Phenylhydrazone and Quinazoline Derivatives from the Cold-Seep-Derived Fungus Penicillium oxalicum

Three new phenylhydrazones, penoxahydrazones A–C (compounds 1–3), and two new quinazolines, penoxazolones A (compound 4) and B (compound 5), with unique linkages were isolated from the fungus Penicillium oxalicum obtained from the deep sea cold seep. Their structures and relative configurations were assigned by analysis of 1D/2D NMR and mass spectroscopic data, and the absolute configurations of 1, 4, and 5 were established on the basis of X-ray crystallography or ECD calculations. Compound 1 represents the first natural phenylhydrazone-bearing steroid, while compounds 2 and 3 are rarely occurring phenylhydrazone tautomers. Compounds 4 and 5 are enantiomers that feature quinazoline and cinnamic acid units. Some isolates exhibited inhibition of several marine phytoplankton species and marine-derived bacteria.


Introduction
Microorganisms of different origin may possess unique genomes and potentials that enable them to produce rare metabolites [1,2]. As an important class of microorganisms, fungi are substantial sources of striking secondary metabolites with diverse structures and bioactivities. The fungal species Penicillium oxalicum is widespread in terrestrial and marine environments, and its secondary metabolites are abundant. In the investigation towards terrestrial-derived P. oxalicum, an azo compound [3], a diterpene [4], a diphenylmethanone [5], a spiro-oxindole alkaloid [6], isochroman carboxylic acids [3], and polyketides [7] were obtained from soil-derived isolates, several limonoids [8], butyrolactones [9], and isocoumarins [10] were discovered from plant-derived isolates, and alkaloids with 1,3-thiazole and 1,2,4-thiadiazole units [11] were separated from an animalderived isolate. On the other hand, more and more marine-derived P. oxalicum strains were chemically examined. A variety of metabolites including chromones [12][13][14][15] from marineanimal-derived strains, phenolic enamides [16], meroterpenoids [16], and alkaloids [17,18] from marine-plant-derived strains, and secalonic acids [19][20][21], anthraquinones [22], alkaloids [23], and a diphenylmethanone [21] from marine-sediment-derived strains were characterized. It is obvious that the metabolic profiles of P. oxalicum strains varied with their habitats. Secondary metabolite biosynthesis genes from fungi have been found to be expressed in deep sea sediments [24,25]. However, chemical surveys were rarely performed on deep-sea-derived P. oxalicum strains, especially cold-seep-derived ones. Penoxahydrazone A (1) was purified as yellow crystals. The molecular formula C35H46N2O4 was determined by interpretation of HRESIMS data. In combination with HSQC correlations, the 1 H NMR spectrum (Table 1) displayed four methyl doublets, two methyl singlets, two double doublets and one singlet ascribable to three olefinic protons, two double doublets and two doublets attributable to four aromatic protons, and a range of signals at δH 1.3-2.9 for seven methylenes and five methines. The 13 C NMR spectrum (Table 1) exhibited 35 resonances, classified into six methyls, seven methylenes, twelve methines, and ten non-protonated carbons by DEPT experiments. The above NMR data partially resembled those for co-isolated dankasterone A [26]. Replacing the signal at δC 199.1 for the C-3 carbonyl group in dankasterone A, a signal at δC 144.4 appeared in the 13 C NMR spectrum of 1, and this carbon atom was located at C-3 by its heteronuclear multiple bond correlation (HMBC)with H-1a. Additionally, the remaining NMR data corresponded to an ortho-substituted benzoic acid unit [27], as supported by the 1 H-1 H chemical shift correlation spectroscopy (COSY) correlations from H-32 thoroughly to H-35 and the HMBC correlations from H-32 to C-36, from H-33 to C-31 and C-35, and from H-35 to C-33 and C-37. To satisfy the elemental composition, an azo unit was situated between C-3 and C-31, which was supported by the HMBC correlations from H-30 to C-3, C-31, C-32, and C-36. Thus, the whole connectivity of 1 was established, which was further validated by other COSY and HMBC correlations ( Figure 2). The absolute configuration was determined by X-ray crystallographic analysis ( Figure 