A new cyclic hexapeptide and a new isocoumarin derivative from the marine sponge-associated fungus Aspergillus similanensis KUFA 0013

Article abstract of DOI:10.3390/md13031432

A new isocoumarin derivative, similanpyrone C (1), a new cyclohexapeptide, similanamide (2), and a new pyripyropene derivative, named pyripyropene T (3) were isolated from the ethyl acetate extract of the culture of the marine sponge-associated fungus Aspergillus similanensis KUFA 0013. The structures of the compounds were established based on 1D and 2D NMR spectral analysis, and in the case of compound 2 the stereochemistry of its amino acid constituents was determined by chiral HPLC analysis of the hydrolysate by co-injection with the D and L amino acids standards. Compounds 2 and 3 were evaluated for their in vitro growth inhibitory activity against MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A373 (melanoma) cell lines, as well as antibacterial activity against reference strains and the environmental multidrug-resistant isolates (MRS and VRE). Only compound 2 exhibited weak activity against the three cancer cell lines, and neither of them showed antibacterial activity.

Full text of DOI:10.3390/md13031432

Mar. Drugs 2015, 13, 1432-1450; doi:10.3390/md13031432
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
A New Cyclic Hexapeptide and a New Isocoumarin
Derivative from the Marine Sponge-Associated Fungus
Aspergillus similanensis KUFA 0013
Chadaporn Prompanya 1,2, Carla Fernandes 2,3, Sara Cravo 2,3, Madalena M. M. Pinto 2,3
,
Tida Dethoup 4, Artur M. S. Silva 5 and Anake Kijjoa 1,2,
*
1
ICBAS-Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto,
Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; E-Mail: chadaporn@buu.ac.th
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Rua dos Bragas 289,
4050-123 Porto, Portugal
2
3
Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP, Laboratório de Química
Orgânica, Departamento de Ciências Químicas, Faculdade de Farmácia, Rua de Jorge Viterbo
Ferreira, 228, 4050-313 Porto, Portugal; E-Mails: cfernandes@ff.up.pt (C.F.);
scravo@ff.up.pt (S.C.); madalena@ff.up.pt (M.M.M.P.)
4
5
Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10240,
Thailand; E-Mail: agrtdd@ku.ac.th
Departamento de Química & QOPNA, Universidade de Aveiro, 3810-193 Aveiro, Portugal;
E-Mail: artur.silva@ua.pt
* Author to whom correspondence should be addressed; E-Mail: ankijjoa@icbas.up.pt;
Tel.: +351-2-2042-8331; Fax: +351-2-2042-8090.
Academic Editor: Johannes F. Imhoff
Received: 27 January 2015 / Accepted: 9 March 2015 / Published: 17 March 2015
Abstract: A new isocoumarin derivative, similanpyrone C (1), a new cyclohexapeptide,
similanamide (2), and a new pyripyropene derivative, named pyripyropene T (3) were
isolated from the ethyl acetate extract of the culture of the marine sponge-associated
fungus Aspergillus similanensis KUFA 0013. The structures of the compounds were
established based on 1D and 2D NMR spectral analysis, and in the case of compound 2 the
stereochemistry of its amino acid constituents was determined by chiral HPLC analysis of
the hydrolysate by co-injection with the D and L amino acids standards. Compounds 2 and
3 were evaluated for their in vitro growth inhibitory activity against MCF-7 (breast
adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A373 (melanoma) cell lines,

Mar. Drugs 2015, 13
as well as antibacterial activity against reference strains and the environmental
1433
multidrug-resistant isolates (MRS and VRE). Only compound 2 exhibited weak activity
against the three cancer cell lines, and neither of them showed antibacterial activity.
Keywords: Aspergillus similanensis; cyclic hexapeptide; similanamide; isocoumarin;
similanpyrone C; pyripyropene T
1. Introduction
In recent years, there has been an increasing interest in marine-derived fungi as a target source of
bioactive marine natural products because many consider them among the world’s greatest untapped
resources for new biodiversity as well as chemodiversity [1–3]. Moreover, through established culture
methods, the compounds can be produced in quantity needed for medicinal chemistry development,
clinical trials and even marketing. Among the marine fungal strains investigated, the fungi of the genus
Aspergillus are the most prolific source of bioactive secondary metabolites, including sterols [4],
cerebrosides [5], sesquiterpenoids [6,7], sesterterpenoids [8,9], diterpenoids [10], meroterpenoids [11],
anthraquinone derivatives [12,13], nucleoside derivatives [14], indole alkaloids [15–17], prenylated
indole alkaloids [18–21], quinazolinone alkaloids [22,23], pyrrolidine alkaloids [8], and cyclic
peptides [24–28].
In our ongoing search for new natural products with antibacterial and anticancer activities produced
by the marine-derived fungi of the genera Neosartorya and Aspergillus, we have recently reported the
isolation of new isocoumarins similanpyrones A and B, a new chevalone (chevalone E), and a new
natural product pyripyrone S; besides the previously reported chevalone B and C, a meroterpenoid
S14-95, and pyripyropene E, from the crude ethyl acetate extract of the undescribed marine
sponge-associated fungus Aspergillus similanensis KUFA 0013 [29]. Reexamination of the
fractions remaining from the previous study of this fungus led to the isolation of a new
8-hydroxy-3-methylisocoumarin derivative, which we have named similanpyrone C (1), a new cyclic
hexapeptide, similanamide (2), and a new pyripyropene analog, pyripyropene T (3) (Figure 1).
Hydrolysis of compound 2, followed by HPLC analysis of its hydrolysate using a chiral column, led to
the elucidation of its amino acid constituents. Compounds 2 and 3 were evaluated for their
antibacterial activity as well their cytotoxicity against three human cancer cell lines.

