Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium

Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium

Biochemical and Biophysical Research Communications xxx (2016) 1e6 Contents lists available at ScienceDirect Biochemical and Biophysical Research Co...

1MB Sizes 0 Downloads 163 Views

Biochemical and Biophysical Research Communications xxx (2016) 1e6

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium Sabrin Ragab Mohamed Ibrahim a, b, *, Ehab Saad Elkhayat c, Gamal Abd Allah Mohamed d, c, Shaukat Mohammed Fat'hi e, f, Samir Anis Ross g a

Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt d Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia e Department of Veterinary Medicine, College of Agricultural and Veterinary Medicine, Qassim University, AL-Qassim, Saudi Arabia f Food Hygiene Department, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt g National Center for Natural Products Research, Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 September 2016 Accepted 9 September 2016 Available online xxx

Four secondary metabolites (1e4), including a new benzamide derivative, namely fusarithioamide A (2(2-aminopropanamido)-N-(1-hydroxy-3-mercaptopropyl) benzamide, 4) and three known compounds; 1-O-acetylglycerol (1), 8-acetylneosolaniol (2), and ergosta-7,22-diene-3b,5a,6b-triol (3) were characterized from the EtOAc extract of Fusarium chlamydosporium isolated from the leaves of Anvillea garcinii (Burm.f.) DC. (Asteraceae). The structures of the isolated metabolites were verified by using 1D and 2D NMR experiments as well as HRESIMS spectral data. Compounds 1e3 were firstly separated from this fungus. Compound 4 has been tested for their antibacterial and antifungal activity against different microorganisms using disc diffusion assay. It showed antibacterial potential towards B. cereus, S. aureus, and E. coli with inhibition zone diameters (IZDs) of 19.0, 14.1, and 22.7 mm, respectively and MICs values of 3.1, 4.4, and 6.9 mg ml1, respectively. Also, it exhibited the most potent antifungal activity towards C. albicans (IZD 16.2 mm) comparable to clotrimazole (IZD 18.5 mm, positive control). Furthermore, compounds 1e4 were evaluated for their in vitro cytotoxic effect against KB, BT-549, SK-MEL, and SKOV3 cell lines. Compounds 4 possessed potent and selective activity towards BT-549 and SKOV-3 cell lines with IC50 values of 0.4 and 0.8 mM, respectively compared to doxorubicin (IC50 0.046 and 0.313 mM, respectively). Moreover, 3 exhibited significant activity towards all tested cell lines. Fusarithioamide A may provide new promising candidates for potential antimicrobial and cytotoxic agent. © 2016 Elsevier Inc. All rights reserved.

Keywords: Fusarithioamide A Fusarium chlamydosporium Benzamide derivative Anvillea garcinii Antimicrobial Cytotoxic activity

1. Introduction Endophytic fungi are symbiotically associated with plants, usually without producing any apparent external symptoms or negative effects [1,2]. They have been known as prominent sources of bioactive and new constituents which are of usefulness for particular medicinal or agrochemical applications [3e5]. Fungi

* Corresponding author. Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia. E-mail addresses: [email protected], [email protected] (S.R.M. Ibrahim).

belonging to the genus Fusarium are wide prevalence pathogens on crops in both semitropical and temperate zones. They can infect vegetables, fruits, small grain cereals and maize, leading to vascular wilt, stem, root, and ear rot, with serious decrease in the yields of crops and severe economic losses [6e8]. In addition, definite strains have also the capability to produce mycotoxins, which can be produced in infected pre-harvest plants and lasting in stored grains or in the field [9]. Previous studies on this genus revealed that it is a wealthy source of biologically effective metabolites, including mycotoxins [10], cyclic depsipeptides [11], integracides [4,5], naphthoquinones [12e15], and pyrone derivatives [16]. In our seeking for discovering bioactive fungal agents, we investigated the fungus Fusarium chlamydosporium

http://dx.doi.org/10.1016/j.bbrc.2016.09.041 0006-291X/© 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: S.R.M. Ibrahim, et al., Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.09.041

2

S.R.M. Ibrahim et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6

isolated from the leaves of Anvillea garcinii (Burm.f.) DC. (Asteraceae). Fusarithioamide A (4), a new benzamide derivative and three known metabolites; 1,3-O-diacetylglycerol (1), 8acetylneosolaniol (2), and ergosta-7,22-diene-3b,5a,6b-triol (3) were isolated from the EtOAc extract of F. chlamydosporium (Fig. 1). Details of the structural characterization and separation of the new compound, as well as its antibacterial and antifungal activities are presented herein. In addition, the in vitro cytotoxic effect of the isolated compounds 1e4 was evaluated.

