Stemodia chilensis Tetracyclic Diterpenoid - ACS Publications

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Proof of the Structure of the Stemodia chilensis Tetracyclic Diterpenoid (+)-19-Acetoxystemodan-12-ol by Synthesis from (+)-Podocarpic Acid: X‑ray Structure Determination of a Key Intermediate Francesca Leonelli,*,† Azzurra Mostarda,‡ Luca De Angelis,‡ Doriano Lamba,§ Nicola Demitri,⊥ Angela La Bella,‡ Francesca Ceccacci,∥ Luisa M. Migneco,‡ and Rinaldo Marini Bettolo*,‡ †

Dipartimento di Biologia Ambientale, ‡Dipartimento di Chimica, and ∥Istituto di Metodologie Chimiche-CNR, Unità Organizzativa di Supporto, Sede di Roma, Università degli Studi di Roma “La Sapienza”, P.le Aldo Moro, 5, I-00185 Roma, Italy § Istituto di Cristallografia-CNR, Unità Organizzativa di Supporto, Sede di Trieste, and ⊥Elettra Sincrotrone Trieste S.C.p.A. di Interesse Nazionale, Area Science Park−Basovizza, Strada Statale 14−Km 163.5, I-34149 Trieste, Italy S Supporting Information *

ABSTRACT: The first synthesis of (+)-19-acetoxystemodan-12-ol (1), a stemodane diterpenoid isolated from Stemodia chilensis, is described. The structure was supported by an X-ray crystallographic analysis of intermediate (+)-9a, which confirmed the proposed structure and excluded the structure of (−)-19-hydroxystemod-12-ene as a possible candidate for the Chilean Calceolaria diterpenoid to which the (−)-19-hydroxystemar-13-ene structure (9b) had been erroneously assigned.

I

n 1991, Garbarino and co-workers described the isolation of a new stemodane diterpenoid, (+)-19-acetoxystemodan-12ol (1), from Stemodia chilensis.1 The structure of this diterpenoid was established on the basis of its 1H and 13C NMR spectroscopic data and on their similarity with those of co-occurring (+)-2-deoxystemodinone (2). The absolute configuration of (+)-1 was considered reasonable because of its co-occurrence with (+)-2. No chemical correlation or X-ray structure determination was performed on (+)-1. Following the work on bicyclo[3.2.1]octane diterpenoids,2 we considered it interesting to confirm the structure of (+)-1. The interest in unambiguously clarifying the structure of Chilean tetracyclic diterpenoids is also shared by other scientists, as indicated in a recent publication.3

The starting material, (+)-podocarpic acid (3), was converted into the known (+)-12-hydroxybicyclo[2.2.2]octan2-one (4) as previously described.2i The secondary C-12 hydroxy group in (+)-4 is properly oriented for rearrangement to the stemodane system (Scheme 1).2a,b Nevertheless, prior to rearrangement, two synthetic operations were necessary: (a) removal of the C-15 carbonyl group, which would prevent the rearrangement to the stemodane system by inhibiting the formation of a positive charge on the adjacent bridgehead carbon, and (b) conversion of the 12-OH into a better leaving group. The latter synthetic operation would imply the selective protection of the 19-OH group. Compound (+)-4 was therefore converted into the corresponding dithioacetal (+)-5 using ethanedithiol and BF3· Et2O at 0 °C. The latter was chemoselectively acetylated at rt with AcCl in the presence of lutidine4 to afford the 19-acetoxy derivative (+)-6. The Raney-Ni reduction of (+)-6 in absolute EtOH under reflux afforded (−)-7, which was treated with MsCl in pyridine to afford (+)-19-acetoxystemod-12-ene 9a via 8. Compound (+)-9a was fully characterized through 1H NMR, 13 C NMR, COSY, HSQC, HMBC, DEPT 135, and NOESY experiments, and its relative configuration was unambiguously Received: September 17, 2015

© XXXX American Chemical Society and American Society of Pharmacognosy

A

DOI: 10.1021/acs.jnatprod.5b00834 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

Scheme 1. Synthesis of (+)-19-Acetoxystemodan-12-ol, 1a

Table 1. NMR Data for (+)-9aa and for an Acetyl Derivative of a Chilean Calceolaria Diterpenoidb acetyl derivative of Chilean Calceolaria diterpenoid

(+)-9a position

Reagents and conditions: (a) 1,2-ethanedithiol, BF3·Et2O, 0 °C, 59% yield; (b) 2,6-lutidine, AcCl, CH2Cl2, rt, 83% yield; (c) Raney-Ni, EtOH, 60 °C, 80% yield; (d) MsCl, NEt3, CH2Cl2, rt, 66% yield; (e) m-chloroperbenzoic acid, CH2Cl2, 89% yield; (f) LiAlH4, Et2O, 40% yield; and (g) 2,6-lutidine, AcCl, CH2Cl2, rt, 51% yield.

