Synthesis and biological evaluation of new chloro/acetoxy substituted isoindole analogues as new tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103421

We have developed a versatile synthetic approach for the synthesis of new isoindole derivatives via the cleavage of ethers from tricyclic imide skeleton compounds. An exo-cycloadduct prepared from the Diels–Alder reaction of furan and maleic anhydride furnished imide derivatives. The epoxide ring was opened with Ac₂O or Ac₂O/AcCl in the presence of a catalytic amount of H₂SO₄ in order to yield new isoindole derivatives 8a-d and 9a-d. The anticancer activity of these compounds was evaluated against the HeLa cell lines. The synthesized compounds showed inhibitory effects on the viability of HeLa cells and the degree of cytotoxicity was increased with the level of bigger branched isoindole derivatives. To better understand the acting mechanism of these molecules, western blot analysis was performed with using mTOR and its downstream substrates. In addition, human mTOR and ribozomal S6 kinase β1 (RS6Kβ1) have been investigated with molecular modelling studies as possible targets for compound series 8 and 9.

Full text of DOI:10.1016/j.bioorg.2019.103421

Journal Pre-proofs
Synthesis and biological evaluation of New Chloro / Acetoxy Substituted Iso-
indole Analogues as new tyrosine kinase inhibitors
Aytekin Köse, Meltem Kaya, Nurhan H. Kishalı, Atilla Akdemir, Ertan Şahin,
Yunus Kara, Gülşah Şanlı-Mohamed
PII:
S0045-2068(19)31330-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103421
YBIOO 103421
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To appear in:
Bioorganic Chemistry
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Revised Date:
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13 August 2019
23 September 2019
2 November 2019
Please cite this article as: A. Köse, M. Kaya, N.H. Kishalı, A. Akdemir, E. Şahin, Y. Kara, G. Şanlı-Mohamed,
Synthesis and biological evaluation of New Chloro / Acetoxy Substituted Isoindole Analogues as new tyrosine
kinase inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103421
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Synthesis and biological evaluation of New Chloro / Acetoxy Substituted Isoindole
Analogues as new tyrosine kinase inhibitors
Aytekin Köse†, Meltem Kaya‡, Nurhan H. Kishalı§, Atilla Akdemir⸹, Ertan Şahin§, Yunus
Kara§*, Gülşah Şanlı-Mohamed‡*
†Department of Chemistry, Faculty of Sciences and Letters, Aksaray University, 68100
Aksaray Turkey
‡Department of Chemistry, Faculty of Sciences, İzmir Institute of Technology, 35430 İzmir
Turkey
§Department of Chemistry, Faculty of Sciences, Ataturk University, 25240 Erzurum Turkey
⸹Computer-Aided Drug Discovery Laboratory, Department of Pharmacology, Faculty of
Pharmacy, Bezmialem Vakif University, 34093 Istanbul, Turkey
Corresponding Author
Prof. Dr. Gülşah Şanlı-Mohamed
Izmir Institute of Technology, Science Faculty, Department of Chemistry
Urla, Izmir, Turkey
Phone: +90 232 7507515; Fax: +90 232 7507509
E-mail: gulsahsanli@iyte.edu.tr, gulsahsanli@hotmail.com
Prof.Dr.Yunus Kara
Atatürk University, Faculty of Sciences, Department of Chemistry, Erzurum Turkey
Phone: +90 442 2314424
E-mail: yukara@atauni.edu.tr

Abstract
We have developed a versatile synthetic approach for the synthesis of new isoindole derivatives via the
cleavage of ethers from tricyclic imide skeleton compounds. An exo-cycloadduct prepared from the
Diels–Alder reaction of furan and maleic anhydride furnished imide derivatives. The epoxide ring was
opened with Ac2O or Ac2O / AcCl in the presence of a catalytic amount of H2SO4 in order to yield new
isoindole derivatives 8a-d and 9a-d. The anticancer activity of these compounds was evaluated against
the HeLa cell lines. The synthesized compounds showed inhibitory effects on the viability of HeLa cells
and the degree of cytotoxicity was increased with the level of bigger branched isoindole derivatives. To
better understand the acting mechanism of these molecules, western blot analysis was performed with
using mTOR and its downstream substrates. In addition, human mTOR and ribozomal S6 kinase β1
(RS6Kβ1) have been investigated with molecular modelling studies as possible targets for compound
series 8 and 9.
Keyword: Norcantharimides; Isoindole; Cytotoxicity; HeLa cells

Introduction
The norcantharimides are known as 3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-
1,3(2H)-dione derivatives, as shown in Fig. 1. Two methods are generally used to derive
norcantharimide or isoindole derivatives. Various substituted groups are bonded to the nitrogen
atom in the imide ring or to any atom in the cyclohexane ring.
Fig.1. The structure of norcantharimide (1), isoindoline-1,3-dione (2), and isoindoline-1,3-
dione derivatives 3.
In recent years, the biological significance of different N-substituted isoindole-1,3-dione
derivatives has been investigated by various research groups [1-7]. One class of N-substituted
isoindole-1,3-dione derivatives contains the norcantharimides, which also have potential
cytotoxic effects [2,4].
The anticancer activities of a great deal of isoindole-1,3-dione derivatives have been
studied in various cell lines, such as human liver carcinoma cell lines (HepG2, Hep3B, and SK-
HEP-1), a bladder cancer cell line (BFTC905), epithelial cells, human lung adenocarcinoma
(A549), Caucasian promyelocytic leukemia cells (HL-60), a human breast adenocarcinoma cell
line (MCF-7), and colon carcinoma cell line SW480 [5-9]. McCluskey et al. reported the
anticancer activity of different groups related to the imide nitrogen in isoindole-1,3-dione [5-
9]. The norcantharimides or isoindole-1,3-dione are known to interact with protein
phosphatases such as PP1 and PP2A. The anticancer activity of norcantharimides is believed to
be due to the inhibition of protein phosphatase 1 and protein phosphatase 2A. Recently,
researchers have been exploring the connection between the protein phosphatase inhibitor and
the anticancer activity [6-11].

