Structural Modifications of Deoxycholic Acid to Obtain Three Known Brassinosteroid Analogues and Full NMR Spectroscopic Characterization.

An improved synthesis route for obtaining known brassinosteroid analogues, i.e., methyl 2α,3α-dihydroxy-6-oxo-5α-cholan-24-oate (11), methyl 3α-hydroxy-6-oxo-7-oxa-5α-cholan-24-oate (15) and methyl 3α-hydroxy-6-oxa-7-oxo-5α-cholan-24-oate (16), from hyodeoxycholic acid (4) maintaining the native side chain is described. In the alternative procedure, the di-oxidized product 6, obtained in the oxidation of methyl hyodeoxycholate 5, was converted almost quantitatively into the target monoketone 7 by stereoselective reduction with NaBH₄, increasing the overall yield of this synthetic route to 96.8%. The complete ¹H- and (13)C-NMR assignments for all compounds synthesized in this work have been made by 1D and 2D heteronuclear correlation gs-HSQC and gs-HMBC techniques. Thus, it was possible to update the spectroscopic information of ¹H-NMR and to accomplish a complete assignment of all (13)C-NMR signals for analogues 5-16, which were previously reported only in partial form.

1. Introduction

Bile acids, such as deoxycholic (1), chenodeoxycholic (2), cholic (3) and hyodeoxycholic (4) (Figure 1), have been used as substrates for the synthesis of a large number of brassinosteroid analogues, keeping the methyl ester or carboxylic function in the side chain [1,2,3,4,5,6,7]. In particular, hyodeoxycholic acid (4) has been used because it contains the modifiable organic functions at suitable positions, satisfying the structural requirements on the A and/or B and A/B trans fusion ring and side chain of active brassinosteroids [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. Hyodeoxycholic acid (HDA) extracted from hog bile was initially used as a precursor for steroid synthesis [27]. It has also been shown that HDA prevents cholesterol-induced gallstones in animals fed with a lithogenic diet [28], whereas oral administration of HDA leads to a decrease in the LDL-cholesterol concentration, a strong stimulation of hepatic cholesterol biosynthesis and an excessive loss of cholesterol in feces [29].

Figure 1

Structure of bile acids used for the synthesis of brassinolide and its analogues.

In this work, we report an improved synthesis route to obtain known brassinosteroid analogues, starting from hyodeoxycholic acid (4), and maintaining the native side chain. Additionally, the full NMR spectroscopic characterization of these derivatives is performed.

2. Results and Discussion

Fischer esterification of hyodeoxycholic acid (4) with the H2SO4-MeOH system gave the methyl hyodeoxycholate (5) in almost quantitative yield (98%). Subsequently, selective oxidation of the C-6 hydroxyl group with PCC/CH2Cl2 afforded the di- and mono-oxidized compounds 6 and 7 with 30% and 68% yield, respectively. These yields are similar to those reported when PDC/CH2Cl2 was the oxidizing agent [13]. However, the yield of compound 7 was 67%–70% when K2CrO4 was used as an oxidizing agent [30,31]. Thus, in order to increase the yield of this reaction, 6 was conveniently converted to the desired monoketone 7 by selective reduction with NaBH4/MeOH at a low temperature (0–5 °C) with 96.8% yield (Scheme 1). This reaction has been used to modify steroids with similar structures [24]. The overall yield of 7 is increased from 68% to 96.8%. The physical properties and spectroscopic data (IR and 1H-NMR) of compounds 6 and 7 were consistent with those previously reported [8,30,31,32]. The 13C-NMR spectroscopic data for compound 7 has been informed but not assigned [32].

Scheme 1

Synthesis of hyodeoxycholic acid derivatives 5–10 and brassinosteroid analogue 11. Reagents and conditions: (a) CH3OH/H2SO4, reflux, 2 h; (b) PCC/DCM, 48 h., r.t., C.C. separation; (c) NaBH4/MeOH, 0–5 °C, 1 h; (d) HCl/CH3OH 2.5%, r.t., 24 h; (e) CH3SO2Cl/DCM, DMAP, 0 °C ≥ r.t. 1 h; (f) Li2CO3/LiBr, DMF, 80 °C, 1.5 h; (g) OsO4 4%/NMO, acetone, r.t., 72 h.

