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porting Information). The a-amine of puromycin is carbamoy-
lated in 16 and 18, and so the intact conjugates should be in-
capable of incorporation into parasite polypeptides. Exposure
to ferrous iron in the parasite, however, is expected to release
free puromycin from 16 (but not from 18). Thus, the detection
of puromycin incorporation in parasites treated with 16 (but
not in those treated with 18) would provide strong evidence
for the trioxolane-mediated release of puromycin from 16. As
detailed below, this is precisely what we observed with conju-
gates 16 and 18.
First, we evaluated the antiplasmodial activity of puromycin
itself (19) as well as its two conjugates 16 and 18. As expected,
puromycin and its trioxolane conjugate 16 exhibited low nano-
molar activity against W2 parasites, whereas nonperoxidic con-
jugate 18 was ꢀ100-fold less potent (Table 1). This result con-
firms that the toxicity of puromycin is largely ablated when
conjugated at the a-amine, as expected. Next, we employed
puromycin (19), trioxolane–puromycin conjugate 16, and non-
peroxidic control 18 to study peroxide-dependent drug release
in live P. falciparum parasites. Synchronized trophozoite stage
parasites, the erythrocytic stage when protein synthesis is
most robust, were incubated with equimolar concentrations
(400 nm) of compound 19, 16, or 18 for up to 12 hours. Para-
sites were periodically released from erythrocytes by saponin
lysis, and the isolated parasites were analyzed for total puro-
mycin incorporation in parasite proteins using a “dot blot”
analysis with a-puromycin antibody (Figure S1 in the Support-
ing Information). Protein samples run on a sodium dodecyl sul-
fate–polyacrylamide gel electrophoresis (SDS–PAGE) gel and
analyzed by Western blotting with a-puromycin antibody con-
firmed that puromycin is incorporated broadly into the para-
site proteome (Figure S2 in the Supporting Information).
Figure 3. Quantification of puromycin incorporation in proteins of Plasmodi-
um falciparum parasites treated with 19, 16, or 18. Negligible puromycin in-
corporation was observed for dioxolane conjugate 18, indicating that puro-
mycin release from 16 is peroxide-dependent.
stages of treatment might reflect a lag in puromycin release
from 16, which is not unexpected given the results of our in
vitro drug release studies, where complete release of free drug
required 60–90 min (Figure 2). Alternatively, the difference
might simply reflect the toxic effects of a trioxolane-based
insult that is conferred to parasites by 16 but not 19. Thus, the
application of trioxolane 16 in excess of its EC50 value might
reasonably be expected to affect rates of parasite growth and
protein synthesis, leading to slower puromycin incorporation
initially. Whatever the case, the dot blot studies with 19, 16,
and 18 combined with the in vitro drug release studies de-
scribed herein serve to validate 3 as a competent scaffold for
drug delivery to P. falciparum parasites.
The results of the puromycin studies were unambiguous
and fully consistent with peroxide-dependent release of puro-
mycin from 16. Thus, parasites treated with either puromycin
(19) or its trioxolane conjugate (16) showed a time-dependent
increase in puromycin incorporation in parasite protein
(Figure 3; see also Figure S1 in the Supporting Information). In
contrast, parasites treated with dioxolane–puromycin conju-
gate 18 showed negligible puromycin incorporation over the
course of the experiment. This finding both confirms the per-
oxide-dependence of puromycin release from 16 and also
demonstrates that puromycin action and toxicity is largely ab-
lated while in conjugated forms (as revealed also by the ꢀ100-
fold higher EC50 value for 18 vs 19). The ability to block the ac-
tivity/toxicity of a drug species prior to release in the parasite
is a key advantage of trioxolane-mediated drug delivery and
distinguishes this approach from hybrid antimalarials, which
must necessarily remain active/toxic in their conjugated forms.
Also of note from these studies was the fact that puromycin
incorporation appeared to be greater in parasites treated di-
rectly with puromycin (19) than in those treated with 16. This
difference appears to be a consequence of more rapid puro-
mycin incorporation during the first hour in 19-treated para-
sites (Figure 3). After this initial burst, rates of incorporation are
similar (nearly equivalent slopes) between parasites treated
with 19 and those treated with 16. The difference in the early
In conclusion, we have described an improved chemical
scaffold for trioxolane-mediated drug delivery. The new mole-
cules 3 are of significantly lower molecular weight, and exhibit
superior drug-like properties when compared with our earlier
systems. An efficient, concise and scalable synthesis of key syn-
thetic intermediate 9 is described and should facilitate addi-
tional studies of these molecules in various applications. Final-
ly, we have demonstrated the utility of the aminonucleoside
puromycin as a chemical–biological marker to study drug re-
lease in P. falciparum. We anticipate that the use of puromycin
as a drug surrogate will enable future studies of trioxolane-
mediated drug delivery in malaria and other disease models.
Acknowledgements
ARR acknowledges the support of the US National Institutes of
Health (NIH) (R21AI0944233 and R01AI105106) and the Bill and
Melinda Gates Foundation (USA). The authors thank Prof. Mat-
thew Bogyo (Stanford University, USA) for helpful discussions and
suggestions.
Keywords: antimalarial agents · drug delivery · Plasmodium
falciparum · puromycin · targeted prodrugs · trioxolanes
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 5
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Fontaine, Shaun D.