Mar. Drugs 2015, 13
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12
18
11
9
1
4
8
5
2
3
7
10
11
8a
4a
10
16
15
6
13
15
9
17
14
12
14
8
13
1
6
19
5
4
7
2
21
20
5"
2"
4"
3"
6"
1"
3
1
22
2'
3'
6'
5'
34
23
4'
30
26
29
13
12
14
33
25
31
24
5
8
32
3
6
7
28
2
1
4
27
9
10
2
15
11
3
Figure 1. Secondary metabolites from Aspergillus similanensis KUFA0013.
2. Results and Discussion
Compound 1 was isolated as pale yellow viscous mass, and its molecular formula C19H20O6 was
established on the basis of the (+)-HRESIMS m/z 345.1342 [M + H]+, indicating ten degrees of
unsaturation. The IR spectrum showed absorption bands for hydroxyl (3443 cm−1), ketone carbonyl
(1730 cm−1), conjugated lactone carbonyl (1683 cm−1), olefin (1647 cm−1) and aromatic (1625, 1572 cm−1)
13
groups. The C NMR (Supplementary Information, Figure S3), DEPTs and HSQC spectra (Table 1,
Supplementary Information, Figure S4) revealed the presence of one ketone carbonyl (δC 205.3), one
conjugated ester carbonyl (δC 166.8), six quaternary sp2 (δC 160.0, 158.3, 153.4, 136.7, 110.0 and
99.6), two methine sp2 (δC 104.4 and 103.0), one ketal (δC 102.9), two sp3 methine (δC 67.2 and 33.9),
two sp3 methylene (δC 48.3 and 47.2), and three methyl (δC 21.6, 19.4 and 15.9) carbons. The 1H NMR
spectrum (Supplementary Information, Figure S1) revealed, besides a singlet of the hydrogen bonded
hydroxyl proton at δH 11.35, one doublet at δH 6.13 (J = 0.9 Hz) and one singlet at δH 6.26, two
multiplets at δH 4.15 and δH 1.98, two double double doublets at δH 2.48 (J = 14.7, 2.9, 1.9 Hz) and δH
2.28 (J = 14.7, 11.3, 0.7 Hz), two double doublets at δH 2.81 (J = 16.8, 5.6 Hz) and δH 2.55 (14.0, 1.9),
two broad doublets at δH 2.81 (J = 14.0 Hz) and δH 2.57 (J = 16.8 Hz), one methyl singlet at δH 2.24,
and two methyl doublets at δH 1.23 (J = 6.2 Hz) and δH 1.21 (J = 6.2 Hz).

Mar. Drugs 2015, 13
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Table 1. 1H and 13C NMR (CDCl3, 500.13 MHz and 125.8 MHz) and HMBC assignment for 1.
Position
δC, Type
166.8, CO
153.4, C
104.4, CH
136.7, C
103.0, CH
158.3, C
110.0, C
160.0, C
99.6, C
δH, (J in Hz)
COSY
HMBC
NOESY
1
3
-
-
4
6.13, d (0.9)
CH3-3
C-3, 5, 8a
CH3-3
4a
-
5
6.26, s
C-4, 6, 7, 8a
6
-
7
-
8
-
-
8a
9α
23.6, CH2
2.81, dd (16.8, 5.6)
2.57, brd (16.8)
1.98, m
H-9β, H-10
H-9α, H-10
C-6, 7, 10, 11
C-7, 10, 11
H-9β, H-10, CH3-10
H-9α, H-10, CH3-10
H-9α,H-9β, CH3-10
9β
10
33.9, CH
102.9, C
47.2, CH2
H-9α, H-9β, CH3-10
11
-
12α
12β
13
2.55, dd (14.0, 1.9)
2.81, brd (14.0)
-
H-12β
H-12α
C-10, 11, 13
C-10, 11, 13
H-12β
H-12α
205.3, CO
48.3, CH2
14α
14β
15
2.28, ddd (14.7, 11.3, 0.7)
2.48, ddd (14.7, 2.9, 1.9)
4.15, m
H-14β, H-15
H-14α, H-15
H-14α, H-14β, CH3-15
H-4
C-13, 15
C-13
H-14β, CH3-15
H-14β, CH3-15
H-14β, CH3-15
H-4
67.2, CH
19.4, CH3
15.9, CH3
21.6, CH3
-
CH3-3
CH3-10
CH3-15
OH-8
2.24, s
C-3, 4
1.23, d (6.2)
1.21, d (6.2)
11.35, s
H-10
C-9, 10, 11
C-14, 15
H-9β, H-10, H-12α, 2β
H-14α, H-14β, H-15
H-15
C-7, 8, 8a

