Article pubs.acs.org/jnp
Structures and Biological Evaluations of Agelasines Isolated from the Okinawan Marine Sponge Agelas nakamurai Delfly B. Abdjul, Hiroyuki Yamazaki,* Syu-ichi Kanno, Ohgi Takahashi, Ryota Kirikoshi, Kazuyo Ukai, and Michio Namikoshi Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai 981-8558, Japan S Supporting Information *
ABSTRACT: Three new N-methyladenine-containing diterpenes, 2oxoagelasines A (1) and F (2) and 10-hydro-9-hydroxyagelasine F (3), were isolated from the Okinawan marine sponge Agelas nakamurai Hoshino together with eight known agelasine derivatives, 2-oxoagelasine B (4), agelasines A (5), B (6), D (7), E (8), F (9), and G (10), and ageline B (11). The structures of 1−3 were assigned on the basis of their spectroscopic data and their comparison with those of the literature. Compounds 3 and 5−11 inhibited the growth of Mycobacterium smegmatis with inhibition zones of 10, 14, 15, 18, 14, 20, 12, and 12 mm at 20 μg/disc, respectively. All compounds were inactive (IC50 > 10 μM) against Huh-7 (hepatoma) and EJ-1 (bladder carcinoma) human cancer cell lines. Three 2-oxo derivatives (1, 2, and 4) exhibited markedly reduced biological activity against M. smegmatis. Moreover, compound 10 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 15 μM.
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performance liquid chromatography (HPLC) using an ODS column and yielded compounds 1 (6.8 mg), 2 (1.5 mg), 3 (3.3 mg), 4 (4.6 mg), 5 (2.8 mg), 6 (2.6 mg), 7 (4.7 mg), 8 (2.3 mg), 9 (3.5 mg), 10 (4.4 mg), and 11 (1.4 mg). Compounds 4−11 were identified as the known agelasine derivatives, 2-oxoagelasine B, agelasines A, B, D, E, F, and G, and ageline B, respectively, by comparing their spectroscopic data with those for the reported values.3 Various kinds of agelasines, including 4−11, have previously been reported from several marine sponges of the genus Agelas.3−5 The 1H and 13C NMR spectra of 1 resembled those of 2oxoagelasine B (4) and agelasine A (5). The molecular formula of 1 was deduced from HRFABMS and NMR data (Table 1) as C26H38N5O, which was the same as that of 4. Although information from COSY data was limited, HMBC data revealed that 1 had the same planar structure as 4 (Figure 1a). The NOESY correlations between H2-12 (δH 2.00)/H-14 (δH 5.48) and H2-15 (δH 5.12)/H3-16 (δH 1.80) showed the E-orientation of the double bond at C-13 (Figure 1b). The cis-configuration of the clerodane moiety was established from NOESY correlations between H-1a (δH 2.73)/H3-19 (δH 1.18), H-1b (δH 2.44)/H3-20 (δH 0.51), and H-10 (δH 1.90)/H3-19. The NOESY correlations between H-7b (δH 1.16)/H3-20 and H3-17 (δH 0.75)/H3-20 revealed the configurations at the C-8 and C-9 positions. These NOESY correlations were similar to those of nakamurol C possessing a cis-clerodane moiety, which has also been isolated from the Okinawan marine sponge Agelas
arine organisms produce a wide variety of metabolites that display diverse biological activities and unique structural features.1 Marine sponges of the genus Agelas have been identified as a rich source of diterpenes with polar functionalities in addition to their chemical markers and bromopyrrole alkaloids.2−5 In the course of our search for bioactive and useful substances from marine organisms, we have reported cancer preventive compounds, protein tyrosine phosphatase 1B (PTP1B) inhibitors, and antimycobacterial substances.6−8 Further investigations on extracts from marine invertebrates revealed that the EtOH extract of the Okinawan marine sponge Agelas nakamurai Hoshino exhibited antibacterial activity against Mycobacterium smegmatis with an inhibition zone of 22 mm at 50 μg/disc. Bioassay-guided isolation afforded three new diterpene alkaloids possessing an N-methyladenine unit, 2oxoagelasine A (1), 2-oxoagelasine F (2), and 10-hydro-9hydroxyagelasine F (3), together with eight known agelasine derivatives: 2-oxoagelasine B (4),3a agelasines A (5),3b B (6),3b D (7),3b E (8),3c F (9),3c and G (10),3d and ageline B (11).3e We herein described the isolation, structure elucidation including absolute configurations, and biological activities of compounds 1−11.
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RESULTS AND DISCUSSION The marine sponge (159.3 g, wet weight) was extracted with EtOH. The obtained extract (4.1 g) inhibited the growth of M. smegmatis (22 mm at 50 μg/disc) and was separated by an octadecylsilane (ODS) column (100 g) into seven fractions. The bioactive fractions were further purified by repeated high© XXXX American Chemical Society and American Society of Pharmacognosy
Received: April 29, 2015
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DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Chart 1
nakamurai.9 Based on the NOE data for 1, the most stable conformer was predicted using Spartan’1410 by Monte Carlo conformational analysis with the semiempirical PM3 molecular orbital method, as shown in Figure 1b. Thus, the structure of compound 1 was assigned and named 2-oxoagelasine A because the configuration of 1 was the same as that of agelasine A (5).3b The molecular formula of 2 was determined as C26H38N5O from HRFABMS and NMR data. 1H and 13C NMR data (Table 1) showed the presence of three trisubstituted double bonds (δH 5.19, 5.55, and 5.85). An analysis of COSY and HMBC spectra revealed the planar structure of 2, as shown in Figure 2a. The NOESY correlations between H-8b (δH 1.66)/H-10 (δH 5.19), H-11a (δH 2.22)/H3-17 (δH 1.64), H2-12 (δH 2.23)/ H-14 (δH 5.55), and H2-15 (δH 5.20)/H3-16 (δH 1.87) established the E-orientation of the two double bonds at C-9 and C-13. The relative configurations at the C-5 and C-6 positions were determined from NOESY correlations between H-1a (δH 2.30)/H3-19 (δH 1.05), H-1a/H3-20 (δH 0.97), and H3-19/H3-20. The most stable conformer of 2 (Figure 2b) was
