Design, synthesis and molecular modeling studies of 2-styrylquinazoline derivatives as EGFR inhibitors and apoptosis inducers

Bioorganic Chemistry 105 (2020) 104358

Contents lists available at ScienceDirect

Bioorganic Chemistry

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

Design, synthesis and molecular modeling studies of 2-styrylquinazoline

derivatives as EGFR inhibitors and apoptosis inducers

,

,d

Noha H. Amin^{a *}, Mohammed T. Elsaadi^{a b}, Shimaa S. Zaki^a, Hamdy M. Abdel-Rahman^c

^aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt

^bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Sinai University-Kantra Branch, Egypt

^cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University, Beni-Suef, Egypt

^dDepartment of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, 71526 Assiut, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords:

Herein, we report the synthesis of novel 2-substituted styrylquinazolines conjugated with aniline or sulfonamide

moieties, anticipated to act as potent anticancer therapeutic agents through preferential EGFR inhibition. In

doing so, all the synthesized compounds were screened for their in vitro anticancer activities (nine subpanels) at

the National Cancer Institute (NCI), USA. The resulting two most active anticancer compounds (7b and 8c) were

then chemically manipulated to investigate feasible derivatives (12a-e and 15a-d). MTT cytotoxicity, in vitro cell

free EGFR and anti-proliferative activity against EGFR/ A549 cell line evaluation for the most active broadly

spectrum candidates (7a/b, 8c/e, 12b and 15d) was conducted. Promising results were obtained for the

styrylquinazoline-benzenesulfonamide derivative 8c (IC₅₀= 8.62 µM, 0.190 µM and = 79.25%), if compared to

lapatanib (IC₅₀= 11.98 µM, 0.190 µM, and 79.25%), respectively. Moreover, its apoptotic induction potential

was studied through cell cycle analysis, Annexin-V and caspase-3 activation assays. Results showed a clear cell

arrest at G2/M phase, a late apoptotic increase (76 folds) and a fruitful caspase-3 expression change (8 folds),

compared to the control. Finally, molecular docking studies of compounds 7a/b, 8c/e, 12b and 15d revealed

proper ﬁtting into the active site of EGFR with a low binding energy score for compound 8c (ꢀ 13.19 Kcal/mole),

compared to lapatanib (ꢀ 14.54 Kcal/mole).

Quinazoline

Styryl

Sulfonamide

Antitumor

EGFR inhibition

Molecular docking

1. Introduction

differentiation and apoptosis [4]. PTKs have been responsible for the

phosphorylation catalysis of tyrosine or serine/threonine residues of

Cancer has been classiﬁed as one of the leading life-threatening

causes worldwide owing to its massive and complicated etiology [1].

That is not to mention the therapeutic struggle against lack of selectivity

and safety, ongoing resistance emergence and various side effects of

anticancer drugs, even of those that have been already approved by

Food and Drug Administration (FDA). Accordingly, vital needs for

developing and discovering potent and selective anticancer agents have

never ceased or has their importance been lessened. [2]. Anti-neoplastic

chemotherapeutic development has passed through several stages

starting from using non-speciﬁc cytotoxic agents to more speciﬁed and

even multi-targeted agents that modulated vital cell proteins including

different kinases [3]. Protein tyrosine kinases (PTKs) have been identi-

ﬁed as essential cellular enzymes that regulate proliferation,

various regulatory proteins [5]. They can be chieﬂy classiﬁed into either

receptor such as epidermal growth factor receptor (EGFR) kinases or

non-receptor ones such as B-RAF. However, EGFR-TK is considered the

prototype of the EGFR (ERBB, class I RTK) family that has been identi-

ﬁed as a trans membrane receptor tyrosine kinase belonging to ErbB

family [6], where its overexpression has been found to be abundant in a

number of various cancers such as non-small cell lung cancer (NSCLC),

head and neck cancer, breast cancer and colorectal cancer [7-10].

Accordingly, the inhibition of tyrosine kinase activity has provided a

rational approach to cancer therapy through the discovery of totally

newly synthesized anti-neoplastic compounds or the structural manip-

ulations of already known molecular cores [11].

A wide range of structural compounds have been reported to act as

Abbreviations: NCI, National Cancer Institute; RTKs, receptor tyrosine kinases; EGFR, epidermal growth factor receptor; MTT, 3-2(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide dye compound; SEM, standard error of the mean.

* Corresponding author.

E-mail address: noha.metri@pharm.bsu.edu.eg (N.H. Amin).

https://doi.org/10.1016/j.bioorg.2020.104358

Received 6 July 2020; Received in revised form 5 September 2020; Accepted 5 October 2020

Available online 8 October 2020

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

potent EGFR inhibitors, notably compounds containing a quinazoline

ring, the ring which has a well-known historical wide range of thera-

peutic efﬁcacy as bioactive antitumor [12-14], analgesic, anti-

inﬂammatory [15], antifungal [16], antibacterial [17,18] or antiviral

agents [19]. Interestingly, several FDA approved antitumor agents that

may act as EGFR inhibitors shared the possession of a quinazoline core.

Further exploration of such cores demonstrated the importance of spe-

ciﬁc quinazoline derivatives over others, namely 4-anilinoquinazoline

derivatives, such as the FDA approved geftinib and erlotinib [20].

However, mutation related resistance has always been a problem to face

and was successfully overcome though developing a second generation

of irreversible covalently bound EGFR inhibitors, such as afatinib, which

in turn led to several dose limiting toxicities in the clinical ﬁled ranging

from skin rash and up to kidney failure [21]. Accordingly and despite of

the arising numbers of such therapeutic agents, the consistent necessity

for a continuous ﬂow of EGFR inhibitors development has been practi-

cally conﬁrmed to catch up with any arising irreversibility, selectivity or

toxicity obstacles [20,21]. Recapturing variant antitumor potencies of

the quinazoline moiety through EGFR inhibition, remarkable anti-

proliferative activities and other multiple mechanisms against various

cancerous cell lines, in accordance with its accessible chemistry

manipulation in the laboratory, roused scientists to invest in such a

molecule [6,22].

linkage) between the quinolone nucleus and the aryl moiety resulted in a

considerable antitumor activity upgrade, exempliﬁed by compound E

(2-methoxystyryl quinolone) [27-29]. Compound E has shown signiﬁ-

cant in vitro antiproliferative activity against lung, colon, liver and

gastric cancerous cell lines (IC₅₀= 0.03 μM, 0.55 μM, 0.33 μM and 1.24

μ

M against H-460, HT-29, HepG2 and SGC-7901 cell lines, respectively),

reaching up to 186 folds more active than geﬁtinib. Accordingly, such

quimoline derivatives could be considered as promising scaffolds for

producing effective antitumor candidates through further structural

modiﬁcations and/ or using other bioisosteric congeners such as qui-

nazolines [28,29].

Wrapping up the above mentioned scientiﬁc literature results, the

present research work proposed a schematic outline for the design and

synthesis of novel quinazoline hybrids (series 7, 8, 12 and 15). The

present quinazoline scaffolds have been synthesized through an effective

covalent hybridization technique between

a 4-substituted styr-

ylquinazoline backbone with various substituted aniline/ sulfonamide

moieties at position 4 of the intended quinazoline, hoping to augment

the overall molecular antitumor activity of the resulting hybrids (7a-e

and 8a-e).Those newly synthesized hybrids have been expected to act as

potent anticancer therapeutic agents through EGFR inhibitory activities

owing to the quinazoline nitrogenous moiety presence, capable of

forming H-bonds with the hinge region of EGFR active site, and thus

resembling the ATP binding site of EGFR. Generation of such novel

chemical hybridized entities has been predicted to demonstrate arising

hydrophobic interaction with the ATP-binding site of EGFR at various

sites. Moreover, bulky substituted aniline/ sulfonamide moieties at C4 of

quinazoline have been expected to serve as an easily reachable lyophilic

part for the rear side of the ATP binding site and thus offering a more

stable and comfortable positioning. Alongside the C-4 quinazoline sub-

stitution, another C-2 quinazoline substitution with a methoxyphenyl

moiety was performed using the previously reported promising spacer

(ethylene linkage), Fig. 1.

In the same direction, it has been expected that the quinazoline ring

can still present a wide range of versatile chemical and biological out-

comes to humanity. Accordingly, the present research was based on the

chemical covalent hybridization of the quinazoline ring at different

positions with other structurally chemical moieties of known effective

anticancer activities in order to augment the overall pharmacological

antitumor outcome of such hybrids, anticipated to exert their action

through EGFR inhibition.

2. Rationale and design

Additionally, the resulting most active antitumor candidates (7b and

8c) have been substituted with a variety of structural styryl side chains

to investigate feasible pharmacological outcomes of such chemical

structural derivatives (12 and 15). Furthermore, evaluation of the

antitumor activity of the synthesized compounds against a panel of

cancerous cell lines, EGFR screening assay, in addition to their ability to

induce apoptosis and cell cycle analysis, were also performed. It has

been conceptualized that the resulting pharmacophoric ﬁgure would act

as a promising template for potent anticancer agents with acceptable

safety margins through EGFR inhibition, Fig. 2.

Literature survey of several potent FDA approved EGFR inhibitors

has been investigated and subsequently correlated to clinical trials.

Observations were taken and it was concluded that 4-anilinoquinazoline

derivatives, such as geﬁtinib A (IC₅₀= 0.090 µM), erlotinib B (IC₅₀

=

0.025 µM) and lapatinib C (IC₅₀= 0.115 µM), have been valuable

commodities for further exploration as anticancer agents [6,20-22].

On the other hand, the power of sulfonyl/ sulfonamide has been

fruitfully explored for their efﬁcacy and considerable safety margins as

anti-tumor agents [23]. Introduction of a sulfonamide functionality onto

various heterocyclic nuclei has produced candidates with a clear anti-

tumor activity spring over the sulfonamide free ones [24].

3. Results and discussion

Nevertheless,

bioisosteric

quinolone

and

quinazoline-

benzenesulfonamide hybrids have been investigated throughout litera-

ture as potent EGFR inhibitors against various cancerous cell lines [12].

Promising antitumor activity results were obtained throughout various

literature studies demonstrated by quinazoline derivatives bearing a

sulfonamide moiety (IC₅₀values in the nanomolar range against MCF-7)

[25,26]. One prominent example was compound D, the most potent

amongst a series of tested compounds (IC50 = 0.13 nmol). Its mecha-

nism of action was illustrated via molecular modelling, where it showed

a similar binding mode to that of geftinib and lapatinib at EGFR tyrosine

kinase binding site. It acted via competing with ATP for binding at the

catalytic domain of EGFR tyrosine kinase [11].

