Enantiomeric chromones from Harrisonia perforata

Phytochemistry Letters 10 (2014) 295–299

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Enantiomeric chromones from Harrisonia perforata Guo Liu a,1, Rong-Rong Zheng a,1, Zhi-Wen Liu a, Wen-Jing Wang a, Guo-Qiang Li a, Chun-Lin Fan a, Xiao-Qi Zhang a,b,**, Wen-Cai Ye a,*, Chun-Tao Che b a

Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China Department of Medicinal Chemistry and Pharmacognosy, and WHO Collaborating Center for Traditional Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA b

A R T I C L E I N F O

Article history: Received 10 August 2014 Received in revised form 13 October 2014 Accepted 14 October 2014 Available online 28 October 2014 Keywords: Harrisonia perforata Prenylated chromone Enantiomer

A B S T R A C T

Six new chromones (1–3 and 7–9), along with 10 known ones, were isolated from the ethanol extract of Harrisonia perforata. The structures of the new compounds were elucidated by extensive spectroscopic and single crystal X-ray diffraction analyses. Compounds 1–3 and 8–9 are all racemates, and the corresponding enantiomers [(+)-1/2/3/8/9 and ()-1/2/3/8/9] were obtained by chiral HPLC separation, respectively. The absolute configuration of (+)-1 was deduced by Mosher’s method. Crown Copyright ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

1. Introduction Harrisonia perforata is the only species of this genus in China, and abounds in Hainan Province of China. The roots of this plant have been used as Chinese folk medicine for the treatment of malaria and boils (Chen et al., 1997). Previous chemical investigations on this plant led to the isolation of limonoids (Tran et al., 1995; Khuong-Huu et al., 2000; Yan et al., 2011; Choodej et al., 2013), polyketides (Yin et al., 2009), and chromones (Choodej et al., 2013; Wang et al., 1983; Thadaniti and Archakunako, 1994; Tanaka et al., 1995; Tuntiwachwuttikul et al., 2006). However, the racemic nature of the prenylated chromones from this plant has not been reported before, although the optical rotation values of several chromones had been reported to be nearly zero (Tanaka et al., 1995). In the current research, six new chromones, ()-perforatin C (1), ()-20 -O-acetylperforatin C (2), ()-erythro-10 -hydroxyperforatin C (3), 5-hydroxy-7-methoxy-2methyl-8-[(1E)-3-oxo-1-butenyl]chromone (7), ()-horriperfin A (8), ()-horriperfin B (9), along with 10 known ones, perforamone C (4) (Tuntiwachwuttikul et al., 2006), heteropeucenin-7-methyl ether (5) (Wang et al., 1983), heteropeucenin-5-methoxy-7-methyl ether (6)

* Corresponding author at: Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China. Tel.: +86 20 85220936; fax: +86 20 85221559. ** Corresponding author. Tel.: +86 20 85220936; fax: +86 20 85221559. E-mail addresses: [email protected] (X.-Q. Zhang), [email protected] (W.-C. Ye). 1 These authors contributed equally.

(Wang et al., 1983), greveiglycol 40 -methyl ether (10) (Dean and Robinson, 1971), greveiglycol (11) (Dean and Robinson, 1971), Omethylalloptaeroxylin (12) (Thadaniti and Archakunako, 1994), prforatic acid (13) (Thadaniti and Archakunako, 1994), perforatic acid methyl ester (14) (Thadaniti and Archakunako, 1994), eugenin (15) (Tuntiwachwuttikul et al., 2006) and saikochromone A (16) (Kobayashi et al., 1990) were isolated from H. perforata. Compounds 1–3 and 8–9 are all racemates, and the corresponding enantiomers [(+)-1/2/3/8/9 and ()-1/2/3/8/9] were obtained by chiral HPLC separation, respectively. The absolute configuration of (+)-1 was deduced by Mosher’s method. Unfortunately, the absolute configuration of the enantiomers of compounds 2, 3, 8 and 9 were not determined due to the limited amount and their small optical rotation values. Details of the isolation and structure elucidation of the new compounds are described herein (Fig. 1). 2. Results and discussion Compound 1 was obtained as yellow amorphous powder. The molecular formula of 1 was established as C16H20O6 by its 13C NMR data and an m/z [M+H]+ 309.1332 ion in the HRESIMS (calcd for C16H21O6, 309.1333). The UV spectrum of 1 showed absorption maxima at 207 and 254 nm. The IR spectrum exhibited characteristic absorptions for hydroxyl (3340 cm1), carbonyl (1659 cm1) and benzene ring (1659, 1430 cm1). The 1H and 13C NMR spectra of 1 (Table 1) revealed the existence of an intramolecularly hydrogen-bonded hydroxyl group [dH 12.69 (1H, s)], a pentasubstituted benzene ring [dH 6.34 (1H, s); dC 163.2, 161.1, 155.4,

