New cyclotetrapeptides and a new diketopiperzine derivative from the marine sponge-associated fungus Neosartorya glabra KUFA 0702

Article abstract of DOI:10.3390/md14070136

Two new cyclotetrapeptides, sartoryglabramides A (5) and B (6), and a new analog of fellutanine A (8) were isolated, together with six known compounds including ergosta-4, 6, 8 (14), 22-tetraen-3-one, ergosterol 5, 8-endoperoxide, helvolic acid, aszonalenin (1), (3R)-3-(1H-indol-3-ylmethyl)-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (2), takakiamide (3), (11aR)-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (4), and fellutanine A (7), from the ethyl acetate extract of the culture of the marine sponge-associated fungus Neosartorya glabra KUFA 0702. The structures of the new compounds were established based on extensive 1D and 2D spectral analysis. X-ray analysis was also used to confirm the relative configuration of the amino acid constituents of sartoryglabramide A (5), and the absolute stereochemistry of the amino acid constituents of sartoryglabramide A (5) and sartoryglabramides B (6) was determined by chiral HPLC analysis of their hydrolysates by co-injection with the D- and L- amino acids standards. Compounds 1-8 were tested for their antibacterial activity against Gram-positive (Escherichia coli ATCC 25922) and Gram-negative (Staphyllococus aureus ATCC 25923) bacteria, as well as for their antifungal activity against filamentous (Aspergillus fumigatus ATCC 46645), dermatophyte (Trichophyton rubrum ATCC FF5) and yeast (Candida albicans ATCC 10231). None of the tested compounds exhibited either antibacterial (MIC > 256 μg/mL) or antifungal activities (MIC > 512 μg/mL).

Full text of DOI:10.3390/md14070136

marine drugs
Article
New Cyclotetrapeptides and a New Diketopiperzine
Derivative from the Marine Sponge-Associated
Fungus Neosartorya glabra KUFA 0702
War War May Zin 1,2, Suradet Buttachon 1,2, Tida Dethoup 3, Carla Fernandes 2,4, Sara Cravo 2,4
,
Madalena M. M. Pinto 2,4, Luís Gales 1,5 José A. Pereira 1, Artur M. S. Silva 6, Nazim Sekeroglu 7
,
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; wwmzin.chem.yu@gmail.com (W.W.M.Z.);
nokrari_209@hotmail.com (S.B.); lgales@ibmc.up.pt (L.G.); jpereira@icbas.up.pt (J.A.P.)
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Rua dos Bragas 289,
4050-313 Porto, Portugal; cfernandes@ff.up.pt (C.F.); scravo@ff.up.pt (S.C.); madalena@ff.up.pt (M.M.M.P.)
Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, 10240 Bangkok, Thailand;
agrtdd@ku.ac.th
Laboratório de Química Orgânica, Departamento de Ciências Químicas, Faculdade de Farmácia,
Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228,
4050-313 Porto, Portugal
Departamento de Química & QOPNA, Universidade de Aveiro, 3810-193 Aveiro, Portugal; artur.silva@ua.pt
Medicinal and Aromatic Plant Programme, Plant and Animal Sciences Department, Vocational School,
Kilis Aralık University, 79000 Kilis, Turkey; nsekeroglu@gmail.com
2
3
4
5
6
7
*
Correspondence: ankijjoa@icbas.up.pt; Tel.: +351-220-428-331; Fax: +351-220-428-090
Academic Editor: Orazio Taglialatela-Scafati
Received: 17 June 2016; Accepted: 15 July 2016; Published: 20 July 2016
Abstract: Two new cyclotetrapeptides, sartoryglabramides A (5) and B (6), and a new
analog of fellutanine A (
ergosta-4, 6, 8 (14), 22-tetraen-3-one, ergosterol 5, 8-endoperoxide, helvolic acid, aszonalenin
), (3R)-3-(1H-indol-3-ylmethyl)-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione ( ), takakiamide ( ),
(11aR)-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione ( ), and fellutanine A
), from the ethyl acetate extract of the culture of the marine sponge-associated fungus Neosartorya
8) were isolated, together with six known compounds including
(
1
2
3
4
(7
glabra KUFA 0702. The structures of the new compounds were established based on extensive 1D and
2D spectral analysis. X-ray analysis was also used to confirm the relative configuration of the amino
acid constituents of sartoryglabramide A (
constituents of sartoryglabramide A ( ) and sartoryglabramides B (
analysis of their hydrolysates by co-injection with the D- and L- amino acids standards. Compounds
were tested for their antibacterial activity against Gram-positive (Escherichia coli ATCC 25922) and
5), and the absolute stereochemistry of the amino acid
5
6) was determined by chiral HPLC
1–8
Gram-negative (Staphyllococus aureus ATCC 25923) bacteria, as well as for their antifungal activity
against filamentous (Aspergillus fumigatus ATCC 46645), dermatophyte (Trichophyton rubrum ATCC
FF5) and yeast (Candida albicans ATCC 10231). None of the tested compounds exhibited either
antibacterial (MIC > 256 µg/mL) or antifungal activities (MIC > 512 µg/mL).
Keywords:
Neosartorya glabra; marine-derived fungus; Mycale sp.; cyclotetrapeptides;
sartoryglabramides A and B; diketopiperazines; fellutanine A epoxide
Mar. Drugs 2016, 14, 136; doi:10.3390/md14070136
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2016, 14, 136
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1. Introduction
Although the chemical constituents of the fungi of the genus Neosartorya, a teleomorphic state of
Aspergillus section Fumigatus, have not previously been intensively investigated [ ], there are currently
1
three reports on the secondary metabolites and their biological activities of Neosartorya glabra (Fennell
& Raper) Kozakiewicz. Jayasuriya et al. first described isolation of three new antibacterial bicyclic
lactones, glabramycins A–C, from N. glabra isolated from a soil sample collected from Candamia, Spain,
by antisense screening [
relative configuration of glabramycin B were achieved [
reverse prenylated indole derivatives, sartoryglabrins A–C, and their in vitro growth inhibitory activity
against three human cancer cell lines, from the Thai collection of a soil-derived N. glabra [ ]. Recently,
2]. However, it is only very recently that the synthesis and revision of the
3
]. Kijjoa et al. described isolation of three new
4
Liu et al. reported isolation of two new polyketides, neosarphenols A and B, together with six known
polyketides and two known meroterpenoids, from the crude ethyl acetate extract of N. glabra CGMCC
32286 [5]. During our ongoing search for bioactive secondary metabolites from members of the genus
Neosartorya and our pursuit for natural antibiotics from marine-derived fungi, we have investigated
the secondary metabolites of a Thai collection of N. glabra KUFA 0702, isolated from the marine
sponge Mycale sp., collected from the coral reef at Samaesarn Island in the Gulf of Thailand. The ethyl
acetate extract of its culture furnished three new compounds including two new cyclotetrapeptides,
sartoryglabramides A (
reported ergosta-4,6,8 (14), 22-tetraen-3-one [
aszonalenin ( ) [ ], (3R)-3-(1H-indol-3-ylmethyl)-3, 4-dihydro-1H-1,4-benzodiazepine-2,5-dione (
takakiamide ( ) [11], (11aR)-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione
) [12], and fellutanine A ( ) [13 14] (Figure 1). Compounds were tested for their antibacterial
5) and B (
6
), and a new analog of fellutanine A (
8
), in addition to the previously
], helvolic acid [ ],
) [10],
6], ergosterol 5, 8-endoperoxide [
7
8
1
9
2
3
(4
7
,
1–8
activity against Gram-positive (Escherichia coli ATCC 25922) and Gram-negative (Staphyllococus aureus
ATCC 25923) bacteria, as well as for their antifungal activity against filamentous (Aspergillus fumigatus
ATCC 46645), dermatophyte (Trichophyton rubrum ATCC FF5) and yeast (Candida albicans ATCC 10231).
2
1
4
3
31
31
32
32
16
27
30
21
24
22
20
25
23
29
29
33
33
26
25
28
28
30
30
29
28
34
2
34
2
19
22
23
26
1
1
24
27
3
3
18
10
21
10
8
8
7
7
17
16
9
4
9
4
20
12
15
19
6
6
5
5
11
12
11
18
17
13
13
14
14
6
5
15
16
4
10
9
8
2'
5
6
12
13
11
13'
3
2
1'
11'
8'
1
12'
3'
7'
6'
9'
4'
7
10'
5'
7
8
Figure 1. Secondary metabolites isolated from the ethyl acetate extract of the culture of N. glabra
KUFA 0702.

