Absolute Configuration of Labdane Diterpenoids from Physalis

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Absolute Configuration of Labdane Diterpenoids from Physalis nicandroides Emma Maldonado,*,† Ana L. Pérez-Castorena,† Yunuen Romero,† and Mahinda Martínez‡ †

Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán, 04510, D. F., México ‡ Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Avenida de las Ciencias s/n, Col. Juriquilla 76230, Querétaro, Qro, México S Supporting Information *

ABSTRACT: A mixture of the new epimeric labdenetriols 1 and 2 was isolated from the aerial parts of Physalis nicandroides. The structures of 1 and 2, including their absolute configurations, were established by analyses of their spectroscopic data, together with the X-ray diffraction analysis of acetonide 3 and chemical correlation with (−)-(13E)-labd-13ene-8α,15-diol (6), whose absolute configuration was also confirmed by X-ray analysis of its dibromo derivative 7. The epimeric labdenediols 8 and 9, the known labdanes 6 and 11, and the acylsucroses 12 and 13 were also isolated. Labdanes 6 and 11 showed moderate anti-inflammatory activities in the induced ear edema model. 1.153/1.150, δC 24.11/24.05, CH3-17; δC 74.69/74.51, C-8), but in their side chain a 13(16)-double bond (δH 5.11 and 4.98, δC 112.15/111.66, CH2-16; δC 149.62/149.42, C-13), together with vicinal primary (δH 3.67/3.65 dd, J = 11.0, 3.0 Hz, H-15a; δH 3.60/3.55 dd, J = 11.0, 1.5 Hz, H-15b; δC 112.15/111.66, CH2-15) and secondary hydroxy groups (δH 4.03/4.01 m, δC 75.59/75.48, CH-14), were present. Thus, the planar structures for 1 and 2 were formulated as 13(16)-labdene-8,14,15-triol. The presence of the 14,15-diol moiety permitted separation of compounds 1 and 2 through derivatization to the isopropylidene derivatives 3 and 4. Subsequent removal of the isopropropylidene group (CuCl2·2H2O in EtOH)19 afforded triol 1 from 3 and 2 from 4. The molecular formula of the less polar isopropylidene derivative (4) was determined as C23H40O3 using HRFABMS (m/z 365.3063 [M + H]+) in conjuction with 13C NMR data. The 1H NMR spectrum of 4 showed resonances for six methyl groups, two corresponding to the isopropylidene (δ 1.44 s, 1.39 s), and the rest to CH3-17 (δ 1.15 s), CH3-18 (δ 0.87 s), CH3-19 (δ 0.785 s), and CH3-20 (δ 0.791 s). The resonances for the vinylic protons H-16a and H16b appeared at δ 5.10 and 4.93, respectively, and those for the oxymethine and oxymethylene protons at δ 4.55 (1H, br t, J = 7.5 Hz, H-14), 4.10 (1H, dd, J = 8.0, 6.5 Hz, H-15a), and 3.59 (1H, t, J = 8.0 Hz, H-15b). COSY correlations of H-16a and H16b with H-14, H-12a (δ 2.18), and H-12b (δ 2.04), and those of H-14 with H-15a and H-15b, together with the HMBC correlations of H3-17 to C-7 (δ 44.2), C-8 (δ 74.1), and C-9 (δ

P

lants of the genus Physalis (Solanaceae) are known to be an important source of C28 steroidal lactones called withasteroids or withanolides, which exhibit a broad range of biological activities.1,2 Several other types of compounds, such as sterols,3 flavonoids,4,5 megastigmane glycosides,6 alkaloids,7,8 ceramides,9 acylsucroses,10,11 and labdane-type diterpenes,4,11 have also been isolated from these species. As part of ongoing studies on the metabolites from Physalis,4,5,10,11 the aerial parts (except the fruits and calixes) of Physalis nicandroides Schltdl., an annual herbaceous plant distributed from Northern Mexico to Costa Rica, were investigated.12 Fruits of this plant are occasionally eaten in Mexico and Guatemala, and their sticky leaves are used to trap insects, primarily fleas.13 The leaves are also used for postpartum baths. 14 Four new labdane diterpenoids (1, 2, 8, and 9) and the known (−)-(13E)-labd13-ene-8α,15-diol (6)15−17 and (+)-(Z)-labda-8(17),13-diene15,16-diol (11)18 were identified. The acylsucroses nicandrose B (12) and nicandrose E (13) were also isolated.10 Compounds 12 and 13 were previously isolated from the fruits of P. nicandroides var. attenuata.10 The structural elucidation of the new compounds and the anti-inflammatory activities of 6 and 11 are described in this paper.

