Biological activities of new monohydroxylated brassinosteroid analogues with a carboxylic group in the side chain

Article abstract of DOI:10.1016/j.steroids.2014.04.007

Thirteen monohydroxylated brassinosteroids analogues were synthesized and tested for their biological activity in plant and animal systems. The cytotoxic activity of the products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control, their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide as standard.

Full text of DOI:10.1016/j.steroids.2014.04.007

Steroids 85 (2014) 58–64
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Biological activities of new monohydroxylated brassinosteroid
analogues with a carboxylic group in the side chain
Miroslav Kvasnica a, Jana Oklestkova a, , Vaclav Bazgier a,b, Lucie Rarova a, Karel Berka c, Miroslav Strnad a
⇑
a Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR & Palacky´ University,
Šlechtitelu˚ 11, 78371 Olomouc, Czech Republic
b
ˇ
´
Department of Physical Chemistry, Faculty of Science, Palacky University, tr. 17. Listopadu 12, 77146 Olomouc, Czech Republic
c
´
Regional Centre of Advanced Technologies and Material, Department of Physical Chemistry, Faculty of Science, Palacky University, 17. Listopadu 12, 77146 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirteen monohydroxylated brassinosteroids analogues were synthesized and tested for their biological
activity in plant and animal systems. The cytotoxic activity of the products was studied using human nor-
mal and cancer cell lines with 28-homocastasterone as positive control, their brassinolide type activity
was established using the bean second-internode test with 24-epibrassinolide as standard.
Ó 2014 Elsevier Inc. All rights reserved.
Received 16 December 2013
Received in revised form 3 April 2014
Accepted 10 April 2014
Available online 21 April 2014
Keywords:
Anticancer activity
Brassinosteroids
Organic synthesis
Molecular docking
Receptor kinase BRI1
Plant bioassays
1. Introduction
brassinolide-bound (PDB ID: 3RJ0, 3BRZ,) forms are available, fol-
lowing independent X-ray diffraction structural determinations
Brassinosteroids (BRs) represent a large group of plant steroids
which include more than 70 structurally and functionally related
compounds [1]. BRs have been found in a wide range of plant spe-
cies, including higher and lower plants, and have been detected in
various plant parts such as pollen, seeds, leaves, stems, roots, flow-
ers and insect galls. They demonstrate various kinds of regulatory
action on the growth and development of plants, such as the stim-
ulation of cell enlargement and cell division, improvement of the
biomass formation, yield and quality of seeds, and plant adaptabil-
ity [2]. 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 hormones. Plants perceive ste-
roids at cell membrane, using the membrane-integral receptor
kinase brassinosteroid insensitive 1 (BRI1) [3–5]. The encoded pro-
tein, BRI1, belongs to a large family of plant LRR (leucine-rich
repeat) receptor-like kinases, characterized by an extracellular
LRR domain, a single-pass transmembrane segment and a cytoplas-
mic kinase domain. BRI1 has been established as an authentic bras-
sinosteroid receptor by genetic and biochemical investigations [6].
Crystal structures of BRI1 in both free (PDB ID: 3RIZ, 3RGX,) and
by two groups [6,7]. The structure of the ligand-binding domain
resembles a superhelix of 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 have indicated that BRs have antiviral,
antiproliferative and antibacterial activity [8]. BRs analogues have
been reported to have antiviral activity against herpes simplex
virus type 1, arenaviruses as well as against replication of vesicular
stomatitis virus in Vero cells [9–11]. Natural types of BRs and their
analogues affected the viability, proliferation, apoptosis and
expression of some cell cycle related proteins in cancer and normal
human cell lines [12–14]. It was shown that natural BRs, 24-epi-
brassinolide and 28-homocastasterone, inhibited in vitro angiogen-
esis of human endothelial cells [15].
The aim of our study relates to the synthesis of new brassinos-
teroid monohydroxylated derivatives and to study of their biolog-
ical and anticancer properties. The plant growth promoting activity
of synthetic analogs was assayed using the bean second internode
bioassay. The antiproliferative activity of the new BRs analogs was
evaluated in vitro using cancer cell lines of different histopatholo-
gical origins and normal human fibroblasts. All derivative
⇑
Corresponding author. Tel.: +420 58563483; fax: +420 585634870.
E-mail address: jana.oklestkova@upol.cz (J. Oklestkova).
http://dx.doi.org/10.1016/j.steroids.2014.04.007
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

M. Kvasnica et al. / Steroids 85 (2014) 58–64
59
structures were further subjected to docking studies using Auto-
Dock Vina [16] in order to find structural patterns responsible for
the results.
calculated for C23H36O4Na (M++Na) 399.25058. Found 399.25059.
Anal. Calcd for C23H36O4: C, 73.37; H, 9.64. Found: C, 73.45; H,
9.60%.
2.2.2. Methyl (20S)-2a-hydroxy-6-oxo-5a-pregnane-20-carboxylate
(2b)
2. Experimental
Yield: 400 mg (46%), m.p. 153–155 °C (MeOH), [
0.62). IR
(cmꢀ1) 3608, 1727, 1708, 1168. 1H NMR (CDCl3) d
0.68, 0.75 (both s, 3H, CH3), 1.20 (d, 3H, J = 6.9 Hz, CH3), 1.95–
2.10 (m, 4H), 2.15 (dd, 1H, J = 11.9, J0 = 2.6 Hz, H-5
), 2.29 (dd,
a
]
D = ꢀ8° (c
2.1. General methods
m
The melting points were determined on a Hund H 600 appara-
tus (Helmut Hund, Germany). The elemental analyses (C, H, N)
were carried out on a Perkin-Elmer 2400 II elemental analyzer.
Optical rotations were measured on an Autopol IV polarimeter
(Rudolf Research Analytical, Flanders, USA) at 25 °C in chloroform
a
1H, J = 13.1, J0 = 4.6 Hz, H-7 ), 2.43 (dq, 1H, J = 10.3, J0 = 6.8 Hz, H-
a
20), 3.65 (s, 3H, OCH3), 3.77 (m, 1H, W = 31.5 Hz, H-2b). 13C
½
NMR d 12.16 (CH3), 14.14 (CH3), 17.06 (CH3), 19.49 (CH2), 21.20
(CH2), 23.96 (CH2), 26.95 (CH2), 34.57 (C), 37.57 (CH), 39.18
(CH2), 42.31 (CH), 42.98 (CH2), 46.56 (CH2), 47.07 (C), 51.35
(CH3), 52.76 (CH), 54.12 (CH), 56.26 (CH), 57.95 (CH), 66.93 (CH),
177.13 (C), 211.58 (C). HRMS: (ESI+) calculated for C23H36O4Na
(M++Na) 399.25058. Found 399.25057. Anal. Calcd for C23H36O4:
C, 73.37; H, 9.64. Found: C, 73.42; H, 9.57%.
and [a]
values are given in 10ꢀ1 deg cm2 g. The infrared spectra
D
were recorded on a Bruker IFS 55 spectrometer in chloroform.
