Biologically Active Metabolites from the Marine Sediment-Derived Fungus Aspergillus flocculosus

Four new compounds were isolated from the Vietnamese marine sediment-derived fungus Aspergillus flocculosus, one aspyrone-related polyketide aspilactonol G (2), one meroterpenoid 12-epi-aspertetranone D (4), two drimane derivatives (7,9), together with five known metabolites (1,3,5,6,8,10). The structures of compounds 1–10 were established by NMR and MS techniques. The absolute stereoconfigurations of compounds 1 and 2 were determined by a modified Mosher’s method. The absolute configurations of compounds 4 and 7 were established by a combination of analysis of ROESY data and coupling constants as well as biogenetic considerations. Compounds 7 and 8 exhibited cytotoxic activity toward human prostate cancer 22Rv1, human breast cancer MCF-7, and murine neuroblastoma Neuro-2a cells.


Results and Discussion
The molecular formula of compound 1 was determined as C9H14O4 by an HRESIMS peak at m/z 209.0785 [M + Na] + , which was supported by the 13 C NMR spectrum.

Results and Discussion
The molecular formula of compound 1 was determined as C 9 H 14 O 4 by an HRESIMS peak at m/z 209.0785 [M + Na] + , which was supported by the 13 C NMR spectrum.
The HMBC correlations ( Figure 2 and Figure S6) from H-4 (δ H 7.27) to C-2 (δ C 174.2), C-3 (δ C 132.8), and C-5 (δ C 84.9) and from H-5 (δ H 4.85) to C-2, C-3, and C-4 (δ C 147.4) suggested the presence of a dihydrofuran ring. The structure of the 1-hydroxyethyl side chain and its location at C-5 in 1 was established by COSY correlations of H-6/H-5 and H-7 and HMBC correlations from H-6 (δ H 4.05) to C-4, C-5, and C-7 (δ C 18.8). The data of COSY spectrum ( Figure S4) and HMBC correlations from H-10 (δ H 1.25) to C-8 (δ C 34.9), C-9 (δ C 66.2), and from both H 2 -8 (δ H 2.52, 2.45) to C-3, C-4, C-9, and C-10 (δ C 23.3) determined the structure of the 2-hydroxypropyl side chain and its location at C-3. 10 (δH 1.25) to C-8 (δC 34.9), C-9 (δC 66.2), and from both H2-8 (δH 2.52, 2.45) to C-3, C-4, C-9, and C-10 (δC 23.3) determined the structure of the 2-hydroxypropyl side chain and its location at C-3. The absolute configuration of the chiral centers C-6 and C-9 of 1 was established using a modified Mosher's method. Esterification of the C-6 and C-9 hydroxy moieties of 1 with (R)-and (S)-MTPA chloride afforded the (S)-and (R)-bis-MTPA-esters, respectively. The observed chemical shift differences Δδ (δS − δR) ( Figure 3A) indicated 6S, 9S configurations. The absolute configuration of C-5 stereocenter in 1 was proven as R on the basis of a characteristic Cotton's effect at λ217 + 11.35 in the CD spectrum (Experimental Section and Figure S8) and a coupling constant value 3 JH5-H6 = 4.4 Hz [14,19]. Compound 1 was recently reported as aspilactonol F, that was a component of unseparated mixture of epimers at C-9. Our study is the first determination of the absolute configurations of all stereocenters of aspilactonol F. The molecular formula of compound 2 was determined as C9H14O4 (the same as 1) on the basis of HRESIMS data and confirmed by 13 C NMR. The NMR data of 2 were very similar to those of 1 (Table 1, Figure S9-16). Thus, the planar structure of 2 was suggested to be the same as that of aspilactonol F (1). The absolute configuration of the chiral centers C-6 and C-9 of 1 was established using a modified Mosher's method. Esterification of the C-6 and C-9 hydroxy moieties of 1 with (R)-and (S)-MTPA chloride afforded the (S)-and (R)-bis-MTPA-esters, respectively. The observed chemical shift differences ∆δ (δ S − δ R ) ( Figure 3A) indicated 6S, 9S configurations. The absolute configuration of C-5 stereocenter in 1 was proven as R on the basis of a characteristic Cotton's effect at λ 217 + 11.35 in the CD spectrum (Experimental Section and Figure S8) and a coupling constant value 3 J H5-H6 = 4.4 Hz [14,19]. Compound 1 was recently reported as aspilactonol F, that was a component of unseparated