Reniochalistatins A–E, Cyclic Peptides from the Marine Sponge

Dec 9, 2014 - Five new cyclic peptides (including four heptapeptides and one octapeptide), reniochalistatins A–E (1–5), were isolated and characte...
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Reniochalistatins A−E, Cyclic Peptides from the Marine Sponge Reniochalina stalagmitis Kai-Xuan Zhan,†,§ Wei-Hua Jiao,‡ Fan Yang,‡ Jing Li,‡ Shu-Ping Wang,‡ Yu-Shan Li,*,† Bing-Nan Han,*,‡ and Hou-Wen Lin*,†,‡,§ †

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Marine Drugs Research Center, Department of Pharmacy, State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, People’s Republic of China § Department of Pharmacy, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Five new cyclic peptides (including four heptapeptides and one octapeptide), reniochalistatins A−E (1−5), were isolated and characterized from the marine sponge Reniochalina stalagmitis collected off Yongxing Island in the South China Sea. Their structures were assigned on the basis of HRESIMS, 1D and 2D NMR spectroscopic data, and MALDI-TOF/TOF data for sequence analysis. The absolute configurations of all of the amino acid residues were determined using chiral-phase HPLC and Marfey’s analysis. The cyclic octapeptide reniochalistatin E showed biological activity in various cytotoxicity assays employing different tumor cell lines (RPMI-8226, MGC-803, HL-60, HepG2, and HeLa).

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of Yongxing Island in the South China Sea. Herein we report the isolation, structure elucidation, and antitumor activities of reniochalistatins A−E (1−5).

s sessile animals, marine sponges rely on chemical defense as a major adaptation strategy to thrive in the extreme environmental conditions of the sea, which may account for the diverse array of bioactive chemicals that they produce.1 More than 5300 chemically diverse compounds have been identified from sponges including unusual nucleosides, terpenes, sterols, cyclic peptides, alkaloids, fatty acids, peroxides, and polyketides.1,2 Among them, cyclic peptides composed of both standard and unusual amino acids have generated considerable interest in the biomedical and pharmaceutical communities1,3 due to their antiviral, antitumor, antimicrobial, and general cytotoxic properties. A noteworthy class of marine cyclic peptides is represented by proline-rich heptapeptides or octapeptides, with the presence of 2−4 proline residues, an array of apolar residues such as Leu, Ile, and Val, and some aromatic residues such as Phe, Tyr, and Trp.4 It has been implied that proline residues offer conformational rigidity to these proline-rich cyclic peptides by restricting the dihedral angles in proline.5,8a Species of the sponge genera Phakellia,6 Axinella,7 Callyspongia,8 and Stylissa9 have been reported to yield bioactive cyclic peptides, with most of these being rich in proline amino acid residues. In our screening program to search for bioactive cyclic peptides from the marine sponges of the South China Sea, we have examined and identified the presence of cyclic peptides rich in proline residues from an extract of a tropical marine sponge, Reniochalina stalagmitis Lendenfeld (class Demospongiae, order Halichondria, family Axnellidae), © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The frozen specimen of R. stalagmitis was exhaustedly extracted with CH2Cl2/MeOH (1:1) to give an organic extract that was partitioned between EtOAc and H2O. The EtOAc-phase extract was subjected to solvent partitioning to yield a petroleum ethersoluble extract and a CH2Cl2-soluble extract. Five cyclic peptides, reniochalistatins A−E (1−5), were obtained from the cytotoxic CH2Cl2-soluble extract by a series of chromatographic separations employing Sephadex LH-20, silica gel, ODS, and reversed-phase HPLC column chromatography. The molecular formula of reniochalistatin A (1) was determined as C37H62N8O8 on the basis of HRESIMS and NMR spectroscopic analysis (Table 1). The peptidic nature of 1 was readily inferred by the presence of eight amide carbonyl resonances, as well as seven typical amide NH resonances in the 1H and 13C NMR spectra. Additionaly, the 13C NMR spectrum exhibited seven α-methine carbons, which were assigned to seven methine proton signals in the HMQC spectrum. Detailed analysis of the HMQC, COSY, HMBC, and TOCSY spectra allowed the identification of seven amino acid Received: August 31, 2014

