Î±-Glucosidase Inhibitory and Cytotoxic Taxane ... - ACS Publications

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α‑Glucosidase Inhibitory and Cytotoxic Taxane Diterpenoids from the Stem Bark of Taxus wallichiana Phu Hoang Dang,† Hai Xuan Nguyen,† Truc Thanh Thi Duong,† Thao Kim Thi Tran,† Phuc Thi Nguyen,† Trang Kieu Thi Vu,† Hung Chi Vuong,‡ Nguyen Huu Trong Phan,† Mai Thanh Thi Nguyen,†,§ Nhan Trung Nguyen,*,†,§ and Suresh Awale⊥ †

Faculty of Chemistry, VNUHCM−University of Science, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam Tay Nguyen Herbals JSC, Tu Tra Ward, Don Duong District, Lam Dong Province Vietnam § Cancer Research Laboratory, Vietnam National University, Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam ⊥ Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan ‡

S Supporting Information *

ABSTRACT: From a CH2Cl2 extract of the bark of Taxus wallichiana, six new taxoids, wallitaxanes A−F (1−6), were isolated, together with 29 known compounds. The structures of the new compounds were elucidated on the basis of spectroscopic data interpretation. Wallitaxane D (4) was identified as an opened oxetane-type taxoid having the first naturally occurring C(H)-20 acetal group, while wallitaxanes E (5) and F (6) are representative of the rare abeo-taxoid class. The isolated compounds were evaluated for their α-glucosidase inhibitory activity and for cytotoxicity against the HeLa human cervical cancer cell line. In the present work, taxanes were found to exhibit α-glucosidase inhibitory activity for the first time, and wallitaxane A (1) showed the most potent effect, with an IC50 value of 3.6 μM. In turn, 7-epi-taxol (16) and 7-epi-10-deacetyltaxol (17) showed IC50 values of 0.05 and 0.085 nM, respectively, against HeLa cells.

T

In a continued study on the screening of medicinal plants for α-glucosidase inhibitory activity,6−10 it was found that a CH2Cl2 extract of the stem bark of T. wallichiana showed strong αglucosidase inhibitory activity, with an IC50 value of 6.0 μg mL−1. In addition, this extract also showed potent cytotoxicity against the HeLa human cervical cancer cell line, with an IC50 value of 0.67 μg mL−1. Thus, an activity-guided fractionation study was carried out on its chemical constituents, leading to the isolation of six new taxoids, wallitaxanes A−F (1−6), together with 29 known compounds (7−35). In this paper, the isolation and structural elucidation of these compounds by spectroscopic methods are described as well as their αglucosidase inhibitory activity evaluation and cytotoxicity determination against the HeLa cancer cell line.

he historic discovery of paclitaxel (known initially as taxol) from Taxus brevifolia as an antineoplastic agent by Wall and Wani,1 and its subsequent development into one of the most versatile anticancer drugs, has inspired many natural product chemists throughout the world to search for new lead compounds.2 Different species of Taxus are distributed throughout the world. Among them, Taxus wallichiana Zucc. grows wild in many countries in Asia including Afghanistan, Pakistan, India, Nepal, Bhutan, the People’s Republic of China, Indonesia, Malaysia, Myanmar, Vietnam, and the Philippines.3 Commonly, this tree is called the Himalayan yew. It is used in Indian folk medicine for the treatment of bronchitis, asthma, epilepsy, snakebites, and scorpion stings.3 In Vietnamese traditional medicine, the wood powder is often used for the treatment of diabetes.4 Previous studies on the chemical constituents of T. wallichiana have led to reports of mainly taxane-type diterpenoids together with lignans, biflavonoids, and phytosterols.3,5 © 2017 American Chemical Society and American Society of Pharmacognosy

Received: January 2, 2017 Published: February 27, 2017 1087

DOI: 10.1021/acs.jnatprod.7b00006 J. Nat. Prod. 2017, 80, 1087−1095

Journal of Natural Products

Article

Chart 1

■

C (25);21 7-β-xylosyltaxol B (26);21 7-β-xylosyl-10-deacetyltaxol B (27);21 taxinine M (28);22 methyl protocatechuate (29);23 (+)−matairesinol (30);24 (−)−matairesinol (31);24 α-conidendrin (32);25 soya-cerebroside II (33);26 oleanolic acid (34);27 and ponasterone A (35).28 Wallitaxane A (1) was isolated as a white, amorphous solid and showed a sodiated molecular ion peak at m/z 557.3094 [M + Na]+, corresponding to the molecular formula C30H46O8. Its IR spectrum demonstrated the presence of hydroxy (3460 cm−1) and ester carbonyl (1740 cm−1) groups. The 1H NMR spectrum displayed signals due to four methyl singlets (δH 1.18, 1.64, 2.00, and 0.83), an exomethylene [δH 4.82 (s) and 5.26 (s)], a 2-methyl-3-hydroxybutyryloxy group [δH 2.41 (m); 3.86 (quint., J = 6.4 Hz); 1.21 (d, J = 6.4 Hz); and 1.17 (d, J = 7.2 Hz)], and two acetoxy groups [δH 2.02 (s) and 2.18 (s)] (Table 1). The 13C NMR spectrum showed the presence of resonances for three carbonyls (δC 175.0, 170.0, 170.0), eight methyl groups (δC 31.7, 25.1, 22.7, 22.6, 21.6, 21.2, 21.0, 14.2), two