δC, type CH3 CH2 CH3 CH3 CH2

20 2 22 17 6

18.4, 18.6, 21.2, 21.9, 22.3,

18 16

28.2, CH3 33.3, CH2

1

35.2, CH2

3

36.4, CH2

7

36.4, CH2

11

37.1, CH2

4

37.3, C

10

38.9, C

14 8

39.3, CH 43.2, CH

2.13 t (6.3) 1.84−1.76, m

15

44.0, CH2

1.88−1.82, m

5

49.3, CH

1.40−1.32, m 1.49−1.42, m

9

50.2, C

19

67.6, CH2

a

confirmed by an X-ray crystallographic analysis using synchrotron radiation data collected at ELETTRA (XRD-1 beamline) (Figure 1).5

δH (J in Hz)

12

117.9, CH

13

146.0, C

21

171.6, C

Figure 1. ORTEP drawing of (+)-9a. Thermal ellipsoids are shown at the 50% probability level.

0.94 s 1.48−1.34, m 2.04 s 1.64 pd (1.4) 1.53−1.43, m 1.36−1.27, m 0.98 s 1.93−1.87, m 1.36−1.31, m 1.71−1.64, m 1.23−1.13, m 1.03−0.92, m 1.83−1.72, m 1.12−1.06, m 1.83−1.72, m 2.36−2.27, m 1.65−1.59, m

4.34 d (10.9) 3.92 dd (10.9, 1.1) 5.05−4.93, m

HMBCc 1, 5 1, 3

δC

14 5

18.0 18.4 21.0 21.7 22.0d

3, 5, 19 8, 15

24.6 27.3

20

28.1

18, 19

31.7

6, 8

34.2

8, 16

36.1d

5, 18, 19 8, 16, 20 16, 17 6, 14, 16 14, 16, 17

36.3 36.8 38.5 41.8 42.2

1, 6, 18, 20 8, 14, 16, 20 5, 19, 22

49.4

11, 14, 17 11, 14, 15, 16, 17 22

123.2

51.4 66.8

138.4 171.1

H and 13C NMR at 400.13 and 100.61 MHz, respectively; δ in ppm relative to the residual solvent peaks of CDCl3 at 7.26 and 77.0 ppm for 1H and 13C, respectively. bThe data reported for the acetyl derivative of an unknown compound from Chilean Calceolaria are those given in ref 5. 13C NMR at 63 MHz; δ in ppm relative to TMS as an internal standard; the signals are reported in decreasing order. c HMBC correlations, optimized for 8 Hz, are from carbons stated to the indicated protons. dInterchangeable within the same column. a1

Obtaining intermediate (+)-9a also offered the opportunity of ascertaining the alternative hypothesis of Garbarino and coworkers (stemodane vs stemarane)6a regarding the structure of a diterpenoid isolated from Chilean Calceolaria whose proposed structure of (−)-19-hydroxystemar-13-ene (9b) was not confirmed by synthesis.2i A comparison of the NMR data of (+)-9a with those reported for the acetyl derivative of a diterpenoid isolated from Chilean Calceolaria showed many differences (Table 1). According to these results, even the structure of (−)-19B



Note

In conclusion, the first synthesis of (+)-19-acetoxystemodan12-ol (1), a stemodane diterpenoid isolated from S. chilensis, was achieved and the structure was confirmed by an X-ray crystallographic analysis of intermediate (+)-9a. This work also demonstrated that the structure of (−)-19-hydroxystemod-12ene could be excluded as a possible candidate for the Chilean Calceolaria diterpenoid to which the structure of (−)-19hydroxystemar-13-ene had been erroneously assigned.2i,6 Further structural work on this unknown Chilean Calceolaria diterpenoid is therefore still necessary.

hydroxy-12-stemod-12-ene cannot be attributed to the diterpenoid isolated from the Chilean Calceolaria species, and thus, further structural work appears to be necessary. Compound (+)-9a dissolved in CH2Cl2 was reacted at rt with m-chloroperbenzoic acid (MCPBA) to diastereoselectively afford the epoxide (+)-10. As previously described by Kelly and co-workers,7 the epoxidation of the stemod-12-ene system proceeds from the less hindered α face. The reduction of the latter with LiAlH4 afforded the diol 11.8 Acetylation with AcCl at rt in the presence of lutidine4 afforded target compound (+)-1.8 Comparison of the NMR data of (+)-1 with those reported for the diterpenoid isolated from S. chilensis showed (Table 2) good overall matching signals, thus suggesting the identity of the two compounds. The stemodane absolute configuration of (+)-1 follows from its derivation from (+)-podocarpic acid, whereas the positive specific rotation follows from the work of Garbarino and co-workers.

