Sulfur-Containing Aristoloxazines and Other ... - ACS Publications

Article Cite This: J. Nat. Prod. 2017, 80, 3112−3119

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Sulfur-Containing Aristoloxazines and Other Constituents of the Roots of Aristolochia orbicularis

María Yolanda Rios,*,† Víctor Navarro,‡ M. Á ngeles Ramírez-Cisneros,† and Enrique Salazar-Rios§ †

Centro de Investigaciones Químicas, IICBA, Universidad Autónoma del Estado de Morelos, Cuernavaca 62209, Morelos, México Laboratorio de Microbiología, Centro de Investigación Biomédica del Sur (IMSS), Xochitepec 62790, Morelos, México § Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Cuernavaca 62350, Morelos, México ‡

S Supporting Information *

ABSTRACT: Six new compounds, aristoloxazine A (1), aristoloxazine B (2), 7-methoxytaliscanine (3), humul-7-en1,4,11-triol (4), 8-hydroxy-β-logipinene (5), and 1β-hydroxy4(14)-eudesmene (6), corresponding to two sulfur-containing aristoloxazines (1 and 2), an aristolactam (3), and three sesquiterpenes (4−6) were isolated, along with 26 known compounds, from the roots of Aristolochia orbicularis. The structures of the new compounds were established based on their spectroscopic and spectrometric data and in the case of aristoloxazine A (1) by single-crystal X-ray crystallography. This is the first report of sulfur-containing aristoloxazines from a natural source. Furthermore, aristoloxazine A (1) was found to possess potent in vitro antimicrobial activity against all resistant Staphylococcus aureus and several fungal strains in which it was evaluated. dimethoxysalicilic acid, β-amyrin,14 α-caryophyllenol,15 transtriacontyl-4-hydroxy-3-methoxycinnamate,16 β-sitosterol,17 stigmasterol,17 2-(4-hydroxyphenyl)-1-nitroethane,18,19 ferulic acid,20 the lignans savinin,21 hinokinin, and pluviatolide, previously isolated from Aristolochia constricta,22 dehydrohinokinin,23 an inseparable diastereomeric mixture of 7′S-parabenzlactone and 7′R-parabenzlactone,24 an inseparable diastereomeric mixture of 8R,8′R,9S-cubebin and 8R,8′R,9R-cubebin, also previously isolated from A. lagesiana and A. pubescens,25 6αhydroxy-β-eudesmol (β-chenopodiol),26 4(15)-eudesmene1β,6α-diol,27 ursolic acid,28 and the aristolochic acid derivatives aristololactam AII,29 aristololactam BI (taliscanine),30 and aristololactam BIII.31 The structures of all these compounds were identified based on a comparison of their physical and spectroscopic data with literature values.

Aristolochia is an important genus from the Aristolochiaceae family, being composed of approximately 600 species.1 Aristolochia species have a versatile and productive biosynthetic machinery, and a variety of natural products have been isolated, including compounds showing skeletal arrangements. Previous studies on Aristolochia species have revealed the presence of aristolochic acids, aristolactams, lignans, neolignans, aporphine and bisaporphine alkaloids, dimeric alkaloids, flavonoids, biflavonoids, tetraflavonoids, alkamides, and mono-, sesqui-, di-, tri-, and tetraterpenes, among other compounds.2,3 In Mexican traditional medicine, the use of plants as crude extracts, infusions, or plasters is a widespread practice to treat some infectious diseases.4 In vitro antimicrobial studies provide the required preliminary observations to select, among the crude plant products, those with useful properties for further chemical and pharmacological studies. Thus, several Aristolochia species have shown antimicrobial activity against panels of microrganisms.5,6 The essential oil from the roots of A. orbicularis, composed by about 40 volatile compounds, showed high repellency against the corn borer Sitophilus zeamais.7 The present study on the antimicrobial activity of Aristolochia orbicularis was performed as a part of an established program to investigate the antimicrobial potential of local medicinal plants. After successive chromatographic procedures on the acetone extract from A. orbicularis roots, 32 compounds were isolated, of which six (compounds 1−6) have not been reported previously. The 26 known compounds were identified as (4S,8R)-p-menth-1-en-9-ol,8 caryophyllene β-oxide,9 madoline R,10 3β-acetyl-Δ7-sitosterol,11,12 syringic acid,13 squalene, 3,5© 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compounds 1 and 2 were isolated as yellow powders. These natural products were deduced as aristolochic acid derivatives showing the same skeleton, in the following manner. First, ring A was assigned as pentasubstituted because of a singlet signal for H-3 at δH 7.64 for compound 1 and δH 7.40 for compound 2 in the 1H NMR spectrum (Table 1; Figures S1 and S17, Supporting Information). For both compounds, ring B proved to be 1,2,3-trisubstituted due to the ABX system [δH 8.16 (d, J = 8.2 Hz, H-11), 7.39 (dd, J = 8.2, 8.2 Hz, H-10), and 7.07 (d, J Received: March 15, 2017 Published: December 6, 2017 3112

DOI: 10.1021/acs.jnatprod.7b00226 J. Nat. Prod. 2017, 80, 3112−3119

Journal of Natural Products

Article

Table 1. 1H [ppm, mult. (J in Hertz)] and 13C NMR Spectroscopic Data for Compounds 1 (500 MHz, CDCl3−DMSO-d6), 2, and 3 (400 MHz, CDCl3) 1 δH

position 1 2 3 3a 4 6a 7 7a 8 9 10 11 11a 11b 11c 1′ 2′ 3′ 1″ 2″ 3″ 4″ 5″ 6″ NHa-1′ NHb-1′ NH OCH3-1 OCH3-2 OCH3-7 OCH3-8 OCH3-3″

7.64, s

5.34, s

7.07, d (8.2) 7.39, dd (8.2, 8.2) 8.16, d (8.2)

