Synthesis and anticancer activity evaluation of new isoindole analogues

Article abstract of DOI:10.1007/s00044-017-1793-1

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 in the presence of a catalytic amount of H₂SO₄ in order to yield new isoindole derivatives (8a and 8b). The anticancer activity of these compounds was evaluated against MCF-7 (breast adenocarcinoma) and A549 (adenocarcinomic human alveolar basal epithelial) cell lines. The synthesized compounds showed concentration- and time-dependent inhibitory effects on the viability of both cell lines. Compound 8a was more toxic compared to 8b in both cancer cell lines, having higher cytotoxicity against A549 cells. Testing the toxicity properties of these compounds on the BEAS 2B (human bronchial epithelial) cell line indicated that while both compounds decreased the cell viability of cancer cells, they were less toxic on healthy lung cells. Microscopy images of A549 cells after treatment with the new isoindole derivatives displayed characteristic apoptotic morphology compared to BEAS 2B cells. 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.

Full text of DOI:10.1007/s00044-017-1793-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1793-1
ORIGINAL RESEARCH
Synthesis and anticancer activity evaluation of new isoindole
analogues
Aytekin Köse1 Yıldız Bal2 Nurhan H. Kishalı1 Gülşah Şanlı-Mohamed2
●
●
●
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Yunus Kara1
Received: 10 June 2016 / Accepted: 18 January 2017
© Springer Science+Business Media New York 2017
Abstract We have developed
a
versatile synthetic
morphology compared to BEAS 2B cells. 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.
approach for the synthesis of new isoindole derivatives via
the cleavage of ethers from tricyclic imide skeleton com-
pounds. An exo-cycloadduct prepared from the Diels–Alder
reaction of furan and maleic anhydride furnished imide
derivatives. The epoxide ring was opened with Ac2O in the
presence of a catalytic amount of H2SO4 in order to yield
new isoindole derivatives (8a and 8b). The anticancer
activity of these compounds was evaluated against MCF-7
(breast adenocarcinoma) and A549 (adenocarcinomic
human alveolar basal epithelial) cell lines. The synthesized
compounds showed concentration- and time-dependent
inhibitory effects on the viability of both cell lines. Com-
pound 8a was more toxic compared to 8b in both cancer
cell lines, having higher cytotoxicity against A549 cells.
Testing the toxicity properties of these compounds on the
BEAS 2B (human bronchial epithelial) cell line indicated
that while both compounds decreased the cell viability of
cancer cells, they were less toxic on healthy lung cells.
Microscopy images of A549 cells after treatment with the
new isoindole derivatives displayed characteristic apoptotic
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Keywords Isoindole Norcantharimide Ether cleavage
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Anticancer Cytotoxicity Cancer cell lines
Introduction
Norcantharidin (2) and norcantharimide (3), which are
derivatives of cantharidin (1), have attracted considerable
attention due to their potential anticancer effects (Fig. 1).
Cantharidin (1), a natural compound has been known to be
an anticancer agent since 13th century. (Nicholls and Teare
1954; Wang 1989; Lin et al. 1998). Because of its sub-
stantial toxicity (Tagwireyi et al. 2000), it is not used in
chemotherapeutical studies. Norcantharidin and nor-
cantharimide have lower toxicity and are known as antic-
ancer agents (Hill et al. 2007). Cantharidin and its analogs
have been found to have inhibitory effects against protein
phosphatase 1 and 2A (PP1 and PP2A) (Li and Casida
1992; Li et al. 1993; Honkanen 1993; Sodeoka et al. 1997).
The anticancer activity of cantharidin (1) and norcantharidin
(2) is thought to come from the inhibition of protein
phosphatase 1 and 2A (Fig. 1). Recently, scientists have
revealed the connection between its protein phosphatase
inhibitor and anticancer activity (McCluskey et al. 2000,
2001, 2003; Sakoff et al. 2002; Hart et al. 2004).
