Cyclopeptides from the Sponge Stylissa flabelliformis - Journal of

Jun 12, 2018 - Chemical Biology Institute, Yale University, West Haven , Connecticut ... Marine Natural Products Laboratory, Korea Institute of Ocean ...
0 downloads 0 Views 1MB Size
Article Cite This: J. Nat. Prod. 2018, 81, 1426−1434

pubs.acs.org/jnp

Cyclopeptides from the Sponge Stylissa f labelliformis Oh-Seok Kwon,† Chang-Kwon Kim,† Woong Sub Byun,† Joonseok Oh,‡,§ Yeon-Ju Lee,⊥ Hyi-Seung Lee,⊥ Chung J. Sim,∥ Dong-Chan Oh,† Sang Kook Lee,† Ki-Bong Oh,*,¶ and Jongheon Shin*,†

Downloaded via KAOHSIUNG MEDICAL UNIV on June 22, 2018 at 18:23:55 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.



Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-742, Korea ‡ Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States § Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States ⊥ 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 ¶ Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-921, Korea S Supporting Information *

ABSTRACT: Three new cyclopeptides, phakellistatins 20−22 (1−3), as well as 10 known cyclopeptides of the same structural class were isolated from the tropical sponge Stylissa f labelliformis. By a combination of chemical and spectroscopic methods, the structures of the new compounds were determined to be an epimeric mixture of cycloheptapeptides (1) and two epimeric cyclodecapeptides (2 and 3) related to the phakellistatins. The cyclopeptides were evaluated for in vitro cytotoxicity against a variety of cancer cell lines, and compounds 2 and 3 exhibited significant activity.

S

of these compounds revealed three new compounds, phakellistatins 20−22 (1−3). The remaining peptides were identified to be the previously reported phakellistatins 2, 4, 8, 9, 11, and 18 (4−9),6,9−12 hymenamide D (10),13 cyclonellin (11),14 and carteritins A and B (12 and 13)15 by a combination of spectroscopic analyses and comparison with the literature. The cytotoxicities of these compounds were measured against a number of cancer cell lines, and several peptides, including the newly isolated 2 and 3, exhibited remarkable in vitro activity.

ponges produce a wide variety of cyclic peptides and depsipeptides.1 The significant structural variations of both the amino acid residues and functionalities of these compounds as well as their potent bioactivities and distinct biochemical properties are of significant interest for biomedical and synthetic applications of sponge-derived cyclic peptides.2,3 Among these peptides, phakellistatins are a well-known group of proline-containing compounds mainly isolated from the tropical sponges of the genus Phakellia. Since the discovery of phakellistatin 1 from Phakellia costata in the early 1990s,4 almost 20 cyclopeptides have been reported that are classified as phakellistatins.1,3,5 Structurally related compounds such as carteritins and hymenamides have also been isolated from a variety of tropical sponges.1,6,7 The significant cytotoxicity of the phakellistatins not only provides important insight into but also raises unanswered questions about the role of proline residues in the bioactivity of cyclopeptides.5,8 During our search for bioactive compounds from tropical sponges, specimens of Stylissa f labelliformis (order Halichondria, family Dictyonellidae) were collected off the shore of Chuuk Island, the Federated States of Micronesia. Prompted by the significant cytotoxicity (ED50 of 89 ppm against the K562 cell line) of the organic extract, combined chromatographybased separations were performed, which led to the isolation of 13 compounds. Combined chemical and spectroscopic analysis © 2018 American Chemical Society and American Society of Pharmacognosy



RESULTS AND DISCUSSION The molecular formula of phakellistatin 20 (1) was deduced to be C35H52N8O9S by HRFABMS analysis. However, both the 13 C and 1H NMR spectra of this compound showed far more signals than anticipated from the mass spectrometric data. These mostly duplicated NMR signals indicated that 1 was indeed a mixture of two very similar compounds (1:1 based on the signal intensity of paired protons in the 1H NMR spectra) (Table 1). Because these compounds were not separated under various chromatographic conditions, the structures were determined as a mixture. The most prominent feature in the Received: February 11, 2018 Published: June 12, 2018 1426

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434

Journal of Natural Products

Article

Chart 1

13

acid residue (Figure 1). By this method, six amino acid residues were readily identified to be alanine (Ala, ×2), asparagine (Asn), isoleucine (Ile), phenylalanine (Phe), and proline (Pro). The remaining NMR signals consisted of an amino acid residue bearing an ethylene (C-22 and C-23) and an isolated methyl group (C-24), which were shown to be connected to each other by the HMBC data (H-23/C-24 and H-24/C-23). Taken together, the moderate shielding of protons and carbons in these groups, the presence of an SO unit in the mass spectrometric data, and a strong absorption band at 1054 cm−1 in the IR data indicated that the remaining amino acid residue was a sulfoxymethionine (Met(O)). Assembly of amino acid residues was accomplished by interresidue HMBC correlations and ROESY-based proton−proton cross-peaks between neighboring residues (Figure 1). By this

C NMR data was the presence of several amide carbonyl carbons around δC 170, indicating the peptidic nature of this compound. This interpretation was consistent with the 1H NMR data, in which signals corresponding to exchangeable NH protons and α-carbonyl protons were detected in the regions δH 8.8−7.2 and 4.5−3.8, respectively. Based on this information, the structure of 1 was determined by a combination of COSY, TOCSY, HSQC, and HMBC experiments. First, all of the protons and the carbons attached to the protons were accurately matched by analyzing the HSQC data. Subsequent tracing of proton spin systems by analyzing the COSY and TOCSY data revealed the side chains of several amino acid residues. Then, based on the three-bond couplings with β-protons in the HMBC data, all of the carbonyl carbons were accurately assigned, thus identifying every amino 1427

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434

Journal of Natural Products

Article

Table 1. 13C and 1H NMR Assignments for Compound 1 in DMSO-d6a unit

position

C (ppm, type)

