marine drugs
Article
Isolation, Structural Characterization and Antidiabetic Activity
of New Diketopiperazine Alkaloids from Mangrove
Endophytic Fungus Aspergillus sp. 16-5c
Geting Ye, Cuiying Huang, Jialin Li, Tao Chen, Jing Tang, Wenbin Liu and Yuhua Long *






Citation: Ye, G.; Huang, C.; Li, J.;
Chen, T.; Tang, J.; Liu, W.; Long, Y.
Isolation, Structural Characterization
and Antidiabetic Activity of New
Diketopiperazine Alkaloids from
Mangrove Endophytic Fungus
Aspergillus sp. 16-5c. Mar. Drugs 2021,
19, 402. https://doi.org/10.3390/
md19070402
Academic Editors: Bin Wu and
Ikuro Abe
Received: 5 July 2021
Accepted: 19 July 2021
Published: 20 July 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Mar. Drugs 2021, 19, 402. https://doi.org/10.3390/md19070402
Guangzhou Key Laboratory of Analytical Chemistry for Biomedicine, School of Chemistry,
South China Normal University, Guangzhou 510006, China; yegeting@m.scnu.edu.cn (G.Y.);
huangcuiying@m.scnu.edu.cn (C.H.); jialinli@m.scnu.edu.cn (J.L.); ct2020@m.scnu.edu.cn (T.C.);
tangjing2020@m.scnu.edu.cn (J.T.); liuwenbin@m.scnu.edu.cn (W.L.)
* Correspondence: longyh@scnu.edu.cn
Abstract: Six new DIKETOPIPERAZINE alkaloids aspergiamides A–F (1–6), together with ten known
alkaloids (7–16), were isolated from the mangrove endophytic fungus Aspergillus sp. 16-5c. The
structures of the new compounds were elucidated based on 1D/2D NMR spectroscopic and HR-
ESIMS data analyses. The absolute configurations of aspergiamides A-F were established based on
the experimental and calculated ECD data. All the compounds were evaluated for the antidiabetic
activity against α-glucosidase and PTP1B enzyme. The bioassay results disclosed compounds 1 and
9 exhibited significant α-glucosidase inhibitory with IC50 values of 18.2 and 7.6 µM, respectively;
compounds 3, 10, 11, and 15 exhibited moderate α-glucosidase inhibition with IC50 values ranging
from 40.7 to 83.9 µM; while no compounds showed obvious PTP1B enzyme inhibition activity.
Keywords: diketopiperazine alkaloids; mangrove fungi; antidiabetic activity; α-glucosidase inhibition;
PTP1B inhibition
1. Introduction
Type 2 diabetes mellitus (T2DM) is a metabolic disorder disease and is characterized
by an imbalance of blood glucose levels due to defects in insulin secretion. Increased blood
glucose levels in T2DM may induce hypertension, atherosclerosis, and other metabolic
disorder diseases [1]. It is estimated that 425 million people have diabetes, and about 90%
account for type 2 diabetes. According to the World Health Organization (WHO), diabetes
will reach the seventh cause of death by 2030 [2]. Although many antidiabetic drugs with
different mechanisms can be available from the market, the inevitable side effects such as
weight gain, liver damage, and allergic reactions hinder their application [3]. PTP1B is an
intracellular non-transmembrane enzyme, which plays a major negative regulator role in
insulin function by dephosphorylation of tyrosine-phosphorylated proteins [4]. Studies
have shown that overexpression of protein tyrosine phosphatase 1B (PTP1B) can lead to the
dephosphorylation of insulin receptors and insulin receptor substrates, resulting in insulin
resistance and ultimately inactivating the entire insulin signaling pathway. Therefore,
PTP1B inhibitors have attracted particular attention as potential therapeutic agents against
diabetes [5]. In addition, α-glucosidase, a traditional antidiabetic target responsible for
the hydrolysis of polysaccharides to monosaccharides, is still considered an important
direction to discover new chemical entities for the treatment of non-insulin-dependent
T2DM [6]. Therefore, the development of PTP1B and α-glucosidase inhibitors could be a
promising strategy in discovering novel antidiabetic drug candidates.
Diketopiperazine (DKP) alkaloids are important secondary metabolites of microbes.
Among these alkaloids, the diketopiperazine moiety is biosynthesized from different amino
acids and formed as the smallest cyclic dipeptide skeleton. Diketopiperazine alkaloids
have many important biological activities, such as antiviral, anti-tumor, antibacterial,
https://www.mdpi.com/journal/marinedrugs
 Diketopiperazine (DKP) alkaloids are important secondary metabolites of microbes.
Among these alkaloids, the diketopiperazine moiety is biosynthesized from different
Mar. Drugs 2021, 19, 402
amino acids and formed as the smallest cyclic dipeptide skeleton. Diketopiperazine2alka- of 12

loids have many important biological activities, such as antiviral, anti-tumor, antibacte-
rial, and so on [7,8]. Indole diketopiperazine alkaloids belong to the subclass of DKPs are
the condensation
and products
so on [7,8]. Indole of a complete alkaloids
diketopiperazine tryptophan with to
belong a second aminoofacid-like
the subclass DKPs are L-
tryptophan, -proline, -phenylalanine, or -leucine [9]. Indole
the condensation products of a complete tryptophan with a second amino acid-like L-
L L L diketopiperazine alkaloids
have attracted
tryptophan, widespread
L-proline, attention not
L-phenylalanine, or only because
L-leucine of theirdiketopiperazine
[9]. Indole unique structurealkaloids
but also
because
have of the widespread
attracted wide range attention
of biological
not activities,
only becausesuchofas antiviral
their unique [10], anticancer
structure [11],
but also
antioxidant
because [12],
of the wideandrange
insecticidal activities
of biological [13]. Marine
activities, such asfungi are important
antiviral sources
[10], anticancer of
[11],
diketopiperazine
antioxidant alkaloids.
[12], and According
insecticidal to statistics,
activities between
[13]. Marine 2000are
fungi and 2019, as many
important sourcesas 155
of
indole diketopiperazine
diketopiperazine alkaloids
alkaloids. were to
According identified from
statistics, marine
between fungi
2000 and alone [14].
2019, as many as
155 indole diketopiperazine
As part of our aims for alkaloids
exploringwere identified
biological active from marine
natural fungi alone
products [14].second-
[15], the
As part of our
ary metabolites aims
from for exploring
fungus biological
Aspergillus active
sp. 16-5c were natural productsChemical
investigated. [15], the secondary
investiga-
metabolites
tion yieldedfrom fungus
six new Aspergillus sp. alkaloids,
diketopiperazine 16-5c wereincluding
investigated. Chemical
five indole investigation
diketopiperazine
yielded
alkaloids,sixaspergiamides
new diketopiperazineA–E (1–5) alkaloids,
and oneincluding five indole
4-quinazolinone like diketopiperazine
diketopiperazine alka- alka-
loids, aspergiamidesFA–E
loids aspergiamide (6), (1–5) andwith
together one 4-quinazolinone
ten known alkaloids like diketopiperazine
(7–16) from the culturealkaloidsex-
aspergiamide F (6), together
tracts of the fungus with ten
16-5c (Figure 1). known alkaloids (7–16)
These compounds werefrom the culture
evaluated extracts of
the antidiabetic
the fungusby16-5c
potential (Figure
screening the1).enzyme
These compounds
inhibition ofwere evaluatedand
α-glucosidase the PTP1B.
antidiabetic
Herein,potential
we de-
by screening the enzyme inhibition of α-glucosidase and PTP1B. Herein,
scribe the isolation and structure elucidation of the new compounds, as well as their bio- we describe the
isolation
activities.and structure elucidation of the new compounds, as well as their bioactivities.