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1793-1) contains supplementary material,
which is available to authorized users.
* Gülşah Şanlı-Mohamed
gulsahsanli@iyte.edu.tr
* Yunus Kara
yukara@atauni.edu.tr
1
Faculty of Science, Department of Chemistry, Atatürk University,
Erzurum, Turkey
In recent years, much effort has been devoted to the
synthesis of N-derivatives of norcantharimide (Robertson
et al. 2011). Some of the synthesized norcantharimide
2
Faculty of Science, Department of Chemistry, İzmir Institute of
Technology, 35430 İzmir, Turkey

Med Chem Res
Fig. 1 Structure of cantharidin
(1), norcantharidin (2), and
norcantharimide (3)
derivatives had an effect on a number of types of cancer
(Robertson et al. 2011). For example, the tumor-inhibitory
action of N-methylcantharimide has been established in
animals. McCluskey et al. (2003) synthesized various nor-
cantharimides and investigated their cytotoxicity against
different carcinomas. They investigated the effects of subtle
skeletal modifications on the anticancer activity of the
compounds. Lin et al. (2000, 2004) also reported the
synthesis and anticancer activity of the N-substituted can-
tharimides (aliphatic, aryl, and pyridyl groups) in vitro
against HepG2 and HL-60 cells. Kok et al. (2007) studied
the synthesis and cytotoxicity of some cantharimide deri-
vatives. They thoroughly explored electronic properties of
the functional group on the cytotoxicity of some canthar-
imide derivatives (Kok et al. 2007).
Recently, we reported a versatile synthetic approach to
the synthesis of new norcantharimide or isoindole deriva-
tives (Tan et al. 2011). We investigated the fluorescence
properties of some of these norcantharimide derivatives
(Tan et al. 2014). In this work, new isoindole derivatives
were synthesized and their antiproliferative properties were
examined on the MCF-7 (breast adenocarcinoma) and A549
(adenocarcinoma human alveolar basal epithelial) cell lines.
The toxicity of the compounds was also examined on the
healthy lung cell line BEAS 2B (human bronchial epithelial
cells). New isoindole derivatives were found to be potential
anticancer agents for the treatment of various cancer types.
Synthesis of (3aR,4S,7R,7aS)-3a,4,7,7a-tetrahydro-4,7-
epoxyisobenzofuran-1,3-dione (6)
In a 250-mL three-necked 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 30 mL of
toluene. The mixture was stirred for 24 h of 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 eva-
poration and crystallized from MeOH/diethyl ether. Yield:
colorless crystals, 8.22 g (97%).
1H NMR (400 MHz, CDCl3, 25 °C) δ = 6.57 (s, 2H, H5,
H6), 5.45 (s, 2H, H4, H7), 3.18 (s, 2H, H3a, H7a). 13C NMR
(100 MHz, CDCl3, 25 °C) δ = 170.1 (2C=O), 137.2 (C5,
C6), 82.4 (C4, C7), 48.9 (C3a, C7a).
Synthesis of 2-((3aR,4S,7R,7aS)-1,3-dioxo-1,3,3a,4,7,7a-
hexahydro-2H-4,7-epoxyisoindol-2-yl)ethyl acetate (7a)
To a 100-mL round-bottom flask with a magnetic stir bar
and reflux condenser was added 3a,4,7,7a-tetrahydro-4,7-
epoxyisobenzofuran-1,3-dione (6) (1.50 g, 9.03 mmol) fol-
lowed by MeOH (15 mL) and ethanolamine (0.55 g, 9.03
mmol). The solution turned dark orange and was brought to
reflux for 24 h. The flask was cooled to room temperature,
and after 2 h the product began to crystallize. The mixture
was stored in a freezer overnight and the precipitate was
collected by vacuum filtration. Yield: 1.0 g (53%) as a white
solid (Heath et al. 2008). This alcohol was then submitted to
acetylation. For the acetylation, to a magnetically stirred
solution of crude product (290 mg, 1.27 mmol) in CH2Cl2
(1 mL) was added acetyl chloride (521 mg, 5.1 mmol). The
mixture was stirred at r.t. for 6 h. Removal of the solvent,
HCl, and excess acetyl chloride under reduced pressure
gave sole product acetate 7a as colorless crystals (M.p:
92–94 °C).
