marine drugs Article Magnificines A and B, Antimicrobial Marine Alkaloids Featuring a Tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione Backbone from the Red Sea Sponge Negombata magnifica Diaa T. A. Youssef 1,* , Hani Z. Asfour 2, Grégory Genta-Jouve 3,4 and Lamiaa A. Shaala 5,6,7,* 1 Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia 2 Department of Medical Parasitology, Faculty of Medicine, Princess Al-Jawhara Center of Excellence in Research of Hereditary Disorders, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] 3 UMR 8038 CiTCoM, Faculté de Pharmacie de Paris, Université Paris Descartes, Avenue de l’observatoire, 75006 Paris, France; [email protected] 4 Molecules of Communication and Adaptation of Microorganisms (UMR 7245), National Museum of Natural History, CNRS, 75231 Paris, France 5 Natural Products Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia 6 Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia 7 Suez Canal University Hospital, Suez Canal University, Ismailia 41522, Egypt * Correspondence: [email protected] (D.T.A.Y.); [email protected] (L.A.S.); Tel.: +966-548535344 (D.T.A.Y.) Abstract: Investigation of the Red Sea sponge Negombata magnifica gave two novel alkaloids, mag- Citation: Youssef, D.T.A.; Asfour, nificines A and B (1 and 2) and a new β-ionone derivative, (±)-negombaionone (3), together with H.Z.; Genta-Jouve, G.; Shaala, L.A. Magnificines A and B, Antimicrobial the known latrunculin B (4) and 16-epi-latrunculin B (5). The analysis of the NMR and HRES- Marine Alkaloids Featuring a IMS spectra supported the planar structures and the relative configurations of the compounds. Tetrahydrooxazolo[3,2-a]azepine- The absolute configurations of magnificines A and B were determined by the analysis of the pre- 2,5(3H,6H)-dione Backbone from the dicted and experimental ECD spectra. Magnificines A and B possess a previously unreported Red Sea Sponge Negombata magnifica. tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione backbone and represent the first natural com- Mar. Drugs 2021, 19, 214. https:// pounds in this class. (±)-Negombaionone is the first β-ionone of a sponge origin. Compounds 1-3 doi.org/10.3390/md19040214 displayed selective activity against Escherichia coli in a disk diffusion assay with inhibition zones up to 22 mm at a concentration of 50 µg/disc and with MIC values down to 8.0 µM. Latrunculin B and Academic Editor: Vassilios Roussis 16-epi-latrunculin B inhibited the growth of HeLa cells with IC50 values down to 1.4 µM. Received: 24 March 2021 Keywords: Red Sea sponge; Negombata magnifica; marine alkaloids; β-ionone; magnificines A and B; Accepted: 9 April 2021 (±)-negombaionone; latrunculin B and 16-epi-latrunculin B; antimicrobial activity; E. coli; cell line Published: 12 April 2021 growth inhibition; HeLa cells Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Sponges belonging to the genus Negombata (formerly Latrunculia)[1] (pp. 698–699) are characterized by diverse secondary metabolites of different classes including macrolides (latrunculins) [2–9], pyrroloiminoquinone alkaloids (discorhabdins) [10–17], terpene per- Copyright: © 2021 by the authors. oxides [18,19], cyclic 2-oxecanone glycosides [20], diterpenes [21], ceramides [22,23], and Licensee MDPI, Basel, Switzerland. peptides [24–26]. Reported latrunculins displayed anticancer, antiviral, antibiotic, an- This article is an open access article tiangiogenic, antimigratory, and microfilament-disrupting activities [2–9]. Pyrroloimino- distributed under the terms and quinone alkaloids exhibited antimicrobial, immunomodulatory, caspase inhibition, antivi- conditions of the Creative Commons ral, feeding deterrence, and antimalarial properties and present potent inhibition potential Attribution (CC BY) license (https:// of mammalian topoisomerase II in vivo [10–17]. Additional pharmacological activities for creativecommons.org/licenses/by/ other chemical entities identified from the genus Negombata include cytotoxicity [18,19,21], 4.0/). Mar. Drugs 2021, 19, 214. https://doi.org/10.3390/md19040214 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, 214 2 of 12 antifeeding [20], antiepileptic, and anti-inflammatory [22,23], potent inotropic effects and inhibition of the cardiac Na/Ca exchanger [24–26]. As a part of our growing interest to discover biologically active leads from ma- rine resources [27–29], the organic extract of the sponge Negombata magnifica was ex- amined. Two alkaloids, magnificines A and B (1 and 2), with a previously unreported tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione skeleton were purified. In addition, a new β-ionone derivative, (±)-negombaionone (3), with the previously reported latrunculin B(4)[2] and 16-epi-latrunculin B (5)[5] were obtained. Structural determinations of 1-5 were accomplished by HRESIMS and NMR spectral analyses. 