3.32, d (15.5) 3.10, d (15.5)

2 δC, type 152.7, C 158.5, C 109.1, CH 119.1, C 163.0, C 152.1, C 37.9, CH 120.0, C 156.1, C 112.0, CH 129.9, CH 120.2, CH 129.4, C 124.4, C 116.2, C 169.9, C 34.5, CH2

δH

3 δC, type

7.40, s

5.19, s

6.73, d (8.2) 7.29, dd (8.2, 8.2) 8.18, d (8.2)

6.29, d (15.7)

7.60, d (15.7)

152.5, C 158.5, C 108.0, CH 117.8, C 164.3, C 152.1, C 44.3, CH 121.3, C 156.9, C 110.9, CH 129.3, CH 120.5, CH 131.7, C 125.8, C 119.0, C 115.9, CH 167.6, C

6.91, d (8.0) 7.08, dd (8.0, 2.0)

144.8, 127.3, 109.5, 147.0, 148.1, 114.9, 123.2,

7.04, d (2.0)

δH

δC, type

7.82, s

7.17, d (8.2) 7.54, dd (8.2, 8.2) 9.02, d (8.2)

151.9, 154.2, 110.6, 120.8, 168.6, 121.4, 136.6, 118.9, 157.3, 111.0, 126.9, 121.1, 131.4, 124.4, 125.3,

C C CH C C C C C C CH CH CH C C C

CH C CH C C CH CH

7.29, br s 7.14, br s 3.89, s 4.03, s

60.7, CH3 56.6, CH3

3.93, s 4.01, s

61.5, CH3 56.9, CH3

3.92, s

56.0, CH3

3.82, s 3.93, s

55.8, CH3 56.2, CH3

8.40 br s 4.08, s 4.06, s 3.94, s 4.03, s

60.5, 57.0, 62.6, 57.2,

CH3 CH3 CH3 CH3

tion) and elemental analysis (Figure S15, Supporting Information). The substituents at C-1, C-2, and C-8 were all assigned as methoxy groups, in accordance with the three CH3 signals observed for each compound in the range δH 3.82 to 4.03. Compounds 1 and 2 were found to contain different substituents on the sulfur atom. Compound 1 includes a modified cysteine residue [δC 34.5 (t, C-2′), δH 3.32 (d, J = 15.5 Hz), and 3.10 (d, J = 15.5 Hz); δC 169.9 (s, C-1′); and δH 7.29 (br s, NHa-1′) and 7.14 (br s, NHb-1′)] (Table 1, Figures 3, S1−S16, Supporting Information), as confirmed by means of the 15N HSQC spectrum (J = 90 Hz) in DMSO-d6 (Figure S11, Supporting Information). In this spectrum, NHa-1′ and NHb-1′ appeared as broad signals at δH 7.32 and 7.11 and showed 1σ H−N correlations with the signal at δN 114.5. Finally, the connectivity of the amide and imine moieties was corroborated by means of the 15N HMBC spectrum (J = 4 Hz) in this same solvent (Figure S12, Supporting Information).33 Cross-peaks of the two AB system protons (H-2′) at δH 3.27 and 3.14 with δN 114.5 and of H-7 at δH 5.29 with δN 366.6 were observed (Figure 1). Compound 2 was assigned as a ferulic acid thioester, in agreement with a trans AB system [δH 6.29 and 7.60 (both d, J = 15.7 Hz), H-2′ and H-3′], an ABX system [δH 7.08 (dd, J = 8.0, 2.0 Hz, H-6″), 7.04 (d, J = 2.0 Hz, H-2″), and

= 8.2 Hz, H-9)] for compound 1 (Figure S1, Supporting Information) and [δH 8.18 (d, J = 8.2 Hz), 7.29 (dd, J = 8.2, 8.2 Hz), and 6.73 (d, J = 8.2 Hz)] for the same protons, respectively, in compound 2 (Figure S17, Supporting Information). The strong absorptions at 1727 cm−1 for 1 and 1712 cm−1 for 2 in the IR spectrum indicated the presence of a carbonyl group in both compounds (C-4, δC 163.0 and 164.3, respectively) (Table 1; Figures S3 and S19, Supporting Information). An imine moiety for each compound was evident from the signal at δC 152.1 (C-6a), which connected to the carbonyl group through an oxygen atom, to make up a 1,2oxazine ring (D ring). Finally, ring C of the phenanthrene core was concluded as having an sp3 C-7, connected to a sulfur atom, in accord with its chemical shift at δC 37.9 (Figures S3 and S5, Supporting Information) and with the observed JC7−H7 of 157.0 Hz in the coupled HSQC experiment (Figures S7 and S7A, Supporting Information). To confirm the oxazine moiety in the structure, a 15N NMR INEPT spectrum (J = 4 Hz, Figure S9, Supporting Information) was performed for compound 1, in which one signal at δN 366.6 was observed. A thioether functionality was confirmed by the 33S NMR spectrum (Figure S10, Supporting Information), which exhibited a broad signal at δS −341.3 ppm.32 The presence of the sulfur atom was corroborated by HRESIMS (Figure S14, Supporting Informa3113



