Synthesis and preliminary evaluation of dimeric-28-homobrassinosteroids for plant growth regulators

Article abstract of DOI:10.1016/j.steroids.2016.08.016

Preparation of synthetic analogues of 28-homobrassinosteroids is reported. Also, the addition of the 28-homocastasterone at the C6 carbonyl group via allyl Gringard reagent followed by olefin cross metathesis resulted in dimeric analogues. Rice lamina inclination assay showed that the replacement of the C6 carbonyl group by 6α-allyl and 6β hydroxyl groups led to a decrease in bioactivity, whereas the dimeric analogues showed a reduced but significant bioactivity when compared to the 28-homocastasterone.

Full text of DOI:10.1016/j.steroids.2016.08.016

Accepted Manuscript
Synthesis and Preliminary Evaluation of Dimeric-28-homobrassinosteroids for
Plant Growth Regulators
Waya S. Phutdhawong, Wanwikar Ruensamran, Weerachai Phutdhawong
PII:
S0039-128X(16)30112-X
http://dx.doi.org/10.1016/j.steroids.2016.08.016
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Reference:
Steroids
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15 July 2016
13 August 2016
Please cite this article as: Phutdhawong, W.S., Ruensamran, W., Phutdhawong, W., Synthesis and Preliminary
Evaluation of Dimeric-28-homobrassinosteroids for Plant Growth Regulators, Steroids (2016), doi: http://
dx.doi.org/10.1016/j.steroids.2016.08.016
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Synthesis and Preliminary Evaluation of Dimeric-28-homobrassinosteroids for
Plant Growth Regulators
Waya S. Phutdhawong1, Wanwikar Ruensamran2, Weerachai Phutdhawong*2,3
1Department of Chemistry, Faculty of Science, Silpakorn University, Nakorn Pathom 73000
Thailand
2 Bioproduct Science program, Department of Science, Faculty of Liberal Arts and Science,
Kasetsart University, Kampang Saen Campus, Nakhon Pathom 73140, Thailand
3
Department of Chemistry, Faculty of Liberal Arts and Science, Kasetsart University
Kampang Saen Campus, Nakhon Pathom 73140, Thailand
*Address correspondence to Weerachai Phutdhawong, Email: faaswcp@ku.ac.th
Abstract
Preparation of synthetic analogues of 28-homobrassinosteroids is reported. Also, the addition
of the 28-homocastasterone at the C6 carbonyl group via allyl Gringard reagent followed by
olefin cross metathesis resulted in dimeric analogues. Rice lamina inclination assay showed
that the replacement of the C6 carbonyl group by 6α-allyl and 6β hydroxyl groups led to a
decrease in bioactivity, whereas the dimeric analogues showed a reduced but significant
bioactivity when compared to the 28-homocastasterone.
Keywords : Brassinosteroids, 28-homocastasterone, dimerization, 6α-allyl-6β hydroxyl
homocastasterone, plant growth promoting activity, rice lamination inclination
1. Introduction
Brassinosteroids (BRs) are unusual steroids with a lactone B-ring structure and a
dihydroxylate side chain [1]. These steroids have been isolated from many plant species and
have been recognized as hormones potentially affecting a wide range of physiological
responses in plants, including stem elongation, pollen tube growth, leaf bending and epinasty,
root growth inhibition, induced synthesis of ethylene, activation of proton pump, xylem
differentiation, synthesis of nucleic acids and proteins, activation of enzymes, and
photosynthesis [2-4]. The brassinolide (1), a C28 brassinosteroid, has been reported to be the
most biologically active of the BRs and widely studied for applications due to their potential
agricultural utility [5]. 28-Homobrassinolide (2) and 28-homocastasterone (3), the most
active C29 BRs [6] have also been intensively investigated for their potential agricultural
uses. As BRs are present in plants only in very small quantities, chemical synthesis of BRs is
the source for scientific and practical use. This results in numerous synthetic efforts and
development of non-natural analogues of BRs that are easier to prepare and which have
similar or even greater activity than the natural BRs [7-10].

Figure 1. Structure of natural brassinolids
The studies on structure-activity relationship in brassinosteroids have shown the most active
brassinosteroids have been shown to possess a trans-fused relationship between A and B-
rings, with the substitutents at C-24 and C-25 also important for bioactivity [11]. The two
vicinal diol moieties with attendant stereochemistry are required for optimum bioactivity
[12]. The conversion of the four hydroxyl groups to the corresponding methyl ethers results
in the loss of activity [13]. The transformation of B-ring lactone moiety suggests the polar
functional groups are necessary for bioactivity [10, 14]. In this article, the preparation of the
novel allyl alcohols and their dimeric-28-homobrassinosteroids are reported along with the
improvement of the synthetic methodology. Also comparison of the bioactivities to 28-
homocastasterone from rice leaf lamina inclination bioassay is shown.
2. Results and Discussion
28-Homobrassinolide (2) and 28-homocastasterone (3) were prepared according to the
synthesis pathway depicted in Scheme 1. The stigmasterol (4) was used as a starting material
and then converted into the ketone (5) with modified protocol of McMorris et al. [5]. Acid-
catalysed opening of the cycolprapane ring of the ketone (5) using HBr and AcOH gave the
3β-bromo-6-ketone (6) in 80% yield. Dehydrobromination using Li2CO3 in DMF dienone (7)
in 38% yield. To form 28-homocastaterone, dienone (7) underwent osium-catalysed
asymmetric dihydroxylation [15] using K3Fe(CN)6 as co-oxidant and dihydroquinidine 4-
chlorobenzoate as chiral ligand. A mixture of the diol (8), the 22S,23S product (9) and 28-
homocastaterone (3) was obtained with yields of 16.1% , 17.1% and 12.8% respectively after
purification by flash column chromatography. The configuration of the side chain diols of 3
and 9 was established by comparison with chemical shift and coupling constants of 1H-NMR
of known structures [16, 17]. Oxidation of the mixture of 3 and 9 via Baeyer-Villiger reaction
using trifluoroacetic acid provided 28- homobrassinolide (2) in 90% yield [17].

