Iheyamides A-C, Antitrypanosomal Linear Peptides Isolated from a Marine Dapis sp. Cyanobacterium

Article abstract of DOI:10.1021/acs.jnatprod.0c00250

Iheyamides A (1), B (2), and C (3), new linear peptides, were isolated from a marine Dapis sp. cyanobacterium. Their structures were elucidated by spectroscopic analyses and degradation reactions. Iheyamide A (1) showed moderate antitrypanosomal activities against Trypanosoma brucei rhodesiense and Trypanosoma brucei brucei (IC₅₀ = 1.5 μM), but the other two analogues, iheyamides B (2) and C (3), did not (IC₅₀ > 20 μM, respectively). The structure-activity relationship clarified that an isopropyl-O-Me-pyrrolinone moiety was necessary for the antitrypanosomal activity. Furthermore, the cytotoxicity of 1 against normal human cells, WI-38, was 10 times weaker than its antitrypanosomal activity (IC₅₀ = 18 μM).

Full text of DOI:10.1021/acs.jnatprod.0c00250

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Article
Iheyamides A−C, Antitrypanosomal Linear Peptides Isolated from a
Marine Dapis sp. Cyanobacterium
Naoaki Kurisawa, Arihiro Iwasaki, Ghulam Jeelani, Tomoyoshi Nozaki, and Kiyotake Suenaga*
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ABSTRACT: Iheyamides A (1), B (2), and C (3), new linear
peptides, were isolated from a marine Dapis sp. cyanobacterium.
Their structures were elucidated by spectroscopic analyses and
degradation reactions. Iheyamide A (1) showed moderate
antitrypanosomal activities against Trypanosoma brucei rhodesiense
and Trypanosoma brucei brucei (IC50 = 1.5 μM), but the other two
analogues, iheyamides B (2) and C (3), did not (IC50 > 20 μM,
respectively). The structure−activity relationship clarified that an
isopropyl-O-Me-pyrrolinone moiety was necessary for the
antitrypanosomal activity. Furthermore, the cytotoxicity of 1
against normal human cells, WI-38, was 10 times weaker than its
antitrypanosomal activity (IC50 = 18 μM).
rypanosomatids are a group of parasites that cause many
neglected tropical diseases all over the world. A species
T
called Trypanosoma brucei (T. b.) is known to be the causative
organism of Nagana disease (T. b. brucei) and sleeping sickness
(T. b. rhodesiense and T. b. gambiense).1 These diseases prevent
economic development and threaten the health of people who
live in regions where trypanosomiasis is epidemic, mainly sub-
Saharan Africa. Although several antitrypanosomal drugs have
been developed and are used in treatment, each of them has
serious problems, such as strong side effects, low permeability
through the blood−brain barrier, and so on.1 Furthermore,
because most of these drugs have been used for decades, there
is a risk of the appearance of resistant strains. Therefore, drug
lead compounds that have a novel mode of action have been
strongly desired.
Meanwhile, natural products from marine cyanobacteria
with antiparasitic activity have been reported, including
symplostatin 4,2 companeramide A,3 and ikoamide4 (anti-
malarial), hoshinolactam5 and janadolide6 (antitrypanosomal),
and almiramide B7 (antileishmanial). Therefore, marine
cyanobacteria are promising resources for finding antiparasitic
agents. Here we describe new linear peptides, iheyamides A
(1), B (2), and C (3), isolated from a marine Dapis sp.
cyanobacterium, which was taxonomically distinguished from
the genus Lyngbya by Paul and co-workers in 2018.8 As for
structural features, the iheyamides (1, 2, and 3) possess an
N,N-dimethylphenylalanine (N,N-diMe-Phe) moiety. Iheya-
mide A (1) further possesses an unusual isopropyl-O-
methylpyrrolinone (iPr-O-Me-pyr) moiety. Regarding bio-
logical activities, 1 exhibited moderate growth-inhibitory
activities against T. b. rhodesiense and T. b. brucei without
showing significant cytotoxicity against normal human cells.
