FITOTE-03124; No of Pages 7 Fitoterapia xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Fitoterapia

F

journal homepage: www.elsevier.com/locate/fitote

3Q2

Shao-Dan Chen a,b,c, Jin-Tao Gao a,b, Jing-Gong Liu a,b, Bo Liu a,b, Rui-Zhi Zhao a,b, Chuan-Jian Lu a,b,⁎

4 5 6

a b c

The Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou 510000, China Guangdong Provincial Hospital of Chinese Medicine, Guangzhou 510000, China Postdoctoral Programme, Guangzhou University of Chinese Medicine, Guangzhou 510000, China

a r t i c l e

10 11 12 13 14

Article history: Received 13 December 2014 Accepted in revised form 8 February 2015 Accepted 12 February 2015 Available online xxxx

20 21 22 23 24 25 26

Keywords: Curcuma kwangsiensis Diarylheptanoids Antiproliferation HH cells HaCaT cells

27

1. Introduction

28 29

Curcuma kwangsiensis S. G. Lee et C. F. Ling (Zingiberaceae), widely distributed in southwest regions of China, has been used as a traditional folk medicine for promoting blood circulation and removing blood stasis. Its roots are one of the most important crude drugs frequently listed in prescriptions of traditional Chinese medicine for the treatment of “Oketsu” [1], various syndromes caused by the obstruction of blood circulation, such as arthralgia, psychataxia, and dysmenorrhea. It has been reported to be rich in diarylheptanoids [2–4] and volatile oil [5–7]. Diarylheptanoids, the main substances in C. kwangsiensis, belong to a class of natural products with a 1,7diarylheptane skeleton possess a variety of biological and pharmacological activities including antioxidant, antihepatotoxic, anti-inflammatory, antiproliferative, antiemetic,

35 36 37 38 39 40 41

15 16 17 18 19

O

R

R

E

C

T

E

Five new diarylheptanoids (1–5), along with nine known ones (6–14), were isolated from the rhizomes of Curcuma kwangsiensis. Their structures were established on the basis of spectroscopic analyses. Compounds 1–3 were cyclic diarylheptanoids rarely discovered from C. kwangsiensis. Of all the isolated compounds, compound 4 showed moderate antiproliferative activity on HH and HaCaT cells. © 2015 Published by Elsevier B.V.

N C

34

a b s t r a c t

U

32 33

i n f o

D

9

P

7

30 31

R O O

2

Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity

1Q1

⁎ Corresponding author at: The Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou 510000, China. Tel.:/Fax: +86 20 8188 7233. E-mail address: [email protected] (C.-J. Lu).

chemopreventive, and antitumor activities [8–14]. During the course of our studies on bioactive diarylheptanoids from 95% EtOH extracts of rhizomes of C. kwangsiensis, 5 new diarylheptanoids (1–5) were isolated along with 9 known ones (6–14) (Fig. 1). Among them, compounds 1–3 were cyclic diarylheptanoids rarely discovered from C. kwangsiensis. This paper describes the isolation and structural elucidation of these new diarylheptanoids and reports the antiproliferative effect of the compounds on HH and HaCaT cells.

42

2. Experimental

51

2.1. General

52

The 1D and 2D NMR spectra were measured with a Bruker AV-500 spectrometer using a CD3OD (δH = 3.31 ppm, δC = 49.3 ppm) and a pyridine-d5 (δH = 8.74 ppm) solutions at room temperature. HR-ESI-MS spectra were acquired using a Thermofisher LTQ-Orbitrap XL hybrid mass spectrometer. Optical rotations were measured on a Rudolph Research Analytical autopol automatic polarimeter at room temperature. UV

53

http://dx.doi.org/10.1016/j.fitote.2015.02.004 0367-326X/© 2015 Published by Elsevier B.V.

