Synthesis of five known brassinosteroid analogs from hyodeoxycholic acid and their activities as plant-growth regulators

Article abstract of DOI:10.3390/ijms18030516

Brassinosteroids (BRs) are plant hormones that promote growth in different plant organs and tissues. The structural requirements that these compounds should possess to exhibit this biological activity have been studied. In this work, a series of known BR analogs 5-15, were synthesized starting from hyodeoxycholic acid 4, and maintaining the alkyl side chain as cholic acid or its methyl ester. The growth-promoting effects of brassinolide (1) and synthesized analogs were evaluated by using the rice lamina inclination assay at concentrations ranging from 1 × 10^-8-1 × 10^-6 M. Our results indicate that in this concentration range the induced bending angle of rice seedlings increases with increasing concentration of BRs. Analysis of the activities, determined at the lowest tested concentration, in terms of BR structures shows that the 2α,3α-dihydroxy-7-oxa-6-ketone moiety existing in brassinolide is required for the plant growing activity of these compounds, as it has been proposed by some structure-activity relationship studies. The effect of compound 8 on cell elongation was assessed by microscopy analysis, and the results indicate that the growth-promoting effect of analog 8 is mainly due to cell elongation of the adaxial sides, instead of an increase on cell number.

Full text of DOI:10.3390/ijms18030516

International Journal of
Molecular Sciences
Article
Synthesis of Five Known Brassinosteroid Analogs
from Hyodeoxycholic Acid and Their Activities as
Plant-Growth Regulators
1
1
2
3
1,
María Isabel Duran , Cesar González , Alison Acosta , Andrés F. Olea , Katy Díaz *
1
,
and Luis Espinoza *
1
Departamento de Química, Universidad Técnica Federico Santa María, Avenida España 1680,
Valparaíso 2340000, Chile; maria.duranp@alumnos.usm.cl (M.I.D.); cesar.gonzalez@usm.cl (C.G.)
Escuela de Ingeniería en Biotecnología, Facultad de Ciencias Biológicas, Universidad Andrés Bello,
2
Quillota 980, Viña del Mar 2520000, Chile; al.acosta@uandresbello.edu
Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile,
3
Santiago 8910339, Chile; andres.olea@uautonoma.cl
*
Correspondence: katy.díaz@usm.cl (K.D.); luis.espinozac@usm.cl (L.E.);
Tel.: +56-032-265-4225; Fax: +56-032-265-4782 (K.D. & L.E.)
Academic Editor: María Serrano
Received: 23 January 2017; Accepted: 23 February 2017; Published: 8 March 2017
Abstract: Brassinosteroids (BRs) are plant hormones that promote growth in different plant
organs and tissues. The structural requirements that these compounds should possess to exhibit
this biological activity have been studied. In this work, a series of known BR analogs
were synthesized starting from hyodeoxycholic acid , and maintaining the alkyl side chain as
cholic acid or its methyl ester. The growth-promoting effects of brassinolide ( ) and synthesized
analogs were evaluated by using the rice lamina inclination assay at concentrations ranging
5–15,
4
1
−
8
−6
M
from 1 × 10 –1 × 10
. Our results indicate that in this concentration range the induced
bending angle of rice seedlings increases with increasing concentration of BRs. Analysis of the
activities, determined at the lowest tested concentration, in terms of BR structures shows that the
2
α α-dihydroxy-7-oxa-6-ketone moiety existing in brassinolide is required for the plant growing
,3
activity of these compounds, as it has been proposed by some structure-activity relationship studies.
The effect of compound on cell elongation was assessed by microscopy analysis, and the results
8
indicate that the growth-promoting effect of analog
sides, instead of an increase on cell number.
8 is mainly due to cell elongation of the adaxial
Keywords: brassinosteroid analogs; synthesis; plant-growth regulators; lamina inclination test
1
. Introduction
Brassinosteroids (BRs) are a naturally occurring polyhydroxysteroidal plant hormone group
that regulates plant growth and development by producing an array of physiological changes [
1
].
−
−
1
Natural BRs occur at low concentrations throughout the plant kingdom, with a range of 1–100 µg·kg
1
fresh weight, while shoots and leaves usually contain even lower amounts, i.e., 0.01–0.1 µg·kg
fresh weight [2]. They have been detected in all plant organs (pollen, anthers, seeds, leaves,
stems, roots, flowers, and grains) and also in the insect and crown galls. Further work has
demonstrated that BRs elicit a broad spectrum of physiological and morphological responses in
plants, including stem elongation, leaf bending and epinasty, induction of ethylene biosynthesis
and proton pump activation, synthesis of nucleic acid and proteins, regulation of carbohydrate
assimilation and allocation, and activation of photosynthesis [1]. The leaf bending caused by extracts
from Distylium racemosum in the rice lamina inclination assay is one of the earliest reports of brassinolide
Int. J. Mol. Sci. 2017, 18, 516; doi:10.3390/ijms18030516
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2017, 18, 516
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(1
) activity in plants [
3
]. In this assay,
1
caused dose-dependent swelling of the adaxial cells in the
joint between the leaf blade and sheath of etiolated rice seedlings. The angle of the leaf bending
caused by the swelling is used as a sensitive bioassay during brassinolide isolation and quantification
procedures [
isolated, was based on brassinolide-mediated stimulation of bean elongation at the 2nd and 3rd
internodes, with higher concentrations causing the splitting of the stem [ ]. Brassinolide and
synthetic analogs were subsequently used in various bioassays, which indicated that in addition to
leaf bending, cell elongation, and cell division, also affects source/sink relationships [ ], proton
pumping and membrane polarization [ ], photosynthesis [10] and stress responses including
thermotolerance [11 12] and senescence [13]. The role of BRs in improving the productivity of various
4]. An early report on growth-promoting activity of “brassin”, from which 1 was
5,6
1
7
8,9
,
crop plants such as vegetables, fruits, and oil seed plants are well documented [14–17].
All natural bioactive BRs possess a vicinal 22R, 23R diol structural functionality (for example
brassinolide (
biological activity. In this sense, several studies have been focused on determining the structural
requirements that these compounds should possess to elicit strong biological activity [18 20]. It has
been concluded that the activity of BRs depends on the oxygen atoms spatial situation, and a new
way to relate the structure of BRs with their activity has been proposed [21 22]. Namely, the activity is
1), castasterone (2) and typhasterol (3), Figure 1), which seems to be essential for high
–
,
attributed to the different conformations that BR molecules can adopt, one of them being the active
conformation. Therefore the structural requirements are not associated to the presence or absence of a
specific functional group in the molecule [18,19], but to the spatial distribution of all the functionalities
present in it [22]. These spatial orientations can be indicated as distances or angles between the
oxygen atoms present in a brassinosteroid. However, in recent decades, efforts have been focused
on the synthesis of new BR analogs, keeping common patterns of organic functions in the A/B rings
and cis-trans fusion between them, as occurs in some natural BRs, but with moderate or dramatic
structural changes (shorter side chains, different oxygenated functions, spirostanic, aromatic and cyclic
substituents, methyl esters, carboxylic acids) [21
presented very important biological activity.
,23–25]. Surprisingly, some of these analogs have
Figure 1. Brassinolide (1), castasterone (2), typhasterol (3) and hyodeoxycholic acid (4).
Therefore, in this work we report the synthesis of a series of known brassinosteroid analogs,
(
6
,
7
,
9
,
10
,
12
,
13 and 15), starting from hyodeoxycholic acid (
11, and 14 (see Figures 1 and 2) [26]. In all these compounds
15 the native side chain is kept as cholic acid or its methyl ester. In addition, the effects of analogs
12 and 15 on elongation in the rice lamina inclination assay were evaluated at concentrations
4) and following a procedure recently
described for the synthesis of analogs
5
5
ranging from 1
compounds in the applied test at the same concentration (1
5, 8,
–
,
6, 8,
−
8
−6
10 M. The results indicate that analogs 12 and 15 are the more active
×
10 –1
×
−
8
×
10 M). Additionally, in this work
we inform the full NMR spectroscopic characterization for these derivatives, which was previously
reported only in partial form.