In our previous papers we described a facile synthesis of isoindoline-1,3-dione
derivatives 3 from 3-sulfolene [12]. Unlike other synthetic methods, we developed the synthesis
of a new class of isoindole derivatives containing substituted groups on the cyclohexane ring.
More recently, we synthesized novel isoindole derivatives and examined their antiproliferative
properties in cell lines MCF-7 (breast adenocarcinoma) and A549 (human alveolar basal
epithelial adenocarcinoma) [13]. The results demonstrated here suggest that these new
compounds might be considered as possible potential anticancer agents for the treatment of
lung and breast cancer.
In light of these findings, we decided to develop an alternative strategy for the synthesis
of chlorine- and acetate-substituted isoindole derivatives. Herein, we report the synthesis and
evaluation of cytotoxicities of isoindole derivatives containing chlorine atom and acetate
functional groups against human cervical carcinoma cells.
Result and Discussion
Chemistry Part
For the synthesis of norcantharimide derivatives, 3a,4,7,7a-tetrahydro-4,7-
epoxyisobenzofuran-1,3-dione (6) was used as the key compound. This key compound was
prepared via an exo-selective cycloaddition of furan and maleic anhydride (Scheme 1) [13-15].
The reaction of the appropriate primer amine with epoxyisobenzofuran-1,3-dione 6 in the
presence of MeOH gave the tricyclic imides in 54-90% yield 7a-d (Scheme 1). Through the use
of four different primary amines under the same reaction conditions we were able to synthesize
corresponding imides 7a-d.
Scheme 1. Synthesis of tricyclic imides 7a-d

In our previous paper we described a facile synthesis of 2-alkyl-1,3-dioxo-2,3,3a,4,7,7a-
hexahydro-1H-isoindole-4,7-diyl diacetate from tricyclic imides 7a by cleavage of ethers from
tricyclic imide skeletal compounds [13]. In this work, treatment of tricyclic imides 7a-d with
acetic anhydride in the presence of H2SO4 at room temperature gave trans-diacetate derivatives
of isoindole-1,3-dione 8a-d as a sole product in a yield of 57-82%. In this reaction, the etheric
bond is cleaved stereospecifically to give trans-diacetate 8a. Furthermore, a series of tricyclic
imides were prepared in order to obtain trans-diacetate derivatives. Under the same reaction
conditions, trans-acetate derivatives 8b-d were obtained from the cleavage of ethers of prepared
tricyclic imides 7b-d (Scheme 2).
Scheme 2. Synthesis and conditions of compounds 8a-d
Based on this reaction, we decided to synthesize chlorine- and acetate-substituted
isoindole derivatives. For this purpose, we used acetyl chloride in the presence of H2SO4 for the
cleavage of ethers. Treatment of tricyclic imide 7a with acetyl chloride in the presence of H2SO4
in acetic anhydride at room temperature gave chloroacetate isoindole 9a as the sole product in
a yield of 67%. We expected formation of 7-chloro-2-methyl-1,3-dioxo-2,3,3a,4,7,7a-
hexahydro-1H-isoindol-4-yl acetate by the H2SO4-assisted ring opening of 7a. However, the
rearrangement product 4-acetoxy-5-chloroisoindole derivative 9a was formed in the presence
of acetyl chloride in this reaction (Scheme 3).

Scheme 3. Synthesis and conditions of compounds 9a-d
1
The most conspicuous features in the H NMR spectrum of chloroacetate 9a were the
presence of the double bond protons and incorporation of the chlorine atom in the molecule.
Double resonance experiments clearly indicated that the HC=CH double bond was located
between the carbon atoms bearing chlorine atom C(5) and bridgehead carbon C(7a).
1
Furthermore, H NMR analysis of the crude product suggested that chloroacetate 9a was a
single stereoisomer, although two stereoisomers would be formed. The exact stereochemistry
of the chlorine atom in 9a was confirmed by single-crystal X-ray analysis of chloroacetate 9a
(Fig. 2).
Compound 9a crystallizes in the monoclinic space group C2/c with two molecules in
the asymmetric unit and each one has the same stereochemistry. The cyclohexene unit has the
half-chair conformation; C-C (cyclohexene) distances are in the range of 1.485(4)-1.530(4) Å
and all have the single bond character. The C2=C3 double bond in the cyclohexene unit is
1.310(4) Å, the pyrrolidine unit has an envelope conformation, and C-N bond lengths are in the
range of 1.372(4)-1.382(4) Å. The C4-Cl1 bond distance is 1.800(3) Å. The structure contains
four asymmetric carbon atoms and the stereogenic centers are as follows: C1(R), C4(R), C5(R),
C6(R). In the solid state, compound 9a is stabilized via numerous intermolecular C-H∙∙∙O
[D∙∙∙A=3.264(3)-3.507 Å] interactions, which leads to the formation of a polymeric structure.
Along with that, C-H∙∙∙Cl [D∙∙∙A=3.487(4) Å] interactions have a contribution in the formation
of a stable structure (Fig. 2b).

(a)
(b)

(c)
Fig. 2. a) Crystal structure of compound 9a with atom labeling scheme (thermal ellipsoids are
drawn at the 40% probability level). b) Intermolecular interactions of 9a indicated by dotted
lines. There are two molecules in the asymmetric unit and the circumference of each is different.
c) Unit cell with the packing of the molecules in the crystal.
The regio- and stereoselective formation of product 9a is remarkable. We suppose that
the formation of product 9a from 8a proceeds by SN2′ reactions [16] as outlined in Scheme 4.
Scheme 4. The proposed mechanism for the synthesis of 4-acetoxy-5-chloroisoindole
derivative 9
In the first step, tricyclic imide converts to diacetate in the presence of acetyl chloride
in acetic anhydride. The free chloride ion (Cl-) is also formed during this reaction. The SN2′
reaction with Cl- can then occur at C(5) on the syn-face with respect to the acetoxy group (in
acidic medium) in the allylic position at C(7) to give chloroacetate 9a. Furthermore, the X-ray
of structure 9a can inform us about the approach of the chloride ion (Cl-). The X-ray crystal

structure indicates clearly that the chloride ion approaches the double bond in 8a exclusively
from the sterically less crowded face with respect to the imide ring of the molecule and this
leads to trans-chloroacetate 9a. To support the proposed reaction mechanism, we carried out
another experiment with tricyclic imide 7a for a better understanding of the reaction. The
1
reaction mixture was monitored by recording H NMR spectra at four different times. As the
halogenation reaction proceeded, monitoring of the product ratio by NMR showed an increase
in the signals corresponding to chloroacetate 9a over the course of time. On the basis of this
observation, the reaction proceeds first with protonation followed by attack of the free chloride
to substitute the acyl group. After this promising result, a series of tricyclic imides were
prepared in order to obtain trans-chloroacetate derivatives. Under the same reaction conditions,
trans-chloroacetate derivatives 9b-d were obtained by acetylation followed by displacement
reaction (Scheme 3).
Biological evaluation
Cytotoxic effects of all newly synthesized compounds (drugs) on HeLa cells were
determined and IC50 values of those drugs were calculated using the MTT activity assay. Cell
viability of HeLa cells using all seven compounds was followed for 24, 48, and 72 h of
incubation time. According to the results of these experiments, the optimum incubation period
was determined as 48 h. Some compounds, and especially 9c, were not effective against HeLa
cells when incubated for 24 h. This might be related to the dissolution of the drug in the cell
medium and transportation into the cell. In 2015, Mondal et al. used 1-amino-4-hydroxy-9,10-
anthraquinone on MDA-MB-231 breast adenocarcinoma cells; they reported 50% inhibitory
rates of this molecule as 200 µM for 24 h of incubation and 140 µM for 48 h of incubation [17].
In another study, 3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (PPDHMP), which was
extracted from a new marine bacterium called Staphylococcus sp. strain MB30, was used on
A549 (lung cancer cells), HeLa (cervical cancer cell line), and normal peripheral blood
mononuclear cells (PBMCs). PPDHMP was used at various concentrations (1, 10, 25, 50, and
100 µg/mL) for 6, 12, 18, and 24 h of incubation. It showed no toxic effects on PBMCs, but it
had 19.94 µg/mL IC50 on HeLa cells and 16.73 µg/mL IC50 onA549 cells for 24 h of incubation.
The most effective inhibition was observed with 24 h of incubation in that research [18].
On the other hand, when drugs were incubated for 72 h, 9a and 8b in particular lost their
stability/effectiveness, and cells might become more resistant to drugs after long incubation
periods. In 2003, Mai and his co-workers investigated the effect of pyrrole-C(2) and -C(4)

