Fitoterapia 92 (2014) 116–126

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Geranylated 2-arylbenzofurans from Morus alba var. tatarica and their α-glucosidase and protein tyrosine phosphatase 1B inhibitory activities Ya-Long Zhang a, Jian-Guang Luo a, Chuan-Xing Wan b, Zhong-Bo Zhou a,b, Ling-Yi Kong a,⁎ a State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China b Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Group, Tarim University, Alaer 843300, People's Republic of China

a r t i c l e

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Article history: Received 22 August 2013 Accepted in revised form 28 October 2013 Available online 9 November 2013 Keywords: Morus alba var. tatarica 2-Arylbenzofuran α-Glucosidase inhibitor PTP1B inhibitor

a b s t r a c t Ten new geranylated 2-arylbenzofuran derivatives, including two monoterpenoid 2-arylbenzofurans (1 and 2), two geranylated 2-arylbenzofuran enantiomers (3a and 3b), and six geranylated 2-arylbenzofurans (4–9), along with four known 2-arylbenzofurans (10–13) were isolated from the root bark of Morus alba var. tatarica. Their structures and relative configurations were established on the basis of spectroscopic data analysis. Compounds 3–7 with one asymmetric carbon at C-7″ were supposed to be enantiomeric mixtures confirmed by chiral HPLC analysis, and the absolute configurations of each enantiomer in 3–7 were determined by Rh2(OCOCF3)4-induced CD and Snatzke's method. The enantiomers with the substituting group at C-2′ exhibited better resolutions on a Chiralpak AD-H column than those with the substituting group at C-4′. Compounds 1–7, 10, 11 and 13, showed α-glucosidase inhibitory activities with IC50 values of 11.9–131.9 μM, and compounds 1 and 9–13 inhibited protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 7.9–38.1 μM. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia due to defects in insulin secretion, or action, or both [1,2]. According to the World Health Organization (WHO) projections, around 300 million or more people will suffer from diabetes by the year 2025 [3]. Type-II diabetes mellitus is the most common form of DM. The control of postprandial blood glucose excursions is an effective treatment for type-II DM. α-Glucosidase inhibitors have been clinically used for managing blood glucose levels. In addition, protein tyrosine phosphatase 1B (PTP1B), an enzyme acting as a key negative regulator of insulin and leptin receptor mediated signaling pathways, has been considered as a therapeutic target for diabetes [4]. With the development of biomedical science, many different types of drugs for type-II DM are now available. ⁎ Corresponding author. Tel./fax: +86 25 8327 1405. E-mail address: [email protected] (L.-Y. Kong). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.10.017

However, these treatments have many undesirable side effects [5]. Therefore, the exploration of new antidiabetes agents having high efficacy but fewer adverse effects remains a pressing challenge. Natural resources provide a huge and highly diversified chemical bank from which we can explore new antidiabetes agents. The genus Morus (Moraceae), consisting of 11 species and 12 varieties in China, is distributed all over the country [6]. Some Morus plants are widely cultivated for their economic value, of which leaves are indispensable food for silk-worms. On the other hand, the root bark of some Morus species has been commonly used as a traditional Chinese Medicine “Sang-Bai-Pi” to treat diabetes, arthritis and rheumatism [7]. Regarding the chemical constituents, a series of 2-arylbenzofurans, flavonoids and other phenolic compounds have been isolated from the bark of M. alba previously [7–9]. Some of these compounds exhibit antioxidant, anti-inflammatory, antihyperglycemic and antihyperpigmentation activities [10–13]. In particular, several prenylated 2-arylbenzofurans showed significant α-glucosidase

Y.-L. Zhang et al. / Fitoterapia 92 (2014) 116–126

and PTP1B inhibitory activities [14,15]. Morus alba var. tatarica is widely cultivated in Xinjiang Province, China [16]. As part of a program to search for antidiabetic agents from natural products [17,18], the chemical constituents of the root bark of M. alba var. tatarica resulted in the isolation of ten new 2-arylbenzofuran derivatives (1–9) (Fig. 1) and four known compounds. Herein, we report the isolation and structure elucidation of compounds 1–9, as well as their inhibitory effects on α-glucosidase and PTP1B. 2. Experimental 2.1. General Optical rotations were measured with a JASCO P-1020 polarimeter. CD spectra were obtained on a JASCO 810 spectropolarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectropolarimeter. IR spectra were measured in KBr-disc on a Bruker Tensor 27 spectrometer. NMR spectra were obtained on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C) in acetone-d6. HRESIMS was carried out on an Agilent UPLC-Q-TOF (6520B). Column chromatography (CC) was performed on silica gel (Qingdao marine Chemical Co., Ltd., China), ODS (40–63 μm, Fuji, Japan), and Sephadex LH-20 (Pharmacia, Sweden). Preparative HPLC was carried out using a Shimadzu LC-6A instrument with a SPD-10A detector using a shim-pack RP-C18 column (20 × 200 mm). Analytical HPLC was measured on an Agilent 1200 Series instrument with a DAD detector using a shim-pack VP–ODS column (250 × 4.6 mm). Chiralpak AD-H columns (0.46 i.d. × 25 cm) were purchased from Daicel Chemical Ltd. (Shanghai, China). α-Glucosidase and PTP1B inhibitory activities were measured spectrophotometrically using a Spectra Max Plus 384 multidetection microplate reader (Molecular Devices, Sunnyvale, CA). Yeast α-glucosidase (EC 3.2.1.20), p-Nitrophenyl-α-D-glucopyranoside (p-NPG), p-nitrophenyl phosphate (pNPP), 1-deoxynojirimycin, and genistein were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). PTP1B and RK-682 were purchased from Enzo Life Sciences, Inc. (NY, USA). 2.2. Plant material The root bark of M. alba var. tatarica was collected in February 2012 from Alaer, Xinjiang Province, China, and was authenticated by Dr. Chuan-Xing Wan, College of Life Sciences, Tarim University. A voucher specimen (No. 20120228) is deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. 2.3. Extraction and isolation The air-dried, powdered root bark of M. alba (3.0 kg) was exhaustively extracted with 75% EtOH (3 × 4 h). After removal of the solvent under reduced pressure, the EtOH extract (228 g) was chromatographed over a silica gel column, eluted with EtOAc, acetone and MeOH successively. The EtOAc portion (101 g) was subjected to a silica gel column (100–200 mesh), eluted with a gradient of CH2Cl2-MeOH (98:2, 95:5, 90:10, 85:15, 80:20, 70:30, 1:1, =v/v), to yield ten fractions (Fr. A–J). Fr. B (6.0 g) was eluted with a gradient of MeOH-H2O (from

