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Article

Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5α-Cholane Type Analogs

by
Jocelyn Oyarce
1,
Vanessa Aitken
1,
César González
1,
Karoll Ferrer
1,
Andrés F. Olea
2,
Teodor Parella
3 and
Luis Espinoza Catalán
1,*
1
Departamento de Química, Universidad Técnica Federico Santa María, Avenida España 1680, Valparaíso 224000, Chile
2
Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, El Llano Subercaseaux 2801, Santiago 8900000, Chile
3
Teodor Parella, Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Catalonia, Spain
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(24), 4612; https://doi.org/10.3390/molecules24244612
Submission received: 22 November 2019 / Revised: 12 December 2019 / Accepted: 13 December 2019 / Published: 17 December 2019
(This article belongs to the Section Organic Chemistry)
Browse Figures
Versions Notes

Abstract

:
Natural brassinosteroids possess a 22R, 23R configuration that appears essential for biological activity. It is, therefore, interesting to elucidate if the activity of brassinosteroids with a short side chain depends on the C22 configuration. Herein, we describe the synthesis of new brassinosteroids analogs with 24-norcholane type of side chain and R configuration at C22. The initial reaction is the dihydroxylation of a terminal olefin that leads to S/R epimers. Three different methods were tested in order to evaluate the obtained S/R ratio and the reaction yields. The results indicate that Upjohn dihydroxylation is the most selective reaction giving a 1.0:0.24 S/R ratio, whereas a Sharpless reaction leads to a mixture of 1.0:0.90 S/R with 95% yield. Using the latter mixture and following a previous reported method, benzoylated derivatives and both S and R brassinosteroids analogs were synthesized. All synthesized compounds were completely characterized by NMR spectroscopy, and HRMS of new compounds are also given. In conclusion, a synthetic route for preparation of new analogs of brassinosteroids of 24-norcholane type and R configuration at C22 were described. It is expected that this will help to elucidate if a configuration at C22 is a structural requirement for hormonal growth activity in plants.

1. Introduction

Since the discovery of brassinolide (1) [1] (Figure 1) a large number of similar compounds have been identified in many plant species [2,3]. This family of polyhydroxylated sterol derivatives, named brassinosteroids (BRs), amounts to almost 80 different molecules which have been recently recognized as the sixth class of plant hormones [4,5]. They act as regulators of plant growth in very low amounts [1,2,3], participating in processes of cell expansion, division, and differentiation in young tissues of growing plants [6,7]. Additionally, they can protect plants from environmental stresses caused by salt, drought, and heavy metals [8,9].
The extremely low amount of BRs present in plants has prompted the chemical synthesis of BRs and 137 analogs have already been reported [4]. Structural variations of BRs arise from the type and position of functions in the A, B rings, fusion A/B ring, and alkyl side chain. One of the main synthetic difficulties is to get the alkyl side chain with the specific configuration found in natural BRs. According to the alkyl substitution in C24, BRs have been classified as C27, C28, and C29, and all natural bioactive BRs possess a vicinal 22R, 23R diol structural functionality that appears essential for high biological activity. It has been suggested that BRs may be derived from sterols with the same side chain, but most of the explored synthetic pathways require several steps with low yields. In recent decades, much effort has been dedicated to the synthesis of new BRs analogs in which common patterns of organic functions in the A/B rings and cis–trans fusion between them are kept intact, but with important structural changes in the side chain. For example, BRs analogs with shorter side chains [10,11,12,13], spirostanic and furostanic [14,15,16,17,18,19,20,21], aromatic and cyclic substituents [22,23,24,25,26,27], esters and carboxylic acids [24,28,29,30] have been reported. Despite these changes, these analogs have presented very important biological activity. Consequently, the structural requirements that these compounds should possess to elicit biological activity have changed and new SAR has been proposed [31,32]. Briefly, these results suggest that activity of BRs analogs depends on the oxygen atoms spatial distribution instead of the presence or absence of one specific functional group in the molecule [33,34]. These spatial orientations can be indicated as distances or angles between the oxygen atoms present in a brassinosteroid.
Following this line, we have previously reported the synthesis of BRs analogs with short side chain and structure (22S)-3α,22,23-trihydroxy-24-nor-5α-cholane (compounds 2–4, Figure 1) and a benzoylated derivative 5 [35]. However, to evaluate the effect of a C22 configuration on biological activity it is necessary to obtain the 22R epimers of these analogs. Herein, we report on the synthesis of a new BRs analog and benzoylated derivatives with R configuration at C22. As the starting reaction is the dihydroxylation of a terminal olefin, synthesized from hyodeoxycholic acid, we have also evaluated the ratio of 22S/22R epimers obtained through three different dihydroxylation reactions. Yields of both possible configurations are compared for all proposed synthetic reactions.

2. Results and Discussion

The most direct synthetic route to BRs analogs with short side alkyl chains carrying a vicinal diol function is the dihydroxylation of terminal olefins. This transformation can be accomplished by either of three different methods, namely: Direct Upjohn dihydroxylation; Epoxidation and subsequent opening of epoxide ring in acid medium; and Sharpless asymmetric dihydroxylation (Scheme 1). Below are described the results obtained by the application of these synthetic paths to dihydroxylation of olefin 9, which was previously synthesized from hyodeoxycholic acid [35]. All tested methods lead to epimeric mixtures of glycols 22S/22R (10a/10b) but in different ratios. In the following, a detailed description of reaction conditions and obtained yields are given for each method.

2.1. Direct Upjohn Dihydroxylation

Upjohn dihydroxylation is a well-known reaction that converts olefins to cis vicinal diol using N-methylmorpholine N-oxide (NMO) and osmium tetroxide. This reaction has been previously used to obtain glycols C22/C23 for 24-norcholane type steroids of similar structure. These studies have shown that the reaction product is a mixture of diastereomers with the 22S epimer as the major product [36,37,38,39]. Alkene 9 was hydroxylated by the Upjohn method and a mixture of epimeric glycols 10a/10b was obtained with a 72% yield (Scheme 1).
The diastereomeric ratio of each glycol in the mixture can be established by integration of 1H-NMR signals assigned to the C21 methyl group, which appear at δH = 0.947 and 0.911 ppm in 10a and 10b epimers, respectively (Figure 2) [35].
Based on these NMR measurements the ratio of 10a:10b obtained by this method was determined as 1.0:0.24.

