Cytotoxic Microcolin Lipopeptides from the Marine Cyanobacterium

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Cytotoxic Microcolin Lipopeptides from the Marine Cyanobacterium Moorea producens Hao-Bing Yu,†,‡ Evgenia Glukhov,‡ Yueying Li,‡ Arihiro Iwasaki,‡,§ Lena Gerwick,‡ Pieter C. Dorrestein,⊥ Bing-Hua Jiao,† and William H. Gerwick*,‡,⊥

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Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, People’s Republic of China ‡ Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093, United States § Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan ⊥ Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States S Supporting Information *

ABSTRACT: Nine new linear lipopeptides, microcolins E−M (1−9), together with four known related compounds, microcolins A−D (10−13), were isolated from the marine cyanobacterium Moorea producens using bioassay-guided and LC-MS/MS molecular networking approaches. Catalytic hydrogenation of microcolins A−D (10−13) yielded two known compounds, 3,4-dihydromicrocolins A and B (14, 15), and two new derivatives, 3,4-dihydromicrocolins C and D (16, 17), respectively. The structures of these new compounds were determined by a combination of spectroscopic and advanced Marfey’s analysis. Structurally unusual amino acid units, 4-methyl-2-(methylamino)pent-3-enoic (Mpe) acid and 2-amino-4methylhexanoic acid (N-Me-homoisoleucine), in compounds 1−3 and 8, respectively, are rare residues in naturally occurring peptides. These metabolites showed significant cytotoxic activity against H-460 human lung cancer cells with IC50 values ranging from 6 nM to 5.0 μM. The variations in structure and attendant biological activities provided fresh insights concerning structure−activity relationships for the microcolin class of lipopeptides.

M

As a continuation of our studies on microcolins and related compounds from marine cyanobacteria,9,20 we investigated a specimen of M. producens that contained new microcolins as detected through the Global Natural Products Social (GNPS) Molecular Networking tool. This led to the isolation of nine new linear lipopeptides, microcolins E−M (1−9), together with four known ones, microcolins A−D (10−13). Moreover, four microcolin derivatives were produced in this study, namely, 3,4-dihydromicrocolins A−D (14−17), which were obtained via catalytic hydrogenation of microcolins A−D (10− 13) and contributed to a growing appreciation of structure− activity relationships in this molecular class. Herein, we report the details of the purification, structure elucidation, and cytotoxic activity evaluation of these linear lipopeptides, as well as new insights into their structure−activity relationships.

arine cyanobacteria have been a rich source of linear lipopeptides,1 such as minnamides,2 microcolins,3 biseokeaniamides,4 grassystatins,5 almiramides,6 and dragonamides,7 and these have a range of biological properties such as antitumor,4,5 immunosuppressive,3 enzyme-inhibitory,4,5 antiparasitic,6 and antimalarial.7 In particular, microcolins A and B showed potent cytotoxic and immunosuppressive activities with IC50 or EC50 values in the subnanomolar to nanomolar range.8 Moreover, the inhibitory activity of microcolin A was time-dependent and reversible and was not associated with a reduction in cell viability.8 To date, several microcolin derivatives have been isolated from Moorea producens (formerly Lyngbya majuscula), including microcolins A− D,3,9−11 majusculamide D,12 and deoxymajusculamide D.12 The potent biological activities of the microcolins13 have inspired their total synthesis,14,15 including production of structural analogues16,17 and structure−activity studies.18,19 Recently, our group synthesized the microcolin analogue majusculamide D, as this compound was found to possess significant cytotoxicity toward PANC-1 (pancreatic carcinoma) and U251N (glioblastoma) cell lines in vitro.20 © XXXX American Chemical Society and American Society of Pharmacognosy



RESULTS AND DISCUSSION A sample of the cyanobacterium M. producens collected from Playa Kalki, Curaçao, in September 10, 1997, was repeatedly Received: June 14, 2019

A

DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

spectrum (Table 1), seven deshielded signals (δC 168.2, 169.8, 169.9, 169.9, 170.8, 172.0, and 177.1) corresponding to amide or ester carbonyl carbons were detected.3 Furthermore, the 1H NMR spectrum (Table 1) of 1 was similar to that of 11 with two singlet N-methyl signals (δH 2.92 and 3.13), five α-protons of amino acid residues (δH 4.97−5.87), one deshielded NH proton signal (δH 6.96), one oxymethine (δH 5.27), and three double-bond protons (δH 5.53, 6.07, and 7.24). The most significant feature differentiating compound 1 from 11 was the appearance of one additional doublet signal (δH 5.53, d, J = 8.0 Hz) that was adjacent to a deshielded α-proton signal (δH 5.87, d, J = 8.0 Hz). These data, together with careful analysis of the MS/MS fragmentation patterns of 1 and 11, suggested that there was a double bond in the leucine (Leu) residue in 1. This partial structure was confirmed by the COSY correlation between H-28 (δH 5.87) and H-30 (δH 5.53) and HMBC correlations from H-28 to C-27 (δC 170.8), C-30 (δC 116.8), and C-31 (δC 141.5) and from H3-32 (δH 1.64) and H3-33 (δH 1.82) to C-30 and C-31. Consistent with the structure of compound 11, interpretation of the COSY and HMBC spectra for 1 revealed the presence of 5-methyl-1,5-dihydro-2H-pyrrol2-one (Mdp), proline (Pro), N-methylvaline (N-Me-Val), 3acetylthreonine (3-Ac-Thr), 4-methyl-2-(methylamino)pent-3enoic acid (Mpe), and 2,4-dimethyloctanoic acid (Dma). The sequences of these partial structures in 1 were determined on the basis of HMBC and NOESY data (Figure 2). The NOESY correlation between H-4 (δH 4.77) and H-8 (δH 5.47) and HMBC correlations from H-4 and H-8 to C-7 (δC 172.0) revealed that an amide bond bridged these two residues. The α-proton of N-Me-Val (δH 5.05) showed NOESY correlation with H-11b (δH 3.80) and HMBC correlations from H-11b to C-13 (δC 168.2) and from H-14 to C-13, revealing the connection between Pro and N-Me-Val. The connectivity between N-Me-Val and 3-Ac-Thr was determined by HMBC correlation from H3-19 (δH 3.13) to C-20 (δC 169.8) and NOESY correlation between H3-19 and H-21 (δH 4.98). Similarly, HMBC correlations were also observed from H-21 to C-27 (δC 170.8) and from H3-34 (δH 2.92) to C-28 (δC 54.5) and C-35 (δC 177.1), establishing the two additional amide linkages between these three amino acids. Thus, the

extracted with CH2Cl2/MeOH (2:1), and the resulting extract was fractionated by silica gel vacuum liquid chromatography (VLC) to produce nine subfractions (A−I). The GNPS Molecular Networking technique revealed a distinct cluster of microcolin nodes (Figure 1).21 The two most polar fractions