Received: March 6, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c00250
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A

Journal of Natural Products
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Table 1. NMR Data for Iheyamide A (1) in CD3OD
b
a
residue
position
δC, type
δH (J in Hz)
3.91, s
COSY
selected TOCSY
selected HMBC
4
selected NOESY
iPr-O-Me-pyrrolinone
1
2
59.6, CH3
172.6, C
3
4
5
6
7
8
9
10
11a
11b
12a
12b
13
95.2, CH
182.0, C
5.24, s
2, 4
4
65.5, CH
29.7, CH
16.0, CH3
18.9, CH3
172.9, CH
61.9, CH
29.9, CH2
4.58, d (2.8)
2.52, m
0.84, d (6.7)
1.10, d (7.2)
6
5, 7, 8
6
6
Pro
5.48, dd (8.8, 4.5)
1.84, m
2.32, m
1.87, m
2.03, m
11a, 11b
10, 11b, 12b
10, 11a,
12b, 13
12a, 13
10
10, 12
25.4, CH2
49.2, CH2
170.5 C
3.68−3.77, m
12a, 12b
15
13
N-Me-Ile
14
15
16
17a
17b
18
19
20
21
59.7, CH
34.3, CH
25.4, CH2
5.08, d (10.3)
2.05, m
1.06, m
16
15, 19
16, 17b, 18
16, 17a, 18
17a, 17b
16
14
17a, 17b
1.53, m
19.9, CH3
15.6, CH3
31.0, CH3
174.7, C
0.88, t (7.4)
0.93, d (6.6)
3.09, s
15, 21
Pro
22
58.7, CH
29.9, CH2
4.79, dd (8.6, 4.6)
1.77, m
2.21, m
2.05, m
1.87, m
23a, 23b
22, 23b,
22, 23a, 24a
23b, 24b, 25a
24a, 25a
23a
23b
24a
24b
25a
25b
26
21
21, 24
25.9, CH2
48.8, CH2
3.72, m
3.65, m
24a, 25b
24a, 25a
27
N-Me-Val
169.7, C
27
28
29
30
31
32
61.5, CH
28.4, CH
18.8, CH3
10.9, CH3
31.1, CH3
173.9, C
4.91, m
2.10, m
0.49, d (6.7)
0.89, d (6.9)
2.96, s
28
26
25a
27, 29, 30
28
28
27, 32
Phe
33
52.0, CH
38.4, CH2
4.91, m
2.54, m
2.86, m
34a, 34b
33, 34b
33, 34a
32, 41
35, 36/40
35, 36/40
34a
34b
35
137.7, C
36/40
37/39
38
130.2, CH
129.4, CH
127.9, CH
172.7, C
7.10, m
7.15, m
7.20, m
37/39
36/40, 38
37/39
38
37/39
N,N-diMe-Phe
41
42
43
44
71.2, CH
37.0, CH2
139.3, C
3.26, dd (10.0, 5.0)
2.84−3.00, m
43
42
41
44, 45/49
45/49
46/48
47
130.3, CH
129.7, CH
127.6, CH
42.6, CH3
7.15, m
7.19, m
7.17, m
2.33, s
46/48
45/49, 47
46/48
47
46/48
42
50/51
a
b
Measured at 400 MHz. Measured at 100 MHz.
H2O. The EtOAc-soluble material was further partitioned
between 90% aqueous MeOH and hexane. The material
obtained from the aqueous MeOH portion was subjected to
fractionation by reversed-phase column chromatography
(ODS silica gel, MeOH−H2O) and repeated reversed-phase
RESULTS AND DISCUSSION
■
The marine Dapis sp. cyanobacterium (5 kg, wet weight) was
collected at Noho Island, Iheya Village, Okinawa, Japan, in
August 2019, and extracted with EtOH. The extract was
filtered, concentrated, and partitioned between EtOAc and
B
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HPLC with trifluoroacetic acid (TFA) as an additive to give
iheyamides A (1, 13.7 mg), B (2, 12.3 mg), and C (3, 1.9 mg)
as TFA salts. We neutralized iheyamide A (1) by partitioning
with K2CO3 aqueous solution and isolated free 1.
Iheyamide C (3) was obtained as a colorless oil. The NMR
data for 3 are summarized in Table 3. The molecular formula
of 3 was found to be C38H56N5O6 by HRESIMS. The 1H NMR
spectrum of iheyamide C (3) in CD3OD is similar to that of 2,
but some signals of α-protons of the amino acids were
overlapped by a signal derived from CD3OD. Therefore, the
NMR analyses of iheyamide C (3) were done by using CD3CN
as a solvent. Although the 13C NMR signal for C-37/C-38 (δC
41.5, overlapped) was broad and almost invisible, the chemical
shift C-37/C-38 was determined by HMQC correlations.
Detailed analyses of the 2D NMR spectra revealed the gross
structure of 3 as shown in Figure 3. Although we could not
observe any HMBC and NOESY correlations that joined C-
30/C-31 and C-28/C-29, the presence of these bonds was
determined based on the NMR chemical shifts of the benzyl
protons at C-30 and the α-methine proton at C-29. As a result,
iheyamide C (3) is an analogue of 1 that lacks the Pro and iPr-
O-Me-pyr moieties.