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In accordance with the suggested structures, the 13C NMR and DEPT spectra for compound 1 showed 20 carbon resonances, corresponding to three OCH3, one CH2, five CH, and 11 quaternary carbons (Figures S3 and S4, Supporting Information). The HSQC experiment established the 1σ H−C correlations as indicated in Table 1 (Figure S5, Supporting Information). In addition, the HMBC (Figure S6, Supporting Information) cross-peaks H-3/C-1, C-2, C-3a, C-11c, C-4 confirmed the A ring functionality (Figure 1). For ring B, the correlations H-10/C-8, C-11a, C-11; H-11/C-11b, C-7a, C-9; and H-9/C-8, C-11 were observed. The H11/C11b HMBC correlation demonstrated the connectivity between both aromatic rings, and the five cross-peaks H-7/C-8, C-7a, C11a, C-11c, and C-6a were used to confirm a 7H-phenanthrene skeleton. The key cross-peak H-7/C-1′ and that of both H-1′/ C-7 and C-2′ supported the presence of a modified cysteine residue (Figure 1). The methoxy groups were located due to their HMBC cross-peaks with their respective quaternary carbons. In the ECD spectrum, for compound 1 (Figure S13, Supporting Information), a positive Cotton effect at 284 nm was observed. In the tridimensional octant distribution, the thioacetamide moiety lies on the lower right, rear octant indicating a 7S absolute configuration (Figure 2).34 These data supported the assignment of compound 1 as (S)-2-(1,2,8trimethoxy-4-oxo-7H-phenanthro[10,1-cd][1,2]oxazin-7-yl)thioacetamide, which has been accorded the trivial name aristoloxazine A. Suitable crystals of compound 1 were obtained from a 1:1 ratio mixture of CH2Cl2/MeOH, and its structure was confirmed by X-ray analysis,35 as shown in Figures 3 and S16, Supporting Information. Crystallographic data for aristoloxazine A (1) have been deposited at the Cambridge Crystallographic Data Centre (CCDC 1572611). The 13C NMR and HSQC spectra for compound 2 showed 28 carbon resonances (Figures S19 and S20, Supporting Information), of which 18 were due to a 1,2,8-trimethoxy-4oxo-7H-phenanthro[10,1-cd][1,2]oxazin-7-yl fragment. The 10 additional signals were supportive of a ferulic acid thioester fragment (Figure 1). This compound (aristoloxazine B) was identified as (S)-2-(1,2,8-trimethoxy-4-oxo-7H-phenanthro[10,1-cd][1,2]oxazin-7-yl)-E-3-(4-hydroxy-3-methoxyphenyl)prop-2-enthioate. On the basis of an m/z 362.0982 [M + Na]+ peak in the positive-ion mode HRESIMS, a molecular formula of C19H17NO5Na and 12 unsaturation degrees were deduced for compound 3. Eight of these unsaturations were due to two aromatic rings. This compound was assigned as being an aristolactam inclusive of a 7H-phenanthrene core. Compound 3 could be determined as a phenanthrene because the 13C NMR data showed a shielding effect for the signal due to C-6a (Δδ = −30.7) and a deshielding effect for C-7 (Δδ = 98.7), C-11c (Δδ = 9.1), and C-4 (Δδ = 5.6) (Figure S23, Supporting Information). The remaining signals for this structural fragment at compound 3 were in accordance with those of compounds 1 and 2, including the C-1, C-2, and C-8 methoxy groups (Table 1; Figures S22−S25, Supporting Information). A fourth methoxy group showed a resonance signal at δH 3.94 (δC 62.6), and its HMBC correlation with C-7 supported the assignment of this compound as 7-methoxytaliscanine (3). Compound 4 was isolated as a colorless oil. Based on the HRFABMS (M − H+, m/z 255.1953), a molecular formula of C15H28O3 and two degrees of unsaturation were deduced. One unsaturation was due to a disubstituted trans double bond [δC

Figure 1. 13C HMBC (blue ↷), 15N HMBC (red ↷), and COSY (bold green lines) correlations of compounds 1−6.

Figure 2. Cotton effect at 284 nm of aristoloxazine A (1) indicating the 7S absolute configuration.

Figure 3. ORTEP projection of aristoloxazine A (1).

6.91 (d, J = 8.0 Hz, H-5″)], and a C-3″ methoxy group [δH 3.93 (δC 56.2)] (Figures S17−S21, Supporting Information).

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Table 2. 1H [ppm, mult (J in Hertz)] and 13C NMR Spectroscopic Data for Compounds 4−6 (400 MHz, CDCl3) 4 δH

position 1

5 δC, type 85.2, C

2a 2b 3

2.15, m 1.52, m 1.84, m

4 5 6 7

3.79, dd (10.0, 7.6) 5.33, d (15.6) 5.15, dd (15.6, 10.4)

88.8, CH 39.4, C 134.6, CH 135.7, CH

8

2.45, m

36.8, CH

9

1.92, m

30.7, CH

10

28.8, CH2

11 12 13 14

2.35, 0.99, 3.53, 1.12, 0.91, 1.13,

15

1.05, d (6.6)

ddd (14.9, 4.8, 4.8) m d (8.0) s s s

35.9, CH2

δH 2.26, 1.33, 2.37, 2.11,

m m ddd (12.4, 6.8, 6.4) m

26.5, CH2

71.1, 19.4, 24.7, 24.3,

CH CH3 CH3 CH3

22.3, CH3

6 δC, type

1.70, dd (1.6,8.0) 1.59, dd (10.8, 10.8) 3.90, ddd (10.8, 9.6, 5.6) 2.48, dd (12.4, 5.6) 1.05, d (12.4)

δC, type

29.3, CH2

3.66, dd (11.4,4.8)

79.0, CH

30.7, CH2

1.83, 1.49, 2.28, 2.15,

31.8, CH2

151.5, C 2.64, ddd (10.4, 8.8, 8.8) 1.93, dd (9.6,9.6)

δH

43.7, 57.4, 34.7, 41.1,

CH CH C CH2

72.3, CH 49.5, CH2

m m ddd (14.0, 5.5, 2.0) m

2.40, d (11.4) 1.28, m 3.19, dd (11.2, 6.2) 2.11, 1.62, 1.80, 1.53,

m m m m

dd (10.8,4.4) s s s

5.05, d (1.2) 4.93, d (1.2)

64.1, 23.0, 32.3, 18.6,

CH CH3 CH3 CH3

113.6, CH2

145.9, C 54.0, CH 29.9, CH2 47.7, CH 27.2, CH2 37.7, CH2

57.0, C 2.90, 1.28, 1.16, 1.24,

34.1, CH2

48.8, C 2.73, 1.12, 1.11, 4.78, 4.40, 0.69,

h (6.9) d (6.9) d (6.9) dd (3.2, 1.6) dd (3.2, 1.6) s

41.0, CH 18.7, CH3 18.6, CH3 106.8, CH2 12.3, CH3

Figure 4. NOESY correlations for compounds 4 and 5.