Figure 1. MS/MS-based molecular network of the microcolins encountered in this study. The cosine level was adjusted to 0.7. Nodes display the measured masses of the molecular ions. The different colors of the nodes represent the different fractions containing the various microcolins. The size of the node is reflective of the relative amount of the indicated compound.

(H and I), which showed cytotoxicity against H-460 cancer cells at 1 μg/mL, were combined and further purified by reversed-phase solid-phase extraction (SPE) and HPLC and analyzed by LC/MS to afford nine new lipopeptides, microcolins E−M (1−9), together with the known microcolins A−D (10−13). Microcolin E (1) was obtained as a colorless oil and had a molecular formula of C39H63N5O8 based on HRESIMS (m/z 752.4560 [M + Na]+) and NMR data, which is smaller than that of microcolin B (11) by 2 amu.3 In the 13C NMR B

DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C NMR Spectroscopic Data of 1 and 2 in CDCl3 1a

position 1 2 3 4 6 7 8 9a 9b 10 11a 11b 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 30 31 32 33 34 35a 36 37a 37b 38 39a 39b 40 41 42 43 44

residue Mdp

Pro

2a

δC, type 169.9, C 125.5, CH 153.8, CH 58.1, CH 17.2, CH3 172.0, C 60.0, CH 28.9, CH2 24.6, CH2 48.0, CH2

N-Me-Val

3-Ac-Thr

Mpe

Dma

168.2, C 59.2, CH 27.3, CH 18.4, CH3 18.9, CH3 30.5, CH3 169.8, C 52.3, CH 68.9, CH 17.2, CH3 169.9, C 21.0, CH3 170.8, C 54.5, CH 116.8, CH 141.5, C 18.7, CH3 25.8, CH3 31.6, CH3 177.1, C 33.3, CH 41.7, CH2 30.6, CH 36.9, CH2 29.1, 22.9, 14.2, 18.1, 19.7,

CH2 CH2 CH3 CH3 CH3

δH (J in Hz) 6.07, 7.24, 4.77, 1.47,

dd (6.0, 2.0) dd (6.0, 2.0) qt (7.0, 2.0) d (6.5)

5.47, 1.87, 2.44, 1.97, 3.70, 3.80,

dd (8.5, 5.5) m m m m m

5.05, 2.27, 0.81, 1.00, 3.13,

d (6.0) m d (6.5) d (6.5) s

4.98, 6.96, 5.27, 1.19,

dd (9.0, 4.0) d (9.0) m d (6.5)

position 1 2 3 4 6 7 8 9a 9b 10 11a 11b 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 30 31 32 33 34 35 36 37a 37b 38 39a 39b 40 41 42 43 44

2.02, s 5.87, d (8.0) 5.53, d (8.0) 1.64, s 1.82, s 2.92, s 2.83, 1.11, 1.86, 1.27, 1.09, 1.30, 1.26, 1.29, 0.89, 1.14, 0.83,

m m m m m m m m t (6.5) d (6.5) d (6.5)

residue Mdp

4-OH-Pro

δC, type 169.9, C 125.3, CH 154.2, CH 58.1, CH 16.9, CH3 174.6, C 59.3, CH 36.6, CH2 71.8, CH 56.9, CH2

N-Me-Val

3-Ac-Thr

Mpe

Dma

168.9, C 59.7, CH 27.1, CH 18.4, CH3 18.8, CH3 30.4, CH3 169.8, C 52.1, CH 68.5, CH 17.4, CH3 169.9, C 21.0, CH3 170.8, C 54.1, CH 116.3, CH 141.5, C 18.7, CH3 25.7, CH3 31.5, CH3 177.1, C 33.8, CH 41.7, CH2 29.8, CH 36.9, CH2 29.1, 22.9, 14.1, 18.1, 19.6,

CH2 CH2 CH3 CH3 CH3

δH (J in Hz) 6.08, 7.27, 4.81, 1.46,

dd (6.0, 2.0) dd (6.0, 2.0) qt (6.5, 2.0) d (6.5)

5.66, 2.02, 2.47, 4.37, 3.81, 3.86,

dd (10.0, 2.0) m m brm dd (11.5, 4.5) m

5.02, 2.25, 0.82, 0.98, 3.01,

d (6.0) m d (6.5) d (6.5) s

4.96, 7.00, 5.25, 1.18,

dd (6.5, 3.5) d (9.0) q (3.5) d (7.0)

2.00, s 5.85, d (7.5) 5.54, d (8.0) 1.63, s 1.81, s 2.91, s 2.82, 1.10, 1.84, 1.28, 1.07, 1.31, 1.26, 1.26, 0.87, 1.12, 0.81,

m m m m m m m m t (6.5) d (6.5) d (6.5)

a

Data recorded at 500 MHz (1H NMR) and 125 MHz (13C NMR).

