New bis([1,2,4]triazolo)[4,3-a:3′,4′-c]quinoxaline derivatives as VEGFR-2 inhibitors and apoptosis inducers: Design, synthesis, in silico studies, and anticancer evaluation

Bioorganic Chemistry 112 (2021) 104949

Contents lists available at ScienceDirect

Bioorganic Chemistry

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

′

New bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline derivatives as VEGFR-2

inhibitors and apoptosis inducers: Design, synthesis, in silico studies, and

anticancer evaluation

a

,

*

b

a

Mohammed M. Alanazi , Hazem A. Mahdy , Nawaf A. Alsaif , Ahmad J. Obaidullah ,

a

Hamad M. Alkahtani , Abdulrahman A. Al-Mehizia , Sultan M. Alsubaie ,

b

,

*

b,*

Mohammed A. Dahab , Ibrahim H. Eissa

a

Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, Saudi Arabia

b

Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo 11884, Egypt

A R T I C L E I N F O

A B S T R A C T

′

Keywords:

A new series of bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline derivatives were designed and synthesized to have

the main essential pharmacophoric features of VEGFR-2 inhibitors. VEGFR-2 inhibitory activities were assessed

for the designed compounds. In addition, cytotoxic activity was evaluated for all derivatives against two human

cancer cell lines namely, HepG-2 and MCF-7. The most cytotoxic compound 20 h was subjected to further

biological investigations including cell cycle, apoptosis, caspase-3, caspase-9, BAX, and Bcl-2 analyses. Different

in silico studies as docking, ADMET and toxicity were carried out. The results exhibited that compounds 20b, 20e,

Anti-proliferative

Apoptosis

′

Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline

Docking studies

VEGFR-2 inhibitors

2

0h and 20m showed promising VEGFR-2 inhibitory activities with IC50 values of 5.7, 6.7, 3.2, and 3.1 µM,

respectively. Moreover, these promising members exhibited the highest antiproliferative activities against the

two cell lines with IC50 values ranging from 3.3 to 14.2 µM, comparing to sorafenib (IC50 = 2.17 and 3.43 µM

against HepG2 and MCF-7, respectively). Additionally, compound 20h induced cell cycle arrest of HepG2 cells at

G2/M phase. Also, such compound increased the progress of apoptosis by 3.5-fold compared to the control. As

well, compound 20h showed a signiﬁcant increase in the level of caspase-3 (2.07-fold), caspase-9 (1.72-fold),

and BAX (1.83-fold), and a signiﬁcant decrease in Bcl-2 level (1.92-fold). The in silico studies revealed that the

synthesized compounds have binding pattern like that of sorafenib.

1

. Introduction

Cancer is still one of the most serious diseases occupying the 2nd

takes place under the effect of PTKs, causing a conformational change

affecting protein function [6]. PTKs have a critical role in cancer

development and progression [7]. Most of cancer diseases involved PTKs

malfunction. More than 60% of all oncoproteins and proto-oncoproteins

(the main players in cancer disease) are PTKs [8]. Because of the rep-

resentation of PTKs as crucial elements in most of cancer diseases, they

take the attention of medicinal chemists and biologists as a good target

for cancer therapy [9,10].

order in the class of life-threatening diseases after cardiovascular ill-

nesses. In addition, the mortality rate due to cancer reached 9.6 million

deaths in 2018 [1]. The chemotherapeutic agents which are used in the

management of cancer suffer from many serious side effects because of

its non-selectivity [2]. The most common side effects caused by

chemotherapy are gastrointestinal, hematological, skin and hair follicle

toxicity, nervous system toxicity, hepatic toxicity, cardiac toxicity, uri-

nary tract toxicity, and pulmonary toxicity [3]. Hence, there is an urgent

need to discover new anticancer drugs or at least to modify the existing

one to get less toxic anticancer agents [4].

Angiogenesis acts

as

a

signiﬁcant factor

in

normal -

physiological conditions [11]. On the other hand, abnormal angiogen-

esis lead to dangerous pathological diseases, particularly, cancer

development [12]. Vascular endothelial growth factor receptor-2

(VEGFR-2), as subtype of PTKs, has the upper hand in angiogenesis

process. Activation of VEGFR-2 by vascular endothelial growth factor

(VEGF) is a serious stage that initiates tumor angiogenesis [13]. So that,

Protein tyrosine kinases (PTKs) are essential key elements of cell

proliferation [5]. Phosphorylation of tyrosine residues of the proteins

*

Corresponding authors.

E-mail addresses: mmalanazi@KSU.EDU.SA (M.M. Alanazi), Mmohammeddahab@azhar.edu.eg (M.A. Dahab), Ibrahimeissa@azhar.edu.eg (I.H. Eissa).

https://doi.org/10.1016/j.bioorg.2021.104949

Received 24 January 2021; Received in revised form 8 April 2021; Accepted 22 April 2021

Available online 30 April 2021

0

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

blocking VEGF/VEGFR-2 signaling pathway and subsequent suppres-

sion of angiogenesis is considered as potential line for cancer manage-

ment [14]. Truly, such line of cancer treatment has led to discovery and

FDA approvals of new VEGFR-2 inhibitors [15].

DFG domain [39]. (iv) The 4th feature is the terminal hydrophobic

moiety, which can form hydrophobic interactions with the allosteric

hydrophobic pocket of ATP binding site [37,40–42] (Fig. 2).

As an extension of our efforts in the ﬁeld of discovery of new anti-

cancer agents [10,36,43–47], especially those targeting VEGFR-2

[48–53], we synthesized a new series of bis([1,2,4]triazolo)[4,3-

Many inhibitors targeting VEGFR-2 have been approved by FDA for

clinical treatment of cancer (Fig. 1). The ﬁrst class of VEGFR-2 is urea

derivatives which includes sorafenib I and regorafenib II [16,17]. These

drugs were indicated for hepatocellular carcinoma, metastatic renal cell

cancer, and thyroid cancer [18,19]. The most common adverse effects of

such drugs are cardiovascular adverse problems, diarrhea, and impair-

ment of renal function [20,21]. The 2nd class is indoline derivatives

which comprises sunitinib III and nintedanib IV [22,23] which were

indicated for non-small-cell lung carcinoma, gastrointestinal stromal

tumor, and renal cell carcinoma [23,24]. Some toxic effects were

attributed to these drugs as hepatotoxicity, reversible acute kidney

injury, hand-foot syndrome, gastrointestinal disturbance, hypertension,

fatigue, and hyperparathyroidism [25]. The 3rd class is quinoline de-

rivatives which comprises lenvatinib V and cabozantinib VI. Such drugs

were approved for prostate cancer, thyroid cancer, melanoma, and renal

cell carcinoma [26–28]. Their administration causes some adverse ef-

fects as fatigue, hypertension, decreased appetite, nausea, diarrhea,

thrombocytopenia, and proteinuria [29–31]. Accordingly, in the recent

years, there are a lot of efforts to ﬁnd out novel anticancer agents

′

a:3 ,4 -c]quinoxaline derivatives as anti-proliferative agents having the

essential pharmacophoric features of VEGFR-2 inhibitors.

1.1. The rationale of molecular design

Our previously synthesized compounds VII and VIII were reported to

possess promising antiproliferative and VEFR-2 inhibitory activities

[51,54]. The rationale of molecular design depended on the lead

modiﬁcation of such compounds to obtain new anti-proliferative agents

targeting VEGFR-2 with potential apoptosis induction.

The modiﬁcation strategy comprised the replacement of 3-(p-tolyl)

quinazolin-4(3H)-one and 3-phenylquinazolin-4(3H)-one moieties of

the lead compounds X and XI, respectively, with bis([1,2,4]triazolo)

′

[4,3-a:3 ,4 -c]quinoxaline moiety. Such replacement was based on some

′

considerations. i) Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline moiety

contains more electron acceptor atoms which may facilitate the binding

interaction with the important amino acids in the hinge region of

VEGFR-2. ii) The high planarity of such moiety may increase the pi-pi

interaction with the hydrophobic amino acids in the hinge region. iii)

[

32–36].

The chemical structure of VEGFR-2 inhibitors should possess four

pharmacophoric features bind efﬁciently with the ATP binding site of

VEGFR-2. i) The 1st feature is a hetero aromatic moiety which is

essential for hydrogen bonding with Cys917 in the hinge region of ATP

binding site [37]. (ii) The 2nd is a spacer group which can occupy the

region allocated between the hinge region and the DFG domain [38],

′

The quinoxaline and bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline moi-

eties was reported to possess promising anti-proliferative activity

[33,45,55]. In addition, the linker moiety (2-sulfanyl-N-phenyl-

acetamide) was retained without modiﬁcation. Moreover, the HBD-HBA

moiety was modiﬁed to be amide (compounds 20a-m), hydrazide

(compound 21), and diamide (compounds 22a,b) moieties Finally,

different substituted aromatic moieties were utilized as a hydrophobic

tail (Fig. 2).

(

iii) The 3rd one is the pharmacophore moiety which consists of a H-

bond donor and a H-bond acceptor groups. Such pharmacophore can

form hydrogen bonding interactions with Glu883 and Asp1044 in the

Fig. 1. Some FDA approved VEGFR-2 inhibitors for clinical treatment of cancer.

2

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Fig. 2. Essential pharmacophoric features of VEGFR-2 inhibitors and schematic summary for the rationale of molecular design.

2

. Results and discussion

according the depicted procedures illustrated in Scheme 2 and Scheme

. 4-Amino benzoic acid 10 was acylated using chloroacetyl chloride

using DMF as solvent and NaHCO as a base to give 4-(2-chlor-

3

2

.1. Chemistry

3

oacetamido)benzoic acid 11. The latter was heated with thionyl chloride

in presence of catalytic amount of DMF to produce the benzoyl chloride

derivative 12. Stirring of compound 12 with appropriate amines

namely, aniline, 2-methylaniline, 3-methylaniline, 4-methylaniline, 2,6-

dimethylaniline, 2-methoxyaniline, 3-methoxyaniline, 4-methoxyani-

line, 2,6-dimethoxyaniline, 4-nitroaniline, benzylamine, 2-phenylethyl-

amine, and 2-aminothiazole at room temperature in acetonitrile / TEA

mixture produced the corresponding intermediates 13a-m, respectively

(Scheme 2).