The absolute configurations of iheyamides A (1), B (2), and
C (3) were determined by acid hydrolysis followed by a
combination of chiral-phase HPLC analyses and ozonolysis.9
For iheyamide A (1), the Phe was determined to be the D-
form, and the other amino acids including N,N-diMe-Phe were
all L-form. To determine the absolute configuration at C5 in
iPr-O-Me-pyr-, we derivatized this moiety to Val by ozonolysis
followed by acid hydrolysis. Next, we compared the HPLC
retention time of the Val derived from 1 with those of
authentic D- and L-Val. As a result, the absolute configuration at
C-5 in iPr-O-Me-pyr was determined to be S. Similarly, the
absolute configurations of iheyamides B (2) and C (3) were
clarified, with the configurations of all of the common amino
acids the same as those of iheyamide A (1).
Iheyamide A (1) was obtained as a colorless oil. The NMR
data for 1 are summarized in Table 1. The molecular formula
1
of 1 was found to be C51H73N7O8 by HRESIMS. In the H
NMR spectrum, one singlet signal at 2.33 ppm, corresponding
to six protons, two singlet methyl signals at 2.96 and 3.09 ppm,
and one relatively deshielded methyl signal at 3.91 ppm
indicated the presence of one N,N-dimethyl amino group, two
N-methyl amides, and one O-methyl moiety, respectively. A
singlet methine signal observed at 5.24 ppm also suggested the
presence of a trisubstituted olefin. In addition, 10 aromatic
proton signals (δH 7.10−7.20) suggested that 1 contained two
monosubstituted benzene rings. This prediction is also
supported by the observation of eight aromatic carbons in
the 13C NMR spectrum (δC 139.3, 137.7, 130.3, 130.2, 129.7,
129.4, 127.9, and 127.6). Furthermore, in the 13C NMR
spectrum, eight deshielded signals (δC 182.0, 174.7, 173.9,
172.9, 172.7, 172.6, 170.5, and 169.7), considered to be amide
or ester carbonyl carbons, were detected. In addition, detailed
analyses of the 2D NMR spectra revealed the presence of seven
partial structures: isopropyl-O-Me-pyrrolinone (iPr-O-Me-
pyr), two prolines (Pro), N-Me-isoleucine (N-Me-Ile), N-
Me-valine (N-Me-Val), phenylalanine (Phe), and N,N-
dimethylphenylalanine (N,N-diMe-Phe). Based on the five
HMBC correlations, H-42/C-41, H-33/C-41, H-33/C-32, H-
31/C-32, and H-31/C-27, the following partial sequence was
clarified: N,N-diMe-Phe-Phe-N-Me-Val. Also, two HMBC
correlations, H-23/C-21 and H-20/C-21, indicated the
presence of the following dipeptide moiety: Pro-N-Me-Ile.
Furthermore, two NOESY correlations, H-25a/H-27 and H-
13/H-15, expanded the sequence as follows: N,N-diMe-Phe-
Phe-N-Me-Val-Pro-N-Me-Ile-Pro. The last remaining moiety,
iPr-O-Me-pyr, could only be located at the C-terminus,
resulting in the gross structure of 1 as shown in Figure 1.
Next, we evaluated the biological activities of 1−3 (Table 4).
We tested antitrypanosomal activities against T. b. rhodesiense
IL-1501 strain and T. b. brucei 221 strain and cytotoxicities
against WI-38 cells (normal human fibroblasts). As a result, 1
showed antitrypanosomal activity with IC50 values of 1.5 μM,
while 2 and 3 did not show such activities (IC50 > 20 μM).
These results showed that iPr-O-Me-pyr was necessary for
antitrypanosomal activity. Further, 1 showed more than 10-
fold weaker cytotoxicity against normal human cells (IC50 = 18
μM) than antitrypanosomal activity.
In conclusion, we isolated three new linear peptides,
iheyamides A (1), B (2), and C (3), from a marine Dapis
sp. cyanobacterium. The genus Dapis has recently been
taxonomically separated from the genus Lyngbya by Paul and
co-workers,8 and iheyamides 1−3 are the first compounds to
be isolated from a marine Dapis sp. cyanobacterium. Their
structures were elucidated by spectroscopic analyses and
degradation reactions. The characteristic N,N-diMe-Phe and
iPr-O-Me-pyr moieties are relatively rare motifs in natural
products. Except for our new compounds (1−3), the former
moiety has only been reported in grassystatin F from a marine
cyanobacterium,10 and the latter moiety has only been
reported in mirabimides from Scytonema mirabile.11 Newly
isolated iheyamide A is the first natural product that has both
moieties. In addition, iheyamide A (1) showed moderate
antitrypanosomal activity. Meanwhile, it showed much weaker
cytotoxicity against normal human cells. This result indicates
that 1 is a promising lead compound for the development of
new antitrypanosomal drugs.