predicted based on the NOE data for 2 using Spartan’1410 by a Monte Carlo conformational analysis with the semiempirical PM3 molecular orbital method. Consequently, the structure of compound 2 was elucidated as shown in Figure 1 and named 2oxoagelasine F. The molecular formula of 3, C26H42N5O, as deduced from HRFABMS and NMR data, was 4 Da (H4) more than that of 2. Although the 1H and 13C NMR spectra of 3 showed the presence of two trisubstituted double bonds, a carbonyl group was not detected. NMR data (Table 1) of the cyclohexyl ring (C-1−C-6 and C-18−C-20) and alkyl adenine moiety (C-13− C-16 and C-1′−C-9′) for 3 were very similar to those for agelasine F (9). The difference in the molecular formulas of 3 and 9 was H2O (18 Da), and therefore, compound 3 was presumed to be a hydrated derivative of 9. As the 13C NMR signal due to the oxygenated carbon (δC 73.2) was not detected in the DEPT spectrum of 3, the OH group was assigned at the C-9 position. The planar structure of 3 was confirmed by an analysis of the COSY and HMBC spectra (Figure 3a). The Eorientation of the C-13 double bond was determined from B
DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H and 13C NMR Data for Compounds 1−3 in CD3OD 1 C#
a
δC, type
2 δH mult. (J in Hz)
1
36.3, CH2
2
202.0, C
3 4 5 6
129.0, CH 172.7, C 40.8, C 37.7, CH2
7
29.4, CH2
8
37.9, CH
2.10, 1.24, 1.31, 1.16, 1.50,
9 10
41.4, C 48.6, CH
1.90, m
11
36.0, CH2
12 13 14 15 16 17 18 19 20 2′ 4′ 5′ 6′ 8′ 9′-Me
33.7, CH2 149.3, C 115.7, CH 48.7, CH2 17.1, CH3 16.3, CH3 20.8, CH3 32.5, CH3 19.7, CH3 157.7, CH 150.9, C 111.2, C 154.2, C 141.6, CH 32.0, CH3
2.73, dd (18.5, 6.6) 2.44, d (18.5)
δC, type
3 δH mult. (J in Hz)
42.7, CH2
2.30, m 2.22, m
201.8, C 5.75, s
m m m m m
1.49, m 1.40, dd (11.8, 5.1) 2.00, ddd (13.4, 13.4, 4.8) 5.48, 5.12, 1.80, 0.75, 1.92, 1.18, 0.51, 8.37,
t (6.8) d (6.8) s d (6.8) s s s s
nda 3.89, s
128.8, CH 172.5, C 43.6, C 34.9, CH 36.1, CH2 35.5, CH2 137.1, C 124.8, CH 27.0, CH2 40.5, CH2 148.8, C 115.8, CH 48.9, CH2 16.9, CH3 16.1, CH3 20.6, CH3 19.8, CH3 15.7, CH3 157.2, CH 150.9, C 111.0, C 154.2, C 141.5, CH 32.0, CH3
5.85, s
2.29, m 1.60, 1.28, 1.96, 1.66,
m m m m
5.19, br s 2.22, m 1.64, m 2.23, m 5.55, 5.20, 1.87, 1.64, 1.96, 1.05, 0.97, 8.45,
t (7.0) d (7.0) s s s s d (6.3) s
nda 3.97, s
δC, type
δH mult. (J in Hz)
28.2, CH2
1.33, m
26.5, CH2
1.90, m 1.80, m 5.30, br s
125.2, CH 140.7, C 41.2, C 34.6, CH
1.63, m
31.3, CH2
1.33, m
36.9, CH2
1.40, m 1.04, m
73.2, C 42.0, CH2 22.8, CH2 41.1, CH2 149.1, C 115.7, CH 48.6, CH2 16.8, CH3 26.9, CH3 19.5, CH3 21.7, CH3 16.3, CH3 157.2, CH 150.9, C 111.2, C 154.2, C 141.6, CH 32.0, CH3
2.20, 1.35, 1.50, 1.30, 2.10,
m m m m m
5.47, 5.12, 1.80, 1.04, 1.51, 0.79, 0.80, 8.37,
t (7.0) d (7.0) s s s s br d (7.1) s
nda 3.88, s
Not detected.
Figure 1. (a) COSY and key HMBC data for 1 and (b) stereostructure and key NOESY correlations of 1.
NOESY correlations between H2-12 (δH 2.10)/H-14 (δH 5.47) and H 2 -15 (δ H 5.12)/H 3 -16 (δ H 1.80). The NOESY correlations between H-1 (δH 1.33)/H3-19 (δH 0.79) and H1/H3-20 (δH 0.80) confirmed the relative configurations at the C-5 and C-6 positions, which were the same as those of agelasine F (9). Therefore, compound 3 was named 10-hydro9-hydroxyagelasine F.
The stereostructure shown in Figure 3b is the most stable conformer of 3 with the 9S*-configuration, which was predicted using Spartan’1410 by a Monte Carlo conformational analysis with the semiempirical PM3 molecular orbital method based on NOE data for 3. The most stable conformation of the 9S*isomer gave an energy level lower than that of 9R*-isomer; however, the configuration at this position could not be C
DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. (a) COSY and key HMBC data for 2 and (b) stereostructure and key NOESY correlations of 2.
Figure 3. (a) COSY and key HMBC data for 3 and (b) stereostructure and key NOESY correlations of 3.
presumed from the conformational analysis because the energy difference was very small (0.38 kcal/mol). The absolute configurations of agelasine derivatives, including agelasine A and ent-agelasine F, were previously established by the chemical degradation method and total synthesis.3b,c,11 The absolute configurations of the diterpene moieties in three new compounds 1−3 were tentatively assigned similarly to those of agelasines A (5) and F (9) because 1−3 were obtained together with known agelasine derivatives from the same marine sponge. Agelasines and their related derivatives have been shown to inhibit the growth of several cancer cell lines and microorganisms, inhibit Na+ and K+-adenosine triphosphatase (ATPase), and suppress RANKL-induced osteoclastogenesis.12−15 In this study, antimicrobial activity against M. smegmatis NBRC 3207 using the paper disc method,8,16 cytotoxicity,17 and the inhibition of PTP1B activity7,18 by the isolated compounds 1−11 were examined (Table 2). Compounds 5−11 exhibited antimycobacterial activity with inhibition zones of 9−14 mm at 10 μg/disc, and a new compound 3 showed 10 mm inhibition at 20 μg/disc (Table 2). The three 2-oxo derivatives (1, 2, and 4) were not active at 20 μg/disc. Therefore, oxidation at the C-2 position, which formed an α,β-unsaturated ketone, appeared to be an unfavorable structural modification for antimycobacterial activity. Because agelasines D (7), E (8), and F (9) were previously reported to inhibit the growth of Mycobacterium tuberculosis,13 the other active compounds 3, 5, 6, 10, and 11 may also be active against M. tuberculosis. Agelasines B (6), C, and D (7) were recently
Table 2. Biological Activities of Compounds 1−11 Against Mycobacterium smegmatis, PTP1B, and Two Human Cancer Cell Lines M. smegmatis (inhibition zone, mm)
compound
10 μg/disc
1 2 3 4 5 6 7 8 9 10 11 streptomycin sulfateb oleanolic acidc doxorubicind
a
20 μg/disc
10 12 12 14 10 14 10 9 30
14 15 18 14 20 12 12
cytotoxicity (IC50, μM) PTP1B (IC50, μM) >23 >23 >23 >23 >24 >24 >24 >24 >24 15 >19
Huh-7 >50 >50 >50 >50 >50 22 36 >50 24 26 >50
EJ-1 >50 >50 >50 >50 42 19 19 >50 16 18 >50
1.0 0.36
0.023
Not active. bPositive control for the antimycobacterial assay (5 μg/ disc). cPositive control for the PTP1B assay. dPositive control for cytotoxicity against Huh-7 and EJ-1 cells. a
