Isolation and Structure Elucidation of New Cytotoxic Macrolides Halosmysins B and C from the Fungus Halosphaeriaceae sp. Associated with a Marine Alga

Two new cytotoxic metabolites, halosmysins B and C, have been isolated from the fungus Halosphaeriaceae sp. OUPS-135D-4 separated from the marine alga Sargassum thunbergii. These chemical structures have been elucidated by 1D and 2D NMR, and HRFABMS spectral analyses. The new compounds had the same 14-membered macrodiolide skeleton as halosmysin A, which was isolated from this fungal strain previously. As the unique structural feature, a diketopiperazine derivative and a sugar are conjugated to the 14-membered ring of halosmysins B and C, respectively. The absolute stereostructures of them were elucidated by the chemical derivatization such as a hydrolysis, the comparison with the known compounds (6R,11R,12R,14R)-colletodiol and halosmysin A, and a HPLC analysis of sugar. In addition, their cytotoxicities were assessed using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines. Halosmysin B was shown to be potent against all of them, with IC50 values ranging from 8.2 ± 1.8 to 20.5 ± 3.6 μM, though these values were slightly higher than those of halosmysin A.

Mar. Drugs 2022, 20, x FOR PEER REVIEW 2 of 12 was established to be 6R,14R upon a comparison with known compound 4 [6]. In the present study, an ongoing search for cytotoxic metabolites from this strain led to the isolation of two new 14-membered macrodiolides designated as halosmysins B (2) and C (3) ( Figure  1). The structural analysis of 2 by 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopic techniques showed that 2 has the same planar structure as 1. On the other hand, the alkaline hydrolysis of 3 gave the enantiomer of 5-hydroxy-(2E)-hexenoic acid derived from 1 and 4. This paper reports the absolute stereostructures of 2 and 3, including nuclear Overhauser effect spectroscopy (NOESY) data, information garnered from the J values in the 1 H NMR spectrum, and a plausible biosynthetic proposal. The cytotoxic activities of these compounds against the murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines are also described.
Halosmysin B (2) had the formula C31H38N2O9S established by m/z 615.2378 [M + H] + (calcd for C31H39N2O9S: 615.2376) by high-resolution fast atom bombardment mass spectrometry (HRFABMS) ( Figure S8). The Fourier transform infrared spectrum revealed peaks at 3378 and 1712 cm −1 , which were assigned to the stretching vibrations of hydroxy

