Two new triterpenoids and a new naphthoquinone derivative isolated from a hard coral-derived fungus Scopulariopsis sp.

    Two new triterpenoids and a new naphthoquinone derivative isolated from a hard coral-derived fungus Scopulariopsis sp. Mohamed S. Elnaggar, Sherif S. Ebada, Mohamed L. Ashour, Weaam Ebrahim, Abdelnasser Singab, Wenhan Lin, Zhen Liu, Peter Proksch PII: DOI: Reference:

S0367-326X(16)30309-4 doi:10.1016/j.fitote.2016.12.003 FITOTE 3540

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

2 September 2016 29 November 2016 1 December 2016

Please cite this article as: Mohamed S. Elnaggar, Sherif S. Ebada, Mohamed L. Ashour, Weaam Ebrahim, Abdelnasser Singab, Wenhan Lin, Zhen Liu, Peter Proksch, Two new triterpenoids and a new naphthoquinone derivative isolated from a hard coral-derived fungus Scopulariopsis sp., Fitoterapia (2016), doi:10.1016/j.fitote.2016.12.003

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ACCEPTED MANUSCRIPT Two new triterpenoids and a new naphthoquinone derivative isolated

a,b

, Sherif S. Ebada b, Mohamed L. Ashour b, Weaam

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Mohamed S. Elnaggar

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from a hard coral-derived fungus Scopulariopsis sp.

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Ebrahim a,c, Abdelnasser Singab b, Wenhan Lin d, Zhen Liu a,*, Peter Proksch a,*

a

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Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-Universität Düsseldorf,

Universitätsstrasse 1, 40225 Düsseldorf, Germany b

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Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Abbassia, Cairo

11566, Egypt c

Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516,

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 100191 Beijing,

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d

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Egypt

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China

*Corresponding authors.

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Tel.: +49 211 81 14163; fax: +49 211 81 11923; e-mail: [email protected] (Z. Liu) [email protected] (P. Proksch)

ACCEPTED MANUSCRIPT ABSTRACT: Scopulariopsis sp. isolated from the Red Sea hard coral Stylophora sp. yielded two

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new triterpenoids (1–2) and a new naphthoquinone derivative (8) when cultured on

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white beans. In addition, fourteen known compounds including three triterpene

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analogues (3–5), two sesquiterpernoids (6–7), two polyketides (9–10) and seven nitrogenous compounds (11–17) were isolated. All structures were determined through extensive analysis of the NMR and MS data as well as by comparison with

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literature data. All isolated compounds were evaluated for their cytotoxic,

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antibacterial and antitubercular activities. However, none of them showed significant

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activity.

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Keywords: Scopulariopsis sp.; Coral-derived fungi; OSMAC; triterpenoids.

ACCEPTED MANUSCRIPT 1. Introduction Marine microorganisms especially fungi isolated from marine sources are

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considered to be continuous prolific sources of bioactive and structurally unique

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natural products and attract a substantial attention of many researchers [1–2]. One of

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the important facets in natural product research focuses on diversifying the chemical profiles of microorganisms that provide bioactive secondary metabolites. Wholegenome sequencing programs of marine microorganisms proved that the biosynthetic

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potential of fungi and bacteria had been widely under estimated. Under standard

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laboratory conditions not all biogenetic gene clusters are transcribed leading to depauperated metabolite profile. Several strategies exist to induce cryptic gene

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clusters thereby taking advantage of the true metabolic capacity of microorganisms

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with regard to the accumulation of secondary products [3]. One of these strategies involves the change of culture parameters such as media composition, pH,

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temperature, oxygen supply level and culture flasks, that is summarized as OSMAC (One Strain Many Compounds) approach [4]. The OSMAC approach has repeatedly

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been shown to induce the formation of new metabolites and/or to enhance the accumulation of constitutively present compounds [5]. In a previous study on this hard coral-derived fungus Scopulariopsis sp., several xanthones, bisabolane-type sesquiterpenoids, alkaloids and polyketides were isolated from solid rice cultures of the fungus [6]. Owing to this remarkable structural diversity of isolated compounds, we initiated now a further study on this fungus in order to explore its metabolic potential when grown on white beans. Switching the culture media from a carbohydrate rich medium such as rice to a protein rich medium like white beans has already in the past caused significant changes of fungal metabolites. For example, when the fungal endophyte Talaromyces wortmannii was

ACCEPTED MANUSCRIPT grown on rice medium it was mainly found to produce dimeric anthracene derivatives whereas the same fungus when grown on white beans yielded cyclic peptides [7–8].