http://dx.doi.org/10.1016/j.phytol.2014.10.014 1874-3900/Crown Copyright ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

G. Liu et al. / Phytochemistry Letters 10 (2014) 295–299

296

Fig. 1. Chemical structures of 1–16.

Table 1 1 H (400 MHz) and

13

C (100 MHz) NMR data of compounds 1–3 (d in ppm, J in Hz).a 1b

Position

dH 2 3 4 4a 5 6 7 8 8a 10 20 30 40 50 100 200 2-CH3 5-OH 7-OCH3 a b c

5.94 s

6.34 s

a b

2.88 dd (14.0, 2.6) 2.81 dd (14.0, 9.4) 3.56 dd (9.4, 2.6) 1.27 s 1.26 s

2.31 s 12.69 s 3.86 s

2c

dC 166.9 108.4 182.9 104.9 161.1 95.2 163.2 105.3 155.4 25.5

dH 6.08 s

6.42 s

a b

78.3 72.9 23.7 26.3

3.10 dd (13.9, 10.7) 2.97 dd (13.9, 2.8) 5.05 dd (10.7, 2.8)

3c

dC 169.7 108.8 184.6 105.9 162.4 96.1 165.2 105.4 156.9 24.0

20.6

1.75 s 2.42 s

80.2 72.9 26.1 25.8 172.4 20.8 20.5

56.3

3.91 s

56.9

1.29 s 1.27 s

dH 6.11 s

6.48 s

5.31 d (6.3) 3.85 d (6.3)

dC 169.9 109.0 184.7 105.7 163.3 96.7 164.9 110.6 156.7 66.7

1.17 s 1.06 s

80.0 73.6 26.4 25.7

2.42 s

20.6

3.92 s

57.0

Overlapped signals were reported without designating multiplicity. Measured in CDCl3. Measured in CD3OD.

105.3, 104.9, 95.2], an a,b-unsaturated ketone [dH 5.94 (1H, s); dC 182.9, 166.9, 108.4], an oxygenated quaternary carbon (dC 72.9), an oxymethine [dH 3.56 (1H, dd, J = 9.4, 2.6 Hz); dC 78.3], a methoxyl [dH 3.86 (3H, s); dC 56.3], a methylene [dH 2.88 (1H, dd, J = 14.0, 2.6 Hz) and 2.81 (1H, dd, J = 14.0, 9.4 Hz); dC 25.5], three methyl [dH 2.31, 1.27 and 1.26 (each 3H, s); dC 26.3, 23.7, 20.6], indicating the existence of prenylated chromone skeleton. The above spectral data are in close agreement with those reported for perforatin C (Tanaka et al., 1995). However, the optical activity of 1 was undetectable, although there was a chiral center (C-20 ), suggesting the racemic nature of 1. Subsequent chiral HPLC of 1 led to the separation of two enantiomers [(+)-1 and ()-1] with opposite optical rotation values. The absolute configuration of the C-20

secondary alcohol in (+)-1 was determined by modified Mosher’s method. (+)-1 was treated with (R)-()- and (S)-(+)-MTPA-Cl, respectively. Interpretation of the 1H NMR chemical shift differences (Dd = dS  dR) between (S)- and (R)-MTPA ester (1a and 1b) established 20 S configuration (Fig. 2). Thus, (+)-1 was deduced as (20 S)-5-hydroxy-7-methoxy-2-methyl-8-(2,3-dihydroxy-3-methylbutyl)chromone [(20 S)-perforatin C], and its enantiomer ()-1 was deduced as (20 R)-5-hydroxy-7-methoxy-2methyl-8-(2,3-dihydroxy-3-methylbutyl)chromone [(20 R)-perforatin C], respectively. Compound 2 was given the molecular formula C18H22O7 on the basis of its 13C NMR and the HRESIMS data ([M+Na]+ at m/z 373.1257, calcd 373.1258), which is 42 mass units more than