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General Experimental Procedures. All solvents were of analytical grade. Melting points: Mettler-FP-61 (uncorrected). Optical rotations: DIP 370 Jasco digital polarimeter. IR spectra: ShimadzuFTIR 8400 S infrared spectrophotometer. 1H and 13C NMR spectra: Bruker AVANCE 400 at 400.13 and 100.61 MHz, respectively; δ in ppm relative to the residual solvent peaks of CDCl3 at 7.26 and 77.0 ppm for 1H and 13C, respectively; J in Hz. HRESIMS data: Micromass Q-TOF spectrometer (Waters) in electrospray-ionization mode. HPLC analysis: Shimadzu LC-10AD; RID detector; analytical columns, Luna 100/5 silicon (2), flow rate of 1 mL/min. GC-MS analysis: Shimadzu GCMS-QP5000. The yields after column chromatography were not optimized. TLC: silica gel 60 F254. Column chromatography (CC): silica gel 60, 70−230 mesh ASTM. Preparation of (+)-5 from (+)-4. To a stirred solution of (+)-4 (0.16 g, 0.51 mmol) in 1,2-ethanedithiol (0.97 mL, 12 mmol) cooled to 0 °C was added BF3·Et2O (0.39 mL, 3.2 mmol). After 5 min, TLC (EtOAc/n-hexane, 1:1) showed the disappearance of (+)-4. The mixture was diluted with CH2Cl2 (30 mL), washed with 2 N NaOH (3 × 6 mL), H2O (until neutral), and brine, dried, and evaporated. The mixture was purified by CC (EtOAc/n-hexane, gradient from 5:95 to 40:60) to afford (+)-5 (0.12 g, 0.31 mmol, 59%): white powder, mp 161.6−163.2 °C; [α]20D = +2.7 (c 4.0, CHCl3); IR (CHCl3) 3684, 3628; 1H NMR (CDCl3) δ 3.78 (1H, A of AB, J = 10.8 Hz, H-19), 3.68 (1H, dd, J = 5.7, 9.3 Hz, H-12), 3.41 (1H, B of AB, J = 10.8 Hz, H-19), 3.39−3.07 (4H, m, 2 × H-21, 2 × H-22), 2.46−2.39 (1H, m, H-11), 2.35−2.25 (2H, m, 2 × H-16), 2.06−1.92 (3H, m, H-14, 2 × OH), 1.91−1.74 (2H, m, H-8, H-13), 1.66−1.54 (2H, m, H-6, H-7), 1.51−1.34 (4H, m, 2 × H-2, H-5, H-11), 1.33−1.13 (3H, m, 2 × H-1, H-6), 1.11 (3H, s, 3 × H-17), 1.10−0.98 (1H, m, H-7), 0.94 (3H, s, 3 × H-18), 0.87−0.82 (4H, m, H-3, 3 × H-20), 0.72 (1H, dd, J = 14.1, 4.8 Hz, H-14); 13C NMR (CDCl3) δ 75.4 (CH, C-12), 72.6 (C, C-15), 65.6 (CH2, C-19), 51.2 (CH2, C-16), 47.8 (CH, C-5), 41.9 (C, C-10), 41.7 (C, C-13), 41.5 (CH2, C-14), 41.3 (CH2, C-21 or C-22), 38.6 (2 × C, C-4 and C-9), 37.8 (CH2, C-21 or C-22), 35.6 (CH2, C-3), 34.2 (CH2, C-11), 33.2 (CH2, C-1 or C-7), 33.0 (CH2, C-1 or C-7), 32.9 (CH, C-8), 27.6 (CH3, C-18), 22.2 (CH2, C-6), 20.0 (CH3, C-17), 18.5 (CH2, C-2), 17.1 (CH3, C-20); HRESIMS m/z 419.2060 (calcd for C22H36O2S2 [M + Na]+, 419.2054); HPLC eluant (EtOAc/nhexane, 25:75), flow (1 mL/min), tr 16.0 min. Preparation of (+)-6 from (+)-5. To a stirred and ice-bath-cooled solution of (+)-5 (0.096 g, 0.24 mmol) in 2,6-lutidine (0.060 mL, 0.52 mmol) was added AcCl (0.050 mL, 0.70 mmol) dropwise under an Ar atmosphere. The mixture was allowed to stir overnight. After TLC monitoring (EtOAc/n-hexane, 4:6) to confirm the completion of the reaction, a few drops of H2O were added, and the mixture was extracted with CH2Cl2 (3 × 20 mL). The organic phase was washed with brine, dried, and evaporated. The residue was purified through SiO2 CC (EtOAc/n-hexane, gradient from 10:90 to 30:70). After crystallization [n-hexane/EtOAc, 1:1], (+)-6 was obtained in 83% yield (0.080 g, 0,18 mmol): mp 170.8−172.5 °C; [α]20D = +2.5 (c 2.9, CHCl3); IR (CHCl3) 1728; 1H NMR (C6D6) δ 4.37 (1H, A of AB, J = 11.0 Hz, H-19), 3.93 (1H, B of AB, J = 11.0 Hz, H-19), 3.62 (1H, dt, J = 9.3, 4.8 Hz, H-12), 2.93−2.61 (4H, m, 2 × H-21, 2 × H-22), 2.47− 2.35 (3H, m, 2 × H-16, OH), 2.26−2.17 (1H, m, H-11), 2.06 (1H, dd, J = 14.1, 10.4 Hz, H-14), 1.83−1.66 (5H, m, H-3, H-8, CH3CO), 1.51−1.31 (4H, m, H-2, H-6, H-7, H11), 1.30−1.17 (5H, m, H-2, H-5,

