Two Novel Aspochalasins from the Gut Fungus Aspergillus sp. Z4

Two novel aspochalasins, tricochalasin A (1) and aspochalasin A2 (2), along with three known compounds (3–5) have been isolated from the different culture broth of Aspergillus sp., which was found in the gut of a marine isopod Ligia oceanica. Compound 1 contains a rare 5/6/6 tricyclic ring fused with the aspochalasin skeleton. The structures were determined on the basis of electrospray ionisation mass spectroscopy (ESIMS), nuclear magnetic resonance (NMR) spectral data, and the absolute configurations were further confirmed by modified Mosher’s method. Cytotoxicity against the prostate cancer PC3 cell line were assayed by the MTT method. Compound 3 showed strong activity while the remaining compounds showed weak activity.


Introduction
Aspochalasins constitute a subgroup within the small group of cytochalasans, which are fungal secondary metabolites known for varieties of biological activities [1]. These include cytotoxic [2][3][4], anti human immunodeficiency virus (HIV) [5], immunomodulatory [6], and nematicidal activity [7]. So far, more than 200 cytochalasan analogues have been reported [1]. Structurally, this group of compounds contains one isoindole unit fused with one macrocyclic ring. Isotope-labeling experiments have revealed that cytochalasans originate from an acetyl-and methionine-derived polyketide chain and the attachment of an amino acid precursors such as Leu, Phe, Ala and Trp [8,9]. With diverse oxygenated regions in the macrocyclic ring, there are several unusual analogues among these known compounds including chaetochalasin A [10], aspergillin PZ [11], spicochalasin A [12], epicochalasines A and B [13]. In our ongoing search for new bioactive metabolites of marine fungi, some new compounds have been purified from the marine-derived fungus Z4 [14][15][16][17], and one strain isolated from the gut of the marine isopod Ligia oceanica. In order to find more novel natural products from this fungus, we employed the OSMAC (one strain, many compounds) approach by varying the culture conditions of Z4. Two new cytochalasans, aspochalasins 1 and 2, in addition to three known cytochalasans, aspochalasins 3-5 ( Figure 1) were purified when cultured in media 2216E and rice. Herein we present the isolation, structure elucidation, and cytotoxic activity of these aspochalasins.

Results and Discussion
Compound 1 was purified as a colorless solid. The molecular formula C32H43NO7 with 12 degrees of unsaturation was established by the positive mode quasi-molecular ion peaks at m Hz)] and other 18 aliphatic protons. The 13 C NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 1 indicated 32 carbon resonances ascribed to two ketone carbonyl, one amide carbonyl, six olefinic carbons, five oxygenated carbons including one acetal carbon, 12 aliphatic carbon atoms and six methyl carbons (Table 1). These features characteristically revealed the structure of 1 to be an aspochalasin skeleton. The excess number of carbon atoms when compared to the previously isolated and reported aspochalasin derivatives [18,19], indicated that 1 was an unusual one.

