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Bio-active sesquiterpenoids and norsesquiternoids from the Red Sea octocoral fulvum fulvum

Seif-Eldin Nasr Ayyad, Mohamed Ali Deyab, Tamer Kosbar, Walied Mohamed Alarif & Ahmed Hassan Eissa

To cite this article: Seif-Eldin Nasr Ayyad, Mohamed Ali Deyab, Tamer Kosbar, Walied Mohamed Alarif & Ahmed Hassan Eissa (2019): Bio-active sesquiterpenoids and norsesquiternoids from the Red Sea octocoral Rhytisma￿fulvum￿fulvum, Natural Product Research, DOI: 10.1080/14786419.2019.1709187 To link to this article: https://doi.org/10.1080/14786419.2019.1709187

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2019.1709187

Bio-active sesquiterpenoids and norsesquiternoids from the Red Sea octocoral fulvum

Seif-Eldin Nasr Ayyada, Mohamed Ali Deyabb, Tamer Kosbara, Walied Mohamed Alarifc and Ahmed Hassan Eissaa aDepartment of Chemistry, Faculty of Science, Damietta University, Damietta, Egypt; bBotany Department, Faculty of Science, Damietta University, Damietta, Egypt; cDepartment of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

ABSTRACT ARTICLE HISTORY Three previously undescribed nardosinane-type sesquiterpenes Received 16 September 2019 (1–3), together with six known compounds (4–9) were isolated Accepted 11 December 2019 from the alcyonacean soft coral Rhytisma fulvum fulvum. 2 and 3 are 13-nornardosinane, and 6,7-seco-13-nornardosinane deriva- KEYWORDS tives, respectively. 2 could be an artifact due to C-6 epimerization Red sea; soft coral; sesquiterpenes; cytotoxic; of the known one 4. Chemical structures were elucidated based antimicrobial on 1 D, 2 D NMR and MS spectral data. Compounds 1-8 showed cytotoxic activity against NCI-H1299, HepG2 and MCF-7 with IC50 values between 0.0352–0.0974, 0.0717–0.3745 and 0.0341–0.1325 mM, respectively. The antibacterial activity of all isolated compounds have examined against a number of Gram- positive (Bacillus cereus, Staphylococcus aureas) and Gram-negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas sp.) bac- teria. Compounds 6 and 7 showed a high degree of inhibition against B. cereus, S. aureus and Pseudomonas sp. Interestingly, neolemnane (6) showed strong inhibition against two fungi, Aspergillus niger and Fusarium oxysporum; while 8 showed positive inhibition against Fusarium oxysporum at 150 mg/mL.

CONTACT Seif-Eldin Nasr Ayyad [email protected] Department of Chemistry, Faculty of Science, Damietta University, Damietta, Egypt. Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1709187. ß 2019 Informa UK Limited, trading as Taylor & Francis Group 2 S.-E. N. AYYAD ET AL.

1. Introduction The Genera Rhytisma, family , an Indopacific soft coral exists in two colour morphs: yellow-brown and grey, was initially identified from the Red Sea waters (Benayahu and Loya 1983;Alderslade2000). Interestingly, in spite of its taxonomic classification as an Alcyoniidae member, the secondary metabolites profile of Rhytisma revealed its close similarity to those of two Genera Lemnalia and Paralemnalia belonging to the family Nephtheidae. Nevertheless, The DNA of Rhytisma showed its closeness to the family Xeniidae rather than the Alcyoniidae (Haverkort-Yeh et al. 2013). To the best of our knowledge, 28 metabolites were reported from the Rhytisma including: sesquiterpenes, norsesquiterpenes and bis-sesquiterpenes as well as steroids (Kashman 1979; Bowden et al. 1980; Green et al. 1992; Jurek and Scheuer 1993; Wessels et al. 2001; Bishara et al. 2008; Trifman et al. 2016). In The present work the secondary metabolite content of the soft coral Rhytisma fulvum fulvum (family Alcyoniidae), collected from the Red Sea Coast at Jeddah, Saudi Arabia was investigated. Fractionation and purification of the organic extract of this were achieved by successive column and preparative Thin-Layer chromatography (TLC), led to isolation and identification of nine compounds (1–9)(Figure 1).