The wave numbers are given in cmꢀ1. The 1H NMR spectra were
taken in CDCl3 on a Bruker AVANCE-400 (at 400 MHz) instrument
with tetramethylsilane as an internal reference. Chemical shifts are
given in ppm (d-scale), coupling constants (J) in Hz. All of the val-
ues were obtained by first-order analysis. The mass spectra (ESI)
were obtained with a LTQ Orbitrap XL (Thermo Fisher Scientific).
2.3. General procedure for Baeyer–Villiger oxidation
For column chromatography, neutral silica gel (60 lm, Fluka)
A solution of trifluoroperoxyacetic acid, freshly prepared from
trifluoroacetic anhydride (0.45 mL, 3.2 mmol) and hydrogen perox-
ide (30%; 0.1 mL) in dichloromethane (3 mL), was added to a solu-
tion of 6-oxocompound (0.40 mmol) in dichloromethane (8 mL).
After standing at room temperature for 5 h, the reaction mixture
was poured into water and taken up in chloroform (50 mL). The
chloroform extract was washed with water, a saturated solution
of potassium hydrogen carbonate, water, and dried over anhydrous
sodium sulfate. Solvents were evaporated under reduced pressure
and products were separated and purified on HPLC in ethyl ace-
tate–hexane 7:3.
was used. The HPLC system consisted of a Gilson semi-preparative
HPLC system including a quaternary pump, liquid handler, UV–VIS,
RI, and ELSD detectors. The semi preparative column was based on
silica gel (Labio Biosphere PSI 200).
Reagents and solvents were purchased from Sigma–Aldrich and
were not purified.
2.2. Hydroboration of 1
Solution of borane in THF (1 M, 20 mL) was added dropwise to
the solution of methyl ester 1 (1 g, 2.49 mmol) in dry THF (30 mL).
The reaction mixture was stirred at room temperature for 6 h.
Then, the reaction mixture was quenched by water (10 mL) and
cooled to 10 °C. Aqueous solution of NaOH (10%, 20 mL) was added
followed by dropwise addition of 30% hydrogen peroxide (30 mL).
This reaction mixture was stirred overnight at room temperature.
It was then poured into a mixture of sodium sulfite (2 g) in water
(50 mL) and acetic acid (1 mL) and stirred for additional 30 min.
After then, the reaction mixture was diluted with ethyl acetate,
extracted twice with water, the extract was dried over anhydrous
sodium sulfate, and solvents evaporated under reduced pressure. A
crude mixture of products was dissolved in a mixture of THF and
acetone (5:1, 10 mL) and 5% aqueous HCl was added. The reaction
mixture was heated under reflux for 3 h. Then it was diluted with
ethyl acetate, extracted twice with water, the extract was dried
over anhydrous sodium sulfate, and the solvents were evaporated
under reduced pressure. Two main products were separated on
HPLC in ethyl acetate–hexane (1:1). Two hydroxy compounds 2a
and 2b were obtained.
2.4. Baeyer–Villiger oxidation of 2a
Ketone 2a (150 mg; 0.40 mmol) was used for Baeyer–Villiger
oxidation as described in general procedure. Reaction afforded
two lactones 3a and 3b.
2.4.1. Methyl (20S)-3a-hydroxy-7-oxa-7a-homo-6-oxo-5a-pregnane-
20-carboxylate (3a)
Yield: 69 mg (44%), m.p. 185–187 °C (MeOH), [
a]D = +10° (c
0.27). IR
m
(cmꢀ1) 3615, 1723, 1182. 1H NMR (CDCl3) d 0.72, 0.90
(both s, 3H, CH3), 1.19 (d, 3H, J = 6.9 Hz, CH3), 1.93 (dt, 1H, J = 6.6,
J0 = 3.0 Hz), 2.14 (ddd, 1H, J = 15.0, J0 = 12.3, J00 = 2.6 Hz, H-4a), 2.43
(dq, 1H, J = 10.3, J0 = 6.9 Hz, H-20), 3.19 (dd, 1H, J = 12.2, J0 = 4.4 Hz,
H-5a), 3.65 (s, 3H, OCH3), 4.07–4.11 (m, 2H, H-7a, H-7b), 4.17 (m,
1H, W = 10.9 Hz, H-3b). 13C NMR d 11.97 (CH3), 14.54 (CH3), 17.02
½
(CH3), 22.11 (CH2), 24.88 (CH2), 26.92 (CH2), 28.27 (CH2), 32.53
(CH2), 32.92 (CH2), 36.34 (C), 39.47 (CH2), 39.50 (CH), 41.79 (CH),
42.35 (CH), 42.68 (C), 51.15 (CH), 51.39 (CH3), 52.80 (CH), 58.36
(CH), 64.87 (CH), 70.32 (CH2), 176.60 (C), 177.02 (C). HRMS:
2.2.1. Methyl (20S)-3
a
-hydroxy-6-oxo-5a-pregnane-20-carboxylate
(ESI+) calculated for C
23H36O5Na (M++Na) 415.24550. Found
(2a)
415.24540. Anal. Calcd for C23H36O5: C, 70.38; H, 9.24. Found: C,
70.29; H, 9.30%.
