Dirchromones: Cytotoxic Organic Sulfur ... - ACS Publications

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Dirchromones: Cytotoxic Organic Sulfur Compounds Isolated from Dirca palustris Alexis St-Gelais, Jean Legault, Vakhtang Mshvildadze, and André Pichette* Chaire de Recherche sur les Agents Anticancéreux d’Origine Naturelle, Laboratoire d’Analyse et de Séparation des Essences Végétales (LASEVE), Département des Sciences Fondamentales, Université du Québec à Chicoutimi, 555 Boulevard de l’Université, Chicoutimi, Québec, Canada, G7H 2B1 S Supporting Information *

ABSTRACT: Eight novel organic sulfur compounds featuring an unprecedented structure among natural organosulfur molecules are reported. The identified compounds, named dirchromones (1−8), were isolated from a dichloromethane extract of the roots, bark, and wood of Dirca palustris, an endemic shrub of eastern North America. Their identification was based on thorough NMR, IR, and HRMS spectroscopic data interpretation. These compounds showed cytotoxic and mild Gram-positive antibacterial activities in vitro, while being inactive against Gram-negative Escherichia coli and the yeast Candida albicans. This is the first report of sulfur-containing compounds from a species of the Thymelaeaceae.

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he North American shrub Dirca palustris L. (Thymelaeaceae; common names moosewood, eastern leatherwood, bois de plomb in French) is found in maple forests of eastern regions of Canada and the United States.1 It has alternate, ovate, slightly acuminate leaves.2,3 Its tubular yellow flowers appear very early in the spring and possess exserted stamens and styles.2,4,5 The wood is brittle, but the fibrous bark is flexible and resistant.3,5,6 It was peeled into long strips and used as ropes and ties by the First Nations of the east coast and by the early European settlers.3,6−8 Indigenous populations also used the crushed bark against cancer.3,6,9 The plant is known to be emetic, laxative, and vesicant.3,6,10−12 D. palustris is the only native member of the Thymelaeaceae in the province of Quebec, Canada.3 This family is noticeable for its daphnane-type diterpenoid orthoesters, which among other biological effects are potent cytotoxic agents.13 Such diterpenoid compounds have been isolated from the related species D. occidentalis, along with cytotoxic lignans and coumarins.14 The chemical composition of D. palustris has not been extensively studied, with only five phenolic glycosides15 and three triglycerides16 reported in the literature. The bioassay-guided fractionation of a cytotoxic extract of this plant led to the isolation of a new group of sulfur-containing secondary metabolites, named dirchromones (1−8, Figure 1). This study presents the isolation, structure elucidation, cytotoxicity, and antibiotic activity of these new compounds.

Figure 1. Structures of dirchromones 1−8, with diagnostic 2D NMR correlations for compounds 1 and 2 (solid lines indicate DQF-COSY correlations, and arrows indicate HMBC correlations).

carbonyl function. A conjugated olefin was inferred from two bands at 1609 (s) and 2928 cm−1 (m). A sulfoxide function was also indicated by a strong peak at 1061 cm−1 (SO stretch17) and a broad band at 3449 cm−1. The 1H NMR, 13C NMR, and DEPT-135 spectra indicated one carbonyl, one methyl, seven sp2 methines, and three quaternary carbons. COSY correlations (Figure 1) showed two separate systems: the trans-alkene formed by the H-11 and H-12 doublets, also apparent from their large coupling constant (J = 14.9 Hz); and the aromatic H-5, H-6, H-7, and H-8, part of an ortho-disubstituted ring, together with quaternary carbons C-9 and C-10 owing to several relevant HMBC correlations. In addition to their coupling pattern, the proper assignments of these protons were confirmed by comparison of their related 13C NMR chemical shifts with those of flavone.18 HMBC correlations (Figure 1) suggested that H-14 is in a β position at C-12. The presence of an α-sulfoxide was inferred from the chemical shift of C-14,

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RESULTS AND DISCUSSION Compound 1 was isolated as an optically inactive amorphous, white solid. Its ESITOFMS analysis showed a pseudomolecular [M + H]+ peak at m/z 235.0415, suggesting the molecular formula C12H10O3S (calcd for C12H11O3S, 235.0423), with an intense [M + H + 2]+ peak (4.7%), also indicative of a sulfur atom. An IR absorption peak at 1643 cm−1 (s) suggested a © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 13, 2015