Figure1.
Figure 1. Chemical
Chemical structures
structuresof
ofcompounds
compounds1–16.
1–16.

2.
2. Results
Results and
and Discussion
Discussion
Structural Elucidation
Structural Elucidation
Compound 1 was obtained as a pale yellow powder. The molecular formula was
Compound 1 was obtained as a pale yellow powder. The molecular formula was es-
established as C21 H25 O3 N3 according to the HRESIMS at m/z 366.1827 [M − H]−− (Calcd
tablished as C21H25O3N3 according to the HRESIMS at m/z 366.1827 [M − H] (Calcd
366.1823 for C21 H24 O3 N3 ), indicating 11 degrees of unsaturation. 13The 13 C NMR and
366.1823 for C21H24O3N3), indicating 11 degrees of unsaturation. The C NMR and HMQC
HMQC spectra (Table 1, Figures S3 and S4) displayed 21 carbon signals, consisting of two
spectra (Table 1, Figures S3 and S4) displayed 21 carbon signals, consisting of two methyls,
methyls, four methylenes (one olefinic carbon), seven methines (including four aromatic
methines), and eight quaternary carbons (including two amide carbons, three olefinic
carbons). The 1 H NMR (Table 1) spectrum of 1 revealed a set of adjacent aromatic protons
at δH 7.08 (m, 1H), 7.13 (m, 1H), 7.24 (d, J = 7.9 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), indicating
the occurrence of two ortho-substituted benzene ring. Four olefinic protons resonances
at δH 5.11 (m, 2H), 6.11 (dd, J = 17.3, 10.6 Hz, 1H), 7.22 (s, 1H), two methyls at δH 1.54
Mar. Drugs 2021, 19, 402 3 of 12

(s, 3H) and 1.55 (s, 3H), three aliphatic methylenes at δH 2.03–1.67 (m, 4H) and 3.63 (t,
J = 6.3 Hz, 2H), one aliphatic methine at δH 4.21 (t, J = 6.3 Hz, 1H) were also recorded
in this spectrum. The 1 H-1 H COSY spectrum of 1 showed cross-peaks that connect the
aromatic protons from H-4 (δH 7.24) through H-7 (δH 7.43), while HMBC correlations from
H-4 to C-7a (δC 136.8)/C-3 (δC 104.3), from H-7 to C-3a (δC 127.1) suggesting an indole ring
(Figure 2). The HMBC correlations from H-12 (δH 4.21) to C-10 (δC 162.5)/C-13 (δC 168.1)
and correlations from H-17 (δH 5.11) to C-15 (δC 40.5), from H3-15a/15b (δH 1.55/1.54)
to C-16 (δC 146.0) combined with the 1 H-1 H COSY correlation between H-16 (δH 6.11)
and H-17, suggesting a diketopiperazine fragment and a prenyl unit, respectively. The
indole ring was further connected with the diketopiperazine and prenyl group to form a
2,3-disubstituted indole diketopiperazine substructure consistent with HMBC correlations
from H3-15a/15b to C-2 (δC 146.2), H-8 (δH 7.22) to C-10/C-3/C-3a. All the above NMR
data suggested the structure of 1 was similar to the known compound neoechinulin A [16],
with the main difference at the presence of an –CH2-CH2-CH2OH moiety in 1. The 1 H-1 H
COSY correlation of H-18 (δH 1.95–2.03)/H-19 (δH 1.67–1.74)/H-20 (δH 3.63) suggested
the existence of –CH2 -CH2 -CH2 – fragment. The chemical shift of C-20 (δC/H 62.5/3.63),
combined with HMBC correlation from H-20 to C-18, indicated a hydroxyl was located
at C-20 to form a –CH2 -CH2 -CH2 OH moiety. Moreover, key HMBC correlations from
H-19 to C-12 suggested the moiety was connected with C-12, as shown in Figure 1. The
Z-geometry of the ∆8 double bond was identified by the lack of NOE effect between
H-8 and NH-14 [17]. The absolute configuration of compound 1 was determined by the
quantum chemical calculation study for electronic circular dichroism (ECD). Consequently,
the calculated ECD for 12R-1 is consistent with the experimental ECD of 1 (Figure 3), which
indicated the absolute configuration of 1 as 12R. Thus, the structure of compound 1 was
identified and named aspergiamide A.
Compound 2 was isolated as a white powder and assigned a molecular formula of
C21 H23 O4 N3 (12 degrees of unsaturation) based on the HRESIMS and 1D NMR
(Figures S8–S14). The 13 C NMR (Table 1) displayed 21 carbon signals, consisting of
two methyls, three methylenes (one olefinic carbon), seven methines, and nine quaternary
carbons (including one carboxyl carbonyl carbon, two amide carbonyl carbons). The 1 H
NMR spectrum (Table 1) of 2 revealed a set of adjacent aromatic protons at δH 7.08 (m, 1H),
7.13 (m, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), indicating the occurrence of
two ortho-substituted benzene ring. Four olefinic protons resonances at δH 5.11 (m, 2H),
6.11 (dd, J = 17.3, 10.6 Hz, 1H), 7.22 (s, 1H), two methyls at δH 1.54 (s, 3H) and 1.55 (s, 3H),
two aliphatic methylenes at δH 2.21 (m, 2H) and 2.48 (m, 2H), one aliphatic methine at
δH 4.25 (t, J = 5.6 Hz, 1H) were also recorded. Cumulative analyses of 1D and 2D NMR
spectroscopic data revealed that 2 possessed a similar planar structure as that of 1. A major
difference was the replacement of a hydroxymethylene group in 1 (δC 62.5) by a carboxyl
group in 2 (δC 176.3), and the chemical shift of H-19 downfield from 1.67 in 1 to 2.84 in
2 due to the deshielding effect of the carboxyl group, indicating that compound 2 is the
oxidation product of 1. Then, the planar structure of 2 was further verified by analyzing
2D NMR, as shown in Figure 2. The 1 H-1 H COSY correlation of H-18 (δH 2.21)/H-19 (δH
2.48) suggested the existence of –CH2 -CH2 – fragment. The chemical shift of C-20 (δC 176.3),
combined with HMBC correlation from H-18 to C-20 and from H-19 to C-20, indicated a
carboxyl group was located at C-20 to form a –CH2 -CH2 -COOH moiety. Moreover, key
HMBC correlations from H-19 to C-12 (δC 56.1) suggested the moiety was connected with
C-12, as shown in Figure 1. ∆8 double bond was identified as Z configuration by the
lack of NOE effect between H-8 and NH-14 [17]. Furthermore, the absolute configuration
of C-12 was determined to be R based on the same negative sign of its specific rota-
tion ( [α]25 ◦ 25 ◦
D − 154.8 , c 0.1, MeOH), as that of compound 1 ([α]D − 171.4 , c 0.1, MeOH)).
Therefore, the structure of compound 2 was identified and named aspergiamide B.
 154.8°, c 0.1, MeOH), as that of compound 1 ([α] − 171.4°, c 0.1, MeOH)). Therefore, the
structure of compound 2 was identified and named aspergiamide B.