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ions at m/z 635.0, 521.7, 424.6, 311.5, and 211.3, corresponding to the successive loss of Leu, Pro, Ile, Val, and the terminal dipeptide ion Pro-Asn (Figure 2).12a,13 The ΔδCβ‑Cγ values of the Pro residues (4.7 and 9.4 ppm for Pro1 and Pro2, respectively) were indicative of a trans and a cis geometry for the proline amide bonds in 1.9b,14 High-resolution ESIMS analysis of reniochalistatin B (2) revealed an [M + Na]+ ion (m/z 866.4798) consistent with a molecular formula of C46H65N7O8, thus requiring 18 degrees of unsaturation. It was also identified as a cyclic heptapeptide on the basis of the presence of seven carbonyl resonances and seven α-methine carbon signals in the 13C NMR spectrum (Table S1). Extensive inspection of the HMQC, COSY, HMBC, and TOCSY spectra allowed the identification of the seven amino acid residues as Pro (2×), Leu (2×), Ile, Phe, and Tyr, of which the Pro (m/z 70.2), Phe (m/z 120.2), and Tyr (m/z 136.2) residues were indicated by their immonium and related ions in the MALDI-TOF/TOF spectrum (Figure S18).12 The existence of two fragments, Pro1-Ile-Phe-TyrLeu1 and Pro2-Leu2, was evidenced by the HMBC correlations of Ile-NH/Pro1-CO and Phe-NH/Ile-CO, as well as the NOESY correlations of Ile-NH/Pro1-Hα, Phe-NH/Ile-Hα, Phe-NH/Tyr-Hα, Tyr-NH/Leu1-Hα, and Leu2-NH/Pro2-Hα, respectively (Figure 1 and Table S1). Furthermore, in the MALDI-TOF/TOF mass analysis (Figure S18), two pairs of a/ b fragment ion peaks were predominantly observed, which covered the whole structure of 2. As a result, the connections of the two fragments as well as the amino acid sequence of cyclic peptide 2 cyclo-(Pro1-Ile-Phe-Tyr-Leu1-Pro2-Leu2) were confirmed by the MALDI-TOF/TOF sequence analysis (Figure 2).12a,13 The ΔδCβ‑Cγ values of the Pro residues (8.9 and 3.9 ppm for Pro1 and Pro2, respectively) were indicative of a cis and a trans geometry for the respective proline amide bonds in 2.9b,14 Reniochalistatin C (3) was isolated as a colorless, glassy, amorphous solid. The molecular formula of 3 was determined to be C49H63N7O8 on the basis of the [M +Na]+ ion at m/z 900.4620 in the HRESIMS spectrum and confirmed by NMR spectroscopic analysis. The 1H NMR spectrum of 3 exhibited α- and amide protons, implying its peptide nature. The 13C NMR spectrum revealed resonances consistent with seven amide carbonyls and seven α-methine carbons, suggesting a heptapeptide (Table S2). A comprehensive interpretation of the 2D NMR spectra (COSY, TOCSY, HMQC, and HMBC) of 3 indicated seven amino acid units: Pro (2×), Phe (2×), Ile (2×), and Tyr. The key HMBC correlations of Phe2-NH/TyrCO, as well as the NOESY correlations of Ile1-NH/Pro1-Hα, Pro1-Hα/Phe2-Hα, Phe2-Hα/Tyr-NH, Tyr-NH/Ile2-NH, Ile2NH/Pro2-Hα, and Pro2-Hα/Phe1-NH, outlined the linear amino acid sequence as Phe1-Pro2-Ile2-Tyr-Phe2-Pro1-Ile1 (Figure 1). Furthermore, the MALDI-TOF/TOF mass analysis of 3 (Figures 2 and S31) supported the connection of Phe1 and Ile1, which confirmed the structure of 3 as cyclo-(Pro1-Ile1-Phe1Pro2-Ile2-Tyr-Phe2).12a,13 The ΔδCβ‑Cγ values of the Pro residues (7.7 and 4.4 ppm for Pro1 and Pro2, respectively) were indicative of a cis and a trans geometry for the respective proline amide bonds in 3.9b,14 Reniochalistatin D (4) was obtained as a colorless, glassy, amorphous solid, and its molecular formula C48H59N7O7 was established from the positive ion HRESIMS peak (m/z 868.4370, [M + Na]+) and the NMR data. Examination of the COSY, TOCSY, HMQC, and HMBC spectra allowed the identification of seven amino acid residues as Pro (3×), Phe