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RESULTS AND DISCUSSION Compounds 1 and 2 were isolated as an epimeric mixture that exhibited IR absorptions for hydroxy groups (3410 cm−1) and double bonds (1647 and 863 cm−1). Analysis of their 1D and 2D NMR spectra indicated that these compounds were labdane diterpenoids closely related to (−)-(13E)-labd-13-ene-8α,15diol (6) because they also possess an 8α-hydroxy group (δH © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 3, 2014

A

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showed significant differences. Finally, the relative configuration of compound 1 was determined using X-ray diffraction analysis of its acetonide 3 (Figure 1). This analysis revealed a 14S configuration in 3 and, consequently, in 1 provided that these compounds belong to the normal labdane series, which could be determined via chemical correlation between 1/2 and (−)-(13E)-labd-13-ene-8α,15-diol (6) (mp 130−131 °C, [α]D −2.0 (c 0.2, CHCl3)), a known compound that also occurs in this plant. However, a review of the literature concerning 6 showed that its low [α]D value has caused confusion about its absolute configuration. Briefly, the chemical correlation between sclareol and labd-13-ene-8,15-diol with [α]D −0.5 (CHCl3) established this compound as the labdane 6.16 Supporting this conclusion, a labd-13-ene-8,15-diol with [α]D +0.7 (c 4.4, CHCl3) was shown to be the enantiomer of 6 via correlation with the ent-labdane 13-epi-(−)-manoyl oxide.17 Subsequently, different signs and values of [α]D (−3.3,4 −2.0,21 −0.5,15 0.0,22 and +0.223) have been described for 6. Structure 6 was recently assigned to a compound with [α]D +1.8 (c 0.9, CHCl3), based on comparison of the vibrational circular dichroism spectrum of (+)-isoabienol with its calculated spectrum obtained by density functional theory (DFT) calculations.24 Compound 6 was treated with bromine in CCl4, to obtain the 13,14-dibromo derivative 7, as indicated by the molecular ion at m/z 468.1073 (C20H36Br2O2) in the HRFABMS and the chemical shift of C-13 (δC 71.0) and CH14 (δH 4.52, dd J = 8.5, 3.0 Hz; δC 65.8) observed in the 1H and 13 C NMR spectra. A crystal of compound 7 was subjected to Xray analysis, and the results established its absolute configuration as 5S,8R,9R,10S,13R,14S (Figure 2), indicating that this compound and, therefore, 6 belong to the normal labdane series. Once confirmed, a chemical correlation of 6 with the mixture of 1 and 2 was performed via photooxidation of 6 and reduction of the resulting hydroperoxides to obtain a mixture of 1 and 2. The 1H NMR spectra of the natural and synthetic mixtures were similar, and the optical rotations of both mixtures showed the same sign {synthetic mixture [α]20D −7 (c 0.3, CHCl3); natural mixture [α]20D −1 (c 0.3, CHCl3)}. The differences in the magnitude of [α]D were attributed to the different ratios of 1 and 2 in each mixture. Thus, the structure of compound 1 was established as (−)-(14S)-labd-13(16)-ene8α,14,15-triol and that of 2 as (−)-(14R)-labd-13(16)-ene8α,14,15-triol. Labdanes 1 and 2 were named physanicantriol and 14-epi-physanicantriol, respectively. A mixture of the epimers 8 and 9 was isolated as a colorless gum. The IR spectrum showed absorptions for hydroxy groups (3440 cm−1) and double bonds (1643 and 894 cm−1). Their HRFABMS showed a pseudomolecular ion at m/z 307.2640 ([M + H]+), in conjunction with 13C NMR data, which was consistent with the molecular formula C20H34O2, indicating an index of hydrogen deficiency of four for these compounds. The 1 H and 13C NMR spectra of 8 and 9 differed from those of 1 and 2 by the resonances of an additional terminal methylene [δH 4.83 m, (H-17a); δH 4.50/4.48 br d, J = 1.5, 1.5 Hz (H17b); δC 106.3/106.2 (C-17), and δC 148.6 (C-8)], which was located at C-8 by the HMBC correlations of H2-17 with C-7 (δC 38.3) and C-9 (δC 56.7/56.4) and those of H2-7, H-9, and H-11a with C-8. The signals at δH 5.13/5.12 (br s, H-16a), 4.98/4.97 (br s, H-16b), 4.19/4.18 (dd, J = 7.0, 3.5 Hz, H-14), 3.69/3.68 (dd, J = 11.5, 3.5 Hz, H-15a), and 3.51/3.50 (dd, J = 11.5, 7.0 Hz, H-15b) indicated that the side chain in 8 and 9 was the same as that of 1 and 2, which was confirmed by COSY and HMBC correlations. Therefore, the structures of 8 and 9

61.7) and those of H-7a and H-7b to C-8, confirmed the proposed structure.