3). A suitable single crystal was obtained from a solution of EtOH with a drop of water and then subjected to the X-ray diffraction analysis using Cu Kα radiation [28]. Consequently, the absolute configuration of 1 was assigned to be 8R, 9R, 10R, 13R, 17R, 20R, and 24R. Although more than 200 natural molecules with a nitrogen-nitrogen bond have been found so far, phenylhydrazone derivatives rarely occurred [27,29]. Furthermore, 1 represents the first natural phenylhydrazone-bearing steroid. Penoxahydrazone A (1) was purified as yellow crystals. The molecular formula C 35 H 46 N 2 O 4 was determined by interpretation of HRESIMS data. In combination with HSQC correlations, the 1 H NMR spectrum (Table 1) displayed four methyl doublets, two methyl singlets, two double doublets and one singlet ascribable to three olefinic protons, two double doublets and two doublets attributable to four aromatic protons, and a range of signals at δ H 1.3-2.9 for seven methylenes and five methines. The 13 C NMR spectrum (Table 1) exhibited 35 resonances, classified into six methyls, seven methylenes, twelve methines, and ten non-protonated carbons by DEPT experiments. The above NMR data partially resembled those for co-isolated dankasterone A [26]. Replacing the signal at δ C 199.1 for the C-3 carbonyl group in dankasterone A, a signal at δ C 144.4 appeared in the 13 C NMR spectrum of 1, and this carbon atom was located at C-3 by its heteronuclear multiple bond correlation (HMBC)with H-1a. Additionally, the remaining NMR data corresponded to an ortho-substituted benzoic acid unit [27], as supported by the 1 H-1 H chemical shift correlation spectroscopy (COSY) correlations from H-32 thoroughly to H-35 and the HMBC correlations from H-32 to C-36, from H-33 to C-31 and C-35, and from H-35 to C-33 and C-37. To satisfy the elemental composition, an azo unit was situated between C-3 and C-31, which was supported by the HMBC correlations from H-30 to C-3, C-31, C-32, and C-36. Thus, the whole connectivity of 1 was established, which was further validated by other COSY and HMBC correlations ( Figure 2). The absolute configuration was determined by X-ray crystallographic analysis ( Figure 3). A suitable single crystal was obtained from a solution of EtOH with a drop of water and then subjected to the X-ray diffraction analysis using Cu Kα radiation [28]. Consequently, the absolute configuration of 1 was assigned to be 8R, 9R, 10R, 13R, 17R, 20R, and 24R. Although more than 200 natural molecules with a nitrogen-nitrogen bond have been found so far, phenylhydrazone derivatives rarely occurred [27,29]. Furthermore, 1 represents the first natural phenylhydrazone-bearing steroid. Penoxahydrazones B(2) and C(3) were obtained as a brown powder. During the purification process, 2 could gradually turn to 3 and vice versa. The sodium adducts ion peaks at m/z 283.0695 and m/z 283.0691 determined by high performance liquid chromatography-electrospray mass spectrometry (HPLC-ESIMS) suggested that 2 and 3 feature the same molecular formula C 13 H 12 N 2 O 4 . On the basis of these characteristics, it is inferred that these two compounds should be a pair of tautomers. The 1 H and 13 C NMR spectra (Table 2) showed two sets of signals with a ratio of 2:1. Aided by HSQC data, they indicated the presence of one oxymethylene, 11 aromatic/olefinic methines, and one carboxyl in each compound. Similar to the analysis of the structure of compound 1, an orthosubstituted benzoic acid unit was deduced to be present in both 2 and 3 based on their NMR data [27], which was further supported by the COSY and the HMBC correlations ( Figure 2). Compared to the NMR data of δ-hydroxymethyl-α-vinylfuran [30], the remaining carbon signals could be assigned to δ-hydroxymethyl furan attached by an olefinic methine group at α position, which was verified by the COSY correlation between H-12 and H-13 and the HMBC correlations from H-10 to C-11 and C-12 and from H 2 -15 to C-13 and C-14. To match the molecular formula, these two moieties were linked via an azo unit, as seen from the HMBC correlation from the de-shielded exchangeable proton to C-7 and C-10 in 2. Although this HMBC correlation was not detected for 3, the similarities of NMR data for C-7 and C-10 between 2 and 3 suggested the