Uvaridacols E–H, Highly Oxygenated Antiausterity Agents from Uvaria

Oct 23, 2012 - Discovery of potential antiausterity agents from the Japanese cypress Chamaecyparis obtusa. Dya Fita Dibwe , Sijia Sun , Jun-ya Ueda ...
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Uvaridacols E−H, Highly Oxygenated Antiausterity Agents from Uvaria dac Suresh Awale,*,†,‡ Jun-ya Ueda,†,‡ Sirivan Athikomkulchai,§ Dya Fita Dibwe,‡ Sherif Abdelhamed,‡ Satoru Yokoyama,‡ Ikuo Saiki,‡ and Ryuta Miyatake⊥ †

Frontier Research Core for Life Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan § Faculty of Pharmacy, Srinakharinwirot University, Nakhon Nayok, 26120, Thailand ⊥ Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan ‡

S Supporting Information *

ABSTRACT: Chemical investigation of the stems of Uvaria dac yielded four new highly oxygenated cyclohexene derivatives named uvaridacols E−H (1−4). Their structures were established through NMR and circular dichroism spectroscopic analysis. Uvaridacols E (1), F (2), and H (4) displayed weak preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions in a concentration-dependent manner, without causing toxicity in normal nutrient-rich conditions.

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he plant Uvaria dac Pierre ex Finet & Gagnep belongs to the Annonaceae family and is a woody tree found mainly in Southeast Asian countries such as Thailand, Burma, and Vietnam. In a continued study on the antiausterity strategybased screening of medicinal plants,1−9 the CH2Cl2 extract of the stems of U. dac from Thailand showed preferential cytotoxic activity against the PANC-1 human pancreatic cancer cell line. The observed preferential cytotoxicity has been attributed to (+)-grandifloracin and cyclohexene derivatives.10 Further study on this bioactive extract resulted in the isolation of four new highly oxygenated cyclohexenyl derivatives, named uvaridacols E−H (1−4). We report herein the structure of these new compounds and their preferential cytotoxic activity against the PANC-1 human pancreatic cancer cell line.

Uvaridacol E (1) was isolated as a white, amorphous solid. Its molecular formula was determined by HRFABMS to be C21H20O7 [m/z 385.1278 (M + H)+]. The IR spectrum of 1 showed the absorptions due to hydroxy (3446 cm−1), ester carbonyl (1636 cm−1), and aromatic ring (1601, 1452 cm−1) functionalities. The 1H NMR spectrum of 1 showed signals due to three oxymethines (δH 4.17, H-2; 4.50, H-4; 5.44, H-3), an oxymethylene (δH 4.50, 4.72, H2-7), and two olefinic methines (δH 5.79, H-6; 5.89, H-5), together with those for two benzoyl groups (Table 1). In turn, the 13C NMR spectrum showed 21 carbon signals including those for five oxygenated sp3 carbons (δC 67.1, 70.1, 74.5, 74.7, and 78.1), two olefinic carbons (δC 129.5 and 130.0), and two benzoyl groups (Table 2). Analysis of the 1H−1H COSY and HMQC spectra revealed the partial connectivities (bold line) between C-2−C-3−C-4−C-5−C-6, which were connected further based on long-range HMBC correlations (Figure 1a). In the HMBC spectrum of 1, the longrange correlations from the oxymethylene protons at δH 4.50, 4.72 (H2-7) to the oxymethine carbon at δC 78.1 (C-2), the olefinic methine carbon at δC 129.5 (C-6), and the quaternary oxygenated carbon at δ 74.7 (C-1) suggested the connectivity of C-2, C-6, and C-7 via the quaternary carbon C-1. Furthermore, significant correlations of the H2-7 oxymethylene protons and the aromatic protons at δH 8.07 (H-2′,6′) with the ester carbonyl carbon at δC 165.9, and of the oxymethine proton at δH 5.44 (H-3) and the aromatic protons at δH 8.12 (H-2″, 6″) with the ester carbonyl carbon at δC 166.9, indicated the locations of the two benzoyl groups to be at C-3 and C-7, Received: August 31, 2012

© XXXX American Chemical Society and American Society of Pharmacognosy

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Table 1. 1H NMR (400 MHz, CDCl3) Spectroscopic Data of Compounds 1−4 position

1

2 3 4 5 6 7

4.17, 5.44, 4.50, 5.89, 5.79, 4.50, 4.72,

OBz-7 2′, 6′ 3′, 5′ 4′

2

d (10.1 Hz) dd (10.1, 6.4 Hz) m dd (10.0, 3.0 Hz) d (10.0 Hz) d (11.5 Hz) d (11.5 Hz)

