Novel HDAC/tubulin dual inhibitor: Design, synthesis and docking studies of α-phthalimidochalcone hybrids as potential anticancer agents with apoptosis-inducing activity

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O R I G I N A L R E S E A R C H

Novel HDAC/Tubulin Dual Inhibitor: Design,

Synthesis and Docking Studies of α-Phthalimido-

Chalcone Hybrids as Potential Anticancer Agents

with Apoptosis-Inducing Activity

This article was published in the following Dove Press journal:

Drug Design, Development and Therapy

1

Ahmed A E Mourad

Introduction: In order to develop novel anticancer HDAC/tubulin dual inhibitors, a novel

series of α-phthalimido-substituted chalcones-based hybrids was synthesized and character-

2

Mai A E Mourad

1

13

3

ized by IR, H NMR, C NMR, mass spectroscopy and X-ray analysis.

Methods: All the synthesized compounds were evaluated for their in vitro anticancer

activity against MCF-7 and HepG2 human cancer cell lines using MTT assay. To explore

the mechanism of action of the synthesized compounds, in vitro β-tubulin polymerization

and HDAC 1 and 2 inhibitory activity were measured for the most potent anticancer hybrids.

Further, cell cycle analysis was also evaluated.

Peter G Jones

¹Pharmacology and Toxicology

Department, Faculty of Pharmacy, Port-

Said University, Port-Said, Egypt;

Medicinal Chemistry Department,

Faculty of Pharmacy, Port-Said University,

2

3

Port-Said, Egypt; Institute of Inorganic

and Analytical Chemistry, Braunschweig,

Germany

Results: The trimethoxy derivative 7j showed the most potent anticancer activity, possessed

the most potent β-tubulin polymerase and HDAC 1 and 2 inhibitory activity and efﬁciently

induced cell cycle arrest at both G2/M and preG1phases in the MCF-7 cell line.

Keywords: chalcones, α-phthalimido, anticancer, cell cycle, histone deacetylase inhibitor,

tubulin polymerase inhibitor

Summary

-

Novel series of α-phthalimido-chalcone hybrids were synthesized and identiﬁed by

different spectroscopic techniques.

The newly synthesized compounds experienced pfotent anticancer activity with IC₅₀at

micromolar concentrations against MCF-7 and Hep G2 cell lines, particularly, 7j hybrid exhibited

.57 and 4.51 fold superior anticancer activity than CA-4 against the tested cell lines, respectively.

-

2

-

The most potent anticancer compounds were investigated as HDAC/tubulin dual inhibitors.

Compound 7j inhibit in vitro β-tubulin polymerization and HDAC 1 and2 activity at

IC₅₀lower than that of CA-4 and entinostat.

Cell cycle analysis was done for the most active compounds and it was clear that the tested

-

compounds successfully arrested cell cycle at G2/M phase and induced preG1 apoptosis

compared to the reference compounds.

-

Molecular docking studies were carried out to explore the binding pattern of the most active

synthesized compounds toward colchicine binding site in tubulin protein and HDAC2 active site.

Introduction

Correspondence: Ahmed A E Mourad

Tel +201069233766

1

Cancer is basically a group of fatal diseases recognized by uncontrolled cell growth.

Email ahmed.mourad@yahoo.com

While chemotherapy remains the main treatment, clinically used anticancer drugs are

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Drug Design, Development and Therapy 2020:14 3111–3130

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http://doi.org/10.2147/DDDT.S256756

Mourad et al

2

generally limited by toxicity, side effects, and resistance.

cancer types. Among HDACs, HDAC2 is the most com-

Liver cancer (hepatocellular carcinoma, HCC), with globally monly overexpressed in many cancer types particularly

rising prevalence remains one of the most common and lethal HCC and breast cancers. Interestingly, HDACi can induce

type of cancer. It ranks as the sixth most frequent cancer type growth arrest in transformed cell, cell death and/or inhibition

and fourth most common cause of cancer-related death of angiogenesis, activation of the extrinsic and/or intrinsic

8

worldwide, as well as being responsible for more than 80% apoptotic pathways and mitotic cell death. Fortunately,

3

of diagnosed primary liver cancer worldwide. Moreover, the normal cells are relatively resistant to HDACi-induced cell

9

therapeutic options remain very limited and restricted on death. Recently, several HDACi such as vorinostat, belino-

liver resection, transplantation and chemoembolization. stat, panobinostat (hydroxamates), romidepsin (cyclic pep-

Notably, a small proportion of patients are suitable candi- tides), chidamide (benzamide) have been approved by FDA

4

dates for these options and the relapse rate is high. On the for treatment of cutaneous T-cell lymphoma, various hema-

10

other hand, breast cancer is the leading cause of death among tological and solid cancers (Figure 1). The benzamide

cancer-related deaths in women, affecting 2.1 million women category can selectively inhibit class I HDACs (HDACs 1,

5

every year. Despite the recent introduction of several pro- 2, 3, and 8) by inducing cell cycle arrest and apoptosis of

mising novel therapies that have shown considerable thera- cancer cell lines, such as entinostat (MS-275) which is in

11,12

peutic success including hormone therapy, radiotherapy and Phase II clinical trial.

chemotherapy, drug resistance remains one of the major

On the other hand, microtubules are polymers of α- and β-

6

challenges in breast cancer treatment.

tubulin heterodimers which play the main role in regulating

Based on the aforementioned aspects, new curative ther- key cellular processes such as cell growth and division,

apeutic strategies are urgently needed that have the advan- preservation of the cell architecture and motility, spindle

13

tages of overcoming the anticancer therapy resistance and formation and intracellular trafﬁcking. Consequently, due

have superior efﬁcacy, lower toxicity as well as better selec- to these key roles tubulin targeting agents constitute an

tivity. Apart from these therapies, histone deacetylase inhi- attractive class of chemotherapeutic drugs and it includes

7

bitors (HDACi) are epigenetic drugs, recently discovered, several agents, such as colchicine, combretastatin (CA-4),

targeting speciﬁc parts of cancer cell signalling, particularly podophyllotoxin, vincristine (VCR), vinorelbine (VRB) and

14

epigenome of cancer cells. Studies have shown that the vinblastine (VBL) (Figure 2). CA-4 is a powerful antimi-

impairment in the balance between the opposing actions of totic compound, possessing potent anticancer activity against

histone deacetylases (HDACs) and histone acetyltrans- a number of human cancer cell lines involving multidrug

15

ferases (HATs) is responsible for epigenetic dysregulation resistant cancer types. It acts as a microtubule destabilizing

of gene expression which results in development of many of agent by speciﬁcally binding to β-subunit of tubulin at the

Figure 1 Examples of some approved HDACi.

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Drug Design, Development and Therapy 2020:14

Mourad et al

Combretastatin (CA-4)

Colchicine

Podophyllotoxin

Figure 2 Examples on some microtubule targeting agents with afﬁnity for colchicine-binding site.

same site of colchicine and thus strongly inhibits tubulin via benzamido-link (Scheme 1) aiming at synergizing the

16

polymerization.

anticancer activity and decreasing the human cytotoxicity by

While searching for a new anticancer agent, chalcones demonstrating their potential activity as promising HDAC-

have shown a successful design of effective antitumor agent. tubulin dual inhibitors. The novel hybrids were tested for their

They are considered as one of the privileged scaffolds dis- in vitro anticancer activity against certain human cancer cell

playing multifarious applications in a wide variety of scien- lines. Moreover, the effects of these hybrids as inhibitors of

1

7,18

19

22

25

tiﬁc domains, such as anticancer,

anti-inﬂammatory,

histone deacetylase and tubulin polymerase were studied and

supported with evaluation of the cell cycle analysis and apop-

totic nature of the most potent compounds. Furthermore,

molecular docking studies were also performed to investigate

the interaction of inhibitors with the colchicine binding site of

tubulin and HDAC2 active site (Figure 3).

20

21

antioxidant, antimicrobial, anti-tubercular, anti-HIV,

23

24

antimalarial, anti-allergic, and leishmanicidal activities.

Notably, different mechanisms of action have been described

for chalcones, including the induction of apoptosis, inhibition

of cell proliferation, inhibition of angiogenesis, blockade of

nuclear factor-kappa B (NF-κB) signaling pathway and

reversal of multidrug resistance or a combination of these

Results and Discussion

Chemistry

26,27

mechanisms.

