Imidazo[2,1-a]isoindole scaffold as an uncharted structure active on Leishmania donovani

Article abstract of DOI:10.1016/j.ejmech.2019.111568

The human protozoan parasites Leishmania donovani and L. infantum are the causative agents of visceral leishmaniasis, as such, responsible for approximately 30,000 deaths annually. The available chemotherapeutic treatments are reduced to a few drugs whose effectiveness is limited by rising drug resistance/therapeutic failure, and noxious side-effects. Therefore, new therapeutic hits are needed. Compounds displaying the imidazo[2,1-a]isoindole skeleton have shown antichagasic, anti-HIV, antimalarial and anorectic activities. Here, we report the leishmanicidal activity of thirty one imidazo[2,1-a]isoindol-5-ol derivatives on promastigotes and intracellular amastigotes of L. donovani. Eight out of thirty one assayed compounds showed EC₅₀ values ranging between 1 and 2 μM with selectivity indexes from 29 to 69 on infected THP-1 cells. Six compounds were selected for further elucidation of their leishmanicidal mechanism. In this regard, compound 29, the imidazoisoindolol with the highest activity on intracellular amastigotes, induced an early decrease of intracellular ATP levels, as well as mitochondrial depolarization, together with a partial plasma membrane destructuration, as assessed by transmission electron microscopy. Consequently, the inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal mechanism of this compound, even when other additional targets cannot be ruled out. In all, the results supported the inclusion of the imidazoisoindole scaffold for the development of new leishmanicidal drugs.

Full text of DOI:10.1016/j.ejmech.2019.111568

Accepted Manuscript
Imidazo[2,1-a]isoindole scaffold as an uncharted structure active on Leishmania
donovani
Sobinson Arsène, Verónica Gómez-Pérez, Ricardo Escarcena, María Ángeles
Abengózar, Raquel García-Hernández, Montserrat Nácher-Vázquez, Arturo San
Feliciano, Francisco Gamarro, Luis Rivas, Esther del Olmo
PII:
S0223-5234(19)30698-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.111568
Article Number: 111568
Reference:
EJMECH 111568
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 April 2019
Revised Date: 26 July 2019
Accepted Date: 27 July 2019
Please cite this article as: S. Arsène, Veró. Gómez-Pérez, R. Escarcena, Marí.Á. Abengózar,
R. García-Hernández, M. Nácher-Vázquez, A. San Feliciano, F. Gamarro, L. Rivas, E.d. Olmo,
Imidazo[2,1-a]isoindole scaffold as an uncharted structure active on Leishmania donovani, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111568.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Micrographs of L. donovani promastigotes
The imidazo[2,1-a]isoindolol (IIOL)
structure present in compounds damaging
Leishmania donovani membrane
Control parasites
After adding IIOL 29

ACCEPTED MANUSCRIPT
Imidazo[2,1-a]isoindole scaffold as an uncharted structure active on
Leishmania donovani
a
b
a
c
Sobinson Arsène , Verónica Gómez-Pérez , Ricardo Escarcena , María Ángeles Abengózar ,
b
c
a
Raquel García-Hernández , Montserrat Nácher-Vázquez , Arturo San Feliciano , Francisco
b
c
a
Gamarro* , Luis Rivas* , Esther del Olmo*
a
Departamento de Ciencias Farmacéuticas: Química Farmacéutica, Facultad de Farmacia, Universidad de
Salamanca, CIETUS, IBSAL, 37007-Salamanca, Spain.
b
c
Instituto de Parasitología y Biomedicina “López-Neyra”, IPBLN-CSIC, Granada, Spain.
Centro de Investigaciones Biológicas, CIB-CSIC, Madrid, Spain.
*
Corresponding authors
E-mail addresses: olmo@usal.es (E. del Olmo, Chemistry), luis.rivas@cib.csic.es (L. Rivas, Mechanism
studies), gamarro@ipb.csic.es (F. Gamarro, in vitro assays).
Abstract
The human protozoan parasites Leishmania donovani and L. infantum are the causative agents of
visceral leishmaniasis, as such, responsible for approximately 30,000 deaths annually. The available
chemotherapeutic treatments are reduced to a few drugs whose effectiveness is limited by rising
drug resistance/therapeutic failure, and noxious side-effects. Therefore, new therapeutic hits are
needed. Compounds displaying the imidazo[2,1-a]isoindole skeleton have shown antichagasic, anti-
HIV, antimalarial and anorectic activities. Here, we report the leishmanicidal activity of thirty one
imidazo[2,1-a]isoindol-5-ol derivatives on promastigotes and intracellular amastigotes of L.
donovani. Eight out of thirty one assayed compounds showed EC values ranging between 1 to 2
5
0
µM with selectivity indexes from 29 to 69 on infected THP-1 cells. Six compounds were selected
for further elucidation of their leishmanicidal mechanism. In this regard, compound 29, the
imidazoisoindolol with the highest activity on intracellular amastigotes, induced an early decrease
of intracellular ATP levels, as well as mitochondrial depolarization, together with a partial plasma
membrane destructuration, as assessed by transmission electron microscopy. Consequently, the
inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal
mechanism of this compound, even when other additional targets cannot be ruled out. In all, the
results supported the inclusion of the imidazoisoindole scaffold for the development of new
leishmanicidal drugs.

ACCEPTED MANUSCRIPT
Keywords: Imidazoisoindolols, antileishmanial activity, energy metabolism.
1. Introduction
Leishmaniasis is a group of diseases caused by protozoan parasites from over 20 species of the
genus Leishmania, transmitted to humans by the bite of infected female phlebotomine sandflies. It
is considered as the third-most-common parasitic disease after schistosomiasis and malaria on the
basis of its morbidity and disability-adjusted life years (DALYs) [1]. With an estimated 700,000 to
1
million new cases and some 26,000 to 65,000 deaths occurring every year, leishmaniasis
continues to be a menace in a large number of countries worldwide [2]. From a clinical perspective,
leishmaniasis is grouped under three major forms. The cutaneous leishmaniasis is its mildest form,
producing ulcers at the site of the bite; the mucocutaneous form courses with lymphatic migration
of Leishmania from the initial bite into mucosal tissues of the nasal, oral and throat cavities, leading
to destruction of the cartilaginous tissue. Nevertheless, the most severe form of the disease is the
visceral leishmaniasis, caused by Leishmania donovani and L. infantum (also known as L. chagasi).
In VL the parasite infects mononuclear phagocyte cells in liver, spleen and bone marrow, causing
the visceral form of leishmaniasis (VL) or kala-azar, fatal in more than 95% of cases if left
untreated. It is characterized by irregular episodes of fever, weight loss, hepatosplenomegaly and
anaemia. VL is endemic in the Indian subcontinent and East Africa; and it is estimated that between
50,000 and 90,000 new cases of VL occur in the world every year.
Treatment options for VL include the use of pentavalent antimonials, amphotericin B (AmB),
paromomycin and miltefosine [1]. Most of these drugs display several drawbacks such as low
efficacy, severe side effects, parental administration, and emergence of drug resistance, or
significant rate of therapeutic failure [3]. For example, miltefosine, the only oral drug for
leishmaniasis treatment produces teratogenic effects, and severe gastrointestinal side effects have
been reported [4].
During the last decades, substantial efforts have been invested to discover more efficacy therapeutic
agents against leishmaniasis [1, 5], fuelled by the significant increase in therapeutic failure and drug
resistance. Research has been focused either on the quest for natural products [6, 7], target-based
drug discovery approaches encompassing, among others, nuclear [8] and kinetoplast
topoisomerases [9], trypanothione reductase (TryR) [10], and biosynthetic pathways for
phospholipids and sterols [11], or new synthetic molecules assayed for phenotypic output [12, 13].
Recently, nitroaromatic drugs like fexinidazole, activated by parasite nitroreductases have been
assayed as new leishmanicidal drugs [14]. Nowadays, Drugs for Neglected Diseases initiative acts
as the best hub for new scaffolds under the last preclinical steps of development or ongoing clinical

ACCEPTED MANUSCRIPT
trials. The oxaborole DNDI-6148, the nitroimidazole DNDi-0690, the aminopyrazole DNDI-5561,
and the GSK3186899/DDD853651 (N-(trans-4-((3-isobutyl-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)amino)-cyclohexyl)-1- phenylmethanesulfonamide 4-trans) are currently in its portfolio [5,15].
In a previous study, we synthesized and tested in vitro against Leishmania spp. some families of
heterocyclic compounds sharing the dihydrostilbene scaffold, including 4-benzylidenephthalazin-1-
ones, phenacetyl-benzamides, 5-benzylimidazo[2,1-a]isoindol-5-ols and 6-benzylpyrimido[2,1-
a]isoindol-6-ols [16]. From these, the last two groups showed a promising leishmanicidal activity,
equivalent to reference clinical drugs. To gain further insight into the leishmanicidal potential of the
5-benzylimidazo[2,1-a]isoindol-5-ol family, new compounds were designed, synthesized and
assayed (compounds 1-31, Scheme 1). The set of new compounds explore the chemical space of
this scaffold, with variation of the electronic nature and size of the substituents, as well as their
positions in the aromatic rings.
In all, we have provided a proof-of-concept for the potentiality of developing new antiparasitic
drugs of the imidazoisoindolol (IIOL) scaffold, until now scarcely studied in medicinal chemistry,
as well as an insight of the molecular basis underlying their antiparasitic potential.
2
. Results and Discussion
.1 Chemistry
2
The imidazoisoindolols (IIOLs) were obtained, by total synthesis through the route depicted in
Scheme 1, as previously reported [16]. Briefly, the synthesis starts by condensation of phthalic
anhydrides with substituted phenyl or naphthylacetic acids to provide benzalphthalides (BPs) and
naphthylmethylphthalides (NPs), through an adaptation of the Nokihara method [17], using toluene
and a Dean-Stark separator to remove the water under inert atmosphere. The resulted BPs (NPs)
were treated with ethylenediamine at 80-90 ºC, to give the benzyl(naphthylmethyl)imidazo[2,1-
a]isoindol-5-ol derivatives BIIOLs (NIIOLs), compounds 1-31. Physicochemical, MS and NMR
spectral data for all the new compounds are here reported.
Scheme 1. General procedure for the synthesis of 5-benzyl(naphthylmethyl)imidazo[2,1-a]isoindol-5-ol
derivatives

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1
The H-NMR spectra of BIIOLs (NIIOLs) showed four multiple signals at δ 2.77/3.21 ppm and
δ 3.70/4.14 ppm (imidazoline protons). The benzylic methylene signals appeared as an AB system
δ 3.10 and 3.38 ppm (J = 13.6-14.0 Hz). The signal corresponding to the hydroxyl proton resonated
as a singlet at δ ~ 4.20 ppm (D O exchangeable). The signals for aromatic protons appeared at δ
2
1
3
6
.81 - 7.55 ppm, depending on the substituents at rings A and B. The C-NMR spectra of BIIOLs
(
NIIOLs) showed signals for three methylene carbons at δ 39.5, 41.7 (C-2, C-3) and 44.0 ppm
benzylic methylene), for a single non protonated oxygenated carbon at δ 89.4 ppm (C-5); and for
(
one amidine carbon around δ 167.8 ppm. The aromatic carbon signals appeared at δ between
22.2-150.2 ppm, depending on the substitution pattern of the rings.
1
Those compounds monosubstituted at ring A (3 to 20, 21 to 24 and 30 to 31) were obtained and
evaluated as mixtures 1:(0.9-0.6) of 7(8)- or 6(9)- substituted derivatives. Both regioisomers were
1
13
properly identified and characterized by their H and C-NMR spectra and, in some cases,
supported by conducting 2D experiments as COSY, HSQC and HMBC NMR correlations.
2.2 Biological analysis
The imidazoisoindole core remains an almost fully uncharted structure in Medicinal Chemistry.
Mazindol, an imidazoisoindole with non-amphetamine anorectic properties, was withdrawn from
the market due to side effects as pulmonary arterial hypertension, also found in other anorexic drugs
[
18]. Five additional works from Olmo et al. rescued the imidazo[2,1-a]isoindolol scaffold as the
basis of antiplasmodial [16, 19], anti-chagasic [20], and anti-HIV [21] activities of heterocyclic
compounds related to stilbenoids, as well as other work unveiling the leishmanicidal activity of this
scaffold on Leishmania promastigotes [22].
In this work a double objective was pursued, first to broaden the knowledge on the structure-
activity relationship for the imidazo[2,1-a]isoindolols on Leishmania, and secondly, to get insight
into their leishmanicidal mechanism. For those purposes, new structures were synthesized and
added to some of the compounds tested on Leishmania promastigotes in the previous work [22],
resulting in a final set of 31 compounds. The chemical space was explored by the diversity of
substituents employed, both in their chemical nature and in their positions in rings A and B of the
IIOL scaffold. In order to get a closer appraisal on the chemotherapeutical potential of these
molecules, they were assayed on intracellular amastigotes, as the most relevant clinical form of the
parasite, and also the closest in vitro model for human Leishmania infection. Secondly, the
leishmanicidal mechanism of the most potent compounds was explored, with a special focus on the