Yield: 380 mg (40%), m.p. 198–200 °C (MeOH), [
0.27). IR
a]
D = ꢀ6° (c
m
(cmꢀ1) 3615, 1727, 1706, 1165. 1H NMR (CDCl3) d
0.68, 0.73 (both s, 3H, CH3), 1.19 (d, 3H, J = 6.9 Hz, CH3), 1.97 (m,
2.4.2. Methyl (20S)-3a-hydroxy-6-oxa-7a-homo-7-oxo-5a-pregnane-
20-carboxylate (3b)
1H), 2.02 (dd, 1H, J = 12.8, J0 = 1.0 Hz, H-7b), 2.29 (dd, 1H, J = 12.8,
J0 = 4.5 Hz, H-7
a
), 2.43 (dq, 1H, J = 10.3, J0 = 6.8 Hz, H-20), 2.73
Yield: 50 mg (32%), m.p. 225–227 °C (MeOH), [
a]D = +10° (c
(dd, 1H, J = 8.9, J0 = 6.9 Hz, H-5
a
), 3.65 (s, 3H, OCH3), 4.16 (t, 1H,
0.22). IR
m
(cmꢀ1) 3614, 1723, 1168. 1H NMR (CDCl3) d 0.71, 0.90
J = 2.1 Hz, H-3b). 13C NMR d 12.17 (CH3), 12.30 (CH3), 17.06
(CH3), 21.01 (CH2), 23.96 (CH2), 26.95 (CH2), 27.69 (CH2), 28.16
(CH2), 31.67 (CH2), 37.90 (CH), 39.27 (CH2), 41.49 (C), 42.35 (CH),
43.00 (C), 46.75 (CH2), 51.34 (CH), 51.66 (CH3), 52.79 (CH), 53.78
(CH), 56.37 (CH), 65.35 (CH), 177.13 (C), 212.37 (C). HRMS: (ESI+)
(both s, 3H, CH3), 1.18 (d, 3H, J = 6.9 Hz, CH3), 1.83 (ddd, 1H,
J = 14.2, J0 = 11.4, J00 = 2.7 Hz, H-4a), 1.92 (d, 1H, J = 12.3, J0 = 3.0 Hz),
2.03 (ddd, 1H, J = 14.2, J0 = 5.1, J00 = 3.1 Hz, H-4b), 2.43 (dq, 1H,
J = 10.3, J0 = 6.9 Hz, H-20), 2.46–2.53 (m, 2H, H-7
a
, H-7b), 3.65 (s,
3H, OCH3), 4.22 (m, 1H, H-3b), 4.62 (dd, 1H, J = 11.4, J0 = 5.1 Hz,

60
M. Kvasnica et al. / Steroids 85 (2014) 58–64
H-5
a
). 13C NMR d 11.56 (CH3), 11.97 (CH3), 16.99 (CH3), 22.17
3430, 1726, 1711, 1182. 1H NMR (CDCl3) d 0.74, 0.88 (both s, 3H,
CH3), 1.21 (d, 3H, J = 6.8 Hz, CH3), 1.90–2.13 (m, 7H), 2.43 (dq,
1H, J = 10.3, J0 = 6.9 Hz, H-20), 3.18 (dd, 1H, J = 12.2, J0 = 4.3 Hz, H-
(CH2), 25.34 (CH2), 26.57 (CH2), 27.99 (CH2), 31.22 (CH2), 34.87
(CH), 35.69 (CH2), 38.10 (CH2), 39.48 (CH2), 39.94 (C), 42.33 (CH),
42.73 (C), 51.33 (CH3), 53.13 (CH), 55.18 (CH), 58.02 (CH), 66.29
(CH), 79.62 (CH), 175.07 (C), 177.05 (C). HRMS: (ESI+) calculated
for C23H36O5Na (M++Na) 415.24550. Found 415.24542. Anal. Calcd
for C23H36O5: C, 70.38; H, 9.24. Found: C, 70.31; H, 9.35%.
5a), 3.95 (m, 1H, H-3b), 4.06–4.10 (m, 2H, H-7a
, H-7b). 13C NMR
d 11.97 (CH3), 14.56 (CH3), 17.00 (CH3), 22.10 (CH2), 24.92 (CH2),
27.08 (CH2), 28.20 (CH2), 32.49 (CH2), 32.91 (CH2), 36.32 (C),
39.48 (CH2), 39.53 (CH), 41.76 (CH), 42.23 (CH), 42.76 (C), 51.20
(CH), 52.37 (CH), 58.32 (CH), 64.94 (CH), 70.29 (CH2), 176.62 (C),
181.14 (C). HRMS: (ESI+) calculated for C22H34O5Na (M++Na)
401.22985. Found 401.22976. Anal. Calcd for C22H34O5: C, 69.81;
H, 9.05. Found: C, 69.77; H, 9.12%.
2.5. Baeyer–Villiger oxidation of 2b
Ketone 2b (150 mg; 0.40 mmol) was used for Baeyer–Villiger
oxidation as described in general procedure. The reaction afforded
two lactones 5a and 5b.
2.6.2. (20S)-3a-Hydroxy-6-oxa-7a-homo-7-oxo-5a-pregnane-20-
carboxylic acid (4b)
2.5.1. Methyl (20S)-2
20-carboxylate (5a)
a
-hydroxy-7-oxa-7a-homo-6-oxo-5
a
-pregnane-
Compound 4b was prepared from methyl ester 3b (70 mg;
0.18 mmol) according to the above general procedure for ester
hydrolysis. Yield: 50 mg (74%), m.p. 272–275 °C (EtOH),
Yield: 62 mg (40%), m.p. 173–175 °C (MeOH), [
a]D = +20° (c
0.31). IR
m
(cmꢀ1) 3610, 1726, 1170. 1H NMR (CDCl3) d 0.71, 0.92
[a]D = +5° (c 0.21; CHCl3:MeOH–1:1). IR (KBr) m
(cmꢀ1) 3428,
(both s, 3H, CH3), 1.19 (d, 3H, J = 6.9 Hz, CH3), 1.58–1.82 (m, 8H),
1.94 (dt, 1H, J = 6.6, J0 = 4.4 Hz), 1.99–2.07 (m, 2H), 2.23 (m, 1H),
2.43 (dq, 1H, J = 10.3, J0 = 6.9 Hz, H-20), 2.67 (dd, 1H, J = 11.9,
1736, 1699, 1151, 1033. 1H NMR (CDCl3 + MeOD) d 0.71, 0.90 (both
s, 3H, CH3), 1.18 (d, 3H, J = 6.9 Hz, CH3), 1.83 (ddd, 1H, J = 14.2,
J0 = 11.4, J00 = 2.7 Hz, H-4a), 1.92 (d, 1H, J = 12.3, J0 = 3.0 Hz), 2.03
(ddd, 1H, J = 14.2, J0 = 5.1, J00 = 3.1 Hz, H-4b), 2.43 (dq, 1H, J = 10.3,
J0 = 4.6 Hz, H-5
a), 3.65 (s, 3H, OCH3), 3.67 (tt, 1H, J = 11.4,
J0 = 4.0 Hz, H-2b). 4.03 (dd, 1H, J = 12.7, J0 = 8.9 Hz, H-7a), 4.09
(dd, 1H, J = 12.7, J0 = 2.1 Hz, H-7b). 13C NMR d 11.95 (CH3), 16.04
(CH3), 17.02 (CH3), 22.19 (CH2), 24.86 (CH2), 24.91 (CH2), 26.91
(CH2), 34.09 (CH2), 38.94 (C), 39.20 (CH), 39.34 (CH2), 42.31 (CH),
42.64 (C), 46.79 (CH), 49.53 (CH2), 51.07 (CH), 51.39 (CH3), 52.77
(CH), 58.89 (CH), 67.02 (CH), 70.33 (CH2), 176.03 (C), 176.98 (C).
