]+)
447.3263, Found 447.3266.
1
compound 10a as a colorless oil: IR ν (cm-1) 2933, 1656, 1593. H
NMR (CDCl3) δ 0.74, 0.89 (both s, 3H, CH3), 1.11 (d, 3H, J = 6.7 Hz,
CH3), 1.71 (m, 2H), 1.78 (m, 2H), 1.92-2.03 (m, 3H), 2.09 (m, 1H),
2.25 (m, 1H, ΣJ = 37.6 Hz), 3.78 (q, 1H, J = 6.8 Hz, OCH), 3.88-4.00
(m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H-2), 6.04 (dd, 1H, J
= 15.7, J´ = 8.9 Hz, H-22), 6.25 (d, 1H, J = 15.7 Hz, H-23), 7.24 (m, 2H,
2×Ar-H), 7.26 (m, 2H, 2×Ar-H). 13C NMR δ 12.24 (CH3), 13.60 (CH3),
20.30 (CH3), 20.86 (CH2), 21.43 (CH2), 24.14 (CH2), 28.36 (CH2), 33.35
(CH), 35.91 (C), 39.68 (CH2), 40.50 (CH), 41.21 (CH2), 41.24 (CH2),
42.67 (C), 48.08 (CH), 53.48 (CH), 55.85 (CH), 55.98 (CH), 64.09
(CH2), 65.58 (CH2), 110.01 (C), 124.79 (CH), 125.70 (CH), 126.06
(CH), 127.11 (2×CH), 128.53 (2×CH), 132.13 (C), 136.56 (C), 138.02
(CH). HRMS: (API+) calculated for C31H42ClO2 ([M+H]+) 481.2873,
Found 481.2878.
(22E)-6,6-Ethylenedioxy-23-(4-bromophenyl)-24-nor-5α-chola-
2,22-diene (11a)
General procedure for WHE reaction with diethyl 4-
bromophenylphosphonate and chromatography on silica
(Et2O/cyclohexane - 1/19) afforded 231 mg (82 %) of the title
1
compound 11a as a colorless oil: IR ν (cm-1) 2933, 1656, 1595. H
NMR (CDCl3) δ 0.74, 0.89 (both s, 3H, CH3), 1.11 (d, 3H, J = 6.7 Hz,
CH3), 1.71 (m, 2H), 1.78 (m, 2H), 1.92-2.03 (m, 3H), 2.09 (m, 1H),
2.25 (m, 1H, ΣJ = 37.6 Hz), 3.78 (q, 1H, J = 6.8 Hz, OCH), 3.88-4.00
(m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H-2), 6.05 (dd, 1H, J
= 15.7, J´ = 8.7 Hz, H-22), 6.23 (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 12.23 (CH3), 13.59 (CH3),
20.25 (CH3), 20.85 (CH2), 21.42 (CH2), 24.13 (CH2), 28.35 (CH2), 33.34
(CH), 35.90 (C), 39.68 (CH2), 40.51 (CH), 41.20 (CH2), 41.23 (CH2),
42.67 (C), 48.07 (CH), 53.47 (CH), 55.82 (CH), 55.97 (CH), 64.08
(CH2), 65.57 (CH2), 110.00 (C), 120.21 (C), 124.78 (CH), 125.69 (CH),
126.10 (CH), 127.46 (2×CH), 131.46 (2×CH), 137.01 (C), 138.15 (CH).
HRMS: (API+) calculated for C31H4279BrO2 ([M+H]+) 525.2368, Found
525.2370.
General procedure for Wadsworth-Horner-Emmons (WHE)
reaction
To a suspension of substituted diethyl phenylphosphonate (1.08
mmol) in dried THF (10 mL) was added n-BuLi (500 µL, 1.6 M
solution in n-hexane, 0.81 mmol) at 0 °C and stirred for 1 h. To the
resultant yellow solution was added a solution of aldehyde 7 (200
mg, 0.54 mmol) in THF (10 mL) and stirred for 4 h at room
temperature. The reaction mixture was quenched by water and
extracted with Et2O (2 × 10 mL). The combined organic fractions
were washed with brine and dried over anhydrous magnesium
sulfate. Evaporation of the volatiles under reduced pressure
followed by column chromatography on silica gel gave the desired
product.
