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Sesterterpenes and phenolic alkenes from the Thai sponge Hyrtios erectus
Tetrahedron 74 (2018) 316e323
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Tetrahedron journal homepage: www.elsevier.com/locate/tet
Sesterterpenes and phenolic alkenes from the Thai sponge Hyrtios erectus Wirongrong Kaweetripob a, Chulabhorn Mahidol a, b, Siriporn Wongbundit a, Pittaya Tuntiwachwuttikul d, Somsak Ruchirawat a, b, c, Hunsa Prawat a, * a
Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Bangkok, 10210, Thailand Chulabhorn Graduate Institute, Chemical Biology Program, Chulabhorn Royal Academy, Kamphaeng Phet 6 Rood, Laksi, Bangkok, 10210, Thailand Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Bangkok, Thailand d Laboratory of Natural Products Chemistry, Faculty of Science and Technology, Phuket Rajabhat University, Phuket, 83000, Thailand b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2017 Received in revised form 20 November 2017 Accepted 27 November 2017 Available online 5 December 2017
Sesterterpene, erectusolide A (1), six phenolic alkenes, erectuseneols AF (2e7) and nine known compounds, luffalactone (8), luffariolide E (9), (6E)- and (6Z)-neomanoalide 24,25-diacetates (10 and 11), 6,6dimethylundecane-2,5,10-trione (12), threo- and erythro-cavernosines (13 and 14), (4E,6E)-dehydromanoalide (15), echinoclerodane A (16), were isolated from the marine sponge Hyrtios erectus. Compound 13 was isolated for the first time from a natural source. The structures of these compounds were elucidated on the basis of spectroscopic analysis. The phenolic alkenes 3 and 7, the sesterterpenes 8 e11 and 15, and compounds 12e14 were evaluated for cytotoxic activities against six cancer cell lines, MOLT-3, HepG2, HeLa, HuCCA-1, A549, and MDA-MB-231. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Hyrtios erectus Thorectidae Sesterterpenes Phenolic alkenes Cytotoxic activities
1. Introduction Marine sponges of the genus Hyrtios (Family Thorectidae) have proven to be a rich source of secondary metabolites such as terpenoids [mainly acyclic triterpenes,1 sesterterpenes (manoalide, scaralane),2e9 sesquiterpenes/quinones10,11], macrolides,12,13 indole alkaloids,7,9,14e20 and phenyl alkenes.21a-d Some of these metabolites have been found to possess multiple biological activities, such as cytotoxicity,2e6,12,13,15 antiplasmodial,9 antifungal,10 and antimicrobial.14 To probe for the use of these compounds as taxonomical and biological marker for the genus Hyrtios, we have undertaken a chemical study of more species of this genus. 2. Results and discussion The sponge Hyrtios erectus (CRI 588) collected from Similan Island, Phangnga province, Thailand was extracted with MeOH and the extract was partitioned between EtOAc and water. The EtOAcsoluble material was subjected to silica gel column
* Corresponding author. E-mail address: [email protected] (H. Prawat). https://doi.org/10.1016/j.tet.2017.11.073 0040-4020/© 2017 Elsevier Ltd. All rights reserved.
chromatography followed by Sephadex LH-20 column chromatography and revered-phase HPLC to yield erectusolide A (1) and erectuseneols AF (2e7) together with nine known compounds. The known compounds were identified as five sesterterpenes, luffalactone (8),22 (3S*,4R* and 3R*,4R*)-luffariolide E (ratio~1:0.1) (9),23e25 (6E)- and (6Z)-neomanoalide 24,25-diacetates (10 and 11),26 and (4E,6E)-dehydromanoalide (15),22 one diterpene, echinoclerodane A (16),27 and three norterpenes, 6,6dimethylundecane-2,5,10-trione (12),28 and threo-cavernosine (13),30 and erythro-cavernosine (14).29,30 All of the known compounds (Fig. 1) were readily identified by extensive study of their spectral data, including HRAPCIMS or HRESIMS, 1D and 2D NMR data, as well as by comparison with those reported in the literatures. The phenolic alkenes 3 and 7, the sesterterpenes 8e11 and 15, and compounds 12e14 were evaluated for cytotoxic activities against six cancer cell lines, MOLT-3, HepG2, HeLa, HuCCA-1, A549, and MDA-MB-231. Compound 1 was obtained as an optically active colorless powder ([a]24 D þ2.5), and its molecular formula was determined to be C25H36O6 (index of hydrogen deficiency ¼ 8) by HRAPCIMS. IR absorption bands at 1772 and 1705 cm1 of 1 suggested the presence of lactone and carbonyl ketone groups, respectively. Six of the
W. Kaweetripob et al. / Tetrahedron 74 (2018) 316e323
317
Fig. 1. Structures of isolated compounds 1e16 and trichotoxin A.21b
eight degrees of hydrogen deficiency implied by the molecular formula of 1 was taken up in four carboneoxygen double bonds and two carbonecarbon double bonds; the molecule was thus bicyclic. The 1H and 13C NMR spectroscopic data of 1 (Table 1) were similar to those of luffariolide E (9)23,24 for the C-1eC-11 region. A pair of two signals (ratio ~ 1:0.3) due to the same carbons and protons were detected for some carbons and protons in the 1H and 13C NMR spectra of 1 (Table 1) similar to the signals of 3S*,4R*- and 3R*,4R*laffariolide E (9), which were synthesized by Kocienski and coworker.24,25 The signals associated with the cyclohexene moiety of 9 were not evident in 1; instead two new singlet signals at dC 208.4 and 214.9 were observed. The presence of the two aliphatic carbonyl signals at dC 208.4 and 214.9 together with a signal for a methyl ketone residue (dC 29.9 and dH 2.13) in the NMR spectrum implied the existence of an open-chain dicarbonyl moiety, which could be derived from an oxidative opening of a cyclohexene ring of 9 at the C-14C-15 olefinic bond.31 This was also compatible with the 32 mass unit differences in the molecular weight of 1 compared to luffariolide E. The structure of 1 was fully supported by 1H1H COSY, HSQC, and HMBC NMR techniques. The 1H1H COSY (Fig. 2) and HSQC data allowed the unambiguous assignment of two spin systems which consisted of two and three methylene units. One methylene group of each unit was adjacent to a ketone functionality, as implied by their chemical shift of dH 2.55 and 2.41 for H2-13
and H2-18, respectively. The first spin system consisted of the methylenes at dH 2.21 (H2-12) and 2.55 (H2-13). The HMBC (Fig. 2) spectra confirmed this system through correlations with the carbonyl at dC 214.9 (C-14). Moreover, the correlation of dH 1.60 (H323) with dC 33.4 (C-12) and of H2-12 (dH 2.21) with the olefinic methine carbon at dC 123.3 (C-10) allowed the extension of the C1C-13. The second spin system consisted of the methylenes at dH 1.42e1.52 (H2-16), 1.38e1.48 (H2-17), and 2.41 (H2-18). The HMBC experiment also confirmed this system through correlations of H217 and H2-18 with the carbonyl signal at dC 208.4 (C-19). The germinal dimethyl groups at dH 1.12 (s, 6H, H3-21/22) correlated with the C-14, C-15, and C-16 in HMBC spectrum, which suggested the attachment of germinal dimethyl (H3-21/22) at C-15. The methyl singlet at dH 2.13 (H3-20) implied that this was the terminus, as it showed a cross-peak with C-18 and C-19. The C-10/C-11 double bond was assigned as E configuration by NOESY experiment (Fig. 2). The 1H and 13C NMR resonances for the segment C-12C-20 of 1 were similar to those found in fasciospongides B and C,31 thus the structure of 1 was established as a mixture of 3S*,4R*- and 3R*,4R*compound 1 (ratio~1:0.3, Fig. 1) and was named erectusolide A. Compound 2 was obtained as an optically active colorless powder ([a]24 D þ2.9), and its molecular formula was determined to be C28H39ClO4 (index of hydrogen deficiency ¼ 9) by HRESIMS (m/z 497.2431 [MþNa]þ; calcd for C28H35 39ClO4, 497.2429). Its UV
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W. Kaweetripob et al. / Tetrahedron 74 (2018) 316e323
Table 1 1 H (600 MHz) and
13
C (150 MHz) NMR (CDCl3) data of compound 1.
Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
3S*,4R*
3R*,4R* (ratio ~ 1:0.3 of 3S*,4R*:3R*,4R*)
dC, type
dH (J in Hz)
dC, type
dH (J in Hz)
175.3, C 30.1, CH2 39.6, CH 77.6, CH 28.0, CH2 137.6, CH 132.7, C 30.8, CH2 26.4, CH2 123.3, CH 135.5, C 33.4, CH2 35.5, CH2 214.9, C 47.5, C 39.2, CH2 19.0, CH2 43.9, CH2 208.4, C 29.9, CH3 24.4, CH3 24.4, CH3 16.2, CH3 164.1, C 69.6, CH2
e 2.43, dd (17.5, 8.5) 2.63, dd (17.5, 9.0) 2.88e2.96, m 4.33e4.39, m 2.28e2.42, m 6.56, brd (4.8) e 2.22e2.32, m 2.11e2.22, m 5.10, t (7.1) e 2.21, t (7.7) 2.55, dd (8.2, 7.7) e e 1.42e1.52, m 1.38e1.48, m 2.41, t (6.8) e 2.13, s 1.12, s 1.12, s 1.60, s e 4.35, dd (9.6, 7.8) 4.54, dd (9.6, 7.1)
175.5, C 30.1, CH2 39.4, CH 76.5, CH 27.5, CH2
e 2.62, dd (17.8, 8.0) 2.70, dd (17.8, 9.0) 2.87e2.96, o 4.35e4.42, o 2.28e2.42, o
68.2, CH2
4.28, dd (9.4, 7.2) 4.42, dd (9.4, 7.9)
spectrum presenting a maximum absorption at 224 nm as well as a shoulder at 279 nm indicated the presence of an aromatic system. The IR spectrum of 2 displayed absorption bands at 3421 and 1733 cm1, suggesting the presence of hydroxyl and ester groups, respectively. The 1H and 13C NMR spectroscopic data of 2 (Table 2) showed the presence of a para-substituted benzene ring [positions 18e23: dH 6.79 (H-20/22), 7.07 (H-19/23) and dC 115.4 (C-20/22), 130.2 (C-18), 130.5 (C-19/23), 154.4 (C-21)]. The diastereotopic benzylic methylene protons (H2-17, dН 2.66/2.75) showed HMBC (Fig. 3) correlations to the aromatic system (C-18, C-19 and C-23), as well as the oxymethine carbon (C-16, dC 72.5), and the allylic methylene carbon (C-15, dC 34.5). The cross-peak in the HMBC spectrum from the olifinic proton (H-14, dH 5.47e5.53) to oxymethine carbon (C-16) was also detected. The consecutive analyses of the 1H1H COSY (Fig. 3) correlations from the allylic methylene protons (H2-11, dH 2.02e2.15) to benzylic methylene protons (H217) indicated a heptenol unit (C-11C-17) comprised the alkyl chain of structure 2 (Fig. 3). The 1H and 13C NMR spectra for the segment C-1C-11 of 2 (Table 2) were closely similar to those of trichotoxin A21b (Fig. 1), except for the spectral signals at C-4. The downfield shift of oxymethine signals at C-4 (dC 78.5 and dH 5.17) relative to the resonance observed for trichotoxin A (dC 76.7 and dH 4.01) indicated that the hydroxyl group at C-4 in 2 was acetylated. This was further confirmed by HMBC (Fig. 3) correlations between H-4 and the carbonyl carbon of the acetate ester (dC 170.5) and satisfying the final index of hydrogen deficiency. The vinyl methine proton (H-24, dН 5.76) showed HMBC correlations to the quaternary carbon (C-10, dC 142.2) and two allylic methylene carbons (C-9, dC 28.4 and C-11, dC 34.7). From the above spectral data, the planar
Fig. 2. Key COSY (
), HMBC (
), and NOESY (
) correlations of 1.
