New Brassinosteroid Analogs with 23,24-Dinorcholan Side Chain, and Benzoate Function at C-22: Synthesis, Assessment of Bioactivity on Plant Growth, and Molecular Docking Study

The synthesis and biological evaluation of brassinosteroids (BRs) analogs with chemical modification in the side alkyl chain is a matter of current interest. Recently, a series of BR analogs with phenyl or benzoate groups in the alkyl chain have been reported. The effect of substitution in the aromatic ring on the biological activities of these new analogs has been evaluated, and the results suggest that the bioactivity is enhanced by substitution with an F atom. In this context, we have synthesized, characterized, and evaluated a series of new analogs of 23,24-bisnorcholenic type in which the benzoate group at the C-22 position is substituted with an F atom at “ortho or para” positions. Plant growth-promoting activities were evaluated by using the rice lamina inclination test and bean second internode biotest. The results obtained with both bioassays indicate that the compound with an F atom in the para position on the aromatic ring is the most active BR analog and in some cases is even more active than brassinolide. The docking study confirmed that compounds with an F atom adopt an orientation similar to that predicted for brassinolide, and the F atom in the “para” position generates an extra hydrogen bond in the predicted binding position.


Introduction
Brassinosteroids (BRs) are an important group of polyhydroxylated naturally occurring steroidal phytohormones found in the plant kingdom in extremely low amounts [1].This phytohormone group regulates plant growth and development by producing an array of physiological changes and eliciting very important functions, such as plant growth regulation and cell division and differentiation in young tissues of growing plants [2][3][4][5][6].They also play an important role in molecular and physiological responses under abiotic stress such as drought, salinity, high temperature, low temperature, and heavy metal stresses [7][8][9].
Due to the low concentrations in which these compounds are found, much effort has been dedicated to synthesize these compounds or their structural analogs using natural and abundant sterols [10].One of the main difficulties found in the synthesis of BRs is the replication of the side alkyl chain due to the presence of at least three chiral centers.Thus, in the quest for new active analogs, various BRs analogs have been obtained by incorporating important structural modifications in their side chain .
in the quest for new active analogs, various BRs analogs have been obtained by incorporating important structural modifications in their side chain .
Recently, the synthesis of BRs analogs carrying phenyl groups, with no or small nonpolar substituents, in the side alkyl chain has been reported [37,38].A comparison of experimental activity on plants (ethylene production or inhibitory effect on Arabidopsis root growth) with a molecular structure indicates that the most active analogs are those with no substitution or substituted with chlorine or fluorine atoms in "ortho, meta and para" positions of the aromatic ring (compounds 2a (o-F), 2b (p-F), and 2c (m-Cl), Figure 1).From this new series of BRs analogs, compound 2a, with a F atom in the ortho position, is the most active and promising compound.These results are confirmed by molecular docking into BRI 1 studies [37,38].In that direction, we have reported the synthesis of new BRs analogs of 24-Norcholanes type, with benzoate functions at the C-23 position of the side chain (compounds 3 and 4, Figure 1) The bioactivity of BRs analogs 3 and 4 was assessed by using the rice lamina inclination test (RLIT) and root elongation in Arabidopsis thaliana.Both compounds show similar biological activity at 1 × 10 −8 , 1 × 10 −7 , and 1 × 10 −6 M concentrations, with 4 being slightly more active in the RLIT test.Interestingly, compound 3 was found to be more active than brassinolide (1) (Figure 1) at all tested concentrations [39].On the other hand, compounds 5 and 6 were also evaluated in RLIT, and the 5/6 diastereoisomer mixture (ratio 1:0/0.44) is significantly more active than brassinolide at 1 × 10 −8 M concentration [40].These results suggest that the incorporation of a benzoate group at the C-23 position of the side chain induces an increase in the biological activity of these compounds.
It is well established that BRs perception occurs in a two-step mechanism, namely binding to the BRI1 (Brassinosteroid Insensitive 1) receptor and induced heterodimerization with a second receptor kinase, BAK1 (BRI1-Associated Receptor Kinase 1) [41,42].The formation of this ternary complex results in full BRI1 activation, initiating a signaling cascade that regulates a series of physiological processes [43,44].Thus, minor modifications to the BR structure may produce important changes in binding to this receptor [37,45].These effects can be evaluated via molecular docking into the BRI1-BAK1 crystallized complex (PDB: 4m7e BRI-1), which provides information about poses and binding energies adopted by BRs analogs in the interaction with this receptor.The bioactivity of BRs analogs 3 and 4 was assessed by using the rice lamina inclination test (RLIT) and root elongation in Arabidopsis thaliana.Both compounds show similar biological activity at 1 × 10 −8 , 1 × 10 −7 , and 1 × 10 −6 M concentrations, with 4 being slightly more active in the RLIT test.Interestingly, compound 3 was found to be more active than brassinolide (1) (Figure 1) at all tested concentrations [39].On the other hand, compounds 5 and 6 were also evaluated in RLIT, and the 5/6 diastereoisomer mixture (ratio 1:0/0.44) is significantly more active than brassinolide at 1 × 10 −8 M concentration [40].These results suggest that the incorporation of a benzoate group at the C-23 position of the side chain induces an increase in the biological activity of these compounds.
It is well established that BRs perception occurs in a two-step mechanism, namely binding to the BRI1 (Brassinosteroid Insensitive 1) receptor and induced heterodimerization with a second receptor kinase, BAK1 (BRI1-Associated Receptor Kinase 1) [41,42].The formation of this ternary complex results in full BRI1 activation, initiating a signaling cascade that regulates a series of physiological processes [43,44].Thus, minor modifications to the BR structure may produce important changes in binding to this receptor [37,45].These effects can be evaluated via molecular docking into the BRI1-BAK1 crystallized complex (PDB: 4m7e BRI-1), which provides information about poses and binding energies adopted by BRs analogs in the interaction with this receptor.
Thus, with the aim of evaluating the effect of the nature and position of a benzoate ring substituent on biological activity, herein, we report the synthesis of three new analogs of 23,24-bisnorcholenic type in which the benzoate group at C-22 position is substituted with a fluorine atom at "ortho or para" positions.Growth-promoting activities were evaluated by using RLIT and the bean second internode (BSI) biotest.Finally, experimental results are compared with those predicted by a study of molecular docking into the BRI1-BAK1 crystallized complex.The discussion of these results could be of great help in the understanding of bioactivity structural requirements associated with the BRs side chain structure.
Thus, with the aim of evaluating the effect of the nature and position of a benzoate ring substituent on biological activity, herein, we report the synthesis of three new analogs of 23,24-bisnorcholenic type in which the benzoate group at C-22 position is substituted with a fluorine atom at "ortho or para" positions.Growth-promoting activities were evaluated by using RLIT and the bean second internode (BSI) biotest.Finally, experimental results are compared with those predicted by a study of molecular docking into the BRI1-BAK1 crystallized complex.The discussion of these results could be of great help in the understanding of bioactivity structural requirements associated with the BRs side chain structure.

