Synthesis of a Microcystis aeruginosa predicted metabolite with antimalarial activity

Article abstract of DOI:10.1016/j.bmcl.2012.06.028

The synthesis of a Microcystis aeruginosa predicted metabolite analog of aerucyclamide B was performed. This hexacyclopeptide was obtained from three heterocyclic building blocks by a convergent macrocycle-assembly methodology. The compound exhibited good in vitro antiplasmodial activity (IC₅₀: 0.18 μM, K1, cholorquine resistant strain).

Full text of DOI:10.1016/j.bmcl.2012.06.028

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4994–4997
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a Microcystis aeruginosa predicted metabolite
with antimalarial activity
a
a
a
b
b
b
Stella Peña , Laura Scarone , Eduardo Manta , Lindsay Stewart , Vanessa Yardley , Simon Croft ,
a,
⇑
Gloria Serra
a
Cátedra de Química Farmacéutica, DQO, Facultad de Química, UDELAR, Gral. Flores 2124, Montevideo CP 11800, Uruguay
Faculty of Infectious & Tropical Disease, LSHTM, Keppel Street, London WC1E 7HT, UK
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a Microcystis aeruginosa predicted metabolite analog of aerucyclamide B was performed.
Received 10 April 2012
Revised 6 June 2012
Accepted 11 June 2012
Available online 16 June 2012
This hexacyclopeptide was obtained from three heterocyclic building blocks by a convergent macrocycle-
assembly methodology. The compound exhibited good in vitro antiplasmodial activity (IC50: 0.18
cholorquine resistant strain).
lM, K1,
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Hexacyclopeptide
Macrocyclization
Oxa/thiazole
Antimalarial
1
Marine natural products play an important role in drug devel-
opment particularly in anticancer, antibiotics and antiparasitics
O
O
H
N
2
N
H
N
S
drugs. Various bioactive cyanobacterial compounds exhibit un-
ique structural features like cyclic peptides containing azole het-
erocycles. It is well known that macrocyclic peptides can
demonstrate drug-like physicochemical and pharmacokinetic
properties such as good metabolic stability, solubility, lipophilicity
and bioavailability.4 In addition, asymmetrical cyclopeptides are
considered as new scaffolds for supramolecular chemistry and pro-
vides an important method for the development and preparation of
S
O
H
O
O
H
N
N
N
3
NH
HN
H
O
NH
HN
O
N
N
O
S
S
Aerucyclamide B (1)
Dendroamide A (2)
5
nanoscale objects.
6
O
N
Examples of azole cyclopeptides are aerucyclamide B (1),
O
7
8
dendroamide A (2), and venturamide A (3), Figure 1. Aerucycla-
mide B was isolated in 2008 by Gademann and co-workers from
the toxic freshwater cyanobacterium Microcystis aeruginosa PCC
S
S
O
O
H
O
N
H
N
N
N
N
H
7
806 and displays potent and selective antiplasmodial activity
NH
NH
HN
O
HN
O
against Plasmodium falciparum K1. This species is the most virulent
of the genus Plasmodium and causes more than 95% of malaria-re-
lated morbidity and mortality. According to the World Health
Organization 2011 report, there were an estimated 216 million epi-
N
N
O
H
S
S
Venturamide A (3)
Macrocycle 4
9
sodes and 655000 deaths of malaria in 2010. Various antimalarial
drugs are presently used for the treatment of this tropical disease,
but the rapid spread of resistance seriously compromises their
efficacy.1
Figure 1. Natural cyclopeptides and predicted macrocycle 4.
0
In spite of the potent antimalarial activity displayed by aerucy-
clamide B, its total synthesis has not been described in the litera-
ture to date. However, aerucyclamide B was obtained through
⇑
Corresponding author. Tel.: +598 2929 0290; fax: +598 2924 1906.
E-mail address: gserra@fq.edu.uy (G. Serra).
oxidation of the natural aerucyclamide A using MnO
2
/benzene.6
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.028