found to exhibit biological activity against dormant mycobacteria, and the target protein was identified for 7.19 D
DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
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2-Oxoagelasine A (1): Yellow oil; [α]D26 −7.5 (c 0.02, CH3OH); UV (CH3OH) λmax (log ε) 212 (4.2), 255 (3.9), 271 (3.9) nm; IR (KBr) νmax 3414, 2929, 1681, 1653, 1617, 1204, 1179, 1132 cm−1; 1H and 13C NMR (CD3OD), Table 1; FABMS m/z 436 [M]+; HRFABMS m/z 436.3060 (calcd for C26H38N5O, 436.3076). 2-Oxoagelasine F (2): Yellow oil; [α]D26 −7.2 (c 0.02, CH3OH); UV (CH3OH) λmax (log ε) 217 (4.0), 239 (3.8), 268 (3.6) nm; IR (KBr) νmax 3435, 2974, 1682, 1651, 1207, 1184, 1135 cm−1; 1H and 13 C NMR (CD3OD), Table 1; FABMS m/z 436 [M]+; HRFABMS m/ z 436.3060 (calcd for C26H38N5O, 436.3076). 10-Hydro-9-hydroxyagelasine F (3): Yellow oil; [α]D26 −6.8 (c 0.02, CH3OH); UV (CH3OH) λmax (log ε) 211 (4.1), 248 (3.6), 271 (3.7) nm; IR (KBr) νmax 3428, 2928, 1680, 1655, 1182, 1134 cm−1; 1H and 13C NMR (CD3OD), see Table 1; FABMS m/z 440 [M]+; HRFABMS m/z 440.3380 (calcd for C26H42N5O, 440.3389). 2-Oxoagelasine B (4): [α]D25 −8.7 (c 0.3, CH3OH); lit. [α]D28 −6.1 (c 0.3, CH3OH);3a [α]D25 −7.4 (c 0.5, CH3OH).4c Agelasine A (5): [α]D23 −25.1 (c 0.03, CH3OH); lit. [α]D25 −31.3 (c 0.59, CH3OH);3b [α]D −29.8 (c 0.22, CH3OH).4a Agelasine B (6): [α]D23 −14.4 (c 0.03, CH3OH); lit. [α]D25 −21.5 (c 1.00, CH3OH);3b [α]D −23.5 (c 1.53, CH3OH).4a Agelasine D (7): [α]D23 +15.4 (c 0.02, CH3OH); lit. [α]D25 +10.4 (c 1.1, CH3OH);3b [α]D20 +11.0 (c 1.0, CH3OH) for synthetic 7.12a Agelasine E (8): [α]D23 −12.5 (c 0.02, CH3OH); lit. [α]D25 −17.1 (c 1.88, CH3OH);3c [α]D −2.1 (c 0.36, CHCl3) for synthetic 8.12b Agelasine F (9): [α]D23 −9.1 (c 0.03, CH3OH); lit. [α]D25 −5.5 (c 2.45, CH3OH);3c [α]D −6.8 (c 0.86, CH3OH).4a Agelasine G (10): [α]D23 −34.9 (c 0.02, CHCl3); lit. [α]D27 −85 (c 0.02, CHCl3).3d Ageline B (11): [α]D25 −10.6 (c 0.1, CH3OH); lit. [α]D −11.6 (c 2.5, CH3OH).3e Antimycobacterial Assay. The antibacterial assay was carried out using M. smegmatis NBRC 3207 by the paper disc method.8,16 The strain NBRC 3207 was obtained from the Biological Resource Center (NBRC), NITE (Chiba, Japan), and maintained in 20% glycerol at −80 °C. The test micro-organism was cultured in Middlebook 7H9 broth containing 0.05% polysorbate 80, 0.5% glycerol, and 10% Middlebook OADC at 37 °C for 2 days and adjusted to 1.0 × 106 CFU/mL. The inoculum was spread on the above medium containing 1.5% agar in a square plate. Each sample in CH3OH was adsorbed to a sterile filter disc (6 mm, Advantec), and after the evaporation of CH3OH, the disc was placed on an agar plate and incubated for 2 days at 37 °C. Streptomycin sulfate (5 μg/disc) and CH3OH were used as positive and negative controls, respectively. WST-1 Assay. Cytotoxicity was assessed by the water-soluble tetrazolium (WST-1; sodium 5-(2,4-disulfophenyl)-2-(4-iodophenyl)3-(4-nitrophenyl)-2H tetrazolium inner salt) assay, which detects metabolically competent cells with an intact mitochondrial electron transport chain.17 Briefly, 1 × 104 cells were seeded into each well of 96-well plastic plates and cultured overnight. Cells were treated with each test compound followed by incubation for 72 h, and the medium containing WST-1 solution (0.5 mM WST-1 and 0.02 mM 1-methoxy5-methylphenazinium methylsulfate; 1-PMS) was added to each well. Cells were incubated for 60 min at 37 °C, and absorption at 438 nm (reference 620 nm) was measured using a SH-1200 microplate reader (Corona Electric). Control cells were treated with 0.1% EtOH. Cell viability was calculated using the following formula: absorbance in the treated sample/absorbance in the control × 100 (%). PTP1B Inhibitory Assay. PTP1B inhibitory activity was determined by measuring the rate of hydrolysis of a substrate (pNPP) according to a previously described method with a slight modification.7,18 Briefly, PTP1B (100 μL of 0.5 μg/mL stock solution) in 50 mM citrate buffer (pH 6.0) containing 0.1 M NaCI, 1 mM dithiothreitol, and 1 mM N,N,N′,N′-ethylenediamine tetraacetate was added to each well of a 96-well plastic plate. Each sample (2.0 μL in MeOH) was added to each well to make a final concentration from 0 to 4.7−5.6 μM and incubated for 10 min at 37 °C. The reaction was initiated by the addition of pNPP (100 μL of 4.0 mM stock solution) in the citrate buffer, incubated at 37 °C for 30 min, and terminated
The cytotoxicities of 1−11 were evaluated against two human cancer cell lines, Huh-7 (hepatoma) and EJ-1 (bladder carcinoma), and are listed in Table 2. None of the compounds was considered active (IC50 > 10 μM). Similar to the results obtained for the antimycobacterial properties for 1−11, 2-oxo derivatives (1, 2, and 4) did not exhibit apparent cytotoxicity at 50 μM. The inhibition of PTP1B activity was induced by compound 10 with an IC50 value of 15 μM (Table 2). PTP1B is an attractive therapeutic target for the treatment of type 2 diabetes and obesity because it plays a key role as a negative regulator in insulin and leptin signaling.20 This is the first study to demonstrate that an agelasine derivative exhibited inhibitory activity against PTP1B. As the debromo derivative 11 was not active, the Br atom seems necessary for this activity. Polybromodiphenyl ethers obtained from marine sponges were found to be potent inhibitors of PTP1B,7a and thus, a Br atom may play a prominent role in the inhibition of PTP1B activity.