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Article

Synthesis and Biological Activity of New Brassinosteroid Analogs of Type 24-Nor-5β-Cholane and 23-Benzoate Function in the Side Chain

by
Nitza Soto
1,
Karoll Ferrer
1,
Katy Díaz
1,
César González
1,
Lautaro Taborga
1,
Andrés F. Olea
2,*,
Héctor Carrasco
2 and
Luis Espinoza
1,*
1
Departamento de Química, Universidad Técnica Federico Santa María, Valparaíso, CP 2340000, Chile
2
Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, Santiago, CP 8900000, Chile
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(9), 4808; https://doi.org/10.3390/ijms22094808
Submission received: 9 April 2021 / Revised: 22 April 2021 / Accepted: 24 April 2021 / Published: 1 May 2021
(This article belongs to the Special Issue Plant Steroids)
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Versions Notes

Abstract

:
Brassinosteroids are polyhydroxysteroids that are involved in different plants’ biological functions, such as growth, development and resistance to biotic and external stresses. Because of its low abundance in plants, much effort has been dedicated to the synthesis and characterization of brassinosteroids analogs. Herein, we report the synthesis of brassinosteroid 24-nor-5β-cholane type analogs with 23-benzoate function and 22,23-benzoate groups. The synthesis was accomplished with high reaction yields in a four-step synthesis route and using hyodeoxycholic acid as starting material. All synthesized analogs were tested using the rice lamina inclination test to assess their growth-promoting activity and compare it with those obtained for brassinolide, which was used as a positive control. The results indicate that the diasteroisomeric mixture of monobenzoylated derivatives exhibit the highest activity at the lowest tested concentrations (1 × 10−8 and 1 × 10−7 M), being even more active than brassinolide. Therefore, a simple synthetic procedure with high reaction yields that use a very accessible starting material provides brassinosteroid synthetic analogs with promising effects on plant growth. This exploratory study suggests that brassinosteroid analogs with similar chemical structures could be a good alternative to natural brassinosteroids.

1. Introduction

Brassinosteroids (BRs) are phytohormones that are found in all plants; they act as plant growth promoters and development regulators at nanomolar to micromolar concentrations [1,2,3,4]. These phytohormones play important roles in cell development and vascular differentiation [5,6]. BRs are also involved in reproductive development, the modulation of gene expression [7,8] and other developmental processes like germination, rhizogenesis, flowering, senescence, abscission and maturation. Additionally, it has been proven that BRs induce plants’ resistance against different abiotic and biotic stresses [9,10,11]. All-natural bioactive BRs possess a vicinal 22R, 23R diol structural functionality (for example brassinolide 1, 24-epibrassinolide 2 and 28-homobrassinolide 3, Figure 1), which seems to be essential for high biological activity. In this sense, several studies have been focused on determining the structural requirements that these compounds should possess to elicit strong biological activity [12,13,14]. Structural variations of BRs principally arise from the type and position of functions in the A, B rings, fusion A/B ring and alkyl side chain.
The extremely low abundance of BRs present in plants (1–100 µg by kg of fresh weight) [15] has prompted the chemical synthesis of BR analogs, and a considerable number of them have already been reported [16]. However, to synthesize molecules with the same stereochemical configuration of natural BRs, both in the rings and side alkyl chain is an enormous synthetic challenge [17]. Consequently, synthetic BRs and BR analogs are generally too expensive for large-scale commercial applications. Therefore, these difficulties have stimulated the search for more accessible and bioactive analogs. The idea is to use natural molecules with chemical structures similar to BRs, such as stigmasterol (4) [18,19,20], pregnenolone (5) [21], hyodeoxycholic acid (6) [22,23] and diosgenin (7) [24,25] as starting material (Figure 1).
Hyodeoxycholic acid (6) is a bile acid extracted from hog bile whose chemical structure contains a similar ring skeleton and a side alkyl chain in the same position as BRs (see Figure 1). Considering that these structural features satisfy the requirements established for natural active BRs, brassinolide and several related compounds have been synthesized by chemical modification of 6 [26,27,28,29,30].
It is worth to mention that natural occurring BRs possess trans-type A/B ring fusion (5α BRs), and this structural factor has been proposed as a key factor for activity. However, studies of synthetic BRs analogs, having cis-type A/B ring fusion (5β BRs), have shown that these compounds exhibit important biological activity in tests such as rice lamina inclination (RLIT) [31], cotyledon expansion and hypocotile elongation radish [32,33,34] and recently in germination effects in tomato [35]. This shows that this structural characteristic of cis A/B ring fusion (5β skeleton) could be considered an alternative for new active BR analogs.
On the other hand, a series of new BR 24-nor-5α-cholane type analogs with a shortest side alkyl chain and an ending group in the form of carboxylic acids, methyl and phenyl esters (Figure 2) have also been synthesized from 6 [33,36,37,38]. Recently, the synthesis of BR analogs with a shortest side alkyl chain of 24-nor-5α-cholane type, oxygenated function in C22 with S/R configuration (Figure 2, compounds 8–14) [39] and a benzoyl ester group at the ending chain (Figure 2, compounds 15–17) were reported [38,39,40].
All these BR analogs were tested for bioactivity using the RLIT and using brassinolide (1) as positive control [35,40]. The results show that analog 8 (C22(S) configuration) was more active than diasteroisomer 9 (C22(R) configuration) and slightly less active than 1. Interestingly, the activities of analogs with benzoyl groups in the side chain (15 and 16) do not depend on the C22 configuration, and both epimers show similar growth-promoting activities, which are almost identical to those exhibited by 1. A molecular docking study suggest that this effect is due to hydrophobic interactions between the ligand and the receptor [40]. As a result, we have suggested that epimeric mixtures of S and R stereoisomers of monobenzoylated derivatives could be used instead of pure compounds [35].
Considering these results, in this work we have synthesized new BR 24-nor-5β-cholane type analogs with A/B ring fusion and acetate function in C6. Even though these analogs are similar to 15, 16 and 17, the synthesis of BR analogs with cis fusion of A/B rings requires fewer synthesis steps (isomerization reaction to convert fusion 5β to fusion 5α of A/B rings is no longer needed) and this is a clear synthetic advantage. In addition, it becomes interesting to evaluate the effect of this structural feature on the biological activity of these new compounds.

