Dragocins A−D, Structurally Intriguing Cytotoxic Metabolites from a

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Cite This: Org. Lett. 2019, 21, 266−270

Dragocins A−D, Structurally Intriguing Cytotoxic Metabolites from a Panamanian Marine Cyanobacterium Hyukjae Choi,*,† Niclas Engene,‡ Tara Byrum,§ Sunghoon Hwang,∥ Dong-Chan Oh,∥ and William H. Gerwick*,§,# †

College of Pharmacy, Yeungnam University, 280 Daehak-ro, Gyeongsan, Gyeongsangbukdo 38541, Republic of Korea Department of Biological Sciences, Florida International University, Miami, Florida 33199, United States § Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093, United States ∥ Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea # Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, United States

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S Supporting Information *

ABSTRACT: Dragocins A−D (1−4) were isolated from a dark-red wooly textured marine cyanobacterium collected in Boca del Drago, Panama. Dragocins A−C (1−3) possessed 2,3-dihydroxypyrrolidine, 1-hydroxy-5-O-Me-benzoyl, and 4′-substitutedβ-ribofuranose moieties that connected to form a nine-membered macrocyclic ring. Dragocins A−C are members of a unique hybrid structural class with substitution at the C-4′ position of a ribofuranose unit. Of the four new compounds, dragocin A was the most potent cytotoxin to human H-460 lung cancer cells

M

cyclization. Additionally, a few cyanobacterial metabolites are present as glycosides with O-methylated furanose or pyranose sugar substituents, such as in lyngbyaloside,9 malyngamide J,10 and several glycolipid species.11

arine cyanobacteria are an extraordinarily rich source of structurally novel secondary metabolites with a wide range of biological activities, including anti-inflammation, antiinfective, and anticancer.1,2 In particular, a number of cytotoxic compounds have been isolated from this group of prokaryotic microorganisms, and many of them, such as largazole,3 apratoxin F,4 and carmaphycin A,5 are currently in the drug discovery pipeline as development candidates. One successful cyanobacterial example is brentuximab vedotin, an FDA approved anticancer agent for anaplastic large cell lymphoma and Hodgkin’s lymphoma.6 The structure of brentuximab vedotin was inspired, in large part, by the cyanobacterial cytotoxin dolastatin 10.7 Most of the marine cyanobacterial cytotoxins described to date can be categorized as deriving from the nonribosomal peptide synthetase or polyketide synthase pathways, or a complex mixture of these two biosynthetic manifolds.8 Furthermore, many of these are highly modified by peripheral decorations, such as halogenation, methylation, oxidation, and © 2018 American Chemical Society

As part of the Panama International Cooperative Biodiversity Group project,12−14 we investigated the cytotoxic constituents of extracts from various cyanobacterial specimens Received: November 20, 2018 Published: December 19, 2018 266

DOI: 10.1021/acs.orglett.8b03712 Org. Lett. 2019, 21, 266−270

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Organic Letters Table 1. NMR Data for Dragocins A (1) and C (3) in CD3OD at 500 MHz (1H) and 125 MHz (13C) 1 position

δC, type

1a 1b 2 3 4 5 6 7 8 9 10 11 1-N-CH3 9-O-CH3 1′ 2′ 3′ 4′ 5′a 5′b 4′-O-CH3

65.1, CH2 73.0, CH 80.1, CH 76.4, CH 77.6, CH 131.1, C 130.8, CH 115.6, CH 162.9, C 115.6, CH 130.8, CH 45.6, CH3 55.5, CH3 105.0, CH 74.7, CH 79.7, CH 106.0, C 70.3, CH2 50.3, CH3

δH (J in Hz)

