Design, synthesis, biological evaluations, molecular docking, and in vivo studies of novel phthalimide analogs

Article abstract of DOI:10.1002/ardp.201700363

A series of novel phthalimide analogs containing an indole or brominated indole moiety were synthesized and their antimicrobial activity was evaluated. Compound 8 showed a broad spectrum activity, revealing 53–67% of erythromycin activity on the tested ba

Full text of DOI:10.1002/ardp.201700363

Received: 16 November 2017
DOI: 10.1002/ardp.201700363
Revised: 7 March 2018
Accepted: 13 March 2018
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FULL PAPER
Design, synthesis, biological evaluations, molecular docking,
and in vivo studies of novel phthalimide analogs
Magdy A. H. Zahran1
Bishoy El-Aarag2
Ahmed B. M. Mehany3
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Amany Belal4
Ali S. Younes1
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1 Faculty of Science, Department of
Abstract
Chemistry, Menoufia University, Shebin
El-Koom, Egypt
A series of novel phthalimide analogs containing an indole or brominated indole moiety
were synthesized and their antimicrobial activity was evaluated. Compound 8 showed a
broad spectrum activity, revealing 53–67% of erythromycin activity on the tested
bacteria and 60–70% of miconazole activity on the tested fungi. Anticancer activity was
evaluated on the cell lines HepG2, MCF-7, A549, H1299, and Caco2. The results
revealed that the new phthalimide analog 8 has broad-spectrum anticancer activity
toward all the tested cancer cell lines, followed by compound 11, which showed good
activity toward all the tested cell lines except for MCF-7. The ability of the promising
analogs5, 8, and11tobindtotopoisomeraseIIDNAgyrase wasinvestigated. Caspase-3
activation and Bcl-2 assay of the best active derivatives 8, 11 in addition to compound 5
were evaluated. The antifibrotic activity was studied in an in vivo model and the
histopathological studies revealed that treatment with the new compound 8 improved
the fibrotic liver tissues to normality.
2 Biochemistry Division, Faculty of Science,
Department of Chemistry, Menoufia
University, Shebin El-Koom, Egypt
3 Faculty of Science, Department of Zoology,
Al-Azhar University, Cairo, Egypt
4 Faculty of Pharmacy, Department of
Medicinal Chemistry, Beni-Suef University,
Beni-Suef, Egypt
Correspondence
Dr. Amany Belal, Faculty of Pharmacy,
Department of Medicinal Chemistry,
Beni-Suef University, Beni-Suef 62514, Egypt.
Email: abilalmoh1@yahoo.com
K E Y W O R D S
caspase-3, gyrase binding mode, indole derivatives, liver fibrosis, phthalimide analogs,
thalidomide
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1
INTRODUCTION
that the phthalimide moiety is a very important biologically active
pharmacophore.[3–6] Antimicrobial properties of phthalimide derivatives
Cancer is a disease characterized by the uncontrolled growth and spread
of abnormal cells. It is caused by external factors, such as smoking,
infectious organisms, an unhealthy diet and internal factors such as
inherited genetic mutations, hormones, and immune conditions. Treat-
ments include surgery, chemotherapy, radiation, hormone, and immune
therapy.[1] It was estimated that the number of deaths attributed to
cancer would rise to an annual 19.3 million by 2025.[2] Therefore, the
research for new cancer-treating agents is an important area in both
organic and medicinal chemistry. Recently, many heterocyclic com-
pounds possessing biologically active properties have been synthesized
and evaluated as anticancer drug candidates. Among these biologically
active heterocycles, phthalimide is an important pharmacophore and
has a privileged structure in drug discovery. It has been reported
were reported.[7–12] Compounds IV and V (Figure 1) exhibited high
antimicrobial activities against the tested microorganisms.[8] Isoindole-
1,3-dione isa building unit inantitumor,[13,14] antiangiogenic[15] as well as
immunomodulatory drug candidates.[16,17] Thalidomide (Figure 1) is a
drug known to have antiangiogenic, immunomodulatory and significant
antitumor activities, its analogs showed significant potency against
several diseases such as prostate cancer, HIV-related ulcers, multiple
myeloma, and Kaposi's sarcoma; this drug consists of two heterocyclic
moieties: the phthalimide, and the piperdine-2,6-dione moiety.[18–20]
Indole-based compounds I–III (Figure 1) are well known in the medicinal
chemistry research as antimicrobial, anticancer,[21–24] antifungal, and
antibacterial[25] active agents. Figure 1 illustrates the structure of indole-
based derivatives and the strategy for designing our target compounds.