Experimental
Chemistry part
All reagents used were commercially available unless
otherwise specified and all solvents were distilled before
use. Melting points were measured with Gallenkamp melt-
1
ing point devices. H and 13C NMR spectra were recorded
on Varian 400 and Bruker 400 spectrometers. Elemental
analysis results were obtained with a LECO CHNS-932
instrument.
1H NMR (400 MHz, CDCl3, 25 °C) δ = 6.47 (s, 2H, H5,
H6), 5.21 (s, 2H, H4, H7), 4.16 (t, 2H, H9, J = 5.4 Hz), 3.69
(t, 2H, H8, J = 5.4 Hz), 2.83 (s, 2H, H3a, H7a), 1.95 (s, 3H,

Med Chem Res
OAc). 13C NMR (100 MHz, CDCl3, 25 °C) δ = 176.2
(2C=O of imide), 171.0 (C=O of acetate), 136.8 (C5, C6),
81.1 (C4, C7), 60.7 (C9), 47.6 (C3a, C7a), 38.0 (C8), 20.9
(CH3). Anal. calcd. for C12H13NO5: C 57.37; H 5.22; N
5.58. Found: C 57.29; H 5.13; N 5.41.
(3aR,4R,7R,7aS)-2-methyl-1,3-dioxo-2,3,3a,4,7,7a-
hexahydro-1H-isoindole-4,7-diyl diacetate (8b)
1H NMR (400 MHz, CDCl3, 25 °C) δ = 6.26 (d, 1H, H6, J
= 10.0 Hz), 6.01 (m, 1H, H5), 5.34 (m, 1H, H7), 5.16 (t, 1H,
H4, J = 4.8 Hz), 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, 25 °C) δ = 175.5 (C1), 175.2 (C3),
169.6 (C4OAc), 169.3 (C7OAc), 126.7 (C6), 125.2 (C5),
67.0 (C7), 64.3 (C4), 40.0 (C7a), 39.6 (C3a), 25.1 (NCH3),
21.0 (COCH3), 20.8 (COCH3). Anal. calcd. for C16H19NO8:
C 55.51; H 5.38; N 4.98. Found: C 55.19; H 5.29; N 5.00.
Synthesis of (3aR,4S,7R,7aS)-2-methyl-3a,4,7,7a-
tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (7b)
To a 100-mL round-bottom flask with a magnetic stir bar
and reflux condenser was added 3a,4,7,7a-tetrahydro-4,7-
epoxyisobenzofuran-1,3-dione (6) (5.0 g, 30.1 mmol) fol-
lowed by MeOH (30 mL) and methylamine (3.74 g, 120.4
mmol). The mixture was refluxed for 24 h. The MeOH was
removed by rotary evaporation, and the crude product was
crystallized from CH2Cl2/hexane. The crystals were col-
lected by filtration. Reducing the filtrate and recrystallizing
again provided a second crop. Yield: colorless crystals (M.
p: 138–140 °C), 4.48 g (83%).
Biological part
Cell cultures
The MCF-7 (Michigan Cancer Foundation-7 breast adeno-
carcinoma) cell line, A549 (adenocarcinoma human alveo-
lar basal epithelial) cell line, and BEAS 2B (human
bronchial epithelial) cell line were kindly provided by the
Biotechnology and Bioengineering Research and Applica-
tion Center, Izmir Institute of Technology, Turkey. The
breast cancer cells were grown in Dulbecco’s modified
Eagle’s medium supplemented with 5% fetal bovine serum
(FBS) and 1% gentamicin sulfate, and lung cancer and
healthy cells were grown in Roswell Park Memorial
Institute-1640 growth medium containing 10% FBS and 1%
gentamicin sulfate at 37 °C in 5% CO2. Medium was
refreshed every 3 days for each cell culture. Typically, cells
were passaged by trypsinization and in growth medium.