2. Results

The synthesis of BR analogs from hyodeoxycholic acid goes through the formation of a terminal olefin.

2.1. Chemistry

Standard acetylation of 6 (Scheme 1) leads to known diacetylated derivative 21 with 91.1% yield (referential: 80% yield [41,42,43]). Oxidative decarboxylation of the side chain of 21, with the PhI(OAc)2/Cu(OAc)2 system [41,42], leads to alkene 22 with 99.6% yield (Scheme 1).
Previously, the synthesis of glycols C22/C23 in steroids of 24-nor-5α-cholane type with shortest side chain (terminal olefins) have been accomplished by Sharpless dihydroxylation reaction [38]. Results indicate that this type of hydroxylation leads to a mixture of C-22 glycols (S/R) with an approximate ratio 1.0:0.9 of both diastereomers, the 22S epimer being the slightly major product [38]. Thus, compound 22 was dihydroxylated by Sharpless reaction and using dihydroquinidine p-chlorobenzoate (DHQD-CLB) as chiral ligand (Scheme 1) [38,44]. The product of this reaction is the 23ab diastereoisomer mixture with a total 95.0% yield. Separation of the 23ab diastereoisomer mixture was not possible, but some 1H signals and all 13C signals in the NMR spectra of the mixture were identified. Thus, it was possible to determine that the C21 methyl group, in 23a and 23b diastereoisomers, appears at δH = 0.94 and 0.90 ppm, respectively (Figure 3). Integration of these signals gives a relative ratio of 23a:23b equal to 1.0:0.7.
Additionally, the structure of compounds 23a and 23b and stereochemistry at C22 was established by simple comparison of 1H and 13C NMR spectra obtained for other 22,23-dihydroxy analogs of similar structure and the 24-norcholan type previously reported [38,39]. Thus, chemical shifts (δ), coupling constants (J) and multiplicities of signals appearing in 1H NMR, and chemical shifts (δ) in 13C NMR spectra, corresponding to H-22, H-23a, H-23b and CH3-21, were compared for both diasteroisomers. The main differences in these spectroscopic parameters were observed between H-21 and carbons C21, C22 and C23 (Figure S1, Supplementary Materials), and they are listed in Table 1.
Benzoylation of glycol mixtures 23a/23b gives mono- and dibenzoylated derivatives, i.e., compounds 18-20 (Scheme 1). Compounds 18 and 20 were purified by C.C. and recrystallized, giving yields of 11.3% and 9.1%, respectively, whereas monobenzoylated derivative 19 could not be isolated in pure form. Additionally, a mixture of monobenzoylated epimers 18/19 were also obtained with 78.2% yield (18:19 = 1.0:0.44). The proportion of each epimer in the mixture was determined from integration of 1H NMR signals at δH = 4.48, 4.39, 4.26 and 4.05 ppm, assigned to H-23a, H-23b and H-22 for 18 and 19 (Figure 4).
The structure of compounds 18 and 19 and stereochemistry at C22 in the mixture was established in a way similar to that used for the mixture 23a/23b, i.e., a simple comparison of 1H and 13C NMR spectra with those previously reported for other 22-hydroxy-23-benzoate and 24-norcholan type [38,39]. In addition, the comparison of 1H NMR spectra of pure compound 18 and the mixture 18/19 allows the assignment of signals for each compound (Figures S2 and S3, Supplementary Materials). A comparison of NMR spectroscopic parameters corresponding to signals assigned to H-22, H-23a, H-23b and CH3-21 in both diasteroisomers is given in Table 2. The main differences in these spectroscopic parameters were observed between H-21 and carbons C21, C22 and C23.
The chemical structure of dibenzoylated derivative 20 was established using 1H, 13C, 2D HMQC, and 2D HMBC NMR techniques. The presence of dibenzoylated function was confirmed by signals observed in the 1H-NMR spectrum (Figure S4, Supplementary Materials), at δH = 8.06 (2H, d, J = 7.8 Hz); 7.97 (2H, d, J = 7.8 Hz); 7.57-7.50 (2H, m); 7.44 (2H, t, J = 7.6 Hz); 7.38 (2H, t, J = 7.6 Hz), which are assigned to protons HAr-2′; HAr-2″, HAr-4′, and HAr-4″; HAr-3′ and HAr-3″ in the aromatic group, respectively (Figure 5). In addition, in the 13C-NMR spectrum (Figure S4, Supplementary Materials) appear signals at 133.04; 132.98; 132.96; 130.38; 129.81; 129.65; 129.62; 128.37 and 128.35 ppm, which were assigned to carbon C4′-Ar; C1′-Ar; C4″-Ar; C2′-Ar; C1″-Ar; C3′-Ar; C2″-Ar; C3″-Ar and C4′-Ar, respectively, of both aromatic rings. These assignments were confirmed by 2D HMQC and HMBC spectra (Figure S4, Supplementary Materials). The main structural information provided by the 2D HMBC spectrum was the assignment and position in the side chain of the two benzoate groups, which showed heteronuclear correlations at 2JCH and 3JCH, as indicated in Figure 5.
To determine the stereochemistry at carbon C22 of compound 20, the monobenzoylated derivative 18 was submitted to an exhaustive benzoylation reaction with PhCOCl/DMAP. A dibenzoylated derivative was obtained with 93% yield, for which NMR data were totally consistent with those obtained for derivative 20. Thus, we can conclude that analogs 18 and 20 have the same configuration at C22.

