Total synthesis of the cyanobacterial metabolite nostodione A: Discovery of its antiparasitic activity against Toxoplasma gondii

Article abstract of DOI:10.1039/c4cc03904a

A total synthesis of the cyanobacterial natural product nostodione A is reported involving a convergent, diversity-oriented route. A small assemblage of structural analogues were prepared and their cytotoxicity and anti-invasion activity against the protozoal parasite Toxoplasma gondii is reported for the first time. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03904a

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Total synthesis of the cyanobacterial metabolite
nostodione A: discovery of its antiparasitic
activity against Toxoplasma gondii†
Cite this:DOI: 10.1039/c4cc03904a
Received 22nd May 2014,
Accepted 12th June 2014
J. McNulty,*a K. Keskar,a C. Bordon,b R. Yolkenb and L. Jones-Brandob
´
DOI: 10.1039/c4cc03904a
www.rsc.org/chemcomm
A total synthesis of the cyanobacterial natural product nostodione product of 4-hydroxyphenylpyruvric acid from L-tyrosine and
A is reported involving a convergent, diversity-oriented route. L-tryptophan.4b Nostodione A belongs to a small family of alkaloids
A small assemblage of structural analogues were prepared and that have been isolated from cyanobacteria in recent years including
their cytotoxicity and anti-invasion activity against the protozoal the dimeric scytonemin (2), (Fig. 1).5 In addition to the anti-
parasite Toxoplasma gondii is reported for the first time.
mitotic and proteosomal activities described above, these highly
UV-absorbing molecules are believed to serve a protective func-
Cyanobacteria (blue-green algae) isolated from marine, fresh- tion against solar radiation within the cyanobacterial colony and
water and terrestrial environments have proven to be a prolific are of interest as potential sunblock ingredients. The chemical
source of biologically active secondary metabolites with a wide synthesis of isoprenostodione was recently reported,6a while a
range of therapeutic potential.1 The indole-containing natural single report of the synthesis of both nostodione A (1)6b and
:
product nostodione A (1) (Fig. 1) was first isolated from the the dimeric scytonemin (2)6c have been reported by Martensson
terrestrial cyanobacterium Nostoc commune in 1994 and shown and co-workers. An enzymatic approach to the Scytonemin
to inhibit mitotic spindle formation.2 The same compound was monomer has also recently been reported.6d
subsequently isolated from the freshwater cyanobacteria Scytonema
We became interested in the synthesis and biological evalua-
hofmanni and shown to possess proteasome inhibitory activity.3 tion of nostodione A (1) for several reasons. Our research groups
Nostodione A exists as a thermodynamic mixture of the (E)- and recently initiated a joint program aimed at the discovery of novel
(Z)-conformational isomers shown (Fig. 1). The compound is believed small-molecules exhibiting biological activity against the parasite
to be biosynthesized from prenostodione,4a an oxidative coupling Toxoplasma gondii, the protozoan responsible for toxoplasmosis.7
From a structural viewpoint, nostodione A resembles several
a Department of Chemistry and Chemical Biology, McMaster University, Hamilton,
known oxidized, condensed indole alkaloids such as indirubin,
tryptanthrin8 and the pyrroloiminoquinones,9 examples of which
Ontario, Canada L8S 4M1. E-mail: jmcnult@mcmaster.ca;
Tel: +1-905-525-9140 ext. 27393
display activity to T. gondii, Fig. 1.
b Stanley Division of Developmental Neurovirology, Department of Pediatrics,
Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore,
Maryland, 21287, USA
Synthetic access to nostodione A would allow for a wider
screening of its biological activity allowing a complete assessment
of its therapeutic potential. In this paper we report a diversity-
oriented approach towards the synthesis of nostodione A itself, as
Fig. 1 Structure of the cyanobacterial secondary metabolite nostodione A (1) and related biosynthetic alkaloids.
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oxalyl chloride in diethyl ether yielding initially the monoacyl
chloride derivative (13). Attempts at intramolecular acylation
initially proved extremely challenging and a summary of select
examples en-route to optimisation of this reaction is collected in
Table S1 (ESI†). Attempted intramolecular acylation on the
immediate acid chloride (13) proved futile with a number of
bases and solvents (Table S1, entries 1 and 2, ESI†). The acid
chloride was converted to the ester (14) in order to probe the
intramolecular ester acylation.10 A wide range of conditions were
investigated for the conversion of (14) into the b-ketophos-
phonate (15) (see Table S1, ESI†). The use of sodium hydride
(NaH) in refluxing THF proved optimal and compound (15)
Fig. 2 Retrosynthetic analysis of nostodione A (1).