Different synthetic pathways were described in Schemes 1-5 for

preparation of both intermediates and ﬁnal designed compounds

Firstly, 2,3-(1H,4H)-quinoxalinedione 3 [56] was prepared by reﬂux

o-phenylenediamine 1 with oxalic acid 2 in 4 N HCl. Heating of com-

pound 3 in a mixture of thionyl chloride and DCE afforded 2,3-

dichloroquinoxaline 4 [56]. The latter was treated with hydrazine hy-

drate in absolute ethanol with continuous stirring at room temp. to

afford 2-chloro-3-hydrazinylquinoxaline 5 [57]. Cyclization of com-

pound 5 was carried out by its reﬂux with triethyl orthoformate to

produce 4-chloro[1,2,4]triazolo[4,3-a]quinoxaline 6 [57], Heating of

compound 6 with hydrazine hydrate in ethanol as a solvent gave 4-

hydrazinyl-[1,2,4] triazolo[4,3-a]quinoxaline 7 [58]. Cyclization of

compound 7 was done by its reﬂux in alcoholic mixture of carbon

disulphide and potassium hydroxide followed by acidiﬁcation using HCl

Next, stirring of compound 12 with phenylhydrazine 17 at room

temperature in acetonitrile / TEA mixture produced the key interme-

diate 18. To prepare the ester derivatives 15a,b, some commercially

available acid derivatives 14a,b namely, benzoic acid and 4-nitroben-

zoic were reﬂuxed in methanol in the presence of sulfuric acid accord-

ing to the reported procedures [47,59,60]. In addition, reﬂux of 15a,b

with hydrazine hydrate afforded the corresponding acid hydrazides 16a,

b [47]. At the end, in acetonitrile and TEA mixture, compound 12 was

stirred with the acid hydrazides 16a,b to produce the corresponding

diamide intermediates 19a,b.

′

to produce bis[1,2,4]triazolo[4,3-a:3 ,4 -c]quinoxaline-3-thiol 8 [33].

Heating of compound 8 with alcoholic solution of potassium hydroxide

gave the corresponding potassium salt 9 [33] (Scheme 1).

Then, the key intermediates 13a-m, 18, and 19a-b, were prepared

3

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Scheme1. Synthetic pathway of potassium salt 9 as a key compound.

Scheme 2. Synthetic pathway of the key intermediates 13a-m.

4

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Scheme 3. Synthetic pathway of the key intermediates 18 and 19a,b.

The structures of the intermediates 13a-m, 18, and 19a,b were

2.2. Biological testing

conﬁrmed by spectral and elemental data. IR spectra of such compounds

ꢀ

1

showed strong bands around 3370–3100 cm corresponding to NHs.

2.2.1. VEGFR-2 inhibitory assay

–

′

Also, IR spectra showed strong C

–

O absorption bands at a range of

770–1624 cm . Moreover, ¹H NMR spectra showed singlet signals

around δ 10.50 and 8.35 ppm corresponding to the two amidic NHs.

All the synthesized bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline-

based amide 20a-m, hydrazide 21, and diamide 22a,b were evaluated

for their VEGFR-2 inhibitory activities. Sorafenib was utilized as a

standard drug and the inhibitory activities were presented as IC50 in

Table 1.

ꢀ 1

1

Additionally, CH protons appeared at around δ 4.30 ppm. Matching

2

with such results, ¹³C NMR spectra also conﬁrmed the validity of sug-

gested structures where characteristic peaks were displayed around δ

Comparing to the inhibitory activity of the reference molecules (IC50

= 3.1 µM), compounds 20b, 20e, 20 h, and 20 m showed promising

VEGFR-2 inhibitory activities with IC50 values of 5.7, 6.7, 3.2, and 3.1

µM, respectively. Additionally, compounds 20f, 20 g, 20i, 20j, 21, and

22b showed moderate activities with IC50 values of 11.4, 13.2, 11.8,

24.1, 17.4, and 23.8 µM, respectively. On the other hand, compounds

20a, 20c, 20d, 20 k, 20 l, and 22a displayed weak activities.

From these results, we can deduce good structure–activity relation-

ships. Comparing the VEGFR-2 inhibitory activities of compounds 21

(incorporating a hydrazide moiety as a HBD-HBA, IC50 = 17.4 µM), 20a

(incorporating an amide moiety as a HBD-HBA, IC50 = 68.7 µM), and

22a (incorporating a diamide moiety as a HBD-HBA, IC50 = 78.3 µM), it

was found that 21 was more active than 20a which was more active than

22a. These results indicated that hydrazide moiety is more favorable

than amide moiety as HBD-HBA group, and the latter is more advanta-

geous than the diamide moiety.

–

1

65.60, 165.05, and 44.00 ppm corresponding to the two C

–

O and CH

2

groups, respectively.

The synthetic pathways of the ﬁnal designed compounds (20a-m,

1, and 22a,b) were illustrated in Scheme 4 and Scheme 5. Compound 9

2

was reﬂuxed with the previously synthesized intermediates (13a-m, 18,

and 19a,b) in dry DMF using catalytic amount of KI to furnish the titled

compounds 20a-m, 21, and 22a,b, respectively.

The spectral and elemental data supported the structures of the ob-

1

tained derivatives, where the H NMR spectra of compounds 20a-m, 21,

and 22a,b displayed characteristic downﬁeld singlet signals around δ

1

0.70 ppm corresponding to the aromatic proton of triazole ring. Taking

compound 20b as a representative example, the IR spectrum demon-

ꢀ 1

strated stretching bands at δ 3273, 3111 cm corresponding to the NH

ꢀ

1

–

groups and 1667, 1644 cm corresponding to C O bonds. The H NMR

1

–

spectrum of this compound showed up-ﬁeld singlet signal at δ 2.28 ppm

corresponding to methyl group. In addition, it exhibited singlet signal at

δ 2.28 ppm 10.05 corresponding to the aromatic proton of triazole ring.

Also, the methylene group was conﬁrmed by the presence of singlet

Then, the activities of different hydrophobic tails were investigated.

From the results, it was found that compound 20 m (IC50 = 3.1 µM)

incorporating thiazole moiety as hydrophobic tail was the most active

member. This revealed that ﬁve-membered heterocyclic ring has the

highest positive effect in VEGFR-2 inhibition. Shifting the hydrophobic

tail into six-membered non-heterocyclic ring such as compound 20a

1

3

signal at δ 4.43 ppm. C NMR spectrum showed the presence of two

peaks at 34.51, 18.41corresponding to CH2 and CH groups.

3

5

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Scheme 4. Synthetic pathway of the titled compounds 20a-m.

incorporating phenyl moiety), led to dramatic decrease in VEGFR-2 inhibition.

(

inhibitory activity (IC50 = 68.7 µM).

Furthermore, substitution of phenyl ring with electron withdrawing

(nitro) group, led to slight increase in VEGFR-2 inhibitory activity

(compound 20j, IC50 = 24.1 µM). Next, we examined the effect of carbon

bridge between HBD/HBA moiety and hydrophobic tail. Comparing the

activity of compound 20a (IC50 = 68.7 µM) incorporating zero carbon

bridge, compound 20 k (IC50 = 59.4 µM) incorporating one carbon

bridge, and compound 20 l (IC50 = 74.2 µM) incorporating two carbon

bridges, indicated that the activity decreased in the order of one carbon

> zero carbon > two carbons.

Substitution of phenyl ring with an electron donating (methoxy)

group at para-position, led to dramatic increase in VEGFR-2 inhibition

(

e.g. compound 20 h, IC50 = 3.2 µM). Oppositely, methoxy substitution

at ortho-position (e.g. compound 20f, IC50 = 11.4 µM) and meta-position

e.g. compound 20 g, IC50 = 13.2 µM), afforded slight decrease in

VEGFR-2 inhibition. Additionally, 2,6-dimethoxy derivative (compound

(

2

0i, IC50 = 11.8 µM) exhibited VEGFR-2 inhibitory activity almost equal

ortho-methoxy derivative (compound 20f).

Another mode of modiﬁcation was carried out, where the phenyl ring

was substituted with methyl group at ortho position to produce com-

pound 20b (IC50 = 5.7 µM). This modiﬁcation led to dramatic increase

in biological activity. In contrary, methyl substitution at meta-position

2.2.2. In vitro anti-proliferative activities

In vitro anti-proliferative activities of the target molecules were

assessed against two human cancer cell lines, hepatocellular carcinoma

(HepG2) and breast cancer (MCF-7), by MTT assay [61]. Sorafenib was

utilized in this test as a positive reference molecule. The results of anti-

(

e.g. compound 20c, IC50 = 91.4 µM) and para-position (e.g. compound

0d, IC50 = 75.5 µM), produced dramatic decrease in VEGFR-2

2

6

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Scheme 5. Synthetic pathway of the titled compounds 21 and 22a,b.

Table 1

Table 2

VEGFR-2. inhibitory activities of the synthesized compounds 20a-m, 21, and

In vitro anti-proliferative activities of the synthesized compounds 20a-m, 21,

2

2a,b fx1.

and 22a,b against HepG2 and MCF-7 cell lines fx2.

a

Comp.

HBA-HBD

R

IC50 (µM) VEGFR-2

Comp.

HBA-HBD

R

IC50 (µM)

2

0a

0b

0c

0d

0e

0f

–NH-CO-

–NH-NH-CO-

–CO –NH-NH-CO-

–

Phenyl

68.7

5.7

HepG2

MCF-7

2-Methylbenzene

3-Methylbenzene

4-Methylbenzene

2,6-Dimethylbenzene

2-Methoxybenzene

3-Methoxybenzene

4-Methoxybenzene

2,6-Dimethoxybenzene

4-Nitrobenzene

Benzyl

2

0a

0b

0c

0d

0e

0f

–NH-CO-

–NH-NH-CO-

–CO –NH-NH-CO-

–

Phenyl

61.2

11.4

68.7

47.8

10.7

19.7

27.4

3.3

62.9

14.2

71.5

78.1

13.7

23.2

37.8

4.4

91.4

75.5

6.7

2-Methylbenzene

3-Methylbenzene

4-Methylbenzene

2,6-Dimethylbenzene

2-Methoxybenzene

3-Methoxybenzene

4-Methoxybenzene

2,6-Dimethoxybenzene

4-Nitrobenzene

Benzyl

11.4

13.2

3.2

0 g

0 h

0i

0 g

0 h

0i

11.8

24.1

59.4

74.2

3.1

0j

14.5

28.4

57.6

68.7

8.7

21.3

41.7

58.7

73.9

11.3

47.2

68.2

48.5

3.43

0 K

0l

0j

Phenethyl

0 K

0l

0 m

1

2-Thiazolyl

Phenethyl

Phenyl

17.4

78.3

23.8

3.1

0 m

1

2-Thiazolyl

2a

2b

Phenyl

38.1

67.4

33.7

2.17

4-Nitrobenzene

–

2a

2b

Phenyl

Sorafenib

4-Nitrobenzene

–

a

All IC50 values are calculated as the mean of at least three different

Sorafenib

experiments.

On the other hand, other compounds exhibited weak antiproliferative

activities.

proliferative activities were presented as IC50 in Table 2.

In general, it was found that HepG2 cells were more sensitive to the

synthesized compounds than MCF-7 cells. The results revealed that

compounds 20b (IC50 ¼ 11.4 and 14.2 µM), 20e (IC50 ¼ 10.7 and 13.7

µM), 20 h (IC50 ¼ 3.3 and 4.4 µM), and 20 m (IC50 ¼ 8.7 and 11.3 µM)

were the most active members against HepG2 and MCF-7, respectively.