Mar. Drugs 2016, 14, 136
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2. Results and Discussion
Compound
5
was isolated as white crystals (mp, 146–148 ˝C), and its molecular formula
C30H30N4O4 was established on the basis of the (+)-HRESIMS m/z 511.2365 [M + H]+, indicating
eighteen degrees of unsaturation. The IR spectrum showed absorption bands for amine (3447 cm´1),
amide carbonyl (1655 cm´1) and aromatic (1622, 1587, and 1526 cm´1). The 13C NMR (Supplementary
Materials, Figure S10), DEPTs and HSQC spectra (Table 1, Supplementary Materials, 12) revealed
the presence of four amide carbonyls (δC 170.2, 169.9, 168.8, 166.5), four quaternary sp2 (δC 138.3,
137.3, 136.5, 124.8), fourteen methine sp2 [δC 130.4, 129.6 (2C), 129.1 (2C), 128.1 (2C), 128.0 (2C), 126.6,
126.3, 126.0, 122.4, 120.4], three methine sp3 (δC 62.2, 55.2, 54.4), and five methylene sp3 (δC 49.4, 37.1,
34.7, 28.3, 24.6). The 1H NMR spectrum (Table 1, Supplementary Materials, Figure S9) revealed three
NH signals at δH 9.40, s, 8.49, d (J = 7.8 Hz) and 7.41, d (J = 9.8 Hz), the signals of four aromatic
protons of anthranilic acid at δH 8.31, dd (J = 7.9, 0.5 Hz, H-6), 7.55, dd (J = 7.7, 1.3 Hz, H-3), 7.48,
ddd (J = 7.9, 7.9, 1.4 Hz, H-5) and 7.16, dd (J = 7.9, 7.7 Hz, H-4) [15]. The anthranilic acid residue was
linked to the phenylalanine residue, through the amino group of the former and the carboxyl group
of the latter, since the HMBC spectrum (Supplementary Materials, Figure S12) showed correlations
of the NH signal at δH 9.40, s (NH-8) to the carbonyl carbon at δC 168.8 (C-9), C-2 (δC 124.8), C-6
(
(
δC 120.4), of the methine proton at δH 4.36, ddd (J = 8.4, 7.8, 5.3 Hz, H-10) to C-9, C-11 (δC 34.7), C-12
δC 138.3), of the methylene protons at δH 2.97, dd (J = 13.9, 8.4 Hz, H-11a) and 3.23, dd (J = 13.9,
5.3 Hz, H-11b) to C-9, C-10 (δC 55.2), C-12, C-13/C-17 (δC 129.6). The COSY spectrum also showed
correlation (Supplementary Materials, Figure S11) of H-10 to H2-11 of this phenylalanine residue
(Table 1 and Figure 2). That this phenylalanine residue (Phe-I) was linked to another phenylalanine
residue (Phe-II) was corroborated by the COSY correlation of H-10 to the proton doublet at δH 8.49, d
(J = 7.8 Hz, NH-18), as well as by the HMBC correlations of NH-18 to C-10 and the carbonyl carbon at
δC 169.9 (C-19), of the methine proton signal at δH 4.58, ddd (J = 9.8, 8.9, 7.3 Hz, H-20) to C-19, C-21
(δC 37.1), C-22 (δC 137.3), of the methylene proton signals at δH 2.71, dd (J = 13.5, 8.9 Hz, H-21a)/2.94
dd (J = 13.5, 7.3 Hz, H-21b) to C-22, C-19, C-20 (δC 54.4), and C-23/C-27 (δC129.1) (Table 1 and Figure 2).
This was further supported by the COSY correlations of H-20 to H2-21 and the proton doublet at δH
7.41 (J = 9.8 Hz, NH-28). The existence of the proline residue was evidenced not only by the COSY
correlations of the double doublet at δH 4.20 (J = 9.8, 2.3 Hz, H-30; δC 62.2) to the multiplets at δH 1.54
and 2.12 (H2-31, δC 28.3), of the multiplet at δH 1.89 (H2-32; δC 24.6) to H2-31 and the double doublet
at δH 3.70 (J = 17.6, 9.6 Hz, H-33a; δC 49.4) and a multiplet at δH 3.63 (H-33b; δC 49.4) but also by the
HMBC correlations of H-30 to the carbon signals at δC 170.2 (CO-29), δC 28.3 (C-31) and δC 24.6 (C-32),
and of H-33a to C-30 (δC 62.2), C-32, of H2-31 to C-29 and C-30, respectively (Table 1 and Figure 2).
That the proline residue was connected to the Phe-II residue, through the carbonyl of the former and
the amino group of the latter, was corroborated by the HMBC correlation of NH-28 to CO-29. Since
there are only three NH signals, the nitrogen of the pyrrolidine ring of the proline residue was linked
to the carbonyl group (C-1) of anthranilic acid. This was corroborated by the HMBC correlations of H-3
to CO-1 (δC 166.5), and of NH-8 to C-2 (δC 124.8) and C-6 (δC 120.4) (Table 1 and Figure 2). Therefore,
combining this information, it was possible to conclude that 5 was cyclo (anthranilic acid-Phe-Phe-Pro).
1
Table 1. H and 13C NMR (DMSO, 500 MHz and 125 MHz) and HMBC assignment for 5.
Position δC, Type
δ , (J in Hz)
H
COSY
HMBC
Anthranilic acid
1
2
3
4
5
6
7
166.5, C
124.8, C
126.6, CH
122.4, CH
130.4, CH
120.4, CH
136.5, C
-
-
-
7.55, dd (7.7, 1.3)
7.16, dd (7.9, 7.7)
7.48, ddd (7.9, 7.9, 1.4)
8.31, dd (7.9, 0.5)
-
H-4
H-3, 5
H-4, 6
H-5
C-1, 5, 7
C-2, 6
C-3, 7
C-2, 4
NH-8
9.40, s
-
C-2, 6, 9