RESULTS AND DISCUSSION

The powdered stem bark of T. wallichiana was refluxed with MeOH. The MeOH-soluble extract was suspended in H2O and successively partitioned with CH2Cl2 and EtOAc to yield CH2Cl2, EtOAc, and a residual aqueous fraction, respectively. Further separation and purification of the CH2Cl2-soluble fraction led to the isolation of 35 compounds including six new taxoids, wallitaxanes A−F (1−6). The known compounds were identified as yunnanxane (7);11 2-deacetoxy-5-decinnamoyltaxinine J (8);12 taxuspinanane K (9);13 taxuspine F (10);14 10βmethoxy-2α,5α,14β-triacetoxytaxa-4(20),11-diene (11);15 2deacetoxytaxinine J (12);16 taxezopidine H (13);17 taxinine B (14);18 10-deacetyl-13-oxobaccatin III (15);16 7-epi-taxol (16);19 7-epi-10-deacetyltaxol (17);16 taxol (paclitaxel) (18);19 10-deacetyltaxol (19);20 7-epi-taxol B (20);19 7-epi10-deacetyltaxol B (21);20 taxol B (22);19 10-deacetyltaxol B (23);20 7-β-xylosyltaxol C (24);21 7-β-xylosyl-10-deacetyltaxol 1088



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Table 1. NMR Spectroscopic Data for Wallitaxanes A (1) and B (2) in CDCl3 wallitaxane A (1) position 1 2 3 4 5 6 7 8 9

δC, type 59.4, 70.8, 42.5, 142.6, 78.6, 29.1, 34.2,

CH CH CH C CH CH2 CH2

39.7, C 45.4, CH2

10 11 12 13

76.1, 137.4, 134.5, 40.0,

CH C C CH2

14 15 16 17 18 19 20

71.1, 37.5, 31.7, 25.1, 21.0, 22.7, 116.9,

CH C CH3 CH3 CH3 CH3 CH2

1′ 2′ 3′ 4′ 5′ OAc-2

175.0, 47.2, 69.7, 21.2, 14.2, 170.0, 21.6, 170.0, 22.1, 55.4,

CO CH CH CH3 CH3 CO CH3 CO CH3 CH3

OAc-2 OMe-10

wallitaxane B (2) δH (J in Hz)

δC, type

1.89, d (2.2) 5.35, dd (6.6, 2.2) 2.93, d (6.6) 5.29, 1.79, 1.21, 1.90,

59.4, 71.2, 40.3, 148.0, 76.6, 31.1, 33.4,

t (2.7) m m m

1.66, dd (15.0, 5.5) 2.29, dd (15.0, 11.8) 4.61, dd (11.8, 5.5)

2.41, m 2.86, dd (19.0, 9.2) 5.03, dd (9.2, 4.8) 1.18, 1.64, 2.00, 0.83, 5.26, 4.82,

s s s s s s

2.41, 3.86, 1.21, 1.17,

m quint. (6.4) d (6.4) d (7.2)

2.02, s 2.18, s 3.28, s

CH CH CH C CH CH2 CH2

40.1, C 45.3, CH2 76.2, 136.5, 135.7, 39.7,

CH C C CH2

71.3, 37.5, 31.8, 25.1, 21.0, 22.5, 113.6,

CH C CH3 CH3 CH3 CH3 CH2

175.3, 47.1, 69.7, 21.0, 14.2, 170.0, 21.6,

CO CH CH CH3 CH3 CO CH3

55.4, CH3

δH (J in Hz) 1.84, d (2.2) 5.35, dd (6.4, 2.2) 3.22, d (6.4) 4.19, 1.72, 1.10, 2.05,

t (3.0) m dt (13.4, 3.7) m

1.63, m 2.23, dd (14.9, 11.8) 4.65, dd (11.8, 5.4)

2.35, dd (18.7, 4.6) 2.81, dd (18.7, 9.4) 5.09, dd (9.4, 4.6) 1.18, 1.63, 2.00, 0.80, 5.10, 4.76,

s s s s s s

2.41, 3.86, 1.21, 1.17,

quint. (7.2) quint. (6.4) d (6.4) d (7.2)

2.02, s

3.28, s

Figure 1. Connectivities (bold lines) deduced by the COSY spectrum and significant HMBC correlations (solid arrows) observed for 1−6.

signals resembled closely those of yunnanxane (7)11 except for the presence of a methoxy group [δH 3.28 (s), δC 55.4] instead of an acetoxy group [δH 2.06 (s), δC 21.4 and 170.1] in 7. The partial connectivity (bold lines) was deduced from the 1H−1H COSY and HSQC spectra, which were connected based on the

pairs of olefinic carbons (δC 142.6, 137.4, 134.5, 116.9), a methoxy group (δC 55.4), three methines (δC 59.4, 47.2, 42.5), five oxymethines (δC 78.6, 76.1, 71.1, 70.8, 69.7), four methylenes (δC 45.4, 40.0, 34.2, 29.1), and two quaternary carbons (δC 39.7, 37.5) (Table 1). These 1H and 13C NMR 1089



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Figure 2. Key NOESY correlations (solid arrows) observed for 1−6.

