A new fused tetracyclic heterocyclic antioxidant from Serratia sp. PAMC 25557 Hari Datta Bhattarai a , Babita Paudel b , Kim Il Chan b , Hyuncheol Oh a , Joung Han Yim b, * a College of Pharmacy, Wonkwang University, Iksan 570-749, Republic of Korea b Division of Life Sciences, Korea Polar Research Institute, KOPRI, Incheon 406-840, Republic of Korea 1. Introduction Several species of marine bacteria (Williams, 2008), including Serratia sp. (Dwivedi et al., 2008), produce biologically active secondary metabolites. We searched for new biologically active natural products produced by marine bacteria and selected Serratia sp. PAMC 25557, because preliminary data suggested that the crude extract had 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity. In the present report, we describe a new tetracyclic heterocycle compound, (4bR,10bR)-4b-hydroxy-10b,12- dihydrodibenzo[c,h][2,6]naphthyridine-5,11(4bH,6H)-dione, and one known compound, spatozoate, which was originally isolated from the brown alga Spatoglossum variabile (Rahman et al., 1999). This is the first report of spatozoate isolation from bacterial sources. 2. Results and discussion Compound 1 (2.5 mg) was isolated as a pale yellow solid from the ethyl acetate extract of the broth culture of Serratia sp. PAMC 25557. The specific rotation of 1 was [a] D 20 +3 (c 0.02, ethanol). The IR spectrum of compound 1 showed absorptions at 3333, 3252 (O– H, N–H), 1727, 1687 (C 55 O), 1621, 1469, 1341, and 1201 cm À1 (C 55 C, Ar). The HRESIMS of 1 showed the molecular ion (M+Na) + at m/z 303.0740, establishing the molecular formula of compound 1 as C 16 H 12 N 2 O 3 with twelve degrees of unsaturation. The number of protons and carbons were supported by the respective NMR data of 1. Eight aromatic protons, one hydroxyl, and two amide protons were observed. The NMR data of 1 are listed in Table 1. The structure of 1 was further elucidated by analyzing data obtained from 2D NMR spectroscopy. The analysis of 1 H, 1 H correlation spectroscopy (COSY) data and splitting patterns of aromatic protons clearly indicated that two di-substituted benzene rings were included in the structure. Two carbonyl (C 55 O) carbons were resonated at 176.7 ppm and 173.9 ppm. Twelve sp2 carbons were observed, suggesting the presence of two benzene rings in the structure. Three exchangeable singlet protons appeared at 6.50 ppm (OH), 10.12 ppm (NH), and 10.27 ppm (NH). Two carbonyl carbons and two di-substituted benzene rings were considered to satisfy the twelve degrees of unsaturation in the molecule. We assigned two fused dihydroquinolinones in such a way that one benzene ring was attached with one heterocycle (Fig. 1). Connections of these two aromatic rings with the remaining structural features were constructed by analyzing HMBC data (Table 1). Particularly, HMBC data from three exchangeable protons provided key correlations for the construction of the dihydrodibenzonaphthyridinedione skeleton. HMBC data for NH- 12 with C-11, C-12a, and C-4a and consideration of the chemical Phytochemistry Letters 6 (2013) 536–538 A R T I C L E I N F O Article history: Received 11 April 2013 Received in revised form 3 July 2013 Accepted 8 July 2013 Available online 24 July 2013 Keywords: Antioxidant DPPH free radical Serratia A B S T R A C T A new fused tetracyclic heterocyclic compound, (4bR,10bR)-4b-hydroxy-10b,12-dihydrodiben- zo[c,h][2,6]naphthyridine-5,11(4bH,6H)-dione (1), and a known compound, butyl 2-[(benzoyloxy)- methyl]benzoate, spatozoate 2, were isolated from the broth culture of Serratia sp. PAMC 25557. The structure of 1 was determined by analyzing spectroscopic data. Compound 1 did not exhibit antimicrobial activity against Escherichia coli, Staphylococcus aureus, or Candida albicans. In addition, up to 100 mg/ml compound 1 did not show any toxicity against Artemia salina larvae. However, compound 1 showed DPPH free radical scavenging activity (IC 50 = 16.7 Æ 0.34 mg/ml). This was the first report of spatozoate isolation from bacterial sources. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl; IR, ionizing radiation; ESIMS, electrospray ionization mass spectrometry; 1D, one-dimensional; 2D, two- dimensional; NMR, nuclear magnetic resonance; DMSO, dimethyl sulfoxide; HMQC, heteronuclear multiple-quantum correlation; HMBC, heteronuclear multi- ple-bond correlation; COSY, correlation spectroscopy; NOE, nuclear Overhauser effect; MS, mass spectrometry; BHA, butylated hydroxyanisole; BST, brine shrimp lethality test. * Corresponding author. Tel.: +82 327605540. E-mail address: jhyim@kopri.re.kr (J.H. Yim). Contents lists available at SciVerse ScienceDirect Phytochemistry Letters jo u rn al h om ep ag e: ww w.els evier.c o m/lo c ate/p hyt ol 1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2013.07.006