Results and Discussion
Compound 1 was purified as a colorless solid. Hz)] and other 18 aliphatic protons. The 13 C NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 1 indicated 32 carbon resonances ascribed to two ketone carbonyl, one amide carbonyl, six olefinic carbons, five oxygenated carbons including one acetal carbon, 12 aliphatic carbon atoms and six methyl carbons (Table 1). These features characteristically revealed the structure of 1 to be an aspochalasin skeleton. The excess number of carbon atoms when compared to the previously isolated and reported aspochalasin derivatives [18,19], indicated that 1 was an unusual one.  Figure S9) from Me-12 (δ H 1.77) to C-5 (δ C 35.9), C-6 (δ C 140.6) and C-7 (δ C 125.6), from Me-25 (δ H 1.57) to C-13 (δ C 125.1), C-14 (δ C 135.9) and C-15 (δ C 36.9) established unit A, possessing a (2-methylpropyl) isoindolone moiety, which had two positions vacant to be linked, R1 and R2 ( Figure 2 , together with HMBC interactions from Me-33 (δ H 1.85) to C-29 (δ C 127.6), C-28 (δ C 158.1) and C-31 (δ C 197.0), from H-20 and OH-32 to C-31 were preliminarily attributed to unit B ( Figure 2). Furthermore, the HMBC correlations from H-19 and H-20 to C-21 (δ C 214.2), from H-18 to C-16 (δ C 29.3), C-17 (δ C 68.6) and C-19 connected R1 with R4, and R2 with R3 respectively. To satisfy the unsaturation, R5 and R6 were linked. Thus, the gross planar structure of 1 was established. The planar structure of 1 was elucidated by 2D NMR spectrum. The 1 H-1 H correlation spectroscopy (COSY) cross peaks ( Figure S8    The relative stereochemistry of 1 was determined with the help of 1 H NMR coupling constants, nuclear Overhauser effect spectroscopy (NOESY) experiments, and comparison with those of reported aspochalasins. The NOESY cross peaks ( Figure S10) among H-5, H-4 and H-8 demonstrated the relative configurations of the isoindolone moiety in accordance with those of reported cytochalasans [20,21]. For the macrocyclic part, the NOESY correlations of H-17, H-18 and H-20 suggested they were cofacial. The double peak of H-18 with big coupling constants between H-19 reflected the trans-orientation of these two atoms. For the penta-heterocycle moiety, both H-19 and H-30 were located at the joint of three cycles, which elucidated the axial bond in these two atoms. Additionally, the NOESY correlations between H-19, H-30, H-26 and H-32 established that these moieties had the same orientation ( Figure 3). The modified Mosher's method using (S)/(R)-α-methoxy-α-(trifluoromethyl) phenylacetyl (MPTA)-Cl was applied to assign the absolute configuration of 1. The positive and negative value disposition (∆δ S-R ) of the Mosher's ester derivatives (1a and 1b) established the absolute configuration of C-17 as S (Figure 4). It is noteworthy that in all natural cytochalasans, so far, the stereochemistry of perhydroisoindol-1-one moiety is the same [4,21] which assigned the absolute configurations for C-3, C-4, C-5,C-8 and C-9 as 3S, 4R, 5S, 8S, 9S, respectively. Therefore, the complete absolute stereochemistry of 1 could be assigned as 3S, 4R, 5S, 8S, 9S, 17S, 18R, 19R, 20R, 26R, 30S, 32S and named as Tricochalasin A.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 4 of 9 The relative stereochemistry of 1 was determined with the help of 1 H NMR coupling constants, nuclear Overhauser effect spectroscopy (NOESY) experiments, and comparison with those of reported aspochalasins. The NOESY cross peaks ( Figure S10) among H-5, H-4 and H-8 demonstrated the relative configurations of the isoindolone moiety in accordance with those of reported cytochalasans [20,21]. For the macrocyclic part, the NOESY correlations of H-17, H-18 and H-20 suggested they were cofacial. The double peak of H-18 with big coupling constants between H-19 reflected the trans-orientation of these two atoms. For the penta-heterocycle moiety, both H-19 and H-30 were located at the joint of three cycles, which elucidated the axial bond in these two atoms. Additionally, the NOESY correlations between H-19, H-30, H-26 and H-32 established that these moieties had the same orientation (Figure 3). The modified Mosher's method using (S)/(R)-α-methoxy-α-(trifluoromethyl) phenylacetyl (MPTA)-Cl was applied to assign the absolute configuration of 1. The positive and negative value disposition (Δδ S-R ) of the Mosher's ester derivatives (1a and 1b) established the absolute configuration of C-17 as S (Figure 4). It is noteworthy that in all natural cytochalasans, so far, the stereochemistry of perhydroisoindol-1-one moiety is the same [4,21] which assigned the absolute configurations for C-3, C-4, C-5,C-8 and C-9 as 3S, 4R, 5S, 8S, 9S, respectively. Therefore, the complete absolute stereochemistry of 1 could be assigned as 3S, 4R, 5S, 8S, 9S, 17S, 18R, 19R, 20R, 26R, 30S, 32S and named as Tricochalasin A.    