3 ROESY

δC, type 65.2, CH2

4, 6, 12

1b, 2 1a, 1-N-CH3 1a, 1b, 3, 1′ 2, 4, 1′ 7, 11, 1-N-CH3 7, 11, 12b, 3′

8 7

5, 6, 9, 11 6, 9, 10

4, 5, 1-N-CH3 9-O-CH3

11 10

11 10

3′ 2′

3′, 2′ 3′, 1′ 2′

6, 9, 8 5, 6, 7, 9 1, 4 9 3, 3′, 4′ 4′

9-O-CH3 4, 5, 1-N-CH3 1b, 4, 5, 11, 5′b 8, 10, 1-N-CH3 2, 3, 9 3′, 1′ 5, 5′a

5′b 5′a

5′b 5′a

5, 3′, 4′ 5, 3′, 4′ 4′

5′b, 4′-O-CH3 5, 5′a, 3′ 5′a, 1′

HMBCa

COSY

TOCSY

1b, 2 1a 1a, 3 2, 4 3, 5 4

1b, 2, 3 1a, 2, 3 1a, 1b, 3, 4, 5 1a, 1b, 2, 4, 5 3, 5 3, 4

7.47, d (8.4) 7.03, d (8.4)

8 7

7.03, 7.47, 2.36, 3.82, 5.27, 4.08, 4.50,

(8.4) (8.4)

(4.5) (4.5)

3.97, 3.17, 4.36, 4.84, 4.04, 4.55,

dd (12.8, 3.3) d (12.8) brd (3.3) m d (10.0, 3.6) d (10.0)

d d s s s d d

3.70, d (13.8) 3.64, d (13.8) 3.38, s

2, 3, 4, 1-N-CH3

72.8, CH 80.7, CH 76.4, CH 77.1, CH 129.4, C 131.0, CH 115.7, CH 162.5, C 115.7, CH 131.0, CH 45.2, CH3 55.7, CH3 106.0, CH 74.6, CH 78.8, CH 107.7, C 75.3, CH2

δH (J in Hz) 3.97, 3.17, 4.36, 4.82, 4.06, 4.70,

dd (12.5, 3.3) d (12.5) brd (3.3) m dd (10.0, 3.0) d (10.0)

7.49, d (8.3) 7.04, d (8.3) 7.04, 7.49, 2.41, 3.83, 5.38, 4.11, 4.77,

d d s s s d d

(8.3) (8.3)

(4.1) (4.1)

4.00, d (13.4) 3.88, d (13.4)

a

From 1H to the indicated 13C.

collected from the Caribbean coast of Panama. In this regard, the extract of a marine cyanobacterium collected near Boca del Drago showed cytotoxicity against H-460 lung cancer cells (2% survival at 30 μg/mL). Subsequent 1H NMR-guided isolation efforts yielded the cytotoxic compounds dragocins A−C (1−3) with intriguing structural features including 4-O-Me or 4-Cl ribofuranose and pyrrolidine subunits, as well as dragocin D (4) which is an anisomycin15 related compound. Herein, we report isolation and structure elucidation of dragocins A−D along with their cytotoxicity to H-460 human lung cancer cells. A dark-red woolly textured marine cyanobacterium was collected by SCUBA near Boca del Drago, Panama (PAB-18May-11−9), and the ethanol-seawater (1:1) material was extracted with CH2Cl2/MeOH (2:1). The specimen was composed of fine filaments with isodiametric cells of ca. 10 μm, a morphology that corresponds with the genus Symploca.16 However, a 16S rDNA phylogenetic analysis (GenBank acc. No. MH795144) indicated that it was not closely related to the Symploca reference strain, but, rather, to uncultured or poorly described specimens (Figure S7). Hence, at this point it is best characterized as an undescribed marine cyanobacterium with a Symploca-like morphology. The crude extract was fractionated into nine primary fractions (A−I) using vacuum liquid chromatography (VLC). The H fraction, eluting with MeOH/EtOAc (1:3), showed brine shrimp toxicity (93% lethality at 50 μg/mL) and cytotoxicity to H-460 human lung cancer cells (2% survival at 30 μg/mL). This fraction was subjected to RP-HPLC with 83% CH3CN to afford four new compounds, dragocins A (1, 1.8 mg, 0.4%), B (2, 1.3 mg, 0.3%), C (3, 4.0 mg, 0.9%), and D (4, 0.6 mg, 0.1%). Dragocin A (1) was isolated as a colorless oil, and the molecular formula was determined as C19H27 NO8 by HRESITOFMS ([M + H]+ m/z 398.1811). The IR spectrum of 1 displayed a prominent absorption band at 3477 cm−1, indicating the presence of hydroxy groups. The 1H and 13C

Figure 1. Planar structures of dragocins A (1) and C (3) based on TOCSY and HMBC analyses.

