Synthesis of new steroidal carbamates with plant-growth-promoting activity: Theoretical and experimental evidence

Article abstract of DOI:10.3390/ijms22052330

A priority of modern agriculture is to use novel and environmentally friendly plant-growth promoter compounds to increase crop yields and avoid the indiscriminate use of synthetic fertilizers. Brassinosteroids are directly involved in the growth and development of plants and are considered attractive candidates to solve this problem. Obtaining these metabolites from their natural sources is expensive and cumbersome since they occur in extremely low concentrations in plants. For this reason, much effort has been dedicated in the last decades to synthesize brassino-steroids analogs. In this manuscript, we present the synthesis and characterization of seven steroi-dal carbamates starting from stigmasterol, β-sitosterol, diosgenin and several oxygenated derivatives of it. The synthesis route for functionalization of diosgenin included epoxidation and epoxy opening reactions, reduction of carbonyl groups, selective oxidation of hydroxyl groups, among others. All the obtained compounds were characterized by¹H and¹³C NMR, HRMS, and their melting points are also reported. Rice lamina inclination test performed at different concentrations es-tablished that all reported steroidal carbamates show plant-growth-promoting activity. A molecular docking study evaluated the affinity of the synthesized compounds towards the BRI1-BAK1 receptor from Arabidopsis thaliana and three of the docked compounds displayed a binding energy lower than brassinolide.

Full text of DOI:10.3390/ijms22052330

International Journal of
Molecular Sciences
Article
Synthesis of New Steroidal Carbamates with
Plant-Growth-Promoting Activity: Theoretical and
Experimental Evidence
Daylin Fernández Pacheco 1 , Leonardo González Ceballos 1 , Armando Zaldo Castro 1,
Marcos R. Conde González 1, Laura González de la Torre 2, Lia Pérez Rostgaard 1, Luis Espinoza 3,
*
,
Katy Díaz 3 , Andrés F. Olea 4 and Yamilet Coll García 1,
*
1
Center for Natural Product Research, Faculty of Chemistry, University of Havana, Zapata and G,
Havana 10400, Cuba; daylinfp@fq.uh.cu (D.F.P.); leo@fq.uh.cu (L.G.C.); zaldo@fq.uh.cu (A.Z.C.);
mrconde@fq.uh.cu (M.R.C.G.); lrostgaard@estudiantes.fq.uh.cu (L.P.R.)
2
3
4
Sierra Maestra Science, Technology and Innovation Entity, Barlovento Complex, 5ta Ave. and 246,
Havana 11300, Cuba; ltorre@bionaturasm.cu
Departamento de Química, Universidad Técnica Federico Santa María, Avenida España 1680,
Valparaíso 2340000, Chile; katy.diaz@usm.cl
Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, El Llano
Subercaseaux 2801, Santiago 8900000, Chile; andres.olea@uautonoma.cl
*
Correspondence: luis.espinozac@usm.cl (L.E.); yamcoll@fq.uh.cu (Y.C.G.); Tel.: +56-32-2654225 (L.E.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A priority of modern agriculture is to use novel and environmentally friendly plant-growth
promoter compounds to increase crop yields and avoid the indiscriminate use of synthetic fertilizers.
Brassinosteroids are directly involved in the growth and development of plants and are considered
attractive candidates to solve this problem. Obtaining these metabolites from their natural sources is
expensive and cumbersome since they occur in extremely low concentrations in plants. For this reason,
much effort has been dedicated in the last decades to synthesize brassinosteroids analogs. In this
manuscript, we present the synthesis and characterization of seven steroidal carbamates starting from
Citation: Pacheco, D.F.; González
Ceballos, L.; Castro, A.Z.; Conde
González, M.R.; González de la Torre,
L.; Rostgaard, L.P.; Espinoza, L.; Díaz,
K.; Olea, A.F.; Coll García, Y.
Synthesis of New Steroidal
Carbamates with Plant-Growth-
Promoting Activity: Theoretical and
Experimental Evidence. Int. J. Mol.
Sci. 2021, 22, 2330. https://doi.org/
10.3390/ijms22052330
stigmasterol, β-sitosterol, diosgenin and several oxygenated derivatives of it. The synthesis route
for functionalization of diosgenin included epoxidation and epoxy opening reactions, reduction of
carbonyl groups, selective oxidation of hydroxyl groups, among others. All the obtained compounds
were characterized by 1H and 13C NMR, HRMS, and their melting points are also reported. Rice
lamina inclination test performed at different concentrations established that all reported steroidal
carbamates show plant-growth-promoting activity. A molecular docking study evaluated the affinity
of the synthesized compounds towards the BRI1-BAK1 receptor from Arabidopsis thaliana and three
of the docked compounds displayed a binding energy lower than brassinolide.
Academic Editor: Jana Oklestkova
Received: 8 February 2021
Accepted: 23 February 2021
Published: 26 February 2021
Keywords: brassinosteroid analogs; plant-growth promoters; molecular docking; carbamates; rice
lamina inclination test
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
The increase of crop yields is a common goal for all research groups dedicated to
supplying agriculture with novel plant-growth enhancers. In this effort, the quest for
biologically active compounds that are also friendly to the environment is of great interest.
The growth and development of plants are regulated by endogenous factors (i.e., phyto-
hormones) and exogenous factors (i.e., plant growth regulators). Within the first group,
hormones and plant growth regulators play an indispensable role in the regulation of de-
velopmental processes. It is known that brassinosteroids (BRs), a family of phytohormones
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
of steroidal nature [
1,2], are involved in promoting cell expansion, cell elongation, cell
division, and vascular differentiation [
3–8], additionally BRs provides protection against
Int. J. Mol. Sci. 2021, 22, 2330. https://doi.org/10.3390/ijms22052330
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 2330
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environmental stress [
9–
11]. Some representative natural occurring BRs are shown in
Figure 1.
OH
OH
OH
OH
OH
OH
HO
HO
HO
O
O
HO
HO
O
HO
H
H
H
O
O
O
28-homobrassinolide (3)
brassinolide (1)
24-epibrassinolide (2)
OH
OH
OH
HO
HO
HO
HO
HO
HO
HO
H
O
teasterone (5)
H
H
O
O
castasterone (4)
typhasterol (6)
Figure 1. Representative natural occurring brassinosteroids.
Since the discovery of BRs in the seventies [1], much attention has been paid to
these molecules because of their recognized participation in physiological processes
beginning in embryonic development up to homeostasis in the adult plant [12]. In
this way they become very promising compounds to be used to increase agricultural
yields. Besides their role as enhancers of agriculture production yields [13,14], BRs
are considered environmentally safe for providing plant protection [10]. However,
the very low concentration of BRs in plants is the main drawback for their extensive
use [4]. Therefore, for practical purposes, chemical synthesis appears as “the only”
source of BRs and/or its analogs. Synthetic advances have allowed obtaining a series
of BRs analogs with novel structures and substituent functions, which exhibit similar
or even higher biological effects as compared to natural molecules [15
last years different research groups have reported the synthesis of new BRs analogs to
study their structure/activity relationship [16 20], resulting in very active and stable
compounds [21 22]. Several studies have proved that some synthetic BRs analogs can
produce a wide range of physiological responses in plants, like potentiate plant growth
and development and protection from several conditions of stress [15 23 24]. Otherwise,
different nitrogen-containing compounds such as polyamines [25], acyl thioureas [26 27],
,16]. Thus, in the
–
,
,
,
,
thiazoles, oxadiazoles, and pyrimidines exhibit a broad biological activity spectrum and
their potential properties as plant growth promoters are of great interest [28]. Besides, the
synthesis of new aryl-brassinosteroid derivatives and their evaluation as plant growth
promoters have been recently reported [29,30].
This work presents the synthesis of a series of BRs carbamates analogs. Various
hydroxysteroids (stigmasterol, β-sitosterol, diosgenin, and oxygenated derivatives) were
condensed with phenyl isocyanate to afford the corresponding carbamates [31]. Post con-
densation reactions, i.e., epoxidation, carbonyl reduction, and oxime formation were used
to introduce new diversification elements. All the obtained compounds were characterized
by NMR (1H and 13C) and HRMS. The growth-promoting activity of these BRs analogs was
determined by using the rice-lamina inclination test due to its simplicity and selectivity [32].
A molecular docking study, using BRs receptor from Arabidopsis thaliana (BRI1-BAK1) as the
model receptor and the final carbamates as ligands, was performed to provide theoretical
insights into the experimental findings.

