Design, synthesis, and biological evaluation of a series of simple and novel potential antimalarial compounds

Article abstract of DOI:10.1016/S0960-894X(01)00308-0

A series of compounds bearing an endocyclic -N-O- moiety with potential antimalarial activity based on simple derivatives of the tropolone purpurogallin was prepared by means of a hetero Diels-Alder reaction using nitrosobenzene as a dienophile. The rationale behind the design of these compounds is presented, together with the synthetic route to derivatives bearing aromatic and aliphatic esters of the C4′-position hydroxyl group of the purpurogallin framework, as well as biological data obtained from in vitro assays against Plasmodium falciparum and Trypanosoma cruzi. Several of the new compounds have activities in the 3-9 μM range, and provide leads for the development of a novel class of antiparasitic drugs with improved biological and pharmacological properties.

Full text of DOI:10.1016/S0960-894X(01)00308-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1851–1854
Design, Synthesis, and Biological Evaluation of a Series of Simple
and Novel Potential Antimalarial Compounds
Hongyu Ren,a Shannon Grady,a Daniela Gamenara,b Horacio Heinzen,b Patrick Moyna,b
Simon L. Croft,c Howard Kendrick,c Vanessa Yardleyc and Guillermo Moynaa,*
aDepartment of Chemistry & Biochemistry, University of the Sciences in Philadelphia, Philadelphia, PA 19104, USA
b
´
´
´
Departamento de Quımica Organica, Facultad de Quımica, CP 1157, Montevideo, Uruguay
cLondon School of Hygiene and Tropical Medicine, Department of Infectious and Tropical Diseases, London WC1E 7HT, UK
Received 12 December 2000; accepted 30 April 2001
Abstract—A series of compounds bearing an endocyclic –N–O– moiety with potential antimalarial activity based on simple deri-
vatives of the tropolone purpurogallin was prepared by means of a hetero Diels–Alder reaction using nitrosobenzene as a dieno-
phile. The rationale behind the design of these compounds is presented, together with the synthetic route to derivatives bearing
aromatic and aliphatic esters of the C40-position hydroxyl group of the purpurogallin framework, as well as biological data
obtained from in vitro assays against Plasmodium falciparum and Trypanosoma cruzi. Several of the new compounds have activities
in the 3–9 mM range, and provide leads for the development of a novel class of antiparasitic drugs with improved biological and
pharmacological properties. # 2001 Elsevier Science Ltd. All rights reserved.
Due to its high morbidity and mortality, human
malaria is an infectious disease of enormous importance
in tropical countries. Despite the eradication programs
that started over 80 years ago, malaria is still a threat
to over 2 billion people living in areas of high incidence.
Although the statistics vary widely, it is estimated that
there are 200 million infected humans, along with 150
million new cases every year. Plasmodium falciparum,
the causative agent of the most malignant forms of
malaria, is a particularly resistant parasite which is
known to have high adaptability by mutation. This
mutability makes quite likely the development of
resistance to vaccines and chemotherapies currently
being introduced.1 Even for drugs with novel modes of
action, such as artemisinin (Fig. 1a) and other natural
and synthetic endoperoxides, there is a high likelihood
that resistant Plasmodia strains will evolve. This would
be especially problematic, because there is high prob-
ability that the mechanism of resistance will involve the
inactivation of the endoperoxide moiety, thus rendering
these strains resistant to drugs with similar chemical
structure.
bond with active iron(II) species present in the proto-
zoan food vacuole, which catalyses the generation of
highly cytotoxic oxygen- and carbon-centered radicals.2
Although this has not been decisively proven to occur in
the parasite, the hypothesis is strongly supported by
results obtained from biomimetic studies.3 We note that
while the standard bond dissociation energy (BDE) of
the peroxo linkage (–O–O–) is 33.2 kcal/mol, the corre-
sponding BDE of the –N–O– linkage is 35.8 kcal/mol.4
Although the BDEs for these bonds are likely to change
when forming part of larger molecules, both moieties
will remain highly labile and prone to undergo homo-
litic cleavage. Therefore, oxazines could follow a reac-
tion pattern (i.e., –N–O– bond opening and radical
generation), and thus biological activity, related to that
of endoperoxides. However, the differences in chemical
structure could make these compounds unaffected by
the potential Plasmodium variants capable of inactivat-
ing endoperoxides which are likely to appear. In this
communication we report the design, preparation, and
biological evaluation of a series of such compounds
(Fig. 1b).