NMR spectra of 1 in CD3OD showed four deshielded carbons (δC 162.9, 131.1, 130.8, and 115.6) with two sets of two aromatic protons each (δH 7.47 and 7.03), indicating the existence of a para-substituted phenyl ring. Additionally, the NMR data revealed compound 1 to contain two dioxygenated carbon atoms (δC 106.0 and 105.0), six N- or O-substituted methines (δH 5.27, 4.84, 4.55, 4.50, 4.36, 4.08, and 4.04), two N- or O- substituted methylenes (δH 3.97/3.17 and 3.70/3.64), and three methyl singlets (two O-methyl and one N-methyl group at δH 3.82, 3.38, and 2.36, respectively) (Table 1). Analysis of 1D and 2D NMR data, including COSY, TOCSY, HSQC, and HMBC, allowed the construction of the

Figure 2. Key ROE correlations used to assign the relative configuration of dragocins A (1) and C (3). 267

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Organic Letters

observed at 105.0 ppm in the 13C NMR spectrum, indicating the ribofuranose was present as the β-anomer.18 Therefore, the relative configuration of this β-ribofuranose group was determined as 1′R*, 2′R*, 3′S*, and 4′R*. The H-2 proton in the dihydroxypyrrolidine ring was present as a broad doublet in the 1H NMR spectrum, resulting from vicinal coupling with H-1a. Therefore, the vicinal coupling between H-2 and H-3 was necessarily smaller than 2 Hz, indicating a trans relationship between these two adjacent protons (e.g., 2S*, 3S*), as typical of such relationships in five-membered ring carbocycles.17 Additional key ROE resonances between H-1′ with H-3 were observed, resulting in placing H-3, H-1′, H-5′a, and 4′-O-CH3 on the same face of the molecule, while H-5/H5′b/H-3′/H-2′ were revealed to be on the opposite face. Furthermore, a large coupling constant was observed between H-4 and H-5 as well as ROE correlations from 1-N-CH3 to H5 and H-7, thus revealing a 4R*, 5R* relative configuration. Upon compiling all of this stereochemical information, the relative configuration of dragocin A (1) was assigned as 2S*, 3S*, 4R*, 5R*, 1′R*, 2′R*, 3′S*, and 4′R*. Dragocin B (2) was also obtained as a colorless oil, and its LRESIMS showed a molecular ion cluster at m/z 388/390 in a ratio of 3:1, indicating the presence of one chlorine atom. The molecular formula of 2 was determined as C17H22ClNO7 by interpretation of HRESITOFMS data ([M + H]+ m/z 388.1161), and the IR spectrum again displayed absorption bands for multiple hydroxy groups. Thus, the structure of 2 was characterized as an analog of 1 based on the similarity of these data along with a close match of most of the 1H and 13C NMR resonances (see Supporting Information, S7). However, both the 1-N-CH3 and 4′-O-CH3 groups in 1 were absent in the 1H and 13C NMR spectra of 2. While it was clear that the N-Me was replaced by an N-H functionality, the substitution at C-4′ was more cryptic, but ultimately deduced to be the site of chlorination by its chemical shift of δC 109.0 and absence of a proton at this position. Dragocin C (3) also displayed an isotope pattern similar to that of 2 by HRESITOFMS ([M + H]+ m/z 402.1318), indicating a molecular formula of C18H24ClNO7. The 1D and 2D NMR spectra of 3 were almost identical to those of 2, although an additional methyl singlet (H-17, δH 2.41) was found in 3 (Table 1). The HMBC correlation from 1-N-CH3 to C-1 and C-4 in 3 indicated that this methyl group was directly attached to the nitrogen of the pyrrolidine ring, as in 1 (Figure 1). The relative configurations of dragocins B (2) and C (3) were determined to be the same as those of compound 1 based on their close similarity in 13C and 1H chemical shifts, 1H−1H coupling constants, and specific rotation (Supporting Information). The 13C and 1H chemical shift differences at C-5′, C-4′, H-5′, H-4′, and H-3′ between 1−3 could result from a stronger electrostatic repulsion by the chlorine atom in 2 and 3 relative to the oxygen atom of 1. The relative configuration of 3 was confirmed by a thorough analysis of its ROESY correlations as described above for dragocin A (1) (Figure 2). The 1D and 2D NMR spectra of dragocin D (4) were relatively simple compared to those of metabolites 1−3. Specifically, all of the proton and carbon resonances assignable to the substituted furanose unit in 1−3 were absent in compound 4, and thus, its structure was assigned as the aglycone metabolite, as depicted in Figure 1. The relative configuration of dragocin D (4) is proposed as a result of the close comparability of 13C and 1H chemical shifts, 1H−1H

