Phenazine derivatives with anti-inflammatory activity from the deep-sea sediment-derived yeast-like fungus cystobasidium laryngis IV17-028

Article abstract of DOI:10.3390/md17080482

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 compou

Full text of DOI:10.3390/md17080482

marine drugs
Article
Phenazine Derivatives with Anti-Inflammatory
Activity from the Deep-Sea Sediment-Derived
Yeast-Like Fungus Cystobasidium laryngis IV17-028
Hwa-Sun Lee 1, Jong Soon Kang 2, Byeoung-Kyu Choi 1, Hyi-Seung Lee 1, Yeon-Ju Lee 1,
Jihoon Lee 1 and Hee Jae Shin 1,
*
1
Marine Natural Products Chemistry Laboratory, Korea Institute of Ocean Science Technology,
385 Haeyang-ro, Yeongdo-gu, Busan 49111, Korea
Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology,
30 Yeongudangi-ro, Ochang-eup, Cheongwon-gu, Cheongju 28116, Korea
Correspondence: shinhj@kiost.ac.kr; Tel.: +82-51-664-3341
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 25 July 2019; Accepted: 16 August 2019; Published: 19 August 2019
Abstract: 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 ( ) were determined
by analysis of spectroscopic data, semi-synthesis and comparison of optical rotation values. All the
isolated compounds ( ), except for , showed nitric oxide (NO) production inhibitory effect against
lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells without cytotoxicity at
concentrations up to 30 g/mL. This is the first report on the yeast-like fungus Cystobasidium laryngis
1–3
1–
7
2
µ
producing phenazines and anti-inflammatory activity of 1–7 including saphenic acid (4).
Keywords: deep-sea sediment; marine yeast-like fungi; Cystobasidium laryngis; phenazine derivatives;
saphenic acid; anti-inflammatory activity
1. 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
]. Among
structurally unique bioactive metabolites and have produced a number of drug candidates [
2
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 (1–7) 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 [
structure and have a broad spectrum of biological activities, such as anti-microbial, anti-cancer, cancer
chemopreventive, anti-malarial and anti-parasitic [ ]. Among them, saphenic acid containing phenazine
4]. Phenazines share a diabenzo annulated pyrazine core
5
core is a common pharmacophore for antibiotics and antitumor agents including saphenamycin and
phenazostatins which were isolated from Streptomyces sp. [6].
Here we report the isolation of Cystobasidium laryngis IV17-028, fermentation, purification of seven
phenazine derivatives (1–7), structure determination of three new compounds 1–3 and biological
Mar. Drugs 2019, 17, 482; doi:10.3390/md17080482
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Mar. Drugs 2019, 17, 482
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activity of all the isolated compounds
1–
7
(Figure 1). This is the first report on the phenazine derivatives
from the marine yeast-like fungus Cystobasidium laryngis and their anti-inflammatory activity.
1
2
3
4
5
6
7
Figure 1. The structures of compounds 1–7.
2. Results and Discussion
Compound
deduced to be C22H17N3O4 from the [M + H]+ peak at m/z 388.1296 (calcd. for 388.1297) in the
HRESIMS, which required 16 degrees of unsaturation. The 1H and HSQC spectra of
showed ten
1 was isolated as a yellowish amorphous solid. The molecular formula of 1 was
1
aromatic proton signals at δH 8.90 (1H, d, J = 7.4 Hz), 8.70 (1H, d, J = 8.6 Hz), 8.23 (1H, d, J = 7.7 Hz),
8.15 (1H, t, J = 7.4 Hz), 7.99 (1H, overlapped), 7.98 (1H, overlapped), 7.89 (1H, d, J = 7.6 Hz), 7.03 (1H, t,