Table 2. NMR Spectroscopic Data for (+)-1a and for a Diterpenoid Isolated from Stemodia chilensisb diterpenoid from S. chilensis

(+)-1 position

δC, type

2 20 22 6 11

18.6, 20.6, 21.2, 22.3, 27.9,

CH2 CH3 CH3 CH2 CH2

18 17 16

28.1, CH3 28.3, CH3 30.2, CH2

12

33.0, CH2

1

36.18, CH2d

3

36.22, CH2d

7

37.0, CH2

4

37.1, C

8 15

37.2, CH 38.2, CH2

10 14 5 9 19

38.5, 46.4, 48.8, 50.4, 67.5,

13 21

72.6, C 171.6, C

C CH CH C CH2

δH (J in Hz) 1.42−1.37, 0.94 s 2.04 s 1.54−1.46, 1.62−1.57, 1.41−1.32, 0.97 s 1.11 s 1.84−1.79, 1.72−1.65, 1.54−1.46, 1.35−1.29, 1.72−1.65, 1.31−1.25, 1.72−1.65, 0.98−0.95, 1.83−1.72, 1.13−1.06,

m

HMBCc

δC

3 5

18.6 20.4 21.0 22.4 28.0

5, 19 14 8, 15

28.2 28.3 30.2

11, 17

33.1

20

36.2

18, 19

36.3

5, 6

37.0

5, 18, 19 16, 11 16

37.2 37.4 38.2

20 17 18, 20 20 18

38.5 46.4 48.9 50.4 67.6

m m m

m m m m m md m md m m

1.72−1.65, m 1.72−1.65, m 1.26−1.19, m 1.98−1.92, m 1.45−1.39, m 4.33 d (10.9) 3.91 dd (10.9, 1.1)

17 22

EXPERIMENTAL SECTION

72.5 171.4

a1 H and 13C NMR data at 400.13 and 100.61 MHz, respectively; δ in ppm relative to the residual solvent peaks of CDCl3 at 7.26 and 77.0 ppm for 1H and 13C, respectively. bThe data reported for the diterpenoid isolated from Stemodia chilensis are those given in ref 1. 13 C NMR at 63 MHz; δ in ppm relative to TMS as an internal standard. cHMBC correlations, optimized for 8 Hz, are from carbons stated to the indicated protons. dInterchangeable within the same column.