Furthermore, chalcones are powerful tubu-

lin assembly inhibitors, with almost similar potency to

The synthesis of the target compounds 7a-j is depicted in

Scheme 1. The synthesis of compounds 4 and 5 followed the

40

28

CA-4.

Of considerable interest, phthalimide and its deriva-

tives have recently emerged as an important class of com-

pounds which are used as the starting synthone for

synthesis of diverse biologically active compounds such

literature procedures using phthalimide as starting material.

Reduction of phthalimide with zinc dust in the presence of

copper sulphate followed by cyclization gave phthalide, which

on reaction with potassium phthalimide in DMF resulted in

formation of α-phthalimido-o-toluic acid (4). Treatment of 4

with thionyl chloride gave α-phthalimido-o-toluoyl chloride

2

9

30

31

as anti-inﬂammatory, antioxidant, anti-angiogenic,

3

2

33

34

hypoglycemic, antitumor, anti-tubercular agents as

well as inhibitor of tumor necrosis factor-α (TNF-α)

(

5). N-(4-Acetylphenyl)-2-(1,3-dioxoisoindolin-2-yl)benza-

35

production. Moreover, α-phthalimido-ketones possess

mide (6) has been synthesized by the reaction of 5 with 4-

amino-acetophenone in the presence of triethyl amine (TEA)

in 92% yield.

3

6

liver X receptor antagonistic, leukotriene D receptor

4

37

38

antagonistic, anti-inﬂammatory, and angiogenesis inhi-

3

9

bitor activities.

The structure of compound 6 was assigned on the basis of

Despite the continuous developments in anticancer drugs, satisfactory elemental analysis as well as spectroscopic data.

new and more effective compounds are still required for this The FT-IR spectrum displayed two bands at 3454 and 3310

−

1

−1

disease to be under control. These considerations and the cm due to NH, a band at 3097 cm due to Ar-CH, a band at

−1

above-mentioned ﬁndings prompted us to design a novel 2926 cm due to Aliph-CH, and three bands at 1766, 1716,

−1

series of α-phthalimido-substituted chalcones-based hybrid 1686 cm due to CO, as well as a band at 1595 cm due to

1

scaffolds by incorporating the two entities in a single molecule C=N. The H NMR Spectrum of compound 6 indicated the

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Drug Design, Development and Therapy 2020:14

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Figure 3 Diagram represents the design strategy of novel HDAC/tubulin inhibitors and the reported anticancer compounds CA-4 and entinostat.

presence of three singlets at 10.64, 5.03, 2.56 ppm, arising from spectra at 10.78–10.51 ppm (NH), however the aromatic

NH, CH and CH resonances, respectively. Moreover, this as well as the oleﬁnic protons appear as complexed multi-

2

3

interpretation is nicely conﬁrmed by MS spectrum, in which plets in the region 9.15–6.11 ppm. The methylene protons

the molecular ion peak and the base peak are the same, and resonate at 4.90–4.63 ppm. Additionally, the structure of

13

appeared at m/z 398. The structure elucidation of compound 6 7a-j is conﬁrmed by some C NMR by giving peaks at

was also evidenced by X-ray crystallographic analysis of sin- (CO-CH=CH) at 202.11–187.29 ppm, CO-N-CO at

gle crystal (Figure 4) (Supplementary Figure S1 and Table S1). 186.11–169.44 ppm, CO-NH at 169.89–158.23 ppm, and

As illustrated in Scheme 1, compound 6 reacted with CH in compounds 7a-j resonate in the region of the

2

substituted aldehydes in the presence of sodium hydroxide solvent. The EI-MS spectra of 7a-e further support the

under Claisen-Schmidt condensation conditions to form given structure as they showed correct molecular ion peak.

the chalcones 7a-j in 93–71% yield. The chemical struc-

ture of the target compounds were elucidated utilizing the Biological Evaluation

spectral data and elemental analyses. The IR spectral data In vitro Anticancer Activity Screening

of compounds 7a-j showed intense peaks at 3468–3320 The novel synthesized hybrids were investigated for their in

−

1

−1

cm (NH) and 1733–1637 cm (CO). Another typical vitro anticancer activity against human MCF-7 (breast can-

1

signal of these compounds was found in the H NMR cer) and human Hep G2 (hepatocellular liver carcinoma) cell

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Drug Design, Development and Therapy 2020:14

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Scheme 1 Synthesis of compounds 6 and 7: Reagents and conditions: (A) Zn dust, NaOH, CuSO

4

; (B) KOH, Acetone; (C) DMF, reﬂux 6 hr, HCl; SOCl

2

, reﬂux, 4 hr; (D)

2 2

4-Aminoacetophenone, CH Cl , TEA, 4 hr, rt; (E) Aromatic aldehyde, 20% NaOH, EtOH, 8–20 hr, rt.

lines by using MTT assay. The CA-4 was used as the refer-

The in vitro screening results revealed that the majority

ence compound. The results are listed in Table 1 and graphi- of the synthesized compounds displayed promising antic-

cally presented in Figure 5A and B.

ancer activity against the tested cancer cell lines at low

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The p-chloro hybrid 7b showed slightly greater antic-

ancer activity compared to CA-4 against MCF-7 cell line.

However, this compound achieved IC₅₀value of 3.21±0.17

µM which represents 2.28 time more potent anticancer

activity than CA-4 against Hep G2 cell line. Meanwhile,

the [2,2]paracyclophanyl 7e and 2-furyl 7h hybrids experi-

enced nearly equal anticancer activity against both MCF-7

and Hep G2 cell lines, which was slightly weaker than the

anticancer activity of CA-4 as indicated by their IC₅₀

values. According to their IC₅₀values, the p-nitro 7f and

1-naphthyl 7d hybrids had weak anticancer activity com-

pared to CA-4. However, the anticancer activity markedly

decreased in the case of the unsubstituted phenyl ring,

which in turn conﬁrmed that the substitution in the phenyl

ring is critical for potentiating the anticancer activity. Also,

the anticancer activity of compound 6 was dramatically

decreased which emphasized the importance of the pre-

sence of the chalcone-based system. Finally, analyzing the

above-mentioned results revealed that the methoxy deriva-

tives experienced the most potent anticancer activity and

this activity decreased in this order: tri-OCH > di-OCH >

Figure 4 X-ray structural analysis of compound 6. Ellipsoids represent 50% prob-

ability levels.

IC₅₀values. In particular, trimethoxy hybrid 7j was the

most active among the synthesized compounds with IC₅₀

values of 1.88 ± 0.06 µM and 1.62 ± 0.02 µM against both

MCF-7 and Hep G2 cell lines, respectively; which repre-

sents 2.57 and 4.51 fold superior activity than the refer-

ence compound. It is noteworthy that, the dimethoxy

derivative 7g and the p-methoxy derivative 7c exhibited

remarkable anticancer activity of 2.32 and 2.01 times the

activity of CA-4 against MCF-7, where the IC₅₀values

were 2.09 ± 0.19 µM and 2.41± 0.11 µM, respectively.

While, the anticancer potency of the two hybrids 7g and 7c

against Hep G2 was nearly equipotent and nearly 4-fold

the reference activity. Introduction of piperonyl group in 7i

hybrid increased the anticancer activity by 1.4 and 3.73

fold compared to the reference compound against MCF-7

and Hep G2 cell lines, respectively.

3

mono-OCH . Of considerable interest, modiﬁcation of the

3

electronic nature of substituents attached to the phenyl ring

of the chalcone moiety inﬂuenced signiﬁcantly the antic-

ancer activity. Where, the piperonyl 7i and p-Cl-phenyl 7b

derivatives possessed remarkable anticancer activity, the

[

2,2] paracyclophanyl 7e and 2-furyl 7h hybrids showed a

slight decrease in the anticancer activity. Moreover, the

anticancer activity was markedly decreased in the p-NO₂-

phenyl 7f and 1-naphthyl 7d derivatives.

Table 1 IC50 Values for Anticancer Activity of Compounds 6,

In vitro Enzyme Inhibition

7a–7j and CA4 Using MTT Assay Against MCF-7, and HepG2

Cell Lines

In vitro Tubulin Polymerization Inhibitory Activity

To predict the possible mechanism of action of the synthe-

sized compounds, β-tubulin (TUBb) polymerization inhi-

bitory activity of the most potent synthesized hybrids 7c,

a

Compound No.