These results showed that compound 20 h is the most promising

2

.2.3. Simple linear regression analysis of VEGFR-2 inhibitory activity

against cytotoxicity

Simple linear regression analysis was performed for two parameters;

VEGFR-2 inhibitory activities (as independent variable) and anti-

proliferative activities (as independent variable) of the synthesized

compounds. Such statistical analysis was carried out to check the extent

of correlation between VEGFR-2 inhibitory activities and anti-

proliferative activities. The results revealed that the coefﬁcients of

members in the synthesized series. In addition, compounds 20f (IC50

9.7 and 23.2 µM), 20i (IC50 ¼ 14.5 and 21.3 µM), and 20j (IC50 ¼ 28.4

and 41.7 µM), showed moderate activities against HepG2 and MCF-7,

respectively. Also, compound 20 showed moderated anti-

proliferative activities against HepG2 cells with IC50 values of 27.4 µM.

¼

1

g

2

determination (R ) were 0.902 and 0.864 against HepG2 and MCF-7,

7

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

respectively. Such values of R²were high enough to indicate the high

correlations between VEGFR-2 inhibitory activities and cytotoxic ac-

tivities of the titled compounds. From these ﬁndings, we can conclude

that the VEGFR-2 inhibition is main cause of cytotoxicity (Fig. 3).

Table 3

Effect of compound 20 h on cell cycle progression in HepG2 cells.

a

Sample

Cell cycle distribution (%)

%

Sub-G1

%G1

%S

% G2/M

HepG2

1.21 ±

.34

57.66 ± 2.17

28.38 ±

2.11

12.72 ± 0.86

2

.2.4. Cell cycle analysis

0

The cell cycle is an effectual mechanism by which eukaryote cells

Comp. 20 h

1.13 ±

37.14 ±

29.56 ±

2.83

32.17 ±

reproduce themselves. There are many checkpoints (phases) at which

any anti-proliferative agent can block the cell growth. Checkpoint

signaling can activate some mechanisms which initiates apoptosis in

case of cellular damage persistence. Detecting such probable phase of

cell arrest is critical in the process of drug discovery [62].

**

***

/

HepG2

0.15

3.42

1.66

a

Values are given as mean ± SEM of three independent experiments. **p <

.01, and ***p < 0.001.

0

[

67–69]. In this test, HepG2 cells were treated with compound 20 h at a

In this work, HepG2 cells were treated with the most cytotoxic

member (compound 20 h) at a concentration of 3.3 µM (equal it IC50

value against HepG2 cells). Investigating the results, it can be noticed

that compound 20 h induced an obvious decrease in the cell population

at G1 phase (37.14%) and a very mild decrease in the cell population at

Sub-G1 phase (1.13%), comparing to the control cells (57.66% and

concentration of 3.3 µM for 24 h. The results showed that the tested

compound afforded marked increase in caspase-3 level (2.07-fold)

compared to the control cells. Moreover, this compound produced a

substantial increase in the level of caspase-9 (1.72-fold) compared to the

control cells (Table 4 & Fig. 6).

1

.21%, respectively). While such compound exerted substantial increase

2

.2.7. Effects on mitochondrial apoptosis pathway (BAX and Bcl-2

in cell population at the G2/M phase (32.17%), and insigniﬁcant in-

crease in cell population at the S phase (29.56%), comparing to the

control cells (12.72% and 28.38%, respectively). These outcomes verify

that the cytotoxicity of compound 20 h against HepG2 cells was due to

arresting the cell growth at G2/M phase (Table 3 and Fig. 4).

determination)

BAX and Bcl-2 are the main regulators of the mitochondrial

apoptosis. These mediators have opposite functions during apoptotic

pathway. BAX has a pro-apoptotic activity while Bcl-2 has anti-apoptotic

effect [70]. So that, BAX / Bcl-2 ratio is a critical factor for mitochon-

drial apoptosis [71].

2

.2.5. Apoptosis analysis

To examine the potential effects of the synthesized compounds on

mitochondrial apoptosis, compounds 20 h (the most cytotoxic member)

was applied on HepG2 at a concentration equal its IC50 value (3.3 µM).

Western blot technique was utilized in this test. The results revealed that

compound 20 h afforded an up-regulation of BAX by 1.83-fold

comparing to the control cells. Additionally, it exhibited down-

regulation of Bcl-2 by 1.92-fold comparing to the control cells.

Furthermore, it was found that BAX / Bcl-2 ratio equal 3.45. Such results

indicated that compound 20 h has a promising positive effect on mito-

chondrial apoptosis (Table 5 & Fig. 7).

Annexin-V/propidium iodide (PI) staining (Annexin-V FTIC) assay

was carried out to assess the apoptotic effect of compound 20 h on the

most sensitive (HepG2) cells at a concentration of 3.3 µM for 24 h.

The results of Annexin-V FTIC assay exhibited that compound 20

h produced an obvious increase in values of Annexin V-FITC positive

apoptotic effect. For early phase, the percent of apoptotic cells increased

from 8.92% (in control cells) to 31.17%. While the late phase comprised

an increase in apoptotic cells from 0.12% (in control cells) to 0.22%.

This denotes 3.5-fold increase in apoptotic cells comparing to the control

(

p < 0.001). Furthermore, necrosis was observed in very small percent

0.34%) (Table 3 & Fig. 5).

2

.3. In silico studies

2

.2.6. Activation of proteolytic caspase cascade (Caspase-3 and caspase-9

determination)

2.3.1. Docking studies

Caspases (a family of cysteine aspartic proteases) have crucial role in

apoptosis [63]. Activation of caspase-3 leads to cleavage of some regu-

latory proteins, which are critical for cell maintenance and survival

To investigate the proposed binding interaction with the target re-

ceptor, docking studies were carried out against VEGFR-2 (PDB ID:

2OH4). Sorafenib was used as a reference ligand. In this study, we

depended on the binding free energy (ΔG) between the docked mole-

cules and the active site, in addition to the correct binding mode. The

reported binding mode against VEGFR-2 comprises important interac-

tion with some crucial amino acid residues including Glu883 and

Asp1044 in the DFG-binding domain and Cys917 in the hinge region

[40,72]. The binding free energies were summarized in Table 6.

At ﬁrst, validation of the docking process was checked through

[

64]. Caspase-3 belongs to executioner caspases and is activated by

intrinsic and extrinsic pathways [65]. Additionally, activation of

caspase-9 cleaves other caspases such as caspase-3, leading to initiation

of the caspase cascade [66].

Effect of the most promising compound 20 h on caspase-3 and

caspase-9 levels was investigated through Western blot analysis

Fig. 3. simple linear regression for the correlation between cytotoxicity and VEGFR-2 inhibition on two cell line models; HepG-2 and MCF-7.

8

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Fig. 4. Cell cycle phases after the treatment of HepG2 Cells with compound 20 h (3.3 µM) for 24 h. Results are presented as mean ± SEM of three different ex-

periments, where **p < 0.01, and ***p < 0.001.

Fig. 5. Apoptosis effect of compound 20 h in HepG2 cells after 24 h treatment stained with Annexin-V/propidium iodide (PI). Results are presented as mean ± SEM

of three different tests, where ***p < 0.001.

9

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Table 4

equal 1.09. This indicates the validity of the docking process (Fig. 8).

Binding mode of sorafenib as a refrence ligand showed a binding free

energy of ꢀ 26.50 kcal/mol. The HBD/HBA (urea) moiety formed three

hydrogen bonds with the two important amino acid residues in the DFG

binding domain (Glu883 and Asp1044). The phenyl group was incor-

porated in hydrophobic interactions in the spacer region forming three

hydrophobic bonds with Lys866 and Val897. In addition, n-methyl-

picolinamide moiety was buried in the hinge region forming two

hydrogen bonds with Cys917 and three hydrophobic interactions with

Leu1033, Cys917, and Ala864. Furthermore, the hydrophobic tail (1-

chloro-2-(triﬂuoromethyl)benzene) formed two hydrophobic in-

teractions with Ile886 and Leu1017. These results were the same as the

reported data [40,72] (Fig. 9).

Apoptosis Effect of compound 20 h in HepG2 cells after 24 h treatment.

a

Sample

Viable (Left

Apoptosis

Necrosis

Bottom)

(Left Top)

Early (Right

Bottom)

Late (Right

Top)

HepG2

90.84 ± 1.41

68.25 ± 2.28

8.92 ± 0.41

31.17 ±

0.12 ±

0.13 ± 0.01

0.34 ± 0.07

0

.01

Comp. 20 h

0.22 ±

*

**

/

HepG2

2.36

0.03

a

Results are presented as mean ± SEM of three different experiments, where

**p < 0.001.

*

The target molecules interacted with active site of VEGFR-2 showing

binding mode almost the same of that of sorafenib. Compound 20b

exerted a binding afﬁnity of ꢀ 29.98 Kcal/mol. The amide group formed

two hydrogen bonds the crucial amino acid residues in the DFG binding

domain (Glu883 and Asp1044). The central phenyl group was incor-

porated in many hydrophobic interactions with Val897, Lys866, Val914,

′

and Glu883. The bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline moiety

was buried in the hinge region to form ﬁve hydrophobic interactions

with Phe1045, Val846, and Leu838. Furthermore, the o-tolyl moiety

occupied the allosteric region forming two hydrophobic interactions

with Ile890 and Ile886 (Fig. 10).

For compound 20e, it exhibited binding energy of ꢀ 31.10 kcal/mol.

The amide group occupied the DFG binding domain and formed two

hydrogen bonds with Glu883 and Asp1044. the central phenyl ring

formed three hydrophobic bonds with Cys1043, Val897 and Val914. The

′

bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline moiety occupied the

hinge region forming two hydrogen bonds with Cys917 through its

hydrogen bond acceptor (nitrogen) atoms. This indicated the beneﬁcial

′

effect of bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline as heterocyclic

moiety. In addition, it formed nine hydrophobic bonds with Leu838,

Leu1033, Phe1045, Val846, and Ala864. The dimethylphenyl moiety

formed four hydrophobic interactions with Leu887, Ile886, Ile890, and

Val897. Additionally, it formed an electrostatic interaction with

Asp1044 (Fig. 11).

Fig. 6. The immunoblotting of caspase-9 and caspase-3 (normalized to β-actin).

All data from three separate experiments are shown as mean ± SEM. **p <

0

.01, ***p < 0.001.

Compound 20 h showed a ΔG value of ꢀ 30.07 kcal/mol. The HBD/

HBA (amide) moiety bound with Glu883 and Asp1044 forming two

Table 5

Effect of compound 20 h on levels of caspases-9 and caspases-3 in HepG2 cells.

a

Sample

Protein expression (normalized to β-actin)

Table 6

Caspases-9

Caspases-3

Effect of compound 20 h on levels of BAX, Bcl-2 proteins expression in HepG2

HepG2

1.00 ± 0.14

1.72 ± 0.09*

1.00 ± 0.09

2.07 ± 0.05

cells.

*

**

2

a

0 h /HepG2

a

Sample

Protein expression (normalized to β-actin)

Results are presented as mean ± SEM of three different experiments, where

BAX

Bcl-2

BAX/Bcl-2 ratio

*

p < 0.05, **p < 0.01, ***p < 0.001.