Please cite this article as: Chen S-D, et al, Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.004

43 44 45 46 47 48 49 50

54 55 56 57 58 59

2

S.-D. Chen et al. / Fitoterapia xxx (2015) xxx–xxx

OR1 OR2

R4 5

3 2

6

1

R3

5

2

R1

2 1

3

4

2 1 2

3

5

4

6

7

1

5 3

1

6 7

1 2

3

HO

R5

R5

3

4

O 1

6

R3

OH

4

6

6

5

R6

5

R4

4

4. R1 = R2 = COCH3 R3 = R4 = R5 = H R6 = OH 4a. R1 = R2 = R3 = R4 = R5 = H R6 = OH 5. R1 = COCH3 R2 = H R3 = OH R4 = R5 = R6 = H 5a. R1 = R2 = H R3 = OH R4 = R5 = R6 = H 8. R1 = COCH3 R2 = H R3 = OH R4 = R5 = H R6 = OH 10. R1 = R2 = COCH3 R3 = OH R4 = R5 = H R6 = OH 13. R1 = COCH3 R2 = H R3 = OH R4 = H R5 = R6 = OH 14. R1 = R2 = COCH3 R3 = OH R4 = H R5 = R6 = OH

R2

F

1. R1 = H R2 = OH R3 = R4 = OCH3 R5 = H 2. R1 = R2 = H R3 = R4 = H R5 = OCH3 3. R1 = OH R2 = R4 = H R3 = R5 = OH OR1 OR2 R3

R5

HO

OH

O

O

4

R O

HO

O

R4

HO

6. R1 = H R2 = H R3 = R4 = OCH3 R5 = H 7. R1 = H R2 = H R3 = OCH3 R4 = R5 = H 11. R1 = H R2 = H R3 = OH R4 = R5 = H 12. R1 =R2 = R3 = R4 = R5 = H

P

HO

OH

OH

9

82

2.2. Plant material

83 84

87

The rhizomes of C. kwangsiensis originating from Guangxi Province, China, were supplied by Kangmei Pharmaceutical Co. Ltd. (Puning, China). A voucher specimen (2013-EZ-0701) was deposited in the Laboratory of Chinese Materia Medica Preparation, Guangdong Academy of Chinese Medicine Science.

88

2.3. Extraction and isolation

89

The rhizomes of C. kwangsiensis (10.0 kg) were refluxed twice with 95% (v/v) aqueous ethanol (2 × 80 L) for 2 h each time. After filtration, the filtrate was concentrated under reduced pressure. The concentrated solution was suspended

73 74 75 76 77 78 79

85 86

90 91 92

C

E

R

R

71 72

O

69 70

C

67 68

N

65 66

U

63 64

in water, centrifuged, and passed through a macroporous resin HP-20 column (10 × 120 cm), successively eluted with 0%, 30% and 95% EtOH-H2O, to afford 40.5, 25.8, and 362.6 g of extracts (Fr.1–Fr.3), respectively. Fr.3 was subjected to open silica gel column chromatography [200–300 mesh, 10 × 120 cm, eluted with cyclohexane and then step gradient CHCl3-MeOH (100:0; 98:2; 95:5; 93:7; 9:1; 8:2; 7:3; 5:5)] to afford subfractions 3.1– 3.8. Subfraction 3.5 (30 g) was applied to ODS column chromatography [5 × 60 cm, eluted with step gradient MeOH-H2O (3:7, 4:6, 5:5, 6:4, 7:3, 10:0)] to afford subfractions 3.5.1–3.5.6. Subfraction 3.5.4 was then applied to open Sephadex LH-20 column chromatography (3 × 100 cm, eluted with MeOH) to afford subfractions 3.5.4.1–3.5.4.5. Subfraction 3.5.4.2 (1.18 g) was subjected to preparative HPLC (eluted with 55% MeOH, 12 mL/min) to yield compounds 6 (109.3 mg, 8 min), 7 (32.4 mg, 10 min), 1 (18.5 mg, 14 min), and a mixture of 8 and 9 (272.5 mg, 19 min). Subfraction 3.5.4.3 (0.89 g) was subjected to preparative HPLC (eluted with 60% MeOH, 12 mL/min) to yield compound 10 (100.7 mg, 15 min). Subfraction 3.5.4.4 (2.53 g) was subjected to preparative HPLC (eluted with 65% MeOH, 12 mL/min) to yield 5 (17.6 mg, 12 min), 4 (8.7 mg, 15 min), and compound 2 (98.5 mg, 20 min). Subfraction 3.6 (20 g) was applied to ODS column chromatography [5 × 60 cm, eluted with step gradient MeOHH2O (3:7, 4:6, 5:5, 7:3,10:0)] to afford subfractions 3.6.1–3.6.5. Subfraction 3.6.3 was then applied to open Sephadex LH-20 column chromatography (3 × 100 cm, eluted with MeOH) to afford subfractions 3.6.3.1–3.6.3.4. Subfraction 3.6.3.2 (2.86 g) was subjected to preparative HPLC (eluted with 40% MeOH, 12 mL/min) to yield compounds 3 (12.4 mg, 10 min), 11 (76.5 mg, 15 min), 12 (68.7 mg, 39 min), 13 (258.7 mg, 46 min), and 14 (122.5 mg, 50 min). (1S,3S,5S)-1,5-epoxy-3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxyphenyl)heptane (1): Yellow oil; [α]25 D + 8.3 (c = 0.20, MeOH). UV (MeOH) λmax 224.0, 282.0 nm; IR (KBr) νmax 3386, 2925, 2854, 1656, 1599, 1362, 1291, 1203, 1023 cm− 1; 1H-NMR (CD3OD, 500 MHz) and