2.2. Bioactivity of BR Analogs Determined by Rice Lamina Inclination Assay (RLIT)

Since the discovery of BRs, it has been well-established that growth-promoting activity in plants is one of the most important functions in which natural BRs are involved [2,45]. Different bioassays have been developed and applied to detect and quantify this effect, i.e., first bean internode, root growth and RLIT. The latter is the most widely used test due to its high specificity and sensitivity, which allows for the detecttion of brassinolide concentrations at a level of 0.05 ng/L [46,47]. Using this test, the growth-promoting activity of a series of natural BRs and synthetic analogs have been evaluated. This data have been analyzed in terms of BR chemical structure, and different structure–activity relationships have been proposed [14,16,31]. In this work, the growth-promoting activities of 18, diasteroisomeric mixture 18/19 and 20 were assessed by RLIT and using 1 as positive control. The reported bending angles (see Table 3) correspond to the mean ± standard deviation of two independent experiments with at least six replicates each (n = 12) for each tested concentration.
The results in Table 3 indicate, that at the lowest tested concentrations (1 × 10−8 and 1 × 10−7 M), the mixture of monobenzoylated compounds 18/19 exhibit activities even higher than that obtained for 1, which is recognized as the most active natural BR. However, at the highest concentration (1 × 10−6 M), 1 becomes the most active once again. On the other hand, no significant differences in activity were observed between analog 18 and mixture 18/19 at concentrations of 1 × 10−7 and 1 × 10−6 M (Table 3). Considering that the ratio 18:19 is 1.0:0.44, this result suggests that, at these concentrations, the S epimer (18) is more active than the R epimer (19). However, at the lowest tested concentration, this behavior is reversed. Finally, the addition of a second benzoate group at the C22 position in the dibenzoylated analog 20 reduces biological activity significantly compared to analog 18 or epimeric mixture 18/19 (Table 3). These results are in line with those previously reported for similar BR 24-norcholane type analogs (compound 17). Molecular docking studies of 15, 16 and 17 have shown that decreasing activity with increasing size in substituent groups in the chain can be attributed to the lowest interaction between the side chain and the BRI1/BAK1 complex [40]. Thus, it seems that substitution in ring B by a carbonyl group (15, 16, 17) or an acetyl group (18, 19, 20) makes no difference to the growth enhancement effect of 24-norcholane type analogs.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Experimental Methods

All reagents were obtained from commercial suppliers at the highest quality grade available and used as received. Conditions and instruments used for measurement of melting points and recording of NMR, FT-IR, and MS spectra are given in detail in previous work [43]. Analytical TLC was run on silica gel 60 in a 0.25 mm layer, and TLC spots were detected by heating after spraying with diluted sulfuric acid (25%). Column chromatographic separations (CC) were carried out, using silica gel 60 (230–400 mesh) as stationary phase EtOAc-hexane gradients of increasing polarity as eluent. All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure, below 40 °C.

