Synthesis and antiproliferative activity of hybrid thiosemicarbazone derivatives bearing coumarin and d-galactose moieties with EGFR inhibitory activity and molecular docking study

MEDICINAL

CHEMISTRY

RESEARCH

Medicinal Chemistry Research (2021) 30:1868–1885

https://doi.org/10.1007/s00044-021-02773-y

ORIGINAL RESEARCH

Synthesis and antiproliferative activity of hybrid thiosemicarbazone

derivatives bearing coumarin and ^D-galactose moieties with EGFR

inhibitory activity and molecular docking study

2

Vu Ngoc Toan¹Nguyen Dinh Thanh

●

Received: 5 May 2021 / Accepted: 19 July 2021 / Published online: 19 August 2021

Abstract

A

series of substituted N-(2,3,4,6-tetra-O-acetyl-β-^D-galactopyranosyl)thiosemicarbazones 5a–5j of substituted

3-acetylcoumarins were synthesized with yields of 45–68%. All synthesized thiosemicarbazones were evaluated for

cytotoxic activity against several cancer cell lines, such as human breast adenocarcinoma cells MCF7, human liver cancer

cells HepG2, human cervical cancer cells HeLa, human melanoma cancer cells SK-Mel-2, and human lung cancer cells LU-

1 by using the standard MTT assay. The IC₅₀values for these cancer cell lines were 1.28–11.81 μM (for MCF-7),

1.53–22.12 μM (for HepG2), 1.43–48.16 μM (for HeLa), 1.82–14.62 μM (for SK-Mel-2), and 1.74–14.62 μM (for LU-1).

Most of the compounds were noncytotoxic against human WI-38 normal cell line (IC₅₀> 16.9 μM). The antiproliferative

mechanisms were studied via EGFR inhibition and molecular docking. Docking studies revealed that there are strong

interactions between a typical compound with the receptor of the EGFR tyrosine kinase domain with Erlotinib.

Graphical Abstract

●

Keywords 3-Acetylcoumarins Antiproliferative D-galactose EGFR tyrosine kinase Thiosemicarbazones Molecular docking

Supplementary information The online version contains

supplementary material available at https://doi.org/10.1007/s00044-

021-02773-y.

* Nguyen Dinh Thanh

2

Faculty of Chemistry, VNU University of Science (Vietnam

nguyendinhthanh@hus.edu.vn

National University, Ha Noi), 19 Le Thanh Tong, Hoan Kiem,

Ha Noi, Vietnam

1

Department of Toxicological Chemistry and Radiation, Institute

for Advanced Technology, Vietnam Academy of Military Science

and Technology, 17 Hoang Sam, Cau Giay, Ha Noi, Vietnam

Medicinal Chemistry Research (2021) 30:1868–1885

1869

Introduction

(7.01, 9.86, 6.22 μM, respectively) [13]. Compounds B, (E)-

8-methoxy-3-(3-(4-methoxyphenyl)acryloyl)-2H-chromen-

2-one, incorporating 3-methoxyphenyl moiety, displays the

best anti-proliferative activity against HepG2 cells (with

IC₅₀= 0.65 μM) [14]. Coumarin C, ethyl 1-((7-((4-nitro-

benzyl)oxy)-2-oxo-2H-chromen-4-yl)methyl)piperidine-4-

carboxylate, is the most potent counterpart against prostate

DU145 and PC-3 cells with IC₅₀= 8.95 and 7.45 μM,

respectively, with no toxic effect against non-tumorigenic

cells [15].

Attaching the thiosemicarbazone group to the coumarin

ring can elicit remarkable effects, most notably anti-cancer

activity, such as anticancer [16], antibacterial [17], anti-

fungal [18], antioxidant [19], and other activity. On the

other hand, some thiosemicarbazones of substituted 3-acetyl

coumarins are synthesized by their reaction with different

thiosemicarbazides, and their properties (such as complex

formations, spectral characteristics) have been studied and

evaluated on their biological activity [19]. Some examples

of these activities of coumarin thiosemicarbazones are dis-

playing in Fig. 2. Compounds D express profound anti-

proliferative activity on MCF-7 (human breast cancer) and

A549 (human lung carcinoma) cell lines [20]. Compounds

Coumarin is one of two important members of benzopyr-

one, including chromone (1-benzopyran-4-one, 4H-chro-

men-4-one) and coumarin (1-benzopyran-2-one, 2H-

chromen-2-one). These rings constitute the core skeleton of

many ﬂavonoid compounds in nature [1]. These compounds

bearing these rings possess diverse pharmacological activ-

ities [2], then they are used as the lead scaffolds in order to

design, synthesize, and evaluate the new bioactive com-

pounds having these rings [2]. Coumarin derivatives have

different remarkable biological activity, such as anti-

bacterial [3], antioxidant [4], and anticancer [5] activity, etc.

Amongst various biological activities of compounds having

coumarin rings, their anticancer activity is interested in

cancer drug discovery [6]. They exhibit this activity via

multiple different mechanisms and targets [7] and inhibit

against different cancer cell lines, such as human liver

cancer HepG2 [8], breast cancer MCF-7 [8], cervical car-

cinoma HeLa [9], melanoma SK-MEL-5 [10], etc. Figure 1

represents some representative examples that have antic-

ancer activity. Geiparvarin, isolated from Geijera parvi-

ﬂora, demonstrates activity in the HL-60 and K562 cell

lines with IC₅₀values of 6.3 and 9.2 μM, respectively [11].

Marmesin, isolated from Gerbera anandria, is found to

have selective antitumor activities against HepG2 cells

when tested against a panel of cancer cell lines [12].

Coumarin-hydrazone derivatives A, N′-(1-(4-dihydroxy-2-

oxo-2H-chromen-3-yl)ethylidene)benzohydrazides, express

IC₅₀values of 6.07 μM (R = OH) against human cervical

cancer cells HeLa, of 7.79 μM (R = OH) and mammary

gland adenocarcinoma cells MCF-7, and of 7.78 μM (R =

H) against human lung adenocarcinoma cells A549 in

comparison with IC₅₀values of standard drug Doxorubicin

E

exhibit the inhibitory activity against the acet-

ylcholinesterase enzyme [21].

Sugar moiety is used as a polar component in the design

and synthesis of bioactive molecules, in general, and in thio-

semicarbazones, in particular [16, 22]. Thiosemicarbazone

having sugar moiety also exhibit various biological properties,

such as antibacterial, antifungal, anticancer, and antioxidant

activity [16, 23], etc. Figure 2 displays some examples of

thiosemicarbazones bearing sugar moiety that have anticancer

activity. Thiosemicarbazone F containing ^D-glucose moiety

has remarkable antiproliferative activity against breast cancer

Fig. 1 Some coumarin

compounds with anticancer

activity having coumarin ring:

Geiparvarin, Marmesin, (A),

(B), and (C) moieties

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Medicinal Chemistry Research (2021) 30:1868–1885

Fig. 2 Some bioactive

thiosemicarbazones bearing

coumarin (D), (E) and sugar

(F), (G) moieties

cell line MDA-MB-231 and prostate cancer cell line PC-3 with

IC₅₀values of 1.60 μM and 2.8 μM, respectively [16]. Com-

pound G exhibits selective cytotoxicity against some cancer

cell lines, such as LU-1, MCF7, HepG2, SW480, P338, and

KB, with IC₅₀values of 2.75–12.75 μM [23].

Results and discussion

Chemistry

Design of thiosemicarbazone derivatives

It is known that epidermal growth factor receptor

(EGFR) is a cell-surface receptor (a transmembrane pro-

tein) belonging to the ErbB family of receptor tyrosine

kinase inhibitors (TKs) [24]. It is activated by binding of its

speciﬁc ligands, including epidermal growth factor and

transforming growth factor α (TGF-α), amphiregulin (AR),

epiregulin (EREG), betacellulin (BTC), heparin-binding

EGF (HB-EGF), and epigen (EPGN) [25]. EGFR has

emerged as an important therapeutic target. EGFR is

involved in cell proliferation and signal transduction and

belongs to the human epidermal growth factor receptor

(HER) family, including HER1 (erbB1, EGFR), HER2

(erbB2, NEU), HER3 (erbB3), and HER4 (erbB4) [26].

The EGFR pathway is responsible and plays an important

role in promoting hepatocellular carcinoma (HCC) metas-

tasis [27]. Overexpression of EGFR is frequently observed

in HCC and EGFR activation has been proven to be a

potential determinant of primary resistance of HCC cells to

sorafenib, Erlotinib, therefore, this may explain the result

of the most potent cytotoxic activity being displayed

against the breast cancer cell line, MCF-7, compared to the

other cell lines [28].

From the above discussions about the antiproliferative

activity of compounds containing coumarin and mono-

saccharide moieties connected by thiosemicarbazone linker,

we have performed the design, synthesis of some thiose-

micarbazones having coumarin ring and ^D-galactose moi-

eties (Fig. 3, Scheme 2). Their antiproliferative activity has

been evaluated on some typical cancer cell lines and the

interaction mode of the most active compounds would be

estimated on the above-mentioned typical enzymes.

In the ﬁrst step, substituted coumarin derivatives con-

sisting of different substitutions like halogens, alkyl,

hydroxyl, methoxy, and the combination of both methyl

and hydroxyl groups were designed. In addition, the

acetyl group was attached to position 3 of the coumarin

ring. Thiosemicarbazide of ^D-galactose was also designed

by addition acetyl group to all hydroxyl ones in sugar

ring, then one of these acetyl groups was replaced by

thiosemicarbazide. Next, thiosemicarbazone derivatives of

coumarins were also designed by replacing the carbonyl

group on the coumarin ring with the thiosemicarbazone

linker. Schemes 1 and 2 display the reaction series in

order to accomplish these designs.

