Design, synthesis and anticancer evaluation of novel 1,3-benzodioxoles and 1,4-benzodioxines

Article abstract of DOI:10.1016/j.ejps.2019.105045

A new set of 1,3-benzodioxoles and 1,4-benzodioxines was designed and synthesized starting from gallic acid as anticancer agents. The antiproliferative effect of the target compounds was evaluated against a panel of cancer cell lines (HepG2, PC-3, MCF-7 and A549) using MTT assay. The 1,4-benzodioxine derivative 11a manifested broad spectrum effect towards the four tested cancer cell lines (IC₅₀ < 10 μM) with lower toxic effect on normal human cell line BJ1. Cell cycle progression of MCF-7 after treatment with compound 11a was studied where it induced cells accumulation at G2/M phase as well as increasing in the percentage of cells at pre-G1. Compound 11a is found to be a tubulin polymerization inhibitor with IC₅₀ = 6.37 μM. Also, flow cytometeric analysis revealed that compound 11a could induce both early and late stage apoptosis in MCF-7 cell line. Moreover, the ability of this compound to stimulate apoptosis in the latter cell line was further confirmed by: increment of Bax/Bcl-2 ratio, increase the expression of tumor suppressor gene p53, boosting the levels of initiator and executioner caspases as well as raise in the amount of cytochrome C. In addition molecular docking study was accomplished on the colchicine binding site of tubulin (pdb: 1SA0) to illustrate the interactions of the most potent compound 11a to the receptor.

Full text of DOI:10.1016/j.ejps.2019.105045

EuropeanJournalofPharmaceuticalSciences139(2019)105045
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Design, synthesis and anticancer evaluation of novel 1,3-benzodioxoles and
1,4-benzodioxines
Rasha Mohamed Hassana, Walaa Hamada Abd-Allahb, Asmaa Mohamed Salmanc,
Aida Abdel-Sattar El-Azzounya, Mohamed Nabil Aboul-Eneina,⁎
a Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618), P.O. 12622,
Dokki, Giza, Egypt
b Pharmaceutical Chemistry Department, College of Pharmaceutical Science and Drug Manufacturing, Misr University for Science and Technology, P.O. 77, 6th of October
City, Giza, Egypt
c Medicinal and Pharmaceutical Chemistry Department, Pharmacology Group, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID:
60014618), P.O. 12622, Dokki, Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Synthesis
1,3-Benzodioxoles
1,4-Benzodioxines
Anticancer
A new set of 1,3-benzodioxoles and 1,4-benzodioxines was designed and synthesized starting from gallic acid as
anticancer agents. The antiproliferative effect of the target compounds was evaluated against a panel of cancer
cell lines (HepG2, PC-3, MCF-7 and A549) using MTT assay. The 1,4-benzodioxine derivative 11a manifested
broad spectrum effect towards the four tested cancer cell lines (IC50 < 10 μM) with lower toxic effect on normal
human cell line BJ1. Cell cycle progression of MCF-7 after treatment with compound 11a was studied where it
induced cells accumulation at G2/M phase as well as increasing in the percentage of cells at pre-G1. Compound
11a is found to be a tubulin polymerization inhibitor with IC50 = 6.37 μM. Also, flow cytometeric analysis
revealed that compound 11a could induce both early and late stage apoptosis in MCF-7 cell line. Moreover, the
ability of this compound to stimulate apoptosis in the latter cell line was further confirmed by: increment of Bax/
Bcl-2 ratio, increase the expression of tumor suppressor gene p53, boosting the levels of initiator and executioner
caspases as well as raise in the amount of cytochrome C. In addition molecular docking study was accomplished
on the colchicine binding site of tubulin (pdb: 1SA0) to illustrate the interactions of the most potent compound
11a to the receptor.
Tubulin
Apoptosis
1. Introduction
first class is microtubule-stabilizing agents such as taxens which pro-
mote microtubule polymerization. The second class is microtubule-de-
Cancer is considered as one of the most serious health minatory
disorders. It is ranked as one of the main reasons of global death (Torre
et al., 2015; Trivedi et al., 2018). Using of available chemotherapeutic
agents is commonly associated with severe side effects and drug re-
sistance (Hao et al., 2016). Therefore, there is still a dire necessity for
developing of new chemotherapeutic agents having fewer side effects.
Microtubules are cytoskeletal structures in eukaryotic cells com-
posed of α, β-tubulin heterodimers. They play an essential role in many
cellular processes such as cell division, maintenance of cell shape and
cell signaling (Downing and Nogales, 1998; Fan et al., 2018). This
renders microtubules an interesting target for anticancer agents. Mi-
crotubule-targeting agents are classified into two classes according to
mechanism by which they interfere with microtubules dynamics. The
stabilizing agents which inhibit microtubule polymerization. The drugs
of the latter class bind to the vinca or the colchicine binding site of
tubulin (Botta et al., 2008; La Regina et al., 2019). It has been reported
that inhibitors which interact with colchicine binding site have ad-
vantages over those targeting other binding sites, such as improved
aqueous solubility, lower toxicity and multidrug resistance effects
(Pettit et al., 1989, 1995; Stengel et al., 2010; Lu et al., 2012; Li et al.,
2019) Many natural and synthetic 1,3-benzodioxole derivatives as po-
dophyllotoxin (Desbène and Giorgi-Renault, 2002), steganacin (Lu
et al., 2012), combretastatin A-2 (Pettit and Singh, 1987) as well as
compounds I and II (Batra et al., 1985; Yi et al., 2012) (Fig. 1) possess
anticancer activity with tubulin polymerization inhibitory effect
through binding to colchicine binding site. Also, some 1,4-benzodioxine
⁎ Corresponding author at: Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical Chemistry Group, Pharmaceutical and Drug Industries Research
Division, National Research Centre, 12622 Dokki, Giza, Egypt.
E-mail address: mnaboulenein@yahoo.com (M.N. Aboul-Enein).
https://doi.org/10.1016/j.ejps.2019.105045
Received 15 June 2019; Received in revised form 21 July 2019; Accepted 13 August 2019
Availableonline14August2019
0928-0987/©2019ElsevierB.V.Allrightsreserved.

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
Fig. 1. Structures of some reported tubulin polymerization inhibitors and the target compounds 8a, b, 9a, b, 10a–c and 11a–c (Red: 1,3-benzodioxole, Blue: 1,3,4-
oxadiazole and Magenta: thiazolidin-4-one). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
congeners have been reported to inhibit tubulin polymerization
(Kiselyov et al., 2010).
2. Experimental
On the other hand, 1,3,4-oxadiazoles possess various biological
activities (Jayashankar et al., 2009; Rashid et al., 2012) due to presence
of –N=C-O- linkage (Rigo and Couturier, 1985; Patel et al., 2013).
Some of 1,3,4- oxadiazoles exhibited their anticancer activity through
inhibition of tubulin polymerization such as compounds III (Kamal
et al., 2011) and IV (Kamal et al., 2016). Also, certain thiazolidin-4-
ones as compounds V (Zhang et al., 2013) and VI (Mu et al., 2015) have
been reported to exhibit good tubulin polymerization inhibition activity
(Fig. 1).
It is well known that hybridization of two or more bioactive scaf-
folds in a single compound is an reliable approach for developing of
novel more effective bio-candidates (Aboul-Enein et al., 2015). With
these aspects in mind, new hybrid structures of 1,3-benzodioxole and its
higher homologue 1,4-benzodioxine with 1,3,4-oxadiazole or thiazo-
lidin-4-one rings have been designed, synthesized and evaluated for
their antiproliferative activity against HepG2, PC-3, MCF-7 and A549
human cancer cell lines. Furthermore, the effect of potent anti-
proliferative candidates on human normal cell was assessed to de-
termine the selectivity of these compounds. Besides, the tubulin poly-
merization inhibition activity of the most active compound was
determined to explore its mechanism of action. In addition, the influ-
ence of the most active compound on cell cycle progression was studied
as well as its effect on the apoptosis regulatory molecules in the cells.
