Isolation and Characterization of Antimicrobial Metabolites from the Sophora tonkinensis-Associated Fungus Penicillium sp. GDGJ-N37

Chemical investigation of Penicillium sp. GDGJ-N37, a Sophora tonkinensis-associated fungus, yielded two new azaphilone derivatives, N-isoamylsclerotiorinamine (1) and 7-methoxyl-N-isoamylsclerotiorinamine (2), and four known azaphilones (3–6), together with two new chromone derivatives, penithochromones X and Y (7 and 8). Their structures were elucidated based on spectroscopic data, CD spectrum, and semi-synthesis. Sclerotioramine (3) showed significant antibacterial activities against B. subtilis and S. dysentery, and it also showed most potent anti-plant pathogenic fungi activities against P. theae, C. miyabeanus, and E. turcicum.

Fungi are a promising source of novel and biologically active natural products for drug discovery [14].Penicillium fungi, recognized for their ability to generate structurally novel and bioactive compounds [15,16], are an important source of antimicrobial agents.In our ongoing search for bioactive metabolites from endophytic fungi [17][18][19], a Sophora tonkinensis-associated fungus, Penicillium sp.GDGJ-N37, was investigated.The EtOAc extract of this fungus showed antibacterial activity to Bacillus subtilis and antifungal activity to Setosphaeria turcica.A follow-up chemical investigation of the extract led to the isolation of two new azaphilones, N-isoamylsclerotiorinamine (1) and 7-methoxyl-Nisoamylsclerotiorinamine (2), together with four known azaphilone derivatives, sclerotioramine (3) [20], isochromophilone VI (4) [21], sclerotiorin (5) [21], and hypocrellone A (6) [22].Two new chromone derivatives, penithochromones X and Y (7 and 8), together with a known one, penithochromone F (9) (Figure 1) [23], were also obtained from the fungus.Among these azaphilones, 3 could be obtained by semi-synthesis from 5 in a yield over 30% by a one-step process.Additionally, azaphilone derivatives 10-12 were semi-synthesized for structure elucidation and structure-activity relationship (SAR) studies.Herein, we described the isolation, structure elucidation, and antimicrobial activity of these compounds.Preliminary SAR of the azaphilone derivatives were also discussed.
new chromone derivatives, penithochromones X and Y (7 and 8), together with a known one, penithochromone F (9) (Figure 1) [23], were also obtained from the fungus.Among these azaphilones, 3 could be obtained by semi-synthesis from 5 in a yield over 30% by a one-step process.Additionally, azaphilone derivatives 10-12 were semi-synthesized for structure elucidation and structure-activity relationship (SAR) studies.Herein, we described the isolation, structure elucidation, and antimicrobial activity of these compounds.Preliminary SAR of the azaphilone derivatives were also discussed.