substitutions on biological activity and they reported some molecules (compounds 3a and 1b
in their article) as antiproliferative agents on Friend murine erythroleukemia (MEL) cells. They
also reported that the cytotoxic activity of these compounds decreased after 48 h of incubation
[19].
Table 1. Structure of drugs as a group with their IC50 values.
GROUP 1 IC50
GROUP 2 IC50
9a
9b
9c
9d
382.82 μM
8b
8c
8d
542.00 μM
171.40 μM
255.86 μM
140.60 μM
366.44 μM
148.59 μM
(The results were obtained using the GraphPad Prism 6 software program [20]).
The IC50 values of 8b, 8c, 8d, 9a, 9b, 9c, and 9d were 542.00 µM, 366.44 µM, 148.59
µM, 382.82 µM, 171.40 µM, 255.86 µM, and 140.60 µM, respectively (Table 1). It can be
concluded that 9d, 8d, and 9b have lower IC50 values. 8b has the maximum IC50 at 542 µM.
In order to determine the relationship between the structures of the synthesized compounds and
their anticancer activity, it would be rational to classify these compounds into two main groups:
i) The substituted groups bonded to the nitrogen atom of the imide. There are three different
groups bonded to the nitrogen atom. These are benzyl, ethyl/metyl and phenyl groups. ii) The
substituted groups bonded to any atom of the cyclohexane ring. For this purpose, two
derivatives were prepared for each group bonded to the nitrogen atom. These are 1-acetoxy-2-
chlorocylohexane (9a-d) (Scheme3) and 1,4-diacetoxy cyclohexane derivatives (8b-d)
(Scheme 2). A total of seven derivatives were prepared. Looking more closely at the structures
and IC50 values of the first group (9a, 9b, 9c, and 9d), 9d (with the lowest IC50) has a benzyl
group, 9b has an ethyl group, 9c has a phenyl group, and 9a has a methyl group in their
respective C(2)-positions. The order of IC50 values is 9d < 9b < 9c < 9a. According to these
results we may suggest that as the -R group in the C(2)-position branches, the IC50 values
decrease. In the second group, 8d has a benzyl group, 8c has a phenyl group, and 8b has an
ethyl group in their respective C(2)-positions. The order of IC50 values is 8d < 8c < 8b, which
also respect to the attached the group to nitrogen atom.

On analysis of this data, it could broadly be concluded that, most active compounds
were derivatives of benzyl and ethyl groups on nitrogen atom compared to compounds are
derivatives of phenyl group on nitrogen atom against HeLa cells. It seems phenyl group on
nitrogen atom does not assist the compounds for improvement of anticancer activity. On the
other hand, as seen from the table, the groups attached to the cyclohexane ring act more effective
on the anticancer activity. The effect of 1-acetoxy-2-chlorocyclohexane derivatives 9b was
better than the effect of 1,4-diacetoxy cyclohexane derivatives 8b against HeLa cells. This is
clear evident when the 1-acetoxy-2-chlorocyclohexane derivative of the ethyl group is attached
to the nitrogen atom, compared to the 1,4-diacetoxycyclohexane derivative. In addition, the
methyl group was replaced with the ethyl group and the activity was determined to decrease.
In summary, in case of HeLa cells, excellent anticancer activity was noticed by three
compounds 8d, 9b and 9d. It was interesting to note that the electron-donating groups such as
benzyl and ethyl substituent which is to bonded nitrogen atom may be responsible for the
improvement of anticancer activity against HeLa cells. Cytotoxic effect was found to be greater
when benzyl group was attached to nitrogen atom.
Western Blot analysis
According to results of MTT assay 9d, 8c, 8d and 9b showed more antiproliferative
effect on HeLa cells and to better understand their acting mechanism, their relation with
autophagy, they were chosen for Western Blot analysis.
For western blot, Cell Cignalling Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit
mAb 9234, Phospho-p70 S6 Kinase (Ser371) Antibody 9208, Phospho-4E-BP1 (Thr37/46)
(236B4) Rabbit mAb 2855 proteins were investigated. For primary and secondary antibody,
Anti-rabbit IgG, HRP-linked Antibody 7074 and ß-Actin (13E5) rabbit mAb were chosen.
Two SDS-PAGE gel were performed for 3 protein, Beta Actin, p-4EBP1 and p-p70S6K.
First gel imaging shows that the phosphorylated level of 4E-BP1 was overexpressed for the
cells which were treated with 9d, 8c and 8d, while control group and 9b had approximately
same protein level. Overexpression order is 9d, 8c and 8d. It is known that if mTOR inhibited,
4E-BP1 should overexpressed according to their inversely proportional relation (Figure 3 and
6). Besides this, there were significant inhibition for phosphorylated level of p70S6K (Thr)
protein, suggesting there were mTOR inhibition in the cells treated with these four drugs

according to the direct proportional relation with mTOR and p70S6K (Figure 3 and 6). Figure
4 and 5 are quantification of the bands in the gel 1.
Figure 3. Imaging of Gel 1
Figure 4. Relative Expression of p-p70S6K (Thr)
Figure 5. Relative Expression of p-4E-BP1
For the second gel, p-4EBP1 and p-p70S6K (Ser) antibodies were used. It was observed
that there were overexpression of phosphorylated level of 4EBP1 protein for the cells treated
with 9d, 8c and 8d (in order of overexpression) according to untreated control group. Also,
significant inhibiton for p-p70S6K (Ser) level in cells treated with these four drugs according

