Columbamides D and E: Chlorinated Fatty Acid Amides from the

Aug 7, 2017 - Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Kota Kinabalu 88450, Sabah, Malaysia. Org. Lett. , 2017, 19 ...
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Columbamides D and E: Chlorinated Fatty Acid Amides from the Marine Cyanobacterium Moorea bouillonii Collected in Malaysia Julius Adam V. Lopez,† Julie G. Petitbois,† Charles S. Vairappan,§ Taiki Umezawa,†,‡ Fuyuhiko Matsuda,†,‡ and Tatsufumi Okino*,†,‡ †

Graduate School of Environmental Science and ‡Faculty of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan § Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Kota Kinabalu 88450, Sabah, Malaysia S Supporting Information *

ABSTRACT: Two new chlorinated fatty acid amides, columbamides D (1) and E (2), along with apratoxins A and C and wewakazole, were isolated from the organic extract of a Moorea bouillonii sample from Sabah, Malaysia. Structure elucidation was accomplished by a combination of MS and NMR analyses. The total synthesis of all four stereoisomers of 1 was completed, and the absolute configuration was determined by chiral-phase HPLC and Marfey’s analysis.

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16S rRNA gene sequence confirmed the sample identity as M. bouillonii (GenBank KY473922). Chemical profiling by LC− MS revealed the presence of the known cytotoxic compounds apratoxins A and C and wewakazole as well as di- and trichlorinated compounds. Dereplication using the MarinLit database showed that the latter were possibly new chlorinated compounds, and thus, they were pursued. This led to the isolation of columbamides D (1) and E (2) along with the known compounds by subjection of the EtOAc fraction to SiO2 and ODS column chromatography followed by RP HPLC (pp S4−S5 in the Supporting Information). The MS and NMR data of the known compounds matched those found in the literature.9−11 The molecular formula of 1 was determined to be C23H43Cl2NO3 ([M + H]+ m/z 452.2685, calcd 452.2693) by ESI-FTMS, suggesting two degrees of unsaturation. The 1D NMR and HSQC data (Table 1 and Figures S3−S5) were used to establish the following: a carbonyl (δH 174.4), two olefinic methines (δC 129.0, 131.2) with identical protons (δH 5.47), a chlorinated methine (δC 64.3, δH 3.88) and methylene (δC 45.1, δH 3.54), an N-methine (δC 57.8, δH 4.39), an N-methyl (δC 33.8, δH 3.02), a methoxy (δC 59.0, δH 3.34), two O-methylenes (δC 71.0, δH 3.68/3.59 and δC 62.3, δH 3.80), and 13 other methylenes (δC 25.9−38.4, δH 1.3−2.4). These signals closely match those of columbamide C except for two extra methylenes.3 At this point, the challenge was to determine the position of these methylenes and the chloromethine. To solve this, 2D NMR data (Table 1 and Figures 1 and S6− S9) were interpreted, including an H2BC experiment as previously reported.3 First, the N,O-dimethylserinol moiety was constructed using the following signals: H-20 to H-21 and H-22 and a hydroxy to H-22 by COSY and H-20 to C-19 and

atty acid amides such as malyngamides and jamaicamides have been recurrently reported from the marine cyanobacterium Moorea producens.1 Their structures consist of a fatty acid chain and a peptide moiety, which are derived from polyketide synthetase (PKS) and nonribosomal peptide synthase (NRPS) pathways, respectively.2 Recently, columbamides A−C, a new group of fatty acid amides that harbor internal and terminal chlorine atoms on the alkyl chain, were isolated from a laboratory culture of Moorea bouillonii.3 Columbamides A and B showed cannabinomimetic activity similar to structurally related fatty acid amides such as the serinolamides, semiplenamides, and mooreamide A.3,4 However, the absolute configuration of the chloromethine on the alkyl chain of the columbamides remains unclear. This information is crucial to fully understand the toxicological and pharmacological aspects of these chiral compounds.5 This problem was also encountered in other compounds bearing a remote chloromethine such as the bartolosides and cylindrofridins.6−8 We report the isolation and structure elucidation of two new columbamides, D (1) and E (2), from M. bouillonii collected in Malaysia. Marfey’s analysis of the N,O-dimethylserinol moiety, total synthesis of all four stereoisomers of 1, and subsequent chiral-phase HPLC were carried out to determine the absolute configuration.