a-c
d
Br
HO
H
O
O
80%
6
4
stigmasterol (
)
5
e
38%
R1
R2
R2
R1
HO
HO
HO
HO
f
+
H
H
O
O
H
O
9
3
R1=H, R2=OH
R1=OH, R2=H
7
8
90%
g
OH
OH
HO
O
HO
O
Scheme 1. Reagents and conditions:(a) MSCl, Pyr.-THF, 0oC; (b) KHCO3, acetone-water, reflux; (c) CrO3,
AcOH-acetone, 0oC; (d) HBr, AcOH, rt..; (e) Li2CO3, DMF, reflux; (f) K3Fe(CN)6, K2CO3, CH3SO3NH2, OsO4,
t-BuOH, rt..
Addition of allylmagnesium bromide to the 6-ketone of 8 proceeded through the si face to
give the triol (8a) as a single product in quantitative yield. The 6α-allyl group in 8a was
determioned by NOESY correlating the 5α-proton and CH2 of 6α-allyl group. This
stereochemistry was also supported by the known 6α-allyl steroid generated by the attack to
the less hindered alpha face of the 6-ketosteroid [18]. Homodimerization of the triol (8a) with
2nd generation Grubbs catalyst in CH2Cl2 at 90 oC for 20 h generated the dimeric compound
8b in 55% yield (Scheme 2). However, the addition of 3 and 9 under the same conditions
were unsuccessful due to their solubility in the reaction conditions. Thus, protections of the
hydroxyl groups of 3 and 11 with acetone and p-TsOH were investigated. Compound 9 was
converted to diacetals (9a) in 85% yield. On the other hand, compound 3 with 22R, 23R-
dihydroxyl groups could not converted to diacetals, instead the acetal 3a was obtained in 43%
yield. Then, the addition of allylmagnesium bromide gave the allyl compounds 3b and 9b in
43% and quantitative yield, respectively. Deprotection followed by homodimerization using
2nd generation Grubbs catalyst gave 3d and 9d in 17% and 20% yield, respectively. (Scheme
3)

Scheme 2. Reagents and conditions: (a) allylmagnesium bromide (0.2 M in Et2O) in Et2O or CH2Cl2 at rt. 40
min; (b) 2nd generation Grubbs catalyst in CH2Cl2, 90 °C 20 h.

Scheme 3. Reagent and conditions: (a) acetone and p-TsOH, rt. 40 h. ; (b) allylmagnesium bromide (0.2 M in
Et2O) in Et2O or CH2Cl2 rt. 40 min; (c) 70% AcOH, reflux 16h.; (d) 2nd generation Grubbs catalyst in CH2Cl2,
90 °C 20 h.
The plant growth promoting activities of the synthetic compounds were evaluated using rice
lamina inclination bioassay with distilled water as a control and co-evaluated with 28-
homocastasterone. Table 1 and graphically Figure 2 are shown the rice lamina inclination of
the new synthetic compounds at a 10 ng/plant dose with the best result from 28-
homocastasterone (3). It can be clearly noted that the 2α, 3α-OH groups and 22R and 23R-
OH groups have significant importance for bioactivity (compounds 3a, 10, and 11). The 6α-
allylated, 6β-OH analogue (3c) did not show high sensitivity to the rice lamina inclination
assay, confirming the carbonyl group is essential for activity [6, 17]. This might be due to the
absence of the hydrogen bond formation between the 6β-OH group and the amino acids in
brassinosteroid receptors [18]. The dimeric analogue (3d) increased slightly in bioactivity
compared to the 6-allyl steroid (3c) but this compound was only 9.2% less active than that of
the 28-homocastasterone (3). On the other hand, the dimeric analogues of 8 and 9 showed the
increase in bioactivity than their monomers. These might be due to the binding sites of the
dimeric molecules to other amino acids in the brassinosteroid receptors.

Table 1. Rice lamina inclination assay at a concentration of 10 ng/plant of 28-homocastasterone and the new
analogs. Mean values from 15-20 replicates standard error.
Compounds
Average angle (°)
distilled water
15
130
78
3
3
8
3a
3b
3c
3d
8
9
5
68
105
118
17
5
6
3
8a
8b
9
23
5
6
7
2
2
3
4
20
42
9a
9b
9c
9d
15
17
35
32
Figure 2. Rice lamina inclination assay of the new synthetic compounds (values are angles in degrees) between
the lamina and sheath, representing the means of 15-20 replicates standard error.