well as the synthesis of a mini-library of analogues and the could be isolated in reasonable yields of 55–60%. With the
discovery of cytotoxicity and anti-invasion activity of these deriva- b-ketophosphonate (15) now on hand, we began initial studies on
tives to T. gondii infection.
the critical HWE reaction as summarized in Scheme 2. Protection
Our retrosynthetic analysis of nostodione A (1) is outlined in of 4-hydroxybenzaldehyde with dihydropyran gave the unstable
Fig. 2. In order to conduct structure–activity evaluation of analogues 4-tetrahydropyranyl ether. This was reacted with the dianion
of (1) we considered a Horner–Wadsworth–Emmons (HWE)-type generated from (15) and the HWE reaction proceeded smoothly
disconnection of the 4-hydroxystyryl unit in (1) leading to the to give the THP-protected derivative of nostodione A (16) which was
b-ketophosphonate (3) and 4-hydroxybenzaldehyde, or protected immediately deprotected, completing the synthesis of nostodione A
derivative thereof. In the synthetic direction, b-ketophosphonate (3) in 68% isolated yield from (15). Synthetic nostodione A was isolated
was envisioned as an ideal nexus that would allow construction of as a yellow pigment with m.p. 285 1C (decomp), lit. m.p. 280 1C
an assemblage of nostodione A analogues via a diversity-oriented (decomp)2 and spectroscopic data in accord with the literature
HWE reaction (with ArCHO or RCHO) in the last step. It was values including an (E):(Z) ratio of 4 : 1 (acetone) for both our
envisioned that the b-ketophosphonate (3) could be accessed synthetic material and the natural product.2,6c This synthesis
through an intramolecular a-phosphonate acylation-type reaction10 constitutes the second reported total synthesis of nostodione A
from the 3-oxalylindole derivative (4), in turn, the product of and was achieved in only 8 chemical steps and 21.6% overall yield
acylation of the indole (5) with oxalyl chloride. Compound (5) from commercial ethyl indole-2-carboxylate (7).
was projected to be the product of a Michaelis–Arbuzov reaction
of a 2-halomethylindole derivative, ultimately derived from reaction from (15) as described. The HWE reaction proved successful
commercially available indole-2-carboxylic acid derivatives. with a variety of substituted benzaldehydes and allowed for the
We were now positioned to exploit the diversity-oriented HWE
The synthesis thus began with ethyl-indole-2-carboxylate (7), as assembly of the mini-library of aryl ring structural analogues (17) to
shown in Scheme 1. In exploratory studies, we determined that (22) summarized in Table 1. NMR analysis of the series of analogues
formation of the necessary 2-halomethylindole derivatives was uncovered a prominent substituent effect on the (E):(Z) ratio of
complicated in the presence of the free indole NH. Protection as these nostodione A derivatives. The (E)-isomer content was observed
the N-tosyl derivative (8) followed by reduction of the ester led to be predominant in all cases, including those containing either
efficiently to the desired alcohol (9). Conversion to the chloro- electron donating or withdrawing groups (ratio of (E)-isomer in
methyl derivative (10) now occurred smoothly and this reactive DMSO-d6: 4-CH3 (88%), 3,4-methylenedioxy (95%), 4-NO2 (498%),
intermediate underwent clean Michaelis–Arbuzov alkylation with tri- 4-OBn (94%), 4-OCH3 (94%), 4-Cl (83%)). These results indicate that
methylphosphite yielding phosphonate (11). Attempts at C3-acylation the free phenolic group in (1) may play a unique role in stabilizing
on intermediate (11) with oxalyl chloride met with failure, which the (Z)-isomer of nostodione A.
we attributed to N-tosyl-induced electronic deactivation at this
With nostodione A itself and the mini-panel of synthetic
position. Removal of the N-tosyl substituent was accomplished analogues available, we now assessed their antiprotozoal activity.
under mild conditions using TBAF.11 Direct acylation on the We employed a published colorimetric assay12 to assess both
deprotected phosphonate (12) now occurred smoothly with in vitro cell cytotoxicity and anti-Toxoplasma capabilities of the
Scheme 1 Synthesis of the 3-oxalyl-indole-2-phosphonomethyl intermediates (13), (14) and ketophosphonate (15).
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Table 1 Assembly of nostodione A mini-panel (1) and (17)–(22) via the HWE
strategy and anti-Toxoplasma biological activity
Scheme 2 Completion of the synthesis of nostodione A (1) employing
the HWE strategy.