4.12 (m); 2.92 (m); 3.14 (td, J = 8.6, 3.0 Hz); 3.25 (m); 3.04 (dd, J = 11.3, 9.7 Hz); 3.67 (dd, J = 11.3, 5.0 Hz)], and a complex side chain of N-tigloylphenylisoserine [δH 7.48−7.35 (m); 4.57 (t, J = 6.8 Hz); 5.30 (dd, J = 8.7, 6.8 Hz); 8.15 (d, J = 8.7 Hz); 6.41 (qd, J = 6.9, 1.4 Hz); 1.72 (dd, J = 6.9, 1.4 Hz); 1.75 (t, J = 1.4 Hz)]. Its 13C NMR spectrum showed signals for six carbonyls (δC 204.7, 194.7, 172.4, 170.8, 168.3, 165.2), seven methyls (δC 26.5, 23.6, 22.7, 13.7, 13.5, 12.4, 9.1), two pairs of olefinic carbons (δC 146.0, 141.6, 132.0, 129.6), a methine (δC 44.9), five oxymethines (δC 83.0, 75.6, 74.1, 73.4, 69.5), a nitrogenated methine (δC 55.6), two methylenes (δC 35.1, 34.5), an oxymethylene (δC 75.0), two quaternary carbons (δC 56.5, 41.1), two oxygenated quaternary carbons (δC 79.3, 77.3), and two aromatic rings (δC 127.4−139.5). These were characteristic of those reported for taxol B derivatives (20−23, 26, and 27).19−21 Comparison of the 1H and 13C NMR spectra of 3 (Table 2) with those of 7-β-xylosyl-10-deacetyltaxol B (27)21 suggested that these compounds differ from each other only due to the presence of a ketone carbonyl at δC 194.7 (C10) in 3 with the loss of an oxymethine carbon (δC‑10 74.2, 27). The occurrence of a C-10 carbonyl was confirmed by an HMBC correlation between the C-18 methyl group and the carbonyl carbon at δC 194.7. The hydroxy, benzoyloxy, and Ntigloylphenylisoserine groups were determined to be at C-1, C2, and C-13, respectively (Figure 1), based on correlations observed in the HMBC spectrum. The NOESY spectrum of 3 (Figure 2) suggested the same relative configuration as those of taxol B derivatives (20−23, 26, and 27).19−21 Wallitaxane D (4) showed a sodiated molecular ion peak at m/z 521.2379 [M + Na]+ (calcd for C25H38O10Na, 521.2363), corresponding to the molecular formula C25H38O10, in the HRESIMS. The 1H NMR spectrum showed resonances for four methyl singlets (δH 1.08, 1.43, 2.01, 1.22), two acetoxy groups (δH 2.04, 2.00), a methoxy group (δH 3.39), an acetal proton [δH 4.92 (d, J = 5.7 Hz)], and a methine proton [δH 2.47 (dd, J = 12.9, 5.7 Hz)] (Table 3). The 13C NMR spectrum exhibited the signals for 30 carbons including those for a pair of olefinic carbons (δC 137.8, 137.3), an acetal carbon (δC 105.9), six oxymethines (δC 86.8, 79.2, 76.0, 72.4, 72.4, 64.8), an

long-range correlations observed in the HMBC spectrum (Figure 1). The HMBC correlation from the methoxy group to the oxymethine carbon at δC 76.1 suggested its location to be at C-10. The locations of two acetoxy groups were determined to be at C-2 and C-5, based on the chemical shifts of H-2 [δH 5.35 (dd, J = 6.6, 2.2 Hz)] and H-5 [δH 5.29 (t, J = 2.7 Hz)] and its HMBC correlations to two overlapped acetoxy carbonyls at δC 170.0. The position of the 2-methyl-3-hydroxybutyryloxy group was confirmed by the HMBC correlation from H-14 (δH 5.03) to the ester carbonyl at δC 175.0 (C-1′) (Figure 1). The relative configuration of 1 was determined on the basis of NOESY correlations (Figure 2). The NOESY correlations of H-2 with H-1β, Me-19, H-9β, and Me-17 indicated their β-orientation with respect to the central eight-membered ring. Furthermore, a NOESY correlation between two acetoxy groups at C-2 and C-5 was supportive of their α-orientation. The methoxy and 2methyl-3-hydroxybutyryloxy groups were assigned to be βoriented based on the NOESY correlations between H-10/H9α, H-10/Me-18, H-3/Me-18, H-3/H-14, and H-14/H-13α. The NMR data of the 2-methyl-3-hydroxybutyryloxy group matched closely with those of the 2R-methyl-3S-hydroxybutyryloxy group of yunnanxane.11 Therefore, the structure of wallitaxane A (1) was assigned as shown. Wallitaxane B (2) was isolated as a yellowish, amorphous solid. Its molecular formula was determined to be C28H44O7 by the HRESIMS. The 1H and 13C NMR spectra of 2 resembled closely those of 1, except for the loss of the signals due to one of the acetoxy groups (Table 1). The HMBC correlation between the H-2 oxymethine proton and the acetoxy carbonyl at δC 170.0 suggested that the location of deacetylation is at C5. Therefore, the structure of wallitaxane B was assigned as 5deacetylwallitaxane A (2). Wallitaxane C (3) was deduced to have the molecular formula C48H57NO17, based on the HRESIMS at m/z 942.3518 [M + Na]+ (calcd for C48H57NO17Na, 942.3524). Its 1H NMR spectrum (Table 2) showed the presence of four characteristic methyl singlets (δH 1.11, 1.05, 1.77, 1.63), an oxymethylene group (δH 4.06), an acetoxy group (δH 2.26), a benzoyloxy group [δH 8.01 (m); 7.72 (m); 7.64 (m)], a xylosyl group [δH 1090



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Table 2. NMR Spectroscopic Data for Wallitaxanes C (3) and E (5) wallitaxane C (3a) position 1 2 3 4 5 6

77.3, 74.1, 44.9, 79.3, 83.0, 35.1,

C CH CH C CH CH2

7 8 9 10 11 12 13

75.6, 56.5, 204.7, 194.7, 141.6, 146.0, 69.5,

CH C C C C C CH

14

34.5, CH2

15 16 17 18 19 20

41.1, 26.5, 23.6, 13.7, 9.1, 75.0,

1′ 2′ 3′ N−H 5′ 6′ 7′ 8′ Me-6′ 1″ 2″ 3″ 4″ 5″ OAc-4 OH-1 OH-2 OBz-2 ipso ortho meta para OBz-15 ipso ortho meta para ipso-Ph-3′ ortho-Ph meta-Ph para-Ph a

