marine drugs

Article
Meirols A–C: Bioactive Catecholic Compounds from the
Marine-Derived Fungus Meira sp. 1210CH-42
Min Ah Lee 1,2 , Jong Soon Kang 3 , Jeong-Wook Yang 3 , Hwa-Sun Lee 1,2 , Chang-Su Heo 1,4 , Sun Joo Park 2 and
Hee Jae Shin 1,4, *

1 Marine Natural Products Chemistry Laboratory, Korea Institute of Ocean Science and Technology,
385 Haeyang-ro, Yeongdo-gu, Busan 49111, Republic of Korea; [email protected] (M.A.L.);
[email protected] (H.-S.L.); [email protected] (C.-S.H.)
2 Department of Chemistry, Pukyong National University, 45 Yongso-ro, Nam-gu, Busan 48513,
Republic of Korea; [email protected]
3 Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology,
30 Yeongudanji-ro, Cheongwon-gu, Cheongju 28116, Republic of Korea; [email protected] (J.S.K.);
[email protected] (J.-W.Y.)
4 Department of Marine Biotechnology, University of Science and Technology (UST), 217 Gajungro, Yuseong-gu,
Daejeon 34113, Republic of Korea
* Correspondence: [email protected]; Tel.: +82-51-664-3341; Fax: +82-51-664-3340

Abstract: Three new catecholic compounds, named meirols A–C (2–4), and one known analog, ar-
govin (1), were isolated from the marine-derived fungus Meira sp. 1210CH-42. Their structures were
determined by extensive analysis of 1D, 2D NMR, and HR-ESIMS spectroscopic data. Their absolute
configurations were elucidated based on ECD calculations. All the compounds exhibited strong an-
tioxidant capabilities with EC50 values ranging from 6.01 to 7.47 µM (ascorbic acid, EC50 = 7.81 µM),
as demonstrated by DPPH radical scavenging activity assays. In the α-glucosidase inhibition assay,
1 and 2 showed potent in vitro inhibitory activity with IC50 values of 184.50 and 199.70 µM, respec-
tively (acarbose, IC50 = 301.93 µM). Although none of the isolated compounds exhibited cytotoxicity
against one normal and six solid cancer cell lines, 1 exhibited moderate cytotoxicity against the
NALM6 and RPMI-8402 blood cancer cell lines with GI50 values of 9.48 and 21.00 µM, respectively.
Compound 2 also demonstrated weak cytotoxicity against the NALM6 blood cancer cell line with a
Citation: Lee, M.A.; Kang, J.S.; Yang, GI50 value of 29.40 µM.
J.-W.; Lee, H.-S.; Heo, C.-S.; Park, S.J.;
Shin, H.J. Meirols A–C: Bioactive Keywords: marine fungus; natural product; Meira sp.; indanone; catechol; meirol; DPPH radical
Catecholic Compounds from the scavenging; α-glucosidase inhibitor; cytotoxicity
Marine-Derived Fungus Meira sp.
1210CH-42. Mar. Drugs 2024, 22, 87.
https://doi.org/10.3390/md22020087

Academic Editor: Tim Bugni 1. Introduction

Received: 23 January 2024
Small-molecule natural products, such as catechol and β-lactam, are widely utilized
Revised: 6 February 2024 in the drug discovery process in various ways [1,2]. Because of their low weight, small-
Accepted: 13 February 2024 molecule drugs can traverse cell membranes, interact with proteins and enzymes within
Published: 14 February 2024 cells, and disrupt specific processes. Alternatively, a biologically active natural product
may inspire the discovery of clinically useful drug agents by offering insights into types
of structural features that may prove valuable. Catechols, benzene derivatives containing
a 3,4-dihydroxyphenyl group, have recently been discovered as diverse derivatives in
Copyright: © 2024 by the authors. microorganisms, plants, insects, and marine organisms [3]. Their ubiquity can be attributed
Licensee MDPI, Basel, Switzerland. to their rich redox chemistry, ability to cross-link through complex and irreversible oxida-
This article is an open access article tion mechanisms, excellent chelating properties, and the various ways in which vicinal
distributed under the terms and hydroxyl groups interact with surfaces of significantly diverse chemical and physical char-
conditions of the Creative Commons
acteristics [4,5]. Aligned with their structural variety, catecholamines have been reported
Attribution (CC BY) license (https://
to exhibit a broad spectrum of biological activities, encompassing antiparasitic, antibacte-
creativecommons.org/licenses/by/
rial, metal-chelating, anti-inflammatory, immune-modulation, wound-healing, antioxidant,
4.0/).

Mar. Drugs 2024, 22, 87. https://doi.org/10.3390/md22020087 https://www.mdpi.com/journal/marinedrugs