ACCEPTED MANUSCRIPT
energy metabolism of the parasite; not only as a feasible ultimate target of these molecules on
Leishmania, but also to monitor variations in parasite viability.
As aforementioned, in a previous work we described the leishmanicidal activity of compounds with
various heterocyclic skeletons on promastigotes of Leishmania braziliensis, L. amazonensis, and L.
donovani. There, those compounds with an imidazoisoindole skeleton without substitution on ring
A, combined with nil or a single para-chloro substitution on ring B, showed the highest potency
[
22]. Therefore, they were taken as reference for the current study, and named here as compounds 1
and 2.
The EC values on intracellular amastigotes of 1 and 2 were compared with those from compounds
5
0
1
3
to 31. Eighteen of these new compounds (3 to 20) have a methyl group (R ) at position 7 or 8 on
ring A. This molecular trait is combined with modifications on ring B, consisting on larger aromatic
groups (α- or β-naphthyl), or groups with effect on the electron density of the ring, either as
electron-donor (Me, OMe, OH) or electron-attracting (F, Cl) substituents. Compound 3, without
substitution at ring B, was the internal control of this series. All these compounds were tested for
leishmanicidal activity as a mixture of regioisomers 1:(0.9-0.6). The activity data for the full set of
compounds against L. donovani intracellular amastigotes in vitro were compiled in Table 1.
Compound 2 showed a good inhibitory activity (EC = 2.1 µM, for amastigotes) together with an
50
appealing selectivity index versus THP-1 cells (SI = 69.1). Compound 1, without substitutions in
rings A or B is ten times less potent than 2, and three times less than 3, with only a single methyl
group in ring A. Besides, the SI of compound 3 was four times lower than that of 2. The
introduction of an electron-attracting substituent (Cl, F) at ortho or para positions had opposite
effects, depending on the halogen. The chloro-substitution, either at position ortho (compound 4,
EC = 1.8 µM) or para (compound 7, EC = 1.4 µM) improved slightly the activity of the parental
50
50
2
. In contrast, the fluoro derivative 6 (EC = 5.7 µM), was two and a half times less potent, and its
50
SI halved respect to compound 2. The introduction of either a strong electron-attracting (NO , 5) or
2
donating groups (OH, 8; OMe, 9) impaired their leishmanicidal activity. The sulfanyl derivative 10
(
EC = 1.3 µM), was slightly more potent than compound 2, whereas compound 7 (EC = 1.4 ±
5
0
5
0
0.3, SI = 62.4) showed similar potency and selectivity. In fact, it was actually the most potent
compound within the para monosubstitued compounds on ring B. The oxidation of the sulfide 10 to
sulfoxide (11) or sulfone (12) led to a reduction in the leishmanicidal activity.
The activity of compounds with a multi-substituted ring B was highly dependent on the nature,
number and position of substituents. The activity decreased for an ortho/para difluoro substitution
(13, EC50 = 7.1 µM), whereas in the case of the dichloro compounds, 14 (ortho/para) and 15
(meta/para) preserved the activity of 7; however, the SI for 15 was better than for 14.

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Table 1. Structure, leishmanicidal activity and cytotoxicity of imidazoisoindolol derivatives.
Intracellular
amastigotes
Promastigotes
1
2
THP-1
MRC-5
Compound
R
R
c
SI
b
b
d
e
CC50 (µM)
CC50 (µM)
EC50 (µM)
IC50 (µM)
a
EC50 (µM)
1
2
3
4
5
6
7
H
H
H
4-Cl
H
27.8 ± 2.9
2.1 ± 0.3
9.2 ± 0.8
1.8 ± 0.0
> 10
>200
182.2 ± 2.1
79.9 ± 3.0
125.6 ± 11.9
58.4 ± 3.7
121.9 ± 5.3
111.1 ± 2.9
64.4 ± 1.6
-
nd
11.8 ± 6.8
nd
nd
nd
nd
145.1 ± 3.1
69.1
17.9
7(8)-Me
164.6 ± 13.2
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
2-Cl
3-NO
4-F
80.7 ± 8.6
182.8 ± 9.6
173.2 ± 3.6
84.6 ± 2.6
44.8
<18.3
30.3
5.9 ± 0.8
nd
16.7 ± 2.5
nd
2
5.7 ± 0.4
1.4 ± 0.3
nd
nd
4-Cl
61.5
6.1 ± 1.3
14.3 ± 1.8
8
9
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
7(8)-Me
6(9)-NO2
4-OH
4-OMe
4-SMe
4-SOMe
7.8 ± 2.6
> 10
>250
176.8 ± 1.3
80.5 ± 5.8
>200
138.9 ± 5.7
120.3 ± 1.9
36.2 ± 3.9
>200
>32.2
<17.7
62.4
-
nd
nd
nd
nd
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
1.3 ± 0.0
>10
5.1 ± 1.0
16.2 ± 1.3
nd
nd
nd
4-SO
2
Me
>10
>200
181.9 ± 22.3
78.5 ± 3.3
15.7 ± 0.4
29.9 ± 2.7
121.2 ± 16.1
187.3 ± 33.4
113.6 ± 11.9
20.5 ± 1.8
20.1 ± 1.8
112.4 ± 11.5
122.0 ± 18.4
110.2 ± 22.1
47.2 ± 1.5
41.0 ± 4.0
33.8 ± 0.3
77.4 ± 8.2
-
nd
2,4-diF
2,4-diCl
3,4-diCl
7.1 ± 1.1
1.3 ± 0.2
1.3 ± 0.2
> 10
162. ± 5.3
22.2 ± 0.0
38.6 ± 0.9
>250
23.0
16.7
29.3
-
nd
nd
12.7 ± 3.4
nd
10.5 ± 1.8
nd
3,4-OCH
2
O
nd
nd
3,4-diOMe
3,4,5-triOMe
α-naphthyl
β-naphthyl
4-Cl
> 10
246.3 ± 5.3
191.6 ±12.3
42.2 ± 4.1
42.4 ± 1.2
146.6 ± 23.0
>200
< 24.6
<19.2
13.5
12.4
<14.6
>20
18.5
35.6
19.7
26.2
12.7
nd
nd
> 10
nd
nd
3.1 ± 0.4
3.4 ± 0.5
>10
nd
nd
nd
nd
nd
nd
7(8)-CH OH
2
4-Cl
>10
nd
nd
7(8)-CHO
7(8)-NO2
6,9-diCl
6,9-diCl
4-Cl
8.3 ± 0.8
1.9 ± 0.4
4.2 ± 0.2
2.6 ± 0.2
6.7 ± 0.8
153.4 ± 11.9
69.0 ± 8.2
82.0 ± 0.6
68.7 ± 2.2
84.4 ± 0.8
nd
nd
4-Cl
7.1 ± 1.1
nd
nd
2-Cl
nd
2
2
6
7
4-Cl
13.3 ± 1.7
nd
14.4 ± 3.6
nd
6,9-diCl
7,8-diCl
7,8-diCl
4-SMe
2
2
3
3
8
9
0
1
4-Cl
3.0 ± 0.4
1.0 ± 0.1
2.5 ± 0.1
1.1 ± 0.3
0.1 ± 0.0
31.9 ± 5.9
31.1 ± 1.1
35.0 ± 2.7
36.0 ± 2.7
16.0 ± 2.1
8.9 ± 2.8
8.5 ± 1.5
31.3 ± 1.8
28.8 ± 1.2
12.0 ± 1.5
10.8
31.1
13.8
33.3
177.3
6.9 ± 1.0
2.1 ± 0.4
9.4 ± 0.9
7.6 ± 1.7
0.1 ± 0.0
nd
7.3 ± 1.5
nd
4-SMe
3,4-diCl
3,4-diCl
6(9)-NO
2
2
7(8)-NO
14.7 ± 2.9
AmB
a)
As specified in Experimental section, activity of the compounds on intracellular amastigotes was measured by the
b)
luciferase test; data were expressed as EC50 ± SD, half maximal effective concentration. Cytotoxic concentration 50
c)
(
CC ) was measured by the inhibition of MTT reduction. Selectivity index (SI) was defined as the ratio: CC50 THP-
5
0
d)
1
/EC50 for intracellular amastigotes.
EC50 stands for the concentration of the respective compound that inhibited
promastigote proliferation by 50%. To this end, MTT reduction was measured after 72 h of promastigote proliferation in
e)
the presence of the respective control. IC50 stands for the concentration of the compound that inhibited MTT reduction
after 4 h incubation of promastigotes with the compound, hence of its short term effects. Data were expressed as the
corresponding parameter ± SD. Three independent experiments were carried out; nd= not determined. Amphotericin B
(
AmB) was used as reference drug. Values highlighted for comparison purposes, in red those of most potent compounds
with EC ≤ 3µM, in blue those compounds with EC values between 3 and 10 µM.
5
0
50

ACCEPTED MANUSCRIPT
In ring B, neither a methylenedioxy substitution (16), nor the presence of two (17) or three methoxy
groups (18), improved the activity respect to compound 9, with a single methoxy substitution. The
presence of an enlarged aromatic group as α−naphthyl, 19 (EC = 3.1 µM), and β-naphthyl, 20
5
0
(
EC = 3.4 µM), led to a leishmanicidal activity similar to compound 2, but with an impaired SI
50
(13.5 and 12.4, respectively versus the SI = 69 of compound 2).
Altogether, we can conclude that the best substitution on ring B (compounds 3 to 20) to kill L.
donovani intracellular amastigotes, was the p-methylsulfanyl (10), followed by the m,p-dichloro
(15), and the p-chloro (7) substitutions. In contrast, the ranking for SI was 10, 7 and 15.
Based on the previous results, some deactivating substitutions on ring A, combined with the best
ones found on ring B (p-Cl, p-SMe and m,p-diCl) were further analysed. Ring A was mono-
substituted at positions 6 or 9 and 7 or 8, or di-substituted at positions 6,9 and 7,8. Among the set of
compounds with a p-chloro on ring B, and monosubstitution at ring A, 21 (6(9)-NO ), 22 [7(8)-
2
CH OH], 23 [7(8)-CHO] and 24 [7(8)-NO ], compound 24, with a nitro group at position 7 or 8
2
2
showed the best activity (EC = 1.9 µM, SI = 35.6), rather similar to 4 (with 7(8)-Me on ring A and
50
o-chloro in ring B).
To note, compound 24, with a 7,(8)-nitro substitution, has better leishmanicidal activity than 21
with a 6(9)-nitro substitution. However, the activity of 30 (EC = 2.5 µM, and SI = 13.8), with the
5
0
6
(9)-NO and two chlorines (m,p-diCl) on ring B, was similar to 2. The 7(8)-nitro derivatives, with
2
one or two chlorine atoms in ring B showed good results (24, EC = 1.9, SI =35.6; 31, EC =1.1
5
0
50
µM, SI=33.3). Thus, the addition of one m-Cl to a p-Cl on ring B improved the leishmanicidal
activity. In fact, compound 31 is the second most potent compound of this series.
The best compound, with a 6,9-dichloro substitution on ring A, was 26 (EC = 2.6, SI = 26.2), with
50
a p-Cl at ring B. Overall, the most potent compound of the series was 29 (EC = 1.0, SI = 31.1)
5
0
with 7,8-diCl on ring A and p-SMe on ring B.
Additionally, the most potent compounds on intracellular amastigotes (EC < 3 µM: 2, 4, 7, 10, 14,
5
0
1
5, 24, 26, 28, 29, 30 and 31) were assayed on L. donovani promastigotes. From these, compound
9 was also the most potent (with EC = 2.1 µM), followed in decreasing order by compounds 10
2
5
0
(
7(8)-Me / p-SMe), 4 (7(8)-Me / o-Cl) ≅ 7 (7(8)-Me / p-Cl), 28 (7,8-diCl / p-Cl) ≅ 24 (7(8)-NO / p-
2
Cl), 30 (6(9)-NO / m,p-diCl) and 31 (7(8)-NO / o,p-diCl). Noteworthy, the IIOLs were more
2
2
potent against the intracellular L. donovani amastigotes than on the promastigote forms of this
parasite.