δC, type

C CH3 CH3 CH3 CH3 CH2

172.4, CO 73.4, CH 55.6, CH 168.3, 132.0, 129.6, 13.5, 12.4, 103.0, 73.3, 75.3, 69.1, 65.2,

C C CH CH3 CH3 CH CH CH CH CH2

170.8, CO 22.7, CH3

wallitaxane E (5b)

δH (J in Hz) 5.56, d (7.0) 3.44, d (7.0) 4.88, 1.71, 2.57, 3.84,

m m m m

5.93, t (8.6) 1.94, m 2.05, m 1.11, 1.05, 1.77, 1.63, 4.06,

δC, type 69.8, 69.4, 45.2, 80.0, 85.2, 37.0,

C CH CH C CH CH2

77.8, 56.2, 206.1, 193.5, 137.3, 166.9, 40.0,

CH C C C C C CH2

27.5, CH2

s s s s s

90.7, 22.8, 23.2, 17.9, 8.9, 76.0,


104.4, 74.3, 76.0, 70.5, 66.0,

CH CH CH CH CH2

δH (J in Hz) 4.58, t (7.3) 3.47, m 4.98, 1.87, 2.64, 4.02,

d (8.5) ddd (15.1, 9.4, 1.3) dt (15.1, 8.5) dd (9.4, 8.5)

2.40, 2.78, 2.25, 2.29,

m m m m

1.59, 1.63, 2.24, 1.75, 4.54, 4.61,

s s s s d (8.2) dd (8.2, 1.0)

4.22, 3.09, 3.35, 3.46, 3.22, 3.82,

d (6.7) ddd (8.2, 6.7, 3.4) td (8.2, 3.4) m dd (11.6, 9.1) dd (11.6, 4.9)

4.57, t (6.8) 5.30, dd (8.7, 6.8) 8.15, d (8.7)

6.41, 1.72, 1.75, 4.12, 2.92, 3.14, 3.25, 3.04, 3.67,

qd (6.9, 1.4) dd (6.9, 1.4) t (1.4) m m td (8.6, 3.0) m dd (11.3, 9.7) dd (11.3, 5.0)

170.8, CO 21.8, CH3

2.26, s 5.09, s

2.14, s 4.09, d (7.3)

165.2, 130.1, 129.2, 128.7, 133.5,

CO C CH CH CH

8.01, m 7.72, m 7.64, m 166.7, 133.5, 130.1, 129.2, 133.4,

139.5, 127.4, 134.4, 128.7,

C CH CH CH

CO C CH CH CH

7.81, dd (8.2, 1.4) 7.48, t (8.2) 7.59, ddt (8.2, 7.0, 1.4)

7.48−7.35, m 7.48−7.35, m 7.48−7.35, m

δ values in DMSO-d6. bδ values in acetone-d6. 1091



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Table 3. NMR Spectroscopic Data for Wallitaxanes D (4) and F (6) wallitaxane D (4a) position

δC, type

1 2

77.1, C 86.8, CH

3 4 5 6

40.9, 50.0, 64.8, 39.5,

CH CH CH CH2

7 8 9 10 11 12 13 14

72.4, 46.1, 79.2, 76.0, 137.8, 137.3, 72.4, 36.4,

CH C CH CH C C CH CH2

15 16 17 18 19 20

42.4, 29.3, 21.2, 15.8, 13.5, 105.9,

C CH3 CH3 CH3 CH3 CH

OMe-20 OAc-7

55.4, CH3

wallitaxane F (6b)

δH (J in Hz)

61.3, C 31.7, CH2

4.22, d (9.6) 2.90, 2.47, 4.10, 1.59, 1.85, 4.30,

dd (12.9, 9.6) dd (12.9, 5.7) brs ddt (13.8, 10.6, 3.0) ddd (13.8, 5.2, 2.6) dd (10.6, 5.2)

4.42, d (10.3) 6.13, d (10.3)

5.61, ddd (10.3, 4.0, 1.6) 2.32, ddd (15.5, 10.3, 1.6) 2.41, dd (15.5, 4.0) 1.08, 1.43, 2.01, 1.22, 4.92,

s s d (1.6) s d (5.7)

OAc-13 a

170.5, 21.8, 170.4, 20.9,

CO CH3 CO CH3

38.5, 149.8, 73.0, 36.1,

CH C CH CH2

69.4, 44.4, 79.3, 75.1, 39.8, 141.3, 126.9, 41.1,

CH C CH CH CH C CH CH2

88.6, 26.9, 29.8, 15.7, 12.1, 111.3,


169.6, 21.3, 170.8, 21.7,

CO CH3 CO CH3

δH (J in Hz) 1.28, m 2.21, m 2.20, m 4.28, 1.62, 2.04, 5.56,

brs m m dd (11.2, 5.1)

5.43, d (10.8) 4.96, d (10.8) 3.01, m 5.50, brs 1.77, m 2.33, m 1.44, 1.25, 2.06, 0.89, 4.68, 5.04,

s s s s s s

3.39, s

OAc-9 OAc-10

δC, type

2.01, s 2.06, s

2.04, s 2.00, s

δ values in acetone-d6. bδ values in CDCl3.