J0 = 6.9 Hz, H-20), 2.46–2.53 (m, 2H, H-7
a
, H-7b), 3.65 (s, 3H,
OCH3), 4.22 (m, 1H, H-3b), 4.62 (dd, 1H, J = 11.4, J0 = 5.1 Hz, H-
). 13C NMR d 11.56 (CH3), 11.97 (CH3), 16.99 (CH3), 22.17
5a
(CH2), 25.34 (CH2), 26.57 (CH2), 27.99 (CH2), 31.22 (CH2), 34.87
(CH), 35.69 (CH2), 38.10 (CH2), 39.48 (CH2), 39.94 (C), 42.33 (CH),
42.73 (C), 51.33 (CH3), 53.13 (CH), 55.18 (CH), 58.02 (CH), 66.29
(CH), 79.62 (CH), 175.07 (C), 177.05 (C). HRMS: (ESI+) calculated
for C22H34O5Na (M++Na) 401.22985. Found 401.22971. Anal. Calcd
for C22H34O5: C, 69.81; H, 9.05. Found: C, 69.77; H, 9.09%.
HRMS: (ESI+) calculated for
Found 415.24534. Anal. Calcd for C23H36O5: C, 70.38; H, 9.24.
Found: C, 70.45; H, 9.31%.
C
23H36O5Na (M++Na) 415.24550.
2.5.2. Methyl (20S)-2
a
-hydroxy-6-oxa-7a-homo-7-oxo-5a-pregnane-
2.6.3. (20S)-2a-Hydroxy-7-oxa-7a-homo-6-oxo-5a-pregnane-20-
20-carboxylate (5b)
carboxylic acid (6a)
Yield: 77 mg (49%), m.p. 201–203 °C (MeOH), [
(cmꢀ1) 3611, 1725, 1168, 1041. 1H NMR (CDCl3) d 0.71, 0.95
(both s, 3H, CH3), 1.19 (d, 3H, J = 6.9 Hz, CH3), 1.90–2.04 (m, 3H),
2.20 (m, 1H, H-1a), 2.40–2.54 (m, 3H, H-7 , H-7b, H-20), 3.65 (s,
3H, OCH3), 3.74 (tt, 1H, J = 11.3, J0 = 4.1 Hz, H-2b). 4.23 (dd, 1H,
J = 11.1, J0 = 5.2 Hz, H-5 ). 13C NMR d 11.96 (CH3), 13.26 (CH3),
a
]
D = +6° (c 0.13).
Compound 6a was prepared from methyl ester 5a (60 mg;
0.15 mmol) according to the above general procedure for ester
hydrolysis. Yield: 42 mg (73%), m.p. 276–278 °C (EtOH),
IR
m
a
[a]D = +19° (c 0.34; CHCl3:MeOH–1:1). IR (KBr) m
(cmꢀ1) 3435,
1728, 1684, 1201, 1177. 1H NMR (CDCl3 + MeOD) d 0.73, 0.91 (both
s, 3H, CH3), 1.21 (d, 3H, J = 6.8 Hz, CH3), 1.91–2.04 (m, 3H), 2.21 (m,
1H), 2.38 (dq, 1H, J = 13.5, J0 = 6.8 Hz, H-20), 2.71 (dd, 1H, J = 11.7,
a
17.00 (CH3), 22.26 (CH2), 25.29 (CH2), 26.58 (CH2), 27.84 (CH2),
33.27 (CH2), 34.44 (CH), 38.10 (CH2), 39.37 (CH2), 41.20 (C),
42.32 (CH), 42.71 (C), 47.14 (CH2), 51.39 (CH3), 53.11 (CH), 55.10
(CH), 58.38 (CH), 66.60 (CH), 82.58 (CH), 174.47 (C), 177.02 (C).
J0 = 4.4 Hz, H-5
4.12 (m, 2H, H-7
a
), 3.67 (tt, 1H, J = 11.3, J0 = 3.9 Hz, H-2b). 4.05–
, H-7b). 13C NMR d 11.63 (CH3), 15.64 (CH3),
a
16.77 (CH3), 21.96 (CH2), 24.64 (2 ꢁ CH2), 26.81 (CH2), 33.32
(CH2), 38.62 (C), 38.92 (CH), 39.19 (CH2), 42.26 (CH), 42.41 (C),
46.60 (CH), 48.90 (CH2), 50.83 (CH), 52.31 (CH), 58.59 (CH), 66.21
(CH), 70.34 (CH2), 177.03 (C), 179.05 (C). HRMS: (ESIꢀ) calculated
for C22H33O5 (M+ꢀH) 377.23335. Found 377.23300. Anal. Calcd for
HRMS: (ESI+) calculated for
C
23H36O5Na (M++Na) 415.24550.
Found 415.24536. Anal. Calcd for C23H36O5: C, 70.38; H, 9.24.
Found: C, 70.37; H, 9.36%.
2.6. General procedure for ester hydrolysis
C22H34O5: C, 69.81; H, 9.05. Found: C, 69.72; H, 9.16%.
Methyl ester (0.15 mmol) in methanol (5 mL) with potassium
hydroxide (80 mg, 1.5 mmol) was heated at reflux under nitrogen
for 10 h. The solution was neutralized with 5% aqueous HCl
(10 mL) and the reaction mixture was left to stand at room temper-
ature for 30 min. The mixture was evaporated to dryness, water
(20 mL) was added, and the product was extracted with chloro-
form (2 ꢁ 50 mL). The chloroform solution was dried with anhy-
drous sodium sulfate and evaporated under reduced pressure.
Product crystallized from ethanol (unless otherwise stated).
2.6.4. (20S)-2
carboxylic acid (6b)
a-Hydroxy-6-oxa-7a-homo-7-oxo-5a-pregnane-20-
Compound 6b was prepared from methyl ester 5b (40 mg;
0.10 mmol) according to the above general procedure for ester
hydrolysis. Yield: 33 mg (85%), m.p. 284–285 °C (EtOH),
[a]D = +12° (c 0.15; CHCl3:MeOH–1:1). IR (KBr) m
(cmꢀ1) 3425,
1720, 1697, 1188, 1038. 1H NMR (CDCl3 + MeOD) d 0.73, 0.94 (both
s, 3H, CH3), 1.21 (d, 3H, J = 6.9 Hz, CH3), 1.89–2.02 (m, 3H), 2.17 (m,
1H, H-1a), 2.39 (dq, 1H, J = 10.3, J0 = 6.9 Hz, H-20), 2.47–2.52 (m,
2H, H-7
a
, H-7b), 3.68 (dd, 1H, J = 14.9, J0 = 7.6, J00 = 3.7 Hz, H-2b).