117

45:55 to 90:10) on an ODS column to give eight subfractions (Fr. B1–B8). Fr. B2 (170 mg) was subjected to CC over silica gel with petroleum ether–acetone (5:2), to give seven subfractions (Fr. B2.1–B2.7). Fr. B2.2 (19 mg) was chromatographed on silica gel and Sephadex LH-20 column, and further purified by preparative HPLC (MeOH-H2O, 65:35) to yield 2 (3 mg). Fr. B2.5 (25 mg) and B2.7 (15 mg) were separated by preparative HPLC (MeOH-H2O, 65:35 and 67:33) to yield 4 (7 mg) and 7 (6 mg), respectively. Fr. B3 (240 mg) was subjected to a silica gel column and an ODS column, and then purified by preparative HPLC (MeOH-H2O, 70:30), to provide 8 (4 mg) and 3 (16 mg). Fr. B4 (100 mg), B6 (200 mg), B7 (800 mg) and B5 (290 mg) were chromatographed on silica gel and further purified by preparative HPLC (MeOH-H2O, 72:28, 75:25, 80:20 and CH3CN-H2O, 60:40) to afford 13 (10 mg), 11 (10 mg), 12 (11 mg) and 9 (12 mg), respectively. Fr. D (10 g) was subjected to a silica gel column, eluted with a gradient of CH2Cl2-MeOH (19:1, 9:1), to yield five subfractions (Fr. D1– D5). Fr. D1 (4 g) was further eluted with a gradient of MeOH-H2O (from 45:55 to 90:10) on an ODS column to give seven subfractions (Fr. D1.1–D1.7). Fr. D1.3 (350 mg) was chromatographed on silica gel and Sephadex LH-20 column, and further separated by preparative HPLC (MeOH-H2O, 67:33) to yield 1 (2 mg). Fr. E (11 g) was eluted with a gradient of increasing MeOH (50–100%) in water on an ODS column to yield seven subfractions (Fr. E1–E7). Fr. E3 (550 mg) was further eluted with MeOH-H2O (55:45) on an ODS column, and five subfractions (Fr. E3.1–E3.5) were collected. Fr. E3.3 (120 mg) was further separated by silica gel CC and Sephadex LH-20 columns, and then further purified by preparative HPLC (MeOH-H2O, 55:45 and CH3CN-H2O, 39:61) to afford 6 (2 mg) and 5 (4 mg), respectively. Fr. E7 (110 mg) was applied to a silica gel column and a Sephadex LH-20 column, and further separated by preparative HPLC (MeOH-H2O, 75:25) to yield 10 (3 mg). 2.3.1. Compound 1 Light brown amorphous powder; [α]25D 0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 203 (sh) (4.18), 218 (4.22), 318 (4.28), 332 (4.21) nm; IR (KBr) νmax 3444, 2963, 2922, 1634, 1489, 1400, 1261, 1145, 1098, 1024, 801 cm−1; 1H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 1; HRESIMS m/z: 395.1856 [M + H]+ (calcd for C24H27O5, 395.1853). 2.3.2. Compound 2 White amorphous powder; [α]25D 0 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 216 (3.95), 306 (3.77) nm; IR (KBr) νmax 3451, 1633, 1436, 1383, 1190, 1140, 1118, 980, 837 cm−1; 1H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 1; HRESIMS m/z: 407.1866 [M − H]− (calcd for C25H27O5, 407.1864). 2.3.3. Compound 3 Yellow amorphous powder; [α]25D −6.6 (c 0.07, acetone, 3a), [α]25D +6.6 (c 0.11, acetone, 3b); UV (MeOH) λmax (log ε) 215 (4.08), 309 (3.89) nm; IR (KBr) νmax 3451, 1628, 1441, 1384, 1189, 1148, 1114, 1043 cm−1; 1H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 1; HRESIMS m/z: 431.1830 [M + Na]+ (calcd for C25H28O5Na, 431.1829).

