Isolation and Total Synthesis of Hoshinolactam, an Antitrypanosomal Lactam from a Marine Cyanobacterium

Article abstract of DOI:10.1021/acs.orglett.7b00047

In the search for new antiprotozoal substances, hoshinolactam, an antitrypanosomal lactam, was isolated from a marine cyanobacterium. The gross structure was elucidated by spectroscopic analyses, and the absolute configuration was determined by the first total synthesis. Hoshinolactam showed potent antitrypanosomal activity with an IC₅₀ value of 3.9 nM without cytotoxicity against human fetal lung fibroblast MRC-5 cells (IC₅₀ > 25 μM).

Full text of DOI:10.1021/acs.orglett.7b00047

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Isolation and Total Synthesis of Hoshinolactam, an Antitrypanosomal
Lactam from a Marine Cyanobacterium
Hidetoshi Ogawa,† Arihiro Iwasaki,† Shimpei Sumimoto,† Masato Iwatsuki,‡,§ Aki Ishiyama,‡,§
Rei Hokari,‡ Kazuhiko Otoguro,‡ Satoshi Omura,‡ and Kiyotake Suenaga*,†
̅
†Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
§
‡Research Center for Tropical Diseases, Kitasato Institute for Life Sciences, and Graduate School of Infection Control Sciences,
Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8641, Japan
S
* Supporting Information
ABSTRACT: In the search for new antiprotozoal substances, hoshinolactam, an
antitrypanosomal lactam, was isolated from a marine cyanobacterium. The gross
structure was elucidated by spectroscopic analyses, and the absolute configuration was
determined by the first total synthesis. Hoshinolactam showed potent antitrypanosomal
activity with an IC50 value of 3.9 nM without cytotoxicity against human fetal lung
fibroblast MRC-5 cells (IC50 > 25 μM).
ropical diseases caused by parasitic protozoa are a threat
Tto human health, mainly in developing countries.
Trypanosomiasis (Chagas disease and sleeping sickness) and
leishmaniasis, inter alia, are classified as neglected tropical
diseases, and over 400 million people are at risk of contracting
these diseases.1 In addition, a parasite of the Trypanosoma
genus, Trypanosoma brucei brucei, is the causative agent of
Nagana disease in wild and domestic animals, and this disease is
a major obstacle to the economic development of affected rural
areas.2 Although some therapeutic agents for these diseases
exist, they have limitations, such as serious side effects and the
emergence of drug resistance.1 Thus, new and more effective
antiprotozoal medicines are needed.1
that 1 exhibited potent antitrypanosomal activity without
cytotoxicity against human fetal lung fibroblast MRC-5 cells.
Here, we report the isolation, structure elucidation, first total
synthesis, and preliminary biological characterization of
hoshinolactam (1).
Marine natural products have recently been considered to be
good sources for drug leads.3 In particular, secondary
metabolites produced by marine cyanobacteria have unique
structures and versatile biological activities,4 and some of these
compounds show antiprotozoal activities. For example, coibacin
A isolated from cf. Oscillatoria sp. exhibited potent anti-
leishmanial activity,5 and viridamide A isolated from Oscillatoria
nigro-viridis showed antileishmanial and antitrypanosomal
activities.6 In our search for new antiprotozoal substances, we
have focused on the constituents of marine cyanobacteria and
reported an antitrypanosomal cyclodepsipeptide, janadolide.7
Recently, we isolated a new antitrypanosomal lactam,
hoshinolactam (1), from a marine cyanobacterium.8 Structur-
ally, 1 contains a cyclopropane ring and a γ-lactam ring. So far,
some metabolites possessing either a cyclopropane ring or a γ-
lactam ring have been discovered from marine cyanobacteria,
such as majusculoic acid9 and malyngamide A.10 To the best of
our knowledge, on the other hand, hoshinolactam (1) is the
first compound discovered in marine cyanobacteria that
possesses both of these ring systems. In addition, we clarified
The marine cyanobacterium was collected at the coast near
Hoshino, Okinawa. The collected cyanobacterium (2 kg, wet
weight) was extracted with MeOH, and the extract was filtered
and concentrated. The residue was partitioned between EtOAc
and H2O. The material obtained from the organic layer was
partitioned between 90% aqueous MeOH and hexanes. The
aqueous MeOH fraction was separated by reversed-phase
column chromatography (ODS silica gel, MeOH−H2O) and
reversed-phase HPLC (Cosmosil 5C18AR-II, MeOH-H2O;
Cosmosil 5C18MS-II, MeOH-H2O; Cosmosil Cholester,
MeCN-H2O) to give hoshinolactam (1) (6.5 mg). The
molecular formula of hoshinolactam (1) was determined on
the basis of positive HRESIMS data (m/z 308.2228, calcd for
Received: January 6, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b00047
Org. Lett. XXXX, XXX, XXX−XXX

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C18H30NO3 [M + H]+ 308.2225). Table 1 shows the NMR data
for 1. An analysis of the H NMR spectrum indicated the
the 1H NMR chemical shifts of (S)-α-methoxy-α-
(trifluoromethyl)phenylacetyl (MTPA) ester 3a and (R)-
MTPA ester 3b revealed that the absolute configuration of
C-3 of 2 was R (Scheme 1; see the Supporting Information).