Mar. Drugs 2024, 22, 87 2 of 10

neuroprotective, nephroprotective, and metabolic regulation activities [3]. Hence, novel

catechol compounds derived from natural sources will serve as a promising foundation for
broadening their potential biological and medical applications.
The novel basidiomycetous fungus Meira was first isolated from citrus leaves and mite
cadavers in 2003 and assigned to M. geulakonigii and M. argovae [6]. Six species in the genus
Meira have been reported to date, namely, M. argovae, M. geulakonigii (Boekhout et al. 2003),
M. nashicola (Yasuda et al. 2006), M. miltonrushii (Rush et al. 2013), M. siamensis (Limtong
et al. 2017), and M. nicotianae (Yichen et al. 2018) [6–9]. These species have been isolated
from plant tissues, fruits, and mites. These Meira species exhibit promising antagonistic
activities against a wide spectrum of fungal and bacterial plant diseases [10]. However, only
one compound, argovin (1), has been reported from the genus Meira [11]. Thus, information
about the active constituents responsible for the various biological activities of Meira sp. is
still limited.
In 2023, the first marine-derived Meira sp. was described in our previous report as
Meira sp. 1210CH-42 [12]. We identified new thiolactones and steroids from the Meira sp.
strain, which exhibited α-glucosidase inhibitory activities [12]. In our ongoing efforts to
find new bioactive small compounds, we have paid attention to the fungus Meira because
the genus has not been studied well, and the strain was the first marine-derived species
showing good bioactivities in our preliminary screening. Therefore, a mass culture was
conducted to isolate bioactive secondary metabolites from the strain. As a result, we discov-
ered novel bioactive catecholic compounds from the culture extract of the fungus Meira sp.
1210CH-42. Herein, we describe the structure determination of catecholic compounds (1–4),
along with their antioxidant, α-glucosidase inhibitory, cytotoxic, tyrosinase inhibitory, and
antimicrobial activities.

2. Results and Discussion

2.1. Structure Elucidation of New Compounds
Compound 1 was isolated as a white amorphous powder, and its molecular formula
was determined to be C9 H8 O3 based on an HR-ESIMS analysis at m/z 165.0552 [M + H]+
(calcd for C9 H9 O3 + , 165.0552), with six degrees of unsaturation. The 1 H and 13 C NMR data
of 1 are summarized in Tables 1 and 2. The 1 H NMR spectrum of 1 in CD3 OD revealed
two olefinic protons (δH 7.15 and 6.84) and two methylene protons (δH 3.01 and 2.63). The
13 C NMR and HSQC spectra showed the presence of nine signals, including one carbonyl

carbon (δC 209.1), two oxygen-bearing sp2 (δC 153.1 and 143.1), two non-protonated sp2
(δC 144.7 and 130.8), two protonated sp2 (δC 117.2 and 116.9), and two methylene (δC 37.6
and 23.3) carbons. The structure of 1 was identified as argovin (4,5-dihydroxyindan-1-one)
by an analysis of its NMR and HRMS data and a comparison of its spectroscopic data with
those previously reported in the literature (Figure 1) [11].
Compound 2 was purified as a white amorphous powder. The molecular formula of 2
was determined to be C8 H7 NO3 by HR-ESIMS analysis at m/z 188.0324 [M + Na]+ (calcd
for C8 H7 NO3 Na+ , 188.0324), which was determined to possess six degrees of unsaturation.
The 1 H NMR spectrum of 2 displayed the signals for two olefinic protons (δH 7.19 and
6.90) and two methylene protons (δH 4.34, overlapped) (Table 1). The 13 C NMR and
HSQC spectra exhibited one carbonyl (δC 174.5), two oxygen-bearing sp2 (δC 150.4 and
141.6), two non-protonated sp2 (δC 132.4 and 125.2), two protonated sp2 (δC 116.9 and
116.5), and one methylene (δC 44.5) carbons (Table 2). The 1 H-1 H COSY correlation of H-6
(δH 6.90)/H-7 (δH 7.16) revealed a pair of ortho-coupled aromatic protons at δH 6.90, d
(J = 8.0 Hz, δC 116.9)/δH 7.16, d (J = 8.0 Hz, δC 116.5). Furthermore, the HMBC correlations,
from H-3 (δH 4.34) to C-1 (δC 174.5)/C-3a (δC 132.4)/C-4 (δC 141.6)/C-7a (δC 125.2); from
H-6 (δH 6.90) to C-4 (δC 141.6)/C-7a (δC 125.2); and from H-7 (δH 7.16) to C-1 (δC 174.5)/C-3a
(δC 132.4)/C-5 (δC 150.4), indicated the presence of a 1-indanone ring system. Additionally,
the HMBC correlation of the singlet at H-3 (δH 4.34, s) with a carbonyl carbon C-1 (δC 174.5)
confirmed the connectivity of an amide carbonyl to the benzene ring (Figure 2). Thus,
Mar. Drugs 2024, 22, 87 3 of 10

the structure of 2 was elucidated as a previously unreported catecholic compound, 4,5-