T

80 81

spectra were recorded on a HITACHI U-2910 UV/Vis spectrometer. IR spectra were obtained using a PerkinElmer Spectrum Two FT-IR spectrometer. The analytical HPLC was performed on a Waters HPLC system equipped with a Waters 600E pump and a 2998 diode array detector (Waters, USA) using a Kromasil 100–5 C18 column (4.6 × 250 mm, 5 μm). The preparative HPLC was carried out on a Waters instrument equipped with a Waters 2545 pump, a Waters 2998 UV detector (Waters, USA), and a Kromasil 100–5 C18 column (21.2 × 250 mm, 5 μm). The semipreparative HPLC was also carried out on a Waters instrument equipped with a Phenomenex Luna C18 column (10.0 × 250 mm, 5 μm). Silica gel (200–300 mesh, Qingdao Haiyang Chemical Ltd., China), macroporous resin HP-20 (Mitsubishi chemical Ltd., Japan), octadecylsilanized (ODS) silica gel (50 μm; YMC Ltd., Japan), and Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden) were used for column chromatography. The (R)- and (S)-MTPA chlorides were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). T-cell lymphoma cell line (HH) was purchased from ATCC, immortal human keratinocyte line (HaCaT) was purchased from CCTCC (Wuhan, China), and cell counting kit-8 (CKK-8) was purchased from DOJINDO Laboratories (Kumamoto, Japan).

61 62

E

60

D

Fig. 1. Structures of compounds 1–14 from the rhizomes of Curcuma kwangsiensis.

Please cite this article as: Chen S-D, et al, Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.004

93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129

S.-D. Chen et al. / Fitoterapia xxx (2015) xxx–xxx 13

Table 2 t2:1 The NMR data of compounds 4 and 5 (in CD3OD, 1H NMR 500 MHz, 13C NMR t2:2 125 MHz). t2:3

2.4. Preparation of the (R)- and (S)-MTPA ester derivatives of 1 and 3

160

163 164

Two aliquots of compound 1 (0.5 mg each) were transferred into two NMR tubes and dried under vacuum overnight at room temperature. Then 20 μL of (R)- or (S)-MTPA chloride and 500 μL of pyridine-d5 were successively added. The NMR tubes were immediately sealed, shake vigorously to ensure

t1:1 t1:2

Table 1 The NMR data of compounds 1–3 (in CD3OD, 1H NMR 500 MHz, 13C NMR 125 MHz).

149 150 151 152 153 154 155 156

161 162

t1:3

Position

1

1 2 3 4 5 6 7 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 3′-OCH3 5′-OCH3

4.71 dd (11.5, 2.0) 1.85 m, 1.75 m 4.24 dddd (3.0, 3.0, 3.0, 3.0) 1.80 m, 1.55 m 3.92 m 1.71 m, 1.55 m 2.65 m

U

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24

N C

δH (J in Hz)

6.67 s

6.67 s 7.00 d (8.5) 6.68 d (8.5) 6.68 d (8.5) 7.00 d (8.5) 3.85 s 3.85 s

δH (J in Hz)

δC

δH (J in Hz)

δC

1 2 3 4 5 6 7 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 3-OAc

2.45 1.76 4.92 1.76 4.92 1.76 2.45

2.50 1.82 5.09 1.70 3.52 1.64 2.62

32.6 38.1 73.1 43.3 68.4 41.3 33.0 135.3 116.8 146.4 144.5 116.5 120.9 143.3 129.7 129.7 127.2 129.7 129.7 21.4 173.4