S. Peña et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4994–4997
4995
O
Our synthesis of thiazole 5, utilized conventional Hantszch’s
1
7
methodology, Scheme 2. Amide formation from Boc-Gly-OH
employing 2,2,2-trichloroethyl chloroformiate/aqueous ammonia
and further reaction with Lawesson’s reagent allowed us to obtain
thioamide 8. Hantszch synthesis using ethyl bromopyruvate in
Py/EtOH at reflux, afforded thiazole 5. This compound was
obtained from Boc-Gly-OH in 40% overall yield.
The cyclodehydration of b-hydroxyamides or b-hydroxythioa-
mides and further oxidation protocol was selected for the prepara-
tion of oxazole 6 and thiazole 7, respectively.
O
OMe
N
H
O
NHBoc
S
O
N
H
N
6
N
H
NH
HN
O
BocHN
5
S
N
N
O
H
First, dipeptide Boc-L-Ile-L-Thr-OMe (9) was synthesized in
very good yield from the corresponding protected aminoacids
employing HBTU as coupling reagent, Scheme 3. Then, the smooth
cyclodehydration reaction with DAST and further oxidation with
S
EtO
O
OMe
Macrocycle 4
NHBoc
H
N
O
1
8
3
BrCCl /DBU allowed us to obtain oxazole 6.
S
High yielding preparation of dipeptide 10 was achieved by
using HBTU, Scheme 4. Attempts to prepare the thioamide 11 from
7
1
9
20
the TBS ether derivative of 10, and Lawesson’s reagent failed. In
contrast, 11 was obtained in good yield using Wipf’s procedure by
cyclodehydration of 10 with DAST, giving oxazoline 12 (68%) and
Scheme 1. Retrosynthetic analysis for macrocycle 4.
2
1
then thiolysis using H
2
S/MeOH/Et
3
N (85%).
This reaction is
From the analyses of genomic data of M. aeruginosa PCC 7806,
high-yielding and offers an alternative to the thionation of pep-
tides using Lawesso n´ s reagent. To produce the desired thiazole 7
from thioamide 11, we investigated the one-pot procedure using
Dittmann and co-workers predicted three hexapeptides containing
thiazole and oxazole rings.11 One of these predicted compounds is
the structural analog of aerucyclamide B, macrocycle 4, containing
an oxazole and L-Ile instead of an oxazoline ring and D-allo-Ile,
respectively, Figure 1. Based on the well-known oxazolines insta-
bility, we hypothesize that macrocycle 4 is a more stable com-
DAST and then BrCCl
1 was obtained in excellent yield (98%).
The next steps were the assembling of the key fragments into
3
/DBU. Following this protocol, compound
1
12
the open intermediate of macrocycle 4. With the purpose of
obtaining a suitable macrocyclization reaction, the less hindered
amide bond formation was selected as the last reaction.
pound than aerucyclamide B.
As part of our search for candidates of antiparasitic new drugs,13
we embarked in the synthesis of cyclohexapeptides that alternate
oxa/thiazole rings, analogs of marine natural products.
In this Letter, we present the synthesis and antimalarial activity
of macrocycle 4, a predicted metabolite that could be isolated from
Microcystis aeruginosa PCC 7806, as a more stable analog of the
antimalarial aerucyclamide B.
Considering that macrocyclizations of linear peptides are often
slow, low yielding procedures accompanied by side reactions, we
planned our synthesis from three heterocyclic building blocks
2
2
Compound 15, was prepared by ethyl ester hydrolysis of 5,
followed by coupling with the N-deprotected thiazole 14 using
HBTU, Scheme 5. Methyl ester hydrolysis of 15, and coupling with
the N-deprotected oxazole 16, rendered the tris-azole compound
2
3
1
7. C- and N- deprotection of the linear precursor 17 was
achieved using aqueous KOH and then HCl/dioxane. Macrocycliza-
tion reaction was performed in diluted conditions (0.001 M) using
2
4
HBTU. The desired macrocycle 4, which resulted in a stable com-
2
5
1
4
15
15
pound, was obtained in 40%.
5,
6, and 7, Scheme 1. The presence of these heterocycles in
the open precursor of 4, could rigidized the hexapeptide and facil-
The antimalarial activity of the macrocycle 4 was determined
in vitro against the chloroquine-resistant K1 strain of P. falciparum
by using the [3H]hypoxanthine incorporation method reported by
itate the macrocycle formation.16
Different methodologies were employed in order to obtain
azoles 5, 6 and 7 using aminoacids as starting materials.
2
6
Desjardins et al. This compound displays an IC50 value of 0.18 lM
O
i)
Cl
O
CCl3
O
Br
Py, EtOH, reflux
5%
OEt
DIPEA/NH3 aq/THF
BocHN
S
O
S
ii) Lawesson reagent
THF, reflux.
N
BocHN
Boc-Gly-OH
NH2
62% (two steps)
EtO
5
O
6
8
Scheme 2. Synthesis of building block 5.
O
O
OMe
OH
O
H
OMe
O
N
L
-Thr-OMe
i) DAST/ CH Cl
2 2
BocHN
N
H
ii) BrCCl / DBU
HBTU, CH Cl
3
2
2
NHBoc
Boc-L-Ile-OH
80 %
71%
9
6
Scheme 3. Synthesis of building block 6.