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In order to define the primary target of imidazoisoindols, their effects on promastigotes were
monitored after a short incubation (t = 4 h) with the selected compounds. Under these experimental
conditions, inhibition of MTT reduction, carried out by mitochondrial dehydrogenases, was
measured immediately after the end of the incubation, and used as a parameter to select those
concentrations of the drug that decreased the viability of parasites. The experimental conditions and
parasite model were selected in order to assess the real effects of IIOLs on the energy metabolism
of Leishmania as their ultimate target: i) short incubations times minimize the variation of energy-
metabolism as a secondary effect of the action on other feasible primary targets; ii) promastigotes
were chosen as their proliferation and metabolic rate are much higher than that of intracellular
amastigotes, for which a severe metabolic quiescence was reported [23, 24], making them easier
cells to highlight anomalies on energy metabolism; iii) the promastigote, as extracellular form, has
free access to the drugs, without the restrictions imposed to intracellular amastigotes by the
macrophage as their host cell. Additionally, a putative modulation of the leishmanicidal effects of
the macrophages by IIOLs was avoided. Throughout its life cycle, Leishmania mostly relies on the
oxidative phosphorylation as the powerhouse for its energy metabolism [24].
As expected, the IC values corresponding to the inhibition of MTT after 4 h incubation were
5
0
consistently higher than their respective EC values, the parameter for inhibition of proliferation
5
0
(Table 1). Consequently, the leishmanicidal mechanism, or at least its ensuing aftermath, did not
reach its final end after the 4 h incubation. As such, IC values were taken as a reference for the
5
0
evaluation of the rest of the energetic parameters.
Next, the variation in the intracellular levels of ATP caused by a selected set of compounds was
assessed on promastigotes of the Leishmania 3-Luc strain. These parasites report ATP variations in
living parasites in real time, due to the expression of a cytoplasmic form of firefly luciferase, unable
to be imported into the glycosomes, as occurs with the native enzyme. Furthermore, the poor
membrane permeability of D-luciferin at pH 7.0 was overcome using a caged luciferin ester
(DMNPE-luciferin) [25]. The variation in luminescence were assessed either immediately after the
addition of the respective IIOL (Figure 1), or after a 4 h incubation (Figure 2). As expected, the
immediate effect of IIOL addition on the inhibition of luminescence (Figure 1) was consistently
lower than that measured after 4 h incubation (Figure 2), and their percentages for luminescence
inhibition were closer to their respective IC (Figure 2). In this experiment, IIOL 29 resulted as the
5
0
most active compound, followed by 26 and 31 for the two incubation time modalities. The decrease
in ATP levels was also observed for the other three tested compounds, although for compound 4 the
drop in ATP levels after the first 30 min of incubation was practically negligible (Figure 1).

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Figure 1. Variation of luminescence of 3-luc L. donovani promastigotes after imidazoisoindolols
4, 7, 10, 26, 29 and 31 addition.
Imidazoisoindolols at the respective concentration were added at a promastigotes suspension in RPMI-Øred
6
medium (20 × 10 cells/mL) in the presence of 50 µM DMNPE-D-luciferin. Afterwards, variation of the
luminescence of living parasites was monitored in a real time basis. The number of the compound is included
inside its corresponding panel. Luminescence was expressed as the percentage respect to untreated parasites. To
improve the clarity of the graphic only the significant concentrations for each imidazoisoindolol were
represented, although the full set of concentrations (5, 10, 20, 30, 40, and 50 µM) were assayed for each of the
selected compounds. Parasites treated with 1.5 µM of 1,4-naphthoquinone were used as internal control. Legend:
Imidazoisoindolol concentration (µM): 50 (p); 40 (
).
ꢀ ); 30 (´); 20 (ô); 10 (q); 1,4-naphthoquinone (1.5 µM,
✩

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Figure 2. Variation of intracellular ATP levels in L. donovani promastigotes after 4h incubation
with imidazoisoindolols 4, 7, 10, 26, 29 and 31.
Imidazoisoindolols at the respective concentrations were added at a promastigotes suspension in RPMI-Øred
6
medium (20 × 10 cells/mL), and incubated for 4 h at 26 ºC. Afterwards DMNPE-luciferin was added (50 µM,
final concentration) and the luminescence recorded for 10 min. Luminescence values were represented as the
percentage respect to untreated controls. Afterwards, variation of the luminescence of living parasites was
monitored in a real time basis. The number of the compound is included inside its corresponding panel.
Luminescence was expressed as the percentage respect to untreated parasites. Parasites treated with 1.5 µM 1,4-
naphthoquinone (1,4-NPHQ) were used as positive control.
In a further step, the origin of the decrease of ATP was approached. In Leishmania, the contribution
of oxidative phosphorylation to fulfil the global demand of ATP is much higher than glycolysis,
especially for intracellular amastigotes [23]. Therefore, to gauge the mitochondrial functionality of

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the parasites, the electrochemical potential of mitochondria (∆Ψm), assessed by the intracellular
accumulation of Rhodamine 123 (Rh123), is a reliable parameter. This probe has a positive charge
delocalized by resonance, behaving as a hydrophobic cation. Consequently, it distributes across
both sides of the organelle membranes in response to the membrane potential following the Nernst
equation [24]. Rh123 is almost exclusively accumulated inside the mitochondrion, as the organelle
with the highest ∆Ψm. As can be seen in Figure 3, compound 29 again produced the highest
inhibition, leading to a full depolarization at 30 µM, followed by compounds 26 and 31. The other
three compounds assayed, 4, 7 and 10, showed a decrease in Rh123 accumulation although at
concentrations considerable higher than the aforementioned compounds.
Figure 3. Rhodamine 123 incorporation to L. donovani promastigotes after incubation with
imidazoisoindolols 4, 7, 10, 26, 29 and 31.
6
Promastigotes were resuspended at 20 × 10 cells/mL in RPMI-Øred and incubated with the corresponding
concentration of imidazoisoindolols for 4 h. Afterwards, parasites were incubated with Rh123, and the
intracellular accumulation of the probe measured by flow cytometry (ʎexc= 488nm, ʎem = 520 nm). Parasites
incubated with 10 mM KCN were used as a control for a dysfunctional mitochondrion.
The induction of membrane permeability is the other most likely alternative to account for the loss
of ATP. Under a permeabilization process, the ionic gradients across the plasma membrane were
dissipated, leading to a futile hydrolysis of ATP by the ionic gradients; additionally, the import of
metabolites carried out by electrochemical transporters is jeopardized. If the membrane lesion is
severe enough, cytoplasmic content, including nucleotides may leak [25]. Plasma membrane
permeabilization was monitored by the increase in fluorescence of SYTOX green, a membrane

ACCEPTED MANUSCRIPT
impermeable intercalating agent. For compounds 26, 29, and 31, a concentration dependent increase
of SYTOX green fluorescence was observed. The most pronounced increase was for compound 29,
evident at 10 µM, whereas compound 31 showed the slowest permeabilization rate (Figure 4).
Figure 4. Entrance of the vital dye SYTOX green in L. donovani promastigotes induced by
imidazoisoindolols 26, 29 and 31.
6
Imidazoisoindolols at their respective concentration was added to a promastigotes suspension (20 x 10
cells/mL) resuspended in RPMI-Øred containing 1.0 µM SYTOX green, and the increase in fluorescence
monitored (ʎ = 485 nm; ʎ = 520 nm). Triton X-100 (0.1%, final concentration) was added at the point indicated
em
by an arrow, and this fluorescence considered as 100%. Only the traces of the significant IIOL concentrations
were included in each panel to avoid confusion due to overlapping traces. Legend: IIOL concentration (µM): 50
(
p); 40 (ꢀ ); 30 (´); 20 (ô); 10 (q).
For concentrations close to their IC , the increase in fluorescence for these three compounds
5
0
remained unnoticed at least 20 min after their addition to the parasites, even for the highest
concentration assayed (50 µM) (Figure 4). Even for compound 29 at 10 µM, this lag was close to 1h
after addition. By electron microscopy some blebbings of the plasma membrane were visualized in
promastigotes treated overnight with compound 29 at 10 µM (~ 70% inhibition of MTT reduction)
(Figure 5). Nevertheless, this damage to the plasma membrane is far less severe than that produced
by typical membrane agents, as some antimicrobial peptides, with massive release of cytoplasmic
material [26]. The micrograph of L. donovani promastigote treated with IIOL 29 (Figure 5, panel B)
showed swelling of the mitochondrial cristae suggesting at least a partial independence of the
mitochondrial damage with a massive membrane permeabilization not observed in the same
micrograph. This hypothesis agrees with the experimental kinetics of the decrease of ATP levels
and entrance of SYTOX green. The intracellular accumulation of SYTOX green is a process close
to irreversibility, due to the high affinity of this probe for DNA. Hence, even transitory and
reversible lesions may lead with time to a significant intracellular concentration of the dye in the
absence of severe membrane permeabilization.

ACCEPTED MANUSCRIPT
Figure 5. Transmission electron micrographs of L. donovani promastigotes treated with
imidazoisoindolol 29.
Parasites were incubated with 10 µM imidazoisoindolol 29 for 12 h in complete growth medium. Panel A.-
Control parasites. Panel B.- Parasites treated with 10 µM of 29. Solid arrows pinpoint damaged mitochondrial
areas. Dashed arrow points out to a blebbing in the plasma membrane of the parasite. Magnification bar = 1µm.
In this regard, a low permeability status of the plasma membrane requires the maintenance of a
phospholipid asymmetry, for which flippases and floppases, both dependent on ATP hydrolysis,
played a major role [27], thus a mild damage to the plasma membrane after a severe ATP depletion
cannot be ruled out. In all, the experimental results support that the plasma membrane
permeabilization is rather an effect than the origin of the leishmanicidal activity of
imidazoisoindolols.
The single mitochondrion of trypanosomatids is an appealing chemotherapeutical target, as
demonstrated by the ample spectrum of chemical structures successfully assayed for leishmanicidal
activity with mitochondrial involvement [28-30]. To note, two of the drugs under current clinical
use, paromomycin and miltefosine, have the mitochondrion of Leishmania as one of their respective
targets [31, 32]. In Leishmania as in other trypanosomatids, there is only a single mitochondrion per
cell [33]; this may ruled out a hypothetical functional compensation by other unharmed
mitochondria [30]. Additionally, a deficit in the mitochondrial contribution to the energy
metabolism of the parasites, can only partially offset by an increase in the glycolytic capacity [34].
The infection of the macrophages by Leishmania shifts the energy metabolism of the host cells from
a glycolytic pattern into a more relevant contribution oxidative phosphorylation [35]. Even when
some of the best IIOLs tested in this work have SI > 30, we cannot discard that the susceptibility of
Leishmania infected macrophages will be higher than the non-infected ones, due to their higher
dependence on oxidative phosphorylation. In this context, under the imidazoisoindolol challenge,
we may surmise a metabolic rewiring of the infected macrophages towards glycolysis, supposedly

ACCEPTED MANUSCRIPT
less favourable for survival and replication of intracellular survival. If this is the case, IIOLs will
not be exclusively targeting the parasite, but the host-parasite interphase as well.
In any case, we cannot rule out the existence of other targets for imidazoisoindolols aside the
mitochondrial energy metabolism of Leishmania, as exemplified with compound 4.
Most potent compounds were selected for the determination of their physicochemical, ADME, and
drug-like properties. These were obtained from OSIRIS [36] and preADMET [37] algorithms
accessed online. Thus, clogP values ranged from 2.37 to 4.57; logS values from -3.20 to -4.33 (at
pH= 7.5 and 25 ºC); human intestinal absorption (HIA) values resulted higher than 95 % and in
vitro plasma protein binding (PPB) values under 90%. In addition, all the selected compounds
accomplished the Lipinski’s Rule of Five [38], and were within the limits established in
Comprehensive Medicinal Chemistry Database (CMC-like rule) [39] and the World Drug Index
(WDI-like rule) [40], to be considered as adequate drug-like molecules. Finally, they attained an
OSIRIS global drug-score qualification in the range 0.52 to 0.79. Even with this favourable profile,
it must keep in mind that Lipinsky rules are frequently violated for antiparasiticidal drugs, including
miltefosine and amphotericin B, two front-line antileishmanial drugs [41].
3. Conclusions
In summary, we have identified several derivatives of imidazo[2,1-a]isoindol-5-ol, that exhibit
promising leishmanicidal activity. Of the compounds tested, the para-monosubstituted 6-
benzylimidazo[2,1-a]isoindol-5-ols containing chloro (Cl) or methylsulfanyl (MeS) groups attached
to the benzyl fragment were the best, with EC values for intracellular amastigotes in the low
5
0
micromolar range of concentrations, and SI values higher than 30. Furthermore, the involvement of
mitochondrion as a target, at least for the most active one (29) was demonstrated, even when
additional targets were also suspected.
All these findings will contribute to establishing structure activity relationships to guide further
design of new compounds, which may include motifs to promote mitochondrial accumulation,
mostly the presence of lipophilic cations, already used for other leishmanicidal molecules, to
promote a selective accumulation in this organelle [25]. The message to be conveyed is the proof-
of-concept for the inclusion of the IIOL scaffold inside the portfolio of new candidates for
preclinical assays against Leishmania.
4
. Experimental
.1. Chemistry
4

