Hybrid Quinazoline 1,3,5-Triazines as Epidermal Growth Factor Receptor (EGFR) Inhibitors with Anticancer Activity: Design, Synthesis, and Computational Study

Accepted Article

Title: Design, synthesis, and computational study of hybrid quinazoline

1,3,5-triazines as epidermal growth factor receptor (EGFR)

inhibitors with anticancer activity

Authors: Prateek Pathak, Hrvoje Rimac, Maria Grishina, Amita Verma,

and Vladimir Potemkin

This manuscript has been accepted after peer review and appears as an

Accepted Article online prior to editing, proofing, and formal publication

of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.202000646

Link to VoR: https://doi.org/10.1002/cmdc.202000646

01/2020

10.1002/cmdc.202000646

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Design, synthesis, and computational study of hybrid quinazoline

1,3,5-triazines as epidermal growth factor receptor (EGFR)

inhibitors with anticancer activity

Prateek Pathak,*^[a]Hrvoje Rimac,*^[a,b]Maria Grishina,^[a]Amita Verma,^[c]Vladimir Potemkin^[a]

[a]

[b]

[c]

Dr. P. Pathak, Dr. H. Rimac, Dr. M. Grishina, Dr. V. Potemkin

Laboratory of Computational Modelling of Drugs

South Ural State University

Chaikovskogo 20A, Chelyabinsk, 454008, Russia

patkhakp@susu.ru, rimatsk@susu.ru, grishinama@susu.ru, potemkinva@susu.ru

Dr. H. Rimac

Department of Medicinal Chemistry

University of Zagreb Faculty of Pharmacy and Biochemistry

Ante Kovacica 1, Zagreb, 10000, Croatia

hrvoje.rimac@pharma.unizg.hr

Dr. A. Verma

Bioorganic and Medicinal Chemistry Research Laboratory, Department of Pharmaceutical Sciences

Sam Higginbottom University of Agriculture, Technology & Sciences

Naini, Prayagraj, Uttar Pradesh 211007, India

amita.verma@shiats.edu.in

Abstract: We report a series of hybrid quinazoline-1,3,5-triazine

derivatives as EGFR inhibitors, which were synthesized and tested

using a variety of in vitro, in silico, and in vivo studies. The derivatives

were found to be active against different cancer and non-toxic against

normal cell lines, with compounds 7c, 7d, 7e, and 7j being the most

potent ones. The derivatives were also evaluated for angiogenesis

inhibition potency in chicken eggs and molecular docking and

dynamics simulation studies were carried out to elucidate

fundamental substituent groups essential for their bioactivity.

Additionally, a SAR study of the derivatives was performed for future

compound optimization. These studies suggested that the derivatives

have a high affinity towards EGFR with favourable pharmacological

properties. The most active compound (7e) was further evaluated for

in vivo anticancer activity against DMBA induced tumour in female

Sprague-Dawley rats as well as its effects on plasma antioxidant

status, biotransformation enzymes, and lipid profile. The study

suggested that the 7e compound has lead properties against breast

cancer and can serve as a starting compound for further development

of anti-EGFR compounds.

molecules. Through their effects on cell proliferation,

differentiation, apoptosis, invasion, and angiogenesis, a normal

organ development takes place. EGFR signalling in tumour cells,

as opposed to normal cells, is changed and often becomes

dysregulated, where EGFR is overexpressed and/or obtains a

gain-of-function mutation.^[6–11]This behaviour leads to

proliferation of tumour cells where they invade the surrounding

tissue. This results in an increased angiogenesis.^[12,13]Ras-Raf-

mitogen activated protein kinase (MAPK) and phosphatidylinositol

3-kinase (PI3K)/Akt pathways^[14]are the two main interconnected

downstream pathways influenced by EGFR intracellular signalling.

The over-expression of these signalling pathways acts as a

driving force in advanced stages of malignant carcinogenesis (e.g.

breast, colon, and bladder cancers, and some chordomas)^[15–17]

(Figure 1). Therefore, in rational drug design, EGFR inhibition is

considered as one of the prime targets in cancer treatment.^[18]As

a result of intensive research, several EGFR inhibitors have been

developed and are divided into generations based on their activity,

e.g. erlotinib (Tarceva^®)^[17], vandetanib (Caprelsa^®)^[19], gefitinib

(Iressa^®)^[20], lapatinib (Tykerb^®, Tyverb^®),^[21]and the current state

of the art, osimertinib (Tagrisso^®)^[22], to name a few (Figure 2A).

EGFR inhibitors can either be reversible or irreversible, with

reversible inhibitors binding in the ATP binding pocket and

irreversible inhibitors binding either to the Cys 797 residue in the

ATP binding pocket or to allosteric binding site(s).^[23–26]

Introduction

Several enzymes are involved in signal recognition, transduction

and amplification and oversee cell growth and division. Among

them, tyrosine kinases (TKs) are considered as one of the most

significant ones^[1,2]due to their versatile activity, such as cell

proliferation, metabolism, and apoptosis. Therefore, irregularity in

TK function is one of the mechanisms of tumour growth and

progression.^[3]

Based on their function, TKs can be categorized either as

epidermal growth factor receptor (EGFR) or non-receptor

kinases.^[4]EGFR is a trans-membrane protein and belongs to the

erbB/HER-family of TKs. These receptors consist of an

extracellular ligand-binding domain, a cysteine-binding domain,

an intracellular domain, and an alpha-helix transmembrane

domain with TK activity within the carboxy-terminus (except for

HER3).^[5]EGFRs participate in transcription of regulatory

Quinazoline and 1,3,5-triazine scaffolds are important classes of

N-heterocyclic compounds that have raised a great interest in

cancer chemotherapeutics.^[27–32]Our team has designed and

synthesized

a number of quinazoline based 1,3,5-triazine

derivatives as effective TK inhibitors^[31,32](Figure 2B), where

entire series of compounds exhibited significant activities (with

IC₅₀ranging from 12.5 ± 0.01 to 43.30 ± 0.51 μM against MCF-7

and 8.8 ± 0.1 to 29.37 ± 0.08 μM against TPC-1 cell lines). It was

found from the SAR studies that introduction of chloro-, bromo-,

and nitro- groups into the phenyl ring improves the anticancer

properties of the derivatives. Additionally, a 3D QSAR study

concluded that the aliphatic bridge between the quinazoline and

the 1,3,5-triazine ring acts as an antipharmacophoric group.

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with a few exceptions,^[23,44]targeting EGFR has not been

extensively studied.^[37]

Results and Discussion

Chemistry

We designed and synthesized a series of compounds by

conjugating two different heterocyclic scaffolds, the 1,3,5-triazine

and the quinazoline rings. The entire synthesis is shown in

Scheme 1. The first step (reaction a) includes an acetylation

reaction, where 2-amino-4,5-dibromo-benzoic acid (1) was

refluxed with acetic anhydride and the intermediate compound

6,7-dibromo-2-methyl-benzo[d][1,3]oxazin-4-one (2) is obtained.

In the next step (reaction b), compound 2 undergoes a

nucleophilic substitution reaction in the presence of p-

phenylenediamine. As a result, the oxygen atom from the oxazin-

4-one ring in compound 2 is replaced by a nitrogen atom and

compound

3

(3-(4-amino-phenyl)-6,7-dibromo-2-methyl-3H-

quinazolin-4-one) is obtained. Another, parallel reaction (reaction

c) is also carried out through a nucleophilic substitution of one of

the chlorine atoms from cyanuric chloride (4) in the presence of

piperidine, resulting in a formation of 2,4-dichloro-6-piperidin-1-yl-

[1,3,5]triazine (compound 5). Reaction d is also a nucleophilic

substitution reaction where another chlorine atom of compound 5

is replaced by various aliphatic and aromatic amines. As a result,

di-substituted 1,3,5-triazine derivatives (6a-n) were obtained. In

the final step (reaction e), 3-(4-amino-phenyl)-6,7-dibromo-2-

methyl-3H-quinazolin-4-one (3) and di-substituted 1,3,5-triazine

Figure 1. EGFR signalling pathway.

derivatives (6a-n) are conjugated through

a nucleophilic

substitution reaction in the presence of tin as a catalyst and the

derivatives (7a-n) are formed. Structural conformations of the

derivatives were done by ¹H NMR, ¹³C NMR and elemental

analysis.

Further docking studies showed that the selectivity of molecules

for TKs would be attained by making use of the supplementary

hydrophobic pocket located in the back of the ATP-binding site by

inclusion of other functional groups. The results were found to be

similar to studies of other researches against kinase

inhibitors.^[33,34]Based on these results, in this research our

attempt was to focus on the development of novel anticancer

agents containing both the quinazoline and the 1,3,5-triazine

rings.^[31,32]This was obtained through molecular hybridization of

the aforementioned rings, while the final molecules were obtained

via substitution on the 1,3,5-triazine ring. The reasoning was that

a substituted 1,3,5-triazine ring would fit nicely into the large

hydrophobic cavity of the EGFR active site.