2.2. Epoxidation and Subsequent Opening of Epoxide Ring in Acid Medium

It has been reported that the epoxidation reaction on steroidal nuclei of similar structure and carrying terminal double bonds C22/C23, mainly produces epoxide 22R [36,37,38]. Thus, epoxidation of alkene 9 with m-CPBA leads to a mixture of epimeric epoxides 11a/11b with 55.7% yield (Scheme 1). The ratio in which these epoxides were obtained was 11a:11b = 0.44:1.0, i.e., the amount of epoxide 22R is twofold larger than that of 22S. This proportion was calculated using the integrals of signals assigned to hydrogens H-22 (δ = 2.73 ppm), H-23a (δ = 2.59 ppm) of major epoxide (compound 11b), and H-23a (δ = 2.64 ppm), H-23b (δ = 2.40 ppm) of the minor epoxide (compound 11a) (see Figure S7, Supplementary Materials). For the determination of the majority epoxide, experimental and NMR spectroscopic data correlations were used [36,37,38]. Namely, it has been shown that comparative thin layer chromatography allows differentiating by polarity both epoxides formed in the epoxidation reaction of steroidal alkenes with 24-norchol-22-ene structures, and the results indicate that less polar epoxide corresponds to 22S configuration [36]. Our results show clearly that the more polar epoxide is preferentially obtained, i.e., epoxide 11b with 22R configuration. Additionally, 1D and 2D NMR spectroscopy, particularly, 2D HSQC with edited phase (2D Edit HSQC), and 2D HMBC spectra, provided essential information to carry out assignments of H-21, H-22, H23a, H23b signals and all 13C signals present in the epoxide mixture 11a/11b.
Subsequently, treatment of epoxides mixture 11a/11b in acidic conditions using the p-TsOH/THF/H2O system (Scheme 1) produces 10a/10b mixture with 75.8% yield [37,38]. Analysis of 1H-NMR spectrum indicates that the major component on this mixture is the less polar glycol 22R in the ratio 10a:10b = 1.0:1.14 (Figure 2). This result indicates that the opening step favors the formation of diastereomeric ratio, changing the proportion of this epimer from 0.44 to 1.0.

2.3. Sharpless Asymmetric Dihydroxylation

Sharpless dihydroxylation reaction has not been used to obtain glycols C22/C23 in steroids with the shortest side chain of 24-nor-5α-cholane type. Thus, olefin 9 was dihydroxylated by using dihydroquinidine p-chlorobenzoate (DHQD-CLB) as a chiral ligand (Scheme 1), following the method described in reference [23]. The reaction product (95% yield) is a mixture of epimeric glycols 10a and 10b in the proportion 1.0:0.9, respectively (Figure 2).
In summary, none of the three tested methods led exclusively to one of the two epimeric glycols. The selectivity follows the order: Upjohn reaction produces mainly the glycol 22S in the ratio 1.0 to 0.24; a slightly reverse proportion is obtained by opening of epoxides, i.e., 1.0 to 1.14, whereas the Sharpless dihydroxylation reaction produced both glycols in similar amounts (1.0 to 0.9). The total yield of glycols mixture is also dependent of the dihydroxylation reaction: 95% for the Sharpless method, 72% for Upjohn, and 43% for expoxidation followed by opening of epoxide ring. Thus, a selection of one of these methods should depend on the expected outcome for hydroxylation reaction.

2.4. Synthesis of Benzoylated Derivatives

As the main goal of this research is to obtain derivatives with 22R configuration, we used the Sharpless dihydroxylation reaction to convert the olefin 9 in the mixture of epimeric glycols 10a/10b. This reaction was selected because the mixture product is obtained with the highest total reaction yield (95%) and glycols 10a/10b are found in similar proportion. The 22S benzoylated derivative (5) has been previously synthesized by reaction of pure compound 10a with PhCOCl/DMAP [35]. Thus, to get compound 7 the glycols mixture 10a/10b obtained by the Sharpless method was submitted to the same benzoylation reaction to obtain both epimers 5 and 7. Interestingly, benzoylation of this mixture leads to a complex mixture of mono and dibenzoylated derivatives, i.e., compounds 5, 7, 8, and 12 (Scheme 2). Compounds 5, 7, and 8 were purified by C.C. and recrystallized, giving yields of 4.0%, 1.2%, and 5.7%, respectively, whereas dibenzoylated derivative 12 could not be isolated in pure form. Additionally, mixtures of monobenzoylated epimers were also obtained, 5/7 with 5.0% yield (5:7 = 0.7:1.0), and 8/12 with 79.9% yield (8:12 = 0.4:1.0). The proportion of dibenzoylated epimers in both mixtures was determined based on the integration signals at δH = 1.22 and 1.16 ppm, assigned to the H-21 of methyl group for 8 and 12, respectively.

Structural Determination of Derivatives 5, 7, and 8

The chemical structure of 5 has been previously determined by 1D and 2D NMR spectroscopy, whereas its absolute configuration 22S was established by using crystal X-ray diffraction techniques [35]. Thus, the structure and stereochemistry at C22 of compound 7 were established by a simple comparison of 1H and 13C-NMR spectra obtained for derivatives 5 and 7. These comparisons considered chemical shifts (δ), coupling constants (J), and multiplicities of signals corresponding to H-22, H-23a, H-23b, and CH3-21. 1H-NMR spectra of both epimers are shown in Figure 3, and these signals are identified.
The main differences in these spectroscopic parameters are listed in Table 1, and the more significant variations in chemical shifts and J values correspond to hydrogen atoms H-23a and CH3-21. On the other hand, differences in chemical shifts were also observed in the 13C-NMR spectra, for signals of C21, C22, and C23. For the latter, the signals appear at δC = 66.39 and 68.84 ppm for 5 and 7, respectively (Table 1). Based on this data the stereochemistry in epimer 7 was assigned as 22R.
The chemical structure of the dibenzoylated derivative 8 was established using 1H, 13C, 2D HSQC, and 2D HMBC NMR techniques. The presence of the dibenzoylated function was confirmed in the 1H-NMR spectrum, by signals observed at δH = 8.06 (2H, dd, J = 8.5 and 1.4 Hz); 7.97 (2H, dd, J = 8.5 and 1.4 Hz); 7.55–7.50 (2H, m); 7.44 (2H, dd, J = 7.9 and 7.9 Hz); 7.38 (2H, dd, J = 7.9 and 7.4 Hz), which are assigned to protons HAr-2″; HAr-2′, HAr-4″, and HAr-4′; HAr-3″ and HAr-5″; HAr-3′ and HAr-5′ in the aromatic group, respectively. In addition, in the 13C-NMR spectrum appear signals at 130.33; 133.06; 129.60; 128.35; 129.74; 133.00; 129.63, and 128.38 ppm, which were assigned to carbon C1′-Ar; C4′-Ar; C2′-Ar and C6′-Ar; C3′-Ar and C5′-Ar; C1″-Ar; C4″-Ar; C2″-Ar and C6″-Ar; C3″-Ar and C5″-Ar, respectively. These assignments were confirmed by 2D HSQC and HMBC spectra. 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 4.
To determine the stereochemistry at carbon C22 of compound 8, a complete benzoylation reaction with PhCOCl/DMAP was carried out for the monobenzoylated derivative 5. In this way, the dibenzoylated derivative was obtained with 95% yield. The spectroscopic data of 1H and 13C-NMR were totally consistent with those obtained for derivative 8 obtained by benzoylation of the glycol mixture 10a/10b.

