Cytotoxic Psammaplysin Analogues from a Suberea sp. Marine

Article pubs.acs.org/jnp

Cytotoxic Psammaplysin Analogues from a Suberea sp. Marine Sponge and the Role of the Spirooxepinisoxazoline in Their Activity Yeon-Ju Lee,*,† Saem Han,† Hyi-Seung Lee,† Jong Soon Kang,‡ Jieun Yun,‡ Chung J. Sim,§ Hee Jae Shin,† and Jong Seok Lee† †

Marine Natural Product Chemistry Laboratory, Korea Institute of Ocean Science and Technology, Ansan 426-744, Republic of Korea ‡ Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon 323-883, Republic of Korea § Department of Biological Science, College of Life Science and Nano Technology, Hannam University, Daejeon 305-811, Republic of Korea S Supporting Information *

ABSTRACT: Seventeen bromotyrosine-derived metabolites, including eight new compounds, were isolated from a Micronesian sponge of the genus Suberea. Four of the new compounds were psammaplysin derivatives (10−13), and the other four were ceratinamine derivatives (14−17). Of the compounds obtained, the psammaplysins exhibited cytotoxicity against human cancer cell lines (GI50 values down to 0.8 μM), while the ceratinamine and moloka’iamine analogues showed almost no activity. These results suggest that the spirooxepinisoxazoline ring system is a requirement for cytotoxicity and, therefore, may serve as an attractive molecular scaffold for the development of a potent anticancer agent.

M

arine sponges of the order Verongida are a rich source of bromotyrosine-derived metabolites, many of which have exhibited diverse biological activities.1,2 The metabolites moloka’iamine (1) and hydroxymoloka’iamine (2), isolated from a Hawaiian sponge of an unidentified genus within the order Verongida,3 and several other Verongida sponges such as Aplysinella sp.4 and Pseudoceratina arabica5 have been shown to exhibit parasympatholytic action, antifouling activity, and cytotoxicity against murine leukemia cells. Ceratinamine (3),6 isolated from Pseudoceratina purpurea, and its 7-hydroxy derivative (4)4 were also reported to exhibit antifouling activity and cytotoxicity against murine leukemia cells. In the case of bromotyrosine derivatives containing a unique spirooxepinisoxazoline ring system, more than 30 psammaplysins (psammaplysin A−W and their 19-hydroxy derivatives) and two ceratinamides (A and B) have been isolated from nine different species of Verongida sponges. Psammaplysin derivatives have been reported to exhibit biological activities that include antifouling, cytotoxic, anti-HIV, and antimalarial activities.7−15 Herein, we report the isolation of psammaplysins (5−13) including the new analogues (10−13), obtained along with new ceratinamine derivatives (14−16), from a Micronesian Suberea sp. marine sponge (order Verongida, family Aplysinellidae).

fraction was then subjected to C18 column chromatography with aqueous MeOH gradient elution, resulting in six fractions. The resulting fractions were further purified by HPLC using Si gel, ODS, or cyano columns to provide known bromotyrosine derivatives (1−9),3−6 including several psammaplysins (5− 9),7,9,14 along with new analogues (10−17). These results were in accordance with previous reports that showed that marine sponges of the genus Suberea are a prolific source of bromotyrosine-derived metabolites, such as ianthelliformisamines,16 subereamines,17 clavatidines,18 and subereaphenols.19

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RESULTS AND DISCUSSION The combined MeOH−CH2Cl2 extract of the lyophilized Suberea sponge was partitioned between n-BuOH and H2O, and the n-BuOH layer was further partitioned between 15% aqueous MeOH and n-hexane. The 15% aqueous MeOH © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 5, 2013

A

dx.doi.org/10.1021/np400448y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 1H and 13C NMR Data (500 and 125 MHz, acetoned6) for Psammaplysin X (10) and 19-Hydroxypsammaplysin X (11) psammaplysin X (10) position

