Two Marine Cyanobacterial Aplysiatoxin Polyketides, Neo

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Two Marine Cyanobacterial Aplysiatoxin Polyketides, Neodebromoaplysiatoxin A and B, with K+ Channel Inhibition Activity Bing-Nan Han,*,†,∥,⊥ Ting-Ting Liang,‡,⊥ Lawrence Jordan Keen,† Ting-Ting Fan,† Xiao-Dan Zhang,† Lin Xu,† Qi Zhao,§ Shu-Ping Wang,∥ and Hou-Wen Lin*,∥ †

Department of Development Technology of Marine Resources, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China ‡ School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China § Faculty of Health Sciences, University of Macau, Macau, China ∥ Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China S Supporting Information *

ABSTRACT: The isolation and structure elucidation of two cyanobacterial debromoaplysiatoxin (DAT) analogues, neodebromoaplysiatoxin A (1) and neo-debromoaplysiatoxin B (2), were reported and found to possess 6/10/6 and 6/6/6 fused-ring systems, respectively, which are rarely seen among aplysiatoxins. Both compounds exhibited potent blocking activity against Kv1.5 with IC50 values of 6.94 ± 0.26 and 0.30 ± 0.05 μM, respectively. These findings suggest the potential of aplysiatoxin analogues in modulating ionic channels and also provide links between the DAT target, protein kinase C, and cell regulation.

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urvival and competition have been the driving force for biological diversity in marine life, enabling marine creatures to develop unique characteristics. Marine cyanobacteria can be seen as exciting examples because of their production of various toxins with different chemotypes,1 including kalkitoxin,2 antillatoxin,3 and aplysiatoxins (ATXs).4−7 ATXs and their related analogues, e.g., oscillatoxins and nhatrangins, are distinct polyketide classes of marine toxins isolated from several cyanobacterial species, including Lyngbya majuscula, Schizothrix calcicola, and Oscillatoria nigro-viridis.7−9 Interestingly, several ATXs are known to possess potent tumor-promoting properties,10,11 and some analogues have shown antiproliferative activity against several human cancer cell lines,12−14 both seemingly through the activation of protein kinase C (PKC).15−17 It is known that PKC activation plays an important role in various regulatory processes through signal pathways involving cell proliferation, differentiation, and apoptosis as well as modulating ion channel functions.23−25 Some potent PKC activators such as bryostatin 1,17 are seen as great therapeutic targets because of the absence of tumor-promoting activity, but their natural availability is limited. Although ATX does show tumor-promoting activity, certain newly synthesized analogues26 show antineoplastic properties with limited tumor promotion. Here we report the isolation and structure elucidation of two new debromoaplysiatoxin (DAT) analogues, neo-debromoaplysiatoxin A (1) and neo-debromoaplysiatoxin B (2) (Figure 1), from the organic extract of a collection of cyanobacteria off the © XXXX American Chemical Society

Figure 1. Structures of 1 and 2.

shore of Hainan Island, China. On the basis of 16S rRNA sequence analysis, this cyanobacterium fell into a stable clade comprising Lyngbya species (Figure S1). Compounds 1 and 2 exhibit unprecedented 6/10/6 and 6/6/6 fused-ring systems, respectively, which are unseen in any other ATXs. Since many toxins can specifically interact with ion channels and ion-pump systems in biomembranes of target cells, we examined the potential of 1 and 2, along with other ATX analogues obtained from this collection, as modulators of ion channel functions. Compounds 1 and 2 are most representative with structures and potassium channel inhibitory activities selectively against Kv1.5 with IC50 values of 6.94 ± 0.26 and 0.30 ± 0.05 μM, respectively. Received: November 29, 2017

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DOI: 10.1021/acs.orglett.7b03672 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Table 1. NMR Data for 1 in Methanol-d4 and 2 in CDCl3 Obtained at 600 and 150 MHz for 1H and 13C, Respectively (J in Hz) 1 1 2 3 4 5a 5b 6 7 8a 8b 9 10 11 12 13a 13b 14a 14b 15 16 17 18 19 20 21 22 23 24 25 26 27 28a 28b 29 30 31 OCH3

2

δH (J)

no. 4.44 s

1.62 d (14.5) 2.08 d (14.5)