dihydroxyisoindolin-1-one, and 2 was named meirol A.
Compound 3 was obtained as a purple amorphous powder. The molecular formula
of 3 was analyzed for C9 H8 O4 on the basis of its parent ion in the HR-ESIMS analysis at
m/z 203.0321 [M + Na]+ (calcd for C9 H8 O4 Na+ , 203.0320), which required six degrees of
unsaturation. The 1 H NMR spectrum of 3 displayed signals of two olefinic protons (δH 7.14
and 6.91), one oxygenated methine proton (δH 5.48), and two methylene protons (δH 3.02
and 2.47) (Table 1). In total, nine carbon resonances were observed in the 13 C NMR spectrum
of 3. These resonances were assigned to one carbonyl (δC 205.6), two oxygen-bearing sp2
(δC 154.2 and 130.0), two non-protonated sp2 (δC 144.1 and 143.5), two protonated sp2 (δC
118.6 and 116.7), one oxymethine (δC 66.9), and one methylene (δC 48.4) carbons, with the
assistance of the HSQC spectrum (Table 2). Analysis of the 1 H-1 H COSY spectrum led to
the construction of an ortho-coupled (J = 8.1) isolated two-proton spin system illustrating
homonuclear coupling correlation between H-6 (δH 6.91, d, J = 8.1) and H-7 (δH 7.14, d,
J = 8.1). The HMBC correlations from H-6 (δH 6.91) to C-4 (δC 130.0)/C-5 (δC 154.2)/C-7a
(δC 144.1) and from H-7 (δH 7.14) to C-5 (δC 154.2)/C-7a (δC 144.1) confirmed the position
of the spin system within an aromatic ring fragment. Additionally, the HMBC correlations
from H-2 (δH 3.02 and 2.47) to C-1 (δC 205.6)/C-3 (δC 66.9)/C-7a (δC 144.1); from H-3
(δH 5.48) to C-1 (δC 205.6)/C-3a (δC 143.5)/C-4 (δC 130.0); and from H-7 (δH 7.14) to C-1
(δC 205.6) allowed for the assembly of a 1-indanone ring system. Furthermore, the presence
of a hydroxy group located at C-3 (δC 66.9) was elucidated through the HMBC correlations
from H-3 (δH 5.48) to C-1 (δC 205.6)/C-3a (δC 143.5)/C-4 (δC 130.0). Therefore, the planar
structure of 3 was elucidated, as shown in Figure 2. To determine the absolute configuration
of 3, the theoretical electronic circular dichroism (ECD) spectra of 3 and its enantiomer
were calculated (Figure 2). The experimental ECD spectrum of 3 showed good agreement
with the calculated ECD spectrum of (3R)-3 (Figure 3). Thus, the absolute configuration of
3 was determined as 3R, and the new indanone derivative, 3, was given the name meirol B.
Compound 4 was isolated as a light purple amorphous powder. The molecular
formula of 4 was determined to be identical to that of 3 (C9 H8 O4 ), as evidenced by the
ion at m/z 203.0322 [M + Na]+ (calcd for C9 H8 O4 Na+ , 203.0320) from the HR-ESIMS
analysis, implying six degrees of unsaturation. However, its 1 H and 13 C NMR spectra
showed different chemical shifts compared with 3. The 1 H NMR spectrum of 4 (Table 1)
showed the signals for two olefinic protons (δH 7.17 and 6.87), one oxygenated methine
proton (δH 4.41), and two methylene protons (δH 3.51 and 2.70). The 13 C NMR and HSQC
spectra of 4 exhibited the presence of nine carbon resonances, including one carbonyl
(δC 207.0), two oxygen-bearing sp2 (δC 153.8 and 128.1), two non-protonated sp2 (δC 143.0
and 139.8), two protonated sp2 (δC 118.0 and 117.3), one oxymethine (δC 75.0), and one
methylene (δC 32.2) carbons (Table 2). The 1 H-1 H COSY correlation of H-2 (δH 4.41)/H-3
(δH 3.51 and 2.70) and H-6 (δH 6.87)/H-7 (δH 7.17) and the strong HMBC correlations
from H-2 (δH 4.41) to C-1 (δC 207.0)/C-3a (δC 139.8) and from H-3 (δH 3.51 and 2.70) to
C-1 (δC 207.0)/C-2 (δC 75.0)/C-3a (δC 139.8)/C-4 (δC 128.1)/C-7a (δC 143.0) indicated that
a hydroxy group was located at C-2 (δC 75.0), and two oxygenated quaternary carbons
were located at C-4 (δC 128.1) and C-5 (δC 153.8). In addition, the presence of a 1-indanone
system was supported by the 1 H-1 H COSY correlation between H-6 (δH 6.87, d, J = 8.1)
and H-7 (δH 7.17, d, J = 8.1), as well as the HMBC correlations from H-6 (δH 6.87) to C-4
(δC 128.1)/C-5 (δC 153.8)/C-7a (δC 143.0) and from H-7 (δH 7.17) to C-1 (δC 207.0)/C-3a
(δC 139.8)/C-5 (δC 153.8). Based on these data analyses, the planar structure of 4 was
determined to be a new positional isomer of 3 (Figure 2). The absolute configuration of
4 was defined by comparing its calculated and experimental ECD spectra. As shown in
Figure 3, the calculated ECD spectrum of 2S-4 closely matched the experimental spectrum,
confirming the 2S absolute configuration for 4. Therefore, the structure of the new natural
product, 4, was clearly determined, and 4 was named meirol C.
 NMR and HSQC spectra showed the presence of nine signals, including one carbonyl car-
bon (δC 209.1), two oxygen-bearing sp2 (δC 153.1 and 143.1), two non-protonated sp2 (δC
non-protonated sp2 (δC 132.4 and2 125.2), two protonated sp2 (δC 116.9 and 116.5), and one
144.7 and 130.8), two protonated sp (δC 117.2 and 116.9), and two methylene (δC 37.6 and
methylene
23.3) carbons. C 44.5) carbons (Table 2). The 1H-1H COSY correlation of H-6 (δH 6.90)/H-7 (δH
(δThe structure of 1 was identified as argovin (4,5-dihydroxyindan-1-one) by
Mar. Drugs 2024, 22, 87 7.16)
an revealed
analysis of itsa NMR
pair ofand
ortho-coupled
HRMS data and aromatic protonsofatitsδHspectroscopic
a comparison 6.90, d (J = 8.0 Hz,
data δC10116.9)/δH
4with
of
7.16, d
those (J = 8.0 Hz,
previously δC 116.5).
reported Furthermore,
in the the HMBC
literature (Figure 1) [11]. correlations, from H-3 (δH 4.34) to C
1 (δC 174.5)/C-3a (δC 132.4)/C-4 (δC 141.6)/C-7a (δC 125.2); from H-6 (δH 6.90) to C-4 (δ
141.6)/C-7a (δC 125.2); and from H-7 (δH 7.16) to C-1 (δC 174.5)/C-3a (δC 132.4)/C-5 (δC 150.4)
indicated the presence of a 1-indanone ring system. Additionally, the HMBC correlation
of the singlet at H-3 (δH 4.34, s) with a carbonyl carbon C-1 (δC 174.5) confirmed the con
nectivity of an amide carbonyl to the benzene ring (Figure 2). Thus, the structure of 2 wa
elucidated as a previously unreported catecholic compound, 4,5-dihydroxyisoindolin-1
one, and 2 was named meirol A.
Figure 1. Structures of 1–4 from the marine fungus strain Meira
fungus strain Meira sp.
sp. 1210CH-42.
Table 1. 1H NMR data of 1–4 (600 MHz for 1H in CD3OD).
Table 1. 1 H NMR data of 1–4 (600 MHz for 1 H in CD3 OD).
Compound 2 was purified as a white amorphous powder. The molecular formula of
1 2 3 4
1 2 was
Positiondetermined to 2 be C8H7NO3 by HR-ESIMS 3 analysis at m/z 188.0324
4 [M + Na]+ (calcd
δH, Mult (J in Hz) δH, Mult (J in Hz) δH, Mult (J in Hz) δH, Mult (J in Hz)
Position for C8H7NO3Na+, 188.0324), which was determined to possess six degrees of unsaturation.
δH , Mult (J in Hz) δ H , Mult (J in Hz) δ H , Mult (J in Hz)
2.47, d δ
(18.6)H , Mult (J in Hz)
The 1H2NMR spectrum
2.63, t (5.6)of 2 displayed the signals for two olefinic protons (δH 7.19 and
4.41, dd6.90)
(7.7, 4.5)
and two methylene protons (δ 4.34, 2.47,
overlapped) d (18.6)
(Table 3.02,
1). dd13(18.6,
The C NMR 6.6)and HSQC spec-
2 2.63, t (5.6) H 4.41, dd (7.7, 4.5)
3.02, dd (18.6, 6.6) 2 (δC 150.4 and2.70, dd two
(16.7, 4.5)
tra exhibited
3 one carbonyl
3.01, t (5.6) (δC 174.5),
4.34,two
s oxygen-bearing 5.48,sp
d (6.6) 141.6),
2.70, dd (16.7, 4.5)
3 3.01, t (5.6) 4.34, s 5.48, d (6.6) 3.51, dd (16.7, 7.7)
3.51, dd (16.7, 7.7)
6 6.84, d (8.0) 6 6.84, d6.90,
(8.0)d (8.0) 6.90, d (8.0)
6.91, d (8.0) 6.91, d (8.0)6.87, d (8.1) 6.87, d (8.1)
7 7.15, d (8.0) 7 7.15, d7.16,
(8.0)d (8.0) 7.16, d (8.0)
7.14, d (8.0) 7.14, d (8.0)7.17, d (8.1) 7.17, d (8.1)