C



Note

3 × H-17), 1.14−1.07 (2H, m, 2 × H-1), 1.06−0.95 (1H, m, H-6), 0.93 (3H, s, 3 × H-18), 0.89−0.79 (2H, m, H-3, H-7), 0.78 (3H, s, 3 × H-20), 0.57 (1H, dd, J = 14.1, 5.2 Hz, H-14); 13C NMR (C6D6) δ 170.3 (C, CO), 75.3 (CH, C-12), 73.2 (C, C-15), 66.8 (CH2, C19), 51.8 (CH2, C-16), 47.9 (CH, C-5), 41.91 (C, C-10 or C-13), 41.89 (CH2, C-14), 41.87 (C, C-10 or C-13), 41.1 (CH2, C-21 or C22), 38.5 (C, C-9), 37.6 (CH2, C-21 or C-22), 37.3 (C, C-4), 36.4 (CH2, C-3), 34.4 (CH2, C-11), 33.2 (CH2, C-1), 32.9 (CH2, C-7), 32.6 (CH, C-8), 28.0 (CH3, C-18), 22.3 (CH2, C-6), 20.5 (CH3CO), 20.4 (CH3, C-17), 18.6 (CH2, C-2), 17.0 (CH3, C-20); HRESIMS m/ z 461.2154 (calcd for C24H38O3S2 [M + Na]+, 461.2160); HPLC eluant (EtOAc/n-hexane, 15:85), flow (1 mL/min), tr 13.8 min. Preparation of (−)-7 from (+)-6. A solution of (+)-6 (0.078 g, 0.18 mmol) in EtOH (13 mL) was stirred at 60 °C with Raney-Ni until TLC (Et2O/n-hexane, 6:4) showed the disappearance of the starting material (3 h). After cooling to rt, the catalyst was removed by filtering through Celite, the solvent was evaporated, and the mixture was purified by CC (Et2O/n-hexane, 1:9) to afford (−)-7 (0.050 g, 0.14 mmol, 80%) as an oil: [α]20D = −7.7 (c 3.5, CHCl3); IR (CHCl3) 1728; 1H NMR (C6D6) δ 4.46 (1H, A of AB, J = 11.0 Hz, H-19), 3.98 (1H, B of AB, J = 11.0 Hz, H-19), 3.31 (1H, ddd, J = 9.1, 5.3, 1.5 Hz, H-12), 2.21−2.11 (1H, m, H-11), 1.82−1.73 (2H, m, H-3, H-15), 1.71 (3H, s, CH3CO), 1.54−1.43 (4H, m, H-2, H-6, H-8, H-14), 1.42−1.26 (4H, m, H-2, H-5, H-7, H-16), 1.25−1.04 (5H, m, 2 × H-1, H-6, H11, H-16), 1.03−0.81 (6H, m, H-3, H-7, H-15, 3 × H-18), 0.77 (6H, s, 3 × H-17, 3 × H-20), 0.58−0.47 (1H, m, H-14); 13C NMR (C6D6) δ 170.4 (C, CO), 73.7 (CH, C-12), 67.0 (CH2, C-19), 48.3 (CH, C5), 41.5 (CH2, C-14), 40.0 (C, C-9 or C-10), 39.0 (C, C-9 or C-10), 37.4 (C, C-4), 36.7 (CH2, C-3), 34.8 (CH2, C-11), 34.1 (CH2, C-7), 34.0 (CH, C-8), 33.1 (CH2, C-1), 32.8 (C, C-13), 28.2 (CH3, C-18), 26.9 (CH2, C-16), 26.1 (CH2, C-15), 23.7 (CH3CO), 22.7 (CH2, C6), 20.6 (CH3, C-17), 18.9 (CH2, C-2), 16.6 (CH3, C-20); HRESIMS m/z 371.2572 (calcd for C22H36O3 [M + Na]+, 371.2562); HPLC eluant (EtOAc/n-hexane, 15:85), flow (1 mL/min), tr 12.5 min. Preparation of (+)-9a from (−)-7. To a