Chemical Synthesis
The synthetic route used to obtain BRs analogs 8-11 is described in Schemes 1 and 2. In Scheme 1 is shown a series of reactions, which, starting from (7) and following reported procedures [46,47], gives compound 17.Experimental details and reaction yields are given in the Experimental Section.Melting points, IR, and 1 H NMR spectroscopic data obtained for these compounds were consistent with those reported [47].Additionally, to complete the available spectroscopic information, 13 C, 13 C DEPT-135, 2D HSQC, and 2D HMBC NMR experiments were carried out (Supplementary Material Figures S1-S24).

Chemical Synthesis
The synthetic route used to obtain BRs analogs 8-11 is described in Schemes 1 and 2. In Scheme 1 is shown a series of reactions, which, starting from (7) and following reported procedures [46,47], gives compound 17.Experimental details and reaction yields are given in the Experimental Section.Melting points, IR, and 1 H NMR spectroscopic data obtained for these compounds were consistent with those reported [47].Additionally, to complete the available spectroscopic information, 13 C, 13 C DEPT-135, 2D HSQC, and 2D HMBC NMR experiments were carried out (Supplementary Material Figures S1-S24).
Thus, with the aim of evaluating the effect of the nature and position of a benzoate ring substituent on biological activity, herein, we report the synthesis of three new analogs of 23,24-bisnorcholenic type in which the benzoate group at C-22 position is substituted with a fluorine atom at "ortho or para" positions.Growth-promoting activities were evaluated by using RLIT and the bean second internode (BSI) biotest.Finally, experimental results are compared with those predicted by a study of molecular docking into the BRI1-BAK1 crystallized complex.The discussion of these results could be of great help in the understanding of bioactivity structural requirements associated with the BRs side chain structure.