to control group were observed. Because of the high exposure time, there was high background
for p-p70S6K (Ser) membrane. Figure 7 and 8 are quantification of gel 2.
Figure 6. Imaging of Gel 2
Figure 7. Relative Expression of p-4E-BP1
Figure 8. Relative Expression of p-p70S6K (Ser)
For the phosphorylated form of the 4EBP1, 9d and 8c had the maximum overexpressive
effect, while they both inhibited the phosphorylated form of p70S6K (Thr). By looking the

effects of each drug on protein levels, it might be a potent inhibitor. In a study published on
2016 suggest that 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione inhibits HeLa
cell growth by causing a G1 arrest and by concomitantly inducing autophagy through the
mediation ofAMPK-mTOR and Akt-mTOR pathways, and may be a promising antitumor agent
against cervical cancer [21]. Overall these findings suggest that drugs may inhibit cervical
cancer cell proliferation by repressing the mTOR pathway.
Molecular modelling studies
Human mTOR and ribozomal S6 kinase β1 (RS6Kβ1) have been investigated as
possible targets for compound series 8 and 9 (Table 1). To this end, crystal structures of human
mTOR (pdb: 4jt5; 3.45 Å) and human RS6Kβ1 (pdb: 4l3j; 2.1 Å), both in complex with an
inhibitor, were retrieved from the RCSB Protein DataBank (www.rcsb.org) [22].
Docking studies indicated that only compound 8c may be able to bind to human mTOR
(Figure 9). Compound 8c forms hydrogen bonds to the side chain of Lys2187 and the backbone
of Val2240. The phenyl group of the ligand is within cation-π interaction distance (~3.5 Å) from
the side chain of Lys2187 and it also forms an arene-H interaction with Ile2356. A benzyl or
ethyl group at this position disturbs these binding interactions. Compound series 9 have a
different scaffold with different relative orientations of the acetyl group with respect to the five-
membered ring. In addition, it has a chlorine substituent. The structural changes also make the
observed docked pose difficult to adopt in the mTOR binding pocket.

Figure 9. The docked pose of compound 8c (turquoise) in the binding pocket of human mTOR.
Hydrogen bonds are indicated in red dashed lines, arene-H interactions are indicated in yellow
dashed lines and the surface of the binding pocket is indicated with a white mesh.
Docking studies into the active site of human RS6Kβ1 indicated that three different
binding poses may be possible (Figure 10). Compound 8d forms hydrogen bonds to the side
chains of Lys100 and Lys218 and an arene-H interaction between the ligand’s phenyl group and
the side chain of Leu74. The two carbonyl groups of the pyrrolidine-2,5-dione moiety do not
form any interactions to the active site, but may form hydrogen bonds to the surrounding
solvent. Compound 9d also forms a hydrogen bond to the side chain of Lys100 and a second
hydrogen bond to the backbone of Leu152. The second carbonyl group of the pyrrolidine-2,5-
dione moiety does not form interactions with the protein but it is solvent exposed. Finally,
compound 9d also forms hydrogen bonds to the side chains of Lys100 and Lys218. All ligands
form hydrophobic interactions with the active site.
Figure 10. The docked poses of compound 8d (panel A; brown) and compounds 9b and 9d
(panel B; purple and turquoise, respectively) in the binding pocket of human RS6Kβ1.
Hydrogen bonds are indicated in red dashed lines, arene-H interactions are indicated in yellow
dashed lines and the surface of the binding pocket is indicated with a white mesh.

Conclusions
Here we have reported an efficient synthesis of new derivatives of isoindole-1,3-dione
bearing chloro/acetoxy groups at the 1,2-position of the cyclohexane ring. This method has the
potential to be widely used in chloro- and acetoxy-substituted isoindole synthesis. The cytotoxic
activities of these compounds were then evaluated in HeLa cells. Based on the results of the
study, we suggest that all the newly synthesized isoindole derivatives might be good potential
anticancer agents for the treatment of cervical cancer due to their antiproliferative activities in
cancer cells. Molecular modelling studies suggested that human mTOR and ribozomal S6
kinase β1 (RS6Kβ1) may act as possible targets for compound series 8 and 9 and that these new
derivatives of isoindole-1,3-dione may inhibit cervical cancer cell proliferation by repressing
the mTOR pathway. However, the cytotoxic properties of these compounds should be
thoroughly investigated in vitro and in vivo in detail for potential clinical utilization.
Furthermore, the synthesis and activities of alternative isoindole derivatives are currently under
investigation.
Experimental
Materials
All reagents used were commercially available unless otherwise specified and all
solvents were distilled before use. Melting points were measured with Gallenkamp melting
1
13
point devices. IR spectra: PerkinElmer Spectrum One FT-IR spectrometer. H and C NMR
spectra: Varian 400 MHz and Bruker 400 MHz spectrometers. Elemental analysis results were
obtained on a LECO CHNS-932 instrument.
Chemistry Part
Synthesis of 3a,4,7,7a-tetrahydro-4,7-epoxyisobenzofuran-1,3-dione (6):
The synthesis was carried out following the literature procedure. [14-16]. In a 250-mL
three-neck round-bottom flask under argon atmosphere, maleic anhydride (5.0 g, 50.99 mmol)
and furan (5.2 g, 76.5 mmol) were dissolved in 20 mL of CH2Cl2. The mixture was stirred for
24 h while heating under reflux. A white precipitate formed during this time. The solid was