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Table 1. Cont.
Position δC, Type
δ , (J in Hz)
H
COSY
HMBC
Phe-I
9
10
11a
b
12
13
14
15
16
17
168.8, CO
55.2, CH
34.7, CH2
-
4.36, ddd (8.4, 7.8, 5.3)
2.97, dd (13.9, 8.4)
3.23, dd (13.9, 5.3)
-
H-11, NH-21
H-10, 11b
H-10, 11a
C-9, 11, 12
C-9, 10, 12, 13, 17
C-9, 10, 12, 13, 17
138.3, C
129.6, CH
128.0, CH
126.0, CH
128.0, CH
129.6, CH
-
7.08, dd (7.4, 1.4)
7.19, dd (7.4, 7.4)
7.18, dd (7.4, 7.4)
7.19, dd (7.4, 7.4)
7.08, dd (7.4, 1.4)
8.49, d (7.8)
H-14
H-14, 15
H-14, 16
H-15, 17
H-16
C-11, 15, 17
C-12, 16
C-13, 17
C-12, 14
C-11, 13, 15
C-10, 19
NH-18
H-20
Phe-II
19
20
21a
b
22
23
24
25
26
27
169.9, CO
54.4, CH
37.1, CH2
-
4.58, ddd (9.8, 8.9, 7.3)
2.71, dd (13.5, 8.9)
2.94, dd (13.5, 7.3)
-
H-21a, b
H-21b, 20
H-21a, 20
C-19, 21, 22
C-19, 20, 22, 23, 27
C-19, 20, 22, 23, 27
137.3, C
129.1, CH
128.1, CH
126.3, CH
128.1, CH
129.1, CH
-
7.14, dd (7.4, 1.4)
7.27, dd (7.4, 7.4)
7.23, dd (7.4, 7.4)
7.27, dd (7.4, 7.4)
7.14, dd (7.4, 1.4)
7.41, d (9.8)
H-24
H-23, 25
H-24, 26
H-25, 27
H-26
C-25, 27
C-22, 26
C-23, 27
C-22, 24
C-23, 25
NH-28
H-20
C-19, 20, 29
Pro
29
30
31a
b
170.2, CO
62.2, CH
28.3, CH2
-
4.20, dd (9.8, 2.3)
1.54, m
H-31a, b
H-30, 31b
C-29, 31, 32
-
2.12, m
H-30, 31a
C-29, 30
32
33a
b
24.6, CH2
49.4, CH2
1.89, m
3.70, dd (17.6, 9.6)
3.63, m
H-31a, b, 32a, b
H-32, 33b
C-30, 32
H-32, 33a
N-34
-
-
31
32
33
28
30
29
34
21
23
20
22
24
19
18
27
1
3
4
5
2
25
26
9
10
7
8
13
14
11
6
12
17
15
16
Figure 2. Key COSY (—) and HMBC (Ñ) correlations of compound 5.
5 was obtained as a suitable crystal for X-ray diffraction, the stereochemistry of its amino acid
Since
residues was tentatively determined by X-ray analysis, and the ORTEP view shown in Figure 3 revealed
that Phe-I, Phe-II and Pro have the same relative configuration. However, since the flack x parameter
(0.3) did not guarantee the absolute confidence of the absolute configurations, the stereochemistry
of the amino acid residues of
5 was confirmed by a chiral HPLC analysis of its acidic hydrolysate,

Mar. Drugs 2016, 14, 136
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using appropriate D- and L-amino acid standards, according to the previously described method [15].
The enantioseparations of the standard amino acids were successfully performed with the Chirobiotic
T column under reversed-phase elution conditions [16]. The elution order of the enantiomers
of all the standards amino acids was confirmed by injecting the solutions of the enantiomeric
mixtures and then each enantiomer separately at a flow rate of 1 mL/min (Supplementary Materials,
Table S1). As predicted, the D-enantiomer was always more strongly retained than the corresponding
L-enantiomer on Chirobiotic column [16]. The retention times (tR min) for standards amino acids,
using MeOH: H2O (80:20 v/v) as mobile phase, at a flow rate of 1.0 mL/min, and with UV detection set
at 210 nm, were L-Phe (3.8) and D-Phe (5.0), L-Pro (6.7) and D-Pro (20.1). Based on mix HPLC analyses
of the acidic hydrolysate with standard D- and L-amino acids (co-injection) (Supplementary Materials,
Figure S27 and Table S1), compound
5 was elucidated as cyclo (anthranilic acid-L-Phe-L-Phe-L-Pro).
Since compound 5 is a new compound, we have named it sartoryglabamide A.
Figure 3. Ortep view of compound 5.
Compound
peak at m/z 550.2501 [(+)-HRESIMS], corresponding to C32H32N5O4 (calcd. 550.2454). Therefore, the
molecular formula C32H31N5O4 was attributed to compound , which indicated twenty degrees of
unsaturation. Like compound , the IR spectrum of
showed absorption bands for amine (3417 cm´1),
amide carbonyl (1649 cm´1) and aromatic (3058, 1620, 1588, 1526 cm´1). With some exceptions,
the general features of the 1H and 13C spectra of compound . The 13C NMR
resembled those of
6
, which was also isolated as white solid (mp, 190–192 ˝C), exhibited the [M + H]+
6
5
6
6
5
(Supplementary Materials, Figure S15), DEPTs and HSQC spectra (Table 2, Supplementary Materials,
Figure S17) displayed signals of four carbonyls (δC 170.2, 170.1, 169.0, 166.4), six quaternary sp2
(
δC 137.4, 136.3, 136.0, 127.7, 125.2, 110.2), fourteen methine sp2 [δC 130.4, 129.0 (2C), 128.1 (2C), 126.5,
126.3, 124.0, 122.6, 120.8, 120.7, 118.5, 118.2, and 111.3], three methine sp3 (δC 62.1, 54.6, 54.3), and five
methylene sp3 (δC 49.4, 37.0, 28.3, 24.9, 24.6). Unlike compound , the 1H NMR spectrum of
(Table 2,
Supplementary Materials, Figure S15), exhibited four NH signals at δH 10.82, d (J = 1.8 Hz), 9.25, s, 8.42,
d (J = 7.9 Hz) and 7.38, d (J = 10.0 Hz). Similar to compound , the presence of the proline residue was
5
6
5
corroborated by the presence of the coupling system of the protons from H-33 to H2-36 [(δH 4.15, dd,
J = 9.0, 2.0 Hz, H-33; δC 62.1), δH 1.45 m and 2.09, m (H2-34; δC 28.3), δH 1.86 m (H2-35; δC 24.6), and δH
3.55 m and 3.67, m (H2-36; δC 49.4)] as well as by the HMBC correlation of H-33 to the carbonyl carbon
at δC 170.2 (C-32), while the presence of the phenylalanine residue was supported by the coupling
system from H2-24 (δH 2.66, dd, J = 13.6, 10.0 Hz, and 2.92, dd, J = 13.6, 6.4 Hz; δC 37.0) through
H-23 (δH 4.61, ddd, J = 10.0, 10.0, 6.4 Hz; δC 54.6) to NH-31 (δH 7.38, d, J = 10.0 Hz), as observed
in the COSY spectrum, as well as by the HMBC correlations from H-23 to C-24 (δC 37.0) and C-25
(δC 137.4), of H2-24 to C-23 (δC 54.6), C-25, C-26/30 (δC 129.0)(Table 2 and Figure 4). Like compound
5
, the HMBC correlation of the amine proton at δH 7.38, d (J = 10 Hz, NH-31) to C-32 confirmed the
linkage of the carbonyl group of the proline residue (C-32) to the amino group of the phenylalanine
residue (N-31). Similarly, the nitrogen of the pyrrolidine ring of the proline residue (N-37) was linked