HepG2

1.00 ± 0.13

1.83 ± 0.11

1.00 ± 0.06

0.52 ± 0.01

1.00 ± 0.18

**

2

a

0 h / HepG2

3.45 ± 0.25

running of the docking procedure for only the co-crystallized ligand

against the active pocket. It was found that the produced RMSD value

between the generated pose of the docked molecule and the original one

Results are presented as mean ± SEM of three different experiments, where

*

p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7. The immunoblotting of BAX and Bcl-2 (normalized to β-actin). All data from three separate experiments are shown as mean ± SEM. *p < 0.05, **p < 0.01,

**p < 0.001.

*

1

0

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Fig. 8. Superimposition of the co-crystallized molecule (pink) and the docking pose (dark green) of the same molecule. (For interpretation of the references to colour

in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 9. A) 3D of sorafenib docked into the active site VEGFR-2. B) 2D of sorafenib docked into the active site VEGFR-2.

hydrogen bonds. In addition, the phenyl group occupied the spacer re-

gion and formed three hydrophobic bonds with Cys1043, Val897 and

hydrophobic interactions with Leu838, Leu1033, and Phe916. The 4-

methoxyphenyl moiety formed two hydrophobic interactions with

Leu887 and Ile886. Additionally, it formed an electrostatic interaction

with Asp1044 (Fig. 12).

′

Val914. The bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxaline moiety occu-

pied the hinge region forming two hydrogen bonds with Cys917 through

its hydrogen bond acceptor (nitrogen) atoms. Also, it formed ﬁve

Regarding Compound 20 m, it exhibited binding free energy of

1

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Fig. 10. A) 3D of compound 20b docked into the active site VEGFR-2. B) 2D of compound 20b docked into the active site VEGFR-2.

ꢀ

26.67 kcal/mol. The HBD/HBA (amide) moiety bound with Glu883

2.3.3. In silico toxicity studies

and Asp1044 forming two hydrogen bonds. In addition, the central

phenyl group formed three hydrophobic bonds with Cys1043, Val897

In this work, eight toxicity parameters were estimated computa-

tionally depending on the constructed toxicity models in Discovery

studio software [73,74]. These models include: carcinogenic potency

TD50 [75], rat maximum tolerated dose [76,77], developmental toxicity

potential [78,79], rat oral LD50 [80], ocular irritancy [81], and skin

irritancy [81].

′

and Val914. Furthermore, the bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]qui-

noxaline moiety occupied the hinge region and formed ﬁve hydrophobic

interactions with Phe1045, Leu838, and Phe916. Also, it formed elec-

trostatic interactions with Arg1049. The thiazole moiety formed one

hydrophobic bond with Leu887 (Fig. 13).

As shown in Table. 8, the calculated toxicity potential of the syn-

thesized compound was low. All compounds were non-carcinogenic for

rat female based on FDA rodent carcinogenicity model. In addition,

compounds 20a, 20 l, and 22a had carcinogenic potency TD50 values of

17.149, 40.080 and 20.144 mg/kg body weight/day, respectively,

which were higher than that of sorafenib (14.244 mg/kg body weight/

day). The other compounds had carcinogenic potency TD50 values less

than sorafenib. Compounds 20b, 20c, 20d, 20 l and 21 had rat

maximum tolerated dose values ranging from 0.090 to 0.211 g/kg body

weight, which were higher than that of sorafenib (0.089 g/kg body

weight). Moreover, all compounds were predicted to be non-toxic

against developmental toxicity potential model. For rat oral LD50

model, compounds 20a, 20c, 20d, 20 g, 20j-20 m, 21, 22a, and 22b

showed oral LD50 values ranging from 0.855 to 2.955 mg/kg body

weight/day which were higher than that of sorafenib (0.823 mg/kg

body weight/day). In addition, all compounds exhibited rat chronic

LOAEL values higher than that of sorafenib. Moreover, all the tested

compounds were predicted to be mild irritant against ocular irritancy

2

.3.2. In silico ADMET analysis

The ADMET parameters (blood brain barrier (BBB) penetration, in-

testinal absorption, aqueous solubility, CYP2D6 inhibition, and plasma

protein binding) were determined computationally using Discovery

studio 4.0. Sorafenib was used as a reference molecule (Fig. 14).

As shown in Table 7, the BBB penetration level of the tested com-

pounds were very low. So that such molecules may have mild CNS

adverse effects. For aqueous solubility, the tested compounds were

predicted to have low or very low levels. Furthermore, compounds 2a,

2

0 k, and 20 m were predicted to have moderate absorption levels,

while the other compounds were in the poor or very poor range. Addi-

tionally, all the tested compounds were predicted as non-inhibitors of

CYP2D6. Finally, all compounds were expected to bind plasma

protein<90%, except compound 20 l.

1

2

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Fig. 11. A) 3D of compound 20e docked into the active site VEGFR-2. B) 2D of compound 20e docked into the active site VEGFR-2.

model, and non-irritant against skin irritancy model. Table 9.

compound upregulated caspase-3, caspase-9, and BAX by 2.07-, 1.72-,

and 1.83-fold, respectively. In contrast, compound 20 h downregulated

Bcl-2 level by1.92-fold. Finally, the computational studies revealed that

our compounds have binding mode almost like that of sorafenib.

3

. Conclusion

In this work, sixteen compounds were designed and synthesized as

modiﬁed analogs of our previously prepared VEGFR-2 inhibitors.

Compounds 20b, 20e, 20 h and 20 m were the most active VEGFR-2

inhibitors with IC50 values of 5.7, 6.7, 3.2, and 3.1 µM, respectively.

SAR revealed that hydrazide moiety is more favorable than amide

moiety as HBD-HBA group, which is more advantageous than the

diamide moiety. Also, ﬁve-membered heterocyclic ring was more

favorable than unsubstituted six-membered non-heterocyclic ring as

hydrophobic tail. In addition, the substituted phenyl ring was more

beneﬁcial than the unsubstituted one for VEGFR-2 inhibition. Moreover,

compounds 20b (IC50 = 11.4 & 14.2 µM), 20e (IC50 = 10.7 & 13.7 µM),

4. Experimental

4.1. Chemistry

All the used chemicals, reagents, and different apparatus were

showed in Supplementary data.

Compounds 3, 4, 5, 6, 7, 8, 9, 11, 12, 15a,b and 16a,b were ob-

tained according to the reported procedures [33,47,56,57,82].

4.1.1. General procedure for synthesis of compounds 13a-m, 18, 19a,b.

A mixture of 4-(2-chloroacetamido)benzoyl chloride 12 (0.002 mol,

0.46 g) and appropriate amines namely, aniline, 2-methylaniline, 3-

methylaniline, 4-methylaniline, 2,6-dimethylaniline, 2-methoxyaniline,

2

0 h (IC50 = 3.3 & 4.4 µM) and 20 m (IC50 = 8.7 & 11.3 µM) were the

most cytotoxic members against HepG2 and MCF-7, respectively. The

most cytotoxic member 20 h arrested the cell cycle at G2/M phase and

induced apoptosis by 3.5-fold compared to the control. Also, this

3-methoxyaniline,

4-methoxyaniline,

2,6-dimethoxyaniline,

4-

1

3

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Fig. 12. A) 3D of compound 20 h docked into the active site VEGFR-2. B) 2D of compound 20 h docked into the active site VEGFR-2.

nitroaniline, benzylamine, 2-phenylethylamine, 2-aminothiazole, phe-

nylhydrazine, benzohydrazide, and 4-nitrobenzohydrazide (0.002 mol)

was stirred in acetonitrile (50 mL) in the presence of TEA (1 mL) for 8 h.

After completion of the reaction, it was quashed by addition of acidiﬁed

water (200 mL) with further ﬁltration and crystallization from ethanol

to produce the target compounds 13a-m,18, 19a,b, respectively.

4.1.1.3. 4-(2-Chloroacetamido)-N-(m-tolyl)benzamide 13c. Yellow crys-

◦

ꢀ 1

tals (yield 80%); mp: 177–179 C; FT-IR (v max, cm ): 3254 (NH), 3041

–

N); ¹H NMR (400 MHz,

(CH aromatic), 1682,1644 (C

DMSO‑d

–

O), 1609(C

–

6

) δ 10.61 (s, 1H), 10.06 (s, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.71

(d, J = 8.8 Hz, 2H), 7.59 (s, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.20 (t, J = 7.8

Hz, 1H), 6.89 (d, J = 7.5 Hz, 1H), 4.29 (s, 2H), 2.28 (s, 3H); Anal. Calcd.

for C16

2 2

H15ClN O (302.76): C, 63.48; H, 4.99; N, 9.25; Found: C63.72;

4

.1.1.1. 4-(2-Chloroacetamido)-N-phenylbenzamide 13a. White crystals

H, 4.56; N, 9.21%.

◦

ꢀ 1

(

yield 70%); mp: 178–180 C; FT-IR (v max, cm ): 3314 (NH), 2975

–

N); ¹H NMR (400 MHz,

(

CH aliphatic), 1666,1628 (C

) δ 10.63 (s, 1H), 10.16 (s, 1H), 7.95 (dd, J = 8.8, 3.3 Hz, 2H),

.78 – 7.70 (m, 4H), 7.33 (q, J = 5.7, 3.1 Hz, 2H), 7.11 – 7.05 (m, 1H),

.31 (s, 2H); ¹³C NMR (101 MHz, DMSO‑d

) δ 165.53, 165.30, 141.85,

39.67, 130.33, 129.43, 129.34, 129.31, 129.04, 128.66, 121.21,

20.40, 119.47, 118.64, 44.0; Anal. Calcd. for C15 13ClN (288.73):

O), 1606 (C

4.1.1.4. 4-(2-Chloroacetamido)-N-(p-tolyl)benzamide

13d. Yellowish

◦

ꢀ 1

DMSO‑d

6

white crystals (yield 82%); mp: 182–184 C; FT-IR (v max, cm ): 3309

–

O), 1599 (C N); H NMR

1

7

4

1

(NH), 2918 (CH aliphatic), 1668, 1643 (C

(400 MHz, DMSO‑d

) δ 10.71 (s, 1H), 10.08 (s, 1H), 7.94 (d, J = 8.7 Hz,

2H), 7.73 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.5

Hz, 3H), 4.31 (s, 2H), 2.25 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d

) δ

–

6

H

2

O

2

6

C, 62.40; H, 4.54; N, 9.70; Found: C, 62.21; H, 4.44; N, 9.44%.

165.52 (2C), 165.08 (2C), 141.79, 137.14, 132.93, 130.37, 129.87,

+

1

29.25, 128.97, 121.32, 120.36, 118.58, 44.03, 20.79; 303 (M + 1,

4

.1.1.2. 4-(2-Chloroacetamido)-N-(o-tolyl)benzamide 13b. Yellow crys-

2 2

10%); Anal. Calcd. for C16H15ClN O (302.76): C, 63.48; H, 4.99; N,

◦

ꢀ 1

tals (yield 75%); mp: 170–172 C; FT-IR (v max, cm ): 3265 (NH), 3065

9.25; Found: C, 63.83; H, 5.37; N, 9.42%.

–

N); Anal. Calcd. for

(

CH aromatic), 1676 (C

15ClN (302.76): C, 63.48; H, 4.99; N, 9.25; Found: C63.66; H,

.76; N, 9.03%.