IC50 (µM)

MCF-7

Hep G2

6

17.59 ± 0.46

14.43 ± 0.51

4.11 ± 0.17

2.41 ± 0.11

11.31 ± 0.43

5.28 ± 0.25

9.78 ± 0.54

2.09 ± 0.19

5.44 ± 0.21

3.45 ± 0.27

1.88 ± 0.06

4.85 ± 0.18

10.82 ± 0.63

12.93 ± 0.26

3.21 ± 0.17

1.87 ± 0.13

8.07 ± 0.39

8.47± 0.34

15.85± 0.71

1.75 ± 0.08

9.05 ± 0.33

1.96 ± 0.07

1.62 ± 0.02

7.32 ± 0.28

b

7

g and 7j was investigated against the tested cancer cell

7

a

b

c

d

e

f

lines. CA-4 was used as reference compound and the

results are presented as IC₅₀values (in μM concentrations)

in Table 2 and Figure 6.

The results represented in Table 2 proved that 7j hybrid

with IC₅₀value of 2.32 ± 0.15 μM had the most potent β-

tubulin polymerization inhibitory activity among tested

and reference compounds. While 7g and 7c hybrids exhib-

ited β-tubulin polymerization inhibition activity compar-

able to that of CA-4 which cleared by their IC₅₀values.

These results supported that 7j hybrid is a potent inhibitor

of tubulin assembly which was in accordance with the in

g

h

i

j

b

CA-4

a

Notes: Mean IC50 value of three independent experiments. CA-4: Positive

reference.

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Drug Design, Development and Therapy 2020:14

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Figure 5 (A) IC50 values for anticancer activity of compounds 6, 7a–7j and CA4 using MTT assay against MCF-7 cell line. (B) IC50 values for anticancer activity of

compounds 6, 7a–7j and CA-4 using MTT assay against Hep G2 cell line.

vitro cytotoxic activity of these compounds against MCF-7 for the selected hybrids were determined against HDAC1

and Hep G2 cell line.

and 2 isoforms using entinostat as a reference compound

(Table 3, Figure 7).

The results presented in Table 3 reveal that all the

In vitro HDAC Inhibitory Activity

selected hybrids possessed potent HDAC1 and HDAC2

To further explore the mechanism of action of the synthe-

sized compounds, compounds 7c and 7g and 7j were

tested for in vitro HDAC inhibitory activity, IC₅₀values

inhibitory activity. In particular, 7j hybrid afforded the

^hⁱ^g^h^e^s^t^H^D^A^C¹^aⁿ^d^H^D^A^C²ⁱⁿ^hⁱ^bⁱ^t^o^r^y^a^c^tⁱ^vⁱ^t^y^wⁱ^t^h^I^C50

value lower than that of entinostat. Meanwhile, compound

7

g exhibited nearly equipotent HDAC1 and HDAC2 inhi-

Table 2 IC₅₀Values for in vitro β-Tubulin Polymerization

Inhibitory Activity of 7c, 7g, 7j, and CA-4 Using ELISA Assay

bitory activity to entinostat which cleared by the proximity

of their IC₅₀values. Moreover, both HDAC1 and HDAC2

inhibitory activities of compound 7c were slightly lower

than entinostat IC₅₀values. In conclusion, the above-men-

tioned results evidenced that 7g and 7j hybrids are potent

inhibitors of both HDAC1 and HDAC2. This was con-

ﬁrmed also by their potent in vitro cytotoxic activity

against the tested cancer cell lines.

a

Compound No.

Tubulin IC50 (μM)

7

c

g

j

3.68 ± 0.21

3.27 ± 0.33

2.32 ± 0.15

2.62 ± 0.14

b

CA-4

a

Notes: Mean IC50 value of three independent experiments. CA-4: Positive

reference.

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Figure 6 IC50 values for in vitro β-tubulin polymerization inhibitory activity of 7c,

Figure 7 IC50 values for in vitro HDAC1 and HDAC2 inhibitory activity of

compounds 7c, 7g, 7j and entinostat.

7

g, 7j and CA-4 using ELISA assay.

Annexin V-FITC/PI Staining Assay

In vitro Cell Cycle Analysis

It was obvious from cell cycle analysis results that the treat-

ment of MCF-7 with the selected hybrids showed consider-

able increase in preG1 phase which is a suggestion of

apoptosis (Table 5, Figure 9). Consequently, to determine

the ability of 7j and 7g hybrids for inducing cell death in

MCF-7 cell line via induction of apoptosis, Annexin V-FITC/

PI staining assay was carried out for the selected compounds

at their IC₅₀(µM) for 24 hours and analyzed.

In vitro DNA-Flow Cytometry

In order to further explore the effect of the most promising

cytotoxic compounds on the cell cycle, cell cycle distribution

using DNA ﬂow cytometry analysis was assessed on 7j and

7c hybrids compared to CA-4 and entinostat (at their IC₅₀

concentration) against MCF-7 cell line. According to the

results presented in Tables 4, 7j and 7g hybrids experienced

the greatest activity by keeping cells in G2/M and preG1

phases compared to untreated control cell and the reference

compounds (Figure 8A and B).

As shown in Table 5, compounds 7j and 7g have the

ability for inducing apoptosis at early apoptotic, late apopto-

tic stages and cell necrosis development, in comparison to

untreated controls. It was found that compounds 7j and 7g

increased cell populations at the early apoptotic stage by

Moreover, the percentage of cells population at G2/M

after 7j and 7g treatment was higher than entinostat by 1.53

and 1.42 folds, respectively. Also, 7j hybrid achieved 1.05

fold higher cell accumulation percentage than CA-4 at G2/M

phase. While, 7c hybrid increased G2/M cells by nearly equal

percentage to CA-4. Interestingly, it is clearly observed that 7j

14.36- and 14.87-fold more than untreated control, respec-

tively. While, 7j and 7g hybrids treatment signiﬁcantly

increased the late apoptotic cells population by 44.60 and

45.24 folds, respectively. Of considerable interest, the

and 7g hybrids exhibited higher apoptosis ability than the selected compounds possessed increasing in both early and

untreated control cell as well as the reference compound late apoptotic cells compared to the reference compounds,

entinostat at preG1 phase by 1.04 and 1.09 times, respectively. CA-4 and entinostat. It is noteworthy that 7j and 7g hybrids

These data established that the selected hybrids have displayed signiﬁcant increase in cell necrosis by 2.09- and

potent anti-proliferative effects and apoptosis-inducing 2.76-fold, respectively, compared to the untreated control

activity by increasing preG1 apoptosis and developing cell. Moreover, the necrosis percent of the selected hybrids

cell growth arrest at G2/M phase.

were nearly equal to CA-4 and entinostat. These ﬁndings

evidenced that the synthesized hybrids cause impairment of

cell division with apoptosis-inducing activity and conﬁrmed

also that these hybrids can potentially inhibit HDAC and

tubulin polymerase enzymes.

Table 3 IC₅₀Values for in vitro HDAC1 & 2 Inhibitory Activity

of Compounds 7c, 7g, 7j and Entinostat

a

Compound No.

HDAC1 IC50 (µM)

HDAC2 IC50 (µM)

7

c

g

j

0.49 ± 0.03

0.32 ± 0.01

0.22 ± 0.01

0.28 ± 0.02

0.85 ± 0.06

0.56 ± 0.03

0.43 ± 0.02

0.55 ± 0.04

b

Molecular Docking Study

Docking at the Colchicine Binding Site in Tubulin

Protein

b

Entinostat

Docking studies have been carried out to explore the

a

Notes: Mean IC50 value of three independent experiments. Entinostat: Positive

reference.

binding ability of the most potent cytotoxic hybrids 7j,

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Drug Design, Development and Therapy 2020:14

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Table 4 Cell Cycle Analysis of Compounds 7j, 7g, CA-4 and Entinostat Against MCF-7 Cell Line

Compound No.

DNA Content

G0-G1

%

% S

% G2/M

%Pre-G1

Comment

7

j

27.58

31.52

27.43

43.29

55.46

24.32

23.94

26.72

25.41

29.71

48.1

23.52

24.63

29.03

22.43

1.78

PreG1 apoptosis and Cell growth arrest at G2/M

–

g

44.54

45.85

31.3

CA-4

Entinostat

Control

14.83

7

g and 7c with colchicine binding site of tubulin. MOE Protein Data Bank, HDAC2 (PDB code: 4LXZ). Entinostat

program was used for docking the selected hybrids and was used as reference compound. The results were expressed

CA4 into the colchicine binding site using the X-ray as docking energy and binding interactions with the active

crystallographic structure of tubulin-colchicine complex site of HDAC2 isoform (Table 7 and Figure 11).