same connectivity of them. Through analysis of the whole structures of 2 and 3, their tautomerization probably arose from the geometric isomerization of the double bond between N-9 and C-10. The 13 C NMR data of 9Z and 9E isomers were computed using the gauge-independent atomic orbital (GIAO) method at the B3LYP/6-31+G(d,p) level via Gaussian 09 software [31] and then were input into Sarotti's DP4+ sheet (https://sarotti-nmr.weebly.com) [32]. According to the DP4+ probabilities for 1 H (100% between 9Z isomer and 2, 100% between 9E isomer and 3, Tables S1 and S2) and 13 C NMR data (99.99% between 9Z isomer and 2, 98.80% between 9E isomer and 3, Tables S3 and S4), 2 and 3 were proposed to possess 9Z and 9E configurations, respectively. These two tautomers are possibly formed by the reaction between 2-hydrazinylbenzoic acid and 5-hydroxymethylfurfural, and the latter is a valuable platform chemical produced mainly by the hydrolysis of saccharides [30].  Penoxahydrazones B(2) and C(3) were obtained as a brown powder. During the purification process, 2 could gradually turn to 3 and vice versa. The sodium adducts ion peaks at m/z 283.0695 and m/z 283.0691 determined by high performance liquid chromatography-electrospray mass spectrometry (HPLC-ESIMS) suggested that 2 and 3 feature the same molecular formula C13H12N2O4. On the basis of these characteristics, it is inferred that these two compounds should be a pair of tautomers. The 1 H and 13 C NMR spectra ( Table 2) showed two sets of signals with a ratio of 2:1. Aided by HSQC data, they indicated the presence of one oxymethylene, 11 aromatic/olefinic methines, and one carboxyl in each compound. Similar to the analysis of the structure of compound 1, an ortho-substituted benzoic acid unit was deduced to be present in both 2 and 3 based on their NMR data [27], which was further supported by the COSY and the HMBC correlations ( Figure 2). Compared to the NMR data of δ-hydroxymethyl-α-vinylfuran [30], the remaining carbon signals could be assigned to δ-hydroxymethyl furan attached by an olefinic methine group at α position, which was verified by the COSY correlation  Penoxahydrazones B(2) and C(3) were obtained as a brown powder. During the purification process, 2 could gradually turn to 3 and vice versa. The sodium adducts ion peaks at m/z 283.0695 and m/z 283.0691 determined by high performance liquid chromatography-electrospray mass spectrometry (HPLC-ESIMS) suggested that 2 and 3 feature the same molecular formula C13H12N2O4. On the basis of these characteristics, it is inferred that these two compounds should be a pair of tautomers. The 1 H and 13 C NMR spectra ( Table 2) showed two sets of signals with a ratio of 2:1. Aided by HSQC data, they indicated the presence of one oxymethylene, 11 aromatic/olefinic methines, and one carboxyl in each compound. Similar to the analysis of the structure of compound 1, an ortho-substituted benzoic acid unit was deduced to be present in both 2 and 3 based on their NMR data [27], which was further supported by the COSY and the HMBC correlations ( Figure 2). Compared to the NMR data of δ-hydroxymethyl-α-vinylfuran [30], the remaining carbon signals could be assigned to δ-hydroxymethyl furan attached by an olefinic methine group at α position, which was verified by the COSY correlation Penoxazolones A (4) and B (5) were originally purified as a colorless oil by a series of achiral isolation. HRESIMS analysis gave the molecular formula C 18 H 16 N 2 O 4 . In accordance with the molecular formula, the 1 H and 13 C NMR spectra (Table 3) displayed just one set of signals assignable to one methyl, one methylene, ten methines, and six non-protonated carbons aided by DEPT and HSQC data. A detailed comparison of NMR data revealed the presence of a quinazolin-4(3H)-one unit [33], which was supported by the COSY correlations from H-6 thoroughly to H-9 and the HMBC correlations from H-2 to C-4 and C-10, from H-6 to C-4, C-8, and C-10, from H-8 to C-6 and C-10, and from H-9 to C-5. However, the signal for an exchangeable proton (H-3) in quinazolin-4(3H)-one was missing. The remaining NMR data corresponded to methyl 3-(4-hydroxyphenyl) propanoate [34], except for the presence of signals for a de-shielded methine group and the lack of signals for