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

DOI:

10.1016/j.ejmech.2014.09.079

Reference:

EJMECH 7386

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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

Activity and DNA binding features

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Novel Phenyl and Pyridyl Substituted Derivatives of Isoindolines: Synthesis, Antitumor

Irena Sovića, Sandra Kraljević Pavelićb, Elitza Markova-Carb, Nataša Ilićb,

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Novel Phenyl- and PyridylSubstituted Derivatives of Isoindolines: Synthesis , Antitumor Activity and DNA binding features

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Raja Nhilic, Sabine Depauwc, Marie-Hélène David-Cordonnierc and Grace Karminski-Zamolaa *

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Novel Phenyl and Pyridyl Substituted Derivatives of Isoindolines: Synthesis, Antitumor Activity and DNA binding features Irena Sovića, Sandra Kraljević Pavelićb, Elitza Markova-Carb, Nataša Ilićb,

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Raja Nhilic, Sabine Depauwc, Marie-Hélène David-Cordonnierc and Grace Karminski-Zamolaa *

[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

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[b] Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, 51000 Rijeka, Croatia,

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[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.

mail: [email protected]

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Abstract

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Box 177, HR-10000 Zagreb, Croatia, Phone No. ++38514597215; Fax No. ++38514597250; e-

Novel phenyl-substituted (3a-3d, 4a, 5, 8a, 8b and 9a) and pyridyl-substituted (3e-3i, 4b,

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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 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

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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

Key

words:

heterocyclic

compounds,

phenyl

and

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sequence selective binding and without DNA intercalation profile.

pyridyl

substituted

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antiproliferative activity, DNA binding, intercalator, sequence-selective binding.

isoindolines,

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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.

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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

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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

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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 IC50 ranging from 0.1 to 1.0 µM in vivo [11]. Some 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

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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

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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

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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

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clinically used HDAC inhibitor suberoylanilide hydroxamic acid (Zolinza(TM)) [21]. Another series of isoindoline derivatives, isoindoline-based hydroxamates was described and several analogues were shown to inhibit HDAC1 with IC50 values in the low nanomolar range and inhibit cellular proliferation of HCT116 human colon cancer cells in the sub-micromolar range. The

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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-

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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.

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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 µM) in inhibiting A549 and HepG2 cell proliferation with IC50 values of 6.76 and 9.44

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µM, 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

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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 1imino-(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. 1.

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Fig. 2.

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(Fig 2) were prepared depending on different reaction conditions.

2. Results and Discussion

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2.1. Chemistry

Prepared compounds were synthesized according to procedures shown in Schemes 1 and 2 by conventional and modified methods of organic synthesis used in preparation of similar heterocyclic compounds from phthalaldehyde and substituted aromatic and heteroaromatic

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amines.

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Scheme 1.

Scheme 2.

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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

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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-

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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

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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. Compound structures were confirmed by 1H and 13C NMR analysis. The formation of the isoindoline ring was confirmed by two-proton singlet at 5.36−4.94 ppm in the 1H 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

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isoindolinone (9a-c) in the 13C NMR spectra. The presence of imino group was supported by the chemical shift at 155.18−153.41 ppm in the

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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

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the 1H NMR spectra and chemical shift of quaternary amidino carbon atom at 168.05−160.02 ppm in the 13C NMR spectra. Compound structures were also analysed by IR spectroscopy. The

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mean feature of IR spectra for isoindoline compounds 3a-i and 8a-e is C=N stretching band of imino group at 1666−1631 cm-1 and for isoinolinone compounds 9a-c is C=O stretching band of carbonyl group at 1720−1680 cm-1. 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 (IC50 ranging from 4,68 – 9,65 µM) were observed

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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 phenylsubstituted isoindoline 3b, 3c, 4a, 5 and the isoindoline with pyridine moiety 3e showed high

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potency on the growth of MCF-7 cells.

Due to selective and strongest effects in comparison with other tested compounds

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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

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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

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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

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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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Table 1.

Table 2.

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2.3. DNA binding properties.

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Fig. 3.

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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

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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

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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).

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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 CTDNA.

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 nonbinding 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

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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 CTDNA. 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

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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

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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.

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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

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topoisomerase I-induced relaxation profile which is in agreement with the ∆Tm measurements

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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

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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

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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

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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

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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.

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3. Conclusion

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Fig. 6.

Novel phenyl-substituted (3a-3d, 4a, 5, 8a, 8b and 9a) and pyridyl-substituted (3e-3i, 4b,

8c-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-2yl)-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 phenylsubstituted isoindoline 8a. Selective effects at micromolar concentrations were observed as well, including good selectivity of 1-imino-(substituted-phenyl)-2-N-(substituted-phenyl) isoindolines

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3a, 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

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compounds 3b and 3g induced apoptosis.

Indeed, DNA binding properties evidenced that compounds 8a, 8c and 8d binds to DNA

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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 (IC50 of 8.1, 0.70, 16, 0.05, 0.73 µM, respectively) through another mechanism, including apoptosis induction. At last, the phenyl-substituted

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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

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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

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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

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spectra were recorded on a Bruker Vertex 70 spectrophotometer with diamond crystal. 1H and 13C NMR spectra were recorded on Varian Gemini 300 or Varian Gemini 600 spectrophotometers at 300, 600, 150 and 75 MHz, respectively. All NMR spectra were measured in DMSO-d6 solutions using TMS as an internal standard. Chemical shifts are reported in ppm (δ) relative to TMS. Mass

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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

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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-

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(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

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product was filtered off and recrystallized from corresponding solvent.

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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).

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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 2a (0.94 g, 8 mmol) after stirring for 2 h and recrystallization from ethanol/toluene 0.69 g (54%) of pale yellow powder was obtained; mp 243-244 °C; IR (diamond) (ν/cm-1): 2211 (C≡N), 1653

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(C=N), 1589 (ar C−C, skeletal vib.), 1509 (ar C−C, skeletal vib.); 1H NMR (300 MHz, DMSOd6) (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 =

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8.52 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 = 7.44 Harom), 7.10 (d, 2H, J = 8.53 Hz, Harom), 6.72 (d, 1H, J = 7.96 Hz, Harom), 5.15 (s, 2H, Hisoind); C NMR (75 MHz, DMSO-d6) (δ/ppm): 154.56 (1C, C=Nimino), 153.23 (1C, Cq-arom), 145.17 (1C,

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Cq-arom), 141.44 (1C, Cq-arom), 134.02 (2C, CHarom), 133.35 (2C, CHarom), 132.00 (1C, CHarom), 130.34 (1C, Cq-arom), 128.07 (1C, CHarom), 125.62 (1C, CHarom), 124.16 (1C, CHarom), 122.12 (2C, CHarom), 120.36 (2C, CHarom), 119.85 (1C, C≡N), 119.45 (1C, C≡N), 105.29 (1C, Cq-arom), 105.01

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(1C, Cq-arom), 53.29 (1C, CH2,isoind); MS (m/z): 335.3 ([M+1]+); elemental analysis calcd. (%) for C22H14N4: C 79.02, H 4.22, N 16.76; found C 78.73, H 4.35, N 16.22. 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