The evolution of EGFR inhibitors, as with all drugs in cancer

treatment, is at least partly driven by cancer resistance to existing

drugs. Therefore, having several structurally different compounds

that bind to the same receptor could be beneficial in preventing

cancer resistance or in cancer treatment after the resistance has

already occurred.^[35,36]Here we also mention in passing that there

are two additional way of targeting EGFRs, by using monoclonal

antibodies directed against the extracellular receptor domain,

such as cetuximab,^[37]or using PROteolysis TArgeting Chimeras

(PROTACs). PROTACs are heterobifunctional molecules, which

consist of a small molecule (binder to the protein target of interest),

a ligand (recruiter of the E3 ligase), and a linker to conjugate the

two moieties.^[38,39]Binding of the PROTAC leads to selective

polyubiquitination of the target protein and its subsequent

degradation at the proteasome. This technology has been

employed in degradation of numerous proteins^[40–43], however,

Anticancer activity

In vitro cytotoxicity of the entire series of the synthesized

derivatives was evaluated for various cell lines, i.e. MCF-7 (breast

cancer), HeLa (cervical cancer), HepG2 (hepatocellular

carcinoma), HL-60 (human promyelocytic leukemia), HFF (human

foreskin fibroblasts), and MCF-12A (normal epithelial cells) using

the MTT assay, with erlotinib as reference. Erlotinib IC₅₀values

are in accordance with its previously reported in-cell target

engagement values,^[15,45,46]which gives validity to the obtained

results. IC₅₀values of all the derivatives were calculated and are

displayed in Table 1. The 7a derivative (with urea substituted

1,3,5-triazine ring) showed a marginal potency against MCF-7

(IC₅₀= 23.4 ± 0.1 μM), HeLa (IC₅₀= 24.2 ± 0.2 μM), HepG2 (IC₅₀

= 35.4 ± 0.6 μM), and HL-60 (IC₅₀= 24.3 ± 0.1 μM), respectively.

This potency was even more decreased by replacing urea with

thiourea (7b). On the other hand, addition of an aromatic amine

such as aniline (7c) improves the anticancer activity against all

the selected cancer cell lines. Additionally, the anticancer potency

was marginally increased (except for HepG2) by introduction of

chlorine in the phenyl ring (7d). This indicates that incorporation

of a group that resonantly donates electrons (e.g. Cl) in the phenyl

ring improves potency against breast and leukemic cancers.

Replacement of the p-chloro-aniline group by a morpholine ring

(7e) additionally improves the activity against all tested cell lines

(MCF-7 (IC₅₀= 17.2 ± 0.1 μM), HeLa (IC₅₀= 17.3 ± 0.1 μM),

HepG2 (IC₅₀= 15.1 ± 0.2 μM), and HL-60 (IC₅₀= 14.3 ± 0.1 μM)),

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F

(A)

F

O

N

O

HN

N

Cl

N

Cl

N

O

N

NH

F

Br

O

N

O

N

HN

N

Gefitinib

NH

Cl

Erlotinib

O

NH

N

U-CH₁IC₅₀= 1.4 µM

U-CH₂IC₅₀= 23 µM

EGFR-K_D= 1.0 nM

U-CH₁IC₅₀= 5.1 µM

U-CH₂IC₅₀= 18 µM

EGFR-K_D= 0.7 nM

N

F

O

NH

N

O

Vandetanib

Afatinib

S

Lapatinib

First generation

GAK-K_D= 86 nM

O

U-CH₁IC₅₀= 0.1 µM

U-CH₂IC₅₀= 6.4 µM

EGFR-K_D= 0.3 nM

U-CH₁IC₅₀= 3.2 µM

U-CH₂IC₅₀= > 50 µM

EGFR-K_D= 2.4 nM

EGFR-K_D= 9.5 nM

Second generation

O

N

S

NH

O

N

F

O

P

NH

N

NH

O

N

NH

Cl

O

N

HO

N

Brigatinib

2-(5-fluoro-2-hydroxyphenyl)-2-

(1-oxoisoindolin-2-yl)-N-(thiazol-2-yl)acetamide

Osimertinib

EGFR-K_i= 2.2 nM

Fourth generation

Third generation

(B)

NH

N

NH

N

Cl

N

O

N

O

Br

NH

N

NH

N

NH

N

6-bromo-N-(4-((4-chlorophenyl)amino)-6-(piperazin-1-yl)-1,3,5-

triazin-2-yl)-2-methyl-4-oxoquinazoline-3(4H)-carboxamide

6-bromo-2-methyl-4-oxo-N-(4-(phenylamino)-6-(piperazin-1-

yl)-1,3,5-triazin-2-yl)quinazoline-3(4H)-carboxamide

8c

8d

Figure 2. (A) Tyrosine kinase (EGFR) inhibitors^[15,47,48], and (B) the most active compounds previously synthesized by our team.

Scheme 1. The reaction scheme for the synthesized derivatives: a) acetic anhydride, reflux for 4 hours, b) p-phenylenediamine, pyridine, reflux for 3 hours, c)

piperidine, acetone, NaHCO₃(10%), stir for 3-4 hours below 5 °C, d) various amines, acetone, NaHCO₃(10%), stir for 4 hours at room temperature, e) 1,4-dioxane,

K₂CO₃, tin granules, reflux for 6-8 hours.

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but a piperidine ring (7f) lowers potency against all tested cell

lines. This further confirms that the presence of electron donating

substituents (in this case oxygen, and, in the case of derivative

7d, chlorine) increases the anticancer potency. A derivative with

a methoxy-aniline substituted 1,3,5-triazine ring (7g) showed a

slight decrease in anticancer potency, compared to compounds

7e and other aniline substituted derivatives (7c and 7d), but was

found to be more potent than compounds with an aliphatic

substitution, such as 7a and 7b. Further, compounds 7h and 7i

(thiosemicarbazide and semicarbazide substituted 1,3,5-triazine

derivatives, respectively) had a significantly lower activity against

all tested cell lines, probably due to their basic nature or simply

due to their polarity. In compounds 7j and 7k (bromo- and nitro-

substituted 1,3,5-triazine derivatives, respectively) we can again

see the role of an aromatic substitution, which increases

anticancer potency (not applicable for the 7n derivative and its p-

amino benzoic acid (PABA) substituent). Briefly, thiourea,

groups with a halogen (fluoro-anilino group) on the triazine

scaffold is necessary for the anticancer activity. Furthermore, they

concluded that the presence of an electron donating group

composed of non-halogen substituents significantly decreases

the anticancer efficacy.^[49]This is in accordance with our study,

which has demonstrated that a halogen containing phenyl ring

(bromo- and nitro-phenyl groups) acts as a key fragment in

increasing the anticancer potential. These results show an

interesting connection between substituents and their anticancer

activity. The selectivity of the derivatives between normal and

cancerous cells was also evaluated using two normal cells lines

(HFF and MCF-12A) and the results showed that most of the

derivatives (not including 7g, 7j, and 7n at higher concentrations)

were non-toxic for normal cell lines (Table 1).

EGFR inhibitory activity

In vitro EGFR inhibition was performed for the entire set of

methylamino, amino, and PABA substituted derivatives 7b, 7l, 7m, derivatives and results are given as IC₅₀and compared to the

and 7n expressed the lowest efficacy among the entire series,

while derivatives 7c, 7d, 7e, and 7j were found to be the most

active. The assays also showed that the potency of some

derivatives (especially derivative 7e) is very similar to erlotinib’s

potency, even though erlotinib still remained the most active

compound. A closer look into the structure-activity relationship

shows that introduction of open aliphatic amino functional groups,

such as in derivatives 7a, 7b, 7h, 7i, 7l, and 7m results in a slightly

lower anticancer efficiency as compared to aromatic substitutions

on the 1,3,5-triazine ring (7c, 7d, 7j, and 7k), which is in

accordance with a previous study.^[15]Additionally, it was illustrated

that the presence of an electronegative group (i.e. Cl, Br, and

NO₂) is crucial for the activity. This was also shown by Srivastava

et al., who determined that presence of electron withdrawing

reference drug (erlotinib) (Table 2). It can be seen that some

derivatives have a moderate to excellent inhibitory potential, with

derivatives 7e, 7j, and 7d being the most potent (with IC₅₀= 36.8

± 0.5 nM, 36.6 ± 0.8 nM, and 41.0 ± 0.7 nM, respectively).

Furthermore, a slightly lower inhibition was reported for the 7c

(IC₅₀= 49.6 ± 0.4 nM) and 7k (IC₅₀= 58.8 ± 0.6 nM) derivatives.

For derivatives 7f and 7g, IC₅₀decreases to 65.2 ± 0.8 nM and

68.9 ± 0.5 nM respectively, followed by derivatives 7a, 7b, 7h, 7i,

7l, 7m, and 7n (IC₅₀> 100 nM in all cases). Erlotinib’s IC₅₀was

found to be 31 nM, which is in accordance with earlier studies,^[32]

and allows comparison of our results with results of other

studies.^[15]This study also suggests that introduction of an

electronegative group helps to improve the anti-EGFR activity,

very similarly to the in vitro anticancer activity assay. Even though

Table 1. In vitro anticancer effect of the synthesized derivatives in selected cell lines.

