Meroterpenoids from a Tropical Dysidea sp. Sponge - ACS Publications

Article pubs.acs.org/jnp

Meroterpenoids from a Tropical Dysidea sp. Sponge Chang-Kwon Kim,† Jung-Kyun Woo,† Seong-Hwan Kim,† Eunji Cho,‡ Yeon-Ju Lee,§ Hyi-Seung Lee,§ Chung J. Sim,⊥ Dong-Chan Oh,† Ki-Bong Oh,*,‡ and Jongheon Shin*,† †

Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-742, Korea ‡ Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-921, Korea § Marine Natural Products Laboratory, Korea Institute of Ocean Science & Technology, P.O. Box 29, Seoul 425-600, Korea ⊥ Department of Biological Science, College of Life Science and Nano Technology, Hannam University, 461-6 Jeonmin, Yuseong, Daejeon 305-811, Korea S Supporting Information *

ABSTRACT: Six new meroterpenoids (1−6), along with arenarol (7), a known rearranged drimane sesquiterpene hydroquinone, were isolated from a Dysidea sp. sponge collected from the Federated States of Micronesia. On the basis of the results of combined spectroscopic analysis, compound 1 was determined to be the cyclic ether derivative of 7, whereas 2 and 3 were assigned as the corresponding sesquiterpene quinones containing taurine-derived substituents. Compounds 4−6 possess a novel tetracyclic skeleton formed by a direct linkage between the quinone and sesquiterpene moieties. The configurations of these new compounds were assigned on the basis of combined NOESY and ECD analysis. These compounds exhibited cytotoxic and antimicrobial activities and weak inhibition against Na+/K+-ATPase.

S

possess the corresponding quinones containing taurine-derived substituents. Compounds 4−6 possess a novel tetracyclic skeleton, including an additional five-membered ring formed by a direct carbon−carbon linkage between the quinone and sesquiterpene moieties. These new compounds exhibited cytotoxicities comparable to those of doxorubicin toward the K562 (human myelogenous leukemia) and A549 (human lung carcinoma) cell lines. Several compounds also exhibited moderate to weak antimicrobial activity toward diverse bacterial and fungal strains and weak inhibition against Na+/K+-ATPase.

ponge-derived meroterpenoids are widely recognized for their various structural features and potent bioactivities.1,2 Since the isolation of avarol, a rearranged drimane sesquiterpene hydroquinone, from Dysidea avara, compounds of this structural class have been obtained from diverse sponges of the order Dictyoceratida.2−5 Structural variations occur at both the quinone (hydroquinone) and polyprenyl moieties, which have resulted in more than 100 novel compounds to date.6 The most frequently encountered bioactivities of these compounds are cytotoxicity and cancer-related activities.7 Moreover, antimicrobial,8a antiproliferative,8b and antiviral8c activities, inhibition against protein tyrosine kinase (PTK)9a and protein tyrosine phosphatase 1B (PTP1B),9b and activation of hypoxia-inducible factor-1 (HIF-1)9c have also been reported. The most notable example of bioactivity of these meroterpenoids might be the significant anti-HIV activity of ilimaquinone, which has attracted considerable biomedical and synthetic interest.10 During the course of a search for bioactive metabolites from tropical sponges, we encountered a purple-colored encrusting Dysidea sp. off the coast of Chuuk Island, Federated States of Micronesia. Motivated by the significant cytotoxicity of the crude extract (IC50 of 14.9 μg/mL against the K562 cell line), the chemical constituents of this sponge were investigated extensively. Herein, we have reported the structure determination of six novel meroterpenoids (1−6) that consist of quinone (hydroquinone) and sesquiterpene moieties. Compound 1 is the cyclic ether derivative of arenarol (7), a known rearranged drimane meroterpene congener,4 whereas 2 and 3 © XXXX American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION The molecular formula of compound 1 was determined to be C21H28O2 through HRFABMS analysis. The 13C NMR data of this compound were very similar to those of arenarol (7): eight aromatic and olefinic carbons were observed at δC 155.8−107.2, and three shielded methyl carbons resonated at δC 28.4, 20.7, and 16.3. Of the deshielded carbons, a methylene carbon at δC 107.2 was indicative of an exo-double bond (Table 1). Corresponding signals were readily assigned from the 1H NMR data, in which three aromatic protons at δH 6.50, 6.49, and 6.40, two exo-double-bond protons at δH 4.91 and 4.77, and three methyl protons at δH 1.29, 1.11, and 0.75 were observed (Table 2). Further examination of the NMR data revealed a significant difference, which was the replacement of a methine Received: September 30, 2015

A

DOI: 10.1021/acs.jnatprod.5b00867 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