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Detailed investigation of Scopulariopsis sp. when grown on white beans yielded now

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two new triterpenoids (1–2) and a new naphthoquinone derivative (8) as well as

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fourteen known compounds (3–7, 9–17) (Fig. 1). None of the metabolites isolated in this study had been detected when the fungus was cultivated on rice medium, thereby

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proving the power of the OSMAC approach as applied in this study.

Fig. 1. Structures of isolated compounds from Scopulariopsis sp.

ACCEPTED MANUSCRIPT 2. Results and discussion Compound 1 was isolated as white amorphous powder. Its molecular formula

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was established as C30H46O6 by the HRESIMS data, missing two H atoms compared

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to 3. The UV spectrum of 1 exhibited absorbances at λmax 249 nm, suggesting the

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presence of a conjugated enone moiety. In the 1H NMR spectrum of 1 (Table 1), an olefinic proton at δH 5.73 (1H, s, H-12), five oxygenated protons at δH 4.21 (1H, dd, J = 11.7, 5.0 Hz, H-15), 4.07 (1H, d, J = 11.1 Hz, H-24a), 4.00 (1H, dd, J = 11.4, 4.6

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Hz, H-7), 3.44 (1H, d, J = 11.1 Hz, H-24b) and 3.37 (1H, dd, J = 12.2, 4.8 Hz, H-3) as

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well as seven methyl groups at δH 1.50 (3H, s, Me-27), 1.24 (3H, s, Me-23), 1.14 (3H, s, Me-25), 1.13 (3H, s, Me-26), 1.06 (3H, s, Me-29), 1.04 (3H, s, Me-28) and 0.89 13

C NMR data of 1 are very similar to

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(3H, s, Me-30) were observed. The 1H and

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those of the known pentacyclic triterpenoid 7β,15α,24-trihydroxyolean-12-ene3,11,22-trione (3) [9], except for the replacement of a carbonyl group by an

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oxygenated methine at the C-3 position in 1, which was confirmed by the COSY correlations between Hab-2 (δH 1.82 and 1.66) and H-3 in addition to the HMBC

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correlations from Me-23 and Hab-24 to C-3 (δC 80.2), C-4 (δC 43.1) and C-5 (δC 52.5). The remaining substructures of 1 were shown to be identical to those of 3 by detailed analysis of the 2D NMR data of 1 (Fig. 2). Based on the similar NOE correlations and close biogenetic relationship, 1 was suggested to posses the same relative configurations at the corresponding chiral centers as 3, whose absolute configuration was determined by X-ray crystallographic analysis. In addition, the NOE correlations between H-3/H-5, H-3/Me-23 and H-5/Me-23 indicated the α-orientation of H-3. Thus, compound 1 was determined as 3β,7β,15α,24-tetrahydroxyolean-12-ene-11,22dione.

ACCEPTED MANUSCRIPT Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data for 1 and 2 in CD3OD.

8 9 10 11 12 13 14 15 16

52.0, C 62.9, CH 38.5, C 200.4, C 130.1, CH 168.7, C 51.4, C 66.1, CH 35.4, CH2

17 18 19

48.1, C 50.1, CH 45.6, CH2

20 21

35.1, C 50.9, CH2

22 23 24

215.5, C 22.9, CH3 64.7, CH2

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25 26 27 28 29 30

0.96, m 1.89, m 1.61, m 4.00, dd (11.4, 4.6)

2.40, s

5.73, s

4.21, dd (11.7, 5.0) 2.22, m 1.43, m 2.56, m 2.27, m 1.43, m

16.9, CH3 12.5, CH3 17.6, CH3 22.0, CH3 31.7, CH3 25.2, CH3

2.63, d (14.4) 2.04, dd (14.4, 2.6) 1.24, s 4.07, d (11.1) 3.44, d (11.1) 1.14, s 1.13, s 1.50, s 1.04, s 1.06, s 0.89, s