3.1.2. Synthesis of Mixture (22S)-22,23-Dihydroxy-24-nor-5β-cholan-3α,6α-diyl Diacetate (23a) and (22R)-22,23-Dihydroxy-24-nor-5β-cholan-3α,6α-diyl Diacetate (23b)

Alquene 22 (250 mg, 0.58 mmol), DHQD-CLB (80 mg, 0.17 mmol), CH3SO2NH2 (120 mg, 1.22 mmol), K2CO3 (500 mg, 3.65 mmol) and K3[Fe(CN)6] (860 mg, 2.61 mmol) were successively added to a t-butanol/water mixture (22 mL, 1:1 v/v). After homogenization by magnetic stirring (5 min), 0.36 mL of an OsO4 solution (1.0 g, 3.933 mmol in 20 mL of t-butanol) was added, and the mixture reaction was stirred at room temperature for 1.5 h. After this period, H2O (10 mL) and a saturated solution of Na2S2O3x5H2O (10 mL) were added. The organic phase was extracted with AcOEt (2 ×x 30 mL), dried over MgSO4, filtered, and the solvent volume was reduced by evaporation under vacuum. The extract, dissolved in DCM (10 mL), was chromatographed on silica gel using hexane/EtOAc mixtures of increasing polarity (19.8:0.2 → 20.0:0.0). A mixture of 23a/23b (257 mg, 95% yield, 23a:23b = 1.0:0.7) was obtained as a colorless powder. IR νmax (cm−1) mixture 23a/23b: 3446 (O-H); 2934 (C-H); 2870 (C-H); 1737 (C=O); 1717 (C=O). 23a 1H NMR (400.1 MHz, CDCl3) (Figure S1, Supplementary Materials): δ (ppm) 5.14 (1H, dt, J = 11.5 and 5.3 Hz, H-6); 4.70 (1H, m, H-3); 3.80 (1H, m, H-22); 3.63 (1H, dd, J = 9.9 and 9.9 Hz, H-23a); 3.51 (1H, m, H-23b); 2.04 (3H, s, CH3CO2-C6); 2,01 (3H, s, CH3CO2-C3); 0.97 (3H, s, H-19); 0.94 (3H, d, J = 6.8 Hz, H-21); 0.66 (3H, s, H-18). 13C NMR (Figure S3, Supplementary Materials) δ (ppm): 170.52 (CH3CO-C3); 170.49 (CH3CO-C6); 74.11 (C22); 73.64 (C3); 70.87 (C6); 62.47 (C23); 56.04 (C14); 55.78 (C17); 45.37 (C5 and C20); 42.72 (C13); 39.89 (C12); 36.04 (C10); 35.01 (C7); 34.68 (C8); 31.25 (C1); 27.78 (C2); 26.41 (C16); 26.22 (C4); 24.17 (C15); 24.02 (C19); 21.40 (CH3CO-C3); 21.37 (CH3CO-C6); 20.67 (C11); 12.62 (C21); 11.75 (C18). 23b 1H NMR (Figure S1, Supplementary Materials) δ (ppm): 5.14 (1H, dt, J = 11.5 and 5.3 Hz, H-6); 4.70 (1H, m, H-3); 3.80 (1H, m, H-22); 3.63 (1H, dd, J = 9.9 and 9.9 Hz, H-23a); 3.51 (1H, m, H-23b); 2.04 (3H, s, CH3CO2-C6); 2.01 (3H, s, CH3CO2-C3); 0.97 (3H, s, H-19); 0.90 (3H, d, J = 5.7 Hz, H-21); 0.66 (3H, s, H-18). 13C NMR (Figure S1, Supplementary Materials) δ (ppm): 170.52 (CH3CO-C3); 170.49 (CH3CO-C6); 73.88 (C22); 73.64 (C3); 70.93 (C6); 66.04 (C23); 56.04 (C14); 55.78 (C17); 45.37 (C5 and C20); 45.32 (C9); 40.10 (C13); 39.82 (C12); 36.01 (C10); 35.01 (C7); 34.68 (C8); 31.25 (C1); 27.54 (C2); 26.41 (C16); 26.22 (C4); 24.17 (C15); 24.01 (C19); 21.40 (CH3CO-C3); 21.37 (CH3CO-C6); 20.67 (C11); 13.01 (C21); 11.87 (C18). MS m/z (%) (23a/23b): 464 (M+ < 1); 313 (25.6); 312 (100); 213 (21.7).

