Goniolanceolatins AâH, Cytotoxic Bis-styryllactones from

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Goniolanceolatins A−H, Cytotoxic Bis-styryllactones from Goniothalamus lanceolatus Nur V. Bihud,† Nurulfazlina E. Rasol,† Syahrul Imran,† Khalijah Awang,‡ Fasihuddin B. Ahmad,§ Chun-Wai Mai,⊥ Chee-Onn Leong,⊥ Geoffrey A. Cordell,∥ and Nor Hadiani Ismail*,†

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†

Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA Selangor Branch, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Malaysia Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia ‡ Department of Chemistry, Faculty of Science, University Malaya, 50603 Kuala Lumpur, Malaysia § Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Semarahan, Sarawak, Malaysia ⊥ School of Pharmacy, International Medical University, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Bukit Jalil, Kuala Lumpur, Malaysia ∥ Natural Products Inc., Evanston, Illinois 60203, United States College of Pharmacy, University of Florida, Gainesville, Florida 32610, United States S Supporting Information *

ABSTRACT: Eight new bis-styryllactones, goniolanceolatins A−H (1−8), possessing a rare α,β-unsaturated δ-lactone moiety with a (6S)-configuration, were isolated from the CH2Cl2 extract of the stembark and roots of Goniothalamus lanceolatus Miq., a plant endemic to Malaysia. Absolute structures were established through extensive 1D- and 2DNMR data analysis, in combination with electronic dichroism (ECD) data. All of the isolates were evaluated for their cytotoxicity against human lung and colorectal cancer cell lines. Compounds 2 and 4 showed cytotoxicity, with IC50 values ranging from 2.3 to 4.2 μM, and were inactive toward human noncancerous lung and colorectal cells. Compounds 1, 3, 6, 7, and 8 showed moderate to weak cytotoxicity. Docking studies of compounds 2 and 4 showed that they bind with EGFR tyrosine kinase and cyclin-dependent kinase 2 through hydrogen bonding interactions with the important amino acids, including Lys721, Met769, Asn818, Arg157, Ile10, and Glu12.

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Goniothalamus lanceolatus Miq. is an endemic plant from the rainforest jungle of Sarawak, Malaysia, and is used by the indigenous population as an alternative medicine to treat cancer. A small amount of either the dried roots or stembark is boiled in water with medium heat for about 30 min, and the decoction is consumed twice a week. In addition, the leaves are boiled in water and used for bathing with the purpose of treating skin infections and to quench fever. The burning leaves are used as a mosquito repellent. Despite the interesting ethnomedical properties, phytochemical and biological studies on G. lanceolatus are limited. In previous chemical investigations of G. lanceolatus, alkaloids and styryllactones were reported from the roots and stembark, with several compounds possessing promising cytotoxic activity against lung and colorectal cancer cell lines.14,15In vivo testing for antiplasmodial activity was

lants in the genus Goniothalamus (Blume) Hook. f. & Thomson, family Annonaceae, can be found in the tropical forests of Southeast Asia and include about 165 species comprising shrubs and trees.1,2 Members of this genus are utilized in many communities as traditional medicines to treat common illnesses;1 consequently, extensive studies have been conducted for their chemical constituents and biological and pharmacological properties. Goniothalamus species are well-known phytochemically as a rich source of styryllactones. More than 100 such derivatives have been reported, with commonly reported skeleta being of the styrylpyrone, furanopyrone, furanofurone, pyranopyrone, butenolide, and heptolide types.3 Bis-styryllactones, however, are a less common occurrence and were previously reported from G. leiocarpus (W.T. Wang) P.T. Li.,4 G. cheliensis Hu,5−7 and G. amuyon (Blanco) Merr.8 Styryllactones were reported to possess significant cytotoxic activity against several tumor cell lines,8−11 as well as having antimycobacterial11,12 and antiplasmodial activities.11−13 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 18, 2018

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DOI: 10.1021/acs.jnatprod.8b01067 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of compounds 1−8.

corresponding to a molecular formula of C26H26O7. The 1H NMR spectrum of 1 showed five aromatic proton signals between δ 7.33−7.42, indicating the presence of a monosubstituted phenyl moiety (H-10 to H-14). Two olefinic protons resonating at δ 6.02 (dd, J = 9.6, 2.4 Hz) and δ 6.94 (ddd, J = 9.6, 6.6, 1.8 Hz) were typical of an α,β-unsaturated δlactone moiety and were assigned as H-3 and H-4, respectively.18 Three oxygenated methine protons at δ 3.69 (dd, J = 8.4, 1.2 Hz), δ 4.41 (d, J = 8.4 Hz), and δ 4.88 (ddd, J = 13.2, 4.2, 1.2 Hz) assigned as H-7, H-8, and H-6 and a pair of geminal methylene protons at δ 2.20 (ddd, J = 18.6, 6.0, 3.6 Hz, H-5a) and δ 2.78 (dddd, J = 18.6, 12,6, 4.8, 2.4 Hz, H-5b) were observed. The 13C NMR spectrum displayed 13 signals, with the most downfield resonance at δ 163.5, characteristic for a carbonyl at C-2 of an α,β-unsaturated δ-lactone moiety.9 Three oxygen-bearing carbons were observed at δ 74.7 (C-7), δ 75.7 (C-6), and δ 77.7 (C-8), and two methine signals for C3 and C-4 were found at δ 120.8 and δ 146.8, respectively. The methylene carbon resonated at δ 26.1, while the quaternary carbon signal of C-9 was observed at δ 137.0. These 1H and 13 C NMR data are summarized in Table 1. The COSY correlations of H-7 with both H-8 and H-6 and the 13JCH HMBC correlations of H-8 with C-10 and C-14 revealed that the monosubstituted phenyl moiety and the α,βunsaturated δ-lactone moiety were connected through a twocarbon bridge, C-7 and C-8. Comparison of the 1H and 13C NMR spectroscopic data of 1 with those of (6S,7S,8S)-

conducted on the n-hexane, CH2Cl2, and MeOH crude extracts.16 A continuation of these studies has led to the isolation of eight new bis-styryllactones, goniolanceolatins A− H (1−8) and four known styryllactones (9−12) possessing the rare (6S)-styrylpyrone and (1S)-pyranopyrone moieties. All of the metabolites were isolated from the stembark, except for goniolanceolatin E 5, which was obtained from the root extract. Interestingly, previous reports of (6S)-styryllactones from nature are rare;17 hence, their discovery in G. lanceolatus enhances interest in this endemic species from Malaysia. Herein, the isolation, structure elucidation, and cytotoxic activities, as well as a biogenetic pathway for the new bisstyryllactones, are described.