Chemical Synthesis
The synthetic route used to obtain BRs analogs 8-11 is described in Schemes 1 and 2. In Scheme 1 is shown a series of reactions, which, starting from (7) and following reported procedures [46,47], gives compound 17.Experimental details and reaction yields are given in the Experimental Section.Melting points, IR, and 1 H NMR spectroscopic data obtained for these compounds were consistent with those reported [47].Additionally, to complete the available spectroscopic information, 13 C, 13 C DEPT-135, 2D HSQC, and 2D HMBC NMR experiments were carried out (Supplementary Material Figures S1-S24).Initially, the keto group of 17 was protected with ethylene glycol/TsOH/C6H6, following a procedure that has been described for steroids of similar structure [48].The expected dioxolane derivative 18 was obtained with a 99.0% yield (Scheme 2), and the presence of a dioxolane ring was established via 1   S25-S29).A subsequent reduction of 18 leads to hydroxy-ketone 19 with a 91.5% yield.Further, 1D and 2D NMR spectra are shown in Figures S30-S34 (Supplementary Material) [48].Sharpless dihydroxylation of the double bond on C-2 in compound 19 produces triol 8 with a 70.9% yield.It is widely known that this reaction is stereo-controlled by A-ring conformation and the presence of a methyl group on C-10.The attack of a reagent (OsO4) is always from the bottom side of the molecule [37].Analog 8 has been previously prepared, but its structure was only characterized by IR spectroscopic data [49].Herein, the complete structural determination of 8 was established by using 1 H and 13 C NMR spectroscopic techniques.So, the signals observed at δH = 3.95 ppm (1H, d, J = 2.8 Hz) and 3.66 ppm (1H, ddd, J = 11.7,4.7, and 3.1 Hz) were assigned to carbynolic hydrogens H-3 and H-2, respectively.On the other hand, the signals observed at dC = 69.58 and 69.23 ppm were assigned to the carbynolic carbons C-3 and C-2, respectively.Additionally, these signals were correlated with 2D HSQC and 2D HMBC spectra (see Supplementary Material Figures S35-S40).The spatial orientation of hydroxyl groups 2α,3α-diol was established through selective 1D Noesy experiments, where the signal of H-2 (δH = 3.66 ppm) showed spatial correlations with H-4β and CH3-19, indicating that these H atoms have the same spatial β-orientation (Figure 3).Initially, the keto group of 17 was protected with ethylene glycol/TsOH/C 6 H 6 , following a procedure that has been described for steroids of similar structure [48].The expected dioxolane derivative 18 was obtained with a 99.0% yield (Scheme 2), and the presence of a dioxolane ring was established via 1   S25-S29).A subsequent reduction of 18 leads to hydroxy-ketone 19 with a 91.5% yield.Further, 1D and 2D NMR spectra are shown in Figures S30-S34 (Supplementary Material) [48].Sharpless dihydroxylation of the double bond on C-2 in compound 19 produces triol 8 with a 70.9% yield.It is widely known that this reaction is stereo-controlled by A-ring conformation and the presence of a methyl group on C-10.The attack of a reagent (OsO 4 ) is always from the bottom side of the molecule [37].Analog 8 has been previously prepared, but its structure was only characterized by IR spectroscopic data [49].Herein, the complete structural determination of 8 was established by using 1 H and 13 C NMR spectroscopic techniques.So, the signals observed at δ H = 3.95 ppm (1H, d, J = 2.8 Hz) and 3.66 ppm (1H, ddd, J = 11.7,4.7, and 3.1 Hz) were assigned to carbynolic hydrogens H-3 and H-2, respectively.On the other hand, the signals observed at d C = 69.58 and 69.23 ppm were assigned to the carbynolic carbons C-3 and C-2, respectively.Additionally, these signals were correlated with 2D HSQC and 2D HMBC spectra (see Supplementary Material Figures S35-S40).The spatial orientation of hydroxyl groups 2α,3α-diol was established through selective 1D Noesy experiments, where the signal of H-2 (δ H = 3.66 ppm) showed spatial correlations with H-4β and CH 3 -19, indicating that these H atoms have the same spatial β-orientation (Figure 3).Benzoylation of a primary alcohol in C-22 of hydroxy-ketone 19 with the corresponding benzoyl chlorides [16,50] produces compounds 20-22 with 85.4%, 65.0%, and 72.5%, yields, respectively.Compounds 20-22 were fully characterized using 1D and 2D NMR spectroscopic techniques (see Figures S41-S55, Supplementary Material).Finally, new BRs analogs 9-11 were obtained by the sharpless dihydroxylation of olefins 20-22, with 90.0%, 82.5%, and 84.9% yields, respectively.Compounds 9-11 were fully characterized using IR, 1D, 2D NMR, and HRMS spectroscopic techniques (see Figures S56-S70, Supplementary Material).

Biological Activity
BRs play important roles in plant growth, development, and responses to abiotic stresses.It is well established that these effects are due mainly to the regulation of cell expansion, cell division, and cell elongation.Thus, BRs activity can be detected and quantified through a series of different plant responses, and, consequently, various bioassays have been proposed.It has been shown that different BRs activities are obtained by using different biological activity tests; this means that the results obtained by using different methods are not equivalent and cannot be compared [40].This is a very important factor to be considered for a comparison of BRs activities and the proposal of structure-activity relationships of synthetic BRs analogs [51,52].Herein, we used the rice lamina inclination test (RLIT), one of the most widely used methods due to its high sensitivity and specificity, and the bean second internode bioassay.