structure 2 (Fig. 1) was thus suggested for the compound. The NOESY (C6D6; Fig. 3) correlations between the methine proton, H24 (dН 5.63) and both methylene groups, H2-11 (dH 1.83e1.94) and H2-12 (dН 1.95e2.05) supported a Z configuration of C-10/C-24 double bond. An E configuration of the C-5/C-6 double bond was supported by examining the chemical shift of the olefinic methyl carbon (C-26, dC 12.3)21c and the NOESY correlations between H-7 and H-26. A Z configuration of the C-13/C-14 double bond was supported by the NOESY correlations between H-12 and H-15 (dH 2.10e2.20) and coupling constant value (JH-13,H-14 ¼ 10.8 Hz). Thus, the structure of 2 was established as (5E,10Z,13Z)-4-acetoxy-10(chloromethylene)-16-hydroxy-17-(4-hydroxyphenyl)-5,7dimethylheptadeca-1,5,13-triene and was named erectuseneol A. Unfortunately insufficient material was available for further experiments to determine the absolute configurations at the chiral centers (C-4, C-7, and C-16). Compound 3 was obtained as an optically active colorless powder ([a]24 D þ4.3), exhibiting the same IR, UV spectra as compound 2. Compound 3 showed molecular formula ion at m/z 539.2539 [MþNa]þ by HRESIMS indicating the molecular formula of 3 was C30H41ClO5 (index of hydrogen deficiency ¼ 10). This difference of 42 amu (C2H2O) compared to the molecular formula of compound 2 could suggest the presence of an additional acetyl group in compound 3. The remarkable resemblance in the 1H and 13 C NMR data of compounds 2 and 3 (Table 2), indicating that both compounds possess similar structural features. The downfield shift of oxymethine group (C-16, dC 74.5 and H-16 dH 5.00e5.05) relative to the resonance observed in compound 2 indicated that the hydroxyl group at C-16 was acetylated in compound 3. This was further confirmed by HMBC correlations between H-16 and the carbonyl carbon of the acetate ester (dC 170.4). Thus, the structure of 3 was established as (5E,10Z,13Z)-10-(chloromethylene)-4,16diacetoxy-17-(4-hydroxyphenyl)-5,7-dimethylheptadeca-1,5,13triene and was named erectuseneol B. Compound 4 was obtained as an optically active colorless powder ([a]24 D 4.0), exhibiting the same UV spectrum as compounds 2 and 3. Its molecular formula was determined to be
W. Kaweetripob et al. / Tetrahedron 74 (2018) 316e323 Table 2 1 H (600 MHz) and
13
319
C (150 MHz) NMR dataa (CDCl3) of compounds 2 and 3.
Position (type)
1 (CH2) 2 (CH) 3 (CH2) 4 (CH) 5 (C) 6 (CH) 7 (CH) 8 (CH2) 9 (CH2) 10 (C) 11 (CH2) 12 (CH2) 13 (CH) 14 (CH) 15 (CH2) 16 (CH) 17 (CH2) 18 (C) 19 (CH) 20 (CH) 21 (C) 22 (CH) 23 (CH) 24 (CH) 25 (CH3) 26 (CH3) 4-OCOMe 4-OCOMe 16-OCOMe 16-OCOMe
2
3
dC
dH (J in Hz)
dH inC6D6 (J in Hz)
dC
dH (J in Hz)
117.3 133.7 37.4 78.5 131.7 134.5 32.3 34.6 28.4 142.2 34.7 25.7 131.4 126.2 34.5 72.5 42.5 130.2 130.5 115.4 154.4 115.4 130.5 112.4 21.3 12.3 170.5 20.7 e e
5.02, brd (10.0) 5.07, dq (17.1, 1.5) 5.67, ddt (17.1, 10.0, 7.0) 2.35, qnt (7.1) 2.41, qnt (7.1) 5.17, t (7.0) e 5.23, d (10.0) 2.33e2.40, o 1.22e1.31, m 1.36e1.44, m 2.01e2.14, m e 2.02e2.15, o 2.08e2.18, o 5.45e5.52, m 5.47e5.53, m 2.18e2.28, m 3.80, brqnt (6.9) 2.66, dd (13.7, 7.7) 2.75, dd (13.7, 5.1) e 7.07, d (8.5) 6.79, d (8.5) e 6.79, d (8.5) 7.07, d (8.5) 5.76, brs 0.97, d (6.7) 1.63, brd (1.2) e 2.01, s e e
5.01, dd (17.0, 1.7) 5.02, brd (10.0) 5.69, ddt (17.0, 10.0, 7.0) 2.27e2.32, m 2.39, brp (7.0) 5.36, t (6.9) e 5.29, brd (10.0) 2.25e2.32, m 1.20e1.33, m 1.34e1.45, m 2.10e2.20, m e 1.83e1.94, m 1.95e2.05, m 5.34e5.40, m 5.50, brdt (10.8, 7.3) 2.10e2.20, m 3.65, brqnt (6.6) 2.57, dd (13.7, 7.2) 2.61, dd (13.7, 5.4) e 6.96, d (8.5) 6.65, d (8.5) e 6.65, d (8.5) 6.65, d (8.5) 5.63, s 0.92, d (6.6) 1.56, brd (1.2)
117.3 133.7 37.4 78.5 131.7 134.5 32.4 34.6 28.5 142.2 34.7 25.6 131.2 125.2 31.0 74.5 39.1 129.3 130.5 115.3 154.5 115.3 130.5 112.4 20.6 12.3 170.5 21.2 170.4 21.1
5.02, brd (9.0) 5.07, dd (17.1, 1.3) 5.67, ddt (17.1, 10.1, 7.0) 2.34, qnt (7.1) 2.41, qnt (7.1) 5.16, t (7.0) e 5.22, d (9.5) 2.32e2.39, o 1.22e1.30, m 1.36e1.43, m 2.00e2.13, m e 2.00e2.10, o 2.02e2.10, o 5.39e5.45, m 5.38e5.43, m 2.25, t (5.8) 5.00e5.05, m 2.74, dd (13.9, 6.5) 2.80, dd (13.9, 6.5) e 7.04, d (8.3) 6.76, d (8.3) e 6.76, d (8.3) 7.04, d (8.3) 5.74, s 0.96, d (6.6) 0.63, s e 2.06, s e 2.00, s
1.68, s e e
qnt ¼ quintet; o ¼ overlap. a Assignments are based on COSY, HSQC, and HMBC experiments.
C22H31ClO2 (index of hydrogen deficiency ¼ 7) by HRAPCIMS. The IR spectrum of 4 displayed absorption bands at 3393 cm1, suggesting the presence of hydroxyl group. Analysis of 1H and 13C NMR spectroscopic data of 4 (Table 3) showed the presence of a parasubstituted benzene ring [dC 130.8 (C-14), 129.8 (C-15/19), 115.3 (C16/18), 154.1 (C-17) and dH 7.06 (H-15/19) and 6.75 (H-16/18)]. The benzylic methylene protons (H2-13, dH 3.48) were substantially deshielded and showed HMBC correlations to the aromatic system (C-14, C-15, and C-19) as well as a moderately polarized double bond (C-12, dC 141.3; C-20, dC 113.1). The polarization of the C-12/C20 double bond and the singlet methine proton (H-20, dH 5.91) was consistent with the presence of a vinyl chloride functionality. The doubly allylic methylene protons (H2-11, dH 2.66) showed HMBC correlations to vinyl chloride moiety and the C-9 quaternary olefinic carbon (dC 138.0) and COSY correlations to the olefinic proton (H-10, dH 5.03). The H2-8 allylic methylene protons (dH 1.95) showed HMBC correlations to both olefinic carbons, C-9 (dC 138.0) and C-10 (dC 120.2), and the C-21 vinyl methyl (dC 15.9). The analyses of both the HMBC and the 1H1H COSY correlations of 4 from the terminal methylene (H2-1, dH 5.14/5.15; dC 117.9) to allylic methylene (H2-8, dH 1.95; dC 39.8) indicated the presence of a 5methyl-4-hydroxyoct-1-en (C-1C-8 and C-22) unit comprised in
Fig. 3. Key COSY (
), HMBC (
), and NOESY in C6D6 (
) correlations of 2.
the structure. The cross-peaks from the oxymethine proton (H-4, dH 3.56) to the olefinic carbon (C-2, dC 135.5), methylene carbon (C-6, dC 32.8) and secondary methyl carbon (C-22, dC 13.9) were also detected in HMBC spectrum. The C-9/C-10 and C-12/C-20 double bonds were assigned as 9E and 12Z configurations by the NOESY correlations (Fig. 4) between olefinic methine proton (H-20, dH 5.91) and doubly allylic methylene protons (H2-11, dH 2.66) and between doubly allylic methylene protons (H2-11) and vinyl methyl protons (H3-21, dH 1.49). Thus, the structure of 4 was established as (9E,12Z)-12-(chloromethylene)-4-hydroxy-13-(4-hydroxyphenyl)5,9-dimethyltrideca-1,9-diene and was named erectuseneol C. To find the absolute configuration at C-4 of 4 was addressed through the application of Mosher's method.32 Both (R)- and (S)-methoxya-(trifluoromethyl)phenyl acetic acid (MTPA) esters of 4 were prepared but did not provide any useful information for assigning the configuration at C-4. Compound 5 was obtained as an optically active pale yellow amorphous solid ([a]25 D þ3.2), exhibiting the same IR, UV spectra as compound 4. Compound 5 showed molecular formula ion at m/z 303.2319 [MH] by HRESIMS indicating the molecular formula of 5 was C20H32O2 (index of hydrogen deficiency ¼ 5). The 1H and 13C NMR spectra of 5 showed the presence of a para-substituted benzene ring [dH 7.00 (H-13/17), 6.74 (H-14/16) and dC 133.9 (C-12), 130.2 (C-13/17), 114.9 (C-14/16), 153.5 (C-15)]. The analyses of both the HMBC and 1H1H COSY correlations of 5 from the terminal methylene (H2-1, dH 5.12/5.14; dC 117.7) to the benzylic methylene (H2-11, dH 2.31/2.53; dC 43.0) indicated the presence of an alkenyl chain, 4-hydroxy-5,7,10-trimethylundec-1-en (C-1C-11, C-18, C19, and C-20) comprised in the structure. The benzylic methylene protons (H2-11 showed HMBC correlations to the aromatic carbons (C-12, C-13, and C-17) and methyl carbon (C-18, dC 19.3). The H-4
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W. Kaweetripob et al. / Tetrahedron 74 (2018) 316e323
Table 3 1 H (600 MHz) and
13
C (150 MHz) NMR data (CDCl3) of compounds 4e7.