The trademark of artemisinin action has been proposed
to be the interaction between its reactive endoperoxide
A facile route for the preparation of oxazines involves
a hetero Diels–Alder reaction between an appropriate
diene and nitrosobenzene derivatives. The procedure
has been extensively documented in the literature and
can be applied to a large range of diene-containing
frameworks,5 and was therefore ideal for our purposes.
*Corresponding author. Fax: +1-215-596-8543; e-mail: g.moyna@
usip.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00308-0

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H. Ren et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1851–1854
For our exploratory series of compounds, we chose
nitrosobenzene as the source for the –N–O– func-
tionality, and derivatives of the benzotropolone
purpurogallin as dienes. These compounds are simple
to prepare and purify,6 and have been previously
employed as Diels–Alder dienes in several studies.7
Starting from trimethylpurpurogallin (1), 10 aliphatic
and aromatic esters of the C40-position hydroxyl group
were prepared in good yield by treatment of a CH2Cl2
solution of the benzotropolone with the appropriate
acyl chloride, triethylamine, and catalytic DMAP
(2a–2j, Fig. 2).
(2k) were also included in the series (3k and 3l), and
prepared following the same protocol employed for
compounds 3a–3j. The regioselectivity of the hetero
Diels–Alder reaction was first established to corre-
1
spond to that shown in Figure 2 from the H and 13C
NMR spectra of the products, and this was later
confirmed by X-ray diffraction studies performed on
oxazine 3l.9
The in vitro biological activity of the new compounds
was assayed against the protozoan parasites P. falci-
parum and Trypanasoma cruzi, and their toxicity eval-
uated against KB cells.10 The results are summarized in
Table 1. Despite the fact that all the compounds are at
least three orders of magnitude less active than chloro-
quine in the P. falciparum assay, most of them still have
activities in the micromolar concentration range. On the
other hand, oxazines 3a, 3f, 3j, and 3k exhibited activ-
ities comparable to benznidazole, one of the current
chemotherapies against T. cruzi.
The resulting esters were then treated with a slight
excess of nitrosobenzene at room temperature using
benzene as solvent. The Diels–Alder reaction pro-
ceeded cleanly, with the products crystallizing out of
solution (3a–3j, Fig. 2).8 In all cases, the starting dienes
were undetectable after 16 h. The Diels–Alder adducts
of 1 and of the simple ether tetramethylpurpurogallin
Figure 1. Chemical structures of artemisinin (a) and oxazines with antiprotozoal activity (b).
Figure 2. Route to oxazines 3a–3l. (i) R-COCl (1.5 equiv), Et3N (1.5 equiv), DMAP (0.5 equiv), CH2Cl2, rt, 4 to 8 h; (ii) Ph–N¼O (1.5 equiv),
benzene, 16 h. PhthN represents the phthalimide group (3j).

H. Ren et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1851–1854
1853
Table 1. In vitro antiprotozoal activity of oxazines 3a–3l against P.
falciparum (PF) and T. cruzi (TC)
absent ketones at the C3-position will be employed with
the goal of reducing the toxicity of the series. Finally, a
CoMFA study correlating electrostatic and steric con-
tributions to the activity and toxicity of the compounds
in the series is also underway. We are hopeful that these
additional studies will allow us to identify the structural
traits responsible for activity against different parasites,
and will ultimately lead us to the development of effec-
tive antiparasitic chemotherapeutic agents with novel
chemical structures. The results from these investiga-
tions will be reported in due course.
