2.1. Chemical investigation of the ethyl acetate extract of the fungus Aspergillus sp.
In depth chemical investigation of the ethyl acetate extract of the fungus Aspergillus sp. isolated from the inner tissues of the soft coral Sinularia sp. collected from the coast of Sharm El-Sheikh, Egypt resulted in the isolation and characterization of one new meroterpenoid austalide X (1), one known austalide W (2)  in addition to six known prenylated indole diketopiperazine alkaloids (3-8) [28, 29] along with phthalic acid and its ethyl derivative (9-10).
Compound (1) was isolated as a pale-yellow amorphous powder. LC-ESI-MS revealed a pseudomolecular ion peak at m/z 505 and 503representing [M+H]+ and [M-H]- respectively, (supplementary S1) and corresponds to the molecular formula C26H32O10. UV spectrum of compound (1) showed a characteristic pattern of austalides with λ max at 270 nm. Its chemical structure was elucidated through 1D and 2D NMR spectroscopic analysis (supplementary S2-S7), alongside with the reported data for the related analogues austalides V and W . Analysis of 1H and APT-NMR spectra displayed a close similarity between compound (1) and austalide W (2), where, APT spectrum (Table 1) revealed the presence of 26 carbons, including one carbonyl group, six olefinic or aromatic carbons and thirteen aliphatic carbons, one tri-oxygenated quaternary carbon, one di-oxygenated quaternary carbon , three oxygenated quaternary carbons, one quaternary aliphatic carbon, one oxygenated methine carbon, one aliphatic methine carbon, two oxygenated methylene carbons, three aliphatic methylenes, two methoxyl carbons and four methyls.
Meanwhile, 1H NMR spectrum of compound (1) (Table 1) displayed two methine protons at δH (ppm) 3.80 (1H, dd, J = 5.5, 0.9 Hz , H-18) and 3.05 (1H, dd, J = 7.5, 1.8 Hz, H-21) in which the former is oxygenated, in addition to five methylene protons at δH (ppm) 5.14 (2H, s, H-1), 4.16, 3.95 (2H, d, J = 9.7Hz, H-25), 2.95 (2H, m, H-22), 2.64, 2.39 (2H, d, J = 14.5 Hz, H-12) and 2.15, 1.85 (2H, dd, J = 15.9, 0.9 Hz; 15.9, 5.5 Hz, H19) in which the former two methylenes are oxygenated in addition to the presence of two methoxyl protons at δH (ppm) 4.16 (3H, s, OMe-29) and 3.53 (3H, s, OMe-28). Besides, four methyl protons at δH (ppm) 2.06 (3H, s, Me-23), 1.69 (3H, s, Me-26), 1.35 (3H, s, Me-24) and 0.87 (3H, s, Me-27) were observed.
The above-mentioned data were found to be in complete accordance with the reported data for austalides class [30-32] in which one known analogues of this group of compounds was also isolated here in our study from the same crude extract, namely austalide W (2) . Compound (2) was found to be in close similarity to the structure of compound (1), moreover, it is worthy to highlight that compound (2) was previously reported to be the first austalide derivative having a 5/6/6/6/6/5/5 heptacyclic ring system including a tetrahydrofuranyl ring . In addition, this ring system arrangement was confirmed also in compound (1) to be in alignment with all previously reported austalides, as well as the affirmation of presence of ring G through the existence of the down-fielded CH2 group (C-25) at δH (ppm) 4.16, 3.95 (2H, d, J = 9.7 Hz) and δC (ppm) 78.2, alongside with the existence of a characteristic down-field carbon shift of C-13 at δC (ppm) 102.0, in which all those characteristic shifts found to be are similar to compound (2)  affirming the presence of the unusual tetrahydrofuranyl ring (ring G) in the structure of compound (1).
By inspection of the NMR data mentioned above for compound (1), it was clear that it differs from austalide W (2) only by the presence of a newly added hydroxyl group at C-18, this hydroxylation is unambiguously deduced through the difference molecular weight of both compound by only 16 units to the molecular weight of compound (1) than compound (2). Moreover, in COSY spectrum, there is a distinct correlation between H-18 and H2-19 confirming the adjacent positioning of both groups. This suggestion was further confirmed through inspection of the reported data for compound (2), in comparison to the data of the newly isolated compound (1), in which instead of the methylene group at C-18 in compound (2), a clearly observed oxygenated methine proton was detected at δH (ppm) 3.8 on C-18. Hence, there is a low field chemical shift from δH (ppm) 1.81-1.94 in compound (2)  to δH (ppm) 3.8 in compound (1) as well as a low field shift of carbon from δC (ppm) 30.92 in compound (2)  to δC (ppm) 69.2 in compound (1), in addition a low field shift is also detected in APT NMR for C-19 from δC (ppm) 30.83 in compound (2)  to δC (ppm) 37.7 in compound (2), side by side with confirmatory HMBC correlations from the newly observed methine proton H-18 to δC 39.2 (C-20) and δC 118.5 (C-17) alongside with correlations from δH (ppm) 0.87 (H3-27) and δH (ppm) 2.15, 1.85 (H2-19) towards δC (ppm) 69.2 (C-18). It is noteworthy to mention that Antipova et al. had isolated an acetylated derivative, austalide V, where the oxygenated methine was placed at C-19, in our case HMBC correlations had affirmed the presence of the oxygenated methine at C-18 instead, as revealed by the absence of strong correlations between the methyl protons of C-27 and the oxygenated methine carbon C-18. Thus, compound (1) must possess a heptacyclic skeleton as other austalide analogues. Hence, the planar structure of (1) was elucidated as shown, for which the trivial name austalide X is proposed.