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Arch Pharm Chem Life Sci. 2018;e1700363.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201700363

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FIGURE 1 Molecular design of the novel phthalimide–indole hybrids
Tryptamine (indole-3-ethylamine) and its derivatives have high pharma-
ceutical significance because of their naturally occurring as well as
their utility in several drugs.[26] Tryptamine derivatives are used in the
treatment of migraine, obesity and behave as antitumor active
analogs.[27–30]
bromination at position 2 of indole to afford compounds 7–11
(Scheme 1). The indolylisoindoldione 1, the alkylated analogs 2 and 4
were prepared according to the previously reported procedures.[37,38]
The analog 3 was obtained by alkylation of 1 by ethyl bromide in the
presence of NaH. The melting point (m.p.) of derivative 3 closely
matches the reported data.[39,40] The 1H NMR spectrum of derivative 5
showed signals at 3.85 ppm for N-CH2, multiplet signal at 5.90 ppm for
olefinic CH, and doublet signal at 5.02 ppm for terminal CH2. The
1H NMR analysis for isoindole derivative 6 revealed triplet signal at
3.84 ppm for N-CH2, multiplet signal at 1.67 ppm for CH2, and triplet
signal at 0.72 ppm for terminal CH3. Characteristic singlet peak of 2-H
of indole ring appears in the 1H NMR spectra of derivatives 5 and 6 at
7.14 and 7.16 ppm, respectively. Derivatives 2–6 were brominated
using NBS in CCl4 under reflux to afford brominated isoindole
derivatives 7–11. The 1H NMR spectra of the derivatives 7–11 have no
singlet peak of 2-H of indole ring in the aromatic area indicating that
the substitution of that proton was successfully done. The 13C NMR,
Based on our previous studies which involved the synthesis and
evaluation of novel thalidomide analogs as anticancer and antiangiogenic
agents[31–36] and the diversity of biological activities of indole derivatives,
the current study aimed to synthesize and evaluate novel phthalimide–
indole hybrids as antimicrobial, antitumor, and anti-hepatic fibrosis agents.
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
The current study includes the synthesis of analogs 2–11 by alkylation
of indole at N1 using different alkylating gents and followed by

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SCHEME 1 Synthesis of N-alkylated and 2-brominated indolyl isoindole analogs 2–11
mass and elemental analysis were also used to elucidate the structures
of all new isoindole derivatives.
revealed that analogs 4, 8, 10, and 11 showed good activity against
C. albicans, their activities were 61, 61, 65, and 69% of miconazole
activity, respectively, followed by compound 3 that showed 59% of
miconazole activity. Analogs 3, 4, 8–11 showed to be active against
Asp. niger, their activities ranged from 60 to 70% of miconazole activity.
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2.2 Biological activities
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2.2.1 Antimicrobial activity
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2.2.2 Antiproliferative activity
The antimicrobial activity of the synthesized analogs against gram
positive bacteria (Bacillus subtilis ATCC 6633, Staphylococcus aureus
ATCC 35556), gram negative bacteria (Escherichia coli ATCC 23282
and Pseudomonas aeruginosa ATCC 10145), fungi (Candida albicans
IMRU 3669), and filamentous fungus (Aspergillus niger ATCC 16404)
was evaluated. Concerning the gram positive bacteria, results in
Table 1 revealed that compounds 5 and 6 have a good activity on
B. subtilis, their activity was 68 and 64% of erythromycin activity,
respectively. Analogs 3, 5, 6, 10, and 11 showed moderate activity
against S. aureus, their activity was 53% of erythromycin activity,
however, analogs 4 and 8 showed better activity against the same
tested microorganism with 60% of erythromycin activity. Concerning
the gram negative bacteria, analogs 4 and 5 showed good activities on
E. coli, their activity was 63% of erythromycin activity. Analog 8
showed two thirds the activity of erythromycin on E. coli but analog 11
showed 59% of erythromycin activity. Analogs 5 and 8 are the best
active on P. aeruginosa followed by analog 6 with a percent of inhibition
equal 65, 65, and 62% of erythromycin activity, respectively. Screening
of synthesized analogs against fungi (C. albicans and Asp. niger)
The synthesized analogs were tested against MCF-7, Caco2, HepG2,
H1299, and A549 cells using SRB assay and thalidomide as a reference
drug. IC50 values are reported in Table 2. The tested analogs showed
activity toward HepG2 cell line, especially analogs 5, 7–11 with
cytotoxicity ranging from 53.3 to 69.6 μM, compared to thalidomide
(193.6 μM). All analogs showed good activity on Caco2 cell line, with
IC50 values less than that of thalidomide except compounds 2 and 6.
Concerning H1299 cell line, analogs 6, 8, and 11 showed activity more
than that of thalidomide, their IC50 values are less than 50 μM. Analogs
8, 10, and 11 showed potent effect on A549 compared to the
reference drug. Analog 8 showed anticancer activity toward MCF-7
cell line comparable with that of thalidomide.
The undesirable damage to normal cells triggered by the high
toxicity of anticancer drugs is one of the chief complications of cancer
chemotherapy. The most active compounds against the studied cancer
cell lines that caused the highest apoptotic effect were selected to be
tested for cytotoxicity against normal liver cell line (THLE-2) and their
IC50 values were determined. Results demonstrated that the selected

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TABLE 1 Antimicrobial activity of phthalimide analogs on different microorganisms
Inhibition zone (mm) of microorganism
Bacteria
Fungi
Analog
Bacillus subtilis
Staph. aureus
Escherichia coli
Pseud. aeruginosa
Candida albicans
Aspergillus niger
2
15
15
16
19
18
12
15
14
16
15
28
–
12
15
17
15
15
13
17
13
15
15
28
–
14
14
17
17
15
12
18
14
14
16
27
–
15
14
16
19
18
15
19
16
15
17
29
–
14
15
16
12
13
12
16
14
17
18
–
12
16
17
12
13
12
19
16
18
19
–
3
4
5
6
7
8
9
10
11
Erythromycin
Miconazole
26
27
The experiments were done in duplicates.
compounds 5, 8, and 11 exhibited IC50 (μM) values of 951.27, 148.72,
and 413.62, respectively. Moreover, these findings indicated that the
IC50 values of compounds against normal liver cells were very high in
comparison to their IC50 doses against the cancer cell lines (Table 3).
activation percent at 47.5%. The obtained results prove the ability of
these new derivatives to act as apoptosis inducers, hence good and
promising anticancer active agents.
