Sulfonic Acid-Containing Flavonoids from the ... - ACS Publications

Article Cite This: J. Nat. Prod. 2018, 81, 2026−2031

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Sulfonic Acid-Containing Flavonoids from the Roots of Phyllanthus acidus Thuc-Huy Duong,†,‡ Mehdi A. Beniddir,§ Van-Kieu Nguyen,‡ Thammarat Aree,‡ Jean-François Gallard,⊥ Dinh-Hung Mac,▽ Huu-Hung Nguyen,∥ Xuan-Hao Bui,† Joël Boustie,# Kim-Phi-Phung Nguyen,□ Warinthorn Chavasiri,*,‡ and Pierre Le Pogam*,§

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†

Department of Chemistry, Ho Chi Minh City University of Education, 280 An Duong Vuong Street, District 5, Ho Chi Minh City 748342, Vietnam ‡ Center of Excellence in Natural Products Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand § ́ Equipe “Pharmacognosie-Chimie des Substances Naturelles”, BioCIS, Université Paris-Sud, CNRS, Université Paris-Saclay, 5 Rue J.-B. Clément, 92290 Châtenay-Malabry, France ⊥ Institut de Chimie des Substances Naturelles, CNRS, ICSN UPR 2301, Université Paris-Saclay, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France ▽ Department of Organic Chemistry, University of Science, Ha Noi National University, 19 Le Thanh Tong Street, District Hoan Kiem, Ha Noi City 748355, Vietnam ∥ Faculty of Biotechnology and Environment, Nguyen Tat Thanh University, 300A Nguyen Tat Thanh, District 4, Ho Chi Minh City 748355, Vietnam # Université Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes)−UMR 6226, F-35000 Rennes, France □ Department of Organic Chemistry, University of Science, National University−Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City 748355, Vietnam S Supporting Information *

ABSTRACT: Six new sulfonic acid-containing flavonoids, acidoflavanone (1), acidoauronol (2), 5-O-methylacidoauronol (3), acidoaurone (4), acidoisoflavone (5), and acidoflavonol (6), were isolated from the EtOH extract of the roots of Phyllanthus acidus. Their structures were unambiguously established by interpretation of their HRESIMS and 1D and 2D NMR data, singlecrystal X-ray diffraction analysis, and comparison to the literature data. These new structures represent the first examples of sulfonic acid-containing flavanones, auronols, aurones, and isoflavones.

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piles, and hepatopathies in the Ayurvedic system of medicine, while leaves are used to alleviate fevers, piles, smallpox, itching, and gum infection.4 These traditional uses have been partly corroborated by in vitro data that demonstrated the antibacterial,5 antiviral,6,7 analgesic, anti-inflammatory,8,9 neuroprotective,10,11 hepatoprotective,4,12 antifibrotic,13 cyto-

he genus Phyllanthus (Phyllanthaceae) includes more than 900 plant species found in tropical and subtropical regions.1 Many of these species are widely used in folk medicine.2 Phyllanthus acidus is a deciduous tree that grows 5 to 8 m tall with slender branches terminated by clustered leaves at their upper ends. The sour edible fruits are pale yellow, fleshy drupes.3 Various ethnobotanical claims are associated with different parts of P. acidus. The fruit is used in folk medicine to treat bronchitis, vomiting, urinary concretions, © 2018 American Chemical Society and American Society of Pharmacognosy

Received: April 23, 2018 Published: September 12, 2018 2026

DOI: 10.1021/acs.jnatprod.8b00322 J. Nat. Prod. 2018, 81, 2026−2031

Journal of Natural Products

Article

Table 1. 1H NMR Spectroscopic Data of Compounds 1−6 (δ in ppm, J in Hz) 1a,c

no. 2 3

3b,c

4b,c

5a,c

5.52, dd, (11.0, 3.5) 3.11, dd, (17.0, 11.5) 2.95, dd, (17.0, 3.5)

5 6 10 2′/6′ 3′/5′ 4-OMe 5-OMe 6-OMe 2-OH 5-OH 6-OH 7-OH 4′-OH

2a,c

6a,d

8.17, s

5.84, s

5.77, s 6.37, s

7.39, d (8.0) 6.75, d (8.0)

2.78, 3.02, 7.04, 6.45, 3.69,

d d d d s

(14.0) (14.0) (8.0) (8.0)

3.31, 3.10, 7.13, 6.68, 3.82, 3.62,

d d d d s s

(14.0) (14.0) (8.0) (8.0)

6.52, s 7.82, d (8.0) 6.85, d (8.0) 3.78, s

7.31, d (8.0) 6.77, d (8.0)

8.37, d (8.0) 6.90, d (8.0)