The relative configuration of those stereocenters included within compound (1) was determined based on NOESY NMR data (Figure 2). A key NOESY correlation between methyl protons at C-26 and C-27 suggested that ring G to be in an exo-position as well as the endo-configuration of the OH group located at C-13, similarly to other previously reported austalide analogues . Meanwhile, the NOE correlations of methyl protons on C-24 with methine proton at C-21 indicate that both rings C & D are being on the same plane in a cis-connection. In addition, the observed cross-peak correlations between the oxygenated methine proton on C-18 with the methine proton at C-21 alongside with an obvious absence of any cross-peaks or correlations with the former proton with either C-26 or C-27, supposed the endo-position of the OH group on C-18.
Moreover, according to the previously reported possible biosynthetic pathway which suggested that they were all originated from the parent 6-[(2E,6E)-farnesyl]-5,7-dihydroxy-4-methylphthalide followed by cyclization and oxidative modification, and thus all austalide members were originated the same way . Additional proposed pathway was reported as another trial performed by Paquette for austalide derivatives in which he reported a full synthesis of austalide B, one of the austalide group of compounds, [33, 34]. Thus, those two reported proposed pathways declared that there is a constancy of carbon atom’s configuration among all austalide group members and suggested having the same stereochemistry at the specified stereocenters. Hence, the absolute configuration of the stereocenters of compound (1) in our study here is coincidence with all other reported austalide derivatives to be 11S*, 13S*, 14S*, 15S*, 17S*, 18R*, 20S*, 21R*.
2.2. Effect of OSMAC approach on secondary metabolites obtained from the ethyl acetate extracts of the fungal strain
Applying OSMAC approach through changing the fermentation culture medium from the conventional solid rice culture medium to solid beans culture medium was investigated. The ethyl acetate extract of Aspergillus sp. obtained from both media was analyzed using LC-PDA-MS. and revealed apparent changes in their UV and MS profiles. From PDA (photodiode array) data it was clearly obvious that major differences both in qualitative and quantitative manner of the secondary metabolites were observed among the two extracts. Figures 3 and 4 illustrate the HPLC/PDA chromatograms recorded at 254 nm and at maximum absorbance 200-400 nm of both extracts. Major peaks existing in both extracts were compared in (table 2) and different peaks were written in italics where λ max., retention times, area % and peak number of the relevant peaks were displayed. For simplification, the comparison included in table 2 had been performed for detection in channel one at 254 nm. Both positive and negative ESI-MS modes of relevant peaks were merged with the PDA data and included in table 3. From merged PDA and MS data, it was concluded that the major effect of changing the culture media was the complete disappearance of the major compounds produced in the original fermentation medium (i.e., Rice medium) at retention time 32.8 min. (18%) and 35.7 min. (21%) when using beans alternatively as a culture media. These two compounds with molecular weights 511 and 479 respectively, corresponding to compounds (3) and (4) (diketopiperazine derivatives), had been isolated and identified. Moreover, the new meroterpenoid, austalide X (1), with molecular weight 504 and λ max 270 nm, was found in both extracts, indicating no significant effect of the culture media on its production by the fungal isolate. Additionally, peak at retention time 22.7 min produced in rice medium with λ max 251, 290 nm, had been disappeared in the beans medium. This peak corresponds to the spiro-diketopiperazine (8) with molecular weight 427.
2.3. In vitro cytotoxic evaluation of the isolated compounds using MTT assay
Cytotoxic evaluation of compounds (3), (4), (6), (8) (diketopiperazine derivatives) as well as compounds (1) and (2) (meroterpenoid derivatives) against Caco-2 (human colorectal carcinoma cell lines) revealed weak to moderate activities displaying IC50 values ranging between 32.5 and 126 µg/mL where the new meroterpenoid, austalide X (1) displayed substantial cytotoxic effect with IC50 of 51.6 µg/mL as illustrated in table 4.