5.92, 5.62, 4.69, 6.14, 5.99, 4.23, 4.49,

8.07, m 7.47, m 7.60, m OBz-3 8.12, d (7.3 Hz) 7.47, m 7.60, m

2″, 6″ 3″, 5″ 4″ OCOCH3

3

d (11.0 Hz) dd (11.0, 4.1 Hz) m dd (10.1, 5.3 Hz) d (10.1 Hz) d (11.2 Hz) d (11.2 Hz)

8.04, m 7.46, m 7.59, q (7.3 Hz) OBz-3 8.04, m 7.46, m 7.59, q (7.3 Hz) 1.96, s

4

5.33, d (10.5 Hz) 4.16, dd (10.5, 7.8 Hz) 4.40, m 5.90, dd (10.5, 2.3 Hz) 5.74, d (10.5 Hz) 4.5, d (11.5 Hz) 4.46, d (11.5 Hz)

5.48, 4.57, 5.57, 5.79, 5.89, 4.57, 4.43,

d (11.0 Hz) br s m dd (10.1, 2.3 Hz) d (10.1 Hz) d (11.5 Hz) d (11.5 Hz)

7.95, d (7.8 Hz) 7.39, m 7.55, m OBz-2 8.03, d (7.8 Hz) 7.39, m 7.55, m

8.12, d (7.8 Hz) 7.46, m 7.59, m OBz-4 8.00, d (7.8 Hz) 7.46, m 7.59, m 1.97, s

Table 2. 13C NMR (100 MHz, CDCl3) Spectroscopic Data of Compounds 1−4 position 1 2 3 4 5 6 7 OBz-7 1′ 2′, 6′ 3′, 5′ 4′ 7′ 1″ 2″, 6″ 3″, 5″ 4″ 7″ OCOCH3 OCOCH3

1

2

3

4

74.5 78.1 70.1 74.7 130.0 129.5 67.1

72.4 68.3 71.1 65.6 130.2 130.9 67.6

74.3 79.6 73.9 72.2 130.2 129.7 67.0

74.1 75.5 71.3 76.5 130.2 129.7 66.6

129.5 128.6 128.6 133.5 165.9 OBz-3 129.2 129.9 128.6 133.8 166.9

129.2 129.9 128.6 133.5 166.5 OBz-3 129.5 129.9 128.7 133.8 165.8 20.9 170.0

129.3 129.7 128.5 133.3 166.2 OBz-2 129.9 130.0 128.7 133.9 167.9

129.0 129.9 128.6 133.5 166.6 OBz-4 129.9 129.9 128.6 133.8 167.4 20.8 171.5

Figure 1. Connectivities (bold line) deduced by the COSY and HMQC spectra and HMBC correlations (→) observed for compounds 1−4.

HRFABMS, corresponding to the molecular formula C23H22O8. The 1H and 13C NMR data of 2 resembled those of 1 and displayed signals due to two benzoyl groups, three oxymethines, one oxymethylene, and two olefinic protons. However, 2 showed one additional signal due to the presence of an acetyl group (Table 1). The locations of the acetyl and two benzoyl groups were determined to be at C-2 and at C-4 and C-7, respectively, based on the HMBC correlations observed (Figure 1b). The relative configuration of 2 was assigned on the basis of the NOESY correlations (Figure 2c) and the coupling constant data. As in 1, uvaridacol F (2) also showed a large coupling constant between H-2/H-3 (J2,3 = 11.0 Hz), suggesting their trans relationships, and a small coupling constant between H-3/H-4 (J2,3 = 4.1 Hz) indicated their cis relationship. Furthermore, NOESY correlations between an acetyl methyl group (δH 1.96) with an oxymethylene (δH 4.23, 4.49; H2-7) and the aromatic protons at δH 7.46 (H-2′,6′) indicated these groups to be similarly oriented. In contrast to 1, the CD spectrum of 2 showed a positive Cotton effect ([θ]244 +11 778, [θ]237 −6560) due to exciton chirality,11,12 suggesting the spatial orientation of the two benzoates at C-3 and C-7 to be in a clockwise fashion (Figure 2d). Therefore, the absolute

respectively. The relative configuration of 1 was assigned on the basis of the coupling constant data and NOESY correlations (Figure 2a). Compound 1 showed a large coupling constant between H-2/H-3 (J2,3 = 10.1 Hz), indicating their trans relationship. Similarly, NOESY correlations between H-2/H-4 and the coupling constant between J3,4 = 6.4 Hz suggested a trans relationship between H-3/H-4. Likewise, a NOESY correlation between H-3/H2-7 suggested that they are oriented axially on the same side of the molecule. The absolute configuration of 1 was established from the negative Cotton effect ([θ]246 −10 254) observed in the CD spectrum, suggesting the spatial orientation of the two benzoates at C-3 and C-7 to be in a counterclockwise manner (Figure 2b).11,12 Therefore, the absolute configuration of uvaridacol E (1) was established as 1R, 2S, 3R, and 4S. Uvaridacol F (2) was obtained as a white, amorphous solid. It showed the molecular ion at m/z 427.1379 (M + H)+ in the B

