Synthesis of marine-derived 3-alkylpyridinium alkaloids with potent antiprotozoal activity

Article abstract of DOI:10.1021/ml200160k

Given the pressing need for new antiprotozoal drugs without cross-resistance with current (failing) chemotherapy, we have explored 3-tridecylpyridinium alkaloids (3TPAs), derivatives of viscosamine, as antiparasitic agents. We have developed a simple synthetic route toward viscosamine and related cyclic and linear monomers and oligomers. Evaluation for cytotoxicity on the protozoan parasites Trypanosoma brucei, Leishmania spp., and Plasmodium falciparum revealed several 3TPAs with antiprotozoal activity in the nanomolar range. Their promising selectivity index in vitro prompted us to study the dynamics of cytotoxicity on trypanosomes in more detail. Parasites were killed relatively slowly at therapeutically safe concentrations, in a process that did not target the cell cycle. Clearance of T. brucei cultures was observed at drug concentrations of 1-10 μM.

Full text of DOI:10.1021/ml200160k

Letter
pubs.acs.org/acsmedchemlett
Synthesis of Marine-Derived 3-Alkylpyridinium Alkaloids with Potent
Antiprotozoal Activity
Boris Rodenko,*,†,‡,§ Mohammed I. Al-Salabi,‡ Ibrahim A. Teka,‡ William Ho,‡ Nasser El-Sabbagh,‡,∥
Juma A. M. Ali,‡ Hasan M. S. Ibrahim,‡,⊥ Martin J. Wanner,† Gerrit-Jan Koomen,†
and Harry P. de Koning*,‡
†Van't Hoff Institute for Molecular Sciences, University of Amsterdam, The Netherlands
‡College of Medical, Veterinary and Life Sciences, Institute of Infection, Immunity and Inflammation, University of Glasgow,
United Kingdom
S
* Supporting Information
ABSTRACT: Given the pressing need for new antiprotozoal
drugs without cross-resistance with current (failing) chemo-
therapy, we have explored 3-tridecylpyridinium alkaloids
(3TPAs), derivatives of viscosamine, as antiparasitic agents.
We have developed a simple synthetic route toward viscos-
amine and related cyclic and linear monomers and oligomers.
Evaluation for cytotoxicity on the protozoan parasites
Trypanosoma brucei, Leishmania spp., and Plasmodium
falciparum revealed several 3TPAs with antiprotozoal activity
in the nanomolar range. Their promising selectivity index in
vitro prompted us to study the dynamics of cytotoxicity on trypanosomes in more detail. Parasites were killed relatively slowly at
therapeutically safe concentrations, in a process that did not target the cell cycle. Clearance of T. brucei cultures was observed at
drug concentrations of 1−10 μM.
KEYWORDS: Alkylpyridinium alkaloids, synthesis, antitrypanosomal activity, antileishmanial activity, antiplasmodial activity,
cytotoxicity
toward viscosamine and related analogues. We have evaluated
bout half of the world's population lives in areas where
Aseriously pathogenic protozoan parasites, including their activity against a panel of parasites and studied aspects of
their effect on Trypanosoma brucei in particular.
Trypanosoma, Leishmania, and Plasmodium sp., are endemic
and cause significant morbidity, mortality, and economic hardship
(Supporting Information, text 1).1 Treatment of these parasitic
infections relies solely on chemotherapy. As these parasites have
evolved intricate immune evasion strategies, effective antiparasite
vaccines are not expected in the near future, despite considerable
efforts in this field.2,3 Severe adverse effects and resistance to
current drugs4−6 articulate the urgent demand for novel, safe, and
effective drugs.
We aim to develop antiprotozoal lead compounds that lack
cross-resistance with current chemotherapy. Marine organisms
are an abundant source of bioactive molecules, and the
3-alkylpyridinium alkaloids isolated from sponges of the order
Haplosclerida display antibacterial7 and anticancer8−10 activity.