collected by filtration and washed two times with cold diethyl ether. The filtrate was reduced
by rotary evaporation to 20 mL and cooled to 4 °C overnight. A second crop crystallized, which
was again collected by filtration and washed with diethyl ether. Finally, the crystals were dried
in a vacuum (≈10–2 mbar) overnight. Yield: 8.22 g (97%) of a white solid. 1H NMR (400 MHz,
CDCl3) δ 6.57 (s, 2H, H5, H6), 5.45 (s, 2H, H4, H7), 3.18 (s, 2H, H3a, H7a). 13C NMR (100 MHz,
CDCl3) δ 170.1 (2 CO), 137.2 (C5, C6), 82.4 (C4, C7), 48.9 (C3a, C7a).
General procedure for the synthesis of 4,7-epoxyisoindole-1,3(2H)-dione derivatives 7a-d:
The synthesis of 4,7-epoxyisoindole-1,3(2H)-dione derivatives started with 3a,4,7,7a-
tetrahydro-4,7-epoxyisobenzofuran-1,3-dione (1.50 g, 9.03 mmol) (6). Compound 6 was
dissolved in MeOH (15 mL) and primary alkyl/aryl amine (9.03 mmol) was added to the
solution. It was refluxed in an oil bath for 24 h, and then it was cooled to room temperature and
the crude product began to crystallize. It was put into a refrigerator overnight and the precipitate
was filtered off. It was washed with hexane to give isoindole-1,3-dione derivatives 7a-d.
Methyl-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (7a): Yield: 82%
colorless crystals, M.p: 138–140 °C, 1H NMR (400 MHz, CDCl3) δ 6.49 (m, 2H, H5, H6), 5.24
(m, 2H, H4, H7), 2.94 (s, 3H, CH3), 2.83 (s, 2H, H3a, H7a). 13C NMR (100 MHz, CDCl3) δ 176.5
(2xCO of imide), 136.7 (C5 and C6), 81.0 (C4 and C7), 47.7 (C3a and C7a), 25.1 (CH3). Anal.
Calcd. for C9H9NO3: C, 60.33; H, 5.06; N, 7.82. Found: C, 60.68; H 4.91; N 7.66.
2-Ethyl-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (7b): Yield: 90%
1
White powder, M.p.:119-121 °C. H NMR (400 MHz, CDCl3) δ 6.49 (s, 2H), 5.23 (s, 2H),
3.50 (q, J = 7.2 Hz, 2H), 2.81 (s, 2H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
176.30, 136.77, 81.09, 47.64, 34.09, 13.14. Anal. Calcd. for C10H11NO3: C, 62.17; H, 5.74; N,
7.25. Found: C, 61.63; H, 5.87; N, 7.45.
4.2.2.3. 2-Phenyl-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (7c): Yield:
55% Yellow crystalline solid, M.p.:134-136 °C. 1H NMR (400 MHz, CDCl3) δ 7.45-7.28 (m,
13
5H), 6.54 (s, 2H), 5.37 (s, 2H), 2.98 (s, 2H). C NMR (100 MHz, CDCl3) δ 175.63, 136.93,
131.93, 129.39, 129.03, 126.81, 81.65, 47.77 Anal. Calcd. for C14H11NO3: C, 69.70; H, 4.60;
N, 5.81. Found: C, 69.77; H, 4.72; N, 5.77.
4.2.2.4. 2-Benzyl-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (7d): Yield:
1
54% White crystalline solid. M.p.:130-132 °C. H NMR (400 MHz, CDCl3) δ 7.29 (m, 5H),
6.47 (s, 2H), 5.25 (s, 2H), 4.62 (s, 2H), 2.82 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 176.17,
136.79, 135.72, 128.86, 128.30, 128.00, 81.15, 47.75, 42.64. Anal. Calcd. for C15H13NO3: C,
70.58; H, 5.13; N, 5.49. Found: C, 70.60; H, 5.27; N, 5.86.

Acid-catalyzed acetolysis of 7 with Ac2O: The synthesis of 1,4-diacetoxy derivatives 8a-d:
Compounds 8a-d were synthesized as described previously [14]. Isoindole derivative
7a (1.0 g, 3.98 mmol) was dissolved in Ac2O (5 mL). After the addition of 3 drops of H2SO4,
the reaction mixture was magnetically stirred at room temperature for 2 days. Ac2O was
removed under reduced pressure. The crude product was recrystallized from CH2Cl2 / hexane
to give 1,4-diacetate derivatives 8a-d.
2-Methyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-isoindole-4,7-diyl-diacetate (8a)14: Yield:
82%. Colorless crystals, M.p.: 135–137 °C. 1H NMR (400 MHz, CDCl3) δ 6.26 (dm, J = 10.0
Hz, 1H, H6) 6.01 (m, 1H, H5), 5.34 (m, 1H, H7), 5.16 (t, J = 4.8 Hz, 1H, H4), 3.49 ( bs, 2H, H3a,
H7a), 2.96 (s, 3H, N-CH3), 2.03 (s, 3H, C7-OAc), 1.91 (s, 3H, C4-OAc). 13C NMR (100 MHz,
CDCl3) δ 175.5 (C1), 175.2 (C3), 169.6 (C4-OAc), 169.2 (C7-OAc), 126.6 (C6), 125.2 (C5), 67.0
(C7), 64.3 (C4), 40.0 (C7a), 39.6 (C3a), 25.1 (N-CH3), 21.0 (COCH3), 20.8 (COCH3). Anal.
Calcd. for C13H15NO6: C 55.51; H 5.38; N 4.98. Found: C 55.19; H 5.29; N 5.00.
2-Ethyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-isoindole-4,7-diyl diacetate (8b): Yield:
62%. White powder, M.p.: 141-143 °C. 1H NMR (400 MHz, CDCl3) δ 6.32 (m, 1H, H6), 6.06
(m, 1H, H5), 5.38 (m, 1H, H7), 5.18 (quasi t, J = 4.8 Hz, 1H, H4), 3.55 (q, J = 7.1 Hz, 2H, N-
CH2), 3.46 (m, 2H, H3a, H7a), 2.05 (s, 3H, C7-OAc), 1.93 (s, 3H, C4-OAc), 1.15 (t, J = 7.2 Hz,
3H, N-CH2CH3). 13C NMR (100 MHz, CDCl3) δ 175.36, 174.97, 169.57, 169.20, 126.92,
124.97, 67.01, 64.07, 39.83, 39.45, 34.14, 20.99, 20.78, 13.24. Anal. Calcd. for C14H17NO6: C,
56.95; H, 5.80; N, 4.74. Found: C, 56.40; H, 5.73; N, 4.67.
1,3-Dioxo-2-phenyl-2,3,3a,4,7,7a-hexahydro-1H-isoindole-4,7-diyl diacetate (8c): Yield
63%. Pale yellow solid. M.p.: 162-164 °C. 1H NMR (400 MHz, CDCl3) δ 7.51-7.24 (m, 5H),
6.42 (dd, J = 10.1, 3.5 Hz, 1H, H6), 6.14 (m, 1H, H5), 5.50 (quasi t, J = 4.2 Hz, 1H, H7), 5.28
(quasi t, J = 4.8 Hz, 1H, H4), 3.68 (m, 2H, H3a, H7a), 2.09 (s, 3H, C7-OAc), 2.00 (s, 3H, C4-
OAc).13C NMR (100 MHz, CDCl3, ppm) δ 174.5, 174.32, 169.56, 169.13, 131.76, 129.53,
129.06, 126.76, 126.32, 125.13, 67.23, 63.95, 39.84, 39.72, 21.01, 20.96. Anal. Calcd. for
C18H17NO6: C, 62.97; H, 4.99; N, 4.08. Found: C, 62.47; H, 5.07; N, 4.04.
2-Benzyl-1,3-dioxo-2,3,3a,4,7,7a-hexahydro-1H-isoindole-4,7-diyl diacetate (8d): Yield
57%. White solid. M.p.: 92-94 °C. 1H NMR (400 MHz, CDCl3) δ 7.43-7.24 (m, 5H), 6.33 (dd,
J = 10.0, 2.9 Hz, 1H, H6), 6.04 (m, 1H, H5), 5.36 (quasi t, J = 4.0 Hz, 1H, H7), 5.11 (quasi t, J
= 4.0 Hz, 1H, H4), 4.63 (dd, JAB = 14.0, 5.5 Hz, 2H, N-CH2 ), 3.48 (m, 2H, H3a, H7a ), 2.04 (s,
13
3H, C7-OAc), 1.39 (s, 3H, C4-OAc). C NMR (100 MHz, CDCl3) δ 175.28, 174.84, 169.45,
169.16, 135.87, 129.44, 128.88, 128.22, 127.01, 124.69, 66.46, 63.86, 42.77, 39.64, 39.42,