2.01 2.16 4.95 1.63 4.18 1.47 1.43

dd (16.0, 4.5) dd (16.0, 2.0) m m dd (10.7, 1.8) m m

1.68 m 1.78 m 4.12 dd (8.8, 4.4) 6.87 dt (7.6, 1.2) 7.15 t (7.8) 6.68 ddd (8.0, 2.6, 1.0) 6.83 0.79 0.75 1.30 0.97 1.25

t (2.1) d (6.4) d (6.9) s s s

2.61 3.07 5.04 3.89 1.21 3.22

dd, (13.5, 4.4) dd (13.5, 8.7) ddd (8.7, 5.5, 4.3) m d (6.4) s

δC, type 169.8, qC 56.9, CH 204.2, qC 75.7, qC 49.8, CH2 42.9, qC 84.2, qC 32.1, CH2 73.1, 34.0, 74.0, 35.4, 31.9,

CH CH CH CH CH2

37.5, CH2 86.0, CH 145.9, qC 119.1, CH 130.3, CH 115.2, CH 158.5, qC 114.3, CH 12.6, CH3 13.5, CH3 24.9, CH3 25.9, CH3 25.4, CH3 172.2, qC 35.7, CH2 74.7, 68.8, 18.8, 57.0,

δH (J)

2.46 m 1.33 dd (13.3, 6.8) 1.83 m

1.79 1.87 4.61 1.66 3.20 1.56 1.46 1.25 1.74 1.53 3.99

dd (13.1, 1.9) dd (13.1, 4.4) m m overlap m m m m m dd (7.4, 5.4)

6.79 brd (7.6) 7.18 t (7.6) 6.74 overlap 6.73 0.81 0.89 0.98 0.86 1.15 3.20

overlap d (6.7) d (6.9) s s d (7.2) s

δC, type 173.6, qC 95.8, qC 181.3, qC 32.1, CH 39.9, CH2 36.1, qC 72.8, qC 30.5, CH2 79.2, CH 36.5, CH 73.6, CH 33.6, CH 30.7, CH2 36.0, CH2 84.2, CH 144.5, qC 119.3, CH 129.7, CH 114.7, CH 156.0, qC 113.3, CH 12.7, CH3 13.7, CH3 23.5, CH3 22.4, CH3 17.9, CH3 56.9, CH3

CH CH CH3 CH3

the above two portions of the structure were determined to be connected through consecutive 1H−1H COSY correlations of H-8/H-9/H-10/H-11/H-12/H-13/H-14/H-15/H3-23 combined with the HMBC correlations from H-8 to C-7 and C10; H-11 to C-9 (δC 73.1) and C-12; and H3-23 to C-10 and C11 (δC 74.0), where C-9 and C-11 are possibly attached with oxides on the basis of the 1H and 13C NMR shift data as well as the structural features of ATXs. Herein, the partially elucidated structure accounted for six degrees of unsaturation. Interpretation of the 1D and 2D NMR spectra indicated the existence of a portion of 3,4-dihydroxyvaleric acid (C-27−C-31), which is connected to the six-membered ring (C-2−C-7) at C-2 through an ester linkage with C-1 (δC 169.8) and C-29 (δC 74.7). According to the NMR data and the molecular formula of compound 1, there remained two ring closures to complete the 10 degrees of unsaturation. Closer comparison of the NMR data of compound 1 and 30-methyloscillatoxin D suggested that a possible oxide linkage of C-7 and C-11 exists in 1 as well, which proposed the presence of tetrahydropyran ring B. Although the ester linkage between C-9 and C-27 was not indicated by the HMBC spectrum, the 1H and 13C NMR data were strongly

Neo-debromoaplysiatoxin A (1) formed colorless crystals and had a molecular formula of C32H46O10 with 10 degrees of unsaturation as established by the HR-ESI-MS ion peak at m/z 613.2991 ([M + Na]+, calcd 613.2989). Its 13C and DEPT NMR spectra exhibited 32 carbon signals (Table 1), attributed to one methoxy, six methyls, five methylenes, 12 methines, and eight quaternary carbons. Interpretation of the 1D and 2D NMR spectra indicated that the planar structure of 1 closely resembles those of DATs, particularly like 30-methyloscillatoxin D. Spectroscopic analysis indicated the presence of a side chain attached with a phenol ring at C-16, a methoxy at C-15, and a methyl at C-12 (C-12 to C-22), which is identical to the corresponding portion in oscillatoxin Ds. HMBC correlations of H-2 to C-3, C-6, and C7; H-5 to C-3, C-4, C-6, C-7, and C-26; H3-24 to C-5, C-6, C-7, and C-25; H3-25 to C-5, C-6, C-7, and C-24; and H3-26 to C-3, C-4, and C-5 displayed the presence of a six-membered ring A with three methyl groups attached (CH3-24, CH3-25, and CH326), and three downfield chemical shifts of C-3 (δC 204.2), C-4 (δC 75.7), and C-7 (δC 84.2) indicated the presence of ketonic, hydroxy, and oxide functionalities on the ring system. Further, B

