Brassinosteroids (Brs) are a naturally occurring steroidal plant hormones group that regulates plant growth and development by producing an array of physiological changes. Brs occur at low concentrations throughout the plant kingdom. They have been detected in all plant organs (pollen, anthers, seeds, leaves, stems, roots, flowers, and grains) and also in the insect and crown galls [1,2]. Further work has demonstrated that Brs not only induce stem elongation but also increase biomass and total crop yield. Moreover, Brs are recognized to have an ameliorative role in plants subjected to various biotic and abiotic stresses, such as high temperature [3], heavy metals excess [4,5], salinity [6], water stress [7,8] and extreme temperatures [9]. Several authors and mainly Hayat et al. have reported structure-activity relationships (SAR) of brassinosteroid [10]. These SAR are based on the functions contained in the A, B ring, A/B ring fusion and in the side chain. However, in recent decades, efforts have been focused on the synthesis of new brassinosteroid analogues, keeping common patterns of organic functions in the A/B rings and cis-trans fusion between these, as in some natural brassinosteroids, but with dramatic structural changes in the side chain (shorter side chains, different oxygenated functions, spirostanic, aromatic and cyclic substituents, esters, carboxylic acids, etc.) and oxygenated functions in C ring. Surprisingly these analogs have presented very important biological activities. On the other hand, the isolation of natural brassinosteroids with oxygenated function in ring C has not been reported. However, the synthesis of this type of analogs is very important for SAR studies of this kind of phytohormone. In this direction, hecogenin (1) is an abundantly available steroidal sapogenin, used as raw material in the production of a large number of Brs spirostanic analogs with oxygenated function in ring C [11-21]. Examples of brassinosteroid analogues oxo-functionalized in C ring are shown in Figure 1. Others active Brs C- oxo and oxa functionalized analogs (6-8) bearing a cholanic acid skeleton were derived from cholic acids [22]. Nevertheless, C-functionalized analogs 9-16 (Figure 2) were obtained from deoxycholic acid [23-25]. The plant growth-promoting activity of compounds 9 and 13 was tested in the hypocotiles elongation and cothyledons expansion of radish bioassay, where the compound 9 showed growth promoting activity at 10^-5 mg/mL concentration in both bioassays, whereas compound 13 showed inhibiter effect in the cothyledons expansion test at the same concentration [24]. Compound 14 showed an increase of 38.6% by weight at 10^-5 mg/mL concentration in cotyledons expansion of radish bioassay [25]. In this work is reported the synthesis and structural determination of four new Brs analogues, obtained from deoxycholic acid, with a cis-A/B ring junction, 24- nor side chain and 11-oxo-12β-hydroxyl/acetate function on C ring containing with 5β-cholanic acid skeleton.

Figure 1: Structure of hecogenin (1) and some Brs spirostanic synthetic analogs (2-5), with oxygenated function in ring C.

Figure 2: Structure of 12-oxo (6-7) and 12-oxa (8) functionalized active Brs analogs bearing a cholanic acid skeleton and 12-oxa and 11-oxa C-functionalized (12-14), 11-oxo-12β-hydroxy (15) and 11-oxo-12β-acetoxy (16) deoxycholic acid derivative.

General

All reagents were purchased from commercial suppliers and used without further purification (Merck, Darmstadt, Germany or Aldrich, St. Louis, MO, USA). Melting points were measured on a Stuart-Scientific SMP3 apparatus (Staffordshire, ST15 OSA, UK) and are uncorrected. Optical rotations were obtained for CHCl₃ or CH₃OH, solutions on a Perkin-Elmer 241 polarimeter (Wellesley, Massachusetts, USA) and their concentrations are expressed in g/100 mL. NMR spectra were recorded on a Bruker AM-200 (Bruker, Rheinstetten, Germany) spectrometer operating at 200.1 MHz for ¹H and 50.3 MHz for ¹³C. Chemical shifts are expressed in ppm downfield relative to TMS (δ scale) in CDC1₃ solutions and coupling constants (J) are given in hertz. Carbon multiplicity were established by a DEPT pulse sequence. IR spectra were recorder as KBr disks in a Bruker FTIR Vector-22 (Bruker, Germany) and frequencies are in cm^-1. Elemental analyses were obtained on a Fisons-Carlo-Erba EA-1108 Automost microanalyzer (Fisons Instruments/Carlo-Erba Instruments, Milano, Italy) For analytical TLC, Merck silica gel 60 in 0.25 mm layer was used and TLC spots were detected by heating after spraying with 25% H₂SO₄ in H₂O. Chromatographic separations were carried out by conventional column on Merck silica gel 60 (230-400 Mesh) using hexane-EtOAc gradients of increasing polarity. All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure, below 40°C.