2. Materials and methods 2.1. General experimental procedures A Digital Melting Point 9100 Electrothermal apparatus was utilized for measuring melting points (Electrothermal Engineering Ltd, Essex, England). For the measurement of optical rotations a Perkin-Elmer polarimeter 341 LC Model was utilized (Perkin-Elmer, Waltham, MA, USA). UV spectra measurement was performed on a 1601 UV/VIS Shimadzu spectrophotometer using MeOH (Shimadzu, Kyoto, Japan). Infrared-400 Shimadzu spectrophotometer was utilized for IR measurement (Shimadzu, Kyoto, Japan). HRESIMS was carried out on an Orbitrap LTQ (ThermoFinnigan, Bremen, Germany). ESIMS spectra were achieved using LCQ DECA spectrometer (ThermoFinnigan, Bremen, Germany). A Bruker Avance DRX 500 MHz spectrometer was used for measuring 1D and 2D NMR spectra (Bruker BioSpin, Billerica, MA, USA). Chromatographic separations were achieved on sephadex LH-20 (0.25e0.1 mm), SiO2 60 (0.04e0.063 mm), and RP-18 (0.04e0.063 mm) (Merck, Darmstadt, Germany). TLC analysis was carried out on SiO2 60 F254 TLC pre-coated plates (0.2 mm) (Merck, Darmstadt, Germany). CHCl3:MeOH (95:5, S1) and CHCl3:MeOH (90:10, S2) were utilized as mobile phases for TLC analyses. The compounds detection was done by UV absorption (lmax 255 and 366 nm) and spraying with ninhydrin or anisaldehyde:H2SO4 spraying reagents.

2.2. Fungal material isolation and identification Anvillea garcinii (Burm.f.) DC. (Asteraceae) was collected in March 2013 from the plant wildly growing at campus of Al-Azhar University. The plant sample was authenticated by Prof. Dr. A .A. Fayed, Prof. of Plant Taxonomy, Faculty of Science, Assiut University, Assiut, Egypt. A herbarium specimen was kept at the Department of Pharmacognosy herbarium, Al-Azhar University (AG-3-2013). Fusarium chlamydosporium was secluded from the inner tissue of A. garcinii leaves. The leaves tissues were strictly sliced under septic conditions and put on potato dextrose agar plates (PDA, Difco), having gentamicin and chloramphenicol as antibacterial agents to prohibit growth of bacteria. The plates were incubated for 4e6 weeks at 27  C. After that, the fungal hyphal tips were regularly taken away and transmitted to PDA fresh plates. The fungus was specified based on its morphological features using light microscopy (CX31RBSF, Olympus), this was genetically fortified by the ITS sequence analysis (Genbank Accession number FC201936.1). It was preserved at the Department of Microbiology, Al-Azhar University, Assiut, Egypt (FuC/March 2013). The fresh culture was transmitted into rice solid cultures (15 Erlenmeyer flasks, 1L each). The cultures have been incubated under septic conditions for 30 days at room temperature. 2.3. Metabolites isolation The culture was extracted using EtOAc and concentrated under vacuum. The obtained extract was suspended in distilled water (200 ml) and fractionated between n-hexane and 90% MeOH. The MeOH extract (4.2 g) was separated to VLC, utilizing n-hexane, EtOAc, and MeOH, which were independently concentrated to get FC-1 (1.2 g), FC-2 (0.9 g), and FC-3 (1.7 g), respectively. FC-2 fraction (0.9 g) was subjected to SiO2 CC using CHCl3:MeOH gradient (100% CHCl3 to 50:50 CHCl3:MeOH), 100 ml fractions were gathered and checked by TLC to get seven sub-fractions: FC2-1 to FC2-7. Subfraction FC2-2 (70 mg) was separated on SiO2 CC (15 g, 50  2 cm)

Fig. 1. Structures of compounds 1-4.

Please cite this article in press as: S.R.M. Ibrahim, et al., Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.09.041