Mar. Drugs 2021, 19, 402 Table 1. 1H (600 MHz) and 13C NMR (150 MHz) data for compounds 1–3. 4 of 12

1a 2a 3a
Position
δC δH (J/Hz) δC δH (J/Hz) δC δH (J/Hz)
2 146.2, C Table 1. 1 H (600 MHz) and 13 C146.2,
NMR C (150 MHz) data for compounds 1–3. C
146.2,
3 104.3, C 104.3, C 104.6, C
1a 2a 3a
3a
Position 127.1, C 127.3, C 126.0, C
4 119.9, CHδC H (J/Hz)
7.24, dδ(7.9) 120.0,δCH
C δH d(J/Hz)
7.26, (7.8) 112.5, δCH
C d (J/Hz)
7.41,δH (8.0)
52 146.2, C
121.2, CH 7.08, m 146.2,
121.2, CHC 7.08, m 146.2,
122.4, CHC 7.12, m
63 104.3, C
122.5, CH 7.13, m 104.3,
122.7, CHC 7.13, m 104.6,
121.1, CHC 7.07, m
3a 127.1, C 127.3, C 126.0, C
74 112.6,119.9,
CH CH 7.43, 7.24,
d (8.0)
d (7.9)
112.6, CH
120.0, CH
7.42, d (8.0)
7.26, d (7.8)
120.2, CH
112.5, CH
7.31, d (7.9)
7.41, d (8.0)
7a5 136.8,121.2,
C CH 7.08, m 136.9, C
121.2, CH 7.08, m 136.7,
122.4,CCH 7.12, m
86 114.5,122.5,
CH CH 7.22,7.13,
s m 114.7, CH
122.7, CH 7.22,
7.13, sm 114.6, CHCH
121.1, 7.23, sm
7.07,
97 112.6,
124.6, C CH 7.43, d (8.0) 112.6,
124.5, C CH 7.42, d (8.0) 127.3, CCH
120.2, 7.31, d (7.9)
107a 136.8,
162.5, C C 136.9,
162.5, C C 136.7,
160.9, CC
8 114.5, CH 7.22, s 114.7, CH 7.22, s 114.6, CH 7.23, s
4.72, dd
129 124.6, C
56.8, CH 4.21, t (5.4) 124.5,
56.1, CHC 4.25, t (5.6) 127.3,
58.7, CH C
10 162.5, C 162.5, C 160.9, C (11.7, 5.9)
13 168.1, C 167.5, C 168.2, C 4.72, dd
12 56.8, CH 4.21, t (5.4) 56.1, CH 4.25, t (5.6) 58.7, CH
15 40.5, C 40.5, C 40.5, C (11.7, 5.9)
13 168.1, C 167.5, C
15a 28.1, CH 3 1.55, s 28.1, CH3 1.55, s 28.0,168.2,
CH3C 1.56, s
15 40.5, C 40.5, C 40.5, C
15b
15a 28.3, CH
28.1,3 CH 1.54, 1.55,
s s 28.2,
28.1,CH
CH3 1.54,
1.55,ss 28.3, CH
28.0, CH3 1.54, s s
1.56,
3 3 3
15b 28.3, CH3 6.11, dd
1.54, s 28.2, CH3 6.11, dds
1.54, 28.3, CH3 1.54, s
16 146.0, CH 146.0, CH 146.2, CH 6.11, dd (17.2,10.8)
(17.3, 10.6)
6.11, dd (17.3, 10.6)
6.11, dd 6.11, dd
16 146.0, CH 146.0, CH 146.2, CH
17 112.7,CH2 (17.3,
5.11, m 10.6) 112.7, CH2 (17.3,
5.11, m10.6) 112.5, CH2 (17.2,10.8)
5.11, m
17 112.7,CH2 5.11, m 112.7, CH 5.11, m 112.5, CH 5.11, m
18 32.6, CH2 1.95, m 31.0, CH2 2 2.21, m 39.0, CH2 2 2.11, m
18 32.6, CH2 1.95, m 31.0, CH2 2.21, m 39.0, CH2 2.11, m
2.34, dddd
2.34,
2.03, 2.03,
m m
(13.0, 5.9)
(13.0, 5.9)
1919 28.4, CH
28.4,2 CH2 1.67, 1.67,
m m 30.2, CH
30.2, CH
2 2 2.48,
2.48,m
m 68.6, CHCH
68.6, 4.51, mm
4.51,
1.74, 1.74,
m m
20 62.5, CH2 3.63, t (6.3) 176.3, C 55.6, CH2 3.53, d (13.2)
20 62.5, CH2 3.63, t (6.3) 176.3, C 55.6, CH2 3.53, d (13.2)
3.92, dd
3.92, dd4.9)
(13.2,
a (13.2, 4.9)
Measured in Methanol-d4 .
a Measured in Methanol-d4.

Figure 2. Key HMBC and 1H-1H COSY correlations of 1–6.

Figure 2. Key HMBC and 1H-1H COSY correlations of 1–6.