(22E)-6,6-Ethylenedioxy-23-(4-iodophenyl)-24-nor-5α-chola-2,22-
diene (12a)
General procedure for WHE reaction with diethyl 4-
(22E)-6,6-Ethylenedioxy-23-(4-fluorophenyl)-24-nor-5α-chola-2,22-
diene (9a)
iodophenylphosphonate
and
chromatography
on
silica
The general procedure for WHE reaction with diethyl 4-
fluorophenylphosphonate and chromatography on silica
(Et2O/cyclohexane - 1/19) yielded? 221 mg (89 %) of the title
compound 9a as a colorless oil: IR ν (cm-1) 2933, 1654, 1599. 1H
NMR (CDCl3) δ 0.74, 0.89 (both s, 3H, CH3), 1.11 (d, 3H, J = 6.7 Hz,
CH3), 1.71 (m, 2H), 1.78 (m, 2H), 1.92-2.03 (m, 3H), 2.09 (m, 1H),
2.23 (m, 1H, ΣJ = 37.6 Hz), 3.77 (q, 1H, J = 6.8 Hz, OCH), 3.87-3.99
(m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H-2), 5.97 (dd, 1H, J
= 15.6, J´ = 8.9 Hz, H-22), 6.25 (d, 1H, J = 15.6 Hz, H-23), 6.94-6.98
(m, 2H, 2×Ar-H), 7.25-7.28 (m, 2H, 2×Ar-H). 13C NMR δ 12.21 (CH3),
13.57 (CH3), 20.36 (CH3), 20.86 (CH2), 21.42 (CH2), 24.12 (CH2), 28.37
(Et2O/cyclohexane - 1/19) afforded 200 mg (65 %) of the title
1
compound 12a as a colorless oil: IR ν (cm-1) 2933, 1656, 1595. H
NMR (CDCl3) δ 0.74, 0.89 (both s, 3H, CH3), 1.11 (d, 3H, J = 6.7 Hz,
CH3), 1.71 (m, 2H), 1.78 (m, 2H), 1.92-2.03 (m, 3H), 2.09 (m, 1H),
2.24 (m, 1H, ΣJ = 37.1 Hz), 3.78 (q, 1H, J = 6.8 Hz, OCH), 3.88-4.00
(m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H-2), 6.06 (dd, 1H, J
= 15.7, J´ = 8.7 Hz, H-22), 6.21 (d, 1H, J = 15.7 Hz, H-23), 7.06 (m, 2H,
2×Ar-H), 7.59 (m, 2H, 2×Ar-H). 13C NMR δ 12.23 (CH3), 13.59 (CH3),
20.24 (CH3), 20.85 (CH2), 21.42 (CH2), 24.13 (CH2), 28.33 (CH2), 33.34
(CH), 35.90 (C), 39.67 (CH2), 40.50 (CH), 41.20 (CH2), 41.23 (CH2),
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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Organic & Biomolecular Chemistry
Page 6 of 11
ARTICLE
Journal Name
42.67 (C), 48.07 (CH), 53.47 (CH), 55.81 (CH), 55.96 (CH), 64.08 (CH3), 55.99 (CH), 56.02 (CH), 64.05 (CH2), 65.54 (CH2), 109.99 (C),
(CH2), 65.57 (CH2), 91.52 (C), 110.00 (C), 124.78 (CH), 125.70 (CH), 113.83 (2×CH), 124.78 (CH), 125.68 (CHD),O1I:2160..14083V9i(/eCCwH6AO)r,tiBc0l1e12O46n7.l99inH2e
126.20 (CH), 127.75 (2×CH), 137.43 (2×CH), 137.60 (C), 138.29 (CH). (2×CH), 130.87 (C), 135.23 (CH), 158.48 (C). HRMS: (API+) calculated
HRMS: (API+) calculated for C31H42IO2 ([M+H]+) 573.2229, Found for C32H45O3 ([M+H]+) 477.3369, Found 477.3371.
573.2230.
(22E)-6,6-Ethylenedioxy-23-(4-isopropylphenyl)-24-nor-5α-chola-
2,22-diene (16a)
(22E)-6,6-Ethylenedioxy-23-(4-nitrophenyl)-24-nor-5α-chola-2,22-
diene (13a)
The general procedure for WHE reaction with diethyl 4-
General procedure for WHE reaction with diethyl 4- isopropylphenylphosphonate and chromatography on silica
nitrophenylphosphonate
and
chromatography
on
silica (Et2O/cyclohexane - 1/19) afforded 231 mg (88 %) of the title
1
(Et2O/cyclohexane - 1/19) afforded 230 mg (87 %) of the title compound 16a as a colorless oil: IR ν (cm-1) 2935, 1656, 1593. H
compound 13a as a colorless oil: IR ν (cm-1) 2930, 1646, 1594, 1510, NMR (CDCl3) δ H NMR (CDCl3) δ 0.74, 0.89 (both s, 3H, CH3), 1.11
1
1346. 1H NMR (CDCl3) δ 0.76, 0.89 (both s, 3H, CH3), 1.15 (d, 3H, J = (d, 3H, J = 6.7 Hz, CH3), 1.229, 1.231 (both d, 3H, J = 7.0 Hz, CH3),
6.5 Hz, CH3), 1.72 (m, 2H), 1.78 (m, 2H), 1.92-2.04 (m, 3H), 2.09 (m, 1.71 (m, 2H), 1.78 (m, 2H), 1.93-2.03 (m, 3H), 2.09 (m, 1H), 2.24 (m,
1H), 2.32 (m, 1H, ΣJ = 37.0 Hz), 3.78 (q, 1H, J = 6.8 Hz, OCH), 3.88- 1H, ΣJ = 37.6 Hz), 2.87 (septet, 1H, J = 7.0 Hz, CH(CH3)2), 3.78 (q, 1H,
4.00 (m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H-2), 6.28 (dd, J = 6.8 Hz, OCH), 3.88-4.00 (m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66
1H, J = 15.7, J´ = 8.6 Hz, H-22), 6.37 (d, 1H, J = 15.7 Hz, H-23), 7.43 (m, 1H, H-2), 6.01 (dd, 1H, J = 15.8, J´ = 8.6 Hz, H-22), 6.27 (d, 1H, J =
(m, 2H, 2×Ar-H), 8.15 (m, 2H, 2×Ar-H). 13C NMR δ 12.26 (CH3), 13.59 15.8 Hz, H-23), 7.15 (d, 2H, J = 8.3 Hz, 2×Ar-H), 7.26 (d, 2H, J = 8.3
(CH3), 20.02 (CH3), 20.85 (CH2), 21.42 (CH2), 24.14 (CH2), 28.32 Hz, 2×Ar-H). 13C NMR δ 12.24 (CH3), 13.60 (CH3), 20.49 (CH3), 20.86
(CH2), 33.34 (CH), 35.90 (C), 39.68 (CH2), 40.75 (CH), 41.21 (CH2), (CH2), 21.43 (CH2), 23.97 (2×CH3), 24.14 (CH2), 28.35 (CH2), 33.35
41.23 (CH2), 42.79 (C), 48.08 (CH), 53.46 (CH), 55.63 (CH), 55.92 (CH), 33.79 (CH), 35.91 (C), 39.68 (CH2), 40.50 (CH), 41.19 (CH2),
(CH), 64.09 (CH2), 65.58 (CH2), 109.97 (C), 123.94 (2×CH), 124.74 41.24 (CH2), 42.61 (C), 48.07 (CH), 53.49 (CH), 55.97 (CH), 56.00
(CH), 125.70 (2×CH), 126.31 (2×CH), 142.50 (CH), 144.67 (C), 146.31 (CH), 64.07 (CH2), 65.56 (CH2), 110.02 (C), 124.81 (CH), 125.69 (CH),
(C). HRMS: (API+) calculated for C31H42NO4 ([M+H]+) 492.3114, 125.86 (2×CH), 126.50 (2×CH), 126.97 (CH), 135.69 (C), 136.44 (CH),
Found 492.3118.