5-OAc

1.95 s

31.8 37.8 71.5 39.5 71.5 37.8 31.8 133.6 130.5 116.3 156.5 116.3 130.5 133.6 130.5 116.3 156.5 116.3 130.5 21.4 173.2 21.4 173.2

m m m m m m m

6.95 d (8.5) 6.71 d (8.5) 6.71 d (8.5) 6.95 d (8.5) 6.95 d (8.5) 6.71 d (8.5) 6.71 d (8.5) 6.95 d (8.5) 1.95 s

D

147 148

t2:4

5

m m m m, 1.59 m m m m

6.62 d (2.0)

6.66 d (8.0) 6.49 dd (8.0, 2.0)

7.15 d (7.0) 7.24 d (7.0) 7.14 m 7.24 d (7.0) 7.15 d (7.0) 1.99 s

E

145 146

4

t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27

even mixing, and stored in a desiccator for 4 h until the reaction was complete. 1H NMR spectra were used to monitor the reaction. The 1H NMR spectra of the final (R)- and (S)-MTPA adducts were recorded directly after each reaction. The (R)- and (S)-MTPA esters of compound 3 were also obtained following the procedure as described for 1.

165

2.5. Hydrolysis of compounds 4 and 5

171

T

143 144

C

141 142

E

139 140

R

137 138

Position

166 167 168 169 170

To a solution of 4 (5.0 mg) in MeOH (2 mL), a drop of HCl 172 (analytical grade) was added. The resulting mixture was stirred 173

R

135 136

O

134

F

158 159

132 133

R O O

157

C-NMR (CD3OD, 125 MHz): see Table 1. HR-ESI-MS: m/z 373.1643 [M − H] − (calc for C21H25O6, 373.1651). (1S,5R)-1,5-epoxy-1-(4-hydroxyphenyl)-7-(3-methoxy-4hydroxyphenyl)heptane (2): Yellow oil; [α]25 D− 42.2 (c = 0.20, CHCl3). UV (MeOH) λmax 228.0, 283.0 nm; IR (KBr) νmax 3385, 2926, 2854, 1656, 1599, 1505, 1453, 1363, 1312, 1189, 1055 cm−1; 1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz): see Table 1. HR-ESI-MS: m/z 327.1590 [M − H] − (calc for C20H23O4, 327.1596). (1R,2S,5S)-1,5-epoxy-2-hydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane (3): Yellow oil. [α]25 D + 9.5 (c =0.20, MeOH). UV (MeOH) λmax 225.0, 282.0 nm; IR (KBr) νmax 3386, 2854, 1656, 1599, cm− 1; 1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz): see Table 1. HR-ESI-MS: m/z 345.1335 [M − H]− (calc for C19H21O6, 345.1338). (3R, 5R)-3,5-diacetyl-1,7-bis(4-hydroxyphenyl)heptane (4): Yellow oil; [α]25 D+ 11.8 (c = 0.20, MeOH). UV (MeOH) λmax 226.0, 272.0 nm; IR (KBr) νmax 3365, 2925, 2855, 1715, 1656, 1599, 1505, 1453, 1357, 1298, 1015 cm−1; 1H-NMR (DMSO-d6, 400 MHz) and 13C-NMR (DMSO-d6, 100 MHz): see Table 2. HRESI-MS: m/z 399.1811 [M − H] − (calc for C23H27O6, 399.1808). (3R,5R)-3-acetyl-5-hydroxyl-1-(3,4-dihydroxyphenyl)-7phenyl-heptane (5): Yellow oil; [α]25 D + 14.7 (c = 0.20, MeOH). UV (MeOH) λmax 224.0, 283.0 nm; IR (KBr) νmax 3356, 2925, 2854, 1722, 1656, 1600, 1500, 1455, 1357, 1299, 1013 cm− 1; 1H-NMR (DMSO-d6, 400 MHz) and 13C-NMR (DMSO-d6, 100 MHz): see Table 2. HR-ESI-MS: m/z 357.1703 [M − H]− (calc for C21H25O5, 357.1702).