S.R.M. Ibrahim et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6

using CHCl3:MeOH (99:1 to 95:5) as an eluent to yield 1 (11.9 mg, colorless oil). SiO2 CC (20 g, 50  2 cm) of sub-fraction FC2-3 (187 mg) using CHCl3:MeOH with rising polarity yielded impure 2, which was purified on RP-18 column (0.04e0.063 mm; 40 g, 50  2 cm) using H2O:MeOH gradient to obtain 2 (6.5 mg, colorless needles). Sub-fraction FC2-4 (209 mg) was handled similar to FC2-3 to get 3 (7.2 mg, colorless needles). Sephadex LH-20 CC (30 g, 50  3 cm) of FC2-5 (114 mg) eluting with MeOH:CHCl3 (95:5) afford impure 4. Its purification was achieved using RP-18 column (0.04e0.063 mm; 40 g, 50  2 cm) eluting with H2O:MeOH gradient to get 4 (5.6 mg, white amorphous powder). The remaining fractions were kept for more study. 2.4. Spectral data 2.4.1. 1-O-acetylglycerol (1) Colorless oil; 1H NMR (CDCl3, 500 MHz): dH 4.04 (1H, dd, J ¼ 12.3, 5.0 Hz, H-1A), 3.88 (1H, dd, J ¼ 12.3, 6.2 Hz, H-1B), 3.63 (1H, m, H-2), 3.35 (2H, m, H-3), 4.89 (1H, d, J ¼ 10.0 Hz, 2-OH), 4.65 (1H, t, J ¼ 5.6 Hz, 3-OH), 2.02 (3H, s, H-20 ); 13C NMR (CDCl3, 125 MHz): dC 63.1 (C-1), 69.7 (C-2), 66.2 (C-3), 170.9 (C-10 ), 21.2 (C-20 ); ESIMS m/z: 135 [MþH]þ. 2.4.2. 8-Acetylneosolaniol (2) Colorless needles, m.p. 190e191 οC; (KBr) nmax 3465, 2989, 1735, 1056 cm1; 1H NMR (CDCl3, 500 MHz): dH 3.69 (1H, d, J ¼ 4.6 Hz, H2), 4.16 (1H, dd, J ¼ 4.6, 2.8 Hz, H-3), 5.21 (1H, d, J ¼ 2.8 Hz, H-4), 2.36 (1H, dd, J ¼ 13.9, 5.4 Hz, H-7A), 2.00 (1H, brd, J ¼ 13.9 Hz, H7B), 5.25 (1H, d, J ¼ 5.4 Hz, H-8), 5.80 (1H, d, J ¼ 4.8 Hz, H-10), 4.26 (1H, d, J ¼ 4.8 H z, H-11), 3.06 (1H, d, J ¼ 14.0 Hz, H-13A), 2.80 (1H, d, J ¼ 14.0 Hz, H-13B), 0.81 (3H, s, H-14), 4.25 (1H, d, J ¼ 13.1 Hz, H15A), 4.06 (1H, d, J ¼ 13.1 Hz, H-15B), 1.74 (3H, s, H-16), 2.02 (3H, s, 4-COCH3), 2.03 (3H, s, 8-COCH3), 2.14 (3H, s, 15-COCH3), 3.23 (1H, brs, 3-OH; 13C NMR (CDCl3, 125 MHz): dC 78.8 (C-2), 78.5 (C-3), 84.7 (C-4), 48.7 (C-5), 43.1 (C-6), 27.4 (C-7), 68.6 (C-8), 136.3 (C-9), 123.9 (C-10), 67.5 (C-11), 64.4 (C-12), 47.3 (C-13), 7.1 (C-14), 172.8 (C-4,15), 20.5 (C-16), 21.1 (4-COCH3), 21.2 (8-COCH3), 21.2 (15-COCH3); ESIMS m/z: 425 [MþH]þ. 2.4.3. Ergosta-7,22-diene-3b,5a,6b-triol (3) Colorless needles, m.p. 167e168 οC; (KBr) nmax 3439, 2953, 1635, 1460, 1381, 1251, 1025 cm1; 1H NMR (DMSO-d6, 500 MHz): dH 1.38 (2H, m, H-1), 1.92 (1H, m, H-2A), 1.35 (1H, m, H-2B), 3.76 (1H, m, H3), 1.85 (1H, m, H-4A), 1.32 (1H, m, H-4B), 3.37 (1H, d, J ¼ 4.8 Hz, H6), 5.08 (1H, d, J ¼ 4.8 Hz, H-7), 1.94 (1H, m, H-9), 1.45 (2H, m, H-11), 2.01 (1H, m, H-12A), 1.65 (1H, m, H-12B), 1.81 (1H, d, J ¼ 7.5 Hz, H14), 1.49 (1H, m, H-15A), 1.25 (1H, m, H-15B), 1.64 (1H, m, H-16A), 1.24 (1H, m, H-16B), 1.25 (1H, m, H-17), 0.90 (3H, s, H-18), 0.55 (3H, s, H-19), 2.01 (1H, m, H-20), 0.99 (3H, d, J ¼ 6.8 Hz, H-21), 5.23 (1H, dd, J ¼ 15.1, 6.8 Hz, H-22), 5.16 (1H, dd, J ¼ 15.1, 7.2 Hz, H-23), 1.98 (1H, m, H-24), 1.42 (1H, m, H-25), 0.78 (3H, d, J ¼ 6.7 Hz, H-26), 0.80 (3H, d, J ¼ 6.7 Hz, H-27), 0.88 (3H, d, J ¼ 6.6 Hz, H-28); 13C NMR (DMSO-d6, 125 MHz): dC 27.7 (C-1), 31.2 (C-2), 65.9 (C-3), 40.2 (C-4), 74.4 (C-5), 72.1 (C-6), 119.4 (C-7), 139.6 (C-8), 42.3 (C-9), 36.5 (C-10), 19.8 (C-11), 38.9 (C-12), 42.1 (C-13), 54.2 (C-14), 21.3 (C-15), 22.6 (C16), 55.3 (C-17), 17.7 (C-18), 12.1 (C-19), 40.4 (C-20), 21.0 (C-21), 135.4 (C-22), 131.3 (C-23), 42.0 (C-24), 32.5 (C-25), 19.4 (C-26), 19.7 (C-27), 17.3 (C-28); ESIMS m/z: 431 [MþH]þ. 2.4.4. Fusarithioamide A (4) White amorphous powder; [a]25D þ56.6 (c 0.5, MeOH); UV (lmax, MeOH) (log ε) 218 (3.89), 310 (3.35) nm; IR nmax (KBr) 3415, 3326, 2566, 1697, 1595, 1515, 718, 675 cm1; NMR data, see Table 1; HRESIMS m/z 298.1229 [MþH]þ (calcd for C13H20N3O3S, 298.1225); 299.1300 [Mþ2H]þ (calcd for C13H21N3O3S, 299.1304).

