Nitrogen-Containing Secondary Metabolites from a Deep-Sea Fungus Aspergillus unguis and Their Anti-Inflammatory Activity

Aspergillus is well-known as the second-largest contributor of fungal natural products. Based on NMR guided isolation, three nitrogen-containing secondary metabolites, including two new compounds, variotin B (1) and coniosulfide E (2), together with a known compound, unguisin A (3), were isolated from the ethyl acetate (EtOAc) extract of the deep-sea fungus Aspergillus unguis IV17-109. The planar structures of 1 and 2 were elucidated by an extensive analysis of their spectroscopic data (HRESIMS, 1D and 2D NMR). The absolute configuration of 2 was determined by comparison of its optical rotation value with those of the synthesized analogs. Compound 2 is a rare, naturally occurring substance with an unusual cysteinol moiety. Furthermore, 1 showed moderate anti-inflammatory activity with an IC50 value of 20.0 µM. These results revealed that Aspergillus unguis could produce structurally diverse nitrogenous secondary metabolites, which can be used for further studies to find anti-inflammatory leads.


Introduction
Deep-sea hydrothermal vents are recognized as one of the most extreme and dynamic habitats on our planet [1]. These hotspot ecosystems are characterized by high temperature, high pressure, low oxygen supply, and the absence of sun light [2]. In addition, hydrothermal vent flows bring fluids with high concentrations of reduced sulfur-containing compounds and heavy metals [2]. Given this fact, microorganisms living in this specific environment are considered as a new frontier for discovery of natural products with unique structures and tremendous pharmacological activities [3].
Aspergillus is renowned as a prolific source of numerous fungal peptides, including lipo-, depsi-, linear-, and cyclic-peptides, which are structurally unique and demonstrated various bioactivities, such as anti-microbial, anti-fungal, anti-inflammatory, and cytotoxic activities [4,5]. Among the peptides derived from Aspergillus spp., unguisins are a unique cyclic heptapetide class commonly produced by Aspergillus unguis, and until now unguisins A-G have been reported [6,7].
Inflammation is a protective response of our body to a wide range of stimuli. This process plays a central role or is an important symptom in the pathogenesis of various chronic diseases for instance Alzheimer's disease, asthma, diabetes, and rheumatoid arthritis [8]. The inflammatory process is characterized by over secretion of nitric oxide (NO) and inflammatory cytokines such as interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6). Therefore, reducing the production of inflammatory mediators is a key indicator for the treatment of various diseases. As part of our study on marine-derived microorganisms isolated near hydrothermal vents, we have reported some anti-inflammatory phenazine alkaloids from a yeast-like fungus Cystobasidium laryngis, and nidulin-related polyketides from A. unguis IV17-109, which showed anti-microbial and cytotoxic activities [9,10]. Based on NMR guided isolation, we found that the 1 H NMR spectra of non-polar fractions from A. unguis IV17-109 showed some minor interesting peaks in the olefinic region, which do not belong to unguisin peptides or nidulin-related polyketides. Further careful purification of these fractions led to the identification of two new compounds, variotin B (1) and coniosulfide E (2) (Figure 1). Anti-inflammatory activity of 1 and 2 was preliminarily evaluated and the result revealed that 1 has moderate activity. Here, we report the details of the isolation, structure identification, and anti-inflammatory nature of these compounds.