P

130 131

3

2

3

δC

δH (J in Hz)

δC

δH (J in Hz)

δC

75.7 41.5 65.9 39.8 73.0 39.5 32.1 135.5 104.9 149.4 136.1 149.4 104.9 134.6 130.7 116.4 156.4 116.4 130.7 57.0 57.0

4.22 dd (11.0, 2.0) 1.73 m, 1.49 m 1.50 m 1.70 m 3.41 m 1.78 m 2.48 m 2.60 m

81.3 34.5 25.4 32.6 79.0 39.8 32.6 135.9 128.8 116.5 157.9 116.5 128.8 135.5 113.3 149.0 145.7 116.4 122.0 56.7

3.83 d (9.5) 3.47 m 2.10 m, 1.51 m 1.78 m, 1.47 m 3.38 m 1.76 m, 1.65 m 2.55 m

86.5 72.0 34.3 32.6 78.6 39.1 32.4 133.8 116.5 146.3 144.4 116.2 121.1 135.5 116.9 146.3 144.4 116.4 121.0

7.19 d (8.5) 6.79 d (8.5) 6.79 d (8.5) 7.19 d (8.5) 6.68 d (2.0)

6.69 d (8.0) 6.58 dd (8.0, 2.0) 3.77 s

6.88 br.s

6.75 br.d 6.75 br.d 6.60 d (2.0)

6.65 d (8.0) 6.49 dd (8.0, 2.0)

Please cite this article as: Chen S-D, et al, Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.004

2.6. Bioactivity assay

180

The antiproliferative effect of the isolated compounds on HH and HaCaT cells was evaluated by CCK-8 (Table 3). HH and HaCaT cells were plated in 96-well plates (2 × 104 cells/well) and exposed to various concentrations of compounds for 24 h, DMSO served as control. After treatment, 10 μL of CKK-8 solution was added to each well, and the 96-well plate was continuously incubated for 4 h. Then the optical density (OD) value was recorded at 450 nm. Experiments were performed in triplicate. IC50 was calculated by SPSS software. The inhibitory rate was calculated as follows:

183 184 185 186 187 188 189

Inhibitory rate ð%Þ ¼



1 – ODexperimental group =ODcontrol group



P

181 Q3 182

 100%

D

191

3. Results and discussion

193

209 210

Compound 1 was obtained as a yellow oil, [α]25 D+ 8.3 (c = 0.20, MeOH). The HR-ESI-MS spectrum exhibited an [MH]− ion peak at m/z 373.1643, corresponding to the molecular formula C21H26O6 (calc for C21H25O6: 373.1651). The 1H NMR spectrum of 1 revealed a 1,4-bissubstituted [δ 7.00 (2H, d, J = 8.5 Hz, H-2″ and H-6″), δ 6.68 (2H, d, J = 8.5 Hz, H-3″ and H5″)], and a 1,3,4,5-tetrasubstituted [δ 6.67 (2H, s, H-2′ and H6′)] benzene rings. The 1H and 13C NMR spectral data of 1 were very close to those of 1,5-epoxy-3-hydroxy-1-(4-hydroxy-3, 5dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)heptane [15] except that the a 1,3,4-trisubstituted phenyl moiety in the latter was changed to 1,3,4,5-tetrasubstituted one in 1 as evidenced from its 1H, 13C and HMBC spectra (Table 1 and Fig. 2). In addition, the NOESY experiment of 1 (Fig. 3) indicated the correlation of H-1 [δ 4.71, (dd, J = 11.5, 2.0 Hz)] with H-5 (δ 3.92, m), suggesting the axial orientations of H-1 and H-5. The coupling constants of H-3 [δ 4.24, (dddd, J = 3.0, 3.0, 3.0 and 3.0 Hz)] demonstrated its equatorial orientation;

t3:1 t3:2

Table 3 Antiproliferative effect of compounds 1–14 on HH and HaCaT cells.

