Pyrazolo[3,4-d]pyrimidine-based dual EGFR T790M/HER2 inhibitors: Design, synthesis, structure–activity relationship and biological activity as potential antitumor and anticonvulsant agents

European Journal of Medicinal Chemistry 214 (2021) 1 13222

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Pyrazolo[3,4-d]pyrimidine-based dual EGFR T790M/HER2 inhibitors:

Design, synthesis, structureeactivity relationship and biological

activity as potential antitumor and anticonvulsant agents

,

Phoebe F. Lamie ^{a *}, Asmaa M. El-Kalaawy ^b, Noha S. Abdel Latif ^c, Laila A. Rashed ^d,

John N. Philoppes ^a

^aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt

^bDepartment of Pharmacology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt

^cDepartment of Medical Pharmacology, Faculty of Medicine, Cairo University, Egypt

^dDepartment of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history:

A new series of pyrazolo[3,4-d]pyrimidine/triazine hybrids 6a-r was designed as antitumor and anti-

convulsant agents. All the prepared compounds were evaluated against colon (HCT-116), breast (MCF-7)

and normal human ﬁbroblast (WI38) cell lines. The most potent derivatives against HCT-116 and MCF-

7 cells were 6o and 6q, with IC₅₀¼ 4.80 and 6.50 nM, respectively, when compared to lapatinib, the

reference drug (IC₅₀¼ 12.00 and 21.00 nM, on HCT-116 and MCF-7, sequentially). All other derivatives

exhibited good to moderate cytotoxic activity. Four compounds 6f, 6j, 6o and 6q were evaluated for their

EGFR T790M/HER2 inhibitory activity. They revealed 81.81e65.70% and 86.66e54.49% inhibitory activity

against EGFR T790M and HER2 in a sequent. The most potent derivatives 6o and 6q were further esti-

mated for cell cycle analysis showing pre G1 apoptotic activity and cell growth arrest at G2/M phase.

Apoptotic marker proteins expression levels (caspase-3/7/9, Bax and Bcl-2) were measured for 6o and 6q.

They showed pro-apoptotic effect by increasing caspase-3/7/9 protein levels and Bax/Bcl-2 ratio.

Moreover, anticonvulsant activity for the prepared compounds 6a-r were evaluated in vivo using

lithium-pilocarpine mice model of Status Epilepticus. EEG changes where recorded and MDA, GSH, GABA

and glutamate were measured in brain tissue of different groups. All tested compounds revealed variable

anti-epileptic effects, the most potent compounds were 6b and 6m. Also 6d, 6e, 6h, 6i, 6k, 6l and 6n

compounds exhibited good anti-seizure activity, while compound 6j showed the lower activity. The rest

of compounds displayed a neutral activity.

Received 16 September 2020

Received in revised form

19 January 2021

Accepted 20 January 2021

Available online 26 January 2021

Keywords:

Pyrazolopyrimidines

Triazines

Antitumor agents

Structure-activity relationship

Anticonvulsant

1. Introduction

expression of EGFR/HER2 such as non-small cell lung cancer

(NSCLC), breast, colon, prostate and ovarian cancer [3,8e1 1].

Protein tyrosine kinases (PTKs) enzymes play an important role

in cell proliferation, metabolism and apoptosis [1,2]. Over expres-

sion or mutation of these enzymes in cancer cells results in un-

controlled cell growth [3]. Epidermal growth factor receptor

protein (EGFR) is one of receptor TKs and belongs to Erb-B family

[4]. It is involved in differentiation and regulation of cell growth.

EGFR family is composed of structurally related RTKs such as HER1,

HER2, HER3 and HER4 [5e7]. Many cancers showed to have over

Therefore, there is a need to inhibit EGFR either by blocking

ligand binding site to the extracellular domain or by using small

molecules to interact with ATP-binding site providing EGFRIs.

A serious problem that develops in cancer patients, is the

resistance to EGFR TKIs, through several mechanisms; mutation of

the gatekeeper residue (T790M), upregulation of alternative

signaling pathways, or bypass mechanisms using other RTKs to

compensate the blockade of EGFR (e.g: HER2 ampliﬁcation)

[12e15].

To overcome the major resistance mechanism, mutation of the

gatekeeper residue (T790M), it was found that in ﬁrst generation

EGFRIs (4-anilinoquinazoline based drugs such as geﬁtinib and

* Corresponding author.

E-mail addresses: feebi.lamey@pharm.bsu.edu.eg, feby.farag@yahoo.com

(P.F. Lamie).

https://doi.org/10.1016/j.ejmech.2021.113222

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

2) two aromatic rings at C-3 and C-5 of triazine ring to increase the

potency of the target compounds,

3) presence of many sites and groups for hydrogen bond formation,

4) substitution on phenyl rings either with electron donating

groups (EDGs) or electron withdrawing groups (EWGs) to

explore the electronic effect on their activities.

erlotinib), threonine was replaced with methionine but that led to

steric repulsion resulting in slight different binding mode of action

and loss of inhibitory activity as well as change in ATP afﬁnity [14].

Second generation EGFRIs, contain also 4-anilinoquinazoline

scaffold (afatinib and dacomitinib), they act by incorporating a

Michael acceptor by covalently modifying a unique cysteine

(Cys797) at EGFR-ATP binding site, but clinically they are not suf-

ﬁcient for patients due to severe side effects [16e18]. Finally, a third

generation of EGFRIs, rociletinib and osimertinib (aminopyrimidine

based structure), were developed to avoid drawbacks of genera-

tions one and two [19,20]. But due to the clinically observed

elevation in blood glucose level, as a result of inhibition in insulin

like growth factor 1, the production has been stopped [21,22]; only

one of third generation inhibitor has FDA approval, osimertinib,

showed excellent clinical results [23,24].

The rational design used to develop the new target compounds

as antitumor and anticonvulsant agents is represented by Fig. 1.

2. Results and discussion

2.1. Chemistry

Screening the literature declared that many pyrazolo[3,4-d]py-

rimidines had potent antitumor activity through inhibition of EGF

tyrosine kinase as represented by compounds I-III in Fig. 1 [3].

Here, we present the development of new pyrazolo[3,4-d]py-

rimidine-based series of dual EGFR mutant T790M/HER2 inhibitors

as well as anticancer evaluation against human colon cancer cell

line HCT-116 and human breast adenocarcinoma cell line MCF-7. As

a result, structure activity relationships (SAR) were investigated.

Cell cycle analysis and apoptotic biomarkers for the most active

derivatives 6o and 6q were determined. The obtained results were

compared to lapatinib, the ﬁrst FDA-approved reversible dual

EGFR/HER2 inhibitor for metastatic and breast cancer.

Another biological activity was studied in this research, epi-

lepsy; it is at the same level of occurrence with breast cancer be-

tween patients in America. It affects 0.5e1% of people world-wide

[25,26].

The periodic unpredictable occurrence of seizures is the most

common symptoms of epilepsy. Initiation and propagation of epi-

lepsy may be due to oxidative injury and leads to neurobiological

abnormality in brain. There are no detectable drugs for prevention

or curing epilepsy, they just control symptoms [27,28].

The intermediate and target compounds were synthesized ac-

cording to the reaction pathways illustrated in Scheme 1. The key

intermediate

5-amino-3-methyl-1-phenyl-1H-pyrazole-4-

carboxamide 2 [35] was cyclized with either diethyl oxalate or

diethyl malonate to yield the pyrazolopyrimidinone ethyl esters 3a

or 3b, respectively [35,36]. Hydrazinolysis of the esters 3a&b with

hydrazine hydrate 99.99% afforded the acid hydrazide derivatives

4a&b [35,36]. Reaction of the acid hydrazides 4a&b with aza-

lactone derivatives 5a-i [37], in glacial acetic acid using anhydrous

sodium acetate, gave the target triazine derivatives 6a-r. Their IR

spectra revealed absorption bands at 3395-3125 cm^ꢀ1due to two

NH groups. Three C¼O stretching vibrations appeared at the range

of 1728-1667 cm^ꢀ1. The ¹H NMR spectra displayed a singlet signal at

d

7.21e7.40 ppm attributed to benzylidene ¼ CH proton. Two D₂O

exchangeable singlet signals were observed at 9.81e12.70 ppm,

d

range, corresponding to both triazine and pyrimidine NHs. No other

evidence for presence of NH₂protons of the parent hydrazides

4a&b was observed.

¹³C NMR (DEPT-Q) for 6a-r showed characteristic peak at

120.25e121.99 cm^ꢀ1corresponding to benzylidene ¼ CH. Con-

struction of the newly formed triazine ring was conﬁrmed from ¹³

C

Both the classical anti-epileptic drugs (AEDs) such as pheno-

barbital, phenytoin, carbamazepine and valproic acid, as well as

newer drugs namely, vigabatrin, zonisamide, tiagabin, topiramate

and lamotrigine were reported to have promising anticonvulsant

activity by reducing partial and generalized seizures [29,30].

Despite the presence of advanced AEDs, many drawbacks still

facing researchers such as dose-related toxicity and adverse drug

reactions (minimal brain impairment, megaloblastic anemia,

aplastic anemia or hepatic failure) which may result in death, in

addition to resistance of some patients to long established AEDs

[31e33].

NMR through the formation of triazine C-5 at d 132.84e136.72 ppm

and triazine C-3 at

peak at a range of

d

146.65e158.70 ppm, beside the presence of a

d

160.05e166.63 ppm corresponding to triazine

C¼O. Another peak appeared at

d 167.26e168.60 ppm attributed to

amidic C¼O.

Additionally, mass spectra for compounds 6a and 6l revealed

molecular ion peaks at m/z ¼ 515 (16.58%) and 564 (15.75%),

respectively.

Reagents and conditions: a, i) Conc. H₂SO₄, stirring, r.t. 7 h, ii)

NH₃; b, diethyl oxalate (for 3a) or diethyl malonate (for 3b), fusion

18 h; c, N₂H₄.H₂O (99.99%), abs. EtOH, reﬂux 4e6 h; d, azalactone

derivative 5a-i, gl. Acetic acid, CH₃COONa, reﬂux 12e14 h.

Prediction of (Z/E) stereochemical conﬁguration of target com-

pounds 6a-r.

As a result, the search for new anticonvulsant drugs continues to

develop safer drugs.

By inspecting lamotrigine structure, a triazine derivative, acting

through prolongation of inactivation of voltage-sensitive Na^þ

channel, and other related triazine analogs and pyrimidine con-

taining compounds (methaqualone, etaqualone, mecloqualone,

mebroqualone and aﬂoqualone), it was found that the pharmaco-

phoric elements responsible for the activity are; lipophilic aryl ring,

hydrogen bond to triazine moiety, and second aryl ring that in-

creases Van der Waal’s bonding at the binding site [25,31,32,34].

Keeping in mind the previous facts, we tried to develop a new

series of pyrazolo[3,4-d]pyrimidines hybrids with triazine scaffold

aiming to have potential anticonvulsant activity as well as anti-

cancer activity. The common features in the prepared compounds

are:

In addition to the used spectroscopic techniques; theoretical

calculations were also adopted to establish the stereochemical

orientation (Z or E-isomer) for the target compounds 6a-r. Chem3D

Pro 12.0 was used to draw both Z and E-isomers and MM2 property

was selected to calculate their total energies, including, stretch,

bending, stretch-bend, torsion, non-1,4 Van Der Waals, 1,4 Van Der

Waals and dipole/dipole interactions. The obtained results revealed

that total energy in E-form exceeded that in Z-form for target

compounds 6a-i, (Table 1). For example, compound 6b was found to

have 51.67 kcal/mol for its Z-form, while 62.13 kcal/mol when

present in its E-form, (Table 1, Fig. 2). On the other hand, com-

pounds 6j-r are more stable in their E-form due to high Van Der

Waals interactions of their Z-form. Thus, compound 6p has

37.75 kcal/mol for its E-form and 963.91 kcal/mol in its Z-form,

(Table 1, Fig. 2).

1) pyrazolo[3,4-d]pyrimidine core combining 1,2,4-triazine ring,

2

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Fig. 1. Represented compounds for pyrazolo[3,4-d]pyrimidines with antitumor activity through EGFRI activity (I-III), anticonvulsant drugs and target compounds 6a-r.

2.2. Biological evaluation

ranging from 4.8 to 93.6 nM and 6.5e126.7 nM, respectively,

(Table 2). The highest cytotoxic activity was observed in com-

pounds 6o and 6q (IC₅₀¼ 4.80 and 6.50 nM on HCT-116 and MCF-

7 cell lines, sequentially). The most potent derivatives were 6f, 6h,

6p, 6q and 6r showing IC₅₀at the range 5.60 nMe12.90 nM on HCT-

116 cells. Moreover compounds 6e, 6g, 6i, 6j, 6l, 6n and 6p had IC₅₀

of 6.90e17.80 nM on MCF-7 cell line compared to lapatinib, the

reference drug.

All other pyrazolopyrimidine analogs exhibited good to mod-

erate cytotoxic activity if compared to lapatinib. Thus, on HCT-

116 cells, IC₅₀values ranged from 14.20 nM to 93.60 nM, and on

MCF-7 cell line, IC₅₀values were at the range of 25.00e61.30 nM.

While, compounds 6a and 6d showed poor cytotoxic activity on

MCF-7 cell line with IC₅₀values equal to 121.30 and 126.70 nM,

2.2.1. Antiproliferative activity

The anticancer activities of the test compounds 6a-r were

evaluated in vitro using MTT assay on human colon adenocarci-

noma (HCT-116) and human breast adenocarcinoma (MCF-7) cell

lines which overexpress EGFR and HER2 [38], in addition to normal

human ﬁbroblast (WI38) cell line. Lapatinib was used as a reference

compound with IC₅₀values 12.00 nM, 21.00 nM and 45.20 nM,

respectively.

2.2.1.1. In vitro cytotoxic activity of compounds 6a-r on HCT-116 and

MCF-7 cells. The prepared compounds 6a-r exhibited cytotoxic

activity against both HCT-116 and MCF-7 cell lines with IC₅₀values

3

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Scheme 1. Synthesis of target compounds 6a-r.

respectively.

compounds showed IC₅₀values between 12.90 nM and 110.80 nM.

The most active derivatives against HCT-116 cell line, 6f and 6o

(IC₅₀¼ 5.60 nM and 4.80 nM, respectively) showed IC₅₀values on

normal cell (26.40 and 29.40 nM, sequentially). Moreover, com-

pounds 6j and 6q, the most potent derivatives on MCF-7 cell line,

Additionally, tested derivatives 6a-r were evaluated against

normal human ﬁbroblast cell line (WI38) to check their cytotoxic

effect. Their IC₅₀values are represented as in Table 2. Lapatinib was

used as a reference drug with IC₅₀value ¼ 45.20 nM. The test

4

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Table 1

and/or p-substituted aryl ring carrying either electon donating

group (EDG) such as methyl group at triazine C-3 or methoxy group

at triazine C-5 or electon withdrawing group (EWG) like Cl at

triazine C-3/5.

Total energy scores (kcal/mol) for Z/E conﬁgurations of compounds 6a-r.

Compd.

No.

Total Energy (Kcal/

mol)

Compd.

No.

Total Energy (Kcal/mol)

Additionally, triazine ring was attached to pyrazolo[3,4-d]py-

rimidine through carbonyl spacer (6a-i) or carboxymethyl spacer

(6j-r).