J = 8.0 Hz), 6.49 (1H, t, J = 7.6 Hz) and 6.42 (1H, d, J = 8.0 Hz), one methine signal at δH 6.06 (1H, q,
J = 6.4 Hz) and one methyl signal at δH 1.81 (3H, d, J = 6.4 Hz) (Figure S1). Moreover, an exchangeable
proton signal of a carboxyl group at δH 15.60 (1H, s) was detected on the 1H NMR spectrum using
CDCl3 (Table 1) (Figure S2). The 13C (Figure S3) and HSQC (Figure S4) spectra displayed ten tertiary
sp2 carbons (δC 172.4, 168.6, 151.5, 145.6, 144.8, 143.9, 143.7, 141.0, 126.6 and 112.2), ten aromatic sp2
carbons (δC 137.8, 136.6, 135.3, 134.7, 133.4, 131.5, 128.7, 128.3, 116.1 and 113.3), an oxygenated methine
carbon (δC 49.5) and a methyl carbon (δC 24.3) (Table 2). The characteristic UV spectrum (
λ
max
256
and 371 nm), distinct deshielded aromatic proton signals at δH 8.90–7.98, an exchangeable proton
signal for a carboxyl group at δH 15.60, a downshifted methine proton signal at δH 6.06 and four
nitrogen-bearing quaternary carbons at δC 144.8, 143.9, 143.7 and 141.0 revealed the presence of a
saphenic acid bearing phenazine moiety [7,8]. Analysis of the COSY spectrum (Figure S5) suggested
four spin systems: From H-2 (δH 8.90) to H-4 (δH 8.70), from H-7 (δH 7.98) to H-9 (δH 8.23), from H-1’
at (δH 6.06) to H-2’ (δH 1.81) and from H-3” (δH 7.89) to H-6” (δH 6.42) (Figure 2). These evidences
revealed that
values of were very similar to
concluded to be a 1,6-disubstituted phenazine derivative (Tables 1 and 2). The molecular formula,
based on the HR-MS result, revealed that has an amino group. The amino group was linked to a
remaining unconnected quaternary carbon C-7” by its chemical shift (δC 151.5). These data clearly
indicated that belongs to the saphenic acid family with 2-aminobenzoic acid moiety. The position of
1
had 1,6- or 1,9-disubstituted phenazine ring. Since 1H and 13C NMR chemical shift
1
4, as well as already reported saphenic acid [9], the structure of 1 was
1
1
the carboxylic acid (1-COOH) was determined by the HMBC correlation between H-2 (δH 8.90) and the
carbonyl carbon (δC 168.6) (Figure S6). The oxygenated methine proton H-1’ (δH 6.06) showed HMBC
cross-peaks with C-5a (δC 143.7), C-6 (δC 145.6) and C-7 (δC 128.7). The HMBC correlation between
the aromatic methine H-3” (δH 7.89) and C-1” (δC 172.4) established the presence of 2-amino benzoic
ester (Figure 2). Though we could not directly ascertain the HMBC correlation between the saphenic
acid moiety and 2-amino benzoic ester, the ROESY correlation between H-1’ (δH 6.06) and H-6” (δH
6.42) established their connection (Figure S7). Furthermore, saphenic acid and 2-amino benzoic ester

Mar. Drugs 2019, 17, 482
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satisfied the unsaturation number and molecular formula. Thereby, the oxygenated methine C-1’ was
found to be connected with C-1” by an ester linkage. Thus, the structure of
6-[1-(2-aminobenzoyloxy)ethyl]-1-phenazinecarboxylic acid.
1
was determined as
Table 1. 1H NMR (600 MHz) data of 1–4.
δ , Mult. (J in Hz)
H
Position
1 a
2 b
3 b
4 b
2
3
4
7
8
8.90
8.15
8.70
7.98
7.99
8.23
6.06
1.81
7.89
6.49
7.03
6.42
d
t
(7.4)
(7.4)
(8.6)
9.05
8.02
8.45
7.87
7.92
8.17
5.78
1.84
d
t
d
d
t
d
q
d
(7.4)
(7.4)
(8.6)
(7.1)
(7.1)
(8.5)
(6.6)
(6.6)
8.94
d
t
d
(7.9)
(7.9)
(9.2)
8.02
8.48
7.94
7.96
8.14
5.82
1.78
d
ovl c
7.78
7.83
8.19
5.71
1.81
d
t
d
q
d
(6.9)
(6.9)
(8.2)
(6.5)
(6.5)
ovl c
ovl c
d
ovl c
9
d
q
d
d
t
(7.7)
(6.4)
(6.4)
(7.6)
(7.6)
(8.0)
(8.0)
(8.4)
(6.5)
(6.5)
1’
2’
3”
4”
5”
6”
q
d
t
d
15.60
1-COOH
1-CONH2
s
15.44
s
b
10.69
6.29
br s
br s
a, b
c
Spectra were recorded in CD3OD and CDCl3, respectively. Signals were overlapped with other signals.
Table 2. 13C NMR (150 MHz) data of 1–4.