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2. Results and Discussion
2.1. Synthesis
Diosgenin (6) has been used to prepare a significant number of spirostanic BRs analogs,
which have shown a promising plant growth-promoting activity [17
,
33–37]. Consider-
ing these antecedents, we were interested in the possibility of preparing new diosgenin
derivatives with the incorporation of carbamate functions in C3, modifications to ring B
and evaluating the effect that these chemical modifications could have on plant growth-
promoting activity.
The synthesis of corresponding carbamates of diosgenin, β-sitosterol and stigmasterol
was carried out by reacting these starting compounds with phenyl isocyanate, using CHCl3
as solvent and HCl as acid catalyst (Scheme 1) [38–40].
Scheme 1. Synthesis of diosgenin, β-sitosterol and stigmasterol carbamates 7, 9 and 11.
The reactions were monitored by thin-layer chromatography (TLC) for 24 h. After
completion, the crudes were purified by column chromatography to afford the respective
carbamates. The reaction yields were 62%, 73% and 71% for compounds 7, 9 and 11, re-
spectively. The main spectroscopic evidence for obtaining of these products comes from
NMR spectroscopy. In the 1H NMR spectra aromatic H give signals at δH = 7.40–7.04 ppm
and H-N produces a signal between δH = 6.58-6.53 ppm, for the three compounds. On
the other hand, in the 13C NMR spectra, the signals observed at δC = 153.16, 153.24
and 153.26 ppm were assigned to the carbamoyl group (NHCO) of compounds 7, 9 and
11, respectively.
Synthesis of carbamate 15 from diosgenin goes through the intermediate 14 that
is obtained by a three-step sequence including epoxidation of diosgenin with meta-
chloroperoxybenzoic acid (m-CPBA) [41], acid-catalyzed epoxide opening [42], and se-
lective oxidation of axial -OH group at position C6 with N-bromosuccinimide (NBS) [43
]
(Scheme 2).
Epoxidation of diosgenin (
yield. The diastereomeric ratio of each epoxide in the mixture was established by
integration of 1H NMR signals assigned to the H-3 epoxide
(compound 12a) which in
the mixture appear at δH = 3.95-3.86 ppm (multiplet) and H-3 epoxide (compound 12b
which appear at δH = 3.73-3.65 ppm (multiplet), and H-6 epoxide (compound 12b) at
δH = 3.06 ppm (d, J = 2.3 Hz) and H-6 epoxide (compound 12a) at δH = 2.90 ppm (d,
6) originates a mixture of α- and β-epoxides with 95%
α
β
)
β
α
J = 4.4 Hz) (Figure 2). Based on these NMR measurements the relative ratio of 12a:12b
was determined as 2:1. Additionally, it was possible to assign most of the 1H and 13
C
NMR signals from the 12a/12b epoxide mixture.

Int. J. Mol. Sci. 2021, 22, 2330
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21
17
25
26
23
O
O
27
18
20
22
24
12
HClO4
m-CPBA
CHCl3 rt. 3 h
11
13
14
O
19
O
16
15
diosgenin (6)
1
4
9
THF/H2O (20:1) rt. 3 h
10
2
3
8
5
7
6
HO
HO
12a (-epoxide)
12b (-epoxide)
(/ 2:1)
13
HO
O
OH
NBS
1,4-dioxane/H2O (7:1) rt. 2 h
O
O
PhNCO
HCl (cat.)
O
O
O
CHCl3 reflux 24 h
N
H
O
HO
HO
HO
O
O
14
15
Scheme 2. Synthesis of a B-ring functionalized derivative of diosgenin carbamate.
Figure 2. 1H NMR spectrum (expansion zone 2.80-4.50 ppm) of diasteroisomeric epoxides mixture
12a/12b.
Opening of the epoxides mixture, in acid medium (HClO4/THF/H2O), generates just
one diastereomer (13, 78% yield) due to steric hindrance induced by the methyl group
at position C19 [42]. Compound 14 was obtained from 13 by stereo and regio-selective
oxidation with NBS (83% yield) [43]. Obtention of 14 was confirmed by 13C NMR, i.e.,
signal at δC = 212.09 ppm assigned to the carbonyl group at C6. Treatment of ketone 14
with phenyl isocyanate under acidic conditions produces carbamate derivative 15 in 68%
yield. The presence of signals at δH = 7.32-7.00 and 6.60 ppm in the 1H NMR spectrum are
assigned to hydrogen atoms from the aromatic ring and the carbamoyl group (NHCO),
respectively, confirming the presence of carbamoyl group in compound 15.
Epoxidation of diosgenin carbamate (
with 94% yield (Scheme 3).
7) with m-CPBA produced the 16a/16b mixture
The diastereomeric ratio of each epoxide in the mixture was established by NMR
spectroscopy in the same way as described above for the mixture 12a 12b. So, integration
/

Int. J. Mol. Sci. 2021, 22, 2330
5 of 19
1
of H NMR signals assigned to the H-3 epoxide
α
β
(compound 16a) which appear at
(compound 16b) which appear at
δH = 4.99-4.90 ppm (multiplet) and H-3 epoxide
δH = 4.82-4.74 ppm (multiplet), and the signals at δH = 3.11 ppm (d, J = 1.7 Hz) and
δH = 2.92 ppm (d, J = 5.7 Hz) assigned to the H-6 and H-6 , respectively (Figure 3). Based
β
α
on these NMR measurements the relative ratio of 16a
:16b obtained was determined as 3:1.
O
O
O
O
m-CPBA
O
O
16a ( -epoxide)
16b ( -epoxide)
CHCl3 rt. 3 h
N
H
O
7
N
H
O
O
(
/ 3:1)
O
O
O
O
NH2OH-HCl
NaOAc.3H2O
O
O
N
H
O
N
H
O
OH
OH
EtOH rt. 3 h
17
OH
15
N
O
O
O
O
O
NaBH4
EtOH 10°C 30 min.
O
O
N
H
O
N
H
O
OH
OH
OH
18
15
O
Scheme 3. Derivatization of diosgenin carbamates, obtaining compound 16–18.
1
Figure 3. H NMR spectrum (expansion zone 2.70-5.10 ppm) of diasteroisomeric epoxides mixture 16a/16b.