The relative stereochemistry of 1 was determined with the help of 1 H NMR coupling constants, nuclear Overhauser effect spectroscopy (NOESY) experiments, and comparison with those of reported aspochalasins. The NOESY cross peaks ( Figure S10) among H-5, H-4 and H-8 demonstrated the relative configurations of the isoindolone moiety in accordance with those of reported cytochalasans [20,21]. For the macrocyclic part, the NOESY correlations of H-17, H-18 and H-20 suggested they were cofacial. The double peak of H-18 with big coupling constants between H-19 reflected the trans-orientation of these two atoms. For the penta-heterocycle moiety, both H-19 and H-30 were located at the joint of three cycles, which elucidated the axial bond in these two atoms. Additionally, the NOESY correlations between H-19, H-30, H-26 and H-32 established that these moieties had the same orientation ( Figure 3). The modified Mosher's method using (S)/(R)-α-methoxy-α-(trifluoromethyl) phenylacetyl (MPTA)-Cl was applied to assign the absolute configuration of 1. The positive and negative value disposition (Δδ S-R ) of the Mosher's ester derivatives (1a and 1b) established the absolute configuration of C-17 as S (Figure 4). It is noteworthy that in all natural cytochalasans, so far, the stereochemistry of perhydroisoindol-1-one moiety is the same [4,21] which assigned the absolute configurations for C-3, C-4, C-5,C-8 and C-9 as 3S, 4R, 5S, 8S, 9S, respectively. Therefore, the complete absolute stereochemistry of 1 could be assigned as 3S, 4R, 5S, 8S, 9S, 17S, 18R, 19R, 20R, 26R, 30S, 32S and named as Tricochalasin A.    Compound 2 was isolated as a white solid. Its molecular formula was deduced to be C 24 13 C NMR spectrum data recorded in CDCl 3 revealed the presence of (2-methylpropyl) isoindolone moiety similar to those found in 1. 13 C NMR and DEPT spectra showed 24 carbon resonances including one ketone carbonyl (δ C 208.9), one ester/lactone carbonyl (δ C 171.9), one amide carbonyl (δ C 172.4), four olefinic carbon (δ C 123. 5 (Table 1). These features characteristically suggested 2 belongs to the same structural family as 1. Comparison with reported aspochalasin derivatives indicated that 2 was similar to aspochalasin A1 [22]. The planar structure of 2 was elucidated by 1 H-1 H COSY and 1 H- 13 1). These features characteristically suggested 2 belongs to the same structural family as 1.
Comparison with reported aspochalasin derivatives indicated that 2 was similar to aspochalasin A1 [22]. The planar structure of 2 was elucidated by 1 H-1 H COSY and 1 H-13 C HMBC experiments ( Figure  5). 1 H-1 H COSY cross peaks ( Figure S20)   The absolute configuration of C-16 in 2 was established by the convenient Mosher's ester. The difference in chemical shift values of the easters 2a and 2b was calculated to assign the absolute configuration at C-16 as R ( Figure 6). Thus, the absolute configuration of 2 was deduced as 3S, 4R, 5S, 8S, 9S, 16R and named as aspochalasin A2. NMR data of compounds 3-5 was in full agreement with the previously reported values for aspochalasins D (3) [23], aspergilluchalasin (4) [24] and aspochalasins T (5) [25].
All compounds were tested for their in vitro cytotoxicity against the prostate cancer PC3 cell lines by the MTT method, using doxorubicin (ADR) as positive control. As shown in Table 2, compound 3 showed strong activity against PC3 cell line, while others showed weak activities against it. Table 2. Growth inhibition of 1-5 against prostate cancer PC3 cell line.
Comparison with reported aspochalasin derivatives indicated that 2 was similar to aspochalasin A1 [22]. The planar structure of 2 was elucidated by 1 H-1 H COSY and 1 H-13 C HMBC experiments ( Figure  5). 1 H-1 H COSY cross peaks ( Figure S20)   The absolute configuration of C-16 in 2 was established by the convenient Mosher's ester. The difference in chemical shift values of the easters 2a and 2b was calculated to assign the absolute configuration at C-16 as R ( Figure 6). Thus, the absolute configuration of 2 was deduced as 3S, 4R, 5S, 8S, 9S, 16R and named as aspochalasin A2. NMR data of compounds 3-5 was in full agreement with the previously reported values for aspochalasins D (3) [23], aspergilluchalasin (4) [24] and aspochalasins T (5) [25].
All compounds were tested for their in vitro cytotoxicity against the prostate cancer PC3 cell lines by the MTT method, using doxorubicin (ADR) as positive control. As shown in Table 2, compound 3 showed strong activity against PC3 cell line, while others showed weak activities against it.  NMR data of compounds 3-5 was in full agreement with the previously reported values for aspochalasins D (3) [23], aspergilluchalasin (4) [24] and aspochalasins T (5) [25].
All compounds were tested for their in vitro cytotoxicity against the prostate cancer PC3 cell lines by the MTT method, using doxorubicin (ADR) as positive control. As shown in Table 2, compound 3 showed strong activity against PC3 cell line, while others showed weak activities against it.