Biological Activity
BRs play important roles in plant growth, development, and responses to abiotic stresses.It is well established that these effects are due mainly to the regulation of cell expansion, cell division, and cell elongation.Thus, BRs activity can be detected and quantified through a series of different plant responses, and, consequently, various bioassays have been proposed.It has been shown that different BRs activities are obtained by using different biological activity tests; this means that the results obtained by using different methods are not equivalent and cannot be compared [40].This is a very important factor to be considered for a comparison of BRs activities and the proposal of structure-activity relationships of synthetic BRs analogs [51,52].Herein, we used the rice lamina inclination test (RLIT), one of the most widely used methods due to its high sensitivity and specificity, and the bean second internode bioassay.

Bioactivity in the Rice Lamina Inclination Test (RLIT) of Brassinosteroid Analogs
The activity of BRs analogs 9-11 was assessed by using RLIT and using brassinolide as a positive control.Measurements were performed at two different concentrations, namely 1 × 10 −7 M and 1 × 10 −6 M (see Table 1).The results are given as the bending angle between laminae and sheaths.The values of bending angles obtained for 1, at both tested concentrations, are in line with those reported in a previous work [17] and indicate that bioactivity of 1 increases with increasing concentration.It can also be observed that analogs 10 and 11, with a benzoate group at C-22 and a fluorine atom in the ortho and para positions, respectively, are more active than analog 8 (no benzoate group) and analog 9 (no F atom in the benzoate group) at both tested concentrations (Table 1).Interestingly, at 1 × 10 −7 M, analog 10 is the most active compound, and its activity is even comparable with that shown by brassinolide.However, at 1 × 10 −6 , M the brassinolide activity is 3 to 9 times higher than that exhibited by BRs analogs 8-11.Thus, a comparison of bending angles measured for 9 with those obtained for 10 and 11 shows that substitution by an F atom in the benzoate group increases plant growth elongation, and this effect depends on the position of the F atom.

Bioactivity in Bean Second Internode Bioassay of Brassinosteroid Analogs
This test has been used in both gibberellins and BRs.For gibberellins, only elongation can be detected, while for BRs curvature, swelling and division of the internode are easily measured [46,47,[53][54][55][56][57][58].The effect on plant tissues depends not only on the structural conformation of the phytohormones but also on the concentration applied, so a doseresponse curve of 1 (1 × 10 −5 -1 × 10 −10 M, Figure 4a,b) was obtained to determine the optimal concentration at which tested molecules should be applied.
namely 1 × 10 −7 M and 1 × 10 −6 M (see Table 1).The results are given as the bending angle between laminae and sheaths.The values of bending angles obtained for 1, at both tested concentrations, are in line with those reported in a previous work [17] and indicate that bioactivity of 1 increases with increasing concentration.It can also be observed that analogs 10 and 11, with a benzoate group at C-22 and a fluorine atom in the ortho and para positions, respectively, are more active than analog 8 (no benzoate group) and analog 9 (no F atom in the benzoate group) at both tested concentrations (Table 1).Interestingly, at 1 × 10 −7 M, analog 10 is the most active compound, and its activity is even comparable with that shown by brassinolide.However, at 1 × 10 −6 , M the brassinolide activity is 3 to 9 times higher than that exhibited by BRs analogs 8-11.Thus, a comparison of bending angles measured for 9 with those obtained for 10 and 11 shows that substitution by an F atom in the benzoate group increases plant growth elongation, and this effect depends on the position of the F atom.

Bioactivity in Bean Second Internode Bioassay of Brassinosteroid Analogs
This test has been used in both gibberellins and BRs.For gibberellins, only elongation can be detected, while for BRs curvature, swelling and division of the internode are easily measured [46,47,[53][54][55][56][57][58].The effect on plant tissues depends not only on the structural conformation of the phytohormones but also on the concentration applied, so a doseresponse curve of 1 (1 × 10 −5 -1 × 10 −10 M, Figure 4a,b) was obtained to determine the optimal concentration at which tested molecules should be applied.The data show that brassinolide activity in the bean second internode bioassay is maximum at 1 × 10 −9 M. Therefore, the effect of BRs analogs was measured at this concentration using 1 as a positive control.
The values obtained from the elongation test of the second internode of the bean are shown in Figure 5 and summarized in Table 2.
The data show that brassinolide activity in the bean second internode bioassay is maximum at 1 × 10 −9 M. Therefore, the effect of BRs analogs was measured at this concentration using 1 as a positive control.
The values obtained from the elongation test of the second internode of the bean are shown in Figure 5 and summarized in Table 2.  Data in Table 2 show that the activity of benzoylated analogs 9 and 10 measured by using the bean second internode bioassay is like that found for brassinolide, whereas analog 11 (F atom in para position) exhibits a surprisingly high activity in elongation.Finally, analog 8 (no benzoate function in the side chain) is inactive at the same concentration.
Therefore, the results obtained with both bioassays indicate that compound 11 with fluorine atom para-substituted on the aromatic ring is the most active BRs analog and in some cases is even more active than brassinolide.

