Novel phenyl and pyridyl substituted derivatives of isoindolines: Synthesis, antitumor activity and DNA binding features

Article abstract of DOI:10.1016/j.ejmech.2014.09.079

Novel phenyl-substituted (3a-3d, 4a, 5, 8a, 8b and 9a) and pyridyl-substituted (3e-3i, 4b, 8c-8e, 9b and 9c) isoindolines were prepared in the reaction of o-phthalaldehyde and corresponding substituted aromatic and heteroaromatic amines by modification of reaction conditions from low to high temperature and from neutral to acidic environment.The antiproliferative activity of chosen substituted isoindolines was assessed on a panel of tumour cell lines and normal human fibroblasts. The majority of tested compounds was active at the highest tested concentrations phenyl-substituted isoindolines 3a and 3b and pyridyl-substituted isoindoline 3g showed a selective effect at micromolar concentrations on HepG2 cell line in comparison with other tested tumour cell lines and normal human fibroblasts. The strongest yet non-selective effect was observed for the pyridyl-substituted isoindoline 8c. These isoindoline derivatives showed diverse mechanism of action on tumour cell death induction as compounds 3a and 8c probably induced mitotic catastrophe while compound 3b induced apoptosis. Indeed, DNA binding properties evidenced that compounds 8a, 8c and 8d bind to DNA as highly potent DNA intercalators. By contrast, compounds 3b, 3e, 3i, 4a and 5 did not target the DNA. At last, the phenyl-substituted compound 8b proved to be a strong DNA binding compound with sequence selective binding and without DNA intercalation profile.

Full text of DOI:10.1016/j.ejmech.2014.09.079

Accepted Manuscript
Novel phenyl and pyridyl substituted derivatives of isoindolines: Synthesis, antitumor
activity and DNA binding features
Irena Sović, Sandra Kraljević Pavelić, Elitza Markova-Car, Nataša Ilić, Raja Nhili,
Sabine Depauw, Marie-Hélène David-Cordonnier, Grace Karminski-Zamola,
Professor
PII:
S0223-5234(14)00898-8
10.1016/j.ejmech.2014.09.079
EJMECH 7386
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 January 2014
Revised Date: 15 September 2014
Accepted Date: 24 September 2014
Please cite this article as: I. Sović, S.K. Pavelić, E. Markova-Car, N. Ilić, R. Nhili, S. Depauw, M.-
H. David-Cordonnier, G. Karminski-Zamola, Novel phenyl and pyridyl substituted derivatives of
isoindolines: Synthesis, antitumor activity and DNA binding features, European Journal of Medicinal
Chemistry (2014), doi: 10.1016/j.ejmech.2014.09.079.
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ACCEPTED MANUSCRIPT
Graphical abstract
Novel Phenyl and Pyridyl Substituted Derivatives of Isoindolines: Synthesis, Antitumor
Activity and DNA binding features
a
b
b
b
Irena Sović , Sandra Kraljević Pavelić , Elitza Markova-Car , Nataša Ilić ,
c
c
c
a *
Raja Nhili , Sabine Depauw , Marie-Hélène David-Cordonnier and Grace Karminski-Zamola
Novel Phenyl- and Pyridyl-
Substituted Derivatives of
Isoindolines: Synthesis ,
Antitumor Activity and
DNA binding features

ACCEPTED MANUSCRIPT
Novel Phenyl and Pyridyl Substituted Derivatives of Isoindolines: Synthesis, Antitumor
Activity and DNA binding features
a
b
b
b
Irena Sović , Sandra Kraljević Pavelić , Elitza Markova-Car , Nataša Ilić ,
c
c
c
a *
Raja Nhili , Sabine Depauw , Marie-Hélène David-Cordonnier and Grace Karminski-Zamola
[
a] Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 20, P. O. Box 177, HR-10000 Zagreb, Croatia
b] Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, 51000 Rijeka,
Croatia,
c] INSERM U837-JPARC (Jean-Pierre Aubert Research Center), Team "Molecular and Cellular
[
[
Targeting for Cancer Treatment“, Université Lille Nord de France, IFR-114, Institut pour la
Recherche sur le Cancer de Lille, Place de Verdun, F-59045 Lille Cedex, France
*
Corresponding author: Professor Grace Karminski-Zamola, Department of Organic Chemistry,
Faculty of Chemical Engineering and Technology, University of Zagreb, Marulićev trg 20, P. O.
Box 177, HR-10000 Zagreb, Croatia, Phone No. ++38514597215; Fax No. ++38514597250; e-
mail: gzamola@fkit.hr
Abstract
Novel phenyl-substituted (3a-3d, 4a, 5, 8a, 8b and 9a) and pyridyl-substituted (3e-3i, 4b,
8
c-8e, 9b and 9c) isoindolines were prepared in the reaction of o-phthalaldehyde and
corresponding substituted aromatic and heteroaromatic amines by modification of reaction
conditions from low to high temperature and from neutral to acidic environment.
The antiproliferative activity of chosen substituted isoindolines was assessed on a panel of tumor
cell lines and normal human fibroblasts. The majority of tested compounds was active at the
highest tested concentrations phenyl-substituted isoindolines 3a and 3b and pyridyl-substituted
isoindoline 3g showed a selective effect at micromolar concentrations on HepG2 cell line in
comparison with other tested tumour cell lines and normal human fibroblasts. The strongest yet

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non-selective effect was observed for the pyridyl-substituted isoindoline 8c. These isoindoline
derivatives showed diverse mechanism of action on tumour cell death induction as compounds 3a
and 8c probably induced mitotic catastrophe while compound 3b induced apoptosis. Indeed,
DNA binding properties evidenced that compounds 8a, 8c and 8d bind to DNA as highly potent
DNA intercalators. By contrast, compounds 3b, 3e, 3i, 4a and 5 did not target the DNA. At last,
the phenyl-substituted compound 8b proved to be a strong DNA binding compound with
sequence selective binding and without DNA intercalation profile.
Key words: heterocyclic compounds, phenyl and pyridyl substituted isoindolines,
antiproliferative activity, DNA binding, intercalator, sequence-selective binding.

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1
. Introduction
Derivatives of isoindolines are nitrogen heterocyclic compounds poorly evaluated in the
scientific literature in comparison with other heterocyclic compounds containing nitrogen.
Natural derivatives of isoindolines were first isolated in the early 60's of the last century [1,2] and
immediately gained interest due to broad biological activity. For example, staurosporine isolated
from the bacteria Streptomyces staurosporeus showed antimicrobial, hypotensive and cytotoxic
activity and acted as thrombocytes aggregation inhibitor or protein kinase inhibitor [3]. These
findings attracted considerable attention of medicinal and synthetic organic chemists. In the last
few years novel synthetic derivatives of isoindolines with different biological activities were
prepared. They showed anxiolytic, antipsychotic, anticonvulsive or anaesthetic activities [4-6]. In
addition, some of them act as inhibitors of dipeptidilpeptidase [7,8] or bind to the dopamine and
serotonine receptors [9,10]. Moreover, some isoindolines exert nonsteroidal anti-inflammatory
activity through selective COX-2 inhibition at IC ranging from 0.1 to 1.0 ꢀM in vivo [11]. Some
5
0
substituted isoindolines act as antihypertensive drugs used against cardiorenal diseases [12,13].
Their application is extended to the coordination chemistry field where they serve as ligands that
enable modelling of three-dimensional structures with desirable areal properties. Because of such
wide application potential and diverse biological properties, novel metal complexes of
isoindoline derivatives were synthesised and described as inhibitors of protein kinase [14] or
inducers of catalase which accounts for a protective role from the harmful attacks of hydrogen
peroxide in cells [15]. Isoindoline derivatives that suppress resistance of colon cancer tumor cells
to other drugs were also described and their activites on epidermal and ovarian cancer [16,17], or
on the inhibition of a number of enzymes responsible for cancer cell reproduction [18] were
documented as well.
A series of isoindolines designed to inhibit VEGF-R2 kinase were synthesized and found to
potently and selectively inhibit VEGF-R2 and Aurora-A kinases [19]. A series of isoindoline
hydroxamic acid derivatives bearing a cyclic amide/imide group as a linker and/or cap structure,
were prepared and showed class-selective potent histone deacetylase (HDAC)-inhibitory activity.
A representative compound showed potent p21 promoter activity, comparable with that of
trichostatin A (TSA), and its cytostatic activity against human prostate cell line LNCaP was more
potent than that of the well-known HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA)

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[20]. Another group of isoindolinone-hydroxamic acid derivatives bearing a 4-(3-pyridyl)phenyl
group as a cap structure, were also synthesized and described as potent histone deacetylase
HDAC) inhibitors. A representative compound showed more potent growth-inhibitory activity
against pancreatic cancer cells and greater upregulation of p21 (WAF1/CIP1) expression than the
(
(
TM)
clinically used HDAC inhibitor suberoylanilide hydroxamic acid (Zolinza ) [21]. Another
series of isoindoline derivatives, isoindoline-based hydroxamates was described and several
analogues were shown to inhibit HDAC1 with IC values in the low nanomolar range and inhibit
5
0
cellular proliferation of HCT116 human colon cancer cells in the sub-micromolar range. The
cellular potency of 1-[2-(3-propyl)indol-2-yl]substituted compound of this series had greatest in
vitro anti-proliferative activity. [22]. In a recent study, novel 5-bromo-2-(5-aryl-1,3,4-thiadiazol-
2
-yl)isoindoline-1,3-dione derivatives were synthesized and docked at the ATP-binding site of
chain B of human topoII alpha. The compounds with best in silico results were further screened
by dye exclusion test for short term cytotoxicity study on Dalton's lymphoma Ascites (DLA) cells
using Trypan blue dye. One of the synthesized compounds exhibited prominent in silico and in
vitro anticancer activity [23]. Finally, a group of authors has recently reported on the synthesis of
a series of novel isoindoline-1,3-diones containing 1,2,4-triazole moiety via one-pot reaction.
Most of them displayed stronger antitumor activities against four human cell lines (HepG2,
A549, PC-3M and MKN45) in comparison with Fluorouracil, used as a positive control. Indeed,
one of the compounds presented a four-fold improvement in comparison with Fluorouracil (35.1
and 46.83 ꢀM) in inhibiting A549 and HepG2 cell proliferation with IC values of 6.76 and 9.44
5
0
ꢀ
M, respectively. Further flow-activated cell sorting analysis revealed this compound displayed
apoptosis-inducing effect on HepG2 cells in a dose-dependent manner [24].
Most of the synthetic methods used for preparation of isoindoline derivatives include
phthalaldehyde and corresponding aliphatic and aromatic amines as starting material [25-27].
Synthetic methods starting from other compounds include phthalonitrile, phthalanhydride and
phthalaldehyde acid as well as multicomponent synthesis [28-30].
With the goal to develop potent isoindolines with diverse biological spectra of activity
and as a part of our continuous research dedicated to the synthesis of novel nitrogen containing
heterocycles as potential anticancer agent, we prepared a series of novel phenyl and pyridyl
substituted isoindoline derivatives. In this work we used a classical synthetic method starting
from phthaldehyde and substituted anilines and aminopyridines for preparation of substituted