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DOI: 10.1021/acs.jnatprod.5b00227 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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carbon at δC 40.4 ppm (C-14), and two trans olefinic protons (H-11 and H-12) were observed, as in compounds 1 and 2. Furthermore, a pyranone ring with 13C NMR chemical shifts closely comparable to those of these aforementioned compounds was also inferred. The aromatic carbons, on the other hand, were more similar to those of compound 2, suggesting the same substitution pattern, with the OMe-6 protons correlating in the HMBC spectrum with C-6. This was confirmed by a COSY correlation between H-7 and H-8, as well as HMBC correlations between H-5 and C-4, C-7, and C-9. These results also found further validation when compared with the reported values for ring A of 6-methoxyflavone.20 Thus, the structure of 3 (6-methoxydirchromone) was concluded to be (E)-6-methoxy-2-[(2-methylsulfinyl)vinyl]-4H-chromen-4-one. Compound 4 showed a similar molecular weight and IR spectrum to those of compound 3, suggesting the same molecular formula and skeleton. Indeed, the NMR spectra indicated the presence of one methoxy group bonded to an aromatic ring, along with the usual pyranone and methylsulfoxide side chain. However, H-5, which correlated with C-4, C7, and C-9 in the HMBC spectrum, was a doublet and showed COSY correlations with H-6. The methoxy group correlated with C-7, while H-8, a doublet with J = 2.4 Hz, exhibited HMBC correlations with C-6 and C-10. The substitution of the aromatic ring by a methoxy group at position 7 was confirmed by a comparison of the aromatic δC with those of ring A of 7methoxyflavone.21 Compound 3 (7-methoxydirchromone) was thus assigned as (E)-7-methoxy-2-[(2-methylsulfinyl)vinyl]4H-chromen-4-one. Compound 5 exhibited a pseudomolecular ion peak [M + H]+ at m/z 295.0635 (calcd for C14H15O5S, 295.0635), suggesting an additional OMe group when compared with 3 and 4, and this was confirmed from the 1H NMR spectrum wherein two singlets resonated at δH 3.99 and 4.01. Apart from the latter signals and those of the typical methylsulfoxide lateral chain and pyranone ring, only two aromatic protons were observed on the 1H NMR spectrum. Since both were singlets, they were deduced to be para to one another. HMBC correlations between H-5 and C-4 and C-7 and between H-8 and C-6 and C-10 allowed their positions to be assigned. The methoxy groups were positioned at C-6 and C-7 from the HMBC spectrum. The structure of 5 (6,7-dimethoxydirchromone) was defined as (E)-6,7-dimethoxy-2-[(2-methylsulfinyl)vinyl]-4H-chromen-4-one and was confirmed by comparing the 13 C NMR chemical shifts of the aromatic portion of the molecule with those reported for 6,7-dimethoxyflavone.22 As a result of its ESITOFMS analysis, compound 6 gave the same molecular formula as 5. As for other compounds isolated, the methylsulfinylvinyl side chain and the pyranone ring were evident from the NMR spectra. The aromatic part comprised two vicinal protons, H-5 and H-6, with the former exhibiting HMBC correlations with C-4 and C-7. Further correlations were observed in the HMBC spectrum between H-6 and C-8 and C-10. Finally, the two methoxy groups correlated with C-7 and C-8, thus indicating the structure of 6 to be (E)-7,8dimethoxy-2-[(2-methylsulfinyl)vinyl]-4H-chromen-4-one. This structure of 7,8-dimethoxydirchromone (6) was confirmed by comparison of the 13C NMR chemical shifts of the aromatic part of the molecule with data reported for 7,8-dimethoxyflavone.21 Compound 7 gave a pseudomolecular ion peak [M + H]+ at m/z 281.0488 (calcd for C13H13O5S, 281.0478), suggesting it to be a dirchromone with a methoxy and a hydroxy group. The