Molecular Docking Studies
To gain a deeper understanding of how plant activity of BRs analogs is influenced by their molecular structure, a molecular docking study was carried out.This process was performed by placing these compounds within the active site of the BRI1-BAK1 crystallized complex (PDB: 4m7e) using AutoDock Vina.
To assess the effectiveness of the analysis, a redocking of brassinolide into the crystallographic structure of BRI1-BAK1 was performed, and the best-resulting pose was compared with the original ligand's pose.The outcomes reveal a remarkable resemblance between the obtained pose with the lowest energy and the crystallographic pose.Therefore, these parameters were deemed appropriate and were used for docking of synthetic ligands.The results indicate that the poses of each ligand within the binding site exhibit a strong alignment with brassinolide, and calculated binding energies for these complexes ranged from −11.9 to −13.2 kcal/mol, which suggests a favorable inclination toward the creation of BRI1-ligand-BAK1 complexes (see Table S1, Supplementary Material).Data in Table 2 show that the activity of benzoylated analogs 9 and 10 measured by using the bean second internode bioassay is like that found for brassinolide, whereas analog 11 (F atom in para position) exhibits a surprisingly high activity in elongation.Finally, analog 8 (no benzoate function in the side chain) is inactive at the same concentration.
Therefore, the results obtained with both bioassays indicate that compound 11 with fluorine atom para-substituted on the aromatic ring is the most active BRs analog and in some cases is even more active than brassinolide.

Molecular Docking Studies
To gain a deeper understanding of how plant activity of BRs analogs is influenced by their molecular structure, a molecular docking study was carried out.This process was performed by placing these compounds within the active site of the BRI1-BAK1 crystallized complex (PDB: 4m7e) using AutoDock Vina.
To assess the effectiveness of the analysis, a redocking of brassinolide into the crystallographic structure of BRI1-BAK1 was performed, and the best-resulting pose was compared with the original ligand's pose.The outcomes reveal a remarkable resemblance between the obtained pose with the lowest energy and the crystallographic pose.Therefore, these parameters were deemed appropriate and were used for docking of synthetic ligands.The results indicate that the poses of each ligand within the binding site exhibit a strong alignment with brassinolide, and calculated binding energies for these complexes ranged from −11.9 to −13.2 kcal/mol, which suggests a favorable inclination toward the creation of BRI1-ligand-BAK1 complexes (see Table S1, Supplementary Material).
As can be seen in Figure 6A, compound 10 adopts an orientation similar to that found for brassinolide.In the predicted binding position of 10 (Figure 6), six hydrogen bonds were identified.Two of these bonds are formed by the carbonyl group of ArCO, interacting with Tyr 597 and Ser 647 at distances of 2.25 Å and 2.27 Å, respectively.The others are created by interactions between the hydroxyl group at position C-2 and His 61 (at 2.04 Å) and Asn 705 (at 2.09 Å).Additionally, the hydroxyl group at C-3 forms a hydrogen bond with Tyr 642 at 2.13 Å.Another hydrogen bond was observed between the fluorine atom and Tyr 597, with a 2.80 Å distance.Furthermore, the Phe 60 residue engages in π-π stacking with the aromatic ring of compound 10.Finally, 10 participates in van der Waals interactions with Trp 564, Met 657, Phe 681, Ile 706, Ile 682, Phe 60, Tyr 597, Tyr 642, and Tyr 599 (Figure S76, Table S2, Supplementary Material).
for brassinolide.In the predicted binding position of 10 (Figure 6), six hydrogen bonds were identified.Two of these bonds are formed by the carbonyl group of ArCO, interacting with Tyr 597 and Ser 647 at distances of 2.25 Å and 2.27 Å, respectively.The others are created by interactions between the hydroxyl group at position C-2 and His 61 (at 2.04 Å) and Asn 705 (at 2.09 Å).Additionally, the hydroxyl group at C-3 forms a hydrogen bond with Tyr 642 at 2.13 Å.Another hydrogen bond was observed between the fluorine atom and Tyr 597, with a 2.80 Å distance.Furthermore, the Phe 60 residue engages in π-π stacking with the aromatic ring of compound 10.Finally, 10 participates in van der Waals interactions with Trp 564, Met 657, Phe 681, Ile 706, Ile 682, Phe 60, Tyr 597, Tyr 642, and Tyr 599 (Figure S76, Table S2, Supplementary Material).S2, Supplementary Material).
It is important to highlight that compound 8, which has the shortest side chain, exhibits low affinity with the BRI1-BAK1 active site, consistent with the results obtained in the biological activity study.Conversely, our results suggest that analogs with a benzoate function at C-22 show improved affinity with the BRI1-BAK1 active site, and the presence of a fluorine atom in the "ortho and para" position strengthens this interaction, as observed in Figure 4C and Figure S76 of Supplementary Material.S2, Supplementary Material).
It is important to highlight that compound 8, which has the shortest side chain, exhibits low affinity with the BRI1-BAK1 active site, consistent with the results obtained in the biological activity study.Conversely, our results suggest that analogs with a benzoate function at C-22 show improved affinity with the BRI1-BAK1 active site, and the presence of a fluorine atom in the "ortho and para" position strengthens this interaction, as observed in Figure 4C and Figure S76 of Supplementary Material.