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2b (1.11 g, 8 mmol) after stirring for 24 h and recrystallization from DMF 1.21 g (81%) of yellow powder was obtained; mp 208-210 °C; IR (diamond) (ν/cm-1): 1655 (C=N), 1582 (NO2 st

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as), 1321 (NO2 st sy); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 8.25 (s, 4H, Harom), 8.22 (d, 2H, J = 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 C20H14N4O4: C 64.17, H 3.77, N 14.97; found C 63.85, H 3.92, N 14.32. 4.1.1.3. 4-(N',N'-dimethylamino)-N-{2-[4-(N',N'-dimethylamino)phenyl]isoindolin-1ylidene}benzenamine 3c Following the general procedure A, from phthalaldehyde 1 (0.33 g, 2.5 mmol) and 4-(N,Ndimethyl)-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) (ν/cm-1): 2897 (CH3 st), 2806 (CH3 st), 1631 (C=N), 1509 (ar C−C, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (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

6H, 2×CH3); arom),

RI PT

(s, 4H, Harom), 6.66 (d, 1H, J = 7.90 Hz, Harom), 4.94 (s, 2H, Hisoind), 2.88 (s, 6H, 2×CH3), 2.87 (s, C NMR (75 MHz, DMSO-d6) (δ/ppm): 153.66 (1C, C=Nimino), 150.26 (1C, Cq-

13

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,

SC

CHarom), 123.74 (1C, CHarom), 122.48 (1C, CHarom), 121.81 (1C, CHarom), 120.84 (1C, CHarom), 114.19 (1C, CHarom), 113.60 (1C, CHarom), 113.12 (1C, CHarom), 112.48 (1C, CHarom), 53.23 (1C,

M AN U

CH2,isoind), 41.37 (2C, 2×CH3), 41.01 (2C, 2×CH3); MS (m/z): 371.3 ([M+1]+); elemental analysis calcd. (%) for C24H26N4: C 77.80, H 7.07, N 15.12; found C 77.23, H 6.65, N 14.96. 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 4acetamidoaniline 2d (1.5 g, 10.0 mmol) after stirring for 24 h and recrystallization from DMF 1.22 g (62%) of pink powder was obtained; mp 228-230 °C; IR (diamond) (ν/cm-1): 3298-3040

TE D

(N–H), 1658 (C=O), 1634 (C=N), 1593 (ar C–C, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (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 = 7.80 Hz, Harom), 6.62 (d, 1H, J = 6.92 Hz, Harom), 5.02 (s, 2H, Hisoind), 2.05 (s, 3H, CH3), 2.04 (s, 13

C NMR (75 MHz, DMSO-d6) (δ/ppm): 168.42 (1C, C=O), 168.23 (1C, C=O),

EP

3H, CH3);

153.43 (1C, C=Nimino), 146.28 (1C, Cq-arom), 141.38 (1C, Cq-arom), 137.06 (1C, Cq-arom), 135.05

AC C

(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), 120.46 (1C, CHarom), 119.70 (2C, CHarom), 53.01 (1C, CH2,isoind), 24.41 (1C, CH3), 24.38 (1C, CH3); MS (m/z): 399.2 ([M+1]+); elemental analysis calcd. (%) for C24H22N4O2: C 72.34, H 5.57, 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-4methylpyridine 2e (0.86 g, 8.0 mmol) after stirring for 5 days and recrystallization from ethanol 0.47 g (37%) of red powder was obtained; mp 174-177 °C; IR (diamond) (ν/cm-1): 3045, 3010,

ACCEPTED MANUSCRIPT

2921 (ar C–H), 1653 (C=N), 1593 (ar C–C, C–N, skeletal vib.), 1544 (ar C–C, C–N, skeletal vib.); 1H NMR (600 MHz, DMSO-d6) (δ/ppm): 8.66 (s, 1H, Harom), 8.30 (d, 1H, J = 5.02 Hz, 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, Harom), 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

RI PT

(d, 1H, J = 7.37 Hz, Harom), 5.20 (s, 2H, Hisoind), 2.34 (s, 3H, CH3), 2.32 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) (δ/ppm): 162.18 (1C, Cq-arom), 154.06 (1C, C=Nimino), 153.09 (1C, Cq-arom), 149.30 (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),

SC

124.28 (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, CH3), 20.96 (1C, CH3); MS (m/z): 315.3 ([M+1]+);

M AN U

elemental analysis calcd. (%) for C20H18N4: C 76.41, H 5.77, N 17.82; found C 75.86, H 5.50, N 17.23.

4.1.1.6. 6-cyano-N-[2-(6-cyanopyridin-3-yl)isoindolin-1-ylidene]pyridin-3-amine 3f Following the general procedure B, from phthalaldehyde 1 (0.73 g, 5.5 mmol) and 5-amino-2cyanopyridine 2f (1.31 g, 11.0 mmol) after refluxing for 10h and recrystallization from DMF 1.33 g (72%) of yellow crystals was obtained; mp 279-281 °C; IR (diamond) (ν/cm-1): 3093 (ar

TE D

C–H), 3034 (ar C–H), 2224 (C≡N), 1647 (C=N), 1550 (ar C–C, C–N, skeletal vib.); 1H NMR (600 MHz, DMSO-d6) (δ/ppm): 9.33 (d, 1H, J = 2.06 Hz, Harom), 8.71 (dd, 1H, J1 = 8.67 Hz, J2 = 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

EP

Hz, Harom), 6.72 (d, 1H, J = 7.88 Hz, Harom), 5.28 (s, 2H, Hisoind); 13C NMR (150 MHz, DMSO-d6) (δ/ppm): 154.00 (1C, C=Nimino), 148.77 (2C, Cq-arom), 144.11 (1C, CHarom), 142.38 (1C, CHarom),

AC C

141.27 (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), 125.63 (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 C20H12N6: 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-5cyanopyridine 2g (0.47 g, 4.0 mmol) after refluxing for 10h and recrystallization from DMF 0.29 g (43%) of yellow powder was obtained; mp 276-278 °C; IR (diamond) (ν/cm-1): 3115 (ar C–H),

ACCEPTED MANUSCRIPT

3055 (ar C–H), 3025 (ar C–H), 2232 (C≡N), 1660 (C=N), 1585 (ar C–C, C–N, skeletal vib.); 1H NMR (600 MHz, DMSO-d6) (δ/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, J1 = 8.94 Hz, J2 = 2.22 Hz, Harom), 8.26 (dd, 1H, J1 = 8.39 Hz, J2 = 2.27 Hz, Harom), 7.71 (d, 1H, J = 7.62 Hz, Harom), 7.61 (t, 1H, J = 7.45 Hz,

RI PT

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, Harom), 5.29 (s, 2H, Hisoind); 13C NMR (75 MHz, DMSO-d6) (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),

