From triazolophthalazines to triazoloquinazolines: A bioisosterism-guided approach toward the identification of novel PCAF inhibitors with potential anticancer activity

Bioorg. Med. Chem. 42 (2021) 116266

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

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

From triazolophthalazines to triazoloquinazolines: A bioisosterism-guided

approach toward the identiﬁcation of novel PCAF inhibitors with potential

anticancer activity

Mohamed H. El-Shershaby^a, Adel Ghiaty^a, Ashraf H. Bayoumi^a, Ahmed A. Al-Karmalawy^b,

,b,*

Ebtehal M. Husseiny^c, Mona S. El-Zoghbi^d, Hamada S. Abulkhair^a

^aPharmaceutical Organic Chemistry Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City 11884, Cairo, Egypt

^bPharmaceutical Chemistry Department, Faculty of Pharmacy, Horus University – Egypt, International Coastal Road, 34518 New Damietta, Egypt

^cDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr City, Cairo, Egypt

^dPharmaceutical Chemistry Department, Faculty of Pharmacy, Menouﬁa University, Gamal Abd El-Nasir Street, Shebin El-Koum, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords:

Inhibition of PCAF bromodomain has been validated as a promising strategy for the treatment of cancer. In this

study, we report the bioisosteric modiﬁcation of the ﬁrst reported potent PCAF bromodomain inhibitor, L-45 to

its triazoloquinazoline bioisosteres. Accordingly, three new series of triazoloquinazoline derivatives were

designed, synthesized, and assessed for their anticancer activity against a panel of four human cancer cells. Three

derivatives demonstrated comparable cytotoxic activity with the reference drug doxorubicin. Among them,

compound 22 showed the most potent activity with IC₅₀values of 15.07, 9.86, 5.75, and 10.79 µM against Hep-

G2, MCF-7, PC3, and HCT-116 respectively. Also, compound 24 exhibited remarkable cytotoxicity effects against

the selected cancer cell lines with IC₅₀values of 20.49, 12.56, 17.18, and 11.50 µM. Compounds 22 and 25 were

Anticancer

PCAF

Bioisosterism

Synthesis

Triazoloquinazoline

Apoptosis

the most potent PCAF inhibitors (IC₅₀, 2.88 and 3.19 μM, respectively) compared with bromosporine (IC₅₀, 2.10

μ

M). Follow up apoptosis induction and cell cycle analysis studies revealed that the bioisostere 22 could induce

apoptotic cell death and arrest the cell cycle of PC3 at the G2/M phase. The in silico molecular docking studies

were additionally performed to rationalize the PCAF inhibitory effects of new triazoloquinazoline bioisosteres.

1. Introduction

including patients with brain metastases ⁹.

Both the quinazoline ^10–12and 1,2,4-triazole ^13–17heterocycles have

Histone acetyltransferase paralogues p300/CBP-associated factor

(PCAF, EC 2.3.1.48) is one member belonging to the subfamily I of the

phylogenetic bromodomain tree ^1,2. Mis-regulation of PCAF has been

individually studied in many articles as interesting classes of anticancer

drugs. Although, the investigation of 1,2,4-triazoloquinazolines as

anticancer has reported in few articles. To the best of our knowledge, six

attempts were done for the development of triazoloquinazoline de-

rivatives with antitumor activity (Fig. 2) ^18–23. Among these, two articles

studied the anticancer activity of [1,2,4]triazolo[1,5-c]quinazolines

^18,19. The third one, reported the potent anticancer activity of [1,2,4]

triazolo[4,3-a]quinazolin-5-one derivatives 8 and 9 against a panel of

human cancer cell lines ²⁰. In 2014, the in vitro cytotoxicity of twenty

two 1,2,4-Triazolo[1,5-a]quinazolines was evaluated against medullo-

blastoma, hepatocellular carcinoma, and melanoma cell lines ²¹. The last

two articles were published in the last year ^22,23and led to the discovery

of new1,2,4-triazolo[4,3-c]quinazolines as a new class of DNA inter-

calators and potential inhibitors of both the EGFR-TK, and

shown to be linked to abnormal cell proliferation and carcinogenesis ^3–5

.

Therefore, PCAF has emerged as an interesting target for the develop-

ment of anticancer chemotherapeutics ^2,6,7

.

Quinazolines are a group of nitrogen-containing heterocyclic com-

pounds that have gained particular importance as they featured in many

8

FDA-approved anticancer agents (Fig. 1). These include geﬁtinib (1;

Iressa®), vandetanib (2; Caprelsa®), erlotinib (3; Tarceva®), lapatinib

(4; Tykreb®), afatinib (5; Gilotrif®), and dacomitinib (6; Vizimpro®).

Soon after, on April 2020, tucatinib (7; Tukysa®) was the last FDA-

approved quinazoline as an anticancer molecule. Tucatinib is used for

the treatment of adult patients with advanced metastatic breast cancer,

* Corresponding author at: Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City 11884, Cairo, Egypt.

E-mail address: hamadaorganic@azhar.edu.eg (H.S. Abulkhair).

https://doi.org/10.1016/j.bmc.2021.116266

Received 8 April 2021; Received in revised form 29 May 2021; Accepted 31 May 2021

Available online 5 June 2021

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

topoisomerase II. These latter studies reported the EGFR inhibition ac-

tivity of 13 and 14 with IC₅₀values ranging from 0.69 to 1.8 µM.

Few years ago, L-45 (15, Fig. 3) was identiﬁed as the ﬁrst potent,

highly selective, and cell-active PCAF bromodomain inhibitor belonging

to the class of [1,2,4]triazolo[3,4-a]phthalazines ⁴. Our recent couple of

research articles reported the design and PCAF bromodomain inhibitory

effects of certain triazolopthalazines as congeners of L-45 ^15,23. The

PCAF antagonistic effects of L-45 was reported to be mediated through

the formation of three key interactions with the binding site of histone

acetyltransferase (Fig. 4): i) a hydrogen bonding interaction between the

side chain NH functionality attached with C-6 of triazolophthalazine

fragment that maintains the interaction with the essential amino acid

residue Asn1436 in the target protein. Two bioisosteric modiﬁcations

were adopted in the newly designed 1,2,4-triazolo[4,3-c]quinazolines

(Fig. 5): i) The pyridazine ring of L-45 was replaced with its isosteric

pyrimidine to keep the favorable π-π stacking interaction with the amino

acid residue Tyr1442 in PCAF protein binding site; ii) The C-6 attached

1,2-ethylenediamine functionality of L-45 was replaced in two series

with other fragments with different number and types of the HBD/HBA

groups (series 1, 2). The signiﬁcance of HBD/HBA groups at C-6 was

investigated in the third series (series 3), via replacement with other

fragments which have no ability of hydrogen bond formation. All the

new compounds were evaluated for their in vitro anticancer activity

against hepatocellular carcinoma (Hep-G2), mammary gland breast

cancer (MCF-7), human prostate cancer (PC3), and colorectal carcinoma

(HCT-116). In addition, follow up in vitro PCAF inhibition assay,

apoptosis induction, and cell cycle analysis studies were conducted to

evaluate the possible underlying anticancer mechanisms of the most

active compounds. Furthermore, an in silico docking study was con-

ducted to rationalize the ability of the designed bioisosteres to bind with

the active site of histone acetyltransferase PCAF as a proposed mode of

their anticancer activity. Finally, ADMET proﬁles of the best effective

derivatives were evaluated to determine their potentials to build up as

good drug candidates.

core and the Glu1389 residue; ii) a π-π stacking interaction between the

pyridazine ring and the Tyr1442 residue; iii) an additional hydrogen

bonding interaction between the N-1 of triazole ring and the Asn1436

residue. The present study aims to use the strategy of bioisosterism for

the development of 1,2,4-triazolo[4,3-c]quinazolines congeners of L-45

as a suggested new scaffold of PCAF inhibitors. This bioisosteric trans-

formation is expected to furnish new derivatives capable to interact with

the PCAF receptor site in a similar pattern with that of the lead com-

pound, L-45.

Bioisosterism is a key concept used by medicinal chemists for the

modiﬁcation of certain lead compounds to safer and more clinically

useful agents. The crucial component for bioisosterism is that bio-

isosteres are expected to affect the same biological target without

considerable perturbation in the biological activity ²⁵. As initially

speciﬁed by Friedman, bioisosteres must include all atoms and mole-

cules which ﬁt the widest deﬁnition for isosteres and have a similar type

of biological activity ²⁶. A more recent deﬁnition of bioisosteres has been

broadened to include compounds or groups that retain nearly equal

molecular shapes, volumes, and approximately the same distribution of

electrons, which exhibit similar physicochemical properties.

2. Results and discussion

2.1. Chemistry

The synthetic approach adopted for the synthesis of the starting

triazoloquinazoline 20 is presented in Scheme 1. Brieﬂy, a solution of

potassium cyanate was added portion-wise into a solution of anthranilic

acid in glacial acetic. The produced quinazoline-2,4(1H,3H)-dione

treated with phosphorus oxychloride to get 2,4-dichloroquinazoline

(18). The addition of hydrazine hydrate dropwise to the latter at

0–5 ^◦C gives 2-chloro-4-hydrazinylquinazoline (19) ^31,32, which was

1.1. Rationale and aim of the work

Considering the biological importance of 1,2,4-triazole and quina-

further treated with phosphorus oxychloride and ﬁnally allowed to react

zoline rings as veriﬁed pharmacophores in FDA approved anticancer

33,34

with propionic anhydride

to obtain the desired starting 3-ethyl-

27–30

agents and as a continuation of our recent studies

of identifying

[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one (20).

new effective anticancer molecules, synthesis of three new sets of 1,2,4-

triazolo[4,3-c]quinazoline derivatives, as bioisosteres of L-45, was car-

ried out. The design of new compounds depended on the concept of

bioisosterism as an interesting approach for the improvement of both the

physicochemical properties and the biological activity. Meanwhile, we

considered keeping the N-1 of the triazole ring in L-45 as an essential

The structure of 20 was established based on its elemental and

spectral data. The disappearance of the NH₂biforked absorption band of

the starting hydrazinyl derivative together with the appearance of a new

ꢀ 1

–

amidic C O absorption band at 1685 cm

in the IR spectrum

conﬁrmed the construction of the tricyclic ring. Also, the ¹H NMR

Fig. 1. Structures of quinazoline-derived FDA approved anticancer agents.

2

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

spectrum revealed a singlet signal at 12.28 ppm which is D₂O

exchangeable due to the amidic NH. The ethyl group at C-3 presented

quartet and triplet signals in the aliphatic region at 2.84 and 1.33 ppm.