this is the first occurrence of the uncommon Mpe residue in a naturally occurring peptide. Microcolin F (2) was also isolated as a colorless oil, and its molecular formula was determined as C39H63N5O9 based on HRESIMS and NMR data; this is larger than compound 1 by 16 amu. The NMR data of compound 2 (Table 1) were almost identical to those of 1, indicating the same carbon skeleton except for the presence of an oxymethine group (δH/δC 4.37/ 71.8). COSY correlations of H-8 (δH 5.66)/H-9b (δH 2.47), H-9b/H-10 (δH 4.37), and H-10/H-11a (δH 3.81) and NOESY correlations of H-4 (δH 4.81)/H-8 and H2-11 (δH 3.81 and 3.86)/H-14 (δH 5.02) confirmed the presence of a 4OH-proline (4-OH-Pro) residue in 2, replacing the Pro residue

residue sequence in 1 could be assigned as Mdp-Pro-N-MeVal-3-Ac-Thr-Mpe-Dma, and this was confirmed by analysis of the MS-MS fragmentation pattern (Figure 3). The configuration at C-4, C-36, and C-38 was suggested to be 4S, 36R, 38R by comparison of the proton chemical shifts and J values to that of natural microcolins,3 as well as to that of synthetic material whose absolute configuration was assigned in a previous study.14 Subsequently, advanced Marfey’s analysis of 1 identified L-Pro, L -N-Me-Val, and L-Thr. 22 The configuration of the nonproteinogenic residue Mpe in 1 was established as L by first hydrogenation (H2/Pd) followed by hydrolysis and Marfey’s analysis (Figure S10).23 Remarkably, C

DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. COSY and key HMBC and NOESY correlations for compounds 1−9.

Figure 3. MS/MS fragmentations for compounds 1−11.

δC 21.0 and 169.9) in 2. A careful analysis of the MS/MS fragmentation pattern for 3 confirmed that a hydroxy group was attached to C-23 (Figure 3). The absolute configurations of the stereocenters in 3 were determined to be the same as in 2 by acid hydrolysis followed by the same Marfey’s analysis as 2 (Figure S27).22 Microcolin H (4) was isolated as a colorless oil with a molecular formula of C38H63N5O9 as determined by HRESIMS and NMR data. Comparison of the 1H NMR data of 4 (Table 2) with those of microcolin A (10) revealed that these two compounds were very similar,3 except for the absence of a methyl doublet (δH 0.82, d, 7.2 Hz) in 4. Taken together with the fragment ion peak at m/z 268.34 in 4 versus m/z 282.18 in

in 1. The absolute configuration at C-4, C-36, and C-38 of 2 was determined to be 4S, 36R, 38R based on the same analysis as described above for compound 1.24 Comparison of the proton−proton coupling constants for the 4-OH-Pro unit matched those for cis-4-OH-L-Pro.12,25 Moreover, the acid hydrolysate of the hydrogenation of 2 was analyzed by Marfey’s method22 and confirmed the presence of cis-4-OH-LPro, L-N-Me-Val, 3-Ac-L-Thr, and L-Mpe (Figure S19). Microcolin G (3) was isolated as a colorless oil and assigned the molecular formula C37H61N5O8, based on NMR and HRESIMS data for the [M + Na]+ ion at m/z 726.4405. The 1 H and 13C NMR spectra of 3 (Table 2) were highly similar to those of 2, except for the absence of the acetyl group (δH 2.00, D

DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C NMR Spectroscopic Data of 3 and 4 in CDCl3 3a

position 1 2 3 4 6 7 8 9a 9b 10 11a 11b 13 14 16 17 18 19 20 21 22 23 24 25 26 28 29 30 31 32 33 34 35a 35b 36 37a 37b 38 39a 39b 40 41 42

residue Mdp

4-OH-Pro

δC, type 169.9, C 125.3, CH 154.4, CH 58.2, CH 16.8, CH3 174.6, C 58.4, CH 36.5, CH2 71.9, CH 57.1, CH2

N-Me-Val

Thr

Mpe

Dma

168.9, C 59.3, CH 27.4, CH 18.4, CH3 18.2, CH3 30.5, CH3 172.8, C 52.3, CH 67.4, CH 18.2, CH3 171.0, C 54.7, CH 116.8, CH 141.6, C 18.7, CH3 25.7, CH3 31.7, CH3 177.3, C 33.5, CH 41.6, CH2 29.5, CH 37.0, CH2 29.3, CH2 22.9, CH2 14.2, CH3 18.3, CH3 19.6, CH3

4b δH (J in Hz) 6.09, 7.28, 4.82, 1.46,

d (5.0) d (5.0) qt (5.5, 1.5) d (6.0)

5.66, 2.02, 2.44, 4.41, 3.80, 3.95,

d (8.0) m m brm dd (9.0, 3.0) m

5.01, 2.27, 0.81, 0.99, 3.07,

d (9.0) m d (5.5) d (5.5) s

4.76, 7.94, 4.09, 1.12,

d d d d

position 1 2 3 4 6 7 8 9a 9b 10 11a 11b 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 30a 30b 31 32 33 34 35 36 37a 37b 38 39 40 41 42 43

(7.5) (8.0) (5.0) (5.0)

5.77, d (6.5) 5.54, d (7.0) 1.62, s 1.81, s 2.94, s 2.81, 1.41, 1.83, 1.27, 1.06, 1.31, 1.26, 1.29, 1.26, 0.87, 1.08, 0.82,

m m m m m m m m m t (5.5) d (5.5) d (5.5)

residue Mdp

4-OH-Pro

δC, type 169.7, C 125.4, CH 154.2, CH 58.1, CH 16.2, CH3 174.6, C 58.6, CH 36.6, CH2 71.9, CH 57.0, CH2

N-Me-Val

3-Ac-Thr

N-Me-Leu

Moa

168.6, C 59.1, CH 27.2, CH 18.4, CH3 18.8, CH3 30.4, CH3 170.2, C 51.9, CH 68.4, CH 16.9, CH3 169.7, C 21.1, CH3 171.2, C 53.8, CH 35.8, CH2 24.8, CH 21.7, CH3 23.4, CH3 30.5, CH3 177.9, C 36.2, CH 34.2, CH2 27.4, 29.3, 31.8, 22.6, 14.1, 17.6,

CH2 CH2 CH2 CH2 CH3 CH3

δH (J in Hz) 6.08, 7.27, 4.81, 1.46,

d (5.4) d (6.0, 2.0) qt (7.2, 2.4) d (6.6)

5.66, d (9.6) 2.01, m 2.47, m 4.37, brm 3.81,dd (11.4, 4.2) 3.86, m 5.01, 2.26, 0.81, 0.98, 3.09,

d (10.8) m d (6.6) d (6.6) s

4.94, 6.98, 5.24, 1.15,

dd (8.4, 2.4) d (8.4) m d (6.6)

