The imidazo[2,1-a]isoindole system. A new skeletal basis for antiplasmodial compounds

Article abstract of DOI:10.1016/S0960-894X(03)00509-2

The in vitro antiplasmodial activity of some dihydrostilbenamides, phtalazinones, imidazo[2,1-a]isoindole and pyrimido[2,1-a]isoindole derivatives related to the natural dihydrostilbenoid isonotholaenic acid is reported. The evaluation was performed on cultures of F32 strain of Plasmodium falciparum and potent representative compounds were also evaluated in the ferriprotoporphyrin IX biomineralization inhibition test (FBIT). Compounds having the imidazo[2,1-a]isoindole skeleton were the most active and one compound of this group resulted to be as potent as chloroquine, but acting through a mechanism different that of the inhibition of heme biomineralization.

Full text of DOI:10.1016/S0960-894X(03)00509-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2769–2772
The Imidazo[2,1-a]isoindole System. A New Skeletal Basis for
Antiplasmodial Compounds
Esther del Olmo,a,* Marlon Garcıa Armas,b Ma Ines Ybarra,c Jose Luis Lopez,a
Patricia Oporto,d Alberto Gimenez,d Eric Deharoe and Arturo San Felicianoa
a
´
´
Departamento de Quımica Farmaceutica, Facultad de Farmacia, 37007-Salamanca, Spain
bUniversidad Privada Antenor Orrego, Trujillo, Peru
c
´
´
Universidad de Tucuman, Tucuman, Argentina
d
´
´
Instituto de Investigaciones Farmaco Bioquımicas, UMSA, La Paz, Bolivia
Institut de Recherche pour le Developpement CP 9214, La Paz, Bolivia
e
´
Received 24 February 2003; revised 14 April 2003; accepted 9 May 2003
In the Memory of Dr. Alicia Seldes, University of Buenos Aires, Argentina.
Abstract—The in vitro antiplasmodial activity of some dihydrostilbenamides, phtalazinones, imidazo[2,1-a]isoindole and pyr-
imido[2,1-a]isoindole derivatives related to the natural dihydrostilbenoid isonotholaenic acid is reported. The evaluation was per-
formed on cultures of F32 strain of Plasmodium falciparum and potent representative compounds were also evaluated in the
ferriprotoporphyrin IX biomineralization inhibition test (FBIT). Compounds having the imidazo[2,1-a]isoindole skeleton were the
most active and one compound of this group resulted to be as potent as chloroquine, but acting through a mechanism different that
of the inhibition of heme biomineralization.
# 2003 Elsevier Ltd. All rights reserved.
Malaria is one of the major health threats to a wide
population across the third world.1 The therapeutic
arsenal of efficacious antimalarial drugs is very limited
and lessened by the spread of resistance.2 Thus, the
development of new alternative agents becomes neces-
sary. In this regard, among other natural products, we
evaluated the activity of isonotholaenic acid (1), a
dihydrostilbenoid isolated from the Andean fern
Notholaena nivea, as well as several of its derivatives as
the piperidide 1a, against Plasmodium falciparum by in
vitro assays. They displayed IC50 values under 20 mg/
mL and this fact prompted us to consider this stilbe-
noids as a source and model for the preparation of new
potential antimalarials. Thus, along with a set of iso-
notholaenic acid derivatives, we prepared several families
of synthetic analogues including some fused heterocyclic
derivatives as phthalazinones, imidazo[2,1-a]isoindoles
and pyrimido[2,1-a]isoindoles, all of them retaining the
main stilbenoid moiety of 1 and incorporating pyrida-
zine, imidazole and pyrimidine related residues, which
could increase their antiprotozoal potency. Some of
these compounds were previously found to be active
against different Leishmania spp3 and Trypanosoma
cruzi4 strains. Here we report the results of the in vitro
evaluation of these types of compounds against Plas-
modium falciparum and, in order to get further insight
into their mechanism of action, we also report the
results of the evaluation of their ability to interfere the
ferriprotoporphyrin (FP) biomineralization process, a
crucial metabolic process for Plasmodium.5
Chemistry
Most compounds were prepared by total synthesis, as
depicted in Scheme 1, through condensation of phthalic
anhydride with several substituted phenylacetic acids,6
to give the corresponding benzalphthalide intermediates
2, which were subsequently treated with piperidine,
hydrazine, ethylenediamine or 1,3-propylenediamine to
give the dihydrostilbenamide (types 3 and 4), phtalazi-
none (types 5 and 6), imidazoisoindole (type 7) and
pyrimidoisoindole (type 8) derivatives, respectively.
Synthetic procedures details, physicochemical and
*Corresponding author. Tel.: +34-923-294528; fax: +34-923-294515;
e-mail: olmo@usal.es
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00509-2