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isoindolines. The main products in these reactions were disubstituted isoindolines of type 1-
imino-(p-substituted-phenyl)-2-N-(p-substituted-phenyl)isoindoline and their pyridine analogs
(3a-3i, 4a, 4b, 5, 8a-8e) (Fig. 1). Amidino substituted phenyl and pyridyl isoindolinones (9a-9c)
(
Fig 2) were prepared depending on different reaction conditions.
Fig. 1.
Fig. 2.
2
2
. Results and Discussion
.1. Chemistry
Prepared compounds were synthesized according to procedures shown in Schemes 1 and
by conventional and modified methods of organic synthesis used in preparation of similar
2
heterocyclic compounds from phthalaldehyde and substituted aromatic and heteroaromatic
amines.
Scheme 1.
Scheme 2.
Reaction was carried out in ethanol at room temperature and 1-imino-(p-substituted-phenyl)-2-N-
(p-substituted-phenyl)isoindolines (3a-3d, 4a and 5) were isolated in good yields. Isoindolines
with pyridine in its moieties (3e-3i and 4b) were prepared at higher temperatures because of
lower amino group nucleophilicity of the pyridine precursor (2e-2i). Phenyl or pyridyl substituted
isoindolines bearing amidino groups (8a-8e) were not reachable by Pinner reaction from cyano
substituted compounds (3a, 3f, 3g). Introducing convergent synthesis for preparation of
compounds 8a-8e, we first prepared amidino and substituted amidinoanilines and
amidinopyridines (7a-7h) in the form of hydrochloride salts from corresponding cyano

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substituted anilines and aminopyridines (6a-6c) by Pinner reaction. Compounds 7a-7h served as
precursors in the condensation reaction with o-phthalaldehyde. Amidinophenyl substituted
isoindolines 8a and 8b and amidinopyridyl substituted isoindolines 8c and 8d were isolated as the
sole products of this reaction. The reaction of o-phthalaldehyde with amidinopyridines where the
nitrogen atom is in ortho position against amino group (7f-h) was unsuccessful in neutral
conditions. However, in acid conditions N-isopropilamidinopyridin substituted isoindoline (8e)
was isolated in the mixture with the corresponding substituted isoindolin-1-one (9b). All attempts
to obtain imino substituted isoindolines from amidino and imidazolinyl substituted 2-
aminopyridines (7f, 7h) were unsuccessful. Isoindolinones 9a and 9c were sole products in the
reaction of 4-amidinoaniline and 5-amidino-2-aminopyridine as precursors. As amino group of
amidino substituted anilines and 3-aminopyridines are less nucleophilic than other substituted
anilines and amino pyridines the reaction of condensation was performed on higher temperature,
and in the case of amidino substituted 2-aminopyridines in acid conditions.
1
13
Compound structures were confirmed by H and C NMR analysis. The formation of the
1
isoindoline ring was confirmed by two-proton singlet at 5.36−4.94 ppm in the H NMR spectra as
well as chemical shift at 53.29−52.33 ppm for isoindoline (3a-i, 8a-e) and 50.87−50.31 ppm for
1
3
isoindolinone (9a-c) in the C NMR spectra. The presence of imino group was supported by the
1
3
chemical shift at 155.18−153.41 ppm in the C NMR spectra of isoindolines 3a-i and 8a-e,
characteristic for the quaternary carbon atom of imino group. The presence of amidino group in
compounds 8a-e and 9a-c was confirmed by broad signal of NH protons at 10.78−9.33 ppm in
1
the H NMR spectra and chemical shift of quaternary amidino carbon atom at 168.05−160.02
1
3
ppm in the C NMR spectra. Compound structures were also analysed by IR spectroscopy. The
mean feature of IR spectra for isoindoline compounds 3a-i and 8a-e is C=N stretching band of
-1
imino group at 1666−1631 cm and for isoinolinone compounds 9a-c is C=O stretching band of
-1
carbonyl group at 1720−1680 cm . Assignations of bands that are characteristic for different
substituents on phenyl and pyridine ring are given in the experimental part.
2
.2. Antitumor activity

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Tested compounds exerted concentration-dependent antiproliferative effects on tested
tumour cells and normal human fibroblasts (Table 1). Compound 3e was the less active while the
most pronounced effect on the cell growth of all tested cell lines was observed for isoindoline 8c.
Selective effect at micromolar concentrations (IC ranging from 4,68 – 9,65 µM) were observed
5
0
for the phenyl substituted isoindolines 3a, 3b and the isoindoline with pyridine moiety 3g on
HepG2 cell line. Comparable IC50 values were previously observed on HepG2 cell line for
isoindoline-1,3-diones group containing 1,2,4-triazole moiety [24]. At last, the group of phenyl-
substituted isoindoline 3b, 3c, 4a, 5 and the isoindoline with pyridine moiety 3e showed high
potency on the growth of MCF-7 cells.
Due to selective and strongest effects in comparison with other tested compounds
observed for compounds 3a, 3b, and 3g on HepG2 cells and normal human fibroblasts and due to
strongest general effect assesed for compound 8c on all cell lines including the metastatic SW620
cells, further mechanistic studies invloving analysis of cell cycle and cell death mechanisms were
pursued on for this group of compounds.
Cell cycle analysis revealed different mechanisms of antiproliferative effect for the
pyridyl-substituted isoindoline 8c and phenyl-substituted isoindolines 3a and 3b on the HepG2
cells. Compounds 3a and 8c induced a strong G2/M arrest which might be indicative for mitotic
catastrophe. This cell death mechanism was confimed for compound 8c by topoisomerase
inhibition assay as well (described in detials in the paragraph “DNA binding properties”), while
compounds 3b and 3g induced an increase of cells in the subG1 phase (Table 2) which might be
indicative of apoptosis. Mitotic catastrophe has recently gained importance as a new strategy in
development of novel anticancer drugs, in particular for treatment of cancers resistant to
traditional chemotherapy. Mitotic catastrophe is a cell death mechanism triggered by some
chemotherapeutics and shares similar features with apoptosis [31]. The morphology of cells
dying by mitotic catastrophe is different from those undergoing apoptosis. This was observed in
our experiments where enlarged cells were visible in treatments of HepG2 with compound 3a and
SW620 with compound 8c, while cytoplasmic shrinkage and apoptotic bodies typical for
apoptosis and confirmed by annexin-V assay were visible for HepG2 cells treated with
compound 3b (Figure 3). Apoptotic bodies and annexin V labelling were not visible in HepG2
cells treated with compound 3g, that suggests a different cell death mechanism.

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Fig. 3.
Table 1.
Table 2.
2
.3. DNA binding properties.
DNA binding properties were assessed for tested compounds. First we used the DNA
melting temperature measurement as a rapid screening for compounds that bind to DNA and that
cause a modification in the DNA double strand helix toward progressive increase of the
temperature. A strong binding of compound to DNA would thus result in stabilisation of the
DNA helix which may be observed by monitoring CT-DNA absorbency at 260 nm. A strong
binding is associated with a strong increase in the temperature that is required to obtain half of
the DNA in a double stranded form and the other half as two: one half representing the double
stranded form and the other half representing two melted single strands of nucleic acids (∆Tm).
The experiment is done with DNA in the presence of tested compounds in comparison with DNA
alone. Measurements for drug/DNA ratio at 0.5 proved that compounds 8a-d strongly stabilized
the DNA helix causing temperature increase higher than 10°C that is required to reach similar
denaturation for control CT-DNA. Compounds 8a and 8c were the most potent ones (Table 3).
Compound 8e was the less efficient with a ∆Tm value around 4 °C whereas other tested
compounds failed to modify DNA melting temperature, suggesting that they failed to bind CT-
DNA.
Table 3.
The mode of binding of the tested compounds to the DNA helix (intercalation or groove binding)
was assessed with circular dichroism spectra method (Figure 4). Intrinsic circular dichroism
(dotted lines) was not evidenced for none of tested compounds. The strongest DNA binding

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compounds (compounds 8a-d, as identified using DNA melting temperature studies) strongly
modified CD spectra’s of CT-DNA (dashed lines) whereas weaker DNA binder 8e or non-
binding compounds did not affect the intrinsic CD of CT-DNA alone. Particularly, compound 8d
presented a typical negative induced CD (ICD) typical for DNA intercalation with maximal
negative peaks around 375 nm. Similar negative ICD (around 355 nm) was obtained for
compound 8b with an increased wavelength from 275 to 285 nm of the positive ICD of CT-
DNA. By contrast, compounds 8a and 8c presented a negative bisignate (but asymmetric) ICD in
the course of titration. Negative bisignate ICD predicts negative helical orientation of the
transition dipoles. Those negative bisignate ICD in the absorption wavelength for 8a and 8c are
associated with a strong modification of CT-DNA circular dichroism with an enhancement of the
positive DNA CD (~280 nm) and a reduction of negative CT-DNA CD (~245 nm). Both changes
are in agreement with DNA intercalation that modifies the DNA topology (base staking, twisting
of the helix). Such strong intercalative binding of compounds 8a and 8c in the DNA helix
correlates with their strong induced ∆Tm values (>15°C) presented in Table 3.
Fig. 4.
Intercalation was additionally evaluated by topoisomerase I-induced DNA relaxation capability
on agarose gels (Figure 5). As expected, compounds 3b, 3i, 4a and 5 did not change the
topoisomerase I-induced relaxation profile which is in agreement with the ∆Tm measurements
showing no binding to DNA (Table 3).
Fig. 5.
Compound 3e showed a very weak relaxation profile modification at the highest tested
concentration which is in agreement with poor DNA binding efficiency and that was not
sufficient to stabilize the DNA helix in the ∆Tm measurement experiment. Interestingly,

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compounds 8a, 8c and 8d strongly changed the DNA relaxation profile of the supercoiled
plasmid DNA, suggesting that these compounds are efficient DNA intercalators, which is in
agreement with circular dichroism analyses (Figure 4). From comparison with camptothecin
(CPT), as a reference drug for topoisomerase I poisoning, none of tested compounds were able to
generate open circular DNA (nicked DNA, Nck localised by a star* in Figure 5) suggesting that
intercalation of compounds 8a, 8c and 8d in the DNA helix is not associated with a poison
activity. By contrast, compound 8b did not change the topoisomerase I-induced relaxation profile
even at the highest drug concentration (50 µM) suggesting that the DNA helix stabilisation
results obtained by ∆Tm measurement experiments is due to another mode of binding to DNA.
In order to obtain additional insights for DNA binding abilities of tested compounds, the
sequence-selective binding was studied by use of DNase I footprinting experiments. None of
tested compounds presented sequence selective binding (exemplified by protected bands on the
gel) except compound 8b that binds to two AT-rich stretches (AATT, ATTA) or three ACAA
sites (two ACAA sites and the reversed sequence TTGT) (Figure 6). This result suggests that
compound 8b is a sequence-selective binder that is in contrast with intercalative properties
assessed for compounds 8a, 8c and 8d. However, sequence-selective binders usually localize in
the minor groove of the DNA helix that leads to a strong positive ICD in the compound
absorption wavelength that was not proved for compound 8b (Figure 4). Therefore the precise
mode of 8b binding to DNA needs further investigation.
Fig. 6.
3
8
. Conclusion
Novel phenyl-substituted (3a-3d, 4a, 5, 8a, 8b and 9a) and pyridyl-substituted (3e-3i, 4b,
c-8e and 9b, 9c) isoindolines were prepared in the reaction of o-phthalaldehyde and
corresponding substituted aromatic and heteroaromatic amines by modification of reaction
conditions from low to high temperature and from neutral to acidic environment.
All tested compounds except 1-imino-[2-(imidazolin-2-yl)-pyridin-5-yl]-1-N-[2-(imidazolin-2-
yl)-pyridin-5-yl]-isoindoline 8c, exerted weak antiproliferative effect on the metastatic cell line
SW620 while the strongest concentration-dependent effects were observed on the breast cancer

