Asperphenins A and B, Lipopeptidyl Benzophenones from a Marine

Apr 7, 2017 - Asperphenins A (1) and B (2), novel diastereomeric lipopeptidyl benzophenone metabolites, were isolated from a marine-derived Aspergillu...
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Asperphenins A and B, Lipopeptidyl Benzophenones from a MarineDerived Aspergillus sp. Fungus Lijuan Liao,† Song Yi Bae,† Tae Hyung Won,† Minjung You,† Seong-Hwan Kim,† Dong-Chan Oh,† Sang Kook Lee,† Ki-Bong Oh,*,‡ and Jongheon Shin*,† †

Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-742, Korea ‡ Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, San 56-1, Sillim, Gwanak, Seoul 151-921, Korea S Supporting Information *

ABSTRACT: Asperphenins A (1) and B (2), novel diastereomeric lipopeptidyl benzophenone metabolites, were isolated from a marinederived Aspergillus sp. fungus. On the basis of the results of combined spectroscopic analyses, the structures of these compounds were determined to be linear assemblies of three motifs: a hydroxy fatty acid, a tripeptide, and a trihydroxybenzophenone. The absolute configurations were assigned using chemical modifications and electronic circular dichroism (ECD) calculations. The novel compounds exhibited significant cytotoxicity on diverse cancer cells.

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interesting bioactivities, such as the inhibition of sortase A, DNA intercalation, and the enhancement of insulin sensitivity in human mesenchymal stem cells, in addition to frequently exhibiting cytotoxicity and antimicrobial activity. In our continuing search, a strain of Aspergillus was collected from marine-submerged decaying wood that had an organic extract with moderate antiproliferative activity (IC50 170 μg/mL) against the K562 leukemia cell line, which led us to the discovery of the new aromatic alkaloids isochaetominines A−C and fumiquinazoline S.6 The results of an antiproliferative assay and LC−ESIMS analysis of chromatographic fractions from the crude extract revealed the presence of metabolites of different structural classes, prompting us to conduct an extensive investigation. Large-scale cultivation of the strain in a semisolid medium, followed sequentially by harvesting and chromatographic separation, afforded two compounds. Here, we report the structures of asperphenins A (1) and B (2), lipopeptidyl benzophenones of a novel skeletal class (Figure 1a). In addition, these compounds exhibited remarkable cytotoxicity against diverse cancer strains. The molecular formula of 1 was deduced to be C42H61N5O11 (15 degrees of unsaturation) by HRFABMS analysis. 13C NMR data for this compound revealed signals of seven carbonyl carbons at δC 201.8, 198.3, 173.9, 171.8, 171.2, 170.9, and 170.2. Given coupling with several exchangeable NH protons at δH 7.92−6.70 in 1H NMR data (Table 1), the latter five carbonyl carbons were thought to be the amide carbonyls, indicating the peptide nature of this compound. The 13C NMR

icroorganisms in marine environments are widely recognized as prolific sources of biologically active and structurally unique secondary metabolites.1 Although chemical investigations of these organisms emerged in the 1980s, much later than reports addressing those from terrestrial microorganisms or even marine macroorganisms, the number of novel natural products isolated from marine microorganisms has significantly increased since the late 1990s.2 With the development of modern microbial genetics and bioinformatics, this trend has accelerated in recent years. Marine fungi, which are among the most frequently encountered marine organisms, have produced more than >3000 novel compounds, several of which have varied and potent bioactivities.3 Because of their diverse biogenetic origins, these compounds exhibit profound structural variations and include molecules in most major structural classes, such as alkaloids, peptides, polyketides, and terpenoids. However, efforts to develop drugs from marine fungal-derived compounds continue to progress extremely slowly, and the only potential drug of this type that has been examined in clinical trials is the anticancer agent plinabulin (NPI-2358), a synthetic derivative of the diketopiperazines halimide and phenylahistin from an Aspergillus sp. fungus; this agent was recently discontinued from a Phase II trial.4 Given their immense biomedical potential, metabolites of marine fungi require extensive investigation. In our search for bioactive metabolites from marine-derived microorganisms, we have recently reported a number of novel compounds, such as acremostrictin, herqueiazole, herqueidiketal, and terrelumamides, that possess unusual carbon frameworks and/or functionalities; in this manner, we have contributed to information regarding the chemical diversity of marine-derived fungi.5 Several of these compounds have © 2017 American Chemical Society