Compound 7 was readily isomerized (94.4% yield) under acid conditions (2.5% HCl/MeOH) to give the derivative 8 possessing 5α-cholestan-6-one skeleton [8,13,32]. This isomerization reaction can be performed under alkaline conditions as well, but under these conditions the obtained compound was isomerized and hydrolyzed (carboxylic acid) with 84% yield [31]. Thus, to get 8, an additional esterification reaction was carried out with 94% yield. The physical properties, IR, and 1H-NMR spectroscopic data of compound 8 have been previously reported, but the signals of 13C-NMR were not assigned [32]. The change of the 5β-cholestan to 5α-cholestan skeleton (A/B ring cis for trans fusion) implies a very important structural modification, and therefore a detailed analysis of NMR spectroscopic data for compounds 7 and 8 must be performed. The main differences found in the 1H- and 13C-NMR data are shown in Figure 2. The signals corresponding to H-3β and H-5α (δH = 4.16 and δH = 2.70 ppm, respectively) in compound 8, are displaced to downfield as compared to compound 7 (δH = 3.62 and δH-5β = 2.33 ppm, respectively). On the other hand, the signal of CH3-19 in the 13C-NMR of compound 8 appears at a higher field (δC = 12.29 ppm) than in the spectrum of compound 7 (δC = 23.16 ppm).

Figure 2

Major differences observed in the 1H- and 13C-NMR data of compounds 7 and 8, associated with the change from the 5β-cholestan to 5α-cholestan skeleton (A/B ring cis for trans fusion).

The reaction of compound 8 with methanesulfonyl chloride (mesyl chloride) in CH2Cl2/DMAP produces the mesylated derivative 9 with 61% yield [28]. The major 1H-NMR spectroscopic evidence for formation of this derivative is the presence of signals at δH = 5.02 and 2.97 ppm assigned to hydrogen H-3β (b.s. 1H) and CH3-SO2- (s, 3H), respectively, whereas in the 13C-NMR spectrum, a signal corresponding to CH3-SO2 was observed at δC = 38.38 ppm. Subsequent treatment of compound 9 with the Li2CO3/LiBr system at 80 °C in dimethylformamide (DMF) produces the alquene 10 with 74.9% yield [33] (Scheme 2). The 1H-NMR spectroscopic data of alquene 10 was consistent with that previously reported [8,32]. Additionally, in the 13C-NMR spectrum the signals at δC = 124.49 (C-2) and 124.95 (C-3) ppm are observed. Both signals were assigned from the 2D HMBC spectrum, where the H-3 (δH = 5.69–5.66 ppm, m, 1H) showed 3JH-C correlation with C-1 (δC = 39.34 ppm) and 2JH-C with C-4 (δC = 27.88 ppm), while H-2 (δH = 5.57–5.55 ppm, m, 1H) showed 3JH-C correlation with C-4 (δC = 27.88 ppm) and 2JH-C with C-1 (δC = 39.34 ppm).

Scheme 2

Synthesis of hyodeoxycholic acid derivatives 8, 12–14 and known 7-oxalactone 15 and 6-oxalactone 16. Reagents and conditions: (a) Ac2O/DMAP, CH2Cl2/py, r.t., 30 min; (b) K2CO3/CH3OH, reflux, 1 h; (c) m-CPBA/CH2Cl2, NaHCO3 r.t., 24 h.

Compound 11 was obtained with 51% yield by stereospecific α-face hydroxylation of olefin 10 using a catalytic amount of osmium tetraoxide (4.0%) in a mixture of (CH3)2CO-H2O (3:1) and 2.0 mL of pyridine, and in the presence of N-methylmorpholine N-oxide [33]. The 1H- and 13C-NMR spectroscopic data of diol 11 were consistent with those reported [8,32]. In addition, the spatial orientations of α-OH in C-2 and α-OH in C-3 were determined by selective-excitation 1D NOESY experiments, where H-2β (3.77 ppm, b.d, J = 11.1 Hz, 1H) showed a long-range spatial correlation with CH3-19 (0.75 ppm, s, 3H), while the observed signals at δC = 68.29 and 68.39 ppm in the 13C-NMR spectrum were assigned to C-2 and C-3, respectively.

In order to get additional deoxycholic acid derivatives, which may be of interest, the chemical transformations described in Scheme 2 were performed. Thus, compound 8 was acetylated under standard conditions (Ac2O/DMAP/CH2Cl2), and commercial compound 12 was obtained with 95% yield. The synthesis of this derivative has been previously reported, but no NMR spectroscopic data was included [25]. The 1H-NMR spectrum of the acetylated derivative 12 shows a singlet at δH = 2.04 ppm (3H, CH3CO) and a multiplet at δH = 5.12 ppm corresponding to H-3β (m, 1H). The latter is shifted to downfield as compared with H-3β (δH = 4.16 ppm, m 1H) in compound 8. Additionally, in the 13C-NMR spectrum the signals appearing at δC = 170.31 (C=O) and 21.39 (CH3CO) ppm (Table 1) confirmed the presence of acetylated derivative 12.