3.1.3. Synthesis of (22S)-22-Hydroxy-24-nor-5β-cholan-3α,6α-diyl Diacetate-23-benzoate (18), (22R)-22-Hydroxy-24-nor-5β-cholan-3α,6α-diyl Diacetate-23-benzoate (19) and (22S)-24-nor-5β-cholan-3α,6α-diyl diacetate-22,23-diyl dibenzoate (20)

To a 23a/23b mixture, (500 mg, 1.08 mmol) dissolved in CH2Cl2 (20 mL), were added DMAP (10 mg, 0.410 mmol) and pyridine (2.0 mL, d = 0.981 g/mL). The temperature was decreased to 0–5 °C under nitrogen atmosphere, and PhCOCl (1.4 mL, d = 1.21 g/mL, 12.05 mmol) was slowly added over 1 h. After addition, the reaction mixture was maintained at room temperature for 36 h. At the end of the reaction, the solvent volume was reduced by rotatory evaporation up to a final volume of 10 mL. The reaction mixture was diluted with EtOAc (40 mL), washed with a saturated solution of KHSO4 (2 × 10 mL) and H2O (3 × 30 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure and the residue was redissolved in DCM (10 mL) and submitted to CC on silica gel with hexane/EtOAc mixtures of increasing polarity (19.8:0.2 -> 9.8:10.2). The obtained mixture (674.3 mg), formed by mono- and dibenzoylated compounds, was submitted to chromatographic separation twice. Three fractions were obtained in the following order of polarity: Fraction I compound 20 (65.7 mg, 9.1% yield); Fraction II mixture of compounds 18 and 19 (478.3 mg, 18:19 = 1.0:0.44, 78.2% yield); and Fraction III compound 18 (68.9 mg, 11.3% yield). Compound 18 was obtained as a colorless powder (m.p. 133 ± 3°C). IR νmax (cm−1): 3523 (O-H); 2942 (C-H); 2890 (C-H); 2866 (C-O); 1735 (C=O); 1716 (C=O); 1276 (OCOAr). 1H NMR (400.1 MHz, CDCl3) (Figure S4, Supplementary Materials) δ (ppm): 8.05 (2H, d, J = 7.8 Hz, HAr-2′); 7.58 (1H, t, J = 7.3 Hz, HAr-4′); 7.45 (2H, t, J = 7.8 Hz, HAr-3′); 5.15 (1H, dt, J = 11.4 and 5.3 Hz, H-6); 4.70 (1H, m, H-3); 4.48 (1H, dd, J = 11.4 and 2.0 Hz, H-23a); 4.21 (1H, dd, J = 11.4 and 10.2, H-23b); 4.07-4.04 (1H, m, H-22); 2.02 (3H, s, 3H, CH3CO-C3); 2.01 (3H, s, CH3CO-C6); 1.04 (3H, d, J = 6.8 Hz, H-21); 0.98 (3H, s, H-19); 0.68 (3H, s, H-18). 13C NMR (Figure S2, Supplementary Materials) δ (ppm): 170.50 (CH3CO-C3); 170.45 (CH3CO-C3); 166.99 (CO-Ar); 133.17 (C4′-Ar); 129.90 (C1′-Ar); 129.62 (C2′-Ar); 128.42 (C3′-Ar); 73.63 (C3); 71.79 (C22); 70.87 (C6); 68.95 (C23); 55.78 (C17); 53.06 (C14); 45.34 (C9); 43.29 (C13); 40.34 (C20); 39.86 (C7); 39.81 (C5); 38.40 (C8); 36.03 (C10); 35.02 (C12); 31.26 (C1); 27.56 (C2); 26.42 (C4); 26.23 (C15); 24.18 (C16); 23.24 (C19); 21.40 (CH3CO-C3); 21.36 (CH3CO-C6); 20.66 (C11); 12.90 (C21); 11.80 (C18). MS m/z (%): 568 (M+ < 1); 327 (32.4); 326 (100); 213 (29.6); 207 (26.1); 145 (19.0); 133 (19.1). Compound 19 1H NMR (400.1 MHz, CDCl3) (Figure S3, Supplementary Materials) δ (ppm): 8.05 (2H, d, J = 7.8 Hz, HAr-2′); 7.58 (1H, t, J = 7.3 Hz, HAr-4′); 7.45 (2H, t, J = 7.8 Hz, HAr-3′); 5.15 (1H, dt, J = 11.4 and 5.3 Hz, H-6); 4.70 (1H, m, H-3); 4.39 (1H, dd, J = 11.4 and 8.4 Hz, H-23a); 4.26 (1H, dd, J = 11.4 and 3.3 Hz, H-23b); 4.07-4.04 (1H, m, H-22); 2.05 (3H, s, CH3CO-C3), 2.02 (3H, s, CH3CO-C6); 1.00 (3H, d, J = 6.4 Hz, H-21); 0,98 (3H, s, H-19); 0.68 (3H, s, H-18). 13C RMN (Figure S3, Supplementary Materials) δ(ppm): 170.50 (CH3CO-C3); 170.45 (CH3CO-C3); 166,68 (CO-Ar); 133.08 (C4′-Ar); 129.88 (C1′-Ar); 129.62 (C2′-Ar); 128.36 (C3′-Ar); 73.63 (C3); 71.86 (C22); 70.87 (C6); 68.88 (C23); 55.78 (C17); 53.06 (C14); 45.34 (C9); 43.29 (C13); 40.34 (C20); 39.86 (C7); 39.81 (C5); 38.40 (C8); 36.03 (C10); 35.02 (C12); 31.26 (C1); 27.56 (C2); 26.42 (C4); 26.23 (C15); 24.18 (C16); 23.24 (C19); 21.40 (CH3CO-C3); 21.36 (CH3CO-C6); 20.66 (C11); 12.39 (C21); 11.90 (C18). Compound 20 was obtained as a colorless powder (m.p. 122 ± 3°C). IR νmax (cm−1): 2946 (C-H); 2870 (C-H); 1282 (C-O); 1249 (C-O); 1723 (C=O); 1723 (C=O); 1283 (OCOAr); 1263 (OCOAr). 1H NMR (400.1 MHz, CDCl3) (Figure S4, Supplementary Materials) δ(ppm: 8.06 (2H, d, J = 7.8 Hz, HAr-2′); 7.97 (2H, d, J = 7.8 Hz, HAr-2″); 7.57-7.50 (2H, m, HAr-4′ and HAr-4″); 7.44 (2H, t, J = 7.6 Hz, HAr-3′); 7.38 (2H, t, J = 7.6 Hz, HAr-3″); 5.56 (1H, bd, J = 9.1 Hz, H-22); 5.16 (1H, dt, J = 11.4 and 5.3 Hz, H-6); 4.71 (1H, m, H-3); 4.64 (1H, d, J = 11.8 Hz, H-23a); 4.50 (1H, dd, J = 11.8 and 9.3 Hz, H-23b); 2.05 (3H, s, CH3CO-C3); 2.02 (3H, s, CH3CO-C6); 1.13 (3H, d, J = 6.0 Hz, H-21); 0.98 (3H, s, H-19); 0.69 (3H, s, H-18). 13C NMR (Figure S4, Supplementary Materials) δ (ppm): 170.50 (CH3CO-C3); 170.45 (CH3CO-C3); 166.61 (CO-Ar′); 165.91 (CO-Ar″); 133.04 (C4′-Ar); 132.98 (C1′-Ar); 132.96 (C4″-Ar); 130.38 (C2′-Ar); 129.81 (C1″-Ar); 129.65 (C3′-Ar); 129.62 (C2″-Ar); 128.37 (C3″-Ar); 128.35 (C4′-Ar); 74.67 (C22); 73.64 (C3); 70.87 (C6); 63.00 (C23); 55.82 (C15 and C17); 53.12 (C9); 45.35 (C5); 43.40 (C13); 39.86 (C7); 38.60 (C8); 36.03 (C10); 35.05 (C20); 34.67 (C12); 31.28 (C1); 27.41 (C2); 26.45 (C4); 26.25 (C15); 24.21 (C16); 23.25 (C19); 21.42 (CH3CO-C3); 21.37 (CH3CO-C6); 20.69 (C11); 13.76 (C21); 11.88 (C18). MS m/z (%): 673 (M+<1); 327 (26.0); 326 (100); 213 (33.3); 145 (22.2); 105 (20.4); 81 (19.1).