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were determined with a JASCO P-2300 digital polarimeter. UV spectra were measured on a U-3310 UV−visible spectrophotometer (Hitachi) and IR spectra on a PerkinElmer Spectrum One Fourier transform infrared spectrometer. NMR spectra were recorded on a JEOL JNMAL-400 NMR spectrometer (400 MHz for 1H and 100 MHz for 13C) in CD3OD (δH 3.30, δC 49.0) or CDCl3 (δH 7.24, δC 77.0). FABMS and EIMS were performed using a JMS-MS 700 mass spectrometer (JEOL). Preparative HPLC was carried out with a Hitachi L-6200 system. Materials. Middlebook 7H9 broth, polysorbate 80, and Middlebook OADC were purchased from BD. PTP1B was purchased from Enzo Life Sciences. p-Nitrophenyl phosphate (pNPP) was purchased from Sigma-Aldrich. Oleanolic acid was purchased from Tokyo Chemical Industry. Plastic plates (96-well) were purchased from Corning Inc. All other chemicals, including organic solvents, were purchased from Wako Pure Chemical Industries Ltd. Marine Sponge and Isolation of Compounds 1−11. The marine sponge was collected by scuba diving at Iriomote Island in Okinawa, Japan, in 2013 and identified as Agelas nakamurai Hoshino by Dr. Kazunari Ogawa (Nakai Laboratory). A voucher specimen was deposited at the Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University as 13-9-7=2-6. The sponge (159.3 g, wet weight) was cut into small pieces and extracted three times with EtOH. The EtOH extract was evaporated, and the residue (4.1 g) was separated into seven fractions (Frs. 1−7) by an ODS column (100 g) with the stepwise elution of CH3OH in H2O. Fr. 5 (800.5 mg, eluted with 85% CH3OH) was subjected to preparative HPLC (Pegasil ODS, 70% CH3OH in H2O containing 0.05% TFA, 2.0 mL/min, UV 210 nm) to give five fractions (Frs. 5.1− 5.5). Fr. 5.3 (280.6 mg) was purified by preparative HPLC (Pegasil ODS, 43% CH3CN in H2O containing 0.05% TFA, 2.0 mL/min, UV 210 nm) to afford compounds 1 (6.8 mg, tR = 20 min) and 4 (4.6 mg, tR = 24 min). Compounds 2 (1.5 mg, tR = 19 min) and 3 (3.3 mg, tR = 38 min) were isolated from Fr. 5.4 (240.2 mg) by preparative HPLC (Pegasil ODS, 2.0 mL/min, UV 210 nm) with 40% CH3CN in H2O (containing 0.05% TFA). A portion (75.0 mg) of Fr. 4 (669.6 mg, 70% CH3OH eluate) was separated by preparative HPLC (Pegasil ODS, 50% CH3CN in H2O containing 0.05% TFA, 2.0 mL/min, UV 210 nm) to give compounds 7 (4.7 mg, tR = 65 min), 8 (2.3 mg, tR = 87 min), 9 (3.5 mg, tR = 80 min), 10 (4.4 mg, tR = 43 min), 11 (1.4 mg, tR = 25 min), and a mixture (14.7 mg) of compounds 5 and 6, which was purified by preparative HPLC (Pegasil ODS, 68% CH3OH in H2O containing 0.05% TFA, 2.0 mL/min, UV 210 nm) to afford compounds 5 (2.8 mg) and 6 (2.6 mg). E
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with the addition of 10 μL of a stop solution (10 M NaOH). The optical density of each well was measured at 405 nm using an MTP500 microplate reader (Corona Electric Co., Ltd.). PTP1B inhibitory activity (%) is defined as [1 − (ABSsample − ABSblank)/(ABScontrol − ABSblank)] × 100. ABSblank was the absorbance of wells containing only the buffer and pNPP. ABScontrol was the absorbance of p-nitrophenol liberated by the enzyme in the assay system without a test sample, whereas ABSsample was that with a test sample. The assays were performed in two independent experiments for all samples. Oleanolic acid, a known phosphatase inhibitor,21 was used as a positive control.
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Gonoi, T.; Fromont, J.; Kashiwada, Y.; Kobayashi, J. Org. Lett. 2014, 16, 3916−3918. (6) (a) Yamazaki, H.; Wewengkang, D. S.; Kanno, S.; Ishikawa, M.; Rotinsulu, H.; Mangindaan, R. E. P.; Namikoshi, M. Nat. Prod Res. 2013, 27, 1012−1015. (b) Kanno, S.; Yomogida, S.; Tomizawa, A.; Yamazaki, H.; Ukai, K.; Mangindaan, R. E. P.; Namikoshi, M.; Ishikawa, M. Int. J. Oncol. 2013, 43, 1413−1419. (c) Kanno, S.; Yomogida, S.; Tomizawa, A.; Yamazaki, H.; Ukai, K.; Mangindaan, R. E. P.; Namikoshi, M.; Ishikawa, M. Oncol. Lett. 2014, 8, 547−550. (7) (a) Yamazaki, H.; Sumilat, D. A.; Kanno, S.; Ukai, K.; Rotinsulu, H.; Wewengkang, D. S.; Ishikawa, M.; Mangindaan, R. E.; Namikoshi, M. J. Nat. Med. 2013, 67, 730−735. (b) Yamazaki, H.; Nakazawa, T.; Sumilat, D. A.; Takahashi, O.; Ukai, K.; Takahashi, S.; Namikoshi, M. Bioorg. Med. Chem. Lett. 2013, 23, 2151−2154. (c) Abdjul, D. B.; Yamazaki, H.; Takahashi, O.; Kirikoshi, R.; Mangindaan, R. E. P.; Namikoshi, M. Bioorg. Med. Chem. Lett. 2015, 25, 904−907. (d) Yamazaki, H.; Takahashi, O.; Kanno, S.; Nakazawa, T.; Takahashi, S.; Ukai, K.; Sumilat, D. A.; Ishikawa, M.; Namikoshi, M. Bioorg. Med. Chem. 2015, 23, 797−802. (8) Bu, Y. Y.; Yamazaki, H.; Ukai, K.; Namikoshi, M. Mar. Drugs 2014, 12, 6102−6112. (9) Shoji, N.; Umeyama, A.; Teranaka, M.; Arihara, S. J. Nat. Prod. 1996, 59, 448−450. (10) Spartan’14; Wavefunction, Inc.: Irvine, CA, 2014. (11) (a) Piers, E.; Breau, M. L.; Han, Y.; Plourde, G. L.; Yeh, W.-L. J. Chem. Soc., Perkin Trans. 1 1995, 963−966. (b) Piers, E.; Roberge, J. Y. Tetrahedron Lett. 1992, 33, 6923−6926. (c) Proszenyáka, Á .; Brændvangb, M.; Charnockc, C.; Gundersena, L.-L. Tetrahedron 2009, 65, 194−199. (12) (a) Stonik, V. A.; Fedorov, S. N. Mar. Drugs 2014, 12, 636−671. (b) Vik, A.; Hedner, E.; Charnock, C.; Tangen, L. W.; Samuelsen, Ø.; Larsson, R.; Bohlin, L.; Gundersen, L. L. Bioorg. Med. Chem. 2007, 15, 4016−4037. (13) (a) Vik, A.; Hedner, E.; Charnock, C.; Samuelsen, O.; Larsson, R.; Gundersen, L.; Bohlin, L. J. Nat. Prod. 2006, 69, 381−386. (b) Bakkestuen, A. K.; Gundersen, L. L.; Petersen, D.; Utenova, B. T.; Vik, A. Org. Biomol. Chem. 2005, 3, 1025−1033. (c) Mangalindan, G. C.; Talaue, M. T.; Cruz, L. J.; Franzblau, S. G.; Adams, L. B.; Richardson, A. D.; Ireland, C. M.; Conception, G. P. Planta Med. 2000, 66, 364−365. (14) Kobayashi, M.; Nakamura, H.; Wu, H. M.; Kobayashi, J.; Ohizumi, Y. Arch. Biochem. Biophys. 1987, 259, 179−184. (15) Kang, M. R.; Jo, S. A.; Yoon, Y. D.; Park, K. H.; Oh, S. J.; Yun, J.; Lee, C. W.; Nam, K. H.; Kim, Y.; Han, S. B.; Yu, J.; Rho, J.; Kang, J. S. Mar. Drugs 2014, 12, 5643−5656. (16) Ericsson, H. Sand. J. Clin. Lab. Invest. 1960, 12, 408−413. (17) Berridge, M. V.; Herst, P. M.; Tan, A. S. Biotechnol. Annu. Rev. 2005, 11, 127−152. (18) Cui, L.; Na, M. K.; Oh, H.; Bae, E. Y.; Jeong, D. G.; Ryu, S. E.; Kim, S.; Kim, B. Y.; Oh, W. K.; Ahn, J. S. Bioorg. Med. Chem. Lett. 2006, 16, 1426−1429. (19) Arai, M.; Yamano, Y.; Setiawan, A.; Kobayashi, M. ChemBiochem 2014, 15, 117−123. (20) (a) Barr, A. J. Future Med. Chem. 2010, 2, 1563−1576. (b) Zhang, S.; Zhang, Z. Y. Drug Discovery Today 2007, 12, 373−381. (c) Lee, S.; Wang, Q. Med. Res. Rev. 2007, 27, 553−573. (21) Liu, J. Ethnopharmacol. 1995, 49, 57−68.
