Antimalarial activity of imidazo[2,1-a]isoindol-5-ol derivatives and related compounds

Article abstract of DOI:10.1016/j.ejmech.2011.08.043

The synthesis of several series of imidazo[2,1-a]isoindol-5-ol derivatives and the results of their evaluation against Plasmodium falciparum are presented and discussed. The effects of electron-withdrawing or-donating substituents on different parts of the molecule, as well as those produced by the incorporation of an additional fused ring, were analyzed. Several compounds showed significant antimalarial activity in vitro with IC₅₀ values as low as 60 nM and a certain efficacy in vivo by reducing parasitemia in Plasmodium berghei mouse models.

Full text of DOI:10.1016/j.ejmech.2011.08.043

European Journal of Medicinal Chemistry 46 (2011) 5379e5386
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antimalarial activity of imidazo[2,1-a]isoindol-5-ol derivatives and related
compounds
,
a
b
c
d
Esther del Olmoa , Bianca Barboza , Louise D. Chiaradia , Alicia Moreno , Juana Carrero-Lérida ,
Dolores González-Pacanowskad, Victoria Muñoze, José L. López-Péreza, Alberto Giméneze,
Agustín Benitoc, Antonio R. Martínezf, Luis M. Ruiz-Pérezd, Arturo San Felicianoa
*
a Departamento de Química Farmacéutica, Facultad de Farmacia, CIETUS, Universidad de Salamanca, Spain
b Departamento de Química, Universidade Federal de Santa Catarina, Campus Universitário Trindade, Florianópolis-SC, Brazil
c Centro Nacional de Medicina Tropical, ISCIII, Madrid, Spain
d Instituto de Parasitología y Biomedicina “López Neyra”, Consejo Superior de Investigaciones Científicas, Armilla, Granada, Spain
e Instituto de Investigaciones Fármaco-Bioquímicas, Universidad Mayor de San Andrés, La Paz, Bolivia
f Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of several series of imidazo[2,1-a]isoindol-5-ol derivatives and the results of their evalu-
ation against Plasmodium falciparum are presented and discussed. The effects of electron-withdrawing
or-donating substituents on different parts of the molecule, as well as those produced by the incorpo-
ration of an additional fused ring, were analyzed. Several compounds showed significant antimalarial
activity in vitro with IC50 values as low as 60 nM and a certain efficacy in vivo by reducing parasitemia in
Plasmodium berghei mouse models.
Received 9 June 2011
Received in revised form
30 August 2011
Accepted 31 August 2011
Available online 7 September 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Synthesis
Imidazoisoindoles
In vitro assays
Plasmodium falciparum
Structureeactivity relationship
In vivo assays
Mouse model
Plasmodium berghei
1. Introduction
development of new alternative agents, especially useful in cases of
multiple drug resistance becomes necessary. During the last
Malaria is a tropical disease mainly caused by Plasmodium fal-
ciparum, that kills more than one million people per year [1]. About
3.3 billion people -half of the world’s population and living in the
poorest countries- are at risk of being infected with malaria. It
represents a major public heath problem in more than 109 coun-
tries [2]. Early diagnosis and prompt treatment are two basic
elements of malaria control. Inappropriate use of antimalarial
drugs in the past century contributed to widespread resistance in
the malaria parasite to drugs such as chloroquine, and sulfadoxine-
pyrimethamine [3] and in South-East Asia resistance to third- and
fourth-line antimalarial drugs has been reported [4]. Therefore, the
decades increasing efforts have been done aiming to discover better
therapeutic agents against malaria. The research has been focused
either on natural products as medicinal plants [5], marine organ-
isms [6] or even bacteria [7], as well as on synthetic processes to
prepare new heterocyclic [8] and organometallic [9] compounds.
In a previous study, we synthesized and tested in vitro against
the F32-Tanzania chloroquine sensitive strain of P. falciparum
several families of heterocyclic compounds, sharing in common the
presence of a natural carbonated dihydrostilbene fragment and
including 4-benzylphthalazin-1-ones, 5-benzylimidazo[2,1-a]iso-
indol-5-ols and 6-benzylpyrimido[2,1-a]isoindolols [10]. Of the
compounds evaluated, the imidazo[2,1-a]isoindolol derivatives
displayed the best antiplasmodial activity, with IC50 values similar
to that of the reference drug chloroquine (CQ). In order to further
explore the antiplasmodial potential of this active series and with
* Corresponding author. Fax: þ34 923294515.
E-mail address: olmo@usal.es (E. del Olmo).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.043

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E.del Olmo et al. / European Journal of Medicinal Chemistry 46 (2011) 5379e5386
the aim of obtaining candidates for in vivo studies, new compounds
of the two main structural types I and II (Fig. 1), were designed and
several small libraries of compounds synthesized and assayed.
Structural changes affected systematically the electronic nature and
size of the substituents and their positions on the corresponding
aromatic rings. On the basis of the in vitro evaluation results, several
representative compounds of two new structural types III and IV,
containing an additional benzene ring fused or attached respec-
tively to the imidazoline fragment (Fig. 1), were further synthe-
sized. In compounds of type III a benzimidazole moiety, which
appears in several clinically used antiparasitic drugs, was inte-
grated into a tetracyclic isoindole[2,1-a]benzimidazole system
expecting to enhance the antiparasitic potential.
observation that the three compounds contained a para-mono-
substitution, as well as that the activity seemed to be independent
of the electron-donating or attracting nature of the substituent. It
was also observed that the presence of other type of substituents (F,
smaller and more electronegative; OH, smaller, acidic, H-bond
promoter) at the para position, as well as the change in position
(ortho: 3 vs 7 and 4 vs 9; meta: 5 vs 9) or in degree of substitution,
di-(11e13, 15e17) or tri-substitution (18), led to compounds with
decreased antiplasmodial potency. There is one exception only, that
of the 3,4-dichloroderivative 14 which retained a high level of
activity similar to those the three para-monosubstituted
compounds mentioned above, 7, 9 and 10.
Apart from the benzylic derivatives, the preparation and the
evaluation of several representative elements of naphthylmethyl
and hetarylmethyl (heterobenzylic) series (compounds type II)
were also accomplished. Structures and results are also included in
Table 1. From a comparative point of view, the naphthylmethyl
derivatives 19 and 20 resulted fairly more potent than those having
thienylmethyl (21, 22) or indolylmethyl (23) substituents. Inter-
2. Chemistry
All the compounds were prepared by total synthesis, through
the main route depicted in Scheme 1, based on the condensation of
phthalic anhydride with substituted phenyl(naphthyl, heteroaryl)
acetic acids, to give the phthalide intermediates 1, which were
subsequently treated with 1,2-diamines at 80e90 ꢀC, to give imi-
dazo[2,1-a]isoindol-5-ol derivatives of types IeIII and, in certain
cases, the N-aryl isoindolinone derivatives of type IV. The main
synthetic procedure to obtain the benzalphthalides 1 was adapted
from that of Nokihara [11] by using toluene and a DeaneStark
separator to remove water, working under inert atmosphere. In
estingly, the most potent compound of this series, the
derivative 20, was significantly better (>25-fold) than its
b
-naphthyl
-naph-
a
thyl analog 19, thus meaning that the steric factor would play an
important role in the mechanism of action or drugetarget
interaction.
Bearing in mind the activity results found for compounds
belonging to series I and II, and with the aim of exploring the role of
the imidazoline (amidine) moiety in activity, the two most potent
compounds of the previously mentioned series, 10 and 20, were
selected as leads for the synthesis of two new classes of compounds
that had never been tested as antiparasitic; namely, the 11-benzyl-
and the 11-naphthylmethyl-isoindolo[2,1-a]benzimidazol-11-ols
(compounds type III). Previously we had observed the negative
influence on the antiplasmodial activity of increasing the size of the
imidazoline toward the tetrahydropyridine ring (pyrimido[2,1-a]
isoindole system) [10].
The reaction between the appropriate benzalphthalide and
naphthylmethylidenephthalide and o-phenylenediamine or its
4-methyl derivative easily led to the desired fused tetracyclic
derivatives 24e27. The methyl derivatives 25 and 27 were obtained
as homogeneous 1:1 regioisomeric mixtures of 7-Me and 8-Me
derivatives. Nevertheless, the attempted condensation of the
same phthalides with 4-chlorophenylen-1,2-diamine failed to
produce the desired tetracyclic compounds, even under stronger
conditions of temperature and reaction time. In this case, probably
due to the electron-withdrawing influence of the presence of the
two chlorine atoms in the aromatic diamine, only one amino group
participated in the condensation with the phthalide, thus leading to
the isoindolinone derivatives 28 and 29, which were recovered and
assayed as mixtures of their E/Z stereoisomers. Unfortunately,
regarding the results of antiplasmodial evaluation of the new series
of fused isoindolobenzimidazolols 24e26 and isoindolinones 28
and 29 (Table 1) clearly demonstrated that the new structural
changes failed to enhance the activity of the imidazo[2,1-a]iso-
indol-5-ol scaffold.
some cases, benzalphthalides
1 were better obtained under
microwave irradiation by the Laçová method with modifications
[12]. Physicochemical, analytical, MS and NMR spectral data for all
the new compounds are here reported.