similar to that of the DMSO-d6 solvent residual peak, in addition to the previous evidence of a sulfur atom obtained. The HMBC spectrum further indicated that H-11 and H-12 correlated with C-2, which thus was determined as the sulfurbearing chain attachment point. The H-11 signal correlated with C-3 as well, with the H-3 proton showing HMBC correlations with the C-4 carbonyl, carbon C-2, and the C-10 aromatic carbon. From the latter correlation, as well as that of H-5 with C-4, it was concluded that the carbon C-10 is bonded to C-4, linked in turn to C-3, which is part of an alkene with C2. The chemical shifts of C-2 and C-9 were explained further by the presence of an ether bond, consistent with the molecular formula of 1. This evidence lead to the assignment of (E)-2-[(2methylsulfinyl)vinyl]-4H-chromen-4-one for the structure of 1, which was given the trivial name dirchromone. Given the lack of any optical rotation for the compound, it was inferred as being a racemic mixture of the two enantiomers of the sulfoxide moiety. Compound 2 was isolated as an optically inactive, amorphous, yellow powder. Its pseudomolecular [M + H]+ ion peak at m/z 251.0371 corresponded to the molecular formula C12H10O4S (calcd for C12H11O4S, 251.0373), again with an intense [M + H + 2]+ peak (4.6%) from the sulfur atom. An IR absorption peak at 3182 cm−1 (br, m) indicated a phenolic compound. A band at 1633 cm−1 (s) once again pointed to a carbonyl function. A sulfoxide group could, here too, be suspected from the medium absorption band at 1025 cm−1. As in 1, the presence of a conjugated olefin resulted in the occurrence of two bands at 1612 cm−1 (s) and 2928 cm−1 (m). The 1H NMR, 13C NMR, and DEPT-135 spectra indicated a carbonyl, a methyl, six sp2 methines, and four quaternary carbons. The nonaromatic portion of the molecule was very similar to that of 1. A common skeleton was indeed found from the corresponding correlations of proton H-14 with C-12, proton H-12 with C-2, and proton H-11 with C-2 and C3, indicating the same methylsulfoxide-alkene-alkene arrangement, with a trans configuration from the large coupling constant. Further correlations of proton H-3 with C-2, C-4, and C-10 and the deshielding of C-2 and C-9 due to the ether bridge suggested the same chromone pattern as in 1 (Figure 1). The multiplicity pattern of aromatic protons indicated a trisubstituted aromatic ring, with two positions bearing oxygen due to downfield chemical shifts. Protons H-7 and H-8 showed mutual COSY correlations, with the former exhibiting a strong meta HMBC correlation with C-9 and the latter correlating with the meta positions of C-6 and C-10, giving information on five out of the six signals. The remaining H-5 doublet, owing to small coupling constant and HMBC meta correlation with C-9, was assigned to the other meta position of H-7, completing the ring assignments. The ring substitution was confirmed by comparison of the observed aromatic chemical shifts with those of 6-hydroxyflavone.19 Analysis of the spectroscopic data pointed to (E)-6-hydroxy-2-[(2-methylsulfinyl)vinyl]-4H-chromen-4-one as being the structure of 2, which was named 6hydroxydirchromone. This compound also was isolated as a racemic mixture of sulfoxides with no discernible optical rotation, as were compounds 3−8. The molecular formula of compound 3 was determined as C13H12O4S from a pseudomolecular ion peak [M + H]+ at m/z 265.0528 (calcd for C13H13O4S, 265.0529). The characteristics of the methylsulfoxide side chain, with a [M + H + 2]+ ion at 4.7% of parent ion abundance, an IR band at 1028 cm−1 (s), a B



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Table 1. 13C NMR Data of Compounds 1−8 (δ in ppm, type) position 2 3 4 5 6 7 8 9 10 11 12 14 6-OMe 7-OMe 8-OMe a

1a 158.0, 113.2, 178.3, 125.9, 125.5, 134.3, 117.8, 155.9, 124.0, 127.4, 141.7, 40.4,

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

2b 158.1, 111.0, 177.2, 107.6, 155.9, 123.8, 119.4, 148.7, 124.3, 125.7, 143.8, 39.6,

3a

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

157.8, 112.4, 178.1, 105.0, 157.2, 124.3, 119.3, 150.7, 124.7, 127.5, 141.4, 40.4, 56.0,

4b

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

158.2, 112.1, 176.6, 126.4, 114.7, 164.1, 100.6, 157.2, 117.2, 125.5, 143.9, 39.6,

5a

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

157.4, 112.8, 177.3, 104.5, 147.9, 154.9, 99.5, 152.0, 117.5, 127.6, 140.7, 40.4, 56.4, 56.5,

56.1, CH3

6a

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

157.8, 112.8, 177.9, 121.3, 110.1, 157.1, 136.7, 150.2, 118.7, 127.5, 141.4, 40.4,

7b

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

56.5, CH3 61.6, CH3

157.6, 111.3, 176.3, 107.1, 146.0, 154.2, 100.1, 150.2, 117.1, 125.8, 143.1, 39.7,

8b

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

56.1, CH3

158.2, 111.4, 177.1, 114.7, 110.1, 151.7, 134.6, 145.2, 118.0, 125.7, 144.1, 39.7,

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

56.3, CH3

In CDCl3, referenced to TMS. bIn DMSO-d6, referenced to main solvent residual peak at 39.5 ppm.