Psammaplysin X (10) was obtained as yellow, amorphous solid. The 1H NMR data for 10 obtained in CD3OD were quite similar to those obtained for psammaplysin A (5); however, the protons at C-20 appear at δH 3.74 (δH 3.14 for psammaplysin A) and signals at δH 7.66, 4.60, 3.14, and 2.62, which were too broad to identify, were observed in the 1H NMR spectrum of 10. From the molecular formula of C27H26Br4ClN3O8 obtained by HRMS analysis, it was assumed that the unassigned signals were from 4-chloro-2-methylenecyclopentane-1,3-dione (C6H4ClO2). As previously reported in the case of wai’anaeamines, 20 broadening of the 1 H NMR signals of a chlorocyclopentanedione is likely caused by E/Z isomerization at C-21/C-22, and the signals can be duplicated and sharpened by lowering the temperature to −30 °C. Instead of lowering the temperature, we used other solvents to reduce the rate of isomerization. 1H NMR signals, sharp enough for unambiguous assignment, were obtained in acetone-d6 (Table 1), enabling various 2D NMR experiments (Figure 1). In one isomer, a 1H signal at δH 3.10 (H2-25) gives strong HMBC correlations to the 13C shifts of δC 200.1 (C-26) and 196.3 (C-23). A singlet at δH 7.82 (H-21) also correlates with carbon signals at δC 200.1 and 106.6 (C-22). A signal at δH 3.10 exhibits a COSY correlation with a signal at δH 2.54 (H-25), which gives a strong HMBC correlation with a carbon shift at δC 55.8 (C-24). In the other isomer, 1H signals at δH 7.84 (H-21) and 2.49 (H-25) correlate with the 13C signal at δC 197.7 (C-26). The specific double-bond configuration for each isomer has not been determined. The structure of compound 11 could be assigned as the 19hydroxy derivative of psammaplysin X because the two methylene signals at δH 3.12 (H-19) and 3.94 (H-20) observed for 10 disappeared and a signal at δH 5.09 (H-19) and two multiplets at δH 3.75 and 3.98 (H-20) appeared in the 1H NMR spectrum.14 In agreement with this observation, the 13C NMR spectrum of 11 showed signals at δC 71.2 (C-19) and 55.8 (C20), which correlated with the aforementioned 1H NMR signals in the HSQC and HMBC spectra (Table 1, Figure 1). Addtionally, HRESIMS data indicated a molecular formula of C27H26Br4ClN3O9. Another psammaplysin analogue with a novel nitrogen substituent is psammaplysin Y (12), which was obtained as a pale yellow, amorphous solid. A proton signal at δH 7.60 (H21) and carbon signals at δC 155.7 (C-21), 107.8 (C-22), 201.1 (C-23), and 205.2 (C-26) suggested the presence of an unsubstituted 2-methylenecyclopentane-1,3-dione moiety (Table 2), which is in accordance with the molecular formula of C27H27Br4N3O8 derived from (+)-HRESIMS. The two doublets that appear at δH 2.38 and 2.34, which show HMBC correlations with two carbonyl signals at δC 205.2 (C-29) and 201.1 (C-21), could be assigned as H2-24 and H2-25 (Figure 1).

δC, typea

1 2 3 4 5

146.3, 103.5, 149.3, 104.1, 37.9,

CH C C C CH2

6 7 8 9 10 11

119.8, 80.1, 158.4, 159.1, 37.5, 30.5,

C CH C C CH2 CH2

12 13 14 15 16 17 18 19 20

72.0, 152.6, 118.7, 134.3, 138.0, 134.3, 118.7, 35.7, 52.0,

CH2 C C CH C CH C CH2 CH2

21

157.2, 157.0, 106.0, 105.6, 196.3, 194.5, 55.8,

CH CH C C C C CH

55.0,

CH

44.9,

CH2

45.5,

CH2

200.1, 197.7, 59.1,

C C CH3

22 23 24

25

26 3-OMe 7-OH C9-NH C20-NH

δH (J in Hz) 7.17, s

3.14, d (16.0) 3.45, d (16.0) 5.10, s

3.64, t (6.0) 2.15, quin (6.0) 4.09, t (6.0)

7.57, s 7.57, s 3.12, m 3.94, m

7.82, brs 7.84, brs

4.54, dd (2.0, 8.5) 4.61, dd (2.0, 8.5) 2.54, dd (2.0, 18.5) 3.10, dd (8.0, 18.5) 2.49, dd (2.0, 18.5) 3.07, dd (8.0, 18.5)