By inspecting in vitro cytotoxic activity results of the prepared

compounds 6a-r against HCT-116 and MCF-7 cell lines, and

comparing them with the reference drug used, lapatinib, some

points can be highlighted, (Fig. 3).

Z-form

E-form

Z-form

E-form

6a

6b

6c

6d

6e

6f

6g

6h

6i

45.8921

51.6796

46.3922

45.4974

52.5985

45.9910

46.9821

54.0893

47.5257

56.4213

62.1321

56.7596

56.4876

61.8872

56.5134

57.3216

63.0306

57.6851

6j

6k

6l

6m

6n

6o

6p

6q

6r

963.9439

969.5242

964.2730

962.8493

968.4254

963.1782

963.9167

969.4907

964.2457

80.9645

46.8927

41.1748

40.0806

45.9470

40.1955

37.7555

43.2380

38.1421

For compounds 6a-i:

i) Improvement of cytotoxic activity of both HCT-116 and MCF-

7 cell lines was achieved in compounds decorated by EWG at

one of the two aryl rings (6g, IC₅₀¼ 13.90 and 12.30 nM) or at

the two rings (6i, IC₅₀¼ 15.00 and 17.80 nM).

with IC₅₀values of 6.90 nM and 6.50 nM, respectively, exerted

cytotoxic activity (IC₅₀¼ 47.20 nM and 54.90 nM, in a sequent) on

WI38 cell line.

Another seven tested compounds, 6a, 6b, 6g, 6l, 6m, 6p and 6q,

could be considered as safe derivatives for normal cells. Their

detectable IC₅₀values were in the range of 49.50 nMe110.80 nM.

ii) Presence of two aryl rings at triazine C-3 and C-5 (6a), increased

HCT-116 inhibitory activity (IC₅₀¼ 14.70 nM) if compared to

lapatinib (IC₅₀¼ 12.00 nM), while caused sharp decrease in

MCF-7 inhibitory activity (IC₅₀¼ 120.30 nM) compacting to

lapatinib (IC₅₀¼ 21.00 nM).

Moreover, Combination between EWG and EDG at triazine C-3/5

aryl rings of (6f, IC₅₀¼ 5.60 and 59.30 nM) and (6h, IC₅₀¼ 12.90 and

32.90 nM), led to increase inhibitory activity of HCT-116 rather than

MCF-7.

2.2.1.2. Structure activity relationships (SAR) of cytotoxic activity of

compounds 6a-r. In this study, a novel series of pyrazolopyr-

imidine/triazine hybrids 6a-r was synthesized. The prepared

compounds characterized by presence of aryl ring at triazine C-3/5

Fig. 2. Theoretical MO calculations for Z/E conﬁguration for compounds 6b and 6p.

5

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Table 2

rings (6j, IC₅₀¼ 68.90 and 6.90 nM) or one aryl ring

substituted with EWG (6l, IC₅₀¼ 93.60 and 7.80 nM) as well

as two p-substituted aryl rings with EDG as in (6n,

IC₅₀¼ 26.40 and 8.00 nM) were observed.

Cytotoxic activity of tested compounds 6a-r and reference drug, lapatinib, on HCT-

116, MCF-7 and WI38 cell lines.

Finally, we can conclude that, compounds with carboxymethyl

spacer (6j-r) were of higher activity than those with carbonyl

spacer (6a-i) on both HCT-116 and MCF-7 cell lines. Thus between

all the tested compounds, compounds 6o and 6q exhibited the

highest cytotoxic activity on HCT-116 and MCF-7 with IC₅₀values

4.8 and 6.5 nM, respectively with 2.5- and 3.2-fold higher than that

of the reference compound lapatinib (IC₅₀¼ 12.00 nM and

21.00 nM on HCT-116 and MCF-7, sequentially).

Compound no.

n

R

R¹

H

IC₅₀results (nM)

^aMean SD

It is worth to notice that both compounds 6o and 6q had p-

chlorophenyl moiety at triazine C-3 or C-5 beside the presence of

carboxymethyl spacer at pyrazolopyrimidine C-6.

HCT-116

MCF-7

WI38

6a

0

1

e

H

14.70 0.41 120.30 3.76 49.50 0.97

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

6m

6n

6o

6p

6q

6r

H

CH₃

OCH₃52.10 1.48 61.30 1.50

97.70 3.87

31.80 0.64

2.2.2. Detection of apoptosis and in vitro DNA-ﬂow cytometry

The most two potent compounds 6o and 6q on HCT-116 and

MCF7 cell lines were selected to determine their effect on cell cycle

progression and apoptosis. HCT-116 and MCF-7 cells were treated

with either 6o or 6q, respectively, at their IC₅₀concentrations for

24 h. Then, ﬂow cytometry was used to analyze the DNA content

and compared to control experiment (without treatment). Deep

sight to ﬂow cytometric analysis showed that treatment HCT-116

and MCF-7 cells with compounds 6o and 6q, sequentially, led to

pre G1 apoptosis denoted by increase in cell population (17.35% and

22.12%, respectively), compared to the control (2.02% and 1.49%,

sequentially), (Fig. 4, Table 3).

Cl

H

28.10 1.44 44.30 0.80

46.50 1.32 126.70 3.46 24.50 0.69

CH₃OCH₃37.70 1.84 17.30 3.05

CH₃Cl 5.60 0.25 59.30 2.20

13.90 0.49 12.30 0.91

OCH₃12.90 0.48 32.90 1.40

36.80 0.47

26.40 0.59

55.00 1.17

25.00 0.35

19.30 0.80

47.20 1.87

17.20 0.54

110.80 2.13

85.60 3.67

18.60 0.83

29.40 1.13

80.20 2.99

54.90 2.16

12.90 0.99

45.20 0.96

Cl

H

Cl

H

15.00 0.30 17.80 0.98

68.90 2.18 6.90 1.40

OCH₃14.20 0.41 31.90 0.90

Cl

H

93.60 2.58 7.80 0.20

18.20 0.48 25.00 1.62

CH₃

CH₃OCH₃26.40 0.31 8.00 0.30

CH₃Cl 4.80 0.10 49.80 1.70

10.30 0.20 12.40 0.80

OCH₃12.90 0.54 6.50 0.10

Cl

e

H

Moreover, it was observed that G2/M cell growth arrest

appeared upon treatment with 6o and 6q, 2.98- and 11.97-fold

increase compared to the control HCT-116 and MCF-7 cells,

respectively, (Fig. 5).

Cl

7.20 0.10

35.00 1.60

Lapatinib

e

12.00 0.48 21.00 0.54

a

All experiments were repeated in triplicate.

In conclusion, the two selected compounds 6o and 6q showed

pre G1 apoptotic activity and cell growth arrest at G2/M phase.

Compound 6q (4-methoxyarylidine/4-chlorophenyl derivative),

showed higher apoptotic activity on MCF-7 cells than 6o (4-

chloroarylidine/4-tolyl derivative) on HCT-116 cells.

iii) Existence of two aryl rings para substituted with EDG (6e,

IC₅₀¼ 37.70 and 17.30 nM), resulted in decreasing HCT-116

inhibitory activity, while increasing MCF-7 inhibitory

activity.

iv) Reduction in inhibitory activity of both HCT-116 and MCF-

7 cell lines was observed in compounds bearing one aryl ring

and one p-substituted aryl ring with EDG or EWG as in (6b,

IC₅₀¼ 52.10 and 61.30 nM), (6c, IC₅₀¼ 28.10 and 44.30 nM)

and (6d, IC₅₀¼ 46.50 and 126.70 nM).

2.2.3. FITC-annexin V-staining for in vitro identiﬁcation of apoptosis

To further conﬁrm that accumulation of cells which was

observed in pre G1 phase was due to apoptosis, FITC-annexin v-

staining technique was used. Thus, HCT-116 and MCF-7 cells were

treated with compounds 6o and 6q, respectively for 24 h, then

analyzed using PI and FITC-annexin V-stain (Fig. 6). It was observed

that both early apoptosis (Q4) and late apoptosis (Q2) stages in

HCT-116 cells treated with compound 6o increased from 0.34%

(control, with no treatment) to 10.37% (30.79-fold increase), and

from 1.31% (control) to 2.72% (2-fold increase), respectively.

Moreover, in MCF-7 cells treated with 6q, late apoptosis was

signiﬁcantly increased from 0.47% (control) to 13.86% (29.48-fold

increase), and in early apoptosis, increased from 0.84% (control)

to 4.98% (5.6-fold increase).

For compounds 6j-r:

i) Highest cytotoxic activity against both HCT-116 and MCF-7 cell

lines was observed in compounds (6p, IC₅₀¼ 10.30 and

12.40 nM) and (6q, IC₅₀¼ 12.90 and 6.50 nM). They are char-

acterized by presence of either EWG at one of the two aryl rings

or their presence together at the two rings.

Very slight reduction in HCT-116 and MCF-7 inhibitory activity

was observed in (6m, IC₅₀¼ 18.20 and 25.00 nM), which has EDG at

one of its two aryl rings.

The results in this experiment were in accordance with that

obtained from the previous one (ﬂow cytometry). That conﬁrms the

cytotoxic effect of compounds 6o and 6q through their apoptotic

activities (Fig. 7).

ii) Increased HCT-116 inhibitory activity and decreased MCF-7

inhibitory activity were displayed in compounds bearing

one aryl and other p-substituted aryl ring with EDG (6k,

IC₅₀¼ 14.20 and 31.90 nM), two aryl groups carrying EDG

and EWG (6o, IC₅₀¼ 4.80 and 49.80 nM) or two aryl groups

with EWG (6r, IC₅₀¼ 7.20 and 35.00 nM).

2.2.4. In vitro ELISA immunoassay measurement of apoptotic

marker proteins expression levels (caspase3/7/9, Bax and Bcl-2)

The apoptotic activity of compounds 6o and 6q was further

investigated by measuring the induction of apoptosis of protein

biomarkers, caspase-3/7/9, Bax and Bcl-2 after 24 h treatment of

HCT-116 and MCF-7 cells with compounds 6o and 6q, respectively,

at their IC₅₀values. Then, the obtained results were compared with

iii) For decreasing inhibitory activity of HCT-116 cell line and

increasing inhibitory activity of MCF-7 cell line, two aryl

6

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Fig. 3. Representative diagram for SAR of test compounds 6a-r.

control HCT-116 and control MCF-7 cells. Activation of caspase-3/7

(due to both intrinsic and extrinsic factors) and caspase-9 (due to

From the results listed in Table 5, it was observed that com-

pounds 6o (76.97%, 72.61%), 6q (81.81%, 86.66%) & 6j (76.35%,

83.61%) had good inhibitory activities on both EGFR and HER2

enzymes, respectively. While, compound 6f (65.70%, 54.49%)

showed moderate inhibitory activities on both EGFR and HER2

enzymes, sequentially.

The inhibitory activities may be attributed to the substitution on

phenyl ring at triazine C-3 and C-5. Thus, unsubstituted phenyl

rings at C-3 and C-5 (6j), or para substituted phenyl rings with EDG

(CH₃/OCH₃) or EWG (Cl) in carboxy methyl derivatives (6o and 6q),

increased inhibitory activity rather than presence of EDG (CH₃) and

EWG (Cl) on phenyl rings of triazine scaffold in carboxy derivative

(6f).

intrinsic factors) leading to cell death as

fragmentation.

a result of DNA

It was observed that compound 6o increased the level of

caspase-3/7/9 by 18.5-, 20- and 16.5-fold, respectively, compared to

the untreated control HCT-116 cells. While, compound 6q increased

the level of caspase-3/7/9 by 12-, 7.9- and 22.9-fold, sequentially,

compared to the untreated control MCF-7 cells.

Tumor cells try to resist apoptosis by increasing the expression

of anti-apoptotic Bcl-2 protein level and decreasing the expression

of pro-apoptotic Bax protein level.

Treatment of HCT-116 and MCF-7 cells with compounds 6o and

6q, respectively, resulted in up regulation in pro-apoptotic protein,

Bax level by 6.9- and 5.6-fold, respectively, compared to the control

HCT-116 and MCF-7 cells.

On the other hand, down regulation in anti-apoptotic protein,

Bcl-2 was observed in HCT-116 and MCF-7 cells treated with 6o and

6q, respectively, by decreasing levels of Bcl-2 by 3.8- and 3.4-fold,

sequentially, compared to control cells HCT-116 and MCF-7.

Finally, we can conclude that the two pyrazolopyrimidine de-

rivatives 6o and 6q showed pro-apoptotic effect through increasing

protein levels of caspase-3/7/9 and Bax/Bcl-2 ratio, (Table 4, Fig. 8).

As a result, kinase IC₅₀for the selected compounds 6f, 6j, 6o and

6q was determined. Four concentrations (0.01

10 M) were measured to determine their IC₅₀values. The order of

reactivity against EGFR was 6q (0.22 M) > 6o (0.42 M) > 6j

M), and that towards HER2 was 6o

M) z 6j (0.87 M) > 6f (11.21 M). Subse-

mM, 0.1 mM,1 mM and

m

(0.65

(0.63

m

M) > 6f (1.88

m

M) > 6q (0.86

m

quently, the three compounds 6j, 6f and 6q were potent as EGFR/

HER2 inhibitors.

2.2.5. In vitro enzyme inhibitory activity assay

2.2.6. In vivo anticonvulsant activity results

To identify new pyrazolopyrimidine derivatives with dual EGFR/

HER2 inhibitory activities, the most potent derivatives on HCT-116

and/or MCF-7 (6f, 6j, 6o and 6q) were selected for further in-vitro

In vivo anticonvulsant activity of the tested compounds was

evaluated using lithium-pilocarpine model of Status Epilepticus in

relation to untreated SE group, phenytoin and lamotrigine as

reference anti-epileptic drugs.

enzyme investigations on EGFR and HER2 at 10 mM.

7

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Fig. 4. Flow cytometry analysis of the most active compounds 6o and 6q on HCT-116 and MCF-7, respectively for 24 h. (A) control HCT-116 cells, (B) control MCF-7 cells, (C) HCT-116

cancer cells treated with compound 6o (4.80 nM), (D) MCF-7 cancer cells treated with compound 6q (6.50 nM).

the mean power frequency spectral edge frequency, amplitude,

amplitude density, power, total power, delta and beta waves as

demonstrated in Tables 5 and 6 Also Compounds 6i and 6n

signiﬁcantly increased (p ˂ 05) total power by 37.32% and 33.82%,

respectively, when compared to the untreated SE group as pre-

sented in Table 7.

Table 3

Cell cycle analysis after treatment with compounds 6o and 6q at their IC₅₀values

(4.80 nM) and (6.50 nM), respectively for 24 h, compared with control HCT-116 and

control MCF-7.

Compound

Results

%Pre-G1

%G1

%S

%G2/M

Compounds 6b and 6m showed more profound improvement

similar to the group received lamotrigine evidenced by non-

signiﬁcant difference (p > 05) in the mean of power frequency,

amplitude, delta waves when compared to mice received lamo-

trigene. Moreover mice administered the compound 6m showed

potent improvement in spectral edge frequency and beta waves as

there was no signiﬁcant change (p > 05) when compared to the

group received lamotrigine as shown in Tables 6 and 7

6o/HCT-116

6q/MCF-7

cont. HCT-116

cont. MCF-7

17.35

22.12

2.02

22.64

25.09

46.95

53.77

31.42

28.34

37.66

42.34

45.94

46.57

15.39

3.89

1.49

2.2.6.1. Effect of the tested compounds on EEG recording changes.

The mean changes in different EEG recording of mice treated by

different compounds and % change from the untreated Status Epi-

lepticus (SE) group were listed in Tables 5 and 6 and represented by

Fig. 9.