Int. J. Mol. Sci. 2021, 22, 2330
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On the other hand, treatment of carbamate 15 with NH2OH.HCl/NaOAc.3H2O system
(according to reported protocols [44,45]), produces oxime 17 with 77% yield. Spectroscopic
evidence of transformation of ketone group into hydroxy oxime function in C6 is the
1
appearance of a singlet signal at δH = 10.51 ppm, assigned at N-OH, in the H NMR
spectrum of compound 17. While in the 13C NMR spectrum of compound 17, the C6
signal was observed at δC = 159.16 ppm, compared to the chemical shift observed for C6
in precursor 15, which was observed at δC = 211.98 ppm. Finally, reduction of the ketone
group (C6) in compound 15 with NaBH4/EtOH, occurs in a stereospecific way producing
compound 18 with 83% yield. The stereospecificity of the reduction could be explained
in terms of the steric hindrance offered by the CH3-19 attached to C10, which force the
reducing agent to be added through the lower face of ring B. The main spectroscopic
evidence that confirms the obtaining of derivative 18 was the observation of the signal
at δH = 4.58 ppm (d, J = 4.2 Hz) assigned to the C6-OH group in the 1H NMR spectrum
of compound 18. While in the 13C NMR spectrum of compound 18, the carbinolic C6
signal was observed at δC = 73.63 ppm, compared to the chemical shift observed for C6 in
precursor 15, which was observed at δC = 211.98 ppm.
2.2. Rice-Lamina Inclination Test
The rice-lamina inclination test is a very simple and selective method that is widely
used to measure activity of BRs analogs as plant-growth promoters [32]. In this assay,
the angle formed between the laminae and sheaths of rice seedlings is determined, and
significant differences observed in treated seedlings are associated to BRs-induced increase
on plant growth. Thus, a higher angle degree indicates higher activity of applied BRs
analogs. This study was conducted at three different concentrations and two bioassays
with several replicas were carried out for statistical purposes. Results obtained are shown
in Table 1. Brassinolide was taken as positive control since it is the most active natural BRs
and distilled water was used as negative control.
Table 1. Effect of brassinolide (1) and BRs analogs 7, 9, 11, 15, 16a/16b, 17 and 18, on the lamina
inclination test of rice seedlings.
BRs Analogs
Number
Inclination between Laminae and Sheaths (Degrees ± Standard Error)
Concentration (µM)
0.01
0.10
1.0
7
9
11
15
21 ± 3 a
35 ± 3 b
31 ± 3 b
29 ± 1 b
25 ± 3 a
13 ± 5 c
13 ± 5 c
34 ± 6 b
16 ± 5 c
30 ± 3 b
31 ± 3 b
33 ± 3 b
51 ± 1 d
36 ± 5 b
29 ± 1 b
55 ± 8 d
11 ± 6 c
21 ± 3 a
11 ± 5 c
14 ± 1 c
40 ± 3 b
41 ± 1 b
38 ± 2 b
29 ± 1 b
78 ± 9 e
16a/16b
17
18
1 (C+)
C- (water)
These values represent the mean
each (n = 12). Values with different superscript letters are statistically different (p < 0.05). Post-Hoc Tukey HSD
±
standard deviation of two independent experiments with at least six replicates
test was applied to show statistically significant differences among the means.
All compounds induce angle degrees higher than the one measured for the negative
control but lower than the respective value obtained for brassinolide. Nonetheless, a
comparison of activities shown by compounds synthesized in this work evidences some
interesting structure-activity results. At low concentrations, the activities of carbamates
derived from stigmasterol and
ity ratio Analog/Brassinolide = 1 to 0.5), and higher than that measured for the diosgenin
carbamate. However, this effect is lost at 1.0 M. The main structural difference between
these compounds lays in the side chain, i.e., the bulky glycosyl group increase the hy-
β-sitosterol are similar to that found for brassinolide (Activ-
µ

Int. J. Mol. Sci. 2021, 22, 2330
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drophobicity and steric effects of any interaction involving this part of the molecule. On
the other hand, at all tested concentrations, analogs 15
activities than that observed for diosgenin-derived carbamate
from just in the substitution in C5 and C6. Thus, it can be concluded that presence of
,
16a
/16b
, 17 and 18 exhibit higher
7. These compounds differ
7
oxygenated functionalities in positions C5 and C6 are key factors in determining growth-
promoting activity of diosgenin carbamates. In this sense, an epoxide group linking C5 and
C6 (mixture 16a
/16b) or carbonyl group in C6 (compound 15) seems to be more effective
than hydroxyl or hydroxymino groups in inducing activity. Interestingly, mixture 16a
/
16b
shows the higher activities of all analogs and at the intermediate concentration is as active
as brassinolide. The advantage for a potential application lays in its synthesis which is
relatively straightforward and with high yields.
2.3. Molecular Docking Study
Previously, a combination of genetic, biochemical, and proteomic approaches has
accelerated the understanding of the BRs signaling pathway in Arabidopsis thaliana [46]. The
signal transduction pathway of BR has been intensively studied the past decade [46–48]
and these studies have established a complex BR signal transduction pathway, that play
an important role in plant growth and development. Structural analysis revealed that
a hydrophobic groove, formed between the inner surface of the helical BRI1-LRR and a
~70-residues island domain, is responsible for the specific recognition of
1 by BRI1 [49].
The binding site of BRI1 is located on the surface of the receptor ectodomain as a nonpolar
cleft lined by nonpolar aromatic and aliphatic residues (I540, I563, W564, Y599, Y642, M657,
F681, I682, I706), whereas hydroxyl groups form the cleft ridge (Y597, Y599, Y642, S647).
Brassinolide fits into the cleft via its nonpolar side and displays its hydroxyl groups towards
the solvent and protein partners [50]. At this point,
1 induces heterodimerization between
BRI1 and the somatic embryogenesis receptor kinases (SERKs) family of co-receptors [51].
This leads to BRI1-SERK trans-phosphorylation and subsequent downstream signaling
mediating plant growth and development.
Molecular docking is a valuable computational tool to predict the interaction geometry
and strength between two macromolecules or, between a small ligand and a macromolecule,
starting from their unbound conformations [52]. It is known that BRs perception occurs in
a two-step mechanism [53]. First, BRs binds to a hydrophobic surface groove on receptor
BRI1-LRR domain, and this is followed by the heterodimerization with BAK1/SERK1-LRR,
fully activating the receptor. To gain further insight into the activity of the new steroidal
carbamates introduced in this study, we performed rigid docking of the ligands into the
active site of the BRI1-BAK1 complex (PDB: 4m7e) using AutoDock Vina [52] since crystal
structures of BRI1 and BAK1 are available.
Redocking of the natural ligand, 1, was performed to assess the quality of the search
parameters. Results show that the lowest energy pose is almost identical to the crystallo-
graphic pose, so the parameters were considered appropriate and docking of synthetic
ligands was carried out. The poses considered representative for each ligand align well
with
1 in the binding site. All Vina binding energies for the complexes were negative
(Figure 4) suggesting that formation of the BRI1–ligand–BAK1 complex is favored.
As can be seen in Figure 5a, the hydrophobic side chains of all carbamates are buried
in the hydrophobic pocket delineated by residues I540, I563, W564, I592, Y597, L615 and
T646. However, since these carbamates are larger than 1, they occupy a larger volume
in the active site, and thus the phenyl groups slightly protrude out of the binding site
(Figure 5b,c). Although it would seem reasonable that the phenyl group could also fit in the
hydrophobic pocket, as has been reported for other analogs [50], the lack of functionalities
that promote the interactions between both proteins and the carbamates in the inverted
conformation, makes it unfavorable. This could be the reason for the modest activities
determined for carbamates as compared to 1.

Int. J. Mol. Sci. 2021, 22, 2330
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Figure 4. Binding energies between 1, 7, 9, 11, 15, 16a, 16b, 17, 18 and BRI1-BAK1 complex.
To gain further insight into the activities of the compounds we also analyzed the
ligand-proteins interactions with the software LigPlot+ (see Figure S12, Supplementary
Materials, for all interaction diagrams) [54,55]. As can be seen in Figure 6, the interaction
diagrams of steroidal carbamates 15 and 18 show the amino acid residues of the active
site participating in the stabilization of complexes BRI1-ligand-BAK1. Circled residues
represent common interactions between
1 and the complexes. As expected, the number
of common interactions observed for carbamate 15 is bigger than that corresponding to
18. This result is in line with the binding energy values obtained for both carbamates since
15 possesses the lowest binding energy and 18 the highest of all synthesized carbamates.
Among all possible interactions between ligands and proteins, hydrophobic packing inter-
actions are predominant. This is not surprising, considering the hydrophobic nature of the
binding pocket formed by the interaction of BRI1 with BAK1. Some important hydrogen
bonds (H41 and V62) are absent for all compounds [53], and this is other fact that could
contribute to the reduced activity of carbamates as compared to
is a hydrogen bond interaction with Y642, which has proved to be important for binding
affinity [54 55], and a new hydrogen bond between the carbonyl moiety of the phenyl
1. On the other hand, there
,
carbamate and the side chain hydroxyl group of T729 [56]. These interactions are present in
several compounds with different activity profiles, which underlines the complexity of BRs
signaling in plants and the intrinsic inaccuracy of Vina scoring function [57]. Assuming
that these poses are appropriate representations of the actual binding mode of steroidal car-
bamates 7, 9, 11, 15, 16a, 16b, 17, and 18, several conclusions regarding functional groups
diversity can be inferred. For example, the presence of oxygenated functionalities on
ring A besides the carbamoyl group can improve the interactions with the BAK1 cofactor,
necessary for proper receptor activation. Moreover, these kinds of functionalities in the
side chains could also contribute to these interactions.
For compounds 16a and 16b, α and β epoxides respectively, the docking studies indicate
that both diastereomers, although presenting two distinct binding modes to the BRI1/BAK1
heterodimer (Figure 7a,b), share almost the same protein contacts (Figure 7c,7d) and bind
with approximately the same strength. Therefore, adaptation to the new geometry of the
steroid due to the
β-epoxide could be invoked to explain the change in binding mode while
retaining the favorable contacts with the proteins.