mixture of epimers at C-9. Our study is the first determination of the absolute configurations of all stereocenters of aspilactonol F. 10 (δH 1.25) to C-8 (δC 34.9), C-9 (δC 66.2), and from both H2-8 (δH 2.52, 2.45) to C-3, C-4, C-9, and C-10 (δC 23.3) determined the structure of the 2-hydroxypropyl side chain and its location at C-3. The absolute configuration of the chiral centers C-6 and C-9 of 1 was established using a modified Mosher's method. Esterification of the C-6 and C-9 hydroxy moieties of 1 with (R)-and (S)-MTPA chloride afforded the (S)-and (R)-bis-MTPA-esters, respectively. The observed chemical shift differences Δδ (δS − δR) ( Figure 3A) indicated 6S, 9S configurations. The absolute configuration of C-5 stereocenter in 1 was proven as R on the basis of a characteristic Cotton's effect at λ217 + 11.35 in the CD spectrum (Experimental Section and Figure S8) and a coupling constant value 3 JH5-H6 = 4.4 Hz [14,19]. Compound 1 was recently reported as aspilactonol F, that was a component of unseparated mixture of epimers at C-9. Our study is the first determination of the absolute configurations of all stereocenters of aspilactonol F. The molecular formula of compound 2 was determined as C9H14O4 (the same as 1) on the basis of HRESIMS data and confirmed by 13 C NMR. The NMR data of 2 were very similar to those of 1 (Table 1, Figure S9-16). Thus, the planar structure of 2 was suggested to be the same as that of aspilactonol F (1). Esterification of the C-6 and C-9 hydroxy moieties of 2 with (R)-and (S)-MTPA chloride afforded the (S)-and (R)-bis-MTPA-esters, respectively. The observed chemical shift differences Δδ (δS − δR) ( Figure 3B) indicated 6R, 9S configurations. The absolute configuration of the C-5 stereocenter in 2 was suggested as S on the basis of a strong negative Cotton's effect at λ216 -11.51 in the CD spectrum (Experimental Section and Figure S17) [19]. Compound 2 was named aspilactonol G.
The molecular formula of compound 4 was established as C22H28O9 on the basis of HRESIMS, containing a peak at m/z 459.1628 [M + Na] + , and was supported by the 13 C NMR spectrum. The molecular formula of compound 2 was determined as C 9 H 14 O 4 (the same as 1) on the basis of HRESIMS data and confirmed by 13 C NMR. The NMR data of 2 were very similar to those of 1 (Table 1, Figures S9-S16). Thus, the planar structure of 2 was suggested to be the same as that of aspilactonol F (1).
Esterification of the C-6 and C-9 hydroxy moieties of 2 with (R)-and (S)-MTPA chloride afforded the (S)-and (R)-bis-MTPA-esters, respectively. The observed chemical shift differences ∆δ (δ S − δ R ) ( Figure 3B) indicated 6R, 9S configurations. The absolute configuration of the C-5 stereocenter in 2 was suggested as S on the basis of a strong negative Cotton's effect at λ 216 -11.51 in the CD spectrum (Experimental Section and Figure S17) [19]. Compound 2 was named aspilactonol G.
The molecular formula of compound 4 was established as C 22 H 28 O 9 on the basis of HRESIMS, containing a peak at m/z 459.1628 [M + Na] + , and was supported by the 13 C NMR spectrum.
signals. The main patterns of the experimental CD spectrum of 4 in methanol (Experimental section, Figure S27) matched well with those of aspertetranone D (5) [5]. The value of the vicinal coupling constant between H-11a and H-12 (9.4 Hz) in 4 instead of 3 JH11a-H12 = 3.9 Hz in aspertetranone D (5) indicated a β orientation of the OH group at C-12 in 4. Thus, the absolute configurations of chiral centers in 4 were suggested as 5aS, 6R, 6aR, 10aR, 11R, 11aS, 12S. Compound 4 was named 12-epiaspertetranone D.   The molecular formula of compound 7 was established as C 15 H 22 O 5 on the basis of an HRESIMS peak at m/z 305.1361 [M + Na] + , which was supported by the 13 C NMR spectrum and corresponded to four double-bond equivalents.
The molecular formula of compound 9 was established as C 15 H 22 O 5 on the basis of an HRESIMS peak at m/z 305.1361 [M + Na] + , which was supported by the 13 C NMR spectrum.
