Novel thienopyrimidine-aminothiazole hybrids: Design, synthesis, antimicrobial screening, anticancer activity, effects on cell cycle profile, caspase-3 mediated apoptosis and VEGFR-2 inhibition

Bioorganic Chemistry 114 (2021) 105137

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Novel thienopyrimidine-aminothiazole hybrids: Design, synthesis,

antimicrobial screening, anticancer activity, effects on cell cycle proﬁle,

caspase-3 mediated apoptosis and VEGFR-2 inhibition

,

*

Yara El-Dash^a, Emad Elzayat^b, Amr M. Abdou^c, Rasha A. Hassan^a

^aPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt

^bZoology Department, Faculty of Science, Cairo University, Cairo, Egypt

^cMicrobiology and Immunology Department, National Research Centre, Giza, Dokki 12622, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords:

A series of novel hybrid compounds of hexahydrobenzo[4,5]thieno[2,3-d]pyrimidine with aminothiazole scaf-

folds were synthesized. The synthesized compounds were evaluated for their cytotoxic activity against the NCI-

60 human tumor cell line panel. Compounds 7c, 7d and 7e exhibited signiﬁcant antiproliferative activities at

10^{ꢀ 5}M dose. Compound 7c exhibited excellent cytotoxic activity against CNS cancer cell lines including SNB-75

and SF-295 as well as renal cancer cell line CAKI-1 when compared with sorafenib as standard anticancer drug. In

addition, compound 7d showed almost comparable anticancer activity to sorafenib against SNB-75 cell line and

displayed moderate activity against SF-295 and CAKI-1 cell lines in comparison to sorafenib. Compound 7c

inhibited the vascular endothelial growth factor receptor 2 (VEGFR-2) with IC₅₀of 62.48 ± 3.7 nM and decreased

both total VEGFR-2 and phosphorylated VEGFR-2 in treated SNB-75 cells suggesting its ability to down regulate

cell proliferation, growth, and survival.. The ﬂow cytometric analysis showed that 7c displayed its cytotoxic

activity through the reduction of the cellular proliferation and induction of cell cycle arrest at the G2/M phase.

Compound 7c clearly boosted the level of the apoptotic caspase-3. All the synthesized compounds were also

screened for their antibacterial and antifungal activity against four pathogenic strains of both Gram-positive and

Gram-negative as well as Candida albicans. Only compound 7d exhibited antifungal activity against Candida

albicans compared to nystatin as the standard antifungal compound.

Thienopyrimidine

Aminothiazole

Synthesis

Anti-proliferative

VEGFR-2

Phosphorylated VEGFR-2, Caspase-3

Cell cycle arrest proﬁle

Antimicrobial activity

1. Introduction

progression [4]. VEGF has been validated as a therapeutic target in

various types of human cancers including colorectal cancer [5], breast

Anti-angiogenesis was suggested in 1971 by Folkman [1] as an

important anticancer strategy based on the ﬁnding that tumor devel-

opment was associated with increased vascularity [2]. The discovery of

the VEGF pathway as a key regulator of angiogenesis led to the creation

of a number of VEGF-targeted agents. VEGFs exert their biological ef-

fects through binding in an overlapping pattern to the kinase domain of

three different, but structurally related VEGF receptors (VEGFRs 1–3).

Upon binding to the extracellular domain of the receptors, they cause

intracellular signaling in endothelial cells, resulting in proliferation and

migration of these cells [3]. Therefore, angiogenesis suppression ap-

proaches based on the inhibition of VEGF/VEGFR signaling pathways

are developing as attractive therapeutic methods for cancer manage-

ment via inhibition of tumor angiogenesis and subsequent tumor growth

cancer [6], non-small cell lung cancer [7], renal cell cancer [8], glio-

blastoma multiforme [9]. VEGFR represent a family of three receptors

including VEGFR-2, which is the key receptor for VEGF in the vascular

endothelium, regulating different intracellular processes [10].

Sorafenib is a potent VEGFR-2 inhibitor and has been approved as a

drug [11]. Most VEGFR-2 inhibitors shared four key pharmacophoric

characteristics, according to a review of sorafenib and other VEGFR-2

inhibitors [12–15]. These features are shown in Fig. 1 and they

include: (a) The main structure of most inhibitors comprises a ﬂat aro-

matic or heteroaromatic ring structure, which occupies the catalytic

ATP-binding domain and contributing in H-bond interaction with

Cys919 residues located in the hinge area of the binding site. (b) A

central aryl ring (hydrophobic spacer), occupying the linker region

* Corresponding author at: Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt (R.A.

Hassan).

https://doi.org/10.1016/j.bioorg.2021.105137

Received 18 March 2021; Received in revised form 12 May 2021; Accepted 27 June 2021

Available online 1 July 2021

Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

among the ATP binding domain and the DFG domain of the receptor. (c)

A linker having a functional group acting as pharmacophore (e.g., amino

or urea) that possesses both H-bond acceptor and donor to bind with two

essential residues (Glu885 and Asp1046) in the DFG (Asp-Phe-Gly)

motif, an essential tripeptide sequence in the active kinase domain. The

NH motifs of the urea or amide moiety typically form one hydrogen

pyrimidines bearing various S-(substituted amino alkyl) moieties at

position 2 rather than position 4, was synthesized. In this series, various

thioether and amide containing linkers were used in the designed

thienopyrimidne-aminothiazole hybrids to study the effect of these

variations on the cytotoxic activity.

–

bond with Glu885, while the C O motif forms another hydrogen bond

–

2. Results and discussion

with Asp1046. (d) The terminal hydrophobic moiety of the inhibitors

occupies the newly created allosteric hydrophobic pocket, revealed

when the phenylalanine residue of the DFG loop ﬂips out of its lipophilic

pocket deﬁning DFG-out or inactive conformation. In this allosteric

binding region, hydrophobic interactions are typically accomplished

[16]. Furthermore, analysis of the X-ray structure of various inhibitors

bound to VEGFR-2 conﬁrmed the required space available for different

substituents around the terminal ring. [17].

2.1. Chemistry

Novel hexahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives 7a-

j were synthesized according to Schemes 1 and 2. Substituted-2-

aminothiazole derivatives 2a-e were prepared by reaction of commer-

cial aryl bromoethanones 1a-e with thiourea according to a previously

reported procedure [38]. 2-Chloro-N-(arylthiazol-2-yl)acetamide 3a-e

and 2-chloro-N-(arylthiazol-2-yl)propanamide derivatives 3f-j were

prepared through alkylation of substituted-aminothiazole derivatives

2a-e with chloroacetyl chloride or 2-chloropropionyl chloride respec-

tively in methylene chloride in presence of triethyl amine [38,39].

The initial intermediate ethyl 2-amino-4,5,6,7-tetrahydro[b]benzo-

thiophene-3-carboxylate (4) was synthesized according to Gewald pro-

cedure [40]. Reacting compound 4 with 4-chlorophenyl isothiocyanate

in absolute ethanol yielded ethyl 2-(3-(4-chlorophenyl) thioureido)-

4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5) [41]. Treat-

ment of compound 5 with potassium hydroxide in ethanol afforded the

corresponding potassium 3-(4-chlorophenyl)-4-oxo-3,4,5,6,7,8-hexahy-

drobenzo[4,5]thieno[2,3-d]pyrimidine-2-thiolate (6) [41]. Reacting

compound 6 with the key intermediates 3a-j in absolute acetone in

presence of anhydrous potassium carbonate afforded the target com-

pounds hexahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives 7a-j

in good to excellent yields.

Recently, thieno[2,3-d]pyrimidin-4-one derivatives, which are ana-

logs of quinazoline alkaloids, have attracted the interest of medicinal

chemistry researchers due to their wide range of biological and phar-

maceutical activities [18]. Many thienopyrimidine based scaffolds have

been developed and evaluated for their anticancer [19,20], antioxidant

[21], antiinﬂammatory [22], antimicrobial [23], antiviral [24], antitu-

berculosis [25], and anti-HIV activities [26]. Furthermore, numerous

studies have been published on the synthesis of various quinazoline

derivatives and their bioisosters; thienopyrimidines as potential anti-

cancer agents acting through various mechanisms including the inhibi-

tion of cyclin dependent kinases (CDKs) [27], epidermal growth factor

receptor (EGFR) [28], methionine synthase [29], 17β-HSD1 [30], matrix

metalloproteinases (MMPs) [31], P-glycoprotein (P-gp) modulation [32]

(Fig. 2).

The tricyclic system, cycloalkylthieno[2,3-d]pyrimidine with

different substitutions at position 4; has been used as a core for the

mechanism-based design and synthesis of numerous anticancer agents

targeting VEGFR-2 [33,34].

All the newly synthesized compounds were characterized by ¹H

NMR, ¹³C NMR, IR and elemental analyses. ¹H NMR, ¹³C NMR spectra

Thiazole nucleus is also an essential part of several clinically applied

anticancer drugs, such as dasatinib, dabrafenib, ixabepilone, patella-

mide A, and epothilone [35]. Many thiazoles in conjugation with het-

erocyclic compounds have been studied for their VEGFR-2 kinase

inhibitory activity as potential antitumor agents [35] (Fig. 3). These

facts, in addition to the fact that amide linkages and thioethers are

considered to contribute to the enhancement of the antitumor activity

[36,37], inﬂuenced the design of our compounds in the current study.