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cell line MCF-7. Antiproliferative effects have been observed on normal diploid human
fibroblasts for pyridyl-substituted isoindolines 3f, 8c, 9b and 9c and for only one phenyl-
substituted isoindoline 8a. Selective effects at micromolar concentrations were observed as well,
including good selectivity of 1-imino-(substituted-phenyl)-2-N-(substituted-phenyl) isoindolines
3
a, 3b and substituted pyridyl isoindolines 3g and 3f on the growth of HepG2 cells and of
compounds 3b, 3g and 3f on the growth of HeLa cells. The strongest yet non-selective effect was
observed for substituted pyridyl-isoindoline 8c. Their mechanism of tumour cell death induction
was however, different as compounds 3a and 8c probably induce mitotic catastrophe while
compounds 3b and 3g induced apoptosis.
Indeed, DNA binding properties evidenced that compounds 8a, 8c and 8d binds to DNA
as highly potent DNA intercalators (Figures 4, 5). Such intercalation profile for the compound 8c
correlates with observed accumulation of SW620 cells in the G2/M phase of the cell cycle (Table
2
). By contrast, other tested compounds (3b, 3e, 3i, 4a and 5) did not target DNA (Table 3,
Figure 4 and data not shown) and may exert their cytotoxic activities, particularly strong
antiproliferative action on MCF-7 cell line (IC of 8.1, 0.70, 16, 0.05, 0.73 µM, respectively)
5
0
through another mechanism, including apoptosis induction. At last, the phenyl-substituted
compound 8b proved to be a strong DNA binding compound (Table 3) with a sequence selective
binding mode (Figure 6) and without a DNA intercalation profile (Figure 5). The bound sequence
is relatively small, in agreement with the tight bent between two isopropylamidinophenyl groups
of compound 8b. Such short bound sequence is in contrast with recent attempts to prepare longer
sequence DNA binders using a more linear heterocyclic dications [37-40] that have a potential in
inhibition of transcription factor binding to DNA [35, 41-42]. Only a small change in the
extremities of imidazoline ring (8a) to obtain the N-isopropylamidine (8b) resulted in a complete
change of DNA binding activity from a starting DNA intercalating compound without DNA
selectivity, to a sequence selective binder. However, observed non-typical CD spectra (negative
ICD instead of strong positive ICD usually observed for groove binders) indicate that precise
interaction modality for compound 8b to the DNA helix needs to be further evaluated in more
details.
4
. Experimental

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4
.1. Chemistry
Melting points were determined on a Koffler hot stage microscope and are uncorrected. IR
1
13
spectra were recorded on a Bruker Vertex 70 spectrophotometer with diamond crystal. H and C
NMR spectra were recorded on Varian Gemini 300 or Varian Gemini 600 spectrophotometers at
3
00, 600, 150 and 75 MHz, respectively. All NMR spectra were measured in DMSO-d solutions
6
using TMS as an internal standard. Chemical shifts are reported in ppm (δ) relative to TMS. Mass
spectra were recorded on an Agilent 1200 series LC/6410 QQQ instrument. Elemental analysis
for carbon, hydrogen and nitrogen were performed on a Perkin-Elmer 2400 elemental analyzer
and a Perkin-Elmer, Series II, CHNS analyzer 2400. All compounds were routinely checked by
thin layer chromatography (TLC) using precoated Merck silica gel 60F-254 plates and the spots
were detected under UV light (254 nm). Column chromatography (CC) was performed using
silica gel (0.063-0.2 mm) Fluka; glass column was slurry-packed under gravity.
4
.1.1. General method for the synthesis of substituted 1-imino(hetero)aryl-2-N-
(hetero)arylisoindolines 3a-i, 8a-e and 9a-c
Method A: A solution of phthalaldehyde 1 and corresponding amines in molar ration 1:2 in
absolute ethanol was prepared. After stirring at room temperature for several hours the resulting
product was filtered off and recrystallized from corresponding solvent.
Method B: A solution of phthalaldehyde 1 and corresponding amines in molar ration 1:2 in
absolute ethanol was prepared. After refluxing for several hours the resulting product was filtered
off and recrystallized from corresponding solvent or purified by column chromatography using
dichloromethane/methanol as eluent (gradient elution from 50:1 to 10:1).
Method C: A solution of phthalaldehyde 1 and corresponding amines in molar ration 1:2 in
absolute ethanol was prepared and few drops of glacial acetic acid were added. After refluxing
for several hours the reaction mixture was evaporated to dryness and resulting mixture was

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purified by column chromatography using dichloromethane/methanol as eluent (gradient elution
from 50:1 to 10:1).
4
.1.1.1. 4-cyano-N-[2-(4-cyanophenyl)isoindolin-1-ylidene]benzenamine 3a
Following the general procedure A, from phthalaldehyde 1 (0.54 g, 4 mmol) and 4-cyanoaniline
2
a (0.94 g, 8 mmol) after stirring for 2 h and recrystallization from ethanol/toluene 0.69 g (54%)
-1
of pale yellow powder was obtained; mp 243-244 °C; IR (diamond) (ν/cm ): 2211 (C≡N), 1653
1
(
C=N), 1589 (ar C−C, skeletal vib.), 1509 (ar C−C, skeletal vib.); H NMR (300 MHz, DMSO-
d
8
7
6
) (δ/ppm): 8.19 (d, 2H, J = 8.98 Hz, Harom), 7.82 (d, 2H, J = 8.98 Hz, Harom), 7.75 (d, 2H, J =
.52 Hz, Harom), 7.64 (d, 1H, J = 7.58 Hz, Harom), 7.55 (t, 1H, J = 7.46 Hz, Harom), 7.22 (t, 1H, J =
.44 Harom), 7.10 (d, 2H, J = 8.53 Hz, Harom), 6.72 (d, 1H, J = 7.96 Hz, Harom), 5.15 (s, 2H, Hisoind);
1
3
C NMR (75 MHz, DMSO-d
Cq-arom), 141.44 (1C, Cq-arom), 134.02 (2C, CHarom), 133.35 (2C, CHarom), 132.00 (1C, CHarom),
30.34 (1C, Cq-arom), 128.07 (1C, CHarom), 125.62 (1C, CHarom), 124.16 (1C, CHarom), 122.12 (2C,
6
) (δ/ppm): 154.56 (1C, C=Nimino), 153.23 (1C, Cq-arom), 145.17 (1C,
1
CHarom), 120.36 (2C, CHarom), 119.85 (1C, C≡N), 119.45 (1C, C≡N), 105.29 (1C, Cq-arom), 105.01
+
(
1C, Cq-arom), 53.29 (1C, CH2,isoind); MS (m/z): 335.3 ([M+1] ); elemental analysis calcd. (%) for
C H N : C 79.02, H 4.22, N 16.76; found C 78.73, H 4.35, N 16.22.
2
2
14
4
4
.1.1.2. 4-nitro-N-[2-(4-nitrophenyl)isoindolin-1-ylidene]benzenamine 3b
Following the general procedure A, from phthalaldehyde 1 (0.54 g, 4 mmol) and 4-nitroaniline
2
b (1.11 g, 8 mmol) after stirring for 24 h and recrystallization from DMF 1.21 g (81%) of
-1
yellow powder was obtained; mp 208-210 °C; IR (diamond) (ν/cm ): 1655 (C=N), 1582 (NO st
2
1
as), 1321 (NO st sy); H NMR (300 MHz, DMSO-d ) (
δ
/ppm): 8.25 (s, 4H, Harom), 8.22 (d, 2H, J
2
6
=
=
8.97 Hz, Harom), 7.68 (d, 1H, J = 7.66 Hz, Harom), 7.58 (t, 1H, J = 7.35 Hz, Harom), 7.25 (t, 1H, J
7.48 Hz, Harom), 7.17 (d, 2H, J = 8.94 Hz, Harom), 6.87 (d, 1H, J = 7.91 Hz, Harom), 5.22 (s, 2H,
+
Hisoind); MS (m/z): 375.1 ([M+1] ); elemental analysis calcd. (%) for C H N O : C 64.17, H
20
14
4
4
3
.77, N 14.97; found C 63.85, H 3.92, N 14.32.
.1.1.3. 4-(N',N'-dimethylamino)-N-{2-[4-(N',N'-dimethylamino)phenyl]isoindolin-1-
4
ylidene}benzenamine 3c
Following the general procedure A, from phthalaldehyde 1 (0.33 g, 2.5 mmol) and 4-(N,N-
dimethyl)-aminoaniline 2c (0.67 g, 5.0 mmol) after stirring for 4 h and recrystallization from

ACCEPTED MANUSCRIPT
ethanol 0.31 g (33%) of golden yellow crystals was obtained; mp 193-195 °C; IR (diamond)
-1
1
(ν/cm ): 2897 (CH st), 2806 (CH st), 1631 (C=N), 1509 (ar C−C, skeletal vib.); H NMR (300
3 3
MHz, DMSO-d ) (
δ
/ppm): 7.77 (d, 2H, J = 8.67 Hz, Harom), 7.55 (d, 1H, J = 7.51 Hz, Harom), 7.43
t, 1H, J = 7.41 Hz, Harom), 7.11 (t, 1H, J = 7.44 Hz, Harom), 6.78 (d, 2H, J = 8.81 Hz, Harom), 6.72
s, 4H, Harom), 6.66 (d, 1H, J = 7.90 Hz, Harom), 4.94 (s, 2H, Hisoind), 2.88 (s, 6H, 2×CH ), 2.87 (s,
6
(
(
3
1
3
6
H, 2×CH ); C NMR (75 MHz, DMSO-d ) (δ/ppm): 153.66 (1C, C=Nimino), 150.26 (1C, Cq-
3 6
arom), 147.39 (1C, Cq-arom), 146.71 (1C, Cq-arom), 141.61 (1C, Cq-arom), 141.47 (1C, Cq-arom), 133.64
1C, CHarom), 131.94 (1C, Cq-arom), 130.42 (1C, CHarom), 127.34 (1C, CHarom), 125.65 (1C,
CHarom), 123.74 (1C, CHarom), 122.48 (1C, CHarom), 121.81 (1C, CHarom), 120.84 (1C, CHarom),
(
1
14.19 (1C, CHarom), 113.60 (1C, CHarom), 113.12 (1C, CHarom), 112.48 (1C, CHarom), 53.23 (1C,
+
CH2,isoind), 41.37 (2C, 2×CH ), 41.01 (2C, 2×CH ); MS (m/z): 371.3 ([M+1] ); elemental analysis
3
3
calcd. (%) for C H N : C 77.80, H 7.07, N 15.12; found C 77.23, H 6.65, N 14.96.
2
4
26
4
4
.1.1.4. 4-acetamido-N-[2-(4-acetamidophenyl)isoindolin-1-ylidene]benzenamine 3d
Following the general procedure A, from phthalaldehyde 1 (0.67 g, 5.0 mmol) and 4-
acetamidoaniline 2d (1.5 g, 10.0 mmol) after stirring for 24 h and recrystallization from DMF
-1
1
.22 g (62%) of pink powder was obtained; mp 228-230 °C; IR (diamond) (ν/cm ): 3298-3040
1
(
(
(
N–H), 1658 (C=O), 1634 (C=N), 1593 (ar C–C, skeletal vib.); H NMR (300 MHz, DMSO-d )
6
δ
/ppm): 9.92 (s, 1H, NHamide), 9.86 (s, 1H, NHamide), 7.94 (d, 2H, J = 7.56 Hz, Harom), 7.60-7.53
m, 5H, Harom), 7.47 (t, 1H, J = 7.42 Hz, Harom), 7.15 (t, 1H, J = 6.92 Hz, Harom), 6.82 (d, 2H, J =
7
3
1
.80 Hz, Harom), 6.62 (d, 1H, J = 6.92 Hz, Harom), 5.02 (s, 2H, Hisoind), 2.05 (s, 3H, CH ), 2.04 (s,
3
1
3
H, CH3); C NMR (75 MHz, DMSO-d ) (
δ
/ppm): 168.42 (1C, C=O), 168.23 (1C, C=O),
6
53.43 (1C, C=Nimino), 146.28 (1C, Cq-arom), 141.38 (1C, Cq-arom), 137.06 (1C, Cq-arom), 135.05
(1C, Cq-arom), 134.49 (1C, Cq-arom), 131.12 (1C, Cq-arom), 130.91 (2C, CHarom), 127.51 (1C,
CHarom), 125.67 (1C, CHarom), 123.90 (2C, CHarom), 121.24 (2C, CHarom), 120.88 (1C, CHarom),
1
3
20.46 (1C, CHarom), 119.70 (2C, CHarom), 53.01 (1C, CH2,isoind), 24.41 (1C, CH ), 24.38 (1C,
+
CH ); MS (m/z): 399.2 ([M+1] ); elemental analysis calcd. (%) for C H N O : C 72.34, H 5.57,
3
24 22
4
2
N 14.06; found C 72.03, H 4.98, N 14.50.
4
.1.1.5. 4-methyl-N-[2-(4-methylpyridin-2-yl)isoindolin-1-ylidene]pyridin-2-amine 3e
Following the general procedure A, from phthalaldehyde 1 (0.54 g, 4.0 mmol) and 2-amino-4-
methylpyridine 2e (0.86 g, 8.0 mmol) after stirring for 5 days and recrystallization from ethanol
-1
0
.47 g (37%) of red powder was obtained; mp 174-177 °C; IR (diamond) (ν/cm ): 3045, 3010,

