Sinningia allagophylla - ACS Publications - American

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Naphthochromenes and Related Constituents from the Tubers of Sinningia allagophylla Dilamara R. Scharf,*,† Maria H. Verdan,† Marcos A. Ribeiro,‡ Edesio L. Simionatto,§ Eduardo L. Sá,† Marcos J. Salvador,⊥ Andersson Barison,† and Maria E. A. Stefanello*,† †

Departamento de Química, Universidade Federal do Paraná, 81530-900, Curitiba, PR, Brazil Instituto de Química, Laboratório de Química de Coordenaçaõ , Universidade Estadual de Campinas, 13083-970, Campinas, SP, Brazil § Departamento de Química, Universidade Regional de Blumenau, 89030-903, Blumenau, SC, Brazil ⊥ Instituto de Biologia, Departamento de Biologia Vegetal, PPG- BTPB, and PPG-BV, Universidade Estadual de Campinas, 13083-970, Campinas, SP, Brazil ‡

S Supporting Information *

ABSTRACT: Chemical investigation of the tubers of Sinningia allagophylla led to the isolation of two new chromenes, (2S)-12-hydroxylapachenole (1) and (3R)-3,4dihydro-3-hydroxy-4-oxo-8-methoxylapachenole (2), and three new dimeric chromenes, allagophylldimers A−C (3−5). Thirteen known compounds, 6-methoxy-7,8-benzocoumarin (6), lapachenole, 8-methoxylapachenole, tectoquinone, 7hydroxytectoquinone, dunniol, α-dunnione, dunnione, 8hydroxydunnione, aggregatin E, cedrol, oleanolic acid, and halleridone, were also identified. 6-Methoxy-7,8-benzocoumarin (6) has been isolated for the first time from a natural source. the Gaussian09 suite.6,7 After geometrical optimization, the optical rotation was calculated for isomers and compared with experimental values, leading to assignment of the absolute configuration.8

Sinningia allagophylla (Mart.) Wiehler (Gesneriaceae) is an herb with perennial tubers and annual shoots, largely distributed in Brazil, Paraguay, and Argentine.1 In Brazilian folk medicine, the tubers are considered useful tonics and emollients, while the leaves and flowers are used as a febrifuge, depurative, and diuretic.2,3 Previous chemical investigations of the ethanolic extract of the tubers of S. allagophylla, guided by cytotoxic activity, led to the isolation of major compounds from the active hexanes-soluble fraction. These were identified as 8methoxylapachenole, lapachenole, tectoquinone, dunniol, and a mixture of steroidal esters. The naphthoquinone dunniol was the compound responsible for the cytotoxic activity observed in the ethanolic extract.4 Another study showed that the ethanolic extract, its hexanes-soluble fraction, and the major compound of this fraction, 8-methoxylapachenole, showed anti-inflammatory and antinociceptive activities.5 The present work reports the isolation and identification of several compounds from the noncytotoxic CH2Cl2-soluble fraction of the ethanolic extract (DCMS) of S. allagophylla tubers. Successive chromatographic fractionation of the DCMS fraction yielded five new (1−5) and 13 known compounds. All products were analyzed by 1D and 2D NMR spectroscopy, and the data were compared with literature data. MS and UV data of the new compounds were also acquired, and conformational searches were performed using molecular modeling, mainly with DFT calculations employing Becke’s three-parameter hybrid functional (B3LYP), as implemented in © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Compound 1 was isolated as a yellow oil with a molecular formula of C17H18O4 (from HRESIMS and NMR data), which is consistent with nine indices of hydrogen deficiency. The 1H NMR data of 1 (Table 1) showed signals of five aromatic protons, i.e., a singlet (δH 6.51) and the four spin system of a 1,2-disubstituted aromatic system (δH 7.44−8.15). Two olefinic protons (δH 5.65, 6.55), one methoxy group (δH 3.96), one hydroxymethylene group (δH 3.70, 3.78), and one methyl group (δH 1.42) were also observed. These data were very similar to those of lapachenole, reported from S. allagophylla4 and reisolated in this work. In comparison with lapachenole, compound 1 differs by replacement of one methyl group at C-2 by a hydroxymethylene group. Analysis of the HSQC and HMBC spectra confirmed the structure of 1 through the following cross-peaks: H-4 (δH 6.55) with C-5 (δC 102.6), C-11 (δC 141.0), and C-2 (δC 79.6) and H-13 (δH 1.42) with C-3 (δC 126.2), C-12 (δC 68.6), and C-2 (Table 1). Received: September 11, 2015