Compound
PF (mM)
TC (mM)
Toxicity (mM)
3a
3b
3c
3d
3e
3f
5.3
6.6
3.9
3.2
3.4
7.5
23.8
>70.5
>68.3
>63.9
>63.4
9.2
6.3
<0.7
3.2
128.2
11.8
6.5
3g
3h
3i
3j
3k
3l
6.3
>61.0
>59.0
>59.6
11.9
4.3
65.3
7.7
>59.0
6.6
6.1
29.1
13.3
89.8
<0.8
<0.8
5.4
8.9
Acknowledgements
Chloroquine (IC50 3.0 nM) and benznidazole (ED50 7.1 mM) were used
as positives for P. falciparum and T. cruzi, respectively. Toxicity was
assayed against KB cells, using podophyllotoxin (ED50 0.2 nM) as a
standard. All determinations were performed in triplicate.
The authors wish to thank Professor Joel M. Kauffman
and Dr. Farzad Kobarfard for insightful discussions
and comments. G.M. greatly acknowledges the financial
support provided by the Office of the Vice-President of
Academic Affairs, USP. Financial support from the
UNDP/World Bank/WHO Special Programme for
Research and Training in Tropical Diseases (T.D.R.) is
also acknowledged (S.L., H.K., and V.Y.).
Several of the compounds display relatively high toxi-
cities in the KB cell assay. This can be associated with
their somewhat low thermal stability and propensity to
liberate highly cytotoxic nitrosobenzene upon under-
going a retro Diels–Alder reaction. The kinetics of the
decomposition process have been studied in detail for
oxazine 3l, for which a half-life of approximately 10 h at
30 ꢀC in chloroform solution has been determined.9 This
could explain why compound 3k, for example, which
has high activity against both P. falciparum and T. cruzi
also shows one of the highest toxicities of the series.
However, several of the compounds with the highest
activities have low or moderate toxicities. This is espe-
cially true for compounds 3d and 3j, and seems to indi-
cate that activity is independent from toxicity. Of these,
oxazine 3d is particularly interesting. This compound
has the lowest toxicity of the series, one of the poorest
activities against T. cruzi, and the highest activity
against P. falciparum, indicating specificity against this
parasite. It is tempting to speculate that this compound
behaves as a prodrug in which the acetoxy group initi-
ally confers high permeability through the membrane,
and is later hydrolyzed in the acidic food vacuole to a
free hydroxyl group that prevents its escape out of the
parasite. A similar absorption mechanism has been
postulated for chloroquine and other aminoquinoline
derivatives.11 The poor activity in all assays and rela-
tively low toxicity of the pCl-benzoate derivative 3h can
also be rationalized in terms of its poor bioavailability.
This compound has very low solubility in both polar
and nonpolar solvents, which can be extrapolated to
low membrane permeability.
References and Notes
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8328. (b) Posner, G. H.; Oh, C. H.; Wang, D.; Gerena, L.;
Milhous, W. K.; Meshnick, S. R.; Asawamahasakda, W. J.
Med. Chem. 1994, 37, 1256.
4. Pauling, L. The Nature of the Chemical Bond, 3rd ed.; Cor-
nell University: New York, 1960; Chapter 3.
5. Boger, D. L.; Weinreb, S. N. In Organic Chemistry, A Ser-
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(b) Forbes, E. J.; Griffiths, J. J. Chem. Soc. 1968, C, 572. (c)
Forbes, E. J.; Griffiths, J. J. Chem. Soc. 1968, C, 575.
