α-Glucosidase Inhibitory Prenylated Anthranols from Harungana

Jan 4, 2016 - Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, Un...
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α‑Glucosidase Inhibitory Prenylated Anthranols from Harungana madagascariensis Oluwatosin O. Johnson,†,‡ Ming Zhao,*,† Jordan Gunn,† Bernard D. Santarsiero,§ Zhi-Qi Yin,†,⊥ Gloria A. Ayoola,‡ H. A. B. Coker,‡ and Chun-Tao Che† †

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States ‡ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos, CMUL Campus, Lagos 100254, Nigeria § Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60607, United States ⊥ Department of Natural Medicinal Chemistry & State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Four new prenylated anthranols, harunganols C−F (1− 4), along with kenganthranol A (5), harunganin (6), and ferruginin A (7), were identified from the leaves of Harungana madagascariensis. The structures of compounds 2, 5, and 7 were confirmed by single-crystal Xray diffraction analysis. Compound 1 is a unique symmetrical anthranol dimer connected via a CH2 group. Compound 4 possesses a unique C10 hemiketal group. All anthranols were evaluated for their αglucosidase inhibitory activities. They displayed a higher potency compared to acarbose except for 3 and 4. In particular, harunganol C (1) showed an IC50 value of 1.2 μM.

(1−4), along with kenganthranol A (5), harunganin (6), and ferruginin A (7), were purified from the n-hexane fraction. Harunganol C (1) was obtained as an orange, amorphous powder. The HRESIMS data showed a deprotonated molecular ion at m/z 931.5167 [M − H]− (calcd for C61H71O8−, 931.5154), suggesting a molecular formula of C61H72O8 with 26 indices of hydrogen deficiency when the 13C NMR data were taken into consideration. The 1H NMR spectrum displayed signals for phenolic hydroxy groups at δH 15.76 (9- and 9′OH), 11.92 (3- and 3′-OH), and 10.01 (8- and 8′-OH) and singlets for methyls at δH 2.41 (CH3-26 and -26′), 1.81 (CH324 and -24′), 1.68 (CH3-25 and -25′), 1.53 (CH3-19 and -19′), 1.46 (CH3-20 and -20′), 1.43 (CH3-14 and -14′), and 1.18 (CH3-15 and -15′) (Table 1). In the 13C NMR spectrum, 31 carbon signals were observed corresponding to seven methyls, four methylenes, five methines, three oxygenated tertiary carbons, a keto-carbonyl carbon, and 11 quaternary carbons. All proton signals could be assigned to the attached carbons through an HSQC experiment (Table 1). On the basis of the 1 H− 1H COSY data, the following spin systems were unambiguously established: H-11,11′ (δH 2.99, 2.54)/H12,12′ (δH 4.39); H-16,16′ (δH 3.06, 2.62)/H-17,17′ (δH

Harungana madagascariensis Lam. ex Poir. (Hypericaceae) is a medicinal plant native to Africa. Among the Yoruba tribe in Nigeria, the plant, known as “Aranje”, is used for the treatment of a wide spectrum of human and veterinary diseases. In Ghana, the stem bark is employed in treating skin diseases and as dressing material for wounds.1,2 The red juice obtained from the leaves and stem bark is reputed for arresting postpartum bleeding in Sierra Leone, while the unopened buds are well known in Liberia for treating puerperal infection.3 Phytochemical studies on H. madagascariensis have led to the isolation of anthraquinones, xanthones, flavonoids, coumarins, and prenylated anthranols, showing antimicrobial, antiplasmodial, and antioxidative activities.4−8 Particularly, prenylated anthranols from the stem bark were reported to show α-glucosidase inhibitory activity.9 The investigation of the leaf extract of H. madagascariensis led to the identification of four new and three known prenylated anthranols. Herein the isolation, structural elucidation, and α-glucosidase inhibitory activities of the anthranols are described.