General Chemical
All reagents were purchased from commercial suppliers and used without further purification.Melting points were measured on an SMP3 apparatus (Stuart-Scientific, now Merck KGaA, Darmstadt, Germany) and are uncorrected. 1H-, 13 C-, 13 C DEPT-135, gs 2D HSQC, and gs 2D HMBC NMR spectra were recorded in CDCl 3 solutions and were referenced to the residual peaks of CHCl 3 at δ = 7.26 ppm and δ = 77.00ppm for 1 H and 13 C, respectively, on an Avance Neo 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C.Chemical shifts are reported in δ ppm, and coupling constants (J) are given in Hz; multiplicities are reported as follows: singlet (s), doublet (d), broad doublet (bd), doublet of doublets (dd), doublet of triplets (dt), triplet (t), broad triplet (bt), quartet (q), doublet of quartet (dq), doublet of double doublets (ddd), triplet of triplets (tt), and multiplet (m).IR spectra were recorded as KBr disks in an FT-IR 6700 spectrometer (Nicolet, Thermo Scientific, San Jose, CA, USA), and frequencies are reported in cm −1 .High-resolution mass spectra (HRMS-ESI) were recorded in a Bruker Daltonik (Bruker, Bremen, Germany).The analysis for the reaction products was performed with the following relevant parameters: dry temperature, 180 • C; nebulizer 0.4 Bar; dry gas, 4 L/min; and spray voltage, 4.5 kV at positive mode.Accurate mass measurements were performed at a resolving power: 140,000 FWHM at range m/z 50-1300.For analytical TLC, silica gel 60 in a 0.25 mm layer was used, and TLC spots were detected by heating after spraying with 25% H 2 SO 4 in H 2 O. Chromatographic separations were carried out by using a conventional column on silica gel 60 (230-400 mesh) using EtOAc-hexane gradients of increasing polarity.All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure below 40 • C. To a solution of bisnorcholenic acid 3β-acetate (7) (3.02 g, 7.77 mmol) in 30 mL of ether, 180 mL of ethereal CH 2 N 2 was added.The reaction mixture was kept under constant stirring and room temperature for 6 h.The end of the reaction was verified via TLC, and the mixture was then concentrated to dryness under reduced pressure.The solid obtained was recrystallized from ether/CH 2 Cl 2 (1:1).Compound 12 (3.12g, 99.8% yield) was obtained as a colorless solid (m.p. = 147.9-152.8-18).IR and 1 H NMR spectroscopic data were consistent with those reported [36,46,47].
3.2.17.2α,3α-Dihydroxy-5α-cholan-6-oxo-23,24-dinor-22-(4-Fluoro)-benzoate-22-yl (11) Compound 11 was synthesized according to the general procedure described in Section 3.2.14 and using the following amounts: olefin 22 (0.30 g, 0.66 mmol); DHQD-CLB (0.08 g, 0.17 mmol); CH 3 SO 2 NH 2 (0.14 g, 1.47 mmol); K 2 CO 3 (0.52 g, 3.76 mmol); K 3 [Fe(CN) 6 ] (1.27 g, 3.86 mmol); and OsO 4 (0.36 mL, 0.07 mmol).Compound 2α,3α-glycol 11 (0.27 g, 0.56 mmol, 84.9% yield) was obtained as a colorless solid (m.p. = 186.5-187.To evaluate the bioactivity of BR analogs (9)(10)(11), we used RLIT [59] and followed the modified procedure previously described by Díaz et al. [17].Seeds of a local rice cultivar (Oryza sativa L.) of the Zafiro variety (provided by INIA-QUILAMAPU, CHILE) were sterilized and then synchronized by soaking in sterile distilled water for 24 h, sown and grown for about 10 days in pots with a substrate, and maintained at 22 • C under a 16 h light/8 h dark photoperiod and 50-60% relative humidity in a plant growth chamber.Uniformly growing rice plants were selected to cut an approximately 8 cm segment containing the second internode of the rice lamina.These segments were then placed in a Petri dish containing sterile distilled water (60 mL) and the respective solutions of each treatment to reach the desired concentration (1 × 10 −7 and 1 × 10 −6 M) of each BR analog and brassinolide (APExBIO) used as a positive control.The negative control contained water and the same amount of dimethyl sulfoxide (DMSO) added to BR analogs.They were then left to incubate for 72 h at 25 • C in the dark to finally measure the angle of inclination of the unrolled sheet between the leaf and the sheath (see Figure S75, Supplementary Material).Each treatment consisted of 10 independent replicates; with these data, significant differences between the positive control and the treatments were determined.Mean values with at least a significant difference (p < 0.05; Student's t-test) were considered.Images were taken with a Leica EZ4HD stereo microscope with camera software (LAS EZ 3.4 DVD 272, Leica Microsystems, Wertzlar, Germany).