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Y.-L. Zhang et al. / Fitoterapia 92 (2014) 116–126

Fig. 1. Structures of compounds 1–9.

2.3.4. Compound 4 Yellow amorphous powder; [α]25D 0 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 218 (4.04), 318 (4.11), 332 (4.03) nm; IR (KBr) νmax 3451, 1634, 1428, 1384, 1146, 1079, 949 cm−1; 1 H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 2; HRESIMS m/z: 395.1850 [M + H]+ (calcd for C24H27O5, 395.1853). 2.3.5. Compound 5 Yellow amorphous powder; [α]25D − 8.9 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 214 (4.04), 310 (3.85) nm; IR (KBr) νmax 3453, 1631, 1490, 1444, 1383, 1148, 1114, 1079, 1011,

949, 828 cm−1; 1H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 2; HRESIMS m/z: 435.1777 [M + Na]+ (calcd for C24H28O5Na, 435.1778).

2.3.6. Compound 6 Yellow amorphous powder; [α]25D −10.4 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 216 (4.11), 317 (4.28), 332 (4.30) nm; IR (KBr) νmax 3442, 1627, 1428, 1384, 1146, 1115, 1015, 967, 950, 821 cm−1; 1H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 2; HRESIMS m/z: 435.1775 [M + Na]+ (calcd for C24H28O5Na, 435.1778).

Y.-L. Zhang et al. / Fitoterapia 92 (2014) 116–126 Table 1 1 H (500 MHz) and

13

C NMR (125 MHz) Data of Compounds 1–3 in acetone-d6. 1

Position 2 3 3a 4 5 6 7 7a 1′ 2′ 3′ 4′ 5′ 6′ 1″a 1″b 2″ 3″ 4″a 4″b 5″a 5″b 6″a 6″b 7″ 8′ 9″a 9″b 10″a 10″b 11″ MeO-3′ a

119

2

δH (J in Hz) 6.87, d (0.5) 7.36, d (8.5) 6.78, dd (8.5, 2.0) 6.94, d (2.0)

6.85, sa

a

6.85, 3.19, 2.89, 2.65,

s dd (13.5, 10.5) dd (13.5, 4.0) dd (10.5, 4.0)

2.54, 1.92, 1.80, 1.57, 3.45,

m m m m dd (8.8, 4.0)

1.07 0.98 4.76, s 4.59, d (1.5)

δC 156.6 101.8 123.3 122.3 113.6 157.3 98.9 157.0 130.1 104.5 158.2 118.1 158.2 104.5 23.6 52.1 150.8 32.5 33.5 77.2

3

δH (J in Hz)

δC

6.82, sa 7.44, d (8.5) 6.83, dd (8.5, 2.0)a 6.99, d (2.0)

6.53, d (2.5) 6.67, 2.98, 2.88, 1.42,

d (2.5) dd (14.5, 7.5) dd (14.5, 7.5)a ma

3.48, d (5.0) 1.59, 1.37, 1.06, 0.98,

m ma ma m

157.2 105.8 122.8 122.5 113.6 157.4 99.0 157.0 133.3 121.5 160.4 101.1 157.5 110.0 24.2

δH (J in Hz) 6.80, sa 7.42, d (8.5) 6.82, dd (8.5, 2.0)a 6.98, d (2.0)

6.54, d (2.5) 6.80, d (2.5)a 3.48, d (6.5)

55.3 46.8 86.8

5.17, m

26.8 40.5

2.05, ma 1.97, m 1.57, ma

87.6

3.97, t (6.0)

1.69, s

41.7 27.9 19.3

0.88, s

24.6

109.3

0.61, s

26.2

4.85, t (1.0) 4.70, s 1.66, s

1.09, s 3.84, s

19.3 56.4

3.83, s

δC 155.8 106.3 123.0 122.5 113.6 157.1 98.9 157.1 133.0 120.8 160.6 100.9 157.7 108.4 26.8 125.6 135.6 16.9 37.0 35.1 75.8 149.9 110.8 18.4

56.6

Overlapped signals.

2.3.7. Compound 7 Yellow amorphous powder; [α]25D 0 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 214 (4.07), 309 (3.87) nm; IR (KBr) νmax 3451, 1636, 1384, 1149, 1114, 1078, 950 cm− 1; 1H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 3; HRESIMS m/z: 449.1938 [M + Na]+ (calcd for C24H28O5Na, 449.1935). 2.3.8. Compound 8 Yellow amorphous powder; [α]25D 2.2 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 215 (4.07), 310 (3.88) nm; IR (KBr) νmax 3452, 1633, 1440, 1384, 1147, 1115, 1012, 950 cm−1; 1H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 3; HRESIMS m/z: 463.2090 [M + Na]+ (calcd for C26H32O6Na, 463.2091). 2.3.9. Compound 9 Yellow amorphous powder; [α]25D 0 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 203 (sh) (3.95), 219 (4.01), 316 (4.06), 330 (3.99) nm; IR (KBr) νmax 3449, 1629, 1440, 1383, 1147, 1113, 1076, 1014, 949, 902, 823 cm− 1; 1H (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 3; HRESIMS m/z: 393.1711 [M − H]− (calcd for C24H25O5, 393.1707).