Figure 1. Gross structure of iheyamide A (1) based on 2D NMR
analyses.
Iheyamide B (2) was obtained as a colorless oil. The NMR
data for 2 are summarized in Table 2. The molecular formula
of 2 was found to be C43H63N6O7 by HRESIMS. The 1H NMR
spectrum of iheyamide B (2) is similar to that of 1 except for
the disappearance of two doublet methyl signals, a singlet
methine signal, and an O-methyl signal. These changes
suggested the absence of the iPr-O-Me-pyr moiety from 1.
Detailed analyses of the 2D NMR spectra revealed the gross
structure of 2 as shown in Figure 2. As a result, iheyamide B
(2) is an analogue of 1 that lacks the iPr-O-Me-pyr moiety.
C
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Table 2. NMR Data for Iheyamide B (2) in CD3OD
b
a
residue
position
δC, type
δH (J in Hz)
COSY
selected HMBC
1
selected NOESY
Pro
1
2
3a
175.1, C
60.5, CH
30.3, CH2
4.37, dd (9.0, 4.9)
1.99, m
3a, 3b
2, 3b
3b
4a
4b
5a
2.25, m
1.94, m
2.05, m
3.63, m
2, 3a, 4a, 4b
3b, 4b, 5a, 5b
3b, 4a, 5a, 5b
4a, 4b
25.8, CH2
48.4, CH2
7
7
5b
3.77, m
4a, 4b
N-Me-Ile
6
7
8
9a
171.1, C
59.6, CH
34.4, CH
25.5, CH2
5.10, d (11.4)
2.07, m
1.08, m
8
6
5a
7, 9, 11
8, 9b, 10
8, 9a, 10
9a, 9b
8
9b
1.56, m
10
11
12
13
10.9, CH3
15.4, CH3
31.0, CH3
174.8, C
0.89, t (7.0)
0.97, d (6.5)
3.13, s
7, 13
13
Pro
14
58.6, CH
29.9, CH2
4.80, dd (8.6, 4.8)
1.78, m
15a, 15b
15b
15a
15b
16a
16b
17a
17b
18
2.25, m
15a, 16a, 16b
25.9, CH2
48.4, CH2
1.94, m
15b, 16b, 17a, 17b
2.05, m
3.63, m
15b, 16a, 17a, 17b
16a, 16b
19
3.77, m
16a, 16b
N-Me-Val
169.7, C
19
20
21
22
23
24
61.8, CH
28.6, CH
18.9, CH3
19.8, CH3
31.2, CH3
173.5, C
4.88, m
2.17, m
0.63, d (6.7)
0.92, d (6.7)
3.01, s
20
18
17a
19, 21, 22
20
20
19, 24
Phe
25
53.4, CH
37.4, CH2
4.71, dd (8.8, 5.9)
2.54, dd (14.7, 8.8)
2.89, dd (14.7, 5.9)
26a, 26b
25, 26b
25, 26a
24, 33
27, 28/32
27, 28/32
26a
26b
27
137.1, C
28/32
29/31
30
NH
33
129.8, CH
129.9, CH
128.1, CH
7.02, m
7.22, m
7.21, m
8.69, d (6.1)
29/31
28/32, 30
29/31
25
30
29/31
N,N-diMe-Phe
167.7, C
70.3, CH
35.6, CH2
34
3.99, dd (10.7, 4.7)
3.02, m
3.29, m
35a, 35b
34, 35b
34, 35a
35a
35b
36
36, 37/41
36, 37/41
135.0, C
37/41
38/40
39
130.3, CH
130.0, CH
128.9, CH
42.5, CH3
7.13, m
7.22, m
7.23, m
2.98, br s
38/40
37/41, 39
38/40
39
38/40
34
42/43
a
b
Measured at 400 MHz. Measured at 100 MHz.
(iheyamides A and B) and CD3CN observed at δC 1.32 (iheyamide
C)) were assigned based on HMBC and HMQC experiments.
HRESIMS spectra were obtained on an LCT Premier XE time-of-
flight (TOF) mass spectrometer (LCT Premier XE, Waters).