3. Conclusion
The 28-homocastasterone (3), and analogues including their dimeric compounds were
prepared from stigmasterol (4) via a key intermediate olefin (7). Evaluation of the rice lamina
inclination bioassay of the synthesized compounds showed they have lower bioactivity than
28-homocastasterone. However, the novel molecules can be further investigated for other
properties, such as persistence, and the results give design considerations for molecules with
structures recognized by brassinosteroid receptors.
4. Experimental
4.1 General Methods:
General: Melting points were determined with a Stuart Scientific SMP 2 melting point
apparatus and are uncorrected. Infrared spectra were recorded on CH2Cl2-film with a Perkin
1
Elmer Spectrum GX FT-IR spectrophotometer. H and 13C NMR spectra were recorded in
1
CDCl3 with a Bruker Avance 300 spectrometer (300 MHz for H, 75 MHz for 13C) using
TMS as an internal standard. Optical rotations were measured on JASCO P-1010 digital
polarimeter. Mass spectra were recorded with a POLARIS Q or HEWLETT PACKARD
5973 mass spectrometer. Reactions were monitored by TLC using aluminium or plastic
sheets precoated with silica gel 60 F254. Column chromatography was performed with
Kieselgel 60. (22E,24S)-3α,5-cyclo-stigmast-22-en-6-one (5) was prepared in 3 steps from
commercial stigmasterol (4).[5]
4.2 Rice lamina inclination assay
o
Rice seedlings were washed in distilled water and then left in water at 30 C for 24 hrs.
Germinated seeds were cultivated on agar under the same growing conditions for 7-8 days
before each of the tested compounds was applied to the top portion of the lamina with a
micropipette (1 µL/plant). Distilled water was used as a control. After incubation at the same
growth condition for 2 more days the rice lamination were obtained and the angle of lamina
inclination between the leaf and the sheath was measured.
4.3 Synthesis
(22E)-3β-bromo-5α-stigmasta-22-en-6-one (6)
To a solution of compound 5 (0.50 g, 0.12 mmol) in AcOH (3 mL) was added 48% HBr (15
mL). The reaction mixture was stirred for 1 h, diluted with water (15 mL) and extracted with
CHCl3 (3x15 mL). The organic layers were combined and washed with sat. NaHCO3 (20 mL)

and brine (20 mL). Evaporation of the solvent and purification of the crude product by
column chromatography (silica gel, 2:1 hexane/EtOAc) yielded 80% of the product (6). Mp.
o
144-146 C; lit. 140-141 C [11]. H NMR (CDC13): δ 5.10 (2H, m, 22-H and 23-H), 3.94
o
1
13
(1H, m, 3α-H), 2.40-1.10 (25H, m), 1.30-1.00 (6H, m), 0.98-0.78 (9H, m), 0.70 (3H, s). C
NMR (CDC13): δ 209.8 (CO), 137.9 (C-22), 129.6 (C-23), 59.0, 56.8, 55.9, 53.9, 51.3, 50.6,
46.6, 42.9, 40.7, 40.4, 39.3, 39.2, 37.8, 33.4, 32.4, 31.9, 28.7, 25.4, 24.0, 21.3, 21.2, 21.1,
19.0, 13.1, 12.3, 12.2. HRES-MS m/z calcd for [M+H]+ C29H48BrO: 491.2883, found:
491.2894.
(22E,24S)-5α-stigmasta-2,22-diene-6-one (7)
A solution of (22E)-3β-bromosigmasta-22-en-6-one (6) (0.30 g, 0.60 mmol) in DMF
(5 mL) was slowly added Li2CO3 (44 mg, 0.60 mmol). The reaction mixture was heated
under reflux for 4 h. Evaporation of the solvent under reduced pressure gave the residue,
water (15 mL) was added and extracted with CH2Cl2 (3x15 mL) . The organic extracts were
combined, dried with anh. Na2SO4 and concentrated to give the crude product which was
purified by column chromatography (silica gel, 2:1 hexane/EtOAc). The compound 7 was
o
o
obtained in 38% yield. Mp. 119-120 C; lit. 119-120 C [5]. 1H NMR (CDCl3): 5.60 (2H, m,
2-H and 3-H), 5.10 (2H, m, 22-H and 23-H), 2.40-1.40 (28H, m), 1.00-0.78 (8H, m), 0.77
(3H, s), 0.75 (3H, s). 13C NMR (CDC13): 211.8 (CO), 138.0 (C-22), 129.5 (C-23), 124.5 and
125.0 (C-2 and C-3), 56.9, 55.9, 53.8, 53.5, 51.3, 47.0, 42.7, 40.4, 40.0, 39.4, 39.3, 37.7,
31.9, 28.7, 25.4, 24.0, 21.7, 21.4, 21.2, 21.1, 19.0, 13.5, 12.3, 12.1. HRES-MS m/z calcd for
[M+H]+ C29H47O: 411.3621, found: 411.3627.
Osium-catalysed asymmetric dihydroxylation
A mixture of (22E,24S)-5α-stigma-2,22-diene-6-one (7) (0.200 g, 0.490 mmol), K3Fe(CN)6
(0.968 g, 2.94 mmol), anh. K2CO3 (0.406 g, 2.94 mmol), methanesulfonamide (0.093 g, 0.98
mmol), dihydroquinidine 4-chlorobenzoate (0.046 g, 0.98 mmol) and OsO4 (0.25 ml 2.5% in
BuOH, 0.020 mmol) in t-BuOH : H2O (1:1, 30 mL) was stirred at room temperature for 10
days. Sodium sulfite (0.50 g) was added, and the mixture was stirred at room temperature for
18 h. t-BuOH was removed under reduced pressure, and the residue was extracted with
EtOAc (3x20 mL). The combined organic extracts were washed with water (20 mL), 0.25 M
H2SO4 (20 mL), brine (20 mL), dried, concentrated in vacuo to give a pale colorless oil which
was purified by flash column chromatography (silica gel, 2:1 hexane/EtOAc) to give the first