¹³C NMR spectrum of this compound revealed eleven carbon types,

among which the carbonyl carbon exhibited its signal as expected at δ

value of 167.83 ppm.

As represented in Scheme 2, our convergent synthesis approach of

ﬁnal target molecules 21–27 started by using a simple and straightfor-

ward strategy. The construction of this set of compounds began by

reacting compound 20 with previously prepared α-chloro-N-arylaceta-

mides ^34–37. The selected approach depended on the highly reliable and

well-established electrophilic substitution reaction, exploiting the good

commercial availability of aniline derivatives and chloroacetyl chloride

precursors. Adopting this approach, seven 2-(3-ethyl-5-oxo-[1,2,4]tri-

azolo[4,3-c]quinazolin-6(5H)-yl)-N-arylacetamide derivatives (21–27)

Fig. 3. L-45, the ﬁrst potent and highly selective PCAF inhibitory

triazolophthalazine.

were afforded in yields ranging from 68 to 86%. All the IR, ¹H NMR, ¹³

C

NMR spectra of these amide derivatives were in accordance with

assumed structures. In all compounds, the ¹H NMR spectra showed

singlet signals of CH₂fragments at the range of 4.45–4.06 ppm. The

purity of synthesized compounds was monitored by TLC and conﬁrmed

by elemental analysis.

Next, to synthesize the ester derivatives of series 2 (28–30), a

mixture of 20 and the appropriate alkyl chloroalkanoate was heated up

to reﬂux in the presence of potassium carbonate to give the desired

compound according to the reported procedure ^27,38,39. Adopting this

approach, three ester derivatives were obtained in a satisfactory yield.

All the IR, ¹H NMR, ¹³C NMR spectra of these derivatives were consistent

with expected structures. In all compounds, the ¹H NMR spectra showed

–

singlet signals of N CH C O fragments around 5.0 ppm which appear

–

2

deshielded by effects of both the nitrogen atom and the carbonyl group.

Afterward, to prepare the 6-alkyltriazoloquinazolinone derivatives

of the third series (31–35), the starting 3-ethyl-[1,2,4]triazolo[4,3-c]

quinazolin-5(6H)-one (20) was allowed to react with the appropriate

alkyl halide in the presence of potassium carbonate according to the

reported procedure ⁴⁰. Progress of all the chemical reactions were

validated by TLC methodology and ﬁnal products were puriﬁed by the

Fig. 4. The key interactions of L-45 (15) with the active site of PCAF.

Fig. 2. Structures of selected [1,2,4]triazoloquinazines with potent anticancer activity.

3

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

Fig. 5. Rational of molecular design of new L-45 bioisosteres as suggested PCAF inhibitors.

Scheme 1. Synthetic protocol of starting triazoloquinazoline.

group at such a position to bind with the Glu1389 fragment in the target

column chromatography method.

protein binding site. The electronic effect of substituents attached with

the terminal phenyl ring had a remarkable impact on the cytotoxic ac-

tivity of this class of compounds. Substituents with + M effects at C-4 of

the terminal phenyl ring displayed signiﬁcant inhibitory potencies with

IC₅₀values ranging from 5.75 to 20.49 µM. Accordingly, the N-aryla-

cetamide derivatives in series 1, compounds 22 and 24 were the most

potent analogs. In particular, compound 22 showed one-third, one-half,

and one-half the activity given by the reference anticancer agent against

Hep-G2, MCF-7, and HCT-116 respectively. The same compound pre-

sented a more potent cytotoxicity effect against the prostate cancer cells

compared with the reference drug, with an IC₅₀value of 5.75 µM. In

addition, the N-arylacetamide derivative 24 presented remarkable

cytotoxic effects against all the selected cancer cells with IC₅₀values of

20.49, 12.56, 17.18, and 11.50 µM respectively. Independently, the para

chloro derivative 25 revealed a moderate activity with IC₅₀values of

36.59, 29.50, 43.54, and 17.65 µM against Hep-G2, MCF-7, PC3, HCT-

116 respectively. Lower activities were observed for other compounds

with no + M group attached at the para position of the terminal phenyl

2.2. Evaluation of biological activity

2.2.1. Cytotoxicity assay

Four cancer cell lines were selected to evaluate the cytotoxicity ef-

fects of new compounds using the MTT colorimetric assay ⁴¹. These cells

include hepatocellular, mammary gland, colorectal, and prostate carci-

noma. The selection of such cancer cells depended on the over-

expression of PCAF in these cell types ^5,42,43. Cytotoxicity of new li-

gands was compared with that of Doxorubicin as a reference anticancer

agent. Results of the preliminary antiproliferative evaluation are shown

in Table 1. The tabulated results showed moderate to good cytotoxicity

for ﬁve of the tested compounds against selected four cancer cell lines.

Concentrations of new ligands necessary for 50% inhibition of tumor cell

proliferation were found to be as low as 5.75 µM. Compounds of series 1

with N-arylacetamide fragment at N-6 of the triazoloquinazoline ring

system were the most effective as anticancer agents. This observation

may reﬂect the importance of the presence of a hydrogen bond donor

4

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

Scheme 2. Synthetic route of new triazoloquinazoline derivatives 21–35.

(HCT-116) were found to be the most sensitive to the cytotoxic effect of

new compounds followed by the breast cancer cells (MCF-7). Hep-

atocarcinoma cells (Hep-G2) were found to be the least sensitive to the

cytotoxic effect of our target compounds as indicated by the higher IC₅₀

values presented in Table 1.

Table 1

In vitro anticancer activity of the designed new triazoloquinazolines.

Compound #

In vitro Cytotoxicity IC₅₀(µM)*

Hep-G2

MCF-7

PC3

HCT-116

Dox^**

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

4.50 ± 0.2

33.35 ± 2.6

31.51 ± 2.4

15.07 ± 1.3

47.36 ± 3.0

36.59 ± 2.9

20.49 ± 1.8

64.60 ± 3.8

64.13 ± 3.5

44.82 ± 2.8

62.79 ± 3.5

81.45 ± 4.3

>100

4.17 ± 0.2

43.89 ± 2.8

36.25 ± 2.5

9.86 ± 0.8

53.84 ± 3.0

29.50 ± 2.5

12.56 ± 1.1

41.32 ± 3.0

72.44 ± 3.7

49.72 ± 2.9

89.36 ± 4.9

78.01 ± 4.2

>100

8.87 ± 0.6

38.62 ± 2.5

29.83 ± 2.3

5.75 ± 0.25

57.83 ± 3.4

43.54 ± 3.2

17.18 ± 1.5

76.37 ± 4.1

69.29 ± 3.7

52.46 ± 3.1

79.16 ± 4.5

75.14 ± 4.4

>100

5.23 ± 0.3

2.2.2. PCAF inhibition enzyme assay

25.39 ± 2.0

20.83 ± 1.8

10.79 ± 1.0

29.12 ± 2.5

17.65 ± 1.5

11.50 ± 1.0

34.06 ± 2.8

40.27 ± 3.2

28.95 ± 2.3

53.55 ± 3.9

45.87 ± 3.4

81.77 ± 4.6

61.45 ± 4.1

44.18 ± 3.3

49.13 ± 3.6

38.20 ± 3.0

The predominant members of the p300/CBP family are appraised as

universal transcriptional coactivators that play a crucial role in cell cycle

regulation and apoptosis. Hence, dysfunction of PCAF is usually corre-

lated with cancer ⁴⁴. Herein, compounds of series 1 with N-arylaceta-

mide fragment at N-6 of the triazoloquinazoline ring system were the

most effective as anticancer agents that encouraged us to select them for

further biological studies to evaluate their inhibitory potentials on the

PCAF enzyme. Compounds of this series have been subjected to in vitro

PCAF ﬂuorometric enzyme inhibition assay in comparison with bro-

45,46

84.19 ± 4.5

59.23 ± 3.4

78.06 ± 4.1

51.40 ± 3.2

93.48 ± 5.3

76.92 ± 4.0

82.33 ± 4.5

66.57 ± 3.5

>100

mosporine as a reference drug following the reported procedure

.

74.28 ± 4.2

71.30 ± 3.9

63.29 ± 3.7

The feedbacks concluded from Table 2 presented that all compounds of

series 1 except 21 showed potent inhibitory activity against PCAF.

Speciﬁcally, compounds 22 and 23 bearing electron-donating groups at

C-4 of the terminal phenyl ring (either + M or + I respectively) exhibited

excellent inhibitory activity toward PCAF with IC₅₀equal 2.88 and 8.96

µM respectively. Furthermore, compounds 25 and 26 containing

Cancer cells treated with the test compound for 72 h and cytotoxicity expressed

as the concentration needed to inhibit 50% of tumor cell proliferation (IC₅₀).

Data here are presented as the means of three independent experiments ± SD.

*

IC₅₀(µM): 1 – 10 (very strong). 11 – 20 (strong). 21 – 50 (moderate). 51 –

100 (weak) and above 100 (non-cytotoxic).

**

Table 2

DOX: Doxorubicin.

IC₅₀of the highest potent derivatives and bromosporine (BSP) on PCAF.

Compound #

IC₅₀(µM)

SD

ring. Among this series of compounds, the N-naphthylacetamide deriv-

ative 27 presented the weakest activity as an anticancer agent with IC₅₀

values above 40.00 µM. Higher doses (≥28.95 µM) of the ester de-

rivatives in series 2 needed to Inhibit 50% of cell proliferation indicating

moderate activity of these derivatives. The N-alkyl derivatives of series 3

were the weakest ones with IC₅₀values above 51.40 µM. In regard to the

tested cancer cells, prostate cancer cells (PC3) and colorectal cancer cells

21

22

> 50

2.88

8.96

47.7

3.19

12.0

19.6

2.1

–

0.13

0.33

1.48

0.19

0.37

0.62

0.08

23

24

25

26

27

BSP

5

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

electron withdrawing groups at C-4 of phenyl ring (either –I or –M

respectively) exerted promising PCAF inhibition activity with IC₅₀equal

3.19 and 12.00 µM, respectively. This suggested that compounds of se-

ries 1 (21–27) may exhibit their anticancer activity through binding to

the active site of PCAF enzyme and disturb its action.

2.3. Molecular docking study

In this work, a molecular docking study was conducted to give

guidance of molecular binding modes of the target compounds inside the

pocket of histone acetyltransferase. Docking was conducted using

MOE2014 to determine the free energy and binding mode. The selection

of promising molecules depended on both the perfect binding mode and

the best binding free energy. With three main key interactions, the

binding mode of the co-crystallized ligand, L-45 with the active site of

histone acetyltransferase PCAF exhibited a binding energy of ꢀ 13.16

kcal/mol. These interactions include: i) a hydrogen bond between

2.2.3. Apoptosis induction study

The induction of apoptotic cell death is a method with which an

anticancer agent kills tumour cells ⁴⁷. In this work, a cytometric quan-

tiﬁcation study was performed to investigate the apoptosis and necrosis

modes of PC3 cell death induced by compound 22, the most active

compound, using PI and annexin-V following the reported procedure ⁴⁸

.