205 206 207 208

t3:3

t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16

C

E

R

R

203 204

O

201 202

C

199 200

N

197 198

Compound

1 2 3 4 5 6 7 8 and 9 10 11 12 13 14

U

195 196

T

192

194

F

179

176 177

hence, the 3-hydroxyl group is axial oriented. The structure was further confirmed by the HMBC, H-H COSY, and NOESY correlations as shown in Figs. 2 and 3. Finally, the advanced Mosher's ester procedure [16–18] was employed to determine the absolute configuration of C-3. Treatment with (R)- and (S)MTPA chlorides led to esterification of the C-3 OH group to afford the (S)- and (R)-MTPA derivatives (1a and 1b), respectively. By observing the 1H NMR chemical shift difference values (ΔδS-R) of the heptane moiety, the absolute configuration of C-3 was determined to be S (Fig. 4). Therefore, compound 1 was assigned as (1S,3S,5S)-1,5-epoxy-3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxyphenyl)heptanes. Compound 2 was obtained as a yellow oil, [α]25 D− 42.2 (c = 0.20, CHCl3). Its molecular formula was determined as C20H24O4 (calc for [M-H]−: 327.1596) on the basis of HR-ESIMS (m/z 327.1590 [M-H]−). The 1H and 13C NMR spectral data of 2 (Table 1) were similar to those of compound 1 except that the substitution at two phenyl moiety and the hydroxyl group at the pyranose ring. The 1H NMR spectrum exhibited the existence of a 1,4-bissubstituted [δ 7.19 (2H, d, J = 8.5 Hz, H-2′ and H-6′), δ 6.79 (2H, d, J = 8.5 Hz, H-3′ and H-5′)] and a 1,3,4trisubstituted [δ 6.68 (1H, d, J = 8.0 Hz, H-2″), δ 6.69 (1H, d, J = 8.0 Hz, H-5″), and δ 6.58 (1H, d, J = 8.0, 2.0 Hz, H-6″)] benzene rings. In the HMBC spectrum of 2, the correlations of H-2′/H-6′ with C-1 (δ 81.3), H-2″ with C-7 (δ 32.6), H-6″ with C-7 (δ 32.6), H-2 with C-1′ (δ 135.9), and H-6 with C-1″ (δ 135.5) suggested the 1,4-bissubstituted benzene ring was connected to C-1 and the 1,3,4-trisubstituted benzene ring was connected to C-7. Additionally, in the NOESY spectrum, the correlation of H-1 (δ 4.22, dd, J =11.5, 2.0 Hz) with H-5 (δ 3.41, m) suggested the axial orientations of H-1 and H-5. Thus, the relative configuration of 2 was the same as that of 1 and (−)centrolobine [19]. Besides, compound 2 exhibited a levorotatory optical activity {[α]25 D− 42.2 (c = 0.20, CHCl3)} and had identical stereochemistry at C-1/C-5 to that of C-2/C-6 of (−)centrolobine [19–21]. Therefore, 2 was assigned as (1S,5R)-1,5epoxy-1-(4-hydroxyphenyl)-7-(3-methoxy-4hydroxyphenyl)heptane. Compound 3 was obtained as a yellow oil. Its molecular formula, C19H22O6, was established by HR-ESI-MS (m/z 345.1335 [M − H]−, calc for C19H21O6: 345.1338). The 1H NMR data indicated that the molecule possessed two 1,3,4trisubstituted benzene rings [δ 6.65 (1H, d, J = 8.0 Hz), 6.49 (1H, dd, J = 8.0, 2.0 Hz), and 6.60 (1H, d, J = 2.0 Hz); δ 6.88 (1H, br.s), 6.75 (1H, br.d), and 6.75 (1H, br.d)]. The 13C NMR spectrum displayed 19 signals consistent with 12 aromatic ring carbons [δ 146.3 (×2), 144.4 (×2), 135.5, 133.8, 121.1, 121.0, 116.9, 116.5, 116.4, and 116.2], three oxymethines (δ 86.5, 78.6, and 72.0), and four methylenes (δ 39.1, 34.3, 32.6, and 32.4), suggesting a diarylheptanoid structure. Since eight of 9 unsaturations were accounted for, compound 3 was inferred to contain one more ring. The 1H and 13C NMR data of compound 3 were similar to those of compound 2. A major difference between them was the substitution pattern and the substituted groups of the two aromatic rings. One 4-hydroxy substituted benzene ring and one 3-methoxy-4-hydroxy substituted benzene ring in 2 were changed into two 3,4dihydroxy substituted benzene rings in 3. Besides, one more hydroxyl group was confirmed to be at C-2 by H-H COSY correlation between H-1 [δ 3.83 (1H, d, J = 9.5 Hz)] and H-2 (δ 3.47, m) and HMBC correlation from H-2 to C-1′ (δ 133.8) and

O

178

for 12 h at room temperature. The reaction solution was then subjected to semipreparative HPLC (eluted with 50% MeOH, 4 mL/min) to yield compound 4a (2.1 mg, 11.7 min). Compound 5a was prepared from compound 5 following the above procedure.