fraction of (22E,24S)-2α,3α-dihydroxy-5α-stigmast-22-en-6-one (8), the second fraction of
(22S,23S)-28-homocastasterone (9) and the last fraction of 28-homocastasterone (3).
(22E,24S)-2α,3α-dihydroxy-5α-stigmast-22-en-6-one (8) as a white solid (0.035 g, 16.1%),
o
1
Mp. 235-237 C; lit. 237-239 [17]. [α]28 = -6.7 (c = 1.00, CHCl3). H NMR (300 MHz,
CDCl3) δ = 5.15 (dd, J = 15.1, 8.4 Hz, 1H, H-22), 5.02 (dd, J = 15.2, 8.3 Hz, 1H, H-23), 4.05
(br s, 1H, H-3), 3.77 (d, J = 10.8 Hz, 1H, H-2), 2.68 (dd, J = 12.6, 3.4 Hz, 1H, H-5), 2.30 (dd,
J = 13.1, 4.6 Hz, 1H, H-7a), 2.20-1.10 (m, 23H), 1.02 (d, J = 6.6 Hz, 3H, H-21), 0.84 (d, J =
6.6 Hz, 3H, H-26), 0.81 (t, J = 7.6 Hz, 3H, H-29), 0.79 (d, J = 6.8 Hz, 3H, H-27), 0.76 (s, 3H,
H-19), 0.68 (s, 3H, H-18). 13C NMR (75 MHz, CDCl3) δ 212.0 (CO), 138.0 (C-22), 129.6 (C-
23), 68.4, 68.3, 56.8, 55.9, 53.8, 51.2, 50.7, 46.8, 42.8, 42.6, 40.4, 40.3, 39.3, 37.7, 31.9, 28.7,
26.3, 25.4, 24.0, 21.2, 21.1, 21.05, 19.0, 13.6, 12.2 (2). HRES-MS m/z calcd for [M+H]+
C29H48O3: 445.3667, found: 445.3676.
o
(22S,23S)-28-Homocastasterone (9) (0.040 g, 17.1%), as a white solid. Mp. 207-208 C; lit.
o
1
206-208 C [17]. [α]28 = -2.1 (c = 1.52, CHCl3). H NMR (300 MHz, CDCl3) δ 4.05 (br s,
1H, ), 3.83 – 3.71 (m, 1H), 3.60 (t, J = 6.6 Hz, 2H), 2.68 (dd, J = 12.4, 3.1 Hz, 1H, H-5), 2.30
(dd, J = 13.0, 4.4 Hz, 1H), 1.04 (d, J = 6.8 Hz, 3H, H-21), 0.96 (t, J = 7.5 Hz, 3H, H-29), 0.95
(d, J = 7.2 Hz, 3H, H-26), 0.88 (d, J = 6.9 Hz, 3H, H-27), 0.76 (s, 3H, H-19), 0.70 (s, 3H, H-
18). 13C NMR (75 MHz, CDCl3) δ 212.5 (CO), 72.1 and 70.5 (C-22 and C-23), 68.3 and 68.2
(C-2 and C-3), 56.2, 53.6, 52.5, 50.8, 49.6, 46.7, 43.5, 42.5, 42.2, 40.1, 39.4, 37.6, 27.8, 26.8,
26.3, 24.2, 21.8, 21.2, 18.5, 17.7, 14.5, 14.2, 13.5, 11.9. HRES-MS m/z calcd for [M+H]+
C29H51O3: 479.3737, found: 479.3733.
(22R,23R)-28-Homocastasterone (3) (0.030 g, 12.8%), as a white solid. Mp. 254-256 oC; lit.
1
253-256 oC [21]. [α]28 = -9.3 (c = 0.13, CHCl3). H NMR (300 MHz, CDCl3) δ 4.04 (d, J =
2.5 Hz, 1H), 3.80-3.73 (m, 1H), 3.72 (d, J = 8.5 Hz, 1H), 3.59 (d, J = 8.7 Hz, 1H), 2.69 (dd, J
= 12.5, 2.9 Hz, 1H), 2.31 (dd, J = 13.0, 4.4 Hz, 1H), 0.97 (d, J = 6.7 Hz, 3H, H-26), 0.96-0.92
(m, 6H, H-27, H-29), 0.92 (d, J = 6.2 Hz, 3H, H-21), 0.76 (s, 3H, H-19), 0.68 (s, 3H, H-18).
13C NMR (75 MHz, CDCl3) δ 212.0 (CO), 74.5 and 72.8 (C-22 and C-23), 68.4 and 68.3 (C-2
and C-3), 56.6, 53.7, 52.5, 50.7, 46.7, 46.4, 42.8, 42.6, 40.6, 39.5, 37.7, 37.0, 28.9, 27.6, 26.3,
23.8, 21.2, 19.4, 18.8, 13.6, 13.5, 11.9x2. HRES-MS m/z calcd for [M+H]+ C29H51O3:
479.3737, found: 479.3733.