Glu1389 residues and the dimethylamino motif of L-45; ii) a π-π stacking

The methodology of annexin-V/PI staining was performed in the

selected cancer cells at a mixed molar concentration of 10 µM of com-

pound 22 for 24 h. As presented in Fig. 6, the treatment of prostate

cancer cells with 22 for 24 h produced an increase in the early apoptosis

from 0.57% in the control sample (DMSO) to 2.21%, and a remarkable

elevation in the percentage of cells in late apoptosis from 0.14% to

21.59%. These results suggested that the N-4-methoxyphenlacetamide

derivative 22 might trigger the programmed cell death, rather than the

necrotic pathway.

interaction between Tyr1442 and the pyridazine ring of the tri-

azolophthalazine motif; iii) a hydrogen bond between the triazole ring

and Asn1436 residue. An outline of free energy of bindings, H-bonding

interactions, and hydrophobic interactions of the newly designed L-45

bioisosteres and that of the re-docked internal co-crystallized ligand is

shown in Table 3.

The binding mode of compound 22, as a representative example of N-

arylacetamide derivatives bearing a substituent with + M effect at C-4 of

the terminal phenyl ring, presented a typical binding interaction pattern

with that of L-45 with the active site of the target protein. Also, the

2.2.4. Cell cycle analysis

distances of hydrogen bond with the Glu1389 and π-π interactions with

Tumour cell growth inhibitors are suggested to trigger an alteration

Tyr1442 are identical for the new triazoloquinazoline bioisostere and

the co-crystallized ligand. A hydrogen bond formed between the

carbonyl group of Glu1389 residue and the NH of terminal acetamide

49

in cell cycle distribution, preferentially the G2/M phase blockade

.

Here, the mechanistic anticancer effect of compound 22 was further

studied by investigating its effect on the cell cycle progression of the PC3

cell line (Figs. 7, and 8). The cell cycle of prostate cancer cells was

assessed by using propidium iodide staining and ﬂow cytometric anal-

ysis ⁵⁰after treatment with compound 22 for 24 h. Compared with that

in control cells, compound 22 produced a remarkable aggregation of

cells in the G2/M and Pre-G1 phases. An eighteen-folds increase in the

PC3 cells percentage in the Pre-G1 phase and about four-folds increase

in the percentage of cells in the G2/M phase could be indicative of

apoptosis. These aggregations were accompanied by a concomitant

decrease in the cell counts in the G0-G1 and S phases of the cell cycle.

The Pre-G1 and G2/M phase results indicated that compound 22

induced apoptosis and arrest the cell cycle at the G2/M phase.

moiety in 22. A favorable hydrophobic interaction in the form of π-π

stacking has also been observed between Tyr1442 of the target protein

and the pyrimidine ring of the triazoloquinazoline motif. This latter

matches the hydrophobic interaction of the pyridazine ring of L-45 and

the same amino acid residue. The third interaction has been revealed as

a hydrogen bond between the triazole ring and the Asn1436 residue.

One additional binding between 22 and PCAF receptor binding site has

been found in the form of a hydrogen bond between the Glu1389 residue

and the terminal acetamide moiety, in which the CH₂fragment played as

a side chain donor. These four desirable interactions may describe the

good anticancer activity of this derivative.

The N-arylacetamide derivative with para bromo substituent of the

terminal benzene ring (24), exhibited an afﬁnity value of ꢀ 10.62 kcal/

mol. Obeying almost the same interaction pattern of L-45 with the

binding site of the receptor protein, the triazole ring formed a hydrogen

bond with Asn1436 residue. The pyrimidine ring interacted with

2.2.5. Selectivity index (SI)

One criterion for the anticancer drug to be good is that it should not

affect the non-cancer cells. A molecule could consider as highly selective

if it presented SI value ≥ 5. A molecule with moderate selectivity pre-

sents an SI value > 2 while low selectivity is considered if the SI is lower

than 2 ⁵¹. In this study, the selectivity index was calculated for the best

effective cytotoxic derivative (22) by assessing its cytotoxicity against

the non-cancer cells of the human microvascular endothelial cells

(HMEC-1) and dividing the obtained IC₅₀value by the IC₅₀value of the

same compound against PC3 cell line. Compound 22 presented an IC₅₀

value of 46.30 µM against HMEC-1, which indicates the good selectivity

of the new L-45 bioisostere toward the cancer cells (SI = 8.05).

Tyr1442 via π-π stacking while the NH of acetamide moiety at position 6

interacted via hydrogen bond with Glu1389 (Fig. 8 & Table 4). Simi-

larly, compound 25 with para chloro substituent of the terminal phenyl

ring revealed a similar virtual binding with the receptor target site. It

exhibited an afﬁnity value of ꢀ 11.04 kcal/mol and interacted with the

active site of histone acetyltransferase PCAF as following: i) a hydrogen

bond is formed between the carbonyl oxygen of Glu1389 and the NH of

the acetamide moiety; ii) a π-π stacking interaction between the Tyr1442

residue and the pyrimidine ring; iii) a hydrogen bond between the tri-

azole ring of 25 and the Asn1436 residue. Following the same pattern of

22, one more hydrogen bond is formed between the Glu1389 residue

and the terminal acetamide moiety, in which the CH₂fragment played as

a side chain donor.

With much lesser binding free energies (-10.05 and ꢀ 10.98), the

other N-arylacetamide derivatives without group possessing + M effect

at C-4 of the terminal phenyl ring (21, and 23), showed binding modes

which are also almost like that of L-45. The N-1 of the triazole rings in

each compound formed a hydrogen bond with the Asn1436 residue. The

pyrimidine ring formed a hydrophobic π-π stacking with Tyr1442 and

the NH of the acetamide moiety interacted with Glu1389 by a hydrogen

bond. Independently, compound 27, a derivative with a bulky aryl ring

attached with the acetamide fragment, exhibited a different virtual

binding mode from that of L-45. Compound 27 revealed an afﬁnity value

of ꢀ 9.07 kcal/mol and showed only two interactions with the binding

Fig. 6. Apoptotic cell death of PC3 induced by DMSO (left side) and compound

22 (right side).

6

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

Fig. 7. Cell cycle phase distribution in PC3 cells treated with DMSO (left side) and compound 22 (right side).

Fig. 8. Cell cycle phase distribution of PC3 cells treated with DMSO (dark bars) and compounds 22 (light bars).

site of histone acetyltransferase PCAF. These interactions involve one

arene-H interaction between the pyrimidine ring of 27 with Tyr1442

and one hydrogen bond between the NH of acetamide moiety at position

6 and the Glu1389 amino acid residue. No interaction formed with the

essential amino acid Asn1436, which may reﬂect the lower activity of

such compound.

(molecular weight and HB acceptors). All the best effective three anti-

cancer derivatives have HB acceptor groups between 6 & 7 and only 1

HB donor, which is agreed with Lipinski’s rules.

Calculation of ADMET proﬁles was conducted using the protocol of

pkCSM descriptors algorithm ⁵³. From the obtained data, it could be

concluded that these derivatives could show lower solubility in water

(ꢀ 4.163 to ꢀ 3.99) compared with ꢀ 2.91 and ꢀ 3.03 in the case of

doxorubicin and bromosporine, respectively. On the other hand, the

intestinal absorptivity of newly designed triazoloquinazoline bio-

isosteres of L-45 in humans is expected to be relatively better than that of

the reference compounds. Evaluating the volume of distribution and the

CNS permeability, the new triazoloquinazoline bioisosteres are sug-

gested to have intermediate VDs values between that of doxorubicin and

bromosporine. In regard to the in silico predicted metabolism, it is

noticeable that the new L-45 bioisosteres and bromosporine could

inhibit the CYP3A4, the key enzyme in the metabolism of doxorubicin

could not. Excretion was evaluated based on the total clearance. The

obtained data revealed that 22 revealed a comparable predicted

maximum total clearance value with that of doxorubicin (0.634

compared with 0.987 respectively). Dissimilar to these two compounds,

24, 25, and bromosporine are expected to have lower total clearance

values (-0.022–0.179). Thus, these latter could be excreted slower, and

accordingly, they have the preference of possible longer dosing in-

tervals. The last parameter analyzed in the pharmacokinetic proﬁles of

our newly synthesized derivatives is their predicted toxicity. All the new

triazoloquinazoline isosteres of L-45 shared the advantage of no ex-

pected AMES toxicity which indicates the preferential of probable non-

mutagenic potentials of these compounds ⁵⁴. Additionally, they also

suggested to not inhibit the hERG I activity, which indicates the safety of

Collectively, the obtained results of molecular docking studies

showed that all the designed acetamide bioisosteres of L-45 have almost

similar positions and orientations inside the binding site of histone

acetyltransferase PCAF (Fig. 9 & Table 4). As well, the distribution of

binding free energies calculated from MOE software showed preferen-

tially that the two most active derivatives; 22 and 24 are highly corre-

lated with the biological activity, Moreover, the presence of electron-

donating group with + M effect attached at the C-4 of terminal ben-

zene ring excreted a good impact on binding afﬁnity and consequently

the anticancer activity. Compounds in series 2 and 3 revealed lower free

energies of binding (from ꢀ 6.06 to ꢀ 8.55 kcal/mol) and all lack the

hydrogen bond with the essential amino acid residue, Glu1389. This

missing interaction may explain the lower activities of compounds in

both series (Table 5).

2.4. In silico pharmacokinetic study

The in silico investigational study of the three most effective cyto-

toxic agents (22, 24, and 25) was conducted (see supporting informa-

tion), to evaluate their physicochemical properties based on directions

of Lipinski’s rule ⁵². In this study, while the newly designed tri-

azoloquinazoline bioisosteres of L-45 did not violate any of Lipinski’s

rules, the reference anticancer drug doxorubicin violated two rules

7

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

ligands were suggested to have higher tolerability than that of doxoru-

bicin, which means the advantage of wide therapeutic indices of the new

derivative. Honorably, the newly designed bioisosteres are expected to

show lower Minnow toxicity values than that of both reference com-

pounds (-0.59, ꢀ 0.35, and 0.21 compared with 4.412 and 0.952, for

doxorubicin and bromosporine, respectively). These lower values of

Minnow toxicities indicate lower hazards and risk of new compounds in

the aquatic environment ⁵⁶. Finally, oral acute toxic doses of the new

compounds (LD₅₀), are expected to be comparable or even slightly

higher than that of the reference anticancer drugs (~2.75 for our new

ligands compared with 2.40 and 2.1 of doxorubicin and bromosporine,

respectively).