Int. J. Mol. Sci. 2021, 22, 2330
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Figure 5. General picture of the ligand poses. (
buried into a hydrophobic pocket. (
all carbamates protrudes out of the binding site. BRI1 (blue), BAK1 (wheat), compounds
16a, 16b, 17 and 18 are depicted as color sticks, whereas brassinolide appears colored black.
a
) Hydrophobic side chains of all carbamates are
b
) Active site with hidden BAK1 for clarity. (
c) Phenyl moiety of
7
,
9
,
11, 15,
15-BRI1/BAK1
18-BRI1/BAK1
Figure 6. Ligand–Protein contacts for steroidal carbamates 15 and 18. Circled residues represent common interactions
between 1 and synthetic ligands. For the other studied compounds see Figure S12.

Int. J. Mol. Sci. 2021, 22, 2330
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Figure 7. Binding modes and protein contacts of diastereomers (a) compound 16a (green sticks), (b) compound 16b (pink
sticks), (c) Protein contacts of compound 16a. (d) Protein contacts of compound 16b. Circled residues represent shared
contacts. BRI1 (blue), BAK1 (wheat). For the other studied compounds see Figures S11 and S12
3. Materials and Methods.
3.1. Chemical
General
All chemical reagents were purchased from Merck, Fluke, or Sigma-Aldrich and used
without previous purification. All solvents were distilled and stored over proper desiccants.
Melting points were measured on a BUCHI M-565 equipment (BÜCHI Labortechnik GmbH,
Essen, Germany) being EtOAc the last solvent used in all cases. NMR spectra were recorded
at 298 K on a Varian Mercury 400 NMR spectrometer (Varian, Palo Alto, CA, USA) at
400 MHz and 101 MHz for 1H and 13C respectively. All spectra were referenced using the
TMS signal or the residual peak of the solvent. Chemical shifts (δ) are reported in ppm
and coupling constants (J) are given in Hz. A TripleToF 6600-1 mass spectrometer (Sciex,
MA, USA) was used for high-resolution mass spectrometry (HRMS). Silica gel (Merck
70–230 mesh) was used for column chromatography (CC) and silica gel plates HF254 for
thin-layer chromatography (TLC). TLC spots were detected by heating after staining with
cerium molybdate in H2SO4.
3.2. Methods of Synthesis
3.2.1. Synthesis of Carbamates
To a solution of diosgenin, β-sitosterol or stigmasterol in CHCl3 (10 mL) was added
dropwise phenyl isocyanate in the ratio 1:4 and some drops of HCl (37% w/w in water).
The reaction mixture was refluxed for 24 h and monitored by TLC (n-hex/EtOAc 3:1). The
obtained solution was concentrated to dryness and the crude product was purified by CC.

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3.2.2. Synthesis of (25R)-5-en-Spirost-3β-yl Phenylcarbamate (7)
Compound was obtained (0.80 g, 62% yield) following the general procedure for
7
synthesis of carbamates. Diosgenin (1.0 g, 2.4 mmol) and isocyanate (1.05 mL, 9.6 mmol).
CC (n-hex/EtOAc 3:1)
1
Compound
7
: white solid, m.p. 219-220 ◦C, Rf = 0.76 (n-hex/EtOAc 3:1). H NMR
(400 MHz, CDCl3) (Figure S7)
δ
= 7.37 (2H, d, J = 8.0 Hz, H-2a), 7.29 (2H, dd, J = 8.6, 7.2 Hz,
H-3a), 7.05 (1H, t, J = 7.3 Hz, H-4a), 6.58 (1H, s, NH), 5.40 (1H, dt, J = 4.2, 1.9 Hz, H-6),
4.60 (1H, tt, J = 11.5, 4.8 Hz, H-3), 4.42 (1H, q, J = 7.5 Hz, H-16), 3.48 (1H, ddd, J = 11.0, 4.5,
2.0 Hz, H-26 eq), 3.38 (1H, t, J = 11.0 Hz, H-26ax), 2.44 (1H, ddd, J = 13.1, 5.1, 2.2 Hz, H-4
eq), 2.34 (1H, td, J = 12.3, 2.8 Hz, H-4 ax), 1.05 (3H, s, H-19), 0.98 (3H, d, J = 7.0 Hz, H-27),
0.79 (6H, d, J = 5.9 Hz, H-18 and H-21). 13C NMR (101 MHz, CDCl3) (Figure S1)
δ = 153.16
(NHCO), 139.77 (C-5), 138.17 (C-1a), 129.16 (C-3a), 123.39 (C-4a), 122.61 (C-6), 118.69 (C-2a),
109.42 (C-22), 80.95 (C-16), 74.97 (C-3), 66.98 (C-26), 62.22 (C-17), 56.58 (C-14), 50.07 (C-9),
41.76 (C-20), 40.41 (C-13), 39.87 (C-12), 38.58 (C-4), 37.09 (C-1), 36.86 (C-10), 32.19 (C-15),
31.98 (C-8), 31.56 (C-7), 31.53 (C-23), 30.44 (C-25), 28.95 (C-24), 28.21 (C-2), 20.98 (C-11),
19.50 (C-19), 17.28 (C-27), 16.44 (C-18), 14.67 (C-21). ESI-HRMS, calculated for C34H48NO4:
534.3583 [M+H]+ found: m/z 534.3638.
3.2.3. Synthesis of 5-en-Stigmast-3β-yl Phenylcarbamate (9)
Compound
8 was obtained (0.47 g, 73% yield) following the general procedure for
synthesis of carbamates.
CC (n-hex/EtOAc 12:1)
β
-sitosterol (0.5 g, 1.2 mmol) and isocyanate (0.52 mL, 4.8 mmol).
1
Compound
8
white solid, m.p. 160-161 ◦C, Rf = 0.77 (n-hex/EtOAc 5:1). H NMR
(400 MHz, CDCl3) (Figure S2)
δ
= 7.37 (2H, d, J = 7.5 Hz, H-2a), 7.29 (2H, d, J = 6.3 Hz,
H-3a), 7.04 (1H, t, J = 7.1 Hz, H-4a), 6.54 (1H, s, NH), 5.40 (1H, s, H-6), 4.68-4.53 (1H, m,
H-3), 0.92 (3H, d, J = 6.2 Hz, H-19), 0.88-0.77 (12H, m, H-21, H-26, H-27 and H-29), 0.67 (3H,
s, H-18). 13C NMR (101 MHz, CDCl3) (Figure S2)
δ = 153.24 (NHCO), 139.92 (C-5), 138.35
(C-1a), 129.19 (C-3a), 123.48 (C-4a), 122.92 (C-6), 118.92 (C-2a), 75.23 (C-3), 57.01 (C-14),
56.43 (C-17), 50.39 (C-9), 46.24 (C-24), 42.62 (C-13), 40.05 (C-12), 38.72 (C-4), 37.27 (C-1),
36.86 (C-10), 36.38 (C-20), 34.30 (C-22), 32.18 (C-7), 32.16 (C-8), 29.60 (C-25), 28.42 (C-16),
28.36 (C-2), 26.63 (C-23), 24.51 (C-15), 23.42 (C-28), 21.32 (C-11), 19.96 (C-26), 19.51 (C-27),
19.31 (C-19), 19.01 (C-21), 12.18 (C-29), 12.07 (C-18). ESI-HRMS, calculated for C36H56NO2:
534.4311 [M+H]+ found: m/z 534.4382.
3.2.4. Synthesis of 5,22-dien-Stigmast-3β-yl Phenylcarbamate (11)
Compound 11 was obtained (0.90 g, 71% yield) following the general procedure for
synthesis of carbamates. Stigmasterol (1.0 g, 2.4 mmol) and isocyanate (1.05 mL, 9.6 mmol).
CC (n-hex/EtOAc 12:).
1
Compound 11 white solid, m.p. 191-193 ◦C, Rf = 0.36 (n-hex/EtOAc 3:1). H NMR
(400 MHz, CDCl3) (Figure S3)
δ = 7.37 (2H, d, J = 7.9 Hz, H-2a), 7.34-7.27 (2H, m, H-3a),
7.05 (1H, t, J = 7.2 Hz, H-4a), 6.53 (1H, s, NH), 5.41 (1H, d, J = 4.5 Hz, H-16), 5.16 (1H, dd,
J = 15.1, 8.5 Hz, H-23), 5.03 (1H, dd, J = 15.1, 8.5 Hz, H-22), 4.70-4.51 (1H, m, H-3), 0.80 (6H,
d, J = 6.3 Hz, H-26 and H-27), 0.70 (3H, s, H-18). 13C NMR (101 MHz, CDCl3) (Figure S3)
δ
= 153.26 (NHCO), 139.94 (C-5), 138.41 (C-1a), 138.37 (C-22), 129.71 (C-23), 129.20 (C-3a),
123.49 (C-4a), 122.91 (C-6), 118.95 (C-2a), 75.25 (C-3), 57.10 (C-14), 56.36 (C-17), 51.48 (C-24),
50.45 (C-9), 42.54 (C-13), 40.52 (C-20), 39.95 (C-12), 38.73 (C-4), 37.29 (C-1), 36.89 (C-10),
32.22 (C-8), 32.16 (C-7), 32.08 (C-25), 29.00 (C-16), 28.37 (C-2), 25.54 (C-28), 24.58 (C-15),
21.40 (C-11), 21.32 (C-21), 21.15 (C-26), 19.51 (C-27), 19.21 (C-19), 12.32 (C-29), 12.28 (C-18).
ESI-HRMS, calculated for C36H54NO2: 532.4154 [M+H]+ found: m/z 532.4196.
3.2.5. Synthesis of Mixture (25R)- 5α,6α-Epoxy-Spirostan-3β-ol (12a) and
(25R)-5β,6β-Epoxy-Espirostan-3β-ol (12b)
Following a procedure previously described [58], a solution of diosgenin (5.0 g,
12.1 mmol) in CHCl3 (30 mL) was cooled in an ice bath. After 5 min, m-chloroperoxybenzoic