1
1
Table 1. H and 13C NMR Data for 1 in C6D6
a
b
unit
position
δC
δH (J in Hz)
Scheme 1. Preparation of MTPA Esters Derived from the
HIMP of Hoshinolactam (1)
HIMP
1
177.8, C
44.1, CH
80.8, CH
57.3, CH
44.6, CH2
2
2.51, dq (5.2, 7.6)
4.94, dd (4.6, 5.2)
3.49, ddd (4.6, 4.7, 9.4)
1.21, m
3
4
5a
5b
6
1.36, m
25.0, CH
21.7, CH3
23.2, CH3
15.0, CH3
1.61, m
7
0.74, d (6.2)
0.76, d (6.3)
1.33, d (7.6)
7.65, s
8
9
NH
1
PCPA
166.0, C
2
117.4, CH
155.0, CH
22.4, CH
23.3, CH
35.7, CH2
22.5, CH2
14.0, CH3
16.1, CH2
5.88, d (15.5)
6.59, dd (10.3, 15.5)
0.91, m
3
4
5
0.59, m
There were four possible combinations of the relative
stereochemistries of C-2, C-3, and C-4: syn and syn, syn and
anti, anti and syn, or anti and anti. To explore the possibility
that the relative stereochemistry of C-3 and C-4 was syn, we
synthesized two possible syn isomers, (2S,3S,4S)-2 and
(2R,3S,4S)-2, from the common intermediate 412 (Scheme
6
0.96, m
7
1.20, tq (7.1, 7.3)
0.78, t (7.3)
8
9a
9b
0.35, ddd (4.5, 6.0, 8.2)
0.42, ddd (4.5, 4.5, 8.8)
1
a
b
2). As a result, the H NMR spectra of the stereoisomers were
Measured at 100 MHz. Measured at 400 MHz.
Scheme 2. Synthesis of Authentic Standards (2S,3S,4S)-2
and (2R,3S,4S)-2
presence of four methyl groups (δH 0.74, 0.76, 0.78 and 1.33),
four protons of the cyclopropane ring (δH 0.35, 0.42, 0.59 and
0.91), and two olefinic protons (δH 5.88 and 6.59). The 13C
NMR and HMQC spectra revealed the existence of two
carbonyl groups (δC 166.0 and 177.8) and two sp2 methines
(δC 117.4 and 155.0). Examination of the COSY and HMBC
spectra established the presence of two fragments derived from
4-hydroxy-5-isobutyl-3-methylpyrrolidin-2-one (HIMP) and 3-
(2-propylcyclopropyl) acrylic acid (PCPA), respectively. The
configuration of the C-2−C-3 olefinic bond in the PCPA was
determined to be trans on the basis of the coupling constant
(3JH2−H3 = 15.5 Hz). The connectivity of the two partial
structures was determined from the HMBC correlation (H-3 of
HIMP/C-1 of PCPA). As a result, the gross structure of 1 was
established as shown in Figure 1.
not consistent with that of 2 from a natural source (see the
Supporting Information). This indicated that the relative
stereochemistry of C-3 and C-4 was anti. Therefore, the
absolute configuration of C-4 was determined to be 4S.
To clarify the absolute configuration of C-2, we synthesized
one of the two possible stereoisomers, (2R,3R,4S)-2, as follows
(Scheme 3). Reduction of known Weinreb amide 613 with
lithium aluminum hydride followed by a non-Evans syn aldol
reaction with the chiral auxiliary 714 afforded the aldol adduct 8
as a single isomer (52% in two steps). Exposure of 8 to DMAP
in MeOH gave the methyl ester 9 (78%). Deprotection of the
methyl ester 9 followed by cyclization under basic conditions
led to the desired lactam (2R,3R,4S)-2 (87% in two steps). The
absolute stereochemistry of C-2 of (2R,3R,4S)-2 was confirmed
to be 2R by application of the phenyl glycine methyl ester
Figure 1. Gross structure of hoshinolactam (1) based on 2D NMR
correlations.