In the present study, the untreated SE group had signiﬁcant

changes (p ˂ 05) when compared to the normal control group in all

EEG recording except theta and alpha waves as there was non-

signiﬁcant effects (p > 05) as shown in Table 6.

2.2.6.2. Effects of the tested compounds on biochemical parameters.

In the current study, the untreated SE group had signiﬁcant changes

(p ˂ 05) when compared to the normal control group in all

biochemical investigations; MDA, GSH, GABA and glutamate levels

that measured in the mice brain tissue.

Also, all groups received the tested compounds indicated sig-

niﬁcant improvement (p ˂ 05) in MDA, GSH, GABA and glutamate

levels in the mice brain tissue in relation to the untreated SE group

In comparison with the untreated SE group; all groups admin-

istered the tested compounds revealed signiﬁcant change (p ˂ 05) in

8

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Fig. 5. Graphical representation of cell cycle analysis of compounds 6o and 6q compared with control cells.

Fig. 6. FIT-annexin-V and PI staining of HCT-116 and MCF-7 cells after treatment with compounds 6o and 6q, respectively, and as control without treatment.

as presented in Table 8.

Moreover all groups administered the tested compounds

signiﬁcantly reduced lipid peroxidation (p ˂ 05) evidenced by

signiﬁcant reduction of MDA as compared to the untreated SE

group and non-signiﬁcant change (p > 05) when compared to

groups received phenytoin and lamotrigine. MDA level was

9

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Fig. 7. Graphical representation of % apoptotic cell in case of treatment with compounds 6o and 6q on HCT-116 and MCF-7 cells, sequentially, against their control.

Table 4

Protein expression level of apoptotic biomarkers caspase 3/7/9, Bax and Bcl-2 in HCT-116 and MCF-7 cells after treatment with compounds 6o (4.80 nM) and 6q (6.50 nM),

respectively, for 24 h.

Compound

Protein expression level conc. (pg./mL)

Caspase- 3

Caspase-7

Caspase-9

Bax

Bcl-2

6o

6q

324 11.3

0.7246 19.71

0.8777 7.71

8.57 16.22

10.29 22.80

258.4 6.97

237.4 5.61

2.55 0.26

3.09 0.29

176.26 6.71

17.489 0.62

14.35 0.47

Control HCT-116

Control MCF-7

0.03675

0.1138

1

0.52

0.45

1

37.03

42.28

1

9.70

1

10.45

1

obviously decreased in groups administered compounds 6a, 6d, 6h,

6i, 6k, 6l, 6m and 6n by 72.13%, 66.18%, 67.54%, 63.24%,66.91%,

74.39%, 69.74% and 69.49%, respectively, in relation to untreated SE

group, beside non-signiﬁcant change (p > 05) with respect to the

normal control group.

glutamate concentration by 76.21%, 72.91%, 75.98%, 76.98% and

82.89%, respectively, with non-signiﬁcant change (p > 05) as

compared to normal control group as shown in Table 8.

Accordingly, compounds 6b and 6m displayed potent anti-

epileptic activity. Also, 6d, 6e, 6h, 6i, 6k, 6l and 6n showed a

good anti-seizure activity, while compound 6j revealed the lower

activity. Rest of compounds exhibited neutral activity.

Additionally GSH level in the groups received all tested com-

pounds was signiﬁcantly increased (p < 05) as compared to the

untreated SE group. Also there was non-signiﬁcant change (p > 05)

in relation to the group received phenytoin except compounds 6j

and 6n; and non-signiﬁcant change (p > 05) as compared to

lamotrigine group except 6g, 6j, and 6n. The improvement of GSH

level was more evident with 6a, 6c, 6d, 6h, 6l, 6m, 6p, 6q and 6r as

it increased by 154.11, 174.47%, 173.91%, 150.97%, 170.04%, 188.85%,

191.86%, 179.51% and 146.06%, respectively, in comparison with the

untreated SE group, added to that there was non-signiﬁcant change

(p > 05) in relation to normal control group as listed in Table 8.

Also in the present study all groups administered the tested

compounds revealed a signiﬁcant increase (p ˂ 05) in GABA con-

centration (inhibitory neurotransmitter). Added to that, all com-

pounds except 6a, 6d, 6f, 6j and 6o showed better improvement in

GABA concentration; similar to phenytoin as there is no signiﬁcant

change (p > 05). While all compounds except 6j and 6p demon-

strated non-signiﬁcant change in GABA level (p > 05) as compared

to the group administered lamotrigine. Compound 6k profoundly

increased GABA level by 104.31% in relation to untreated SE group

and there was non-signiﬁcant change (p > 05) in comparison with

the normal control group (Table 8).

2.3. ADME study

2.3.1. In silico ADME prediction

The most active derivatives against HCT-116 and MCF-7 cell lines

6o and 6q, respectively, and the reference lapatinib were subjected

to in silico ADME prediction study to indicate their pharmacokinetic

properties such as absorption, distribution, metabolism and

excretion and comparing them with lapatinib, (Table 9). Thus, the

two target derivatives 6o and 6q showed good intestinal absorption

values 96.59% and 96.67%, sequentially, which are nearly equal to

lapatinib (96.86%).

Moreover, moderate permeability was observed for 6o, 6q and

lapatinib toward in vitro human colorectal carcinoma cells (CaCo-2)

(21.81, 21.68 and 17.99, respectively). While, low permeability was

appeared for the latter compounds towards in vitro Madin Darby

Canine Kidney cells (MDCK). Compounds 6o and 6q exerted strong

bound effect to plasma protein (91.15% and 91.22%, in a sequent) as

well as lapatinib (97.56%). Moreover, the target compounds 6o and

6q couldn’t penetrate the CNS because they had low absorption

into the CNS and their predicted blood brain barrier (BBB) values

were less than 0.40.

In addition, all tested compounds exhibited signiﬁcant reduc-

tion (p ˂ 05) in glutamate level (excitatory neurotransmitter) in

relation to the untreated SE group. moreover, glutamate level in the

groups administered compounds 6b,6d,6e,6h,6k, 6l and 6m not

only signiﬁcantly reduced (p ˂ 05) as compared to the untreated SE

group but also showed non-signiﬁcant change (p > 05) when

compared to mice received phenytoin and lamotrigine. Also com-

pounds 6b, 6d, 6k, 6l and 6m exhibited potent decrease in

Maximum skin permeability was observed for the target de-

rivatives 6o and 6q and also for the reference drug which ranged

from ꢀ2.91 to ꢀ2.32. From the obtained results, we concluded that

the target derivatives 6a and 6q had good predictable pharmaco-

kinetic properties.

10

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Fig. 8. Graphical representation of apoptotic biomarkers protein expression level (Caspase-3/7/9), Bax and Bcl-2 after treatment with compounds 6o and 6q at their IC₅₀for 24 h

using ELISA immunoassay technique.

2.3.2. Predicted pharmacokinetic properties

3. Conclusion

A molecule with high bioactivity should have scores more than

0.00. While, those with values ꢀ0.50 to 0.00, exhibited moderate

biological activities. The compound classiﬁed as biologically inac-

tive if its obtained score was less than ꢀ0.50.

In an attempt to overcome the resistance against EGFR in-

hibitors, a new series of pyrazolo[3,4-d]pyrimidine-based dual

EGFR T790M/HER2 was developed. All the prepared compounds

6a-r were screened for antitumor activity against two cell lines,

colon (HCT-116) and breast (MCF-7), in which EGFR is overex-

pressed, and on human ﬁbroblast cells (WI38). The tested de-

rivatives showed good to moderate activity. The highest cytotoxic

activity on HCT-116 and MCF-7 cells were observed in compounds

6o and 6q with IC₅₀value of 4.80 and 6.50 nM, respectively, if

compared with reference drug lapatinib (IC₅₀¼ 12.00 and 21.00 nM

on HCT-116 and MCF-7, sequentially). Both 6o and 6q shared in

By inspecting data listed in Table 10, it was revealed that com-

pounds 6o and 6q had moderate activity on GPCR ligand afﬁnity

and kinase inhibitory activity. Additionally, they had certain activity

to act as protease inhibitor and enzyme inhibitor. Whereas, they

gave low scores when ion channel modulator and nuclear receptor

ligand parameters were tested. Lapatinib showed good biological

activity as kinase inhibitor and moderate activity for all the other

remaining tested parameters.

11

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

NMR and ¹³C NMR (DEPT-Q) were carried out using the Bruker

instrument at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR

spectrophotometer, (Faculty of Pharmacy, Beni-Suef University,

Beni-Suef, Egypt), in DMSO‑d₆, D₂O using TMS as an internal

Table 5

% inhibitory effect 10

the reference lapatinib (10

m

M and IC₅₀

(mM) values of compounds 6f, 6j, 6o and 6q and

mM) on EGFR T790M and HER2 kinase activities.

standard and chemical shifts were recorded in ppm on the

d scale

using DMSO‑d₆(2.5) as a solvent. Coupling constant (J) values were

estimated in Hertz (Hz). Splitting patterns are designated as fol-

lows: s, singlet; d, doublet, t, triplet; q, quartet; dd, doublet of

doublet; m, multiplet. The electron impact (EI) mass spectra were

recorded on a Hewlett Packard 5988 spectrometer (Palo Alto, CA).

Microanalysis was performed for C, H, N on PerkinElmer 2400 at

the Microanalytical center, Cairo University, Egypt and was

within þ0.4% of theoretical values. Analytical thin layer chroma-

tography (TLC): pre-coated plastic sheets, 0.2 mm silica gel with UV

indicator (Macherey-Nagel) was employed routinely to follow the

course of reactions and to check the purity of products. All other

reagents, solvents were purchased from the Aldrich Chemical

Company (Milwaukee, WI) and, were used without further

puriﬁcation.

Compd. no.

N

R

R¹

% inhibition (10

m

M)

IC₅₀

EGFR-T790M HER2

54.49% 1.8872 11.2114

(mM)

EGFR-T790M HER2

6f

6j

6o

6q

0

1

CH₃Cl

H

CH₃Cl

Cl

M)

65.70%

76.35%

76.97%

H

83.61% 0.6556

72.61% 0.42225

86.66% 0.2267

94%

0.8723

0.6337

0.8657

OCH₃81.81%

77%

4.1.1. General procedure for synthesis of compounds 6a-r

Lapatinib (10

m

A mixture of the acid hydrazide 4a or 4b (0.01 mol), the

appropriate azalactone derivative 5a-i (0.01 mol), sodium acetate

(0.02 mol) in glacial acetic acid (10 mL) was reﬂuxed for 12e14 h

(monitored by TLC). The precipitated that formed on hot was

ﬁltered, dried and crystalized from 95% ethanol to afford 6a-r.

having p-chlorophenyl moiety and carboxymethyl part in their

structures.

The two compounds were further selected to determine their

effect on cell cycle progression and apoptosis. They showed pre G1

apoptotic activity beside cell growth arrest at G2/M phase.

Moreover, it was observed that both early and late apoptosis

stages in HCT-116 cells treated with 6o showed 30.79-fold and 2-

fold increase, respectively. While, compound 6q exhibited 29.48-

fold increase in late apoptosis in MCF-7 cells.

By measuring apoptotic marker protein expression levels (cas-

pase-3/7/9, Bax and Bcl-2), the two pyrazolopyrimidine derivatives

6o and 6q showed pro-apoptotic effect by increasing caspase-3/7/9

protein levels and Bax/Bcl-2 ratio.

Among tested compounds, four selected derivatives 6f, 6j, 6o

and 6q were selected for further in vitro enzyme investigations on

mutant EGFR T790M and HER2. They exhibited good inhibitory

activities on both the enzymes (81.81e65.70% and 86.66e54.49%,

respectively). As a result, their IC₅₀on the tested enzymes were

determined.

In silico ADME prediction and pharmacokinetic study for com-

pounds 6o and 6q were recorded resulting in good to moderate

scores.

Merging pyrazolo[3,4-d]pyrimidine scaffold with triazine ring in

one structure encouraged us to evaluate their anticonvulsant ac-

tivity, Where lithium-pilocarpine mice model of Status Epilepticus

was used. EEG recording changes where evaluated in addition to

measurement of MDA, GSH, GABA and glutamate in the brain tissue

of different groups. All compounds showed anti-epileptic effects

with different efﬁcacy as the most potent compounds were 6b and

6m. additionally 6d, 6e, 6h, 6i, 6k, 6l, 6n compounds displayed

good anti-seizure activity, while compound 6j showed the lower

activity. Other compounds revealed a neutral activity.

4.1.1.1. (Z)-6-(5-Benzylidene-6-oxo-3-phenyl-1,2,5,6-tetrahydro-

1,2,4-triazine-2-carbonyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]

pyrimidin-4(5H)-one (6a). Yellow solid; 49% yield; mp 368e370 ^ꢁC;

IR (KBr) 3210, 3140 (2NH), 3032 (CH-aromatic), 2928 (CH-

aliphatic), 1720-1670 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz, DMSO‑d₆,

d

¼ ppm) ¼ 2.58 (s, 3H, CH₃), 7.37 (s, 1H, ¼ CH), 7.53e7.65 (m, 7H,

d

benzylidene H-3, H-4, H-5, pyrazolopyrimidine phenyl H-4 and

phenyl H-3, H-4, H-5), 8.12 (d, J ¼ 7.6 Hz, 2H, benzylidene H-2, H-6),

8.19e8.25 (m, 4H, pyrazolopyrimidine phenyl H-3, H-5, phenyl H-2,

H-6), 8.39 (d, J ¼ 7.6 Hz, pyrazolopyrimidine phenyl H-2, H-6), 11.20

(s, 1H, NH, D₂O exchangeable), 12.68 (s, 1H, NH, D₂O exchangeable);

¹³C NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.74 (CH₃), 107.19

(pyrazolopyrimidine C-3a), 121.94 (¼CH), 127.31 (pyrazolopyr-

imidine phenyl C-2, C-6),127.47 (pyrazolopyrimidine phenyl C-4),

128.81 (benzylidene C-4), 129.45 (phenyl C-2, C-6), 129.75 (phenyl

C-3, C-5),129.82 (benzylidene C-2, C-6),129.88 (benzylidene C-3, C-

5), 131.59 (phenyl C-4), 133.09 (pyrazolopyrimidine phenyl C-3, C-

5),133.20 (phenyl C-1),136.15 (triazine C-5),138.48 (benzylidene C-

1), 141.01 (pyrazolopyrimidine phenyl C-1), 146.82 (pyrazolopyr-

imidine C-7a), 150.95 (pyrazolopyrimidine C-3), 151.20 (triazine C-

3), 158.11 (pyrazolopyrimidine C-6), 159.06 (pyrazolopyrimidine

C¼O), 160.05 (triazine C¼O), 168.17 (C¼O); EIMS (m/z) 516 (Mþ1,

4.50%), 515 (M^þ., 16.58%), 77 (100%). Anal.Calcd for C₂₉H₂₁N₇O₃

(515.17): C, 67.56; H, 4.11; N, 19.02. Found: C, 67.49; H, 4.15; N, 19.13.