Bean Second Internode Bioassay
The bean second internode test for compounds 8-11 was carried out using the procedure reported by Slavikova et al., 2008 [60], with some modifications.Bean seeds (Phaseolus vulgaris L., cv.Pinto) were sown and germinated for three days, and then the uniformly germinated seeds were transplanted into pots containing perlite, vermiculite, and a substrate.The pots were kept in a plant growth chamber at 22 • C, with 48 W/m 2 light and a 16 h/8 h light/dark photoperiod.When the second internode of bean plants was 1-2 mm long (approx.after 7 days), they were treated with each of the tested compounds dissolved in DMSO and water at a final concentration of 1 × 10 −8 M via application on a small scar generated once the bract was removed from the base of the second internode.At the time of application, a 5 µL drop of each solution was mixed with a 2 µL drop of TWEEN ® 20 (AMRESCO ® ) for adhesion.
Control plants were treated with water and TWEEN 20 ® only.Measurements of the second internode length were made after 5 days.The difference between the length of the second internode of treated and control plants was used as a measure of activity.

Molecular Docking
The crystallographic structure of the protein Brassinosteroid Insensitive 1 (BRI1), in complex with BRI1-Associated Receptor Kinase 1 (BAK1) and the natural ligand (brassinolide) (PDB ID: 4M7E), resolved at 3.60 Å, was retrieved from the Protein Data Bank (http://www.rcsb.org/).The structure was optimized using pdb2pqr.py(Version 3.6.0),implemented on the web server PDB2PQR (http://server.poissonboltzmann.org/pdb2pqr,accessed on 7 August 2023), and utilized the AMBER force field.The protonation state of ionizable groups at pH 8 was assigned using PROPKA 4.5 [61,62].For each of the analyzed compounds, a molecular model was generated through their SMILES string using UCSF Chimera [63].Energy minimization of the models was performed by employing Chimera's default conditions with MMTK and Antechamber parameters [64].AutoDock Vina 1.1.2was employed as the docking algorithm, utilizing a grid box of dimensions 20 × 20 × 20 Å, with the center of brassinolide in the crystal structure serving as the center of the docking box.For each analyzed molecular model, five docking runs were conducted, generating ten poses each, with an exhaustiveness of eight.The maximum energy difference between modes was limited to 2 kcal/mol.The docking analyses were inspected based on binding affinity values (kcal/mol) as well as hydrogen, hydrophobic, and electrostatic bonding interactions.Following docking, a comparison was made between the docked ligand and re-docked brassinolide poses.The visualization of the docked poses for their analysis was performed using UCSF Chimera [63] and Discovery Studio Visualizer (BIOVIA, San Diego, CA, USA) [65].

Figure 3 .
Figure 3. (a) 1 H NMR (MeOD) spectrum of compound 8.(b) Selective 1D NOE spectrum, showing the selective excitation of H-2 and the NOE effects observed with H-4β and CH3-19.The other signal observed corresponds to the scalar coupling of H-2 with H-3.

Figure 3 .
Figure 3. (a) 1 H NMR (MeOD) spectrum of compound 8.(b) Selective 1D NOE spectrum, showing the selective excitation of H-2 and the NOE effects observed with H-4β and CH 3 -19.The other signal observed corresponds to the scalar coupling of H-2 with H-3.

Figure 4 .
Figure 4. Dose-response curve of compound 1 used as positive control 4 days after application at different concentrations: (a) visual representation; (b) effect of compound 1 on the prolongation of the second internode of the bean.Error bars represent S.D.