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DOI: 10.1021/acs.jnatprod.5b00799 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. NMR Data of Compounds 1 (400 MHz, CDCl3) and 2 (600 MHz, CDCl3) 1 position 2 3 4 4a 5 6 6a 7 8 9 10 10a 11 12 13 OMe-6 OMe-8 OH

δC, mult. 79.6, 126.2, 125.5, 114.7, 102.6, 149.7, 125.4, 121.4, 126.3, 125.8, 122.0, 126.4, 141.0, 68.6, 22.3, 55.8,

C CH CH C CH C C CH CH CH CH C C CH2 CH3 CH3

2

δH (J in Hz)

HMBC

5.65 d (9.8) 6.55 d (9.8)

2, 4a, 12, 13 2, 5, 11

6.51 s

4, 6, 6a

8.15 7.48 7.44 8.12

6, 9, 10a 6a, 10 7, 10a 8, 11

m m m m

3.70 d (11.5) 3.78 d (11.5) 1.42 s 3.96 s

2, 3, 13 2, 3, 12 6

δC, mult. 84.6, 76.7, 193.3, 110.5, 97.8, 149.0, 132.7, 101.7, 161.1, 118.6, 125.5, 120.8, 153.5, 27.1, 17.1, 55.8, 55.6,

C CH C C CH C C CH C CH CH C C CH3 CH3 CH3 CH3

δH (J in Hz)

HMBC

4.50 d (2.1)

2, 4, 12, 13

7.03 s

4, 4a, 6, 6a, 11

7.51 d (2.5)

6, 8, 9, 10a

7.18 dd (9.2, 2.5) 8.18 d (9.2)

7, 8, 10a 6a, 8, 11

1.76 1.27 3.89 3.96 3.83

2, 3, 13 2, 3, 12 6 8 2, 3, 4

s s s s d (2.1)

S to be related to the levorotatory isomer. Therefore, 1 was identified as (2S)-12-hydroxylapachenole. Compound 2 was isolated as a colorless solid, with the molecular formula C17H18O5 (from NMR and HRESIMS), compatible with nine indices of hydrogen deficiency. Its 1H NMR data (Table 1) showed signals of four aromatic protons, one being isolated (δH 7.03) and three (δH 7.18−8.18) in a spin

Conformational analysis of 1 showed that the energy minimum is achieved when the 2H-pyran ring adopts a halfchair conformation with the methyl group in the equatorial position (Figure 1). This is in agreement with data obtained from 1D NOE experiments, since irradiation of H-3 (δH 5.65) caused an NOE enhancement of the methyl group singlet. The calculated optical rotation indicated the absolute configuration B



