molecules Article Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5α-Cholane Type Analogs 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; jocelyn.oyarce@sansano.usm.cl (J.O.); vanessa.aitken.13@sansano.usm.cl (V.A.); cesar.gonzalez@usm.cl (C.G.); Karoll.ferrer.14@sansano.usm.cl (K.F.) 2 Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, El Llano Subercaseaux 2801, Santiago 8900000, Chile; andres.olea@uautonoma.cl 3 Teodor Parella, Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Catalonia, Spain; teodor.parella@uab.cat * Correspondence: luis.espinozac@usm.cl; Tel.: +56-32-2654425  Received: 22 November 2019; Accepted: 13 December 2019; Published: 17 December 2019  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. Keywords: synthesis; brassinosteroid; analogues; 24-nor-5α-cholane 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 [1–3], 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 Molecules 2019, 24, 4612; doi:10.3390/molecules24244612 www.mdpi.com/journal/molecules Molecules 2019, 24, 4612 2 of 14 obaMolecules 2019, 24, x 2 of 14 BRs possess a vicinal 22R, 23R diol structural functionality that appears essential for high biological for highItbiological activity. has been activity. suggested It has thatbeen suggested BRs may that BRs be derived from may be derived sterols with thefromsamesterols with the side chain, butsame most side of thechain, explored butsynthetic most of pathways the explored synthetic require severalpathways steps withrequire several low yields. steps decades, In recent with lowmuch yields. In effort recent has been decades, dedicated much effort to the has been synthesis of dedicated to the synthesis new BRs analogs in whichof new BRs common analogsofinorganic patterns which functions common patterns in the A/B ofrings organicand functions cis–transinfusion the A/B rings and between them cis–trans are kept fusion between intact, themimportant but with are kept intact, but structural with important changes in the side structural chain. For changes example,in the BRsside chain. analogs withForshorter example,sideBRs analogs chains [10–13],with shorter side spirostanic and chains [10–13], furostanic spirostanic [14–21], aromaticand andfurostanic [14–21], aromatic cyclic substituents and cyclic [22–27], esters substituents and carboxylic [22–27], acids estershave [24,28–30] and carboxylic been acids reported. [24,28–30] Despite thesehave been these changes, reported. Despite analogs havethese changes, presented verythese analogs important have presented biological activity. very importantthe Consequently, biological structural activity. Consequently, requirements the structural that these compounds requirements should possess that to these compounds elicit biological should possess activity to elicitand have changed biological new SAR activity haveproposed has been changed and newBriefly, [31,32]. SAR has been these proposed results suggest [31,32]. that Briefly, these results suggest that activity of BRs analogs depends on activity of BRs analogs depends on the oxygen atoms spatial distribution instead of the presence orthe oxygen atoms spatial distribution absence of one instead specificof functional the presence or absence group of one specific in the molecule [33,34]. functional These spatial group in the molecule orientations can be [33,34]. These indicated spatial orientations as distances can be the or angles between indicated oxygenasatomsdistances or angles present between the oxygen atoms in a brassinosteroid. present in a brassinosteroid. Following this line, we have previously reported the synthesis of BRs analogs with short side chainFollowing and structure this line, we have previously reported the synthesis (22S)-3α,22,23-trihydroxy-24-nor-5α-cholane of BRs analogs (compounds 2–4, with Figure short sidea 1) and chain and structure (22S)-3α,22,23-trihydroxy-24-nor-5α-cholane (compounds benzoylated derivative 5 [35]. However, to evaluate the effect of a C22 configuration on biological 2–4, Figure 1) and a benzoylated derivative 5 [35]. However, to evaluate the effect of a C22 configuration activity it is necessary to obtain the 22R epimers of these analogs. Herein, we report on the synthesis of on biological aactivity new BRs it is necessary analog to obtain thederivatives and benzoylated 22R epimers of these with analogs. Herein, R configuration at C22.we Asreport on the reaction the starting synthesis is of a new BRs analog and benzoylated derivatives with R configuration at C22. the dihydroxylation of a terminal olefin, synthesized from hyodeoxycholic acid, we have also evaluated As the starting reaction is the the ratiodihydroxylation of 22S/22R epimers of aobtained terminalthrough olefin, synthesized