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

Abstract

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 ¹³C-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 ¹³C-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].

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].

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 (δ_H = 4.16 and δ_H = 2.70 ppm, respectively) in compound 8, are displaced to downfield as compared to compound 7 (δ_H = 3.62 and δ_H-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 (δ_C = 12.29 ppm) than in the spectrum of compound 7 (δ_C = 23.16 ppm).

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 δ_H = 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 δ_C = 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 δ_C = 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 (δ_H = 5.69–5.66 ppm, m, 1H) showed ³J_H-C correlation with C-1 (δ_C = 39.34 ppm) and ²J_H-C with C-4 (δ_C = 27.88 ppm), while H-2 (δ_H = 5.57–5.55 ppm, m, 1H) showed ³J_H-C correlation with C-4 (δ_C = 27.88 ppm) and ²J_H-C with C-1 (δ_C = 39.34 ppm).

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 δ_C = 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 δ_H = 2.04 ppm (3H, CH₃CO) and a multiplet at δ_H = 5.12 ppm corresponding to H-3β (m, 1H). The latter is shifted to downfield as compared with H-3β (δ_H = 4.16 ppm, m 1H) in compound 8. Additionally, in the ¹³C-NMR spectrum the signals appearing at δ_C = 170.31 (C=O) and 21.39 (CH₃CO) ppm (

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.

) confirmed the presence of acetylated derivative 12.

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 δ_C = 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 δ_H = 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 δ_C = 170.33 and 21.41 ppm (Table 1) were assigned to the acetyl group, while the signal at δ_C = 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 δ_H = 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 δ_C = 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., ²J_HC correlations between H-5α (δ_H = 3.17 ppm, dd, J = 4.4 and 12.2 Hz) with the signals at δ_C = 176.76 (C=O of lactone function), δ_C = 36.29 and δ_C = 30.81 ppm which were assigned to the carbons C-6, quaternary C-10 and C-4, respectively. Additionally, H-5α showed ³J_HC correlation with signals at δ_C = 30.81, δ_C = 14.52, δ_C = 32.88 and δ_C = 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.

A similar analysis was performed for the structure determination of 6-oxolactone 16; thus, in the ¹H-NMR spectrum for 16 a signal at δ_H = 4.60 ppm (dd, 1H, J = 5.3 and 11.3 Hz) was observed, correlated by 2D ¹H-¹³C HSQC with the signal at δ_C = 79.65 ppm (C-7) with impair multiplicity (from DEPT-135 analysis) and assigned to H-5α. Additionally, H-5 α showed ²J_HC correlation with signals at δ_C = 35.63 ppm and ³J_HC correlation with signals at δ_C = 11.53, δ_C = 57.97 and δ_C = 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. 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 (7). 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.