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acid (m-CPBA) (3.34 g, 19.4 mmol) was added in small portions. The reaction mixture was
stirred at room temperature for 3 h and consumption of starting material was monitored by
TLC (n-hex/EtOAc 3:1). At the end of reaction, CHCl3 (80 mL) was added and the diluted
mixture transferred to a separation funnel. This solution was washed (3x) with Na2SO3 (5%
solution w/w), Na2CO3 (sat. solution) and water, respectively. The organic layer was dried
over anhydrous Na2SO4 and then concentrated to dryness. The crude product was puri-
fied by recrystallization from acetone. Compounds 12a
/12b were obtained as an epoxide
mixture of epimers at C5-C6 positions (4.92 g, 95% yield, 12a:
α
/
β
12b = 2:1). ESI-HRMS,
calculated for C27H43O4: 431.3161 [M+H]+ found: m/z 431.3171. The signals assignment
was carried out from the 1H and 13C NMR spectra in the 12a/12b mixture:
1
Compound 12a: Rf = 0.19 (n-hex/EtOAc 3:1). H NMR (400 MHz, CDCl3) (Figure S4)
δ
= 4.38 (1H, q, J = 7.5 Hz, H-16), 4.17-3.61 (1H, m, H-3), 3.46 (1H, ddd, J = 10.9, 4.5, 2.0 Hz,
H-26 eq), 3.36 (1H, td, J = 10.9, 2.1 Hz, H-26 ax), 2.90 (1H, d, J = 4.4 Hz, H-6), 1.09-0.94
(6H, m, H-19 and H-27), 0.79-0.65 (6H, m, H-18 and H-21). 13C NMR (101 MHz, CDCl3)
(Figure S4)
δ = 109.22 (C-22), 80.59 (C-16), 68.63 (C-3), 66.81 (C-26), 66.83 (C-5), 63.67 (C-17),
62.09 (C-6), 61.84 (C-14), 56.61 (C-9), 42.52 (C-20), 41.54 (C-13), 40.27 (C-12), 39.81 (C-4),
37.17 (C-10), 34.96 (C-1), 32.37 (C-15), 31.61 (C-2), 31.34 (C-8), 31.05 (C-25), 30.25 (C-7),
29.43 (C-23), 28.78 (C-24), 21.74 (C-11), 20.46 (C-27), 17.10 (C-19), 16.26 (C-18), 14.45 (C-21).
ESI-HRMS, calculated for C27H43O4: 431.3161 [M+H]+ found: m/z 431.3171.
1
Compound 12b: H NMR (400 MHz, CDCl3) (Figure S4)
δ = 4.38 (1H, q, J = 7.5 Hz,
H-16), 3.73-3.66 (1H, m, H-3), 3.46 (1H, ddd, J = 10.9, 4.5, 2.0 Hz, H-26 eq), 3.36 (1H, td,
J = 10.9, 2.1 Hz, H-26 ax), 3.06 (1H, d, J = 2.3 Hz, H-6), 1.09-0.94 (6H, m, H-19 and H-27),
0.79-0.65 (6H, m, H-18 and H-21). 13C NMR (101 MHz, CDCl3) (Figure S4)
δ = 109.26 (C-22),
80.64 (C-16), 69.39 (C-3), 66.81 (C-26), 65.61 (C-5), 63.67 (C-17), 62.86 (C-6), 61.84 (C-14),
55.95 (C-9), 42.18 (C-20), 41.58 (C-13), 40.22 (C-12), 39.84 (C-4), 37.17 (C-10), 34.99 (C-1),
32.72 (C-15), 31.71 (C-2), 31.32 (C-8), 31.01 (C-25), 30.27 (C-7), 29.29 (C-23), 28.75 (C-24),
21.74 (C-11), 20.46 (C-27), 17.07 (C-19), 16.15 (C-18), 15.96 (C-21).
3.2.6. Synthesis of (25R)-Spirostan-3β,5α,6β-Triol (13)
Following a procedure previously described [59,60], the mixture 12a/12b was cooled
in an ice bath. After 5 min, HClO4 (3.9 mL, 65.1 mmol) was added dropwise. The reaction
mixture was stirred at room temperature for 3 h and followed by TLC (EtOAc). Next, the
solution was concentrated to one-third of the starting volume and the concentrated solution
was poured over water. The resulting suspension was filtered and the obtained solid dried
in a desiccator. Compound 13 was obtained (3.25 g, 78%) without further purification.
1
Compound 13: white solid, m.p. 285-286 ◦C, Rf = 0.13 (EtOAc). H NMR (400 MHz,
DMSO-d6) (Figure S5) δ = 4.43 (1H, s, C6-OH), 4.26 (1H, q, J = 7.8 Hz, H-16), 4.16 (1H, d,
J = 5.6 Hz, C3-OH), 3.79 (1H, dt, J = 16.2, 5.5 Hz, H-3), 3.65 (1H, s, C5-OH), 3.40 (1H, dd,
J = 9.9, 2.8 Hz, H-26 eq), 3.30 (1H, d, J = 3.8 Hz, H-6), 3.20 (1H, t, J = 11.0 Hz, H-26 ax),
1.03 (3H, s, H-19), 0.89 (3H, d, J = 6.9 Hz, H-27), 0.77-0.66 (6H, m, H-18 and H-21). 13C
NMR (101 MHz, DMSO) (Figure S5)
δ = 108.36 (C-22), 80.23 (C-16), 74.27 (C-5), 74.04 (C-6),
65.89 (C-3), 65.70 (C-26), 61.91 (C-17), 55.51 (C-14), 44.63 (C-9), 41.10 (C-20), 40.86 (C-4),
40.17 (C-13), 37.89 (C-10), 34.66 (C-7), 32.01 (C-15), 31.45 (C-1), 31.08 (C-8), 30.88 (C-2), 29.81
(C-23), 29.66 (C-25), 28.48 (C-24), 20.52 (C-11), 17.07 (C-27), 16.27 (C-19), 16.24 (C-18), 14.61
(C-21). ESI-HRMS, calculated for C27H45O5: 449.3267 [M+H]+ found: m/z 449.3325.
3.2.7. Synthesis of (25R)-3β,5α-Dihydroxy-Espirostan-6-ona (14)
Compound 13 (2.0 g, 4.6 mmol) was dissolved in 160 mL of a 1,4-dioxane/water (7:1)
mixture. Then, NBS (3.36 g, 18.9 mmol) was added at room temperature under no light
conditions. The reaction mixture was stirred for 2 h and monitored by TLC (EtOAc). After
this time, Na2SO3 10% w/w was added and the mixture extracted with CHCl3. The organic
layer was washed with water, dried over anhydrous Na2SO4 and then concentrated to
dryness. Compound 14 was obtained (1.7 g, 83%) without further purification.