(

PDB code: 1SA0) as tubulin protein template. The results

of energy binding scores and binding interactions are selected compounds conﬁrmed that the selected hybrids pos-

depicted in Table 6 and Figure 10.

sessed higher binding energy than the reference compound

Interestingly, the estimated docking energy of the

The energy binding scores recorded in Table 6 which means better binding at the HDAC2 active site.

showed that the selected hybrids displayed higher energy Moreover, the order of binding energy was; tri-OCH (7j) >

3

binding scores than the reference ligand, consequently, di-OCH (7g) > mono-OCH (7c), which was consistent with

3

better binding at the colchicine binding site. The highest the HDAC inhibition assay. Compound 7j showed an energy

energy binding score of −9.10 Kcal/mol was observed binding value of −9.01 Kcal/mol, while compounds 7g and

with the trimethoxy hybrid 7j, followed by dimethoxy 7g

and mono-methoxy 7c hybrids, respectively, which jus-

tiﬁed the cytotoxicity results and the estimated tubulin

inhibitory activity. The binding interaction of 7j hybrid

with colchicine binding site is shown in Figure 10B,

where two hydrogen bonding interactions were formed

with GLN 11 and GLY 144 with bond length of 3.30 Å

and 2.80 Å, respectively. In addition to π-cation interac-

tions with LYS 254 with bond length of 3.74 Å

Moreover, compound 7g achieved two π- hydrogen inter-

actions with GLN 11 and ALA 12 with bond length of

7

c showed energy binding values of −8.43 and −8.39 Kcal/

mol, respectively. Compound 7j showed two hydrogen bond-

ing interactions between oxygen of both phthalimido- and

chalcone-based moiety with ARG 197, ASN 331 of distances

of 2.82 Å and 3.19 Å, respectively Figure 11B. In addition,

one hydrogen bonding interaction was formed between oxy-

gen of chalcone nucleus of 7g hybrid and ASN 331 of

distance of 3.15 Å. Alsq, π-cation interaction was formed

between phthalimido aromatic ring and NH group of ARG

2

3

11 with distance of 5.00 Å Figure 11D. However, 7c hybrid

exhibited one hydrogen bonding interaction with ASN 331 of

.94 Å Figure 11F. On the other hand, entinostat showed one

4

.23 Å and 3.63 Å, respectively. In addition, another π-π

2

interaction was formed with TYR 224 (bond length: 3.81

Å) Figure 10D. Meanwhile, one hydrogen bonding inter-

action was formed between NH group of 7c hybrid and

ASP 329 (bond length: 2.87 Å) Figure 10F. The refer-

ence ligand CA4 showed one hydrogen bonding interac-

tion with SER 140 (3.03 Å) and π-hydrogen interaction

with GLY 144 (3.80 Å) Figure 10H.

hydrogen bonding interaction with ASN 331 (3.09 Å) and

another π-hydrogen interaction with PRO 344 (4.60 Å) simi-

lar to 7g hybrid and 7j hybrid, respectively Figure 11H.

Conclusion

A novel series of α-phthalimido-substituted chalcones-based

hybrids as dual HDAC/tubulin inhibitors was synthesized

and evaluated for its in vitro anticancer activity by MTT

Docking at the HDAC2 Active Site

In order to investigate the possible binding modes between method against MCF-7 and HepG2 human cell lines, using

the synthesized compounds and HDAC active site, the dock- CA-4 as reference compound. Moreover, the in vitro tubulin

ing studies on the most potent cytotoxic compounds 7j, 7g polymerase and HDAC 1 and 2 inhibitory activity were

and 7c were performed using MOE program where the 3D evaluated for the most potent compounds 7c, 7g and 7j

structure of the HDAC active sites is available at RCSB using CA-4 and entinostat as reference compounds. The

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Figure 8 (A) Cell cycle analysis in MCF-7 cell line treated with 7j and 7g hybrids at their IC50 (µM). (B) Cell cycle analysis and apoptosis effect in MCF-7 cell line treated

with 7j, 7g, CA-4 and entinostat compounds.

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Table 5 Results of Apoptotic Assay of 7j, 7g, CA-4 and (Thermo Scientiﬁc Gcms) Model (ISQ LT) using Thermo

Entinostat

X-Caslibur Software at the Regonal Center for Mycology

Compound No.

Apoptosis

Total

Necrosis

and Biotechnology (RCMB) Al-Aazhar University, Naser

City, Cairo. Elemental analyses were performed on Perkin–

Elmer 2400 CHN Elemental analyzer by the Microanalytical

Center, Faculty of Science at the University of Cairo and are

within ± 0.4% of theoretical values unless otherwise

speciﬁed.

Early

Late

7

j

23.52

24.63

29.03

22.43

1.78

6.75

6.99

4.92

8.67

0.47

14.72

14.93

21.65

10.68

0.33

2.05

2.71

2.46

3.08

0.98

g

CA-4

Entinostat

Control

Materials

Chemicals and solvents used in the preparation of the

results revealed that the three substituted methoxy deriva- target compounds are of commercial grade, and purchased

tives 7c, 7g and 7j experienced greater anticancer activity from Alfa Aesar, Cambrian chemicals, Aldrich, Acros

than that of CA-4; particularly the trimethoxy derivative 7j Organics, Fluka, Merck, and El-Nasr Pharmaceutical

which experienced the most potent anticancer activity and Chemicals Companies. Chemicals were used without pur-

potently inhibited β-tubulin polymerase and HDAC 1 and 2 iﬁcation. Solvents were puriﬁed following the .reported

activities. Additionally, the cell cycle analysis proved that

Synthesis of α-Phthalimido-o-Toluoyl Chloride (5)

A mixture of potassium phthalimide, 50 g (0.26 mol) and

these promising hybrids can induce apoptosis and prevent the

proliferation of MCF-7 by sequestering higher cell percen-

34.8 g phthalide (0.26 mol) in 150 mL DMF was reﬂuxed for

tage at G2/M and pre G1 phases than untreated control cells,

CA-4 and entinostat. The molecular docking studies showed

that these potent anticancer hybrids could efﬁciently bind to

colchicine binding site of both tubulin polymerase enzyme

and HDAC2 active sites with energy scores higher than the

reference ligands. The results of our study proved that α-

phthalimido-substituted chalcones-based hybrids may serve

as novel promising dual HDAC/tubulin inhibitors in search-

ing for more effective anticancer agents.

5

hours, and then was cooled to r.t. and poured into a mixture

of 100 mL glacial acetic acid and 150 mL water. The sepa-

rated solid was washed with water and 95% ethanol.

Recrystallization from acetic acid gave 41.35 g of α-phthali-

40

midi-o-toluic acid (4) (57%), m.p. 264–6°C, Lit. 266–7°. A

suspension of 50 g (0.178 mol) of 4 and 93 g (0.78 mol)

thionyl chloride was reﬂuxed for 3 hours. Dry benzene (30

mL) was added to the mixture and distilled with the excess

thionyl chloride under reduced pressure. This later process

was repeated twice to remove the excess thionyl chloride.

