Penicamide A, A Unique N,N′-Ketal Quinazolinone Alkaloid from Ascidian-Derived Fungus Penicillium sp. 4829

Previously unreported N,N′-ketal quinazolinone enantiomers [(−)-1 and (+)-1] and a new biogenetically related compound (2), along with six known compounds, 2-pyrovoylaminobenzamide (3), N-(2-hydroxypropanoyl)-2 amino benzoic acid amide (4), pseurotin A (5), niacinamide (6), citreohybridonol (7), citreohybridone C (8) were isolated from the ascidian-derived fungus Penicillium sp. 4829 in wheat solid-substrate medium culture. Their structures were elucidated by a combination of spectroscopic analyses (1D and 2D NMR and Electron Circular Dichroism data) and X-ray crystallography. The enantiomeric pair of 1 is the first example of naturally occurring N,N′-ketal quinazolinone possessing a unique tetracyclic system having 4-quinazolinone fused with tetrahydroisoquinoline moiety. The enantiomeric mixtures of 1 displayed an inhibitory effect on NO production in lipopolysaccharide-activated RAW264.7 cells, while the optically pure (–)-1 showed better inhibitory effect than (+)-1.


Introduction
Ascidians have continued to attract a widespread interest for a long time as an important resource for marine natural products with novel structures and potent pharmacological activities [1,2]. One of the most famous marine natural products is the anticancer drug trabectedin (ET-743), which was initially isolated from the marine ascidian Ecteinascidia turbinata [3,4]. More and more research suggests that the real producer of ET-743 is the endosymbiont γ-proteobacterium Candidatus Endoecteinascidia frumentensis [5]. Currently, more scientists have paid attention to ascidian-derived microorganisms, from which more than 150 secondary metabolites with promising bioactivities have been discovered up to now [6]. For example, novel antitumor antibiotics lomaiviticins A and B containing unique dimeric diazobenzofluorene glycoside structures were obtained from ascidian-associated actinomycetes Micromonospora lomaivitiensis [7]; the antimycobacterial oxazinin A, with an intricate dimeric structure, was discovered from a new strain of filamentous ascidian-associated fungus, Eurotiomycetes strain 110162 [8].
Our research group has focused on the secondary metabolites of ascidian-derived fungi isolated from the South China Sea in recent years [9,10].

Results and Discussion
Penicamide A (1) was obtained as a white solid. The molecular formula was determined as C18H18N2O3 on the basis of the positive HR-ESIMS (High Resolution Mass Spectrometry) ( Figure S1

Results and Discussion
Penicamide A (1) was obtained as a white solid. The molecular formula was determined as C 18 Figure S3) and HSQC data (Table 1) of 1 revealed the presence of 18 carbons for 13 sp 2 hybridized carbons and five sp 3 hybridized carbons including one carbon (δ C 68.9) connected with heteroatoms.  Figure S5) between H-3 and H-4, H-4 and H-5, H-5 and H-6, and the HMBC correlations ( Figure S6) from aromatic proton H-3 to C-1 and C-2, H-6 to C-2 and C-7, and the amide proton 18-NH to C-1 and C-17, as well as their chemical shift ( Figure 2). The HMBC correlations of H-12 with C-11 and C-13, H-14 with C-10 and C-15, 13-OH with C-13, and H-20 with C-11 established a 1,2,3,5-tetrasubstituted benzene group. The key HMBC correlations from H-9 to C-7, C-10, and C-11, H-16 to C-10, C-14, C-15, and C-17, together with the chemical shift (δ C 42.1, C-9; 41.7, C-16; 68.9, C-17) suggested that the phenyl group (ring D) was connected to the N-8 of 4-quinazolinone moiety through methylene C-9. At the same time, HMBC correlations from H-16 to C-10, C-14, C-15, and C-17 indicated that C-15 of phenyl group was linked to C-17 of 4-quinazolinone moiety via methylene C-16 to form the C ring. The remaining methyl group was located at C-17, supported by the HMBC correlation of H 3 -19 with C-16 and C-17. of 1 revealed the presence of 18 carbons for 13 sp 2 hybridized carbons and five sp 3 hybridized carbons including one carbon (δC 68.9) connected with heteroatoms.  Figure S5) between H-3 and H-4, H-4 and H-5, H-5 and H-6, and the HMBC correlations ( Figure S6) from aromatic proton H-3 to C-1 and C-2, H-6 to C-2 and C-7, and the amide proton 18-NH to C-1 and C-17, as well as their chemical shift ( Figure 2). The HMBC correlations of H-12 with C-11 and C-13, H-14 with C-10 and C-15, 13-OH with C-13, and H-20 with C-11 established a 1,2,3,5-tetrasubstituted benzene group. The key HMBC correlations from H-9 to C-7, C-10, and C-11, H-16 to C-10, C-14, C-15, and C-17, together with the chemical shift (δC 42.1, C-9; 41.7, C-16; 68.9, C-17) suggested that the phenyl group (ring D) was connected to the N-8 of 4-quinazolinone moiety through methylene C-9. At the same time, HMBC correlations from H-16 to C-10, C-14, C-15, and C-17 indicated that C-15 of phenyl group was linked to C-17 of 4-quinazolinone moiety via methylene C-16 to form the C ring. The remaining methyl group was located at C-17, supported by the HMBC correlation of H3-19 with C-16 and C-17.  Compound 1 was crystallized from methanol by slow evaporation method to afford a crystal of the triclinic space group P −1 , so the structure of 1 was further confirmed by X-ray crystallography ( Figure 3). The P −1 space group of the X-ray structure suggested that compound 1 should be an enantiomer in the solid crystals (Figure 3), which agreed with the result of no signal in the CD spectrum and no optical Mar. Drugs 2019, 17, 522 4 of 10 activity sign in methanol. Subsequently, (±)-1 was subjected to chiral HPLC for purification, and the two enantiomers, (+)-1 (t R = 28.7 min) and (−)-1 (t R = 31.5 min) were obtained, respectively. They displayed opposite Cotton effects in their CD spectra and opposite optical rotations ( Figure 4). The calculated ECD data of (R)-1 were comparable to the experimental one of (+)-1. This result suggested that the absolute configuration of (+)-1 was R and (−)-1 was S. Thus, (+)-1 and (-)-1 was named as (+)-penicamide A and (−)-penicamide A, respectively.