E

174 175

S.-D. Chen et al. / Fitoterapia xxx (2015) xxx–xxx

R O

4

IC50 (μM) HH

HaCaT

N200 N200 N200 74.35 N200 N200 N200 N200 N200 N200 N100 N100 N100

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Please cite this article as: Chen S-D, et al, Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.004

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C-4 (δ 32.6) (Fig. 2). The relative configuration of 3 was determined by the analyses of coupling constants and NOESY data (Fig. 3). The large coupling constant (J = 10.0 Hz) between H-1 and H-2 indicate the two protons to have axial orientation. The NOESY correlations between H-1 and H-5, between H-4 eq and H-5, between H-3eq and H-2, between H-3eq and H-4ax, and between H-2 and H-4ax were observed, suggesting the axial orientations of H-1, H-2, and H-5. Using the same method described for 1, 1H NMR analysis of the (S)- and (R)-MTPA derivatives (3a and 3b), the absolute configuration of C-2 in 3 was determined to be S. Thus, compound 3 was assigned as (1R,2S,5S)-1,5-epoxy-2-hydroxy-1,7-bis(3,4dihydroxyphenyl)heptane. Compound 4 was isolated as yellow oil. The HR-ESI-MS spectrum exhibited a unique [M-H]− ion peak at m/z 399.1811 corresponding to the molecular formula of C23H28O6 (calc for [M-H]−: 399.1808). The 1H NMR spectrum of 4 (Table 2) exhibited the presence of two 4-hydroxyphenyl groups at δ 6.95 (4H, d, J = 8.5 Hz, H-2′, H-6′, H-2″, and H-6″) and 6.71 (4H, d, J = 8.5 Hz, H-3′, H-5′, H-3″and H-5″). The 13C NMR spectrum of 3 revealed the existence of two 1,4-substituted aromatic ring [δ 133.6, 130.5 (×2), 116.3 (×2), 156.5], two acetyl groups (δ 173.2 and 21.4), two methenyl groups [δ 71.5 (×2)], and five methylene [δ 31.8 (×2), 37.8 (×2), and 39.5] (Table 2). The 1H and 13C NMR spectroscopic data and optical rotation of 4 were similar to those of compound (3R, 5R)-3,5-dihydroxy-1,7bis(4-hydroxyphenyl)heptane [22], which had been obtained from Tacca chantrieri. However, distinctive differences in the 13 C NMR spectrum were observed between these two molecules. The chemical shifts of C-2, C-4, and C-6 were upfield 3.6, 6.2 (3.1 × 2), and 3.6 ppm respectively, while both of the chemical shifts of C-3 and C-5 were downfield 2.8 ppm in the

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Fig. 2. Key HMBC (→) and COSY ( ) correlations of 1–5.

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C NMR spectrum of 4, which indicated that C-3 and C-5 were acetoxylated. The structure was further confirmed by the HMBC and H-H COSY correlations as shown in Fig. 2. Diarylheptanoids with C-3 acetylated/C-5 hydroxylated or C-3/C-5 diacetylated were common in C. kwangsiensis [3]. The absolute configuration of C-3 and C-5 of most of compounds were 3R and 5R, which indicated that the absolute configuration of this type of diarylheptanoids in C. kwangsiensis was conservative and relatively fixed. In order to determine the absolute configuration at C-3 and C-5, compound 4 was deacetylated with HCl/MeOH to gave the corresponding 3,5dihydroxyl derivative 4a [3,22], by comparing their 1H NMR spectroscopic data and optical rotation with the reported in the literature. Besides, the positive specific rotation of 4, [α]25 D+ 11.8 (c = 0.20, MeOH), further confirmed the 3R and 5R configurations since the corresponding 3S and 5S forms were reported to show an opposite [α]D value [23]. Thus, the structure of 4 was established as (3R,5R)-3, 5-diacetyl-1,7bis(4-hydroxyphenyl)heptane. Compound 5 was also obtained as yellow oil. The molecular formula of 5 was established as C21H26O5 by its HR-ESI-MS (m/z 357.1703 [M − H]−, calc for C21H25O5: 357.1702). The 1H NMR spectrum of 5 revealed a 1,3,4-trisubstituted phenyl moiety [δH 6.66 (1H, d, J = 8.0 Hz, H-5′), 6.62 (1H, d, J = 2.0 Hz, H-2′), and 6.49 (1H, dd, J = 8.0, 2.0 Hz, H-6′)] and a phenyl moiety [δH 7.24 (2H, d, J = 7.0 Hz, H-2″ and H-6″), 7.15 (2H, d, J = 7.0 Hz, H-3″ and H-5″), and 7.14 (1H, m, H-4″)]. The 1H and 13C NMR spectroscopic data of 5 (Table 2) were similar to those of (3R,5R)-3-acetoxy-5-hydroxy-1, 7-bis(3,4-dihydroxyphenyl) heptane [3], which was also isolated from C. kwangsiensis except one of a 1,3,4-trisubstituted phenyl moiety in the latter was changed to a phenyl one. The phenyl moiety could be