Various cycloalkylthieno[2,3-d]pyrimidine derivatives combining

these bioactive moieties were designed and evaluated for their anti-

cancer activity. The goal of our research was to develop novel de-

rivatives with the essential pharmacophoric features of the reported

VEGFR-2 inhibitors with bioisosteric modiﬁcations at four different

are provided in the supplementary materials. IR spectra of derivatives

–

7a-j revealed the presence of C O stretching as a broad band in the

range 1678–1693 cm^{ꢀ 1}. The ¹H NMR of compounds 7a-j were recorded

in DMSO‑d₆for all the derivatives. ¹H NMR spectra of 7a-j revealed the

presence of aromatic proton of thiazole ring as a singlet signal in the

range δ 7.63–7.97 ppm. Derivatives 7a-e showed S-CH₂protons as

singlet signal at δ 4.18–4.19 ppm in ¹H NMR. While the ¹H NMR spectra

of compounds 7f-j showed a quartet at δ 4.71 ppm corresponding to S-

CH-CH₃and a doublet at δ 1.49–1.50 ppm corresponding to the methyl

protons. ¹³C NMR spectra of 7a-j showed the presence of two carbonyl

carbons in the range of δ 161.4–166.7 ppm and δ 166.7–170.7 ppm

corresponding to the amidic linker and pyrimidone carbonyl groups

respectively. Moreover, derivatives 7a-e showed S-CH₂carbon at δ 36.3

ppm. While, S-CH-CH₃of compounds 7f-j appeared as two signals at the

positions.

A

series of 4-chlorophenyl cycloalkylthieno[2,3-d]

Fig. 1. The basic structural requirements for sorafenib as reported VEGFR-2 inhibitor.

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Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

Fig. 2. Structures of some reported quinazoline derivatives and thieno[2,3-d]pyrimidne derivatives with cytotoxic activity.

range δ 17.3–17.6 ppm for CH₃and at the range δ 45.8–45.9 ppm for S-

breast. The growth inhibition percentages obtained from the single dose

test are shown in Table 1. In light of the NCI screening results, the

following observations could be outlined. Compounds 7c and 7d

exhibited the most potent anticancer activity against CNS cancer cell

lines SF-295, SNB-75 and renal cell line CAKI-1 with growth inhibition

percentages 57.59, 58.35 and 50.99 respectively for 7c and 45.35, 46.94

and 52.39 respectively for 7d. In addition, compounds 7c and 7d

exhibited moderate anticancer activity against CNS cancer cell line SF-

268 with cell growth inhibition of 30.38% and 29.7% respectively.

Compound 7e showed moderate anticancer activity against CNS cancer

cell line SF-295 with cell growth inhibition 35.87%. Compounds 7c and

7d exhibited moderate anticancer activity against four non-small cell

lung cancer cell lines, including A549/ATCC, NCI-H226, NCI-H23 and

NCI-H522 with cell growth inhibition 29.63%, 36.03%, 43.13% and

CH carbon.

2.2. Biological evaluation

2.2.1. Anticancer activity

2.2.1.1. In vitro cytotoxic activity against a panel of 60 human tumor cell

lines. In this study, all the newly synthesized compounds were selected

by National Cancer Institute (USA) [42] for anticancer evaluation under

the Developmental Therapeutic Program (DTP) [43–46]. The selected

compounds were evaluated at one dose (10^{ꢀ 5}M) against 60 different

human tumor cell lines, representing leukemia, melanoma and tumor of

lung, colon, central nervous system (CNS), ovary, kidney, prostate and

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Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

Fig. 3. Thiazole containing anticancer drugs and some thiazole derivatives, as VEGFR/EGFR inhibitors.

Scheme 1. Synthesis of aryl thiazole derivatives 3a-j and intermediates 4,5 and 6 Reagents and conditions: a) Thiourea ethanol anhydrous Na aetate, R.T, 4h; b)

Chloroaceyl chloride or 2-chloropropionylchloride CH₂Cl₂, TEA R.T 4h; c) 4-Chlorophenyl isothicyanate, ethnol reﬂux 9h; d) KOH ehanol reﬂux 6h.

30.21% respectively for 7c and 42.47%, 28.25%, 37.6% and 31.51%

respectively for 7d. Compound 7c exhibited also moderate anticancer

activity against one colon cancer cell line HT29 with growth inhibition

38.34%. Likewise, compound 7f demonstrated moderate anticancer

activity against colon cancer cell line HCT-15 with growth inhibition

43.9%. Compound 7d showed moderate anticancer activity against two

colon cancer cell lines HCT-116 and HT29 with cell growth inhibition

39.28% and 32.98% respectively. Compound 7c displayed moderate

anticancer activity against one ovarian cancer cell line OVCAR-8 with

growth inhibition percentage 31.86 and lower inhibitory activity

against ovarian cell lines IGROV1 and OVCAR-4 with cell growth inhi-

bition 27.37% and 27.14% respectively, while compound 7d showed

moderate anticancer activity against two ovarian cancer cell lines

IGROV1 and OVCAR-8 with cell growth inhibition 29.76% and 32.89%

respectively. Compound 7c was effective moderately against two pros-

tate cancer cell lines including PC-3 and DU-145 with growth inhibition

percentage 42.45 and 31.45 respectively, while compound 7d showed

moderate anticancer activity against prostate cancer cell line PC-3 with

cell growth inhibition 41.63% and lower inhibition against prostate

cancer cell line DU-145 with cell growth inhibition 25.48%. Only

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Bioorganic Chemistry 114 (2021) 105137

Thienopyrimidine and aminothiazole scaffolds are parts of numerous

potent VEGFR-2 kinase inhibitors. Therefore, to conﬁrm the VEGFR-2

kinase binding site as a possible target of action to the synthesized de-

rivatives, the most active antiproliferative derivatives 7c, 7d and 7e

were selected to evaluate their inhibitory activity against VEGFR-2 using

the VEGFR-2 kinase inhibitory kit assay. The results were reported as a

50% inhibition concentration value (IC₅₀) calculated from the concen-

tration–inhibition response curve and summarized in Fig. 5. Doxoru-

bicin and sorafenib were used as positive controls in this assay. The

tested compounds 7c and 7e showed better inhibitory activity than

doxorubicin (IC₅₀= 86.01 ± 5.0 nM) with IC₅₀values 62.48 ± 3.7 and

79.71 ± 4.7 nM respectively. Meanwhile, compound 7d showed the

least inhibitory activity with IC₅₀value 128.1 ± 7.5 nM. All tested

compounds 7c, 7d and 7e displayed less inhibitory activity compared to

sorafenib.

2.2.1.4. Measurement of the effect of compound 7c on the total concen-

tration of VEGFR-2. The total concentration of VEGFR-2 was evaluated

in SNB-75 CNS cancer cell line using the IC₅₀of compound 7c (7.12 µM)

to assess its ability to down-regulate this key receptor. The results

indicate that compound 7c can down-regulate VEGFR-2 by 78.5%

compared to the untreated SNB-75 as shown in Fig. 6. These results

suggest that 7c has a down-regulating effect on VEGFR-2 expression.

2.2.1.5. Measurement of the effect of compound 7c on the phosphorylation

of VEGFR-2. The ability of compound 7c to directly inhibit VEGFR-2

phosphorylation (the active form of VEGFR-2) at its IC₅₀concentra-

tion (7.12 µM) was evaluated in SNB-75 cells using an in vitro kinase

assay with VEGFR-2 (Phospho-Tyr951) Colorimetric Cell-Based ELISA

Kit. The results showed that compound 7c can interfere with VEGFR-2

phosphorylation by 58.44% compared to the untreated SNB-75 cells

(Fig. 7). All the previous ﬁndings suggest that the thienopyrimidine

derivative 7c will lower both total VEGFR-2 and phosphorylated

VEGFR-2 in treated SNB-75 cells compared to untreated SNB-75 cells

suggesting that 7c has down-regulating effect on cell proliferation,

growth, and survival.

Scheme 2. Sysnhesis of the target compounds hexahydrobenzo[4,5]thieno[2

3-d]pyrimidine 7a-j Reagents and conditions a) Absolute aceone, reﬂux 15-20h.

compound 7c displayed moderate anticancer activity against breast

cancer cell line MDA-MB-468 and HS 578T with cell growth inhibition

34.92% and 27.44% respectively. Finally, it is worth mentioning, that all

the tested compounds showed inhibition activity against renal cancer

cell line UO-31 with cell growth inhibition between (14.97–25.89%).

Compounds 7a,b and 7 g-j showed no activity against most investigated

cancer cell lines.

2.2.1.2. In vitro antiproliferative activity. Antiproliferative activity of

compounds 7c and 7d was examined against three human tumor cell

lines, namely, CNS cancer; SF-295 and SNB-75 and renal cancer; CAKI-1.

