Phenazine Derivatives with Anti-Inflammatory Activity from the Deep-Sea Sediment-Derived Yeast-Like Fungus Cystobasidium laryngis IV17-028

Three new phenazine derivatives (1–3), along with known compounds (4–7) of saphenic acid derivatives, were isolated from a deep-sea sediment-derived yeast-like fungus Cystobasidium larynigs collected from the Indian Ocean. The structures of the new compounds (1–3) were determined by analysis of spectroscopic data, semi-synthesis and comparison of optical rotation values. All the isolated compounds (1–7), except for 2, showed nitric oxide (NO) production inhibitory effect against lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells without cytotoxicity at concentrations up to 30 μg/mL. This is the first report on the yeast-like fungus Cystobasidium laryngis producing phenazines and anti-inflammatory activity of 1–7 including saphenic acid (4).


Introduction
Natural products and their derivatives have been recognized as an attractive source of drug discovery [1]. In particular, microorganisms from the marine environment are a rich source of structurally unique bioactive metabolites and have produced a number of drug candidates [2]. Among them, marine microbes from the deep-sea are a relatively untapped reservoir of metabolites with structural and biological diversity waiting to be discovered because of lack of technology and the difficulty of collecting samples [3].
In our continuing search for bioactive substances from deep-sea microorganisms, we encountered a rare marine-derived yeast-like fungus Cystobasidium laryngis isolated from a deep-sea sediment sample collected from the Indian Ocean Ridge in 2017. This yeast-like fungus was designated as IV17-028 and cultivated in a large scale. Phenazine compounds (1)(2)(3)(4)(5)(6)(7) including saphenic acid (4) were isolated from the extract of the culture broth by chromatographic methods and recrystallization. Phenazines are a large group of redox-active secondary metabolites produced by various bacteria (Streptomyces and Pseudomonas) and some archea [4]. Phenazines share a diabenzo annulated pyrazine core structure and have a broad spectrum of biological activities, such as anti-microbial, anti-cancer, cancer chemopreventive, anti-malarial and anti-parasitic [5]. Among them, saphenic acid containing phenazine core is a common pharmacophore for antibiotics and antitumor agents including saphenamycin and phenazostatins which were isolated from Streptomyces sp. [6].
It was supposed that this strain produces phenazine derivatives through the same biosynthesis pathway [6]. So, we assumed that the stereocenter of C-1' has the same configuration with (R)-saphenic ). Thus, the stereochemistry of C-1' was determined to be R-configuration. Peculiarly, 1 was easily changed under the light. However, it could be changelessly stored in the dark and fridge for several weeks. It was supposed that this strain produces phenazine derivatives through the same biosynthesis pathway [6]. So, we assumed that the stereocenter of C-1' has the same configuration with (R)-saphenic acid (4). To determine the stereochemistry of C-1' in 1, 1 was semi-synthesized with 4 and 2-aminobenzoic acid ( Figure 3). Semi-synthesized 1 showed identical 1 H NMR and MS spectra with natural 1 (Figures S26-S28). In addition, natural and semi-synthesized 1 had the same negative optical rotation values (R-saphenic acid:  Compound 2 was purified as a yellowish needle. The molecular formula of 2 was deduced to be C15H13N3O2 with 11 degrees of unsaturation from the [M + H] + peak at m/z 268.1088 (calcd for 268.1086) in the HRESIMS. Based on the HRMS data ( Figure S8), 2 had one more nitrogen and one less oxygen than saphenic acid (4). Chemical shifts and splitting patterns of 2 (Figures S9 and S10) were closely similar to those of 4. The main difference was the appearance of two exchangeable proton signals at δH 10.69 and δH 6.29 for amide protons in 2 instead of the exchangeable proton signal at δH 15.44 of a carboxyl group in 1. The amide protons showed correlations each other in the COSY spectrum ( Figure S12). Therefore, the planer structure of 2 was elucidated as a new derivative of saphenic acid and named as saphenic amide. 2 also had a negative optical rotation value ([α] 25 D −13, c 0.1, CHCl3), which was in agreement with that of 4 ([α] 25 D −13 (c 0.1, CHCl3), suggesting that the stereochemistry of C-1' was R-form.