Int. J. Mol. Sci. 2017, 18, 516
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Figure 2. Brassinosteroid analogs obtained from hyodeoxycholic acid (4).
. Results and Discussion
2
2
.1. Chemical Synthesis
Hyodeoxycholic acid (
4) has been used for the synthesis of brassinolide and a number of analogs
because it contains the organic functions, which can be chemically modified, at suitable positions in
order to fulfill the structural requirements for their activity [27].
Compound
methods [26 28
previously described by us [26]. Compound
in which the alcohol group at C-3 position of compound
5
, with 5
32], but we have obtained it from hyodeoxycholic acid (
is obtained from through a dehydration reaction
is transformed into the double bond
α
-cholestan-6-one skeleton (Scheme 1), has been synthesized by different
,
–
1
) following a procedure
9
5
5
at C-2 position of compound
conversion of 3 or 3
9
. A large number of reports have shown that the indirect or direct
2
α
β
-hydroxy steroid in
∆
alkenes can take place by following at least three different
strategies. The indirect path transforms the alcohol into a good leaving group such as tosylate, which
in a subsequent treatment with Li CO /LiBr/DMF in reflux system [25 28 33 35] or treatment of
alkenes [26 36 39]. On the other
-hydroxy steroid that is carried
out by adsorbing the substrate on silica gel in a reaction catalyzed by copper (II) sulfate [32 40 42].
was performed by using a mild one-pot method
was obtained with 83% yield (Scheme 1) [43].
were consistent
and respectively,
,
, –
, –
2
3
2
mesylate derivatives under the same conditions produces the
∆
hand, an efficient alternative is the direct dehydration of 3 or 3
α
β
,
–
In this work, the conversion of compound
5 to 9
◦
with Tf O/DMAP in CH Cl at 0 C, and the alkene
9
2
2
2
1
13
The spectroscopic data (IR, H and C-NMR) and physical properties of compound
with those reported [26 28 32]. Compounds and were synthesized from
according to the methodology previously described by us [26].
The synthesis of lactones 11 and 14 from 3 -hydroxy-6-oxo-5
reported [26] (Figure 2). In this case, the Baeyer-Villiger oxidation of
9
,
,
6
7
9
5
α
α
-cholanate
5
5 has been previously
with m-CPBA/CH Cl gave a
2
2
mixture of known 7-oxalactone 11 and 6-oxalactone 14 with 10.8% and 14.9% yields, respectively [26].
Other authors reported the synthesis of these compounds with 26% and 44% yields respectively [29].
However, it is known that oxidation of 5
formation of the 7-oxalactone when electron-withdrawing substituents are present in the C-3 positions.
Also, the use of CF CO H as an oxidant agent has a marked effect upon the 6-oxa:7-oxa ratio, and
α-6-keto-steroid would result in the stereo and regioselective
3
3
can lead to the preferential formation of the desired 7-oxa isomer [44
–46]. For these reasons we
carry out the Baeyer-Villiger oxidation of compound with CF CO H/CHCl system. In this case
7
3
3
3
the known 7-oxalactone 10 and 6-oxalactone 13 (Scheme 1) were obtained with 44% and 8% yields,
respectively. Even though the synthesis of compounds 10 and 13 was previously reported, no NMR
and IR spectroscopic data of these compounds were included [15]. Thus, the full structural assignment
1
13
of compound 10 was mainly assigned by H, C, 2D Heteronuclear Single Quantum Coherence
1
(
HSQC) and 2D Heteronuclear Multiple Bond Correlation (HMBC) NMR spectroscopy. In the H-NMR
spectrum of compound 10 a signal was observed at
the two hydrogens H-7 and correlated by 2D H- C HSQC with the signal at
δ = 4.10–4.07 ppm (m, 2H), which was assigned to
H
1
13
δ = 70.47 ppm (CH -7
C 2

Int. J. Mol. Sci. 2017, 18, 516
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from 13C and DEPT-135 spectra, Table 1). Additionally, from 2D H– C HMBC spectrum important
1
13
3
heteronuclear correlations were observed: (i) H-7 shows J
correlations with the signal at
δ
= 176.11
δ = 58.43
C
HC
C
that was assigned to the carbon C-6 (C=O of lactone function, Table 1), and the signal at
ppm, assigned to the carbon C-9; (ii) H-5
α
at
δ
= 3.02 ppm (1H, dd, J = 12.1 and 4.0 Hz) showed
H
3
JHC correlation with signals at
δ
= 14.58, and
δ
= 58.43 ppm (Table 1), which were assigned to
C
C
2
carbons CH -19 and C-9 respectively; (iii) H-5
α
showed J correlation with signals at
δ
= 29.79,
3
HC
C
3
6.18 and 176.11 ppm assigned to carbons C-4, C-10 and C-6, respectively (Figure 3a). These 2D HMBC
observations confirm unequivocally the 7-oxalactone position for compound 10.
CO H
2
CO CH3
CO CH3
2
2
CO
2
CH
3
ref [26]
a
b
HO
HO
5
9
6
HO
HO
H
H
H
H
OH
hyodeoxycholic acid (4)
O
O
O
c
CO CH3
2
CO2CH3
CO CH3
2
d
+
7
AcO
1
0
13
H
O
O
AcO
AcO
O
H
H
O
O
e
e
CO H
2
CO H
2
1
2
15
HO
O
HO
O
H
H
O
O
Scheme 1. Synthesis of hyodeoxycholic acid derivatives
and 15. Reagents and conditions: ( ) Tf O/DMAO/CH Cl , 0 C, 24 h.; (
2 h.; ( ) Ac O/DMAP, CH Cl /py, r.t., 30 min.; (
5
,
7
,
9
and brassinosteroid analogs
6
,
10
,
12
,
13
◦
a
b
) OsO 4%/NMO, acetone, r.t.,
2
2
2
4
◦
7
c
d
) (CF SO ) /H O /CHCl , 0 C, 24 h.; (
e
) K CO
3
2
2
2
3
2 2
2
2
3
2
1
5% (aqueous solution)/CH OH, reflux, 1 h. Derivative 5 has been previously reported [26].
3
Figure 3. Major observed 2JHC
and H-7 for compound 10 (7-oxalactone derivative (
(
red arrows) and 3JHC
)) and compound 13 (6-oxolactone derivative (
(
blue arrows) correlations of hydrogens H-5
α
a
b
)).
A similar analysis was performed to determine the structure of 6-oxolactone 13. Thus, in the
H-NMR spectrum a signal at = 4.46 ppm (1H, dd, J = 11.2 and 5.4 Hz) was assigned to H-5 , and
= 79.53 ppm (CH with impair multiplicity from
1
δ
α
H
1
13
correlated by 2D H- C HSQC with the signal at
δ
2
C
DEPT-135 spectrum). Additionally, H-5
α
showed J correlation with signal at
δ
= 32.86 ppm that
C
HC
3
was assigned to the carbon C-4 and J correlation with signals at δ = 11.48, 57.91 and 174.73 ppm,
HC
C
that were assigned to the carbons CH -19, C-9 and C-6 (C=O, of lactone function, Table 1) respectively.
3
2
While the signal of H-7 (2H, m) at
δ
= 2.51–2.39 ppm showed J
correlation with signals appearing
HC
H
at δ = 34.70 and 174.73 ppm, which were assigned to carbons C-8 and C-6 respectively (Figure 3b).
C