H [δ, mult, (J in Hz)]

unit

cis-Pro

Phe

Met(O) 1 2 3 4 5 6 7 8

169.76, C 60.34, CH 29.90, CH2 20.85, CH2 45.34, CH2 170.96, C 57.64, CH 37.03, CH2

9 10/14 11/13 12 7-NH

137.63, 128.58, 128.42, 126.53,

15 16 17b 17c 18b 18c 16-NH 18-NH2 19b 19c 20 21b 21c 20-NH

171.50, C 49.57, CH 36.05, CH2 36.01, CH2 172.55, C 172.52, C

22b 22c

170.10, C 170.08, C

C CH CH CH

4.43, 2.16, 1.55, 3.24,

dd m; m; m;

(7.4, 2.3) 1.60, m 0.73, m 2.62, m

4.17, ddd (12.1, 7.7, 4.1) 3.17, dd (13.9, 4.0) 3.04, m 7.13, 7.29, 7.22, 8.61,

d (7.4) t (7.4) t (7.4) d (7.7)

4.32, m 3.07, m 3.01, m

52.02, 52.29, 24.72, 24.55, 50.29,

25c

50.28, CH2

26b 26c 23-NHb 23-NHc

38.07, CH3 37.89, CH3

27 28b 28c 29b 29c 28-NHb 28-NHc

172.37,C 47.75, CH 47.49, CH 15.98, CH3 15.95, CH3

30 31 32 33 34 35 31-NHb 31-NHc

170.31, C 56.89, CH 35.36, CH 24.63, CH2 10.67, CH3 14.50, CH3

CH CH CH2 CH2 CH2

H [δ, mult, (J in Hz)] 4.33, 4.31, 2.18, 2.20, 2.78, 2.60, 2.72, 2.69, 2.52, 2.51, 8.23, 8.23,

m m m; 1.98, m m; 1.90, m ddd (13.2, 9.7, 6.5) m ddd (13.1, 10.5, 5.5) ddd (13.1, 10.5, 5.5) s s d (9.6) d (9.6)

4.47, 4.47, 1.16, 1.16, 7.34, 7.35,

dq (7.2, 6.4) dq (7.2, 6.4) d (6.4) d (6.4) d (7.2) d (7.2)

4.00, 1.73, 1.53, 0.81, 0.79, 8.76, 8.76,

dd (8.4, 4.9) m m; 1.28, m t (7.3) d (7.1) d (4.8) d (4.8)

Ile 7.75, d (5.4) 7.82, d (7.5); 7.28, d (7.5)

172.26, C 172.23, C 51.44, CH 16.83, CH3 16.81, CH3

C (ppm, type)

Ala2

Asn

Ala1

position 23b 23c 24b 24c 25b

3.85, 1.32, 1.32, 8.56,

dq (3.0, 7.4) d (7.4) d (7.4) d (3.0)

a

Data were measured at 200 and 800 MHz for 13C and 1H NMR, respectively. b,cData were signals of 1:1 epimers, respectively.

Met(O)

method, the amino acid sequence was determined to be cyclo[Pro-Phe-Asn-Ala1-Met(O)-Ala2-Ile]. This NMR-based assembly was confirmed by high-resolution LC/MS-MS analysis, by which several key fragments were adequately identified (Figure 2 and Supporting Information Figure S32). The 13C and 1H NMR data of compound 1 indicated that 1 was a mixture of two very similar compounds. Based on the results of combined 2D NMR analyses, the assignments of all of the carbons and protons revealed the same planar structure for both constituents (Table 1), suggesting an isomeric relationship between the two constituents. It is unlikely that the compound exists as a mixture of conformers at the Pro1 residue not only because the chemical shifts of the protons and carbons at this residue are the same but also because there is a large shift between the β- and γ-carbons of both constituents (ΔδC‑3−δC‑4 = 9.05 ppm), indicating a cis conformation.15−17 Furthermore, a Marfey’s analysis of 1 to determine the absolute configurations of the amino acid residues assigned L configurations to all the residues (Figures S35 and S36). The distinction between L-Ile and L-allo-Ile was also accomplished by similar analysis, which unambiguously assigned L-Ile for 1 (Figure S40).18 Consequently, the two constituents of 1 were defined to be isomeric at the sulfoxide group of the Met(O) residue, the only remaining stereogenic center, and this result was consistent with the noticeable differences in the NMR chemical shifts around this group (C-23−C-26). This interpretation was

supported by an oxidation of 1 to a sulfone-bearing derivative as a single reaction product, followed by a Marfey’s analysis for the Met(O2) residue (Experimental Section and Figures S30, S31, and S42).19 Thus, the structure of compound 1, designated as phakellistatin 20, was determined to be a mixture of cycloheptapeptides that were epimeric at the sulfoxide. The isolation of both Met(O) epimers from a specimen may indicate an abiotic oxidation of the Met residue during the storage of the sponge specimen or in the separation process.20 A literature study revealed that 1 is structurally related to phakellistatins 5 and 14, in which Met(O) and Asn units of 1 replace Met and β-OMe-Asp of the latter compounds, respectively.21,22 The molecular formula of phakellistatin 21 (2) was deduced to be C60H84N10O14S by HRFABMS analysis. The 13C NMR data of this compound showed signals for 11 carbonyl carbons, suggesting that this compound has the same peptidic nature as 1 but is considerably larger in molecular size. As described for 1, the structure of 2 was determined by combined spectroscopic methods, by which 10 amino acid residues were unambiguously identified to be glutamic acid (Glu), Ile, leucine (Leu), Met(O), Phe, Pro (×4), and tyrosine (Tyr). Subsequent assembly of these units by combined HMBC and ROESY data analysis determined the cyclodecapeptide sequence to be cyclo-[Pro1-Pro2-Met(O)-Phe-Glu-Leu-Pro3Pro4-Tyr-Ile] (Figure 1), which was confirmed by high1428