Potent cytotoxicity at 1 μg/mL against MCF7 breast cancer cells was found in the EtOAc fraction of a brownish-red, matforming cyanobacterium collected near Mantanani Island in Sabah, Malaysia. Phylogenetic analysis (Figure S1) using the © XXXX American Chemical Society

Received: June 20, 2017

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DOI: 10.1021/acs.orglett.7b01869 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters Table 1. NMR Spectroscopic Data for 1 and 2 in CDCl3e

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Measured at 100 MHz. bMeasured at 400 MHz. cMeasured at 600 MHz. Values in red are from TOCSY or H2BC experiments. dDerived from HSQC, HMBC, and H2BC data. eValues in brackets correspond to signals from the rotamer.

was positioned at C-10, as supported by COSY, HMBC, and H2BC signals toward the neighboring atoms (Figure 1). COSY and TOCSY signals from H-13 to H-16 could not be distinguished because of heavy overlap. Nonetheless, the connections from C-13 to C-16 were made on the basis of the following data: HMBC signals to C-14 from H-12 and H-16 and H2BC signals from H-15 to C-14 and C-16 as well as from H-16 to C-15. Finally, a terminal chloromethylene was established by COSY signals through H-16 to H-18, reinforced by H2BC and HMBC signals, to complete the planar structure. The molecular formula of columbamide E (2) was determined to be C23H42Cl3NO3 ([M + H]+ m/z 486.2286, calcd 486.2303) by ESI-FTMS. It differed from 1 by only 34 Da, suggesting the replacement of a hydrogen atom by a chlorine atom. Indeed, the NMR data for 2 (Table 1 and Figures S10−S16) were identical to those for 1 except for two new signals (δC 73.8, δH 5.75 and δC 44.0, δH 2.19), which corresponded to a terminal dichloromethine and its adjacent methylene, respectively, similar to those of columbamide B.3 However, it was discovered that 2 was actually a 3:4 mixture of

Figure 1. 2D NMR correlations for 1.

H-23 to C-21 by HMBC. H2BC signals also supported the C21/C-20/C-22/−OH fragment. Connection to the carbonyl C1 was made by HMBC signals from H-19 and H-20. Next, the sequence from C-1 to C-13 was accomplished. C-2 was connected to C-1 via 2J and 3J correlations from H-2 and H-3, respectively. COSY signals through H-2 to H-4 were found, as well as corresponding H2BC signals. The double bond was located between C-4 and C-5, as established by H2BC signals from H-4/5 to C-3 and C-6 and vice versa. The chain was extended from C-5 to C-13 with the following data: COSY through H-5 to H-7 and H-9 to H-11 and TOCSY through H-7 to H-9 and H-11 to H-13. The internal chlorine B