2.1. Chemistry
All melting points were measured by the Electrothermal Capillary
apparatus and were uncorrected. Infrared spectra were recorded as KBr
pellets with [JASCO FT/IR-6100 spectrometer] and the values are
characterized in cm−1 1H NMR (400 MHz) and 13C NMR (100 MHz)
.
spectra were performed on [Jeol ECA 500 MHz spectrometer] and
chemical shift values were recorded in ppm. Mass spectral data were
measured with electron impact ionization technique at 70 eV.
Elemental microanalyses were performed at Microanalytical Units in
the National Research Centre as well as Cairo University. Column
chromatography was done using silica gel as a stationary phase.
2.1.1. Synthesis of methyl 3,4,5-trihydroxybenzoate (2)
A solution of 17 g (0.1 mol) gallic acid and 0.002 g (0.01 mmol) of p-
toluene sulfonic acid in 100 mL methanol was refluxed under stirring
overnight. The mixture was cooled, the solvent was evaporated under
vacuum and the residual was dissolved in EtOAc. Thereafter, the or-
ganic phase was shaken with 10% NaHCO3 solution, dried (Na2SO4)
and evaporated to obtain 2 in 75% yield as buff solid m.p. 198 °C [lit.
(Mostafa et al., 2006) 200 °C].
2

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
2.1.2. Synthesis of methyl 3,4-dihydroxy-5-methoxybenzoate (3)
Methyl 3,4,5-trihydroxybenzoate (50 g, 270 mmol) (2) and borax
(54 g, 140 mmol) were dissolved in distilled water (1 L) and stirred at
room temperature for 1 h. A solution of 13% NaOH was added and the
solution was stirred for 10 min followed by addition of dimethylsulfate
(135 mL, 370 mmol) and further stirred for 15 h. The reaction was
neutralized to pH 7–8 using 10% sulfuric acid, extracted with ethyl
acetate, dried (Na2SO4) and evaporated in vacuo to afford compound 3
as a crude brown solid which was subjected to purification through
column chromatography using petroleum ether (40–60): EtOAc 70:30
as mobile phase to obtain methyl 3,4-dihydroxy-5-methoxybenzoate
(42.5 g, yield 79%) as a white solid m.p. 112 °C [lit. (Pettit and Singh,
1987) 110–111 °C].
CH2-benzodioxole), 7.12–7.23 (m, 2H, Har.), 7.99 (s, 1H, NH), 9.27 (s,
1H, NH), 10.18 (s, 1H, NH); 13C NMR (DMSO d6, δ, ppm): 31.4 (CH3),
56.9 (OCH3), 102.4 (CH2-benzodioxole), 102.6, 108.9, 127.1, 138.3,
143.2, 148.7 (aromatic carbons), 165.6 (C=O), 182.3 (C=S); MS (EI)
m/z (%): 283 (M+, 2.28), 179 (100); Anal. calcd. for C11H13N3O4S: C,
46.64; H, 4.63; N, 14.83; S, 11.32. Found: C, 46.82; H, 4.76; N, 14.95; S,
11.50.
2.1.5.2. N-Butyl-2-(7-methoxybenzo[d][1,3]dioxole-5-carbonyl)
hydrazine-1-carbothioamide (6b). White solid, m.p. 210 °C, yield 88%;
IR (KBr, cm−1) 3436, 3316, 3265 (3NH), 1620 (C=O); 1H NMR (DMSO
d6, δ, ppm): 0.82–0.85 (t, 3H, J = 7.65 Hz, CH3-CH2-CH2-CH2-), 1.23
(m, 2H, CH3-CH2-CH2-CH2-), 1.46 (s, 2H, CH3-CH2-CH2-CH2-),
3.41–3.42 (t, 2H, J = 6.4 Hz, CH3-CH2-CH2-CH2-), 3.86 (s, 3H,
OCH3), 6.07 (s, 2H, CH2-benzodioxole), 7.14–7.25 (m, 2H, Har.), 8.04
(s, 1H, NH), 9.16 (s, 1H, NH) 10.19 (s, 1H, NH); 13C NMR (DMSO d6, δ,
ppm): 14.3 (CH3-CH2-CH2-CH2-), 19.9 (CH3-CH2-CH2-CH2-), 31.3 (CH3-
CH2-CH2-CH2-), 43.8 (CH3-CH2-CH2-CH2-), 56.9 (OCH3), 102.4 (CH2-
benzodioxole), 102.6, 109.1, 127.1, 138.3, 143.2, 148.7 (aromatic
2.1.3. General procedure for synthesis of methyl 7-methoxybenzo[d][1,3]
dioxole-5-carboxylate (4a) and methyl 8-methoxy-2,3-dihydrobenzo[b]
[1,4]dioxine-6-carboxylate (4b)
To 0.108 mol of dichloromethane or dichloroethane and 19.9 g
(0.144 mol) of K2CO3 in 300 mL DMF, 14.4 g (0.0727 mol) of 3 in
250 mL DMF was added dropwise under stirring and the mixture was
heated at 90 °C for 12 h. After cooling to room temperature, the reaction
mixture was filtered and the remaining residue was washed with
EtOAc. The filtrate was shaken with water, dried (Na2SO4) and eva-
porated. The obtained solid was column chromatographed using pet-
roleum ether (40–60): EtOAc 60:40 as mobile phase to give the desired
compounds.
carbons), 165.5 (C=O), 181.8 (C=S); MS (EI) m/z (%): 324 (M+
,
2.1); Anal. calcd. for C14H19N3O4S: C, 51.68; H, 5.89; N, 12.91; S, 9.85.
Found: C, 51.76; H, 5.98; N, 13.12; S, 9.99.
2.1.5.3. 2-(7-Methoxybenzo[d][1,3]dioxole-5-carbonyl)-N-
phenylhydrazine-1-carbothioamide (6c). White solid, m.p. 218 °C, yield
87%; IR (KBr, cm−1) 3312, 3277, 3218 (3NH), 1617 (C=O); 1H NMR
(DMSO d6, δ, ppm): 3.84 (s, 3H, OCH3), 6.06 (s, 2H, CH2-benzodioxole),
7.12–7.29 (m, 7H, Har.), 9.67–9.74 (m, 2H, 2NH), 10.38 (s, 1H, NH);
13C NMR (DMSO d6, δ, ppm): 56.9 (OCH3), 102.6 (CH2-benzodioxole),
109.0, 125.6, 126.6, 127.2, 128.4, 138.4, 139.7, 143.3, 148.7 (aromatic
carbons), 165.6 (C=O), 181.5 (C=S); MS (EI) m/z (%): 345 (M+, 0.2),
179 (100); Anal. calcd. for C16H15N3O4S: C, 55.64; H, 4.38; N, 12.17; S,
9.28. Found: C, 55.82; H, 4.59; N, 12.25; S, 9.35.
4a: White solid, yield 65%, m.p. 92 °C [lit. (Zhang et al., 2007)
89–90 °C].
4b: White solid, yield 60%, m.p. 104 °C [lit. (Tsyganov et al., 2013)
106–108 °C].
2.1.4. Synthesis of 7-methoxybenzo[d][1,3]dioxole-5-carbohydrazide (5a)
and 8-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazide (5b)
A mixture of 5 mmol of methyl ester 4a or 4b and 25 mmol of hy-
drazine hydrate (98%) in 5 mL of methanol was refluxed for 6 h (to get
5a) or 5 days (to get 5b). After cooling a white precipitate of the desired
hydrazide 5a or 5b was formed and filtered off.
2.1.5.4. N-(4-Chlorophenyl)-2-(7-methoxybenzo[d][1,3]dioxole-5-
carbonyl)hydrazine-1-carbothioamide (6d). White solid, m.p. 208 °C,
yield 90%; IR (KBr, cm−1) 3439, 3315, 3223 (3NH), 1617 (C=O); 1H
NMR (DMSO d6, δ, ppm): 3.87 (s, 3H, OCH3), 6.07 (s, 2H, CH2-
benzodioxole), 7.18–7.38 (m, 6H, Har.), 9.77 (s, 1H, NH), 9.82 (s, 1H,
NH), 10.45 (s, 1H, NH); 13C NMR (DMSO d6, δ, ppm): 56.9 (OCH3),
102.4 (CH2-benzodioxole), 102.9, 109.0, 127.0, 128.0, 128.3, 129.4,
138.4, 138.7, 143.3, 148.7 (aromatic carbons), 165.5 (C=O), 182.2
5a: White solid, yield 85%, m.p. 204 °C [lit. (Semenov et al., 2010)
205–206 °C].