–

O), 1601 (C

C

16

H

2

O

2

4.1.1.5. 4-(2-Chloroacetamido)-N-(2,6-dimethylphenyl)benzamide 13e.

◦

ꢀ 1

4

White crystals (yield 77%); mp: 150–152 C; FT-IR (v max, cm ): 3268,

1

4

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Fig. 13. A) 3D of compound 20 m docked into the active site VEGFR-2. B) 2D of compound 20 m docked into the active site VEGFR-2.

–

1

◦

3

113 (NH), 3048 (CH aromatic), 1690, 1645 (C

–

O), 1602 (C

–

N); H

Yellowish white crystals (yield 72%); mp: 186–188 C; FT-IR (v max,

ꢀ 1

NMR (400 MHz, DMSO‑d

6

) δ 10.64 (s, 1H), 9.68 (s, 1H), 7.98 (d, J = 8.5

cm ): 3324 (NH), 3004 (CH aromatic), 2959,2831 (CH aliphatic),

–

6

O), 1607 (C N); H NMR (400 MHz, DMSO‑d ) δ 10.60

1

Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.10 (m, 3H), 4.31 (s, 2H), 2.16 (s,

1666, 1649 (C

–

6

H).

¹³C NMR (101 MHz, DMSO‑d

(s, 1H), 10.12 (s, 1H), 7.94 (d, J = 8.7 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H),

6

) δ 165.49, 164.82 (2C), 141.77,

36.11, 129.85, 129.12 (2C), 128.86, 128.83, 128.59, 127.71, 127.51,

7.45 (t, J = 2.2 Hz, 1H), 7.35 (ddd, J = 8.1, 2.0, 0.9 Hz, 1H), 7.22 (t, J =

1

8.1 Hz, 1H), 6.65 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H), 4.29 (s, 2H), 3.23 (s,

+

13

18.71, 44.05, 18.71, 18.38; 317 (M + 1, 100%); Anal. Calcd. for

6

3H); C NMR (101 MHz, DMSO‑d ) δ 165.53, 165.31, 159.82, 141.85,

C

17

H

2

17ClN O

2

(316.79): C, 64.46; H, 5.41; N, 8.84; Found: C, 64.25; H,

140.90, 130.30, 129.31, 129.01, 119.47, 118.62, 113.38, 112.56,

5

.30; N, 8.50%.

109.25, 106.13, 55.36, 44.05; Anal. Calcd. for C16 15ClN (318.76):

H

2 3

O

C, 60.29; H, 4.74; N, 8.79; Found: C, 60.65; H, 4.75; N, 8.65%.

4

.1.1.6. 4-(2-Chloroacetamido)-N-(2-methoxyphenyl)benzamide 13f. Off

◦

ꢀ 1

white crystals (yield 70%); mp: 180–182 C; FT-IR (v max, cm ): 3422,

3

4.1.1.8. 4-(2-Chloroacetamido)-N-(4-methoxyphenyl)benzamide 13 h.

–

N);; ¹H NMR

◦

ꢀ 1

284, 3196, 3119 (NH), 1677, 1645 (C

–

O), 1603 (C

–

White crystals (yield 77%); mp: 177–179 C; FT-IR (v max, cm ): 3314

–

N); H

1

(

400 MHz, DMSO‑d

6

) δ 10.60 (s, 1H), 9.33 (s, 1H), 7.97 – 7.91 (m, 2H),

(NH), 1951, 2835 (CH aliphatic), 1671, 1639 (C

NMR (400 MHz, DMSO‑d

) δ 10.80 (s, 1H), 10.06 (s, 1H), 7.94 (d, J =

8.8 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 9.0 Hz, 3H), 6.90 (d, J

O), 1600 (C

–

7

1

.76 (dd, J = 7.9, 1.7 Hz, 1H), 7.73 – 7.66 (m, 2H), 7.15 (ddd, J = 8.2,

6

.3, 1.7 Hz, 1H), 7.06 (dd, J = 8.3, 1.4 Hz, 1H), 6.94 (td, J = 7.6, 1.4 Hz,

H), 4.29 (s, 2H), 3.80 (s, 3H); ¹³C NMR (101 MHz, DMSO‑d

) δ 165.52,

= 9.1 Hz, 2H), 4.32 (s, 2H), 3.72 (s, 3H); C NMR (101 MHz, DMSO‑d

13

6

)

64.73, 151.78, 141.88, 129.88, 129.11, 128.83, 127.31, 126.45,

20.86, 119.57, 118.73, 112.13, 111.39, 56.11, 44.05; Anal. Calcd. for

δ 165.53, 164.87, 155.91, 141.76, 132.73, 130.35, 129.17, 128.84,

124.47, 122.91, 121.99, 114.86, 114.30, 113.44, 55.55, 44.02; Anal.

C

16

H

2

15ClN O

3

(318.76): C, 60.29; H, 4.74; N, 8.79; Found: C, 60.65; H,

2 3

Calcd. for C16H15ClN O (318.76): C, 60.29; H, 4.74; N, 8.79; Found: C,

4

.70; N, 8.82%.

60.47; H, 5.02; N, 8.89%.

4

.1.1.7. 4-(2-Chloroacetamido)-N-(3-methoxyphenyl)benzamide 13 g.

4.1.1.9. 4-(2-Chloroacetamido)-N-(2,6-dimethoxyphenyl)benzamide 13i.

1

5

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Fig. 14. computational prediction of ADMET parameters for the synthesized compounds and sorafenib.

◦

ꢀ 1

Yellow crystals (yield 82%); mp: 172–173 C; FT-IR (v max, cm ):

Table 7

–

3

428, 3289 (NH), 3062 (CH aromatic), 1677, 1646 (C

O), 1603

–

6

C N); H NMR (400 MHz, DMSO‑d ) δ 10.65 (s, 1H), 9.40 (s, 1H), 8.03

Binding free energies (ΔG in Kcal/mol) of the synthesized compounds and sor-

afenib against VEGFR-2.

1

(

–

1

7.94 (m, 2H), 7.94 – 7.88 (m, 1H), 7.75 (d, J = 7.5 Hz, 2H), 7.38 (s,

Comp. No.

ΔG [Kcal/mol]

Compound

ΔG [Kcal/mol]

13

H), 7.33 (d, J = 8.4 Hz, 1H), 4.32 (s, 2H), 3.96 (s, 6H) ; C NMR (101

2

0a

0b

0c

0d

0e

0f

ꢀ 29.03

ꢀ 29.98

ꢀ 30.75

ꢀ 29.84

ꢀ 31.10

ꢀ 30.21

ꢀ 30.25

ꢀ 30.07

ꢀ 31.51

20j

ꢀ 29.91

ꢀ 30.01

ꢀ 29.33

ꢀ 26.67

ꢀ 30.91

ꢀ 29.46

ꢀ 31.14

ꢀ 26.50

6

MHz, DMSO‑d ) δ 165.55, 164.74, 151.89, 141.97, 137.73, 129.83,

20 k

20 l

1

29.16, 128.93, 126.80, 119.55, 119.37, 119.14, 118.76, 109.72, 56.44,

5

6.39, 44.08; Anal. Calcd. for C17 17ClN (348.78): C, 58.54; H, 4.91;

H

2 4

O

20 m

21

N, 8.03; Found: C, 58.34; H, 4.77; N, 7.88%.

22a

0 g

0 h

0i

22b

4

.1.1.10. 4-(2-Chloroacetamido)-N-(4-nitrophenyl)benzamide 13j. Or-

Sorafenib

◦

ꢀ 1

ange crystals (yield 72%); mp: 188–190 C; FT-IR (v max, cm ): 3331

–

6

N); H NMR (400 MHz, DMSO‑d ) δ

1

(

NH), 1770, 1681 (C

–

O), 1597 (C

–

1

7

0.71 (s, 1H), 10.64 (s, 1H), 8.27 – 8.22 (m, 2H), 8.07 – 8.02 (m, 2H),

1

3

.99 – 7.95 (m, 2H), 7.78 – 7.71 (m, 2H), 4.30 (s, 2H); C NMR (101

) δ 167.29, 165.97, 165.61, 146.06, 142.79, 142.41,

29.70, 129.46, 129.42, 125.35, 125.14, 120.35, 120.11, 118.67, 44.05;

Anal. Calcd. for C15 12ClN (333.73): C, 53.99; H, 3.62; N, 12.59;

Found: C, 53.75; H, 3.49; N, 12.47%.

Table 8

ADMET parameters for the synthesized compounds and sorafenib.

MHz, DMSO‑d

6

1

Comp.

BBB

Solubility

Absorption

CYP2D6

PPB

H

3 4

O

a

b

c

d

e

level

prediction

2

0a

0b

0c

0d

0e

0f

4

1

2

1

2

1

2

3

1

2

1

2

3

0

FALSE

TRUE

4

.1.1.11. N-Benzyl-4-(2-chloroacetamido)benzamide 13 k. White crys-

tals (yield 80%); mp: 181–183 ^◦C; FT-IR (v max, cm ): 3188, 3106

ꢀ 1

–

(NH), 3054 (CH aromatic), 2942 (CH aliphatic), 1702, 1634 (C

O),

(302.76): C, 63.48; H, 4.99;

N, 9.25; Found: C, 63.22; H, 4.78; N, 9.11%.

–

1

603 (C N); Anal. Calcd. for C16H15ClN O

–

2 2

0 g

0 h

0i

0j

4.1.1.12. 4-(2-Chloroacetamido)-N-phenethylbenzamide 13 l. White

◦

ꢀ 1

0 K

0l

crystals (yield 85%); mp: 177–179 C; FT-IR (v max, cm ): 3314 (NH),

2

–

N); H NMR (400

1

975 (CH aliphatic), 1666, 1628 (C

–

O), 1606 (C

–

0 m

1

FALSE

TRUE

MHz, DMSO‑d

6

) δ 10.84 (s, 1H), 8.55 (t, J = 5.6 Hz, 1H), 8.13 (s, 2H),

7

.83 – 7.79 (m, 2H), 7.71 – 7.66 (m, 2H), 7.22 (d, J = 1.6 Hz, 3H), 4.32

2a

2b

13

(s, 2H), 3.02 – 2.97 (m, 2H), 2.82 (dd, J = 8.4, 6.5 Hz, 2H); C NMR

Sorafenib

(

101 MHz, DMSO‑d ) δ 166.02, 165.48, 141.50, 140.01, 130.06, 129.55,

6

a

129.51, 129.25, 129.21, 128.63, 128.59, 128.30, 119.42, 118.49, 44.00,

BBB means blood brain barrier which may be very high (0), high (1), me-

dium (2), low (3), or very low (4).

+

41.31, 35.61; 317 (M + 1, 20%); Anal. Calcd. for C17

2 2

H17ClN O

b

Solubility level may be very low (1), low (2), good (3), or optimal (4).