3.1. Chemistry

The target compounds were synthesized according to the depicted

Schemes 1-3. The structures of the newly synthesized compounds were

established on their elemental analyses and spectral data (Supplemen-

tary). As a starting point in Scheme 1, the 2-methylquinazolin-4(3H)-

one nucleus 3 was synthesized following Niementowski synthesis

through the direct anhydrous fusion of anthranilic acid 1 with thio-

acetamide 2 in a molar ratio of 1: 1.5 [30]. Then compound 3 was used

as the main synthetic backbone for the newly synthesized target com-

pounds 7a-e and 8a-e. The key intermediates 5 and 6 were prepared

through the reaction of 2-methylquinazolin-4(3H)-one 3 with p-

methoxybenzaldehyde 4 in acetic acid to afford 2-methylquinazolin-4

(3H)-one 5, followed by its chlorination reaction with phosphorus

oxychloride in ether to result in 4-chloro-2-methylquinazoline 6 under

controlled conditions of temperature and time [31]. Furthermore, the

target compounds 7a-e were synthesized through the condensation re-

action of the building block, 4-chloro-2-(4-methoxystyryl) quinazoline

6, with different amines in DMF in presence of potassium carbonate,

except for compounds 7a and 7c that were stirred in isopropanol and

Meanwhile, the worldwide immune stimulatory chloroquine (4-

anlinoquinoline skeleton) has aroused increasing attention owing to its

antiproliferative potency and excellent antiangiogenesis activities

against different types of cancer. Furthermore, some 2-substituted-4-ani-

linoquinoline derivatives (2-arylsufonyl/ styrylquinolone) were syn-

thesized and evaluated for their antiproliferative activity against four

human cancerous cell lines, where highly improved anticancer activities

were noticed for 2-styrylquinolone derivatives over other analogues.

Also, it was observed that the introduction of a suitable spacer (ethylene

2

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

Fig. 1. Design strategy of the present compounds 7a-e and 8a-e.

Fig. 2. Chemical modiﬁcations of the most active candidates 7b and 8c and further biological evaluation.

ethylene glycol, respectively. Similarly, condensing 4-chloro-2-(4-

methoxystyryl) quinazoline 6 with different sulfonamides in absolute

ethanol in presence of anhydrous potassium carbonate, as a suitable acid

eliminating reagent, afforded sulfonyl derivatives 8a-e in good yields.

In Scheme 2, Endicot modiﬁed synthetic method was adopted to

obtain the key intermediate, 4-chloro-2-methylquinazoline 9 through

keeping temperature and work-up time to minimum and immediately

purifying the crude in neutral medium in order to avoid any possible

instability of 4-chloroquinazoline to an acid. The synthesis was con-

ducted through the activation of position 4 of 2-methylquinazolin-4

(3H)-one 3 by chlorination with phosphorus oxychloride at minimum

temperature for minimum time [32] and then followed by a condensa-

tion reaction with p-chloroaniline 10 in DMF in presence of potassium

carbonate to afford N-(4-chlorophenyl)-2-methylquinazolin-4-amine

11. Moreover, the target substituted styryl derivatives 12a-e were syn-

thesized through the reaction of appropriate substituted aldehydes with

compound 11 in glacial acetic acid in presence of sodium acetate under

classical condensation conditions of appropriate aldehydic dervitisation.

Furthermore in Scheme 3, N-(5-methylisoxazol-3-yl)-4-((2-methyl-

quinazolin-4-yl)amino) benzenesulfonamide 14, another key interme-

diate that was prepared through the reaction of 4-chloro-2-

methylquinazoline 9 with sulfamethoxazole 13 in ethanol in presence

of anhydrous potassium carbonate. Last but not least, the target com-

pounds 15a-e were prepared through a condensation reaction of com-

pound 14 with different aldehydes in glacial acetic acid in presence of

sodium acetate.

3.2. In vitro cytotoxic screening and SAR discussion

The obtained screening results of all the present newly synthesized

compounds were obtained from the National Cancer Institute (NCI),

Bethesda, Maryland, USA, represented as mean graph of the percent

growth of treated cells (Supplementary tables S1 and S2) and then

demonstrated as percent of growth inhibition (GI %) in Figs. 3a and 3b

3

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

Scheme 1. Synthesis of compounds 3, 5, 6,7a-e and 8a-e. Reagents and reaction conditions: a. Reﬂux, 2 h; b. Acetic acid, Na acetate, reﬂux, 24 h; c. POCl₃,

reﬂux, 2 h; d. Appropriate amine, DMF/ K₂CO₃or isopropanol or ethylene glycol, reﬂux, 24 h; e. Appropriate sulfonamide, ethanol, K₂CO₃, reﬂux, 24 h.

[33-35]. The obtained results showed that the tested quinazoline de-

rivatives possessed a broad-spectrum anti-tumor activity, particularly

against non-small cell lung cancer A549\ ATCC and breast cancer MCF7

(known for EGFR over expression), ranging from GI % = 64.79 to

20.21% and from GI % = 69.32 to 21.55%, respectively.

to compounds 7b,c and 8a,c,e (GI % = 85.68, 63.74, 74.4, 82.48 and

79.18%, respectively).

Concerning renal cancer, A498 cell lines presented powerful sensi-

tivity against compounds 7b,e and 8a,c,e (GI % = 83.37, 90.34, 90.42,

98.27 and 92.2%, respectively).

It was observed that the tested quinazoline derivatives exhibited a

distinctive pattern of selectivity. Leukemia CCRF-CEM cell lines were

found to be remarkably sensitive to compounds 7a and 7b (GI % = 87.14

and 91.94%, respectively). In addition, leukemia TB cell lines showed

sensitivity to compound 7a (GI % = 89.9%). Also, compounds 8c and 8e

exhibited considerable activities against leukemia SR cells (GI % =

54.57 and 50.53%, respectively).

Moreover, Breast cancer, MCF7 cell lines proved to be sensitive to

compounds 7b and 8a,c,e (GI % = 55.99, 53.34, 67.92 and 57.84%,

respectively), while MDA-MB-468 cell lines showed moderate sensitivity

to compounds 7b and 8c,e (GI % = 71.92, 70.06 and 75.98%,

respectively).

In an attempt to discover more anti-tumor active agents, compounds

7b and 8c were used as lead compounds and were selected for further

derivativization by different aldehydic compounds at position two to

give series 12a-e and 15a-d, which were later screened also at NCI and

the results obtained were demonstrated as percent of inhibition. (Sup-

plementary; table 2s)

Regarding non-small lung cancer, A549\ATCC cell lines showed

sensitivity towards a variety of compounds such as 7b and 8a,c,e (GI %

= 64.97, 57.99, 60.86 and 57.75%, respectively). Also, NCI-H322M cell

lines were sensitive to compounds 7b and 8a,c,e (GI % = 55.73, 61.39,

62.66 and 70.98%). Additionally, strong anticancer activities of com-

pounds 7b and 7a against NCI-H460 and NCI-H522 cell lines were

recorded (GI % = 81.8 and 84.74%, respectively).

Unfortunately, compound 15d showed remarkably low cell growth

promotion against melanoma MDA-MB-435 cell line (GI % = ꢀ 44.14%),

but on the other hand it demonstrated a broad spectrum activity against

leukemia K-562, SR cell lines (GI % = 82.21 and 80.58%, respectively),

colon cancer HCT-15, KM12 (GI % = 68.24 and 68.22%, respectively)

and breast cancers MCF7 (GI % = 69.32%). Also, compounds 12b,d and

In respect to melanoma, LOX IMVI cell line, diverse compounds

showed various activities such as 7b and 8a,c,e (GI % = 81.63, 61.71,

68.53 and 73.69%, respectively). UACC-62 cell line displayed sensitivity

4

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

Scheme 2. Synthesis of compounds 9, 11 and 12a-e. Reagents and reaction conditions: a. POCl₃, reﬂux, 2 h; b. DMF, K₂CO₃, reﬂux, 24 h; c. Appropriate

aldehyde, glacial acetic acid, Na acetate, reﬂux, 24 h.

Scheme 3. Synthesis of compounds 14 and 15a-d. Reagents and reaction conditions: a. Ethanol, anhydrous K₂CO₃, reﬂux, 24 h; b. Appropriate aldehyde, glacial

acetic acid, Na acetate, reﬂux, 24 h.

15b exhibited moderate anti-tumor activity against leukemia (K-562

and SR cell lines), melanoma (LOX IMVI and UACC-62) and breast

cancers (MCF7).

position as 3-chloro-4-(3-ﬂuorobenzyl)oxy moiety of lapatinib, where

such lipophyllic substitutions should lead to better ﬁtting into the EGFR

binding site. Accordingly, the present synthesized compounds were ex-

pected to act similarly and was practically demonstrated via their sig-

niﬁcant GI% against variable cell lines, such as the quinazoline hybrids

between substituted aniline moieties at C-4 and methoxystyryl at C-2,

exempliﬁed by compounds 7a (GI % = 89.9%) and 7b (GI % = 91.94%)

against leukemia cell lines (CCRF-CEM and TB). Similarly, the quina-

zoline hybrids between benzenesaulfonamide moities at C-4 and

methoxystyryl at C-2, exempliﬁed by compounds 8c (GI % = 98.27%)

and 8e (GI % = 92.2%) showed considerable anticancer activities

against renal cancer cell lines (A498) [11]. Furthermore, variable sub-

stituents have been incorporated on the aniline/ sulfonamide of C-4 and

styryl of C-2 moieties of the quinazoline ring to investigate their

Structure activity results correlation was based on several literature

reviews about effective EGFR inhibitors, where these compounds

compete with ATP for binding with the catalytic domain of tyrosine

kinase, exempliﬁed by quinazoline cores, especially those substituted at

position 4 like the reference drug, lapatinib. It has been noticed that such

cores effectively bind with ATP binding cleft through hydrophobic in-

teractions with hydrophobic pocket in N-terminal domain of EGFR

enzyme via its two nitrogen atoms, acting as hydrogen bond acceptors.

Similarly, the present newly synthesized quinazoline hybrids that were

substituted with aniline/sulfonamide moieties at C-4 and variable

substituted styryl moieties at C-2 were expected to interact at the same

5

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

Fig. 3a. GI % of compounds 7a, 7b, 8a, 8c, 8e and 15d over the most sensitive cell lines (Leukemia, lung and melanoma subpanels).

Fig. 3b. GI % of compounds 7a, 7b, 8a, 8c, 8e and 15d over the most sensitive cell lines (Renal, breast and colon subpanels).

inﬂuence on pharmacological activity. Generally 2-substituted

methoxystyryl qunazoline derivatives 7a (hydroxyaniline), 7b (chlor-

oaniline), 8c (isoxazolyl benzenesulfonamide) and 8e (dimethylpyr-

imidine) have proved a dramatic activity improvement over other non

methoxystyryl quinazoline derivatives such as compounds 12b (chlor-

ostyryl) or 12d (dimethylaminoatyryl) derivatives. However, compound

15d was an exception as it was a methoxystyryl derivative that showed a

remarkably low GI % = ꢀ 44.14 against melanoma (MDA-MB-435)

subpanel.

Table 1

Cytotoxicity assay (MTT) and log P values of compounds 7a, 7b, 8c, 8e, 12b and

15 d and lapatanib against non-small lung cancer cell lines (A549) and normal

lung ﬁbroblasts (WI38).