2.4. Chiral HPLC analysis of 1–9 Compounds 1–9 were analyzed by a Chiralpak AD-H column (detection at 230 nm, eluted with a mixture of n-hexane and isopropanol at different flow rates). Compounds 1–3, 5 and 7–9 were performed on chiral HPLC using n-hexane-isopropanol (70:30) as eluent at a flow rate of 1 ml/min, respectively; compound 4 was separated by chiral HPLC using n-hexaneisopropanol (65:35) as eluent at a flow rate of 0.6 ml/min; compound 6 was separated by chiral HPLC at a flow rate of 0.8 ml/min with n-hexane-isopropanol (70:30).

2.5. Absolute configuration of C-7″ in 3a, 3b and 8 The in situ formed [Rh2(OCOCF3)4] complex method was used according to the published procedure [19,20]. Compound 3a (3b or 8) (0.3 mg) was dissolved in dried solution of the dirhodium trifluoroacetate [Rh2(OCOCF3)4] complex (1.0 mg) in CH2Cl2 (600 μL). After mixing, the first CD spectrum was recorded immediately, and the time evolution was monitored until stationary (about 10 min). The inherent CD spectrum was subtracted. The sign of the E band at around 350 nm in the induced CD data was correlated to the absolute configuration of the secondary alcohol [19,20].

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Table 2 1 H (500 MHz) and

13

C NMR (125 MHz) Data of Compounds 4–6 in acetone-d6. 4

Position 2 3 3a 4 5 6 7 7a 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″a 5″b 6″a 6″b 7″ 8″ 9″a 9″b 10″ a

5

δH (J in Hz) 6.90, sa 7.37, d (8.0) 6.79, dd (8.0, 2.0) 6.95, d (2.0)

6.91, sa

a

6.91, s 3.40, d (7.0) 5.35, m 1.79, 2.05, 1.97, 1.59,

s ma ma ma

3.97, t (6.0) 4.86, d (1.0) 4.71, s 1.67, s

δC 156.4 102.0 123.2 122.3 113.6 157.2 98.9 157.0 130.4 104.4 157.7 116.9 157.7 104.4 23.6 124.3 135.5 16.9 37.1 35.2 75.8 149.9 110.8 18.4

6

δH (J in Hz)

δC

6.80, sa 7.42, d (8.0) 6.82, dd (8.0, 2.0)a 6.98, d (2.0)

6.50, d (2.5) 6.74, d (2.5) 3.52, d (6.0) 5.23, m 1.69, 2.25, 2.00, 1.64, 1.32, 3.24,

s m m m m m

156.1 106.1 123.1 122.5 113.6 157.1 98.9 157.1 133.3 119.2 158.1 104.5 157.4 108.4 26.8 125.7 135.9 17.1 38.2 31.3

δH (J in Hz) 6.90, sa 7.37, d (8.5) 6.79, dd (8.5, 2.0) 6.95, d (2.0)

6.91, sa

6.91, sa 3.40, dd (7.0, 4.0) 5.36, m 1.79, 2.25, 2.00, 1.66, 1.34, 3.25,

s m m m m m

δC 156.4 102.0 123.3 122.4 113.7 157.3 99.0 157.2 130.5 104.6 157.9 117.1 157.9 104.6 23.6 124.3 135.9 17.0 38.4 31.5

1.09, sa

79.1 73.4 26.8

1.09, sa

79.3 73.4 26.5

1.09, sa

25.6

1.09, sa

25.6

Overlapped signals.

2.6. Absolute configurations of each peak in the chromatograph of 5 and 6

performed in triplicate. 1-Deoxynojirimycin and genistein were used as positive controls [24,25].

Snatzke's method was used according to published literature [21,22]. DMSO, spectroscopy grade, was dried with 4 Å molecular sieves, and mixtures of 1:1.3 diol/Mo2(OAc)4 were submitted to CD measurement at the concentration of 0.4 mg/mL for 5 (6). After mixing, the first CD was recorded at once, and the time evolution was surveyed until stationary (about 30–60 min). The inherent CD spectrum was subtracted. The absolute configuration of the 7″,8″-diol moiety was determined by the sign at around 310 nm in CD spectra observed [21,22].

2.8. PTP1B inhibitory assay

2.7. α-Glucosidase inhibitory assay The inhibitory effect on α-glucosidase was measured using the spectrophotometric method described previously with slight modifications [23]. The assay was performed in a 96 well microplate. The α-glucosidase (3.0 U/ml) and substrate (1.0 mM p-nitrophenyl-α-D-glucopyranoside) were dissolved in 50 mM pH 6.8 sodium phosphate buffer. The inhibitor was preincubated with α-glucosidase at 37 °C for 30 min, and then the substrate was added to the reaction mixture. After 30 min of incubation at 37 °C, the amount of released p-nitrophenyl was measured in terms of the absorbance at 405 nm using a microplate reader. The percent inhibition (%) was obtained using the following equation: Inhibition (%) = (Ac−As) / Ac × 100, where Ac is the absorbance of the control, and As is the absorbance of the sample. The IC50 value was calculated from three independent assays,