Preparation of MTPA Esters
Compound 1 was transferred into two clean NMR tubes (0.5 mg in each tube) and dried completely under vacuum. Deuterated pyridine (400 µL) was added to dissolve the sample and (R)-MTPA-Cl/(S)-MTPA-Cl (8µL) were quickly added into the tubes, respectively. All contents were mixed thoroughly by shaking the tubes carefully. The reaction was performed at room temperature for 4h to obtained the S-MTPA and the R-MTPA ester (1a and 1b), respectively. The chemical shift differences (∆δ = δ S − δ R ) calculated from the 1 H NMR spectra of the two diastereomeric esters 1a and 1b, obtained without purification, enabled us to determine the absolute configuration of C-17 as S.
Similarly, the S-MTPA and R-MTPA ester (2a and 2b) of compound 2 was obtained. 1 H NMR data of 2a and 2b were run without further purification and the chemical shift differences (∆δ = δ S − δ R ) enabled us to determine the absolute configuration of C-16 as R.

Cytotoxicity Bioassays
The cytotoxicity was measured by the MTT assay against the prostate cancer PC3 cell line. Tumor cell lines were seeded in 96-well plates (4 × 10 3 per well in 100 µL). After 24 h of incubation, the cells were treated with gradient concentrations (100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM) for another 72 h. Afterwards, MTT solution (5.0 mg/mL in RPMI-1640 media, Sigma, St. Louis, MO, USA) was added (20 µL/well) and the plates were incubated for another 4 h at 37 • C. The compounds were dissolved in DMSO and cell growth inhibition assay was performed as reported previously [26]. The growth inhibitory ability of the compounds were calculated and expressed using the IC 50 value by dose-effect analysis software. Doxorubicin (ADR) was used as a positive control.

Conclusions
A chemical investigation was carried out on the marine fungus Z4, which resulted in the isolation of two novel aspochalasins (compounds 1 and 2) along with three known aspochalasins (compounds 3-5). The planar structures were determined and compound 1 contains a rare 5/6/6 tricyclic ring fused with the aspochalasin skeleton. Furthermore, the absolute configurations were established by modified Mosher's method. Cytotoxicity against the prostate cancer PC3 cell line were assayed by MTT method. Compound 3 showed strong activity while others showed weak activity.