δC, Type
Position
1 a
2 b
3 b
4 b
1
2
3
4
4a
5a
6
7
8
9
9a
10a
1’
2’
1”
2”
C
CH
CH
CH
C
C
C
CH
CH
CH
C
C
CH
CH
CH
C
C
C
CH
CH
CH
C
124.9 e
137.6
130.6
135.1
141.8 e
142.4
144.0
127.9
133.4
127.2
140.4
139.9
68.0
C
CH
CH
CH
C
C
C
CH
CH
CH
C
126.6 c,f
137.8 f
131.5
129.2 d
136.3
130.3
134.3
141.8 d
142.5
143.3
127.4
131.9
128.5
143.1
140.8
68.8
136.6 f
143.9 c,f
143.7 f
145.6
141.0
142.8
127.7
131.4
128.6
141.4
C
C
CH
CH
CH
C
128.7
134.7 f
128.3
144.8 f
141.0
C
C
CH
CH3
C
CH
CH3
49.5 g
24.3
CH
CH3
C
68.9
23.8
CH
CH3
23.9
24.1
172.4 f
112.2
C
133.4 f
116.1
3”
4”
5”
6”
7”
CH
CH
CH
CH
C
135.3
113.3
151.5
168.6 f
1-COOH
1-CONH2
C
166.0
C
166.5 f
C
a, b
c–e
f
Spectra were recorded in CD3OD and CDCl3, respectively.
Assignments interchangeable. Chemical shifts
g
were obtained from the HMBC spectrum. Chemical shift was overlapped with solvent signals.
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

Mar. Drugs 2019, 17, 482
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acid (
4
). To determine the stereochemistry of C-1’ in
1
,
1
was semi-synthesized with
4
and
1
2-aminobenzoic acid (Figure 3). Semi-synthesized
1
showed identical H NMR and MS spectra
with natural
optical rotation values (R-saphenic acid: [
semi-synthetic
1
(Figures S26–S28). In addition, natural and semi-synthesized
1
α
had the same negative
25
25
α
]
−
73, (c 0.05, CHCl3); natural
1: [
]
−
80, (c 0.05, CHCl3);
1: [α
]
−
40, (c 0.05, CHCl3)D). Thus, the stereochemistry of C-1’ Dwas determined to be
25
D
R-configuration. Peculiarly, 1 was easily changed under the light. However, it could be changelessly
stored in the dark and fridge for several weeks.
2
3
1
Figure 2. The Key COSY and HMBC correlations of 1–3.
Figure 3. Semi-synthesis of 1.
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),
less oxygen than saphenic acid ( ). Chemical shifts and splitting patterns of
were closely similar to those of . The main difference was the appearance of two exchangeable proton
signals at δH 10.69 and δH 6.29 for amide protons in instead of the exchangeable proton signal at
δH 15.44 of a carboxyl group in . The amide protons showed correlations each other in the COSY
spectrum (Figure S12). Therefore, the planer structure of
2
had one more nitrogen and one
4
2
(Figures S9 and S10)
4
2
1
2
was elucidated as a new derivative of
25
saphenic acid and named as saphenic amide.
(c 0.1, CHCl3), which was in agreement with that of
stereochemistry of C-1’ was R-form.
2
also had a negative optical rotation value [
α
]
−
13,
D
25
D
4
[α
]
−
13 (c 0.1, CHCl3), suggesting that the
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 1H 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). 13C NMR and HSQC spectra displayed
three quaternary carbons (δC 142.8, 141.4 and 141.0), three aromatic sp2 carbons (δC 131.4, 128.6 and
127.7), an oxygenated methine carbon (δC 68.9) and a methyl carbon (δC 23.8). The pattern of 1H NMR
spectrum and UV maximum at 255 and 366 nm revealed the presence of a typical phenazine moiety.

Mar. Drugs 2019, 17, 482
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The main difference between
3
and the other isolated compounds was the disappearance of a carboxyl
1
group in
3
. H and 13C NMR data of
3
showed only a half set of signals and
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 and
, the stereochemistry of C-1’ in was determined to be the same as that in . Thus, the structure of
was determined to be a new saphenic acid derivative and named as saphenol.