Position 4
dC, type dH (J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 OH
117.9, CH2 135.5, CH 39.1, CH2 73.9, CH 37.7, CH 32.8, CH2 25.6, CH2 39.8, CH2 138.0, C
5
6
7
dC, type dH (J in Hz)
dC, type dH (J in Hz)
dC, type dH (J in Hz)
5.14, brd (10.0) 5.15, brd (17.1) 5.83, dddd (17.1, 10.0, 8.0, 6.3) 2.13e2.21, m 2.23 e2.30, m 3.56, dt (8.4, 4.5)
117.7, CH2 135.6, CH 39.3, CH2 73.6, CH 1.49e1.58, o 35.0, CH 1.11e1.18, o 1.38e1.46, 41.1, o CH2 1.30e1.38, o 1.40e1.47, 30.2, o CH 1.95, brt (6.2) 33.70, CH2 e 33.73, CH2 120.2, 5.03, brtd (7.2, 1.0) 35.4, CH CH 33.1, 2.66, d (7.1) 43.0, CH2 CH2 141.3, C e 133.9, C 35.6, 3.48, s CH2 130.8, C e 129.8, 7.06, d (8.4) CH 115.3, 6.75, d (8.4) CH 154.1, C e 115.3, CH 129.8, CH 113.1, CH 15.9, CH3 13.9, CH3 e
6.75, d (8.4) 7.06, d (8.4) 5.91, s
5.12, br d (10.1) 5.14, br d (17.1) 117.8, CH2 5.83, dddd (17.1, 10.1, 7.8, 6.4) 135.6, CH 2.18, dt (14.1, 8.2) 2.25, brdt 39.1, (14.1, 5.2) CH2 3.52, dt (8.9, 3.9) 74.1, CH 1.56e1.66, m 38.1, CH 1.00, ddd (13.4, 8.1, 6.5) 1.37, dd 30.5, (13.4, 6.5) CH2 1.42e1.50, m 34.2, CH2 1.03e1.38, o 35.3, CH 1.03e1.38, o 42.9, CH2 1.56e1.66, m 133.7, C
5.12, brd (9.9) 5.14, brd (17.1) 5.82, dddd (17.1, 10.0, 8.0, 6.5) 2.15, dt (14.2, 8.3) 2.27, ddd (14.2, 5.1, 4.9) 3.52, dt (8.9, 3.7) 1.44e1.51, m 1.41e1.48, m 1.14e1.27, m 1.29e1.35, m 1.60e1.68, m 2.33, dd (13.5, 8.0) 2.55, dd (13.5, 6.2) e
2.31, dd (13.6, 8.0) 2.53, dd (13.6, 130.2, 7.00, d (8.4) 6.3) CH e 114.9, 6.74, d (8.4) CH 7.00, d (8.4) 153.5, C e
130.2, CH 114.9, 6.74, d (8.4) CH 153.5, C e 114.9, CH 130.2, CH 19.3, CH3 20.4, CH3 14.2, CH3
6.74, d (8.4)
e
4.66, s
7.00, d (8.4)
114.9, CH 130.2, CH 19.3, CH3 13.8, CH3
6.74, d (8.4) 7.00, d (8.4) 0.84, d (6.6) 0.87, d (6.6)
0.83, d (6.6)
114.9, CH2 138.1, CH 33.6, CH2 26.8, CH2 132.7, CH 125.2, CH 35.5, CH2 70.0, CH 37.3, CH2 139.9, C
4.97, brdd (10.2, 1.0) 5.02, brdd (17.1, 1.5) 5.82, ddt (17.0, 10.2, 6.5) 2.06e2.15, m 2.11e2.19, m 5.57, brdt (10.8, 6.8) 4.42, brdt (10.8, 7.4) 2.19e2.31, m 3.85, brs 2.32, dd (13.7, 4.5) 2.37, dd (13.7, 8.5) e
41.1, 3.39, s CH2 130.2, C e 130.2, 7.03, d (8.4) CH 115.4, 6.77, d (8.4) CH 154.3, C e 115.4, CH 130.2, CH 115.9, CH
6.77, d (8.4) 7.03, d (8.4) 5.95, s
0.84, d (6.6) 0.88, d (6.6)
1.49, s 0.91, d (6.8) e
oxymethine proton (dH 3.52) showed HMBC correlations to C-6 (dC 41.1), C-2 (dC 135.6), and C-20 (dC 14.2) and the H2-3 allylic methylene (dH 2.18/2.25) showed the HMBC cross-peak to C-1 (dC 117.7). Compound 5 was thus established as 4-hydroxy-11-(4hydroxyphenyl)-5,7,10-trimethylundeca-1-ene and was named erectuseneol D. Compound 6 was obtained as an optically active colorless powder ([a]25 D þ3.0), exhibiting the same IR, UV spectra as compound 5. Compound 6 showed molecular formula ion at m/z 261.1862 [MH] by HRESIMS indicating the molecular formula of
Fig. 4. Key COSY (
), HMBC (
), and NOESY (
) correlations of 4.
e
4.72, brs
e
4.86, brs
6 was C17H26O2 (index of hydrogen deficiency ¼ 5). Analyses of 1D and 2D NMR spectra clearly showed 6 was an analog of 5. The spectral signals from C-1C-5 and C-8C-17 of the carbon backbone of 6 were identical to C-1C-5 and C-10C-18 and C-20 of 5. The benzyl methylene protons (H2-9, dH 2.33/2.55) showed HMBC correlations to the aromatic system (C-10, dC 133.7; C-11/15, dC 130.2), secondary methyl (C-16, dC 19.3) and methylene carbon (C-7, dC 34.2). The methine proton (H-8, dH 1.60e1.68) showed COSY correlations to benzylic methylene protons (H2-9, dH 2.33/2.55), methyl protons (H3-16, dH 0.84) and methylene protons (H2-7, dH 1.14e1.24/1.29e1.35). The H2-7 methylene showed COSY correlations to methylene protons (H2-6, dH 1.41e1.48). The methylene carbon (C-6, dC 30.5), methyl carbon (C-17, dC 13.8) and olefinic carbon (C-2, dC 135.6) and the allylic methylene carbon (C-3, dC 39.1) showed HMBC correlations to oxymethine proton (H-4, dH 3.52) and terminal methylene protons (H2-1), respectively. The 1 H1H COSY correlations from the methylene protons at dH 1.41/ 1.48 (H2-6) to the terminal olefinic protons at dH 5.12/5.14 (H2-1) and the secondary methyl at dH 0.87 (H3-17) were also observed. Thus, compound 6 was identified as 4-hydroxy-9-(4-
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321
4. Experimental section 4.1. General experimental procedures
Fig. 5. Key COSY (
), HMBC (
), and NOESY (
) correlations of 7.
hydroxyphenyl)-5,8-dimethylnon-1-ene and was named erectuseneol E. Compound 7 was obtained as an optically active colorless powder ([a]25 D 5.4), exhibiting the same UV and IR spectra as compounds 4e6. Compound 7 showed molecular formula ion at m/ z 305.1315 [MH] by HRESIMS indicating the molecular formula of 7 was C18H23ClO2 (index of hydrogen deficiency ¼ 7). The 1H and 13 C NMR spectroscopic data of 7 (Table 3) showed the presence of a para-substituted benzene ring [dC 130.2 (C-12), 130.2 (C-13/17), 115.4 (C-14/16), 154.3 (C-15) and dH 7.03 (H-13/17) and 6.77 (H-14/ 16)]. The benzylic methylene protons (H2-11, dH 3.39) were substantially deshielded and showed HMBC correlations to the aromatic system (C-12, C-13, and C-17) as well as a moderately polarized olefinic carbons (C-10, dC 139.9; C-18, dC 115.9). The polarization of the C-10/C-18 double bond and the singlet methine proton (H-18, dH 5.95) was consistent with the presence of a vinyl chloride functionality. The 1H1H COSY correlations of 7 from the terminal olefinic methylene protons at dH 4.97/5.02 (H2-1) to the allylic methylene protons at dH 2.32/2.37 (H2-9) indicated the presence to a 8-hydroxynona-1,5-diene unit in the structure. The allylic methylene protons (H2-9) showed HMBC correlations to the vinyl chloride moiety and COSY correlation to the oxymethine proton (H-8, dH 3.85). The position of hydroxyl group at C-8 was confirmed by HMBC correlation between dH 3.85 (H-8) and dC 139.9 (C-10) and 125.2 (C-6). The NOESY (Fig. 5) correlation between H-18 vinyl methine proton (dH 5.95) and H211 (dH 3.39) benzylic methylene protons supported a Z configuration of C-10/C18 double bond. A Z configuration of the C-5/C-6 double bond was supported by the NOESY correlation between H2-4 allylic methylene protons (dH 2.11e2.19) and H2-7 allylic methylene protons (dH 2.19e2.31) and coupling constant value (JH-5,H-6 ¼ 10.8 Hz). Thus, the structure of 7 was established as (5Z,10Z)-10-(chloromethylene)-8-hydroxy11-(4-hydroxyphenyl)-undeca-1,5-diene and was named erectuseneol F. Cytotoxicity of the isolated compounds 3, 7, 9e16 were evaluated against six cancer cell lines (Table 4), MOLT-3 (acute lymphoblastic leukemia), HepG2 (hepatocarcinoma), HeLa (human cervical carcinoma), HuCCA-1 (human chlolangiocarcinoma), A549 (human lung cancer), and MDA-MB-231 (hormone independent breast cancer). Sestertserpene, (4E,6E)-dehydromanoalide (15), and diterpene, echinoclerodane A (16), showed good cytotoxic activity against MOLT-3 cell line with an IC50 values of 3.79 and 5.82 mM, respectively and showed weakly cytotoxic activity against HuCCA1 cell line with an IC50 values of 21.76 and 16.57 mM, respectively. Phenolic alkene, erectuseneol B (3) showed weakly cytotoxic activity against MOLT-3 cell line with IC50 values of 18.43 mM. 3. Conclusions The secondary metabolites of Hyrtios erectus (CRI 588) again show that sesterterpene manoalide derivatives 1, 8e11, and 15 are widespread in this genus.6,7,9 The isolation of the six new phenolic alkenes, 2e7, which were rare natural products, may be of chemotaxonomic relevance.