residues as Pro (2×), Leu (2×), Ile, Val, and Asn. Eight carbonyls and the two proline rings accounted for 10 of the 11 degrees of unsaturation implied by the molecular formula, which suggested 1 was a cyclic peptide and further supported by the NOESY experiment (Figure 1) as well as MALDI-TOF/ TOF sequence analysis (Figure 2). Two fragments, Pro1-Asn-Val and Leu1-Leu2, were outlined by the HMBC correlations of Asn-Hα/Pro1-CO, Val-NH/AsnCO, and Leu2-NH/Leu1-CO, respectively (Figure 1). The NOESY correlations between Leu2-Hα/Pro1-Hδ further allowed the connection of the fragments of Leu1-Leu2 and Pro1-Asn-Val. The NOESY correlations between Ile-NH/Pro2Hδ revealed the Ile-Pro2 fragment. However, due to the significant overlap of two carbonyl signals (Pro2, Val) in the 13C NMR spectrum (δC 170.21 and 170.25), the connectivities of Pro2/Leu1 and Val/Ile could not be unambiguously assigned by the observed HMBC correlations of Pro2-Hα/Pro2-CO (δC 170.21 or 170.25) and Val-Hα/Val-CO (δC 170.21 or 170.25) (Table 1 and Figure 1). Ultimately, the sequence of 1 was confirmed by MALDI-TOF/TOF sequence analysis. Although there was more than one possible ring-opening position for the cyclic peptide, the preferred ring-opening of 1 occurred at the Leu2-Pro1 amide bond due to the high protonation affinity associated with the proline nitrogen (Supporting Information, Figure S4).10,11 The immonium and related ions indicated the existence of Leu or Ile (m/z 86.2) and Pro (m/z 70.2) residues.12 A linearized peptide 1, Pro1-Asn-Val-Ile-Pro2-Leu1Leu2, was demonstrated by a main series of adjacent bn(+1) B

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Table 1. 1H (600 MHz) and 13C NMR (150 MHz) Data for Reniochalistatin A (1) in DMSO-d6a position Pro1 CO α β

δC, mult 170.9, C 62.8, CH 30.0, CH2

γ

25.3, CH2

δ

47.8, CH2

Asn CO α β γ-CO α-NH γ-NH2 Val CO α β γ γ′ NH Ile CO α β γ

170.6, 49.3, 36.3, 172.2,

C CH CH2 C

δH (J in Hz)

4.01, dd (9.2, 8.0) a: 2.26, m b: 1.55, m a: 1.85, m b: 1.78, m a: 3.73, m b: 3.42, m

4.63, dd (10.3, 5.4) 2.79, m

170.0, 57.7, 35.3, 24.9,

C CH CH CH2

3.83, 2.20, 0.89, 0.90, 7.99,

δC, mult

δ β-Me NH Pro2 CO α β

11.0, CH3 16.0, CH3

21.5, CH2

δ

46.1, CH2

γ δ δ′ NH Leu2 CO α β

dd (8.0, 7.5) m d (7.2) d (7.2) d (8.4)