SC

128.32 (1C, CHarom), 125.74 (1C, CHarom), 124.58 (1C, CHarom), 118.27 (1C, C≡N), 117.89 (1C, C≡N), 116.77 (1C, CHarom), 114.01 (1C, CHarom), 103.77 (1C, Cq-arom), 103.35 (1C, Cq-arom), 52.80

M AN U

(1C, CH2,isoind); MS (m/z): 337.3 ([M+1]+); elemental analysis calcd. (%) for C20H12N6: C 71.42, 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-5nitropyridine 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): 1663 (C=N), 1594 (ar C–C, C–N, skeletal vib.), 1568 (NO2 st as), 1330 (NO2 st sy); 1H NMR

TE D

1

(300 MHz, DMSO-d6) (δ/ppm): 9.22 (d, 1H, J = 2.75 Hz, Harom), 9.17 (d, 1H, J = 2.80 Hz, Harom), 8.86 (d, 1H, J = 9.38 Hz, Harom), 8.57 (dd, 1H, J1 = 9.38 Hz, J2 = 2.81 Hz, Harom), 8.51 (dd, 1H, J1 = 8.85 Hz, J2 = 2.84 Hz, Harom), 7.70 (d, 1H, J = 7.61 Hz, Harom), 7.60 (t, 1H, J = 7.49 Hz, Harom),

EP

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 C18H12N6O4: C 57.45, H 3.21, N 22.33; found C

AC C

57.73, H 3.41, N 22.57.

4.1.1.9. 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-acetamido5-aminopyridine 2i (1.00 g, 6.6 mmol) after stirring for 24h and recrystallization from ethanol/H2O 0.70 g (53%) of beige powder was obtained; mp 245-250 °C; IR (diamond) (ν/cm-1): 3313-3003 (N–H), 1689 (C=O), 1639 (C=N), 1596 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 10.47 (s, 1H, NHamide), 10.40 (s, 1H, NHamide), 8.95 (s, 1H, Harom), 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

(d, 1H, J = 6.19 Hz, Harom), 5.11 (s, 2H, Hisoind), 2.09 (s, 6H, 2×CH3);

13

C NMR (75 MHz,

DMSO-d6) (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), 139.59 (2C, CHarom), 133.53 (1C, Cq-arom), 130.92 (2C, CHarom), 130.15 (1C, CHarom), 127.38 (1C,

RI PT

CHarom), 124.88 (1C, CHarom), 123.69 (1C, CHarom), 113.65 (1C, CHarom), 112.94 (1C, CHarom), 52.36 (1C, CH2,isoind), 23.76 (2C, 2×CH3); MS (m/z): 401.2 ([M+1]+); elemental analysis calcd. (%) for C22H20N6O4: C 65.99, H 5.03, N 20.99; found C 65.58, H 4.80, N 20.62.

4.1.1.10. 4-(imidazolin-2-yl)-N-{2-[4-(imidazolin-2-yl)phenyl]isoindolin-1-ylidene}benzenamine

SC

dihydrochloride 8a

Following the general procedure B, from phthalaldehyde 1 (0.027 g, 0.2 mmol) and 4-(2-

M AN U

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 (diamond) (ν/cm-1): 3370-2782 (N–H), 1652 (C=N), 1591 (C=Namidine); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 10.61 (bs, 4H, NHamidine), 8.29 (s, 2H, Harom), 8.15-8.07 (m, 4H, Harom), 7.68 (s, 1H, Harom), 7.59 (s, 1H, Harom), 7.23 (bs, 3H, Harom), 6.65 (s, 1H, Harom), 5.24 (s, 2H, Hisoind), 4.00 (s, 8H, 4×CH2); 13C NMR (75 MHz, DMSO-d6) (δ/ppm): 164.82 (1C, Cq-amidine), 164.49 (1C,

TE D

Cq-amidine), 156.11 (1C, Cq-arom), 153.19 (1C, C=Nimino), 146.31 (1C, Cq-arom), 141.51 (1C, Cq-arom), 132.23 (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), 116.17 (1C, Cq-arom), 116.08 (1C, Cq-arom), 53.24 (1C, CH2,isoind), 44.66 (4C, 4×CH2); MS (m/z):

EP

211.2 ([(M-2Cl-)/2]+); elemental analysis calcd. (%) for C26H26Cl2N6: C 63.29, H 5.31, N 17.03; found C 63.65, H 5.01, N 16.76.

AC C

4.1.1.11. 4-(N'-isopropylamidino)-N-[2-(4-N'-isopropylamidinophenyl)isoindolin-1ylidene]benzenamine dihydrochloride 8b Following the general procedure B, from phthalaldehyde 1 (0.158 g, 1.2 mmol) and 4-Nisopropylamidinoaniline 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 (diamond) (ν/cm-1): 3376-2935 (N–H ), 1654 (C=N), 1592 (C=Namidine), 1509 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 9.47 (bs, 6H, NHamidine), 8.22 (d, 2H, J = 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, 4×CH3); 13C NMR (75 MHz, DMSO-d6) (δ/ppm): 161.75 (1C, Cq-amidine), 161.53 (1C, Cq-amidine), 155.05 (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,

RI PT

CHarom), 125.70 (1C, CHarom), 124.30 (1C, CHarom), 123.24 (1C, Cq-arom), 122.86 (1C, Cq-arom), 121.22 (2C, CHarom), 119.72 (2C, CHarom), 53.23 (1C, CH2,isoind), 45.45 (2C, 2×CH), 21.83 (2C, 2×CH3), 21.79 (2C, 2×CH3); MS (m/z): 227.3 ([(M-2Cl-)/2]+); elemental analysis calcd. (%) for C28H34Cl2N6: C 63.99, H 6.52, N 15.99; found C 63.70, H 6.43, N 15.82.

SC

4.1.1.12. 5-(imidazolin-2-yl)-N-{2-[5-(imidazolin-2-yl)pyridin-2-yl]isoindolin-1-ylidene}pyridin2-amine dihydrochloride 8c

M AN U

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 (diamond) (ν/cm-1): 3370-2940 (N–H), 1651 (C=N), 1608 (C=Namidine), 1567 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 10.78 (bs, 4H, NHamidine), 9.44 (d, 1H, J = 2.34 Hz, Harom), 8.81 (dd, 1H, J1 = 8.83 Hz, J2 = 2.46 Hz, Harom), 8.55 (d, 1H, J = 2.27 Hz,

TE D

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, Harom), 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, Harom), 5.36 (s, 2H, Hisoind), 4.04 (s, 4H, 2×CH2), 4.03 (s, 4H, 2×CH2);

13

C NMR (75 MHz,

DMSO-d6) (δ/ppm): 163.56 (1C, Cq-amidine), 163.22 (1C, Cq-amidine), 154.60 (1C, C=Nimino), 150.31

arom),

EP

(1C, Cq-arom), 143.50 (1C, CHarom), 141.87 (1C, Cq-arom), 141.72 (1C, CHarom), 141.31 (1C, Cq134.99 (1C, Cq-arom), 134.68 (1C, Cq-arom), 132.73 (1C, CHarom), 129.66 (1C, Cq-arom), 128.98