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2.2.4 Bcl-2 evaluation
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2.2.3 Caspase-3 activation assay
Bcl-2 proteins have been previously reported as an essential factor for
apoptosis resistance in hepatocellular carcinoma, additionally, Bcl-2
inhibited apoptosis and promoted the process of tumorigenesis and
chemoresistance.[42] Results in Figure 3 revealed that the treatment of
HepG2 cells with compounds 5, 8, 11, and thalidomide significantly
(P < 0.001) reduced the expression levels of the anti-apoptotic protein
Bcl-2 by 62.76, 76.21, 68.54, and 75.77%, respectively, compared to
Caspase-3 activation for the most active anticancer derivatives 8, 11
and compound 5 was evaluated. As caspase-3 plays a major role in
apoptosis process,[41] the obtained results are shown in Figure 2. All
the three derivatives activated caspase-3 with a percent exceeding
that of thalidomide (30%). Compounds 8 and 11 revealed activation
percent at 58 and 64.5%, respectively, followed by compound 5
TABLE 2 Anticancer activity of phthalimide analogs on different human cancer cells
IC50 (µM)a
Analog
HepG2
MCF-7
A549
H1299
Caco2
2
>200
>200
69.002 2.64
87.074 3.68
69.102 2.79
64.4 2.76
55.09 1.59
46.63 1.32
65.75 2.81
48.83 1.44
79.25 3.2
59.62 2.53
40.85 1.2
52.67 1.43
93.317 3.76
86.06 3.53
77.28 2.98
84.75 3.54
107.7 3.99
54.53 1.69
58.15 2.36
60.96 2.79
56.44 1.87
49.6 1.54
88.68 3.9
3
74.13 3.02
78.59 3.32
69.62 2.88
123.65 5.04
63.41 2.55
61.67 2.34
53.34 1.53
59.37 2.45
57.38 1.73
193.63 10.04
>200
100.83 3
4
81.75 3.23
144.38 5.43
79.72 3.34
61.319 1.78
62.93 2.54
108.85 4.05
78.19 3.15
106.49 3.89
65.83 2.78
59.93 2.46
83.24 3.44
58.06 2.34
>200
5
6
7
8
38.51 0.99
68.8 2.76
28.59 0.54
41.33 1.23
44.53 1.32
9
10
11
Thalidomide
aIC50 values are the mean SD of three independent experiments.

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TABLE 3 Effect of the new promising compounds 5, 8, 11, and
thalidomide on normal human liver cells (THLE-2)
slides of fibrotic mice revealed congestion and multifocal interstitial
lymphocytic aggregates (Figure 4C) as well as inflammatory cells
aggregation in portal area and fibroblastic proliferation. Additionally,
marked hydropic degeneration was shown in the surrounding hepatic
parenchyma (Figure 4D). Thalidomide-treated mice indicated highly
dilated hepatic veins and cystically dilated bile ducts surrounded by
fibroblastic proliferation (Figure 4E) as well as telangiectatic sinusoids
and mitotically active hepatocytes (Figure 4F). The histopathology of
mice treated with analog 11 showed that the central veins and the
main hepatic vein radicals were severely dilated and some of them are
free of blood others engorged with erythrocytes with a ruptured walls
and parenchymal hemorrhage as presented in Figure 4G. Moreover,
they showed apparently normal hepatic parenchyma, hypertrophic
Kupffer cells, and mitotically active hepatocytes with bluish–red
cytoplasm, enlarged nuclei, and double nucleation (Figure 4H).
Figure 4I indicated that mice treated with analog 5 revealed apparently
normal hepatic parenchyma, mild portal inflammatory reaction, dilated
hepatic venous branches, and cystified bile duct. Also, they showed
central vein and hepatic sinusoids with normal shapes and structures.
Additionally, the hepatocytes are in a good healthy condition with
preserved cytoplasm and active prominent nuclei as well as the
Kupffer cells are hypertrophied as shown in Figure 4J. The treatment
with analog 8 presented apparently normal hepatic parenchyma and
vascular tree with preserved biliary and lymphatic structures and
minimal portal inflammatory reactions. Moreover, most of hepatocytes
are highly activated with prominent enlarged hyperchromatic nuclei as
well as the Kupffer cells are modemiceely hypertrophied and showed
preserved normal morph–histology structures (Figure 4K and L).