Compound 3 was obtained as a yellowish amorphous solid. Based on the HRESIMS spectrum ( Figure S20), the molecular formula was established as C16H16N2O2 with 10 degrees of unsaturation It was supposed that this strain produces phenazine derivatives through the same biosynthesis pathway [6]. So, we assumed that the stereocenter of C-1' has the same configuration with (R)-saphenic acid (4). To determine the stereochemistry of C-1' in 1, 1 was semi-synthesized with 4 and 2-aminobenzoic acid ( Figure 3). Semi-synthesized 1 showed identical 1 H NMR and MS spectra with natural 1 (Figures S26-S28). In addition, natural and semi-synthesized 1 had the same negative optical rotation values (R-saphenic acid:  Compound 2 was purified as a yellowish needle. The molecular formula of 2 was deduced to be C15H13N3O2 with 11 degrees of unsaturation from the [M + H] + peak at m/z 268.1088 (calcd for 268.1086) in the HRESIMS. Based on the HRMS data ( Figure S8), 2 had one more nitrogen and one less oxygen than saphenic acid (4). Chemical shifts and splitting patterns of 2 (Figures S9 and S10) were closely similar to those of 4. The main difference was the appearance of two exchangeable proton signals at δH 10.69 and δH 6.29 for amide protons in 2 instead of the exchangeable proton signal at δH 15.44 of a carboxyl group in 1. The amide protons showed correlations each other in the COSY spectrum ( Figure S12). Therefore, the planer structure of 2 was elucidated as a new derivative of saphenic acid and named as saphenic amide. 2 also had a negative optical rotation value ([α] 25 D −13, c 0.1, CHCl3), which was in agreement with that of 4 ([α] 25 D −13 (c 0.1, CHCl3), suggesting that the stereochemistry of C-1' was R-form.
Compound 3 was obtained as a yellowish amorphous solid. Based on the HRESIMS spectrum ( Figure S20), the molecular formula was established as C16H16N2O2 with 10 degrees of unsaturation from the [M + H] + peak at m/z 269.1290 (calcd for 269.1290). The 1 H NMR ( Figure S15) and HSQC ( Figure S17) spectra of 3 showed three aromatic proton signals at δH 8.19 (1H, d, J = 8.2 Hz), 7.83 (1H, t, J = 6.9 Hz) and 7.78 (1H, d, J = 6.9 Hz), an oxygenated methine proton signal at δH 5.71 (1H, q, J = 6.5 Hz) and a methyl signal at δH 1.81 (1H, d, J = 6.5 Hz). 13  Compound 2 was purified as a yellowish needle. The molecular formula of 2 was deduced to be C 15 H 13 N 3 O 2 with 11 degrees of unsaturation from the [M + H] + peak at m/z 268.1088 (calcd. for 268.1086) in the HRESIMS. Based on the HRMS data ( Figure S8), 2 had one more nitrogen and one less oxygen than saphenic acid (4). Chemical shifts and splitting patterns of 2 (Figures S9 and S10) were closely similar to those of 4. The main difference was the appearance of two exchangeable proton signals at δ H 10.69 and δ H 6.29 for amide protons in 2 instead of the exchangeable proton signal at δ H 15.44 of a carboxyl group in 1. The amide protons showed correlations each other in the COSY spectrum ( Figure S12). Therefore, the planer structure of 2 was elucidated as a new derivative of saphenic acid and named as saphenic amide. 2 also had a negative optical rotation value [α] 25 D −13, (c 0.1, CHCl 3 ), which was in agreement with that of 4 [α] 25 D −13 (c 0.1, CHCl 3 ), suggesting that the stereochemistry of C-1' was R-form.