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1
Compound 14: white solid, m.p. 271-272 ◦C, Rf = 0.46 (EtOAc). H NMR (400 MHz,
DMSO-d6) (Figure S6) = 5.32 (1H, s, C5-OH), 4.34 (1H, d, J = 5.6 Hz, C3-OH), 4.28 (1H, q,
δ
J = 7.4 Hz, H-16), 3.75-3.62 (1H, m, H-3), 3.45-3.36 (1H, m, H-26 eq), 3.20 (1H, t, J = 11.0 Hz,
H-26 ax), 2.68 (1H, t, J = 12.3 Hz, H-7 ax), 0.90 (3H, d, J = 6.8 Hz, H-27), 0.73 (3H, d,
J = 6.3 Hz, H-21), 0.68 (6H, d, J = 9.3 Hz, H-18 and H-19). 13C NMR (101 MHz, DMSO)
(Figure S6)
δ = 212.09 (C-6), 108.37 (C-22), 80.02 (C-5), 79.02 (C-16), 65.90 (C-3), 65.16 (C-26),
61.67 (C-17), 55.55 (C-14), 43.83 (C-9), 41.96 (C-10), 41.45 (C-20), 41.04 (C-7), 40.58 (C-13),
39.19 (C-4), 36.31 (C-12), 35.72 (C-8), 31.10 (C-15), 30.87 (C-2), 30.44 (C-1), 29.78 (C-23),
29.56 (C-25), 28.45 (C-24), 20.81 (C-11), 17.06 (C-27), 16.10 (C-18), 14.58 (C-19), 13.64(C-21).
ESI-HRMS, calculated for C27H43O5: 447.3110 [M+H]+ found: m/z 447.3177.
3.2.8. Synthesis of (25R)- 5α- Hydroxy-6-oxo-Spirostan-3β-yl Phenylcarbamate (15)
Compound 15 was obtained (0.86 g, 68% yield) following the general procedure
for synthesis of carbamates. Compound 14 (1.0 g, 2.2 mmol) and isocyanate (1.12 mL,
8.8 mmol). CC (n-hex/EtOAc 3:1).
1
Compound 15 white solid, m.p. 264-266
(400 MHz, CDCl3) (Figure S7)
º
C, Rf = 0.32 (n-hex/EtOAc 3:1). H NMR
δ
= 7.32 (2H, d, J = 7.7 Hz, H-2a), 7.29 (2H, d, J = 7.1 Hz,
H-3a), 7.09-7.00 (1H, m, H-4a), 6.60 (1H, s, NH), 5.04 (1H, tt, J = 10.7, 5.0 Hz, H-3), 4.41 (1H,
q, J = 7.8, 6.3 Hz, H-16), 3.47 (1H, ddd, J = 11.0, 4.6, 1.9 Hz, H-26 eq), 3.36 (1H, t, J = 10.9 Hz,
H-26 ax), 2.90 (1H, s, C5-OH), 2.81-2.68 (1H, m, H-7 ax), 0.97 (3H, d, J = 6.8 Hz, H-27), 0.84
(3H, s, H-19), 0.79 (3H, d, J = 6.3 Hz, H-21), 0.75 (3H, s, H-18). 13C NMR (101 MHz, CDCl3)
(Figure S7)
δ = 211.98 (C-6), 153.39 (NHCO), 137.83 (C-1a), 129.19 (C-3a), 123.69 (C-4a),
119.00 (C-2a), 109.42 (C-22), 80.64 (C-5), 80.48 (C-16), 71.70 (C-3), 67.00 (C-26), 62.18 (C-17),
56.20 (C-14), 44.48 (C-9), 42.65 (C-10), 41.96 (C-20) 41.76 (C-7), 41.22 (C-13), 39.71 (C-12),
36.92 (C-4), 32.96 (C-8), 31.70 (C-15), 31.49 (C-23), 30.42 (C-1), 29.70 (C-25), 28.93 (C-24),
26.76 (C-2), 21.37 (C-11), 17.27 (C-19), 16.56 (C-27), 14.59 (C-18), 14.18 (C-21). ESI-HRMS,
calculated for C34H48NO6: 566.3481 [M+H]+ found: m/z 566.3527.
3.2.9. Synthesis of Mixture (25R)-5
α,6α-epoxy-Spirostan-3β-yl Phenylcarbamate (16a) and
(25R)- 5β,6β-Epoxy-Spirostan-3β-yl Phenylcarbamate (16b)
A solution of compound (0.1 g, 0.19 mmol) in CHCl3 (15 mL) was placed in an ice
7
bath. After 5 min, m-CPBA (53 mg, 0.30 mmol) was added. The reaction mixture was
stirred at room temperature for 3 h based on TLC (n-hex/EtOAc 3:1) consumption of
starting material. Next, CHCl3 (55 mL) was added and the diluted mixture transferred to a
separation funnel. This solution was washed (3x) with Na2SO3 (5% solution w/w), Na2CO3
(sat. solution) and water, respectively. The organic layer was dried over anhydrous Na2SO4
and then concentrated to dryness. The crude product was purified by CC (n-hex/EtOAc
6:1). Compounds 16a and 16b were obtained as a mixture of
α/β epimers at C5-C6
positions (0.1 g, 94% yield, 16a:16b = 3:1). ESI-HRMS, calculated for C34H48NO5: 550.3532
[M+H]+ found: m/z 550.3567. The signals assignment was carried out from the 1H and 13
C
NMR spectra in the 16a/16b mixture (Figure S8).
1
Compound 16a: Rf = 0.59 (n-hex/EtOAc 3:1). H NMR (400 MHz, CDCl3) (Figure S8)
= 7.33 (4H, dd, J = 16.6, 9.1 Hz, H-2a and H-3a), 7.11-6.98 (1H, m, H-4a), 6.55 (1H, s, NH),
δ
5.04-4.68 (1H, m, H-3), 4.38 (1H, q, J = 7.3 Hz, H-16), 3.53-3.41 (1H, m, H-26 eq), 3.36 (1H,
td, J = 10.8, 2.2 Hz, H-26 ax), 2.91 (1H, d, J = 5.7 Hz, H-6), 1.07 (3H, s, H-19), 0.95 (3H, d,
J = 6.8 Hz, H-27), 0.78 (3H, d, J = 6.2 Hz, H-21), 0.74 (3H, s, H-18). 13C NMR (101 MHz,
CDCl3) (Figure S8) δ = 152.95 (NHCO), 138.29 (C-1a), 129.19 (C-3a), 123.37 (C-4a), 118.94
(C-2a), 109.38 (C-22), 80.83 (C-16), 72.62 (C-3), 67.08 (C-26), 65.26 (C-5), 62.36 (C-17), 59.12
(C-6), 56.88 (C-14), 42.82 (C-9), 41.91 (C-20), 40.59 (C-13), 39.67 (C-12), 36.68 (C-4), 35.41
(C-10), 32.45 (C-1), 31.91 (C-15), 31.71 (C-8), 30.54 (C-23), 29.80 (C-7), 29.25 (C-25), 29.12
(C-24), 27.84 (C-2), 20.73 (C-11), 17.24 (C-27), 16.42 (C-19), 16.16 (C-18), 14.59 (C-21).
1
Compound 16b: H NMR (400 MHz, CDCl3) (Figure S8)
δ = 7.33 (4H, dd, J = 16.6,
9.1 Hz, H-2a and H-3a), 7.11-6.98 (1H, m, H-4a), 6.58 (1H, s, NH), 4.82-4.74 (1H, m, H-3),
4.38 (1H, q, J = 7.3 Hz, H-16), 3.53-3.41 (1H, m, H-26 eq), 3.36 (1H, td, J = 10.8, 2.2 Hz,
H-26 ax), 3.11 (1H, d, J = 1.7 Hz, H-6), 1.07 (3H, s, H-19), 0.95 (3H, d, J = 6.8 Hz, H-27),

