Isoquinoline Alkaloids as Protein Tyrosine Phosphatase Inhibitors from a Deep-Sea-Derived Fungus Aspergillus puniceus

Puniceusines A–N (1–14), 14 new isoquinoline alkaloids, were isolated from the extracts of a deep-sea-derived fungus, Aspergillus puniceus SCSIO z021. Their structures were elucidated by spectroscopic analyses. The absolute configuration of 9 was determined by ECD calculations, and the structures of 6 and 12 were further confirmed by a single-crystal X-ray diffraction analysis. Compounds 3–5 and 8–13 unprecedentedly contained an isoquinolinyl, a polysubstituted benzyl or a pyronyl at position C-7 of isoquinoline nucleus. Compounds 3 and 4 showed selective inhibitory activity against protein tyrosine phosphatase CD45 with IC50 values of 8.4 and 5.6 µM, respectively, 4 also had a moderate cytotoxicity towards human lung adenocarcinoma cell line H1975 with an IC50 value of 11.0 µM, and 14, which contained an active center, -C=N+, exhibited antibacterial activity. An analysis of the relationship between the structures, enzyme inhibitory activity and cytotoxicity of 1–14 revealed that the substituents at C-7 of the isoquinoline nucleus could greatly affect their bioactivity.

Puniceusine C (3) was found to have the molecular formula C 21 H 18 N 2 O 4 by HRESIMS that was nearly twice that of 2. The 1 H and 13 C NMR data (Tables 1 and 2) showed great similarity to those of 2, and the clearest difference between them was the disappearance of a methyl signal and the additional presence of a methylene signal (δ H 4.32, 2H, s; δ C 19.4) in 3. The 1 H NMR spectrum of 3 showed a double increase in the integral areas for H-1, H-3, H-4, H-5, and a methoxy group. The HMBC correlations ( Figure 2) from δ H 4.32 (H 2 -9) to C-6/C-7/C-8 suggested a methylene instead of a methyl attached at C-7. These data indicated that 3 was a symmetrical dimer of 1, connected at positions C-7 and C-7 by a methylene C-9. Thus, the structure of 3 was determined as shown. Mar. Drugs 2022, 20, 78 4 of 15 Puniceusine B (2) was assigned the molecular formula C11H11NO2 by HRESIMS. The 1 H NMR and 13 C NMR data (Tables 1 and 2) showed great similarity to those of 1, and the main difference between them was the additional presence of one methyl (δH 2.25, 3H, s; δC 9.2) and the disappearance of one aromatic hydrogen in 2. The HMBC correlations from δH 2.25 to C-6/C-7/C-8 suggested the additional methyl attached at C-7. Hence, the structure of 2 was determined to be 6-methoxy-7-methyl-8-hydroxy-isoquinolin.
Puniceusine C (3) was found to have the molecular formula C21H18N2O4 by HRESIMS that was nearly twice that of 2. The 1 H and 13 C NMR data (Tables 1 and 2) showed great similarity to those of 2, and the clearest difference between them was the disappearance of a methyl signal and the additional presence of a methylene signal (δH 4.32, 2H, s; δC 19.4) in 3. The 1 H NMR spectrum of 3 showed a double increase in the integral areas for H-1, H-3, H-4, H-5, and a methoxy group. The HMBC correlations ( Figure 2) from δH 4.32 (H2-9) to C-6/C-7/C-8 suggested a methylene instead of a methyl attached at C-7. These data indicated that 3 was a symmetrical dimer of 1, connected at positions C-7′ and C-7 by a methylene C-9. Thus, the structure of 3 was determined as shown. Puniceusine D (4) showed the same molecular formula of C21H18N2O4 as that of 3 by analysis of its HRESIMS and NMR data (Tables 1 and 2). The 1 H NMR spectrum showed the presence of two downfield hydrogens at δH 9.59 (1H, s) and 9.52 (1H, s); six aromatic hydrogens at δH 8.58 (1H, d, J = 7.0 Hz), 8.31 (1H, d, J = 7.8 Hz), 8.29 (1H, d, J = 7.0 Hz), 8.05 (1H, d, J = 6.5 Hz), 7.11 (1H, s), and 7.00 (1H, s); two methoxys groups at δH 3.79 (3H, s) and 3.98 (3H, s); and one methylene at δH 4.47 (2H, s). The 13 C NMR spectrum showed 21 carbon signals including one methylene, two methoxyls, eight aromatic methines, and ten aromatic non-protonated carbons. These data showed similarity to those of 1-3, which indicated that 4 was also a dimer of 1. The HMBC correlations from H-5 to C-4/C-8a, from H-16 to C-10/C-14a/C-15/C-17, and from H2-9 to C-6/C-7/C-8/C-10/C-10a/C-17 ( Figure 2) suggested that 4 was an asymmetric dimer of 1 connected at positions C-7 and C-10 by a methylene C-9. The two methoxy groups were attached at C-6 and C-17 based on the HMBC correlations of δH 3.98 (3H, s) with C-17 and δH 3.79 (3H, s) with C-6, respectively. Therefore, the structure of 4 was established as shown.