ACCEPTED MANUSCRIPT
2
921 (ar C–H), 1653 (C=N), 1593 (ar C–C, C–N, skeletal vib.), 1544 (ar C–C, C–N, skeletal
1
vib.); H NMR (600 MHz, DMSO-d ) (
δ
/ppm): 8.66 (s, 1H, Harom), 8.30 (d, 1H, J = 5.02 Hz,
6
Harom), 8.22 (d, 1H, J = 5.10 Hz, Harom), 7.65 (d, 1H, J = 7.57 Hz, Harom), 7.52 (t, 1H, J = 7.25 Hz,
arom), 7.16 (t, 1H, J = 7.62 Hz, Harom), 6.98 (t, 2H, J = 5.45 Hz, Harom), 6.84 (s, 1H, Harom), 6.31
H
13
(
d, 1H, J = 7.37 Hz, Harom), 5.20 (s, 2H, Hisoind), 2.34 (s, 3H, CH ), 2.32 (s, 3H, CH ); C NMR
3 3
(
75 MHz, DMSO-d ) (
δ
/ppm): 162.18 (1C, Cq-arom), 154.06 (1C, C=Nimino), 153.09 (1C, Cq-arom),
49.30 (1C, Cq-arom), 148.80 (1C, CHarom), 148.75 (1C, Cq-arom), 147.85 (1C, CHarom), 141.49 (1C,
Cq-arom), 131.67 (1C, CHarom), 130.75 (1C, Cq-arom), 127.66 (1C, CHarom), 125.55 (1C, CHarom),
6
1
1
24.28 (1C, CHarom), 120.26 (1C, CHarom), 120.18 (1C, CHarom), 116.70 (1C, CHarom), 114.66 (1C,
+
CHarom), 52.33 (1C, CH2,isoind), 21.58 (1C, CH
3
), 20.96 (1C, CH
3
); MS (m/z): 315.3 ([M+1] );
elemental analysis calcd. (%) for C H N : C 76.41, H 5.77, N 17.82; found C 75.86, H 5.50, N
2
0
18
4
1
7.23.
.1.1.6. 6-cyano-N-[2-(6-cyanopyridin-3-yl)isoindolin-1-ylidene]pyridin-3-amine 3f
4
Following the general procedure B, from phthalaldehyde 1 (0.73 g, 5.5 mmol) and 5-amino-2-
cyanopyridine 2f (1.31 g, 11.0 mmol) after refluxing for 10h and recrystallization from DMF
-1
1
.33 g (72%) of yellow crystals was obtained; mp 279-281 °C; IR (diamond) (
ν
/cm ): 3093 (ar
1
C–H), 3034 (ar C–H), 2224 (C≡N), 1647 (C=N), 1550 (ar C–C, C–N, skeletal vib.); H NMR
(600 MHz, DMSO-d
6
) (
1 2
δ/ppm): 9.33 (d, 1H, J = 2.06 Hz, Harom), 8.71 (dd, 1H, J = 8.67 Hz, J =
2
.18 Hz, Harom), 8.44 (d, 1H, J = 2.18 Hz, Harom), 8.09 (d, 1H, J = 8.74 Hz, Harom), 8.02 (d, 1H, J
=
8.25 Hz, Harom), 7.71 (d, 1H, J = 7.60 Hz, Harom), 7.64-7.61 (m, 2H, Harom), 7.30 (t, 1H, J = 7.61
1
3
6
Hz, Harom), 6.72 (d, 1H, J = 7.88 Hz, Harom), 5.28 (s, 2H, Hisoind); C NMR (150 MHz, DMSO-d )
(
δ
/ppm): 154.00 (1C, C=Nimino), 148.77 (2C, Cq-arom), 144.11 (1C, CHarom), 142.38 (1C, CHarom),
1
41.27 (1C, Cq-arom), 140.01 (1C, Cq-arom), 132.11 (1C, CHarom), 129.82 (1C, CHarom), 129.13 (1C,
CHarom), 128.50 (1C, CHarom), 128.00 (1C, CHarom), 126.68 (1C, CHarom), 125.88 (1C, Cq-arom),
1
25.63 (1C, Cq-arom), 125.07 (1C, CHarom), 123.92 (1C, CHarom), 118.16 (1C, C≡N), 117.77 (1C,
+
C≡N), 52.44 (1C, CH2,isoind); MS (m/z): 337.3 ([M+1] ); elemental analysis calcd. (%) for
C
20
H
12
N
6
: C 71.42, H 3.60, N 24.99; found C 71.13, H 3.35, N 24.22.
4
.1.1.7. 5-cyano-N-[2-(5-cyanopyridin-2-yl)isoindolin-1-ylidene]pyridin-2-amine 3g
Following the general procedure B, from phthalaldehyde 1 (0.28 g, 2.0 mmol) and 2-amino-5-
cyanopyridine 2g (0.47 g, 4.0 mmol) after refluxing for 10h and recrystallization from DMF 0.29
-1
g (43%) of yellow powder was obtained; mp 276-278 °C; IR (diamond) (ν/cm ): 3115 (ar C–H),

ACCEPTED MANUSCRIPT
1
3
055 (ar C–H), 3025 (ar C–H), 2232 (C≡N), 1660 (C=N), 1585 (ar C–C, C–N, skeletal vib.); H
NMR (600 MHz, DMSO-d ) ( /ppm): 8.92 (d, 1H, J = 1.83 Hz, Harom), 8.88 (d, 1H, J = 8.94 Hz,
Harom), 8.84 (d, 1H, J = 1.81 Hz, Harom), 8.30 (dd, 1H, J = 8.94 Hz, J = 2.22 Hz, Harom), 8.26 (dd,
δ
6
1
2
1
1 2
H, J = 8.39 Hz, J = 2.27 Hz, Harom), 7.71 (d, 1H, J = 7.62 Hz, Harom), 7.61 (t, 1H, J = 7.45 Hz,
Harom), 7.26 (t, 1H, J = 7.65 Hz, Harom), 7.23 (d, 1H, J = 8.36 Hz, Harom), 6.53 (d, 1H, J = 7.99 Hz,
1
3
Harom), 5.29 (s, 2H, Hisoind); C NMR (75 MHz, DMSO-d ) (
δ
/ppm): 163.91 (1C, Cq-arom), 154.51
1C, C=Nimino), 154.45 (1C, Cq-arom), 153.54 (1C, CHarom), 152.34 (1C, CHarom), 142.07 (1C,
CHarom), 141.86 (1C, CHarom), 141.75 (1C, Cq-arom), 132.80 (1C, CHarom), 129.90 (1C, Cq-arom),
28.32 (1C, CHarom), 125.74 (1C, CHarom), 124.58 (1C, CHarom), 118.27 (1C, C≡N), 117.89 (1C,
6
(
1
C≡N), 116.77 (1C, CHarom), 114.01 (1C, CHarom), 103.77 (1C, Cq-arom), 103.35 (1C, Cq-arom), 52.80
+
(
1C, CH2,isoind); MS (m/z): 337.3 ([M+1] ); elemental analysis calcd. (%) for C H N : C 71.42,
20
12
6
H 3.60, N 24.99; found C 71.05, H 3.47, N 24.41.
4
.1.1.8. 5-nitro-N-[2-(5-nitropyridin-2-yl)isoindolin-1-ylidene]pyridin-2-amine 3h
Following the general procedure B, from phthalaldehyde 1 (0.67 g, 5.0 mmol) and 2-amino-5-
nitropyridine 2h (1.40 g, 10.0 mmol) in DMF after refluxing for 8h and recrystallization from
-
DMF 0.60 g (32%) of yellow-green powder was obtained; mp 277-279 °C; IR (diamond) (ν/cm
1
1
)
: 1663 (C=N), 1594 (ar C–C, C–N, skeletal vib.), 1568 (NO st as), 1330 (NO st sy); H NMR
2 2
(
300 MHz, DMSO-d
6
) (
δ
/ppm): 9.22 (d, 1H, J = 2.75 Hz, Harom), 9.17 (d, 1H, J = 2.80 Hz, Harom),
= 9.38 Hz, J = 2.81 Hz, Harom), 8.51 (dd, 1H, J
8.85 Hz, J = 2.84 Hz, Harom), 7.70 (d, 1H, J = 7.61 Hz, Harom), 7.60 (t, 1H, J = 7.49 Hz, Harom),
8
.86 (d, 1H, J = 9.38 Hz, Harom), 8.57 (dd, 1H, J
1
2
1
=
2
7
.28-7.21 (m, 2H, Harom), 6.75 (d, 1H, J = 7.99 Hz, Harom), 5.35 (s, 2H, Hisoind); MS (m/z): 377.3
+
(
[M+1] ); elemental analysis calcd. (%) for C H N O : C 57.45, H 3.21, N 22.33; found C
1
8
12
6
4
5
4
7.73, H 3.41, N 22.57.
.1.1.9. 6-acetamido-N-[2-(6-acetamidopyridin-3-yl)isoindolin-1-ylidene]pyridin-3-amine 3i
Following the general procedure A, from phthalaldehyde 1 (0.44 g, 3.3 mmol) and 2-acetamido-
5
-aminopyridine 2i (1.00 g, 6.6 mmol) after stirring for 24h and recrystallization from
-
1
ethanol/H O 0.70 g (53%) of beige powder was obtained; mp 245-250 °C; IR (diamond) (
ν
/cm ):
2
1
3
313-3003 (N–H), 1689 (C=O), 1639 (C=N), 1596 (ar C–C, C–N, skeletal vib.); H NMR (300
MHz, DMSO-d ) (
δ
/ppm): 10.47 (s, 1H, NHamide), 10.40 (s, 1H, NHamide), 8.95 (s, 1H, Harom),
6
8
.37 (s, 1H, Harom), 8.08 (s, 2H, Harom), 7.93 (s, 1H, Harom), 7.64 (d, 1H, J = 7.52 Hz, Harom), 7.54
(t, 1H, J = 7.41 Hz, Harom), 7.37 (d, 1H, J = 7.47 Hz, Harom), 7.24 (t, 1H, J = 6.39 Hz, Harom), 6.68