The yield of the entitled compound was 47.92 g (90%) as

Experimental

Chemistry

Melting points were determined on Mel-Temp hot stage

40

white prisms, m. p. 181–183°C, Lit. 183.5–184.8°C.

apparatus and were uncorrected. Reactions were routinely Synthesis of N-(4-Acetylphenyl)-2-(1,3-

monitored by thin-layer chromatography on Merck silica gel Dioxoisoindolin-2-Yl)Benzamide (6)

PF₂₅₄plates and spots were visualized with UV light at 254 To a magnetically stirred solution of acid chloride 5 (5 g,

nm. IR spectra were recorded as KBr disks on a Fourier 0.0167 mol) in abs. CH Cl (20 mL) was added 4-amino-

2

Transform (FT-IR) Bruker spectrophotometer at Central acetophenone (2.25 g, 0.0167 mol) in one portion and

Lab, Faculty of Science, Minia University, El-Minia, 1.686 g (0.0167 mol) TEA. The reaction mixture was

1

13

Egypt. H NMR and C NMR spectra were carried out on stirred for a further 2 hours at r.t. and then treated with a

a Bruker apparatus operating at 400 MHz and 100 MHz, saturated solution of NaCl (20 mL), and extracted with

respectively using TMS as internal reference at the Faculty of ethyl acetate (30 mL). The organic layer was washed with

Science, Sohag University, Sohag, Egypt. Chemical shifts (δ 1N HCl, NaHCO solution and ﬁnally with H O, then

3

2

values) are given in parts per million (ppm) using CDCl₃dried over anhyd. Na SO . The solvent was removed

2

4

(

7.19) or DMSO-d (2.5) as solvents and coupling constants under reduced pressure and the residue was recrystallized

6

J) in Hertz. Splitting patterns are designated as follows: s, from ethyl acetate/n-hexane to give the entitled compound.

singlet; bs, broad singlet; d, doublet; t, triplet; m, multiplet.

White solid, 4.2 g (92%), m.p. 238–240 °C; FTIR

−1

EI-MS: Mass spectrum was carried out on direct probe con- (KBr, cm ) ν: 3454, 3310 (NH), 3097 (Ar-CH), 2926

troller inlet part to single quadruple mass analyzer in (Aliph.-CH), 1766, 1716, 1686 (CO), 1595 (C=N).

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Figure 9 Flow cytometric analysis of Annexin V-FITC/PI induced by compounds 7j, 7g, CA-4, entinostat at their IC50 (µM).

1

Supplementary data page 1) H NMR (400 MHz, DMSO- 130 (54), 120 (15), 116 (21), 104 (11), 90 (7), 77 (20), 76

d₆): δ 10.64 (s, 1H, NH), 7.95 (d, 2H, J= 8.32 Hz, Ar-H)), (20), 63 (3) (Supplementary data page 31). Anal. Calcd.

7

.87 (d, 2H, J= 8.32 Hz, Ar-H), 7.84 (br. S, 4H, Ar-H), for C H N O (398.41): C, 72.35; H, 4.55: N, 7.03.

24 18 2 4

.61 (d, J= 8.32 Hz, 1H, Ar-H), 7.47 (t, J= 8.32 Hz, 2H, Found: C, 72.05; H, 4.75; N, 6.82.

Ar-H), 7.42 (t, J= 8.32 Hz, 2H, Ar-H), 7.32 (d, J= 8.32 Hz,

H, Ar-H), 5.03 (s, 2H, CH ), 2.56 (s, 3H, CH ) Synthesis of (E)-2-((1,3-Dioxoisoindolin-2-yl)Methyl)-

2

3

Supplementary data page 12, 13). MS: m/z (rel. intensity), N-(4-(3-(Arylacroyloyl)Phenyl)Benzamides 7

99 (M ₊1, 33), 398 (M , 100), 397 (19), 381 (11), 264 General Procedure

+

3

(22), 263 (9), 265 (4), 251 (2), 133 (4), 131 (6), 132 (3), A solution of 20% NaOH (0.5 mL) was added to a well

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Table 6 Energy Binding Scores (Kcal/mol) for Compounds 7j, Ar-H, CH=CH), 6.66 (s, 1H, Ar-H), 4.64 (s, 2H, CH₂)

g, 7c and CA4 with the Colchicine Binding Site

7

(

Supplementary data page 17, 18 ). MS: m/z (rel. inten-

+

Compound No.

Energy Binding Scores

sity) 520 (M , 9), 512 (3), 510 (19), 492 (7), 469 (7),

4

3

52 (10), 410 (6), 393 (7), 387 (100), 364 (18), 358 (44),

40 (17), 226 (7), 208 (7), 201 (14), 178 (12), 160 (4),

7

j

−9.10

−8.87

−7.20

−6.76

g

c

133 (20), 113 (25), 104 (34), 101 (51), 91 (37), 70 (29),

4 (34) (Supplementary data page 33). Anal. Calcd. for

C H ClN O (520.96): C, 71.47; H, 4.06;: N, 5.38.

CA4

5

3

1

21

2 4

Found: C, 71.80; H, 3.85; N, 5.30.

stirred mixture of compound 6 (0.16 g, 0.402 mmol) and

different aldehydes (0.402 mmol) in 15 mL absolute etha-

nol at r.t. Stirring was continued for 8–20 hours. (the

reaction was monitored by tlc). The reaction mixture was

extracted with ethyl acetate, then treated with brine, 1N

HCl, NaHCO , and washed with H O. The solvent was

(

E)-2-((1,3-Dioxo-2,3-Dihydro-1H-Inden-2-Yl)Methyl)-N-

4-(3-(4-Methoxyphenyl)Acryloyl)Phenyl)Benzamide (7c)

White solid, 0.115g (74%), m.p. 191–193 °C. FTIR (KBr,

−

1

cm ) ν: 3422, 3326 (NH), 3066 (Ar-CH), 1704, 1678, 1654

CO),1597, 1516 (C=N) (Supplementary data page 4).

3

2

(

removed and the residue was recrystallized from dioxane-

ethanol to afford the title compound.

1

HNMR (400 MHz, DMSO-d ): δ 10.73 (s, 1H, NH),

6

8

3

.80–7.02 (m, 18H, Ar-H, CH=CH), 4.63 (s, 2H, CH ),

2

13

.85 (s, 3H, OCH ) (Supplementary data page 19);

C

3

(

E)-N-(4-Cinnamoylphenyl)-2-((1,3-Dioxo-2,3-Dihydro-

H-Inden-2-Yl)Methyl)Benzamide (7a)

Pale yellow solid, 0.128 g (83%), m.p. 163–165 °C. FTIR

NMR (100 MHz, DMSO-d ): δ 197.02 (CO-CH=CH),

6

1

69.43 (CONCO), 168.55 (CONH), 168.45 (COCH=CH),

−1

158.19 (Ar-C), 144.86 (Ar-C), 144.66 (Ar-C), 138.49 (Ar-

C), 137.99 (Ar-C), 136.33 (2Ar-C), 136.19 (2Ar-C), 132.19

(Ar-C), 131.07 (Ar-C), 130.81 (Ar-C), 130.05 (2Ar-C),

(

KBr, cm ) ν: 3343 (NH), 3061 (Ar-CH), 1700, 1653

(

1

CO), 1600, 1523 (C=N) (Supplementary data page 2).

H NMR (400 MHz, DMSO-d ): δ 10.74 (s, 1H, NH),

6

1

29.88 (2Ar-C), 128.39 (2Ar-C), 127.07 (Ar-C), 120.35

Ar-C), 119.22 (Ar-C), 119.71 (2Ar-C), 114.55 (2Ar-C),

55.87 (OCH ) (Supplementary data page 43 ). MS: m/z

8

.70–7.39 (m, 17 H, Ar-H, CH=CH), 6.65 (s, 2H, Ar-H),

(

4

1

.65 (s, 2H, CH ) (Supplementary data page 14, 15, 16);

2

3

C NMR (100 MHz, DMSO-d ): δ 188.24 (COCH=CH),

6

+

(

rel. intensity), 516 (M , 19), 515 (M -1, 100), 500 (4),

86 (5), 473 (5), 461 (3), 448 (1), 410 (2), 399 (6), 398

24), 383 (20), 368 (11), 310 (2), 296 (2), 264 (85), 250 (10),

36 (10), 204 (4), 179 (3), 177 (4), 150 (2), 132 (12), 130

1

69.44 (CONCO), 168.38 (CONH), 168.29 (COCH=CH),

43.62 (Ar-C), 138.99 (2Ar-C), 138.02 (Ar-C), 137.19

4

(

Ar-C), 136.07 (Ar-C), 134.11 (Ar-C), 130.19 (Ar-C),

29.16 (2Ar-C), 129.02 (2Ar-C), 127.46 (Ar-C), 127.37

Ar-C), 126.92 (Ar-C), 123.36 (Ar-C), 122.69 (Ar-C),

19.97 (2Ar-C), 119.81 (2Ar-C), 115.02 (2Ar-C), 113.27

2Ar-C) (Supplementary data page 42 ). MS: m/z (rel.