3. Biology
The in vitro antiplasmodial activity of compounds 2e29 was
evaluated against the CQ sensitive strain of P. falciparum 3D7. The
IC50 values were determined and compared with that of CQ taken
as the reference drug [13] (Table 1). The in vivo antimalarial efficacy
of selected representatives was evaluated in mice challenged with
Plasmodium berghei according to a reported procedure [14].
4. Results and discussion
The IC50 values obtained for the compound type I, 9 against the
3D7 strain in vitro, was somewhat higher than the previously re-
ported using a different assay against the F32-Tanzania strain [10].
Several modifications were further performed on this parent
compound in order to analyze the influence of the nature of the
substituent attached to the benzyl fragment. Thus compounds
2e18 were synthesized and evaluated for antiplasmodial activity
according to a reported procedure [15] with minor modifications.
The results are shown in Table 1.
As it can be seen, the three most potent antiplasmodial
compounds (in Type I) 7, 9 and 10 displayed IC50 values ranging
between 60 and 240 nM. The order of antiparasitic potency was 10
(R1 ¼ eSCH3) > 7 (R1 ¼ -Cl) > 9 (R1 ¼ eOCH3) and of interest is the
A selection of the active compounds was analyzed in vitro for
cytotoxicity against monkey kidney CV-1 cells. All tested
compounds exhibited low cytotoxicity in this assay and, conse-
quently, the two main types of imidazoisoindol-5-ols were
considered as potential candidates for assaying the antimalarial
efficacy in vivo on infected mice. The selected compounds showed
physicochemical, ADME, and drug-like properties within those
considered globally adequate for a drug candidate according to
OSIRIS [16] and preADMET [17] databases and predictions accessed
online. Thus, clogP values ranged from 2.80 to 4.34; logS values
from ꢁ2.14 to ꢁ3.73 (at pH ¼ 7.5 and 25 ꢀC); human intestinal
type I: phenyl
R1
R1
type II: naphthyl, hetaryl
HO
R2
N
N
N
R2
type III
H2N
O
type IV
Fig. 1. The structural basis of this research.

E.del Olmo et al. / European Journal of Medicinal Chemistry 46 (2011) 5379e5386
5381
naphthyl
phenyl-
hetaryl
R
R1
O
R1
(naphthyl-
hetaryl-)
HO
R2
1,2-diamines
acetic acids
N
O
or
O
N
N
R2
K2CO3
H2N
O
IV
O
O
1
I - III
Scheme 1. General procedure for the synthesis of imidazo[2,1-a]isoindol-5-ol, isoindolo[2,1-a]benzimidazol-11-ol and isoindolin-1-one derivatives.
absorption (HIA) values resulted higher than 95% and in vitro
plasma protein binding (PPB) values under 90%. In addition, the
compounds were evaluated to accomplish the Lipinski’s Rule of
Five [18] and to lye within the limits to be considered adequate
drug-like molecules according to the Comprehensive Medicinal
Chemistry Database (CMC-like rule) [19] and the World Drug Index
(WDI-like rule) [20]. Finally, they attained a global drug-score
qualification in the range 0.53e0.87 [16].
The in vivo antimalarial efficacy of the seven selected imida-
zoisoindol-5-ols was determined by the classical 4-day suppressive
test, commonly known as the Peters test [14], on Swiss male mice
infected with P. berghei NK65. Mice were firstly treated intraperi-
toneally with daily doses of 50 mg/kg/during four days, and in the
light of the experimental results, some of the compounds were
then assayed at doses of 25 mg/kg. In this assay, compounds
decreasing the parasitemia level measured on the fifth day by 50%
or more with respect to the control group are considered as effi-
cacious antimalarial drugs at the administered dose. The antima-
larial efficacy results are presented in Table 2, along with data
corresponding antiplasmodial activity in vitro and cytotoxicity
values on monkey kidney CV-1 cells.
dose of 50 mg/kg/day. Interestingly, though a certain parallelism
was generally observed between in vitro and in vivo results, the
treatment with an in vitro less potent compound 16, resulted in
a 53% reduction of the parasitemia. On the other hand, the
dichlorinated compound 14, fairly more potent in vitro than 16,
gave rise to only a 20% reduction of the parasitemia at 25 mg/kg/
day, while resulting toxic for mice at high doses. This fact could be
justified taken into account the rather high lipophilicity
(cLogP ¼ 4.34) of compound 14 and its ability for penetrating the
CNS, with a calculated in vivo bloodebrain barrier penetration
(BBBP) index of 2.55 (conc in brain/conc in blood) [17]. Despite its
low efficacy, that could be due to its accumulation in brain and
lipidic tissues rather than blood, compound 14 might be considered
of interest for evaluation in cases of cerebral malaria [21]. Finally,
compound 9, with relevant in vitro antiplasmodial potency,
rendered a discrete 23% reduction of parasitemia at high doses. All
these facts will be taken into account for future in vivo efficacy and
toxicity evaluation experiments.
In summary, we have identified several derivatives of imidazo
[2,1-a]isoindol-5-ol, that exhibit promising antimalarial activity. Of
the compounds tested the para-monosubstituted 6-benzylimidazo
[2,1-a]isoindol-5-ols containing Cl- or MeS- groups attached to the
benzyl fragment were favored. In addition we have established that
the
b-naphthylmethyl derivative also exhibits significant activity
and may serve as drug lead in the search for new agents against
cerebral malaria. All these findings will contribute to establishing
The
b-naphthyl derivative 20, though resulting toxic at the
highest dose tested, resulted also the most efficacious at the dose of
25 mg/kg/day, reducing the parasitemia to the residual level of 13%,
with respect to the untreated control. Compound 10 rendered
a similar level of parasitemia (16% with regard to controls) at the
Table 1
In vitro activity of compounds of Type IeIV on Plasmodium falciparum (strain 3D7).
R1
R1
R1
R1
Cl
HO
HO
1
HO
3
1
11
5
9
7
N
N
N
N
N
N1
R2
N 5
H2N
O
Type I
Type II
Type III
Type IV
Comp. type I
R1
IC50
37.8
15.2
>35
8.36
CQI
Comp. type II
R1
IC50
CQI
2
3
4
5
H
2-Cl
2-OMe
3-OMe
4-F
< 1
< 1
< 1
< 1
<1
19
20
21
22
23
1-naphthyl
2-naphthyl
2-thienyl
3-thienyl
1-methylindol-2-yl
16.3
0.06
19.6
6.6
<1
16.7
<1
<1
<1
6
4.20
13.5
7
8
9
4-Cl
0.12
>35
0.24
0.06
33.3
>35
8.80
0.30
>35
2.20
27.6
1.49
8.3
< 1
4.2
16.7
< 1
< 1
< 1
3.3
< 1
<1
< 1
<1
4eOH
4-OMe
4-SMe
2,4-F2
3,5-F2
2,4-Cl2
3,4-Cl2
3-Cl,4eOH
3,4eOCH2O
3,4-(OMe)2
3,4,5-(OMe)3
Type III
24
R1
R2
H
Me
H
Me
4eCH3SPh
4eCH3SPh
2-naphthyl
2-naphthyl
>27
15.6
>30
nt
<1
<1
<1
10
11
12
13
14
15
16
17
18
25a
26
27a
Type IV
28b
R1
IC50
13.9
10.0
CQI
<1
<1
4eCH3SPh
2-naphthyl
29b
Chloroquine (CQ)
0.010
100
Values under 1mM are bolded between IC50 (mM), media from triplicate experiments; nt: not tested.
CQI (chloroquine index.) ¼ (IC50 mM chloroquine/IC50
m
M compound) ꢂ 100. Equivalent to antiplasmodial potency of compounds relative to CQ ¼ 100.
a
Mixture of Me-regioisomers.
b
Mixture of Z/E steroisomers.