The PTP1B inhibitory assay was performed in a 96 well microplate according to a published procedure with slight modifications [15,26]. The enzyme activity was measured in a reaction mixture containing 2 mM p-nitrophenyl phosphate (pNPP) and PTP1B (0.1 μg) in 50 mM citrate, pH 6.0, 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT). The final volume of the reaction mixture was 200 μL. The reaction was initiated by the addition of PTP1B, incubated at 37 °C for 30 min, and terminated by adding 20 μL of 10 M NaOH. The amount of released p-nitrophenyl was estimated by measuring the absorbance at 405 nm using a microplate reader. The nonenzymatic hydrolysis of 2 mM pNPP was corrected by measuring the increase in the absorbance at 405 nm obtained in the absence of PTP1B enzyme. RK-682 was used as a positive control [15,26]. 3. Results and discussion Compound 1 was obtained as light brown amorphous powder. The molecular formula was determined to be C24H26O5 by HRESIMS (m/z 395.1856 [M + H]+, calcd for 395.1853). Absorption maxima in the UV spectrum of 1 were observed at λmax 203 (sh), 218, 318, and 332 nm, which was indicative of a 2-arylbenzofuran [27]. Its IR spectrum showed absorptions for OH (3444 cm−1) and aromatic (1634 and 1489 cm−1) moieties. In the 1H NMR spectrum (Table 1), one

Y.-L. Zhang et al. / Fitoterapia 92 (2014) 116–126 Table 3 1 H (500 MHz) and

13

C NMR (125 MHz) Data of Compounds 7–9 in acetone-d6. 7

Position 2 3 3a 4 5 6 7 7a 1′ 2′ 3′ 4′ 5′ 6′ 1″a 1″b 2″ 3″ 4″ 5″a 5″b 6″a 6″b 7″ 8″ 9″ 10″ MeO-3′ MeO-8″ a

121

8

δH (J in Hz) 6.81, sa 7.42, d (8.0) 6.82, dd (8.0, 2.0)a 6.98, s

6.54, d (2.5) 6.80, d (2.5)a 3.48, d (6.5) 5.18, m 1.69, s 2.23, m 2.00, m 1.65, m 1.32, m 3.25, m a

1.09, s 1.09, sa 3.83, s

δC 155.8 106.4 123.1 122.5 113.6 157.2 98.9 157.1 133.1 120.8 160.6 100.9 157.7 108.4 26.8 125.6 135.9 17.0 38.2 31.3 78.9 73.2 26.5 25.5 56.6

9

δH (J in Hz)

δC

6.81,sa 7.42, d (8.5) 6.82, dd (8.0, 2.0)a 6.98, d (2.0)

6.54, d (2.0) 6.81, d (2.0)a 3.48, d (6.0) 5.18, m 1.69, s 2.23, m 2.00, m 1.63, m 1.29, m 3.32, m 1.07, s 1.04, s 3.83, s 3.13, s

155.8 106.4 123.1 122.5 113.6 157.2 98.9 157.1 133.1 120.8 160.6 100.9 157.7 108.4 26.8 125.6 135.9 17.0 38.1

δH (J in Hz) 7.00, s 7.39, d (8.5) 6.80, dd (8.5, 2.0)a 6.97, d (2.0)

6.81, d (1.5)a 6.91, d (1.5) 3.00, dd (17.0, 5.5) 2.60, dd (17.0, 8.0) 3.91, m

δC 156.0 102.3 123.2 122.4 113.7 157.3 99.0 157.2 131.2 110.1 156.2 105.9 157.4 103.8 27.9

1.24, s 1.73, m

68.5 79.9 18.7 39.3

30.9

2.22, m

22.8

76.8 78.4 21.3 20.8 56.6 49.7

5.16, t (7.0) 1.67, s 1.62, s

126.0 132.3 26.3 18.2

Overlapped signals.

set of ABX coupling system at δH 7.36 (1H, d, J = 8.5 Hz), 6.94 (1H, d, J = 2.0 Hz), and 6.78 (1H, dd, J = 8.5, 2.0 Hz), and the signal δH 6.87 (1H, d, J = 0.5 Hz), along with the singlet at δH 6.85 (2H, s) were assigned to the 2-arylbenzofuan moiety. Two proton signals at δH 4.76 (1H, s) and 4.59 (1H, d, J = 1.5Hz) and a signal at δH 3.45 (1H, dd, J = 8.5, 2.0 Hz) suggested that a terminal methylene group and an oxygen-bearing methine group were present in the cyclized geranyl group (Table 1) [28]. These revealed the presence of a monoterpene unit of a cyclized group in 1, and compound 1 was a hybrid of a 2-arylbenzofuran and a cyclized monoterpene. The 1H and 13C NMR data of 1 (Table 1) were accomplished by a combination of HSQC, HMBC and ROESY experiments. The HMBC correlations from the methylene signals at δH 3.19 (H-1″a) and 2.89 (H-1″b) to C-3′, C-5′ (δC 158.2, overlapped), and C-4′ (δC 118.1) indicated the cyclized geranyl group to be linked to the C-4′. In addition, a hydroxyl group was located at C-6″, which was suggested from the HMBC correlations between δH 1.07 (3H, s, H-8″) and 0.98 (3H, s, H-9″) and δC 77.2 (C-6″). The relative configuration of 1 was established by the analysis of the ROESY spectrum, which showed a ROESY correlation between the axial protons H-2″ and H-6″ (Fig. 2). Thus, the structure of 1 was assigned as 4′-(6,6-dimethyl-5-hydroxyl-2-methylenecyclohexylmethyl)-3′,5′,6- trihydroxy-2-arylbenzofuran. Compound 2 was isolated as white amorphous powder. Its HRESIMS at m/z 407.1866 [M-H]− (Calcd for 407.1864), consistent with the molecular formula as C25H28O5, corresponding to 12° of unsaturation. Similar to 1, the 1H NMR spectrum of 2 (Table 1), showed that compound 2 also