Table 2.1313C NMR data of 1–4 (150 MHz13for 13C, in CD3OD).

Table 2. C NMR data of 1–4 (150 MHz for C, in CD3 OD).
1 2 3 4
Position 1 2 3 4
Position δC, Type δC, Type δC, Type δC, Type
1 209.1, δCC , Type δC , Type
174.5, C C , TypeC
δ205.6, δC , Type
207.0, C
2 1 209.1,
37.6, CH 2 C NH174.5, C 205.6,
48.4,CCH2 207.0,75.0,
C CH
3 2 37.6,
23.3, CH2 CH 2 NHCH2
44.5, 48.4,
66.9,CH
CH2 75.0, 32.2,
CH CH2
3 23.3, CH2 44.5, CH2 66.9, CH 32.2, CH2
3a3a 144.7, 144.7,
C C 132.4, CC
132.4, 143.5,
143.5, CC 139.8,139.8,
C C
4 4 143.1, 143.1,
C C 141.6, CC
141.6, 130.0, C C
130.0, 128.1,
128.1, C C
5 5 153.1, 153.1,
C C 150.4 C C
150.4 154.2,C C
154.2, 153.8,153.8,
C C
6 6 116.9, CH CH
116.9, 116.9,
116.9, CHCH 118.6,
118.6,CHCH 117.3,117.3,
CH CH
7 117.2, CH 116.5, CH 116.7, CH 118.0, CH
7 117.2, CH 116.5, CH 116.7, CH 118.0, CH
7a 130.8, C 125.2, C 144.1, C 143.0, C
7a 130.8, C 125.2, C 144.1, C 143.0, C

1 1 COSY and HMBC correlations of 1–4.

Figure KeyH-
Figure2.2.Key 1H-H
1H COSY and HMBC correlations of 1–4.

Compound 3 was obtained as a purple amorphous powder. The molecular formula

of 3 was analyzed for C9H8O4 on the basis of its parent ion in the HR-ESIMS analysis a
m/z 203.0321 [M + Na]+ (calcd for C9H8O4Na+, 203.0320), which required six degrees o
Mar. Drugs 2024, 22, 87
x FOR PEER REVIEW 5 5of
of 10
10

Figure 3. Experimental and calculated ECD spectra of 3 and 4.