General procedure for the protection of diols
To a solution of compounds 3 or 9 (1 equiv.) in acetone was added p-TsOH (2.5 mol%) and
stirred at room temperature for 40 h. The solvent was removed by evaporatation and the
residue was dissolved in dichloromethane. The organic layer was washed with brine, dried
(MgSO4) and concentrated in vacuo to give a brown oil. Purification by column
chromatography gave the acetonide (3a, 9a).
(22R,23R,24S)-2α,3α,22,23-Tetrahydroxy-5α, stigmastan-6-one 2,3-acetonide (3a)
Prepared from compound 3 (60.0 mg, 0.12 mmol) in acetone (5.0 mL), after the protection
reaction compound 3a was obtained as a white solid (27.0 mg, 43.3%) with recovery of some
o
compound 3 (25.0 mg, 41.7%) Rf 0.19 (hexane/EtOAc, 1:1). Mp. 248-250 C. IR (cm-1):
1
3054, 2986, 1706, 1421, 1262. H NMR (300 MHz, CDCl3) δ 4.05 (s, 1H, H-3), 3.85-3.66
(m, 3H, H-2, H-22, H-23), 2.69 (d, J = 10.5 Hz, 1H, H-5), 2.30 (dd, J = 13.0, 4.3 Hz, 1H, H-
7a), 1.35 (s, 6H, CH3x2), 0.98 (d, J = 6.4 Hz, 3H, H-26), 0.96 (d, J = 6.8 Hz, 3H, H-27), 0.91
(d, J = 6.8 Hz, 3H, H-21), 0.90 (t, J = 6.8 Hz, 3H, H-29), 0.76 (s, 3H, H-19), 0.67 (s, 3H, H-
18). 13C NMR (75 MHz, CDCl3) δ 212.1 (CO), 107.5 (C-acetal), 80.3, 77.3, 68.4, 68.3, 56.5,
53.7, 53.4, 50.7, 46.8, 46.7, 42.9, 42.6, 40.3, 39.2, 37.8, 35.7, 28.9, 27.8, 27.4, 27.2, 26.4,
23.8, 21.2, 20.2, 19.6, 19.2, 14.2, 13.6, 12.6, 11.8. HRES-MS m/z calcd for [M+H]+
C32H55O5: 519.4044, found: 519.4039.
(22S,23S,24S)-2α,3α,22,23-Tetrahydoxy-5α-stigmastan-6-one 2,3:22,23-diacetonide (9a)
Prepared from compound 11 (60.0 mg, 0.12 mmol) in acetone (5.0 mL), after the protection
reaction compound 11a was obtained as a white solid (60.0 mg, 85.0%). Mp. 186-187 oC. Rf
1
0.79 (hexane/EtOAc, 1:1). IR (cm-1): 2952, 2872, 1711, 1462, 1378, 1367, 1241, 1055. H
NMR (300 MHz, CDCl3) δ 4.20 (br s, 1H, H-3), 4.07-3.98 (m, 1H, H-2), 3.92 (dd, J = 8.5,
1.8 Hz, 1H, H-22), 3.83 (dd, J = 8.5, 3.2 Hz, 1H, H-23), 2.46 (dd, J = 12.4, 4.0 Hz, 1H, H-5),
2.25 (dd, J = 12.9, 4.3 Hz, 1H, H-7a), 1.42 (s, 3H, CH3), 1.30 (s, 6H, CH3x2), 1.27 (s, 3H,
CH3), 0.96 (d, J = 6.8 Hz, 3H, H-21), 0.90 (t, J = 6.5 Hz, 3H, H-29), 0.89 (d, J = 5.6 Hz, 3H,
H-26), 0.88 (d, J = 6.9 Hz, 3H, H-27), 0.61 (s, 3H, H-19), 0.60 (s, 3H, H-18).13C NMR (75
MHz, CDCl3) δ 210.3 (CO), 106.9 and 105.7 (C-acetals), 78.7, 76.3, 71.3, 71.1, 55.3, 52.3,
51.8, 50.5, 45.8, 45.5, 42.5, 41.4, 40.1, 38.3, 37.3, 36.5, 27.6, 27.0, 26.5, 26.2, 25.8, 25.5,
23.2, 22.4, 21.5, 20.1, 18.6, 17.6, 13.0, 12.3, 11.6, 10.5. HRES-MS m/z calcd for [M+H]+
C35H59O5: 559.4357, found: 559.4346.