Activity of compound 7e was only examined against CNS cancer SF-295

in light of its growth inhibition percentages obtained from the single

dose screening. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetra-

zolium bromide) colorimetric assay protocol was used as described by

Mosmann [47]. Sorafenib and doxorubicin were included in the exper-

iments as reference cytotoxic drugs. The results were expressed as

growth inhibitory concentration (IC₅₀) values, which represent the

compound’s concentration required to produce 50% inhibition of cell

growth after 72hr of incubation. IC₅₀values were calculated from the

concentration–inhibition response curve and summarized in Fig. 4. In-

vestigations of the cytotoxic activity showed that compound 7c was the

most potent derivative against SNB-75, SF-295 and CAKI-1 cell lines

2.2.1.6. Molecular docking in the active site of VEGFR-2. In order to

study the binding features of the newly synthesized compounds in the

binding site of VEGFR-2, the molecular modeling studies were carried

out using Molecular Operating Environment (MOE, 2019.0102) soft-

ware. All minimizations were performed with MOE until an RMSD

gradient of 0.05 kcal∙mol^ꢀ1Å^ꢀ1 with MMFF94x force ﬁeld and the

partial charges were automatically calculated. The X-ray crystallo-

graphic structure of VEGFR-2 co-crystalized with sorafenib (PDB ID:

4ASD) was downloaded from the protein data bank (https://www.rcsb.

org/structure/4ASD). Through examination of the binding interactions

of sorafenib to the active site of the receptor, it shows H-bond in-

teractions with the key amino acids Glu885, Cys919, Cys1045 and

Asp1046 (Fig. 8A).

Docking setup was ﬁrst validated by self-docking of the co-

crystallized ligand (sorafenib) in the vicinity of the binding site of the

receptor, the docking score (S) was ꢀ 10.2499 kcal/mol. and root mean

square deviation (RMSD) was 0.1511 Å (Fig. 8B and 8C).

with IC₅₀= 7.12 ± 0.33, 7.36 ± 0.39 and 4.84 ± 0.22 μM respectively as

compared to the IC₅₀of sorafenib of 14.77 ± 0.68, 14.3 ± 0.76 and 7.23

± 0.33 µM respectively. Compound 7d showed almost equipotent anti-

cancer activity (IC₅₀= 16.36 ± 0.76 μM) to sorafenib against SNB-75

The validated setup was then used in predicting the ligand-receptor

interactions at the binding site for compounds 7c, 7d and 7e. The ability

of the target compounds to interact with the key amino acids in the

binding site rationalizes their good activity as indicated by their docking

pattern and docking scores compared to that of sorafenib. The three

tested compounds showed good binding scores ranging from ꢀ 9.8363

kcal/mol. to ꢀ 8.7776 kcal/mol. and they exhibited similar ﬁt to sor-

afenib into the binding pocket of the target receptor with similar binding

interactions. The results are summarized in Table 2 and Figs. 9, 10 and

11.

cell line. Compound 7d also displayed moderate activity (IC₅₀= 20.1

± 1.08 M) against SF-295 cancer cell line in comparison to sorafenib

and weak activity (IC₅₀= 22.52 ± 1.04 M) against CAKI-1 renal cancer

cell line in comparison to sorafenib. Compound 7e displayed almost

equipotent activity (IC₅₀= 17.7 ± 0.94 M) compared to sorafenib (IC₅₀

μ

= 14.3 ± 0.76

μM) against SF-295 cell line. All tested compounds

showed weak activity compared to doxorubicin against SNB-75, SF-295

and CAKI-1 cell lines with exception to compound 7c which showed

0.75-fold the activity of doxorubicin against SF-295 CNS cell line.

2.2.1.3. Measurement of the inhibitory activity on VEGFR-2.

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Bioorganic Chemistry 114 (2021) 105137

Table 1

Growth inhibition percentages obtained from the single dose (10^{ꢀ 5}M) test of compounds 7a-j.

Panel/ cell line

Compound

7a

7b

7c

7d

7e

7f

7 g

7 h

7i

7j

Leukemia

CCRF-CEM

HL-60(TB)

K-562

–

12.25

16.39

–

MOLT-4

RPMI-8226

SR

12.95

–

13.33

–

19.42

18.19

15.07

Non-Small Cell Lung Cancer

A549/ATCC

EKVX

–

29.63

25.02

–

42.47

19.80

–

14.09

15.32

15.7

11.8

17.47

–

HOP-62

–

HOP-92

–

NCI-H226

–

36.03

43.13

12.18

–

28.25

37.6

17.7

–

14.43

–

NCI-H23

–

12.2

14.19

–

NCI-H322M

NCI-H460

–

NCI-H522

14.11

30.21

31.51

15.03

18.15

13.44

14.76

17.94

Colon Cancer

COLO 205

HCC-2998

HCT-116

HCT-15

–

14.86

–

18.4

12.23

38.34

16.17

21.15

–

39.28

12.25

32.98

15.17

24.77

–

10.00

43.9

HT29

11.98

–

KM12

–

SW-620

CNS Cancer

SF-268

–

30.38

57.59

–

15.24

58.35

21.24

29.7

45.35

–

13.18

46. 94

21.91

–

12.51

16.22

–

11.55

–

SF-295

23.37

–

35.87

–

SF-593

–

SNB-19

SNB-75

U251

–

12.04

11.69

14.84

10.32

14.74

–

Melanoma

LOX IMVI

MALME-3M

M14

–

15.72

–

26.63

–

10.29

–

23.44

–

MDA-MB-435

SK-MEL-2

SK-MEL-28

SK-MEL-5

UACC-257

UACC-62

–

15.59

17.18

–

14.11

–

12.05

14.92

19.13

19.04

13.68

11.05

Ovarian Cancer

IGROV1

–

12.34

27.37

10.96

27.14

24.35

31.86

19.52

–

29.76

14.32

12.89

–

32.89

24.23

–

15.07

–

13.97

–

OVCAR-3

–

OVCAR-4

–

OVCAR-5

10.59

13.46

OVCAR-8

–

NCI/ADR-RES

SK-OV-3

Renal Cancer

786-0

–

ACHN

–

12.97

52.39

–

CAKI-1

RXF 393

SN12C

11.16

–

50.99

–

15.17

10.07

10.83

–

20.46

–

11.55

–

25.35

–

TK-10

–

12.51

23.36

–

UO-31

17.93

14.79

25.36

25.89

20.25

24.47

15.95

18.54

Prostate Cancer

PC-3

–

42.45

31.45

41.63

25.48

–

10.4

–

DU-145

–

Breast Cancer

MCF7

–

12.65

19.28

27.44

–

23.07

34.92

17.76

19.65

10.05

–

23.81

–

15.23

17.37

10.39

10.09

–

MDA-MB-231/ATCC

HS 578T

–

10.94

–

BT-549

–

T-47D

–

MDA-MB-468

10.55

12.27

Mean Inhibition*

0

18.30

16.83

4.35

5.14

3.57

1.66

2.62

1.87

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Bioorganic Chemistry 114 (2021) 105137

– Growth inhibition percentage produced by the compound is lower than 10%.

* Mean inhibition was calculated according to GI% values over all NCI- 60 cell lines

Fig. 4. Graphical representation for concentrations required for 50% inhibition of cell viability (IC₅₀) of compounds 7c, 7d and 7e compared to doxorubicin

and sorafenib.

Fig. 7. Graphical representation to show that 7c treated SNB-75 cells will

Fig. 5. Graphical representation for concentrations required for 50% inhibition

of VEGFR-2 (IC₅₀) of compounds 7c, 7d and 7e compared to doxorubicin

and sorafenib.

lower the phosphorylated VEGFR-2 compared to control untreated cells.

2.2.1.7. Structure activity relationship. Structure Activity Relationship

studies were performed for the two synthesized series of thienopyr-

imidine–aminothiazole hybrids 7a-e and 7f-j. In the ﬁrst series the

percentage of inhibition pattern for the compounds in Table 1 showed

that substitution with weak electron withdrawing halogen atom (4-

chlorophenyl 7c, 3-bromophenyl 7d or 4-bromophenyl 7e) yields more

potent cytotoxic agents. Particularly the 4-chloro derivative 7c which

exhibited the highest mean of inhibition percentage over the 60 cell line

panel equal to 18.32% (Table 1). The unsubstituted phenyl derivative 7a

and the derivative substituted with strong electron withdrawing 3-nitro

group 7b showed low cytotoxic activity along all tested cell lines.

Conﬁrming the importance of the 4-chloro substitution; compound

7c displayed the highest inhibition percentage against SNB-75, SF-295

and CAKI-1 cell lines in the single dose in vitro cytotoxic screening

(Table 1). Moreover, compound 7c showed sensitivity against higher

number of tested cell lines among halogen substituted derivatives (7c-

7e). Investigations of the in vitro anti proliferative assay indicated that

compound 7c was the most potent derivative against SNB-75, SF-295

and CAKI-1 cell lines in comparison to soreﬁnib as reference drug

(Fig. 4). In addition, the favorable interactions displayed by compound

7c with the key amino acids at the VEGFR-2 active site was comparable

Fig. 6. Graphical representation to show that 7c treated SNB-75 cells showed

decrease in the total concentration of VEGFR-2 compared to the control un-

treated cells.

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Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

Fig. 8. A) 2D interactions of sorafenib within VEGFR-2 active site; B) and C) 2D representation and 3D representation of the superimposition of the co-crystallized

(red) and the docking pose (green) of sorafenib in the active site of VEGFR-2. (For interpretation of the references to color in this ﬁgure legend, the reader is referred

to the web version of this article.)

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Bioorganic Chemistry 114 (2021) 105137

percentages than the halogenated derivatives 7c-e. On the other hand,

the unsubstituted phenyl derivative 7f and 3-nitrophenyl derivative 7 g

showed an increase in the mean of inhibition percentages compared to

compounds 7a and 7b.