147.43 (C). HRMS: (API+) calculated for C34H49O2 ([M+H]+) 489.3733,
Found 489.3736.
(22E)-6,6-Ethylenedioxy-23-(4-methylphenyl)-24-nor-5α-chola-
2,22-diene (14a)
(22E)-6,6-Ethylenedioxy-23-(4-cyanophenyl)-24-nor-5α-chola-2,22-
General procedure for WHE reaction with diethyl 4- diene (17a)
methylphenylphosphonate and chromatography on silica The general procedure for WHE reaction with diethyl 4-
(Et2O/cyclohexane - 1/19) afforded 200 mg (81 %) of the title cyanophenylphosphonate and chromatography on silica
1
compound 14a as a colorless oil: IR ν (cm-1) 2934, 1656, 1595. H (Et2O/cyclohexane - 1/19) afforded 220 mg (87 %) of the title
NMR (CDCl3) δ δ 0.74, 0.89 (both s, 3H, CH3), 1.11 (d, 3H, J = 6.7 Hz, compound 17a as an amorphous solid: IR ν (cm-1) 2933, 2224, 1646,
CH3), 1.71 (m, 2H), 1.77 (m, 2H), 1.92-2.03 (m, 3H), 2.09 (m, 1H), 1604. 1H NMR (CDCl3) δ 0.75, 0.89 (both s, 3H, CH3), 1.13 (d, 3H, J =
2.23 (m, 1H, ΣJ = 37.2 Hz), 2.31 (s, 3H, CH3), 3.77 (q, 1H, J = 6.5 Hz, 6.7 Hz, CH3), 1.71 (m, 2H), 1.78 (m, 2H), 1.93-2.03 (m, 3H), 2.09 (m,
OCH), 3.87-3.98 (m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H- 1H), 2.30 (m, 1H, ΣJ = 37.2 Hz), 3.78 (q, 1H, J = 6.8 Hz, OCH), 3.88-
2), 6.00 (dd, 1H, J = 15.6, J´ = 8.9 Hz, H-22), 6.26 (d, 1H, J = 15.6 Hz, 4.00 (m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H-2), 6.21 (dd,
H-23), 7.08 (d, 2H, J = 7.9 Hz, 2×Ar-H), 7.21 (d, 2H, J = 7.9 Hz, 2×Ar- 1H, J = 15.7, J´ = 8.6 Hz, H-22), 6.31 (d, 1H, J = 15.7 Hz, H-23), 7.39
H). 13C NMR δ 12.20 (CH3), 13.56 (CH3), 20.42 (CH3), 20.84 (CH2), (m, 2H, 2×Ar-H), 7.56 (m, 2H, 2×Ar-H). 13C NMR δ 12.25 (CH3), 13.59
21.07 (CH3), 21.40 (CH2), 24.11 (CH2), 28.32 (CH2), 33.32 (CH), 35.87 (CH3), 20.07 (CH3), 20.84 (CH2), 21.42 (CH2), 24.13 (CH2), 28.32
(C), 39.66 (CH2), 40.42 (CH), 41.16 (CH2), 41.21 (CH2), 42.58 (C), (CH2), 33.34 (CH), 35.90 (C), 39.67 (CH2), 40.66 (CH), 41.20 (CH2),
48.04 (CH), 53.47 (CH), 55.96 (2×CH), 64.03 (CH2), 65.53 (CH2), 41.22 (CH2), 42.76 (C), 48.08 (CH), 53.45 (CH), 55.65 (CH), 55.92
109.97 (C), 124.77 (CH), 125.67 (CH), 125.76 (2×CH), 126.96 (CH), (CH), 64.09 (CH2), 65.58 (CH2), 109.77 (C), 109.97 (C), 119.21 (C),
129.08 (2×CH), 135.24 (C), 136.25 (CH), one aromatic C not 124.75 (CH), 125.70 (CH), 125.99 (CH), 126.36 (2×CH), 132.30
detected. HRMS: (API+) calculated for C32H45O2 ([M+H]+) 461.3420, (2×CH), 141.46 (CH), 142.62 (C). HRMS: (API+) calculated for
Found 461.3422.
C32H42NO2 ([M+H]+) 472.3216, Found 472.3218.
(22E)-6,6-Ethylenedioxy-23-(4-methoxyphenyl)-24-nor-5α-chola-
2,22-diene (15a)
General procedure for dihydroxylation of dienes
To a solution of diene (160 mg), hydroquinidine 4-chlorobenzoate
(45 mg; 0.097 mmol), methansulfonamide (65 mg; 0.68 mmol),
potassium carbonate (280 mg; 2.03 mmol), and potassium
ferricyanide (700 mg; 2.13 mmol) in the mixture of t-butanol and
water (15 mL; 1:1 v/v) was added 0.2 mL of osmium tetroxide in t-
butanol (1 g/20 mL; 0.039 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 minutes 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 chromatography on silica gel gave
the desired product.