Compound ID

IC₅₀^a(µM)

SI ^(c)

CLog P

(Mean ± SEM) ^b

A549

WI38

7a

26.69 ± 1.60

31.35 ± 1.80

8.62 ± 0.50

28.85 ± 1.70

43.38 ± 2.50

27.98 ± 1.60

11.98 ± 0.70

65.5 ± 3.68

28.4 ± 1.6

1<

1>

1 <

1>

1 <

5.81

7.22

5.81

6.35

8.01

5.08

NT^(d)

7b

8c

52.6 ± 2.95

27.3 ± 1.53

18.1 ± 1.02

45.0 ± 2.53

17.0 ± 0.96

3.3. Cytotoxicity potential assay study

8e

12b

15d

Lapatinib

3.3.1. Cytotoxicity potential assay study (MTT assay)

Representative most active compounds showing broad spectrum

activity (7a, 7b, 8c, 82, 12b and 15d) were selected to be further tested

for their cytotoxicity potency using MTT test and results were expressed

as IC_50%, Table 1 and Fig. 4. Non-small cell lung cancer cell lines (A549,

obtained from the American Type Culture Collection, Rockville, MD,

USA) were chosen for the assay for their well-known fact of EGFR

overexpression [36]. Interestingly, 2,4-disubstituted quinazoline deriv-

ative 8c (IC₅₀= 8.62 µM) showed potent cytotoxicity against A549 cell

line, almost double that of lapatinib (IC₅₀= 11.98 µM). On the other

hand, moderate activity was shown by compounds 7a, 8e and 15d

(nearly reaching half that of lapatinib), while compounds 7b and 12b

possessed the lowest anti-tumor activity. These ﬁndings have been

attributed to the presence of both the methoxystyryl group and the

oxazolyl moiety as a sulphonamide derivative rather than any other

substitution at position 4. Also, a general positive effect of the lipophilic

impact of such derivatives upon interacting with EGFR active binding

(a): IC₅₀values are the mean of three separate experiments and is a concentration that

causes 50% growth inhibition, (b): Standard error of the mean, Values are the mean

± SD (n = 3).

(c): SI = Selectivity Index = IC₅₀value normal cell/IC₅₀value cancer cell.

(d): Not tested.

site has been expected to contribute in their relative overall antitumor

effects as shown by log P values, Table 1. Moreover, MTT IC₅₀values and

selectivity index (SI) for the same compounds were performed on

normal lung ﬁbroblasts (WI38), noncancerous cell lines used as a

reference to check for a possible tumor selectivity and thus expected

toxicity. Compounds 7b, 8e and 12b showed good selectivity results

(less than 1), even better than those of lapatinib, so proving greater

safety margins. On the other hand, compounds 7a, 8c and 15d showed

mild to moderate toxicity (SI more than one and less than 25), Table 1.

6

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

methoxy group was changed to a chloro moiety.

Table 2

IC50 values and growth inhibition percent of target compounds 7a, 7b, 8c, 82,

12b and 15 d and lapatinib against EGFR.

3.4.2. In vitro EGFR enzyme inhibition assay of target compounds against

EGFR-expressed in A549 cell line

Compound ID

EGFR

Further investigation was performed for the compounds 7a, 7b, 8c,

8e, 12b and 15d, which showed good antitumor activity results against

EGFR enzyme, using a more speciﬁc expressed EGFR activity in A549

cell line at conﬁrmatory diagnostic unit at VACSERA, Cairo [37]. NSCLC

cell lines (A549) were treated with the desired compounds with their

IC₅₀concentrations for accurate assay then lysed and subjected to

enzyme-linked-immunosorbent assay (ELISA) to determine their activity

against EGFR in this cell line. The results were presented as percentage

inhibition. The results were consistent with in vitro EGFR assay, which

also showed expected high potency of compound 8c (% inhibition of

EGFR enzyme in cell line A549 = 79.25%) and that was pretty compa-

rable to that of lapatinib (83.33%). These ﬁndings conﬁrmed the

importance of p-methoxystyryl moiety for EGFR inhibition. On the other

hand, moderate activity was presented by compounds 7a, 7b, 8e and

12b, but low activity was possessed by compound 15d, Table 3 and

Fig. 4.

% Inhibition at 1 µg/ml

IC₅₀^a(µM)

(Mean ± SEM) ^b

7a

63.49

62.58

69.71

62.70

66.05

60.86

75.09

NT^c

0.355 ± 10.15

0.369 ± 10.50

0.190 ± 5.44

0.307 ± 8.79

0.269 ± 7.70

0.634 ± 18.11

0.115 ± 0.003

NT

7b

8c

8e

12b

15d

Lapatinib

Control

(a): IC₅₀values are the mean of ﬁve separate experiments and is a concentration that

causes 50% growth inhibition, (b): Standard error of the mean, (c): not tested.

3.4. Epidermal growth factor receptor enzyme inhibition assay

3.4.1. In vitro EGFR enzyme inhibition assay

A further EGFR kinase assay was carried out against A549 cell lines,

known for their EGFR overexpression, to study the postulated mecha-

nism of action of the present synthesized compounds. With reference to

the preceding considerable antiproliferative screening results against 60

cell lines and expressive cytotoxicity results, compounds 7a (p-hydrox-

yphenyl derivative) and 7b (para-chloro phenyl derivative) for aniline-

methoxystyrylquinazolines, 8c (methylisoxazole derivative) and 8e

(dimethyl pyrimidine) for sulfonamide–methoxystyrylquinazoline, in

addition to compounds 12b and 15d were all subjected to in vitro cell

free EGFR enzyme inhibition determination using EnzyChromTM Kinase

Assay Kit (EKIN-400) according to manufacturer’s instructions by ELISA

assay method and using lapatinib as a reference [36,37]. Table 2 and

Fig. 4.

3.5. Cellular mechanism of action study

3.5.1. Cell cycle inhibition

The most active antitumor compound 8c was selected for further

Table 3

Percentage inhibition values of target compounds 7a, 7b, 8c, 82, 12b and 15

d and lapatinib against EGFR expressed in A549.

Compound ID

EGFR/ A549

% inhibition

Conc. ± SEM ^a(ng/ml)

7a

70.05

73.32

79.25

73.01

74.41

68.39

83.33

0

0.523 ± 7.6

0.474 ± 14.1

0.369 ± 13.8

0.479 ± 9.6

0.455 ± 8.9

0.562 ± 3.5

0.296 ± 3.06

1.777 ± 78.400

The obtained results that compound 8c exhibited showed potent

inhibition of EGFR enzyme (IC₅₀= 0.190 µM) if compared to lapatinib

(EGFR IC₅₀= 0.115 µM). This inhibitory activity was attributed to the

presence of a p-methoxystyryl moiety as previously mentioned, in

addition to the oxazole ring of sulphonamide moiety. Furthermore,

compounds 7a, 7b, 8e and 12b showed moderate EGFR inhibition. Also,

it could be concluded that the sulfonamide derivative 8e was favored

over other substituted aniline derivatives in position 4 (7a, 7b). More-

over, compounds 15d possessed low EGFR inhibition activity when the

7b

8c

8e

12b

15d

Lapatinib

Control

(a): IC₅₀values are the mean of seven separate experiments and is a concentration

that causes 50% growth inhibition, (b): Standard error of the mean, (c): not tested.

Fig. 4. Cytotoxicity IC₅₀% (µM), cell free EGFR assay (%inhibition) and EGFR (%inhibition) expressed in A549 cells of target compounds 7a, 7b, 8c, 82, 12b and 15

d and lapatinib.

7

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

study regarding its effect on cell cycle progression and induction of

apoptosis in A549 cell line at Vacsera using Propidium Iodide kit and

analyzed by Flow Cytometry technique using FACS Calibur reader [38].

The cell line was treated with tested compounds at their IC₅₀values for

24 h. Its effect on the normal cell cycle proﬁle and induction of apoptosis

was analyzed. From these results, it was observed that compound 8c

increased concentration at pre G1 (apoptosis) phase (23.87%), if

compared to control (1.69%). A signiﬁcant increase in the percentage at

G2/M phase was observed after treatment of cell line with tested com-

pound, indicating that A549 cell line arrested at G2/M phase of cell

cycle and contributed to cell cytotoxicity owing to the effect of the tested

compound 8c. On other hand, cell population at G0/G1 and S phase was

decreased after treatment with the same compound, if compared to

control, Table 4.

(1.276, 1.286 and 1.436 for compounds 7a, 7b and 12b, respectively,

thus predicting considerable absorption for orally administered dosage

forms of the respective compounds [42], Table 7. The volume of dis-

tribution calculated using a steady-state volume of distribution (VDss)

showed that compounds 8e, 12b and 15d had lower theoretical dose

required for uniform distribution in the plasma than compounds 7a, 7b

and 8c, while the degree of diffusing across plasma membrane increased

in this order; 12b < 15d < 7b < 7a < 8c < 8e, measured as the fraction

that is in the unbound state.

On the other hand, BBB permeability of all the compounds was

considered promising as the log BB values were below 0.3, thus can be

safely used with minimal side effects on the brain, Table 7. Furthermore,

a group of enzymes that play signiﬁcant roles in drug metabolism is the

CYP isozymes. The tested compounds showed variable range of CYP

interactions, Table 7. Moreover, predicted in-silico toxicity was assessed,

where no mutagenic potential of all the tested compounds was observed

through AMES toxicity testing. Generally, all the compounds were

recorded to act as effective hERG II inhibitors, but not hERG I, the fact

that might correlate to subsequent therapeutic cardiac effects [42]. All

the compounds showed acceptable safety towards skin sensitization

with a liability towards hepatotoxicity induction, Table 7.

3.5.2. Cell apoptosis assay

The consequent aptitude of compound 8c for apoptosis induction

was further measured using annexin-V-FITC assay [39]. It was observed

that some treated cells were in a late apoptotic stage (upper right

quadrant) after a 24 h of treatment of A549 cells (at their IC₅₀concen-

tration) with compound 8c, Fig. 5. This was indicated by the signiﬁcant

increase in the percentage of annexin V positive and PI positive cells.

Compound 8c showed an increase of 76 folds more than control in late

apoptosis. Also, an increase in late apoptosis was noticed over the early

one (lower right quadrant), making recovery of apoptotic cells to be

healthy more difﬁcult. In conclusion, the tested compound 8c was able

to induce late apoptosis with necrosis percentage = 2.36%, Table 5.

Collectively, the predicted pharmacokinetic properties and toxicity

of the target compounds results conﬁrmed that those newly synthesized

compounds did not only show good preferential EGFR inhibitory ac-

tivities, but also possessed a variable range of promising pharmacoki-

netic properties that could be used as a versatile tool for future

development of promising safely used anticancer agents.

3.5.3. Active Caspase-3 expression level assay

3.7. Molecular docking study

Induction of apoptosis of the tested compound 8c was further

conﬁrmed through assessing caspase-3 expression levels in A549 cell

after 24 h treatment using Invitrogen Caspase-3 (active) Human ELISA

kit [40]. CASP-3 protein activation plays a key role in the execution-

phase of cell apoptosis, Table 6. Caspase-3 activity (in fold change,

FLD) signiﬁcantly increased after cell treatment with compound 8c

(7.94 folds) if compared to control. As expected, the tested compound

activated caspase-3 as a mechanism of apoptosis induction, indicating

that it not only inhibited proliferation of cancer cells via EGFR inhibi-

tion, but also promoted caspase-3 activation and subsequent efﬁcient

cell death.

Molecular docking study was performed in an attempt to explain the

kinase activity of the most active compounds through illustrating their

positing and ﬁtting into EGFR active binding site using ‘Molecular

Operating Environment 2019.0101’ software. Moreover, the crystal

structure of the enzyme with the reference drug (lapatinib) was obtained

from the protein data bank; PDB code: 1XKK [43]. Docking of lapatinib

showed the binding mode of lead compound through interaction with

two amino acids: Leu718 and Met793 through pi-bonds and hydrogen

bond acceptor, respectively, into the active site of EGFR enzyme, Fig. 6.