The absolute configuration of hoshinolactam (1) was
established as follows. Regarding the HIMP moiety, there
were three stereocenters: C-2, C-3, and C-4. This indicated that
there were eight possible stereoisomers for the HIMP moiety.
To determine the absolute configuration of C-3 of the HIMP
moiety, the modified Mosher’s method11 was applied to 2,
which was prepared by the acid hydrolysis of 1. Comparison of
B
DOI: 10.1021/acs.orglett.7b00047
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Scheme 3. Synthesis of Authentic Standard (2R,3R,4S)-2
Scheme 4. Synthesis of (4S,5S)-1 and (4R,5R)-1
(PGME) method15 to the hydrolysate of 9 (see the Supporting
Information). Moreover, the 1H NMR spectrum of this
compound did not match that of (2R,3S,4S)-2. This indicated
that the absolute stereochemistry of C-3 was 3R. The 1H NMR
spectrum of (2R,3R,4S)-2 was in agreement with that of the
natural lactam 2 (see the Supporting Information). Thus, the
absolute configuration of C-2 of 2 was determined to be 2R,
and the stereochemistry of the HIMP moiety was clarified to be
(2R,3R,4S).
Meanwhile, the relative stereochemistry of the two stereo-
genic centers of the PCPA moiety was determined on the basis
of analyses of the coupling constants and NOESY correlations,
as described below (Figure 2). Based on detailed decoupling
11, respectively. Next, hydrolysis of (4S,5S)-12 and (4R,5R)-12
and condensation with HIMP (2) using the Shiina reagent
afforded (4S,5S)-1 and (4R,5R)-1, respectively. Surprisingly,
their 1H NMR spectra were very similar to each other, and only
proton signals corresponding to the cyclopropane ring were
slightly different. In addition, their 13C NMR spectra were
completely consistent. On the other hand, their specific optical
rotations were completely different [(4S,5S)-1: [α]26.6 +59 (c
D
0.50, CHCl3), (4R,5R)-1: [α]22.7 −54 (c 0.80, CHCl3)]. As a
D
result, the spectroscopic data (1H and 13C NMR spectra, and
specific optical rotation) of (4S,5S)-1 were fully consistent with
those of natural hoshinolactam (1) (see the Supporting
Information), and thus, the absolute configuration of
hoshinolactam (1) was established as shown in 1.
Figure 2. Relative stereochemistry of the PCPA moiety based on
NOESY correlations and coupling constants.
Hoshinolactam (1) showed potent antitrypanosomal activity
against Trypanosoma brucei brucei GUTat 3.1 strain with an IC50
value of 3.9 nM, which was equivalent to the commonly used
therapeutic drug pentamidine (IC50 4.7 nM) (Table 2). On the
experiments, the following coupling constants were observed:
3
3
3JH‑4/H‑9a = 8.2 Hz, JH‑4/H‑9b = 4.5 Hz, JH‑5/H‑9a = 6.0 Hz, and
3JH‑5/H9‑b = 8.8 Hz. The two large coupling constants indicated
that H-4/H-9a and H-5/H-9b were in a cis relationship,
respectively. On the other hand, the small values indicated that
H-4/H-9b and H-5/H-9a were in a trans relationship,
respectively. In addition, this conclusion was supported by
the four NOESY correlations, H-2/H-4, H-4/H-9a, H-3/H-5,
and H-5/H-9b. Thus, the relative stereochemistry of C-4 and
C-5 of PCPA was determined to be trans. However, the
absolute stereochemistry remained to be clarified. Therefore,
we sought to determine the stereochemistry through the total
synthesis of hoshinolactam (1) as follows (Scheme 4).
Table 2. Antitrypanosomal Activity of Hoshinolactam (1)
IC50 (nM)
compd
antitrypanosomal activity
cytotoxicity
hoshinolactam (1)
synthetic 1
6.1
3.9
4.7
>25000
>25000
16800
a
pentamidine
a
Drug used to treat trypanosomal disease.
other hand, 1 did not show any cytotoxicity against human fetal
lung fibroblast MRC-5 cells (IC50 > 25 μM). This indicated
that hoshinolactam (1) recognized specific biomolecules for
Trypanosoma protozoa.