The structures of the known compounds were identified as the previously reported (R)-saphenic
acid ( ), phenazine-1-carboxylic acid ( ), 6-(1-hydroxyehtyl)phenazine-1-carboxylic acid ( ) and
6-acetyl-phenazine-1-carboxylic acid ( ) by the comparison of NMR, MS and optical rotation values
with the literature [ 10]. When comparing with saphenic acid ( ), was a lack of 1-hydroxy-ethyl
group, was methylated form of saphenic acid ( ), and was oxidized saphenic acid. As previously
reported [ ], saphenic acid ( ) of yellow needle form turned brown after several hours in day light
as well as fluorescent light. Moreover, was quickly turned from yellow into orange in hydrophilic
solvents like methanol and dimethyl sulfoxide under the light within a few minutes.
The anti-inflammatory activity of 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 have never been
reported [ ]. Even though saphenic acid is a common pharmacophore for antibiotics and antitumor
reagents, its biological activity has never been reported [11].
, except for , 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 g/mL (Figure 4). Among the isolated compounds, inhibited NO production with the highest
EC50 value of 19.6 M. Saphenic acid ( ) and their derivatives ( and ) showed a similar inhibition
tendency (Table 3). Methylated ( ) and oxidized ( ) 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 ( , EC50 = 46.8 M). Furthermore, when there is no functional group at C-6
of phenazine ( , EC50 = 76.1 M), the activity was lowest. We could not carry out further biological
3 had no optical activity,
indicating that
3
3
4
3
4
3
4–7
4
5
6
7
9,
4 5
6
4
7
9
4
4
1–7
4–7
9
1–
7
2
µ
6
µ
4
6
7
6
7
1
µ
5
µ
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.
Figure 4. Effects of
1–7 on production of nitric oxide (NO) in LPS-induced macrophage RAW 264.7
cells (A) and cell viability of RAW 264.7 cells (B).

Mar. Drugs 2019, 17, 482
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Table 3. Effective Concentrations (EC50, µg/mL and µM) of 1–7 against NO assay.
Compounds
4
1
2
3
5
6
7
(
µ
g/mL) 18.10
>30
-
14.67
54.7
6.15
22.9
17.06
76.1
5.53
19.6
5.32
20.0
EC50
(µM)
3. Materials and Methods
3.1. General Experimental Procedures
46.8
Optical rotation was measured on a Rudolph research analytical Autopol III S2 polarimeter
(Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were obtained on a Shimadzu
UV-1650PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR spectra were recorded
on a JASCO FT/IR-4100 spectrophotometer (JASCO Corporation, Tokyo, Japan). NMR spectra were
collected on a Bruker AVANCE III 600 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany)
operating at 600 MHz (1H) and 150 MHz (13C). LRMS data were acquired on an Agilent 6100 single
quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with electron ionization
(EI). HRESIMS was obtained with a Waters SYNPT G2 Q-TOF mass spectrometer (Waters Corporation,
Milford, CT, USA) at Korea Basic Science Institute (KBSI) in Cheongju, Republic of Korea. HPLC was
performed using a PrimeLine binary pump (Analytical Scientific Instruments, Inc., El Sobrante, CA,
USA) with Shodex RI-101 refractive index detector (Shoko Scientific Co. Ltd., Yokohama, Japan) and
S3210 variable UV detector (Schambeck SFD GmbH, Bad Honnef, Germany). Columns used for HPLC
were YMC-Pack Silica (250 mm
YMC-Triart C18 (250 mm 10 mm i.d., 5
230–400 mesh) and RP-C18 silica gel (YMC-Gel ODS-A, 12 nm S-75
×
10 mm i.d., 5
µ
m), YMC ODS-A (250 mm
4.6 mm i.d., 5
m) were used for open column
×
10 mm i.d., 5
µm). Silica gel 60 (Merck,
µm) and
×
µm and 250 mm
×
µ
chromatography. Mass culture was carried out in a 100 L fermenter (Fermentec Corporation, Cheongju,
Republic of Korea). All solvents used were either HPLC grade or distilled prior to use.
3.2. 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◦ 07.51210 S,
longtitude: 068◦ 06.60330 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.