3.81, s

3.63, s

3.75, s 7.64, s

12.29, s 12.54, s 12.53, s

12.64, s

9.06, s

a

Measured in DMSO-d6. bMeasured in D2O. cRecorded at 500 MHz. dRecorded at 600 MHz.

toxic,14 and hypotensive15,16 properties of the crude extracts and/or isolated constituents. Recent phytochemical investigations of the nonpolar constituents of P. acidus afforded new cleistanthane diterpenes, i.e., phyllanes A and B17 and phyllaciduloids A−D.18 Likewise, former studies on P. acidus yielded numerous new norbisabolane sesquiterpene glycosides, some of which exhibited potent antihepatitis B activity.7,19 Although flavonoids are one of the main groups of specialized metabolites that are produced by Phyllanthus species, and unique sulfonic acid-containing flavones20,21 and flavonols22 have already been isolated from various plant species, the flavonoid constituents of P. acidus have not been investigated,23 paving the way for the current phytochemical investigation of its polar fractions. The current study reports the isolation and structure elucidation of six new sulfonic acid-containing flavonoids, acidoflavanone (1), acidoauronol (2), 5-O-methylacidoauronol (3), acidoaurone (4), acidoisoflavone (5), and acidoflavonol (6), from the EtOH extract of the roots of P. acidus. These new metabolites were isolated along with the known compounds phyllanthacidoid methyl ester and phyllanthacidoid acid.7 The major compound, 1, was tested for the in vitro inhibition of cell proliferation of the human hepatoma HepG2 and human breast MCF-7 cancer cell lines.

NMR spectrum, in conjunction with the HSQC spectrum, revealed the presence of a carbonyl (δC 197.9), four aromatic methines (δC 128.1 and 115.3, each 2 carbons), an oxygenated methine (δC 78.0), an sp3 methylene (δC 42.0), a methoxy (δC 60.2), six tertiary carbons (including five oxygenated carbons at δC 157.6, 157.5, 156.5, 155.6, and 128.1), and two aromatic quaternary carbons (δC 129.5 and 111.8) (Table 2). Table 2. 13C NMR Spectroscopic Data of Compounds 1−6 (δ in ppm)

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RESULTS AND DISCUSSION Compounds 1−6 were isolated from the ethanol extract of P. acidus roots by repetitive chromatographic separations, including column chromatography and preparative TLC. Compound 1 was isolated as a light yellow, amorphous solid. Its molecular formula was determined to be C16H14O9S based on the deprotonated molecular ion at m/z 381.0295 (calcd for C16H13O9S, 381.0286), indicating 12 indices of hydrogen deficiency. The 1H NMR spectrum of 1 revealed three phenolic groups (δH 13.07, 12.29, and 9.46), an AA′BB′ spin system comprising two pairs of two-proton doublets at δH 7.39 (2H, d, J = 8.0 Hz) and 6.75 (2H, d, J = 8.0 Hz), an ABX spin system comprising a diastereotopic methylene (δH 3.11, 1H, dd, J = 17.0, 11.5 Hz and δH 2.95, 1H, dd, J = 17.0, 3.5 Hz), and an oxygenated methine (δH 5.52, 1H, dd, J = 11.0, 3.5 Hz), along with a methoxy group (δH 3.63, s) (Table 1). The 13C

a

no.

1a,c

2a,c

3b,c

4b,c

5a,c

6a,d

2 3 4 5 6 7 8 9 10 1′ 2′/6′ 3′/5′ 4′ 4-OMe 5-OMe 6-OMe

78.0 42.0 197.9 157.6 128.1 155.6 101.8 157.5 111.8 129.5 128.1 115.3 156.5

106.3 192.5 158.7 92.4 164.2 101.9 169.4 108.3 40.5 124.0 131.5 114.4 155.7 55.6

107.6 195.0 152.0 131.7 161.0 ND 169.8 107.3 40.5 124.6 131.7 114.9 154.5 61.0 60.7

146.0 179.0 160.8 96.4 ND 101.2 163.4 103.8 110.3 122.8 132.4 114.2 157.5 55.0

150.3 124.8 174.1 160.9 96.2 159.0 111.0 156.1 108.7 122.2 130.2 115.0 157.1

147.3 135.5 175.9 152.2 130.2 154.6 102.9 148.2 109.6 121.9 130.2 115.3 159.3

56.0

60.2

59.6 b

c

Measured in DMSO-d6. Measured in D2O. Recorded at 125 MHz. Recorded at 150 MHz.