Compound 3 was isolated as a pale yellow powder. It gave a molecular formula

of C21 H23 O3 N3 , as established by the HRESIM and NMR data, showing 12 degrees of
unsaturation. The 13 C NMR and HSQC spectra displayed 21 carbon signals, consisting of
two methyls, three methylenes (one olefinic carbon), seven methines, and nine quaternary
carbons (including one carboxyl carbonyl carbon, two amide carbonyl carbons). Analysis
of the 1 H NMR spectrum of 3 revealed a set of adjacent aromatic protons at δH 7.07 (m),
 Compound
Compound 3 was3 isolated
was isolated as a pale yellow
as a pale powder.
yellow powder. It gave a molecular
It gave a molecular formula formulaof of
C21HC 23O3N3, as established by the HRESIM and NMR data, showing 12 degrees of unsat-
21H23O3N3, as established by the HRESIM and NMR data, showing 12 degrees of unsat-
uration. The 13The
uration. C NMR 13C NMR and HSQC and HSQC spectra displayed
spectra displayed21 carbon
21 carbon signals, consisting
signals, consistingof two of two
Mar. Drugs 2021, 19, 402 methyls, three methylenes (one olefinic carbon), seven methines, and nine quaternary car-5 of 12
methyls, three methylenes (one olefinic carbon), seven methines, and nine quaternary car-
bonsbons
(including
(includingone carboxyl
one carboxyl carbonyl carbon,
carbonyl two amide
carbon, two amide carbonyl
carbonyl carbons). Analysis
carbons). Analysisof of
the 1HtheNMR
1H NMR spectrum spectrumof 3 revealed
of 3 revealed a set aofsetadjacent aromatic
of adjacent protons
aromatic at δHat
protons 7.07
δH (m),
7.07 7.12
(m), 7.12
(m), 7.12
7.31 (d,
(m),(m), J
7.317.31= 7.9),
(d, (d, 7.41,
J = J7.9),
= 7.9),
(d,
7.41, J = 8.0),
(d,(d,
7.41,
indicating
J =J =
8.0),
8.0),indicating
the
indicatingthe
occurrence
theoccurrence
of two ortho-substituted
occurrence of two ortho-substituted
ortho-substituted
benzene
benzenering.ring.
benzene FourFour
ring. olefinic
Four protons
olefinic
olefinic at δHat
protons
protons 5.11 (m),
atδHδH5.11 6.11
(m),
5.11 (m),(dd,
6.11 J =(dd,
(dd,
6.11 17.2, 10.8),
J = J17.2, 7.23
10.8),
= 17.2, (s), two
7.23
10.8), (s), two
7.23 (s),
methylenes
methylenes
two at δH
methylenes 2.11
at δH (m,
at2.11
δH (m,1H), 2.34
2.111H), (dd,
(m, 2.34 J
1H), (dd,= 13.0,
2.34 J(dd, 5.9),
= 13.0, 3.53
J =5.9), (d,
13.0,3.53 J
5.9), = 13.2),
(d,3.53 3.92
J = 13.2), (dd,
(d, J =3.9213.2),J = 3.92
(dd, 13.2,
J = (dd,
13.2,
4.9),J4.9),
two aliphatic
two
= 13.2, aliphatic
4.9), two methines
methines
aliphatic atmethines
δHat4.51δH (m)at δand
4.51 (m) 4.72
and
4.51 (m)(dd,
4.72 J 4.72
and(dd, = 11.7,
J(dd, 5.9).
= 11.7, All the
J = 5.9).
11.7, All above
5.9). the
Allabovedata
the data
above
H
weredata
indicative
werewere of
indicative a prenyl
of a prenyl
indicative containing
containing
of a prenyl 2, 3-disubstituted
2, 3-disubstituted
containing indole diketopiperazine
indole diketopiperazine
2, 3-disubstituted skeleton.
indole diketopiperazine skeleton.
The skeleton.
1H-1H COSY correlation
The 1H-1HThe 1 H-1 H COSY of H-18 (δH 2.11,
correlation (δHof 2.34)/H-19
H-18 (δH (δ H 4.51)/H-20 (δH 3.53, 3.92) sug-
2.11, (δ2.34)/H-19 (δ(δ
COSY correlation of H-18 2.11, 2.34)/H-19 H 4.51)/H-20 H H4.51)/H-20
3.53, 3.92) sug- (δH
gested
3.53,an
gested aliphatic
3.92)ansuggested fragment
aliphatic fragment of
an aliphatic–CH 2-CH-CH2–, combined with key HMBC correlations
fragment
of –CH 2-CH-CH of –CH2–, 2 -CH-CH2 –,
combined combined
with key HMBC with correlations
key HMBC
fromcorrelations
H-19H-19
from to C-12 from
to (δCH-19
C-12 58.7),
(δC to from
C-12from
58.7), H-18 to C-13
(δC 58.7),
H-18 from (δH-18
to C-13 C 168.2), from H-20 to C-10 (δC 160.9)
(δCto168.2),
C-13 (δ C 168.2),
from H-20from to C-10H-20(δto C-10
C 160.9)
suggested
(δ the aliphatic
C 160.9) suggested
suggested the aliphatic fragment
the fragment was connected
aliphatic fragment
was connected with diketopiperazine
was connected
with ring ring
with diketopiperazine
diketopiperazine at C-12 and
at C-12 ringandat
N-11C-12
to form
N-11 and aN-11
to form five-member
a to form aring.
five-member The chemical
five-member
ring. ring.shift
The chemical ofchemical
Theshift C-19
of C-19 68.6,
(δC/Hshift
(δ 4.51;
C/Hof C-19
68.6, CH),
(δC/H
4.51; together
CH), 68.6, 4.51;
together
withCH),
the HRESIMS
with together
the HRESIMS result,
with the indicated
HRESIMS
result, a hydroxyl
indicated result,
a hydroxyl was connected
indicated was a hydroxyl
connected with C-19.
was
with The geometry
connected
C-19. Thewith C-19.
geometry
of the
TheΔ8 double
geometry bond
of thewas ∆8 Z configuration
double bond wasaccording
Z to the
configuration
of the Δ8 double bond was Z configuration according to the chemical shift of H-8 (δH 7.23)chemical
according shifttoof H-8
the (δ H 7.23)shift
chemical
together
of H-8with
together the
(δHwith lack
7.23) of
thetogether
lack NOE effecteffect
with
of NOE between
the lack ofH-8
between NOE and
H-8 NH-14
effect
and between[17]. [17].
NH-14 TheH-8NOESY
andNOESY
The correlation
NH-14 [17]. The
correlation
observed
NOESY between
observed correlationH-12
between H-12 and
observedH-19
and H-19indicated
between that
H-12 and
indicated H-12
thatH-19and H-19
H-12indicated were
and H-19that on
were the
H-12 same
andsame
on the face
H-19of were
face of
the molecule
on
thethe same
molecule(Figure
face of4),the
(Figure implying
molecule
4), implying the existence
(Figure of two
4), implying
the existence of possible
the existence
two possiblerelative
of configurations.
two
relative possible relative
configurations.
Therefore,
Therefore,compound
configurations. compound 3 had3 only
Therefore, had one pair
compound
only one of pair enantiomers
3 had ofonly one (12S,
enantiomers 19S-3
pair (12S,
of or 12R,
enantiomers
19S-3 19R-3).
or 12R, (12S, The The
19S-3
19R-3). or
absolute
12R, configuration
19R-3). The of
absolute 3 was determined
configuration by
of 3 ECD
was calculation
determined
absolute configuration of 3 was determined by ECD calculation (Figure 3). The calculated (Figure
by ECD 3). The
calculationcalculated
(Figure 3).
dataTheof 12S, 19S-3
calculated showed
data of good
12S, 19S-3agreement
showed with
good the experimental
agreement
data of 12S, 19S-3 showed good agreement with the experimental ECD of 3. Thus, the with theECD of 3.
experimental Thus, ECDthe of 3.
structure
Thus, of
the3 was assigned
structure of 3 and
was named
assigned
structure of 3 was assigned and named aspergiamide C. aspergiamide
and named C.
aspergiamide C.

FigureFigure 3. Comparison
3. Comparison
Figure between
between the experimental
thebetween
3. Comparison experimental and calculated
and calculated
the experimental andECD ECD ECD
spectra
spectra
calculated of 3.
1 and
of 1 spectra
and of 13.and 3.

Figure 4. Key4.NOESY
Figure correlations
KeyNOESY
NOESY of 3 and
correlations of334.
and4.4.
Figure 4. Key correlations of and