N-Isoamylsclerotiorinamine
The absolute configuration at C-7 of 1 and 2 was elucidated by comparing their CD spectra with that of isochromophilone VI (4) (Figure 3).Compounds 1, 2, and 4 had similar CD spectra, which showed a positive Cotton effect at 380 nm and a negative Cotton effect at 300 nm.It revealed that the absolute configuration of C-7 was R in 1 and 2 [24,25].The absolute configuration of the C-13 stereocenter in 1 and 2 was determined by semi-synthesis.Isoamylamine was employed to provide 1 and the deacetylate analogue 11 from the known sclerotiorin (5).Compound 11 was further methylated with CH 3 I to give 2. The 1 H NMR spectra of the semisynthetic products 1 and 2 were identical to those of the natural products 1 and 2, respectively.On the other hand, compounds 1 and 2 are most likely derived from the same biogenetic pathway as 3 and 4. It meant that the absolute configuration of C-13 in 1 and 2 was an S-configuration, just the same as the absolute configuration of C-13 in 3 and 4. Thus, the absolute configurations of 1 and 2 were 7R, 13S.The absolute configuration at C-7 of 1 and 2 was elucidated by comparing their CD spectra with that of isochromophilone VI (4) (Figure 3).Compounds 1, 2, and 4 had similar CD spectra, which showed a positive Cotton effect at 380 nm and a negative Cotton effect at 300 nm.It revealed that the absolute configuration of C-7 was R in 1 and 2 [24,25].The absolute configuration of the C-13 stereocenter in 1 and 2 was determined by semi-synthesis.Isoamylamine was employed to provide 1 and the deacetylate analogue 11 from the known sclerotiorin (5).Compound 11 was further methylated with CH3I to give 2. The 1 H NMR spectra of the semisynthetic products 1 and 2 were identical to those of the natural products 1 and 2, respectively.On the other hand, compounds 1 and 2 are most likely derived from the same biogenetic pathway as 3 and 4. It meant that the absolute configuration of C-13 in 1 and 2 was an S-configuration, just the same as the absolute configuration of C-13 in 3 and 4. Thus, the absolute configurations of 1 and 2 were 7R, 13S.The absolute configuration at C-7 of 1 and 2 was elucidated by comparing their CD spectra with that of isochromophilone VI (4) (Figure 3).Compounds 1, 2, and 4 had similar CD spectra, which showed a positive Cotton effect at 380 nm and a negative Cotton effect at 300 nm.It revealed that the absolute configuration of C-7 was R in 1 and 2 [24,25].The absolute configuration of the C-13 stereocenter in 1 and 2 was determined by semi-synthesis.Isoamylamine was employed to provide 1 and the deacetylate analogue 11 from the known sclerotiorin (5).Compound 11 was further methylated with CH3I to give 2. The 1 H NMR spectra of the semisynthetic products 1 and 2 were identical to those of the natural products 1 and 2, respectively.On the other hand, compounds 1 and 2 are most likely derived from the same biogenetic pathway as 3 and 4. It meant that the absolute configuration of C-13 in 1 and 2 was an S-configuration, just the same as the absolute configuration of C-13 in 3 and 4. Thus, the absolute configurations of 1 and 2 were 7R, 13S.2) of H-9/H-10/H-11/H-12/H-13, and the HMBC correlations (Figure 2) from H-12 to C-14, and from 14-OCH 3 to C-14 defined the side chain.The HMBC correlation from H-9 to C-2 confirmed that the chain was located at C-2.The NMR data of 7 were similar to that of penithochromone F (9) [23], except for the disappearance of a CH 2 unit in the side chain in 7. Hence, the structure of 7 was assigned as shown in Figure 1.Penithochromone Y (8) was isolated as a light-yellow oil.Its molecular formula was determined as C 17 H 20 O 6 on the basis of HRESIMS analysis.Its NMR data resembled those of 7 (Table 2).The only distinction was the absence of 14-OCH 3 in 8.It was confirmed by the HMBC correlation from H-12 (δ H 1.53) to C-14 (δ C 174.7) (Figure 2).The structure of 8 is shown in Figure 1.
Semi-synthesis plays a pivotal role in providing enough material for further biological studies, determination of the absolute configurations, as well as investigation of the structure-activity relationship.During the study, the structure-activity relationship of these azaphilone derivatives was investigated.The semisynthetic transformation of 5 into 3 was achieved by one step using NH 3 •H 2 O [26], deacelysclerotioramine (10) was also obtained as a byproduct.N-methylsclerotiorinamine (12) was semi-synthesized from 3 by methylating with CH 3 I [27].
The antibacterial activities of the natural products 1-9 and the semi-synthetic analogs 10-12 against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus megaterium, and Shigella dysentery were evaluated.As shown in Table S1, 3 showed antibacterial activities against S. aureus, B. subtilis, B. megaterium, and S. dysentery with MIC values of 12.5, 3.125, 3.125, and 6.25 µg/mL, respectively, while 5 was inactive to these five strains except for B. subtilis (MIC value 100 µg/mL).In light of the structures and antibacterial activity results, we could see that when the O-atom at the 2-position was replaced by a N-atom, just like compounds 5 and 3, the bacterial activities would increase.It suggested that a N-atom at the 2-position in 3 was essential for its antibacterial activity.In addition, a comparison of the activities of 3 with 1, 4 and 12 revealed that an alkyl group substitution of 2-NH might lose or decrease their antibacterial activities.It should be mentioned that all the tested compounds showed no inhibitory effect on E. coli.
The antifungal activities of all compounds except 9 against the five plant pathogenic fungi, Alternaria citri, A. oleracea, Pestalotiopsis theae, Cochliobolus miyabeanus, and Exserohilum turcicum, were tested.As shown in Table S2, 3 was found to exhibit significant antifungal activity against these fungi with MIC values ranging from 3.125 to 25 µg/mL surpassing the efficacy of the positive control carbendazim.Notably, 3 showed the most potent activity against P. theae, C. miyabeanus, and E. turcicum.Compound 5 exhibited a potent effect on C. miyabeanus and E. turcicum with MIC values of 6.25 and 12.5 µg/mL but showed inhibitory to A. citri, A. oleracea, and P. theae with MICs ranging from 50 to 100 µg/mL.These results indicated that a N-atom at the 2-position in 3 played a positive role in their antifungal activity.On the other hand, 3 displayed better activity than 1, 4 and 12, suggesting that the presence of 2-NH might increase its antifungal activity.
In the antimicrobial screening, sclerotioramine (3) exhibited significant antifungal efficacy, which is better than carbendazim.Compound 3 is a N-containing azaphilone, which was mainly obtained from Penicillium sp. and Chaetomium sp.[13].It has demonstrated diverse biological activities, including anti-inflammatory [24], cytotoxic [28], and antibacterial activities [29].However, few studies on the antifungal activity of 3 against plant pathogens have been reported.The present results contribute valuable insights into the potential applications of compound 3 as an effective antifungal agent.