d

The AA′BB′ spin system defined the B ring as a paradisubstituted aromatic ring. The presence of the hydroxy substituent was confirmed by the long-range heteronuclear correlations between the phenolic proton at δH 7.39 and C-3′/ 5′ (δC 115.3) and C-4′ (δC 156.5). The diastereotopic methylene at δH 3.11/2.95 [(dd, J = 17.0, 11.5 Hz)/(dd, J = 17.0, 3.5 Hz)] was assigned to C-3 based on its HMBC correlations with C-2 (δC 78.0), C-1′ (δC 129.5), and C-4 (δC 197.9). The hydrogen-bonded hydroxy group at δH 13.07 was located at C-5 based on its HMBC correlations to C-10 (δC 2027



Article

C-6 (δC 128.1) is consistent with it being flanked by two oxy substituents, leading to assignment of this hydroxy group at C7.24−26 The chemical shift of the methoxy carbon at δC 60.2 is also consistent with such a substitution pattern.27 Accordingly, the sulfonic acid group must be linked to C-8. This assignment was supported by a comparison of the 13C NMR spectroscopic data of 1 with those of its analogue, 6-methoxynaringenin, which lacks the sulfonic acid moiety.28 The 13C NMR chemical shift of C-8 in 1 (δC 101.8) appeared approximately 10 ppm downfield from that of the equivalent carbon in 6methoxynaringenin, and this difference is within the range typical of the downfield chemical shift associated with the introduction of a sulfonic acid group in similar flavonoid scaffolds.20−22 Acidoflavanone (1) was optically inactive and thus racemic. Single-crystal X-ray analysis with Mo Kα (λ = 0.710 73 Å) corroborated the 2D structure deduced from the spectroscopic data and confirmed the presence of the two enantiomers (Figure 3). Compound 2 was obtained as a pale yellow, amorphous solid with a molecular formula of C16H14O9S, established by the HRESIMS ion at m/z 381.0303 [M − H]− (calcd for C16H13O9S, 381.0286), indicating 2 is an isomer of 1. However, the NMR data revealed some important structural differences between these two compounds, including the presence of a hemiketal moiety based on the carbon resonance at δC 106.3. An AA′BB′ benzenoid spin system was suggested by the signals at δH 7.04 (2H, J = 8.0 Hz, H-2′/H-6′) and 6.45 (2H, d, J = 8.0 Hz, H-3′/H-5′), and a hydroxy group was assigned based on the downfield shift of the HO-carrying carbon (δC 155.7), while a −CH2R group was revealed by the HMBC correlations between the diastereotopic methylene protons at δH 3.02/2.78 (H2-10) and C-1′ (δC 124.0) and C2′/C-6′ (131.5). Additionally, these diastereotopic benzylic methylene proton signals showed HMBC cross-peaks to C-2 (δC 106.3) and C-3 (δC 192.5) (Figure 2), revealing the presence of a hemiketal function at C-2. These structural features indicated that 2 was a rare auronol-type compound. As for the rest of the structure, the A ring was deduced to be trisubstituted, and it contained sulfonic acid, hydroxy, and methoxy substituents along with an aromatic methine (δH 5.84, δC 92.4). Its 13C chemical shift is consistent with the presence of ortho oxygen functionalities.29,30 Considering the polyketide origin of the A ring allowed the assignment of the aromatic methine at C-5 based on the general oxygenation pattern of all

111.8), C-6 (δC 128.1), and C-5 (δC 157.6). Likewise, the methoxy group was located at C-6 based on the HMBC correlation between the protons at δH 3.63 and C-6 (δC 128.1) (Figure 2).

Figure 1. Structures of compounds 1−6.

Figure 2. Key HMBC correlations of compounds 1−6.

Based upon the molecular formula, the hydrogen deficiency requirements, and structural constraints, the structure of 1 was determined as a flavanone with the last two substituents assigned as a third hydroxy group and a sulfonic acid moiety that can be located at either C-7 or C-8. The chemical shift of

Figure 3. ORTEP diagram of the sodium salt of the (2S)-enantiomer of compound 1 (A); the unit cell of 1 showing a 1:1 racemic mixture (B). 2028