Synthesis

24-oic 3α, 12β-diacetoxy-11-oxo-5β-cholan acid (17)

A solution of 16 (3.2 g, 7.61 mmol) and K₂CO₃ (0.8 g, 7.55 mmol) in MeOH (150 mL) was stirred at room temperature for 1.5 h. The end of the reaction was verified by TLC. Then the solvent was removed (until a 40 mL approximate volume) and the residue acidified with 2% HCl (15 mL) and extracted with EtOAc (3 × 20 mL). The organic layer was washed with 5% NaHCO₃ (30 mL) and water (2 × 15 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 petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→10.2:9.8). Compound 17 (3.17 g, 85% yield) was as a colorless solid: m.p. 85.5-91.7°C (MeOH/Et₂O); [α] _D ²⁵+46.2° (c=0.405, CHCl₃); IR: 3432-2516; 1735; 1243; 1028. ¹H NMR: 0.69 (s, 3H, H-18); 0.93 (d, J=6.3 Hz, 3H, H-21); 1.16 (s, 3H, H-19); 2.02 (s, 3H, CH₃CO); 2.15 (s, 3H, CH₃CO); 2.35 (m, 2H, H-23); 2.49 (d, J=10.6 Hz, 1H, H-9); 4.70 (m, ¹H, H-3); 4.90 (s, 1H, H-12). Elemental analysis: found C, 68.22%; H, 8.57%; C₂₈H₄₂O₇ requires C, 68.54%; H, 8.63%.

3α, 12β-diacetoxy-24-nor-5β-cholan-22-en-11-one (18)

To a solution of 17 (3.42 g, 7.69 mmol) in anhydrous benzene (300 mL) were added Cu(OAc)₂ (0.25 g, 1.38 mmol) and pyridine (1.0 mL). Then refluxed and Pb(OAc)₄ (8.34 g, 18.81 mmol) was added in four portions at hourly intervals. After the addition was completed, the reaction was continued for 1 h. The end of reaction was verified by TLC, and then the mixture was filtered and the solvent was evaporated under reduced pressure. The crude was re-dissolved in CH₂Cl₂ (5 mL) and chromatographed on silica gel with petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→16.4:3.6). Compound 18 (2.24 g, 72.3% yield) was obtained as a colorless solid: m.p. 170.9-172.8°C (hexane/ Et₂O); [α]_D²⁵+64.8° (c=0.466, CHCl₃); IR: 3069; 1732; 1723; 1637; 1450; 1364; 1237; 1029; 910. ¹H NMR: 0.66 (s, 3H, H-18); 0.99 (d, J=6.9 Hz, 3H, H-21); 1.16 (s, 3H, H-19); 2.02 (s, 3H, CH₃CO); 2.18 (s, 3H, CH₃CO); 2.46 (m, 2H, H-1 and H-20); 2.48 (d, J=10.2 Hz, 1H, H-9); 4.70 (m, ¹H, H-3); 4.90 (dd, J=10.1 and 2.1 Hz, ¹H, H-23); 4.92 (s, ¹H, H-12); 4.95 (ddd, J=17.1, 2.1 and 0.8 Hz, 1H, H-23); 5.77 (ddd, J=17.1, 10.1 and 8.8 Hz, 1H, H-22). Elemental analysis: found C, 72.87%; H, 9.13%; C₂₇H₄₀O₅ requires C, 72.94%; H, 9.07%.

3α, 12β-dihydroxy-24-nor-5β-cholan-22-en-11-one (19)