Optical rotations were measured on a JASCO P-1020 polarimeter in CHCl3. UV spectra were recorded using a Shimadzu UV-1700 Phasmaspect UVeVis spectrophotometer in MeOH. Infrared spectra were taken on a Perkin Elmer Spectrum One spectrophotometer as ATR technique. HRMS were performed on a Bruker (Micro ToF) spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 solution containing Me4Si as internal standard on a Bruker AVANCE 600 spectrometer. HPLC was carried out on a Waters 600 system equipped with a Waters Delta 600 pump, a Waters 600 Controller, a Waters 2998 photodiode array detector, and Waters Empower 2 software. Sephadex™ LH-20 (GE Healthcare BioSciences AB) was used for a column gel filtration. All commercial grade solvents were distilled prior to use and spectral grade solvents were used for spectroscopic measurements. 4.2. Sponge material The sponge, Hyrtios erectus, was collected from Similan Island in the Andaman Sea (Phangnga province, Thailand) in February 2011, at a depth of 30e40 feet by hand via SCUBA diving. This sponge was identified by Dr. Sumaitt Putchakarn, Head of Marine Biodiversity Research, Unit Curator of Porifera and Echinodermata, Institute of Marine Science, Burapha University, Bangsaen, Chonburi, Thailand. A voucher specimen (CRI 588) is presently deposited at the Laboratory of Natural Products, Chulabhorn Research Institute, Bangkok, Thailand. 4.3. Extraction and isolation A frozen sample (wet wt 5.8 kg) of Hyrtios erectus (CRI 588, 14.0 kg) was cut into small pieces and extracted repeatedly with MeOH (20 L 3). After evaporation of the solvent, the concentrated MeOH extracts was partitioned between EtOAc and water. The EtOAc fraction (105 g) was chromatographed on a silica gel column with CH2Cl2hexane (1:1) containing increasing proportions of acetone as eluent, to give 7 fractions (A-G). Fraction B [8.0 g, eluted with acetoneCH2Cl2hexane (1:3:3)] was further separated by a Sephadex LH-20 column chromatography with MeOHCH2Cl2 (1:1) as eluent, to give 4 fractions (B1-B4). B1 (561 mg) were purified by HPLC column (Sunfire C18, 5 mm, 10 250 mm) with CH3CNH2O (85:15), flowrate 2.5 mL/min, l 224 nm, to afford 8 (2.1 mg, at 18.3 min) and 9 (2.1 mg of 3S*,4R* and 3R*,4R*)-luffariolide E, ratio~1:0.1, at 24.8 min). B2 (7.0 g) was fractionated by Sephadex LH-20 with MeOH to yield 4 fractions (B2.1-B2.4). B2.3 (2.7 g) was repeatedly fractionated by Sephadex LH-20 with MeOHCH2Cl2 (1:1) as eluent, to give a mixture compounds (1.0 g) that was separated using on a HPLC column (Sunfire C18, 5 mm, 10 250 mm) with CH3CNH2O (77:23), flowrate 2.5 mL/min, l 224 nm, to afford compounds 1 (3.4 mg, at 6.2 min), 2 (2.1 mg, at 21.5 min), 3 (2.3 mg, at 38.3 min), 10 (37.7 mg, at 54.5 min), and 11 (28.4 mg, at 56.3 min). B2.4 (4.0 g) was repeatedly fractionated by Sephadex LH-20 with MeOHCH2Cl2 (1:1) as eluent, to give 3 subfractions (B2.4.1B2.4.3) and each subfraction was further separated by HPLC column (Sunfire C18, 5 mm, CH3CNH2O, l 224 nm) to afford compounds 12 (7.6 mg), 13 (4.6 mg), 14 (76.0 mg), 15 (70.0 mg), 4 (3.0 mg), 16 (5.4 mg), 5 (1.3 mg), 6 (1.4 mg), and 7 (2.0 mg). 4.3.1. Erectusolide A (1) Colorless powder; [a]24 D þ2.5 (c 0.39, CHCl3); UV lmax (MeOH) nm (log ε) 202 (3.9), 225 (3.6); IR (ATR) nmax 2928, 1772, 1705, 1413,
322
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Table 4 Cytotoxicity of compounds 3, 7, 9e16 (IC50, mM). Compound
MOLT-3
HepG2
Hela
HuCCA-1
A549
MDA-MB-231
3 7 9 10 11 12 13 14 15 16 Doxorubicin Etoposide
18.43 ± 0.39 56.00 ± 0.87 37.78 ± 0.63 33.27 ± 0.71 49.26 ± 4.19 I 129.07 ± 3.26 124.32 ± 3.35 3.79 ± 0.18 5.82 ± 0.15 ea 0.04 ± 0.01
61.89 ± 3.46 I 90.63 ± 3.10 I I I 73.21 ± 3.54 I 93.79 ± 3.21 35.63 ± 1.15 0.36 ± 0.05 ea
ea I 81.88 ± 0.49 72.41 ± 4.74 83.72 ± 2.72 I 97.86 ± 3.32 77.54 ± 2.80 13.72 ± 0.05 73.89 ± 4.95 0.19 ± 0.00 ea
I I I I I I 23.29 ± 1.71 I 21.76 ± 0.69 16.57 ± 0.92 0.40 ± 0.03 ea
83.08 ± 4.17 I I I I I I I 40.78 ± 2.15 19.06 ± 0.45 0.35 ± 0.02 ea
75.44 ± 1.00 I I 99.44 ± 1.53 I I I I 83.74 ± 2.89 47.17 ± 0.00 1.64 ± 0.17 ea
I ¼ inactive at IC50 > 50 mg/mL. a not determined; etoposide and doxorubicin were used as the reference compounds.
1367, 1227, 1176, 1100, 1080, 1027, 846, 734, 702 cm1; 1H and 13C NMR: (Table 1); HRAPCIMS m/z 433.2593 [M þ H]þ (calcd for C25H37O6, 433.2585). 4.3.2. Erectuseneol A (2) Colorless powder; [a]24 D þ2.9 (c 0.34, CHCl3); UV lmax (MeOH) nm (log ε) 224 (4.0), 279 (sh); IR (ATR) nmax 3421 (br), 2928, 2866, 1733, 1541, 1515, 1457, 1372, 1238, 1172, 1022, 735, 703 cm1; 1H and 13 C NMR: (Table 2); HRESIMS m/z 497.2431 [M þ Na]þ (calcd for C28H35 39ClNaO4, 497.2429). 4.3.3. Erectuseneol B (3) Colorless powder; [a]24 D þ4.3 (c 0.21, CHCl3); UV lmax (MeOH) nm (log ε) 224 (3.9), 278 (sh); IR (ATR) nmax 3421 (br), 2951, 2925, 2857, 1733, 1714, 1615, 1542, 1516, 1444, 1372, 1236, 1172, 1022, 918, 835, 753 cm1; 1H and 13C NMR: (Table 2); HRESIMS m/z 539.2539 [M þ Na]þ (calcd for C30H35 41ClNaO5, 539.2535). 4.3.4. Erectuseneol C (4) Colorless powder; [a]24 D 4.0 (c 0.32, CHCl3); UV lmax (MeOH) nm (log ε) 224 (2.8), 279 (sh); IR (ATR) nmax 3393 (br), 2927, 2856, 1702, 1616, 1542, 1514, 1444, 1377, 1232, 1170, 1100, 1024, 918, 832, 736 cm1; 1H and 13C NMR: (Table 3); HRAPCIMS m/z 363.2090 [M þ H]þ (calcd for C22H35 32ClO2, 363.2085). 4.3.5. Erectuseneol D (5) Pale yellow amorphous solid; [a]25 D þ3.2 (c 0.13, CHCl3); UV lmax (MeOH) nm (log ε) 224 (3.7), 280 (sh); IR (ATR) nmax 3362 (br), 2951, 2922, 2853, 1716, 1615, 1515, 1457, 1377, 1231, 1171, 1111, 981, 914, 811, 773, 720 cm1; 1H and 13C NMR: (Table 3); HRESIMS m/z 303.2319 [MH] (calcd for C20H31O2, 303.2329). 4.3.6. Erectuseneol E (6) Colorless powder; [a]25 D þ3.0 (c 0.12, CHCl3); UV lmax (MeOH) nm (log ε) 224 (3.9), 279 (sh); IR (ATR) nmax 3362 (br), 2951, 2923, 2853, 1716, 1640, 1615, 1542, 1515, 1457, 1377, 1233, 1171, 1111, 979, 914, 849, 813, 773, 720 cm1; 1H and 13C NMR: (Table 3); HRAPCIMS m/z 261.1862 [MH] (calcd for C17H25O2, 261.1860). 4.3.7. Erectuseneol F (7) Colorless powder; [a]25 D 5.4 (c 0.20, CHCl3); UV lmax (MeOH) nm (log ε) 226 (4.1), 277 (sh); IR (ATR) nmax 3351 (br), 2924, 2853, 1715, 1600, 1513, 1442, 1367, 1227, 1170, 1039, 913, 809 cm1; 1H and 13 C NMR: (Table 3); HRAPCIMS m/z 305.1315 [M H] (calcd for þ C18H35 22ClO2, 305.1314), HRESIMS m/z 329.1290 [M þ Na] (calcd for C18H35 23ClNaO2, 329.1279).
4.4. Cytotoxicity assay Cytotoxic activity for adhesive cell lines including HepG2, HeLa, HuCCA-1, A549, and MDA-MB-231 cancer cell lines was evaluated with the MTT assay.33,34 For the non-adhesive MOLT-3 cell line, the cytotoxicity was assessed using the XTT assay.35 The reference substances used were doxorubicin and etoposide (Table 4). Acknowledgements We thank Pakamas Intachote, Suchada Sengsai, and Busakorn Saimanee for cytotoxic assays. We thank the Mahidol University for partial financial support. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.tet.2017.11.073. References 1. Williams DE, Tahir A, Andersen RJ. J Nat Prod. 1999;62:653e654. 2. Mahidol C, Prawat H, Sangpetsiripan S, Ruchirawat S. J Nat Prod. 2009;72: 1870e1874. 3. Elhady SS, El-Halawany AM, Alahdal AM, Hassanean HA, Ahmed SA. Molecules. 2016;21:82. 4. Elhady SS, Al-Abd AM, El-Halawany AM, Alahdal AM, Hassanean H,A, Ahmed SA. Mar Drugs. 2016;14:130. 5. Kobayashi M, Okamoto T, Hayashi K, Yokoyama N, Sasaki T, Kitagawa I. Chem Pharm Bull. 1994;42:265e270. 6. Li J, Du L, Kelly M, Zhou YD, Nagle DG. J Nat Prod. 2013;76:1492e1497. 7. Yin L, Li H, Chen X, Qiu Y. Rec Nat Prod. 2014;8:417e421. 8. Doi Y, Shigemori H, Ishibashi M, et al. Chem Pharm Bull. 1993;41:2190e2191. €ng GM, Wright AD, Kaminsky R. J Nat Prod. 2000;63:825e829. 9. Kirsch G, Ko 10. Xu WH, Ding Y, Jacob MR, et al. Bioorg Med Chem Lett. 2009;19:6140e6143. 11. Salmoun M, Devijver C, Daloze D, et al. J Nat Prod. 2000;63:452e456. 12. Kobayashi M, Aoki S, Gato K, Kitagawa I. Chem Pharm Bull. 1996;44: 2142e2149. 13. Kobayashi M, Aoki S, Sakai H, Kihara N, Sasaki T, Kitagawa I. Chem Pharm Bull. 1993;41:989e991. 14. Kubota T, Nakamura K, Sakai K, Fromont J, Gonoi T, Kobayashi J. Chem Pharm Bull. 2016;64:975e978. 15. Khokhar S, Feng Y, Campitelli MR, et al. Bioorg Med Chem Lett. 2014;24: 3329e3332. 16. He WF, Xue DQ, Yao LG, Li JY, Li J, Guo YW. Mar Drugs. 2014;12:3982e3993. 17. Salmoun M, Devijver C, Daloze D, Braekman JC, van Soest RWM. J Nat Prod. 2002;65:1173e1176. 18. Imada K, Sakai E, Kato H, et al. Tetrahedron. 2013;69:7051e7055. 19. Tanaka N, Momose R, Takahashi-Nakaguchi A, Gonoi T, Fromont J, Kobayashi J. Tetrahedron. 2014;70:832e837. 20. Takahashi Y, Iinuma Y, Kubota T, et al. Org Lett. 2011;13:628e631. 21. a) Sata N, Galario MA, Sitachitta N, Scheuer PJ, Kelly M. J Nat Prod. 2005;68: 262e263; b) Bertin MJ, Zimba PV, He H, Moeller PDR. Tetrahedron Lett. 2016;57: 5864e5867; c) Hwang IH, Oh J, Kochanowska-Karamyan A, Doerksen RJ, Na M, Hamann MT.
