Dragmacidins G and H, Bisindole Alkaloids Tethered by a

LCMS analysis of the extract and a cytotoxicity assay of the HPLC fractions generated from a small-scale extract of a Lipastrotethya sp. marine sponge...
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Dragmacidins G and H, Bisindole Alkaloids Tethered by a Guanidino Ethylthiopyrazine Moiety, from a Lipastrotethya sp. Marine Sponge Yuki Hitora,† Kentaro Takada,*,† Yuji Ise,‡ Shigeru Okada,† and Shigeki Matsunaga*,† †

Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan ‡ Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Toba, Mie 517-0004, Japan S Supporting Information *

ABSTRACT: LCMS analysis of the extract and a cytotoxicity assay of the HPLC fractions generated from a small-scale extract of a Lipastrotethya sp. marine sponge demonstrated the presence of bisindole alkaloids that were associated with the cytotoxic activity. Two bisindole alkaloids tethered by a guanidino ethylthiopyrazine moiety, dragmacidins G (1) and H (2), were isolated, and their structures were assigned by analysis of the MS and NMR data. They showed moderate cytotoxic activity against HeLa cells.

B

isindole alkaloids such as topsentins, hamacanthins, and dragmacidins are dimers of tryptamine and unique to marine sponges.1 A few related metabolites were also reported from phyla outside of the Porifera: 2,5-bis(6-bromo-1H-indol3-yl)piperazine from the tunicate Didemnum candidum and alocasin A (3,3′-(pyrazine-2,5-diyl)bis(1H-indol-5-ol)) from the terrestrial plant Alocasia macrorrhiza.2,3 Uneven distribution of these metabolites across the phyla suggests their microbial origin.4 We examined the extract of a Lipastrotethya sp. marine sponge by LCMS and HPLC fractions from a small-scale extract by a cytotoxicity test.5 This analysis showed the presence of dimeric indoles that were associated with cytotoxic activity. We then isolated the active compounds and identified them as new compounds dragmacidin G (1)6 and dragmacidin H (2) and the known compounds, toxic to fish, topsentin B1 (3, topsentin) and topsentin B2 (4, bromotopsentin).7,8

three mutually coupled aromatic protons (H-4′, H-5′, and H7′; H-4″, H-5″, and H-7″) and another pair of aromatic protons coupled to exchangeable protons (H-2′, 1′-NH; H-2″, 1″-NH). There were another aromatic proton (H-6), two pairs of methylene protons (H2-8 and H2-9), an exchangeable proton (10-NH), and a very broad exchangeable signal centered at δH 7.2 (12-NH and 13-NH2). Interpretation of the COSY, HSQC, and HMBC spectra permitted the assignment of two 3,6disubstituted indoles and two contiguous methylenes. The 13C NMR chemical shifts of the two indoles showed that substituents at the 6-position were bromine in both cases.7,8 The 1JCH values of 138 and 141 Hz for the two methylenes (C8 and C-9, respectively) suggested that they were both substituted by a heteroatom.9 An HMBC cross-peak between H2-9 and a carbon at δC 156.9 (C-11) indicated that C-9 was substituted by a guanidyl group. The remaining aromatic proton (H-6) was attached to a carbon at δC 135.4 and exhibited a 1JCH value of 182 Hz, suggesting that this carbon was in a six-membered aromatic ring and adjacent to a heteroatom.10 H-6 correlated intensely with carbons at δC 143.3 (C-2) and 145.9 (C-5) and weakly with a carbon at δC 150.2 (C-3) in the HMBC spectrum. Although these carbon chemical shift values suggested that H-6 was in a

In the ESIMS spectrum of dragmacidin G (1), the protonated molecules were observed at m/z 584, 586, and 588 in a ratio of 1:2:1, suggesting the presence of two bromine atoms. The molecular formula of C 23 H 20 Br 2 N 7 S was determined by HRESIMS. Analysis of the 1H and 13C NMR data in conjunction with the HSQC spectrum revealed a pair of © 2016 American Chemical Society and American Society of Pharmacognosy