AC C

(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 C24H24Cl2N8: C 58.19, H 4.88, N 22.62; found C 58.31, H 4.48, N 22.44. 4.1.1.13. 6-amidino-N-[2-(6-amidinopyridin-3-yl)isoindolin-1-ylidene]pyridin-3-amine dihydrochloride 8d Following the general procedure B, from phthalaldehyde 1 (0.039 g, 0.29 mmol) and 5-amino-2amidino-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

obtained; mp 278-280 °C; IR (diamond) (ν/cm-1): 3123 (N–H), 3041 (N–H), 1676 (C=Namidine), 1633 (C=N), 1567 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (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, J1 = 8.83 Hz, J2 = 2.16 Hz, Harom), 8.54-8.51 (m, 2H, Harom), 8.46 (d, 1H, J = 8.49 Hz, Harom), 7.77-

RI PT

7.72 (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, J = 7.91 Hz, Harom), 5.35 (s, 2H, Hisoind); 13C NMR (150 MHz, DMSO-d6) (δ/ppm): 162.13 (1C, 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),

SC

137.69 (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),

M AN U

124.20 (1C, CHarom), 53.03 (1C, CH2,isoind); MS (m/z): 186.2 ([(M-2Cl)/2]+); elemental analysis calcd. (%) for C20H20Cl2N8: C 54.18, H 4.55, N 25.28; found C 54.33, H 4.64, N 25.02. 4.1.1.14. 5-(N'-isopropylamidino)-N-[2-(5-N'-isopropylamidinopyridin-2-yl)isoindolin-1ylidene]pyridin-2-amine dihydrochloride 8e and 2-N-[(5-N'-isopropylamidino)pyridin-2yl]isoindolin-1-one hydrochloride 9b

Following the general procedure C, from phthalaldehyde 1 (0.067 g, 0.50 mmol) and 2-amino-5-

TE D

(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:

8e: 0.030 g (12%) of light yellow powder; mp 260-262 °C; IR (diamond) (ν/cm-1): 3389-2927

EP

(N–H), 1666 (C=N), 1617 (C=Namidine), 1591 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 9.35 (bs, 6H, NHamidine), 8.88 (d, 1H, J = 8.98 Hz, Harom), 8.82, (d, 1H, J =

AC C

2.13 Hz, Harom), 8.76 (d, 1H, J = 2.36 Hz, Harom), 8.21 (dd, 1H, J1 = 9.04 Hz, J2 = 2.50 Hz, Harom), 8.16 (dd, 1H, J1 = 8.52 Hz, J2 = 2.50 Hz, Harom), 7.75 (d, 1H, J = 7.63 Hz, Harom), 7.62 (t, 1H, J = 7.47 Hz, Harom), 7.26-7.21 (m, 2H, Harom), 6.53 (d, 1H, J = 7.96 Hz, Harom), 5.33 (s, 2H, Hisoind), 4.06 (q, 2H, J = 6.23 Hz, 2×CH), 1.32 (d, 6H, J = 6.68 Hz, 2×CH3), 1.29 (d, 6H, J = 6.66 Hz, 2×CH3);

13

C NMR (75 MHz, DMSO-d6) (δ/ppm): 164.78 (1C, Cq-arom), 160.33 (1C, Cq-amidine),

160.00 (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), 120.52 (1C, Cq-arom), 120.25 (1C, Cq-arom), 115.77 (1C, CHarom), 113.20 (1C, CHarom), 52.76 (1C,

ACCEPTED MANUSCRIPT

CH2,isoind), 45.63 (2C, 2×CH), 21.80 (2C, 2×CH3), 21.77 (2C, 2×CH3); MS (m/z): 228.3 ([(M2Cl)/2]+); elemental analysis calcd. (%) for C26H32Cl2N8: C 59.20, H 6.11, N 21.24; found C 59.03, H 6.02, N 21.32. 9b: 0.113 g (68%) of beige powder; mp >300 °C; IR (diamond) (ν/cm-1): 3502 (N–H), 3332 (N– 1

RI PT

H), 3155 (N–H), 2959 (N–H), 1680 (C=O), 1619 (C=Namidine), 1602 (ar C–C, C–N, skeletal vib.); H NMR (300 MHz, DMSO-d6) (δ/ppm): 9.57 (bs, 2H, NHamidine), 8.81 (d, 1H, J = 2.03 Hz,

Harom), 8.68, (d, 1H, J = 8.85 Hz, Harom), 8.26 (dd, 1H, J1 = 8.90 Hz, J2 = 2.52 Hz, Harom), 7.86 (d, 1H, 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,

2×CH3);

13

SC

1H, NHamidin), 5.16 (s, 2H, Hisoind), 4.12 (sep, 1H, J = 6.35 Hz, CH), 1.30 (d, 6H, J = 6.38 Hz, C NMR (75 MHz, DMSO-d6) (δ/ppm): 167.99 (1C, C=O), 160.02 (1C, Cq-amidine),

M AN U

154.71 (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×CH3); MS (m/z): 295.2 ([M-Cl]+); elemental analysis calcd. (%) for C17H19ClN4O: C 61.72 H 5.79, N 16.94; found C 61.49, H 5.88, N 16.78.

4.1.1.15. 2-N-(4-Amidinophenyl)isoindolin-1-one hydrochloride 9a

TE D

Following the general procedure B, from phthalaldehyde 1 (0.155 g, 1.1 mmol) and 4amidinoaniline hydrochloride 7i (0.312 g, 2.3 mmol) after refluxing for 6h and recrystallization from ethanol 0.025 g (9%) of white powder was obtained; mp >300 °C; IR (diamond) (ν/cm-1): 3306-3010 (N–H), 1680 (C=O), 1607 (C=Namidine), 1491 (ar C–C, skeletal vib.); 1H NMR (300

EP

MHz, DMSO-d6) (δ/ppm): 9.36 (s, 2H, Hamidine), 9.12 (s, 2H, Hamidin), 8.16 (d, 2H, J = 9.03 Hz, 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, 13

C NMR (75 MHz, DMSO-d6) (δ/ppm):

AC C

Harom), 7.60-7.55 (m, 1H, Harom), 5.12 (s, 2H, Hisoind);

167.79 (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); MS (m/z): 252.1 ([(M-Cl-)]+); elemental analysis calcd. (%) for C15H14ClN3O: C 62.61 H 4.90, N 14.60; found C 62.22 H 4.73, N 14.32. 4.1.1.16. 2-N-(5-Amidinopyridin-2-yl)isoindolin-1-one hydrochloride 9c Following the general procedure C, from phthalaldehyde 1 (0.041 g, 0.30 mmol) and 2-amino-5amidinopyridine 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/cm1

): 3382-2927 (N–H), 1720 (C=O), 1673 (C=Namidine), 1598 (ar C–C, C–N, skeletal vib.); 1H

NMR (300 MHz, DMSO-d6) (δ/ppm): 9.33 (bs, 4H, NHamidine), 8.91 (d, 1H, J = 2.24 Hz, Harom), 8.70 (d, 1H, J = 8.95 Hz, Harom), 8.33 (dd, 1H, J1 = 8.94 Hz, J2 = 2.53 Hz, Harom), 7.87 (d, 1H, J =

Hisoind);

RI PT

7.59 Hz, Harom), 7.76 (s, 1H, Harom), 7.75 (s, 1H, Harom), 7.63-7.56 (m, 1H, Harom), 5.17 (s, 2H, C NMR (75 MHz, DMSO-d6) (δ/ppm): 168.05 (1C, C=O), 163.86 (1C, Cq-amidine),

13

155.18 (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,

SC

CHarom), 119.92 (1C, Cq-arom), 112.62 (1C, CHarom), 50.31 (1C, CH2,isoind); MS (m/z): 253.1 ([MCl]+); elemental analysis calcd. (%) for C14H13ClN4O: C 58.24 H 4.54, N 19.40; found C 58.38,

M AN U

H 4.59, N 19.55.