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72.2, CH

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217.4, C 56.3, C 57.8, CH 20.5, CH2

2 δH (J in Hz) 2.24, m 1.45, m 2.77, m 2.34, m

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80.2, CH 43.1, C 52.5, CH 28.8, CH2

34.9, CH2

36.5, CH2

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3 4 5 6

δC, type 40.4, CH2

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28.5, CH2

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2

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δC, type 38.0, CH2

1 δH (J in Hz) 1.72, m 1.00, m 1.82, m 1.66, m 3.37, dd (12.2, 4.8)

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position

48.4, C 54.5, CH 37.5, C 127.2, CH 126.6, CH 136.0, C 42.3, C 66.4, CH 43.2, CH2 42.9, C 137.9, C 37.8, CH2 32.6, C 44.0, CH2 77.5, CH 20.2, CH3 65.0, CH2 18.5, CH3 17.1, CH3 14.7, CH3 18.7, CH3 32.3, CH3 25.2, CH3

1.42, m 1.66, m 1.60, m 1.65, m 1.41, m 2.03, dd (3.0, 1.4) 6.39, dd (10.6, 3.0) 5.64, dd (10.6,1.4)

4.02, dd (12.4, 4.1) 1.90, dd (12.8, 4.1) 1.58, dd (12.8, 12.4)

2.39, dd (14.6, 1.9) 1.74, d (14.6) 1.52, m 1.44, m 3.39, dd (12.1, 4.7) 1.16, s 4.01, d (11.4) 3.53, d (11.4) 1.14, s 0.81, s 0.99, s 1.07, s 1.00, s 0.81, s

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Fig. 2. Key COSY and HMBC correlations of 1 (left) and 2 (right).

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Compound 2 exhibited the molecular formula C30H46O4 as determined by the HRESIMS data, containing eight degrees of unsaturation. Its 1H and

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C data (Table

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1) resembled those of the known compound 15α,24-dihydroxyolean-12-ene-3,11,22trione (4) [10], except for the disappearance of two carbonyl groups and the presence

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of two additional olefinic protons at δH 6.39 (dd, H-11) and 5.64 (dd, H-12) as well as

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an additional oxygenated methine at δH 3.39 and δC 77.5 (CH-22) in 2. The COSY

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correlations between H-10 (δH 2.03, dd)/H-11 and H-11/H-12, and key HMBC correlations from H-12 to C-13 (δC 136.0), C-14 (δC 42.3) and C-18 (δC 137.9), and

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from Hab-19 (δH 2.39 and 1.74) to C-13 and C-18 indicated a conjugated diene system at the C-11/C-12 and C-13/C-18 positions. In addition, the COSY correlations between Hab-21 (δH 1.52 and 1.44) and H-22 and the HMBC correlations from Me-28 (δH 2.03, s) to C-16 (δC 43.2), C-17 (δC 42.9), C-18 and C-22 indicated a hydroxy group attached at C-22. Detailed interpretation of the 2D NMR spectra of 2 revealed that the remaining substructures of 2 were identical to those of 4 (Fig. 2). The absolute configuration of 4 was determined by X-ray diffraction method [8]. Considering the similarity of NOE correlations and relationship in biosynthesis between 2 and 4, it was suggested that they shared the same configurations at the corresponding positions. Moreover, key NOE cross peaks from H-22 to Hα-16 (δH 1.58), Hα-21 (δH

ACCEPTED MANUSCRIPT 1.44) and Me-30 (δH 0.81), and from Me-28 (δH 1.07) to Hβ-16 (δH 1.90), Hβ-19 (δH 1.74) and Hβ-21 (δH 1.52) indicated the α-orientation of H-22. Thus, compound 2 was

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determined as 15α,22β,24-trihydroxyolean-11,13-diene-3-one.