three different from hyodeoxycholic dihydroxylation acid, we reactions. have Yields also of both evaluated possible the ratio of 22S/22R configurations epimers are compared forobtained through all proposed three reactions. synthetic different dihydroxylation reactions. Yields of both possible configurations are compared for all proposed synthetic reactions. Figure 1. Structure Figure 1. Structure of of brassinolide brassinolide (1) (1) and and analogs of 24-nor-5α-cholan 2–8 of analogs 2–8 24-nor-5α-cholan type. type. 2. Results and Discussion 2. Results and Discussion The most direct synthetic route to BRs analogs with short side alkyl chains carrying a vicinal The most direct synthetic route to BRs analogs with short side alkyl chains carrying a vicinal diol diol function is the dihydroxylation of terminal olefins. This transformation can be accomplished by function is the dihydroxylation of terminal olefins. This transformation can be accomplished by either either of three different methods, namely: Direct Upjohn dihydroxylation; Epoxidation and subsequent 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 opening of epoxide ring in acid medium; and Sharpless asymmetric dihydroxylation (Scheme 1). are described the results obtained by the application of these synthetic paths to dihydroxylation of Below are described the results obtained by the application of these synthetic paths to olefin 9, which was previously synthesized from hyodeoxycholic acid [35]. All tested methods lead dihydroxylation of olefin 9, which was previously synthesized from hyodeoxycholic acid [35]. All to epimeric mixtures of glycols 22S/22R (10a/10b) but in different ratios. In the following, a detailed tested methods lead to epimeric mixtures of glycols 22S/22R (10a/10b) but in different ratios. In the description of reaction conditions and obtained yields are given for each method. following, a detailed description of reaction conditions and obtained yields are given for each method. 2.1. Direct Upjohn Dihydroxylation Upjohn 2.1. Direct dihydroxylation Upjohn is a well-known reaction that converts olefins to cis vicinal diol using Dihydroxylation N-methylmorpholine N-oxide (NMO) and osmium tetroxide. This reaction has been previously used to 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 Molecules 2019, 24, 4612 3 of 14 obtain glycols C22/C23 for 24-norcholane type steroids of similar structure. These studies have shown obaMolecules that the reaction 2019, 24, x product is a mixture of diastereomers with the 22S epimer as the major product 3 of [36–39]. 14 Alkene 9 was hydroxylated by the Upjohn method and a mixture of epimeric glycols 10a/10b was shown that the reaction product is a mixture of diastereomers with the 22S epimer as the major obtained with product a 72%Alkene [36–39]. yield (Scheme 1). 9 was hydroxylated by the Upjohn method and a mixture of epimeric glycols The diastereomeric ratio of 10a/10b was obtained with a 72% yieldeach glycol mixture can be established by integration of 1 H-NMR in the 1). (Scheme signals assigned to the C21 ratio The diastereomeric methyl group, of each which glycol appear in the mixture δH = at can be0.947 and 0.911 established ppm in 10a by integration of 1and H- 10b epimers, NMRrespectively signals assigned (Figure to the2)C21 [35]. methyl group, which appear at δH = 0.947 and 0.911 ppm in 10a and 10b epimers, respectively (Figure 2) [35]. 2. Spectra 1 Figure Figure 2. SpectraH-NMR 1H-NMRof of epimeric mixture epimeric mixture of of glycols glycols 10a:10b 10a:10b obtained obtained by a) Upjohn by a) Upjohn method;method; b) b) Epoxidation and subsequent opening of epoxide ring in acid medium; c) Sharpless Epoxidation and subsequent opening of epoxide ring in acid medium; c) Sharpless method.method. Based on these Based NMRNMR on these measurements the ratio measurements the of 10a:10b ratio obtained of 10a:10b by thisby obtained method was determined this method was as 1.0:0.24. determined as 1.0:0.24. 2.2. Epoxidation andand 2.2. Epoxidation Subsequent Opening Subsequent OpeningofofEpoxide RingininAcid Epoxide Ring AcidMedium Medium It hasIt has beenbeen reported reported that that theepoxidation the epoxidation reaction reaction ononsteroidal nuclei steroidal nucleiof similar structure of similar and and structure carrying terminal double bonds C22/C23, mainly produces epoxide 22R [36–38]. carrying terminal double bonds C22/C23, mainly produces epoxide 22R [36–38]. Thus, epoxidation of Thus, epoxidation of alkene alkene 9 with9m-CPBA with m-CPBA leadsleads to a to a mixture mixture of epimeric of epimeric epoxides11a/11b epoxides 11a/11b with with 55.7% 55.7%yield yield(Scheme (Scheme 1). 