Synthesis and characterization of thiosemicarbazone

derivatives

The reaction of different substituted salicylaldehydes 1a-1f

with ethyl acetoacetate in the presence of piperidine (as the

catalyst) led to producing corresponding substituted

3-acetylcoumarins 3a-3f (Scheme 1_(I)) [29–31]. The con-

densation process was carried out under ultrasound condi-

tions at room temperature. 3-Acetyl-4-methylcoumarin (3g)

was prepared by condensation reaction between o-hydroxy

acetophenone and ethyl acetoacetate in the presence of

piperidine (as the catalyst) under microwave-assisted reﬂux

conditions for 40 min (Scheme 1_(III)). Microwave power was

400 W. Another 3-acetyl coumarin, 3-acetyl-7-hydroxy-4-

methylcoumarin (3h), was prepared from resorcinol by two-

step synthesis: ﬁrstly, 7-hydroxy-4-methyl-2H-chromen-2-

Medicinal Chemistry Research (2021) 30:1868–1885

1871

Fig. 3 Rationally designed

coumarin−^D-galactose hybrid

compound through

thiosemicarbazone linker and

designed structures of

synthesized hybrid compounds

in this work

one (1′h) was produced by condensation of resorcinol with

ethyl acetoacetate in 70% sulfuric acid [32]; after that,

acetyl group was introduced into position 3 of coumarin

ring by acetylation using glacial acetic acid in the presence

of POCl₃(Scheme 1_(II)) [29].

Spectral data, including IR and NMR spectra, supported

the formation of the imine link in the target molecules

5a–5j. The characteristic IR absorption band appeared at

1618–1603 cm⁻¹for >C=N bond, the carbon atom in this

bond had a chemical shift at δ = 178.9–178.2 ppm. The

thiourea component in thiosemicarbazone linkage group

(−NHCSNH−N=C<) was speciﬁed by characteristic IR

absorption bands at ν = 3354–3447 and 3328–3234 cm⁻¹

(stretching vibrations of N−H bond), and by proton che-

mical shifts at δ = 11.36–11.02 (singlet) and δ =

8.77–8.76 ppm (doublet, J = 6.5–7.0 Hz) in ¹H NMR

spectra for N–H groups.

Two other 3-acetyl-4-hydroxy coumarins (3i, R = H, and

3j, R = 8-Me) were prepared by acetylation on position 3 of

corresponding 4-hydroxy coumarins (3′i,3′j) using glacial

acetic acid and PPA or acetyl chloride in pyridine. These

coumarins were synthesized by two-step synthesis from phe-

nol or o-cresol by treating them with Meldrum’s acid to afford

substituted 3-oxo-3-phenoxypropanoic acids 1′i,1′j, respec-

tively. By treating these acids with Eaton’s reagent according

to modiﬁed literature procedures, the mentioned-above cou-

marins (3′i,3′j) were obtained (Scheme 1_IV[29–31]). N-

(2,3,4,6-Tetra-O-acetyl-β-^D-galactopyranosyl)thiosemicarba-

zides (4) in Scheme 2 were prepared from corresponding

isothiocyanates and 100% hydrazine hydrate using absolute

ethanol as solvent using our previous procedure [23].

The condensation reaction between N-(2,3,4,6-tetra-O-

acetyl-β-^D-galactopyranosyl)-thiosemicarbazide (4) and

substituted 3′-acetylcoumarin (3a–3j) resulted in the for-

mation of corresponding thiosemicarbazones (5a–5j)

(Scheme 2). We performed this reaction by using the

microwave-assisted heating method (at the power of 400 W

with irradiation times of 30–45 min). Both absolute

methanol and ethanol could be used as a solvent for

this process, but the former was favorable because thiose-

micarbazide was easily soluble in methanol at

room temperature but the target product did not dissolve,

even at the reaction temperature. 3-Acetylcoumarins were

dissolved with difﬁculty in these solvents in the same

conditions, but they dissolved better under the reaction

conditions (under microwave-assisted heating conditions).

Therefore, absolute methanol was used favorably as a sol-

vent for this reaction, and glacial acetic acid was applied as

a catalyst. The obtained products were soluble better in hot

ethanol, even 96% ethanol.

Biological activity

In vitro cytotoxic activity

All synthesized thiosemicarbazones 5a–5j were screened

against four typical human cancer cell lines, including

breast adenocarcinoma cells MCF7, liver cancer cells

HepG2, cervical cancer cells HeLa, melanoma cancer

cells SK-Mel-2, and lung cancer cells LU-1. The standard

3-(4,5-dimethylthiadiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay was applied to determining cyto-

toxicity of these thiosemicarbazones [33]. Three reference

drugs, including doxorubicin (DOX), Lapatinib, and

Erlotinib, were used in these assays. Their IC₅₀values

against each appropriate cancer cell line were presented in

Table 1. The obtained IC₅₀values for all tested cancer cell

lines were also listed in this table.

These IC₅₀values suggested that most of compounds

5a–5j had remarkable cytotoxic activity to all of the tested

cancerous cell lines. In general, our compounds were more

selective towards MCF-7, HepG2, SK-Mel-2, and LU-1 than

HeLa cell lines. Some compounds had good activity against

tested cancer cell lines with IC₅₀< 3 μM, such as 5a, 5b, 5i,

and 5j against MCF-7 cancer cell line, 5a, 5b, 5c, and 5h

against HepG2, 5e, and 5h against HeLa, 5d, 5h, 5i, and 5j

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Medicinal Chemistry Research (2021) 30:1868–1885

Scheme 1 Synthesis of some

substituted 3-acetylcoumarins

3a–3j. Reaction conditions: I (i)

Piperidine, under ultrasound

condition, 35 min, 25 °C. II (ii)

Ethyl acetoacetate, CH₃COONa,

under microwave-assisted

conditions, 90 min, 400 W. III

(iii) 70% Sulfuric acid, 0 °C then

25 °C, 24 h; (iv) Glacial acetic

acid, POCl₃, under reﬂux,

90 min. IV (v) 90 °C, 5 h; (vi)

Eaton’s reagent, 70 °C, 1.5 h;

(vii) Glacial acetic acid, POCl₃,

under reﬂux, 50 min

Scheme 2 Synthetic path to

substituted 3-acetylcoumarin

(tetra-O-acetyl-β-^D-

galactopyranosyl)

thiosemicarbazones 5a–5j.

Reaction conditions: (i) Glacial

acetic acid (catalyst), MeOH

(solvent), 30−45 min. under

MW-assisted conditions at

400 W

against SK-Mel-2, 5e, 5f, 5i, and 5j against LU-1 cancer cell

lines. The order of the inhibitory effect against tested

cancer cell lines was below. The comparison between the

antiproliferative activity of our potent compounds and

Doxorubicin was represented graphically in Fig. 4.

* MCF-7 cell lines: 5a > 5b > 5i > 5j > 5f > 5d > 5g > 5h

> 5c > 5e, of which compound 5a (R = H, IC₅₀= 1.28 μM)

had the best activity in the series.

* HepG2 cell lines: 5a > 5b > 5c > 5h > 5i > 5j > 5e > 5d

> 5f > 5g. Compounds 5a and 5b showed the highest

activity in this sequence (with IC₅₀values of 1.53 and

1.75 μM, respectively).

* HeLa cell lines: 5h > 5e > 5f > 5a > 5b > 5c > 5j > 5g >

5i > 5d, of which two compounds 5h and 5e had better

antiproliferative activity against these cell lines (with IC₅₀

values of 1.83 and 1.43 μM, respectively).

Medicinal Chemistry Research (2021) 30:1868–1885

Table 1 Antiproliferative

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Compd.

R

IC₅₀(μM)

activity of thiosemicarbazones

5a–5j

MCF-7

HepG2

HeLa

SK-Mel-2

LU-1

WI-38

5a

5b

5c

5d

5e

5f

H

1.28 0.08 1.53 0.05 3.75 0.24 11.25 0.41 14.62 0.18 325 1.43

1.81 0.07 1.75 0.07 4.79 0.32 5.52 0.21 6.21 0.11 364 1.49

7.18 0.23 2.21 0.08 7.16 0.42 9.21 0.32 5.24 0.21 201 1.35

5.18 0.23 9.93 0.12 48.16 0.31 2.97 0.08 9.52 0.28 189 1.56

11.81 0.31 8.25 0.12 1.83 0.09 8.24 0.42 2.25 0.22 169 1.28

4.65 0.32 12.21 0.11 2.18 0.19 5.31 0.32 1.98 0.08 287 1.65

5.28 0.42 22.12 0.19 32.52 0.31 5.47 0.31 8.26 0.32 227 1.99

6-Cl

6-Br

6-Me

7-Me

8-OMe

4-Me

5g

5h

5i

7-OH-4-Me 6.28 0.12 3.03 0.15 1.43 0.07 2.25 0.08 5.73 0.21 271 1.46

4-OH 1.92 0.11 7.22 0.09 35.52 0.31 1.93 0.07 3.75 0.22 188 1.87

4-OH-8-Me 2.12 0.11 7.92 0.14 12.67 0.32 1.82 0.43 1.74 0.07 210 1.39

5j

DOX

1.28 0.08 0.92 0.06 1.27 0.07 1.23 0.07 1.34 0.07

5.02 0.17 11.71 1.31 7.12 0.17 16.32 1.31 14.16 1.31

3.11 0.14 6.73 0.33 5.15 0.14 12.45 0.33 9.75 0.33

−

Lapatinib

Erlotinib

* SK-Mel-2 cell lines: 5j > 5i > 5h > 5d > 5f > 5g > 5b >

5e > 5c > 5a. Compounds 5i and 5j also exhibited the

highest activity in this sequence, with IC₅₀values of 1.93

and 1.83 μM, respectively.

* LU-1 cell lines: 5j > 5f > 5e > 5i > 5c > 5h > 5b > 5g >

5d > 5a, in which compounds 5j and 5f expressed the

highest cytotoxicity (with IC₅₀values of 1.74 and 1.98 μM,

respectively).

Some conclusions on the structure-activity relationship

of tested thiosemicarbazones could be drawn as follows.

Both compounds 5a (unsubstituted coumarin ring, R = H)

and 5b (having 6-chloro group) exhibited good inhibitory

activity against two cancer cell lines MCF-7 and HepG2,

whereas compound 5c bearing bromo group on position 6

of coumarin ring inhibited only cancer cell line HepG2

and had unremarkable antiproliferative activity against

MCF-7 cell line. Compounds 5i and 5j incorporating

hydroxyl group at position 4 of coumarin ring, possessed

strong antiproliferative activity against two investigated

cell lines against MCF-7 and SK-Mel-2 cancer cell lines.