Mar. Drugs 2015, 13
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1
Analysis of the H, 13C NMR, HSQC and HMBC spectra (Table 1) revealed the presence of a
6,7-disubstituted 3-methyl-1H-isochromen-1-one nucleus, similar to that of similanpyrone B [29].
Thus, another portion of the molecule consisted of one ketone (δC 205.3), one ketal (δC 102.9), one
methine (δH 1.98, m; δC 33.9), one oxymethine (δH 4.15, m; δC 67.2), three methylene (δH 2.81, dd,
J = 16.8, 5.6 Hz and δH 2.57, brd, J = 16.8 Hz, δC 23.6; δH 2.81, brd, J = 14.0 Hz and 2.55, dd,
J = 14.0, 1.9 Hz; δC 47.2; δH 2.48, ddd, J = 14.7, 2.9, 1.9 Hz and 2.28, ddd, J = 14.7, 11.3, 0.7 Hz,
δC 48.3), two methyl (δH 1.21, d, J = 6.2 Hz; δC 21.6 and δH 1.23, d, J = 6.2 Hz, δC 15.9) groups. That
this portion was 2,10-dimethyl-1,7-dioxaspiro[5.5]undec-8-en-4-one was evidenced by the COSY
correlations (Table 1, Supplementary Information, Figure S2) of H2-9 (δH 2.81, dd, J = 16.8, 5.6 Hz
and 2.57, brd, J = 16.8 Hz) to H-10 (δH 1.98, m), of H-10 to CH3-10 (δH 1.23, d, J = 6.2 Hz), of H2-14
(δH 2.48, ddd, J = 14.7, 2.9, 1.9 Hz and 2.28, ddd, J = 14.7, 11.3, 0.7 Hz) to H-15 (δH 4.15, m), and of
H-15 to CH3-15 (δH 1.21, d, J = 6.2 Hz), as well as by the HMBC cross peaks (Table 1, Supplementary
Information, Figure S5) of H2-9 to C-10 and C-11 (δC 102.9), of H2-12 (δH 2.81, brd, J = 14.0 Hz
and 2.55, dd, J = 14.0, 1.9 Hz) to C-10, 11, 13 (δC 205.3), and of H-14 to C-13 and 15, respectively
(Figure 2). That the 1,7-dioxaspiro[5.5]undec-8-en-4-one ring system was fused with the
3-methyl-1H-isochromen-1-one nucleus, through C-8 and C-9 of the methyldihydropyran ring of the
former and C-6 and C-7 of the latter, was supported by the HMBC correlations of H2-9 to C-6, 7
(Table 1, Supplementary Information, Figure S5). Literature search revealed that 1 is a new compound,
and in compliance with our previous work, we have therefore named it similanpyrone C. Since 1 was
isolated as viscous mass, it was not possible to obtain suitable crystals for the X-ray analysis.
Consequently, the absolute configuration of C-10, C-11 and C-15 is still undetermined.
1
8
9
2
7
6
10
11
8a
4a
12
3
13
14
5
4
15
Figure 2. Key HMBC correlations of compound 1.
In an attempt to determine the stereochemistry of C-10 and C-15, we have sorted out the coupling
constants of both H2-9 and H2-14. The fact that H2-9 appeared as a double doublet at δH 2.81 with a
germinal coupling of 16.8 Hz, and a small coupling at δH 5.6 Hz, typical of axial-equatorial coupling,
and a broad doublet at δH 2.57 with a germinal coupling constant of 16.8 Hz, we concluded that
CH3-10 was in the β-axial position of the half-chair conformation of the tetrahydropyran ring. This was
corroborated by the correlations of H-10, which was in α equatorial position, to both H-9β (δH 2.57,
brd, J = 16.8 Hz) and H-9α (δH 2.81, dd, J = 16.8, 5.6 Hz) in the NOESY spectrum (Table 1,
Supplementary Information, Figure S6). On the contrary, one of H-14 appeared as a double double
doublet at δH 2.28, with a germinal coupling of 14.7 Hz and a diaxial coupling of 11.3 Hz, as well as a
small coupling (long range) of 0.7 Hz, while another appeared also as a double double doublet at

Mar. Drugs 2015, 13
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δH 2.48, with a germinal coupling of 14.7 Hz and an axial-equatorial coupling of 2.9 Hz, as well as a
small coupling (long range) of 1.9 Hz. These patterns of couplings revealed that CH3-15 was in α
equatorial position of the chair conformation of the tetrahydro-4H-pyran-4-one ring. This analysis was
corroborated by the NOESY spectrum (Table 1, Supplementary Information, Figure S6), which
exhibited a correlation only between H-15 and H-14β (δH 2.48, ddd, J = 14.7, 2.9, 1.9 Hz) and not
between H-15 and H-14α (δH 2.28, ddd, J = 14.7, 11.3, 0.7 Hz). This assignment was also supported by
the higher chemical shift value of H-14β than that of H-14α since the former is under the anisotropic
deshielding of the C-13 carbonyl group. Based on the same reasoning, we assigned the broad doublet
at δH 2.81 (J = 14.0 Hz) as H-12β, and the double doublet at δH 2.55 (J = 14.0, 1.9 Hz) as H-12α.
Consequently, the relative configuration of C-10 and C-15 was tentatively assigned as 10S* and 15R*.
The relative configuration of C-11 was tentatively assigned as 11S* based on the fact that the NOESY
spectrum (Table 1, Supplementary Information, Figure S6) did not exhibit any correlation between
H-10 and H-15. According to the molecular model, when the configuration of C-11 is S*, the
substituents on C-11 are arranged in a way that H-10 and H-15 are pointing toward the opposite
directions. On the contrary, the R* configuration of C-11 would have H-10 and H-15 close enough to
give a strong NOESY correlation.
Similanpyrone C (1) can be assumed to be derived from the acetate-malonate pathway
(Scheme 1). Cyclization and enolization of the pentaketide (I) leads to the formation of
6,8-dihydroxy-3-methylisocoumarin (II), which, after Claisen condensation with the tetraketide (III),
gives rise to IV. Enolization of the side chain, together with a formation of the hemiketal by the
phenolic hydroxyl group on C-6 of the isocoumarin nucleus and the ketone carbonyl of the side chain,
leads to the formation of a hemiketal V. Formation of the ketal and methylation by SAM in the side
chain finally gives rise to similanpyrone C (1).
Compound 2 was isolated as pale yellow viscous mass, and its molecular formula C34H52N6O6 was
established on the basis of the (+)-HRESIMS m/z 641.4053 [M + H]+, indicating twelve degrees of
unsaturation. The IR spectrum showed absorption bands for amine (3335 cm−1), carbonyl (1682,
13
1644 cm−1) and aromatic (1594, 1519 cm−1). The C NMR (Supplementary Information, Figure S8),
DEPTs and HSQC spectra (Table 2, Supplementary Information, Figure S10) revealed the presence of
six amide carbonyls (δC 174.3, 174.2, 170.7, 170.2, 169.3, 168.9), two quaternary sp2 (δC 137.0,
122.7), four methine sp2 (δC 131.7, 127.1, 123.9, 123.4), eight methine sp3 (δC 65.1, 61.4, 59.3, 50.9,
47.9, 29.9, 25.5, 24.4), six methylene sp3 (δC 52.5, 37.8, 36.2, 28.1, 27.4, 24.5) and eight methyl
1
(δC 37.9, 23.3, 23.2, 22.1, 21.7, 19.8, 18.4, 16.2) carbons. The H NMR spectrum (Table 2,
Supplementary Information, Figure S7) revealed, besides four NH signals at δH 7.43, d (J = 7.4 Hz),
7.64, d (J = 9.8 Hz), 8.02, d (J = 7.9 Hz) and 9.41, brs, the signals of the aromatic protons of the
1,2-disubstituted benzene ring at δH 7.20, dd (J = 7.7, 1.5 Hz), 7.13, ddd (J = 7.9, 7.9, 1.0 Hz), 7.47,
ddd, (J = 7.9, 7.9, 1.6 Hz) and 8.29, d (J = 8.3 Hz). That the 1,2-disubstituted benzene ring belonged to
the anthranilic acid residue was corroborated by the HMBC correlations of the NH signal at δH 9.41,
brs to the carbon signal at δC 123.9 (C-6), and of the double doublet at δH 7.20 (J = 7.7, 1.5 Hz, H-3) to
the carbons at δC 131.7 (C-5), 137.0 (C-7) and 170.2 (CO-1) (Table 2, Figure 3, Supplementary
Information, Figure S11). The anthranilic acid residue was linked to the valine residue, through the
amino group of the former and the carboxyl group of the latter, since the HMBC spectrum
(Supplementary Information, Figure S11) showed correlations of the NH signal at δH 9.41, brs to the