Compound
Compound
Compound 4 was obtained
44 was
was as aas
obtained
obtained pale
as yellow
aa pale
pale powder
yellow
yellow powderwithwith
powder a molecular
with formula
aa molecular
molecular formula
formula
C21HCC O
2521 3N
H based on HRESIMS (Figure S22), indicating 11 degrees of unsaturation.
25O33N33based on HRESIMS (Figure S22), indicating 11 degrees of unsaturation.The
21H25
3 O N based on HRESIMS (Figure S22), indicating 11 degrees of The The
unsaturation.
13 C NMR and HMQC spectra (Table 2) showed signals for two amide carbons, four aro-

matic carbons, two olefinic carbons, three methylenes, three oxygen- or nitrogen-bonded
methines, two methyls, and five quaternary carbons, combined with a set of adjacent aro-
matic protons at δH 7.07 (m), 7.12 (m), 7.31 (d, J = 7.9), 7.41, (d, J = 8.0) in 1 H NMR spectrum,
suggesting high similarity to the known compound brevianamide W (9) [18]. However,
the degree of unsaturation for 9 was 12, one more than that of 4. Moreover, two olefinic
Mar. Drugs 2021, 19, 402 6 of 12

carbons [δC 112.5 (CH), 126.2 (C)] in 9 were replaced by two oxygen- or nitrogen-bonded
methines [δC 68.9 (CH), 62.7 (CH)] in 4 according to the 13 C NMR and HMQC spectra. The
1 H-1 H COSY correlations between OH-8 (δ 5.33)/H-8 (δ 5.30)/H-9 (δ 4.0) indicated a
H H H
-CH-CH-OH unit. The key HMBC (Table 2) correlations from OH-8 to C-8 (δC 68.9)/C-9
(δC 62.7)/C-3 (δC 111.1), from H-8 to C-10 (δC 164.4) /C-3a (δC 127.3), combined with the
chemical shift of C-8 [δC/H 68.9/5.30 (dd, J = 6.2, 3.5)], suggesting that ∆8 double bond of
9 was reduced, and a proton at C-8 was replaced by a hydroxyl group. Thus, the planar
structure of 4 was shown in Figure 1. The correlation between H-9 and H-12 in the NOESY
spectrum (Figure 3) suggested that they were located on the same side. Therefore, the
ECD spectra of the four possible absolute configurations (8R, 9S, 12S-4 or 8R, 9R, 12R-4;
8S, 9S, 12S-4 or 8S, 9R, 12R-4) were calculated (Figure 5). The experimental ECD curve
was consistent with the calculated configurations of 8R, 9S, 12S-4. Thus, the structure of
compound 4 was identified and named aspergiamide D.

Table 2. 1 H (600 MHz) and 13 C NMR (150 MHz) data for compounds 4–6.

4a 5b 6b
Position
δC δH (J/Hz) δC δH (J/Hz) δC δH (J/Hz)
1 10.5, s
2 140.7, C 143.6, C 77.2, CH 5.71, s
3 111.1, C 106.5, C 151.4, C
3a 127.3, C 128.7, C
4 110.3, CH 7.30, d (8.0) 112.4, CH 7.40, d (8.1)
5 118.0, CH 6.86, m 121.0, CH 7.03, m 148.4, C
6 120.2, CH 6.97, m 122.3, CH 7.09, m 128.2, CH 7.73, d (8.1)
7 121.6, CH 7.70, d (8.0) 121.2, CH 7.74, d (8.0) 136.0, CH 7.84, ddd
7a 135.0, C 136.6, C (8.0, 7.0, 1.4)
5.30, dd 7.56, ddd
8 68.9, CH 77.8, CH 5.60, d (2.4) 128.7, CH
(3.5, 6.2) (8.0, 7.0, 0.8)
4.0, dd 8.14, dd
9 62.7, CH 63.4, CH 4.39, s 127.6, CH
(4.7, 6.2) (1.1, 8.0)
10 164.4, C 165.8, C 121.6, C
11 161.8, C
3.86, dd
12 58.4, CH 60.1, CH 4.16, m
(6.9, 9.6)
5.37, dd
13 169.5, C 170.6, C 58.9, CH
(7.1, 8.4)
14 8.16, d (4.5) 171.8, C
3.37, dd
15 38.7, C 40.1, C 40.4, CH2
(7.1, 13.5)
3.44, dd
(8.4, 13.5)
15a 28.1, CH3 1.46, s 28.2, CH3 1.60, s
15b 28.2, CH3 1.44, s 28.5, CH3 1.57, s
6.16, dd 6.16, dd
16 146.3, CH 146.9, CH 128.4, C
(17.5, 10.5) (17.5, 10.5)
5.18, ddd
17 110.7, CH2 5.09, m 112.6, CH2 131.7, CH 7.06, d (8.4)
(17.5,10.5,0.8)
18 28.8, CH2 2.09, m 30.0, CH2 1.95, m 116.1, CH 6.64, d (8.4)
2.34, dd
1.73, m
(7.9, 3.4)
19 21.9, CH2 1.83, m 22.9, CH2 1.95, m 157.6, C
2.06, m
20 45.3, CH2 3.26, m 46.1, CH2 3.47, m 116.1, CH 6.64, d (8.4)
3.40, m 3.76, m
21 131.7, CH 7.06, d (8.4)
8-OH 5.33, d (3.5)
8-OCH3 56.9, CH3 3.17, s
a Measured in DMSO-d6 . b Measured in Methanol-d4 .
 planar structure of 4 was shown in Figure 1. The correlation between H-9 and H-12 in the
14 8.16, d (4.5) 171.8, C
NOESY spectrum (Figure 3) suggested that they were located on the same side. Therefore,
3.37, dd
15 38.7, C the ECD spectra of the40.1,
fourC possible absolute configurations
40.4, CH(8R,
2
9S, 12S-4 or 8R, 9R, 12R-
(7.1, 13.5)
4; 8S, 9S, 12S-4 or 8S, 9R, 12R-4) were calculated (Figure 5). The experimental
3.44, dd ECD curve
Mar. Drugs 2021, 19, 402 was consistent with the calculated configurations of 8R, 9S, 12S-4. Thus, the structure
(8.4, 13.5) 7 of 12 of
15a 28.1, CH3 1.46, s 28.2, CH 1.60, s
compound 4 was identified and named aspergiamide D.
3

15b 28.2, CH3 1.44, s 28.5, CH3 1.57, s

6.16, dd 6.16, dd
16 146.3, CH 146.9, CH 128.4, C
(17.5, 10.5) (17.5, 10.5)
17 110.7, CH2 5.09, m 112.6, CH2 5.18, ddd (17.5,10.5,0.8) 131.7, CH 7.06, d (8.4)
18 28.8, CH2 2.09, m 30.0, CH2 1.95, m 116.1, CH 6.64, d (8.4)
2.34, dd
1.73, m
(7.9, 3.4)
19 21.9, CH2 1.83, m 22.9, CH2 1.95, m 157.6, C
2.06, m
20 45.3, CH2 3.26, m 46.1, CH2 3.47, m 116.1, CH 6.64, d (8.4)
3.40, m 3.76, m
21 131.7, CH 7.06, d (8.4)
8-OH 5.33, d (3.5)
8-OCH3 56.9, CH3 3.17, s
Measured in DMSO-d6. b Measured in Methanol-d4.
a

Figure 5. Comparison between the experimental and calculated ECD spectra of 4.