Mar. Drugs 2016, 14, 136
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to the carbonyl group of anthranilic acid (C-1, δC 166.4). That one of the phenyl residues of
5 was
replaced by a tryptophan residue in 6 was substantiated by the presence of the indole system, which
was characterized by the coupling system of H-14 (δH 7.58, d, J = 7.9 Hz, δC 118.5) through H-17 (δH
7.34, d, J = 8.0 Hz, δC 111.3), as observed in the COSY spectrum (Table 2 and Figure 4, Supplementary
Materials, Figure S16), and also by the HMBC correlations from NH-19 (δH 10.82, brs) to C-12 (δC 110.2),
C-13 (δC 127.7), C-18 (δC 136.0) and C-20 (δC 124.0) (Table 2 and Figure 4, Supplementary Materials,
Figure S18), as well as of the ethylamino moiety, as evidenced by the coupling system from H2-11 (δH
3.14, dd, J = 14.7, 6.7 Hz and 3.32 dd, J = 14.7, 5.9 Hz; δC 24.9) through H-10 (δH 4.52, ddd, J = 7.9, 6.7,
5.9 Hz; δC 54.3) to NH-21 (8.42, d, J = 7.9 Hz) (Table 2 and Figure 4). That the tryptophan residue was
linked to the phenylalanine residue, through the amino group of the former and the carbonyl group of
the latter, was corroborated by the HMBC correlations of NH-21 to the carbonyl carbons at δC 170.1
(C-22) and 169.0 (C-9), as well as of H-10 to C-9, C-11, C-12 and C-22. Finally, the amino group of the
anthranilic acid residue was linked to the carbonyl group of the tryptophan residue was supported by
the HMBC correlations of NH-8 (δH 9.25, s) to C-2 (δC 125.2), C-6 (δC 120.7) and C-9. Therefore,
6 was
identified as cyclo (anthranilic acid-Trp-Phe-Pro).
1
Table 2. H and 13C NMR (DMSO, 500 MHz and 125 MHz) and HMBC assignment for 6.
Position
δC, Type
δ , (J in Hz)
H
COSY
HMBC
Anthranilic acid
1
2
3
4
5
6
7
166.4, CO
125.2, C
126.5, CH
122.6, CH
130.4, CH
120.7, CH
136.3, C
-
-
-
7.53, d (7.6)
7.16, dd (7.6, 7.6)
7.48, ddd (8.3, 7.6)
8.27, d (8.3)
-
H-4
H-3, 5
H-4, 6
H-5
C-1, 5, 7
C-2, 6
C-3, 7
C-2, 4
NH-8
9.25, s
C-2, 6, 9
Trp
9
10
11a
b
12
13
14
15
16
17
169.0, CO
54.3, CH
24.9, CH2
-
4.52, ddd (7.9, 6.7, 5.9)
3.32, dd (14.7, 5.9)
3.14, dd (14.7, 6.7)
-
H-11, NH-21
H-10, 11b
H-10, 11a
C-9, 11, 12, 22
C-9, 10, 12, 13, 20
C-9, 10, 12, 13, 20
110.2, C
127.7, C
118.5, CH
118.2, CH
120.8, CH
111.3, CH
136.0, C
-
-
7.58, d (7.9)
6.98, dd (7.9, 7.5)
7.06, dd (8.0, 7.5)
7.34, d (8.0)
-
10.82, brs
7.04, d (1.8)
8.42, d (7.9)
H-15
H-14, 16
H-15, 17
H-16
C-16, 18
C-13, 17
C-14, 18
C-13, 15
18
NH-19
20
H-20
NH-19
H-10
C-12, 13, 18, 20
C-13
124.0, CH
-
NH-21
C-9, 22
Phe
22
23
24a
b
25
26
27
28
29
30
170.1, CO
54.6, CH
37.0, CH2
-
4.61, ddd (10.0, 10.0, 6.4)
2.66, dd (13.6, 10.0)
2.92, dd (13.6, 6.4)
-
H-24a, b
H-23, 24b
H-23, 24a
C-24, 32
C-22, 23, 25, 26, 30
C-22, 23, 25, 26, 30
134.4, C
129.0, CH
128.1, CH
126.3, CH
128.1, CH
129.0, CH
-
7.10, dd (7.7, 1.0)
7.20, m
7.18, m
7.20, m
7.10, dd (7.7, 1.0)
7.38, d (10.0)
H-27
H-26, 28
H-27, 29
H-28, 30
H-29
C-25
C-25
C-28
C-25
C-32
NH-31
H-23
Pro
32
33
34a
b
35
36a
b
170.2, CO
62.1, CH
28.3, CH2
-
4.15, dd (9.0, 1.2)
1.45, m
2.09, m
1.86, m
3.55, m
3.67, m
-
H-34a, b
H-33, 34b
C-32
H-33, 34a
H-34a,b, 36a, b
H-35, 36b
24.6, CH2
49.4, CH2
H-35, 36a
N-37
-