Mar. Drugs 2021, 19, 9 5 of 10 a methylene group. This methine group was adjacent to the ester carbonyl group as seen from their HMBC correlation, and it was bonded to N-3 of the quinazolin-4(3H)-one moiety on the basis of its HMBC correlations with C-2 and C-4. Thus, the planar structure was established, validated by other COSY and HMBC correlations (Figure 2). Before assignment of the absolute configuration, a chiral HPLC was used to detect the enantiomeric purity due to the presence of a stereogenic carbon atom (C-12). As a result, enantiomers 4 and 5 with a ratio of 1:2 were obtained ( Figure S26), and they exhibited opposite optical rotations. To ascertain the absolute configurations of 4 and 5, their ECD spectra were determined in MeOH and simulated via the time-dependent density function theory method with the same solvent. Based on the similarities between experimental and calculated ECD spectra (Figure 4), the absolute configurations of 4 and 5 were proposed as 12R and 12S, respectively. These two enantiomers might be yielded by adding quinazolin-4(3H)-one to methyl 3-(4-hydroxyphenyl)acrylate through a carbocation intermediate.   Mariculture is often threatened by harmful algal blooms and pathogenic bacteria. Compounds 1-5 and dankasterone A were assayed for inhibition of the three microalgae Chattonella marina, Heterosigma akashiwo, and Prorocentrum donghaiense [35], and the results are shown in Table 4. Isolates 1, 4, 5, and dankasterone A could inhibit the three microalga species with the IC50 values ranging from 0.57 to 9.1 μg/mL. The noticeable activities of 1 and dankasterone A suggested that the steroid moiety seemed to be the key pharmacophore. In combination with the weak activities of 2/3, the slightly higher activities of 1 than those of dankasterone A indicated that the 2-hydrazinylbenzoic acid unit appeared weak to increase the activities. In addition, their inhibition against four marine-derived bacterial pathogens including Vibrio anguillarum, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio splendidus was detected using the disk diffusion method [36]. We found that 1, 4, 5, and dankasterone A showed moderate inhibition against V. harveyi and V. parahaemolyticus, and their inhibition zone diameters exceeded 10 mm at 20 μg/disk. The MIC values of active molecules were also measured, but only those of 4 and 5 (8 μg/mL) appeared lower than 10 μg/mL. A structure-activity relationship analysis revealed that both enantiomerization of 4 and 5 and addition of 2-hydrazinylbenzoic acid Mariculture is often threatened by harmful algal blooms and pathogenic bacteria. Compounds 1-5 and dankasterone A were assayed for inhibition of the three microalgae Chattonella marina, Heterosigma akashiwo, and Prorocentrum donghaiense [35], and the results are shown in Table 4. Isolates 1, 4, 5, and dankasterone A could inhibit the three microalga species with the IC 50 values ranging from 0.57 to 9.1 µg/mL. The noticeable activities of 1 and dankasterone A suggested that the steroid moiety seemed to be the key pharmacophore. In combination with the weak activities of 2/3, the slightly higher activities of 1 than those of dankasterone A indicated that the 2-hydrazinylbenzoic acid unit appeared weak to increase the activities. In addition, their inhibition against four marine-derived bacterial pathogens including Vibrio anguillarum, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio splendidus was detected using the disk diffusion method [36]. We found that 1, 4, 5, and dankasterone A showed moderate inhibition against V. harveyi and V. parahaemolyticus, and their inhibition zone diameters exceeded 10 mm at 20 µg/disk. The MIC values of active molecules were also measured, but only those of 4 and 5 (8 µg/mL) appeared lower than 10 µg/mL. A structure-activity relationship analysis revealed that both enantiomerization of 4 and 5 and addition of 2-hydrazinylbenzoic acid to dankasterone A had almost no influence on the antibacterial activities. In view of the above inhibitory effects of 1, 4, 5, and dankasterone A on the microalgal and bacterial species, their toxicities to the marine zooplankton Artemia salina were also tested. All the isolates possess low toxicities, with LC 50 values being higher that 40 µg/mL.