2

1

46), 129 (9), 116 (21), 104 (30), 91 (15), 76 (59), 51 (14)

Supplementary data page 34). Anal. Calcd. for

(

1

C H N O (516.54): C, 74.41; H, 4.68; N, 5.42. Found

32 24 2 5

(

+

C, 74.20; H, 4.33; N, 5.77.

intensity) 487 (M +1, 7), 486 (M , 22), 486 (50), 453

(

100), 250 (2), 133 (9), 120 (21), 117 (6), 104 (47), 103

54), 97 (11), 92 (16), 78 (11), 77 (56), 65 (42), 51 (9) (E)-2-((1,3-Dioxo-2,3-Dihydro-1H-Inden-2-Yl) Methyl)-N-

Supplementary data page 32). Anal. Calcd. for (4-(3-(Naphthylen-1-Yl)Acryloyl)Phenyl)Benzamide (7d)

C H N O (486.52): C, 76.53; H, 4.56; N, 5.76. Found: Pale orange solid, 0.141g (91%), m.p. 141–142 °C. FTIR

31 22 2 4

−

1

C, 76.21; H, 4.72; N, 5.55.

(

KBr, cm ) ν: 3328, 3320 (NH), 3046 (Ar-CH), 1686,

639 (CO), 1602, 1584 (C=N) (Supplementary data page

(

E)-N-4-(3-(4-Clorophenyl)Acryloyl)Phenyl)-2-((1,3-

Dioxo-2,3-Dihydro-1H-Inden-2-Yl)Methyl)Benzamide (7b)

Yellow solid, 0.142 g (92%), m.p. 142–143 °C. FTIR 4.65 (s, 2H, CH

5). H NMR (400 MHz, DMSO-d

9.15–7.42 (m, 20H, Ar-H, CH=CH), 6.69 (s, 1H, Ar-H),

) (Supplementary data page 20). MS: m/z

6

): δ 10.77 (s, 1H, NH),

2

−

1

+

(

KBr, cm ) ν: 3458, 3341 (NH), 3065 (Ar-CH), 2924 (rel. intensity), 537 (M +1, 5) 536 (M , 18), 526 (36), 512

Aliph.-CH), 1702, 1653 (CO), 1601, 1523 (C=N) (10), 491 (12), 461 (2), 434 (7), 413 (10), 411 (100), 409

1

Supplementary data page 3). H NMR (400 MHz, (23), 402 (16), 383 (15), 367 (3), 455 (2), 279 (2), 258 (30,

DMSO-d ): δ 10.75 (s, 1H, NH), 8.22–7.39 (m, 17 H, 175 (3), 152 (18), 126 (3), 105 (2), 79 (4) (Supplementary

6

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Figure 10 Docking and binding pattern of compounds 7j, 7g, 7c and CA-4 showing interactions with different amino acid residues found in the active site of colchicine

binding site in tubulin protein (PDB code: 1SA0). (A) 3D structure of compound 7j (yellow) (B) 2D structure of compound 7j. (C) 3D structure of compound 7g (yellow)

(

D) 2D structure of compound 7g. (E) 3D structure of compound 7c (yellow) (F) 2D structure of compound 7c. (G) 3D structure of reference compound CA-4 (yellow)

H) 2D structure of reference compound CA-4.

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Table 7 Energy Binding Scores (Kcal/mol) for Compounds 7j, (4), 447 (3), 426 (3), 403 (2), 398 (14), 382 (19), 369 (7),

g, 7c and Entinostat at the Active Site of HDAC2

7

3

49 (11), 340 (4), 321 (6), 306 (7), 286 (18), 250 (15), 236

(10), 224 (4),205 (4), 193 (2), 175 (5), 167 (7), 150 (3),

32 (6), 77 (42), 76 (91), 65 (100) (Supplementary data

page 37). Anal. Calcd. for C H N O (531.51): C, 70.05;

Compound No.

Energy Binding Scores

7

j

−9.01

−8.43

−8.39

−7.67

g

c

31 21 3 6

H, 3.98; N, 7.91. Found C, 70.24; H, 3.74; N, 7.63.

Entinostat

(

E)-N-4-(3-(2,4-Dimethoxyphenyl)Acryloyl)Phenyl)-2-

(1,3-Dioxo-2,3-Dihydro-1H-Inden-2-Yl)Methyl)

(

data page 35 ). Anal. Calcd. for C H N O (536.58): C,

3

5 24 2 4

Benzamide (7g)

7

8.34; H, 4.51; N, 5.22. Found C, 78.54; H, 4.51; N, 5.39.

Yellow solid, 0.115 g (74%), m.p. 162–164 °C. FTIR

−

1

(

KBr, cm ) ν: 3421 (NH), 2933, 2839 (Aliph.-

(

E)-2-((1,3-Dioxo-2,3-Dihydro-1H-Inden-2-Yl) Methyl)-N-

4-(3-([2.2]Paracyclophan-4-Yl)Acryloyl)Phenyl)Benzamide

7E)

CH), 1733, 1653 (CO), 1596, 1506 (Aliph.-CH)

1

(

Supplementary data page 8 ). H NMR (400 MHz,

DMSO-d ): δ 10.71 (s, 1H, NH), 8.07–7.41 (m, 13H,

6

Pale yellow solid, 0.144g (93%), m.p. 153–154 °C.

−

1

Ar-H), 6.65 (m, 4H, Ar-H), 4.63 (s, 2H, CH

2

), 3.89 (s,

FTIR (KBr, cm ) ν: 3441 (NH), 3010 (Ar-CH), 2926,

890 (Aliph.-CH), 1682 (CO), 1626, 1593 (C=N)

3

H, OCH ), 3.85 (s, 3H, OCH ) (Supplementary data

3

2

+

1

page 24, 25 ). MS: m/z (rel. intensity), 547 (M +1, 4),

(

Supplementary data page 6 ). H NMR (400 MHz,

+

5

46 (M , 2), 493 (2), 458 (2), 446 (11), 430 (8), 428

12), 414 (46), 400 (36), 382 (88), 357 (3), 340 (7),

14 (3), 308 (5), 283 (5), 262 (7), 254 (9), 236 (2), 250

(5), 190 (3), 183 (4), 178 (5), 166 (5), 163 (20), 149

DMSO-d ): δ 10.53 (s, 1H, NH), 7.04–6.08 (m, 21 H.

6

(

Ar-H, CH=CH), 4.64 (s, 2H, CH ), 3.17 –2.95 (m, 8H, 2

2

3

CH -CH ) (Supplementary data page 21). MS: m/z (rel.

2

+

intensity), 645 (M +1, 6) 644 (M , 5), 425 (8), 353

(

25), 133 (9), 121 (21), 104 (9), 92 (13), 76 (100), 69

16), 65 (21), 63 (17) (Supplementary data page 38).

(

15), 351 (15), 292 (5), 286 (7), 247 (8), 245 (9), 238

5), 236 (12), 234 (7), 210 (7), 207 (15), 203 (8), 160

6), 152 (4), 144 (11). 131 (18), 112 (10), 104 (100),

Anal. Calcd. for C H N O (546.57): C, 72.52; H,

3

26

2

6

4

.79; N, 5.13. Found C, 72.21; H, 4.58; N, 5.37.

1

03 (99), 100 (15), 89 (16), 78 (60), 72 (27), 63 (9), 52

19) (Supplementary data page 36 ). Anal. Calcd. for

C H N O (644.76): C, 80.10; H, 5.63; N, 9.69. (E)-2-((1,3-Dioxo-2,3-Dihydro-1H-Inden-2-Yl) Methyl)-N-

(

4

3 36 2 4

Found C, 79.83; H, 5.88; N, 9.98.