Table 1

Chemical shifts, δ, of 13C-NMR (CDCl3, 100.6 MHz) for compounds 12–16.

C	12	13 *	14	15	16
1	32.38	32.87	32.36	32.88	27.46
2	27.89	28.96	27.88	22.13	30.75
3	68.87	66.01	68.85	64.88	66.31
4	25.26	28.70	25.28	30.81	35.63
5	52.58	52.83	52.56	41.78	79.65
6	211.86	215.65	211.84	176.76	175.35
7	46.71	47.59	46.73	70.44	38.07
8	37.91	39.41	37.93	39.45	35.27
9	53.73	55.03	53.72	58.35	57.97
10	41.26	43.71	41.24	36.29	39.85
11	21.06	22.17	21.09	24.80	22.17
12	39.44	40.78	39.47	39.68	39.66
13	43.02	44.16	43.07	42.66	42.71
14	55.79	57.18	55.76	55.72	56.00
15	25.00	28.42	25.00	28.23	25.24
16	23.88	24.92	23.87	27.82	27.93
17	56.74	57.89	56.75	51.45	55.48
18	12.01	12.41	12.01	11.79	11.78
19	12.39	12.68	12.40	14.51	11.53
20	35.24	36.60	35.25	35.31	34.83
21	18.19	18.72	18.17	18.14	18.12
22	30.67	32.01	30.65	31.01	30.99
23	30.84	32.24	30.83	32.49	31.15
24	174.67	178.19	179.20	174.60	174.64
CH3O	51.45	-	-	51.50	51.52
CH3CO-	170.31	-	170.33	-	-
CH3CO-	21.39	-	21.41	-	-

* The 13C-NMR spectrum of compound 13 was recorded in CD3OD solution.

The synthesis of compound 13 by saponification of 8 with NaOH/dioxane under reflux conditions has been previously informed [31]. Saponification of compound 8 under mild conditions (K2CO3/MeOH, reflux) produces the carboxylic acid 13 with similar yields (82.2% yield) (Scheme 2). The physical property data, IR and 1H-NMR were consistent with those reported [31]. The 13C-NMR spectrum shows a signal at δC = 178.19 ppm (C=O) (Table 1), confirming the presence of the carboxylic function. Standard acetylation (Ac2O/DMAP/CH2Cl2) of 13 produces compound 14 with 72.8% yield. Compound 14 showed signals at 3373–2495 (OH), 1736 (C=O) and 1708 (C=O) cm−1 in the IR spectrum, while in 1H-NMR the signals observed at δH = 5.12 ppm (m, 1H) and 2.03 ppm (s, 3H) were assigned to the H-3β and CH3CO, respectively. In the 13C-NMR spectrum the observed signals at δC = 170.33 and 21.41 ppm (Table 1) were assigned to the acetyl group, while the signal at δC = 179.20 ppm (Table 1) was assigned to the carboxylic function. These spectroscopic data confirmed the structure of compound 14.

The Baeyer-Villiger oxidation of 3α-hydroxy-6-oxo-5α-cholanate 8 with m-CPBA/CH2Cl2 gave a mixture of known 7-oxalactone 15 and 6-oxalactone 16 with 10.8% and 14.9% yields, respectively. The physical and IR, 1H-NMR spectroscopic data of 15 and 16 were consistent with those reported for these compounds [14,25]. Considering that the original 1H-NMR spectra were recorded with a 60 MHz spectrometer, and no 13C-NMR data was reported, we believe that it is worth updating the analysis of NMR data for these compounds.

The full structural assignment of compound 15 and in particular the 7-oxalactone position was mainly assigned by 1H, 13C, 2D HSQC and 2D HMBC NMR spectroscopy. In the 1H-NMR spectrum of compound 15 appears a signal at δH = 4.08–4.06 ppm (m, 2H), which was assigned to the two hydrogens H-7 and correlated by 2D 1H-13C HSQC with the signal δC = 70.44 ppm (C-7) with pair multiplicity (from DEPT-135 analysis). Additionally, from the 2D 1H-13C HMBC spectrum important heteronuclear correlations were observed, i.e., 2JHC correlations between H-5α (δH = 3.17 ppm, dd, J = 4.4 and 12.2 Hz) with the signals at δC = 176.76 (C=O of lactone function), δC = 36.29 and δC = 30.81 ppm which were assigned to the carbons C-6, quaternary C-10 and C-4, respectively. Additionally, H-5α showed 3JHC correlation with signals at δC = 30.81, δC = 14.52, δC = 32.88 and δC = 58.35 ppm, which were assigned to the carbons C-4, CH3-19, C-1 and C-9, respectively (Figure 3a). These 2D HMBC observations unequivocally confirm the 7-oxalactone position for compound 15.