Article

A-ring dioxygenated aurones published to date.31 The oxygenated aromatic carbons were observed at δC 158.7, 164.2, and 169.4, and the HMBC cross-peak of the protons at δH 3.69 to the carbon at δC 158.7 placed the methoxy group at the carbon resonating at δ C 158.7. The 13 C NMR spectroscopic data of A-ring-unsubstituted aurones (e.g., dalmaisione D32 and 4′-chloro-2-hydroxyaurone/4′-chloroaurone33) showed that the chemical shift of C-6 was 7 to 9 ppm downfield compared to that of C-4 in the more commonly reported flavanone core.34 Accordingly, oxygen-bearing aromatic carbons at C-6 resonate in the 164−172 ppm range, whereas those at C-4 are upfield shifted to ca. 156−160 ppm in various A-ring mono- or meta-disubstituted aurones and auronols30,35−40 (the chemical shielding effect from the substituent is considered mild to negligible in such orientation for oxygen substituents41). These considerations led to the unambiguous assignment of the methoxy and hydroxy substituents at C-4 and C-6, respectively. Consequently, the sulfonic acid group was located at C-7. To validate this structure, the 13C NMR spectroscopic data of 2 were compared to those of its analogues that lacked sulfonic acid moieties. Although the NMR data of carpusin, an analogue of 2 lacking the sulfonic acid moiety, were reported in 1983,29 the NMR assignments of this structure are suspect42 and therefore might not represent a relevant model for comparison. Accordingly, the 13C NMR spectroscopic data of 2 were instead compared to those of the closely related compound cudrauronol, revealing excellent agreement except for the chemical shift values of C-7.30 The 13C NMR chemical shift of C-7 in 2 appeared 10 ppm downfield relative to that of the corresponding carbon of cudrauronol (respectively δC of 101.9 and 91.9) due to the introduction of the sulfonic acid moiety. The lack of optical rotation confirmed the racemic nature of 2, which could be related to the epimerization of the hemiketal function.31 Thus, the structure of acidoauronol (2) was established as indicated in Figure 1. Compound 3, isolated as a pale yellow, amorphous, and optically inactive solid, gave a molecular formula of C17H16O10S based on its negative-ion HRESIMS data, which showed an [M − H]− peak at m/z 411.0374 (calcd for C 17 H 15 O 10 S, 411.0391). Both its 1 H and 13 C NMR spectroscopic data were similar to those of 2, with the only major difference being the presence of an aromatic methoxy group in lieu of the aromatic methine at H-5. This assignment was supported by the upfield chemical shifts of the carbons at the ortho-positions (C-4 and C-6) compared to those of 2. Lastly, the chemical shift values of the methoxy groups are consistent with the presence of an o-disubstituted methoxy group. Indeed, whereas chemical shift values of methoxy groups are typically between 55.0 and 56.6 ppm, these carbons are reported to be deshielded when another methoxy group is present in an ortho-position, and the signals generally shift to the 59.5−63.6 ppm range.34 Therefore, the chemical shift values of C-4 and C-5, 61.0, and 60.7 ppm, respectively, are consistent with the proposed substitution pattern. Thus, the structure of 3 was determined to be 5-O-methylacidoauronol. Compound 4 was assigned a molecular formula of C16H12O8S (HRESIMS, m/z 363.0178 [M − H]−, calcd for C16H11O8S, 363.0180), which differs from 1 and 2 by 18 mass units (loss of H2O). Examination of the 1H NMR spectrum of 4 revealed signal patterns similar to those of 2, except the diastereotopic methylene protons of 2 were replaced with a signal for an olefinic proton at δH 6.52 (1H, s). These

spectroscopic features suggested that 4 differed from 2 by the presence of a Δ2(10) double bond. Accordingly, the vinylic proton at δH 6.52 could be located at C-10 based on its HMBC cross-peaks with C-3 (δC 179.0), C-2 (δC 146.0), and C-2′/6′ (δC 132.4). The presence of a Δ2(10) double bond was confirmed by the downfield shifts of both C-2 and C-10 (146.0 and 110.3 ppm in 4 vs 40.5 and 106.3 ppm in 2, respectively). Collectively, these spectroscopic features defined the aurone scaffold of 4. The Z-geometry of the olefinic bond was determined based on the chemical shift of the exocyclic olefinic methine (δC 110.3) that lies within the range observed for Zaurones (104−113 ppm), whereas in E-aurones these carbons are downfield shifted to approximately 120 ppm.27,36 The small amount of 4 prevented the acquisition of a 13C NMR spectrum of satisfactory quality, but inverse-detection heteronuclear NMR spectra provided all of the carbon chemical shift values. The NMR chemical shifts associated with the aromatic methine (δH 5.77, δC 96.4) confirmed the meta relationship between the methoxy and phenolic hydroxy groups,32,33,41 and the methoxy group was located at C-4 based on the HMBC cross-peak between the protons at δH 3.78 and C-4 (δC 159.0), as previously discussed in the structure elucidation of 2, thereby also establishing that the phenolic hydroxy group was located at the C-6 position. The sulfonic acid group could only be located at C-7. The structural assignment of 4 based on its NMR data is fully supported by comparison to the data of its analogue, (Z)-4-methoxy-6,4′-dihydroxyaurone,39 which lacks the sulfonic acid group, resulting in the structure of acidoaurone (4) as displayed in Figure 1. This structure determination is supported by the joint isolation of the corresponding aurone and auronol metabolites in the same vegetal material reported from other sources.33 Compound 5 was obtained as a yellowish, amorphous solid. Its molecular formula, C16H12O8S, was established from the prominent deprotonated molecular ion peak at m/z 363.0196 (calcd for C16H11O8S, 363.0180) in its HRESIMS data. The 1 H NMR spectrum was similar to that of 4, and the main difference was the downfield chemical shift of the olefinic proton to δH 8.17 (vs δH 6.52 in 4), which was reminiscent of an H-2 isoflavone signal. This assignment was further strengthened by the upfield chemical shift of the carbonyl carbon (δC 174.1 in 5 vs 192.5 to 197.9 for compounds 1−3) and by the chemical shift values of C-2 (δC 150.3) and C-3 (δC 124.8) that also appear at shifts typical for isoflavones.36 The long-range heteronuclear correlations between the olefinic proton at δH 8.17 and C-1′ (δC 122.2), C-3 (δC 124.8), C-9 (δC 156.1), and C-4 (δC 174.1) confirmed the isoflavone scaffold of 5. The key NMR shifts of the A ring revealed close structural similarities between this compound and both 2 and 4. In particular, the chemical shifts of the aromatic methine (δH 6.37, δC 96.2) indicated it was located between two oxygen substituents, placing this group at C-627 and leaving the methoxy and phenolic hydroxy groups to be assigned at either C-5 or C-7. The hydroxy group at C-5 has a notable influence on the C-ring carbon resonances through intramolecular hydrogen bond interactions. In particular, C-4 appears between 179 and 182 ppm in 5-hydroxyisoflavones, whereas this carbon resonates at approximately 174−175.5 ppm in 5-methoxyisoflavones.27 Accordingly, the chemical shift of C-4 in 5 (δC 174.1) unambiguously placed the methoxy group at the C-5 position, leaving C-8 as the only possible location for the sulfonic acid moiety. This substitution pattern was confirmed by the HMBC cross-peaks from OH-7 to C-6 (δC 96.2), C-7 2029