stirred and ice-bathcooled solution of (−)-7 (0.034 g, 0.097 mmol) in Et3N (0.034 mL, 0.24 mmol) was added a solution of MsCl (0.019 mL, 0.24 mmol) in anhydrous CH2Cl2 (1 mL). After stirring for 2 h at rt, TLC monitoring (Et2O/n-hexane, 4:6) revealed the complete disappearance of the starting material. After cooling to 0 °C using an ice bath, the reaction mixture was acidified with 1 N HCl and extracted with CH2Cl2 (3 × 20 mL). The combined organic phase was then washed with a saturated NaHCO3 solution and brine, dried, filtered, and evaporated to afford (+)-9a. The crude material was purified by SiO2 CC (Et2O/ n-hexane, 0.5:9.5) to afford, after crystallization from EtOH/H2O, (+)-9a (21 mg, 0.064 mmol, 66%): mp 101.4−102.2 °C; [α]20D = +18 (c 0.97, CHCl3); IR (CHCl3) 1728; 1H NMR (C6D6) δ 5.05−4.93 (1H, m, H-12), 4.34 (1H, A of AB, J = 10.9 Hz, H-19), 3.92 (1H, B of ABX, JAB = 10.9, JBX = 1.1 Hz, H-19), 2.36−2.27 (1H, m, H-11), 2.13 (1H, t, J = 6.3 Hz, H-14), 2.04 (3H, s, CH3CO), 1.94−1.60 (7H, m, H-1, H-3, H-7, H-8, H-11, H-15, H-16), 1.64 (3H, pd, J = 1.4 Hz, 3 × H-17), 1.59−1.05 (9H, m, H-1, 2 × H-2, H-5, 2 × H-6, H-7, H-15, H16), 1.04−0.91 (1H, m, H-3), 0.97 (3H, s, 3 × H-18), 0.94 (3H, s, 3 × H-20); 13C NMR (C6D6) δ 171.6 (CO), 146.0 (C, C-13), 117.9 (CH, C-12), 67.6 (CH2, C-19), 50.2 (C, C-9), 49.3 (CH, C-5), 44.0 (CH2, C-15), 43.2 (CH, C-8), 39.3 (CH, C-14), 38.9 (C, C-10), 37.3 (C, C4), 37.1 (CH2, C-11), 36.4 (2 × CH2, C-3 and C-7), 35.2 (CH2, C-1), 33.3 (CH2, C-16), 28.2 (CH3, C-18), 22.3 (CH2, C-6), 21.9 (CH3, C17), 21.2 (CH3CO), 18.6 (CH2, C-2), 18.4 (CH3, C-20); GC-MS m/z 330 [M]+ (6), 289 (4), 275 (29), 257 (30), 246 (5), 215 (7), 201 (10), 187 (16), 175 (14), 159 (20), 161 (18), 159 (20), 147 (19), 133 (20), 123 (72), 119 (31), 105 (47), 95 (45), 81 (100), 79 (54), 55 (68); HRESIMS m/z 353.2444 (calcd for C22H34O2 [M + Na]+, 353.2457); HPLC eluant (EtOAc/n-hexane, 5:95), flow (1 mL/min), tr 5.9 min. Preparation of (+)-10 from (+)-9a. To a solution of (+)-9a (7.7 mg, 0.023 mmol) in anhydrous CH2Cl2 (2 mL) was added mchloroperbenzoic acid (16 mg, 0.092 mmol). After stirring for 1 h at rt, TLC monitoring (Et2O/n-hexane, 2:8) revealed the complete disappearance of the starting material. After cooling to 0 °C using