In the present study, it was found that compound 8c was bound to EGFR

enzyme active site (Energy afﬁnity ꢀ 13.19 kcal/mol) in a similar mode

as that of the reference drug, lapatinib, through hydrogen bond acceptor

to Leu792 and key amino acid Met793 via sulphonyl oxygen, respec-

tively, in addition to an extra pi-bond with Met793 via phenyl ring of

sulphonamide moiety (Supplementary tables, S3). Its proper interac-

tion explained its potency as EGFR inhibitor. However, a moderate

EGFR inhibitory activity for compounds 7a,b and 8e and which was

contributed to their retention of pi-bond interaction of phenyl ring of

quinazoline nucleus with key amino acid Leu718. However, their

interaction with different amino acids for each compound was clear

except for compound 12b, which did not interact with the main amino

acid, but instead it showed seven binding interactions with ﬁve different

amino acids. Also, binding interactions of compound 15d with key

amino acids (Leu718 or Met793) were not visible, the fact that coincided

with preceding in vitro biological results. On the other hand, compounds

12b and 15d showed two binding interaction via H-donor and H-

acceptor binding with Asp855 in addition to Arg841 interaction with pi-

bond (Supplementary ﬁgures, S1). The overall docking results of the

tested compounds came out to conﬁrm all the above mentioned bio-

logical in vitro EGFR inhibitory activities. Collectively, compound 8c

showed potent EGFR inhibition activity similar to that of lapatinib,

where other tested compounds possessed moderate to weak activities.

The activity of compound 8c could be attributed to good binding afﬁnity

of sulphonyl oxygen via H-bond acceptor to Leu792 and Met793, the key

amino acids of EGFR enzyme, in addition to an extra pi-bond between

the phenyl ring of sulphonamide moiety and Met793 amino acid. Also,

3.6. ADMET studies

Computational studies for predicted pharmacokinetic properties and

toxicity of the target compounds 7a, 7b, 8c, 8e, 12b and 15d have been

performed and results were presented in Table 7. ADMET studies were

predicted using pkCSM tool (http://biosig.unimelb.edu.au/pkcsm/p

rediction) [41]. The SMILE molecular structures of the compounds

were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov). The

prediction was carried out as a methodological virtual screening, based

on various criteria, where all the compounds showed excellent human

intestinal absorption results ranging from 86 to 99% absorption.

Moreover, human colon adenocarcinoma-2 parameter (Caco2; high

Caco2 permeability would translate in values >0.9) showed good results

Table 4

Cell cycle analysis of tested compounds in A549 cell line.

Compound

ID

Results

DNA content %

% G0-

G1

%S

% G2-

M

%Pre G1 (%

apoptosis)

Comment

8c/ A549

38.15

26.59

34.28

35.26

23.87

cell cycle arrest

at G2/M

Control/

A549

57.83

7.89

1.69

–

8

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

Fig. 5. Effect of compound 8c (A) and control (B) on DNA-ploidy ﬂow cytometric analysis of A549. Representative dot plots of A549 cells treated with compound 8c

(C) and control (D) for 24 h and analyzed by ﬂow cytometry after double staining of the cells with Annexin-V FITC and PI.

d) have been designed and synthesized. Those hybrids ﬁrstly started

Table 5

with synthesizing two new series of 2,4-disubstituted quinazoline de-

Effect of tested compounds on apoptosis and necrosis in A549.

rivatives 7a-e and 8a-e through ﬁxing a p-methoxystyryl moiety at po-

Compound. ID

Apoptosis

Necrosis

sition 2 of the quinazoline backbone, then followed by the incorporation

of either substituted anilino derivatives or sulfonamide derivatives,

respectively, at position 4 of the quniazoline core. Further experimental

chemistry work was conducted on two most potent representatives from

each series; 7b (4- p-chlorophenylanilino) and 8c (4-oxazolylbenzene-

sulfonamide) to result in another two new quinazoline series 12a-e and

15a-d, respectively. All the compounds of the four series were evaluated

for their in vitro anticancer activities (nine subpanels) at the National

Cancer Institute (NCI), USA. Accordingly, it was concluded that com-

pound 8c, a quinazoline backbone hybridized with a methoxystyryl

moiety at position 2 and an isoxazolylbenzenzensulfonamide moiety at

position 4, exhibited the best pharmacological results. It showed broad

spectrum anti-cancer activity with potent efﬁcacy against A549 cell line

(IC₅₀= 8.62 µM), almost double that of lapatinib (IC₅₀= 11.98 µM),

while compounds 7a,b, 8e and 12b revealed moderate activities.

Further mechanistic biological investigation was conducted through

cytotoxicity studies against NSLC (A549), non-cell EGFR, EGFR

expressed in A549 cell line assays, in addition to molecular modeling

studies. The research observation has recorded that generally substitu-

tion of the quinazoline nucleus at position 2 with p-methoxystyryl

moiety derivative resulted in potent broad spectrum anti-tumor activity

that was enhanced by an oxazolyl benzensulfonamide moiety at position

4 as in compound 8c. Otherwise, replacement of p-methoxystyryl moiety

at position 2 or other substituted anilino derivatives at position 4 led to

moderate or low activities. The observed good activity of compound 8c

Total

Early

Late

8c/A549

23.87

1.69

4.79

0.43

16.72

0.22

2.36

1.04

Control. A549

Table 6

Effect of tested compounds on Caspase-3 expression level in A549.

Compound ID

Results

Casp3/ A549

Conc (pg/ml)

FLD

8c

319.9 ± 9.55

40.28 ± 6.2

7.94

1

Control

the methoxystyryl moiety, which has been embedded in hydrophobic

region of EGFR enzyme, has signiﬁcantly contributed to the consider-

able anticancer efﬁcacy of compound 8c through implied EGFR

inhibition.

4. Conclusion

Wrapping up the present research work and results, it could be

brieﬂy stated that novel quinazoline hybrids (7a-e, 8a-e, 12a-e and 15a-

9

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

Table 7

Predicted pharmacokinetic properties and toxicity of the target compounds 7a, 7b, 8c, 8e, 12b and 15d.

Model Name (Unit)

Predicted Value

7a

7b

8c

8e

12b

15d

Lipophilicity (LogP)

5.561

93.759

1.276

ꢀ 0.635

0.164

0.020

No

6.206

92.093

1.286

ꢀ 0.532

0.157

0.108

No

5.649

99.694

ꢀ 0.248

ꢀ 0.756

0.169

ꢀ 1.441

No

5.452

96.647

ꢀ 0.065

ꢀ 0.839

0.182

ꢀ 1.494

No

5.903

90.926

1.436

ꢀ 0.937

0.029

0.052

No

5.052

86.744

ꢀ 0.096

ꢀ 1.032

0.044

ꢀ 1.936

No

Intestinal absorption- human (%)

Caco2 permeability (log Papp in 10–6 cm/s)

VDss- human (log L/kg)

Fraction unbound- human

BBB permeability (log BB)

CYP2D6 substrate/ inhibitor

CYP3A4 substrate/ inhibitor

Yes

AMES toxicity (Categorical: Yes/No)

hERG I inhibitor (Categorical: Yes/No)

hERG II inhibitor (Categorical: Yes/No)

Hepatotoxicity (Categorical: Yes/No)

Skin Sensitisation (Categorical: Yes/No)

No

Yes

No

Caco2: Human colon adenocarcinoma-2, VDss: Steady-state volume of distribution, BBB: Blood-brain barrier, AMES: Salmonella typhimurium reverse mutation assay,

hERG: Human ether-a-go-go-related gene.

Fig. 6. 2D and 3D Docking of (a) lapatinib and (b) compound 8c into active binding site of EGFR (using PDB code: 1XKK).

has been attributed to good binding afﬁnity of the sulphonyl oxygen via

H-bond acceptor to Leu792 and Met793, the key amino acids of EGFR

enzyme, in addition to an extra Pi-bond between phenyl ring of sul-

phonamide moiety and Met793 amino acid, not to mention the effect of

the methoxystyryl moiety through its pretty inclusion within the hy-

drophobic region of EGFR enzyme. Collectively, it could be safely

concluded that compound 8c possessed a good apoptosis induction ac-

tivity in addition to considerable EGFR inhibitory activity. Moreover, it

was hypothesized that further derivatization of the most active com-

pound 8c in the upcoming future could result in promising potent anti-

tumor activity with improved EGFR selectivity and thus higher safety

proﬁles.

10

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

5. Experimental

5.1.2.3. (E)-2-(4-Methoxystyryl)-N-(4-nitrophenyl)quinazolin-4-amine

(7c). Yield 57%; mp 241–243 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3448 (NH) 3119,

5.1. Chemistry

2924 (CH aromatic, CH aliphatic). ¹H NMR δ ppm: 3.80 (s, 3H, OCH₃),

–

6.60 (d, 2H, CH aromatic, J = 9.2 Hz), 6.89 (d, 1H, CH CH, J = 16.0

–

Melting points were carried out by open capillary tube method using

IA 9100 MK-Digital Melting Point Grifﬁn Apparatus and were uncor-

rected. Elemental Microanalyses were carried out using Vario El III,

CHNSO analyzer (Germany) at Faculty of Pharmacy, Al-azhar Univer-

sity. Infrared Spectra were done on Bruker FT-IR spectrophotometer

Vector 22, Schimadzu 435, Perkin-Elmer 457 and Jasco FT.IR plus 460

Japan and expressed in wave number (cm^{ꢀ 1}), using KBr discs. ¹H NMR

Spectra were carried out using Bruker Avance III at 400 MHz (Bruker

AG, Switzerland) using DMSO‑d₆or otherwise stated as a solvent. The

chemical shifts were expressed in δ ppm units using trimethylsilane as

the internal standard. ¹³C NMR spectra were carried out using Bruker

Avance III at 100 MHz (Bruker AG, Switzerland) using DMSO‑d₆as a

solvent. Mass Spectra was done on Shimadzu Qp- 2010 plus. All the

reactions were monitored by thin layer chromatography. Silica gel/

TLC-cards DC- Alufolien-Kieselgel with ﬂuorescent indicator 254 nm;

layer thickness 0.2 mm. Petroleum ether: ethyl acetate (1:1) or (1:2) was

the adopted solvent system.

Hz), 7.00 (d, 2H, CH aromatic, J = 8.8 Hz), 7.11 (d, 2H, CH aromatic, J

= 8.8 Hz), 7.62 (t, 1H, CH aromatic, J = 10.4 Hz), 7.86 (d, 1H, CH ar-

omatic, J = 7.6 Hz), 7.92 (t, 1H, CH aromatic, J = 9.8 Hz), 8.08 (d, 1H,

–

CH aromatic, J = 7.6 Hz), 8.18 (d, 1H, CH CH, J = 8.4 Hz), 8.25 (d, 2H,

–

CH aromatic, J = 8.8 Hz), 9.85 (s, 1H, NH, exch. D₂O). ¹³C NMR δ ppm:

55.78 (OCH₃), 112.84, 115.00, 121.51, 122.37, 126.31, 126.86, 127.20,

128.21, 128.94, 129.71, 134.63, 138.07, 152.06, 157.16, 160.96,

162.07 (aromatic Cs), 123.05, 134.18 (2 oleﬁnic Cs). Mass (m/z): 398

(M⁺, 11.77), 277 (100). Anal. Calcd for C₂₃H₁₈N₄O₃(398.41): C, 69.34;

H, 4.55; N, 14.06. Found: C, 69.01; H, 4.79; N, 14.32%.