1). The ratio in which these epoxides were obtained was 11a:11b = 0.44:1.0, i.e., the amount of epoxide 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 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), assigned to hydrogens and H-23a (δ = 2.64 ppm), (δ = 2.73 H-22 H-23b (δ =ppm), H-23a 2.40 ppm) of the = 2.59epoxide (δ minor ppm) of(compound major epoxide (compound 11a) (see Figure S7, 11b), and H-23a (δ = 2.64 ppm), H-23b (δ = 2.40 ppm) of the minor epoxide (compound Supplementary Materials). For the determination of the majority epoxide, experimental and 11a) (seeNMR Figure S7, Supplementary spectroscopic data correlations were used [36–38]. Namely, it has been shown that comparative thinNMR Materials). For the determination of the majority epoxide, experimental and layer chromatography spectroscopic allowswere data correlations differentiating by polarity used [36–38]. Namely, both epoxides it has been formed shown in thecomparative that epoxidation thin layerreaction of steroidal alkenes chromatography allowswith 24-norchol-22-ene differentiating structures, by polarity bothand the results epoxides indicateinthat formed theless polar epoxidation epoxide corresponds to 22S configuration [36]. Our results show clearly that the more reaction of steroidal alkenes with 24-norchol-22-ene structures, and the results indicate that less polarpolar epoxide is preferentially obtained, i.e., epoxide 11b with 22R configuration. Additionally, 1D and 2D NMR epoxide corresponds to 22S configuration [36]. Our results show clearly that the more polar epoxide spectroscopy, particularly, 2D HSQC with edited phase (2D Edit HSQC), and 2D HMBC spectra, is preferentially obtained, i.e., epoxide 11b with 22R configuration. Additionally, 1D and 2D NMR provided essential information to carry out assignments of H-21, H-22, H23a, H23b signals and all spectroscopy, 13C signals particularly, 2D HSQC with edited phase (2D Edit HSQC), and 2D HMBC spectra, present in the epoxide mixture 11a/11b. provided essential information to carry out assignments of H-21, H-22, H23a, H23b signals and all 13 C signals present in the epoxide mixture 11a/11b. Molecules 2019, 24, 4612 4 of 14 obaMolecules 2019, 24, x 4 of 14 Subsequently, treatment of epoxides mixture 11a/11b in acidic conditions using the Subsequently, treatment of epoxides mixture 11a/11b in acidic conditions using the p- p-TsOH/THF/H2 O system (Scheme 1) produces 10a/10b mixture with 75.8% yield [37,38]. Analysis of TsOH/THF/H 2O system (Scheme 1) produces 10a/10b mixture with 75.8% yield [37,38]. Analysis of 1 H-NMR spectrum indicates that the major component on this mixture is the less polar glycol 22R in 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 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. of diastereomeric ratio, changing the proportion of this epimer from 0.44 to 1.0. 2.3. Sharpless Asymmetric Dihydroxylation 2.3. Sharpless Asymmetric Dihydroxylation Sharpless dihydroxylation reaction has not been used to obtain glycols C22/C23 in steroids with Sharpless the shortestdihydroxylation reaction has not been side chain of 24-nor-5α-cholane used type. to obtain Thus, olefinglycols C22/C23 in steroids 9 was dihydroxylated with by using the shortest side chain of 24-nor-5α-cholane type. Thus, olefin 9 was dihydroxylated dihydroquinidine p-chlorobenzoate (DHQD-CLB) as a chiral ligand (Scheme 1), following the method by using dihydroquinidine described p-chlorobenzoate in reference (DHQD-CLB) [23]. The reaction product (95% asyield) a chiral ligand (Scheme is a mixture 1),glycols of epimeric following the 10a and method 10b described in the proportionin reference [23]. The reaction 1.0:0.9, respectively (Figureproduct 2). (95% yield) is a mixture of epimeric glycols 10a and 10b in the proportion 1.0:0.9, respectively (Figure 2). 21 OH O 20 22 23 OH m-CPBA OsO4/NMO CH2Cl2/NaHCO3 (CH3)2CO/H2O 11a: C-22 (S) 10a: C-22 (S) AcO AcO 9 (Upjohn) AcO H 11b: C-22 (R) H H 10b: C-22 (R) O 11a:11b = 0.44:1.0 (55.7%) O O 10a:10b = 1.0:0.24 (72%) DHQD-CLB/CH 3SO2NH2 K2CO3/K3[Fe(CN)6] p-TsOH OsO4 THF/H2O (CH3)3COH/H2O (Sharpless) 10a:10b = 1.0:1.14 (75.8%) 10a:10b = 1.0:0.9 (95%) Scheme 1. Dihydroxylation of olefin 9 to get a mixture of epimeric glycols 10a/10b by using different 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 synthetic pathways: 1) Direct Upjohn dihydroxylation; 2) Epoxidation and subsequent opening of epoxide ring in acid medium and; 3) Direct asymmetric Sharpless dihydroxylation. epoxide ring in acid medium and; 3) Direct asymmetric Sharpless dihydroxylation. In summary, none of the three tested methods led exclusively to one of the two epimeric glycols. 