Figure 1. Isolated compounds from Rhytisma fulvum fulvum. NATURAL PRODUCT RESEARCH 3

2. Results and discussion 2.1. Structure elucidation Compound 1 was isolated as optical active yellow oil. HRESIMS and 13C NMR assigned the 1 13 molecular formula as, C17H26O3,andfivedouble-bondequivalents.The H- and CNMR spectra revealed the presence of one carbonyl (dC 212.9 C), an acetyl (dC 170.7 C, 20.7 CH3; dH 2.02 s) and a tri-substituted double-bond (dC 137.5 C, 123.8 CH; dH 5.56 d) (cf. exp., Figure S2). The aforementioned functionalities account for three unsaturation degrees, sug- gesting a bicyclic structure. In addition to the methyl of acetate group, 1HNMRspectra indicated the presence two secondary methyls (dH 0.94, d and 0.81, d) and a tertiary one 13 3 (dH 0.90, s). Further analysis of the C and DEPT NMR experiments displayed one sp qua- 3 ternary carbon (dC 42.7), three sp methines (dC 61.9, 32.8 and 31.3), one oxygenated- methylene (dC 66.1), as well as four upfield-methylenes (40.5, 30.6, 26.6 and 25.5). With the aid of HSQC experiments, all protons were accounted and the compound contains no hydroxyl function. From the 1H-1H COSY spectrum, three separate couplings in the form of cross-peaks were observed between; the first one being between H-12 and H-11, H-11 and H-13, the second H-2 coupled with H-3, H-3 coupled with H-4, and H-4 coupled with H-14, while the third one noted started from the olefinic proton H-7 resonating at dH 5.56 to H- 8, H-8 to H-9 and H-9 to H-10 (As depicted in Figure S1). HMBC correlations established the connections of CH3-14 to C-4 and CH3-15 to C-5, as well as H-11 to C-6, C-13 and C12 (Figure S1). The above-mentioned observations and reviewing literatures on metabolites isolated from the genus Rhytisma reflect the presence of a nardosinane-type sesquiterpene. The locations of the carbonyl and the acetate functions were established from the HMBC spectra as shown in Figure S1.Thestructureofcompound1, namely, 12-O-acetyl-nardo- sinan-6-en-1-one is shown in Figure 1. The relative stereochemistry of 1 was concluded by studying the NOESY cross-peaks. NOEs observed between all the methyl protons (Me-13, 14 and 15) suggest their co-facial orientation, the b-side; consequently, the a-side is occu- pied by H-4 and H-10. The absolute stereochemistry of 1 was concluded as depicted in Figure 1, on the basis biogenetic considerations and on comparison of the obtained 13C NMR data with those in the literature (Bishara et al. 2008;Dalozeetal.1977). Compound 2 was isolated as optical active yellow oil. HRESIMS and 13C NMR assigned the molecular formula as, C14H20O3, and five double-bond equivalents. Analysis of its 13C NMR spectrum and DEPT spectra showed the presence of two car- bonyl functions (dC 207.3 and 206.1 ppm), which accounted for two oxygen-atoms (cf. exp., Figure S3). The absence absorption due to hydroxyl functions in the IR spectrum; assigned the third oxygen atom to an etheric function. Based on the absence of reso- nances in the low-field region, compound 2 is a tricyclic skeleton. The presence of 1 13 oxirane ring was evidenced from the H- and C NMR absorptions (dC 65.1 C and 61.8; 1 1 dH 3.16 d). Extensive studies of HSQC, H- H COSY and HMBC spectral data (Figure S1) indicated a 13-nornardosinane structure (Izac et al. 1982; Jurek and Scheuer 1993, Bishara et al. 2008). On comparing the obtained spectral data of 2 with these of the known co-occurred 6a-acetyl-4b,5b-dimethyl-1(10)-a-epoxy-7-oxodecalin (4) (Izac et al. 1982; Jurek and Scheuer 1993), the orientation of the H-6 proton is being the only dif- ference. The NOESY correlation cross-peaks were observed between the methyl pro- tons of CH3-14 and CH3-15, as well as H-1. No NOEs observed between the latter 4 S.-E. N. AYYAD ET AL. protons (b-side) and H-6, which assigns a-configuration to H-6. Analysis of coupling con- stants revealed the absence of w-coupling between H-6 and Heq-8, confirmed the C-6 b-substituent. Compound 2 was assigned as 6b-acetyl-1(10)-a-13-nornardosin-7-one. The new compound 2 is an isomer to 4 (which previously isolated from Paralemnalia thyrsoide) (Izac et al. 1982). Compound 4, also isolated in this article from R. fulvum ful- vum and the 1Hand13C NMR spectral data of 4 was in good agreement with those reported in the literature (Izac et al. 1982). However, adsorption of 4 on Si gel for hours resulted in epimerization at C-6 and provided a mixture of both compounds 2 and 4. Therefore, generation of 2 from 4 during isolation cannot be excluded. Compound 3 was isolated as faint yellow oil. EIMS, HRESIMS and 13C NMR assigned the molecular formula of 3 as, C15H24O4, with four unsaturation equivalents. Two sites of unsaturation were accounted as two carbonyl groups; a ketone and an ester functions