In vitro anticancer activity (IC₅₀in μM)

Derivative

MCF-7

HeLa

HepG2

HL60

HFF

MCF-12A

7a

7b

23.4 ± 0.1

36.0 ± 0.4

18.7 ± 0.2

18.2 ± 0.5

17.2 ± 0.1

19.6 ± 0.2

21.5 ± 0.2

23.7 ± 0.4

22.5 ± 0.2

17.8 ± 0.4

19.4 ± 0.1

39.5 ± 0.5

25.7 ± 0.2

25.3 ± 0.2

16.7 ± 0.7

24.2 ± 0.2

24.7 ± 0.1

19.2 ± 0.2

18.0 ± 0.5

17.3 ± 0.1

20.5 ± 0.4

22.0 ± 0.6

23.2 ± 0.2

24.4 ± 0.1

17.5 ± 0.1

20.3 ± 0.1

32.2 ± 0.1

24.3 ± 0.3

25.4 ± 0.4

17.3 ± 0.2

35.4 ± 0.6

43.3 ± 0.2

15.8 ± 0.3

16.5 ± 0.1

15.1 ± 0.2

18.5 ± 0.1

19.0 ± 0.1

36.8 ± 0.4

30.8 ± 0.2

15.2 ± 0.1

12.7 ± 0.3

45.4 ± 0.4

32.5 ± 0.6

36.7 ± 0.4

12.6 ± 0.1

24.3 ± 0.1

26.3 ± 0.2

16.9 ± 0.2

16.5 ± 0.3

14.3 ± 0.1

18.2 ± 0.1

21.2 ± 0.1

24.8 ± 0.4

22.2 ± 0.2

14.8 ± 0.1

18.6 ± 0.1

30.2 ± 0.1

22.8 ± 0.4

25.0 ± 0.1

13.7 ± 0.3

> 100

7c

> 100

7d

> 100

7e

> 100

7f

> 100

7g

74.0 ± 0.1

> 100

78.9 ± 0.7

> 100

7h

7i

> 100

7j

78.6 ± 0.4

> 100

7k

> 100

7l

> 100

7m

7n

> 100

72.6 ± 0.5

70.1 ± 0.5

78.1 ± 0.5

> 100

Erlotinib

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the inhibition of other kinases (off-target effects) was not tested

and therefore cannot be excluded, based on the similarity of anti-

cancer activity and EGFR inhibition results, it can be safe to say

that the synthesized derivatives engage the EGFR and produce

their anticancer activity mostly through its inhibition. While the

EGFR IC₅₀values reflect the intrinsic sensitivity of the enzyme to

the inhibitors, the IC₅₀values obtained for different cell lines are a

result of a complicated interplay between the inhibitors and the

cells, involving different physical, chemical and biological factors,

resulting in lower inhibitor activity.^[15,50–52]Additional effects of the

7e compound are discussed in the “In vivo pharmacology” section.

Antiangiogenic activity in MCF-7 inoculated chicken CAM

Since migration of cancer cells enhances the number of newly

formed blood vessel branch points compared to the PBS‐treated

control group, the synthesized 1,3,5-triazine derivatives (at

concentration of 0.01 nM) were evaluated for their antiangiogenic

activity against MCF-7 inoculated chicken embryos. The results

are reported in term of score (Figure 3), as described in the

Experimental Section. The entire set of derivatives showed

moderate to significant angiogenesis inhibition scores (from 0.6 ±

0.1 to 1.9 ± 0.1), compared to erlotinib, which had a score of 1.87

± 0.03. Briefly, the 7e derivative, which was found to be the most

promising compound in the MTT and EGFR assays, was also the

most potent in this assay, with a score of 1.90 ± 0.06. This was

followed by derivatives 7j (1.90 ± 0.02), 7d (1.84 ± 0.02), 7c (1.82

± 0.07), and 7k (1.50 ± 0.07). The rest of the derivatives showed

either moderate or low inhibition (~50%) compared to erlotinib,

with derivative 7l being the least potent one (0.6 ± 0.1). As in the

previous results, these results also demonstrate the importance

of cyclic amine substituents (such as morpholine and piperidine

Figure 3. Antiangiogenic activity of synthesized derivatives at 0.01 nM against

MCF-7 inoculated CAM. Derivatives marked with asterisk (*) have a statistically

different (p < 0.05) inhibition score than erlotinib.

rings) or electron donating groups in the 1,3,5-triazine scaffold, as

opposed to the open chain aliphatic amino groups.

Docking studies

First, redocking of erlotinib was performed using the 4HJO PDB

structure (resolution 2.75 Å). However, a relatively weak binding

constant (-7.9 kcal/mol) with a non-correct pose was found, and

erlotinib in the correct crystallographic pose was found to bind

with a binding constant of -6.6 kcal/mol. Docking of erlotinib was

also tried with the 1XKK structure, but yielded another incorrect

pose, but with identical binding constant of -7.9 kcal/mol. This

shows that the docking procedure was not able to correctly

determine the correct pose and binding energy of erlotinib,

probably due to its flexible aliphatic chains. Since the tested

derivatives are more similar to lapatinib, redocking of lapatinib

was also tried for the 1XKK complex. Comparison of the redocked

pose with the crystallographic structure is shown in Figure 4. As

can be seen, there is virtually no difference between lapatinib

redocked poses obtained under different docking settings (tan)

and their energies (ranging from -11.0 to -10.7 kcal/mol) and the

crystallographic pose (light blue). The most significant difference

is in the orientation of the flexible chain which is located outside

Table 2. IC₅₀values and docking results for EGFR inhibition by synthesized

derivatives.

K_BIND

(kcal/mol)

Derivative

IC₅₀(nM)

7a

7b

197.2 ± 0.8

255.1 ± 0.8

49.6 ± 0.4

41.0 ± 0.7

36.8 ± 0.5

65.2 ± 0.8

68.9 ± 0.5

201.1 ± 0.6

108.1 ± 0.4

36.6 ± 0.8

58.8 ± 0.6

108.2 ± 0.4

178.4 ± 0.9

246.2 ± 0.6

31.1 ± 0.5

-10.0

-9.6

7c

-11.4

-11.6

-11.4

-11.1

-9.9

7d

7e

7f

7g

7h

7i

-10.4

-11.7

-11.2

-9.4

7j

7k

7l

7m

7n

-10.1

-9.9

Figure 4. Docked poses of lapatinib in its charged and neutral state and without

water molecules and in presence of HOH 4 and HOH 22 water molecules (tan)

compared to the lapatinib’s crystallographic orientation (light blue).

erlotinib

-10.9

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Figure 5. Comparison of the best docking poses for ligands 7j (tan), 7d (light

blue), 7c (pink), and 7e (green) with crystallographic structure of lapatinib (red)

and five of the most important amino acid residues (gray).

of the binding pocket. Due to this, docking of other ligands was

conducted in their most prevalent moieties at pH 7.4, but in

absence of all water molecules. Under these conditions, the

calculated lapatinib binding energy was -10.9 kcal/mol. Docking

results for other ligands are shown in Figure 5 and Table 2. It is

immediately noticeable that there is no significant difference in

their poses. This is expected, as the difference in their structures

is also minimal. The most significant difference can be seen in the

case of ligand 7e and its morpholine ring. Compared to the other

three ligands, which have an (un)-substituted phenyl ring,

morpholine ring is neither planar nor aromatic and is also bulkier.

However, this does not pose a major disadvantage in binding

because, as can be seen from Figure 5, the part of the binding

site which accommodates these substituent groups is not buried

inside the protein. These obtained docking poses (erlotinib and

ligands 7c, 7d, 7e, and 7j) were then used in MD simulations to

check the validity of the obtained results, as docking can

sometimes deliver unstable poses,^[32]as well as to determine the

contribution of individual amino acid residues to ligand binding.

Figure 6. Mass weighted root-mean-square deviation (RMSD) through time for

ligands 7j (black), 7d (red), 7c (green), and 7e (blue) bound to EGFR.

case of the complex with ligand 7e, the number of H-bonds

increases over time (also discussed later). Binding energies for all

complexes were calculated using the MM-GBSA protocol (Table

3) to obtain a further insight into the EGFR-ligand binding. While

in the docking studies, binding affinity decreased in the order 7j >

7d > 7c > 7e, in the MD simulations the order is 7e > 7j > 7d > 7c

(while erlotinib’s affinity was calculated to be -49.82 kcal/mol, and

is situated between compounds 7e and 7j, for complete MM-

GBSA results see Supplementary Information).

From Table 3 it can be seen that all 4 derivatives have very similar

interactions with nearby amino acid residues, but what

differentiates the 7e derivative are its stronger hydrophobic

interactions with the two most contributing residues, Leu 718 and

Val 726, as well as a much stronger interaction (mostly van der

Waals and electrostatic in nature) with the Cys 797 residue (0.8 –

1.18 kcal/mol stronger interaction compared to the other three

residues). This residue was previously identified as the key

residue and is the target of covalent EGFR inhibitors.^[24,53–55]

Interactions with other key residues, such as Lys 745, Thr 790,

and Thr 854 seem to be approximately of the same strength.

These residues were recognized as key residues in lapatinib

binding,^[56]as well as binding of other EGFR inhibitors.^{[15,45,46,57]}

As a side note, it has to be pointed out that the calculated ΔG_bind

is overestimated in absolute terms, which is a known limitation of

the MM-GBSA method.^[58]This fact was extensively discussed by

Homeyer and Gohlke,^[59]which pointed out that, despite this

drawback, the method has a huge potential in predicting relative

binding energies in biomolecular complexes and can be used to

compare relative binding energies of structurally similar

compounds, as it is in this case. For this reason, the obtained

ΔG_bindof tested compounds should only be analysed relative to

each other.^[24,53–55]

MD simulations

Both docked erlotinib poses (using 1XKK and 4HJO EGFR

conformations) underwent MD simulations to see if they will

converge and be comparable with the crystallographic structure.

This was found to be true (data not shown), further emphasizing

the fact that one should be careful while interpreting docking

results. Additionally, these results validate the approach of using

MD simulations in obtaining correct binding poses for both

erlotinib and synthesized ligands, as well as in obtaining further

insight into their interactions with EGFR. Figure 6 shows mass-

weighted root-mean-square deviation (RMSD) of all four

complexes through time. It can be seen that some complexes

achieve equilibrium state earlier (7j and 7d), while for other

complexes it takes more time (7c and 7e). The graph also

indicates that in the case of the complex with the 7e ligand, the

most significant protein conformation change occurs, compared

to other ligands (discussed later). The lowest RMSD values are

found for the complex with the 7j ligand. This can also be seen in

the intermolecular H-bond graph (Figure 7). For the 7j ligand the

number of intermolecular H-bonds is relatively stable for the entire

duration of the simulation (with a fast drop in number of H-bonds

at the beginning of the simulation). The behaviour of complexes

with ligands 7d and 7c is similar, the only difference is that it takes

them more time to achieve the equilibrium state. However, in the

The discrepancy in the binding affinities obtained through docking

and MD simulations can be explained by the RMSD and H-bond

data. The protein conformation obtained by X-ray crystallography

favours binding of ligand 7e the least (compared to the other three

ligands), but if we let EGFR obtain its natural conformation in a

water solution through MD simulations, its conformation will

change in a way that favours 7e binding the most. This

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tested ligands. However, in the MD simulations, since the enzyme

is not constrained, it will adapt a conformation that lowers the

energy of the whole complex by, among other things, forming

additional H-bonds which are not present in the initial

crystallographic enzyme conformation, as can be seen in Figure

8. This fact also emphasizes the importance of choosing the most

appropriate protein conformation for docking studies, i.e. among

other points to consider (such as resolution of the PDB structure,

missing side chains, clashes, Ramachandran outliers, etc.), one

should ensure that the ligand from the PDB structure is

structurally as similar as can be with the tested ligands to ensure

the appropriateness of the binding site conformation and the

validity of the results.