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297

Fig. 2. 1H NMR chemical shift difference of MTPA esters of (+)-1.

1. Detailed examination of the 1H and 13C NMR spectra of 2 and comparison with those of 1 revealed considerable structural similarity between the two compounds, except for the existence of signals for an additional acetoxyl [dH 1.75 (3H, s), dC 172.4, 20.8] in 2. The HMBC correlations between H-20 (dH 5.05) and C-100 (dC 172.4) indicated that the acetoxyl was located at C-20 . The undetectable optical activity of 2 indicated it was also a racemate, which was further confirmed by chiral HPLC analysis. Thus, compound 2 was deduced as ()-20 -O-acetylperforatin C. Compound 3 was assigned a molecular formula of C16H20O7 according to the HRESIMS ([M+Na]+ at m/z 347.1104, calcd 347.1101) and 13C NMR data, which is 16 mass units more than 1. Detailed examination of the 1H and 13C NMR spectra of 3 revealed that its structural fragments resembled those in 1, except that the signals for a methylene [dH 2.88 (1H, dd, J = 14.0, 2.6 Hz), 2.81 (1H, dd, J = 14.0, 9.4 Hz); dC 25.5] were replaced by the signals for an oxymethine [dH 5.31 (1H, d, J = 6.3 Hz); dC 66.7]. The 1H–1H COSY correlation between H-10 (dH 5.31) and H-20 (dH 3.85) in addition to the HMBC correlations between H-10 and C-7/C-8a/C-30 revealed that C-10 was substituted by a hydroxy group. The erythro configuration of the vicinal diol was deduced by the large coupling constant for H-10 and H-20 (J = 6.3 Hz) (Braga et al., 2012), as well as the NOESY correlations between H-10 and H-40 /H-50 . Compound 3 was also a racemate, which was evidenced by the lack of optical activity. Thus, compound 3 was deduced as ()-erythro-10 hydroxyperforatin C. Compound 7 possesses the molecular formula of C15H14O5 determined by its 13C NMR and HRESIMS data ([M+H]+ at m/z 275.0921, calcd 275.0914). The NMR spectra of 7 also resembled those of 1, except that the signals for the C5 side chain were replaced by those for 3-oxo-1-butenyl. This was supported by the HMBC correlations between H-10 (dH 7.91) and C-7 (dC 164.9)/C-8a (dC 156.7)/C-30 (dC 199.6). The E form of the double bond C10 –C20 was determined by the large coupling constant for H-10 and H-20 (J = 16.5 Hz). Thus, compound 7 was identified as 5-hydroxy-7methoxy-2-methyl-8-[(1E)-3-oxo-1-butenyl]chromone. The molecular formula of compound 8 was determined to be C17H20O6 by its 13C NMR and HRESIMS data ([M+H]+ at m/z 321.1331, calcd 321.1333). The NMR spectra of 8 were almost identical to those of greveiglycol 40 -methyl ether (10) (Dean and Robinson, 1971), except for some differences in the C5 side chain. The HMBC correlations between 5-OCH3 (dH 3.71) and C-5 (dC 161.9), and between 10 -OCH3 (dH 3.85) and C-10 (dC 73.1) suggested that the methoxyls were also located at C-5 and C-10 , respectively. Based on the molecular formula, C-30 and C-7 were also anticipated to be connected through an oxygen atom. However, the coupling constant for H-10 (J = 4.5 Hz) and its NOESY correlation with H-20 suggested these two protons were cofacial, which was further confirmed by the X-ray crystallographic analysis (Fig. 3). The crystals of 8 had the space group C2/c, indicative of the racemic