Figure 5. Comparison between the experimental and calculated ECD spectra of 4.
Compound 5 was isolated as a light yellow powder. The molecular formula was es-
Compound 5 was isolated as a light yellow powder. The molecular formula was
tablished
Table 2.established
H (600 asMHz)
C22H27and
O3N133 according toMHz)
the HRESIMS, showing 114–6. degrees of unsaturation.
as C22 H27C O3NMR (150 data for compounds
1
N3 according to the HRESIMS, showing 11 degrees of unsaturation.
The 13C 13 NMR and HMQC spectra (Table 2) showed signals for seven quaternary carbons,
The C NMR and HMQC spectra (Table 2) showed signals for seven quaternary carbons,
4 afour methylenes, eight methines, and two 5 b methyls, together with a set of adjacent 6 b aromatic
Position four methylenes, eight methines, and two methyls, together with a set of adjacent aromatic
δC δH (J/Hz)
protons at δH 7.03 (m), 7.09 δC (m), 7.40 (d, Jδ=H 8.1),
(J/Hz) 7.74 (d, J = 8.0)δin δH (J/Hz)
C 1H NMR spectrum,
protons at δH 7.03 (m), 7.09 (m), 7.40 (d, J = 8.1), 7.74 (d, J = 8.0) in 1 H NMR spectrum,
1 indicating
10.5, s high similarity to 4, except one more methoxyl signal at δH 3.17 (s, 3H) and δC
indicating high similarity to 4, except one more methoxyl signal at δH 3.17 (s, 3H) and δC
2 140.7, C 56.9 was observed in the143.6, 1D NMRC NMR data spectra of 5. HMBC correlations 77.2, CH from 3-8 (δs
OCH5.71, H
56.9 was observed in the 1D data spectra of 5. HMBC correlations from OCH 3 -8 (δH
3 111.1, C 3.17) to C-8 (δ 77.8), from
3.17) to C-8 (δC 77.8),106.5,
C H-8 to C-3a (δ 128.7)/C-10 (δ
fromCH-8 to C-3a (δC 128.7)/C-10 (δC 165.8)
C C 165.8) indicated the
151.4, indicated
C methoxyl
the methoxyl
3a 127.3, C waswaslocated at C-8.
located The absolute
at C-8. 128.7, C configuration
The absolute configurationof 5 wasof 5tentatively assigned
was tentatively as 9R and
assigned 12Rand
as 9R
by the negative sign of its specific rotation ([α] − 22.1°, c 0.1, MeOH) which was compa-
4 110.3, CH 7.30,
12R dby(8.0)
the negative112.4,sign CH
of its specific7.40, d (8.1)
rotation ( [α]25 ◦
D − 22.1 , c 0.1, MeOH) which was
5 118.0, CH rable to
6.86, mthat of rubrumline121.0,F ([α]
CH − 22.7°) [19]. Based on the above configurations of C-
comparable to that of rubrumline F ([α]D 7.03,
25
− 22.7 m ) [19]. Based148.4,
◦ on theC above configurations
6 120.2, CH 9 and C-12, compound 5 gave only one pair of enantiomers (8R, 9R, 12R-5 or 8S, 7.73,12R-
9R,
of6.97,
C-9mand C-12, compound122.3, CH 5 gave only 7.09,
onempair of enantiomers128.2, CH(8R, 9R, 12R-5 dor(8.1)
8S,
7 121.6, CH 5). Consequently,
7.70, d (8.0) the calculated
121.2, CH ECD (Figure
7.74,6)d for 8R,
(8.0) 9R, 12R-5 showed
136.0, CH good agreement
7.84, ddd
9R, 12R-5). Consequently, the calculated ECD (Figure 6) for 8R, 9R, 12R-5 showed good
with the experimental ECD of 5. Thus, the structure of 5 was identified and named asper-
7a 135.0, C agreement with the 136.6, C
experimental ECD of 5. Thus, the structure of 5 was identified (8.0, 7.0, and
1.4)
giamide E.
named
5.30, ddaspergiamide E. 7.56, ddd
8 68.9, CH 77.8, CH 5.60, d (2.4) 128.7, CH
(3.5, 6.2) (8.0, 7.0, 0.8)
4.0, dd 8.14, dd
9 62.7, CH 63.4, CH 4.39, s 127.6, CH
(4.7, 6.2) (1.1, 8.0)
10 164.4, C 165.8, C 121.6, C
11 161.8, C
3.86, dd
12 58.4, CH 60.1, CH 4.16, m
(6.9, 9.6)
13 169.5, C 170.6, C 58.9, CH 5.37, dd

Figure 6. Comparison
Figure between
6. Comparison the the
between experimental andand
experimental calculated ECD
calculated spectra
ECD of 5.of 5.
spectra

Compound 6 was obtained as a white powder. It gave a molecular formula of

C18 H15 O4 N3 , as established by HRESIMS and NMR data, indicating 13 degrees of unsatu-
ration. The 13 C NMR spectrum of 6 showed one methylene signal, ten methines (including
eight aromatic methines and one oxygenated methine), and seven quaternary carbons.
Analysis of the 1 H NMR spectrum of 6 (Table 2) revealed a set of adjacent aromatic protons
at 7.56, (dd, J = 4.0, 11.1), 7.73 (d, J = 8.1), 7.84 (m), and 8.14 (dd, J = 1.1, 8.0), and a set of
opposite aromatic protons at δH 6.64 (d, J = 8.4, 2H), 7.04 (d, J = 8.4, 2H), indicating the
occurrence of one ortho-substituted benzene ring and one para-substituted benzene ring.
The 1 H-1 H COSY spectrum of 6 showed cross-peaks that connect the aromatic protons
from H-6 (δH 7.73) through H-9 (δH 8.14), while HMBC correlations from H-6 to C-10
(δC 121.6), from H-9 to C-5 (δC 128.2)/C-11 (δC 161.8) suggested a quinazoline ring. The
 opposite aromatic protons at δH 6.64 (d, J = 8.4, 2H), 7.04 (d, J = 8.4, 2H), indicating the
occurrence of one ortho-substituted benzene ring and one para-substituted benzene ring.
The 1H-1H COSY spectrum of 6 showed cross-peaks that connect the aromatic protons
from H-6 (δH 7.73) through H-9 (δH 8.14), while HMBC correlations from H-6 to C-10 (δC
Mar. Drugs 2021, 19, 402 8 of 12
121.6), from H-9 to C-5 (δC 128.2)/C-11 (δC 161.8) suggested a quinazoline ring. The
quinazoline ring was further fused with a pyrazine to form a tricyclic pyrazino-
quinazolinedione [20], which was consistent with HMBC correlations from H-13 (δH 5.37)
quinazoline
to C-11/C-3 (δCring was further
151.4)/C-14 fused with
(δC 171.8), fromaH-2pyrazine to form
(δH 5.71) a tricyclic
to C-3/C-14. pyrazinoquina-
Moreover, the linkage
between the para-substituted benzene ring and pyrazinoquinazolinedione moietytoby C-
zolinedione [20], which was consistent with HMBC correlations from H-13 (δ H 5.37)
15 C-11/C-3 (δC 151.4)/C-14 (δC 171.8), from H-2 (δH 5.71) to C-3/C-14. Moreover, the linkage
was deduced based on HMBC correlations from H-13 to C-6, from H-17/H-19 to C-15
between the para-substituted benzene ring and pyrazinoquinazolinedione moiety by C-15
(Figure 2). The planar structure of 6 showed a close similarity to that of brevianamide M
was deduced based on HMBC correlations from H-13 to C-6, from H-17/H-19 to C-15
(15)(Figure
[21], with
2). The theplanar
mainstructure
differenceof 6being
showedonea close
moresimilarity
hydroxyltoin 6, of
that which was deduced
brevianamide
based on [21],
M (15) the lack
with ofthean aromatic
main proton,
difference beingand
onethe
moredownfield
hydroxyl shift of C-19
in 6, which was(δCdeduced
157.6) in 1D
NMRbasedspectra.
on the Compound 6 has two
lack of an aromatic chiral
proton, andcenters (C-2/C-13);
the downfield therefore,
shift of the ECD
C-19 (δC 157.6) in 1Dspectra
of four
NMRpossible absolute configurations
spectra. Compound 6 has two chiral(2S,13S-6 or 2R,13R-6;
centers (C-2/C-13); 2S,13R-6
therefore, theor
ECD2R,13S-6)
spectra were
of four possible
calculated as shown absolute configurations
in Figure (2S,13S-6 or 2R,13R-6;
7. The experimental ECD data 2S,13R-6 or 2R,13S-6)
was consistent withwerethe cal-
calculated as shown in Figure 7. The experimental ECD data was consistent
culated configurations of 2S,13S-6. Thus, the structure of compound 6 was identified with the and
calculated configurations of 2S,13S-6. Thus, the structure of compound 6 was identified
named aspergiamide F.
and named aspergiamide F.