ACCEPTED MANUSCRIPT
1
3
(
3
d, 1H, J = 6.19 Hz, Harom), 5.11 (s, 2H, Hisoind), 2.09 (s, 6H, 2×CH ); C NMR (75 MHz,
DMSO-d ) (
δ
/ppm): 168.84 (1C, C=O), 168.61 (1C, C=O), 153.84 (1C, C=Nimino), 147.50 (1C,
Cq-arom), 147.14 (1C, Cq-arom), 142.23 (1C, Cq-arom), 142.19 (1C, Cq-arom), 141.26 (1C, Cq-arom),
39.59 (2C, CHarom), 133.53 (1C, Cq-arom), 130.92 (2C, CHarom), 130.15 (1C, CHarom), 127.38 (1C,
6
1
CHarom), 124.88 (1C, CHarom), 123.69 (1C, CHarom), 113.65 (1C, CHarom), 112.94 (1C, CHarom),
+
5
2.36 (1C, CH2,isoind), 23.76 (2C, 2×CH
3
); MS (m/z): 401.2 ([M+1] ); elemental analysis calcd.
(
%) for C H N O : C 65.99, H 5.03, N 20.99; found C 65.58, H 4.80, N 20.62.
22 20 6 4
4
.1.1.10. 4-(imidazolin-2-yl)-N-{2-[4-(imidazolin-2-yl)phenyl]isoindolin-1-ylidene}benzenamine
dihydrochloride 8a
Following the general procedure B, from phthalaldehyde 1 (0.027 g, 0.2 mmol) and 4-(2-
imidazolinyl)-aniline hydrochloride 7a (0.10 g, 0.4 mmol) after refluxing for 8h and
recrystallization from ethanol 0.02 g (20%) of yellow powder was obtained; mp 272-275 °C; IR
-1
1
(
diamond) (
ν
/cm ): 3370-2782 (N–H), 1652 (C=N), 1591 (C=N
); H NMR (300 MHz,
amidine
DMSO-d ) (
δ
/ppm): 10.61 (bs, 4H, NHamidine), 8.29 (s, 2H, Harom), 8.15-8.07 (m, 4H, Harom), 7.68
6
(s, 1H, Harom), 7.59 (s, 1H, Harom), 7.23 (bs, 3H, Harom), 6.65 (s, 1H, Harom), 5.24 (s, 2H, Hisoind),
1
3
4
.00 (s, 8H, 4×CH
Cq-amidine), 156.11 (1C, Cq-arom), 153.19 (1C, C=Nimino), 146.31 (1C, Cq-arom), 141.51 (1C, Cq-arom),
32.23 (1C, CHarom), 130.80 (2C, CHarom), 130.24 (1C, Cq-arom), 130.03 (2C, CHarom), 128.07 (1C,
CHarom), 125.58 (1C, CHarom), 124.34 (1C, CHarom), 121.80 (2C, CHarom), 119.62 (2C, CHarom),
2 6
); C NMR (75 MHz, DMSO-d ) (δ/ppm): 164.82 (1C, Cq-amidine), 164.49 (1C,
1
1
2
16.17 (1C, Cq-arom), 116.08 (1C, Cq-arom), 53.24 (1C, CH2,isoind), 44.66 (4C, 4×CH ); MS (m/z):
2
-
+
2 6
11.2 ([(M-2Cl )/2] ); elemental analysis calcd. (%) for C26H26Cl N : C 63.29, H 5.31, N 17.03;
found C 63.65, H 5.01, N 16.76.
.1.1.11. 4-(N'-isopropylamidino)-N-[2-(4-N'-isopropylamidinophenyl)isoindolin-1-
4
ylidene]benzenamine dihydrochloride 8b
Following the general procedure B, from phthalaldehyde 1 (0.158 g, 1.2 mmol) and 4-N-
isopropylamidinoaniline hydrochloride 7b (0.50 g, 2.4 mmol) after refluxing for 7h and
recrystallization from ethanol 0.064 g (10%) of yellow powder was obtained; mp 275-276 °C; IR
-1
(
diamond) (
ν
/cm ): 3376-2935 (N–H ), 1654 (C=N), 1592 (C=Namidine), 1509 (ar C–C, C–N,
1
skeletal vib.); H NMR (300 MHz, DMSO-d ) (
δ
/ppm): 9.47 (bs, 6H, NHamidine), 8.22 (d, 2H, J =
6
8
.90 Hz, Harom), 7.86-7.78 (m, 4H, Harom), 7.69 (d, 1H, J = 7.58 Hz, Harom), 7.58 (t, 1H, J = 7.42
Hz, Harom), 7.21 (t, 1H, J = 7.89 Hz, Harom), 7.15 (d, 2H, J = 8.53 Hz, Harom), 6.74 (d, 1H, J = 8.02

ACCEPTED MANUSCRIPT
Hz, Harom), 5.21 (s, 2H, Hisoind), 4.13 (qv, 2H, J = 6.10 Hz, 2×CH), 1.30 (t, 12H, J = 5.85 Hz,
1
3
4
1
×CH ); C NMR (75 MHz, DMSO-d ) (
δ
/ppm): 161.75 (1C, Cq-amidine), 161.53 (1C, Cq-amidine),
3
6
55.05 (1C, Cq-arom), 153.09 (1C, C=Nimino), 145.37 (1C, Cq-arom), 141.56 (1C, Cq-arom), 132.02
(1C, CHarom), 130.43 (1C, Cq-arom), 130.35 (2C, CHarom), 129.60 (2C, CHarom), 127.94 (1C,
CHarom), 125.70 (1C, CHarom), 124.30 (1C, CHarom), 123.24 (1C, Cq-arom), 122.86 (1C, Cq-arom),
1
2
21.22 (2C, CHarom), 119.72 (2C, CHarom), 53.23 (1C, CH2,isoind), 45.45 (2C, 2×CH), 21.83 (2C,
-
+
×CH ), 21.79 (2C, 2×CH ); MS (m/z): 227.3 ([(M-2Cl )/2] ); elemental analysis calcd. (%) for
3
3
C H Cl N : C 63.99, H 6.52, N 15.99; found C 63.70, H 6.43, N 15.82.
2
8
34
2
6
4
2
.1.1.12. 5-(imidazolin-2-yl)-N-{2-[5-(imidazolin-2-yl)pyridin-2-yl]isoindolin-1-ylidene}pyridin-
-amine dihydrochloride 8c
Following the general procedure B, from phthalaldehyde 1 (0.042 g, 0.31 mmol) and 5-amino-2-
2-imidazolinyl)-pyridine hydrochloride 7c (0.126 g, 0.62 mmol) after refluxing for 24h and
recrystallization from ethanol 0.030 g (20%) of yellow powder was obtained; mp 270-272 °C; IR
(
-1
(
diamond) (
ν
/cm ): 3370-2940 (N–H), 1651 (C=N), 1608 (C=Namidine), 1567 (ar C–C, C–N,
1
skeletal vib.); H NMR (300 MHz, DMSO-d
6
) (
δ
/ppm): 10.78 (bs, 4H, NHamidine), 9.44 (d, 1H, J
=
2.34 Hz, Harom), 8.81 (dd, 1H, J = 8.83 Hz, J = 2.46 Hz, Harom), 8.55 (d, 1H, J = 2.27 Hz,
1 2
Harom), 8.51 (d, 1H, J = 8.81 Hz, Harom), 8.43 (d, 1H, J = 8.44 Hz, Harom), 7.78-7.72 (m, 2H,
arom), 7.64 (t, 1H, J = 7.43 Hz, Harom), 7.28 (t, 1H, J = 7.51 Hz, Harom), 6.72 (d, 1H, J = 8.06 Hz,
H
13
Harom), 5.36 (s, 2H, Hisoind), 4.04 (s, 4H, 2×CH ), 4.03 (s, 4H, 2×CH ); C NMR (75 MHz,
2
2
DMSO-d ) (δ/ppm): 163.56 (1C, Cq-amidine), 163.22 (1C, Cq-amidine), 154.60 (1C, C=Nimino), 150.31
6
(1C, Cq-arom), 143.50 (1C, CHarom), 141.87 (1C, Cq-arom), 141.72 (1C, CHarom), 141.31 (1C, Cq-
arom), 134.99 (1C, Cq-arom), 134.68 (1C, Cq-arom), 132.73 (1C, CHarom), 129.66 (1C, Cq-arom), 128.98
1C, CHarom), 128.43 (1C, CHarom), 127.11 (1C, CHarom), 126.28 (1C, CHarom), 125.55 (1C,
(
CHarom), 125.47 (1C, CHarom), 124.52 (1C, CHarom), 53.08 (1C, CH2,isoind), 45.00 (4C, 4×CH2);
+
MS (m/z): 212.1 ([(M-2Cl)/2] ); elemental analysis calcd. (%) for C H Cl N : C 58.19, H 4.88,
2
4
24
2
8
N 22.62; found C 58.31, H 4.48, N 22.44.
.1.1.13. 6-amidino-N-[2-(6-amidinopyridin-3-yl)isoindolin-1-ylidene]pyridin-3-amine
4
dihydrochloride 8d
Following the general procedure B, from phthalaldehyde 1 (0.039 g, 0.29 mmol) and 5-amino-2-
amidino-pyridine hydrochloride 7e (0.100 g, 0.58 mmol) after refluxing for 4h, stirring at the
room temperature for 24h and recrystallization from ethanol 0.054 g (41%) of orange powder was