Mar. Drugs 2015, 13
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carbonyl carbon at δC 170.7 (C-8), of the methine proton at δH 4.32 dd, J = 7.4, 3.3 Hz (H-9) to the
methine carbon at δC 29.9 (C-10), the methyl carbon at δC 16.2 (C-12) and C-8, and of the NH signal at
δH7.43, d (J = 7.5 Hz) to C-9 (δC 59.3) and C-10 (Table 2, Figure 3). The presence of the leucine
residue was supported by the coupling system from CH-14 (δH 4.57, m; δC 50.9) through CH3-17
(δH 0.97, d, J = 6.5 Hz; δC 23.3) and CH3-18 (δH 0.88, d, J = 6.4 Hz; δC 21.1), and of NH at δH 8.02 d
(J = 7.9 Hz) to H-14, as observed in the COSY spectrum (Table 2, Supplementary Information,
Figure S9), as well as by the HMBC correlations of the NH signal at δH 8.02 d (J = 7.9 Hz) to C-14
(Table 2, Figure 3). That the valine residue was linked to the leucine residue was supported by the
HMBC cross peak between the NH signal of the former (δH7.43, d, J = 7.5 Hz) to the signal of the
carbonyl carbon (δC174.2, C-13) of the latter. In turn, the leucine residue was linked to the alanine
residue, as evidenced by the HMBC cross peaks of the NH signal of the former to the carbonyl carbon
signal (δC174.3, C-19) of the latter, and of the proton signal at δH 4.82, dd, J = 9.7, 7.3 Hz (H-20) to
C-19 and the methyl carbon at δC 18.4 (C-21), as well as by the COSY cross peaks of H-20 to CH3-21
(δH 1.29, d, J = 7.3 Hz), and of H-20 to NH at δH 7.64, d (J = 7.9 Hz). The presence of the N- methyl
leucine moiety was evidenced by the coupling system from H-23 (δH 3.49, dd, J = 9.0, 4.7 Hz) through
CH3-26 (δH 0.97d, J = 6.5; δH 23.2) and CH3-27 (δH 0.99, d, J = 6.5; δH 22.1), as observed in the COSY
spectrum (Table 2, Supplementary Informatscheion, Figure S9), as well as by the HMBC correlations
of H-23 to C-22 (δC 169.3) and CH3-28 (δC 37.9). Finally, the presence of the pipecolic acid residue
was supported by the coupling system from CH-30 (δH 3.71, dd, J = 11.3, 2.5; δC 61.4) through CH2-34
(δH 3.16, dd, J = 13.2, 2.3; 4.14, dd, J = 14.4, 2.4; δC 52.5), as observed in the COSY spectrum
(Table 2, Supplementary Information, Figure S9). Since both H-30 and CH3-28 gave HMBC cross
peaks to C-29 (δC 168.9), the pipecolic acid residue was linked to the N-methyl leucine residue through
the carboxyl group of the former and a nitrogen atom of the latter. Since 2 presents twelve degrees of
unsaturation, the nitrogen atom of the piperidine ring of the pipecolic acid residue was linked to the
carbonyl of the anthranilic acid residue. The proposed structure was supported by the NOESY
correlations which showed cross peaks of NH at δH 9.41, brs to H-9, CH3-12, of NH at δH 7.43
(d, J = 7.5 Hz) to H-9, CH3-11 (δH 1.06, d, J = 6.9 Hz), CH3-12, H-14, of NH at δH 8.02 (d, J = 7.9 Hz)
to H-14, H-15 (δH 2.02, m; 1.77, m), CH3-17, of NH at δH 7.64 (d, J = 7.9 Hz) to CH3-21, H-23,
CH3-28, of H-3 to H-34β (δH 4.14, dd, J = 14.4, 2.4 Hz), and of H-30 to CH3-28 (Table 2,
Supplementary Information, Figure S12). Combining this information, it was possible to conclude that
2 was cyclo (anthranilic acid-Val-Leu-Ala-N-methyl-Leu-pipecolic acid).