(dC 208.6 and 173.7; respectively), implies a bicyclic skeleton. DEPT experiments showed the presence of 4 methyl, 5 methylene, 2 methine and 4 quaternary carbons. 1H- and 13C NMR spectra, as well as HSQC experiments (Figure S4), evidenced the presence of a tri-substituted epoxide ring (dC 62.9 C, 55.6 CH; dH 2.89 t), an oxygenated methyl (dC 51.4 CH3; dH 3.64 s), one secondary methyl (dC 15.8 CH3; dH 0.84 d) and two tertiary methyls (dC/dH 18.0 CH3/1.00 s and 32.4 CH3/2.19 s). After accounting for all oxygen- atoms, compound 3 is a monocarbocyclic structure fused with an oxirane ring (cf. exp.). The 1H-1H COSY spectrum indicated the presence of isolated methylene protons, H-6 protons and two separate couplings in the form of cross-peaks between; the first one being between proton signals from H-1 to H-14 and the second between H-8 and H-9 protons (Figure S1). HMBC correlations established the position of the: oxirane ring (H-1 to C-2 and C-10), Me-14 and Me-15 (H-15 to C-10, C-4 and C-6), and the two carbonyls (H-6 to C-5 and C-11; H-8 to C-7 and C-10, respectively). After careful examination the spectral data of 3 and on comparing with those reported in the literature (Jurek and Scheuer 1993,Bisharaetal.2008;Dalozeetal.2010), compound 3 is a 6,7-seco-13- nornardosinane. The relative stereochemistry of 3 was concluded by studying the NOESY cross-peaks. NOEs observed are closely similar to those of compound 2,indicatingthe b-orientation of H-14 and H-15, along with the a-position of the epoxide ring. In addition to three new compounds (1–3), we have isolated several known compounds (Figure 1) from R. fulvum fulvum:6a-acetyl-1(10)-a-13-nornardosin-7-one (4) (which previously isolated from Paralemnalia thyrsoide) (Izac et al. 1982), 12-Acetoxy-l(10)-aristolene (5) and 4-Acetoxy-2,8-neolemnadien-5-one (6) (which previ- ously isolated from Lemnalia africana) (Jurek and Scheuer 1993), besides, a cembra- noid-diterpene Nephthenol (7) (which previously isolated from Nephthea sp.) (Schmitz et al. 1974) and two steroids 24-Methylcholesterol (8) and 23,24-methylenecholesterol (9) (which previously isolated from diverse natural sources, in particular from soft corals such as Sinularia sp., Sinularia dura, Lobophytum sp. and others) (Subrahmanyam et al. 1992; Radwan et al. 2008; Putra et al. 2012).