Fungal Material and Fermentation
Penicillium oxalicum 13-37 was isolated from deep sea cold seep sediments. The species was identified by morphology and by analysis of the ITS regions of its rDNA, whose sequence data have been deposited at GenBank with the accession number MT898464. Its fermentation was carried out statically at room temperature for 30 days in 200 × 1 L Erlenmeyer flasks, each containing 50 g rice, 2 g glucose, 0.6 g peptone, 0.5 g yeast extract, 0.3 g monosodium glutamate, 0.1 g NaBr, 50 mL pure water, and 50 mL natural seawater from the coast of Yantai, China.

X-ray Crystallographic Analysis
Yellow crystals of 1 were obtained from a solution of EtOH with a drop of water. Crystallographic data were collected at 100 K using Cu Kα radiation (λ = 1.54178Å) on a Bruker APEX DUO diffractometer equipped with an APEX II CCD. The structure of 1 was solved by direct methods with the SHELXS-97 software package. All non-hydrogen atoms were refined anisotropically with SHELXL-97 and SHELXL-2014 using full-matrix least-squares, and refinements of the H atoms in calculated positions were performed using a riding model. Molecular graphics were calculated with PLATON [37]. Crystallographic data have been deposited in the Cambridge Crystallographic Data Centre as CCDC 2,024,007 for 1. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif (or from the CCDC, 12 Union Road, Cambridge CB21EZ, U.K.; fax: + 44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).

Crystal Data for Penoxahydrazone A (1)
Crystal data is as follows: 2(C 35

Computational Details
Conformational searches for compounds 2-5 were operated via the Dreiding force field in MarvinSketch software (optimization limit = normal, diversity limit = 0.1; MarvinSketch with Calculator Plugins for Structure Property Prediction and Calculation, Marvin, Version 6.2.2, 2014, ChemAxon, Budapest, Hungary). Regardless of the rotation of methyl and hydroxy groups, the energy-minimized conformers (Figures S1-S4) within a 3 kcal/mol energy threshold from the global minimum were generated after conformational optimization at the B3LYP/6-31+G(d,p) level in DMSO for 2 and 3 and at the B3LYP/6-31G(d) level in MeOH for 4 and 5 using Gaussian 09 software [31]. The 1 H and 13 C NMR data of each conformer of 2 and 3 were computed at the B3LYP/6-31+G(d,p) level in DMSO through the gauge-independent atomic orbital (GIAO) method, while the ECD spectrum of each conformer of 4 and 5 was calculated at the B3LYP/6-31G(d) level in MeOH via the time-dependent density function theory (TD-DFT) method and then drawn by SpecDis software with sigma = 0.25 and UV-shift = −8 nm. The overall calculated 1 H and 13 C NMR data and ECD curves of each compound were produced by Boltzmann weighting. All of the above calculations were performed with the integral equation formalism variant (IEF) of the polarizable continuum model (PCM) as implemented in Gaussian 09.

Conclusions
Five new dinitrogen-bearing metabolites have been isolated and identified from a coldseep-derived strain of P. oxalicum, and they display high novelty due to the unprecedented linkages. As phenylhydrazone derivatives, 1 features a special steroid framework, while tautomers 2 and 3 contain a furan ring bonding to the azo group. It seems that this fungal strain has the ability to form phenylhydrazones through adding 2-hydrazinylbenzoic acid to ketones or aldehydes. The discovery of penoxahydrazones A-C (1-3) adds greatly to the diversity of phenylhydrazone derivatives. As quinazoline derivatives, enantiomers 4 and 5 possess a unique linkage between quinazoline alkaloid and cinnamic acid units. These metabolites with more or less antimicroalgal and antibacterial activities are structurally different from those isolated from the P. oxalicum strains of other origin, which demonstrates that deep-sea-derived fungi actually feature the potential to produce novel metabolites and further underpins the significance of chemically exploring the extreme-environmentderived fungi, such as those from the deep sea cold seep.