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0.78 (3H, d, J = 6.2 Hz, H-21), 0.74 (3H, s, H-18). 13C NMR (101 MHz, CDCl3) (Figure S8)
δ
= 152.98 (NHCO), 137.98 (C-1a), 129.03 (C-3a), 123.52 (C-4a), 118.88 (C-2a), 109.22 (C-22),
80.73 (C-16), 72.37 (C-3), 66.92 (C-26), 63.49 (C-5), 62.49 (C-17), 58.97 (C-6), 56.09 (C-14),
42.65 (C-9), 41.75 (C-20), 40.37 (C-13), 39.94 (C-12), 36.67 (C-4), 35.35 (C-10), 32.79 (C-1),
31.85 (C-15), 31.56 (C-8), 30.73 (C-23), 29.42 (C-7), 29.09 (C-25), 28.96 (C-24), 27.61 (C-2),
21.79 (C-11), 17.08 (C-27), 16.26 (C-19), 15.99 (C-18), 14.42 (C-21).
3.2.10. Synthesis of ((25R)-5α-Hydroxy-6-Hydroxymino-3β-yl Phenylcarbamate (17)
Compound 15 (0.2 g, 0.4 mmol) was dissolved in ethanol (20 mL). Then, NH2OH
·
HCl
(0.11 g, 1.6 mmol) and NaOAc 3H2O (0.22 g, 1.6 mmol) were added. The resulting mix-
·
ture was refluxed for 3 h while been monitored by TLC (n-hex/EtOAc 3:1). The solvent
volume was reduced to one-third of the initial volume and the concentrated solution was
poured over water. The formed solid was filtered and purified by CC (n-hex/EtOAc 3:1).
Compound 17 was obtained (0.18 g, 77%).
1
Compound 17 white solid, m.p. 257-259 ◦C, Rf = 0.36 (n-hex/EtOAc 3:1). H NMR
(400 MHz, DMSO-d6) (Figure S9)
δ = 10.51 (1H, s, N-OH), 9.50 (1H, s, NH), 7.45 (2H, d,
J = 7.9 Hz, H-2a), 7.25 (2H, t, J = 7.8 Hz, H-3a), 6.96 (1H, t, J = 7.3 Hz, H-4a), 5.09-4.97
(2H, m, H-3 and C5-OH), 4.30 (1H, q, J = 7.1 Hz, H-16), 3.46-3.37 (1H, m, H-26 eq), 3.22
(1H, t, J = 11.0 Hz, H-26 ax), 2.98 (1H, dd, J = 13.3, 4.6 Hz, H-7 ax), 0.91 (3H, d, J = 6.8 Hz,
H-27), 0.81-0.67 (9H, m, H-18, H-19 and H-21). 13C NMR (101 MHz, DMSO) (Figure S9)
δ
= 159.16 (C-6), 153.10 (NHCO), 139.30 (C-3a), 128.65 (C-4a), 122.13 (C-2a), 118.06 (C-22),
108.41 (C-16), 80.13 (C-5), 75.42 (C-3), 70.92 (C-26), 65.91 (C-17), 61.65 (C-14), 55.47 (C-9),
44.22 (C-10), 41.07 (C-20), 40.56 (C-4), 40.40 (C-13), 34.61 (C-12), 33.98 (C-8), 31.22 (C-15),
30.84 (C-1), 29.81 (C-23), 29.35 (C-25), 28.45 (C-7), 26.83 (C-24), 24.66 (C-2), 20.76 (C-11),
17.05 (C-19), 16.19 (C-27), 14.57 (C-18), 14.02 (C-21). ESI-HRMS, calculated for C34H49N2O6:
581.3590 [M+H]+ found: m/z 581.3623.
3.2.11. Synthesis of (25R)-5α,6β-Dihydroxy-Spirostan-3β-yl Phenylcarbamate (18)
A solution of compound 15 (1.0 g, 1.8 mmol) in ethanol (85 mL) was placed in an ice
bath and NaBH4 (0.1 g, 2.7 mmol) dissolved in EtOH (5 mL) was added under stirring.
Reaction was stopped after 30 min when TLC (n-hex/EtOAc 3:1) showed no more starting
material. Next, reaction crude was poured over water and the resulting suspension filtered.
The solid was purified by CC (n-hex/EtOAc 4:1). Compound 18 was obtained (0.85 g,
83% yield).
1
Compound 18 white solid, m.p. 258-260 ◦C, Rf = 0.21 (n-hex/EtOAc 3:1). H NMR
(400 MHz, DMSO-d6) (Figure S10)
δ = 9.49 (1H, s, NH), 7.45 (2H, d, J = 8.0 Hz, H-2a), 7.25
(2H, dd, J = 8.5, 7.3 Hz, H-3a), 6.96 (1H, t, J = 7.3 Hz, H-4a), 5.08 (1H, tt, J = 11.0, 5.4 Hz,
H-3), 4.58 (1H, d, J = 4.2 Hz, C6-OH), 4.28 (1H, q, J = 13.4, 6.3 Hz, H-16), 4.03 (1H, s, C5-OH),
3.45-3.36 (2H, m, H-6 and H-26 eq), 3.21 (1H, t, J = 11.0 Hz, H-26 ax), 1.09 (3H, s, H-19), 0.90
(3H, d, J = 6.9 Hz, H-27), 0.77-0.70 (6H, m, H-18 and H-21). 13C NMR (101 MHz, DMSO)
(Figure S10) δ = 153.19 (NHCO), 139.36 (C-1a), 128.63 (C-3a), 122.08 (C-4a), 118.02 (C-2a),
108.37 (C-22), 80.20 (C-16), 74.24 (C-5), 73.63 (C-6), 71.27 (C-3), 66.92 (C-26), 65.88 (C-17),
61.84 (C-14), 55.41 (C-9), 54.88 (C-20), 44.55 (C-13), 41.10 (C-12), 37.87 (C-10), 37.14 (C-4),
34.51 (C-7), 31.65 (C-8), 31.43 (C-15), 30.85 (C-1), 29.80 (C-23), 29.59 (C-25), 28.46 (C-24),
27.07 (C-2), 20.47 (C-11), 17.07 (C-27), 16.25 (C-19), 16.19 (C-18), 14.60 (C-21). ESI-HRMS,
calculated for C34H50NO6: 568.3638 [M+H]+ found: m/z 568.3681.
3.3. Rice-Lamina Inclination Assay
The biological activity of the compounds was evaluated by the tilt test of the sheet
according to the procedure described [30]. After soaking the rice seeds (Oryza sativa) Zafiro
variety provided by the Institute of Agricultural Research (INIA-Quilamapu) in sterile
distilled water for 24 h, the seeds were selected and cultivated at 22 ◦C in a plant growing
chamber growing under a photoperiod of 16 h of light/8 h of darkness in pots with soil and
plenty of water. Etiolated rice seedlings were grown so far, which were homogeneous and