Article

separation of the closely related sulfonic acid-containing flavonoids could be achieved. TLC analyses were carried out on precoated silica gel 60 F254 or silica gel 60 RP-18 F254S plates (Merck), and spots were visualized by spraying with 10% H2SO4 solution followed by heating. Plant Material. The roots of Phyllanthus acidus (Phyllanthaceae) were collected from Hong Son Village, Ham Thuan Bac, in Binh Thuan Province (Vietnam) in July 2014. The botanical identification was confirmed by Dr. Pham Van Ngot, Department of Botany, Faculty of Biology, Ho Chi Minh (Vietnam) University of Pedagogy. A voucher specimen (No. UP-B01) was deposited in the herbarium of the Department of Organic Chemistry, Faculty of Chemistry, Ho Chi Minh University of Pedagogy (Vietnam). Extraction and Isolation. The dried roots of P. acidus (35 kg) were crushed and extracted with EtOH (3 × 20 L) at 70 °C for 8 h. The filtered solution was evaporated to dryness under reduced pressure to obtain a crude extract (2.0 kg). This crude extract was separated by normal-phase silica gel column chromatography eluted with a solvent system of n-hexane/EtOAc (stepwise gradient, 10:0 to 0:10) to afford fractions H (41.1 g), HA (10.2 g), EA1 (114.2 g), and EA2 (170.6 g). Fraction EA2 was dissolved in MeOH/H2O (9:1). The filtered solution was concentrated in vacuo to obtain fraction S (15.2 g), which was subsequently subjected to silica gel column chromatography using an isocratic mobile phase consisting of a CHCl 3 /EtOAc/acetone/MeOH/HOAc/H 2 O solvent system (53:7:6.5:0.4:0.2:0.05) to obtain fractions S1−S4. Further purification of fraction S1 (951.0 mg) using the same isocratic elution solvent with open-air column chromatography led to the isolation of 1 (2.6 mg) and 6 (1.1 mg). Fraction S2 (325.0 mg) was submitted to preparative TLC using the same solvent system. Six consecutive runs were required to sufficiently separate the compounds. Four bands were desorbed from the plates, yielding 2 (1.4 mg), 3 (1.2 mg), 4 (1.2 mg), and 5 (1.3 mg). The TLC Rf values obtained with the solvent system given above were 0.41, 0.28, 0.30, 0.70, 0.29, and 0.43 for compounds 1, 2, 3, 4, 5, and 6, respectively. (±)-Acidoflavanone (1): pale yellow, amorphous solid; [α]20D 0 (c 0.1, H2O); UV (H2O), λmax (log ε) 291 (3.80), 241 (4.27) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 381.0295 [M − H]− (calcd for C16H13O9S, 381.0286). (±)-Acidoauronol (2): pale yellow, amorphous solid; [α]20D 0 (c 0.1, H2O); UV (H2O), λmax (log ε) 327 (2.78), 285 (3.02) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 381.0303 [M − H]− (calcd for C16H13O9S, 381.0286). (±)-5-O-methylacidoauronol (3): pale yellow, amorphous solid; [α]20D 0 (c 0.1, H2O); UV (H2O), λmax (log ε) 336 (3.19), 282 (3.50) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 411.0374 [M − H]− (calcd for C17H15O10S, 411.0391). Acidoaurone (4): pale yellow, amorphous solid; UV (H2O), λmax (log ε) 384 (3.39) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 363.0178 [M − H]− (calcd for C16H13O9S, 363.01801). Acidoisoflavone (5): pale yellow, amorphous solid; UV (H2O), λmax (log ε) 250 (2.96) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 363.01962 [M − H]− (calcd for C16H13O9S, 363.01801). Acidoflavonol (6): pale yellow, amorphous solid; UV (H2O), λmax (log ε) 368 (4.20), 267 (4.14) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 397.02340 [M + H]+ (calcd for C16H13O10S, 397.02290). Cytotoxicity Assay. The cytotoxicity of compound 1 was evaluated against HepG2 (liver hepatocellular carcinoma) and MCF-7 (breast cancer) cell lines using the MTT assay with doxorubicin as the positive control. The experimental details were similar to those previously reported.17