The molecular formula of puniceusine E (5) was determined as C22H21N2O4 by HRESIMS. The 1 H and 13 C NMR data (Tables 1 and 2) of 5 were greatly similar to those of 4, and the only obvious difference between them was the absence of one aromatic hydrogen and the additional presence of one methyl signal (δH 2.38, 3H, s; δC 10.8) in 5. The HMBC correlations from H3-18 (δH 2.38) to C-15/C-16/C-17 suggested a methyl located at C-16 instead of a hydrogen. Thus, the structure of 5 was established as shown.
The molecular formula of puniceusine E (5) was determined as C 22 H 21 N 2 O 4 by HRES-IMS. The 1 H and 13 C NMR data (Tables 1 and 2) of 5 were greatly similar to those of 4, and the only obvious difference between them was the absence of one aromatic hydrogen and the additional presence of one methyl signal (δ H 2.38, 3H, s; δ C 10.8) in 5. The HMBC correlations from H 3 -18 (δ H 2.38) to C-15/C-16/C-17 suggested a methyl located at C-16 instead of a hydrogen. Thus, the structure of 5 was established as shown.
Puniceusine L (12) had a molecular formula of C 18 H 17 NO 5 on the basis of its HRESIMS and NMR data. Its 1 H and 13 C NMR data (Tables 3 and 4) showed a similarity to those of 8-11. A detailed analysis of HSQC and HMBC spectra suggested that 12 contained the same isoquinoline unit as 8-11. In addition, considering the molecular formula and unsaturation degrees of 12, the HMBC correlations from H 2 -9 to C-6/C-7/C-8/C-10/C-11/C-14, from H-12 to C-10/C-11/C-13/C-15, from H 2 -15 to C-12/C-13/C-16, and from H 3 -16 to C-13/C-15 (Figure 4), suggested a 6-ethyl-4-hydroxy-2H-pyran-2-one unit attached at the methylene C-9 of isoquinoline unit. The above assignment was further confirmed by a single crystal X-ray diffraction analysis (Figure 3). Puniceusine M (13) had the molecular formula of C 19 H 19 NO 5 on the basis of its HRESIMS. The 1 H and 13 C NMR data (Tables 3 and 4) were very similar to those of 12. The only difference between them was the disappearance of one aromatic hydrogen and the additional presence of a methyl (δ H 2.01 (3H, s), δ C 9.9). The HMBC correlations from H 3 -17 (δ H 2.01) to C-11/C-12/C-13 suggested the additional methyl attached at C-12. Therefore, the structure of 13 was established as shown.
Puniceusine N (14) had the molecular formula C 22 H 32 NO 4 + , as determined by HRES-IMS. The 1 H and 13 C NMR (Tables 3 and 4) data of 14 showed a similarity to those of 2, and the clearest difference between them was the additional presence of two methyls, seven methylenes (one oxygenated), one methine, and one carboxyl in 14. Detailed analysis of the HMBC and COSY spectra proved that 14 contained the same isoquinoline unit as that of 2. In addition, combining with the COSY correlation of H 2 -10 (δ H 4.88, t, J = 6.3 Hz) with H 2 -11 (δ H 3.18, t, J = 6.3 Hz) (Figure 4), the HMBC correlations from H 2 -10 to C-1/C-3/C-11/C-12 (δ C 171.8, C), and from H 2 -11 to C-9/C-12 (Figure 4) (Figure 4), suggested that the 2ethylhexanol group connected with the carboxyl of the -CH 2 -CH 2 -COO-group to form an ester. The optical rotation and measured CD data of 14 were zero, which indicated 14 was a racemic mixture. However, an HPLC analysis of 14 with a chiral column (CHIRALPAK IA and IB, respectively, 4.6 mm × 250 mm column), eluting with n-hexane/ethanol/TFA ( Figures S99 and S100), showed a big trailing peak. The reason for this could be that the two kinds of chiral columns were not suitable for the chiral separation of 14. Thus, the structure of 14 was determined as shown.
All of the 14 compounds were evaluated for their enzyme inhibitory activity against five PTPs including CD45, SHP1, TCPTP, PTP1B and LAR, cytotoxicity towards human lung adenocarcinoma cell line H1975, and antibacterial activity. The results of protein phosphatase inhibition assays (Table 5) showed that only 3 and 4 selectively exhibited significant inhibitory activity against CD45 with IC 50 values of 8.4 and 5.6 µM, respectively, and 1, 8, 9, 10, 12 and 13 showed a mild inhibitory activity against several PTPs. A cytotoxicity assay (Table 5) showed that only 4 had a moderate cytotoxicity towards H1975 cell lines with an IC 50 value of 11.0 µM. The analysis of the relationship of their structures, enzyme inhibitory activity and cytotoxicity displayed that the substituents at C-7 of the isoquinoline nucleus could greatly affect their bioactivity. In addition, antibacterial assays exhibited that 14 had medium antibacterial activity towards Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Escherichia coli, with MIC values of 100 µg/mL, and 4 could inhibit the growth of E. coli with a MIC value of 100 µg/mL, while other compounds did not show clear antibacterial activity towards the three indicators. The results indicated that -C=N + unit was an active center for the antibacterial activity of 14.  (2-[(4-acetylphenyl)amino]-3-chloronaphthoquinone); "-": Not tested.