ACCEPTED MANUSCRIPT
-1
obtained; mp 278-280 °C; IR (diamond) (
ν
/cm ): 3123 (N–H), 3041 (N–H), 1676 (C=Namidine),
1
1
633 (C=N), 1567 (ar C–C, C–N, skeletal vib.); H NMR (300 MHz, DMSO-d
6
) (
δ
/ppm): 9.58
(
bs, 4H, NHamidine), 9.48 (d, 1H, J = 1.98 Hz, Harom), 9.44 (bs, 4H, NHamidine), 8.73 (dd, 1H, J =
1
8
7
.83 Hz, J = 2.16 Hz, Harom), 8.54-8.51 (m, 2H, Harom), 8.46 (d, 1H, J = 8.49 Hz, Harom), 7.77-
2
.72 (m, 2H, Harom), 7.64 (t, 1H, J = 7.43 Hz, Harom), 7.29 (t, 1H, J = 7.73 Hz, Harom), 6.75 (d, 1H,
1
3
J = 7.91 Hz, Harom), 5.35 (s, 2H, Hisoind); C NMR (150 MHz, DMSO-d ) (δ/ppm): 162.13 (1C,
6
Cq-amidine), 161.83 (1C, Cq-amidine), 154.51 (1C, C=Nimino), 150.20 (1C, Cq-arom), 142.96 (1C,
CHarom), 141.87 (1C, Cq-arom), 141.48 (1C, CHarom), 141.26 (1C, Cq-arom), 137.86 (1C, Cq-arom),
1
37.69 (1C, Cq-arom), 132.64 (1C, CHarom), 129.72 (1C, Cq-arom), 129.09 (1C, CHarom), 128.45 (1C,
CHarom), 127.87 (1C, CHarom), 125.54 (1C, CHarom), 124.86 (1C, CHarom), 124.49 (1C, CHarom),
+
1
24.20 (1C, CHarom), 53.03 (1C, CH2,isoind); MS (m/z): 186.2 ([(M-2Cl)/2] ); elemental analysis
calcd. (%) for C H Cl N : C 54.18, H 4.55, N 25.28; found C 54.33, H 4.64, N 25.02.
2
0
20
2
8
4
.1.1.14. 5-(N'-isopropylamidino)-N-[2-(5-N'-isopropylamidinopyridin-2-yl)isoindolin-1-
ylidene]pyridin-2-amine dihydrochloride 8e and 2-N-[(5-N'-isopropylamidino)pyridin-2-
yl]isoindolin-1-one hydrochloride 9b
Following the general procedure C, from phthalaldehyde 1 (0.067 g, 0.50 mmol) and 2-amino-5-
(N-isopropyl)-amidinopyridine hydrochloride 7g (0.200 g, 1.00 mmol) after refluxing for 2h,
stirring at the room temperature for 24h and column chromatography two compounds were
obtained:
-1
8
e: 0.030 g (12%) of light yellow powder; mp 260-262 °C; IR (diamond) (ν/cm ): 3389-2927
1
(
N–H), 1666 (C=N), 1617 (C=Namidine), 1591 (ar C–C, C–N, skeletal vib.); H NMR (300 MHz,
DMSO-d ) (
δ
/ppm): 9.35 (bs, 6H, NHamidine), 8.88 (d, 1H, J = 8.98 Hz, Harom), 8.82, (d, 1H, J =
6
2
8
7
4
2
1
.13 Hz, Harom), 8.76 (d, 1H, J = 2.36 Hz, Harom), 8.21 (dd, 1H, J = 9.04 Hz, J = 2.50 Hz, Harom),
1
2
1 2
.16 (dd, 1H, J = 8.52 Hz, J = 2.50 Hz, Harom), 7.75 (d, 1H, J = 7.63 Hz, Harom), 7.62 (t, 1H, J =
.47 Hz, Harom), 7.26-7.21 (m, 2H, Harom), 6.53 (d, 1H, J = 7.96 Hz, Harom), 5.33 (s, 2H, Hisoind),
.06 (q, 2H, J = 6.23 Hz, 2×CH), 1.32 (d, 6H, J = 6.68 Hz, 2×CH ), 1.29 (d, 6H, J = 6.66 Hz,
3
13
×CH
3
); C NMR (75 MHz, DMSO-d
6
) (
δ
/ppm): 164.78 (1C, Cq-arom), 160.33 (1C, Cq-amidine),
60.00 (1C, Cq-amidine), 155.23 (1C, Cq-arom), 154.40 (1C, C=Nimino), 149.62 (1C, CHarom), 148.49
(1C, CHarom), 141.76 (1C, Cq-arom), 139.07 (1C, CHarom), 138.78 (1C, CHarom), 132.65 (1C,
CHarom), 129.98 (1C, Cq-arom), 128.16 (1C, CHarom), 125.80 (1C, CHarom), 124.63 (1C, CHarom),
1
20.52 (1C, Cq-arom), 120.25 (1C, Cq-arom), 115.77 (1C, CHarom), 113.20 (1C, CHarom), 52.76 (1C,

ACCEPTED MANUSCRIPT
3 3
CH2,isoind), 45.63 (2C, 2×CH), 21.80 (2C, 2×CH ), 21.77 (2C, 2×CH ); MS (m/z): 228.3 ([(M-
+
2
5
9
Cl)/2] ); elemental analysis calcd. (%) for C H Cl N : C 59.20, H 6.11, N 21.24; found C
2
6
32
2
8
9.03, H 6.02, N 21.32.
b: 0.113 g (68%) of beige powder; mp >300 °C; IR (diamond) (
-1
ν
/cm ): 3502 (N–H), 3332 (N–
H), 3155 (N–H), 2959 (N–H), 1680 (C=O), 1619 (C=Namidine), 1602 (ar C–C, C–N, skeletal vib.);
1
H NMR (300 MHz, DMSO-d ) (
δ
/ppm): 9.57 (bs, 2H, NHamidine), 8.81 (d, 1H, J = 2.03 Hz,
6
H
arom), 8.68, (d, 1H, J = 8.85 Hz, Harom), 8.26 (dd, 1H, J = 8.90 Hz, J = 2.52 Hz, Harom), 7.86 (d,
1
2
1
1
2
1
H, J = 7.58 Hz, Harom), 7.76 (s, 1H, Harom), 7.75 (s, 1H, Harom), 7.63-7.56 (m, 1H, Harom), 5.76 (s,
H, NHamidin), 5.16 (s, 2H, Hisoind), 4.12 (sep, 1H, J = 6.35 Hz, CH), 1.30 (d, 6H, J = 6.38 Hz,
13
×CH
3 6
); C NMR (75 MHz, DMSO-d ) (δ/ppm): 167.99 (1C, C=O), 160.02 (1C, Cq-amidine),
54.71 (1C, C=Nimino), 148.62 (1C, CHarom), 142.14 (1C, Cq-arom), 138.99 (1C, CHarom), 133.83
(1C, CHarom), 132.08 (1C, Cq-arom), 128.90 (1C, CHarom), 124.39 (1C, CHarom), 124.16 (1C,
CHarom), 121.16 (1C, Cq-arom), 112.48 (1C, CHarom), 50.30 (1C, CH2,isoind), 45.62 (1C, CH), 21.76
+
(
2C, 2×CH ); MS (m/z): 295.2 ([M-Cl] ); elemental analysis calcd. (%) for C H ClN O: C
3
17 19
4
6
4
1.72 H 5.79, N 16.94; found C 61.49, H 5.88, N 16.78.
.1.1.15. 2-N-(4-Amidinophenyl)isoindolin-1-one hydrochloride 9a
Following the general procedure B, from phthalaldehyde 1 (0.155 g, 1.1 mmol) and 4-
amidinoaniline hydrochloride 7i (0.312 g, 2.3 mmol) after refluxing for 6h and recrystallization
-1
from ethanol 0.025 g (9%) of white powder was obtained; mp >300 °C; IR (diamond) (ν/cm ):
1
3
306-3010 (N–H), 1680 (C=O), 1607 (C=Namidine), 1491 (ar C–C, skeletal vib.); H NMR (300
MHz, DMSO-d ) (
δ
/ppm): 9.36 (s, 2H, Hamidine), 9.12 (s, 2H, Hamidin), 8.16 (d, 2H, J = 9.03 Hz,
6
Harom), 7.97 (d, 2H, J = 9.01 Hz, Harom), 7.83 (d, 1H, J = 7.52 Hz, Harom), 7.74-7.69, (m, 2H,
1
3
Harom), 7.60-7.55 (m, 1H, Harom), 5.12 (s, 2H, Hisoind); C NMR (75 MHz, DMSO-d ) (
δ
/ppm):
67.79 (1C, C=O), 165.18 (1C, Cq-amidine), 144.61 (1C, C=Nimino), 141.62 (1C, Cq-arom), 133.41
1C, CHarom), 132.31 (1C, Cq-arom), 129.75 (2C, CHarom), 128.89 (1C, CHarom), 124.02 (1C,
CHarom), 123.92 (1C, CHarom), 122.82 (1C, Cq-arom), 118.88 (2C, CHarom), 50.87 (1C, CH2,isoind);
6
1
(
-
+
MS (m/z): 252.1 ([(M-Cl )] ); elemental analysis calcd. (%) for C H ClN O: C 62.61 H 4.90, N
1
5
14
3
1
4.60; found C 62.22 H 4.73, N 14.32.
.1.1.16. 2-N-(5-Amidinopyridin-2-yl)isoindolin-1-one hydrochloride 9c
4
Following the general procedure C, from phthalaldehyde 1 (0.041 g, 0.30 mmol) and 2-amino-5-
amidinopyridine hydrochloride 7h (0.100 g, 0.60 mmol) after refluxing for 24h and column

ACCEPTED MANUSCRIPT
-
chromatography 0.025 g (29%) of white powder was obtained; mp >300 °C; IR (diamond) (
ν
/cm
1
1
)
: 3382-2927 (N–H), 1720 (C=O), 1673 (C=Namidine), 1598 (ar C–C, C–N, skeletal vib.); H
NMR (300 MHz, DMSO-d ) (
δ
/ppm): 9.33 (bs, 4H, NHamidine), 8.91 (d, 1H, J = 2.24 Hz, Harom),
6
8
7
.70 (d, 1H, J = 8.95 Hz, Harom), 8.33 (dd, 1H, J = 8.94 Hz, J = 2.53 Hz, Harom), 7.87 (d, 1H, J =
1 2
.59 Hz, Harom), 7.76 (s, 1H, Harom), 7.75 (s, 1H, Harom), 7.63-7.56 (m, 1H, Harom), 5.17 (s, 2H,
1
3
Hisoind); C NMR (75 MHz, DMSO-d ) (
δ
/ppm): 168.05 (1C, C=O), 163.86 (1C, Cq-amidine),
55.18 (1C, C=Nimino), 148.76 (1C, CHarom), 142.18 (1C, Cq-arom), 138.73 (1C, CHarom), 133.90
1C, CHarom), 132.02 (1C, Cq-arom), 128.91 (1C, CHarom), 124.39 (1C, CHarom), 124.21 (1C,
6
1
(
CHarom), 119.92 (1C, Cq-arom), 112.62 (1C, CHarom), 50.31 (1C, CH2,isoind); MS (m/z): 253.1 ([M-
+
Cl] ); elemental analysis calcd. (%) for C14
H 4.59, N 19.55.
4
H13ClN O: C 58.24 H 4.54, N 19.40; found C 58.38,
1
4
.1.2. Synthesis of N -[2-(4-aminophenyl)isoindolin-1-ylidene]benzene-1,4-diamine 4a
A suspension of 3d (1.00 g, 2.5 mmol) in 2M H SO (20 ml) was refluxed for 2h. After cooling
2
4
the reaction mixture was neutralized with NaOH and product was extracted with dichlormethan.
The solvent was removed under reduced pressure and 0.55 g (70%) of brown powder was
-1
obtained; mp 202-204 °C; IR (diamond) (ν/cm ): 3420-3028 (NH ), 1626 (C=N), 1506 (ar C–C,
2
1
skeletal vib.); H NMR (300 MHz, DMSO-d ) (
δ
/ppm): 7.58 (d, 2H, J = 8.67 Hz, Harom), 7.53 (d,
6
1
1
4
H, J = 7.61 Hz, Harom), 7.41 (t, 1H, J = 7.40 Hz, Harom), 7.11 (t, 1H, J = 7.57 Hz, Harom), 6.68 (d,
H, J = 7.90 Hz, Harom), 6.60 (d, 2H, J = 8.65 Hz, Harom), 6.55 (s, 4H, Harom), 4.91 (s, 2H, NH2),
13
.88 (s, 2H, Hisoind), 4.69 (s, 2H, NH ); C NMR (150 MHz, DMSO-d ) (δ/ppm): 153.41 (1C,
2
6
C=Nimino), 144.74 (1C, Cq-arom), 143.35 (1C, Cq-arom), 141.00 (1C, Cq-arom), 140.47 (1C, Cq-arom),
31.00 (1C, Cq-arom), 130.74 (1C, Cq-arom), 129.82 (1C, CHarom), 126.68 (1C, CHarom), 125.30 (1C,
1
CHarom), 123.16 (1C, CHarom), 122.45 (2C, CHarom), 121.71 (1C, CHarom), 121.37 (1C, CHarom),
+
1
14.94 (2C, CHarom), 113.74 (2C, CHarom), 53.00 (1C, CH2,isoind); MS (m/z): 315.2 ([M+1] );
elemental analysis calcd. (%) for C H N : C 76.41, H 5.77, N 17.82; found C 76.15, H 5.45, N
2
0
18
4
1
7.53.
4
4
.1.3. Synthesis of 6-amino-N-[2-(6-aminopyridin-3-yl)isoindolin-1-ylidene]pyridin-3-amine
b