General procedure for WHE reaction with diethyl 4-
methoxyphenylphosphonate and chromatography on silica
(Et2O/cyclohexane - 1/19) afforded 203 mg (80 %) of the title
compound 15a as a colorless oil: IR ν (cm-1) 2934, 1656, 1594. H
1
NMR (CDCl3) δ 0.74, 0.89 (both s, 3H, CH3), 1.11 (d, 3H, J = 6.7 Hz,
CH3), 1.71 (m, 2H), 1.78 (m, 2H), 1.92-2.03 (m, 3H), 2.09 (m, 1H),
2.22 (m, 1H, ΣJ = 37.4 Hz), 3.77 (q, 1H, J = 6.7 Hz, OCH), 3.79 (s, 3H,
OCH3), 3.87-3.99 (m, 3H, 3×OCH), 5.54 (m, 1H, H-3), 5.66 (m, 1H, H-
2), 5.91 (dd, 1H, J = 15.7, J´ = 8.7 Hz, H-22), 6.24 (d, 1H, J = 15.7 Hz,
H-23), 6.82 (m, 2H, 2×Ar-H), 7.25 (m, 2H, 2×Ar-H). 13C NMR δ 12.21
(CH3), 13.58 (CH3), 20.50 (CH3), 20.85 (CH2), 21.41 (CH2), 24.12
(CH2), 28.38 (CH2), 33.33 (CH), 35.88 (C), 39.67 (CH2), 40.43 (CH),
41.18 (CH2), 41.22 (CH2), 42.58 (C), 48.06 (CH), 53.48 (CH), 55.24
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Organic & Biomolecular Chemistry
Page 7 of 11
Journal Name
ARTICLE
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-phenyl-24-nor-5α-cholan- 505.2721, Found 505.2723. Anal. Calcd for C29H41ClOV5i:ewC,Ar6ti8cl.e9O6n;liHne,
6-one (8c)
8.18. Found: C, 68.90; H, 8.28 %.
DOI: 10.1039/C6OB01479H
The general procedure for dihydroxylation of 8b and
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 144 mg (77
%) of the title compound 8c as a white solid: m. p. 268-270 °C
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-bromophenyl)-24-nor-
5α-cholan-6-one (11c)
1
(EtOH), IR ν (cm-1) 3344, 2940, 1708, 1496. H NMR (DMSO-d6) δ
The general procedure for dihydroxylation of 11b and
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 146 mg (80
%) of the title compound 11c as a white solid: m. p. 247-249 °C (i-
PrOH), IR ν (cm-1) 3237, 2941, 1709, 1600, 1490. 1H NMR (DMSO-d6)
δ 0.34, 0.60 (both s, 3H, CH3), 0.84 (d, 3H, J = 6.7 Hz, CH3), 1.79 (m,
1H), 1.87 (m, 1H), 1.98 (dd, 1H, J = 13.0, J´ = 4.6 Hz), 2.07 (t, 1H, J =
12.5Hz), 2.58 (dd, 1H, J = 12.2, J´ = 3.1 Hz), 3.42-3.47 (m, 2H), 3.74
(m, 1H), 4.19 (d, 1H, J = 2.8 Hz, OH), 4.33 (d, 1H, J = 6.1 Hz, OH), 4.36
(dd, 1H, J = 8.6, J´ = 3.8 Hz), 4.58 (d, 1H, J = 4.3 Hz, OH), 5.20 (d, 1H,
J = 3.8 Hz, OH), 7.23 (d, 2H, J = 8.6 Hz, 2×Ar-H), 7.51 (d, 2H, J = 8.6
Hz, 2×Ar-H). 13C NMR δ 11.49 (CH3), 12.50 (CH3), 13.38 (CH3), 20.81
(CH2), 23.31 (CH2), 26.84 (CH2), 27.30 (CH2), 36.50 (CH), 37.06 (CH),
39.15 (C), 41.87 (C), 42.08 (CH2), 45.99 (CH2), 50.32 (CH), 51.89 (CH),
52.89 (CH), 55.98 (CH), 67.13 (CH), 67.52 (CH), 74.51 (CH), 76.16
(CH), 120.08 (C), 129.26 (2×CH), 131.01 (2×CH), 142.83 (C), 211.62
(C). One CH2 covered by DMSO multiplet. HRMS: (API+) calculated
for C29H4279BrO5 ([M+H]+) 549.2216, Found 549.2216. Anal. Calcd for
C29H41BrO5: C, 63.38; H, 7.52. Found: C, 63.29; H, 7.55 %.
0.30, 0.59 (both s, 3H, CH3), 0.86 (d, 3H, J = 6.4 Hz, CH3), 1.80 (m,
1H), 1.86 (m, 1H), 1.97 (dd, 1H, J = 13.1, J´ = 4.6 Hz), 2.07 (t, 1H, J =
12.6Hz), 2.58 (dd, 1H, J = 12.1, J´ = 3.2 Hz), 3.44 (m, 1H), 3.48 (dd,
1H, J = 8.4, J´ = 4.4 Hz), 3.74 (m, 1H), 4.19 (d, 1H, J = 2.8 Hz, OH),
4.32 (d, 1H, J = 6.1 Hz, OH), 4.35 (dd, 1H, J = 8.6, J´ = 3.9 Hz), 4.51 (d,
1H, J = 4.3 Hz, OH), 5.14 (d, 1H, J = 3.9 Hz, OH), 7.22-7.27 (m, 3H),
7.31 (m, 2H). 13C NMR δ 11.39 (CH3), 12.47 (CH3), 13.36 (CH3), 20.79
(CH2), 23.28 (CH2), 26.82 (CH2), 27.22 (CH2), 36.29 (CH), 37.02 (CH),
39.14 (C), 41.84 (C), 42.03 (CH2), 45.95 (CH2), 50.28 (CH), 51.88 (CH),
52.85 (CH), 55.95 (CH), 67.10 (CH), 67.49 (CH), 75.16 (CH), 76.30
(CH), 127.01 (2×CH), 127.21 (CH), 128.07 (2×CH), 143.28 (C), 211.57
(C). One CH2 covered by DMSO multiplet. HRMS: (API+) calculated
for C29H43O5 ([M+H]+) 471.3110, Found 471.3108. Anal. Calcd for
C29H42O5: C, 74.01; H, 8.99. Found: C, 73.95; H, 9.06 %.