(316.79): C, 64.46; H, 5.41; N, 8.84; Found: C, 64.28; H, 5.22; N, 8.69%.

c

Absorption level may be good (0), moderate (1), poor (2), or very poor (3).

d

CYP2D6 means cytochrome P2D6 which may be inhibitor (TRUE) or non-

4

.1.1.13. 4-(2-Chloroacetamido)-N-(thiazol-2-yl)benzamide 13 m. Yel-

inhibitor (FALSE).

e

◦

ꢀ 1

low crystals (yield 70%); mp: 160–162 C; FT-IR (v max, cm ): 3315

PBB means plasma protein binding which may be<90% (FALSE) or more

1

–

6

N); H NMR (400 MHz, DMSO‑d ) δ

(

NH), 1691, 1645 (C

–

O), 1601 (C

–

than 90% (TRUE)

1

2.54 (s, 1H), 10.72 (s, 1H), 8.08 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.8 Hz,

1

6

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

Table 9

In silico toxicity studies of the synthesized compounds and sorafenib.

Comp.

FDA Rodent

Carcinogenic Potency

Rat Maximum

Tolerated Dose

Developmental

Rat Oral

Rat Chronic

Ocular

Skin

a

b

Carcinogenicity (Rat-

Female)

TD50 (Rat)

Toxicity Potential

LD50

LOAEL

Irritancy

b

(Feed)

2

0a

0b

0c

0d

0e

0f

Non-Carcinogen

Single-Carcinogen

17.149

6.070

5.840

5.369

1.239

2.067

2.081

3.559

11.459

40.080

8.492

12.530

20.144

3.995

14.244

0.110

0.090

0.085

0.061

0.058

0.080

0.082

0.095

0.074

0.211

0.119

0.086

0.089

Non-Toxic

Toxic

0.855

0.589

1.993

2.815

0.596

0.397

1.071

0.685

0.293

1.566

1.390

1.613

2.848

2.085

1.680

2.955

0.823

0.091

0.104

0.098

0.104

0.072

0.051

0.033

0.043

0.049

0.080

0.085

0.118

0.166

0.158

0.142

0.119

0.005

Mild

Non-

Irritant

Non-

Irritant

Non-

Irritant

Non-

Irritant

Non-

Irritant

Non-

Irritant

Non-

0 g

0 h

0i

Irritant

Non-

Irritant

Non-

Irritant

Non-

0j

Irritant

Non-

0k

0l

Irritant

Non-

Irritant

Non-

0 m

1

Irritant

Non-

Irritant

Non-

2a

2b

Irritant

Non-

Irritant

Non-

Sorafenib

Irritant

a

Unit: mg/kg body weight/day

Unit: g/kg body weight

b

2

H), 7.54 (s, J = 7.0 Hz, 1H), 7.26 (s, J = 7.0 Hz, 1H), 4.32 (s, 2H); Anal.

Calcd. for C12 10ClN S (295.74): C, 48.74; H, 3.41; N, 14.21; Found:

C, 49.06; H, 3.61; N, 14.32%.

8.6 Hz, 2H), 4.31 (s, 2H); Anal. Calcd. for C16

H

13ClN

4

O

5

(376.75): C,

H

3

O

2

51.01; H, 3.48; N, 14.87; Found: C, 50.86; H, 3.40; N, 14.79%.

4

.1.2. General procedure for synthesis of compounds 20a-m, 21, and 22a,b

′ ′

A mixture of potassium salt of bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]qui-

4

.1.1.14. 2-Chloro-N-(4-(2-phenylhydrazine-1-carbonyl)phenyl)acet-

◦

amide 18. Yellowish white crystals (yield 72%); mp: 166–168 C; FT-IR

noxaline-3-thiol 9 (0.5 g, 0.001 mol) and the prepared intermediates

13a-m, 18, and 19a,b (0.001 mol) in DMF (30 mL) was heated on a

water bath for 8 h. After cooling to room temperature, reaction mixture

was poured on crushed ice. The obtained precipitates were collected by

ﬁltration, dried and crystalized from ethanol to give the target com-

pounds 20a-m, 21, and 22a,b, respectively.

ꢀ 1

–

N); H NMR

1

(

v max, cm ): 3322 (NH), 1681, 1640 (C

400 MHz, DMSO‑d

) δ 10.62 (s, 1H), 10.31 – 10.25 (m, 1H), 7.91 – 7.87

m, 2H), 7.69 (dd, J = 8.7, 3.0 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 6.78 –

.74 (m, 2H), 6.69 (d, J = 9.6 Hz, 2H), 4.30 (s, 2H); Anal. Calcd. for

14ClN (303.75): C, 59.31; H, 4.65; N, 13.83; Found: C, 59.09; H,

O), 1600 (C

–

6

C

15

H

3 2

O

4

.50; N, 13.69%.

′

4

.1.2.1. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

4

.1.1.15. N-(4-(2-benzoylhydrazine-1-carbonyl)phenyl)-2-chlor-

acetamido)-N-phenylbenzamide (20a).. White crystal (yield, 68%); m.

◦

ꢀ 1

oacetamide 19a. White crystals (yield 77%); mp: 185–187 C; FT-IR (v

p. = 220–222 C. FT-IR (v max, cm ): 3273, 3118 (NH), 1702, 1648

ꢀ

1

–

1

–

N); ¹H NMR (700 MHz, DMSO‑d

max, cm ): 3280 (NH), 1676, 1644 (C

MHz, DMSO‑d

) δ 10.65 (s, 1H), 10.48 (s, 1H), 10.44 (s, 1H), 7.93 – 7.90

m, 4H), 7.72 (dd, J = 8.0, 3.1 Hz, 2H), 7.60 – 7.55 (m, 1H), 7.52 (d, J =

O), 1603 (C

N); H NMR (400

(C

–

O), 1597 (C

–

6

) δ 10.77 (s, 1H),

10.14 (s, 1H), 10.02 (s, 1H), 8.64 – 8.58 (m, 1H), 8.47 (dd, J = 7.8, 1.7

6

(

Hz, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.79 – 7.71 (m, 6H), 7.35 (t, J = 7.8

1

3

13

8

1

.1 Hz, 2H), 4.31 (s, 2H); C NMR (101 MHz, DMSO‑d

65.72, 165.58, 142.08, 133.00, 129.53, 129.27, 129.05, 127.99 (2C),

27.77, 119.56, 119.11, 118.75, 44.06; Anal. Calcd. for C16 14ClN

6

) δ 166.35 (2C),

Hz, 2H), 7.09 (t, J = 7.3 Hz, 1H), 4.60 (s, 2H); C NMR (176 MHz,

DMSO‑d ) δ 166.03, 165.28, 147.72, 142.13, 142.12, 139.72, 139.34,

6

H

3

O

3

138.78, 130.10, 129.20 (2C), 129.04 (2C), 128.37, 128.35, 124.08,

(

331.76): C, 57.93; H, 4.25; N, 12.67; Found: C, 57.77; H, 4.05; N,

123.98, 123.19, 120.79 (2C), 118.83 (2C), 118.59, 118.06, 38.91; MS

+

1

2.49%.

(m/z): 495 (M + 1, 30%), 268 (55%); Anal. Calcd. for C25

18 8 2

H N O S

(

494.53): C, 60.72; H, 3.67; N, 22.66. Found: C, 60.43; H, 3.55; N,

2.43%.

2

4

.1.1.16. 2-chloro-N-(4-(2-(4-nitrobenzoyl)hydrazine-1-carbonyl)phenyl)

◦

acetamide 19b. Red crystals (yield 75%); mp: 192–194 C; FT-IR (v max,

ꢀ

1

–

1

′

cm ): 3276 (NH), 1678, 1648 (C

DMSO‑d

) δ 10.86 (s, 1H), 10.69 (s, 1H), 10.58 (s, 1H), 8.37 (d, J = 7.8

Hz, 2H), 8.14 (d, J = 6.0 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 7.74 (d, J =

O), 1602 (C

N); H NMR (400 MHz,

4.1.2.2. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

acetamido)-N-(o-tolyl)benzamide (20b). Faint yellow crystal (yield,

6

◦

ꢀ 1

70%); m.p. = 240–242 C. FT-IR (v max, cm ): 3273, 3111 (NH), 1667,

1

7

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

–

N); ¹H NMR (700 MHz, DMSO‑d

–

1

7

644 (C

–

O), 1598 (C

6

) δ 10.77 (s,

127.39, 125.98, 124.53, 124.11, 123.22, 120.67, 118.94 (2C), 118.59,

+

H), 10.12 (s, 1H), 10.05 (s, 1H), 8.35 (dd, J = 8.1, 1.4 Hz, 1H), 7.99 –

118.08, 111.80, 56.19, 38.92; MS (m/z): 525 (M + 1, 20%); Anal.

.93 (m, 2H), 7.87 (dd, J = 8.1, 1.5 Hz, 1H), 7.80 – 7.75 (m, 2H), 7.68

20 8 3

Calcd. for C26H N O S (524.56): C, 59.53; H, 3.84; N, 21.36 Found: C,

(

ddd, J = 8.2, 7.3, 1.5 Hz, 1H), 7.66 – 7.61 (m, 3H), 7.14 (d, J = 8.4 Hz,

59.36; H, 3.66; N, 21.21%.

2

1

H), 4.43 (s, 2H), 2.28 (s, 3H); ¹³C NMR (176 MHz, DMSO‑d

6

) δ 166.79,

′

65.14, 151.65, 142.36, 141.86, 138.30, 136.99, 135.60, 134.13,

30.75 (2C), 129.63, 129.16 (2C), 128.73, 128.34, 127.03, 126.44 (2C),

26.33, 124.07, 118.88 (2C), 117.13, 34.51, 18.41; Anal. Calcd. for

4.1.2.7. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

acetamido)-N-(3-methoxyphenyl)benzamide (20 g). Yellow crystal

◦

ꢀ 1

(yield, 68%); m.p. = 234–236 C. FT-IR (v max, cm ): 3428, 3109

1

C

26

H

20

8

N O

2

S (508.56): C, 61.41; H, 3.96; N, 22.03. Found: C, 61.22; H,

(NH), 1649 (C

(s, 1H), 10.11 (s, 1H), 10.03 (s, 1H), 8.62 (dd, J = 8.0, 1.5 Hz, 1H), 8.48

dd, J = 7.9, 1.8 Hz, 1H), 7.98 – 7.93 (m, 2H), 7.80 – 7.70 (m, 4H), 7.47

(t, J = 2.3 Hz, 1H), 7.39 – 7.34 (m, 1H), 7.25 (t, J = 8.1 Hz, 1H), 6.68

–

O), 1598 (C N); H NMR (700 MHz, DMSO‑d ) δ 10.77

6

3

.89; N, 21.85%.