Compounds 8a and 8b were dissolved in dimethyl sulfoxide
(DMSO) before all analyses.
1H NMR (400 MHz, CDCl3, 25 °C) 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, 25 °C) δ = 176.5
(2×CO of imide), 136.7 (C5, C6), 81.0 (C4, C7), 47.7 (C3a,
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).
Acid-catalyzed acetolysis of 7 with Ac2O
Norcantharimide derivative 7a (2.3 g, 9.15 mmol) was dis-
solved in Ac2O (5 mL). After the addition of three 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. Yield (8a): white powder (M.p: 111–113 °C),
2.15 g (66%). The same procedure was applied to 0.116 g
(0.65 mmol) of 7b in 3 mL of Ac2O for 24 h. Compound 8b
was obtained as colorless crystals (M.p: 135–137 °C).
Yield: 0.15 g (82%).
Cell viability assays
The cytotoxicities of the synthesized norcantharimide deri-
vatives (compounds 8a and 8b) were evaluated against the
MCF-7, A549, and BEAS 2B cell lines using the MTT (3-
(3aR,4R,7R,7aS)-2-(2-acetoxyethyl)-1,3-dioxo-2,3,3a,4,7,7a-
hexahydro-1H-isoindole-4,7-diyl diacetate (8a)
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide) proliferation assay as a fast and sensitive quantifica-
tion of cell proliferation and viability. MTT, a yellow
tetrazole, is reduced to purple formazan in a living cell’s
mitochondria by mitochondrial dehydrogenases of the
viable cell (Mosmann 1983). This reduction only occurs if
mitochondrial reductase enzymes are active; thus, conver-
sion is directly related to the number of viable cells. Briefly,
MCF-7, A549, and BEAS 2B cells were seeded onto 96-
well microassay plates at a density of 1 × 104 cells/cm2 and
incubated at 37 °C in 95% air, 5% CO2 environments for 24
h. Synthesized compounds 8a and 8b were added in various
concentrations (0–250 µg/mL) and incubated for 24, 48, and
1H NMR (400 MHz, CDCl3, 25 °C) δ = 6.25 (d, 1H, H6, J
= 10.0 Hz), 6.00 (m, 1H, H5), 5.34 (bs, 1H, H7), 5.17 (t, 1H,
H4, J = 4.8 Hz), 4.15 (m, A of AB, 1H, H9a), 4.11 (m, B of
AB, 1H, H9b), 3.71 (m, 2H, H8), 3.48 (m, 2H, H3a, H7a),
2.01 (s, 3H, OAc), 1.94 (s, 3H, OAc), 1.92 (s, 3H, OAc).
13C NMR (100 MHz, CDCl3, 25 °C) δ = 175.3 (C3), 175.0
(C1), 171.0 (NCH2CH2OAc), 169.6 (C4OAc), 169.3
(C7OAc), 126.5 (C6), 125.2 (C5), 66.8 (C7), 64.1 (C4), 61.1
(C9), 39.9 (C7a), 39.4 (C3a), 38.1 (C8), 21.0 (COCH3), 20.9
(COCH3), 20.8 (COCH3). Anal. calcd. for C16H19NO8: C
54.39; H 5.42; N 3.96. Found: C 54.13; H 5.13; N 4.00.