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-fluorophenyl)-24-nor-
5α-cholan-6-one (9c)
The general procedure for dihydroxylation of 9b
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 149 mg (80
and
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-iodophenyl)-24-nor-
5α-cholan-6-one (12c)
%) of the title compound 9c as a white solid: m. p. 277-279 °C
1
(EtOH), IR ν (cm-1) 3251, 2937, 1709, 1607, 1513. H NMR (DMSO-
The general procedure for dihydroxylation of 12b and
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 150 mg (83
%) of the title compound 12c as a white solid: m. p. 253-255 °C
(EtOH), IR ν (cm-1) 3193, 2943, 1710, 1590. 1H NMR (DMSO-d6) δ
0.35, 0.60 (both s, 3H, CH3), 0.84 (d, 3H, J = 6.7 Hz, CH3), 1.79 (m,
1H), 1.86 (m, 1H), 1.98 (dd, 1H, J = 13.2, J´ = 4.9 Hz), 2.07 (t, 1H, J =
12.5Hz), 2.58 (dd, 1H, J = 12.2, J´ = 3.4 Hz), 3.41-3.48 (m, 2H), 3.74
(m, 1H), 4.19 (d, 1H, J = 2.8 Hz, OH), 4.32 (d, 1H, J = 6.1 Hz, OH), 4.34
(dd, 1H, J = 8.6, J´ = 3.7 Hz), 4.56 (d, 1H, J = 4.3 Hz, OH), 5.18 (d, 1H,
J = 3.8 Hz, OH), 7.09 (m, 2H, 2×Ar-H), 7.67 (m, 2H, 2×Ar-H). 13C NMR
δ 11.49 (CH3), 12.48 (CH3), 13.36 (CH3), 20.80 (CH2), 23.29 (CH2),
26.83 (CH2), 27.28 (CH2), 36.51 (CH), 37.03 (CH), 39.14 (C), 41.85 (C),
42.05 (CH2), 45.96 (CH2), 50.30 (CH), 51.87 (CH), 52.86 (CH), 55.95
(CH), 67.11 (CH), 67.49 (CH), 74.60 (CH), 76.07 (CH), 92.95 (C),
129.40 (2×CH), 136.83 (2×CH), 143.19 (C), 211.57 (C). One CH2
covered by DMSO multiplet. HRMS: (API+) calculated for C29H42IO5
([M+H]+) 597.2077, Found 597.2076. Anal. Calcd for C29H41IO5: C,
58.39; H, 6.93. Found: C, 58.32; H, 7.01 %.
d6) δ 0.33, 0.60 (both s, 3H, CH3), 0.84 (d, 3H, J = 6.7 Hz, CH3), 1.80
(m, 1H), 1.85 (m, 1H), 1.98 (dd, 1H, J = 13.0, J´ = 4.8 Hz), 2.07 (t, 1H,
J = 12.6Hz), 2.58 (dd, 1H, J = 12.2, J´ = 3.1 Hz), 3.42-3.47 (m, 2H),
3.74 (m, 1H), 4.19 (d, 1H, J = 2.4 Hz, OH), 4.32 (d, 1H, J = 6.1 Hz, OH),
4.37 (dd, 1H, J = 8.7, J´ = 3.5 Hz), 4.54 (d, 1H, J = 4.3 Hz, OH), 5.14 (d,
1H, J = 4.0 Hz, OH), 7.14 (m, 2H), 7.30 (m, 2H). 13C NMR δ 11.42
(CH3), 12.44 (CH3), 13.38 (CH3), 20.80 (CH2), 23.30 (CH2), 26.84
(CH2), 27.28 (CH2), 36.35 (CH), 37.04 (CH), 39.15 (C), 41.86 (C), 42.06
(CH2), 45.97 (CH2), 50.31 (CH), 51.89 (CH), 52.88 (CH), 55.97 (CH),
67.11 (CH), 67.51 (CH), 74.40 (CH), 76.34 (CH), 114.84 (d, J = 21.6
Hz, 2×CH), 128.86 (d, J = 8.4 Hz, 2×CH), 139.58 (d, J = 2.4 Hz, C),
161.26 (d, J = 242.3 Hz, C), 211.62 (C). One CH2 covered by DMSO
multiplet. 19F NMR {1H} δ -115.37 (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, 71.88; H, 8.55 %.
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-chlorophenyl)-24-nor-
5α-cholan-6-one (10c)
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-nitrophenyl)-24-nor-
5α-cholan-6-one (13c)
The general procedure for dihydroxylation of 10b and
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 152 mg (82
%) of the title compound 10c as a white solid: m. p. 251-253 °C
The general procedure for dihydroxylation of 13b
and
1
(EtOH), IR ν (cm-1) 3220, 2940, 1712, 1598, 1494. H NMR (DMSO-
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 144 mg (78
%) of the title compound 13c as a white solid: m. p. 244-246 °C
(EtOH), IR ν (cm-1) 3180, 2940, 1711, 1605, 1523, 1349. 1H NMR
(DMSO-d6) δ 0.34, 0.59 (both s, 3H, CH3), 0.87 (d, 3H, J = 6.7 Hz,
CH3), 1.80 (m, 1H), 1.86 (m, 1H), 1.97 (dd, 1H, J = 13.2, J´ = 4.6 Hz),
2.06 (t, 1H, J = 12.5Hz), 2.58 (dd, 1H, J = 12.2, J´ = 3.4 Hz), 3.45 (m,
1H), 3.50 (m, 1H), 3.74 (m, 1H), 4.19 (d, 1H, J = 2.8 Hz, OH), 4.33 (d,
1H, J = 6.1 Hz, OH), 4.53 (dd, 1H, J = 7.9, J´ = 3.5 Hz), 4.74 (d, 1H, J =
4.6 Hz, OH), 5.47 (d, 1H, J = 3.5 Hz, OH), 7.57 (m, 2H), 8.19 (m, 2H).
13C NMR δ 11.46 (CH3), 12.64 (CH3), 13.37 (CH3), 20.80 (CH2), 23.30
(CH2), 26.83 (CH2), 27.32 (CH2), 36.77 (CH), 37.03 (CH), 39.14 (C),
41.86 (C), 42.09 (CH2), 45.97 (CH2), 50.31 (CH), 51.92 (CH), 52.87
(CH), 55.95 (CH), 67.11 (CH), 67.51 (CH), 74.46 (CH), 76.09 (CH),
123.31 (2×CH), 128.25 (2×CH), 146.62 (C), 151.53 (C), 211.61 (C).