1.99, s 5.27, 1.57, 1.72, 1.44, 0.86, 0.94, 2.94,

(7.2, 1.0) m m m d (6.6) d (6.6) s

2.71, 1.40, 1.74, 1.40, 1.42, 1.41, 1.44, 0.88, 1.13,

m m m m m m m t (6.6) d (6.6)

a

Data recorded at 500 MHz (1H NMR) and 125 MHz (13C NMR). bData recorded at 600 MHz (1H NMR) and 150 MHz (13C NMR).

to literature data for synthetic (R)-2-methyloctanoic acid ([α]25 D −15.3, MeOH, c 1.6) and (S)-2-methyloctanoic acid ([α]25 D +16.2, MeOH, c 1.1) (differences in measured versus literature values for (R)-2-methyloctanoic acid likely stem from inaccuracies in the weight of this hydrolysis fragment).26 Subsequently, the remaining stereocenters in 4 were determined to be the same as those in 10 by Marfey’s analysis and a closely similar NMR data set (Figure S36).22 The molecular formula of microcolin I (5), isolated as a colorless oil, was found to be C38H63N5O8 by HRESIMS and NMR data. In combination with the NMR data, the 14 mass unit difference between 5 and 11 and 16 mass unit difference between 5 and 4 suggested that compound 5 lacked the C-38 methyl group but was otherwise identical to microcolin B (11).

10, compound 4 was deduced to possess a 2-methyloctanoic acid (Moa) group attached to N-29 instead of the Dma group in 10. This deduction was supported by COSY correlations between H2-37 (δH 1.40 and 1.74)/H-36 (δH 2.71), H-36/H343 (δH 1.13), and H2-41 (δH 1.44)/H3-42 (δH 0.88), as well as HMBC correlations from H-28 (δH 5.27), H3-34 (δH 2.94), H2-37, and H3-43 to C-35 (δC 177.9), from H2-37 to C-38 (δC 27.4) and C-39 (δC 29.3), from H2-38 (δH 1.40) to C-39 and C-40 (δC 31.8), and from H2-41 and H3-42 to C-40 (Figure 2). To establish the absolute configuration at C-36 in compound 4, the specific rotation of the 2-methyloctanoic acid was determined after hydrolysis and HPLC purification ([α]25 D −2.4, MeOH, c 0.1). Thus, the C-36 stereocenter in the Moa unit could be assigned as R configuration by comparison E

DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C NMR Spectroscopic Data of 5 and 6 in CDCl3 5a

position 1 2 3 4 6 7 8 9a 9b 10 11a 11b 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 30 31 32 33 34 35 36 37a 37b 38 39 40 41 42 43

residue Mdp

Pro

6a

δC, type 169.8, C 125.5, CH 153.8, CH 58.1, CH 17.2, CH3 172.1, C 60.0, CH 28.9, CH2 24.6, CH2 47.9, CH2

N-Me-Val

3-Ac-Thr

N-Me-Leu

Moa

169.0, C 59.2, CH 27.4, CH 18.4, CH3 18.8, CH3 30.5, CH3 169.7, C 52.0, CH 68.7, CH 17.2, CH3 169.9, C 21.0, CH3 171.3, C 53.9, CH 35.9, CH2 24.8, CH 21.7, CH3 23.3, CH3 30.4, CH3 177.9, C 36.2, CH 34.2, CH2 27.3, 29.3, 31.8, 22.6, 14.1, 17.6,

CH2 CH2 CH2 CH2 CH3 CH3

δH (J in Hz) 6.06, 7.23, 4.76, 1.46,

dd (6.0, 1.2) dd (6.0, 2.4) qt (6.6, 1.8) d (6.5)

5.46, 1.85, 2.42, 1.94, 2.02, 3.71, 3.79,

dd (8.4, 5.4) m m m m m m

position 1 2 3 4 6 7 8 9a 9b 10 11a 11b 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 29a 29b 30 31 32 33 34 35 36a 36b 37 38 39 40 41 42 43

5.03, d (10.8) 2.26, 0.80, 1.15, 3.11,

m d (6.6) d (6.6) s

4.96, 6.94, 5.24, 1.15,

dd (9.0, 3.6) d (9.0) m d (6.6)

2.00, s 5.26, dd (10.2, 6.0) 1.60, m 1.70, m 1. 41, m 0.87, d (6.6) 0.94, d (6.6) 2.93, s 2.71, 1.38, 1.73, 1.23, 1.26, 1.25, 1.28, 0.88, 1.23,

m m m m m m m t (6.6) d (6.6)

residue Mdp

Pro

δC, type 169.8, C 125.5, CH 153.9, CH 58.0, CH 17.2, CH3 173.8, C 60.0, CH 28.8, CH2 24.7, CH 47.9, CH2

Val

3-Ac-Thr

N-Me-Leu

Dma

168.4, C 58.0, C 27.1, CH 18.2, CH3 18.9, CH3 170.0, C 53.0, CH 69.8, CH 17.2, CH3 169.8, C 21.1, CH3 171.6, C 53.9, CH 35.5, CH2 24.8, CH 21.7, CH3 23.3, CH3 30.4, CH3 177.3, C 33.8, CH 42.1, CH2 30.8, 37.1, 29.1, 22.9, 14.2, 18.2, 19.5,

CH CH2 CH2 CH2 CH3 CH3 CH3

δH (J in Hz) 6.07, 7.24, 4.76, 1.43,

dd (6.0, 1.8) dd (6.0, 1.8) qt (6.6, 1.8) d (7.2)

5.46, 1.87, 2.46, 2.02, 3.70, 3.81,

d (8.4, 6.0) m m m m m

4.60, 6.82, 2.10, 0.87, 1.00,

dd (6.0, 8.4) dd (9.6, 4.2) m d (6.0) d (6.6)

4.51, 7.06, 5.38, 1.11,

dd (9.0, 2.4) d (8.4) m d (6.6)

2.01, s 5.24, 1.62, 1.75, 1.43, 0.83, 0.95, 2.94,

dd (10.2, 6.0) m m m d (6.0) d (6.6) s

2.83, 1.21, 1.84, 1.27, 1.26, 1.25, 1.23, 0.88, 1.08, 0.89,

m m m m m m m t (6.6) d (7.2) d (6.6)

a

Data recorded at 600 MHz (1H NMR) and 150 MHz (13C NMR).