IC50 (μM) SD
Analog
THLE-2
5
951.27 52.06
148.72 9.06
413.62 15.34
1598.66 57.24
8
11
Thalidomide
FIGURE 2 Caspase-3 activation % for the new compounds and
thalidomide
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2.3 Molecular docking studies
Results showed that analog 8 exhibited a broad spectrum activity. So
that, these results motivated us to investigate its binding mode and
possible interaction with topoisomerase II DNA gyrase. The co-
crystallized ligand (clorobiocin) was redocked into the pocket site of
topoisomerase II DNA. It showed a docking score energy −14.5 kcal/
mol at root mean square deviation (RMDS) value equal 1.2. Also, it has
formed three hydrogen bonding with Arg 136, Asn 46, and Asp 73 in
addition to arene-cation interaction with Arg 76 amino acid as shown
in Figure 5. Compound 8 was drawn in 2D then transformed to 3D,
energy minimized and saved in a molecular data base (MDB) file to be
docked into the active site of topoisomerase II DNA enzyme. It showed
score energy lower than the co-crystallized ligand (−16.4 kcal/mol)
with two arene-cation interactions with Arg 76 amino acid through its
indole moiety (Figure 6). Additional investigations for affinity, binding
mode, and possible interactions of the promising new derivatives 5
and 11 were also performed, their docking score energy was −8.2
and −10.01 kcal/mol, respectively, they showed less binding affinity
than compound 8, this might help in explaining why activity of
compound 8 is better. Figures 7 and 8 represented their binding and
interactions with topoisomerase II DNA binding site. Both of them 5
and 11 showed one arene-cation interaction with Arg 76 amino
acid, and both showed ligand exposure through the phenyl and alkyl
FIGURE 3 Effect of compounds 5, 8, 11, and thalidomide on the
expression levels of Bcl-2 in HepG2 cancer cells treated with the
compounds at their IC50 concentrations. Data are expressed as the
mean SD of three separate experiments. *Significantly different
from control at P < 0.001
the control untreated cells (Figure 3). The ability of the tested
compounds to significantly down-regulate Bcl-2 levels indicates their
potential to enhance apoptosis.
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2.2.5 Anti-hepatic fibrosis evaluation
The effect of analogs 5, 8, and 11 in a model of liver fibrosis induced in
mice by carbon tetrachloride (CCl4) action was histopathologically
investigated using hematoxylin and eosin (H&E) staining. As shown in
Figure 4A and B, liver slides of normal mice showed regular lobulation,
central veins, and structures of the portal area. Conversely, liver

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FIGURE 4 Histopathological analysis of liver tissues: (A and B) Liver section of normal group observed under microscope (100 and 400,
respectively); (C and D) liver section of CCl4 control observed under microscope (100 and 400, respectively); (E and F) liver section of
CCl4 + thalidomide observed under microscope (100 and 400, respectively); (G and H) liver section of CCl4 + analog 11 observed under
microscope (100 and 400, respectively); (I and J) liver section of CCl4 + analog 5 observed under microscope (100 and 400, respectively); (K
and L) liver section of CCl4 + analog 8 observed under microscope (100 and 400, respectively)
derivative as shown in the 2D interaction figures, compound 11 with
lower docking score energy and with additional ligand exposure with
the receptor through its Br atom as like as compound 8. These results
are in agreement with the biological screening results as compound 8
was the best active one of them, followed by compound 11, then
compound 5.
against S. aureus. Bromination of derivative 3 increased its activity
against E. coli while decreased the antibacterial activity of the other
analogs against the same bacteria. The effect of allyl and n-propyl
groups appears again in the activity of derivatives 5 and 6, they showed
the highest activity against P. aeruginosa. The brominated derivative 8
showed higher activity against the same bacteria than the non-
brominated one 3. As for C. albicans, the brominated derivatives 10 and
11 exhibited the highest antifungal activity, these compounds have
allyl and n-propyl groups, respectively. The brominated derivatives 8,
10, and 11 are the best antifungal active agents against Asp. niger.
Derivative 8 has the highest antimicrobial activity against five types of
microorganisms, so this attracts us to investigate more about such
compound. The linker between phthalimide and indole moieties might
have an important role in the anticancer activity of the synthesized
compounds. This appears clearly against HepG2 and Caco2 cells.
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2.4 Structure–activity relationship (SAR)
It is noticed that the R group and Br atom affected the antimicrobial
activity of the synthesized compounds. Concerning B. subtilis,
derivatives containing allyl 5 and n-propyl 6 groups are the most
active antibacterial analogs. Bromination decreases their activity
against such gram positive bacteria. Analog containing benzyl group 4
and the brominated analog 8 have the highest antibacterial activity

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FIGURE 5 (A) 2D interactions for the co-crystallized ligand
(clorobiocin) in the binding site of topoisomerase II DNA gyrase. (B)
3D interactions for the co-crystallized ligand (clorobiocin) in the
binding site of topoisomerase II DNA gyrase
FIGURE 6 (A) 2D interactions of analog 8 with topoisomerase II
DNA gyrase binding sites. (B) 3D interactions of analog 8 with
topoisomerase II DNA gyrase binding sites
Except derivative 2, all tested compounds are more potent than
thalidomide, which does not have a linker, against HepG2 cells. Among
the non-brominated compounds, compound 5, containing unsaturated
allyl substituent, has the most potency against HepG2 cells
(IC50 = 69.617 μM). Compound 6, containing the saturated n-propyl
group, has the lowest potency. Bromination increases the potency of
the compounds against liver carcinoma cells. These data support the
synthesis of potent anti-liver cancer compounds containing spacer
attached to phthalimide moiety in the future. Bromination increases
the cytotoxicity of the analogs, except for analogs 4 and 6, against
MCF-7 cancer cells. Introduction of Br atom increases the anticancer
activity of the derivatives, except for 2 and 4, toward A549 cells.