Med Chem Res
72 h. Untreated cells were used as a control group. After
incubation, the medium was removed and replaced with
MTT-containing medium. Plates were incubated for an
additional 4 h at 37 °C. MTT medium was removed and
100 µL of DMSO was added to dissolve the formazan
crystal. The absorbance was determined using a plate reader
at a wavelength of 540 nm. Each experiment was assayed
three times in triplicate. Cytotoxicity assessments of com-
pounds 8a and 8b on MCF-7, A549, and BEAS 2B cells
were represented as percentage of metabolically active cells
and metabolic activity under standard growth conditions
was considered as 100%.
For the visual comparison of cancer and healthy cells
treated with the synthesized norcantharimide derivatives
(compounds 8a and 8b), optical microscopy was used. For
this purpose, BEAS 2B and A549 cells were inoculated in
96-well plates at a density of 1 × 104 cells/cm2 and incu-
bated overnight at 37 °C, 5% CO2, and 95% relative
humidity. After waiting overnight, compounds 8a and 8b
were added to wells of each cell line separately at a con-
centration of 10 µg/mL and incubated for 24 h. A control
group of cells that were not treated with either of the
compounds was also prepared. After incubation, they were
examined using optical microscopy (Olympus CKX41,
Tokyo, Japan).
Treatment of tricyclic imide 7a–b with acetic anhydride
in the presence of H2SO4 at room temperature gave the
trans-diacetate derivative of norcantharimide 8a–b as a sole
product in 66 and 82% yield, respectively. In this reaction,
the etheric bond is stereospecifically cleaved to give trans-
diacetate (8a or 8b) (Baran et al. 2003; Kose et al. 2013).
Compounds were purified by crystallization from CH2Cl2/
hexane.
Biological evaluation
In view of the anticancer activity and potential clinical use
of both synthesized isoindole derivatives (8a and 8b), the
cytotoxic properties of 8a and 8b were evaluated by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) test on two cancer cell lines (MCF-7: human breast
adenocarcinoma; A549: human alveolar basal epithelial
adenocarcinoma) and one healthy cell line (BEAS 2B:
human bronchial epithelial cells). The MTT test is a col-
orimetric assay for assessing the tested cell viability in
which MTT, a yellow tetrazolium, is reduced to purple
formazan by mitochondrial dehydrogenases in living cells.
Therefore, quantification of the metabolic activity of MCF-
7, A549, and BEAS 2B cells in the presence of various
concentrations (0.5, 2, 10, 50, and 250 µg/mL) of com-
pounds 8a and 8b was obtained by spectrophotometer for
24, 48, and 72 h. It was observed that, when compounds 8a
and 8b were applied to MCF-7 and A549 cells, the cell
viability of both cancer cell lines decreased with the
Results and discussion
Chemistry
increase in both compound concentrations in
concentration-dependent manner (Fig. 2).
a
Although both synthesized compounds showed
concentration-dependent inhibitory effects on the viability
of both cell lines, compound 8a had slightly better anti-
proliferative activity compared to 8b on the tested cell lines.
After 24 h, cells treated with compound 8a had approxi-
mately 39% metabolic activity at a compound concentration
of 50 µg/mL for A549 and 55% for MCF-7 cells. However,
cells treated with compound 8b had metabolic activity of
53% for A549 and 64% for MCF-7 cells after 24 h at a
compound concentration of 50 µg/mL. It was also observed
that when the incubation time was increased up to 72 h, the
cytotoxicity of both compounds increased for both cancer
cell lines. Moreover, comparison of the metabolic activity
of A549 and MCF-7 cancer cells in the presence of the
newly isoindole compounds showed that compounds 8a and
8b displayed better cytotoxic effects on A549 cells com-
pared to MCF-7 cells (Fig. 2). After 72 h, MCF-7 cells
treated with compound 8a at 50 µg/mL had approximately
14% metabolic activity, and those treated with compound
8b at 50 µg/ml had approximately 33% metabolic activity.
Under the same conditions, metabolic activity of A549 cells
was 5% for compound 8a and 13% for compound 8b.