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RESULTS AND DISCUSSION

The crude CH2Cl2 extracts from the dried stembark and roots of G. lanceolatus were deemed of interest based on preliminary cytotoxicity assessment against lung and colorectal cancer cell lines which revealed cell viability of less than 20% at 100 μM after 72 h. Chromatographic separation of the extracts utilizing MPLC, HPLC, and recycling HPLC afforded 12 styryllactones, of which 1−8 were new compounds (Figure 1). The IR spectra of compounds 1−8 indicated the presence of hydroxy and carbonyl groups through absorptions in the range of 3389−3466 cm−1 and 1687−1745 cm−1, respectively. Compound 1, a white amorphous powder, gave an [M + Na]+ ion at m/z 473.1556 (calcd 473.1571) in OBITRAP-MS, B


6 7 8 9 10, 14 11, 13 12 2′ 3′ 4′ 5a′ 5b′

C

a

7.26−7.44, m

6.02 dd (9.6, 2.4) 6.94, ddd (9.6, 6.6, 1.8) 2.20, ddd (18.6, 6.6, 3.6) 2.78, dddd (18.6, 13.2, 4.8, 2.4) 4.88, ddd (13.2, 4.8, 1.2) 3.69, dd (8.4, 1.2) 4.41, d (8.4)

7.26−7.44, m

6.02, dd (9.6, 2.4) 6.94, ddd (9.6, 6.6, 1.8) 2.20, ddd (18.6, 6.6, 3.6) 2.78, dddd (18.6, 13.2, 4.8, 2.4) 4.88, ddd (13.2, 4.8, 1.2) 3.69, dd (8.4, 1.2) 4.41, d (8.4)

δH (J in Hz)

1

δC

76.6 75.7 82.6 138.3 127.2 128.2 128.0

76.1 76.5 83.7 138.2 127.6 128.3 128.1 163.5 120.6 145.9 25.9

163.7 120.6 145.7 26.1

7.17−7.25, m

b

6.02, dd (9.6, 2.4) 6.88, ddd (9.6, 6.6, 2.4) 2.11, ddd (18.6, 6.6, 3.6) 2.62, dddd (18.6, 12.6, 4.8, 2.4) 4.84, ddd (12.6, 7.2, 3.0) 3.89, dd (6.0, 3.0) 4.95 d (6.0)

7.17−7.25, m


δH (J in Hz)

2a δC

76.9 78.9 72.0 140.8 126.4 128.8 128.6

76.3 74.4 79.2 137.5 126.4 128.6 128.8 163.1 120.5 145.6 25.9

163.7 120.9 145.7 26.1

7.37−7.44, m

5.90, dd (9.6, 3.0) 6.73, ddd (9.6, 6.6, 2.4) 1.66, ddd (18.6, 6.6, 4.2) 2.44, dddd (18.6, 12.0, 4.8, 2.4) 4.44, m 3.54, dd (5.4, 3.0) 5.25, d (5.4)

7.37−7.44, m

6.04, dd (9.6, 2.4) 6.95, ddd (9.6, 6.6, 2.4) 2.27, ddd (18.6, 6.6, 3.6) 2.73, dddd (18.6, 12.6, 4.8, 2.4) 4.92, ddd (12.6, 3.6, 1.8) 3.81, d (8.4) 4.58, d (8.4)

δH (J in Hz)

3a δC

74.4 74.4 75.3 136.2 128.1 128.5 128.9 20.1 169.7

75.6 74.7 77.9 136.7 128.2 128.9 129.0 163.1 121.3 144.6 26.1

163.3 120.9 145.6 26.2

1.67, s

13

7.22−7.37, m

5.04, ddd (11.0, 6.0, 1.5) 5.20, dd (9.5, 1.5) 4.61, d (9.5)

6.04, dd (8.5, 1.8) 6.88, ddd (8.5, 3.5, 1.8) 2.30, m

7.27−7.45, m


δH (J in Hz)

4b

13

1

Data were obtained in CDCl3(600 MHz for H, 150 MHz for C, δ in ppm) Data was obtained in CDCl3(500 MHz for H, 125 MHz for C, δ in ppm)

75.7 74.7 77.7 137.0 128.1 129.0 129.0

75.7 74.7 77.7 137.0 128.1 129.0 129.0 163.5 120.8 146.8 26.1

2 3 4 5a 5b

6′ 7′ 8′ 9′ 10′, 14′ 11′, 13′ 12′ 7′-COCH3 7′-COCH3 -OCH3

δC

163.5 120.8 146.8 26.1

position

1a

Table 1. 1H and 13C NMR Data of Compounds 1−5 δC

56.5

73.9 75.5 78.4 137.3 128.5 129.3 129.3

76.1 75.1 78.0 137.5 128.5 129.2 129.2 169.8 35.8 71.9 30.1

163.8 121.2 146.1 26.4

d (4.2) quin (4.2) ddd (15.0, 4.2, 3.0) ddd (15.0, 12.0, 3.6)

7.22−7.33, m

4.87, ddd (12.0, 3.0, 1.2) 3.59, dd (8.4, 1.2) 4.33, d (8.4)

2.63, 3.80, 1.88, 2.05,

7.22−7.33, m


δH (J in Hz)

5a

Journal of Natural Products Article



Article

Figure 2. Selected 1H−1H COSY and1H−13C HMBC correlations of compounds 1−8.

between H-7 and H-8, as well as H-7′ and H-8′.9 The 3J6/7 and 3J6′/7′ values were smaller (1.2 Hz), which indicated that the bond angles of H-6/H-7 and H-6′/H-7′ were close to 90°.7 The NOESY spectrum showed correlations between H-6/H-6′ and the aromatic protons H-10/H-10′ and H-14/H-14′, suggesting their close proximity. The absolute configuration was determined using electronic circular dichroism (ECD) data according to the reported model for related styrylpyrones.3,20,21 The negative Cotton effect in the 250−272 nm region is consistent with a (6S) configuration (Figure 4).15,17 Based on biosynthetic considerations and the above observations, the configuration of 1 was determined as (6S,7S,8S:6′S,7′S,8′S). This is the first report of a symmetrical bis-styryllactone with a 6S/6′S configuration from nature. Thus, the structure of goniolanceolatin A 1 was defined as shown in Figure 1. Compound 2 showed the same molecular formula C26H26O7 as 1, as deduced by its QTOF-MS, indicating the compounds to be isomeric. However, unlike 1, the 1D NMR spectra of 2 clearly shows that it is an unsymmetrical bis-styryllactone, revealing 19 proton and 26 carbon signals (Table 1), attributable to two goniodiol-type monomers. The two α,βunsaturated δ-lactone moieties were confirmed through