contained the 2-arylbenzofuan moiety. Additionally, two groups of unequivalent proton signals at δH 6.53 (1H, d, J = 2.5 Hz, H-4′) and 6.67 (1H, d, J = 2.5 Hz, H-6′) suggested 2-aryl was substituted asymmetrically. The presence of a methoxy group was evidenced from the three proton singlet at δH 3.84. It was linked to position C-3′ of the 2-arylbenzofuan nucleus which was indicated by the HMBC correlations between H3-12″ and C-3′. The absence of carbon resonances appearing at field lower than 90 ppm indicated that the C10 side chain of 2 was saturated. Considering the 10 units of unsaturation required for 2-arylbenzofuan nucleus, the other two unsaturations were attributed to two rings in the side chain. These two rings consist of a 7-oxo-[2.2.1]system, as determined by 1H–1H COSY and HMBC spectroscopy (Fig. 3). This was further confirmed by comparison of the NMR data of analogous partial structure in the parvixanthone I [29]. The C-4″/C-5″/C-6″ connectivity was deduced from the 1H-1H COSY cross-peaks between H-4″/H-5″ and H-5″/H-6″. From this partial structure, the six-membered ring from C-2″ to C-7″ could be confirmed from the following HMBC correlations: Me-9″/C-2″,3″,4″,10″; Me-10″/C-2″,3″,4″,9″; and Me-11″/C-2″,6″,7″. The C-4″ and C-7″ positions were further deduced to be connected by an oxygen bridge (O-8″) by the key HMBC correlation of H-4″/C-7″ and the fact that both C-4″ (δC 86.8) and C-7″ (δC 87.6) were oxygenated. Thus, a cyclized monoterpenoid substitution in 2 was determined as a 7-oxo-[2.2.1]-system, which was linked to position C-2′ of the 2-arylbenzofuan nucleus, as indicated by the HMBC correlations between H-1″/C-1′,2′,3′. Therefore, the structure of 2 was

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Fig. 2. Key HMBC and ROESY correlations of 1.

determined as 2′-(1,3,3-trimethyl-7- oxabicyclo[2.2.1]hept-2ylmethyl)-3′-methoxy-5′,6-dihydroxy-2-arylbenzofuran. Due to the optical rotation of 2 was measured to be 0, 2 was a racemic mixture of enantiomers, which was further confirmed by chiral HPLC analysis (Fig. 4). Compound 3 was obtained as yellow amorphous powder that gave a molecular formula of C25H28O5, as deduced by HRESIMS. Compared to the 1H and 13C NMR spectrum of compound 2 (Table 1), compound 3 was assigned to contain the same 3′-methoxy-5′,7-dihydroxy-2-arylbenzofuran nucleus. Signals in the up field of the 1H NMR spectrum were deduced to be a changed geranyl group, one of whose double bonds was hydrated. The 13C NMR signal at δC 75.8 further supported the presence of a hydroxy group. In the HMBC spectrum (Fig. 3), long-range correlations of H-9″/C-7″,10″ and H-7″/C-5″,9″,10″, demonstrated that the geranyl group was replaced by a 7″-hydroxy-3″,8″-dimethylbut-2″,8″-dioctenyl group. Furthermore, H-1″ showed long-range correlations with C-1′ and C-3′, supporting that the changed geranyl group was located at C-2′. Thus, the structure of 3 was elucidated as 2′-(6-hydroxy-3,7-dimethyl-2,7-octadien-1-yl)-3′-methoxy5′,6-dihydroxy-2-arylbenzofuran. Although there was only one chiral center (C-7″) in 3, the optical activity was undetectable. Compound 3 was supposed to be a racemic mixture, which was further confirmed by chiral HPLC analysis (Fig. 4). Finally, a pair of enantiomers

Fig. 3. (A) Key HMBC (H → C) and 1H-1H COSY (thick black lines) correlations of 2. (B) Key HMBC correlations (H → C) of 3. (C) Key HMBC correlations (H → C) of 5. (D) Key HMBC correlations (H → C) of 9.