DOI: 10.1021/acs.orglett.7b01869 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters 1 and 2 on the basis of MS (Figure S17) and the presence of the chloromethylene signal (δC 45.2, δH 3.54) of 1 in the NMR spectra (Figures S11−S16). Nevertheless, this issue does not dispute the planar structure of 2. Experiments to determine the configurations of 1 and 2 were performed. An E geometry for the double bond in 1 and 2 was found by obtaining the coupling constant (3JH,H = 16 Hz) from 13 C satellite signals observed using non-decoupled HSQC analysis (Figure S18).12 (R)- and (S)-N,O-dimethylserinol (3) (Figure S19) were synthesized13 and used as standards for Marfey’s analysis (Figure S20) of the hydrolysates of 1 and 2, which indicated an R configuration at C-20. Finally, utilizing (R)- and (S)-3, the total synthesis of (10R,20S)-, (10R,20R)-, (10S,20R)-, and (10S,20S)-1 (Scheme 1) was conducted to determine the absolute configuration at C-10. Monoetherification of 1,10-decanediol (4) with 2-naphthylmethyl bromide (NAPBr) and subsequent TEMPO oxidation gave aldehyde 5 (Scheme 1). For enantioselective installation of a chlorine atom at C-10, the modified MacMillan’s conditions using the imidazolidinone catalyst (S)-6 were applied to 5, furnishing labile α-chloroaldehyde 7.14,15 The absolute configuration of 7 was unambiguously assigned to be S by the modified Mosher’s method (Figure S21). The enantiomeric excess was determined from the 1H NMR data of the MTPA esters (Figure S22). Then treatment of 7 with Wittig reagent in a one-pot operation produced unsaturated ester 8. After reduction of 8, a five-step sequence toward a four-carbon homologation provided α,β-unsaturated ester 10. The unsaturated ester moiety of 10 was successfully converted into allyl bromide 11 in three steps via a mesylate.16 Then 11 was subjected to enolate alkylation conditions using EtOAc and LDA in the presence of HMPA to give γ,δ-unsaturated ester 12.17 Next, the terminal chlorine atom was introduced in two steps, deprotection of NAP ether with DDQ followed by Appel reaction and hydrolysis of the ester group, to afford carboxylic acid 14. Finally, the amide bond was introduced by treatment of the acyl chloride derived from from 14 with Et3N, N,Obis(trimethylsilyl)acetamide (BSA), and (S)-3 to provide (10S,20S)-1 in 71% yield. 18 The other stereoisomers [(10S,20R)-1, (10R,20S)-1, and (10R,20R)-1] were similarly prepared by using (S)-3 or (R)-3 and (S)-6 or (R)-6. Chiral-phase HPLC results showed that the 20S isomers elute later and have a broad peak (Figure S23). By a coelution experiment, natural 1 eluted as a single peak only with the synthetic (10R,20R) isomer (Figure S24), leading to its unambiguous configurational assignment. Chiral recognition at C-10 by the Chiralpak AD-H column was remarkable and was possibly driven by a halogen-bonding interaction between chlorine and the carbamate carbonyl group of the stationary phase.19 ECD spectra were also taken but could provide information about C-20 only (Figure S25). Although 13C NMR data deviations (0.03−0.1 ppm) on the serinol moiety (C-19 to C-22) among the diastereomers of 1 were initially observed, consistent results could not be obtained in three trials. Moreover, no significant change in the 13C NMR chemical shift at C-10 was detected because of its distance to C-20. While the use of chiral fluorescent reagents for compounds with remote stereocenters has been well-reported, diastereomers were not always distinguished by NMR data alone, and their separation by HPLC may require complex conditions, as in the case of miyakosyne A.20,21 The biological activities of natural 1 and 2 could not be assessed because of trace amounts of the highly cytotoxic

Scheme 1. Total Synthesis of the (10S,20S) Stereoisomer of Columbamide D (1)

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Organic Letters apratoxins detected by MS.9,10 Nevertheless, the synthetic compounds were tested and were noncytotoxic to human MCF7 breast and H460 lung cancer cells at 22 μM. Meanwhile, wewakazole was finally confirmed as a nonsiderophore akin to wewakazole B.22 It was inactive at 88 μM (Figure S26). The synthesized isomers of 1 were not tested for iron-binding activity since the crude fraction of natural 1 was inactive. In summary, two new columbamides, D (1) and E (2), were isolated from M. bouillonii collected in Malaysia. The planar structures were established by MS and NMR data. The absolute configuration of 1 was unambiguously assigned as (10R,20R) by Marfey’s analysis, total synthesis, and chiral-phase HPLC analysis. This work provides a significant addition to our knowledge about columbamides, particularly on stereochemistry, and the natural products arsenal of the genus Moorea.