Mar. Drugs 2015, 13
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+
4
I
3
II
III
V
IV
SAM
VI
1
Scheme 1. Proposed biogenesis of similanpyrone C (1).

Mar. Drugs 2015, 13
1440
Table 2. 1H and 13C NMR (CDCl3, 500.13 MHz and 125.8 MHz) and HMBC assignment for 2.
Position
1
δC, Type
170.2, CO
122.7, C
127.1, CH
123.4, CH
131.7, CH
123.9, CH
137.0, C
-
δH, (J in Hz)
-
COSY
HMBC
NOESY
2
-
3
7.20, dd (7.7, 1.5)
7.13, ddd (7.9, 7.9, 1.0)
7.47, ddd (7.9, 7.9, 1.6)
8.29, d (8.3)
-
H-4
H-3, 5
H-4, 6
H-5
C-1, 5, 7
C-2, 6
H-34
4
Anthranilic acid
5
C-3, 7
6
C-2, 4
H-12
7
NH
8
9.41, brs
C-6, 7, 8
NH (Val), H-9, 12
170.7, CO
59.3, CH
29.9, CH
19.8, CH3
16.2, CH3
-
-
9
4.32, dd (7.4, 3.3)
2.68, m
H-10, NH
H-9, 11, 12
H-10
C-8, 10, 11, 12
H-10, 11
10
11
12
NH
13
14
15
16
17
18
NH
19
20
21
NH
H-9, 11, 12
Val
1.06, d (6.9)
0.94, d (7.0)
7.43, d (7.5)
-
C-9, 10, 12
C-9, 10, 11
C-9, 10, 13
H-10
H-9
H-9, 11, 12, 14
H-15, 18
174.2, CO
50.9, CH
36.2, CH2
24.4, CH
23.3, CH3
21.7, CH3
-
4.57, m
H-15, NH
H-14, 16
H-15, 17, 18
H-16
2.02, m; 1.77, m
1.77, m
Leu
Ala
0.97, d (6.5)
0.88, d (6.4)
8.02, d (7.9)
-
C-15, 16, 18
C-15, 16, 17
C-13, 19
H-16
H-14
NH (Ala), H-14, 15, 17
H-21
174.3, CO
47.9, CH
18.4, CH3
-
4.82, dd (9.7, 7.3)
1.29, d (7.3)
7.64, d (7.9)
H-21, NH
H-20
C-19, 21
C-19, 20
C-22
H-20
C-21, 23, 28

Mar. Drugs 2015, 13
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Table 2. Cont.
22
23
24
25
26
27
28
29
30
31
32
33
34α
34β
169.3, CO
65.1, CH
37.8, CH2
25.5, CH
23.2, CH3
22.1, CH3
37.9, CH3
168.9, CO
61.4, CH
28.1, CH2
24.5, CH2
27.4, CH2
52.5, CH2
-
3.49, dd (9.0, 4.7)
1.95, m; 2.20, m
1.65, m
H-24
H-23, 25
H-24, 26, 27
H-25
C-22, 24, 28, 29
N-Me Leu
0.97, d (6.5)
0.99, d (6.5)
3.20, s
C-24, 25, 27
C-24, 25, 26
C-23, 29
H-25
23, 30, 32, 34α
-
3.71, dd (11.3, 2.5)
2.05, m
H-31
C-1, 29
H-34α
H-30, 32
H-31, 33
H-32, 34
H-33
Pipecolic acid
2.07, m
1.56, m
3.16, dd (13.2 2.3)
4.14, dd (14.4, 2.4)
H-34β
H-34α

Mar. Drugs 2015, 13
1442
12
18
11
10
13
16
15
9
14
17
8
6
19
5
4
7
21
2
20
3
1
22
34
23
26
30
31
33
29
25
24
32
28
27
Figure 3. Key HMBC correlations of compound 2.
1
The H and 13C NMR data of compound 2 resembled those of PF1171C, a cyclic hexapeptide
isolated from extracts of the unidentified ascomycete OK-128 fermented with okara by Kai et al. [30],
and later by a total synthesis by Masuda et al. [31]; however, its value of optical rotation was
quite different from that of PF1171C [30,31]. Moreover, PF1171C was reported as white solid
(mp 138–140 °C) [31], while compound 2 is pale yellow viscous mass. This observation suggested that
compound 2 and PF1171C could be diastereomers.
The stereochemistry of the amino acid residues of compound 2 was determined by chiral HPLC
analysis of its acidic hydrolysate, using appropriate D and L amino acids standards, according to the
previously described method [32]. The enantioseparations of the standard amino acids were successfully
performed with the Chirobiotic T column under reversed-phase elution conditions [33]. Actually, the
teicoplanin selector of this column has several characteristic features that make it suitable for amino
acid analysis [33–35]. The elution order of the enantiomers of all the standard amino acids was
confirmed by injecting the solutions of the racemic or enantiomeric mixtures and then each enantiomer
separately at a flow rate of 1 mL/min (Supplementary Information, Figure S18). As predicted, the D
enantiomer was always more strongly retained than the corresponding L enantiomer on the Chirobiotic
T column [33]. Based on mix HPLC analyses of the acidic hydrolysate with standard amino acids
(co-injection) (Supplementary Information, Figure S19 and Table S1), compound 2 was elucidated
unambiguously as cyclo (anthranilic acid-L-Val-D-Leu-L-Ala-N-methyl-L-Leu-D-pipecolic acid).
Although the amino acid sequence of compound 2 is the same as that of PF1171C, the stereochemistry
of its amino acid constituents is different from that of the amino acids constituent of PF1171C. While
PF1171C contains D-Ala, L-Leu, D-Val, and L-pipecolic acid, compound 2 contains L-Ala, D-Leu,
L-Val and D-pipecolic acid. Thus compound 2 is a new compound, and we have named it similanamide.
It is interesting to note that Kai et al. [30] has firstly assigned the stereochemistry of the amino acid
constituents of PF1171C using the reversed phase HPLC analysis of the Marfey derivatives of the
amino acids. Since the retention times for the Marfey derivatives of D-Ala (19.4 min) and L-Ala