Mar. Drugs 2022, 20, x 2 of 8 toid arthritis [8]. The inflammatory process is characterized by over secretion of nitric oxide (NO) and inflammatory cytokines such as interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6). Therefore, reducing the production of inflammatory mediators is a key indicator for the treatment of various diseases. As part of our study on marine-derived microorganisms isolated near hydrothermal vents, we have reported some anti-inflammatory phenazine alkaloids from a yeast-like fungus Cystobasidium laryngis, and nidulin-related polyketides from A. unguis IV17-109, which showed anti-microbial and cytotoxic activities [9,10]. Based on NMR guided isolation, we found that the 1 H NMR spectra of non-polar fractions from A. unguis IV17-109 showed some minor interesting peaks in the olefinic region, which do not belong to unguisin peptides or nidulin-related polyketides. Further careful purification of these fractions led to the identification of two new compounds, variotin B (1) and coniosulfide E (2) (Figure 1). Anti-inflammatory activity of 1 and 2 was preliminarily evaluated and the result revealed that 1 has moderate activity. Here, we report the details of the isolation, structure identification, and anti-inflammatory nature of these compounds.

Results and Discussion
Compound 1 was isolated as pale-yellow needles with the molecular formula of C20H27NO2 based on its HRESIMS peak at m/z 336.1938, ([M+Na] + , calculated for C20H27NO2Na, 336.1939), requiring 8 indices of hydrogen deficiency. The 1 H NMR spectrum of 1 showed signals attributed to a methyl group at δH 1.62 (d, J = 4.5, H3-21), seven methylene groups at δH 2.04-3.82, and ten olefinic protons at δH 5.42-7.37. The 13 C NMR spectrum in combination with HSQC data revealed signals of 20 resonances belonging to a methyl at δC 18.1, seven methylene carbons at δC 18.1-47.0, ten olefinic carbons at δC 121.7-146.9, and two carbonyl carbons at δC 168.2 and 177.8. Two carbonyl and ten sp 2 carbons, accounting for 7 out of 8 degrees of unsaturation, indicated 1 is a monocyclic compound. The structure of a five-membered lactam ring was determined by continuous 1 H-1 H COSY correlations from H2-2 to H2-4, and the HMBC correlation from H2-4 to C-1. A substructure was identified as a C-16 polyunsaturated fatty acid by continuous 1 H-1 H COSY correlations from H-7 to H3-21, and the HMBC correlations from H-7 and H-8 to C-6 ( Figure 2). The connection of the fatty acid and the lactam ring was corroborated by the HMBC correlation from H2-4 to C-6.

Results and Discussion
Compound 1 was isolated as pale-yellow needles with the molecular formula of C 20 H 27 NO 2 based on its HRESIMS peak at m/z 336.1938, ([M+Na] + , calculated for C 20 H 27 NO 2 Na, 336.1939), requiring 8 indices of hydrogen deficiency. The 1 H NMR spectrum of 1 showed signals attributed to a methyl group at δ H 1.62 (d, J = 4.5, H 3 -21), seven methylene groups at δ H 2.04-3.82, and ten olefinic protons at δ H 5.42-7.37. The 13 C NMR spectrum in combination with HSQC data revealed signals of 20 resonances belonging to a methyl at δ C 18.1, seven methylene carbons at δ C 18.1-47.0, ten olefinic carbons at δ C 121.7-146.9, and two carbonyl carbons at δ C 168.2 and 177.8. Two carbonyl and ten sp 2 carbons, accounting for 7 out of 8 degrees of unsaturation, indicated 1 is a monocyclic compound. The structure of a five-membered lactam ring was determined by continuous 1 H-1 H COSY correlations from H 2 -2 to H 2 -4, and the HMBC correlation from H 2 -4 to C-1. A substructure was identified as a C-16 polyunsaturated fatty acid by continuous 1 H-1 H COSY correlations from H-7 to H 3 -21, and the HMBC correlations from H-7 and H-8 to C-6 ( Figure 2). The connection of the fatty acid and the lactam ring was corroborated by the HMBC correlation from H 2 -4 to C-6.