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RESULTS AND DISCUSSION The MeOH extract of the leaves of H. madagascariensis was successively partitioned into n-hexane, EtOAc, and n-BuOH fractions. Four new prenylated anthranols, harunganols C−F © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 17, 2015

A

DOI: 10.1021/acs.jnatprod.5b00924 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data for Compound 1a position 1, 1′ 2, 2′ 2,2′CH2 3, 3′ 4, 4′

δC, type 191.2, C 114.5, C 16.7, CH2

δH (J in Hz)

3.47, s

180.6, C

position

δC, type

13, 13′ 14, 14′ 15, 15′

134.8, C 18.0, CH3 25.8, CH3

16, 16′

40.6, CH2

50.3, C

4a, 4′a 5, 5′ 6, 6′ 7, 7′ 8, 8′

139.3, 118.9, 141.9, 122.5, 153.9,

8a, 8′a

110.9, C

22, 22′

122.3, CH

9, 9′ 9a, 9′a 10, 10′ 10a, 10′a 11, 11′

161.6, 107.9, 115.9, 136.4,

23, 24, 25, 26,

131.7, 18.1, 25.7, 20.6,

12, 12′

C CH C C C

17, 18, 19, 20, 21,

C C CH C

41.5, CH2

117.9, CH

2.99, dd (9.2, 13.4) 2.54, dd (4.2, 13.4) 4.39, br m

17′ 18′ 19′ 20′ 21′

23′ 24′ 25′ 26′

118.2, 134.6, 18.0, 25.8, 25.0,

CH C CH3 CH3 CH2

C CH3 CH3 CH3

δH (J in Hz) 1.43, s 1.18, s 3.06, dd (8.1, 14.0) 2.62, dd (5.9, 14.0) 4.53, br m 1.53, s 1.46, s 3.48, partial overlap 5.16, dd/tlike (7.0, 7.0) 1.81, s 1.68, s 2.41, s

3-OH, 3′-OH

11.92, s

8-OH, 8′-OH

10.01, s

9-OH, 9′-OH

15.76, s

a

The coupling constants (J) are in parentheses and reported in Hz; chemical shifts (δ) are given in ppm.

4.53); and H-21,21′ (δH 3.48)/H-22,22′ (δH 5.16) (Figure 1). When the 13C NMR spectroscopic data of 1 were compared to those of psorantin,10 high similarities were revealed except for those of two additional prenyl groups in 1, indicating 1 was a symmetrical anthranol dimer linked via a CH2 bridge. Detailed interpretation of the HMBC data then led to the proposed structure of 1. Thus, the CH2 group at δH 3.47 (2H, s) showed HMBC correlations to carbons at δC 114.5 (C-2,2′), 180.6 (C3,3′), and 191.2 (C-1,1′). Both H-11,11′ and H-16,16′ correlated to C-3,3′, C-4,4′ (δC 50.3) and C-4a,4′a (δC 139.3), supporting the presence of two prenyl groups at C4,4′. The H-21,21′ (δH 3.48) showed long-range correlations to C-6,6′ (δC 141.9), C-7,7′ (δC 122.5), and C-8,8′ (δC 153.9), leading to the assignment of an additional prenyl group at C7,7′. Consequently, the structure of 1 was established as shown and given the trivial name harunganol C. Harunganol D (2) was obtained as a yellow, amorphous powder. A molecular formula of C25H28O6 with 12 indices of hydrogen deficiency was suggested by HRESIMS (m/z 425.1935 [M + H]+, calcd for C25H29O6+, 425.1959), when the 13C NMR data were taken into consideration. The 1H NMR spectrum displayed resonances for two phenolic hydroxy groups at δH 12.23 (s, 8-OH) and 12.21 (s, 1-OH) and five

Figure 1. 1H−1H COSY and selected HMBC correlations for 1−4.