20.89, 19.96. Anal. Calcd. for C19H19NO6: C, 63.86; H, 5.36; N, 3.92. Found: C, 63.64; H, 5.17;
N, 3.92.
General procedure for the synthesis of 1,2-chloroacetate derivatives 9a-d:
In a round-bottom flask, tricyclic compounds 7a-d (0.5 g) were dissolved with 4 mL of
Ac2O and then 2 mL of AcCl was added to this slurry solution at 25 °C. After that, three drops
of H2SO4 were added drop by drop. After that addition, the color of the reaction began to darken.
The resulting solution was stirred for 48 h. The reaction was concentrated under vacuum and
unreacted AcCl was removed. The crude product was precipitated and the liquid layer was
concentrated under vacuum. It was crystallized from CH2Cl2 / hexane to give 1,2-chloroacetate
derivatives 9a-d.
5-Chloro-2-methyl-1,3-dioxo-2,3,3a,4,5,7a-hexahydro-1H-isoindol-4-yl acetate (9a): Yield
67%. Colorless crystal, M.p.: 89-91 °C. 1H NMR (400 MHz, CDCl3, ppm): δ 6.28 (dd, J = 9.9,
3.7 Hz, 1H, A part of AB system, H6), 6.02 (m, 1H, B part of AB system, H7), 5.47 (quasi t, J
=4.1 Hz, 1H, H4) 4.47 (dd, J = 5.6, 3.5 Hz, 1H, H5), 3.68 (dd, J = 8.9, 4.4 Hz, 1H, H3a), 3.51
(dt, J = 8.8, 3.3 Hz, 1H, H7a), 2.98 (s, 3H, C4-OAc), 1.90 (s, 3H, N-CH3). 13C NMR (100 MHz,
CDCl3, ppm): δ 175.50, 175.36, 169.13, 125.81, 125.57, 69.19, 48.30, 39.28, 39.01, 25.09,
20.71. Anal. Calcd. for C11H12ClNO4: C, 51.27; H, 4.69; N, 5.44. Found: C, 50.98; H, 4.73; N,
5.40.
5-Chloro-2-ethyl-1,3-dioxo-2,3,3a,4,5,7a-hexahydro-1H-isoindol-4-yl acetate (9b): Yield
58%, pale yellow solid. M.p.:105-107 °C. 1H NMR (400 MHz, CDCl3) δ 6.32 (dd, J = 9.9, 3.7
Hz, 1H, A part of AB system, H6), 6.03 (m, 1H, B part of AB system, H7), 5.50 (quasi t, J = 3.9
Hz, 1H, H4), 4.50 (dd, J = 5.9, 3.3 Hz, 1H, H5), 3.67 (dd, J = 9.0, 4.5 Hz, 1H, A part of AB
system, H3a), 3.58 (qd, J = 7.3, 1.5 Hz, 2H, N-CH2), 3.50 (dt, J = 9.0, 3.3 Hz, 1H, B part of AB
system, H7a), 1.92 (s, 3H, C4-OAc), 1.16 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3)
δ 175.30, 175.13, 169.26, 125.70, 125.66, 69.16, 47.97, 39.03, 38.52, 34.01, 20.53, 13.25. Anal.
Calcd. for C12H14ClNO4: C, 53.05; H, 5.19; N, 5.16. Found: C, 52.91; H, 5.19; N, 5.33.
5-Chloro-1,3-dioxo-2-phenyl-2,3,3a,4,5,7a-hexahydro-1H-isoindol-4-yl acetate (9c): Yield
60%, white solid. M.p.:125-127 °C. 1H NMR (400 MHz, CDCl3) δ 7.50-7.23 (m, 5H), 6.40 (dd,
J = 9.8, 3.7 Hz, 1H, A part of AB system, H6), 6.10 (m, 1H, B part of AB system, H7), 5.59
(quasi t, J = 3.7 Hz, 1H, H4), 4.57 (dd, J = 5.8, 3.3 Hz. 1H, H5), 3.85 (dd, J = 9.2, 4.4 Hz, 1H,
A part of AB system, H3a), 3.72 (dd, J = 9.2, 3.3 Hz, 1H, B part of AB system, H7a), 1.99 (s,
3H, C4-OAc). 13C NMR (100 MHz, CDCl3) δ 174.45, 174.44, 169.11, 131.74, 129.56, 129.11,

126.32, 125.92, 125.43, 69.56, 48.09, 39.25, 38.97, 20.90. Anal. Calcd. for C16H14ClNO4: C,
60.10; H, 4.41; N, 4.38. Found: C, 60.34; H, 4.61; N, 4.38.
2-Benzyl-5-chloro-1,3-dioxo-2,3,3a,4,5,7a-hexahydro-1H-isoindol-4-yl acetate (9d): Yield
51%, white solid. M.p:81-83 °C. 1H NMR (400 MHz, CDCl3) δ 7.43-7.24 (m, 5H), 6.31 (dd, J
= 9.9, 3.7 Hz, 1H, A part of AB system, H6), 6.01 (m, 1H, B part of AB system, H7), 5.44 (quasi
t, J = 4.4 Hz, 1H, H4), 4.63 (d, JAB = 20.9 Hz, 2H, N-CH2), 4.42 (dd, J = 5.9, 3.3 Hz, 1H, H5),
3.66 (dd, J = 9.2, 4.4 Hz, 1H, A part of AB system, H3a), 3.52 (dt, J = 9.2, 3.3 Hz, 1H, B part
of AB system, H7a), 1.37 (s, 3H, C4-OAc). 13C NMR (100 MHz, CDCl3) δ 175.2, 174.9, 169.3,
135.8, 129.7, 129.0,128.4, 125.7, 125.6, 68.9, 48.1, 43.0, 39.2, 38.7, 19.9. Anal. Calcd. for
C17H16ClNO4: C, 61.18; H, 4.83; N, 4.20. Found: C, 61.47; H, 4.93; N, 4.24.
Biological part
Cell cultures
HeLa cell lines were kindly provided by IZTECH Biotechnology and Bioengineering
Research and Application Center, Izmir Institute of Technology, Turkey. Media for HeLa cells
was Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) with 4500 mg glucose/L,
pyridoxine, HCl and NaHCO3, without L-glutamine. HeLa cells were cultured in DMEM
growth medium with 10% fetal bovine serum (Sigma), 10% pen-strep solution (Biological
Industries) and 10% L-glutamine solution (Biological Industries). Cells were incubated in an
incubator which provides 37°C and 5% CO2. Medium was refreshed every 3 days for each cell
culture. Typically, cells were passaged by trypsinization and in growth medium. All compounds
were dissolved in dimethyl sulfoxide (DMSO) before all the analyses.
Cell Viability Assays
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation
assay was performed to evaluate the cytotoxicity of the synthesized norcantharimide
compounds reflecting the cell viability of HeLa cell lines in presence of the compounds. MTT,
a yellow tetrazole, is reduced to purple formazan in living cell’s mitochondria by mitochondrial
dehydrogenases of viable cells [20]. This reduction only occurs if mitochondrial reductase
enzymes are active; thus, conversion is directly related to the number of viable cells. In the
experiment, cells were cultured in 96-well plates (SPL Life Sciences) with 3x103 cell/well and
waited for 24 hours in order to attach surface of the plate bottom. Compounds were solved in
sterile DMSO and stock solution of each seven compounds were prepared as 200 mM in 100