Then, we investigated the effect of the compounds 1-10 on the viability and colony formation ability of human drug-resistant prostate cancer 22Rv1 cells (Table 4). MTT assay revealed the compounds 7 and 8 to be cytotoxic in 22Rv1 cells, with IC50 values of 31.5 μM and 3.0 μM, respectively. Compounds 1-6, 9, and 10 were non-cytotoxic against these cells at concentrations up to 100 μM. In this model, docetaxel (positive control) showed cytotoxicity, with IC50 of 0.02 μM. At the same time, compounds 4 and 9 were able to inhibit the colony formation of 22Rv1 prostate cancer cells (in vitro prototype of in vivo anti-metastatic activity) for 41% and 36%, respectively, at 100 μM. It is known that 22Rv1 cells are resistant to hormone therapy because they express the androgen receptor splice variant AR-V7 [20]. The compounds which demonstrated cytotoxic activity toward AR-V7-positive 22Rv1 cells therefore may be promising for the therapy of human drug-resistant prostate cancer.
Then, we investigated the effect of the compounds 1-10 on the viability and colony formation ability of human drug-resistant prostate cancer 22Rv1 cells (Table 4). MTT assay revealed the compounds 7 and 8 to be cytotoxic in 22Rv1 cells, with IC 50 values of 31.5 µM and 3.0 µM, respectively. Compounds 1-6, 9, and 10 were non-cytotoxic against these cells at concentrations up to 100 µM. In this model, docetaxel (positive control) showed cytotoxicity, with IC 50 of 0.02 µM. At the same time, compounds 4 and 9 were able to inhibit the colony formation of 22Rv1 prostate cancer cells (in vitro prototype of in vivo anti-metastatic activity) for 41% and 36%, respectively, at 100 µM. It is known that 22Rv1 cells are resistant to hormone therapy because they express the androgen receptor splice variant AR-V7 [20].
The compounds which demonstrated cytotoxic activity toward AR-V7-positive 22Rv1 cells therefore may be promising for the therapy of human drug-resistant prostate cancer.
Finally, the new compounds 7 and 9 were tested for cytotoxicity toward human breast cancer cells MCF-7 and did not show any effect up to 100 µM (Table 4). Additionally, the known compounds 8 and 10 were examined in this experiment as reference substances. Compound 8 showed a weak cytotoxic effect, with IC 50 of 59.6 µM, whereas, previously, a higher cytotoxicity of 8 toward MCF-7 cells was reported (IC 50 = 6.08 µM) [11]. This could be explained by different treatment times used by us (24 h) in comparison with those used by Fang and colleagues (72 h) [11]. Moreover, different amounts of cells per well were used. Note, compound 10 was non-cytotoxic up to 100 µM.
The analysis of structure-activity relationships of compounds 7-10, together with literature data, showed that these compounds have three relevant structural sites. First, a double bond at C7=C8 as part of an α,β-unsaturated lactone. Previously, it was shown that the cytotoxicity of such moiety can be explained by a nucleophilic Michael addition reaction with biological nucleophiles [8,21]. In the case of the non-cytotoxic compounds 9 and 10, the double bond of the α,β-unsuturated lactone may be inaccessible for a nucleophile attack because of steric obstacles. Second, a hydroxyl group at C-9 in the drimane core is also essential for cytotoxicity. In fact, a recent report of a series of similar compounds revealed the most pronounced cytotoxicity for compounds possessing a 9-OH group [9]. Finally, our results strongly suggest that the presence of a p-nitrobenzoyl moiety significantly enhances the cytotoxic activity. Previously, Tan et al. [9] demonstrated that the nitrobezoylation of 6-OH increased the cytotoxicity of related compounds towards human renal cell carcinoma cells compared with that of 14-OH-derivatives. At the same time, it should be noted that another study of 6-and 14-nitrobenzoate derivatives cytotoxicity toward other cancer cell lines did not support this observation [11].