Compound 5j also exhibited strong activity against the

LU-1 cancer cell line (IC₅₀= 1.74 μM) and bad activity

against the HeLa cancer cell line. Replacement of H by

other substituents, except chloro group, led to a decrease

in the antiproliferative activity of synthesized compounds

against MCF-7 and HepG2 cell lines. Attaching of methyl

group alone at position 7 (in 5e) or hydroxy and methyl

groups at positions 7 and 4 (in 5h) simultaneously on

coumarin ring made this activity increase for HeLa cell

lines, whereas hydroxy group alone on position 4 (in 5i)

or along with methyl group at position 8 (in 5j) of cou-

marin ring increased the inhibitory activity against both

SK-Mel-2 and LU-1 cell lines. The methyl group alone at

positions 4, 6, or 7 on coumarin rings the worst inhibitory

activity against HepG2, HeLa, and MCF-7 cell lines,

Fig. 4 Comparison between the antiproliferative activity of potent

synthesized compounds 5a–5j with DOX, Lapatinib, and Erlotinib

respectively. Coumarin thiosemicarbazone (5a) had the

worst inhibitions for two cancer cell lines SK-Mel-1

and LU-1.

In order to evaluate the safety as a potential drug of all

synthesized thiosemicarbazones 5a–5j, their cytotoxicity on

normal cells was determined for the human WI-38 normal cell

line. The obtained investigation results showed that most of

these compounds were noncytotoxic against this cell line with

IC₅₀> 169 μM (Table 1), whereas the IC₅₀values against

MCF-7 cell line were less than 1.28 μM, against HepG-2 cell

line were less than 1.53 μM, against HeLa were less than

1.43 μM, against SK-Mel-2 were less than 1.82 μM, and

against LU-1 cell line were less than 1.34 μM. Therefore,

based on these obtained biological results, it means that the

most active compounds on the non-cancerous cells were safe

toward normal cells.

EGFR inhibition assay

In order to investigate the mode of antiproliferative activity of

the synthesized compounds, the most active four compounds,

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Medicinal Chemistry Research (2021) 30:1868–1885

Table 2 IC₅₀values against EGFR

Table 4 Percent of cell death induced by compounds 5a and 5b on

HepG2 cells (in %)

Compound

EGFR IC₅₀(μM)

Compound

Apoptosis

Early

Necrosis

Total

5a

0.512

0.258

1.873

1.624

0.063

0.105

Late

5b

5c

5a

7.38

6.42

0.28

12.61

18.65

0.16

9.15

12.54

1.43

29.14

37.61

1.87

5h

5b

Lapatinib

Erlotinib

Control

The obtained results presented in Table 4 showed that

compounds 5a and 5b had the ability to induce apoptosis at

early, late apoptotic stages and necrosis development,

compared to untreated control. In the early stage, the

apoptosis percentages induced by compounds 5a and 5b

were 7.38 and 6.42%, respectively whereas the apoptosis

percentage of control (0.1% DMSO) was 0.28%. In the late

stage, compared to control, the apoptosis cells percentages

of tested compounds 5a and 5b were increased to 12.61

and 18.65%, respectively. In addition, the necrosis cells

percentages were changed remarkably, reaching 9.15 and

12.54%, respectively. These two compounds increased cell

population at the early apoptotic stage by 26.36 and 22.93

folds, respectively, compared to control cells. Interestingly,

these compounds signiﬁcantly increased the late apoptotic

cell population by 78.81 and 116.56 folds, respectively,

compared to control cells. In addition, compounds 5a and

5b signiﬁcantly increased cell necrosis by 6.40 and 8.77

folds, respectively, compared to control cells. The total

apoptosis (including the early and late stages) and necrosis

percentages were 29.14 and 37.61%, respectively, indi-

cated that the treatment of HepG2 cells with compounds 5a

and 5b leading to an increase in the apoptotic cells per-

centages from 1.87% to 29.14 and 37.61%, respectively. It

is clear that the tested compounds, 5a, and 5b, caused

remarkable apoptosis in the HepG2 cell line. Finally, the

potential antiproliferative activity together with EGFR

inhibitory activity exerted by compounds 5a and 5b

prompted us to discover their possible binding interactions

with EGFR active sites by carrying out a molecular dock-

ing study.

Table 3 Inhibitory activities of compounds 5a and 5b against EGFR-

TKs

Compound

IC₅₀(μM)

EGFR-L858R-TK

EGFR-T790M-TK

5a

102.63 0.34

85.92 0.32

61.16 0.26

91.23 0.41

461.61 3.12

432.65 2.91

377.42 5.23

422.17 2.66

5b

Lapatinib

Erlotinib

5a, 5b, 5c, and 5h against HepG2 cancer cell line were further

subjected to EGFR kinase activity assay [34, 35]. Lapatinib

and Erlotinib were used as standard drugs (which inhibit

EGFR) [36] and the HepG2 cancer cells were used for this

test. The obtained results were displayed in Table 2.

The biological data in this table showed that compounds

5a and 5b had better inhibitory activity than the two

remained ones, but worse activity than standard drugs,

Lapatinib and Erlotinib. So, these compounds were chosen

for further evaluation of their inhibition effect against other

types of EGFR tyrosine kinase, EGFR T790M-TK, and

EGFR L858R-TK. The inhibitor activities of compounds 5a

and 5b against EGFR-L858R- and EGFR-T790M-tyrosine

kinases were displayed in Table 3. The potent compounds

5a and 5b showed about two-third of the activities of

Lapatinib on the two types of mutants. Compound 5a

showed activity near to Erlotinib but less than Lapatinib.

Also, compound 5b showed activity near Lapatinib and had

better activity than Erlotinib and better than compound 5a.

Annexin V-FITC apoptosis assay

Molecular docking

The programmed cell death in biological systems included

autophagy, necrosis, and apoptosis [9, 14, 15]. Apoptosis is

a series of biochemical events leading to morphological

changes and cell death. It is known that many cytotoxic

drugs show anticancer activities by inducing cell apoptosis.

To further understand the antiproliferative mechanism of

compounds 5a and 5b, their effects on cell cycle progres-

sion in HepG2 cells were examined (at the IC₅₀of this cell

line) using the ﬂow cytometry method with annexin V/

propidium iodide double staining [34].

A molecular docking study was carried out to obtain a better

understanding of EGFR higher inhibitory activity results of

compounds 5a, 5b, 5c, and 5h in order to rationalize and

describe their binding mode into EFGR active site. It’s

known that Erlotinib was a tyrosine kinase inhibitor used to

block EGFR signaling in cancer. This drug exhibited its

activity thought to bind the active conformation of the

EGFR-TKD (tyrosine kinase domain) [35]. The target for

docking simulations was the crystallographic structure of

Medicinal Chemistry Research (2021) 30:1868–1885

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Table 5 Molecular docking analysis of protein target 4HJO with selected compounds 5a, 5b, 5c, 5h in comparison with Erlotinib

Compd. Binding free energy (kcal/ Hydrogen bonding

mol)

5a

5b

−6.847

−6.804

+) C=O group of the 6-acetyl group with HS group of cysteine (residue Cys773, l = 2.30 Å).

+) Oxygen atom of C−O bond in 4-acetyl with 2-amino group of cysteine (residue Cys773, l = 2.71 Å).

+) C=O group of coumarin ring with a 2-amino group of methionine (residue Met769) (l = 2.00 Å).

+) Aromatic H-bonding of the oxygen atom of 3-OH group in threonine (residue Thr766) with hydrogen

atoms H-4 and H-5 of coumarin ring (l = 3.28 and 3.50 Å, respectively) and of C=O carboxylate of

asparagine (residue Asp831) with hydrogen atom H-8 of coumarin ring (l = 3.16 Å).

+) C=O group of 6-acetyl with HS group of cysteine (residue Cys773, l = 2.15 Å).

+) Oxygen atom of pyranose ring with a 2-amino group of cysteine (residue Cys773, l = 2.65 Å).

+) C=O group of 3-acetyl with 2-amino group of lysine (residue Lys692, l = 2.33 Å).

+) C=S of thiosemicarbazone with a 2-amino group of methionine (residue Met769, l = 2.44 Å).

+) Chloro substituent with a 2-amino group of lysine (residue Lys721, l = 3.00 Å)

5c

−6.760

−6.256

+) C=O group of 6-acetyl with 2-amino group of lysine (residue Lys692, l = 2.60 Å) and with a 2-amino

group of lysine (residue Lys704, l = 2.17 Å).

+) Aromatic H-bonding of C=O carboxylate asparagine (residue Asp831) with hydrogen atom H-7 of

coumarin ring (l = 3.31 Å).

5h

+) 7-OH group of coumarin ring with the negative-charged oxygen atom of carboxylate group of

asparagine (residue Asp831, l = 1.96 Å).

+) NH group of hydrazone moiety with a 2-amino group of methionine (residue Met769, l = 2.18 Å).

+) NH group of thiourea moiety with C=O carboxyl group of Proline (residue Pro770, l = 2.71 Å).

+) Nitrogen atom N-1 of quinazoline with a 2-amino group of methionine (residue Met769, l = 1.96 Å).

Erlotinib −8.984

+) Oxygen atom of terminal methoxy group of Erlotinib with 2-amino group of cysteine (residue Cys773,

l = 2.00 Å).

+) Aromatic H-bonding of oxygen atom in C=O of methionine with hydrogen atom H-2 of Erlotinib

(residue Met769, l = 3.23 Å).

+) Aromatic H-bonding of oxygen atom in C=O carboxylic of glutamine with hydrogen atom H-8 of

Erlotinib (residue Gln767, l = 3.38 Å).