Saturated n-propyl group might have a role in the potency of the
derivatives against H1299 cells. Derivatives 6 and 11 have the lowest
IC50 values (46.631 and 40.847 μM, respectively). The brominated
derivative 8 is more potent than its non-brominated analog 3 against
H1299. Regarding Caco2 cells, bromination increases the cytotoxicity
of the compounds. This suggests that Br atom might have a role in the
cytotoxicity against Caco2 cells. So, brominated indole scaffolds can be
taken in consideration in the future to build up new cytotoxic
compounds against Caco2 cancer cells. The brominated derivative 8 is
more potent than the drug thalidomide against all tested cancer cell
lines. The potency of the brominated derivative 11 is higher than that
of thalidomide against HepG2, A549, H1299, and Caco2 cell lines.
These two compounds 8 and 11 attract us to understand more about
their activity.
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3
CONCLUSION
A series of novel thalidomide analogs were synthesized. Compound 8
showed a broad spectrum antimicrobial activity. The new analogs
exhibited anticancer activity toward different cancer cell lines,
especially HepG2 and Caco2 cancer cells. Compounds 8 and 11
showed to be the best active analogs as anticancer agents. Caspase-3
activation for these compounds (58 and 64.5%) was much better than
for thalidomide (30%) and compound 5 (47.5%) that showed to be

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FIGURE 8 (A) 2D interactions of analog 11 with topoisomerase II
DNA gyrase binding sites. (B) 3D interactions of analog 11 with
topoisomerase II DNA gyrase binding sites
FIGURE 7 (A) 2D interactions of analog 5 with topoisomerase II
DNA gyrase binding sites. (B) 3D interactions of analog 5 with
topoisomerase II DNA gyrase binding sites
DMSO-d6 as solvent at 300 MHz using TMS as internal standard and
the chemical shifts were reported as δ values in ppm. The 13C NMR
spectra for synthesized compounds 5, 6, 8, and 11 were recorded on a
Bruker 400 MHz (Ulm University, Germany) in DMSO-d6 as solvent
at 101 MHz, while the 13C NMR spectra for the compounds 7, 9, and
10 were recorded on JEOL JNM-ECZ500R-500 MHz (Mansoura
University, Egypt) in DMSO-d6 as solvent at 400 MHz using TMS as
internal standard and the chemical shifts were reported as δ values in
ppm. Mass spectra were recorded with a Shimadzu QP-2010 Plus mass
spectrometer in EI (70 eV) mode. The elemental analyses were
performed at the Micro Analytical Center, Cairo University, Egypt.
The NMR spectra as well as the InChI codes together with some
biological activity data are provided as Supporting Information.
active on two cancer cell lines only. Furthermore, compound 8 and
thalidomide inhibited the Bcl-2 concentration level (76.21 and 75.77%)
followed by compound 11 (68.54%) and compound 5 (62.76%).
Moreover, compound 8 improved the fibrotic liver tissues to normal
ones. Further investigations are recommended especially on analogs 8
and 11 as they are promising candidates with potential anticancer
activity.
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4
EXPERIMENTAL
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4.1 Chemistry
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4.1.1 General
Melting points for the synthesized compounds were measured using a
Stuart melting point apparatus and are uncorrected. The reaction
progress was monitored using TLC aluminum sheets silica gel (Merck
60F254) and visualized using UV lamp. The 1H NMR spectra for all new
derivatives were recorded on Varian Gemini NMR spectrometer in
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4.1.2 General procedure for the synthesis of
isoindole derivatives 2–6
To a solution of isoindole 1 (1 mmol) in dry DMF (3 mL), sodium hydride
(NaH) (1.5 mmol) was added, and kept stirring at room temperature for

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30 min, then the appropriate halide derivative (1.5 mmol) was added
dropwise. The reaction mixture was heated under reflux for 1 h. After
completion of the reaction as monitored by TLC, the reaction was
diluted with water and the product was extracted with diethyl ether.
The organic layer was dried over anhydrous Na2SO4, filtered off, and
concentrated to give a residue, which was purified by column
chromatography on silica gel to afford the isoindole derivatives 2–6.
119.51, 121.72, 122.96, 126.51, 131.57, 134.34, 136.52, 167.71. MS
(m/z, relative abundance %): 384 (M++2, 4), 382 (M+, 4). Anal. calcd.
for C19H15BrN2O2: C, 59.55; H, 3.95; N, 7.31. Found: C, 59.71; H, 3.67;
N, 7.41.