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E. del Olmo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2769–2772
Scheme 1. Compounds tested as antiplasmodial agents.
Results and Discussion
spectroscopic data and structure assignments for these
compounds, will be reported in a complete paper.
The results of the antiplasmodial evaluation are
shown in Table 1. Data for isonotholaenic acid 1 and
its piperidide 1a, and for chloroquine are included for
comparison and as that a reference drug, respectively.
The intermediate benzalphthalides 2 were also eval-
uated, but they resulted practically inactive. In the
case of phthalazinones, derivatives with other sub-
stituents at position N-2 (ethyl, allyl, butyl, phenyl,
etc.) were also assayed giving results close to those
found for compounds 5a–f and 6a–f. An index describ-
ing the antiplasmodial potency of the compounds, rela-
tive to chloroquine, has been calculated for each
Biological Assays
Assay against Plasmodium falciparum
F32-Tanzania (chloroquine sensitive) strains of Plasmo-
dium falciparum7 were cultured according to Trager and
Jensen,8 on glucose-enriched RPMI 1640 medium sup-
plemented with 10% human serum at 37 ꢀC in aꢀcandle
jar incubator.9 After 24 h of incubation at 37 C, the
medium was replaced by fresh medium containing the
compound to be evaluated, and incubation was con-
tinued for a further 48 h period. On the third day of the
test, a blood smear was taken from each well, para-
sitaemia counted and the inhibition percentage of para-
sitaemia calculated for each concentration of sample in
relation with the control. IC50 values were determined
graphically by plotting the inhibition percentage versus
concentration. Each test also included an untreated con-
trol with solvent and a positive control with Chloroquine
(IC50=40 nM).
Table 1. In vitro antiplasmodial activity of compounds 1, 1a and 3–8
(Plasmodium falciparum, strain F32)
Compd
IC50
Index Compd
(%)
IC50
mg/mL
6.0
Index
(%)
mg/mL
mM
mM
16.3
1
16.0
53.0
<0.1
—
1a
4a
4b
4c
4d
4e
4f
0.25
—
3a
nt
nt
3b
>100
>100
98
<0.1
<0.1
<0.1
<0.1
17
32
8.5
48
<0.1
<0.1
0.2
3c
36
>100
100
84
3d
>100
>100
35
20.7
82
3e
30
12
5
<0.1
0.12
0.2
3f
12
0.11
33.8
20
FBIT
5a
>100
>100
<0.1
1.1
6a
6b
6c
6d
6e
6f
5b
1.0
3.8
1.0
26
3.8
3.6
84
1.1
The procedure for testing FPbiomineralization was
described by Deharo et al.5 and consisted of incubating
a solution containing the compound to be evaluated (or
solvent for control), haemin chloride and sodium ace-
tate buffer. After incubation, the plate was centrifuged,
the remaining pellet resuspended in DMSO to remove
unreacted FPand centrifuged once again. The pellet,
consisting of precipitate of b-haematin, was dissolved
for direct spectroscopic quantification.10 The data were
expressed as the percentage of inhibition of FPbiomi-
neralization calculated by the equation: % inhibi-
tion=100 Â [(O.D. control À O.D. compound)/(O.D.
control)]. Chloroquine showed IC50=28mM.
5c
95
>100
>100
6
>100
>100
>100
22
<0.1
<0.01
<0.01
0.18
<0.1
0.4
5d
11
5e
32
15
1
>100
<0.1
52.8 <0.1
5f
7a
2.5
9.5
0.4
8a
8b
8c
8d
8e
8f
3.6
7.5
1.1
0.5
7b
0.017
4.9
0.04 100
2.3
7c
9.3
0.4
35
38
104
104
16.1
11.5
<0.1
<0.1
0.2
7d
0.067
0.022
0.036
0.013
0.19 21
0.07 57
0.11 36
0.04 100
7e
5.2
3.6
7f
0.3
Chloroquine
Index=IC50 mM chloroquine/IC50 mM compound (antiplasmodial potency,
relative to chloroquine=100).
nt=not tested.