Fungal Strain
The strain of A. flocculosus was isolated from a sediment sample (Nha Trang Bay, South China Sea, Vietnam) and identified as described earlier [12]. The strain is stored at the collection of microorganisms of Nha Trang Institute of Technology and Research Application VAST (Nha Trang, Vietnam) under the code 01NT.1.12.3.

Cultivation of the Fungus
The fungus was cultured at 28 • C for three weeks in 50 × 500 mL Erlenmeyer flasks, each containing rice (20.0 g), yeast extract (20.0 mg), KH 2 PO 4 (10 mg), and natural sea water from Nha Trang Bay (40 mL).

Cell Culture
All cell lines used in this investigation were purchased from ATCC. The neuroblastoma cell line Neuro-2a and the human breast cancer cell line MCF-7 were cultured in DMEM medium containing 10% fetal bovine serum (Biolot, St. Petersburg, Russia) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).
The human prostate cancer cell line 22Rv1 was cultured according to the manufacturer's instructions in 10% FBS/RPMI medium (Invitrogen). Cells were continuously kept in culture for a maximum of 3 months, were routinely inspected microscopically for stable phenotype, and regularly checked for contamination with mycoplasma. Cell line authentication was performed by DSMZ (Braunschweig, Germany) using highly polymorphic short tandem repeat loci [22].
All cells were incubated at 37 • C in a humidified atmosphere containing 5% (v/v) CO 2 .

Cytotoxicity Assay
The in vitro cytotoxicity of individual substances was evaluated using the MTT assay, which was performed as previously described [23]. Docetaxel was used as a control.

Colony Formation Assay
The colony formation assay was performed as described before with slight modifications [22]. 22Rv1 cells were treated with the testing compounds for 48 h and then were trypsinized. The number of alive cells was counted with the trypan blue exclusion assay as described before [24]. In total, 100 viable cells were plated into each well of six-well plates in complete fresh medium (3 mL/well) and were incubated for 14 days. Then, the medium was aspirated, and the surviving colonies were fixed with 100% MeOH, followed by washing with PBS, and air-drying at RT. Next, the cells were incubated with a Giemsa staining solution was for 25 min at RT, the staining solution was aspirated, and the wells were rinsed with dH 2 O and air-dried. The number of cell colonies was counted by naked eye.

Conclusions
A new aspyrone-related polyketide, aspilactonol G (2), a new meroterpenoid, 12-epi-aspertetranone D (4), two new drimane derivatives (7,9), together with six known metabolites were isolated from the Vietnamese marine sediment-derived fungus A. flocculosus. The structures of compounds 1-10 were established using spectroscopic methods. The absolute configurations of chiral centers were determined using either a modified Mosher's method (for compounds 1 and 2) or a combination of ROESY data, coupling constants analysis and biogenetic considerations for compounds 4, 7 and 9. Drimane sesquiterpenoid derivatives 7 and 8 showed cytotoxicity toward human prostate cancer 22Rv1, human breast cancer MCF-7, and murine neuroblastoma Neuro-2a cells. The analysis of structure-activity relationships of compounds 7-10 together with literature data showed that these compounds have three sites in their structures related to cytotoxicity, i.e., a double bond at C7=C8, a hydroxyl group at C-9, and a p-nitrobenzoyl moiety.