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; The selectivity follows the order: Upjohn reaction produces mainly the glycol 22S in the ratio 1.0 to a slightly reverse proportion is obtained by opening of epoxides, i.e., 1.0 to 1.14, whereas the Sharpless 0.24; a slightly reverse proportion is obtained by opening of epoxides, i.e., 1.0 to 1.14, whereas the dihydroxylation reaction produced both glycols in similar amounts (1.0 to 0.9). The total yield of Sharpless dihydroxylation reaction produced both glycols in similar amounts (1.0 to 0.9). The total glycols mixture is also dependent of the dihydroxylation reaction: 95% for the Sharpless method, 72% yield of glycols mixture is also dependent of the dihydroxylation reaction: 95% for the Sharpless for Upjohn, and 43% for expoxidation followed by opening of epoxide ring. Thus, a selection of one of method, 72% for Upjohn, and 43% for expoxidation followed by opening of epoxide ring. Thus, a these methods should depend on the expected outcome for hydroxylation reaction. selection of one of these methods should depend on the expected outcome for hydroxylation reaction. 2.4. Synthesis of Benzoylated Derivatives 2.4. Synthesis of Benzoylated Derivatives. As the main goal of this research is to obtain derivatives with 22R configuration, we used the As the Sharpless main goal of this dihydroxylation research reaction is to obtain to convert derivatives the olefin 9 in thewith 22R of mixture configuration, we used epimeric glycols the 10a/10b. Sharpless dihydroxylation reaction to convert the olefin 9 in the mixture of epimeric This reaction was selected because the mixture product is obtained with the highest total reaction glycols 10a/10b. This reaction yield (95%) and was selected glycols because 10a/10b the mixture are found product in similar is obtained proportion. The with the highest derivative 22S benzoylated total reaction (5) yield (95%) and glycols 10a/10b are found in similar proportion. The 22S benzoylated has been previously synthesized by reaction of pure compound 10a with PhCOCl/DMAP [35]. Thus, derivative (5) hasget to been previously compound synthesized 7 the by reaction glycols mixture 10a/10bof pure compound obtained by the 10a with PhCOCl/DMAP Sharpless [35]. Thus, method was submitted to to get compound 7 the glycols mixture 10a/10b obtained by the Sharpless method the same benzoylation reaction to obtain both epimers 5 and 7. Interestingly, benzoylation of thiswas submitted to the same benzoylation reaction to obtain both epimers 5 and 7. Interestingly, benzoylation mixture leads to a complex mixture of mono and dibenzoylated derivatives, i.e., compounds 5, 7, 8, of this mixture and leads to2). 12 (Scheme a complex Compoundsmixture 5, 7,of mono and andpurified 8 were dibenzoylated by C.C. derivatives, i.e., compounds and recrystallized, 5, 7, of giving yields 8, and 12 (Scheme 2). Compounds 5, 7, and 8 were purified by C.C. and recrystallized, giving 4.0%, 1.2%, and 5.7%, respectively, whereas dibenzoylated derivative 12 could not be isolated in pure yields of 4.0%, 1.2%, form. and 5.7%,mixtures Additionally, respectively, whereas dibenzoylated of monobenzoylated epimers derivative were also12obtained, could not5/7 bewith isolated 5.0%inyield pure form. (5:7 Additionally, = 0.7:1.0), mixtures and 8/12 of monobenzoylated with 79.9% epimers yield (8:12 = 0.4:1.0). The were also obtained, proportion 5/7 with epimers of dibenzoylated 5.0% yield in (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. Molecules 2019, 24, 4612 5 of 14 both mixtures obaMolecules 24, xdetermined based on the integration signals at δH = 1.22 and 1.16 ppm, assigned 2019,was 5 of to 14 the H-21 of2019, obaMolecules methyl 24, x group for 8 and 12, respectively. 5 of 14 Scheme 2. Benzoylated derivatives 5, 7, 8, and 12 derivatives obtained by benzoylation reaction of Scheme 2. Benzoylated derivatives 5, 7, 8, and 12 derivatives obtained by benzoylation reaction of Scheme 2. Benzoylated glycols mixture 10a/10b.derivatives 5, 7, 8, and 12 derivatives obtained by benzoylation reaction of glycols mixture 10a/10b. glycols mixture 10a/10b. 2.4.1. Structural Structural Determination Determination of Derivatives of Derivatives 5, 7, 5, 7, and 8 and 8 2.4.1. Structural Determination of Derivatives 5, 7, and 8 The chemical The chemical structure structure of of 55 has has been been previously previously determined determined by by 1D 1D and and 2D2D NMR NMR spectroscopy, spectroscopy, whereasThe chemical its absolute structure of configuration5 has been 22S waspreviously establisheddetermined by using by 1D crystal and X-ray2D whereas its absolute configuration 22S was established by using crystal X-ray diffraction techniques NMR spectroscopy, diffraction techniques [35]. whereas [35]. Thus, Thus, its absolute configuration the structure the structure 22S was and stereochemistry and stereochemistry established at C22 ofatcompound by using C22 of compound crystal X-ray diffraction 7 were established 7 were established by a simple bytechniques a simple comparison [35]. 