However, there have been no reports on their antiprotozoal
potential. Here, we focus on the synthesis and antiprotozoal
evaluation of 3-tridecylpyridinium alkaloids (3TPAs) of the
viscosamine family, consisting of N-alkylated pyridinium units
and saturated C13 alkyl chains (Supporting Information, text 2).
Naturally occurring 3TPAs isolated to date include linear
and cyclic oligomers such as cyclostelletamine C (dimer)11 or
viscosamine (trimer).12 The first total synthesis of viscosamine
was based on the Zincke reaction.13 We present a simpler route
We based the synthesis of 3TPAs on a versatile protection
strategy of the pyridine nitrogen with a p-methoxybenzyl
(PMB) group.14 Pyridyl alkanol 1 is readily converted to
its PMB-protected iodide 3 (Scheme 1). Dimerization of
3-alkylpyridine 1 with 3 in refluxing acetonitrile gave alcohol 4,
which was similarly converted to corresponding iodide 5 and
condensed with a third molecule of 1 to yield trimeric alcohol
6. After transfer-deprotection of the PMB group in refluxing
pyridine macrocyclization, precursor 7 was obtained in pure
form by crystallization (57% yield starting from 4). The
corresponding iodide 8 was cyclized by refluxing in acetonitrile
(1 mM), which gave viscosamine 9 in 43% yield over two steps.
Unlike all of the dimeric bis-3-alkylpyridinium macrocycles that
we prepared before,14 trimeric iodide salt 9 refused to
crystallize and was purified by chromatography over Al2O3.
Ion exchange of the triiodide salt with Dowex 1X2-200 gave the
better water-soluble trichloride, also as an amorphous solid. A
comparable sequence was used to prepare cyclic tetramer 13.
Received: August 15, 2011
Accepted: October 5, 2011
Published: October 5, 2011
© 2011 American Chemical Society
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a
Scheme 1. Synthesis of 3-TPAs
a
Reagents and conditions: (a) PBMCl, KI, acetonitrile. (b) I2, TPP, imidazole, toluene-acetonitrile. (c) Acetonitrile, reflux. (d) Pyridine, reflux. (e)
R-I, ethyl acetate (for 14) or acetonitrile (for 15), reflux. TPP, triphenylphosphine.
PMB-alcohol 4 was deprotected to 10 in refluxing pyridine and
dimerized with iodide 5 to the corresponding tetrapyridine 11.
Deprotection, iodination, and macrocyclization gave the novel,
64-membered macrocycle 13 in crystalline form. Monomeric
3TPAs containing C1 or C4 alkyl chains on the pyridinium
nitrogen were easily synthesized by N-alkylation of pyridine 1,
yielding alcohols 14 and 15, and, following iodination, their
corresponding iodides 16 and 17, respectively. In short, we
generated viscosamine 9 in 21% yield in eight steps starting
from pyridyl alcohol 1 employing only four reaction types. The
convergent route reported before required nine steps and eight
different reaction types yielding viscosamine in 3% yield starting
from pyridyl alcohol 1.13 Our strategy constitutes an easy entry
toward the controlled synthesis of higher order (cyclic)
oligomers, for example, the novel tetrameric macrocycle 13.
The antitrypanosomal activity was tested against bloodstream
form (BF) T. brucei (Table 1). Cationic analogues alkylated on
the pyridine nitrogen all displayed submicromolar EC50 values,
except 14, which displayed an EC50 value of just over 2 μM.
Pyridyl alcohol 1, lacking a substituent on the pyridine nitrogen,
appeared to be much less toxic to these parasites. Cyclic
oligomers (9 and 13) displayed similar antitrypanosomal activity
as linear oligomers (7, 10, and 11) with EC50 values between
0.22 and 0.56 μM. Monomers 2, 3, 15, 16, and 17 were even
more active than the reference drug diminazene aceturate; the
most active compounds were 2 (EC50 = 50 nM) and 17 (EC50 =
14 nM); reference drugs pentamidine and cymelarsan displayed
at least an order of magnitude higher activity.