4.1.1.2. (Z)-6-[5-(4-Methoxybenzylidene)-6-oxo-3-phenyl-1,2,5,6-

tetrahydro-1,2,4-triazine-2-carbonyl]-3-methyl-1-phenyl-1H-pyr-

azolo[3,4-d]pyrimidin-4(5H)-one (6b). Yellow solid; 35% yield; mp

275e277 ^ꢁC; IR (KBr) 3252, 3229 (2NH), 3009 (CH-aromatic), 2924

(CH-aliphatic), 1723-1670 (3C ¼ O) cm^ꢀ1

;

¹H NMR (400 MHz,

¼ ppm) ¼ 2.60 (s, 3H, CH₃), 3.82 (s, 3H, OCH₃), 7.05 (d,

4. Experimental

DMSO‑d₆,

d

J ¼ 8.4 Hz, 2H, p-methoxyphenyl H-3, H-5), 7.40e7.42 (m, 3H, ¼CH,

pyrazolopyrimidine phenyl H-4 and phenyl H-4), 7.54e7.58 (m, 4H,

pyrazolopyrimidine phenyl H-3, H-5, phenyl H-3 and H-5), 7.66 (d,

J ¼ 8.4 Hz, 2H, p-methoxyphenyl H-2, H-6), 8.17e8.21 (m, 4H,

pyrazolopyrimidine phenyl H-2, H-6, phenyl H-2 and H-6), 10.95 (s,

4.1. Chemistry

Melting points were determined using a Grifﬁn apparatus and

are uncorrected. Infrared spectra (IR) were recorded, using KBr

discs, on a Shimadzu IR-435 spectrophotometer and values were

represented in cm^ꢀ1(Faculty of Pharmacy, Cairo University). ¹H

1H, NH, D₂O exchangeable), 12.17 (s, 1H, NH, D₂O exchangeable);¹³

C

NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.76 (CH₃), 55.82 (OCH₃),

12

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Table 6

EEG recording changes in different studied groups.

EEG recording changes (Mean SD)

Groups

Mean power frequency Hz

Spectral edge frequency Hz SEF (95%)

Total Power (V2)

Amplitude

mV

Amplitude density mV/Hz

N

17.05 1.04

48.96 5.69

7.05 0.78

6.49 0.45

2.25 0.16^a

9.62 0.49

SE

Ph

La

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

97.21 1.95^a

193.90 4.21^a

3.43 0.15^a

5.20 0.19^a,b

4.57 .48^a,b

3.88 0.29^a,c

4.48 0.17^a

4.10 0.11^a,c

3.96 0.04^a,c

4.07 0.05^a,c

3.96 0.12^a,c

3.78 0.27^a,c

3.94 0.14^a,c

4.71 0.02^a,b

3.51 0.23^a,c,d

4.00 0.01^a,c

4.22 0.10^a,c

4.23 0.20^a,c

4.59 0.21^a,b

3.34 0.17^a,c,d

3.42 0.23^a,c,d

3.88 0.25^a,c

3.45 0.30^a,c,d

3.29 0.08^a

40.30 1.70^a,b

118.79 8.23^a,b

5.82 0.40^b,d

9.05 0.25^b,d

48.04 1.46^a,b,c

67.81 1.53^a,b,c,d

50.07 0.19^a,b,c

70.41 0.76^a,b,c,d

60.34 1.42^a,b,c,d

65.53 0.69^a,b,c,d

68.56 1.70^a,b,c,d

75.02 1.42^a,b,c,d

57.92 1.56^a,b,c,d

60.65 1.71^a,b,c,d

73.90 1.62^a,b,c,d

58.95 0.67^a,b,c,d

54.33 0.70^a,b,c,d

52.27 2.46^a,b,c

65.13 3.69^a,b,c,d

73.49 2.03^a,b,c,d

69.49 2.21^a,b,c,d

69.42 1.02^a,b,c,d

72.86 1.72^a,b,c,d

127.84 3.46^a,b

5.01 0.11^a,b,c

3.76 0.37^a,b,c,d

4.49 0.15^a,b,c

3.96 0.16^a,b,c,d

3.65 0.25^a,b,c,d

4.03 0.22^a,b,c,d

3.74 0.23^a,b,c,d

3.48 0.07^a,b,c,d

3.85 0.15^a,b,c,d

4.27 0.26^a,b,c,d

3.67 0.26^a,b,c,d

3.74 0.28^a,b,c,d

4.11 0.12^a,b,c,d

4.29 0.16^a,b,c

4.06 0.18^a,b,c,d

3.38 0.12^a,b,c,d

3.49 0.13^a,b,c,d

3.32 0.23^a,b,c,d

3.28 0.09^a,b,c,d

7.98 0.55^a,b,c

5.87 0.38^a,b,c,d

6.19 0.24^a,b,c,d

5.83 0.31^a,b,c,d

4.48 0.11^a,b,c,d

5.16 0.23^a,b,c,d

5.65 0.25^a,b,c,d

5.80 0.15^a,b,c,d

4.83 0.24^a,b,c,d

5.07 0.08^a,b,c,d

5.33 0.06^a,b,c,d

4.86 0.19^a,b,c,d

5.37 0.44^a,b,c,d

6.92 0.44^a,b,c,d

6.02 0.14^a,b,c,d

5.33 0.18^a,b,c,d

5.53 0.23^a,b,c,d

5.51 0.26^a,b,c,d

5.57 0.28^a,b,c,d

162.27 2.44^a,b,c,d

144.88 1.32^a,b,c,d

160.40 3.06^a,b,c,d

150.74 2.20^a,b,c,d

157.54 1.85^a,b,c,d

154.17 2.35^a,b,c,d

167.54 2.20^a,b,c,d

150.26 1.55^a,b,c,d

152.31 2.33^a,b,c,d

160.06 1.46^a,b,c,d

151.50 1.86^a,b,c,d

139.43 1.71^a,b,c,d

137.66 2.25^a,b,c

142.63 4.03^a,b,c,d

162.61 3.32^a,b,c,d

160.29 0.73^a,b,c,d

154.72 2.73^a,b,c,d

167.93 2.27^a,b,c,d

6m

6n

6o

6p

6q

6r

EEG recording changes (Mean SD)

Groups

Power pV2

Total Power density pV2/Hz

Delta

m

V2/Hz

Theta

m

V2/Hz

Alpha mV2/Hz

N

32.49 2.79

79.66 3.96

63.20 4.47

18.50 0.64

14.12 0.26

16.91 0.94

15.83 1.03

18.20 0.64

18.26 0.42

18.93 0.09

18.64 0.99

18.68 0.76

19.05 0.65

16.39 5.17

16.98 0.28

19.93 0.26

18.61 0.98

17.57 0.71

16.99 0.31

15.79 0.37

17.95 0.37

20.45 0.69

19.06 0.68

21.91 1.55

21.77 1.21

14.34 0.37

16.87 0.51

14.37 0.54

15.42 0.33

18.96 0.61

15.94 0.67

20.06 0.16

18.41 0.37

17.71 0.77

19.19 1.23

21.46 0.95^a,c,d

16.07 0.42

17.65 1.04

22.52 0.75^a,c,d

17.30 0.34

16.89 0.04

15.86 0.68

16.99 0.75

22.26 1.07^a,c,d

22.75 1.37^a,b,c,d

21.60 1.18^a,c,d

22.92 1.64^a,b,c,d

SE

Ph

La

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

5.96 0.77^a

13.88 0.45^a

9.08 0.39^a

25.80 .89^a,b,d

66.85 1.37^a,b,d

62.03 0.61^a,b,c

45.71 0.78^a,b,c,d

50.70 2.23^a,b,c,d

42.01 0.58^a,b,c,d

43.05 0.80^a,b,c,d

44.76 1.08^a,b,c,d

42.80 1.46^a,b,c,d

41.71 0.76^a,b,c,d

47.95 1.40^a,b,c,d

46.34 1.74^a,b,c,d

41.97 0.29^a,b,c,d

43.81 0.59^a,b,c,d

48.41 1.27^a,b,c,d

55.84 1.71^a,b,c,d

50.61 1.05^a,b,c,d

38.42 1.22^a,b,c,d

38.23 0.54^a,b,c,d

38.14 1.43^a,b,c,d

39.14 0.90^a,b,c,d

53.20 3.00^a,b,d

47.71 1.60^a,b,c

37.62 2.07^a,b,c,d

42.58 3.16^a,b,c

33.68 1.25^a,b,c,d

31.04 0.50^a,b,c,d

35.87 1.29^a,b,c,d

32.40 0.92^a,b,c,d

32.15 1.29^a,b,c,d

39.10 1.54^a,b,c,d

37.57 0.84^a,b,c,d

33.08 1.70^a,b,c,d

33.29 0.46^a,b,c,d

38.81 1.03^a,b,c,d

42.35 0.97^a,b,c

42.02 1.21^a,b,c,d

29.94 1.41^a,b,c,d

33.14 0.66^a,b,c,d

32.52 0.82^a,b,c,d

30.86 1.31^a,b,c,d

22.21 0.37^a,b,c

14.23 0.47^a,b,c,d

19.67 1.02^a,b,c

13.96 0.19^a,b,c,d

14.53 0.55^a,b,c,d

17.79 0.42^a,b,c,d

15.02 0.37^a,b,c,d

13.92 0.15^a,b,c,d

17.33 0.65^a,b,c,d

19.42 0.51^a,b,c,d

14.11 0.26^a,b,c,d

15.95 0.13^a,b,c,d

18.14 0.18^a,b,c,d

20.47 0.85^a,b,c

19.01 0.69^a,b,c,d

13.19 0.70^a,b,c,d

12.99 0.18^a,b,c,d

13.85 0.33^a,b,c,d

13.50 0.83^a,b,c,d

6m

6n

6o

6p

6q

6r

Each value represents the mean SD.

Statistical analysis was performed using one-way ANOVA test followed by Tukey post-hoc test.

Differences between group means were considered signiﬁcant at p < 0.05.

^aSigniﬁcantly different from normal control group value at p < 0.05.

^bSigniﬁcantly different from untreated SE group value at p < 0.05.

^cSigniﬁcantly different from phenytoin received group value at p < 0.05.

^dSigniﬁcantly different from lamotrigine received group value at p < 0.05.

N: normal control group; SE: untreated Status Epilepticus group model; Ph: phenytoin; La: lamotrigine; 6a-6r: the tested compounds.

102.21 (pyrazolopyrimidine C-3a), 114.89 (p-methoxybenzylidene

C-3, C-5), 121.97 (¼CH), 126.88 (pyrazolopyrimidine phenyl C-2, C-

6), 127.25 (pyrazolopyrimidine phenyl C-4), 128.41(p-methox-

ybenzylidene C-1), 129.73 (phenyl C-3, C-5), 131.91 (phenyl C-2, C-

6), 132.00 (phenyl C-1), 133.66 (p-methoxybenzylidene C-2, C-6),

135.00 (triazine C-5), 135.40 (pyrazolopyrimidine phenyl C-3, C-5),

137.37 (phenyl C-4), 141.01 (pyrazolopyrimidine C-1), 143.48 (pyr-

azolopyrimidine C-7a), 148.29 (pyrazolopyrimidine C-3), 156.15

(triazine C-3), 157.24 (pyrazolopyrimidine C-6), 160.08 (p-methox-

ybenzylidene C-4), 162.27 (pyrazolopyrimidine C¼O), 166.63

(triazine C¼O), 168.38 (C¼O); Anal. Calcd for C₃₀H₂₃N₇O₄(545.18):

C, 66.05; H, 4.25; N, 17.97. Found: C, 66.17; H, 4.15; N, 17.89.

4.1.1.3. (Z)-6-[5-(4-Chlorobenzylidene)-6-oxo-3-phenyl-1,2,5,6-

tetrahydro-1,2,4-triazine-2-carbonyl]-3-methyl-1-phenyl-1H-pyr-

azolo[3,4-d]pyrimidin-4(5H)-one (6c). Yellow solid; 58% yield; mp

230e232 ^ꢁC; IR (KBr) 3264, 3148 (2NH), 3024 (CH-aromatic), 2932

(CH-aliphatic), 1728-1670 (3C ¼ O) cm^ꢀ1

;

¹H NMR (400 MHz,

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.58 (s, 3H, CH₃), 7.39e7.45 (m, 3H, ¼CH

and p-chlorophenyl H-3, H-5), 7.53e7.66 (m, 6H, phenyl H-3, H-4,

H-5, pyrazolopyrimidine phenyl H-3, H-4 and H-5), 8.12 (d,

J ¼ 7.2 Hz, 2H, phenyl H-2, H-6), 8.21 (d, J ¼ 8 Hz, 2H, p-chlor-

ophenyl H-2, H-6), 8.41 (d, J ¼ 8.8 Hz, 2H, pyrazolopyrimidine

phenyl H-2, H-6), 12.09 (s, 1H, NH, D₂O exchangeable), 12.65 (s, 1H,

NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

13

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Fig. 9. EEG recording signal changes in different studied groups. N: normal control group; SE: untreated Status Epilepticus group model; Ph: phenytoin; La: lamotrigine; 6a-6r: the

tested compounds.

Table 7

The mean % Change of EEG recording in different groups.

% of Change From untreated SE group

Groups

Mean power frequency Hz

Spectral edge frequency Hz SEF (95%)

Total Power (V2)

Amplitude

m

V

Amplitude density mV/Hz

Ph

La

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

6m

6n

6o

6p

6q

6r

ꢀ58.54%

ꢀ50.58%

ꢀ30.24%

ꢀ48.49%

ꢀ27.57%

ꢀ37.93%

ꢀ32.59%

ꢀ29.47%

ꢀ22.83%

ꢀ40.42%

ꢀ37.61%

ꢀ23.98%

ꢀ39.36%

ꢀ44.11%

ꢀ46.23%

ꢀ33.00%

ꢀ24.40%

ꢀ28.52%

ꢀ28.59%

ꢀ25.05%

ꢀ38.74%

ꢀ34.07%

ꢀ16.31%

ꢀ25.28%

ꢀ17.28%

ꢀ22.26%

ꢀ18.75%

ꢀ20.49%

ꢀ13.59%

ꢀ22.51%

ꢀ21.45%

ꢀ17.45%

ꢀ21.87%

ꢀ28.09%

ꢀ29.00%

ꢀ26.44%

ꢀ16.14%

ꢀ17.33%

ꢀ20.21%

ꢀ13.39%

51.60%

33.24%

13.12%

30.61%

19.53%

15.45%

18.66%

15.45%

10.20%

14.87%

37.32%

2.33%

16.62%

23.03%

23.32%

33.82%

2.62%

158.67%

122.67%

67.11%

99.56%

76.00%

62.22%

79.11%

66.22%

54.67%

71.11%

89.78%

63.11%

66.22%

82.67%

90.67%

80.44%

50.22%

55.11%

47.56%

45.78%

175.08%

142.55%

78.42%

88.15%

77.20%

36.17%

56.84%

71.73%

76.29%

46.81%

54.10%

62.01%

47.72%

63.22%

110.33%

82.98%

62.01%

68.09%

67.48%

69.30%

0.29%

13.12%

0.58%

% of Change From untreated SE group

Groups

Power pV2

Total Power density pV2/Hz

Delta

m

V2/Hz

Theta

m

V2/Hz

Alpha mV2/Hz

Ph

La

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

6m

6n

6o

6p

6q

6r

332.89%

272.65%

138.76%

230.03%

134.23%

143.79%

198.49%

152.01%

133.56%

190.77%

225.84%

136.74%

167.62%

204.36%

243.46%

218.96%

121.31%

117.95%

132.38%

126.51%

381.63%

346.90%

229.32%

265.27%

202.67%

210.16%

222.48%

208.36%

200.50%

245.46%

233.86%

202.38%

215.63%

248.78%

302.31%

264.63%

176.80%

175.43%

174.78%

181.99%

485.90%

425.44%

314.32%

368.94%

270.93%

241.85%

295.04%

256.83%

254.07%

330.62%

313.77%

264.32%

266.63%

327.42%

366.41%

362.78%

229.74%

264.98%

258.15%

239.87%

19.76%

12.11%

28.90%

29.32%

34.07%

32.01%

32.29%

34.92%

16.08%

20.25%

41.15%

31.80%

24.43%

20.33%

11.83%

27.12%

44.83%

34.99%

55.17%

54.18%

ꢀ14.82%

ꢀ8.6%

12.39%

ꢀ5.51%

18.91%

9.13%

4.98%

13.75%

27.21%

ꢀ4.74%

4.62%

33.49%

2.55%

0.12%

ꢀ5.99%

0.71%

31.95%

34.85%

28.04%

35.86%

Percentage (%) changes were calculated by comparing groups received the tested compounds with the untreated SE group.