d6) δ 0.34, 0.60 (both s, 3H, CH3), 0.85 (d, 3H, J = 6.7 Hz, CH3), 1.80
(m, 1H), 1.85 (m, 1H), 1.98 (dd, 1H, J = 13.0, J´ = 4.8 Hz), 2.07 (t, 1H,
J = 12.6Hz), 2.58 (dd, 1H, J = 12.2, J´ = 3.4 Hz), 3.42-3.48 (m, 2H),
3.74 (m, 1H), 4.19 (d, 1H, J = 2.4 Hz, OH), 4.33 (d, 1H, J = 6.1 Hz, OH),
4.37 (dd, 1H, J = 8.4, J´ = 3.8 Hz), 4.58 (d, 1H, J = 4.6 Hz, OH), 5.20 (d,
1H, J = 3.8 Hz, OH), 7.29 (m, 2H), 7.37 (m, 2H). 13C NMR δ 11.46
(CH3), 12.49 (CH3), 13.38 (CH3), 20.80 (CH2), 23.30 (CH2), 26.84
(CH2), 27.29 (CH2), 36.47 (CH), 37.05 (CH), 39.15 (C), 41.87 (C), 42.07
(CH2), 45.98 (CH2), 50.31 (CH), 51.89 (CH), 52.89 (CH), 55.97 (CH),
67.12 (CH), 67.51 (CH), 74.44 (CH), 76.21 (CH), 128.10 (2×CH),
128.87 (2×CH), 131.52 (C), 142.41 (C), 211.62 (C). One CH2 covered
by DMSO multiplet. HRMS: (API+) calculated for C29H42ClO5 ([M+H]+)
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Organic & Biomolecular Chemistry
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Journal Name
One CH2 covered by DMSO multiplet. HRMS: (ESI-) calculated for (CH), 52.89 (CH), 55.96 (CH), 67.12 (CH), 67.52 (CH), 75.05 (CH),
View Article Online
C29H41NO7 ([M•]-) 515.2883, Found 515.2888. Anal. Calcd for 76.18 (CH), 125.97 (2×CH), 127.04 (2×CH),D1O4I:01.60.610(3C9)/,C164O7B.021147(9CH),
C29H41NO7: C, 67.55; H, 8.01. Found: C, 67.45; H, 8.09 %.
211.62 (C). One CH2 covered by DMSO multiplet. HRMS: (API+)
calculated for C32H49O5 ([M+H]+) 513.3580, Found 513.3585. Anal.
Calcd for C32H48O5: C, 74.96; H, 9.44. Found: C, 74.89; H, 9.51 %.
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-methylphenyl)-24-nor-
5α-cholan-6-one (14c)
The general procedure for dihydroxylation of 14b and (22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-cyanophenyl)-24-nor-
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 145 mg (78 5α-cholan-6-one (17c)
%) of the title compound 14c as a white solid: m. p. 271-272 °C The general procedure for dihydroxylation of 17b and
1
(EtOH), IR ν (cm-1) 3215, 2937, 1710, 1610, 1516. H NMR (DMSO- chromatography on silica (MeOH/CHCl3 - 1/16) afforded 139 mg (75
d6) δ 0.32, 0.59 (both s, 3H, CH3), 0.84 (d, 3H, J = 6.7 Hz, CH3), 1.80 %) of the title compound 17c as a white solid: m. p. 260-262 °C
(m, 1H), 1.86 (m, 1H), 1.98 (dd, 1H, J = 13.2, J´ = 4.6 Hz), 2.07 (t, 1H, (EtOH), IR ν (cm-1) 3198, 2936, 2230, 1706, 1611, 1510. 1H NMR
J = 12.5Hz), 2.27 (s, 3H, CH3), 2.58 (dd, 1H, J = 12.2, J´ = 3.4 Hz), (DMSO-d6) δ 0.33, 0.59 (both s, 3H, CH3), 0.85 (d, 3H, J = 6.7 Hz,
3.42-3.48 (m, 2H), 3.74 (m, 1H), 4.19 (d, 1H, J = 2.8 Hz, OH), 4.31 CH3), 1.74-1.88 (m, 2H), 1.98 (dd, 1H, J = 13.2, J´ = 4.8 Hz), 2.06 (t,
(dd, 1H, J = 8.6, J´ = 3.8 Hz), 4.34 (d, 1H, J = 6.1 Hz, OH), 4.46 (d, 1H, 1H, J = 12.5Hz), 2.57 (dd, 1H, J = 12.2, J´ = 3.4 Hz), 3.46-3.50 (m, 2H),
J = 4.4 Hz, OH), 5.00 (d, 1H, J = 3.8 Hz, OH), 7.10-7.15 (m, 4H). 13C 3.74 (m, 1H), 4.20 (d, 1H, J = 2.4 Hz, OH), 4.34 (d, 1H, J = 6.1 Hz, OH),
NMR δ 11.50 (CH3), 12.44 (CH3), 13.40 (CH3), 20.83 (CH2, CH3), 23.32 4.46 (dd, 1H, J = 8.1, J´ = 4.3 Hz), 4.69 (d, 1H, J = 4.3 Hz, OH), 5.04 (d,
(CH2), 26.85 (CH2), 27.28 (CH2), 36.38 (CH), 37.07 (CH), 39.15 (C), 1H, J = 4.0 Hz, OH), 7.48 (d, 2H, J = 8.3 Hz, 2×Ar-H), 7.79 (d, 2H, J =
41.89 (C), 42.07 (CH2), 46.00 (CH2), 50.33 (CH), 51.90 (CH), 52.91 8.3 Hz, 2×Ar-H). 13C NMR δ 11.47 (CH3), 12.64 (CH3), 13.41 (CH3),
(CH), 56.00 (CH), 67.14 (CH), 67.52 (CH), 74.96 (CH), 76.36 (CH), 20.83 (CH2), 23.33 (CH2), 26.87 (CH2), 27.33 (CH2), 36.71 (CH), 37.08
126.99 (2×CH), 128.71 (2×CH), 136.15 (C), 140.20(C), 211.68 (C). (CH), 39.14 (C), 41.89 (C), 42.11 (CH2), 46.00 (CH2), 50.35 (CH), 51.94
One CH2 covered by DMSO multiplet. HRMS: (API+) calculated for (CH), 52.91 (CH), 55.98 (CH), 67.15 (CH), 67.54 (CH), 74.74 (CH),
C30H45O5 ([M+H]+) 485.3267, Found 485.3270. Anal. Calcd for 76.12 (CH), 109.86 (C), 119.03 (C), 128.05 (2×CH), 132.14 (2×CH),
C30H44O5: C, 74.34; H, 9.15. Found: C, 74.30; H, 9.20 %.