methyl singlets at δH 2.36 (CH3-21), 1.88 (CH3-19), 1.70 (CH3-20), 1.39 (CH3-14), and 1.30 (CH3-15), respectively (Table 2). The 13C and DEPT spectra revealed 25 carbons corresponding to five methyls, two methylenes, five methines, four oxygenated tertiary carbons, a carbonyl carbon, and eight quaternary carbons (Table 3). The 13C NMR spectroscopic data suggested a structure similar to that of kenganthranol C.9 Interpretation of 1H−1H COSY, HSQC, and HMBC spectra (Figure 1) led to the elucidation of the 2D structure of 2 as shown, in which a six-membered ring was generated via a C3,13B

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Table 2. 1H (400 MHz) NMR Spectroscopic Data for Compounds 2−4 (δ in ppm) 2a position 2 7 10 11 12 14 15 16 17 19 20 21 1-OH 8-OH 10-OH 12-OH

3a

δH (J in Hz)

δH (J in Hz)

6.26, s 6.79, s 5.84, d (9.2) 3.48, dd (5.2, 16.4) 2.83, dd (7.6, 16.4) 3.89−3.93,c m 1.39, s 1.30, s 3.89, dd (7.5, 17.0) 3.42, dd (5.2, 17.0) 5.13, m 1.88, s 1.70, s 2.36, s 12.21, s 12.23, s 4.56, d (9.2) 4.42, d (5.2)

6.26, s 6.80, s 5.72, d (9.1) 3.41−3.48c 3.32, dd (7.2, 16.0) 4.86, dd (7.2, 9.5) 1.28,d s 1.24,d s 3.87, dd (8.1, 15.7) 3.37−3.45c 5.12, m 1.86, s 1.60, s 2.35, s 12.59, s 12.34, s 4.63, d (9.2) 3.79, s

4b δH (J in Hz) 6.36, s 6.77, s 3.50, dd (7.2, 16.7) 3.37, dd (9.5, 16.7) 4.71, dd (7.2, 9.5) 1.23,e s 1.35,e s 4.48, br d (10.0) 4.67c 1.09, s 1.46, s 2.40, s 12.82, s 12.10, s

a Data measured in acetone-d6. bData measured in CDCl3. cSignal was partially obscured. d,eData are interchangeable

Table 3. 13C (100 MHz) NMR Spectroscopic Data for Compounds 2−4 (δ in ppm) position 1 2 3 4 4a 5 6 7 8 8a 9 9a 10 10a 11 12 13 14 15 16 17 18 19 20 21

2a

3a

4b

δC, type

δC, type

δC, type

163.3, 104.5, 162.3, 113.2, 144.7, 132.1, 148.7, 119.6, 161.5, 113.2, 193.0, 109.3, 61.5, 141.2, 28.2, 69.3, 79.3, 25.9, 21.1, 27.7, 123.8, 132.3, 18.2, 25.7, 20.8,

C CH C C C C C CH C C C C CH C CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH3

166.4, C 97.6, CH 168.8, C 121.4, C 140.8,c C 132.3, C 148.5, C 119.7, CH 161.6, C 113.3, C 192.2, C 108.1, C 62.6, CH 140.7,c C 28.1, CH2 92.6, CH 71.4, C 25.5, CH3 25.9, CH3 27.8, CH2 124.0, CH 132.0, C 18.0, CH3 25.7, CH3 20.7, CH3

166.3, 99.5, 168.0, 119.7, 132.9, 121.9, 148.2, 120.0, 162.0, 109.8, 189.6, 108.9, 99.4, 137.7, 29.8, 91.3, 71.9, 24.5, 25.7, 63.6, 87.6, 78.3, 23.4, 29.9, 18.7,

C CH C C C C C CH C C C C C C CH2 CH C CH3 CH3 CH CH C CH3 CH3 CH3

Figure 2. Key NOESY correlations for compounds 2−4.