The 1H and 13C NMR data of the more polar isopropylidene 3 were similar to those of 4 (Tables 1 and 2), and the correlations in the COSY, HSQC, and HMBC spectra were the same. Only small differences in the chemical shifts of some proton and carbon resonances were observed. The most relevant were the resonances for the C-12 protons, which in 3 appeared as a multiplet at δH 2.12 (2H), and those of C-12, C14, and C-16, which exhibited a Δδ ≥ 0.5 ppm with respect to those of 4, indicating that 3 and 4 and, therefore, 1 and 2 are C14 epimers. This was confirmed via the oxidative cleavage of the mixture of 1 and 2 with sodium periodate,20 which gave the α,β-unsaturated aldehyde 5. The molecular formula of 5 was determined as C19H32O2 based on the molecular ion at m/z 292 in the EIMS. Its 1H NMR spectrum showed the resonance for the formyl proton at δ 9.53 (H-14) and those for the vinylic protons H-16a and H-16b at δ 6.29 and 5.98. The triols 1 and 2 exhibited similar IR and NMR spectra with only small differences in the chemical shifts of the proton and carbon resonances around C-14 (Table 1). Thus, the 1H NMR spectrum of 1 showed the resonances for H-12a and H-12b as broad ddd’s at δ 2.25 and 2.09. The resonance of H-14 was observed at δ 4.23 (br dd, J = 7.0, 4.0 Hz), and those of H-15a, H-15b, H-16a, and H-16b appeared at δ 3.67, 3.60, 5.11, and 4.97, respectively. In the 1H NMR spectrum of 2, the resonances of H-12a and H-12b appeared at δ 2.14 as a triplet (J = 7.5 Hz), those of H-14 appeared at δ 4.22 (br dd, J = 7.5, 3.0 Hz), and those of H-15a, H-15b, H-16a, and H-16b were observed at δ 3.65, 3.55, 5.11, and 4.97, respectively. In their 13 C NMR spectra, only the resonances for C-12 (δ 35.5/36.3, 1/2), C-14 (δ 75.6/74.5, 1/2), and C-16 (δ 112.1/111.6, 1/2) B

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Table 1. 1H NMR Data for Compounds 1−5 and 7−10 (500 MHz, CDCl3) position 1a 1b

3a

2

4b

5

7

8/9

1.64 m 0.96 td (13.0, 3.5) 1.59 m

1.67 m 0.95 td (13.0, 4.0) 1.59 m

1.66 m 0.97 td (13.0, 3.5) 1.60 m

1.69 m 0.95 td (13.0, 4.0) 1.58 m

1.76 br d (12.5) 0.96 td (13.0, 3.5) 1.60 m

1.75 m 1.02 tt (13.0, 3.0)

2a

1.66 m 0.96 td (13.0, 3.5) 1.59 m

2b 3a

1.44 m 1.38 m

1.43 m 1.38 m

1.45 m 1.37 m

1.44 m 1.38 m

1.44 m 1.38 m

1.45 m 1.40 m

3b

1.15 m

1.15 m

0.93 dd (12.5, 2.0) 1.66 m 1.27 dtd (13.5, 12.5, 3.0) 1.87 dt (12.0, 3.0) 1.39 m

0.93 dd (12.5, 2.0) 1.66 m 1.27 dtd (14.0, 12.5, 3.5) 1.87 dt (12.0, 3.0) 1.40 m

1.14 td (13.0, 4.0) 0.92 dd (12.5, 2.5) 1.66 m 1.27 dtd (13.5, 12.0, 3.5) 1.86 dt (12.5, 3.0) 1.42 m

1.15 m

5

1.15 td (13.0, 4.0) 0.93 dd (12.0, 2.5) 1.66 m 1.26 dtd (13.5, 12.5, 3.0) 1.86 dt (12.5, 3.0) 1.39 m

1.15 td (13.5, 4.0) 0.93 dd (12.0, 2.5) 1.63 m 1.26 m

1.48 m 1.39 dddd (13.5, 5.0, 3.0, 1.5) 1.17 td (13.5, 4.0)

1.13 1.58 1.46 2.14

1.09 1.57 1.44 2.12

1.12 1.49 1.42 2.36

2.14 t (7.5)

2.12 m

4.22 br dd (7.5, 3.0) 3.65 dd (11.5, 3.0) 3.55 dd (11.5, 7.5) 5.11 t (1.5)

4.55 br t (7.0)

1.09 t (4.0) 1.58 m 1.42 m 2.18 br ddd (14.5, 11.5, 5.5) 2.04 br ddd (14.5, 11.5, 5.5) 4.55 br t (7.5)

4.09 dd (8.0, 6.5) 3.62 t (8.0)

4.10 dd (8.0, 6.5)

16a

1.15 m 1.63 m 1.46 m 2.25 br ddd (15.0, 10.0, 5.5) 2.09 br ddd (15.0, 9.0, 7.0) 4.23 br dd (7.0, 4.0) 3.67 dd (11.0, 4.0) 3.60 dd (11.0, 7.0) 5.11 br s

5.12 br s

5.10 t (1.5)

16b 17

4.97 br s 1.15 s

4.97 br d (1) 1.14 s

4.94 br s 1.16 s

4.93 m 1.15 s

6a 6b 7a 7b 9 11a 11b 12a 12b 14 15a 15b

18 19 20 a

1

0.88 s 0.79 s 0.80 s

t (4.0) m m t (7.5)

0.87 s 0.79 s 0.80 s

t (4.0) m m m

0.87 s 0.79 s 0.80 s

0.93 dd (12.0, 2.5) 1.64 m 1.26 dtd (13.5, 12.0, 3.5) 1.87 dt (12.0, 3.0) 1.41 m t (4.0) m m m

2.36 m 9.53 s

3.59 t (8.0) 6.29 dt (1.0, 1.0) 5.98 br s 1.15 d (0.5)

0.87 s 0.785 s 0.791 s

0.87 s 0.79 s 0.77 s

1.85 dt (12.5, 3.5) 1.42 m

1.56 tt (13.5, 3.5)