ACCEPTED MANUSCRIPT
All commercial chemicals (Aldrich, Alpha, Fischer, SDS) were used as purchased and solvents
Fischer, SDS, Scharlau) purified by the standard procedures prior to use [42]. Reactions were
monitored by Thin Layer Chromatography (TLC) (Kieselgel 60 F254 precoated plates, E. Merck,
Germany), the spots were detected by exposure to UV lamp at 254 nm, and colorization with 10%
(
λ
phosphomolybdic acid or ninhydrin spray, and further heating of the plate. Melting points (Mp)
were determined with a Büchi apparatus in open capillaries and were uncorrected. Separations by
flash column chromatography were performed on neutral alumina Merck 60 silica gel (0.063-0.2
mesh). Infrared spectra were recorded on a FT-IR spectrometer Perkin Elmer, System BX using
neat samples, without solvent, or KBr disks. NMR spectra were recorded either on a Bruker ARX-
1
13
1
4
00 (400 MHz for H, 100 MHz for C) or a Bruker AC 200 MHz (200 MHz for H, 50 MHz for
1
3
C). The spectra were measured either in CDCl , C D or DMSO-d , using tetramethylsilane
3
6
6
6
(TMS) as internal standard, chemical shifts (δ) are given in ppm and coupling constants (J) in
Hertz. High resolution mass spectra (HRMS) were obtained by electron spray ionisation-mass
spectrometry (ESI-MS) technique (5 kV) on a QSTAR XL mass spectrometer.
4.1.1. General procedure for the synthesis of intermediate benzal(naphthylmethylidene)phthalides
(BPs, NPs)
2.2 mmol of phthalic anhydride or derivative, 2.7 mmol of the corresponding phenyl/
naphthylacetic acid and 0.26 mmol of sodium acetate with 5 mL of toluene were placed into in a
round bottom flask plugged to a Dean-Stark apparatus. The mixture was maintained under N2
atmosphere at 210-245 ºC, with magnetic stirring, for 9-33 h. After cooling, the reaction mixture
was extracted with ethyl acetate and washed successively with (sat) Na CO , brine and water, dried
2
3
over Na SO and evaporated under reduced pressure to give the crude product. Solid products were
2
4
purified by crystallization, whereas oils were subjected to flash chromatography on silica gel. The
ratio between the 6(7)-regioisomers was determined by comparison of the integral of their
1
respective olefinic protons (signal H-8), in H-NMR. All phthalides were obtained as Z isomers.
Their configuration was confirmed through NOE or ROESY experiments, and final yields ranged
between 40 - 95%.
We report here the data of benzalphthalides BP-1, BP-2 and BP-3, as precursors to the three most
representative IIOLs 26, 29 and 31, respectively.
4.1.1.1. (Z)-4,7-Dichloro-3-(4-chlorobenzylidene)isobenzofuran-1(3H)-one
, BP-1
Reaction time 16 h. Compound crystallized from MeOH/hexane to give a pale yellow solid, Mp:
-1 1
2
07-209 ºC, yield 46%. IR (KBr): νmax 3076, 1774, 1647, 1465, 1224, 1152, 996, 903, 819 cm . H

ACCEPTED MANUSCRIPT
NMR (400 MHz, C D ): δ 6.50 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 1H), 6.87 (s, 1H), 7.15 (d, J
6
6
13
=
8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H). C NMR (100 MHz, C D ): δ 111.1, 122.4, 125.7, 129.0
6 6
+
(
2C), 130.7, 131.6, 131.8 (2C), 131.9, 134.8, 135.6, 137.1, 141.8, 161.2. HRMS (ESI ) calcd. for
+
C H Cl O [M+Na] : 346.9404, found. 346.9408.
15
7
3
2
4
.1.1.2. (Z)-5,6-Dichloro-3-(4-methylsulfanylbenzylidene)isobenzofuran-1(3H)-one, BP-2
Reaction time 12 h. Compound crystallized from MeOH/hexane to give a yellow solid, Mp: 200-
02 °C. yield: 59%, IR (KBr): νmax 2907, 1757, 1659, 1589, 1312, 1284, 1153, 1082, 929, 888, 816
2
−1 1
cm . H NMR (200 MHz, CDCl ): δ 2.52 (s, 3H), 6.36 (s, 1H), 7.25 (d, J = 8.6 Hz, 2H), 7.73 (d, J
3
1
3
=
8.6 Hz, 2H), 7.85 (s, 1H), 8.00 (s, 1H). C NMR (50 MHz, CDCl ): δ 15.3, 108.6, 121.6, 122.6,
3
+
1
25.3, 126.1 (2C), 127.2, 130.6 (2C), 134.0, 139.4, 139.7, 139.9, 141.2, 165.1 HRMS (ESI ) calcd.
+
for C H Cl O S [M+H] : 336.9851, found. 336.9840.
1
6
11
2
2
4.1.1.3. (Z)-3-(3,4-Dichlorobenzylidene)-5(6)-nitroisobenzofuran-1(3H)-one, BP-3
Reaction time 14 h. Compound crystallized from MeOH/hexane to give an orange solid, Mp: 190-
-1
1
94 ºC, yield: 38%. IR (KBr): νmax 2921, 1778, 1616, 1536, 1344, 1100, 1001, 855, 763 cm .
+
+
HRMS (ESI ) calcd. for C H Cl NO [M+H] : 335.9830, found 335.9839.
15
8
2
4
1
(
Z)-3-(3,4-Dichlorobenzylidene)-5-nitroisobenzofuran-1(3H)-one, BP-3a. H NMR (400 MHz,
DMSO-d ): δ 7.26 (s, 1H), 7.71 (dd, J = 8.8, 1.2 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 1.2
6
1
3
Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.66 (dd, J = 8.4, 2.0 Hz, 1H), 8.93 (d, J = 2.0 Hz, 1H). C NMR
(
100 MHz, DMSO-d ): δ 106.5, 120.8, 122.5, 127.0, 129.9, 130.0, 131.1 (2C), 131.4, 131.6, 133.5,
6
1
44.2 (2C), 148.6, 164.4.
Z)-3-(3,4-Dichlorobenzylidene)-6-nitroisobenzofuran-1(3H)-one, BP-3b. H NMR (400 MHz,
DMSO-d ): δ 7.19 (s, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.73 (dd, J = 8.8, 1.2 Hz, 1H), 7.95 (d, J = 1.2
1
(
6
1
3
Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.37 (dd, J = 8.4, 2.0 Hz, 1H), 8.60 (d, J = 2.0 Hz, 1H). C NMR
100 MHz, DMSO-d ): δ 108.1, 116.6, 123.8, 125.2, 127.1, 129.8, 129.9, 130.3, 131.2, 131.6,
(
6
133.3, 140.7, 144.1, 151.8, 164.2.
4.1.2. General procedure for the preparation of 5-benzyl(naphthylmethyl)imidazo[2,1-a]isoindol-5-
ol derivatives (compounds 1 - 32)
.0 mmol of the corresponding benzyl(naphthylmethylidene)phthalide was dissolved into
1
ethylenediamine (4 mL) and heated at 70 – 80 ºC for 7 – 12 h with magnetic stirring. After cooling,
the reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was

ACCEPTED MANUSCRIPT
dried over Na SO and concentrated under reduced pressure to give the crude product, which was
2
4
further purified by column chromatography on silica gel. Reaction yields ranged within 80 - 100%.
Compounds 3 to 20, 22 to 24 and 31 were obtained as mixtures of 7(8)-regioisomers, and
compounds 21 and 30 as mixtures of 6(9)-regioisomers, in relation 1:(0.9-0.6) approximately.
4.1.2.1. 5-Benzyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 1.
The compound was prepared and characterized as previously described in [23]. Yield: 91%.
4.1.2.2. 5-(4-Chlorobenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 2.
The compound was prepared and characterized as previously described in [23]. Yield: 93%.
4.1.2.3. 5-Benzyl-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 3.
-1
Yield: 85%. Oil. IR (NaCl): νmax 3359, 3294, 2925, 1684, 1621, 1393, 1083, 703 cm . HRMS
+
+
(
ESI ) calcd. for C H N O [M+H] : 279.1492, found 279.1504.
18 19 2
5-Benzyl-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 3a. Differential assignment of
1
NMR data for pairs of regioisomers based on homo- and heteronuclear 2D-NMR correlations. H
NMR (200 MHz, CDCl ): δ 2.35 (s, 3H), 2.77-3.10 (m, 2H), 3.12 (d, J = 14.0 Hz, 1H), 3.41 (d, J =
3
1
4.0 Hz, 1H), 3.67 (bs, 1H, D O), 3.95-4.05 (dt, J = 14.5, 2.6 Hz, 2H), 6.77-7.03 (m, 5H), 7.09 (d, J
2
1
3
=
7.9 Hz, 1H), 7.20 (s, 1H), 7.37 (d, J = 7.9 Hz, 1H). C NMR (50 MHz, CDCl ): δ 22.0, 39.4,
3
41.8, 44.6, 89.2, 122.9, 126.8, 127.9 (3C), 129.8, 130.0 (2C), 130.9, 135.2, 142.6, 148.3, 167.8.
1
5
-Benzyl-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 3b. H NMR (200 MHz, CDCl ): δ
3
2.29 (s, 3H), 2.77-3.10 (m, 2H), 3.11 (d, J = 14.0 Hz, 1H), 3.22 (d, J = 14.0 Hz, 1H), 3.67 (bs, 1H,
D O), 3.95-4.05 (dt, J = 14.5, 2.6 Hz, 2H), 6.77-7.03 (m, 5H), 7.12 (d, J = 7.5 Hz, 1H), 7.19 (d, J =
2
1
3
7
1
.5 Hz, 1H), 7.30 (s, 1H). C NMR (50 MHz, CDCl ): δ 21.4, 39.4, 41.8, 44.6, 89.3, 122.2, 123.3,
3
27.9 (3C), 130.0 (2C), 130.9, 132.8, 138.9, 135.2, 145.2, 167.8.
4.1.2.4. 5-(2-Chlorobenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 4.
-
1
Yield: 78%. Oil. IR (NaCl): νmax 3292, 2925, 1681, 1620, 1442, 1392, 1080, 1053, 755, 732 cm .
+
+
HRMS (ESI ) calcd. for C H ClN O [M+H] : 313.1110, found 313.1105.
18
18
2
1
5
-(2-Chlorobenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 4a. H NMR (200 MHz,
CDCl ): δ 2.32 (s, 3H), 2.82-3.15 (m, 2H), 3.32 (d, J = 14.0 Hz, 1H), 3.54 (d, J = 14.0 Hz, 1H),
3
4
1
1
.02-4.12 (dt, J = 14.5, 2.2 Hz, 2H), 6.90 (m, 3H), 7.11 (d, J = 7.9 Hz, 1H), 7.12 (m, 1H), 7.17 (bs,
1
3
H), 7.45 (d, J = 7.9 Hz, 1H). C NMR (50 MHz, CDCl ): δ 19.3, 36.8, 37.9, 39.1, 86.3, 120.0,
3
20.4, 123.7, 125.3, 125.5, 126.7, 127.1, 128.9, 130.6, 132.2, 139.9, 145.3, 165.0.