Ph: phenytoin; La: lamotrigine; 6a-6r: the tested compounds.

14

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Table 8

Biochemical analysis in different studied groups.

Parameters (Mean SD)

Groups

MDA (nmol/mg protein)

GSH (mmol/mg protein)

GABA (ng/mg) protein

Glutamate (ng/mg) protein

N

8.36 2.15

76.23 13.15

23.23 2.41^a

59.56 14.78^b

63.26 9.26^b

59.03 1.20^b

56.06 3.16^a,b

63.76 1.05^b

63.63 6.46^b

46.00 2.15^a,b

49.53 1.85^a,b

41.90 3.70^a,b,c

58.30 3.10^b

56.70 7.10^a,b

39.30 3.30^a,b,c,d

54.93 9.75^a,b

63.66 2.41^b

67.10 8.87^b

35.00 2.32^a,b,c,d

54.50 1.75^a,b

67.80 3.81^b

64.93 0.25^b

57.16 0.95^b

221.63 7.71

1.46 0.15

SE

Ph

La

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

54.40 14.59^a

23.46 1.80^a,b

23.03 3.56^a,b

15.16 2.85^b

25.00 1.15^a,b

25.30 2.85^a,b

18.40 1.27^b

28.96 1.30^a,b

26.80 1.65^a,b

24.33 0.77^a,b

17.66 3.25^b

20.00 3.85^b

23.43 1.75^a,b

18.00 1.75^b

13.93 3.65^b

16.46 2.50^b

16.60 1.70^b

23.03 2.80^a,b

28.90 2.40^a,b

27.20 0.30^a,b

21.43 3.2^a,b

97.06 3.98^a

13.03 0.70^a

2.43 0.30^b

187.20 2.19^a,b

177.66 13.65^a,b

156.96 10.23^a,b,c

163.70 14.01^a,b

169.63 16.27^a,b

155.03 11.75^a,b,c

174.36 9.42^a,b

153.50 6.00^a,b,c

183.80 2.40^a,b

181.53 0.95^a,b

169.36 6.71^a,b

148.16 4.60^a,b,c,d

198.30 7.26^b

2.46 0.32^b

5.43 1.35^a,b,c,d

3.10 0.60^b

6.43 0.05^a,b,c,d

3.53 0.65^b

4.20 0.10^a,b

8.33 1.76^a,b,c,d

7.83 1.30^a,b,c,d

4.36 0.30^a,b

5.50 0.65^a,b,c,d

6.40 0.10^a,b,c,d

3.13 1.00^b

175.30 9.26^a,b

185.60 2.00^a,b

166.83 1.55^a,b

152.16 8.85^a,b,c

135.93 3.55^a,b,c,d

166.50 3.15^a,b

176.86 9.45^a,b

3.00 0.96^b

6m

6n

6o

6p

6q

6r

2.23 0.15^b

6.46 0.20^a,b,c,d

8.13 0.95^a,b,c,d

7.10 0.20^a,b,c,d

8.33 1.66^a,b,c,d

5.40 0.70^a,b,c,d

% of Change From untreated SE group

Groups

MDA

GSH

GABA

Glutamate

N

e

SE

Ph

La

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

e

ꢀ56.88%

ꢀ57.67%

ꢀ72.13%

ꢀ54.04%

ꢀ53.49%

ꢀ66.18%

ꢀ46.76%

ꢀ50.74%

ꢀ55.28%

ꢀ67.54%

ꢀ63.24%

ꢀ56.93%

ꢀ66.91%

ꢀ74.39%

ꢀ69.74%

ꢀ69.49%

ꢀ57.67%

ꢀ46.88%

ꢀ50%

156.39%

172.32%

154.11%

141.33%

174.47%

173.91%

98.02%

113.22%

80.37%

150.97%

144.08%

69.18%

136.46%

174.04%

188.85%

50.67%

134.61%

191.86%

179.51%

146.06%

92.87%

83.04%

61.71%

68.66%

74.77%

59.73%

79.64%

58.15%

89.37%

87.03%

74.49%

52.65%

104.31%

80.61%

91.22%

71.88%

56.77%

40.05%

71.54%

82.22%

ꢀ81.35%

ꢀ81.12%

ꢀ58.33%

ꢀ76.21%

ꢀ50.65%

ꢀ72.91%

ꢀ67.77%

ꢀ36.07%

ꢀ39.91%

ꢀ66.54%

ꢀ57.79%

ꢀ50.88%

ꢀ75.98%

ꢀ76.98%

ꢀ82.89%

ꢀ50.42%

ꢀ37.61%

ꢀ45.51%

ꢀ36.07%

ꢀ58.56%

6m

6n

6o

6p

6q

6r

ꢀ60.61%

Each value represents the mean SD.

Statistical analysis was performed using one-way ANOVA test followed by Tukey post-hoc test.

Differences between group means were considered signiﬁcant at p < 0.05.

^aSigniﬁcantly different from normal control group value at p < 0.05.

^bSigniﬁcantly different from untreated SE group value at p < 0.05.

^cSigniﬁcantly different from phenytoin received group value at p < 0.05.

^dSigniﬁcantly different from lamotrigine received group value at p < 0.05.

Percentage (%) changes were calculated by comparing groups received the tested compounds with untreated SE group.

N: normal control group; SE: untreated Status Epilepticus group model; Ph: phenytoin; La: lamotrigine; 6a-6r: the tested compounds; MDA: malondialdehyde; GSH:

glutathione reduced; GABA: gamma-aminobutyric acid.

d

¼ 13.73 (CH₃), 107.40 (pyrazolopyrimidine C-3a), 121.99 (¼CH),

(549.13): C, 63.33; H, 3.67; N, 17.83. Found: C, 63.27; H, 3.71; N,

17.85.

127.19 (phenyl C-1), 127.29 (pyrazolopyrimidine phenyl C-2, C-6),

127.45 (pyrazolopyrimidine phenyl C-4), 128.31 (phenyl C-2, C-6),

128.87 (p-chlorophenyl C-3, C-5), 129.46 (phenyl C-3, C-5), 129.57

(p-chlorophenyl C-2, C-6), 133.03 (p-chlorophenyl C-1), 133.22

(phenyl C-4), 134.72 (pyrazolopyrimidine phenyl C-3, C-5), 136.19

(p-chlorophenyl C-4), 136.51 (triazine C-5), 138.49 (pyrazolopyr-

imidine phenyl C-1), 146.85 (pyrazolopyrimidine C-7a), 150.96

(pyrazolopyrimidine C-3), 151.20 (triazine C-3), 158.10 (pyr-

azolopyrimidine C-6), 159.07 (pyrazolopyrimidine C¼O), 160.45

4.1.1.4. (Z)-6-[5-Benzylidene-6-oxo-3-(p-tolyl)-1,2,5,6-tetrahydro-

1,2,4-triazine-2-carbonyl]-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]

pyrimidin-4(5H)-one (6d). Yellow solid; 37% yield; mp 337e339 ^ꢁC;

IR (KBr) 3325, 3194 (2NH), 3009 (CH-aromatic), 2924 (CH-

aliphatic),1722-1673 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 2.36 (s, 3H, CH₃), 2.56 (s, 3H, CH₃), 7.31e7.49 (m,

2H, ¼CH, benzylidene H-4), 7.51e7.58 (m, 7H, benzylidene H-3, H-

5, p-tolyl H-3, H-5, phenyl H-3, H-4, H-5), 8.04 (d, J ¼ 8.4 Hz, 2H,

(triazine C¼O), 168.05 (C¼O); Anal. Calcd for

C₂₉H₂₀ClN₇O₃

15

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

Table 9

Predicted in silico ADME properties for compounds 6o and 6q, and lapatinib.

Compd.

No.

^aHIA

^bCaCo-2

^cMDCK

^dBPB

^eBBB

^fSkinP

Acceptable value

70e100% (good absorption)

˃ 90 (high permeability)

˃ 90% strong binding

˃ 0.40 CNS active compound

ꢂ ꢀ3

6^ꢁ

6q

Lapatinib

96.59

96.67

96.86

21.81

21.68

17.99

0.04

0.05

91.15

91.22

97.56

0.20

0.18

0.03

ꢀ2.71

ꢀ2.91

ꢀ2.32

a

Human intestinal absorbtion (%).

b

c

d

e

f

in vitro CaCo cell permeability (nm/sec).

in vitro MDCK cell permeability (nm/sec).

in vitro plasma protein binding (%).

in vitro blood brain barrier penetration (C. brain/C. blood).

Skin permeability.

Table 10

Predicted pharmacokinetic properties of target compounds 6o, 6q and lapatinib.

Compd.

No.

GPCR ligand

Ion channel modulator

Kinase inhibitor

Nuclear receptor ligand

Protease inhibitor

Enzyme inhibitor

6o

6q

Lapatinib

ꢀ0.28

ꢀ0.32

ꢀ0.04

ꢀ1.07

ꢀ1.16

ꢀ0.52

ꢀ0.17

ꢀ0.24

0.36

ꢀ0.91

ꢀ0.96

ꢀ0.35

ꢀ0.44

ꢀ0.46

ꢀ0.21

ꢀ0.38

ꢀ0.45

ꢀ0.08

benzylidene H-2, H-6), 8.20 (d, J ¼ 7.2 Hz, 2H, p-tolyl H-2, H-6), 8.25

(d, J ¼ 8 Hz, 2H, phenyl H-2, H-6), 9.98 (s, 1H, NH, D₂O exchange-

able), 12.65 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz,

4.1.1.6. (Z)-6-[5-(4-Chlorobenzylidene)-6-oxo-3-(p-tolyl)-1,2,5,6-

tetrahydro-1,2,4-triazine-2-carbonyl]-3-methyl-1-phenyl-1H-pyr-

azolo[3,4-d]pyrimidin-4(5H)-one (6f). Yellow solid; 54% yield; mp

378e380 ^ꢁC; IR (KBr) 3368, 3194 (2NH), 3013 (CH-aromatic), 2928

DMSO‑d₆,

d

¼ ppm)

d

¼ 13.77 (CH₃), 21.64 (CH₃), 103.87 (pyr-

azolopyrimidine C-3a), 121.98 (¼CH), 127.22 (phenyl C-2, C-6),

127.50 (phenyl C-4), 128.83 (benzylidene C-4), 129.21 (p-tolyl C-1),

129.39 (benzylidene C-2, C-6), 129.71 (p-tolyl C-3, C-5), 129.94 (p-

tolyl C-2, C-6), 131.26 (benzylidene C-3, C-5), 133.04 (phenyl C-3, C-

5), 134.34 (triazine C-5), 135.86 (benzylidene C-1), 137.46 (p-tolyl C-

4), 138.68 (phenyl C-1), 146.55 (pyrazolopyrimidine C-7a), 150.75

(pyrazolopyrimidine C-3), 155.69 (triazine C-3), 158.24 (pyr-

azolopyrimidine C-6), 160.11 (pyrazolopyrimidine C¼O), 165.78

(triazine C¼O), 168.37 (C¼O); EIMS (m/z) 530 (Mþ1, 0.85%), 529

(M^þ., 4.63%), 77 (100%). Anal.Calcd for C₃₀H₂₃N₇O₃(529.19): C,

68.04; H, 4.38; N, 18.52. Found: C, 68.13; H, 4.34; N, 18.49.

(CH-aliphatic),1723-1678 (3C ¼ O) cm^ꢀ1

;

¹H NMR (400 MHz,

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.35 (s, 3H, CH₃), 2.55 (s, 3H, CH₃), 7.21 (s,

1H,¼CH), 7.55e7.60 (m, 7H, p-tolyl H-3, H-5, p-chlorobenzylidene

H-3, H-5 and phenyl H-3, H-4, H-5), 8.09e8.11 (m, 4H, p-tolyl H-2,

H-6 and p-chlorobenzylidene H-2, H-6), 8.30 (d, J ¼ 8 Hz, 2H,

phenyl H-2, H-6), 9.98 (s, 1H, NH, D₂O exchangeable), 11.10 (s, 1H,

NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 14.00 (CH₃), 21.64 (CH₃), 104.56 (pyrazolopyrimidine C-3a),

121.91 (¼CH), 127.19 (phenyl C-2, C-6), 128.96 (phenyl C-4), 129.50

(p-tolyl C-1), 129.71 (p-chlorobenzylidene C-3, C-5), 129.77 (p-

chlorobenzylidene C-2, C-6), 130.15 (p-tolyl C-3, C-5), 131.11 (p-tolyl

C-2, C-6), 132.43 (p-chlorobenzylidene C-1), 133.07 (p-chlor-

obenzylidene C-4), 134.40 (phenyl C-3, C-5), 135.46 (triazine C-5),

138.72 (phenyl C-1), 138.96 (p-tolyl C-4), 146.54 (pyrazolopyr-

imidine C-7a), 151.78 (pyrazolopyrimidine C-3), 154.84 (triazine C-

3), 158.77 (pyrazolopyrimidine C-6), 160.08 (pyrazolopyrimidine

C¼O), 163.14 (triazine C¼O), 168.51 (C¼O); Anal. Calcd for

4.1.1.5. (Z)-6-[5-(4-Methoxybenzylidene)-6-oxo-3-(p-tolyl)-1,2,5,6-

tetrahydro-1,2,4-triazine-2-carbonyl]-3-methyl-1-phenyl-1H-pyr-

azolo[3,4-d]pyrimidin-4(5H)-one (6e). Yellow solid; 56% yield; mp

380e382 ^ꢁC; IR (KBr) 3198 (broad, 2NH), 3008 (CH-aromatic), 2924

(CH-aliphatic),1720-1675 (3C ¼ O) cm^ꢀ1

;

¹H NMR (400 MHz,

¼ 2.35 (s, 3H, CH₃), 2.51 (s, 3H, CH₃), 3.86 (s,

C

₃₀H₂₂ClN₇O₃(563.15): C, 63.89; H, 3.93; N, 17.38. Found: C, 63.92;

DMSO‑d₆,

d

¼ ppm)

d

H, 3.87; N, 17.35.