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E.del Olmo et al. / European Journal of Medicinal Chemistry 46 (2011) 5379e5386
Table 2
5.1.1.1. (Z)-3-(4-Methylsulfanylbenzylidene)isobenzofuran-1-one,
1a. Yellow solid; mp 168e170 ꢀC; IR (KBr):
max 2919, 1785, 1658,
1588, 1493, 1471, 1271, 1181, 1079, 975, 811, 761 cm-1 1H NMR
(200 MHz, CDCl3):
Cytotoxicity and antimalarial efficacy of selected imidazo[2,1-a]isoindol-5-ols in
mice infected with Plasmodium berghei.
n
Compound
P. falciparum
Cytotoxicitya
CC50 (mM)
Parasitemiab
inhibition (%)
at 50 mg/kg
Parasitemia
inhibition (%)
at 25 mg/kg
d
2.50 (s, 3H), 6.35 (s, 1H), 7.24 (d, J ¼ 8.8 Hz, 2H),
IC50
(m
M)
7.53 (ddd, J ¼ 7.7, 7.3, 2.2 Hz, 1H), 7.75 (d, J ¼ 8.8 Hz, 2H), 7.75 (m,
1H), 7.77 (m, 1H), 7.91 (d, J ¼ 7.7 Hz, 1H); 13C NMR (50.3 MHz,
7
9
0.12
0.24
0.06
0.30
2.20
1.49
0.06
>16
nt
>24
nt
>32
nt
nt
7
23
84
nt
nt
nt
19
nt
nt
87
CDCl3):
d 15.2, 106.5, 119.6, 123.2, 125.5, 126.0 (2C), 126.6, 129.5,
130.3 (2C), 134.4, 139.4, 140.5, 144.1, 166.9; ESI-MS: m/z 269.3301
[M þ H]þ; Anal. Calcd for C16H12O2S: C, 71.62; H, 4.51; S, 11.95.
Found: C, 71.65; H, 4.53; S, 11.90.
10
14
16
18
20
Toxicc
53
<5
Toxicc
5.1.1.2. (Z)-3-(2-Naphthylmethylene)isobenzofuran-1-one,
1b. White solid; mp 176e178 ꢀC; IR (KBr):
n
max 3046, 2926, 1778,
1662, 1473, 1350, 1273, 1171, 1079, 979, 898, 818, 741 cm-1; 1H NMR
(400 MHz, CDCl3):
Values under 1mM are bolded between IC50 (mM), media from triplicate
experiments.
a
Values for other compounds tested: 17 (>30
n ¼ 5. nt: not tested.
mM), 19 (>15 mM).
b
c
d
6.57 (s, 1H), 7.49 (m, 2H), 7.55 (ddd, J ¼ 8.0, 7.5,
1.5 Hz, 1H), 7.73 (ddd, J ¼ 7.5, 7.5, 1.5 Hz, 1H), 7.80 (dd, J ¼ 7.5, 1.5 Hz,
1H), 7.80 (d, J ¼ 8.0 Hz, 1H), 7.81 (m, 1H), 7.87 (m, 1H), 7.95 (dd,
J ¼ 8.0; 1.5 Hz, 1H), 8.04 (dd, J ¼ 8.0; 1.5 Hz, 1H), 8.24 (bs, 1H); 13C
Death of more than half of the animals.
structure activity relationships that will guide further design of
new active compounds. Due to the previous observation that
antimalarial activity of these compounds could not be correlated
with ferriprotoporphyrin biopolymerisation inhibition [10,22],
mode of action studies are underway in order to understand the
global response of the parasite to these imidazo[2,1-a]isoindol-5-ol
derivatives. In addition to acute and subchronic toxicity determi-
nations, further efficacy assays, with lower and higher i.p. doses and
with oral administration will be necessary to define the respective
ED50 values and the actual therapeutic indexes and windows for
compounds 10 and 20.
NMR (100.6 MHz, CDCl3): d 107.1, 119.8, 123.3, 125.6, 126.4, 126.7,
127.2, 127.6128.3, 128.4, 129.7, 129.8, 130.7, 133.0, 133.4, 134.6, 140.6,
144.7, 167.0; ESI-MS: m/z 273.0837 [M þ H]þ; Anal. Calcd for
C19H12O2: C, 83.81; H, 4.44. Found: C, 83.85; H, 4.40.
5.1.2. General procedure for the preparation of imidazo[2,1-a]
isoindol-5-ol derivatives (Types IeIV)
1.0 mmol of the corresponding benzalphthalide was solved into
ethylenediamine (4 mL) and heated at 70e80 ꢀC with magnetic
stirring for 7e12 h. After cooling the reaction mixture was extracted
with ethyl acetate and washed with water to pH ¼ 7. The organic
layer was dried over Na2SO4 and concentrated under reduced
pressure to give the crude product which was purified by column
chromatography on silica gel. All the products were obtained as
oils. Reaction yields ranged within 80e100%.
5. Experimental
5.1. Chemistry
5.1.2.1. 5-Benzyl-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-ol, 2. IR
(NaCl):
n
max 3292, 2928, 1688, 1682, 1600, 1469, 1081, 910, 770,
2.79 (m, 1H), 2.88 (m, 1H),
Melting points (mp) were determined by a Büchi apparatus in
open capillaries and were uncorrected. All commercial chemicals
were used as purchased and solvents purified by the standard
procedures prior to use [23]. Thin-layer chromatography was per-
formed on Merck 60 Silica Gel GF-254 precoated plates and the
identification was done with UV light and colorization with 10%
phosphomolybdic acid or ninhydrin spray followed by heating.
Flash column chromatography was performed on Merck 60 silica
gel (0.063e0.2 mesh). Infrared spectra were recorded by using neat
samples, without solvent or KBr, on an FT-IR spectrometer Nicolet
Impact 410 model. NMR spectra were recorded on a Bruker AC-200
(200 MHz), or ARX-400 (400 MHz). The spectra were measured in
700 cm-1 1H NMR (200 MHz, CDCl3):
d
3.16 (d, J ¼ 13.9 Hz, 1H), 3.39 (d, J ¼ 13.9 Hz, 1H), 3.45 (m, 1H), 4.04
(m, 1H), 4.25 (bs, OH), 6.81 (m, 2H), 7.04 (m, 3H), 7.29e7.51 (m, 4H);
13C NMR (50.3 MHz, CDCl3):
d 39.5, 41.7, 44.0, 89.4, 122.2, 122.6,
126.5, 127.7 (2C), 128.6, 129.7 (2C), 130.6, 131.6, 134.8, 147.5, 167.3;
ESI-MS: m/z 265.1263 [M þ H]þ; Anal. Calcd for C17H16N2O: C,
77.25; H, 6.10; N, 10.60. Found: C, 77.21; H, 6.09; N, 10.61.
5.1.2.2. 5-(2-Chlorobenzyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-
5-ol, 3. IR (NaCl):
n
max 3295, 2929, 1689, 1599, 1470, 1123, 1086,
2.84 (m, 1H), 2.95
758, 730, 701 cm-1 1H NMR (200 MHz, CDCl3):
d
(m, 1H), 3.21 (m, 1H), 3.36 (d, J ¼ 13.6 Hz, 1H), 3.60 (d, J ¼ 13.6 Hz,
1H), 3.95 (bs, OH), 4.19 (m, 1H), 7.00 (m, 3H), 7.15 (m, 1H), 7.18 (m,
1H), 7.25 (m, 1H), 7.40 (m, 1H), 7.59 (dd, J ¼ 7.9, 1.4, 1H); 13C NMR
CDCl3 relative to TMS (d 0.00 ppm). High resolution mass spectra
(HRMS) were recorded on QSTAR XL mass spectrometer, by elec-
tron spray ionization (ESI-MS) technique (5 kV).
(50.3 MHz, CDCl3): d 39.3, 40.6, 41.8, 89.0, 122.6, 122.8, 126.3, 128.2,
128.9, 129.4, 130.5, 131.6, 131.8, 133.2, 134.9, 147.7, 167.5; ESI-MS: m/
z 299.0873 [M þ H]þ; Anal. Calcd for C17H15ClN2O: C, 68.34; H, 5.06;
N, 9.38. Found: C, 68.31; H, 5.04; N, 9.36.
5.1.1. General procedure for the synthesis of benzalphthalides 1
2.2 mmol of phthalic anhydride, 2.7 mmol of the corresponding
phenylacetic acid and 0.26 mmol of sodium acetate with 5 mL of
toluene were adapted in a round bottom flask to a DeaneStark
apparatus. The mixtures were maintained under Nitrogen at
210e245 ꢀC, with magnetic stirring, for 9e33 h. After cooling the
reaction mixture was treated with ethyl acetate and washed with
(sat.) Na2CO3, brine and water, dried over Na2SO4 and concentrated
under reduced pressure to give the crude products. Solid products
were purified by crystallization and oils by flash chromatography
on silica gel. All phthalides were obtained as Z-isomers, configu-
ration confirmed through NOE or ROESY experiments, and yields
ranged 40e95%.
