Potent algicides based on the cyanobacterial alkaloid nostocarboline

Article abstract of DOI:10.1021/ol052968b

Nostocarboline and seven derivatives were prepared and displayed minimal inhibitory concentration (MIC) values ≥100 nM against the growth of Microcystis aeruginosa PCC 7806, Synechococcus PCC 6911, and Kirchneriella contorta SAG 11.81, probably via the in

Full text of DOI:10.1021/ol052968b

ORGANIC
LETTERS
2006
Vol. 8, No. 4
737-740
Potent Algicides Based on the
Cyanobacterial Alkaloid Nostocarboline
Judith F. Blom,† Tobias Brutsch,‡ Damien Barbaras,‡ Yann Bethuel,‡
1
Hans H. Locher,§ Christian Hubschwerlen,§ and Karl Gademann*,‡
Laboratorium fu¨r Organische Chemie, ETH Zurich, CH-8093 Zu¨rich,
Limnological Station, Institute of Plant Biology, UniVersity of Zu¨rich, Seestr. 187,
CH-8802 Kilchberg, and Actelion Percurex AG, Schwarzwaldallee 215,
WRO-1058, CH-4058 Basel
gademann@org.chem.ethz.ch
Received December 8, 2005
ABSTRACT
Nostocarboline and seven derivatives were prepared and displayed minimal inhibitory concentration (MIC) values
g
100 nM against the growth
of Microcystis aeruginosa PCC 7806, Synechococcus PCC 6911, and Kirchneriella contorta SAG 11.81, probably via the inhibition of
photosynthesis. The natural product hybrid nostocarboline/ciprofloxacin displayed additional antibacterial activity against several Gram-
negative bacteria (MICs
g0.7 µM). Nostocarboline can thus be considered a potent, selective, readily available, natural algicide.
The unspecific adsorption of bacteria, cyanobacteria, algae,
and higher organisms to submerged surfaces (i.e., biofouling)
constitutes a significant challenge in marine and freshwater
environments.1 The impact of biofouling on ships, pipelines,
oil platforms, nuclear power plants, and the like causes
significant financial consequences.1,2 These problems are
accentuated as the use of organotin antifouling paints is
increasingly banned all over the world.2 In addition, resis-
tance of aquatic species to Cu contributes to the need of novel
antifouling compounds.1,3 Many benthic aquatic organisms
secrete such compounds for deterrence purposes, and the use
of such natural products offers the possibility of identifying
novel lead structures.3 We recently reported the isolation of
the cholinesterase inhibitor nostocarboline (4),4 a carbolinium
alkaloid from the cyanobacterium Nostoc 78-12A.5 In this
communication, we demonstrate that nostocarboline (4) and
derivatives are quickly inhibiting the growth of phytoplanktal
organisms (i.e., cyanobacteria and green algae) and thus
possess potential antifouling activities.
The synthesis of derivatives of nostocarboline is straight-
forward (Scheme 1). Following modified literature proce-
dures,6 chlorination using different conditions of norharmane
gives both the 6-chloronorharmane (1) and the 6,8-dichlo-
ronorharmane (2), which are both separable by flash chromato-
† Institute of Plant Biology, University of Zu¨rich.
‡ Laboratorium fu¨r Organische Chemie der ETH Zu¨rich.
(4) Becher, P. G.; Beuchat, J.; Gademann, K.; Ju¨ttner, F. J. Nat. Prod.
2005, 68, 1793-1795.
§ Actelion Percurex AG.
(1) Review: Fusetani, N. Nat. Prod. Rep. 2004, 21, 94-104.
(2) Overview: Champ, M. A. Sci. Total EnViron. 2000, 258, 21-71.
(3) Abarzua, S.; Jakubowski, S.; Eckert, S.; Fuchs, P. Bot. Mar. 1999,
42, 459-465. Reviews: Bhadury, P.; Wright, P. C. Planta 2004, 219, 561-
578. Gross, E. M. Crit. ReV. Plant Sci. 2003, 22, 313-319. Armstrong, E.;
Boyd, K. G.; Burgess, J. G. Biotechnol. Annu. ReV. 2000, 6, 221-241.
Smith, G. D.; Doan, N. T. J. Appl. Phycol. 1999, 11, 337-344.
(5) This Nostoc strain, isolated from a wastewater lagoon, was reported
as anticyanobiotic, with the responsible compound not being determined;
see: Flores, E.; Wolk, C. P. Arch. Microbiol. 1986, 145, 215-219.
(6) Nakano, K.; Suyama, K.; Fukazawa, H.; Uchida, M.; Wakabayashi,
K.; Shiozawa, T.; Terao, Y. Mutat. Res. 2000, 470, 141-146. Ponce, M.
A.; Tarzi, O. I.; Erra-Balsells, R. J. Heterocycl. Chem. 2003, 40, 419-
426.
10.1021/ol052968b CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/25/2006