Figure 4 .
Figure 4. Dose-response curve of compound 1 used as positive control 4 days after application at different concentrations: (a) visual representation; (b) effect of compound 1 on the prolongation of the second internode of the bean.Error bars represent S.D.

Figure 5 .
Figure 5.Effect of BRs analogs 8-11 and positive control (1) on the elongation bean second internode bioassay at a concentration of 1 × 10 −8 M.

Figure 5 .
Figure 5.Effect of BRs analogs 8-11 and positive control (1) on the elongation bean second internode bioassay at a concentration of 1 × 10 −8 M.

Figure 6 .
Figure 6.Binding modes of compound 10 into BRI1-BAK1 heterodimer.Binding modes of 10 are represented as turquoise and red sticks.(A) Poses of 10 and brassinolide (brown sticks) within the BRI1-BAK1 binding site; (B) pose of 10 and its interaction within the BRI1-BAK1 binding site; (C) a 2D view of binding interactions of 10 within the binding site.Hydrogen bonds are represented in green segmented lines.π-π stacking is represented in dark pink segmented lines.Hydrophobic interactions are represented in pink segmented lines.On the other hand, compound 11 adopts a similar orientation to that of compound 10 and brassinolide (Figure S76, Supplementary Material).At the predicted binding position of 11, six hydrogen bonds were identified, five of which are consistent with those identified in compound 10 (Tyr 597 at 2.25 Å, Ser 647 at 2.33 Å, His 61 at 1.96 Å, Asn 705 at 2.57 Å, and Tyr 642 at 2.21 Å).However, the fluorine atom of the benzoate group in the "para" position generates a hydrogen bond with Ser 48 at 2.88 Å.Furthermore, the Phe 60 residue participates in π-π stacking with the aromatic ring of compound 11.On the other hand, 11 generates van der Waals interactions with Trp 564, Met 657, Phe 681, Ile 706, Ile 682, Phe 60, Tyr 597, Tyr 642, and Tyr 599 (Figure S76, TableS2, Supplementary Material).It is important to highlight that compound 8, which has the shortest side chain, exhibits low affinity with the BRI1-BAK1 active site, consistent with the results obtained in the biological activity study.Conversely, our results suggest that analogs with a benzoate function at C-22 show improved affinity with the BRI1-BAK1 active site, and the presence of a fluorine atom in the "ortho and para" position strengthens this interaction, as observed in Figure4Cand FigureS76of Supplementary Material.

Figure 6 .
Figure 6.Binding modes of compound 10 into BRI1-BAK1 heterodimer.Binding modes of 10 are represented as turquoise and red sticks.(A) Poses of 10 and brassinolide (brown sticks) within the BRI1-BAK1 binding site; (B) pose of 10 and its interaction within the BRI1-BAK1 binding site; (C) a 2D view of binding interactions of 10 within the binding site.Hydrogen bonds are represented in green segmented lines.π-π stacking is represented in dark pink segmented lines.Hydrophobic interactions are represented in pink segmented lines.On the other hand, compound 11 adopts a similar orientation to that of compound 10 and brassinolide (Figure S76, Supplementary Material).At the predicted binding position of 11, six hydrogen bonds were identified, five of which are consistent with those identified in compound 10 (Tyr 597 at 2.25 Å, Ser 647 at 2.33 Å, His 61 at 1.96 Å, Asn 705 at 2.57 Å, and Tyr 642 at 2.21 Å).However, the fluorine atom of the benzoate group in the "para" position generates a hydrogen bond with Ser 48 at 2.88 Å.Furthermore, the Phe 60 residue participates in π-π stacking with the aromatic ring of compound 11.On the other hand, 11 generates van der Waals interactions with Trp 564, Met 657, Phe 681, Ile 706, Ile 682, Phe 60, Tyr 597, Tyr 642, and Tyr 599 (Figure S76, TableS2, Supplementary Material).It is important to highlight that compound 8, which has the shortest side chain, exhibits low affinity with the BRI1-BAK1 active site, consistent with the results obtained in the biological activity study.Conversely, our results suggest that analogs with a benzoate function at C-22 show improved affinity with the BRI1-BAK1 active site, and the presence of a fluorine atom in the "ortho and para" position strengthens this interaction, as observed in Figure4Cand FigureS76of Supplementary Material.

Angle between Lamina and Sheaths (Degrees ± Standard Error) 1
These values represent the mean ± standard deviation of two independent experiments with at least ten replicates each.Average angle of negative control: 3 ± 2.6.Letters represent experiments with a significant difference between positive control(1)and analog treatments at the 0.05 significance level (Student's t-test). 1