Figure 7. Comparison between the experimental and calculated ECD spectra of 6.

Figure 7. Comparison between the experimental and calculated ECD spectra of 6.
In addition to the isolation of the above new compounds, ten known analogues
including
In additionbrevianamide Q (7) [22],
to the isolation of thebrevianamide
above new Rcompounds,
(8) [22], brevianamide
ten knownKanalogues
(9) [21], in-
brevianamide
cluding brevianamide Q (7)N-Prenyl-cyclo-
W (10) [18], L -tryptophyl-
[22], brevianamide L -proline
R (8) [22], (11) [23], brevianamide
brevianamide K (9) [21], brevi-
F (12) [24], epi-deoxybrevianamide E (13) [25], cyclo-(tryptophyl-phenylalanyl) (14) [26],
anamide W (10) [18], N-Prenyl-cyclo-L-tryptophyl-L-proline (11) [23], brevianamide F (12)
brevianamide M (15) [21] and brevianamide N (16) [21] were isolated and identified from
[24], epi-deoxybrevianamide E (13) [25], cyclo-(tryptophyl-phenylalanyl) (14) [26], brevi-
this fungus. Their structures were determined by comparing their NMR and MS data with
anamide M (15)
the reported in [21] and brevianamide N (16) [21] were isolated and identified from this
literatures.
fungus.Investigation
Their structures on thewere determined
biological by of
activities comparing
prenylatedtheir
DKPNMR and disclosed
alkaloids MS data withan- the
reported
tioxidantin activity
literatures.
in the literature [27,28]. It is reported that excessive accumulation of free
Investigation
radical on the biological
and synchronous reduced the activities of prenylated
antioxidant DKP alkaloids
defense mechanisms and candisclosed
induce anti-
complications
oxidant activity ofin diabetes mellitus
the literature [15]. InItorder
[27,28]. to find potent
is reported antidiabetic
that excessive natural prod-
accumulation of free
ucts, all the compounds (1–16) were screened for the enzyme inhibitory
radical and synchronous reduced the antioxidant defense mechanisms and can induce activities against
α-glucosidase and PTP1B (Table 3). Compared with the positive control acarbose (408 µM),
complications of diabetes mellitus [15]. In order to find potent antidiabetic natural prod-
all of the tested compounds, except compounds 5, 12, and 13, showed good to moderate
ucts, all the compounds (1–16) were screened for the enzyme inhibitory activities against
inhibitory activity to α-glucosidase. Especially, compounds 1 and 9 exhibited significant
α-glucosidase
α-glucosidaseand PTP1Bwith
inhibitory (Table
IC503). Compared
values of 18.2 andwith
7.6the
µM,positive control
respectively. acarbose (408
Compounds
μM), all 11,
3, 10, of the
andtested compounds,
15 showed moderate except compounds
α-glucosidase 5, 12, and
inhibition with13,
ICshowed good to mod-
50 values ranging
from 40.7 to 83.9 µM. However, these compounds have no obvious PTP1B inhibition at a
concentration of 100 µg/mL (Table 3).
Mar. Drugs 2021, 19, 402 9 of 12

Table 3. Inhibitory potency of DPKs to α-glucosidase and PTP1B.

α-glucosidase PTP1B (Inhibition α-glucosidase PTP1B (Inhibition

Compd. Compd.
(IC50 /µM) Ratio/%) a (IC50 /µM) Ratio/%) a
1 18.2 29 10 40.7 <10
2 130.7 <10 11 37.5 <10
3 83.9 <10 12 1086.6 <10
4 144.2 <10 13 480.5 <10
5 1093.5 <10 14 353.2 <10
6 267.3 <10 15 67.8 <10
7 198.2 <10 16 362.6 <10
8 364.3 <10 Acarbose b 408.0 -
9 7.6 <10 Oleanolic acid c - 99.1
aThe inhibition ratio screening concentration was 100 µg/mL for the PTP1B test; b Acarbose was used as a positive control for the
α-glucosidase inhibitory test; c Oleanolic acid was used as a positive control for PTP1B inhibitory test.

3. Materials and Methods

3.1. General Experimental Procedures
HRESIMS data were recorded with a Finnigan LTQ-Orbitrap Elite (Thermo Fisher,
Waltham, MA, USA). NMR spectra were reported by Bruker AVANCE NEO 600 MHz
spectrometer (Bruker BioSpin, Fällanden, Switzerland). CD spectrum was obtained on a
Chirascan spectropolarimeter (Applied Photophysics, Leatherhead, UK). Optical rotation
was determined on an MCP 500 (Anton Paar, Austria). UV spectra were obtained on
a PERSEE TU-1990 spectrophotometer. Sephadex LH-20 (25−100 µm; GE Healthcare,
Sweden) and silica gel (200−300 mesh; Qingdao Shenghai Chemical Co. Ltd., Qingdao,
China) were used for CC. Thin-layer chromatography (TLC) was detected on the Silica gel
GF254 plate (Qingdao Marine Chemical Ltd., Qingdao, China).

3.2. Fungal Material

The fungus (16-5c) used in this research was isolated from the leaves of S. apetala, a
mangrove plant that was collected from Hainan Island, China. The fungus was identified
as Aspergillus sp. by the ITS region (deposited in GenBank, accession no JX993829) [29].

3.3. Fermentation, Extraction, and Isolation

The fungus Aspergillus sp. 16-5c was fermented on solid autoclaved rice medium
using eighty 1 L Erlenmeyer flasks; each contained 50 g rice and 50 mL 0.3% saline water,
culturing in room temperature under static condition for 28 days. The mycelia and solid rice
medium were extracted with methanol three times. The organic solvents were evaporated
under reduced pressure; we obtained 110 g of organic extract. The extract was isolated by
column chromatography over silica gel eluting with a gradient of petroleum ether/ethyl
acetate from 1/0 to 0/1 to afford four fractions (Fractions 1–5). Fraction 3 (253 mg) was
applied to silica gel CC and purified by Sephadex LH-20 CC to obtain compounds 7–10, 13,
and 16. Fraction 4 (203 mg) was applied to column chromatography over silica gel, eluting
with CH2Cl2/MeOH (100:1), and then further purified by Sephadex LH-20 CC eluted with
MeOH to give compounds 1–6, 11–12, and 14–15.
Aspergiamide A: pale yellow powder; [α]25 ◦
D − 171.4 (c 0.1, MeOH); (c 0.1,MeOH) ;
00