EGFR (see “Molecular docking”). The free energies and

binding mode of the selected (most active against HepG2

cell) compounds 5a, 5b, 5c, and 5h with EGFR in com-

parison with Erlotinib were performed through Schrödinger

software [37] and represented in Table 5. Compounds 5

consisted of two different parts in terms of polarity: two

coumarin ring was the non-polar part, while the sugar

moiety was the polar one. The docking results revealed that

the compounds 5a, 5b, 5c, and 5h had similar orientations

inside the ATP binding site (Fig. 5) as Erlotinib was in this

active site. Table 5 also displayed the molecular docking

analysis of protein target 4HJO with selected compounds

5a, 5b, 5c, 5h in comparison with Erlotinib. As shown in

this table, the designed thiosemicarbazone derivatives

showed good binding energies (from −6.847 to

−6.256 kcal/mol), compared with the energy binding of

Erlotinib that was −8.984 kcal/mol. Figure 5 displayed the

2D interactions with EGFR of selected compounds and

Erlotinib and Fig. 6 represented the 3D interactions with

EGFR of these ligands.

ones, which took place when the tested bioactive com-

pounds approached the active site (at the binding cavity of

EGFR tyrosine kinase domain). For example, compound 5a

formed two hydrogen bonds only with amino acid residue

cysteine Cys773, and hydrophobic interactions at the

hydrophobic cavity containing six amino acid residues,

including six amino acid residues Leu753, Leu764, Thr766,

Leu768, Met769, and Pro779. Other hydrophobic interac-

tions were also found to occur at the hydrophobic cavity

containing three amino acid residues, including three amino

acid residues Thr830, Asp831, and Leu834 (Figs. 5A and

6A). Compound 5b formed ﬁve hydrogen bonds with four

amino acid residues Lys721, Met769, Cys773, and Lys692,

and hydrophobic interactions at the hydrophobic cavity

containing four amino acid residues Leu764, Ile765,

Thr766, Leu768. Other hydrophobic interactions were also

found to occur at the hydrophobic cavity containing three

amino acid residues Thr830, Asp831, and Leu834 (Figs. 5B

and 6B). Compound 5c formed two hydrogen bonds with

two amino acid residues Lys692 and Lys705, and hydro-

phobic interactions at the hydrophobic cavity containing

four amino acid residues Leu764, Thr766, Gln767, Leu768.

Figure 5 displayed some important and responsible

active interactions, including hydrophilic and hydrophobic

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Medicinal Chemistry Research (2021) 30:1868–1885

(A) 5a (R=H)

(B) 5b (R = 6-Cl)

(C) 5c (R = 6-Br)

(D) 5h (R = 7-OH-4-Me)

(E) Erlotinib

Legends

Fig. 5 The 2D interactions with EGFR of compound 5a (A), of compound 5b (B), of compound 5c (C), of compound 5h (D), and Erlotinib (E)

Medicinal Chemistry Research (2021) 30:1868–1885

1877

(A) 5a (R = H)

(B) 5b (R = 6-Cl)

(C) 5c (R = 6-Br)

(D) 5h (R = 7-OH-4-Me

(E) Erlotinib

(F)

Fig. 6 The 3D interaction with EGFR of compound 5a (A), of compound 5b (B), of compound 5c (C), of compound 5h (D), and Erlotinib (E).

Section (F) displayed the superimpositions of Erlotinib (red), 5a (green), 5b (blue), 5c (magenta) and 5h (orange) in receptor cavity

Other hydrophobic interactions were also found to occur at

the hydrophobic cavity containing three amino acid residues

Thr830, Asp831, and Leu834 (Figs. 5C and 6C). Com-

pound 5h formed three hydrogen bonds with three amino

acid residues Met769, Pro770, and Asp831, and hydro-

phobic interactions at the hydrophobic cavity containing

four amino acid residues Leu764, Thr766, Gln767, Leu768.

Other hydrophobic interactions were also found to occur at

the hydrophobic cavity containing three amino acid residues

Thr830, Asp831, and Leu834 (Figs. 5D and 6D). As shown

in Figs. 5 and 6, quinazoline moiety of Erlotinib was

inserted into the adenine pocket of the EGFR receptor and

its nitrogen atom number 1 formed a hydrogen bond with

Met769 with bond length 1.96 Å. Erlotinib formed two

hydrogen bonds with Met769 and Cys773, and hydrophobic

interactions at the hydrophobic cavity containing ﬁve amino

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Medicinal Chemistry Research (2021) 30:1868–1885

acid residues Leu764, Ile765, Thr766, Gln767, Leu768.

Other hydrophobic interactions were also found to occur at

the hydrophobic cavity containing three amino acid residues

Thr830, Asp831, and Leu834 (Figs. 5E and 6E). The

binding energy values and the hydrogen-bond binding in

Table 5 showed that the hydrogen-bond binding of the

C=O group of 6-acetyl with HS group of cysteine (residue

Cys773) played the important role in the docking interac-

tions of selected ligands with the active site of EGFR

receptor. Besides, ligand 5a (R = H) had additional

hydrogen-bond binding of C=O group of coumarin ring

with a 2-amino group of methionine (residue Met769), and

aromatic H-bonding of the oxygen atom of 3-OH group in

threonine (residue Thr766) with hydrogen atoms H-4 and

H-5 of coumarin ring, and of C=O carboxylate of aspar-

agine (residue Asp831) with hydrogen atom H-8 of cou-

marin ring. It is these interactions that enhance the active

interaction of 5a with EGFR receptors.

As shown in Table 5, Figs. 3 and 4, D-galactopyranose

and coumarin moieties of compounds 5a and 5b inserted

better into the adenine pocket of the EGFR receptor. This

increases the EGFR inhibitory activity of compounds 5a

and 5b. Both of these compounds have hydrogen-bonding

interactions with amino acids just as Erlotinib did. These

binding interactions may be responsible for the EGFR

inhibitory potency of these compounds. In addition, com-

pound 5b formed a hydrogen bond with lysine Lys721,

which possibly makes its inhibitory activity higher than that

of compound 5a. However, in cases of compounds 5c and

5h, the absence of these hydrogen-bonding interactions with

the key amino acid residue in the ATP binding site could

explain the decrease in IC₅₀of ligands 5c and 5h. Thus,

compounds 5a and 5b had a similar binding mode like

Erlotinib on the EGFR enzyme and have good binding

energies. Amongst hydrophilic and hydrophobic interac-

tions at the active site of the EGFR enzyme, the hydrogen-

bond bindings between acetate function and appropriate

amino acid residues contributed the most in intensifying

their potency against this enzyme.

results of the annexin VFITC apoptosis assay, it is clear that

the treatment of HepG2 cells with compounds 5a and 5b

causing to increase in apoptotic cells percentage, and

these compounds induced apoptosis in the HepG2 cell line.

The resulted docking studies showed that compounds 5a,

5b, 5c, 5h had similar binding modes to Erlotinib on the

EGFR enzyme and have good binding energies. Amongst

hydrophilic and hydrophobic interactions at the active site

of the EGFR enzyme, the hydrogen-bond bindings between

acetate function and appropriate amino acid residues had the

most important contribution in intensifying their potency

against this enzyme.

Experimental

Melting points were determined by the open capillary

method on STUART SMP3 instrument (BIBBY STER-

ILIN, UK) and are uncorrected. IR spectra (KBr disc) were

recorded on an Impact 410 FT-IR Spectrometer (Nicolet,

1

USA). H and ¹³C NMR spectra were recorded on Bruker

Avance Spectrometer AV500 (Bruker, Germany) at

500 MHz and 125 MHz, respectively, using DMSO-d₆as

solvent and TMS as an internal standard; ESI/HR-mass

spectra were recorded on LC-ESI-HRMS LTQ Orbitrap XL

or Thermo Scientiﬁc Exactive Plus Orbitrap spectrometers

(ThermoScientiﬁc, USA) in methanol using ESI method.

The analytical thin-layer chromatography (TLC) was per-

formed on silica gel 60 WF₂₅₄S aluminum sheets (Merck,

Germany) and was visualized with UV light. Chemical

reagents in high purity were purchased from the Merck

Chemical Company (in Viet Nam). All materials were of

reagent grade for organic synthesis.

Synthesis of substituted 3-acetylcoumarins (3a–3j)

Synthesis of substituted 3-acetylcoumarins (3a–3h) from

substituted salicylaldehydes under ultrasound irradiation

General procedure Appropriately substituted salicylalde-

hydes (1a–1h, 10 mmol), ethyl acetoacetate (2, 15 mmol,

1.95 g, 1.91 mL) were dissolved in methanol (5 mL). To this

mixture piperidine (1 mol%, 0.01 mL) was added. The

reaction mixture was irradiated under ultrasound conditions

at room temperature for a suitable time (see Table 1), then

cooled to room temperature. The precipitate was collected

by suction ﬁltration, washed with cold methanol (2 × 5 mL),

and recrystallized from 96% ethanol to afford substituted

3-acetylcoumarins (3a-h).

Conclusions

Thiosemicarbazones 5a–5j bearing coumarin and D-galac-

tose moieties were produced from appropriate 3-acetyl

derivatives and thiosemicarbazide, respectively. The

microwave-assisted method showed the efﬁcient and con-

venient method for the synthesis of these thiosemicarba-

zones. These compounds exhibited potent antiproliferative

activity against tested cancer cells, including MCF7,

HepG2, HeLa, SK-Mel-2, and LU-1. Amongst tested

compounds, 5a and 5b showed good activity against

EGFR-L858R and EGFR-T790M tyrosine kinase. From the

3-Acetylcoumarin (3a) From salicylaldehyde 1a (R = H,

10 mmol, 1.0 mL), ethyl acetoacetate 2 (15 mmol, 1.9 mL).

Yield: 1.5 g (79%, method B); 1.7 g (92%, method C) of 3a

Medicinal Chemistry Research (2021) 30:1868–1885

1879

as shining yellow crystals. M.p.: 119−120 °C, ref.: 119

−120 °C [38]. IR (KBr, cm⁻¹): 1738 (ν_C=Oketone), 1675

(ν_C=Olactone), 1610, 1595 and 1551 (ν_C=Carene), 1214

and 1085 (ν_COC).

under microwave-assisted conditions of 400 W for 90 min,

then cooled to room temperature. The reaction mixture was

poured into ice water, separated yellow precipitate was

collected by suction ﬁltration, washed with cold methanol

(2 × 5 mL), and recrystallized from 96% ethanol to afford

3-acetyl-4-methylcoumarin (3g) as yellow needle crystals.