Compound 3 was obtained as a yellowish amorphous solid. Based on the HRESIMS spectrum ( Figure S20), the molecular formula was established as C 16 H 16 N 2 O 2 with 10 degrees of unsaturation from the [M + H] + peak at m/z 269.1290 (calcd. for 269.1290). The 1 H NMR ( Figure S15) and HSQC ( Figure S17) spectra of 3 showed three aromatic proton signals at δ H 8.19 (1H, d, J = 8.2 Hz), 7.83 (1H, t, J = 6.9 Hz) and 7.78 (1H, d, J = 6.9 Hz), an oxygenated methine proton signal at δ H 5.71 (1H, q, J = 6.5 Hz) and a methyl signal at δ H 1.81 (1H, d, J = 6.5 Hz). 13 C NMR and HSQC spectra displayed three quaternary carbons (δ C 142.8, 141.4 and 141.0), three aromatic sp 2 carbons (δ C 131.4, 128.6 and 127.7), an oxygenated methine carbon (δ C 68.9) and a methyl carbon (δ C 23.8). The pattern of 1 H NMR spectrum and UV maximum at 255 and 366 nm revealed the presence of a typical phenazine moiety. The main difference between 3 and the other isolated compounds was the disappearance of a carboxyl group in 3. 1 H and 13 C NMR data of 3 showed only a half set of signals and 3 had no optical activity, indicating that 3 consisted of two equivalent molecular portions and had a symmetric structure of 1-(2,3-diaminophenyl)ethanol. By considering NMR chemical shifts and biosynthetic pathway of 3 and 4, the stereochemistry of C-1' in 3 was determined to be the same as that in 4. Thus, the structure of 3 was determined to be a new saphenic acid derivative and named as saphenol.
The anti-inflammatory activity of 1-7 was assessed by NO assay, and their cytotoxicity on RAW 264.7 cells was evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The biological activities of the previously reported compounds 4-7 have never been reported [9]. Even though saphenic acid is a common pharmacophore for antibiotics and antitumor reagents, its biological activity has never been reported [11]. 1-7, except for 2, exhibited inhibitory effect against LPS-induced NO production in RAW 264.7 cells (Figure 4). Moreover, these compounds did not affect the viability of RAW 264.7 cells at concentrations up to 30 µg/mL (Figure 4). Among the isolated compounds, 6 inhibited NO production with the highest EC 50 value of 19.6 µM. Saphenic acid (4) and their derivatives (6 and 7) showed a similar inhibition tendency (Table 3). Methylated (6) and oxidized (7) saphenic acids showed no significant difference in the activity. However, these compounds were about twice stronger than saphenic acid substituted with 2-amino benzoic acid (1,EC 50 = 46.8 µM). Furthermore, when there is no functional group at C-6 of phenazine (5, EC 50 = 76.1 µM), the activity was lowest. We could not carry out further biological activity tests due to insufficient amount. However, our results suggest that NO inhibitory activity is influenced by the carboxylic acid at C-1 as well as the presence of simple functional group at C-6 of phenazine ring.
an oxygenated methine carbon (δC 68.9) and a methyl carbon (δC 23.8). The pattern of 1 H NMR spectrum and UV maximum at 255 and 366 nm revealed the presence of a typical phenazine moiety. The main difference between 3 and the other isolated compounds was the disappearance of a carboxyl group in 3. 1 H and 13 C NMR data of 3 showed only a half set of signals and 3 had no optical activity, indicating that 3 consisted of two equivalent molecular portions and had a symmetric structure of 1-(2,3-diaminophenyl)ethanol. By considering NMR chemical shifts and biosynthetic pathway of 3 and 4, the stereochemistry of C-1' in 3 was determined to be the same as that in 4. Thus, the structure of 3 was determined to be a new saphenic acid derivative and named as saphenol.
1-7, except for 2, exhibited inhibitory effect against LPS-induced NO production in RAW 264.7 cells (Figure 4). Moreover, these compounds did not affect the viability of RAW 264.7 cells at concentrations up to 30 μg/mL (Figure 4). Among the isolated compounds, 6 inhibited NO production with the highest EC50 value of 19.6 μM. Saphenic acid (4) and their derivatives (6 and 7) showed a similar inhibition tendency (Table 3). Methylated (6) and oxidized (7) saphenic acids showed no significant difference in the activity. However, these compounds were about twice stronger than saphenic acid substituted with 2-amino benzoic acid (1, EC50 = 46.8 μM). Furthermore, when there is no functional group at C-6 of phenazine (5, EC50 = 76.1 μM), the activity was lowest. We could not carry out further biological activity tests due to insufficient amount. However, our results suggest that NO inhibitory activity is influenced by the carboxylic acid at C-1 as well as the presence of simple functional group at C-6 of phenazine ring.