General Experimental Procedure
The procedures were the same as previously reported [13,14].

Fungal Material
The strain Aspergillus puniceus SCSIO z021 was isolated from a deep-sea sediment of Okinawa Trough (27 • 34.01 N and 126 • 55.59 E,~1589 depth), which was located approximately 4.7 km from an active hydrothermal vent. The strain (GenBank accession number KX258801) was identified as Aspergillus puniceus through DNA extraction, ITS sequence amplification and sequence alignment, which has a 99% similarity to A. puniceus (GenBank accession number GU456970). The strain A. puniceus SCSIO z021 was deposited in the RNAM Center, South China Sea Institute of Oceanology, Chinese Academy of Science.

Fermentation and Extraction
The fungus strain was cultivated on potato glucose agar (PDA) plate containing 3% sea salt at 28 • C for 7 days. The spores were selected and transferred to a complex culture medium (glucose 1%, D-mannitol 2%, maltose 2%, corn meal 0.05%, monosodium glutamate 1%, KH 2 PO 4 0.05%, MgSO 4 ·7H 2 O 0.03%, yeast extract 0.3%, sea salt 3%) to obtain a spore suspension that was cultured in a shaker at 28 • C for 3 days at a rotating speed of 180 rmp. The fungus was cultured in 1 L Erlenmeyer flasks each containing 300 mL of 3# medium (glucose 1%, D-mannitol 2%, maltose 2%, corn meal 0.05%, monosodium glutamate 1%, KH 2 PO 4 0.05%, MgSO 4 ·7H 2 O 0.03%, yeast extract 0.3%, sea salt 3%) at 28 • C for 33 days under static condition. After fermentation, the broth and mycelia were separated with gauze. The broth was extracted with XAD-16 resin and sequentially eluted with H 2 O and EtOH to obtain crude extract (61.7 g). The mycelia was extracted three times with acetone, and further extracted three times with EtOAc to yield a crude extract (48.2 g).

X-ray Crystallographic Analysis of 6 and 12
The crystal data were obtained on a Rigaku MicroMax 007 diffractometer (Rigaku Corporation, Tokyo, Japan)with Cu Kα radiation and a graphite monochromator. The crystal structures of 6 and 12 were solved by direct methods with the SHELXTL and refined by full-matrix, least-squares techniques. Crystallographic data for 6 and 12 were deposited with the Cambridge Crystallographic Data Centre as supplementary publication numbers, CCDC 2112471 and 2112479, respectively.