3H, OCH₃), 7.11 (d, J ¼ 8 Hz, 2H, p-methoxybenzylidene H-3, H-5),

7.30e7.40 (m, 3H, p-tolyl H-3, H-5 and ¼ CH), 7.53e7.59 (m, 3H,

phenyl H-3, H-4, H-5), 8.02 (d, J ¼ 8 Hz, 2H, p-methoxybenzylidene

H-2, H-6), 8.19 (d, J ¼ 8 Hz, 2H, p-tolyl H-2, H-6), 8.36 (d, J ¼ 8 Hz,

2H, phenyl H-2, H-6), 12.05 (s, 2H, 2NH, D₂O exchangeable); ¹³C

4.1.1.7. (Z)-6-(5-Benzylidene-3(4-chlorophenyl)-6-oxo-1,2,5,6-

tetrahydro-1,2,4-triazine-2-carbonyl)-3-methyl-1-phenyl-1H-pyr-

azolo[3,4-d]pyrimidin-4(5H)-one (6g). Yellow solid; 31% yield; mp

297e299 ^ꢁC; IR (KBr) 3217, 3132 (2NH), 3032 (CH-aromatic), 2928

NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.77 (CH₃), 21.52 (CH₃),

55.93 (OCH₃), 107.33 (pyrazolopyrimidine C-3a), 115.10 (p-

methoxybenzylidene C-3, C-5), 121.94 (¼CH), 124.92 (p-methox-

ybenzylidene C-1), 127.07 (phenyl C-2, C-6), 127.19 (p-tolyl C-1),

128.64 (phenyl C-4), 129.23 (p-tolyl C-3, C-5), 129.70 (p-tolyl C-2, C-

6), 129.93 (p-methoxybenzylidene C-2, C-6), 134.42 (triazine C-5),

135.22 (phenyl C-3, C-5), 138.63 (p-tolyl C-4), 138.76 (phenyl C-1),

142.97 (pyrazolopyrimidine C-7a), 146.65 (pyrazolopyrimidine C-

3), 151.56 (triazine C-3), 158.16 (pyrazolopyrimidine C-6), 158.77 (p-

methoxybenzylidene C-4), 160.95 (pyrazolopyrimidine C¼O),

162.03 (triazine C¼O), 168.20 (C¼O); Anal. Calcd for C₃₁H₂₅N₇O₄

(559.20): C, 66.54; H, 4.50; N, 17.52. Found: C, 66.57; H, 4.61; N,

17.58.

(CH-aliphatic),1720-1673 (3C ¼ O) cm^ꢀ1

;

¹H NMR (400 MHz,

¼ 2.58 (s, 3H, CH₃), 7.39e7.45 (m, 3H, ¼ CH

DMSO‑d₆,

d

¼ ppm)

d

and p-chlorophenyl H-2, H-6), 7.53e7.66 (m, 6H, benzylidene H-3,

H-4, H-5 and phenyl H-3, H-4, H-5), 8.12 (d, J ¼ 7.6 Hz, 2H, ben-

zylidene H-2,H-6), 8.23 (d, J ¼ 8.4 Hz, 2H, phenyl H-2, H-6), 8.41 (d,

J ¼ 8.4 Hz, 2H, p-chlorophenyl H-3, H-5), 12.09 (s, 1H, NH, D₂O

exchangeable), 12.70 (s, 1H, NH, D₂O exchangeable); ¹³C NMR

(100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.74 (CH₃), 107.41 (pyr-

azolopyrimidine C-3a), 121.89 (¼CH), 127.22 (p-chlorophenyl C-1),

127.25 (phenyl C-2, C-6), 128.31 (benzylidene C-4), 128.88 (benzy-

lidene C-3, C-5), 129.46 (benzylidene C-2, C-6), 129.56 (p-chlor-

ophenyl C-2, C-6),129.79 (p-chlorophenyl C-3, C-5),133.06 (triazine

16

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

C-5), 133.20 (phenyl C-4), 134.73 (phenyl C-3, C-5), 136.17 (benzy-

lidene C-1), 136.52 (p-chlorophenyl C-4), 138.52 (phenyl C-1),

146.83 (pyrazolopyrimidine C-7a), 147.43 (pyrazolopyrimidine C-

3), 151.20 (triazine C-3), 158.08 (pyrazolopyrimidine C-6),

159.07(pyrazolopyrimidine C¼O), 160.45 (triazine C¼O), 168.04

(C¼O); Anal. Calcd for C₂₉H₂₀ClN₇O₃(549.13): C, 63.33; H, 3.67; N,

17.83. Found: C, 63.21; H, 3.54; N, 17.81.

1H, ¼CH), 7.52 (t, J ¼ 7.6 Hz,1H, benzylidene H-4), 7.57e7.68 (m, 5H,

phenyl H-3, H-4, H-5 and benzylidene H-3 and H-5), 7.75 (t,

J ¼ 7.6 Hz,1H, pyrazolopyrimidine phenyl H-4), 7.96 (d, J ¼ 8 Hz, 2H,

benzylidene H-2, H-6), 8.03 (d, J ¼ 8 Hz, 2H, phenyl H-2, H-6), 8.15

(d, J ¼ 8 Hz, 2H, pyrazolopyrimidine phenyl H-3, H-5), 8.35 (d,

J ¼ 8 Hz, 2H, pyrazolopyrimidine phenyl H-2, H-6),11.41 (s, 1H, NH,

D₂O exchangeable), 12.45 (s, 1H, NH, D₂O exchangeable); ¹³C NMR

(100 MHz, DMSO‑d₆,

d

¼ ppm) ¼ 13.76 (CH₃), 49.07 (CH₂), 104.75

d

4.1.1.8. (Z)-6-[3-(4-Chlorophenyl)-5-(4-methoxybenzylidene)-6-oxo-

1,2,5,6-tetrahydro-1,2,4-triazine-2-carbonyl]-3-methyl-1-phenyl-1H-

pyrazolo[3,4-d]pyrimidin-4(5H)-one (6h). Yellow solid; 59% yield;

mp 335e337 ^ꢁC; IR (KBr) 3294, 3132 (2NH), 3048 (CH-aromatic),

2928 (CH-aliphatic),1722-1679 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

(pyrazolopyrimidine C-3a), 121.82 (¼CH), 125.47 (pyrazolopyr-

imidine phenyl C-2, C-6),127.08 (pyrazolopyrimidine phenyl C-4),

127.86 (benzylidene C-4), 128.16 (phenyl C-2, C-6), 128.57 (phenyl

C-3, C-5),128.66 (benzylidene C-2, C-6),128.72 (benzylidene C-3, C-

5), 128.98 (phenyl C-4), 131.82 (phenyl C-1), 132.84 (triazine C-5),

133.10 (pyrazolopyrimidine phenyl C-3, C-5), 134.59 (benzylidene

C-1), 139.72 (pyrazolopyrimidine phenyl C-1), 146.48 (pyr-

azolopyrimidine C-7a), 152.83 (pyrazolopyrimidine C-3), 155.13

(triazine C-3), 158.83 (pyrazolopyrimidine C-6), 163.89 (pyr-

azolopyrimidine C¼O), 166.44 (triazine C¼O), 167.26 (C¼O); Anal.

Calcd for C₃₀H₂₃N₇O₃(529.19): C, 68.04; H, 4.38; N, 18.52. Found: C,

68.13; H, 4.24; N, 18.56.

DMSO‑d₆,

d

¼ ppm) ¼ 2.56 (s, 3H, CH₃), 3.86 (s, 3H, OCH₃), 7.11 (d,

d

J ¼ 7.4 Hz, 2H, p-methoxybenzylidene H-3, H-5), 7.32 (s, 1H, ¼ CH),

7.53e7.60 (m, 5H, p-chlorophenyl H-2, H-6 and phenyl H-3, H-4, H-

5), 8.13 (d, J ¼ 7.4 Hz, 2H, p-methoxybenzylidene H-2,H-6), 8.22 (d,

J ¼ 8 Hz, 2H, phenyl H-2, H-6), 8.36 (d, J ¼ 8.8 Hz, 2H, p-chlor-

ophenyl H-3, H-5),12.02 (s,1H, NH, D₂O exchangeable),12.06 (s,1H,

NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.78 (CH₃), 55.95 (OCH₃), 107.17 (pyrazolopyrimidine C-3a),

115.13 (p-methoxybenzylidene C-3, C-5), 121.46 (¼CH), 121.98

(phenyl C-2, C-6), 126.67 (p-methoxybenzylidene C-1), 127.19

(phenyl C-4), 129.46 (p-chlorophenyl C-3, C-5), 129.63 (p-chlor-

ophenyl C-2, C-6), 130.44 (p-methoxybenzylidene C-2, C-6), 132.20

(p-chlorophenyl C-1), 134.03 (triazine C-5), 135.30 (phenyl C-3, C-

5), 137.38 (p-chlorophenyl C-4), 146.21 (phenyl C-1), 148.91 (pyr-

azolopyrimidine C-7a), 152.42 (pyrazolopyrimidine C-3), 156.22

(triazine C-3), 157.81 (pyrazolopyrimidine C-6), 160.58 (p-

methoxybenzylidene C-4), 162.12 (pyrazolopyrimidine C¼O),

166.28 (triazine C¼O), 168.10 (C¼O); Anal. Calcd for C₃₀H₂₂ClN₇O₄

(579.14): C, 62.13; H, 3.82; N,16.90. Found: C, 62.27; H, 3.75; N,17.11.

4.1.1.11. (E)-6-{2-[5-(4-Methoxybenzylidene)-6-oxo-3-phenyl-5,6-

dihydro-1,2,4-triazine-2(1H)-yl]-2-oxoethyl}-3-methyl-1-phenyl-1H-

pyrazolo[3,4-d]pyrimidin-4(5H)-one (6k). Yellow solid; 32% yield;

mp 277e279 ^ꢁC; IR (KBr) 3198 (broad, 2NH), 3009 (CH-aromatic),

2928 (CH-aliphatic), 1720-1680 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.52 (s, 3H, CH₃), 3.68 (s, 2H, CH₂), 3.84 (s,

3H, OCH₃), 7.08 (d, J ¼ 8.2 Hz, 2H, p-methoxybenzylidene H-3, H-5),

7.26 (s, 1H, ¼CH), 7.35e7.38 (m, 4H, phenyl H-3, H-4, H-5, pyr-

azolopyrimidine phenyl H-4), 7.48e7.55 (m, 4H, p-methox-

ybenzylidene H-2, H-6, pyrazolopyrimidine phenyl H-3, H-5), 7.94

(d, J ¼ 7.2 Hz, 2H, phenyl H-2, H-6), 8.33 (d, J ¼ 8.8 Hz, 2H, pyr-

azolopyrimidine phenyl H-2, H-6), 10.11 (s, 1H, NH, D₂O

exchangeable), 12.33 (s, 1H, NH, D₂O exchangeable);¹³C NMR

4.1.1.9. (Z)-6-[5-(4-Chlorobenzylidene)-3-(4-chlorophenyl)-6-oxo-

1,2,5,6-tetrahydro-1,2,4-triazine-2-carbonyl]-3-methyl-1-phenyl-1H-

pyrazolo[3,4-d]pyrimidin-4(5H)-one (6i). Yellow solid; 43% yield;

mp 292e294 ^ꢁC; IR (KBr) 3395, 3194 (2NH), 3021 (CH-aromatic),

2940 (CH-aliphatic),1724-1670 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

(100 MHz, DMSO‑d₆,

d

¼ ppm) ¼ 13.77 (pyrazolopyrimidine CH₃),

d

40.59 (CH₂), 55.92 (OCH₃), 104.59 (pyrazolopyrimidine C-3a),

115.08 (p-methoxybenzylidene C-3, C-5), 121.71 (¼CH), 126.93

(pyrazolopyrimidine phenyl C-2, C-6), 126.99 (p-methox-

ybenzylidene C-1), 127.05 (phenyl C-2, C-6), 127.71 (pyrazolopyr-

imidine phenyl C-4), 128.44 (phenyl C-3, C-5), 129.10 (phenyl C-4),

129.48 (p-methoxybenzylidene C-2, C-6), 132.38 (phenyl C-1),

134.28 (pyrazolopyrimidine C-3, C-5), 135.23 (triazine C-5), 138.78

(pyrazolopyrimidine phenyl C-1), 146.28 (pyrazolopyrimidine C-

7a), 152.86 (pyrazolopyrimidine C-3), 155.21 (triazine C-3), 156.34

(pyrazolopyrimidine C-6), 158.85 (p-methoxybenzylidene C-4),

165.83 (pyrazolopyrimidine C¼O), 166.42 (triazine C¼O), 168.24

(C¼O); Anal. Calcd for C₃₁H₂₅N₇O₄(559.20): C, 66.54; H, 4.50; N,

17.52. Found: C, 66.48; H, 4.61; N, 17.57.

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.50 (s, 3H, CH₃), 7.07e7.12 (m, 4H, p-

chlorobenzylidene H-3, H-5 and p-chlorophenyl H-2, H-6), 7.23 (s,

1H, ¼CH), 7.37 (t, J ¼ 7.2 Hz, 1H, phenyl H-4), 7.53 (t, J ¼ 8 Hz, 2H,

phenyl H-3, H-5), 7.84 (d, J ¼ 8.4 Hz, 2H, p-chlorobenzylidene H-2,

H-6), 8.05 (d, J ¼ 8 Hz, 2H, phenyl H-2, H-6), 8.32 (d, J ¼ 8.8 Hz, 2H,

p-chlorophenyl H-3, H-5), 11.36 (s, 1H, NH, D₂O exchangeable),

12.45 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO‑d₆,

d

¼ ppm) ¼ 13.77 (CH₃), 104.73 (pyrazolopyrimidine C-3a), 115.06

d

(phenyl C-2, C-6), 121.68(¼CH), 124.87 (p-chlorophenyl C-1), 126.91

(phenyl C-4), 127.08 (p-chlorobenzylidene C-1), 128.39 (p-chlor-

obenzylidene C-3, C-5), 128.91 (p-chlorophenyl C-3, C-5), 129.59 (p-

chlorobenzylidene C-2, C-6), 129.66 (p-chlorophenyl C-2, C-6),

134.33 (triazine C-5), 135.13 (phenyl C-3, C-5), 138.79 (p-chlor-

obenzylidene C-4), 142.70 (p-chlorophenyl C-4), 146.29 (phenyl C-

1), 152.86 (pyrazolopyrimidine C-7a), 155.20 (pyrazolopyrimidine

C-3), 158.70 (triazine C-3), 158.86 (pyrazolopyrimidine C-6), 161.94

(pyrazolopyrimidine C¼O), 166.28 (triazine C¼O), 168.32 (C¼O);

4.1.1.12. (E)-6-{2-[5-(4-Chlorobenzylidene)-6-oxo-3-phenyl-5,6-

dihydro-1,2,4-triazine-2(1H)-yl]-2-oxoethyl}-3-methyl-1-phenyl-1H-

pyrazolo[3,4-d]pyrimidin-4(5H)-one (6l). Yellow solid; 33% yield;

mp 310e312 ^ꢁC; IR (KBr) 3229, 3198 (2NH), 3009 (CH-aromatic),

2947 (CH-aliphatic), 1716-1674 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.73 (s, 3H, CH₃), 3.94 (s, 2H, CH₂), 7.32 (s,

EIMS (m/z) 583 (M^þ.

C

,

1.12%), 91 (100%). Anal.Calcd for

₂₉H₁₉Cl₂N₇O₃(583.09): C, 59.60; H, 3.28; N, 16.78. Found: C, 59.58;

H, 3.14; N, 16.88.