5.1.2.3. 5-(2-Methoxybenzyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
indol-5-ol, 4. IR (NaCl):
n
max 3350, 2928, 1685, 1610, 1511, 1245,
2.89 (m, 2H), 3.25
1176, 1031, 751 cm-1 1H NMR (200 MHz, CDCl3):
d
(m,1H), 3.28 (d, J ¼ 13.9 Hz,1H), 3.41 (d, J ¼ 13.9 Hz,1H), 3.55 (s, 3H),
4.02 (m,1H), 4.05 (bs, OH), 6.61 (m, 2H), 6.70 (dd, J ¼ 7.2,1.8 Hz,1H),
7.02 (ddd, J ¼ 7.5, 7.2,1.8 Hz,1H), 7.21e7.42 (m, 3H), 7.50 (d, J ¼ 7.3 Hz,
1H); 13C NMR (50.3 MHz, CDCl3):
d 39.6, 41.5, 43.2, 89.2, 114.9, 116.1,
122.6,123.0,124.3,127.9,128.9,129.0,129.6,130.1,131.7,148.7,147.8,
167.3; ESI-MS: m/z 295.1368 [M þ H]þ; Anal. Calcd for C18H18N2O2: C,
73.45; H, 6.16; N, 9.52. Found: C, 73.38; H, 6.11; N, 9.49.

E.del Olmo et al. / European Journal of Medicinal Chemistry 46 (2011) 5379e5386
5383
5.1.2.4. 5-(3-Methoxybenzyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
indol-5-ol, 5. IR (NaCl): max 3352, 2928, 1683, 1610, 1512, 1243,
1178, 1031, 880, 770, 700 cm-1 1H NMR (200 MHz, CDCl3):
2.89 (m,
(dd, J ¼ 7.7, 1.5 Hz, 1H), 7.53 (d, J ¼ 7.3, 1H); 13C NMR (50.3 MHz,
n
CDCl3): d 15.7, 39.4, 41.5, 43.8, 89.5, 122.4, 123.0, 126.0 (2C), 128.9,
d
130.4 (2C), 130.8, 131.8, 131.9, 136.7, 147.7, 167.6; ESI-MS: m/z
311.4143 [M þ H]þ; Anal. Calcd for C18H18N2OS: C, 69.65; H, 5.84; N,
9.02. Found: C, 69.63; H, 5.81; N, 9.00.
2H), 3.14 (d, J ¼ 13.9 Hz, 1H), 3.15 (m, 1H), 3.37 (d, J ¼ 13.9 Hz, 1H),
3.53 (s, 3H), 4.00 (m, 1H), 4.02 (bs, OH), 6.28 (dd, J ¼ 2.6, 0.9 Hz, 1H),
6.40 (dd, J ¼ 7.3, 0.9 Hz, 1H), 6.59 (dd, J ¼ 8.0, 2.6 Hz, 1H), 6.95 (d,
J ¼ 8.0, 7.3 Hz,1H), 7.30 (ddd, J ¼ 7.3, 7.3, 1.8 Hz,1H), 7.40 (dd, J ¼ 7.3,
1.8 Hz, 1H), 7.45 (ddd, J ¼ 7.3, 7.3, 1.8 Hz, 1H), 7.53 (dd, J ¼ 7.3, 1.8 Hz,
5.1.2.10. 5-(2,4-Difluorobenzyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
indol-5-ol, 11. IR (NaCl):
1080, 876, 812, 756, 698 cm-1 1H NMR (200 MHz, CDCl3):
2.93e3.12 (m, 2H), 3.11e3.30 (m,1H), 3.31 (d, J ¼ 14.0 Hz,1H), 3.41
(d, J ¼ 14.0 Hz, 1H), 4.15 (dt, J ¼ 12.2, 2.5 Hz, 1H), 6.53e6.69 (m, 3H),
7.34e7.60 (m, 4H); 13C NMR (50.3 MHz, CDCl3):
35.4, 39.6, 41.4,
n max 3295, 2928, 1687, 1583, 1468, 1120,
1H), 13C NMR (50.3 MHz, CDCl3):
d 39.6, 41.9, 44.3, 55.0, 89.5, 112.5,
115.4, 122.4 (2C), 122.9, 128.9 (2C), 130.9, 131.9, 136.5, 147.8, 159.0,
167.5; ESI-MS: m/z 295,1368 [M þ H]þ; Anal. Calcd for C18H18N2O2:
C, 73.45; H, 6.16; N, 9.52. Found: C, 73.37; H, 6.08; N, 9.53.
d
d
89.8, 103.4 (t, J ¼ 25.9), 110.7 (d, J ¼ 18.5), 117.7 (d, J ¼ 11.1), 122.3,
122.8, 129.2, 130.6, 131.1, 132.4, 147.1, 159.0 (m), 163.7 (m), 167.9;
ESI-MS: m/z 301.1103 [M þ H]þ; Anal. Calcd for C17H14F2N2O: C,
67.99; H, 4.70; N, 9.33. Found: C, 67.891; H, 7.61; N, 9.24.
5.1.2.5. 5-(4-Fluorobenzyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-
5-ol, 6. IR (NaCl):
n
max 3351, 2928, 1688, 1596, 1490, 1082, 821,
2.83 (m, 1H), 2.92 (m, 1H),
762 cm-1 1H NMR (200 MHz, CDCl3):
d
3.11 (m, 1H), 3.08 (d, J ¼ 14.0 Hz, 1H), 3.31 (d, J ¼ 14.0 Hz, 1H), 3.98
(dt, J ¼ 14.0, 2.9 Hz, 1H), 6.69 (m, 4H), 7.30 (m, 2H), 7.41 (d,
5.1.2.11. 5-(3,5-Difluorobenzyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
J ¼ 6.6 Hz, 2H); 13C NMR (50.3 MHz, CDCl3):
d
39.4, 41.7, 43.6, 89.3,
indol-5-ol, 12. IR (NaCl):
n
max 3354, 2929, 1687, 1590, 1471, 1130,
2.99e3.19 (m,
114.8 (d, J ¼ 20.3 Hz, 2C), 122.3, 123.0, 129.0, 130.6, 130.8, 131.9 (d,
J ¼ 7.4 Hz, 2C),132.0,147.7,161.7 (d, J ¼ 243.9 Hz),167.5; ESI-MS: m/z
283.1168 [M þ H]þ; Anal. Calcd for C17H15FN2O: C, 72.32; H, 5.36; N,
9.92. Found: C, 72.21; H, 5.29; N, 9.85.
1081, 910, 882, 750 cm-1 1H NMR (200 MHz, CDCl3):
d
3H), 3.17 (d, J ¼ 14.0 Hz, 1H), 3.40 (d, J ¼ 14.0 Hz, 1H), 3.50 (m, OH),
4.15 (dt, J ¼ 14.0, 2.5 Hz, 1H), 6.33 (m, 2H), 6.50 (m, 1H), 7.36e7.55
(m, 3H), 7.63 (d, J ¼ 7.2 Hz, 1H); 13C NMR (50.3 MHz, CDCl3):
d 39.2,
41.7, 44.3, 88.8, 102.5 (t, J ¼ 25.9 Hz,), 112.9 (d, J ¼ 24.0 Hz), 122.2,
123.2, 129.2, 130.6, 132.2, 138.7, 138.9, 147.4, 159.9 (d, J ¼ 12.9 Hz),
164.9 (d, J ¼ 12.9 Hz), 167.5; ESI-MS: m/z 301.1074 [M þ H]þ; Anal.
Calcd for C17H14F2N2O: C, 67.99; H, 4.70; N, 9.33. Found: C, 67.90; H,
4.68; N, 9.19.
5.1.2.6. 5-(4-Chlorobenzyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-
5-ol, 7. IR (NaCl):
n
max 3359, 2927, 1687, 1598, 1492, 1083, 819,
2.85 (m, 1H), 2.96 (m,
762, 700 cm-1; 1H NMR (200 MHz, CDCl3):
d
1H), 3.14 (d, J ¼ 13.5 Hz 1H), 3.18 (m, 1H), 3.37 (d, J ¼ 13.5 Hz, 1H),
3.50 (bs, OH), 4.06 (dt, J ¼ 14.6, 2.9 Hz, 1H), 6.74 (d, J ¼ 8.4 Hz, 2H),
7.02 (d, J ¼ 8.4 Hz, 2H), 7.35 (m, 2H), 7.47 (d, J ¼ 7.3 Hz, 1H), 7.55 (dd,
5.1.2.12. 5-(2,4-Dichlorobenzyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
J ¼ 8.0, 1.5 Hz, 1H); 13C NMR (50.3 MHz, CDCl3):
d
39.4, 41.7, 43.7,
indol-5-ol, 13. IR (NaCl):
n
max 3361, 2929, 1687, 1588, 1470, 1120,
2.97 (m,
89.4, 122.3, 123.0, 128.1 (2C), 129.1, 130.7, 131.3, 131.2, 132.0, 132.7,
133.5, 147.5, 167.7; ESI-MS: m/z 299.0873 [M þ H]þ; Anal. Calcd for
C17H15ClN2O: C, 68.34; H, 5.06; N, 9.38; Found: C, 68.31; H, 5.03; N,
9.39.