Table 3

Results of in silico docking for new compounds and the co-crystallized ligand (L-

45) with the binding site of histone acetyltransferase PCAF.

Comp.

ΔG (kcal/

RMSD

H-bonding interactions

Hydrophobic

interactions

mol)

(Å)

Residue

Distance

(Å)

Residue

Distance

(Å)

L-45

ꢀ 11.52

ꢀ 10.05

0.81

Glu1389

Asn1436

3.47

2.82

3.40

TYR

3.63

1442

21

22

0.93

1.18

Glu1389

Asn1436

Tyr1442

3.47

2.85

2.93

2.5. Structure-activity relationship study

As mentioned above, studying the SAR of new triazoloquinazoline

bioisosteres of L-45 as anticancer agents is a major objective of the

present study. Comparing the cytotoxic activity of compounds 21–27

incorporating an N-arylacetamide moiety at N-6 (Series 1) with that of

compounds 28–30 incorporating ester group at the same position (Series

2), indicated that there is a marked decrease in the activity upon

replacing HBD/HBA fragments with a hydrogen bond acceptor frag-

ment. This observation may reﬂect the importance of the presence of a

hydrogen bond donor group at such a position to bind with the Glu1389

fragment in the target protein binding site. A dramatic fall in the activity

has also been observed upon attachment of an alkyl group instead of the

last two fragments (Fig. 10). The electronic effect of substituents

attached with the terminal phenyl ring of N-arylacetamide moiety had a

remarkable impact on the cytotoxicity. Substituents with + M effects at

the para position displayed a remarkable positive impact on the anti-

cancer activity. Accordingly, the N-arylacetamide derivatives in series 1,

compounds 22 and 24 were the most potent analogs. Among the N-

arylacetamide derivatives, the bulky N-naphthylacetamide derivative

revealed the least activity as an anticancer agent. The relative lower

cytotoxic inhibitory effects of such compounds may be due to the

inability of such compounds to accommodate the binding site of the

target receptor.

TYR

3.64

1442

ꢀ 11.31

Glu1389

Asn1436

3.47

2.82

3.40

TYR

3.63

3.77

1442

23

24

ꢀ 10.98

ꢀ 10.62

1.15

1.16

Asn1436

3.16

TYR

1442

Glu1389

Asn1436

3.67

3.13

TYR

3.79

1442

25

ꢀ 11.04

0.93

Glu1389

Asn1436

3.47

2.82

3.40

TYR

3.63

3.65

1442

27

28

ꢀ 9.07

ꢀ 8.55

1.20

1.07

Glu1389

2.84

TYR

1442

Glu1389

Asn1436

3.40

3.34

VAL

3.71

3.65

3. Conclusion

1385

TYR

In summary, we are here reporting the bioisosterism-guided

approach for the design of three novel series of triazoloquinazolines

derivatives with the objective of developing new analogous structures of

the ﬁrst reported PCAF inhibitor L-45. The target compounds were

designed based on keeping essential structural fragments that are

essential for binding with the PCAF receptor binding site with two

bioisosteric modiﬁcation of the lead compound: pyridazine ring of L-45

1442

29

30

31

32

35

ꢀ 8.43

ꢀ 6.39

ꢀ 5.25

ꢀ 6.06

ꢀ 6.50

1.20

1.12

0.93

1.10

0.61

Asn1436

3.18

2.99

2.86

2.99

TYR

3.65

3.66

3.89

3.77

1442

TYR

1442

was replaced with its isosteric pyrimidine to keep the favorable π-π

stacking interaction with the Tyr1442 residue; different substitution

patterns were introduced to the position 6 of triazoloquinazoline scaf-

folds to investigate the effect on both the cytotoxic activity and binding

afﬁnities with the PCAF target protein. A follow up enzymatic assay was

conducted on the best effective compounds to determine their potentials

as PCAF inhibitors. Results of the PCAF enzyme assay revealed the

powerful inhibitory effects of 22 and 25. Additionally, compound 22

was selected to investigate its ability to induce apoptotic cell death and

to arrest the cell cycle of PC3 cancer cells. The most active candidates in

the quest for effective cytotoxic agents will serve as useful leads and

merit further investigations.

TYR

1442

TYR

1442

TYR

4.00

3.81

1442

TYR

1442

these new agents on the human’ heart electrical activity ⁵⁵. unfortu-

4. Experimental section

nately, they are expected to share the disadvantage of both reference

`

drugs as they could inhibit the human ether-a-go-go-related gene, hERG

4.1. General

II, which indicates the possibility of cardiac arrhythmia. As well, all the

new ligands and reference drugs shared the obstacle of expected hepa-

totoxicity. Regarding the maximum tolerated dose in humans, the new

Melting points were measured using electrothermal (Stuart SMP30)

apparatus and were uncorrected. Infrared spectra were recorded on Pye

8

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

Table 4

The 3D binding interactions and the 3D positioning of co-crystallized ligand L-45, 22, 24, and 25 with the active site of histone acetyltransferase.

Comp.

3D Interaction

3D Protein positioning

L-45

22

24

25

Unicam SP 1000 IR spectrophotometer at the Pharmaceutical Analytical

Unit, Faculty of Pharmacy, Al-Azhar University. ¹H NMR and ¹³C NMR

spectra were recorded in DMSO‑d₆at 300 and 100 MHz respectively on a

Varian Mercury VXR-300 NMR spectrometer at NMR Lab, Faculty of

Science, Cairo University. TMS was used as an internal standard,

chemical shift and coupling constant values are listed in ppm and Hz,

respectively. Mass spectra and elemental analyses were carried out at the

Regional Center of Mycology and Biotechnology, Al-Azhar University,

Cairo, Egypt. Reaction progress were monitored with Merck silica gel

IB2-F plates (0.25 mm thickness) and were visualized under a UV lamp

9

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

Fig. 9. 2D interactions of the co-crystallized ligand L-45 (upper left panel), 22 (upper right panel), 24 (lower left panel), and 25 (lower right panel) with the active

site of histone acetyltransferase (PDB ID 5tpx).

Table 5

ADMET proﬁle of the three most active derivatives, the reference anticancer, and PCAF inhibitor.

Parameter

22

24

25

Doxorubicin

Bromosporine

Molecular Properties

Molecular Weight

LogP

377.404

426.274

381.823

543.525

404.452

2.2539

3.0078

2.8987

0.0013

2.34804

Rotatable Bonds

HB Acceptors

HB Donors

5

4

5

7

6

12

8

1

6

2

Surface Area

159.937

162.326

158.762

222.081

161.528

Absorption

Water solubility

Intestinal abs. (human)

ꢀ 3.99

ꢀ 4.163

ꢀ 4.106

ꢀ 2.915

ꢀ 3.035

97.919

95.478

95.545

62.372

82.254

Distribution

VDss (human)

BBB permeability

CNS permeability

ꢀ 0.155

ꢀ 0.615

ꢀ 2.536

ꢀ 0.221

ꢀ 0.586

ꢀ 2.224

ꢀ 0.24

1.647

ꢀ 0.361

ꢀ 1.473

ꢀ 3.277

ꢀ 0.577

ꢀ 2.247

ꢀ 1.379

ꢀ 4.307

Metabolism

CYP3A4 substrate

CYP3A4 inhibitor

Yes

No

Yes

No

Yes

No

Excretion

Total Clearance

0.634

ꢀ 0.179

ꢀ 0.158

0.987

0.022

Toxicity

AMES toxicity

No

Max. tolerated dose

hERG I inhibitor

hERG II inhibitor

Oral Rat Acute Toxicity (LD₅₀

0.156

No

0.115

No

0.113

No

0.081

No

0.606

No

Yes

)

2.738

1.084

Yes

2.751

1.713

Yes

2.75

1.723

Yes

2.408

3.339

Yes

2.148

1.783

Yes

Oral Rat Chronic Toxicity (LOAEL)

Hepatotoxicity

Minnow toxicity

ꢀ 0.592

ꢀ 0.358

ꢀ 0.212

4.412

0.952

using different solvent systems as mobile phases. Reagents and starting

anthranilic acid, phosphorus oxychloride, hydrazine hydrate, propionic

and aniline derivatives were purchased from Aldrich chemical company

and were used as received. Compounds 19 and 20 were synthesized ac-

anhydride, alkyl halides, alkyl-

α

-chloroacetate, chloroacetyl chloride,

cording to the directions of previously reported procedures ³¹

.

10

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

4.3.1. 2-(3-Ethyl-5-oxo-[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)-N-

phenylacetamide (21)

Yellowish white solid, Yield: 86%; m.p.

255–257 ^◦C. IR (KBr) cm^{ꢀ 1}: 3332 (NH), 3062 (CH aromatic), 2974 (CH

1

–

aliphatic), 1693 (C O), 1620 (C C aromatic). H NMR (DMSO‑d ) δ

–

ppm: 10.43 (S, 1H, NH, D₂O exchangeable), 8.29 (d, 1H, J = 8.0 ⁶Hz,

quinazoline-H10), 7.79 (dd, 1H, J = 7.6 Hz, quinazoline-H8), 7.60

(d,1H, J = 8.4 Hz, quinazoline-H7), 7.56 (d, 2H, J = 7.6 Hz, phenyl-H2,

H6), 7.49 (dd, 1H, J = 8.4 Hz, quinazoline-H9), 7.33 (t, 2H, phenyl-H3,

H5), 7.10 (t, 1H, phenyl-H4), 5.19 (S, 2H, N-CH₂), 2.90 (q, 2H, J = 8.0,

CH₂CH₃), 1.36 (t, 3H, J = 8.0, CH₂-CH₃). MS (m/z): 347 (C₁₉H₁₇N₅O_2,

Fig. 10. Summary of SAR study of the synthesized triazoloquinazolines

bioisosteres.

4.2. Synthesis of (3-ethyl -[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one

(20)

100%, M^+.), 255 (C₁₃H₁₁N₄O_2,52.04%), 227 (C₁₂H₁₁N₄O, 43.34%).

12

–

C NMR (DMSO‑d ) δ ppm: 168.54 (C O), 165.50 (quinazoline-C4),

6

–

152.43 (triazole-C3), 145.10 (quinazoline-C O), 138.85 (phenyl-C1),

138.19 (quinqzoline-C8a), 133.66 (quinazoline-C7), 129.34 (phenyl-C3,

C5), 125.14 (phenyl-C4), 124.58 (quinqzoline-C5), 124.25 (quinqzo-

line-C6), 119.87 (phenyl-C2, C4), 116.11 (quinqzoline-C8), 111.22

(quinazoline-C4a), 47.32 (CH₂CO), 22.01 (CH₂), and 12.62 (CH₃). Anal.