Norcantharimide derivatives have an isoindole-1,3-dione
skeleton structure. To obtain new isoindole or norcanthar-
imide derivatives, different functional groups can be
attached to the imide nitrogen or cyclohexane ring. In this
study, we first attempted to synthesize new isoindole deri-
vatives via the cleavage of ethers from tricyclic imide
compounds. For this purpose, the key compound 3a,4,7,7a-
tetrahydro-4,7-epoxyisobenzofuran-1,3-dione was used. We
then explored the biological evaluation of this new series of
isoindole derivatives in vitro.
The key compound used in this work, 3a,4,7,7a-tetra-
hydro-4,7-epoxyisobenzofuran-1,3-dione (6), was prepared
via an exo-selective cycloaddition of furan to maleic
anhydride (Sodeoka et al. 1997; Goh et al. 2008). Con-
densation of the appropriate primer amine (2-aminoethanol
or methylamine) with 3,6-3a,4,7,7a-tetrahydro-4,7-epox-
yisobenzofuran-1,3-dione (6) in the presence of CH2Cl2
gave the corresponding imide in good yield (Fig. 2) (Tan
et al. 2011, 2014). With the use of two different primary
amines in the same reaction conditions, we were able to
obtain compounds 7a and 7b (Scheme 1).

Med Chem Res
Fig. 2 Cell viability of A549 and MCF-7 cells after the treatment with compound 8a and 8b for 24, 48, and 72 h
The cytotoxicities of compounds 8a and 8b were also
tested on a healthy lung cell line, BEAS 2B, and the results
were compared with the lung cancer cell line A549 treated
with compounds 8a and 8b (Fig. 3).
Both synthesized compounds showed concentration-
dependent inhibitory effects on the viability of BEAS 2B
cells, but in comparison to A549 cells, the metabolic
activity of BEAS 2B was better when treated with both

Med Chem Res
Scheme 1 . Synthesis and
conditions of compounds 6–8
compounds, 8a and 8b. After 24 h, BEAS 2B cells treated
with compounds 8a and 8b had approximately 61 and 66%
metabolic activity, respectively, at a compound concentra-
tion of 50 µg/mL. However, under the same conditions, the
metabolic activity of A549 cells was 39% with compound
8a and 53% with compound 8b. These findings show that
the newly synthesized isoindole derivatives (8a and 8b)
have more cytotoxic effects on cancer cells in comparison to
healthy cells and therefore they could be considered as new
candidate anticancer agents in medicine.
For the visual comparison of cancer (A549) and healthy
(BEAS 2B) cells treated with the newly synthesized isoindole
derivatives (compounds 8a and 8b), optical microscopy was
used (Fig. 4). It was observed that A549 cells treated with
compounds 8a and 8b were highly damaged and the number
of whole cells was fewer compared to BEAS 2B cells. After
treatment with a 10 µg/mL concentration of compounds 8a
and 8b for 24 h of incubation at 37 °C, 8a treated cells of the
A549 line showed more characteristics of apoptotic mor-
phology compared to 8b treated A549 cells. The observed
images were consistent with the results of cytotoxicity of
compounds 8a and 8b on A549 and BEAS 2B cells.
Conclusion
Fig. 3 Cytotoxicity assessments of compound 8a and 8b on A549 and
BEAS 2B cells represented as percentage metabolically active cells
versus compound concentration for 24 h
As can be seen from the data presented, subtle modifications
of norcantharimide skeleton permitted the development of a

Med Chem Res
Fig. 4 Optical microscopy images of BEAS 2B and A549 cells treated with 10 μg/mL 8a and 8b after 24 h of incubation at 37 °C
Compliance with ethical standards
new series of isoindole analogs. Cytotoxic activities of these
compounds were then evaluated in two different cancer cell
lines, A549 and MCF-7 and one healthy cell line, BEAS
Conflict of interest The authors declare no competing interests.
2B. Isoindole analogs, N-2-acetoxyetyl 8a and N-Me 8b
displayed cytotoxicity depending on the functional groups
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