2.6.1. (20S)-3a-Hydroxy-7-oxa-7a-homo-6-oxo-5a-pregnane-20-
4.28 (dd, 1H, J = 10.7, J0 = 4.7 Hz, H-5 ). 13C NMR d 11.64 (CH3),
a
carboxylic acid (4a)
12.85 (CH3), 16.78 (CH3), 22.01 (CH2), 25.09 (CH2), 26.47 (CH2),
27.60 (CH2), 32.55 (CH2), 34.26 (CH), 37.79 (CH2), 39.20 (CH2),
40.82 (C), 42.26 (CH), 42.47 (C), 46.49 (CH2), 52.65 (CH), 54.85
(CH), 58.04 (CH), 65.73 (CH), 82.87 (CH), 175.44 (C), 179.08 (C).
Compound 4a was prepared from methyl ester 3a (50 mg;
0.13 mmol) according to the above general procedure for ester
hydrolysis. Yield: 35 mg (73%), m.p. 129–131 °C (lyophilized from
t-BuOH), [
a
]D = +25° (c 0.14; CHCl3:MeOH–1:1). IR (KBr)
m
(cmꢀ1
)
HRMS: (ESI+) calculated for C
22H34O5Na (M++Na) 401.22985.

M. Kvasnica et al. / Steroids 85 (2014) 58–64
61
Found 401.22968. Anal. Calcd for C22H34O5: C, 69.81; H, 9.05.
Found: C, 69.71; H, 9.20%.
130.58 (2 ꢁ CH), 135.72 (C), 150.56 (C), 163.71 (C), 174.31 (C),
176.95 (C). HRMS: (ESI+) calculated for C30H39NO8Na (M++Na)
564.25679. Found 564.25612. Anal. Calcd for
C30H39NO8: C,
2.6.5. Methyl (20S)-3
a
-(4-nitrobenzoyloxy)-6-oxo-5
a-pregnane-20-
66.52; H, 7.26; N, 2.59. Found: C, 66.43; H, 7.35; N, 2.50%.
carboxylate (8)
Diethyl azodicarboxylate (40% in toluene; 0.6 mL; 1.53 mmol)
was added to a solution of hydroxyketone 7 (200 mg; 0.53 mmol),
triphenylphosphine (400 mg; 1.53 mmol), and 4-nitrobenzoic acid
(260 mg; 1.56 mmol) in dry THF. Reaction was stirred at 80 °C for
4 h. THF was evaporated under reduced pressure and the mixture
was subjected to column chromatography in ethyl acetate-hexane
1:3 to afford 4-nitrobenzoate 8 (245 mg; 88%), m.p. 193–195 °C
2.8. Hydrolysis of 4-nitrobenzoates 4a and 4b
Compound 4a and 4b were prepared from methyl esters 9a and
9b (each 50 mg; 0.09 mmol) according to general procedure for
hydrolysis of ester. Spectral characteristics for both acids were in
agreement with the data reported in experiments above.
(MeOH), [
a
]
D = ꢀ23° (c 0.26). IR
m
(cmꢀ1) 1720, 1609, 1600, 1530,
1277. 1H NMR (CDCl3) d 0.63, 0.74 (both s, 3H, CH3), 1.13 (d, 3H,
J = 6.9 Hz, CH3), 1.85–2.01 (m, 4H), 2.27 (dd, 1H, J = 13.2, J0 = 4.5 Hz,
2.9. Molecular docking
H-7
a
), 2.37 (dq, 1H, J = 10.3, J0 = 6.8 Hz, H-20), 2.56 (dd, 1H, J = 12.4,
Docking was performed to obtain prediction of conformation
and energy ranking between BRI1 receptor (PDB ID: 3RGZ) and
the steroid molecule. The docking studies were carried out using
AutoDock Vina 1.05 [16]. All 3D structures of BRI1 ligands were
obtained with Marvin 5.10.3 [7], 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) [17] pro-
gram 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
the docked ligand with brassinolide crystal-like poses and the best
crystal-like poses of each ligand were analyzed.
J0 = 3.1 Hz, H-5
a), 3.58 (s, 3H, OCH3), 5.35 (m, 1H, W = 5.3 Hz, H-
½
3b), 8.12 (m, 2H, 2 ꢁ HAr), 8.24 (m, 2H, 2 ꢁ HAr). 13C NMR d 12.20
(CH3), 12.43 (CH3), 17.09 (CH3), 21.09 (CH2), 23.97 (CH2), 25.10
(CH2), 25.40 (CH2), 26.98 (CH2), 32.69 (CH2), 37.88 (CH), 39.19
(CH2), 41.30 (C), 42.32 (CH), 43.02 (C), 46.61 (CH2), 51.39 (CH),
52.83 (CH3), 52.87 (CH), 53.94 (CH), 56.32 (CH), 70.93 (CH),
123.58 (2 ꢁ CH), 130.59 (2 ꢁ CH), 136.14 (C), 150.51 (C), 163.75
(C), 177.12 (C), 210.99 (C). HRMS: (ESI+) calculated for C30H39NO7-
Na (M++Na) 548.26187. Found 548.26169. Anal. Calcd for
C30H39NO7: C, 68.55; H, 7.48; N, 2.66. Found: C, 68.50; H, 7.56;
N, 2.54%.
2.7. Baeyer–Villiger oxidation of 8
Ketone 8 (200 mg; 0.38 mmol) was used for Baeyer–Villiger oxi-
dation as described in general procedure. The reaction afforded
two lactones 9a and 9b.
2.10. 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,
2.7.1. Methyl (20S)-3
-pregnane-20-carboxylate (9a)
Yield: 109 mg (53%), m.p. 227–230 °C (MeOH), [
1.27). IR
(cmꢀ1) 1724, 1609, 1600, 1531, 1278. 1H NMR (CDCl3)
a-(4-nitrobenzoyloxy)-7-oxa-7a-homo-6-oxo-
5
a
a]D = +25° (c
m
d 0.73, 0.97 (both s, 3H, CH3), 1.19 (d, 3H, J = 6.9 Hz, CH3), 2.33
(ddd, 1H, J = 15.1, J0 = 12.3, J00 = 2.6 Hz, H-4a), 2.44 (dq, 1H,
2 mM L-glutamine, and 1% penicillin–streptomycin. The cell lines
J = 10.2, J0 = 6.8 Hz, H-20), 3.10 (dd, 1H, J = 12.2, J0 = 4.4 Hz, H-5
a),
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.
3.65 (s, 3H, OCH3), 4.06–4.17 (m, 2H, H-7
a, H-7b), 5.39 (m, 1H,
W
= 5.2 Hz, H-3b), 8.20 (m, 2H, 2 ꢁ HAr), 8.32 (m, 2H, 2 ꢁ HAr).
½
13C NMR d 11.95 (CH3), 14.57 (CH3), 17.01 (CH3), 22.16 (CH2),
24.85 (CH2), 25.30 (CH2), 26.88 (CH2), 29.77 (CH2), 33.99 (CH2),
36.33 (C), 39.32 (CH2), 39.46 (CH), 42.27 (CH), 42.63 (C), 42.89
(CH), 51.07 (CH), 51.38 (CH3), 52.79 (CH), 58.78 (CH), 70.46
(CH2), 70.52 (CH), 123.61 (2 ꢁ CH), 130.54 (2 ꢁ CH), 135.99 (C),
150.52 (C), 163.78 (C), 175.55 (C), 176.91 (C). HRMS: (ESI+) calcu-
lated for C30H39NO8Na (M++Na) 564.25679. Found 564.25603.
Anal. Calcd for C30H39NO8: C, 66.52; H, 7.26; N, 2.59. Found: C,
66.46; H, 7.36; N, 2.55%.
2.11. Calcein AM cytotoxicity assay
Suspensions with approximately 1.0 ꢁ 105 cells/mL were dis-
tributed in 96-well microtiter plates and after 24 h of stabilization
the BRs analogues tested were added at the desired concentrations
in DMSO. Control cultures were treated with DMSO alone, and the
final concentration of DMSO in the reaction mixture never
exceeded 0.6%. In most cases six serial 4-fold dilutions of the test
2.7.2. Methyl (20S)-3
-pregnane-20-carboxylate (9b)
Yield: 70 mg (34%), m.p. 205–208 °C (MeOH), [
1.64). IR
(cmꢀ1) 1725, 1609, 1600, 1531, 1272. 1H NMR (CDCl3)
a
-(4-nitrobenzoyloxy)-7-oxo-7a-homo-6-oxa-
substances were added at time zero in 20
titer plate wells and the highest final concentration in the wells
was 50 M. After incubation for 72 h, Calcein AM solution (2 M,
lL aliquots to the micro-
5
a
a]
D = ꢀ10° (c
l
l
m
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 divid-
ing 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.
d 0.73, 0.98 (both s, 3H, CH3), 1.18 (d, 3H, J = 6.9 Hz, CH3), 2.19–
2.31 (m, 2H), 2.43 (dq, 1H, J = 10.3, J0 = 6.9 Hz, H-20), 2.46–2.58
(m, 2H, H-7
a
, H-7b), 3.65 (s, 3H, OCH3), 4.55 (dd, 1H, J = 11.3,
J0 = 5.3 Hz, H-5
a
), 5.44 (m, 1H, W = 5.1 Hz, H-3b), 8.19 (m, 2H,
½
2 ꢁ HAr), 8.33 (m, 2H, 2 ꢁ HAr). 13C NMR d 11.60 (CH3), 11.92
(CH3), 16.96 (CH3), 22.20 (CH2), 24.95 (CH2), 25.29 (CH2), 26.53
(CH2), 32.07 (CH2), 32.93 (CH2), 34.76 (CH), 38.15 (CH2), 39.31
(CH2), 39.86 (C), 42.24 (CH), 42.67 (C), 51.35 (CH3), 53.11 (CH),
55.04 (CH), 58.30 (CH), 71.61 (CH), 79.60 (CH), 123.62 (2 ꢁ CH),

62
M. Kvasnica et al. / Steroids 85 (2014) 58–64
Table 1
uniformity from a large population of seedlings and then trans-
Activity in the bean second-internode bioassay.
ferred into pots containing perlite and 1/10 diluted Hoagland solu-
tion (half concentration, pH 5.7). Seedlings were grown in a light-
controlled cultivation room (25–27 °C, light 48 W mꢀ2, light/dark
period 16/8 h). Seven days old plants with second internodes
2 mm long were treated with different amounts of tested com-
Compound
Maximal prolongation
of the second
internode (mm)
SD
(mm)
Amount applied
for maximal
prolongation (mol)
24-Epibrassinolide
54.8
13.1
17.1
14.5
9.7
10.1
3.9
8.1
6.6
11.6
8.3
3.4
7.3
10.8
6.6
7.7
4.6
3.6
6.1
1.1
5.3
3.2
2.2
4.8
0.9
6.3
0.8
10ꢀ10
10ꢀ9
10ꢀ10
10ꢀ11
10ꢀ11
10ꢀ10
10ꢀ9
10ꢀ8
10ꢀ8
10ꢀ9
10ꢀ9
10ꢀ12
10ꢀ10
10ꢀ9
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
8
pounds in 5 lL fractionated lanolin. The substances were applied
in microdrops to the scar left after the removal of bract from the
base of the second internode. 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 length of the
second internodes was measured after 5 days and the difference in
length between treated and control plants provided a measure of
activity (Table 1).
9a
9b
3. Results and discussion
4.4
3.1. Chemistry
2.12. The bean second-internode bioassay
For the preparation of above mentioned compounds we set out
Brassinolide-type activity was measured by the bean second-
internode bioassay modified by us [18]. Seeds of bean (Phaseolus
vulgaris L., cv. Pinto) germinated for 2 days were selected for
from the known [19] methyl (20S)-6,6-ethylenedioxy-5a-pregn-2-
en-20-carboxylate (1). This olefin was used for hydroboration reac-
tion [20] was followed by acidic hydrolysis of the ketal group. Two
Scheme 1. (i) (a) B2H6, THF, r.t.; (b) 30% H2O2, NaOH, H2O, r.t.; Na2SO3, AcOH, H2O, r.t.; (ii) TFAA, 30% H2O2, CH2Cl2, r.t.; (iii) (a) KOH, MeOH, refl.; (b) 5% HCl, H2O, r.t.
Scheme 2. (i) DEAD, Ph3P, 4-nitrobenzoic acid, THF, 80 °C; (ii) TFAA, 30% H2O2, CH2Cl2, r.t.; (iii) (a) KOH, MeOH, refl.; (b) 5% HCl, H2O, r.t.

M. Kvasnica et al. / Steroids 85 (2014) 58–64
63
main products were isolated and characterized as 3
a-hydroxyester
even when tested in concentrations of up to 50
lM. No BRs deriv-
2a and 2 -hydroxyester 2b. Their structure was determined by 1D
a
ative mediated loss of viability was observed in the BJ fibroblasts.
Brassinolide-type activity was measured by the second inter-
node bean bioassay [24]. 24-Epibrassinolide showed very good
biological activity and therefore was used as a positive control. This
compound elicited nearly 200% increase in elongation of the
and 2D NMR spectra. Both hydroxyesters were then used for Bae-
yer–Villiger oxidation. In both cases, two isomers of lactone were
obtained, 3a and 3b from hydroxyester 2a, and 5a and 5b from
hydroxyester 2b. Unlike 2a,3a-dihydroxy derivatives when the
mostly desired 7-oxa-6-oxo isomer is obtained in very high yield,
monohydroxy derivatives afforded both isomers in ratio almost
1:1, even a higher yield of 6-oxa-7-oxo isomer 5b in case of 2a-
Table 2
Resulting binding free energies for best poses and for crystal-like pose and its ranking
between all poses. Star (*) denotes structures with common first pose binding more
deeply in the receptor groove similarly as shown on Fig. 2.
hydroxyester 2b. This fact proved that presence of hydroxy group
on A ring is essential for oxidation to a less sterically hindered posi-
tion. [21] All four lactones were then hydrolyzed with potassium
hydroxide to prepare four acids 4a, 4b, 6a, and 6b.
Acids 4a and 4b were prepared also by alternative way using
Mitsunobu acylation. As a starting material we used known [22]
Compound
D
Gbind
(kcal/mol)
D
Gbind
(kcal/mol)
X-tal
best
Brassinolide
24-Epibrassinolide
28-Homocastasterone
ꢀ10.6
ꢀ9.8
ꢀ10.6 (1.)
ꢀ9.8 (1.)
ꢀ9.3 (2.)
ꢀ9.1 (2.)
ꢀ9.0 (2.)
ꢀ8.9 (4.)
ꢀ9.0 (4.)
ꢀ9.0 (4.)
ꢀ8.2 (11.)
ꢀ8.9 (2.)
ꢀ8.0 (10.)
ꢀ9.0 (3.)
ꢀ9.0 (3.)
ꢀ8.6 (2.)
ꢀ8.3 (3.)
ꢀ7.7 (14.)
ꢀ9.6
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
8
ꢀ10.1⁄
ꢀ10.1⁄
ꢀ9.7⁄
ꢀ10.5⁄
ꢀ9.8⁄
ꢀ10.4⁄
ꢀ9.8⁄
ꢀ10.2⁄
ꢀ9.9⁄
ꢀ10.1⁄
ꢀ8.9
methyl (20S)-3b-hydroxy-6-oxo-5a-pregnan-20-carboxylate (7).
Standard Mitsunobu procedure [23] used also for steroidal alcohols
afforded 4-nitrobenzoate 8 in a very high yield. The following reac-
tions were the same as in previous experiments. Baeyer–Villiger
oxidation was followed with alkaline hydrolysis of lactones 9a
and 9b prepared. Both products were identical with acids 4a and
4b. All chemical reactions and conditions can be seen in Scheme 1
and 2. All compounds were characterized by NMR, IR and MS tech-
niques, together with elemental analysis and optical rotation.
9a
9b
ꢀ8.9
ꢀ8.9
3.2. Biological activity and docking
Antiproliferative activity was determined by comparing human
normal fibroblasts (BJ) and cancer cell lines (T-lymphoblastic leu-
kemia CEM, breast carcinoma MCF7, cervical carcinoma cell line
HeLa). Cells of all these lines 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 were calculated. 28-
homocastasterone was used as a positive control, which is most
potent natural brassinosteroid towards CEM cells (IC50 13 lM,
[12]). All tested monohydroxylated BRs had no detectable activity,
Fig. 1. Comparison of brassinolide analogues with brassinolide crystal pose (black;
PDB ID: 3RGZ). Brassinolide interacts with mostly nonpolar groove in a receptor.
Only one hydrogen bond is present between the 23-hydroxyl group and main chain
nitrogen in S647 residue. Brassinolide docked into the same pose as in crystal (cf.
white and black). Both 24-epibrassinolide (yellow) and 28-homocastasterone
(orange) showed similar pose with slightly less binding affinity accountable to
the small variation in the tail region, while still retaining the hydrogen bond found
in crystal. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
Fig. 2. Comparison of monosubstituted brassinolide analogue 3b (yellow) with
brassinolide crystal pose (black; PDB ID: 3RGZ). Two major poses were detected –
(A) more deeply immersed pose with best binding free energy and with hydrogen
bonding pattern to S647, (B) brassinolide-like binding pose with lower affinity. (For
interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)

64
M. Kvasnica et al. / Steroids 85 (2014) 58–64
second internode, causing its elongation, swelling, twisting and
splitting [25]. The newly prepared monohydroxylated BRs analogs
were very weakly active inducing elongation of the second bean
internode only (Table 1); the best activity was found in 2b
(+17.1 mm) and in 3a (+14.5 mm). In the case of 2b, applied
amount was 10ꢀ10 mol and in the case of 3a 10ꢀ11 mol per plant.
The weak activity of derivatives was further analyzed with use
of molecular docking of their structures into the receptor domain
of kinase brassinosteroid insensitive 1 (BRI1). Firstly, we docked
into the structure of brassinolide, 24-epibrassinolide and 28-
homocastasterone in order to analyze their best fir. All these com-
pounds docked into the receptor groove in a similar fashion to
brassinolide crystal pose (Fig. 1) with only a slightly less affinity
to the receptor than brassinolide itself (Table 2). Binding pose is
mainly dictated by the number of nonpolar residues in a receptor
groove forming a hydrophobic pocket interacting with nonpolar
parts of rings and tail of brassinolide and its derivatives. Hydroxyl
groups are mostly pointing outward from the receptor groove to
the solvent, whereas 23-hydroxyl group interacts with the main
chain nitrogen in S647 residue. Brassinolide, 24-epibrassinolide
and 28-homocastasterone all have the nonpolar tail pointing dee-
per into the hydrophobic pocket close to W564 and L615 residues.
In such a pose, 2,3-dihydroxy group, thought to be important for
biological response [7], positioned in the same area pointing out-
ward from the binding pocket. It was thus shown, that Autodock
Vina is able to dock ligands into the binding pocket in crystal-like
pose within energetically best poses (Table 2).
References
[1] (a) Bajguz A. Metabolism of brassinosteroids in plants. Plant Physiol Biochem
2007;45:95–107;
(b) Khripach VA, Zabinskij VN, de Groot AE. Brassinosteroids – A New Class of
Plant Hormones. Academic Press; 1999;
(c) Back TG, Pharis RP. Structure–activity studies of brassinosteroids and the
search for novel analogues and mimetics with improved bioactivity. J Plant
Growth Regul 2003;22:350–61;
(d) Sakurai A, Yokota T, Clouse SD. Brassinosteroids
Hormones. Tokyo: Springer-Verlag; 1999.
– Steroidal Plant
[2] Clouse SD, Sasse JM. Brassinosteroids: essential regulators of plant growth and
development. Ann Rev Plant Physiol Plant Mol Biol 1998;49:427–51.
[3] Li JM, Chory J. A putative leucine-rich repeat receptor kinase involved in
brassinosteroid signal transduction. Cell 1997;90:929–38.
[4] Kinoshita T, Cano-Delgado A, Seto H, Hiranuma S, Fujioka S, Yoshida S, Chory J.
Binding of brassinosteroids to the extracellular domain of plant receptor
kinase BRI1. Nature 2005;433:167–71.