Chromatographic analyses were performed using an HPLC system
consisting of a pump (model PU-2080, JASCO) and a UV detector
(model UV-2075, JASCO). All chemicals and solvents used in this
study were the best grade available and obtained from a commercial
source (Nacalai Tesque).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a JASCO DIP-1000 polarimeter. UV spectra were
recorded on a JASCO V730-BIO instrument. IR spectra were
recorded on a JASCO RT/IR-4200 instrument. All NMR data were
1
recorded on a JEOL JNM-ECS400 spectrometer for H (400 MHz)
and 13C (100 MHz). 1H NMR chemical shifts (referenced to residual
CD3OD observed at δH 3.31 (iheyamides A and B) and CD3CN
observed at δH 1.94 (iheyamide C)) were assigned using a
combination of data from COSY and HMQC experiments. Similarly,
13C NMR chemical shifts (referenced to CD3OD observed at δC 49.0
Identification of the Marine Cyanobacterium. A cyanobacte-
rial filament was isolated under a microscope and crushed with
D
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Figure 2. Gross structure of iheyamide B (2) based on 2D NMR
correlations.
Figure 3. Gross structure of iheyamide C (3) based on 2D NMR
data.
freezing and thawing. The 16S rDNA genes were PCR-amplified from
the isolated DNA using the primer set CYA359F (a cyanobacterial-
specific primer) and CYA1371R (a universal primer). The PCR
reaction contained DNA derived from a cyanobacterial filament, 0.5
μL of KOD-Multi & Epi- (Toyobo), 1.0 μL of each primer (0.5 μM,
respectively), 12.5 μL of 2× PCR buffer for KOD-Multi & Epi-, and
H2O for a total volume of 25 μL. The PCR reaction was performed as
Table 3. NMR Data for Iheyamide C (3) in CD3CN
b
a
residue
position
δC, type
δH (J in Hz)
COSY
selected HMBC
1
selected NOESY
N-Me-Ile
1
2
3
4a
4b
5
6
7
172.6, C
62.5, CH
33.6, CH
25.6, CH2
4.65, d (10.6)
2.01, m
1.46, m
3
4, 6
3, 4b, 5
3, 4a, 5
9a, 9b
3
2.03, m
10.8, CH3
16.0, CH3
32.6, CH3
174.1, C
57.8, CH
29.2, CH2
0.84, t (7.0)
0.94, d (6.7)
3.04, s
2, 8
13
9
7
Pro
8
9
4.73, dd (8.6, 4.6)
1.74, m
2.14, m
1.82, m
3.56, m
10a, 10b
10a
10b
11
12a
12b
13
15b
15a, 16a, 16b
10a, 10b, 12a, 12b
25.5, CH2
48.2, CH2
11
11
14
14
3.74, m
N-Me-Val
168.9, C
14
15
16
17
18
19
60.8, CH
28.3, CH
18.9, CH3
19.5, CH3
31.0, CH3
172.4, C
4.85, d (10.9)
2.13, m
0.63, d (6.4)
0.87, d (6.4)
2.98, s
15
13
12a, 12b
14, 16, 17
15
15
14, 19
Phe
20
52.7, CH
37.3, CH2
4.80, m
2.62, dd (14.2, 9.1)
2.86, m
21a, 22b, NH
20, 21b
20, 21a
21a
21b
22
19, 22, 23/27
19, 22, 23/27
137.1, C
23/27
24/26
25
NH
28
129.5, CH
129.8, CH
127.7, CH
7.20, m
7.02, m
7.19, m
7.57, d (6.6)
24/26
23/27, 25
24/26
20
24/26
25
N,N-diMe-Phe
166.5, C
29
30
31
68.4, CH
34.7, CH2
135.6, C
4.00, m
3.14, m,
30
29
32/36
33/35
34
129.6, CH
130.1, CH
128.3, CH
41.5, CH3
7.22, m
7.13, m
7.23, m
2.82, br, s
33/35
32/36, 34
33/35
31, 33/35
34
37/38
29
a
b
Measured at 400 MHz. Measured at 100 MHz.
E
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solution (50 mL), and the organic layer was evaporated to give
Table 4. Growth-Inhibitory Activities of Iheyamides A (1),
B (2), and C (3)
iheyamide A (1, 13.7 mg).
Iheyamide A (1): colorless oil; [α]25 −117 (c 1.06, MeOH); UV
D
IC50 values (μM)
(MeOH) λmax (log ε) 236 nm (4.17); IR (neat) 3300, 1718, 1636,
1437, 1320, 1249 cm−1; 1H NMR, 13C NMR, COSY, HMBC,
NOESY, and TOCSY data, Table 1; HRESIMS m/z 912.5595 [M +
H]+ (calcd for C51H74N7O8, 912.5599).