Mar. Drugs 2015, 13
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(20.0 min) were too close for resolution, they had wrongly assigned D-Ala for L-Ala. However, in our
chiral HPLC analysis using the Chirobiotic T column under reversed-phase elution conditions, not
only the retention time of L-Ala (7.16 min) was very different from that of D-Ala (9.36 min),
but also the retention times of the D/L pair of other amino acid constituents (Supplementary
Information, Table S1).
So far, only few cyclohexapeptides have been reported from marine-derived microorganisms.
Wu et al. [36] have isolated two cyclohexapeptides, nocardiamides A and B, from the culture broth of
the marine-derived actinomycete Nocardiopsis sp. CNX037, isolated from sediment. Cai et al. [37]
have isolated two anti-Mycobacterium tuberculosis cyclohexapeptides from a Streptomyces
hygroscopicus Strain, while Song et al. [38] reported isolation of three new cyclopeptides, desotamides
B–D, from the deep South China Sea-derived Streptomyces scopuliridis SCSIO ZJ46. To the best of
our knowledge, compound 2 is the first cyclopeptide containing D-pipecolic acid residue ever isolated
from marine fungi.
Compound 3 was isolated as pale yellow viscous mass, and its molecular formula C29H33NO8 was
established on the basis of (+)-HRESIMS m/z 524.2287 [M + H]+, indicating fourteen degrees of
unsaturation. The IR spectrum showed absorption bands for hydroxyl (3418 cm−1), ester carbonyl
(1732 cm−1), conjugated ester carbonyl (1667cm−1), olefin (1643 cm−1), aromatic (1557, 1507 cm−1).
The 13C NMR (Supplementary Information, Figure S15), DEPTs and HSQC spectra (Table 3,
Supplementary Information, Figure S16) revealed the presence of two ester carbonyl (δC 170.1 and
169.8), one conjugated carbonyl (δC 161.6), five quaternary sp2 (δC 160.2, 156.8, 146.1, 126.9, 100.3),
six methine sp2 (δC 151.3, 146.5, 132.8, 123.9, 109.4, 98.7), one oxyquaternary sp3 (δC 85.9), two
oxymethine sp3 (δC 75.7 and 72.8), one oxymethylene sp3 (δC 64.4), two quaternary sp3 (δC 40.3, 38.4),
one methine sp3 (δC 40.7), three methylene sp3 (δC 35.2, 27.3, 22.9), and five methyl (δC 23.8, 20.8,
1
20.5, 20.1 and 12.7) carbons. The general feature of 13C and H spectra of compound 3 closely
resembled those of pyripyropene S, previously isolated from the same fungus [29]. Analysis of the 1H
(Supplementary Information, Figure S13), 13C, HSQC and HMBC spectra (Table 3, Supplementary
Information, Figure S17) revealed the presence of the ring system comprising of the
decahydronaphthalene fused, on C-5 and C-6, with the 2H,5H-pyrano[4,3-b]pyran-5-one, which
connected to the pyridine ring through C-6′ of the former and C-3″ of the latter, similar to
pyripyropene S [29]. However, there were only two acetoxyl groups (δC 170.1, CO; δC 20.5, CH3, δH
2.00, s and δC 169.8, CO; δC 20.8, CH3; δH 2.00, s) in compound 3. That the acetoxyl groups were on
C-1 and C-11 was supported by the fact that the chemical shift values of H-1 (δH 4.64, t, J = 8.5 Hz)
and H-11 (δH 3.75, s) were very similar to those of pyripyropene S, whereas the chemical shift of H-7
(δH 3.85, dd, J = 10.6, 4.2 Hz) was nearly 1.4 ppm less than that of pyripyropene S. On the other hand,
the 13C chemical shift values of C-6 (δH 85.9) and C-8 (δC 27.4) of compound 3 were 2.00 and
3.00 ppm, respectively, higher than those of the corresponding carbons in pyripyropene S, while the
13C chemical shift value of C-7 (δC 75.5) of compound 3 was 2.00 ppm lower than that of C-7 of
pyripyropene S. Since H-7 appeared as a double doublet with coupling constants of 10.6 and 4.2 Hz,
the position of the hydroxyl group on C-7 was β. Thus, compound 3 is 7-deacetylpyripyropene S. In
order to prove the stereochemistry of compound 3, the NOESY experiment was carried out. As the
NOESY spectrum (Table 3) clearly exhibited correlations of CH3-15 to CH3-12, but not to H-1 and
H-9; of CH3-12 to CH3-14 and CH3-15, and of CH3-14 to CH3-12, but not to H-7 (Table 3,