8-Methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazide
(5b)
(C=S); MS (EI) m/z (%): 379.8 (M+
16H14ClN3O4S: C, 50.60; H, 3.72; N, 11.06; S, 8.44. Found: C, 50.82;
H, 3.91; N, 11.19; S, 8.65.
, 8); Anal. calcd. for
C
White solid, m.p. 178–180 °C, yield 80%; IR (KBr, cm−1) 3436,
3298 (NH, NH2), 1611 (C=O); 1H NMR (DMSO d6, δ, ppm): 3.89 (s, 3H,
OCH3), 4.20 (s, 4H, 2× CH2-benzdioxine), 4.39 (s, 2H, NH2), 6.98–7.03
(m, 2H, Har.), 9.58 (s, 1H, NH); 13C NMR (DMSO d6, δ, ppm): 56.32
(OCH3), 64.39, 64.511 (2× CH2-benzdioxine), 103.87, 109.27, 125.37,
136.05, 143.79, 148.88 (aromatic carbons), 165.84 (C=O); MS (EI) m/
z (%): 224 (M+, 14); Anal. calcd. for C10H12N2O4: C, 53. 57; H, 5.39; N,
12.49. Found: C, 53.71; H, 5.50; N, 12.32.
2.1.5.5. 2-(7-Methoxybenzo[d][1,3]dioxole-5-carbonyl)-N-(4-
methoxyphenyl)hydrazine-1-carbothioamide (6e). White solid, m.p.
210 °C, yield 90%; IR (KBr, cm−1) 3750, 3447, 3314 (3NH), 1622
(C=O); 1H NMR (DMSO d6, δ, ppm): 3.75 (s, 3H, OCH3), 3.89 (s, 3H,
OCH3), 6.10 (s, 2H, CH2-benzodioxole), 6.89–6.91 (m, 2H, Har.),
7.22–7.32 (m, 4H, Har.), 9.58 (s, 1H, NH), 9.67 (s, 1H, NH), 10.38 (s,
1H, NH); 13C NMR (DMSO d6, δ, ppm): 55.6, 56.9 (2× OCH3), 102.4
(CH2-benzodioxole), 102.8, 109.0, 113.7, 127.0, 128.0, 132.3, 138.3,
143.2, 148.6, 157.2 (aromatic carbons), 165.6 (C=O), 181.9 (C=S);
MS (EI) m/z (%): 375.8 (M+, 21); Anal. calcd. for C17H17N3O5S: C,
54.39; H, 4.56; N, 11.19; S, 8.54. Found: C, 54.49; H, 4.65; N, 11.28; S,
8.75.
2.1.5. General procedure for synthesis of N-alkyl/arayl-2-(7-methoxybenzo
[d][1,3]dioxole-5-carbonyl)hydrazine-1-carbothioamide (6a–e) and 2-(8-
methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)-N-alkyl/arayl
hydrazine-1-carbothioamide (7a–e)
The appropriate isothiocyanate (0.01 mol) was added to a stirred
ethanolic solution of 0.01 mol of 5a or 5b. The reaction mixture was
refluxed for 1 h. The obtained solid was filtered, washed with diethyl
ether and dried.
2.1.5.6. 2-(8-Methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)-N-
methylhydrazine-1-carbothioamide (7a). White solid, m.p. 232 °C, yield
94%; IR (KBr, cm−1) 3438, 3289, 3229 (3NH), 1667 (C=O); 1H NMR
(DMSO d6, δ, ppm): 2.86 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 4.24–4.29
(m, 4H, 2× CH2-benzodioxine),7.13–7.14 (m, 2H, Har), 8.00 (s, 1H,
NH), 9.26 (s, 1H, NH), 10.16 (s, 1H, NH); 13C NMR (DMSO d6, δ, ppm):
30.9 (CH3), 55.9 (OCH3), 63.9, 64.1 (2× CH2-benzodioxine), 104.2,
2.1.5.1. 2-(7-Methoxybenzo[d][1,3]dioxole-5-carbonyl)-N-
methylhydrazine-1-carbothioamide (6a). White solid, m.p. 224 °C, yield
93%; IR (KBr, cm−1) 3702, 3687, 3440 (3NH), 1620 (C=O); 1H NMR
(DMSO d6, δ, ppm): 2.96 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 5.87 (s, 2H,
3

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
109.8, 123.9, 136.1, 143.1, 148.3 (aromatic carbons), 165.2 (C=O),
bromoacetate (0.0055 mol) and anhydrous sodium acetate (0.04 mol)
were added and the mixture was refluxed while stirring for 5 h.
Thereafter, the mixture was evaporated in vacuo, the crude product was
column chromatographed using a mixture of DCM:EtOAC (7:3) as a
mobile phase to give the desired compounds.
182.1 (C=S); MS (EI) m/z (%): 297 (M+, 0.16); Anal. calcd. for
C
12H15N3O4S: C, 48.48; H, 5.09; N, 14.13; S, 10.78. Found: C, 48.67; H,
5.18; N, 14.24; S, 10.97.
2.1.5.7. N-butyl-2-(8-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-
carbonyl)hydrazine-1-carbothioamide (7b). White solid, m.p. 200 °C,
yield 90%; IR (KBr, cm−1) 3746, 3442, 3279 (3NH), 1664 (C=O); 1H
NMR (DMSO d6, δ, ppm): 0.82–0.85 (t, 3H, J = 7.65 Hz, CH3-CH2-CH2-
CH2-), 1.25 (m, 2H, CH3-CH2-CH2-CH2-),1.46 (s, 2H, CH3-CH2-CH2-
CH2-), 3.41–3.42 (t, 2H, J = 6 Hz, CH3-CH2-CH2-CH2-), 3.79 (s, 3H,
OCH3), 4.24–4.25 (m, 4H, 2× CH2-benzodioxine), 7.11–7.13 (m, 2H,
2.1.6.1. 7-Methoxy-N′-(3-methyl-4-oxothiazolidin-2-ylidene)benzo[d]
[1,3]dioxole-5-carbohydrazide (8a). Yellowish white solid, m.p. 198 °C,
yield 75%; IR (KBr, cm−1) 3426 (NH), 1719, 1622 (2 C=O); 1H NMR
(DMSO d6, δ, ppm): 3.15 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 4.00 (s, 2H,
CH2-thiazolidine), 6.07 (s, 2H, CH2-benzodioxole), 7.06–7.18 (m, 2H,
Har.), 10.78 (s, 1H, NH); 13C NMR (DMSO d6, δ, ppm): 29.7 (CH2, CH2-
thiazolidine), 33.0 (CH3), 56.9 (OCH3), 101.7 (CH2, CH2-benzodioxole),
102.5, 108.5, 127.7, 138.0, 143.3, 148.7 (aromatic carbons), 162.8,
163.9, 172.3 (C]N, 2× C]O); MS (EI) m/z (%): 323 (M+, 100); Anal.
calcd. for C13H13N3O5S: C, 48.29; H, 4.05; N, 13.00; S, 9.92. Found: C,
48.21; H, 3.91; N, 12.88; S, 9.71.
H
ar.), 8.01 (s, 1H, NH), 9.13 (s, 1H, NH), 10.16 (s, 1H, NH); 13C NMR
(DMSO d6, δ, ppm): 14.2 (CH3-CH2-CH2-CH2-), 19.8 (CH3-CH2-CH2-
CH2-), 31.3 (CH3-CH2-CH2-CH2-), 43.8 (CH3-CH2-CH2-CH2-), 56.3
(OCH3), 64.3, 64.5 (2× CH2, CH2-benzodioxine), 104.5, 110.2,
124.2, 136.6, 143.6, 148.7 (aromatic carbons), 165.9 (C=O), 184.2
(C=S); MS (EI) m/z (%): 339 (M+, 100); Anal. calcd. for C15H21N3O4S:
C, 53.08; H, 6.24; N, 12.38; S, 9.45. Found: C, 53.26; H, 6.35; N, 12.45;
S, 9.67.