135.7 (d), δH 5.15 (dd, J = 15.6, 10.4 Hz) and 134.6 (d), δH 5.33 (d, J = 15.6 Hz)] (Table 2; Figures S26 and S28, Supporting Information). Next, a tertiary [δC 85.2 (s)] and two secondary alcohols [δC 88.8 (d), δH 3.79 (dd, J = 9.5, 7.6 Hz) and δC 71.1 (d), δH 3.53 (d, J = 8.0 Hz)] were confirmed because of the strong absorption at 3448 cm−1 in the IR spectrum. The HSQC experiment (Figure S30, Supporting Information) demonstrated the 1σ H−C correlations as indicated in Table 2. The DEPT spectrum (Figure S29, Supporting Information) showed four CH3, four CH2, five CH, and two quaternary carbons, suggesting that the second unsaturation is an 11-membered ring and that this compound is a humulene analogue. In the same manner, CH3-12, CH3-13, and CH3-14 showed singlet multiplicity, and CH3-15 appeared as a doublet in the 1H NMR spectrum (Figure S26, Supporting Information). The positions of the double bond and of the hydroxy groups were established from the HMBC spectrum of 4 (Figure 1). In this spectrum (Figure S31, Supporting Information), the cross-peaks H-6/C-7, C-8, C-13, and C-14

helped establish that the double bond occurs between C-6 and C-7, and this was corroborated by the cross-peaks between CH3-15 and C-7, C-8, and C-9. The gem-dimethyl group correlations of CH3-13 and CH3-14 with C-4, C-5, and C-6 showed that one of the three hydroxy groups occurs at C-4. Additionally, the cross-peaks CH3-12 with C-11, C-1, and C-2 demonstrated that the remaining hydroxy groups are at C-1 and C-11. Finally, correlations of H-11 with C-9, C-1, and C-12 were also observed. The structure of compound 4 agreed with the spin systems H-2, H-3, and H-4; H-6, H-7, H-8, and H-15; and H-10 and H-11 in the COSY spectrum (Figure 1, Figure S27; Supporting Information). NOESY cross-peaks for compound 4 supported its proposed relative configuration, with selected cross-peaks shown in Figure 4. The correlations between H-7 and H-8 and H-11 and between H-11 and CH312 were used to locate all these groups on the same molecular face, and therefore, CH3-15 and OH-11 were determined as occurring on the opposite face. Finally, cross-peaks between H4 and CH3-13 and CH3-14 were also observed (Figure S31, 3115



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methylene-10-methyldecahydronaphthalene [1β-hydroxy4(14)-eudesmene, 6]. All other HMBC cross-peaks observed for this compound agreed with this structure (Figure 1). All the NMR data obtained for compounds 3−6 agreed with those reported for related compounds (Tables S3−S6, Supporting Information). Aristoloxazine A (1) was tested for inhibitory activity against four Gram-positive bacteria, namely, one methicillin-sensitive (MSSA) and three methicillin-resistant (MRSA) strains of Staphylococcus aureus. It was also evaluated against three Gramnegative bacteria (Escherichia coli, Proteus mirabilis, and Salmonella typhi), as well as three fungi (Trichophyton mentagrophytes, T. rubrum, and Aspergillus niger), and one yeast (Candida albicans). The results are summarized in Tables 3 and 4. Compound 1 exhibited potent in vitro antimicrobial