an ice bath, the reaction mixture was basified with a Na2CO3-saturated solution and poured into a separatory funnel; the two phases were separated, and the organic phase was washed with a Na2CO3-saturated solution until no peroxides were present. The combined organic phases were then washed with brine, dried, filtered, and evaporated to afford (+)-10. The crude material was purified by SiO2 CC (Et2O/nhexane, 5:95) to afford, after crystallization from n-hexane, (+)-10 (7.1 mg, 0.021 mmol, 89%): mp 99.4−101.2 °C; [α]20D = +19 (c 0.13, CHCl3); IR (CHCl3) 1730; 1H NMR (CDCl3) 4.28 (1H, A of AB, J = 10.9 Hz, H-19), 3.89 (1H, B of ABX, JAB = 10.9, JBX = 1.2 Hz, H-19), 2.82 (1H, d, J = 4.1 Hz, H-12), 2.18−2.10 (2H, m, H-11, H-14), 2.03 (3H, s, CH3CO), 1.85−1.64 (6H, m, H-1, H-3, H-7, H-8, H-15, H16), 1.55−1.48 (2H, m, H-6, H-16), 1.46−1.02 (8H, m, H-1, 2 × H-2, H-5, H-6, H-7, H-11, H-15), 1.27 (3H, s, 3 × H-17), 1.00−0.89 (1H, m, H-3), 0.95 (3H, s, 3 × H-18), 0.90 (3H, s, 3 × H-20); 13C NMR (CDCl3) δ 171.6 (C, CO), 67.5 (CH2, C-19), 63.3 (C, C-13), 58.1 (CH, C-12), 49.4 (C, C-9), 48.4 (CH, C-5), 42.3 (CH, C-8), 38.5 (C, C-10), 38.0 (CH, C-14), 37.2 (C, C-4), 36.8 (CH2, C-15), 36.3 (CH2, C-3), 35.7 (CH2, C-1 or C-7), 35.2 (CH2, C-1 or C-7), 33.7 (CH2, C11), 28.1 (CH3, C-18), 27.1 (CH2, C-16), 22.2 (CH2, C-6), 21.2 (CH3CO), 19.9 (CH3, C-17), 18.5 (CH2, C-2), 18.3 (CH3, C-20). Preparation of 11 from (+)-10. To a solution of (+)-10 (6.9 mg, 0.020 mmol) in anhydrous THF (2 mL) was added LiAlH4 (1.0 mg, 0.026 mmol). After stirring for 4 h at 60 °C, TLC monitoring (Et2O/ n-hexane, 7:3) revealed the complete disappearance of the starting material. After quenching with wet Et2O, the mixture was poured into a separatory funnel, and the two phases were separated; the organic phase was washed with H2O and brine, dried, filtered, and evaporated to afford 11. The crude material was purified by SiO2 CC (Et2O/nhexane gradient from 15:85 to 35:65) to afford 11 (2.4 mg, 0.0080 mmol, 40%). Because of the small amount of material, no mp nor α could be recorded. 1H NMR (CDCl3) 3.87 (1H, A of AB, J = 10.7 Hz, H-19), 3.47 (1H, B of ABX, JAB = 10.7, JBX = 1.0 Hz, H-19), 1.99−1.86 (2H, m, H-7, H-14), 1.85−1.46 (2H, m, H-3, H-16), 1.74−1.56 (5H, m, H-1, H-8, H-11, H-15, H-16), 1.55−1.37 (6H, m, 2 × H-2, H-5, 2 × H-6, H-12), 1.36−1.17 (4H, m, H-1, H-11, H-12, H-15), 1.17−1.03 (1H, m, H-7), 1.11 (3H, s, 3 × H-17), 1.02−0.84 (1H, m, H-3), 0.99 (3H, s, 3 × H-18), 0.92 (3H, s, 3 × H-20); 13C NMR (CDCl3) δ 72.6 (C, C-13), 66.0 (CH2, C-19), 50.3 (C, C-9), 48.8 (CH, C-5), 46.3 (CH, C-14), 38.7 (C, C-4 or C-10), 38.5 (C, C-4 or C-10), 38.2 (CH2, C-1 or C-15), 37.3 (CH, C-8), 37.1 (CH2, C-7), 36.4 (CH2, C-1 or C15), 35.5 (CH2, C-3), 33.0 (CH2, C-12), 30.2 (CH2, C-16), 28.3 (CH3, C-17), 28.0 (CH2, C-11), 27.7 (CH3, C-18), 22.4 (CH2, C-6), 20.6 (CH3, C-20), 18.7 (CH2, C-2); GC-MS m/z 288 [M − H2O]+ (9), 275 (48), 257 (53), 245 (6), 229 (7), 217 (17), 201 (11), 187 (14), 175 (20), 161 (18), 147 (26), 135 (33), 123 (76), 119 (43), 105 (84), 94 (90), 81 (100), 67 (68), 55 (96). Preparation of (+)-1 from (+)-11. The acetylation of (+)-11 (2.4 mg, 0.0078 mmol) to afford (+)-1 was performed as described for the preparation of (+)-6 (see above). The crude material was purified by SiO2 CC (Et2O/n-hexane gradient from 10:90 to 15:85) to afford (+)-1 (1.4 mg, 0.0040 mmol, 51%). Because of the small amount of material, no mp nor α could be recorded. IR (CHCl3) 1726; 1H NMR (CDCl3) δ 4.33 (1H, A of AB, J = 10.9 Hz, H-19), 3.91 (1H, B of ABX, JAB = 10.9, JBX = 1.1 Hz, H-19), 2.04 (3H, s, CH3CO), 1.99−1.86 (2H, m, H-7, H-14), 1.85−1.78 (1H, m, H-16), 1.77−1.63 (5H, m, H1, H-3, H-8, H-15, H-16), 1.63−1.46 (4H, m, 2 × H-6, H-11, H-12), 1.45−1.17 (7H, m, H-1, 2 × H-2, H-5, H-11, H-12, H-15), 1.16−1.03 (1H, m, H-7), 1.12 (3H, s, 3 × H-17), 1.02−0.78 (1H, m, H-3), 0.97 (3H, s, 3 × H-18), 0.94 (3H, s, 3 × H-20); 13C NMR (CDCl3) δ 171.6 (CO), 72.6 (C, C-13), 67.5 (CH2, C-19), 50.4 (C, C-9), 48.8 (CH, C5), 46.4 (CH, C-14), 38.5 (C, C-10), 38.2 (CH2, C-15), 37.2 (CH, C8), 37.1 (C, C-4), 37.0 (CH2, C-7), 36.22 (CH2, C-1 or C-3), 36.18 (CH2, C-1 or C-3), 33.0 (CH2, C-12), 30.2 (CH2, C-16), 28.3 (CH3, C-17), 28.1 (CH3, C-18), 27.9 (CH2, C-11), 22.3 (CH2, C-6), 21.2 (CH3CO), 20.6 (CH3, C-20), 18.6 (CH2, C-2); GC-MS m/z 348 [M]+ (8), 330 (100), 315 (23), 301 (5), 270 (30), 257 (73), 217 (36), 201 (12), 187 (24), 175 (45), 161 (26), 148 (50), 133 (41), 119 (41), 105 (62), 93 (51), 81 (49), 67 (21), 55 (27); HRESIMS m/z 371.2573 (calcd for C22H36O3 [M + Na]+, 371.2562). D