ECD Calculations
The ECD calculation for 9 was performed by Gaussian 16 program package. The procedures were the same as described in our previous study [13,14]. Briefly, the conformational search was performed by a MMFF model, then the conformers with lower relative energies (<10 kcal/mol) were subjected to geometry optimization with the DFT method at the B3LYP/6-311G(d) level. Vibrational frequency calculations were carried out at the same level to evaluate their relative thermal (∆E) and free energies (∆G) at 298.15 K. The geometryoptimized conformers were further calculated at the M06-2X/def2-TZVP level and the solvent (methanol) effects were taken into consideration by using SMD. The optimized conformers with a Boltzmann distribution of more than 1% population were subjected to ECD calculation, which were performed by TDDFT methodology at the PBE1PBE/TZVP level. The ECD spectrum was generated by the software SpecDis using a Gaussian band shape with 0.3 eV exponential half-width from dipole-length dipolar and ratational strengths. The calculated spectrum of 9 was generated from the low-energy conformers according to the Boltzmann distribution of each conformer in MeOH solution. Details regarding optimized conformation geometries, thermodynamic parameters, and Boltzmann distributions (Tables S1-S3) of all conformations are provided in the Supporting Information.

Protein Phosphatase Inhibition Assays
The same methods as described in our previous study [13,14] were applied to test the inhibition activity of compounds 1-14 against five human protein tyrosine phosphatases (CD45, SHP1, TCPTP, PTP1B and LAR).

Cytotoxicity
Cytotoxic activity was evaluated using human lung adenocarcinoma cell line H1975 by CCK-8 method. Briefly, each of the test compounds was dissolved in DMSO and further diluted to give final concentrations of 80, 40, 20, 10, 5, 2.5, and 1.25 µg/mL, respectively. H1975 cells (5 × 10 3 cells/plate) were seeded in 96-well plates and treated with compounds at the indicated concentration for 24 h, and then 10 µL CCK-8 reagent was added to each well, and the plates were incubated at 37 • C for another 4 h. Next, the optical density was measured at a wavelength of 450 nm with the Bio-Rad (Hercules, CA, USA) microplate reader. Dose-response curves were generated, and the IC 50 values were calculated from the linear portion of log dose-response curves.

Antibacterial Assays
Antibacterial activities of 1-14 against E. coli, S. aureus and MRSA were evaluated using the 2-fold dilution assay in 96-microwell plates. Briefly, all the indicator bacteria were cultured on Luria−Bertani (LB) agar plates at 37 • C for 12 h, and then a single colony was picked into LB liquid medium and cultivated on a rotary shaker at 37 • C for 12 h. Then, the bacterial suspensions with LB medium were diluted until the difference of the OD 600 values between the bacterial suspensions and the medium was 0.01~0.02. Each of the tested compounds was dissolved in DMSO to give an initial concentration of 5 mg/mL, and further diluted with the bacterial suspensions by twofold serial dilution to give a final concentration of 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL, respectively. The 96-well plates were incubated at 37 • C for 12 h. MIC value was determined as the lowest concentration with no visible bacterial growth. Ampicillin was used as the positive control and DMSO as the negative control. All experiments were performed three times.

Conclusions
Summarily, 14 new isoquinoline alkaloids (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) were obtained from the deep-seaderived fungus, A. puniceus SCSIO z021. Compounds 3-5 and 8-13 unprecedentedly contained an isoquinolinyl, a polysubstituted benzyl or a pyronyl at position C-7 of the isoquinoline nucleus, which was different from 1-benzylisoquinoline analogues and other isoquinoline alkaloids from plants commonly containing substituents at positions C-1, N-2, C-3, C-4 or C-8 of isoquinoline skeleton. In addition, 3 and 4 showed selective inhibitory activity against CD45; 4 also had moderate cytotoxicity towards human lung adenocarcinoma cell line H1975, and 14 contained an active center -C=N + which had evident antibacterial activity towards three indicator bacteria. An analysis of the relationship of the structures, enzyme inhibitory activity and cytotoxicity of 1-14 displayed that the substituents at C-7 of the isoquinoline nucleus could greatly affect their bioactivity. The results greatly enrich the structural diversity of isoquinoline alkaloids from fungi, and provide a potential lead compound for the development of a selective CD45 inhibitor and anticancer drug.