Calc. for: (C₁₉H₁₇N₅O₂) (M.W. = 347): C, 65.69; H, 4.93; N, 20.16%;

Found: C, 65.83; H, 5.06; N, 20.40%.

A solution of 2-chloro-4-hydrazinylquina-

zoline 19 (2.25 g, 0.012 mol) in propionic anhydride (15 ml) was

heated under reﬂux for 3 h. After the reaction goes to completion

(monitored by TLC), the reaction mixture was concentrated under

reduced pressure. The obtained solid product was washed with three

portions of n-hexane, 30 ml each, and dried to give the desired product

4.3.2. 2-(3-Ethyl-5-oxo-[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)-N-(4-

methoxyphenyl)acetamide (22)

as a yellowish white solid, Yield: 96%, m.p. 263–265 ^◦C. IR (KBr) cm^{ꢀ 1}

:

–

3425 (NH), 3043 (CH aromatic), 2970 (CH aliphatic), 1755 (C O),

–

1

–

1600 (C C aromatic). H NMR (DMSO‑d ) δ ppm: 12.28 (S, 1H, NH,

6

D₂O exchangeable), 8.11–8.13 (d, J = 8.4 Hz, 1H, quinazoline-H10),

7.66–7.68 (dd, J = 7.6 Hz, 1H, quinazoline-H8), 7.39–7.41 (d, J = 8.4

Hz, 1H, quinazoline-H7), 7.35–7.37 (dd, J = 8.4 Hz, 1H, quinazoline-

Brown solid, Yield: 75%; m.p.

H9), 2.84 (q, J = 8.4 Hz, 2H, CH₂CH₃), 1.33 (t, J = 8.4 Hz, 3H,

13

–

CH₂CH₃). C NMR (DMSO‑d ) δ ppm: 167.83 (C O), 153.16 (quina-

–

6

zoline-C4), 144.26 (triazole-C3), 137.39 (quinqzoline-C8a), 133.07

(quinazoline-C7), 124.48 (quinqzoline-C5), 123.99 (quinqzoline-C6),

116.50 (quinqzoline-C8), 110.65 (quinazoline-C4a), 22.04 (CH₂), and

12.57 (CH₃). MS (m/z): 214 (C₁₁H₁₀N₄O,73.34%), 213 (C₁₁H₉N₄O,

100%, M^+.), 199 (C₁₀H₇N₄O, 3.46%),185 (C₉H₅N₄O, 2.04%). Anal.

Calc. for: (C₁₁H₁₀N₄O) (M.W. = 214): C, 61.67; H, 4.71; N, 26.15%;

Found: C, 61.89; H, 4.85; N, 25.98%.

262–264 ^◦C. IR (KBr) cm^{ꢀ 1}:3194 (NH), 3062 (CH aromatic), 2970 (CH

1

–

aliphatic), 1674 (C O), 1589 (C C aromatic). H NMR (DMSO‑d ) δ

–

ppm: 10.05 (S, 1H, NH, D₂O exchangeable), 8.67 (d, 1H, J = 8.4 ⁶Hz,

quinazoline-H10), 8.04 (dd, 2H, J = 8.0 Hz, phenyl-H2, H6), 7.52 (dd,

1H, J = 7.6 Hz, quinazoline-H8), 6.88 (dd, 2H, J = 8.0 Hz, phenyl-H3,

H5), 6.68 (d,1H, J = 8.4 Hz, quinazoline-H7), 6.59 (dd, 1H, J = 8.0

Hz, quinazoline-H9), 4.06 (S, 2H, N-CH₂₎, 3.72 (S, 3H, OCH₃), 2.79 (q,

2H, J = 8.0, CH₂CH₃), 1.31 (t, 3H, J = 8.0, CH₂CH₃). ¹³C NMR

4.3. General procedure for synthesis of 2-(3-ethyl-5-oxo-[1,2,4]triazolo

[4,3-c]quinazolin-6(5H)-yl)-N-arylacetamide derivatives (21–27)

Into a stirred solution of 3-ethyl-[1,2,4]triazolo[4,3-c]quinazolin-5

(6H)-one (20) (2.14 g, 0.01 mol) in dry DMF (20 ml), few crystals of

anhydrous K₂CO₃were added followed by the appropriate 2-chloro-N-

arylacetamide derivative (0.01 mol). The reaction mixture was heated to

reﬂux temperature on a water bath for 6 h. After cooling to room tem-

perature, the reaction mixture was poured onto ice-cooled water (100

ml) and stirred for one more hour. The formed precipitate was ﬁltered

off under vacuum and washed with a copious amount of water until the

ﬁltrate became neutral to litmus paper, washed with three repetitive

portions of ice-cold water (20 ml), and then ﬁnally recrystallized from

ethanol to afford the corresponding N-arylacetamide derivative.

–

(DMSO‑d ) δ ppm: 168.33 (C O), 161.47 (quinazoline-C4), 157.65

–

6

–

(triazole-C3), 155.69 (quinazoline-C O), 146.04 (phenyl-C1), 132.47

(quinazoline-C8a), 130.21 (quinazoline-C7), 128.63 (phenyl-C3, C5 and

quinazoline-C4a), 121.17 (phenyl-C4), 114.85 (quinazoline-C5), 114.48

(quinazoline-C6), 111.09 (phenyl-C2, C6), 111.09 (quinazoline-C8),

47.37 (CH₂CO), 21.69 (CH₃O), 19.75 (CH₂) and 11.84 (CH₃).MS (m/z):

377 (C₂₀H₁₉N₅O₃, 4.56%, M^+.), 348 (C₁₈H₁₄N₅O_3,1.94%). Anal. Calc.

for: (C

H N₅O₃) (M.W. = 377): C, 63.65; H, 5.07; N, 18.56%; Found:

20 19

C, 63.49; H, 5.23; N, 18.72%.

11

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

4.3.3. 2-(3-Ethyl-5-oxo-[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)-N-(p-

tolyl)acetamide (23)

4.3.5. N-(4-Chlorophenyl)-2-(3-ethyl-5-oxo-[1,2,4]triazolo[4,3-c]

quinazolin-6(5H)-yl)acetamide (25)

White solid, Yield: 73%; m.p.

Faint greenish white solid, Yield:

258–260 ^◦C. IR (KBr) cm^{ꢀ 1}: 3314 (NH), 3040 (CH aromatic), 2981 (CH

78%; m.p. 253–255 ^◦C. IR (KBr) cm^{ꢀ 1}: 3294 (NH), 3094 (CH aromatic),

1

–

aliphatic), 1711 (C O), 1518 (C C aromatic). H NMR (DMSO‑d ) δ

–

2925 (CH aliphatic), 1650 (C O), 1591 (C C aromatic). ¹H NMR

ppm: 10.08 (S, 1H, NH, D₂O exchangeable), 8.09 (d, 1H, J = 8 ⁶Hz,

quinazoline-H10), 7.95 (dd, 1H, J = 7.6 Hz, quinazoline- H8), 7.54 (dd,

2H, J = 8.0 Hz, phenyl-H2, H6), 7.48 (dd, 2H, J = 8.0 Hz, phenyl-H3,

H5), 6.67 (d,1H, J = 8.4 Hz, quinazoline- H7), 6.58 (dd, 1H, J = 8.4

Hz, quinazoline- H9), 4.45 (S, 2H, N-CH₂), 2.84 (q, 2H, J = 8.0,

CH₂CH₃), 2.62 (S, 1H, CH₃), 1.34 (t, 3H, J = 8.0, CH₂CH₃). ¹³C NMR

–

(DMSO‑d₆) δ ppm: 10.08 (S, 1H, NH, D₂O exchangeable), 8.00 (d, 1H, J

= 8.0 Hz, quinazoline-H10), 7.56 (dd, 2H, J = 7.6 Hz, phenyl-H2, H6)

7.42 (dd, 2H, J = 8.0 Hz, phenyl-H3, H5), 7.25 (dd, 1H, J = 7.6 Hz,

quinazoline-H8), 7.15 (d,1H, J = 8.4 Hz, quinazoline-H7), 6.90 (dd, 1H,

J = 8.4 Hz, quinazoline-H9), 4.02 (S, 2H, N-CH₂), 2.81 (q, J = 8.4 Hz,

2H, CH₂CH₃), 1.27 (t, J = 8.4 Hz, 3H, CH₂CH₃). ¹³C NMR (DMSO‑d₆) δ

–

(DMSO‑d ) δ ppm: 168.58 (C O), 158.29 (quinazoline-C4), 155.44

–

6

–

ppm: 170.99 (C O), 161.45(quinazoline-C4), 159.01 (triazole-C3),

–

(triazole-C3), 146.14 (C O), 137.62 (phenyl-C4), 136.84 (phenyl-C1),

–

157.56 (C O), 145.98 (phenyl-C1), 138.72 (quinazoline-C8a), 129.18

134.31 (quinazoline-C8a), 129.74 (phenyl-C3, C5), 129.67 (quinazo-

line-C7), 128.44 (quinazoline-C6), 126.71 (quinazoline-C5), 119.73

(phenyl-C2, C6), 119.67 (quinazoline-C5), 115.91 (quinazoline-C4a),

113.90 (quinazoline-C8), 53.07 (N-CH₂), 21.13 (p-CH₃), 20.93 (CH₂)

and 11.83 (CH₃). MS (m/z): 361 (C₂₀H₁₉N₅O₂, 1041%, M^+.), 348

(C₁₉H₁₈N₅O_2,15.84%), 319 (C₁₇H₁₃N₅O_2,5.12%). Anal. Calc. for:

(C₂₀H₁₉N₅O₂) (M.W. = 361): C, 66.47; H, 5.30; N, 19.38%; Found: C,

66.31; H, 5.48; N, 19.60%.

(phenyl-C3, C5), 126.88 (quinazoline-C7), 122.56 (quinazoline-C6),

121.18 (quinazoline-C5), 120.88 (phenyl-C4), 117.07 (phenyl-C2, C6),

115.38 (quinazoline-C8), 114.56 (quinazoline-C4a), 57.54 (N-CH2),

19.75 (CH2), and 12.58 (CH3). MS (m/z): 383 (C₁₉H₁₆ClN₅O_2,1.78%,

M + 2), 381 (C₁₉H₁₆Cl N₅O_2,13.51%, M^+.), 352 (C₁₇H₁₁ClN₅O_2,

18.03%). Anal. Calc. for: (C₁₉H₁₆ClN₅O₂) (M.W. = 381): C, 59.77; H,

4.22; N, 18.34%; Found: C, 59.96; H, 4.37; N, 18.51.