Table 2

Docking scores, hydrogen bonds and interactions of tested compounds and

sorafenib inside VEGFR-2 binding site (PDB entry 4ASD).

Compound

S (kcal/

mol)

Amino

acids

Interacting

groups

Type of

Length

interaction

2.2.1.8. Cell cycle analysis. Anticancer mediators exert their cytotoxic

action by terminating cellular proliferation at deﬁnite checkpoints

which are distinct stages in the cell cycle. Suppression of these phases

results in termination of the cell proliferation. Cell cycle analysis em-

ploys ﬂow cytometry to discriminate cells within the different cell cycle

phases. In this work, the most active compound in cytotoxic assay 7c

was further exposed to cell cycle analysis to explore the deﬁnite phase at

which cell cycle arrest takes place in the SNB-75 CNS cancer cell line.

Sorafenib

ꢀ 10.2499

Glu885

Cys919

Cys1045

Asp1046

NH (urea)

NH (amide)

N (pyridine)

H-bond donor

H-bond

2.76

3.16

2.86

3.07

3.32

2.82

–

O (C O)

acceptor

–

O (C O)

H-bond

acceptor

H-bond

acceptor

7c

ꢀ 9.0323

Glu855

Glu885

Leu889

Cys919

Cys1045

Asp1046

S (thiazole)

Non-classical

4.40

4.05

3.87

4.03

3.67

2.92

SNB-75 cells were exposed to compound 7c at its IC₅₀(7.12 μM) for 24 h

S

(σ hole)

and its effect on the cell population in different cell phases was recorded

and presented in Fig. 12. Exposure of SNB-75 cells to compound 7c

resulted in signiﬁcant drop in the cell population at the G0-G1 and S

phases with 14.26% and 21.68%, respectively. Moreover, marked in-

crease in the proportion of cells in the G2/M phase by 2.5-fold, and in

the pre-G1 phase by 23.8-fold, in comparison to the control (DMSO) was

observed. This evidently shows that the target thienopyrimidine deriv-

ative 7c stopped the cell cycle proliferation of SNB-75 cells in the G2/M

phase.

(thiophene)

H-bond

S

acceptor

Cl

H-bond

N (thiazole)

NH

acceptor

Electrostatic

H-bond

acceptor

H-bond donor

Non-classical

7d

7e

ꢀ 8.7776

ꢀ 9.8363

Glu885

Val899

Cys919

Leu1053

Ile1044

Asp1046

S

3.82

4.05

3.52

4.04

3.75

3.25

(thiophene)

N (thiazole)

Br

(σ hole)

H-bond

acceptor

N (thiazole)

Cl

Halogen bond

H-bond

2.2.1.9. Apoptosis determination by Annexin V-FITC assay. Annexin V-

based ﬂow cytometry test represents a helpful approach for etermining

whether death of cells is attributed to programmed apoptosis or to un-

controlled necrosis. To ensure the ability of compound 7c to induce

apoptosis, a cytoﬂuorimetric analysis was performed using propidium

iodide (PI), which stains DNA and enters only dead cells, and ﬂuorescent

immunolabeling of the Annexin-V protein. Annexin-V binds to phos-

phatidylserine (PS) expressed only on the surface of the apoptotic cells

and ﬂuoresces green after interacting with the ﬂuorochrome labeled

Annnexin-V. On the other hand, PI stains DNA and enters only dead cells

[48]. Dual staining for Annexin-V and PI permits discrimination be-

tween live cells, early apoptotic cells, late apoptotic cells and necrotic

cells. Compound 7c was selected to be tested for its effect on the cell

cycle of the SNB-75 cell line since it showed the highest anticancer ac-

tion toward this cell line. As shown in Fig. 13, after 24 h of treatment of

CH₂

acceptor

Electrostatic

H-bond donor

H-bond

Glu885

Val899

NH

3.53

3.96

4.23

3.13

2.95

3.99

N (thiazole)

S (thiazole)

Val914

acceptor

–

Cys1045

Asp1046

O (C O)

Non-classical

–

O (C O)

(σ hole)

CH₂

H-bond

acceptor

H-bond

acceptor

Electrostatic

to that of soreﬁnib (Fig. 9) which may explain its remarkable VEGFR-2

inhibitory activity.

SNB-75 cells with compound 7c at its IC₅₀concentration (7.12 μM), a

Replacing the chlorine atom in compound 7c with bromine atom in

compound 7e at para position leads to decline in the activity. Compound

7e exhibited mean of inhibition equals 4.35%. Also, inhibition per-

centages among SNB-75, SF-295 and CAKI-1 cell lines were reduced, in

addition to reduction in number of affected cell lines in comparison to

compound 7c. The decline in activity was also observed in the in vitro

anti proliferative assay against SF-295 cell line with IC₅₀= 17.7 ± 0.94

decrease in the percentage of the survived cells was observed. The re-

sults revealed an elevation in the apoptotic cells percentage in early

apoptosis phase from 0.27% to 4.71% and a signiﬁcant elevation in late

apoptosis phase from 0.07% to 21.3%. In late stage of apoptosis, caspase

and independent routes are mediated leading to DNA fragmentation,

and nuclear condensation. Furthermore, an elevation in the cells per-

centage in necrosis phase from 1.39% to 15.26% was observed. This

corresponds to an increase in the total apoptosis percentage from 1.73%

to 41.27%.

μ

M compared to 7c with IC₅₀= 7.36 ± 0.39

μM against the same tested

cell line (Fig. 4).

Furthermore, switching the position of the bromo group from para in

7e to meta position in 7d led to increase in the mean of inhibition from

4.35% for 7e to 16.83% for 7d (Table 1), indicating the important

cytotoxic effect of the meta position of bromo group in 7d. Besides, it is

noticeable that compound 7d was nearly as active as the most potent

cytotoxic derivative 7c against most cell lines in the single dose in vitro

cytotoxic screening. On the other hand, 3-bromophenyl derivative 7d

showed decrease in activity against SNB-75, SF-295 and CAKI-1 cell

lines in comparison to 4-chlorophenyl derivative 7c upon conducting in

vitro anti proliferative assay.

2.2.1.10. The effect of compound 7c on the apoptotic marker level (Cas-

pase-3). The stimulation of caspases as caspase-3 is responsible for

apoptosis. Caspase-3 is an effector caspase that has a vital role in

apoptosis. It is activated through one of the initiator caspases which

leads to activation of certain enzymes responsible for fragmentation of

DNA [49]. Therefore, the apoptosis induction by compound 7c in SNB-

75 cells was examined via a caspase-3 assay, compared to doxorubicin

and sorafenib as reference drugs. Compound 7c clearly boosted the level

of the apoptotic caspase-3 by 9.29-fold compared to sorafenib which

increased the level of caspase-3 by 8.16-fold (Fig. 14). On the other

hand, doxorubicin increased the level of caspase-3 by 12.8-fold. This

proposed that compound 7c might prompt apoptosis via a caspase-

dependent mechanism.

Substitution of R²with a methyl group in the thienopyrimidine de-

rivatives led to synthesis of the second series 7f-j. Comparing in vitro

cytotoxic activity with the ﬁrst series revealed that halogen substituted

derivatives including 4-chlorophenyl 7 h, 3-bromophenyl 7i or 4-bro-

mophenyl 7j, showed marked decrease in inhibitory activity with

most cell lines and marked decrease in the mean of inhibition

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Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

Fig. 9. A) 2D interactions of compound 7c within VEGFR-2 active site; B) 3D diagram of compound 7c interactions within VEGFR-2 binding site; C) 3D diagram of

compound 7c superimposed with sorafenib interactions within VEGFR-2 binding site.

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Bioorganic Chemistry 114 (2021) 105137

Fig. 10. A) 2D interactions of compound 7d within VEGFR-2 active site; B) 3D diagram of compound 7d interactions within VEGFR-2 binding site; C) 3D diagram of

compound 7d superimposed with sorafenib interactions within VEGFR-2 binding site.

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Bioorganic Chemistry 114 (2021) 105137

Fig. 11. A) 2D interactions of compound 7e within VEGFR-2 active site; B) 3D diagram of compound 7e interactions within VEGFR-2 binding site; C) 3D diagram of

compound 7e superimposed with sorafenib interactions within VEGFR-2 binding site.

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Bioorganic Chemistry 114 (2021) 105137

Fig. 12. A) Graphical representation of the cell cycle analysis of compound 7c. B) Effect of compound 7c (7.12 µM) on DNA-ploidy ﬂow cytometric analysis of SNB-

75 cells after 24 h.

2.2.2. Antibacterial activity

against CNS cancer cell lines SNB-75 and SF-295 and the renal cancer

line CAKI-1 with IC₅₀values of 7.12 ± 0.33 and 7.36 ± 0.39 and 4.84 ±

0.22 µM, respectively. Compound 7c showed inhibition of VEGFR-2

with IC₅₀= 62.48 ± 3.7 nM. In addition, compound 7c lowered both

total VEGFR-2 and phosphorylated VEGFR-2 in treated SNB-75 cells

suggesting that 7c has down-regulating effect on cell proliferation,

growth, and survival. The results of molecular docking study conﬁrmed

that the binding pattern of compound 7c was consistent with its VEGFR-

2 inhibitory activity. The most potent compound 7c induced signiﬁcant

elevation in late apoptosis phase from 0.07% to 21.3% in SNB-75 cells as

shown by Annexin V-FITC/PI assay. This evidence was reinforced by an

increase in the level of apoptotic caspases-3 by 9.29-fold. Moreover,

analyzing the results of cell cycle analysis showed that derivative 7c

arrested the cell cycle proliferation of SNB-75 cancer cells in the G2/M

phase. These results support the cytotoxic activity of 7c and may present

this compound as a candidate for further biological evaluation.