1.09 dd (13.0, 2.5) 1.73 m 1.32 dtd (13.0, 13.0, 4.5) 2.39 br dt (13.0, 3.0) 1.97 td (13.0, 5.0)

1.07 t (4.0) 1.58 m 1.55 m 2.29 ddd (15.0, 10.5, 7.0) 1.96 ddd (15.0, 10.0, 7.0) 4.52 dd (8.5, 3.0) 4.39 dd (12.5, 3.0) 3.99 dd (12.5, 8.5) 1.79 s

1.63 m/1.61 m 1.69 m/1.66 m 1.48 m/1.51 m 2.23 ddd/2.14 ddd (15.0, 11.0, 4.5) 1.78 m/1.88 ddd (15.0, 9.0, 7.0) 4.19 dd/4.18 dd (7.0, 3.5) 3.69 dd/3.68 dd (11.5, 3.5) 3.51 dd/3.50 dd (11.5, 7.0) 5.13 br s/5.12 br s

1.22 s

4.98 br s/4.97 br s 4.83 dt (1.5, 1.5)

0.87 s 0.80 s 0.67 s

4.50 br d/4.48 br d (1.5) 0.87 s 0.80 s 0.69 s

10 1.72 m 0.99 td (13.0, 4.0) 1.54 tt (13.5, 3.5) 1.48 m 1.38 dtd (13.0 3.0, 1.5) 1.17 td (13.0, 4.0) 1.08 dd (13.0, 3.0) 1.72 m 1.32 dtd (13.0, 13.0, 4.0) 2.39 ddd (13.0, 4.0, 2.5) 1.97 br dt (13.0, 5.0) 1.62 m 1.64 m 1.48 m 2.43 m 2.03 m 9.54 s

6.23 dt 1.0) 5.97 br 4.85 dt 1.0) 4.58 dt 1.0) 0.87 s 0.80 s 0.67 s

(1.5, s (1.5, (1.0,

Isopropylidene: δ 1.45, s, 3H; δ 1.40, s, 3H. bIsopropylidene: δ 1.44, s, 3H; δ 1.39, s, 3H. 500 MHz; 13C at 125 MHz) spectrometer with TMS as an internal standard. EIMS and ESIMS were recorded on a JEOL JMS-AX505HA and an ESI ion trap Bruker Esquire 6000 mass spectrometer, respectively. HRFABMS (using polyethylene glycol 600 as a standard) were measured on a JEOL JMS-SX102A spectrometer. Column chromatography (CC) was performed on silica gel 60 (Merck G) and assisted with vacuum. TLC was performed on precoated Alugram Sil G/UV254 plates with thicknesses of 0.25 mm or Alugram RP-18 W/ UV254 plates with thicknesses of 0.15 mm. Preparative TLC was performed on precoated Alugram SIL G 200/UV254 or SIL RP-18W/ UV254 plates with thicknesses of 2.0 mm. Plant Material. The aerial parts of Physalis nicandroides Schltdl. were collected in Delegación Santa Rosa Jáuregui, La Barreta, Querétaro State, México, in October 2006. A voucher specimen of ́ the plant (M. Martinez 5742) was identified by one of the authors (M.M.) and deposited at the Herbarium of the Universidad Autónoma de Querétaro. Extraction and Isolation. Dried and ground leaves, flowers, and stems of the plant (960 g) were successively extracted with acetone (12 L) and MeOH (9 L) to obtain 37.3 and 43.8 g of extracts, respectively. These extracts were combined and partitioned (EtOAc− H2O) to give 40.2 and 40.5 g of the EtOAc and aqueous fractions, respectively. The EtOAc fraction was fractioned using CC (column A)

were elucidated as labda-8(17),13(16)-diene-14,15-diol, and as in the case of 1 and 2, it was proposed that 8 and 9 were epimers at C-14. The epimeric relationship was confirmed via the NaIO4 oxidation of 8 and 9, to afford the aldehyde 10. Compounds 8 and 9 were named physanicandiol and 14-epiphysanicandiol. The anti-inflammatory activities of compounds 6 and 11 were evaluated on TPA-induced mouse ear edema. Doses of 1 μmol/ear of compounds 6 and 11 inhibited edema at 64.32% and 41.35%, respectively. Therefore, only the IC50 of compound 6 was determined (IC50 0.67 μmol/ear), which was twice that of the control, indomethacin (IC50 0.297 μmol/ ear).

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting points (uncorrected) were determined on a Fisher-Johns melting point apparatus (Fisher Scientific, U.S.A.). The optical rotations were measured on a PerkinElmer 343 polarimeter. The IR spectra were recorded on a Nicolet FTIR-Magna 750 spectrophotometer. 1H and 13 C NMR spectra were recorded on a Varian Unity Plus 500 (1H at C

DOI: 10.1021/np500688c J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Data for Compounds 1−5 and 7−10 (125 MHz, CDCl3)

a

position

1

2

3a

4b

5

7

8/9

10

1 CH2 2 CH2 3 CH2 4C 5 CH 6 CH2 7 CH2 8C 9 CH 10 C 11 CH2 12 CH2 13 C 14 CH 15 CH2 16 CH2 17 CH3 18 CH3 19 CH3 20 CH3

39.7 18.4 41.9 33.2 56.1 20.5 44.5 74.7 60.4 39.2 24.4 35.5 149.4 75.6 65.7 112.1 24.0 33.4 21.5 15.5

39.7 18.4 41.9 33.2 56.1 20.5 44.2 74.5 60.6 39.2 24.0 36.3 149.5 74.5 65.8 111.6 24.1 33.4 21.5 15.5

39.7 18.4 42.0 33.3 56.1 20.5 44.5 74.2 61.6 39.1 24.5 35.0 147.4 79.1 69.0 111.3 24.1 33.4 21.5 15.4

39.7 18.5 42.0 33.2 56.1 20.5 44.2 74.1 61.7 39.1 24.4 35.6 147.6 78.6 69.2 110.7 24.1 33.4 21.5 15.5

39.7 18.5 42.0 33.3 56.1 20.5 44.2 74.2 61.6 39.0 23.9 31.6 150.9 195.1

39.6 18.5 42.0 33.3 56.2 20.4 43.9 74.4 61.2 39.3 20.8 48.7 71.0 65.8 65.9 27.8 24.6 33.4 21.5 15.4

39.1 19.4 42.2 33.6 55.57/55.56 24.5 38.3 148.6 56.7/56.4 39.8/39.7 22.3/22.0 31.59/31.57 149.3/149.1 75.3/74.9 65.7/65.6 110.5/110.3 106.3/106.2c 33.6 21.7 14.52/14.48

39.1 19.4 42.2 33.6 55.6 24.4 38.3 148.2 56.5 39.6 22.0 27.3 151.0 194.8

134.1 24.0 33.4 21.5 15.4

133.8 106.5c 33.6 21.7 14.5

Isopropylidene δ 109.2, 26.4, 25.7. bIsopropylidene δ 109.2, 26.5, 25.7. cCH2.

Figure 1. ORTEP drawing of isopropylidene derivative 3.

Figure 2. ORTEP drawing of (8R,13R,14S)-13,14-dibromolabdane8,15-diol (7).

eluted with mixtures of hexanes−EtOAc of increasing polarity as follows: Fr. A1 (1:0); A2, A3 (19:1); A4 (9:1 to 17:3); A5 (17:3 to 7:3); A6 (4:1); A7 (1:1); A8 (1:1); A9 (1:4); A10 (0:1). CC (hexanes−acetone, 49:1 to 17:3) of Fr. A2 gave a mixture of βsitosterol and stigmasterol (321 mg). Fr. A4 (2.84 g) was separated using CC eluted with hexanes−EtOAc (9:1) to obtain five fractions (B1−B5). Fr. B2 (623 mg) was discolored with activated charcoal and subjected to two successive CC (hexanes−acetone, 9:1; CH2Cl2− acetone, 1:0 to 19:1) and preparative TLC (silica gel, CH2Cl2− acetone, 49:1, 4 runs) to obtain a mixture of 8 and 9 (69 mg). Fr. A5 (6.7 g) was discolored with activated charcoal and purified using CC eluted with hexanes−acetone (17:3 to 3:1) to obtain fractions C1−C4. Fr. C1 (5.12 g) was subjected to repeated Si gel CC (hexanes−iPrOH, 97:3 to 95:5) followed by crystallization from EtOAc−hexanes to afford compounds 6 (385 mg) and 11 (2.12 g). Fr. A6 (2.6 g) was chromatographed over a Si gel column eluted with hexanes−acetone

(4:1 to 3:2) to afford six fractions (D1−D6). Fr. D2 (847 mg) was discolored using activated charcoal and subjected to CC (CH2Cl2− acetone, 9:1) to obtain a mixture of compounds 1 and 2 (516 mg). Fr. A7 (2.6 g) was subjected to CC eluted with a hexanes−EtOAc gradient (1:1 to 3:2) to give five fractions (E1−E5). Fr. E2 was chromatographed over a Si gel column eluted with hexanes−acetone (3:1) to give 552 mg of a mixture containing compound 12. A portion of this mixture (39.5 mg) was purified using preparative RP-TLC (EtOH−H2O, 3:2, 7 runs) to obtain 7.3 mg of 12. Fraction A8 (1.5 g) was discolored with activated charcoal and purified by CC eluted with hexanes−acetone mixtures of increasing polarity (7:3 to 3:2) to afford five fractions (F1−F5). Frs. F2 and F3 were combined (389 mg) and subjected to CC (CHCl3−acetone, 4:1) to give a mixture containing compound 13 (172.3 mg), which was purified using preparative RPTLC (MeOH−H2O, 7:3, 5 runs) to afford 15.4 mg of 13. D

DOI: 10.1021/np500688c J. Nat. Prod. XXXX, XXX, XXX−XXX

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and 13C NMR (CDCl3) see Tables 1 and 2; HRFABMS m/z 468.1073 [M]+ (calcd for C20H36Br2O2, 468.1062). Photooxidation of Compound 6. Methylene blue (32.1 mg) was added to a solution of 6 (125.6 mg) in EtOH. The mixture was stirred in sunlight for 6 h and cooled (0 °C), and NaBH4 (53 mg) added. The mixture was stirred for 0.5 h, quenched with glacial HOAc (0.5 mL), concentrated, diluted with H2O (8 mL), and extracted with EtOAc (5 mL, 4 times). The organic fraction was washed with brine and H2O, dried with anhydrous Na2SO4, concentrated, and purified using CC (hexanes−acetone, 7:3) to give 48.2 mg of the mixture of 1 and 2 ([α]20D −7 (c 0.3, CHCl3). Preparation of Compound 10. Si gel supported NaIO4 reagent20 (117 mg) was added to a stirred suspension of the mixture of compounds 8 and 9 (19.8 mg) in CH2Cl2 (3 mL). The suspension was left at room temperature for 1 h and filtered. The filtrate was purified using CC (hexanes−EtOAc, 9:1) to give compound 10 (13.7 mg) as a pale yellow gum; IR (CHCl3) νmax 1716 (second derivative 1718, 1690), 1643, 1461, 1386, 1368, 1082, 908 cm−1; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; EIMS m/z 274 [M]+ (10), 259 (11), 241 (5), 189 (16), 177 (21), 149 (31), 137 (100), 123 (42), 109 (36), 95 (54), 81 (55), 69 (49), 55 (42), 43 (53). X-ray Crystallographic Data of Physanicantriol Isopropylidene Derivative (3). The data were collected from a colorless prism (0.388 × 0.273 × 0.099 mm) at 296(2) K on a Bruker APEX DUO diffractometer equipped with an Apex II CCD detector using Incoatec IμS with multilayer optic, Cu Kα radiation (λ 1.541 78 Å): C23H40O3 (formula weight 364.55); monoclinic, space group P21; a = 11.1197(2) Å, b = 8.0038(2) Å, c = 12.2820(3) Å; α = γ = 90°; β = 91.670(1)°, V = 1092.63(4) Å3; Z = 2; Dcalc = 1.108 Mg/m3; F(000) = 404. A total of 12 849 reflections were collected in the theta range of 3.60° to 70.14°, with 3648 independent reflections [R(int) = 0.0128]; completeness to θ = 70.14° was 98.8%. The structure was solved by direct methods using SHELXL-97 and refined using full matrix least-squares on F2 using SHELXL-97 with anisotropic temperature factors for nonhydrogen atoms converging at final R indices [I > 2σ(I)], R1 = 0.0333, wR2 = 0.0940. Absolute structure parameter: 0.36(18). Hydrogen atoms, except those bonded to oxygen, were included at calculated positions and were not refined. X-ray Crystallographic Data of (8R,13R,14S)-13,14-Dibromolabdane-8,15-diol (7). The data were collected from a colorless needle (0.496 × 0.301 × 0.048 mm) at 298(2) K on a Bruker Smart Apex CCD diffractometer with monochromated Mo Kα radiation (λ 0.710 73 Å): C20H36Br2O2, (formula weight 468.31); orthorhombic, space group P21212; a = 10.9195(3) Å, b = 27.8841(8) Å, c = 7.1858(2) Å; α = β = γ = 90°, V = 2187.94(11) Å3; Z = 4; Dcalc = 1.422 Mg/m3; F(000) = 968. A total of 31 220 reflections were collected in the theta range of 2.003° to 27.495°, with 5010 independent reflections [R(int) = 0.0611]; completeness to θmax was 99.7%. The structure was solved by direct methods using SHELXL-97 and refined by full matrix least-squares on F2 using SHELXL-97 with anisotropic temperature factors for non-hydrogen atoms converging at final R indices [I >2σ(I), R1 = 0.0394, wR2 = 0.0713]. Absolute structure parameter: 0.018(6). Hydrogen atoms, except those bonded to oxygen, were included at calculated positions and were not refined. Crystallographic data for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre, CCDC 1045714 (3) and CCDC 1045715 (7). Copies of these data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-336-033 or e-mail: [email protected]). 12-O-Tetradecanoylphorbol 13-Acetate (TPA)-Induced Ear Edema. The anti-inflammatory activity was evaluated as previously described.11 Groups of five male CD-1 mice weighing 25−30 g were anaesthetized with sodium pentobarbital. TPA (2.5 μg) dissolved in EtOH (10 μL) was topically applied to both sides of the right ear of the mice (5 μL each side). The left ear received only EtOH (10 μL). After 10 min, doses of 0.1 to 1.0 μmol of the test compounds or indomethacin (reference compound) dissolved in 20 μL of EtOH− CH2Cl2 (1:1) were applied to the right ear (10 μL each face). The control animals received only the vehicle (20 μL). After 4 h, the

Physanicantriol (1): colorless crystals (EtOAc−hexanes); mp 94− 96 °C; [α]20D −1 (c 0.2, CHCl3); IR (CHCl3) νmax 3398, 1648, 1603, 1462, 1389, 1084, 910 cm−1; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; ESIMS m/z 363 [M + K]+, 347 [M + Na]+; HRFABMS m/z 325.2757 [M + H]+ (calcd for C20H37O3, 325.2743). 14-epi-Physanicantriol (2): colorless gum; [α]20D −12 (c 0.2, CHCl3); IR (CHCl3) νmax 3380, 1646, 1603, 1463, 1388, 1086, 910 cm−1; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; FABMS m/z 347 [M + Na]+; ESIMS m/z 363 [M + K]+, 347 [M + Na]+. Physanicandiol (8)/14-epi-Physanicandiol (9): pale yellow gum; [α]20D +4 (c 0.3, CHCl3); IR (CHCl3) νmax 3434, 1643, 1603, 1460, 1388, 1081, 894 cm−1; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; EIMS m/z 306 [M]+ (10), 291 (19), 273 (14), 255 (18), 245 (12), 205 (10), 191 (11),177 (23), 149 (32), 137 (100), 123 (44), 109 (49), 95 (63), 81 (65), 69 (49), 55 (33), 41 (30); HRFABMS m/z 307.2640 [M + H]+ (calcd for C20H35O2, 307.2637). Isopropylidene Derivatives 3 and 4. p-Toluensulfonic acid (12.4 mg) was added to a stirred solution of 1 and 2 (288 mg) in acetone (10 mL). The reaction mixture was left at room temperature for 30 min, and the solvent was evaporated using an air stream. The reaction mixture was subjected to CC (hexanes−EtOAc, 3:1) to obtain 264 mg of a mixture of 3/4 and recovered material. This mixture was subjected to repeated silica gel CC eluted with hexanes−EtOAc (88:12) to afford 73.3 mg of the more polar acetonide (3), 64.7 mg of the less polar one (4), 27 mg of a mixture of 3 and 4, and 53 mg of 1/2. Acetonide 3: colorless crystals (EtOAc−hexanes); mp 99−101 °C; [α]20D +21 (c 0.2, CHCl3); IR (CHCl3) νmax 3498, 1648, 1460, 1385, 1156, 1064, 909, 860 cm−1; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; FABMS m/z 364 [M]+, 347, 289, 271, 245, 205, 191, 177, 163, 137, 123, 109, 95, 81, 69, 55, 43. Acetonide 4: colorless gum; [α]20D −21 (c 0.2, CHCl3); IR (CHCl3) νmax 3497, 1648, 1603, 1460, 1385, 1156, 1065, 909, 859 cm−1; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; FABMS m/z 364 [M]+, 347, 329, 289, 271, 245, 205, 191, 177, 163, 137, 123, 109, 95, 81, 69, 55, 43; HRFABMS m/z 365.3063 [M + H]+ (calcd for C23H41O3, 365.3056). Hydrolysis of Compound 3. A solution of 3 (31.3 mg) and CuCl2· 2H2O (74.7 mg) in EtOH (4 mL)19 was stirred for 48 h; then 20.7 mg of CuCl2·2H2O was added, and the mixture was stirred for 24 h. NaHCO3 (149 mg) was added, and the mixture was stirred until its color changed from dark green to turquoise blue. H2O (5 mL) was added, and the precipitate was filtered. The filtrate was extracted with EtOAc (4 × 5 mL) and dried over anhydrous Na2SO4, and the solvent was evaporated and separated by preparative TLC eluted with hexanes−acetone (3:2) to obtain 26.4 mg of 1. Hydrolysis of Compound 4. A solution of 4 (35.1 mg) and CuCl2· 2H2O (83.8 mg) in EtOH (5 mL)19 was stirred for 48 h; then 42 mg of CuCl2·2H2O was added, and the mixture was stirred for 24 h. NaHCO3 (148 mg) was added, and the mixture was worked up as for 3 to obtain 25.3 mg of 2. Preparation of Compound 5. Si gel supported NaIO4 reagent20 (170 mg) was added to a stirred solution of the mixture of 1 and 2 (60 mg) in CH2Cl2 (3 mL). The suspension was left at room temperature for 1 h and filtered off. The filtrate was purified using CC eluted with hexanes−EtOAc (9:1) to give the aldehyde 5 (21.6 mg) as a pale yellow gum; [α]20D −6 (c 0.1, CHCl3); IR (CHCl3) νmax 3492, 1688 (second derivate 1719, 1687), 1628, 1604, 1462, 1388, 1123, 1081, 942 cm−1; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; EIMS m/z 292 [M]+ (8), 262 (12), 238 (19), 222 (37), 191 (73), 177 (50), 149 (22), 137 (58), 123 (68), 109 (76), 95 (83), 91 (89), 81 (84), 69 (100), 55 (73), 43 (94). Bromination of Compound 6. A stirred solution of compound 6 (38.7 mg) in CCl4 (3 mL) containing NaHCO3 (14 mg) was cooled to 0 °C. A solution of Br2 in CCl4 (4% v/v) was added dropwise, until persistence of the red color. The solution was stirred for 20 min and the solvent eliminated using an air stream. CC (hexanes−EtOAc, 17:3) of the reaction mixture led to the isolation of 12.2 mg of (8R,13R,14S)-13,14-dibromolabdane-8,15-diol (7): colorless crystals (EtOAc−hexanes); mp 124−126 °C; [α]20D +1 (c 0.3, CHCl3); IR (CHCl3) νmax 3592, 1602, 1459, 1388, 1071, 936, 908 cm−1; 1H NMR E

DOI: 10.1021/np500688c J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(16) Asselineau, C.; Bory, S.; Fétizon, M.; Laszlo, P. Bull. Soc. Chim. Fr. 1961, 1429−1431. (17) Jefferies, P. R.; Payne, T. G. Aust. J. Chem. 1965, 18, 1441−1450. (18) Villamizar, J.; Fuentes, J.; Salazar, F.; Tropper, E.; Alonso, R. J. Nat. Prod. 2003, 66, 1623−1627. (19) Iwata, M.; Ohrui, H. Bull. Chem. Soc. Jpn. 1981, 54, 2837−2838. (20) Zhong, Y. L.; Shing, T. K. M. J. Org. Chem. 1997, 62, 2622− 2624. (21) Carman, R. M. Aust. J. Chem. 1973, 26, 879−881. (22) Calabuig, M. T.; Cortés, M.; Francisco, C. G.; Hernández, R.; Suárez, E. Phytochemistry 1981, 20, 2255−2258. (23) Demetzos, C.; Harvala, C.; Philianos, S. M. J. Nat. Prod. 1990, 53, 1365−1368. (24) Gómez-Hurtado, M. A.; Torres-Valencia, J. M.; ManríquezTorres, J.; del Río, R. E.; Motilva, V.; García-Mauriño, S.; Á vila, J.; Talero, E.; Cerda-García-Rojas, C. M.; Joseph-Nathan, P. Phytochemistry 2011, 72, 409−414.

animals were sacrificed by cervical dislocation, and a plug (7 mm diameter) was removed from each ear. The edematous response was measured as the weight difference between the two punches. The percent inhibition was calculated by the following equation: % = [(A − B)/A] × 100; A = edema induced by TPA; B = edema induced by TPA plus sample. The data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. The IC50 value (μmol/ ear) was estimated from the linear regression equation.

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of compounds 1−5 and 7−10 are available free of charge via Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 525556224412. Fax: 525556162217. E-mail: emmaldon@ unam.mx. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to R. A. Toscano for solving the X-ray structures of compounds 3 and 7 and to M. Reyes for the collection of the X-ray data of compound 3. We also thank H. ́ I. Chávez, B. Quiroz, R. Gaviño, A. Peña, and E. Huerta Rios, for the NMR spectra; R. Patiño for the IR and optical rotations; L. Velasco, C. Márquez, and J. Pérez for the MS; and A. Nieto for the biological assay.

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REFERENCES

(1) Ray, A. B.; Gupta, E. In Progress in the Chemistry of Organic Natural Products; Herz, W.; Kirby, G. W.; Moore, R. E.; Steglich, W.; Tamm, Ch., Eds.; Springer-Verlag: Wien, 1994; Vol. 63, pp 1−106. (2) Veleiro, A. S.; Oberti, J. C.; Burton, G. In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier Science Pub.: Amsterdam, 2005; Vol. 32, pp 1019−1052. (3) Sinha, S. C.; Ali, A.; Bagchi, A.; Sahai, M.; Ray, A. B. Planta Med. 1987, 53, 55−57. (4) Pérez-Castorena, A. L.; Oropeza, R. F.; Vázquez, A. R.; Martínez, M.; Maldonado, E. J. Nat. Prod. 2006, 69, 1029−1033. (5) Maldonado, E.; Gutiérrez, R.; Pérez-Castorena, A. L.; Martínez, M. J. Mex. Chem. Soc. 2012, 56, 128−130. (6) Qiu, L.; Zhao, F.; Liu, H.; Chen, L.; Jiang, Z.; Liu, H.; Wang, N.; Yao, X.; Qiu, F. Chem. Biodiversity 2008, 5, 758−763. (7) Asano, N.; Kato, A.; Oseki, K.; Kizu, H.; Matsui, K. Eur. J. Biochem. 1995, 229, 369−376. (8) Basey, K.; McGaw, B. A.; Woolley, J. G. Phytochemistry 1992, 31, 4173−4176. (9) Su, B. N.; Misico, R.; Park, E. J.; Santarsiero, B. D.; Mesecar, A. D.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D. Tetrahedron 2002, 58, 3453−3466. (10) Maldonado, E.; Torres, F. R.; Martínez, M.; Pérez-Castorena, A. L. J. Nat. Prod. 2006, 69, 1511−1513. (11) Pérez-Castorena, A. L.; Martínez, M.; Maldonado, E. J. Nat. Prod. 2010, 73, 1271−1276. (12) Cuevas-Arias, C. T.; Vargas, O.; Rodríguez, A. Rev. Mex. Biodiversity 2008, 79, 67−79. (13) Martínez, M. Revision of Physalis Section Epeteiorhiza (Solanaceae). Anales del Instituto de Biologia,́ Universidad Nacional Autónoma de México, Serie Botánica; 1998; Vol. 69, pp 71−117. (14) Aguilar, A.; Camacho, J. R.; Chino, S.; Jácquez, P.; López, M. E. Herbario Medicinal del Instituto Mexicano del Seguro Social; México, 1994; p 196. (15) Forster, P. G.; Ghisalberti, E. L.; Jefferies, P. R. Phytochemistry 1985, 24, 2991−2993. F

DOI: 10.1021/np500688c J. Nat. Prod. XXXX, XXX, XXX−XXX