ΔTbat1, lacking the TbAT1/P2 aminopurine transporter15 and
consequently displaying reduced sensitivity to diamidines and
melaminophenyl arsenicals,16,17 and (b) B48, derived from
ΔTbat1, lacking activity of a second drug transporter, HAPT1,
and consequently highly resistant to diminazene, pentamidine,
and the melaminophenyl arsenicals.18,19 The trypanocidal
activity of the 3TPAs was not, as a class, substantially different
between wild-type and the two drug-resistant lines, suggesting
that uptake of this compound class is not dependent on the
known drug transporters and that cross-resistance with the
diamidine and melaminophenyl arsenical drugs does not occur.
The antileishmanial activity was tested against Leishmania
major promastigotes and Leishmania mexicana axenic amasti-
gotes. The data on L. major promastigotes revealed similar
trends as those with the African trypanosomes: monomers (2
and 15−17) generally show higher activity than oligomers (7,
9−11, and 13). On amastigotes, linear 3TPAs were more active
than cyclic derivatives. Among oligomers, the presence of three
heterocycles, as in 7, appeared optimal for toxicity against all
kinetoplastids studied. Cationic 3TPAs showed higher
leishmanicidal activity than the reference drug pentamidine,
currently in clinical use to treat leishmaniasis. The strong correla-
tion between the activities of the 3TPAs against Trypanosoma
and Leishmania sp. (r2 = 0.86, linear regression using T. brucei
wt and L. mexicana amastigote EC50 values) suggests a similar
mode of action against the different kinetoplastids.
Screening against the apicomplexan parasite Plasmodium
falciparum revealed viscosamine 9 and its linear precursor 7 as
the alkaloids with the highest antiplasmodial activity, with EC50
values of 53 and 68 nM, respectively2 orders of magnitude
less active than reference drug chloroquine. The trend observed
To assess potential cross-resistance with the current first-line
trypanocidal drugs, the 3TPAs were also tested against two
drug-resistant clonal lines, derived from T. brucei s427: (a)
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a
Table 1. Antiprotozoal and Cytotoxic Activities of 3-TPAs
T. brucei clone
L. major
promastigotes
L. mexicana
amastigotes
compd
T. brucei wt
T. brucei ΔTbAt1
B48
P. falciparum
HEK293
1
16 ± 3
0.049 ± 0.005
0.19 ± 0.04
0.22 ± 0.03
0.41 ± 0.02
0.40 ± 0.06
0.31 ± 0.04
0.56 ± 0.04
2.3 ± 0.1
0.16 ± 0.01
0.086 ± 0.019
0.014 ± 0.002
0.26 ± 0.05
0.0014 ± 0.0005
34 ± 4
0.084 ± 0.011
0.48 ± 0.10
0.27 ± 0.003
0.49 ± 0.03
0.62 ± 0.04
0.41 ± 0.03
0.66 ± 0.05
35 ± 1
>50
>50
7.5 ± 2.6
0.24 ± 0.05
0.22 ± 0.04
0.068 ± 0.015
0.053 ± 0.012
0.14 ± 0.04
0.16 ± 0.06
0.19 ± 0.02
1.5 ± 0.4
204 ± 10
21 ± 1
41 ± 1
16 ± 2
26 ± 2
25 ± 1
11 ± 2
14 ± 3
183 ± 4
48 ± 2
47 ± 3
26 ± 2
2
0.062 ± 0.010
0.20 ± 0.01
0.25 ± 0.01
0.28 ± 0.02
0.30 ± 0.02
0.33 ± 0.004
0.42 ± 0.05
2.7 ± 0.5
0.32 ± 0.01
1.0 ± 0.04
0.85 ± 0.08
0.72 ± 0.04
1.6 ± 0.08
1.1 ± 0.04
0.64 ± 0.04
2.7 ± 0.2
0.65 ± 0.03
0.40 ± 0.06
0.19 ± 0.04
0.81 ± 0.03
0.34 ± 0.19
0.23 ± 0.03
1.1 ± 0.02
4.8 ± 0.2
1.7 ± 0.08
0.28 ± 0.01
0.29 ± 0.04
24 ± 4
3
7
9
10
11
13
14
15
16
17
2.3 ± 0.1
0.26 ± 0.01
0.18 ± 0.03
0.20 ± 0.02
0.11 ± 0.02