General procedure for the Grignard reaction
To a solution of allylmagnesium bromide (0.2 M in Et2O, 5 equiv.) at 0 °C was added a
solution of ketone-brassinosteroids (8, 3a, 19a) (1 equiv.) in Et2O or CH2Cl2. The mixture
was stirred at room temperature for 40 min and quenched with saturated aqueous NH4Cl. The
solvent was evaporated and the residue was dissolved in dichloromethane. The combined
organic extracts were washed with brine, dried (MgSO4) and concentrated in vacuo to give a
pale colorless oil which was prified by column chromatography giving the allyl products (8a,
3b, 9b).
(22E,24S)-6α-Allyl-2α,3α,6β-trihydroxy-5α-sitostane (8a)
Prepared from compound 8 (80.0 mg, 0.18 mmol) in CH2Cl2 (10.0 mL), after the Grignard
reaction compound 8a was obtained as a white wax like-solid (88.0 mg, quantitative yield).
[α]28 = -20.4 (c = 1.77, CHCl3). Rf 0.35 (hexane/EtOAc, 1:1). IR (cm-1): 3054, 2987, 1421,
1265, 896. 1H NMR (300 MHz, CDCl3) δ 5.89-5.72 (m, 1H, H-2′), 5.26-4.91 (m, 4H, H-22,
H-23, H-3′), 4.08 (br s, 1H, H-3), 3.81 (br s, 1H, H-2), 2.40-1.05 (m), 1.04 (s, 3H, H-19), 1.03
(d, J = 6.6 Hz, 3H, H-21), 0.86 (d, J = 6.4 Hz, 3H, H-26), 0.82 (t, J = 6.6 Hz, 3H, H-29), 0.81
(d, J = 6.6 Hz, 3H, H-27), 0.72 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 138.2 (C-22), 133.9 (C-
2′), 129.4 (C-23), 118.6 (C-3′), 73.2 (C-6), 69.1, 68.7, 56.1, 56.0, 53.8, 51.2, 46.9, 42.8, 42.6,
42.5, 40.5, 39.7, 37.6, 31.9, 30.8, 29.7, 28.9, 26.5, 25.4, 24.3, 21.2, 21.1, 20.7, 19.0, 15.8,
12.3, 12.2. HRES-MS m/z calcd for [M+Cl]+ C32H54ClO3: 521.3761, found: 521.3758.
(22R,23R,24S)- 6α-Allyl-2α,3α,6β,22,23-pentahydroxy-5α-sitostane 2,3-acetonide (3b)
Prepared from compound 3a (52.0 mg, 0.10 mmol) in CH2Cl2 (5.0 mL), after the Grignard
reaction compound 3b was obtained as a white solid (23.0 mg, 43%) and with recovery of
o
compound 3a (20.0 mg, 36%). Mp. 109-110 C. Rf 0.24 (hexane/EtOAc, 1:1). IR (cm-1):
3419, 2942, 1377, 1235. 1H NMR (300 MHz, CDCl3) δ 5.90-5.71 (m, 1H, H-2′), 5.13 (d, J =
8.0 Hz, 1H, H-3′a), 5.09 (d, J = 15.3 Hz, 1H, H-3′b), 4.06 (br s,1H, H-2), 3.83-3.76 (m, 3H,
H-2, H-22, H-23) 1.35 (s, 6H, CH3x2), 1.03 (s, 3H, H-19), 0.98-0.95 (m, 6H), 0.92-0.88 (m,
6H), 0.69 (s, 3H, H-18). 13C NMR (75 MHz, CDCl3) δ 133.9 (C-2), 118.7 (C-3′), 107.4, 80.4,
77.3, 73.2, 69.1, 68.6, 55.8, 53.8, 53.5, 46.9, 46.6, 43.3, 42.8, 42.5, 39.6, 37.6, 35.7, 30.9,
28.9, 28.0, 27.4, 27.2, 26.5, 24.1, 20.8, 20.2, 19.5, 19.2, 15.8, 14.1, 12.6, 11.8. HRES-MS m/z
calcd for [M+H]+ C35H61O5: 561.4514, found: 561.4516.