between H-11α (δH 3.48) and CH3-14; between H-11β (δH 2.83) and H-10 (δH 5.84); and between H-12 (δH 3.89−3.93) and CH3-14 and CH3-15. The relative configuration of compound 2 was thus determined as shown. Finally, the structure of 2 was confirmed by single-crystal X-ray diffraction (Figure 3) and given the trivial name harunganol D. Harunganol E (3) was obtained as a yellow, amorphous powder. The HRESIMS data showed a protonated molecular ion at m/z 425.1938 [M + H]+ (calcd for C25H29O6+, 425.1959), and 25 signals were observed in the 13C NMR spectrum, suggesting a molecular formula of C25H28O6 with 12 indices of hydrogen deficiency. The 13C NMR data of 3 were highly similar to those of kenganthranol C,9 except for the signals of C-4a, C-10, and C-10a. Using 1H−1H COSY, HSQC, and HMBC analyses (Figure 1), the 2D structure of 3 was determined as shown. Compared to kenganthranol C, 3 has a 10-OH instead of a 10-OCH3 in the structure. In the HMBC spectrum, H-12 (δH 4.86) correlated with C-3 (δC 168.8), supporting the presence of a C3,12-ethereal bridge. The observation of NOESY correlations between H-10 (δH 5.72) and H-11α (δH 3.32) and between H-12 and H-11β (δH 3.41− 3.48) suggested the presence of 10α-H and 12β-H, establishing the relative configuration of the molecule. Compound 3 was given the trivial name harunganol E. Harunganol F (4) was obtained as a yellow, amorphous powder. The HRESIMS displayed a protonated molecular ion at m/z 455.1686 [M + H]+ (calcd for C25H27O8+, 455.1700), suggesting a molecular formula of C25H26O8 with 14 indices of

a

Data measured in acetone-d6. bData measured in CDCl3. cData are interchangeable.

ether-type linkage. Indeed, a weak correlation from CH3-14 to C-3 (δC 163.2) was observed in the HMBC spectrum. The presence of the 10β-H and the 12α-H was determined on the basis of NOESY results (Figure 2), as shown by correlations C

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moiety was proposed based on the following evidence: HMBC correlation between H-17 (δH 4.67) and C-10 (δC 99.4); chemical shifts of C-10, C-17 (δC 87.6), and C-18 (δC 78.3); and the HRESIMS data of the compound. In the NOESY spectrum, CH3-20 (δH 1.46) correlated with H-11α (δH 3.37), supporting the signal assignment of CH3-20, which was spatially closer to H-11 than CH3-19. Furthermore, the following NOESY correlations were observed: H-16 (δH 4.48) with CH3-19 and CH3-21; H-11α with H-12 (δH 4.71) and H-17 (δH 4.67); and H-17 with CH3-20. Taken together, the α-orientations of H-12, H-16, and H-17 were proposed, and the relative configuration of the compound was thus established. Consequently, the structure of 4 was identified as shown and given the trivial name harunganol F. Compounds 5 and 6 were identified as kenganthranol A9 and harunganin,11 respectively, based on the NMR spectroscopic data. Kenganthranol A (5) has been previously isolated from the stem bark of H. madagascariensis. Single-crystal X-ray diffraction data of 5 confirmed the structure and further revealed it as a mixture of 10R- and 10S-enantiomers (Figure 2). Compound 7 was identified as ferruginin A5,11,12 by singlecrystal X-ray diffraction and HRESIMS. Compounds 1−7 were evaluated for α-glucosidase inhibitory activity (Table 4). Except for 3 and 4, all compounds are more Table 4. α-Glucosidase Inhibitory Activity of 1−7 α-glucosidase inhibition compound

IC50 ± SEM (μM)

1 2 3 4 5 6 7 acarbose

1.2 ± 0.05 36.9 ± 1.73 206.6 ± 15.43 116.7 ± 7.03 44.1 ± 5.83 37.7 ± 4.65 47% inhibition at 5 μM 119.7 ± 10.44

potent than the positive control acarbose. In particular, compound 1 displayed an IC50 value of 1.2 μM. Prenylated anthranols from the stem bark of H. madagascariensis were also previously reported to possess potent α-glucosidase inhibitory properties.9 These prenylated anthranols could be potential leads for the development of α-glucosidase inhibitors. In summary, seven prenylated anthranols, including the four new structures harunganols C−F (1−4), were identified from the leaves of H. madagascariensis. Harunganol C (1) is a unique symmetrical anthranol dimer connected via a methylene group. Harunganol F (4) possesses a unique hemiketal group at C-10. Harunganol C (1) exhibited potent α-glucosidase inhibitory activity with an IC50 value of 1.2 μM.