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Saponification reaction of compounds 10 and 13 with K CO /MeOH in reflux and subsequent
2
3
acidification with aqueous 5% HCl produced carboxylic acids 12 and 15 with 83% and 75% yields,
respectively (Scheme 1). These compounds were previously reported, but only partial NMR
spectroscopy data was provided [31]. Thus, a deeper NMR analysis for 12 and 15 is provided in
Figure S1 of Supplementary Materials.
Table 1. δ13C-RMN (CDCl , 100.6 MHz) for compounds 10, 12, 13 and 15.
3
C
10
12 *
13
15 *
1
2
3
4
5
6
7
8
9
33.69
27.84
68.48
29.79
42.64
176.11
70.47
39.43
58.43
36.18
22.15
39.64
42.66
55.81
25.17
24.80
51.52
11.79
14.58
35.32
18.14
30.81
31.03
174.59
51.46
170.32
21.38
34.04
28.90
65.49
33.39
42.88
179.68
71.75
40.79
59.49
37.25
23.26
41.01
43.82
57.12
28.83
25.76
52.54
12.21
14.93
36.62
18.63
31.95
32.15
178.09
-
31.74
27.37
69.43
32.86
79.53
174.73
38.02
34.70
57.91
39.58
22.09
39.52
42.61
56.00
25.15
24.75
55.37
11.67
11.48
35.18
18.04
30.66
30.90
174.39
51.39
170.02
21.19
32.40
28.51
66.95
36.57
81.59
178.28
38.79
36.36
59.09
40.88
23.27
40.97
43.87
57.46
28.51
26.32
56.54
12.15
11.83
36.62
18.62
31.97
32.12
178.20
-
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
1
1
1
1
1
1
1
1
1
2
2
2
2
2
CH O
3
CH CO-
-
-
-
-
3
CH CO-
3
*
The 13C-NMR spectrum of compounds 12 and 15 was recorded in CD3OD solution.
2
.2. Bioactivity in the Rice Lamina Inclination Assay of Brassinosteroid Analogs
A number of bioassays, such as first bean internode, root growth, and rice lamina inclination, has
been developed to evaluate brassinolide activity [33
activity of BR analogs was evaluated by the rice lamina inclination bioassay, because of its specificity
and high sensitivity for brassinolide and related compounds [33 47 49], using a local rice (ZAFIRO)
cultivar. The results are presented in Figure 4 and listed in Table 2.
,47–52]. In this work, plant growth-promoting
,
–

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Table 2. Effect of brassinolide and analogs 5, 6, 8, 12 and 15, on the lamina inclination of rice seedlings.
◦
BRs
Angle Degrees between Laminae and Sheaths ( ) (± Standard Error)
Concentration (M)
−
6
−7
−8
Compounds
1 × 10
1 × 10
1 × 10
5
6
8
11 ± 2.5
47 ± 4.7
49 ± 2.0
26 ± 2.5
23 ± 6.0
64 ± 4.8
1 ± 0.0
29 ± 4.5
29 ± 6.3
26 ± 2.5
18 ± 8.7
30 ± 4.1
20 ± 0
0 ± 0.0
0 ± 1.0
7 ± 2.4
15 ± 0.0
10 ± 0.0
28 ± 2.9
1
1
2
5
Brassinolide (C+)
Control (C−)
The angles given in the table correspond to the difference between the induced angle produced
by treatment with each compound and that found for the negative control. Values are means of angles
measured for four segments per treatment considering two assays.
Angle between Laminae and Sheaths
Compounds
−
6
−7
−8
1
× 10
M
1 × 10
M
1 × 10
M
Figure 4. Rice-lamina assay using the second leaf lamina joints of excised leaf segments treated with
−
6
−7
−8
brassinolide (
1) and more active BR analogs at 1
×
10 , 1
×
10 and 1
×
10 M. Scale bars = 2 cm.
The values of Table 2 indicate that the effect is dose-dependent, i.e., for compounds
5, 6 and 8 the
activity increases steadily with increasing concentration, whereas compounds 12 and 15 exhibit an
−
6
initial twofold increase in activity, which reach a maximum value at 1
×
10 M. Finally, the effect of
−
8
−6
brassinolide seems to level off above 1 × 10 M and then increases in a factor of two at 1 × 10 M.
The concentration effect on growth-promoting activity of BRs has been studied by several
authors [53
–56]. Wada et al. found a linear correlation between the bending of leaf segments and
−
10
−8
10 M [53]. The effect
concentrations of brassinolide and castasterone in the range 1
×
10 –1
10 M. It has also been shown that application of exogenous
BRs induces an inhibitory effect on root-growth in Arabidopsis when a threshold concentration is
reached [54 55]. Similar results have been found in rice, where at relatively higher concentration BRs
inhibit both root and seedling growth [57]. The effect of increasing concentrations of brassinolide
×
−
8
leveled off at concentrations above 1
×
,