γ δ δ′ NH

4.10, d (9.6, 7.2) 1.92, m a: 1.31, m b: 1.07, m

170.25, Cb 60.5, CH 30.9, CH2

γ

Leu1 CO α β

7.81, d (6.0) a: 7.92, s b: 7.17, s 170.21, Cb 61.5, CH 29.5, CH 19.8, CH3 18.4, CH3

position

171.8, C 50.3, CH 39.9, CH2 24.6, CH 23.2, CH3 22.5, CH3

171.2, C 51.3, CH 38.3, CH2 24.3, CH 23.6, CH3 20.9, CH3

δH (J in Hz) 0.74, d (7.2) 0.80, d (7.2) 7.83, d (9.6)

4.24, d (7.8) a: 2.30, m b: 1.97, m a: 1.85, m b: 1.47, m a: 3.42, m b: 3.26, dd (10.2, 10.2)

4.39, m a: 1.55, m b: 1.16, m 1.41, m 0.86, d (6.6) 0.78, d (7.0) 7.40, d (7.2)

4.14, m a: 1.55, m b: 1.18, m 1.72, m 0.80, d (6.3) 0.86, d (6.6) 8.59, d (5.4)

a

Assignments of the 13C and 1H signals were made on the basis of DEPT, COSY, TOCSY, HMQC, HMBC, and NOESYspectroscopic data; data reported in ppm. bAssignments may be interchanged.

(3×), and Ile (Table S3). The HMBC correlations of Phe3NH/Ile-CO and Phe1-NH/Pro1-CO established Phe3-Ile and Phe1-Pro1 fragments. Due to the significant overlap of two carbonyl signals (δC 169.92 and 169.98 for Phe2 or Pro2) in the 13 C NMR spectrum (Table S3) and the lack of the insightful HMBC correlations of the mutiple proline residues (Figure 1), the connectivity for each amino acid was mainly established by the NOESY correlations of 4 (Figure 1 and Table S3). Furthermore, the MALDI-TOF/TOF mass analysis (Figure S44) confirmed the structure of 4 as cyclo-(Pro1-Phe1-Pro2Phe2-Ile-Phe3-Pro3).12a,13 The ΔδCβ‑Cγ values of the Pro residues (9.7, 9.2, and 9.5 ppm for Pro1, Pro2, and Pro3, respectively) were indicative of cis geometries for all proline amide bonds in 4.9b,14 Reniochalistatin E (5) showed a major adduct ion peak at m/ z 938.5473 [M + Na] + in the HRESIMS spectrum, corresponding to a molecular formula of C49H73N9O8 (18 degrees of unsaturation). The 13C NMR spectrum showed eight carbonyl peaks at δC 170.0−172.3, thus leaving unaccounted 10 additional double bonds or rings in the molecule (Table S4). The HMQC correlations between eight α-proton signals and eight α-carbon resonances indicated that 5 was a peptide containing eight amino acid residues (Table S4). Detailed analysis of the 2D NMR data obtained from COSY, TOCSY, HMQC, and HMBC experiments revealed the presence of Val, Leu, Ile (2×), and Pro (3×) residues, accounting for three more degrees of unsaturation. Moreover,