Supporting Information). Assuming H-8 as beta, the structure of this natural product (humul-7-en-1,4,11-triol, 4) was deduced as (1S*,4R*,8S*,11R*)-trihydroxyhexahydrohumul6,7-ene. Compound 5 was isolated as a pale yellow oil, and a [M − H]+ peak occurring at m/z 219.1751 was observed in the HRFABMS. Its molecular formula, C15H24O, indicated four degrees of unsaturation. One of these was due to an exocyclic gem-disubstituted double bond [δC 113.6 (t), δH 5.05 (d, J = 1.2 Hz) and δH 4.93 (d, J = 1.2 Hz); and δC 151.5 (s)] (Table 2; Figures S32 and S33, Supporting Information). The three additional degrees of unsaturation were consistent with a tricyclic system occurring in the molecule. A secondary hydroxy group [δC 72.3 (d), δH 3.90 (ddd, J = 10.8, 9.6, 5.6 Hz)] was evident from the strong absorption at 3435 cm−1 in the IR spectrum. The DEPT experiment (Figure S34, Supporting Information) showed 15 carbon resonances: three CH3, five CH2, four CH, and three quaternary carbons. The three methyl groups could be located on quaternary carbons because of their singlet multiplicity in the 1H NMR spectrum (Figure S32, Supporting Information). The HSQC correlations are indicated in Table 2 (Figure S35, Supporting Information). Thus, a longipinene skeleton was inferred for compound 5 in accordance with all these data and the cross-peaks observed in the HMBC spectrum (Figure 1, Figure S36, Supporting Information). In addition, in this experiment, correlations of both hydrogens H2-15 with C-2, C-3, and C-4 and of H2-2 with C-15, C-3, C-4, C-1, and C-11 were observed. Other HMBC correlations were of H2-1 with C-2, C-3, C-10, and C-11, of CH3-12 and CH3-13 with C-5, C-6, and C-7, of H-5 with C-3, C-4, C-6, C-12, and C-13, and of H-11 with C-1. Additionally, the cross-peaks of H2-7 with C-6, C-12, and C-13, of CH3-14 with C-11, C-10, and C-9, and of H2-9 with C-8, C-10, and C11 were also observed. All these data supported the structure of 5 as 8-hydroxylongipin-3,15-ene. If H-8 is beta, the NOESY cross-peaks H-8/H-5 were consistent with an S configuration for C-5 (Figure 4, Figure S37, Supporting Information), while the correlation H-5/H-4 supported an R configuration at C-4, and, finally, the cross-peak between H-4 and H-7 corresponded to a boat conformation for a seven-membered ring. In this manner, the structure of this natural product (8-hydroxy-βlongipinene, 5) was deduced to be (4R*,5S*,8S*,10S*,11R*)8-hydroxylongipin-3,15-ene. Compound 6 was purified as a colorless oil. From the HRFABMS (m/z 221.1911) a molecular formula of C15H26O, corresponding to three degrees of unsaturation, was determined. An exocyclic gem-disubstituted double bond was evident [δC 106.8 (t), δH 4.78 (dd, J = 3.2, 1.6 Hz) and δH 4.40 (dd, J = 3.2, 1.6 Hz); and δC 145.9 (s)] (Table 2; Figures S38 and S40, Supporting Information). A secondary hydroxy group [δC 79.0 (d), δH 3.66 (dd, J = 11.4, 4.8 Hz)], a quaternary methyl [δC 12.3 (q), δH 0.69 (s)], and an isopropyl group [δC 41.0 (d), δH 2.73 (h, J = 6.9 Hz); δC 18.7 (q), δH 1.12 (d, J = 6.9 Hz); and δC 18.6 (q), δH 1.11 (d, J = 6.9 Hz)] were also found to be part of the structure. In the HSQC spectrum (Figure S41, Supporting Information), the remaining eight signals were a quaternary carbon, two CH carbons, and five CH2 carbons, indicating that the last two degrees of unsaturation corresponded to a decalin ring and that 6 is a eudesmane derivative. Based on the CH3-15/C-1 HMBC correlation (Figure S42, Supporting Information), the hydroxy group could be located at C-1, with this compound accordingly assigned as (1R*,5S*,7R*,10R*)-1-hydroxy-7-isopropyl-4-

Table 3. Minimum Inhibitory Concentration (MIC) Values of Aristoloxazine A (1) and Standard Antibiotics against Methicillin-Sensitive (MSSA) and Methicillin-Resistant (MRSA) Gram-PositiveStaphylococcus aureus Strains MIC (μg/mL)a compound/antibiotic

MSSA

MRSA1

MRSA2

MRSA3

aristoloxazine A (1) chloramphenicol oxacillin ampicillin

0.5 1.0 2.0 2.0

0.25 2.0 32 64

0.25 2.0 128 128

0.25 2.0 128 128

MSSA (ATCC 6358), MRSA1 (ATCC 11632 β-lactamase resistant), MRSA2 (ATCC 33591 methicilin resistant), MRSA3 (ATCC 43300 methicilin resistant). a

Table 4. Minimum Inhibitory Concentration (MIC) Values of Aristoloxazine A (1) against Four Fungal Strains MIC (μg/mL)a compound/antibiotic

Tm

Tr

An

Ca

aristoloxazine A (1) nystatin miconazole

1.0 nt 2.0

1.0 nt 2.0

25 nt 16.0

25 8.0 nt

a

Tm = Trichophyton mentagrophytes ATCC 28185, Tr = Trichophyton rubrum ATCC 28188, An = Aspergillus niger ATCC 10335, Ca = Candida albicans ATCC 10231, nt = not treated.

activity against all these methicillin-resistant S. aureus strains, including MSSA, with MIC values from 0.25 to 0.50 μg/mL (Table 3). The highest concentration of compound 1 assayed against the Gram-negative bacteria for this study was 128 μg/ mL. No activity was observed at this concentration. Compound 1 also exhibited substantial inhibitory activities against the dermatophytes T. mentagrophytes and T. rubrum, affording MIC values of 1.0 μg/mL in each case, which were lower than the control values used (Table 4). The MIC values for filamentous fungus A. niger and yeast C. albicans were close to those of the two control substances used (Table 4). Compounds 1 and 2 are the first aristolochic acid derivatives including a thioether and a thioester, respectively. Both compounds also are the first to exhibit an oxazine moiety. Their occurrence in A. orbicularis is interesting because this is the first report of sulfur and oxazine groups in natural products isolated from a species in the genus Aristolochia. The acetamide fragment in compound 1 seems to originate from a cysteine residue via oxidative decarboxylation. The acetamide fragment could be transformed to the corresponding thiol and then 3116