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on rice seedlings growth has also been evaluated, and the inhibitory effect has been attributed to
inactivation of gibberellins, another growth hormone [58].
The opposite effects on growth of different tissues in the same plant, reported for high
concentrations of exogenous BRs, has been explained in terms of differences in the concentration of
endogenous BRs. Application of exogenous BRs induces growth inhibition in those tissues where the
endogenous BR concentration is near to the threshold concentration, but promotes growth in those
containing the lowest endogenous BR concentration [55].
As our results indicate that the concentration effect on growth activity depends on the structure
of BR analogs, the activities measured at the lowest tested concentration (see Table 2) will be used to
analyze the relation between activity and chemical structures of assayed compounds.
Brassinolide is commonly used as positive control to evaluate the activity of BR analogs, and
therefore the effect of
collected in Table 2 indicate that, at the lowest assayed concentration, compounds 12 and 15 are the
most active BR analogs. The lowest activities are shown by deoxybrassinosteroids and , in which
the oxa group is not present in ring B. These results are in line with other works where it has been
suggested that the 2 ,3 -dihydroxy-7-oxa-6-ketone moiety existing in brassinolide is a structural
requisite for a compound manifesting the plant growth-promoting activity [33]. On the other hand,
compounds and are synthetic analogs of typhasterol, which is considered to be a biosynthetic
1 on bending angle in the lamina assay has been taken as a reference. The results
5,
6
8
α
α
5
8
intermediate to castasterone and brassinolide [59], and whose activity in the rice-lamina inclination
bioassay is only one tenth of that shown by brassinolide [60]. A comparison of activities of compounds
5
and
induces a large increase in activity. On the other hand, a comparison of activities exhibited by analogs
and 15 shows that the 7-oxalactone is slightly more active than the 6-oxalactone. Similar results have
8 indicates that a minor change in the structure of the side alkyl chain (COOCH to COOH)
3
1
2
been reported for other synthetic analogs of typhasterol, but the activity changes were 40–50 times
larger for the 7-oxalactone [61]. In other words, a change in the spatial distribution of the oxa-lactone
moiety in ring B produces a slight change in activity for these compounds.
Interestingly, at the higher assayed concentration deoxybrassinosteroids
6 and 8 become twofold
more active than compounds 12 and 15. However, as discussed above, these results are more difficult to
explain because at higher concentrations there are other factors affecting the growth-promoting activity.
In order to determine the effect of BRs on cell elongation we have evaluated the effect of compound
8
on cell elongation by microscopy analysis. Frotis of vegetal tissue of second leaf lamina joints
was performed, and the inner mesophyll cells were observed through an optical microscope 40
The shape and size of cells showed that those cells submitted to treatment with compound were
stretched longitudinally as compared to the negative control (Figure 5a,b). These results indicate that
×
.
8
growth-promoting effect of analog
of an increase on cell number.
8 is mainly due to changes of cell length of the adaxial sides, instead
a
b
Figure 5. Changes induced by analog
8 in mesophyll cell length in second lamina of rice plants.
Longitudinal sections of adaxial sides of the second leaf lamina joint: (a) negative control; (b) in the
presence of analog 8. Optic Microscope 40×, Scale of bars = 20 µm.

Int. J. Mol. Sci. 2017, 18, 516
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3
. Materials and Methods
3
.1. General
Chemicals were obtained from Merck (Darmstadt, Germany) or Aldrich (St. Louis, MO, USA) and
were used without further purification. A detailed description of conditions used to register Fourier
1
13
13
transform infrared (FT-IR) spectra, H, C, C DEPT-135, 2D HSQC and 2D HMBC spectra has been
given elsewhere [26]. IR spectra were recorded as KBr disks in a FT-IR Nicolet 6700 spectrometer
−
1
(
Thermo Scientific, San Jose, CA, USA) and frequencies are reported in cm . High-resolution mass
spectra (HRMS-ESI) were recorded in a Thermo Fisher Scientific Exactive Plus mass spectrometer.
The analysis for the reaction products was performed with the following relevant parameters: heater
◦
temperature, 50 C; sheath gas flow, 5 (arbitrary unit); sweep gas flow rate, 0 (arbitrary unit) and spray
voltage, 3.0 kV at negative mode. The accurate mass measurements were performed at a resolving
power: 140,000 FWHM at range m/z 300–500. Elemental analyses were obtained on a Carlo Erba
Fisons EA-1108 Elemental Analyzer (Fisons Instruments/Carlo Erba Instruments, Milano, Italy). Silica
gel (200–300 mesh, Merck, Darmstadt, Germany) was used for Column Chromatography (C.C.) and
silica gel plates HF-254 for thin layer chromatography (TLC). TLC spots were detected by heating
after spraying with 25% H SO in H O. Melting points were measured on a Stuart-Scientific SMP3
2
4
2
apparatus (Stone, Staffordshire ST15 OSA, UK) and are uncorrected.
3
.2. Synthesis
3
.2.1. Methyl 2-en-6-oxo-5α-cholan-24-oate (9)
To a solution of compound
DMAP 10 mg (0.082 mmol) were added. Then the mixture was cooled to 0 C in an ice-water
bath. Later 1.2 mL (7.14 mmol) of trifluoromethanesulfonic anhydride (triflic anhydride) were added.
5 (1.0 g, 0.207 mmol) in 40 mL of CH Cl , 0.5 mL of pyridine and
2 2
◦
◦
The reaction mixture was kept under slow stirring at 0 C for 24 h. The end of reaction was verified by
TLC, the mixture was then concentrated in a rotary evaporator to a volume of approximately 10 mL.
Then AcOEt (30 mL) was added and the organic layer was washed with NaHCO saturated solution
3
(
2
× 20 mL) and water (2 × 20 mL), dried over Na SO , and filtered. The solvent was evaporated in
2
4
a rota-vapor and the crude was re-dissolved in CH Cl (5 mL) and chromatographed on silica gel
2
2
with EtOAc/hexane mixtures of increasing polarity (0.2:9.8
The H, C-NMR and IR spectroscopic data and physical properties of alkene 9 were consistent with
those reported [26,28,32].
→
3.0:7.0) Compound 9 0.793 g, 83% yield.
1
13
3
.2.2. Methyl 2α, 3α-dihydroxy-6-oxo-5α-cholan-24-oate (6)
Compound
6
was synthesized from
9
according to the methodology previously described by
1
us [26]. From 700 mg (1.81 mmol) of compound
9, 517 mg (68% yield) were obtained. The H,
13
C
-
NMR and IR spectroscopic data and physical properties of compound
6
were consistent with
those reported [26
H, 9.59%.
,28,32]. Elemental analysis: found C, 71.45%; H, 9.67%; C H O requires C, 71.39%;
25
40
5
3
.2.3. Methyl 3α-acetoxy-6-oxo-5α-cholan-24-oate (7)
Compound was synthesized from by standard acetylation according to the methodology
previously described by us [26]. From 3.5 g (8.65 mmol) of compound , 3.78 g (98% yield) were
NMR and IR spectroscopic data and physical properties of compound were
consistent with those reported [26]. Elemental analysis: found C, 72.68%; H, 9.55%; C H O requires
7
5
7
1
13
obtained. The H,
C-
7
27
42
5
C, 72.61%; H, 9.48%.