Mar. Drugs 2016, 14, 136
7 of 15
34
35
36
31
33
37
25
32
26
24
23
22
27
1
30
3
2
28
21
29
9
7
4
8
14
10
11
15
5
13
6
12
20
16
19
18
17
Figure 4. Key COSY (—) and HMBC (Ñ) correlations of compound 6.
The absolute stereochemistry of the amino acid residues of compound
6 was also determined
by chiral HPLC analysis of its acidic hydrolysate, using appropriate D- and L-amino acids standards.
The retention times (tR min) for standards amino acids, using MeOH: H2O (80:20 v/v) as mobile phase,
at a flow rate of 1.0 mL/min, and with UV detection set at 210 nm, were L-Phe (3.8) and D-Phe (5.0),
L-Pro (6.7) and D-Pro (20.1), L-Trp (4.5) and D-Trp (5.2). Based on mix HPLC analyses of the acidic
hydrolysate with standard D- and L-amino acids (co-injection) (Supplementary Materials, Figure S28,
Table S1), compound
6 is also a new compound, we have named it sartoryglabamide B.
Compound was isolated as pale yellow viscous mass, and its molecular formula C22H20N4O3
6 was elucidated as cyclo (anthranilic acid-L-Trp-L-Phe-L-Pro). Since compound
8
was established on the basis of the (+)-HRESIMS m/z 389.1626 [M + H]+, indicating fifteen degrees
of unsaturation. The IR spectrum showed absorption bands for amine (3420 cm´1), amide carbonyl
(1649 cm´1) and aromatic (1418 cm´1). The 13C NMR (Supplementary Materials, Figure S22), DEPTs
and HSQC spectra (Table 3, Supplementary Materials, Figure S25) revealed the presence of two amide
carbonyls (δC 169.8 and 167.7), five quaternary sp2 (δC 148.4, 136.0, 131.1, 127.4, 109.5), nine methine
sp2 (δC 128.9, 124.1, 122.5, 120.9, 118.5, 118.3, 117.8, 111.3, 109.8), one oxygen bearing quaternary
sp3 (δC 85.9), one oxygen bearing methine sp3 (δC 84.0), two methine sp3 (δC 58.6, 55.1) and two
methylene sp3 (δC 41.3, 24.7). The 1H NMR spectrum (Table 3, Supplementary Materials, Figure S21),
exhibited, besides four NH signals at δH 10.88, brd (J = 1.4 Hz), 7.72, brs, 6.68, brs, and 6.05, s, and,
in conjunction with the COSY and HSQC spectra (Table 3, Supplementary Materials, Figures S23
and S24), the proton signals of two 1,2-disubstituted benzene rings at δH 7.60, d (J = 7.9 Hz, H-4;
δC 118.5), 7.33, d (J = 7.9 Hz, H-7, δC 111.3), 7.07, ddd (J = 7.9, 7.9, 1.1 Hz, H-6, δC 120.9), 6.99, ddd
(J = 7.9, 7.9, 0.5 Hz, H-5, δC 118.3), and at δH 7.18, d (J = 7.4 Hz, H-41; δC 122.5), 7.05, ddd (J = 7.8,
7.4, 1.3 Hz, H-61, δC 128.9), 6.61, ddd (J = 7.8, 7.4, 0.5 Hz, H-5’, δC 117.8) and 6.54, d (J = 7.8 Hz,
H-7’, δC 109.8). That one of the 1,2-disubstituted benzene rings was part of the indole moiety was
corroborated by the HMBC correlations of H-4 to C-3 (δC 109.5), C-6 (δC 120.9) and C-8 (δC 136.0), of
the amine proton at δH 10.88, brd (J = 1.4 Hz, NH-1) to C-2 (δC 124.1), C-3, C-8, C-9 (δC 127.4), and of
H-2 (δH 7.25, d, J = 2.3 Hz) to C-3 and C-9 (Table 3 and Figure 5). The presence of a 2,5-disubstituted
1,4-diketopiperazine was supported by the HMBC correlations of NH-13’ (δH 7.72, brs) to the carbonyl
1
at δC 167.7 (C-12), the methine carbons at δC 58.6 (C-11 ) and δC 55.1 (C-11) and the methylene carbon
at δC 24.7 (C-10), of NH-13 (δH 6.05, s) to the methylene carbon at δC 41.3 (C-101), of H-11 (δH 4.46,
t, J = 5.1 Hz) to C-10 and C-12, of H-111 (δH 4.66, dd, J = 11.6, 6.7 Hz) to C-101 and C-121. Moreover,
the COSY correlations of H-11 to H2-10 (δH 3.06, dd, J = 15.7, 6.5 Hz and 3.40, m), and of H-111 to
H2-101 (δH 1.83, dd, J = 13.0, 11.6 Hz and 2.43, dd, J = 13.0, 6.7 Hz) (Table 3 and Figure 5) confirmed

Mar. Drugs 2016, 14, 136
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1
that the substituents on C-11 and C-11 are methylene groups. The indole ring system was connected
to the 1, 4-diketopiperazine moiety through CH2-10 since the HMBC spectrum exhibited correlations
of H-11 to C-3, and of H-10 to C-9 (Table 3, Figure 5, Supplementary Materials, Figure S25). The second
1,2-disubstituted benzene ring was part of the 2,3-disubstituted 2,3-dihydro-1H-indole ring system
1
since the HMBC spectrum showed correlations of NH-1 (δH 6.68,d, J = 4.1 Hz) to the oxygenated sp3
1
1
quaternary carbon at δC 85.9 (C-3 ) and to the quaternary aromatic carbon at δC 131.1 (C-9 ), and also
1
1
1
1
of H-2 (δH 5.33, d, J = 4.1 Hz,) to C-3 . Since the HMBC spectrum showed correlations of H-2 to C-10 ,
as well as of H-101 to C-31, it was concluded that the 2, 3-disubstitute1d 2, 3-dihydro-1H-indole ring
system was linked to the 1, 4-diketopiperazine moiety through CH2-10 . As all of the 1H and 13C data
so far mentioned accounted only for C22H20N4O2, which is1one oxygen atom less than the molecular
1
formula, the epoxide functionality was placed between C-2 and C-3 .
4
10
12
5
13
11'
3
2
11
9
2'
3'
1'
8'
13'
8
1
9'
10'
12'
6
7'
7
4'
6'
5'
Figure 5. Key COSY (—) and HMBC (Ñ) correlations of compound 8.
1
Table 3. H and 13C NMR (DMSO, 300 and 75 MHz), HMBC assignment and NOESY for 8.
Position
δC, type
δ , (J in Hz)
H
COSY
HMBC
NOESY
2
3
124.1, CH
109.5, C
7.25, d (2.3)
NH-1
C-3, 9
H-10a, 11 (str), NH-131
-
4
5
6
7
8
9
10a
b
118.5, CH
118.3, CH
120.9, CH
111.3, CH
136.0, C
7.60, d (7.9)
6.99, ddd (7.9, 7.9, 0.5)
7.07, ddd (7.9, 7.9, 1.1)
H-5
H-4, 6
H-5, 7
H-6
C-3, 6, 8
C-7, 9
C-4, 8
C-5, 9
H-10a, 11 (str)
7.33, d (7.9)
-
-
127.4, C
24.7, CH2
3.06, dd (15.7, 6.5)
H-10b, 11
H-10a, 11
H-10a, 10b
C-3, 9, 11, 12
C-3, 9, 11, 12
C-3, 10, 12
H-4, 10b, 11, NH-131
H-10a
3.40, m
4.46, t (5.1)
-
5.33, d (4.1)
-
11
55.1, CH
167.7, CO
84.0, CH
85.9, C
122.5, CH
117.8, CH
128.9, CH
109.8, CH
148.4, C
H-2, 4, 10a, 111, NH-131
12
21
NH-11
C-31, 101
H-11, NH-13, NH-11 (str)
NH-13
31
41
7.18, d (7.4)
6.61, ddd (7.8, 7.4, 0.5)
7.05, ddd (7.8, 7.8, 1.3)
6.54, d (7.8)
-
H-51
H-41, 61
H-51, 71
H-61
C-61, 81
C-71, 91
C-41, 81
C-51, 91
51
61
71
81
91
131.1, C
41.3, CH2
-
101a
b
1.83, dd (13.0, 11.6)
2.43, dd (13.6, 6.7)
4.66, dd (11.6, 6.7)
-
10.88, brd (1.4)
6.68, d (4.1)
6.05, s
H-101b, 11
H-101a, 11
H-101a, 10b
C-111, 121
C-31
H-101b
H-101a, 111
H-11, 21, 101b
111
121
NH-1
NH-11
NH-13
NH-131
58.6, CH
169.8, CO
C-101, 121
-
-
-
-
H-2
C-2, 3, 8, 9
C-31, 91
H-2, 4
H-21
-
-
C-101
H-21, 41
H-10a (str), 11 (str), H-2
7.72, brs
C-10, 11, 111, 12