3

Table 1 NMR spectral data of 4 (DMSO-d6, 500 and 125 MHz). No.

dH [mult., J (Hz)]

dC (mult.)

HMBC

1 2 3 4 5 6 7 8 9 10

8.57 dd (8.2, 1.5) 7.47 dt (8.2, 1.5) 7.11 dt (8.2, 1.5) 7.74 dd (8.2, 1.5) 7.59 brs 4.10 brddt (8.0, 6.5, 5.6) 1.77 m 1.35 m 2.62 m 11.71 brs 8.16 brs 11.99 s 3.73 m 5.97 d (8.0) 1.30 d (7.0)

120.9 C 138.7 C 119.8 CH 131.8 CH 122.4 CH 128.5 CH 170.2 C 77.6 CH 31.7 CH2

5 2, 6 1, 3 2, 4, 7 1, 7, 10 7, 11 11

20.5 CH2 174.1 C 67.9 CH 20.9 CH3

9 10, 11 9, 10 2, 3, 15, 16

11 12 13 14 15 16 17 18

15, 18 15, 16 15, 16

2.5. Microbial strains The organisms used in this study were Gram-positive bacteria, Staphylococcus aureus (AUMC-B-54) and Bacillus cereus (AUMC-B5), Gram-negative bacteria, Escherichia coli (AUMCB-53) and Pseudomonas aeurginosa (AUMC-B-73), and fungus Candida albicans (AUMC-418), which were secured by Assiut University Mycology Center, Assiut, Egypt. 2.6. Evaluation of the antimicrobial activity The antimicrobial effect of 4 was assessed by agar disc diffusion assay according to the previously described method [17,18]. Adjusted inoculums of each microorganism, equivalent to the turbidity of 0.5 McFarland standards, were streaked separately using sterile swab over the surface of Muller Hinton agar plates. Sterile filter paper discs (6 mm diameter) were impregnated with 20 ml of 4 (in DMSO) prepared in concentration (20 mg/disc) and applied on the inoculated plates. The plates were incubated at 37 οC for 24 h. Control discs impregnated with DMSO (20 ml/disc) were used to determine the solvent activity. Ciprofloxacin (10 mg/disc) and clotrimazole (5 mg/disc) were used as standard antibacterial and antifungal, respectively. The plates were incubated at 30  C for 48 h for yeast and 37  C for 24 h for bacteria. The diameters of inhibitory zones were measured after the incubation period. All experiments were performed in triplicate and the antimicrobial activity was expressed as the mean of inhibition zone diameters (Table 2). 2.7. Minimal inhibitory concentrations determination Minimal inhibitory concentrations (MICs) of 4 were assessed as previously outlined for Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Candida albicans [19,20]. The two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) values of isolated compounds against microorganisms. 100 ml of 4 (100 mg ml1) and 50 mg ml1 of antimicrobial standards (ciprofloxacin, clotrimazole) were serially diluted two-fold in triplicate with Mueller-Hinton broth for antibacterial test and Sabouraud broth for yeast test in 96-well microplates to make eight concentrations of 4 (0.78e100 mg ml1) and standards (0.39e50 mg ml1). 100 ml of fresh culture of bacteria (106 CFU ml1) and yeast

Please cite this article in press as: S.R.M. Ibrahim, et al., Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.09.041

4

S.R.M. Ibrahim et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6

Table 2 Results of antimicrobial activity and MIC values of fusarithioamide A (4). MICs (mg ml-1)

Inhibition zone diameter (IZD) (mm) Strains

4

Cipro.a

Clot.b

4

Cipro.