If we compare ligand poses after docking (Figure 5) and after MD

simulations (Figure 8), we can see that the quinazoline part of the

ligands, which is inside the binding pocket, has virtually the same

conformation in all the ligands. As for the parts which are located

outside of the binding site, for ligands 7j, 7d and 7c, they remain

in the same orientation as in the docking studies, but in the case

of ligands 7e, this part of the molecule is rotated by approximately

180°. This is enabled by the change in the secondary structure of

protein residues 991–1012 (Figure 8A) and 718–723 (Figure 8B).

In the case of the complex with ligand 7j, residues 991–1012 are

located the closest to the binding pocket, compared to the other

complexes. Complexes with ligands 7c and 7d are intermediate

cases, and in the case of the 7e ligand these residues are located

the farthest away, enabling the rotation of the molecule and much

more favourable interactions. This rotation is additionally

supported by a change in conformation of residues 718–723

(Figure 8B). In the case of complex with the 7e ligand, these

residues are located closer to the ligand, resulting in bad contacts

and favouring the rotation of this part of the ligand. This can be

seen especially in the case of Gly 719: while its contribution to

ligand binding is significant for ligands 7j, 7d and 7c (-0.85 – -1.21

kcal/mol), for ligand 7e binding it has a less significant role (-0.16

kcal/mol). Additionally, all other residues in this segment

(residues 718–723) slightly disfavour ligand 7e binding.

Figure 7. Number of intermolecular hydrogen bonds through time for the best

four complexes.

conformational change is reflected in higher RMSD values for the

7e complex. Additionally, at the beginning of the simulation, the

number of intermolecular H-bonds for the 7e complex is the

lowest (the least favourable binding), but we can see that the

number of intermolecular H-bonds increases over time (while for

the other 3 complexes it decreases from the initial value). If we

consider the entire time of the simulations, the average number of

H-bonds for the 7e ligand is more than 3 times higher than for the

rest of the complexes, regardless of the initial lowest value (the

average number of intermolecular H-bonds decreases in order 7e

(1.07 ± 0.95) > 7c (0.29 ± 0.64) > 7d (0.28 ± 0.59) > 7j (0.23 ±

0.54)). This confirms the importance of enzyme conformation:

crystallographic conformation of the enzyme was suboptimal for

binding of the 7e ligand, resulting in the lowest affinity among the

Table 3. Contribution of top ten individual residues to ΔG_bind(kcal/mol) of tested ligands to EGFR.

Derivative 7c Derivative 7d Derivative 7e

Derivative 7j

Total

-41.8

Total

-43.27

Total

-52.79

Total

-46.65

Leu 718

Val 726

Lys 745

Cys 797

Leu 844

Thr 854

Leu 788

Gly 719

Ala 743

Thr 790

-2.9

-2.2

-2.0

-1.7

-1.6

-1.3

-1.1

-1.0

-0.9

Leu 718

Lys 745

Val 726

Cys 797

Leu 844

Thr 854

Thr 790

Leu 788

Ala 743

Gly 719

-2.83

-2.22

-2.13

-1.59

-1.54

-1.34

-1.11

-1.03

-0.91

-0.85

Leu 718

Val 726

Cys 797

Leu 844

Lys 745

Gly 796

Thr 790

Thr 854

Ile 744

-2.95

-2.62

-2.50

-1.67

-1.66

-1.65

-1.26

-1.19

-1.18

-1.04

Leu 718

Val 726

Lys 745

Leu 844

Thr 854

Cys 797

Gly 719

Thr 790

Leu 788

Ser 720

-2.60

-2.51

-2.16

-1.55

-1.32

-1.31

-1.21

-1.08

-1.06

Ala 743

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Figure 8. Poses of EGFR-ligand complexes after MD simulations with

secondary structure of protein residues 718–723 and 991–1012 shown. 7j

complex is depicted in tan, 7d in light blue, 7c in pink, and 7e in green: A) side

view, B) top view.

In summary, stronger binding of the 7e ligand compared to the

other three derivatives is mostly a result of a much stronger

interactions with the key Cys 797 residue, as well as

conformational EGFR changes brought about by the introduction

of the morpholine ring, which is not present in the case of the other

ligands.

Using SAR, docking and MD results, a valuable insight into the

pharmacophoric and physicochemical characteristics was

obtained. This will form a basis for further development of novel

hybrid quinazoline 1,3,5-triazines with even higher potency.

Figure 9. Effect of the 7e derivative on the body weight (A), tumour incidence

(B), and tumour volume (C) in different animal groups. A statistically significant

difference in tumour volume and incidence (p < 0.001) from the control is

marked by an asterisk (*).

In vivo pharmacology

Effect on body weight, percentage of tumour incidence and

tumour volume

In vitro anticancer activity, EGFR inhibitory activity, in ovo

angiogenesis inhibition, and docking and molecular dynamics

results of the 7e derivative encouraged us to further examine its

(Figure 10(A-D) and 11(A-D), respectively). These results clearly

show that the level of these enzymes in DMBA induced group was

significantly reduced (p < 0.001), compared to the control.

Additionally, the treated groups showed significant improvement

(p < 0.001) in the antioxidant status. Further, the correlation study

with tumour incidence and volume suggested that the 7e

derivative displays extensive protective properties against breast

cancer probably via scavenging of free radicals. Lipid

peroxidation parameters such as lipid hydroperoxides (LOOH)

and thiobarbituric acid reactive substances (TBARS) extensively

influence production of reactive oxygen species (ROS), play a

considerable role in the tumour progression, and are considered

critical in peroxidation of membrane lipids. For that reason, levels

of LOOH and TBARS were also measured (Figures 10(E-F) and

11(E-F), respectively). It can be clearly seen that in the DMBA

treated animal groups, LOOH and TBARS levels are significantly

elevated (p < 0.001), compared to the control group. So, the 7e

derivative actively participates in lowering the levels of these

markers and is correlated with an increase of antioxidant

parameters. In a study by Karki et al., it was established that the

free radical generation is a major cause of cell damage, with

enzymatic and non-enzymatic antioxidants acting as protective

mechanisms.^[60]In the present study, we also observed this

phenomenon. As the 7e derivative decreases the levels of free

radicals and oxidative stress, it has an additional benefit, and can

also serve as an indicator of cancer chemotherapy efficiency.

in

vivo

activity.

Therefore,

we

utilized

7,12-

dimethylbenz(a)anthracene (DMBA) to induce mammary cancer

in experimental animals to validate the defensive effect of the 7e

derivative. These effects were shown as an influence on body

weight of experimental animals (Figure 9A). The DMBA induced

group displayed a substantial decline in body weight as compared

to the control group. In the treated groups, body weight of the

animals has notably improved in a dose-dependent manner.

Additionally, the 7e derivative was also evaluated based on the

tumour volume and tumour incidence percentage (Figure 9B and

9C), which showed that the 7e derivative has exceptional

efficiency in reducing both tumour incidence and its volume. This

suggests that it has a broad and significant activity against

mammary cancer.

Effect on enzymatic and non-enzymatic antioxidants

This study aimed to determine the effect of the 7e derivative on

the antioxidant status in plasma and mammary tissue of

experimental animals. Various studies established that

antioxidants act as a crucial defence system against cancer.^[60]

Therefore, anti-breast cancer efficiency of the 7e derivative can

be also examined through the study of the endogenous

antioxidant system because in various stages of cancer, the

antioxidant system is greatly deregulated and compromised. The

results showed significant changes (p < 0.001) in chloramphenicol

acetyltransferase (CAT), glutathione peroxidase (GPX),

glutathione (GSH), and superoxide dismutase (SOD) levels

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Figure 11. Effect of the 7e derivative on the antioxidant status in the mammary

tissue of treated animals and control. A statistically significant difference in

means in comparison to control is marked by a symbol: * p < 0.001, ** p < 0.01,

^‡p > 0.05.

significantly higher (p < 0.001) in the DMBA treated group,

compared to the control group. However, level of GST and GR

enzymes were found to be significantly reduced (p < 0.001). The

drug treated group of animals illustrated a dose dependent

decline in the level of phase I enzymes (CYP and Cyt-b5) in liver

and in mammary tissue. Moreover, a significant rise (p < 0.001) in

GST and GR levels was reported in liver and mammary tissue.

These results imply that the 7e derivative has a notable role in the

modulation of levels of phase I and phase II enzymes.

Figure 10. Effect of the 7e derivative on the plasma antioxidant status of treated

animals. A statistically significant difference in means in comparison to control

is marked by a symbol: * p < 0.001, ** p < 0.01, ^‡p > 0.05.

Effects on biotransformation enzymes in liver microsomes and in

mammary tissue

Effect on the lipid profile level

This segment of the study included a comparison of lipid profiles

such as triglycerides (TG), total cholesterol (TC), high-density

lipoproteins (HDL), low-density lipoproteins (LDL), and very-low-

density lipoprotein (VLDL) (Figure 14). Since higher level of

triglycerides and LDL are associated with cancer and

metastasis,^[61]levels of these parameters were measured and

found to be significantly elevated (p < 0.001) in the DMBA treated

group, whereas they were decreased in the 7e treated group in a

dose dependent manner.