Table 2. 1H NMR Data of Compounds 1−8 [δ in ppm, multiplicity (J in Hz)] position

1a

3 5 6 7 8 11 12 14 6-OMe 7-OMe 8-OMe

6.41, s 8.20, dd (8.0, 1.7) 7.43, t (8.4) 7.71, ddd (8.4, 7.2, 1.7) 7.48, d (7.6) 7.06, d (14.9) 7.55, d (14.9) 2.80, s

a

2b

3a

6.59, s 7.25, d (3.1)

6.40, s 7.56, d (3.1)

7.24, dd (9.3, 3.1) 7.47, d (9.3) 6.98, d (15.2) 7.97, d (15.2) 2.77, s

7.30, dd (9.2, 3.1) 7.42, d (9.2) 7.05, d (15.0) 7.52, d (15.0) 2.79, s 3.91, s

4b

5a

6.63, s 7.92, d (8.8) 7.07, dd (8.8, 2.4)

6.38, s 7.52, s

7.11, 7.00, 7.98, 2.78,

6.91, 7.03, 7.48, 2.78, 3.99, 4.01,

d (2.4) d (15.1) d (15.1) s

3.92, s

s d (15.0) d (15.0) s s s

6a 6.35, s 7.93, d (9.0) 7.05, d (9.0)

7.06, d (15.0) 7.56, d (15.0) 2.80, s 4.01, s 4.00, s

7b 6.54, s 7.25, s

7.09, 6.97, 7.92, 2.76,

s d (15.2) d (15.2) s

3.90, s

8b 6.57, s 7.47, d (8.3) 7.18, d (8.3)

6.99, d (15.1) 8.12, d (15.1) 2.78, s 3.93, s

In CDCl3, referenced to TMS. bIn DMSO-d6, referenced to main solvent residual peak at 2.50 ppm.

Dirchromones are the first sulfur-containing secondary metabolites reported from a plant in the Thymelaeaceae. Indeed, sulfur compounds are relatively scarce among higher plants. They are noticeably found within two major groups. Alliaceae family members produce cysteine-derived sulfoxides, which includes alliin, for which the degradation products following enzymatic cleavage have attracted considerable research efforts due, in good part, to their putative cancer chemopreventive properties.24−27 Similarly, members of the order Brassicales, as well as plants from the Pittosporaceae, Phytolaccaceae, and the genus Drypetes, contain sulfur-bearing glucosinolates, which are converted into isothiocyanates and related products by myrosinases.28 Glucosinolates have also drawn interest given their proapoptotic29−31 and potential chemopreventive properties.32−34 The cytotoxicity of compounds 1−8 was evaluated against human cancer and healthy cell lines including lung adenocarcinoma (A549), colorectal adenocarcinoma (DLD1), and skin fibroblasts (WS1), using the Hoechst assay.35 The results presented in Table 3 are expressed as the concentration inhibiting 50% of cell growth (IC50). Etoposide when used as a positive control selectively inhibited cancer cell growth with IC50 values of 0.48 μM for A549 cells and 3.3 μM for DLD-1 cells. The results showed that dirchromone 1 and the monomethoxydirchromones 3 and 4 were the most active compounds against all tested cell lines. In addition, they were found to be 5 to 10 times more active against DLD-1 (IC50: 1.0

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C NMR chemical shifts were very similar to those of 5, suggesting that it had a similar substitution pattern. This was further supported by the HMBC correlations and the presence of two aromatic proton singlets. HMBC correlations were observed between H-5 and C-4, C-7, and C-9, confirming their locations. Since the methoxy group showed HMBC correlations with C-7, its position, along with that of the hydroxy group, could be deduced as C-7 and C-6, respectively. Consequently, the structure of 6-hydroxy-7-methoxydirchromone (7) was assigned as (E)-6-hydroxy-7-methoxy-2-[(2methylsulfinyl)vinyl]-4H-chromen-4-one. Compound 8 exhibited the same molecular formula as compound 7, and on analysis of the HMBC and DQF-COSY spectra, the compound was found to be consistent with those of compounds 1−7 in terms of the side chain and the pyranone ring. The two aromatic protons appeared as doublets with coupling constants indicative of their ortho arrangement. Strong HMBC correlations between H-5 and C-4, C-7, and C-9, along with a correlation between the methoxy group with C-7, allowed the assigment of their positions, and it could be deduced that the phenol group is linked to C-8. This led to the determination of 8 as (E)-8-hydroxy-7-methoxy-2-[(2methylsulfinyl)vinyl]-4H-chromen-4-one (8-hydroxy-7-methoxydirchromone), which was confirmed by comparison of the 13C NMR chemical shift data in the aromatic section with those reported for 8-hydroxy-7-methoxyflavone.23 C