The antibacterial activity of the prepared compounds was evaluated

in vitro against four pathogenic strains of both Gram-positive and Gram-

negative. Gram-positive strains were represented by Staphylococcus

aureus and Streptococcus pneumoniae while Gram-negative strains were

represented by Escherichia coli and Salmonella Typhimurium using disc

diffusion method on Muller-Hinton Agar (MHA). All tested compounds

did not show any antibacterial activity against the tested bacteria.

2.2.3. Antifungal activity

The antifungal activity of the prepared compounds was evaluated in

vitro against Candida albicans. Only compound 7d exhibited antifungal

activity against Candida albicans (Fig. 15) with inhibition zones of 1.8 ±

0.2, compared to an inhibition zone of 2.3 ± 0.1 cm for nystatin as the

standard antifungal compound.

3. Conclusion

4. Experimental

A series of novel thienopyrimidine-aminothiazole hybrids 7a-j was

prepared. The synthesized compounds were screened for their anti-

cancer activity against the NCI-60 human tumor cell line panel. Com-

pounds 7c and 7d showed broad antiproliferative activity on numerous

cell lines. Compound 7c was found to be the most potent derivative

4.1. Chemistry

4.1.1. General

Melting points were obtained on a Grifﬁn apparatus and were

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Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

Fig. 13. A). Percentage and stages of induced apoptosis in control SNB-75 and SNB-75 treated with compound 7c; B) Representative dot plots of SNB-75 cells treated

with 7c (7.12 µM) for 24 h and analyzed by ﬂow cytometry after double staining of the cells with Annexin-V-FITC and PI.

spectrophotometer (Shimadzu Corp., Kyoto, Japan) Faculty of Phar-

macy, Cairo University, Cairo, Egypt and values were represented in

cm^{ꢀ 1}

.

¹H NMR spectra were carried out on Bruker 400 MHz (Bruker

Corp., Billerica, MA, USA) spectrophotometer, Faculty of Pharmacy,

Cairo University, Cairo, Egypt. Chemical shifts were recorded in ppm on

δ scale, coupling constants (J) were given in Hz and peak multiplicities

are designed as follows: s, singlet; d, doublet; dd, doublet of doublet; t,

triplet; m, multiplet. ¹³C NMR spectra were carried out on Bruker 100

MHz spectrophotometer, Faculty of Pharmacy, Cairo University, Cairo,

Egypt. Progress of the reactions were monitored by TLC using precoated

aluminum sheet silica gel MERCK 60F 254 and was visualized by UV

lamp. Compounds 2a-e, 3a-j, 4, 5 and 6 were prepared according to the

reported procedures. [38–41]

4.1.2. General procedure for the preparation of 2-((3-(4-chlorophenyl)-4-

oxo-3,4,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)-N-

(4-arylthiazol-2-yl)acetamide derivatives (7a-e) and 2-((3-(4-

Fig. 14. Graphical representation for active caspase-3 assay of compound 7c

chlorophenyl)-4-oxo-3,4,5,6,7,8-hexahydrobenzo [4,5]thieno[2,3-d]

pyrimidin-2-yl)thio)-N-(4-arylthiazol-2-yl)propanamide derivatives (7f-j)

To a solution of compound 6 (0.36 g, 0.001 mol) in absolute acetone

(20 mL), derivatives 3a-j (0.001 mol) were added. The mixture was

heated under reﬂux for 15–20 h. The precipitate formed on hot was

compared to doxorubicin and sorafenib.

uncorrected. Microanalyses for C, H and N were carried out at the

Regional Center for Mycology and Biotechnology, Faculty of Pharmacy,

Al-Azhar University. IR spectra were recorded on Shimadzu IR 435

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Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

Fig. 15. Antifungal activity of the synthesized derivatives against C. albicans using nystatin as control.

ﬁltered, dried and recrystallized from ethyl acetate/ ethanol mixture

(1:1).

C, 54.09; H, 3.36; N, 9.34, found C, 54.23; H, 3.49; N, 9.51.

4.1.2.4. N-(4-(3-bromophenyl)thiazol-2-yl)-2-((3-(4-chlorophenyl)-4-

oxo-3,4,5,6,7,8-hexahydro benzo[4,5]thieno[2,3-d]pyrimidin-2-yl) thio)

4.1.2.1. 2-((3-(4-chlorophenyl)-4-oxo-3,4,5,6,7,8-hexahydrobenzo[4,5]

thieno[2,3-d]pyrimidin-2-yl)thio)-N-(4-phenylthiazol-2-yl)

acetamide

acetamide (7d). White solid: 82% yield; mp 266–268 ^◦C; IR (KBr, cm^{ꢀ 1}

)

(7a). White solid: 75% yield; mp 170–172 ^◦C; IR (KBr, cm^{ꢀ 1}) 3298,

3113–3028, 2981–2931, 1685 (br.), 1550, 794; ¹H NMR (400 MHz,

DMSO‑d₆) δ; 12.57 (s, 1H, NH, D₂O exchangeable), 7.91 (d, J = 8.0 Hz,

2H, ArH), 7.68 (d, J = 8.8 Hz, 2H, ArH), 7.63 (s, 1H, ArH thiazole), 7.53

(d, J = 8.8 Hz, 2H, ArH), 7.45 (t, J = 8.0 Hz, 2H, ArH), 7.34 (t, J = 8.0

Hz, 1H, ArH), 4.18 (s, 2H, S-CH₂), 2.79 (t, J = 6.0 Hz, 2H, CH₂), 2.69 (t,

J = 6.0 Hz, 2H, CH₂), 1.78–1.72 (m, 4H, 2CH₂); ¹³C NMR (100 MHz,

DMSO‑d₆) δ: 170.7, 166.6, 161.5, 158.2, 157.6, 156.8, 149.3, 135.2,

134.9, 134.6, 131.8, 131.7, 131.3, 130.1, 129.2, 128.2, 126.1, 119.0,

108.6, 36.3, 25.5, 24.8, 22.9, 22.1. Anal. Calcd for C₂₇H₂₁ClN₄O₂S₃

(565.13): C, 57.38; H, 3.75; N, 9.91, found C, 57.51; H, 3.89; N, 10.12.

3194, 3086–3001, 2935–2839, 1681 (br.), 1560, 794; ¹H NMR (400

MHz, DMSO‑d₆) δ; 12.61 (s, 1H, NH, D₂O exchangeable), 8.12 (s, 1H,

ArH), 7.92 (d, J = 8.0 Hz, 1H, ArH), 7.78 (s, 1H, ArH thiazole), 7.68 (d, J

= 8.6 Hz, 2H, ArH), 7.53 (d, J = 8.6 Hz, 3H, ArH), 7.41 (t, J = 8.0 Hz,

1H, ArH), 4.18 (s, 2H, S-CH₂), 2.79 (t, J = 6.0 Hz, 2H, CH₂), 2.69 (t, J =

6.0 Hz, 2H, CH₂). 1.80–1.72 (m, 4H, 2CH₂), ¹³C NMR (100 MHz,

DMSO‑d₆) δ:172.6, 166.7, 161.5, 158.4, 157.7, 156.7, 147.6, 136.8,

135.2, 134.9, 131.8, 131.4, 131.3, 130.9, 130.2, 128.7, 125.0, 122.6,

118.9, 110.1, 36.3, 25.5, 24.8, 22.8, 22.1.Anal. Calcd for

C

₂₇H₂₀BrClN₄O₂S₃(644.03): C, 50.35; H, 3.13; N, 8.70, found C, 50.42;

H, 3.38; N, 8.97.