149.34 (C), 211.68 (C). One CH2 covered by DMSO multiplet. HRMS:
(API+) calculated for C30H42NO5 ([M+H]+) 496.3063, Found 496.3064.
Anal. Calcd for C30H41NO5: C, 72.70; H, 8.34. Found: C, 72.69; H, 8.39
%.
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-methoxyphenyl)-24-
nor-5α-cholan-6-one (15c)
The general procedure for dihydroxylation of 15b and
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 150 mg (81 Molecular docking
%) of the title compound 15c as a white solid: m. p. 242-244 °C
Docking was performed to obtain prediction of conformation and
1
(EtOH), IR ν (cm-1) 3197, 2934, 1710, 1616, 1513. H NMR (DMSO-
energy ranking between BRI1 receptor (PDB ID: 3RGZ) and the
steroid molecule. The docking studies were carried out using
AutoDock Vina 1.05.31 All 3D structures of BRI1 ligands were
obtained with Marvin 5.10.332, software which can be used for
drawing, displaying and characterization of chemical structure,
substructures and reactions. Ligands were prepared as derivatives
of natural ligand brassinolide (BLD). Polar hydrogens were added to
all ligands and proteins with the AutoDock Tools (ADT)16 program
prior to docking with Autodock Vina program. Grid box with size of
40 Å were centered on active site of protein. The exhaustiveness
parameter was set to 20 (default 8). After docking, we compared
the docked ligand with brassinolide crystal-like poses and the best
crystal-like poses of each ligand were analyzed.
d6) δ 0.33, 0.60 (both s, 3H, CH3), 0.84 (d, 3H, J = 6.7 Hz, CH3), 1.80
(m, 1H), 1.86 (m, 1H), 1.98 (dd, 1H, J = 13.2, J´ = 4.6 Hz), 2.07 (t, 1H,
J = 12.7Hz), 2.58 (dd, 1H, J = 12.2, J´ = 3.4 Hz), 3.42-3.49 (m, 2H),
3.72 (s, 3H, CH3), 3.74 (m, 1H), 4.18 (d, 1H, J = 2.5 Hz, OH), 4.30 (dd,
1H, J = 8.6, J´ = 3.8 Hz), 4.32 (d, 1H, J = 6.4 Hz, OH), 4.44 (d, 1H, J =
4.1 Hz, OH), 4.96 (d, 1H, J = 3.8 Hz, OH), 6.87 (m, 2H), 7.17 (m, 2H).
13C NMR δ 11.48 (CH3), 12.38 (CH3), 13.37 (CH3), 20.80 (CH2), 23.30
(CH2), 26.83 (CH2), 27.26 (CH2), 36.31 (CH), 37.04 (CH), 39.14 (C),
41.86 (C), 42.04 (CH2), 45.97 (CH2), 50.30 (CH), 51.89 (CH), 52.88
(CH), 54.97 (CH3), 55.98 (CH), 67.11 (CH), 67.50 (CH), 74.59 (CH),
76.29 (CH), 113.43 (2×CH), 128.12 (2×CH), 135.17 (C), 158.29 (C),
211.59 (C). One CH2 covered by DMSO multiplet. HRMS: (API+)
calculated for C30H45O6 ([M+H]+) 501.3218, Found 501.3216. Anal.
Calcd for C30H44O6: C, 71.97; H, 8.86. Found: C, 71.91; H, 8.97 %.
The pea inhibition biotest
(22R, 23R)-2α,3α,22,23-tetrahydroxy-23-(4-isopropylphenyl)-24-
nor-5α-cholan-6-one (16c)
Pea seedlings (Pisum arvense L. sort Arvica) germinating for 2 days
were selected for uniformity from a large population and then
transferred into pots containing perlite and 1/10 diluted Hoagland
solution (half concentration, pH 5.7) After 24 h in a dark cultivation
room (24 oC, humidity 75%) the seedlings were treated with
different amounts of tested compounds in 5 µl fractionated lanolin.
The substances 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.