ACCEPTED MANUSCRIPT
1
5
-(2-Chlorobenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 4b. H NMR (200 MHz,
CDCl ): δ 2.31 (s, 3H), 2.82-3.15 (m, 2H), 3.32 (d, J = 14.0 Hz, 1H), 3.54 (d, J = 14.0 Hz, 1H),
3
4.02-4.12 (dt, J = 14.5, 2.2 Hz, 2H), 6.90 (m, 3H), 7.11 (m, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.18 (d, J
1
3
=
8.0 Hz, 1H), 7.38 (s, 1H). C NMR (50 MHz, CDCl ): δ 18.7, 36.8, 37.9, 39.1, 86.3, 119.7,
3
120.6, 123.7, 125.3, 125.5, 126.7, 128.9, 130.0, 130.6, 132.2, 143.0, 145.0, 165.0.
4.1.2.5. 7(8)-Methyl-5-(3-nitrobenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 5.
-1
Yield: 68%. Oil. IR (NaCl): νmax 3354, 3294, 2923, 1684, 1528, 1350, 1338, 1085, 824 cm .
+
+
HRMS (ESI ) calcd. for C H N O [M+H] : 324.1343, found 324.1338.
18
18
3
3
1
7
-Methyl-5-(3-nitrobenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 5a. H NMR (200 MHz,
CDCl ): δ 2.31 (s, 3H), 3.10 (d, J = 14.0 Hz, 1H), 3.35 (d, J = 14.0 Hz, 1H), 3.72 (m, 2H), 3.79 (m,
3
2H), 6.96 (m, 3H), 7.22 (m, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.61 (dd, J = 8.0, 7.8 Hz, 1H), 7.79 (d, J
13
=
8.0 Hz, 1H), 7.80 (m, 1H), 8.09 (m, 1H), 8.14 (m, 1H). C NMR (50 MHz, CDCl ): δ 22.0, 39.2,
3
4
1.8, 44.2, 88.8, 121.9, 122.0, 122.8, 125.1, 128.1, 128.8, 130.2, 136.0, 139.5, 143.1, 147.7, 147.8,
67.6.
1
1
8
-Methyl-5-(3-nitrobenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 5b. H NMR (200 MHz,
CDCl ): δ 2.28 (s, 3H), 3.10 (d, J = 14.0 Hz, 1H), 3.35 (d, J = 14.0 Hz, 1H), 3.60 (bs, 1H, D O),
3
2
3.72 (m, 2H), 3.79 (m, 2H), 6.96 (m, 3H), 7.30 (d, J = 8.1 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.61
13
(
dd, J = 8.0, 7.8 Hz, 1H), 7.77 (bs, 1H), 7.80 (m, 1H), 8.09 (m, 1H), 8.14 (m, 1H). C NMR (50
MHz, CDCl ): δ 21.4, 39.2, 41.8, 44.2, 88.8, 121.9 (2C), 123.0, 125.1, 128.8, 130.8, 133.1, 136.0,
3
137.3, 139.5, 144.5, 147.5, 167.6.
4.1.2.6. 5-(4-Fluorobenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 6.
Yield: 98%. Oil. IR (NaCl): νmax 3352, 3293, 2926, 1683, 1620, 1511, 1223, 1077, 1053, 832, 781,
-1
+
+
7
26 cm . HRMS (ESI ) calcd. for C H FN O [M+H] : 297.1397, found 297.1381.
18 18 2
1
5
-(4-Fluorobenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 6a. H NMR (200 MHz,
CDCl ): δ 2.42 (s, 3H), 2.36-3.09 (m, 2H), 3.28 (d, J = 14.0 Hz, 1H), 3.39 (d, J = 14.0 Hz, 1H),
3
3
7
1
1
.63 (bs, 1H, D O), 3.90-4.14 (m, 2H), 6.72-6.82 (m, 4H), 7.32 (bs, 1H), 7.33 (m, 1H), 7.46 (d, J =
2
1
3
.5 Hz, 1H). C NMR (50 MHz, CDCl ): δ 22.0, 39.2, 41.8, 43.8, 89.0, 114.9 (d, J = 7.4 Hz, 2C),
3
22.9, 123.3, 128.2, 130.9, 131.3, 131.5 (d, J = 28.1 Hz, 2C), 142.7, 148.2, 162.4 (d, J = 244 Hz),
67.7.
1
5
-(4-Fluorobenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 6b. H NMR (200 MHz,
CDCl ): δ 2.37 (s, 3H), 3.09-2.36 (m, 2H), 3.28 (d, J = 14.0 Hz, 1H), 3.39 (d, J = 14.0 Hz, 1H),
3
3.63 (s, 1H, D O), 4.14-3.90 (m, 2H), 6.82-6.72 (m, 4H), 7.18 (d, J = 8.0 Hz, 1H), 7.30 (m, 1H),
2
13
7
.39 (bs, 1H). C NMR (50 MHz, CDCl ): δ 21.4, 39.2, 41.8, 43.8, 89.0, 114.9 (d, J = 7.4 Hz, 2C),
3

ACCEPTED MANUSCRIPT
1
1
22.0, 122.8, 128.2, 130.9, 131.5 (d, J = 28.1 Hz, 2C), 132.8, 139.0, 145.0, 162.4 (d, J = 244 Hz),
67.7.
4.1.2.7. 5-(4-Chlorobenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 7.
-1
Yield: 86%. Oil. IR (NaCl): νmax 3258, 2925, 1679, 1620, 1490, 1115, 1080, 815, 728 cm . HRMS
+
+
(
ESI ) calcd. for C H ClN O [M+H] : 313.1110, found 313.1106.
18 18 2
1
5
-(4-Chlorobenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 7a. H NMR (200 MHz,
CDCl ): δ 2.37 (s, 3H), 2.74-3.36 (m, 2H), 3.10 (d, J = 14.0 Hz, 1H), 3.33 (d, J = 14.0 Hz, 1H),
3
3
1
4
.81 (bs, 1H, D O), 3.96-4.09 (m, 2H), 6.72 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H), 7.14 (s,
2
1
3
H), 7.16 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H). C NMR (50 MHz, CDCl ): δ 21.9, 39.3,
3
1.7, 43.8, 89.0, 122.7, 128.0 (3C), 129.8, 130.8, 131.2 (2C), 132.7, 133.6, 142.7, 148.0, 167.6.
1
5
-(4-Chlorobenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 7b. H NMR (200 MHz,
CDCl ): δ 2.32 (s, 3H), 2.74-3.36 (m, 2H), 3.10 (d, J = 14.0 Hz, 1H), 3.33 (d, J = 14.0 Hz, 1H),
3
3
.81 (bs, 1H, D O), 3.96-4.09 (m, 2H), 6.72 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H), 7.12 (d, J
2
1
3
=
8.8 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 7.32 (s, 1H). C NMR (50 MHz, CDCl ): δ 21.3, 39.3,
3
4
1.7, 43.8, 88.9, 122.8, 123.3, 128.0 (2C), 130.8, 131.2 (2C), 132.5, 132.7, 133.6, 139.0, 144.8,
67.6.
1
4.1.2.8. 5-(4-Hydroxybenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 8.
-1
Yield: 53%. Oil. IR (NaCl): νmax 3354, 2921, 1668, 1616, 1440, 1231, 1072, 831 cm . HRMS
+
+
(
ESI ) calcd. for C H N O [M+H] : 295.1441, found 295.1442.
18 19 2 2
1
5
-(4-Hydroxybenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 8a. H NMR (200
MHz, CDCl ): δ 2.35 (s, 3H), 2.79-2.96 (m, 2H), 3.25 (d, J = 14.0 Hz, 1H), 3.40 (d, J = 14.0 Hz,
3
1
7
4
H), 3.79-3.88 (m, 2H), 6.43 (d, J = 8.3 Hz, 2H), 6.54 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 1.1, 1H),
13
.24 (dd, J = 7.9, 1.1 1H), 7.34 (d, J = 7.9, 1H). C NMR (50 MHz, CDCl ): δ 21.9, 39.9, 41.3,
3
2.6, 90.6, 114.8 (2C), 122.9, 123.0, 125.4, 128.1, 130.0, 130.8 (2C), 143.0, 147.6, 155.5, 168.5.
1
5
-(4-Hydroxybenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 8b. H NMR (200
MHz, CDCl ): δ 2.28 (s, 3H), 2.79-2.96 (m, 2H), 3.25 (d, J = 14.0 Hz, 1H), 3.40 (d, J = 14.0 Hz,
3
1
7
1
H), 3.79-3.88 (m, 2H), 6.43 (d, J = 8.3 Hz, 2H), 6.53 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 7.9 Hz, 1H),
13
.20 (d, J = 7.9, 1H), 7.26 (bs, 1H). C NMR (50 MHz, CDCl ): δ 21.2, 39.9, 41.3, 42.6, 90.6,
3
14.8 (2C), 122.3, 122.6, 125.4, 128.1, 130.8 (2C), 133.0, 139.3, 144.5, 155.5, 168.5.
4.1.2.9. 5-(4-Methoxybenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 9.
-1
Yield: 94%. Oil. IR (NaCl): νmax 3357, 2929, 1680, 1612, 1513, 1249, 1035, 819, 756 cm . HRMS
+
+
(
ESI ) calcd. for C H N O [M+H] : 309.1598, found 309.1609.
19 21 2 2

ACCEPTED MANUSCRIPT
1
5
-(4-Methoxybenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 9a. H NMR (400
MHz, CDCl ): δ 2.42 (s, 3H), 2.71-2.93 (m, 2H), 3.22 (d, J = 13.9 Hz, 1H), 3.36 (d, J = 13.9 Hz,
3
1
H), 3.57 (bs, 1H, D O), 3.68 (s, 3H), 3.98-4.08 (m, 2H), 6.60 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8
2
1
3
Hz, 2H), 7.18 (bs, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H). C NMR (100 MHz,
CDCl ): δ 21.9, 39.5, 41.7, 43.2, 55.0, 89.6, 113.2 (2C), 122.6, 122.8, 128.2, 129.7, 130.4, 130.8
3
(
2C), 148.1, 142.5, 158.2, 167.9.
-(4-Methoxybenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 9b. H NMR (400
MHz, CDCl ): δ 2.36 (s, 3H), 3.00-3.20 (m, 2H), 3.23 (d, J = 13.9 Hz, 1H), 3.36 (d, J = 13.9 Hz,
1
5
3
1
H), 3.57 (bs, 1H, D O), 3.68 (s, 3H), 3.98-4.08 (m, 2H), 6.60 (d, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8
2
1
3
Hz, 2H), 7,16 (d, J = 7.7 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.36 (bs, 1H). C NMR (100 MHz,
CDCl ): δ 21.3, 39.5, 41.7, 43.2, 55.0, 89.5, 113.2 (2C), 122.1, 123.1, 127.0, 128.2, 130.9 (2C),
3
132.6, 138.8, 144.9, 158.2, 167.8.
4.1.2.10. 7(8)-Methyl-5-(4-methylsulfanylbenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 10.
-1
Yield: 94%. Oil. IR (NaCl): νmax 3294, 2922, 1682, 1620, 1405, 1080, 819, 732, 703 cm . HRMS
+
+
(
ESI ) calcd. for C H N OS [M+H] : 325.1369, found 325.1376.
19 21 2
1
7
-Methyl-5-(4-methylsulfanylbenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 10a. H NMR
(
200 MHz, CDCl ): δ 2.34 (s, 3H), 2.38 (s, 3H), 2.98-3.21 (m, 2H), 3.11 (d, J = 14.0 Hz, 1H), 3.33
3
(
d, J = 14.0 Hz, 1H), 3.95-4.15 (m, 2H), 4.20 (bs, 1H, D O), 6.73 (d, J = 8.2 Hz, 2H), 6.93 (d, J =
2
1
3
8
.2 Hz, 2H), 7.19 (bs, 1H), 7.26 (d, J = 7.5 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H). C NMR (50 MHz,
CDCl ): δ 15.6, 21.6, 39.5, 41.7, 43.8, 89.4, 122.2, 122.8, 126.0 (2C), 128.1, 130.4 (2C), 130.9,
3
131.9, 136.6, 142.7, 148.1, 167.8.
1
8
-Methyl-5-(4-methylsulfanylbenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 10b. H NMR
(
200 MHz, CDCl ): δ 2.33 (s, 3H), 2.35 (s, 3H), 2.81-2.98 (m, 2H), 3.09 (d, J = 14.0 Hz, 1H), 3.32
3
(
d, J = 14.0 Hz, 1H), 3.95-4.15 (m, 2H), 4.20 (bs, 1H, D O), 6.73 (d, J = 8.3 Hz, 2H), 6.93 (d, J =
2
1
3
8
.3 Hz, 2H), 7.12 (d, J = 7.2 Hz, 1H), 7.20 (d, J = 7.2 Hz, 1H), 7.32 (bs, 1H). C NMR (50 MHz,
CDCl ): δ 15.6, 21.4, 39.5, 41.7, 43.8, 89.4, 122.8, 123.3, 126.0 (2C), 128.1, 130.4 (2C), 130.9,
3
132.8, 136.6, 139.0, 144.9, 167.8.
4.1.2.11. 7(8)-Methyl-5-(4-methylsulfinylbenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 11.
Yield: 89%. Oil. IR (NaCl): νmax 3422, 2924, 1685, 1619, 1406, 1303, 1148, 1088, 959, 821, 733
-1
+
+
cm . HRMS (ESI ) calcd. for C H N O S [M+H] : 341.1318, found 341.1322.
1
9
21
2
2
1
7
-Methyl-5-(4-methylsulfinylbenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 11a. H NMR
(
200 MHz, CDCl ): δ 2.36 (s, 3H), 2.57 (s, 3H), 2.99-3.18 (m, 2H), 3.13 (d, J = 13.6 Hz, 1H), 3.40
3
(d, J = 13.6 Hz, 1H), 4.02-4.12 (m, 2H), 6.98 (d, J = 2.0 Hz, 1H), 7.12 (dd, J = 8.0, 2.0 Hz, 1H),