Metabolic enzymes are involved in the biotransformation process

and excretion of by-products generated by cancer causing agents.

It was observed that DMBA metabolism is influenced by CYP and

other phase I enzymes. These metabolites have a major role in

the induction of cancer and its related effects. Additionally,

biotransformation enzymes, such as GST and GR, which are

involved in the phase II metabolism, convert DMBA into water

soluble conjugates.^[49]Due to this, excretion of these metabolites

increases, and this acts as a protective mechanism in cancer

prevention. So, it was crucial to also see if the 7e derivative has

any influence on these enzymes. Expression of liver and

mammary tissues biotransformation enzymes, such as phase I

(cytochrome P450 (CYP), cytochrome-b5 (Cyt-b5)) and phase II

(glutathione S-transferase (GST), glutathione reductase (GR))

enzymes, in the treated and control animals are displayed in

Figures 12 and 13. The level of CYP and Cyt-b5 enzymes were

Conclusion

We report a synthesis of a series of hybrid quinazoline-1,3,5-

triazine derivatives and their anticancer activity. Pharmacological

evaluation indicated that substitution of the 1,3,5-triazine ring by

morpholine and aniline rings and their derivatives increases

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Figure 12. Effect of the 7e derivative on the level of biotransformation enzymes

in mammary tissue microsomes of treated and control group. A statistically

significant difference in means in comparison to control is marked by a symbol:

* p < 0.001, ** p < 0.01, ^‡p > 0.05.

Figure 14. Effect of the 7e derivative on the lipid level profile of treated and

control group A statistically significant difference in means in comparison to

control is marked by a symbol: * p < 0.01, ^#p < 0.05, ^‡p > 0.05.

mechanisms of action, such as modulating the antioxidant status

and biotransformation enzymes. In this regard, the 7e derivative

is an excellent lead compound with efficiency similar to that of

erlotinib. Since this compound is based on a different scaffold, it

represents an opportunity to develop structurally different EGFR

inhibitors, which could be used in the case of cancer resistance

to drugs already in use. Additional studies are in progress to

further optimise these drug-like characteristics and will be

reported in due course.

Figure 13. Effect of the 7e derivative on the level of biotransformation enzymes

in liver microsomes of treated and control animal groups

A statistically

significant difference in means in comparison to control is marked by a symbol:

** p < 0.001, * p < 0.01, ^#p < 0.05, and ^‡p > 0.05.

ligand potency in EGFR inhibition. Further, 3D QSAR studies

confirmed that introduction of aniline and its respective variants is

a potential way to increase the biological activity through

improving pharmacophoric properties. MD studies have shown

that all the tested ligands bind very strongly in the EGFR active

pocket, which is in accordance with the experimental results.

Their orientation in the active site is also quite similar, with only

exception being the 7e ligand. MD simulations have also shown a

drawback of docking studies in considering protein as a rigid

molecule. In vivo anticancer evaluation of 7e showed that it has a

potential to inhibit tumour growth and incidence through additional

Experimental Section

Chemistry

General

All experiments were carried out using commercial reagents and analytical

grade solvents without further purification. Melting points of the derivatives

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was determined using Veego (MPI melting point instrument). ¹H NMR and

¹³C NMR analysis were performed using JEOL 300 and Bruker Avance II

100 NMR spectrometers respectively, with CDCl3 as solvent and TMS as

the internal standard. Mass spectra was obtained using the VG-

AUTOSPEC spectrometer equipped with ESI source (Fisons Instruments,

Manchester, UK). Elemental analysis was carried out on Vario EL-III

CHNOS elemental analyser (Elementar Analysensysteme, Hanau,

Germany).

1-(4-((4-(6,7-dibromo-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)amino)-

6-(piperidin-1-yl)-1,3,5-triazin-2-yl)thiourea (7b)

Light brown solid; yield 75% (4.8 g, 7.44 mmol); molecular formula:

C₂₄H₂₃Br₂N₉OS; melting point: 210-212 ºC; molecular weight: 645.38; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

7.25 (d, 2H, J = 7.5 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 7.2 Hz, 2xCH, Ar), 7.49

(s, 1H, quinazoline), 8.06 (s, 1H, quinazoline), 8.34 (s, 1H, NH), 9.13 (s,

2H, NH₂), 9.65 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm: 23.7, 24.5,

25.5, 54.8, 117.7, 122.4, 124.5, 127.4, 128.8, 130.6, 134.5, 148.1, 154.1,

160.6, 165.7, 171.7, 176.0, 181.4; MS-ESI (m/z): 645.6 [M+H]⁺; elemental

analysis: calculated C: 44.67%, H: 3.59%, N: 19.53%; found C: 45.05%;

H: 3.25%; N: 19.90%.

Synthesis of 6,7-dibromo-2-methyl-4H-benzo[d][1,3]oxazin-4-one (2)

A mixture of 2-amino-4,5-dibromobenzoic acid (1) (0.05 mol) and acetic

anhydride (0.1 mol) was refluxed under anhydrous conditions for 4 hours.

The excess amount of acetic anhydride was distilled under reduced

pressure and reaction mixture was cooled to room temperature. The

intermediate benzoxazine-4-one was obtained as a solid mass and was

used immediately in the next step (melting point: 173-175 ºC, yield: 84%).

6,7-dibromo-2-methyl-3-(4-((4-(phenylamino)-6-(piperidin-1-yl)-1,3,5-

triazin-2-yl)amino)phenyl) quinazoline-4(3H)-one (7c)

Black solid; yield 75% (4.9 g, 7.40 mmol); molecular formula:

C₂₉H₂₆Br₂N₈O; melting point: 228-230 ºC; molecular weight: 662.39; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

7.01 (t, 1H, CH, Ar), 7.2 (d, 2H, J = 7.2 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 7.2

Hz, 2xCH, Ar), 7.35 (t, 2H, 2xCH, Ar), 7.49 (s, 1H, CH, quinazoline), 7.74

(d, 2H, J = 8.4 Hz, 2xCH, Ar), 8.34 (s, 1H, NH), 8.91 (s, 1H, NH); ¹³C NMR

(100 MHz, CDCl₃) δ ppm: 23.7, 24.5, 25.5, 54.8, 117.7, 122.4, 122.4, 124.5,

127.4, 128.8, 129.5, 130.6, 134.5, 134.5, 138.9, 148.1, 154.1, 160.6, 165.7,

168.9, 176; MS-ESI (m/z): 662.6 [M+H]⁺; elemental analysis: calculated C:

52.59%, H: 3.96%, N: 16.92%; found C: 52.94%, H: 3.65%, N: 16.72%.

Synthesis of 3-(4-aminophenyl)-6,7-dibromo-2-methylquinazolin-4(3H)-

one (3)

6,7-dibromo-2-methyl-4H-benzo[d][1,3]oxazin-4-one (2) (0.01 mol) was

refluxed with p-phenylenediamine (0.01 mol) in presence of pyridine (15

ml) for 3 hours with occasional shaking. The reaction mixture was then

cooled to room temperature. The crystals were filtered, rinsed with water,

and dried. The product formed was recrystallized from ethyl acetate. The

obtained product was a dark brown solid. Yield: 79% (3.2 g, 7.82 mmol);

molecular formula: C₁₅H₁₁Br₂N₃O; melting point: 220-222 ºC; molecular

weight: 409.08; ¹H NMR (300 MHz, CDCl₃, TMS) δ ppm: 2.26 (s, 3H, CH₃),

4.52 (s, 2H, NH₂), 6.85 (d, 2H, J = 6.9 Hz, 2xCH, Ar); 7.14 (d, 2H, J = 7.2

Hz, 2xCH, Ar); 7.49 (s, 1H, quinazoline), 8.062 (s, 1H, quinazoline); ¹³C

NMR (100 MHz, CDCl₃) δ ppm: 24.5, 117.7, 122.0, 124.5, 127.4, 124.5,

128.8, 130.6, 134.5, 148.1, 154.1, 156.1, 160.6.

6,7-dibromo-3-(4-((4-((4-chlorophenyl)amino)-6-(piperidin-1-yl)-1,3,5-

triazin-2-yl)amino)phenyl)-2-methylquinazolin-4(3H)-one (7d)

Black solid: yield 85% (5.9 g, 8.47 mmol); molecular formula:

1

C₂₉H₂₅Br₂ClN₈O; melting point: 270-272 ºC; molecular weight: 696.83; H

Synthesis of 4,6‐dichloro‐2‐piperazin‐1‐yl‐[1,3,5]triazine (5) and

disubstituted 1,3,5‐triazine (6a-n)

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

7.20 (d, 2H, J = 6.6 Hz, 2xCH, Cl-Ar), 7.25 (d, 2H, J = 6.8 Hz, 2xCH, Ar),

7.30 (d, 2H, J = 6.9 Hz, 2xCH, Ar), 7.49 (s, 1H, CH, quinazoline), 7.53 (d,

2H, J = 6.6 Hz, 2xCH, Cl-Ar), 8.06 (s, 1H, CH, quinazoline), 8.34 (s, 1H,

NH), 8.91 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm: 23.7, 24.5, 25.5,

54.8, 117.7, 122, 122.1, 122.4, 124.5, 127.4, 128.8, 129.6, 130.6, 134.5,

137, 148.1, 154.1, 160.6, 165.7, 168.9, 176; MS-ESI (m/z): 696.2 [M+H]⁺;

elemental analysis: calculated C: 49.99%, H: 3.62%, N: 16.08%; found C:

49.45%, H: 3.25%, N: 15.86%.

The derivatives 5 and 6(a-n) were synthesized as described in our

previous research.^[31,32,62]

Synthesis

of

6,7-dibromo-2-methyl-3-(4-((4-(substitutedamino)-6-

(piperidin-1-yl)-1,3,5-triazine-2yl)amino)phenyl)quinazolin-4(3H)-one (7a-

n)

0.01 mol of 3-(4-aminophenyl)-6,7-dibromo-2-methylquinazolin-4(3H)-one

(3) and 0.01 mol of previously synthesized disubstituted 1,3,5‐triazine

derivatives (6a‒n) were dissolved in 25 ml of 1,4‐dioxane. Further, 0.01

mol of K₂CO₃and a pinch of tin granules (as catalyst) were added into the

reaction mixture and refluxed for 6-8 hours with continuous stirring. The

excess solvent was evaporated under reduced pressure and recrystallized

from ethanol to obtain the pure product.

6,7-dibromo-2-methyl-3-(4-((4-morpholino-6-(piperidin-1-yl)-1,3,5-triazin-

2-yl)amino)phenyl) quinazoline-4(3H)-one (7e)

White solid; yield 76% (4.9 g, 7.47 mmol); molecular formula:

C_{2 28}Br₂N₈O₂; melting point: 260-262 ºC; molecular weight: 656.38; ¹H

7h

NMR (300 MHz, CDCl₃, TMS) δ ppm: : 1.57 (m, 4H, 2xCH₂, piperidine),

1.68 (m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.68 (t, 4H, 2xCH₂,

morpholine), 3.79 (t, 4H, 2xCH₂, piperidine), 3.75 (t, 4H, 2xCH₂,

morpholine), 7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 6.6 Hz,

2xCH, Ar), 7.69 (s, 1H, CH, quinazoline), 8.06 (s, 1H, CH, quinazoline),

8.34 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm: 23.7, 24.5, 25.5, 25.5,

48.7, 54.8, 66.3, 117.7, 122, 122.4, 124.5, 127.4, 128.8, 130.6, 134.5,

148.1, 154.1, 160.6, 168.9, 176, 179.2; MS-ESI (m/z): 656.2 [M+H]⁺;

elemental analysis: calculated C: 49.41%, H: 4.30%; N: 17.07%; found C:

49.10%; H: 4.60%; N: 17.15%.

1-(4-((4-(6,7-dibromo-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)amino)-

6-(piperidin-1-yl)-1,3,5-triazin-2-yl)urea (7a)

Dark brown solid; yield 72% (4.5 g, 7.15 mmol); molecular formula:

C₂₄H₂₃Br₂N₉O₂; melting point: 230-232 ºC; molecular weight: 629.32; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

6.20 (s, 2H, NH₂), 7.25 (d, 2H, J = 7.5 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 7.2

Hz, 2xCH, Ar), 7.49 (s, 1H, quinazoline), 8.062 (s, 1H, quinazoline), 8.34

(s, 1H, NH), 8.95 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm: 23.7,

24.5, 25.5, 54.8,117.7, 122.0, 127.4, 124.5, 128.8, 130.6, 134.5, 148.1,

154.1; MS (m/z): 629.7 [M+H]⁺; elemental analysis: calculated C: 45.81%,

H: 3.68%, N: 20.03%; found C: 45.94%, H: 3.32%, N: 20.10%.

6,7-dibromo-3-(4-((4,6-di(piperidin-1-yl)-1,3,5-triazin-2-yl)amino)phenyl)-

2-methylquinazolin-4(3H)-one (7f)

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Dark brown solid; yield 69% (4.5 g, 6.88 mmol); molecular formula:

C₂₈H₃₀Br₂N₈O; melting point: 220-222 ºC; molecular weight: 654.41; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 8H, 4xCH₂, piperidine), 1.68

(m, 4H, 2xCH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 8H, 4xCH₂,

piperidine), 7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 6.9 Hz,

2xCH, Ar), 7.49 (s, 1H, CH, quinazoline), 8.06 (s, 1H, CH, quinazoline),

8.34 (s, 1H, NH); ¹³C NMR(100 MHz, CDCl₃) δ ppm: 23.7, 24.5, 25.5, 54.8,

54.8, 117.7, 122, 122.4, 127.4, 128.8, 130.6, 134.5, 148.1, 154.1, 160.6,

168.9, 177.6; MS-ESI (m/z): 654.4 [M+H]⁺; elemental analysis: calculated

C: 51.39%, H: 4.62%, N: 17.12%; found C: 51.20%, H: 4.60%; N: 17.05%.

7.30 (d, 2H, J = 6.9 Hz, 2xCH, Ar), 7.408 (d, 2H, J = 7.2 Hz, 2xCH, Br-Ar),

7.49 (s, 1H, CH, quinazoline), 8.06 (s, 1H, CH, quinazoline), 8.34 (s, 1H,

NH), 8.91 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm: 23.7, 24.5, 25.5,

54.8, 116.7, 117.7, 118.5, 122, 122.4, 124.5, 127.4; MS-ESI (m/z): 640.9

[M+H]⁺; elemental analysis: calculated C: 46.99%, H: 3.40%, N:15.12%;

found C: 46.64%, H: 3.50%, N: 15.50%.

6,7-dibromo-2-methyl-3-(4-((4-((4-nitrophenyl)amino)-6-(piperidin-1-yl)-

1,3,5-triazin-2-yl)amino)phenyl)quinazolin-4(3H)-one (7k)

Yellowish-white solid; yield 74% (5.2 g, 7.35 mmol); molecular formula:

C₂₉H₂₅Br₂N₉O₃; melting point: 265-267 ºC; molecular weight: 707.39; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 6.6 Hz, 2xCH, Ar), 7.46

(d, 2H, J = 6.3 Hz, 2xCH, nitro-Ar), 7.49 (s, 1H, CH, quinazoline), 8.032 (d,

2H, J = 6.3 Hz, 2xCH, nitro-Ar), 8.06 (s, 1H, CH, quinazoline), 8.34 (s, 1H,

NH), 9.13 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm: 23.7, 24.5, 25.5,

54.8, 117.7, 117.7, 119.2, 122.4, 124.5, 124.7, 127.4, 128.8, 130.6, 134.5,

137.9, 145, 148.1, 154.1, 160.6, 165.7, 168.9, 176; MS-ESI (m/z): 707.9

[M+H]⁺; elemental analysis: calculated C: 49.24%, H: 3.56%, N: 17.82;

found C: 49.28%, H: 3.82%, N: 17.70%.

6,7-dibromo-3-(4-((4-((4-methoxyphenyl)amino)-6-(piperidin-1-yl)-1,3,5-

triazin-2-yl)amino)phenyl)-2-methylquinazolin-4(3H)-one (7g)

Brown solid; yield 82% (5.6 g, 8.09 mmol); molecular formula:

C₃₀H₂₈Br₂N₈O₂; melting point: 265-267 ºC; molecular weight: 692.42; ¹H

NMR (300 MHz, CDCl₃, TMS): 1.57 (m, 4H, 2xCH₂, piperidine), 1.68 (m,

2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.81 (s, 3H, OCH₃), 3.75 (t, 4H,

2xCH₂, piperidine), 6.93 (d, 2H, J = 7.8 Hz, methoxy-Ph), 7.25 (d, 2H, J =

6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 6.6 Hz, 2xCH, Ar), 7.49 (s, 1H, CH,

quinazoline), 7.66 (d, 2H, J = 6.9 Hz, methoxy-Ph), 8.06 (s, 1H, CH,

quinazoline), 8.34 (s, 1H, NH), 8.91 (s, 1H, NH); ¹³C NMR (100 MHz,

CDCl₃) δ ppm: 24.5, 54.8, 55.8, 115.1, 117.7, 122, 122.4, 124.5, 127.4,

128.8, 130.6, 131.2, 134.5, 148.1, 153.3, 154.1, 160.6, 165.7, 168.9, 176; ;

MS-ESI (m/z): 692.8 [M+H]⁺; elemental analysis: calculated C: 52.04%, H:

4.08%, N: 16.18%; found C: 52.10%, H: 4.10%, N: 16.40%.

6,7-dibromo-2-methyl-3-(4-((4-(methylamino)-6-(piperidin-1-yl)-1,3,5-

triazin-2-yl)amino)phenyl) quinazolin-4(3H)-one (7l)

Light brown solid; yield 69% (4.1 g, 6.83 mmol); molecular formula:

C₂₄H₂₄Br₂N₈O; melting point: 206-208 ºC; molecular weight: 600.32; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 2.92 (s, 3H, CH₃), 3.75 (t, 4H,

2xCH₂, piperidine), 7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 7.2

Hz, 2xCH, Ar), 7.49 (s, 1H, CH, quinazoline), 7.96 (s, 1H, NH), 8.06 (s, 1H,

CH, quinazoline), 8.34 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm:

23.7, 24.5, 25.5, 28.5, 54.8, 117.7, 122, 122.4, 124.5, 127.4, 128.8, 130.6,

134.5, 148.1, 154.1, 160.6, 165.7, 165.7, 176; MS-ESI (m/z): 600.1

[M+H]⁺; elemental analysis: calculated C: 48.02%, H: 4.03%, N: 18.67%;

found C: 48.08%, H: 3.90%, N: 18.60%.

2-(4-((4-(6,7-dibromo-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)amino)-

6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazine-1-carbothioamide (7h)

Light brown solid; yield 74% (4.8 g, 7.27 mmol); molecular formula:

C₂₄H₂₄Br₂N₁₀OS; melting point: 258-260 ºC; molecular weight: 660.39; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 7.8 Hz, 2xCH, Ar), 7.49

(s, 1H, CH, quinazoline), 8.06 (s, 1H, CH, quinazoline), 8.34 (s, 1H, NH),

9.13 (s, 2H, NH₂), 9.65 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm:

21.7, 24.5, 25.5, 54.8, 117.7, 122, 124.5, 127.4, 128.8, 130.6, 134.5, 134.5,

148.1, 154.1, 160.6, 165.7, 174.3, 182.5; MS-ESI (m/z): 660.9 [M+H]⁺;

elemental analysis: calculated C: 52.65%, H: 4.66%, N: 21.21%; found C:

52.40%, H: 4.42%, N: 21.45%.