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assigned as R and the structure was defined as (3R)-3,4dihydro-3-hydroxy-4-oxo-8-methoxylapachenole. Compound 3 was isolated as a yellow solid with a molecular formula of C34H36O6, consistent with 17 indices of hydrogen deficiency. The 1H NMR data of 3 (Table 2) showed signals for six aromatic protons in the spin systems of two 1,2,4trisubstituted aromatic moieties (δH 7.13−8.13), three isolated protons (δH 6.09, 6.36, and 6.58), four methoxy groups (δH 3.82, 3.87, 3.93, and 3.94), four methyl groups (δH 1.31, 1.55, 1.62, and 1.70), two diastereotopic methylene protons (δH 1.99 and 2.22), and a benzylic proton (δH 3.77). These data resembled those of 8-methoxylapachenole,4 but the molecular mass and the duplication of several signals indicated a dimeric structure. The presence of benzylic and methylene protons suggested hydrogenation of an olefinic bond of the 2H-pyran ring. The location of the linkage between the moieties was determined by analysis of correlations in the HSQC and HMBC spectra. The most relevant cross-peaks were H-4′ (δH 3.77) with C-2 (δC 79.2), C-4 (δC 122.1), C-4′a (δC 113.9), and C-11′ (δC 143.9); H-5′ (δH 6.58) with C-4′ (δC 36.0), C-6′ (δC 147.6), and C-11′ (δC 143.9); and H-12′ (δH 1.55) with C-3′ (δC 43.8), C-2′ (δC 74.3), and C-13′ (δC 23.8). The 1D NOE experiment showed correlations for H-4′/H-13, H-4′/H-13′, and H-13′/H-3′a. The remaining HSQC and HMBC data (Table 2) supported the structure 3, a dimeric compound probably formed by the acid-catalyzed coupling of two 8methoxylapachenole4 molecules at C-3/C-4′. Due to high steric hindrance, the monomeric moieties must be orthogonally arranged, as shown by conformational analysis (Figure 3). Compound 3 (allagophylldimer A) is levorotatory, and, according to the calculated optical rotation, its absolute configuration was assigned as 4′S. Compound 4, allagophylldimer B, was isolated as a yellow solid with the molecular formula C34H34O7, indicating 18 indices of hydrogen deficiency. Its 1H NMR data (Table 2) were similar to those of 3, but showing an allylic proton as a singlet and lacking the methylene proton signals. In the HMBC spectrum the allylic proton (δH 3.20) had cross-peaks with carbons at δC 25.8(C-12′), 79.5 (C-2), 111.0 (C-4′a), 122.8 (C4), 132.9 (C-3), and 191.4 (C-4′). These correlations indicate a linkage between C-3/C-3′. The NOE data showed correlations for H-3′/H-13 and H-3′/H-13′. The structure of 4 was confirmed by X-ray diffraction analysis (Figure 4), but it was not possible to determine the absolute configuration by this technique. Applying DFT optical rotation calculations, the absolute configuration of 4 was assigned as 3′S. Compound 5, a yellow solid, had the same molecular formula as 4. Its 1H NMR data (Table 3) showed signals for eight aromatic protons and four methoxy groups, one olefinic proton (δH 6.62), three methyl groups (δH 1.38, 1.64, and 2.07), a singlet at δH 9.45, typical of a formyl proton, and three methinic protons (δH 2.49, 4.12, and 5.51). Analyses of the HSQC and HMBC data evidenced a carbonyl group (δC 195.4), two methine carbons (δC 34.6 and 45.0), and one oxymethine carbon (δC 68.9). The correlations in the HMBC spectrum led to structure 5. The most significant cross-peaks were H-3 (δH 2.49) with C-12 (δC 23.5), C-4 (δC 68.9), and C-3′ (δC 157.2); H-4 with C-4′ (δC 34.6), C-2 (δC 77.6), and C-5 (δC 103.8); H5′ with C-4′, C-6′ (δC 148.9), and C-11′ (δC 142.0); and H-4′ with C-2, C-2′ (δC 137.3), C-5′ (δC 104.6), and C-11′ (Table 3). The 3J3,4 and 3J3,4′ values of 4.7 and 3.8 Hz, respectively, suggested a cis relationship between H-3, H-4, and H-4′. The conformational analysis showed H-4 and H-4′ in axial positions,

Figure 1. Conformation and key NOE 1D (solid lines) and HMBC (dashed lines) correlations for 1.

system characteristic of a 1,2,4-trisubstituted aromatic system. Signals of two methoxy groups (δH 3.96 and 3.99), two methyl groups (δH 1.27 and 1.76), and two doublets at δH 4.50 and 3.83 (J = 2.1 Hz) were also observed. The latter doublet disappeared upon addition of D2O, indicating a hydroxy group. From HSQC and HMBC data the presence of 17 carbons, including a carbonyl group (δC 193.3) and two oxygenated carbons (δC 84.6 and 76.7), was deduced. The structure was confirmed by the following HMBC correlations: H-10 (δH 8.18) with C-8 (δC 161.1) and C-11 (δC 153.5); H-5 with C-4 (δC 193.3), C-6 (δC 149.0), and C-11; H3-12 with C-2 (δC 84.6), C-3 (δC 76.7), and C-13 (δC 17.1), and H-3 (δH 4.50) with C-2, C-4, C-12, and C-13 (Table 1). In the 1D NOE experiment, irradiation of H3-12 (δH 1.76) caused an NOE effect on H-3, but irradiation of H3-13 did not. This is consistent with a half-chair conformation of the chromanone ring with the hydroxy group in the equatorial position (Figure 2). Compound 2 is levorotatory, and, according to the calculated optical rotation, its absolute configuration was

Figure 2. Conformation and key NOE 1D (solid lines) and HMBC (dashed lines) correlations for 2. C



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Table 2. NMR Data (400 MHz, CDCl3) of Compounds 3 and 4 3 position

a

δC, mult.