two linear assemblies of shielded protons from H2-1 to H2-3 and from H2-6 to H-8 including the H3-13. The HMBC correlations of these protons, particularly those of the exomethylene protons and methyl protons with neighboring carbons, connected all of the partial moieties to be a rearranged drimane-type sesquiterpene similar to 7 (Figure 1). Finally, the ether bridge, which was anticipated from the preliminary interpretation of the spectroscopic data, was constructed between C-10 and C-17 based on the crucial HMBC correlations at H-15/C-10 and H-15/C-17. Thus, the planar structure of 1 was defined as a cyclic ether derivative of 7, such as that found in aureol A and dactyloquinones A−E from the sponges Smenospongia aurea11 and Dactylospongia elegans,12 respectively. The relative configurations of the asymmetric carbon centers at C-5, C-8, C-9, and C-10 were assigned based on NOESY analysis (Figure 2). The mutual cross-peaks among H-1α, H3α, and H3-12 were used to assign the C-12 methyl group as αoriented to the bicyclic moiety. Consequently, the cross-peaks at H-1β/H3-14, H-1β/H-15β, and H-2β/H3-14, between the protons across the A/B ring juncture, led to the assignment of a cis ring juncture between these rings. Additional cross-peaks at H-7β/H3-14, H3-13/H3-14, H3-13/H-15α, and H3-14/H-15β were supportive of β-orientations for both the C-13 and C-14 methyl groups to the bicyclic moiety. Thus, the overall relative configuration was determined to be 5S*, 8S*, 9R*, and 10R*. However, the approaches proposed for the absolute configuration of 1, first by ECD calculations on 3-D models and then by chemical conversion from 7 under various conditions, were unable to acquire the desired results. Finally, the same 5S, 8S, 9R, and 10R configuration as for 7 was deduced by comparison of the signs of specific rotations ([α]25 D +2 and +14 for 1 and 7, respectively), and the biogenetic relationship was run between

of 7 with an oxygenated nonprotonated carbon at δC 83.5 in 1. In conjunction with an additional degree of unsaturation inherent in the mass spectrometric data, these NMR differences suggested that 1 possesses a cyclic ether moiety. Given this information, the planar structure of 1 was determined through a combination of 1H−1H COSY, HSQC, and HMBC analysis. First, all of the protons and their attached carbons were precisely matched by the HSQC data. An ABXtype spin system consisting of three aromatic protons was defined based on the COSY data. Subsequently, their HMBC correlations with neighboring protons revealed a hydroquinone moiety analogous to 7. Similarly, the COSY data also defined

Table 1. 13C NMR (ppm, Type) Assignments for Compounds 1−6 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

1a 29.9, 24.3, 31.5, 155.8, 45.7, 33.9, 28.5, 34.6, 40.2, 83.5, 107.2, 28.4, 16.3, 20.7, 36.4, 122.4, 147.5, 117.7, 115.2, 151.1, 115.6,

CH2 CH2 CH2 C C CH2 CH2 CH C C CH2 CH3 CH3 CH3 CH2 C C CH CH C CH

2a 22.9, 25.7, 32.7, 154.4, 40.4, 39.0, 28.6, 39.5, 45.0, 48.3, 106.6, 33.5, 17.7, 19.5, 35.9, 143.9, 184.2, 149.1, 97.4, 187.2, 140.2, 39.6, 49.5,

CH2 CH2 CH2 C C CH2 CH2 CH C CH CH2 CH3 CH3 CH3 CH2 C C C CH C CH CH2 CH2

3b 21.0, 24.1, 31.2, 152.7, 38.7, 37.4, 26.9, 37.9, 43.2, 47.0, 105.9, 32.6, 16.7, 18.5, 35.1, 144.6, 185.6, 153.0, 102.4, 183.4, 135.9, 50.3, 48.6, 39.2,

4a

CH2 CH2 CH2 C C CH2 CH2 CH C CH CH2 CH3 CH3 CH3 CH2 C C C CH C CH CH2 CH2 CH3

31.4, 24.3, 33.0, 156.8, 43.5, 33.1, 26.9, 35.7, 50.3, 61.2, 107.9, 26.9, 18.2, 24.0, 45.3, 154.5, 186.5, 96.5, 150.9, 182.5, 149.6, 39.8, 49.5,

CH2 CH2 CH2 C C CH2 CH2 CH C C CH2 CH3 CH3 CH3 CH2 C C CH C C C CH2 CH2

5a 27.9, CH2 24.8, CH2 124.6, CH 141.4, C 41.3, C 31.9, CH2 27.8, CH2 33.1, CH 50.9, C 61.2, C 19.8, CH3 25.6, CH3 18.4, CH3 19.6, CH3 43.8, CH2 155.1, C 186.1, C 95.7, CH 152.6, C 183.5, C 148.8, C 29.5, CH3

6a 31.5, 24.0, 32.9, 156.8, 43.6, 32.9, 26.8, 35.4, 50.9, 61.4, 107.9, 26.4, 18.0, 24.1, 44.1, 147.5, 182.6, 159.6, 109.5, 188.4, 155.3, 56.8,

CH2 CH2 CH2 C C CH2 CH2 CH C C CH2 CH3 CH3 CH3 CH2 C C C CH C C CH3

a,b

Data were obtained in MeOH-d4 and DMSO-d6, respectively. B



Article

Table 2. 1H NMR [δ, mult, (J in Hz)] Assignments for Compounds 1−6 1a

position 1α 1β 2α 2β 3α 3β 6α 6β 7α 7β 8 10 11 12 13 14 15α 15β 17 18 19 20 21 22 23 24

1.83, 2.00, 1.88, 1.71, 2.63, 2.20, 1.86, 1.92, 1.25, 1.66, 1.68,

m br dd (13.7, 3.5) m m m br dd (13.7, 4.6) m m m m m

4.91, 4.77, 1.29, 0.75, 1.11, 2.57, 2.63,

s s s d (6.3) s d (17.4) d (17.4)

6.50, d (8.4) 6.49, dd (8.4, 2.5) 6.40, d (2.5)