0.034 ± 0.010
0.33 ± 0.04
0.31 ± 0.04
0.36 ± 0.002
0.51 ± 0.06
0.26 ± 0.06
0.31 ± 0.03
0.063 ± 0.008
7.9 ± 0.7
0.0032 ± 0.0007
0.00045 ± 0.00004
0.0078 ± 0.0014
diminazene
pentamidine
0.066 ± 0.018
0.0013 ± 0.003
0.21 ± 0.04
4.3 ± 0.02
8.8 ± 0.9
phenylarsine oxide 0.00066 ± 0.00013
cymelarsan
6.4 ± 2.3
0.0039 ± 0.0009
chloroquine
0.00067 ± 0.00018
a
Data are EC50 values in μM ± standard error of means (n ≥ 3). EC50 values were determined after at least 72 h of drug treatment.
for the kinetoplastids that monomers in general displayed
higher activity than oligomers is not seen with P. falciparum.
Yet, as also seen for the kinetoplastid parasites, linear trimer 7
was the most active compound among the oligomeric 3TPAs
(7, 10, and 11). The data suggest that either the 3TPAs act on
a different target in kinetoplastid and apicomplexan parasites or
that these compounds act on similar targets but that these are
differentially essential to either phylum. Nevertheless, the strong
in vitro activity seen against these evolutionary distinct phyla is
remarkable.
the drug induces growth arrest rather than direct cell lysis. We
investigated this by performing a series of flow cytometry
experiments scoring for total DNA content (as a cell cycle
indicator) and cell lysis and DNA fragmentation (as a marker
for apoptosis). Cultures were incubated for up to 48 h with
various drug concentrations, and at 24 and 48 h, duplicate
samples were taken. In one sample, incubated directly with PI,
the fluorescence correlated to the amount of DNA but only in
cells permeable to the dye (Figure 2, “lysis” panel). The other
sample was fixed and permeabilized with digitonin before
PI incubation so that all cells revealed their DNA content
(“DNA content” panel). The drug-free controls show a normal
distribution of DNA content,21 with the majority of cells having
one diploid set of chromosomes (2C) and those undergoing
mitosis two sets (4C), representing an unperturbed cell cycle.
At 24 h, the drug-free controls show no permeability to PI, and
even at 48 h, only a small number of cells had become
permeable, reflecting normal aging of a newly passaged culture
(“lysis” panel).21 Treatment with 0.3 μM monomer 17, shown
as a representative example, had almost no measurable effect
at 24 h, but a significant proportion of cells had become
PI-permeable at 48 h (“lysis” panel). Some of the lysed cells
display fluorescence below 100 units, indicating some DNA
degradation,21 but this likely occurred following cell death,
rather than causing it. The “DNA content” panel showed little
Without exception, the cytotoxicity of 3TPAs on human
HEK293 cells was much lower than their antiprotozoal activity.
Monomers showed the most favorable selectivity index
(EC50(HEK)/EC50(parasite) ratio) related to antitrypanosomal
activity: 2 (437-fold), 15 (299-fold), 16 (540-fold), and 17
(>1000-fold). The selectivity index for antileishmanial activity
was lower than for antitrypanosomal activity, yet with reasonable
values remaining for monomers 15 (145-fold) and 16 (149-fold).
The antiplasmodial selectivity was found to be promising for
viscosamine 9 (493-fold) and its linear precursor 7 (237-fold).