Received: August 2, 2016 Published: October 25, 2016 2973

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pyrazine ring, it was not possible to exclude the possibility of the pyrimidine ring system, i.e., structure 1′. The presence of the pyrazine ring was confirmed by the 1H−15N HMBC data, in which H-6 was correlated intensely with a nitrogen at δN 323.3 and weakly with a nitrogen at δN 298.7;11−13 only one HMBC correlation from H-6 to an 15N signal was expected for a pyrimidine ring. The positions of the above-mentioned substituents in the pyrazine ring were assigned on the basis of the HMBC correlations: H-2″ was correlated with C-5, whereas H-6 was correlated with C-3″, demonstrating that one indole was attached to the carbon adjacent to C-6. H-2′ was correlated with C-2, indicating that the other indole was attached to C-2. H2-8 was correlated with C-3 in the HMBC spectrum. In order to fulfill the molecular formula, C-8 was linked to C-3 via a sulfur atom. This assignment was in agreement with the chemical shifts of H2-8 and C-8.14,15 Compound 1 was previously reported in a patent without characterization data.6

The molecular formula of dragmacidin H (2), C23H20BrN7S, suggested that it was a debromo derivative of dragmacidin G (1). Instead of the 1H and 13C NMR signals assigned to one of the two 3,6-disubstituted indoles, those assignable to a 3substituted indole were present in 2 (Table 1). Interpretation of the 2D NMR data demonstrated that the bromine atom at C-6′ in 1 was replaced by a hydrogen atom in 2. The assignment of the position of the debromination was demonstrated by an HMBC correlation between H-6 and a carbon at δC 112.4 (C-3″) which comprised the nonbrominated indole ring. Dragmacidin G (1), dragmacidin H (2), topsentin B1 (3), and topsentin B2 (4) showed cytotoxicity against HeLa cells with IC50 values of 4.2, 4.6, 4.4, and 1.7 μM, respectively. A variety of bisindole alkaloids related to the topsentins and dragmacidins have been isolated from marine sponges.1 Dragmacidins G and H are the first examples from marine sponges with a pyrazine tether between the indoles. The guanidino ethylthio group in 1 and 2 is rare among marine natural products. This group was found only in the phloeodictines from marine sponges Phloeodictyon sp. and Oceanapia f istulosa.14−16 Recent studies on the biosynthetic gene clusters of a fungus showed that dipeptide-derived alkaloids tethered by a thiopyrazine could be produced by nonribosomal peptide synthetases (NRPS).12 The products of this system are different from the dragmacidines, which are dimerization products of tryptamine, because the former dipeptides retain all of the carbon atoms of the amino acid residues used as the starting material. Similarly, the biosynthesis

Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for 1 (DMSO-d6/TFA (1000:1)) and 2 (DMSO-d6) 1 position 2 3 5 6 8 9 10-NH 11 1′-NH 2′ 3′ 3a′ 4′ 5′ 6′ 7′ 7a′ 1″-NH 2″ 3″ 3a″ 4″ 5″ 6″ 7″ 7a″

δC, type 143.3, 150.2, 145.9, 135.4, 28.8, 40.4,

C C C CH CH2 CH2

2

δH, mult (J in Hz)

8.97, 3.56, 3.55, 7.90,

s m m brs

HMBC

2, 3, 5, 3″ 3, 9 8, 11

156.9, C 127.9, CH 111.9, C 125.4, C 123.2, CH 122.9, CH 114.8c, C 114.5, CH 137.2, C 127.8, CH 112.6, C 124.1, C 122.7, CH 123.4, CH 114.8c, C 114.7c, CH 138.0, C

δC, type 142.9, 150.2, 146.5, 135.2, 28.6, 40.4,

C C C CH CH2 CH2

δH, mult (J in Hz)

8.96, s 3.54, t (6.4) 3.48, t (6.4) 9.51 brs

157.6, C 11.81, brd (2.1) 8.08, brd (2.1)