2a 1.82, 2.05, 1.66, 1.84, 2.51, 2.12, 1.12, 2.05, 1.22, 1.54, 1.24, 1.19, 4.71,

m br dd (13.7, 3.4) m m td (13.2, 5.5) br dd (13.4, 4.4) ddd (14.2, 13.7, 3.4) m m m m br d (5.8) br s (2H)

1.06, 0.90, 0.91, 2.43, 2.60,

s d (6.2) s d (13.8) d (13.8)

3b 1.79, 1.93, 1.63, 1.70, 2.40, 2.08, 1.09, 1.98, 1.22, 1.43, 1.16, 1.02, 4.70, 4.67, 1.02, 0.82, 0.83, 2.35, 2.62,

4a

m m m m br dd (13.2, 5.3) br dd (13.6, 4.3) m m m m m m s s s d (6.3) s d (13.7) d (13.7)

1.84, 1.86, 2.14, 1.70, 2.32, 2.43, 1.01, 2.21, 1.95, 1.44, 1.84,

m m m m td (13.7, 5.6) m td (13.5, 4.7) m m m m

4.66, 4.62, 1.09, 1.10, 1.06, 2.81, 2.25,

s s s d (7.4) s d (19.3) d (19.3)

5.37, s 5.48, d (2.1)

5.44, d (2.3)

6.35, d (2.1) 3.54, t (6.6) 3.08, t (6.6)

6.17, 3.56, 2.76, 2.93,

d (2.3) t (7.5) t (7.5) s

5a 1.80, 1.83, 1.86, 1.91, 5.48,

m m m m br s

1.54, 1.59, 1.81, 1.22, 1.82,

m m m m m

6a

1.67, s 1.03, 0.94, 1.08, 2.90, 2.22,

s d (6.3) s d (19.7) d (19.7)

1.85, 1.88, 2.17, 1.69, 2.32, 2.43, 0.99, 2.22, 1.99, 1.43, 1.87,

m m m m td (13.8, 5.4) m td (13.7, 4.4) m m dq (13.8, 4.7) m

4.67, 4.60, 1.07, 1.10, 1.06, 2.80, 2.25,

s s s d (6.9) s d (18.3) d (18.3)

5.27, s 5.80, s

3.52, t (6.8) 3.07, t (6.8)

2.81, s

3.78, s

a,b

Data were obtained in MeOH-d4 and DMSO-d6, respectively.

Figure 2. Selected NOESY (arrows) correlations of compounds 1 and 4.

between this compound and compounds 1 and 7, such as the presence of two additional methylenes, a nitrogenous ore group and a sulfurous ore group. On the basis of the strong absorption band at 1376 cm−1 in the IR data, the sulfurcontaining functionality was determined to be a sulfonic acid. There were resonances in the data for 2 that matched the terpene resonances in 7, indicating the presence of the same rearranged drimane-type sesquiterpene moiety. Six of the eight remaining carbons were hypothesized to form a disubstituted quinone moiety based on their characteristic chemical shifts in the 13C NMR data: δC 187.2 (C), 184.2 (C), 149.1 (C), 143.9 (C), 140.2 (CH), and 97.4 (CH) (Table 1). The corresponding proton signals in the 1H NMR data were also supportive of the quinone moiety: δH 6.35 (1H, d, J = 2.1 Hz) and 5.48 (1H, d, J

Figure 1. COSY (bold lines) and selected HMBC (JCH = 8 Hz, arrows) and D-HMBC (JCH = 4 Hz, dashed arrows) correlations of compounds 1, 2, 4, and 6.

these compounds. Thus, the structure of 1, designated as aureol B, was determined to be a new cyclic ether-containing meroterpenoid.11 The molecular formula of compound 2 was deduced as C23H33NO5S based on HRFABMS analysis. The mass spectrometric data suggested significant structural differences C



Article

through its HMBC correlations with H2-1, H3-12, H3-14, and H2-15. Due to the significant shifts of the quinone carbons in the 13 C NMR data from the other compounds, both the structure determination and NMR assignments of this portion of 4 were accomplished through HMBC analysis (Table 1). The longrange correlations with H2-15 were used to place the nonprotonated carbons at δC 186.5, 154.5, and 149.6 at the neighboring C-16, C-17, and C-21 positions (Figure 1). An additional long-range correlation with H2-1 not only secured the signal at δC 149.6 at C-21 of the quinone unit but also directly linked this carbon at C-10 of the sesquiterpene moiety, thus constructing a five-membered ring corresponding to an additional degree of unsaturation (Figure 1). Accordingly, the other carbons at δC 186.5 and 154.5 were placed at C-17 and C16, respectively. The HMBC correlations of the methine proton at δH 5.37 (δC 96.5) with C-16 and a carbonyl carbon at δC 182.5 secured these at C-18 and C-20, respectively. Finally, a long-range correlation with the taurine H2-22 placed a nonprotonated carbon at δC 150.9 at C-19, fully assigning a 1,4-quinone moiety. Since there are sponge-derived sesquiterpene quinones possessing either a C-18 or a C-19, the substitution pattern of this moiety of 4 was further investigated through a D-HMBC experiment.13 Using the parameters optimized for 4 Hz of the H−C coupling constant, this experiment revealed four-bond correlations at H-18/C-15 and H2-22/C-20, thus eliminating the alternative possibility of the taurine attachment at C-18 (Figure 1). This interpretation was supported further by the shielding of a quinone carbonyl carbon (C-20, δC 182.5) bearing an electron-withdrawing α-substituent when compared with the other alternative (C-17, δC 186.5), consistent with those in the literature.14 Compound 4 was found to possess asymmetric carbon centers at C-5, C-8, C-9, and C-10, identical to 1. The NOESY analysis also showed virtually identical cross-peaks between these compounds, assigning the same cis A/B and trans B/C ring junctions and a syn orientation between H3-13 and H3-14 for 4 (Figure 2). Thus, the overall relative configuration was assigned as 5S*, 8S*, 9R*, and 10R*. Although the absolute configuration was not directly assigned by either spectroscopic or chemical analysis, the biogenetic relations with 6 enabled assignments of the 5S, 8S, 9R, and 10R configuration to be proposed, as discussed below. Thus, the structure of cycloaurenone A (4) was determined to be a taurine-containing sesquiterpene quinone of a new skeletal class. A literature study revealed a number of sponge-derived meroterpenoids with an additional carbon−carbon linkage between the sesquiterpene and quinone (hydroquinone) moieties.15 To the best of our knowledge, however, this is the first example of a fivemembered ring formed by a direct carbon−carbon linkage between the A/B ring junction and the quinone moiety among sponge-derived meroterpenoids. The molecular formula of cycloaurenone B (5) was deduced to be C22H29NO2 based on HRFABMS analysis. Although the 13 C and 1H NMR data of this compound were similar to those of 4, detailed examination revealed several significant differences. First, the signals of the C-22 and C-23 methylenes were replaced by those of a methyl group: δC 29.5; δH 2.81, 3H, s. In conjunction with the loss of sulfonic acid from the mass spectrometric data, this was hypothesized to be a N-methyl group. Second, the C-4 exo-methylene double bond was replaced with a trisubstituted one: δC 141.4 (C) and 124.6