Given the promising selectivity indices of 3TPAs on T. brucei,
we investigated their trypanocidal dynamics in more detail. The
EC50 values in Table 1 were determined after 72 h of drug
treatment; to study the early toxic effects of 3TPAs on
trypanosomes, we used a real-time cell lysis assay based on
propidium iodide (PI) fluorescence.20 PI is unable to cross the
intact cell membrane; hence, it can only bind to DNA and RNA
(and thus fluoresce) when the cell membrane is compromised
(indicative of cell death). At high 3TPA concentrations
(50−100 μM), all trypanosomes died within the first 30 min
(Figure 1). We found that for monomers (2, 3, and 15−17),
only concentrations ≥100 × EC50 caused cell lysis within the 8
h of duration of the experiment. In contrast, as little as 780 nM
dimer 10 (2 × EC50) caused full cell lysis within 3 h, whereas
higher order oligomers (7, 9, 11, and 13) showed rapid lysis at
10−15 × EC50.
It appears that monomeric 3TPAs (2, 3, and 15−17), even at
concentrations well above their EC50 values (Table 1;
determined after 72 h of drug incubation), kill trypanosomes
slowly. This could be due to induction of apoptosis or because
Figure 1. Time to reach 80% lysis of BF T. brucei vs concentration of
3TPA as determined by the PI-based rapid lysis assay.
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Figure 2. Flow cytometry analysis of BF T. brucei cultures incubated in the presence or absence (controls) of monomer 17. “Lysis” panel: Cells were
incubated with PI for 10 min after exposure for 24 or 48 h to 17. “DNA content” panel: Cells were fixed with 70% methanol after incubation with 17
and treated with RNase prior to staining with PI and analysis by flow cytometry. Samples for DNA content and for lysis analysis were taken from the
same culture. Each panel represents the counting of 10000 cells. The experiment shown is representative of three similar experiments.
Figure 3. Reversibility of the effect of various concentrations of monomers 2 and 15−17 on the growth of BF T. brucei. Cultures (seeded at 2 × 104
cells) exposed to test compounds at the indicated concentrations were monitored over 72 h. Compounds were added at t = 0 h and removed by
washing cultures after 24 h (■), 48 h (▲), or not at all (○). Controls are drug-free cultures (▽).
evidence of DNA fragmentation, indicating that only the dead
cells had less than 2C DNA content and that no significant
DNA degradation occurred prior to cell death. This conclusion
is much strengthened by analysis of the culture incubated
with 1 μM 17: Even at 24 h, most of the cells had become
PI-permeable, and indeed, the lower panel shows that there are
relatively few cells left with 2C or 4C DNA content but that
nonetheless very few cells with fragmented DNA are
identifiable. As DNA fragmentation is believed to be an
inherent consequence of apoptosis, it is unlikely that the 3TPAs
exert their antikinetoplastid actions in this way.
The same experiments were performed with various other
alkaloids used in this study (see Figures S1 and S2 in the
Supporting Information). In no case was evidence for DNA
fragmentation observed after incubation with the 3TPAs, in
contrast to a series of choline-derived compounds that we
recently reported.21 Nor did the flow cytometry provide any
indication of specific cell cycle arrest, as the proportion of
cells 2C or 4C DNA content remained constant throughout a
48 h of incubation with concentrations of 3 × or 10 × EC50
(Figures 2 and S1 and S2 in the Supporting Information). This
result was confirmed using fluorescence microscopy after DAPI
staining (not shown). We conclude that unlike some
antitrypanosomal compounds,22 3TPAs do not cause growth
arrest at a particular check point in the cell cycle.