2′, 3′, 3a′, 7a′ 2, 3′, 3a′, 7a′

8.09, d (8.6) 7.25, dd (8.6, 1.9)

3′, 5′, 6′, 7a′ 3a′, 7′

7.70, d (1.9)

3a′, 5′, 6′, 7a′

11.93, brd (2.5) 8.32, d (2.8)

2″, 3″, 3a″, 7a″ 5, 3″, 3a″, 7a″

8.25, d (8.5) 7.31, dd (8.5, 1.9)

3″, 5″, 6″, 7a″ 3a″, 7″

7.70, brd (1.9)

3a″, 5″, 6″, 7a″

127.9, 111.9, 125.5, 123.2, 122.8, 114.8, 114.5, 137.2,

CH C C CH CH C CH C

127.9, CH 112.4, C 125.0, C 121.0, CH 120.7, CH 112.1, CH 112.2,, CH 137.1, C

NDa,b 8.10, brs

8.11, d (8.6) 7.24, dd (1.8, 8.6) 7.69, brd (1.8) ND 8.30, m

8.31, 7.20, 7.20, 7.50,

m m m m

Not determined. bThe following exchangeable broad signals were observed: 1, δH 7.20 (4H); 2, δH 11.90 (2H), 9.51 (1H), 8.10 (3H). Assignments may be interchanged.

a c

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well) were seeded in 96-well plates and incubated overnight. Microscopic images of cells were collected every 2 h for 3 days of cell culture at 37 °C. Extraction and Isolation. The sponge (380 g) was cut into small pieces and extracted with EtOH (700 mL × 2) and a mixture of CHCl3 (350 mL) and MeOH (350 mL). The extracts were combined and concentrated in vacuo and partitioned between H2O and EtOAc. A part of the organic layer was concentrated and subjected to ODS column chromatography eluting with 40% MeOH, 60% MeOH, 80% MeOH, and MeOH. The 40% MeOH and 60% MeOH fractions were purified by ODS HPLC (Cosmosil MS-II, 20 mm × 250 mm) eluting with 65% MeOH containing 0.5% AcOH to afford dragmacidin G (1) (280 mg) and dragmacidin H (2) (19 mg) together with topsentin B1 (3, 180 mg) and topsentin B2 (4, 13 mg). Dragmacidin G (1): yellow solid; UV (MeOH) λmax (log ε) 220 (4.3), 290 (4.2), 390 (4.1); IR (neat) νmax 3170, 1680, 1550, 1450, 1410 cm−1; 1H and 13C NMR data (DMSO-d6/TFA (1000:1)), Table 1; HRESIMS m/z 585.9821 [M + H]+ (calcd for C23H20Br2N7S, 585.9842). Dragmacidin H (2): yellow solid; UV (MeOH) λmax (log ε) 223 (4.2), 281 (4.1), 320 (4.0), 393 (4.1); IR (neat) νmax 3170, 1670, 1550, 1450, 1410 cm−1; 1H and 13C NMR data (DMSO-d6), Table 1; HRESIMS m/z 506.0754 [M + H]+ (calcd for C23H20Br2N7S, 506.0758). Cell Culture and MTT Assay. HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin at 37 °C under an atmosphere of 5% CO2. To each well of a 96-well microplate containing 200 μL of tumor cell suspension (1 × 104 cells/mL) was added samples after overnight preincubation, and the plate was incubated for 72 h. Then 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) saline solution (1 mg/mL, 50 μL) was added to each well. After 3 h incubation, the supernatant was discarded, DMSO (150 μL) was added, and the absorbance at 570 nm was measured.

and function of dipeptide-derived pyrazinones by an NRPS in the bacterium Staphylococcus aureus was studied in detail;17,18 the products of this system also maintain the carbon atoms of the amino acids. Considering that the dragmacidins and topsentins are dimers of tryptamine, which lacks one carbon from tryptophan, it is hard to believe that they are produced by the NRPS pathway. In the extract of the Lipastrotethya sp. sponge, dragmacidins G and H and two topsentin derivatives were the predominant constituents: positional isomers derived from alternate modes of bromination or hydroxylation were not detected. This implies that the incorporation of substituted tryptamine-like precursors into the products was meticulously controlled, implying the enzymatic production of 1 and 2 as well as topsentin B1(3) and topsentin B2 (4). The idea of a non-NRPS origin of this class of metabolites is supported by the presence of regioisomers among hamacanthins differing in the positions of indoles with respect to the piperazinone ring.1 Only two other sponges of the genus Lipastrotethya have been chemically analyzed. Those from Hainan Province, China, and Chuuk State, Federation of Micronesia, afforded sesquiterpene dimers and triterpene glycosides.19−21

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were measured on a Shimadzu Biospec 1600. IR spectra were measured on a PerkinElmer Frontier IR spectrometer. NMR spectra were recorded on a JEOL alpha 600 NMR spectrometer. Chemical shifts were referenced to solvent peaks: δH 2.49 and δC 39.5 for DMSO. HRESI mass spectra were measured on a JEOL JMS-T100LC. HPLC separation of each extract into a 96-well plate was conducted on a Waters Alliance 2695 with 2996 PDA detector HPLC system with ChromScience fraction collector Hummingbird. HPLC purification was carried out on a Shimadzu LC 20AT with an SCL-10Avp controller and an SPD-10Avp detector. Animal Material. The sponge Lipastrotethya sp. was collected by dredging at Kurose (between 33°20.9082 N, 139°41.1862 E and 33°21.0722 N, 139°40.5142 E), north of Hachijo Island, at a depth of 185−213 m, during the KT-07-31 cruise of R/V Tansei-maru of the Japan Agency for Marine-Earth Science and Technology (JAMSTEC), November 2007. The sample was frozen after collection and stored at −20 °C until used. The sponge was columnar in shape, measured ca. 20 cm in maximum diameter and 30 cm in height, and partly covered by a Vulcanella (Vulcanella) sp. sponge. The color was yellow in life and dark brown in EtOH. The surface was smooth or partly slightly grooved, fleshy in appearance; numerous oscules were opened on top of the sponge; the texture was firm and elastic. Spicules were composed of oxeas in a very wide size range (55−1100 μm in length, 1−45 μm in width) and few styles clearly originated from oxea. Larger oxeas composed loose bundles vertically ascending to the surface. Smaller oxeas were disorganized or intercrossed each other in the choanosome. No special ectosomal spicule was present. Larger oxeas or styles were usually slightly bent at midpoint, rarely straight, with sharp or blunt tips. Smaller oxeas were usually straight with sharp tips. The geographically closest species is Lipastrotethya hilgendolfi once collected from Hakodate, Japan,22 but in that species smaller categories of oxeas are totally lacking. The specimen (NSMT-Po-248) was deposited at the National Museum of Nature and Science, Tokyo. HPLC Fractionation of the Extract and Time-Lapse Imaging Analysis. A 20 g portion of the sponge was homogenized and extracted with EtOH (60 mL). The extract was concentrated and partitioned between H2O and CHCl3. The organic layer was dried and applied to a short ODS column, and the column was eluted with MeOH. A 5 mg portion of the MeOH fraction was subjected to reversed-phase HPLC (Cosmosil MS-II, 4.6 × 250 mm, 20% MeCN + 0.8% AcOH to MeCN in 60 min) and fractionated into a 96-well deep-well plate. The plate was dried in vacuo, and MeOH (50 μL) was added to each well prior to the assay. HeLa/Fucci2 cells (2000 cells/

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00710. NMR data for 1 and 2 and the result of the cytotoxicity assay of 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (K. Takada): [email protected]. *Tel (S. Matsunaga): 81-3-5841-5297. Fax: 81-3-5841-8166. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” (23102007) and JSPS KAKENHI Grant Numbers 25252037, 25712024, 25660163, 24-7872, 15K14800, and 15K14799 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank the captain and crew of the R/V Tansei-maru for sample collection.

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REFERENCES

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