= 2.1 Hz) (Table 2). This interpretation was confirmed by combined 2D NMR analysis, including the crucial long-range correlations at the linkage between the quinone and sesquiterpene moieties in the HMBC data: H-15/C-16, C-17, C-21, H-19/C-17, C-21, and H-21/C-15, C-17, C-19. The two new methylenes that appeared (C-22, δC 39.6, δH 3.54; C-23, δC 49.5, δH 3.08) were directly connected to each other based on the COSY and HMBC correlations at H2-22/H2-23 and H222/C-23 and H2-23/C-22, respectively. Also the proton and carbon chemical shifts suggested the methylenes were linked to sulfonic acid and amine functionalities; thus a taurine-derived moiety was revealed. Subsequently, the attachment of this moiety at C-18 of the quinone ring was demonstrated by the HMBC correlation at H-22/C-18. This interpretation was also supported by the long-range coupling (J = 2.1 Hz) between H19 and H-21 that eliminated the alternative possibility of the taurine being attached at C-19.9a The relative configuration of 2 was assigned as 5S*, 8S*, 9R*, and 10R*, identical to 7, based on the similar NMR data between these compounds and the NOESY cross-peaks at H1α/H-3α, H-1α/H3-12, H-1β/H3-14, H-1β/H-15β, H-2β/H314, H-3α/H3-12, H-7β/H3-14, H3-13/H-15α, and H3-14/H15β in 2. The similar specific rotations between these compounds ([α]25 D +37 and +14 for 2 and 7, respectively) also assisted in assigning the relative configuration of 2. Thus, the structure of 2, designated as melemeleone C, was determined to be a sesquiterpene quinone containing a taurine-derived substituent. Previous examples of spongederived taurine-containing sesquiterpene quinones are melemeleones A and B from Dysidea avara9a and dysidine from D. villosa.9b The molecular formula of compound 3 was determined to be C24H35NO5S based on HRFABMS analysis. The NMR data of this compound were very similar to those of 2, with the signals of an additional methyl group as the most notable difference. The chemical shifts (C-24, δC 39.2; δH 2.93, 3H, s) of this methyl group suggested its attachment at the taurine nitrogen (Tables 1 and 2), which was confirmed by a combined 2-D NMR analysis including the crucial HMBC correlations at H22/C-24, H-24/C-18, and H-24/C-22. Thus, the structure of 3, designated as melemeleone D, was determined to be the Nmethyl derivative of 2. Cycloaurenone A (4) was isolated as a violet-colored, amorphous solid, and it was determined to be C23H31NO5S through HRFABMS analysis. The spectroscopic data of this compound were reminiscent of those obtained for 2, indicating the same taurine-containing sesquiterpene quinone composition. However, detailed examination of the 1H and 13C NMR data revealed significant differences in both the sesquiterpene and quinone portions (Tables 1 and 2). In conjunction with an additional degree of unsaturation inherent in the mass data, these differences prompted us to determine the structure in detail. For the meroterpenoid congeners 1−3, a combination of HSQC and 1H−1H COSY data revealed two linear spin systems of shielded protons for 4 (Figure 1). Subsequently, extensive HMBC analysis, including the correlations of the conspicuous H2-11 exo-methylenes, the H3-13 and H3-14 methyls, and the H2-15 methylenes with neighboring carbons, supported a rearranged drimane sesquiterpene moiety identical to 1−3. In this way, a nonprotonated carbon at δC 61.2 in the 13 C NMR spectrum was assigned at the C-10 ring junction D



