**1. Introduction**

Marine fungi are rich sources of new biologically active compounds [1]. Fungi of the genus *Aspergillus*, section *Circumdati* (*Aspergillus insulicola*, *Aspergillus flocculosus*, *Aspergillus ochraceus*, *Aspergillus ochraceopetaliformis,* and others) [2], are known to produce metabolites belonging to various chemical classes: aspyrone-related pentaketides [3,4], meroterpenoids [5,6], diketopiperazine alkaloids [7], drimane sesquiterpenoids and their nitrobenzoyl derivatives [8,9], steroids, and cerebrosides [10]. Many of them possess antimicrobial [4,10], antiviral [11], cytotoxic [8,11], and neuroprotective [12] activities.

Aspyrone-related pentaketides are polyketide metabolites commonly found in this fungal group [13]. Usually, they are divided into three structural types: linear (aspinonene) [3], δ-lactones (aspyrone) [3], and γ-lactones (iso-aspinonene, aspilactonols) [3,14]. Meroterpenoid metabolites of *Aspergillus,* section *Circumdati* fungi are represented mainly by triketidesesquiterpenoids with rare α-pyrone-contained linear or angular skeleton. To date, only several representatives of this chemical class belonging to the aspertetranones [5] and ochraceopones [6] series were reported. Nitrobenzoyl derivatives of drimane-sesquiterpenoids were initially found in *A. insulicola* species but can also be produced by other related fungi [15]. These compounds are characterized by a small structural diversity with two isomeric backbones (cinnamolide- and confertifolin-based) and various locations of acyl groups. A residue of *p*-nitrobenzoic acid usually can be found at positions 9-OH or 14-OH. Nitrobenzoyl derivatives are relatively unstable compounds that cannot be hydrolyzed to form the corresponding sesquiterpenoids [8]. Acetylation of these compounds with acetic anhydride results in rearrangemen<sup>t</sup> and formation of several products [16].

Recently, we have started a project focusing on the search for producers of novel bioactive compounds among fungi isolated from various substrates found in the Vietnamese waters of the South China Sea [17,18]. Thus, from a sediment sample collected in Nha Trang Bay, we have isolated a strain of fungus *A. flocculosus*. Recently, we described the new neuroprotective alkaloid mactanamide produced by this strain [12]. Herein, we report the isolation, structure elucidation and cytotoxic activity of four new (**2**,**4**,**7**,**9**) and six known (**1**,**3**,**5**,**6**,**8**,**10**) metabolites produced by the same fungus (Figure 1).

**Figure 1.** Chemical structures of the isolated compounds **1**–**10**.

### **2. Results and Discussion**

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

A close inspection of the 1H and 13C NMR data of **1** (Table 1, Figures S1–S3) revealed the presence of two methyls (δC 23.3, 18.8; δH 1.31, 1.25), one methylene (δC 34.9; δH 2.52, 2.45), three oxygen-bearing *sp*3-methines (δC 84.9, 67.8, 66.2; δH 4.85, 4.08, 4.05) and one *sp*2-methine (δC 147.4; δH 7.27). Two remaining signals at δC 132.8 and 174.2 ppm corresponded to a quaternary *sp*2-carbon and a carboxyl carbon, respectively.

The HMBC correlations (Figure 2 and Figure S6) from H-4 (δH 7.27) to C-2 (δC 174.2), C-3 (δC 132.8), and C-5 (δC 84.9) and from H-5 (δH 4.85) to C-2, C-3, and C-4 (δC 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 (δH 4.05) to C-4, C-5, and C-7 (δC 18.8). The data of COSY spectrum (Figure S4) and HMBC correlations from H-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.


**Table 1.** 1H and 13C NMR data (δ in ppm, CDCl3) for aspilactonols G (**1**) and F (**2**).

1H NMR and 13C NMR spectroscopic data were measured at 500 MHz and 125 MHz, respectively.

**Figure 2.** The key HMBC correlations of **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 Δδ (δS − δR) (Figure 3A) indicated 6*S*, *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 <sup>3</sup>*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.

**Figure 3.** Δδ (δS−δR) values (in ppm) for the MTPA ester of **1** (**A**) and **2** (**B**).

The molecular formula of compound **2** was determined as C9H14O4 (the same as **1**) on the basis of HRESIMS data and confirmed by 13C 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 Δδ (δS − δR) (Figure 3B) indicated 6*R, 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]<sup>+</sup>, and was supported by the 13C NMR spectrum.