goniodiol, obtained previously from the stembark of the same plant,15 indicated high similarity, except for the upfield shift of H-8 by 0.54 ppm in 1. In the 13C NMR spectrum, C-8 of 1 (δ 77.7) was shifted downfield by 4.00 ppm when compared with C-8 of (6S,7S,8S)-goniodiol.15 Combining the 1D NMR data of 1, together with the observed molecular weight at m/z 473.1556 [M + Na]+, suggested the presence of two monosubstituted phenyl moieties and two α,β-unsaturated δlactone units, implying that 1 was composed of two goniodioltype monomers in a symmetrical arrangement. The HMBC spectrum showed an unusual correlation of H-8/H-8′ (δ 4.41) with C-8′/C-8 (δ 77.7), which can be explained by the 3JCH correlations of H-8 to C-8′ and the H-8′ to C-8. Therefore, it was concluded that two goniodiol-type monomers were connected through a dehydration process, forming an ether bridge between C-8 and C-8′ to produce a bis-styryllactone. The relative configuration was established from the coupling constants and NOESY data, followed by a fully energyminimized structure to observe the stereochemical relationships (Figure 3). The 3J7/8 and 3J7′/8′(H−C−C−H) coupling constants of 8.4 Hz, indicating that H-7 and H-8 and H-7′ and H-8′ were trans-oriented, with a dihedral angle of 160° (Karplus correlation)19 suggesting an erythro relationship D



Article

Figure 3. Three-dimensional perspective of the fully energy-minimized structures of compounds 1, 4, 5, 6, 7, and 8 and the observed selective 1 H−1H NOESY correlations.

carbonyl signals resonating at δ 163.5 (C-2′) and δ 163.7 (C2). The HMBC correlations between H-8 (δ 4.87) with C-8′ (δ 82.6) and of H-8′ (δ 4.95) with C-8 (δ 83.7) indicated that the two monomeric units were condensed through dehydration to form the ether bridge between C-8 and C-8′.

Analysis of the coupling constants involving H-6, H-6′, H-7, H-7′, H8, and H-8′ suggested that 2 differs from 1 only at the configuration of C-8′. The 3J7/8 coupling constant of 9.0 Hz, indicated an erythro conformation of H-7 and H-8, while the smaller vicinal coupling of 6.0 Hz for 3J7′/8′ in 2 suggested that H-7′ and H-8′ are in a slightly staggered threo arrangement. E



Article

Conformational analysis of 2 revealed that there are five preferred conformations having these erythro-threo arrangements with a total Boltzmann distribution of 90% (Table S1, Supporting Information). Consequently, the configuration of goniolanceolatin B 2 was assigned as (6S,7S,8S:6′S,7′S,8′R). The spectroscopic data of compound 3 were closely similar to those of 1 and 2. However, unlike 1 and 2, the HMBC spectrum showed a 3JCH correlation between H-7′ (δ 3.54) with C-8 (δ 79.2), indicating that 3 was ether-linked at C-7′ and C-8 (Figure 2). In 3, H-8 showed a NOESY correlation with H-7′, suggesting that these protons were cofacial (Figure S48, Supporting Information). Therefore, based on biosynthetic considerations, the absolute configuration of goniolanceolatin C 3 was assigned as (6S,7S,8S:6′S,7′S,8′S). This is the first reported bis-styryllactone having an ether bridge between C-8 and C-7′ of two styrylpyrone moieties. Compound 4 has a molecular formula of C28H28O8 as deduced by QTOF-MS. The spectroscopic data showed similarities with those of 2; however, the protonated molecular ion of 4 showed 42 mass units more than 2, indicative of an acetyl fragment. This was confirmed by a three-proton singlet at δ 1.67 in the 1H NMR spectrum and resonances at δ 20.1 and δ 169.7, respectively, in the 13C NMR spectrum. The H-7′ resonance (δ 5.20) in 4 was deshielded compared to 2 (δ 3.83), suggesting acetylation of the C-7′ hydroxy group. The NOESY experiment showed correlation of H-7′ with the methyl protons of the acetate group at δ 1.67 (Figure 3), while the HMBC experiment supported the substitution through a 3 JCH correlation of H-7′ with the ester carbonyl of the acetyl group at δ 169.7. The HMBC experiment also showed an ether linkage between C-8 (δ 77.9) and C-8′ (δ 75.3) (Figure 2). On the basis of the aforementioned analyses and biosynthetic considerations, the structure of goniolanceolatin D 4 was proposed to have a (6S,7S,8S:6′S,7′R,8′R) absolute configuration. Compound 5, with a molecular formula of C27H30O8 as deduced by OBITRAP-MS, showed an additional three-proton singlet at δ 3.34 (δC 56.5), characteristic of a methoxy group, and explaining the 31 mass units difference compared with 1− 3. The 13C NMR spectrum exhibited a signal at δ 163.8, in accordance with an α,β-unsaturated δ-lactone unit in a goniodiol-type moiety, and another resonance was observed at δ 169.8, suggesting a saturated δ-lactone moiety.22 The COSY correlations of the methylene protons H-3a′, H-3b′ (δ 2.63) and H-5a′ (δ 1.88) with an oxygenated methine proton at δ 3.80 (H-4′) indicated that the methine carbon was adjacent to two methylene carbons (Figure 2). The 3JCH HMBC correlations between the methoxy protons and the methine carbon C-4′, confirmed the location of the methoxy group. Methoxylation at C-4′ could have taken place biogenetically through a 1,4-Michael addition on the enone system. A correlation was also noted between the methylene protons (H-3a′, H-3b′) and a carbonyl resonance at δ 169.8, hence establishing that one fragment consisted of a saturated δlactone moiety. The coupling constant of H-4′/H-5′ (4.2 Hz), indicated that these protons possess a dihedral angle close to 60°.19 The NOESY spectrum showed no correlation between H-4′ and H-6′ indicating an opposite orientation. The HMBC correlations were also observed between H-8 (δ 4.36) and C-8′ (δ 78.0) and H-8′ (δ 4.33) with C-8 (δ 78.4), indicating that the two fragments were linked between C-8 and C-8′ through an ether bridge (Figure 2). On the basis of biosynthetic considerations, the absolute configuration of goniolanceolatin