ACCEPTED MANUSCRIPT
1
3
7
.25 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H). C NMR (50 MHz,
CDCl ): δ 21.4, 22.0, 39.1, 41.8, 43.7, 88.8, 122.7, 122.8, 126.9, 123.3 (2C), 132.9 (2C), 129.9,
3
130.8, 143.7, 143.8, 148.0, 167.6.
1
8
-Methyl-5-(4-methylsulfinyl)benzyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 11b. H NMR
(
200 MHz, CDCl ): 2.27 (s, 3H), 2.57 (s, 3H), 2.83-3.02 (m, 2H), 3.13 (d, J = 13.6 Hz, 1H), 3.40 (d,
3
J = 13.6 Hz, 1H), 4.02-4.12 (m, 2H), 7.00 (d, J = 7.6, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.21 (bs, 1H),
1
3
7
4
1
.28 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H). C NMR (50 MHz, CDCl ): δ 21.4, 22.0, 39.1,
3
1.8, 43.7, 88.8, 122.0, 123.1, 123.3 (2C), 127.2, 130.8, 131.1, 132.9 (2C), 138.7, 143.8, 144.8,
67.6.
4.1.2.12. 7(8)-Methyl-5-(4-methylsulfonylbenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 12.
Yield: 91%. Oil. IR (NaCl): νmax 3372, 2924, 1685, 1597, 1409, 1302, 1149, 1090, 959, 826, 729
-1
+
+
cm . HRMS (ESI ) calcd. for C H N O S [M+H] : 357.1267, found 357. 1267.
1
9
21
2
3
1
7
-Methyl-5-(4-(methylsulfonyl)benzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 12a. H NMR
(
200 MHz, CDCl ): 2.37 (s, 3H), 2.98 (s, 3H), 3.00-3.18 (m, 2H), 3.08 (d, J = 13.6 Hz, 1H), 3.39 (d,
3
J = 13.6 Hz, 1H), 4.00-4.12 (m, 2H), 7.01 (d, J = 8.0 Hz, 2H), 7.20 (bs, 1H), 7.26 (d, J = 7.6 Hz,
1
3
1
3
1
H), 7.40 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H). C NMR (50 MHz, CDCl ): δ 22.7, 45.1,
3
9.7, 42.5, 45.2, 89.2, 122.6, 124.1, 127.5, 127.6 (2C), 131.7 (2C), 131.6, 133.7, 140.0, 142.5,
45.3, 168.2.
1
8
-Methyl-5-(4-(methylsulfonyl)benzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 12b. H NMR
(
200 MHz, CDCl ): δ 2.32 (s, 3H), 2.98 (s, 3H), 3.00-3.18 (m, 2H), 3.08 (d, J = 13.6 Hz, 1H), 3.39
3
(d, J = 13.6 Hz, 1H), 4.00-4.12 (m, 2H), 7.03 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 7.6 Hz, 1H), 7.16 (d,
1
3
J = 7.6 Hz, 1H), 7.33 (bs, 1H), 7.61 (d, J = 8.0 Hz, 2H). C NMR (50 MHz, CDCl ): δ 22.0, 45.1,
3
3
1
9.7, 42.5, 45.2, 89.2, 123.3, 123.7, 127.6 (2C), 130.7, 131.6, 131.7 (2C), 131.3, 139.4, 140.0,
43.6, 168.2.
4.1.2.13. 5-(2,4-Difluorobenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 13.
-1
Yield: 65%. Oil. IR (NaCl): νmax 3360, 2930, 1680, 1620, 1490, 1249, 1080, 820, 720, 760 cm .
+
+
HRMS (ESI ) calcd. for C H F N O [M+H] : 315.1309, found 315.1305.
18
17
2
2
1
5
-(2,4-Difluorobenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 13a. H NMR (200
MHz, CDCl ): δ 2.44 (s, 3H), 2.86-3.18 (m, 2H), 3.28 (d, J = 14.0 Hz, 1H), 3.39 (d, J = 14.0 Hz,
3
1H), 4.10-4.17 (m, 2H), 6.56-6.67 (m, 3H), 7.27 (bs, 1H), 7.29 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 7.5
1
3
Hz, 1H), C NMR (50 MHz, CDCl ): δ 22.0, 36.0, 39.1, 41.6, 88.8, 103.5 (t, J =25.7 Hz), 110.7 (d,
3
J = 20.2 Hz), 118.0 (d, J = 14.7 Hz), 123.2, 128.1, 129.9, 130.8, 132.3 (m), 142.8, 148.0, 160.5 (dd,
J = 247.7, 12.9 Hz), 161.5 (dd, J = 247.7, 12.9 Hz), 167.6.

ACCEPTED MANUSCRIPT
1
5
-(2,4-Difluorobenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 13b. H NMR (200
MHz, CDCl ): δ 2.38 (s, 3H), 2.86-3.18 (m, 2H), 3.28 (d, J = 14.0 Hz, 1H), 3.35 (d, J = 14.0 Hz,
3
1H), 4.10-4.17 (m, 2H), 6.56-6.67 (m, 3H), 7.18 (d, J = 7.9 Hz, 1H), 7.32 (s, 1H), 7.37 (d, J = 7.9
1
3
Hz, 1H). C NMR (50 MHz, CDCl ): δ 21.4, 36.0, 39.1, 41.6, 88.8, 103.5 (t, J =25.7 Hz), 110.7 (d,
3
J = 20.2 Hz), 118.0 (d, J = 14.7 Hz), 122.0, 123.2, 130.9, 132.3 (m), 139.2, 142.8, 144.9, 160.5 (dd,
J = 247.7 Hz, 12.9), 161.5 (dd, J = 247.7 Hz, 12.9), 167.6.
4.1.2.14. 5-(2,4-Dichlorobenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 14.
-1
Yield: 47%. Oil; IR (NaCl): νmax 3354, 2926, 1682, 1621, 1473, 1080, 1051, 831 cm . HRMS
+
+
(
ESI ) calcd. for C H Cl N O [M+H] : 347.0712, found 347. 0718.
18 17 2 2
1
5
-(2,4-Dichlorobenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 14a. H NMR (200
MHz, CDCl ): δ 2.34 (s, 3H), 2.85-3.15 (m, 2H), 3.25 (d, J = 14.1 Hz, 1H), 3.40 (bs, 1H, D O),
3
2
3.40 (d, J = 14.1 Hz, 1H), 4.02-4.10 (m, 2H), 6.86 (d, J = 8.3 Hz, 1H), 6.94 (dd, J = 8.3, 1.8 Hz,
1
3
1
H), 7.11 (bs, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.19 (s, 1H), 7.46 (d, J = 8.3 Hz, 1H). C NMR (50
MHz, CDCl ): δ 22.0, 39.1, 40.4, 41.9, 88.6, 123.0, 126.8 (2C), 128.0, 129.3, 130.0, 130.8, 132.4,
3
133.3, 135.6, 142.8, 148.3, 167.7.
1
5
-(2,4-Dichlorobenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 14b. H NMR (200
MHz, CDCl ): δ 2.32 (s, 3H), 2.85-3.13 (m, 2H), 3.19 (d, J = 14.1 Hz, 1H), 3.40 (bs, 1H, D O),
3
2
3.47 (d, J = 14.1 Hz, 1H), 4.02-4.13 (m, 2H), 6.86 (d, J = 8.3 Hz, 1H), 6.94 (dd, J = 8.3, 1.8 Hz,
1
3
1
H), 7.11 (bs, 1H), 7.16 (d, J = 7.0 Hz, 1H), 7.22 (d, J = 7.0 Hz, 1H), 7.39 (s, 1H). C NMR (50
MHz, CDCl ): δ 21.5, 39.1, 40.4, 41.9, 88.6, 122.3, 123.4, 126.8, 128.0, 129.3, 132.1, 132.4, 132.9,
3
133.3, 135.6, 139.2, 145.2, 167.7.
4.1.2.15. 5-(3,4-Dichlorobenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 15.
-1
Yield: 39%. Oil. IR (NaCl): νmax 3293, 2926, 1684, 1620, 1442, 1081, 909, 810, 733 cm . HRMS
+
+
(
ESI ) calcd. for C H Cl N O [M+H] : 347.0712, found 347.0707.
18 17 2 2
1
5
-(3,4-Dichlorobenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 15a. H NMR (200
MHz, CDCl ): δ 2.38 (s, 3H), 2.89-2.98 (m, 2H), 3.00 (d, J = 13.8 Hz, 1H), 3.30 (d, J = 13.8 Hz,
3
1
7
H), 4.00 (sa, 1H, D O), 3.96-4.05 (m, 2H), 6.62 (dd, J = 8.0, 2.2 Hz, 1H), 6.93 (d, J = 2.2 Hz, 1H),
2
1
3
.12 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H). C NMR
(
50 MHz, CDCl ): δ 19.3, 36.7, 38.9, 40.8, 86.2, 120.1, 120.3, 125.4, 126.7, 127.1, 127.4, 128.2,
3
129.1, 129.3, 132.8, 140.3, 145.0, 165.0.
1
5
-(3,4-Dichlorobenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 15b. H NMR (200
MHz, CDCl ): δ 2.34 (s, 3H), 2.98-2.89 (m, 2H), 3.00 (d, J = 13.8 Hz, 1H), 3.30 (d, J = 13.8 Hz,
3
1
H), 4.00 (sa, 1H, D O), 3.96-4.05 (m, 2H), 6.62 (dd, J = 8.0, 2.2 Hz, 1H), 6.93 (d, J = 2.2 Hz, 1H),
2

ACCEPTED MANUSCRIPT
1
3
7
.12 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 7.8 Hz, 1H), 7.35 (s, 1H). C NMR
(
50 MHz, CDCl ): δ 18.7, 36.7, 38.9, 40.8, 86.3, 119.4, 120.7, 125.4, 126.6, 127.1, 128.2, 129.0,
3
129.3, 130.3, 132.8, 136.7, 141.8, 165.0.
4.1.2.16. 5-(3,4-Methylendioxybenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 16.
-1
Yield: 39%. Oil. IR (NaCl): νmax 3356, 2923, 1683, 1620, 1493, 1247, 1039, 909, 731 cm . HRMS
+
+
(
ESI ) calcd. for C H N O [M+H] : 323.1396, found 323.1390.
19 19 2 3
1
5
-(3,4-Methylendioxybenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 16a. H NMR
(
200 MHz, CDCl ): δ 2.43 (s, 3H), 2.93-3.10 (m, 2H), 3.13 (d, J = 14.0 Hz, 1H), 3.33 (d, J = 14.0
3
Hz, 1H), 4.02-4.11 (m, 2H), 3.72 (sa, 1H, D O), 5.83 (s, 2H), 6.27 (d, J = 2.2 Hz, 1H), 6.32 (dd, J =
2
8.1, 2.2 Hz, 1H), 6.52 (d, J = 8.1Hz, 1H), 7.30 (bs, 1H), 7.25 (d, J = 7.5 Hz, 1H), 7.48 (d, J = 7.5
13
Hz, 1H). C NMR (50 MHz, CDCl ): δ 22.0, 39.6, 41.6, 43.8, 89.7, 100.8, 107.7, 110.2, 122.7,
3
122.9, 123.2, 128.7, 129.9, 130.9, 142.7, 146.3, 147.1, 148.1, 167.8.
1
5
-(3,4-Methylendioxybenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 16b. H NMR
(
200 MHz, CDCl ): δ 2.37 (s, 3H), 3.10-2.93 (m, 2H), 3.13 (d, J = 14.0 Hz, 1H), 3.33 (d, J = 14.0
3
Hz, 1H), 4.02-4.11 (m, 2H), 3.72 (bs, 1H, D O), 5.83 (s, 2H), 6.27 (d, J = 2.2 Hz, 1H), 6.32 (dd, J =
2
8.1, 2.2 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.41
1
3
(
bs, 1H). C NMR (50 MHz, CDCl ): δ 21.4, 39.6, 41.6, 43.8, 89.6, 100.8, 107.7, 110.2, 122.2,
3
123.2, 128.3, 129.9, 130.9, 132.8, 139.0, 144.9, 147.1, 148.1, 167.8.
4.1.2.17. 5-(3,4-Dimethoxybenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 17.
-
1
Yield: 58%. Oil. IR (NaCl): νmax 3360, 2932, 1683, 1616, 1515, 1263, 1147, 1028, 905, 731 cm .
+
+
HRMS (ESI ) calcd. for C H N O [M+H] : 339.1703, found 339.1683.
20
23
2
3
1
5
-(3,4-Dimethoxybenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 17a. H NMR (200
MHz, CDCl ): δ 2.44 (s, 3H), 3.03-2.89 (m, 2H), 3.19 (d, J = 14.0 Hz, 1H), 3.35 (d, J = 14.0 Hz,
3
1H), 3.58 (s, 3H), 3.77 (s, 3H), 4.05-4.14 (m, 2H), 6.24 (d, J = 2.2 Hz, 1H), 6.46 (dd, J = 8.3, 2.1
1
3
Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H), 7.32 (bs, 1H), 7.35 (m, 1H), 7.48 (d, J = 7.5 Hz, 1H). C NMR
50 MHz, CDCl ): 21.9, 39.6, 41.7, 43.8, 55.5, 55.7, 89.6, 110.6, 113.0, 122.2, 122.8, 123.2, 127.5,
(
3
128.5, 129.7, 142.5, 147.7, 148.1, 148.3, 167.6.
1
5
-(3,4-Dimethoxybenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 17b. H NMR (200
MHz, CDCl ): δ 2.37 (s, 3H), 3.03-2.89 (m, 2H), 3.16 (d, J = 14.0 Hz, 1H), 3.34 (d, J = 14.0 Hz,
3
1H), 3.58 (s, 3H), 3.77 (s, 3H), 4.05-4.14 (m, 2H), 6.24 (d, J = 2.1 Hz, 1H), 6.45 (dd, J = 8.2, 2.1
Hz, 1H), 6.61 (d, J = 8.2 Hz, 1H), 7.19 (bd, J = 7.9 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.40 (bs, 1H),
1
3
C NMR (50 MHz, CDCl ): 21.3, 39.6, 41.7, 43.8, 55.5, 55.7, 89.4, 110.6, 113.0, 122.2, 122.8,
3
123.2, 128.6, 129.7, 132.6, 138.9, 145.2, 148.1, 148.3, 167.6.