three organisms tested) and induced rapid killing of
cyanobacterial cells at 10 µM within 24 h. A change in the
phenotype of Synechococcus could already be observed
at a 100 nM concentration of nostocarboline (4). Structure
activity studies revealed that the quaternary group
appears to be essential for biological activity, as the
demethylated compounds 1 and 2 were found to be
inactive.8 The degree of chlorination (e.g., 3 vs 4 vs 7)
had a small impact on biological activity, as a 10-fold
decrease in activity for 3 and 7 against M. aeruginosa
was observed. The replacement of the substituent on the 2-N
atom led to an increase in activity of the Bn derivative
6 against Synechococcus (MIC ) 100 nM) compared to
the parent natural product. Replacing the methyl in 4
with the allyl in 5 results in a 10-fold loss of activity
against M. aeruginosa but retained activity against
Synechococcus. It is interesting to note that all compounds
induced rapid killing of cells above the MPC concen-
tration.
Scheme 1. Preparation of Nostocarboline (4) and Derivatives
The activity of nostocarboline and derivatives can be
compared to ciprofloxacin, a known and potent inhibitor of
cyanobacterial growth.9 Nostocarboline (4) displayed in-
creased activity against Synechococcus and is also active
against the eukaryote K. contorta compared to ciprofloxacin
which is selective for prokaryotic organisms.
graphy. Alkylation of the mono-Cl species 1 with different
electrophiles resulted in nostocarboline (4), the allyl deriva-
tive 5, and the Bn derivative 6. Similarly, 8-Cl-nostocarboline
(7) as well as the deschloronostocarboline 3 were also
obtained in good yields.
Compounds 1-7 were then evaluated against the growth
of the toxic cyanobacterium Microcystis aeruginosa PCC
7806 and the nontoxic cyanobacterium Synechococcus PCC
6911. In addition, Kirchneriella contorta SAG 11.81 was
selected as a member of the eukaryotic green algae. Growth
curves were measured over 280 h, and the minimal inhibitory
concentration (MIC) and the minimal phytotoxic concentra-
tion (MPC) were determined at 170 h after addition of the
different compounds (Table 1).7
Moreover, IC50 values were obtained to better characterize
the effect of nostocarboline (4) on growth (Figure 1). The
Table 1. Biological Activity of Compounds 1-7, 9, and
Ciprofloxacin to the Growth of M. aeruginosa (Ma),
Synechococcus (SC), and K. contorta (KC)
MIC (µM)a
MPC (µM)b
compound
MA
SC
KC
MA
SC
KC
1
2
3
4
5
6
7
9
>100 >100 ndc
>100 >100 ndc
>100 >100 ndc
>100 >100 ndc
10
1
10
10
10
1
1
1
1
0.1
1
ndc
1
10
10
10
10
10
1
50
10
10
10
10
50
ndc
>100
ndc
ndc
ndc
10
ndc
ndc
ndc
10
Figure 1. Effect of nostocarboline (4) on Microcystis aeruginosa
(white squares), Synechococcus (black circles), and Kirchneriella
contorta (white triangles). The graph shows the inhibition of the
growth of the phytoplankton species in comparison to the control
on day 7 after addition of nostocarboline.
1
ciprofloxacin
1
10
>100 >100 >100 >100
a MIC refers to the minimal inhibitory concentration at which reduction
of growth (OD675nm) vs the control is observed. b MPC refers to the minimal
phytotoxic concentration at which reduction of the OD675nm compared to
the time of addition was observed. c nd ) not determined.
IC50 values for the inhibition of the growth of the three
different phytoplankton species were determined after 7 days
(8) In the literature, harmane was shown to possess cyanobacteriocidal
activity (30 µg/disk); see: Kodani, S.; Imoto, A.; Mitsutani, A.; Murakami,
M. J. Appl. Phycol. 2002, 14, 109-114.
(9) Robinson, A. A.; Belden, J. B.; Lydy, M. J. EnViron. Toxicol. Chem.
2005, 24, 423-430. Halling-Sorensen, B.; Lutzhoft, H. C. H.; Andersen,
H. R.; Ingerslev, F. J. Antimicrob. Chemother. 2000, 46, 53-58.
Nostocarboline (4) was shown to be a potent inhibitor of
cyanobacterial and algal growth (MIC ) 1 µM against the
(7) See Supporting Information for full growth curves.
738
Org. Lett., Vol. 8, No. 4, 2006