Results and Discussion
Halosphaeriaceae sp., a fungal strain from S. thunbergii, was incubated at 27 • C for six weeks in a medium (70 L) containing 1% glucose, 1% malt extract, and 0.05% peptone in artificial seawater adjusted to pH 7.5. The EtOAc extract of the culture filtrate after incubation was purified on a silica gel column, followed by reverse-phase high-performance liquid chromatography (HPLC), affording halosmysins B (2) (0.8 mg) and C (3) (5.5 mg). The structural determination of halosmysin A (1) has been reported [6], and (6R,11R,12R,14R)colletodiol (4) was identified by comparison with the data reported by MacMillan et al. [7].
The relative configuration and the conformation of 2 were investigated by NOESY experiments (Table S1 and Figure 2 and Figure S5). For the 14-membered ring moiety of 2, the NOESY correlations between H-3 and H-5β and between H-4 and H-6, and the large coupling constants (J 5β,6 = 12.6 Hz and J 5β,4 = 10.8 Hz) showed that the angle between bond C-6-C-5 and bond C-4-C-3 was as that of 1. Furthermore, the NOESY correlations from H-16 to H-13α and H-13β and the large coupling constants (J 13β,14 = 11.4 Hz) revealed the angle between bond C-14-C-16 and bond C-13-C-12 ( Figure 2 and Tables S1 and S2). The NOESY correlations between H-9 and H-4 (NH) and between H-10 and H-4 (NH) were observed in 1. However, the correlation observed in 2 was only between H-10 and H-4 (NH), and H-9 correlated with H-1 (NH). The NOESY correlations (H-9/H-3, H-10/H-12, and H-10/3 -SCH 3 ) suggested that the orientation for H-10 was opposite to that of H-9. The above evidence showed that the stereo-arrangements of H-3 and H-6 in the diketopiperazine ring of 2 were opposite to those of 1. Thus, the relative configuration of 2 was established, as shown in Figure 2, which was a stereoisomer of 1 at C-10, C-3 , and C-6 . As described elsewhere [6], the absolute configuration of 1 was determined by alkaline hydrolysis, i.e., the 1 H NMR spectrum and the specific rotation of the reaction product, 5-hydroxy-(2E)-hexenoic acid, were identified with those of the hydrolyzed product from (6R,11R,12R,14R)-colletodiol (4). The reaction gave (-)-5-hydroxy-(2E)-hexenoic acid when the same procedure was applied to 2, as expected. Hence, the absolute stereostructure of 2 was 6R,9S,10R,12R,14R,3 S,6 R ( Figure 2).
Alkaline hydrolysis, which was the same procedure used with 1 and 2, was carried out to determine the absolute configuration of 3. The purification of the reaction mixture gave two carboxylic acids. The 1 H NMR spectral data of them were in perfect agreement with those of the carboxylic acids obtained by the hydrolysis of 4, 5-hydroxy-(2E)-hexenoic acid and 4,5,7-trihydroxy-(2E)-octenoic acid, respectively (Scheme 2). Furthermore, the specific rotations of 4,5,7-trihydroxy octenoic acid obtained from 3 and 4 were in agreement. However, the specific rotations of 3-derived 5-hydroxy hexenoic acid showed a positive sign ([α] D + 9.1), and that of 4-derived showed a negative sign ([α] D − 14.3). The evidence showed that the absolute configuration in the 14-membered macrodiolide moiety of 3 is 6S,11R,12R,14R. The absolute stereostructure of the sugar conjugated to C-11 in the 14-membered ring was determined by the discrimination between the aldose enantiomers using reversed-phase HPLC. In producing the standard samples, D-and L-glucose were treated with L-cysteine methyl ester hydrochloride and o-torylisothiocyanate in pyridine, respectively. Each reaction mixture was analyzed by HPLC, which showed retention times for the D-glucose and L-glucose derivatives of 17.7 min and 12 min, respectively [22]. After the hydrolysis by hydrochloric acid of 3, the same procedure as standard gave the HPLC peak at 17.7 min. In addition, the 1 H NMR spectral data of the sugar derivative from 3 were in good agreement with those of the D-glucose derivative. Therefore, 3 was confirmed to be the glycoside with α-D-glucose.  Alkaline hydrolysis, which was the same procedure used with 1 and 2, was carried out to determine the absolute configuration of 3. The purification of the reaction mixture gave two carboxylic acids. The 1 H NMR spectral data of them were in perfect agreement with those of the carboxylic acids obtained by the hydrolysis of 4, 5-hydroxy-(2E)-hexenoic acid and 4,5,7-trihydroxy-(2E)-octenoic acid, respectively (Scheme 2). Furthermore, the specific rotations of 4,5,7-trihydroxy octenoic acid obtained from 3 and 4 were in agreement. However, the specific rotations of 3-derived 5-hydroxy hexenoic acid showed a positive sign ([α]D + 9.1), and that of 4-derived showed a negative sign ([α]D − 14.3). The evidence showed that the absolute configuration in the 14-membered macrodiolide moiety of 3 is 6S,11R,12R,14R. The absolute stereostructure of the sugar conjugated to C-11 in the 14-membered ring was determined by the discrimination between the aldose enantiomers using reversed-phase HPLC. In producing the standard samples, D-and L-glucose were treated with L-cysteine methyl ester hydrochloride and o-torylisothiocyanate in pyridine, respectively. Each reaction mixture was analyzed by HPLC, which showed retention times for the D-glucose and L-glucose derivatives of 17.7 min and 12 min, respectively [22]. After the hydrolysis by hydrochloric acid of 3, the same procedure as standard gave the HPLC peak at 17.7 min. In addition, the 1 H NMR spectral data of the sugar derivative from 3 were in good agreement with those of the D-glucose derivative. Therefore, 3 was confirmed to be the glycoside with α-D-glucose.  As a primary screen for the antitumor activity, the cancer cell growth inhibitory properties of halosmysins B (2) and C (3) isolated in this study were examined using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines. A previous study reported that 1 had potent cytotoxicity against these cell lines, whereas 4 did not inhibit cell growth [6]. As expected, 2, having the same piperazinedione derivative, showed potent activity comparable to 5-fluorouracil against all these cells, particularly the Scheme 2. The hydrolysis of 3 and 4, and the derivatization of the sugar in 3.
As a primary screen for the antitumor activity, the cancer cell growth inhibitory properties of halosmysins B (2) and C (3) isolated in this study were examined using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines. A previous study reported that 1 had potent cytotoxicity against these cell lines, whereas 4 did not inhibit cell growth [6]. As expected, 2, having the same piperazinedione derivative, showed potent activity comparable to 5-fluorouracil against all these cells, particularly the HL-60 cell line. Compound 3, colletodiol glycoside, did not inhibit cell growth ( Table 2). The addition of various functional groups to the double bond between C-9 and C-10 provides information on the structure-activity relationship and the detailed mechanism of activity. Therefore, the search for 14-membered macrolide analogs from this fungal metabolite will continue.