E 5 was defined as (6S,7S,8S:4′R,6′S,7′S,8′S). This compound was isolated from the CH2Cl2 extract of the roots and was detected previously in the LCMS profile of the crude plant extract. Hence, it is believed that 5 is a true natural product. Compound 6 gave the same molecular formula C26H26O7 as 1−3, based on the OBITRAP-MS data. However, a comparison of the 1D NMR spectra with those of (6S,7S,8S)-goniodiol and (1S,5S,7S,8S)-deoxygoniopyrone B, isolated previously from the stembark and roots of this plant,15 indicated that 6 was composed of styrylpyrone and pyranopyrone-type moieties. This was confirmed by signals at δ 163.0 (C-2) and δ 169.3 (C-3′), indicating the presence of the α,β-unsaturated δ-lactone moiety of a styrylpyrone and the saturated δ-lactone moiety of a pyranopyrone.8 A W-type coupling, characteristic of a pyranopyrone skeleton,22 was observed between the oxymethine protons H-1′ (δ 4.42) and H-5′ (δ 4.54) in the COSY experiment, thus establishing a pyranopyrone-type monomer. The HMBC spectrum showed 3 JCH correlations of H-8 (δ 4.38) with C-8′ (δ 83.5), and H-8′ (δ 4.04) with C-8 (δ 80.9), establishing an ether bridge between C-8 and C-8′ (Figure 2). The C-7C-8 bond assumed an erythro conformation based on the 3J7/8 coupling constant of 9.0 Hz,9 while the C-6C-7 bond showed a threo conformation (1.2 Hz). The coupling constant between H-7′ and H-8′ in the pyranopyrone-type monomer was 8.4 Hz and thus in a trans-diaxial configuration, while the substituent groups were in equatorial orientations.22 The NOESY experiment showed a correlation between H-8′ and H-8, suggesting that both protons were spacially aligned (Figure 3). The experiment also showed a strong correlation between the axial H-7′ and the axial H-9b′, but no correlation was observed with H-4a′ and H-4b′. In addition, no correlation was observed between H-9a′/H-9b′ with either H-4a′/H-4b′ or H-8′. The above evidence inferred that both the pyrano and lactone rings were in boat (boat−boat) conformations. The ECD spectrum of 6 had a negative Cotton effect in the 250− 272 nm range (Figure 4), indicating that C-6 of the α,β-

Figure 4. Experimental ECD spectra for 1−4 and 6−8.

unsaturated δ-lactone moiety possessed an S configuration.17 On the basis of the biosynthetic pathway described previously, pyranopyrones are derived from (6S,7S,8S)- goniodiol through nucleophilic attack of the hydroxy group on the lactone ring followed by cyclization to produce two six-membered linkages.17 The C-1′ in the pyranopyrone was initially the C6 from the (6S,7S,8S)-goniodiol moiety; thus, on the basis of biosynthetic considerations, the configuration of C-1′ in 6 was F


Journal of Natural Products Table 2. 1H and

13

Article

C NMR Data of Compounds 6−8a 6

position

δC

2 3 4 5a 5b 6 7 8 8-COCH3 8-COCH3 9 10,14 11,13 12 1′ 3′ 4a′ 4b′ 5′ 7′ 8′ 9a′ 9b′ 10′ 11′,15′ 12′,14′ 13′

163.0 120.8 144.9 25.8 75.3 74.7 80.9

138.8 127.4 128.9 129.0 75.9 168.0 40.7 65.3 75.6 83.5 23.7 139.2 128.0 128.8 128.8

7 δH (J in Hz)

5.88, 6.78, 1.97, 2.63, 4.18, 3.48, 4.38,

dd (9.5, 2.8) ddd (9.5, 6.3, 2.0) ddd (18.6, 6.3, 3.9) dddd (18.6, 13.0, 5.0, 2.5) ddd (13.0, 4.2, 1.2) br s d (9.0)

7.21−7.40, m 4.42, br d (4.8) 2.47, 2.86, 4.54, 4.45, 4.04, 1.82, 2.52,

dd (18.0, 2.4) ddd (18.0, 3.0, 2.4) br.d (3.0) d (8.4) d (8.4) dd (15.0, 4.8) ddd (15.0, 4.8, 1.5)

7.35−7.45, m

δC 163.2 120.9 144.7 25.8 75.2 73.4 82.5

139.0 125.9 127.8 127.9 74.2 168.9 36.1 65.9 70.0 77.2 23.9 137.8 128.5 129.0 129.1

δH (J in Hz) 5.91, 6.74, 1.87, 2.52, 3.48, 3.46, 4.14,

dd (9.7, 2.2) ddd (9.7, 6.0, 1.8) ddd (18.0, 6.0, 3.6) dddd (18.0, 13.0, 5.0, 2.4) dd (13.0, 3.6) dd (8.7, 3.6) d (8.7)

7.33−7.41, m 4.19, br s 2.76, 2.89, 4.50, 4.95, 3.86, 1.50, 2.37,

dd (19.4, 5.4) d (19.4) br.s s br s m ddd (14.0, 4.0, 2.0)

7.33−7.41, m

8 δC 163.2 121.0 145.2 26.1 76.1 74.0 74.6 21.1 169.6 137.1 126.9 127.5 127.5 78.9 168.5 40.8 65.3 76.1 75.9 23.8 139.3 128.6 128.7 128.7

δH (J in Hz) 6.03, 6.94, 2.27, 2.78, 4.65, 3.92, 5.89, 2.11,

ddd (9.8, 2.8, 0.8) ddd (9.8, 6.6, 2.0) dddd (18.0, 6.6, 4.2, 0.8) dddd (18.0, 13.2, 5.4, 3.0) ddd (13.2, 4.2, 2.4) dd (7.8, 2.4) d (7.8) s

7.35−7.45, m 4.76, br d (4.4) 2.60, 2.96, 4.61, 4.36, 4.02, 2.02, 2.67,

dd (18.0, 2.4) ddd (18.0, 3.0, 1.2) br.d (3.0) d (9.0) dd (9.0, 1.2) dd (14.7, 4.2) ddd (14.7, 4.2, 1.6)

7.35−7.45, m

Data for 6−8 were obtained in CDCl3(600 MHz for 1H, 150 MHz for 13C, δ in ppm)

a

deduced as S. The absolute configuration of goniolanceolatin F 6 thus defined as (6S,7S,8S:1′S,5′S,7′S,8′R). The spectroscopic data of 7 were closely similar to those of 6 (Table 2), indicating a comparable scaffold. However, the dihedral angle for the pyranopyrone-type monomer could not be determined because the four oxymethine protons resonated as broad singlets, implying that the coupling constants between the protons were small (100 >100 >100 >100 >100 5.9 ± 0.5b 8.0 ± 2.2 4.1 ± 0.6b 9.5 ± 2.7 10.9 ± 2.1 4.1 ± 0.5b 5.5 ± 1.4b 4.1 ± 0.5b >10.0 13.0 ± 3.1 >10.0 >10.0 7.0 ± 1.1b >10.0 17.0 ± 1.8 a