An analysis of NMR data of **4** (Table 2, Figures S20–S24) revealed the presence of six methyl groups (δC 25.1, 24.0, 18.5, 17.3, 10.8, 9.5; δH 2.24, 1.89, 1.43, 1.41, 1.39, 1.31), one *sp*3-methylene group (δC 45.6; δH 2.86, 2.76), two *sp*3-methines (δC 39.5, 39.3; δH 2.32, 2.00), two oxygen-bearing ones (δC 75.15, 63.5; δH 4.63, 4.36), one quaternary *sp*3-carbon (δC 55.5), three oxygen-bearing quaternary *sp*3-carbons (δC 83.0, 76.5, 75.07), two quaternary *sp*2-carbons (δC 107.3, 102.2), three oxygen-bearing quaternary *sp*2-carbons (δC 164.4, 162.5, 157.9), and two ketone groups (δC 211.4, 209.1).


**Table 2.** 1H and 13C NMR data (δ in ppm, CDCl3) for 12-*epi*-aspertetranone D (**4**).

1H NMR and 13C NMR spectroscopic data were measured at 500 MHz and 125 MHz, respectively.

The HMBC correlations of **4** (Figure 4 and Figure S25, Table 2) suggested the presence of a linear tetracyclic backbone like in the recently reported merosesquiterpenoids aspetetranones A-D [5]. The general features of the 13C NMR spectrum of **4** (Table 2, Figures S21–S22) were similar to those of aspertetranone D (**5**) [5], with the exception of the C-6, C-11, C-11a, C-12, C-15, and C-18 carbon 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 <sup>3</sup>*J*H11a-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 5a*S*, 6*R*, 6a*R*, 10a*R*, 11*R*, 11a*S*, 12*S*. Compound **4** was named 12-*epi*-aspertetranone D.

**Figure 4.** The key HMBC correlations of **4.**

The molecular formula of compound **7** was established as C15H22O5 on the basis of an HRESIMS peak at *m*/*z* 305.1361 [M + Na]<sup>+</sup>, which was supported by the 13C NMR spectrum and corresponded to four double-bond equivalents.

A close inspection of the 1H and 13C NMR data of **7** (Table 3, Figures S30–S32) revealed the presence of two methyl groups (δC 26.8, 20.8; δH 1.23, 1.15), three *sp<sup>3</sup>*-methylene groups (δC 42.0, 32.6, 17.6; δH 2.13, 1.63, 1.50 (2H), 1.38, 1.24), two oxygen-bearing *sp*3-methylene groups (δC 75.0, 68.4; δH 4.44, 4.41, 4.24, 3.42), two *sp*3-methine groups (δC 63.5, 47.1; δH 4.62, 2.00), including one oxygen-bearing, one *sp*2-methine group (δC 139.1; δH 6.96), three quaternary *sp*3-carbons (δC 77.5, 39.0, 38.3), including one oxygen-bearing, and two quaternary *sp*2-carbons (δC 169.6, 130.1).



1H NMR and 13C NMR spectroscopic data were measured a in CDCl3 at 500 MHz and 125 MHz, respectively, and b in DMSO-d6 at 700 MHz and 176 MHz, respectively.

The 13C NMR data of **7** were similar to those of the drimane moiety of insulicolide A (**8**) [15], also reported as 9α-14-dihydroxy-6β-*p*-nitrobenzoylcinnamolide [8], with the exception of the C-3, C-6, C-7, C-8, and C-14 carbon signals. The COSY spectrum data (Figure S33) and HMBC correlations (Figure S35, Table 3) from H-6 (δH 4.62) to C-7 (δC 139.1), C-8 (δC 130.1), and C-10 (δC 39.0), from H-7 (δH 6.96) to C-5 (δC 47.1), C-9, and C-12 (δC 169.6), from H3-13 (δH 1.15) to C-3 (δC 42.0), C-4 (δC 38.3), C-5 (δC 47.1), and C-14 (δC 68.4), and from H3-15 (δH 1.23) to C-1 (δC 32.6), C-5, C-9, and C-10 proved the drimane framework of **7** the same as in insulicolide A (**8**).

The ROESY correlations (Figure S36) of H3-13 with H-5 (δH 2.00) and H-6, long-range COSY correlation H3-15/H-5, together with the vicinal coupling constant <sup>3</sup>*J*H5-H6 = 4.4 Hz established the relative configurations of the C-4, C-5, C-6, and C-10 chiral centers. The absolute configurations of the stereocenters in **7** were suggested as depicted in Figure 1 from CD spectra similarity (Figures S37 and

S38) and biogenetic relationship with insulicolide A (8), whose absolute configurations were determined previously by X-ray analysis [15]. Compound **7** was named 6β,9<sup>α</sup>,14-trihydroxycinnamolide.

The molecular formula of compound **9** was established as C15H22O5 on the basis of an HRESIMS peak at *m*/*z* 305.1361 [M + Na]<sup>+</sup>, which was supported by the 13C NMR spectrum.