Fungal Material
The fungus Halosphaeriaceae sp. was isolated from the surface of the marine alga Sargassum thunbergii. Halosphaeriaceae sp. collected at Osaka bay, Japan in July 2017. The fungal strain was identified based on the result of ITS rDNA nucleotide sequence analysis by Techno Suruga Laboratory Co., Ltd. (Shizuoka, Japan). The alga Sargassum thunbergii was wiped with EtOH and a cutting was applied to the surface of nutrient agar layered in a Petri dish. Serial transfers of one of the resulting colonies provided a pure strain of Halosphaeriaceae sp.

Culturing and Isolation of Metabolites
The fungus was cultured at 27 • C for four weeks in a medium (70 L) containing 1% glucose, 1% malt extract, and 0.05% peptone in artificial seawater adjusted to pH 7.5. Then, the culture filtrate was extracted thrice with AcOEt. The combined extracts were evaporated in vacuo to afford a mixture of crude metabolites (12.2 g Table 1 and Table S1.

Sugar Analysis of 3
Glycoside 3 was analyzed with reference to the method described in Ref. [22]. 3 (4.8 mg) and 0.5 N HCl (1 mL) were stirred at 80 • C for 2 h. The reaction mixture was evaporated in vacuo, and the residue was dissolved in pyridine (1 mL) containing L-cysteine methyl ester hydrochloride (5.0 mg), and was heated at 60 • C for 1 h. o-torylisothiocyanate (5.0 mg) was added to the reaction mixture, and was heated at 60 • C for 1 h. The reaction mixture was directly analyzed by ODS HPLC using MeOH/H 2 O (70:30). The peak at 17.7 min was coincident with derivative of D-glucose.

Assay for Cytotoxicity
Cytotoxic activities of 1, 2 and 3 were examined by the same procedure to date [6], the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. P388, HL-60, and L1210 cells were cultured in RPMI 1640 Medium (10% fetal calf serum) at 37 • C in 5% CO 2 . The test materials were dissolved in dimethyl sulfoxide (DMSO) to give a concentration of 10 mM, and the solution was diluted with the Essential Medium to yield concentrations of 200, 20, and 2 µM, respectively. Each solution was combined with each cell suspension (1 × 10 5 cells/mL) in the medium, respectively. After incubating at 37 • C for 72 h in 5% CO 2 , grown cells were labeled with 5 mg/mL MTT in phosphate-buffered saline (PBS), and the absorbance of formazan dissolved in 20% sodium dodecyl sulfate (SDS) in 0.1 N HCl was measured at 540 nm with a microplate reader (MTP-310, CORONA electric). Each absorbance value were expressed as a percentage relative to that of the control cell suspension that was prepared without the test substance using the same procedure as that described above. All assays were performed three times, and semilogarithmic plots were constructed from the averaged data, and the effective dose of the substance required to inhibit cell growth by 50% (IC 50 ) was determined.

The Origin of the Cell Lines
The P388 cell line was obtained from Dr. Numata (death, Osaka Medical and Pharmaceutical University, Japan). The HL-60 cell line was obtained from Dr. Kawai (death, Fuso Pharmaceutical Industries, Ltd., Osaka, Japan). The L1210 cell line was from Dr. Endo (Kanazawa University, Japan).

Conclusions
In conclusion, we have isolated two new cytotoxic metabolites, halosmysins B and C, from the marine-alga-derived fungus Halosphaeriaceae sp., which had the same 14membered macrodiolide skeleton as halosmysin A. We determined the absolute configuration of them using the chemical technique. 2 exhibited a potent cytotoxicity against the HL-60 cell line. In order to study the structure-activity relationship and the biosynthetic pathway, the search for 14-membered ring macrolide analogs from this fungal metabolite will be continued.