Mar. Drugs 2015, 13
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Supplementary Information, Figure S18), the stereochemistry of compound 3 is the same as that of
pyripyropene S [29], i.e., 1S*, 4R*, 6S*, 7S*, 9R*, 10R*. Since it is a new compound we have named
it pyripyropene T.
Table 3. 1H and 13C NMR (DMSO, 300.13 MHz and 75.47 MHz) and HMBC assignment for 3.
Position
δC, Type
72.8, CH
22.9, CH2
35.2, CH2
38.4, C
δH, (J in Hz)
COSY
H-2
HMBC
NOESY
1
2
4.64, t (8.5)
1.79, m
H-1, 3
H-2
3
1.98, m
4
-
5
146.1, C
85.9, C
-
6
-
7
75.7, CH
27.3, CH2
40.7, CH
40.3, C
3.85, dd (10.6, 4.2)
H-8
H-7
H-8
8
1.70, m
9
1.48, m
10
11
12
13
14
15
2′
-
64.4, CH2
23.8, CH3
109.4, CH
20.1, CH3
12.7, CH3
161.6, C
100.3, C
160.2, C
98.7, CH
156.8, C
146.5, CH
126.9, C
132.8, CH
123.9, CH
151.3, CH
170.1, CO
20.5, CH3
169.8, CO
20.8, CH3
3.75, s
C-1, 9
C-3, 4, 5
H3-15
1.19, s
H3-14, 15
6.16, s
C-4, 6, 2″, 4″
C-5, 6, 7
1.45, s
H3-12
0.84, s
C-1, 9, 10, 11
H2-11, H3-12
-
3′
-
4′
-
5′
7.11, s
C-3′, 4′, 6′, 3″
C-3″, 6″
6′
-
2″
3″
4″
5″
6″
OAc-1
9.0, d (1.7)
H-4″
H-5′
-
8.25, dt (8.7, 2.2)
H-2″, 5″
H-4″, 6″
H-2″, 5″
H-5′, 5″
H-4″, 6″
H-5″
7.54, dd (7.9, 4.8)
C-3″
8.68, dd (4.8, 1.5)
C-2″, 4″
-
2.00, s
-
CO (Ac)
CO (Ac)
OAc-11
2.00, s
Since compound 1 was isolated in a very small amount, only compounds 2 and 3 were evaluated for
their cytotoxicity and antibacterial activity. Compounds 2 exhibited weak in vitro growth inhibitory
activity, by Sulforhodamine B (SRB) assay [39], against the MCF-7 (breast adenocarcinoma,
GI50 = 125 ± 0), NCI-H460 (non-small cell lung cancer, GI50 = 117.50 ± 3.55) and A373 (melanoma,
GI50 = 115 ± 7.07) cell lines. Compounds 2 and 3 were also tested for their antibacterial activity against
four reference strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas
aeruginosa), as well as the environmental multidrug-resistant isolates, according to the previously
described method [40], and neither of them showed activity (MIC values higher than 256 µg/mL).

Mar. Drugs 2015, 13
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3. Experimental Section
3.1. General Procedure
Melting points were determined on a Bock monoscope and are uncorrected. Optical rotations were
determined on an ADP410 Polarimeter (Bellingham + Stanley Ltd., Tunbridge Wells, Kent, UK).
Infrared spectra were recorded in a KBr microplate in a FTIR spectrometer Nicolet iS10 from Thermo
Scientific (Waltham, MA, USA.) with Smart OMNI-Transmission accessory (Software 188 OMNIC 8.3).
1H and 13C-NMR spectra were recorded at ambient temperature on a Bruker AMC instrument (Bruker
Biosciences Corporation, Billerica, MA, USA) operating at 500.13 and 125.8 MHz or at 300.13 and
75.4 MHz, respectively. High-resolution mass spectra were measured with a Waters Xevo QToF mass
spectrometer (Waters Corporations, Milford, MA, USA) coupled to a Waters Aquity UPLC system. A
Merck (Darmstadt, Germany) silica gel GF254 was used for preparative TLC, and a Merck Si gel 60
(0.2–0.5 mm) was used for analytical chromatography.
3.2. Extraction and Isolation
Isolation and identification of the fungus as well as fractionation of the crude extract of the culture
of A. similanensis KUFA0013 have been previously described by us [29]. Frs 185–196 were combined
(654 mg) and purified by TLC (Si gel, CHCl3:Me2CO:HCO2H, 97:3:0.1) to give 7.4 mg of 1.
Frs 310–327 were combined (1.19 g), applied on a Sephadex H-20 column (10 g) and eluted MeOH,
wherein ten sfrs of 1 mL were collected. Sfrs 1–7 were combined and purified by TLC (Si gel,
CHCl3:Me2CO:HCO2H, 19:1:0.01) to give 108 mg of 2. Frs 336–345 were combined (165 mg) and
purified by TLC (Si gel, CHCl3:Me2CO:HCO2H, 17:3:0.01) to give additional 60 mg of 2. Frs
354–398 were combined (1.15 g), applied on a Sephadex LH-20 column (10 g) and eluted with
MeOH, wherein thirty four sfrs of 1 mL were collected. Sfrs 7–15 were combined (150 mg) and
purified by TLC (Si gel, CHCl3:Me2CO:HCO2H, 4:1:0.01) to give 67 mg of pyripyropene S [29]. Frs
435–443 were combined (377 mg), applied on a Sephadex LH-20 column (10 g) and eluted with a
mixture of 1:1 v/v of CHCl3: MeOH, wherein fourteen sfrs of 1 mL were collected. Sfrs 8–11 (62 mg)
were combined and purified by TLC (Si gel, CHCl3:Me2CO:HCO2H, 19:1:0.01) to give 35 mg of 3.
3.2.1. Similanpyrone C (1)
20
Pale yellow viscous mass; [α]D = −80.0 (c 0.01, CHCl3); λmax (log ε) 239 (4.57), 245(4.59), 332
(2.10) nm; IR (KBr) νmax 3443, 2923, 2852, 1730, 1683, 1647, 1625, 1572, 1508, 1457, 1429, 1352,
1
1251 cm−1; H and 13C NMR (Table 1); HRESIMS m/z 345.1342 (M + H)+ (calculated for
C19H21O6, 345.1338).
3.2.2. Similanamide (2)
20
Pale yellow viscous mass; [α]D = +30.3 (CHCl3, c 0.03), IR (KBr) νmax 3335, 3054, 2958, 2870,
1
1682, 1644, 1594, 1519, 1449, 1292 cm−1; H and 13C NMR (Table 2); HRESIMS m/z 641.4053
(calculated for C34H53N6O6, 641.4027).