3.3. 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 H2O and hexane (Hex). 85% MeOH layer (1.72 g)

Mar. Drugs 2019, 17, 482
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was subjected to ODS vacuum column chromatography followed by stepwise gradient elution with
MeOH/H2O (1:4, 2:3, 3:2, 4:1 and 5:0, v/v) as eluent. Insoluble part (23.1 mg) containing obtained
from the 60% MeOH fraction was recrystallized from acetone to give a pure (9.9 mg) as yellow
4
4
needles. The 60% MeOH fraction was again applied to an ODS flash column chromatography with a
MeOH/H2O solvent system (4:6, 5:5, 6:4, 7:3 and 8:2, v/v). The subfraction eluted with MeOH/H2O (6:4)
was purified by a reversed-phase HPLC (YMC ODA-S, 250
flow rate: 2.0 mL/min; detector: RI) to yield crude (2.3 mg, tR 50 min). The crude
in MeOH to afford a pure (1.1 mg) as yellow crystals. The 80% MeOH fraction was purified by a
RP-HPLC (YMC ODS-A, 250 10 mm i.d., 5 m; 28% MeCN in H2O containing 0.01% TFA; flow rate:
3.5 mL/min; detector: UV; wavelength: 254 nm) to give (0.7 mg, tR 30 min). The remaining crude
×
10 mm i.d., 5
µ
m; 48% MeOH in H2O;
2
2
was recrystallized
2
×
µ
3
extract (33.7 g) was suspended in H2O and sequentially partitioned with Hex, CHCl3, EtOAc and
BuOH. The CHCl3 soluble fraction (5.94 g) was separated on silica gel flash column chromatography
with MC/MeOH (10:0, 20:1, 10:1 and 5:1, v/v). The MC:MeOH (20:1) fraction was collected into nine
fractions. Subfractions 3–5 (34.1 mg) from the MC:MeOH (20:1) fraction was purified by a normal
phase HPLC (YMC-Pack SIL; 250
×
10 mm i.d., 5
µm; EtOAc:Hex (2:8); flow rate: 2.0 mL/min; detector:
RI) to yield (0.7 mg, tR 35 min) and
5
6
(0.5 mg, tR 25 min). Subfractions 6 and 7 (4.8 g) were again
applied to an ODS-A vacuum column chromatography by stepwise gradient elution with MeOH/H2O
(1:4, 2:3, 3:2, 4:1 and 5:0, v/v). Each fraction was divided into three subfractions. The third 80%MeOH
fraction (468.5 mg) was separated with a normal phase HPLC (YMC-Pack SIL; 250
×
10 mm i.d., 5
µm;
EtOAc:Hex:MeOH (1:1:0.02); flow rate: 2.0 mL/min; detector: RI) to afford
(2.1 mg, tR 14 min) and 7 (1.3 mg, tR 12 min).
1
(1.7 mg, tR 8 min), 4
25
Compound
1
: Yellowish amorphous; [
α]
−
13 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 371 (1.25), 256
D
(1.83) nm; IR (CHCl3) νmax 3727, 3625, 2883, 1222, 770 cm−1; 1H and 13C NMR data (CD3OD), see Tables 1
and 2; 1H NMR (CDCl3, 600 MHz) δH 15.60 (1H, s), 8.99 (1H, d, J = 6.7 Hz), 8.62 (1H, d, J = 8.6, 6.7 Hz),
8.15 (1H, d, J = 8.3 Hz), 8.05 (1H, t, J = 7.6 Hz), 7.98 (1H, d, J = 7.8 Hz), 7.95 (1H, d, J = 6.8 Hz), 7.91 (1H, t,
J = 8.5 Hz), 7.10 (1H, t, J = 8.1 Hz), 6.56 (1H, t, J = 7.4 Hz), 6.39 (1H, d, J = 8.6 Hz), 6.03 (1H, q, J = 14.2,
7.6 Hz), 1.82 (3H, d, J = 6.7 Hz); HRESIMS m/z 388.1296 [M + H]+ (calcd. for C22H18N3O4, 388.1297).
25
Saphenic amide (
2
): Yellowish needle; [
α
]
−
13 (c 0.1, CHCl3); UV (CHCl3)
λ
max
(log ε) 370 (0.87),
255 (1.52) nm; IR (CHCl3) νmax 3328, 2922,D1664, 1218, 770 cm−1; 1H and 13C NMR data (CDCl3), see
Tables 1 and 2; HRESIMS m/z 268.1088 [M + H]+ (calcd. for C15H14N3O2, 268.1086).
25
Saphenol (
3
): Yellowish amorphous; [
α
]
0 (c 0.1, CHCl3); UV (CHCl3)
λ
max
(log ε) 366 (0.61), 255
D
(1.41) nm; IR (CHCl3) νmax 3413, 2957, 1218, 770 cm−1; 1H and 13C NMR data (CDCl3), see Tables 1
and 2; HRESIMS m/z 269.1290 [M + H]+ (calcd. for C16H17N2O2, 269.1290).