compound
T. b. rhodesiense
T. b. brucei
WI-38 cells
iheyamide A (1)
iheyamide B (2)
iheyamide C (3)
1.5
1.5
18
>20
>20
>20
>20
0.005
>20
>20
0.001
The fraction that contained iheyamides B (2) and C (3) was
further purified by HPLC [Cosmosil PBr (φ 20 × 250 mm); flow rate
5 mL/min; detection, UV 215 nm; solvent 88% MeOH containing
0.1% TFA] in four batches to give two fractions that contained
iheyamide B (2, tR = 31.0 min) and iheyamide C (3, tR = 28.0 min),
respectively. Each fraction was further purified by HPLC [Cosmosil
Cholester (φ 20 × 250 mm); flow rate 5 mL/min; detection, UV 215
nm; solvent 40% MeCN containing 0.1% TFA] to give iheyamide B
(2) (12.3 mg, tR = 32.0 min) and iheyamide C (3) (1.9 mg, tR = 45.6
min).
a
pentamide
a
Positive control.
follows: initial denaturation for 2 min at 94 °C and amplification by
40 cycles of 10 s at 98 °C and 1.5 min at 66 °C. PCR products were
analyzed on agarose gel (1%) in TBE buffer and visualized by
ethidium bromide staining. The obtained DNA was sequenced with
CYA359F and CYA1371R primers. This sequence is available in the
DDBJ/EMBL/Genbank databases under accession number
LC514420. The nucleotide sequence of the 16S rRNA gene obtained
in this study was used for phylogenetic analysis with the sequences of
related cyanobacterial 16S rRNA genes.8 All sequences were aligned
by the SINA web service (version 1.2.11)12 with default settings. The
poorly aligned positions and divergent regions were removed by
Gblocks Server (version 0.91b),13 implementing the options for a less
stringent selection, including the ‘Allow smaller final blocks’, ‘Allow
gap positions within the final blocks’, and ‘Allow less strict flanking
positions’ options. The obtained 887 nucleotide positions were used
for phylogenetic analysis. JModeltest (version 2.1.7)14,15 with default
settings was used to select the best model of DNA substitution for the
maximum likelihood (ML) analysis and Bayesian analysis according
to the Akaike information criterion (AIC). The ML analysis was
conducted by PhyML (version 20131016),15 using the TIM1+I+G
model with a gamma shape parameter of 0.4280, a proportion of
invariant sites of 0.4490, and nucleotide frequencies of F(A) = 0.2519,
F(C) = 0.2261, F(G) = 0.2963, and F(T) = 0.2257. Bootstrap
resampling was performed on 1000 replicates. The ML tree was
visualized with Njplot (version 2.3).16 The Bayesian analysis was
conducted by MrBayes (version 3.2.5)17 using the GTR+I+G model.
The Markov chain Monte Carlo process was set at two chains, and
1 000 000 generations were conducted. Sampling frequency was
assigned at every 500 generations. After analysis, the first 100 000
trees were deleted as burn-in, and the consensus tree was constructed.
The Bayesian tree was visualized with FigTree (version 1.4.0, http://
tree.bio.ed.ac.uk/software/figtree). As a result, the cyanobacterium
(accession no. LC514420) formed a clade with Dapis sp. Therefore,
the cyanobacterium was classified into Dapis sp.
Collection, Extraction, and Isolation. The Dapis sp. cyano-
bacterium (5 kg, wet weight) was collected at Noho Island, Okinawa,
Japan, in August 2019. The collected cyanobacterium was extracted
with EtOH (2 × 3 L) for 3 days. The extract was filtered, and the
filtrate was concentrated. The residue was partitioned between EtOAc
(3 × 0.6 L) and H2O (0.6 L). The material obtained from the organic
layer was partitioned between 90% aqueous MeOH (0.3 L) and
hexane (3 × 0.3 L) and evaporated to give an aqueous MeOH
fraction (2.59 g). A 1.21 g amount of this aqueous MeOH fraction
was separated by column chromatography on ODS (12.1 g) eluted
with 40% MeOH, 60% MeOH, 80% MeOH, MeOH, and CHCl3−
MeOH (1:1). A 240 mg amount of the fraction (544.6 mg) eluted
with 80% MeOH was subjected to HPLC [Cosmosil 5C18-MS-II (φ
20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent
80% MeOH] in five batches to give two fractions that contained
iheyamide A (1) (63.8 mg, last collected fraction) and iheyamide B
(2) and iheyamide C (3) (48.8 mg, tR = 21.0 min). A 21.3 mg sample
of the fraction that contained iheyamide A (1) was further purified by
HPLC [Cosmosil Cholester (φ 20 × 250 mm); flow rate 5 mL/min;
detection, UV 215 nm; solvent 55% MeCN containing 0.1% TFA] to
give iheyamide A as a TFA salt (1, tR = 27.1 min). The fraction was
partitioned between EtOAc (3 × 50 mL) and K2CO3 aqueous
Iheyamide B (2): colorless oil; [α]25 −80 (c 0.92, MeOH); IR
D
1
(neat) 3263, 1636, 1457, 1201, 1137 cm−1; H NMR, 13C NMR,
COSY, HMBC, and NOESY data, Table 2; HRESIMS m/z 775.4756
[M + H]+ (calcd for C43H63N6O7, 775.4758).