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Compound 8 was obtained as a yellow amorphous solid, showing UV absorption

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at λmax 203, 269 and 351 nm. Based on the HRESIMS data, its molecular formula was determined to be C12H10O3, indicating eight degrees of unsaturation. The 1H NMR spectrum of 8 (Table 2) displayed three olefinic protons at δH 7.81 (1H, s, H-8), 7.30

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(1H, s, H-5) and 6.74 (1H, q, J = 1.6 Hz, H-3) as well as two methyl groups at δH 2.29

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(3H, s, Me-12) and 2.12 (3H, d, J = 1.6 Hz, Me-11). The HMBC correlation from Me12 to C-6 (δC 162.3), C-7 (δC 132.6) and C-8 (δC 130.6), from H-5 to C-6, C-7, C-9

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(δC 125.8) and C-4 (δC 186.7), and from H-8 to C-6, C-10 (δC 133.8) and C-1 (δC

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186.0) indicated the presence of a tetrasubstituted benzene ring with a hydroxy group, a methyl group and two carbonyl groups attached at the C-6, C-7, C-9 and C-10

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positions, respectively (Fig. 3). Together with the HMBC correlations from Me-11 to C-1, C-2 (δC 149.8) and C-3 (δC 136.0) and from H-3 to C-4, C-10 and C-11 (δC 16.3),

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compound 8 was determined as 6-hydroxy-2,7-dimethyl-1,4-naphthoquinone as shown in Figure 1. Table 2. 1H (700 MHz) and 13C (175 MHz) NMR data for compound 8 in CD3OD. position 1 2 3 4 5 6 7 8 9 10 11 12

δC, type 186.0, C 149.8, C 136.0, CH 186.7, C 111.6, CH 162.3, C 132.6, C 130.6, CH 125.8, C 133.8, C 16.3, CH3 16.4, CH3

δH (J in Hz)

6.74, q (1.6) 7.30, s

7.81, s

2.12, d (1.6) 2.29, s

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Fig. 3. Key HMBC correlations of 8.

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In addition to the latter new compounds, 14 known compounds were identified as 7β,15α,24-trihydroxyolean-12-ene-3,11,22-trione (3) [9], 15α,24-dihydroxyolean-12ene-3,11,22-trione (4) [10], soyasapogenol B (5) [11], (2E, 4E)-4′-dihydrophaseic

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acid (6) [12], (2Z, 4E)-4′-dihydrophaseic acid (7) [13], 6-hydroxy-2,2-dimethyl-2H-

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chromene (9) [14], scoparone (10) [15], 5-methyluracil (11) [16], 4-hydroxy-3methoxy-2(1H)-quinolinone (12) [17], 4-hydroxyphenylglyoxylic acid amide (13)

(16) [21], and N-acetyl-β-oxotryptamine (17) [22], by

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indol-3-yl) oxoacetamide

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[18], indole-3-carboxaldehyde (14) [19], indole-3-carboxylic acid (15) [20], (1H-

comparison of their NMR and MS data with those in literature.

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All isolated compounds were evaluated for their cytotoxicity against the mouse lymphoma cell line L5178Y, for their antibacterial activities against three pathogenic

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bacteria Staphylococcus aureus ATCC 25923, S. aureus ATCC 700699 and Acinetobacter baumannii ATCC BAA 1605 as well as for their antitubercular activity against Mycobacterium tuberculosis. However, none of them showed significant activity when tested at a dose of 10 µg/mL. Previous reports on the secondary metabolites from genus Scopulariopsis were very few, in which two cyclodepsipeptides scopularides A and B [23], two naphthalene

derivatives

1-(4'-hydroxy-3',5'-dimethoxy-phenyl)-1,8-

dimethoxynaphthalen-2(1H)-one and 1,8-dimethoxynaphthalen-2-ol [24] as well as an alkaloid fumiquinazoline L [25] were isolated. In present study, 17 metabolites including five triterpenoids (1–5), two sesquiterpernoids (6–7), three polyketides (8–

ACCEPTED MANUSCRIPT 10) and seven nitrogenous compounds (11–17) were isolated from the white bean culture of Scopulariopsis sp. Among them, 1, 2 and 8 are new while the remaining

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known compounds are all isolated from this fungus for the same time. All these

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metabolites (1–17) isolated from the solid bean culture of Scopulariopsis sp. in this

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study clearly differ from those of the same fungus grown on solid rice media [6], proving the power of the OSMAC approach for diversifying the metabolite profiles of

3. Experimental section

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3.1. General experimental procedures

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fungi.