4.1.2. Synthesis of N1-[2-(4-aminophenyl)isoindolin-1-ylidene]benzene-1,4-diamine 4a A suspension of 3d (1.00 g, 2.5 mmol) in 2M H2SO4 (20 ml) was refluxed for 2h. After cooling 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

TE D

obtained; mp 202-204 °C; IR (diamond) (ν/cm-1): 3420-3028 (NH2), 1626 (C=N), 1506 (ar C–C, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 7.58 (d, 2H, J = 8.67 Hz, Harom), 7.53 (d, 1H, 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, 1H, J = 7.90 Hz, Harom), 6.60 (d, 2H, J = 8.65 Hz, Harom), 6.55 (s, 4H, Harom), 4.91 (s, 2H, NH2),

EP

4.88 (s, 2H, Hisoind), 4.69 (s, 2H, NH2);

13

C NMR (150 MHz, DMSO-d6) (δ/ppm): 153.41 (1C,

C=Nimino), 144.74 (1C, Cq-arom), 143.35 (1C, Cq-arom), 141.00 (1C, Cq-arom), 140.47 (1C, Cq-arom),

AC C

131.00 (1C, Cq-arom), 130.74 (1C, Cq-arom), 129.82 (1C, CHarom), 126.68 (1C, CHarom), 125.30 (1C, CHarom), 123.16 (1C, CHarom), 122.45 (2C, CHarom), 121.71 (1C, CHarom), 121.37 (1C, CHarom), 114.94 (2C, CHarom), 113.74 (2C, CHarom), 53.00 (1C, CH2,isoind); MS (m/z): 315.2 ([M+1]+); elemental analysis calcd. (%) for C20H18N4: C 76.41, H 5.77, N 17.82; found C 76.15, H 5.45, N 17.53.

4.1.3. Synthesis of 6-amino-N-[2-(6-aminopyridin-3-yl)isoindolin-1-ylidene]pyridin-3-amine 4b

ACCEPTED MANUSCRIPT

A suspension of 3i (0.10 g, 0.25 mmol) in 2M H2SO4 (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 obtained; mp 228-231 °C; IR (diamond) (ν/cm-1): 3439 (NH2), 3288 (NH2), 3155 (NH2), 1623

RI PT

(C=N), 1502 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 8.33 (s, 1H, 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), 13

C NMR (75 MHz, DMSO-d6)

SC

5.82 (s, 2H, NH2), 5.52 (s, 2H, NH2), 4.94 (s, 2H, Hisoind);

(δ/ppm): 156.91 (1C, Cq-arom), 155.85 (1C, Cq-arom), 154.91 (1C, C=Nimino), 142.07 (1C, CHarom),

M AN U

141.88 (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), 125.45 (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 C18H16N6: C 68.34, H 5.10, N 26.56; found C 68.71, H 5.25, N 26.23.

dihydrochloride 5

TE D

4.1.4. Synthesis of N1-[2-(4-aminophenyl)isoindolin-1-ylidene]benzene-1,4-diamine

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

EP

under reduced pressure to obtain 0.08 g (95%) gray powder; mp 276-278 °C; IR (diamond) (ν/cm-1): 2934-2719 (NH3+), 2554 (NH3+), 1638 (C=N), 1509 (ar C–C, skeletal vib.); 1H NMR

AC C

(300 MHz, DMSO-d6) (δ/ppm): 7.85 (s, 2H, Harom), 7.69 (s, 1H, Harom), 7.45 (s, 1H, Harom), 7.35 (bs, 2H, Harom), 7.28 (s, 1H, Harom), 7.19 (bs, 2H, Harom), 7.06 (s, 3H, Harom), 5.38 (s, 2H, Hisoind); C NMR (75 MHz, DMSO-d6) (δ/ppm): 153.22 (1C, C=Nimino), 144.35 (1C, Cq-arom), 135.27 (2C,

13

CHarom), 132.40 (1C, Cq-arom), 132.31 (1C, Cq-arom), 132.02 (1C, Cq-arom), 131.97 (1C, Cq-arom), 131.67 (1C, Cq-arom), 129.05 (2C, CHarom), 128.29 (1C, CHarom), 125.31 (1C, CHarom), 124.65 (2C, CHarom), 124.12 (2C, CHarom), 123.96 (2C, CHarom), 53.28 (1C, CH2,isoind); MS (m/z): 316.2 ([M2Cl]+); elemental analysis calcd. (%) for C20H20Cl2N4: C 62.02, H 5.20, N 14.47; found C 61.80, 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

RI PT

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

SC

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.

M AN U

Unsubstituted amidines: the suspension was treated with anhydrous ammonia gas and stirred for 24h 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 (mplit >300 °C) [32]. 4.1.5.2. 4-N-isopropylamidinoaniline hydrochloride 7b

TE D

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]. 4.1.5.3. 5-Amino-2-(imidazolin-2-yl)pyridine hydrochloride 7c From 5-amino-2-cyanopyridine 6b (0.60 g, 5.0 mmol) and ethylenediamine (1.00 ml, 15.0 mmol)

EP

after reprecipitation from ethanol and diethylether 0.32 g (32%) of white powder was obtained; mp 255-257 °C; IR (diamond) (ν/cm-1): 2959-2687 (NH2, NHamidine), 2049 (NH2, NHamidine), 1600

AC C

(C=N), 1500 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 9.03 (bs, 2H, NHamidine), 8.11 (d, 1H, J = 2.55 Hz, Hpy), 8.04 (d, 1H, J = 8.70 Hz, Hpy), 7.03 (dd, 1H, J1 = 8.71 Hz, J2 = 2.65 Hz, Hpy), 6.74 (s, 2H, NH2), 3.91 (s, 4H, 2×CH2); 13C NMR (75 MHz, DMSOd6) (δ/ppm): 162.35 (1C, Cq-amidine), 151.72 (1C, Cq-arom), 141.75 (1C, CHarom), 137.54 (1C, Cqarom),

124.32 (1C, CHarom), 118.52 (1C, CHarom), 44.54 (2C, 2×CH2); MS (m/z): 164.1 ([(M-