(

′ ′

.1.2.3. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

4

1

3

acetamido)-N-(m-tolyl)benzamide (20c). Yellow crystal (yield, 65%);

(ddd, J = 8.2, 2.5, 0.9 Hz, 1H), 4.59 (s, 2H), 3.76 (s, 3H); C NMR (176

◦

ꢀ 1

m.p. = 246–248 C. FT-IR (v max, cm ): 3271, 3127 (NH), 2904 (CH

MHz, DMSO‑d ) δ 166.04, 165.30, 159.87, 147.71, 142.15, 142.14,

6

–

N); ¹H NMR (700 MHz,

–

aliphatic), 1702, 1648 (C

DMSO‑d

) δ 10.76 (s, 1H), 10.06 (s, 1H), 10.03 (s, 1H), 8.62 (d, J = 8.1

Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.75 (dt, J =

6.3, 8.1 Hz, 4H), 7.62 (s, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.23 (t, J = 7.8

O), 1606 (C

140.92, 139.36, 138.78, 130.08, 129.82, 129.20 (2C), 128.38, 128.36,

124.10, 123.22, 118.83 (2C), 118.60, 118.07, 112.97, 109.49, 106.44,

6

+

55.46, 38.89; MS (m/z): 525 (M + 1, 50%); Anal. Calcd. for

2

26 20 8 3

C H N O S (524.56): C, 59.53; H, 3.84; N, 21.36 Found: C, 59.22; H,

1

3

Hz, 1H), 6.92 (d, J = 7.7 Hz, 1H), 4.59 (s, 2H), 2.31 (s, 3H); C NMR

176 MHz, DMSO‑d ) δ 166.03, 165.20, 147.71, 142.13, 142.09, 139.63,

39.36, 138.78, 138.16, 130.15, 129.17 (2C), 128.88, 128.37, 124.68,

3.66; N, 21.11%.

(

6

′

1

3

4.1.2.8. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

acetamido)-N-(4-methoxyphenyl)benzamide (20 h). White crystal

24.10, 123.21, 121.33 (2C), 118.83 (2C), 118.59, 118.07, 117.98,

+

ꢀ 1

(yield, 78%); m.p. = 238–240 ^◦C. FT-IR (v max, cm ): 3274, 3125

8.90, 21.71; MS (m/z): 509 (M + 1, 40%); Anal. Calcd. for

1

C

26

H

20

8

N O

2

S (508.56): C, 61.41; H, 3.96; N, 22.03. Found: C, 61.28; H,

(NH), 2904 (CH aliphatic), 1702, 1645 (C

–

O) , 1608 (C

–

N); H NMR

3

.79; N, 21.85%.

(700 MHz, DMSO‑d ) δ 10.75 (s, 1H), 10.02 (s, 2H), 8.61 (d, J = 8.0 Hz,

6

1

H), 8.49 – 8.45 (m, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.78 – 7.70 (m, 4H),

7.66 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 4.59 (s, 2H), 3.75 (s,

3H); ¹³C NMR (176 MHz, DMSO‑d

) δ 166.00, 164.85, 155.92, 147.72,

′

4

.1.2.4. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

acetamido)-N-(p-tolyl)benzamide (20d). Yellowish white crystal

6

ꢀ 1

(

yield, 75%); m.p. = 250–252 ^◦C. FT-IR (v max, cm ): 3271, 3128

142.12, 141.97, 139.35, 138.78, 132.76, 130.20, 129.05 (2C), 128.37,

NH), 2904 (CH aliphatic), 1699, 1646 (C^–

–

O), 1607 (C N); H NMR

) δ 10.76 (s, 1H), 10.07 (s, 1H), 10.03 (s, 1H), 8.62

1

128.35, 124.09, 123.20, 122.40 (2C), 118.82 (2C), 118.59, 118.06,

–

+

700 MHz, DMSO‑d

dd, J = 8.1, 1.5 Hz, 1H), 8.48 (dd, J = 7.7, 1.8 Hz, 1H), 7.97 – 7.93 (m,

H), 7.77 (ddd, J = 9.4, 7.6, 1.6 Hz, 2H), 7.73 – 7.71 (m, 2H), 7.67 – 7.63

6

114.17 (2C), 55.64, 38.90; MS (m/z): 525 (M + 1, 45%); Anal. Calcd.

20 8 3

for C26H N O S (524.56): C, 59.53; H, 3.84; N, 21.36 Found: C, 59.26;

2

H, 3.99; N, 21.56%.

1

3

(

m, 2H), 7.15 (d, J = 8.2 Hz, 2H), 4.59 (s, 2H), 2.29 (s, 3H); C NMR

176 MHz, DMSO‑d ) δ 166.02, 165.07, 147.71, 142.15, 142.04, 139.37,

38.79, 137.18, 132.92, 130.19, 129.44 (2C), 129.13 (2C), 128.38 (2C),

′

(

6

4.1.2.9. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

acetamido)-N-(2,6-dimethoxyphenyl)benzamide (20i). Off white

1

2

◦

1

ꢀ 1

24.12, 123.23, 120.82 (2C), 118.82 (2C), 118.60, 118.08, 38.89,

crystal (yield, 70%); m.p. = 250–252 C. FT-IR (v max, cm ): 3418,

+

0.97; MS (m/z): 509 (M + 1, 35%); Anal. Calcd. for C26

H

20

8

N O

2

S

3108 (NH), 1662 (C

–

O), 1600 (C N); H NMR (700 MHz, DMSO‑d ) δ

6

(

508.56): C, 61.41; H, 3.96; N, 22.03. Found: C, 61.32; H, 3.75; N,

10.79 (s, 1H), 10.02 (s, 1H), 9.38 (s, 1H), 8.62 (d, J = 8.1 Hz, 1H), 8.47

(d, J = 7.9 Hz, 1H), 7.99 – 7.92 (m, 4H), 7.76 – 7.72 (m, 3H), 7.34 (d, J

2

2.25%.

.1.2.5. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

=

8.5 Hz, 2H), 4.60 (s, 2H), 3.98 (s, 6H); ¹³C NMR (176 MHz, DMSO‑d

)

6

′

4

δ 166.06, 164.75, 151.85, 147.73, 142.13, 139.36, 138.78, 137.69,

129.05 (2C), 128.38 (2C), 126.85, 124.37, 124.10, 123.21, 118.99 (2C),

118.97 (2C), 118.59, 118.09, 110.10 (2C), 56.46 (2C), 38.92 ; Anal.

acetamido)-N-(2,6-dimethylphenyl)benzamide (20e). White crystal

◦

ꢀ 1

(

yield, 80%); m.p. = 230–232 C. FT-IR (v max, cm ): 3261, 3114

–

1

NH), 1644 (C

O), 1602 (C

N); H NMR (700 MHz, DMSO‑d

6

) δ 10.76

22 8 4

Calcd. for C27H N O S (554.59): C, 58.48; H, 4.00; N, 20.21. Found: C,

s, 1H), 10.02 (s, 1H), 9.68 (s, 1H), 8.61 (d, J = 8.1 Hz, 1H), 8.46 (d, J =

58.19; H, 3.80; N, 20.01%.

7

1

.9 Hz, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.75 (td, J = 12.4, 9.3, 5.4 Hz, 4H),

.12 (s, 3H), 4.60 (s, 2H), 2.19 (s, 6H); ¹³C NMR (176 MHz, DMSO‑d

) δ

′

6

4.1.2.10. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-

ylthio)acetamido)-N-(4-nitrophenyl)benzamide (20j). Reddish crys-

66.00, 164.85, 147.73, 142.10, 142.02, 139.33, 138.78, 136.14 (2C),

35.89, 129.67, 129.01 (2C), 128.36, 128.34, 128.16 (2C), 127.07,

◦

ꢀ 1

tal (yield, 67%); m.p. = 260–262 C. FT-IR (v max, cm ): 3302 (NH),

1

24.07, 123.18, 118.95 (2C), 118.57, 118.05, 38.92, 18.57 (2C); MS (m/

3092 (CH aromatic), 1656 (C

–

O), 1597 (C N); H NMR (700 MHz,

–

+

z): 523 (M + 1, 100%); Anal. Calcd. for C27

H

22

N

8

O

2

S (522.59): C,

DMSO‑d ) δ 10.71 (s, 1H), 10.31 (s, 1H), 10.02 (s, 1H), 8.32 (d, J = 8.1

6

2.06; H, 4.24; N, 21.44 Found: C, 61.80; H, 4.09; N, 21.25%.

Hz, 1H), 7.97 (dt, J = 6.8, 2.1 Hz, 3H), 7.73 (dd, J = 8.8, 2.1 Hz, 2H),

7

.70 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.58 (t, J = 7.9 Hz,

′

4

.1.2.6. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)

1H), 7.49 – 7.45 (m, 1H), 7.38 (td, J = 8.1, 2.0 Hz, 1H), 7.15 (d, J = 7.9

acetamido)-N-(2-methoxyphenyl)benzamide (20f). Orange crystal

Hz, 1H), 5.23 (s, 2H); ¹³C NMR (176 MHz, DMSO‑d

6

) δ 166.91, 166.01,

ꢀ 1

(

yield, 74%); m.p. = 242–246 ^◦C. FT-IR (v max, cm ): 3422, 3116

151.64, 146.12, 142.98, 142.78, 141.87, 138.33, 135.62, 130.96,

130.57, 129.60 (2C), 129.10, 128.78, 128.36 (2C), 125.26 (2C), 124.12,

–

NH), 3035 (CH aromatic), 2938(CH aliphatic), 1660 (C O) , 1603

–

1

C

N); H NMR (700 MHz, DMSO‑d

6

) δ 10.77 (s, 1H), 10.03 (s, 1H),

17 9 4

120.20 (2C), 118.88, 117.18, 34.49; Anal. Calcd. For C25H N O S

9

1

7

8

.32 (s, 1H), 8.62 (dd, J = 8.1, 1.5 Hz, 1H), 8.48 (dd, J = 7.9, 1.7 Hz,

H), 7.97 – 7.94 (m, 2H), 7.78 (dddd, J = 18.4, 10.4, 7.8, 1.6 Hz, 3H),

.74 – 7.71 (m, 2H), 7.18 (ddd, J = 8.9, 7.5, 1.7 Hz, 1H), 7.10 (dd, J =

.3, 1.3 Hz, 1H), 6.97 (td, J = 7.6, 1.3 Hz, 1H), 4.60 (s, 2H), 3.85 (s, 3H);

(539.53): C, 55.65; H, 3.18; N, 23.37. Found: C, 55.43; H, 3.29; N,

23.14%.