2.1.6.2. N′-(3-butyl-4-oxothiazolidin-2-ylidene)-7-methoxybenzo[d][1,3]
dioxole-5-carbohydrazide (8b). Yellowish white solid, m.p. 204 °C, yield
78%; IR (KBr, cm−1) 3211 (NH), 1736, 1621 (2 C=O); 1H NMR (CDCl3,
δ, ppm): 0.89–92 (t, J = 6.7 Hz, 3H, CH3-CH2-CH2-CH2-), 1.32 (m, 2H,
CH3-CH2-CH2-CH2-), 1.64 (m, 2H, CH3-CH2-CH2-CH2-), 3.79–3.83 (m,
4H, CH3-CH2-CH2-CH2-, CH2-thiazolidine), 3.91 (s, 3H, OCH3), 6.02 (s,
2H, CH2-benzodioxole), 7.13–7.33 (m, 2H, Har.), 8.21 (s, 1H, NH); 13C
NMR (DMSO d6, δ, ppm): 13.7 (CH3-CH2-CH2-CH2-), 20.0 (CH3-CH2-
CH2-CH2-), 29.1 (CH3-CH2-CH2-CH2-), 32.9 (CH2, CH2-thiazolidine),
43.5 (CH3-CH2-CH2-CH2-), 56.9 (OCH3), 101 (CH2, CH2-benzodioxole),
102.3, 108.4, 127.2, 138.5, 143.7, 148.9 (aromatic carbons), 159.6,
163.9, 170.8 (C]N, 2× C]O); MS (EI) m/z (%): 365 (M+, 100); Anal.
calcd. for C16H19N3O5S: C, 52.59; H, 5.59; N, 11.50; S, 8.77. Found: C,
52.57; H, 5.60; N, 11.52; S, 8.79.
2.1.5.8. 2-(8-Methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)-N-
phenylhydrazine-1-carbothioamide (7c). White solid, m.p. 194 °C, yield
92%; IR (KBr, cm−1) 3439, 3300, 3167 (3NH), 1630 (C=O); 1H NMR
(DMSO d6, δ, ppm): 3.88 (s, 3H, OCH3), 4.24–4.25 (m, 4H, 2× CH2-
benzodioxine), 7.09–7.32 (m, 7H, Har.), 8.01 (s, 1H, NH), 9.15 (s, 1H,
NH), 9.63 (s, 1H, NH); 13C NMR (DMSO d6, δ, ppm): 56.2 (OCH3), 64.3,
64.4 (2× CH2, CH2-benzodioxine), 103.8, 109.2, 123.9, 124.5, 125.3,
128.5, 136.0, 139.7, 143.7, 148.8 (aromatic carbons), 165.8 (C=O),
179.8 (C=S); MS (EI) m/z (%): 360 (M+ +1, 35); Anal. calcd. for
C
17H17N3O4S: C, 56.81; H, 4.77; N, 11.69; S, 8.92. Found: C, 56.92; H,
4.86; N, 11.80; S, 9.13.
2.1.5.9. N-(4-chlorophenyl)-2-(8-methoxy-2,3-dihydrobenzo[b][1,4]
dioxine-6-carbonyl)hydrazine-1-carbothioamide (7d). White solid, m.p.
218 °C, yield 91%; IR (KBr, cm−1) 3884, 3746, 3295 (3NH), 1670
(C=O); 1H NMR (DMSO d6, δ, ppm): 3.80 (s, 3H, OCH3), 4.25–4.26 (m,
4H, 2× CH2-benzodioxine), 7.16–7.17 (m, 2H, Har.), 7.36–7.45 (m, 4H,
2.1.6.3. 8-Methoxy-N′-(3-methyl-4-oxothiazolidin-2-ylidene)-2,3-
dihydrobenzo[b][1,4]dioxine-6-carbohydrazide (9a). Yellowish white
solid, m.p. 218 °C, yield 78%; IR (KBr, cm−1) 3434 (NH), 1716, 1629
(2 C=O); 1H NMR (DMSO d6, δ, ppm): 2.96 (s, 3H, CH3), 3.83 (s, 3H,
OCH3), 4.00 (s, 2H, CH2-thiazolidine), 4.22 (s, 4H, 2× CH2-
benzodioxine), 6.90–7.03 (m, 2H, Har.), 10.64 (s, 1H, NH); 13C NMR
(DMSO d6, δ, ppm): 29.7 (CH2, CH2-thiazolidine), 32.1 (CH3), 56.3
(OCH3), 64.4, 64.5 (2× CH2, CH2-benzodioxine), 104.2, 109.7, 126.9,
125.4, 136.4, 143.7, 148.9 (aromatic carbons) 163.0, 163.7, 172.2 (C]
N, 2× C]O); MS (EI) m/z (%): 336.9 (M+, 100); Anal. calcd. for
H
ar.), 9.76 (s, 1H, NH), 9.82 (s, 1H, NH), 10.43 (s, 1H, NH); 13C NMR
(DMSO d6, δ, ppm): 56.3 (OCH3), 64.3, 64.6 (2× CH2, CH2-
benzodioxine), 104.6, 110.3, 118.2, 124.1, 128.3, 129.7, 136.7,
138.5, 143.6, 148.7 (aromatic carbons), 166.0 (C=O), 183.2 (C=S);
MS (EI) m/z (%): 392.8 (M+, 3); Anal. calcd. for C17H16ClN3O4S: C,
51.84; H, 4.10; N, 10.67; S, 8.14. Found: C, 51.98; H, 4.32; N, 10.78; S,
8.25.
C
14H15N3O5S: C, 49.85; H, 4.48; N, 12.46; S, 9.50. Found: C, 49.96; H,
4.67; N, 12.65; S, 9.71.
2.1.5.10. 2-(8-Methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)-N-
(4-methoxyphenyl)hydrazine-1-carbothioamide (7e). White solid, m.p.
185 °C, yield 90%; IR (KBr, cm−1) 3884, 3746, 3295 (3NH), 1670
(C=O); 1H NMR (DMSO d6, δ, ppm): 3.75 (s, 3H, OCH3), 3.82 (s, 3H,
OCH3), 4.27–4.28 (m, 4H, 2× CH2-benzodioxine), 6.89–6.91 (m, 2H,
2.1.6.4. N′-(3-butyl-4-oxothiazolidin-2-ylidene)-8-methoxy-2,3-
dihydrobenzo[b][1,4]dioxine-6-carbohydrazide (9b). Yellowish white
solid, m.p. 224 °C, yield 72%; IR (KBr, cm−1) 3437 (NH), 1719, 1645
(2 C=O); 1H NMR (CDCl3, δ, ppm): 0.89–0.91 (t, J = 6.7 Hz, 3H, CH3),
1.22–1.32 (m, 2H, CH3-CH2-CH2-CH2-), 1.64–1.66 (m, 2H, CH3-CH2-
CH2-CH2-), 3.79–3.89 (m, 4H, CH3-CH2-CH2-CH2-, CH2-thiazolidine),
3.93 (s, 3H, OCH3), 4.25–4.33 (m, 4H, 2× CH2-benzodioxine),
6.94–7.25 (m, 2H, Har.), 8.20 (s, 1H, NH); 13C NMR (DMSO d6, δ,
ppm): 13.7 (CH3-CH2-CH2-CH2-), 20.0 (CH3-CH2-CH2-CH2-), 29.1 (CH3-
CH2-CH2-CH2-), 32.9 (CH2, CH2-thiazolidine), 43.5 (CH3-CH2-CH2-CH2-
), 56.4 (OCH3), 64.2, 64.8 (2× CH2, CH2-benzodioxine), 104.1, 108.6,
125.0, 136.5, 143.6, 149.2 (aromatic carbons), 159.1, 164.0, 170.8
(C]N, 2× C]O); MS (EI) m/z (%): 379 (M+, 60); Anal. calcd. for
H
ar.), 7.19–7.30 (m, 4H, Har.), 9.57 (s, 1H, NH), 9.67 (s, 1H, NH), 10.35
(s, 1H, NH); 13C NMR (DMSO d6, δ, ppm): 55.67, 56.3 (2× OCH3),
64.3, 69.9 (2× CH2, CH2-benzodioxine), 104.7, 110.4, 113.6, 124.4,
128.0, 132.5, 136.6, 143.6, 148.7, 157.2 (aromatic carbons), 165.8
(C=O), 182.2 (C=S); MS (EI) m/z (%): 388.4 (M+, 16); Anal. calcd. for
C
18H19N3O5S: C, 55.52; H, 4.92; N, 10.79; S, 8.23. Found: C, 55.71; H,
5.14; N, 10.87; S, 8.44.