Biological Material
The strain was isolated from the ascidian Styela plicata, which was collected in the Bay of Da'ao, Shenzhen City, Guangdong, Province, China, in April 2016. The fungus was obtained using the standard protocol for isolation [27]. Fungal identification was carried out using a molecular biological protocol by DNA amplification and sequencing of the ITS (Internal Transcribed Spacer) region. The sequence data obtained from the fungal strain have been deposited at GenBank with an accession no. MH465534. A BLAST search result showed that the sequence was most similar (99%) to the sequence of Penicillium canescens (compared to KJ027992.1). A strain was deposited in the Guangdong Microbial Culture Center (GDMCC) under the patent depository number GDMCC No. 60402.

X-ray Crystallographic Analysis of Compound 1
Compound 1 was acquired as colorless crystals from CH 3 OH using the vapor diffusion method. The single crystal X-ray diffraction data was obtained on a Rigaku Oxford Diffraction with Cu-Kα radiation (λ = 1.54178 A). The structures were solved by direct methods using the SHELXS-97 program (University of Göttingen, Göttingen, Germany) and refined using full-matrix least-squares difference Fourier techniques using Olex2 software (Durham University, Durham, UK). Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: 44-(0)1223-336033, or e-mail: deposit@ccdc.cam.ac.uk).

Calculation of the ECD Spectra
Molecular Merck force field (MMFF) and DFT/TD-DFT calculations were carried out with Spartan 14 software (Wavefunction Inc., Irvine, CA, USA) and Gaussian 09 program, respectively [28]. Conformers within 10 kcal/mol energy window were generated and optimized using DFT calculations at the B3LYP/6-31G(d) level. Conformers with Boltzmann distribution over 1% were chosen for ECD calculations in methanol at the B3LYP/6-311+g(2d,p) level. The IEF-PCM solvent model for MeOH was used. ECD spectra were generated using the program SpecDis 3.0 (University of Würzburg, Würzburg, Germany) and OriginPro 8.5 (OriginLab, Ltd., Northampton, MA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.30 eV. All calculations were performed by Tianhe-2 in the National Supercomputer Center in Guangzhou.

Cytotoxic Assay
All the isolated compounds were evaluated for their cytotoxicity against three human cancer cell lines, A549 (lung cancer), HepG2 (liver cancer), and MCF-7 (breast cancer). The three tumor cell lines were generously provided by the cell bank of the Chinese Academy of Sciences (Shanghai, China).
The cytotoxic activity of the tested compounds were assayed by the MTT method using 96 well plates according to the previously reported procedure [29].

Anti-Inflammation Bioassays
The anti-inflammatory activity of the isolated compounds was evaluated according to the previously reported method [30].