Mar. Drugs 2016, 14, 136
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Since compound
8
was obtained as pale yellow viscous mass, its stereochemistry could not be
determined by X-ray crystallography. However, as compounds
A (7) [13 14], it is reasonable to assume that the stereochemistry of C-11 and C-11 of both compounds
are the same. Like compounds 1–3, 5 and , fellutanine A ( ) and compound must be derived from
the same biosynthetic precursor, i.e., L-tryptophan. Consequently, the absolute configurations of C-11
and C-111 of fellutanine A (
) and compound are presumed to be S. In an effort to unravel the
stereochemistry of C-11, C-111, and the epoxide bearing carbons (C-21 and C-31) of compound
, the
NOESY experiments and molecular dynamic simulations were carried out. The NOESY spectrum of
8 was isolated together with fellutanine
1
,
6
7
8
7
8
8
compound
8
(Table 3 and Figure 6, Supplementary Materials, Figure S26) exhibited correlations o1f
1
1
H-11 to H-2, H-4, H-10a, H-11 , NH-13 , therefore confirming the cis-relation between H-11 and H-11 .
Since the coupling constant between H-11 and H-10a is 6.5 Hz, H-10a m1ust be in an equatorial and
H-11 in axial positions in the major conformation. On the other hand, H-11 exhibited only correlations
to H-11 and H-101b (δH 2.43, dd, J = 13.0, 6.7 Hz), and H-21, but not with H-101a (δH 1.83, dd, J = 13.0,
11.6 Hz) and NH-13, while H-21 gave correlations to only H-111 and NH-13, but not to H-101a or
101b. The values of1the coupli1ng constants of H-111 to H-101a (J = 11.6 Hz) and to H-101b ((J = 6.7 Hz)
1
1
indicated that H-11 and H-10 a are “anti”, while H-11 and H-10 b are “gauche”. These data indicated
1
1
1
1
that H-2 is in the same face as H-11 and points to the opposite direction from H-10 a/10 b. However,
these correlations do not allow us to determine the ste1reochemistry of the epoxide. Surprisingly, the
NOESY spectrum also shows strong correlation of H-4 to NH-13.
H
H
O
H
H
H
H
4
H
H
H
H
10
12
13 N
N
5
3
2
11
9
2'
3'
1'
8'
O
N13'
H
H
8
H
11'
1
9'
10'
12'
6
N
H
7'
7
H
4'
H
H
H
O
6'
H
5'
H
Figure 6. Key NOESY (Ø) correlations of compound 8.
Furthermore, a strong NOESY cross-peak between H-11 and H-111 of compound
8, and in
conjunction with conformational search, molecular dynamics and ab initio molecular modeling,
showed that both amide bonds in the diketopiperazine ring are cis and that both amino acids have
the same stereochemistry for their α-carbons. This type of six-membered ring is thermodynamically
stable because resonance compensates for the extra energy of the amide cis configurations [17], when
compared to the more normal trans configuration. Nevertheless, cis peptide bonds occur naturally
even in linear biological proteins [18]. NOESY cross-peaks and molecular modeling also aided the
assignment of the absolute configurations to the epoxide carbon atoms of 8. The minimal energy
conformations for the R/R and S/S models are presented in Figure 7, showing how the epoxide oxygen
points outwards in both cases for minimal repulsion. Conformational analysis was based mainly on the
combinations of the three staggered conformations for C-101/C-111 bond and two for the C-31/C-101
bond. These six conformers differ by less than 7 kcal/mol (RHF/6-21G total energy), independently of
1
1
the configuration of the epoxide. Of all the conformations, the most stable conformation of the 2 S/3 S
epoxide actually explains simultaneously the observed NOESY correlations of H-41 to NH-13 and of
H-21 to H-111 (Table 3 and Figure 6, Supplementary Materials, Figure S26). On the other hand, none
of the R/R conformations justifies the NOESY data without assuming unreasonable spin-diffusion.
The assignment of the S/S isomer for the epoxide carbons of
8 has to assume, however, that there

Mar. Drugs 2016, 14, 136
10 of 15
is a spin-diffusion during the mixing time; otherwise, given the proposed structure for
8
, it would
not be possible to explain also the H-21/H-13 NOESY cross-peak. The proximities H-41/NH-13
and H-21/H-111 are physically incompatible with direct H-21/H-13 NOE proximity. The fact that
H-21/H-111/NH-13 form a coupled dipolar spin system is perhaps an explanation for the very week
1
H-11 /NH-13 NOESY cross-peak (Table 3 and Figure 6, Supplementary Materials, Figure S26), which
is expected to be strong unless some polarization transfer is at play between the three spins.
Figure 7. The two possible epoxide configurations for
8 in their lowest RHF/6-21G total energy
conformation. Solid lines indicate direct NOESY correlations, explained by the S/S stereoisomer
and not by the R/R. The discontinuous line shows how spin diffusion gives rise to an H-21/NH-13
NOESY cross-peak.
Taking all of the evidence together, the structure of compound
8
was proposed as fellutanine A
2 S, 3 S-epoxide. To the best of our knowledge, compound 8 is also a new compound.
Compounds were tested for their antibacterial activity against Gram-positive (Escherichia coli
1
1
1–8
ATCC 25922) and Gram-negative (Staphyllococus aureus ATCC 25923) bacteria, as well as for
their antifungal activity against filamentous (Aspergillus fumigatus ATCC 46645), dermatophyte
(Trichophyton rubrum ATCC FF5) and yeast (Candida albicans ATCC 10231), according to the previously
described protocols [19
,20]; however, none of the tested compounds exhibited either antibacterial
(MIC > 256 µg/mL) or antifungal activities (MIC > 512 µg/mL).

