Hatsusamides A and B: Two New Metabolites Produced by the Deep-Sea-Derived Fungal Strain Penicillium steckii FKJ-0213

Two new nitrogen-containing metabolites, designated hatsusamide A (1) and B (2), were isolated from a culture broth of Penicillium steckii FKJ-0213 together with the known compounds tanzawaic acid B (3) and trichodermamide C (4) by physicochemical (PC) screening. The structures of 1 and 2 were determined as a tanzawaic acid B-trichodermamide C hybrid structure and a new analog of aspergillazines, respectively. The absolute configuration of 1 was determined by comparing the values of tanzawaic acid B and trichodermamide C in the literatures, such as 1H-nuclear magnetic resonance (1H-NMR) data and optical rotation, after hydrolysis of 1. Compounds 1–4 were evaluated for cytotoxicity and anti-malarial activities. Compounds 1 and 3 exhibited weak anti-malarial activity at half-maximal inhibitory concentration (IC50) values of 27.2 and 78.5 µM against the K1 strain, and 27.9 and 79.2 µM against the FCR3 strain of Plasmodium falciparum, respectively. Furthermore, 1 exhibited cytotoxicity against HeLa S3, A549, Panc1, HT29 and H1299 cells, with IC50 values of 15.0, 13.7, 12.9, 6.8, and 18.7 μM, respectively.


Introduction
Fungal metabolites have been utilized as drugs and as agricultural and chemical reagents. Fungal metabolites containing nitrogen atoms are especially important due to their strong and unique activities. For example, oxaline and neoxaline, discovered from the culture broth of Aspergillus japonicus Fg-551, exhibit mitotic activity and have been used to guide the development of anti-mitotic agents [1][2][3][4]. Moreover, in the Kyoto Encyclopedia of Genes and Genomes (KEGG) MEDICUS (https://www.kegg.jp/kegg/medicus/) database, which presents information on commercially available

