Marine invertebrates are a rich source of new metabolites as reported in marine libraries[1,2]. Till date majority of these compounds have been identifed from marine invertebrates sources predominantly sponges.Marine sponges are widely distributed from intertidal zones to thousands of meters deep in the ocean. The demosponge, Ircinia fusca (Carter 1880) is a commonly found on the intertidal rocky shores of the Arabian Sea,India. Sponges of the genus Iricina have been proven to be a rich source of diverse secondary metabolites like Cheilanthane sesterterpenoids, Irciniastatins, quinones  and Ircinialactams . Some of the metabolites from Iricinia sps exhibited antifouling, antiinﬂammatory and antimicrobial activities. In addition to this, a number of cytotoxic compounds have also been reported from Iricinia genus[10,11,12].
Material and Methods
General experimental details
Optical rotations were determined on a Rudolph Research Analytical (AUTOPOL V) polarimeter at a wavelength of 589 nm (sodium D line) using a1.0-decimeter cell with a total volume of 1.0mL. The UV spectra were measured on Agilent technologies carry series UV-VIS spectrophotometer and Infrared spectra on Bruker ALPHA. All solvents were of analytical grade. Column chromatography was performed on Merck silica gel (120-200 mesh) and Sephadex LH-20 (Sigma-Aldrich Chemie GmbH). Thin layer chromatography was carried out with silica gel GF254 plates, Merck, USA. The 1H and 13C, DEPT-135, COSY, TOCSY, HSQC, HMBC, ROESY and 400 MHz (or 100 MHz for 13C) at Bruker 400 MHz (Internal standard: TMS). The chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz. The positive ion HR-ESI-MS spectrum was recorded on Mass Q-TOF-LC-MS spectrometer (Bruker Daltonics).
Collection of sponge
The sponge Ircinia fusca (Carter, 1880) was collected from Vayngani (N 16055.827, E 073016.973),West coast of Maharashtra, INDIA, in Feb 2016. The sponge was identifed by Dr. Satish S.Mokashe, Associate Professor, Department of Zoology, Dr. Babasaheb Ambedkar Marathwada University, INDIA.
Extraction and isolation
In the laboratory, the sponge was washedwith distilled water to remove surface salts, sand, and epiphytes. The sponge was dabbed with tissue paper to remove excess water, cut into small pieces and placed in a lyophilizer to completely dry. The dried material was (2g) reduced to small pieces and extracted with methanol. Desalting of sponge methanolic extract with acetone. The methanolic extracts were concentrated under vacuum using a rotavapor at 400 C followed by a partition with hexane. All the partition layers were subjected to preliminary bioactivity studies (antibacterial & antifungal) by disc diﬀusion method .
The isolated compounds 1–2 were tested against antibacterial i.e., Escherichia coli (NCIM 2065),Salmonella typhimurium (NCIM 2501), Bacillus subtilis (NCIM 2063), Staphylococcus aureus (NCIM 2079), Mycobacterium smegmatis (NCIM 5138) and antifungal strains Aspergillus niger (NCIM 1207), Penicillium chrysogenum (NCIM 1315), Alternaria sp (NCIM 900),and Fusarium sp (NCIM 1372). The crude extracts weredissolved in DMSO at a concentration of 1 mg/mL. The discs were loaded with diﬀerent concentration’s (10- 500μg/disk) of the pure compound, to fnd out the inhibitory potential. The diameters of the inhibition zones generated around the discs were measured (Ø in mm). The tests were performed in triplicate and the mean values are given in Table 1. DMSO used to dissolve the extracts and the compounds were checked for the absence of antimicrobial activity. The diameters of the halos of inhibition can be rationalized on a qualitative basis as follows: Ø < 7 mm: inactive, 7 mm ≤ Ø < 8 mm: slightly active, 8 mm ≤ Ø <9 mm: signifcantly active, Ø ≥ 9 mm: very active. Thecompound which showed ≥ 9 mm was selected for MIC studies.
Cytotoxicity was evaluated against HeLa, SiHa (cervical cancer cells) and MDA-MB-231 (Breast cancercell line) lines. Cell proliferation was followed by the colorimetric MTT test .
During the course of our search for bioactive substances from marine sponges, we collected Iricnia fusca from the Arabian Sea, West coast of Maharashtra, India. The crude methanolic extract of the specimen exhibited cytotoxicity in preliminary studies was selected for further purifcation. Chromatographic separation of the MeOH extracts using C18 semi-preparative reverse phase HPLC yielded lead two new compounds i.e.: compound 1 (10mg), 2 (13 mg), as showed in Fig. S1. Herein we describe the structure elucidation and bioactivity metabolites.