2.2. Biological activities All isolated compounds showed cytotoxic activity against different human cancer cell lines HepG2 (hepatocellular liver carcinoma), NCI-H1299 (lung carcinoma) and MCF-7 NATURAL PRODUCT RESEARCH 5

(breast carcinoma). Compounds 1, 5, 6 and 7 displayed strong significant cytotoxicity against cell line NCI-H1299 and moderate cytotoxicity against HepG2 cell line. Compounds 2-4 showed a moderate cytotoxicity against NCI-H1299 and weak response for HepG2. Only compounds 7 and 8 have been evaluated against MCF-7 cell, where they showed strong and weak responses, respectively (Table S1). Compounds 18 were tested for their antimicrobial activity, against two Gram- positive bacteria (Bacillus cereus and staphylococcus aureus), three bacteria of Gram- negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas sp.) and three fungi (Aspergillus niger, Fusarium oxysporum and Candida albicans). Compounds 6 and 7 showed a high degree of inhibition against B. cereus, staphylococcus aureus and Pseudomonas sp. while showed a mild inhibition against E. coli and K. pneumoniae in comparison with penicillin G as a standard drug (Table S2). For compounds 3 and 8,no inhibition activities were observed, while compounds 1, 2, 4 and 5 showed appreciated activities in some bacteria (Table S2). Compound 6 showed good inhibitions against two fungi (Aspergillus niger, Fusarium oxysporum), while compound 8 showed reason- able inhibition against only the fungus, Fusarium oxysporum at 150 mg/mL. For all other compounds, no activity was observed in the other three tested fungi (Table S3).

3. Experimental 3.1. General

1D and 2D NMR spectra were recorded in CDCl3 on Joel ECA- II 500 MHz NMR spec- trometer and Bruker Avance III 400 MHz NMR spectrometer, at NMR Unit Center, Faculty of Science and Faculty of Pharmacy, Mansoura University. EIMS analyses were carried out on a Thermo, USA Spectrometer with detector MS (Ultra DSQ II) at Central lab, National Institute of Oceanography and Fisheries, Alexandria, Egypt. Thin-layer chromatography (TLC) was performed on silica gel G60 F254 (Merck, Darmstadt, Germany) of 0.2 mm layer thickness and Silica gel 60 for column chromatography (60–120 mesh LR). P-anisaldehyde was the spray reagent.

3.2. Animal material The soft coral R. fulvum fulvum was collected from the Red Sea Coast at Jeddah, Saudi Arabia, in April 2017, and was identified by Prof. Mohsen El-Sherbiny, Marine biology department, Faculty of Marine Sciences, King Abdulaziz University. A voucher specimen (RFF18-77) was deposited at Marine biology department, Faculty of Marine Sciences, King Abdulaziz University.

3.3. Extraction and isolation The partially dried (200.0 g) were extracted with a mixture of equal volume of

CHCl3/MeOH (3 1 L) at room temperature. Evaporation to dryness yielded 15.0 g of an oily residue. The residue was homogenised with an amount of silica gel 60 A, and was loaded in column chromatography (60 2.5 cm) using silica gel 60 A (400.0 g) and eluted using petroleum ether with increasing proportions of CH2Cl2 and EtOAc to yield 6 S.-E. N. AYYAD ET AL.

fractions, each of 50 ml. The fraction eluted with petroleum ether-CH2Cl2 (9:1) afforded compound 5, with petroleum ether-CH2Cl2 (6:4) afforded compound 6 and 7, with pet- roleum ether-CH2Cl2 (5:5) to give compound 1, with petroleum ether-CH2Cl2 (4:6) afforded compounds 8 and 9, and the fraction eluted with petroleum ether- EtOAc (9:1) gave compounds 2–4. All fractions were purified by preparative TLC plate (20 20 cm, 0.25 mm thickness) with appropriate solvent systems to give nine compounds (1–9).