Results are presented as mean (n = 3). bp < 0.05 compares to IC50 of Cisplatin in the respective cell lines

2.9 ± 0.3b >10.0 >10.0 >10.0 21.0 ± 5.6 3.2 ± 0.5b 5.2 ± 0.8 2.6 ± 0.2b 9.3 ± 1.0 11.9 ± 2.1 5.4 ± 0.6b 7.5 ± 1.2b 2.2 ± 0.07b >10.0 32.7 ± 4.8 7.8 ± 1.1 >10.0 6.4 ± 1.1 >10.0 12.4 ± 0.8 goniolanceolatin B 2 goniolanceolatin C 3 goniolanceolatin D 4 goniolanceolatin H 8 cisplatin

7.1 ± 1.3b 9.5 ± 1.9b 4.6 ± 0.03b >10.0 25.5 ± 6.3

>100 >100 >100 >100 >100

SW 48 Caco2 HCC 2998

noncancer lung cell

MRC5 NCI-H23 NCI-H1299

lung cancer cells

Calu1 A549 compounds

Table 3. Cytotoxicity Data of Compounds 2, 3, 4, and 8a

cytotoxicity (IC50, μM)

colorectal cancer cells

HCT116

noncancer colorectal cell

previously reported from the root extract of G. lanceolatus; however, this is the first report from the stembark of the plant (Figure S49, Supporting Information). Compound 12, (1S,5S,7R,8S)-3-exo,7-endo-(+)-8-epi-9-deoxygoniopypyrone (12),22 has never been reported from this plant. It is worth noting that while the known compounds 9−12 are monomeric, the new bis-styryllactones 1−8 are composed of two monomeric units (isolated from the plant15): either a pyranopyrone and a styrylpyrone unit or two styrylpyrone units with an ether bridge linkage through C-8/C-8′, C-8/C7′, or C-7/C-8′. The biosynthetic pathway for the styryllactones was proposed to occur from shikimic acid with the incorporation of two acetate-malonate units to form the basic carbon skeleton.2,23 Sequential tailoring reactions, including reductions, oxidations, and cyclizations at different positions then generated the different styryllactones. A plausible biogenetic pathway for the known compounds 9−12 was proposed previously. 17 The bis-styryllactones 1−8 comprise two structural moieties: either a pyranopyrone unit and a styrylpyrone unit or two styrylpyrone units with an ether bridge linkage through C-8/C-8′, C-8/C-7′, or C-7/C-8′. On the basis of biosynthetic considerations, it is considered that these compounds are derived from a pyranopyrone or styrylpyrone precursor condensing with another styrylpyrone, and they are linked through an ether bond formed through an overall enzymatic dehydration. Acid catalysis of a styrylpyrone containing secondary hydroxy groups at C-7 and C-8 protonates the hydroxy group to become a good leaving group, H2O, thus forming a 2° carbocation. Through an SN1 reaction, the carbocation combines with another nucleophile (hydroxy group from another styrylpyrone/pyranopyrone) leading to the formation of a new C−O−C bond. The final step is deprotonation of the product by a weak base, resulting in the ether-bridge linkage between the two moieties. A plausible biosynthetic pathway for 1 is depicted in Figure S50 (Supporting Information), and the isolated bis-styryllactones are considered to be derived through analogous reactions. Compounds 1−3 are formed from two (6S,7S,8S)-goniodiol moieties, which were isolated abundantly from the stembark of G. lanceolatus in a previous study,15 while compounds 4, 6, 7, and 8 are derived from a (6S,7S,8S)-goniodiol with the 8-Oacetyl derivative of (6S,7S,8S)-goniodiol, deoxygoniopypyrone B, and parvistone D, also isolated from the same plant.15 Compounds 1−8 (excluding 5) were evaluated against a panel of human lung and colorectal cancer cell lines (Table 3). The anticancer drug cisplatin was used as the positive control. Compounds 2, 3, 4, and 8 showed significant cytotoxicity with IC50 values less than 10 μM. Comparing to the IC50 value of cisplatin in the respective cancer cells, the compounds induced a statistical significant lower IC50 (p < 0.05), warranting further mechanistic study. Two of the compounds, 2 and 4, showed strong cytotoxicity toward most of the tested human lung and colorectal cancer cells, with IC50 values in the range of 2.2−7.8 μM. Both compounds possess configurational similarity at C8/C-8′ (8R- and 8′S), and the orientation of H-8/H-8′ on the ether linkage were in the opposite directions, hence indicating that a nonsymmetrical dimer produced enhanced cytotoxicity compared to the symmetrical dimer 1, which might possess a conformational constraint. It is interesting to note that most of the IC50 values of 2 and 4 were lower and also statistically significant (p < 0.05) compared to the IC50 of cisplatin in respective cell lines. In addition, 4 induced a lower IC50 than 2,

CCD 841 CoN

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able to position itself within the hydrophobic pocket behind the ATP binding site consisting of Val702, Ala719, Lys721, and Leu764. In addition, one of the key interactions observed is between the C-8 hydroxy group with the side chain of Lys721 at 2.69 Å, which in turn was recognized as a gatekeeper of the hydrophobic pocket. On the other hand, it was observed that the C-8′ hydroxy group forms a hydrogen bond interaction to the main chain (NH) of Met769 in the hinge region at 2.31 Å. Docking results for compound 4 (Figure 5) showed binding with EGFR tyrosine kinase through hydrogen bonding interactions with amino acids like Lys721 and Asn818. The docking pose of 4 reveals the presence of hydrophobic (π−π and π-alkyl) interactions with the protein residues Val702, Leu694, Ala719, Met769, Leu820, Met769, Lys721, Leu764, and Met742, located near the gatekeeper Thr799. Residues like Val702, Leu694, Ala719, Met769, and Leu820 form electrostatic π−π interactions with both phenyl rings that further stabilizes the ligand−enzyme complex for 4. Additionally, the α,β-unsaturated conjugated bonds also contribute to the complex stability through π-alkyl interactions with the residues Met769, Lys721, Leu764, and Met742. The side chain (NH) of Lys721 establishes hydrogen bonding with the carbonyl oxygen of the C-8 ester at 2.48 Å. This interaction is important, as Lys721, which is located in the ATP-binding site of EGFR, is considered to be one of the key residues to influence the biological activity of EGFR.30 Another hydrogen bonding interaction was observed between the carbonyl oxygen of the α,β-unsaturated δ-lactone and the side chain (HD21) of the catalytic residue Asn818 at 2.18 Å. Docking studies of the active compounds 2 and 4 against cyclin-dependent kinase 2 (PDB: 2R3J) showed that they interact well with residues within the active site. Figure 6 shows that 2 has fewer hydrogen bonding interactions; however, the most significant interaction involved a residue in the inhibitory phosphorylation site, Thr14.31 A hydrogen bonding interaction was observed between the carbonyl oxygen on the α,β-unsaturated δ-lactone unit with the side chain (NH) of Thr14 at 2.09 Å. Another hydrogen bonding was observed between the C-8′ hydroxy group with the side chain (Hε) of Arg157 at 2.33 Å, which is expected to further downregulate the activity of CDK2. Compound 4 binds to the enzyme through hydrogen bonding with active site residues such as Arg157, Ile10, and Glu12, while being stabilized by other residues through electrostatic interactions. The carbonyl oxygen on the α,βunsaturated δ-lactone moiety is able to form a hydrogen bond with the NH2 side chain (NH21) of Arg157 at 1.76 Å. The oxygen of the ester carbonyl of 4 forms another hydrogen bonding interaction with the side chain (NH) in subdomain I (Glu 12) at 1.98 Å, which is important for the correct localization of the ATP triphosphate.32 Another hydrogen bond is formed between the C-8′ hydroxy group and the side chain of Ile10 at 2.04 Å. The ligand−enzyme complex of 4 is further stabilized by π−π interactions with residues in the functional region of the ATP binding site involving Ile10, Leu83, Asp86, Gln131, and Leu134. As these are essential residues that form hydrogen bonds with ATP, the π−π interactions are necessary to obtain stronger and more selective binding interactions.33 Further stabilization of the binding site is achieved through the ability of 4 to bury the α,β-unsaturated δ-lactone moiety within the protein core, thus creating a hydrophobic core area. This allows the α,β-unsaturated conjugation on the α,β-unsaturated δ-lactone to develop a