Compound 1 was obtained as white color. The specifc rotation was [α]25D = 0.1 (c 1.0, CH3OH). The ESI-MS-QTOF exhibited a pseudo molecular ion peak at m/z 251.18 [M+H] + (Fig. S2), corresponding to the molecular formula of C 11H17N5O2, indicating six degrees of unsaturation. The chemical shifts of three carbons at δ C 138.7 (CH, H-2), 131.8 (C), and 123.4 (CH, H-5) was a characteristic feature of imidazole ring with C-4 sum tuition . The presence of methylene at δH 3.94 (2H, s, H-6) and HMBC correlations H-6 to δ C 171.1 (amide carbon), C-2 (δC 138.7 ), C-5 (δC 123.4) revealed N-methyl-Imidazole- 4-yl- acetamide moiety in compound 1. Further 1H NMR data showed two singlets at δH 3.08 & 3.87 as N-methyl protons, one methine proton at δH 3.68 (1H, t, J = 6.5 Hz, 4ꞌ), single methylene at δH 3.10 (2H, d, J = 6.5 Hz, 5ꞌ) as showed in Table 1. The COSY correlations between H-4ꞌ/ H-3ꞌ, as well as the HMBC correlations δH 3.08 (N- methyl proton) and H-4ꞌ to a quaternary carbon δC 157.8, (C-2ꞌ) was a characteristic feature of the 1,3 dimethyl -2-Oxoimidazole as showed in Fig. 2.
The long range HMBC correlations of H-4ꞌ with C-6 (δC 29.6) confrmed that compound1 has N-(1,3-dimethyl- 2-oxoimidazolidin-4-yl)-2-(1-methyl-1H-imidazol-4-yl) acetamide, this was supported by the NOE cross peaks H-4ꞌ to H-6 as showed in Fig. 2. Thus, the planar structure of 1 was elucidated as shown in Fig. 1 named as Iricimidazole.
Compound 2 was obtained as pale yellow viscous, and its molecular formula was calculated as C 11H12O6Na from HR-ESI-MS at m/z 263.21 [M+Na] + (Fig. S 11) indicating six degrees of unsaturation. The specifc rotation was [α]25D = -1.6 (c 1.0, CH3OH).The IR spectrum showed absorption bands for 1675, 1195, 1136 cm-1 suggested the presence of ester and ether groups in the molecule, respectively. The 13C NMR spectrum of 2 exhibited total of 11 carbons (Table 2) including two carbonyl carbons δ C 169.2(C-2), δC 164.4(C-6ꞌ), one quaternary carbon C-3ꞌ (δC 152.5), six methine sp2 carbons δC 86.5 (C-4), 71.1 (C-3), 75.1(C-1ꞌ), 90.4 (C-2ꞌ), 142.7 (C-4ꞌ), 102.3 (C-5ꞌ), oxymethylene sp2 carbons at δC 62.1 (C-5), methoxy sp2 carbon at δ C 53.3.
The COSY correlations of H-3/H-4/H-5 indicate an isolated spin system, as well as HMBC correlations of H-5 to C-2 Fig. 3 confrmed the dihydrofuran 2-one framework in compound 2. The presence of COSY correlations H-4ꞌ/H-5ꞌ and H1ꞌ/H-2ꞌ exhibited AB spin system as showed in Fig. 3, and HMBC correlations of H-4ꞌ to C-3ꞌ, C-6ꞌ revealed the presence of cyclohexene ring. The long range HMBC correlations of methoxy proton at δH 3.35 to C-3ꞌ asserted the location of a methoxy group at C-3ꞌ position and H-2ꞌ/H-5ꞌ to C-4ꞌ, C-3ꞌ confrmed the 4 hydroxy - 3-methoxy-6-oxocyclohexa- 2,4-dien- 1-yl moiety. Further, HMBC cross peaks of H-1ꞌ and to C-3 (δC 71.1) and H-4 to C-1ꞌ establishes that compound1 has 4-hydroxy-3-((3-methoxy-6-oxocyclohexa-2,4- dien-1-yl)oxy) dihydrofuran-2(3H)-one. Thus, the planar structure of 1 was elucidated as shown in Fig.1, named as ‘Dihydrofuranolꞌ The NOE cross peaks of δH 3.35 to H-5ꞌ and H-1ꞌ supports the above confrmation as showed in Fig. 3.
Compounds 1 & 2 have been reported as new metabolites from I.fusca and its structure was elucidated by NMR and mass spectroscopic analysis. Isolated Compound 1 and 2 were also evaluated for their antifungal and antibacterial activity, but none of them showed signifcant inhibitory activity. Compounds 1 & 2 exhibited cytotoxic activity against HeLa, SiHa (cervical cancer cells), and MDA-MB-231(Breast cancer cell line) lines with IC 50 values 200 µg/mL.
Compound 1: 1H NMR (400 MHz, MeOD) δ 8.87 (s, 1H), 7.53 (s, 1H), 3.93 (s, 2H), 3.87 (s, 3H), 3.68 (t, J = 6.5 Hz, 1H), 3.10 (d, J = 6.5 Hz, 2H), 3.08 (s, 3H).
Compound 2:1H NMR (500 MHz, MeOD) δ 8.01 (d, J = 8.3 Hz, 1H), 5.90 (d, J = 4.8 Hz, 1H), 5.70 (d, J = 8.3 Hz, 1H), 4.18 (d, J = 4.8 Hz, 1H), 4.15 (d, J = 4.7 Hz, 1H), 4.01 (dd, J = 7.2, 4.7 Hz, 1H), 3.80 (m, 2H), 3.35 (s, 3H).