The geometries of ∆ 7,9,13,15 were deduced as E-form by their large coupling constants (Table 1) and the chemical shift of terminal methyl (C-21) was δ C 18.1, revealing the geometry of ∆ 19 was E-form [11,12]. Therefore, 1 was determined as a new variotin derivative with a non-branched side chain and named variotin B [13]. Mar. Drugs 2022, 20, x 3 of 8 The geometries of ∆ 7,9,13,15 were deduced as E-form by their large coupling constants (Table 1) and the chemical shift of terminal methyl (C-21) was δC 18.1, revealing the geometry of ∆ 19 was E-form [11,12]. Therefore, 1 was determined as a new variotin derivative with a non-branched side chain and named variotin B [13].
Compound 2 was isolated as a colorless solid and its molecular formula was determined as C22H40N2O4S, with four indices of hydrogen deficiency based on its HRESIMS peak at m/z 451. The structure of a cysteinol unit was determined by sequential 1 H-1 H COSY correlations of H-8a,b/H-7/H-25a,b. A partial structure of N-acetylglycine, which was connected to the cysteinol moiety via a peptide bond, was determined by the HMBC correlations of H-7/C-5, H2-4/C-2, and H3-1/C-2. The remaining 15 carbons were assigned as a 10-hydro-11hydroxyfarnesyl moiety based on a detailed analysis of 1 H-1 H COSY and HMBC data (Figure 2), and the connection of this moiety with the cysteinol residue via a thioether bond was determined by the HMBC correlations of H-8 a,b/C-10 and H-10a,b/C-8. The geometry of ∆ 11 was deduced as E-form by the strong NOESY correlations from H3-24 to H-10a,b and H-13a,b; and no-observed NOESY correlation from H3-24 to H-11. Similarly, ∆ 15 was also determined as E-form ( Figure 2). Consequently, the gross structure of 2 was determined as shown in Figure 1.
To determine the absolute configuration of 2, we synthesized its analogs (4 and 5, a pair of enantiomers synthesized from L-and D-cysteine and farnesyl chloride, Scheme 1) from commercially available substances. By comparing the optical rotation sign of 2 [] ̶ 100 (c 0.3, MeOH) with that of 4 [] ̶ 110 (c 0.3, MeOH) and 5 [] +120 (c 0.3, MeOH), the absolute configuration of 2 was determined to be the same as that of 4 (7R). Thus, 2 was determined as a new derivative of sulfur-containing natural products, coniosulfides A-D [14], and named coniosulfide E.
A co-isolated known compound was identified as unguisin A (3) by comparing its spectroscopic data with the corresponding literature values [6].   The structure of a cysteinol unit was determined by sequential 1 H-1 H COSY correlations of H-8 a,b /H-7/H-25 a,b . A partial structure of N-acetylglycine, which was connected to the cysteinol moiety via a peptide bond, was determined by the HMBC correlations of H-7/C-5, H 2 -4/C-2, and H 3 -1/C-2. The remaining 15 carbons were assigned as a 10-hydro-11-hydroxyfarnesyl moiety based on a detailed analysis of 1 H-1 H COSY and HMBC data (Figure 2), and the connection of this moiety with the cysteinol residue via a thioether bond was determined by the HMBC correlations of H-8 a,b /C-10 and H-10 a,b /C-8. The geometry of ∆ 11 was deduced as E-form by the strong NOESY correlations from H 3 -24 to H-10 a,b and H-13 a,b ; and no-observed NOESY correlation from H 3 -24 to H-11. Similarly, ∆ 15 was also determined as E-form ( Figure 2). Consequently, the gross structure of 2 was determined as shown in Figure 1.
To determine the absolute configuration of 2, we synthesized its analogs ( , the absolute configuration of 2 was determined to be the same as that of 4 (7R). Thus, 2 was determined as a new derivative of sulfur-containing natural products, coniosulfides A-D [14], and named coniosulfide E. Since some fungal peptides were reported to show anti-inflammatory activity [4], 1 and 2 were evaluated for their anti-inflammatory activity. Subsequently, 1 showed moderate anti-inflammatory activity with an IC50 value of 20.0 µM. Even though a literature A co-isolated known compound was identified as unguisin A (3) by comparing its spectroscopic data with the corresponding literature values [6].