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characterized by 1H, 13C, DEPT-135, DQF-COSY, HSQC, and HMBC experiments. Compounds 1−6 were analyzed using standard NMR tubes, and compounds 7 and 8 were analyzed using highprecision Shigemi NMR tubes, given the small quantities available. ESITOFMS analyses were performed on an Agilent LC 1200 Series/ 6210 TOF LC-MS in the positive mode. Preparative HPLC separations were performed using either a Shimadzu system (two LC-20AP pumps, SIL-10AP automatic injector, CTO-20AC column oven, FRC-10A fraction collector, and SPD-M20A diode-array detector) thermostated at 25 °C with a flow rate of 20 mL/min or an Agilent 1100 system (two pumps, automatic injector, fraction collector, and multiple-wavelength detector) running at ambient temperature with a flow rate of 20 mL/min. Preparative columns used were either 22 × 250 mm Vydac ODS 218TP preparative columns (10 μm particle size) or 21.2 × 250 mm XDB-C18 prep HT columns (7 μm particle size). Solvents were purchased from Fisher (Canada). TLC plates (aluminum sheets of ultrapure silica gel 250 μm, with indicator F254) and ultrapure silica gel (40−63 μm) were supplied from Silicycle (Canada). Good TLC conditions for visualization of compounds 1−8 were CHCl3−MeOH (15:1) developed with H2SO4 20% in MeOH followed by heating at 100 °C and observation at 365 nm under UV light. Plant Material. Whole Dirca palustris plants were collected by Alexis St-Gelais, Hubert Marceau, and Myriam Gauthier in May 2012 in Repentigny, Québec, Canada (45°45′28″ N, 73°30′55″ W). A specimen of the collected plants was identified by Patrick Nadeau (biology technician, Université du Québec à Chicoutimi) and deposited in the herbarium Louis-Marie at Université Laval, Québec (reference number QFA0375681). The defoliated plants were air-dried and coarsely ground. Extraction and Isolation. The dried and ground roots, bark, and wood of D. palustris (12.5 kg) were defatted with hexanes and extracted with CH2Cl2 (0.57%). The CH2Cl2 extract was partitioned between hexanes and MeOH. The polar fraction (50 g) was submitted to low-pressure liquid chromatography over silica gel using a CH2Cl2− MeOH gradient (100:0 to 0:100), yielding subfractions A−E. Subfraction B (23.9 g) was refined over silica gel with hexanes− EtOAc as a gradient (3:1 to 1:1), then MeOH, giving subfractions B1−B7. Subfraction B7 (4.28 g) was purified over silica gel with a hexanes−acetone gradient (2:1 to 0:100), then MeOH, affording subfractions B7A−B7H. Subfraction B7D (344 mg) was further purified using preparative HPLC (Agilent system, Vydac column, 13% isocratic CH3CN for 45 min) to yield compounds 1 (tR = 20.1 min, 35.9 mg), 3 (tR = 40.1 min, 29.7 mg), 4 (tR = 36.7 min, 6.7 mg), and 6 (tR = 29.8 min, 10.9 mg). Preparative HPLC purification (Shimadzu system, Vydac column, 13−16% CH3CN in 30 min followed by 16% CH3CN for 7 min) of subfraction B7E (287 mg) afforded compound 2 (tR = 13.8 min, 15.9 mg), 4 (tR = 33.0 min, 6.9 mg), and 6 (tR = 29.5 min, 5.3 mg). Compounds 5 (tR = 21.9 min, 10.5 mg) and 8 (tR = 20.4 min, 5.6 mg) were purified from subfraction B7F (424 mg), again using preparative HPLC (Shimadzu system, Vydac column, 15−40% MeOH in 60 min). Finally, HPLC purification (Shimadzu system, Vydac column, 20−40% MeOH in 60 min) of subfraction B7G (167 mg) afforded compounds 1 (tR = 30.9 min, 0.5 mg) and 7 (tR = 21.4 min, 0.9 mg). Some compounds containing impurities were further purified by preparative HPLC (Agilent system, XDB-C18 column): compound 1 (40% isocratic MeOH, tR = 9.2 min), compound 2 (36% isocratic MeOH, tR = 7.7 min), compound 3 (38% isocratic MeOH, tR = 16.6 min), and compound 6 (45% isocratic MeOH, tR = 6.6 min). All compounds appeared as yellow-green spots on TLC when examined under UV light at 365 nm; the color was enhanced by spraying with H2SO4 and heating 5 min at 110 °C. Dirchromone (1): white, amorphous powder; [α]20D 0 (c 0.02, CH2Cl2); UV (IPA) λmax (log ε) 204 (4.46), 251 (4.27), 302 (4.27); IR (film) νmax 3449, 3049, 2923, 2854, 1643, 1609, 1562, 1515, 1465, 1387, 1332, 1305, 1246, 1223, 1122, 1061, 968, 880, 857, 817, 779, 756, 677 cm−1; 1H NMR (CDCl3, 400 MHz) data, see Table 2; 13C NMR (CDCl3, 100 MHz) data, see Table 1; ESITOFMS m/z 235.0415 (calcd for C12H11O3S, 235.0423).

