Article Cite This: J. Nat. Prod. 2018, 81, 1666−1672 pubs.acs.org/jnp Macrophilones from the Marine Hydroid Macrorhynchia philippina Can Inhibit ERK Cascade Signaling † ‡ § ⊥ † ∥ Pengcheng Yan, , Daniel A. Ritt, Katherine Zlotkowski, Heidi R. Bokesch, , # ⊥ § William C. Reinhold, John S. Schneekloth, Jr., Deborah K. Morrison,*, † and Kirk R. Gustafson*, † Molecular Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States ‡ School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, People’s Republic of China § Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States ⊥ Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States ∥ Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, United States # Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, United States *S Supporting Information ABSTRACT: Six new macrophilone-type pyrroloimino- quines were isolated and identified from an extract of the marine hydroid Macrorhynchia philippina. The proton- deficient and heteroatom-rich structures of macrophilones B−G(2−7) were elucidated by spectroscopic analysis and comparison of their data with those of the previously reported metabolite macrophilone A (1). Compounds 1−7 are the first pyrroloiminoquines to be reported from a hydroid. The macrophilones were shown to inhibit the enzymatic conjugation of SUMO to peptide substrates, and macro- philones A (1) and C (3) exhibit potent and selective cytotoxic properties in the NCI-60 anticancer screen. Bioinformatic analysis revealed a close association of the cytotoxicity profiles of 1 and 3 with two known B-Raf kinase inhibitory drugs. While compounds 1 and 3 showed no kinase inhibitory activity, they resulted in a dramatic decrease in cellular protein levels of selected components of the ERK signal cascade. As such, the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads that target the ERK signal Downloaded via NATL INST OF HEALTH on November 19, 2018 at 19:45:28 (UTC). transduction pathway. See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ydroids are widely distributed in the world’s oceans, but detailed chemical investigation. According to the World H they have only sparsely been studied chemically Register of Marine Species the genus Macrorhynchia is compared to other colonial marine invertebrate organisms synonymous with Lytocarpus,19 and there is a report in the such as sponges, corals, and ascidians. However, previous literature of 14-membered macrolides that were isolated from a ff chemical examinations of hydroids have a orded a diverse hydroid described as Lytocarpus philippinus.13 While there is array of bioactive metabolites including anthracenes (anthra- 1−6 7 some ambiguity in the literature concerning hydroid taxonomic cenones), polyhalogenated monoterpenoids, brominated fi 20,21 β 8 9 10,11 descriptions and classi cation, it is likely that the hydroid -carbolines, piperidinols, dithiocarbamates, homoeico- examined in the current study is the same as the hydroid that sanoids,12 macrolactones,13 a pentapeptide,14 a 4-hydroxyben- − provided the macrolides. No macrolides were detected during zoyl derivative,15 and polyhydroxylated sterols.16 18 Some of these metabolites exhibited significant cytotoxic activ- fractionation of the M. philippina extract; rather, new − ities;1,13 15 thus hydroids represent an understudied resource pyrroloiminoquinone derivatives were isolated. We recently of chemical diversity for potential anticancer discovery efforts. reported the structure elucidation and synthesis of macro- An extract from an Australian collection of the hydroid Macrorhynchia philippina showed significant activity in the Received: May 1, 2018 NCI-60 cell line anticancer screen and, thus, was selected for Published: July 6, 2018 © 2018 American Chemical Society and American Society of Pharmacognosy 1666 DOI: 10.1021/acs.jnatprod.8b00343 J. Nat. Prod. 2018, 81, 1666−1672 Journal of Natural Products Article philone A (1), a potent cytotoxic metabolite that also arrests 212, 233, 257, 310, and 359 nm, differed from the spectrum the small ubiquitin-like modifier (SUMO) conjugation cascade recorded for 1, which indicated a modified chromophore. A in vitro.22 Post-translational attachment of the SUMO-protein notable difference in the 13C NMR spectrum of 2 was the loss δ to other protein substrates is