2.2. Bioactivity
2.2. Bioactivity Evaluation
Evaluation ofof Compounds
Compounds
The antioxidant
The antioxidant activities
activitiesof 1–4were
of1–4 wereassessed
assessedusing
usingthe the1,1-diphenyl-2-picrylhydrazyl
1,1-diphenyl-2-picrylhydra-
(DPPH) radical scavenging assay. As indicated in
zyl (DPPH) radical scavenging assay. As indicated in Table1–4Table 3, exhibited
3, 1–4 considerable
exhibited free
considerable
radical scavenging activities with
free radical scavenging activities with EC values ranging from 6.01 ± 0.07 to 7.47
50 EC50 values ranging from 6.01 ± 0.07 to 7.47 ± 0.13± 0.13 µM,
showing
µM, betterbetter
showing activities than the
activities thanpositive control,
the positive ascorbic
control, acid (EC
ascorbic acid = 7.81
50 (EC ± 0.25 µM).
50 = 7.81 ± 0.25
Also, 1–4
µM). were
Also, 1–4evaluated for α-glucosidase
were evaluated inhibitory activities
for α-glucosidase inhibitory (Table 3). Compounds
activities and 2
(Table 3).1 Com-
exhibited significant inhibitory effects with IC 50 values of 184.50 ± 2.93 and 199.70
pounds 1 and 2 exhibited significant inhibitory effects with IC50 values of 184.50 ± 2.93 and ± 1.87 µM,
respectively. Meanwhile, 3 showed weaker activity (IC 50 = 367.43 ± 3.01 µM)
199.70 ± 1.87 µM, respectively. Meanwhile, 3 showed weaker activity (IC50 = 367.43 ± 3.01 than the pos-
itive than
µM) control,
theacarbose
positive (IC 50 = 301.93
control, ± 3.55
acarbose µM).
(IC50 A structure–activity
= 301.93 relationship analysis
± 3.55 µM). A structure–activity re-
of 1–4 indicated that the hydroxy groups at C-2 or C-3 in diol-indanone impacted their an-
lationship analysis of 1–4 indicated that the hydroxy groups at C-2 or C-3 in diol-indanone
tioxidant properties but did not have a significant impact on their α-glucosidase inhibitory
impacted their antioxidant properties but did not have a significant impact on their α-
activity. Furthermore, all the compounds were screened for their cytotoxic activity against
glucosidase inhibitory activity. Furthermore, all the compounds were screened for their
six solid and seven blood cancer cell lines (Table 3). Compounds 1 and 2 showed selective
cytotoxic activity against six solid and seven blood cancer cell lines (Table 3). Compounds
cytotoxicity against two out of the seven blood cancer cell lines (HL-60, acute myelogenous
1 and 2 showed selective cytotoxicity against two out of the seven blood cancer cell lines
Mar. Drugs 2024, 22, 87 6 of 10

leukemia; Raji, Burkitt’s lymphoma; K562, chronic myelogenous leukemia; RPMI-8402, T cell
acute lymphocytic leukemia; NALM6, B cell acute lymphocytic leukemia; U266, multiple
myeloma; WSU-DLCL2, diffuse large B cell lymphoma). Among the tested compounds,
only 1 showed weak cytotoxicity (GI50 = 21.00 ± 0.47 µM) against the RPMI-8402 cell line.
Compounds 1 and 2 exhibited selective cytotoxicity against the NALM6 blood cancer cell line
(GI50 = 9.47 ± 0.41 and 29.55 ± 2.27 µM, respectively), while the other compounds did not
demonstrate significant cytotoxicity (GI50 > 30 µM). Additionally, all compounds showed no
cytotoxicity against one normal cell (MRC-9) and six solid cancer cell lines (PC-3, prostate;
NCI-H23, lung; HCT-15, colon; NUGC-3, stomach; ACHN, renal; MDA-MB-231, breast) even
at a concentration of 30 µM.

Table 3. Antioxidant, α-glucosidase inhibitory, and cytotoxic activities of 1–4.

DPPH Radical α-Glucosidase Cytotoxic Activity

Scavenging Activity Inhibitory Activity (GI50 ± SD, µM) c
Compound
(EC50 ± SD, µM) a (IC50 ± SD, µM) b RPMI-8402 d NALM6 d
1 7.47 ± 0.13 184.50 ± 2.93 21.00 ± 0.47 9.47 ± 0.41
2 6.82 ± 0.06 199.70 ± 1.87 >30 29.55 ± 2.27
3 6.01 ± 0.07 367.43 ± 3.01 >30 >30
4 6.20 ± 0.12 >555.00 >30 >30
Ascorbic acid 7.81 ± 0.25 – – –
Acarbose – 301.93 ± 3.55 – –
Doxorubicin – – 0.015 ± 0.13 × 10−2 0.003 ± 0.06 × 10−2
a The half maximal effective concentration (µM); b the 50% inhibitory concentration (µM); c the 50% growth
inhibitory concentration (µM) values represent the means ± standard deviation based on triplicate experiments;
d RPMI-8402, T cell acute lymphocytic leukemia; NALM6, B cell acute lymphocytic leukemia.