4.3.6. 2-(3-Ethyl-5-oxo-[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)-N-(4-

nitrophenyl)acetamide (26)

4.3.4. N-(4-Bromophenyl)-2-(3-ethyl-5-oxo-[1,2,4]triazolo[4,3-c]

quinazolin-6(5H)-yl)acetamide (24)

Yellowish white solid, Yield: 76%; m.

Faint brown solid, Yield: 68%; m.p.

p. 266–268 C. IR (KBr) cm^{ꢀ 1}: 3200 (NH), 3094 (CH aromatic), 2976

◦

263–265 ^◦C. IR (KBr) cm^{ꢀ 1}: 3318 (NH), 3094 (CH aromatic), 2926 (CH

1

–

(CH aliphatic), 1661 (C O), 1589 (C C aromatic). H NMR (DMSO‑d )

δ ppm: 10.32 (S, 1H, NH, D₂O exchangeable), 8.04 (d, 1H, J = 8 H⁶z,

quinazoline-H10), 7.59 (dd, 2H, J = 8.0 Hz, phenyl-H2, H6) 7.50 (dd,

2H, J = 7.6 Hz, phenyl-H3, H5), 7.26 (dd, 1H, J = 7.6 Hz, quinazoline-

H8), 6.70 (d,1H, J = 8.4 Hz, quinazoline-H7), 6.50 (dd, 1H, J = 8.4 Hz,

quinazoline- H9), 4.11 (S, 2H, N-CH₂), 2.82 (q, J = 8.4 Hz, 2H, CH₂CH₃),

1.33 (t, J = 8.4 Hz, 3H, CH₂CH₃). ¹³C NMR (DMSO‑d₆) δ ppm: 169.13

1

–

aliphatic), 1657 (C O), 1583 (C C aromatic). H NMR (DMSO‑d ) δ

–

ppm: 10.86 (S, 1H, NH, D₂O exchangeable), 8.28 (d, 1H, J = 8 ⁶Hz,

quinazoline-H10), 8.04 (dd, 2H, J = 8.0 Hz, phenyl-H2, H6) 7.88 (dd,

2H, J = 8.0 Hz, phenyl-H3, H5), 7.24 (dd, 1H, J = 7.6 Hz, quinazoline-

H8), 6.76 (d,1H, J = 8.4 Hz, quinazoline-H7), 6.55 (dd, 1H, J = 8.4 Hz,

quinazoline-H9), 4.17 (S, 2H, N-CH₂), 2.85 (q, J = 8.4 Hz, 2H, CH₂CH₃),

1.33 (t, J = 8.4 Hz, 3H, CH₂CH₃). ¹³C NMR (DMSO‑d₆) δ ppm: 159.81

–

(C O), 161.46 (quinazoline-C4), 159.01 (triazole-C3), 157.65 (C O),

–

(C O), 157.35 (quinazoline-C4), 154.12 (triazole-C3), 146.67 (C O),

–

145.98 (phenyl-C1), 138.73 (quinazoline-C8a), 137.51 (phenyl-C3, C5),

132.08 (quinazoline-C7), 128.62 (quinazoline-C6), 122.91 (quinazo-

line-C5), 121.55 (phenyl-C4), 117.09 (phenyl-C2, C6), 115.31 (quina-

zoline-C8), 114.53 (quinazoline-C4a), 47.47 (N-CH₂), 19.76 (CH₂) and

12.50 (CH₃). MS (m/z): 427 (C₁₉H₁₆Br N₅O_2,6.61%, M + 2), 425

(C₁₉H₁₆Br N₅O_2,15.79%, M^+.), 255 (C₁₂H₉N₅O_2,4.25%). Anal. Calc. for:

(C₁₉H₁₆BrN₅O₂) (M.W. = 425): C, 53.54; H, 3.78; N, 16.43%; Found: C,

53.75; H, 3.94; N, 16.61%.

144.85 (phenyl-C4), 134.17 (phenyl-C1), 128.48 (quinazoline-C8a),

127.43 (quinazoline-C7), 126.34 (quinazoline-C6), 124.27 (quinazo-

line-C5), 117.81 (phenyl-C3, C5), 116.75 (phenyl-C2, C6), 112.12

(quinazoline-C8), 111.41 (quinazoline-C4a), 58.03 (N-CH₂), 19.26

(CH₂), and 12.17 (CH₃). MS (m/z): 392 (C₁₉H₁₆N₆O_4,14.13%, M^+.).

Anal. Calc. for: (C₁₉H₁₆N₆O₄) (M.W. = 392): C, 58.16; H, 4.11; N,

21.42%; Found: C, 58.37; H, 4.25; N, 21.09%.

12

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Bioorganic & Medicinal Chemistry 42 (2021) 116266

4.3.7. 2-(3-Ethyl-5-oxo-[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)-N-

(naphthalen-1-yl)acetamide (27)

2.89 (q, J = 8.4 Hz, 2H, CH₂CH₃), 1.34 (t, J = 8.4 Hz, 3H, CH₂CH₃). ¹³

C

–

NMR (DMSO‑d ) δ ppm: 168.81 (C O), 168.70 (quinazoline-C4),

–

6

–

152.40 (triazole-C3), 144.85 (C O), 137.66 (quinazoline-C8a), 133.76

–

(quinazoline-C7), 125.27 (quinazoline-C5), 124.82 (quinazoline-C6),

116.10 (quinazoline-C8), 111.18 (quinazoline-C4a), 53.09 (N-CH₂),

45.55 (OCH₃), 22.01 (CH₂) and 12.56 (CH₃). MS (m/z): 286 (C₁₄H₁₄

N₄O₃, 100%, M^+.), 227 (C₁₂H₁₁N₄O, 6.16%). Anal. Calc. for: (C_{14 14}

H

N₄O₃) (M.W. = 286): C, 58.74; H, 4.93; N, 19.57%; Found: C, 58.94; H,

Brownish solid, Yield: 85%; m.p.

5.16; N, 19.75%.

4.4.2. Ethyl 2-(3-ethyl-5-oxo-[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)

acetate (29)

287–289 ^◦C. IR (KBr) cm^{ꢀ 1}: 3258 (NH), 3062 (CH aromatic), 2978 (CH

aliphatic), 1687 (C O), 1592 (C C aromatic). ¹H NMR (DMSO-d_δ)

–

White solid, Yield: 91%; m.p. 241–242 ^◦C.

ppm: 10.16 (S, 1H, NH, D₂O exchangeable), 8.20 (d, 2H, naphthalene-

H5, H8), 7.99 (d, 2H, naphthalene-H2, H4), 7.93 (d, 1H, quinazoline-

H10), 7.84 (d, 1H, quinazoline-H7), 7.55 (t, 3H, naphthalene- H3,H6,

H7), 7.28 (dd, 1H, quinazoline-H8), 6.72 (dd, 1H, quinazoline-H9), 4.30

(S, 2H, N-CH₂), 2.79 (q, J = 8.4 Hz, 2H, CH₂CH₃), 1.29 (t, J = 8.4 Hz, 3H,

13

–

IR (KBr) cm^{ꢀ 1}: 3051 (CH aromatic), 2989 (CH aliphatic), 1724 (C O),

CH₂CH₃). C NMR (DMSO‑d ) δ ppm: 169.79 (C O), 159.98 (quina-

–

6

–

1

zoline-C4), 157.67 (triazole-C3), 146.09 (C O), 135.91 (naphthalene-

–

1620 (C C aromatic). H NMR (DMSO‑d ) δ ppm: 8.27 (d, 1H, J = 8.0

6

C1), 134.18 (quinazoline-C8a), 134.16 (naphthalene-C4a), 128.97

(naphthalene-C5), 127.28 (quinazoline-C7), 126.54 (naphthalene-C3),

126.02 (naphthalene-C6), 125.80 (naphthalene-C7), 124.84 (naphtha-

lene-C8a), 123.71 (quinazoline-C5), 123.11 (quinazoline-C6), 121.95

(naphthalene-C8), 120.40 (napthalene-C4), 115.97 (quinazoline-C8),

114.61 (quinazoline-C4a), 111.20 (naphthalene-C2), 47.42 (N-CH₂),

19.76 (CH₂), and 12.21 (CH₃). MS (m/z): 397 (C₂₃H₁₉N₅O₂, 95.72%,

M^+.), 255 (C₁₃H₁₁N₄O_2,29.19%), 240 (C₁₂H₈N₄O_2,34.04%)^.Anal. Calc.

for: (C₂₃H₁₉N₅O₂) (M.W. = 397): C, 69.51; H, 4.82; N, 17.62%; Found:

C, 69.75; H, 4.89; N, 17.84%.

Hz, quinazoline-H10), 7.78 (dd, 1H, J = 7.6 Hz, quinazoline-H8), 7.62

(d,1H, J = 8.4 Hz, quinazoline-H7), 7.49 (dd, 1H, J = 8.0 Hz,

quinazoline-H9), 5.19 (S, 2H, N-CH2), 4.19 (q, 2H, O-CH₂CH3), 2.89 (q,

J = 8.4 Hz, 2H, CH₂CH₃), 1.34 (t, 3H, O-CH₂CH₃), 1.23 (t, J = 8.4 Hz,

13

–

3H, CH₂CH₃). C NMR (DMSO‑d ) δ ppm: 168.86 (C O), 168.27

–

6

–

(quinazoline-C4), 152.36 (triazole-C3), 144.83 (quinazoline-C O),

–

137.65 (quinazoline-C8a), 133.72 (quinazoline-C7), 125.24 (quinazo-

line-C5), 124.77 (quinazoline-C6), 116.05 (quinazoline-C8), 111.13

(quinazoline-C4a), 62.02 (OCH₂), 45.62 (N-CH₂), 22.00 (CH₂), 14.47

(CH₃) and 12.53 (CH₃).MS (m/z): 300 (C₁₅H₁₆N₄O_3,32.27%, M^+.)_,271

(C₁₃H₁₁N₄O₃, 1.88%), 227 (C₁₂H₁₁N₄O, 23.86%). Anal. Calc. for:

(C₁₅H₁₆N₄O₃) (M.W. = 300): C, 59.99; H, 5.37; N, 18.66%; Found: C,

60.15; H, 5.49; N, 18.84%.