ACCEPTED MANUSCRIPT
4
.1.2.18. 5-(3,4,5-Trimethoxybenzyl)-7(8)-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 18.
-1
Yield: 50%. Oil. IR (NaCl): νmax 3357, 2928, 1681, 1590, 1457, 1241, 1185, 836, 784 cm . HRMS
+
+
(
ESI ) calcd. for C H N O [M+H] : 369.1809, found 369.1789.
21 25 2 4
1
5
-(3,4,5-Trimethoxybenzyl)-7-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 18a. H NMR
(
200 MHz, CDCl ): δ 2.40 (s, 3H), 2.80-3.07 (m, 2H), 3.13 (d, J = 14.0 Hz, 1H), 3.32 (d, J = 14.0
3
Hz, 1H), 3.59 (s, 6H), 3.72 (s, 3H), 3.98-4.10 (m, 2H), 6.01 (s, 2H), 7.33 (d, J = 7.5 Hz, 1H), 7.38
1
3
(
bs, 1H), 7.46 (d, J = 7.5 Hz, 1H). C NMR (50 MHz, CDCl ): δ 21.9, 39.4, 41.7, 44.6, 55.9 (2C),
3
60.8, 89.3, 107.1 (2C), 122.2, 128.5, 129.7 (2C), 131.2, 136.7, 142.6, 148.4, 152.5 (2C), 167.7.
1
5
-(3,4,5-Trimethoxybenzyl)-8-methyl-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 18b. H NMR
(
200 MHz, CDCl ): δ 2.35 (s, 3H), 2.80-3.07 (m, 2H), 3.13 (d, J = 14.0 Hz, 1H), 3.11 (d, J = 14.0
3
Hz, 1H), 3.59 (s, 6H), 3.72 (s, 3H), 3.98-4.10 (m, 2H), 6.01 (s, 2H), 7.16 (bd, J = 7.9 Hz, 1H), 7.36
1
3
(
d, J = 7.9 Hz 1H), 7.38 (bs, 1H). C NMR (50 MHz, CDCl ): δ 21.3, 39.4, 41.7, 44.6, 55.9 (2C),
3
60.8, 89.2, 107.1 (2C), 122.8, 123.3, 130.7, 131.2, 132.6, 136.7, 139.1, 145.3, 152.5 (2C), 167.7.
4.1.2.19. 7(8)-Methyl-5-(naphthalen-1-ylmethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 19.
-1
Yield: 96%. Oil. IR (NaCl): νmax 3294, 2927, 1685, 1620, 1455, 1396, 1273, 1084, 732 cm .
+
+
HRMS (ESI ) calcd. for C H N O [M+H] : 329.1648, found 329.1654.
22
21
2
1
7
-Methyl-5-(naphthalen-1-ylmethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 19a. H NMR
(
200 MHz, CDCl ): δ 2.33 (s, 3H), 2.84 (m, 2H), 3.61 (d, J = 13.8 Hz, 1H), 3.79 (d, J = 13.8 Hz,
3
1H), 3.58-3.89 (m, 2H), 6.96 (m, 2H), 7.30 (s, 1H), 7.38 (m, 2H), 7.49 (d, J = 7.9 Hz, 1H), 7.71 (d,
1
3
J = 7.9 Hz, 1H), 7.77 (m, 1H), 7.79 (m, 1H), 7.93 (m, 1H). C NMR (50 MHz, CDCl ): δ 21.7,
3
3
9.0, 41.1, 42.0, 89.0, 122.8, 123.4, 124.4, 125.1, 125.4, 125.6, 127.6, 127.8, 128.6, 128.9, 129.6,
32.6, 132.9, 133.7, 142.5, 149.0, 167.8.
1
1
8
-Methyl-5-(naphthalen-1-ylmethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 19b. H NMR
(
200 MHz, CDCl ): δ 2.21 (s, 3H), 2.84 (m, 2H), 3.61 (d, J = 13.8 Hz, 1H), 3.79 (d, J = 13.8 Hz,
3
1
2
3
1
H), 3.58-3.89 (m, 2H), 6.96 (m, 2H), 7.27 (d, J = 7.0 Hz, 1H), 7.36 (d, J = 7.0 Hz, 1H), 7.38 (m,
1
3
H), 7.55 (s, 1H), 7.77 (m, 1H), 7.79 (m, 1H), 7.93 (m, 1H). C NMR (50 MHz, CDCl ): δ 21.3,
3
9.0, 41.1, 42.0, 89.0, 122.5, 123.4, 124.4, 125.1, 125.4, 125.6, 127.6, 127.8, 128.6, 129.6, 132.0,
32.6, 132.9, 133.7, 138.9, 146.2, 167.8.
4.1.2.20. 7(8)-Methyl-5-(naphthalen-2-ylmethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 20.
-1
Yield: 96%. Oil. IR (NaCl): νmax 3290, 2926, 1684, 1618, 1396, 1078, 825, 779, 742 cm . HRMS
+
+
(
ESI ) calcd. for C H N O [M+H] : 329.1654, found 329.1655.
22 21 2

ACCEPTED MANUSCRIPT
1
7
-Methyl-5-(naphthalen-2-ylmethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 20a. H NMR
(
200 MHz, CDCl ): δ 2.32 (s, 3H), 3.09-3.21 (m, 2H), 3.28 (d, J = 13.5 Hz, 1H), 3.52 (d, J = 13.5
3
Hz, 1H), 3.93-4.00 (m, 2H), 4.28 (s, 1H, D O), 7.31 (s, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.55-6.98 (m,
2
1
3
7
1
1
H), 7.70 (d, J = 8.0 Hz, 1H). C NMR (50 MHz, CDCl ): δ 22.0, 39.8, 41.6, 44.4, 89.8, 122.7,
3
25.6, 125.9, 127.4, 127.6, 128.3, 129.0, 129.9, 130.0, 130.9, 131.0, 132.8 (2C), 133.1, 142.6,
48.1, 167.8.
1
8
-Methyl-5-(naphthalen-2-ylmethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 20b. H NMR
(
200 MHz, CDCl ): δ 2.26 (s, 3H), 2.77-2.82 (m, 2H), 3.24 (d, J = 13.5 Hz, 1H), 3.52 (d, J = 13.5
3
Hz, 1H), 3.93-4.00 (m, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.52 (s, 1H), 7.55-
1
3
6
.98 (m, 7H). C NMR (50 MHz, CDCl ): δ 21.3, 39.8, 41.6, 44.4, 89.7, 122.4, 123.1, 125.6,
3
127.4, 127.6, 128.3, 129.0, 129.9, 130.9, 131.0, 132.3, 132.8 (2C), 133.1, 139.0, 144.9, 167.8.
4.1.2.21. 5-(4-Chlorobenzyl)-6(9)-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 21.
-
1
Yield: 60%. Oil. IR (NaCl): νmax 3422, 2927, 1701, 1622, 1537, 1358, 1080, 1015, 815, 725 cm .
+
+
HRMS (ESI ) calcd. for C H ClN O [M+H] : 344.0796, found 344.0790.
17
15
3
3
5-(4-Chlorobenzyl)-6-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 21a.
1
H NMR (400 MHz, CDCl ): δ 3.31-3.50 (m, 2H), 3.33 (d, J = 14.0 Hz, 1H), 3.41 (d, J = 14.0 Hz,
3
1H), 3.93-4.12 (m, 2H), 6.62 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 7.52 (dd, J = 8.0, 7.9 Hz,
1
3
1
4
H), 7.61 (d, J = 7.9, 1H), 8.10 (d, J = 8.0 Hz, 1H). C NMR (100 MHz, CDCl ): δ 38.9, 41.1,
3
2.7, 92.1, 123.8, 126.9, 127.7, 128.4 (4C), 131.4, 132.6, 134.2, 140.0, 149.6, 164.8.
5-(4-Chlorobenzyl)-9-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 21b.
1
H NMR (400 MHz, CDCl ): δ 3.07-3.10 (m, 2H), 3.33 (d, J = 14.0 Hz, 1H), 3.41 (d, J = 14.0 Hz,
3
1H), 3.93-4.12 (m, 2H), 6.78 (d, J = 7.6 Hz, 2H), 6.92 (d, J = 7.6 Hz, 2H), 7.36 (d, J = 7.2, Hz, 1H),
1
3
7
.46 (dd, J = 7.2, 7.3, 1H), 7.71 (d, J = 7.3 Hz, 1H). C NMR (100 MHz, CDCl ): δ 38.8, 40.3,
3
41.1, 89.6, 122.6, 123.8, 128.4 (4C), 130.6, 131.4, 132.9, 133.2, 144.7, 145.5, 163.1.
4.1.2.22. 5-(4-Chlorobenzyl)-7(8)-(hydroxymethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 22.
Yield: 87%. Oil. IR (NaCl): νmax 3368, 2925, 1677, 1620, 1422, 1285, 1210, 1015, 815, 812, 720
-1
+
+
cm . HRMS (ESI ) calcd. for C H ClN O [M+H] : 329.1051, found 329.1041.
1
8
18
2
2
1
5
-(4-Chlorobenzyl)-7-(hydroxymethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 22a. H NMR
(
400 MHz, CDCl ): δ 2.89-3.13 (m, 2H), 3.14 (d, J = 14.0 Hz, 1H), 3.29 (d, J = 14.0 Hz, 1H), 3.89-
3
4
1
8
.10 (m, 2H), 4.57 (s, 2H), 6.99 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.0 Hz,
1
3
H), 7.28 (s, 1H), 7.40 (d, J = 8.0 Hz, 1H). C NMR (100 MHz, CDCl ): δ 39.2, 41.2, 42.7, 63.5,
3
9.3, 119.7, 122.4, 126.8, 127.5 (2C), 130.2, 130.8, 130.6 (2C), 132.8, 142.4, 146.7, 167.2.