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434

Journal of Natural Products

Article

determined to be a cyclopeptide closely related to phakellistatin 8 (6) with the difference at the replacement of Ile and Val units with Met(O) and Glu residues, respectively. The molecular formula of phakellistatin 22 (3) was established to be C60H84N10O14S, identical to 2, by HRFABMS analysis. The spectroscopic data of this compound were also very similar to those of 2 (Table 2). A combination of 1D and 2D NMR analyses deduced that these compounds had the same planar structure, which was supported by a highresolution LC/MS-MS analysis to determine the amino acid sequence (Figure 2 and Figure S34). Furthermore, the conformations of the four Pro units of 3 were also found to be the same as those of 2 (ΔδCβ−δCγ = 3.47, 8.91, 3.31, and 8.84 ppm for Pro1−4, respectively, Table 2). Finally, a Marfey’s analysis assigned L configurations to all the amino acid residues, as well as the 57S configuration at the Ile residue in 3 (Figure S41). Thus, the structure of phakellistatin 22 (3) was determined to be an epimer of phakellistatin 21 (2) at the sulfoxide. Having determined the structures of 2 and 3, detailed comparison of the 13C and 1H NMR data revealed noticeable shifts in the peaks of several protons and carbons concentrated at the 14-SO group of the Met(O) unit, suggesting an epimeric relationship between these compounds. The isolation of 2 and 3, two sulfoxide epimers, as an individual compound prompted us to determine the absolute configurations of this stereogenic center. However, the ROESY data for these compounds were virtually identical to each other and failed to provide crucial information for configuration assignment. Additionally, attempts at chemical analysis using dabsyl chloride also failed since the sulfoxide groups of the natural products rapidly isomerized during hydrolysis.24 Furthermore, our CP3-based computational approach also did not produce conclusive results, and the configurations of sulfoxides remain unassigned.25 In addition to phakellistatins 20−22, chromatography-based separation of the extract afforded 10 known cyclopeptides, namely, phakellistatins 2, 4, 8, 9, 11, and 18 (4−9),6,9−12 hymenamide D (10),13 cyclonellin (11),14 and carteritins A and B (12 and 13),15 which possess 2−4 units of Pro as key structural components, suggesting an exceptionally cyclopeptide-rich composition of the tropical S. f labelliformis sponge. Proline-rich phakellistatins are widely recognized due to their significant cytotoxicity, primarily against the P388 leukemia cell line.4,5 However, the somewhat controversial results obtained

Figure 1. Key correlations of COSY and TOCSY (bold), HMBC (arrows), and ROESY (dashed arrows) experiments for compounds 1 and 2.

resolution LC/MS-MS analysis (Figure 2 and Figure S33). Then, the conformations of the Pro units were assigned as trans for Pro1 and Pro3 and cis for Pro2 and Pro4 based on the diagnostic carbon chemical shifts (ΔδCβ−δCγ = 3.43, 8.85, 3.71, and 8.70 ppm for Pro1−4, respectively; Table 2) of these residues. Similar opposite conformations of neighboring Pro units have been found in a number of similar cyclopeptides, such as phakellistatins 7−9 and 12.11,23 Finally, a Marfey’s analysis assigned L configurations to all the amino acid residues, as well as the 57S configuration at the Ile residue in 2 (Figure S41). Thus, the structure of phakellistatin 21 (2) was

Figure 2. High-resolution LC/MS-MS fragmentation analyses of compounds 1−3 (a−c). 1429

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434

Journal of Natural Products

Article

Table 2. 13C and 1H NMR Assignments for Compounds 2 and 3 in DMSO-d6a 2 unit

position

C (ppm, type)

3 H [δ, mult, (J in Hz)]

unit

trans-Pro1

position

C (ppm, type)

H [δ, mult, (J in Hz)]

trans-Pro1 1 2 3 4 5

169.56, C 58.85, CH 28.41, CH2 24.99, CH2 47.66, CH2

3.93, 2.23, 2.00, 3.82,

brs m; 1.68, m m; 1.67, m m; 3.56, m

cis-Pro2

1 2 3 4 5

169.42, C 58.87, CH 28.42, CH2 24.95, CH2 47.74, CH2

3.85, 2.23, 2.00, 3.80,

brs m; 1.68, m m; 1.64, m m; 3.56, m

6 7 8 9 10

171.43, C 60.67, CH 30.97, CH2 22.06, CH2 46.53, CH2

4.42, 2.31, 1.83, 3.42,

d (9.0) m; 2.16, m m; 1.62, m m; 3.37, m

11 12 13 14 15 12-NH

169.99, C 51.32, CH 23.38, CH2 50.46, CH2 38.43, CH3

4.62, 2.19, 2.84, 2.48, 8.85,

brd (2.9) m; 1.90, m m; 2.50, m s brd (6.7)

16 17 18 19 20/24 21/23 22 17-NH

171.21, C 55.38, CH 37.45, CH2 136.17, C 129.27, CH 128.43, CH 126.76, CH

25 26 27 28 29 26-NH

169.97, C 52.70, CH 26.84, CH2 30.59, CH2 174.11, C

30 31 32 33 34 35 31-NH

168.82, C 47.79, CH 41.61, CH2 24.07, CH 23.56, CH3 21.24, CH3

36 37 38 39 40

cis-Pro2 6 7 8 9 10

171.55, C 60.57, CH 30.90, CH2 22.05, CH2 46.48, CH2

11 12 13 14 15 12-NH

170.15, C 51.72, CH 20.50, CH2 47.46, CH2 36.32, CH3

16 17 18 19 20/24 21/23 22 17-NH

171.15, C 55.18, CH 37.39, CH2 136.27, C 129.28, CH 128.42, CH 126.74, CH

25 26 27 28 29 26-NH

169.92, C 52.57, CH 26.75, CH2 30.44, CH2 173.93, C

4.44, 2.32, 1.84, 3.40,

d (8.6) m; 2.14, m m; 1.66, m m (2H)

Met(O)

Met(O) 4.59, 2.12, 2.79, 2.48, 8.85,

brd (5.8) m; 2.00, m m; 2.70, m s brd (7.8)