The general procedure for dihydroxylation of 16b and
chromatography on silica (MeOH/CHCl3 - 1/16) afforded 146 mg (79
%) of the title compound 16c as a white solid: m. p. 247-249 °C
1
(EtOH), IR ν (cm-1) 3215, 2938, 1710, 1617, 1513. H NMR (DMSO-
d6) δ 0.31, 0.59 (both s, 3H, CH3), 0.84 (d, 3H, J = 6.7 Hz, CH3), 1.17
(d, 6H, J = 6.9 Hz, 2×CH3), 1.77-1.86 (m, 2H), 1.98 (dd, 1H, J = 13.2, J´
= 4.6 Hz), 2.07 (t, 1H, J = 12.5Hz), 2.58 (dd, 1H, J = 12.1, J´ = 3.2 Hz),
2.86 (septet, 1H, J = 6.9 Hz), 3.44 (m, 1H), 3.50 (dt, 1H, J = 8.3, J´ =
4.1 Hz), 3.74 (m, 1H), 4.19 (d, 1H, J = 2.8 Hz, OH), 4.31 (dd, 1H, J =
8.6, J´ = 3.8 Hz), 4.33 (d, 1H, J = 6.1 Hz, OH), 4.44 (d, 1H, J = 4.0 Hz,
OH), 5.00 (d, 1H, J = 3.8 Hz, OH), 7.17 (s, 4H). 13C NMR δ 11.46 (CH3),
12.50 (CH3), 13.37 (CH3), 20.80 (CH2), 23.32 (CH2), 23.92 (CH3), 24.02
(CH3), 26.85 (CH2), 27.20 (CH2), 33.11 (CH), 36.33 (CH), 37.05 (CH),
39.14 (C), 41.87 (C), 42.07 (CH2), 45.98 (CH2), 50.32 (CH), 51.97
8 | J. Name., 2012, 00, 1-3
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Determination of ethylene production
substances were added at time zero in 20 μl aliquots to the
microtiter plate wells and the highest finDalOcI:o1n0c.1e0n3Vt9ri/eaCwt6iAoOrntiBc0lein1O4n7tl9hinHee
wells was 50 μM. After incubation for 72 h, Calcein AM solution (2
μM, Molecular Probes) was added and the cells were incubated for
a further hour. The fluorescence of viable cells was then quantified
using a Fluoroskan Ascent instrument (Labsystems, Finland). The
percentage of surviving cells in each well was calculated by dividing
the intensity of the fluorescence signals from the exposed wells by
the intensity of signals from control wells and multiplying by 100.
These ratios were then used to construct dose-response curves
from which IC50 values, the concentrations of the respective
compounds that were lethal to 50 % of the tumor cells, were
calculated.
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 chromatograph (Agilent Technologies, GC System)
equipped with a flame 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; 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 quantitative
measure of ethylene concentration.The measurements were done
in triplicates and data were statistically analyzed using the Student’s
t test.
Conclusions
Several novel brassinosteroid 23-phenyl analogues were
synthesized based on molecular docking into BRI1 receptor.
The introduction of a phenyl group with no or small non-polar
substituents (fluorine, chlorine, methyl) resulted in new
compounds with plant growth promoting activities comparable
with natural brassinosteroids. Results of biological screenings
showed that molecular docking into BRI1 is a powerful tool for
prediction and design of new compounds with strong
brassinosteroid activities. New active compounds might be
good candidates for potential application in agriculture to
improve growth and yield or to increase resistance of plants
against various biotic and abiotic stresses. Because recent
progress in the chemical synthesis also leads to overcome
economically restriction, which are currently the major
constraints to use BRs at large scale in the fields.
Arabidopsis brassinosteroid sensitivity assays
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 BL (Fuji
Chemical Industries) and BR derivatives at 22 °C in a 16 h/8 h light-
dark cycle for 5 d. For the hypocotyl assay, after stratification,
plants were exposed to light for 6 h and grown in dark for 5 days.
Roots and hypocotyls were then straightened on solid media plates,
scanned with an Epson high-resolution scanner and the entire root
and
hypocotyl
length
measured
with
ImageJ
(http://rsbweb.nih.gov/ij/). For each treatment, more than 25
seedlings were analyzed in two biological repeats. P values were
calculated with a two-tailed Student t-test using Excel software.
Acknowledgements
BES-1 dephosphorylation assay
We thank Yanhai Yin for sharing BES1 antibodies. This work was
For BES1 dephosphorylation studies, thirty to sixty 5-day-old supported by projects of the Ministry of Education, Youth and
Arabidopsis seedlings grown on BL and new BR derivatives in Sports CR NPUI LO1204, by grant GJ15-08202Y and 14-27669P of
continuous light were used. DMSO was used as the control solvent. the Grant Agency of the Czech Republic. The work was also
The protein extraction and Western blot analysis were carried out supported by student projects IGA_PrF_2016_018 and
as previously described.33 Endogenous BES1 was detected using IGA_PrF_2016_028 of Palacky University in Olomouc. This work was
rabbit polyclonal anti-BES1 antibodies (1:1000)34 and HRP- supported by ELIXIR CZ research infrastructure project (MEYS Grant
conjugated anti-rabbit antibodies (1:10000; NA934, GE Healthcare). No: LM2015047) including access to computing and storage
Signals were detected using ECL (ECL plus, GE healthcare).
facilities. Qing Lu is indebted to the Chinese Scholarship Council
(CSC) for his predoctoral fellowship.
Cell Cultures
The screening cell lines: T-lymphoblastic leukemia CEM; breast
carcinoma MCF7 (estrogen-sensitive); cervical carcinoma cell line
HeLa; and human foreskin fibroblasts BJ were obtained from the
American Type Culture Collection (Manassas, VA, USA). Cells were
cultured in DMEM (Dulbecco's Modified Eagle Medium, Sigma, MO,
USA). Media used were supplemented with 10% fetal bovine serum,
2 mM L-glutamine, and 1% penicillin-streptomycin. The cell lines
were maintained under standard cell culture conditions at 37 °C and
5% CO2 in a humid environment. Cells were subcultured twice or
three times a week using the standard trypsinization procedure.
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