2.5. Synthesis of BRs Analogs

Synthesis of BRs analogs 4 and 6 was carried out by saponification reaction of the monobenzoylated derivatives 5 and 7, respectively, with 90% and 82.5% yield, respectively (Scheme 3).
The spectroscopic data of 1H and 13C-NMR obtained for compound 4 were consistent with those previously reported [35,40]. The structural and stereochemical determination at C22 for triol 6 was established by comparison of NMR data between epimers 4 and 6. These comparisons considered chemical shifts (δ), coupling constants (J), and multiplicities of signals corresponding to H-21, H-22, H-23a, and H-23b. 1H-NMR spectra of both epimers are shown in Figure 5, where these signals are identified.
The main differences observed among the indicated hydrogens are listed in Table 2. It is seen that hydrogen atoms H-21, H-23a, and H-23b experience variations in chemical shifts and J values, whereas H-22 shows also changes in multiplicity. On the other hand, in 13C-NMR spectra differences in chemical shifts for signals assigned to C21, C22, and C23, but mainly in carbon C21 and C23, are observed (Table 2). From this data the stereochemistry in C22 can be assigned as R for the epimer 6.

3. Materials and Methods

3.1. General Experimental Methods

All reagents were purchased from commercial suppliers and used without further purification. Melting points were measured on a SMP3 apparatus (Stuart-Scientific, now Merck KGaA, Darmstadt, Germany) and are uncorrected. 1H, 13C, 13C DEPT-135, gs 2D HSQC, and gs 2D HMBC NMR spectra were recorded in CDCl3 or MeOD solutions, and are referenced to the residual peaks of CHCl3 at δ = 7.26 ppm and δ = 77.00 ppm for 1H and 13C, respectively, and CD3OD at δ = 3.30 ppm and δ = 49.00 ppm for 1H and 13C, respectively, on an Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for 1H and 100.6 MHz for 13C, and Avance 600 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 600.13 MHz for 1H and 150.90 for 13C. Chemical shifts are reported in ppm and coupling constants (J) are given in Hz, multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet (t), quartet (q), multiplet (m), broad singlet (bs). IR spectra were recorded as KBr disks in a 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 an Exactive Plus mass spectrometer (Thermo Scientific, Waltham, MA, USA). The analysis for the reaction products was performed with the following relevant parameters: heater temperature, 100 °C; sheath gas flow, 5 (arbitrary unit); sweep gas flow rate, 0 (arbitrary unit); and spray voltage, 3.0 kV at negative or positive mode. The accurate mass measurements were performed at a resolving power: 140,000 FWHM at range m/z 300–700. For analytical TLC, silica gel 60 in 0.25 mm layer was used and TLC spots were detected by heating after spraying with 25% H2SO4 in H2O. Chromatographic separations were carried out by 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.

3.2. Synthesis

3.2.1. Upjohn Dihydroxylation of Alkene 9. Obtaining (22S)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10a) and (22R)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10b)

To a solution of alkene 9 (2.50 g, 6.47 mmol) in acetone (150 mL) NMO (0.45 g, 3.84 mmol) was added. After mixture homogenization by magnetic stirring, OsO4 (210 mmol) was added dropwise with stirring and reacted for 36 h at room temperature. The end of the reaction was verified by TLC. Then the solvent was removed (up to 25 mL approximate volume) and water (25 mL) and Na2S2O3.5H2O (25 mL saturated solution) were added. The organic layer was extracted with EtOAc (2 × 30 mL), washed with water (2 × 50 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (10 mL) and chromatographed on silica gel with hexane/EtOAc mixtures of increasing polarity (19.8:0.2 → 9.8:10.2). A mixture of 10a/10b = 1.0/0.24 was obtained (1.97 g, 72% yield).

3.2.2. Epoxidation of Alkene 9. Obtaining (22S)-6-oxo-22,23-epoxy-24-nor-5α-cholan-3α-yl acetate and (11a) and (22R)-6-oxo-22,23-epoxy-24-nor-5α-cholan-3α-yl acetate (11b)

To a solution of alkene 9 (4.0 g, 10.36 mmol) in CH2Cl2 (100 mL) NaHCO3 (1.13 g, 13.45 mmol) and mCPBA (77%, 2.32 g, 13.45 mmol) were added. Then the mixture was stirred for 48 h and the end of the reaction was verified by TLC. Later, the reaction mixture was filtered to eliminate the m-chlorobenzoic acid formed and the liquid was evaporated under reduced pressure. The residue was dissolved in AcOEt (30 mL) and the solution was washed with a saturated solution of NaHCO3 (2 × 10 mL), then with water (3 × 10 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (10 mL) and chromatographed on silica gel with hexane/EtOAc mixtures of increasing polarity (19.8:0.2 → 12.8:7.2). A mixture of 11a/11b = 0.44/1.0 was obtained (2.32 g, 55.7% yield).
Compound 11a: 1H-NMR (600 MHz, CDCl3) δ (ppm): 5.13–5.11 (1H, m, H-3), 2.64 (1H, dd, J = 5.1 and 4.1 Hz, H-23β), 2.63 (1H, ddd, J = 6.9, 4.1, and 2.8 Hz, H-22), 2.40 (1H, dd, J = 5.1 and 2.8 Hz, H-23α), 2.56 (1H, dd, J = 12.7 and 2.1 Hz, H-5), 2.33 (1H, dd, J = 13.2 and 4.6 Hz, H-7α), 2.03 (3H, s, CH3CO), 0.970 (3H, d, J = 6.8 Hz, H-21), 0.744 (3H, s, H-19), 0.667 (3H, s, H-18). 13C-NMR (150 MHz, CDCl3) see Table S1.
Compound 11b: 1H-NMR (600 MHz, CDCl3) δ (ppm): 5.13–5.11(1H, m, H-3), 2.80 (1H, dd, J = 4.8 and 3.8 Hz, H-23β), 2.73 (1H, ddd, J = 6.5, 3.8, and 2.5 Hz, H-22), 2.59 (1H, dd, J = 4.8 and 2.7 Hz, H-23α), 2.56 (1H, dd, J = 12.7 and 2.1 Hz, H-5), 2.32 (1H, dd, J = 13.3 and 4.6 Hz, H-7α), 2.03 (3H, s, CH3CO), 1.11 (3H, d, J = 5.6 Hz, H-21), 0.744 (3H, s, H-19), 0.659 (3H, s, H-18). 13C-NMR (150 MHz, CDCl3) see Table S1.