Structure Elucidation of 1 and 2
Compound 1 was isolated as a yellow amorphous solid and determined to have the molecular formula C 39 H 46 N 2 O 10 by high-resolution electrospray ionization (HR-ESIMS) (m/z 703.3221 [M + H] + , calcd. for 703.3225). The 1 H NMR (nuclear magnetic resonance) ( Figure S1 and Table 1) and HMQC (Heteronuclear Multiple Quantum Coherence) spectra in (CD 3 ) 2 CO showed the presence of two aromatic protons, nine olefinic protons, three oxymethine protons, two methoxy protons, an N-methyl proton, and three methyl protons. The 13 C ( Figure S2 and Table 1) NMR spectrum showed the presence of four oxygenated carbons, seventeen unsaturated carbons (including five carbons seemingly adjacent to a hetero atom), and three carbonyl carbons. The gross structure of 1 was deduced from detailed analyses of 2D NMR data, including 1 H-1 H COSY (Correlation Spectroscopy), HMQC, and HMBC (Heteronuclear Multiple Bond Correlation) pectra in (CD 3 ) 2 CO (Figures S3-S5). The 1 H-1 H COSY spectra revealed the presence of three partial structures a, b, and c, as shown in Figure 1. The planar structure of 1 was deduced to have a tanzawaic acid B unit by the partial structure a and the HMBC cross-peaks of H-3" and H-2" to C-1", H-8" to C-10", H-9" to C-7" and C-15", H-10" to C-8", and H-15" to C-9". The partial structure b and HMBC cross-peaks of H-3 to C-1, C-2, C-5 and C-9, H-5 to C-3, C-7 and C-9, and H-6 to C-4 and C-8 revealed the presence of a coumarin unit. The positions of two methoxy groups were determined by the HMBC cross-peaks of C-7-methoxy protons to C-7 and C-8-methoxy protons to C-8. The HMBC cross-peaks of N-methyl protons to C-1 and C-2 revealed the presence of an N-methyl amide group at C-2. The partial structure of c and HMBC cross-peaks of H-5 to C-4 , C-6 and C-7 , H-6 to C-4 and C-8 , H-7 to C-5 and C-9 , and H-9 to C-4 revealed the presence of 2-cyclohexene-1-ol. When the structure, including N-methyl amide coumarin and 2-cyclohexene-1-ol, was searched using the Dictionary of Natural Products database, it was found to correspond with trichodermamide C. As 1 was considered to consist of tanzawaic acid B ester-linked trichodermamide C, the structure of 1 was confirmed from hydrolysis products. Compound 1 was hydrolyzed by 1 M NaOH at room temperature for 2 h. After neutralization, the hydrolysate was analyzed by LC/MS (Liquid chromatography-mass spectrometry) ( Figure S6) and purified by preparative HPLC (High Performance Liquid Chromatography). The two isolated products were identified as tanzawaic acid B and trichodermamide C by comparison with the 1 H-NMR ( Figures S7 and S8) and optical rotation values in the literature. Therefore, the structure of 1 was suggested to be a hybrid structure of tanzawaic acid B and trichodermamide C. Finally, the connectivity of these two known compounds was determined by the HMBC cross peak of H-5 to C-1". spectrometry) ( Figure S6) and purified by preparative HPLC (High Performance Liquid Chromatography). The two isolated products were identified as tanzawaic acid B and trichodermamide C by comparison with the 1 H-NMR ( Figures S7 and S8) and optical rotation values in the literature. Therefore, the structure of 1 was suggested to be a hybrid structure of tanzawaic acid B and trichodermamide C. Finally, the connectivity of these two known compounds was determined by the HMBC cross peak of H-5′ to C-1″. The relative configuration of tanzawaic acid B and the absolute configuration of trichodermamide C have been reported previously [12][13][14]. To determine the absolute configuration of 1, tanzawaic acid B (3) was crystalized to carry out a single a crystal X-ray structure analysis. As a result of the analysis, the absolute configuration of tanzawaic acid B was determined, as shown in Figure S9. Therefore, the absolute configuration of 1 was determined to be 4′S, 5′R, 8′R, 9′S, 6″R, 7″R, 10″S, 12″R, 14″S, and 15″R ( Figure 2).  The relative configuration of tanzawaic acid B and the absolute configuration of trichodermamide C have been reported previously [12][13][14]. To determine the absolute configuration of 1, tanzawaic acid B (3) was crystalized to carry out a single a crystal X-ray structure analysis. As a result of the analysis, the absolute configuration of tanzawaic acid B was determined, as shown in Figure S9. Therefore, the absolute configuration of 1 was determined to be 4 S, 5 R, 8 R, 9 S, 6"R, 7"R, 10"S, 12"R, 14"S, and 15"R ( Figure 2). hydrolysis products. Compound 1 was hydrolyzed by 1 M NaOH at room temperature for 2 h. After neutralization, the hydrolysate was analyzed by LC/MS (Liquid chromatography-mass spectrometry) ( Figure S6) and purified by preparative HPLC (High Performance Liquid Chromatography). The two isolated products were identified as tanzawaic acid B and trichodermamide C by comparison with the 1 H-NMR ( Figures S7 and S8) and optical rotation values in the literature. Therefore, the structure of 1 was suggested to be a hybrid structure of tanzawaic acid B and trichodermamide C. Finally, the connectivity of these two known compounds was determined by the HMBC cross peak of H-5′ to C-1″. The relative configuration of tanzawaic acid B and the absolute configuration of trichodermamide C have been reported previously [12][13][14]. To determine the absolute configuration of 1, tanzawaic acid B (3) was crystalized to carry out a single a crystal X-ray structure analysis. As a result of the analysis, the absolute configuration of tanzawaic acid B was determined, as shown in Figure S9. Therefore, the absolute configuration of 1 was determined to be 4′S, 5′R, 8′R, 9′S, 6″R, 7″R, 10″S, 12″R, 14″S, and 15″R ( Figure 2).   Table 1) and HMQC spectra in CD 3 OD showed the presence of two aromatic protons, three olefinic protons, two oxymethine protons, two methoxy protons, and an N-methyl proton. The 13 C NMR ( Figure S11 and Table 1) spectrum showed the presence of four oxygenated carbons, ten unsaturated carbons (including three carbons seemingly adjacent to a hetero atom), and two carbonyl carbons. The gross structure of 2 was deduced from detailed analyses of 2D NMR data, including 1 H-1 H COSY, HMQC, and HMBC spectra in CD 3 OD (Figures S12-S14). The 1 H-1 H COSY spectra revealed the presence of three partial structures a, b, and c, as shown in Figure 3A. The presence of 1,2,3-trihydroxy-4-cyclohexene was revealed by the partial structures a and b and the HMBC cross-peaks of H-5 to C-4 and C-7 , H-6 to C-7 and C-8 , H-7 to C-5 and C-9 , H-8 to C-4 and C-6 , and H-9 to C-7 . The HMBC cross-peaks of H-3 to C-2 , C-4 , and C-9 , H-5 to C-3 , and H-9 to C-2 , and C-4 revealed the presence of a tetrahydrofuran unit connecting at C-4 and C-9 of the cyclohexene unit. The partial structure c and HMBC cross-peaks of H-3 to C-1, C-5, and C-9, H-5 to C-2 ( 4 J), C-3, C-7, and C-9, and H-6 to C-4 and C-8 revealed the presence of a coumarin unit. The positions of two methoxy groups were determined by the HMBC cross-peaks of C-7-methoxy protons to C-7 and C-8-methoxy protons to C-8. The HMBC cross-peaks of N-methyl protons to C-1 and C-2 revealed the presence of an N-methyl amide group at C-2. The gross structure was determined by the HMBC cross-peaks of H-3 to C-1 . Finally, the position of an amino group was determined at C-2 by a chemical shift. Thus, the planar structure of 2 was determined as a new N-methyl analog of aspergillazines D and E ( Figure 3A) [15]. of 2 was determined as a new N-methyl analog of aspergillazines D and E ( Figure 3A) [15].
The partial relative configuration of 2 was deduced by the ROESY (rotating-frame nuclear Overhauser effect correlation spectroscopy) spectrum, coupling constant and putative biogenetic grounds. From the ROESY correlation between H-6′ (δH 4.14) and H-3′ (δH 3.04), these protons had a β-orientation ( Figure 3B). The coupling constant between H-5′ (δH 3.70) and H-6′ was 8.0 Hz, indicating that the proton at C-5 has an α-orientation ( Figure 3B). Aspergillazines D and E are reported to be biogenetically related to penicillazine and epimerize at C-2 position. The equilibrium ratio of C-2 epimers is 1:0.85 [15,16]. Detail analyses of 1D and 2D-NMR showed that 2 also epimerized at C-2′ position. However, the equilibrium ratio of C-2′ epimers was 1:0.13 ( Figure S10). The only difference from aspergillazines D and E, the N-methyl group, is probably involved in the suppression of the epimerization at C-2′ position. Compound 2 is also considered to be produced in the process of producing penicillazine and aspergillazines. Thus, the partial relative configuration of 2 was deduced, as shown in Figure 3B.   The partial relative configuration of 2 was deduced by the ROESY (rotating-frame nuclear Overhauser effect correlation spectroscopy) spectrum, coupling constant and putative biogenetic grounds. From the ROESY correlation between H-6 (δ H 4.14) and H-3 (δ H 3.04), these protons had a β-orientation ( Figure 3B). The coupling constant between H-5 (δ H 3.70) and H-6 was 8.0 Hz, indicating that the proton at C-5 has an α-orientation ( Figure 3B). Aspergillazines D and E are reported to be biogenetically related to penicillazine and epimerize at C-2 position. The equilibrium ratio of C-2 epimers is 1:0.85 [15,16]. Detail analyses of 1D and 2D-NMR showed that 2 also epimerized at C-2 position. However, the equilibrium ratio of C-2 epimers was 1:0.13 ( Figure S10). The only difference from aspergillazines D and E, the N-methyl group, is probably involved in the suppression of the epimerization at C-2 position. Compound 2 is also considered to be produced in the process of producing penicillazine and aspergillazines. Thus, the partial relative configuration of 2 was deduced, as shown in Figure 3B.