ACCEPTED MANUSCRIPT
1
5
-(4-Chlorobenzyl)-8-(hydroxymethyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 22b. H NMR
(
400 MHz, CDCl ): δ 2.79-3.35 (m, 2H), 3.14 (d, J = 14.0 Hz, 1H), 3.29 (d, J = 14.0 Hz, 1H), 3.85-
3
4
2
8
.10 (m, 2H), 4.49 (s, 2H), 6.73 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.0 Hz,
1
3
H), 7.33 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H). C NMR (100 MHz, CDCl ): δ 39.1, 41.2, 42.6, 63.4,
3
9.2, 120.6, 121.8, 127.5 (2C), 130.1, 132.1, 130.6 (2C), 132.8, 145.6 (2C), 167.1.
4.1.2.23. 5-(4-Chlorobenzyl)-5-hydroxy-3,5-dihydro-2H-imidazo[2,1-a]isoindole-7(8)-
carbaldehyde, 23.
Yield: 79%. Oil. IR (NaCl): νmax 3296, 2928, 1704, 1682, 1620, 1347, 1088, 815, 779, 732 cm .
-1
+
+
HRMS (ESI ) calcd. for C H ClN O [M+H] : 327.0900, found 327.0907.
18
16
2
2
1
5-(4-Chlorobenzyl)-5-hydroxy-3,5-dihydro-2H-imidazo[2,1-a]isoindole-7-carbaldehyde, 23a.
H
NMR (200 MHz, CDCl ): δ 3.12 (d, J = 14.0 Hz, 1H), 3.33-2.80 (m, 2H), 3.39 (d, J = 14.0 Hz, 1H),
3
4.15-3.96 (m, 2H), 6.86 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 7.9 Hz, 1H), 7.31
1
3
(
s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 10.10 (s, 1H). C NMR (50 MHz, CDCl ): δ 39.7, 40.9, 43.5,
3
90.0, 128.1 (2C), 128.9, 129.6, 130.0, 130.9, 131.2 (2C), 131.7, 133.4, 139.7, 143.8, 168.0, 190.8.
1
5-(4-Chlorobenzyl)-5-hydroxy-3,5-dihydro-2H-imidazo[2,1-a]isoindole-8-carbaldehyde, 23b.
H
NMR (200 MHz, CDCl ): δ 3.12 (d, J = 14.0 Hz, 1H), 3.33-2.80 (m, 2H), 3.39 (d, J = 14.0 Hz, 1H),
3
4.15-3.96 (m, 2H), 6.88 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.17 (s, 1H), 7.19 (d, J = 8.1
13
Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 10.17 (s, 1H). C NMR (50 MHz, CDCl ): δ 39.7, 40.9, 43.5,
3
90.0, 128.1 (2C), 128.6, 128.9, 130.9, 131.2 (2C), 131.7, 132.0, 133.4, 139.6, 143.5, 168.0, 190.6.
4.1.2.24. 5-(4-Chlorobenzyl)-7(8)-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 24.
Yield: 85%. Oil. IR (NaCl): νmax 3360, 2925, 1699, 1533, 1430,1348, 1235 1112, 1086, 903, 813,
-1
+
+
7
23 cm . HRMS (ESI ) calcd. for C H ClN O [M+H] : 344.0796, found 344.0788.
17 15 3 3
1
5
-(4-Chlorobenzyl)-7-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 24a. H NMR (400 MHz,
CDCl ): δ 2.92-3.13 (m, 2H), 3.27 (d, J = 14.1 Hz, 1H), 3.41 (d, J = 14.1 Hz, 1H), 4.08-4.21 (m,
3
2H), 6.82 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.2 Hz, 1H), 8.26 (bs, 1H), 8.36
1
3
(
d, J = 8.2 Hz, 1H). C NMR (100 MHz, CDCl ): δ 38.9, 42.0, 43.6, 89.1, 117.9, 124.1, 128.3
3
(2C), 127.0, 128.4, 131.1 (2C), 132.2, 133.1, 144.8, 149.0, 165.2.
1
5
-(4-Chlorobenzyl)-8-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 24b. H NMR (400 MHz,
CDCl ): δ 2.92-3.13 (m, 2H), 3.26 (d, J = 14.1 Hz, 1H), 3.41 (d, J = 14.1 Hz, 1H), 4.08-4.21 (m,
3
2
1
1
H), 6.78 (d, J = 7.9 Hz, 2H), 7.07 (d, J = 7.9 Hz, 2H), 7.49 (d, J = 8.2 Hz, 1H), 8.24 (d, J = 8.2 Hz,
1
3
H), 8.41 (bs, 1H). C NMR (100 MHz, CDCl ): δ 38.9, 41.8, 43.6, 89.0, 118.6, 123.6, 124.6,
3
28.3 (2C), 128.4, 131.1 (2C), 132.2, 132.6, 141.6, 150.2, 165.2.

ACCEPTED MANUSCRIPT
4
.1.2.25. 6,9-Dichloro-5-(2-chlorobenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 25.
-1
Yield: 92%. Oil. IR (NaCl): νmax 3196, 2925, 1687, 1612, 1396, 1235, 1088, 895, 736 cm . HRMS
+
+
1
(
ESI ) calcd. for C H Cl N O [M+H] : 367.0166, found 367.0160. H NMR (400 MHz, CDCl ):
17 14 3 2 3
δ 2.82-3.13 (m, 2H), 3.63 (d, J = 14.4 Hz, 1H), 3.78 (d, J = 14.4 Hz, 1H), 3.93-3.98 (m, 2H), 4.24
(
bs, 1H, D O), 6.76 (dd, J = 7.2, 1.2, 1H), 6.87 (ddd, J = 8.0, 7.2, 1.6, 1H), 6.98 (ddd, J = 8.4, 8.0,
2
1
3
1
.2, 1H), 7.16 (dd, J = 8.4, 1.6, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H). C NMR
(
100 MHz, CDCl ): δ 37.9, 38.3, 42.3, 88.6, 126.3, 128.0, 128.4, 128.8, 129.3, 129.7, 131.4, 132.0,
3
132.4, 133.9, 134.7, 146.2, 163.8.
4.1.2.26. 6,9-Dichloro-5-(4-chlorobenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 26.
-1
Yield: 91%. Oil. IR (NaCl): νmax 3234, 2930, 1685, 1598, 1376, 1225, 1093, 815, 732 cm . HRMS
+
+
1
(
ESI ) calcd. for C H Cl N O [M+H] : 367.0172, found 367.0173. H NMR (400 MHz, CDCl ):
17 14 3 2 3
δ 2.96-3.05 (m, 2H), 3.21 (d, J = 14.0 Hz, 1H), 3.76 (d, J = 14.0 Hz, 1H), 4.04-4.08 (m, 2H), 4.32
(
bs, 1H, D O), 6.70 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 7.36 (d, J
2
1
3
=
8.8 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H). C NMR (100 MHz, CDCl ): δ 38.4, 40.1, 41.4, 89.1,
3
127.6, 128.4 (2C), 129.4, 130.4 (2C), 132.1, 132.8, 132.9, 133.9, 145.2, 164.1.
4.1.2.27. 6,9-Dichloro-5-(4-methylsulfanylbenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 27.
-1
Yield: 95%. Oil. IR (NaCl): νmax 3367, 2923, 1700, 1594, 1459, 1161, 1089, 822, 734 cm . HRMS
+
+
1
(
ESI ) calcd. for C H Cl N OS [M+H] : 379.0433, found 379.0411. H NMR (400 MHz, CDCl ):
18 17 2 2 3
δ 2.34 (s, 3H), 2.97-3.05 (m, 2H), 3.24 (d, J = 14.0 Hz, 1H), 3.80 (d, J = 14.0 Hz, 1H), 4.07-4.12
m, 2H), 6.73 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.0
(
1
3
Hz, 1H). C NMR (100 MHz, CDCl ): δ 15.1, 38.4, 40.2, 41.5, 89.2, 126.2 (2C), 127.6 (2C),
3
128.8, 129.3, 129.5, 131.0, 131.9, 133.8, 137.0, 145.5, 164.1.
4.1.2.28. 7,8-Dichloro-5-(4-chlorobenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 28.
-1
Yield: 55%. Oil. IR (NaCl): νmax 3297, 2929, 1693, 1604, 1406, 1297, 1088, 895, 814, 732 cm .
+
+
1
HRMS (ESI ) calcd. for C H Cl N O [M+H] : 367.0172, found 367.0143. H NMR (200 MHz,
1
7
14
3
2
CDCl ): δ 2.83-3.09 (m, 2H), 3.09 (d, J = 14.0 Hz, 1H), 3.31 (d, J = 14.0 Hz, 1H), 3.99-4.06 (m,
3
2
1
H), 4.33 (bs, 1H, D O), 6.78 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.2 Hz, 2H), 7.39 (s, 1H), 7.58 (s,
2
1
3
H). C NMR (50 MHz, CDCl ): δ 39.0, 41.9, 43.7, 88.8, 124.7, 125.0, 128.4 (2C), 130.5, 131.3
3
(2C), 132.9, 133.1, 133.7, 136.5, 147.0, 165.4.
4.1.2.29. 7,8-Dichloro-5-(4-methylsulfanylbenzyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 29.
-1
Yield: 92%. Oil. IR (NaCl): νmax 3296, 2924, 1692, 1604, 1406, 1298, 1087, 896, 812, 732 cm .
+
+
1
HRMS (ESI ) calcd. for C H Cl N OS [M+H] : 379.0433, found 379.0403. H NMR (200 MHz,
18
17
2
2

ACCEPTED MANUSCRIPT
CDCl ): δ 2.38 (s, 3H), 2.85-3.25 (m, 2H), 3.08 (d, J = 14.0 Hz, 1H), 3.31 (d, J = 14.0 Hz, 1H),
3
4
1
.02-4.16 (m, 2H), 4.18 (bs, 1H, D O), 6.76 (d, J = 7.5 Hz, 2H), 6.99 (d, J = 7.5 Hz, 2H), 7.44 (s,
2
1
3
H), 7.61 (s, 1H). C NMR (50 MHz, CDCl ): δ 15.6, 39.3, 42.0, 43.7, 89.1, 124.8 (2C), 126.1
3
(2C), 130.4 (2C), 130.6, 131.0, 133.4, 136.2, 137.2, 147.1, 165.4.
4.1.2.30. 5-(3,4-Dichlorobenzyl)-6(9)-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 30.
-1
Yield: 89%. Oil. IR (NaCl): νmax 3427, 2926, 1701, 1537, 1471, 1356, 1090, 823, 769, 725 cm .
+
+
HRMS (ESI ) calcd. for C H Cl N O [M+H] : 378.0407, found 378.0407.
17
14
2
3
3
5-(3,4-Dichlorobenzyl)-6-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 30a.
1
H NMR (400 MHz, CDCl ): δ 2.86-3.03 (m, 2H), 3.29 (d, J = 14.0 Hz, 1H), 3.92 (d, J = 14.0 Hz,
3
1H), 4.06-4.10 (m, 2H), 6.57 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H), 7.05 (d, J = 8.0 Hz, 1H), 7.52 (dd, J
1
3
=
7.6, 8.0 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H). C NMR (100 MHz, CDCl ):
3
δ 39.3, 42.4, 42.5, 91.8, 127.2, 128.4, 129.0, 130.8, 131.2, 131.8, 132.0, 132.7, 134.8, 135.7, 140.9,
145.4, 165.1.
5-(3,4-Dichlorobenzyl)-9-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 30b.
1
H NMR (400 MHz, CDCl ): δ 2.86-3.03 (m, 2H), 3.29 (d, J = 14.0 Hz, 1H), 4.06-4.10 (m, 2H),
3
4.16 (d, J = 14.0 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 7.07 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.41 (d, J
1
3
=
7.6 Hz, 1H), 7.60 (dd, J = 7.6, 7.7 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H). C NMR (100 MHz,
CDCl ): δ 39.4, 42.5, 44.2, 88.7, 123.1, 124.3, 127.2, 130.1, 131.7, 132.7, 132.8, 133.5, 134.7,
3
135.5, 146.4, 150.8, 163.0.
4.1.2.31. 5-(3,4-Dichlorobenzyl)-7(8)-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 31.
-1
Yield: 91%. Oil; IR (NaCl): νmax 3427, 2925, 1698, 1533, 1472, 1348, 1031, 902, 820, 728 cm .
+
+
HRMS (ESI ) calcd. for C H Cl N O [M+H] : 378.0407, found 378.0407.
17
14
2
3
3
1
5
-(3,4-Dichlorobenzyl)-7-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 31a. H NMR (400
MHz, CDCl ): δ 2.84-2.90 (m, 2H), 3.15 (d, J = 14.0 Hz, 1H), 3.35 (d, J = 14.0 Hz, 1H), 4.05-4.16
3
(
m, 2H), 4.47 (s, 1H, D O), 6.46 (d, J = 2.0, 1H), 6.63 (dd, J = 8.0, 2.0, 1H), 7.14 (d, J = 8.0 Hz,
2
1
3
1
H), 7.68 (dd, J = 8.4, 1.6 Hz, 1H), 8.14 (d, J = 1.6 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H). C NMR
(
100 MHz, CDCl ): δ 39.0, 41.9, 43.4, 88.8, 117.9, 124.7, 127.1, 129.9, 130.1, 131.3, 131.8, 132.1,
3
132.2, 134.7, 135.8, 150.2, 165.1.
1
5
-(3,4-Dichlorobenzyl)-8-nitro-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol, 31b. H NMR (400
MHz, CDCl ): δ 2.84-2.90 (m, 2H), 3.15 (d, J = 14.0 Hz, 1H), 3.35 (d, J = 14.0 Hz, 1H), 4.05-4.16
3
(
m, 2H), 4.47 (s, 1H, D O), 6.67 (dd, J = 8.0, 1.6, 1H), 7.01 (d, J = 1.6, 1H), 7.15 (d, J = 8.0 Hz,
2
1
3
1
H), 7.39 (d, J = 8.4 Hz, 1H), 8.19 (dd, J = 8.4, 2.0 Hz, 1H), 8.34 (d, J = 2.0 Hz, 1H). C NMR
(
100 MHz, CDCl ): δ 39.0, 42.1, 43.4, 88.8, 118.7, 123.7, 124.8, 129.9, 130.1, 131.3, 131.9, 132.1,
3