2. Results and Discussion 2.1. Purification of 1-5 Fractionation of the methanolic extract of N. magnifica [30] (Figure1) using partition (on silica gel), size exclusion (Sephadex LH 20), and purification of active fractions on HPLC afforded 1-5. Figure 1. Underwater photograph of the Red Sea Negombata magnifica. 2.2. Structure of Magnificine A (1) 25 Magnificine A (1) (Figure2) obtained as an optically active ([ α] = + 70◦) oil. D The chemical structure of 1 was determined from interpretation of its MS and NMR + spectra (Figures S1–S10). The HRESIMS data (m/z = 282.0961, C11H17NNaO6, [M + Na] ) supported molecular formula C11H17NO6, suggesting four degrees of unsaturation. Its 13C NMR spectrum and HSQC experiment exhibited 11 signals including four quaternary carbons, two oxygenated methines, two methylenes and three methyls (Figure2 and Table1 ). The combined 1H NMR spectrum and COSY experiment supported the existence 1 1 of a single H- H coupling system from H2-7 to H2-9 (CH2-7–CH-8–CH2-9) (Figure3). 2 Beside the geminal coupling between the protons at C-7 (δH 2.03 and 1.33, J7a,7b = 11.5 Hz), 3 3 vicinal couplings from H-7a ( J7a,8 = 4.2 Hz) and H-7b ( J7b,8 = 11.5 Hz) to the oxygenated methine H-8 (δH 4.13, tt, J = 11.5, 4.2 Hz) were observed. Furthermore, H-8 exhibited 3 additional vicinal ( JHH) couplings to H-9a (δH 2.54, ddd, J = 11.5, 4.2, 1.8 Hz) and H-9b (δH 1.51, t, J = 11.5 Hz) completing the coupling system. Mar. Drugs 2021, 19, 214 3 of 12 Figure 2. Chemical structures of 1-5. 1 13 Table 1. NMR data of 1 (600 MHz for H and 150 for C, CDCl3). No. δC (mult.) δH [mult., J (Hz)] HMBC NOESY 2 171.5, qC H-3 3 113.3, CH 5.72 (s) H3-11 5 180.7, qC H-3, H2-7, H3-11, H3-12 6 35.0, qC H3-11, H3-12, H2-7 7a 49.8, CH2 2.03 (ddd, 11.5, 4.2, 2.4) H3-11, H3-12, H2-9 7b 1.33 (t, 11.5) 8 65.1, CH 4.13 (tt, 11.5, 4.2) H2-7, H2-9 H-7b, H3-12, H3-13 9a 47.9, CH2 2.54 (ddd, 11.5, 4.2, 1.8) H2-7, H3-13 9b 1.51 (t, 11.5) 10 86.4, qC H3-13, H-3, H2-9 11 29.9, CH3 1.31 (s) H3-12 H-3 12 25.1, CH3 1.27 (s) H3-11 H-8, H3-13 13 25.6, CH3 1.59 (s) H-8, H3-12 Figure 3. Subunits of 1 and 3, and COSY and HMBC of 1-3. 13 The C NMR resonances at δC 49.8 (CH2, C-7), 65.1 (CH, C-8), and 47.9 (CH2, C-9) are correlated to the protons at δH 2.03/1.33 (H-7a and H-7b), 4.13 (H-8), and 2.54/1.51 (H-9a and H-9b) in the HSQC experiment, supporting the assignment of these signals and the placement of OH group at C-8. The interruption of the spin-coupling system of H2-7– H-8–H2-9 on both sides suggests the quaternary nature of C-6 and C-10. The substituents at C-6 and C-10, and the existence of an amidic carbonyl (δC 180.7, C-5) were confirmed from the HMBC of H3-11/C-6, H3-12/C-6, H3-11/C-7, H3-12/C-7, H3-11/C-5, H3-12/C-5, H3-13/C-9, H3-13/C-10, H2-7/C-5, and H2-9/C-10 (Table1 and Figure3) , completing the structure of the seven-membered ring (Fragment A). The remaining elements of C2H2O4 1 13 (Fragment B) displayed two signals in the H and C NMR spectra at δH/C 171.5 (qC, Mar. Drugs 2021, 19, 214 4 of 12 C-2) and 5.72/113.3 (CH, s, H-3/C-3) corresponding to a carbonyl of a lactone moiety and an oxygenated methine. The downfield chemical shift of C-10 at δC 86.4 (qC) supported its attachment to the heteroatoms (O and N) of the lactone and amide functionalities. The HMBC of H-3/C-10, H2-9/C-10 and H3-10/C-10 supported this assignment. The appearance of the NMR signals of H-3/C-3 at δH/C 5.72/113.3 supported the attachment C-3 to the N atom of the amidic group of the seven-membered ring as well as the presence of the remaining elements (OOH) at C-3, completing the molecular formula of 1.