Int. J. Mol. Sci. 2017, 18, 516
9 of 13
3
.2.4. Methyl 3α-acetoxy-6-oxo-7-oxa-5α-cholan-24-oate (10) and
Methyl 3α-acetoxy-6-oxa-7-oxo-5α-cholan-24-oate (13)
Preparation of oxidant: 0.40 mL of H O (30%), (26.5 mmol) was slowly dripped into a solution of
2
2
◦
CF SO ) O, (16.9 mL, 119.9 mmol) at 0 C, diluted with CHCl (3 mL) and stirred for 30 min.
3 2 2 3
(
The oxidant mixture was slowly added over the solution of compound 7 (0.8 g, 1.79 mmol in 10 mL
◦
of CHCl ) at 0 C and slowly stirred in N atmosphere for 24 h. The end of reaction was verified by
3
2
TLC, the mixture was filtered, then concentrated in a rotary evaporator to a volume of approximately
0 mL. Then AcOEt (40 mL) was added and the organic layer was washed with saturated NaHCO3
solution (2 20 mL), water (2 15 mL), then dried over Na SO , and filtered. The solvent was
evaporated and the crude was re-dissolved in CH Cl (5 mL) and chromatographed on silica gel with
1
×
×
2
4
2
2
EtOAc/hexane mixtures of increasing polarity (0.2:50.0
Fraction I: 0.35 g (44% yield) compound 10. Fraction II: 0.27 g of mixture compounds 10 and 13.
Fraction III: 0.066 g (8% yield) compound 13.
→
23.8:26.2) Three fractions were obtained.
◦
−1
1
Compound 10. Colorless solid (m.p. = 131
±
2 C, Hexane/Et O) IR (cm ): 2949 (C–H); 2867 (C–H);
2
2
849 (C–H); 1738 (C=O); 1366 (CH ); 1240 (C–O); 1189 (C–O); 1080 (C–O). H-NMR (CDCl ): 5.09 (1H,
3
3
m, H-3); 4.10–4.07 (2H, m, H-7); 3.66 (s, 3H, CH O); 3.02 (1H, dd, J = 12.1 and 4.0 Hz, H-5); 2.35 (1H,
3
m, H-23); 2.26–2.13 (2H, m, H-23 and H-4); 2.06 (3H, s, CH CO); 0.92 (3H, d, J = 6.5 Hz, H-21); 0.89
3
(
3H, s, H-19); 0.69 (3H, s, H-18). 13C-NMR: See Table 1. Elemental analysis: found C, 70.16%; H, 9.23%;
C H O requires C, 70.10%; H, 9.15%.
27
42
6
◦
−1
1
Compound 13. Colorless solid (m.p. = 125
2
±
3 C, Hexane/Et O) IR (cm ): 2978 (C–H); 2955 (C–H);
2
863 (C–H); 1738 (C=O); 1720 (C=O); 1365 (CH ); 1254 (C–O); 1038 (C–O). H-NMR (CDCl ): 5.06 (1H,
3
3
m, H-3); 4.46 (1H, dd, J = 11.2 and 5.4 Hz, H-5); 3.66 (3H, s, CH O); 2.51–2.39 (2H, m, H-7); 2.35–2.27
3
(
(
1H, m, H-23); 2.22–2.18 (1H, m, H-23); 2.03 (3H, s, CH CO); 0.870 (3H, d, J = 4.8 Hz, H-21); 0.865
3
3H, s, H-19); 0.650 (3H, s, H-18). 13C-NMR: See Table 1. HRMS-ESI (negative mode): Calculated
4
05.2719 found 405.2762. Elemental analysis: found C, 70.18%; H, 9.25%; C H O requires C, 70.10%;
27 42 6
H, 9.15%.
3
.2.5. Acid-3α-hydroxy-6-oxo-7-oxa-5α-cholan-24-oic (12)
To a solution of compound 10 (100 mg, 0.22 mmol) in 20 mL of CH OH, 10 mL of 15% aqueous
3
solution of K CO were added. The reaction mixture was maintained with constant stirring and
2
3
reflux for 1 h. The end of reaction was verified by TLC, then the mixture was concentrated in a rotary
evaporator and evaporated to dryness. The crude was acidified with 15 mL of aqueous 5% HCl (until
pH = 2–3). The solid obtained was filtered and washed with water (10–15 mL) to neutral pH and
then it was dried under a vacuum. 73 mg (83% yield) of compound 12 were obtained. Compound 12
:
◦
◦
−1
colorless solid (m.p. = 177–180 C, MeOH/Et O; referential: 177–181 C [24]) IR (cm ): 3480–2540
2
(
(
O–H); 2926 (C–H); 2869 (C–H); 2853 (C–H); 1725 (C=O); 1704 (C=O); 1380 (CH ); 1189 (C–O); 1128
3
1
C–O); 1074 (C–O). H-NMR (CD OH): 4.21 (1H, dd, J = 12.5 and 9.8 Hz, H-7a), 4.08–4.03 (2H, m, H-7b
3
and H-3); 3.24 (1H, dd, J = 12.5 and 4.2 Hz, H-5
.95 (3H, d, J = 6.4 Hz, H-21); 0.86 (3H, s, H-19); 0.740 (3H, s, H-18). 13C-NMR: See Table 1. HRMS-ESI
negative mode): Calculated 405.2719 found 405.2650. Elemental analysis: found C, 70.98%; H, 9.50%;
C H O requires C, 70.90%; H, 9.42%.
α); 2.33–2.29 (1H, m, H-23); 2.23–2.07 (1H, m, H-23);
0
(
24
38
5
3
.2.6. Acid-3α-hydroxy-6-oxa-7-oxo-5α-cholan-24-oic (15)
Compound 15 was obtained from 13 by the same method described above. To a solution of
compound 13 (50 mg, 0.11 mmol) in 15 mL of CH OH, 5 mL of 15% aqueous solution of K CO
3
3
2
were added. Then 33 mg (75% yield) of compound 15 were obtained. Compound 15: colorless solid
◦ ◦ −1
m.p. = 275–279 C, MeOH/Et O; referential: 276–278 C [31]) IR (cm ): 3457–2538 (O–H); 2956
2
(
(
(
C–H); 2943 (C–H); 2870 (C–H); 2852 (C–H); 1730 (C=O); 1680 (C=O); 1380 (CH ); 1250 (C–O); 1199
3
1
C–O); 1140 (C–O); 1074 (C–O). H-NMR: (CD OD) 4.68 (1H, dd, J = 11.0 and 5.2 Hz, H-5
α
); 4.09 (1H,
3