Compounds 1–4 did not show significant tyrosinase inhibitory activity at a concentra-
tion of 100 µM (kojic acid, IC50 = 41.85 µM). Also, 1–4 were evaluated for their antimicrobial
properties against three Gram-positive bacteria (Bacillus subtilis KCTC 1021, Micrococcus
luteus KCTC 1915, and Staphylococcus aureus KCTC 1927) and three Gram-negative bacteria
(Escherichia coli KCTC 2441, Salmonella typhimurium KCTC 2515, and Klebsiella pneumonia
KCTC 2690). However, none of the compounds inhibited the growth of Gram-positive and
Gram-negative bacteria at a concentration of 32.0 µg/mL.

3. Materials and Methods

3.1. General Experimental Procedures and Reagents
NMR spectra were acquired with a Bruker AVANCE III 600 spectrometer (Bruker
Biospin GmbH, Rheinstetten, Germany) with a 3 mm probe operating at 600 MHz (1 H)
and 150 MHz (13 C). Chemical shifts were expressed in ppm with reference to the solvent
peaks (δH 3.31 and δC 49.15 ppm for CD3 OD). UV spectra were recorded with a Shimadzu
UV-1650PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR spectra were
obtained on an OPUS FT/IR-ALPHA II spectrophotometer (Bruker OPTIK GmbH & Co.
KG, Ettlingen, Germany). Optical rotations were measured with a Rudolph analytical
Autopol III S2 polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). LR-
ESIMS data were obtained with an ISQ EM mass spectrometer. HR-ESIMS data were
obtained with a Waters SYNPT G2 Q-TOF mass spectrometer (Waters Corporation, Milford,
MA, USA) at the Korea Basic Science Institute (KBSI) in Cheongju, Republic of Korea.
ECD spectra were recorded with a JASCO J-1500 circular dichroism spectrometer (JASCO
Corporation, Tokyo, Japan) at the Center for Research Facilities, Changwon National
University, in Changwon, Republic of Korea. HPLC was performed using a BLS-Class
pump (Teledyne SSI, Inc., State College, PA 16803, USA) with a Shodex RI-201H refractive
index detector (Shoko Scientific Co., Ltd., Yokohama, Japan). Columns for HPLC were YMC-
Triart C18 (250 mm × 10 mm, 5 µm), YMC-Triart C8 (250 mm × 10 mm, 5 µm), and YMC-
CHIRAL PREP CD PM (250 mm × 4.6 mm, S-10 µm). RP silica gel (YMC-Gel ODS-A, 12 nm,
S-75 µm) was used for open-column chromatography. Organic solvents were purchased
Mar. Drugs 2024, 22, 87 7 of 10

as HPLC grade, and ultrapure waters were obtained from the Milipore Mili-Q Direct 8
system. The reagents used in the bioassay were purchased from Sigma-Aldrich and Tokyo
Chemical Industry. Cancer cell lines were purchased from the Japanese Cancer Research
Resources Bank (JCRB) (NUGC-3, JCRB Cell Bank/Cat. # JCRB0822), the DSMZ-German
Collection of Microorganisms and Cell Cultures (RPMI-8402, DSMZ/Cat # ACC 290; WSU-
DLCL2, DSMZ/Cat # ACC 575), and the American Type Culture Collection (ATCC) (PC-3,
ATCC/Cat. # CRL-1435; MDA-MB-231, ATCC/Cat. # HTB-26; ACHN, ATCC/Cat. #
CRL-1611; NCI-H23, ATCC/Cat. # CRL-5800; HCT-15, ATCC/Cat. # CCL-225; HL-60,
ATCC/Cat. # CCL-240; Raji, ATCC/Cat # CCL-86; K562, ATCC/Cat # CCL-243; NALM6,
ATCC/Cat # CRL-3273; U266, ATCC/Cat # TIB-196).

3.2. Fungal Strain and Fermentation

The fungal strain Meira sp. 1210CH-42 was obtained from a seawater sample collected
at the Chuuk Islands, Federated States of Micronesia, in 2010, as described previously [12].
The strain was identified as Meira sp. (GenBank accession number OQ693946) through
DNA amplification and by sequencing the ITS region of the rRNA gene, as described
earlier [12]. The cultures of strain 1210CH-42 were performed in modified Bennett’s broth
medium (1% D-glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol,
sea salt 10 g/L, pH 7.0). A seed culture was prepared from a spore suspension of strain
1210CH-42 by inoculating it into 2 L flasks and incubating it at 28 ◦ C for 7 days in a rotary
shaker at 120 rpm. The seed culture was inoculated aseptically into a 100 L fermenter
containing 70 L of sterilized culture medium (0.1% v/v). Large-scale fermentation was
conducted at 28 ◦ C, 40 rpm, and with an airflow rate of 10 L/min (LPM) for a duration of
21 days before being harvested.