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Tetrahedron Lett. 2013;54:3872e3876; d) Schock TB, Huncik K, Beauchesne KR, Villareal TA, Moeller PDR. Environ Sci Technol. 2011;45:7503e7509. Potts BCM, Capon RJ, Faulkner DJ. J Org Chem. 1992;57:2965e2967. Tsuda M, Shigemori H, Ishibashi M, Sasaki T, Kobayashi J. J Org Chem. 1992;57: 3503e3507. Hareau-Vittini G, Kocienski PJ. Synlett. 1995:893e894. Hareau-Vittini G, Kocienski PJ, Reid G. Synthesis. 1995:1007e1013. Wonganuchitmeta S, Yuenyongsawad S, Keawpradub N, Plubrukarn A. J Nat Prod. 2004;67:1767e1770. Cheng CH, Chung HM, Hwang TL, et al. J Mol. 2012;17:9443e9450. Chao CH, Chou KJ, Wang GH, et al. J Nat Prod. 2010;73:1538e1543.
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Sesterterpenes and phenolic alkenes from the Thai sponge Hyrtios erectus Wirongrong Kaweetripob a, Chulabhorn Mahidol a, b, Siriporn Wongbundit a, Pittaya Tuntiwachwuttikul d, Somsak Ruchirawat a, b, c, Hunsa Prawat a, * a
Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Bangkok, 10210, Thailand Chulabhorn Graduate Institute, Chemical Biology Program, Chulabhorn Royal Academy, Kamphaeng Phet 6 Rood, Laksi, Bangkok, 10210, Thailand Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of Education, Bangkok, Thailand d Laboratory of Natural Products Chemistry, Faculty of Science and Technology, Phuket Rajabhat University, Phuket, 83000, Thailand b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2017 Received in revised form 20 November 2017 Accepted 27 November 2017 Available online 5 December 2017
Sesterterpene, erectusolide A (1), six phenolic alkenes, erectuseneols AF (2e7) and nine known compounds, luffalactone (8), luffariolide E (9), (6E)- and (6Z)-neomanoalide 24,25-diacetates (10 and 11), 6,6dimethylundecane-2,5,10-trione (12), threo- and erythro-cavernosines (13 and 14), (4E,6E)-dehydromanoalide (15), echinoclerodane A (16), were isolated from the marine sponge Hyrtios erectus. Compound 13 was isolated for the first time from a natural source. The structures of these compounds were elucidated on the basis of spectroscopic analysis. The phenolic alkenes 3 and 7, the sesterterpenes 8 e11 and 15, and compounds 12e14 were evaluated for cytotoxic activities against six cancer cell lines, MOLT-3, HepG2, HeLa, HuCCA-1, A549, and MDA-MB-231. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Hyrtios erectus Thorectidae Sesterterpenes Phenolic alkenes Cytotoxic activities
1. Introduction Marine sponges of the genus Hyrtios (Family Thorectidae) have proven to be a rich source of secondary metabolites such as terpenoids [mainly acyclic triterpenes,1 sesterterpenes (manoalide, scaralane),2e9 sesquiterpenes/quinones10,11], macrolides,12,13 indole alkaloids,7,9,14e20 and phenyl alkenes.21a-d Some of these metabolites have been found to possess multiple biological activities, such as cytotoxicity,2e6,12,13,15 antiplasmodial,9 antifungal,10 and antimicrobial.14 To probe for the use of these compounds as taxonomical and biological marker for the genus Hyrtios, we have undertaken a chemical study of more species of this genus. 2. Results and discussion The sponge Hyrtios erectus (CRI 588) collected from Similan Island, Phangnga province, Thailand was extracted with MeOH and the extract was partitioned between EtOAc and water. The EtOAcsoluble material was subjected to silica gel column
* Corresponding author. E-mail address: [email protected] (H. Prawat). https://doi.org/10.1016/j.tet.2017.11.073 0040-4020/© 2017 Elsevier Ltd. All rights reserved.
chromatography followed by Sephadex LH-20 column chromatography and revered-phase HPLC to yield erectusolide A (1) and erectuseneols AF (2e7) together with nine known compounds. The known compounds were identified as five sesterterpenes, luffalactone (8),22 (3S*,4R* and 3R*,4R*)-luffariolide E (ratio~1:0.1) (9),23e25 (6E)- and (6Z)-neomanoalide 24,25-diacetates (10 and 11),26 and (4E,6E)-dehydromanoalide (15),22 one diterpene, echinoclerodane A (16),27 and three norterpenes, 6,6dimethylundecane-2,5,10-trione (12),28 and threo-cavernosine (13),30 and erythro-cavernosine (14).29,30 All of the known compounds (Fig. 1) were readily identified by extensive study of their spectral data, including HRAPCIMS or HRESIMS, 1D and 2D NMR data, as well as by comparison with those reported in the literatures. The phenolic alkenes 3 and 7, the sesterterpenes 8e11 and 15, and compounds 12e14 were evaluated for cytotoxic activities against six cancer cell lines, MOLT-3, HepG2, HeLa, HuCCA-1, A549, and MDA-MB-231. Compound 1 was obtained as an optically active colorless powder ([a]24 D þ2.5), and its molecular formula was determined to be C25H36O6 (index of hydrogen deficiency ¼ 8) by HRAPCIMS. IR absorption bands at 1772 and 1705 cm1 of 1 suggested the presence of lactone and carbonyl ketone groups, respectively. Six of the
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Fig. 1. Structures of isolated compounds 1e16 and trichotoxin A.21b
eight degrees of hydrogen deficiency implied by the molecular formula of 1 was taken up in four carboneoxygen double bonds and two carbonecarbon double bonds; the molecule was thus bicyclic. The 1H and 13C NMR spectroscopic data of 1 (Table 1) were similar to those of luffariolide E (9)23,24 for the C-1eC-11 region. A pair of two signals (ratio ~ 1:0.3) due to the same carbons and protons were detected for some carbons and protons in the 1H and 13C NMR spectra of 1 (Table 1) similar to the signals of 3S*,4R*- and 3R*,4R*laffariolide E (9), which were synthesized by Kocienski and coworker.24,25 The signals associated with the cyclohexene moiety of 9 were not evident in 1; instead two new singlet signals at dC 208.4 and 214.9 were observed. The presence of the two aliphatic carbonyl signals at dC 208.4 and 214.9 together with a signal for a methyl ketone residue (dC 29.9 and dH 2.13) in the NMR spectrum implied the existence of an open-chain dicarbonyl moiety, which could be derived from an oxidative opening of a cyclohexene ring of 9 at the C-14C-15 olefinic bond.31 This was also compatible with the 32 mass unit differences in the molecular weight of 1 compared to luffariolide E. The structure of 1 was fully supported by 1H1H COSY, HSQC, and HMBC NMR techniques. The 1H1H COSY (Fig. 2) and HSQC data allowed the unambiguous assignment of two spin systems which consisted of two and three methylene units. One methylene group of each unit was adjacent to a ketone functionality, as implied by their chemical shift of dH 2.55 and 2.41 for H2-13
and H2-18, respectively. The first spin system consisted of the methylenes at dH 2.21 (H2-12) and 2.55 (H2-13). The HMBC (Fig. 2) spectra confirmed this system through correlations with the carbonyl at dC 214.9 (C-14). Moreover, the correlation of dH 1.60 (H323) with dC 33.4 (C-12) and of H2-12 (dH 2.21) with the olefinic methine carbon at dC 123.3 (C-10) allowed the extension of the C1C-13. The second spin system consisted of the methylenes at dH 1.42e1.52 (H2-16), 1.38e1.48 (H2-17), and 2.41 (H2-18). The HMBC experiment also confirmed this system through correlations of H217 and H2-18 with the carbonyl signal at dC 208.4 (C-19). The germinal dimethyl groups at dH 1.12 (s, 6H, H3-21/22) correlated with the C-14, C-15, and C-16 in HMBC spectrum, which suggested the attachment of germinal dimethyl (H3-21/22) at C-15. The methyl singlet at dH 2.13 (H3-20) implied that this was the terminus, as it showed a cross-peak with C-18 and C-19. The C-10/C-11 double bond was assigned as E configuration by NOESY experiment (Fig. 2). The 1H and 13C NMR resonances for the segment C-12C-20 of 1 were similar to those found in fasciospongides B and C,31 thus the structure of 1 was established as a mixture of 3S*,4R*- and 3R*,4R*compound 1 (ratio~1:0.3, Fig. 1) and was named erectusolide A. Compound 2 was obtained as an optically active colorless powder ([a]24 D þ2.9), and its molecular formula was determined to be C28H39ClO4 (index of hydrogen deficiency ¼ 9) by HRESIMS (m/z 497.2431 [MþNa]þ; calcd for C28H35 39ClO4, 497.2429). Its UV
318
W. Kaweetripob et al. / Tetrahedron 74 (2018) 316e323
Table 1 1 H (600 MHz) and
13
C (150 MHz) NMR (CDCl3) data of compound 1.
Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
3S*,4R*
3R*,4R* (ratio ~ 1:0.3 of 3S*,4R*:3R*,4R*)
dC, type
dH (J in Hz)
dC, type
dH (J in Hz)
175.3, C 30.1, CH2 39.6, CH 77.6, CH 28.0, CH2 137.6, CH 132.7, C 30.8, CH2 26.4, CH2 123.3, CH 135.5, C 33.4, CH2 35.5, CH2 214.9, C 47.5, C 39.2, CH2 19.0, CH2 43.9, CH2 208.4, C 29.9, CH3 24.4, CH3 24.4, CH3 16.2, CH3 164.1, C 69.6, CH2
e 2.43, dd (17.5, 8.5) 2.63, dd (17.5, 9.0) 2.88e2.96, m 4.33e4.39, m 2.28e2.42, m 6.56, brd (4.8) e 2.22e2.32, m 2.11e2.22, m 5.10, t (7.1) e 2.21, t (7.7) 2.55, dd (8.2, 7.7) e e 1.42e1.52, m 1.38e1.48, m 2.41, t (6.8) e 2.13, s 1.12, s 1.12, s 1.60, s e 4.35, dd (9.6, 7.8) 4.54, dd (9.6, 7.1)
175.5, C 30.1, CH2 39.4, CH 76.5, CH 27.5, CH2
e 2.62, dd (17.8, 8.0) 2.70, dd (17.8, 9.0) 2.87e2.96, o 4.35e4.42, o 2.28e2.42, o
68.2, CH2
4.28, dd (9.4, 7.2) 4.42, dd (9.4, 7.9)
spectrum presenting a maximum absorption at 224 nm as well as a shoulder at 279 nm indicated the presence of an aromatic system. The IR spectrum of 2 displayed absorption bands at 3421 and 1733 cm1, suggesting the presence of hydroxyl and ester groups, respectively. The 1H and 13C NMR spectroscopic data of 2 (Table 2) showed the presence of a para-substituted benzene ring [positions 18e23: dH 6.79 (H-20/22), 7.07 (H-19/23) and dC 115.4 (C-20/22), 130.2 (C-18), 130.5 (C-19/23), 154.4 (C-21)]. The diastereotopic benzylic methylene protons (H2-17, dН 2.66/2.75) showed HMBC (Fig. 3) correlations to the aromatic system (C-18, C-19 and C-23), as well as the oxymethine carbon (C-16, dC 72.5), and the allylic methylene carbon (C-15, dC 34.5). The cross-peak in the HMBC spectrum from the olifinic proton (H-14, dH 5.47e5.53) to oxymethine carbon (C-16) was also detected. The consecutive analyses of the 1H1H COSY (Fig. 3) correlations from the allylic methylene protons (H2-11, dH 2.02e2.15) to benzylic methylene protons (H217) indicated a heptenol unit (C-11C-17) comprised the alkyl chain of structure 2 (Fig. 3). The 1H and 13C NMR spectra for the segment C-1C-11 of 2 (Table 2) were closely similar to those of trichotoxin A21b (Fig. 1), except for the spectral signals at C-4. The downfield shift of oxymethine signals at C-4 (dC 78.5 and dH 5.17) relative to the resonance observed for trichotoxin A (dC 76.7 and dH 4.01) indicated that the hydroxyl group at C-4 in 2 was acetylated. This was further confirmed by HMBC (Fig. 3) correlations between H-4 and the carbonyl carbon of the acetate ester (dC 170.5) and satisfying the final index of hydrogen deficiency. The vinyl methine proton (H-24, dН 5.76) showed HMBC correlations to the quaternary carbon (C-10, dC 142.2) and two allylic methylene carbons (C-9, dC 28.4 and C-11, dC 34.7). From the above spectral data, the planar
Fig. 2. Key COSY (
), HMBC (
), and NOESY (
) correlations of 1.
structure 2 (Fig. 1) was thus suggested for the compound. The NOESY (C6D6; Fig. 3) correlations between the methine proton, H24 (dН 5.63) and both methylene groups, H2-11 (dH 1.83e1.94) and H2-12 (dН 1.95e2.05) supported a Z configuration of C-10/C-24 double bond. An E configuration of the C-5/C-6 double bond was supported by examining the chemical shift of the olefinic methyl carbon (C-26, dC 12.3)21c and the NOESY correlations between H-7 and H-26. A Z configuration of the C-13/C-14 double bond was supported by the NOESY correlations between H-12 and H-15 (dH 2.10e2.20) and coupling constant value (JH-13,H-14 ¼ 10.8 Hz). Thus, the structure of 2 was established as (5E,10Z,13Z)-4-acetoxy-10(chloromethylene)-16-hydroxy-17-(4-hydroxyphenyl)-5,7dimethylheptadeca-1,5,13-triene and was named erectuseneol A. Unfortunately insufficient material was available for further experiments to determine the absolute configurations at the chiral centers (C-4, C-7, and C-16). Compound 3 was obtained as an optically active colorless powder ([a]24 D þ4.3), exhibiting the same IR, UV spectra as compound 2. Compound 3 showed molecular formula ion at m/z 539.2539 [MþNa]þ by HRESIMS indicating the molecular formula of 3 was C30H41ClO5 (index of hydrogen deficiency ¼ 10). This difference of 42 amu (C2H2O) compared to the molecular formula of compound 2 could suggest the presence of an additional acetyl group in compound 3. The remarkable resemblance in the 1H and 13 C NMR data of compounds 2 and 3 (Table 2), indicating that both compounds possess similar structural features. The downfield shift of oxymethine group (C-16, dC 74.5 and H-16 dH 5.00e5.05) relative to the resonance observed in compound 2 indicated that the hydroxyl group at C-16 was acetylated in compound 3. This was further confirmed by HMBC correlations between H-16 and the carbonyl carbon of the acetate ester (dC 170.4). Thus, the structure of 3 was established as (5E,10Z,13Z)-10-(chloromethylene)-4,16diacetoxy-17-(4-hydroxyphenyl)-5,7-dimethylheptadeca-1,5,13triene and was named erectuseneol B. Compound 4 was obtained as an optically active colorless powder ([a]24 D 4.0), exhibiting the same UV spectrum as compounds 2 and 3. Its molecular formula was determined to be
W. Kaweetripob et al. / Tetrahedron 74 (2018) 316e323 Table 2 1 H (600 MHz) and
13
319
C (150 MHz) NMR dataa (CDCl3) of compounds 2 and 3.
Position (type)
1 (CH2) 2 (CH) 3 (CH2) 4 (CH) 5 (C) 6 (CH) 7 (CH) 8 (CH2) 9 (CH2) 10 (C) 11 (CH2) 12 (CH2) 13 (CH) 14 (CH) 15 (CH2) 16 (CH) 17 (CH2) 18 (C) 19 (CH) 20 (CH) 21 (C) 22 (CH) 23 (CH) 24 (CH) 25 (CH3) 26 (CH3) 4-OCOMe 4-OCOMe 16-OCOMe 16-OCOMe
2
3
dC
dH (J in Hz)
dH inC6D6 (J in Hz)
dC
dH (J in Hz)
117.3 133.7 37.4 78.5 131.7 134.5 32.3 34.6 28.4 142.2 34.7 25.7 131.4 126.2 34.5 72.5 42.5 130.2 130.5 115.4 154.4 115.4 130.5 112.4 21.3 12.3 170.5 20.7 e e
5.02, brd (10.0) 5.07, dq (17.1, 1.5) 5.67, ddt (17.1, 10.0, 7.0) 2.35, qnt (7.1) 2.41, qnt (7.1) 5.17, t (7.0) e 5.23, d (10.0) 2.33e2.40, o 1.22e1.31, m 1.36e1.44, m 2.01e2.14, m e 2.02e2.15, o 2.08e2.18, o 5.45e5.52, m 5.47e5.53, m 2.18e2.28, m 3.80, brqnt (6.9) 2.66, dd (13.7, 7.7) 2.75, dd (13.7, 5.1) e 7.07, d (8.5) 6.79, d (8.5) e 6.79, d (8.5) 7.07, d (8.5) 5.76, brs 0.97, d (6.7) 1.63, brd (1.2) e 2.01, s e e
5.01, dd (17.0, 1.7) 5.02, brd (10.0) 5.69, ddt (17.0, 10.0, 7.0) 2.27e2.32, m 2.39, brp (7.0) 5.36, t (6.9) e 5.29, brd (10.0) 2.25e2.32, m 1.20e1.33, m 1.34e1.45, m 2.10e2.20, m e 1.83e1.94, m 1.95e2.05, m 5.34e5.40, m 5.50, brdt (10.8, 7.3) 2.10e2.20, m 3.65, brqnt (6.6) 2.57, dd (13.7, 7.2) 2.61, dd (13.7, 5.4) e 6.96, d (8.5) 6.65, d (8.5) e 6.65, d (8.5) 6.65, d (8.5) 5.63, s 0.92, d (6.6) 1.56, brd (1.2)
117.3 133.7 37.4 78.5 131.7 134.5 32.4 34.6 28.5 142.2 34.7 25.6 131.2 125.2 31.0 74.5 39.1 129.3 130.5 115.3 154.5 115.3 130.5 112.4 20.6 12.3 170.5 21.2 170.4 21.1
5.02, brd (9.0) 5.07, dd (17.1, 1.3) 5.67, ddt (17.1, 10.1, 7.0) 2.34, qnt (7.1) 2.41, qnt (7.1) 5.16, t (7.0) e 5.22, d (9.5) 2.32e2.39, o 1.22e1.30, m 1.36e1.43, m 2.00e2.13, m e 2.00e2.10, o 2.02e2.10, o 5.39e5.45, m 5.38e5.43, m 2.25, t (5.8) 5.00e5.05, m 2.74, dd (13.9, 6.5) 2.80, dd (13.9, 6.5) e 7.04, d (8.3) 6.76, d (8.3) e 6.76, d (8.3) 7.04, d (8.3) 5.74, s 0.96, d (6.6) 0.63, s e 2.06, s e 2.00, s
1.68, s e e
qnt ¼ quintet; o ¼ overlap. a Assignments are based on COSY, HSQC, and HMBC experiments.