The C(H)-20 methine proton also showed a NOESY correlation with H-4 (Figure 2). Therefore, the relative configuration of C-20 in 4 was assigned as shown. Wallitaxane E (5) showed the molecular formula C34H42O13, as deduced from the positive HRESIMS at m/z 657.2544 [M + Na]+. The 1H NMR spectrum showed the presence of a 11(15→1)abeo-taxoid with signals for four methyl singlets (δH 1.59, 1.63, 2.24, 1.75), an exomethylene [δH 4.54 (d, J = 8.2 Hz); 4.61 (dd, J = 8.2, 1.0 Hz)], an acetoxy group (δH 2.14), a benzoyloxy group [δH 7.81 (dd, J = 8.2, 1.4 Hz); 7.48 (t, J = 8.2 Hz); 7.59 (ddt, J = 8.2, 7.0, 1.4 Hz)], and a xylosyl group [δH 4.22 (d, J = 6.7 Hz); 3.09 (ddd, J = 8.2, 6.7, 3.4 Hz); 3.35 (td, J = 8.2, 3.4 Hz); 3.46 (m); 3.22 (dd, J = 11.6, 9.1 Hz); 3.82 (dd, J = 11.6, 4.9 Hz)] (Table 2). Its 13C NMR spectrum showed resonances for four carbonyls (δC 206.1, 193.5, 170.8, 166.7), four methyls (δC 23.2, 22.8, 17.9, 8.9), a pair of olefinic carbons (δC 166.9, 137.3), a methine (δC 45.2), an anomeric carbon (δC 104.4), six oxymethines (δC 85.2, 77.8, 76.0, 74.3, 70.5, 69.4), three methylenes (δC 40.0, 37.0, 27.5), two oxymethylenes (δC 76.0, 66.0), two quaternary carbons (δC 69.8, 56.2), two oxygenated quaternary carbons (δC 90.7, 80.0), and an aromatic ring (δC 129.2−133.5) (Table 2). The NMR data of the aglycone moiety of 5 resembled closely those of 15-benzoyl-10deacetyl-2-debenzoyl-10-dehydro-11(15→1)abeo-baccatin III,31

oxygenated quaternary carbon (δC 77.1), two methines (δC 50.0, 40.9), two methylenes (δC 39.5, 36.4), and two quaternary carbons (δC 46.1, 42.4) (Table 3). These signals resembled those of opened oxetane-taxoids having a 6/8/6 ring system.29 In the 1H−1H COSY spectrum, correlations were observed between H-2, H-3, H-4, and H-20; H-4, H-5, H-6, and H-7; H9 and H-10; and H-13 and H-14 (Figure 1). The HMBC correlations between H-2/C-20 and H-20/C-2 suggested the presence of a C-2(20) tetrahydrofuran ring.29,30 The HMBC correlations between H-20/OCH3 and OCH3/C-20 indicated the location of a methoxy group at C-20. The locations of two acetoxy groups were determined to be at C-10 and C-13, based on the chemical shifts of H-10 [δH 6.13 (d, J = 10.3 Hz)] and H-13 [δH 5.61 (ddd, J = 10.3, 4.0, 1.6 Hz)] and HMBC correlations of the two corresponding acetoxy carbonyls. This compound was assigned as the first naturally occurring opened oxetane-taxoid with a C(H)-20 methine group. Two oxymethine protons, H-2 and H-9, were suggested to be β-oriented by their NOESY correlations to two methyl groups, Me-17 and Me-19. The NOESY correlations between H-10/H-3, H-10/ Me-18, H-10/H-7, and H-7/H-3 indicated their α-orientation. The C-2(20) tetrahydrofuran ring and the C-5 hydroxy and C13 acetoxy groups were α-oriented, as deduced by the NOESY correlations between H-4/H-5, H-4/Me-19, and H-13/Me-16. 1092



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20−25, 30−32, 34, and 35 were found to possess α-glucosidase inhibitory activity (Table 4). Wallitaxane A (1) showed the

except for the presence of a methylene in 5 instead of an oxymethine proton assigned for C-13.32 Therefore, 5 was suggested to be a 9,10-diketo-abeo-taxoid glycoside. The sugar unit was deduced to be a β-xylosyl moiety by comparison of its 13 C NMR data with those reported in the literature.33 The positions of the acetoxy, hydroxy, and benzoyloxy groups were determined to be at C-4, C-2, and C-15 from the HMBC spectrum (Figure 1). The location of the xylosyl group was determined to be at C-7 based on the HMBC correlations between H-1″/C-7 and H-7/C-1″. The relative configuration of 5 was deduced by NOESY spectroscopic analysis, in which NOESY correlations of H-2 with Me-16, Me-17, and Me-19 indicated the β-orientation of the H-2 oxymethine proton and the [C(OBz)Me2]-1 group. Moreover, the C-4 acetoxy group and the H-7 oxymethine proton were indicated to be αoriented by the NOESY correlations between H-3/H-7, H-5/ H-7, and Me-19/H-20 (Figure 2). Thus, the structure of wallitaxane E (5) was assigned as shown. Wallitaxane E (5) represents the first naturally occurring example of an abeotaxane glycoside. Wallitaxane F (6) showed a sodiated molecular ion peak at m/z 441.2256 [M + Na]+ (calcd for C24H34O6Na, 441.2253) corresponding to the molecular formula C24H34O6 in the HRESIMS. Its 1H NMR spectrum exhibited signals for four methyl singlets (δH 1.44, 1.25, 2.06, 0.89), an exomethylene (δH 4.68, 5.04), two acetoxy groups (δH 2.01, 2.06), and an olefinic proton at δH 5.50 (brs) (Table 3). The 13C NMR spectrum showed the signals of 24 carbons including those for two carbonyls (δC 170.8, 169.6), six methyls (δC 29.8, 26.9, 21.7, 21.3, 15.7, 12.1), two pairs of olefinic carbons (δC 149.8, 141.3, 126.9, 111.3), two methines (δC 39.8, 38.5), four oxymethines (δC 79.3, 75.1, 73.0, 69.4), three methylenes (δC 41.1, 36.1, 31.7), two quaternary carbons (δC 61.3, 44.4), and an oxygenated quaternary carbon (δC 88.6) (Table 3). Comparison of these data with those in the literature34 suggests the structure of 6 to be a 11(15→1)abeo-taxa-4(20)-ene-type taxoid, as evident from the 13C NMR chemical shifts of an oxygenated quaternary carbon (C-15; δC 88.6) and a quaternary carbon (C-1; δC 61.3).34 The characteristic olefinic signals at δ 5.50 (brs, H-13) and 126.9 (C-13) and the 1H−1H COSY correlations (bold lines) between an olefinic proton H13 and CH2-14 suggested the presence of an unusual double bond at C-12(13) in ring A,35 which was confirmed by the HMBC correlations between H-13/C-10 and H-13/C-11. The HMBC correlation between the H-10 oxymethine proton and quaternary oxygenated carbon at δC 88.6 (C-15) indicated the presence of a C-10(15)-oxido bridge in 6. The locations of one hydroxy and two acetoxy groups were determined to be at C-5, C-7, and C-9, respectively, based on the observed HMBC correlations shown in Figure 1. The C-10(15) tetrahydrofuran ring and the C-7 acetoxy group were indicated to be β-oriented based on the NOESY correlations between H-3/H-10 and H7/H-10 (Figure 2). Furthermore, the NOESY correlations between H-5/OAc-7, OAc-7/Me-19, Me-19/H-9, and H-9/H11 indicated their β-orientation. Therefore, the structure of wallitaxane F (6) was assigned as shown. Wallitaxane F (6) represents the first example of a taxoid having the 11(15→1) abeo-taxa-4(20),12-diene feature. All isolated compounds were tested for their α-glucosidase inhibitory activity. Acarbose, which is currently used clinically in combination with either diet or antidiabetic agents to control the blood glucose level of patients, was used as the positive control in this study. Compounds 1, 2, 4−8, 11−14, 16, 17,