μl DMSO. Working concentrations were prepared by diluting that stock solutions with
complete media and also DMSO (final DMSO concentration was 1% in a well) before the
experiment. All compounds were added to wells according to 20, 50, 100, 200, 500, 1000, 2000
μM with triplicate assay. After all compound added, cells were incubated 24h, 48h and 72h in
order to determine cytotoxic effects. At the end of each incubation period, media was removed
from wells and MTT stock solution (5 mg/ml PBS) was diluted 1:10 ratio with complete media
and added to wells. After 4 hours incubation, plates were centrifuged at 1800 rpm 10 minutes
at room temperature. Supernatant was removed; DMSO was added to each well and shaked 150
rpm 15 minutes at room temperature in order to solve all formazan crystals. Absorbance was
determined at 540 nm (Thermo Electron Type 1500 Multiskan Spectrum). Metabolic activity
at standard growth conditions was considered as 100%. The concentration inhibiting cell
viability by 50% (IC50 values) was calculated.
Western Blot Analysis
Whole cell protein extracts of compound treated HeLa cells were prepared using
CelLytic™ MEM Protein Extraction Kit (Sigma). Cell Cignalling Phospho-p70 S6 Kinase
(Thr389) (108D2) Rabbit mAb 9234, Phospho-p70 S6 Kinase (Ser371) Antibody 9208,
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb 2855 proteins were detected using primary
and secondary antibody, Anti-rabbit IgG, HRP-linked Antibody 7074 and ß-Actin (13E5) rabbit
mAb. The protein bands visualization was conducted using the Pierce™ ECL Western Blotting
Substrate (Life Technologies) and a G:BOX imaging system.
Statistical Analysis
The results represented the mean ± standard deviation from at least three independent
experiments. Statistical significance was assessed by One-way ANOVA using GraphPad Prism
5, and with the Dunnett's multiple comparison tests.
Molecular modelling studies
The three-dimensional structures of all ligands were prepared in their lowest energy
conformation using the MOE software package (v2019.01, Chemical Computing Group, Inc,
Montreal, Canada) and the ligands were energy minimized (MMFF94x force field).
All protein structures were obtained from the RCSB protein databank: human mTOR
in complex with inhibitor (pdb: 4jt5; 3.45 Å) and human RS6Kβ1 in complex with inhibitor
(pdb: 4l3j; 2.1 Å). The protein and inhibitor atoms were retained and all other atoms were

omitted. The remaining structure was protonated using the protonate 3D functionality of MOE
and subsequently the obtained structure was energy-minimized (AMBER14:EHT) [23].
Docking calculations were performed using the FlexX docking tool (v2.3.2; BioSolveIT
GmbH, St. Augustin, Germany) within MOE. The binding pocket was defined as all residues
within 6.5 Å of the crystallized inhibitors. All ligands were docked fifty times and the best
scoring three poses were subjected to refinement calculations. To this end, the ligand and
binding pocket residues were energy minimized and rescored using GBVI/WSA force field
[24].
Conflicts of Interest
We declare that we have no conflict of interest
Acknowledgments
The authors would like to thank Atatürk University for financial support (Grant No: FCD-2018-
7009 and FFM-2017-6349). The authors also thank the Biotechnology and Bioengineering
Center laboratory staff of Izmir Institute of Technology and for its financial and technical
support.
References
1
N.S. El-Gohary, M.I. Shaaban, Synthesis, Antimicrobial, Antiquorum-Sensing, and
Cytotoxic Activities of New Series of Isoindoline1,3-dione, Pyrazolo[5,1-a]- isoindole,
and Pyridine Derivatives, Arch. Pharm. Chem. Life. Sci. 348 (2015), 666–680.
C.E.P. Galvis, L. Y. V. Méndez, V. K. Kouznetsov, Cantharidin‐ Based Small Molecules
as Potential Therapeutic Agents, Chem. Biol. Drug. Des. 82 (2013), 477-499.
P.F. Lamie, J.N. Philoppes, A.O. El-Gendy, L. Rarova, J. Gruz, Design, Synthesis and
Evaluation of Novel Phthalimide Derivatives as in Vitro Anti-Microbial, Anti-Oxidant
and Anti-Inflammatory Agents, Molecules, 20 (2015), 16620-16642.
2
3
4
J.Y. Wu, C.D. Kuo, C.Y. Chu, M.S. Chen, J.H. Lin, Y.J. Chen, H.F. Liao, Synthesis of
Novel Lipophilic N-Substituted Norcantharimide Derivatives and Evaluation of Their
Anticancer Activities, Molecules, 19 (2014), 6911-6928.