C22H31ClO2 (index of hydrogen deficiency ¼ 7) by HRAPCIMS. The IR spectrum of 4 displayed absorption bands at 3393 cm1, suggesting the presence of hydroxyl group. Analysis of 1H and 13C NMR spectroscopic data of 4 (Table 3) showed the presence of a parasubstituted benzene ring [dC 130.8 (C-14), 129.8 (C-15/19), 115.3 (C16/18), 154.1 (C-17) and dH 7.06 (H-15/19) and 6.75 (H-16/18)]. The benzylic methylene protons (H2-13, dH 3.48) were substantially deshielded and showed HMBC correlations to the aromatic system (C-14, C-15, and C-19) as well as a moderately polarized double bond (C-12, dC 141.3; C-20, dC 113.1). The polarization of the C-12/C20 double bond and the singlet methine proton (H-20, dH 5.91) was consistent with the presence of a vinyl chloride functionality. The doubly allylic methylene protons (H2-11, dH 2.66) showed HMBC correlations to vinyl chloride moiety and the C-9 quaternary olefinic carbon (dC 138.0) and COSY correlations to the olefinic proton (H-10, dH 5.03). The H2-8 allylic methylene protons (dH 1.95) showed HMBC correlations to both olefinic carbons, C-9 (dC 138.0) and C-10 (dC 120.2), and the C-21 vinyl methyl (dC 15.9). The analyses of both the HMBC and the 1H1H COSY correlations of 4 from the terminal methylene (H2-1, dH 5.14/5.15; dC 117.9) to allylic methylene (H2-8, dH 1.95; dC 39.8) indicated the presence of a 5methyl-4-hydroxyoct-1-en (C-1C-8 and C-22) unit comprised in
Fig. 3. Key COSY (
), HMBC (
), and NOESY in C6D6 (
) correlations of 2.
the structure. The cross-peaks from the oxymethine proton (H-4, dH 3.56) to the olefinic carbon (C-2, dC 135.5), methylene carbon (C-6, dC 32.8) and secondary methyl carbon (C-22, dC 13.9) were also detected in HMBC spectrum. The C-9/C-10 and C-12/C-20 double bonds were assigned as 9E and 12Z configurations by the NOESY correlations (Fig. 4) between olefinic methine proton (H-20, dH 5.91) and doubly allylic methylene protons (H2-11, dH 2.66) and between doubly allylic methylene protons (H2-11) and vinyl methyl protons (H3-21, dH 1.49). Thus, the structure of 4 was established as (9E,12Z)-12-(chloromethylene)-4-hydroxy-13-(4-hydroxyphenyl)5,9-dimethyltrideca-1,9-diene and was named erectuseneol C. To find the absolute configuration at C-4 of 4 was addressed through the application of Mosher's method.32 Both (R)- and (S)-methoxya-(trifluoromethyl)phenyl acetic acid (MTPA) esters of 4 were prepared but did not provide any useful information for assigning the configuration at C-4. Compound 5 was obtained as an optically active pale yellow amorphous solid ([a]25 D þ3.2), exhibiting the same IR, UV spectra as compound 4. Compound 5 showed molecular formula ion at m/z 303.2319 [MH] by HRESIMS indicating the molecular formula of 5 was C20H32O2 (index of hydrogen deficiency ¼ 5). The 1H and 13C NMR spectra of 5 showed the presence of a para-substituted benzene ring [dH 7.00 (H-13/17), 6.74 (H-14/16) and dC 133.9 (C-12), 130.2 (C-13/17), 114.9 (C-14/16), 153.5 (C-15)]. The analyses of both the HMBC and 1H1H COSY correlations of 5 from the terminal methylene (H2-1, dH 5.12/5.14; dC 117.7) to the benzylic methylene (H2-11, dH 2.31/2.53; dC 43.0) indicated the presence of an alkenyl chain, 4-hydroxy-5,7,10-trimethylundec-1-en (C-1C-11, C-18, C19, and C-20) comprised in the structure. The benzylic methylene protons (H2-11 showed HMBC correlations to the aromatic carbons (C-12, C-13, and C-17) and methyl carbon (C-18, dC 19.3). The H-4
320
W. Kaweetripob et al. / Tetrahedron 74 (2018) 316e323
Table 3 1 H (600 MHz) and
13
C (150 MHz) NMR data (CDCl3) of compounds 4e7.
Position 4
dC, type dH (J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 OH
117.9, CH2 135.5, CH 39.1, CH2 73.9, CH 37.7, CH 32.8, CH2 25.6, CH2 39.8, CH2 138.0, C
5
6
7
dC, type dH (J in Hz)
dC, type dH (J in Hz)
dC, type dH (J in Hz)
5.14, brd (10.0) 5.15, brd (17.1) 5.83, dddd (17.1, 10.0, 8.0, 6.3) 2.13e2.21, m 2.23 e2.30, m 3.56, dt (8.4, 4.5)
117.7, CH2 135.6, CH 39.3, CH2 73.6, CH 1.49e1.58, o 35.0, CH 1.11e1.18, o 1.38e1.46, 41.1, o CH2 1.30e1.38, o 1.40e1.47, 30.2, o CH 1.95, brt (6.2) 33.70, CH2 e 33.73, CH2 120.2, 5.03, brtd (7.2, 1.0) 35.4, CH CH 33.1, 2.66, d (7.1) 43.0, CH2 CH2 141.3, C e 133.9, C 35.6, 3.48, s CH2 130.8, C e 129.8, 7.06, d (8.4) CH 115.3, 6.75, d (8.4) CH 154.1, C e 115.3, CH 129.8, CH 113.1, CH 15.9, CH3 13.9, CH3 e
6.75, d (8.4) 7.06, d (8.4) 5.91, s
5.12, br d (10.1) 5.14, br d (17.1) 117.8, CH2 5.83, dddd (17.1, 10.1, 7.8, 6.4) 135.6, CH 2.18, dt (14.1, 8.2) 2.25, brdt 39.1, (14.1, 5.2) CH2 3.52, dt (8.9, 3.9) 74.1, CH 1.56e1.66, m 38.1, CH 1.00, ddd (13.4, 8.1, 6.5) 1.37, dd 30.5, (13.4, 6.5) CH2 1.42e1.50, m 34.2, CH2 1.03e1.38, o 35.3, CH 1.03e1.38, o 42.9, CH2 1.56e1.66, m 133.7, C
5.12, brd (9.9) 5.14, brd (17.1) 5.82, dddd (17.1, 10.0, 8.0, 6.5) 2.15, dt (14.2, 8.3) 2.27, ddd (14.2, 5.1, 4.9) 3.52, dt (8.9, 3.7) 1.44e1.51, m 1.41e1.48, m 1.14e1.27, m 1.29e1.35, m 1.60e1.68, m 2.33, dd (13.5, 8.0) 2.55, dd (13.5, 6.2) e
2.31, dd (13.6, 8.0) 2.53, dd (13.6, 130.2, 7.00, d (8.4) 6.3) CH e 114.9, 6.74, d (8.4) CH 7.00, d (8.4) 153.5, C e
130.2, CH 114.9, 6.74, d (8.4) CH 153.5, C e 114.9, CH 130.2, CH 19.3, CH3 20.4, CH3 14.2, CH3
6.74, d (8.4)
e
4.66, s
7.00, d (8.4)
114.9, CH 130.2, CH 19.3, CH3 13.8, CH3
6.74, d (8.4) 7.00, d (8.4) 0.84, d (6.6) 0.87, d (6.6)
0.83, d (6.6)
114.9, CH2 138.1, CH 33.6, CH2 26.8, CH2 132.7, CH 125.2, CH 35.5, CH2 70.0, CH 37.3, CH2 139.9, C
4.97, brdd (10.2, 1.0) 5.02, brdd (17.1, 1.5) 5.82, ddt (17.0, 10.2, 6.5) 2.06e2.15, m 2.11e2.19, m 5.57, brdt (10.8, 6.8) 4.42, brdt (10.8, 7.4) 2.19e2.31, m 3.85, brs 2.32, dd (13.7, 4.5) 2.37, dd (13.7, 8.5) e
41.1, 3.39, s CH2 130.2, C e 130.2, 7.03, d (8.4) CH 115.4, 6.77, d (8.4) CH 154.3, C e 115.4, CH 130.2, CH 115.9, CH
6.77, d (8.4) 7.03, d (8.4) 5.95, s
0.84, d (6.6) 0.88, d (6.6)
1.49, s 0.91, d (6.8) e
oxymethine proton (dH 3.52) showed HMBC correlations to C-6 (dC 41.1), C-2 (dC 135.6), and C-20 (dC 14.2) and the H2-3 allylic methylene (dH 2.18/2.25) showed the HMBC cross-peak to C-1 (dC 117.7). Compound 5 was thus established as 4-hydroxy-11-(4hydroxyphenyl)-5,7,10-trimethylundeca-1-ene and was named erectuseneol D. Compound 6 was obtained as an optically active colorless powder ([a]25 D þ3.0), exhibiting the same IR, UV spectra as compound 5. Compound 6 showed molecular formula ion at m/z 261.1862 [MH] by HRESIMS indicating the molecular formula of
Fig. 4. Key COSY (
), HMBC (
), and NOESY (
) correlations of 4.
e
4.72, brs
e
4.86, brs
6 was C17H26O2 (index of hydrogen deficiency ¼ 5). Analyses of 1D and 2D NMR spectra clearly showed 6 was an analog of 5. The spectral signals from C-1C-5 and C-8C-17 of the carbon backbone of 6 were identical to C-1C-5 and C-10C-18 and C-20 of 5. The benzyl methylene protons (H2-9, dH 2.33/2.55) showed HMBC correlations to the aromatic system (C-10, dC 133.7; C-11/15, dC 130.2), secondary methyl (C-16, dC 19.3) and methylene carbon (C-7, dC 34.2). The methine proton (H-8, dH 1.60e1.68) showed COSY correlations to benzylic methylene protons (H2-9, dH 2.33/2.55), methyl protons (H3-16, dH 0.84) and methylene protons (H2-7, dH 1.14e1.24/1.29e1.35). The H2-7 methylene showed COSY correlations to methylene protons (H2-6, dH 1.41e1.48). The methylene carbon (C-6, dC 30.5), methyl carbon (C-17, dC 13.8) and olefinic carbon (C-2, dC 135.6) and the allylic methylene carbon (C-3, dC 39.1) showed HMBC correlations to oxymethine proton (H-4, dH 3.52) and terminal methylene protons (H2-1), respectively. The 1 H1H COSY correlations from the methylene protons at dH 1.41/ 1.48 (H2-6) to the terminal olefinic protons at dH 5.12/5.14 (H2-1) and the secondary methyl at dH 0.87 (H3-17) were also observed. Thus, compound 6 was identified as 4-hydroxy-9-(4-
W. Kaweetripob et al. / Tetrahedron 74 (2018) 316e323
321
4. Experimental section 4.1. General experimental procedures
Fig. 5. Key COSY (
), HMBC (
), and NOESY (
) correlations of 7.