Received: March 5, 2017 Published: April 7, 2017 2066

DOI: 10.1021/acs.orglett.7b00661 Org. Lett. 2017, 19, 2066−2069

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Table 1. NMR Data of 1 and 2 (in DMSO-d6, Values in ppm, J in Hz)a

Figure 1. (a) Structures of asperphenins A (1) and B (2). (b) COSY/ TOCSY spectra and key HMBC correlations of 1.

data also revealed 12 aromatic carbons at δC 161.6−106.8 which may correspond to two phenyls that account for the remaining eight degrees of unsaturation in the molecular formula. In addition, the presence of a long hydrocarbon chain was revealed by severely overlapped signals in shielded regions in both the 13C and 1H NMR data (δC 32−22 and δH 1.21− 1.19). Thus, 1 was determined to be a mixed biogenetic product that consisted of two phenyls, a lipid chain and amino acid residues. Given this information, the planar structure of 1 was determined by a combination of 2-D NMR analyses such as 1 H−1H COSY, TOCSY, HSQC, and HMBC experiments (Figure 1b). First, all protons and their attached carbons were adequately matched using HSQC data. Subsequently, two highly substituted aromatic rings (C-1-C-6, C-14; C-8-C-13) were deduced on the basis of a combination of COSY and HMBC correlations. The construction of a benzophenone moiety that connected these rings was accomplished using HMBC correlations between the carbonyl carbon at δC 201.8 (C-7) and the ring protons H-2, H-4, H-10, and H-12. The COSY and TOCSY data also revealed a proton spin system (H16−H-21) that included an amide proton at δH 7.66 (17-NH). The connection of this 2-amino-4-methylpentanyl group to the benzophenone was verified by HMBC correlations between a carbonyl carbon at δC 198.3 (C-15) and H-2, H-16, and H-17. A combination of 2-D NMR data was used to establish the amino acid residues of asparagine (Asn, C-27-C-30) and glutamine (Gln, C-22-C-26), whose linkage was verified by the H-17/C-22, 17-NH/C-22, and 23-NH/C-27 HMBC correlations. The remaining structural motif of 1 was determined to be a 3-hydroxydodecanoic acid residue (C-31−C-42) based on COSY and TOCSY data. Finally, the amide linkage between this residue and Asn was established by the 28-NH/C-31 HMBC correlation. Thus, asperphenin A (1) is a lipopeptidyl benzophenone that belongs to a unique class of mixed biogenetic products with three distinct origins. A literature study revealed that the lipopeptide portion (C-22−C-42) of 1 is present in a number of fungal metabolites.7 To our knowledge, however, the benzophenone and a β-amino acid residue of 1 are novel structural motifs, while other compounds either completely lack similar motifs or have α-amino groups.

a d

Data were recorded on a Bruker AV-600 spectrometer. Overlapped signals.

b−

Asperphenin A (1) includes four asymmetric carbon centers at C-17, C-23, C-28, and C-33; determinations of the absolute configurations at these centers required extensive analyses. First, the configuration of the hydroxy-bearing center C-33 was determined to be R by Mosher’s method (Figure 2). To prevent instability attributed to the trihydroxybenzophenone unit, MTPA-esters (3aS and 3aR) were obtained using the 2067

DOI: 10.1021/acs.orglett.7b00661 Org. Lett. 2017, 19, 2066−2069

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Scheme 2. Stepwise Reduction of Asperphenins A (1) and B (2) with NaBH4