Please cite this article as: Chen S-D, et al, Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.004

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Fig. 4. Δδ values (in ppm) = δS − δR for (S)- and (R)-MPTA esters of 1 and 3.

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This research was financially supported by the Guangdong Natural Science Fund (S2013030011515), the Guangdong Financial Industry Technology Research Development Fund [2011(285)05], the Guangdong Science and Technology Department-Guangdong Province Academy of Chinese Medicine Science Joint Special Fund (2011B032200009), and the Guangdong Provincial Hospital of Chinese Medicine Special Fund (YK2013B1N11).

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bis(4-hydroxyphenyl)heptane (12) [25], (3R,5R)-3-acetoxy-5hydroxy-1,7-bis(3,4-dihydroxyphenyl) heptane (13) [3], and (3R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)heptane (14) [26], were isolated and identified by comparison of their spectroscopic data with those reported in the literature. Among them, compounds 8 and 9 were obtained as a pair of inseparable enantiomers. All isolated compounds (1–14) were evaluated for their inhibitory effects on proliferation of HH cells and HaCaT cells. Compound 4 exhibited moderate inhibitory effects on proliferation of HH and HaCaT cells and could serve as a possible candidate for future psoriasis chemotherapy.

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further evidenced from the HMBC correlations from H-4″/to C2″ and C-6″ and the H-H COSY correlation from H-2″ to H-3″ and from H-5″ to H-6″. Thus, the planar structure of compound 5 was assigned. Deacetylation of compound 5 with HCl/MeOH gave the corresponding 3-deacetyl derivative 5a, which was identified as (3R,5R)-3,5-dihydroxyl-1-(3,4-dihydroxyphenyl)-7-phenyl-heptane, by comparing their 1H NMR spectroscopic data and optical rotation with the reported in the literature [24]. Besides, the positive specific rotation of 5, [α]25 D+ 14.7 (c = 0.20, MeOH), further confirmed the 3R and 5R configurations. Thus, compound 5 was identified as (3R,5R)-3acetyl-5-hydroxyl-1-(3,4-dihydroxyphenyl)-7-phenyl-heptane. In addition to four new diarylheptanoids (1–5), nine known ones, (3R, 5S)-3, 5-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxyphenyl)heptane (6) [25], (3R,5S)-3,5dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl) heptane (7) [3], (3R, 5R)-3-acetoxy-5-hydroxy-1(3,4-dihydroxyphenyl)-7-(4-hydroxy phenyl)heptane (8) [3], (3S,5S)-3-acetoxy-5-hydroxy-1-(3,4-dihydroxyphenyl)-7(4-hydroxyphenyl)heptane (9) [3],(3R,5R)-3,5-diacetoxy1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane (10) [3], (3R,5S)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane (11) [3], (3R,5S)-3,5-dihydroxy-1,7-

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Fig. 3. Key NOESY connectivities for compounds 1–3.

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Please cite this article as: Chen S-D, et al, Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.004

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Please cite this article as: Chen S-D, et al, Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.004

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Five new diarylheptanoids from the rhizomes of Curcuma kwangsiensis and their antiproliferative activity.

Five new diarylheptanoids (1-5), along with nine known ones (6-14), were isolated from the rhizomes of Curcuma kwangsiensis. Their structures were est...
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