Table 3. Cytotoxic Activity of Compounds 1 and 3−6 IC50 (μM) compound 1 3 4 5 6 etoposide

A549 10 9 10.5 >10 >10 0.48

±1 ±1 ± 0.6

± 0.08

DLD-1

WS1

± ± ± ± ± ±

9±1 6±1 9±1 >10 >10 >10

1.0 1.1 1.8 4.2 3.3 3.3

0.1 0.1 0.2 0.3 0.4 0.7

to 1.8 μM) cells in comparison with A549 (IC50: 9 to 10.5 μM) cells and WS1 (IC50: 6 to 9 μM) cells, suggesting some selectivity toward colon cancer cells. Interestingly, the presence of a hydroxy group (compounds 2, 7, and 8; with IC50 > 10 μM for all cell lines) reduced the cytotoxicity of the dirchromones. The presence of two methoxy groups in positions R1 and R2 (5) or R2 and R3 (6) also decreased the cytotoxicity when compared to compounds 1, 3, and 4. Furthermore, sulfur-containing compounds from the Alliaceae and Brassicales have well-known antimicrobial properties.36−39 Therefore, the antibacterial activity of compounds 1− 8 was also evaluated against the Gram-negative Escherichia coli and the Gram-positive Staphylococcus aureus using a modified microdilution method.40 Gentamycin was used as a positive control. The results presented in Table 4, expressed as the Table 4. Antibacterial Activity of Compounds 1−8 against S. aureus compound

MIC90 (μg/mL)

1 2 3 4 5 6 7 8 gentamycin

3.7 7.8 4.1 8.3 18.4 18.4 14.0 14.0 0.04

dilution at which at least 90% of bacterial proliferation was inhibited (MIC90), showed that all compounds were slightly active against S. aureus with MIC90 values ranging from 3.8 to 18.7 μg/mL. Compound 1 was the most active. The presence of a methoxy group in position R2 seems to be detrimental to the antibacterial activity, since compound 3 was more active than 4 despite sharing the same substituent. Furthermore, compounds 1−3 were all more active than 4−8, for which all possess a methoxy group at position C-7. In contrast, none of the compounds showed activity against E. coli or Candida albicans for the tested concentration range (data not shown).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an Autopol IV, model A21200 APIV/1W, digital polarimeter (Rudolph Research Analytical). UV spectra were measured on an Agilent 8453 UV−visible spectrophotometer from 190 to 400 nm using a 1 cm cell. FTIR spectra were measured on a PerkinElmer Spectrum One instrument. Samples were deposited in solution on NaCl disks and dried as films before measurement. NMR spectra were recorded at 292 K on a Bruker Avance 400 spectrometer (5 mm QNP with Z-gradient probe) operating at 400.13 MHz for 1H NMR or 100.61 MHz for 13C NMR. Each compound was D