1 HThus, and 13the comparison of of structure C-NMR 1H and 13 and spectra stereochemistry C-NMR obtained spectra at C225 and obtained for derivatives of forcompound derivatives7 were 5 andestablished 7. These comparisons by chemical 7. considered a simple These comparisons comparison considered of 1H and 13C-NMR spectra obtained for derivatives 5 and 7. These comparisons chemical shifts (δ), coupling constants (J), and multiplicities of signals corresponding shifts (δ), coupling constants (J), and multiplicities of signals corresponding to H-22, H-23a, H-23b, and to considered H-22, CH 1 chemical H-23a, H-23b, andshiftsCH(δ), coupling 3-21. 1 H-NMR constants spectra (J), and multiplicities of both of signals epimers are shown corresponding in Figure to 3, and these 3 -21. H-NMR spectra of both epimers are shown in Figure 3, and these signals are identified. H-22, signals H-23a, H-23b, and CH3-21. 1H-NMR spectra of both epimers are shown in Figure 3, and these are identified. signals are identified. Figure 3. Comparison between 11 H-NMR spectra of epimers 5 and 7, and their main differences in Figure 3. Comparison between H-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. shifts chemical Comparison between for signals H-NMR H-21, 1H-22, andspectra of epimers 5 and 7, and their main differences in H-23a–b. chemical shifts for signals H-21, H-22, and H-23a–b. The main differences in these spectroscopic parameters are listed in Table 1, and the more The main significant differences variations in these in chemical spectroscopic shifts parameters and J values correspondare to listed in Table hydrogen atoms1,H-23a and the andmore CH3- significant variations in chemical shifts and J values correspond to hydrogen atoms H-23a and CH3- Molecules 2019, 24, 4612 6 of 14 The main obaMolecules differences 2019, 24, x in these spectroscopic parameters are listed in Table 1, and the 6more of 14 significant variations in chemical shifts and J values correspond to hydrogen atoms H-23a and CH3 -21. 21. On Onthethe other other hand, hand, differences differences in in chemical chemical shifts shifts werealso were alsoobserved the1313C-NMR observedininthe C-NMR spectra, for signals of C21, C21, C22, C22, and andC23. C23. For Forthe thelatter, latter,the thesignals appearatatδCδC==66.39 signalsappear 66.39and and68.84 68.84ppm ppmfor for 5 and 5 and 7, 7, respectively respectively (Table (Table 1).1). Based Based onon this this data data thethe stereochemistry stereochemistry in in epimer epimer 7 was 7 was assigned assigned as as 22R. 22R. Comparison between 1 (400.1 MHz, CDCl ) and 13 Table 1. Table 1. Comparison between signals signalsof of 1H C (100.6 H (400.1 MHz, CDCl33) and 13C (100.6 MHz, MHz, CDCl CDCl33)) NMR NMR for for H/C21, H/C22, and H/C23a–b, for epimers 5 and 7. H/C21, H/C22, and H/C23a–b, for epimers 5 and 7. H/C H/C Signal Signal Compound Compound 5 5 Compound 7 Compound 7 H-21H-21 1.06 ppm (3H, (3H, 1.06 ppm J = Hz) d, J =d,6.9 6.9 Hz) 0.990 0.990ppm ppm(3H, (3H, d,d, J = 6.1 Hz) Hz) H-22H-22 4.06 ppm 4.06 ppm (1H,(1H, m) m) 4.04 4.04 ppm(1H, ppm dd,JJ ==8.4 (1H,dd, 8.4 and and 3.6 3.6Hz) Hz) H-23a 4.50 (1H, H-23a 4.50 ppm ppm dd, (1H,Jdd, J = 11.4 = 11.4 andand1.7 1.7 Hz)Hz) 4.36 4.36 ppmppm(1H, dd,JJ ==11.2 (1H,dd, 11.2 and and 8.4 8.4Hz) Hz) H-23b 4.20 ppm (1H, dd, J = 11.4 and 4.2 Hz) 4.23 ppm (1H, dd, J = 11.2 and 3.6 Hz) H-23bC21 4.20 ppm (1H, dd, 12.90 J = 11.4 ppm and 4.2 Hz) 4.23 ppm (1H, dd, J = 11.2 and 3.6 Hz) 12.30 ppm C21C22 12.9071.77 ppmppm 12.30 71.40ppm ppm C22C23 71.7766.39 ppmppm 71.40 68.84ppm ppm C23 66.39 ppm 68.84 ppm The chemical structure of the dibenzoylated derivative 8 was established using 1 H, 13 C, 2D HSQC, The and 2D HMBC chemical NMR structure of the techniques. Thedibenzoylated presence of thederivative 8 was function dibenzoylated established wasusing 1H, 13C, 2D confirmed in the 1HSQC, and 2D HMBC NMR techniques. The presence of the dibenzoylated function H-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 was confirmed in andthe1.4H-NMR 1 spectrum, Hz); 7.55–7.50 (2H, by m);signals observed 7.44 (2H, dd, J = at 7.9δand H = 8.06 (2H, dd, J = 8.5 and 1.4 Hz); 7.97 (2H, dd, J 7.9 Hz); 7.38 (2H, dd, J = 7.9 and 7.4 Hz), which =are 8.5assigned and 1.4 toHz); 7.55–7.50 protons (2H, HAr-2 HAr-2”; m); 7.44 (2H, dd,and 0 , HAr-4”, J = 7.9 and0 ;7.9 HAr-4 Hz); 7.38 HAr-3” and (2H, dd, HAr-3 HAr-5”; J = 7.9 0and and7.4 Hz),0 HAr-5 which are assigned to protons HAr-2′′; HAr-2′, HAr-4′′, and 13 HAr-4′; HAr-3′′ and in the aromatic group, respectively. In addition, in the C-NMR spectrum appear signals at 130.33; HAr-5′′; HAr-3′ and HAr-5′ in the aromatic group, respectively. In addition, in the 13C-NMR spectrum appear signals 133.06; 129.60; 128.35; 129.74; 133.00; 129.63, and 128.38 ppm, which were assigned to carbon C1 -Ar; 0 at 130.33; C40 -Ar;133.06; C20 -Ar129.60; and C6 128.35; 129.74; 0 -Ar; C3 133.00; 0 -Ar and C50129.63, and 128.38 -Ar; C1”-Ar; ppm, C4”-Ar; whichand C2”-Ar were assigned C6”-Ar; to carbon C3”-Ar and C1′-Ar; C4′-Ar; C2′-Ar and C6′-Ar; C3′-Ar and C5′-Ar; C1′′-Ar; C4′′-Ar; C5”-Ar, respectively. These assignments were confirmed by 2D HSQC and HMBC spectra. The C2′′-Ar and C6′′-Ar; C3′′-Ar main and C5′′-Ar, structural respectively. information These assignments provided by the 2D HMBC were spectrum confirmedwas by 2Dthe HSQC and HMBC assignment spectra. and position inThe the main structural information provided by the 2D HMBC spectrum was the assignment side chain of the two benzoate groups, which showed heteronuclear correlations at JCH and JCH as 2 and position 3 in the sideinchain indicated Figureof4.the two benzoate groups, which showed heteronuclear correlations at 2JCH and 3JCH as indicated in Figure 4. 2 3 4. Main heteronuclear Figure 4. Figure heteronuclear correlations correlations 2D 2D HMBC HMBC to to 2JJCH CH (blue) and 3JJCH (red) observed CH (red) observed for for the dibenzoylated derivative dibenzoylated derivative8,8,which whichconfirm confirmthe thepositions positionsofofboth benzoate both groups benzoate groupsin C22 andand in C22 C23C23 of the of sideside the chain. chain. To determine the stereochemistry at carbon C22 of compound 8, a complete benzoylation 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 reaction with PhCOCl/DMAP was carried out for the monobenzoylated derivative 15. In this13way, the dibenzoylated derivative was obtained with 95% yield. The spectroscopic data of H and C-NMR 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 were totally consistent with those obtained for derivative 8 obtained by benzoylation of the glycol mixture 10a/10b. 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). obaMolecules obaMolecules 2019, 2019, 24, 24, xx 77 of of 14 14 2.5. 2.5. Synthesis Synthesis of of BRs BRs Analogs Analogs Synthesis of BRs Synthesis BRs analogs ofanalogs 44 and and 66 was was carried carried out out by by saponification saponification reaction reaction of of7 ofthe the Molecules 2019, 24, 4612 14 monobenzoylated derivatives 5 and 7, respectively, with 90% and 82.5% yield, respectively (Scheme monobenzoylated derivatives 5 and 7, respectively, with 90% and 82.5% yield, respectively (Scheme 3). 3). Scheme 3. Synthesis of of brassinosteroids (BRs) (BRs) analogs 44 and and 6 saponification reaction reaction the Scheme 3. Synthesis Synthesis of brassinosteroids brassinosteroids (BRs) analogs analogs 4 and 66 by by saponification saponification reaction of of the monobenzoylated derivatives 55 and 7, 7, respectively. respectively. monobenzoylated derivatives 5 and 7, respectively. The spectroscopic spectroscopic data of 1 H and 13 C-NMR obtained for compound 4 were consistent with those The The spectroscopic data data of of 1H 1 H and and 13C-NMR 13 C-NMR obtained obtained for for compound compound 44 were were consistent consistent with with those those previously previously reported [35,40]. The structural and stereochemical determination at C22 for triol 6 was previously reported [35,40]. The structural and stereochemical determination at C22 for triol 6 was reported [35,40]. The structural and stereochemical determination at C22 for triol 6 was established by established by comparison of of NMR data between epimers 4 and 6. These comparisons considered established by comparison comparison of NMR NMR data data between between epimers epimers 44 and and 6. 6. These These comparisons comparisons considered considered chemical shifts chemical shifts (δ), coupling coupling constants constants (J), (J), and and multiplicities of of signals corresponding corresponding to H-21, H-21, H-22, chemical shifts (δ), (δ), coupling 1 H-NMRconstants (J), and multiplicities multiplicities of signals signals corresponding to to H-21, H-22, H-22, H-23a, H-23a, and H-23b. spectra of both epimers are shown in Figure 5, where these signals and H-23b. H-23a, and H-23b. H-NMR spectra of both epimers are shown in Figure 5, where these signals are H-NMR spectra of both epimers are shown in Figure 5, where these signals 1 1 are are identified. identified. identified. Figure Comparison between 11 H-NMR spectra spectra of of glycol glycol epimers epimers 444 and and 6, and their 6, and their main main differences differences Figure 5. 