5
6
S.H.L. Kok, C.H. Chui, W.S. Lam, J. Chen, F.Y. Lau, R.S.M. Wong, G.Y.M. Cheng,
P.B.S. Lai, T.W.T. Leung, Synthesis and structure evaluation of a novel cantharimide and
its cytotoxicity on SK-Hep-1 hepatoma cells, Bioorg. Med. Chem. Lett., 17 (2007), 1155-
1159.
T.A. Hill, S.G. Stewart, S.P. Ackland, J. Gilbert, B. Sauer, J.A. Sakoff, A. McCluskey,
Synthesis of new norcantharidin analogues and their anticancer evaluation,.Bioorg. Med.
Chem., 15 (2007), 6126-6134.
7
P.Y. Lin, S.J. Shi, H.L. Shu, H.F. Chen, C.C. Lin, P.C. Liu, L.F. Wang, A simple
procedure for preparationofN-thiazolyl and N-thiadiazolylcantharidinimides and
evalution of their cytotoxicities against humanhepatocellular carcinoma cell, Bioorg.
Chem. 28 (2000), 266-272.
8
T.A. Hill, S.G. Stewart, B. Sauer, J. Gilbert, S.P. Ackland, J.A. Sakoff, A. McCluskey,
Heterocyclic substituted cantharidin and norcantharidin analogues-synthesis, protein
phosphatase (1 and 2A) inhibition and anticancer activity Bioorg. Med. Chem. Lett., 17
(2007), 3392-3397.
9
L.H. Lin, H.S. Huang, C.C. Lin, L.W. Lee, P.Y. Lin, Effects of cantharidins on human
carcinoma cell Chem. Pharm. Bull, 52 (2004), 855-857.
10 A. McCluskey, C. Walkom, M.C. Bowyer, S.P. Ackland, E. Gardiner, J.A. Sakoff,
Effects of cantharidins on human carcinoma cell, Bioorg. Med. Chem. Lett. 11 (2001),
2941-2946.
11 J.A. Sakoff, S.P. Ackland, M.L. Baldwin, M.A. Keane, A. McCluskey, Anticancer
activity and protein phosphatase 1 and 2A inhibition of a new generation of cantharidin
analogues Invest New Drugs, 20 (2002), 1-11.
12 a) A. Tan, B. Koc, E. Sahin, N.H. Kishali, Y. Kara, Synthesis of New Cantharimide
Analogues Derived from 3-Sulfolene, Synthesis, 7 (2011), 1079-1084. b) A. Tan, M.Z.
Kazancioglu, D. Aktas, O. Gundogdu, E. Sahin, N.H. Kishali, Y. Kara, Convenient
synthesis of new polysubstituted isoindole-1,3-dione analogues, Turk. J. Chem., 38
(2014), 629-637. c) A. Tan, E. Bozkurt, N.H. Kishali, Y. Kara, New and Convenient
Synthesis of Amino Phthalimide Derivatives and their Photoluminescent Properties,
Helv. Chim. Acta, 97 (2014), 1107-1114.
13 A. Köse, Y. Bal, N.H. Kishalı, G. Sanlı-Mohamed, Y. Kara, Synthesis and anticancer
activity evaluation of new isoindole analogues, Med. Chem. Res., 26 (2017), 779-786.

14 Y.W. Goh, B.R. Pool, J.M. White, Structural Studies on Cycloadducts of Furan, 2-
Methoxyfuran, and 5-Trimethylsilylcyclopentadiene with Maleic Anhydride and N-
Methylmaleimide, Org. Chem., 73 (2008), 151-156.
15 M. Sodeoka, Y. Baba, S. Kobayashi, N. Hirukawa, Structure-Activity Relationship of
Cantharidin Derivatives to Protein Phosphatases 1, 2A1, and 2B, Bioorg Med Chem Lett.,
7 (1997), 1833-1836.
16 N.H. Kishali, E. Sahin, Y. Kara, Regio- and Stereocontrolled Synthesis of
(2R*,3R*,4R*)-3,4-Dichloro-1,2,3,4,5,8-hexahydronaphthalen-2-yl Acetate via Tandem
SN2′ Reactions Helv. Chim. Acta, 89 (2006), 1246-1253.
17 P. Mondal, S. Roy, G. Loganathan, B. Mandal, D. Dharumadurai, M.A. Akbarsha, P.S.
Sengupta, S. Chattopadhyay, P.S. Guin, 1-Amino-4-hydroxy-9,10-anthraquinone - An
analogue of anthracycline anticancer drugs, interacts with DNA and induces apoptosis in
human MDA-MB-231 breast adinocarcinoma cells: Evaluation of structure-activity
relationship using computational, spectroscopic and biochemical studies, Biochemistry
and Biophysics Reports, 4 (2015), 312-323.
18 P. Lalitha, V. Veena, P. Vidhyapriya, P. Lakshmi, R. Krishna, N. Sakthivel, Anticancer
potential of pyrrole (1, 2, a) pyrazine 1, 4, dione, hexahydro 3-(2-methyl propyl)
(PPDHMP) extracted from a new marine bacterium, Staphylococcus sp. strain MB30,
Apoptosis, 21 (2016), 566-577.
19 A. Mai, S. Massa, I. Cerbara, S. Valente, R. Ragno, P. Bottoni, R. Scatena‖, P. Loidl, G.
Brosch, 3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides as a new class
of synthetic histone deacetylase inhibitors. 2. Effect of pyrrole-C2 and/or -C4
substitutions on biological activity, J. Med. Chem., 47 (2004), 1098-1109.
20 T.J. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays, Immunol. Meth., 65 (1983), 55-63.
21 J.H. Tsai, L.S. Hsu, H.C. Huang, C.L. Lin, M.H. Pan, H.M. Hong and W.J. Chen. 1-(2-
Hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione induces G1 cell cycle arrest and
autophagy in hela cervical cancer cells. International Journal of Molecular Sciences. 17
(2016), 1274-1284.
22 a) H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N.
Shindyalov and P. E. Bourne, The Protein Data Bank, Nucleic Acids Research., 28
(2000), 235-242. b) H. Yang, D. G. Rudge, J. D. Koos, B. Vaidialingam, H. J. Yang and
N. P. Pavletich, mTOR kinase structure, mechanism and regulation, Nature, 497 (2013),
217-223. c) J. Wang, C. Zhong, F. Wang, F. Qu and J. Ding, Crystal structures of S6K1

provide insights into the regulation mechanism of S6K1 by the hydrophobic motif,
Biochem. J., 454 (2013), 39-47.
23 P. Labute, Protonate3D: Assignment of ionization states and hydrogen coordinates to
macromolecular structures, Proteins Struct. Funct. Bioinforma, 75 (2009), 187-205.
24 P. Labute, The generalized Born/volume integral implicit solvent model: Estimation of the
free energy of hydration using London dispersion instead of atomic surface area, J.
Comput. Chem., 29 (2008), 1693-1698.

Graphical Abstract
8d IC50: 148.59 μM
9b IC50: 171.40 μM
9d IC50: 140.60 μM

Highlights
 A versatile synthetic approach for the synthesis of new isoindole derivatives was
developed.
 The anticancer activity of these compounds was evaluated.
 The synthesized compounds showed inhibitory effects on the viability of HeLa cells.
 The degree of cytotoxicity was increased with the level of bigger branched isoindole
derivatives.

Conflicts of Interest
We declare that we have no conflict of interest.