2.4. In silico molecular docking to evaluate COVID-19 inhibitory potential of isolated compounds
In silico molecular docking was performed on three critical proteins involved in the growth, replication, and invasive character of SARSCoV-2 virus namely 2019-nCoV main protease 2019-nCoV spike glycoprotein and Angiotensin-Converting Enzyme 2(ACE2) (Table 5). Noteworthy to highlight that protease enzyme plays an important role in the process of protein maturation in many viruses via cleansing proproteins after being translated into host cell cytosol. Thus inhibition of viral proteases could serve as therapeutic targets for antiviral agents via reduction of mature viral particles assembly . Meanwhile, S glycoprotein is crucially important in the process of virus attachment, fusion and entrance within the host cell acting as the main target for host immune system via neutralizing the antibodies. Thus, S glycoprotein serves as the main target vaccine preparation and in many therapeutic approaches . Besides, the effectiveness of binding of SARS viral spike (S) protein with ACE2 was elucidated and turned to play the main role in SARS-CoV transmissibility. Hence, the inhibition of ACE2 catalytic pocket using bioactive drug leads could change ACE2 conformation so that it effectively prevents the entrance of SARS-CoV into the host cells .
Results displayed in Table 5 revealed that compound (10) followed by compound (9) highly inhibited all the examined proteins with high fitting scores. Compound (10) showed binding energy (∆G) equals = -25.20 kcal/mole with 2019-nCoV main protease active site owing to the formation of four conventional H-bonds with Asp187, Arg188, Gln189 in addition to the formation of two π-alkyl bonds with Met165 and Met49, one C-H bond with Asp187 and many Van der Waals interactions with the amino acid residues present at the active center (Figure 5A). However, Compound (10) greatly inhibited 2019-nCoV spike glycoprotein displaying high fitting score (∆G = -12.17 kcal/mole) exceeding that of 2019-nCoV spike glycoprotein co-crystalized ligand that showed ∆G of -2.61 kcal/mole. This mainly attributed to the formation of firm three H-bonds with Thr236 and Asn234 in addition to the formation of C-H bond with Thr236 and Van der Waals interaction with Thr108 (Figure 5B). Regarding ACE2, compound (10) displayed the highest fitting within its active site with (∆G = -15.86 kcal/mole) showing superior fitting when compared to ACE2 co-crystalized ligand (∆G = -6.65 kcal/mole). This firm fitting can be interpreted by the virtue of formation of three conventional H-bonds with Ala193, His195, Asn194 in addition to the formation of C-H bond with His195 and Van der Waals interaction with Val107 and Asn103 (Figure 5C).
2.5 ADME / TOPKAT prediction
Prediction of the pharmacokinetic and pharmacodynamic potential as well as the toxicity properties of the isolated compounds was performed using ADME/TOPKAT protocol in Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA). Regarding human intestinal descriptor, compounds (1), (2), (4), (7), (8) (9) and (10), showed good human intestinal absorption level and thus they lie within the 99% absorption ellipse as displayed in ADMET plot (Figure 6). Concerning the solubility level, compound (10) showed optimal solubility while the rest of compounds revealed possible or good solubility levels. Compounds (4), (9) and (10) revealed medium to low BBB penetration level and thus lie inside the 99% BBB confidence eclipse in ADMET plot (Figure 6). On the contrary, the rest of compounds have undefined BBB penetration level taking level 4 and lies outside the 99% BBB confidence eclipse. Regarding the binding of compounds with plasma protein, compounds (3-8) and (10) displayed less than 90% PPB while the rest of compounds showed more than 90% PPB. Additionally, all of the isolated compounds showed no inhibition to CYP2D6 but some of the compounds showed certain hepatotoxic effect as compounds (3-8) and (10) (Table 6).
Regarding TOPKAT prediction, all the examined isolated compounds displayed no mutagenicity as predicted by chemical Ames mutagenicity protocol done in silico. Furthermore, most of the compounds showed no carcinogenic effect to both male and female rat (NTP) except for compounds (9-10) for female rat and compounds (1-2) and (6-8) for male rats. They displayed rat oral LD50 values between 0.16-9.68 g/kg-body-weight with compounds (9-10) showed the highest LD50 of 9.68 and 4.05 g/kg-body-weight, respectively. Similarly for the rat chronic LOAEL level, all the isolated compounds displayed values between 0.0017-1.6549 g/kg-body-weight with compounds (9-10) showed the highest LOAEL of 0.9889 and 1.6549 g/kg-body-weight, respectively. Regarding skin irritancy, most of the isolated compounds showed no to mild skin irritation. For ocular irritation, most of the isolated compounds showed no to mild skin irritation except compound (6-8) and (10) that showed moderate eye irritation. Thus, it can be concluded that most of the isolated compounds showed reasonable pharmacokinetic, pharmacodynamic and toxicity properties so could be incorporated in pharmaceutical dosage forms to prevent cancer and combat COVID-19 infection. Additionally, compounds (9) and (10) that showed the highest fitting scores revealed the best pharmacokinetic and pharmacodynamic properties with slight toxicity that can be controlled by the given doses when formulated in pharmaceutical preparations (Table 7).