Figure 3. Proposed biosynthetic origin of dragocins A (1), C (3), and D (4).

Figure 4. Cytotoxicity of the dragocins A−D (1−4) against the H460 human lung cancer cell line at 30 and 3.0 μg/mL.

planar structure of dragocin A (1) (Figure 1). A spin system of C-1/C-2/C-3/C-4/C-5 was observed in both the COSY and TOCSY spectra, and the HMBC correlations from 1-N-Me to C-1 and C-4 led to the construction of an N-Me-2,3dihydroxypyrrolidine unit. HMBC correlations from H-7 to C-5/C-6 and from H-5 to C-6 indicated that the parasubstituted phenyl ring was connected to oxygenated carbon C-5. An additional spin system could be constructed by COSY and TOCSY correlations between an oxygenated methine (H2′, δH 4.08) with one bis-oxygenated methine (H-1′, δH 5.27) and one oxygen-bearing methine (H-3′, δH 4.50). HMBC correlations from an additional oxygen-bearing methylene (H5′, δH 3.70 and 3.64) to a quaternary dioxygenated carbon (C4′), along with additional HMBC correlations from H-1′, H-2′, and H-3′ to C-4′, allowed construction of a C-4′ substituted furanose ring. The bis-oxygenated methine (H-1′) and the oxygenated ring methylene (H-5′), representing the two termini of the furanose moiety showed HMBC correlations to C-3 and C-5, respectively, and supported the presence of a nine-membered macrocyclic ring in dragocin A (Figure 1). The positions of the two O-methyl groups were assigned by HMBC correlations from the 9-O-CH3 (δH 3.82) to C-9 and from the 4′-O-CH3 (δH 3.38) to C-4′, as well as the 13C chemical shifts of C-9 (δC 162.9) and C-4′ (δC 106.0) (Figure 1). The relative configuration of the stereocenters in dragocin A (1) were examined using coupling constants and ROE correlations. Because H-1′ was observed as a broadened singlet in the 1H NMR spectrum, the 3JH‑1′, H‑2′ was deduced to be less than 2 Hz, indicating a trans relationship of H-1′ and H-2′.17 By ROESY, the 4′-O-CH 3 resonance showed correlations with H-5′a and H-1′, while H-5′b showed correlations with both H-3′ and H-5 (Figure 2). Based on these NMR features, the sugar unit in dragocin A (1) was revealed to be a ribofuranose. The anomeric carbon, C-1′, was 268

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the pure compounds, accurate IC50 values could not be established, and the brine shrimp toxicity of the pure compounds was not determined. Although structurally related compounds such as AB3217-A have been reported to possess antimite activity, the dragocins were found to have no activity in this assay (data not shown).