3.1.4. Synthesis of (22S)-24-Nor-5β-cholan-3α,6α-diyl Diacetate-22,23-diyl Dibenzoate (20)

DMAP (5 mg, 0.205 mmol) and pyridine (0.5 mL, d = 0.981 g/mL) were successively added to a solution of 18 (25.0 mg, 0.044 mmol) in CH2Cl2 (10 mL) with stirring under nitrogen atmosphere. Then PhCOCl 0.5 mL (d = 1.21 g/mL, 4.30 mmol) was added, and the reaction mixture was maintained at room temperature for 3 h. At the reaction ending (verified by TLC), the solvent volume was taken to 2 mL by evaporation under reduced pressure. The resulting mixture was diluted with EtOAc (15 mL), washed successively with a saturated solution of KHSO4 (2 × 3 mL) and H2O (3 × 5 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was redissolved in DCM (5 mL) and purified by CC using hexane/EtOAc mixtures of increasing polarity (19.8:0.2 -> 9.8:10.2). Dibenzoylated compound 20 was obtained (24.7 mg) with 93% yield. The 1H and 13C-NMR spectroscopic data were consistent with those described above.

3.2. In Vitro Rice Lamina Inclination Test (RLIT)

The growth-promoting activity of all new synthesized BR analogs was evaluated, and compared with that shown by brassinolide, using the rice lamina inclination test. The experimental protocol has been described elsewhere [37,48]. Briefly, segments of rice leaf of approximately 6 cm were excised from growing rice plants that have reached a size of which the second internode can be obtained. Six of these segments per treatment were incubated for 48 h at 22 °C in the dark on Petri plates (50 mL) containing different concentrations (1 × 10−8 M, 1 × 10−7 M and 1 × 10−6 M) of testing samples (18, diasteroisomeric mixture 18/19 and 20). Brassinolide (APE BIO, Boston, MA, USA) was used as a positive control, whereas pure sterile water was used as a negative control. After incubation, the opening angle formed between the leaf and the sheath was measured with a protractor. The reported magnitude of angle values are given as the mean of twelve measurements with standard deviations (n = 12). This test was performed twice for each treatment