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2769–2772
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Table 2. Inhibition of ferriprotoporphyrin biopolymerization (FBIT)
by selected compounds
action, some selected compounds were tested to check
their ability to block the heme biomineralization pro-
cess, but none of them was fairly active. The results of
the FBIT are shown in Table 2. As it can be seen, only
compounds 5b and 7e show a very low degree of inhib-
ition, which is much far from the values observed for
quinine and chloroquine. Thus, the antiplasmodial
activity evidenciated here for these types of compounds
must clearly proceed through another mechanism.
Compd
FBIT IC50
Index (%)
mg/mL
mM
5b
6b
7b
7e
7f
8a
1530
5752
0.5
—
—
1.6
—
—
11
na
na
590
1735
na
na
It can be concluded that a novel antimalarial type of
compounds, based on the imidazo[2,1-a]isoindole sys-
tem and represented principally by compound 7b, has
been discovered. In addition, it must be considered that
compounds of type 7 and 8 have been evaluated as
racemic mixtures and their resolution or enantio-
selective synthesis could give access to pure enantio-
mers, with probable better antiplasmodial properties for
one of each pair of them; unless the stereochemical
lability of the hemi-ketoaminal moiety would provoke
the equilibration of each enantiomer towards the race-
mic form. While the synthesis and evaluation of new
related compounds, their evaluation against resistant
strains and the in vivo antimalarial assays with several
selected substances are already in progress, cytotoxicity,
acute toxicity and mechanistic studies on these
compounds are scheduled.
Quinine
Chloroquine
80
8.5
250
28
100
Index [IC50 chloroquine /IC50 compound]; na=not active
compound displaying appreciable activity and included
in Table 1.
As it can be observed in Table 1, the series of keto-
stilbenamides (3), diketostilbenamides (4), phtalazi-
nones (5) and 2-methylphthalazinones (6) show discrete
activity against Plasmodium, with representative mem-
bers showing IC50 values within the 1–50 mM range (4d
and 4f; 5b and 5f; 6a, 6b, 6d and 6f). Their clearly lower
potency compared to chloroquine, in addition to the
small number of compounds evaluated do not permit to
establish fair structure–activity correlations. Never-
theless, it could be noted that for these series the size and
position of the substituent(s) on the phenyl group, rather
than their electron-donating or withdrawing nature,
would have an influence on the antiplasmodial potency.
Acknowledgements
M.G.A. thanks AECI (Spain) for a fellowship. M.I.Y.
thanks the University of Tucuman (Argentina) for the
support. Financial support of the chemical part came
from Spanish DGICYT (SAF 98/0103). Biological eva-
luations were supported by the Institut de Recherche
pour le Developpement (IRD-France) and the OAS
(OEA-Regional Flora). This collaborative work has
been performed under the auspices of the Ibero-American
Program of Science and Technology for Development
(CYTED), Sub-Program X.
The series of pyrimidoisoindoles (8) seems to be slightly
more potent. Four (8a, 8b, 8e and 8f) of six compounds
of this series display IC50 values under 20 mM and, in an
overall sense, they behave similarly to those of the other
series mentioned above.
Surprisingly, the reduction in size of the fused six-member
tetrahydropyrimidine ring towards the close analogue
five-member imidazoline ring, promotes a considerable
increase of the anti-Plasmodium activity in the imidazoi-
soindole series (7). Most compounds of this series, while
maintaining the amidine moiety at an equivalent position
and having identical substitution pattern on the phenyl
group, display activities from one to more than two orders
of magnitude higher than those corresponding pyr-
imidoisoindoles (7b/8b, 7d/8d, 7e/8e and 7f/8f).
References and Notes
´
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7. Kindly provided by Prof. H. Ginsburg, Hebrew University
of Jerusalem.
The p-methoxyphenyl group promotes the highest activity
in compound 7b, with IC50 value and anti-Plasmodium
Index equivalent to those of chloroquine. Other symmetric
p-ClPh- or 3,4,5-triMeOPh-substituents, though being
three/four times less potent than 7b, retain the order of
activity. In contrast with the other series the 3,4-methyle-
nedioxyphenyl group, present in compound 7e, increases
the antiplasmodial activity up to more than 55% of the
potency of chloroquine. Nevertheless, a complete SAR
analysis must wait to the results of evaluation of a larger
number of substances with a greater variety of substituents.
8. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
9. DMSO (50 mL) was added to the product to be evaluated
and the solution dispersed in RPMI 1640 medium with the aid
of mild sonication in a sonicleaner bath (Branson Ltd), and
On the light of these in vitro results, looking for corre-
lating the activity with an established mechanism of

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E. del Olmo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2769–2772
then diluted as required in culture medium. The final DMSO
concentration was never greater than 0.1%. A total volume of
150 mL of total culture medium with the diluted products and
the suspension of human red blood cells in medium (0+ group,
5% haematocrit) with 1% parasitaemia, was placed into the
wells of 96-well microtitre plates. All tests were performed in
triplicate. Chemicals were obtained from Sigma Chemicals
Co., USA
10. Incubation was performed in a normal flat bottom 96-well
plate at 37 ꢀC for 18–24 h. The mixture contained: 50 mL of a
10 mg/mL of compound solution (or 50 mL of solvent for
control), 50 mL of 0.5 mg/mL freshly solution of haemin
chloride (Sigma H 5533) in DMSO and 100 mL of 0.5 M
sodium acetate buffer pH 4.4, prepared according to
Deutscher.11 The final pH of the mixture was 5–5.2. It is
important to adhere to the following order of addition: first
the haemin chloride solution, second the buffer, and finally the
solvent or the compound solution. After incubation, the plate
was centrifuged at 1600Âg for 5 min. The supernatant was
discarded by vigorously flipping of the plate upside down the
plate twice. The remaining pellet was resuspended with 200 mL
of DMSO and centrifuged once again and the supernatant
similarly discarded. The pellet, was dissolved in 150 mL of 0.1 M
NaOH in the same plate and its absorption at 405 nm measured
with a micro-ELISA reader (Tittered Multiskan MCC/340).
11. Deutscher, M. P. Methods in Enzymology. Guide to Pro-
tein Purification. Deutscher, M. P. Ed. Academic Press, 1990,
182, 32.