Int. J. Mol. Sci. 2017, 18, 516
10 of 13
b.s, H-3); 2.61 (1H, dd, J = 12.6 and 12.0 Hz, H-7a); 2.40 (1H, d, J = 12.0 Hz, H-7b); 2.30 (1H, m, H-23);
.19 (1H, m, H-23); 0.940 (3H, d, J = 6.4 Hz, H-21); 0.880 (3H, s, H-19); 0.730 (3H, s, H-18). 13C-NMR:
See Table 1. Elemental analysis: found C, 70.96%; H, 9.49%; C H O requires C, 70.90%; H, 9.42%.
2
24
38
5
3
.3. Biological Activity: A Rice Lamina Inclination Assay
The lamina joint bending assay using excised leaf segments was performed as previously
described [33] with minor modifications. After soaking for 24 h in distilled water, seeds of a rice
Oryza sativa) cultivar Zafiro (obtained from the National Agricultural Research Institute (INIA)) were
grown in pots (10 cm length 15 cm diameter) filled with substrate + vermiculite + perlite (in the ratio
:1:1) inside a greenhouse maintained at a 16 h day (22 C)/8 h night (20 C) cycle, with 50%–60% of
(
×
◦
◦
2
relative humidity (RH). Uniform seedlings were selected and leaf segments of approximately 8 cm,
consisting of the second leaf lamina and the second lamina joint and sheath, were excised. For each
treatment, four of these segments were incubated in 60 mL of sterile distilled water for 24 h inside
−
8
−7
−6
10 M) of the test sample
a petri dish containing a finite amount (1
×
10 , 1
×
10 and 1
×
(
BR analogs: 12 and 15). Brassinolide, used as positive control, was purchased from Sigma
5, 6, 8,
Chemical Co., St. Louis, USA. The negative control contains only physiological saline. After incubating
◦
for 48 h at 25 C in darkness, the magnitude of the angle between the leaf and sheath was measured.
Images were taken using a Leica EZ4HD Stereo Microscope with camera software. This test was made
in duplicate for each treatment.
Tissue sections from leaf segments treated with compound 8 were fixed, stained with Lactophenol
Cotton Blue (BD, Franklin Lakes, NJ, USA), and observed by optical microscopy. Images were captured
with a camera ICC50HD coupled to a DM500 microscope (Leica Microsystems, Wetzlar, Germany),
and cell lengths were measured using Leica software ((Leica Microsystems, Wetzlar, Germany).
4
. Conclusions
Therefore, active BR analogs synthesized and described in this work might be used as an
alternative to manipulate plant height and offer the potential to improve production of crops. However,
the optimal growth-promoting concentration must be determined.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/3/516/s1.
Acknowledgments: The authors thank to FONDECYT (grant No.1160446) and the Dirección General de
Investigación y Postgrado (DGIP-USM grant No. 116.13.12 of Universidad Técnica Federico Santa María.
Author Contributions: María Isabel Duran collaborated in the synthesis, separation and purification of
compounds 5–9. Cesar González collaborated in the synthesis, separation, and purification of compounds
1
0
–
13 and structure determination of hyodeoxycholic acid derivatives by spectroscopic methods (1D, 2D
NMR and IR). Katy Díaz performed the bioassays and wrote the biological component of this manuscript.
Alison Acosta collaborated with the synthesis, separation, and purification of compound 12 15 and bioassay.
,
Andrés F. Olea collaborated in the discussion and interpretation of the results, manuscript redaction and
corrections. Luis Espinoza supervised the whole work, collaborated in the syntheses, structure determination of
hyodeoxycholic acid derivatives by spectroscopic methods (1D, 2D NMR and IR), and manuscript redaction.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
MDPI
DOAJ
TLA
Multidisciplinary digital Publishing Institute
Directory of open access journals
Three letter acronym
LD
Linear dichroism
BRs
Brassinosteroids
DCM
PCC
DMAP
Dichloromethane
Pyridinium ChloroChromate
4-Dimethylaminopyridine

Int. J. Mol. Sci. 2017, 18, 516
11 of 13
DMF
Dimethylformamide
Tf O
2
Trifluoromethanesulfonic anhydride
m-CPBA
DEPT-135
HSQC
3-Chloro or metha-chloroperoxybenzoic acid
Distortionless Enhancement by Polarization Transfer with flip angle of 135
Heteronuclear Single Quantum Coherence
◦
HMBC
Heteronuclear Multiple Bond Correlation
References
1.
2.
3.
4.
Bajguz, A. Brassinosteroids—Occurence and Chemical Structures in Plants. In Brassinosteroids: A Class of
Plant Hormone; Hayat, S., Ahmad, A., Eds.; Springer: Dordrecht, The Netherlands, 2016.
Fujioka, S. Natural Occurrence of Brassinosteroids in the Plant Kingdom. In Brassinosteroids: Steroidal Plant
Hormones; Sakurai, A., Yokota, T., Clouse, S.D., Eds.; Springer-Verlag: Tokyo, Japan, 1999.
Marumo, S.; Hattori, H.; Abe, H.; Nonoyama, Y.; Munakata, K. Presence of Novel Plant Growth Regulators
in Leaves of Distylium Racemosum Sieb et Zucc. Agric. Biol. Chem. 1968, 32, 528–529.
Crozier, A.; Yokota, T.; Bishop, G.J.; Kamiya, Y. Biosynthesis of Hormones and Elicitor Molecules.
In Biochemistry and Molecular Biology of Plants; Buchanan, B.B., Gruissem, W., Jones, R.L., Eds.; American
Society of Plant Physiologists: Rockville, MD, USA, 2000.
5
6
.
.
Mitchell, J.W.; Mandava, N.; Worley, J.F.; Plimmer, J.R.; Smith, M.V. Brassins—A New Family of Plant
Hormones from Rape Pollen. Nature 1970, 225, 1065–1066. [CrossRef] [PubMed]
Grove, M.D.; Spencer, G.F.; Rohwedder, W.K.; Mandava, N.; Worley, J.F.; Warthen, J.D.; Steffens, G.L.;
Flippenanderson, J.L.; Cook, J.C. Brassinolide, A Plant Growth-Promoting Steroid Isolated from
Brassica-Napus Pollen. Nature 1979, 281, 216–217. [CrossRef]
7
.
Krizek, D.T.; Mandava, N.B. Influence of Spectral Quality on the Growth-Response of Intact Bean-Plants
to Brassinosteroid, A Growth-Promoting Steroidal Lactone. 2. Chlorophyll Content and Partitioning of
Assimilate. Physiol. Plant. 1983, 57, 324–329. [CrossRef]
8
9
1
1
1
1
.
Cerana, R.; Bonetti, A.; Marre, M.T.; Romani, G.; Lado, P.; Marre, E. Effects of A Brassinosteroid on Growth
and Electrogenic Proton Extrusion in Azuki Bean Epicotyls. Physiol. Plant. 1983, 59, 23–27. [CrossRef]
Romani, G.; Marre, M.T.; Bonetti, A.; Cerana, R.; Lado, P.; Marre, E. Effects of A Brassinosteroid on Growth
and Electrogenic Proton Extrusion in Maize Root Segments. Physiol. Plant. 1983, 59, 528–532. [CrossRef]
.
0. Braun, P.; Wild, A. The Influence of Brassinosteroid on Growth and Parameters of Photosynthesis of Wheat
and Mustard Plants. J. Plant Physiol. 1984, 116, 189–196. [CrossRef]
1. Mandava, N.B.; Sasse, J.M.; Yopp, J.H. Brassinolide, A Growth-Promoting Steroidal Lactone. 2. Activity in
Selected Gibberellin and Cytokinin Bioassays. Physiol. Plant. 1981, 53, 453–461. [CrossRef]
2. Wilen, R.W.; Sacco, M.; Gusta, L.V.; Krishna, P. Effects of 24-Epibrassinolide on Freezing and Thermotolerance
of Bromegrass (Bromus-Inermis) Cell-Cultures. Physiol. Plant. 1995, 95, 195–202. [CrossRef]
3. Dhaubhadel, S.; Chaudhary, S.; Dobinson, K.F.; Krishna, P. Treatment with 24-epibrassinolide, a
brassinosteroid, increases the basic thermotolerance of Brassica napus and tomato seedlings. Plant Mol. Biol.
1
999, 40, 333–342. [CrossRef] [PubMed]
1
1
1
1
1
4. Khripach, V.; Zhabinskii, V.; de Groot, A. Twenty years of brassinosteroids: Steroidal plant hormones warrant
better crops for the XXI century. Ann. Bot. 2000, 86, 441–447. [CrossRef]
5. Vardhini, B.V.; Anuradha, S.; Rao, S.S.R. Brassinosteroids—New class of plant hormone with potential to
improve crop productivity. Indian J. Plant Physiol. 2006, 11, 1–12.
6. Divi, U.K.; Krishna, P. Brassinosteroid: A biotechnological target for enhancing crop yield and stress tolerance.
New Biotechnol. 2009, 26, 131–136. [CrossRef] [PubMed]
7. Vriet, C.; Russinova, E.; Reuzeau, C. Boosting Crop Yields with Plant Steroids. Plant Cell 2012, 24, 842–857.
[CrossRef] [PubMed]
8. Thompson, M.J.; Meudt, W.J.; Mandava, N.B.; Dutky, S.R.; Lusby, W.R.; Spaulding, D.W. Synthesis of
Brassinosteroids and Relationship of Structure to Plant Growth-Promoting Effects. Steroids 1982, 39, 89–105.
[CrossRef]
1
9. Takatsuto, S.; Yazawa, N.; Ikekawa, N.; Takematsu, T.; Takeuchi, Y.; Koguchi, M. Structure Activity
Relationship of Brassinosteroids. Phytochemistry 1983, 22, 2437–2441. [CrossRef]