Int. J. Mol. Sci. 2021, 22, 2330
15 of 19
presented the second internode of the rice blade for cutting, this segment was deposited in
sterile distilled water in Petri dishes for 24 h. Subsequently, six segments per treatment
were incubated in a Petri dish containing 60 mL of distilled water and compounds
11 15 17 18 and mixture 16a 16b at concentrations 0.01, 0.1 and 1 M, for 48 h in the
dark. Segments incubated with under the same conditions were used as positive control,
7, 9,
,
,
,
/
µ
1
whereas sterile distilled water was added to segments used as negative control. Finally,
the magnitude of the angle induced between the blade and the sheath was measured. Two
bioassays were evaluated.
3.4. Molecular Docking
3.4.1. Ligand/Molecular Target Selection and Preparation
2D structures of the steroids for the study were obtained using ChemDraw Profes-
sional 15.0 (Perkin Elmer, Waltham, MA, USA). Three-dimensional structure coordi-
nates were generated and preliminarily optimized with the MM2 force-field—Steepest
Descent Algorithm [61] implemented in Chem3D 15.0 (Perkin Elmer). All structures
were further optimized through PM7 semi-empirical method implemented in MOPAC
2016 code (http://OpenMOPAC.net; accessed on 1–10 July 2020). All ligand PDB
files were converted into PDBQT format (input for AutoDock Vina) [52]. The charges
on the ligand atoms were generated using the Gasteiger model, nonpolar hydrogens
were merged, and default rotatable bonds were retained using TORSDOF utility in
AutoDockTools (http://mgltools.scripps.edu; accessed on 2 February 2020) [62].
The crystal structure of the protein Brassinosteroid Insensitive 1 (BRI1) in complex
with BRI1-Associated Receptor Kinase 1 (BAK1) and the natural ligand 1, PDB ID: 4m7e,
resolved at 3.60 Å was retrieved from Protein Data Bank (http://www.rcsb.org; accessed
on 2–15 January 2019). The structure was optimized using pdb2pqr.py (Version 2.0.0)
implemented in the web server PDB2PQR (http://nbcr-222.ucsd.edu/pdb2pqr_2.0.0/;
accessed on 3–22 February 2019), using the AMBER force-field, and the protonation state of
ionizable groups at pH 8 was assigned using PROPKA [58,59]. The grid search was selected
of size 22 × 22 × 22 Å [62] with the center of simulation box matching the center of 1.
3.4.2. Docking Procedure and Analysis of Protein-Ligand Complexes.
Docking simulations were performed using AutoDock Vina 1.1.2. The active site of
the proteins heterodimer was kept rigid, and non-flexible docking was carried out. The
docking parameters were set to default except the following: exhaustiveness = 32 and
num_modes = 2. The Vina code predicts the adopted conformations with the binding
affinity (kcal/mol). The best two docked conformations, according to the binding affinity
from 15 independent runs, were analyzed to produce 30 final docked poses. These poses
were clustered according to the Root-Mean-Square-Deviation (RMSD), with a cut-off of
1.5 Å among poses. The binding energy of the cluster is the average binding energy of all the
conformations present in it. The cluster with the lowest binding energy and higher number
of conformations was selected as the representative binding mode of that particular ligand.
Graphical representations of the protein-ligand complexes (Figure S11) were prepared
using PyMolTM version 2.1.0 (Schrödinger, New York, NY, USA, https://pymol.org/;
accessed on 10 February 2020).
4. Conclusions
In this study we reported the synthesis of seven new N-phenyl carbamates BRs analogs,
starting from natural diosgenin, stigmasterol, β-sitosterol and synthetic hydroxysteroids.
Several functional groups such as double bonds, epoxy, hydroxy, keto, and oxime were
combined with the carbamate moiety in the same chemical structure. A rice-lamina
inclination assay was conducted for all compounds demonstrating their effect as plant-
growth enhancers. Among the five diosgenin-derived carbamates, compounds 15 with a
keto group in C6, and mixture of epoxides 16a
/16b (α and β isomers) were more active
in vitro than compounds 17 and 18, which carry double bond, hydroxyl or hydroxymino
7,

Int. J. Mol. Sci. 2021, 22, 2330
16 of 19
group in positions C5 and/or C6, respectively. The mixture of epoxides, 16a
/
16b, showed
activity similar to that of natural brassinolide. Finally, a docking study showed essential
aspects related to the interaction of carbamates and BRI1-BAK1, BRs receptor proteins in
Arabidopsis thaliana.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422
-0067/22/5/2330/s1, Figure S1: NMR spectra of (25R)-spirost-3-N-phenyl-carbamoyl-5-en-3
); Figure S2: NMR spectra of 3-N-phenyl-carbamoyl-stigmast-5-en-3 -ol ( ); Figure S3: NMR
spectra of 3-N-phenyl-carbamoyl-stigmast-5,22-dien-3 -ol (11); Figure S4: NMR spectra of mix-
ture (25R)-spirostan-5 ,6 -epoxi-3 -ol (12a) and (25R)-spirostan-5 ,6 -epoxi-3 -ol (12b); Figure S5:
NMR spectra of (25R)-spirostan-3 ,5 ,6 -triol (13); Figure S6: NMR spectra of (25R)-espirostan-
,5 -dihidroxi-6-ona (14); Figure S7: NMR spectra of (25R)-spirostan-3-N-phenyl-carbamoyl-3 ,5
dihydroxy-6-one (15); Figure S8: NMR spectra of mixture (25R)-spirostan-5 ,6 -epoxi-3-N-phenyl-
carbamoyl-3 -ol (16a) and (25R)-spirostan-5 ,6 -epoxi-3-N-phenyl-carbamoyl-3 -ol (16b); Figure S9:
NMR spectra of (25R)-espirostan-3-N-fenil-carbamoil-3 ,5 -dihydroxy-6-oxime (17); Figure S10:
NMR spectra of (25R)-spirostan-3-N-phenyl-carbamoyl-3 ,5 ,6 -triol (18); Figure S11: Represen-
tations of ligand-BRI1/BAK1 complexes. Crystallographic poses of carbamates steroids 11
12a 12b 13 14 15 16a 16b 17 and 18. Crystallographic pose of is in black sticks; Figure S12:
Comparison between the interactions of heterodimer BRI1/BAK1 with and steroidal carbamates
β-ol
(7
β
9
β
α
α
β
β
β
β
β
α β
3
β
α
β α-
α
α
β
β
β
β
β
α
β
α β
7,
9,
,
,
,
,
,
,
,
,
1
1
ligands; Table S1: Energy (kcal/mol) and conformation clusters of the docked ligands.
Author Contributions: This work was developed as follows: conceptualization and methodology,
A.Z.C., L.E., Y.C.G.; synthesis, purification and characterization D.F.P., L.G.C., L.G.d.l.T., L.P.R.;
experiments biological activity, K.D.; docking calculations, M.R.C.G., resources, A.Z.C., L.E., K.D.,
Y.C.G.; writing—original draft preparation, D.F.P., L.G.C., M.R.C.G., K.D.; figures and schemes, D.F.P.,
L.G.C., M.R.C.G.; writing—review and editing, L.E., A.F.O., Y.C.G.; supervision, A.Z.C., Y.C.G.;
funding acquisition, L.E., K.D., A.F.O., Y.C.G. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by FONDECYT (Fondo Nacional de Desarrollo Científico y
Tecnológico) (Grant No. 1191330).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in Supplementary Material.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
BRI1
BAK1
BRs
CC
TLC
m-CPBA
NBS
LRR-RLKs
SERK
NMR
ppm
s
Brassinosteroid Insensitive 1
BRI-Associated Kinase 1
Brassinosteroids
Column Chromatography
Thin-Layer Chromatography
meta-Chloroperoxybenzoic Acid
N-Bromosuccinimide
Leucine-Rich Repeat Receptor-Like Kinases
Somatic Embryogenesis Receptor Kinase
Nuclear Magnetic Resonance
parts per millions
singlet
d
doublet
t
triplet
q
quartet
dd
double doublets
dt
double triplets
td
tripe doublets

Int. J. Mol. Sci. 2021, 22, 2330
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ddd
tt
doublet of double doublets
triplet of triplets
m
multiplet
eq
ax
equatorial H disposition
axial H disposition
High Resolution Electrospray Ionization Mass Spectrometry
n-hexane
Ethyl acetate
Tetrahydrofuran
Dimethyl sulfoxide
phenyl isocyante
room temperature
ESI-HRMS
n-hex
EtOAc
THF
DMSO
PhNCO
rt.
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