5.1.2.4. (E)-Ethyl 4-((2-(4-methoxystyryl)quinazolin-4-yl)amino)benzo-

ate (7d). Yield 62%; mp 267–269 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3441 (NH), 3150,

2920 (CH aromatic, CH aliphatic), 1666 (CO). ¹H NMR δ ppm: 1.25 (t,

3H, CH₃, J = 10.6 Hz), 3.86 (s, 3H, OCH3), 4.17 (q, 2H, CH₂, J = 8.0 Hz),

–

6.55 (d, 1H, CH CH, J = 8.4 Hz), 7.02 (d, 2H, CH aromatic, J = 8.8 Hz),

–

7.11 (d, 2H, CH aromatic, J = 8.4 Hz), 7.46 (t, 1H, CH aromatic, J =

10.12 Hz), 7.60–7.65 (m, 4H, CH aromatic), 7.72 (d, 1H, CH aromatic, J

–

5.1.1. Materials

= 8.4 Hz), 7.86 (t, 1H, CH aromatic, J = 10.0 Hz), 8.09 (d, 1H, CH CH,

–

Compounds 3 [30], 5,6 [31] and 9 [32] were synthesized acorrding

to the reported procedures.

J = 8.4 Hz), 8.20 (d, 1H, CH aromatic, J = 7.6 Hz), 12.21 (s, 1H, NH,

exch. D₂O). ¹³C NMR δ ppm: 14.66 (CH3), 55.79 (OCH3), 78.57 (CH2),

113.08, 114.38, 115.02, 115.45, 119.18, 121.44, 126.32, 127.37,

5.1.2. General procedure for the synthesis of compounds (7a-e)

128.14, 128.92, 129.75, 130.46, 138.41, 149.62, 152.52, 161.07 (aro-

–

The appropriate amino derivative (10 mmole) was added to a solu-

tion of (E)-4-chloro-2-(4-methoxystyryl) quinazoline 6 (1.23 g, 10

mmole) in dry DMF (15 mL) in presence of potassium carbonate (1.23 g,

10 mmole), except for compounds 7a and 7c that were stirred in iso-

propanol and ethylene glycol, respectively. The reaction mixture was

then reﬂuxed for 24 h and poured onto ice/water, then the solution was

acidiﬁed with acetic acid (pH 7). The resulting precipitate was ﬁltered,

washed with water, dried and re-crystallized from aqueous ethanol.

matic carbons), 121.11, 134.85 (2 oleﬁnic Cs), 162.64 (C O). Mass (m/

–

z): 425 (M⁺, 1.06), 70 (100). Anal. Calcd for C₂₅H₂₁N₃O₃(425.17): C,

72.98; H, 5.14; N, 10.21. Found: C, 72.69; H, 5.82; N, 10.44%.

5.1.2.5. (E)-N-(4-((2-(4-Methoxystyryl)quinazolin-4-yl)amino)phenyl)

acetamide (7e). Yield 60%; mp 250–252 ^◦C IR (ν_max/cm^{ꢀ 1}): 3444, 3280

(NHs), 3120, 2924 (CH aromatic, CH aliphatic), 1674 (CO). ¹H NMR δ

ppm: 2.00 (s, 3H, CH₃), 3.82 (s, 3H, OCH₃), 6.65 (d, 2H, CH aromatic, J

–

= 8.0 Hz), 6.86 (d, 1H, CH CH, J = 16.0 Hz), 6.91 (d, 2H, CH aromatic,

–

5.1.2.1. (E)-4-((2-(4-Methoxystyryl)quinazolin-4-yl)amino)phenol (7a).

Yield 64%; mp 202–204 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3363, 3248 (OH, NH),

3132, 2924 (CH aromatic, CH aliphatic). ¹H NMR δ ppm: 3.63 (s, 3H,

J = 8.0 Hz), 7.03 (d, 2H, CH aromatic, J = 8.0 Hz), 7.44 (t, 1H, CH

aromatic, J = 16.0 Hz), 7.62–7.67 (m, 3H, CH aromatic), 7.78 (t, 1H, CH

–

aromatic, J = 16.0 Hz), 7.90 (d, 1H, CH CH, J = 16.0 Hz), 8.10 (d, 1H,

–

CH aromatic, J = 8.0 Hz), 9.62 (s, 1H, NH, exch. D₂O). ¹³C NMR δ ppm:

24.51 (CH₃), 55.80 (OCH₃), 114.76, 115.04, 119.00, 121.42, 123.93,

126.32, 127.50, 128.09, 129.28, 129.77, 137.39, 139.29, 152.34,

157.89, 161.11, 162.83 (aromatic Cs), 122.52, 134.93 (2 oleﬁnic Cs),

OCH₃), 5.92 (s, 1H, OH, exch. D₂O), 6.59 (d, 2H, CH aromatic, J = 8.4

–

Hz), 6.66 (d, 1H, CH CH, J = 14.4 Hz), 6.78 (d, 2H, CH aromatic, J =

8.4 Hz), 7.02 (d, 2H, CH aromatic, J = 8.8 Hz), 7.16 (d, 2H, CH aromatic,

J = 8.8 Hz), 7.42 (t, 1H, CH aromatic, J = 9.8 Hz), 7.56 (d, 1H, CH

aromatic, J = 8.0 Hz), 7.74 (t, 1H, CH aromatic, J = 15.34 Hz), 8.06 (d,

1H, CH aromatic, J = 7.8 Hz), 9.46 (s, 1H, NH, exch. D₂O). ¹³C NMR δ

ppm: 55.86 (OCH₃), 115.35, 115.95, 116.25, 121.09, 124.69, 124.88,

126.14, 126.31, 127.04, 130.58, 142.58, 149.43, 152.89, 154.58,

155.65, 162.22 (aromatic Cs), 123.34, 134.73 (2 oleﬁnic Cs). Mass (m/

z): 369 (M⁺, 9.74), 159 (100). Anal. Calcd for C₂₃H₁₉N₃O₂(369.42): C,

74.78; H, 5.18; N, 11.37. Found: C, 75.01; H, 5.34; N, 11.63%.

+

–

168.20 (C O). Mass (m/z): 395 (M , 9.07), 54 (100). Anal. Calcd for

–

C

₂₅H₂₁N₃O₂(395.45): C, 75.93; H, 5.35; N, 10.63. Found: C, 75.69; H,

5.57; N, 10.86%.

5.1.3. General procedure for the synthesis of compounds (8a-e)

A mixture of 4-chloro-2-(4-methoxystyryl)quinazoline (6) (0.89 g,

10 mmole) and appropriate sulphonamide derivative (10 mmole) was

reﬂuxed in ethanol (10 mL) in presence of anhydrous potassium car-

bonate (1.38 g, 10 mmole) for 24 h. The reaction mixture was then

cooled, poured onto ice/water and acidiﬁed with acetic acid (pH 6). The

resulting precipitate was ﬁltered, washed with water, dried and re-

crystallized from aqueous ethanol.

5.1.2.2. (E)-N-(4-Chlorophenyl)-2-(4-methoxystyryl)quinazolin-4-amine

(7b). Yield 65%; mp 226–228 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3263 (NH), 3186,

2927 (CH aromatic, CH aliphatic). ¹H NMR δ ppm: 3.82 (s, 3H, OCH₃),

–

6.79 (d, 1H, CH CH, J = 16.4 Hz), 6.96 (d, 2H, CH aromatic, J = 8.4

–

Hz), 7.49 (d, 2H, CH aromatic, J = 8.8 Hz), 7.55 (t, 1H, CH aromatic, J =

15.6 Hz), 7.61 (d, 2H, CH aromatic, J = 7.6 Hz), 7.75 (d, 1H, CH aro-

5.1.3.1. (E)-N-((4-((2-(4-Methoxystyryl)quinazolin-4-yl)amino)phenyl)

sulfonyl)acetamide (8a). Yield 72%; mp 238–240 ^◦C. IR (ν_max/cm^{ꢀ 1}):

3368, 3186 (NHs), 3132, 2954 (CH aromatic, CH aliphatic), 1674 (CO),

–

matic, J = 8.0 Hz), 7.79–7.86 (m, 2H, CH aromatic, CH CH), 7.97 (d,

2H, CH aromatic, J = 8.0 Hz), 8.06 (d, 1H, CH aromatic, J = 7.6 Hz),

9.85 (s, 1H, NH, exch. D₂O). ¹³C NMR δ ppm: 55.71 (OCH₃), 114.81,

115.01, 121.48, 123.58, 126.11, 126.91, 127.35, 128.16, 128.82,

129.72, 134.75, 138.99, 151.04, 157.55, 160.38, 161.05 (aromatic Cs),

123.53, 133.62 (2 oleﬁnic Cs). Mass (m/z): 387 (M⁺, 86.3), 386 (100).

Anal. Calcd for C₂₃H₁₈ClN₃O (387.86): C, 71.22; H, 4.68; N, 10.83.

Found: C, 70.89; H, 4.84; N, 11.15%.

1300, 1149 (SO₂).¹H NMR δ ppm: 2.09 (s, 3H, CH₃), 3.81 (s, 3H, OCH₃),

–

6.86 (d, 1H, CH CH, J = 16.0 Hz), 7.01 (d, 2H, CH aromatic, J = 8.8

–

Hz), 7.43 (t, 1H, CH aromatic, J = 14.8 Hz), 7.61–6.66 (m, 5H, CH ar-

omatic), 7.75 (d, 2H, CH aromatic, J = 8.4 Hz), 7.78 (t, 1H, CH aromatic,

–

J = 14.4 Hz), 7.88 (d, 1H, CH CH, J = 16.00 Hz), 8.09 (d, 1H, CH

–

aromatic, J = 7.6 Hz), 10.40 (s, 1H, NH, exch. D₂O). ¹³C NMR δ ppm:

11

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

–

24.57 (CH₃), 55.78 (OCH₃), 115.02, 118.89, 119.08, 121.44, 126.14,

126.32, 127.13, 127.36, 128.11, 129.75, 134.45, 138.46, 149.59,

152.41, 161.08, 162.43 (aromatic Cs), 122.50, 134.85 (2 oleﬁnic Cs),

CH CH, J = 15.6 Hz), 6.96 (d, 2H, CH aromatic, J = 8.8 Hz), 7.10 (d,

–

2H, CH aromatic, J = 8.8 Hz), 7.43 (t, 1H, CH aromatic, J = 14.4 Hz),

7.55–7.62 (m, 4H, CH aromatic), 7.65 (d, 1H, CH aromatic, J = 8.0 Hz),

+

–

170.02 (C O). Mass (m/z): 475 (M , 6.19), 255 (100). Anal. Calcd for

7.70 (t, 1H, CH aromatic, J = 17.6 Hz), 7.88 (d, 1H, CH CH, J = 12.4

–

C₂₅H₂₂N₄O₄S (474.53): C, 63.28; H, 4.67; N, 11.81. Found: C, 63.06; H,

Hz), 8.11 (d, 1H, CH aromatic, J = 7.6 Hz), 9.86, 10.31 (s, 2H, NH, exch.

D₂O). ¹³C NMR δ ppm: 24.54 (2CH₃), 55.74 (OCH₃), 114.90, 115.03,

118.30, 118.94, 124.33, 126.32, 127.39, 128.08, 128.73, 129.57,

129.77, 132.28, 138.54, 149.55, 152.26, 160.68, 161.11, 162.31,

172.56 (aromatic Cs), 121.42, 134.92 (2 oleﬁnic Cs). Mass (m/z): 539

(M⁺, 34.16), 55 (100). Anal. Calcd for C₂₉H₂₆N₆O₃S (538.62): C, 64.76;

H, 4.87; N, 15.60. Found: C, 64.52; H, 5.12; N, 15.89%.