8. Compounds 3a–3l were fully characterized by IR, MS, and
1H and 13C NMR. For example: Oxazine 3a: mp 140–142 ꢀC
1
(dec.). H NMR d 2.41 (s, 3H), 3.64 (s, 3H), 3.81 (s, 3H), 3.90
(s, 3H), 5.31 (dd, J=1.0, 6.8 Hz, 1H), 6.12 (dd, J=1.0, 9.6 Hz,
1H), 6.72 (dd, J=6.8, 9.6 Hz, 1H), 6.78 (s, 1H), 7.17 (m, 5H);
13C NMR d 21.3, 53.5, 56.9, 61.8, 69.3, 103.1, 111.7, 118.9,
122.2, 124.1, 127.4, 129.9, 136.5, 138.6, 143.4, 147.5, 151.3,
157.4, 170.4, 189.9. ESI-MS: 413.3 (M+H). Oxazine 3d: mp
154–155 ꢀC (dec.). H NMR d 2.19 (s, 3H), 3.64 (s, 3H), 3.82
1
(s, 3H), 3.90 (s, 3H), 5.06 (s, 2H), 5.34 (dd, J=0.9, 6.6 Hz,
1H), 6.10 (dd, J=0.9, 9.3 Hz, 1H), 6.73 (dd, J=6.6, 9.3 Hz,
1H), 6.80 (s, 1H), 7.21 (m, 5H); 13C NMR d 20.0, 52.6, 56.0,
60.7, 61.1, 68.2, 102.1, 111.3, 118.1, 120.8, 123.2, 126.5, 129.0,
135.8, 138.0, 142.6, 146.1, 150.3, 156.8, 166.8, 170.6, 189.1.
ESI-MS: 470.5 (M+H). Oxazine 3k: mp 129–131 ꢀC (dec.). 1H
NMR d 3.73 (s, 3H), 3.87 (s, 3H), 3.93 (s, 3H), 5.32 (dd,
J=1.0, 6.7 Hz, 1H), 6.19 (dd, J=1.0, 9.5 Hz, 1H), 6.77 (dd,
J=6.7, 9.5 Hz, 1H), 6.54 (s, 1H), 7.17 (m, 5H), 12.29 (s, 1H);
13C NMR d 52.6, 56.0, 60.5, 68.3, 101.1, 105.8, 109.4, 118.1,
123.4, 126.6, 129.1, 134.4, 135.7, 138.1, 150.3, 157.5, 159.8,
196.3. ESI-MS: 369.9 (M+H). Oxazine 3l: mp 105–107 ꢀC
Additional studies to explain in more detail the obser-
vations described above are currently in progress. First,
kinetic studies of the decomposition rates of all the
compounds in different media are being performed, and
these will be correlated with their biological properties.
Second, since one of the principal driving forces of the
retro Diels–Alder reaction responsible for the release of
nitrosobenzene is the return to stable pseudo-aromatic
starting materials, tropolone derivatives with reduced or

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H. Ren et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1851–1854
1
(dec.). H NMR d 3.69 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 3.93
(s, 3H), 5.35 (dd, J=0.8, 6.7 Hz, 1H), 6.17 (dd, J=0.8, 9.3 Hz,
1H), 6.64 (dd, J=6.7, 9.3 Hz, 1H), 6.77 (s, 1H), 7.14 (m, 5H);
13C NMR d 52.7, 55.9, 60.8, 61.5, 68.7, 102.1, 108.7, 117.6,
120.6, 122.7, 126.9, 128.6, 135.2, 136.2, 143.4, 150.2, 155.8,
156.1, 189.6. ESI-MS: 384.2 (M+H). All NMR spectra
were recorded in CDCl3. Chemical shifts (d) are in ppm and
coupling constants (J) in hertz.
9. Gamenara, D.; Dıas, E.; Tancredi, N.; Heinzen, H.;
Moyna, P.; Forbes, E. J. J. Braz. Chem. Soc. 2001, in press.
10. (a) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chu-
lay, J. D. Antimicrob. Agents Chemother. 1979, 16, 710. (b)
Buckner, F. S.; Verlinde, C. L.; La Flamme, A. C.; Van
Voorhis, W. C. Antimicrob. Agents Chemother. 1996, 40, 2592.
11. Bray, P. G.; Ward, S. A. FEMS Microbiol. Lett. 1993, 13, 1.