coupling constants, and specific rotation to those of compounds 1−3 and anisomycin.15 Because the supply of dragocins A−D (1−4) was limited, the absolute configurations were deduced by comparing the calculated specific rotations with the observed values. Calculation of this value for dragocin A (1) was performed with the configuration 2S, 3S, 4R, 5R, 1′R, 2′R, 3′S, and 4′R. The observed and calculated specific rotations were of the same sign and close to each other in magnitude (calculated [α]D −50.6; observed [α]D25 −28.3), indicating this was the correct stereoconfiguration. Similarly, the absolute configurations of the other dragocins were assigned as 2S, 3S, 4R, 5R, 1′R, 2′R, 3′S, 4′S for 2 and 3 and 2S, 3S, 4R, 5R for 4, signifying that the sugar units of 1−3 are derived from 4′substituted β-D-ribofuranose. Structurally, dragocins A−C (1−3) are related to the known antimite compound AB3217-A isolated from Streptomyces platensis AB3217.19 AB3217-A possesses a D-xylose rather than D-ribose sugar, and its two analogs have acyl groups attached to the 2′-OH of D-ribose. By contrast, the dragocins A−C (1−3) have intriguing structural features, namely an OMe or Cl attached to the C-4′ position of D-ribose. In fact, to our knowledge, this is the first report of a C-4′-substituted ribofuranose-containing natural product. Dragocin D (4), the structurally truncated analog of dragocins A and C, is similar to the known antibiotic anisomycin, isolated from Streptomyces griseolus.15 Compared to the structure of anisomycin, dragocin D (4) has an additional N-Me group, a hydroxy group at the C-5 position, and lacks an acetoxy group at the 3-OH position. Dragocins A−C (1−3) are members of a new structure class that appears to derive from a mixed biosynthetic pathway containing modules of NRPS, PKS, and carbohydrate metabolism. These metabolites are also modified by N- and O-methylation as well as halogenation, all of which are frequently observed tailoring reactions in cyanobacterial secondary metabolites.8 The biogenesis of dragocin D (4) likely begins with N- and O-methylation of a tyrosine residue, β-hydroxylation, followed by PKS extension with acetate, and cyclization to the pyrrolidone derivative. The oxidation levels of two carbons (C-1 and C-2) need adjustment; this may occur via a dehydration−rehydration sequence (Figure 3). Perhaps the most interesting structural feature in dragocins A−C (1− 3) is the presence of a ribose moiety attached to C-3 of dragocin D (4) and subsequent macrocyclic ring formation between C-5 and C-5′. It is alluring to consider phosphoribosyl pyrophosphate (PRPP), the most common activated form of ribose, as a potential intermediate in this process, as it provides rational mechanisms for both of these bond connections (Figure 3). However, less clear is how oxidization at C-4′ occurs with either an additional pendant methoxy- or chloro- group. While for the former a 2,3,5-trihydroxy-4-oxopentanal is a conceivable intermediate, along with S-adenosylmethionine (SAM), to form a 4-methoxy form of PRPP, the corresponding chlorocompound is more difficult to rationalize. The VLC fraction containing the dragocins (34.6 mg) showed potent cytotoxicity against H-460 human lung cancer cells (2% survival at 30 μg/mL) and against brine shrimp (7% survival at 50 μg/mL). The cytotoxicity of each of the purified dragocins was subsequently tested at both 3 and 30 μg/mL. Among the four compounds, dragocin A (1) was the most potent [23% survival at 3 μg/mL (7.6 μM) and 10% survival at 30 μg/mL (75.5 μM), Figure 4]. Due to the limited supply of



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03712.



Full experimental details and spectroscopic data (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hyukjae Choi: 0000-0002-7707-4767 Dong-Chan Oh: 0000-0001-6405-5535 William H. Gerwick: 0000-0003-1403-4458 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research is supported by the Panama ICBG project (NIH FIC U01 TW006634) and CA100851 and by the National Research Foundation of Korea grant (NRF-2017R1A2B4006110).



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