ACCEPTED MANUSCRIPT
2 4
A suspension of 3i (0.10 g, 0.25 mmol) in 2M H SO (5 ml) was refluxed for 2h. After cooling
the reaction mixture was neutralized with NaOH and product was extracted with dichlorethan.
The solvent was removed under reduced pressure and 0.04 g (57%) of brown powder was
-1
obtained; mp 228-231 °C; IR (diamond) (
ν
/cm ): 3439 (NH ), 3288 (NH ), 3155 (NH ), 1623
2
2
2
1
(
C=N), 1502 (ar C–C, C–N, skeletal vib.); H NMR (300 MHz, DMSO-d ) (
δ
/ppm): 8.33 (s, 1H,
6
Harom), 7.95 (d, 1H, J = 8.74 Hz, Harom), 7.58 (d, 1H, J = 7.56 Hz, Harom), 7.50 (s, 1H, Harom), 7.46
(d, 1H, J = 7.45 Hz, Harom), 7.20 (t, 1H, J = 7.52 Hz, Harom), 6.99 (d, 1H, J = 8.50 Hz, Harom), 6.79
(d, 1H, J = 7.86 Hz, Harom), 6.50 (d, 1H, J = 8.63 Hz, Harom), 6.46 (d, 1H, J = 8.37 Hz, Harom),
1
3
5
.82 (s, 2H, NH ), 5.52 (s, 2H, NH ), 4.94 (s, 2H, Hisoind); C NMR (75 MHz, DMSO-d6)
2 2
(
δ
/ppm): 156.91 (1C, Cq-arom), 155.85 (1C, Cq-arom), 154.91 (1C, C=Nimino), 142.07 (1C, CHarom),
1
41.88 (1C, Cq-arom), 139.40 (1C, CHarom), 137.31 (1C, Cq-arom), 132.61 (1C, CHarom), 131.42 (1C,
CHarom), 130.96 (1C, Cq-arom), 130.78 (1C, CHarom), 128.21 (1C, Cq-arom), 127.48 (1C, CHarom),
1
25.45 (1C, CHarom), 124.00 (1C, CHarom), 108.69 (1C, CHarom), 107.80 (1C, CHarom), 53.45 (1C,
+
CH2,isoind); MS (m/z): 317.2 ([M+1] ); elemental analysis calcd. (%) for C H N : C 68.34, H
18
16
6
5
.10, N 26.56; found C 68.71, H 5.25, N 26.23.
1
4
.1.4. Synthesis of N -[2-(4-aminophenyl)isoindolin-1-ylidene]benzene-1,4-diamine
dihydrochloride 5
A stirred suspension of compounds 4a (0.05 g, 0.2 mmol) in absolute ethanol was cooled to 0°C
and saturated with HCl(g). After stirring for 3h at room temperature the solvent was removed
under reduced pressure to obtain 0.08 g (95%) gray powder; mp 276-278 °C; IR (diamond)
-1
+
+
1
(ν
/cm ): 2934-2719 (NH ), 2554 (NH ), 1638 (C=N), 1509 (ar C–C, skeletal vib.); H NMR
3 3
(
300 MHz, DMSO-d ) (
δ
/ppm): 7.85 (s, 2H, Harom), 7.69 (s, 1H, Harom), 7.45 (s, 1H, Harom), 7.35
6
(bs, 2H, Harom), 7.28 (s, 1H, Harom), 7.19 (bs, 2H, Harom), 7.06 (s, 3H, Harom), 5.38 (s, 2H, Hisoind);
1
3
6
C NMR (75 MHz, DMSO-d ) (δ/ppm): 153.22 (1C, C=Nimino), 144.35 (1C, Cq-arom), 135.27 (2C,
CHarom), 132.40 (1C, Cq-arom), 132.31 (1C, Cq-arom), 132.02 (1C, Cq-arom), 131.97 (1C, Cq-arom),
31.67 (1C, Cq-arom), 129.05 (2C, CHarom), 128.29 (1C, CHarom), 125.31 (1C, CHarom), 124.65 (2C,
1
CHarom), 124.12 (2C, CHarom), 123.96 (2C, CHarom), 53.28 (1C, CH2,isoind); MS (m/z): 316.2 ([M-
+
2
Cl] ); elemental analysis calcd. (%) for C H Cl N : C 62.02, H 5.20, N 14.47; found C 61.80,
2
0
20
2
4
H 5.35, N 14.31.

ACCEPTED MANUSCRIPT
4
.1.5. General method for the synthesis of amidino substituted amines 7a-h
A suspension of cyano substituted amines in absolute ethanol was cooled to 0°C and saturated
with dry HCl gas. The suspension was stirred at room temperature until IR spectra indicated the
lack of the cyano peak (3-5ds). The imidoester hydrochloride intermediates were then
precipitated from the solution by addition of dry diethylether, filtered off, washed with dry
diethylether and dried under reduced pressure. The crude product was suspended in absolute
ethanol and the appropriate amine (3× equimolar) was added. Imidazolinyl amidines: the reaction
mixture was refluxed for 24h and after cooling the product was filtered off and washed with dry
diethylether. Isopropyl amidines: the reaction mixture was stirred at room temperature for 24h,
dry diethylether was added and the product was filtered off and washed with dry diethylether.
Unsubstituted amidines: the suspension was treated with anhydrous ammonia gas and stirred for
2
4h at room temperature. The product was then filtered off and washed with dry diethylether.
4
.1.5.1. 4-(Imidazolin-2-yl)aniline hydrochloride 7a
From 4-cyanoaniline 6a (0.90 g, 7.6 mmol) and ethylenediamine (1.52 ml, 22.8 mmol) 1.50 g
97%) of white powder was obtained; mp >300 °C (mp >300 °C) [32].
(
lit
4
.1.5.2. 4-N-isopropylamidinoaniline hydrochloride 7b
From 4-cyanoaniline 6a (0.90 g, 7.6 mmol) and isopropylamine (1.87 ml, 22.8 mmol) 1.55 g
93%) of white powder was obtained; mp >300 °C (mplit >300 °C) [32].
.1.5.3. 5-Amino-2-(imidazolin-2-yl)pyridine hydrochloride 7c
(
4
From 5-amino-2-cyanopyridine 6b (0.60 g, 5.0 mmol) and ethylenediamine (1.00 ml, 15.0 mmol)
after reprecipitation from ethanol and diethylether 0.32 g (32%) of white powder was obtained;
-1
mp 255-257 °C; IR (diamond) (
ν
/cm ): 2959-2687 (NH , NH
), 2049 (NH , NHamidine), 1600
amidine 2
2
1
(
C=N), 1500 (ar C–C, C–N, skeletal vib.); H NMR (300 MHz, DMSO-d ) (
δ
/ppm): 9.03 (bs,
6
2
8
H, NHamidine), 8.11 (d, 1H, J = 2.55 Hz, H ), 8.04 (d, 1H, J = 8.70 Hz, H ), 7.03 (dd, 1H, J =
py py 1
1
3
.71 Hz, J = 2.65 Hz, H ), 6.74 (s, 2H, NH ), 3.91 (s, 4H, 2×CH ); C NMR (75 MHz, DMSO-
2
py
2
2
d6) (
δ/ppm): 162.35 (1C, Cq-amidine), 151.72 (1C, Cq-arom), 141.75 (1C, CHarom), 137.54 (1C, Cq-
arom), 124.32 (1C, CHarom), 118.52 (1C, CHarom), 44.54 (2C, 2×CH ); MS (m/z): 164.1 ([(M-
2
+
Cl)+1] ).
4
.1.5.4. 5-Amino-2-(N-isopropylamidino)pyridine hydrochloride 7d
From 5-amino-2-cyanopyridine 6b (0.60 g, 5.0 mmol) and isopropylamine (0.64 ml, 15.0 mmol)
after reprecipitation from ethanol and diethylether 0.39 g (36%) of white powder was obtained;

ACCEPTED MANUSCRIPT
-1
mp 251-253 °C; IR (diamond) (
ν
/cm ): 3379-2877 (NH , NHamidine), 1624 (C=N), 1580 (ar C–C,
2
1
C–N, skeletal vib.); H NMR (300 MHz, DMSO-d
6
) (δ/ppm): 9.08 (bs, 3H, NHamidine), 8.10 (d,
1
H, J = 2.55 Hz, H ), 8.07 (d, 1H, J = 8.78 Hz, H ), 7.04 (dd, 1H, J = 8.74 Hz, J = 2.64 Hz,
py py 1 2
13
H ), 6.54 (s, 2H, NH ), 4.12 (quint, 1H, J = 6.23 Hz, CH), 1.27 (d, 6H, J = 6.37 Hz, 2×CH ); C
py
2
3
NMR (75 MHz, DMSO-d
6
) (δ
/ppm): 157.83 (1C, Cq-amidine), 149.49 (1C, Cq-arom), 136.17 (1C,
CHarom), 130.30 (1C, Cq-arom), 124.88 (1C, CHarom), 118.65 (1C, CHarom), 44.83 (1C, CH), 21.83
+
(
2C, 2×CH ); MS (m/z): 180.1 ([(M-Cl)+1] ).
3
4
.1.5.5. 2-Amidino-5-aminopyridine hydrochloride 7e
From 5-amino-2-cyanopyridine 6b (0.60 g, 5.0 mmol) after reprecipitation from ethanol and
-
1
diethylether 0.24 g (33%) of white powder was obtained; mp 259-262 °C; IR (diamond) (
359-2898 (NH
NMR (300 MHz, DMSO-d ) (
ν
/cm ):
1
3
2
, NHamidine), 1682 (C=N), 1656, 1637, 1580 (ar C–C, C–N, skeletal vib.); H
δ
/ppm): 8.10 (d, 1H, J = 2.36 Hz, H ), 8.08 (d, 1H, J = 8.67 Hz,
py
6
H ), 7.48 (bs, 4H, NH
py
), 7.04 (dd, 1H, J = 8.73 Hz, J = 2.64 Hz, H ), 6.65 (s, 2H, NH );
1 2 py 2
amidine
1
3
C NMR (75 MHz, DMSO-d ) (
δ
/ppm): 162.20 (1C, Cq-amidine), 149.82 (1C, Cq-arom), 136.50 (1C,
6
CHarom), 129.45 (1C, Cq-arom), 125.19 (1C, CHarom), 118.39 (1C, CHarom); MS (m/z): 138.1 ([(M-
+
Cl)+1] ).
4
.1.5.6. 2-Amino-5-(imidazolin-2-yl)pyridine hydrochloride 7f
From 2-amino-5-cyanopyridine 6c (1.00 g, 8.3 mmol) and ethylenediamine (1.66 ml, 24.9 mmol)
after reprecipitation from ethanol and diethylether 0.51 g (32%) of white powder was obtained;
-1
mp 256-258 °C; IR (diamond) (
ν
/cm ): 3484-2741 (NH , NHamidine), 1680 (C=N), 1618 (NH2),
2
1
1
2
8
589 (ar C–C, C–N, skeletal vib.); H NMR (300 MHz, DMSO-d
6
) (
δ
/ppm): 8.63 (d, 1H, J =
.28 Hz, H ), 7.99 (dd, 1H, J = 8.89 Hz, J = 2.42 Hz, H ), 7.15 (s, 2H, NH ), 6.54 (d, 1H, J =
py
1
2
py
2
13
.91 Hz, H ), 3.85 (s, 4H, 2×CH ); C NMR (75 MHz, DMSO-d ) (
δ
/ppm): 163.63 (1C, Cq-
py
2
6
amidine), 163.32 (1C, Cq-arom), 151.14 (1C, CHarom), 136.95 (1C, CHarom), 107.81 (1C, CHarom),
+
1
06.85 (1C, Cq-arom), 44.73 (2C, 2×CH
2
); MS (m/z): 164.1 ([(M-Cl)+1] ).
4
.1.5.7. 2-Amino-5-(N-isopropylamidino)pyridine hydrochloride 7g
From 2-amino-5-cyanopyridine 6c (1.00 g, 8.3 mmol) and isopropylamine (2.04 ml, 24.9 mmol)
after reprecipitation from ethanol and diethylether 0.52 g (30%) of white powder was obtained;
-1
mp 232-238 °C; IR (diamond) (
ν
/cm ): 3363-2848 (NH , NHamidine), 1680 (C=N), 1637, 1604 (ar
2
1
C–C, C–N, skeletal vib.), 1573 (ar C–C, C–N, skeletal vib.); H NMR (300 MHz, DMSO-d )
6
(δ/ppm): 9.23 (bs, 1H, NHamidine), 9.16 (bs, 1H, NHamidine), 8.80 (bs, 1H, NHamidin), 8.35 (d, 1H, J