between compounds 6 and 11 showed them to be identical through the majority of the structure,3 except for the absence of an N-Me group (δH/δC 2.95/30.4). The N-Me-Val residue in compound 11 was thus deduced to be replaced by a valine (Val) residue in 6; this was supported by COSY correlations between NH-15 (δH 6.82)/H-14 (δH 4.60), H-14/H-16 (δH 2.10), H-16/H3-17 (δH 0.87), and H-16/H3-18 (δH 1.00). COSY, HMBC, and NOESY correlations and MS/MS fragmentations confirmed the residue sequence in compound 6 to be Mdp-Pro-Val-3-Ac-Thr-N-Me-Leu-Dma. A combination of highly similar NMR data sets and the advanced Marfey’s method were used to determine that the absolute

This could be confirmed by diagnostic COSY, NOESY, and HMBC correlations (Figure 2) that established the sequence Mdp-Pro-N-Me-Val-3-Ac-Thr-N-Me-Leu-Moa; this was further supported by key MS/MS fragmentations (Figure 3). Following Marfey’s analysis and NMR data comparisons with compounds 4 and 11, the absolute configuration of microcolin I (5) could be determined as shown (Figure S45). Microcolin K (6) was obtained as a colorless oil and had a molecular formula of C38H63N5O8 based on HRESIMS (m/z 740.4567 [M + Na]+) and NMR data, which is smaller than that of microcolin B (11) by 14 amu. Indeed, a careful comparison of 1H and 13C NMR chemical shifts (Table 3) F

DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 4. 1H and position 1 2 3 4 6 7 8 9a 9b 10 11a 11b 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 29 31a 31b 32a 33 34 35 36 37 38a 38b 39 40a 40b 41 42 43 44 45

13

Article

C NMR Spectroscopic Data of 7 and 8 in CDCl3

residue Mdp

4-OH-Pro

δC, type 169.9, C 125.4, CH 154.1, CH 58.1, CH 16.9, CH3 174.7, C 58.6, CH 36.6, CH2 71.9, CH 57.0, CH2

N-Me-Val

3-Pr-Thr

N-Me-Leu

Dma

168.9, C 59.2, CH 27.1, CH 18.8, CH3 18.8, CH3 30.3, CH3 169.8, C 51.8, CH 68.4, CH 17.4, CH3 173.3, C 27.4, CH2 9.1, CH3 171.3, C 53.7,CH 35.7, CH2 24.8, CH 21.5, CH3 23.4, CH3 30.4, CH3 177.9, C 33.8, CH2 41.9, CH2 30.7, CH 37.1, CH2 29.1, 22.9, 14.1, 18.2, 19.5,

CH2 CH2 CH3 CH3 CH3

δH (J in Hz) 6.08, 7.27, 4.81, 1.46,

dd (6.0, 1.2) dd (6.0, 1.2) qt (7.2, 2.4) d (6.5)

5.80, 2.04, 2.48, 4.37, 3.80, 3.86,

d (6.0) m m brm dd (11.4, 3.6) m

5.01, 2.25, 0.81, 0.98, 3.09,

d (11.4) m d (6.6) d (6.6) s

4.95, 6.98, 5.25, 1.24,

dd (8.4, 1.2) d (8.4) m d (6.6)

position 1 2 3 4 6 7 8 9a 9b 10 11a 11b 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 30a 30b 31 32a 32b 33 34 35 36 37 38a 38b 39 40a 40b 41 42 43 44 45

2.28, q (7.8) 1.09, t (7.8) 5.28, 1.57, 1.74, 1.40, 0.84, 0.94, 2.95,

dd (13.2, 7.8) m m m d (6.6) d (6.6) s

2.84, 1.09, 1.86, 1.33, 1.08, 1.28, 1.25, 1.27, 0.88, 1.11, 0.86,

m m m m m m m m t (6.6) d (6.6) d (6.6)

residue Mdp

4-OH-Pro

δC, type 169.9, C 125.4, CH 154.2, CH 58.1, CH 16.9, CH3 174.7, C 58.6, CH 36.6, CH2 71.9, CH 57.0, CH2

N-Me-Val

3-Ac-Thr

Hil

168.9, C 59.2, CH 27.1, CH 18.4, CH3 18.8, CH3 30.3, CH3 169.8, C 51.8, CH 68.4, CH 17.4, CH3 169.7, C 21.1, CH3 171.3, C 53.7, CH 40.0, CH2 31.1, CH 28.4, CH2

Dma

11.0, CH3 19.3, CH3 30.4, CH3 177.9, C 33.8, CH 41.9, CH2 30.7, CH 37.1, CH2 29.1, 22.9, 14.1, 18.2, 19.5,

CH2 CH2 CH3 CH3 CH3

δH (J in Hz) 6.08, 7.27, 4.80, 1.46,

dd (6.0, 1.8) dd (6.6, 2.4) qt (6.6, 1.8) d (6.6)

5.66, 2.03, 2.47, 4.37, 3.79, 3.86,

dd (4.2, 1.8) m m brm dd (11.4, 3.0) m

5.01, 2.25, 0.81, 0.98, 3.09,

d (10.8) m d (6.6) d (7.2) s

4.94, 7.01, 5.25, 1.15,

dd (9.0, 3.0) d (9.0) m d (6.6)

1.99, s 5.27, 1.64, 1.74, 1.21, 1.04, 1.42, 0.82, 0.91, 2.96,

dd (10.2, 6.0) m m m m m t (6.6) t (6.6) s

2.84, 1.08, 1.88, 1.25, 1.09, 1.30, 1.26, 1.27, 0.88, 1.12, 0.84,

m m m m m m m m t (6.6) d (6.6) d (6.6)

a

Data recorded at 600 MHz (1H NMR) and 150 MHz (13C NMR).