Clot.

S. aureus B. cereus E. coli P. aeurginosa C. albicans

14.1±0.13 19.0±0.22 22.7±0.19 16.2±0.11

16.6±0.09 20.7±0.21 26.9±0.11 16.5±0.08 N.T.

N.T. N.T. N.T. N.T. 18.5±0.07

4.4±0.08 3.1±0.12 6.9±0.06 100±1.36 2.6±0.52

3.8±0.05 2.6±0.18 3.5±0.10 6.2±0.06 N.T.

N.T. N.T. N.T. N.T. 3.7±0.09

a b

Ciprofloxacin as antibacterial standard; Clotrimazole as antifungal standard; N.T. not tested.

(2  105 CFU ml1) were added to each well. DMSO (2.5%) was used as negative control. A relation between the square inhibition distance and Logarithmic concentration (log C) was plotted. MIC values were listed as the log C at which showed no apparent growth of the microorganism. 2.8. Cytotoxic assay The cytotoxic effect was evaluated in vitro towards ovarian (SKOV-3), epidermoid (KB), malignant melanoma (SK-MEL), and ductal (BT-549) carcinomas. Cells (25,000 cells/well) were incubated for 24 h. Tested samples were added at different concentrations and cells were incubated for 48 h. Neutral Red dye was utilized to determine the cell viability [21]. Doxorubicin (positive control) and DMSO (negative control) were used [1,3]. 2.9. Statistical analysis All assays were performed in triplicate. The results were expressed as mean ± SD (standard deviation) using the student ttest. The statistical significance was evaluated by one-way analysis of variance (ANOVA). The values were considered to be significantly different when p values were less than 0.05 (p < 0.05).

128.5 in the HSQC spectrum, respectively. These signals in conjunction with the two NH groups at dH 7.59 (brs, H-8) and 11.99 (s, H-14) and carbonyl group dC 170.2 (C-7) indicated the presence 2-aminobenzamide moiety in 4 [24]. This was established by the observed HMBC relations of H-3 to C-5, H-4 to C-2 and C-6, H-5 to C-1 and C-3, H-6 to C-2 and C-4, H-8 to C-1, and C-7, and H-14 to C-2 and C-3 (Fig. 2A) and further assured by the ESIMS fragment peak at m/z 135. The signals at dH 3.73 (1H, m, H-16)/dC 67.9 (C-16), 1.30 (d, J ¼ 7.0 Hz, H-18)/20.9 (C-18), 5.97 (d, J ¼ 8.0 Hz, H-17), and 174.1 (C15) were attributable to an 2-aminopropanal moiety, which was confirmed by the HMBC relations (Fig. 2A). Its connectivity with the amino group of the 2-aminobenzamide moiety was proved by the HMBC cross peaks of H-14 to C-15 and C-16. Furthermore, the 1H NMR showed two multiplet methylene protons at dH 1.77 (H-10A) and 1.35 (H-10B), correlating with an oxymethine at dH 4.10 (brddt, J ¼ 8.0, 6.5, 5.6 Hz, H-9) and methylene proton at dH 2.62 (m, H-11) in the 1H-1H COSY spectrum. These signals showed HSQC cross peaks to the carbons at dC 77.6 (C-9), 31.7 (C-10), and 20.5 (C-11). Additionally, two heteroatoms signals at dH 8.16 (brs, H-13) and 11.71 (brs, H-12) were observed. They showed 1H-1H COSY cross peaks with H-9 and H-11, respectively, indicating the presence of 3mercaptopropan-1-ol moiety, which was confirmed by the observed correlations in the HMBC spectrum. Moreover, the ESIMS