3a (1.1 mg) and 3b (1.1 mg) were successfully obtained by chiral HPLC, and the measured specific rotation values of them were −6.6° and + 6.6°, respectively. The absolute configuration of each enantiomer was determined by the induced CD of the in situ formed [Rh2(OCOCF3)4] complex [19,30]. The Rh complex of 3a exhibited a positive E band at around 350 nm, while that of 3b exhibited a negative E band at around 350 nm (Fig. 5). According to the bulkiness rule [19,20,30], the absolute configurations at C-7″ of 3a and 3b were assigned as R and S, respectively. Compound 4 was isolated as yellow, amorphous powder, and its molecular formula, C24H26O5, was assigned by HRESIMS. Comparison of the 1H and 13C NMR spectroscopic data of 4 and 3 indicated the changed geranyl group at C-4′ and the absence of MeO-3′ in 4 (Table 2). Thus, the structure of 4 was determined as 4′-(6- hydroxy-3,7-di-methyl-2,7octadien-1-yl)-3′,5′,6-trihydroxy-2-arylbenzofuan. Lack of any optical rotation indicated that 4 was also a racemic mixture, confirmed by chiral HPLC analysis (Fig. 4). Compound 5, yellow, amorphous powder, was assigned a molecular formula of C24H28O6 by HRESIMS. Compared to the 1 H and 13C NMR spectra of 3 (Table 2), compound 5 was assigned to contain the 3′,5′,7-trihydroxy-2-arylbenzofuan nucleus and a geranyl group, one of whose double bonds was hydroxylated. The 13C NMR signals at δC 73.4 and 79.1 further supported the presence of two hydroxy groups. The structure was confirmed with the aid of HSQC and HMBC experiments. In the HMBC spectrum (Fig. 3), the correlations of H-9″/ C-7″,8″,10″ and H-7″/C-5″,6″,8″,9″,10″, demonstrated that the geranyl group was 7″,8″-dihydroxylated. Furthermore, H-1″ showed correlations with C-1′, C-2′, and C-3′, indicating that the changed geranyl group located at C-2′. Thus, the structure of 5 was elucidated as 2′-(6,7- dihydroxy-3,7-dimethyl-2octen-1-yl)-3′,5′,6-trihydroxy-2-arylbenzofuan. Compound 6 was isolated as yellow amorphous powder. Its molecular formula of C24H28O6 was established by HRESIMS. Its 1 H NMR spectrum was similar to that of compound 5, except for a characteristic signal of symmetrically substituted 2-aryl at δH 6.91(2H, s, H-2′,6′) instead of signals of asymmetrically substituted 2-aryl. It was confirmed by 13C NMR signals at δC 157.9(C-3′, C-5′) and 104.6(C-2′, C-6′) (Table 2). Thus, the structure of 6 was assigned as 4′-(6,7-dihydroxy-3,7dimethyl-2- octen-1-yl)-3′,5′,6-trihydroxy-2-arylbenzofuan. Although the optical rotations of 5 and 6 was measured to be −8.9° and −10.4°, respectively, the optical rotation values were much smaller than the values of those compounds containing the analogous partial structure [31]. Therefore, 5 and 6 were deduced to be racemic mixtures with different ratios of S and R enantionmers, respectively. The negative optical rotations of 5 and 6 in MeOH indicated that the S enantiomer predominated in 5 and 6 [31]. It was further confirmed using an in situ dimolybdenum CD method [21,22]. According to the empirical rule proposed by Snatzke, the positive sign at around 310 nm of 5 and 6 indicated that the S enantiomer predominated in 5 and 6 (Fig. 6) [21,22]. Compound 7, which was obtained as yellow, amorphous powder, was assigned as C25H30O6 on the basis of its positive HRESIMS results (m/z 449.1938 [M + Na]+, calcd for 449.1935). Its 1H NMR spectrum was also similar to that of compound 5, except for an additional methoxy group at δH 3.83 (Table 3). Furthermore, the HMBC correlation between H-11″

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123

Fig. 4. The chromatographs of compounds 1–9 (1–9) on a Chiralpak AD-H column.

and C-3′ indicated that the methoxy group was linked to position C-3′. Thus, the structure of 7 was assigned as 2′(6,7-dihydroxy-3,7-dimethyl- 2-octen-1-yl)-3′-methoxy-5′,6dihydroxy-2-arylbenzofuran. Lack of any optical rotation

indicated that 7 was also a racemic mixture, which was further confirmed by chiral HPLC analysis (Fig. 4). Compound 8 was obtained as yellow amorphous powder and analyzed for a molecular formula of C26H32O6 by

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(Fig. 4). The results showed that there were two peaks with equal peak area in each chromatograph of compounds 2–4, 7 and 9, while only one peak in those of 1 and 8, respectively. The chromatographs of 5 and 6 showed that the S enantiomer predominated in 5 and 6 with different ratios of S and R enantionmers (1.4:1 for 5a and 5b, and 2:1 for 6a and 6b). The structure of compound 7 was similar to that of 5 and 6, so peak 1 was assigned as S enantiomer (7a), and peak 2 was assigned as R enantiomer (7b) in the chromatograph of 7. In addition, the structure of compound 4 was similar to that of 3, so peak 1 was assigned as R enantiomer (4a), and peak 2 was assigned as S enantiomer (4b) in the chromatograph of 4. Interestingly, these 2-arylbenzofuran enantiomers with the substituent group at C-4′ were found to be more difficult to be separated by a Chiralpak AD-H column than those with the substitution at C-2′ (Fig. 4). The absolute configurations of each peak in the chromatographs of 2 and 9 remained unknown. Unfortunately, after the pharmacological assays, there were not enough materials to obtain each enantiomer from these racemic mixtures except for 3. Four known compounds were identified as albafuran A (10) [32], albafuran B (11) [32], mulberrofuran A (12) [33], and moracin I (13) [34] by comparison of their spectroscopic data with those reported. All isolated compounds were tested for inhibitory activities against α-glucosidase. As shown in Table 4, all the compounds investigated apart from 8, 9 and 12 exhibited a certain degree of α-glucosidase inhibitory activity. Compounds 1 and 2, two Fig. 5. A: CD spectrum of the Rh complex of 3a with the inherent CD spectrum subtracted; B: CD spectrum of the Rh complex of 3b with the inherent CD spectrum subtracted.