4. Conclusions

The synthesis of BR 24-nor-5β-cholane type analogs with 23-benzoate function (18–19) and 22- and 23-benzoate groups (20) was accomplished with high reaction yields (18/19, 78%; 18, 11%; and 20, 9.1%), using hyodeoxycholic acid as starting material. All products and intermediate compounds were fully characterized, mainly by 1D and 2D NMR spectroscopic techniques.
Growth-promoting activity was assessed by RLIT and the results show that, at the lowest tested concentrations (1×10−8–1×10−7 M), the mixture 18/19 exhibit the highest activity, including higher than brassinolide. The high activity of similar 24-nor-5β-cholane type analogs carrying bulky groups at the end of the shortest side chain has been attributed to hydrophobic interaction between the aromatic ring with the BRI1/BAK1 complex [40]. In this case, the synthetic analogs are even simpler because the hydroxyl group in ring B of hyodeoxycholic acid was just acetylated instead of oxidized to a carbonyl group. In addition, the direct use of a diasteroisomers mixture avoids an arduous separation step. Thus, the results of growth-promoting activity exhibited by the mixture 18/19 along with its straightforward synthesis validate the synthetic approach described herein, as a potential method for the obtention of BR synthetic analogs with promising effects on plant growth.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ijms22094808/s1.

Author Contributions

N.S., K.F. and C.G. carried out the synthesis, separation and purification of compounds. K.D. supervised all bioactivity experiments, participated in the interpretation, discussion and writing of the manuscript. L.T. and H.C. recorded the NMR spectra and correlated the data with chemical structures. L.E. performed project administration, supervision, investigation and manuscript redaction. A.F.O. collaborated in the discussion and interpretation of the results, manuscript redaction and corrections. 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

Not applicable.

Acknowledgments

Nitza Soto thanks the financial support from Dirección General de Postgrado (DGIIE-USM) of Universidad Técnica Federico Santa María.

Conflicts of Interest

The authors declare no conflict of interest.