Figure 3

(a) Inverse detection heteronuclear-correlated 2D 1H-13C HMBC contour plot and major observed 2JHC and 3JHC correlations for H-5α in 7-oxalactone 15 and (b) for 6-oxolactone 16.

A similar analysis was performed for the structure determination of 6-oxolactone 16; thus, in the 1H-NMR spectrum for 16 a signal at δH = 4.60 ppm (dd, 1H, J = 5.3 and 11.3 Hz) was observed, correlated by 2D 1H-13C HSQC with the signal at δC = 79.65 ppm (C-7) with impair multiplicity (from DEPT-135 analysis) and assigned to H-5α. Additionally, H-5 α showed 2JHC correlation with signals at δC = 35.63 ppm and 3JHC correlation with signals at δC = 11.53, δC = 57.97 and δC = 175.35 ppm, which were assigned to the carbons C-4, CH3-19, C-9 and C-6 (C=O, of lactone function), respectively (Figure 3b).

3. Materials and Methods

3.1. General

All reagents were purchased from commercial suppliers (Merck, Darmstadt, Germany or Aldrich, St. Louis, MO, USA), and used without further purification. Melting points were measured on a Stuart-Scientific SMP3 apparatus (Staffordshire, UK) and are uncorrected. 1H, 13C, 13C DEPT-135, sel. gs1D 1H NOESY, 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 a Bruker Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany), operating at 400.1 MHz for 1H and 100.6 MHz for 13C. Chemical shifts are reported in δ ppm and coupling constants (J) are given in Hz. IR spectra were recorded as KBr disks in a FT-IR Nicolet 6700 spectrometer (Thermo Scientific, San Jose, CA, USA) and frequencies are reported in cm−1. For analytical TLC, silica gel 60 (Merck) 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, Merck) 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

Methyl 3α, 6α-dihydroxy-5β-cholan-24-oate (5). A solution of 4 (20.0 g, 50.95 mmol) in MeOH (150 mL) and 1.0 mL of H2SO4 was refluxed for 2.0 h. The end of reaction was verified by TLC. Then the solvent was removed (until a 40 mL approximate volume) and diluted with EtOAc (90 mL). The organic layer was washed with saturated solution of NaHCO3 (40 mL) and water (2 × 30 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (15 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 7.6:2.4). Compound 5 (19.50 g 98% yield) was a colorless solid (m.p. = 60–65 °C, MeOH/Et2O); IR (cm−1): 3385 (O-H); 2938 (C-CH3); 2858 (C-CH2-C); 1743 (C=O); 1452 (CH2); 1376 (CH3); 1169 (C-O). 1H-NMR: 4.05 (ddd, J = 4.8, 4.8 and 11.9 Hz, 1 H, H-6); 3.66 (s, 3H, CH3O); 3.62 (m, 1H, H-3); 2.35 (ddd, J = 5.1, 10.1 and 15.0 Hz, 1H, H-23); 2.21 (ddd, J = 6.6, 9.6 and 15 Hz, 1H, H-23); 0.91 (d, J = 5.1 Hz, 3H, H-21); 0.90 (s, 3H, H-19); 0.63 (s, 3H, H-18). 13C-NMR: See Table 2.

Table 2

Chemical shifts, δ, of 13C-RMN (CDCl3, 100.6 MHz) for compounds 5–11.