Mar. Drugs 2015, 13
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3.2.3. Pyripyropene T (3)
20
Pale yellow viscous mass; [α]D = +106 (CHCl3, c 0.03), IR (KBr) νmax 3418, 2949, 1732, 1667,
1
1643, 1557, 1507, 1480, 1246, 1028 cm−1; H and 13C NMR (Table 3); HRESIMS m/z 524.2287
(M + H)+, calcd for C29H34NO8, 524.2284.
3.3. Amino Acids Analysis of Acidic Hydrolysate of Compound 2
3.3.1. Acid Hydrolysis
The stereochemistry of the amino acids was determined by analysis of the acidic hydrolysate from
compound 2. Compound 2 (5.0 mg) was dissolved in 6 N HCl (5 mL) and heated at 110 °C, in a
furnace, for 24 h in a sealed glass tube. After cooling to room temperature, the solution was dried
under N2 for 24 h, reconstituted in methanol for HPLC-MS (200 µL), filtered through a 4 mm PTFE
Syringe Filter F2504-4 of 0.2 µm pore size (Thermo Scientific, Mumbai, India), and then analyzed by
HPLC equipped with a chiral column.
3.3.2. Chiral HPLC Analysis
The HPLC system consisted of Shimadzu LC-20AD pump, equipped with a Shimadzu DGV-20A5
degasser, a Rheodyne 7725i injector fitted with a 20 µL loop, and a SPD-M20A DAD detector (Kyoto,
Japan). Data acquisition was performed using Shimadzu LCMS Lab Solutions software, version 3.50
SP2. The chiral column used in this study was Chirobiotic T (15 cm × 4.6 mm I.D., particle size 5 µm)
manufactured by ASTEC (Whippany, NJ, USA). The mobile phase composition was
MeOH:H2O:CH3CO2H (70:30:0.02, v/v/v), all were LC-MS grade solvents obtained from
Sigma-Aldrich Co (St. Louis, MO, USA). The flow rate was 0.5 mL/min and the UV detection
wavelength was 210 nm. Analyses were performed at room temperature in an isocratic mode.
All standards of racemic amino acids and pure amino acid enantiomers were purchased from
Sigma-Aldrich Co (St. Louis, MO, USA). The elution order of the enantiomers of all the standards
amino acids was confirmed by injecting the solutions of the racemic or enantiomeric mixtures, and
then each enantiomer separately (or only L- amino acid in the case of N-methyl leucine) at a flow rate
of 1 mL/min or 0.5 mL/min. Working solutions of single enantiomeric amino acids were prepared by
dissolution in MeOH at the concentration of 1 mg/mL (10 µL sample injection), while the
enantiomeric mixtures were prepared by mixing equal aliquots of each enantiomer (20 µL sample
injection). Mix HPLC analyses of the acidic hydrolysate with standard amino acids (co-injection)
confirmed the stereochemistry of the amino acids of compound 2.
4. Conclusions
Following our first report of the isolation of new isocoumarin derivatives and merotepenoids from
the ethyl acetate crude extract of the culture of the undescribed marine sponge-associated fungus
Aspergillus similanensis KUFA 0013, we have reexamined its remaining column fractions and have
isolated a new isocoumarin derivative containing an unusual 1,7-dioxaspiro-undecenone moiety,
together with a new cyclohexapeptide and a new pyripyropene analog. Although several cyclopeptides

Mar. Drugs 2015, 13
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have been reported from many fungi of the genus Aspergillus, this is the first report of isolation of
cyclohexapeptide from the marine-derived fungus. The fact that these new cyclohexapeptide and
pyripyropene analog did not exhibit relevant antibacterial and the in vitro growth inhibitory activities
on human cancer cell lines does not mean that they are void of other interesting biological activities. In
order to prove this hypothesis, it is necessary to explore their potential in a broader biological or
pharmacological assay system.
Acknowledgments
This work was partially supported by the Project MARBIOTECH (reference NORTE-07-0124-
FEDER-000047) within the SR&TD Integrated Program MARVALOR—Building research and
innovation capacity for improved management and valorization of marine resources, supported by the
Programa Operacional Regional do Norte (ON.2—O Novo Norte), the European Regional Development
Fund, and FCT-Fundação para a Ciência e a Tecnologia under the project CEQUIMED-PEst-OE/
SAU/UI4040/2014. We thank Michael Lee of the Department of Chemistry, Leicester University (UK),
for providing the HRESIMS. C.P. thanks the Faculty of Pharmaceutical Sciences, Burapha University,
Thailand for her scholarship to the University of Porto. We thank Júlia Bessa for technical support.
Author Contributions
Chadaporn Prompanya performed isolation, purification and structure elucidation of some compounds;
Carla Fernandes and Sara Cravo performed HPLC analysis of amino acids by chiral column;
Tida Dethoup isolated, identified, cultured the fungi, and prepared the crude extract; Artur M.S. Silva
provided 1D and 2D NMR spectra. Madalena M.M. Pinto, and Anake Kijjoa conceived, designed the
research, elucidated the structure of the compounds and wrote the paper.
Conflicts of Interest
The authors declare no conflict of interest.
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