4.1.2.2. 2-((3-(4-chlorophenyl)-4-oxo-3,4,5,6,7,8-hexahydrobenzo[4,5]

thieno[2,3-d]pyrimidin-2-yl)thio)-N-(4-(3-nitrophenyl)thiazol-2-yl)acet-

4.1.2.5. N-(4-(4-bromophenyl)thiazol-2-yl)-2-((3-(4-chlorophenyl)-4-

oxo-3,4,5,6,7,8-hexahydro benzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)

amide (7b). White solid: 85% yield; mp 198–200 ^◦C; IR (KBr, cm^{ꢀ 1}

)

acetamide (7e). Buff solid: 98% yield; mp 258–260 ^◦C; IR (KBr, cm^{ꢀ 1}

)

3197, 3086–3032, 2997–2843, 1681 (br.), 1562, 794; ¹H NMR (400

MHz, DMSO‑d₆) δ; 12.71 (s, 1H, NH, D₂O exchangeable), 8.75 (s, 1H,

ArH), 8.37 (d, J = 8.0 Hz, 1H, ArH), 8.19 (dd, J = 2.4, 8.0 Hz, 1H, ArH)

7.96 (s, 1H, ArH thiazole), 7.75 (t, J = 8.0 Hz, 1H, ArH), 7.68 (d, J = 8.4

Hz, 2H, ArH), 7.54 (d, J = 8.4 Hz, 2H, ArH), 4.19 (s, 2H, S-CH₂), 2.79 (t,

J = 6.0 Hz, 2H, CH₂), 2.69 (t, J = 6.0 Hz, 2H, CH₂). 1.76–1.72 (m, 4H,

2CH₂), ¹³C NMR (100 MHz, DMSO‑d₆) δ: 166.9, 161.5, 158.7, 157.6,

156.7, 148.8, 146.9, 136.1, 135.2, 134.9, 132.2, 131.8, 131.3, 130.9,

130.2, 120.5, 118.9, 111.2, 36.3, 25.6, 24.8, 22.8, 22.1. Anal. Calcd for

3213, 3100–3074, 2989–2839, 1681 (br.), 1562, 833; ¹H NMR (400

MHz, DMSO‑d₆) δ; 12.59 (s, 1H, NH, D₂O exchangeable), 7.86 (d, J =

8.4 Hz, 2H, ArH), 7.70 (s, 1H, ArH thiazole), 7.68 (d, J = 8.4 Hz, 1H,

ArH), 7.64 (d, J = 8.4 Hz, 2H, ArH), 7.61–7.58, 7.55–7.52 (m, 1H, ArH),

7.46–7.44 (m, 1H, ArH), 4.18 (s, 1H, S-CH₂), 4.16 (s, 1H, S-CH₂), 2.79 (t,

J = 6.0 Hz, 2H, CH₂), 2.69 (t, J = 6.0 Hz, 2H, CH₂), 1.71–1.71 (m, 4H,

2CH₂), ¹³C NMR (100 MHz, DMSO‑d₆) δ: 166.8, 166.7, 161.5, 158.4,

157.7, 156.7, 148.1, 135.2, 134.9, 133.8, 132.1, 131.8, 130.5, 130.2,

129.7, 128.1, 121.3, 119.0, 109.4, 36.3, 25.5, 24.8, 22.9, 22.1. Anal.

Calcd for C₂₇H₂₀BrClN₄O₂S₃(644.03): C, 50.35; H, 3.13; N, 8.70, found

C, 50.61; H, 3.40; N, 8.96.

C

₂₇H₂₀ClN₅O₄S₃(610.13): C, 53.15; H, 3.30; N, 11.48, found C, 53.44;

H, 3.62; N, 11.70.

4.1.2.3. 2-((3-(4-chlorophenyl)-4-oxo-3,4,5,6,7,8-hexahydrobenzo[4,5]

thieno[2,3-d]pyrimidin-2-yl)thio)-N-(4-(4-chlorophenyl)thiazol-2-yl)acet-

4.1.2.6. 2-((3-(4-chlorophenyl)-4-oxo-3,4,5,6,7,8-hexahydrobenzo[4,5]

thieno[2,3-d]pyrimidin-2-yl)thio)-N-(4-phenylthiazol-2-yl)propanamide

(7f). Buff solid: 83% yield; mp 250–252 ^◦C; IR (KBr, cm^{ꢀ 1}) 3190,

3062–3024, 2981–2839, 1693 (br.), 1560, 793; ¹H NMR (400 MHz,

DMSO‑d₆) δ; 12.63 (s, 1H, NH, D₂O exchangeable), 7.92 (d, J = 8.0 Hz,

2H, ArH), 7.68–7.64 (m, 3H, ArH + ArH thiazole), 7.54–7.43 (m, 4H,

ArH), 7.36–7.32 (m, 1H, ArH), 4.73 (q, J = 8.0 Hz, 1H, S-CH-CH₃), 2.78

(t, J = 6.0 Hz, 2H, CH₂), 2.69 (t, J = 6.0 Hz, 2H, CH₂), 1.79–1.70 (m, 4H,

2CH₂), 1.50 (d, J = 8.0 Hz, 3H, S-CH-CH₃) ¹³C NMR (100 MHz,

DMSO‑d₆) δ: 170.3, 161.5, 158.1, 157.6, 156.2, 149.4, 135.2, 134.8,

134.6, 131.8, 131.7, 131.3, 130.1, 129.1, 128.2, 126.1, 119.1, 108.8,

◦

amide (7c). Greyish white solid: 88% yield; mp 270–272 C; IR (KBr,

1

cm^{ꢀ 1}) 3209, 3100–3074, 2935–2843, 1678 (br.), 1560, 837; H NMR

(400 MHz, DMSO‑d₆) δ; 12.59 (s, 1H, NH, D₂O exchangeable), 7.93 (d, J

= 8.6 Hz, 2H, ArH), 7.70 (s, 1H, ArH thiazole), 7.68 (d, J = 8.6 Hz, 2H,

ArH), 7.52 (t, J = 8.8 Hz, 4H, ArH), 4.18 (s, 2H, S-CH₂), 2.79 (t, J = 6.0

Hz, 2H, CH₂), 2.69 (t, J = 6.0 Hz, 2H, CH₂), 1.78–1.73 (m, 4H, 2CH₂),

¹³C NMR (100 MHz, DMSO‑d₆) δ: 166.7, 161.5, 158.4, 157.7, 156.7,

148.1, 135.2, 134.8, 133.4, 131.7, 130.2, 129.2, 127.8, 118.9, 109.3,

36.3, 25.5, 24.8, 22.8, 22.1. Anal. Calcd for C₂₇H₂₀Cl₂N₄O₂S₃(599.57):

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Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

45.9, 25.5, 24.8, 22.9, 22.1, 17.6. Anal. Calcd for C₂₈H₂₃ClN₄O₂S₃

(579.16): C, 58.07; H, 4.00; N, 9.67, found C, 58.24; H, 4.16; N, 9.88.

4.2. Biological testing

4.2.1. Anticancer activity

4.1.2.7. 2-((3-(4-chlorophenyl)-4-oxo-3,4,5,6,7,8-hexahydrobenzo[4,5]

thieno[2,3-d]pyrimidin-2-yl)thio)-N-(4-(3-nitrophenyl)thiazol-2-yl)prop-

anamide (7g). Yellowish white solid: 90% yield; mp 270–272 ^◦C; IR

4.2.1.1. Measurement of anticancer activity against a panel of 60 cell

lines. Screening of the anticancer activity of the newly synthesized

compounds 7a-j was performed by the US National Cancer Institute

(NCI) using 60 different human tumor cell lines as in accordance with

1

(KBr, cm^{ꢀ 1}) 3178, 3100–3074, 2981–2854, 1681 (br.), 1566, 794; H

NMR (400 MHz, DMSO‑d₆) δ; 12.74 (s, 1H, NH, D₂O exchangeable),

8.77 (t, J = 4.0 Hz, 1H, ArH), 8.38–8.36 (m, 1H, ArH), 8.21–8.18 (m, 1H,

ArH) 7.97 (s, 1H, ArH thiazole), 7.76 (t, J = 8.0 Hz, 1H, ArH), 7.69–7.65

(m, 2H, ArH), 7.55–7.48 (m, 2H, ArH), 4.71 (q, J = 8.0 Hz, 1H, S-CH-

CH₃), 2.78 (t, J = 6.0 Hz, 2H, CH₂), 2.69 (t, J = 6.0 Hz, 2H, CH₂),

1.78–1.71 (m, 4H, 2CH₂), 1.50 (d, J = 8.0 Hz, 3H, S-CH-CH₃) ¹³C NMR

(100 MHz, DMSO‑d₆) δ: 170.6 161.4, 158.6, 157.6, 148.7, 146.9, 136.1,

135.3, 134.8, 132.1, 131.9, 131.8, 131.7, 131.3, 130.9, 130.2, 122.8,

120.5, 119.0, 111.3, 45.8, 25.5, 24.8, 22.8, 22.1, 17.3. Anal. Calcd for

the standard procedure as formerly reported [43–46]. In brief, 100 μL of

each cell type were inoculated into 96 well microplates which were then

incubated at 37 ^◦C, 5% CO₂, 95% air and 100% relative humidity for 24

h. Two plates of each cell line were ﬁxed in situ with TCA and constitutes

the cell population measurement at zero time (Tz). Tested compounds

were dissoluted in dimethyl sulfoxide and were added to the microplate

wells containing 100 μL of complete medium with 50 mg/mL genta-

micin and the plates were then incubated for 48 h at 37 ^◦C, 5% CO₂, 95%

air, and 100% relative humidity. For adherent cells, the experiment was

C

₂₈H₂₂ClN₅O₄S₃(624.15): Elemental Analysis: C, 53.88; H, 3.55; N,

11.22; found C, 54.16; H, 3.72; N, 11.43.

ended and cells were ﬁxed by adding 50 μL of cold 50% (w/v) TCA with

incubation at 4 ^◦C for 60 min. The supernatant was removed, and plates

were washed ﬁve times with tap water and air dried. Then, 100 L of

μ

4.1.2.8. 2-((3-(4-chlorophenyl)-4-oxo-3,4,5,6,7,8-hexahydrobenzo[4,5]

thieno[2,3-d]pyrimidin-2-yl)thio)-N-(4-(4-chlorophenyl)thiazol-2-yl)prop-

0.4% (w/v) sulforhodamine B (SRB) solution in 1% acetic acid was

added to all wells, and plates were incubated for 10 min at room tem-

perature. Later, unbound dye was washed away ﬁve times with 1%

acetic acid and the plates were air dried. Bound stain was then dissolved

in 10 mM trizma base, and the absorbance was measured at 515 nm

using an automated microplate reader. For suspension cells, the same

procedure is carried out except that the experiment was ended by ﬁxing

ꢀ 1

◦

anamide (7h). Buff solid: 77% yield; mp 266–268 C; IR (KBr, cm

)