C	5	6	7	8	9	10	11
1	35.56	35.71	34.37	31.65	27.80	39.34	40.18
2	29.20	36.42	34.86	28.16	31.64	124.49	68.29
3	71.48	208.58	70.15	65.41	78.71	124.95	68.39
4	34.86	39.83	29.85	27.90	26.56	27.88	26.28
5	48.40	59.67	59.40	51.65	51.78	53.35	50.69
6	67.98	210.75	213.88	212.70	211.08	211.96	214.15
7	30.11	42.08	37.97	46.83	46.52	46.95	46.73
8	34.79	36.61	37.06	37.94	37.79	37.67	35.65
9	39.81	40.83	39.60	53.77	53.35	53.82	53.66
10	35.90	38.22	39.99	41.53	40.99	40.00	42.98
11	20.72	21.23	20.83	21.03	20.94	21.08	21.17
12	39.92	39.41	42.91	39.45	39.25	39.44	39.35
13	42.80	43.02	43.09	42.89	42.90	42.84	42.55
14	55.89	55.74	55.79	55.71	56.58	56.69	56.61
15	28.10	27.88	27.98	27.67	51.79	53.35	23.91
16	24.17	23.85	23.96	23.88	23.79	21.70	27.91
17	56.14	56.70	56.82	56.73	55.62	55.75	55.69
18	11.98	11.92	11.96	12.00	11.92	11.92	12.01
19	23.48	22.40	23.16	12.29	12.39	13.48	13.55
20	35.32	35.19	35.28	35.29	35.19	35.29	35.30
21	18.21	18.18	18.23	18.21	18.13	18.23	18.22
22	30.91	30.80	30.90	30.89	30.79	30.90	31.04
23	31.02	30.95	31.05	31.03	30.94	31.03	30.89
24	174.71	174.50	174.67	174.67	174.56	174.64	174.72
CH3O-	51.48	51.44	51.52	51.50	51.42	51.49	51.54
MsO-	-	-	-	-	38.38	-	-