Figure 3. ORTEP representation of 2, 5, and 7 (the C atom labeling for 7 shown here is different from the atom numbering used for structures of 1−6).

hydrogen deficiency. The 1H NMR spectrum showed signals for two phenolic hydroxy groups at δH 12.10 (s, 8-OH) and 12.82 (s, 1-OH) and five methyls at δH 2.40 (CH3-21), 1.46 (CH3-20), 1.23 (CH3-14), 1.35 (CH3-15), and 1.09 (CH3-19), respectively (Table 2). The 13C and DEPT spectra exhibited 25 carbon signals corresponding to five methyls, a methylene, five methines, a dioxygenated secondary carbon, five oxygenated tertiary carbons, a carbonyl carbon, and seven quaternary carbons (Table 3). Based on the interpretation of 1H−1H COSY, HSQC, and HMBC data (Tables 2 and 3; Figure 1), compound 4 was identified to be a prenylated anthranol derivative. In the HMBC spectrum, H-12 (δH 4.71) correlated with C-3 (δC 168.0), allowing the establishment of a C3,12ethereal bridge. The presence of a unique C-10 hemiketal

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations at the sodium D line were measured with a PerkinElmer 241 digital polarimeter using a quartz cell with a path length of 100 mm at room temperature. Concentrations (c) are given in g/100 mL. IR spectra were measured on a Jasco Fourier Transform IR spectrometer (FT-IR model 410) loaded with OMNIC software. NMR spectra were recorded on a Bruker Advance-400 spectrometer. All chemical shifts were quoted on the δ scale in ppm using residual solvent as the internal standard (acetone-d6: 2.04 ppm for 1H NMR, 29.8 and 206.0 ppm for 13C NMR; CDCl3: 7.24 ppm for 1H NMR, 77.0 ppm for 13C NMR). Coupling constants (J) are reported in hertz. HRESIMS were D