3.3. Extraction and Isolation of Compounds 1–4

A culture broth of strain 1210CH-42 (total, 70 L) was harvested via high-speed cen-
trifugation at 60,000 rpm. Subsequently, the supernatant was extracted two times with
ethyl acetate (EtOAc, 140 L). The EtOAc layer was evaporated under reduced pressure
to yield a crude extract (4.61 g). The crude extract was subjected to ODS open column
chromatography (YMC Gel ODS-A, 12 nm, S75 µm) followed by stepwise gradient elution
with MeOH/H2 O (v/v) (20:80, 40:60, 60:40, 80:20, and 100:0) as an eluent. The 20% MeOH
(0.89 g) was subjected to ODS open column chromatography (YMC Gel ODS-A, 12 nm,
S75 µm) followed by stepwise gradient elution with MeOH/H2 O (v/v) (5:95, 10:90, 15:85,
20:80, and 100:0) to provide five fractions (F1–F5). Fraction F4 (293 mg) was purified by
a semi-preparative reversed-phase HPLC (YMC-Triart C18 column 250 mm × 10 mm i.d.,
5 µm; 15% MeOH in H2 O; flow rate: 2.0 mL/min; detector: RI) to yield 1 (18.1 mg, tR
62.0 min). Fraction F2 (140 mg) was further purified by a semi-preparative RP HPLC (YMC-
Triart C18 column 250 mm × 10 mm i.d., 5 µm; 5% MeOH in H2 O; flow rate: 2.0 mL/min;
detector: RI) to obtain eighteen subfractions (F2.SF1–F2.SF18). Compound 2 was re-purified
from subfraction F2.SF16 by a semi-preparative RP HPLC (YMC-Triart C8 column 250 mm
× 10 mm i.d., 5 µm; 5% MeOH in H2 O; flow rate: 2.0 mL/min; detector: RI) to yield
2 (8.2 mg, tR 38.0 min). Fraction F3 (266 mg) was purified using a semi-preparative RP
HPLC (YMC-Triart C18 column 250 mm × 10 mm i.d., 5 µm; 10% MeOH in H2 O; flow rate:
2.0 mL/min; detector: RI) to yield eighteen subfractions (F3.SF1–F3.SF18). Subsequently,
compounds 3 and 4 were isolated from subfraction F3.SF4 using an analytical HPLC (YMC-
CHIRAL PREP CD PM 250 mm × 4.6 mm, S-10 µM; flow rate: 0.8 mL/min; detector: RI)
with an isocratic elution of 5% MeOH in H2 O to yield 3 (4.3 mg, tR 11.0 min) and 4 (5.2 mg,
tR 13.0 min).
Argovin (1): White amorphous powder; UV (MeOH) λmax (log ε) 213 (3.71), 236 (3.95),
283 (3.82) nm; IR (MeOH) νmax 3200, 1651, 1589, 1471, 1299 cm−1 ; 1 H and 13 C NMR data
(CD3 OD), see Tables 1 and 2; HR-ESIMS m/z 165.0552 [M + H]+ (calcd. for C9 H9 O3 + ,
165.0552; 187.0371) and [M + Na]+ (calcd for C9 H8 O3 Na+ , 187.0371).
Mar. Drugs 2024, 22, 87 8 of 10

Meirol A (2): White amorphous powder; UV (MeOH) λmax (log ε) 219 (3.96), 261 (3.83),
290 (3.39) nm; IR (MeOH) νmax 3233, 1626, 1448, 1419, 1292 cm−1 ; 1 H and 13 C NMR data
(CD3 OD), see Tables 1 and 2; HR-ESIMS m/z 166.0503 [M + H]+ (calcd. for C8 H8 NO3 + ,
166.0504; 188.0324) and [M + Na]+ (calcd for C8 H7 NO3 Na+ , 188.0324).
Meirol B (3): Purple amorphous powder; [α]25 D +23.3 (c 0.1, MeOH); UV (MeOH) λmax
(log ε) 214 (3.81), 235 (3.98), 287 (3.74) nm; ECD (MeOH, λ [nm] (∆ε), c = 1.58 mM) 309
(−0.83), 282 (+1.44), 271 (+1.10), 237 (+3.00), 209 (0.56); IR (MeOH) νmax 3270, 1681, 1592,
1488, 1280 cm−1 ; 1 H and 13 C NMR data (CD3 OD), see Tables 1 and 2; HR-ESIMS m/z
203.0321 [M + Na]+ (calcd for C9 H8 O4 Na+ , 203.0320).
Meirol C (4): Light purple amorphous powder; [α]25 D +23.3 (c 0.1, MeOH); UV (MeOH)
λmax (log ε) 212 (3.72), 237 (3.95), 291 (3.77) nm; ECD (MeOH, λ [nm] (∆ε), c = 1.58 mM) 343
(+0.71), 314 (−0.50), 290 (+0.94), 270 (+0.59), 242 (+2.57); IR (MeOH) νmax 3256, 1686, 1611,
1286 cm−1 ; 1 H and 13 C NMR data (CD3 OD), see Tables 1 and 2; HR-ESIMS m/z 203.0322
[M + Na]+ (calcd for C9 H8 O4 Na+ , 203.0320).

3.4. Computational Analysis

The initial geometry optimization and conformational searches were generated using
Conflex 8 (rev. B, Conflex Corp., Tokyo, Japan). The optimization and calculation for
electronic circular dichroism (ECD) were conducted utilizing the Gaussian 16 program
(rev. B.01, Gaussian Inc., Wallingford, CT, USA). Conformational searches were executed
through MMFF94s force field calculations, with a search limit set at 5 kcal/mol. The
conformers were optimized using the ground state method at the CAM-B3LYP/6-31 G+
(d, p) level in MeOH with an IEFPCM model for ECD. The theoretical calculations of
ECD spectra were performed using TD-SCF at the CAM-B3LYP /6-31 G+ (d, p). The ECD
spectrum was derived by calculating the Boltzmann-weighted sum of conformer spectra.
The final ECD spectra were simulated using SpecDis (v. 1.71) with σ values ranging from
0.20 to 0.30 eV. All calculated curves were UV-shifted by +10 to +15 nm to better simulate
experimental spectra.