(δC 159.0), and C-8 (δC 111.0); this latter chemical shift was consistent with that of a sulfonic acid-substituted carbon.43 The overall substitution pattern of the A ring was confirmed by the long-range heteronuclear correlations from the aromatic methine H-6 (δH 6.37) to C-5, C-7, C-8, and C-10 (δC 108.7). These spectroscopic data defined the structure of acidoisoflavone (5), as displayed in Figure 1. Compound 6 was isolated as a yellow, amorphous solid. Its molecular formula was determined to be C16H12O10S (13 indices of hydrogen deficiency) from its HRESIMS ion at m/z 397.0234 [M + H]+ (calcd for C16H13O10S, 397.0229), indicating that it differs from 1 by the absence of two hydrogen atoms and the addition of an oxygen atom. Initially, the 1H NMR data revealed the disappearance of both the H-2 methine and H-3 diastereotopic methylene signals, which, along with the requirements of the molecular formula, suggested that the flavanone core of 1 might have been replaced by a flavonol scaffold in 6. Likewise, the 13C NMR data of 6 were highly reminiscent of those of 1, mainly differing in the downfield shifts of C-2 and C-3 (δC 147.3 and 135.5, respectively) to shifts typical of flavonols.44 Thus, the structure of acidoflavonol (6) was elucidated as shown in Figure 1. The 13 C NMR data of 6-methoxykaempferol, an analogue of 6 that lacks the sulfonic acid moiety, were in excellent agreement with the chemical shifts reported herein, except for the chemical shift of C-8 (δC 102.9), which was reported to be 9 ppm upfield in 6-methoxykaempferol (approximately δC 94.0),45,46 revealing the expected deshielding from the sulfonic acid substituent. Aurones and auronols are uncommon classes of flavonoids. We herein report the first aurones and auronols isolated from a plant in the family Phyllanthaceae, and these compounds have not been reported in plants of the closely related family Euphorbiaceae.31 Likewise, neither sulfonic acid-substituted aurones nor auronols have previously been reported. These two points might be related because all representatives of the small group of naturally occurring sulfonic acid-containing flavonoids have been obtained from plants of the genus Phyllanthus. The current article is also the first report of sulfonic acid-substituted flavanone and isoflavone derivatives. Owing to the shortage of material, only compound 1 could be assayed for its in vitro cytotoxicity against HepG2 and MCF-7 cancer cell lines using the MTT method; 17 doxorubicin was used as the positive control. This compound was found to be inactive against these cell lines.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded using a Jasco DIP-370 digital polarimeter at 293 K at the sodium line; [α]D values are given in 10−1 deg·cm−2. 1D and 2D NMR spectra were acquired using Bruker AM-500 MHz and Bruker AM-600 spectrometers. Chemical shifts in ppm are referenced to the residual solvent signal (D2O: δH = 4.79; DMSO-d6: δH = 2.50, δC = 39.5). HRESIMS data were recorded using a Bruker microTOF Q-II with the measurements being performed in negative-ion mode. The crystallographic analyses of suitable single crystals were conducted using a Bruker X8 APEXII Kappa CCD area-detector diffractometer with Mo Kα radiation (λ = 0.710 73 Å) at 100 K. UV spectra were obtained with a PerkinElmer Lambda 25 UV−vis spectrometer. Open-column chromatography separations were performed on silica gel (40−63 μm, Himedia) in normal phase and at atmospheric pressure. The preparative TLC separations were run in presaturated development chambers, and the plates were allowed to fully air-dry prior to being rerun in the same solvent system until a good