Mar. Drugs 2016, 14, 136
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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 measured 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
1
8.3). H and 13C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument
(Bruker Biosciences Corporation, Billerica, MA, USA) operating at 300.13 or 500.13 MHz, and 75.4 or
125.8 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 column chromatography.
3.2. Extraction and Isolation
The strain KUFA 0702 was isolated from the marine sponge Mycale sp., which was collected,
by scuba diving at a depth of 15–20 m, from the coral reef at Samaesarn Island (12˝34136.6411
N
100˝56159.6911 E) in the Gulf of Thailand, Chonburi Province, in February 2015. The sponge was
washed with 0.06% sodium hypochlorite solution for 1 min, followed by sterilized seawater 3 times,
and then dried on sterile filter paper, cut into small pieces (5
ˆ
5 mm), and placed on a malt extract
agar (MEA) medium containing 70% seawater and 300 mg/L of streptomycin sulfate. After incubation
˝
at 28 C for 7 days, the hyphal tips were transferred onto a slant MEA and maintained as pure culture
for further identification. The fungus was identified as Neosartorya glabra (Fennell & Raper) Kozak
based on morphological characteristics such as colony growth rate and growth pattern on standard
media, namely Czapek1s agar, Czapek yeast autolysate agar and malt extract agar. Microscopic
characteristics including size, shape and ornamentation of ascospores were examined under light
and scanning electron microscopes. This identification was supported by sequence analysis of the
β-tubulin, calmodulin and actin genes as described in the previous report [21]. Neosartorya glabra
was also confirmed by sequence analysis of the internal transcribed spacer (ITS) gene, according
the procedure previously described by us [11]. Its gene sequences were deposited in GenBank
with accession numbers KU955860. The pure cultures were deposited as KUFA 0702 at Kasetsart
University Fungal Collection, Department of Plant Pathology, Faculty of Agriculture, Kasetsart
˝
University, Bangkok, Thailand. The fungus was cultured for one week at 28 C in 5 Petri dishes
(i.d. 90 mm) containing 15 mL of potato dextrose agar. In order to obtain the mycelial suspension,
the mycelial plugs were transferred to two 500 mL Erlenmeyer flasks containing 250 mL of potato
dextrose broth, and then incubated on a rotary shaker at 150 rpm at 28 ˝C for 7 days. Forty 1000-mL
Erlenmeyer flasks, each containing 300 g of cooked rice, were autoclaved at 121 ˝C for 15 min, and
then inoculated with 25 mL of mycelial suspension of N. glabra, and incubated at 28 ˝C for 30 days,
after which the moldy rice was macerated in ethyl acetate (20 L total) for 7 days, and then filtered
with filter paper. The ethyl acetate solution was concentrated under reduced pressure to yield 98.2 g
of crude ethyl acetate extract, which was dissolved in 1000 mL of CHCl3, and then washed with
H2O (3 ˆ 500 mL). The organic layers were combined and dried with anhydrous Na2SO4, filtered and
evaporated under reduced pressure to give 71.2 g of the crude chloroform extract, which was applied
on a column of silica gel (420 g), and eluted with mixtures of petrol-CHCl3 and CHCl3–Me2CO, 250 mL
fractions were collected as follows: Frs 1-80 (petrol–CHCl3, 1:1), 81-144 (petrol–CHCl3, 3:7), 145-201
(petrol–CHCl3, 1:9), 202-356 (CHCl3–Me2CO, 9:1), 357-398 (CHCl3–Me2CO, 7:1), and 399-410 (Me2CO).
Frs 85-105 were combined (2.04 g) and purified by TLC (silica gel G254, CHCl3–Petrol–EtOAc–HCO2H,
8:1:1:0.01) to give 11 mg of ergosta-4,6,8 (14), 22-tetraen-3-one. Fr 207 (1.14 g) was applied over a
column chromatography of Sephadex LH-20 (10 g) and eluted with MeOH and a mixture of MeOH:
CH2Cl2 (1:1), wherein 20 mL subfractions were collected as follows: sfrs 1–90 (MeOH), and 91–145

Mar. Drugs 2016, 14, 136
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(MeOH: CH2Cl2, 1:1). Sfrs 53–61 were combined (19.5 mg) and recrystallized in MeOH to give
16.8 mg of ergosterol 5,8-endopeoxide. Sfrs 62-90 were combined (53.2 mg) and purified by TLC
(silica gel G254, CHCl3-Petrol-EtOAc-HCO2H, 8:1:1:0.01) to give 11.2 mg of 1. Frs 206–212 were
combined (4.88 g) and applied over a column chromatography of Si gel (45 g) and eluted with
mixture of petrol–CHCl3, CHCl3–Me2CO and Me2CO, wherein 100 mL subfractions were collected
as follows: sfrs 1–51 (petrol–CHCl3, 1:1), 52-107 (petrol–CHCl3, 3:7), 108–164 (petrol–CHCl3, 1:9),
165–190 (CHCl3–Me2CO, 9.5:0.5), 191–310 (CHCl3–Me2CO, 9:1). Sfrs 83–164 were combine (53.4 mg)
and recrystallized in MeOH to give 27.6 mg of ergosterol 5,8-endopeoxide. Sfr 166 (38.8 mg) was
recrystallized in Me2CO to give 8.7 mg of 1. Frs 213–245 were combined (3.61 g) and applied over a
column chromatography of Sephadex LH-20 (10 g) and eluted with MeOH, wherein 60 sfrs of 20 mL
were collected. Sfrs 31–51 were combined and purified by TLC (silica gel G254, CHCl3–Me2CO–HCO2H,
4:1:0.01) to give 9.7 mg of 3 and 13.1 mg of 4. Frs 246–257 were combined (1.44 g) and recrystallized
in MeOH to give 23.7 mg of helvolic acid. Frs 273–287 were combined (621.0 mg) and purified by
TLC (silica gel G254, CHCl3–Me2CO–HCO2H, 7:3:0.03) to give 12.1 mg of helvolic acid and 32.3 mg
of 5. Frs 363–373 were combined (1.26 g) and applied over a column chromatography of Sephadex
LH-20 (10 g) and eluted with MeOH, wherein 60 subfractions of 20 mL were collected. Sfrs 22–54
were combined (91.2 mg) and purified by TLC (silica gel G254, CHCl3–Me2CO–HCO2H, 9.5:0.5:0.03) to
give 14.7 mg of
G254, CHCl3–Me2CO–HCO2H, 3:2:0.03) to give 32.8 mg of
6
and 10 mg of
2
. Frs 374–398 were combined (1.37 g) and purified by TLC (silica gel
. Frs 403–405 were combined (2.49 g) and
8
applied over a column chromatography of Sephadex LH-20 (10 g) and eluted with MeOH, wherein
112 sfrs of 20 mL were collected. Sfrs 90–112 were combined (24.9 mg) and purified by TLC (silica gel
G254, CHCl3–Me2CO–HCO2H, 9.5:0.5:0.03) to give 20.7 mg of 7.
3.2.1. Satoryglabramide A (5)
White crystal, mp 146–148 ˝C (CHCl3-Me2CO); rαsD20 +34.6 (c 0.06, Me2CO); IR (KBr)
νmax 3447,
1
3060, 3028, 2920, 2850, 1655, 1622, 1587, 1526, 1453, 1415, 1300, 1261, 1173 cm´1; H and 13C NMR
(see Table 1); HRESIMS m/z 511.2365 (M + H)+ (calculated for C30H31N4O4, 511.2345).
3.2.2. Satoryglabramide B (6)
20
White solid, mp 190–192 ˝C (CHCl3–Me2CO); rαsD +42.8 (c 0.05, Me2CO); IR (KBr)
ν
3417,
max
3058, 2924, 2852, 1649, 1620, 1588, 1526, 1454, 1418, 1302, 1263, 1101 cm´1; 1H and 13C NMR (see Table 2);
HRESIMS m/z 550.2501 (M + H)+ (calculated for C32H32N5O4, 550.2454).
3.2.3. Fellutanine A Epoxide (8)
20
Pale yellow viscous mass; rαsD +13.9 (c 0.07, Me2CO); IR (KBr)
ν
3420, 2922, 1649, 1416, 1188,
max
1047, 1025, 996 cm´1; 1H and 13C NMR (see Table 3); HRESIMS m/z 389.1626 (M + H)+ (calculated for
C22H21N4O3, 389.1614).
3.3. X-ray Crystal Structure of Sartoryglabramide A (5)
A single crystal of sartoryglabamide A was mounted on a cryoloop using paratone. X-ray
diffraction data was collected at room temperature with a Gemini PX Ultra equipped with CuKα
radiation (λ = 1.54184 Å). The crystal was orthorhombic, space group P212121, cell volume 5459.8(2)
Å3 and unit cell dimensions a = 15.1792(3) Å, b = 18.7674(5) Å and c = 19.1659(3) Å (uncertainties
in parentheses). There are two molecules per unit cell with calculated density of 1.242 g/cm3.
The structure was solved by direct methods using SHELXS-97 and refined with SHELXL-97 [22].
Carbon, nitrogen and oxygen atoms were refined anisotropically. Hydrogen atoms were either
placed at their idealized positions using appropriate HFIX instructions in SHELXL and included
in subsequent refinement cycles or were directly found from difference Fourier maps and were refined
freely with isotropic displacement parameters. The refinement converged to R (all data) = 10.02%