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

mp 251-253 °C; IR (diamond) (ν/cm-1): 3379-2877 (NH2, NHamidine), 1624 (C=N), 1580 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 9.08 (bs, 3H, NHamidine), 8.10 (d, 1H, J = 2.55 Hz, Hpy), 8.07 (d, 1H, J = 8.78 Hz, Hpy), 7.04 (dd, 1H, J1 = 8.74 Hz, J2 = 2.64 Hz, Hpy), 6.54 (s, 2H, NH2), 4.12 (quint, 1H, J = 6.23 Hz, CH), 1.27 (d, 6H, J = 6.37 Hz, 2×CH3); 13C

RI PT

NMR (75 MHz, DMSO-d6) (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×CH3); MS (m/z): 180.1 ([(M-Cl)+1]+). 4.1.5.5. 2-Amidino-5-aminopyridine hydrochloride 7e

SC

From 5-amino-2-cyanopyridine 6b (0.60 g, 5.0 mmol) after reprecipitation from ethanol and diethylether 0.24 g (33%) of white powder was obtained; mp 259-262 °C; IR (diamond) (ν/cm-1):

M AN U

3359-2898 (NH2, NHamidine), 1682 (C=N), 1656, 1637, 1580 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 8.10 (d, 1H, J = 2.36 Hz, Hpy), 8.08 (d, 1H, J = 8.67 Hz, Hpy), 7.48 (bs, 4H, NHamidine), 7.04 (dd, 1H, J1 = 8.73 Hz, J2 = 2.64 Hz, Hpy), 6.65 (s, 2H, NH2); C NMR (75 MHz, DMSO-d6) (δ/ppm): 162.20 (1C, Cq-amidine), 149.82 (1C, Cq-arom), 136.50 (1C,

13

CHarom), 129.45 (1C, Cq-arom), 125.19 (1C, CHarom), 118.39 (1C, CHarom); MS (m/z): 138.1 ([(MCl)+1]+).

TE D

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; mp 256-258 °C; IR (diamond) (ν/cm-1): 3484-2741 (NH2, NHamidine), 1680 (C=N), 1618 (NH2),

EP

1589 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 8.63 (d, 1H, J = 2.28 Hz, Hpy), 7.99 (dd, 1H, J1 = 8.89 Hz, J2 = 2.42 Hz, Hpy), 7.15 (s, 2H, NH2), 6.54 (d, 1H, J =

AC C

8.91 Hz, Hpy), 3.85 (s, 4H, 2×CH2); amidine),

13

C NMR (75 MHz, DMSO-d6) (δ/ppm): 163.63 (1C, Cq-

163.32 (1C, Cq-arom), 151.14 (1C, CHarom), 136.95 (1C, CHarom), 107.81 (1C, CHarom),

106.85 (1C, Cq-arom), 44.73 (2C, 2×CH2); 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; mp 232-238 °C; IR (diamond) (ν/cm-1): 3363-2848 (NH2, NHamidine), 1680 (C=N), 1637, 1604 (ar C–C, C–N, skeletal vib.), 1573 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (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, J1 = 8.83 Hz, J2 = 2.49 Hz, Hpy), 6.97 (s, 2H, NH2), 6.52 (d, 1H, J = 8.85 Hz, Hpy), 4.04 (bs, 1H, CH), 1.25 (d, 6H, J = 6.35 Hz, 2×CH3);

13

C NMR (75 MHz,

DMSO-d6) (δ/ppm): 163.15 (1C, Cq-arom), 160.45(1C, Cq-amidine), 149.86 (1C, CHarom), 137.21 (1C, CHarom), 112.47 (1C, Cq-arom), 107.33 (1C, CHarom), 45.11 (1C, CH), 21.89 (2C, 2×CH3); MS

RI PT

(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 diethylether 0.49 g (35%) of white powder was obtained; mp 243-245 °C; IR (diamond) (ν/cm-1):

SC

3335-2886 (NH2, NHamidine), 1664 (C=N), 1634, 1604 (ar C–C, C–N, skeletal vib.), 1575 (ar C–C, C–N, skeletal vib.); 1H NMR (300 MHz, DMSO-d6) (δ/ppm): 8.85 (s, 4H, NHamidine), 8.50 (d, 1H,

M AN U

J = 2.43 Hz, Hpy), 7.85 (dd, 1H, J1 = 8.91 Hz, J2 = 2.55 Hz, Hpy), 7.09 (s, 2H, NH2), 6.53 (d, 1H, J = 8.91 Hz, Hpy); 13C NMR (75 MHz, DMSO-d6) (δ/ppm): 164.11 (1C, Cq-arom), 163.48 (1C, Cqamidine),

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]+).

TE D

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

EP

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 µg/ml

AC C

streptomycin in a humidified atmosphere with 5% CO2 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

RI PT

on the same plate at the assay end point − the results are therefore a percentile difference from the calculated expected value.

The IC50 values for each compound were calculated from dose-response curves using linear regression analysis by fitting the mean test concentrations that give PG values above and below

SC

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

M AN U

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.

TE D

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

EP

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

AC C

the analysis, the cells were washed again with PBS and stained with 1 µg/mL of propidium iodide (PI) with the addition of 0.2 µg/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, 3b at concentration 25 µM and with compound 3g at concentration 50 µM while SW620 cells

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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 µL/well of incubation buffer, containing Annexin V-Fluos labelling reagent and propidium iodide (PI) 15 min. The

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cells were then washed with PBS and analysed under the fluorescent microscope.

4.6. DNA melting temperature studies.

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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

described [34].

4.7. Circular dichroism

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of the various tested compounds at drug/base pair ratio (R) 0.25 or 0.5 in 1 mL BPE buffer as

EP

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

AC C

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 265 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[32P]-dATP (3000 Ci/mmol, PerkinElmer, France). This 265-bp radio-labeled DNA fragment was

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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),

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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,

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BioRad). Each base position was localized from comparison with a G-track obtained using

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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

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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.

EP

5. Acknowledgements

We greatly appreciate the financial support of the Croatian Ministry of Science Education and

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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.

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6. References

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of biologically active mould metabolites, Chem. Commun. (London) (1967) 26-27.

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Bousquet, G. A. Cunha, M. D. Moskey, A. A. Ahmed, L. J. Pease, K. B. Glaser, K. D. Stewart, S. K. Davidsen, M. R. Michaelides, Isoindolinone ureas: a novel class of KDR kinase inhibitors, Bioorg. Med. Chem. Lett. 14 (2004) 4505-4509.

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Holmqvist, M. Hsu, L. Jiang, G. Liu, Q. Lu, C. Patel, J.R. Suresh, M. Selvaraj, L. Urban, P. Wang, Y. Yan-Neale, L. Whitehead, H. Zhang, L. Zhou, P. Atadja, The design, synthesis and structure–activity relationships of novel isoindoline-based histone deacetylase inhibitors, Bioorg. Med. Chem. Lett. 21 (2011) 4909-4912.