Yield: 0.81 g (40%). M.p.: 101−103 °C, ref.: 101−103 °C

[42]. IR (KBr, cm⁻¹): 1742 (ν_C=Olactone), 1616 (ν_C=N),

1522 and 1495 (ν_C=Carene), 1372 (ν_C=S), 1224 & 1062

(ν_COCester).

3-Acetyl-6-chlorocoumarin

(3b) From

5-

chlorosalicylaldehyde 1b (10 mmol, 1.57 g), ethyl acet-

oacetate 2 (10 mmol, 1.30 g, 1.24 mL). Yield: 1.16 g

(52%, method A), 1.85 g (83%, method B), 2.07 g (93%,

method C) of 3b as yellow needle crystals. M.p.: 204

−205 °C, ref.: 206 °C [39]. IR (KBr, cm⁻¹): 1736 (ν_C=O

ketone), 1676 (ν_C=Olactone), 1610, 1596 and 1552 (ν_C=C

arene), 1204 and 1083 (ν_COC).

Synthesis of 3-acetyl-7-hydroxy-4-methylcoumarin (3h)

from resorcinol

3-Acetyl-6-bromocoumarin (3c) From 5-bromosalicylaldehyde

1c (10 mmol, 2.01 g), ethyl acetoacetate 2 (10 mmol, 1.30 g,

1.24 mL). Yield: 1.47 g (55%, method A), 2.24 g (84%,

method B), 2.51 g (94%, method C) of 3c as lemon yellow

needle crystals. M.p.: 221−222 °C, ref.: 220−221 °C [38]. IR

(KBr, cm⁻¹): 1742 (ν_C=Oketone), 1675 (ν_C=Olactone), 1596,

1546 and 1504 (ν_C=Carene), 1211 and 1109 (ν_COClactone).

A mixture of resorcinol (1h, 0.1 mol, 11.0 g) and ethyl

acetoacetate (0.015 mol, 19.5 g, 18.6 mL) in 70% sulfuric

acid (75 mL) was heated on a water bath at 70 °C for 3 h.

The resulting reddish-brown colored solution was decom-

posed with crushed ice (150 g). The separated bright

yellow-colored solid was ﬁltered, washed with an excess of

cold water, dried, and crystallized from methanol to get the

pure product, 4-methyl-7-hydroxycoumarin (1′h) as dark

brown crystals. Yield: 22.6 g (85%). M.p. 184−185 °C,

refs.: 185 °C [32], 189–191 °C [38].

Compound 1′h (37 mmol, 6.51 g) was dissolved in gla-

cial acetic acid (56 mmol, 3.36 g, 3.23 mL). To this solu-

tion, POCl₃(12 mmol, 1.84 g, 1.12 ml) was added. The

reaction mixture was heated under reﬂux for 90 min, then

was allowed to cool to room temperature, poured onto

crushed ice, collected the solid product by suction ﬁltration,

washed with water, dried at room temperature, and recrys-

tallized from a mixture of 96% ethanol and toluene (4:1 by

volume) to afford 3-acetyl-4-methyl-7-hydroxycoumarin

(3h) as yellowish solids. Yield: 4.84 g (60%). M.p.: 157

−159 °C. IR (KBr, cm⁻¹): 1742 (ν_C=Olactone), 1616

(ν_C=N), 1522 and 1495 (ν_C=Carene), 1372 (ν_C=S), 1224 &

1062 (ν_COCester); ¹H NMR (500 MHz, DMSO-d₆), δ

(ppm): 7.81 (d, J = 8.5 Hz, 1H, H-5), 7.25 (d, J = 2.3 Hz,

1H, H-8), 7.16 (dd, J = 8.75, 2.25 Hz, 1H, H-6), 6.39 (d,

J = 1.0 Hz, 1H, H-8), 2.31 (s, 3H, C=NCH₃).

3-Acetyl-6-methylcoumarin (3d) From 5-methylsalicylaldehyde

1d (10 mmol, 1.36 g), ethyl acetoacetate 2 (10 mmol, 1.30 g,

1.24 mL). Yield: 0.95 g (47%, method A), 1.42 g (70%, method

B), 1.82 g (90%) of 3d as lemon yellow needle crystals. M.p.:

127–128 °C, ref.: 127.5–128.3 °C [38]. IR (KBr, cm⁻¹): 1740

(ν_C=Oketone), 1675 (ν_C=Olactone), 1597, 1548, and 1504 (ν_C=C

arene), 1212 and 1109 (ν_COClactone).

3-Acetyl-7-methylcoumarin (3e) From 4-methylsalicylaldehyde

1e (0 mmol, 1.36 g), ethyl acetoacetate 2 (10 mmol, 1.30 g,

1.24 mL). Yield: 0.95 g (47%, method A), 1.42 g (70%, method

B), 1.82 g (90%) of 3e as lemon yellow needle crystals. M.p.:

160−161 °C, ref.: 160−161 °C [40]. IR (KBr, cm⁻¹): 1741

(ν_C=Oketone), 1676 (ν_C=Olactone), 1597, 1546 and 1504 (ν_C=C

arene), 1211 and 1109 (ν_COClactone).

3-Acetyl-8-methoxycoumarin (3f) From ortho-vanillin 1f

(3-methoxysalicylaldehyde, R = 3-OMe, 10 mmol, 1.52 g),

ethyl acetoacetate 2 (10 mmol, 1.30 g, 1.24 mL). Yield:

1.42 g (65%, method A) of 3f as pale-yellow needle crys-

tals. M.p.: 173−174 °C, ref.: 172 °C [41]. IR (KBr, cm⁻¹):

1734 (ν_C=Oketone), 1685 (ν_C=Olactone), 1601, 1567 and

1472 (ν_C=Carene), 1200 and 1091 (ν_COClactone).

Synthesis of substituted 3-acetyl-4-hydroxycoumarins (3i

and 3j)

These 3-acetyl derivatives of coumarins were synthesized

according to literature procedures [29–31] with some mod-

iﬁcations. A mixture of phenol (1i) or o-cresol (1j) (20 mmol)

and Meldrum’s acid (20 mmol, 3.17 g) was stirred at 90 °C

for 5 h, then cooled to room temperature. Ethyl acetate and

saturated sodium bicarbonate solution were added to the

reaction mixture. The separated aqueous layer was acidiﬁed

to pH = 1−2 with conc. HCl and extracted with methylene

chloride several times. The combined extracts were dried

Synthesis of 3-acetyl-4-methylcoumarin (3g) from

o-hydroxyacetophenone

A reaction mixture consisted of o-hydroxyacetophenone

(1g, 0.1 mol, 13.6 g, 1.20 mL), and ethyl acetoacetate

(0.15 mol, 19.5 g, 18.6 mL). Anhydrous sodium acetate

(0.15 mol, 12.5 g) was added and the mixture was heated

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Medicinal Chemistry Research (2021) 30:1868–1885

over CaCl₂and concentrated to give crude compounds 1′i or

1′j, respectively, that enough pure for the next conversion.

A mixture of 1′i or 1′j and Eaton’s reagent (30 mL) was

stirred at 70 °C for 1.5 h and then water was added to this

mixture while stirring vigorously. The precipitate was ﬁl-

tered by suction, washed with water, and dried in air to give

the yellow solids, which were recrystallized from 96%

ethanol to afford title compounds 3′i or 3′j, respectively.

These compounds were converted into corresponding

3-acetyl derivatives 3i or 3j, respectively, by using the

above-mentioned procedure for the synthesis of 3-acetyl-4-

methyl-7-hydroxycoumarin 3h.

of 5a as pale-yellow crystals from 96% ethanol. M.p.: 114

−115 °C; ½α²_D⁵+77.8 (c 0.23, CHCl₃). IR (KBr), ν (cm⁻¹):

3354 and 3332 (ν_NHthiosemicarbazone), 1742 (ν_C=O

ester), 1677 (ν_C=Olactone), 1721 (ν_C=Olactone), 1614

(ν_C=N), 1557 (ν_C=Carene), 1366 (ν_C=S), 1229 and 1065

1

(ν_COCester); H NMR (500 MHz, DMSO-d₆), δ (ppm):

11.08 (s, 1H, H-b), 8.76 (d, J = 6.5 Hz, 1H, H-a), 8.26 (s,

1H, H-4′), 7.71 (d, J = 7.0 Hz, 1H, H-5′), 7.67 (t, J =

7.0 Hz, 1H, H-6′), 7.54 (t, J = 7.5 Hz, 1H, H-7′), 7.48 (d,

J = 7.5 Hz, 1H, H-8′), 5.58−5.57 (m, Hz, 1H, H-1),

5.33–5.32 (m, 1H, H-4), 5.25–5.24 (m, 1H, H-3),

4.98–4.96 (m, 1H, H-2), 4.20–4.19 (m, 2H, H-6a and H-

6b), 4.04–4.02 (m, 1H, H-5), 2.39 (s, 3H, C=N–CH₃),

2.01–1.94 (s, 4 × 3H, 4 × CH₃CO ester); ¹³C NMR

(125 MHz, DMSO-d₆), δ (ppm): 178.21, 171.03, 170.05,

169.73, 169.45, 155.15, 151.45, 150.44, 135.73, 132.76,

127.90, 125.31, 118.36, 117.12, 116.50, 79.53, 71.82,

70.96, 69.62, 67.18, 62.06, 20.82, 20.80, 20.71, 20.66,

14.83; ESI-HRMS(+): C₂₆H₂₉N₃O₁₁S, calcd. for M + H

= 592.1601 Da, M + Na = 614.1420 Da, found: m/z

592.1607 ([M+H]⁺), 614.1428 ([M+Na]⁺).

4-Hydroxycoumarin (3′i, R = H) From phenol 1i (20 mmol,

1.88 g). Yield: 2.44 g (75%) as pale-yellow crystals. M.p.

210−212 °C, ref.: 213.0−214.3 °C [30].

4-Hydroxy-8-methylcoumarin (3′j, R = Me) From o-cresol

1j (20 mmol, 2.16 g). Yield: 2.46 g (70%) as yellow solids.