Phe

Phe 4.21, dd (12.7, 5.9) 3.04, dd (12.7, 5.9); 2.67, m 7.17, 7.31, 7.24, 7.39,

d (7.3) t (7.3) t (7.3) brs

Glu

4.14, m 3.06, m; 2.63, m 7.15, 7.31, 7.24, 7.28,

d (7.3) t (7.3) t (7.3) brs

Glu 4.27, m 2.05, m; 1.69, m 2.12, m; 2.07, m 8.19, brs

Leu

4.25, m 2.03, m; 1.68, m 2.13, m; 2.10, m 8.14, brs

Leu 30 31 32 33 34 35 31-NH

168.78, C 47.76, CH 41.32, CH2 23.98, CH 23.51, CH3 21.27, CH3

36 37 38 39 40

169.66, C 57.92, CH 27.70, CH2 24.38, CH2 46.26, CH2

41 42 43 44 45

170.07, C 60.28, CH 30.31, CH2 21.74, CH2 46.25, CH2

46 47 48

170.48, C 55.35, CH 31.73, CH2

4.56, 1.25, 1.54, 0.86, 0.93, 6.90,

ddd (11.3, 7.3, 2.1) m; 1.18, m m d (6.6) d (6.6) brd (7.3)

trans-Pro3

4.54, 1.22, 1.52, 0.85, 0.93, 6.89,

m m (2H) m d (6.6) d (6.6) brd (6.3)

169.62, C 57.95, CH 27.70, CH2 24.39, CH2 46.18, CH2

3.00, 1.97, 1.85, 3.50,

brs m; 1.41, m m (2H) m; 3.26, m

41 42 43 44 45

170.23, C 60.21, CH 30.59, CH2 21.75, CH2 46.16, CH2

4.10, 2.28, 1.73, 3.28,

d (7.6) m; 1.89, m m; 1.47, m m; 3.26, m

46 47 48

170.51, C 55.31, CH 31.74, CH2

4.24, m 3.20, m 2.84, dd (13.5, 4.8)

trans-Pro3 3.00, 1.97, 1.84, 3.50,

brs m; 1.42, dt (19.3, 8.2) m (2H) m; 3.27, m

cis-Pro4

cis-Pro4 4.09, 2.28, 1.79, 3.29,

d (7.6) m; 1.88, m m; 1.47, m m; 3.25, m

Tyr

Tyr 4.16, m 3.20, m 2.84, dd (13.6, 4.9)

1430

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434

Journal of Natural Products

Article

Table 2. continued 2 unit

position

C (ppm, type)

49 50/54 51/53 52 47-NH 52-OH

128.57, 130.26, 114.25, 155.17,

C CH CH C

55 56 57 58 59 60 56-NH

170.01, C 53.79, CH 37.90, CH 23.93, CH2 10.78, CH3 14.19, CH3

3 H [δ, mult, (J in Hz)]

unit

6.73, d (8.1) 6.44, d (8.1) 8.87, brs 9.11, brs

Ile

a

position

C (ppm, type)

49 50/54 51/53 52 47-NH 52-OH

128.59, 130.25, 114.26, 155.14,

C CH CH C

55 56 57 58 59 60 56-NH

170.00, C 53.66, CH 38.10, CH 23.82, CH2 10.79, CH3 14.20, CH3

H [δ, mult, (J in Hz)] 6.75, d (8.1) 6.45, d (8.1) 8.98, brs 9.11, brs

Ile 4.38, 1.49, 1.57, 0.77, 0.90, 6.82,

t (9.1) m m; 0.89, m t (7.3) d (6.7) brs

4.41, 1.47, 1.57, 0.78, 0.90, 6.58,

t (9.3) brs m; 0.89, m t (7.3) d (6.7) brs

Data were measured at 200 and 800 MHz for 13C and 1H NMR, respectively.

Table 3. Results of Cytotoxicity Tests IC50 (μM) compound

A549

HCT116

K562

MDA-MB-231

SK-HEP-1

SNU638

1 2 3 4 5 6 7 8 9 10 11 12 13 etoposide

15 0.19 0.023 0.66 7.4 0.54 1.4 2.8 6.7 >50 >50 19 >50 0.72

12 0.14 0.011 0.71 4.5 0.10 1.0 1.6 3.9 >50 >50 6.2 >50 0.68

11 0.17 0.021 3.2 30 0.29 1.1 5.6 33 >50 >50 3.9 >50 1.3

12 0.40 0.35 1.3 10 2.5 8.6 3.2 9.1 >50 >50 17 >50 4.4

13 0.041 0.014 0.77 7.2 2.3 12 1.7 7.7 >50 >50 11 >50 0.93

12 0.16 0.010 1.6 24 1.7 2.2 6.9 37 >50 >50 11 >50 0.43

compounds (2−4, 6−9, and 12) bearing three or four Pro units were generally more potent than those bearing one or two Pro units (1, 10, 11, and 13), which may provide more insight on the cytotoxicity of phakellistatins. In addition to cytotoxicity, our measurements of antimicrobial activity and selected enzyme inhibition (isocitrate lyase, Na+/K+-ATPase, sortase A) activities did not exhibit positive results for any of these cyclopeptides (MIC > 128 μM and IC50 > 100 μM for antimicrobial and enzyme-inhibition activity, respectively). In summary, 13 cyclopeptides, including three new cyclopeptides (1−3) of the phakellistatin class, were isolated from the tropical sponge S. f labelliformis. The new compounds were structurally elucidated to be an epimeric mixture of cycloheptapeptides (1) and two epimeric cyclodecapeptides (2 and 3) by a combination of chemical and spectroscopic methods. The cyclopeptides were evaluated for in vitro cytotoxicity against a variety of cancer cell lines, and several compounds exhibited significant inhibition.

with synthetic compounds render this cytotoxicity an unsolved puzzle.5,8 Therefore, the isolation of several cyclopeptides led us to evaluate the cytotoxicity of these cyclopeptides against a number of cancer cell lines. In our measurements using a panel of six cell lines, compounds 2, 3, 4, and 6 exhibited significant cytotoxicity against all of the tested cell lines (Table 3). Among these compounds, the newly isolated 2 and 3 were the most active and were significantly more potent than etoposide, the positive control. Notably, between these sulfoxide-epimeric compounds, 3 was noticeably more active than 2 against several cell lines. In contrast, 1, a mixture of epimeric sulfoxides, was totally inactive against all of the cell lines, indicating the significance of specific amino acid residues for cytotoxicity. A number of known compounds such as 5, 7, 8, 9, and 12 were also active against a number of cell lines. However, the inhibitory activity of these compounds was noticeably weaker than that of etoposide. The lack of cytotoxicity of the known compounds 10, 11, and 13 against all of the cell lines was consistent with the literature.13−15 Despite the substantial amount of data obtained from our tests, crucial information regarding the structure−activity relationship was not determined, including features such as ring size, presence of specific amino acid residues, and conformations of proline residues. An exception would be the number of Pro residues in which



EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a JASCO P-1020 polarimeter with a 1 cm cell. UV spectra were acquired using a Hitachi U-3010 spectrophotometer. IR spectra were recorded on a JASCO 4200 FT-IR spectrometer using a

1431

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434

Journal of Natural Products

Article

purified metabolites were isolated in the following amounts: 163.0, 7.7, 6.5, 3.2, 19.2, 24.2, 12.6, 14.3, 15.5, 12.6, 47.6, 11.3, and 7.6 mg of 1− 13, respectively. Phakellistatin 20 (1): pale yellow, amorphous solid; [α]25 D −47 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (3.48), 266 (2.40) nm; IR (ZnSe) νmax 3383, 2973, 1644, 1520, 1054, 1032, 1011 cm−1; 1H and 13 C NMR data, Table 1; HRFABMS m/z 761.3657 [M + H]+ (calcd for C35H53N8O9S, 761.3656). Phakellistatin 21 (2): pale yellow, amorphous solid; [α]25 D +44 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.69), 277 (2.76) nm; IR (ZnSe) νmax 3331, 2972, 1638, 1510, 1455, 1318, 1055 cm−1; 1H and 13 C NMR data, Table 2; HRFABMS m/z 1201.5966 [M + H]+ (calcd for C60H85N10O14S, 1201.5967). Phakellistatin 22 (3): pale yellow, amorphous solid; [α]25 D +47 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.67), 278 (2.79); IR (ZnSe) νmax 3272, 2966, 1630, 1518, 1454, 1346, 1055 cm−1; 1H and 13 C NMR data, Table 2; HRFABMS m/z 1201.5969 [M + H]+ (calcd for C60H85N10O14S, 1201.5967). Oxidation of Phakellistatin 20 (1). To a solution of 1 (5 mg) in MeOH (5 mL) was added oxone (50 mg in 1 mL of H2O), and the mixture was stirred at room temperature for 2 h. The solution was diluted with H2O, filtered (0.2 μm), and washed with MeOH. Further purification by analytical HPLC (YMC-ODS column, 4.6 × 250 mm; 0.7 mL/min; H2O−MeCN (45:55)) afforded 1-Met(O2), the sulfone derivative (tR = 30.1 min), as a pure compound: 1H NMR (DMSO-d6) δH cis-Pro, 4.42 (1H, d, J = 7.4 Hz), 3.24 (1H, m), 2.62 (1H, m), 2.16 (1H, dd, J = 12.0, 6.6 Hz), 1.60 (1H, m), 1.55 (1H, m), 0.73 (1H, m); Phe, 8.60 (1H, d, J = 7.7 Hz), 7.29 (2H, t, J = 7.4 Hz), 7.22 (1H, t, J = 7.4 Hz), 7.13 (2H, d, 7.4 Hz), 4.17 (1H, m), 3.21−3.02 (2H, m); Asn, 7.82 (1H, brs), 7.74 (1H, d, J = 5.6 Hz), 7.28 (1H, brs), 4.32 (1H, m), 3.07−3.01 (2H, m); Ala1, 8.58 (1H, d, J = 3.2 Hz), 3.85, (1H, dq, J = 3.2, 7.4 Hz), 1.33 (3H, d, J = 7.4 Hz); Met(O2): 8.29 (1H, d, J = 9.6 Hz), 4.33 (1H, m), 3.18−3.00 (2H, m), 2.97 (3H, s), 2.25 (1H, m), 1.98 (1H, m); Ala2, 7.34 (1H, d, J = 7.2 Hz), 4.46 (1H, dq, J = 7.2, 6.7 Hz), 1.16 (3H, d, J = 6.7 Hz); Ile: 8.78 (1H, d, J = 4.6 Hz), 4.00 (1H, dd, J = 8.6, 4.7 Hz), 1.73 (1H, m), 1.53 (1H, m), 0.81 (3H, t, J = 7.3 Hz), 0.79 (3H, d, J = 7.1 Hz); 13C NMR (DMSO-d6) δC cis-Pro, 169.8 (C), 60.3 (CH), 45.3 (CH2), 29.9 (CH2), 20.8 (CH2); Phe, 170.1 (C), 137.6 (CH), 128.6 (CH), 128.6 (CH), 128.4 (CH), 128.4 (CH), 126.5 (CH), 57.6 (CH), 37.0 (CH2); Asn, 172.6 (C), 171.6 (C), 49.6 (CH2), 36.1 (CH2); Ala1, 172.3 (C), 51.6 (CH), 16.8 (CH3); Met(O2), 169.8 (C), 51.6 (CH), 51.0 (CH3), 40.2 (CH2), 24.3 (CH2); Ala2, 172.3 (C), 47.8 (CH), 15.9 (CH3); Ile, 170.3 (C), 56.9 (CH), 35.3 (CH), 24.6 (CH2), 14.5 (CH3), 10.7 (CH3); HRFABMS m/z 777.3615 [M + H]+ (calcd for C35H53N8O10S, 777.3605). Marfey’s Analysis of Compounds 1−3. Each of compounds 1− 3 (0.5 mg each) was hydrolyzed with 6 N HCl (0.5 mL) for 2 h at 110 °C. After cooling to room temperature, the hydrolysate mixtures were evaporated to dryness, and traces of HCl were removed by repeated drying in vacuo. Acid hydrolysates (suspended in 50 μL of H2O) and aqueous solutions (50 mM) of each authentic L- and D-amino acid were used as starting materials for the synthesis of L-DAA-derivatized amino acids. Each portion (50 μL) was treated with 1 M NaHCO3 (20 μL) and then with L-FDAA (100 μL of a 10 mg/mL solution in acetone), and the mixture was heated to 50 °C for 1 h. The reaction was quenched with 1 N HCl (20 μL) and dried under vacuum. The residue was redissolved in MeCN (300 μL) and analyzed by analytical HPLC (YMC ODS-A column, 4.6 × 250 mm, 5 μm; 0.7 mL/min; UV detector (340 nm)) based on retention time (tR, min). The retention times of the derivatized amino acids were measured under two reversed-phase conditions due to polarity considerations: (1) H2O−MeCN gradient (from 75:25 with 0.1% trifluoroacetic acid (TFA) to 35:65 with 0.1% TFA in 40 min); standard amino acids: LMet(O) (12.5 min), D-Met(O) (11.9), L-Asn (14.4), D-Asn (15.0), LAla (18.5), D-Ala (21.4), L-Pro (19.3), D-Pro (20.6), L-Phe (30.0), DPhe (33.4), L-Ile (29.2), D-Ile (34.5); compound 1: L-Met(O) (12.5), L-Asn (14.5), L-Ala (18.7), L-Pro (19.4), L-Phe (30.0), L-Ile (29.2); (2) H2O−MeCN gradient (from 90:10 with 0.1% TFA to 46:54 with 0.1% TFA in 8 min) and then isocratic (46:54 with 0.1% TFA); standard amino acids: L-Met(O) (12.9), D-Met(O) (12.7), L-Glu (13.7), D-Glu