To a solution of 18 (1.58 g, 3.55 mmol) in MeOH (60 mL) was added K₂CO₃ (0.79 g, 7.54 mmol), then the suspension was stirred at room temperature for 6 h. The end of the reaction was verified by TLC. Then the solvent was removed (until a 10 mL approximate volume) and the residue acidified with 2% HCl (10 mL) and extracted with EtOAc (2 × 20 mL). The organic layer was washed with 5% NaHCO₃ (20 mL) and water (2 × 10 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 petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→13.8:6.2). Compound 19 (1.26 g, 98% yield) was as a colorless solid: m.p. 120.3- 121.2°C (MeOH/Et₂O); [α]_D ²⁵+61.4° (c=0.44, CHCl₃); IR: 3490; 3258; 3066; 1709; 1638; 1453; 1020; 922; 906. ¹H NMR: 0.43 (s, 3H, H-18); 1.06 (d, J=6.9 Hz, 3H, H-21); 1.13 (s, 3H, H-19); 2.43 (m, 1H, H-1α); 2.55 (m, 1H, H-20); 3.57 (m, 1H, H-3); 3.84 (s, 1H, H-12); 4.82 (dd, J=10.2 and 2.2 Hz, 1H, H-23); 4.91 (ddd, J=17.1, 2.2 and 0.8 Hz, 1H, H-23); 5.73 (ddd, J=17.1, 10.2 and 8.7 Hz, 1H, H-22). Elemental analysis: found C, 76.49%; H, 10.15%; C23H36O3 requires C, 76.62%; H, 10.06%.

3α, 12β-diacetoxy-22(S), 23-dihydroxy-24-nor-5β-cholan-11-one (20)

To a solution of alkene 18 (2.05 g, 4.28 mmol) in acetone (150 mL) was added NMO (0.45 g, 3.84 mmol). Then the mixture was homogenized by magnetic stirring and 1.5 mL of 4% OsO₄ (0.157 mmol) were added dropwise with stirring for 12 h at room temperature. The end of the reaction was verified by TLC. Then the solvent was removed (until a 25 mL approximate volume) and water (25 mL) and Na₂S₂O₃.5H₂O (25 mL saturated solution) were added. The organic layer was extracted with EtOAc (2 × 30 mL), washed with water (2 × 50 mL), dried over 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 petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→15.6:4.4), by subsequent recrystallization (MeOH/Et₂O) compound 20 was obtained as a colorless solid (1.50 g, 68% yield): m.p. 183.7-186.6°C (MeOH/Et₂O); [α]_D ²⁵+27.6° (c=0.156, CHCl₃); IR: 3423; 1747; 1723; 1248; 1026. ¹H NMR: 0.67 (s, 3H, H-18); 0.91 (d, J=6.3 Hz, 3H, H-21); 1.15 (s, 3H, H-19); 2.00 (s, 3H, CH₃CO); 2.14 (s, 3H, CH₃CO); 2.47 (d, J=11.1 Hz, 1H, H-9); 3.47 (m, 1H, H-23); 3.63 (m, 1H, H-23); 3.72 (m, 1H, H-22); 4.68 (m, 1H, H-3); 4.88 (s, 1H, H-12). Elemental analysis: found C, 67.60%; H, 8.87%; C₂₇H₄₂O₇ requires C, 67.75%; H, 8.85%.

3α, 12β, 22(S), 23-tetrahydroxy-24-nor-5β-cholan-11-one (21)

To a solution of 20 (0.07 g, 0.146 mmol) in MeOH (50 mL) was added K₂CO₃ (0.035 g, 0.215 mmol), then the suspension was stirred at room temperature for 6 h. The end of the reaction was verified by TLC. Then the solvent was removed (until a 10 mL approximate volume) and the residue acidified with 2% HCl (2 mL) and extracted with EtOAc (2 × 10 mL). The organic layer was washed with 5% NaHCO₃ (10 mL) and water (2 × 10 mL), dried over Na₂SO₄, and filtered. The solvent was evaporated under reduced pressure. Subsequent recrystallization (MeOH/Et₂O) gave compound 21 (0.0485 g, 84.1% yield) which it was identified as a colorless solid: m.p. 152.8-154°C (MeOH/Et₂O); [α]_D ²⁵+99.5° (c=0.20, MeOH); IR: 3428; 1700; 1066; 1022. Elemental analysis: found C, 69.71%; H, 9.61%; C₂₃H₃₈O₅ requires C, 70.02%; H, 9.71%.