In conclusion, hoshinolactam (1), an antitrypanosomal
lactam, was isolated from a marine cyanobacterium. The
gross structure was elucidated by spectroscopic analyses, and
Regarding the PCPA moiety, two possibilities for the
stereochemistry remained, (4S,5S) or (4R,5R). To clarify the
absolute configuration of the PCPA moiety, we synthesized two
ethyl esters, (4S,5S)-12 and (4R,5R)-12, from known alcohols
(2S,3S)-1016 and (2R,3R)-1017 by Swern oxidation followed by
the Horner−Wadsworth−Emmons reaction with phosphonate
C
DOI: 10.1021/acs.orglett.7b00047
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(13) Fehrentz, J. A.; Castro, B. Synthesis 1983, 1983, 676−678.
the absolute configuration was determined by the first total
synthesis. This synthetic process should provide routes for
obtaining synthetic analogues. In addition, hoshinolactam (1)
showed potent antitrypanosomal activity without cytotoxicity
against human fetal lung fibroblast MRC-5 cells. Studies on the
structure−activity relationships of synthetic analogues should
contribute to the development of new antitrypanosomal drugs.
(14) Crimmins, M. T.; King, B. W.; Tabet, E. A. J. Am. Chem. Soc.
1997, 119, 7883−7884.
(15) Nagai, Y.; Kusumi, T. Tetrahedron Lett. 1995, 36, 1853−1856.
(16) Charette, A. B.; Juteau, H.; Lebel, H.; Molinaro, C. J. Am. Chem.
Soc. 1998, 120, 11943−11952.
(17) Charette, A. B.; Brochu, C. J. Am. Chem. Soc. 1995, 117, 11367−
11368.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b00047.
1H, 13C, COSY, HMQC, HMBC, and NOESY NMR
spectra in C6D6 and 1H and 13C NMR spectra in
1
CD3OD for hoshinolactam (1); H NMR spectra in
1
CD3OD for lactam 2 from 1; H NMR spectra in C6D6
for MTPA esters and PGME amides; spectroscopic data
and 1H and 13NMR spectra for synthetic products;
detailed experimental procedures (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: suenaga@chem.keio.ac.jp.
ORCID
Kiyotake Suenaga: 0000-0001-5343-5890
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI Grant No.
16H03285 and the Naito Foundation.
■
REFERENCES
■
(1) Hotez, P. J.; Molyneux, D. H.; Fenwick, A.; Kumaresan, J.; Sachs,
S. E.; Sachs, J. D.; Savioli, L. N. Engl. J. Med. 2007, 357, 1018−1027.
(2) WHO. Trypanosomiasis, human African (sleeping sick-
ness).www.who.int/mediacentre/factsheets/fs259/en/ (accessed Dec
2, 2016).
(3) Molinski, T. F.; Dalisay, D. S.; Lievens, S. L.; Saludes, J. P. Nat.
Rev. Drug Discovery 2009, 8, 69−85.
(4) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.;
Prinsep, M. R. Nat. Prod. Rep. 2016, 33, 382−431.
(5) Balunas, M. J.; Grosso, M. F.; Villa, F. A.; Engene, N.; McPhail, K.
L.; Tidgewell, K.; Pineda, L. M.; Gerwick, L.; Spadafora, C.; Kyle, D.
E.; Gerwick, W. H. Org. Lett. 2012, 14, 3878−3881.
(6) Simmons, T. L.; Engene, N.; Urena, L. D.; Romero, L. I.; Ortega-
̃
Barría, E.; Gerwick, L.; Gerwick, W. H. J. Nat. Prod. 2008, 71, 1544−
1550.
(7) Ogawa, H.; Iwasaki, A.; Sumimoto, S.; Iwatsuki, M.; Ishiyama, A.;
Hokari, R.; Otoguro, K.; Omura, S.; Suenaga, K. J. Nat. Prod. 2016, 79,
̅
1862−1866.
(8) The marine cyanobacterium was morphologically classified into
the genus Oscillatoria. However, we failed to identify the genus of the
cyanobacterium based on the 16S rRNA gene (see the Supporting
Information).
(9) MacMillan, J. B.; Molinski, T. F. J. Nat. Prod. 2005, 68, 604−606.
(10) Cardellina, J. H., II; Marner, F.-J.; Moore, R. E. J. Am. Chem. Soc.
1979, 101, 240−242.
(11) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092−4096.
(12) Jouin, P.; Castro, B.; Nisato, D. J. Chem. Soc., Perkin Trans. 1
1987, 1177−1182.
D
DOI: 10.1021/acs.orglett.7b00047
Org. Lett. XXXX, XXX, XXX−XXX