DOI: 10.1021/acs.orglett.7b03672 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters supportive of this remaining linkage, accounting for the final degree of unsaturation and thereby completing the closure of ring C. Furthermore, the planar structure of 1 was established as depicted in Figure 2 and confirmed by X-ray analysis.

compound 1 and 30-methyloscillatoxin D, 1H and 13C NMR data suggested a possible oxide linkage of C-7 and C-11 to form ring B, resulting in a 6/6/6 fused-ring system for the completed planar structure of 2 (Figure 3). The relative configuration of 2 was determined by detailed analysis of the NOESY spectrum and deduction from the biogenetically relevant configuration of compound 1. As shown in Figure S25, the observed key NOESY correlations of H-4 with H-5a and H3-25 implicated their relative syn relationships in the ring A system as well as the axial positions of H-4 and CH3-25.5 The cis relationship of H-9 with H-8a was deduced from the NOESY correlation, which was consistent with the small couplings of H-9 to H-8a and H-8b (J8a,9 = 1.9 Hz, J8b,9 = 4.4 Hz),5 and the NOESY correlation of H-9 with H-10 suggested their syn relationship oriented to the geometry of ring B. The trans relationships of H3-23 and H-11 to H-9 on ring B were deduced from the NOESY correlations of H3-23 and H-11 as well as their elucidated orientations on ring B.7 In addition, the cis configuration of H-11, H-12, and H3-23 was determined on the basis of their corresponding NOESY correlations and their oriented geometry attached to ring B (Figure S25). The relative configurations at C7 and C15 were proposed to remain the same as in compound 1 on the basis of the correlated structure formation via the same origin seen in our plausible biosynthetic pathway of both compounds (Figure S8). The absolute configuration was deduced by comparison of the experimental circular dichroism (CD) spectrum with the electronic CD (ECD) spectrum calculated using time-dependent density functional theory,20 and the good agreement between the calculated ECD spectrum and the experimental CD spectrum allowed the assignment of 2 as (4R,7R,9S,10S,11R,12S,15S) (Figure S25). A plausible biosynthetic pathway for compounds 1 and 2 starting from debromoaplysiatoxin is postulated in Figure S8. Compounds 1 and 2 did not show apparent cytotoxicity toward HepG2 and PC9 cancer cells at 10 μM, but they exhibited potent inhibitory effects on the potassium channel Kv1.5 expressed in Chinese hamster ovary (CHO) cells with IC50 values of 6.94 ± 0.26 and 0.30 ± 0.05 μM, respectively (Figure 4). The voltage-gated potassium channels (hERG,

Figure 2. Key 2D correlations and X-ray structure of 1.

X-ray diffraction analysis of 1 with Cu Kα radiation resulted in a Flack parameter of 0.07(8) and a Hooft parameter of 0.11(8) for 1926 Bijvoet pairs,18,19 allowing an explicit assignment of the absolute configuration of 1 as (2R,4R,7S,9S,10S,11R,12S,15S,29R,30R), as shown in Figure 2, consistent with the sequential correlations of H3-26/H-5b/H3-25, H-2/H3-24/H-5a, and H12/H-11/H3-23 in the NOESY spectrum (Figure S16). Neo-debromoaplysiatoxin B (2) has a molecular formula of C27H38O6 as determined by the HR-ESI-MS peak at m/z 481.2560 ([M + Na]+, calcd 481.2566), requiring nine degrees of unsaturation. Spectroscopic analysis indicated the presence of a side chain with a phenol ring attached that is identical to the corresponding portion in compound 1 (C-12 to C-22). NMR analysis indicated that the ketone feature at C-3 of compound 1 is replaced by an enol functionality at C-2 (δC 95.8)/C-3 (δC 181.3) and that an ester carbonyl at C-1 (δC 173.6) is attached to C-2 (Figure 3 and Table 1). 1H−1H COSY correlations of H-

Figure 3. Key 2D correlations and ECD spectra showing experimental and calculated analysis of 2.