(4-(3-(Furan-2-Yl)Acryloyl)Phenyl)Benzamide (7h)

Pale yellow solid, 0.109g (71%), m.p. 170–172 °C. FTIR

−

1

(

KBr, cm ) ν : 3411 (NH), 1711, 1647 (CO), 1601, 1530

(

E)-2-((1,3-Dioxo-2,3-Dihydro-1H-Inden-2-Yl) Methyl)-N-

1

(

Aliph.-CH) (Supplementary data page 9). H NMR (400

4-(3-(4-Nitrophenyl)Acryloyl)Phenyl)Benzamide (7f)

MHz, DMSO-d ): δ 10.72 (s, 1H, NH), 8.30 –6.66 (m,

Orange solid, 0.139g (90%), m.p. 145–146 °C. FTIR (KBr,

6

−1

7H, Ar-H, furyl), 4.90 (s, 2H, CH ) (Supplementary

2

cm ) ν: 3468, 3382 (NH), 2927 (Aliph.-CH), 1637 (CO),

13

1

data page 26, 27); C NMR (100 MHz, DMSO-d ): δ

1

595, 1517 (C=N) (Supplementary data page 7). H NMR

6

2

1

02.11 (COCH=CH), 186.11 (CONCO), 169.89 (CONH),

47.33, 146.73, 139.52, 138.18, 138.02, 135.09, 134.11,

30.00, 128.42, 128.22, 126.82, 124.33, 120.87, 119.89,

(

400 MHz, DMSO-d ): δ 10.78 (s, 1H, NH), 8.26–7.53 (m,

6

1

6H, Ar-H, CH=CH), 6.68 (s, 2H, Ar-H), 4.62 (s, 2H,

1

3

CH ) (Supplementary data page 22, 23 ); C NMR (100

2

MHz, DMSO-d₆):

δ

187.29 (COCH=CH), 166.22 119.77, 116.82, 115.90, 114.61, 114.11 (Ar-C, furyl-C,

(

CONCO), 158.23 (CONH), 148.11 (Ar-C), 145.01 (Ar- CH=CH) (Supplementary data page 45 ). MS: m/z (rel.

+

C), 144.34 (Ar-C), 142.12 (Ar-C), 139.01 (Ar-C), 138.72 intensity), 477 (M +1, 4) 476 (M , 7), 475 (45), 474

Ar-C), 132.32 (2Ar-C), 132.07 (2Ar-C), 130.01 (Ar-C), (30), 473 (100), 458 (26), 456 (57), 440 (12), 264 (13),

28.29 (Ar-C), 128.04 (2Ar-C), 127.12 (Ar-C), 126.14 213 (3), 195 (2), 185 (2), 159 (5), 156 (7), 133 (4), 130

2Ar-C), 125.90 (2Ar-C), 125.01 (2Ar-C), 124.72 (2Ar- (12), 120 (29), 105 (2), 102 (7), 92 (21), 78 (7), 65 (44)

C), 124.32 (Ar-C), 123.10 (Ar-C), 122.90 (Ar-C) (Supplementary data page 39). Anal. Calcd. for

(

1

(

Supplementary data page 44). MS: m/z (rel. intensity),

C

29

H

20

N

2

O

5

(476.48): C, 73.10; H, 4.23; N, 5.88. Found

+

5

32 (M +1,), 531 (M , 8), 519 (8), 515 (4), 479 (3), 470 C, 72.72; H, 4.55; N, 6.09.

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Figure 11 Docking and binding pattern of compounds 7j, 7g, 7c and CA-4 showing interactions with different amino acid residues found in HDAC2 active site (PDB code:

LXZ). (A) 3D structure of compound 7j (yellow) (B) 2D structure of compound 7j. (C) 3D structure of compound 7g (yellow) (D) 2D structure of compound 7g. (E) 3D

4

structure of compound 7c (yellow) (F) 2D structure of compound 7c. (G) 3D structure of entinostat reference compound (yellow) (H) 2D structure of entinostat

reference compound.

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(

E)-N-4-(3-(Benzo[D][1,3]Dioxol-5-Yl)Acryloyl)Phenyl)-2- to the manufacturer’s instructions (1000–2000 cells/well)

(1,3-Dioxoisoindolin-2-Yl)Methyl)Benzamid (7i) using 96-well microtiter plates. The cells were incubated in

Pale yellow solid, 0.114g (74%), m.p. 244–246 °C. FTIR a humidiﬁed atmosphere containing 5% CO in air at 37°C

2

−1

(

KBr, cm ) ν: 3450, 3349, 3241 (NH), 2908 (Aliph.-CH), for 34 hours. The synthesized hybrids 6 and 7a–j at different

1

642 (CO), 1588, 1552 (C=N) (Supplementary data page 10). concentrations (0.1, 1, 10, 25, 100 μM), in addition to CA4,

1

H NMR (400 MHz, DMSO-d ): δ 10.51 (s, 1H, NH), 7.91– were added to culture media for 72 hours. Media were

6

6.43 (m, 15H, Ar-H, CH=CH), 6.11 (s, 2H, Ar-H), 4.59 (s, 2H, removed. 200 μL of 5% MTT reagent was added to each

CH ) (Supplementary data page 28, 29 ); MS. m/z (rel. inten- well and plates were incubated in CO incubator at 37°C for

2

+

sity) 531 (M +1, 2), 530 (M , 20), 518 (13), 516 (2), 500 (15), 2 hours. After an incubation period, the formed formazan

86 (23), 453 (3), 268 (40), 267 (100), 239 (19), 180 (7), 152 crystals were dissolved in 200 μL isopropanol/well with

6), 144 (28), 133 (6), 120 (36), 117 (9), 104 (15), 89 (48), 77 continuous shaking using MaxQ 2000 plate shaker

19), 65 (61) (Supplementary data page 40). Anal. Calcd. for (Thermo Fisher Scientiﬁc Inc, MI, USA) at room tempera-

C H N O (530.53): C, 72.45; H, 4.18; N, 5.28. Found C, ture. Absorbance was measured spectrophotometrically at

4

(

32 22 2 6

7

2.31; H, 4.33; N, 5.47.

wavelength 570 nm using a Stat FaxR 4200 plate reader

41

(

Awareness Technology, Inc., FL, USA).

Cell viability were calculated as percentage of control

and the concentration that inhibits 50% of maximum cell

Yellow solid, 0.181 g (78%), m.p. 187–189 °C. FTIR (KBr, proliferation were determined and expressed as IC₅₀using

(

E)-2-((1,3-Dioxoisoindolin-2-Yl)Methyl)-N-(4-(3-(3,4,5-

Trimethoxyphenyl)Acryloyl)Phenyl)Benzamide (7j)

−1

cm ) ν: 3454 (NH), 2932, 2853 (Aliph.-CH), 1733, 1637 Graph Pad Prism 5 software (Graph Pad software Inc,

CO), 1563, 1538 (Aliph.-CH) (Supplementary data CA, USA).

1

page 11). H NMR (400 MHz, DMSO-d ): δ 10.69 (s, 1H,

6

NH), 8.57–6.61 (m, 16H, Ar-H, CH=CH), 4.61 (s, 2H, CH ₂ ), In vitro Enzyme Inhibitory Assays

3

.89 (s, 3H, 1OCH ), 3.85 (s, 6H, 2 OCH ) (Supplementary To investigate the effect of synthesized hybrids on the

data page 30). MS: m/z (rel. intensity), 576 (M , 12), 462 (8), enzymatic activity of HDAC and tubulin, MCF-7 line

3 3

+

4

44 (8), 430 (9), 416 (55), 409 (12), 383 (87), 355 (2), 340 (9), was used and incubated in DMEM supplemented with

12 (8). 297 (3), 279 (8), 264 (4). 236 (5), 193 (4). 167 (6), 160 10% fetal bovine serum, 1% penicillin-streptomycin and

3

6), 132 (9), 104 (11), 76 (100), 77 (34) (Supplementary data 10 μg/mL insulin (Sigma). All of the other chemicals and

page 41). Anal. Calcd. for C H N O (576.60): C, 70.82; H, reagents were from Sigma, or Invitrogen Cancer cells were

3

4 28 2 7

5

4

.89; N, 4.86. Found C, 70.51; H, 5.11; N, 4.65.

seeded at density 1.2–1.8 x 10 /well in 100 μL DMEM

using 96-well plates. To each well, 100 μL of the synthe-

sized compounds 7c, 7g and 7j were added 18–24 hours

prior to HDAC and tubulin inhibition assay.

Biological Evaluation

In vitro Anti-Proliferative Activity Assay

The anti-proliferative activity of the synthesized hybrids was

tested using MTT assay method against MCF-7 and Hep G2. Tubulin Polymerization Inhibitory Assay

The principle of assay based on the ability of viable cell to The assay was done on MCF-7 cell line using TUBb

cleave tetrazolium ring of MTT reagent yielding purple for- ELISA kit (Cloud-Clone Corp.). Cancer cells were seeded

mazan crystals which are insoluble in aqueous solutions.

into 96-well microtiter plates and kept in a humidiﬁed

The amount of the formazan dye generated by dehy- atmosphere at 37°C for 1 day. After dilution to the speci-

drogenase in cells is directly proportional to the number of ﬁed concentration, 100 μL of the synthesized hybrids 7c,

living cells.