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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.8b00322. 2030



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Article

Copies of 1D (1H and 13C) and 2D NMR spectra for compounds 1−6; experimental details for X-ray diffraction analyses of 1 (PDF)

(18) Zheng, X.-H.; Yang, J.; Lv, J.-J.; Zhu, H.-T.; Wang, D.; Xu, M.; Yang, C.-R.; Zhang, Y.-J. Fitoterapia 2018, 125, 89−93. (19) Vongvanich, N.; Kittakoop, P.; Kramyu, J.; Tanticharoen, M.; Thebtaranonth, Y. J. Org. Chem. 2000, 65, 5420−5423. (20) Than, N. N.; Fotso, S.; Poeggeler, B.; Hardeland, R.; Laatsch, H. Z. Z. Naturforsch., B: J. Chem. Sci. 2006, 61, 57−60. (21) Thanh, N. V.; Huong, P. T. T.; Nam, N. H.; Cuong, N. X.; Thao, N. P.; Dejaegher, B.; Gordien, A.; Heyden, Y. V.; QuetinLeclercq, J.; Minh, C. V. Phytochem. Lett. 2014, 7, 182−185. (22) Huang, Y.-L.; Chen, C.-C.; Hsu, F.-L.; Chen, C.-F. J. Nat. Prod. 1998, 61, 1194−1197. (23) Nahar, L.; Sarker, S. D.; Delazar, A. In Phyllanthus Species: Scientific Evaluation and Medicinal Applications; Kuttan, R., Harikumar, K. B., Eds.; CRC Press: Boca Raton, 2011; pp 119−138. (24) Wenkert, E.; Gottlieb, H. E. Phytochemistry 1977, 16, 1811− 1816. (25) Agrawal, P. K.; Rastogi, R. P. Heterocycles 1981, 16, 2181− 2236. (26) Collado, I. G.; Macias, F. A.; Massanet, G. M.; Luis, F. R. J. Nat. Prod. 1985, 48, 819−822. (27) Agrawal, P. K. Carbon-13 NMR of Flavonoids; Elsevier: Amsterdam, 1989. (28) Lee, S. R.; Kim, M.-S.; Kim, S.; Hwang, K. W.; Park, S.-Y. Chem. Biodivers. 2017, 14, No. e1700231. (29) Mathew, J.; Rao, A. S. Phytochemistry 1983, 22, 794−795. (30) Lin, C.-F.; Chen, Y.-J.; Huang, Y.-L.; Chiou, W.-F.; Chiu, J.-H.; Chen, C.-C. J. Asian Nat. Prod. Res. 2012, 14, 704−707. (31) Boucherle, B.; Peuchmaur, M.; Boumendjel, A.; Haudecoeur, R. Phytochemistry 2017, 142, 92−111. (32) Kobayashi, S.; Miyase, T.; Noguchi, H. J. Nat. Prod. 2002, 65, 319−328. (33) Atta-Ur-Rahman; Choudhary, M. I.; Hayat, S.; Kahn, A. M.; Ahmed, A. Chem. Pharm. Bull. 2001, 49, 105−107. (34) Iinuma, M.; Matsuura, S.; Kusuda, K. Chem. Pharm. Bull. 1980, 28, 708−716. (35) Seabra, R. M.; Silva, A. M.; Andrade, P. B.; Moreira, M. M. Phytochemistry 1998, 48, 1429−1432. (36) Pelter, A.; Ward, R. S.; Gray, T. I. J. Chem. Soc., Perkin Trans. 1 1976, 23, 2475−2483. (37) Li, X.-C.; El Sohly, H. N.; Nimrod, A. C.; Clark, A. M. J. Nat. Prod. 1999, 62, 767−769. (38) Sayed, H. M.; Mohamed, M. H.; Farag, S. F.; Mohamed, G. A.; Ebel, R.; Omobuwajo, O. R. M.; Proksch, P. Bull. Pharm. Sci.-Assiut Univ. 2006, 29, 9−32. (39) Huang, H.-Q.; Li, H.-L.; Tang, J.; Lv, Y.-F.; Zhang, W.-D. Biochem. Syst. Ecol. 2008, 36, 590−592. (40) Xiao, C.-J.; Zhang, Y.; Qiu, L.; Dong, X.; Jiang, B. Planta Med. 2014, 80, 1727−1731. (41) Stothers, J. B. Carbon-13 NMR Spectroscopy; Academic Press: New York, USA, 1972. (42) Lima, A. F.; Mileo, P. G. M.; Andrade-Neto, M.; Braz-Filho, R.; Silveira, E. R.; Pessoa, O. D. L. Magn. Reson. Chem. 2009, 47, 165− 168. (43) Kulesh, N. I.; Vasilevskaya, N. A.; Veselova, M. V.; Denisenko, V. A.; Fedoreev, S. A. Chem. Nat. Compd. 2008, 44, 712−714. (44) Ternai, B.; Markham, K. R. Tetrahedron 1976, 32, 565−569. (45) Fuchino, H.; Satoh, T.; Tanaka, N. Chem. Pharm. Bull. 1996, 44, 1748−1753. (46) De Andrade, F. D.; Dos Santos, L. C.; Dokkedal, A. L.; Vilegas, W. Phytochemistry 1999, 51, 411−415.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +33 1 46 83 55 97. *E-mail: [email protected]. ORCID