2-[2-(2-Bromo-1-ethyl-1H-indol-3-yl)ethyl]-1H-isoindole-
1,3(2H)-dione (8)
Pale yellow solids; m.p. 127–129°C; yield: 70%; 1H NMR (DMSO-d6,
300 MHz), δ ppm: 1.13 (t, J = 7.2 Hz, 3H), 3.02 (t, J = 7.2 Hz, 2H),
3.79 (t, J = 6.9 Hz, 2H), 4.19 (q, J = 6.9 Hz, 2H), 7.02 (t, J = 7.2 Hz, 1H),
7.13 (t, J = 7.2 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H),
7.81 (s, 4H). 13C NMR (DMSO-d6, 101 MHz, ppm): δ 14.73, 23.74,
37.13, 39.49, 109.84, 110.76, 112.32, 117.59, 119.43, 121.67, 122.81,
126.81, 131.55, 134.23, 135.43, 167.56. MS (m/z, relative abundance %):
398 (M++2, 15), 396 (M+, 15). Anal. calcd. for C20H17BrN2O2: C, 60.47;H,
4.31; N, 7.05. Found: C, 59.94; H, 4.00; N, 7.23.
2-[2-[1-(Prop-2-en-1-yl)-1H-indol-3-yl]ethyl]-1H-isoindole-
1,3(2H)-dione (5)
Greenish-yellow crystals; m.p. 126–128°C; yield: 52%; 1H NMR
(DMSO-d6, 300 MHz), δ ppm: 3.03 (t, J = 7.8 Hz, 2H), 3.85 (t, J = 7.8 Hz,
2H), 4.72 (d, J = 5.1 Hz, 2H), 4.90 (d, J = 17.1 Hz, 1H), 5.04
(d, J = 10.2 Hz, 1H), 5.86–5.92 (m, 1H), 6.99 (t, J = 7.5 Hz, 1H), 7.10
(t, J = 8.1 Hz, 1H), 7.14 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.58
(d, J = 7.2 Hz, 1H), 7.83–7.84 (m, 4H). 13C NMR (DMSO-d6,
101 MHz, ppm): δ 23.60, 38.18, 47.67, 109.93, 110.36, 116.20,
118.29, 118.55, 121.14, 122.91, 126.46, 127.48, 131.57, 134.29,
134.36, 136.01, 167.71. MS (m/z, relative abundance %): 331 (M++1,
25), 330 (M+, 100). Anal. calcd. for C21H18N2O2: C, 76.34; H, 5.49; N,
8.48. Found: C, 76.49; H, 5.87; N, 8.60.
2-[2-(1-Benzyl-2-bromo-1H-indol-3-yl)ethyl]-1H-isoindole-
1,3(2H)-dione (9)
White solids; m.p. 129–131°C; yield: 80%; 1H NMR (DMSO-d6,
300 MHz), δ ppm: 3.09 (t, J = 6.6 Hz, 2H), 3.85 (t, J = 6.6 Hz, 2H),
5.41 (s, 2H), 6.92 (d, J = 6.9 Hz, 1H), 7.06–7.12 (m, 3H), 7.19–7.28 (m,
3H), 7.37 (d, J = 8.1 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.79 (s, 4H).
13C NMR (DMSO-d6, 500 MHz, ppm): δ 23.80, 37.22, 47.28, 110.36,
111.32, 113.41, 117.79, 119.78, 122.00, 122.93, 126.00, 126.88,
127.11, 128.52, 131.54, 134.30, 136.17, 137.44, 167.71. MS (m/z,
relative abundance %): 460 (M++2, 1), 458 (M+, 1). Anal. calcd. for
2-[2-(1-Propyl-1H-indol-3-yl)ethyl]-1H-isoindole-1,3(2H)-dione
(6)
Green crystals; m.p. 110–112°C; yield: 65%; 1H NMR (DMSO-d6,
300 MHz), δ ppm: 0.72 (t, J = 7.5 Hz, 3H), 1.63–1.70 (m, 2H), 3.03
(t, J = 7.5 Hz, 2H), 3.84 (t, J = 7.5 Hz, 2H), 4.03 (t, J = 6.9 Hz, 2H), 6.99
(t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 7.16 (s, 1H), 7.38
(d, J = 7.5 Hz, 1H), 7.55 (d, J = 6.9 Hz, 1H), 7.8–7.86 (m, 4H).
13C NMR (DMSO-d6, 101 MHz, ppm): δ 11.03, 23.05, 23.61, 38.19,
46.74, 109.74, 109.85, 118.29, 118.34, 120.99, 122.92, 126.44,
127.38, 131.58, 134.29, 136.02, 167.74. MS (m/z, relative abundance
%): 333 (M++1, 25), 332 (M+, 100). Anal. calcd. for C21H20N2O2:
C, 75.88; H, 6.06; N, 8.43. Found: C, 75.60; H, 5.81; N, 8.38.
C25H19BrN2O2: C, 65.37; H, 4.17; N, 6.10. Found: C, 65.16; H, 3.85;
N, 6.22.