Fig. 3. ORTEP drawing of 8.

nature was also evidenced by the lack of optical activity. Thus, the structure of compound 8 was established as shown and named as ()-horriperfin A. Compound 9 gave a pseudomolecular ion [M+H]+ m/z 291.1227 (calcd 291.1227) by its HRESIMS, consistent with a molecular formula of C16H18O5. The NMR spectra of 9 resembled those of 8, except for the absence of a methoxyl and an oxymethine, the presence of a methylene, as well as some difference at C-10 . The HMBC correlations between H-10 (dH 5.12) and C-7 (dC 160.6)/C-8a (dC 160.1)/C-30 (dC 77.6) and the 1H–1H COSY correlation between H-10 (dH 5.12) and H-20 a (dH 2.15) suggested that the hydroxy group was located at C-10 . Chiral HPLC analysis revealed compound 9 was also a racemate. Thus, the structure of compound 9 was established as shown and named as ()-horriperfin B.

3. Experimental 3.1. General Optical rotations were measured on a Jasco P-1020 digital polarimeter at 25 8C. UV spectra were obtained on a Jasco V-550 UV/VIS spectrophotometer with a 1 cm length cell. IR spectra were recorded on a Jasco FT/IR-480 plus infrared spectrometer with KBr discs. HRESIMS data were measured on an Agilent 6210 ESI/TOF mass spectrometer. NMR experiments were performed on Bruker AV-400 and AV-500 spectrometers. Single-crystal data were performed using an Agilent Gemini S Ultra diffractometer and Cu Ka radiation (l = 1.54184 A˚). Column chromatography (CC) was performed on silica gel (200–300 mesh; Qingdao Marine Chemical Inc., Qingdao, PR China), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) and ODS (YMC, Kyoto, Japan), respectively. TLC analyses were carried out using precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, PR China). Preparative high-performance liquid chromatography (HPLC) was carried out on an Agilent 1260 system equipped with a G1310B Iso pump, a G1365D MWD VL detector, a Cosmosil 5C18-MS-II reversed-phase column (20 mm  250 mm, 5 mm, Nacalai Tesque, Kyoto, Japan). Chiral separation was performed on an Agilent 1260 system equipped with a G1311C Quat pump, a G1315D DAD VL detector and Phenomenex Lux 5u Cellulose-4 and Cellulose-2 columns (4.6 mm  250 mm, 5 mm, Phenomenex, Torrance, USA). All solvents used in column chromatography and HPLC were of analytical grade (Shanghai Chemical Plant, Shanghai, China) and chromatographic grade (Fisher Scientific, New Jersey, USA), respectively.

G. Liu et al. / Phytochemistry Letters 10 (2014) 295–299

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(R)-()- and (S)-(+)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) were obtained from Sigma–Aldrich.