Methyl 3, 6-dioxo-5β-cholan-24-oate (6) and Methyl 3α-hydroxy-6-oxo-5β-cholan-24-oate (7). To a solution of 5 (16.50 g, 40.58 mmol) in DCM (100 mL), 8.75 g, (40.58 mmol) of PCC in 60 mL of DCM, were added by slow dripping. The reaction mixture was slowly stirred for 48 h at room temperature and the end of reaction was verified by TLC, filtered on silica (pore size 60 Å, 220–440 mesh) with DCM 30 mL. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 5.8:4.2). Two fractions were obtained: Fraction I, 4.95 g (30.0% yield) of compound 6; Fraction II, 11.2 g (68% yield) of compound 7. Compound 6 was a colorless solid (m.p. = 106–107 °C, MeOH/Et2O). IR (cm−1): 2947 (C-CH3); 2874 (C-CH3); 1743 (C=O); 1717 (C=O); 1689 (C=O); 1469 (CH2); 1383 (CH3); 1164 (C-O). 1H-NMR: 3.64 (s, 3H, CH3O); 2.63 (dd, J = 13.4 and 14.7 Hz, 1H, H-12); 0.93 (s, 3H, H-19); 0.91 (d, J = 6.5 Hz, 3H, H-21); 0.67 (s, 3H, H-18). 13C-NMR: See Table 2. Compound 7 was a colorless solid (m.p. = 95–100 °C, MeOH/Et2O). IR (cm−1): 3301 (O-H); 2943 (C-CH3); 2867 (C-CH2-C); 2849 (C-CH2-C); 2828 (CH3O); 1740 (C=O); 1709 (C=O); 1436 (CH2); 1326 (CH3); 1171 (C-O). 1H-NMR: 3.66 (s, 3H, CH3O); 3.62 (m, 1H, H-3); 2.33 (ddd, J = 5, 10 and 15.2 Hz, 1H, H-23); 0.92 (d, J = 6.4 Hz, 3H, H-21); 0.83 (s, 3H, H-19); 0.64 (s, 3H, H-18). 13C-NMR: See Table 2. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Methyl 3α-hydroxy-6-oxo-5β-cholan-24-oate (). A solution of compound 6 (4.95 g, 12.3 mmol) was prepared in 100 mL of MeOH. This solution was placed in a bath of ice-water between 0–5 °C. Subsequently 0.47 g (12.3 mmol) of NaBH4 were added in four portions (approximately 0.118 g each) maintaining the temperature and with slow stirring. The end of reaction was verified by TLC, so 20 mL of acetone and 5 mL of HCl 2.5% were added, maintaining the reaction temperature. The reaction mixture was concentrated by evaporation under reduced pressure to a volume of about 15 mL, and then AcOEt (50 mL) was added. The organic layer was washed with saturated solution of NaHCO3 (20 mL) and water (2 × 30 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 5.8:4.2) Two fractions were obtained: Fraction I, 2.88 g of unreacted compound 6; Fraction II, 2.04 g (41.2%) of compound 7. Later reduction with NaHB4/CH3OH at 0–5 °C of recovered compound 6 (2.88 g), produced 2.75 g of 7 with 95.5% yield. The total yield in both reductions for compound 7 was 96.8%. The physical and spectroscopic properties of compound 7 were identical to those reported above for oxidation of compound 5. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Methyl 3α-hydroxy-6-oxo-5α-cholan-24-oate (8). Compound 7 (4.79 g, 11.8 mmol) was dissolved in 100 mL of 2.5% v/v HCl-MeOH, at room temperature and constant agitation for 24 h. The end of reaction was verified by TLC. The solvent was evaporated under reduced pressure and the crude was re-dissolved in 40 mL of AcOEt. The organic layer was washed with saturated solution of NaHCO3 (30 mL) and water (2 × 30 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 6.0:4.0). Compound 8 (4.79 g, 94.4% yield) was a colorless solid (m.p. = 128–132 °C, MeOH/Et2O) IR (cm−1): 3302 (O-H); 2943 (C-CH3); 2867 (C-CH2-C); 2849 (C-CH2-C); 2828 (CH3O); 1740 (C=O); 1709 (C=O); 1436 (CH2); 1375 (CH3); 1257 (C-O); 1172 (C-O). 1H-NMR: 4.16 (m, 1H, H-3); 3.66 (s, 3H, CH3O); 2.70 (t, J = 7.9 Hz, 1H, H-5); 2.33 (ddd, J = 5.3, 10.3 and 15.5 Hz, 1H, H-23); 0.91 (d, J = 6.4 Hz, 3H, H-21); 0.72 (s, 3H, H-19); 0.65 (s, 3H, H-18). 13C-NMR: See Table 2. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Methyl 3α-methanesulfonyl-6-oxo-5α-cholan-24-oate (9). Compound 8 (1.00 g, 2.47 mmol) was dissolved in 20 mL of DCM, 5 mg of DMAP and 1 mL of pyridine were added, then the mixture was cooled to 0–5 °C. Later 1.0 mL (12.9 mmol) of CH3SO2Cl slowly was added with gentle agitation. The mixture was kept at 0 °C for 30 min and subsequently left at room temperature. The end of reaction was verified by TLC. The solvent was evaporated under reduced pressure and the crude was re-dissolved in 20 mL of AcOEt. The organic layer was washed with saturated solution of NaHCO3 (40 mL) and water (2 × 30 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 5.8:4.2). Two fractions were obtained: Fraction I, 726 mg (61.0% yield) of compound 9, and Fraction II, 230 mg of unreacted compound 8. Compound 9 was a colorless solid (m.p. = 129–131 °C, MeOH/Et2O) IR (cm−1): 2945 (C-CH3); 2867 (C-CH2-C); 1736 (C=O); 1709 (C=O); 1435 (CH2); 1351 (CH3); 1255 (C-O); 1171 (C-O). 1H-NMR: 5.02 (b.s, 1H, H-3); 3.64 (s, 3H, CH3O); 2.97 (s, 3H, CH3SO3); 2.60 (dd, J = 2.3 and 12.4 Hz, 1H, H-5); 0.901 (d, J = 6.4 Hz, 3H, H-21); 0.713 (s, 3H, H-19); 0.639 (s, 3H, H-18). 13C-NMR: See Table 2. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Methyl 2-en-6-oxo-5α-cholan-24-oate (10). To a solution of compound 9 (1.30 g, 0.269 mmol) in 40 mL of DMF, 200 mg Li2CO3 (2.71 mmol) and LiBr (235 mg, 2.71 mmol) were added. Then the reaction mixture was stirred and refluxed at 80 °C for 1.5 h. The end of reaction was verified by TLC, the mixture was then filtered and the mother liquor concentrated to a volume of approximately 15 mL under reduced pressure. Then AcOEt (30 mL) were added. The organic layer was washed with water (2 × 20 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 2.4:7.6). Compound 10 (0.900 g, 74.9% yield) was a colorless solid (m.p. = 67–69 °C, hexane/Et2O) IR (cm−1): 3020 (CH=); 2968 (CH3-); 2939 (CH3-); 2899 (C-CH2-C); 2867 (C-CH2-C); 1737 (C=O); 1703 (C=O); 1635 (C=C); 1435 (CH2); 1383 (CH3); 1250, (C-O); 1168 (C-O). 1H-NMR: 5.69–5.66 (m, 1H, H-3); 5.57–5.55 (m, 1H, H-2); 3.66 (s, 3H, CH3O); 0.924 (d, J = 6.4 Hz, 3H, H-21); 0.701 (s, 3H, H-19); 0.665 (s, 3H, H-18). 13C-NMR: See Table 2. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Methyl 2α,3α-dihydroxy-6-oxo-5α-cholan-24-oate (11). A solution of compound 10 (500 mg, 1.29 mmol) in 30 mL of acetone and 10 mL water was prepared. Later 10 mg of NMMNO, 2 mL 4.0% (0,314 mmol) OsO4 solution and 2 mL of pyridine were added. The mixture was kept under constant stirring at room temperature for 72 h. The end of reaction was verified by TLC, and then 25 mL of Na2S2O3 saturated solution were added and allowed to stir for 30 min. The reaction was concentrated to a volume of approximately 15 mL under reduced pressure. Then AcOEt (40 mL) were added. The organic layer was washed with water (2 × 25 mL), dried over anhydrous Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 8.2:1.8). Two fractions were obtained: Fraction I, 218 mg of unreacted compound 10 and Fraction II, 277 mg (51.0% yield) of compound 11. Compound 11 was a colorless solid (m.p. = 178–179 °C, MeOH/Et2O). IR (cm−1): 3392 (OH); 2944 (CH3-); 2867 (C-CH2-C); 1738 (C=O); 1709 (C=O); 1435 (CH2); 1376 (CH3); 1255, C-O); 1170 (C-O). 1H-NMR: 4.05 (b.s, 1H, H-3); 3.77 (b.d, J = 11.1 Hz, 1H, H-2); 3.66 (s, 3H, CH3O); 2.67 (dd, J = 3.0 and 12.6 Hz, 1H, H-5); 0.926 (d, J = 6.4 Hz, 3H, H-21); 0.752 (s, 3H, H-19); 0.659 (s, 3H, H-18). 13C-NMR: See Table 2. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Methyl 3α-acetoxy-6-oxo-5α-cholan-24-oate (12). To a solution of compound 8 (2.00g, 4.94 mmol) in 20 mL of DCM, 5 mg of DMAP, 1 mL of pyridine and 0.5 mL (5.3 mmol) of Ac2O were added. The reaction mixture was kept under constant stirring and room temperature for 30 min. The end of reaction was verified by TLC, the mixture was then concentrated to a volume approximately 5 mL under reduced pressure. Then AcOEt (30 mL) were added. The organic layer was washed with water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 4.7:5.3). Compound 12 (1.9 g, 95% yield) was a colorless solid (m.p. = 174–176 °C, MeOH/Et2O) IR (cm−1): 2943 (CH3); 2869 (CH2); 1737 (C=O); 1708 (C=O); 1435 (CH2); 1376 (CH3); 1262 (C-O); 1221 (C-O); 1172 (C-O). 1H-NMR: 5.12 (m, 1H, H-3); 3.66 (s, 3H, CH3O); 2.56 (dd, J = 3.3 and 12.0 Hz, 1H, H-5); 2.38 (ddd, J = 5.4, 10.3 and 15.4 Hz, 1H, H-23); 2.04 (s, 3H, CH3CO); 0.94 (d, J = 6.4 Hz, 3H, H-21); 0.74 (s, 3H, H-19); 0.67 (s, 3H, H-18). 13C-NMR: See Table 1. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Acid 3α-hydroxy-6-oxo-5α-cholan-24-oic (13). A solution of compound 8 (5.0 g, 12.36 mmol) in 60 mL of MeOH was prepared. Later 20 mL of 15% P/V K2CO3 solution were added, and the reaction mixture was refluxed for 1 h. The end of reaction was verified by TLC, the mixture was then concentrated to a volume approximately 15 mL under reduced pressure. This was acidified with 20 mL of 10% HCl solution. Then AcOEt (40 mL) were added. The organic layer was washed with water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and a colorless solid (4.11 g, 82.2% yield) was obtained. Compound 13 (m.p. = 102–107 °C, MeOH/Et2O) IR (cm−1): 3403–2500 (O-H); 2945 (CH3); 2869 (CH2); 1712 (C=O); 1689 (C=O); 1442 (CH2); 1380 (CH3). 1H-NMR (CD3OD): 4.04 (m, 1H, H-C3); 2.75 (t, J = 8.1 Hz, 1H, H-5); 2.31 (ddd, J = 5.3, 9.9 and 15.2 Hz, 1H, H-23); 0.96 (d, J = 6.5 Hz, 3H, H-21); 0.730 (s, 3H, H-19); 0.71 (s, 3H, H-18). 13C-NMR: See Table 1. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Acid 3α-acetoxy-6-oxo-5α-cholan-24-oic (14). To a solution of compound 13 (5.0 g, 12.8 mmol) in 60 mL of DCM, 10 mg of DMAP, 1 mL of pyridine and 1.2 mL (12.8 mmol) of Ac2O were added. The reaction mixture was kept under constant stirring and room temperature for 1 h. The end of reaction was verified by TLC, the mixture was then concentrated to a volume approximately 10 mL under reduced pressure. Then AcOEt (40 mL) were added. The organic layer was washed with 5% HCl (1 × 10 mL), water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 8.8:11.2). Compound 14 (3.64 g, 72.8% yield) was a colorless solid (m.p. = 171–177 °C, MeOH/Et2O) IR (cm−1): 3373-2495 (O-H); 2947 (CH3); 2869 (CH2); 1736 (C=O); 1708 (C=O); 1444 (CH2); 1376 (CH3). 1H-NMR: 5.12 (m, 1H, H-3); 2.56 (dd, J = 3.3 and 12.0 Hz, 1H, H-5); 2.38 (ddd, J = 5.4, 10.3 and 15.4 Hz, 1H, H-23); 2.03 (s, 3H, CH3CO); 0.94 (d, J = 6.4 Hz, 3H, H-21); 0.74 (s, 3H, H-19); 0,67 (s, 3H, H-18). 13C-NMR: See Table 1. 1H-NMR and 13C-NMR are shown in Supplementary Materials.