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Ijms 22 04808 g001 550
Figure 1. Structure of naturally occurring brassinosteroids and precursors.
Figure 1. Structure of naturally occurring brassinosteroids and precursors.
Ijms 22 04808 g001
Ijms 22 04808 g002 550
Figure 2. Structure of synthetic BRs 24-norcholan type 8–14 and 24-norcholan type 15–20 benzoylated analogs.
Figure 2. Structure of synthetic BRs 24-norcholan type 8–14 and 24-norcholan type 15–20 benzoylated analogs.
Ijms 22 04808 g002
Ijms 22 04808 sch001 550
Scheme 1. Synthesis of BR 24-nor-5β-cholane type analogs with function 23-benzoate 18–20. Reagents and conditions: first two steps are described in Ref. [43] (a) DHQD-CLB/CH3SO2NH2, K2CO3/K3[Fe(CN)6], OsO4/(CH3)3COH/H2O, r.t, 1.5 h 23a/23b (1.0:0.7), 95% yield; (b) C6H5COCl/DMAP/CH2Cl2/py, r.t, 36 h, 18 (11.3% yield), 18/19 (1.0:0.44, 78.2% yield), 20 (9.1% yield).
Scheme 1. Synthesis of BR 24-nor-5β-cholane type analogs with function 23-benzoate 18–20. Reagents and conditions: first two steps are described in Ref. [43] (a) DHQD-CLB/CH3SO2NH2, K2CO3/K3[Fe(CN)6], OsO4/(CH3)3COH/H2O, r.t, 1.5 h 23a/23b (1.0:0.7), 95% yield; (b) C6H5COCl/DMAP/CH2Cl2/py, r.t, 36 h, 18 (11.3% yield), 18/19 (1.0:0.44, 78.2% yield), 20 (9.1% yield).
Ijms 22 04808 sch001
Ijms 22 04808 g003 550
Figure 3. Amplification zone 0.5–2.2 ppm of 1H NMR of diasteroisomeric mixture 23a/23b.
Figure 3. Amplification zone 0.5–2.2 ppm of 1H NMR of diasteroisomeric mixture 23a/23b.
Ijms 22 04808 g003
Ijms 22 04808 g004 550
Figure 4. Amplification of 1H NMR spectrum of mixture 18/19 in the zone 3.95–5.20 ppm.
Figure 4. Amplification of 1H NMR spectrum of mixture 18/19 in the zone 3.95–5.20 ppm.
Ijms 22 04808 g004
Ijms 22 04808 g005 550
Figure 5. Amplification zone 8.15–4.45 ppm of 1H NMR for dibenzoylated derivative 20 and the main heteronuclear correlations 2D HMBC to 2JCH (blue) and 3JCH (red) observed for 20, which confirm the positions of both benzoate groups in C22 and C23 of the side chain. HAr-2′; HAr-2″, HAr-4′, and HAr-4″; HAr-3′ and HAr-3″ are hydrogen atoms in the aromatic rings
Figure 5. Amplification zone 8.15–4.45 ppm of 1H NMR for dibenzoylated derivative 20 and the main heteronuclear correlations 2D HMBC to 2JCH (blue) and 3JCH (red) observed for 20, which confirm the positions of both benzoate groups in C22 and C23 of the side chain. HAr-2′; HAr-2″, HAr-4′, and HAr-4″; HAr-3′ and HAr-3″ are hydrogen atoms in the aromatic rings
Ijms 22 04808 g005
Table
Table 1. Comparison between signals of 1H (400.1 MHz, CDCl3) and 13C (100.6 MHz, CDCl3) NMR for H/C21, H/C22 and H/C23a-b, for the diasteroisomers 23a and 23b.
Table 1. Comparison between signals of 1H (400.1 MHz, CDCl3) and 13C (100.6 MHz, CDCl3) NMR for H/C21, H/C22 and H/C23a-b, for the diasteroisomers 23a and 23b.
H/C SignalCompound 23aCompound 23b
H-210.94 ppm (d, J = 6.8 Hz)0.90 (d, J = 5.7 Hz)
H-223.81–3.79 ppm (m)3.81–3.79 ppm (m)
H-23a3.63 (dd, J = 9.9 and 9.9 Hz)3.63 (dd, J = 9.9 and 9.9 Hz)
H-23b3.51 (m)3.51 (m)
C2112.62 ppm13.01 ppm
C2274.11 ppm73.88 ppm
C2362.47 ppm66.04 ppm
Table
Table 2. Comparison between signals of 1H (400.1 MHz, CDCl3) and 13C (100.6 MHz, CDCl3) NMR for H/C21, H/C22 and H/C23a-b, for the diasteroisomers 18 and 19.
Table 2. Comparison between signals of 1H (400.1 MHz, CDCl3) and 13C (100.6 MHz, CDCl3) NMR for H/C21, H/C22 and H/C23a-b, for the diasteroisomers 18 and 19.
H/C SignalCompound 18Compound 19
H-211.04 ppm (d, J = 6.8 Hz)1.00 (d, J = 6.4 Hz)
H-224.07-4.04 ppm (m)4.07–4.04 ppm (m)
H-23a4.48 (dd, J = 11.4 and 2.0 Hz)4.39 (dd, J = 11.4 and 8.4 Hz)
H-23b4.21 (dd, J = 11.4 and 10.2 Hz)4.26 (dd, J = 11.4 and 3.3 Hz)
C2112.90 ppm12.39 ppm
C2271.79 ppm71.86 ppm
C2368.95 ppm68.88 ppm
Table
Table 3. Effect of compound 18, epimeric mixture 18/19 and compound 20 on lamina inclination of rice seedlings.
Table 3. Effect of compound 18, epimeric mixture 18/19 and compound 20 on lamina inclination of rice seedlings.
Bending Angle between Lamina and Sheaths
(Degrees ± Standard Error)
CompoundStructure Concentration [M]
1 × 10−81 × 10−71 × 10−6
18 Ijms 22 04808 i00120 ± 1.048 ± 2.949 ± 2.5
18/19
(1.0:0.44)		 Ijms 22 04808 i00268 ± 2.550 ± 2.546 ± 2.9
20 Ijms 22 04808 i0036 ± 2.525 ± 1.023 ± 2.1
1 (C+) * Ijms 22 04808 i00431 ± 1.141 ± 4.570 ± 7.6
Water (C−) 7 ± 4.5
* 1 was used as positive control (C+).
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Soto, N.; Ferrer, K.; Díaz, K.; González, C.; Taborga, L.; Olea, A.F.; Carrasco, H.; Espinoza, L. Synthesis and Biological Activity of New Brassinosteroid Analogs of Type 24-Nor-5β-Cholane and 23-Benzoate Function in the Side Chain. Int. J. Mol. Sci. 2021, 22, 4808. https://doi.org/10.3390/ijms22094808

AMA Style

Soto N, Ferrer K, Díaz K, González C, Taborga L, Olea AF, Carrasco H, Espinoza L. Synthesis and Biological Activity of New Brassinosteroid Analogs of Type 24-Nor-5β-Cholane and 23-Benzoate Function in the Side Chain. International Journal of Molecular Sciences. 2021; 22(9):4808. https://doi.org/10.3390/ijms22094808

Chicago/Turabian Style

Soto, Nitza, Karoll Ferrer, Katy Díaz, César González, Lautaro Taborga, Andrés F. Olea, Héctor Carrasco, and Luis Espinoza. 2021. "Synthesis and Biological Activity of New Brassinosteroid Analogs of Type 24-Nor-5β-Cholane and 23-Benzoate Function in the Side Chain" International Journal of Molecular Sciences 22, no. 9: 4808. https://doi.org/10.3390/ijms22094808

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