preparative HPLC using CH3OH-H2O (60:40, 6 mL/min) to afford 7 (1.8 mg, tR = 38.7 min), 4 (10.9 mg, tR = 41.7 min) and 14 (2.3 mg, tR = 68.4 min). Fr. B (14.3 g) was subjected to silica gel column (5 cm  30 cm) eluted with cyclohexane-EtOAc (100:0 ! 0:100, v/v) to afford four subfractions (Fr. B1–Fr. B4). Fr. B2 was further separated by Sephadex LH-20 CC (CHCl3-CH3OH, 1:9) to yield 15 (2.7 mg) and 16 (3.8 mg). Racemic mixture 1 was separated into enantiomers (+)-1 (3.9 mg, tR = 13.7 min) and ()-1 (3.7 mg, tR = 15.1 min) by chiral HPLC (Phenomenex Lux 5u cellulose-2; CH3OH-H2O, 68:32; 25 8C; 1.0 mL/min). Compounds (+)-2 (0.9 mg, tR = 44.6 min) and ()-2 (1.2 mg, tR = 41.1 min) were isolated from 2 by chiral HPLC (Phenomenex Lux 5u cellulose-4; CH3CN-H2O with 0.5% trifluoroacetic acid, 27:73; 30 8C; 0.8 mL/min). Racemic mixture 3 was separated into enantiomers (+)-3 (0.5 mg, tR = 20.5 min) and ()-3 (0.6 mg, tR = 21.4 min) by chiral HPLC (Phenomenex Lux 5u cellulose-2; CH3OH-H2O, 60:40; 25 8C; 1.0 mL/min). Compounds ()-8 (2.0 mg, tR = 10.5 min) and (+)-8 (1.8 mg, tR = 11.4 min) were isolated from 8 by chiral HPLC (Phenomenex Lux 5u cellulose-4; CH3OH-H2O, 70:30; 30 8C; 1.0 mL/min). Racemic mixture 9 was separated into enantiomers (+)-9 (1.1 mg, tR = 6.7 min) and ()-9 (0.7 mg, tR = 8.6 min) by chiral HPLC (Phenomenex Lux 5u cellulose-2; CH3CN-H2O, 68:32; 25 8C; 1.0 mL/min). Perforatin C (1): yellow amorphous powder; UV (CH3OH) lmax (log e) 207 (4.10), 254 (4.08) nm; IR (KBr) nmax 3340, 2972, 1659, 1430, 1332, 1274, 1202, 1124 cm1; HRESIMS m/z 309.1332 [M+H]+ (calcd for C16H21O6, 309.1333); 1H and 13C NMR data see Table 1. (+)-1: 25 ½a25 D +22.3 (c 1.2, CH3OH); (–)-1: ½aD 21.5 (c 1.1, CH3OH). Preparation of (S)-(1a) and (R)-(1b) MTPA esters. The (S)- and (R)-MTPA ester derivatives of compound (+)-1 were prepared in a manner described previously (Su et al., 2002; Pan et al., 2013; Takeshige et al., 2012). In brief, two portions of the compound (1.5 mg) were added into two NMR tubes and dried completely. Pyridine-d5 was added to both tubes (each 0.5 mL). Then, (S)MTPA-Cl (10 mL) or (R)-MTPA-Cl (10 mL) was injected into the NMR tubes separately under N2 gas protection and quickly mixed with the dissolved sample. The 1H NMR data of the (S)- and (R)MTPA esters (1a and 1b) of (+)-1 were recorded after the reactions were complete (3 h). 1H NMR data for 1a (400 MHz, pyridine-d5; data were obtained from the reaction NMR tube directly): d 6.90

3.2. Plant material The leaves and twigs of H. perforata were collected in Jianfengling, Hainan Province of P. R. China, in July 2010, and authenticated by Senior Engineer Shi-Man Huang (Hainan University). A voucher specimen (No. 2010070501) deposited in the herbarium of the College of Pharmacy, Jinan University, Guangzhou, PR China. 3.3. Extraction and isolation The air-dry leaves and twigs of H. perforata (19.3 kg) were powdered and extracted at room temperature with 95% EtOH (3  50 L, 24 h each), and the solution was concentrated under reduced pressure to afford a brownish residue (0.9 kg), which was suspended in H2O (3 L), then successively extracted with EtOAc (3  3 L) and n-BuOH (3  3 L), respectively. The EtOAc extract was evaporated to give a residue (274 g), which was then subjected to silica gel column (15 cm  80 cm) eluted with cyclohexane-EtOAc (100:0 ! 0:100, v/v) to afford six major fractions (Fr. A–Fr. F). Fr. E (17.5 g) was subjected to a silica gel column (5 cm  30 cm) eluted with gradient CHCl3-acetone (100:0 ! 0:100, v/v) to afford five subfractions (Fr. E1–Fr. E5). Fr. E1 was further separated by Sephadex LH-20 column chromatography (CC) (CHCl3-CH3OH, 1:1) to yield 2 (3.1 mg), 5 (27.4 mg) and 12 (25.1 mg). Compounds 10 (2.1 mg), 11 (6.5 mg) and 13 (11.8 mg) were obtained from Fr. E4 by Sephadex LH-20 CC (CHCl3-CH3OH, 1:1). Fr. E3 was then subjected to silica gel column (3 cm  20 cm) eluted with cyclohexane-acetone (100:0 ! 0:100, v/v), and followed by Sephadex LH-20 CC (CHCl3-CH3OH, 1:1) to yield 1 (14.7 mg), 3 (2.9 mg) and 6 (28.6 mg). Fr. E5 was purified by preparative HPLC on a reversedphase C18 column (20 mm  250 mm, 5 mm) using CH3OH-H2O (45:55, 6 mL/min) as eluent to yield 8 (10.6 mg, tR = 39.6 min) and 9 (2.5 mg, tR = 59.0 min). Fr. D (19.8 g) was subjected to silica gel column (5 cm  35 cm) eluted with CHCl3-acetone (100:0 ! 0:100, v/v) to afford seven subfractions (Fr. D1–Fr. D7). Fr. D2 was further separated by Sephadex LH-20 CC (CHCl3-CH3OH, 1:1), followed by