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Note

(7) Kelly, R. B.; Harley, M. L.; Alward, S. J.; Rej, R. N.; Gowda, G.; Mukhopadhyay, A.; Manchand, P. S. Can. J. Chem. 1983, 61, 269−275. (8) Because of the small amount of material, no mp nor α could be recorded.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00834. 1 H and 13C NMR spectra of all synthesized compounds; X-ray structure and crystallographic data for (+)-9a (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Dr. F. Sciubba for helpful discussions on NMR spectra and to the XRD-1 beamline staff at ELETTRA Trieste (Italy) for experimental assistance. Financial support from Università degli Studi di Roma “La Sapienza” (Ateneo) and MIUR (FIRB 2012) is also gratefully acknowledged.

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DEDICATION Dedicated to the Memory of Professor Juan A. Garbarino. REFERENCES

(1) Chamy, M. C.; Piovano, M.; Garbarino, J. A.; Gambaro, V. Phytochemistry 1991, 30, 1719−1721. (2) (a) Bravetti, D.; Marini Bettolo, R.; Lupi, A. Helv. Chim. Acta 1982, 65, 371−376. (b) Marini Bettolo, R.; Tagliatesta, P.; Lupi, A.; Bravetti, D. Helv. Chim. Acta 1983, 66, 760−770. (c) Marini Bettolo, R.; Tagliatesta, P.; Lupi, A.; Bravetti, D. Helv. Chim. Acta 1983, 66, 1922−1928. (d) Lupi, A.; Patamia, M.; Grgurina, I.; Marini Bettolo, R.; Di Leo, O.; Gioia, P.; Autnaroli, S. Helv. Chim. Acta 1984, 67, 2261− 2263. (e) Lupi, A.; Patamia, M.; Marini Bettolo, R. Helv. Chim. Acta 1988, 71, 872−875. (f) Berettoni, M.; Marini Bettolo, R.; Montanari, V.; Prencipe, T.; Romeo, S. Helv. Chim. Acta 1991, 74, 1671−1678. (g) Di Stefano, S.; Leonelli, F.; Garofalo, B.; Mandolini, L.; Marini Bettolo, R.; Migneco, L. M. Org. Lett. 2002, 4, 2783−2785. (h) Leonelli, F.; Caschera, B.; Silvestri, L.; Prastaro, A.; Corso, G.; Ceccacci, F.; La Bella, A.; Migneco, L. M.; Marini Bettolo, R. Helv. Chim. Acta 2008, 91, 598−607. (i) Leonelli, F.; Blesi, F.; Dirito, P.; Trombetta, A.; Ceccacci, F.; La Bella, A.; Migneco, L. M.; Marini Bettolo, R. J. Org. Chem. 2011, 76, 6871−6876. (j) Leonelli, F.; Latini, V.; Trombetta, A.; Bartoli, G.; Ceccacci, F.; La Bella, A.; Sferrazza, A.; Lamba, D.; Migneco, L. M.; Marini Bettolo, R. J. Nat. Prod. 2012, 75, 1944−1950. (3) Muñoz, M. A.; Chamy, C.; Bucio, M. A.; Hernández-Barragán, A.; Joseph-Nathan, P. Tetrahedron Lett. 2014, 55, 4274−4277. (4) Ishihara, K.; Kurihara, H.; Yamamoto, H. J. Org. Chem. 1993, 58, 3791−3793. (5) Crystallographic data for the structure of (+)-19-acetoxystemod12-ene (9a) reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC 1026508). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44-(0)1223-336033 or e-mail: [email protected]). (6) (a) Chamy, M. C.; Piovano, M.; Garbarino, J. A.; Miranda, C.; Gambaro, V. Phytochemistry 1990, 29, 2943−2946. (b) Garbarino, J. A.; Molinari, A. Phytochemistry 1990, 29, 3037−3039. (c) Chamy, M. C.; Piovano, M.; Garbarino, J. A.; Gambaro, V. Phytochemistry 1991, 30, 3365−3368. E


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