Isolation of bioactive endophytic secondary metabolites
The extract of the strain cultivated in MPG medium showed potent cytotoxic effects against HeLa (CC50 = 6.2 µg mL−1) in addition to antiproliferative effects against K-562 and HUVEC cell lines (GI50 = 5.9 and 4.8 µg mL−1 respectively). Column chromatographic fractionation of the defatted methanolic extract started on Silica gel using chloroform and methanol (9:1) as a solvent mixture followed by gradual increase of polarity till elution with 100% methanol. Fractions of similar chromatographic properties were combined, and final fractions obtained were assayed for anticancer activity. Active fractions were purified using Sephadex LH-20. Final purification was performed on preparative column chromatography using 25% acetonitrile in water on reversed phase silica as a stationary phase.
Structure elucidation of bioactive metabolites
The first secondary metabolite obtained appeared as white powder and a molecular formula of C40H37N6O4 has been indicated for it by HRESIMS and a molecular weight of m/z 665.3190 [M + H]+ calcd, 665.3193. The 1H NMR spectrum revealed the presence of five aromatic proton signals corresponding to protons H-19–H-23 and their aromatic carbons C-19–C-23 were detected in the 13C NMR and DEPT spectra as well, thus indicating the presence of a substituted aromatic ring. The HMBC spectrum revealed a correlation between the proton at δ 7.05 ppm and the carbon signal at δ 36.6 ppm in addition to a correlation between the proton signal at δ 7.1 ppm and the same carbon signal at δ 36.7 ppm thus confirming connection of this aromatic ring to a methylene group at δ 36.7 ppm. Accordingly, the first part of the structure of this natural product was revealed to be a phenyl alanyl group.
Furthermore, four methine carbons of another aromatic ring (C-5–C-8) were in the 13C NMR and DEPT spectra as well as four corresponding aromatic protons (H-5–H-8), thus representing a disubstituted aromatic ring.
HMBC correlations indicated that the substituted carbons of this aromatic ring are the quaternary carbons at δ 148.5 ppm (C-9) and 134.1 ppm (C-4) which are connected to an olefinic quaternary carbon at δ 59.2 ppm (C-3) that is correlated in HMBC with H-6 at δ 6.50 ppm. The connection of a heteroatom to the quaternary carbon at δ 148.5 ppm (C-9) of this aromatic ring was concluded from its high chemical shift. These NMR data led to the deduction of a tryptophan subunit for the structure of this natural product. The quaternary carbon at δ 59.2 ppm (C-3) is correlated with the protons attached to C-12 (δ 3.24 and 2.41 ppm) which is itself connected to the aromatic ring moiety containing the aromatic quaternary carbon (C-24) at δ 126.2 ppm.
From these correlations, it was concluded that this aromatic ring belonged to a second tryptophan unit of the structure. This was confirmed by HMBC correlations (Fig. 2) observed for H-27 at δ 7.51 ppm to the quaternary carbon C-32 at δ 108.8 ppm, the high chemical shift of C-29 (δ 135.4 ppm) supporting its connection to an imine group, as well as the correlation of the methylene protons H-33 at δ 3.04 and 3.08 ppm with C-29 (δ 135.4 ppm). By comparison of the structure suggested in Fig. 1 for the compound and its NMR data (Additional file 1: Table S1) with literature its identity was confirmed as asperazine, the diketopiperazine dimer previously isolated from A. niger obtained from a Caribbean sponge (Varoglu 1997).
Analysis of the NMR data of the second metabolite isolated from A. neoniger endophyte revealed it to be a homologue of asperazine (1) with the same molecular weight (HRESIMS, m/z 665.3190 [M + H]+ calcd 665.3193) and molecular formula (C40H37N6O4). The main differences between the two metabolites were the observed higher chemical shift of C-3 at δ 72.6 ppm indicating its attachment to a nitrogen atom and the lower chemical shift of C-24 at δ 111.3 ppm. Additionally, the DEPT spectrum revealed C-24 to be a primary carbon, while it is a quaternary carbon in compound 1. Accordingly, a connection of the two subunits of the structure elucidated for (1) through a C-N bond from C-3 to the inolic nitrogen was concluded for the dimer of compound (2). The suggested structure for 2 was confirmed by HMBC correlations (Fig. 2) observed from the methylenic protons (H-12) to C-11, C-13, C-28, and C-29 in addition to correlations observed from H-31 to C-3, C-28, and C-29. These data led to the deduction of the structure of asperazine A presented in Fig. 3 for this compound which was matching with previously reported literature data of the compound (Lia 2015).