Table 2 1 H (400 MHz) and

13

C (100 MHz) NMR data of compounds 7–9 (d in ppm, J in Hz).a 7b

Position

dH 2 3 4 4a 5 6 7 8 8a 10 20 30 40 50 2-CH3 5-OH 5-OCH3 7-OCH3 10 -OCH3 a b c

8c

dC

7.91 d (16.5) 7.07 d (16.5)

167.2 109.3 182.7 105.2 164.5 95.6 164.9 103.1 156.7 132.2 128.8

2.38 s

199.6 27.7

6.12 s

6.41 s

2.46 s 13.39 s

20.7

3.98 s

56.6

dH 6.06 s

6.34 s

4.57 d (4.5) 3.87 d (4.5)

9c

dC 166.0 112.1 179.8 109.0 161.9 97.8 160.0 104.4 159.6 73.1 73.6

dH 6.06 s

6.38 s

a b

5.12 dd (4.9, 3.0) 2.15 dd (14.7, 3.0) 2.04 dd (14.7, 4.9)

dC 166.6 112.1 180.3 109.0 161.7 98.2 160.6 105.5 160.1 59.6 42.1

1.45 s 1.42 s 2.40 s

80.8 27.1 21.3 19.7

1.44 s 1.50 s 2.39 s

77.6 29.9 26.6 19.9

3.71 s

56.5

3.86 s

56.6

3.85 s

61.2

Overlapped signals were reported without designating multiplicity. Measured in CDCl3. Measured in CD3OD.