M.p. 231−232 °C, refs.: 231.1−233.4 °C [30].

3-Acetyl-4-hydroxycoumarin (3i) From coumarin 3′i

(10 mmol, 1.62 g). Yield: 1.43 g (70%) of 3j as pale-yellow

crystals. M.p. 133−135 °C, refs.: 133−135 °C [29].

3-Acetyl-6-chlorocoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

galactopyranosyl)thiosemicarbazone (5b)

3-Acetyl-4-hydroxy-8-methylcoumarin (3j) From coumarin

3′j (20 mmol, 2.16 g). Yield: 1.48 g (68%) of 3j as yellow

solids. M.p. 115−117 °C, ref.: 116 °C [43].

From 3b (1 mmol, 445 mg) and 4 (1 mmol, 421 mg).

Microwave irradiation time: 30 min. Yield: 325 mg (52%)

of 5b as yellow crystals from 96% ethanol. M.p.: 160

25

D

−161 °C; ½α +78.1 (c 0.24, CHCl₃). 3313 (ν_NH), 1738

General procedure for the synthesis of substituted

3-acetylcoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

galactopyranosyl)thiosemicarbazones (5a–5j)

(ν_C=Oester), 1623 (ν_C=Olactone), 1610 (ν_C=N), 1520 and

1490 (ν_C=Carene), 1371 (ν_C=S), 1230 and 1051 (ν_COC

1

ester); H NMR (500 MHz, DMSO-d₆), δ (ppm): H NMR

(500 MHz, DMSO-d₆), δ (ppm): 11.08 (s, 1H, H-b), 8.76 (d,

J = 6.5 Hz, 1H, H-a), 8.26 (s, 1H, H-4′), 7.97 (d, J =

1.0 Hz, 1H, H-5′), 7.58 (t, J = 8.5 Hz, 1H, H-7′), 7.48 (d,

J = 8.5 Hz, 1H, H-8′), 5.58−5.57 (m, Hz, 1H, H-1), 5.33

−5.32 (m, 1H, H-4), 5.25−5.24 (m, 1H, H-3), 4.97−4.96

(m, 1H, H-2), 4.20–4.19 (m, 2H, H-6a and H-6b),

4.04–4.01 (m, 1H, H-5), 2.38 (s, 3H, C=N-CH₃), 2.01–1.94

(s, 4 × 3H, 4 × CH₃CO ester); ¹³C NMR (125 MHz, DMSO-

d₆), δ (ppm): 178.88, 171.02, 169.73, 169.33, 169.04,

158.60, 151.52, 149.45, 135.15, 132.90, 130.31, 128.28,

118.94, 118.45, 118.40, 79.87, 72.09, 70.33, 70.03, 66.44,

62.84, 21.24, 21.14, 20.71, 20.59, 14.99; EI-HRMS(+):

A suspension mixture consisted of appropriate substituted

3-acetylcoumarins (3a–3j, 1 mmol) and N-(2,3,4,6-tetra-O-

acetyl-β-^D-galactopyranosyl)thiosemicarbazide (4, 1 mmol)

in methanol (5 mL). Glacial acetic acid (0.05 mL) was

added to this mixture. The reaction was performed by

irradiating under reﬂux for 30 min in a home-hold micro-

wave oven. After irradiating for 30−45 min, the suspension

mixture became a clear solution. The irradiation was con-

tinued in the given time. At the end of the reaction, the

precipitate has appeared. The reaction mixture was cooled

to room temperature; the colorless precipitate was ﬁltered

with suction. The crude product was recrystallized from

95% ethanol or toluene: ethanol (1:1 in volume) to afford

the title compounds 5a–5j.

C₂₆H₂₈³⁵ClN₃O₁₁S/

C₂₆H₂₈³⁷ClN₃O₁₁S,

625.1133/

627.1104 Da, calcd. for M+H = 626.1211/628.1182 Da,

found: m/z 626.1217/628.1189 ([M+H]⁺).

3-Acetylcoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

3-Acetyl-6-bromocoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

galactopyranosyl)thiosemicarbazone (5a)

galactopyranosyl)thiosemicarbazone (5c)

From 3a (1 mmol, 188 mg) and 4 (1 mmol, 421 mg).

From 3c (1 mmol, 267 mg) and 4 (1 mmol, 421 mg).

Microwave irradiation time: 45 min. Yield: 266 mg (45%)

Microwave irradiation time: 30 min. Yield: 390 mg (58%)

Medicinal Chemistry Research (2021) 30:1868–1885

1881

25

D

of 5c as yellow crystals from 96% ethanol. M.p.:

194–195 °C; ½α +80.3 (c = 0.26, CHCl₃). IR (KBr), ν

25

159–160 °C; ½α +79.3 (c 0.25, CHCl₃). IR (KBr),

(cm⁻¹): 3441 (ν_NH), 1736 (ν_C=Oester & ν_C=Olactone), 1616

(ν_C=N), 1521 and 1470 (ν_C=Carene), 1371 (ν_C=S), 1227 and

1057 (ν_COCester); ¹H NMR (500 MHz, DMSO-d₆), δ (ppm):

11.14 (s, 1H, H-b), 8.77 (d, J = 6.5 Hz, 1H, H-a), 8.25

(s, 1H, H-4′), 7.50 (s, 1H, H-5′), 7.48 (d, J = 8.75 Hz, 1H,

H-5′), 7.09 (d, J = 8.75 Hz, 1H, H-6′), 7.01 (s, 1H, H-8′),

5.58−5.57 (m, Hz, 1H, H-1), 5.33–5.32 (m, 1H, H-4),

5.25–5.24 (m, 1H, H-3), 4.97–4.95 (m, 1H, H-2), 4.20–4.19

(m, 2H, H-6a and H-6b), 4.05–4.01 (m, 1H, H-5), 2.38 (s,

3H, C=N–CH₃), 2.33 (s, 3H, 7-CH₃), 2.01–1.95 (s, 4 × 3H,

4 × CH₃CO ester); ¹³C NMR (125 MHz, DMSO-d₆), δ

(ppm): 178.64, 171.33, 170.05, 169.73, 169.45, 161.34,

151.51, 151.45, 136.50, 135.72, 128.23, 125.79, 118.32,

116.74, 113.74, 80.20, 72.13, 70.96, 68.54, 67.18, 61.41,

21.50, 21.13, 20.82, 20.71, 20.66, 15.00.; ESI-HRMS(+):

C₂₇H₃₁N₃O₁₁S, calcd. for M+H = 606.1758 Da, M+Na =

628.1577 Da, found: m/z 606.1767 ([M+H]⁺), 628.1584

([M+Na]⁺).

D

ν (cm⁻¹): 3447 and 3316 (ν_NH), 1730 (ν_C=Oester), 1671

(ν_C=Olactone), 1607 (ν_C=N), 1524 (ν_C=Carene), 1370

1

(ν_C=S), 1230 and 1055 (ν_COCester); H NMR (500 MHz,

DMSO-d₆), δ (ppm): 11.02 (s, 1H, H-b), 8.76 (d, J =

7.0 Hz, 1H, H-a), 8.20 (s, 1H, H-4′), 7.97 (d, J = 8.5 Hz,

1H, H-7′), 7.90 (s, 1H, H-5′), 7.18 (d, J = 8.5 Hz, 1H, H-

8′), 5.58–5.57 (m, Hz, 1H, H-1), 5.33–5.32 (m, 1H, H-4),

5.25–5.24 (m, 1H, H-3), 4.97–4.96 (m, 1H, H-2),

4.20–4.19 (m, 2H, H-6a and H-6b), 4.04–4.01 (m, 1H, H-

5), 2.36 (s, 3H, C=N–CH₃), 2.01−1.95 (s, 4 × 3H, 4 ×

CH₃CO ester); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm):

178.24, 170.97, 170.05, 169.73, 169.45, 158.54, 151.53,

150.28, 135.38, 135.09, 130.49, 118.65, 118.25, 117.69,

117.16, 79.67, 71.97, 71.18, 69.62, 67.11, 62.48, 21.68,

20.66,

20.29,

20.02,

15.47;

ESI-HRMS(+):

C₂₆H₂₈⁷⁹BrN₃O₁₁S/C₂₆H₂₈⁸¹BrN₃O₁₁S, calcd. for M+H

= 670.0706/672.0686 Da, found: m/z 670.0715/672.0694

([M+H]⁺).

3-Acetyl-8-methoxycoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

3-Acetyl-6-methylcoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

galactopyranosyl)thiosemicarbazone (5f)

galactopyranosyl)thiosemicarbazone (5d)

From 3f (1 mmol, 218 mg) and 4 (1 mmol, 421 mg).

From 3d (1 mmol, 202 mg) and 4 (1 mmol, 421 mg).