UV (MeOH) λmax (logε) 222 (3.30), 284 (0.90), 337 (1.05) nm; ECD λext (∆ε) (MeOH),
220 (−18.75), 240 (+13.50), 275 (+16.51), 340 (−10.18) nm; HRESIMS at m/z 366.1827
[M − H]− (calcd for C21 H24 N3 O3 , 366.1823). 1 H and 13 C NMR see Table 1.
Aspergiamide B: white powder; [α]25 ◦ 00
D − 154.8 (c 0.1, MeOH); (c 0.1,MeOH) ; UV
(MeOH) λmax (log ε) 224 (2.16), 284 (0.59),338 (0.76) nm; HRESIMS at m/z 382.1766
[M + H]+ (calcd for C21 H24 N3 O4 , 382.1761). 1 H and 13 C NMR see Table 1.
Aspergiamide C: pale yellow powder; [α]25 ◦
D + 64.5 (c 0.1, MeOH); (c 0.1,MeOH) ;
00

UV (MeOH) λmax (log ε) 225 (2.36), 284 (0.671), 338 (0.90) nm; ECD λext (∆ε) (MeOH),
210 (+38.95), 254 (35.40), 345 (+19.85) nm.; HRESIMS at m/z 364.1670 [M − H]− (calcd for
C21 H22 O3 N3 , 364.1666). 1 H and 13 C NMR see Table 1.
Mar. Drugs 2021, 19, 402 10 of 12

Aspergiamide D: pale yellow powder; [α]25 ◦

D + 78.5 (c 0.1, MeOH); (c 0.1,MeOH) ;
00

UV (MeOH) λmax (log ε) 213 (1.63) nm; ECD λext (∆ε) (MeOH), 217.5 (−11.51), 263 (+6.15)
nm; HRESIMS at m/z 366.1828 [M − H]− (calcd for C21 H24 O3 N3 , 366.1823). 1 H and 13 C
NMR see Table 2.
Aspergiamide E: light yellow powder; [α]25 ◦
D − 22.1 (c 0.1, MeOH); (c 0.1,MeOH) ;
00

UV (MeOH) λmax (log ε) 213 (2.13) nm; ECD λext (∆ε) (MeOH), 210 (−3.45), 245 (−1.12),
260 (−0.36), 271 (−0.78) nm; HRESIMS at m/z 404.1946 [M + Na]+ (calcd for C22 H27 O3 N3 Na,
404.1944). 1 H and 13 C NMR see Table 2.
Aspergiamide F: white powder; [α]25 ◦
D − 136.8 (c 0.1, MeOH); (c 0.1,MeOH) ; UV
00

(MeOH) λmax (log ε) 213 (2.13), 261 (0.67) nm; ECD λext (∆ε) (MeOH), 210 (+20.12), 237.3
(−20.05), 260 (−6.58), 305 (−9.85) nm; HRESIMS at m/z 336.0993 [M − H]− (calcd for
C18 H15 O4 N3 , 336.0989). 1 H and 13 C NMR see Table 2.

3.4. Antidiabetic Bioassays

The α-glucosidase inhibition assay was performed in a 96-well microplate reader
method as described in a previous report [24]. The α-glucosidase assay was performed at
37 ◦ C with a final volume of 200 µL. For each well, 10 µL samples (dissolved in DMSO), fol-
lowed by the addition of 50 µL phosphate buffer (pH 6.86), 20 µL 0.2 U/mL α-glucosidase
solutions and incubated for 5 min. 20 µL substrate p-nitrophenyl-α-D-glucopyranoside
(p-NPG) was added to each well to start the reaction. The reaction was incubated at 37 ◦ C
for 15 min and stopped by the addition of 50 µL 0.4 M Na2 CO3 . The α-Glucosidase activity
was determined by measuring the release of p-nitrophenol from p-NPG at 405 nm.
PTP1B inhibitory activity was determined by measuring the enzymatic hydrolysis
rate of p-nitrophenyl phosphate (p-NPP) as reported in the study [30]. This assay was
performed at 37 ◦ C with a final volume of 100 µL, using a Tris-HCl buffer (25 mM Tris-HCl,
1 mM EDTA, 1 mM DTT, pH 7.5). Samples were dissolved in 10 µL DMSO followed by the
addition of 30 µL buffer, 40 µL 0.075 U/µL PTP1B stock solution, and then added 20 µL
22.26 µg/µL p-NPP solution to start the reaction. After 30 min, the reaction ended by
adding 50 µL 2.5 M NaOH solution. The absorbance was measured at 405 nm.
The purity of the tested compounds by HPLC analysis for bioassay was 96% for 1,
96% for 2, 97% for 3, 98% for 4, 96% for 5, 98% for 6, 98% for 7, 98% for 8, 98% for 9, 99%
for 10, 96% for 11, 96% for 12, 97% for 13, 98% for 14, 97% for 15, and 95% for 16.

3.5. ECD Calculation

Conformational searches were carried out by means of the Spartań14 software using
the Molecular Merck force field (MMFF). All density functional theory (DFT) and time-
dependent (TD)-DFT calculations were performed with Gaussian 09 program. Conformers
within a 10 kcal/mol energy window were generated and optimized by DFT calculations at
the B3LYP/6-31+G (d, p) level. Conformers with a Boltzmann distribution of over 5% were
chosen for ECD calculations by the TD-DFT method at the B3LYP/6-31+G (d, p) level [31].
The polarizable continuum model for MeOH was used. The calculated ECD curves were
generated using the SpecDis 3.0 (University of Würzburg, Würzburg, Germany) and Origin
Pro 8.0 (Origin Lab, Ltd., Northampton, MA, USA) from dipole-length rotational strengths
by applying Gaussian band shapes with sigma = 0.30 eV.

4. Conclusions
In conclusion, chemical investigation of the mangrove endophytic fungus Aspergillus
sp. 16-5c led to isolation and identification of six new alkaloids, aspergiamides A–F (1–6),
with ten known analogs (7–16). The potential of DKPs alkaloids as antidiabetic agents
was evaluated by α-glucosidase inhibition and PTP1B inhibition assay. The results of
the α-glucosidase inhibition assay showed compounds 1 and 9 exhibited significant α-
glucosidase inhibitory with IC50 values of 18.2 and 7.6 µM, respectively. Meanwhile, all the
tested DKPs compounds showed no obvious PTP1B inhibition at 100 µg/mL concentration.
Mar. Drugs 2021, 19, 402 11 of 12

These outcomes expanded the chemical and biological diversity of DKPs alkaloids and
may provide new molecules for antidiabetic drug discovery.

Supplementary Materials: The HRESIMS and NMR spectrum (Figures S1–S61) are available online
at https://www.mdpi.com/article/10.3390/md19070402/s1.
Author Contributions: Conceptualization, Y.L.; methodology, G.Y. and C.H.; software, G.Y. and
C.H.; validation, Y.L.; formal analysis, G.Y., C.H., T.C.; investigation, G.Y., C.H., J.L., W.L., J.T.;
resources, Y.L.; data curation, G.Y., C.H., T.C.; writing—original draft preparation, G.Y. and C.H.;
writing—review and editing, Y.L. and G.Y.; supervision, Y.L.; project administration, Y.L.; funding
acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China, grant
number 41876153.
Data Availability Statement: Not applicable.
Acknowledgments: The authors gratefully acknowledge a grant from the National Natural Science
Foundation of China (No. 41876153).
Conflicts of Interest: The authors declare no conflict of interest.

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