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Figure 2. NOESY correlations (green →), CD spectra, and exciton chirality (blue →) for compounds 1−4.

and two olefinic protons. However, there was evidence of an upfield shift of H-3 to δH 4.57 (δH 5.62, 2) and a downfield shift of H-4 to δH 5.57 (δH 4.69, 2) in uvaridacol H (4). Therefore, a hydroxy group at C-3 and a benzoyloxy group at C-4 were evident, which were confirmed from the HMBC spectrum (Figure 1d). Uvaridacol H (4) showed large coupling constants between H-2/H-3 (J2,3 = 11.0 Hz) and H-3/H-4 (J3,4 = 11.0 Hz), suggesting their trans relationship. In the NOESY spectrum, correlations were observed between H2-7/H-2, H27/H-6, and H-2/H-4, suggesting H2-7, H-2, and H-4 all to be in the same orientation (Figure 2g). Uvaridacol H (4) displayed a negative Cotton effect (Figure 2h) in its CD spectrum ([θ]242 −17 250), attributable to exciton coupling of the allylic benzoyl group at C-4.12 Therefore, the absolute configuration of uvaridacol H (4) was determined as 1R, 2R, 3S, and 4R. The compounds isolated were tested for their cytotoxic activity against a PANC-1 human pancreatic cancer cell line in normal nutrient-rich medium (DMEM) and nutrient-deprived medium (NDM), utilizing an antiausterity strategy.13 When compared to normal cells, human pancreatic tumor cells such as PANC-1 cells show an extraordinary capacity of tolerance to nutrition starvation that enable them to survive in the hypovascular (austerity) tumor microenvironment.14 Moreover, these cell lines are extremely resistant to conventional anticancer drugs in clinical use. Therefore, the search for agents that preferentially inhibit the survival of cancer cells under low nutrition conditions (antiausterity agent) is a novel approach in anticancer drug discovery. While conventional anticancer drugs are nonselective and often toxic to normal cells growing in nutrient-sufficient conditions, antiausterity agents are selectively toxic to cancer cells surviving under a low nutrition state, a characteristic condition of the tumor microenvironment. Among the test compounds, 1 (PC50, 135 μM), 2 (PC50, 124 μM), and 4 (PC50, 156 μM) exhibited a weak preferential cytotoxicity against the PANC-1 cell line,

configuration of uvaridacol F (2) was established as 1S, 2R, 3S, and 4S. Uvaridacol G (3) was isolated as a colorless, amorphous solid. Its molecular formula was deduced to be the same as uvaridacol E (1) from the HRFABMS, as C21H20O7 [m/z 385.1285 (M + H)+]. The 1H and 13C NMR spectra of 3 also closely resembled those of 1 (Tables 1 and 2) and showed signals due to three oxymethines, an oxymethylene, two olefinic methines, and two benzoyl groups. However, they were characterized by the downfield shift of H-2 to δH 5.33 (δH‑2 4.17, 1) and the upfield shift of H-3 to δH 4.16 (δH‑3 5.44, 1). Therefore, the presence of a benzoyl at C-2 and a hydroxy at C3 was assumed. HMBC correlations were observed between the ester carbonyl carbon at δC 167.9 (OCO-2) and the protons at δH 8.04 (H-2″, 6″) and 5.33 (H-2), confirming the location of the benzoyl substituent at C-2 (Figure 1c). The relative configuration of 3 was assigned on the basis of the NOESY correlations and the coupling constant data. Compound 3 also showed large coupling constants between H-2/H-3 (J2,3 = 10.5 Hz) and H-3/H-4 (J3,4 = 7.8 Hz), suggesting their trans relationship to each other. On the other hand, NOESY correlations were observed between H-2 and H-4, suggesting that they are axially oriented toward the same direction (Figure 2e). Compound 3 showed a negative Cotton effect ([θ]242 −6248) due to exciton chirality (Figure 2b), suggesting the spatial orientation of two benzoates at C-2 and C-7 to be in a counterclockwise manner (Figure 2f).11,12 Therefore, the absolute configuration of 3 was concluded to be 1R, 2R, 3S, and 4R. Uvaridacol H (4) was obtained as a white, amorphous solid. Its molecular formula was deduced to be the same as uvaridacol F (2) from the HRFABMS, as C23H22O8 [m/z 427.1393 (M + H)+]. The 1H and 13C NMR data of 4 closely resembled those of uvaridacol F (2) and displayed signals due to two benzoyl groups, an acetyl group, three oxymethines, one oxymethylene, C

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while 3 was inactive (PC50, >200 μM). Arctigenin, an antiausterity strategy-based anticancer agent,1 was used as positive control and showed the most potent preferential activity (PC50, 1.0 μM). In contrast, gemcitabine and 5-FU, which are clinically used anticancer drugs for the treatment of pancreatic cancer,15 were inactive in both NDM and DMEM (PC50, >200 μM) at the maximum dose used, when tested for 24 h.