in the COSY and TOCSY spectra, a downfield NH resonance at δH 10.78 (1H, d, J = 2.7 Hz, H-1) correlated with an aromatic proton signal at δH 6.98 (1H, d, J = 2.7 Hz, H-2), along with the continuous spin correlations of proton signals characteristic of a 1,2-disubstituted benzene at δH 7.47 (1H, d, J = 10.1 Hz, H-4), 6.96 (1H, m, H-5), 7.05 (1H, t, J = 10.1 Hz, H-6), and 7.32 (1H, d, J = 10.1 Hz, H-7), indicated the presence of an indole ring, adding six more degrees of unsaturation. Subsequently, long-range correlations from the βmethylene protons at δH 3.61 and 3.37 to the carbon resonances at δC 56.8 (Trp-Cα), 111.5 (Trp-C-3), 124.1 (Trp-C-2), and 127.3 (Trp-C-9) allowed us to assign the eighth residue as tryptophan (Table S4). The key HMBC correlations of Ile2-NH/Pro3-CO, Ile1-NH/Val-CO, Val-NH/Pro2-CO, and Leu-NH/Pro1-CO, as well as the NOESY correlations of Pro3Hα/Ile1-Hα, Val-NH/Pro2-Hα, Pro2-Hγb/Trp-H-2, and LeuNH/Pro1-Hα, displayed the existence of the two fragments Ile2Pro3-Ile1-Val-Pro2-Trp and Pro1-Leu (Figure 1). However, due to the significant overlap of two carbonyl signals of Ile2 and Leu (δC 171.84 and 171.98) in the 13C NMR spectrum, the connectivities of Leu/Trp could not be unambiguously assigned on the basis of the observed HMBC correlations of Trp-NH/ Leu-CO (δC 171.84 or 171.98). Ultimately, the MALDI-TOF/ TOF sequence analysis was used to elucidate the connections of the fragments (Figure S57). A linearized peptide, Pro1-LeuTrp-Pro2-Val-Ile1-Pro3-Ile2, was indicated by a series of adjacent bn(+1) ions, which suggested that peptide 5 was cyclo-(Pro1C

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Figure 1. Key correlations of HMBC (solid arrow) and NOESY (dashed arrow) experiments for reniochalistatins A−E (1−5).

Figure 2. MALDI-TOF/TOF sequence ions (m/z) for the protonated molecules [M + H]+ of reniochalistatins A−E (1−5).

Leu-Trp-Pro2-Val-Ile1-Pro3-Ile2), and the preferred ring-opening occurred at the Ile2-Pro1 amide bond (Figure 2).12a,13 Thus, the macrocyclic structure was completed, accounting for the final degree of unsaturation of reniochalistatin E (5). The ΔδCβ‑Cγ values of the Pro residues (4.8, 4.3, and 4.1 ppm for Pro1, Pro2, and Pro3, respectively) were indicative of trans geometries for all proline amide bonds in 5.9b,14 A chiral-phase HPLC analysis of the hydrolyzed amino acids of the reniochalistatins A−E (1−5) clarified that all of the Pro, Phe, Val, Leu, Ile, and Tyr residues were L-form amino acids (Figures S13, S27, S40, S53, and S66). Marfey’s analysis was used to confirm the L-form of the Asn and Trp residues (Figures S14 and S67). Compared to the previously reported proline-rich cyclic peptides, such as phakellistatins,6 euryjanicins,4a stylissamides,15 stylopeptides,16 and stylisins,9a reniochalistatins C (3) and D (4) are closely related to phakellistatin 18 and stylissamide C, with over 70% similarity in terms of peptide sequences. Interestingly, reniochalistatins A−E (1−5) share less than 50% similarity within the sequences. In the present work, reniochalistatins A−E (1−5) were tested againist five different human cancer cell lines (RPMI-8226, MGC-803, HL-60, HepG2, and HeLa). Surprisingly, the four heptapeptides (1− 4) showed no cytotoxicity against the tested cell lines. However, the octapeptide (5) exhibited cytotoxic activity

against myeloma RPMI-8226 and gastric MGC-803 cells with IC50 values of 4.9 and 9.7 μM, respectively, but with no activity against leukemia HL-60 and hepatoma HepG2 (IC50 > 20.0 μM) and cervical HeLa (IC50 17.3 μM) cells. It is premature to draw any general conclusions regarding a structure−activity relationship among the proline-rich cyclic peptides, owing to conflicting reports of naturally occurring, proline-rich cyclic peptides that were initially described as having cytotoxic activity, but subsequent synthetic samples were not active.5c,9a,17 This study extends our knowledge of secondary metabolites from the marine sponges of the genus Reniochalina previously dominated by acetylenic alcohols, dihydrothiopyranone, and fatty acids.8a,18 Questions of whether these cyclic peptides are endogenous secondary metabolites of the sponge or its symbionts have drawn attention in the marine natural products community.7b,19