Mar. Drugs 2016, 14, 136
13 of 15
and wR2 (all data) = 15.26%. The absolute structure could not be established with confidence (flack x
parameter 0.3(4)).
Full details of the data collection and refinement and tables of atomic coordinates, bond lengths
and angles, and torsion angles have been deposited with the Cambridge Crystallographic Data Centre
(CCDC 1483750).
3.4. Amino Acids Analysis of Acidic Hydrolysate of Sartoryglabramide A (5) and Sartoryglabramide B (6)
3.4.1. Acid Hydrolysis
The stereochemistry of the amino acids was determined by analysis of the acidic hydrolysate from
˝
6. Compound 5 or 6 (5.0 mg) was dissolved in 6 N HCl (5 mL) and heated at 110 C, in a furnace,
5
and
for 24 h in a sealed glass tube. After cooling to room temperature, the solution was dried under N2
for 24 h, reconstituted in MeOH for HPLC-MS (200 L), filtered through a 4 mm PTFE Syringe Filter
F2504-4 of 0.2 m pore size (Thermo Scientific, Mumbai, India), and then analyzed by HPLC equipped
µ
µ
with a chiral column.
3.4.2. 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
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
m) manufactured by ASTEC (Whippany, NJ, USA). The mobile phase composition was MeOH:
µL loop, and a SPD-M20A DAD detector (Kyoto,
ˆ
5
µ
H2O (80:20 v/v), all were LC-MS grade solvents obtained from Sigma-Aldrich Co. (St. Louis, MO,
USA). The flow rate was 1.0 mL/min and the UV detection wavelength was 210 nm. Analyses were
performed at room temperature in an isocratic mode. All standards of 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 enantiomeric mixtures,
and then each enantiomer separately. 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 5 and 6.
3.4.3. Molecular Mechanics Conformation Analysis of Fellutanine A Epoxide (8)
Molecular simulations for structure
Waltham, MA, USA). Stochastic and dihedral driver conformational search, with MMFF force
field energy minimization, was done for both S/S and R/R isomers of , followed by ab initio
8 were carried out in ChemBio3D Ultra 14 (Perkin-Elmerm,
8
RHF/6-21G energy re-minimization of the lowest energy conformations using CS GAMESS interfaced
by ChemBio3D. The PCM solvent model for DMSO was used on the ab initio minimizations.
4. Conclusions
Although there are few reports of the constituents of N. glabra, this is the first study of the
secondary metabolites from the marine-derived strain of this fungus. It is interesting to point out that
even though some common fungal metabolites previously isolated from other members of this and
related genera, such as ergosta-4,6,8 (14), 22-tetraen-3-one, ergosterol 5,8-endoperoxide, helvolic acid,
aszonalenin, takakiamide, (3R)-3-(1H-indol-3-ylmethyl)-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione
(2) and fellutanine A (7), compound 4 was only described as a synthetic intermediate obtained by
cyclocondensation of L-proline with isatoic acid anhydride [12]. Moreover, this is the first report on
isolation of the cyclopeptides (sartoryglabramides A and B) from the genus Neosartorya. In addition,
despite the fact that compounds 1–8 did not exhibit antimicrobial activities in our assay protocols,

Mar. Drugs 2016, 14, 136
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it does not mean that they do not possess any other relevant biological activities. It is also worth
mentioning that several cyclopeptides have been shown to possess antifungal and antibacterial
activities, however, their potencies depend on the stereochemical configurations of the amino acids
constituents [23]. Therefore, it is not surprising that the stereochemistry of the amino acids constituents
of both sartoryglabramides A (5) and B (6) could play an important role in their (lack of) antimicrobial
activities. Therefore, it is necessary to further examine the isolated metabolites in other target-based
assay protocols.
Supplementary Materials: The supplementary materials are available online at www.mdpi.com/1660-3397/14/
7/136/s1.
Acknowledgments: This work was developed in the Natural Products Research Laboratory of the Department
of Chemistry, Instituto de Ciências Biomédicas Abel Salazar (ICBAS) of the University of Porto, and
partially supported through national funds provided by FCT—Foundation for Science and Technology and
European Regional Development Fund (ERDF) and COMPETE, under the projects PEst-C/MAR/LA0015/2013,
PTDC/MAR-BIO/4694/2014 as well as by the project INNOVMAR—Innovation and Sustainability in
the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, within
Research Line NOVELMAR/INSEAFOOD/ECOSERVICES), supported by North Portugal Regional Operational
Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional
Development Fund (ERDF)”. We thank Michael Lee of the Department of Chemistry, Leicester University (UK) for
providing the HRESIMS. War War May Zin thanks the Lotus Unlimited Project under the ERASMUS MUNDUS
ACTION 2-EU-Asia Mobility Project for a Ph.D. scholarship. We thank Júlia Bessa for technical support.
Author Contributions: A.K. and M.P. conceived, designed the experiments; W.W.M.Z. performed isolation,
purification and structure elucidation of the compounds; S.B. participated in structure elucidation of the
compounds; T.D. collected, identified, cultured the fungus and prepared the extract; L.G. performed X-ray
analysis; J.P. performed molecular mechanics conformation analysis; A.S. provided NMR spectra; C.F. and S.C.
performed chiral HPLC analyses of the amino acids; N.S. participated in discussion of the results; A.K. wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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2016 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
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