an important regulatory of a signal for an amino-bearing carbon ( C 156.4 in 1) and the 2 δ component of critical cellular processes, and disruption of appearance of a signal for an oxygenated sp carbon ( C 173.1, SUMOylation is often associated with cancer and other C-6), which suggested that the amino group in 1 was replaced 23−25 by a hydroxy group in 2. In addition, significant deshielding diseases. Further investigation of the M. philippina extract δ was observed for both the sulfur-bearing methyl ( C 37.9 in 2; provided six additional iminoquinone derivatives, macro- 2 δ − − 17.1 in 1) and C-5 sp ( C 101.2 in 2; 95.7 in 1) carbons. philones B G(2 7), and their isolation, structural character- These data and molecular formula considerations indicated the ization, and biological evaluations are described below. thioether in 1 was oxidized to a sulfoxide in 2. An HMBC δ correlation from H3-9 ( H 2.97 s) to C-5 showed the position of the sulfoxide was consistent with that of the thiomethyl group in 1, and this was supported by an additional four-bond correlation to C-6 when the HMBC experiment was optimized for 2 Hz couplings (Figure 1). Thus, macrophilone B (2) was elucidated as 6-hydroxy-3-(hydroxymethyl)-4-imino-5-(meth- ylsulfinyl)-1,4-dihydro-7H-indol-7-one. Macrophilone C (3) had a molecular formula of C11H11N3O2S as determined by HRESIMS data, requiring seven degrees of unsaturation. The UV spectrum of 3 with absorptions at 213, 259, 325, and 390 nm was similar to the UV spectrum of 1, but its molecular formula revealed one ■ RESULTS AND DISCUSSION additional carbon and an additional unsaturation equivalent. 1 Sequential chromatography of the M. philippina extract on The H NMR spectrum of 3 in CD3OD showed signals for an olefinic proton (δ 7.20 s, H-2), an oxymethylene (δ 4.70 s, DIOL and Sephadex LH-20 supports, followed by repeated C18 H H 22 δ reversed-phase HPLC, provided macrophilone A (1) along H2-10), and two mutually coupled methylenes ( H 3.86 t, J = ff with six additional structural analogues (2−7). These 4.9 Hz, H2-7; 3.04 t, J = 4.9 Hz, H2-6). Numerous e orts to compounds all have a high heteroatom content and relatively observe exchangeable protons in 3 by acquiring spectra in dry 13 few protons observable by NMR, so the total synthesis of 1 DMSO-d6 were unsuccessful. The C NMR spectrum in 22 2 δ was undertaken to confirm its structure. The molecular CD3OD displayed seven nonprotonated sp carbons [ C 167.2 formula of macrophilone B (2) was established as (C-9), 159.6 (C-4), 144.6 (C-8a), 128.1 (C-9a), 125.5 (C-3), 2 δ C H N O S based on HRESIMS data, and this differed 121.6 (C-3a), 91.6 (C-4a)], a protonated sp ( C 126.1, C-2), 10 10 2 4 δ from the molecular formula of 1 by the addition of two and a hydroxymethylene carbon ( C 55.7, C-10), which oxygens and the loss of NH. The 1H(Table 1) and 13C NMR corresponded closely with the pyrroloiminoquinone skeleton 22 δ data (Table 2)of2 corresponded closely with those of 1, of 1. Two additional methylene carbons ( C 43.4, C-7; 22.3, showing all the characteristic signals for a hydroxymethyl- C-6) and the requirement of one more unsaturation equivalent substituted pyrroloiminoquinone moiety. A full suite of HMBC indicated they bridged the amino and thio groups to form a δ correlations, including correlations from H2-8 ( H 4.69 s) to 3,4-dihydro-2H-1,4-thiazine ring. The deshielded chemical δ δ δ δ the C-4 imino carbon ( C 160.3) and H-2 ( H 7.22 s) to the C- shifts of H2-7 ( H 3.86) and C-7 ( C 43.4) in comparison δ δ δ 7 oxo carbon ( C 170.2), supported this assignment (Figure 1). with those of H2-6 ( H 3.04) and C-6 ( C 22.3) suggested that However, the UV spectrum of 2, with absorbance maxima at C-7 was N-substituted and C-6 was S-substituted. HMBC 1 δ J − a Table 1. H NMR Data, H ( in Hz), for Compounds 2 7 pos. 23 3b 45 67 2 7.22, s 7.20, s 7.09, s 7.38, s 7.26, s 7.07, s 7.25, s 3 2.92, t (7.5) 2.98, m 4 3.89, t (7.5) 4.00, m 6 3.04, t (4.9) 2.87, t (4.7) 3.42, br d 3.21, dt (14.0) (14.1, 2.7) 2.76, td 2.54, td (14.0, 4.1) (14.1, 3.9) 7 3.86, t (4.9) 3.56, t (4.7) 4.07, br d 3.95, ddd 3.01, t (4.9) 3.33, m (15.6) (14.9, 3.9, 2.7) 2.69, td 3.96, br t 3.74, td (14.0, 3.7) (14.0) (14.6, 2.7) 8 4.69, s 3.86, t (4.9) 4.07, m 3.95, m 9 2.97, s 10 4.70, s 4.48, s 4.76, s 4.77 s a b 600 MHz, CD3OD.