ACCEPTED MANUSCRIPT
=
=
2.32 Hz, Hpy), 7.74 (dd, 1H, J
1 2 2
= 8.83 Hz, J = 2.49 Hz, Hpy), 6.97 (s, 2H, NH ), 6.52 (d, 1H, J
1
3
8.85 Hz, H ), 4.04 (bs, 1H, CH), 1.25 (d, 6H, J = 6.35 Hz, 2×CH ); C NMR (75 MHz,
py
3
DMSO-d ) (
δ
/ppm): 163.15 (1C, Cq-arom), 160.45(1C, Cq-amidine), 149.86 (1C, CHarom), 137.21 (1C,
6
3
CHarom), 112.47 (1C, Cq-arom), 107.33 (1C, CHarom), 45.11 (1C, CH), 21.89 (2C, 2×CH ); MS
+
(
m/z): 180.1 ([(M-Cl)+1] ).
4
.1.5.8. 5-Amidino-2-aminopyridine hydrochloride 7h
From 2-amino-5-cyanopyridine 6c (1.00 g, 8.3 mmol) after reprecipitation from ethanol and
-1
diethylether 0.49 g (35%) of white powder was obtained; mp 243-245 °C; IR (diamond) (
ν/cm ):
3
335-2886 (NH , NHamidine), 1664 (C=N), 1634, 1604 (ar C–C, C–N, skeletal vib.), 1575 (ar C–C,
2
1
C–N, skeletal vib.); H NMR (300 MHz, DMSO-d
6
) (δ/ppm): 8.85 (s, 4H, NHamidine), 8.50 (d, 1H,
J = 2.43 Hz, H ), 7.85 (dd, 1H, J = 8.91 Hz, J = 2.55 Hz, H ), 7.09 (s, 2H, NH ), 6.53 (d, 1H,
py
1
2
py
2
1
3
J = 8.91 Hz, H ); C NMR (75 MHz, DMSO-d ) (δ/ppm): 164.11 (1C, Cq-arom), 163.48 (1C, Cq-
py
6
amidine), 150.35 (1C, CHarom), 136.80 (1C, CHarom), 110.89 (1C, Cq-arom), 107.56 (1C, CHarom); MS
+
(
m/z): 138.1 ([(M-Cl)+1] ).
4
.2. Cell culturing
The cell lines HeLa (cervical carcinoma), SW620 (colorectal adenocarcinoma, metastatic),
HEpG2 (hepatocellular carcinoma), MCF-7 (breast epithelial adenocarcinoma, metastatic), WI38
(transformed human lung fibroblasts) and BJ (normal diploid human fibroblasts), were cultured
as monolayers and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 U/ml penicillin and 100 ꢀg/ml
2
streptomycin in a humidified atmosphere with 5% CO at 37 °C.
4
.3. Proliferation assays
The panel cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0,
at 5000 per well. Test agents were freshly added after 24h in five, serial dilutions (0,01 to 100
µM) and incubated for further 72 hours. Working dilutions were freshly prepared on the day of
testing in the growth medium. The solvent (DMSO) was also tested for eventual inhibitory
activity by adjusting its concentration to be the same as in the working concentrations (DMSO
concentration never exceeded 0.1%). After 72 hours of incubation, the cell growth rate was

ACCEPTED MANUSCRIPT
evaluated by performing the MTT assay: experimentally determined absorbance values were
transformed into a cell percentage growth (PG) using the formulas proposed by NIH and
described previously [33]. This method directly relies on comparsion bewteen the number of
control cells at the day of assay with the growth of treated cells or untreated cells in control wells
on the same plate at the assay end point − the results are therefore a percentile difference from the
calculated expected value.
The IC values for each compound were calculated from dose-response curves using linear
5
0
regression analysis by fitting the mean test concentrations that give PG values above and below
the reference value. If, however, all of the tested concentrations produce PGs exceeding the
respective reference level of effect (e.g. PG value of 50) for a given cell line, the highest tested
concentration is assigned as the default value (in the screening data report that default value is
preceded by a ">" sign). Each test point was performed in quadruplicate in three individual
experiments. The results were statistically analyzed (ANOVA, Tukey post-hoc test at p < 0.05).
Finally, the effects of the tested substances were evaluated by plotting the mean percentage
growth for each cell type in comparison to control on dose response graphs.
4
.4. Cell cycle analysis
A total of 25000 cells were seeded in 6-well plates (Falcon, Germany). After 24 h, HepG2 were
treated with compounds 3a, 3b at concentrations 10 µM and 25 µM and with compound 3g at
concentrations 20 µM 50 µM while SW620 cells were treated with compound 8c at
concentrations 20 µM and 50 µM. After 24 and 48 h, the attached cells were trypsinized,
combined with floating cells, washed with PBS, and fixed with 70% ethanol. Immediately before
the analysis, the cells were washed again with PBS and stained with 1 ꢀg/mL of propidium iodide
(PI) with the addition of 0.2 ꢀg/mL of RNase A. The stained cells were then analysed with
Becton Dickinson FACScalibur flow cytometer (10 000 counts were measured). Each test point
was performed in duplicate. The percentage of the cells in each cell cycle phase was based on the
obtained DNA histograms and determined by using the FCSExpress 4 Flow Research Edition
(De Novo Software). Statistical analysis was performed in Microsoft Excel by using the ANOVA
at p < 0.05.
4
.5. Detection of apoptosis

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Annexin V-Fluos staining kit (Roche) assay was used to assess apoptosis induction according to
the manufacturer’s recommendations. The HepG2 and SW620 cells were seeded on chamber
slides (4000 cells/well, Lab-Tk II Chamber slide, Nunc, SAD) and treated with compounds 3a,
3
b at concentration 25 µM and with compound 3g at concentration 50 µM while SW620 cells
were treated with compound 8c at concentration 50 µM for 24 h. Upon treatment, the growth
medium was removed from wells. Attached cells were covered with 100 ꢀL/well of incubation
buffer, containing Annexin V-Fluos labelling reagent and propidium iodide (PI) 15 min. The
cells were then washed with PBS and analysed under the fluorescent microscope.
4
.6. DNA melting temperature studies.
The absorbency of DNA was measured at 260 nm in quartz cells using an Uvikon XL
spectrophotometer thermostated with a PTC-424S/L Peltier type cell changer (Jasco) cryostat
every min over a range of 20 to 100 °C with an increment of 1 °C per min. Tm values
correespond to the termperature at the midpoint of the hyperchromic transition deduced from
first-derivative plots. The variation of melting temperature (∆Tm) was obtained by substracting
the melting temperature measurement of CT-DNA (control Tm) to that obtained in the presence
of the various tested compounds at drug/base pair ratio (R) 0.25 or 0.5 in 1 mL BPE buffer as
described [34].
4
.7. Circular dichroism
The various drugs (50 µM) were incubated in BPE in the abscence (control) or presence of
increasing concentrations of CT-DNA specified in the figure legend. The CD spectra were
collected in a quartz cell of 10 mm path length from 480 to 230 nm using a J-810 Jasco
spectropolarimeter at a controled temperature of 20 °C fixed by a PTC-424S/L peltier type cell
changer (Jasco) as described previously [34].
4
.8. DNase I footprinting
DNase I footprinting experiments were conducted essentially as previously described [35] using a
65 bp DNA fragment obtained from EcoRI and PvuII double digestion of the pBluscript plasmid
Stratagene, La Jolla, CA) followed by 3’-end labeled at the EcoRI site upon incorporation of α-
2
(
[
3
2
P]-dATP (3000 Ci/mmol, PerkinElmer, France). This 265-bp radio-labeled DNA fragment was

ACCEPTED MANUSCRIPT
incubated with increasing concentrations of the tested compounds (15 min, 37 °C) prior to
controled digestion with DNase I. The reaction was stopped by ethanol precipitation, drying
under vaccum and subsequent dissolution of the DNA pellet in 4 µL of denaturing loading buffer
(80% formamide solution containing tracking dyes). Heated DNA samples (90 °C for 4 min),
were chilled on ice prior to be loaded on a 8% denaturing polyacrylamide gels containing 8 M
urea. After electrophoretic migration, gels were soaked in 10% acetic acid and dried on Whatman
3
MM paper under vacuum. After an exposition of the gels to storage screen for the appropriated
delay at room temperature, the results were collected using a Personal Molecular Imager (PMI
,
BioRad). Each base position was localized from comparison with a G-track obtained using
dimethyl-sulfate (DMS) and piperidine treatment of the same DNA fragment.
4
.9. Topoisomerase I-mediated DNA relaxation and cleavage assay
Topoisomerase I-mediated DNA relaxation experiment was performed as previously described
36] using graded concentrations of the tested compounds incubated with supercoiled pUC19
[
plasmid DNA and human topoisomerase I (Topogen, USA). The reactions were stopped upon
addition of SDS and proteinase K. The various DNA forms were separated on a 1% agarose
gel electrophoresis (2 h at 120 V) in TBE buffer. Gels were stained post-migration in a bath
containing ethidium bromide, washed and photographed under UV light. Camptothecin (CPT)
was used as a positive control for topoisomerase I poisoning effect.
5
. Acknowledgements
We greatly appreciate the financial support of the Croatian Ministry of Science Education and
Sports (projects 125-0982464-1356 and 335-0000000-3532). M. H. David-Cordonnier is grateful
to the Ligue Nationale Contre le Cancer (Comité du Nord, Septentrion) and the Association pour
la Recherche sur le Cancer for grants; the Institut pour la Recherche sur le Cancer de Lille
(
IRCL), the CHRU de Lille and the Région Nord/Pas-de-Calais for a post-doctoral fellowship to
Raja Nhili and the IRCL for technical expertise (Sabine Depauw). ). The IMPRT-IFR114 in Lille
is acknowledged for giving access to the Personal Molecular Imager (PMI) equipment.