4.85; N, 12.04%.

5.1.3.2. (E) - N- carbamimidoyl ꢀ 4-((2- (4-methoxystyryl) quinazolin- 4-

yl) amino) benzene sulfonamide (8b). Yield 68%; mp 260–262 ^◦C. IR

(

ν_max/cm^{ꢀ 1}): 3402, 3344 (NHs, NH2), 3224, 2954 (CH aromatic, CH

aliphatic), 1678 (CO), 1307, 1130 (SO₂).¹H NMR δ ppm: 3.79 (s, 3H,

OCH₃), 5.70 (s, 1H, NH, exch. D₂O), 6.56 (d, 2H, CH aromatic, J = 4.2

–

5.1.4. Synthesis of N-(4-Chlorophenyl)-2-methylquinazolin-4-amine (11)

Para-chloroaniline (1.23 g, 10 mmole) was added to a solution of 4-

chloro-2-methylquinazoline 9 (1.23 g, 10 mmole) in DMF (15 mL) in

presence of potassium carbonate (1.23 g, 10 mmole). The reaction

mixture was reﬂuxed for 24 h and poured onto ice/water, then the so-

lution was acidiﬁed with acetic acid (pH 7). The resulting precipitate

was ﬁltered, washed with water, dried and recrystallized from aqueous

ethanol. Yield 70%; mp 270–272 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3398 (NH), 3186,

2931 (CH aromatic, aliphatic). ¹H NMR δ ppm: 2.35 (s, 3H, CH₃), 7.37

(d, 2H, CH aromatic, J = 8.8 Hz), 7.42 (t, 1H, CH aromatic, J = 14.8 Hz),

7.55 (d, 1H, CH aromatic, J = 8.4 Hz), 7.62 (d, 2H, CH aromatic, J = 8.8

Hz), 7.74 (t, 1H, CH aromatic, J = 15.2 Hz), 8.06 (d, 1H, CH aromatic, J

= 7.6 Hz), 8.55 (s, 1H, NH, exch. D₂O). Mass (m/z): 270 (M+, 45), 167

(100). Anal. Calcd for C₁₅H₁₂ClN₃(269.73): C, 66.79; H, 4.48; N, 15.58.

Found: C, 66.51; H, 4.67; N, 15.84%.

Hz), 6.93 (d, 1H, CH CH, J = 16.0 Hz), 6.98 (d, 2H, CH aromatic, J =

8.4 Hz), 7.41(d, 2H, CH aromatic, J = 8.4 Hz), 7.61 (d, 2H, CH aromatic,

J = 8.8 Hz), 7.70 (t, 1H, CH aromatic, J = 16.0 Hz), 7.78 (d, 1H, CH

aromatic, J = 8.0 Hz), 7.86 (t, 1H, CH aromatic, J = 16.0 Hz), 7.95 (d,

–

1H, CH CH, J = 16.0 Hz), 8.08 (d, 1H, CH aromatic, J = 8.4 Hz), 8.53

(s, 1H, NH₂, exch. D₂O), 9.86 (s, 1H, NH, exch. D₂O). ¹³C NMR δ ppm:

55.75 (OCH₃), 112.82, 114.81, 114.93, 121.37, 126.28, 127.29, 127.70,

129.55, 129.70, 131.27, 132.30, 134.48, 137.95, 151.79, 158.53,

160.75 (aromatic Cs), 121.57, 133.94 (2 oleﬁnic Cs), 175.57 (C = NH).

Mass (m/z): 475 (M⁺, 10.40), 81 (100). Anal. Calcd for C₂₄H₂₂N₆O₃S

(474.53): C, 60.75; H, 4.67; N, 17.71. Found: C, 60.54; H, 4.80; N,

17.53%.

5.1.3.3. (E)- 4- ((2- (4- Methoxystyryl) quinazolin- 4- yl) amino)- N- (5-

methylisoxazol- 3- yl) benzenesulfonamide (8c). Yield 70%; mp

272–274 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3471, 3387 (NHs), 3186, 2927(CH aro-

matic, CH aliphatic), 1465, 1172 (SO₂).¹H NMR δ ppm: 2.31 (s, 3H,

5.1.5. General procedure for the synthesis of compounds (12a-e)

The appropriate aldehyde (20 mmole) was added to a solution of N-

(4-chlorophenyl)-2-methylquinazolin-4-amine 11 (2.7 g, 10 mmole) in

glacial acetic acid (10 mL) in presence of sodium acetate (1.6 g, 20

mmole) and reﬂuxed for 24 h. The product was poured onto ice/water,

ﬁltered off, dried and crystallized from ethanol to give the desired

compound.

CH₃), 3.83 (s, 3H, OCH₃), 6.22 (s, 1H, CH isoxazole), 6.85 (d, 1H,

–

CH CH, J = 16.0 Hz), 7.03 (d, 2H, CH aromatic, J = 8.8 Hz), 7.14 (d,

–

2H, CH aromatic, J = 8.0 Hz), 7.46 (t, 1H, CH aromatic, J = 14.4 Hz),

7.62 (d, 2H, CH aromatic, J = 8.8 Hz), 7.66 (d, 2H, CH aromatic, J = 8.4

Hz), 7.72 (d, 1H, CH aromatic, J = 8.4 Hz), 7.79 (t, 1H, CH aromatic, J =

–

14.8 Hz), 7.91 (d, 1H, CH CH, J = 15.6 Hz), 8.10 (d, 1H, CH aromatic,

–

J = 8.00 Hz), 12.29 (s, 1H, NH, exch. D₂O). ¹³C NMR δ ppm: 12.70

(CH₃), 55.78 (OCH₃), 99.89, 112.70, 114.86, 115.02, 119.08, 126.32,

127.36, 127.82, 128.11, 129.75, 135.17, 138.25, 138.46, 149.58,

152.42, 161.09, 162.43 (aromatic Cs), 121.44, 134.86 (2 oleﬁnic Cs).

Mass (m/z): 514 (M⁺, 4.22), 67 (100). Anal. Calcd for C₂₇H₂₃N₅O₄S

(513.57): C, 63.14; H, 4.51; N, 13.64. Found: C, 63.47; H, 4.78; N,

13.92%.

5.1.5.1. (E)-4-(2-(4-((4-Chlorophenyl)amino)quinazolin-2-yl)vinyl)

phenol (12a). Yield 64%; mp 388–390 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3417, 3341

(NH, OH), 3126, 2924 (CH aromatic, CH aliphatic). ¹H NMR δ ppm: 6.37

(d, 2H, CH aromatic, J = 8.4 Hz), 6.72 (d, 2H, CH aromatic, J = 8.0 Hz),

–

6.95 (d, 1H, CH CH, J = 15.6 Hz), 7.07 (d, 2H, CH aromatic, J = 8.4

Hz), 7.40–7.44 (m, 3H, CH aromatic), 7.54 (d, 1H, CH aromatic, J = 7.6

–

Hz), 7.72 (t, 1H, CH aromatic, J = 16.0 Hz), 7.90 (d, 1H, CH CH, J =

16.0 Hz), 8.05 (d, 1H, CH aromatic, J = 7.6 Hz), 8.52 (s, 1H, NH, exch.

D₂O), 9.40 (s, 1H, OH, exch. D₂O). ¹³C NMR δ ppm: 115.50, 119.02,

121.83, 125.96, 126.15, 126.76, 129.76, 131.04, 132.92, 149.60,

163.22, 167.06, 175.57, 188.23 (aromatic Cs), 121.05, 134.42 (2

oleﬁnic Cs). Mass (m/z): 374 (M⁺, 18.9), 124 (100). Anal. Calcd for

5.1.3.4. (E) ꢀ 4- ((2- (4 -Methoxystyryl) quinazolin- 4-yl) amino)- N-

(pyrimidin-2- yl) benzenesulfonamide (8d). Yield 68%; mp 268–270 ^◦C.

IR (ν_max/cm^{ꢀ 1}): 3425, 3356 (NHs), 3105, 2935 (CH aromatic, CH

aliphatic), 1442, 1153 (SO₂). ¹H NMR δ ppm: 3.80 (s, 3H, OCH₃), 6.02

(d, 2H, CH aromatic, J = 8.0 Hz), 6.56 (t, 1H, CH aromatic, J = 16.0 Hz),

–

C

₂₂H₁₆ClN₃O (373.83): C, 70.68; H, 4.31; N, 11.24. Found: C, 70.94; H,

6.85 (d, 1H, CH CH, J = 16.0 Hz), 7.03 (d, 2H, CH aromatic, J = 8.0

–

4.47; N, 11.56%.

Hz), 7.45 (t, 1H, CH aromatic, J = 16.0 Hz), 7.62–7.67 (m, 5H, CH ar-

–

omatic), 7.79 (t, 1H, CH aromatic, J = 16.0 Hz), 7.91 (d, 1H, CH CH, J

–

5.1.5.2. (E)-N-(4-Chlorophenyl)-2-(4-chlorostyryl)quinazolin-4-amine

= 16.0 Hz), 8.10 (d, 1H, CH aromatic, J = 8.0 Hz), 8.49 (d, 2H, CH

aromatic, J = 4.4 Hz), 11.29 (s, 1H, NH, exch. D₂O), 12.28 (s, 1H, NH,

exch. D₂O). ¹³C NMR δ ppm: 55.79 (OCH₃), 112.51, 114.10, 114.86,

115.02, 118.97, 126.32, 126.39, 127.35, 127.87, 128.09, 129.79,

129.84, 132.29, 138.54, 149.52, 152.55, 158.34, 159.72, 161.10 (aro-

matic Cs), 121.41, 134.92 (2 oleﬁnic Cs). Mass (m/z): 511 (M⁺, 5.14), 69

(100). Anal. Calcd for C₂₇H₂₂N₆O₃S (510.57): C, 63.52; H, 4.34; N,

16.46. Found: C, 63.19; H, 4.47; N, 16.80%.

(12b). Yield 65%; mp 301–303 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3309 (NH), 3186,

1

–

2931 (CH aromatic, CH aliphatic). H NMR δ ppm: 7.00 (d, 1H, CH CH,

J = 16.4 Hz), 7.17 (d, 2H, CH aromatic, J = 8.2 Hz), 7.24 (d, 2H, CH

aromatic, J = 8.0 Hz), 7.47–7.53 (m, 3H, CH aromatic), 7.68–7.70 (m,

3H, CH aromatic), 7.81 (t, 1H, CH aromatic, J = 15.2 Hz), 7.91 (d, 1H,

–

CH CH, J = 16.0 Hz), 8.11 (d, 1H, CH aromatic, J = 7.6 Hz), 12.33 (s,

–

1H, NH, exch. D₂O). ¹³C NMR δ ppm: 115.90, 122.32, 126.35, 126.83,

127.59, 129.58, 129.78, 130.34, 134.68, 135.04, 137.35, 151.72,

156.26, 162.23 (aromatic Cs), 121.57, 134.39 (2 oleﬁnic Cs). Mass (m/

z): 392 (M⁺, 12.5), 75 (100). Anal. Calcd for C₂₂H₁₅Cl₂N₃(392.28): C,

67.36; H, 3.85; N, 10.71. Found: C, 67.04; H, 4.12; N, 11.04%.