3.4. Spectral data 3.4.1. 12-O-acetyl-nardosinan-6-en-1-one (1) 22 1 Yellowish oil (4.2 mg, 0.0021%); [a] D - 128.0 (c 0.01, CH2Cl2); IR Vmax (film) cm : 2964 and 2927 (C-H), 1738 (Ac), 1704 (C ¼ O), 1460, 1368, 1234, 1181, 1034; 1H NMR

(CDCl3, 500 MHz): 2.41 (1 H, ddd, 15.0, 3.5, 2.0 Hz, H-2a), 2.30 (1 H, ddd, 15.0, 6.5, 4.0 Hz, H-2b), 1.46 (1 H, m, H-3a), 1.42 (1 H, m, H-3b), 2.14 (1 H, m, H-4), 5.56 (1 H,d, 5.5 Hz, H- 7), 2.63 (1 H, m, H-8a), 2.45 (1 H, m, H-8b), 2.12 (1 H, m, H-9a), 1.97 (1 H, m, H-9b), 2.33 (1 H, dd, 3.5, 1.5 Hz, H-10), 2.15 (1 H, m, H-11), 4.51 (1 H, dd, 10.5, 3.5 Hz, H-12a), 3.68 (1 H, dd, 10.5, 10.5 Hz, H-12b), 0.94 (3 H, d, 7.0 Hz, H-13), 0.81 (3 H, d, 7.0 Hz, H-14), 0.90 13 (3 H, s, H-15), 2.02 (3 H, s, CH3C ¼ O), C NMR (CDCl3, 125 MHz): 212.9 (C, C-1), 40.5

(CH2, C-2), 26.6 (CH2, C-3), 32.8 (CH, C-4), 42.7 (C, C-5), 137.5 (C, C-6), 123.8 (CH, C-7), 30.6 (CH2, C-8), 25.5 (CH2, C-9), 61.9 (CH, C-10), 31.3 (CH, C-11), 66.1 (CH2, C-12), 17.6 (CH3, C-13), 15.1 (CH3, C-14), 21.6 (CH3, C-15), 170.7 (C ¼ O, Ac), 20.7 (CH3, Ac); EIMS m/ þ þ z 278 (6) [M, C17H26O3] , 263 (8) [M – CH3], 203 (44) [M–C3H7O2] , 135 (89) þ þ [M–C8H15O2] , 107 (100) [M–C10H19O2] ; HRESIMS (positive mode) m/z ¼ 279.1952 þ [M þ H] (Calculated m/z ¼ 279.1960 for C17H27O3).

3.4.2. 6-b-1(10)-a-13-nornardosin-7-one (2) 1 Yellowish oil, isolated as mixture with 4 (4.0 mg, 0.0020%); IR Vmax (film) cm : 2964 1 and 2935 (C-H), 1728 and 1701 (C ¼ O), 1464, 1357, 1242, 1165, 1038; H NMR (CDCl3, 500 MHz): 3.16 (1 H, brd, 2.0 Hz, H-1), 1.91-1.97 (2 H, m, H-2), 1.25 (1 H, m, H-3a), 1.15 (1 H, m, H-3b), 1.92 (1 H, m, H-4), 3.74 (1 H, s, H-6), 2.70 (1 H, ddd, 15.0, 11.0, 6.0 Hz, H- 8a), 2.45 (1 H, ddd, 15.0, 8.5, 5.5 Hz, H-8b), 1.33-1.37 (2 H, m, H-9), 2.20 (3 H, s, H-12), 13 0.65 (3 H, d, 6.5 Hz, H-14), 1.21 (3 H, s, H-15), C NMR (CDCl3, 125 MHz): 61.8 (CH, C-1), 22.2 (CH2, C-2), 25.4 (CH2, C-3), 33.9 (CH, C-4), 45.0 (C, C-5), 69.1 (CH, C-6), 206.1 (CH,

C-7), 38.4 (CH2, C-8), 29.9 (CH2, C-9), 65.1 (C, C-10), 207.3 (CH, C-11), 34.1 (CH2, C-12), þ 18.0 (CH3, C-14), 14.1 (CH3, C-15); EIMS m/z 236 (46) [M, C14H20O3] , 221 (12) [M–CH3], þ þ 163 (62) [M–C4H9O] , 109 (100) [M–C7H11O2] ; HRESIMS (positive mode) m/ þ z ¼ 237.1483 [M þ H] (Calculated m/z ¼ 237.1491 for C14H21O3).