Microwave irradiation time: 35 min. Yield: 285 mg (46%)

Microwave irradiation time: 40 min. Yield: 345 mg (57%)

of 5f as yellow crystals from 96% ethanol. M.p.:

25

of 5d as yellow crystals from 96% ethanol. M.p.: 309

217–218 °C; ½α +81.4 (c 0.24 CHCl₃). IR (KBr),

D

25

D

−310 °C; ½α +82.3 (c 0.25, CHCl₃). IR (KBr), ν (cm

ν (cm⁻¹): 3348 and 3220 (ν_NH), 1764 (ν_C=Oester), 1729

(ν_C=Olactone), 1610 (ν_C=N), 1570 and 1523 (ν_C=Carene),

1371 (ν_C=S), 1234 and 1042 (ν_COCester); ¹H NMR

(500 MHz, DMSO-d₆), δ (ppm): 11.11 (s, 1H, H-b), 8.76

(d, J = 7.0 Hz, 1H, H-a), 8.09 (s, 1H, H-4′), 7.46 (d, J =

8.5 Hz, 1H, H-5′), 7.23 (t, J = 8.25 Hz, 1H, H-6′), 7.10 (d,

J = 8.25 Hz, 1H, H-7′), 5.59–5.56 (m, Hz, 1H, H-1),

5.34–5.31 (m, 1H, H-4), 5.26–5.24 (m, 1H, H-3),

4.98–4.95 (m, 1H, H-2), 4.20–4.19 (m, 2H, H-6a and H-

6b), 4.04–4.01 (m, 1H, H-5), 3.92 (s, 3H, 8-OCH₃), 2.38

(s, 3H, C=N–CH₃), 2.01–1.94 (s, 4 × 3H, 4 × CH₃CO

ester); ¹³C NMR (125 MHz, DMSO-d₆), δ (ppm): 178.50,

171.04, 170.08, 169.73, 169.45, 161.70, 151.44, 147.48,

141.89, 133.13, 126.68, 122.65, 118.94, 117.55, 115.33,

80.20, 72.70, 70.96, 69.62, 67.18, 62.06, 56.10, 21.61,

⁻¹): 3443 (ν_NH), 1734 (ν_C=Oester and ν_C=Olactone), 1618

(ν_C=N), 1561 and 1478 (ν_C=Carene), 1374 (ν_C=S), 1228

1

and 1058 (ν_COCester); H NMR (500 MHz, DMSO-d₆), δ

(ppm): 11.11 (s, 1H, H-b), 8.76 (d, J = 6.5 Hz, 1H, H-a),

8.06 (s, 1H, H-4′), 7.50 (s, 1H, H-5′), 7.19 (d, J =

8.25 Hz, 1H, H-8′), 7.14 (d, J = 8.25 Hz, 1H, H-7′),

5.58–5.57 (m, Hz, 1H, H-1), 5.33–5.32 (m, 1H, H-4),

5.25–5.24 (m, 1H, H-3), 4.97–4.96 (m, 1H, H-2),

4.20–4.19 (m, 2H, H-6a and H-6b), 4.04–4.02 (m, 1H, H-

5), 2.38 (s, 3H, C=N–CH₃), 2.35 (s, 3H, 6-CH₃), 2.01

−1.95 (s, 4 × 3H, 4 × CH₃CO ester); ¹³C NMR (125 MHz,

DMSO-d₆), δ (ppm): 178.66, 171.66, 170.02, 169.73,

169.45, 158.50, 151.41, 148.72, 136.11, 135.55, 135.48,

129.54, 118.83, 116.93, 116.83, 79.40, 72.17, 71.05,

69.51, 67.11, 61.91, 20.96, 20.82, 20.79, 20.73, 20.65,

14.72; ESI-HRMS(+): C₂₇H₃₁N₃O₁₁S, calcd. for M+H =

606.1758 Da, M+Na = 628.1577 Da, found: m/z

606.1766 ([M+H]⁺), 628.1584 ([M+Na]⁺).

21.24,

20.82,

20.71,

14.24;

ESI-HRMS(+):

C₂₇H₃₁N₃O₁₂S, calcd. for M+H = 622.1707 Da, M+Na

= 644.1526 Da, found: m/z 622.1716 ([M+H]⁺),

644.1531 ([M+Na]⁺).

3-Acetyl-7-methylcoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

3-Acetyl-4-methylcoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

galactopyranosyl)thiosemicarbazone (5e)

galactopyranosyl)thiosemicarbazone (5g)

From 3e (1 mmol, 202 mg) and 4 (1 mmol, 421 mg).

Microwave irradiation time: 40 min. Yield: 240 mg (40%)

of 5e as yellow crystals from 96% ethanol. M.p.:

From 3g (1 mmol, 202 mg) and 4 (1 mmol, 421 mg).

Microwave irradiation time: 40 min. Yield: 205 mg (34%)

of 5g as yellow crystals from 96% ethanol. M.p.:

1882

Medicinal Chemistry Research (2021) 30:1868–1885

25

D

203–204 °C; ½α +80.3 (c 0.23, CHCl₃). IR (KBr),

3384 (ν_NH), 1723 (ν_C=Oester), 1648 (ν_C=Olactone), 1603

(ν_C=N), 1552 (ν_C=Carene), 1327 (ν_C=S), 1240 and 1098

(ν_COCester); ¹H NMR (500 MHz, DMSO-d₆), δ (ppm):

14.48 (s, 1H, 4-OH), 11.36 (s, 1H, H-b), 8.76 (d, J =

7.5 Hz, 1H, H-a), 7.91 (d, J = 8.0 Hz, 1H, H-5′), 7.50

(t, J = 8.0 Hz, 1H, H-7′), 7.28 (d, J = 8.0 Hz, 1H, H-8′),

7.20 (t, J = 8.0 Hz, 1H, H-6′), 5.59–5.56 (m, Hz, 1H, H-1),

5.34–5.31 (m, 1H, H-4), 5.27–5.24 (m, 1H, H-3), 4.98

−4.95 (m, 1H, H-2), 4.20–4.19 (m, 2H, H-6a and H-6b),

4.04–4.01 (m, 1H, H-5), 2.28 (C=N–CH₃), 2.01–1.94 (s,

4 × 3H, 4×CH₃CO ester); ¹³C NMR (125 MHz, DMSO-d₆),

δ (ppm): 178.19, 171.03, 170.05, 169.73, 169.45, 166.88,

161.36, 155.74, 152.50, 132.86, 124.65, 124.10, 116.39,

112.50, 103.10, 80.72, 73.80, 70.64, 69.03, 68.73, 62.00,

20.92, 20.80, 20.71, 20.59, 17.12; ESI-HRMS(+):

C₂₇H₃₁N₃O₁₂S, calcd. for M+H = 608.1550 Da, M+Na =

630.1370 Da, found: m/z 608.1555 ([M+H]⁺), 630.1376

([M+Na]⁺).

ν (cm⁻¹): 3443 (ν_NH), 1737 (ν_C=Oester and ν_C=Olactone),

1617 (ν_C=N), 1521 and 1470 (ν_C=Carene), 1371 (ν_C=S),

1

1227 and 1057 (ν_COCester); H NMR (500 MHz, DMSO-

d₆), δ (ppm): 11.24 (s, 1H, H-b), 8.76 (d, J = 7.0 Hz, 1H,

H-a), 7.76 (d, J = 8.0 Hz, 1H, H-5′), 7.57 (t, J = 8.0 Hz,

1H, H-7′), 7.41 (d, J = 8.0 Hz, 1H, H-8′), 7.25 (t, J =

8.0 Hz, 1H, H-6′), 5.59–5.56 (m, Hz, 1H, H-1), 5.34–5.31

(m, 1H, H-4), 5.27–5.24 (m, 1H, H-3), 4.98–4.95 (m, 1H,

H-2), 4.20–4.19 (m, 2H, H-6a and H-6b), 4.05–4.02 (m,

1H, H-5), 2.94 (s, 3H, 4-CH₃), 2.35 (C=N–CH₃),

2.01–1.94 (s, 4 × 3H, 4 × CH₃CO ester); ¹³C NMR

(125 MHz, DMSO-d₆), δ (ppm): 178.97, 171.03, 170.48,

169.73, 169.02, 160.73, 153.85, 150.74, 143.94, 133.38,

124.26, 124.17, 118.97, 117.58, 114.39, 80.31, 72.10,

71.60, 68.45, 67.18, 61.43, 21.65, 20.92, 20.69, 19.96,

17.08, 16.79; ESI-HRMS(+): C₂₇H₃₁N₃O₁₁S, calcd. for M

+H = 606.1758 Da, M+Na = 628.1577 Da, found: m/z

606.1767 ([M+H]⁺), 628.1585 ([M+Na]⁺).

3-Acetyl-4-hydroxy-8-methylcoumarin N-(2,3,4,6-tetra-O-

acetyl-β-^D-galactopyranosyl)thiosemicarbazone (5j)

3-Acetyl-7-hydroxy-4-methylcoumarin N-(2,3,4,6-tetra-O-

acetyl-β-^D-galactopyranosyl)thiosemicarbazone (5h)

From 3j (1 mmol, 218 mg) and 4 (1 mmol, 421 mg).

From 3h (1 mmol, 218 mg) and 4 (1 mmol, 421 mg).

Microwave irradiation time: 30 min. Yield: 405 mg (65%)

Microwave irradiation time: 40 min. Yield: 315 mg (51%)

of 5j as yellow crystals from 96% ethanol. M.p.: 161

25

of 5h as yellow crystals from 96% ethanol. M.p.: 153

−162 °C; ½α +78.9 (c 0.23, CHCl₃). IR (KBr), ν (cm⁻¹):

D

25

−154 °C; ½α +82.2 (c 0.25, CHCl₃). IR (KBr), ν (cm⁻¹):

3499 and 3234 (ν_NH), 1748 (ν_C=Oester and ν_C=Olactone),

1606 (ν_C=N), 1574, 1500 and 1486 (ν_C=Carene), 1373

D

3354 and 3328 (ν_NH), 1751 (ν_C=Oester and ν_C=Olactone),

1616 (ν_C=N), 1522 and 1495 (ν_C=Carene), 1372 (ν_C=S),

1

(ν_C=S), 1225 and 1050 (ν_COCester); H NMR (500 MHz,

1

1224 and 1062 (ν_COCester); H NMR (500 MHz, DMSO-

DMSO-d₆), δ (ppm): 14.28 (s, 1H, 4-OH), 11.36 (s, 1H, H-

b), 8.76 (d, J = 6.5 Hz, 1H, H-a), 7.84 (d, J = 8.0 Hz, 1H,

H-5′), 7.32 (d, J = 8.0 Hz, 1H, H-7′), 7.09 (t, J = 8.0 Hz,

1H, H-6′), 5.59–5.57 (m, Hz, 1H, H-1), 5.33–5.32 (m, 1H,

H-4), 5.25–5.24 (m, 1H, H-3), 4.98–4.95 (m, 1H, H-2),

4.20–4.19 (m, 2H, H-6a and H-6b), 4.04–4.01 (m, 1H, H-

5), 2.32 (s, 3H, 8-CH₃), 2.28 (C=N–CH₃), 2.01–1.94 (s,

4 × 3H, 4 × CH₃CO ester); ¹³C NMR (125 MHz, DMSO-

d₆), δ (ppm): 178.43, 171.22, 170.05, 169.73, 169.45,

166.25, 160.67, 155.70, 151.42, 133.20, 127.84, 124.67,

121.39, 110.52, 105.96, 79.07, 73.16, 70.98, 69.62, 67.50,

62.71, 21.34, 20.82, 20.71, 19.72, 17.85, 17.04; ESI-HRMS

(+): C₂₇H₃₁N₃O₁₂S, calcd. for M+H = 622.1707 Da, M

+Na = 644.1526 Da, found: m/z 622.1714 ([M+H]⁺),

644.1531 ([M+Na]⁺).