Article

(CH); δH 5.48, 1H, br s. A vinyl methyl group also appeared, δC 19.8; δH 1.67, 3H, s (Tables 1 and 2), suggesting an endodouble bond. All of these interpretations were readily confirmed by combined 2-D NMR data, in which the crucial long-range correlations at H-11/C-3, C-4, C-5 and H-22/C-19 were observed in the HMBC data. Thus, the structure of cycloaurenone B (5) was determined as a derivative of 4 with modified sesquiterpene and quinone moieties. The molecular formula of cycloaurenone C (6) was established as C22H28O3 through HRFABMS analysis. On the basis of the comparison of the 13C and 1H NMR data, the sesquiterpene portion of this compound was hypothesized to be the same as that of 4. The presence of a methoxy group replacing the methylamino group of 5 was also apparent from the following data: δC 56.8; δH 3.78, 3H, s. However, detailed examination of the NMR data revealed significant differences of the quinone portion, which prompted a more extensive investigation. A combined 2-D NMR experiment confirmed the same sesquiterpene moiety as 4, assigning H2-15 at δH 2.80 and 2.25. Subsequently, the HMBC correlations of these protons were used to place the carbons at δC 182.6, 155.3, and 147.5 at the nearby C-16, C-17, and C-21. However, additional correlation with H2-1 assigned not the carbon at δC 147.5 but that at δC 155.3 at C-21 instead, in contrast to the trend observed for 4 and 5 (Figure 1). Moreover, the HMBC correlations of a quinone proton at δH 5.80 with both C-17 and C-21 supported the location of this methine at C-19 (δC 109.5). Accordingly, a three-bond correlation with the newly appearing methoxy proton assigned the carbon at δC 159.6 at C-18, whereas the remaining carbonyl carbon at δC 188.4 must be assigned to C-20. This interpretation was in good agreement with the shielding of a quinone carbonyl carbon bearing an αelectron-withdrawing substituent other than those in 4 and 5 and in the literature.15 The NOESY data of 6 exhibited very similar proton−proton cross-peaks to those of 4, including all of the crucial ones across the A/B ring junctions, thereby supporting the same 5S*, 8S*, 9R*, and 10R* relative configuration. Then, the absolute configuration was determined through ECD calculations (Figure 3). The CD profile of 6 was in good agreement with the ECD calculation on a DFT-based 3-D model around the UV maximum at 283 nm, assigning the 5S, 8S, 9R, and 10R

absolute configuration. Thus, the structure of cycloaurenone C (6) was determined to be a modified drimane sesquiterpene quinone bearing a methoxy group.16 On the basis of their biogenetic relationship, the absolute configurations of congeners 4 and 5 were also hypothesized to be the same as that of 6. Sponge-derived meroterpenoids exhibit diverse and potent bioactivities.1 In the present investigations, compounds 1−7 exhibited cytotoxicities comparable to those of doxorubicin toward the K562 and A549 cell lines (Table 3). However, there were no significant differences of bioactivities that could be attributed to the structural variation on the quinone moieties. In bioassays against diverse bacterial strains, these compounds exhibited moderate to weak inhibitions. Notably, the hydroquinone-bearing compounds 1 and 7 were considerably more active than other compounds bearing a variety of quinones. Compound 7 was also active against a number of pathogenic fungal strains. In bioassays against selected enzymes, these compounds were either inactive or negligeable against microbial sortase A (SrtA) and isocitrate lyase (ICL). However, these compounds exhibited moderate to weak inhibition against Na+/ K+-ATPase. In summary, six new meroterpenoids were isolated from a tropical Dysidea sp. sponge. On the basis of the results of combined spectroscopic analysis, aureol B (1) was determined to be the cyclic ether derivative of arenarol (7), a rearranged drimane meroterpene congener, whereas melemeleones C (2) and D (3) represent the corresponding quinones containing taurine-derived substituents. Cycloaurenones A−C (4−6) belong to a new class of meroterpenoids with an additional five-membered ring. The new compounds exhibited cytotoxic activities comparable to those of doxorubicin toward the K562 and A549 cell lines. Several of the compounds also exhibited moderate to weak antimicrobial activity and weak inhibition against Na+/K+-ATPase.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter using a 1 cm cell. UV spectra were acquired using a Hitachi U-3010 spectrophotometer. CD spectra were recorded using an Applied Photophysics Chirascan-plus circular dichroism spectrometer. IR spectra were recorded on a JASCO 300E FT-IR spectrometer using a ZnSe cell. NMR spectra were recorded in MeOH-d4 and DMSO-d6 with solvent peaks as the δH 3.30/δC 49.0 and δH 2.50/δC 39.5 internal standards on Bruker Avance 400, 500, and 600 MHz spectrometers. Proton and carbon NMR spectra were measured at 400 and 100 MHz (3), 500 and 125 MHz (1, 2, 4, and 6), and 600 and 150 MHz (5), respectively. Highresolution FAB mass spectrometry data were obtained at the Korea Basic Science Institute (Daegu, Korea) and acquired using a JEOL JMS 700 mass spectrometer with meta-nitrobenzyl alcohol (NBA) as the matrix for the FABMS. HPLC was performed on a SpectraSYSTEM p2000 equipped with a SpectraSYSTEM RI-150 refractive index detector. All solvents used were of spectroscopic grade or were distilled from glass prior to use. Animal Material. Specimens of a Dysidea sp. sponge (sample number 102CH-416) were collected by hand using scuba offshore of Chuuk Island in the Federated States of Micronesia at a depth of 15 m on February 15, 2010. The sponge was thin and encrusting and had dimensions of 4 × 4.5 cm. The surface was a thin membrane with sand crust and large sharp conules. The texture was soft and compressible, and the color was purple in life. The skeleton was composed of large, thick, sand-cored primary fibers and dense sand-cored secondary fibers with diameters of 280−700 and 100−250 μm, respectively. This species is morphologically similar to Dysidea avara (Schmidt, 1862) in shape but differs in the arrangements and sizes of fibers and in the