2.1.6. General method for synthesis of 7-methoxy-N′-(3-alkyl-4-
C17H21N3O5S: C, 53.81; H, 5.58; N, 11.07; S, 8.45. Found: C, 54.10; H,
oxothiazolidin-2-ylidene)benzo[d][1,3]dioxole-5-carbohydrazide
(8a,
5.67; N, 11.19; S, 8.66.
b),8-methoxy-N′-(3-alkyl-4-oxothiazolidin-2-ylidene)-2,3-dihydrobenzo[b]
[1,4]dioxine-6-carbohydrazide (9a, b), N-aryl-5-(7-methoxybenzo[d][1,3]
dioxol-5-yl)-1,3,4-oxadiazol-2-amine (10a–c) and N-4-aryl-5-(8-methoxy-
2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-amine (11a–c)
To 0.0035 mol of 6a–e or 7a–e in absolute ethanol (30 mL), ethyl
2.1.6.5. 5-(7-Methoxybenzo[d][1,3]dioxol-5-yl)-N-phenyl-1,3,4-
oxadiazol-2-amine (10a). White solid, m.p. 230 °C, yield 87%; IR (KBr,
cm−1) 3427 (NH); 1H NMR (DMSO d6, δ, ppm): 3.87 (s, 3H, OCH3),
6.09 (s, 2H, CH2-benzodioxole), 6.97–7.10 (m, 3H, Har.), 7.32 (m, 2H,
4

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
2.2. Single-crystal X-ray study
Har.), 7.56 (m, 2H, Har.); 13C NMR (DMSO d6, δ, ppm): 56.8 (OCH3), 100
(CH2, CH2-benzodioxole), 102.7, 106.1, 117.5, 118.3, 122.3, 129.5,
137.7, 139.2, 144.1, 149.5 (aromatic carbons), 157.9, 160.1 (2× C]
N); MS (EI) m/z (%): 311(M+, 53); Anal. calcd. for C16H13N3O4: C,
61.73; H, 4.21; N, 13.50. Found: C, 61.95; H, 4.42; N, 13.71.
A suitable crystal of compound 10a for single crystal X-ray mea-
surements was obtained through crystallization of an ethanolic solution
of the compound. The data were collected at T = 298 K on Enraf Nonius
590 Kappa CCD single crystal diffractometer equipped with graphite
monochromated MoKα (λ = 0.71073 Å) radiation using ψ–ω scan
technique at room temperature. The crystal structure was determined
by direct method using SIR92, SUPERFLIP, SHELXS (Altomare et al.,
1994; Palatinus and Chapuis, 2007; Sheldrick, 2008) which revealed
the positions of all non-hydrogen atoms then refined by the full matrix
least squares refinement based on F2 using maXus package (Mackay
et al., 1999).
2.1.6.6. N-(4-chlorophenyl)-5-(7-methoxybenzo[d][1,3]dioxol-5-yl)-
1,3,4-oxadiazol-2-amine (10b). White solid, m.p. 230 °C, yield 82%; IR
(KBr, cm−1) 3437 (NH); 1H NMR (DMSO d6, δ, ppm): 3.80 (s, 3H,
OCH3), 6.08 (s, 2H, CH2-benzodioxole), 7.02–7.09 (m, 2H, Har.), 7.36
(d, 2H, J = 8.6 Hz, Har.), 7.58 (d, 2H, J = 8.6, Har.); 13C NMR (DMSO
d6, δ, ppm): 56.8 (OCH3), 100.4 (CH2, CH2-benzodioxole), 102.6,
106.01, 118.3, 119.0, 125.6, 129.5, 137.7, 138.3, 144.0, 149.4
(aromatic carbons), 157.5, 159.5 (2× C]N); MS (EI) m/z (%):
344.97 (M+, 100); Anal. calcd. for C16H12ClN3O4: C, 55.58; H, 3.50;
N, 12.15. Found: C, 55.79; H, 3.71; N, 12.27.
2.3. Biological evaluation
2.3.1. Cell lines and cell culture
Human HepG2, PC-3, MCF-7 as well as A549 carcinoma cell lines
were purchased VACSERA-EGYPT. HepG2, PC3 and A549 cells were
kept in RPMI-1640 medium [Biowest] whereas MCF-7 was cultured in
[DMEM-F12 (Gibco)]. Media was augmented with 10% (v/v) fetal bo-
vine serum (FBS; Gibco) and 1% Penicillin/Streptomycin (Sigma). Cells
were protected at 37 °C in tissue culture flasks with 5% CO2 completely
humidified incubator.
2.1.6.7. 5-(7-Methoxybenzo[d][1,3]dioxol-5-yl)-N-(4-methoxyphenyl)-
2,5-dihydro-1,3,4-oxadiazol-2-amine (10c). White solid, m.p. 210 °C,
yield 85%; IR (KBr, cm−1) 3423 (NH); 1H NMR (DMSO d6, δ, ppm):
3.73 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.11 (s, 2H, CH2-benzodioxole),
6.93–6.95 (d, 2H, J = 7.9 Hz, Har.), 7.02–7.1 (m, 2H, Har.), 7.52–7.54
(d, 2H, J = 7.9 Hz, Har.), 10.39 (s, 1H, NH); 13C NMR (DMSO d6, δ,
ppm): 55.5, 56.7 (2× OCH3), 99.9 (CH2, CH2-benzodioxole), 102.6,
106, 114.7, 118.4, 118.8, 132.3, 137.6, 144.6, 149.4, 154.0 (aromatic
carbons), 157.7, 160.2 (2×C=N); MS (EI) m/z (%): 341(M+, 100);
Anal. calcd. for C17H15N3O5: C, 59.82; H, 4.43; N, 12.31. Found: C,
59.91; H, 4.64; N, 12.53.
2.3.2. In vitro cytotoxic assay
The target compounds were evaluated for their antiproliferative
activity against panel of cell lines HepG2, PC-3, MCF-7 and A549
human carcinoma cell lines using colorimetric MTT [3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide]
assay
(Mosmann, 1983). Concisely, cells were sowed at density of 1 × 104
cells/well in a 96 well flat bottom plate. Media was aspirated then
180 μL fresh medium was added to the cells. After that, the compounds
under investigation were added in triplicate to the wells at a range of
concentrations (100, 50, 25, 12.5 μM). Wells which were treated with
0.5% DMSO was used as negative control while cells incubated with
doxorubicin was used as positive control. The medium was detached
and exchanged with 50 μL MTT solution [0.5 mg/mL; Sigma] after 48 h
which is freshly diluted from a 5 mg/mL stock solution and the plates
were incubated for 4 h at 37 °C till crystals became observable. After
that 200 μL of 10% sodium dodecyl sulfate in deionized water was
added for each well for discontinuation of the reaction and dissolving
the formed crystals. Graph-Pad PRISM version-5 software was used to
2.1.6.8. 5-(8-Methoxy-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-phenyl-
1,3,4-oxadiazol-2-amine (11a). White solid, m.p. 198 °C, yield 79%; IR
(KBr, cm−1) 3432 (NH); 1H NMR (DMSO d6, δ, ppm): 3.80 (s, 3H,
OCH3), 4.26 (s, 4H, 2× CH2-benzodioxine), 6.93–7.01 (m, 2H, Har.),
7.31–7.58 (m, 4H, Har.); 13C NMR (DMSO d6, δ, ppm): 56.4 (OCH3),
64.4, 64.6 (2× CH2, CH2-benzodioxine), 102.2, 107.7, 116.1, 117.5,
122.1, 125.91, 129.5, 136.0, 139.4, 144.5, 149.7 (aromatic carbons),
157.9, 160.2 (2× C]N); MS (EI) m/z (%): 324.99 (M+, 100); Anal.
calcd. for C17H15N3O4: C, 62.76; H, 4.65; N, 12.92. Found: C, 62.97; H,
4.86; N, 13.11.