7g and 7j and CA4 were added to each well, then the plate

Cancer cells, (MCF-7) and human hepatocellular carci- was incubated for 2 hours at 37°C.

noma (Hep G2) were purchased from American type Cell

After discarding the supernatant, 100 μL of kit detec-

Culture collection (ATCC, Manassas, USA). The tested cells tion reagent A was added to each well, then the plate was

were maintained in Dulbecco’s Modiﬁed Eagle’s Medium re-incubated for 2 hours at 37°C. Kit detection reagent B

(DMEM, Invitrogen/Life Technologies) supplemented with was added to each well and the plate incubated for 30

1

0% fetal bovine serum (FBS, Hyclone) and antibiotics (100 minutes at 37°C. After ﬁve washing cycles, 90 μL of

units/mL penicillin and 100 mg/mL streptomycin). After 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) solution was added

detachment with trypsin, cancer cells were seeded according to each well, then the plate was incubated for 15–25

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Drug Design, Development and Therapy 2020:14

3127

Mourad et al

minutes at 37°C, followed by the addition of 50 μL of stop CA-4 and entinostat at their IC50 in DMSO (1% v/v). The

solution. Different concentrations (0.01, 0.1, 1, 10 μM) of cell plates were incubated for 24 hours. The cells were

the synthesized hybrids 7c, 7g and 7j and CA4 were then ﬁxed with 70% ice-cold ethanol after washing twice

incubated with the cells for 72 hours.

with cold phosphate buffered saline (PBS). Cells washed

Absorbance was measured spectrophotometrically at with PBS for 30 minutes at 37°C and recovered by cen-

wavelength 450 nm using a Stat FaxR 4200 plate reader trifugation at 2000 rpm for 5 minutes.

4

2

(

Awareness Technology, Inc., FL, USA). The concentra-

Cells were stained with DNA ﬂuorochrome propidium

tion that inhibits 50% of maximum TUBb activity were iodide (PI). The plates were incubated at room temperature

determined and expressed as IC₅₀using Graph Pad Prism in a dark place for 20 minutes. Then the cells were inves-

5

software (Graph Pad software Inc, CA, USA ).

tigated with a FACS Caliber ﬂow cytometer (Becton

42

Dickinson, Heidelberg, Germany).

HDAC Inhibitory Assay

Annexin V-FITC/PI Staining Assay

The effect of the synthesized hybrids 7c, 7g and 7j on

HDAC (1 and 2) were investigated using ELISA assay kit

6

MCF-7 cells at a density of 4 x 10 /well were incubated with

synthesized compounds 7g, 7j in addition to CA-4 and entino-

stat at their IC50 for 24 hours. Cells were subjected to three

washing cycles with ice cold PBS, then suspended in PBS.

Cell apoptosis was detected by Annexin V-FITC Apoptosis

Detection Kit (BioVisionResearch Products, USA). The cells

were stained with PI staining solution, Annexin V-FITC and

incubated at room temperature for 15 minutes in a dark place.

Cells were investigated by ﬂow cytometry using FACS caliber

(Mybiosource, Inc.). According to manufacturer’s instruc-

tions, MCF-7 cell was seeded into 96-well microtiter

plates and kept in a humidiﬁed atmosphere at 37°C for 1

day. After dilution to the speciﬁed concentration, 100 μL

of the synthesized compounds 7c, 7g and 7j and positive

control entinostat were added to each well, then the plate

incubated for 2 hours at 37°C.

After discarding the supernatant, 100 μL of kit detec-

tion reagent A was added to each well, then the plate re-

incubated for 2 hours at 37°C. Kit detection reagent B was

added to each well and the plate incubated for 30 minutes

at 37°C. After ﬁve washing cycles, 90 μL of 3,3ʹ,5,5ʹ-

tetramethylbenzidine (TMB) solution was added to each

well, then the plate was incubated for 15–25 min at 37°C,

followed by the addition of 50 μL of stop solution.

Different concentrations (0.01, 0.1, 1, 10 μM) of the

synthesized hybrids 7c, 7g and 7j and entinostat were

incubated with the cells for 72 hours.

44

(Becton Dickinson, Heidelberg, Germany).

Molecular Docking Study

Molecular modelling studies of the selected derivatives 7c,

g, 7j, CA-4 and entinostat were performed using MOE

7

software. Ligands were built into the builder interface of

the MOE program and their energies were minimized until

a root mean square deviation (RMSD) gradient of 0.01

kcal/mol and a root mean square (RMS) distance of 0.1 Å

with MMFF94X (Merck molecular force ﬁeld 94X) force-

ﬁeld and the partial charges were automatically calculated.

The X-ray crystallographic structure of tubulin-colchicine

complex (PDB code: 1SA0) and HDAC2 active site (PDB

code: 4LXZ) were downloaded from protein data bank

Absorbance was measured spectrophotometrically at

wavelength 405 nm using a Stat FaxR 4200 plate reader

4

3

(Awareness Technology, Inc., FL, USA). The concentra-

tion that inhibits 50% of maximum HDAC activity were

determined and expressed as IC50 using Graph Pad Prism

(www.rcsb.org). The enzymes were prepared, the hydro-

gens were added then the atoms connection and type were

checked with automatic correction. The obtained poses

were studied and the poses showing the best ligand-

enzyme interactions were selected and stored for energy

5

software (Graph Pad software Inc, CA).

In vitro Cell Cycle Analysis

In vitro DNA Flow Cytometry Assay

5

MCF-7 cells at density of 2 x 10 were seeded into each calculations.

well of a 6-well plate. The cells were kept in Dulbecco’s

Modiﬁed Eagle’s Medium (DMEM), supplemented with Crystal Structure Determinations

10% fetal bovine serum, and incubated in a humidiﬁed The crystal was mounted in inert oil and transferred to the

atmosphere containing 5% CO in air at 37°C for 24 cold gas stream of a Rigaku-Oxford XtaLAB Synergy

2

hours. The old medium was replaced with a fresh one diffractometer using mirror-focussed Cu Kα radiation.

containing the synthesized hybrids 7g, 7j in addition to Absorption corrections were based on multi-scans. The

submit your manuscript | www.dovepress.com

3

128

Drug Design, Development and Therapy 2020:14

doi:10.1016/S0959-440X(98)80099-7

Mourad et al

2

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gram SHELXL-2017. The hydrogen atom of the NH group

was reﬁned freely. Other hydrogens were included using a

riding model or rigid methyl groups. Complete crystal-

lographic data have been deposited at the Cambridge

Crystallographic Data Centre as CCDC 1,948,416.

Copies of the data can be obtained free of charge from

www.ccdc.cam.ac.uk/data_request/cif.

2

005;14(12):1497–1511. doi:10.1517/13543784.14.12.1497

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Crystallographic data for compound 6: C H N O , M =

24

18

2

4

r

13543784.13.1.21

3

98.40, Temperature (K) = 100, Crystal habit: colorless plate,

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Crystal size (mm): 0.15 × 0.10 × 0.04, Crystal system:

Monoclinic, Space group: P2 /c, Cell dimensions: [a (Å) =

1

6

.5120(3), b (Å) = 33.6736(13), c (Å) = 8.5409(3),

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β (°) = 95.237(4)], Cell volume (Å ) = 1865.06, Z = 4, D (g

x

−3

cm ) = 1.419, Radiation, wavelength (Å): Cu Kα, 1.54184 Å,

−1

μ (mm ) = 0.80, 2θ(max) (°) =154.8, Reﬂections collected =

9,381, Independent reﬂections = 3927, R(int) = 0.031,

3

Transmissions = 0.728–1.000, No. of parameters = 276,

2

Goodness-of-ﬁt on F = 1.04, wR2 (all reﬂections) = 0.092,

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Acknowledgment

The authors are deeply grateful for Professor H. Hopf. Institut

fuer Organische Chemie, TU Braunschweig, Germany, for

critical reading of the manuscript.

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Disclosure

The authors reports no conﬂicts of interest in this work.

2

0. Singh N, Kumar N, Rathee G, et al. Privileged scaffold chalcone:

synthesis, characterization and its mechanistic interaction studies

with BSA employing spectroscopic and chemoinformatics

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