4.4. General procedure for synthesis of alkyl 2-(3-ethyl-5-oxo-[1,2,4]

triazolo[4,3-c]quinazolin-6(5H)-yl)alkanoate derivatives (28–30)

Into a stirred solution of 3-ethyl-[1,2,4]triazolo[4,3-c]quinazolin-5

(6H)-one (20) (2.14 g, 0.01 mol) in dry DMF (20 ml), few crystals of

anhydrous K₂CO₃were added followed by the appropriate alkyl-2-

chloroacetate derivative (0.01 mol). The reaction mixture was heated

to reﬂux temperature on a water bath for 4 h. After cooling to room

temperature, the reaction mixture was poured onto ice-cooled water

(100 ml) and stirred for one more hour. The formed solid product was

ﬁltered off under vacuum and washed with a copious amount of water

until the ﬁltrate became neutral to litmus paper, washed with three

repetitive portions of ice-cold water (20 ml), and then ﬁnally recrys-

tallized from methanol to afford the corresponding ester derivative.

4.4.3. Ethyl 2-(3-ethyl-5-oxo-[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)

propanoate (30)

Brown solid, Yield: 76%; m.p. 246–248 ^◦C.

4.4.1. Methyl 2-(3-ethyl-5-oxo-[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-

yl)acetate (28)

IR (KBr) cm^{ꢀ 1}: 3082 (CH aromatic), 2981 (CH aliphatic), 1747 (C O),

–

1

–

1620 (C C aromatic). H NMR (DMSO‑d ) δ ppm: 8.28 (d, 1H, J = 8.0

6

Hz, quinazoline-H10), 7.81 (dd, 1H, J = 7.6 Hz, quinazoline-H8), 7.80

(d,1H, J = 8.0 Hz, quinazoline-H7), 7.52 (dd, 1H, J = 8.4 Hz,

quinazoline-H9), 5.80 (S, 1H, N-CH), 4.13 (q, 2H, OCH₂CH₃), 2.86 (q,

2H, CH₂CH₃), 1.60 (d, 3H, J = 8.0 HzN-CHCH₃), 1.33 (t,3H, O-

CH₂CH₃), 1.10 (t, J = 8.4 Hz, 3H, CH₂CH₃). ¹³C NMR (DMSO‑d₆) δ ppm:

Light brown solid, Yield: 89%; m.p.

–

169.84 (C O), 168.57 (quinazoline-C4), 152.30 (triazole-C3), 144.04

–

(quinazoline-C O), 137.29 (quinazoline-C8a), 133.29 (quinazoline-

–

C7), 125.60 (quinazoline-C5), 124.75 (quinazoline-C6), 115.68 (qui-

nazoline-C8), 111.54 (quinazoline-C4a), 61.62 (OCH₂), 53.52 (N-CH),

21.99 (CH₂), 14.54 (CH₃), 14.34 (CH₃) and 12.53 (CH₃). MS (m/z): 314

(C₁₆H₁₈N₄O_3,100%, M^+.), 285 (C₁₄H₁₃N₄O₃, 4.15%), 241(C₁₃H₁₃

N₄O, 3.88%). Anal. Calc. for: (C₁₆H₁₈N₄O₃) (M.W. = 314): C, 61.13; H,

5.77; N, 17.82%; Found: C, 61.29; H, 5.89; N, 18.04%.

238–240 ^◦C. IR (KBr) cm^{ꢀ 1}: 3078 (CH aromatic), 2981 (CH aliphatic),

1

–

1724 (C O), 1620 (C C aromatic). H NMR (DMSO‑d ) δ ppm: 8.27

–

(d,1H, J = 8.0 Hz, quinazoline-H10), 7.79 (dd, 1H,⁶J = 7.6 Hz,

quinazoline-H8), 7.63 (d,1H, J = 8.4 Hz, quinazoline-H7), 7.50 (dd, 1H,

J = 8.0 Hz, quinazoline-H9), 5.21 (S, 2H, N-CH₂), 3.69 (S, 3H, OCH₃),

13

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

IR (KBr) cm^{ꢀ 1}: 3051 (CH aromatic), 2958 (CH aliphatic),1708 (C O),

–

4.5. General procedure for synthesis of 3-ethyl-6-alkyl-[1,2,4]triazolo

–

1

–

[4,3-c]quinazolin-5(6H)-one derivatives (31–35)

1620 (C C aromatic). H NMR (DMSO‑d ) δ ppm: 8.20–8.22 (d, J = 8.0

Hz1H, quinazoline-H10), 7.78–7.79 (dd,⁶J = 7.6 Hz, 1H, quinazoline-

H8), 7.67–7.69 (d, J = 8.4 Hz, 1H, quinazoline-H7), 7.41–7.45 (dd, J

= 8.4 Hz, 1H, quinazoline-H9), 4.26 (t, 2H, N-CH₂), 2.84 (q, J = 8.4 Hz,

2H, CH₂CH₃), 1.67 (m, 2H, N-CH₂CH₂), 1.55 (t, J = 8.4 Hz, 3H,

CH₂CH₃), 1.28 (m, 6H, CH₂CH₂CH₂), 0.86 (t, 3H, CH₃hexyl). MS (m/z):

298 (C₁₇H₂₂N₄O, M^+.)_,241 (C₁₃H₁₃N₄O, 51.86%). Anal. Calc. for:

(C₁₇H₂₂N₄O) (M.W. = 298): C, 68.43; H, 7.43; N, 18.78%; Found: C,

68.17; H, 7.60; N, 18.91%.

Into a stirred solution of 3-ethyl-[1,2,4]triazolo[4,3-c]quinazolin-5

(6H)-one (20) (2.14 g, 0.01 mol) in dry DMF (20 ml) containing a cat-

alytic amount of potassium iodide, few crystals of anhydrous K₂CO₃

were added followed by the appropriate alkyl halide (0.01 mol). The

reaction mixture was heated to reﬂux temperature on a water bath for

2–5 h. After cooling to room temperature, the reaction mixture was

poured onto ice-cooled water (100 ml) and stirred for one more hour.

The formed solid product was ﬁltered off under vacuum and washed

with a plentiful amount of water until the ﬁltrate became neutral to

litmus paper, washed with three repetitive portions of ice-cold water

(20 ml), and then ﬁnally recrystallized from methanol.

4.5.4. 6-Allyl-3-ethyl-[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one (34)

4.5.1. 3-Ethyl-6-ethyl-[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one (31)

White solid, Yield: 82%; m.p. 273–275 ^◦C.

Yellowish white solid, Yield: 86%; m.p.

IR (KBr) cm^{ꢀ 1}: 3112 (CH allyl), 3082 (CH aromatic), 2966 (CH

aliphatic),1712 (C O), 1620 (C C aromatic). ¹H NMR (DMSO‑d₆) δ

–

ppm: 8.23–8.25 (d, J = 8.0 Hz1H, quinazoline-H10), 7.76–7.78 (dd, J =

7.6 Hz, 1H, quinazoline-H8), 7.56–7.58 (d, J = 8.4 Hz, 1H, quinazoline-

H7), 7.43–7.47 (dd, J = 8.4 Hz, 1H, quinazoline-H9), 5.96 (m, 1H, CH =

CH₂₎, 5.15–5.20 (dd, J = 10 Hz, cis, 2H, CH₂= CH), 4.95 (d, 2H, N-CH₂),

246–248 ^◦C. IR (KBr) cm^{ꢀ 1}

: 3089 (CH aromatic), 2974 (CH

aliphatic),1712 (C O), 1642 (C C aromatic). ¹H NMR (DMSO‑d₆) δ

–

ppm: 8.21–8.23 (d, J = 8.0 Hz1H, quinazoline-H10), 7.75–7.77 (dd, J =

7.6 Hz, 1H, quinazoline-H8), 7.70–7.72 (d, J = 8.4 Hz, 1H, quinazoline-

H7), 7.42 (dd, J = 8.4 Hz, 1H, quinazoline-H9), 4.32 (q, 2H, N-CH₂),

2.82 (q, 2H, CH₂), 1.31 (t, 6H, 2CH₃). ¹³C NMR (DMSO‑d₆) δ ppm:

168.07 (quinazoline-C4), 152.15 (triazole-C3), 144.42 (quinazoline-

2.87 (q, J = 8.4 Hz, 2H, CH₂CH₃), 1.36 (t, J = 8.4 Hz, 3H, CH₂CH₃). ¹³

C

NMR (DMSO‑d₆) δ ppm: 168.13 (quinazoline-C4), 152.35 (triazole-C3),

–

144.71 (quinazoline-C O), 137.55 (quinazoline-C8a), 133.30 (allylic-

–

CH), 132.20 (quinazoline-C7), 132.20 (quinazoline-C5), 125.04 (qui-

nazoline-C6), 124.28 (allylic-CH), 117.38 (quinazoline-C8), 111.55

(quinazoline-C4a), 46.12 (N-CH₂), 22.02 (CH₂) and 12.60 (CH₃). MS

(m/z): 254 (C₁₄H₁₄N₄O, M^+.), 253 (C₁₄H₁₃N₄O, 100%) 213, (C₁₁H₉N₄O,

3.62%). Anal. Calc. for: (C₁₄H₁₄N₄O) (M.W. = 254): C, 66.13; H, 5.55; N,

22.03%; Found: C, 65.87; H, 5.72; N, 21.97%.

–

C

O), 137.21 (quinazoline-C8a), 133.54 (quinazoline-C7), 125.19

(quinazoline-C5), 124.14 (quinazoline-C6), 116.03 (quinazoline-C8),

111.54 (quinazoline-C4a), 40.53 (N-CH₂), 22.00 (CH₂), 12.92 (CH₃) and

12.62 (CH₃). MS (m/z): 242 (C₁₃H₁₄N₄O, 100%, M^+.), 213 (C₁₁H₉N₄O,

30.38%). Anal. Calc. for: (C₁₃H₁₄N₄O) (M.W. = 242): C, 64.45; H, 5.82;

N, 23.13%; Found: C, 64.31; H, 5.98; N, 22.87%.

4.5.5. 6-(4-Chlorobutyl)-3-ethyl-[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-

one (35)

4.5.2. 3-Ethyl-6-propyl-[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one (32)

White solid, Yield: 85%; m.p. 248–250 ^◦C.

Yellowish white solid, Yield: 88%; m.p.

IR (KBr) cm^{ꢀ 1}: 3074 (CH aromatic), 2958 (CH aliphatic),1708 (C O),

–

1

–

1620 (C C aromatic). H NMR (DMSO‑d ) δ ppm: 8.23–8.25 (d, J = 8.0

Hz1H, quinazoline-H10), 7.79–7.81 (dd,⁶J = 7.6 Hz, 1H, quinazoline-

H8), 7.71–7.74 (d, J = 8.4 Hz, 1H, quinazoline-H7), 7.44 (dd, J = 8.4

Hz, 1H, quinazoline-H9), 4.26 (t, 2H, N-CH₂CH₂CH₃), 2.84 (q, J = 8.4

Hz, 2H, CH₂CH₃), 1.74 (m, 2H, N-CH₂CH₂CH₃), 1.33 (t, 3H, N-

CH₂CH₂CH₃), 1.00 (t, J = 8.4 Hz, 3H, CH₂CH₃). MS (m/z): 256

(C₁₄H₁₆N₄O, M^+.)_,213 (C₁₁H₉N₄O, 26.18%), 172 (C₈H₄N₄O, 100%).