Since some fungal peptides were reported to show anti-inflammatory activity [4], 1 and 2 were evaluated for their anti-inflammatory activity. Subsequently, 1 showed moderate anti-inflammatory activity with an IC 50 value of 20.0 µM. Even though a literature review revealed many synthetic analogs of 2 demonstrated inhibitory effects on human isoprenylcysteine carboxyl methyltransferase (hIcmt) [15] or the inflammation process [16], unfortunately, 2 showed no anti-inflammatory activity at a concentration of 30.0 µM. Due to the limited amount of 2, we were unable to check its effect on hIcmt. Therefore, further studies are needed to find the bioactivities of 2.
To further investigate the anti-inflammatory activity of 1, we examined the inhibitory effect of 1 on lipopolysaccharide (LPS)-induced production of inflammatory mediators, including NO, IL-6, and iNOS, in RAW 264.7 cells. The treatment of RAW 264.7 cells with LPS led to the accumulation of nitrite and IL-6, and 1 dose-proportionally inhibited LPS-induced production of nitrite and IL-6 in LPS-stimulated RAW 264.7 cells (Figure 3A,B). To further examine whether the effect of 1 were due to its effects on the mRNA expression of cognate genes, we investigated the effect of 1 on the mRNA expression of inducible nitric oxide synthase (iNOS) and IL-6 by quantitative polymerase chain reaction (qPCR). The mRNA levels of iNOS and IL-6 were induced by LPS treatment, and this induction was suppressed by 1 in a concentration-dependent manner ( Figure 3C,D). Considering the above-mentioned data, it is noticeable that 1 showed anti-inflammatory activity by suppressing the production of NO and the expression of iNOS and IL-6 with no cytotoxicity at the treated concentrations. The results revealed that fungal natural products could be an important source of leads for the development of new anti-inflammatory drugs with minimal side effects.
isoprenylcysteine carboxyl methyltransferase (hIcmt) [15] or the inflammation process [16], unfortunately, 2 showed no anti-inflammatory activity at a concentration of 30.0 µM. Due to the limited amount of 2, we were unable to check its effect on hIcmt. Therefore, further studies are needed to find the bioactivities of 2.
To further investigate the anti-inflammatory activity of 1, we examined the inhibitory effect of 1 on lipopolysaccharide (LPS)-induced production of inflammatory mediators, including NO, IL-6, and iNOS, in RAW 264.7 cells. As shown in Figure 3A,B, The treatment of RAW 264.7 cells with LPS led to the accumulation of nitrite and IL-6, and 1 doseproportionally inhibited LPS-induced production of nitrite and IL-6 in LPS-stimulated RAW 264.7 cells (Figure 3A,B). To further examine whether the effect of 1 were due to its effects on the mRNA expression of cognate genes, we investigated the effect of 1 on the mRNA expression of inducible nitric oxide synthase (iNOS) and IL-6 by quantitative polymerase chain reaction (qPCR). The mRNA levels of iNOS and IL-6 were induced by LPS treatment, and this induction was suppressed by 1 in a concentration-dependent manner ( Figure 3C,D). Considering the above-mentioned data, it is noticeable that 1 showed antiinflammatory activity by suppressing the production of NO and the expression of iNOS and IL-6 with no cytotoxicity at the treated concentrations. The results revealed that fungal natural products could be an important source of leads for the development of new anti-inflammatory drugs with minimal side effects.  and IL-6 (B) in culture supernatants were determined by Griess reaction and ELISA, respectively. The mRNA levels of IL-6 (C) and iNOS (D) were examined by qPCR. Data are represented as the mean ± SD of quadruplicate determinations. An asterisk (*) denotes that the response is significantly different from vehicle-treated group as determined by Dunnett's multiple comparison test at p < 0.05. The results shown are representatives of more than two independent experiments (UN: Untreated; VH: Vehicle (0.1% DMSO)).