′

4.1.2.11. N-Benzyl-4-(2-(bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinox-

alin-3-ylthio) acetamido)benzamide (20 k). White crystal (yield,

1

3

C NMR (176 MHz, DMSO‑d ) δ 166.04, 164.72, 151.78, 147.71,

6

◦

ꢀ 1

1

42.17, 142.14, 139.36, 138.78, 129.68, 129.00 (2C), 128.38, 128.36,

68%); m.p. = 255–257 C. FT-IR (v max, cm ): 3265, 3127 (NH), 1698,

1

8

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

–

N); ¹H NMR (700 MHz, DMSO‑d

′

1

2

6

644 (C

–

O), 1606 (C

–

6

) δ 10.71 (s,

4.1.2.16. 2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)-

H), 10.02 (s, 1H), 8.95 (t, J = 6.1 Hz, 1H), 8.61 (dd, J = 8.0, 1.6 Hz,

H), 8.47 (dd, J = 7.8, 1.8 Hz, 1H), 7.90 – 7.87 (m, 2H), 7.78 – 7.73 (m,

H), 7.67 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 6.4 Hz, 4H), 7.25 (td, J = 6.4,

N-(4-(2-(4-nitrobenzoyl)hydrazine-1-carbonyl)phenyl)acetamide

◦

(22b). Faint yellow crystal (yield, 68%); m.p. = 277–279 C. FT-IR (v

ꢀ 1

–

6

max, cm ): 3402 (NH), 1602 (C N); H NMR (700 MHz, DMSO‑d ) δ

1

–

1

3

.0, 2.8 Hz, 1H), 4.58 (s, 2H), 4.48 (d, J = 5.9 Hz, 2H); C NMR (176

11.97 (s, 1H), 10.80 (s, 1H), 10.67 (s, 1H), 10.60 (s, 1H), 10.12 (s, 1H),

8.35 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H), 7.94 (d, J = 2.7 Hz,

1H), 7.86 (dd, J = 7.9, 1.4 Hz, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.69 – 7.66

(m, 1H), 7.65 – 7.62 (m, 1H), 7.47 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 6.99

MHz, DMSO‑d

6

) δ 166.06, 165.94, 147.71, 142.13, 141.80, 140.25,

1

4

39.36, 138.78, 129.68, 128.73 (2C), 128.71 (2C), 128.37, 128.36 ,

27.68 (2C), 127.17, 124.10, 123.22, 118.82 (2C), 118.59, 118.07,

1

3

3.03, 38.89.

(d, J = 8.2 Hz, 1H), 6.96 (t, J = 7.5 Hz, 1H), 4.44 (s, 2H); C NMR (176

+

;

MS (m/z): 509 (M + 1, 75%); Anal. Calcd. For C26

H

20

8

N O

2

S

6

MHz, DMSO‑d ) δ 166.09, 165.67, 164.99, 147.71, 142.40, 142.13,

(

508.56): C, 61.41; H, 3.96; N, 22.03. Found: C, 61.29; H, 3.66; N,

139.35, 138.78, 135.00, 133.84, 132.23, 131.11, 129.03 (2C), 128.38,

2

2.26%.

.1.2.12. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-

128.36, 127.72 (2C), 127.66, 126.66, 124.09, 123.21, 118.96, 118.59,

+

1

18.07, 38.92; MS (m/z): 583 (M + 1, 100%); Anal. Calcd. for

′

4

C

26

H

18

N

10

O

5

S (582.56): C, 53.61; H, 3.11; N, 24.04. Found: C, 53.44; H,

ylthio)acetamido)-N-phenethylbenzamide (20 l). White crystal (yield,

3.01; N, 23.88%.

◦

ꢀ 1

7

1%); m.p. = 248–250 C. FT-IR (v max, cm ): 3309 (NH), 1673

–

6

N); H NMR (700 MHz, DMSO‑d ) δ 10.69 (s, 1H),

1

(

C

O), 1627 (C

–

4

.2. Biological testing

1

0.02 (s, 1H), 8.62 (dd, J = 7.9, 1.6 Hz, 1H), 8.51 – 8.45 (m, 2H), 7.81

(

d, J = 8.5 Hz, 2H), 7.79 – 7.73 (m, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.30 (t,

.2.1. In vitro VEGFR-2 kinase assay

J = 7.5 Hz, 2H), 7.25 (d, J = 7.5 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H), 4.57 (s,

VEGFR-2inhibitory activity of the synthesized compounds were

1

3

2

H), 3.47 (dt, J = 8.1, 6.0 Hz, 2H), 2.85 (t, J = 7.5 Hz, 2H); C NMR

tested using VEGFR-2 ELISA kit according to the reported method

(

176 MHz, DMSO‑d ) δ 165.99, 165.92, 147.72, 142.13, 141.66, 140.04,

6

described in Supplementary data [83].

1

3

39.36, 138.78, 129.94, 129.12 (2C), 128.81 (2C), 128.55 (2C), 128.38,

28.36, 126.55, 124.11, 123.22, 118.79 (2C), 118.59, 118.08, 41.33,

4

.2.2. In vitro anti-proliferative activity

+

8.88, 35.65; MS (m/z): 523 (M + 1, 50%); Anal. Calcd. For

Anti-proliferative activity activities of the synthesized compounds

27

C H22N8O2S (522.59): C, 62.06; H, 4.24; N, 21.44. Found: C, 61.88; H,

were assessed against MCF-7 and HepG-2 using MTT assay protocol

61,84–86] as described in Supplementary data.

4

.01; N, 21.35%.

[

′

4

.1.2.13. 4-(2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-

4

.2.3. Flow cytometry analysis for cell cycle

ylthio)acetamido)-N-(thiazol-2-yl)benzamide (20 m). Greenish white

Cell cycle analysis was performed using propidium iodide (PI)

◦

ꢀ 1

crystal (yield, 70%); m.p. = 244–246 C. FT-IR (v max, cm ): 3413

staining and ﬂow cytometry analysis for compound 20 h as described in

Supplementary data [87,88].

–

6

N); H NMR (700 MHz, DMSO‑d ) δ 12.50

1

(

NH), 1679 (C

s, 1H), 10.91 (s, 1H), 10.80 (s, 1H), 8.09 (m, J = 2.0 Hz, 2H), 7.78 (m, J

2.0 Hz, 2H), 7.56 (m, J = 3.6 Hz, 2H), 7.27 (m, J = 3.6 Hz, 2H), 7.05 –

.04 (m, 1H), 7.03 – 7.00 (m, 1H), 4.33 (s, 2H); Anal. Calcd. For

(501.54): C, 52.69; H, 3.01; N, 25.14. Found: C, 52.49; H,

.16; N, 25.35%.

O), 1601 (C

–

=

4

.2.4. Flow cytometry analysis for apoptosis

7

Flow cytometry cell apoptosis analysis was used to investigate the

22 15 9 2 2

C H N O S

apoptotic effect of the compound 20 h as described in Supplementary

data [89,90].

3

′ ′

.1.2.14. 2-(Bis([1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)-

4

.2.5. Western blot analysis

N-(4-(2-phenyl hydrazine-1-carbonyl)phenyl)acetamide (21). Off

The effect of compound 20 h on the expression of cleaved caspase-3,

◦

ꢀ 1

white (yield, 65%); m.p. = 225–227 C. FT-IR (v max, cm ): 3263,

caspase-9, BAX, and Bcl-2 were determined using Western blot analysis

–

6

O), 1599 (C N); H NMR (700 MHz, DMSO‑d ) δ

1

3

1

7

1

108 (NH), 1673 (C

–

as described in Supplementary data [67–69].

0.74 (s, 1H), 10.02 (s, 2H), 8.60 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.6 Hz,

H), 7.95 – 7.89 (m, 4H), 7.67 (d, J = 8.6 Hz, 2H), 7.55 – 7.50 (m, 1H),

.16 (t, J = 7.6 Hz, 1H), 6.78 (dd, J = 21.7, 11.7 Hz, 2H), 6.73 (q, J =

4

2

.3. In silico studies

.3, 6.6 Hz, 1H), 4.58 (s, 2H); ¹³C NMR (176 MHz, DMSO‑d

) δ 166.24,

6

.3.1. Docking studies

65.98, 147.71, 142.08, 139.32, 138.78 (2C), 130.80, 130.26, 129.20,

Crystal structure of VEGFR-2 [PDB ID: PDB ID: 2OH4, resolution:

.05 Å] was obtained from Protein Data Bank. The docking investigation

28.79, 128.34 (2C), 124.05, 123.63, 123.16, 119.05, 118.97, 118.76,

18.57 (2C), 118.05 (2C), 112.79, 38.91; Anal. Calcd. For C25

19 9 2

H N O S

was accomplished using MOE2014 software. At ﬁrst, the crystal struc-

ture of VEGFR-2 was prepared by removing water molecules. Only one

chain was retained beside the co-crystallized ligand. Then, the selected

chain was protonated and subjected to minimization of energy process.

Next, the active site of the target protein was deﬁned.

(

509.55): C, 58.93; H, 3.76; N, 24.74. Found: C, 58.65; H, 3.55; N,

2

4.55%.

4

.1.2.15. N-(4-(2-Benzoylhydrazine-1-carbonyl)phenyl)-2-(bis

′

(

[1,2,4]triazolo)[4,3-a:3 ,4 -c]quinoxalin-3-ylthio)acetamide (22a).

◦

Structures of the synthesized compounds and sorafenib (as reference

standard) were drawn using ChemBioDraw Ultra 14.0 and saved as

MDL-SD format. Such ﬁle was opened using MOE to display the 3D

structures which were protonated and subjected to energy minimiza-

tion. Formerly, validation of the docking process was performed by

docking the co-crystallized ligand against the isolated pocket of active

site. The produced RMSD value indicated the validity of process. Finally,

docking of the tested compounds was done through the dock option

inserted in compute window. For each docked molecule, 30 docked

poses were produced using ASE for scoring function and force ﬁeld for

reﬁnement. The results of the docking process were then visualized

using Discovery Studio 4.0 software [91–94].

Pale white crystal (yield, 70%); m.p. = 280–282 C. FT-IR (v max,

ꢀ

1

–

_N₎_;1H NMR

–

cm ): 3262, 3130 (NH), 1703, 1636 (C

–

O), 1603 (C

(

700 MHz, DMSO‑d ) δ 10.77 (s, 1H), 10.47 (s, 1H), 10.42 (s, 1H), 10.03

6

s, 1H), 8.63 (dd, J = 8.2, 1.5 Hz, 1H), 8.48 (dd, J = 7.8, 1.7 Hz, 1H),

7

.94 – 7.91 (m, 4H), 7.79 – 7.74 (m, 2H), 7.73 (d, J = 8.7 Hz, 2H), 7.63 –

13

.59 (m, 1H), 7.54 (t, J = 7.7 Hz, 2H), 4.59 (s, 2H); C NMR (176 MHz,

) δ 166.25, 166.08, 165.40, 147.70, 142.36, 142.15, 139.37,

38.79, 135.18, 131.98, 130.93, 130.38, 129.90, 129.06 (2C), 128.37

DMSO‑d

6

1

(

2C), 127.64 (2C), 124.12, 123.23, 118.91(2C), 118.60, 118.10, 38.94.

+

MS (m/z): 538 (M + 1, 100%); Anal. Calcd. for C26

19 9 3

H N O S (537.56):

C, 58.09; H, 3.56; N, 23.45. Found: C, 58.33; H, 3.33; N, 23.22%.

1

9

M.M. Alanazi et al.

Bioorganic Chemistry 112 (2021) 104949

4

.3.2. ADMET studies

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28] R. Elisei, M.J. Schlumberger, S.P. Müller, P. Sch o ¨ ffski, M.S. Brose, M.H. Shah,

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The authors extend their appreciation to the Deanship of Scientiﬁc

Research at King Saud University for funding this work through research

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New bis([1,2,4]triazolo)[4,3-a:3′,4′-c]quinoxaline derivatives as VEGFR-2 inhibitors and apoptosis inducers: Design, synthesis, in silico studies, and anticancer evaluation

DOI: 10.1016/j.bioorg.2021.104949

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