5/H-4/H3-26 together with HMBC couplings from H-4 to C-2, C-3, and C-5 depicted the partial structure from C-1 to C-5. According to 1D and 2D NMR data, C-5 is connected to a quaternary carbon C-6 substituted with a pair of gem-dimethyl groups (C-25/C-24). As well as this, HMBC correlations of H324 to C-5, C-6, and C-7 and H-5 to C-4, C-6, and C-7 suggested a bond connection between C-6 and C-7. Furthermore, a long consecutive 1H−1H COSY correlation from H-8 to H-15 connected the C-8−C-11 fragment with the portion of the phenol side chain. In addition, HMBC correlations of H-8 to C7 and C-2 connected C-7 with the C-8−C-15 fragment, completing the closure of ring A. The ester linkage between C-9 (δC 79.2) and C-1 (δC 173.6) was proposed by the HMBC corrections of H-9 to C-1. Thereby, a six-membered lactone ring C was shown to be fused with ring A. According to the molecular formula of compound 2, another ring closure was needed to account for the last unsaturated degree. As in

Figure 4. Dose−response study of 1 and 2 with Kv1.5 expression in CHO cells at HP of −80 mV. Data points represent mean ± SEM of three to five measurements. Solid curves are fits to the Hill equation. (A) Inhibitory effect of 1 showed an IC50 value of 6.94 ± 0.26 μM. (B) Inhibitory effect of 2 showed an IC50 value of 0.30 ± 0.05 μM.

Kv1.5, and Kir2.1) and sodium channels (Nav1.5, Nav1.7, and Nav1.8) were tested, among which only Kv1.5 was strongly affected (Table S1 and Figures S2 and S3). Additionally, a comparative study to observe the effects of the two compounds and debromoaplysiatoxin on PKC phosphorylation was undertaken. Interestingly, compound 1 showed strong PKCδ activation mimicking that of debromoaplysiatoxin, C

DOI: 10.1021/acs.orglett.7b03672 Org. Lett. XXXX, XXX, XXX−XXX

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for their contributions to the collection of cyanobacteria, pharmacological experiments, and chemical separation.

but compound 2 did not present any increased phospho-PKCδ expression up to 10 μM (Figure S5). The ultrarapid activating delayed rectifier K+ current (IKur) carried by the Kv1.5 channel is a major repolarizing current in human atria but has no effect in the ventricle.21,22 Thus, Kv1.5 has arisen as an important molecular target for the treatment of atrial tachyarrhythmias with fewer side effects. Aplysiatoxin (ATX) has been known to have tumor-promoting activity, which hampers its therapeutic potential. However, new analogues have been reported to possess antiproliferative effects as well as being strong PKC activators, similar to another marine-derived product, bryostatin 1.17 Here we report for the first time that two new debromoaplysiatoxin (DAT) analogues, neo-debromoaplysiatoxin A (1) and B (2), selectively inhibit Kv1.5 with IC50 values of 6.94 ± 0.26 and 0.30 ± 0.05 μM, respectively. Surprisingly, phosphorylation of some specific K+ channels by protein kinases, such as PKC, can modulate the K+ channel activity.23−25 Therefore, we believe further investigation of the mechanism of compounds 1 and 2 in modulating the potassium activity correlated with PKC activation should be conducted to develop a better understanding of using ATX/ DAT analogues.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03672. Experimental procedures, bioassays, crystallographic data, and spectra (PDF) Accession Codes

CCDC 1570939 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by e-mailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*(B.-N.H.) E-mail: [email protected]. *(H.-W.L.) E-mail: [email protected]. ORCID

Bing-Nan Han: 0000-0003-3616-9409 Qi Zhao: 0000-0002-5969-6407 Hou-Wen Lin: 0000-0002-7097-0876 Author Contributions ⊥

B.-N.H. and T.-T.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Special Fund for Agro-scientific Research in the Public Interest of Zhejiang Province (LGN18C190011), the National Natural Science Foundation of China (Grants 81373321, 41729002, 41476121, 81402844, and U1605221), and the National Key Research and Development Program of China (2016YFF0202300). In addition, we are very grateful to Yan-Kai Jiao, Zhen Yang, and Yang-Hua Tang D

DOI: 10.1021/acs.orglett.7b03672 Org. Lett. XXXX, XXX, XXX−XXX