Anti-Malarial Activity of 1-4
Compounds 1-4 were tested for anti-malarial activity against both a chloroquine-resistant K1 strain and chloroquine-sensitive FCR3 strain of Plasmodium falciparum (Table 2). Compounds 1 and 3 showed anti-malarial at half-maximal inhibitory concentration (IC 50 ) values of 27.2 and 78.5 µM against the K1 strain and 27.9 and 79.2 µM against the FCR3 strain of P. falciparum, respectively. Compounds 2 and 4 were inactive against both strains. Chloroquine was used as a positive control.

General Experimental Procedures
All solvents were purchased from Kanto Chemical (Tokyo, Japan). Silica gel and octa-decanoylsilicon (ODS) were purchased from Fuji Silysia Chemical (Aichi, Japan).

Isolation and Identification of Strain FKI-0213
Fungal strain FKJ-0213 (JAMSTEC ID: NT10-19 S01-8) was isolated from a deep-sea sediment sample at the bottom of the Sagami Bay off Hatsushima island, Shizuoka, Japan. This strain was identified as a member of the genus Penicillium based on its verticillate conidiophores and conidial chains. The internal transcribed spacer (ITS) sequence of FKJ-0213 was compared with sequences in the GenBank database using BLASTN 2.8.1 analyses [18]. The sequence of FKJ-0213 was in complete agreement with the sequence of CBS 260.55 (ex-type of Penicillium steckii, GenBank accession number NR_111488). Therefore, the producing strain FKJ-0213 was identified as Penicillium steckii based on its morphology and DNA barcoding.
The EtOAc extract (6.0 g) obtained by the procedure described above from the other-day-cultured broth (6 L) was separated by silica gel column chromatography using the same solvent system. The CHCl 3 /CH 3 OH = 9/1 fraction was separated by ODS column chromatography (10% to 100% CH 3 OHaq gradient system). The collected fraction (59.3 mg) containing hatsusamide B was purified by reversed-phase preparative HPLC using a Triart-PFP (pentafluorophenyl) column with a solvent system of 45% CH 3 OH aq containing 0.1% formic acid to yield hatsusamide B (2) (28.3 mg). Hatsusamide

Hydrolysis of 1
A sample of 1 (10.3 mg) was dissolved in 2 M NaOH and stirred at room temperature for 2 h. After stirring, the solution was neutralized with 10% H 2 SO 4 and evaporated. The residue was dissolved in CH 3 OH and separated by preparative thin layer chromatography (TLC) to yield two crude fractions containing tanzawaic acid B and trichodermamide C derived from hatsusamide A. The fraction containing tanzawaic acid B was purified by reversed-phase preparative HPLC using a Triart-PFP column with a solvent system of 85% CH 3 OHaq containing 0.1% formic acid to yield tanzawaic acid B (0.6 mg) derived from hatsusamide A. The fraction containing trichodermamide C was purified by