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measured on a Waters SYNAPT Q-TOF high-definition mass spectrometer using positive electrospray ionization. Plant Material. Fresh leaves of Harungana madagascarensis were collected from the forest in the Igbesa area of Ogun state, Nigeria, and authenticated by O. O. Oyebanji (Department of Botany, University of Lagos). A voucher specimen (LUH 6112) was deposited in the Department of Botany, Faculty of Science, University of Lagos, Nigeria. Extraction and Isolation. The powdered air-dried leaves of H. madagascariensis (2.23 kg) were extracted with MeOH by percolation at room temperature to obtain 675 g of crude extract. The crude extract was partitioned into n-hexane-soluble (110 g), ethyl acetatesoluble (54 g), and n-butanol-soluble (196 g) fractions. The n-hexanesoluble fraction (110 g) was subjected to flash column chromatography on silica gel (60 mesh, 300 g) and eluted with mixtures of petroleum ether−EtOAc (100:0−50:50, v/v). By TLC monitoring, elutes were combined into four fractions (Fr. A−D). Fr. B (24 g) was further separated on a silica gel column (60 mesh, 180 g), using petroleum ether−EtOAc (95:5−70:30) as mobile phase. Precipitates from fractions eluted by petroleum ether−EtOAc (85:15) were collected, dissolved in MeOH, and purified on Sephadex LH-20 (eluted with MeOH) to yield compound 5 (65 mg). The remaining part of the petroleum ether−EtOAc (85:15)-eluted fractions were combined and separated on a silica gel column (100 g silica gel; eluted with n-hexane−EtOAc, 9:1, v/v) followed by chromatography on Toyopearl HW-40F (eluted with MeOH) to obtain compound 1 (20 mg). All remaining fractions from the Toyopearl HW-40F column were combined and applied to a C18 column eluted with 80% aqueous MeOH to obtain compound 7 (1.2 mg). Compound 6 was precipitated from fractions eluted by petroleum ether−EtOAc (80:20). Fr. C (30 g) was separated into four subfractions on a silica gel column (200 g) eluted with mixtures of n-hexane and EtOAc (75:25−40:60). The second subfraction (2 g) was rechromatographed over silica gel (100 g) using n-hexane−acetone (85:5) as mobile phase to yield compounds 2 (9 mg) and 3 (32 mg). The EtOAc-soluble portion (54 g) was separated into six subfractions on a silica gel column (60 mesh, 210 g) eluted with n-hexane−EtOAc (100:0−20:80, v/v). The sixth subfraction (11 g) was further purified on a silica gel column (60 mesh, 280 g; eluted with n-hexane−acetone, 6:4, v/v) followed by Sephadex LH-20 chromatography (eluted with MeOH) to afford compound 4 (4 mg). Harunganol C (1): orange, amorphous powder; IR (film) νmax 3400, 2923, 1634, 1593, 1434, 1354, 1306, 1275, 1224, 1176, 1103, 1089, 1072, 1042, 876 cm−1; 1H and 13C NMR (CDCl3, 400 and 100 MHz, respectively), see Table 1; (−)-HRESIMS m/z 931.5167 [M − H]− (calcd for C61H71O8−, 931.5154). Harunganol D (2): yellow, amorphous powder; [α]20 D −0.8 (c 0.3, acetone); IR (film) νmax 3438, 2976, 2928, 1640, 1612, 1594, 1575, 1478, 1364, 1323, 1303, 1286, 1250, 1196, 1171, 1134, 1097, 1073, 945 cm−1; 1H NMR (acetone-d6, 400 MHz), see Table 2; 13C NMR (acetone-d6, 100 MHz), see Table 3; (+)-HRESIMS m/z 425.1935 [M + H]+ (calcd for C25H29O6+, 425.1959). Harunganol E (3): yellow, amorphous powder; [α]20 D −1.1 (c 0.2, acetone); IR (film) νmax 3458, 2974, 1639, 1615, 1580, 1472, 1403, 1329, 1296, 1279, 1241, 1196, 1163, 1134, 1094, 948 cm−1; 1H NMR (acetone-d6, 400 MHz), see Table 2; 13C NMR (acetone-d6, 100 MHz), see Table 3; (+)-HRESIMS m/z 425.1938 [M + H]+ (calcd for C25H29O6+, 425.1959). Harunganol F (4): yellow, amorphous powder; [α]20 D −5.0 (c 0.1, acetone); IR (film) νmax 3425, 2977, 2923, 1641, 1614, 1585, 1479, 1406, 1361, 1303, 1245, 1235, 1211, 1166, 1135, 1087, 1068, 1034, 998, 965, 925, 890, 842, 824 cm−1; 1H NMR (CDCl3, 400 MHz), see Table 2; 13C NMR (CDCl3, 100 MHz), see Table 3; (+)-HRESIMS m/z 455.1686 [M + H]+ (calcd for C25H27O8+, 455.1700). Ferruginin A (7): yellow, amorphous powder; (−)-HRESIMS m/z 459.2556 [M − H]− (calcd for C30H35O4−, 459.2541). Single-Crystal X-ray Structure Determination. X-ray diffraction intensity data were collected at the Advanced Photon Source, Argonne National Laboratory, on LS-CAT (beamline 21-ID-D) with a 50 μm X-ray beam, at a distance of 100 mm and a temperature of 100