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hexane−CH2Cl2, 7:3, 160 fractions, 50 mL each) yielded (4S,8R)-pmenth-1-en-9-ol (7, 42 mg, 0.15%) and caryophyllene β-oxide (176 mg, 0.62%); group G-2 (n-hexane−CH2Cl2, 7:3, 95 fractions, 30 mL each) yielded madoline R (26 mg, 0.09%) and 3β-acetyl-Δ7-sitosterol (276 mg, 0.97%); group G-3 (n-hexane−CH2Cl2, 7:3, 190 fractions, 30 mL each) yielded syringic acid (23 mg, 0.08%); group G-4 (n-hexane− AcOEt, 9:1, 180 fractions, 50 mL each) yielded squalene (43 mg, 0.17%), 3,5-dimethoxysalicylic acid (14 mg, 0.05%), and β-amyrin (67 mg, 0.23%); group G-5 (n-hexane−AcOEt, 85:15, 179 fractions, 50 mL each) yielded α-caryophyllenol (26 mg, 0.09%), triacontyl ferulate (286 mg, 1.00%), (1S*,4R*,8S*,11R*)-trihydroxyhexahydrohumul6,7-ene (humul-7-en-1,4,11-triol, 4, 29 mg, 0.10%), (4R*,5S*,8S*,10S*,11R*)-8-hydroxylongipin-3,15-ene (8-hydroxy-βlongipinene, 5, 33 mg, 0.12%), and (1R*,5S*,7R*,10R*)-1-hydroxy7-isopropyl-4-methylene-10-methyldecahydronaphthalene (1β-hydroxy-4(14)-eudesmene, 6, 52 mg, 0.18%); group G-6 (n-hexane− AcOEt, 85:15, to n-hexane−AcOEt, 8:2, 103 fractions, 50 mL each) yielded a β-sitosterol−stigmasterol mixture (102 mg, 0.36%), 2-(4hydroxyphenyl)-1-nitroethane (16 mg, 0.06%), ferulic acid (52 mg, 0.18%), savinin (81 mg, 0.28%), and hinokinin (146 mg, 0.51%); group G-7 (n-hexane−AcOEt, 8:2, to n-hexane−AcOEt, 75:25, 119 fractions, 20 mL each) yielded pluviatolide (293 mg, 1.03%), dehydrohinokinin (122 mg, 0.43%), and an inseparable diastereomeric mixture of 7′S-parabenzlactone and 7′R-parabenzlactone (114 mg, 0.40%); group G-8 (n-hexane−AcOEt, 75:25, to n-hexane−AcOEt, 7:3, 119 fractions, 20 mL each) yielded an inseparable diastereomeric mixture of 8R,8′R,9S-cubebin (1077 mg, 3.79%), 6α-hydroxy-βeudesmol (β-chenopodiol, 85 mg, 0.30%), and 4(15)-eudesmene1β,6α-diol (106 mg, 0.37%); group G-9 (CH2Cl2−acetone, 9:1, 63 fractions, 10 mL each) yielded ursolic acid (69 mg, 0.24%), aristololactam BIII (186 mg, 0.65%), and (S)-2-(1,2,8-trimethoxy-4oxo-7H-phenanthro[10,1-cd][1,2]oxazin-7-yl)-E-3-(4-hydroxy-3methoxyphenyl)prop-2-en-thioate (aristoloxazine B, 2, 56 mg, 0.20%); group G-10 (CH2Cl2−acetone, 9:1, 82 fractions, 20 mL each) yielded 7-methoxytaliscanine (3, 27 mg, 0.09%) and aristololactam BI (taliscanine, 356 mg, 1.25%); group G-11 (CH2Cl2−acetone, 85:15, 56 fractions, 20 mL each) yielded aristololactam AII (216 mg, 0.76%); group G-12 (CH2Cl2−acetone, 8:2, 56 fractions, 30 mL each) yielded (S)-2-(1,2,8-trimethoxy-4-oxo-7H-phenanthro[10,1-cd][1,2]oxazin-7yl)thioacetamide (aristoloxazine A, 1, 1200 mg, 4.22%). Aristoloxazine A (1): pale yellow powder (AcOEt); mp 184−186 °C; [α]25D +140.8 (c 0.1, acetone); UV (MeOH) λmax (log ε) 278 (6.24), 205 (7.34) nm; ECD (MeOH, 1 × 10−4 M) λmax (Δε) 284 (+120) nm; IR (film) νmax 3405, 2919, 2849, 1727, 1679, 1571, 1462, 1413, 1369, 1259, 1023, 754 cm−1; 1H NMR (CDCl3−DMSO-d6, 500 MHz) and 13C NMR (CDCl3-DMSO-d6, 125 MHz) data, see Table 1; 1 H NMR (DMSO-d6, 500 MHz) δ 8.09 (1H, d, J = 8.2 Hz, H-11), 7.62 (1H, s, H-3), 7.41 (1H, dd, J = 8.2, 8.2 Hz, H-10), 7.32, 7.11 (each 1H, br s, NH2), 7.11 (1H, d, J = 8.2 Hz, H-9), 5.29 (1H, s, H-7), 4.04 (3H, s, OCH3-2), 3.91 (3H, s, OCH3-8), 3.87 (3H, s, OCH3-1), 3.27, 3.14 (each 1H, d, J = 15.1 Hz, H-1′); 13C NMR (DMSO-d6, 125 MHz) δ 169.5 (C, C-2′), 162.9 (C, C-4), 158.4 (C, C-2), 156.0 (C, C8), 152.5 (C, C-1), 152.0 (C, C-6a), 129.9 (C, C-11a), 129.1 (CH, C10), 124.0 (C, C-11b), 120.0 (CH, C-11), 120.0 (C, C-7a), 118.9 (C, C-3a), 116.0 (C, C-11c), 112.3 (CH, C-9), 109.3 (CH, C-3), 60.7 (CH3, OCH3-1), 56.8 (CH3, OCH3-2), 56.1 (CH3, OCH3-8), 37.8 (CH, C-7), 34.6 (CH2, C-1′); HRESIMS (positive-ion mode) m/z 437.0797 (calcd for C20H18N2O6SNa, 437.0783); anal. C 57.3025, H 4.3311, N 6.6685, O 24.2908, S 7.4071 (calcd C 57.96, H 4.38, N 6.76, O 23.16, S 7.74). X-ray Crystal Data for Compound 1. The crystal structure of 1 was determined by crystallography. Single crystals of aristoloxazine A (1) (C20H18N2O6S) were obtained. A suitable crystal was selected and diffracted on a SuperNova, Dual, Cu at zero, EosS2 diffractometer. The crystal was kept at 100.0(3) K, with CuKα radiation (λ = 1.542) during data collection. Using Olex2,37 the structure was solved with the ShelXT38 structure solution program using intrinsic phasing and refined with the ShelXL39 refinement package using least squares minimization. Molecular graphics were computed with ORTEP. Crystal data for C20H18N2O6S (M = 414.42 g/mol): monoclinic,