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[23] A. Gupta, P. Singh, B. Kamble, A. Kulkarni, M. Joghee Nanjan Chandrasekar Synthesis, Docking and Biological Evaluation of Some Novel 5-bromo-2- (5-aryl-1,3,4-thiadiazol-2-

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and mechanistic disclosure on the rearrangement reaction of o-phthalaldehyde with amide/thioamide analogs, Tetrahedron 63 (2007) 9338–9344. [26] I. Takahashi, R. Miyamoto, K. Nishiuchi, M. Hatanaka, A. Yamano, A. Sakushima, S. Hosoi, Studies of isoindoles. 12. A structural revision of the 1:2 Mannich type condensation

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reaction product formed from o-phthalaldehyde and substituted aniline, Heterocycles 63 (2004)

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Acids Res. 36 (2008) 3341-3353.

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heterocyclic diamidines, Nucleic Acids Res. 42 (2014) 1379-1390.

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Figure caption Figure 1. Prepared substituted isoindoline compounds 3a-8e Figure 2. Prepared substituted isoindolinone compounds 9a-9c

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Figure 3. Apoptotic HepG2 cells treated with compound 3b at 25 µM for 48h (magnification 20x).

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Figure 4. Circular dichroism spectra. CT-DNA (50 µM, dashed lines) was incubated with increasing concentrations of indicated compounds from 1 to 60 µM (8a) or to 80 µM (8b-d) (bold lines, respectively). Control for the absence of intrinsic CD (60 µM of 8a or 80 µM of 8bd) is presented as dotted lines.

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Figure 5. Topoisomerase I-induced DNA relaxation. pUC19 supercoiled (Sc) plasmid DNA in the absence (lanes DNA) or presence of topoisomerase I (Topo I) to generate topoisomers (Topo) and increasing concentrations of the indicated compounds (µM). Camptothecin (CPT) was used as a control for topoisomerase I-induced poisoning activity evidenced by the appearance of a nicked form (Nck, *). Rel, relaxed DNA

AC C

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Figure 6. DNase I footprinting experiments. Increasing concentrations of the radiolabeled 265 bp DNA fragment was incubated with increasing concentrations (µM) of compounds 8a or 8b prior to mild digestion by DNase I. Samples were separated on a denaturing polyacrylamide gel (A) and densitometric analysis was performed for compound 8b (B). G-track lane (G) was used to precisely localize the protected sites located using black boxes.

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R2

N

N

R1 X

R X

Y

R2

X

X Y

Y

R1

R

H+ClN

3a X = CH, Y = CH, R1 = CN, R2 = H 3c X = CH, Y = CH, R1 = N(CH3)2, R2 = H

3f X = CH, Y = N, R1 = CN, R2 = H 3g X = N, Y = CH, R1 = CN, R2 = H 3h X = N, Y = CH, R1 = NO2, R2 = H

N H NH2+Cl-

8d X = CH, Y = N, R = NH2 NH2+Cl-

8e X = N, Y = CH, R=

5 X = CH, Y = CH, R1 = NH3+Cl-, R2 = H

TE D

HN

EP

Fig 1.

X

HN H+ClN

4a X = CH, Y = CH, R1 = NH2, R2 = H

N

NH2+Cl-

8c X = CH, Y = N, R =

3i X = CH, Y = N, R1 = NHCOCH3, R2 = H 4b X = CH, Y = N, R1 = NH2, R2 = H

N H

8b X = CH, Y = CH, R=

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3d X = CH, Y = CH, R1 = NHCOCH3, R2 = H

SC

8a X = CH, Y = CH, R =

3b X = CH, Y = CH, R1 = NO2, R2 = H

3e X = N, Y = CH, R1 = H, R2 = CH3

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N

N

AC C

Y

NH2+Cl-

9a X = CH, Y = CH, R = NH2

R

Y

NH2+Cl-

9b X = N, Y = CH, R= HN

O

9c X = N, Y = CH, R=

NH2+ClNH2

Fig 2.

SC

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ACCEPTED MANUSCRIPT

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Fig 3.

40

CD (mdeg)

0 -20

20

0

-20

250

TE D

CD (mdeg)

20

-40

300

350

400

-40

450

250

Wavelength (nm)

300

350

400

40

0

-20

8d CD (mdeg)

EP

20

450

Wavelength (nm)

8c

40

20

0

-20

-40

AC C

CD (mdeg)

8b

8a

40

250

-40 300

350

400

450

250

Wavelength (nm)

300

350

400

Wavelength (nm)

Fig 4.

450

ACCEPTED MANUSCRIPT

3i

8c

8d

0 20 50 5 10 20 50 5 10 20 50 5 10 20 50 5 10 20 50 5 10 20 50 0

* *

CPT

3b

4a

5

8a

8b

0 20 50 5 10 20 50 5 10 20 50 5 10 20 50 5 10 20 50 5 10 20 50 0

SC

DNA

Topo I

* *

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Rel or Nck* Topo Sc

8e

DNA

3e

DNA

Rel or Nck* Topo Sc

CPT

RI PT

DNA

Topo I

* * *

AC C

Sc

8d

Fig 5.

DNA

8c

0 10 20 50 0 0.5 1 2 3 4 5 0 1 2 5 10 20 50 0 0.5 1 2 5 10 20 50 0

EP

Rel or Nck* Topo

8a

CPT

TE D

DNA

Topo I

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8b

SC

RI PT

0 1 2.5 5 10 25 50 0 1 2.5 5 10 25 50 G

A

8a

150

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140 130 120 110

90

1.5

B

0.5 µM

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Differential Cleavage

80

1

1 µM

2.5 µM

ATAA

AACA

ATTA

TE D

100

AATT

5 µM

10 µM

25 µM

8b

0.5

AC C

0

-0.5

-1

-1.5

5'-GTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCT 140 130 120 110 90 100

nucleotide position

Fig 6.

ACCEPTED MANUSCRIPT

R2 R2

N

CHO

R1

X

RI PT

H 2N

R2

2a-i

3a-i

X Y

R1 2a X = CH, Y = CH, R1 = CN, R2 = H 2b X = CH, Y = CH, R1 = NO2, R2 = H 2c X = CH, Y = CH, R1 = N(CH3)2, R2 = H 2d X = CH, Y = CH, R1 = NHCOCH3, R2 = H 2e X = N, Y = CH, R1 = H, R2 = CH3 2f X = CH, Y = N, R1 = CN, R2 = H 2g X = N, Y = CH, R1 = CN, R2 = H 2h X = N, Y = CH, R1 = NO2, R2 = H 2i X = CH, Y = N, R1 = NHCOCH3, R2 = H

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SC

3a X = CH, Y = CH, R1 = CN, R2 = H 3b X = CH, Y = CH, R1 = NO2, R2 = H 3c X = CH, Y = CH, R1 = N(CH3)2, R2 = H 3d X = CH, Y = CH, R1 = NHCOCH3, R2 = H 3e X = N, Y = CH, R1 = H, R2 = CH3 3f X = CH, Y = N, R1 = CN, R2 = H 3g X = N, Y = CH, R1 = CN, R2 = H 3h X = N, Y = CH, R1 = NO2, R2 = H 3i X = CH, Y = N, R1 = NHCOCH3, R2 = H

2M H2SO4 ∆

N

X

NH2

Y

TE D

N

4a-b X 4a X = CH, Y = CH 4b X = CH, Y = N

Y

H 2N

AC C

EP

HCl(g) EtOHaps

NH3+Cl-

N

N 5

-

Y

N

r.t. or ∆

Y 1

X

EtOHaps

2 CHO

R1

Cl+H3N

Scheme 1.