Microbial Material
The strain IV17-028 was isolated from a deep-sea sediment sample collected using a multi-corer (MC) mounted on the R/V ISABU from the Indian Ocean (date: 9 August 2017, latitude: 008 • 07.5121 S, longtitude: 068 • 06.6033 E, depth: 4317 m). A total of 1 g of the heated sediment sample in the dry oven at 60 • C for 30 min, was spread onto the surface of Bennett (BN)'s agar plates (1% glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, 3.2% artificial sea salt and 1.8% agar, pH 7.02 before sterilization). The plates were incubated for 5 days at room temperature and then 20 days at 4 • C fridge. The resulting colony of dark green color was transferred and maintained on the BN agar plate. The colors of the colonies formed were beige to green on the BN agar medium. The strain was identified as Cystobasidium laryngis on the basis of 26S rRNA sequence analysis. The sequence of IV17-028 was submitted to GenBank under accession number MK131277.

Fermentation and Isolation of Metabolites
The seed and production cultures were carried out in the BN broth medium (1% glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, 3.2% artificial sea salt, pH 7.02 before sterilization). The seed culture was performed in a 2 L baffled Erlenmeyer flask containing 600 mL of autoclaved medium at 28 • C for 5 days on a rotary shaker at 140 rpm. The seed culture broth was inoculated into a 100 L fermenter containing 60 L of the broth medium under the aseptic condition. The fermenter was operated at 28 • C, 50 rpm and airflow rate of 20 liter per minute (LPM) in the dark for 10 days. After cultivation, the broth was extracted with ethyl acetate (EtOAc, 60 L) twice. The EtOAc extract was evaporated to obtain crude extract (46.5 g). A portion of crude extract (12.8 g) was partitioned with 85% methanol (MeOH) in H 2 O and hexane (Hex). 85% MeOH layer (1.72 g)

Semi-Synthesis of 1
To a solution of 2-aminobenzoic acid (50.0 mg, 0.36 mM) in toluene (1.2 mL) was added thionyl chloride solution (1.8 mL, 1.8 mM) at room temperature and the mixture was refluxed for 3 h. After completion of the reaction was confirmed by TLC, the solvent was removed under vacuum to obtain the crude acid chloride as yellow oil, which was used for further reaction without purification [12].

NO 2
− accumulation was used as an indicator of NO production as described previously [13].
RAW 264.7 cells were plated at 5 × 10 5 cells/mL, pre-treated with various concentrations (0.3, 1, 3, 10 or 30 µg/mL) of 1-7 and stimulated with LPS (200 ng/mL) for 24 h. The supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2% phosphoric acid) and incubated at room temperature for 10 min. NaNO 2 was used to generate a standard curve, and the concentration of nitrite in the medium was determined by measuring optical density at 540 nm [13]. Cell viability was determined by XTT assay as described previously [14]. And it was confirmed that the concentration of compounds used in this study has no significant effect on cell viability.

Conclusions
Three new compounds (1)(2)(3) and four known compounds (4-7) of phenazine class were isolated from a rare marine yeast-like fungus Cystobasidium laryngis. The structures of the new compounds were elucidated by detailed spectroscopic data analysis. Moreover, the absolute stereochemistry of 1-4 and 6 was determined by comparison of optical rotation values with literature. All the isolated compounds 1-7, except for 2, exhibited moderate NO production inhibition activity without cytotoxicity. This is the first report on the isolation of the new and known phenazine derivatives from the yeast-like fungus Cystobasidium laryngis and their anti-inflammatory activity.  Figure S4: HSQC spectrum of compound 1 in CD 3 OD, Figure S5: COSY spectrum of compound 1 in CD 3 OD, Figure S6: HMBC spectrum of compound 1 in CD 3 OD, Figure S7: ROESY spectrum of compound 1 in CD 3 OD, Figure S8: HRESI-MS spectrum of compound 1, Figure S9: 1 H NMR spectrum of compound 2 (600 MHz, CDCl 3 ), Figure S10: 13 C NMR spectrum of compound 2 (150 MHz, CDCl 3 ), Figure S11: HSQC spectrum of compound 2 in CDCl 3 , Figure S12: COSY spectrum of compound 2 in CDCl 3 , Figure S13: HMBC spectrum of compound 2 in CDCl 3 , Figure S14: HRESI-MS spectrum of compound 2, Figure S15: 1 H NMR spectrum of compound 3 (600 MHz, CDCl 3 ), Figure S16: 13 C NMR spectrum of compound 3 (150 MHz, CDCl 3 ), Figure S17: HSQC spectrum of compound 3 in CDCl 3 , Figure S18: COSY spectrum of compound 3 in CDCl 3 , Figure S19: HMBC spectrum of compound 3 in CDCl 3 , Figure S20: HRESI-MS spectrum of compound 3, Figure S21