Perkin-Elmer-241 polarimeter was used for optical rotations measurement. 1H, 13

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C and 2D NMR spectra were recorded on Bruker Avance 600 or 700 spectrometer.

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HRESIMS data were obtained from a FTHRMS-Orbitrap (Thermo Finnigan) mass spectrometer. HPLC analyses were carried out using Dionex Ultimate 3000 LC

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system coupled with a photodiode array detector (UVD340S). The analytical column (125 × 4 mm, L × ID) was prefilled with Eurospher-10 C18 (Knauer). Semi-

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preparative HPLC was carried out using a LaChrom-Merck Hitachi system (L7100 pump, L7400 UV detector and a 300 × 8 mm Eurospher C18 column) at a flow rate of 5 mL/min. Merck MN Silica gel 60 M or Sephadex LH-20 was applied for column chromatography. Precoated silica gel 60 F254 plates (Merck) were used for TLC analysis with detection at 254 and 365 nm following by spraying with anisaldehyde reagent. All solvent was distilled before use and spectroscopic grade solvent was used for spectroscopic measurements. 3.2. Fungal material and identification The hard coral Stylophora sp. was collected in November 2012 near the coastline of Ain El-Sokhna area, Red Sea, Egypt. Isolation of the fungal strain from the coral

ACCEPTED MANUSCRIPT was carried out according to the standard procedure as described before [26]. Based on DNA amplification and sequencing of the ITS region (GenBank accession no.

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KP027401) [26], the fungus was identified as Scopulariopsis sp. and the next relative

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strain was S. brevicaulis strain AS2L 15-2 (GenBank accession no. KP117309). A

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voucher plate for this fungal strain is kept in one of the authors’ laboratory (P.P.) with the code ST-F1. 3.3. Cultivation, extraction and isolation

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Cultivation of the strain was performed on a solid white bean medium, which

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was prepared by autoclaving 100 g beans and 110 mL water at 121 °C for 20 min, in 12 1L-Erlenmeyer flasks for 30 days at room temperature under static conditions.

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After fermentation, the fungal culture in each flask was extracted with EtOAc (3 ×

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500 mL). The obtained crude extract (12 g) was partitioned between n-hexane and 90% aqueous MeOH, giving 8 g of 90% aqueous MeOH extract. The latter was

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subjected to vacuum liquid chromatography (VLC) on silica gel 60 eluting with gradient mobile phase (n-hexane-EtOAc 100:0 to 0:100, DCM-MeOH 100:0 to 0:100)

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to give twelve fractions (V1-V12). Fraction V4 (130 mg) was chromatographed over a Sephadex LH-20 column (80 × 3 cm) using MeOH to yield 9 (1.2 mg) and 8 (1.0 mg). Fraction V5 (320 mg) was fractionated on a Sephadex LH-20 column (100 × 5 cm) with MeOH to give 10 subfractions (V5-S1 to S10). Compounds 5 (16 mg) and 10 (1.1 mg) were crystallized from subfractions V5-S3 and V5-S4, respectively. Compounds 13 (1.3 mg) and 14 (1.0 mg) were obtained from V5-S5 (16 mg) through semi-preparative RP-HPLC with 35% MeOH/H2O as eluting system. Fractions V6 (580 mg) was separated by a VLC column as described above to give 10 subfractions (V6-S1 to S10). Compound 16 (1.5 mg) was recovered through

ACCEPTED MANUSCRIPT further purification of subfraction V6-S5 (120 mg) over a Sephadex LH-20 column (80 × 3 cm) using MeOH, while 4 (1.0 mg) was obtained from V6-S6 (13 mg) using

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semi-preparative RP-HPLC with 78% MeOH/H2O as mobile phase. Subfraction V6-

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S8 (30 mg) was subjected to a Sephadex LH-20 column (60 × 2 cm) with MeOH as

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eluent to give 15 (1.4 mg).