25
1
Saphenic acid (
4
): Yellowish needle; [
α
]
−
13 (c 0.1, CHCl3); H and 13C NMR data (CDCl3), see
Tables 1 and 2; LREIMS m/z 269.1 [M + HD]+.
Phenazine-1-carboxylic acid (5
): Yellowish amorphous; 1H NMR (CDCl3, 600 MHz) δH 15.58 (1H, s),
8.97 (1H, d, J = 7.0 Hz), 8.52 (1H, d, J = 8.7 Hz), 8.34 (1H, d, J = 8.3 Hz), 8.28 (1H, d, J = 8.64 Hz),
8.04–7.96. (1H × 3, overlapped); LREIMS m/z 225.1 [M + H]+.
25
6-(1-hydroxyehtyl)phenazine-1-carboxylic acid (
6): Yellowish amorphous; [
α
]
−
60◦ (c 0.05, CHCl3);
1H NMR (CDCl3, 600 MHz) δH 15.62 (1H, s), 8.95 (1H, d, J = 7.0 Hz), 8.53 (1H, Dd, J = 8.7 Hz), 8.17 (1H,
d, J = 8.3 Hz) 8.05 (1H, br d), 8.03–8.00 (1H
×
2, overlapped), 5.76 (1H, q, J = 12.9, 6.5 Hz), 3.42 (3H, s),
1.62 (3H, d, J = 6.4 Hz); LREIMS m/z 283.1 [M + H]+.
6-acetylphenazine-1-carboxylic acid (
): Yellowish amorphous; 1H NMR (CDCl3, 600 MHz) δH 15.26
7
(1H, s), 9.01 (1H, dd, J = 7.0, 1.3 Hz), 8.55 (1H, dd, J = 8.7, 1.3 Hz), 8.41 (1H, dd, J = 8.7, 1.3 Hz), 8.29
(1H, dd, J = 6.9, 1.3 Hz), 8.08 (1H, dd, J = 8.6, 7.1 Hz), 8.05 (1H, dd, J = 8.6, 6.9 Hz), 3.07 (3H, s); LREIMS
m/z 267.1 [M + H]+.

Mar. Drugs 2019, 17, 482
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3.4. 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].
To a solution of (R)-saphenic acid (
4
) (5.4 mg, 0.02 mM) in DCM (200
µ
L) was added triethylamine
(5
µ
L) at 0 ◦C and stirred for 10 min. To this mixture, the solution of acid chloride (150
µL, 0.02 mM) in
DCM (2.7 mL) was added drop wise at 0 ◦C and stirred at room temperature for 16 h. The reaction
solution was evaporated in vacuo and separated on PTLC (Hex-EtOAc, 4:6). The fractions containing
a yellowish spot (Rf 0.1 ~ 0.4) were combined and further purified by RP-HPLC (YMC Triart-C18,
250
×
4.6 mm i.d., 5
µ
m; 45% MeCN in H2O containing 0.01% TFA; flow rate: 1.0 mL/min; detector:
1
RI) to give semi-synthesized
1
(0.2 mg, tR 32 min). H NMR (CDCl3, 600 MHz) δH 15.61 (1H, s), 8.99
(1H, d, J = 5.8 Hz), 8.61 (1H, d, J = 7.5 Hz), 8.14 (1H, d, J = 8.6 Hz), 8.05 (1H, t, J = 7.26 Hz), 7.95 (1H,
d, J = 7.8 Hz), 7.94 (1H, d, J = 5.5 Hz), 7.89 (1H, t, J = 8.5 Hz), 7.08 (1H, d, J = 7.1 Hz), 6.54 (1H, t,
J = 7.4 Hz), 6.35 (1H, d, J = 8.6 Hz), 6.03 (1H, q, J = 12.7, 5.9 Hz), 1.80 (3H, d, J = 6.7 Hz) (Figure S26).
3.5. Nitrite Quantification
−
NO2 accumulation was used as an indicator of NO production as described previously [13].
RAW 264.7 cells were plated at 5
30 g/mL) of and stimulated with LPS (200 ng/mL) for 24 h. The supernatants were mixed with an
×
105 cells/mL, pre-treated with various concentrations (0.3, 1, 3, 10 or
µ
1–7
equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride
and 2% phosphoric acid) and incubated at room temperature for 10 min. NaNO2 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.