confirmed by COSY correlation between H3-27 (δH 1.09, t, 7.8) and H2-26 (δH 2.28, q, 7.8), as well as HMBC correlation from H2-26 to C-25 (δC 173.3). Diagnostic 2D NMR (Figure 2) correlations and MS-MS fragmentations (Figure 3) established the residue sequence as shown. These data, together with the NMR and Marfey’s analysis, permitted assignment of the structure of compound 7 as shown (Figure S59). Microcolin K is biosynthetically interesting because propionate is a rare metabolic building block in marine cyanobacteria.27

configurations of all stereocenters in 6 were identical to those of 11 (Figure S51). Microcolin J (7), a colorless oil, showed a sodium ion adduct by HRESIMS that was consistent with a molecular formula of C40H67N5O9 [e.g., an additional CH2 compared with microcolin A (10)] and confirmed using NMR data. However, Marfey’s analysis of 7 (Figure S65) identified the same suite of amino acid residues as for 10. Comparison of the 1D NMR data (Table 4) for 7 with 10 revealed the only significant difference as the replacement of the acetyl group resonances with those for a propionyl (Pr) group. This was G

DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 5. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data of 9 in CDCl3 position

residue

δC, type

1 2 3a 3b 4 5a 5b 7 8 10 11 12 13 14 15 16 17 18 19 20

4-OH-pro

169.9, C 58.5, CH 35.6, CH2 70.7, CH 55.3, CH2

N-Me-Val

3-Ac-Thr

170.1, C 58.7, CH 27.2, CH 18.6, CH3 18.8, CH3 30.5, CH3 169.9, C 51.9, CH 67.9, CH 17.5, CH3 169.7, C 21.1, CH3

δH, (J in Hz) 4.75, 2.32, 2.45, 4.45, 3.67, 3.85,

brm m m brm m m

4.93, 2.22, 0.87, 0.96, 3.16,

d (9.5) m d (6.0) d (6.5) s

4.94, 7.07, 5.24, 1.15,

d d d d

position

residue

δC, type

21 22 24a 24b 25 26 27 28 29 30 31a 31b 32 33a 33b 34 35 36 37 38

N-Me-Leu

171.4, C 53.7, CH 35.6, CH2

(9.5) (9.0) (6.5) (6.5)

1.95, s

δH, (J in Hz)

24.7, CH 21.5, CH3 23.4, CH3 30.3, CH3 178.1, C 33.8, CH 41.9, CH2

Dma

30.7, CH 37.1, CH2 29.1, 22.9, 14.1, 18.2, 19.5,

CH2 CH2 CH3 CH3 CH3

5.28, 1.54, 1.77, 1.40, 0.84, 0.91, 2.96,

dd (10.5, 5.0) m m m t (6.5) t (6.5) s

2.84, 1.10, 1.88, 1.29, 1.07, 1.23, 1.24, 1.25, 0.88, 1.12, 0.85,

m m m m m m m m t (6.5) d (6.5) d (6.5)

resonances for the Mdp unit were missing. The sequence and connectivity of amino acid residues, determined as 4-OH-ProN-Me-Val-3-Ac-Thr-N-Me-Leu-Dmo, were supported by NOESY correlation between H-5b (δH 3.85) and H-8 (δH 4.93) and HMBC correlations from H-8, H3-13 (δH 3.16), and H-15 (δH 4.94) to C-14, from NH-16 (δH 7.07) and H-22 (δH 5.28) to C-21 (δC 171.4), and from H-22, H3-28 (δH 2.96), and H3-37 (δH 1.12) to C-29 (δC 178.1). The configuration at C-30 and C-32 was unambiguously deduced to be 30R,32R by comparison of the proton chemical shifts and J values to that of Dma in the microcolins.3,14 The remaining amino acid residues in 9 were all determined to be L-configured using the advanced Marfey’s analysis (Figure S77).22 In addition to the nine new compounds 1−9, the four known microcolins A−D (10−13) were also isolated and identified.3 Additionally, four semisynthetic derivatives, 3,4dihydromicrocolins A−D (14−17), were also obtained and characterized in the course of these studies.18 All the isolated compounds, except 8, were evaluated against the H-460 non-small-cell lung carcinoma cell line. All compounds, except 3,4-dihydromicrocolin C (16) (IC50 value >30 μM), showed excellent cytotoxicity (Table 6), with IC50 values in the range of 6 nM to 5.0 μM. Comparing the relative potency of these derivatives revealed that a hydroxy

Microcolin L (8), also a colorless oil, showed by HRESIMS an [M + Na]+ ion at m/z 784.4826, suggestive of a molecular formula of C40H67N5O9 [e.g., a second analogue with an additional CH2 group compared to microcolin A (10)]; this formula was confirmed using NMR data. Comparison of the 1D NMR data (Table 4) and MS/MS data for 8 with 10 (Figure 3) revealed the loss of resonances for the N-Me-Leu residue and the appearance of resonances for an N-Mehomoisoleucine (N-Me-Hil) residue.27,28 This deduction was supported by COSY correlations between H-28 (δH 5.27)/H230 (δH 1.62 and 1.74), H2-30/H-31 (δH 1.21), H-31/H2-32 (δH 1.04 and 1.42), H2-32/H3-33 (δH 0.82), and H-31/H3-34 (δH 0.91), as well as HMBC correlations from H-28 to C-27 (δC 171.3) and C-36 (δC 177.9) and from H3-35 (δH 2.96) to C-28 (δC 53.7) and C-36. Extensive NMR analysis and Marfey’s amino acid analysis (Figure S67) revealed compound 8 to possess 4S-Mdp, cis-4-OH-L-Pro, L-N-Me-Val, 3-Ac-L-Thr, and 36R,38R-Dma units.21 In order to determine the configuration of the uncommon Hil amino acid unit in 8, two groups of stereoisomers [group 1 = (2S,4S)-N-Me-Hil + (2R,4S)-N-Me-Hil; group 2 = (2S,4S)-N-Me-Hil + (2R,4S)-NMe-Hil + (2S,4R)-N-Me-Hil + (2R,4R)-N-Me-Hil] were synthesized.28,29 Moreover, the (2S,4S)-N-Me-Hil was slightly predominant over the (2R,4S)-N-Me-Hil in group 1.28 Also, the retention time of the (2S,4S)-N-Me-Hil-D-FDAA isomer is longer than the (2S,4S)-N-Me-Hil-D-FDAA isomer.30 The configuration of the Hil amino acid unit was thus determined to be 2S,4S by this Marfey’s analysis (Figures S67 and S68). Microcolin M (9), a colorless oil, analyzed for C34H60N4O9 by HRESIMS of the sodium adduct ion at m/z 691.4250 and confirmed using NMR data. The 1H and 13C NMR data (Table 5) showed typical microcolin characteristic signals: two singlet N-methyl signals (δH 2.96 and 3.16), four α-protons (δH 4.75, 4.93, 4.94, and 5.28), two oxymethines (δH 4.45 and 5.24), one deshielded NH signal (δH 7.07), and six amide or ester carbonyls (δC 169.9−178.1). These data together with MS/MS fragmentations identified most of the same amino acid residues in compound 9 as in other microcolins, namely, 4OH-Pro, N-Me-Val, 3-Ac-Thr, N-Me-Leu, and Dmo; however,