5. Comparison Comparison between 1H-NMR H-NMR spectra of glycol epimers and 6, and their main differences in chemical shifts for signals H-21, H-22, and H-23a–b. in chemical shifts for signals H-21, H-22, and H-23a–b. The main The differences observed among the indicated hydrogens are listed in Table 2. It is It seenseen that The main main differences differences observed observed among among the the indicated indicated hydrogens hydrogens areare listed listed in in Table Table 2.2. It is is seen hydrogen that atoms H-21,H-21, H-23a, and H-23b experience variations in chemical shifts and J values, whereas that hydrogen hydrogen atoms atoms H-21, H-23a, H-23a, and and H-23b H-23b experience experience variations variations in in chemical chemical shifts shifts and and JJ values, values, H-22 shows whereas also H-22 changes shows alsoinchanges multiplicity. in On the other multiplicity. On hand, the in 13 C-NMR other hand, inspectra 13C-NMR differences spectra in chemical differences whereas H-22 shows also changes in multiplicity. On the other hand, in 13C-NMR spectra differences shifts in for signals assigned to C21, C22,toand C23, but mainlybutin carbon C21 and C23, are observed in chemical chemical shifts shifts for for signals signals assigned assigned to C21, C21, C22, C22, and and C23, C23, but mainly mainly in in carbon carbon C21 C21 and and C23, C23, are are (Table 2). (Table observed From this 2). data the From stereochemistry this data the in C22 caninbeC22 stereochemistry assigned can beas R for the assigned asepimer R for 6. epimer 6. the observed (Table 2). From this data the stereochemistry in C22 can be assigned as R for the epimer 6. Molecules 2019, 24, 4612 8 of 14 Table 2. Comparison between the signals of 1 H (400.1 MHz, MeOD) and 13 C (100.6 MHz, MeOD) NMR for H/C21, H/C22, and H/C23a–b, for the epimers glycol 4 and 6. H/C Signal Triol 4 Triol 6 H-21 0.943 ppm (3H, d, J = 6.9 Hz) 0.925 ppm (3H, d, J = 6.3 Hz) H-22 3.71 ppm (1H, dt, J = 8.9 and 3.2 Hz) 3.73 ppm (1H, ta, J = 6.5 Hz) H-23a 3.60 ppm (1H, dd, J = 11.3 and 2.7 Hz) 3.53 ppm (1H, dd, J = 10.9 and 7.1 Hz) H-23b 3.40 ppm (1H, dd, J = 11.3 and 8.9 Hz) 3.46 ppm (1H, dd, J = 10.9 and 6.5 Hz) C21 13.42 ppm 12.37 ppm C22 75.19 ppm 74.45 ppm C23 63.19 ppm 65.53 ppm 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. 1 H, 13 C, 13 C 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 1 H and 13 C, respectively, and CD3 OD at δ = 3.30 ppm and δ = 49.00 ppm for 1 H and 13 C, respectively, on an Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C, and Avance 600 Digital NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 600.13 MHz for 1 H and 150.90 for 13 C. 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% H2 SO4 in H2 O. 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 Na2 S2 O3 . 5H2 O (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). Molecules 2019, 24, 4612 9 of 14 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 CH2 Cl2 (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: 1 H-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, CH3 CO), 0.970 (3H, d, J = 6.8 Hz, H-21), 0.744 (3H, s, H-19), 0.667 (3H, s, H-18). 13 C-NMR (150 MHz, CDCl3 ) see Table S1. Compound 11b: 1 H-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, CH3 CO), 1.11 (3H, d, J = 5.6 Hz, H-21), 0.744 (3H, s, H-19), 0.659 (3H, s, H-18). 13 C-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 H2 O (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 Et2 O/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), CH3 SO2 NH2 (228 mg, 2.33 mmol), K2 CO3 (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 H2 O (10 mL) and a saturated solution of Na2 S2 O3 ·5H2 O (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). Molecules 2019, 24, 4612 10 of 14 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 CH2 Cl2 (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), H2 O (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, 1 H, 13 C-NMR) were consistent with those previously reported [35]. Compound 7: colorless solid, m.p. 174.6 ± 1 ◦ C (Hexane/Et2 O), 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). 1 H-NMR (400 MHz, CDCl3 ) δ (ppm): 8.02 (2H, d, J = 7.4 Hz, HAr-20 ), 7.54 (1H, t, J = 7.4 Hz, HAr-40 ), 7.41 (1H, t, J = 7.8 Hz, HAr-30 ), 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, CH3 CO), 0.990 (3H, d, J = 6.1 Hz, H-21), 0.712 (3H, s, H-19), 0.668 (3H, s, H-18). 13 C-NMR see Table S1. HRMS-ESI (positive mode): m/z calculated for C H O : 524.3138 [M]+ , found 32 44 6 525.3204 [M + 1]+ . Compound 8: colorless solid, m.p. 205.9 ± 2 ◦ C (Hexane/Et2 O), 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). 