Iheyamide C (3): colorless oil; [α]25 −57 (c 0.32, MeOH); IR
D
1
(neat) 3263, 1634, 1456, 1202, 1136 cm−1; H NMR, 13C NMR,
COSY, HMBC, and NOESY data, Table 3; HRESIMS m/z 678.4231
[M + H]+ (calcd for C38H56N5O6, 678.4231).
Determination of the Absolute Configuration of Iheyamide
A (1). Iheyamide A (1, 0.3 mg) was treated with 6 M HCl (100 μL)
for 20 h at 110 °C. The hydrolyzed product was evaporated to
dryness and could be separated into each component. [Conditions for
HPLC separation: column, Cosmosil 5C18-PAQ (φ 4.6 × 250 mm);
flow rate, 1.0 mL/min; detection at 215 nm; solvent H2O. Retention
times (min) of components; Pro (3.2), N-Me-Val (3.7), N-Me-Ile
(5.3), Phe (10.4), and N,N-diMe-Phe (21.4).]
The N-Me-Ile fraction was dissolved in H2O (50 μL) and analyzed
by HPLC, and the retention times were compared to those of
authentic standards [column, Cosmosil PBr (φ 4.6 × 250 mm); flow
rate, 1.0 mL/min; detection 254 nm; solvent, 5% MeCN with 0.1%
TFA]. The retention times for authentic standards were 7.0 min (N-
Me-allo-Ile) and 7.7 min (N-Me-Ile). The retention time of N-Me-Ile
from natural 1 was 7.7 min, indicating the presence of N-Me-Ile.
Each fraction was dissolved in H2O (50 μL) and analyzed by chiral-
phase HPLC, and the retention times were compared to those of
authentic standards [column, DAICEL CHIRALPAK MA(+) (φ 4.6
× 50 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent,
several conditions]. With 2 mM CuSO4 as a solvent, the retention
times for authentic standards were 2.6 min (D-Pro) and 4.7 min (L-
Pro), 3.2 min (N-Me-D-Val) and 5.0 min (N-Me-L-Val), and 8.0 min
(N-Me-D-Ile) and 14.0 min (N-Me-L-Ile). The retention times of each
amino acid from natural 1 were 4.7, 5.0, and 14.0 min, indicating the
presence of L-Pro, N-Me-L-Val, and N-Me-L-Ile, respectively. With
10% MeCN 2 mM CuSO4 solution as a solvent, the retention times
for authentic standards were 4.2 min (D-Phe) and 5.5 min (L-Phe),
and 8.1 min (N,N-diMe-D-Phe) and 13.3 min (N,N-diMe-L-Phe). The
retention times of each amino acid from natural 1 were 4.2 and 13.3
min, indicating the presence of D-Phe and N,N-diMe-L-Phe,
respectively.
Iheyamide A (1, 0.5 mg) was dissolved in 2.5 mL of MeOH and
ozonized at −78 °C for 20 min. The solvent was evaporated, and the
product was treated with 6 M HCl (100 μL) for 17 h at 110 °C. The
hydrolyzed product was evaporated to dryness and separated into Val
from iPr-O-Me-pyrrolinone. [Conditions for HPLC separation:
column, Cosmosil 5C18-PAQ (φ 4.6 × 250 mm); flow rate, 1.0
mL/min; detection at 215 nm; solvent H2O. Retention times (min) of
components; Val (3.4).] The fraction was dissolved in H2O (50 μL)
and analyzed by chiral-phase HPLC, and the retention time was
compared to those of authentic standards [column, DAICEL
CHIRALPAK MA(+) (φ 4.6 × 50 mm); flow rate, 1.0 mL/min;
detection at 254 nm; solvent, several conditions]. With 2 mM CuSO4
as a solvent, the retention times for authentic standards were 3.2 min
(D-Val) and 6.1 min (L-Val). The retention time of Val from iPr-O-
F
https://dx.doi.org/10.1021/acs.jnatprod.0c00250
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Me-pyrrolinone of natural 1 was 6.1 min, indicating the presence of
(5S)-iPr-O-Me-pyrrolinone.