ZnSe cell. NMR spectra were recorded in DMSO-d6, with the solvent peaks δH 2.50/δC 39.50 as internal standards, on a Bruker Avance 800 MHz spectrometer. Proton and carbon NMR spectra were measured at 800 and 200 MHz, respectively. High-resolution FABMS spectrometric data were obtained at the Korea Basic Science Institute and acquired using a JEOL JMS 700 mass spectrometer with metanitrobenzyl alcohol (NBA) as a matrix. High-resolution LC-MS/MS data were obtained at the National Instrumentation Center for Environmental Management (Seoul, Korea) on a Q-TOF 5600 instrument equipped with a Dionex U-3000 HPLC system. HPLC was performed on a SpectraSYSTEM p2000 equipped with a refractive index detector (SpectraSYSTEM RI-150) and a UV−vis detector (Gilson UV−vis-151). All solvents used were of spectroscopic grade or were distilled prior to use. Animal Material. Specimens of the Stylissa f labelliformis sponge (sample number 122CH-703) were collected by hand using scuba offshore of Chuuk Island in the Federated States of Micronesia at a depth of 15 m on February 7, 2012. The red sponge was fan-shaped, 5 mm thick, and up to 23.5 cm long. The skeletal structures and sizes of the spicules were consistent with those of S. f labelliformis (Hentschel, 1912). A voucher specimen (registry no. spo. 74) was deposited in the MBIK (Marine Biodiversity Institute of Korea), Seocheon, 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 (3 × 2 L). The combined extracts (151.1 g) were successively partitioned between H2O (91.9 g) and n-BuOH (54.9 g); the latter layer was repartitioned between H2O−MeOH (15:85, 24.2 g) and n-hexane (30.1 g). Then, the H2O−MeOH layer was separated by C18 reversed-phase flash chromatography using sequential mixtures of MeOH and H2O as the eluents (six fractions in a H2O−MeOH gradient, from 50:50 to 0:100) followed by acetone and finally EtOAc. Based on the results of the 1H NMR spectroscopy and cytotoxicity analyses, the fractions that eluted with 50:50 H2O−MeOH (11.15 g), 40:60 H2O−MeOH (1.40 g), 20:80 H2O−MeOH (0.87 g), and 10:90 H2O−MeOH (0.90 g) were chosen for separation. The 50:50 H2O− MeOH fraction was separated by Sephadex LH-20 gel permeation chromatography, eluting with 1:1 CH2Cl2−MeOH (column size, 500 × 20 mm; fraction size, 20 mL), to yield 15 fractions. Based on the results of the 1H NMR analyses, the second fraction was separated by semipreparative reversed-phase HPLC (YMC-ODS column, 10 × 250 mm; 2.0 mL/min; H2O−MeOH, 55:45), yielding, in order of elution, compounds 13 (tR = 18.7 min), 11 (tR = 22.0 min), and 1 (tR = 34.5 min). The former two compounds were further purified by analytical HPLC (YMC-ODS column, 4.6 × 250 mm; 0.7 mL/min; H2O− MeCN gradient (from 90:10 to 40:60 in 40 min); tR = 14.1 and 19.2 min, respectively). The 40:60 H2O−MeOH fraction was separated by Sephadex LH-20 gel permeation chromatography, eluting with 1:1 CH2Cl2−MeOH, to yield 15 fractions. The first fraction was separated by semipreparative reversed-phase HPLC (2.0 mL/min; H2O−MeCN gradient (from 90:10 with 0.1% formic acid to 100:0 with 0.1% formic acid in 40 min)), yielding compounds 10 (tR = 22.5 min), 2 (tR = 24.1 min), and 3 (tR = 24.6 min). The latter two compounds were further purified by analytical HPLC (0.7 mL/min; H2O−MeOH gradient (from 90:10 with 0.1% formic acid to 0:100 with 0.1% formic acid in 40 min); tR = 29.4 and 29.8 min, respectively). The 20:80 H2O−MeOH fraction was separated by semipreparative reversed-phase HPLC (2.0 mL/min; H2O−MeOH, 35:65), yielding compounds 12 (tR = 21.5 min), 4 (tR = 50.2 min), 9 (tR = 50.3 min), 5 (tR = 66.5 min), and 8 (tR = 64.9 min). The former three compounds were further purified by analytical HPLC (0.7 mL/min; H2O−MeCN, 38:62; tR = 23.4, 28.0, and 35.0 min, respectively). The latter two compounds were also purified by analytical HPLC (YMC−CN column, 4.6 × 250 mm; 0.7 mL/min; H2O−MeOH, 68:32; tR = 32.0 and 37.3 min, respectively). The 10:90 H2O−MeOH fraction was separated by semipreparative reversed-phase HPLC (2.0 mL/min; H2O−MeOH, 27:73), yielding compounds 7 (tR = 26.5 min) and 6 (tR = 30.3 min), which were then purified by analytical HPLC (0.7 mL/ min; H2O−MeOH, 35:65; tR = 34.7 and 42.0 min, respectively). The 1432