produces a thioester with a ferulic acid residue to produce compound 2.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 MC polarimeter (Beaconsfield, UK). UV spectra were recorded at 25 °C on a Hewlett-Packard 8453 diode array spectrophotometer (Waldbronn, Germany), using CHCl3 as solvent. Electronic circular dichroism (ECD) measurements were recorded on a JASCO J-1500 circular dichroism spectrophotometer (Tokyo, Japan). IR spectra were obtained as films (CHCl3) on a Thermo Scientific Nicolet 6700 IR spectrometer (Thermo Electron Corporation, Madison, WI, USA). NMR spectra were recorded on a Varian Inova 400 MHz spectrometer (Varian, Inc., Palo Alto, CA, USA) or Bruker Avance III HD 500 MHz spectrometer (Bruker, Rheinstetten, Germany) at 400 or 500 MHz for 1H NMR, 1H−1H COSY, HSQC, and HMBC and at 100 or 125 MHz for 13C NMR and DEPT, using CDCl3 or DMSO-d6 as solvent, as indicated. Chemical shifts are reported in ppm (δ) relative to the tetramethylsilane signal. 15 N and 33S NMR measurements were made at 50.62 and 38.38 MHz, respectively. A liquid ammonia scale was used as a reference for the 15 N NMR chemical shift. Ammonium sulfate in a D2O sample was used as the 33S chemical shift reference. The mass spectrometric data for compounds 1−3 were recorded on an Agilent 6545 Series Q-TOF LC/MS system (1290 Infinity II) dual AJS ESI source (Agilent Technologies, Inc., Waldbronn, Germany). In turn, the MS data for compounds 4−6 were recorded on a JEOL JMStation-JM 700 mass spectrometer (JEOL, Tokyo, Japan) in a matrix of glycerol. X-ray data were collected using an Agilent Technologies Super Nova X-ray singlecrystal diffractometer using Cu Kα radiation (Abingdon, UK). Plant Material. The roots of A. orbicularis were collected in Xochicalco, Morelos, México, in October 2014. The plant (voucher INAHM-2032) was identified by Margarita Avilés and Macrina Fuentes from the Instituto Nacional de Antropologiá e Historia Morelos (INAHM) and was deposited at the Herbarium of INAHM, Cuernavaca, Morelos, México. Laboratory Safety Guidance. Most Aristolochia species biosynthesize aristolochic acids and their derivatives, which have been shown to possess carcinogenic properties.2 The isolation and structural characterization of the Aristolochia compounds was performed under the following special laboratory precautions: work under a fume hood and over disposable plastic-backed paper, identify the work and storage areas, limit access to these areas, wear disposable gloves and fully closed lab coats, do not wear these items outside of the work area, avoid direct contact with eyes and skin, wash both hands after completing the work.36 Extraction and Isolation. The air-dried and powdered roots from A. orbicularis (670 g) were extracted with acetone (3 L × 48 h × 3 times) at room temperature. The extraction solvent was concentrated to dryness in vacuo to obtain 28.4 g of a brown dark residue. Fractionation of this extract by open column chromatography over silica gel 60 was performed using a pure CH2Cl2 to 2:8 CH2Cl2− AcOEt gradient, as indicated below, collecting 312 fractions of 150 mL each. The composition of the fractions was monitored by thin-layer chromatography (TLC), and the compounds were visualized using a UV lamp and spraying them with a 1% solution of (NH4)4Ce(SO4)4· H2O in 2 N H2SO4. On the basis of TLC, the fractions were pooled into 12 groups: G-1 (fractions 3 and 4, 2.0 g, CH2Cl2), G-2 (fractions 5 and 6, 1.4 g, CH2Cl2), G-3 (fractions 7 and 8, 1.5 g, CH2Cl2), G-4 (fractions 9−18, 2.3 g, CH2Cl2), G-5 (fractions 19−45, 2.4 g, CH2Cl2), G-6 (fractions 46−101, 3.0 g, CH2Cl2−AcOEt, 95:5), G-7 (fractions 102−124, 2.8 g, CH2Cl2−AcOEt, 9:1); G-8 (fractions 125−150, 0.58 g, CH2Cl2−AcOEt, 8:2); G-9 (fractions 151−175, 0.73 g, CH2Cl2− AcOEt, 7:3); G-10 (fractions 176−210, 0.87 g, CH2Cl2−AcOEt, 6:4); G-11 (fractions 211−261, 1.8 g, CH2Cl2−AcOEt, 4:6), and G-12 (fractions 262−312, 1.9 g, CH2Cl2−AcOEt, 2:8). Each group was further separated into pure compounds using column chromatography over silica gel 60 and a gradient of n-hexane−CH2Cl2, n-hexane− AcOEt, or CH2Cl2−acetone as eluent, as indicated. Group G-1 (n3117