2 3 4 4a 5 6 6a 7 8 9 10 10a 11 12 13 2′ 3′

79.2, C 143.1, C 122.1, CH n.o.a 103.0, CH 148.4, C 127.0, C 100.5, CH 157.7, C 118.5, CH 123.4, CH 120.8, C 140.8 C 26.6, CH3 26.0, CH3 74.3, C 43.8, CH2

4′ 4′a 5′ 6′ 6′a 7′ 8′ 9′ 10′ 10′a 11′ 12′ 13′ OMe-6 OMe-6′ OMe-8 OMe-8′

36.0, 113.9, 106.0, 147.6, 126.6, 100.4, 157.9, 118.1, 123.7, 121.7, 143.9, 29.9, 23.8, 55.7, 55.8, 55.5, 55.4,

CH C CH C C CH C CH CH C C CH3 CH3 CH3 CH3 CH3 CH3

4

δH (J in Hz)

δC, mult.

HMBC

6.09 s

2, 5

6.36 s

4, 6, 6a, 11

7.44 d (2.5)

6, 8, 9, 10a

7.13 dd (9.1, 2.5) 8.07 d (9.1)

7, 10a 6a, 8, 11

1.62 s 1.70 s

2, 3, 13 2, 3, 12

1.99 dd (13.5, 11.6) 2.22 dd (13.5, 6.4) 3.77 dd (11.6, 6.4)

2′, 3, 4′a, 12′, 13′,

6.58 s

4′, 6′a, 11′

7.47 d (2.5)

6′, 9′, 10′a

7.15 dd (9.1, 2.5) 8.13 d (9.1)

7′, 10′a 6′a, 8′, 11′

1.55 1.31 3.87 3.82 3.93 3.94

2′, 3′, 13′ 2′, 3′, 12′ 6 6′ 8 8′

s s s s s s

2, 4, 4′a, 11′

79.5, 132.9, 122.8, 113.9, 103.6, 148.3, 127.1, 101.6, 157.9, 118.4, 123.5, 120.7, 141.4, 25.4, 26.2, 81.8, 54.9,

C C CH C CH C C CH C CH CH C C CH3 CH3 C CH

191.4, 111.0, 99.3, 148.6, 132.3, 100.6, 160.9, 118.8, 125.7, 120.7, 152.0, 25.8, 26.4, 55.9, 55.7, 55.3, 55.7,

C C CH C C CH C CH CH C C CH3 CH3 CH3 CH3 CH3 CH3

δH (J in Hz)

HMBC

6.32 s

2, 3′, 4a, 5, 11

6.22 s

4, 6a

7.38 d (2.6)

9, 6, 10a

7.24 dd (9.0, 2.6) 8.02 d (9.0)

7, 10a 6a, 8, 11

1.53 s 1.67 s

2, 3, 13 2, 3, 12

3.20 s

2, 3, 4, 4′, 4′a, 12′

7.18 s

4′, 6′, 6′a, 11′

7.58 d (2.5)

6′, 8′, 9′, 10′a

7.10 dd (9.1, 2.5) 8.29 d (9.1)

7′, 10′a 6′a, 8′, 11′

1.69 1.55 3.80 4.01 3.90 4.01

2′, 3′, 13′ 2′, 3′, 12′ 6 6′ 8 8′

s s s s s s

n.o., not observed.

dunnione,10 lapachenole,11 8-methoxylapachenole,4 tectoquinone,12 7-hydroxytectoquinone,13 dunniol,14 aggregatin E,15 cedrol,16 oleanolic acid,17 and halleridone.18 Compound 6 had been synthesized,9,19 but its NMR data were not reported. Therefore, the complete assignments of 1H and 13C NMR data are collated in Table 4. This is the first report of compound 6 from a natural source.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in CHCl3 on a Rudolph Research polarimeter. The UV spectra were obtained in MeOH on a Shimadzu UV-2401PC spectrophotometer. 1D and 2D NMR experiments were carried out in CDCl3 at 295 K on a Bruker AVANCE 400 or AVANCE III 600 NMR spectrometer, observing 1H at 400 or 600 MHz and 13C at 100 or 150 MHz, respectively. All 1H and 13C NMR chemical shifts are given in ppm (δ), using TMS as internal reference, with coupling constants (J) in Hz. HRESIMS data were obtained on a Micromass ESI-Qq Tof or a Thermo Scientific LTQ-Orbitrap XL mass spectrometer, using the positive-ion mode. X-ray data were collected on an Bruker D8 Venture Photon 100 by using graphitemonochromated Cu Kα radiation (λ = 1.5418 Å) at 100(2) K. Accurate unit cell dimensions and orientation matrices were


while H-3 occupies the equatorial position (Figure 5). Accordingly, in the NOE experiments correlations between H-3/H-4, H-3/H-12, H-4/H-12, and H-3/H4′ were observed. Therefore, H-3, H-4, and H-4′ are cofacial. Compound 5 is levorotatory, and, according to the calculated optical rotation, its absolute configuration was assigned as (3S, 4R, 4′S). The known compounds were identified as 6-methoxy-7,8benzocoumarin (6),9 α-dunnione,10 dunnione,10 8-hydroxyD



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Figure 4. View of compound 4 with thermal ellipsoid displacement at the 50% probability level.

Table 3. NMR Data (400 MHz, CDCl3) of Compound 5 position 2 3 4 4a 5 6 6a 7 8 9 10 10a 11 12 13 2′ 3′ 4′ 4′a 5′ 6′ 6′a 7′ 8′ 9′ 10′ 10′a 11′ 12′ 13′ OMe-6 OMe-6′ OMe-8 OMe-8′

δC, mult. 77.6, 45.0, 68.9, 111.0, 103.8, 148.4, 127.8, 100.7, 158.2, 118.2, 124.0, 121.1, 143.4, 23.5, 28.0, 137.3, 157.2, 34.6, 112.6, 104.6, 148.9, 126.9, 100.8, 158.0, 118.6, 123.2, 120.6, 142.0, 195.4, 10.2, 55.7, 55.6, 55.4, 55.4

C CH CH C CH C C CH C CH CH C C CH3 CH3 C CH CH C CH C C CH C CH CH C C CH CH3 CH3 CH3 CH3

δH (J in Hz)

HMBC

2.49 dd (4.4, 4.4) 5.51 d (4.4)

2, 2′, 4, 4a, 4′,12, 2, 4a, 4′, 5

6.84 s

4, 6a, 6, 11

7.44 d (2.6)

6, 8, 9, 10a

7.13 dd (9.1, 2.6) 8.10 d (9.1)

10a 6a, 8, 11

1.38 s 1.64 s

2, 3, 13 2, 3, 12

6.62 dd (10.4, 1.1) 4.12 dd (10.4, 4.4)

12′, 13′ 2, 2′, 3, 4′a, 5′, 11′

6.18 s

4′, 6′, 6′a, 11′

7.43 d (2.6)

6′, 8′, 9′, 10′a

7.18 dd (9.1, 2.6) 8.20 d (9.1)

10′a 6′a, 8′, 11′

9.45 2.07 3.84 3.82 3.91 3.92

2′, 13′ 2′, 3′, 12′ 6 6′ 8 8′

s d (1.1) s s s s


Table 4. NMR Data (400 MHz, CDCl3) of Compound 6 position

δC, mult.

2 3 4 4a 5 6 7 8 8a 9 10 11 12 OMe-6

161.2, C 116.2, CH 144.2, CH 114.3, C 100.0, CH 152.3, C 127.3, C 124.0, C 146.1, C 122.2, CH 127.8, CH 128.2, CH 122.5, CH 56.0, CH3

δH (J in Hz)

HMBC

6.51 d (9.5) 7.79 d (9.5)

2, 4a 2, 5, 8a

6.71 s

4, 6, 7, 8a

8.51, m 7.66 m 7.66 m 8.28 m 4.05, s

8a, 11 8, 12 7, 9 7, 8, 10 6

Bruker Saint and Bruker SADABS were used to integrate and scale data, respectively. The structure was solved with Olex2,21 using the ShelXT22 structure solution program using direct methods and refined with the ShelXL23 by a full-matrix least-squares technique on F2. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms in the compound were added to the structure in idealized positions and further refined according to the riding model; Olex2 was

determined by least-squares refinement of the reflections obtained by θ−χ scans. The data were indexed and scaled with the ApexII Suite.20 E



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100.0 K, μ(Cu Kα) = 0.746 mm−1, Dcalc = 1.319 g cm−3, 158 195 reflections measured (7.49° ≤ 2θ ≤ 148.986°), 5710 unique (Rint = 0.0311, Rsigma = 0.0083), which were used in all calculations. The final R1 was 0.0325 (I > 2σ(I)), wR2 was 0.0883 (all data), and the goodness of fit was 1.03. The crystallographic data can be requested from the CSD with the deposit number CCDC 1408676. Allagophylldimer C (5): yellow solid; [α]20D −97 (c 0.01, MeOH); UV−vis (MeOH) λmax (log ε) 215 (5.38); 259 (5.29); 337 (4.53) nm; 1 H and 13C NMR data see Table 3; HRESIMS m/z 555.2388 [M + H]+ (calcd for C34H35O7 555.2383). 6-Methoxy-7,8-benzocoumarin (6): yellow solid; UV−vis (MeOH) λmax (log ε) 218 (4.34); 272 (4.16); 283 (4.19); 313 (3.61); 376 (3.53) nm; 1H and 13C NMR data see Table 4; HRESIMS m/z 227.0699 [M + H]+ (calcd for C14H11O3 227.07083).

also used for the drawing of molecular graphics and the preparation of figures for publication. Geometry optimization and density functional theory (DFT) calculations on the electronic structure of the compounds employed the B3LYP hybrid functional, using the LANL2DZ basis set as implemented in the Gaussian09 suite.7 All HPLC separations were performed in a Waters apparatus equipped with PDA detector and a semipreparative Nucleosil 100-5 C18 column (250 × 4.6 mm). Silica gel (Merck, 230−400 mesh) was used for column chromatographic (CC) separation, while silica gel 60 PF254 (Merck) was used for analytical (0.25 mm) and preparative (1.0 mm) TLC. Compounds were visualized by exposure under UV254/366 light and spraying with 5% (v/v) H2SO4 in ethanol solution, followed by heating on a hot plate. Plant Material. Tubers of S. allagophylla were collected in Palmeira, Paraná State, Brazil (25°26′3″ S, 49°59′60″ W). The plant was identified by Clarisse Bolfe Poliquesi, who deposited a voucher in the herbarium of Museu Botânico Municipal de Curitiba (MBM 3530). Extraction and Isolation. Dried and powdered tubers (166.2 g) were extracted at room temperature with EtOH (3 × 1 L). After solvent removal, the extract (27.2 g) was dissolved in EtOH/H2O (1:1, 300 mL) and partitioned successively with hexanes and CH2Cl2 (3 × 100 mL, each solvent). The CH2Cl2-soluble portion (5.45 g) was subjected to silica gel CC, using CH2Cl2 and mixtures of CH2Cl2/ acetone (95:5, 8:2, 1:1), to give 13 fractions (F1−F13) after TLC analyses. F1 (10.5 mg) yielded 8-methoxylapachenole. Fraction F2 (104.6 mg) was subjected to further CC, eluted with a gradient of acetone in hexanes (1−50%), to give six subfractions (F2.1−F2.6). Subfraction F2.2 (46.7 mg) yielded lapachenole (2.2 mg), 8methoxylapachenole (16.1 mg), dunniol (5.4 mg), and tectoquinone (1.0 mg) after PTLC in hexanes/acetone (95:5). Subfraction F2.5 (12.3 mg) was purified by HPLC (100% MeCN) to give α-dunnione (tR = 3.6 min, 1.6 mg) and 3 (tR = 5.7 min, 1.6 mg). Fraction F5 (17.0 mg) was subjected to PTLC in hexanes/CH2Cl2/MeOH (4:1:0.1), yielding cedrol (3.2 mg) and a mixture (10.5 mg) that was separated by HPLC (100% MeCN) to give 4 (tR = 10.2 min, 3.2 mg) and 5 (tR = 9.43 min, 4.8 mg). F6 (34.4 mg) was purified by HPLC (100% MeCN) yielding 1 (tR = 7.10 min, 6.1 mg), 5 (tR = 9.49 min, 2.1 mg), and a mixture that was separated by HPLC (MeCN/H2O, 80:20) to give 2 (tR = 5.33 min, 2.1 mg) and 6 (tR = 5.00 min, 1.1 mg). F8 (59.7 mg) was purified by PTLC in benzene/EtOAc (95:5) giving dunnione (3.8 mg) and 8hydroxydunnione (2.8 mg). F9 (49.7 mg) yielded 8-hydroxydunnione (9.8 mg) and aggregatin E (3.0 mg) after repeated PTLC in hexanes/ EtOAc (8:2). F11 (200.0 mg) was submitted to CC and eluted with mixtures of hexanes/acetone (9:1, 8:2, 7:3, 6:4, 1:1), acetone, and MeOH to give eight subfractions (F11.1−F11.8). F11.3 (28.9 mg) yielded oleanolic acid (4.1 mg) after PTLC in hexanes/acetone (9:1). F11.5 (13.4 mg) gave 7-hydroxytectoquinone (1.2 mg) after PTLC in CH2Cl2. An aliquot (40 mg) of fraction F12 (1.17 g) yielded halleridone (12.2 mg) after PTLC in hexanes/acetone (1:1). (2S)-12-Hydroxylapachenole (1): yellow oil; [α]20D −64 (c 0.01, MeOH); UV−vis (MeOH) λmax (log ε) 215 (4.04); 248 (3.93); 319 (3.45) nm; 1H and 13C NMR data see Table 1; HRESIMS m/z 257.11304 [M + H]+ (calcd for C16H17O3 257.11781). (3R)-3,4-Dihydro-3-hydroxy-4-oxo-8-methoxylapachenole (2): colorless solid; [α]20D −41 (c 0.02, MeOH); UV−vis (MeOH) λmax (log ε) 216 (3.51); 273 (3.52); 317 (3.19); 375 (3.30) nm; 1H and 13 C NMR data see Table 1; HRESIMS m/z 303.1224 [M + H]+ (calcd for C17H19O5 303.12327). Allagophylldimer A (3): yellow solid; [α]20D −18 (c 0.01, MeOH); UV−vis (MeOH) λmax (log ε) 216 (3.69), 259 (3.54), 277 (3.37), 345 (2.91) nm; 1H and 13C NMR data see Table 2; HRESIMS m/z 541.2587 [M + H]+ (calcd for C34H37O6 541.2591). Allagophylldimer B (4): yellow solid; [α]20D −32 (c 0.01, MeOH); UV−vis (MeOH) λmax (log ε) 231 (3.67), 277 (3.56), 374 (2.96) nm; 1 H and 13C NMR data see Table 2; HRESIMS m/z 555.2376 [M + H]+ (calcd for C34H35O7 555.2383). Crystallographic data: monoclinic, space group P21/n (no. 14), a = 9.9747(10) Å, b = 15.9545(16) Å, c = 17.8555(18) Å, β = 100.710(2)°, V = 2792.0(5) Å3, Z = 4, T =

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00799. Chemical structures of known compounds; UV and 1H and 13C NMR spectra of compounds 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +55 47 3221-6099. E-mail: [email protected] (D. R. Scharf). *Tel: +55 41 3361-3177. E-mail: [email protected] (M. E. A. Stefanello). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to C. B. Poliquesi, at Museu Botânico Municipal de Curitiba, for collection and identification of the plant and to CNPq for financial support (process 462392/ 2011-5), scholarships, and authorization for access of samples from the Brazilian genetic heritage (process 010087/2012-5). M.J.S. is grateful to CAPES and FAPESP for financial support. Thanks also are due to CCAD-UFPR, LCPAD-UFPR, and Dr. R. A. Burrow at UFSM for computational facilities.

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REFERENCES

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Sinningia allagophylla - ACS Publications - American

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