Fungal Material
The fungus Penicillium sp.GDGJ-N37 was a Sophora tonkinensis-associated fungus obtained from Baise, Guangxi Province, China in 2017.The genomic DNA extraction was carried out using the Fungal DNA kits (E.Z.N.A., Omega, Norcross, GA, USA) in accordance with the manufacturer's guidelines.The internal transcribed spacer (ITS1-5.8S-ITS2)regions of the fungi were amplified utilizing the polymerase chain reaction (PCR) with universal ITS primers, ITS1F (5 ′ -CTTGGTCATTTAGAGGAAGTAA-3 ′ ) and ITS4 (5 ′ -TCCTCCGCTTATTGATATGC-3 ′ ) [30].The PCR involved an initial denaturation at 94 • C for 5 min, followed by 30 cycles of 94 • C denaturation for 40 s, 52 • C annealing for 40 s, and a 72 • C extension for 1 min, concluding with a final extension at 72 • C for 10 min.Subsequently, the amplified products underwent sequencing (Invitrogen, Shanghai, China), and a BLASTN search was employed to identify sequences with the closest match in the GenBank using the Basic Local Alignment Search Tool (NCBI).The sequence of its rDNA ITS region had been submitted to GenBank (the GenBank accession number OP622861).The strain was preserved at the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University.

Fermentation, Extraction and Isolation
The fungal strain was cultivated on rice solid medium in 270 Erlenmeyer flasks at room temperature for 30 days, each containing 80 g of rice and 100 mL of water.The fermented material was extracted with EtOAc (3 × 10 L) to afford the crude extract (90.0 g).

Antimicrobial Assay
Antimicrobial evaluation against bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus megaterium, and Shigella dysentery was carried out by the serial-dilution method following reports found in the literature [18,31,32].Anti-phytopathogenic activities against Alternaria citri, A. oleracea, Pestalotiopsis theae, Cochliobolus miyabeanus, and Exserohilum turcicum were assessed using a modified version of the two-fold serial dilutions method as the literature described [33,34].The test compounds were dissolved in DMSO to prepare a stock solution.Ciprofloxacin and carbendazim were used as the positive controls with respect to bacteria and plant pathogenic fungi.

Conclusions
In summary, we described a chemical investigation of the fungus Penicillium sp.GDGJ-N37.Two new nitrogenated azaphilones, N-isoamylsclerotiorinamine (1) and 7methoxyl-N-isoamylsclerotiorinamine (2), together with four known azaphilones (3)(4)(5)(6), and two new chromone derivatives, penithochromones X and Y (7 and 8), were obtained from the fermentation culture of the fungus.Remarkably, compound 3 exhibited significant anti-plant pathogenic fungi activities.The present research not only expands the structural diversity of azaphilones, but also provides inspiration for the discovery of antifungal leading compounds.

1 a 2 b δ C δ H (J in Hz) δ C δ H (J in Hz)
The molecular formula was determined as C 25 H 34 ClNO 3 with 9 degrees of unsaturation by the HRESIMS at m/z 432.2322 [M + H] + (calcd.for C 25 H 35 ClNO 3 + , 432.2305).An isotope peak at m/z 434.2298 (calcd.for C 25 H 35 37 ClNO 3 + , 434.2271) indicated the presence of a chlorine atom in 2. The 1 H NMR and 13 C NMR data of 2 (Table a 1 H NMR measured at 400 MHz;13C NMR measured at 100 MHz.b 1 H NMR measured at 600 MHz;13C NMR measured at 150 MHz.7-Methoxyl-N-isoamylsclerotiorinamine (2) was isolated as a red powder.