Table 6. Cytotoxic Activity of 1−17 to H-460 Lung Cancer Cells (IC50, nM)

a

H

compound

H-460

compound

H-460

1 2 3 4 5 6 7 8 9

1000 ± 20 37 ± 4 160 ± 20 47 ± 5 550 ± 50 69 ± 10 200 ± 50 not tested 510 ± 60

10 11 12 13 14 15 16 17 doxorubicin

6±1 910 ± 70 650 ± 30 75 ± 10 2800 ± 700 5000 ± 160 −a 2000 ± 140 180 ± 10

“−” indicates inactive with an IC50 value of >30 μM. DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

493.0 mg). The most cytotoxic fractions against the H-460 cell line were Fr. H and I. Fraction H exhibited 96.2% and 86.7% inhibition at 1 and 10 μg/mL, respectively, while fraction I exhibited 95.5% and 88.4% inhibition at 1 and 10 μg/mL, respectively. These subfractions were prepared as described previously for GNPS Molecular Networking.22 Briefly, the crude extract and fractions A−I were dissolved at 0.3 mg/mL in MeOH and passed through a Bond ElutC18 OH cartridge (Agilent Technologies, USA). A 20 μL aliquot of each sample was injected and analyzed via LC-UV-MS/MS (monitoring 200−600 nm and m/z 20−1000 in positive ion mode) eluted at 0.6 mL/min by a gradient program of CH3CN/H2O (0.1% formic acid): 0−2 min for 30%, 2−22 min for 30% to 99%, 22−26 min for 99%, 26−26.5 min for 99% to 30%, 26.5−30 min for 30%. The MS/MS spectra of the nine fractions were used to generate a molecular network using the GNPS Web site and visualized using Cytoscape 3.7 software for the purpose of dereplication and molecular targeting. All data have been uploaded to the MassIVE Database at the GNPS Web site (https://gnps.ucsd.edu/ProteoSAFe/static/gnpssplash2.jsp) and is publicly available through access number MSV000083976. Accordingly, fractions H and I were selected for further isolation studies because they had microcolin network nodes and clusters, and they exhibited the highest levels of cancer cell cytotoxicity. Combination of fractions H and I resulted in a new fraction named “NH” (693.0 mg); this was dissolved in 40% MeOH/60% H2O and subjected to chromatography over a C18 SPE and eluted sequentially with 40% MeOH/60% H2O (Fr. NH1, 62.7 mg), 60% MeOH/40% H2O (Fr. NH2, 156.3 mg), 80% MeOH/20% H2O (Fr. NH3, 221.7 mg), 100% MeOH (Fr. NH4, 177.3 mg), and 100% CH2Cl2 (Fr. NH5, 43.8 mg). Fr. NH3 was further purified by HPLC using a Kinetex C18 semipreparative column and isocratic elution using 65% CH3CN/30% H2O at the flow rate of 2 mL/min, which yielded five subfractions: Fr. NH3A (10.0−12.5 min, 7.2 mg), Fr. NH3B (14.0− 15.0 min, 9.9 mg), Fr. NH3C (15.5−17.5 min, 13.5 mg), Fr. NH3D (19.0−22.0 min, 60.3 mg), and Fr. NH3E (contained compound 11, 30.0−32.5 min, 13.6 mg). Subfraction NH3A was further separated by reversed-phase HPLC with the Kinetex C18 semipreparative column eluting with 45% CH3CN to afford compound 3 (2.0 mL/min, 233 nm, tR = 39.5 min, 0.2 mg). Subfraction NH3B was also separated by reversed-phase HPLC using a YMC column eluting with 80% CH3CN to afford compounds 13 (2.0 mL/min, 233 nm, tR = 35.4 min, 2.4 mg) and 9 (2.0 mL/min, 233 nm, tR = 37.9 min, 0.9 mg). Subfraction NH3C was purified by reversed-phase HPLC using a YMC column eluting with 90% CH3CN/10% H2O, detected at 269 nm, giving compound 4 (0.7 mg) at 11.8 min and compound 2 (0.7 mg) at 12.4 min. Subfraction NH3D was also purified by reversedphase HPLC using a YMC column with an elution of 85% CH3CN detected at 233 nm to give compounds 10 (2.0 mL/min, tR = 19.8 min, 12.9 mg) and 12 (2.0 mL/min, tR = 20.5 min, 2.1 mg). Similarly, Fr. NH4 was further purified by reversed-phase HPLC using a Kinetex C18 semipreparative column and isocratic elution using 80% CH3CN/20% H2O at a flow rate of 2.0 mL/min, which yielded three subfractions: Fr. NH4A (13.0−15.5 min, 7.2 mg), Fr. NH4B (22.0−23.6 min, 9.9 mg), and Fr. NH4C (also compound 11, 30.2− 33.5 min, 1.5 mg). Subfraction NH4A was purified by reversed-phase HPLC using a YMC column with 95% CH3CN/5% H2O (2.0 mL/ min), detected at 233 nm, giving compounds 5 (0.7 mg) at 12.8 min, 6 (0.1 mg) at 13.4 min, and 7 (0.2 mg) at 14.3 min. Subfraction NH4B was also purified by a reversed-phase HPLC YMC column with an elution of 95% CH3CN detected at 233 nm to give compounds 1 (2.0 mL/min, tR = 0.9 min, 0.4 mg) and 8 (2.0 mL/ min, tR = 15.2 min, 0.3 mg). 1 Microcolin E (1): colorless oil; [α]25 D −27 (c 0.10, MeOH); H and 13 + C NMR data, Table 1; HRESIMS m/z 752.4560 [M + Na] (calcd for C39H63N5O8Na, 752.4569). 1 Microcolin F (2): colorless oil; [α]25 D −27 (c 0.10, MeOH); H and 13 + C NMR data, Table 1; HRESIMS m/z 768.4515 [M + Na] (calcd for C39H63N5O9Na, 768.4518).