2-[2-[2-Bromo-1-(prop-2-en-1-yl)-1H-indol-3-yl]ethyl]-
1H-isoindole-1,3(2H)-dione (10)
Yellow solids; m.p. 145–147°C; yield: 65%; 1H NMR (DMSO-d6,
300 MHz), δ ppm: 3.05 (t, J = 7.2 Hz, 2H), 3.82 (t, J = 6.9 Hz, 2H), 4.56
(d, J = 17.1 Hz, 1H), 4.77 (d, J = 4.5 Hz, 2H), 4.99 (d, J = 10.2 Hz, 1H),
5.79–5.85 (m, 1H), 7.02 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 8.4 Hz, 1H), 7.39
(d, J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.79 (s, 4H). 13C NMR
(DMSO-d6, 500 MHz, ppm): δ 23.79, 37.18, 46.23, 110.19, 110.99,
113.11, 115.65, 117.68, 119.65, 121.85, 122.91, 126.70, 131.57,
133.21, 134.30, 136.00, 167.69. MS (m/z, relative abundance %): 410
(M++2, 2), 408 (M+, 2). Anal. calcd. for C21H17BrN2O2: C, 61.63;
H, 4.19; N, 6.84. Found: C, 61.32; H, 3.93; N, 6.75.
|
4.1.3 General procedure for the synthesis of
isoindole derivatives 7–11
To a suspension of isoindole derivatives 2–6 (1 mmol) in CCl4
(5 mL), N-bromosuccinimide (NBS) (1.5 mmol) was added. The reaction
mixture was stirred at reflux for 1 h and monitored by TLC. After
completion of the reaction, the precipitate was filtered off to remove
the unreacted NBS. The solvent was evaporated under reduced
pressure and the residue was recrystallized from ethanol to afford the
isoindole derivatives 7–11.
2-[2-(2-Bromo-1-propyl-1H-indol-3-yl)ethyl]-1H-isoindole-
1,3(2H)-dione (11)
White solids; m.p. 101–103°C; yield: 75%; 1H NMR (DMSO-d6,
300 MHz), δ ppm: 0.74 (t, J = 7.8 Hz, 3H), 1.57–1.62 (m, 2H), 3.04
(t, J = 7.2 Hz, 2H), 3.81 (t, J = 6.9 Hz, 2H), 4.09 (t, J = 7.5 Hz, 2H),
7.01 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 8.1 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H),
7.53(d, J = 7.8 Hz, 1H), 7.77–7.80 (m, 4H). 13CNMR(DMSO-d6,101 MHz,
ppm): δ 10.76, 22.70, 23.75, 37.15, 45.58, 110.10, 110.62, 112.91,
117.58, 119.39, 121.62, 122.83, 126.63, 131.56, 134.23, 136.01,
167.59. MS (m/z, relative abundance %): 412 (M++2, 4), 410 (M+, 4).
2-[2-(2-Bromo-1-methyl-1H-indol-3-yl)ethyl]-1H-isoindole-1,3
(2H)-dione (7)
Yellow solids; m.p. 162–164°C; yield: 60%; 1H NMR (DMSO-d6,
300 MHz), δ ppm: 3.03 (t, J = 6.9 Hz, 2H), 3.69 (s, 3H), 3.78 (t, J = 7.5 Hz,
2H), 7.02 (t, J = 7.5 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 8.1 Hz,
1H), 7.53 (d, J = 7.2 Hz, 1H), 7.81–7.82 (m, 4H). 13C NMR (DMSO-d6,
500 MHz, ppm): δ 23.86, 31.35, 37.22, 10.09, 110.45, 113.55, 117.46,

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ZAHRAN ET AL.
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Anal. calcd. for C21H19BrN2O2: C, 61.33;H, 4.66; N, 6.81. Found: C, 61.62;
H, 4.88; N, 6.60.
caspase activity were calculated as the ratio of values between mock
treated and treated cells.[44]
|
4.2 Biological assays
|
4.2.4 Bcl-2 assay
HepG2 cells (ATCC) were seeded in a 96-well plate at density
1.8 × 10000 cells/well in RPMI 1640 containing 10% fetal bovine
serum. Cells were incubated for overnight and then the culture
medium was replaced with fresh medium (100 μL) and cells were
treated with the tested compounds at their IC50 values and incubated
at 37°C for 24 h. After incubation period, cells were lysed with cell
extraction buffer and then the lysates were diluted in standard diluent
buffer. Bcl-2 concentration was measured using ELISA kit (Invitrogen
Zymed® Bcl-2 ELISA Kit (96 tests) according to the supplier's protocol.
The Bcl-2 concentrations (ng/mL) in cell lysates were obtained from
the constructed standard curve. The experiment was repeated three
independent times and the concentration of Bcl-2 were expressed as
mean SD.
|
4.2.1 Antimicrobial activity
The antimicrobial activity was performed using the agar diffusion
technique.[43] All examinations were done in duplicates against gram
positive bacteria (B. subtilis ATCC 6633, S. aureus ATCC 35556), gram
negative bacteria (E. coli ATCC 23282 and P. aeruginosa ATCC 10145),
fungi (C. albicans IMRU 3669) and filamentous fungus (Asp. niger ATCC
16404). The bacteria were grown on nutrient agar while the fungi were
grown on Czapek's Dox agar medium. The bacteria and fungi were
exposed to 5 mg/mL of each analog for 24 and 48 h, respectively.