1081, 879, 815, 758, 699 cm-1 1H NMR (200 MHz, CDCl3):
d
1H), 3.06 (m, 1H), 3.21 (m, 1H), 3.33 (d, J ¼ 13.9 Hz, 1H), 3.56 (d,
J ¼ 13.9 Hz, 1H), 4.00 (bs, OH), 4.18 (m, 1H), 6.96 (d, J ¼ 8.4 Hz, 1H),
7.03 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.25 (d, J ¼ 1.8 Hz, 1H), 7.25 (d,
J ¼ 7.3 Hz, 1H), 7.45 (m, 1H) 7.48 (m, 1H) 7.66 (d, J ¼ 7.7, 1H); 13C
5.1.2.7. 5-(2-Hydroxybenzyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
NMR (50.3 MHz, CDCl3): d 39.1, 40.4, 41.9, 88.7, 122.5, 123.1, 126.7,
indol-5-ol, 8. IR (NaCl):
n
max 3302, 2929, 1683, 1585, 1471, 1210,
129.1,129.3,130.6 (2),132.0, 132.5, 133.6, 135.6,147.8,167.5; ESI-MS:
m/z 333.0481 [M þ H]þ; Anal. Calcd for C17H14Cl2N2O: C, 61.28; H,
4.23; N, 8.41. Found: C, 61.25; H, 4.24; N, 8.39.
1083, 1280, 762, 700 cm-1 1H NMR (200 MHz, CDCl3 þ CD3OD):
d
2.90 (m, 2H), 3.06 (d, J ¼ 14.0 Hz,1H), 3.26 (d, J ¼ 14.0 Hz,1H), 3.28
(m, 1H), 3.83 (m, 1H), 6.41 (d, J ¼ 8.6 Hz, 2H), 6.51 (d, J ¼ 8.6 Hz, 2H),
7.25e7.35 (m, 2H), 7.41e7.47 (m, 2H); 13C NMR (50.3 MHz,
5.1.2.13. 5-(3,4-Dichlorobenzyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
CDCl3 þ CD3OD):
d
39.9, 41.3, 42.6, 90.8, 114.8 (2C), 122.5, 122.7,
indol-5-ol, 14. IR (NaCl):
n
max 3352, 2929, 1688, 1599, 1470, 1130,
2.95 (m,
125.3, 129.2, 130.6, 130.8 (2C), 132.1, 147.2, 155.6, 168.4; ESI-MS: m/z
281.1212 [M þ H]þ; Anal. Calcd for C17H16N2O2: C, 72.84; H, 5.75; N,
9.99. Found: C, 72.81; H, 7.69; N, 10.01.
1080, 880, 820, 740, 700 cm-1 1H NMR (200 MHz, CDCl3):
d
1H), 3.05 (m, 1H), 3.10 (d, J ¼ 13.9 Hz, 1H), 3.14 (m, 1H), 3.36 (d,
J ¼ 13.9 Hz, 1H), 4.05 (bs, OH), 4.15 (m, 1H), 6.68 (dd, J ¼ 8.2, 2.2 Hz,
1H), 6.99 (d, J ¼ 2.2 Hz, 1H), 7.16 (d, J ¼ 8.2 Hz, 1H), 7.36 (bd, J ¼ 7.6,
1H), 7.41 (ddd, J ¼ 7.6, 7.3, 1.2 Hz, 1H), 7.53 (ddd, J ¼ 7.6, 7.3, 1.2 Hz,
5.1.2.8. 5-(4-Methoxybenzyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
indol-5-ol, 9. IR (KBr):
n
max 3351, 2929, 1681, 1612, 1513, 1248,
2.87
1H), 7.62 (d, J ¼ 7.6, 1H); 13C NMR (50.3 MHz, CDCl3):
d 39.2, 41.7,
1179, 1033, 825, 760, 701 cm-1 1H NMR (200 MHz, CD3OD):
d
43.5, 88.9, 122.3, 123.0, 129.1, 129.2, 129.3, 130.6 (2), 131.7, 131.9,
132.1, 135.4, 147.3, 167.4; ESI-MS: m/z 333.048 [M þ H]þ; Anal. Calcd
for C17H14Cl2N2O: C, 61.28; H, 4.23; N, 8.41. Found: C, 61.22; H, 4.20;
N, 8.40.
(m, 1H), 2.97 (m, 1H), 3.13 (d, J ¼ 13.9 Hz, 1H), 3.19 (m, 1H), 3.34 (d,
J ¼ 13.9 Hz, 1H), 3.67 (s, 3H), 4.06 (dt, J ¼ 14.2, 2.9 Hz, 1H), 6.58 (d,
J ¼ 8.8 Hz, 2H), 6.73 (d, J ¼ 8.8 Hz, 2H), 7.32 (m, 1H), 7.37 (m, 1H),
7.48 (m, 1H), 7.55 (dd, J ¼ 7.3, 1.1 Hz, 1H); 13C NMR (50.3 MHz,
CD3OD):
d
41.5, 43.2, 44.5, 55.5, 92.8, 114.2 (2), 123.5, 124.0, 127.9,
5.1.2.14. 5-(3-Chloro-4-hydroxybenzyl)-2,5-dihydro-3H-imidazo[2,1-
130.4, 132.4 (2C), 133.2 (2C), 148.6, 160.0, 169.6; ESI-MS: m/z
295.1368 [M þ H]þ; Anal. Calcd for C18H18N2O2: C, 73.45; H, 6.16; N,
9.52. Found: C, 73.40; H, 6.15; N, 9.51.
a]isoindol-5-ol, 15. IR (NaCl):
n max 3350, 2929, 1687, 1595, 1460,
1150, 1075, 880, 821, 745, 719 cm-1 1H NMR (200 MHz,
CDCl3 þ CD3OD): 2.80e3.42 (m, 3H), 3.07 (d, J ¼ 14.0 Hz, 1H), 3.31
d
(d, J ¼ 14.0 Hz, 1H), 3.99 (dt, J ¼ 14.3, 4.5 Hz, 1H), 6.51 (dd, J ¼ 8.3,
2.2 Hz, 1H), 6.62 (d, J ¼ 8.3 Hz, 1H), 6.71 (d, J ¼ 2.2 Hz, 1H), 7.40 (m,
2H), 7.48e7.54 (m, 2H); 13C NMR (50.3 MHz, CDCl3 þ CD3OD):
5.1.2.9. 5-(4-Methylsulfanylbenzyl)-2,5-dihydro-3H-imidazo[2,1-a]
isoindol-5-ol, 10. IR (NaCl):
n
max 3349, 2921, 1687, 1598, 1494,
2.35 (s,
1118, 1078, 816, 767, 700 cm-1 1H NMR (200 MHz, CDCl3):
d
d 39.8, 40.3, 42.2, 90.8, 115.9, 119.7, 122.5, 122.8, 126.6, 129.1, 129.4,
3H), 2.73 (m, 1H), 2.92 (m, 1H), 3.12 (d, J ¼ 13.9 Hz, 1H), 3.18 (m, 1H),
3.35 (d, J ¼ 13.9 Hz, 1H), 4.02 (dt, J ¼ 14.1, 2.8 Hz, 1H), 4.22 (bs, OH),
6.72 (d, J ¼ 8.4 Hz, 2H), 6.93 (d, J ¼ 8.4 Hz, 2H), 7.33 (m, 2H), 7.45
130.5, 131.0, 132.3, 146.8, 151.4, 168.4; ESI-MS: m/z 315.0823
[M þ H]þ; Anal. Calcd for C17H15ClN2O2: C, 64.87; H, 4.80; N, 8.90.
Found: C, 64.83; H, 8.68; N, 8.85.

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E.del Olmo et al. / European Journal of Medicinal Chemistry 46 (2011) 5379e5386
5.1.2.15. 5-(1,3-Benzodioxol-5-ylmethyl)-2,5-dihydro-3H-imidazo
[2,1-a]isoindol-5-ol, 16. IR (NaCl): max 3359, 3295, 2925, 2778,
1686, 1503, 1490, 1249, 1194, 1039, 928, 880, 814, 767 cm-1 1H NMR
(200 MHz, CDCl3):
1407, 1313, 1250, 1197, 1073, 1034, 909, 766 cm-1 1H NMR (200 MHz,
CDCl3):
n
d
3.49 (d, J ¼ 10.0 Hz, 1H), 3.51 (d, J ¼ 10.0 Hz, 1H), 3.63 (d,
J ¼ 15.1 Hz, 1H), 3.74 (d, J ¼ 15.1 Hz, 1H), 4.43 (d, J ¼ 12.2 Hz, 1H),
4.55 (d, J ¼ 12.2 Hz, 1H), 6.43 (dd, J ¼ 3.6, 1.1 Hz, 1H), 6.69 (dd,
J ¼ 5.0, 3.6 Hz, 1H), 6.75 (bs, OH), 6.97 (dd, J ¼ 5.0, 1.1 Hz, 1H),
d
2.87 (m, 1H), 2.93 (m, 1H), 3.09 (d, J ¼ 13.9 Hz,
1H), 3.15 (m, 1H), 3.30 (d, J ¼ 13.9 Hz, 1H), 3.75 (bs, OH), 4.05 (dt,
J ¼ 14.6, 2.6 Hz, 1H), 5.80 (s, 2H), 6.27 (dd, J ¼ 8.0, 1.8 Hz, 1H), 6.30
(d, J ¼ 1.8 Hz,1H), 6.50 (d, J ¼ 8.0 Hz, 1H), 7.34 (dd, 1H, J ¼ 6.3,1.1 Hz,
1H), 7.39 (ddd, 1H, J ¼ 8.3, 7.3, 1.1 Hz, 1H), 7.49 (ddd, J ¼ 7.3, 6.3,
1.1 Hz, 1H), 7.58 (d, J ¼ 8.3, 1.1, 1H); 13C NMR (50.3 MHz, CDCl3):
7.22e7.34 (m, 2H), 7.53 (m, 2H); 13C NMR (50.3 MHz, CDCl3):
d 37.0,
39.1 (2), 92.1, 122.3, 122.8, 124.9, 126.3, 127.2, 129.4, 130.4, 132.7,
135.6, 146.9, 169.3; ESI-MS: m/z 271.0827 [M þ H]þ; Anal. Calcd for
C15H14N2OS: C, 66.64; H, 5.22; N, 10.36. Found: C, 66.62; H, 5.23; N,
10.35.