A close inspection of the 1H and 13C NMR data of **9** (Table 3, Figures S39–S41) revealed the presence of two methyl groups (δC 27.9, 21.6; δH 1.40, 0.97), three *sp*3-methylene groups (δC 37.8 (2C), 18.0; δH 1.71, 1.59, 1.54, 1.45, 1.32, 1.10), two oxygen-bearing *sp<sup>3</sup>*-methylene groups (δC 68.1, 65.6; δH 4.94, 4.79, 3.94, 3.26), three *sp*3-methine groups (δC 70.0, 64.1, 48.6; δH 4.00, 3.99, 1.57), including two oxygen-bearing, two quaternary *sp*3-carbons (δC 38.3, 36.3), and three quaternary *sp*2-carbons (δC 173.4, 173.1, 122.1).

The HMBC correlations (Table 3, Figure S42) from H-6 (δH 3.99) to C-5 (δC 48.6), C-7 (δC 64.1), C-8 (δC 122.1), C-9 (δC 173.1), and C-10 (δC 36.3), from H-7 (δH 4.00) to C-12 (δC 173.4), from H2-11 (δH 4.94, 4.79) to C-8, C-9, and C-12, from H3-13 (δH 0.97) to C-3 (δC 37.8), C-4 (δC 38.3), C-5, and C-14 (δC 65.6), from H3-15 (δH 1.40) to C-1 (δC 37.8), C-5, C-9, and C-10 indicated the drimane moiety in **9** being the same as in <sup>7</sup>α,14-dihydroxy-6β-p-nitrobenzoylconfertifolin [8].

The ROESY correlations (Figure 5 and Figure S43) of H3-13 with H-5 (δH 1.57), H-6 (δH 3.99), and H-7 (δH 4.00), of H3-15 with H2-14 (δH 3.94, 3.26), together with the coupling constant <sup>3</sup>*J*H6-H7 = 2.1 Hz indicated the related configurations of the chiral centers in **9** as depicted (Figure 1). Compound **9** was named 6β,7β,14-trihydroxyconfertifolin.

**Figure 5.** Key ROESY correlations of **9**.

Besides the new compounds **1,2,4,7**, and **9**, the known dihydroaspirone (**3**) [14], aspertetranones D (**5**) [5,6] and A (**6**) [5], insulicolide A (**8**) [15], and <sup>7</sup>α,14-dihydroxy-6β-p-nitrobenzoylconfertifolin (**10**) [8] were isolated from this fungal strain.

All isolated compounds were tested for cytotoxicity toward murine neuroblastoma Neuro-2a cells (Table 4). Compound **7** demonstrated cytotoxic activity toward Neuro-2a cell, with the IC50 of 24.1 μM, while its analogue **9** was non-cytotoxic up to 100 μM. The highest activity was demonstrated for 9<sup>α</sup>,14-dihydroxy-6β-p-nitrobenzoylcinnamolide (**8**), with IC50 of 4.9 μM, while its analogue **10** did not affect the viability of Neuro-2a cells. Compounds **1**–**6** were non-cytotoxic against Neuro-2a cells at concentrations up to 100 μM.

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.


**Table 4.** Cytotoxic effects of the isolated compounds **1**–**10**.

"nt": compound was not tested; "-": compound did not demonstrate any effect at the concentration of 100 μM.

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 IC50 of 59.6 μM, whereas, previously, a higher cytotoxicity of **8** toward MCF-7 cells was reported (IC50 = 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 <sup>α</sup>,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 <sup>α</sup>,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 sugges<sup>t</sup> 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].

### **3. Materials and Methods**

### *3.1. General Experimental Procedures*

Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA). UV spectra were recorded on a Specord UV−vis spectrometer (Carl Zeiss, Jena, Germany) in methanol. NMR spectra were recorded in CDCl3, acetone-d6 and DMSO-*d*6 with Bruker DPX-500 (Bruker BioSpin GmbH, Rheinstetten, Germany) and Bruker DRX-700 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometers, using TMS as an internal standard. HRESIMS spectra were measured on a Maxis impact mass spectrometer (Bruker Daltonics GmbH, Rheinstetten, Germany).

Low-pressure liquid column chromatography was performed using silica gel (50/100 μm, Imid, Russia). Plates (4.5 cm × 6.0 cm) precoated with silica gel (5–17 μm, Imid) were used for thin-layer chromatography. Preparative HPLC was carried out with a Shimadzu LC-20 chromatograph (Shimadzu

USA Manufacturing, Canby, OR, USA) using YMC ODS-AM (YMC Co., Ishikawa, Japan) (5 μm, 10 mm × 250 mm) and YMC SIL (YMC Co., Ishikawa, Japan) (5 μm, 10 mm × 250 mm) columns with a Shimadzu RID-20A refractometer (Shimadzu Corporation, Kyoto, Japan).