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434

Journal of Natural Products

Article

(2) (a) Fusetani, N.; Matsunaga, S. Chem. Rev. 1993, 93, 1793−1806. (b) Lambert, J. N.; Mitchell, J. P.; Roberts, K. D. J. Chem. Soc., Perkin Trans. 2001, 1, 471−484. (c) Kohli, R. M.; Walsh, C. T.; Burkart, M. D. Nature 2002, 418, 658−661. (d) White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509−524. (e) Joseph, B.; Nair, V. M.; Sujatha, S. Int. J. Pharm. Sci. Res. 2012, 3, 4689−4696. (3) Fang, W.-Y.; Dahiya, R.; Qin, H.-L.; Mourya, R.; Maharaj, S. Mar. Drugs 2016, 14, 194. (4) Pettit, G. R.; Cichacz, Z.; Barkoczy, J.; Dorsaz, A.-C.; Herald, D. L.; Williams, M. D.; Doubek, D. L.; Schmidt, J. M.; Tackett, L. P.; Brune, D. C. J. Nat. Prod. 1993, 56, 260−267. (5) Meli, A.; Tedesco, C.; Della Sala, G.; Schettini, R.; Albericio, F.; De Riccardis, F.; Izzo, I. Mar. Drugs 2017, 15, 78. (6) Zhang, H.-J.; Yi, Y.-H.; Yang, G.-J.; Hu, M.-Y.; Cao, G.-D.; Yang, F.; Lin, H.-W. J. Nat. Prod. 2010, 73, 650−655. (7) Recent examples: (a) Ibrahim, S. R. M.; Min, C. C.; Teuscher, F.; Ebel, R.; Kakoschke, C.; Lin, W.; Wray, V.; Edrada-Ebel, R.; Proksch, P. Bioorg. Med. Chem. 2010, 18, 4947−4956. (b) Arai, M.; Yamano, Y.; Fujita, M.; Setiawan, A.; Kobayashi, M. Bioorg. Med. Chem. Lett. 2012, 22, 1818−1821. (c) Aviles, E.; Rodriguez, A. D. Tetrahedron 2013, 69, 10797−10804. (d) Kita, M.; Gise, B.; Kawamura, A.; Kigoshi, H. Tetrahedron Lett. 2013, 54, 6826−6828. (e) Pelay-Gimeno, M.; Meli, A.; Tulla-Puche, J.; Albericio, F. J. Med. Chem. 2013, 56, 9780−9788. (f) Wang, X.; Morinaka, B. I.; Molinski, T. F. J. Nat. Prod. 2014, 77, 625−630. (g) Daletos, G.; Kalscheuer, R.; Koliwer-Brandl, H.; Hartmann, R.; de Voogd, N. J.; Wray, V.; Lin, W.; Proksch, P. J. Nat. Prod. 2015, 78, 1910−1925. (h) Sun, J.; Cheng, W.; de Voogd, N. J.; Proksch, P.; Lin, W. Tetrahedron Lett. 2016, 57, 4288−4292. (8) (a) Pettit, G. R.; Xu, J.-p.; Cichacz, Z. A.; Williams, M. D.; Chapuis, J. C.; Cerny, R. L. Bioorg. Med. Chem. Lett. 1994, 4, 2677− 2682. (b) Tan, L. T.; Williamson, R. T.; Gerwick, W. H.; Watts, K. S.; McGough, K.; Jacobs, R. J. Org. Chem. 2000, 65, 419−425. (c) Saviano, G.; Rossi, F.; Benedetti, E.; Pedone, C.; Mierke, D. F.; Maione, A.; Zanotti, G.; Tancredi, T.; Saviano, M. Chem. - Eur. J. 2001, 7, 1176− 1183. (d) Tabudravu, J. N.; Jaspars, M.; Morris, L. A.; Kettenes-van den Bosch, J. J.; Smith, N. J. Org. Chem. 2002, 67, 8593−8601. (9) Pettit, G. R.; Tan, R.; Williams, M. D.; Tackett, L.; Schmidt, J. M.; Cerny, R. L.; Hooper, J. N. A. Bioorg. Med. Chem. Lett. 1993, 3, 2869− 2874. (10) Pettit, G. R.; Xu, J.-p.; Cichacz, Z.; Schmidt, J. M.; Dorsaz, A.-C.; Boyd, M. R.; Cerny, R. L. Heterocycles 1995, 40, 501−506. (11) Pettit, G. R.; Xu, J.-p.; Dorsaz, A.-C.; Williams, M. D.; Boyd, M. R.; Cerny, R. L. Bioorg. Med. Chem. Lett. 1995, 5, 1339−1344. (12) Pettit, G. R.; Tan, R.; Ichihara, Y.; Williams, M. D.; Doubek, D. L.; Tackett, L. P.; Schmidt, J. M.; Cerny, R. L.; Boyd, M. R.; Hooper, J. N. A. J. Nat. Prod. 1995, 58, 961−965. (13) Tsuda, M.; Shigemori, H.; Mikami, Y.; Kobayashi, J. Tetrahedron 1993, 49, 6785−6796. (14) Milanowski, D. J.; Rashid, M. A.; Gustafson, K. R.; O’Keefe, B. R.; Nawrocki, J. P.; Pannell, L. K.; Boyd, M. R. J. Nat. Prod. 2004, 67, 441−444. (15) Afifi, A. H.; El-Desoky, A. H.; Kato, H.; Mangindaan, R. E. P.; de Voogd, N. J.; Ammar, N. M.; Hifnawy, M. S.; Tsukamoto, S. Tetrahedron Lett. 2016, 57, 1285−1288. (16) Dorman, D. E.; Bovey, F. A. J. Org. Chem. 1973, 38, 2379−2383. (17) Siemion, I. Z.; Wieland, T.; Pook, K.-H. Angew. Chem., Int. Ed. Engl. 1975, 14, 702−703. (18) Hess, S.; Gustafson, K. R.; Milanowski, D. J.; Alvira, E.; Lipton, M. A.; Pannell, L. K. J. Chromatogr. A 2004, 1035, 211−219. (19) (a) Takada, K.; Irie, R.; Suo, R.; Matsunaga, S. J. Nat. Prod. 2017, 80, 2845−2849. (b) Stewart, A. K.; Ravindra, R.; Van Wagoner, R. M.; Wright, J. L. C. J. Nat. Prod. 2018, 81, 349−355. (20) (a) Glass, R. S.; Schöneich, C.; Wilson, G. S.; Nauser, T.; Yamamoto, T.; Lorance, E.; Nichol, G. S.; Ammam, M. Org. Lett. 2011, 13, 2837−2839. (b) Mevers, E.; Byrum, T.; Gerwick, W. H. J. Nat. Prod. 2013, 76, 1810−1814. (c) Kim, G.; Weiss, S. J.; Levine, R. L. Biochim. Biophys. Acta, Gen. Subj. 2014, 1840, 901−905. (d) Chen, M.; Shao, C.-L.; Fu, X.-M.; Kong, C.-J.; She, Z.-G.; Wang, C.-Y. J. Nat. Prod. 2014, 77, 1601−1606.