suggesting that an acetate group at C-7′ in 4 enhances the cytotoxicity. However, the IC50 valuesof 2 and 4 were not statistically different (p < 0.05, data not shown). Compound 3, which has an ether linkage at H-7′/H-8 showed mild cytotoxicity, with IC50 values in the range of 5.2−9.5 μM. Compound 8 showed significant cytotoxicity toward two cancer cell lines (NCI-H23 and HCT116) with IC50 values 9.3 and 9.5 μM. Compounds 1, 6, and 7 showed weak cytotoxicity. The dramatic change in the cytotoxicity of 6−8 indicates that the respective moieties in a bis-styryllactone are also important. The “styrylpyrone-styrylpyrone” type is more favorable for cytotoxicity than the corresponding “styrylpyrone-pyranopyrone” type. The decreased cytotoxicity may be due to the rigidity of the pyranopyrone scaffold and the absence of a center for Michael addition. It is interesting to note that the cytotoxicity induced by the bis-styryllactones was selective to cancer cells, without affecting the noncancerous lung and colorectal cell lines, MRC5 and CCD 841 CoN. Active compounds 2 and 4 were subjected to docking studies against the potential targets, EGFR-TK (1M17) and CDK2 (2R3J). These enzymes are considered promising targets in combating lung and colorectal cancer, as the overexpression of these kinases commonly takes place in many tumors.24−26 While EGFR-TK is capable of retarding the activity of epidermal growth factor receptor (EGFR), CDK2 is involved in the prevention of cell cycle progression.27,28 EGFRTK inhibitors display their anticancer mechanism through the inhibition of EGFR-TK phosphorylation by reversible competitive binding at the ATP site.27 On the other hand, CDK2 inhibitors usually regulate CDK2 activity by preventing phosphorylation of CDK2.29 The results for Libdock (Table 4) showed that 2 and 4 are compounds with the highest Libdock values for both EGFRTable 4. Libdock value of compounds 1-4 and 6-8 docked against EGFR-TK (1M17) and CDK2 (2R3J) Libdock score compounds

EGFR-TK (1M17)

CDK2 (2R3J)

1 2 3 4 6 7 8

115.500 124.267 122.739 124.393 144.644 111.409 115.732

77.237 125.100 107.281 124.389 91.322 106.925 92.187

TK and CDK2. On the other hand, compounds that are not active, such as compounds 7 and 8 had lower Libdock values. The active compounds 2 and 4 were able to bind almost ideally in the active site of EGFR tyrosine kinase (PDB: 1M17). Docking results showed that 2 binds with EGFR tyrosine kinase, through hydrogen bonding with amino acids like Lys721 and Met769 (Figure 5). In addition, the aromatic rings of 2 form several electrostatic π-alkyl interactions with Val702, Leu694, Ala719, and Leu820 to further stabilize the ligand− enzyme complex. Additionally, the α,β-unsaturated bond conjugations also contribute to the complex stability through π-alkyl interactions with Cys751, Gln767, Met769, Lys721, Leu764, and Met742. One of the hydrogen bonds observed is between the carbonyl oxygen of the α,β-unsaturated δ-lactone and the side chain of Lys721 at the distance of 4.99 Å. On the basis of these results, the α,β-unsaturated δ-lactone moiety is I



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Figure 5. Docking alignments for compounds 2 and 4 in the binding cavity of EGFR.

Figure 6. Docking alignments for compounds 2 and 4 in the binding cavity of CDK2.

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stable ligand enzyme complex through π-alkyl interactions with Val18, Ala31, and Leu134. In summary, a phytochemical study of the stembark and root extracts of G. lanceolatus Miq. led to the isolation of eight new compounds, goniolanceolatins A−H (1−8), along with four known compounds (9−12). The goniolanceolatin-type compounds are composed of either a pyranopyrone unit and a styrylpyrone unit or two styrylpyrone units, which are linked by an ether bridge at C-8/C-8′, C-8/C-7′, or C-7/C-8′. The new compounds are unusual because they are composed of rare (6S)-styrylpyrones and (1S)-phenylpyranopyrones. Among the new compounds, 2 and 4 had strong cytotoxicity against selected human lung and colorectal cancer cells. These findings support the further exploration of the cytotoxicity of styryllactones from a mechanistic perspective.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a JASCO DIP-370 automatic polarimeter using MeOH as solvent. Electronic circular dichroism (ECD) spectra were recorded on a JASCO J-815 spectrometer with MeCN as solvent. IR spectra were obtained using a PerkinElmer Spectrum 100 in a KBr pellet. MS data were measured using an Agilent Technologies 6520 Accurate-Mass Q-TOF and LTQ Obitrap Discovery Hybrid Ion Trap-Obitrap mass spectrometer. The 1D and 2D NMR spectra were recorded in CDCl3 on Bruker Ascend 600 MHz and Bruker Ultrashield Plus 500 MHz spectrometers. Chemical shifts are reported on the δ ppm scale, and coupling constants are given in Hz. Fractionation of the crude extract was carried out using MPLC on a Yamazen Flash Liquid Chromatography W-Prep 2XY instrument, with a 120 g silica gel 40 μm (46 × 130 cm) and a 250 g silica gel 40 μm (46 × 180 cm) column. TLC was performed on NP F254 plates (20 cm × 20 cm) from Merck (Darmstadt, Germany), and the spots J