Figure 2. ΔδH (δ3aS − δ3aR) values of MTPA esterifications for triacetylcycloasperphenin A (3a).

triacetyl derivative cycloasperphenin A (3a) (Scheme 1). The absolute configurations at the C-23 and C-28 centers of the Asn Scheme 1. MTPA Esterification of Asperphenins A (1) and B (2) via Cycloasperphenins A (3) and B (4)

reduction, possibly due to the steric interactions of the 7hydroxy group with the newly formed 15-hydroxy group.) Based on the 1H−1H COSY and proton-decoupling experiments, vicinal proton−proton coupling constants (3JHH) were measured around the C-15 and C-17 asymmetric centers. Additionally, long-range carbon−proton coupling constants (2JCH and 3JCH) were measured from HETLOC experiments. A combination of these analyses, along with the ROESY analyses, were used to assign the relative configurations of 9 and 10, derived from 1, as 15S*,17R* and 15R*,17R*, respectively (Figures S3−S6). Contrarily those of 11 and 12, derived from 2, were assigned as 15S*, 17S* and 15R*, 17S*, respectively (Figures S7−S10). Thus, the 17-epimeric relationship between asperphenins A (1) and B (2) was unambiguously defined. The absolute configuration at C-17 was eventually determined using CD measurements and ECD calculations. Notably, 1 and 2 did not exhibit distinct CD profiles, possibly due to free folding in the tested solutions (Figure S11). Consequently, CD measurements were performed on cycloasperphenins A (3) and B (4), which possessed additional structural rigidity at C-17 and neighboring positions. The CD profiles of 3 and 4 had opposite profiles at approximately 250 nm that were highly consistent with ECD calculations for the models 3m and ent-3m, respectively (Figure 3). Accordingly, the absolute configurations at the C-17 asymmetric center were determined to be R and S for 1 and 2, respectively. Thus, asperphenins A (1) and B (2) were structurally determined to be epimeric lipopeptidyl benzophenones of a novel structural class. The antiproliferative activity of the crude extract prompted us to investigate such activity for asperphenins. Both 1 and 2 exhibited significant antiproliferative activity against diverse human cancer cell lines, with IC50 values ranging from 0.8 to 9.7 μM (Table S1). Among the tested cell lines, RKO colorectal carcinoma cells were the most sensitive to both 1 and 2, with IC50 values of 0.8 and 1.1 μM, respectively. The detailed mode of action and structure−activity relationships will be reported in the future. In summary, asperphenins A and B, cytotoxic lipopeptidyl benzophenone metabolites, were isolated and structurally

and Gln residues of 1 were determined to be L via advanced Marfey’s analysis (Figure S2). The configuration at the asymmetric center C-17 is discussed below. The molecular formula of the congener asperphenin B (2) was deduced to be C42H61N5O11, which was identical to 1. The planar structure of this compound was determined to be the same as 1 based on a combination of 1-D and 2-D NMR analyses (Table 1). A comparison of 1H and 13C NMR data for 1 and 2 revealed that the protons and carbons around the asymmetric center C-17 were noticeably shifted, suggesting that these compounds exhibited an epimeric relationship at this center. This interpretation was supported by the assignments of absolute configurations at the dipeptide (C-23 and C-28) and fatty acid (C-33) asymmetric centers of 2 that were identical to the corresponding configurations for 1 (Figures S1 and S2). The determination of configurations at the C-17 β-amino acid center of asperphenins required elaborate efforts since this amide group was unable to be chemically reduced to a reactive free amine for the MTPA or similar analyses. The NMR-based approaches, such as NOESY experiments, also did not produce the reliable proton−proton or proton−carbon correlations around this center. Furthermore, the CD-based approaches were unsuccessful due to the weak absorption of the natural asperphenins. Consequently, this problem was solved by Jbased configurational analysis and ECD calculations on synthetic derivatives. First, compound 1 was readily reduced to epimeric 7-hydroxy derivatives 5 and 6 with NaBH4. Further reduction of 5 provided the epimeric 7,15-dihydroxy derivatives 9 and 10. Similarly, compound 2 was reduced to 7,15dihydroxy derivatives 11 and 12 via the 7-hydroxy derivative 7 (Scheme 2). (The epimeric 7-hydroxy derivatives 6 and 8 from 1 and 2, respectively, were completely decomposed during the 2068