(13.9), L-Pro (14.6), D-Pro (14.9), L-Ile (17.4), D-Ile (19.8), L-Phe (17.6), D-Phe (19.2), L-Leu (17.8), D-Leu (21.1), L-Tyr (20.0), D-Tyr (23.1); compound 2: L-Met(O) (12.9), L-Glu (13.7), L-Pro (14.6), LIle (17.4), L-Phe (17.5), L-Leu (17.8), L-Tyr (19.9); compound 3: LMet(O) (12.9), L-Glu (13.7), L-Pro (14.6), L-Ile (17.4), L-Phe (17.5), L-Leu (17.8), L-Tyr (19.9). For the β-carbon position of the Ile unit, the hydrolysates were analyzed under different reversed-phase conditions:21 (1) H2O−acetic acid (95:5)−MeCN−MeOH (90:10) gradient (75:25 isocratic in 15.0 min and then from 75:25 to 50:50 in 50 min); standard amino acids: LIle (53.2 min), L-allo-Ile (53.3 min), compound 1: L-Ile (53.1 min); (2) H2O−acetic acid (95:5)−MeCN−MeOH (90:10) gradient (from 65:35 to 55:45 in 50 min); standard amino acids: L-Ile (26.5 min), Lallo-Ile (26.8 min), compound 2: L-Ile (26.6 min); compound 3: L-Ile (26.6 min). The Met(O) residue was further analyzed for the sulfone derivatives, 1-Met(O2) for 1 and similarly prepared ones from 2 and 3 under different reversed-phase conditions: H2O−MeCN gradient (from 60:40 with 0.1% TFA to 50:50 with 0.1% TFA in 30.0 min); standard amino acids: L-Met(O2) (7.44 min), D-Met(O2) (7.35 min), compound 1: L-Met(O2) (7.48 min); compound 2: L-Met(O2) (7.49 min); compound 3: L-Met(O2) (7.49 min). Biological Assays. The cytotoxicity assays were performed in accordance with published protocols.26a Antimicrobial and enzymeinhibition assays were performed according to a method described previously.26b



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00121. 1 H, 13C, and 2D NMR spectra of compounds 1−3 (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]. ORCID

Dong-Chan Oh: 0000-0001-6405-5535 Sang Kook Lee: 0000-0002-4306-7024 Jongheon Shin: 0000-0002-7536-8462 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank the Basic Science Research Institute in Daegu, Korea, for providing mass spectrometric 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 Ministry of Oceans and Fisheries, Korea (Grant PM 58482), and the Medical Research Center (Grant No. 2009-0083533) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning, Korea.



REFERENCES

(1) Blunt, J. W.; Carroll, A. R.; Copp, B. R.; Davis, R. A.; Keyzers, R. A.; Prinsep, M. R. Nat. Prod. Rep. 2018, 35, 8−53, and earlier reports in the series. 1433

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434

Journal of Natural Products

Article

(21) (a) Pettit, G. R.; Xu, J.-p.; Cichacz, Z. A.; Williams, M. D.; Dorsaz, A.-C.; Brune, D. C.; Boyd, M. R.; Cerny, R. L. Bioorg. Med. Chem. Lett. 1994, 4, 2091−2096. (b) Pettit, G. R.; Toki, B. E.; Xu, J.P.; Brune, D. C. J. Nat. Prod. 2000, 63, 22−28. (22) Pettit, G. R.; Tan, R. J. Nat. Prod. 2005, 68, 60−63. (23) Pettit, G. R.; Tan, R. Bioorg. Med. Chem. Lett. 2003, 13, 685− 688. (24) (a) Minetti, G.; Balduini, C.; Brovelli, A. Ital. J. Biochem 1994, 43, 273−283. (b) Gennaris, A.; Ezraty, B.; Henry, C.; Agrebi, R.; Vergnes, A.; Oheix, E.; Bos, J.; Leverrier, P.; Espinosa, L.; Szewczyk, J.; Vertommen, D.; Iranzo, O.; Collet, J.-F.; Barras, F. Nature 2015, 528, 409−412. (25) Smith, S. G.; Goodman, J. M. J. Org. Chem. 2009, 74, 4597− 4607. (26) (a) Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112−1116. (b) Kim, C.-K.; Woo, J.-K.; Kim, S.-H.; Cho, E.; Lee, Y.-J.; Lee, H.-S.; Sim, C. J.; Oh, D.-C.; Oh, K.-B.; Shin, J. J. Nat. Prod. 2015, 78, 2814− 2821.

1434

DOI: 10.1021/acs.jnatprod.8b00121 J. Nat. Prod. 2018, 81, 1426−1434