3.4.3. 6,7-seco-13-nornardosinane (3) 22 1 Pale yellow oil, (7.0 mg, 0.0035%); [a] D þ 181.0 (c 0.01, CH2Cl2); IR Vmax (film) cm : 2954 and 2931 (C-H), 1738 (C ¼ O), 1704 (C ¼ O), 1438, 1358, 1261, 1169, 1039; 1H

NMR (CDCl3, 500 MHz): 2.89 (1 H, t, 2.5 Hz, H-1), 1.81-1.85 (2 H, m, H-2), 1.22-1.29 (1 H, m, H-3a), 1.12-1.20 (1 H, m, H-3b), 1.83 (1 H, m, H-4), 2.57 (2 H, dd, 13.5, 13.5 Hz, H-6), 2.23 (1 H, m, H-8a), 2.14 (1 H, m, H-8b), 2.25 (1 H, m, H-9a), 2.14 (1 H, m, H-9b), 2.19 13 (3 H, s, H-12), 0.84 (3 H, d, 7.0 Hz, H-14), 1.00 (3 H, s, H-15), 3.64 (3 H, s, OCH3), C NMR NATURAL PRODUCT RESEARCH 7

(CDCl3, 125 MHz): 55.6 (CH, C-1), 21.6 (CH2, C-2), 24.0 (CH2, C-3), 31.1 (CH, C-4), 40.8 (C, C-5), 48.2 (CH, C-6), 173.7 (C, C-7), 28.0 (CH2, C-8), 25.0 (CH2, C-9), 62.9 (C, C-10), 208.6 (CH, C-11), 32.4 (CH3, C-12), 15.8 (CH3, C-14), 18.0 (CH3, C-15), 51.4 (CH3, OCH3); EIMS þ þ þ m/z 268 (4) [M, C15H24O4] , 195 (54) [M–C3H5O2] , 135 (40) [M–C6H13O3] , 109 (100) þ þ [M–C8H15O3] ; HRESIMS (positive mode) m/z ¼ 269.1753 [M þ H] (Calculated m/ z ¼ 269.1746 for C15H25O4).

3.5. Biological evaluation 3.5.1. Cytotoxicity assay Human tumour carcinoma cell lines HepG2 (hepatocellular carcinoma) NCI-H1299 (lung carcinoma), MCF-7 (human breast carcinoma) used in this study were obtained from American Type Culture Collection (ATCC, Minnesota, USA). The tumour cell lines were maintained at the National Cancer Institute, Cairo, Egypt, by serial sub-culturing. The cytotoxicity potential was carried out using sulforhodamine B (SRB) colorimetric assay (Vichai and Kirtikara 2006).

3.5.2. Antimicrobial activity assay Agar well diffusion method was applied to investigate the antimicrobial activity of the compounds at concentrations of 50, 100 and 150 lg/mL in DMSO, which was also applied alone as a control. The antibacterial activities against (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumo- niae) were tested using nutrient agar medium. The antifungal activities were also tested against three local fungal (Aspergillus niger, Fusarium oxysporum and Candida sp.) using DOX agar medium. Wells (10 mm) were made by a sterile cork borer in agar medium that was inoculated with the tested microorganisms. In each well, we applied 200 lL of the tested compound. We kept the agar plates at 4 C for at least 30 minutes, until the complete diffusion of the tested derivative; then plates were incubated at 37 Cor30C for bacteria and fungi, respectively. Penicillin G and miconazole were used as standard antibacterial and antifungal, respectively. The anti- microbial activities were assayed in terms of inhibition zone diameters after 24 hours and 7 days for both bacteria and fungi, respectively.

4. Conclusion Three new nardosinane-type sesquiterpenes along with six known compounds were isolated for the first time from Rhytisma fulvum fulvum. The diversity of metabolites refers to the richness of the genus Rhytisma in secondary metabolites and the highly effect of the environment on the production of such metabolites. Generally, the iso- lated compounds displayed antiproliferation and antimicrobial activities, amongst these; the antifungal activity of the lemnane- derivative (6) is of considerable interest.

Acknowledgments The authors wish to thank Mr. Kamal Al-dahodi, Faculty of Maritime studies, King Abdulaziz University, for sample collection. 8 S.-E. N. AYYAD ET AL.

Disclosure statement No potential conflict of interest was reported by the authors.

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