ASSOCIATED CONTENT
* Supporting Information S
1 H, 13C, DEPT, COSY, HMQC, HMBC, and NOESY NMR spectra for 1−3 and experimental data for known compounds. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00375.
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AUTHOR INFORMATION
Corresponding Author
*Phone/Fax: +81 22 727 0218. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the Foundation for Japanese Chemical Research to H.Y. We are grateful to the Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, for providing the human cancer cell lines, Dr. K. Ogawa of Z. Nakai Laboratory for identifying the marine sponge, and to Mr. T. Matsuki and S. Sato of Tohoku Pharmaceutical University for measuring mass and NMR spectra.
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REFERENCES
(1) (a) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H.; Prinsep, M. R. Nat. Prod. Rep. 2015, 32, 116−211 and previous reports in this series. (b) Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1−48 and previous reports in this series. (2) Gordaliza, M. Mar. Drugs 2009, 7, 833−849. (3) (a) Calcul, L.; Tenney, K.; Ratnam, J.; McKerrow, J. J.; Crews, P. Aust. J. Chem. 2010, 63, 915−921. (b) Nakamura, H.; Wu, H.; Ohizumi, Y.; Hirata, Y. Tetrahedron Lett. 1984, 25, 2989−2992. (c) Wu, H.; Nakamura, H.; Kobayashi, J.; Ohizumi, Y.; Hirata, Y. Tetrahedron Lett. 1984, 25, 3719−3722. (d) Ishida, K.; Ishibashi, M.; Shigemori, H.; Sasaki, T.; Kobayashi, J. Chem. Pharm. Bull. 1992, 40, 766−767. (e) Capon, R. J.; Faulkner, D. J. J. Am. Chem. Soc. 1984, 106, 1819−1822. (4) (a) Fu, X.; Schmitz, F. J.; Tanner, R. S.; Kelly-Borges, M. J. Nat. Prod. 1998, 61, 548−550. (b) Appenzeller, J.; Mihci, G.; Martin, M. T.; Gallard, J. F.; Menou, J. L.; Boury-Esnault, N.; Hooper, J.; Petek, S.; Chevalley, S.; Valentin, A.; Zaparucha, A.; Al-Mourabit, A.; Debitus, C. J. Nat. Prod. 2008, 71, 1451−1454. (c) Stout, E. P.; Yu, L. C.; Molinski, T. F. Eur. J. Org. Chem. 2012, 5131−5135. (d) Kubota, T.; Iwai, T.; Nakaguchi, A. T.; Fromont, J.; Gonoi, T.; Kobayashi, J. Tetrahedron 2012, 68, 9738−9747. (5) (a) Wu, H.; Nakamura, H.; Kobayashi, J.; Kobayashi, M.; Ohizumi, Y.; Hirata, Y. Bull. Chem. Soc. Jpn. 1986, 59, 2495−2504. (b) Iwagawa, T.; Kaneko, M.; Okamura, H.; Nakatani, M.; Van Soest, R. W. M. J. Nat. Prod. 1998, 61, 1310−1312. (c) Hertiani, T.; EdradaEbel, R.; Ortlepp, S.; Van Soest, R. W. M.; De Voogd, N. J.; Wray, V.; Hentschel, U.; Kozytska, S.; Muller, W. E. G.; Proksch, P. Bioorg. Med. Chem. 2010, 18, 1297−1311. (d) Kusama, T.; Tanaka, N.; Sakai, K.; F
DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
Structures and Biological Evaluations of Agelasines Isolated from the Okinawan Marine Sponge Agelas nakamurai Delfly B. Abdjul, Hiroyuki Yamazaki,* Syu-ichi Kanno, Ohgi Takahashi, Ryota Kirikoshi, Kazuyo Ukai, and Michio Namikoshi Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai 981-8558, Japan S Supporting Information *
ABSTRACT: Three new N-methyladenine-containing diterpenes, 2oxoagelasines A (1) and F (2) and 10-hydro-9-hydroxyagelasine F (3), were isolated from the Okinawan marine sponge Agelas nakamurai Hoshino together with eight known agelasine derivatives, 2-oxoagelasine B (4), agelasines A (5), B (6), D (7), E (8), F (9), and G (10), and ageline B (11). The structures of 1−3 were assigned on the basis of their spectroscopic data and their comparison with those of the literature. Compounds 3 and 5−11 inhibited the growth of Mycobacterium smegmatis with inhibition zones of 10, 14, 15, 18, 14, 20, 12, and 12 mm at 20 μg/disc, respectively. All compounds were inactive (IC50 > 10 μM) against Huh-7 (hepatoma) and EJ-1 (bladder carcinoma) human cancer cell lines. Three 2-oxo derivatives (1, 2, and 4) exhibited markedly reduced biological activity against M. smegmatis. Moreover, compound 10 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 15 μM.
M
performance liquid chromatography (HPLC) using an ODS column and yielded compounds 1 (6.8 mg), 2 (1.5 mg), 3 (3.3 mg), 4 (4.6 mg), 5 (2.8 mg), 6 (2.6 mg), 7 (4.7 mg), 8 (2.3 mg), 9 (3.5 mg), 10 (4.4 mg), and 11 (1.4 mg). Compounds 4−11 were identified as the known agelasine derivatives, 2-oxoagelasine B, agelasines A, B, D, E, F, and G, and ageline B, respectively, by comparing their spectroscopic data with those for the reported values.3 Various kinds of agelasines, including 4−11, have previously been reported from several marine sponges of the genus Agelas.3−5 The 1H and 13C NMR spectra of 1 resembled those of 2oxoagelasine B (4) and agelasine A (5). The molecular formula of 1 was deduced from HRFABMS and NMR data (Table 1) as C26H38N5O, which was the same as that of 4. Although information from COSY data was limited, HMBC data revealed that 1 had the same planar structure as 4 (Figure 1a). The NOESY correlations between H2-12 (δH 2.00)/H-14 (δH 5.48) and H2-15 (δH 5.12)/H3-16 (δH 1.80) showed the E-orientation of the double bond at C-13 (Figure 1b). The cis-configuration of the clerodane moiety was established from NOESY correlations between H-1a (δH 2.73)/H3-19 (δH 1.18), H-1b (δH 2.44)/H3-20 (δH 0.51), and H-10 (δH 1.90)/H3-19. The NOESY correlations between H-7b (δH 1.16)/H3-20 and H3-17 (δH 0.75)/H3-20 revealed the configurations at the C-8 and C-9 positions. These NOESY correlations were similar to those of nakamurol C possessing a cis-clerodane moiety, which has also been isolated from the Okinawan marine sponge Agelas
arine organisms produce a wide variety of metabolites that display diverse biological activities and unique structural features.1 Marine sponges of the genus Agelas have been identified as a rich source of diterpenes with polar functionalities in addition to their chemical markers and bromopyrrole alkaloids.2−5 In the course of our search for bioactive and useful substances from marine organisms, we have reported cancer preventive compounds, protein tyrosine phosphatase 1B (PTP1B) inhibitors, and antimycobacterial substances.6−8 Further investigations on extracts from marine invertebrates revealed that the EtOH extract of the Okinawan marine sponge Agelas nakamurai Hoshino exhibited antibacterial activity against Mycobacterium smegmatis with an inhibition zone of 22 mm at 50 μg/disc. Bioassay-guided isolation afforded three new diterpene alkaloids possessing an N-methyladenine unit, 2oxoagelasine A (1), 2-oxoagelasine F (2), and 10-hydro-9hydroxyagelasine F (3), together with eight known agelasine derivatives: 2-oxoagelasine B (4),3a agelasines A (5),3b B (6),3b D (7),3b E (8),3c F (9),3c and G (10),3d and ageline B (11).3e We herein described the isolation, structure elucidation including absolute configurations, and biological activities of compounds 1−11.