Authors
Naoaki Kurisawa − Department of Chemistry, Faculty of Science
and Technology, Keio University, Yokohama, Kanagawa 223-
8522, Japan
Arihiro Iwasaki − Department of Chemistry, Faculty of Science
and Technology, Keio University, Yokohama, Kanagawa 223-
8522, Japan; orcid.org/0000-0002-3775-5066
Ghulam Jeelani − Department of Biomedical Chemistry,
Graduate School of Medicine, The University of Tokyo, Tokyo
113-0033, Japan
Determination of the Absolute Configurations of Iheya-
mide B (2) and Iheyamide C (3). Iheyamide B (2, 0.2 mg) and
iheyamide C (3, 0.2 mg) were treated with 6 M HCl (100 μL) for 18
h at 110 °C, respectively. The hydrolyzed product was evaporated to
dryness and could be separated into each component, as with
iheyamide A (1).
Each fraction was dissolved in H2O (50 μL) and analyzed by chiral-
phase HPLC, and the retention times were compared to those of
authentic standards [column, DAICEL CHIRALPAK MA(+) (φ 4.6
× 50 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent,
several conditions]. With 2 mM CuSO4 as a solvent, the retention
times for authentic standards were the same as those for iheyamide A
(1). The retention times of each amino acid from natural 2 and 3
were 4.7, 5.0, and 14.0 min, indicating the presence of L-Pro, N-Me-L-
Val, and N-Me-L-Ile, respectively. With 10% MeCN 2 mM CuSO4
solution as a solvent, the retention times for authentic standards were
4.2 min (D-Phe) and 5.5 min (L-Phe), and 8.1 min (N,N-diMe-D-Phe)
and 13.3 min (N,N-diMe-L-Phe). The retention times of each amino
acid from natural 2 and 3 were 4.2 and 14.0 min, indicating the
presence of D-Phe and N,N-diMe-L-Phe, respectively.
In Vitro Antitrypansomal Assay. The Trypanosoma brucei
rhodesiense strains IL-150118 and Trypanosoma brucei brucei strains
22119 were cultured at 37 °C under a humidified 5% CO2 atmosphere
in HMI-9 medium20 supplemented with 10% heat-inactivated fetal
bovine serum (FBS). For in vitro studies, compounds were dissolved
in DMSO and diluted in culture medium prior to being assayed. The
maximum DMSO concentration in the in vitro assays was 1%.
The compounds were tested in an AlamarBlue serial drug dilution
assay21 to determine the 50% inhibitory concentrations (IC50). Serial
drug dilutions were prepared in 96-well microtiter plates containing
the culture medium, and wells were inoculated with 4.0 × 104 cells/
mL T. b. rhodesiense IL-1501 parasites or T. b. brucei 221 parasites.
Cultures were incubated for 69 h at 37 °C under a humidified 5%
CO2 atmosphere. After this time, 10 μL of resazurin (12.5 mg
resazurin [Sigma] dissolved in 100 mL of phosphate-buffered saline)
was added to each well. The plates were incubated for an additional 3
h. The plates were read in a SpectraMax Gemini XS microplate
fluorescence scanner (Molecular Devices) using an excitation
wavelength of 536 nm and an emission wavelength of 588 nm.
Cell Growth Analysis. WI-38 cells were cultured at 37 °C with
5% CO2 in DMEM (Nissui) supplemented with 10% heat-inactivated
FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL
amphotericin, 300 μg/mL L-glutamine, and 2.25 mg/mL NaHCO3.
Cells were seeded at 4 × 103 cells/well in 96-well plates (Iwaki) and
cultured overnight. Various concentrations of compounds were then
added, and cells were incubated for 72 h. Cell proliferation was
measured by the MTT assay.
Tomoyoshi Nozaki − Department of Biomedical Chemistry,
Graduate School of Medicine, The University of Tokyo, Tokyo
113-0033, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00250
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by JSPS KAKENHI (Grant Numbers
18K14346, 19H04662, and 16H03285) and Keio Gijuku
Academic Development Funds.
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00250.
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NMR spectra for iheyamides (1−3); HPLC chromato-
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phylogenetic tree (PDF)
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AUTHOR INFORMATION
■
̈
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(20) Raz, B.; Iten, M.; Grether-Buhler, Y.; Kaminsky, R.; Brun, R.
Acta Trop. 1997, 68, 139−147.
Corresponding Author
(21) Huber, W. Acta Trop. 1993, 55, 257−261.
Kiyotake Suenaga − Department of Chemistry, Faculty of
Science and Technology, Keio University, Yokohama, Kanagawa
223-8522, Japan; orcid.org/0000-0001-5343-5890;
Email: suenaga@chem.keio.ac.jp
G
https://dx.doi.org/10.1021/acs.jnatprod.0c00250
J. Nat. Prod. XXXX, XXX, XXX−XXX