d
39.5, 41.8, 44.0, 89.4, 100.8, 107.7, 110.2, 122.4, 123.0, 123.3, 128.7,
129.0, 130.8, 132.0, 146.3, 147.1, 147.8, 167.7; ESI-MS: m/z 309,3312
[M þ H]þ; Anal. Calcd for C18H16N2O3 C, 70.12; H, 5.23; N, 9.09.
Found: C, 70.10; H, 5.20; N, 9.10.
5.1.2.21. 5-(3-Thienylmethyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
indol-5-ol, 22. IR (NaCl):
1470, 1410, 1316, 1241, 1157, 1072, 1030, 790, 769, 713 cm-1 1H NMR
(400 MHz, CDCl3):
n max 3254, 3102, 2934, 2855, 1677, 1614,
5.1.2.16. 5-(3,4-Dimethoxybenzyl)-2,5-dihydro-3H-imidazo[2,1-a]
d
3.35 (d, J ¼ 9.9 Hz, 1H), 3.41 (d, J ¼ 9.9 Hz, 1H),
isoindol-5-ol, 17. IR (NaCl):
1467, 1262, 1238, 1143, 859, 814, 767, 704 cm-1 1H NMR (200 MHz,
CDCl3):
n
max 3361, 2929, 2836, 1682, 1516,
3.49 (d, J ¼ 11.0 Hz, 1H), 3.55 (d, J ¼ 11.0 Hz, 1H), 4.41 (d, J ¼ 12.1 Hz,
1H), 4.46 (d, J ¼ 12.1 Hz, 1H), 6.47 (m, 1H), 6.62 (m, 1H), 7.01 (dd,
J ¼ 4.8, 2.9 Hz, 1H), 7.21 (m, 1H), 7.27 (m, 1H), 7.45 (m, 1H), 7.52 (m,
d
2.89 (m,1H), 2.93 (m, 1H), 3.14 (d, J ¼ 13.9 Hz,1H), 3.16 (m,
1H), 3.35 (d, J ¼ 13.9 Hz, 1H), 3.52 (s, 3H), 3.73 (s, 3H), 3.80 (bs, OH),
4.03 (dt, J ¼ 14.3, 2.9 Hz,1H), 6.16 (d, J ¼ 2.0 Hz,1H), 6.42 (dd, J ¼ 8.4,
2.0 Hz, 1H), 6.57 (d, J ¼ 8.4 Hz, 1H), 7.34 (ddd, J ¼ 7.3, 6.9, 1.1, 1H),
7.44 (dd, J ¼ 7.3, 1.1 Hz, 1H), 7.46 (ddd, J ¼ 7.3, 6.9, 1.1, 1H), 7.54 (dd,
1H); 13C NMR (100.6 MHz, CDCl3):
d 37.3, 38.8 (2C), 92.1, 122.1,
122.7, 123.3, 123.9, 125.0, 128.5, 129.1, 132.4, 134.3, 147.4, 168.9; ESI-
MS: m/z 271.0827 [M þ H]þ; Anal. Calcd for C15H14N2OS: C, 66.64;
H, 5.22; N, 10.36. Found: C, 66.60; H, 5.20; N, 10.33.
J ¼ 7.3, 1.1, 1H); 13C NMR (50.3 MHz, CDCl3):
d 39.5, 41.8, 44.0, 55.7
(2C), 89.5, 110.6, 113.0, 122.3, 122.3, 123.0, 127.4, 128.9, 131.0, 131.8,
147.8 (2), 148.0, 167.5; ESI-MS: m/z 325.3737 [M þ H]þ; Anal. Calcd
for C19H20N2O3 C, 70.35; H, 6.21; N, 8.64. Found: C, 70.32; H, 6.20; N,
8.61.
5.1.2.22. 5-[(1-methyl-1H-indol-2-yl)methyl]-2,5-dihydro-3H-imi-
dazo[2,1-a]isoindol-5-ol, 23. IR (NaCl):
2856, 1703, 1675, 1614, 1537, 1469, 1407, 1333, 1250, 1155, 1072,
1036, 741 cm-1 1H NMR (200 MHz, CDCl3):
3.50 (m, 3H), 3.56 (s,
n max 3435, 3245, 2926,
d
3H), 3.68 (d, J ¼ 15.0 Hz,1H), 4.40 (m, 1H), 6.21 (s, 1H), 6.68 (bs, OH),
7.00 (m, 1H), 7.02e7.17 (m, 3H), 7.20e7.34 (m, 2H), 7.36 (d, J ¼ 7.9
Hz, 1H) 7.47 (m, 1H), 7.56 (d, J ¼ 7.5 Hz, 1H); 13C NMR (50.3 MHz,
5.1.2.17. 5-(3,4,5-Trimethoxybenzyl)-2,5-dihydro-3H-imidazo[2,1-a]
isoindol-5-ol, 18. IR (NaCl):
n max 3357, 3297, 2937, 2837, 1693,
1591,1466,1243, 836, 880, 770, 712 cm-1 1H NMR (200 MHz, CDCl3):
CDCl3):
d 32.3, 32.7, 39.1 (2), 92.5, 106.9, 109.1, 118.6, 118.8, 121.4,
d
2.83 (m, 1H), 2.96 (m, 1H), 3.14 (d, J ¼ 13.9 Hz, 1H), 3.19 (m, 1H),
122.4, 122.6, 127.4, 128.3, 129.1, 130.5, 132.5, 136.3, 148.1, 169.2; ESI-
MS: m/z 318.1528 [M þ H]þ; Anal. Calcd for C20H19N3O: C, 75.69; H,
6.03; N, 13.24. Found: C, 75.63; H, 6.00; N, 13.25.
3.37 (d, J ¼ 13.9 Hz, 1H), 3.69 (s, 6H), 3.73 (s, 3H), 3.80 (bs, OH), 4.06
(m, 1H), 5.99 (s, 2H), 7.36 (dd, J ¼ 6.8, 1.1 Hz, 1H), 7.47 (m, 1H), 7.51
(m,1H), 7.58 (d, J ¼ 7.7,1H); 13C NMR (50.3 MHz, CDCl3):
d 39.6, 41.8,
44.4, 55.9 (2), 60.8, 89.6, 107.4 (2), 122.4, 122.9, 128.9, 130.5, 131.1,
131.8, 147.8, 152.5(3), 167.5; ESI-MS: m/z 355,1580 [M þ H]þ; Anal.
Calcd for C20H22N2O4: C, 67.78; H, 6.26; N, 7.90. Found: C, 67.75; H,
6.24; N, 7.85.