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RESULTS AND DISCUSSION The marine sponge (159.3 g, wet weight) was extracted with EtOH. The obtained extract (4.1 g) inhibited the growth of M. smegmatis (22 mm at 50 μg/disc) and was separated by an octadecylsilane (ODS) column (100 g) into seven fractions. The bioactive fractions were further purified by repeated high© XXXX American Chemical Society and American Society of Pharmacognosy
Received: April 29, 2015
A
DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Chart 1
nakamurai.9 Based on the NOE data for 1, the most stable conformer was predicted using Spartan’1410 by Monte Carlo conformational analysis with the semiempirical PM3 molecular orbital method, as shown in Figure 1b. Thus, the structure of compound 1 was assigned and named 2-oxoagelasine A because the configuration of 1 was the same as that of agelasine A (5).3b The molecular formula of 2 was determined as C26H38N5O from HRFABMS and NMR data. 1H and 13C NMR data (Table 1) showed the presence of three trisubstituted double bonds (δH 5.19, 5.55, and 5.85). An analysis of COSY and HMBC spectra revealed the planar structure of 2, as shown in Figure 2a. The NOESY correlations between H-8b (δH 1.66)/H-10 (δH 5.19), H-11a (δH 2.22)/H3-17 (δH 1.64), H2-12 (δH 2.23)/ H-14 (δH 5.55), and H2-15 (δH 5.20)/H3-16 (δH 1.87) established the E-orientation of the two double bonds at C-9 and C-13. The relative configurations at the C-5 and C-6 positions were determined from NOESY correlations between H-1a (δH 2.30)/H3-19 (δH 1.05), H-1a/H3-20 (δH 0.97), and H3-19/H3-20. The most stable conformer of 2 (Figure 2b) was
predicted based on the NOE data for 2 using Spartan’1410 by a Monte Carlo conformational analysis with the semiempirical PM3 molecular orbital method. Consequently, the structure of compound 2 was elucidated as shown in Figure 1 and named 2oxoagelasine F. The molecular formula of 3, C26H42N5O, as deduced from HRFABMS and NMR data, was 4 Da (H4) more than that of 2. Although the 1H and 13C NMR spectra of 3 showed the presence of two trisubstituted double bonds, a carbonyl group was not detected. NMR data (Table 1) of the cyclohexyl ring (C-1−C-6 and C-18−C-20) and alkyl adenine moiety (C-13− C-16 and C-1′−C-9′) for 3 were very similar to those for agelasine F (9). The difference in the molecular formulas of 3 and 9 was H2O (18 Da), and therefore, compound 3 was presumed to be a hydrated derivative of 9. As the 13C NMR signal due to the oxygenated carbon (δC 73.2) was not detected in the DEPT spectrum of 3, the OH group was assigned at the C-9 position. The planar structure of 3 was confirmed by an analysis of the COSY and HMBC spectra (Figure 3a). The Eorientation of the C-13 double bond was determined from B
DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H and 13C NMR Data for Compounds 1−3 in CD3OD 1 C#
a
δC, type
2 δH mult. (J in Hz)
1
36.3, CH2
2
202.0, C
3 4 5 6
129.0, CH 172.7, C 40.8, C 37.7, CH2
7
29.4, CH2
8
37.9, CH
2.10, 1.24, 1.31, 1.16, 1.50,
9 10
41.4, C 48.6, CH
1.90, m
11
36.0, CH2
12 13 14 15 16 17 18 19 20 2′ 4′ 5′ 6′ 8′ 9′-Me
33.7, CH2 149.3, C 115.7, CH 48.7, CH2 17.1, CH3 16.3, CH3 20.8, CH3 32.5, CH3 19.7, CH3 157.7, CH 150.9, C 111.2, C 154.2, C 141.6, CH 32.0, CH3
2.73, dd (18.5, 6.6) 2.44, d (18.5)
δC, type
3 δH mult. (J in Hz)
42.7, CH2
2.30, m 2.22, m
201.8, C 5.75, s
m m m m m
1.49, m 1.40, dd (11.8, 5.1) 2.00, ddd (13.4, 13.4, 4.8) 5.48, 5.12, 1.80, 0.75, 1.92, 1.18, 0.51, 8.37,
t (6.8) d (6.8) s d (6.8) s s s s
nda 3.89, s
128.8, CH 172.5, C 43.6, C 34.9, CH 36.1, CH2 35.5, CH2 137.1, C 124.8, CH 27.0, CH2 40.5, CH2 148.8, C 115.8, CH 48.9, CH2 16.9, CH3 16.1, CH3 20.6, CH3 19.8, CH3 15.7, CH3 157.2, CH 150.9, C 111.0, C 154.2, C 141.5, CH 32.0, CH3
5.85, s
2.29, m 1.60, 1.28, 1.96, 1.66,
m m m m
5.19, br s 2.22, m 1.64, m 2.23, m 5.55, 5.20, 1.87, 1.64, 1.96, 1.05, 0.97, 8.45,
t (7.0) d (7.0) s s s s d (6.3) s
nda 3.97, s
δC, type
δH mult. (J in Hz)
28.2, CH2
1.33, m
26.5, CH2
1.90, m 1.80, m 5.30, br s
125.2, CH 140.7, C 41.2, C 34.6, CH
1.63, m
31.3, CH2
1.33, m
36.9, CH2
1.40, m 1.04, m
73.2, C 42.0, CH2 22.8, CH2 41.1, CH2 149.1, C 115.7, CH 48.6, CH2 16.8, CH3 26.9, CH3 19.5, CH3 21.7, CH3 16.3, CH3 157.2, CH 150.9, C 111.2, C 154.2, C 141.6, CH 32.0, CH3
2.20, 1.35, 1.50, 1.30, 2.10,
m m m m m
5.47, 5.12, 1.80, 1.04, 1.51, 0.79, 0.80, 8.37,
t (7.0) d (7.0) s s s s br d (7.1) s
nda 3.88, s
Not detected.
Figure 1. (a) COSY and key HMBC data for 1 and (b) stereostructure and key NOESY correlations of 1.
NOESY correlations between H2-12 (δH 2.10)/H-14 (δH 5.47) and H 2 -15 (δ H 5.12)/H 3 -16 (δ H 1.80). The NOESY correlations between H-1 (δH 1.33)/H3-19 (δH 0.79) and H1/H3-20 (δH 0.80) confirmed the relative configurations at the C-5 and C-6 positions, which were the same as those of agelasine F (9). Therefore, compound 3 was named 10-hydro9-hydroxyagelasine F.