d₆), δ (ppm): 11.24 (s, 1H, H-b), 10.93 (s, 1H, 7-OH), 8.76

(d, J = 7.0 Hz, 1H, H-a), 7.63 (d, J = 8.5 Hz, 1H, H-5′),

6.83 (d, J = 8.5 Hz, 1H, H-6′), 6.82 (s, 1H, H-8′), 5.58–5.57

(m, Hz, 1H, H-1), 5.33–5.32 (m, 1H, H-4), 5.27–5.25 (m,

1H, H-3), 4.97–4.96 (m, 1H, H-2), 4.20–4.19 (m, 2H, H-6a

and H-6b), 4.05–4.02 (m, 1H, H-5), 3.92 (s, 3H, 8-OCH₃),

2.83 (s, 3H, 4-CH₃), 2.35 (C=N–CH₃), 2.01–1.94 (s, 4 ×

3H, 4 × CH₃CO ester); ¹³C NMR (125 MHz, DMSO-d₆), δ

(ppm): 178.27, 171.12, 170.26, 169.73, 169.26, 161.58,

160.62, 153.78, 152.49, 143.99, 125.71, 114.15, 113.16,

112.93, 102.63, 79.55, 71.90, 70.58, 69.62, 67.04, 61.83,

21.91, 20.82, 20.71, 20.45, 17.08, 16.9; ESI-HRMS(+):

C₂₇H₃₁N₃O₁₂S M, calcd. for M+H = 622.1707 Da, M+Na

= 644.1526 Da, found: m/z 622.1713 ([M+H]⁺), 644.1531

([M+Na]⁺).

Biological assays

3-Acetyl-4-hydroxycoumarin N-(2,3,4,6-tetra-O-acetyl-β-^D-

galactopyranosyl)thiosemicarbazone (5i)

In vitro cytotoxic assay

From 3i (1 mmol, 204 mg) and 4 (1 mmol, 421 mg).

Microwave irradiation time: 35 min. Yield: 460 mg (76%)

Dilution series (128, 32, 16, 8, 4, 2, 1, and 0.5 µg/mL of each

compound 5a-5j) were prepared and used for MTT assay [33].

Two cancer cell lines were seeded at a density of 3 × 10⁴cells/

well and treated with a range of concentrations in triplicate in

of 5i as yellow crystals from 96% ethanol. M.p.: 171

25

−172 °C; ½α +79.8 (c 0.23, CHCl₃). IR (KBr), ν (cm⁻¹):

D

Medicinal Chemistry Research (2021) 30:1868–1885

1883

96-well cell culture plates, whereupon cell proliferation was

assessed using a standard MTT assay. Speciﬁcally, the growth

inhibitory activity of benzothiazepines was determined using

MTT, which correlates the cell number with the mitochondrial

reduction of MTT to a blue formazan precipitate. In brief, the

cells were plated in 96-well plates and allowed to attach

overnight. The medium was then replaced with serum-free

medium containing the test compounds and cells were incu-

bated at 37 °C for 72 h. The medium was then replaced with a

fresh medium containing 1 mg/mL MTT. Following incuba-

tion at 37 °C for 2–4 h, the wells were aspirated, the dye was

solubilized in DMSO and the absorbance was measured at

540 nm using a Tecan™ GENios® Microplate Reader (Con-

quer Scientiﬁc, USA). The viability of cells was compared

with that of the control cells. The slope of the absorbance

change was used for calculating the reaction rate. Negative

controls were performed in the absence of enzyme and com-

pound, and positive controls in the presence of enzyme and

100% DMSO. The percentage of residual activity was calcu-

lated as the difference in absorbance between the time 6 and

2 min, obtained by the average of two experiments carried out

in triplicate. The obtained rate was related to the rate when the

inhibitor was absent. IC₅₀values were calculated from linear

extrapolations of reaction rate (as a function of the logarithm

of the concentration). The IC₅₀values were determined with

increasing concentrations of inhibitor versus % of inhibition,

in triplicate in two independent experiments. The experiment

was done in triplicate and the IC₅₀values were obtained

through non-linear regression using the software GraphPad

Prism version 7.0.

Annexin-V assay

HepG2 cells were seeded in a 96-well plate (1 × 10⁵cells/

well), incubated for 24 h, then treated with vehicle (0.1%

DMSO) or 10 mM of tested compounds, 5a and 5b, for

24 h. The cells were then harvested, washed using PBS, and

stained for 15 min at room temperature in the dark using

annexin V-FITC and PI in binding buffer (10 mM HEPES,

140 mM NaCl and 2.5 mM CaCl₂at pH 7.4), then analyzed

by the ﬂow cytometer [45].

Molecular docking

The two-dimensional structures (.mae) of selected com-

pounds (ligands) 5a, 5b, 5c, and 5h, and Erlotinib were

drawn and the structure was analyzed by using a 2D

sketcher and 3D builder of Maestro version 12.5 (Schrö-

dinger suite 2020-3) [37] according to a previous method

[46]. The three-dimensional structures of these compounds

(ligands) were generated from three-dimensional structures

prepared by conformational search tool using OPLS-2005

force ﬁeld for geometrically minimizing with MacroModel

version 12.9 followed by conformational analysis using

MMFFs force ﬁeld. Monte Carlo Multiple Minimum

(MCMM) conformational search was used with 2500

iterations and a convergence threshold of 0.05 kJ/mol.

Water was chosen as solvent. Truncated Newton Conjugate

Gradient minimization was used with 2500 iterations and a

convergence threshold of 0.05 kJ/mol. Other parameters

were used as default.

Crystallographic structure of EGFR tyrosine kinase

domain with Erlotinib was retrieved from Protein Data

Bank (PDB ID: 4HJO, resolution 2.75 Å, https://www.

rcsb.org/structure/4HJO) and considered as a target for

docking simulations. Coordinates of the protein-ligand

complex were ﬁxed for errors in atomic representations

and optimized using Protein Preparation Wizard Maestro

version 11.5 (Maestro, v. 11.5: Schrödinger, LLC, New

York, NY, USA). The bond orders were assigned to

residues, hydrogen atoms were added at pH 7.0 2.0. The

restrained minimizations were carried out using the OPLS

2005 force ﬁeld with an RMSD cut-off value of 0.3 Å for

heavy atom convergences. The molecular docking was

accomplished and analyzed via the Glide version 8.8

docking tool. The receptor grid was located in the center

based on the active site of the protein, using the receptor

grid generation tool. The Glide HTVS 8.8 algorithm

(High-Throughput Virtual Screening Mode) was

employed using a grid box volume of 10 × 10 × 10 Å.

Brieﬂy, Glide approximates a systematic search of posi-

tions, orientations, and conformations of the ligand in the

receptor-binding site using a series of hierarchical ﬁlters.

The bond orders were assigned to residues, hydrogen

EGFR inhibition assay and annexin-V assay

EGFR inhibition assay was performed on HepG2 cells using

cloud clone SEA757Hu 96 Kit, following the same instruc-

tions from the manufacturer protocol through homogeneous

time-resolved ﬂuorescence assay. The fraction of the EGFR

was measured in the presence of the tested compounds using

the equation: E_(%)= E_max/(1 + [I]/ID₅₀), E_maxis the activity in

the absence of the inhibitor, [I] is the inhibitor concentration

and ID₅₀is the inhibitor concentration when E_(%)= 0.5 E_max

,

then the dose-response curve was plotted. Inhibitory activity

against EGFR L858R-TK and EGFR T790M-TK was per-

formed using HTRF (homogeneous time-resolved ﬂuores-

cence) assay method [44]. Different kinases and ATP were

obtained from Sigma. The speciﬁed kinase and its substrate

were incubated with tested compounds /reference drug for

5 min in buffer solution to start the enzymatic reaction, ATP

was then added to the reaction mixture and maintained for

30 min at room temperature. Stopping the reaction was per-

formed by adding detection reagents containing EDTA for 1 h

then the IC₅₀values were determined by GraphPad Prism

version 7.0.

1884

Medicinal Chemistry Research (2021) 30:1868–1885

atoms were added at pH 7.0 2.0. The restrained mini-

mizations were carried out using the OPLS 2005 force

ﬁeld with an RMSD cut-off value of 0.3 Å for heavy atom

convergences.

12. Jain M, Kapadia R, Jadeja RN, Thounaojam MC, Devkar RV,

Mishra SH. Hepatoprotective activity of Feronia limonia root. J

Pharm Pharm. 2012;64:888–96. https://doi.org/10.1111/j.2042-

7158.2012.01481.x.

13. Govindaiah P, Dumala N, Mattan I, Grover P, Jaya Prakash M.

Design, synthesis, biological and in silico evaluation of coumarin-

hydrazone derivatives as tubulin targeted antiproliferative agents.

Bioorg Chem. 2019;91:103143 https://doi.org/10.1016/j.bioorg.

2019.103143.

Compliance with ethical standards

14. Elshemy HAH, Zaki MA. Design and synthesis of new coumarin

hybrids and insight into their mode of antiproliferative action.

Bioorg Med Chem. 2017;25:1066–75. https://doi.org/10.1016/j.

bmc.2016.12.019.

Conﬂict of interest The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional afﬁliations.

15. Goud NS, Kanth Makani VK, Pranay J, Alvala R, Qureshi IA,

Kumar P, et al. Synthesis, ¹⁸F-radiolabeling and apoptosis

inducing studies of novel 4, 7-disubstituted coumarins. Bioorg

Chem. 2020;97:103663 https://doi.org/10.1016/j.bioorg.2020.

103663.

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Article Doi

Synthesis and antiproliferative activity of hybrid thiosemicarbazone derivatives bearing coumarin and d-galactose moieties with EGFR inhibitory activity and molecular docking study

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