G. Liu et al. / Phytochemistry Letters 10 (2014) 295–299

(1H, s, H-6), 6.10 (1H, s, H-3), 6.04 (1H, dd, J = 10.7, 3.0 Hz, H-20 ), 3.77 (3H, s, 7-OCH3), 3.61 (1H, overlapped, H-10 a), 3.38 (1H, dd J = 14.1, 3.0 Hz, H-10 b), 2.14 (3H, s, 2-CH3), 1.60 (3H, s, 40 -CH3), 1.56 (3H, s, 50 -CH3); 1H NMR data for 1b (400 MHz, pyridine-d5; data were obtained from the reaction NMR tube directly): d 6.79 (1H, s, H-6), 6.06 (1H, dd, J = 11.1, 2.5 Hz, H-20 ), 6.04 (1H, s, H-3), 3.65 (3H, s, 7-OCH3), 3.53 (1H, overlapped, H-10 a), 3.27 (1H, dd, J = 13.7, 2.5 Hz, H-10 b), 2.09 (3H, s, 2-CH3), 1.66 (3H, s, 40 -CH3), 1.64 (3H, s, 50 -CH3). 20 -O-acetylperforatin C (2): yellow oil; UV (CH3OH) lmax (log e) 207 (4.01), 254 (3.97) nm; IR (KBr) nmax 3440, 2981, 1734, 1658, 1616, 1411, 1330, 1245, 1120 cm1; HRESIMS m/z 373.1257 [M+Na]+ (calcd for C18H22O7Na, 373.1258); 1H and 13C 25 NMR data see Table 1. (+)-2: ½a25 D +15.4 (c 0.6, CH3OH); (–)-2: ½aD 16.5 (c 0.8, CH3OH). Erythro-10 -hydroxyperforatin C (3): yellow oil; UV (CH3OH) lmax (log e) 205 (4.58), 252 (4.55), 289 (4.18) nm; IR (KBr) nmax 3421, 2975, 1660, 1596, 1385, 1274, 1207, 1120 cm1; HRESIMS m/ z 347.1104 [M+Na]+ (calcd for C16H20O7Na, 347.1101); 1H and 13C 25 NMR data see Table 1. (+)-3: ½a25 D +16.1 (c 0.3, CH3OH); (–)-3: ½aD 16.5 (c 0.4, CH3OH). 5-Hydroxy-7-methoxy-2-methyl-8-[(1E)-3-oxo-1-butenyl]chromone (7): yellow amorphous powder; UV (CH3OH) lmax (log e) 206 (3.96), 254 (3.89), 297 (3.68), 331 (3.76) nm; IR (KBr) nmax 2924, 1663, 1587, 1429, 1383, 1337, 1256, 1211, 1180 cm1; HRESIMS m/z 275.0921 [M+H]+ (calcd for C15H15O5, 275.0914); 1H and 13C NMR data see Table 2. Horriperfin A (8): colorless blocks, mp 237–238 8C; UV (CH3OH) lmax (log e) 208 (4.42), 248 (4.40), 289 (4.01) nm; IR (KBr) nmax 3398, 1663, 1610, 1453, 1391, 1351, 1200 cm1; HRESIMS m/z 321.1331 [M+H]+ (calcd for C17H21O6, 321.1333); 1H and 13C NMR 25 data see Table 2. (+)-8: ½a25 D +19.2 (c 1.2, CH3OH); (–)-8: ½aD 18.1 (c 1.3, CH3OH). Horriperfin B (9): yellow amorphous powder; UV (CH3OH) lmax (log e) 203 (4.84), 255 (4.64) nm; IR (KBr) nmax 3401, 2973, 1660, 1613, 1446, 1356, 1126 cm1; HRESIMS m/z 291.1227 [M+H]+ (calcd for C16H19O5, 291.1227); 1H and 13C NMR data see Table 2. 25 (+)-9: ½a25 D +12.7 (c 0.7, CH3OH); (–)-9: ½aD 11.0 (c 0.5, CH3OH). 3.4. X-ray crystallographic analysis of horriperfin A (8) Crystals suitable for X-ray diffraction were grown from CHCl3/ MeOH (1:2). A colorless block of approximate dimensions of 0.33 mm  0.28 mm  0.23 mm was mounted on a glass fiber for the diffraction experiment. Intensity data were collected on an Agilent Gemini S Ultra CCD diffractometer with Cu Ka radiation (l = 1.54184 A˚). Crystal data: C17H20O6, M = 320.33, monoclinic, space group: C2/c; unit cell dimensions were determined to be a = 9.4767(6) A˚, b = 16.1607(8) A˚, c = 20.6748(9) A˚, a = 90.008, b = 98.347(5)8, g = 90.008, V = 3132.8(3) A˚3, Z = 8, Dx = 1.358 mg/ m3, F (000) = 1360.0. A total of 2472 reflections were collected (2156 unique, Rint = 0.0499) in the range 5.618–62.898 of u. The structure was solved by the direct methods and refined by fullmatrix least squares on F2 using the SHELXL-97 package software. All non-hydrogen atoms were refined anisotropically. The final refinement gave R1 = 0.0499 (wR2 = 0.1520) for observed reflections