3.64, s 6.12, brsb 7.91, brsb 10.36, brsb 10.28, brsb

19-hydroxypsammaplysin X (11) δC, typea 146.3, 103.5, 149.3, 104.1, 37.9,

CH C C C CH2

119.8, 80.1, 158.4, 159.1, 37.5, 31.9,

C CH C C CH2 CH2

72.1, 153.2, 118.7, 131.4, 141.9, 131.4, 118.7, 71.2, 55.8,

CH2 C C CH C CH C CH CH2

157.8, 158.0, 106.1, 105.8, 196.3, 194.5, 55.0,

CH CH C C C C CH

55.1,

CH

45.3,

CH2

45.5,

CH2

200.2, 197.8, 59.1,

C C CH3


3.14, d (16.0) 3.45, d (16.0) 5.10, s

3.66, t (6.0) 2.16, quin (6.0) 4.11, t (6.0)

7.71, s 7.71, s 5.09, brs 3.75, brd (13.5) 3.98, brd (13.5) 7.90, brs 7.91, brs

4.55, dd (3.0, 8.5) 4.63, dd (3.0, 8.5) 2.54, dd (3.0, 18.5) 3.14, dd (8.5, 18.5) 2.50, dd (3.0, 18.5) 3.09, dd (8.5, 18.5)

3.64, s 6.10, brsb 7.89, brsb 10.45, brsb 10.36, brsb

a

Carbons correlating with the corresponding proton. bExchangeable signals.

The 2-methylenecyclopentene-1,3-dione moiety, found in the structure of psammaplysin E9 and its 19-hydroxy derivative,14 is a unique nitrogen substituent that had never been found in the structure of any other natural compounds. A hydrochlorinated version of this moiety has been encountered only in the structures of two bromotyrosine derivatives, wai’anaeamines B



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stituent that has never been found in the structures of natural products. Compound 13 had a proton signal at δH 8.13 (H-21) that gives an HSQC correlation with a carbon signal at δC 162.4 (C21), indicating the presence of a formamide functionality, a characteristic of ceratinamide A. The other 1H and 13C NMR signals were also similar to those of ceratinamide A except for the signals attributed to H-19 and H-20 (Table 2). A hydroxymethine signal at δH 4.82 (H-19), which has a COSY correlation with two methylene signals at δH 3.41 and 3.56 (H20), suggests 13 is 19-hydroxyceratinamide A, which agrees with the molecular formula of C22H23Br4N3O8 derived from (+)-HRESIMS analysis. The specific rotations of all of the isolated psammaplysins are negative and of similar magnitude; thus, it is highly likely that psammaplysin analogues, including the new compounds, share the same absolute configuration and biosynthetic pathway of the spirooxepinisoxazoline ring system. In addition, the specific rotations of psammaplysins A (5) and B (6), obtained from this sponge extract, were in agreement with the reported values in the literature.7 The absolute configurations of the psammaplysins have never been established, although attempts have been made for psammaplysin A;7,14 thus, data provided up to now for the psammaplysins have defined only the relative configurations. The configuration of the C-19 stereogenic center for the 19-hydroxypsammaplysins (6, 7, 9, 11, and 13) was not established, although there have been experiments that suggest psammaplysin B (6) might exist as a mixture of diastereomers at C-19.14

Figure 1. Selected COSY and HMBC correlations for 10−12.

Table 2. 1H and 13C NMR Data (500 and 125 MHz, acetoned6) for Psammaplysin Y (12) and 19-Hydroxyceratinamide A (13) psammaplysin Y (12) position

δC, typea

1 2 3 4 5

146.4, 103.5, 149.3, 104.2, 38.0,

CH C C C CH2

6 7 8 9 10

119.8, 80.2, 158.6, 159.1, 37.5,

C CH C C CH2

11

30.5,

CH2

12

72.1,

CH2

13 14 15 16 17 18 19

152.7, 118.7, 134.5, 138.3, 134.5, 118.7, 36.0,

C C CH C CH C CH2

20

51.7,

CH2

21 22 23 24

155.7, 107.8, 201.1, 33.6,

CH C C CH2

25

34.3,

CH2

205.2, 59.1,

C CH3

26 3-OMe 7-OH C9-NH C19-OH C20-NH


3.14, d (16.0) 3.46, d (16.0) 5.10, s

3.64, t (6.0) 2.14, quin (6.0) 4.09, t (6.0)

7.56, s 7.56, s 3.06, t (7.0) 3.86, t (7.0)

7.60, s

19-hydroxyceratinamide A (13) δC, typea 146.4, 103.4, 149.3, 104.1, 37.9,

CH C C C CH2

119.8, 80.1, 158.5, 159.1, 37.5,

C CH C C CH2

30.4,

CH2

72.0,

CH2

152.8, 118.5, 131.4, 143.3, 131.4, 118.5, 71.6,

C C CH C CH C CH

46.2,

CH2

162.4,

CH

59.1,

CH3


3.14, d (16.0) 3.46, d (16.0) 5.10, s

3.64, t (6.0) 2.16, quin (6.0) 4.11, t (6.0)

7.65, s 7.65, s 4.82, brs 3.41, brd (14.0) 3.56, brd (14.0) 8.13 (s)

The next bromotyrosine derivatives obtained from this sponge extract were the subereamides (14−17), derivatives of ceratinamine. Subereamide A (14) was obtained as a pale yellow, amorphous solid. The 1H NMR data for 14 were identical to those of ceratinamine (4) except for three additional doublets at δH 6.65 (H-17), 6.74 (H-18), and 7.34 (H-14), which had HSQC correlations with the carbon signals at δC 142.5 (C-17), 142.7 (C-18), and 149.8 (C-14), respectively (Table 3). Another three additional 13C signals at δC 99.4 (C-15), 194.0 (C-19), and 197.4 (C-16), along with the signals mentioned above, suggested the presence of the 2methylenecyclopentene-1,3-dione moiety previously found in the structure of psammaplysin E (8). From these observations, the structure of 14 was determined, and the HRMS spectrum, which indicated the molecular formula of C19H17Br2N3O4, correlated with this assignment. The 1H NMR spectrum of subereamide B (15) also contained all of the characteristic signals of ceratinamine. An additional doublet of doublets at δH 4.60 (H-17), coupled with signals at δH 3.03 (H-18a) and 2.47 (H-18b), and another

2.38, d (9.0) 2.34, d (9.0) 3.64, s 6.37, brsb 7.93, brsb 10.20, brsb

3.64, 6.23, 7.93, 6.18, 7.43,

s brsb brsb brsb brsb

a


and psammaplysin X. The hydrogenated version, 2-methylenecyclopentane-1,3-dione, is another unique nitrogen subC



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Table 3. 1H and 13C NMR Data (500 and 125 MHz, acetoned6) for Subereamide A (14) and Subereamide B (15) subereamide A (14) position

δC, typea

1 2 3

113.0, 144.2, 38.2,

C C CH2

4

29.6,

CH2

5

71.5,

CH2

6 7 8 9 10 11 12

152.4, 118.6, 134.4, 138.7, 134.4, 118.6, 36.4,

C C CH C CH C CH2

13

51.2,

CH2

14

149.8,

CH

15

99.4,

C

16

197.4,

C

17

142.5,

CH

18

19 C2-NH C13-NH

142.7,

194.0,

CH

δH (J in Hz)

3.64, t (7.0) 2.15, quin (7.0) 4.09, t (7.0)

7.59, s 7.59, s 3.02, t (7.0) 3.77, t (7.0) 7.34, s

6.65, d (6.5)

6.74, d (6.5)

C

subereamide B (15) δC, typea

δH (J in Hz)

112.6, 143.8, 37.7,

C C CH2

29.4,

CH2

71.1,

CH2

152.2, 118.3, 134.0, 137.8, 134.0, 118.3, 35.3,

C C CH C CH C CH2

51.6,

CH2

3.95, t (7.0)

156.6, 156.8, 104.4, 107.4, 195.6, 193.9, 54.7,

CH CH C C C C CH

7.79, s 7.75, s

55.4,

CH

45.2,

CH2

44.6,

CH2

197.2, 199.6, 8.78, brsb 8.60, brsb

respectively, which suggested that both have a fatty acid side chain, and 17 has an additional hydroxy group compared to 16. A doublet at δH 0.86 (6H) in the 1H NMR spectra of both compounds indicated the presence of an iso-branched fatty acid substituent, previously found in the structure of psammaplysin D (7). From these observations and the results of 2D NMR analysis, the structures of 16 and its hydroxy derivative 17 were determined. Selected compounds were screened for cytotoxicity against six human tumor cell lines. As shown in Table 4, molok’iamines (1, 2) and ceratinamines (3, 4) showed no inhibitory growth activity against human cancer cell lines at concentrations up to 70 μM. A previous report also has shown that compounds 1−3 exhibit no cytotoxicity against the human colon cancer cell line HCT-116 at 10 μg/mL.5 On the other hand, most of the psammaplysin analogues (5, 6, 8−11) exhibited variable cytotoxicity against several human cancer cell lines. Psammaplysin D (7), however, lacked activity (GI50 > 10 μM), which might be explained by its high lipophilicity. The cytotoxicity of the psammaplysins compared to 1−4 suggests that the spirooxepinisoxazoline ring system is a requirement for activity and, thus, might serve as an attractive molecular scaffold for the development of potent anticancer agents.

3.64, t (6.5) 2.15, quin (6.5) 4.09, t (6.5)

7.59, s 7.59, s 3.10, t (7.0)

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured on a JASCO digital polarimeter using a 5 cm cell. UV spectra were measured in MeOH using a Shimadzu spectrophotometer (UV-1650PC). IR spectra were recorded on a JASCO FT/IR4100. 1H NMR spectra were recorded on a Varian Unity 500 (500 MHz) spectrometer. Chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance resulting from incomplete deuteration as the internal references (acetone-d6: δH 2.050 ppm). 13C NMR spectra were recorded on a Varian Unity 500 (125 MHz) spectrometer with complete proton decoupling. Chemical shifts are reported in ppm from tetramethylsilane with the solvent as the internal reference (acetone-d6: δC 206.68, 29.92 ppm). High-resolution mass spectrometry was performed on a hybrid ion-trap time-of flight mass spectrometer (Shimadzu LCMS-IT-TOF) equipped with an ESI source (ESI-IT-TOFMS) at Korea Basic Science Institute. HPLC was performed with YMC-Pack Pro C18, YMC-Pack Cyano, or YMC-Pack silica columns using a Shodex RI-101 detector. Biological Material. The sponge Suberea sp. was collected by hand using scuba at a 10 m depth offshore of Chuuk, Federated States of Micronesia, on February 18, 2010. The voucher specimens were deposited at the Natural History Museum, Hannam University, Korea (registry no. Spo. 69), by one of the authors (C.J.S.). The growth form of this specimen erect is 30 mm in size and 15 mm in thickness. When alive the color is purple, and greenish-purple post-mortem. The surface is smooth with pronounced conules; the texture is firm, but compressible. The skeleton is composed of sparse dendritic fibers, which are composed of both bark and pith elements. The fibers are very brittle and have diameters ranging from 250 to 800 μm. The specimen was similar to Suberea creba (Bergquist, 1995) in skeletal structure, except for the color and habitat. Extraction and Isolation. The collection (634.2 g, wet wt) was immediately freeze-dried and kept at −20 °C until investigated. The lyophilized sponge was extracted with MeOH (1 L × 3) and CH2Cl2 (1 L × 1) at room temperature. The combined extract (102.7 g) was partitioned between n-BuOH and H2O, and the organic layer (50.8 g) was further partitioned between 15% aqueous MeOH and n-hexane. The aqueous MeOH fraction (29.2 g) was then subjected to reversedphase column chromatography (YMC Gel ODS-A, 60 Å, 230 mesh) with a stepped gradient solvent system of 50%, 40%, 30%, 20%, 10% aqueous MeOH, and 100% MeOH. The fraction that eluted with 50% aqueous MeOH (6.5 g) was then subjected to reversed-phase MPLC, followed by final purification on reversed-phase HPLC (YMC-Pack

4.60, dd (4.0, 8.0) 4.52, dd (4.0, 8.0) 2.47, dd (4.0, 19.0) 3.03, dd (8.0, 19.0) 2.51, dd (4.0, 19.0) 3.10, dd (8.0, 19.0)

C C 8.81, brsb 8.64, brsb

a


doublet of doublets at δH 4.52 (H-17), coupled with signals at δH 3.10 (H-18a) and 2.51 (H-18b), suggested the presence of geometrical isomers of 4-chloro-2-methylenecyclopentane-1,3dione (Table 3). As seen in the case of wai’anaeamines and psammaplysin X (10), 15 was also detected as a 1:1 mixture of E/Z isomers in acetone-d6. In one isomer, the proton signal at δH 3.03 (H-18a) had HMBC correlations with carbon signals at δC 195.6 (C-16) and 197.2 (C-19). This enabled the assignment of the proton and carbon signals that corresponded to the chlorocyclopentanedione moiety. The NMR signals of the other isomer were assigned in the same way. Subereamide C (16) and 12-hydroxysubereamide C (17), which were obtained with psammaplysin D (7) from the fraction that eluted with 100% MeOH, were also expected to be derivatives of ceratinamine, judging by the 1H and 13C NMR spectra (Experimental Section). Compounds 16 and 17 had molecular formulas of C28H43Br2N3O3 and C28H43Br2N3O4, D



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Table 4. Growth Inhibition (GI50, μM) of Compounds 1−11 against a Panel of Human Tumor Cell Linesa cell line (GI50 μM)b compound

HCT-15

PC-3

ACHN

MDA-MB-231

NUGC-3

NCI-H23

1 2 3 4 5 6 7 8 9 10 11 doxorubicin

>70 >70 >70 >70 3.9 4.0 24 7.4 3.8 3.3 3.5 1.4

>70 >70 >70 >70 6.9 2.7 25 3.7 1.4 2.3 2.1 0.52

>70 >70 >70 >70 5.1 1.6 27 10.3 2.3 3.3 2.5 2.0

>70 >70 >70 >70 4.3 0.53 21 3.9 0.51 1.2 0.8 1.8

>70 >70 >70 >70 3.8 2.5 26 4.0 2.3 3.5 4.0 0.51

>70 >70 >70 >70 12.4 3.7 27 7.0 3.6 6.4 3.5 1.9

a

HCT-15, colon cancer; PC-3, prostate cancer; ACHN, renal cancer; MDA-MB-231, breast cancer; NUGC-3, stomach cancer; NCI-H23, lung cancer. bGI50 values are the concentration corresponding to 50% growth inhibition. Data are an average of at least two tests. Subereamide B (15): pale yellow, amorphous solid; [α]25D +63 (c 0.5, acetone); UV (MeOH) λmax (log ε) 309 (4.48), 208 (4.90) nm; 1 H and 13C NMR (acetone-d6, 500 and 125 MHz), see Table 3; (+)-LRESIMS m/z (rel int) 546 (40), 548 (100), 550 (65), 552 (14) [M + H]+; (+)-HRESIMS m/z 547.9410 [M + H]+ (calcd for C19H1981Br79Br ClN3O4, 547.9452). Subereamide C (16): pale yellow, amorphous solid; UV (MeOH) λmax (log ε) 285 (2.55), 208 (4.83) nm; 1H NMR (CDCl3, 500 MHz) δ 7.35 (2H, s, H-8 and H-10), 7.32 (1H, t, J = 6.0 Hz, C2-NH), 5.54 (1H, t, J = 7.0, C13-NH), 4.12 (2H, t, J = 6.0 Hz, H-5), 3.70 (2H, q, J = 6.0 Hz, H-3), 3.46 (2H, q, J = 7.0 Hz, H-13), 2.75 (2H, t, J = 7.0 Hz, H-12), 2.15 (2H, t, J = 7.5 Hz, H-15), 2.12 (2H, quin, J = 6.0 Hz, H4), 1.60 (2H, m, H-16), 1.50 (1H, m, H-26), 1.25−1.29 (16H, m, H17−H24), 1.15 (2H, m, H-25), 0.85 (6H, d, J = 6.5 Hz, H-27 and H28); 13C NMR (CDCl3, 125 MHz) δ 173.7 (C-14), 151.1 (C-6), 143.4 (C-2), 138.7 (C-9), 133.3 (C-8 and C-10), 118.2 (C-7 and C-11), 112.0 (C-1), 72.0 (C-5), 40.6 (C-13), 39.2 (C-3), 39.2 (C-25), 37.1 (C-15), 34.8 (C-12), 29.9−29.7 (C-17−C-24), 28.6 (C-4), 28.2 (C26), 26.0 (C-16), 22.9 (C-27 and C-28); (+)-LRESIMS m/z (rel int) 628 (50), 630 (100), 632 (53) [M + H]+; (+)-HRESIMS m/z 630.1741 [M + H]+ (calcd for C28H4481Br79Br N3O3, 630.1731). 12-Hydroxysubereamide C (17): pale yellow, amorphous solid; [α]25D +60 (c 0.5, acetone); UV (MeOH) λmax (log ε) 284 (2.29), 208 (4.89) nm; 1H NMR (CDCl3, 500 MHz) δ 7.54 (2H, s, H-8 and H10), 7.08 (1H, t, J = 6.0 Hz, C2-NH), 5.85 (1H, t, J = 7.0, C13-NH), 4.82 (1H, dd, J = 6.0, 3.0 Hz, H-12), 4.12 (2H, t, J = 6.0 Hz, H-5), 3.71 (2H, q, J = 6.0 Hz, H-3), 3.68 (1H, ddd, J = 13.0, 6.0, 3.0 Hz, H-13), 3.32 (1H, ddd, J = 13.0, 6.0, 6.0 Hz, H-13), 2.21 (2H, t, J = 7.0, H-15), 2.12 (2H, quin, J = 6.0 Hz, H-4), 1.63 (2H, m, H-16), 1.51 (1H, m, H26), 1.25−1.31 (16H, m, H17−H24), 1.15 (1H, m, H-25), 0.86 (6H, d, J = 6.5 Hz, H-27 and H-28); 13C NMR (CDCl3, 125 MHz) δ 176.1 (C-14), 151.8 (C-6), 143.3 (C-2), 141.7 (C-9), 130.5 (C-8 and C-10), 118.4 (C-7 and C-11), 111.9 (C-1), 73.1 (C-12), 72.2 (C-5), 48.2 (C13), 39.3 (C-3), 36.7 (C-15), 36.7 (C-25), 29.6−29.6 (C-17−C-24), 28.5 (C-4), 28.2 (C-26), 26.0 (C-16), 22.9 (C-27 and C-28); (+)-LRESIMS m/z (rel int) 644 (50), 646 (100), 648 (50) [M + H]+; (+)-HRESIMS m/z 646.1691 [M + H]+ (calcd for C28H4481Br79Br N3O4, 646.1681). Cytotoxicity Assays. The growth inhibition assays against human cancer cell lines, in particular, ACHN (renal), NCI-H23 (lung), MDAMB0231 (breast), HCT-15 (colon), NUGC (stomach), and PC-3 (prostate), were carried out according to a published protocol.21 In brief, cancer cells were added to a 96-well plate containing control (doxorubicin) or test compounds. After being incubated for 48 h, cultures were fixed with 50% trichloroacetic acid (50 μg/mL) and stained with 0.4% sulforhodamine B in 1% acetic acid. Unbound dye was removed by washing with 1% acetic acid, and protein-bound dye was extracted with 10 mM Tris base (pH 10.5) for determination of

Pro C18) to afford 2 (28.3 mg), 1 (54.0 mg), 4 (67.1 mg), and 3 (21.3 mg). The 40% aqueous MeOH fraction (1.9 g) was subjected to reversed-phase MPLC, followed by reversed-phase HPLC to afford 6 (147.2 mg), and the 30% aqueous MeOH fraction (2.1 g) was purified by reversed-phase MPLC to afford 5 (1.2 g). The 20% aqueous MeOH fraction (710.0 mg) was subjected to reversed-phase MPLC, followed by final purification on reversed-phase HPLC to afford 15 (1.0 mg), 14 (1.3 mg), and 13 (2.6 mg). The 10% aqueous MeOH fraction (1.1 g) was subjected to MPLC using a silica column, followed by final purification on reversed-phase HPLC (YMC-Pack Cyano) to afford 11 (29.3 mg), 9 (13.5 mg), 10 (37.2 mg), 8 (29.8 mg), and 12 (1.6 mg). Finally, the 100% MeOH fraction (710.0 mg) was purified by silica MPLC using a silica column, followed by reversed-phase HPLC (YMC-Pack Cyano) to afford 16 (3.3 mg), 17 (2.2 mg), and 7 (49.5 mg). Psammaplysin A (5): white, amorphous solid; [α]25D −71 (c 0.5, MeOH) (lit.7 [α]25D −65 (c 0.5, MeOH)). Psammaplysin B (6): white, amorphous solid; [α]25D −75 (c 0.5, MeOH) (lit.7 [α]25D −60 (c 0.6, MeOH)). Psammaplysin X (10): yellow, amorphous solid; [α]25D −64 (c 0.5, acetone); UV (MeOH) λmax (log ε) 311 (4.38), 207 (4.83) nm; 1H and 13C NMR (acetone-d6, 500 and 125 MHz), see Table 1; (+)-LRESIMS m/z (rel int) 872 (20), 874 (65), 876 (100), 878 (82), 880 (35) [M + H]+; (+)-HRESIMS m/z 875.8170 [M + H]+ (calcd for C27H2781Br279Br2ClN3O8, 875.8181). 19-Hydroxypsammaplysin X (11): pale yellow, amorphous solid; [α]25D −82 (c 0.5, acetone); UV (MeOH) λmax (log ε) 309 (4.36), 206 (4.80) nm; 1H and 13C NMR (acetone-d6, 500 and 125 MHz), see Table 1; (+)-LRESIMS m/z (rel int) 888 (20), 890 (60), 892 (100), 894 (77), 896 (33) [M + H]+; (+)-HRESIMS m/z 891.8098 [M + H]+ (calcd for C27H2781Br279Br2ClN3O9, 891.8130). Psammaplysin Y (12): pale yellow, amorphous solid; [α]25D −77 (c 0.5, acetone); UV (MeOH) λmax (log ε) 307 (4.35), 207 (4.87) nm; 1 H and 13C NMR (acetone-d6, 500 and 125 MHz), see Table 2; (+)-LRESIMS m/z (rel int) 838 (29), 840 (73), 842 (100), 844 (70), 846 (21) [M + H]+; (+)-HRESIMS m/z 841.8614 [M + H]+ (calcd for C27H2881Br279Br2N3O8, 841.8572). 19-Hydroxyceratinamide A (13): pale yellow, amorphous solid; [α]25D −62 (c 0.5, acetone); UV (MeOH) λmax (log ε) 305 (4.06), 208 (4.83) nm; 1H and 13C NMR (acetone-d6, 500 and 125 MHz), see Table 2; (+)-LRESIMS m/z (rel int) 774 (20), 776 (67), 778 (100), 780 (68), 782 (21) [M + H]+; (+)-HRESIMS m/z 777.8254 [M + H]+ (calcd for C22H2481Br279Br2N3O8, 777.8258). Subereamide A (14): pale yellow, amorphous solid; UV (MeOH) λmax (log ε) 298 (4.39), 207 (4.73) nm; 1H and 13C NMR (acetone-d6, 500 and 125 MHz), see Table 3; (+)-LRESIMS m/z (rel int) 510 (53), 512 (100), 514 (50) [M + H]+; (+)-HRESIMS m/z 511.9669 [M + H]+ (calcd for C19H1881Br79BrN3O4, 511.9645). E



Article

optical density. The absorbance at 540 nm was determined using a VersaMax microplate reader (Molecular Devices, LLC.).

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(18) Buchanan, M. S.; Carroll, A. R.; Wessling, D.; Jobling, M.; Avery, V. M.; Davis, R. A.; Feng, Y.; Hooper, J. N. A.; Quinn, R. J. J. Nat. Prod. 2009, 72, 973−975. (19) Shaker, K. H.; Zinecker, H.; Ghani, M. A.; Imhoff, J. F.; Schneider, B. Chem. Biodiversity 2010, 7, 2880−2887. (20) Lacy, C.; Scheuer, P. J. J. Nat. Prod. 2000, 63, 119−121. (21) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenny, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112.

ASSOCIATED CONTENT

S Supporting Information *

Figure S1−S8. 1H, 13C, and selected 2D NMR data for compounds 10−17. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-31-400-6171. Fax: +82-31-400-6170. E-mail: yjlee@ kiost.ac. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant of the Korea Institute of Ocean Science and Technology (PE98932) and the Ministry of Oceans and Fisheries. We thank the Department of Marine Resources, State of Chuuk, Federated States of Micronesia, for allowing marine organism research. We also thank Y. H. Kim (Korea Basic Science Institute, Ochang, Korea) for providing mass data.

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REFERENCES

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Cytotoxic Psammaplysin Analogues from a Suberea sp. Marine

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