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ASSOCIATED CONTENT

S Supporting Information *

1 H and 13C NMR spectra of compounds 1−4. This information is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-76-434-7640. Fax: +81-76-434-7640. E-mail: [email protected].

EXPERIMENTAL SECTION

Notes

The authors declare no competing financial interest.

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General Experimental Procedures. Optical rotations were measured on a JASCO P2100 digital polarimeter. CD measurements were carried out on a JASCO J-805 spectropolarimeter. IR spectra were measured with a JASCO FT/IR-460 Plus spectrophotometer. NMR spectra were recorded on a JEOL ECX400 Delta spectrometer with TMS as internal standard, and chemical shifts are expressed in δ values. HRFABMS measurements were carried out on a JEOL JMSAX505HAD mass spectrometer, and glycerol was used as matrix. Analytical and preparative TLC were carried out on precoated silica gel 60F254 and RP-18F254 plates (Merck, 0.25 or 0.50 mm thickness). Plant Material. The stems of Uvaria dac were collected at Sakaerat Environmental Research Station, Nakhon Ratchasima Province, Thailand, in May 2011 and were authenticated by Dr. Atchara Teerawatananon (Natural Research Division, National Science Museum, Thailand). A voucher specimen (TMPW 27320) was deposited at the Museum for Materia Medica, Institute of Natural Medicine, University of Toyama, Japan. Extraction and Isolation. A CH2Cl2 extract (4.0 g) of the stems of U. dac was chromatographed on silica gel using a MeOH−CH2Cl2 gradient system (Buchi MPLC, C-601/C-605 dual pump), to give nine fractions. Fraction 7 (eluted with 12% MeOH−CHCl3; 55 mg) was subjected to reversed-phase preparative TLC with CH3CN−acetone− H2O (1:1:1) followed by purification with normal-phase preparative TLC, with 2% MeOH−CH2Cl2, to afford 1 (2 mg) and 2 (3 mg). Fraction 8 (eluted with 14% MeOH−CHCl3, 102 mg) was subjected to reversed-phase preparative TLC with CH3CN−acetone−H2O (2:2:1) to give 4 (2 mg). Fraction 9 (an eluent of 20% MeOH− CHCl3, 250 mg) was purified by reversed-phase preparative TLC with CH3CN−acetone−H2O (1:1:1), to give 3 (3 mg). Uvaridacol E (1): white, amorphous solid; [α]23D −16.4 (c 1.0, CHCl3); CD (c 2.60 × 10−4 M, EtOH) [θ]246 −11 395; IR νmax (KBr) 3446, 2923, 1636, 1452, 1217 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRFABMS m/z 385.1278 [M + H]+ (calcd for C21H21O7, 385.1287). Uvaridacol F (2): white, amorphous solid; [α]25D −52 (c 1.0, CHCl3); CD (c 2.34 × 10−4 M, EtOH) [θ]244 +11 778, [θ]237 −6560; IR νmax (KBr) 3450, 2923, 1720, 1452, 1279 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRFABMS m/z 427.1379 [M + H]+ (calcd for C23H23O8, 427.1393). Uvaridacol G (3): white, amorphous solid; [α]25D −12 (c 1.0, CHCl3); CD (c 2.60 × 10−4 M, EtOH) [θ]242 −6248; IR νmax (KBr) 3651, 2925, 2365, 1648 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRFABMS m/z 385.1285 [M + H]+ (calcd for C21H21O7, 385.1287). Uvaridacol H (4): white, amorphous solid; [α]23D−123 (c 1.0, CHCl3); CD (c 2.27 × 10−4 M, EtOH) [θ]242 −17 250; IR νmax (KBr) 3446, 1723, 1542, 1374, 1271 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRFABMS m/z 427.1393 [M + H]+ (calcd for C23H23O7, 427.1393). Preferential Cytotoxic Activity. The PANC-1 (RBRCRCB2095) human pancreatic cancer cell line was purchased from Riken BRC cell bank and maintained in standard Dulbecco’s modified Eagle's medium (D-MEM) with 10% FBS supplement, 0.1% NaHCO3, and 1% antibiotic−antimycotic solution. All the biological materials as well as in vitro preferential cytotoxicity of the isolated compounds were the same as described previously.10

ACKNOWLEDGMENTS This work was supported by a grant from Toyama Support Center for Young Principal Investigators in Advanced Life Sciences and a Grant-in-Aid for Scientific Research C (No. 24510314) JSPS KAKENHI, Grant-in-Aid for Scientific Research C (No. 24510314).

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