299

[with I > 2s(I)], and the goodness of fit on F2 was equal to 1.059. The crystallographic data had been deposited at the Cambridge Crystallographic Data Centre as CCDC 1017313. Acknowledgments Financial support of this research was provided by grants from Ministry of Science and Technology of China (Nos. 2013BAI11B05, 2013DFM30080), the Program for New Century Excellent Talents in University (No. NCET-12-0676), the Fundamental Research Funds for the Central Universities (No. 21612203), and Educational Commission of Guangdong Province (No. gjhz1003).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2014. 10.014. References Braga, P.A.C., Severino, V.G.P., Freitas, S.D.L., Silva, M.F.G.F., Fernandes, J.B., Vieira, P.C., Pirani, J.R., Groppo, M., 2012. Dihydrocinnamic acid derivatives from Hortia species and their chemotaxonomic value in the Rutaceae. Biochem. Syst. Ecol. 43, 142–151. Chen, S.K., Chen, B.Y., Li, H., 1997. Flora of China (Zhongguo Zhiwu Zhi), vol. 43. Science Press, Beijing, , pp. 15. Choodej, S., Sommit, D., Pudhom, K., 2013. Rearranged limonoids and chromones from Harrisonia perforata and their anti-inflammatory activity. Bioorg. Med. Chem. Lett. 23, 3896–3900. Dean, F.M., Robinson, M.L., 1971. The heartwood chromones of Cedrlopsis grevei. Phytochemistry 11, 3221–3227. Khuong-Huu, Q., Chiaroni, A., Riche, C., Nguyen-Ngoc, H., Nguyen-Viet, K., KhuongHuu, F., 2000. New rearranged limonoids from Harrisonia perforata. J. Nat. Prod. 63, 1015–1018. Kobayashi, M., Tawara, T., Tsuchida, T., Mrrsuhashi, H., 1990. Studies on the constituents of Umbelliferae plants. XVIII. Minor constituents of bupleuri radix: occurrence of saikogenins, polyhydroxysterols, a trihydroxy C18 fatty acid, a lignan and a new chromone. Chem. Pharm. Bull. 38, 3169–3171. Pan, L., Acuna, U.M., Li, J., Jena, N., Ninh, T.N., Pannell, C.M., Chai, H., Fuchs, J.R., Blanco, E.J.C., Soejarto, D.D., Kinghorn, A.D., 2013. Bioactive flavaglines and other constituents isolated from Aglaia perviridis. J. Nat. Prod. 76, 394–404. Su, B.N., Park, E.J., Mbwambo, Z.H., Santarsiero, B.D., Mesecar, A.D., Fong, H.H.S., Pezzuto, J.M., Kinghorn, A.D., 2002. New chemical constituents of Euphorbia quinquecostata and absolute configuration assignment by a convenient Mosher ester procedure carried out in NMR tubes. J. Nat. Prod. 65, 1278–1282. Takeshige, Y., Kawakami, S., Matsunami, K., Otsuka, H., Lhieochaiphant, D., Lhieochaiphant, S., 2012. Oblongionosides A–F, megastigmane glycosides from the leaves of Croton oblongifolius Roxburgh. Phytochemistry 80, 132–136. Tanaka, T., Koike, K., Mitsunaga, K., Narita, K., Takano, S., Kamioka, A., Sase, E., Ouyang, Y., Ohmoto, T., 1995. Chromones from Harrisonia perforata. Phytochemistry 40, 1787–1790. Thadaniti, S., Archakunako, W., 1994. Chromones from the branches of Harrisonia perforate (Blanco.) Merr. J. Sci. Soc. Thailand 20, 183–187. Tran, V.S., Nguyen, M.P., Kamperdick, C., Adam, G., 1995. Perforatinolone, a limonoid from Harrisonia perforata. Phytochemistry 38, 213–215. Tuntiwachwuttikul, P., Phansa, P., Pootaeng-on, Y., Taylor, W.C., 2006. Chromones from the Branches of Harrisonia perforate. Chem. Pharm. Bull. 54, 44–47. Wang, M.X., Zhang, M.S., Zhu, Y.L., 1983. Studies on the chemical constituents of a Chinese folk medicine Niu-Jin-Guo (Harrisonia perforate Blanco Merr). Acta. Pharm. Sin. 18, 113–118. Yan, X.H., Di, Y.T., Fang, X., Yang, S.Y., He, H.P., Li, S.L., Lu, Y., Hao, X.J., 2011. Chemical constituents from fruits of Harrisonia perforata. Phytochemistry 72, 508–513. Yin, S., Chen, X., Su, Z.S., Yang, S.P., Fan, C.Q., Ding, J., Yue, J.M., 2009. Harrisotones A– E, five novel prenylated polyketides with a rare spirocyclic skeleton from Harrisonia perforata. Tetrahedron 65, 1147–1152.