to be IC50 ) 2.1 µM for M. aeruginosa, IC50 ) 5.8 µM for
Synechococcus, and IC50 ) 29.1 µM for K. contorta. These
results show the sensitivity of the toxic cyanobacterium M.
aeruginosa to nostocarboline (4).
The conjugate 9 was thus obtained via the known product 8
of the reaction of ciprofloxacin with 1,4-di(chloromethyl)-
benzene13 and subsequent alkylation of 6-Cl-norharmane (1),
shown in Scheme 2. Compound 9 retained the broad activity
Nostocarboline (4) was also tested against the growth of
its producer, Nostoc 78-12A, and showed a pronounced
reduced activity as a MIC value of 50 µM and a high MPC
value of 100 µM were determined. This large difference in
MIC values for autoinhibition (50 µM) to the MPC values
for competing organisms (10 µM) results in sustained growth
of the producing organism with concomitant killing of
competing phytoplankton. Moreover, significant amounts of
nostocarboline (4) were detected in standing cultures of
Nostoc 78-12A by HPLC-MS. Thus, these results point to
the presumed ecological role of nostocarboline (4) by
inducing competitive advantage via secretion.
Scheme 2. Preparation of the Quinolone Carboline Hybrid 9
To elucidate the mode of action of nostocarboline (4),
growth experiments were performed with an initial growth
phase in the dark. Virtually no inhibitory action was observed
in that period for nostocarboline (4) for concentrations of
up to 50 µM, indicating that nostocarboline activity is
correlated to the photosynthesis of M. aeruginosa.3,10 This
notion is supported by the fact that nostocarboline (4) was
not active against the growth of pathogenic nonphotosyn-
thetic bacteria (different strains of Staphylococcus, Entero-
coccus, Pseudomonas, Streptococcus, Haemophilus, Mo-
raxella, Escherichia) and yeast up to concentrations of 93
µM (see also Table 2). This selective inhibitory activity to
of nostocarboline (4) against photoautotrophs and gained
activity against eukaryotic organisms when compared to
ciprofloxacin (Table 1). Moreover, the hybrid 9 displays
antibacterial activity against several Gram-negative strains
(Table 2), whereas the parent nostocarboline (4) was inactive.
However, a loss of 1 to 2 orders of magnitude compared to
ciprofloxacin was observed. Some of this reduced activity
can be explained by efflux phenomena, as the hybrid 9
displayed increased activity against an efflux deficient
Escherichia coli strain (0.7 µM).
In conclusion, nostocarboline and its derivatives 3-7
display potent cyanobacteriocidal and algicidal activity
against photosynthetic aquatic organisms. The benefits of
nostocarboline (4) include (a) potent and fast reduction of
phytoplankton growth, (b) cheap and simple preparation, (c)
the biogenic nature offering benefits in its potential registra-
tion (“natural algicide”), (d) selectivity to photosynthetic
organisms, and (e) a structure which is amenable to easy