3.5. Antioxidant Activity Assay

The DPPH radical scavenging activities of 1–4 were determined by the reported
method [13]. In a 96-well plate, 100 µL of sample solution (in MeOH) was mixed with
100 µL of DPPH solution (0.16 mM in MeOH), shaken several times, and then incubated at
room temperature for 30 min. The absorbance at 517 nm was recorded. Ascorbic acid was
used as the positive control, and the experiments were performed in triplicate.

3.6. α-Glucosidase Inhibitory Activity Assay

The α-glucosidase inhibitory activity was determined by measuring the absorbance
increase resulting from the hydrolysis of p-nitrophenyl-α-D-glucopyranoside (pNPG, TCI)
by α-glucosidase at 405 nm using a microplate reader, according to the reference to pre-
viously reported literature [12]. The 130 µL sample solution (in 0.1 mM PBS) with the
30 µL α-glucosidase solution (0.2 U/mL) was incubated at 37 ◦ C for 10 min. Subsequently,
40 µL of 5 mM pNPG was added. The reaction mixture was further incubated at 37 ◦ C for
20 min. The α-glucosidase inhibitory activity was determined using a microplate reader at
405 nm. The negative control was prepared by substituting PBS buffer for the sample in the
same way as the test. Acarbose served as the positive control, and the experiments were
conducted in triplicate.

3.7. Cytotoxicity Assay

The cytotoxic activities of 1–4 were measured using the CellTiter-Glo luminescent cell
viability assay (Promega, Madison, WI, USA) and conducted by the SRB (sulforhodamine
B) assay, as previously described [14,15]. The luminescence signal was quantified using
a GloMax-Multi Detection System (Promega, Madison, WI, USA), and GI50 values were
Mar. Drugs 2024, 22, 87 9 of 10

determined utilizing a relative GI50 model in GraphPad Prism (GraphPad, San Diego, CA,
USA). Doxorubicin was used as the positive control.

3.8. Tyrosinase Inhibitory Activity Assay

The tyrosinase inhibitory activity was assessed using L-DOPA and the 96-well mi-
croplate method, as previously reported [16]. Briefly, 140 µL of 20 mM phosphate buffer
(pH 6.8) and 20 µL of mushroom tyrosinase (480 U/mL) in the same buffer were added
to wells containing 20 µL of the test compounds. After incubation for 10 min at 25 ◦ C,
20 µL of L-DOPA (0.85 mM) was added to the 200 µL reaction system. The incubation was
continued for another 20 min at 25 ◦ C; then, the colored end product’s absorbance was
measured at 475 nm using a microplate reader. Kojic acid was utilized as a positive control
in the reference sample experiment. All experiments were performed in triplicate for each
compound.

3.9. Antibacterial Assay

The antibacterial activities were determined using the 96-well microplate method,
as described in a published report, against three Gram-positive and three Gram-negative
bacteria [17].

4. Conclusions
In summary, four catecholic compounds (1−4), including three new ones (2–4), were
identified from Meira sp. 1210CH-42. Their structures were elucidated by a detailed analy-
sis of NMR and HRESIMS data. The absolute configurations of 3 and 4 were determined
by calculating the ECD spectra of their possible isomers. The antioxidant-DPPH assay
showed that all compounds exhibited more significant free radical scavenging activity
(EC50 = 6.01–7.47 µM) than ascorbic acid. Additionally, 1 and 2 exhibited moderate α-
glucosidase inhibitory activity (IC50 = 184.50 and 199.70 µM, respectively) and selective
cytotoxicity against blood cancer cell lines (RPMI-8402 and NALM6, GI50 = 9.47–29.40 µM).
As a result, the catecholic indanones from Meira sp. 1210CH-42 could serve as a po-
tential agent for antioxidative, α-glucosidase inhibitory, and anticancer leads. Further
investigation is needed to determine the biological mechanism of compounds from the
marine-derived fungus Meira.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/md22020087/s1: Figures S1–S6: 1 H, 13 C NMR, HSQC, COSY, HMBC, and HR-ESIMS data
of 1. Figures S7–S30: 1 H, 13 C NMR, HSQC, COSY, HMBC, HR-ESIMS, UV, and IR data of 2–4.
Figures S31–S32 and Tables S1–S4: TDSCF-ECD calculation data of 3–4.
Author Contributions: Conceptualization, H.J.S.; investigation, M.A.L., J.S.K., J.-W.Y., H.-S.L., C.-S.H.
and S.J.P.; resources, M.A.L.; writing—original draft preparation, M.A.L.; writing—review and
editing, H.J.S. and M.A.L.; project administration, H.J.S.; funding acquisition, H.J.S. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was supported by the Korea Institute of Marine Science and Technology Pro-
motion (KIMST) Grant funded by the Ministry of Oceans and Fisheries, Korea (Grant No. 20220027)
and by the Korea Institute of Ocean Science and Technology (PEA0211).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The data presented in the article are available in the Supplementary
Materials.
Acknowledgments: The authors express gratitude to Jung Hoon Choi, Korea Basic Science Institute,
Ochang, Korea, for providing mass data.
Conflicts of Interest: The authors declare no conflicts of interest.
Mar. Drugs 2024, 22, 87 10 of 10

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