1H, ¼CH), 7.36e7.40 (m, 3H, phenyl H-3, H-4, H-5), 7.52e7.56 (m,

3H, pyrazolopyrimidine phenyl H-3, H-4, H-5), 7.57 (d, J ¼ 7.6 Hz,

2H, phenyl H-2, H-6), 7.97 (d, J ¼ 7.6 Hz, 2H, pyrazolopyrimidine

phenyl H-2, H-6), 8.03 (d, J ¼ 8 Hz, 2H, p-chlorophenyl H-3, H-5),

8.36 (d, J ¼ 8 Hz, 2H, p-chlorophenyl H-2, H-6), 11.41 (s, 1H, NH, D₂O

exchangeable), 12.45 (s, 1H, NH, D₂O exchangeable); ¹³C NMR

4.1.1.10. (E)-6-[2-(5-Benzylidene-6-oxo-3-phenyl-5,6-dihydro-1,2,4-

triazine-2(1H)-yl)-2-oxoethyl]-3-methyl-1-phenyl-1H-pyrazolo[3,4-

d]pyrimidin-4(5H)-one (6j). Yellow solid; 43% yield; mp

242e244 ^ꢁC; IR (KBr) 3294, 3148 (2NH), 3067 (CH-aromatic), 2994-

2924 (CH-aliphatic), 1721-1668 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

(100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.76 (CH₃), 49.06 (CH₂), 104.70

(pyrazolopyrimidine C-3a), 121.81 (¼CH), 127.07 (pyrazolopyr-

imidine phenyl C-2, C-6), 127.36 (phenyl C-1), 127.67 (pyr-

azolopyrimidine phenyl C-4), 128.66 (phenyl C-2, C-6), 129.16 (p-

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.52 (s, 3H, CH₃), 4.09 (s, 2H, CH₂), 7.36 (s,

17

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

chlorophenyl C-3, C-5), 129.50 (phenyl C-3, C-5), 129.64 (p-chlor-

ophenyl C-2, C-6), 133.83 (p-chlorophenyl C-1), 133.12 (phenyl C-4),

134.60 (pyrazolopyrimidine phenyl C-3, C-5), 135.96 (p-chlor-

ophenyl C-4), 136.66 (triazine C-5), 138.75 (pyrazolopyrimidine

phenyl C-1), 146.33 (pyrazolopyrimidine C-7a), 152.84 (pyr-

azolopyrimidine C-3), 155.15 (triazine C-3), 158.81 (pyrazolopyr-

imidine C-6), 160.75 (pyrazolopyrimidine C¼O), 166.43 (triazine

C¼O), 168.25 (C¼O); EIMS (m/z) 564 (M^þ., 15.75%), 129 (100%).

Anal.Calcd for C₃₀H₂₂ClN₇O₃(563.15): C, 63.89; H, 3.93; N, 17.38.

Found: C, 63.78; H, 4.01; N, 17.27.

mp 308e310 ^ꢁC; IR (KBr) 3368, 3194 (2NH), 3021 (CH-aromatic),

2943 (CH-aliphatic),1724-1689 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.26 (s, 3H, CH₃), 2.53 (s, 3H, CH₃), 3.80 (s,

2H, CH₂), 7.12 (d, J ¼ 8 Hz, 2H, p-tolyl H-3, H-5), 7.27 (s, 1H, ¼CH),

7.38 (t, J ¼ 8 Hz, 1H, phenyl H-4), 7.52 (t, J ¼ 8 Hz, 2H, phenyl H-3, H-

5), 7.58 (d, J ¼ 8.4 Hz, 2H, p-chlorophenyl H-3, H-5), 7.86 (d, J ¼ 8 Hz,

2H, p-tolyl H-2, H-6), 8.05 (d, J ¼ 8 Hz, 2H, phenyl H-2, H-6), 8.35 (d,

J ¼ 8.4 Hz, 2H, p-chlorophenyl H-2, H-6), 11.41 (s, 1H, NH, D₂O

exchangeable), 12.24 (s, 1H, NH, D₂O exchangeable); ¹³C NMR

(100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.78 (CH₃), 21.60 (CH₃), 39.99

(CH₂), 104.74 (pyrazolopyrimidine C-3a), 121.66 (¼CH), 124.54 (p-

tolyl C-1), 127.00 (phenyl C-2, C-6), 127.05 (phenyl C-4), 128.62 (p-

chlorophenyl C-3, C-5), 129.48 (p-chlorophenyl C-2, C-6), 129.62 (p-

tolyl C-3, C-5), 129.73 (p-tolyl C-2, C-6), 133.22 (p-chlorophenyl C-

1), 134.51 (phenyl C-3, C-5), 135.80 (p-chlorophenyl C-4), 136.72

(triazine C-5), 138.80 (phenyl C-1), 143.25 (p-tolyl C-4), 146.33

(pyrazolopyrimidine C-7a),152.85 (pyrazolopyrimidine C-3), 155.16

(triazine C-3), 158.80 (pyrazolopyrimidine C-6), 160.59 (pyr-

azolopyrimidine C¼O), 166.27 (triazine C¼O), 168.32 (C¼O); Anal.

Calcd for C₃₁H₂₄ClN₇O₃(577.16): C, 64.41; H, 4.19; N, 16.96. Found:

C, 64.37; H, 4.15; N, 17.11.

4.1.1.13. (E)-6-[2-(5-Benzylidene-6-oxo-3-(p-tolyl)-5,6-dihydro-

1,2,4-triazine-2(1H)-yl)-2-oxoethyl]-3-methyl-1-phenyl-1H-pyrazolo

[3,4-d]pyrimidin-4(5H)-one (6m). Buff solid; 32% yield; mp

294e296 ^ꢁC; IR (KBr) 3275, 3125 (2NH), 3009 (CH-aromatic), 2986,

2924 (CH-aliphatic),1715-1668 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.34 (s, 3H, CH₃), 2.54 (s, 3H, CH₃), 3.68 (s,

2H, CH₂), 7.15 (d, J ¼ 7.6 Hz, p-tolyl H-3, H-5), 7.24 (s, 1H, ¼CH),

7.48e7.56 (m,4H, phenyl H-4, benzylidene H-3, H-4, H-5), 7.79 (t,

J ¼ 8 Hz, 2H, phenyl H-3, H-5), 8.02 (d, J ¼ 8.2 Hz, 2H, benzylidene

H-2, H-6), 8.17 (d, J ¼ 7.6 Hz, 2H, p-tolyl H-2, H-6), 8.25 (d, J ¼ 7.4 Hz,

2H, phenyl H-2, H-6), 9.81 (s, 1H, NH, D₂O exchangeable), 12.03 (s,

1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO‑d₆,

4.1.1.16. (E)-6-[2-(5-(4-Benzylidene)-3-(4-chlorophenyl)-6-oxo-5,6-

dihydro-1,2,4-triazine-2(1H)-yl)-2-oxoethyl]-3-methyl-1-phenyl-1H-

pyrazolo[3,4-d]pyrimidin-4(5H)-one (p). Buff solid; 48% yield; mp

291e293 ^ꢁC; IR (KBr) 3294, 3148 (2NH), 3067 (CH-aromatic), 2994,

2928 (CH-aliphatic),1720-1668 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

d

¼ ppm)

d

¼ 13.95 (CH₃), 22.63 (CH₃), 39.90 (CH₂), 106.06 (pyr-

azolopyrimidine C-3a), 120.52 (¼CH), 121.28 (phenyl C-2, C-6),

121.83 (phenyl C-4), 125.43 (benzylidene C-2, C-6), 126.30 (benzy-

lidene C-3, C-5), 127.08 (benzylidene C-4), 127.97 (p-tolylC-3, C-5),

128.58 (p-tolyl C-2, C-6), 129.43 (p-tolyl C-1), 129.59 (phenyl C-3, C-

5), 134.75 (phenyl C-1),136.71 (triazine C-5),137.81 (benzylidene C-

1), 139.41 (p-tolyl C-4), 146.05 (pyrazolopyrimidine C-7a), 149.69

(pyrazolopyrimidine C-3), 154.31 (triazine C-3), 155.71 (pyr-

azolopyrimidine C-6), 161.39 (pyrazolopyrimidine C¼O), 164.67

(triazine C¼O), 168.60 (C¼O); Anal. Calcd for C₃₁H₂₅N₇O₃(543.20):

C, 68.50; H, 4.64; N, 18.04. Found: C, 68.37; H, 4.71; N, 17.98.

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.47 (s, 3H, CH₃), 3.61 (s, 2H, CH₂), 7.22 (s,

1H, ¼CH), 7.33 (t, J ¼ 7.8 Hz, 1H, benzylidene H-4), 7.39 (d, J ¼ 8 Hz,

2H, p-chlorophenyl H-2, H-6), 7.40 (t, J ¼ 7.8 Hz, 2H, benzylidene H-

3, H-5), 7.45 (t, J ¼ 7.2 Hz, 1H, phenyl H-4), 7.52 (d, J ¼ 8 Hz, 2H, p-

chlorophenyl H-3, H-5), 7.58 (t, J ¼ 7.2 Hz, 2H, phenyl H-3, H-5), 7.60

(d, J ¼ 7.2 Hz, 2H, benzylidene H-2, H-6), 8.04 (d, J ¼ 7.6 Hz, 2H,

phenyl H-2, H-6), 11.37 (s, 1H, NH, D₂O exchangeable), 12.26 (s, 1H,

NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO‑d₆,

¼ 13.40 (CH₃), 38.19 (CH₂), 105.34 (pyrazolopyrimidine C-3a),

d

¼ ppm)

4.1.1.14. (E)-6-[2-(5-(4-Methoxybenzylidene)-6-oxo-3-(p-tolyl)-5,6-

dihydro-1,2,4-triazine-2(1H)-yl)-2-oxoethyl]-3-methyl-1-phenyl-1H-

pyrazolo[3,4-d]pyrimidin-4(5H)-one (6n). Yellow solid; 57% yield;

mp 311e313 ^ꢁC; IR (KBr) 3368, 3194 (2NH), 3021 (CH-aromatic),

2943 (CH-aliphatic), 1724-1670 (3C ¼ O) cm^ꢀ1; ¹H NMR (400 MHz,

d

121.66 (¼CH), 123.90 (phenyl C-2, C-6), 126.25 (phenyl C-4), 127.93

(benzylidene C-4), 128.50 (benzylidene C-2, C-6), 128.61 (benzyli-

dene C-3, C-5), 128.92 (p-chlorophenyl C-3, C-5), 129.18 (p-chlor-

ophenyl C-2, C-6), 129.32 (phenyl C-3, C-5), 130.4 (p-chlorophenyl

C-1), 134.5 (triazine C-5), 135.20 (phenyl of triazine C-1), 135.76 (p-

chlorophenyl C-4), 139.73 (phenyl C-1), 144.43 (pyrazolopyrimidine

C-7a), 149.25 (pyrazolopyrimidine C-3), 156.26 (triazine C-3),

158.40 (pyrazolopyrimidine C-6), 160.89 (pyrazolopyrimidine

C¼O), 165.87 (triazine C¼O), 168.62 (C¼O); Anal.Calcd for

DMSO‑d₆,

d

¼ ppm)

d

¼ 2.25 (s, 3H, CH₃), 2.53 (s, 3H, CH₃), 3.68 (s,

3H, OCH₃), 3.85 (s, 2H, CH₂), 7.07e7.12 (m, 4H, p-tolyl H-3, H-5 and

p-methoxybenzylidene H-3, H-5), 7.23 (s, 1H, ¼CH), 7.38 (t,

J ¼ 7.6 Hz, 1H, phenyl H-4), 7.53 (d, J ¼ 7.6 Hz, 2H, phenyl H-3, H-5),

7.84 (d, J ¼ 8 Hz, 2H, p-methoxybenzylidene H-2, H-6), 8.05 (d,

J ¼ 8.4 Hz, 2H, phenyl H-2, H-6), 8.31 (d, J ¼ 8.8 Hz, 2H, p-tolyl H-2,

H-6), 11.35 (s, 1H, NH, D₂O exchangeable), 12.45 (s, 1H, NH, D₂O

C

₃₀H₂₂ClN₇O₃(563.15): C, 63.89; H, 3.93; N, 17.38. Found: C, 63.77;

H, 4.07; N, 17.43.

exchangeable); ¹³C NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.77

(CH₃), 21.52 (CH₃), 39.97 (CH₂), 55.93 (OCH₃), 107.33 (pyr-

azolopyrimidine C-3a), 115.10 (p-methoxybenzylidene C-3, C-5),

121.71 (¼CH), 121.94 (phenyl C-2, C-6), 124.92 (p-methox-

ybenzylidene C-1), 127.07 (p-tolyl C-1), 127.19 (p-methox-

ybenzylidene C-2, C-6), 128.64 (phenyl C-4), 129.23 (p-tolyl C-3, C-

5),129.70 (p-tolyl C-2, C-6),129.93 (phenyl C-1),134.42 (phenyl C-3,

C-5), 135.22 (triazine C-5), 138.63 (p-tolyl C-4), 138.76 (pyr-

azolopyrimidine C-7a), 142.97 (pyrazolopyrimidine C-3), 146.65

(triazine C-3), 158.16 (pyrazolopyrimidine C-6), 158.77 (p-methox-

ybenzylidene C-4), 160.09 (pyrazolopyrimidine C¼O), 162.03

(triazine C¼O), 168.20 (C¼O); EIMS (m/z) 574 (Mþ1, 7.17%), 573

(M^þ., 10.14%), 77 (100%). Anal.Calcd for C₃₂H₂₇N₇O₄(573.21): C,

67.01; H, 4.74; N, 17.09. Found: C, 67.13; H, 4.65; N, 17.11.

4.1.1.17. (E)-6-[2-(3-(4-Chlorophenyl)-5-(4-methoxybenzylidene)-6-

oxo-5,6-dihydro-1,2,4-triazine-2(1H)-yl)-2-oxoethyl]-3-methyl-1-

phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (q). Yellow solid;

52% yield; mp299-301 ^ꢁC; IR (KBr) 3368, 3190 (2NH), 3071 (CH-

aromatic), 2936 (CH-aliphatic),1724-1670 (3C ¼ O) cm^ꢀ1

;

¹H NMR

¼ 2.53 (s, 3H, CH₃), 3.81 (s, 2H,

(400 MHz, DMSO‑d₆,

d

¼ ppm)

d

CH₂), 3.85 (s, 3H, OCH₃), 7.09 (d, J ¼ 8.4 Hz, 2H, p-methox-

ybenzylidene H-3, H-5), 7.29 (s, 1H, ¼CH), 7.38e7.40 (m, 3H, phenyl

H-4 and p-chlorophenyl H-3, H-5), 7.53 (t, J ¼ 8 Hz, 2H, phenyl H-3,

H-5), 7.95 (d, J ¼ 8.4 Hz, 2H, p-methoxybenzylidene H-2, H-6), 8.04

(d, J ¼ 7.6 Hz, 2H, phenyl H-2, H-6), 8.32 (d, J ¼ 8.8 Hz, 2H, p-

chlorophenyl H-2, H-6), 11.37 (s, 1H, NH, D₂O exchangeable), 12.45

(s, 1H, NH, D₂O exchangeable);¹³C NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.77 (CH₃), 40.59 (CH₂), 55.94 (OCH₃), 104.72 (pyr-

4.1.1.15. (E)-6-[2-(5-(4-Chlorobenzylidene)-6-oxo-3-(p-tolyl)-5,6-

dihydro-1,2,4-triazine-2(1H)-yl)-2-oxoethyl]-3-methyl-1-phenyl-1H-

pyrazolo[3,4-d]pyrimidin-4(5H)-one (6o). Yellow solid; 42% yield;

azolopyrimidine C-3a), 115.12 (p-methoxybenzylidene C-3, C-5),

121.69 (¼CH), 126.48 (p-chlorophenyl C-1), 126.94 (p-methox-

ybenzylidene C-1),127.06 (phenyl C-2, C-6),129.29 (p-chlorophenyl

18

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

C-3, C-5), 129.64 (p-chlorophenyl C-2, C-6), 129.96 (phenyl C-4),

130.15 (p-methoxybenzylidene C-2, C-6), 134.10 (triazine C-5),

135.32 (phenyl C-3, C-5), 137.38 (p-chlorophenyl C-4), 138.75

(phenyl C-1), 146.34 (pyrazolopyrimidine C-7a), 152.82 (pyr-

azolopyrimidine C-3), 155.17 (triazine C-3), 157.82 (pyrazolopyr-

imidine C-6), 158.81 (p-methoxybenzylidene C-4), 162.16

(pyrazolopyrimidine C¼O), 166.46 (triazineC ¼ O), 168.11(C¼O);

EIMS (m/z) 595 (Mþ2, 0.91%), 593 (M þ ., 2.16%), 57 (100%).