Nostodione A
and analogues
Isolated
yield (%)
IC50
(mM)
IC90
(mM)
TD50
(mM)
Entry
1
TI
absorbance (570–650 nm) in wells containing drug, Toxoplasma,
and CPRG was compared to that in parasite control wells. In the
cytotoxicity wells, the bioreduction of the cell viability reagent by
viable cells into a soluble, colored formazan product was captured
by reading the plates at 490–650 nm. The median and 90%
inhibitory concentrations (IC50, IC90 respectively) and the median
cytotoxic dose (TD50) were calculated using CalcuSyn software
(Biosoft, Cambridge, U.K.). For each compound, a therapeutic index
(TI) was calculated with the formula TI = TD50/IC50. This number
reflects the specific activity of a compound against Toxoplasma.
Atovaquone, a broad spectrum anti-parasitic drug used therapeuti-
cally to treat many parasitic protozoan diseases including malaria,
was used as the assay positive control. The overall biological
activity data is summarized in Table 1.
Nostodione A (1) proved to have quite low specific activity
against T. gondii displaying an IC50 of 85 mM and a TI of 1. Of the
structural analogues of nostodione A, the lowest IC50 values were
displayed by the mono-substituted 4-alkoxy-substituted derivatives
alone (Table 1, entries 3 and 4). The 4-benzyloxy- and 4-methoxy-
derivatives exhibited IC50 values of 4.6 mM and of 5.6 mM respec-
tively. In contrast, the 4-methyl- and 3,4-methylenedioxy derivatives
were less potent, as were those containing electron withdrawing
groups 4-chloro- and 4-nitro (entries 2 and 5–7). More importantly,
the 4-benzyloxy-containing derivative (18) demonstrated low cyto-
toxicity with a therapeutic index of 470, significantly less cytotoxic
than the methoxy-containing analog (19). The results also show
clearly that the substituent effect on biological activity is not a
simple electronic effect, demonstrating no discernible quantitative
structure–activity correlation. The results point to the 4-benzyloxy
substituent as a key fragment on the anti-Toxoplasma pharmaco-
phore of nostodione A that is amenable to further optimisation of
both potency and selectivity. A preliminary evaluation of direct
effects of the compounds on extracellular tachyzoites was per-
formed. The commonly used red/green invasion assay12b allowed
68
82
68
72
55
42
44
85
183
114
30
108
1
8
2
3
4
5
6
21.6
4.6
5.6
27.8
19
172
Z320
25
70
5
44
103
78
161
6
23
1
7
8
50.6
0.2
166
0.6
219
21
4
Atovaquone
111
nostodione mini-library. Briefly, diluted compounds were added us to evaluate nostodione A and the mini-panel of structural
to human foreskin fibroblast (HFF; ATCC) cells growing in 96-well analogues for inhibition of host cell invasion by the tachyzoites
tissue culture plates. Beginning at 320 mM, the compounds were using fluorescent labels to distinguish tachyzoites that had actively
serially diluted across the plate by dilutions of 0.5 log 10. T. gondii penetrated (green bars) the cells from those that were attached but
RH-2F tachyzoites that constitutively express b-galactosidase unable to enter (red bars) the host cells. A decrease in the number
(b-gal) were then added to most wells, leaving 2 wells in each of penetrated parasites (Fig. 3, green bars) relative to the vehicle
column parasite-free for cytotoxicity testing. The substrate for [DMSO (VHL)] is indicative of invasion inhibition.
b-gal, chlorophenol red-b-D-galactopyranoside (CPRG), was added
As shown in Fig. 3 all of the compounds tested (20 mM)
to the Toxoplasma wells after 4 days of incubation at 37 1C/5% significantly inhibited tachyzoite invasion of the host cell. Further, in
CO2. Further incubation for 20 h was followed by addition of the this assay, an effect on attachment is defined as a difference in the
cell viability reagent, CellTiter 96 Aqueous One Solution Reagent total numbers of both penetrated and attached parasites relative to
(Promega Corp., WI) to the uninfected cytotoxicity wells. Color same of the vehicle.12b With the exception of the 4-methyl derivative
reactions in all wells were then read in a Vmax microplate reader (20), all of the test compounds significantly inhibited tachyzoite
(Molecular Devices, CA) after 3 h of incubation. The amount of attachment. The inhibition of attachment and the inhibition of
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Further elaboration on this lead compound toward the development
of a novel potent and selective anti-toxoplasmosis agent and
investigation of related antiprotozoal activity of nostodione A
and analogues from functionalised indole-2-carboxylates13 is
currently in progress. These preliminary results also indicate
interesting diversity in the structure–activity relationships
(SAR) of the compounds relevant to the inhibition of host cell
attachment and invasion by T. gondii tachyzoites. Further
biological studies to delineate the nature of such SARs in more
detail are in progress.
We thank NSERC (JMcN, KK) and the Stanley Medical Research
Institute (CB, RY, LJB, JMcN) for financial support of this work.
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inhibit invasion while not inhibiting attachment as is displayed
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Chem. Commun.
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