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation measurements were conducted on an Autopol I polarimeter (No. 30575, manufactured by Rudolph Research Analytical) with a 10 cm length cell at room temperature. UV and IR (KBr) spectra were recorded on a Hitachi U-3010 spectrophotometer and Jasco FTIR-400 spectrometer, respectively. 1H, 13C, DEPT, COSY, HMQC, HMBC, TOCSY,

D

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MALDI-TOF/TOF data, Figure 2; HRESIMS m/z 868.4370 [M + Na]+ (calcd for C48H59N7O7Na, 868.4374). Reniochalistatin E (5, cyclo-(L-Pro-L-Leu-L-Trp-L-Pro-L-Val-L-Ile-LPro-L-Ile)): colorless, glassy, amorphous solid; [α]25D −100 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 221 (4.38), 283 (3.61), 291 (3.55) nm; IR (KBr) νmax 3292, 2962, 2931, 1681, 1623, 1520, 1449 cm−1; 1H and 13C NMR data, Table S4; MALDI-TOF/TOF data, Figure 2; HRESIMS m/z 938.5473 [M + Na]+ (calcd for C49H73N9O8Na, 938.5480). Chiral-Phase HPLC Analysis of the Acid Hydrolysate of 1−5. Each of the peptides 1−5 (0.5 mg) was dissolved in 6 N HCl (1 mL) and heated at 110 °C for 18 h. After cooling to room temperature (rt), the solvent was evaporated, and traces of HCl were removed by repeated drying under vacuum with distilled H2O. The dried hydrolysate was dissolved in 500 μL of 2 mM CuSO4/H2O solution. The hydrolysates of peptides 1−5 and authentic L- and D-amino acids were analyzed with a chiral-phase column (MCI GEL CRS10W, 4.6 × 50 mm, Mitsubishi Chemical Corporation) using 2 mM CuSO4/H2O solution as the mobile phase with UV detection at 254 nm on a Waters 1525/2998 HPLC instrument. All amino acid residues in reniochalistatins A−E (1−5) were found to correspond to the L-configuration by comparison of retention time values (tR, min) with those of standard amino acids: (1) aqueous 2 mM CuSO4, flow rate at 1 mL/ min, L-Ile (19.9), D-Ile (8.7), L-allo-Ile (14.2), D-allo-Ile (7.6), L-Leu (17.3), D-Leu (9.2), L-Tyr (28.2), D-Tyr (16.1), L-Pro (6.2), D-Pro (3.3), L-Phe (45.7), D-Phe (25.8); (2) aqueous 2 mM CuSO4, flow rate at 0.8 mL/min, L-Ile (26.2), D-Ile (10.9), L-allo-Ile (18.6), D-allo-Ile (10.4), L-Leu (23.0), D-Leu (12.1), L-Pro (7.9), D-Pro (4.2), L-Val (9.7), D-Val (5.2) (Figures S13, S27, S40, S53, and S66). HPLC Analysis of the Acid Hydrolysates of 1 and 5 Using Marfey’s Method. To determine the absolute configurations of the Asn in 1 and the Trp in 5, HPLC analyses of the acid hydrolysates were conducted. Compounds 1 (0.36 mg) and 5 (0.50 mg) were hydrolyzed as mentioned above. The hydrolysate was processed using a published protocol20 with the following exceptions: (1) 20 μL of 1 M HCl was used; (2) the resulting residue after solvent removal was resuspended in 100 μL of MeOH; (3) a YMC-Park Pro C18, 4.6 × 250 mm, 5 μm column was used. Under typical acid hydrolysis conditions, Asn residues will be converted to Asp residues.21 The retention times for FDAA derivatives of L-Asp, D-Asp, L-Trp, and D-Trp were 11.6, 12.5, 23.6, and 24.5 min, respectively. The FDAA derivatives of the amino acids from the hydrolysate of 1 and 5 showed peaks at 11.7 (LAsp) and 23.7 (L-Trp) min, respectively (Figures S14, S67), and the amino acids were assigned to be L-Asn in 1 and L-Trp in 5. Cell Culture. The human myeloma RPMI-8226 cell line was provided by Dr. Jian Hou, Changzheng Hospita (Second Military Medical University, Shanghai, China). The human gastric cancer MGC-803 cell line, human cervical cancer HeLa cell line, human leukemia HL-60 cell line, and human hepatoma HepG2 cell line were purchased from the Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). The RPMI-8266 and MGC-803 cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in an atmosphere of 5% CO2. The HL-60 cell line was cultured in RPMI-1640 medium supplemented with 20% FBS and incubated at 37 °C in an atmosphere of 5% CO2. The HeLa and HepG2 cell lines were cultured in HGDMEM, supplemented with 10% FBS in 5% CO2 at 37 °C. Biological Assays. The cytotoxic activities of these comounds were assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS) assay as described previously.22 RPMI-8266, HeLa, MGC-803, HepG2, and HL-60 cells were seeded in 96-well microplates at a density of 1 × 104 cells/well in 100 μL of growth media and incubated for 24 h. Culture media containing different concentrations of the samples were then added. After incubation for 72 h, 20 μL of MTS was added to each well, and the plate was incubated for 2 h. The absorbance was measured spectrophotometrically at a wavelength of 490 nm (with a reference wavelength of 690 nm) using a SpectraMax 340 plate reader/spectrophotometer (Molecular Devices Corp.). The positive controls were SAHA against RPMI-8226 (IC50 2.4 μM) and