Int. J. Mol. Sci. 2017, 18, 516
12 of 13
2
2
0. Takatsuto, S.; Yazawa, N.; Ikekawa, N.; Morishita, T.; Abe, H. Synthesis of (24R)-28-Homobrassinolide
Analogs and Structure Activity Relationships of Brassinosteroids in the Rice-Lamina Inclination Test.
Phytochemistry 1983, 22, 1393–1397. [CrossRef]
1. Brosa, C.; Soca, L.; Terricabras, E.; Ferrer, J.C.; Alsina, A. New synthetic brassinosteroids:
A 5 α-hydroxy-6-ketone analog with strong plant growth promoting activity. Tetrahedron 1998, 54, 12337–12348.
[CrossRef]
2
2
2
2. Brosa, C.; Capdevila, J.M.; Zamora, I. Brassinosteroids: A new way to define the structural requirements.
Tetrahedron 1996, 52, 2435–2448. [CrossRef]
3. Iglesias-Arteaga, M.A.; Martinez, C.P.; Manchado, F.C. Spirostanic analogues of castasterone. Steroids 2002
7, 159–163. [CrossRef]
4. Romero-Avila, M.; de Dios-Bravo, G.; Mendez-Stivalet, J.M.; Rodriguez-Sotres, R.; Iglesias-Arteaga, M.A.
,
6
Synthesis and biological activity of furostanic analogues of brassinosteroids bearing the 5 α-hydroxy-6-oxo
moiety. Steroids 2007, 72, 955–959. [CrossRef] [PubMed]
2
2
2
5. Rosado-Abon, A.; de Dios-Bravo, G.; Rodriguez-Sotres, R.; Iglesias-Arteaga, M.A. Synthesis and plant growth
promoting activity of polyhydroxylated ketones bearing the 5α-hydroxy-6-oxo moiety and cholestane side
chain. Steroids 2012, 77, 461–466. [CrossRef] [PubMed]
6. Herrera, H.; Carvajal, R.; Olea, A.F.; Espinoza, L. Structural modifications of deoxycholic acid to obtain three
known brassinosteroid analogues and full NMR spectroscopic characterization. Molecules 2016, 21, 1139.
[CrossRef] [PubMed]
7. Jiang, B.; Huang, L.; Tian, W.; Zhou, W. Methods for Construction of Sidechain of Brassinosteroids and
Application to Syntheses of Brassinosteroids. In Structure and Chemistry (Part E); Rahman, A., Ed.; Elsevier:
Amsterdam, The Netherlands, 2016.
2
2
3
8. Zhou, W.S.; Tian, W.S. The Synthesis of Steroids Containing Structural Unit of A, B Ring of Brassinolide and
Ecdysone from Hyodesoxycholic Acid. Acta Chim. Sin. 1984, 42, 1173–1177.
9. Zhou, W.S.; Jiang, B.; Pan, X.F. Studies on Steroidal Plant-Growth Hormones. 8. the Regioselective Synthesis
of Methyl 3-α-Hydroxy-7-Oxa-6-Oxo-B-Homo-5-α-Cholanate. Acta Chim. Sin. 1989, 47, 182–185.
0. Iida, T.; Tamaru, T.; Chang, F.C.; Niwa, T.; Goto, J.; Nambara, T. Potential Bile-Acid Metabolites. 20.
A New Synthetic Route to Stereoisomeric 3,6-Dihydroxy-5-α-Cholanoic and 6-Hydroxy-5-α-Cholanoic Acids.
Steroids 1993, 58, 362–369. [CrossRef]
3
3
1. Zhou, W.; Jiang, B.; Shen, J. Synthesis of Cholesteric Lactones and Analogs as Plant Growth Regulators.
Patent CN 1184113 A, 10 June 1998.
2. Yang, Y.X.; Zheng, L.T.; Shi, J.J.; Gao, B.; Chen, Y.K.; Yang, H.C.; Chen, H.L.; Li, Y.C.; Zhen, X.C. Synthesis
of 5
Discovery of a novel neuroinflammation inhibitor. Bioorg. Med. Chem. Lett. 2014, 24, 1222–1227. [CrossRef]
PubMed]
α-cholestan-6-one derivatives and their inhibitory activities of NO production in activated microglia:
[
3
3
3
3
3
3. Wada, K.; Marumo, S. Synthesis and Plant Growth-Promoting Activity of Brassinolide Analogs.
Agric. Biol. Chem. 1981, 45, 2579–2585.
4. Robaina, C.; Zullo, M.A.; Coll, F. Síntesis y Caracterización Espectroscópica de Análogos de Brasinoesteroides
a partir de Ácido Cólico. Revista Cubana de Química XIII 2001, 2, 15–20.
5. Rivera, D.G.; Leon, F.; Coll, F.; Davison, G.P. Novel 5β-hydroxyspirostan-6-ones ecdysteroid antagonists:
Synthesis and biological testing. Steroids 2006, 71, 1–11. [CrossRef] [PubMed]
6. Takatsuto, S.; Yazawa, N.; Ikekawa, N. Synthesis and Biological-Activity of Brassinolide Analogs,
2
6,27-Bisnorbrassinolide and Its 6-oxo Analog. Phytochemistry 1984, 23, 525–528. [CrossRef]
7. Takatsuto, S.; Ikekawa, N. Synthesis and Activity of Plant Growth-Promoting Steroids, (22R,23R,24S)-28-
Homobrassinosteroids, with Modifications in Ring-A and Ring-B. J. Chem. Soc. Perkin Trans. 1 1984, 439–447.
[
CrossRef]
8. Iglesias-Arteaga, M.A.; Martinez, C.S.P.; Manchado, F.C. Synthesis and characterization of (25R)-2
α-spirostan-12,23-dione. Synth. Commun. 1999, 29, 1811–1818. [CrossRef]
9. Takatsuto, S.; Yazawa, N.; Ishiguro, M.; Morisaki, M.; Ikekawa, N. Stereoselective Synthesis of Plant
Growth-Promoting Steroids, Brassinolide, Castasterone, Typhasterol, and Their 28-Nor Analogs. J. Chem.
Soc. Perkin Trans. 1 1984, 139–146. [CrossRef]
3
3
α,3α-epoxy-
5