Figure 3. Experimental CD spectrum of 6 (black) and calculated ECD spectra of 6 (blue) and ent-6 (red). E


5.9 4.2 6.1 2.3 4.9 3.3 3.7 0.8

4.8 6.9 5.7 1.1 4.0 0.7 3.4 1.6

1 2 3 4 5 6 7 doxorubicin ampicillin amphotericin B berberine chloride 3-NPb ouabain 0.13

2 64 128 >128 >128 >128 4

A

128 >128 16 4

B

Gram(+) bacteria

128 >128 16 4

D

128 >128 16 2

E

Gram(−) bacteria

MIC (μg/mL)

4

>128 >128 >128 >128 >128 >128 >128

F

0.5

>128 16 128 >128 >128 >128 4

G

1

>128 >128 >128 >128 >128 >128 4

H

fungi

4

8 128 128 >128 128 >128 8

I

8

>128 >128 >128 >128 >128 >128 >128

J

40.6

>128 118.5 >128 >128 >128 ND >128

SrtA

2.5

NDc 43.5 93.3 ND ND >128 ND

3.4

21.6 35.8 42.5 123.5 >128 >128 3.6

ICL Na+/K+−ATPase

IC50 (μg/mL)

A: Staphylococcus aureus (ATCC 6538p), B: Bacillus subtilis (ATCC 6633), C: Kocuria rhizophila (NBRC 12708), D: Salmonella enterica (ATCC 14028), E: Proteus hauseri (NBRC 3851), F: Escherichia coli (ATCC 35270), G: Candida albicans (ATCC 10231), H: Trichophyton rubrum (IFO 9185), I: Trichophyton mentagrophytes (IFO 40996), J: Aspergillus f umigatus (HIC 6094). b3-Nitropropionic acid. c Due to the high initial absorbance, inhibition was not accurately measured.

a

A549

K562

compound

IC50 (μM)

Table 3. Results of Bioactivity Tests of Compounds 1−7a

Journal of Natural Products Article

F



Article

2.9 × 10−4 M, MeOH) λmax (Δε) 236 (−0.27), 279 (+2.56), 321 (−1.87) nm; IR (ZnSe) νmax 3395, 2922, 1666, 1644, 1594 cm−1; 1H and 13C NMR data, Tables 1 and 2, respectively; HRFABMS m/z 341.2130 [M + H]+ (calcd for C22H29O3, 341.2132). Computational Analysis. The ground-state geometries were optimized with density functional theory (DFT) calculations using TURBOMOLE 6.5 at the basis set def-SV(P) for all atoms and the functional B3LYP/DFT level; the ground states were further confirmed by harmonic frequency calculations. The calculated ECD data corresponding to the optimized structures were obtained using TDDFT at the functional B3LYP/DFT level at the basis set def2TZVPP for all atoms. The CD spectra were simulated by overlapping for each transition, where σ is the width of the band at 1/e height. ΔEi and Ri are the excitation energies and rotatory strengths for transition i, respectively. In the current work, the value of σ was 0.10 eV.