and NOESY NMR spectra were acquired at room temperature on Bruker AVANCE-600 and Bruker AMX-500 instruments with DMSOd6 as the solvent. Spectra were referenced to residual solvent signals with resonances at δH 2.49 and δC 39.7. High-resolution ESIMS spectra were acquired with a Waters Q-Tof micro YA019 mass spectrometer. MALDI-TOF/TOF spectra were recorded on a 4700 Proteomics analyzer (Applied Biosystems). Reversed-phase HPLC was performed on a YMC-Pack Pro C18 column (250 × 10 mm, 5 μm) using a Waters 600 HPLC instrument with a Waters 996 UV detector. Column chromatography (CC) was performed on ODS (15 μm, YMC Co.) and Sephadex LH-20 (18−110 μm, Pharmacia Co.). The fractions were monitored by TLC (HSGF 254, Yantai, China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in H2O. The enantioselective HPLC of the amino acids was conducted with a chiral-phase column (MCI GEL CRS10W, 4.6 × 50 mm, Mitsubishi Chemical Corporation). Amino acid standards used for enantio selective analysis were purchased from Sigma-Aldrich Chemical Corporation. Animal Material. Specimens of Reniochalina stalagmitis Lendenfeld were collected around Yongxing Island in the South China Sea during the month of July in 2009 and were identified by Mrs. Shirley Sorokin (Centre of Marine Bioproducts Development, Flinders University, Australia). A voucher sample (No. SZH) was deposited in the Laboratory of Marine Drugs, Department of Pharmacy, Changzheng Hospital, Second Military Medical University, China. The sponge R. stalagmitis is of great abundance in the Xisha Islands, and therefore this collection was considered to have no significant adverse ecological effect. Extraction and Purification. The extracts from the sponge (72 g, dry wt) were obtained with CH2Cl2/MeOH (1:1), and combined extracts were concentrated under vacuum. The concentrated extract was then suspended in H2O, and EtOAc was used to extract the organic components. The EtOAc-soluble extract was partitioned between MeOH/H2O (9:1) and petroleum ether to afford the petroleum ether-soluble extract (1.1 g). The MeOH/H2O (9:1) phase was diluted to 3:2 with water, which was partitioned by CH2Cl2 to yield the CH2Cl2-soluble extract (1.3 g). This CH2Cl2-soluble extract was subjected to CC on Sephadex LH-20 using CH2Cl2/MeOH (1:1) as eluent to give three subfractions (A−C). Subfraction B (700 mg) was subjected to CC on ODS silica and further purified by HPLC (YMC-Pack Pro C18, 5 μm, 10 × 250 mm, 2.0 mL/min, UV detection at 210 and 275 nm) eluting with CH3CN/H2O (50:50) to yield pure peptides reniochalistatin B (2, 9.0 mg, t R 40.9 min) and reniochalistatin D (4, 5.1 mg, tR 49.7 min); CH3CN/H2O (45:55) to yield pure reniochalistatin A (1, 5.8 mg, tR 17.1 min); CH3CN/H2O (60:40) to yield pure reniochalistatin C (3, 3.0 mg, tR 26.6 min); and CH3CN/H2O (55:45) to yield reniochalistatin E (5, 2.8 mg, tR 29.2 min). Reniochalistatin A (1, cyclo-(L-Pro-L-Asn-L-Val-L-Ile-L-Pro-L-Leu-LLeu)): colorless, glassy, amorphous solid; [α]25D −130 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.13) nm; IR (KBr) νmax 3312, 2959, 1643, 1529, 1432 cm−1; 1H and 13C NMR data, Table 1; MALDITOF/TOF data, Figure 2; HRESIMS m/z 769.4586 [M + Na]+ (calcd for C37H62N8O8Na, 769.4588). Reniochalistatin B (2, cyclo-(L-Pro-L-Ile-L-Phe-L-Tyr-L-Leu-L-Pro-LLeu)): colorless, glassy, amorphous solid; [α]25D −110 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.25), 279 (3.02) nm; IR (KBr) νmax 3320, 2958, 1645, 1517, 1447 cm−1; 1H and 13C NMR data, Table S1; MALDI-TOF/TOF data, Figure 2; HRESIMS m/z 866.4798 [M + Na]+ (calcd for C46H65N7O8Na, 866.4792). Reniochalistatin C (3, (cyclo-(L-Pro-L-Ile-L-Phe-L-Pro-L-Ile-L-Tyr-LPhe)): colorless, glassy, amorphous solid; [α]25D −110 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.34), 277 (3.25) nm; IR (KBr) νmax 3342, 3244, 2964, 2931, 1653, 1622, 1516, 1446 cm−1; 1H and 13C NMR data, Table S2; MALDI-TOF/TOF data, Figure 2; HRESIMS m/z 900.4620 [M + Na]+ (calcd for C49H63N7O8Na, 900.4636). Reniochalistatin D (4, cyclo-(L-Pro-L-Phe-L-Pro-L-Phe-L-Ile-L-Phe-LPro)): colorless, glassy, amorphous solid; [α]25D −66 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.29) nm; IR (KBr) νmax 3275, 2965, 1646, 1550, 1446, 1431 cm−1; 1H and 13C NMR data, Table S3; E

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doxorubicin against MGC-803, HL-60, HepG2, and HeLa (IC50 0.1, 0.2, 0.4, and 0.5 μM, respectively).

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ASSOCIATED CONTENT

* Supporting Information S

Copies of 1D and 2D NMR, HRESIMS, UV, IR, MALDITOF/TOF spectra, and chiral HPLC and Marfey’s analysis for compounds 1−5. These materials can be accessed free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(Y. S. Li) E-mail: [email protected]. *(B. N. Han) E-mail: [email protected]. *(H. W. Lin) Tel: +86-21-58732594. Fax: +86-21-68383346. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Mrs. S. Sorokin (Centre of Marine Bioproducts Development, Flinders University, Australia) for identifying the sponge. This research was supported by the National Natural Science Fund for Distinguished Young Scholars of China (81225023), the National Natural Science Fund of China (Nos. 41476121, 81402844, 81302691, 81373321, 41106127, 81172978, 81072573, and 81001394), and Shanghai Subject Chief Scientist (12XD1400200). We are also grateful for the financial support of the National High Technology Research and Development Program of China (863 Projects, Nos. 2013AA092902 and 2011AA09070107).

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REFERENCES

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