Int. J. Mol. Sci. 2017, 18, 516
13 of 13
4
4
0. Zhou, W.S.; Huang, L.F. Studies on Steroidal Plant-Growth Regulator. 25. Concise Stereoselective
Construction of Side-Chain of Brassinosteroid from the Intact Side-Chain of Hyodeoxycholic Acid-Formal
Syntheses of Brassinolide, 25-Methylbrassinolide, 26,27-Bisnorbrassinolide and Their Related-Compounds.
Tetrahedron 1992, 48, 1837–1852.
1. Huang, L.F.; Zhou, W.S. Studies on Steroidal Plant-Growth Regulators. 33. Novel Method for Construction
of the Side-Chain of 23-Arylbrassinosteroids via Heck Arylation and Asymmetric Dihydroxylation as Key
Steps. J. Chem. Soc. Perkin Trans. 1 1994, 24, 3579–3585. [CrossRef]
4
4
4
4
4
2. Nishiguchi, T.; Machida, N.; Yamamoto, E. Dehydration of Alcohols Catalyzed by Copper(II) Sulfate
Adsorbed on Silica-Gel. Tetrahedron Lett. 1987, 28, 4565–4568. [CrossRef]
3. Kumar, R.R.; Haveli, S.D.; Kagan, H.B. A Mild-One Pot Method for Conversion of Various Steroidal
Secondary Alcohols into the Corresponding Olefins. Synlett 2011, 12, 1709–1712. [CrossRef]
4. Takatsuto, S.; Ikekawa, N. Remote Substituent Effect on the Regioselectivity in the Baeyer-Villiger Oxidation
of 5-α-Cholestan-6-One Derivatives. Tetrahedron Lett. 1983, 24, 917–920. [CrossRef]
5. Back, T.G. Stereoselective Synthesis of Brassinosteroids. In Stereoselective Synthesis (Part J); Rahman, A., Ed.;
Elsevier: Amsterdam, The Netherlands, 1997.
6. Voigt, B.; Schmidt, J.; Adam, G. Synthesis of 24-epiteasterone, 24-epityphasterol and their
B-homo-6a-oxalactones from ergosterol. Tetrahedron 1996, 52, 1997–2004. [CrossRef]
4
4
7. Maeda, E. Rate of Lamina Inclination in Excised Rice Leaves. Physiol. Plant. 1965, 18. [CrossRef]
8. Takeno, K.; Pharis, R.P. Brassinosteroid-Induced Bending of the Leaf Lamina of Dwarf Rice Seedlings—An
Auxin-Mediated Phenomenon. Plant Cell Physiol. 1982, 23, 1275–1281. [CrossRef]
4
5
5
9. Ramirez, J.A.; Centurion, O.M.T.; Gros, E.G.; Galagovsky, L.R. Synthesis and bioactivity evaluation of
brassinosteroid analogs. Steroids 2000, 65, 329–337. [CrossRef]
0. Takematsu, T.; Takenchi, Y.; Koguchi, M. New plant growth regulators, Brassinolide analogues: Their biological
effects and application to agriculture and biomass production. Chem. Regul. Plants 1983, 18, 2–15.
1. Kim, S.K.; Asano, T.; Marumo, S. Biological-Activity of Brassiosteroid Inhibitor Km-01 Produced by A Fungus
Drechslera-Avenae. Biosci. Biotechnol. Biochem. 1995, 59, 1394–1397. [CrossRef]
5
5
2. Adam, G.; Marquardt, V. Brassinosteroids. Phytochemistry 1986, 25, 1787–1799. [CrossRef]
3. Wada, K.; Marumo, S.; Abe, H.; Morishita, T.; Nakamura, K.; Uchiyama, M.; Mori, K. A Rice Lamina
Inclination Test—A Micro-Quantitative Bioassay for Brassinosteroids. Agric. Biol. Chem. 1984, 48, 719–726.
4. Clouse, S.D.; Langford, M.; McMorris, T.C. A brassinosteroid-insensitive mutant in Arabidopsis thaliana
exhibits multiple defects in growth and development. Plant Physiol. 1996, 111, 671–678. [CrossRef] [PubMed]
5. Müssig, C.; Shin, G.-H.; Altmann, T. Brassinosteroids Promote Root Growth in Arabidopsis. Plant Physiol.
5
5
5
5
2
003, 133, 1261–1271. [CrossRef] [PubMed]
6. Jeong, D.H.; Lee, S.; Kim, S.L.; Hwang, I.; An, G. Regulation of brassinosteroid responses by phytochrome B
in rice. Plant Cell Environ. 2007, 30, 590–599. [CrossRef] [PubMed]
7. Tong, H.; Jin, Y.; Liu, W.; Fang, J.; Yin, Y.; Qian, Q.; Zhu, L.; Chu, C. Dwarf and low-tillering, a new member of
the gras family, plays positive roles in brassinosteroid signaling in rice. Plant J. 2009, 58, 803–816. [CrossRef]
[PubMed]
5
5
8. Tong, H.; Xiao, Y.; Liu, D.; Gao, S.; Liu, L.; Yin, Y.; Jin, Y.; Qian, Q.; Chu, C. Brassinosteroid Regulates
Cell Elongation by Modulating Gibberellin Metabolism in Rice. Plant Cell 2014, 26, 4376–4393. [CrossRef]
[PubMed]
9. Suzuki, H.; Fujioka, S.; Takatsuto, S.; Yokota, T.; Murofushi, N.; Sakurai, A. Biosynthesis of Brassinolide from
Teasterone Via Typhasterol and Castasterone in Cultured-Cells of Catharanthus-Roseus. J. Plant Growth Regul.
1
994, 13, 21–26. [CrossRef]
6
0. Abe, H.; Morishita, T.; Uchiyama, M.; Takatsuto, S.; Ikekawa, N. A New Brassinolide-Related Steroid in the
Leaves of Thea-Sinensis. Agric. Biol. Chem. 1984, 48, 2171–2172. [CrossRef]
6
1. Abe, H.; Asakawa, S.; Ando, T.; Mouri, T.; Aburatani, M.; Takeuchi, T. Effect of Introducing A Lactone Group
Into Typhasterol and Teasterone to Promote Rice Lamina Inclination. Biosci. Biotechnol. Biochem. 1992, 56,
1
312–1313. [CrossRef]
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