3.2.3. Opening of Epoxide Ring of Mixture 11a/11b in Acid Medium. Obtaining (22S)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10a) and (22R)-22,23-dihydroxy-6-oxo-24-nor-5 α-cholan-3 α-yl acetate (10b)

To a solution of mixture 11a/11b (2.28 g, 5.66 mmol) in THF (60 mL) and H2O (15 mL), p-TsOH (0.19 g, 1.10 mmol) was added. The mixture was stirred for 3 h and the end of the reaction was verified by TLC. Then the solvent was evaporated under reduced pressure and removed (15 mL approximate volume). The residue was dissolved in AcOEt (50 mL) and the solution was washed with a saturated solution of NaHCO3 (3 × 10 mL), then with water (3 × 10 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure and the solid residue was crystallized from a 3:1 of Et2O/MeOH mixture. A mixture of 10a/10b = 1.0:1.14 was obtained (1.80 g, 75.8% yield).

3.2.4. Direct Asymmetric Sharpless Dihydroxylation of Alkene 9. Obtaining (22S)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10a) and (22R)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10b)

To a mixture of t-butanol/water (40 mL, 1:1 v/v) alkene 9 (500 mg, 1.29 mmol), DHQD-CLB (157 mg, 0.331 mmol), CH3SO2NH2 (228 mg, 2.33 mmol), K2CO3 (993 mg, 7.11 mmol), and K3[Fe(CN)6] (2.494 g, 7.50 mmol) were added, then the mixture was homogenized by magnetic stirring for 5 min. Later, 700 μL of OsO4 solution (1.0 g, 3.933 mmol in 20 mL of t-butanol) were added, and the mixture reaction was stirred at room temperature for 6 h. The end of the reaction was verified by TLC, then H2O (10 mL) and a saturated solution of Na2S2O3·5H2O (10 mL) were added. The reaction mixture was extracted with AcOEt (2 × 30 mL), both organic phases were combined, dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (10 mL) and chromatographed on silica gel with hexane/EtOAc mixtures of increasing polarity (19.8:0.2 → 9.8:10.2). A mixture of 10a/10b = 1.0/0.9 was obtained (515 mg, 95% yield).

3.2.5. Synthesis of (22S)-22-hydroxy-6-oxo-24-nor-5α-cholan-3α,23-diyl 3-acetate 23-benzoate (5), (22R)-22-hydroxy-6-oxo-24-nor-5α-cholan-3α,23-diyl 3-acetate 23-benzoate (7) and (22S)-6-oxo-24-nor-5α-cholan-3α,22,23-triyl 3-acetate 22,23-dibenzoate (8)

To a solution of mixture 10a/10b (3.702 g, 8.216 mmol) in CH2Cl2 (75 mL), DMAP (20 mg, 0.819 mmol) and pyridine (5.0 mL, d = 0.981 g/mL) were added. The mixture was cooled to 0–5 °C under nitrogen atmosphere.
PhCOCl 1.10 mL (9.47 mmol, d = 1.21 g/mL) was added by slow drip during 1 h, then the reaction mixture was maintained at room temperature for 4 h. The end of the reaction was verified by TLC, the solvent was evaporated under reduced pressure until concentrated to a volume of 10 mL. Subsequently, the mixture was diluted with EtOAc (40 mL). The organic layer was washed with a saturated solution of KHSO4 (2 × 10 mL), H2O (3 × 30 mL), dried over MgSO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (10 mL) and chromatographed on silica gel with hexane/EtOAc mixtures of increasing polarity (19.8:0.2 → 9.8:10.2), a mixture of 4.966 g corresponding to mono and dibenzoylated compounds was obtained. The chromatographic separation was repeated three times, and five fractions were obtained in the following order of polarity. Fraction I: compound 8 (283 mg, 5.7% yield); Fraction II: mixture of compounds 8 and 12 (3.960 g, 8:12 = 0.4:1.0, 79.9% yield); Fraction III: mixture of compounds 5 and 7 (248 mg, 5:7 = 0.7:1.0, 5.0% yield); Fraction IV: compound 7 (59 mg, 1.2% yield); Fraction V: compound 5 (198 mg, 4% yield).
Compound 5: Physical properties (melting point) and spectroscopic data (IR, 1H, 13C-NMR) were consistent with those previously reported [35].
Compound 7: colorless solid, m.p. 174.6 ± 1 °C (Hexane/Et2O), IRνmax (cm−1): 3509 (O–H), 2948–2869 (C–H), 1731 (C=O), 1718 (C=O), 1703 (C=O), 1603 (C=C Ar), 1580 (C=C Ar), 1451 and 1379 (C–H), 1277 (C–O), 1157 (C–O), 1088 (C–O), 712 (CH-Ar). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.02 (2H, d, J = 7.4 Hz, HAr-2′), 7.54 (1H, t, J = 7.4 Hz, HAr-4′), 7.41 (1H, t, J = 7.8 Hz, HAr-3′), 5.08 (1H, m, H-3), 4.36 (1H, dd, J = 11.2 and 8.4 Hz, H-23a), 4.23 (1H, dd, J = 11.2 and 3.6 Hz, H-23b), 4.04 (1H, dd, J = 8.4 and 3.4 Hz, H-22), 2.54 (1H, dd, J = 11.7 and 2.9 Hz, H-5), 2.28 (1H, dd, J = 13.4 and 4.6 Hz, H-7β), 2.01 (3H, s, CH3CO), 0.990 (3H, d, J = 6.1 Hz, H-21), 0.712 (3H, s, H-19), 0.668 (3H, s, H-18). 13C-NMR see Table S1. HRMS-ESI (positive mode): m/z calculated for C32H44O6: 524.3138 [M]+, found 525.3204 [M + 1]+.
Compound 8: colorless solid, m.p. 205.9 ± 2 °C (Hexane/Et2O), IRνmax (cm−1): 2943–2867, (C–H), 1736 (C=O), 1717 (C=O), 1710 (C=O), 1602 (C=C Ar), 1380 (CH3), 1284 (C–O), 1112 (C–O), 1070 (C–O), 713 (CH-Ar). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.06 (2H, dd, J = 8.5 and 1.4 Hz, HAr-2″), 7.97 (2H, dd, J = 8.5 and 1.4 Hz, HAr-2′), 7.55–7.50 (2H, m, HAr-4″ and HAr-4′), 7.44 (2H, dd, J = 7.9 and 7.9 Hz, HAr-3″ and HAr-5″), 7.38 (2H, dd, J = 7.9 and 7.4 Hz, HAr-3′ and HAr-5′), 5.56 (1H, dt, J = 9.4 and 3.1 Hz, H-22), 5.12 (1H, bt, J = 2.6 Hz, H-3), 4.66 (1H, dd, J = 12.0 and 2.3 Hz, H-23a), 4.49 (1H, dd, J = 12.0 and 9.4 Hz, H-23b), 2.57 (1H, dd, J = 12.5 and 3.2 Hz, H-5), 2.34 (1H, dd, J = 13.0 and 4.6 Hz, H-7α); 2.05 (3H, s, CH3CO), 1.16 (3H, d, J = 7.0 Hz, H-21), 0.748 (3H, s, H-19), 0.710 (3H, s, H-18). 13C-NMR see Table S1. HRMS-ESI (positive mode): m/z calculated for C39H48O7: 628.3400 [M]+, found 629.3461 [M + 1]+.

3.2.6. Synthesis of (22S)-24-nor-5α-cholan-6-oxo-3α,22,23-triyl 3-acetate 22,23-dibenzoate (8) from 5

To obtain compound 8 from 5 the same procedure described above was followed, with 5 (100 mg, 0.191 mmol) in CH2Cl2 (20 mL), pyridine (1.0 mL), DMAP (0.5 mg), PhCOCl (0.5 mL, 4.40 mmol). Compound 8 (114 mg, 95% yield). The spectroscopic data of 1H and 13C-NMR were consistent with those described above.

3.2.7. Synthesis of (22S)-3α,22,23-trihydroxy-24-nor-5α-cholan-6-one (4) from 5

To a solution of 5 (50 mg, 2.46 mmol) in MeOH (30 mL) K2CO3 (10 mL, 15% w/w) was added. Then the suspension was stirred at reflux for 1.5 h. The end of the reaction was verified by TLC. Then the solvent was removed to dry and the residue acidified with 2.5% w/w HCl (5 mL). The obtained solid was filtered and washed with 5% NaHCO3 (10 mL) and water (2 × 10 mL) and dried. Compound 4 (32.5 mg, 90% yield). The physical properties (melting point) and spectroscopic IR, 1H, 13C-NMR were consistent with those previously reported [35,40].

3.2.8. Synthesis of (22R)-3α,22,23-trihydroxy-24-nor-5α-cholan-6-one (6) from 7

To obtain compound 6 from 7 the same procedure described above was followed, with 6 (87 mg, 0.17 mmol) in MeOH (30 mL), K2CO3 (15 mL, 15% w/w), reflux 1.5 h. Compound 6 (52 mg, 82.5% yield).
Compound 6: colorless solid, m.p. 210.5 ± 1 °C (Et2O/MeOH), IRνmax (cm−1): 3421 (O–H), 2941–2867, (C–H), 1703 (C=O), 1461 (C–H), 1380 (CH3), 1284 (C–O), 1051 (C–O). 1H-NMR (400 MHz, MeOD) δ (ppm): 4.04 (1H, bs, H-3), 3.73 (1H, bt, J = 6.5 Hz, H-22), 3.53 (1H, dd, J = 10.9 and 7.1 Hz, H-23a), 3.46 (1H, dd, J = 10.9 and 6.5 Hz, H-23b), 2.78 (1H, t, J = 7.9 Hz, H-5), 2.24 (1H, dd, J = 13.0 and 4.7 Hz, H-7α), 2.13 (1H, bt, J = 13.0 Hz, H-7β), 0.925 (3H, d, J = 6.3 Hz, H-21), 0.762 (3H, s, H-19), 0.753 (3H, s, H-18). 13C-NMR see Table S1. HRMS-ESI (negative mode): m/z calculated for: C23H38O4: 378.2770 [M]+; found 377.2702 [M − 1]+.

4. Conclusions

In this study, BRs analogs and benzoylated derivatives with R stereochemistry at C22 were synthesized. The first synthetic step involves the transformation of the terminal olefin 9 to vicinal diol function. This reaction was carried out by three different dihydroxylation methods, and the yields and mixture composition of glycol mixtures with configuration R/S in C22 were determined. The results indicate that all tested methods lead to mixtures of both epimeric glycols. The selectivity is highest for the Upjohn reaction, but the total yield of glycols mixture is largest for the Sharpless method, 95%. Thus, a selection of one of these methods should depend of the expected outcome for hydroxylation reaction. The benzoylation reaction of the mixture of glycols obtained by Sharpless dihydroxylation produced a complex mixture of mono- and dibenzoylated derivatives, where the known analogue 5 and the new analogs 7 and 8 were separated and purified. Additionally, the new BRs analog 6 was obtained by saponification of derivative 7. All chemical structures of the compounds synthesized in this research were fully characterized by 1D and 2D NMR experiments. Finally, we hope that these new analogs of brassinosteroids with short lateral side chain of 24-nor-5α-cholane type, and oxygenated functions in the form of diols in C22S/R and C23 benzoates, will allow to elucidate if a configuration at C22 is a structural requirement for hormonal growth activity in plants.

Supplementary Materials

The following are available online, Figure S1: NMR spectra of (22R)-3α,22,23-trihydroxy-24-nor-5α-cholan-6-one (6), Figure S2: NMR spectra (22R)-22-hydroxy-6-oxo-24-nor-5α-cholan-3α,23-diyl 3-acetate 23-benzoate (7), Figure S3: NMR spectra of (22S)-6-oxo-24-nor-5α-cholan-3α,22,23-triyl 3-acetate 22,23-dibenzoate (8), Figure S4: 1H NMR spectrum of (22S)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10a) and (22R)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10b) mixture obtained by Upjohn dihydroxylation, Figure S5: 1H NMR spectrum of (22S)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10a) and (22R)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10b) mixture obtained by opening of epoxide ring mixture 11a/11b, Figure S6: 1H NMR spectrum of (22S)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10a) and (22R)-22,23-dihydroxy-6-oxo-24-nor-5α-cholan-3α-yl acetate (10b) mixture obtained by Sharpless dihydroxylation, Figure S7: NMR spectra of (22S)-6-oxo-22,23-epoxy-24-nor-5α-cholan-3α-yl acetate and (11a) and (22R)-6-oxo-22,23-epoxy-24-nor-5α-cholan-3α-yl acetate (11b) mixture, Figure S8: 1H NMR spectrum of (22S)-22-hydroxy-6-oxo-24-nor-5α-cholan-3α,23-diyl 3-acetate 23-benzoate (5) and (22R)-22-hydroxy-6-oxo-24-nor-5α-cholan-3α,23-diyl 3-acetate 23-benzoate (7) mixture, Figure S9: 1H NMR spectrum of (22S)-6-oxo-24-nor-5α-cholan-3α,22,23-triyl 3-acetate 22,23-dibenzoate (8) and (22R)-6-oxo-24-nor-5α-cholan-3α,22,23-triyl 3-acetate 22,23-dibenzoate (12) mixture, Figure S10: HRMS-ESI of (22R)-3α,22,23-trihydroxy-24-nor-6-oxo-5α-cholan (6), Figure S11: HRMS-ESI of (22R)-22-hydroxy-24-nor-6-oxo-5α-cholan-3α,23-diyl 3-acetate 23-benzoate (7), Figure S12: HRMS-ESI of (22S)-6-oxo-24-nor-5α-cholan-3α,22,23-triyl 3-acetate 22,23-dibenzoate (8). Table S1: 13C NMR signals for compounds 4–8, 10a–10b, 11a and 11b.

Author Contributions

J.O., V.A., K.F., and C.G. carried out the synthesis, separation, and purification of compounds. L.E.C. Project Administration, supervised the whole research, collaborated on the synthesis, structure determination by spectroscopic methods (1D, 2D NMR, HRMS, and IR), and manuscript redaction. T.P. collaborated on the spectra recording 1D, 2D NMR of epoxide mixture 11a/11b and structural determination discussion. A.F.O. collaborated in the discussion and interpretation of the results, manuscript redaction, and corrections.

Funding

This research was funded by FONDECYT (Fondo Nacional de Desarrollo Científico y Tecnológico) (grant No. 1160446 and No. 1191330).

Acknowledgments

Jocelyn Oyarce wants to thank Dirección General de Investigación y Postgrado (DGIP-USM) of Universidad Técnica Federico Santa María. Karoll Ferrer wants to thank Dirección General de Investigación y Postgrado (DGIP-USM) and the Programa de Iniciación de Investigación Científica (PIIC) N°072/2016 of Universidad Técnica Federico Santa María for the financial support. Teodor Parella acknowledges the financial support provided by Spanish MINECO (project PGC2018-095808-B-I00).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Mitchell, J.W.; Mandava, N.; Worley, J.F.; Plimmer, J.R.; Smith, M.V. Brassins - A New Family of Plant Hormones from Rape Pollen. Nature 1970, 225, 1065–1066. [Google Scholar] [CrossRef] [PubMed]
  2. Fujioka, S. Natural Occurrence of Brassinosteroids in the Plant Kingdom. In Brassinosteroids: Steroidal Plant Hormones; Sakurai, A., Yokota, T., Clouse, S.D., Eds.; Springer-Verlag: Tokyo, Japan, 1999; pp. 21–45. [Google Scholar]
  3. Bajguz, A. Brassinosteroids – Occurence and Chemical Structures in Plants. In Brassinosteroids: A Class of Plant Hormone; Hayat, S., Ahmad, A., Eds.; Springer: Dordrecht, The Netherlands, 2016; pp. 1–27. [Google Scholar]
  4. Liu, J.; Zhang, D.; Sun, X.; Ding, T.; Lei, B.; Zhang, C. Structure-Activity Relationship of Brassinosteroids and their Agricultural Practical Usages. Steroids 2017, 124, 1–17. [Google Scholar] [CrossRef] [PubMed]
  5. Peres, A.L.G.L.; Soares, J.S.; Tavares, R.G.; Righetto, G.; Zullo, M.A.T.; Mandava, N.B.; Menossi, M. Brassinosteroids, the Sixth Class of Phytohormones: A Molecular View from the Discovery to Hormonal Interactions in Plant Development and Stress Adaptation. Int. J. Mol. Sci. 2019, 20, 331. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Oklestkova, J.; Rarova, L.; Kvasnica, M.; Strnad, M. Brassinosteroids: Synthesis and Biological Activities. Phytochem. Rev. 2015, 14, 1053–1072. [Google Scholar] [CrossRef]
  7. Clouse, S.D. A History of Brassinosteroid Research from 1970 through 2005: Thirty-Five Years of Phytochemistry, Physiology, Genes, and Mutants. J. Plant. Growth. Regul. 2015, 34, 828–844. [Google Scholar] [CrossRef]
  8. Bajguz, A.; Hayat, S. Effects of Brassinosteroids on the Plant Responses to Environmental Stresses. Plant. Physiol. Biochem. 2009, 47, 1–8. [Google Scholar] [CrossRef]
  9. Hayat, S.; Alyemeni, M.N.; Hasan, S.A. Foliar Spray of Brassinosteroid Enhances Yield and Quality of Solanum Lycopersicum under Cadmium Stress. Saudi. J. Biol. Sci. 2012, 19, 325–335. [Google Scholar] [CrossRef] [Green Version]
  10. Černý, V.; Strnad, M.; Kamínek, M. Preparation of 2α,3α-dihydroxy-7-oxa-6-oxo-23,24-dinor-B-homo-5α-Cholanic Acid, its Esters and Amides as Brassinolide Analogues. Collect. Czech. Chem. Commun. 1986, 51, 687–697. [Google Scholar] [CrossRef]
  11. Kvasnica, M.; Oklestkova, J.; Bazgier, V.; Rarova, L.; Berka, K.; Strnad, M. Biological Activities of New Monohydroxylated Brassinosteroid Analogues with a Carboxylic Group in the Side Chain. Steroids 2014, 85, 58–64. [Google Scholar] [CrossRef]
  12. Uesusuki, S.; Watanabe, B.; Yamamoto, S.; Otsuki, J.; Nakagawa, Y.; Miyagawa, H. Synthesis of Brassinosteroids of Varying Acyl Side Chains and Evaluation of Their Brassinolide-like Activity. Biosci. Biotechnol. Biochem. 2004, 68, 1097–1105. [Google Scholar] [CrossRef] [Green Version]
  13. Šíša, M.; Hniličková, J.; Swaczynová, J.; Kohout, L. Syntheses of New Androstane Brassinosteroids with 17β-Ester Groups—Butyrates, Heptafluorobutyrates, and Laurates. Steroids 2005, 70, 755–762. [Google Scholar] [CrossRef] [PubMed]
  14. Gil, R.P.; Iglesias-Arteaga, M.A.; Martinez, C.P.; Manchado, F.C.; Garcia, D.C.; Rosado, A. Synthesis of Analogues of Brassinosteroids From Chenodeoxycholic Acid. Eur. J. Org. Chem. 1998, 2405–2407. [Google Scholar]
  15. Iglesias-Arteaga, M.; Gil, R.P.; Leliebre-Lara, V.; Martinez, C.S.P.; Manchado, F. Synthesis and Biological Activity of (22R,25R)-5 alpha-furostan-2 alpha,3 alpha,26-triol. J. Chem. Res. 1996, 504–505. [Google Scholar]
  16. Iglesias-Arteaga, M.; Gil, R.; Leliebre-Lara, V.; Manchado, F.; Pérez, C.S.; Rosado, A. Synthesis of (25R)-5α-Spirostan-2α,3α,6β-triol Triacetate. Synth. Commun. 1998, 28, 75–81. [Google Scholar] [CrossRef]
  17. Iglesias-Arteaga, M.; Gil, R.P.; Leliebre-Lara, V.; Martinez, C.S.P.; Manchado, F.; Perez, A.R.; Rios, L.P. Synthesis of (22R,25R)-3 beta,26-dihydroxy-5 alpha-furostan-6-one. Synth. Commun. 1998, 28, 1381–1386. [Google Scholar] [CrossRef]
  18. Iglesias-Arteaga, M.; Gil, R.P.; Leliebre-Lara, V.; Martinez, C.S.P.; Manchado, F. Synthesis of (22R,25R)-2 alpha,3 alpha,26-trihydroxy-5 alpha-furostanaone-6-one. Synth. Commun. 1998, 28, 1779–1784. [Google Scholar] [CrossRef]
  19. Iglesias-Arteaga, M.A.; PérezGil, R.; LeliebreLara, V.; CollManchado, F.; PérezMartínez, C.S. Synthesis of (25R)-2α,3α-Epoxy-5α-Spirostan-6,23-Dione. Synth. Commun. 1998, 28, 4387–4392. [Google Scholar] [CrossRef]
  20. Iglesias-Arteaga, M.A.; Martinez, C.S.P.; Manchado, F.C. Synthesis and Characterization of (25R)-2 alpha,3 alpha-epoxy-5 alpha-spirostan-12,23-dione. Synth. Commun. 1999, 29, 1811–1818. [Google Scholar] [CrossRef]
  21. Iglesias Arteaga, M.A.; Gil, R.P.; Pérez Martínez, C.S.; Manchado, F.C. Synthetic Steroidal Sapogenins. Part III1 23-Ketohecogenin and 23-Ketoisochiapagenin. Synth. Commun. 2000, 30, 163–170. [Google Scholar] [CrossRef]
  22. Huang, L.F.; Zhou, W.S. Studies on Steroidal Plant-Growth Regulators. Part 33. Novel Method for Construction of the Side-Chain of 23-Arylbrassinosteroids Via Heck Arylation and Asymmetric Dihydroxylation As Key Steps. J. Chem. Soc. Perkin. Trans. 1 1994, 3579–3585. [Google Scholar] [CrossRef]
  23. Kvasnica, M.; Oklestkova, J.; Bazgier, V.; Rárová, L.; Korinkova, P.; Mikulík, J.; Budesinsky, M.; Béres, T.; Berka, K.; Lu, Q.; et al. Design, Synthesis and Biological Activities of New Brassinosteroid Analogues with a Phenyl Group in the Side Chain. Org. Biomol. Chem. 2016, 14, 8691–8701. [Google Scholar] [CrossRef] [PubMed]
  24. Zhou, W.S.; Tian, W.S. The Synthesis of Steroids Containing Structural Unit of A, B Ring of Brassinolide and Ecdysone from Hyodesoxycholic Acid. Acta. Chim. Sinica. 1984, 42, 1173–1177. [Google Scholar]
  25. Back, T.; Pharis, R. Structure-Activity Studies of Brassinosteroids and the Search for Novel Analogues and Mimetics with Improved Bioactivity. J. Plant. Growth. Regul. 2004, 22, 350–361. [Google Scholar] [CrossRef] [PubMed]
  26. Kovganko, N.V.; Ananich, S.K. Advances in the Chemical Synthesis of Brassinosteroids. Chem. Nat. Compd. 1997, 33, 389–416. [Google Scholar] [CrossRef]
  27. Korinkova, P.; Bazgier, V.; Oklestkova, J.; Rarova, L.; Strnad, M.; Kvasnica, M. Synthesis of Novel Aryl Brassinosteroids through Alkene Cross-Metathesis and Preliminary Biological Study. Steroids 2017, 127, 46–55. [Google Scholar] [CrossRef]
  28. Duran, M.I.; Gonzalez, C.; Acosta, A.; Olea, A.F.; Diaz, K.; Espinoza, L. Synthesis of Five Known Brassinosteroid Analogs from Hyodeoxycholic Acid and Their Activities as Plant-Growth Regulators. Int. J. Mol. Sci. 2017, 18, 516. [Google Scholar] [CrossRef] [Green Version]
  29. Zhou, W.; Jiang, B.; Shen, J. Synthesis of Cholesteric Lactones and Analogs as Plant Growth Regulators. Patent CN 1184113 A, 10 June 1998. [Google Scholar]
  30. Tian, W.S.; Zhou, W.S. Studies on Steroidal Plant Growth Hormone VI. Conversion of Methyl 3,6-Diketoallocholanate into Methyl Δ^2-6-ketoallocholenate. Acta. Chim. Sinica. 1988, 46, 824–826. [Google Scholar]
  31. Brosa, C.; Capdevila, J.M.; Zamora, I. Brassinosteroids: A New Way to Define the Structural Requirements. Tetrahedron 1996, 52, 2435–2448. [Google Scholar] [CrossRef]
  32. Brosa, C.; Soca, L.; Terricabras, E.; Ferrer, J.C.; Alsina, A. New Synthetic Brassinosteroids: A 5 alpha-hydroxy-6-ketone Analog with Strong Plant Growth Promoting Activity. Tetrahedron 1998, 54, 12337–12348. [Google Scholar] [CrossRef]
  33. Thompson, M.J.; Meudt, W.J.; Mandava, N.B.; Dutky, S.R.; Lusby, W.R.; Spaulding, D.W. Synthesis of Brassinosteroids and Relationship of Structure to Plant Growth-Promoting Effects. Steroids 1982, 39, 89–105. [Google Scholar] [CrossRef]
  34. Takatsuto, S.; Yazawa, N.; Ikekawa, N.; Takematsu, T.; Takeuchi, Y.; Koguchi, M. Structure Activity Relationship of Brassinosteroids. Phytochem 1983, 22, 2437–2441. [Google Scholar] [CrossRef]
  35. Carvajal, R.; Gonzalez, C.; Olea, A.F.; Fuentealba, M.; Espinoza, L. Synthesis of 2-Deoxybrassinosteroids Analogs with 24-nor, 22(S)-23-Dihydroxy-Type Side Chains from Hyodeoxycholic Acid. Molecules 2018, 23, 1306. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Ishiguro, M.; Kajikawa, A.; Haruyama, T.; Ogura, Y.; Okubayashi, M.; Morisaki, M.; Ikekawa, N. Synthetic studies of Withanolides. Part 1. Synthesis of 5,6beta-epoxy-4beta-hydroxy-5beta-cholest-2-en-1-one and Related Compounds. J. Chem. Soc. Perkin. Trans. 1 1975, 2295–2302. [Google Scholar] [CrossRef] [PubMed]
  37. Espinoza, L.; Cortes, M. Synthesis and biological Activities of Two New Brassinosteroids Functionalized in Ring C. Bol. Soc. Chil. Quim. 2002, 47, 335–347. [Google Scholar]
  38. Espinoza, L.; Cortes, M. Synthesis and Biological Activity of Brassinosteroids Analogues. Bol. Soc. Chil. Quim. 2002, 47, 511–516. [Google Scholar]
  39. Espinoza, L. Synthesis of Four New Brassinosteroids Analogues 11-Oxo-Functionalized on C Ring, with 24-Nor Side Chain and Containing 5â-Cholanic Acid Skeleton. Organic. Chem.Curr.Res. 2015, 4, 1–6. [Google Scholar]
  40. Yang, Y.X.; Zheng, L.T.; Shi, J.J.; Gao, B.; Chen, Y.K.; Yang, H.C.; Chen, H.L.; Li, Y.C.; Zhen, X.C. Synthesis of 5 alpha-cholestan-6-one Derivatives and their Inhibitory Activities of NO Production in Activated Microglia: Discovery of A Novel Neuroinflammation Inhibitor. Bioorg. Med. Chem. Lett. 2014, 24, 1222–1227. [Google Scholar] [CrossRef]
Sample Availability: Samples of the compounds 4–8 are available from the authors.
Molecules 24 04612 g001 550
Figure 1. Structure of brassinolide (1) and analogs 2–8 of 24-nor-5α-cholan type.