HRESIMS. In the 1H NMR spectrum (Table 3), besides proton signals of two methoxy groups at δH 3.83 and 3.13, it was similar to compound 5. In addition, the HMBC correlations between δH 3.83 and δC 160.6(C-3′) and between δH 3.13 and δC 78.4(C-8″) suggested that the hydroxyl groups at C-3′ and C-8″ were methylated. The absolute configuration at C-7″ was determined by the induced CD of the in situ formed [Rh2 (OCOCF3)4] complex [19,20]. The Rh complex of 8 exhibited a negative E band at around 350 nm, and the absolute configuration at C-7″ was assigned as S according to the bulkiness rule (Fig. 7) [19,20,30]. Thus, the structure of 8 was determined as 2′-[(6S)-6-hydroxy-7-methoxy-3,7-dimethyl-2-octen- 1-yl]-3′methoxy-5′,6-dihydroxy-2-arylbenzofuran. Compound 9 was obtained as yellow amorphous powder, with the molecular formula of C24H26O5, as deduced by HRESIMS. Comparison of the 1H and 13C NMR spectroscopic data of 9 and 5 indicated that their structural difference was the side chain (Table 3). The 1H NMR signal at δH 3.91 (1H, m, H-2″) and two 13C NMR signals at δC 68.5(C-2″) and 79.9(C-3″) were assigned to a 2″,3″-epoxy group, which was further confirmed by the HMQC correlation of H-2″/C-2″ and the HMBC correlations of H-2″/C-2′,1″,3″,4″,5″ (Fig. 3). Thus, the structure of 9 was assigned as 2′-[[3-methyl-3-(4-methyl-3-penten-1-yl)-2oxiranyl]methyl]-3′,5′,6-trihydroxy-2-arylbenzofuran. The absence of ROESY correlation between H-2″ and H3-4″ indicated a 2″,3″-trans-configuration, while lack of any optical rotation points to a racemic mixture of enantiomers. Compounds 1–9 were analyzed by a Chiralpak AD-H column using n-hexane-isopropanol mixtures as eluents

Fig. 6. A: CD spectrum of 5 in DMSO solution of Mo2(OAc)4 with the inherent CD spectrum subtracted; B: CD spectrum of 6 in DMSO solution of Mo2(OAc)4 with the inherent CD spectrum subtracted.

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Table 5 Inhibition effects of isolated compounds 1 and 9–13 against PTP1Ba. PTP1Bb Compound

IC50 ± SD (μM)

1 9 10 11 12 13 RK-682c

38.1 35.1 7.9 8.9 13.3 21.3 3.2

a b

Fig. 7. CD spectrum of the Rh complex of 8 with the inherent CD spectrum subtracted.

monoterpenoid 2-arylbenzofurans, showed the highest inhibitions at 11.9 ± 1.3 and 21.5 ± 0.9 μM, respectively. Comparing the inhibitory activities of 3a, 3b and 4, 5 and 7, and 10 and 12, respectively, it seemed that 3′-methoxyl substitution on the B ring would decrease the inhibitory activity, which was similar to that about flavonoids in the earlier reports [35,36]. The inhibitory effect of 3a (R enantiomer) was not significantly different from that of 3b (S enantiomer). In addition, compounds 8–12 showed obviously weaker inhibitory activities than those compounds (3–7) containing more hydroxyl groups in the geranyl group. It seemed that increasing the number of hydroxyl groups in the straight side chain of geranylated 2-arylbenzofurans would improves potential inhibitory effects against α-glucosidase. Compounds 1–13 were also screened for their inhibitory activity against PTP1B (Table 5). Of the 14 compounds screened, albafuran A (10) and albafuran B (11) exhibited strong inhibitory effects against PTP1B, with IC50 values of 7.9 ± 0.4 and 8.9 ± 1.1 μM, respectively. Albafuran A (10) showed almost the same inhibitory activity as the earlier report [15]. Compounds 12 and 13 exhibited moderate inhibition, with IC50 values of 13.3 ± 0.7 and 21.3 ± 1.5 μM, respectively. Compounds 1 and 9 exhibited weak inhibition, with IC50 values of 38.1 ± 1.8 and 35.1 ± 2.1 μM, respectively. However, other compounds containing hydroxyl groups in the straight side chain showed weaker inhibitions, with IC50 values of more than 50 μM. It seemed that increasing the lipophilicity of straight side chain leads to stronger inhibitory activities against PTP1B [15].

Table 4 Inhibition effects of isolated compounds against α-glucosidase. Compound

IC50 ± SD (μM)a

Compound

IC50 ± SD (μM)

1 2 3a 3b 4 5 6 7

11.9 21.5 85.3 67.2 33.1 21.9 29.5 101.5

8 9 10 11 12 13 1-DNJb Genisteinb

N150 N150 123.6 ± 7.1 131.9 ± 4.5 N150 49.5 ± 2.8 234.4 ± 19.4 17.8 ± 1.1

a b

± ± ± ± ± ± ± ±

1.3 0.9 2.7 3.2 1.9 1.7 3.4 6.5

Values present mean ± SD of triplicate experiments. Positive controls.

c

± ± ± ± ± ± ±

1.8 2.1 0.4 1.1 0.7 1.5 0.3

IC50 values of other compounds were more than 50 μM. Values present mean ± SD of triplicate experiments. Positive control.

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