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styryllactones, 6 (7.5 mg) and 3 (3.2 mg) were obtained. Subfraction F10k (89.4 mg) was subjected to semipreparative HPLC over ODS, which led to the isolation of subsubfractions F10k-i to F10k-iii. The last peak (F10k-iii) appeared at retention time 9.5 to 10.5 min, using a gradient of MeOH/H2O (80:20). Through 1H NMR data analysis, F10k-iii (14.6 mg) was a mixture and was subjected to recycling HPLC over ODS (isocratic solvent system MeOH/H2O, 70:30). After eight recycles over a period of 240 min, the pure bisstyryllactone 2 (5.7 mg) was collected. A sample of the roots’ extract (30.0 g) was fractionated by MPLC on a silica gel column eluted with a n-hexane/EtOAc:MeOH gradient system to give nine fractions (R1-R9). Subfraction R6 (232.0 mg) was subjected to preparative HPLC over ODS using a gradient of MeOH/ H2O to afford six subsubfractions. By 1H NMR data analysis, the second subsubfraction was a mixture and processed through recycling HPLC over ODS (isocratic solvent system MeOH/H2O, 40:60). After three recycles over 75 min, pure bis-styryllactones 1 (46.7 mg) and 5 (5.0 mg) were collected. Other isolates from the root extract were described previously.14,15 Goniolanceolatin A (1). White amorphous powder; [α]20 D +5 (c 0.4, MeOH); ECD (CH3CN) λmax (Δε) 258 (−5.72), 216 (+16.96) nm; IR (KBr) νmax 3417 (OH), 1703 and 1687 cm−1 (CO), 1399, 1270, 1252, 1080, 1055, 824, and 702 cm−1; 1H and 13C NMR spectra, see Table 1; OBITRAP-MS m/z 473.1556 [M + Na]+ (calcd for C26H26O7 473.1571) Goniolanceolatin B (2). White amorphous powder; [α]20 D +153 (c 0.5, MeOH); ECD (CH3CN) λmax (Δε) 256 (−9.16), 220 (+1.05) nm; IR (KBr) νmax 3434 cm−1 (OH), 2914, 1719, and 1706 (CO), 1384, 1257, 1068, 817, and 701 cm−1; 1H and 13C NMR data, see Table 1; QTOF-MS m/z 451.1759 [M + H]+ (calcd for C26H26O7, 451.1751) Goniolanceolatin C (3). White amorphous powder; [α]20 D −107 (c 0.5, MeOH); ECD (CH3CN) λmax (Δε) 257 (−5.42), 219 (+1.88) nm; IR (KBr) νmax; 3412 (OH), 2917, 1709, and 1695 (CO), 1385, 1259, 1053, 817, and 706 cm−1; 1H and 13C NMR data, see Table 1; QTOF-MS m/z 451.1755 [M + H]+ (calcd for C26H26O7, 451.1751) Goniolanceolatin D (4). White amorphous powder; [α]20 D +88 (c 0.5, MeOH); ECD (CH3CN) λmax (Δε) 258 (−3.54), 2197 (+9.78) nm; IR (KBr) νmax 3445 cm−1 (OH), 2923, 1727 (CO), 1375, 1229, 1056, 1034, 817, and 702 cm−1; 1H and 13C NMR data, see Table 1; QTOF-MS m/z 493.1862 [M + H]+ (calcd for C28H28O8, 493.1857) Goniolanceolatin E (5). White amorphous powder; [α]20 D +8 (c 0.5, MeOH); IR (KBr) νmax 3389 (OH), 1745, and 1694 (CO), 1386, 1260, and 1235 cm−1; 1H and 13C NMR data, see Table 1; OBITRAP-MS m/z 505.1815 [M + Na]+ (calcd for C26H26O7, 505.1833) Goniolanceolatin F (6). White amorphous powder; [α]20 D +159 (c 0.5, MeOH); ECD (CH3CN) λmax (Δε) 257 (−8.05), 226 (−4.10), 213 (+0.64) nm; IR (KBr) νmax 3466 (OH), 1735, and 1694 (CO), 1393, 1245, 1086, 1070, 1048, 816, 764, and 700 cm−1; 1H and 13C NMR data, see Table 2; OBITRAP-MS m/z 451.1735 [M + H]+ (calcd for C26H26O7, 451.1751) Goniolanceolatin G (7). White amorphous powder; [α]20 D +102 (c 0.5, MeOH); ECD (CH3CN) λmax (Δε) 256 (−5.94); IR (KBr) νmax3428 (OH), 2928, 1728 (CO), 1382, 1231, 1078, 1039, 816, and 702 cm−1; 1H and 13C NMR data, see Table 2; QTOF-MS m/z 451.1749 [M + H]+ (calcd for C26H26O7, 451.1751) Goniolanceolatin H (8). White amorphous powder; [α]20 D +189 (c 0.5, MeOH); ECD (CH3CN) λmax (Δε) 256 (−4.75); IR (KBr) νmax3401 (OH), 2917, 1719 (CO), 1384, 1222, 1064, 1043, 816, 757, and 703 cm−1; 1H and 13C NMR data, see Table 2; QTOF-MS m/z 493.1842 [M + H]+ (calcd for C28H28O8, 493.1857) Cell Culture and Cytotoxicity Assay. All human cell lines, except NCI-H1299 and NCI-H23, were obtained from the American Type Culture Collection (ATCC) and were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The NCI-H1299 and NCI-H23 cell lines were gifts from the National Cancer Institute, Selangor, Malaysia. The noncancerous human colorectal cells, CCD