[5] She J, Han ZF, Zhou B, Chai JJ. Structural basis for differential recognition of
brassinolide by its receptors. Protein Cell 2013;4:475–82.
[6] She J, Han ZF, Kim TW, Wang JJ, Cheng W, Chang JB, et al. Structural insight into
brassinosteroid perception by BRI1. Nature 2011;474:472–6.
[7] Hothorn M, Belkhadir Y, Dreux M, Dabi T, Noel JP, Wilson IA, Chory J. Structural
basis of steroid hormone perception by the receptor kinase BRI1. Nature
2011;474:467–71.
[8] Bhardwaj R, Arora N, Uppal P, Sharma I, Kanwar MK. Prospects of
brassinosteroids in medicinal applications. In: Hayat S, Ahmad A, editors.
Brassinosteroids:
439–59.
A class of Plant Hormone. Germany: Springer; 2011. p.
[9] Wachsman MB, Lopez EM, Ramirez JA, Galagovsky LR, Coto CE. Antiviral effect
of brassinosteroids against herpes virus and arenaviruses. Antivir Chem
Chemother 2000;11:71–7.
[10] Wachsman MB, Lopez EM, Ramirez JA, Galagovsky LR, Coto CE. Antiviral
activity of brassinosteroids derivatives against measles virus in cell cultures.
Antivir Chem Chemother 2002;13:61–6.
[11] Wachsman MB, Castilla V, Talarico LB, Ramirez JA, Galagovsky LR, Coto CE.
However in the case of the monohydroxylated derivatives syn-
thetized in this work, we have found that these compounds
showed two distinct binding poses: (i) one with the best binding
free energy characterized with a more deeply immersed pose and
retained hydrogen bonding pattern to S647, and (ii) a brassino-
lide-like binding pose with a lower affinity lacking interactions
with hydrophobic pocket due to the shorter and more polar tail
(Fig. 2). Moreover, the introduction of 4-nitrobenzoic acid on 3-
hydroxyl group further lowered the affinity towards the BRI recep-
tor. As most of the interactions between the receptor binding
groove and the ligand are mediated through nonpolar interactions,
the larger the area of contact between ligand and pocket would
govern the affinity. In such case, the shorter aliphatic tail is moving
prepared the monohydroxylated brassinosteroid derivatives out of
the productive pose more deeply into the receptor groove, thus
effectively blocking 2- or 3-hydroxyl groups, which are thought
to be important for the biological response of the receptor [7]
and diminishing their proposed function in brassinolide-type
activity. In future, we will focus on the aliphatic tail elongation
in the brassinosteroid derivatives synthesis, as this seems to be
the key feature of the brassinolide-like substrates interacting with
BRI receptor.
Antiherpetic
mode
of
action
of
(22S,23S)-3b-bromo-5a,22,23-
trihydroxystigmastan-6-one in vitro. Int J Antimicrob Agents 2004;5:524–6.
[12] Malikova J, Swaczynova J, Kolar Z, Strnad M. Anticancer and antiproliferative
activity of natural brassinosteroids. Phytochemistry 2008;69:418–26.
[13] Steigerova J, Oklestkova J, Levkova M, Rarova L, Kolar Z, Strnad M.
Brassinosteroids cause cell cycle arrest and apoptosis of human breast
cancer cells. Chem Biol Interact 2010;188:487–96.
[14] Steigerova J, Rarova L, Oklestkova J, Krizova K, Levkova M, Svachova M, Kolar Z,
Strnad M. Mechanisms of natural brassinosteroid-induced apoptosis of
prostate cancer cells. Food Chem Toxicol 2012;50:4068–76.
ˇ
[15] Rarova L, Zahler S, Liebl J, Kryštof V, Sedlák D, Bartu˚ nek P, Kohout L, Strnad M.
Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells.
Steroids 2012;77:1502–9.
[16] Trott O, Olson AJ. Software News and Update AutoDock Vina: improving the
speed and accuracy of docking with
a new scoring function, efficient
optimization, and multithreading. J Comput Chem 2010;31:455–61.
[17] Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ.
Autodock4 and AutoDockTools4: automated docking with selective receptor
flexibility. J Comput Chem 2009;16:2785–91.
[18] Sisa M, Hnilickova J, Swaczynova J, Kohout L. Syntheses of new androstane
brassinosteroids with 17b-ester group – butyrates, heptafluorobutyrates, and
laurates. Steroids 2005;70:755–62.
[19] Eignerova B, Slavikova B, Budesinsky M, Dracinsky M, Klepetarova B, Stastna E,
Kotora M. Synthesis of fluorinated brassinosteroids based on alkene cross-
metathesis and preliminary biological assessment.
2009;52:5753–7.
J
Med Chem
[20] Hanson JR, Liman MD, Nagaratnam S. Regio- and stereochemical effects in the
hydroboraton of
1997;22:282–3.
D
2-steroidal allylic and homoallylic alcohols. J Chem Res (S)
[21] Takatsuto S, Ikekawa N. Remote substituent effect on the regioselectivity in
Acknowledgments
the Baeyer–Villiger oxidation of 5
Lett 1983;24:917–20.
a-cholestan-6-one derivatives. Tetrahedron
The program ‘‘Návrat’’ for Research, Development, and Innova-
tions’’ (LK21306), which is funded by the Ministry of Education,
Youth and Sports of the Czech Republic, is highly appreciated.
This work was supported by project of the Ministry of Educa-
tion, Youth and Sports CR NPUI LO1204 and by the Czech Science
Foundation (GACR 14-27669P).
V.B. acknowledges support by the Student Project PrF_2013_
028 of Palacky University.
K.B. acknowledges support by Operational Program Research
and Development for Innovations – European Regional Develop-
ment Fund (CZ.1.05/2.1.00/03.0058).
[22] Cerny V, Strnad M, Kaminek M. Preparation of 2
a, 3a-dihydroxy-7-oxa-6-oxo-
23,24-dinor-B-homo-5 -cholanic acid, its esters and amides as brassinolide
a
analogs. Collect Czech Chem Commun 1986;51:687–97.
[23] Ismaili J, Boisvert M, Longpre F, Carange J, Gall CL, Martinoli MG, Daoust B.
Brassinosteroids and analogs as neuroprotectors: synthesis and structure–
activity relationships. Steroids 2012;77:91–9.
[24] Mitchell JW, Mandava N, Worley JF, Plimmer JR, Smith MV. Brassins a new
family of plant hormones from rape pollen. Nature 1970;225:1065–6.
[25] Swaczynová J, Novák O, Hauserová E, Fuksová K, Šíša M, Strnad M. New
techniques for the estimation of naturally occurring brassinosteroids. J Plant
Growth Regul 2006;26:1–14.