group at C-4 of Pro and a double bond in the Mdp moiety are important for this biological activity, an observation consistent with previously reported results.18 Losing the N-methyl group from the Val residue as found in compound 6 compared with 11 led to increased cytotoxic activity (from 910 nM in 11 to 69 nM in 6); this may be an important design feature to consider for increasing the potency of synthetic microcolin analogues. In addition, the presence of an acetyl group at C-3 of Thr (e.g., comparing compounds 2 with 3, and 10 with 13) improved the cytotoxic activity. However, by contrast a propionate group at this position decreased the cytotoxic activity [compound 7 is 32-fold less potent than microcolin A (10)]. Moreover, the addition of a double bond in the N-Me-Leu residue of microcolins E−G (1−3) correlated with a loss of inhibitory activity. However, elimination of one of the pendant methyl groups in the fatty acid side chain had relatively little effect on the cytotoxicity and thus does not likely have a significant role in determining the bioactivity of the series. In general, the significant cancer cell cytotoxic activities of these new microcolins support their continued investigation so as to develop a deeper understanding of structure−activity relationships and mechanism of cytotoxic action.



EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation data were recorded using a JASCO P-2000 polarimeter. UV was measured using a diode array detector (model Survegor PDA Plus). 1D NMR and 2D NMR spectra were acquired at room temperature using a Bruker Avance III DRX-600 NMR (compounds 4−8) with a 1.7 mm dual tune TCI cryoprobe and a JEOL ECZ 500 NMR spectrometer (compounds 1−3 and 9) equipped with a 3 mm inverse detection probe with tetramethylsilane as the internal standard. HRESIMS data were obtained using an Agilent 6530 Accurate-Mass QTOF mass spectrometer (positive ion mode). All nominal mass resolution LC/ MS data were obtained on a Phenomenex Kinetex C18 analytical column (100 × 4.6 mm × 5 μm, Phenomenex) using a Thermo Finnigan Surveyor Autosampler-Plus/LC-Pump-Plus/PDA-Plus system and a Thermo Finnigan LCQ Advantage Plus mass spectrometer. Reversed-phase HPLC was performed on a Kinetex C18 semipreparative column (150 × 10.0 mm × 5 μm, Phenomenex) or YMC column (YMC-Pack Pro C18 RS, 5 μm) using a Thermo Fisher Scientific HPLC system comprising a Thermo Dionex UltiMate 3000 pump, RS autosampler, RS diode array detector, and automated fraction collector. Column chromatographic purifications were performed using silica gel (type H, 10−40 μm, Sigma-Aldrich) and C18 SPE (5000 mg/20 mL, SEClute). All solvents were HPLC grade as purchased from Thermo Fisher Scientific. Cyanobacterial Material. The cyanobacteria samples were collected off Playa Kalki, Curaçao (N 12°22′32.4, W 69°09′28.3; 17−18 m depth), in September 10, 1997, by scuba diving, and identified as a red-colored Moorea producens on the basis of morphological characteristics. The organism (no. NAK10SEP97-01; voucher specimen available from the corresponding author) was preserved in 1.0 L of 1:1 seawater/2-propanol solution, transported to the laboratory, and frozen at −20 °C until extraction. Extraction, Molecular Networking, and Isolation. The preserved cyanobacterium was filtered through cheesecloth, and the biomass extracted repeatedly by soaking in 1.0 L of 2:1 CH2Cl2/ MeOH with warming (99%, and this was confirmed by NMR data. The cells (purchased from ATCC in August 2017) were routinely monitored by microscopy and assessed for microbial contamination. Batches of cells were frozen in DMSO and subsequently used in no more than 28 passages for each batch. The cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 1% penicillin/ streptomycin. The suspended cells (180 μL) at an initial density of 3.33 × 104 cells/mL were seeded into each well of a 96-well microplate and allowed to adhere for 24 h before drug addition. Before staining with MTT (thiazolyl blue tetrazolium bromide 98%; Sigma-Aldrich), the cells were treated with the indicated test compounds for 48 h. Doxorubicin and 1% DMSO in RPMI 1640 without fetal bovine serum were used as positive and negative controls, respectively. Pure compounds were initially dissolved in DMSO and brought to a starting concentration of 30 μM, and then nine more working solutions were made through serial dilution using a factor of 0.3164 with RPMI 1640 media without phosphate-buffered saline. The optical density (OD) of the stained lysate was measured at 630 and 570 nm in a 96-well SpectraMax M12 microplate reader from Molecular Devices. IC50 values were obtained from the curves of average OD values of the triplicate tests versus drug concentrations using GraphPad Prism 8.1.2. For the most potent compounds, the initial screening concentrations were reduced in additional experiments in order to build full dose response curves.



Spectrometry Facility) for some of the HRESIMS data and B. Duggan for assistance with NMR technical support.



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00549.



REFERENCES

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1D and 2D NMR and HRESIMS spectra for compounds 1−9 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: (858)-534-0578. E-mail: [email protected]. ORCID

Hao-Bing Yu: 0000-0003-0516-4664 Arihiro Iwasaki: 0000-0002-3775-5066 Lena Gerwick: 0000-0001-6108-9000 Pieter C. Dorrestein: 0000-0002-3003-1030 William H. Gerwick: 0000-0003-1403-4458 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was supported by the National Key Research and Development Program of China (No. 2018YFC0310900) (H.B.Y.), the National Institutes of Health CA100851 (W.H.G.), and the International Postdoctoral Exchange Fellowship Program (No. 20170092) (H.B.Y.). We also thank Y. Su (UCSD Chemistry and Biochemistry Mass K

DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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DOI: 10.1021/acs.jnatprod.9b00549 J. Nat. Prod. XXXX, XXX, XXX−XXX