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6-Hydroxydirchromone (2): yellow, amorphous powder; [α]20D 0 (c 0.02, CH2Cl2); UV (IPA) λmax (log ε) 203 (4.22), 225 (3.97), 266 (3.95), 307 (3.84) nm; IR (film) νmax 3379, 3182, 3060, 2961, 2928, 2857, 1633, 1613, 1470, 1404, 1359, 1325, 1250, 1193, 1163, 1122, 1025, 977, 959, 823, 773 cm−1; 1H NMR (DMSO-d6, 400 MHz) data, see Table 2; 13C NMR (DMSO-d6, 100 MHz) data, see Table 1; ESITOFMS m/z 251.0373 (calcd for C12H11O4S, 251.0373). 6-Methoxydirchromone (3): beige, amorphous powder; [α]20D 0 (c 0.02, CH2Cl2); UV (IPA) λmax (log ε) 205 (4.61), 228 (sh) (4.23), 272 (4.08), 301 (3.88) nm; IR (film) νmax 3395, 3062, 3005, 2960, 2926, 2852, 1633, 1614, 1515, 1485, 1460, 1438, 1381, 1363, 1313, 1291, 1282, 1243, 1209, 1119, 1156, 1028, 973, 860, 830, 733 cm−1; 1H NMR (CDCl3, 400 MHz) data, see Table 2; 13C NMR (CDCl3, 100 MHz) data, see Table 1; ESITOFMS m/z 265.0528 (calcd for C13H13O4S, 265.0529). 7-Methoxydirchromone (4): brownish, amorphous powder; [α]20D 0 (c 0.03, CH2Cl2); UV (IPA) λmax (log ε) 208 (4.57), 238 (4.30), 310 (4.24) nm; IR (film) νmax 3390, 3059, 3003, 2954, 2926, 2854, 1638, 1613, 1517, 1461, 1440, 1390, 1361, 1327, 1283, 1238, 1204, 1163, 1118, 1089, 1025, 963, 922, 836, 733 cm−1; 1H NMR (DMSO-d6, 400 MHz) data, see Table 2; 13C NMR (DMSO-d6, 100 MHz) data, see Table 1; ESITOFMS m/z 265.0516 (calcd for C13H13O4S, 265.0529). 6,7-Dimethoxydirchromone (5): beige, amorphous powder; [α]20D 0 (c 0.1, CH2Cl2); UV (IPA) λmax (log ε) 210 (4.53), 241 (4.27), 260 (4.07), 310 (4.09) nm; IR (film) νmax 3412, 3056, 3008, 2955, 2928, 2850, 1641, 1613, 1509, 1476, 1459, 1380, 1327, 1272, 1219, 1201, 1166, 1122, 1080, 1029, 1007, 960, 861, 826, 731 cm−1; 1H NMR (CDCl3, 400 MHz) data, see Table 2; 13C NMR (CDCl3, 100 MHz) data, see Table 1; ESITOFMS m/z 295.0635 (calcd for C14H15O5S, 295.0635). 7,8-Dimethoxydirchromone (6): brownish, amorphous powder; [α]20D 0 (c 0.03, CH2Cl2); UV (IPA) λmax (log ε) 209 (4.69), 256 (4.37), 310 (4.29) nm; IR (film) νmax 3417, 3053, 3002, 2950, 2926, 2852, 1634, 1603, 1584, 1562, 1513, 1464, 1455, 1429, 1391, 1333, 1287, 1234, 1212, 1177, 1128, 1099, 1059, 1031, 979, 808, 778, 719 cm−1; 1H NMR (CDCl3, 400 MHz) data, see Table 2; 13C NMR (CDCl3, 100 MHz) data, see Table 1; ESITOFMS m/z 295.0635 (calcd for C14H15O5S, 295.0635). 6-Hydroxy-7-methoxydirchromone (7): orange, amorphous powder; [α]20D 0 (c 0.01, CH2Cl2); UV (IPA) λmax (log ε) 205 (4.39), 239 (4.07), 268 (3.91), 308 (3.74) nm; IR (film) νmax 3395, 2956, 2922, 2852, 1734, 1615, 1506, 1457, 1382, 1306, 1279, 1216, 1159, 1134, 1074, 973, 865, 833, 789, 755 cm−1; 1H NMR (DMSO-d6, 400 MHz) data, see Table 2; 13C NMR (DMSO-d6, 100 MHz) data, see Table 1; ESITOFMS m/z 281.0488 (calcd for C13H13O5S, 281.0478). 8-Hydroxy-7-methoxydirchromone (8): yellow, amorphous powder; [α]20D 0 (c 0.01, CH2Cl2); UV (IPA) λmax (log ε) 207 (4.44), 274 (4.07), 297 (sh) (3.91) nm; IR νmax (film) 3380, 2955, 2925, 2854, 1736, 1634, 1611, 1583, 1517, 1462, 1403, 1379, 1327, 1289, 1209, 1158, 1118, 1093, 1037, 1009, 964, 810, 767 cm−1; 1H NMR (DMSOd6, 400 MHz) data, see Table 2; 13C NMR (DMSO-d6, 100 MHz) data, see Table 1; ESITOFMS m/z 281.0473 (calcd for C13H13O5S, 281.0478). Cell Culture. The A549 human lung carcinoma (#CCL-185), DLD-1 human colorectal adenocarcinoma (#CCL-221), and WS1 skin fibroblast (#CRL-1502) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were grown in minimum essential medium containing Earle’s salt (Mediatech Cellgro, Herndon, VA, USA), supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA), 1× solution of vitamins, 1× sodium pyruvate, 1× nonessential amino acids, 100 IU of penicillin, and 100 μg/mL of streptomycin (Mediatech Cellgro). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Cytotoxicity Assay. Exponentially growing cells were plated at a density of 5 × 103 cells per well in 96-well microplates (BD Falcon) in culture medium (100 μL) and were allowed to adhere for 16 h before treatment. Then, cells were incubated for 48 h in the presence or absence of 100 μL of increasing concentrations of extract, fraction, or pure compounds dissolved in culture medium and DMSO. The final concentration of DMSO in the culture medium was maintained at