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(4) (a) Mayer, A. M. S.; Glaser, K. B.; Cuevas, C.; Jacobs, R. S.; Kem, W.; Little, R. D.; McIntosh, J. M.; Newman, D. J.; Potts, B. C.; Shuster, D. E. Trends Pharmacol. Sci. 2010, 31, 255−265. (b) Martins, A.; Vieira, H.; Gaspar, H.; Santos, S. Mar. Drugs 2014, 12, 1066−1101. (5) (a) Julianti, E.; Oh, H.; Jang, K. H.; Lee, S. K.; Oh, D.-C.; Oh, K.B.; Shin, J. J. Nat. Prod. 2011, 74, 2592−2594. (b) Jeon, J.-e.; Julianti, E.; Oh, H.; Park, W.; Oh, D.-C.; Oh, K.-B.; Shin, J. Tetrahedron Lett. 2013, 54, 3111−3115. (c) Julianti, E.; Lee, J.-H.; Liao, L.; Park, W.; Park, S.; Oh, D.-C.; Oh, K.-B.; Shin, J. Org. Lett. 2013, 15, 1286−1289. (d) Liao, L.; Lee, J.-H.; You, M.; Choi, T. J.; Park, W.; Lee, S. K.; Oh, D.-C.; Oh, K.-B.; Shin, J. J. Nat. Prod. 2014, 77, 406−410. (e) You, M.; Liao, L.; Hong, S. H.; Park, W.; Kwon, D. I.; Lee, J.; Noh, M.; Oh, D.C.; Oh, K.-B.; Shin, J. Mar. Drugs 2015, 13, 1290−1303. (6) Liao, L.; You, M.; Chung, B. K.; Oh, D.-C.; Oh, K.-B.; Shin, J. J. Nat. Prod. 2015, 78, 349−354. (7) (a) Shigemori, H.; Wakuri, S.; Yazawa, K.; Nakamura, T.; Sasaki, T.; Kobayashi, J. Tetrahedron 1991, 47, 8529−8534. (b) Lee, Y. M.; Dang, H. T.; Hong, J.; Lee, C.-O.; Bae, K. S.; Kim, D.-K.; Jung, J. H. Bull. Korean Chem. Soc. 2010, 31, 205−208. (c) Lee, Y. M.; Dang, H. T.; Li, J.; Zhang, P.; Hong, J.; Lee, C.-O.; Jung, J. H. Bull. Korean Chem. Soc. 2011, 32, 3817−3820. (d) Xu, D.; Ondeyka, J.; Harris, G. H.; Zink, D.; Kahn, J. N.; Wang, H.; Bills, G.; Platas, G.; Wang, W.; Szewczak, A. A.; Liberator, P.; Roemer, T.; Singh, S. B. J. Nat. Prod. 2011, 74, 1721−1730.

Figure 3. Experimental CD spectra of cycloasperphenins A (3) and B (4) and calculated ECD spectra of 3m and ent-3m.

elucidated from a culture broth of marine-derived Aspergillus sp. fungus.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00661. Detailed experimental procedures, 1H, 13C, and 2D NMR and ECD spectra (PDF)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *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 This study was supported by the BK21 Plus Program (L.L. and T.H.W.) in 2014 and by National Research Foundation of Korea (NRF) grants funded by the Korean government (Ministry of Science, ICT and Future Planning) (Nos. 20090083533 and 2010-0020429, J.S.).



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DOI: 10.1021/acs.orglett.7b00661 Org. Lett. 2017, 19, 2066−2069