appearance of conules. A voucher specimen (registry No. spo. 74) was deposited at the Natural History Museum, Hannam University, Daejeon, Korea. Extraction and Isolation. Freshly collected specimens were immediately frozen and stored at −25 °C until use. Lyophilized specimens were macerated and repeatedly extracted with MeOH (3 × 2 L) and CH2Cl2 (2 × 2 L). The combined extracts (16.62 g) were successively partitioned between H2O (8.91 g) and n-BuOH (6.81 g); the latter fraction was repartitioned between H2O−MeOH (15:85, 4.35 g) and n-hexane (2.29 g). The former layer was separated by C18 reversed-phase flash chromatography using sequential mixtures of MeOH and H2O as the eluents (six fractions in H2O−MeOH gradient, from 50:50 to 0:100), followed by acetone and finally EtOAc. On the basis of the results of 1H NMR spectroscopic and cytotoxicity analyses, the fractions eluted with 30:70 H2O−MeOH (0.25 g) and MeOH (0.50 g) were chosen for separation. The 30:70 H2O−MeOH fraction was separated by semipreparative reversedphase HPLC (YMC-ODS column, S-5 μm, 10 mm × 250 mm; H2O− MeOH, 40:60, tR = 30.2 min), yielding compound 2. Further purification of the subfractions (2 and 3) by analytical HPLC (YMCODS column, S-5 μm, 4.6 mm × 250 mm; H2O−MeCN, 72:28, tR = 17.1 and 20.2 min) afforded compounds 4 and 3, respectively, as amorphous solids. The MeOH fraction from flash chromatography was separated by semipreparative reversed-phase HPLC (H2O−MeOH, 10:90, tR = 15.7 min), yielding compound 7. Further purification of other subfractions (4 and 7) by reversed-phase HPLC (H2O−MeCN, 40:60, tR = 52.3, 75.1, and 86.2 min) afforded, in order of elution, compounds 5, 6, and 1 as amorphous solids. The purified metabolites were isolated in the following amounts: 6.6, 36.4, 20.4, 4.4, 1.8, 1.1, and 22.8 mg of 1 to 7, respectively. Aureol B (1): pale yellow, amorphous solid; [α]25 D +2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 211 (4.29), 229 (4.12), 299 (3.88) nm; CD (c 3.1 × 10−4 M, MeOH) λmax (Δε) 232 (−0.75), 300 (+0.57), 338 (−0.06) nm; IR (ZnSe) νmax 3322, 2931, 1670, 1637, 1612 cm−1; 1H and 13C NMR data, Tables 1 and 2, respectively; HRFABMS m/z 313.2127 [M + H]+ (calcd for C21H29O2, 313.2123). Melemeleone C (2): violet, amorphous solid; [α]25 D +37 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (4.41), 287 (3.97), 485 (3.55) nm; CD (c 2.3 × 10−4 M, MeOH) λmax (Δε) 205 (−2.67), 268 (+0.86), 310 (−0.36) nm; IR (ZnSe) νmax 3370, 2933, 1673, 1638, 1588, 1509, 1376, 1212 cm−1; 1H and 13C NMR data, Tables 1 and 2, respectively; HRFABMS m/z 436.2162 [M + H]+ (calcd for C 23 H 34 NO 5 S, 436.2158), 458.1975 [M + Na] + (calcd for C23H33NO5SNa, 458.1977). Melemeleone D (3): violet, amorphous solid; [α]25 D +10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.21), 295 (3.63), 487 (3.19) nm; CD (c 2.2 × 10−4 M, MeOH) λmax (Δε) 201 (−4.51), 274 (+0.72), 313 (−0.78) nm; IR (ZnSe) νmax 3388, 2933, 1673, 1638, 1585, 1458, 1379, 1205 cm−1; 1H and 13C NMR data, Tables 1 and 2, respectively; HRFABMS m/z 450.2311 [M + H]+ (calcd for C 24 H 36 NO 5 S, 448.2314), 472.2136 [M + Na] + (calcd for C24H35NO5SNa, 472.2134). Cycloaurenone A (4): violet, amorphous solid; [α]25 D +30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 211 (4.17), 304 (3.67), 494 (3.17) nm; CD (c 2.3 × 10−4 M, MeOH) λmax (Δε) 232 (−0.75), 300 (+0.57), 338 (−0.06) nm; IR (ZnSe) νmax 3334, 2933, 1666, 1627, 1577, 1505, 1316, 1212 cm−1; 1H and 13C NMR data, Tables 1 and 2, respectively; HRFABMS m/z 434.1999 [M + H]+ (calcd for C 23 H 32 NO 5 S, 434.2001), 456.1823 [M + Na] + (calcd for C23H31NO5SNa, 456.1821). Cycloaurenone B (5): violet, amorphous solid; [α]25 D +2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.02), 300 (3.50), 499 (2.81) nm; CD (c 2.9 × 10−4 M, MeOH) λmax (Δε) 204 (−10.13), 226 (+4.70), 310 (+0.14), 410 (−2.96) nm; IR (ZnSe) νmax 3341, 2926, 1670, 1643, 1588 cm−1; 1H and 13C NMR data, Tables 1 and 2, respectively; HRFABMS m/z 340.2273 [M + H]+ (calcd for C22H30NO2, 340.2277). Cycloaurenone C (6): pale yellow, amorphous solid; [α]25 D +2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.73), 283 (3.37) nm; CD (c

Δϵ(E) =

1 2.297 × 10−39

1 2πσ

A

[−(E −ΔEi)2 /(2σ )2 ]

∑ ΔEiR ie i

Biological Assays. Cytotoxicity assays were performed in accordance with the literature protocols.17 Na+/K+−ATPase,18a sortase A,18b isocitrate lyase,18c and antimicrobial19 inhibition assays were performed according to previously described methods.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00867. 1 H, 13C, and 2-D NMR spectra of compounds 1−6 and detailed ECD data of compound 6 (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*Tel (K.-B. Oh): 82 2 880 4646. Fax: 82 2 873 2039. E-mail: [email protected]. *Tel (J. Shin): 82 2 880 2484. Fax: 82 2 762 8322. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the Basic Science Research Institute in Daegu, Korea, for providing the mass spectrometry data. Particular thanks are given to the Department of Marine Resources, State of Chuuk, Federated States of Micronesia, for allowing this marine organism research. This study was supported by the BK21 Plus Program in 2015 and financially supported by the Ministry of Oceans and Fisheries, Korea (Grant PM 58482), and the Medical Research Center (No. 2009-0083533) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning.

■

REFERENCES

(1) Menna, M.; Imperatore, C.; D’Aniello, F.; Aiello, A. Mar. Drugs 2013, 11, 1602−1643. (2) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2015, 32, 116−211 and earlier reports in this series. (3) Minale, L.; Riccio, R.; Sodano, G. Tetrahedron Lett. 1974, 38, 3401−3404. (4) Schmitz, F. J.; Lakshmi, V.; Powell, D. R.; van der Helm, D. J. Org. Chem. 1984, 49, 241−244. (5) Hirsch, S.; Rudi, A.; Kashman, Y. J. Nat. Prod. 1991, 54, 92−97. (6) Gordaliza, M. Mar. Drugs 2010, 8, 2849−2870. G