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space group P21 (no. 4), a = 4.70819(5) Å, b = 18.20421(17) Å, c = 10.60117(11) Å, β = 95.3229(9)°, V = 904.696(15) Å3, Z = 2, T = 100.0(3) K, μ(Cu Kα) = 1.979 mm−1, Dcalc = 1.521 g/cm3, 12 596 reflections measured (8.376° ≤ 2θ ≤ 145.526°), 3542 unique (Rint = 0.0246, Rsigma = 0.0224), which were used in all calculations (Tables S1 and S2, Supporting Information). The final R1 was 0.0228 (I > 2σ(I)), and wR2 was 0.0598. The refinement model description is included (Tables S1 and S2, Supporting Information). Aristoloxazine B (2): yellow powder (AcOEt); mp 239−242 °C; [α]25D +279.6 (c 0.01, acetone); UV λmax (log ε) 285 (6.25), 205 (7.34) nm; IR (film) νmax 3400, 2918, 2850, 1712, 1598, 1465, 1365, 1264, 1169, 1030 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; HRESIMS (positive-ion mode) m/z 517.1248 (calcd for C28H23NO7S, 517.1195). 7-Methoxytaliscanine (3): yellow powder (AcOEt); mp 101−103 °C; UV λmax (log ε) 242 (4.98) nm; IR (film) νmax 2919, 2850, 1687, 1660, 1462, 1267, 1029, 747 cm−1; 1H NMR (CDCl3, 400 MHz) and 13 C NMR (CDCl3, 100 MHz), see Table 1; HRESIMS (positive-ion mode) m/z 362.0982 (calcd for C19H17NO5Na, 362.1004). Humul-7-en-1,4,11-triol (4): colorless oil; [α]25D −7.5 (c 0.1, acetone); IR (film) νmax 3448, 2927, 2869, 1457, 1379, 1163, 1061 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Table 2; FABMS (positive-ion mode) m/z 257 [M + H]+ (15), 255 (100), 253 (93), 237 (83), 221 (61), 219 (52), 203 (44); HRFABMS (positive-ion mode) m/z 255.1953 [M − H]+ (calcd for C15H27O3, 255.1960). 8-Hydroxy-β-longipinene (5): colorless oil; [α]25D −18.9 (c 0.1, acetone); IR (film) νmax 3436, 2928, 2866, 1708, 1460, 1367, 1172, 1054 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Table 2; FABMS (positive-ion mode) m/z 221 [M + H]+ (23%), 219 (100), 201 (53); HRFABMS (positive-ion mode) m/z 219.1751 [M − H]+ (calcd for C15H23O, 219.1749). 1β-Hydroxy-4(14)-eudesmene (6): colorless oil; [α]25D −27.3 (c 0.1, acetone); IR (film) νmax 3430, 2922, 2870, 1314, 1173, 1048 cm−1; 1 H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Table 2; FABMS (positive-ion mode) m/z 223 [M + H]+ (17%), 221 (100), 204 (72), 189 (72), 146 (34); HRFABMS (positive-ion mode) m/z 221.1911 [M − H]+ (calcd for C15H25O, 221.1905). Antimicrobial Activity. Bacterial Strains. A set of Gram-positive and Gram-negative bacteria [Staphylococcus aureus-sensitive ATCC 6538, S. aureus ATCC 11632 (β-lactamase producer), S. aureus ATCC 33591 (methicillin resistant), S. aureus ATCC 43300 (methicillin resistant), Escherichia coli ATCC 25922, Proteus mirabilis ATCC 43071, and Salmonella typhi ATCC 06539] was used. The cultures were maintained in Mueller-Hinton (MH) agar [Merck, (Germany), AMH] at 4 °C prior to their use. Bacterial strains were inoculated in a MH broth (Merck) at 37 °C, 18 h prior to initiating the test. Antibacterial Activity (MIC Determination). Microbial activity was determined following the standard broth microdilution method described by the National Committee for Clinical Laboratory Standards. 40 Aristoloxazine A (1) and a standard antibiotic (chloramphenicol, oxacillin, and ampicillin) were serially diluted in microplate wells until a concentration of 0.125−128 μg/mL was obtained. The cultures were adjusted to 105 colony-forming unit (CFU/mL) inoculum size, employing the standard 0.5 MacFarland scale. Five microliters of culture was added to each well, which contained culture medium and the diluted samples. The microplates were incubated at 37 °C for 24 h. After this period, 20 μL of piodonitrotetrazolium violet (0.5 mg/mL) was added to each well. The formation of a red color indicated cellular viability. The MIC value was determined as the lowest concentration of the sample assayed that did not form the red color in the microplate well. Antifungal Activity. The following four strains of fungi were used during the test: Trychophyton mentagrophytes ATCC 28185, Trychophyton rubrum ATCC 28188, Aspergillus niger ATCC 10535, and Candida albicans ATCC10231. The filamentous fungi were maintained on potato dextrose agar (Merck, Germany) at 27 °C. Sabouraud glucose agar (Merck) was used to keep up the yeast and as an assay medium. The antifungal assay was performed by the agar dilution method41,42 using Petri dishes (Falcon, MA, USA). The stock

solution of aristoloxazine A (1) and the reference compounds, nystatin (Merck) and miconazole (Sigma, México), were 2-fold serial diluted, yielding concentrations in the range from 128 to 0.5 μg/mL. Final concentrations of DMSO in the test were less than 2% (v/v). A final inoculum of 105 cell/mL for Candida albicans and 106 spore/mL for filamentous fungi were each placed on top of the solidified agar with a loop calibrated to deliver 0.005 mL. Experiments were carried out in duplicate and incubated at 29 °C. The fungal growth was checked first in control plates prepared without any test sample after 24, 48, and 72 h, depending on the incubation period required for a visible growth: 24 h for Candida albicans, 24 h for Aspergillus niger, and 72 h for dermatophytes.

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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.7b00226. 1D and 2D NMR spectra of 1−6; Tables S1−S6; ECD, MS, and elemental analysis data of 1 (PDF) Supporting figure (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (M. Y. Rios): +52 (777) 329-7000, ext. 6024. Fax: +52 (777) 329-7997. E-mail: [email protected]. ORCID

María Yolanda Rios: 0000-0002-8875-8734 M. Á ngeles Ramírez-Cisneros: 0000-0003-4696-7359 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by CONACyT (grant number 241044) and IMSS (grant FIS/IMSSPROT/G12/ ́ 1140). We are grateful to B. Dominguez, M. Medina, P. Román, and G. Vargas for their technical assistance. All spectroscopic and spectrometric analyses were obtained from LANEM (grant numbers 279905 and 254145).

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REFERENCES

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