Erythromycin was used as positive control for bacteria and miconazole
for fungi.
|
4.2.2 Anticancer activity method
The five cell lines, mammary gland breast cancer cell line (MCF-7),
human intestinal cancer cells (Caco2), human hepatocellular carcinoma
cell line (HepG2) and human lung cancer cells (H1299 and A549) were
obtained from VACSERA-Cell Culture Unit, Cairo, Egypt. The cells
were cultured in RPMI-1640 medium with 10% fetal bovine serum.
Antibiotics were added (100 units/mL penicillin and 100 μg/mL
streptomycin) at 37°C in a 5% CO2 incubator.
Thecellswereseededina96-wellplateatadensityof104 cells/well
for 48 h at 37°C under 5% CO2. After incubation, the cells were treated
with phthalimide analogsin a range of concentration(6.25–200 μM) and
incubated for 24 h, then the medium was discarded. Fix with 10%
trichloroacetic acid (TCA) 150 µL/well for 1 h at 4°C. Wash by water
three times (TCA reduce SRB protein binding). Wells were stained by
SRB 70 µL/well for 10 min at room temperature with 0.4%. Wash with
acetic acid 1% to remove unbound dye. The plates will be 24 h air dried.
The dye will be solubilized with 50 µL/well of 10 mM Tris base (pH 7.4)
for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well
was measured at 570 nm with an ELISA microplate reader (EXL 800
USA). The relative cell viability in percentage was calculated as (A570 of
treated samples/A570 of untreated sample) × 100 and the IC50 values
were calculated using sigmoidal concentration response curve fitting
models (SigmaPlot software).
|
4.2.5 In vivo liver fibrosis model
Experimental animals
Sixty male albino mice (22 3 g) were obtained from National Research
Centre (Cairo, Egypt). The mice were selected for the study and
maintained at a controlled temperature of 25°C and constant humidity
(50–70%) under a 12-h light/dark cycle, with free access to diet and
water. The animal study was performed according to the international
rules considering animal experiments and the internationally accepted
ethical principles for laboratory animal use and care.
Experimental design for antifibrotic activity
Liver fibrosis was induced in male albino mice by intraperitoneal
injection (IP) of CCl4 at a dose of 0.4 mL/kg dissolved in olive oil twice a
week for 8 weeks. All the groups were injected with CCl4 except group
1 (negative control group) animals were taken olive oil twice a week for
8 weeks. Eight weeks after fibrosis induction male albino mice were
divided into five groups (10 mice each) and treated with 10 mg/kg of
analogs 5, 8, and 11 once daily for 8 weeks. At the end of the study, the
animals were anesthetized with ether and sacrificed. Liver tissues of all
animals were removed and stored in −80°C in nuclease free tubes for
the histopathological analysis.
|
4.2.3 Caspase-3 activation assay
Histopathological examination
The formalin preserved hepatic tissues of the tested mice were
processed in an automated tissue processor. The processing
consisted of an initial two steps fixation and dehydration. Fixation
comprising tissue immersion in 10% buffered formalin for 48 h,
followed by removal of fixative in distilled water for 30 min.
Dehydration was then carried out by running the tissues through
a graded series of alcohol (70, 90, and 100%). The tissue was initially
exposed to 70% alcohol for 120 min followed by 90% alcohol for
After treating the HepG2 cells with selected compounds for 48 h,
harvested cells were lysed with caspase lysis buffer at pH 7.5 and
then incubated with Ac-DEVD-AMC/Ac-LEHD-AFC substrate in
20 mM HEPES (pH 7.5), 0.1% CHAPS, 2 mM EDTA, and 5 mM DTT
at 37°C for 2 h. The released AMC and AFC are directly proportional
to caspase-3 activity, they were detected at wavelength of
380/460 nm (for AMC), 400/505 nm (for AFC). The obtained values
were normalized with total protein concentration and the relative

ZAHRAN ET AL.
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90 min and then two cycles of absolute alcohol, each for 1 h.
Dehydration was then followed by clearing the samples in several
changes of xylene. It consisted of tissue immersion for an hour in a
mixture comprising 50% alcohol and 50% xylene, followed by pure
xylene for one and a half hour. Samples were then impregnated with
molten paraffin wax, then embedded and blocked out. Paraffin
sections (4–5 μm) were stained with hematoxylin and eosin and then
examined for liver fibrosis.
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4.3 Molecular docking study
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chemical computing group, Canada. 1KZN represents clorobiocin
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derivatives 5, 8, and 11 were converted to 3D by ChemDraw, then
protonated, energy minimized by Merck Molecular force field
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CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
[27] R. F. Salikov, A. Y. Belyy, N. S. Khusnutdinova, Y. V. Vakhitova, Y. V.
Tomilov, Bioorg. Med. Chem. Lett. 2015, 25, 3597.
ORCID
[28] J. L. Brandes, D. Kudrow, S. R. Stark, C. P. O’Carroll, J. U. Adelman, F. J.
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Amany Belal
http://orcid.org/0000-0003-1045-0163
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How to cite this article: Zahran MAH, El-Aarag B, Mehany
[45] http://www.rcsb.org/pdb/explore.do?structureId=1kzn.
ABM, Belal A, Younes AS. Design, synthesis, biological
evaluations, molecular docking, and in vivo studies of novel
phthalimide analogs. Arch Pharm Chem Life Sci. 2018;1–12.
https://doi.org/10.1002/ardp.201700363
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