2.1.6.9. N-(4-chlorophenyl)-5-(8-methoxy-2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)-1,3,4-oxadiazol-2-amine (11b). White solid, m.p. 248 °C,
yield 80%; IR (KBr, cm−1) 3440 (NH); 1H NMR (DMSO d6, δ, ppm):
3.80 (s, 3H, OCH3), 4.26 (s, 4H, 2× CH2, CH2-benzodioxine), 6.93–700
(m, 1H, Har.), 7.38 (d, 2H, J = 8.6 Hz, Har.), 7.58 (d, 2H, J = 8.6 Hz,
determine the IC50
.
2.3.3. In vitro cytotoxicity on human normal skin fibroblast cell line BJ1
Compound 11a was evaluated on human normal skin fibroblast cell
line BJ1 (ATCC number CRL-2522) for their cytotoxic effect using the
same mentioned MTT assay protocol (Mosmann, 1983).
H
ar.), 10.77 (s, 1H, NH); 13C NMR (DMSO d6, δ, ppm): 56.4 (OCH3),
64.5 (2× CH2, CH2-benzodioxine), 102.2, 107.4, 116.0, 119, 125.91,
129.46, 136.15, 138.23, 144.5, 149.78 (aromatic carbons), 158.2,
159.9 (2× C]N); MS (EI) m/z (%): 358.88 (M+, 100); Anal. calcd.
for C17H14ClN3O4: C, 56.76; H, 3.92; N, 11.68. Found: C, 56.87; H, 4.14;
N, 11.87.
2.3.4. Cell cycle analysis
The MCF-7 cells (2 × 105/well) were incubated with compound 11a
at its IC50. After 24 h ice-cold PBS was used to for washing the cells
twice then the cells were collected and fixed ice-cold ethanol 70% (v/v)
ethanol. The cells were washed again with PBS followed by re-sus-
pending with [0.1 mg/mL RNase], staining with [40 mg/mL PI] and
examination using flow cytometry [FACScalibur (Becton Dickinson)].
2.1.6.10. 5-(8-Methoxy-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-(4-
methoxyphenyl)-2,5-dihydro-1,3,4-oxadiazol-2-amine (11c). White solid,
m.p. 214 °C, yield 82%; IR (KBr, cm−1) 3440 (NH); 1H NMR (DMSO d6,
δ, ppm): 3.73 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.30 (s, 4H, 2× CH2,
CH2-benzodioxine), 6.94–7.04 (m, 4H, Har.), 7.52–7.54 (d, 2H,
J = 8 Hz, Har.), 10.49 (s, 1H, NH); 13C NMR (DMSO d6, δ, ppm):
55.6, 56.3 (2× OCH3), 64.4, 64.5 (2× CH2, CH2-benzodioxine), 102.0,
107.5, 114.7, 116.1, 118.9, 132.4, 135.8, 144.4, 149.6, 154.8 (aromatic
carbons), 157.7, 160.3 (2× C]N); MS (EI) m/z (%): 355 (M+, 100);
Anal. calcd. for C18H17N3O5: C, 60.84; H, 4.82; N, 11.83. Found: C,
61.05; H, 4.93; N, 12.07.
2.3.5. Tubulin polymerization assay
MCF-7 cells were cultured with DMEM and augmented with [10%
FBS and 1% penicillin-streptomycin]. Plate cells with volume of 100 μL,
complete growth medium and 100 μL of the tested compound per well
were prepared in a 96-well plate for 18–24 h before the enzyme assay
for tubulin. The microtiter plate which was used for this kit was pre-
coated with specific antibody to TUBb. Then standards or samples were
added to the suitable microtiter plate wells having a biotin-conjugated
5

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
Scheme 1. Synthesis of target compounds 8a–b, 9a–b, 10a–c and 11a–c.
Reagents and conditions: i) PTSA, MeOH, reflux, overnight. ii) Borax, NaOH, dimethyl sulfate, r.t. 15 h. iii): CH2Cl2 or ClCH2CH2Cl, K2CO3, DMF, 90 °C, 12 h. iv) 100%
NH2NH2·H2O, methanol, reflux, 6 h. v) Appropriate isothiocynate, ethanol, reflux 1 h. vi) Ethyl bromoacetate, anhydrous sod. acetate, reflux 5 h.
antibody that is particular to TUBb. Subsequently, each microplate well
was provided with Avidin conjugated to [Horseradish Peroxidase
(HRP)] and incubated. After adding TMB substrate solution, only those
wells containing TUBb, biotin-conjugated antibody and enzyme-con-
jugated Avidin will show color change. The reaction was terminated
through adding sulfuric acid solution and the change in color was
determined spectrophotometrically at a wavelength of [450 nm
10 nm]. The TUBb concentration was measured through comparing the
O.D. of the samples to the standard curve (Liliom et al., 1995).
2.3.6. Annexin V-FITC apoptosis assay
The MCF-7cells were sowed as mentioned above, incubated with
6

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
Fig. 2. ORTEP diagram of 10a with thermal ellipsoids drawn at 50% probability.
compound 11a at its IC50, collected, washed twice with PBS and then
centrifuged. Treatment of cells [105 of cells] with Annexin V-FITC and
propidium iodide (PI) through apoptosis detection kit [BD Biosciences,
San Jose, CA]. The binding of both Annexin V-FITC and PI was in-
vestigated using flow cytometry on FACScalibur [BD Biosciences, San
Jose, CA]. Logarithmic amplification for FL1 (FITC) and FL2 (PI)
channels was used for data gathering. CellQuest software was used for
performing quadrant analysis of co-ordinate dot plots. Cells which are
unstained were used for adjusting the photomultiplier voltage and
abolishing spectral overlap among the FL1 and the FL2 signals (Eldehna
et al., 2015).
downloaded from PDB followed by protein preparation and addition of
hydrogen atoms. The binding site of the selected co-crystallized ligand
(colchicine) was created as volumes. Before docking water molecules
were omitted from the protein and the tested compounds were mini-
mized energetically using CHARMm Force Field through ligand mini-
mization tool. Docking was accomplished according to protocol settings
defaulting.
3. Results and discussion
3.1. Chemistry
2.3.7. In vitro measurement for the concentration of p53, Bax and Bcl-2
proteins
The synthetic strategy of the target candidates 1,3-benzodioxoles
and 1,4-benzodioxines and their intermediates is shown in Scheme 1.
Gallic acid (1) was esterified using p-toluene sulfonic acid as a catalyst
to obtain the methyl ester 2 (Mostafa et al., 2006). The latter was
subjected to selective etherification using borax and dimethyl sulfate to
obtain methyl 5-methoxygallate (3) (Pettit and Singh, 1987). Sub-
sequent cyclization of 3 using dichloromethane or 1,2-dichloroethane
gave the corresponding 1,3-benzodioxole and 1,4-benzodioxine deri-
vatives 4a (Zhang et al., 2007) and 4b (Tsyganov et al., 2013), re-
spectively. Hydrazinolysis of 4a or 4b gave the corresponding hy-
drazides 5a (Semenov et al., 2010) and 5b. The synthesis of the new
thiosemicarbazides 6a–e and 7a–e was achieved through the reaction
of hydrazides 5a or 5b with the appropriate isothiocyanate in ethanol.
IR spectra of thiosemicarbazides 6a–e and 7a–e showed three bands at
3746–3167 cm−1 for three NH. Also the structures of the latter com-
pounds were proved using 1H NMR by the presence of singlet signals of
NH at 8.00–10.45 ppm.