3. Results and discussion Chromatographic separation of the EtOAc extract of F. chlamydosporium, using SiO2, sephadex LH-20, and RP-18 column chromatography, yielded one new (4) and three known compounds (1e3) (Fig. 1). Their structures were verified by spectral data analysis, including UV, IR, 1D and 2D NMR, and HRMS. Compound 4 was separated as a white amorphous powder and possessed positive ninhydrin test, indicating the presence of terminal NH2 [22]. Its molecular formula was deduced as C13H19N3O3S based on the HRESIMS pseudo-molecular ion peaks at m/z 298.1229 [MþH]þ (calcd for C13H20N3O3S, 298.1225) and 299.1300 [Mþ2H]þ (calcd for C13H21N3O3S, 299.1304) in a 20:1 ratio, compatible with a sulfur containing compounds [23]. It had UV absorptions at 218 and 310 nm. The IR spectrum displayed absorptions at 3415, 3326, 2566, 1697, 1595 and 1515 cm1 for hydroxyl, amine, mercapto, amide carbonyl, and aromatic C-H functionalities, respectively. Inspection of the 1H and 13C NMR spectra together with HSQC experiment revealed the existence of 13 carbon signals: one methyl, two methylenes, 6 methines one of them for oxymethine at dC 77.6 (C-9) and one for NH-bonded methine at dC 67.9 (C-16), and 4 quaternary carbons, consisting of two carbonyls at dC 170.2 (C-7) and 174.1 (C15) and 2 non-protonated aromatic carbons (Fig. 3, Table 1). The 1H and 1H-1H COSY spectra of 4 exhibited four correlated aromatic protons for a 1,2-di-substituted phenyl moiety at dH 8.57 (dd, J ¼ 8.2, 1.5 Hz, H-3), 7.47 (dt, J ¼ 8.2, 1.5 Hz, H-4), 7.11 (dt, J ¼ 8.2, 1.5 Hz, H-5), and 7.74 (dd, J ¼ 8.2, 1.5 Hz, H-6) (Fig. 3). They correlated with the carbons, resonating at dC 119.8, 131.8, 122.4, and

Fig. 2. Some key 1H-1H COSY and HMBC correlations (A) and possible fragmentation pattern (B) of 4.

Please cite this article in press as: S.R.M. Ibrahim, et al., Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.09.041

S.R.M. Ibrahim et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6

Fig. 3. 1H and

13

5

C NMR spectra of compound 4.

fragment peak indicated the loss of 207 mass units, which is compatible with the foreseeable fragment peak [MþH-C3H7OS]þ (Fig. 2B). The HMBC cross peaks of H-9 to C-7 and H-8 to C-10 proved its attachment of at position 8. Thus, the structure of 4 was unambiguously illustrated as 2-(2-aminopropanamido)-N-(1hydroxy-3-mercaptopropyl) benzamide and named fusarithioamide A. 2-Aminobenzamide derivatives had been reported previously from F. sambucinum [24]. The structures of 1e3 were identified and confirmed by combined spectroscopic analyses as 1-O-acetylglycerol (1) [25], 8acetylneosolaniol (2) [26], ergosta-7,22-diene-3b,5a,6b-triol (3) [27], respectively. The spectroscopic data for these compounds were in good correspondence with those cited in the literature.

Sulfur containing compounds and 2-aminobenzamide derivatives are known to exhibit remarkable antimicrobial activity [28e30]. Thus, 4 was evaluated for antibacterial potential towards the Gram positive (B. cereus and S. aureus) and Gram negative strains (P. aeruginosa and E. coli), as well as antifungal effect towards C. albicans using agar disc diffusion assay. In addition, its MIC was assessed. Compound 4 possessed potent antibacterial activity with inhibition zone diameters (IZDs) of 19.0,14.1, and 22.7 mm, respectively and MICs values of 3.1, 4.4, and 6.9 mg ml1, respectively against B. cereus, S. aureus, and E. coli, respectively compared to ciprofloxacin (IZDs 20.7, 16.6, and 26.9 mm, respectively). It's noteworthy that it exhibited potent antifungal activity with IZD 16.2 mm and MIC 2.6 mg ml1 towards C. albicans comparable to the

Please cite this article in press as: S.R.M. Ibrahim, et al., Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.09.041