Table 4. α-Glucosidase Inhibitory Activity of the Isolated Compounds compound

IC50 (μM)

compound

IC50 (μM)

compound

IC50 (μM)

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

3.6 113 142 97.5 83.6 88.5 194 193 >250 >250 31.8 38.0

13 14 15 16 17 18 19 20 21 22 23 24

15.0 111 >250 42.7 48.8 >250 >250 99.5 112 82.5 121 94.1

25 26 27 28 29 30 31 32 33 34 35 acarbosea

86.9 >250 82.6 >250 231 175 46.5 194 >250 87.2 115 215

a

Positive control.

most potent inhibitory activity, with an IC50 value of 3.6 μM, which was 60 times more potent than acarbose (IC50, 215 μM). The occurrence of a C-10 methoxy group in compounds 1, 2, and 11 was found to enhance activity. The presence of the C-1 acetoxy group decreased the inhibition activity (1 ≫ 2), and the C-14 2-methyl-3-hydroxybutyryloxy group increases activity significantly (1 ≫ 11). In this work, taxanes were found to have α-glucosidase inhibitory activity for the first time. The major isolated compounds were also evaluated for their cytotoxicity against the HeLa human cervical cancer cell line (Table 5). Compound 16, 17, and 19−22 exhibited remarkable Table 5. Cytotoxicity of the Isolated Compounds against the HeLa Cancer Cell Linea compound

IC50 (μM)

4 5 6 7 9 10 12 13 15

>10 4.9 >10 >10 >10 >10 5.7 >10 >10

compound

IC50 (μM)

compound

IC50 (μM)

16 17 19 20 21 22 24 paclitaxelb

0.000 050 0.000 085 0.000 75 0.000 65 0.000 88 0.000 82 0.0015 0.0014

25 26 27 28 30 32 33 35

0.05 0.14 0.72 >10 >10 >10 >10 >10

a

Compounds 1−3, 8, 11, 14, 18, 23, 29, 31, and 34 were not tested due to the insufficient amounts isolated. bPositive control.

cytotoxicity against the HeLa cell line with subnanomolar IC50 values ranging from 0.05 to 0.88 nM. Compounds 16 and 17 showed the most potent cytotoxic activity, with IC50 values of 0.05 and 0.085 nM, respectively, more potent than that of paclitaxel, a positive control (IC50, 1.4 nM). The epimers with an OH-7α substituent were found to be more potently cytotoxic than their OH-7β epimers (16 > 18; 17 > 19; 20 > 22). The presence of a 7-O-β-xyloxyl group decreased the resultant activity (22 ≫ 27). Moreover, 7-O-acetylation was found to enhance cytotoxicity (16 > 17; 20 > 21; 24 > 25; 26 > 27). These conclusions were consistent with a previous report on the structure−activity relationships of taxol derivatives.36 1093