Anal.Calcd for C₃₁H₂₄ClN₇O₄(593.16): C, 62.68; H, 4.07; N, 16.51.

Found: C, 62.59; H, 4.13; N, 16.55.

absorbance at a wave length 570 nm using a micro plate reader

[38].

4.2.2. Cell cycle analysis using DNA-ﬂow cytometry

Both HCT-116 and MCF-7 cells were treated with compounds 6o

and 6q, respectively, at their IC₅₀values and incubated for 24 h.

After incubation, the cells were washed with cold phosphate

buffered saline (PBS) and ﬁxed with 70% ethanol. The ﬁxed cells

were rinsed with PBS and then stained with propidium iodide (PI)

in a solution containing Triton X-100 as well as RNase, keep 15 min

at 37 ^ꢁC according the instruction manual. Then the samples were

analyzed with a FACS Caliber ﬂow cytometer (Becton Dickinson &

Co., Franklin Lakes, NJ) [38].

4.1.1.18. (E)-6-[2-(5-(4-Chlorobenzylidene)-3-(4-chlorophenyl)-6-

oxo-5,6-dihydro-1,2,4-triazine-2(1H)-yl)-2-oxoethyl]-3-methyl-1-

phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (r). Yellow solid;

55% yield; mp314-316 ^ꢁC; IR (KBr) 3213 (broad, 2NH), 3071 (CH-

4.2.3. Measurement of apoptosis using Annexin-V-FITC/PIstaining

To assess apoptosis and necrosis using Annexin V-FITC/PI

apoptosis detection kit, the cells were stained with Annexin V-

ﬂuorescein isothiocyanate (FITC) and counterstaining with propi-

dium iodide (PI). 2 ꢃ 10⁵Cells were exposed to compounds 6o and

6q at IC₅₀values (4.8 and 6.5 nM) for 24 h and 48 h. Then, the cells

were trypsinized and washed in cold phosphate-buffered saline

(PBS), stained with Annexin V-FITC and PI for 15 min at 37 ^ꢁC in the

dark. Finally, the samples were analyzed using ﬂow cytometer

(FACS Caliber, Becton Dickinson Biosciences & Co., Franklin Lakes,

NJ). For each sample, the number of analyzed cells was 10000 [38].

aromatic), 2932 (CH-aliphatic),1724-1667 (3C ¼ O) cm^ꢀ1

;

¹H NMR

¼ 2.53 (s, 3H, CH₃), 3.81 (s, 2H,

(400 MHz, DMSO‑d₆,

d

¼ ppm)

d

CH₂), 7.34 (s, 1H, ¼CH), 7.37e7.42 (m, 3H, phenyl H-4, p-chlor-

ophenyl H-2, H-6), 7.53 (t, J ¼ 8.4 Hz, 2H, phenyl H-3, H-5), 7.58 (d,

J ¼ 8.8 Hz, 2H, p-chlorobenzylidene H-3, H-5), 7.97 (d, J ¼ 8.8 Hz,

2H, p-chlorophenyl H-3, H-5), 8.05 (d, J ¼ 8.8 Hz, 2H, p-chlor-

obenzylidene H-2, H-6), 8.35 (d, J ¼ 8.4 Hz, 2H, phenyl H-2, H-6),

11.43 (s, 1H, NH, D₂O exchangeable), 12.44 (s, 1H, NH, D₂O

exchangeable);¹³C NMR (100 MHz, DMSO‑d₆,

d

¼ ppm)

d

¼ 13.77

(pyrazolopyrimidine CH₃), 40.56 (CH₂), 104.71 (pyrazolopyrimidine

C-3a), 121.69 (¼CH), 126.12 (p-chlorophenyl C-1), 127.08 (phenyl C-

2, C-6), 128.13 (phenyl C-4), 129.35 (p-chlorobenzylidene C-3, C-5),

129.52 (p-chlorophenyl C-3, C-5), 129.64 (p-chlorobenzylidene C-2,

C-6),130.39 (p-chlorophenyl C-2, C-6),133.03 (p-chlorobenzylidene

C-1), 134.65 (phenyl C-3, C-5), 136.11 (p-chlorobenzylidene C-4),

136.48 (triazine C-5), 137.87 (phenyl C-1), 138.73 (p-chlorophenyl

C-4), 146.35 (pyrazolopyrimidine C-7a), 152.81 (pyrazolopyr-

imidine C-3),155.11 (triazine C-3),158.82 (pyrazolopyrimidine C-6),

159.77 (pyrazolopyrimidine C¼O), 166.47 (triazine C¼O), 168.13

(C¼O); Anal.Calcd for C₃₀H₂₁Cl₂N₇O₃(597.11): C, 60.21; H, 3.54; N,

16.38. Found: C, 60.11; H, 3.60; N, 16.43.

4.2.4. Measurment of apoptotic biomarkers using ELISA

immunoassay technique

4.2.4.1. Measurement of human active caspase-3/7/9, Bax and Bcl2

content. To allow cells of HCT-116 and MCF-7 that obtained from

ATCC to grow at 37 ^ꢁC, RPMI 1640 containing 10% FBS was used.

Stimulated with the compounds 6^ꢁand 6q to be tested for caspase-

3, caspase-7, and caspase-9; Bax or Bcl2 and lysed with cell

extraction buffer. To dilute the lysate over the range of the assay and

measure human active caspase-3, caspase-7, caspase-9, Bax or Bcl2

content, Standard Diluent Buffer was used (BIORAD iScriptTM One-

Step Real-Time RT-PCR Kit with SYBR® Green). The procedure for

the used kit was in accordance with the manufacturer’s instructions

[38].

4.2. Biological evaluation

4.2.1. Measurment of cytotoxic activity

4.2.1.1. Materials and methods. In this research, breast adenocar-

cinoma (MCF-7), colon carcinoma (HCT-116) and human ﬁbroblast

(WI38) cell lines were obtained from American Type Culture

Collection (ATCC) and maintained at VACSERA, Giza, Egypt. Lapa-

tinib was used as a reference drug. Cells were cultured in Dulbecco’s

Modiﬁed Eagle’s medium (DMEM) (Invitrogen/Life Technologies)

supplemented with 10% FBS (Hyclone), 10 mg/ml of insulin (Sigma),

and 1% penicillin-streptomycin. Sigma or Invitrogen was used as a

source for all of the other chemicals and reagents.

4.2.4.2. mRNA isolation. RNeasy extraction kit was used to isolate

m RNA in up to 1 ꢃ 10⁷cells. RNeasyLysisBuffer (RLT) was used to

disrupt and homogenize cells. Ethanol was added to the lysate to

promote selective binding of RNA to the RNeasy membrane. The

sample was applied to the RNeasy Mini spin column until total RNA

bounds to the membrane. High quality of RNA was eluted by the

action of RNase-free water. All binds were centrifuged using a

micro-centrifuge [38].

Before the MTT assay, plate cells were maintained (cells density

4.2.4.3. Preparation of master mix and ampliﬁcation protocol.

1.2e1.8 ꢃ 10,000 cells/well) in a volume of 100

m

l complete growth

A reaction mix of total volume (50

the following recipe: 2X SYBR®Green RT-PCR Master (25

ward primer (10 M) (1.5 L), reverse primer (10 M) (1.5

Nuclease-free H₂O (11 L), RNA template (1 pge100 ng total RNA)

(10 L) and iScriptreverse transcriptase for One-Step RT-PCR (1 L).

Ampliﬁcation was performed using 7500 Fast RT-PCR Systems

(AppliedBiosystems, USA) 10 ng of cDNA using a Power Sybr Green

PCR Master MIX (Applied Biosystems).The ampliﬁcation protocol

was as follows: cDNA synthesis: 50 ^ꢁC for 10 min, iScript Reverse

transcriptase inactivation: 95 ^ꢁC for 5 min, polymerase chain re-

action cycling and detection (40 cycles): 95 ^ꢁC for 10 s, data

collection step: 60 ^ꢁC for 30 s, melt curve analysis: 95 ^ꢁC for 1 min,

55 ^ꢁC for 1 min and 55 ^ꢁC for 10 s (80 cycles, increasing each by

0.5 ^ꢁC each cycle). Dissociation curve software was used to check

the products routinely [38].

m

L) was prepared according to

L), for-

L),

medium þ 100

m

l of the tested compound per well in a 96-well

m

plate for 24 h.

m

4.2.1.2. Cytotoxic activity using MTT assay. MTT [3-(4,5-

dimrthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay

was used to measure the cytotoxic activity for the tested com-

pounds and the reference drug according to the described method

[32]. For best results, cells were plated in 96-multiwell plate in

DMEM containing 10% FBS (fetal bovine serum) for 24 h. Then, cells,

test compounds and reference drug were treated with vehicle

DMSO for 48 h. After that, the media was replaced with 200 ml

DMEM containing 0.5 mg/ml of MTT and were incubated for 2 h.

After the incubation period, dissolve the resulting formazan crys-

m

tals using 200

mL of DMSO. Spectrophotometrically measure

19

P.F. Lamie, A.M. El-Kalaawy, N.S. Abdel Latif et al.

European Journal of Medicinal Chemistry 214 (2021) 113222

4.2.5. In vitro EGFR/HER2 inhibitory activity assay

for further biochemical analysis of Malondialdehyde (MDA),

reduced glutathione (GSH), gamma-aminobutyric acid (GABA) and

glutamate in brain tissue.

A cell-free assay was performed to measure the percentage of

inhibition of EGFR T790M and HER2 kinases according to the re-

ported method. The used kit for immune-assay was cloud clone

SEA757Hu 96 Tests. The following equation: E (%) ¼ E max/(1þ [I]/

ID₅₀) was applied, where E (%) is the fraction of the enzyme activity

measured in the presence of the inhibitor, E max is the activity in

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

ID₅₀is the inhibitor concentration at which E (%) ¼ 0.5 E max, then a

dose-response curve was generated. For each experiment, mean

values of two independent replicates were calculated [38].

4.2.6.3. Biochemical analysis. MDA [41] and GSH [42] were ob-

tained from Biodiagnostic Co. (Giza, Egypt), and measured calori-

metrically in the brain tissue. GABA was available from (Bioassay

Technology Laboratory) and Glutamate (obtained from (CusaBio

co.) both were assessed in the brain tissue using ELISA kits based on

manufacturer’s protocol.

4.2.6.4. Statistical analysis. All obtained data were expressed as

4.2.6. In vivo anticonvulsant study

mean

SD. Statistical analysis was achieved by using one-way

4.2.6.1. Animals. One hundred and ten male mice weighing

(20e30 g) were received from the animal facility at Faculty of

Medicine (Kasr Al-Aini), Cairo University, Egypt. Mice were adapted

to laboratory conditions for two weeks before the start of the

experimental work, they were housed in large wire mesh cages (5

per cage) under standard laboratory conditions with temperature

(24 2 ^ꢁC), humidity (about 60e70%), 12 h of dark/light cycle and

free access to food and tap water. All animal dealings were in

respect to the National Institutes of Health (NIH) guide for the care

and use of laboratory animals (NIH Publications No. 8023, revised

1978).

ANOVA followed by Tukey post-hoc test by the aid of the com-

puter software program SPSS 22 (SPSS, Chicago, Ill, USA), where the

value of p < 0.05 was regarded as statistically signiﬁcant.

4.3. ADME study

4.3.1. In silico ADME prediction

In order to predict pharmacokinetic properties for test com-

pounds, PreADME online server (https://preadmet.bmdrc.kr/) was

used. Many characters were calculated such as human intestinal

absorption (HIA), cell permeability of CaCo-2 cell and Maden Darby

Canine Kideny (MDCK) cell, plasma protein binding (PPB), blood

brain barrier (BBB) and skin permeability (SP). The obtained results

were listed in Table 9.

4.2.6.2. Experimental design. After the accommodation period,

mice were randomly arranged into twenty two groups (ﬁve mice in

each group) as follows:

4.3.2. Predicted pharmacokinetic properties

Group 1 (Normal control group): Mice administered orally 1 ml

distilled water daily for 10 days.

Bioactivity properties such as G-protein coupling receptor

(GPCR) ligand, ion channel modulator, kinase inhibitor, nuclear

receptor ligand, protease inhibitor and enzyme inhibitor of com-

pounds 6o and 6q and lapatinib were checked using molinspiration

(https://www.molinspiration.com/). The obtained results were lis-

ted in Table 10.

Group 2 (Status Epilepticus (SE) untreated model group): in which

lithium-pilocarpine mice model of Status Epilepticus was

induced (Lithium Carbonate (LiCa)/pilocarpine) where SE was

induced according to the lithium-pilocarpine SE model [39,40].

Brieﬂy, mice were administered pilocarpine hydrochloride

powder (300 mg/kg i.p.; El-Gomhouria Co. for trading chem-

icals, Cairo, Egypt) 2 h after LiCa injection (3 mEq/kg i.p.; Nile

Pharma Co. for Pharmaceuticals and Chemical Industries, Cairo,

Egypt). In addition, mice were injected with atropine sulfate

powder (1 mg/kg i.p.; Fluka (Chemika, Switzerland). 30 min

prior to pilocarpine administration to reduce its peripheral

cholinergic side effects.

Contribution

All authors contributed equally to this work.

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Group (3) (phenytoin þ SE): Mice received oral phenytoin (Nile

Pharma Co. Cairo, Egypt) at a dose of (20 mg/kg).

Group (4) (lamotrigene þ SE): Mice were given oral lamotrigine

(GlaxoSmithKline Co. Cairo, Egypt) at a dose of (20 mg/kg).

References

Groups (5e22 group) (tested compounds þ SE): Mice were

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6h, 6i, 6j, 6k, 6L, 6m, 6n, 6o, 6p, 6q and 6r respectively at a dose of

(20 mg/kg).

All drugs and tested compounds were given to mice for 10 days

before induction of Status Epilepticus. After induction of SE in the

groups administered phenytoin, lamotrigine and tested com-

pounds, mice were continuously monitored to detect signs of

seizure activity. Then mice in all groups were anesthetized by

urethane (1 g/kg) for Electroencephalography (EEG) recording,

using (AD Instruments Power Lab v.7.3.7) followed by ofﬂine

analysis of EEG waves that illustrated and stored in a PC through the

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Pyrazolo[3,4-d]pyrimidine-based dual EGFR T790M/HER2 inhibitors: Design, synthesis, structure–activity relationship and biological activity as potential antitumor and anticonvulsant agents

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