were observed under UV light (254 and 366 nm) and under visible light after spraying with 10% sulfuric acid and Dragendorff’s reagent. Analytical HPLC was performed on an Agilent HPLC 1100 HPLC series system, equipped with a Diode Array Detector 1200 series (G1315B), Micro degasser (G1379A), and a quaternary pump (G1311A) with a C18 reversed phase column (Sunfire 5 μm, 4.6 × 250 mm. Semipreparative HPLC was done using an Agilent Technologies 1200 Binary Pump system equipped with a Multiple Wavelength Detector (G1364B) with a C18 reversed phase column Sunfire 5 μm (10 × 250 mm). A preparative Recycling HPLC (JaigelLC-9103) paired with Diode Array Detector was used for further purification, using a Jaigel-ODS-AP, SP-120−15 (20 × 250 mm) column. Plant Material. The stembark and roots of G. lanceolatus were collected from Lundu, Sarawak, Malaysia in June 2012, and the voucher specimens were deposited in the herbarium of the Universiti Malaysia Sarawak before being couriered to the Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA Selangor Branch, Bandar Puncak Alam, Selangor, Malaysia. Extraction and Isolation. Plant extractions were carried out by cold percolation. Dried, ground stembark (ca. 2.0 kg) and roots (4.0 kg) were defatted in n-hexane at room temperature, followed by extraction three times with CH2Cl2 (10−15 L) over 3 days. Evaporation of the solvent under vacuum gave the CH2Cl2 crude extracts of stembark (25.9 g) and roots (60.4 g). A sample of the stembark extract (12 g) was fractionated using MPLC, eluting with a gradient system of n-hexane/EtOAc (70:30 to 0:100) from 0 to 90 min to afford 16 fractions (F1 to F16) on the basis of TLC analysis. Fraction F9 was refractionated using MPLC, utilizing a gradient system of n-hexane/EtOAc (90:10 to 0:100) from 0 to 90 min, and yielded 13 subfractions (F9a-F9m). Subfraction F9b was composed of 9 (225.3 mg) as determined by its 1H NMR spectrum. Separation of subfraction F9j (58.6 mg) by semipreparative HPLC using a solvent gradient from MeOH/H2O (70:30) produced a broad single peak labeled as subsubfraction F9j-i (9.7 mg). Analysis of 1D and 2D NMR data revealed a mixture, which was further purified using recycling HPLC over ODS, and an isocratic solvent system of MeOH/H2O (60:40). After eight cycles over 190 min, compound 8 (3.4 mg) was obtained. Fraction F10 was refractionated using MPLC, employing a gradient system of n-hexane/EtOAc (40:60 to 0:100) from 0 to 60 min to afford 16 subfractions (F10a-F10p). Subfraction F10c (225.7 mg) was subjected to purification by semipreparative HPLC over ODS using a solvent gradient from MeOH/H2O (60:40), and at retention time 9.5 min, 10 was obtained (66.6 mg). Subfraction F10e (279.5 mg) was purified using semipreparative HPLC over ODS, and gave four subsubfractions (F10e-i, F10e-ii, F10e-iii, F10e-iv). Subsubfraction F10e-ii (6.9 mg) was obtained with a gradient of MeOH/H2O (45:65), and further purified through recycling HPLC over ODS, utilizing an isocratic solvent system of MeOH/H2O (50:50). At about 720 min (20 cycles), compound 12 (1.1 mg) was collected. Subfraction F10f (216.3 mg) was subjected to semipreparative HPLC over ODS and afforded seven subsubfractions (F10f-i to F10f-vii). Subsubfractions F10f-iii and F10f-iv were collected at a gradient of MeOH/H2O (40:60) and identified as 11 (41.3 mg) and 12 (3.5 mg), respectively. Subsubfraction F10f-vii was collected at a gradient of MeOH/H2O (60:40), and bis-styryllactone 7 (1.93 mg) was identified. Purification of subfraction F10h (245.5 mg) by semipreparative HPLC over ODS afforded three subsubfractions, F10h-i to F10h-iii. Subsubfraction F10h-iii (3.4 mg) was collected at a gradient of MeOH/H2O (60:40) and identified as the bisstyryllactone 4 (3.4 mg). Subfraction F10j (156.0 mg) yielded a total of six peaks (F10j-i to F10j-vi) after isolation by semipreparative HPLC over ODS. Bis-styryllactone 1 (3.2 mg) was identified from F10j-iii at a gradient of MeOH/H2O (40:60). The major peak (F10jiv), which occurred within 11 to 12 min, eluted with MeOH/H2O (70:30), was a mixture by 1H NMR data analysis. For further purification, a sample of F10j-iv (31.8 mg) was injected into a recycling HPLC over ODS utilizing an isocratic solvent system of MeOH/H2O (70:30), and after 233 min, on the ninth cycle, two bisK



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841 CoN were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin. All cells were kept in an incubator at 37 °C and 5% carbon dioxide. Culture conditions were optimized on the basis of previous studies. The cytotoxicity of the compounds was determined by calculating the IC50 values, the concentration at which 50% of the cells were killed, using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA).34 Briefly, human lung cancer cells (A549, Calu1, NCIH1299, NCI-H23), human colorectal cancer cells (HCC2998, Caco2, SW48, HCT116), noncancerous human lung cells (MRC5), and noncancerous human colorectal cells (CCD 841 CoN) were plated in 384-well plates for 24 h followed by treatment for 72 h. Luminescence was measured using SpectraMax M3Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, U.S.A.). The experiments were validated using cells treated with cisplatin (Sigma-Aldrich, St. Louis, MO, U.S.A.). The IC50 values of all data were reported as mean ± standard deviation (S.D.) from a minimum of three independent experiments. Statistically significant differences were analyzed using independent t test through SPSS (version 18.0) for Windows, comparing the IC50 values induced by the compounds and the IC50 values of cisplatin in the respective cell lines.35 A p value less than 0.05 (p < 0.05) was considered a statistically significant differences. Docking Method. All compounds were prepared using Chem3D by CambridgeSoft (Cambridge, MA, U.S.A.). The geometry and energy of the structures were optimized using MMFF94. Libdock (Discovery Studio, Version 4.0) with default settings was employed to identify the binding modes of compounds 2 and 4 responsible for their activity. The protein used for the docking study was Epidermal Growth Factor Receptor (EGFR) tyrosine kinase domain with 4anilinoquinazoline inhibitor erlotinib (PDB ID: 1M17) and Cyclin Dependent Kinase 2 (CDK-2) (PDB ID: 2R3J),36,37 obtained from the protein data bank Web site (http://www.rcsb.org/pdb/). The protein was optimized by removing the ligand and water molecules before being minimized and used for docking purposes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01067. The 1D NMR, 2D NMR, and OBITRAP-MS spectra of compounds 1−8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 6013-2675756. E-mail: [email protected]. ORCID

Nor Hadiani Ismail: 0000-0002-2374-4630 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by Malaysian Government grants through the Fundamental Research Grant Scheme, FRGS/1/ 2016/STG01/UiTM/1 and FRGS/1/2017/SKK08/IMU/03/ 1. N.V.B. and N.E.R. acknowledge with gratitude, the Ministry of Education Malaysia and Universiti Teknologi MARA for Ph.D. scholarships. Geoffrey A. Cordell is associated with Universiti Teknologi MARA as a Visiting Professor.

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REFERENCES

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