6

S.R.M. Ibrahim et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6

Table 3 Results of cytotoxic activity of compounds 1-4. Compd

1 2 3 4 Doxorubicin

IC50 (mM), Mean ± SEM SK-MEL

KB

BT-549

SKOV-3

23.9±0.41 14.0±0.90 1.70±0.11 9.3±0.86 0.171±0.19

28.0±1.10 1.68±0.06 1.90±0.09 7.7±0.95 0.027±0.27

16.0±0.51 9.6±0.22 1.4±0.15 0.4±0.07 0.046±0.33

12.4±0.39 1.40±0.10 1.10±0.09 0.8±0.01 0.313±0.08

positive control substance clotrimazole (IZD 18.5 mm and MIC 3.7 mg ml1). However, 4 showed no activity towards P. aeurginosa. These results were in agreement with that reported by Kyung (2011) which stated that the sulfur compounds showed more potent inhibitory effects towards fungi than bacteria [31]. Additionally, compounds 1e4 were evaluated for their in vitro cytotoxic effect towards KB, BT-549, SK-MEL, and SKOV-3 cell lines. It is noteworthy that compounds 4 showed potent and selective activity towards BT-549 and SKOV-3 cell lines with IC50 values of 0.4 and 0.8 mM, respectively compared to doxorubicin (IC50 0.046 and 0.313 mM, respectively). Moreover, 3 exhibited significant activity with IC50 values of 1.7, 1.9, 1.4, and 1.1 mM, respectively towards SK-MEL, KB, BT-549, and SKOV-3 cell lines, respectively. However, 2 showed activity towards KB and SKOV-3 cell lines with IC50 1.68 and 1.40 mM, respectively. Compound 1 had weak activity towards all tested cancer cell lines (Table 3). Therefore, compound 4 may represent a simple model of natural antimicrobial and cytotoxic agents, and its unique effect deserves further consideration, which might procure to discover of new therapeutic agents. In summary, four compounds (1e4) were isolated and characterized from the EtOAc extract of F. chlamydosporium isolated from the leaves of Anvillea garcinii (Asteraceae), one of them is new natural product (4). Compounds 1e3 were reported for the first time from this fungus. Compound 4 exhibited potent antibacterial, antifungal, and cytotoxic activities. Conflict of interest There is no conflict of interest associated with the authors of this paper. References [1] S.R.M. Ibrahim, E.S. Elkhayat, G.A. Mohamed, A.I.M. Khedr, M.A. Fouad, M.H.R. Kotb, S.A. Ross, Aspernolides F and G, new butyrolactones from the endophytic fungus Aspergillus terreus, Phytochem. Lett. 14 (2015) 84e90. [2] S.R.M. Ibrahim, G.A. Mohamed, A.M. Moharram, D.T.A. Youssef, Aegyptolidines A and B: new pyrrolidine alkaloids from the fungus Aspergillus aegyptiacus, Phytochem. Lett. 12 (2015) 90e93. [3] E.S. Elkhayat, S.R.M. Ibrahim, G.A. Mohamed, S.A. Ross, Terrenolide S, a new anti-leishmanial butenolide from the endophytic fungus Aspergillus terreus, Nat. Prod. Res. 30 (2016) 814e820. [4] S.R.M. Ibrahim, H.M. Abdallah, G.A. Mohamed, S.A. Ross, Integracides H-J: new tetracyclic triterpenoids from the endophytic fungus Fusarium sp. Fitoterapia 112 (2016) 161e167. [5] S.R.M. Ibrahim, G.A. Mohamed, S.A. Ross, Integracides F and G: new tetracyclic triterpenoids from the endophytic fungus Fusarium sp. Phytochem. Lett. 15 (2016) 125e130.