3α, 12β, 22(S), 23-tetraacetoxy-24-nor-5β-cholan-11-one (22)

Compound 20 (0.09 g, 0.188 mmol) was dissolved in CH₂Cl₂ (30 mL) and pyridine (3.0 mL). Later 4-N,N-dimethylaminopyridine (DMAP, 5 mg) and Ac₂O (3 mL) were added to the solution. The end of the reaction was verified by TLC (2 h), and then the solvent was reduced to a volume about 5 mL, extracted with EtOAc (2 × 10 mL). The organic layer was washed with 5% KHSO₄ (2 × 5 mL) and water (2 × 10 mL), dried over Na₂SO₄ and filtered. The solvent was evaporated under reduced pressure. The crude was redissolved in CH₂Cl₂ (3 mL) and chromatographed on silica gel with petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2 → 14.2:5.8), to yield pure 20 (0.09 g, 85.1% yield) as a colorless solid: m.p. 78.1-79.5°C (hexane/EtOAc); [α] _D ²⁵+41.5° (c=0.412, CHCl₃); IR: 1740; 1451; 1370; 1243; 1028. ¹H NMR: 0.65 (s, 3H, H-18); 0.99 (d, J=6.7 Hz, 3H, H-21); 1.15 (s, 3H, H-19); 2.01 (s, 3H, CH₃CO); 2.04 (s, 3H, CH₃CO); 2.07 (s, 3H, CH₃CO); 2.14 (s, 3H, CH₃CO); 2.50 (d, J=11.1 Hz, 1H, H-9); 3.97 (dd, J=12.1 and 9.0 Hz, 1H, H-23); 4.29 (dd, J=12.1 and 2.0 Hz, 1H, H-23); 4.69 (m, ¹H, H-3); 4.85 (s, 1H, H-12); 5.14 (m, 1H, H-22). Elemental analysis: found C, 66.10%; H, 8.13%; C₃₁H₄₆O₉ requires C, 66.17%; H, 8.24%.

3α, 12β-diacetoxy-[2,2-dimethyl-22(S), 23-dioxolane]-24-nor-5β- cholan-11-one (23)

To a solution of 20 (0.08 g, 0.167 mmol) in dry acetone (50 mL) was added anhydrous CuSO₄ (0.250 g, 1.57 mmol), then the suspension was stirred in N₂ atmosphere at room temperature for 5 days. The end of the reaction was verified by TLC. Then the mixture was filtered and the solvent was evaporated under reduced pressure. The crude was re-dissolved in CH₂Cl₂ (3 mL) and chromatographed on silica gel with petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→16.8:3.2) to yield pure 23 (0.06 g, 69.2% yield) as a colorless oil; [α]_D ²⁵+32.5° (c=0.520, CHCl₃); IR: 1734; 1699; 1245; 1066; 1030. ¹H NMR: 0.67 (s, 3H, H-18); 0.85 (d, J=6.9 Hz, 3H, H-21); 1.13 (s, 3H, H-19); 1.29 (s, 3H, O₂CCH₃); 1.36 (s, 3H, O₂CCH₃); 2.01 (s, 3H, CH₃CO); 2.14 (s, 3H, CH₃CO); 2.45 (m, 1H, H-9); 3.53 (dd, J=7.8 Hz and 7.8 Hz, 1H, H-23); 3.89 (dd, J=7.8 and 6.3 Hz, 1H, H-23); 4.07 (m, 1 H, H-22); 4.67 (m, 1H, H-3); 4.89 (s, 1H, H-12). Elemental analysis: found C, 69.50%; H, 8.97%; C₃₀H₄₆O₇ requires C, 69.47%; H, 8.94%.