0.5% (v/v) to avoid toxicity. Cytotoxicity was assessed using Hoechst (bis-benzimide).35 It was expressed as the concentration of drug inhibiting cell growth by 50% (IC50). Antibacterial and Antifungal Assays. Activities were evaluated using a modified microdilution method.40 Exponentially growing bacteria were plated in 96-well round-bottom microplates (Costar, Corning Inc.) at a density of 5 × 103 Gram-negative Escherichia coli (ATCC 25922) or 3.5 × 104 Gram-positive Staphylococcus aureus (ATCC 25923) per well in 100 μL of nutrient broth (Difco) or 2 × 103 Candida albicans per well in 100 μL of Sabouraud dextrose (Difco). Increasing concentrations of compounds (solubilized in Biotech DMSO, then diluted in nutrient broth or Sabouraud dextrose) were then added (100 μL per well). The final concentration of DMSO in the culture medium was maintained at 0.1% (volume/volume) to avoid solvent toxicity. The plates were incubated for 24 h at 37 °C. Absorbance was read using a Varioskan Ascent plate reader (Thermo Electron) at 600 nm for bacteria and 540 nm for yeasts.

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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.5b00227. NMR, ESITOFMS, and IR spectra of compounds 1−8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +1-418-545-5011, ext. 5081. Fax: +1-418-545-5012. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the Chaire de Recherche sur les Agents Anticancéreux d’Origine Naturelle and the Canadian Institutes of Health Research for funding and the FRQNT and NSERC for scholarships. They also thank Sentiers de la ̂ Inc. for allowing the collection of plant specimens Presqu’ile from its private maple forest, Dr. S. Lantagne (Direction du Laboratoire d’Expertises et d’Analyses Alimentaires, Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec) for locating the first D. palustris test samples, and S. Lavoie, F. Simard, C. Grenon, C. Dussault, and K. Lalancette for support and advice.

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REFERENCES

(1) United States Department of Agriculture. Dirca palustris L. Eastern Leatherwood http://plants.usda.gov/java/profile?symbol= dipa9 (accessed Jun 26, 2012). (2) Gleason, H. A.; Cronquist, A. Manual of Vascular Plants of Northeastern United States and Adjacent Canada, 2nd ed.; The New York Botanical Garden: Bronx, NY, 1993. (3) Marie-Victorin. Flore Laurentienne; 3rd ed.; Gaëtan Morin éditeur: Montréal, QC, 2002; p 362. (4) Williams, C. Plant Species Biol. 2004, 19, 101−106. (5) Gray, A.; Fernald, M. L. Gray’s Manual of Botany; American Book: New York, NY, 1970; p 1044. (6) Erichsen-Brown, C. Medicinal and Other Uses of North American Plants - A Historical Survey with Special Reference to the Eastern Indian Tribes; Dover Publications, Inc.: New York, NY, 1979; p 179. (7) Core, E. L. Econ. Bot. 1967, 21, 199−214. (8) Moerman, D. E. Native American Ethnobotany; Timber Press, Inc.: Portland, OR, 1998; p 201. E



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