3275, 3116–3062, 2935–2839, 1681 (br.), 1590, 794; ¹H NMR (400

MHz, DMSO‑d₆) δ; 12.64 (s, 1H, NH, D₂O exchangeable), 7.93 (d, J =

8.8 Hz, 2H, ArH), 7.70 (s, 1H, ArH thiazole), 7.68–7.64 (m, 2H, ArH),

7.54–7.47 (m, 4H, ArH), 4.71 (q, J = 7.2 Hz, 1H, S-CH-CH₃), 2.78 (t, J =

5.8 Hz, 2H, CH₂), 2.68 (t, J = 5.8 Hz, 2H, CH₂), 1.78–1.70 (m, 4H,

2CH₂), 1.49 (d, J = 7.2 Hz, 3H, S-CH-CH₃) ¹³C NMR (100 MHz,

DMSO‑d₆) δ: 170.4, 161.5, 158.3, 157.6, 156.1, 148.2, 135.2, 134.8,

133.4, 132.7, 131.8, 131.8, 131.7, 131.3, 130.2, 129.2, 127.8, 119.0,

109.5, 45.8, 25.5, 24.8, 22.9, 22.1, 17.5. Anal. Calcd for

settled cells at the bottom of the wells by adding 50 μL of 80% TCA.

Calculation of growth inhibition percentages was as follows:

[(Ti ꢀ Tz)/(C ꢀ Tz)] × 100 for concentrations for which Ti ≥ Tz

C

₂₈H₂₂Cl₂N₄O₂S₃(613.60): C, 54.81; H, 3.61; N, 9.13, found C, 55.08;

[(Ti ꢀ Tz)/Tz] × 100 for concentrations for which Ti < Tz

H, 3.87; N, 9.40.

Where, (Tz) is the absorbance at time zero, (C) is the absorbance of

control growth, and (Ti) is the absorbance of test after the addition of the

drug. The one-dose data are reported as a mean graph of the percent

growth of treated cells. The reported number is growth relative to the

no-compound control and relative to the time zero number of cells. This

permits recognition of both growth inhibition and lethality.

4.1.2.9. N-(4-(3-bromophenyl)thiazol-2-yl)-2-((3-(4-chlorophenyl)-4-

oxo-3,4,5,6,7,8-hexa hydro benzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)

propanamide (7i). White solid: 86% yield; mp 268–270 ^◦C; IR (KBr,

1

cm^{ꢀ 1}) 3174, 3109–3059, 2935–2839, 1693 (br.), 1573, 794; H NMR

(400 MHz, DMSO‑d₆) δ; 12.64 (s, 1H, NH, D₂O exchangeable), 8.13 (s,

1H, ArH), 7.94–7.91 (m, 1H, ArH), 7.80 (s, 1H, ArH thiazole), 7.69–7.65

(m, 2H, ArH), 7.55–7.48 (m, 3H, ArH), 7.42 (t, J = 8.0 Hz, 1H, ArH),

4.71 (q, J = 8.0 Hz, 1H, S-CH-CH₃), 2.79 (t, J = 6.0 Hz, 2H, CH₂), 2.69 (t,

J = 6.0 Hz, 2H, CH₂), 1.80–1.71 (m, 4H, 2CH₂), 1.50 (d, J = 8.0 Hz, 3H,

S-CH-CH₃) ¹³C NMR (100 MHz, DMSO‑d₆) δ: 170.4, 161.4, 158.3, 157.6,

156.1, 147.6, 136.8, 135.2, 134.8, 131.9, 131.8, 131.7, 131.4, 131.3,

130.9, 130.2, 128.7, 124.9, 122.6, 119.0, 110.3, 45.8, 25.5, 24.8, 22.8,

22.1, 17.4. Anal. Calcd for C₂₈H₂₂BrClN₄O₂S₃(658.05): C, 51.11; H,

3.37; N, 8.51, found C, 51.34; H, 3.53; N, 8.79.

4.2.1.2. In vitro anti-proliferative activity

4.2.1.2.1. Cell culture. Cell line cells were obtained from American

Type Culture Collection, cells and were cultured using Dulbecco’s

Modiﬁed Eagle’s Medium (DMEM) (Invitrogen/Life Technologies) sup-

plemented with 10% fetal bovine serum (FBS) (Hyclone), 10 μg/mL of

insulin (Sigma), and 1% penicillin–streptomycin. All of the added

chemicals and reagents were obtained from Sigma, or Invitrogen. Plate

cells (cells density 1.2–1.8 × 10,000 cells/well) in a volume of 100 μL

complete growth medium and 100 μL of the tested compound per well in

4.1.2.10. N-(4-(4-bromophenyl)thiazol-2-yl)-2-((3-(4-chlorophenyl)-4-

oxo-3,4,5,6,7,8-hexa hydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)

propanamide (7j). White solid: 67% yield; mp 250–252 ^◦C; IR (KBr,

a 96-well plate for 24 h before the MTT assay.

4.2.1.2.2. Cell culture protocol. Culture medium was removed to a

centrifuge tube. The cell layer was rinsed with 0.25% (w/v) Trypsin

1

cm^{ꢀ 1}) 3275, 3107–3062, 2935–2839, 1685 (br.), 1546, 794; H NMR

0.53 μM ethylenediaminetetraacetic acid (EDTA) solution to eliminate

(400 MHz, DMSO‑d₆) δ; 12.64 (s, 1H, NH, D₂O exchangeable), 7.87 (d, J

= 8.0 Hz, 2H, ArH), 7.72 (s, 1H, ArH thiazole), 7.68–7.63 (m, 4H, ArH),

7.54–7.47 (m, 2H, ArH), 4.71 (q, J = 7.2 Hz, 1H, S-CH-CH₃), 2.78 (t, J =

6.0 Hz, 2H, CH₂), 2.69 (t, J = 6.0 Hz, 2H, CH₂), 1.78–1.71 (m, 4H,

2CH₂), 1.49 (d, J = 7.2 Hz, 3H, S-CH-CH₃) ¹³C NMR (100 MHz,

DMSO‑d₆) δ: 170.4, 161.5, 158.3, 157.6, 156.1, 148.2, 135.2, 134.8,

133.8, 132.1, 131.8, 131.7, 131.7, 131.3, 130.2, 128.1, 121.3, 119.0,

109.6, 45.8, 25.5, 24.8, 22.8, 22.1, 17.5. Anal. Calcd for

all traces of serum which contains Trypsin inhibitor. Trypsin EDTA so-

lution (2.0–3.0 mL) was added to ﬂask and cells were observed under an

inverted microscope until cell layer is dispersed. Growth medium

(6.0–8.0 mL) was added and cells were aspirated by gently pipetting.

The cell suspension was transferred to the centrifuge tube with the

medium and was centrifuged for 5–10 min. The supernatant was dis-

carded. The cell pellet was suspended in new growth medium. Appro-

priate aliquots of the cell suspension were added to new culture vessels.

Cultures were incubated at 37 ^◦C for 24 h. After treatment of cells with

the serial concentrations of the compound to be tested incubation is

carried out for 48 h at 37 ^◦C, then the plates were examined under the

inverted microscope and were preceded for the MTT assay.

C

₂₈H₂₂BrClN₄O₂S₃(658.05): C, 51.11; H, 3.37; N, 8.51, found C, 51.40;

H, 3.52; N, 8.78.

16

Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

4.2.1.2.3. MTT assay protocol. The 3-(4,5-dimethylthiazol-2-yl)-

ligands which are not involved in the binding were removed, the protein

was prepared for the docking study using Protonate 3D protocol in MOE

with default options. The co-crystalized ligand (sorafeinib) was used to

deﬁne the binding site for docking. Triangle Matcher placement method

and London dG scoring function were used for docking.

2,5-diphenyltetrazolium bromide (MTT) method [47] of monitoring in

vitro cytotoxicity is suitable for use with multiwell plates. The evaluation

of cell population growth is based on the capability of living cells to

reduce the yellow product MTT to a blue product, formazan, by a

reduction reaction occurring in the mitochondria. The three cell lines

were incubated for 24 h in 96-microwell plates. The number of living

cells in the presence or absence (control) of the various test compounds

is directly proportional to the intensity of the blue color, measured by

spectrophotometry using (ROBONIK P2000 Spectrophotometer) at a

wavelength of 570 nm. Measure the background absorbance of multi-

well plates at 690 nm and subtract from the 570 nm measurement. Five

4.2.1.7. Measurement of the effect of compound 7c on the level of caspase-

3 protein (Marker of apoptosis). The level of the apoptotic marker

caspase-3 was measured using BIORAD iScript TM One-Step RT-PCR kit

with SYBR® Green. The procedure of the used kit was performed ac-

cording to the manufacturer’s instructions.