ACCEPTED MANUSCRIPT

CN

R 1. HCl(g)/EtOHaps Y H2N

X

X

7b X = CH, Y = CH, R =

7a-h

6a X = Y = CH 6b X = CH, Y = N 6c X = N, Y = CH

NH2

N H NH2+Cl-

RI PT

H2N

NH2+Cl-

7e X = CH, Y = N, R =

7a X = CH, Y = CH, R =

2. RNH2/EtOHaps

Y

H+ClN

H+ClN

7f X = N, Y = CH, R =

HN

H+ClN

N H NH2+Cl-

7g X = N, Y = CH, R =

7c X = CH, Y = N, R =

N H NH2+Cl-

HN

SC

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HN

NH2

H+ClN

8a X = CH, Y = CH, R = N H

N

R X

NH2+Cl-

8b X = CH, Y = CH, R =

Y

HN H+ClN

N

CHO

R 2

EtOHaps or EtOHaps, acetic acid ∆

X

N H

8a-e 8d X = CH, Y = N, R =

NH2+ClNH2

R

NH2+Cl-

7a-h

8e X = N, Y = CH, R =

NH2+Cl-

HN

7i X = Y = CH, R =

EP

NH2

AC C

1

H2N

8c X = CH, Y = N, R =

Y

TE D

Y CHO

X

NH2+Cl-

7h X = N, Y = CH, R =

7d X = CH, Y = N, R =

NH2+Cl-

9a X = CH, Y = CH, R = NH2

N

R X

O

9b X = N, Y = CH, R = HN

Y 9c X = N, Y = CH, R =

9a-c

Scheme 2.

NH2+Cl-

NH2+ClNH2

ACCEPTED MANUSCRIPT

Table 1. The inhibition effects of compounds 3a-9c on the growth of tumour cells and normal fibroblasts in vitro. The results are presented as IC50 values in µM.

1

HeLa 50.03 7.69 4.46 8.81 92.47 >100 97.08 >100 >100 >100 62.39 78.04 >100 6.25 68.83 >100 >100 60.04

SC

RI PT

BJ 75.24 75.26 9.00 63.81 >100 92.38 80.07 >100 >100 >100 80.46 8,77 53.86* 4.26 >100 >100 18.10 15.82

M AN U

MCF-7 46.10 8.14 40.54 71.24 8.99 38.18 0.70 15.99 0.05 80.65 0.73 20.01 53.43 3.76 42.12 >100 6.43 35.63

TE D

Substance No. 3a 3b 3f 3g 3c 3d 3e 3i 4a 4b 5 8a 8b 8c 8d 8e 9b 9c

IC50a (µM) Cell lines SW620 HepG2 77.17 4.68 33.38 9.65 83.82 5.51 >100 9.41 >100 82.30 >100 70.95 >100 43.23 >100 30.39 >100 >100 >100 88.85 51.15 >100 >100 83.24 >100 62.17 5.26 20.50 >100 33.85 >100 >100 79.87 51.51 44.80 40.62

AC C

EP

IC50; 50% inhibitory concentration, or compound concentration required to inhibit tumour cell proliferation by 50%. * tested on normal human fibroblasts WI38

ACCEPTED MANUSCRIPT

Cell percentage (% ± standard deviation)

Compound 3a control 24 h 10 µM 25 µM control 10 µM 48 h 25 µM

4.8 ± 0.6 13.3 ± 3.9 15.7 ± 4.6 1.9 ± 0.0 5.2 ± 0.1 5.7 ± 0.5

Compound 3b control 24 h 20 µM 50 µM control 48 h 20 µM 50 µM

3.1 ± 0.9 6.6 ± 2.4 8.3 ± 0.3 2.8 ± 0.3 4.4 ± 0.2 4.3 ± 0.3

Compound 3g control 24 h 20 µM 50 µM control 48 h 20 µM 50 µM

61.4 ± 2.0 54.1 ± 1.7 45.9 ± 5.8 62.1 ± 4.9 0.0 ± 0.0 0.0 ± 0.0

S

12.0 ± 10.2 26.7 ± 0.0 16.8 ± 14.8 6.8 ± 5.3 0.0 ± 0.0 0.0 ± 0.0

G2/M

26.6 ± 12.2 19.2 ± 1.7 37.3 ± 20.7 31.1 ± 0.4 100.0 ± 0.0 100.0 ± 0.0

8.6 ± 2.8 25.6 ± 1.8 27.6 ± 0.1 0.0 ± 0.0 1.3 ± 1.9 0.8 ± 1.2

31.7 ± 1.9 15.6 ± 0.3 17.4 ± 0.4 45.0 ± 4.8 44.9 ± 1.6 39.4 ± 0.3

2.9 ± 1.1 6.6 ± 0.5 6.7 ± 0.6 1.7 ± 0.5 5.7 ± 0.6 8.4 ± 1.0

66.5 ± 1.4 58.1 ± 2.3 57.4 ± 2.1 62.7 ± 0.5 61.0 ± 2.0 61.2 ± 0.0

17.0 ± 2.9 21.5 ± 1.2 26.4 ± 2.2 9.3 ± 13.2 19.5 ± 5.6 18.5 ± 4.4

16.5 ± 1.6 20.4 ± 3.5 16.2 ± 0.1 28.0 ± 13.8 19.5 ± 7.6 20.3 ± 4.5

12.1 ± 0.3 7.5 ± 0.3 7.8 ± 0.2

38.1 ± 1.1 19.6 ± 0.7 22.1 ± 0.3

39.4 ± 0.2 40.4 ± 2.0 36.2 ± 2.5

22.5 ± 0.9 40.0 ± 2.8 41.7 ± 2.8

EP

TE D

59.7 ± 0.9 58.8 ± 1.5 55.0 ± 0.3 55.0 ± 4.8 53.7 ± 3.5 59.7 ± 0.9

AC C

Cell line SW620 Compound 8c control 24 h* 20 µM 50 µM

G1

SC

subG1

M AN U

Treatment Cell line HepG2

RI PT

Table 2. Cell cycle analysis of HepG2 cells treated with compound 3a at concentrations 10 µM and 25 µM and compounds 3b and 3g at concentrations 20 µM and 50 µM after 24-h and 48-h treatemnts as well as of SW620 treated with compound 8c at concentrations 10 µM and 25 µM for 24 h and 48 h, however after 48 h all treated cells were found to be dead. Statistically significant changes (p

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

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 reactio...
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