Mehdi A. Beniddir: 0000-0003-2153-4290 Thammarat Aree: 0000-0002-7298-7401 Kim-Phi-Phung Nguyen: 0000-0003-0868-2213 Pierre Le Pogam: 0000-0002-3351-4708 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Rachadapisek Sompote Fund for Postdoctoral Fellowship, Chulalongkorn University, for T.H.D. The authors would like to thank Dr. P. V. Ngot, Department of Botany, Faculty of Biology, Ho Chi Minh University of Education, for the botanical authentication of the studied plant. The authors are also indebted to K. Leblanc for performing the HRMS measurements of 6.

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REFERENCES

(1) Unander, D. W.; Webster, G. L.; Blumberg, B. S. J. Ethnopharmacol. 1995, 45, 1−18. (2) Unander, D. W.; Webster, G. L.; Blumberg, B. S. J. Ethnopharmacol. 1991, 34, 97−133. (3) Tharakan, S. T. T. In Phyllanthus Species: Scientific Evaluation and Medicinal Applications; Kuttan, R., Harikumar, K. B., Eds.; CRC Press: Boca Raton, 2011; pp 1−22. (4) Jain, N. K.; Lodhi, S.; Jain, A.; Nahata, A.; Singhai, A. K. J. Chin. Integr. Med. 2011, 9, 49−56. (5) Meléndez, P. A.; Capriles, V. A. Phytomedicine 2006, 13, 272− 276. (6) Direkbusarakom, S.; Herunsalee, A.; Yoshimizu, M.; Ezura, Y. Gyobyo Kenkyu 1996, 31, 209−213. (7) Lv, J.-J.; Yu, S.; Wang, Y.-F.; Wang, D.; Zhu, H.-T.; Cheng, R.-R.; Yang, C.-R.; Xu, M.; Zhang, Y.-J. J. Org. Chem. 2014, 79, 5432−5447. (8) Chakraborty, R.; Biplab, D.; Devanna, N.; Sen, S. Asian Pac. J. Trop. Biomed. 2012, 2, S953−S961. (9) Hossen, M. J.; Jeon, S. H.; Kim, S. C.; Kim, J. H.; Jeong, D.; Sung, N. Y.; Yang, S.; Baek, K.-S.; Kim, J. H.; Yoon, D. H. J. Ethnopharmacol. 2015, 168, 217−228. (10) Ingkaninan, K.; Temkitthawon, P.; Chuenchom, K.; Yuyaem, T.; Thongnoi, W. J. Ethnopharmacol. 2003, 89, 261−264. (11) Uddin, M. S.; Al Mamun, A.; Hossain, M. S.; Ashaduzzaman, M.; Noor, M. A. A.; Hossain, M. S.; Uddin, M. J.; Sarker, J.; Asaduzzaman, M. Adv. Alzheimer's Dis. 2016, 5, 53−72. (12) Jain, N. K.; Singhai, A. K. Asian Pac. J. Trop. Med. 2011, 4, 470−474. (13) Sousa, M.; Ousingsawat, J.; Seitz, R.; Puntheeranurak, S.; Regalado, A.; Schmidt, A.; Grego, T.; Jansakul, C.; Amaral, M. D.; Schreiber, R. Mol. Pharmacol. 2007, 71, 366−376. (14) Mahidol, C.; Prawat, H.; Prachyawarakorn, V.; Ruchirawat, S. Phytochem. Rev. 2002, 1, 287−297. (15) Leeya, Y.; Mulvany, M. J.; Queiroz, E. F.; Marston, A.; Hostettmann, K.; Jansakul, C. Eur. J. Pharmacol. 2010, 649, 301−313. (16) Chongsa, W.; Kanokwiroon, K.; Jansakul, C. J. Ethnopharmacol. 2015, 176, 79−89. (17) Duong, T.-H.; Bui, X.-H.; Le Pogam, P.; Nguyen, H.-H.; Tran, T.-T.; Chavasiri, W.; Boustie, J. Tetrahedron 2017, 73, 5634−5638. 2031


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