Methyl 3α-hydroxy-6-oxo-7-oxa-5α-cholan-24-oate (15) and methyl 3α-hydroxy-6-oxa-7-oxo-5α-cholan-24-oate (16). To a solution of 8 (100 mg, 0.247 mmol) in 60 mL of DCM, 55.3 mg (0.320 mmol) of m-CPBA (77%) and 269 mg (0.320 mmol) of NaHCO3 were added and the mixture was stirred at room temperature by 24 h. The end of reaction was verified by TLC, the mixture was filtered then concentrated to a volume approximately 5 mL under reduced pressure. Then AcOEt (40 mL) were added. The organic layer was washed with saturated NaHCO3 solution (2 × 20 mL), water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 → 6.0:4.0) Three fractions were obtained. Fraction I: 37 mg of unreacted compound 8. Fraction II: 11.2 mg (10.8% yield) of compound 15 and Fraction III: 15.5 mg (14.9% yield) of compound 16. Compound 15 was a colorless solid (m.p. = 140–142 °C, MeOH/Et2O) IR (cm−1): 3446 (O-H); 2947 (C-CH3); 2871 (C-CH2-C); 2849 (C-CH2-C); 1732 (C=O); 1436 (CH2); 1373 (CH3); 1251 (C-O); 1168 (C-O). 1H-NMR: 4.15 (b.s. 1H, H-3); 4.08-4.06 (m, 2H, H-7); 3.66 (s, 3H, CH3O); 3.17 (dd, J = 4.4 and 12.2 Hz, 1H, H-5); 0.907 (d, J = 6.5 Hz, 3H, H-21); 0.878 (s, 3H, H-19); 0.700 (s, 3H, H-18). 13C-NMR: See Table 1. Compound 16 was a colorless solid (m.p. = 155–157 °C). IR (cm−1): 3446 (O-H); 2946 (C-CH3); 1732 (C=O); 1444 (CH2); 1377 (CH3); 1250 (C-O); 1166 (C-O). 1H-NMR: 4.60 (dd, J = 5.3 and 11.3 Hz, 1H, H-5); 4.20 (b.s. 1H, H-3); 3.65 (s, 3H, CH3O); 0.897 (d, J = 6.6 Hz, 3H, H-21); 0.878 (s, 3H, H-19); 0.678 (s, 3H, H-18). 13C-NMR: See Table 1. 1H-NMR and 13C-NMR are shown in Supplementary Materials

4. Conclusions

An improved synthesis of known brassinosteroid analogues 7-oxalactone 15, 6-oxalactone 16 and the 2α,3α-diol 11 from hyodeoxycholic acid has been described (4). These compounds and intermediates were obtained through modification of hyodeoxycholic acid as described previously. In the alternative procedure described here, the co-product 6 obtained in the oxidation of methyl hyodeoxycholate 5 was converted into the target monoketone 7 by stereoselective reduction with NaBH4, increasing the overall yield of this synthetic route to 96.8%.

Additionally, using mono- and bi-dimensional NMR techniques, we have updated the 1H-NMR spectroscopic information for derivatives of hyodeoxycholic acid 5–16, and a complete assignment of all 13C-NMR signals has also been made.

Derivatives 8, 13 and 14 will be used in the synthesis of new brassinosteroid analogues, while derivatives 8, 11, 15 and 16 will be evaluated as plant-growth regulators in the Rice Lamina Inclination Assay and for antifungal activity in plants. These results will be reported later.