Anal. Calc. for: (C₁₄H₁₆N₄O) (M.W. = 256): C, 65.61; H, 6.29; N,

21.86%; Found: C, 65.39; H, 6.41; N, 22.05%.

241–243 ^◦C. IR (KBr) cm^{ꢀ 1}: 3065 (CH aromatic), 2970 (CH aliphatic),

1

–

1693 (C O), 1620 (C C aromatic). H NMR (DMSO‑d ) δ ppm: 8.23 (d,

–

6

1H, J = 8.0 Hz, quinazoline-H10), 7.80 (dd, 1H, J = 7.6 Hz, quinazoline-

H8), 7.78 (d, 1H, J = 8.0 Hz, quinazoline-H7), 7.44 (dd, 1H, J = 8.4 Hz,

quinazoline-H9), 4.34 (t, 2H, N-CH₂), 3.71 (t, 2H, CH₂Cl), 2.85 (q, J =

8.4 Hz, 2H, CH₂CH₃), 1.88 (t, J = 8.4 Hz, 3H, CH₂CH₃), 1.3 (t, 4H,

2CH₂). MS (m/z): 304 (C₁₅H₁₇Cl N₄O, 75.20%, M^+.)_,269 (C₁₅H₁₇N₄O,

16.90%), 241 (C₁₃H₁₃N₄O, 57.93%), 213 (C₁₁H₉N₄O, 51.67%). Anal.

Calc. for: (C₁₅H₁₇Cl N₄O) (M.W. = 304): C, 59.11; H, 5.62; N, 18.38%;

Found: C, 59.35; H, 5.84; N, 18.50%.

4.5.3. 3-Ethyl-6-hexyl-[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one (33)

4.6. In vitro cytotoxic activity

Four human cancer cell lines namely; Hepatocellular carcinoma

(Hep-G2), Mammary gland breast cancer (MCF-7), Human prostate

cancer (PC3), and Colorectal carcinoma (HCT-116) were obtained from

VACSERA, Cairo, Egypt. Doxorubicin was used as a standard anticancer

Yellow solid, Yield: 88%; m.p. 268–270 ^◦C.

14

M.H. El-Shershaby et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116266

4 Moustakim M, Clark PGK, Trulli L, et al. Discovery of a PCAF Bromodomain

Chemical Probe. Angew Chemie. 2017;129(3):845–849. https://doi.org/10.1002/

ange.201610816.

drug for comparison. The inhibitory effects of our target compounds on

cell growth of the above-mentioned cell lines were determined using the

MTT assay ⁴¹

.

5 Liu T, Wang X, Hu W, et al. Epigenetically Down-Regulated Acetyltransferase PCAF

Increases the Resistance of Colorectal Cancer to 5-Fluorouracil. Neoplasia. 2019;21

(6):557–570. https://doi.org/10.1016/j.neo.2019.03.011.

4.7. PCAF inhibition enzyme assay

6 Tallen G, Riabowol K. Keep-ING balance: Tumor suppression by epigenetic

regulation. FEBS Lett. 2014;588(16):2728–2742. https://doi.org/10.1016/j.

febslet.2014.03.011.

A ﬂuorescent histone acetyltransferase estimation was applied for

compounds of series 1 according to the previously reported procedure

^45,58. Three independent experiments were performed and bromospor-

ine was used as a positive control.

7 Shagufta Ahmad I. An insight into the therapeutic potential of quinazoline

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4.8. Apoptosis induction study

The effects of compound 22 as an apoptosis inducer was analyzed

using Annexin V-FITC/PI apoptosis detection kit. PC3 cells were stained

with Annexin-V ﬂuorescein and counterstained with propidium iodide.

Then, cells in a density of 2X10⁵/well were incubated with the test

compound for 48 h. Next, the cells were trypsinized, washed with

phosphate-buffered saline, and ﬁnally stained for 15 min at 37 ^◦C in the

10 El-Azab AS, Al-Omar MA, Abdel-Aziz AA-M, et al. Design, synthesis and biological

evaluation of novel quinazoline derivatives as potential antitumor agents: Molecular

docking study. Eur J Med Chem. 2010;45(9):4188–4198. https://doi.org/10.1016/j.

ejmech.2010.06.013.

11 Al-Suwaidan IA, Abdel-Aziz AA-M, Shawer TZ, et al. Synthesis, antitumor activity

and molecular docking study of some novel 3-benzyl-4(3H)quinazolinone analogues.

J Enzyme Inhib Med Chem. 2016;31(1):78–89. https://doi.org/10.3109/

14756366.2015.1004059.

dark. FACS Caliber ﬂow cytometer was used in the analysis process ⁴⁸

.

12 Ezzat HG, Bayoumi AH, Sherbiny FF, et al. Design, synthesis, and molecular docking

studies of new [1,2,4]triazolo[4,3-a]quinoxaline derivatives as potential A2B

receptor antagonists. Mol Divers. 2021;25(1):291–306. https://doi.org/10.1007/

s11030-020-10070-w.

4.9. Cell cycle analysis

The effect of compound 22 on the cell cycle distribution was

13 Turky A, Sherbiny FF, Bayoumi AH, Ahmed HEA, Abulkhair HS. Novel 1,2,4-triazole

derivatives: Design, synthesis, anticancer evaluation, molecular docking, and

pharmacokinetic proﬁling studies. Arch Pharm (Weinheim). 2020;353(12):2000170.

https://doi.org/10.1002/ardp.202000170.

analyzed with a FACS Caliber ﬂow cytometer following the reported

procedures (see supporting information) ⁵⁰

.

14 Turky A, Bayoumi AH, Ghiaty A, El-Azab AS, Abdel-Aziz A-MA, Abulkhair HS.

Design, synthesis, and antitumor activity of novel compounds based on 1,2,4-tria-

zolophthalazine scaffold: Apoptosis-inductive and PCAF-inhibitory effects. Bioorg

Chem. 2020;101:104019. https://doi.org/10.1016/j.bioorg.2020.104019.

15 Turky A, Bayoumi AH, Sherbiny FF, El-Adl K, Abulkhair HS. Unravelling the

anticancer potency of 1,2,4-triazole-N-arylamide hybrids through inhibition of

STAT3: synthesis and in silico mechanistic studies. Mol Divers. 2021;25(1):403–420.

https://doi.org/10.1007/s11030-020-10131-0.

4.10. Docking studies

In the present work, all docking experiments were performed for all

the ﬁnal target hybrid structures using Molecular Operating Environ-

ment software (MOE 2014) to evaluate the free energy of binding and to

explore the binding mode toward histone acetyltransferase PCAF.

Redocking of the co-crystallized ligand and docking of its newly

designed ﬁnal bioisosteres were performed using a default protocol

against the target receptor according to the direction of our previously

16 Syed YY. Selinexor: First Global Approval. Drugs. 2019;79(13):1485–1494. https://

doi.org/10.1007/s40265-019-01188-9.

17 Kovalenko SI. Synthesis and Anticancer Activity of 2-(Alkyl-, Alkaryl-, Aryl-, Hetaryl-

)[1,2,4]triazolo[1,5-c]quinazolines. Sci Pharm. 2013;81(2):359–391. https://doi.

org/10.3797/scipharm.1211-08.

reported procedures ^15,24

.

18 Antypenko OM, Kovalenko SI, Karpenko OV, Nikitin VO, Antypenko LM. Synthesis,

Anticancer, and QSAR Studies of 2-Alkyl(aryl, hetaryl)quinazolin-4(3 H)-thione’s

and [1,2,4]Triazolo[1,5- c ]quinazoline-2-thione’s Thioderivatives. Helv Chim Acta.

2016;99(8):621–631. https://doi.org/10.1002/hlca.201600062.

Declaration of Competing Interest

19 Driowya M, Leclercq J, Verones V, et al. Synthesis of triazoloquinazolinone based

compounds as tubulin polymerization inhibitors and vascular disrupting agents. Eur

J Med Chem. 2016;115:393–405. https://doi.org/10.1016/j.ejmech.2016.03.056.

20 Al-Salahi R, Marzouk M, Ashour AE, Alswaidan I. Synthesis and Antitumor Activity

of 1,2,4-Triazolo[1,5-a]quinazolines. Asian J Chem. 2014;26(7):2173–2176. https://

doi.org/10.14233/ajchem.2014.16849.

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.

21 Ewes WA, Elmorsy MA, El-Messery SM, Nasr MNA. Synthesis, biological evaluation

and molecular modeling study of [1,2,4]-Triazolo[4,3-c]quinazolines: New class of

EGFR-TK inhibitors. Bioorg Med Chem. 2020;28(7), 115373. https://doi.org/

10.1016/j.bmc.2020.115373.

Acknowledgement

The authors would like to express their appreciation to Prof. Dr.

Magda A. El-Sayed, Professor of Pharmaceutical Chemistry at Horus

University in Egypt for her unlimited support throughout writing and

revision of the article.

22 Alesawy MS, Al-Karmalawy AA, Elkaeed EB, et al. Design and discovery of new 1,2,4-

triazolo[4,3- c ]quinazolines as potential DNA intercalators and topoisomerase II

inhibitors. Arch Pharm (Weinheim). November 2020. https://doi.org/10.1002/

ardp.202000237.

23 Abulkhair HS, Turky A, Ghiaty A, Ahmed HEA, Bayoumi AH. Novel

triazolophthalazine-hydrazone hybrids as potential PCAF inhibitors: Design,

synthesis, in vitro anticancer evaluation, apoptosis, and molecular docking studies.

Bioorg Chem. 2020;100, 103899. https://doi.org/10.1016/j.bioorg.2020.103899.

24 Patani GA, LaVoie EJ. Bioisosterism: A Rational Approach in Drug Design. Chem Rev.

1996;96(8):3147–3176. https://doi.org/10.1021/cr950066q.

Appendix A. Supplementary material

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

org/10.1016/j.bmc.2021.116266.

25 Martin YC. A practitioner’s perspective of the role of quantitative structure-activity

analysis in medicinal chemistry. J Med Chem. 1981;24(3):229–237. https://doi.org/

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From triazolophthalazines to triazoloquinazolines: A bioisosterism-guided approach toward the identification of novel PCAF inhibitors with potential anticancer activity

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