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(5) Thayer, A. M. Chem. Eng. News 2007, 85, 11−19. (6) Leão, P. N.; Nakamura, H.; Costa, M.; Pereira, A. R.; Martins, R.; Vasconcelos, V.; Gerwick, W. H.; Balskus, E. P. Angew. Chem., Int. Ed. 2015, 54, 11063−11067. (7) Afonso, T. B.; Costa, M. S.; Rezende de Castro, R.; Freitas, S.; Silva, A.; Schneider, M. P. C.; Martins, R.; Leão, P. N. J. Nat. Prod. 2016, 79, 2504−2513. (8) Preisitsch, M.; Niedermeyer, T. H. J.; Heiden, S. E.; Neidhardt, I.; Kumpfmüller, J.; Wurster, M.; Harmrolfs, K.; Wiesner, C.; Enke, H.; Müller, R.; Mundt, S. J. Nat. Prod. 2016, 79, 106−115. (9) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett, T. H. J. Am. Chem. Soc. 2001, 123, 5418−5423. (10) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. Bioorg. Med. Chem. 2002, 10, 1973−1978. (11) Nogle, L. M.; Marquez, B. L.; Gerwick, W. H. Org. Lett. 2003, 5, 3−6. (12) Luy, B.; Hauser, G.; Kirschning, A.; Glaser, S. J. Angew. Chem., Int. Ed. 2003, 42, 1300−1302. (13) Gao, Y. R.; Guo, S. H.; Zhang, Z. X.; Mao, S.; Zhang, Y. L.; Wang, Y. Q. Tetrahedron Lett. 2013, 54, 6511−6513. (14) Brochu, M. P.; Brown, S. P.; MacMillan, D. W. C. J. Am. Chem. Soc. 2004, 126, 4108−4109. (15) Umezawa, T.; Shibata, M.; Kaneko, K.; Okino, T.; Matsuda, F. Org. Lett. 2011, 13, 904−907. (16) Stork, G.; Baine, N. H. Tetrahedron Lett. 1985, 26, 5927−5930. (17) Odejinmi, S. I.; Wiemer, D. F. J. Nat. Prod. 2005, 68, 1375− 1379. (18) Pettit, G. R.; Smith, T. H.; Arce, P. M.; Flahive, E. J.; Anderson, C. R.; Chapuis, J. C.; Xu, J. P.; Groy, T. L.; Belcher, P. E.; Macdonald, C. B. J. Nat. Prod. 2015, 78, 476−485. (19) Peluso, P.; Mamane, V.; Aubert, E.; Cossu, S. J. Chromatogr. A 2014, 1345, 182−192. (20) Ohrui, H.; Terashima, H.; Imaizumi, K.; Akasaka, K. Proc. Jpn. Acad., Ser. B 2002, 78, 69−72. (21) Mori, K.; Akasaka, K.; Matsunaga, S. Tetrahedron 2014, 70, 392−401. (22) Lopez, J. A. V.; Al-Lihaibi, S. S.; Alarif, W. M.; Abdel-Lateff, A.; Nogata, Y.; Washio, K.; Morikawa, M.; Okino, T. J. Nat. Prod. 2016, 79, 1213−1218.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01869. Full experimental details and spectral data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel. +81 11 706 4519. Fax: +81 11 706 4867. E-mail: okino@ ees.hokudai.ac.jp. ORCID

Julius Adam V. Lopez: 0000-0002-1812-8104 Julie G. Petitbois: 0000-0002-5602-0591 Taiki Umezawa: 0000-0003-4280-6574 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant 16H04975. We thank Dr. Y. Kumaki of the High-Resolution NMR Laboratory, Hokkaido University, for the 2D NMR analyses. We are grateful to Dr. M. Morikawa and Dr. M. Kurasaki of the Faculty of Environmental Earth Science, Hokkaido University, for the use of genetics and cell culture facilities, respectively. C.S.V. and T.O. thank the Sabah Biodiversity Centre for the collection permits and support rendered during the course of this investigation (permit no. JKM/MBS.1000-2/2).

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DOI: 10.1021/acs.orglett.7b01869 Org. Lett. XXXX, XXX, XXX−XXX