Fraction V8 (300 mg) was subjected to a Sephadex LH-20 column (100 × 5 cm) eluting with MeOH to give 10 subfractions (V8-S1 to S10). Subfraction V8-S2 (48

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mg) was purified by semi-preparative RP-HPLC using 70% MeOH/H2O as eluting

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system to afford 3 (4.2 mg), 1 (2.5 mg) and 2 (2.8 mg). Following the same procedure, 11 (3.2 mg) and 12 (1.6 mg) were obtained from V8-S5 (16 mg).

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Subfraction V8-S7 (30 mg) was purified on a Sephadex LH-20 column (60 × 2 cm)

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using DCM-MeOH (1:1) as mobile phase to yield 17 (1.3 mg). Fraction V10 (310 mg) was fractionated over a Sephadex LH-20 column (100 ×

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5 cm) using MeOH to give 8 subfractions (V10-S1 to S8). Purification of subfraction V10-S5 (28 mg) using semi-preparative HPLC with 20% MeOH/H2O gave 6 (1.7 mg)

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and 7 (1.3 mg).

3.3.1. 3β,7β,15α,24-tetrahydroxyolean-12-ene-11,22-dione (1) White amorphous powder;  20 D + 21 (c 0.2, MeOH); UV (MeOH) λmax: 249 nm; 1H and 13C NMR data see Table 1; HRESIMS [M+H]+ m/z 503.3368 (calcd. for C30H47O6, 503.3367). 3.3.2. 15α,22β,24-trihydroxyolean-11,13-diene-3-one (2) White amorphous powder;  D + 28 (c 0.3, MeOH); UV (MeOH) λmax: 243 and 20

249 nm; 1H and 13C NMR data see Table 1; HRESIMS [M+H]+ m/z 471.3466 (calcd. for C30H47O4, 471.3469). 3.3.3. 6-hydroxy-2,7-dimethyl-1,4-naphthoquinone (6)

ACCEPTED MANUSCRIPT Yellow amorphous solid; UV (MeOH) λmax: 203, 269 and 351 nm; 1H and

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NMR data see Table 2; HRESIMS [M+H]+ m/z 203.0701 (calcd. for C12H11O3,

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203.0703).

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3.4. Cytotoxicity assay

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Cytotoxicity assay against the L5178Y mouse lymphoma cell line was performed using the MTT method with kahalalide F as positive control and media with 0.1% DMSO as negative control as described before [27].

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3.5. Antimicrobial assay

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Antimicrobial activity against S. aureus ATCC 25923, S. aureus ATCC 700699, A. baumannii ATCC BAA 1605 and M. tuberculosis was evaluated by the broth

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micro dilution method according to the recommendations of the Clinical and

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Laboratory Standards Institute (CLSI) as previously described [28]. Moxifloxacin and DMSO were used as positive and negative control, respectively.

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Acknowledgments

A scholarship granted and financed by the Egyptian government (Ministry of

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High Education) to M.S.E. is gratefully acknowledged. We wish to thank Prof. W.E.G. Müller (Univ. Mainz) for carrying out cytotoxicity assays and Prof. R. Kalscheuer (Univ. Düsseldorf) for antimicrobial assay. P.P. wants to thank the Manchot-Foundation for support. References [1] J.W. Blunt, B.R. Copp, R.A. Keyzers, M.H.G. Munro, M.R. Prinsep, Marine natural products, Nat. Prod. Rep. 31 (2014) 160–258. [2] J.F. Imhoff, A. Labes, J. Wiese, Bio-mining the microbial treasures of the ocean: new natural products, Biotechnol. Adv. 29 (2011) 468–82. [3] A. Marmann, A.H. Aly, W. Lin, B. Wang, P. Proksch, Co-Cultivation–A

ACCEPTED MANUSCRIPT powerful emerging tool for enhancing the chemical diversity of microorganisms, Mar. Drugs 12 (2014) 1043–1065.

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ACCEPTED MANUSCRIPT 4 (1998) 107–113. [11] R.L. Baxter, K.R. Price, G.R. Fenwick, Sapogenin structhure: analysis of the 13C-

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and 1H-NMR spectra of soyasapogenol B, J. Nat. Prod. 53 (1990) 298–302.

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Graphical abstract