5.1.3.5. (E)-N- (4, 6- dimethylpyrimidin ꢀ 2-yl) ꢀ 4- ((2- (4- methox-

ystyryl) quinazolin-4- yl) amino) benzenesulfonamide (8e). Yield 65%;

mp 229–231 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3471, 3356 (NHs), 3186, 2931 (CH

aromatic, CH aliphatic), 1419, 1172 (SO₂).¹H NMR δ ppm: 2.04 (s, 6H,

2CH₃), 3.84 (s, 3H, OCH₃), 6.80 (s, 1H, CH aromatic), 6.83 (d, 1H,

12

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

5.1.5.3. (E)-N-(4-Chlorophenyl)-2-(4-nitrostyryl)quinazolin-4-amine

benzenesulfonamide 14 (3.9 g, 10 mmole) in glacial acetic acid (10 mL)

in presence of sodium acetate (1.6 g, 20 mmole). The reaction mixture

was reﬂuxed for 24 h. The product was then poured onto ice/water,

ﬁltered off, dried and re-crystallized from ethanol to give the desired

compound.

(12c). Yield 60%; mp 356–358 ^◦C. IR (

νmax/cm-1): 3305 (NH), 3124,

2927 (CH aromatic, CH aliphatic). ¹H NMR δ ppm: 7.19–7.25 (m, 3H,

–

CH CH, CH aromatic), 7.54 (d, 2H, CH aromatic, J = 8.0 Hz), 7.72 (t,

–

1H, CH aromatic, J = 16.0 Hz), 7.85 (d, 1H, CH aromatic, J = 8.0 Hz),

–

7.94 (d, 2H, CH aromatic, J = 8.4 Hz), 8.02–8.09 (m, 2H, CH CH, CH

–

aromatic), 8.14 (d, 1H, CH aromatic, J = 8.0 Hz), 8.30 (d, 2H, CH ar-

omatic, J = 8.0 Hz), 12.44 (s, 1H, NH, exch. D₂O). ¹³C NMR δ ppm:

116.86, 124.69, 125.89, 126.39, 127.21, 127.80, 129.11, 131.12,

135.11, 136.26, 141.95, 148.04, 151.26, 162.09 (aromatic Cs), 121.76,

134.04 (2 oleﬁnic Cs). Mass (m/z): 402 (M+, 5.96), 293 (100). Anal.

Calcd for C₂₂H1₅ClN₄O₂(402.38): C, 65.59; H, 3.75; N, 13.91. Found: C,

65.31; H, 3.98; N, 4.25%.

5.1.7.1. (E)-4- ((2-(4-Hydroxystyryl) quinazolin ꢀ 4- yl) amino) –N- (5-

methylisoxazol ꢀ 3- yl) benzenesulfonamide (15a). Yield 64%; mp

283–285 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3468, 3332 (NHs, OH), 3066, 2924(CH

aromatic, CH aliphatic), 1465, 1126 (SO₂).¹H NMR δ ppm: 2.36 (s, 3H,

CH₃), 6.08 (s, 1H, CH aromatic), 6.58 (d, 2H, CH aromatic, J = 8.8 Hz),

–

6.83 (d, 1H, CH CH, J = 12.8 Hz), 6.93 (d, 2H, CH aromatic, J = 8.4

–

Hz), 7.12 (s, 1H, OH, exch. D₂O), 7.44–7.52 (m, 3H, CH aromatic), 7.57

(d, 1H, CH aromatic, J = 7.6 Hz), 7.62 (t, 1H, CH aromatic, J = 10.8 Hz),

–

5.1.5.4. (E)-N-(4-Chlorophenyl)-2-(4-dimethylaminostyryl)quinazolin-4-

7.76 (d, 2H, CH aromatic, J = 8.4 Hz), 7.86 (d, 1H, CH CH, J = 14.4

–

amine (12d). Yield 62%; mp 275–277 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3479 (NH),

Hz), 8.07 (d, 1H, CH aromatic, J = 7.2 Hz), 9.33, 9.79 (s, 2H, 2NH, exch.

D₂O). ¹³C NMR δ ppm: 12.50 (CH₃), 95.74, 113.05, 115.60, 115.90,

116.32, 123.14, 123.52, 126.15, 126.31, 128.88, 129.02, 129.25,

130.95, 142.89, 158.56, 163.83, 170.25, 191.41 (aromatic Cs), 120.67,

132.58 (2 oleﬁnic Cs). Mass (m/z): 500 (M⁺, 11.5), 57 (100). Anal. Calcd

for C₂₆H₂₁N₅O₄S (499.54): C, 62.51; H, 4.24; N, 14.02. Found: C, 62.83;

H, 4.51; N, 14.13%.

3055, 2931 (CH aromatic, CH aliphatic). ¹H NMR δ ppm: 2.85 (s, 6H,

–

2CH ), 6.55 (d, 2H, CH aromatic, J = 8.8 Hz), 6.63 (d, 1H, CH CH, J =

–

12.0³Hz), 6.67 (d, 2H, CH aromatic, J = 8.8 Hz), 7.04 (d, 2H, CH aro-

matic, J = 8.4 Hz), 7.15 (d, 2H, CH aromatic, J = 8.4 Hz), 7.44 (t, 1H, CH

aromatic, J = 14.8 Hz), 7.57 (d, 1H, CH aromatic, J = 7.6 Hz), 7.68 (d,

–

1H, CH CH, J = 16.0 Hz), 7.75 (t, 1H, CH aromatic, J = 14.8 Hz), 8.07

–

(d, 1H, CH aromatic, J = 8.0 Hz), 9.68 (s, 1H, NH, exch. D₂O).¹³C NMR δ

ppm: 30.77 (2CH3), 112.90, 114.65, 115.02, 119.45, 119.84, 120.82,

126.02, 126.99, 128.41, 128.73, 129.10, 131.80, 142.80, 149.33,

161.77, 163.12 (aromatic carbons), 121.48, 134.22 (2 oleﬁnic Cs). Mass

(m/z): 401 (M⁺, 16.0), 134 (100). Anal. Calcd for C₂₄H₂₁ClN₄(400.90):

C, 71.90; H, 5.28; N, 13.98. Found: C, 71.76; H, 5.49; N, 13.71%.

5.1.7.2. (E)-4- ((2-(4- Chlorostyryl) quinazolin- 4-yl) amino) -N- (5-

methylisoxazol-3- yl) benzenesulfonamide (15b). Yield 62%; mp

290–292 ^◦C. IR (

νmax/cm-1): 3464, 3179 (NHs), 3061, 2920 (CH aro-

matic, CH aliphatic), 1404, 1118 (SO2).¹H NMR δ ppm: 2.30 (s, 3H,

CH3), 6.12 (s, 1H, CH aromatic isoxazole), 6.58 (d, 2H, CH aromatic, J

–

= 8.4 Hz), 7.01 (d, 1H, CH CH, J = 16.4 Hz), 7.46 (d, 2H, CH aromatic,

–

5.1.5.5. (E)-4-(2-(4-((4-Chlorophenyl)amino)quinazolin-2-yl)vinyl)-2-

methoxyphenol (12e). Yield 58%; mp 204–206 ^◦C. IR (ν_max/cm^{ꢀ 1}):

3402, 3271 (NH, OH), 3127, 2924 (CH aromatic, CH aliphatic). ¹H NMR

δ ppm: 3.67 (s, 3H, OCH₃), 4.43 (s, 1H, OH, exch. D₂O), 6.24 (d, 2H, CH

aromatic, J = 8.0 Hz), 6.55 (d, 1H, CH aromatic, J = 8.0 Hz), 6.67 (d,

J = 8.4 Hz), 7.50 (m, 3H, CH aromatic), 7.69 (m, 3H, CH aromatic), 7.80

–

(t, 1H, CH aromatic, J = 14.8 Hz), 7.92 (d, 1H, CH CH, J = 16.0 Hz),

–

8.11 (d, 1H, CH aromatic, J = 7.6 Hz), 10.95, 12.38 (s, 2H, 2NH, exch.

D2O). ¹³C NMR δ ppm: 12.53 (CH3), 98.80, 113.49, 115.77, 116.10,

116.53, 123.04, 125.82, 126.35, 126.55, 127.46, 129.56, 129.73,

130.96, 134.75, 137.02, 149.52, 154.41, 162.81, 175.42 (aromatic Cs),

121.67, 134.55 (2 oleﬁnic Cs). Mass (m/z): 518 (M+, 36.72), 144 (100).

Anal. Calcd for C₂₆H₂₀ClN₅O₃S (517.99): C, 60.29; H, 3.89; N, 13.52.

Found: C, 60.18; H, 4.17; N, 13.41%.

1H, CH aromatic, J = 8.0 Hz), 6.89 (s, 1H, CH aromatic), 7.02 (d, 1H,

–

CH CH, J = 16.0 Hz), 7.10 (d, 2H, CH aromatic, J = 8.0 Hz), 7.40–7.43

–

(m, 2H, CH aromatic, CH CH), 7.52 (d, 1H, CH aromatic, J = 8.0 Hz),

7.71 (t, 1H, CH aromatic, J = 15.2 Hz), 8.06 (d, 1H, CH aromatic, J =

8.0 Hz), 9.28 (s, 1H, NH, exch. D₂O).¹³C NMR δ ppm: 55.04 (OCH₃),

107.78, 114.34, 115.69, 117.58, 118.69, 125.86, 126.15, 126.70,

127.21, 128.94, 131.10, 149.64, 151.66, 156.19, 163.56, 170.12,

186.92 (aromatic Cs), 121.04, 134.31 (2 oleﬁnic Cs). Mass (m/z): 404

(M⁺, 15.1), 151 (100). Anal. Calcd for C₂₃H₁₈ClN₃O₂(403.86): C, 68.40;

H, 4.49; N, 10.40. Found: C, 68.53; H, 4.62; N, 10.73%.

5.1.7.3. (E)-N- (5-Methylisoxazol ꢀ 3-yl) -((2 -(4- nitrostyryl) quinazolin-

4- yl) amino) benzenesulfonamide (15c). Yield 60%; mp 320–322 ^◦C. IR

(

ν_max/cm^{ꢀ 1}): 3479, 3178 (NHs), 3055, 2924 (CH aromatic, CH

aliphatic), 1512, 1103 (SO2). ¹H NMR δ ppm: 2.35 (s, 3H, CH₃), 5.33 (s,

–

1H, CH aromatic), 7.18 (d, 1H, CH CH, J = 16.0 Hz), 7.49 (d, 2H, CH

aromatic, J = 6.4 Hz), 7.62 (d, 1H, CH aromatic, J = 7.2 Hz), 7.71 (d,

5.1.6. Synthesis of N-(5-methylisoxazol-3-yl) ꢀ 4- ((2-methylquinazolin-4-

yl) amino) benzenesulfonamide (14)

2H, CH aromatic, J = 7.2 Hz), 7.79 (t, 1H, CH aromatic, J = 15.2 Hz),

–

7.91 (d, 1H, CH aromatic, J = 8.4 Hz), 8.01 (d, 1H, CH CH, J = 16.0

A mixture of 4-chloro-2-methylquinazoline 9 (0.89 g, 10 mmole) and

sulfamethoxazole (0.56 g, 10 mmole) was reﬂuxed in ethanol (10 mL) in

presence of anhydrous potassium carbonate (1.38 g, 10 mmole) for 24 h.