(22S,23S,24S)-6α-Allyl-2α,3α,6β,22,23-pentahydoxy-5α-stigmastan-6-one
diacetonide (9b)
2,3:22,23-
Prepared from compound 9a (40.0 mg, 0.072 mmol) in Et2O (5.0 mL), after the Grignard
reaction compound 9b was obtained as a white solid (43.0 mg, quantitative yield). Mp. 97-99
oC. Rf 0.83 (hexane/EtOAc, 1:1). IR (cm-1): 2986, 2873, 2305, 1421, 1379, 1264, 1047, 895.
1H NMR (300 MHz, CDCl3) δ 5.91-5.74 (m, 1H, H-2′), 5.14 (d, J = 8.1 Hz, 1H, H-3′a), 5.10
(d, J = 15.8 Hz, 1H, H-3′b), 4.31 (t, J = 3.9 Hz, 1H, H-3), 4.16-4.05 (m, 1H, H-2), 4.00 (dd, J
= 8.5, 1.6 Hz, 1H, H-22), 3.93 (dd, J = 8.6, 3.0 Hz, 1H, H-23), 1.48 (s, 3H, CH3), 1.38 (s, 6H,
CH3x2), 1.35 (s, 3H, CH3), 1.04 (d, J = 6.9 Hz, 3H, H-21), 1.00-0.94 (m, 9H, H-26, H-27, H-
29), 0.93 (s, 3H, H-19), 0.71 (s, 3H, H-18). 13C NMR (75 MHz, CDCl3) δ 133.7 (C-2′), 118.9
(C-3′), 107.4, 106.6, 79.6, 77.2, 73.0, 72.8x2, 55.7, 53.3, 52.8, 46.4, 44.2, 43.4, 43.2x2, 39.8,
38.4, 37.2, 30.4, 29.7, 28.6, 28.3, 27.5, 27.3, 26.8, 26.6, 24.4, 23.2, 23.1, 20.8, 19.6, 18.6,
14.6, 13.9, 13.3, 11.5. HRES-MS m/z calcd for [M+H]+ C38H65O5: 601.4827, found:
601.4819.
General procedure for the deprotection of acetals
A solution of allyl (3b, 9b) (0.10 mmol) in 70% AcOH (5.0 mL) was heated at 90 °C for 16
h. After cooling, ice-water was added, the solid filtered, then washed with cold water and
dried, giving the tetraol (3c, 9c).
(22R,23R,24S)- 6α-Allyl-2α,3α,6β,22,23-pentahydroxy-5α-sitostane (3c)
Prepared from compound 3b (55.0 mg, 0.10 mmol), after the deprotection reaction compound
o
3c was obtained as a white solid (30.0 mg, 57%). Mp. 106-107 C. [α]28 = +3.8 (c = 0.52,
1
CHCl3). Rf 0.61 (hexane/EtOAc, 1:20). IR (cm-1): 3419, 2935, 2871, 1462, 1381, 1043. H
NMR (300 MHz, CDCl3) δ 5.88-5.75 (m, 1H, H-2′), 5.13 (d, J = 7.6 Hz, 1H, H-3′a), 5.09 (d,
J = 15.4 Hz, 1H, H-3′b), 4.07 (br s, 1H, H-3), 3.80 (d, J = 11.5 Hz, 1H, H-2), 3.72 (d, J = 8.4
Hz, 1H, H-23), 3.61 (d, J = 8.5 Hz, 1H, H-22), 1.03 (s, 3H, H-19), 1.00-0.86 (m, 12H), 0.70
(s, 3H, H-18). 13C NMR (75 MHz, CDCl3) δ 133.8 (C-2′), 118.6 (C-3′), 74.8, 73.2, 72.8,
69.1, 55.9, 53.7, 52.6, 46.9, 46.3, 43.3, 42.8, 42.7, 42.4, 39.9, 37.6, 37.0, 30.9, 29.7, 28.9,
27.8, 26.5, 24.1, 21.2, 20.8, 19.5, 18.9, 15.8, 13.5, 11.9. HRES-MS m/z calcd for [M+NH4]+
C32H60NO5: 538.4466, found: 538.4456.
(22S,23S,24S)-6α-Allyl-2α,3α,6β,22,23-pentahydoxy-5α-stigmastan-6-one (11c)

Prepared from compound 11b (60.0 mg, 0.10 mmol), after the deprotection reaction
compound 11c was obtained as a white solid (35.0 mg, 67%). Mp. 127-129 oC. [α]28 = -4.7 (c
= 1.52, CHCl3). Rf 0.66 (hexane/EtOAc, 1:20). IR (cm-1): 3410, 2986, 2305, 1421, 1264,
1
1156, 896. H NMR (300 MHz, CDCl3) δ 5.88-5.72 (m, 1H, H-2′), 5.12 (d, J = 6.2 Hz, 1H,
H-3′a), 5.08 (d, J = 15.0 Hz, 1H, H-3′b), 4.06 (br s, 1H, H-3), 3.85 – 3.75 (m, 1H, H-2), 3.60-
3.59 (m, 2H, H-22, H-23), 1.03 (s, 3H, H-18), 1.02 (d, J = 6.2 Hz, 3H, H-21), 0.96 (t, J = 6.7
Hz, 3H, H-29), 0.94 (d, J = 7.3 Hz, 3H, H-26), 0.88 (d, J = 6.8 Hz, 3H, H-27), 0.72 (s, 3H, H-
19). 13C NMR (75 MHz, CDCl3) δ 133.9 (C-2′), 118.6 (C-3′), 73.2, 72.2, 70.6 69.1, 68.6,
55.7, 53.8, 52.7, 49.7, 46.9, 43.4, 43.1, 42.8, 42.5, 42.4, 39.8, 37.6, 30.8, 28.0, 26.9, 26.5,
24.4, 21.8, 20.7, 18.6, 17.7, 15.7, 14.4, 14.1, 12.0. HRES-MS m/z calcd for [M+NH4]+
C32H60NO5: 538.4466, found: 538.4456.
General procedure for dimerization
To a solution of allyl-brassinosteroids (8a, 3c, 9c) (1 equiv.) in anh. CH2Cl2 was added 2nd
generation Grubbs catalyst 2.5 %mol at the room temperature under argon. The mixture was
heated under pressured tube at 90 °C for 20 h and purified by column chromatography giving
the dimer products (8b, 3d, 9d).
Dimeric compound of 8a
Prepared from compound 8a (50.0 mg, 0.10 mmol) in CH2Cl2 (10.0 mL) following the
general procedure, compound 8b was obtained as a white solid (12.0 mg, 13 %) with
o
recovery of compound 8a (27.0 mg, 55.5 %). Mp. 155-157 C. [α]28 = -65.6 (c = 0.52,
1
CHCl3). Rf 0.40 (hexane/EtOAc, 1:20). IR (cm-1): 3410, 2987, 1421, 1265, 896. H NMR
(300 MHz, CDCl3-MeOD) δ 5.43 (br s, 2H, H-2′), 5.17 (dd, J = 15.1, 8.4 Hz, 2H, H-22), 5.02
(dd, J = 15.1, 8.4 Hz, 2H, H-23), 4.01 (br s, 2H, H-2), 3.73 (d, J = 10.6 Hz, 2H, H-3), 2.27 (d,
J = 10.2 Hz, 2H, H-5), 1.03 (d, J = 6.4 Hz, 6H, H-21), 1.02 (s, 6H, H-19), 0.85 (d, J = 6.4 Hz,
6H, H-26), 0.81 (t, J = 7.4 Hz, 6H, H-29), 0.80 (d, J = 6.7 Hz, 6H, H-27), 0.71 (s, 6H, H-18).
13C NMR (75 MHz, CDCl3-MeOD) δ 138.2 (C-22), 129.3 (C-2′), 129.2 (C-23), 73.7 (C-6),
69.0 (C-2), 68.3 (C-3), 56.2, 56.1, 54.0, 51.2, 45.1, 43.1, 42.5, 42.4, 41.5, 40.4, 39.8, 37.4,
31.8, 30.8, 28.8, 26.4, 25.3, 24.4, 21.1, 21.0, 20.7, 18.9, 15.7, 12.2, 12.1. HRES-MS m/zcalcd
for [M+Cl]+ C62H104ClO6: 979.7521, found: 979.7516.