Figure 1. Structure of brassinolide (1) and analogs 2–8 of 24-nor-5α-cholan type.
Molecules 24 04612 g001
Molecules 24 04612 g002 550
Figure 2. Spectra 1H-NMR of epimeric mixture of glycols 10a:10b obtained by a) Upjohn method; b) Epoxidation and subsequent opening of epoxide ring in acid medium; c) Sharpless method.
Figure 2. Spectra 1H-NMR of epimeric mixture of glycols 10a:10b obtained by a) Upjohn method; b) Epoxidation and subsequent opening of epoxide ring in acid medium; c) Sharpless method.
Molecules 24 04612 g002
Molecules 24 04612 sch001 550
Scheme 1. Dihydroxylation of olefin 9 to get a mixture of epimeric glycols 10a/10b by using different synthetic pathways: 1) Direct Upjohn dihydroxylation; 2) Epoxidation and subsequent opening of epoxide ring in acid medium and; 3) Direct asymmetric Sharpless dihydroxylation.
Scheme 1. Dihydroxylation of olefin 9 to get a mixture of epimeric glycols 10a/10b by using different synthetic pathways: 1) Direct Upjohn dihydroxylation; 2) Epoxidation and subsequent opening of epoxide ring in acid medium and; 3) Direct asymmetric Sharpless dihydroxylation.
Molecules 24 04612 sch001
Molecules 24 04612 sch002 550
Scheme 2. Benzoylated derivatives 5, 7, 8, and 12 derivatives obtained by benzoylation reaction of glycols mixture 10a/10b.
Scheme 2. Benzoylated derivatives 5, 7, 8, and 12 derivatives obtained by benzoylation reaction of glycols mixture 10a/10b.
Molecules 24 04612 sch002
Molecules 24 04612 g003 550
Figure 3. Comparison between 1H-NMR spectra of epimers 5 and 7, and their main differences in chemical shifts for signals H-21, H-22, and H-23a–b.
Figure 3. Comparison between 1H-NMR spectra of epimers 5 and 7, and their main differences in chemical shifts for signals H-21, H-22, and H-23a–b.
Molecules 24 04612 g003
Molecules 24 04612 g004 550
Figure 4. Main heteronuclear correlations 2D HMBC to 2JCH (blue) and 3JCH (red) observed for the dibenzoylated derivative 8, which confirm the positions of both benzoate groups in C22 and C23 of the side chain.
Figure 4. Main heteronuclear correlations 2D HMBC to 2JCH (blue) and 3JCH (red) observed for the dibenzoylated derivative 8, which confirm the positions of both benzoate groups in C22 and C23 of the side chain.
Molecules 24 04612 g004
Molecules 24 04612 sch003 550
Scheme 3. Synthesis of brassinosteroids (BRs) analogs 4 and 6 by saponification reaction of the monobenzoylated derivatives 5 and 7, respectively.
Scheme 3. Synthesis of brassinosteroids (BRs) analogs 4 and 6 by saponification reaction of the monobenzoylated derivatives 5 and 7, respectively.
Molecules 24 04612 sch003
Molecules 24 04612 g005 550
Figure 5. Comparison between 1H-NMR spectra of glycol epimers 4 and 6, and their main differences in chemical shifts for signals H-21, H-22, and H-23a–b.
Figure 5. Comparison between 1H-NMR spectra of glycol epimers 4 and 6, and their main differences in chemical shifts for signals H-21, H-22, and H-23a–b.
Molecules 24 04612 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 epimers 5 and 7.
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 epimers 5 and 7.
H/C SignalCompound 5Compound 7
H-211.06 ppm (3H, d, J = 6.9 Hz)0.990 ppm (3H, d, J = 6.1 Hz)
H-224.06 ppm (1H, m)4.04 ppm (1H, dd, J = 8.4 and 3.6 Hz)
H-23a4.50 ppm (1H, dd, J = 11.4 and 1.7 Hz)4.36 ppm (1H, dd, J = 11.2 and 8.4 Hz)
H-23b4.20 ppm (1H, dd, J = 11.4 and 4.2 Hz)4.23 ppm (1H, dd, J = 11.2 and 3.6 Hz)
C2112.90 ppm12.30 ppm
C2271.77 ppm71.40 ppm
C2366.39 ppm68.84 ppm
Table
Table 2. Comparison between the signals of 1H (400.1 MHz, MeOD) and 13C (100.6 MHz, MeOD) NMR for H/C21, H/C22, and H/C23a–b, for the epimers glycol 4 and 6.
Table 2. Comparison between the signals of 1H (400.1 MHz, MeOD) and 13C (100.6 MHz, MeOD) NMR for H/C21, H/C22, and H/C23a–b, for the epimers glycol 4 and 6.
H/C SignalTriol 4Triol 6
H-210.943 ppm (3H, d, J = 6.9 Hz)0.925 ppm (3H, d, J = 6.3 Hz)
H-223.71 ppm (1H, dt, J = 8.9 and 3.2 Hz)3.73 ppm (1H, ta, J = 6.5 Hz)
H-23a3.60 ppm (1H, dd, J = 11.3 and 2.7 Hz)3.53 ppm (1H, dd, J = 10.9 and 7.1 Hz)
H-23b3.40 ppm (1H, dd, J = 11.3 and 8.9 Hz)3.46 ppm (1H, dd, J = 10.9 and 6.5 Hz)
C2113.42 ppm12.37 ppm
C2275.19 ppm74.45 ppm
C2363.19 ppm65.53 ppm

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Oyarce, J.; Aitken, V.; González, C.; Ferrer, K.; Olea, A.F.; Parella, T.; Espinoza Catalán, L. Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5α-Cholane Type Analogs. Molecules 2019, 24, 4612. https://doi.org/10.3390/molecules24244612

AMA Style

Oyarce J, Aitken V, González C, Ferrer K, Olea AF, Parella T, Espinoza Catalán L. Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5α-Cholane Type Analogs. Molecules. 2019; 24(24):4612. https://doi.org/10.3390/molecules24244612

Chicago/Turabian Style

Oyarce, Jocelyn, Vanessa Aitken, César González, Karoll Ferrer, Andrés F. Olea, Teodor Parella, and Luis Espinoza Catalán. 2019. "Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5α-Cholane Type Analogs" Molecules 24, no. 24: 4612. https://doi.org/10.3390/molecules24244612

APA Style

Oyarce, J., Aitken, V., González, C., Ferrer, K., Olea, A. F., Parella, T., & Espinoza Catalán, L. (2019). Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5α-Cholane Type Analogs. Molecules, 24(24), 4612. https://doi.org/10.3390/molecules24244612

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