5.1.2.23. 11-(4-Methylsulfanylbenzyl)-11H-isoindolo[2,1-a]benzimi-
dazol-11-ol, 24. IR (NaCl):
1499, 1463, 1389, 1313, 1262, 1152, 1089, 1051, 748 cm-1 1H NMR
(400 MHz, CDCl3):
n max 3361, 3064, 2922, 2855,1684,1621,
d
2,48 (s, 3H), 2.87 (d, J ¼ 14.0 Hz, 1H), 3.39 (d,
J ¼ 14.0 Hz, 1H), 6.70 (d, J ¼ 8.2 Hz, 1H), 6.88 (d, J ¼ 7.9 Hz, 1H), 7.02
(d, J ¼ 8.3 Hz, 2H), 7.12 (d, J ¼ 8.3 Hz, 2H), 7.23 (m, 1H), 7.29 (m, 1H),
7.41 (m, 1H), 7.43 (m, 1H), 7.46 (m, 1H), 7.79 (d, J ¼ 7.2 Hz, 1H); 13C
5.1.2.18. 5-(1-Naphthylmethyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
indol-5-ol, 19. IR (NaCl):
n
max 3359, 3049, 2928, 1684, 1597, 1469,
2.84 (m,
1110,1086,1021, 781, 767 cm-1 1H NMR (200 MHz, CDCl3):
d
NMR (100.6 MHz, CDCl3): d 15.7, 43.4, 92.3,118.5,123.0, 123.7, 123.8,
1H), 2.92 (m, 2H), 3.61 (d J ¼ 14.0 Hz, 1H), 3.70 (bs, OH), 3.81 (d
J ¼ 14.0 Hz,1H), 3.89 (m,1H), 7.15 (m,1H), 7.26 (m,1H), 7.35 (m,1H),
7.37 (m, 2H), 7.40 (m, 1H), 7.63 (m, 1H), 7.75 (m, 1H), 7.78 (m, 1H),
7.90 (m, 1H), 7.92 (dd, J ¼ 7.3; 1.8, 1H); 13C NMR (50.3 MHz, CDCl3):
123.9, 126.0 (2), 126.9, 129.6, 130.3, 131.4 (2), 131.8, 132.2, 137.0,
144.4, 146.7 (2), 168,9. ESI-MS: m/z 359.1141 [M þ H]þ; Anal. Calcd
for C22H18N2OS: C, 73.71; H, 5.06; N, 7.82. Found: C, 73.69; H, 5.08;
N, 7.80.
d
39.0, 41.1, 42.1, 89.2, 122.8, 123.0, 124.4, 125.1, 125.4, 125.8, 127.7,
128.7, 128.8, 129.0, 130.5, 131.8, 131.9, 132.8, 133.7, 148.7, 167.7; ESI-
MS: m/z 315.1419 [M þ H]þ; Anal. Calcd for C21H18N2O: C, 80.23; H,
5.77; N, 8.91. Found: C, 80.21; H, 5.73; N, 8.90.
5.1.2.24. 7(8)-Methyl-11-(4-methylsulfanylbenzyl)-11H-isoindolo
[2,1-a]benzimidazol-11-ol, 25. IR (NaCl):
1620, 1511, 1464, 1384, 1311, 1261, 1087, 1045, 1021, 804 cm-1 1H
NMR (200 MHz, CDCl3): 2,32 (s, 3H), 2,47 (s, 3H), 2.81 (d,
n max 3356, 2920, 1683,
d
5.1.2.19. 5-(2-Naphthylmethyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
J ¼ 14.0 Hz, 1H), 3.37 (d, J ¼ 14.0 Hz, 1H), 6.68 (s, 1H), 6.72 (d,
J ¼ 7.5 Hz,1H), 6.79 (d, J ¼ 7.5 Hz,1H), 7.02 (d, J ¼ 8.0 Hz, 2H), 7.04 (d,
J ¼ 7.5 Hz, 1H), 7.11 (d, J ¼ 8.0 Hz, 2H), 7.38 (dt, J ¼ 7.5, 1.4 Hz, 1H),
7.43 (dt, J ¼ 7.5, 1.4 Hz, 1H), 7.71 (dd, J ¼ 7.5, 1.4 Hz, 1H); 13C NMR
indol-5-ol, 20. IR (NaCl):
n
max 3360, 3054, 2926, 1690, 1560, 1468,
2.95 (m, 1H),
1078, 860, 820, 753 cm-1 1H NMR (200 MHz, CDCl3):
d
3.03 (m, 1H), 3.28 (m, 1H), 3.29 (bs, OH), 3.44 (d, J ¼ 13.9, 1H), 3.60
(d, J ¼ 13.9, 1H), 4.24 (m, 1H), 7.02 (dd, J ¼ 8.4, 1.1, 1H), 7.35e7.74 (m,
(50.3 MHz, CDCl3):
d 14.6 (CH3), 15.8 (CH3), 21.2 (CH3), 21.3 (CH3),
10H); 13C NMR (50.3 MHz, CDCl3):
d
39.4, 41.9, 44.6, 89.3, 122.4,
43.5, 92.4, 119.0, 123.7, 123.8, 123.9, 125.9 (2), 126.0, 129.5, 130.2,
131.5 (2),131.9 (2),132.0,136.9,139.6,144.2,146.7,169,1; ESI-MS: m/
z 373.1296 [M þ H]þ; Anal. Calcd for C23H20N2OS: C, 74.16; H, 5.41;
N, 7.52. Found: C, 74.13; H, 5.38; N, 7.47.
122.9, 125.5, 125.8, 127.3, 127.4, 127.5, 128.1, 128.8, 128.9, 130.7,
131.8, 132.2, 132.7, 133.0, 147.8, 167.4; ESI-MS: m/z 315.1419
[M þ H]þ; Anal. Calcd for C21H18N2O: C, 80.23; H, 5.77; N, 8.91.
Found: C, 80.19; H, 5.74; N, 8.89.
5.1.2.25. 11-(2-Naphthylmethyl)-11H-isoindolo[2,1-a]benzimidazol-
5.1.2.20. 5-(2-Thienylmethyl)-2,5-dihydro-3H-imidazo[2,1-a]iso-
11-ol, 26. IR (NaCl):
n max 3357, 3052, 2922, 1683, 1620, 1502, 1463,
indol-5-ol, 21. IR (NaCl):
n max 3251, 2942, 2853, 1677, 1615, 1470,
1392, 1313, 1261, 1151, 1090, 1052, 750 cm-1 1H NMR (200 MHz,

E.del Olmo et al. / European Journal of Medicinal Chemistry 46 (2011) 5379e5386
5385
CDCl3):
d
3.04 (d, J ¼ 14.0 Hz, 1H), 3.59 (d, J ¼ 14.0 Hz, 1H), 6.61 (d,
5.2. Biological studies
J ¼ 7.5, 1H), 6.86 (d, J ¼ 7.5 Hz, 1H), 6,92 (bs, OH), 7.22 (m, 1H), 7,28
(m, 1H), 7.32 (dd, J ¼ 7.9, 1.1 Hz, 1H), 7,39 (m, 1H), 7.42 (m, 1H), 7.46
(m, 1H), 7.53 (bs, 1H), 7.66 (d, J ¼ 7.9 Hz, 1H), 7,69 (m, 1H), 7.71 (dd,
J ¼ 7.9 Hz, 1H), 7,80 (m, 2H), 7.82 (m, 1H); 13C NMR (50.3 MHz,
5.2.1. Antiplasmodial activity in vitro
Parasites of the P. falciparum strain 3D7 were cultured using
standard procedures as described [15] with minor modifications.
Cultures were maintained in fresh group 0 positive human eryth-
rocytes, obtained from the “Centro Regional de Transfusión San-
guínea-SAS” (Granada, Spain) and suspended at 5% hematocrit in
RPMI 1640 containing 2% human serum, 0.2% NaHCO3, 0.5% Albu-
CDCl3):
d 44.3, 92.6, 118.4, 119.7, 123.7, 123.8, 125.7, 125.9, 127.3,
127.5, 127.7 (2), 129.1 (2), 129.6 (2), 129.9, 130.3, 132.0, 132.4, 132.7,
133.1, 144.1, 146.7, 167,5; ESI-MS: m/z 363.1419 [M þ H]þ; Anal.
Calcd for C25H18N2O: C, 82.85; H, 5.01; N, 7.73. Found: C, 82.80; H,
4.94; N, 7.75.
max II, 150
mM hypoxanthine, and 12.5 mg/mL gentamicin. Flasks
were incubated at 37 ꢀC, under 5% CO2.
5.1.2.26. 7(8)-Methyl-11-(2-naphthylmethyl)-11H-isoindolo[2,1-a]
The method used to assess parasite growth was based on the
fluorescent assay by using the SYBR Green I nucleic acid staining
dye (10,000 ꢂ stock concentration, purchased from Molecular
Probes, Inc. Eugene, Oreg.), a dye with marked fluorescence
enhancement upon contact with Plasmodium DNA [15]. This
method was used to determine the effective concentration of drug
that resulted in a 50% reduction in the observed fluorescence
(EC50), after 48 h of parasite growth in the presence of different
concentrations of imidazoisoindolol and related compounds.
benzimidazol-11-ol, 27. IR (NaCl):
1620, 1502, 1463, 1392, 1261, 1151, 1090, 1052, 750 cm-1 1H NMR
(200 MHz, CDCl3):
n max 3357, 2922, 2853, 1683,
d
2.31 (s, 3H), 2.27 (s, 3H), 3.05 (d, J ¼ 13.0 Hz,
1H), 3.65 (d, J ¼ 13.0 Hz, 1H), 4.38 (bs, OH), 6.62 (d, J ¼ 7.5 Hz, 1H),
6.70 (bs, 2H), 7.24e7.27 (m, 4H), 7.34e7.51 (m, 3H), 7.62e7.72 (m,
2H), 7.79e7,83 (m, 2H), 13C NMR (50.3 MHz, CDCl3):
d 21.2 (CH3),
20.6 (CH3), 44.2, 92.8, 117.9, 119.7, 123.8, 124.0, 125.8, 126.0, 127.4
(2C), 127.6, 127.8, 129.2 (2C), 129.6 (2C), 129.9, 130.1, 132.3, 132.5,
132.7, 133.1, 140.2, 146.6, 167.8. ESI-MS: m/z 377.1576 [M þ H]þ;
Anal. Calcd for C26H20N2O: C, 82.95; H, 5.35; N, 7.44. Found: C,
82.90; H, 5.29; N, 7.35.