Dimeric compound of 3c
Prepared from compound 3c (100.0 mg, 0.20 mmol) in CH2Cl2 (10.0 mL) following the
general procedure, compound 3d was obtained as a white solid (35 mg, 17 %) with recovery
of compound 3c (52.0 mg, 52 %). Mp. 212-214 oC. [α]28 = +22.0 (c = 0.55, CHCl3). Rf 0.21
1
(hexane/EtOAc, 1:20). IR (cm-1): 3419, 3056, 2986, 2842, 1650, 1404, 1016. H NMR (300
MHz, CDCl3-MeOD) δ 5.48 (br s, 2H, H-2’), 4.02 (br s, 2H, H-3), 3.78 (br s, 2H, H-2), 3.69
(d, J = 8.7 Hz, 2H, H-23), 3.54 (d, J = 8.5 Hz, 2H, H-22), 1.01 (s, 6H, H-19), 0.99-0.84 (m,
24H), 0.70 (s, 6H, H-18). 13C NMR (75 MHz, CDCl3-MeOD) δ 129.9 (C-2′), 77.3, 74.5, 74.4,
72.2, 68.7, 68.3, 56.7, 54.0, 52.6, 46.2, 45.1, 42.8, 42.0, 41.1, 39.9, 37.1, 30.7, 29.6, 29.0,
27.7, 26.2, 23.9, 21.1, 20.7, 19.4, 18.8, 15.6, 13.5, 11.9, 11.8. HRES-MS m/z calcd for
[M+Na]+ C62H108O10Na: 1035.7835, found: 1035.7825.
Dimeric compound of 8c
Prepared from compound 8c (100.0 mg, 0.20 mmol) in CH2Cl2 (10.0 mL) following the
general procedure, compound 8d was obtained as a white solid (40 mg, 20 %) with recovery
o
of compound 8c (55.0 mg, 55.5 %). Mp. 182-184 C. [α]28 = -76.5 (c = 0.48, CHCl3). Rf
1
0.24 (hexane/EtOAc, 1:20). IR (cm-1): 3410, 2949, 2839, 1650, 1404, 1016. H NMR (300
MHz, CDCl3-MeOD) δ 5.43 (br s, 2H, H-2’), 4.00 (br s, 2H, H-3), 3.74-3.70 (m, 2H, H-2),
3.52-3.49 (m, 4H, H-22, H-23), 1.02 (s, 6H, H-19), 1.00-0.93 (m, 18H, H-21, H-26, H-29),
0.94 (d, J = 7.1 Hz, 6H), 0.86 (d, J = 6.8 Hz, 6H, H-27), 0.73 (s, 6H, H-18). 13C NMR (75
MHz, CDCl3-MeOD) δ 133.2 (C-2′), 77.8, 76.4, 74.3, 72.9, 72.1, 59.8, 57.9, 56.5, 53.0, 49.1,
47.0, 46.7, 46.4, 46.2, 45.2, 43.8, 41.2, 34.7, 32.1, 30.7, 30.4, 28.4, 25.4, 24.6, 22.6, 21.2,
19.6, 18.4, 18.2, 15.7. HRES-MS m/z calcd for [M+Na]+ C62H108O10Na: 1035.7835, found:
1035.7818.
Acknowledgements
We thank the Kasetsart University and CITO Co.Ltd. for the financial supports. We
also thank Department of Chemistry, Silpakorn University for spectroscopic determinations.
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Graphic Abstract
Synthesis and Preliminary Evaluation of Dimeric-28-homobrassinosteroids for Plant
Growth Regulators
Waya S. Phutdhawong, Wanwikar Ruensamran, Weerachai Phutdhawong*
The synthesis and bioactivity of the synthetic analogues of 28-homobrassinosteroids
including the dimeric compounds of 28-homocastasterone were studied. Evaluation of the
rice lamina inclination activity revealed that the dimeric analogues possessed a reduced but
significant bioactivity when compared to the nature 28-homocastasterone.

Research Highlights
-Preparation of (22R, 23R)- and (22S, 23S)-homocastasterone are reported.
-Nucleophilic additions of C6 carbonyl group by allylic Grignard reagent were
designed.
-Olefin metathesis of the allyl analogues using Grubbs catalyst resulted in the dimeric
analogues.
-Rice lamina inclination assay of the dimeric analogues showed the significant activity
compared to the natural 28-homocastasterone.