Slow-acting drugs may still induce a rapid cellular effect that
eventually leads to cell death. Diamidines, for example,
accumulate rapidly to high concentrations in the cell, leading
to cell death many hours later.18 Pentamidine, with an EC50
value of 1.4 nM on wild type T. brucei, has been shown to act
slowly on the parasite at therapeutic concentrations (i.e., ≤10 ×
EC50).20 Whether an agent needs only relatively short contact
at effective concentrations with the target cell or must be
maintained at effective levels until the entire cell population has
died has obvious consequences for the therapeutic efficacy of
the compound class in vivo. To investigate whether the 3TPAs
require continuous exposure, growth of BF T. brucei treated
with various concentrations of monomers was monitored over
72 h, while drugs were washed out after 24 and 48 h. Treatment
with 2 and 15−17 at 10 μM for 24 h showed complete
trypanocidal action (not shown), while at 1 μM only 17 and 2
cleared a trypanosome culture completely, requiring 24 and 48
h of exposure, respectively (Figure 3). At lower concentrations,
treatment with 2 and 17 was incomplete even at 72 h of drug
incubation. These results seem to confirm that the effect of
monomeric 3TPAs was reversible at low concentrations, as in
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reversibility at low concentrations represents the recovery and
resumed growth of affected organisms or whether the increase in
the number of parasites on withdrawal of the alkaloids is solely
due to replication of unaffected cells within the population.
Summarizing, we have developed an efficient route toward
3TPA monomers and oligomers, including viscosamine 9 and
the novel, cyclic tetramer 13. Cheap starting materials, easy
crystallization (except 9), and the absence of expensive
chromatographic steps make this method an attractive entry
to novel antiprotozoal agents. The 3TPAs showed submicro-
molar and several, even nanomolar, antiprotozoal activity. The
evolutionary distance between Plasmodium en Kinetoplastida is
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required exposure to 1−10 μM 3TPA. Given the small number
of compounds tested here, the hit rate is remarkable, and we are
confident that further chemical modification and systematic
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ASSOCIATED CONTENT
■
S
* Supporting Information
Additional text, compound synthesis, and characterization;
biological assays; and FACS histograms. This material is
available free of charge via the Internet at http://pubs.acs.org.
(16) de Koning, H. P.; Anderson, L. F.; Stewart, M.; Burchmore,
R. J.; Wallace, L. J.; Barrett, M. P. The trypanocide diminazene
aceturate is accumulated predominantly through the TbAT1 purine
transporter: Additional insights on diamidine resistance in African
trypanosomes. Antimicrob. Agents Chemother. 2004, 48, 1515−1519.
(17) Bray, P. G.; Barrett, M. P.; Ward, S. A.; de Koning, H. P.
Pentamidine uptake and resistance in pathogenic protozoa: Past,
present and future. Trends Parasitol. 2003, 19, 232−239.
(18) Bridges, D. J.; Gould, M. K.; Nerima, B.; Maser, P.; Burchmore,
R. J.; de Koning, H. P. Loss of the high-affinity pentamidine
transporter is responsible for high levels of cross-resistance between
arsenical and diamidine drugs in African trypanosomes. Mol.
Pharmacol. 2007, 71, 1098−1108.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: b.rodenko@nki.nl (B.R.) or Harry.De-Koning@
glasgow.ac.uk (H.P.d.K.).
Present Addresses
§Department of Chemical Biology, The Netherlands Cancer
Institute, Amsterdam, The Netherlands.
∥Department of Veterinary Pharmacology, Faculty of Veteri-
nary Medicine, Alexandria University, Egypt.
⊥Faculty of Science, Department of Zoology, Sebha University,
Libya.
(19) de Koning, H. P. Ever-increasing complexities of diamidine and
arsenical cross-resistance in African trypanosomes. Trends Parasitol.
2008, 24, 345−349.
Funding
(20) Gould, M. K.; Vu, X. L.; Seebeck, T.; de Koning, H. P.
Propidium iodide-based methods for monitoring drug action in the
Kinetoplastidae: comparison with the Alamar Blue assay. Anal.
Biochem. 2008, 382, 87−93.
(21) Ibrahim, H. M.; Al-Salabi, M. I.; El Sabbagh, N.; Quashie, N. B.;
Alkhaldi, A. A.; Escale, R.; Smith, T. K.; Vial, H. J.; de Koning, H. P.
M.I.A.-S., I.A.T., J.A.M.A., and H.M.S.I. were supported by
personal studentships from the Libyan government.
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