Previously we have reported the synthesis, and structural determination, of compounds 15 and 16 from deoxycholic acid [25]. Selective saponification of 16 in K₂CO₃/MeOH at room temperature afforded acid 17 with 85% yield (Scheme 1). The presence of the carboxylic function was confirmed by the signal at δC=179.27 ppm in 13C NMR spectrum (see Table 1). The olefinic intermediate 18 (Scheme 1) was obtained with 72.3% yield by decarboxylation reaction of 17 in Pb(OAc)₄/Cu(AcO)₂ system, as reported for the preparation of other derivatives [24,26-28]. The presence of terminal exocyclic double bond was confirmed by the signals observed in the ¹H NMR spectrum at δ_H=4.90 ppm (dd, J=10.1 and 2.1 Hz, 1H, H-23); 4.95 ppm (ddd, J=17.1, 2.1 and 0.8 Hz, 1H, H-23) and 5.77 ppm (ddd, J=17.1, 10.1 and 8.8 Hz, 1H, H-22). While two signals at δC=113.36 and 142.79 ppm were observed in the ¹³C NMR, assigned to at C-22 and C-23 respectively (see Table 1). Saponification of olefin 18 under mild conditions (K₂CO₃/ MeOH at room temperature for six hours) produces the ketol 19 with 98% yield (Scheme 1) The presence of two signals at δ_H=3.57 ppm (m, 1H) and δ_H=3.84 ppm (s, 1H) in the ¹H NMR spectrum confirms the removal of acetate groups. These carbinolic hydrogens were assigned as H-C3 and H-C12, respectively, according to their observed hydrogen multiplicities. The next step was the dihydroxylation reaction of olefin 18, however, some authors have reported that the electrophilic reactions at the steroidal C-22 double bond with OsO₄, RuCl₃/NaIO₄, Prevost-Woodward reaction with I2/AgOAc produces predominantly (S) configuration at C-22 [29-31]. Then treatment of the alkene 18, with catalytic OsO₄ in NMO and purification of reaction crude by C.C. and subsequent crystallization, afforded compound 20 with 68% yield (Scheme 1). The presence of three carbinolic proton signals at δ_H=3.47 ppm (m, 1H); 3.63 ppm (m, 1H) and 3.72 ppm (m, 1H) in the 1H NMR spectrum that were assigned to the hydrogens H-23a, H-23b and H-22, respectively by 2D heteronuclear correlations with C-23 and C-22 and ¹³C DEPT-135 experiment. The configuration at C-22 was established as (S) considering referential aspects for this reaction and comparing NMR spectroscopic data for similar compounds previously reported by us for other derivatives [23,24]. The new derivatives 21, 22 and 23 were prepared from compound 20, according to Scheme 2. Compound 21 was obtained by treatment of 20 under mild saponification conditions (K₂CO₃/MeOH, r.t.) with 84.1% yield, whereas standard acetylation (Ac₂O/DMAP/CH₂Cl₂) of compound 20 produces the tetra-acetylated derivative 22 with 85.1% yield. 1H NMR spectroscopic evidence indicates the presence of four singlets signals at δ_H=2.01, 2.04, 2.07 and 2.14 ppm (3H, CH₃CO each), while six signals at δC=20.86, 20.90, 21.25, 21.40, 169.94, 2 x 170.46 and 170.94, ppm were observed in 13C NMR (Table 1), so confirming the presence of four acetate groups in the molecule. Finally, by ketalization reaction of 20 with (CH₃)₂CO/CuSO₄ anhydrous system, the ketal derivative 23 was obtained with 69.2% yield, according to the methodology previously described. In the ¹H NMR spectrum of compound 23 two singlets signals at δ_H=1.29 and 1.36 ppm were observed, these were assigned to the methyl of acetonide group [O₂C(CH₃₎₂]. While in the ¹³C NMR spectrum, the signals observed at δC=25.33 (CCH3), 26.39 (CCH₃) and 108.30 ppm [O₂C(CH₃₎₂], confirming the presence of acetonide group.

Scheme 1: Synthetic route developed for intermediates 17-19 and Brs analog 20. Conditions and reagents: a. K2CO3/MeOH, r.t. 1.5 h, 85%. b. Pb(OAc)₄/Cu(OAc)₂/C₆H₆, reflux, 2.5 h, 72.3%. c. K₂CO₃/MeOH, r.t. 6 h, 98%. d. OsO₄ 4%, (CH₃)₂CO/NMO, r.t. 12 h, 68%.

Scheme 2: Synthetic route developed for Brs analog 21-23. Conditions and reagents: a. K₂CO₃/MeOH, r.t. 6 h, 84.1%. b. Ac₂O/DMAP/py/CH₂Cl₂, 2 h, 85.1%. c.CuSO₄/(CH₃)₂CO, r.t. 5 days, 69.2%.

Table 1: δ 13C NMR (CDCl₃, 50.3 MHz) for compounds 17-20 and 22-23.

We have designed a synthetic sequence, which allows the obtainment of three new synthetic derivatives (compounds 17-19) from commercial deoxycholic acid and four new brassinosteroids analogues (compounds 20-23) with 24-nor side chain, oxygenated function at C-22 (S) and C-23 and 11-oxo functionalized on C ring, containing 5β-cholanic acid skeleton. Bioassays in Rice Lamina Inclination Test (RLIT) and growth in Arabidopsis thaliana, to detect possible biological activity of compounds 20-23, are under being developed and these results will be reported later.

The author thank to the Dirección General de Investigación y Postgrado (DGIP-USM Grant No. 27.15.83.) of Universidad Técnica Federico Santa María for financial support of this work.