For the fluorescence assay, 90
mL of the cell suspension was
introduced into each test well. Several wells containing non-
parasitised erythrocytes at 5% hematocrit served as reference
controls. Stock solutions of the test drugs were prepared at
a concentration of 100 mg/mL in >99.9% DMSO (Merck), serially
diluted in complete medium, and dispensed into triplicate test
5.1.2.27. (Z/E) 2-(2-amino-5-chlorophenyl)-3-(4-methylsulfanylben-
zylidene)isoindolin-1-one, 28. IR (NaCl):
n max 3434, 3336, 2919,
1704, 1628, 1588, 1495, 1426, 1384, 1339, 1262, 1184, 1088, 1050,
wells to yield final concentrations ranging from 0 to 0.2
mg/mL.
908, 758 cm-1. ESI-MS: m/z 393.0751 [M þ H]þ.
Final well volume was 100 L for the fluorescence assays with
m
Z-isomer : 1H NMR (400 MHz, CDCl3):
d
2.43 (s, 3H), 3.88 (bs,
a final concentration of DMSO ꢃ 0.001%. The stock culture was
synchronized with 5% sorbitol, and then approximately 96 h later,
the parasites were determined to be mostly late ring stages and
early trophozoites. The stock culture was then diluted with
complete medium and nonparasitised erythrocytes to yield
a hematocrit of 5% and a parasitemia of 0.5%. Cultures were added
NH2), 6.80 (s, 1H), 6.85 (d, J ¼ 8.3 Hz, 2H), 6.85 (m, 1H), 6.90 (d,
J ¼ 8.3 Hz, 2H), 7.12 (d, J ¼ 8.4 Hz, 1H), 7.27 (d, J ¼ 8.4 Hz, 1H), 7.50
(dd, J ¼ 7,8. 7.5, 1H), 7.68 (dd, J ¼ 7,8. 7.5, 1H), 7.84 (d, J ¼ 7.8 Hz, 1H),
7.92 (d, J ¼ 7.8 Hz, 1H); 13C NMR (100.6 MHz, CDCl3):
d 16.1, 108.1,
118.6, 119.2, 119.5, 123.8, 125.6 (2), 126.2, 127.4, 129.1 (2), 129.2131.2,
131.3, 132.5, 133.7, 135.5135.8, 137.8, 145.1, 167.7.
to each well reaching a final volume of 100 mL per well. Plates were
E-isomer: 1H NMR (400 MHz, CDCl3):
d
2.54 (s, NH2), 3.94 (s,
incubated at 37 ꢀC in 5% CO2. After 48 h of growth, SYBR Green I in
lysis buffer was added to each well, and the contents were mixed
until no visible erythrocyte sediment remained. After 1 h of incu-
bation in the dark at room temperature, fluorescence was
measured with a fluorescence multiwell plate reader (SpectraMax
Gemini EM) from Molecular Devices, with excitation and emission
wavelength bands centered at 485 and 530 nm, respectively. By
using the accompanying SOFTmaxPro software, the background
reading for an empty well was subtracted to yield fluorescence
counts for analysis.
3H), 6.22 (s, 1H), 6.34 (dd, J ¼ 8.4, 2.1 Hz, 1H), 6.57 (d, J ¼ 8.4 Hz,
1H), 6.63 (d, J ¼ 2.1 Hz, 1H), 6.85 (d, J ¼ 8.3 Hz, 2H), 6.90 (d,
J ¼ 8.3 Hz, 2H), 7.33 (d, J ¼ 8.3 Hz, 1H), 7.43 (t, J ¼ 7.4 Hz, 1H), 7.57
(t, J ¼ 8.3 Hz, 1H), 7.94 (d, J ¼ 8.3 Hz, 1H); 13C NMR (100.6 MHz,
CDCl3):
d 15.5, 112.3, 116.4, 116.7, 118.4, 121.6, 123.2, 123.7, 125.6
(2C), 126.8, 129.1 (2C), 129.6, 129.8, 132.1, 134.2, 135.1, 138.7, 138.8,
144.2, 165.9.
5.1.2.28. (Z/E) 2-(2-amino-5-chlorophenyl)-3-(2-naphthylmethyli-
dene)isoindolin-1-one, 29. IR (NaCl):
n
max 3456, 3375, 3330, 2954,
The counts were plotted against the logarithm of the drug
concentration and curve fitting by nonlinear regression to yield the
drug concentration that produced 50% of the observed decline from
the maximum counts in the drug-free control wells (IC50). The
fluorescence of parasitized erythrocytes in time 0 was substracted,
and the counts were then plotted and analyzed by linear regression
using Sigma Plot.
1701, 1621, 1497, 1466, 1427, 1269, 1175, 1120, 1074, 909, 749 cm-
cm-1. ESI-MS: m/z 397.1029 [M þ H]þ.
1
Z-isomer: 1H NMR (200 MHz, CDCl3):
d 3.93 (s, NH2), 6.13 (dd,
J ¼ 8.4, 2.2 Hz, 1H), 6.51 (d, J ¼ 2.2 Hz, 1H), 6.55 (m, 1H), 6.58 (d,
J ¼ 8.4 Hz, 1H), 7.00 (s, 1H), 7.11 (dd, J ¼ 8.4, 1.6 Hz, 1H), 7.40 (m,
1H), 7.41 (m, 1H), 7.50 (m, 2H), 7.51 (m, 3H), 7.71 (dt, J ¼ 7.6,
0.9 Hz, 1H), 7.82 (bs, 1H), 7.90 (d, J ¼ 7.6 Hz, 1H), 7.96 (d,
Each compound was evaluated per triplicate, including an
untreated control with solvent and culture medium and a positive
J ¼ 7.5 Hz, 1H); 13C NMR (50.3 MHz, CDCl3):
d 108.8, 116.4, 118.4,
118.5, 119.6, 121.6, 123.9, 126.0, 126.1, 126.3, 126.7, 127.4, 127.8,
128.4, 128.5, 129.2, 129.3, 130.2, 132.1, 132.6, 134.1, 134.2, 138.9,
144.3, 167.9.
control with CQ (IC50 ¼ 0.010
mM) as the reference drug for potency
comparisons.
E-isomer: 1H NMR (200 MHz, CDCl3):
d
4.00 (s, NH2), 6.43 (s,
5.2.2. In vivo antimalarial assays
1H), 6.89 (dd, J ¼ 8.4, 2.2 Hz, 1H), 6.93 (d, J ¼ 2.2 Hz, 1H), 7.18 (d,
J ¼ 8.4 Hz,1H), 7.35 (dt, J ¼ 7.7, 0.6 Hz,1H), 7.41 (m,1H), 7.50 (m,1H),
7.51 (m, 2H), 7.55 (m, 2H), 7.56 (m, 1H), 7.69 (m, 1H), 7.80 (bs, 1H),
The 4-day suppressive test was performed against P. berghei
NK65 in infected mice according to the procedure detailed by
Deharo et al. [24]. Swiss male mice (20 ꢄ 2 g) were infected with
107 parasitized cells on day 0. Groups of five mice were treated
intraperitoneally from day 0 (2 h after infection) to day 3 with doses
of 50 (25) mg/kg/day of compounds. The parasitemia was estimated
on day 4 examining, at the same hour of administration, Giemsa
7.88 (d, J ¼ 7.6 Hz, 1H); 13C NMR (50.3 MHz, CDCl3):
d 112.8, 116.8,
119.2, 123.2, 123.8, 126.3, 126.4, 126.5, 127.2, 127.3, 127.8, 127.9,
128.3, 129.7, 129.9, 131.4, 132.2, 132.4, 132.8, 133.2, 135.1, 135.6,
136.1, 145.1, 165.2.

5386
E.del Olmo et al. / European Journal of Medicinal Chemistry 46 (2011) 5379e5386
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sitized red blood cells was counted on at least 9000 red blood cells
for each concentration. Percent growth inhibition of the parasite
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Inhibition (%) ¼ (parasitemia in control - parasitemia in treated
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for complimentary access to preADMET database. BB and LDC thank
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and ALBAN (E07D401988BR) EC Programs. Financial support from
FIS-PI-060782, FIS-PI-052026, SAF2010-20059, the “Plan Andaluz
de Investigación (BIO199)” and ISCIII-RICET (RD06/0021: 0000,
0018, 0022) is also acknowledged.
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