The stereostructure shown in Figure 3b is the most stable conformer of 3 with the 9S*-configuration, which was predicted using Spartan’1410 by a Monte Carlo conformational analysis with the semiempirical PM3 molecular orbital method based on NOE data for 3. The most stable conformation of the 9S*isomer gave an energy level lower than that of 9R*-isomer; however, the configuration at this position could not be C
DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. (a) COSY and key HMBC data for 2 and (b) stereostructure and key NOESY correlations of 2.
Figure 3. (a) COSY and key HMBC data for 3 and (b) stereostructure and key NOESY correlations of 3.
presumed from the conformational analysis because the energy difference was very small (0.38 kcal/mol). The absolute configurations of agelasine derivatives, including agelasine A and ent-agelasine F, were previously established by the chemical degradation method and total synthesis.3b,c,11 The absolute configurations of the diterpene moieties in three new compounds 1−3 were tentatively assigned similarly to those of agelasines A (5) and F (9) because 1−3 were obtained together with known agelasine derivatives from the same marine sponge. Agelasines and their related derivatives have been shown to inhibit the growth of several cancer cell lines and microorganisms, inhibit Na+ and K+-adenosine triphosphatase (ATPase), and suppress RANKL-induced osteoclastogenesis.12−15 In this study, antimicrobial activity against M. smegmatis NBRC 3207 using the paper disc method,8,16 cytotoxicity,17 and the inhibition of PTP1B activity7,18 by the isolated compounds 1−11 were examined (Table 2). Compounds 5−11 exhibited antimycobacterial activity with inhibition zones of 9−14 mm at 10 μg/disc, and a new compound 3 showed 10 mm inhibition at 20 μg/disc (Table 2). The three 2-oxo derivatives (1, 2, and 4) were not active at 20 μg/disc. Therefore, oxidation at the C-2 position, which formed an α,β-unsaturated ketone, appeared to be an unfavorable structural modification for antimycobacterial activity. Because agelasines D (7), E (8), and F (9) were previously reported to inhibit the growth of Mycobacterium tuberculosis,13 the other active compounds 3, 5, 6, 10, and 11 may also be active against M. tuberculosis. Agelasines B (6), C, and D (7) were recently
Table 2. Biological Activities of Compounds 1−11 Against Mycobacterium smegmatis, PTP1B, and Two Human Cancer Cell Lines M. smegmatis (inhibition zone, mm)
compound
10 μg/disc
1 2 3 4 5 6 7 8 9 10 11 streptomycin sulfateb oleanolic acidc doxorubicind
a
20 μg/disc
10 12 12 14 10 14 10 9 30
14 15 18 14 20 12 12
cytotoxicity (IC50, μM) PTP1B (IC50, μM) >23 >23 >23 >23 >24 >24 >24 >24 >24 15 >19
Huh-7 >50 >50 >50 >50 >50 22 36 >50 24 26 >50
EJ-1 >50 >50 >50 >50 42 19 19 >50 16 18 >50
1.0 0.36
0.023
Not active. bPositive control for the antimycobacterial assay (5 μg/ disc). cPositive control for the PTP1B assay. dPositive control for cytotoxicity against Huh-7 and EJ-1 cells. a
found to exhibit biological activity against dormant mycobacteria, and the target protein was identified for 7.19 D
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2-Oxoagelasine A (1): Yellow oil; [α]D26 −7.5 (c 0.02, CH3OH); UV (CH3OH) λmax (log ε) 212 (4.2), 255 (3.9), 271 (3.9) nm; IR (KBr) νmax 3414, 2929, 1681, 1653, 1617, 1204, 1179, 1132 cm−1; 1H and 13C NMR (CD3OD), Table 1; FABMS m/z 436 [M]+; HRFABMS m/z 436.3060 (calcd for C26H38N5O, 436.3076). 2-Oxoagelasine F (2): Yellow oil; [α]D26 −7.2 (c 0.02, CH3OH); UV (CH3OH) λmax (log ε) 217 (4.0), 239 (3.8), 268 (3.6) nm; IR (KBr) νmax 3435, 2974, 1682, 1651, 1207, 1184, 1135 cm−1; 1H and 13 C NMR (CD3OD), Table 1; FABMS m/z 436 [M]+; HRFABMS m/ z 436.3060 (calcd for C26H38N5O, 436.3076). 10-Hydro-9-hydroxyagelasine F (3): Yellow oil; [α]D26 −6.8 (c 0.02, CH3OH); UV (CH3OH) λmax (log ε) 211 (4.1), 248 (3.6), 271 (3.7) nm; IR (KBr) νmax 3428, 2928, 1680, 1655, 1182, 1134 cm−1; 1H and 13C NMR (CD3OD), see Table 1; FABMS m/z 440 [M]+; HRFABMS m/z 440.3380 (calcd for C26H42N5O, 440.3389). 2-Oxoagelasine B (4): [α]D25 −8.7 (c 0.3, CH3OH); lit. [α]D28 −6.1 (c 0.3, CH3OH);3a [α]D25 −7.4 (c 0.5, CH3OH).4c Agelasine A (5): [α]D23 −25.1 (c 0.03, CH3OH); lit. [α]D25 −31.3 (c 0.59, CH3OH);3b [α]D −29.8 (c 0.22, CH3OH).4a Agelasine B (6): [α]D23 −14.4 (c 0.03, CH3OH); lit. [α]D25 −21.5 (c 1.00, CH3OH);3b [α]D −23.5 (c 1.53, CH3OH).4a Agelasine D (7): [α]D23 +15.4 (c 0.02, CH3OH); lit. [α]D25 +10.4 (c 1.1, CH3OH);3b [α]D20 +11.0 (c 1.0, CH3OH) for synthetic 7.12a Agelasine E (8): [α]D23 −12.5 (c 0.02, CH3OH); lit. [α]D25 −17.1 (c 1.88, CH3OH);3c [α]D −2.1 (c 0.36, CHCl3) for synthetic 8.12b Agelasine F (9): [α]D23 −9.1 (c 0.03, CH3OH); lit. [α]D25 −5.5 (c 2.45, CH3OH);3c [α]D −6.8 (c 0.86, CH3OH).4a Agelasine G (10): [α]D23 −34.9 (c 0.02, CHCl3); lit. [α]D27 −85 (c 0.02, CHCl3).3d Ageline B (11): [α]D25 −10.6 (c 0.1, CH3OH); lit. [α]D −11.6 (c 2.5, CH3OH).3e Antimycobacterial Assay. The antibacterial assay was carried out using M. smegmatis NBRC 3207 by the paper disc method.8,16 The strain NBRC 3207 was obtained from the Biological Resource Center (NBRC), NITE (Chiba, Japan), and maintained in 20% glycerol at −80 °C. The test micro-organism was cultured in Middlebook 7H9 broth containing 0.05% polysorbate 80, 0.5% glycerol, and 10% Middlebook OADC at 37 °C for 2 days and adjusted to 1.0 × 106 CFU/mL. The inoculum was spread on the above medium containing 1.5% agar in a square plate. Each sample in CH3OH was adsorbed to a sterile filter disc (6 mm, Advantec), and after the evaporation of CH3OH, the disc was placed on an agar plate and incubated for 2 days at 37 °C. Streptomycin sulfate (5 μg/disc) and CH3OH were used as positive and negative controls, respectively. WST-1 Assay. Cytotoxicity was assessed by the water-soluble tetrazolium (WST-1; sodium 5-(2,4-disulfophenyl)-2-(4-iodophenyl)3-(4-nitrophenyl)-2H tetrazolium inner salt) assay, which detects metabolically competent cells with an intact mitochondrial electron transport chain.17 Briefly, 1 × 104 cells were seeded into each well of 96-well plastic plates and cultured overnight. Cells were treated with each test compound followed by incubation for 72 h, and the medium containing WST-1 solution (0.5 mM WST-1 and 0.02 mM 1-methoxy5-methylphenazinium methylsulfate; 1-PMS) was added to each well. Cells were incubated for 60 