Article

(7) (a) Müller, W. E. G.; Maidhof, A.; Zahn, R. K.; Schröder, H. C.; Gasić, M. J.; Heidemann, D.; Bernd, A.; Kurelec, B.; Eich, E.; Seibert, G. Cancer Res. 1985, 45, 4822−4826. (b) Rodriguez, J.; Quinoa, E.; Riguera, R.; Peters, B. M.; Abrell, L. M.; Crews, P. Tetrahedron 1992, 32, 6667−6680. (c) Longley, R. E.; McConnell, O. J.; Essich, E.; Harmody, D. J. Nat. Prod. 1993, 56, 915−920. (d) Radeke, H. S.; Digits, C. A.; Casaubon, R. L.; Snapper, M. L. Chem. Biol. 1999, 6, 639−647. (e) Daletos, G.; de Voogd, N. J.; Müller, W. E. G.; Wray, V.; Lin, W. H.; Feger, D.; Kubbutat, M.; Aly, A. H.; Proksch, P. J. Nat. Prod. 2014, 77, 218−226. (8) (a) Shigemori, H.; Madono, T.; Sasaki, T.; Mikami, Y.; Kobayashi, J. Tetrahedron 1994, 50, 8347−8354. (b) Sladić, D.; Gašić. Molecules 2006, 11, 1−33. (c) Sagar, S.; Kaur, M.; Minneman, K. P. Mar. Drugs 2010, 8, 2619−2638. (9) (a) Alvi, K. A.; Diaz, M. C.; Crews, P. J. Org. Chem. 1992, 57, 6604−6607. (b) Zhang, Y.; Li, Y.; Guo, Y.-w.; Jiang, H.-l.; Shen, X. Acta Pharmacol. Sin. 2009, 30, 333−345. (c) Du, L.; Zhou, Y.-D.; Nagle, D. G. J. Nat. Prod. 2013, 76, 1175−1181. (10) (a) Luibrand, R. T.; Erdman, T. R.; Vollmer, J. J.; Scheuer, P. J.; Finer, J.; Clardy, J. Tetrahedron 1979, 35, 609−612. (b) Takizawa, P. A.; Yucel, J. K.; Velt, B.; Faulkner, D. J.; Deerinck, T.; Soto, G.; Ellisman, M.; Malhotra, V. Cell 1993, 73, 1079−1090. (c) Bruner, S. D.; Radeke, H. S.; Tallarico, J. A.; Snapper, M. L. J. Org. Chem. 1995, 60, 1114−1115. (d) Bankaitis, V. A. Science 2002, 295, 290−291. (e) Lu, P.-H.; Chueh, S.-C.; Kung, F.-L.; Pan, S.-L.; Shen, Y.-C.; Guh, J.-H. Eur. J. Pharmacol. 2007, 556, 45−54. (11) (a) Djura, P.; Stierle, D. B.; Sullivan, B.; Faulkner, D. J. J. Org. Chem. 1980, 45, 1435−1441. (12) (a) Mitome, H.; Nagasawa, T.; Miyaoka, H.; Yamada, Y.; van Soest, R. W. M. J. Nat. Prod. 2001, 64, 1506−1508. (b) Mitome, H.; Nagasawa, T.; Miyaoka, H.; Yamada, Y.; van Soest, R. W. M. Tetrahedron 2002, 58, 1693−1696. (13) Furihata, K.; Seto, H. Tetrahedron Lett. 1995, 36, 2817−2820. (14) Jiao, W.-H.; Xu, T.-T.; Yu, H.-B.; Mu, F.-R.; Li, J.; Li, Y.-S.; Yang, F.; Han, B.-N.; Lin, H.-W. RSC Adv. 2014, 4, 9236−9246. (15) (a) Jiao, W.-H.; Xu, T.-T.; Yu, H.-B.; Chun, G.-D.; Huang, X.-J.; Yang, F.; Li, Y.-S.; Han, B.-N.; Liu, X.-Y.; Lin, H.-W. J. Nat. Prod. 2014, 77, 346−350. (b) Jiao, W.-H.; Xu, T.-T.; Zhao, F.; Gao, H.; Shi, G.-H.; Wang, J.; Hong, L.-L.; Yu, H.-B.; Li, Y.-S.; Yang, F.; Lin, H.-W. Eur. J. Org. Chem. 2015, 960−966. (16) (a) Bourguet-Kondracki, M.-L.; Martin, M.-T.; Guyot, M. Tetrahedron Lett. 1992, 33, 8079−8080. (b) Aoki, S.; Kong, D.; Matsui, K.; Rachmat, R.; Kobayashi, M. Chem. Pharm. Bull. 2004, 52, 935−937. (17) (a) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (b) Ulukaya, E.; Ozdikicioglu, F.; Oral, A. Y.; Demirci, M. Toxicol. In Vitro 2008, 22, 232−239. (18) (a) Johansson, M.; Karlsson, L.; Wennergren, M.; Jansson, T.; Powell, T. L. J. Clin. Endocrinol. Metab. 2003, 88, 2831−2837. (b) Oh, K.-B.; Kim, S.-H.; Lee, J.; Cho, W.-J.; Lee, T.; Kim, S. J. Med. Chem. 2004, 47, 2418−2421. (c) Lee, H.-S.; Lee, T.-H.; Yang, S. H.; Shin, H. J.; Shin, J.; Oh, K.-B. Bioorg. Med. Chem. Lett. 2007, 17, 2483−2486. (19) Oh, K.-B.; Lee, J. H.; Chung, S.-C.; Shin, J.; Shin, H. J.; Kim, H.K.; Lee, H.-S. Bioorg. Med. Chem. Lett. 2008, 18, 104−108.

H


Meroterpenoids from a Tropical Dysidea sp. Sponge - ACS Publications

Recommend Documents