hydroxyphenyl)-5,8-dimethylnon-1-ene and was named erectuseneol E. Compound 7 was obtained as an optically active colorless powder ([a]25 D 5.4), exhibiting the same UV and IR spectra as compounds 4e6. Compound 7 showed molecular formula ion at m/ z 305.1315 [MH] by HRESIMS indicating the molecular formula of 7 was C18H23ClO2 (index of hydrogen deficiency ¼ 7). The 1H and 13 C NMR spectroscopic data of 7 (Table 3) showed the presence of a para-substituted benzene ring [dC 130.2 (C-12), 130.2 (C-13/17), 115.4 (C-14/16), 154.3 (C-15) and dH 7.03 (H-13/17) and 6.77 (H-14/ 16)]. The benzylic methylene protons (H2-11, dH 3.39) were substantially deshielded and showed HMBC correlations to the aromatic system (C-12, C-13, and C-17) as well as a moderately polarized olefinic carbons (C-10, dC 139.9; C-18, dC 115.9). The polarization of the C-10/C-18 double bond and the singlet methine proton (H-18, dH 5.95) was consistent with the presence of a vinyl chloride functionality. The 1H1H COSY correlations of 7 from the terminal olefinic methylene protons at dH 4.97/5.02 (H2-1) to the allylic methylene protons at dH 2.32/2.37 (H2-9) indicated the presence to a 8-hydroxynona-1,5-diene unit in the structure. The allylic methylene protons (H2-9) showed HMBC correlations to the vinyl chloride moiety and COSY correlation to the oxymethine proton (H-8, dH 3.85). The position of hydroxyl group at C-8 was confirmed by HMBC correlation between dH 3.85 (H-8) and dC 139.9 (C-10) and 125.2 (C-6). The NOESY (Fig. 5) correlation between H-18 vinyl methine proton (dH 5.95) and H211 (dH 3.39) benzylic methylene protons supported a Z configuration of C-10/C18 double bond. A Z configuration of the C-5/C-6 double bond was supported by the NOESY correlation between H2-4 allylic methylene protons (dH 2.11e2.19) and H2-7 allylic methylene protons (dH 2.19e2.31) and coupling constant value (JH-5,H-6 ¼ 10.8 Hz). Thus, the structure of 7 was established as (5Z,10Z)-10-(chloromethylene)-8-hydroxy11-(4-hydroxyphenyl)-undeca-1,5-diene and was named erectuseneol F. Cytotoxicity of the isolated compounds 3, 7, 9e16 were evaluated against six cancer cell lines (Table 4), MOLT-3 (acute lymphoblastic leukemia), HepG2 (hepatocarcinoma), HeLa (human cervical carcinoma), HuCCA-1 (human chlolangiocarcinoma), A549 (human lung cancer), and MDA-MB-231 (hormone independent breast cancer). Sestertserpene, (4E,6E)-dehydromanoalide (15), and diterpene, echinoclerodane A (16), showed good cytotoxic activity against MOLT-3 cell line with an IC50 values of 3.79 and 5.82 mM, respectively and showed weakly cytotoxic activity against HuCCA1 cell line with an IC50 values of 21.76 and 16.57 mM, respectively. Phenolic alkene, erectuseneol B (3) showed weakly cytotoxic activity against MOLT-3 cell line with IC50 values of 18.43 mM. 3. Conclusions The secondary metabolites of Hyrtios erectus (CRI 588) again show that sesterterpene manoalide derivatives 1, 8e11, and 15 are widespread in this genus.6,7,9 The isolation of the six new phenolic alkenes, 2e7, which were rare natural products, may be of chemotaxonomic relevance.
Optical rotations were measured on a JASCO P-1020 polarimeter in CHCl3. UV spectra were recorded using a Shimadzu UV-1700 Phasmaspect UVeVis spectrophotometer in MeOH. Infrared spectra were taken on a Perkin Elmer Spectrum One spectrophotometer as ATR technique. HRMS were performed on a Bruker (Micro ToF) spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 solution containing Me4Si as internal standard on a Bruker AVANCE 600 spectrometer. HPLC was carried out on a Waters 600 system equipped with a Waters Delta 600 pump, a Waters 600 Controller, a Waters 2998 photodiode array detector, and Waters Empower 2 software. Sephadex™ LH-20 (GE Healthcare BioSciences AB) was used for a column gel filtration. All commercial grade solvents were distilled prior to use and spectral grade solvents were used for spectroscopic measurements. 4.2. Sponge material The sponge, Hyrtios erectus, was collected from Similan Island in the Andaman Sea (Phangnga province, Thailand) in February 2011, at a depth of 30e40 feet by hand via SCUBA diving. This sponge was identified by Dr. Sumaitt Putchakarn, Head of Marine Biodiversity Research, Unit Curator of Porifera and Echinodermata, Institute of Marine Science, Burapha University, Bangsaen, Chonburi, Thailand. A voucher specimen (CRI 588) is presently deposited at the Laboratory of Natural Products, Chulabhorn Research Institute, Bangkok, Thailand. 4.3. Extraction and isolation A frozen sample (wet wt 5.8 kg) of Hyrtios erectus (CRI 588, 14.0 kg) was cut into small pieces and extracted repeatedly with MeOH (20 L 3). After evaporation of the solvent, the concentrated MeOH extracts was partitioned between EtOAc and water. The EtOAc fraction (105 g) was chromatographed on a silica gel column with CH2Cl2hexane (1:1) containing increasing proportions of acetone as eluent, to give 7 fractions (A-G). Fraction B [8.0 g, eluted with acetoneCH2Cl2hexane (1:3:3)] was further separated by a Sephadex LH-20 column chromatography with MeOHCH2Cl2 (1:1) as eluent, to give 4 fractions (B1-B4). B1 (561 mg) were purified by HPLC column (Sunfire C18, 5 mm, 10 250 mm) with CH3CNH2O (85:15), flowrate 2.5 mL/min, l 224 nm, to afford 8 (2.1 mg, at 18.3 min) and 9 (2.1 mg of 3S*,4R* and 3R*,4R*)-luffariolide E, ratio~1:0.1, at 24.8 min). B2 (7.0 g) was fractionated by Sephadex LH-20 with MeOH to yield 4 fractions (B2.1-B2.4). B2.3 (2.7 g) was repeatedly fractionated by Sephadex LH-20 with MeOHCH2Cl2 (1:1) as eluent, to give a mixture compounds (1.0 g) that was separated using on a HPLC column (Sunfire C18, 5 mm, 10 250 mm) with CH3CNH2O (77:23), flowrate 2.5 mL/min, l 224 nm, to afford compounds 1 (3.4 mg, at 6.2 min), 2 (2.1 mg, at 21.5 min), 3 (2.3 mg, at 38.3 min), 10 (37.7 mg, at 54.5 min), and 11 (28.4 mg, at 56.3 min). B2.4 (4.0 g) was repeatedly fractionated by Sephadex LH-20 with MeOHCH2Cl2 (1:1) as eluent, to give 3 subfractions (B2.4.1B2.4.3) and each subfraction was further separated by HPLC column (Sunfire C18, 5 mm, CH3CNH2O, l 224 nm) to afford compounds 12 (7.6 mg), 13 (4.6 mg), 14 (76.0 mg), 15 (70.0 mg), 4 (3.0 mg), 16 (5.4 mg), 5 (1.3 mg), 6 (1.4 mg), and 7 (2.0 mg). 4.3.1. Erectusolide A (1) Colorless powder; [a]24 D þ2.5 (c 0.39, CHCl3); UV lmax (MeOH) nm (log ε) 202 (3.9), 225 (3.6); IR (ATR) nmax 2928, 1772, 1705, 1413,
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Table 4 Cytotoxicity of compounds 3, 7, 9e16 (IC50, mM). Compound
MOLT-3
HepG2
Hela
HuCCA-1
A549
MDA-MB-231
3 7 9 10 11 12 13 14 15 16 Doxorubicin Etoposide
18.43 ± 0.39 56.00 ± 0.87 37.78 ± 0.63 33.27 ± 0.71 49.26 ± 4.19 I 129.07 ± 3.26 124.32 ± 3.35 3.79 ± 0.18 5.82 ± 0.15 ea 0.04 ± 0.01
61.89 ± 3.46 I 90.63 ± 3.10 I I I 73.21 ± 3.54 I 93.79 ± 3.21 35.63 ± 1.15 0.36 ± 0.05 ea
ea I 81.88 ± 0.49 72.41 ± 4.74 83.72 ± 2.72 I 97.86 ± 3.32 77.54 ± 2.80 13.72 ± 0.05 73.89 ± 4.95 0.19 ± 0.00 ea
I I I I I I 23.29 ± 1.71 I 21.76 ± 0.69 16.57 ± 0.92 0.40 ± 0.03 ea
83.08 ± 4.17 I I I I I I I 40.78 ± 2.15 19.06 ± 0.45 0.35 ± 0.02 ea
75.44 ± 1.00 I I 99.44 ± 1.53 I I I I 83.74 ± 2.89 47.17 ± 0.00 1.64 ± 0.17 ea
I ¼ inactive at IC50 > 50 mg/mL. a not determined; etoposide and doxorubicin were used as the reference compounds.
1367, 1227, 1176, 1100, 1080, 1027, 846, 734, 702 cm1; 1H and 13C NMR: (Table 1); HRAPCIMS m/z 433.2593 [M þ H]þ (calcd for C25H37O6, 433.2585). 4.3.2. Erectuseneol A (2) Colorless powder; [a]24 D þ2.9 (c 0.34, CHCl3); UV lmax (MeOH) nm (log ε) 224 (4.0), 279 (sh); IR (ATR) nmax 3421 (br), 2928, 2866, 1733, 1541, 1515, 1457, 1372, 1238, 1172, 1022, 735, 703 cm1; 1H and 13 C NMR: (Table 2); HRESIMS m/z 497.2431 [M þ Na]þ (calcd for C28H35 39ClNaO4, 497.2429). 4.3.3. Erectuseneol B (3) Colorless powder; [a]24 D þ4.3 (c 0.21, CHCl3); UV lmax (MeOH) nm (log ε) 224 (3.9), 278 (sh); IR (ATR) nmax 3421 (br), 2951, 2925, 2857, 1733, 1714, 1615, 1542, 1516, 1444, 1372, 1236, 1172, 1022, 918, 835, 753 cm1; 1H and 13C NMR: (Table 2); HRESIMS m/z 539.2539 [M þ Na]þ (calcd for C30H35 41ClNaO5, 539.2535). 4.3.4. Erectuseneol C (4) Colorless powder; [a]24 D 4.0 (c 0.32, CHCl3); UV lmax (MeOH) nm (log ε) 224 (2.8), 279 (sh); IR (ATR) nmax 3393 (br), 2927, 2856, 1702, 1616, 1542, 1514, 1444, 1377, 1232, 1170, 1100, 1024, 918, 832, 736 cm1; 1H and 13C NMR: (Table 3); HRAPCIMS m/z 363.2090 [M þ H]þ (calcd for C22H35 32ClO2, 363.2085). 4.3.5. Erectuseneol D (5) Pale yellow amorphous solid; [a]25 D þ3.2 (c 0.13, CHCl3); UV lmax (MeOH) nm (log ε) 224 (3.7), 280 (sh); IR (ATR) nmax 3362 (br), 2951, 2922, 2853, 1716, 1615, 1515, 1457, 1377, 1231, 1171, 1111, 981, 914, 811, 773, 720 cm1; 1H and 13C NMR: (Table 3); HRESIMS m/z 303.2319 [MH] (calcd for C20H31O2, 303.2329). 4.3.6. Erectuseneol E (6) Colorless powder; [a]25 D þ3.0 (c 0.12, CHCl3); UV lmax (MeOH) nm (log ε) 224 (3.9), 279 (sh); IR (ATR) nmax 3362 (br), 2951, 2923, 2853, 1716, 1640, 1615, 1542, 1515, 1457, 1377, 1233, 1171, 1111, 979, 914, 849, 813, 773, 720 cm1; 1H and 13C NMR: (Table 3); HRAPCIMS m/z 261.1862 [MH] (calcd for C17H25O2, 261.1860). 4.3.7. Erectuseneol F (7) Colorless powder; [a]25 D 5.4 (c 0.20, CHCl3); UV lmax (MeOH) nm (log ε) 226 (4.1), 277 (sh); IR (ATR) nmax 3351 (br), 2924, 2853, 1715, 1600, 1513, 1442, 1367, 1227, 1170, 1039, 913, 809 cm1; 1H and 13 C NMR: (Table 3); HRAPCIMS m/z 305.1315 [M H] (calcd for þ C18H35 22ClO2, 305.1314), HRESIMS m/z 329.1290 [M þ Na] (calcd for C18H35 23ClNaO2, 329.1279).
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