4.2.1.7.1. RNA isolation and reverse transcription. mRNA isolation is

carried out using RNeasy extraction kit, up to 1 × 107 cells, depending

on the cell line. Cells are disrupted in RNeasy Lysis Buffer (RLT buffer)

and homogenized, ethanol is then added to the lysate, generating con-

ditions that stimulate selective binding of RNA to the RNeasy mem-

brane. The sample is then applied to the RNeasy mini spin column, total

RNA binds to the membrane, contaminants are efﬁciently ﬂew through,

and high quality RNA is eluted in RNase-free water.

concentrations ranging from 0.01 μM to 100 μM (with semi-log decrease

in concentration) were tested for each of the compounds under study.

Each experiment was carried out in triplicate. The IC₅₀values [the

concentration required for 50% inhibition of cell viability] were calcu-

lated using sigmoidal dose response curve-ﬁtting models.

4.2.1.3. Measurement of inhibitory activity against VEGFR-2. In vitro

VEGFR-2 inhibitory activity of compounds 7c, 7d and 7e was evaluated

4.2.1.7.2. Master mix preparation. All the following reagents were

using ten-fold serial dilutions (1, 0.1, 0.01, 0.001

μM) against VEGFR-2

mixed together to give total volume (50 μL). 2X SYBR® Green RT-PCR

(KDR) Kinase Assay Kit Catalog # 40,325 according to the manufac-

turer’s instructions. Brieﬂy, the master mixture (25 L per well) was

prepared and added to each well. 5 L of inhibitor solution of each well

labeled as “Test Inhibitor” was added. For the “Positive Control“ and

“Blank”, 5 L of the same solution without inhibitor (Inhibitor buffer)

was added. 3 mL of kinase buffer was prepared by mixing 600 L of

kinase buffer with 2400 L water. To the wells designated as ”Blank“, 20

L of kinase buffer was added. The amount of VEGFR-2 needed for the

assay was calculated and diluted to 1 ng/ L with kinase buffer. The

reaction was initiated by adding 20 L of diluted VEGFR-2 to the wells

labelled “Positive Control” and ”Test Inhibitor Control“. The mixtures

reaction mixture (25

primer (10 M) (1.5

to 100 ng total RNA) (10

Step RT-PCR (1 L).

μ

L), forward primer (10

L), nuclease-free H₂O (11

L) and iScript reverse transcriptase for One-

μ

M) (1.5

μL), reverse

μ

μL), RNA template (1 pg

μ

4.2.1.7.3. Ampliﬁcation protocol. Incubate whole reaction mixture

in a real-time thermal detection system (Rotorgene) as follows: cDNA

synthesis: 10 min at 50 ^◦C, iScript reverse transcriptase inactivation: 5

μ

◦

μ

min at 95 C, polymerase chain reaction (PCR) cycling and detection

μ

(30–45 cycles): 10 s at 95 ^◦C and 30 s at 55 ^◦C to 60 ^◦C (data collection

step) and melt curve analysis: 1 min at 95 ^◦C and 1 min at 55 ^◦C and 10 s

at 55 ^◦C (80 cycles, increasing each by 0.5 ^◦C each cycle).

μ

were incubated at 30 ^◦C for 45 min. After the 45 min, 50

μL of Kinase-Glo

Max reagent was added to each well. The plate was incubated at room

temperature for 15 min. Luminescence was measured using the micro-

plate reader.

4.2.1.8. Cell cycle analysis of compound 7c. The SNB-75 cells were

treated with compound 7c at its IC₅₀concentration for 24 h. After

treatment, the cells were then washed with ice-cold phosphate buffered

◦

saline (PBS) twice, collected by centrifugation and ﬁxed at ꢀ 20 C in

4.2.1.4. Measurement of total VEGFR-2 concentration. The effect of

compound 7c on total VEGFR-2 expression in SNB-75 CNS cancer cell

line was evaluated at the IC₅₀of compound 7c (7.12 µM) using Human

VEGF Receptor 2 Simple Step ELISA (Enzyme-Linked Immunosorbent

Assay) Kit according to the manufacturer’s instructions. Brieﬂy the assay

in 96-well plates employs an afﬁnity tag labelled capture antibody and a

reporter conjugated detector antibody which immunocapture the sam-

ple analyte in solution. The complex (captured antibody/analyte/de-

tector antibody) is, in turn, immobilized via immunoafﬁnity of an anti-

tag antibody coating the well. The concentration of total VEGFR-2 was

measured by recoding the OD at 450 nm followed by quantiﬁcation

using the standard curve.

70% (v/v) ethanol, washed with PBS, re-suspended with 0.1 mg/mL

RNase, stained with 40 mg/mL propidium iodide (PI), incubated for 1 h

and analyzed by ﬂow cytometry using FACS Calibur (Becton Dickinson)

[52]. The cell cycle distributions were calculated using Cell- Quest

software (Becton Dickinson).

4.2.1.9. Measurement of apoptosis using Annexin-V-FITC apoptosis detec-

tion kit. Annexin V ﬂuorescein isothiocyanate (FITC) /PI apoptosis

detection kit (BD Biosciences, San Diego, CA) was used according to the

manufacturer’s instructions to measure the apoptotic activity of com-

pounds 7c. Brieﬂy, 4 × 10⁶cell/T 75 ﬂask were exposed to compound 7c

at its IC₅₀concentration for 24 h. The cells then were collected by

trypsinization and 0.5 × 10⁶cells were washed twice with PBS and

4.2.1.5. Measurement of phosphorylated VEGFR-2. The ability of com-

pound 7c to inhibit VEGFR-2 phosphorylation in SNB-75 cells was

performed using VEGFR-2 (Phospho-Tyr951) Colorimetric Cell-Based

ELISA Kit, Aviva Systems Biology Corporation San Diego, USA (Cata-

log #: OKAG02083) according to the manufacturer’s directions.

stained with 5 μL Annexin V-FITC and 5 μL PI in 1 × binding buffer for

15 min at room temperature in the dark. Annexin V-FITC binding was

analyzed using a BD FACS Calibur ﬂow cytometer (BD Biosciences, San

Jose, CA).

4.2.2. Antibacterial activity

The antibacterial activity of the prepared compounds 7a-j was

evaluated in vitro against four pathogenic strains of both Gram-positive

and Gram-negative using disc diffusion method on Muller-Hinton Agar

(MHA). Gram-positive strains were represented by Staphylococcus aureus

and Streptococcus pneumoniae while Gram-negative strains were repre-

sented by Escherichia coli and Salmonella Typhimurium.

4.2.1.6. Molecular docking of the active compounds in the active site of

VEGFR-2. All the molecular modeling studies were carried out using

Molecular Operating Environment (MOE, 2019.0102) software. All

minimizations were performed with MOE until an RMSD gradient of

0.05 kcal∙mol^{ꢀ 1}Å^{ꢀ 1}with MMFF94x force ﬁeld and the partial charges

were automatically calculated. The X-ray crystallographic structure of

vascular endothelial growth factor receptor 2 (VEGFR-2) co-crystalized

with sorafenib (PDB ID: 4ASD) [50] was downloaded from the protein

data bank [51]. For each co-crystallized enzyme; water molecules and

The test organisms were maintained on agar slant at 4 ^◦C and sub-

cultured on a fresh agar plates. For disc diffusion assay, bacterial liquid

cultures were initiated by placing a loop of bacteria from the slant into

17

Y. El-Dash et al.

Bioorganic Chemistry 114 (2021) 105137

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10 mL of Lysogeny Broth (LB) medium [53]. Agar diffusion test was

conducted to detect the bacterial susceptibility to the prepared com-

pounds. A volume of 100 μL of cell culture suspension matching with 0.5

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medium. The prepared compounds were adjusted to a concentration of

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paper discs (5

μ

g) were incubated at 37 ± 0.1 ^◦C for 24 h to allow

bacterial growth. After incubation, the inhibition of bacterial growth

was evaluated by measuring the diameter (cm) of the clear zone around

each disc. The resulting inhibition zones were compared with the inhi-

bition zones of the standard antibiotics Ampicillin (AM-10) and Levo-

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were also used as control for the solvent. The experiment was carried out

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The antifungal activity of the prepared compounds 7a-j was evalu-

ated in vitro against Candida albicans NRRL-Y 477. Well diffusion method

was conducted to detect the C. albicans susceptibility to the prepared

compounds [54]. The test organism was maintained on Sabouraud

Dextrose Agar (SDA) slant at 4 ^◦C. The prepared compounds were

adjusted to a concentration of 50 mg/mL using DMSO as a solvent. For

well diffusion assay, an inoculum was subcultured on SDA plate and the

plate was incubated for 24 h at 35 ^◦C. One colony obtained from agar

was inoculated into Sabouraud Dextrose broth followed by incubation at

35 ^◦C to prepare a suspension of a concentration matching 0.5 McFar-

land standard. A total of 1 mL of the prepared suspension was swabbed

on Potato Dextrose Agar (PDA) plate and left to dry. Wells with a

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◦

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Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

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The authors are grateful to all associates of National Institutes of

Health, USA for carrying out the anticancer screening. The authors

thank Dr. Esam Rashwan, Head of the conﬁrmatory diagnostic unit

VACSERA-EGYPT, for carrying out cell cycle analysis, VEGFR-2 assays

and caspase-3 marker.

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Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.105137.

`

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Novel thienopyrimidine-aminothiazole hybrids: Design, synthesis, antimicrobial screening, anticancer activity, effects on cell cycle profile, caspase-3 mediated apoptosis and VEGFR-2 inhibition

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