In agreement with Cesur et al. (1992) reaction of thiosemicarba-
zides 6a, b or 7a, b (where R = alkyl) with ethyl bromoacetate in
presence of anhydrous sodium acetate yielded the target thiazolidi-
nones 8a, b and 9a, b, respectively. Meanwhile, the same reaction with
4-arylthiosemicarbazides 6c–e or 7c–e gave 1,3,4-oxadiazoles 10a–c
and 11a–c, respectively. The structures of the new thiazolidinones 8a, b
and 9a, b were confirmed in 1H NMR by presence of singlet signal of
CH2 between 3.6 and 4 ppm as well as in 13C NMR by appearance of
new signal at 30 ppm. The single crystal X-ray study of compound 10a
(CCDC: 1907714) also proved the formation of 1,3,4-oxadiazole ring
(Fig. 2) (Table 1). The structures of the new intermediates and target
compounds were further substantiated using different spectral data and
elemental analyses.
In vitro measurement for the concentration of p53, Bax and Bcl-2
proteins p53 ELISA kit, Human Bax ELISA kit and Bcl-2 Elisa kit, re-
spectively were used to measure the tumor suppressor gene p53,
apoptotic markers Bax and the antiapoptotic marker Bcl-2 levels.
Concisely, Cell lysates were set from control and MCF-7 cells [2 × 105/
mL] after treatment with compound 11a at IC50 concentration. Then,
the same amounts of cell lysates were loaded and probed with specific
antibodies. Samples were measured at [450 nm in ROBONEK P2000
ELISA reader], All experiments were performed in triplicates (Nowar
et al., 2018).
2.3.8. Measurement of caspases levels
The effect of compound 11a on the level of caspases 3/7, 8 and 9 in
MCF-7 cell line was evaluated at its IC50 concentration using [Caspase-
glo 3/7 Assay kit (Promega), Caspase 8 Human ELISA kit (BMS2024)
and Caspase 9 Human ELISA kit (BMS2025)] measured on ROBONIK
P2000 (India) spectrophotometer at 450 nm against untreated control
cells (negative control) using standard protocols of the manufacturer
(Nowar et al., 2018).
2.3.9. Determination of cytochrome C level
The level of cytochrome C on MCF-7 cell line after treatment with
compound 11a at its IC50 concentration was determined using
ab119521 –Cytochrome c Human ELISA Kit according to manufac-
turer's procedure.
2.4. Docking studies
Molecular docking study was performed using discovery studio
domain 2016. The 3D crystal structure of tubulin (PDB code: ISA0), was
7

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
Table 3
Selectivity index values for compounds 10a and 11a relative to BJ1 cell line.
Table 1
Crystal data and structure refinement for 10a.
CCDC number
1907714
Compound
A549
MCF-7
PC-3
HepG2
Crystal data
10a
11a
16.82
5.64
1.63
7.59
4.75
5.25
3.21
5.98
Chemical formula
Molecular weight
Temperature
C16H13N3O4
311.297
298 K
Wavelength
Crystal system
Space group
0.71073
Triclinic
Pī
congeners having 1,3,4-oxadiazole ring 10a–c and 11a–c exhibited
variable anticancer effect. The substitution on the phenyl ring in 1,3,4-
oxadiazole derivatives 10a–c and 11a–c with either electron donating
or withdrawing group decreased the anticancer potential. Conclusively,
the 1,4-benzodioxine oxadiazole hybrids 11a–c displayed better cyto-
toxic effect than the 1,3-benzodioxole homologues 10a–c on most of
the tested cell lines.
On the other hand, the derivatives which displayed remarkable
anticancer activity 10a and 11a were selected to determine their cy-
totoxicity on human normal cell line BJ1 (skin fibroblasts) (Table 3).
Selectivity indices of these compounds on each cancer cell line were
calculated relative to BJ1 cell line and presented in Table 3. According
to Suffness findings (Suffness, 1990) the compound is considered to be
selective if it has selectivity index > 2. From the calculated values
compound 10a has the highest selectivity on A549, however its se-
lectivity on MCF-7 is poor. Meanwhile, compound 11a has good se-
lectivity on all the tested cancer cell lines relative to normal cell line
(Table 3).
Cell dimensions
a = 6.0093 (3) Å
b = 10.3447 (5) Å
c = 12.8800 (7) Å
ɑ = 110.276 (3)°
β = 96.473 (3)°,
γ = 97.463 (3)°
733.91 (6) Å3
2
Volume
Z
Radiation type
Absorption coefficient
Density
Mo Kα
0.104 mm−1
1.409 Mg m−3
0.17 × 0.48 × 0.18 mm
Crystal size
Data collection
T min, T max
F000
0.842, 0.983
324
−8 ≤ h ≤ 8, −14 ≤ k ≤ 13,
0 ≤ l ≤ 18
Multi scan
Index range
Absorption correction
No. of measured, independent and
observed [I > 2.0 σ (I)] reflections
4186, 4186, 1109
Refinement
3.2.2. Cell cycle analysis
Refinement method
No. of parameters
R [F2 > 2.0 σ (F2)], wR (F2), S
Δρmax, Δρmin
Full matrix least square on F2
208
Compound 11a which is the most active one was selected to in-
vestigate its effect on the cell cycle progression of MCF-7 cells at its IC50
using flowcytometry. DNA content analysis using propidium iodide
showed that compound 11a produced a raise in the percentage of cells
at pre-G1 by 16.8-folds in comparison with the untreated cells which is
a significant marker of apoptosis. In addition, compound 11a interfered
with the cell cycle at G2/M phase causing cells accumulation 4.4 folds
more than the control (Fig. 3).
0.0575, 0.2016, 0.648
0.161, −0.169 eÅ3
3.2. Biological evaluation
3.2.1. Cytotoxic activity
The antiproliferative activity of 1,3-benzodioxoles and 1,4-benzo-
dioxines 8a, b, 9a, b, 10a–c and 11a–c is presented in Table 2. This
study was performed on four human cancer cell lines A549, MCF-7, PC-
3 and HepG2 which are derived from human lung, breast, prostate and
liver tumors, respectively using MTT assay (Mosmann, 1983). The 1,4-
benzodioxine oxadiazole hybrid 11a exhibited the highest cytotoxic
effect against all the tested cell lines with IC50 < 10 μM, while its 1,3-
benzodioxole homologue 10a had good to moderate anticancer activity
with IC50 ranged between 6.56 and 67.70 μM with higher selectivity on
A549 cell line. Generally, compounds bearing the 4-thiazolidinone ring
8a, b and 9a, b were devoid of anticancer activity. Meanwhile, the
3.2.3. Effect on tubulin polymerization
Antimitotic drugs are widely used in management of metastatic
breast cancer (Davidson, 1995; Kennedy et al., 1995). It has been re-
ported that compounds which cause arrest in cell cycle at G2/M phase
have an influence on tubulin assembly (Kanthou et al., 2004). Thus, the
mode of action of the most active derivative 11a was investigated by
determination of its tubulin polymerization inhibitory effect. Colchicine
was used as a reference compound and the IC50 values were calculated
as shown in Table 4. Compound 11a exhibited tubulin polymerization
inhibition activity with IC50 6.37 μM.
Table 2
Anticancer effect of the new 1,3-benzodioxole and 1,4-benzodioxine derivatives on different human cell lines.
Compound
IC50 (μM)a
A549
MCF-7
> 100
PC-3
> 100
HepG2
> 100
BJ1
8a
8b
9a
9b
> 100
> 100
> 100
> 100
ND
ND
ND
ND
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
10a
10b
10c
11a
11b
11c
Doxorubicin
6.56
66.05
27.62
7.94
56.21
14.11
3.01
0.19
67.70
1.12
23.23
> 100
57.86
8.53
> 100
42.55
6.77
1.66
34.40
> 100
54. 09
7.49
> 100
27.92
3.52
0.16
110.40
ND
ND
44.80
4.66
2.64
1.91
0.18
0.15
2.88
1.23
0.18
> 100
> 100
5.90
> 100
24.87
4.21
0.81
0.64
0.20
0.32
0.51
ND
ND
ND
0.07
0.21
0.19
0.24
1.11
0.11
ND: not determined.
a
IC50 values are the mean
S.D. of three separate experiments.