3-(4-((4-amino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)amino)phenyl)-6,7-

dibromo-2-methylquinazolin-4(3H)-one (7m)

Yellowish solid; yield 64% (3.75 g, 6.40 mmol); molecular formula:

C₂₃H₂₂Br₂N₈O; melting point: 267-269 ºC; molecular weight: 586.29; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

6.94 (s, 2H, NH₂), 7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 6.9

Hz, 2xCH, Ar), 7.49 (s, 1H, CH, quinazoline), 8.06 (s, 1H, CH, quinazoline),

8.34 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm: 23.7, 24.5, 25.5, 54.8,

117.7, 122, 122.4, 124.5, 127.4, 128.8, 130.6, 134.5, 148.1, 154.1, 160.6,

165.7, 176, 182.4; MS-ESI (m/z): 586.4 [M+H]⁺; elemental analysis:

calculated C: 47.12%, H: 3.78%, N: 19.11%; found C: 47.02%, H: 3.50%,

N: 19.25%.

2-(4-((4-(6,7-dibromo-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)amino)-

6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazine-1-carboxamide (7i)

Light brown solid; yield 71% (4.5 g, 6.98 mmol); molecular formula:

1

C₂₄H₂₄Br₂N₁₀O₂; melting point: 262-264 ºC; molecular weight: 644.33; H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

6.22 (s, 2H, NH₂), 7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 6.9

Hz, 2xCH, Ar), 7.49 (s, 1H, CH, quinazoline), 8.06 (s, 1H, CH, quinazoline),

8.34 (s, 1H, NH), 9.13 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ ppm:

23.7, 24.5, 25.5, 54.8, 117.7, 122, 122.4, 124.5, 127.4, 128.8, 130.6, 134.5,

148.1, 154.1, 157.4, 160.6, 165.7, 182.9; MS-ESI (m/z): 644.8 [M+H]⁺;

elemental analysis: calculated C: 44.74%, H: 3.75%, N: 24.74%; found C:

44.45%, H: 3.50%, N: 24.85%.

4-((4-((4-(6,7-dibromo-2-methyl-4-oxoquinazolin-3(4H)-yl)phenyl)amino)-

6-(piperidin-1-yl)-1,3,5-triazin-2-yl)amino)benzoic acid (7n)

Brown solid; yield 68% (4.8 g, 6.80 mmol); molecular formula:

C₃₀H₂₆Br₂N₈O₃; melting point: 272-274 ºC; molecular weight: 706.40; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH3), 3.75 (t, 4H, 2xCH₂, piperidine),

7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.30 (d, 2H, J = 6.3 Hz, 2xCH, Ar), 7.49

(s, 1H, CH, quinazoline), 7.73 (d, 2xCH, J = 6.6 Hz, Ar), 8.03 (d, 2H, J =

6.3 Hz, 2xCH, Ar), 8.06 (s, 1H, CH, quinazoline), 8.34 (s, 1H, NH), 8.914

(s, 1H, NH), 12.712 (s, 1H, OH, Ar); ¹³C NMR (100 MHz, CDCl₃) δ ppm:

23.7, 24.5, 25.5, 54.8, 111.2, 117.7, 120.2, 122, 122.4, 124.5, 127.4, 128.8,

6,7-dibromo-3-(4-((4-((4-bromophenyl)amino)-6-(piperidin-1-yl)-1,3,5-

triazin-2-yl)amino)phenyl)-2-methylquinazolin-4(3H)-one (7j)

Brown solid; yield 79% (5.8 g, 7.82 mmol); molecular formula:

C₂₉H₂₅Br₃N₈O; melting point: 276-278 ºC; molecular weight: 741.29; ¹H

NMR (300 MHz, CDCl₃, TMS) δ ppm: 1.57 (m, 4H, 2xCH₂, piperidine), 1.68

(m, 2H, CH₂, piperidine), 2.26 (s, 3H, CH₃), 3.75 (t, 4H, 2xCH₂, piperidine),

7.01 (d, 2H, J = 6.3 Hz, 2xCH, Br-Ar), 7.25 (d, 2H, J = 6.3 Hz, 2xCH, Ar),

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130.6, 131.1, 134.5, 144.1, 148.1, 154.1, 160.6, 165.7, 168.9, 169.3, 176;

MS-ESI (m/z): 706.2 [M+H]⁺; elemental analysis: calculated C: 51.01%, H:

3.71%, N: 15.86%; found C: 51.02%, H: 3.40%, N: 15.60%.

incubating them, and adding 50 mL of the kinase detection reagent.

Reading of the plates was performed using the ADP-Glo Luminescence

reader. The same procedure was also performed for the control. The

corrected activity results were calculated as the difference between the

tested and control samples for each protein kinase target and IC₅₀were

calculated by the standard equation.^[32,49,63]

Pharmacological assays

In vitro anticancer assays

The synthesized derivatives were screened for their anti-cancer activity

against four different cell lines: MCF-7 (human breast cancer cell line),

HeLa (human cervical cancer cell line), HL-60 (human promyelocytic

leukaemia cell line), and HepG2 (human hepatic cancer cell line). As a

cytotoxicity control, two normal human cell lines, HFF (human foreskin

fibroblast cell line) and MCF-12A (normal epithelial breast cell line) were

used.

In ovo antiangiogenic activity on cancer induced chorioallantoic membrane

(CAM)

Fertilized chicken eggs (480 in total) were obtained from

a local

commercial hatchery and the dirt was removed mechanically using a 70%

denatured ethanol. Eggs were then placed into a rotating incubator (37 °C,

60% relative humidity for 10 days, 4 rotation/hour) for embryogenesis.

Each of the incubation days was termed as an embryo development day

(EDD). On the third EDD, the eggs were placed on their side in an egg

rack. A torch was used to candle for the blunt end of the air sac. The

chorioallantoic vein and its large blood vessels junctions were marked. A

1 cm²box was drawn approximately 1 cm away from the branch point of

the vein. The marked area was then cleaned with iodine and drills into the

marked area were made using a rotating cutting tool. A small hole was

made in the eggshell membrane using a 25 gauge syringe, being careful

not to tear the underlying CAM. The CAM was subsequently detached from

the eggshell membrane using gentle suction created by an automatic

pipette (fitted with a piece of ¼” tygon tubing) by placing it against the hole

in the air sac. The process of suction was carried out in such a way that

the air pocket was underneath the hole in the square, signifying that the

CAM has been successfully detached from the eggshell. The air sac and

the square near the chorioallantoic vein were then sealed with a semi-

transparent laboratory tape. On EDD 8, the seal was removed, and a 4

mm radial disc was placed on the chorioallantoic membrane of each egg.

The eggs were then resealed and incubated for the next 24 hours at the

same conditions.

Cell cultures

The selected cancer and normal cell lines were maintained (1×10⁴

cells/well) in monolayer cultures in a supplemented Dulbecco’s modified

eagle’s medium (DMEM) with 10% heat-inactivated foetal bovine serum

(FBS), 1% L-glutamine, and 50 µg/mL gentamycin sulphate, at 37 ºC, in a

CO₂incubator in an atmosphere of humidified 5% CO₂and 95% air.

Preparation of samples

Derivatives were dissolved in DMSO and 1 mg/mL concentration solutions

were prepared. The derivatives were then diluted with DMEM and 10%

FBS, after which the solutions were filtered and sterilized, using a 0.2 µM

syringe filter to prepare working solutions. Further dilutions (1–100 µM)

were made, again followed by filtration and sterilization.

Cytotoxic activity measurement

In vitro cytotoxic activities of synthesized derivatives were determined

using the MTT assay. Different concentrations of derivatives (diluted in the

culture medium), were added into the wells with respective controls and

incubated for 48 hours. After that, 10 mL of MTT (3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added into each well

and the plates were further incubated for 4 hours. Then the supernatant

from each well was carefully removed, and formazan crystals were

dissolved in 100 mL of DMSO, after which absorbance at 540 nm was

recorded.^[31,32,49]All calculations were carried out in duplicate.

Determination of cell viability (%) is calculated using the following formula:

A selected tumour cell line (MCF-7) was detached from its culture dish

using EDTA:PBS (1:10 v/v) to remove the excess residual media. MCF-7

cells (4×10⁴cells) were placed on the top of uncoated 12 μm ring inserts.

Eggs were put in the upright position for at least 6 hours, allowing the

migration of the cancer cells into the lower chamber.

Synthesized derivatives (1.0 μM) were prepared in PBS. 10 μL of these

solutions was added directly to the disk on top of CAM. At higher doses of

tested and reference compounds the immunity of the chicken embryos was

found to be compromised and the integrity of the CAM was at risk.

Therefore, lower concentration (1 μM) of the test and standard compounds

were used.^[31,32,64]

(

푀푒푎푛 푂퐷 푠푎푚푝푙푒

(

)

퐶푒푙푙 푣푖푎푏푖푙푖푡푦 % =

⁾× 100

(1)

(

)

푀푒푎푛 푂퐷 푐표푛푡푟표푙

Where OD represents optical density. Determination of IC₅₀values was

obtained by statistical analysis.

Activity measurements were done by counting the number of blood vessels

in the area under the disk using a stereo microscope. Eggs that showed

inflammation and embryo-toxicity due to experimental error were excluded.