4
996
S. Peña et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4994–4997
O
OMe
OH
O
OMe
L
-Ser-OMe
O
DAST
HBTU, CH Cl
BocHN
CH Cl
2
2
BocHN
2
2
N
N
H
O
Boc-L-Ile-OH
91 %
68 %
1
0
12
O
OMe
OH
OMe
N
H S (g)
NHBoc
H
2
S
i) DAST/ CH Cl
2
2
MeOH:Et N
BocHN
O
3
ii)BrCCl /DBU
3
N
H
S
8
5%
98% (two steps)
11
7
Scheme 4. Synthesis of building block 7.
5
7
KOH aq
THF
HCl (g)
Dioxane
quant.
quant.
S
N
BocHN
S
N
OMe
BocHN
NH
H
2
.HCl
N
O
O
2
i) HBTU/CH Cl2
HN
15
S
N
HO
O
65%
MeO C
H
2
S
13
14
OMe
O
TFA
N
NH .TFA
i) KOHaq/THF
6
2
quant.
ii) HBTU/CH Cl
2
2
O
H
52 %
16
O
NHBoc
CO Me
S
2
S
N
O
H
N
O
H
N
i) KOH aq/THF
ii) HCl/Dioxane
N
N
H
NH
NH
HN
O
HN
H
O
N
iii) HBTU/DCM
N
O
O
4
0%
H
S
S
4
17
Scheme 5. Synthesis of macrocycle 4.
showing enhanced activity when compared with its analog aerucy-
clamide B (IC50 = 0.7 M).
Supplementary data
l
In summary, the total synthesis of the previously predicted ana-
log of aerucyclamide B, compound 4, was accomplished through a
convergent macrocycle-assembly strategy from oxa/thiazole frag-
ments in good yield. The experimental data of the synthesized
macrocycle should assist in identification of this metabolite from
Mycrocycstis aeruginosa.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.028. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
This stable compound shows 4 times more in vitro antimalarial
activity than aerucyclamide B.
We are currently investigating the synthesis and biological
evaluation of new analogs of macrocycle 4.
1
2
3
.
.
.
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(a) Qin, S. Y.; Xu, X. D.; Chen, C. S.; Chen, J. X.; Li, Z. Y.; Zhuo, R. X.; Zhang, X. Z.
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Acknowledgments
This work was supported by Grants from ANII (FCE2720), CSIC
Grupos (UdelaR) and PEDECIBA.
3
7, 664; (e) Jollife, K. A. Supramol. Chem. 2005, 17, 81; (f) Singh, Y.; Sokolenko,