K, using a MAR CCD 300 mm area detector. All structures were solved by SHELXS and refined with SHELX-2014. For compound 2, a needle crystal, roughly 10 × 10 × 50 μm, was used for data collection. The space group was determined to be P1̅ (No. 2) by the absence of systematic absences and successful structure determination. A total of 72 images were collected with an ω-scan width of 5°. They were indexed and integrated by HKL2000 and yielded a total of 32 019 intensities. They were averaged to yield a total of 5135 reflections with R(int) = 0.046, 91.3% complete to a resolution of 0.75 Å, average I/σ = 15.0, and average redundancy of 1.8 over 1̅ symmetry. A disordered MeOH molecule, located near a center of inversion, was included in the model. Hydrogen atoms were evident in the difference electron density map and added to the model with geometric restraints and isotropic displacement parameters related to the attached carbon or oxygen atom. For compound 5, a small crystal block, roughly 20 × 50 × 50 μm, was used for data collection. The space group was determined to be P21/c (No. 14) by identification of the systematic absences. A total of 144 images were collected, two full sets of 360° at different exposures, and each with an ω-scan width of 5°. They were indexed, integrated, and scaled using HKL2000. The data were averaged over 2/m symmetry to a total of 5984 reflections with R(int) = 0.037, completeness of 99.0%, and redundancy of 13.1 at a resolution of 0.73 Å. Hydrogen atoms were evident from the difference electron density map and added to the model with geometric restraints and isotropic displacement parameters related to the attached carbon or oxygen atom. For compound 7, a crystal block, 5 × 50 × 100 μm, was used for data collection. The space group was determined to be P21/c (No. 14) by identification of the systematic absences and successful refinement of the structure. A total of 291 images were collected, 111 with a scan width of 2° and another 180 with a scan width of 2°. They were indexed and integrated by HKL2000 and yielded a total of 375 989 intensities. They were averaged to yield a total of 13 247 reflections with R(int) = 0.120, 98.0% complete to a resolution of 0.75 Å, average overall I/σ = 4.9, and average redundancy of 9.6 over 2/m symmetry. Hydrogen atoms were evident in the difference electron density map and added to the model with geometric restraints and isotropic displacement parameters related to the attached carbon or oxygen atom. Two independent molecules, different only by a C−C singlebond rotation in the prenyl group, were observed in the crystal structure. The data for X-ray diffraction analyses of compounds 2, 5, and 7 have been deposited at the Cambridge Crystallographic Data Centre. CCDC 1430592 (for 2), CCDC 1430593 (for 5), and CCDC 1430594 (for 7) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www. ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax + 44-1223-336033; e-mail [email protected]). Enzyme Inhibition Assay. The α-glucosidase inhibition assay was performed according to a slightly modified method of the Sigma assay kit (Sigma MAK123). α-Glucosidase from Saccharomyces cerevisiae was purchased from Sigma (G0660-750UN). The assay was performed in a 96-well format consisting of dual sample/blank groupings done in triplicate. Sample wells included 140 μL of 67 mM K3PO4 buffer in deionized H2O (adjusted to pH 6.8 at 37 °C using 1 M NaOH), 20 μL of 3 mM glutathione in deionized H2O, 20 μL of 0.7 units/mL αglucosidase enzyme in cold deinoized H2O, and 20 μL of sample prepared in DMSO (serially diluted with deionized H2O to the required concentrations). For the blank wells, enzyme was substituted with 20 μL of deionized H2O. Immediately preceding analysis, 20 μL of 10 mM p-nitrophenyl α-D-glucoside solution in deionized H2O was added to all wells. Absorbance readings were taken once a minute for 40 min at 400 nm on a Biotek Synergy H4 Hybrid microplate reader using Gen 5.1.11 software. Acarbose, a widely used clinical antidiabetic drug, was used as positive control. E

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00924. 1D and 2D NMR spectroscopic data and IR spectra for compounds 1−4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +1-312-9965234. Fax: +1-312-9967107. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The use of the Advanced Photon Source, Argonne National Laboratory, operated for the U.S. Department of Energy Office of Science was supported by Contract No. DE-AC0206CH11357; station 21-ID-D was supported by the Life Sciences Collaborative Access Team. We thank the Mass Spectrometry, Metabolomics & Proteomics Facility of the University of Illinois at Chicago for assistance in HRMS data acquisition.

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DOI: 10.1021/acs.jnatprod.5b00924 J. Nat. Prod. XXXX, XXX, XXX−XXX