8

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
Fig. 3. Flow cytometry analysis for MCF-7 cells. A) Control cells, B) cells after treatment with compound 11a at its IC50 concentration.
release (Youle and Strasser, 2008). The Bcl-2 and Bax levels were
determined in MCF-7 cells after treatment with compound 11a to study
its ability to enhance apoptosis. It was found that levels of Bcl-2 protein
were down-regulated to its half value compared to control after
treatment with compound 11a. On the other hand, exposure of the
tested cells to compound 11a yielded about 10 folds increase of Bax
protein expression. From these results it was found that Bax/Bcl-2 ratio
was elevated comparing with control indicating the ability of
compound 11a to induce apoptosis (Table 5).
Table 4
Inhibition of tubulin polymerization (IC50) of com-
pound 11a.
Comp.
IC50
SD (μM)a
11a
Colchicine
6.37
4.13
0.29
0.22
a
IC50 values are the mean
S.D. of three separate
experiments.
3.2.4. Annexin V-FITC apoptosis assay
3.2.5.2. Effect on level of p53. It has been reported that expression of
Bcl-2 and Bax genes is controlled through tumor suppressor gene p53
(Miyashita et al., 1994). After treatment with compound 11a, the level
of tumor suppressor gene p53 expression was determined in MCF-7
cells. The tested compound could promote the expression of tumor
suppressor gene p53 by 11.8 folds compared to control (Table 5).
During apoptosis phosphatidylserine (PS) in the inner plasma
membrane loses its asymmetric distribution and translocates to the
outer membrane. Annexin V/PI assay can be used to identify apoptotic
cells because annexin V preferentially binds to PS (Andree et al., 1990).
The plasma membrane of the cells at early apoptosis excludes viability
dyes such as propidium iodide (PI). Thus, cells in early apoptosis will be
stained by annexin V but not with PI (Annexin V positive, PI-negative).
However, the cell membrane loses integrity in late-stage apoptosis and
the cells will be stained with both types of dyes (Annexin V positive, PI-
positive). Treatment of MCF-7 cell line with 5.90 μM of compound 11a
resulted in an increase in cells at early-stage apoptosis by 8.7 folds
comparing to control. Also the percentage of cells in late apoptosis
increased from 0.33% in control cells to 19.02% in treated ones (Fig. 4).
3.2.5.3. Measurement of caspases levels. Caspases are cysteine
containing proteases which hydrolyze substrate at aspartic acid
residues. This group of enzymes provides a vital role in controlling
cell death. There are two pathways of apoptosis either receptor-
mediated (extrinsic) or mitochondrial mediated (intrinsic). Caspases
is classified as either initiator caspases (caspase-8 and -9) or executioner
ones (caspase-3, -6, and -7). Extrinsic and intrinsic apoptosis lead to
activation of initiator caspases 8 and 9, respectively (Chen and Wang,
2002). Consequently, these two initiator caspases 8 and 9 trigger the
activation of executioner caspases-3/6/7 (Parrish et al., 2013). Elevated
Bax/Bcl2 ratio for compound 11a encouraged to measure its effect on
the levels of both initiator caspases 8 and 9 and executioner caspases-3/
7 in MCF-7 cells. Treatment of the cells with IC50 concentration of
compound 11a caused significant increase in levels of caspases 8, 9, and
3/7 by 4.5, 20.2, 3.9 folds, respectively comparing with negative
control. These findings suggest that compound 11a elicit both
extrinsic and intrinsic apoptotic pathways in MCF-7 cells.
3.2.5. Measurement of apoptosis regulatory molecules
3.2.5.1. Effect on Bcl-2 family members. The Bcl-2 proteins are divided
into proteins that promote apoptosis like Bax and others inhibit
apoptosis like Bcl-2 (Perlman et al., 1999; Frenzel et al., 2009).
Generally Bcl-2 family proteins play a role in regulating apoptosis by
controlling the mitochondrial membrane permeability (Brunelle and
Letai, 2009). Apoptosis is attenuated by antiapoptotic members of this
group of proteins such as Bcl-2 through decreasing mitochondrial
apoptogenic factors release such as cytochrome c into cytoplasm.
While, Bcl-2 family pro-apoptotic members as Bax cause caspases
9

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
Fig. 4. Effect of compound 11a on percentage of apoptotic cells in MCF-7 cells using Annexin V/PI double staining.
3.2.5.4. Determination of cytochrome C level. Upon increase of the Bax/
Bcl-2 ratio the cytochrome C is released into the cytoplasm and it
triggers the executioner caspases (Shimizu et al., 1999). Measurement
of cytochrome C level in MCF-7 cells after incubation with derivative
11a revealed significant increase in its amount by 7.6 folds compared to
untreated cells. This result is in accordance with the above-mentioned
increase in executioner caspases level.
4. Conclusion
In this study novel derivatives of 1,3-benzodioxole and its higher
homologue 1,4-benzodioxine hybridized with 1,3,4-oxadiazole or
thiazolidin-4-one rings were synthesized and evaluated as anticancer
agents against HepG2, PC-3, MCF-7 and A549 human cancer cell lines.
The 1,4-benzodioxine derivative 11a is the most active compound with
a broad spectrum activity towards the four tested cancer cell lines
(IC50 < 10 μM) and it displayed lower toxic effect on normal human
cell line BJ1. Flow cytometeric analysis showed that compound 11a
induced accumulation of MCF-7 cells at G2/M phase as well as con-
siderable increase in the cells percentage at pre-G1. Compound 11a is
proved to possesses a tubulin polymerization inhibitory effect with
IC50 = 6.37 μM. This compound could induce both early and late stage
apoptosis in MCF-7 cells. Also, the ability of this compound to stimulate
apoptosis in the latter cell line was further confirmed by increment of
Bax/Bcl-2 ratio, increase the expression of tumor suppressor gene p53,
boosting the levels of initiator and executioner caspases as well as raise
in the amount of cytochrome C. Furthermore, molecular docking of
compound 11a was performed on the colchicine binding site of tubulin
to investigate its binding mode to the target receptor. These findings
encouraged the future profound investigation of 1,3-benzodioxole and
1,4-benzodioxine derivatives in order to produce anticancer agents
having either of these moieties.
3.3. Molecular modeling study
Molecular docking study was performed to understand the binding
and interactions of derivative 11a inside the colchicine binding site of
tubulin (PDB ID: 1SA0) using Discovery studio 2016 (Ahmed et al.,
2017). DAMA-colchicine binding site contains two hydrophobic cavities
bind to the phenyl and tropone rings of the co-crystallized ligand.
Analysis of docking results of compound 11a showed that it had a
comparable CDOCKER energy with that of the reference ligand. Be-
sides, it interacted with various amino acids reported previously in
docking studies of CSIs (Ahmed et al., 2017; El-Sherief et al., 2018). The
most active compound 11a showed good shape complimentarily in the
active binding site and having five hydrogen bonds (two hydrogen
bonds with Asn 258, two hydrogen bond with Asn 350 and one hy-
drogen with val 315 residues). Moreover, it forms van der Waals in-
teraction with Leu 242, Val 238, Val 318, Val 351, Met 259, Ile 378 and
pi-alkyl interaction with Leu 248, Lys 254, Lue 255 and Ala 250 as well
as pi‑sulfur interaction with Cys241 (Fig. 5).
Acknowledgments
The authors would like express their thanks to the National
Research Centre, Dokki, Giza, Egypt for supporting this work under the
Table 5
Influence of compound 11a on the expression levels of apoptosis regulatory molecules in MCF-7 cell line.a
Comp.
Bax
ng/mL
Bcl-2
ng/mL
p53
ng/mL
Caspase 8
ng/mL
Caspase 9
ng/mL
Caspase 3/7
ng/mL
Cytochrome C
ng/mL
11a
Control
0.21
0.02
2.91
5.83
0.53
0.04
0.73
0.16
13.33
0.65
0.47
0.12
0.76
0.10
a
All experiments were performed at IC50 concentration of 11a on MCF-7 cell line.
10

R.M. Hassan, et al.
EuropeanJournalofPharmaceuticalSciences139(2019)105045
Fig. 5. 2D Interaction diagram (A) for co-crystallized ligand colchicine (B) for compound 11a.
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Declaration of competing interest
Authors have no conflict of interest to declare.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ejps.2019.105045.
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