The experiment was done in triplicate and each test sample was tested on

10 different eggs to measure cumulative inhibition score.^[65,66]

Antiangiogenic scores were calculated using the formula:

EGFR inhibitory activity

Kinase-Glo^®Plus (luminescent kinase assay) kit was used to determine

EGFR inhibitory activity of the synthesized derivatives. The assay is based

upon quantification of the remaining ATP in the solution of the kinase

reaction, which is inversely correlated to the luminescent signal.^[63]In the

assay, a stock solution of the derivatives (100 mM in 10% DMSO) were

prepared and serially diluted (10 nM to 500 nM). Further, 5 mL of the

diluted solution was added to a 50 mL of the reaction mixture (10 mM

MgCl₂, 40 mM Tris, pH 7.4, 0.1 mg/mL BSA, 0.2 mg/mL Poly (Glu, Tyr)

substrate, 10 mM ATP and EGFR (50 nM)). Enzymatic reactions were

allowed to proceed for 40 minutes at 30 °C, after which the plates were

incubated for 5 minutes at room temperature and 50 mL of Kinase-Glo^®

Plus kit solution was added into each 96-well plate. The reaction mixture

was again incubated at 30 °C for 30 minutes. After the incubation period,

25 mL of ADP-Glo reagent was added to terminate the assay, which was

followed by shaking of the plates for 30 minutes at ambient temperature,

(푁 + 푁 )×2

1

2

퐴푣푒푟푎푔푒 푆푐표푟푒 =

(2)

푁

Where N is a total number of eggs where a derivative showed weak, strong

or no effect, N₁is a total number of eggs where a derivative showed a

medium effect, N₂is a total number of eggs where a derivative showed a

strong effect. The score patterns of semi-quantitative results are as

follows:

0: no or weak effect (more than 150 new blood vessels).

1: medium effect (between 50 and 150 new blood vessels).

2: strong effect (between 0 and 50 new blood vessels or the capillary‐free

zone is at least twice as large as the pallet).

13

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Where the symbol < > represents the average value over 1000 snapshots

collected from the last 30 ns of the corresponding MD trajectories. The

calculated MM-GBSA binding free energies were decomposed into

Docking studies

Docking studies were performed using AutoDock Vina,^[67]which uses

dispersion, hydrogen bonds, electrostatic, and desolvatation components

for determination of the most probable complex conformation. 3D

coordinates of the EGFR kinase domain complexed with lapatinib (PDB

entry 1XKK)^[56]and erlotinib (PDB entry 4HJO)^[68]were taken from RCSB

Protein Data Bank. Water molecules were omitted from the structures,

hydrogen atoms were added were necessary, and all Lys, Arg, His and

Cys side chains were protonated, while all Asp and Glu side chains were

deprotonated and both amino and carboxyl ends were charged using the

UCSF Chimera 1.14. program (University of California, USA),^[69]which was

also used for results visualization and interpretation. Additionally, all

missing amino acid residues were added according to Šali and Bundell’s

Modeller service^[50]accessed through UCSF Chimera 1.14. Initial 3D

conformation of all ligands (7a-7n) was determined using HyperChem 8.0

(Hypercube, Inc., Gainesville, FL, USA). A grid map of size 22.5 × 22.5 ×

22.5 Å was generated by the AutoGrid program^[70]and centred on the

coordinates of one of the nitrogen atoms of lapatinib’s quinazoline ring

(12.683, 33.305, 37.276). The receptor molecule was regarded as rigid

while all ligands’ single bonds could rotate freely. Docking procedure was

run with exhaustiveness set to 100. The validity of the approach was

assessed by lapatinib redocking using 4 different conditions (neutral

lapatinib moiety and the charged moiety most prevalent at pH 7.4, without

water molecules and in presence of HOH 4 and HOH 22 water molecules,

which are located inside the binding site). The charge of the ligand was

calculated according to Ionescu et al.^[71]

specific residue contribution on

a per-residue basis according to

established procedures. This protocol calculates the contributions to

ΔG_bindarising from each amino acid side chains and identifies the nature

of the energy change in terms of interaction and solvation energies.^[78,79]

Due to the similarity of the compounds, the entropic component of ΔG_bind

was not calculated.

In vivo anticancer pharmacology

Animal model

Female Sprague-Dawley (SD) rats, weighing between 100 and 150 g,

were used for the study after approval from the Institutional Animal Ethics

Committee. The animals were kept in suitable cages at 25 °C, with 45–

55% humidity and 12/12 hours light/dark cycles under standard

atmosphere with free access to food and water. The entire protocol for

conducting the animal studies was reviewed and approved by the

Institutional Animal Ethics Committees (IAEC) for the Purpose of Control

and Supervision of Experiments on Animals, Government of India

(1546/PO/E/S/11/CPCSEA).

Oral toxicity study

The dose of the 7e derivative was estimated using an acute toxicity study

in healthy SD rats (6–8 weeks old) as per OECD Guideline-423. The rats

were fasted overnight with water ad libitum. A dose of 5, 10, 20 30, and 40

mg of the 7e derivative per kilogram of body weight was introduced orally

to the rats. After administration of the drug, the rats were observed at least

once during the first 2 hours and then again after 24 and 48 hours for any

behavioural changes or death. Since animals did not exhibit any toxic

symptoms, behaviour changes or mortality up to 30 mg/kg of body weight

during the observation period, doses of 5 mg/kg, 10 mg/kg, and 15 mg/kg

were selected for the study.

Molecular dynamics (MD) simulations

Docked positions of erlotinib and four ligands with the highest affinities for

binding to EGFR were used as starting points for MD simulations. AMBER

ff14SB force field was used to model the enzyme and GAFF force field

was used in the case of ligands. Such protein-ligand complexes were

solvated in a truncated octahedral box of TIP3P water molecules spanning

a 12 Å thick buffer, neutralized by Na⁺ions and submitted to geometry

optimization in AMBER16 program,^[72]employing periodic boundary

conditions in all directions. For the first 1500 cycles the complex was

restrained and only water molecules were optimized, after which another

2500 cycles of optimization followed where both water molecules and the

complex were unrestrained. Optimized systems were gradually heated

from 0 to 300 K and equilibrated during 30 ps using NVT conditions,

followed by productive and unconstrained MD simulations of 300 ns

employing a time step of 2 fs at a constant pressure (1 atm) and

temperature (300 K), the latter held constant using Langevin thermostat

with a collision frequency of 1 ps^-1. Bonds involving hydrogen atoms were

constrained using the SHAKE algorithm,^[73]while the long-range

electrostatic interactions were calculated employing the Particle Mesh

Ewald method.^[74]The nonbonded interactions were truncated at 11.0 Å.

Analysis of the trajectories was performed using the cpptraj module of

AmberTools16.^[75]

Induction of breast cancer

7,12-dimethylbenz(a) anthracene (DMBA) is

a polycyclic aromatic

hydrocarbon which induces cancer through formation of epoxides, which

bind to DNA and form DNA adducts and lead to erroneous DNA

transcription.^[49,80]Breast cancer was induced with a single 25 mg

subcutaneous DMBA injection (emulsified in sunflower oil and saline) near

the mammary gland.

Experimental design

A total number of 30 animals was randomly divided into five groups, with

each group containing six animals. Different groups received different

treatments as follows:

Group I: Animals were treated with a 0.9% w/v of NaCl solution and served

as the untreated control.

Groups II: Animals were treated with a single subcutaneous injection of

DMBA (25 mg) near the mammary gland. This group served as a negative

control without any drug treatment.

Binding free energy calculations and decomposition

Group III: DMBA + derivative (7e) at a dose of 5 mg/kg once per day

throughout the experimental period.

The binding energy, ΔG_bind, of simulated complexes was calculated using

the MM-GBSA (Molecular Mechanics – Generalized Born Surface Area)

protocol,^[58,76]available as a part of AmberTools16.^[72]MM-GBSA is a

method for the calculation of ΔG_bindfrom snapshots of MD trajectory^[77]with

an estimated standard error of 1–3 kcal/mol.^[58]ΔG_bindis calculated in the

following manner:

Group IV: DMBA + derivative (7e) at a dose of 10 mg/kg once per day

throughout the experimental period.

Group V: DMBA + derivative (7e) at a dose of 15 mg/kg once per day

throughout the experimental period.

A free access to water and food was supplied during the entire experiment

and the body weight of all animals was regularly monitored. On the final

day (end of the 16^thweek), all animals were kept fasted overnight and

sacrificed by cervical dislocation under ether anaesthesia. Blood samples

ΔG_bind= < G_complex> – < G_protein> – < G_ligand

>

(3)

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of all animals were collected by puncturing the retro-orbital sinus plexus

and were kept in labelled centrifuge tubes. Plasma was separated by

centrifuge (10,000 rpm for 15 minutes). Selected tissues (liver and

mammary glands) were immediately excised and homogenized using 0.1

M Tris-HCl buffer (pH 7.4) at 4 °C and centrifuged. The resulting clear

homogenates were collected for biochemical assays.^[81,82]

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A part of the work was supported by Act 211 Government of the

Russian Federation, contract 02.A03.21.0011 and by the Ministry

of Science and Higher Education of Russia (Grant FENU-2020-

0019). Authors are also sincerely thankful to Department of

Pharmaceutical sciences, SHUATS, Allahabad, Uttar Pradesh,

India and Laboratory of Computational Modelling of Drugs, Higher

Medical and Biological School, South Ural State University,

Chelyabinsk, Russia for experimental and computational

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Keywords: EGFR inhibitors • synthesis • molecular modelling •

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Entry for the Table of Contents

In silico study

Anti-angiogenic activity

In vivo anticancer activity

In vitro anticancer activity

18

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Hybrid Quinazoline 1,3,5-Triazines as Epidermal Growth Factor Receptor (EGFR) Inhibitors with Anticancer Activity: Design, Synthesis, and Computational Study

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