1 H-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-20 ), 7.55–7.50 (2H, m, HAr-4” and HAr-40 ), 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-30 and HAr-50 ), 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, CH3 CO), 1.16 (3H, d, J = 7.0 Hz, H-21), 0.748 (3H, s, H-19), 0.710 (3H, s, H-18). 13 C-NMR see Table S1. HRMS-ESI (positive mode): m/z calculated for C39 H48 O7 : 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 CH2 Cl2 (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 1 H and 13 C-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) K2 CO3 (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 Molecules 2019, 24, 4612 11 of 14 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, 1 H, 13 C-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), K2 CO3 (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 (Et2 O/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). 1 H-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). 13 C-NMR see Table S1. HRMS-ESI (negative mode): m/z calculated for: C23 H38 O4 : 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: 1 H 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: 1 H 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: 1 H 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: 1 H 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: 1 H 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: 13 C NMR signals for compounds 4–8, 10a–10b, 11a and 11b. Molecules 2019, 24, 4612 12 of 14 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. [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. 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. 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. [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. [CrossRef] [PubMed] 6. Oklestkova, J.; Rarova, L.; Kvasnica, M.; Strnad, M. Brassinosteroids: Synthesis and Biological Activities. Phytochem. Rev. 2015, 14, 1053–1072. [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. [CrossRef] 8. Bajguz, A.; Hayat, S. Effects of Brassinosteroids on the Plant Responses to Environmental Stresses. Plant. Physiol. Biochem. 2009, 47, 1–8. [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. [CrossRef] 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. [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. [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. [CrossRef] 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. [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. 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. 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. [CrossRef] Molecules 2019, 24, 4612 13 of 14 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. [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. [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. [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. [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. [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. [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. [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. 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. [CrossRef] [PubMed] 26. Kovganko, N.V.; Ananich, S.K. Advances in the Chemical Synthesis of Brassinosteroids. Chem. Nat. Compd. 1997, 33, 389–416. [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. [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. [CrossRef] 29. Zhou, W.; Jiang, B.; Shen, J. Synthesis of Cholesteric Lactones and Analogs as Plant Growth Regulators. Patent CN 1184113 A, 10 June 1998. 30. Tian, W.S.; Zhou, W.S. Studies on Steroidal Plant Growth Hormone VI. Conversion of Methyl 3,6-Diketoallocholanate into Methyl ∆ˆ2-6-ketoallocholenate. Acta. Chim. Sinica. 1988, 46, 824–826. 31. Brosa, C.; Capdevila, J.M.; Zamora, I. Brassinosteroids: A New Way to Define the Structural Requirements. Tetrahedron 1996, 52, 2435–2448. [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. [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. [CrossRef] 34. Takatsuto, S.; Yazawa, N.; Ikekawa, N.; Takematsu, T.; Takeuchi, Y.; Koguchi, M. Structure Activity Relationship of Brassinosteroids. Phytochem 1983, 22, 2437–2441. [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. [CrossRef] [PubMed] 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. [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. 38. Espinoza, L.; Cortes, M. Synthesis and Biological Activity of Brassinosteroids Analogues. Bol. Soc. Chil. Quim. 2002, 47, 511–516. Molecules 2019, 24, 4612 14 of 14 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. 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. [CrossRef] Sample Availability: Samples of the compounds 4–8 are available from the authors. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).