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(CH3)2CO−CHCl3 mixtures and recrystallized in a (CH3)2CO− CHCl3 (50:50) mixture to afford compounds 26 (30.0 mg) and 3 (4.0 mg). Wallitaxane A (1): white, amorphous solid; [α]25 D +38.4 (c 0.1, MeOH); IR νmax (KBr) 3460, 1740, 1645, 1450, 1020 cm−1; 1H and 13 C NMR (CDCl3, 500 MHz, see Table 1); HRESIMS m/z 557.3094 [M + Na]+ (calcd for C30H46O8Na, 557.3090). Wallitaxane B (2): yellowish, amorphous solid; [α]25 D +36.6 (c 0.1, MeOH); IR νmax (KBr) 3458, 1745, 1650, 1442, 1018 cm−1; 1H and 13 C NMR (CDCl3, 500 MHz, see Table 1); HRESIMS m/z 515.2992 [M + Na]+ (calcd for C28H44O7Na, 515.2985). Wallitaxane C (3): white, amorphous solid; [α]25 D −23.8 (c 0.1, MeOH); IR νmax (KBr) 3470, 1730, 1660, 1605, 1380, 1070 cm−1; 1H and 13C NMR (DMSO-d6, 500 MHz, see Table 2); HRESIMS m/z 942.3518 [M + Na]+ (calcd for C48H57NO17Na, 942.3524). Wallitaxane D (4): white, amorphous solid; [α]25 D +17.5 (c 0.1, MeOH); IR νmax (KBr) 3535, 1735, 1652, 1070, 1035 cm−1; 1H and 13 C NMR (acetone-d6, 500 MHz, see Table 3); HRESIMS m/z 521.2379 [M + Na]+ (calcd for C25H38O10Na, 521.2363). Wallitaxane E (5): white, amorphous solid; [α]25 D −30.6 (c 0.1, MeOH); IR (KBr) νmax 3410, 1725, 1605, 1380, 1070 cm−1; 1H and 13 C NMR (acetone-d6, 500 MHz, see Table 2); HRESIMS m/z 657.2544 [M + Na]+ (calcd for C34H42O13Na, 657.2547). Wallitaxane F (6): yellowish, amorphous powder; [α]25 D −18.2 (c 0.1, MeOH); IR (KBr) νmax 3445, 2980, 1730, 1255, 1026 cm−1; 1H and 13C NMR (CDCl3, 500 MHz, see Table 3); HRESIMS m/z 441.2256 [M + Na]+ (calcd for C24H34O6Na, 441.2253). α-Glucosidase Inhibitory Assay. The inhibitory activity of αglucosidase was determined according to a previous method.10 Thus, 3 mM p-nitrophenyl-α-D-glucopyranoside (25 μL) and 0.2 U/mL αglucosidase (25 μL) in 0.01 M phosphate buffer (pH = 7.0) were added to the sample solution (625 μL) to start the reaction. Each reaction was carried out at 37 °C for 30 min and stopped by adding 0.1 M Na2CO3 (375 μL). Enzymatic activity was quantified by measuring absorbance at 401 nm. One unit of α-glucosidase activity was defined as the amount of enzyme liberating p-nitrophenol (1.0 μM) per minute. The IC50 value was defined as the concentration of a α-glucosidase inhibitor that inhibited 50% of α-glucosidase activity. Acarbose, a known α-glucosidase inhibitor, was used as a positive control. Cytotoxicity Testing. Cell viability in the presence or absence of tested compounds was determined using the Cell Counting Kit-8 kit (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). The HeLa cell line (RCB0007) was purchased from the Riken BRC cell bank (Tsukuba, Japan) and maintained in standard Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) supplement, 0.1% NaHCO3, and 1% antibiotic-antimycotic solution. For the cytotoxicity experiment, exponentially growing cells were harvested and plated in 96-well plates (2 × 103/well) in DMEM at 37 °C under humidified 5% CO2 and 95% air for 24 h. After the cells were washed with phosphate-buffered saline (PBS), the medium was changed to serially diluted test samples in DMEM, with the control and blank in each plate. After 72 h of incubation, cells were washed twice with PBS, and 100 μL of DMEM containing 10% WST-8 cell counting kit solution was added to each well. After 3 h of incubation, the absorbance at 450 nm was measured on an EnSpire Multimode plate reader (PerkinElmer, Inc., Waltham, MA, USA). Cell viability was calculated from the mean values from three wells using the following equation:

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an A. Krüss Optronic polarimeter P3000 (A. Krüss Optronic GmbH, Hamburg, Germany). The IR spectra were measured with a Shimadzu IR-408 infrared spectrometer (Shimadzu Pte., Ltd., Singapore). The NMR spectra were taken on a Bruker Avance III 500 spectrometer (Bruker BioSpin AG, Bangkok, Thailand) with tetramethylsilane as internal standard, and the chemical shifts are expressed in δ values. HRESIMS was performed on a Bruker micrOTOF QII mass spectrometer (Bruker Singapore Pte., Ltd., Singapore). Column chromatography was carried out using silica gel 60, 0.06−0.2 mm (Scharlau, Barcelona, Spain) and LiChroprep RP-18, 40−63 μm (Merck KGaA, Darmstadt, Germany). Analytical and preparative TLC were carried out on precoated Kieselgel 60F254 or RP18 plates (Merck KGaA, Darmstadt, Germany). Other chemicals were of the highest grade available. Plant Material. The stem bark of T. wallichiana was collected in the Don Duong district, Lam Dong province, Vietnam, in March 2010 and was identified by Dr. Hung Chi Vuong, Director of Tay Nguyen Herbals JSC. A voucher specimen (MDE0040) has been deposited at the Division of Medicinal Chemistry, Faculty of Chemistry, VNUHCM−University of Science. Extraction and Isolation. The powdered stem bark of T. wallichiana (4.8 kg) was refluxed with MeOH, and the MeOH-soluble extract (1 kg) was suspended in H2O and successively partitioned with CH2Cl2 and EtOAc to give CH2Cl2-soluble (80 g), EtOAc-soluble (190 g), and residual aqueous (15 g) fractions. The CH2Cl2 fraction (80 g) was subjected to silica gel column chromatography (12 × 150 cm) and eluted with EtOAc−petroleum ether mixtures (v/v, 0:100 → 100:0) to obtain 13 major fractions (Fr.1−Fr.13). Fractions Fr.4 (2.4 g) and Fr.5 (2.6 g) were chromatographed over a silica gel column (3 × 80 cm) with EtOAc−petroleum ether and CHCl3−petroleum ether mixtures and were then purified by preparative TLC using EtOAc−nhexane (10:90) or CHCl3−petroleum ether (30:30) to afford compounds 11 (2.7 mg), 12 (19.4 mg), 13 (3.5 mg), 14 (2 mg), 29 (10.0 mg), and 34 (5.0 mg). Fraction Fr.6 (3.4 g) was passed over a silica gel column (4 × 80 cm), eluted with CHCl3−n-hexane and CHCl3−petroleum ether mixtures, to yield 10 subfractions (Fr.6.1− 6.10). Subfractions Fr.6.6 and Fr.6.10 were purified using preparative TLC with CHCl3−n-hexane (10:90) to afford compounds 7 (10.7 mg), 1 (5.3 mg), and 2 (2.5 mg). Subfraction Fr.6.8 was purified with MeOH−CHCl3 (5:95) to obtain compounds 6 (6.5 mg) and 31 (2.6 mg). Fraction Fr.7 (6.1 g) was subjected to silica gel column chromatography (5 × 100 cm) with MeOH−CHCl3 mixtures to yield eight subfractions (Fr.7.1−7.8). Subfraction Fr.7.1 was purified by preparative TLC using EtOAc−petroleum ether (10:90) to afford compounds 30 (6 mg) and 32 (5 mg). Subfractions Fr.7.4−7.5 were chromatographed with (CH3)2CO−petroleum ether, EtOAc−nhexane, and (CH3)2CO−n-hexane mixtures to afford compounds 8 (2.7 mg), 9 (9.0 mg), 10 (20.0 mg), and 15 (9.0 mg). Subfractions Fr.7.6−7.8 were subjected to ODS silica gel column chromatography using H2O−MeOH mixtures (80:20 → 70:30) as eluents, to afford compounds 16 (8.0 mg), 17 (3.5 mg), 20 (5.0 mg), and 21 (6.0 mg). Fraction Fr.8 (16.3 g) was passed over a silica gel column (8 × 150 cm) with MeOH−CHCl3 mixtures, to yield 12 subfractions (Fr.8.1− 8.12). Subfractions Fr.8.2−8.4 were chromatographed with MeOH− CHCl3 mixtures (5:95 → 20:80) to afford compounds 18 (3.2 mg), 19 (5.7 mg), and 28 (4.5 mg). Subfractions Fr.8.5−8.9 were subjected to ODS silica gel column chromatography with H2O−MeOH mixtures (100:0 → 50:50) and purified by preparative TLC using MeOH− CHCl3 (5:95) to afford compounds 22 (4.3 mg), 23 (3.0 mg), 24 (2.6 mg), and 35 (3.8 mg). Fractions Fr.9 (3.6 g) and Fr.11 (1.8 g) were chromatographed with (CH3)2CO−CHCl3, MeOH−CHCl3, and (CH3)2CO−MeOH mixtures to afford compounds 25 (5.0 mg), 26 (20.0 mg), 27 (16.0 mg), 4 (10.0 mg), 5 (3.0 mg), and 33 (20.0 mg). Fraction Fr.10 (21.3 g) was subjected to silica gel column chromatography with EtOAc−petroleum ether and MeOH−CHCl3 mixtures, to yield seven subfractions (Fr.10.1−10.7). Subfraction Fr.10.5 (9.1 g) was separated by column chromatography with

Cell viability (%) = [(Abs test sample − Abs blank )

■

/(Abscontrol − Abs blank )] × 100%

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00006. 1094



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Article

(26) Kim, S. Y.; Choi, Y.-H.; Huh, H.; Kim, J.; Kim, Y. C.; Lee, H. S. J. Nat. Prod. 1997, 60, 274−276. (27) Irungu, B. N.; Orwa, J. A.; Gruhonjic, A.; Fitzpatrick, P. A.; Landberg, G.; Kimani, F.; Midiwo, J.; Erdélyi, M.; Yenesew, A. Molecules 2014, 19, 14235−14246. (28) Vokác,̌ K.; Buděsň ský, M.; Harmatha, J.; Kohoutová, J. Phytochemistry 1998, 49, 2109−2114. (29) Wang, X.-X.; Shigemori, H.; Kobayashi, J. Tetrahedron 1996, 52, 2337−2342. (30) Shen, Y.-C.; Wang, S.-S.; Pan, Y.-L.; Lo, K.-L.; Chakraborty, R.; Chien, C.-T.; Kuo, Y.-H.; Lin, Y.-C. J. Nat. Prod. 2002, 65, 1848−1852. (31) Zhang, J.; Sauriol, F.; Mamer, O.; Zamir, L. O. Phytochemistry 2000, 54, 221−230. (32) Wang, S.-S.; El-Razek, M. H. A.; Chen, Y.-C.; Chien, C.-T.; Guh, J.-H.; Kuo, Y.-H.; Shen, Y.-C. Chem. Biodiversity 2009, 6, 2255−2262. (33) Agrawal, P. K. Phytochemistry 1992, 31, 3307−3330. (34) Appendino, G.; Ö zen, H. Ç .; Gariboldi, P.; Torregiani, E.; Gabetta, B.; Nizzola, R.; Bombardelli, E. J. Chem. Soc., Perkin Trans. 1 1993, 1563−1566. (35) Ni, Z.-Y.; Wu, Y.-B.; Zhang, M.-L.; Wang, Y.-F.; Dong, M.; Sauriol, F.; Huo, C.-H.; Shi, Q.-W.; Gu, Y.-C.; Kiyota, H.; Cong, B. Biosci., Biotechnol., Biochem. 2011, 75, 1698−1701. (36) Kingston, D. G. I. Phytochemistry 2007, 68, 1844−1854.

Copies of spectroscopic data for 1−6 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail (N. T. Nguyen): [email protected]. Tel: +84-907426-331. Fax: +84-838-353-659. ORCID

Nhan Trung Nguyen: 0000-0001-5142-4573 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant from Vietnam National University, Ho Chi Minh City (No. A2015-18-02), to M.T.T.N.

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