4. Conclusions
Three new compounds (
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 and
was determined by comparison of optical rotation values with literature. All the isolated compounds
, except for , exhibited moderate NO production inhibition activity without cytotoxicity. This is
1–3) and four known compounds (4–7) of phenazine class were isolated
1–4
6
1
–7
2
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.
5. Patents
Shin, H.J.; Lee, H.-S.; Choi, B.-K.; Lee, H.-S.; Lee, Y.-J.; Lee, J. S.; Lee, J. Method of producing
saphenic acid and its derivatives and use thereof. KR 10-2018-0167792, 21 December 2018.
Supplementary Materials: The followings are available online at http://www.mdpi.com/1660-3397/17/8/482/s1,
1
1
Figure S1: H NMR spectrum of compound
1
(600 MHz, CD3OD), Figure S2: H NMR spectrum of compound
(150 MHz, CD3OD), Figure S4: HSQC
in CD3OD, Figure S5: COSY spectrum of compound in CD3OD, Figure S6: HMBC
in CD3OD, Figure S7: ROESY spectrum of compound in CD3OD, Figure S8: HRESI-MS
, Figure S9: 1H NMR spectrum of compound (600 MHz, CDCl3), Figure S10: 13C NMR
(150 MHz, CDCl3), Figure S11: HSQC spectrum of compound
in CDCl3, Figure S13: HMBC spectrum of compound
, Figure S15: 1H NMR spectrum of compound
3 (600 MHz, CDCl3), Figure S16:
1
(600 MHz, CDCl3), Figure S3: 13C NMR spectrum of compound
1
spectrum of compound
spectrum of compound
spectrum of compound
spectrum of compound
COSY spectrum of compound
1
1
1
1
1
2
2
2
in CDCl3, Figure S12:
2
2 in CDCl3, Figure S14:
HRESI-MS spectrum of compound
2
13C NMR spectrum of compound
3
(150 MHz, CDCl3), Figure S17: HSQC spectrum of compound
in CDCl3, Figure S19: HMBC spectrum of compound
Figure S20: HRESI-MS spectrum of compound
, Figure S21: 1H NMR spectrum of compound
3
3
in CDCl3,
in CDCl3,
Figure S18: COSY spectrum of compound
3
3
4
(600 MHz,

Mar. Drugs 2019, 17, 482
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CDCl3), Figure S22: 13C NMR spectrum of compound
4
(150 MHz, CDCl3), Figure S23: 1H NMR spectrum of
compound
NMR spectrum of compound
5 6
(600 MHz, CDCl3), Figure S24: 1H NMR spectrum of compound (600 MHz, CDCl3), Figure S25: 1H
7
(600 MHz, CDCl3), Figure S26: 1H NMR spectrum of semi-synthesized
1
(600
MHz, CDCl3), Figure S27: LR-MS spectrum of semi-synthesized
between semi-synthesized 1 and natural 1.
1
, Figure S28: Comparison of 1H NMR data
Author Contributions: H.J.S. was the principal investigator, who proposed ideas for the present work, managed
and supervised the whole research work, prepared and corrected the manuscript and contributed to the structure
elucidation of the new and known compounds. H.-S.L. (Hwa-Sun Lee) achieved all experiments for compounds
1–7, including fermentation, isolation, and structure elucidation, semi-synthesis and prepared the manuscript. J.S.K.
evaluated the cytotoxicity of 1–7. B.-K.C., H.-S.L. (Hyi-Seung Lee), Y.-J.L. and J.L. contributed to analyzing data.
Funding: This research was a part of the project titled ‘Understanding the deep-sea biosphere on seafloor
hydrothermal vents in the Indian Ridge (No. 20170411)’ funded by the Ministry of Oceans and Fisheries, Korea
and the Korea Institute of Ocean Science and Technology (grant PE99752).
Acknowledgments: The authors express gratitude to Y.H. Kim and J.H. Choi (Korea Basic Science Institute,
Cheongju, Republic of Korea) for providing high resolution mass data and to D.-B. Sung, Y.J. Kim and S.-J. Kim
(Marine natural products chemistry lab, KIOST) for their advice on the semi-synthesis of 1.
Conflicts of Interest: The authors declare no conflict of interest.
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