[6] A. Bottalico, Fusarium diseases of cereals. Species complex and related mycotoxin profiles in Europe, J. Plant Pathol. 80 (1998) 85e103. [7] E.R. Champaco, R.D. Martyn, M.E. Miller, Comparison of Fusarium solani and Fusarium oxysporum as causal agents of fruit rot and root rot of muskmelon, Hort. Sci. 28 (1993) 1174e1177. [8] M.H. Hashmi, U. Thrane, Mycotoxins and other secondary metabolites in species of Fusarium isolated from seeds of capsicum, coriander and fenugreek, Pak. J. Bot. 22 (1990) 106e116. [9] A. Visconti, Problems associated with Fusarium mycotoxins in cereals, Bull. Inst. Compr. Agric. Sci. Kinki Univ. 9 (2001) 39e55. [10] J. Fostso, J.F. Leslie, J.S. Smith, Production of beauvericin, moniliformin, fusaproliferin, and fumonisins B1, B2, and B3 by fifteen ex-type strains of Fusarium species, Appl. Environ. Microbiol. 68 (2002) 5195e5197. [11] H. Song, H. Lee, C. Lee, A new cytotoxic cyclic pentadepsipeptide, neo-Nmethylsansalvamide produced by Fusarium solani KCCM90040, isolated from potato, Food Chem. 126 (2011) 472e478. [12] A.G. Medentsev, V.K. Akimenko, Naphthoquinone metabolites of the fungi, Phytochemistry 47 (1998) 935e959. [13] A.G. Medentsev, A.Y. Arinbasarova, V.K. Akimenko, Biosynthesis of naphthoquinone pigments by fungi of the genus Fusarium, Appl. Biochem. Microbiol. 41 (2005) 503e507. [14] K. Tadpetch, C. Chukong, L. Jeanmard, A. Thiraporn, V. Rukachaisirikul, S. Phongpaichit, J. Sakayaroj, Cytotoxic naphthoquinone and a new succinate ester from the soil fungus Fusarium solani PSU-RSPG227, Phytochem. Lett. 11 (2015) 106e110. [15] J.H. Tatum, R.A. Baker, R.E. Berry, Three further naphthoquinones produced by Fusarium solani, Phytochemistry 24 (1985) 3019e3021. [16] K. Trisuwan, V. Rukachaisirikul, K. Borwornwiriyapan, S. Phongpaichit, J. Sakayaroj, Pyrone derivatives from the soil fungus Fusarium solani PSURSPG37, Phytochem. Lett. 6 (2013) 495e497. [17] S.R.M. Ibrahim, Diacarperoxide S, new norterpene cyclic peroxide from the sponge Diacarnus megaspinorhabdosa, Nat. Prod. Commun. 7 (2012) 9e12. [18] CLSI, Performance Standards for Antimicrobial Disk Susceptibility TestsTwelfth Edition. CLSI Document M02ea12, Clinical and Laboratory Standards Institute, Wayne, PA, 2015. [19] G.A. Mohamed, S.R.M. Ibrahim, M.I.A. Shaaban, S.A. Ross, Mangostanaxanthones I and II, new xanthones from the pericarp of Garcinia mangostana, Fitoterapia 98 (2014) 215e221. [20] R.D. Pearson, R.T. Steigbigel, H.T. Davis, S.W. Chapmann, Method for reliable determination of minimal lethal antibiotic concentrations, Antimicrob. Agents Chemother. 18 (1980) 699e708. [21] E. Borenfreund, H. Babich, N. Martin-Alguacil, Rapid chemosensitivity assay with human normal and tumor cells in vitro, Vitro Cell Dev. Biol. 26 (1990) 1030e1034. [22] M. Friedman, Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences, J. Agric. Food Chem. 52 (2004) 385e406. [23] Z. Liu, G. Xia, S. Chen, Y. Liu, H. Li, Z. She, Eurothiocin A and B, sulfurcontaining benzofurans from a soft coral-derived fungus Eurotium rubrum SH-823, Mar. Drugs 12 (2014) 3669e3680. [24] D. Niederer, C. Tamm, W. Zuercher, Nitrogen containing metabolites of Fusarium sambucinum, Tetrahedron Lett. 33 (1992) 3997e4000. [25] H.J. Yan, S.S. Gao, C.S. Li, X.M. Li, B.G. Wang, Chemical constituents of a marinederived endophytic fungus Penicillium commune, G2M, Molecules 15 (2010) 3270e3275. [26] J.A. Lansden, R.J. Cole, J.W. Dorner, R.H. Cox, H.G. Cutler, J.D. Clark, A new trichothecene mycotoxin isolated fromFusarium tricinctum, J. Agric. Food Chem. 26 (1978) 246e249. [27] L. Jinwei, Z. Xinxin, L. Yuanfa, Supercritical carbon dioxide extraction of Ganoderma lucidumspore lipids, LWT- Food Sci. Technol. 70 (2016) 16e23. [28] Y.N. Mabkhot, A.M. Al-Majid, A. Barakat, S.S. Al-Showiman, M.S. Al-Har, S. Radi, M.M. Naseer, T.B. Hadda, Synthesis and biological evaluation of 2aminobenzamide derivatives as antimicrobial agents: opening/closing pharmacophore site, Int. J. Mol. Sci. 15 (2014) 5115e5227. [29] S. Kim, R. Kubec, R.A. Musah, Antibacterial and antifungal activity of sulfurcontaining compounds from Petiveria alliacea L. J. Ethnopharmacol. 104 (2006) 188e192. [30] S.R.M. Ibrahim, H.M. Abdallah, A.M. El-Halawany, G.A. Mohamed, Naturally occurring thiophenes: isolation, purification, structural elucidation, and bioactivities, Phytochem. Rev. 15 (2016) 197e220. [31] K.H. Kyung, Volatile sulfur compounds in food, ACS Symp. Ser. 1068 (2011) 323e338.

Please cite this article in press as: S.R.M. Ibrahim, et al., Fusarithioamide A, a new antimicrobial and cytotoxic benzamide derivative from the endophytic fungus Fusarium chlamydosporium, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/ j.bbrc.2016.09.041