The reaction mixture was then cooled, poured onto ice/water and

acidiﬁed with acetic acid (pH 6). The resulting precipitate was ﬁltered,

washed with water, dried and re-crystallized from ethanol. Yield 72%;

mp 237–239 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3468, 3394 (NHs), 3066, 2981(CH

aromatic, CH aliphatic), 1465, 1138 (SO₂). ¹H NMR δ ppm: 2.26 (s, 3H,

CH₃-CO), 2.36 (s, 3H, CH₃), 6.04 (s, 1H, CH aromatic), 6.56 (d, 2H, CH

aromatic, J = 8.4 Hz), 7.44–7.59 (m, 3H, CH aromatic), 7.57 (d, 1H, CH

aromatic, J = 8.0 Hz), 7.76 (t, 1H, CH aromatic, J = 16.0 Hz), 8.07 (d,

1H, CH aromatic, J = 8.00 Hz), 12.26 (s, 1H, NH, exch. D₂O). Mass (m/

z): 395 (M⁺, 1.8), 92 (100). Anal. Calcd for C₁₉H₁₇N₅O₃S (395.43): C,

57.71; H, 4.33; N, 17.71. Found: C, 58.04; H, 4.57; N, 17.62%.

Hz), 8.09 (t, 1H, CH aromatic, J = 16 Hz), 8.21 (d, 1H, CH aromatic, J =

8.4 Hz), 8.27 (d, 2H, CH aromatic, J = 8.4 Hz), 10.15 (s, 1H, NH, exch.

D₂O). ¹³C NMR δ ppm: 12.55 (CH3), 95.37, 113.59, 114.33, 116.00,

116.38, 119.08, 124.29, 125.33, 126.00, 126.66, 127.36, 129.10,

133.55, 135.65, 141.45, 146.26, 147.99, 151.46, 166.87 (aromatic Cs),

122.25, 134.60 (2 oleﬁnic Cs). Mass (m/z): 529 (M+, 3.26), 90 (100).

Anal. Calcd for C₂₆H₂₀N₆O₅S (528.54): C, 59.08; H, 3.81; N, 15.90.

Found: C, 59.27; H, 4.03; N, 16.14%.

5.1.7.4. (E)-4-((2-(4-Hydroxy-3-methoxystyryl)quinazolin-4-yl)amino)-

N-(5-methylisoxazol-3-yl)benzenesulfonamide (15d). Yield 58%; mp

345–347 ^◦C. IR (ν_max/cm^{ꢀ 1}): 3417, 3332 (NHs, OH), 3066, 2962(CH

aromatic, CH aliphatic), 1465, 1126 (SO₂). ¹H NMR δ ppm: 2.30 (s, 3H,

CH₃), 3.82 (s, 3H, OCH₃), 6.11 (s, 1H, CH aromatic), 6.60 (m, 3H, CH

–

aromatic), 6.86 (d, 1H, CH CH, J = 16.0 Hz), 7.04 (d, 1H, CH aromatic,

–

5.1.7. General procedure for the synthesis of compounds (15a-d)

The appropriate aldehyde (20 mmole) was added to a solution of N-

(5-methylisoxazol-3-yl)-4-((2-methylquinazolin-4-yl)amino)

J = 8.0 Hz), 7.40–7.49 (m, 3H, CH aromatic), 7.59–7.65 (m, 2H, CH

–

aromatic), 7.80 (t, 1H, CH aromatic, J = 15.2 Hz), 7.91 (d, 1H, CH CH,

–

J = 16.0 Hz), 8.12 (d, 1H, CH aromatic, J = 8.0 Hz), 10.96 (s, 1H, NH,

13

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

exch. D₂O), 12.29 (s, 1H, OH, exch. D₂O). ¹³C NMR δ ppm: 12.52 (CH3),

55.79 (OCH3), 95.76, 113.05, 113.91, 115.04, 118.89, 124.59, 126.34,

126.44, 127.49, 128.06, 129.30, 129.79, 132.62, 138.55, 149.62,

152.22, 153.75, 158.46, 161.12, 162.27, 170.31 (aromatic carbons),

121.42, 134.98 (oleﬁnic Cs). Mass (m/z): 529 (M⁺, 10.5), 43 (100).

Anal. Calcd for C₂₇H₂₃N₅O₅S (529.27): C, 61.24; H, 4.38; N, 13.22.

Found: C, 60.98; H, 4.50; N, 13.45%.

the standard reference drug (Lapatinib) were added to the cells. The

control cells without the test compounds were also cultured, then the

plate was incubated. Cells survival was evaluated at the end of the in-

cubation period via MTT colorimetric assay. After incubation, media

were removed and each vial of MTT [M-5655] were reconstituted to be

used with 3 mL of medium or balanced salt solution without phenol red

and serum. Reconstituted MTT was added in an amount equal to 10% of

the culture medium volume and incubated for an additional 4 h. The

crystals of formazan were dissolved by adding an amount of MTT Sol-

ubilization Solution [M-8910] and the optical density was measured

spectrophotometrically at 570 nm. The experiments were performed in

three replicates for each concentration of compound; the results were

normalized to the control value and expressed as percentage of control.

The compound concentrations which give 50% growth inhibition are

5.2. In vitro cytotoxic screening

All of the synthesized quinazoline derivatives were chosen for in vitro

anti-proliferative screening at single concentration of 10 mM for

approximately 60 human tumor cell lines panel derived from nine

neoplastic diseases (lung, blood, CNS, colon, skin, kidney, ovary, pros-

tate and breast) at the U.S. National Cancer Institute. The detailed

procedure of the NCI anticancer screening has been described (Sup-

plementary M) according to the reference article [33-35]. Brieﬂy,

human tumor cells are inoculated into 96 well microtiter plates in 100

referred to as the IC₅₀

.

Cell viability (% of control) = Absorbance of test - Absorbance of

medium × 100/ Absorbance of control

μ

L at plating densities ranging from 5000 to 40,000 cells/well depend-

5.4. Epidermal growth factor receptor enzyme inhibition assay

ing on the doubling time of individual cell lines. After cell inoculation,

the microtiter plates are incubated for 24 h prior to addition of experi-

mental drugs. After 24 h, two plates of each cell line are ﬁxed in situ with

trichloroacetic acid (TCA). Experimental drugs are solubilized in

dimethyl sulfoxide and at the time of addition, an aliquot of frozen

concentrate is thawed and diluted to twice the desired ﬁnal maximum

5.4.1. In vitro EGFR enzyme inhibition assay

The in vitro EGFR enzyme inhibition assay was performed at conﬁr-

matory diagnostic unit, VACSERA, Egypt for selected compounds (7a,

7b, 8c, 8e, 12e and 15b). The evaluation was carried out by ELISA assay

method using EnzyChromTM Kinase Assay Kit (EKIN-400) with lapati-

nib as a reference drug [36,37].

test concentration with complete medium containing 50 μg/mL genta-

micin. Additional four, 10-fold or ½ log serial dilutions are made to

provide a total of ﬁve drug concentrations plus control. Aliquots of these

different drug dilutions are added to the appropriate microtiter wells

5.4.2. In vitro EGFR enzyme inhibition assay of target compounds against

EGFR-expressed in A549 cell line

already containing 100

μ

L of medium, resulting in the required ﬁnal

The assay was carried out according to ELISA assay using Human Bax

ELISA (EIA-4487) kits at conﬁrmatory diagnostic unit at VACSERA.

Brieﬂy, A549 cell line was incubated with IC₅₀of each compound for 2 h

then EGFR concentration and the concentration of each sample was

determined by comparing the optical density of the samples to the

standard curve using lapatinib as a reference drug; the exact method-

ology was mentioned in supplementary M [37].

drug concentrations. Following drug addition, the plates are incubated

for an additional 48 h. For adherent cells, the assay is terminated by the

addition of cold TCA. Cells are ﬁxed in situ by the gentle addition of 50

μ

L of cold 50% (w/v) TCA (ﬁnal concentration, 10% TCA) and incubated

for 60 min at 4 ^◦C. The supernatant is discarded, and the plates are

washed ﬁve times with tap water and air dried. Sulforhodamine B (SRB)

solution is added to each well, and plates are incubated for 10 min at

room temperature. After staining, unbound dye is removed and the

plates are air dried. Bound stain is subsequently solubilized with 10 mM

trizma base, and the absorbance is read on an automated plate reader at

a wavelength of 515 nm. For suspension cells, the methodology is the

same except that the assay is terminated by ﬁxing settled cells at the

5.5. Cellular mechanism of action study

5.5.1. Cell cycle analysis

Cell cycle analysis was carried out for the most active compound 8c

at conﬁrmatory diagnostic unit at VACSERA using Propidium Iodide kit

and analyzed by ﬂow cytometry technique. A549 cell line was treated

with tested compounds with their IC₅₀for 24 h, then washed, collected

by centrifugation, suspended with RNase, stained with (PI) and analyzed

by ﬂow cytometry using FACS Calibur reader, compared to the control

(cell line without drug treatment) [38].

bottom of the wells by gently adding 50 μL of 80% TCA (ﬁnal concen-

tration, 16% TCA). Using the seven absorbance measurements [time

zero, (Tz), control growth, (C), and test growth in the presence of drug at

the ﬁve concentration levels (Ti)], the percentage growth is calculated at

each of the drug concentrations levels followed by growth inhibition of

50% (GI₅₀) calculation. The drug concentration resulting in total growth

inhibition (TGI). Values are calculated for each of the three parameters if

the level of activity is reached; however, if the effect is not reached or is

exceeded, the value for that parameter is expressed as greater or less

than the maximum or minimum concentration tested.

5.5.2. Cell apoptosis assay

Apoptosis assay was consequently carried out at conﬁrmatory diag-

nostic unit at VACSERA using Annexin V-FITC Apoptosis detection kit. A

biparametric cytoﬂuorimetric analysis was performed using Propidium

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

immunolabeling of the protein annexin-V, which binds to phosphati-

dylserine (PS) expressed on the surface of the apoptotic cells and ﬂuo-

resces green after interacting with the labeled annexin-V. In brief, A549

was treated with tested compounds at their IC₅₀, incubated for 24 h,

5.3. Cytotoxicity potential assay study (MTT assay)

The most active compounds (7a, 7b, 8c, 8e, 12e and 15b) were

further tested for cytotoxicity on non-small lung cancer cell line (A549)

and normal lung ﬁbroblasts (WI38), obtained from the American Type

Culture Collection (Rockville, MD, USA) at conﬁrmatory diagnostic unit

in VACSERA-Egypt for IC₅₀determination. The detailed procedure was

explained (Supplementary M), according to Mosmann methodology

[36]. Brieﬂy, tumor cells were cultured and then plated in a volume of

collected, washed with cold PBS, then stained with 5 μL of Annexin V-

FITC and 5 μL of propidium iodide, incubated at room temperature for 5

min and ﬁnally analyzed by ﬂow cytometry using FACS Caliber detector

[39].

100

μ

L, growth medium + 100 ul of the tested compound per well in a

5.5.3. Active Caspase-3 expression level assay

96-well plate were completed for 24 h before the MTT (3-(4,5-dime-

thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Then, various

To assess caspase-3 activity, the manufacturer’s instructions for the

human active caspase-3 content assay kit (supplementary M) were

followed carefully at conﬁrmatory diagnostic unit at VACSERA through

concentration of test compounds (0.39, 1.56, 6.25, 25 and 100 μg) and

14

N.H. Amin et al.

Bioorganic Chemistry 105 (2020) 104358

injection of cells with IC₅₀of tested compound 8c using ROBONIK

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Design, synthesis and molecular modeling studies of 2-styrylquinazoline derivatives as EGFR inhibitors and apoptosis inducers

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