modification resulting in more potent derivatives such as 6
or the natural product hybrid 9. For all these reasons,
nostocarboline (4) can thus be considered a promising lead
structure for the development of algicides addressing the
worldwide need for effective, biogenic, and simple antifoul-
ing agents. Current efforts are directed toward the elucidation
of the mode of action of nostocarboline (4), a general
evaluation of its antifouling properties, and the related in
Table 2. Antibacterial Evaluation of Nostocarboline (4), the
Nostocarboline Ciprofloxacin Hybrid 9, and Ciprofloxacina
strainsb
4
hybrid 9
cipro
S. aureus
H. influenzae
M. catarrhalis
E. coli ATCC 25922
E. coli A-337
>93
>93
>93
>93
>93
42.4
10.6
21.2
10.6
0.7
1.5
e0.09
e0.09
e0.09
e0.09
a Values are given in µM. b The strains surveyed were Staphylococcus
aureus ATCC 29213, Haemophilus influenzae A-921, Moraxella catarrhalis
A-894, Escherichia coli ATCC 25922, and Escherichia coli A-337 (TolC
efflux deficient).
photoautotrophs can be rationalized by the structural similar-
ity of nostocarboline (4) to known and potent algicides such
as diuron/monuron or herbicides such as paraquat. Detailed
mechanistic studies on the photosynthesis inhibition of
nostocarboline (4) are thus warranted.
The natural product hybrid11 9 was targeted next, as this
quinolone chimera12 could possess a dual mode of action.
(10) Several cyanobacterial metabolites inhibit photosynthesis in compet-
ing organisms. For examples, see: Hagmann, L.; Ju¨ttner, F. Tetrahedron
Lett. 1996, 36, 6539-6542. Todorova, A. K.; Ju¨ttner, F. J. Org. Chem.
1995, 60, 7891-7895.
(11) Review on natural product hybrids: Tietze, L. F.; Bell, H. P.;
Chandrasekhar, S. Angew. Chem., Int. Ed. 2003, 42, 3996-4028.
(12) For other quinolone chimeras, see, for example: Hubschwerlen, C.;
Specklin, J.-L.; Sigwalt, C.; Schroeder, S.; Locher, H. H. Bioorg. Med.
Chem. 2003, 11, 2313-2319.
(13) The electrophile was prepared according to: Kerns, R. J.; Rybak,
M. J.; Kaatz, G. W.; Vaka, F.; Cha, R.; Grucz, R. G.; Diwadkar, V. U.
Bioorg. Med. Chem. Lett. 2003, 13, 2109-2112.
Org. Lett., Vol. 8, No. 4, 2006
739

vivo properties such as stability and biodegradation proper-
ties.
Nationalfonds (Grant Nr. 200021-101601/1) and the ETH
(Grant TH-13/04-3) is gratefully acknowledged.
Supporting Information Available: Synthetic proce-
dures, full characterization of compounds, copies of spectra,
biological experiments, and growth curves. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. K.G. thanks Prof. Dr. Erick M.
Carreira for financial support in the context of his habilitation.
We thank Prof. Dr. Friedrich Ju¨ttner for valuable discussions.
M. Gaertner is acknowleged for excellent technical as-
sistance. Financial support from the Schweizerischer
OL052968B
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Org. Lett., Vol. 8, No. 4, 2006