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6395.
6
.
22. Experimental data for compound 15: R
f
= 0.3 (EtOAc:hexane, 1:1); [
3
a
]
D
= À2.8 (c
1
1
2
2.45, CH Cl
2
); H NMR (400 MHz, CDCl ) d 0.95 (t, 3H, J = 7.4 Hz), 0.97 (d, 3H,
K. J. Nat. Prod. 1891, 2008, 71.
J = 6.8 Hz), 1.22–1.33 (m, 1H), 1.47 (s, 9H), 1.56–1.67 (m, 1H), 2.32–2.42 (m,
1H), 3.94 (s, 3H), 4.63 (d, 2H, J = 5.9 Hz), 5.32–5.40 (m, 2H), 7.97 (d, 1H,
7
8
.
.
Ogino, J.; Moore, R. E.; Patterson, G. M. L.; Smith, C. D. J. Nat. Prod. 1996, 59, 581.
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J = 9.3 Hz), 8.05 (s, 1H), 8.11 (s, 1H); 13C NMR (100 MHz, CDCl
) d 11.3, 16.0,
24.8, 28.3 (3C), 39.4, 42.3, 52.5, 55.6, 80.6, 124.3, 127.3, 147.1, 149.1, 155.6,
160.7, 161.8, 169.6, 171.8. HRMS m/z calcd for NaO (M+Na)
3
9
.
World Malaria Report, 2011; World Health Organization: 2011.
C
20
H
28
N
4
5 2
S
1
1
0. Burrows, N. J.; Chibale, K.; Wells, T. N. C. Curr. Top. Med. Chem. 2011, 11, 1226.
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Wiley: New Jersey, 2003. Vol. 60, Part B.
491.1399, found 491.1414. IR
1481, 1276, 1248, 1217, 1167 cm
m
max (liquid film) 2968, 2933, 1718, 1701, 1670,
À1
.
23. Experimental data for compound 17: R
f
= 0.38 (EtOAc:hexane, 3:2); [
a
]
D
= À5.0
1
1
(c 0.4, MeOH); 1H NMR (400 MHz, CDCl
3
) d 0.92–1.02 (m, 12H), 1.24–1.33 (m,
2H), 1.46 (s, 9H), 1.58–1.66 (m, 2H), 2.11–2.18 (m, 1H), 2.24–2.34 (m, 1H), 2.62
(s, 3H), 3.89 (s, 3H), 4.62–4.67 (m, 1H), 5.26 (dd, 1H, J = 7.6 Hz, J = 9.3 Hz), 5.40
3. (a) Peña, S.; Scarone, L.; Manta, E.; Serra, G. Chem. Heterocycl. Compd. 2011, 47,
7
03; (b) Sellanes, D.; Campot, F.; Nuñez, I.; Lin, G.; Espósito, P.; Saldaña, J.;
(dd, 1H, J = 6.2 Hz, J = 9.1 Hz) 5.54 (dd,
J = J = 5.8 Hz, 1H), 7.91 (dd,
1 2
13
Domínguez, L.; Manta, E.; Serra, G. Tetrahedron 2010, 66, 5384; c) Scarone, L.;
Fajardo, J.; Saldaña, J.; Domínguez, L.; Espósito, P.; Dematteis, S.; Wipf, P.;
Manta, E.; Serra, G. Lett. Drug Des. Disc. 2009, 6, 413.
J
1
2
= J = 9.6 Hz, 1H), 8.03 (s, 1H), 8.09 (s, 1H); C NMR (100 MHz, CDCl ) d
3
11.2, 11.5, 12.1, 15.5, 15.9, 24.8, 25.3, 28.3 (3C), 39.1, 39.5, 42.4, 51.5, 52.0, 55.5,
80.5, 123.6, 124.5, 127.5, 149.0, 149.4, 155.7, 156.2, 160.6, 160.7, 161.8, 162.7,
1
1
1
4. Houssin, R.; Lohez, M.; Bernier, J. L.; Henichart, J. P. J. Org. Chem. 1985, 50,
42 6 7 2
170.1, 171.1. HRMS m/z calcd for C30H N NaO S [M+Na]+ 685,2454 found
2
787.
5. Wagner, B.; Schumann, D.; Linne, U.; Koert, U.; Marahiel, M. A. J. Am. Chem. Soc.
006, 128, 10513.
685.2447. IR
1130, 1101 cm
m
max (liquid film) 2959, 2928, 2872, 2857, 1719, 1654, 1544, 1182,
.
À1
2
24. Experimental data for macrocycle 4: R
(c 0.4, MeOH); 1H NMR (400 MHz, CDCl
2H), 1.50–1.59 (m, 1H), 1.63- 1.74 (m, 1H), 2.10–2.18 (m, 2H), 2.68 (s, 3H), 4.71
(dd, 1H, J = 2.6 Hz, J = 18.0 Hz), 5.07 (dd, 1H, J = 5.6 Hz, J = 18.0 Hz), 5.31 (dd,
1H, J = 3.8 Hz, J = 8.5 Hz), 5.44 (dd, 1H, J = 6.0 Hz, J = 8.5 Hz), 8.08 (s, 1H),
8.16 (s, 1H), 8.42–8.50 (m, 3H); C NMR (100 MHz, CDCl ) d 11.6, 11.7, 11.8,
f
= 0.37 (EtOAc:hexane, 3:2); [
a
]
D
= À56.0
6. (a) Ehrlich, A.; Heyne, H.-U.; Winter, R.; Beyermann, M.; Haber, H.; Carpino, L.
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3
) d 0.93–1.04 (m, 12H), 1.26–1.37 (m,
1
2
1
2
1
2
1
2
13
3
14.8, 15.2, 25.1, 26.0, 39.9, 40.8, 41.0, 52.1, 55.0, 123.1, 124.3, 128.3, 148.7,
1
1
7. Hantzsch, A. Ann. Chem. 1888, 249, 1.
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C
24
H
30
N
6
NaO S
4 2
(M+Na) 553.1668, found 553.1649. IR
m
max (liquid film) 3400,
À1
2
, 1165.
3123, 2965, 2930, 2860, 1670, 1539, 1491 cm
.
1
2
9. Nishio, T. J. Chem. Soc., Perkin Trans. 1 1993, 1113.
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25. Macrocycle 4 was stable even at room temperature.
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Chemother. 1979, 16, 710.