min at 37 °C, and absorption at 438 nm (reference 620 nm) was measured using a SH-1200 microplate reader (Corona Electric). Control cells were treated with 0.1% EtOH. Cell viability was calculated using the following formula: absorbance in the treated sample/absorbance in the control × 100 (%). PTP1B Inhibitory Assay. PTP1B inhibitory activity was determined by measuring the rate of hydrolysis of a substrate (pNPP) according to a previously described method with a slight modification.7,18 Briefly, PTP1B (100 μL of 0.5 μg/mL stock solution) in 50 mM citrate buffer (pH 6.0) containing 0.1 M NaCI, 1 mM dithiothreitol, and 1 mM N,N,N′,N′-ethylenediamine tetraacetate was added to each well of a 96-well plastic plate. Each sample (2.0 μL in MeOH) was added to each well to make a final concentration from 0 to 4.7−5.6 μM and incubated for 10 min at 37 °C. The reaction was initiated by the addition of pNPP (100 μL of 4.0 mM stock solution) in the citrate buffer, incubated at 37 °C for 30 min, and terminated
The cytotoxicities of 1−11 were evaluated against two human cancer cell lines, Huh-7 (hepatoma) and EJ-1 (bladder carcinoma), and are listed in Table 2. None of the compounds was considered active (IC50 > 10 μM). Similar to the results obtained for the antimycobacterial properties for 1−11, 2-oxo derivatives (1, 2, and 4) did not exhibit apparent cytotoxicity at 50 μM. The inhibition of PTP1B activity was induced by compound 10 with an IC50 value of 15 μM (Table 2). PTP1B is an attractive therapeutic target for the treatment of type 2 diabetes and obesity because it plays a key role as a negative regulator in insulin and leptin signaling.20 This is the first study to demonstrate that an agelasine derivative exhibited inhibitory activity against PTP1B. As the debromo derivative 11 was not active, the Br atom seems necessary for this activity. Polybromodiphenyl ethers obtained from marine sponges were found to be potent inhibitors of PTP1B,7a and thus, a Br atom may play a prominent role in the inhibition of PTP1B activity.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were determined with a JASCO P-2300 digital polarimeter. UV spectra were measured on a U-3310 UV−visible spectrophotometer (Hitachi) and IR spectra on a PerkinElmer Spectrum One Fourier transform infrared spectrometer. NMR spectra were recorded on a JEOL JNMAL-400 NMR spectrometer (400 MHz for 1H and 100 MHz for 13C) in CD3OD (δH 3.30, δC 49.0) or CDCl3 (δH 7.24, δC 77.0). FABMS and EIMS were performed using a JMS-MS 700 mass spectrometer (JEOL). Preparative HPLC was carried out with a Hitachi L-6200 system. Materials. Middlebook 7H9 broth, polysorbate 80, and Middlebook OADC were purchased from BD. PTP1B was purchased from Enzo Life Sciences. p-Nitrophenyl phosphate (pNPP) was purchased from Sigma-Aldrich. Oleanolic acid was purchased from Tokyo Chemical Industry. Plastic plates (96-well) were purchased from Corning Inc. All other chemicals, including organic solvents, were purchased from Wako Pure Chemical Industries Ltd. Marine Sponge and Isolation of Compounds 1−11. The marine sponge was collected by scuba diving at Iriomote Island in Okinawa, Japan, in 2013 and identified as Agelas nakamurai Hoshino by Dr. Kazunari Ogawa (Nakai Laboratory). A voucher specimen was deposited at the Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University as 13-9-7=2-6. The sponge (159.3 g, wet weight) was cut into small pieces and extracted three times with EtOH. The EtOH extract was evaporated, and the residue (4.1 g) was separated into seven fractions (Frs. 1−7) by an ODS column (100 g) with the stepwise elution of CH3OH in H2O. Fr. 5 (800.5 mg, eluted with 85% CH3OH) was subjected to preparative HPLC (Pegasil ODS, 70% CH3OH in H2O containing 0.05% TFA, 2.0 mL/min, UV 210 nm) to give five fractions (Frs. 5.1− 5.5). Fr. 5.3 (280.6 mg) was purified by preparative HPLC (Pegasil ODS, 43% CH3CN in H2O containing 0.05% TFA, 2.0 mL/min, UV 210 nm) to afford compounds 1 (6.8 mg, tR = 20 min) and 4 (4.6 mg, tR = 24 min). Compounds 2 (1.5 mg, tR = 19 min) and 3 (3.3 mg, tR = 38 min) were isolated from Fr. 5.4 (240.2 mg) by preparative HPLC (Pegasil ODS, 2.0 mL/min, UV 210 nm) with 40% CH3CN in H2O (containing 0.05% TFA). A portion (75.0 mg) of Fr. 4 (669.6 mg, 70% CH3OH eluate) was separated by preparative HPLC (Pegasil ODS, 50% CH3CN in H2O containing 0.05% TFA, 2.0 mL/min, UV 210 nm) to give compounds 7 (4.7 mg, tR = 65 min), 8 (2.3 mg, tR = 87 min), 9 (3.5 mg, tR = 80 min), 10 (4.4 mg, tR = 43 min), 11 (1.4 mg, tR = 25 min), and a mixture (14.7 mg) of compounds 5 and 6, which was purified by preparative HPLC (Pegasil ODS, 68% CH3OH in H2O containing 0.05% TFA, 2.0 mL/min, UV 210 nm) to afford compounds 5 (2.8 mg) and 6 (2.6 mg). E
DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
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with the addition of 10 μL of a stop solution (10 M NaOH). The optical density of each well was measured at 405 nm using an MTP500 microplate reader (Corona Electric Co., Ltd.). PTP1B inhibitory activity (%) is defined as [1 − (ABSsample − ABSblank)/(ABScontrol − ABSblank)] × 100. ABSblank was the absorbance of wells containing only the buffer and pNPP. ABScontrol was the absorbance of p-nitrophenol liberated by the enzyme in the assay system without a test sample, whereas ABSsample was that with a test sample. The assays were performed in two independent experiments for all samples. Oleanolic acid, a known phosphatase inhibitor,21 was used as a positive control.
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ASSOCIATED CONTENT
* Supporting Information S
1 H, 13C, DEPT, COSY, HMQC, HMBC, and NOESY NMR spectra for 1−3 and experimental data for known compounds. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00375.
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AUTHOR INFORMATION
Corresponding Author
*Phone/Fax: +81 22 727 0218. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the Foundation for Japanese Chemical Research to H.Y. We are grateful to the Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, for providing the human cancer cell lines, Dr. K. Ogawa of Z. Nakai Laboratory for identifying the marine sponge, and to Mr. T. Matsuki and S. Sato of Tohoku Pharmaceutical University for measuring mass and NMR spectra.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX
