Novel triazolophthalazine-hydrazone hybrids as potential PCAF inhibitors: Design, synthesis, in vitro anticancer evaluation, apoptosis, and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2020.103899

Three novel series of triazolophthalazine derivatives bearing hydrazone moiety were designed, synthesized, and evaluated for their anticancer activity against four human cancer cell lines by MTT assay. Six derivatives demonstrated comparable activity with Doxorubicin reference drug against the selected cancer cells. Especially, compound 16 showed the most potent activity with IC₅₀ values of 5.70, 8.04, 11.15, and 4.25, μM against HePG2, MCF-7, PC3, and HCT-116 respectively. Also, compound 26 exhibited comparable inhibitory effect with that of Doxorubicin against the selected cancer cell lines with IC₅₀ values of 6.45, 8.63, 12.28, and 7.03 μM against HePG2, MCF-7, PC3, and HCT-116 respectively. Investigation of the apoptotic activity of the two most active compounds revealed that compounds 16 and 26 could induce both the early and the late apoptosis of HePG2. Further mechanistic study of the HePG2 cell cycle confirmed the spectacular cytotoxic and apoptotic effects of both compounds. Compounds 16 and 26 showed a pronounced increase in cells in G2/M and Pre G1 phases with a concomitant reduction of cells in G0-G1 and S phases. A follow up enzymatic assay indicated that these two compounds have comparable activities with that of bromosporine as PCAF inhibitors with IC₅₀ values of 8.13 and 5.31 μM respectively. Moreover, molecular docking study for all the synthesized compounds was performed to predict their binding affinities toward the active site of histone acetyltransferase GCN5. Results of molecular docking were strongly correlated with that of the cytotoxicity study.

Full text of DOI:10.1016/j.bioorg.2020.103899

BioorganicChemistry100(2020)103899
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Bioorganic Chemistry
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Novel triazolophthalazine-hydrazone hybrids as potential PCAF inhibitors:
Design, synthesis, in vitro anticancer evaluation, apoptosis, and molecular
docking studies
⁎
Hamada S. Abulkhaira,b, , Abdallah Turkya, Adel Ghiatya, Hany E.A. Ahmeda,c
,
Ashraf H. Bayoumia
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City 11884, Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Horus University – Egypt, International Costal Road, New Damietta, Egypt
c Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College, Taibah University, Al-Madinah Al-Munawarah 41477, Saudi Arabia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three novel series of triazolophthalazine derivatives bearing hydrazone moiety were designed, synthesized, and
evaluated for their anticancer activity against four human cancer cell lines by MTT assay. Six derivatives de-
monstrated comparable activity with Doxorubicin reference drug against the selected cancer cells. Especially,
compound 16 showed the most potent activity with IC50 values of 5.70, 8.04, 11.15, and 4.25, µM against
HePG2, MCF-7, PC3, and HCT-116 respectively. Also, compound 26 exhibited comparable inhibitory effect with
that of Doxorubicin against the selected cancer cell lines with IC50 values of 6.45, 8.63, 12.28, and 7.03 µM
against HePG2, MCF-7, PC3, and HCT-116 respectively. Investigation of the apoptotic activity of the two most
active compounds revealed that compounds 16 and 26 could induce both the early and the late apoptosis of
HePG2. Further mechanistic study of the HePG2 cell cycle confirmed the spectacular cytotoxic and apoptotic
effects of both compounds. Compounds 16 and 26 showed a pronounced increase in cells in G2/M and Pre G1
phases with a concomitant reduction of cells in G0-G1 and S phases. A follow up enzymatic assay indicated that
these two compounds have comparable activities with that of bromosporine as PCAF inhibitors with IC50 values
of 8.13 and 5.31 µM respectively. Moreover, molecular docking study for all the synthesized compounds was
performed to predict their binding affinities toward the active site of histone acetyltransferase GCN5. Results of
molecular docking were strongly correlated with that of the cytotoxicity study.
[1,2,4]Triazolo[3,4-a]phthalazine
Hydrazones
Anticancer
Molecular hybridization
PCAF
Apoptosis
Molecular docking
1. Introduction
acetyltransferase paralogues p300/CBP-associated factor (PCAF) is a
member of the subfamily I of the phylogenetic bromodomain tree [7].
Post-translational modifications of histones and methylation of DNA
are the two main levels of epigenesis and are crucial to chromatin
function [1]. The aberrant histone acetylation is involved in the per-
manent changes in gene expression [2]. Consequently, histone acetyl-
transferases (HATs) have emerged as interesting prime targets for an-
ticancer chemotherapeutics [3]. Likewise, the bromodomain family of
proteins is frequently dysregulated in cancer [4], and because of that
has gained special focus as a novel target for pharmacological inter-
vention [5]. Recently, aberrant expression of bromodomain-containing
proteins has been linked to the development of abnormal cell pro-
liferation and tumorigenesis [5]. New avenues for developing antic-
ancer drugs have been highlighted by the proven potent cytotoxic ac-
tivity of selective bromodomain inhibitors [6]. The histone
Mis-regulation of PCAF has been shown to be associated with a variety
of human diseases including HIV infection [8,9], neuroinflammation
[10], and cancer [11–13]. Mis-regulation of PCAF signaling pathway
leads to its over-expression in different types of cancer cells including
hepatocellular [14], mammary gland [15], colorectal [13], and prostate
carcinoma [16].
A promising class of PCAF inhibitors that inhibit histone acetyl
transferases with a micromolar range of IC50′s is the 1,2,4-triazoles
[17]. Triazolopthalazines represent one of the structural classes of po-
tent bromodomain and extra-terminal motif (BET) inhibitors [18,19].
Few years ago, compound 1 (L-45) was discovered as the first potent
[1,2,4]triazolo[3,4-a]phthalazine as PCAF bromodomain inhibitor
[12]. Also, bromosporine is a 1,2,4-triazolopthalazines derivative that
⁎ 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.bioorg.2020.103899
Received 2 April 2020; Received in revised form 27 April 2020; Accepted 28 April 2020
Availableonline17May2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
Fig. 1. Structures of selected [1,2,4]triazolo[3,4-a]phthalazines with potent anticancer activity against hepatocellular carcinoma.
has the ability of broadly targeting BRDs (including PCAF) with nano-
molar affinity [20]. Additionally, [1,2,4]triazolo[3,4-a]phthalazine is a
pharmacophore ring system, presented in certain anticancer agents
[21–23]. In the last decade, many synthetic [1,2,4]triazolo[3,4-a]
phthalazines (Fig. 1) have been reported to exhibit potent anticancer
activities against hepatocellular carcinoma with IC50 values range of
1.60–6.04 [21,22,24].
cell death [21]. Hydrazone incorporating anticancer agents proven to
induce apoptosis via a caspase-independent pathway. They originate
the activation of pro-apoptotic proteins and induced the generation of
reactive oxygen species [27,34].
1.1. Rationale and ligand-based design
On the other hand, hydrazones are an important class of organic
compounds with diverse biological activities, particularly as anticancer
[25–27] Hydrazone derivatives 5 & 6 (Fig. 2) showed high cytotoxic
activity and superior selectivity against malignant over non-malignant
cells. In addition, compound 7 (CPTH2) was reported as an apoptosis
inducer with a dual inhibitory effect of GCN5 and PCAF [28,29]. Also,
the hydrazone derivative 8 revealed, with good selectivity index, potent
cytotoxicity effect against MGC-803, MCF-7, and EC-109 cell lines with
IC50 values of 0.85, 0.95, and 1.77 µM respectively [30]. Further me-
chanistic investigation of compound 8 confirmed that it could induce
apoptosis of MGC-803 cells through the mitochondrial pathway. The
apoptotic effect of 8 is accompanied by activations of caspase-9/3, up-
regulation of the expression of Bax, Bak, as well as down-regulation of
that of Bcl-2 and Mcl-1.
The key interactions of compound 1 in the histone acetyltransferase
GCN5 binding site (PDB: 5TPX) involve three main features as shown in
Fig. 3 a) a hydrogen bond between the NH attached with C-6 of tria-
zolophthalazine core and Glu1389 residue b) a π-π stacking interaction
between the pyridazine ring of the triazolophthalazine motif and
Tyr1442 c) an additional hydrogen bond between the N-1 of triazole
ring and Asn1436 residue.
In light of the afore-mentioned facts and as a continuation of our
previous studies on identifying new antiproliferative agents [35–42],
molecular hybridization between triazolophthalazine scaffold and hy-
drazone moiety was carried out in an attempt to get new effective an-
ticancer molecules with increased potency. Molecular hybridization is
one of the well-known approaches adopted in drug design which in-
volves the combination of pharmacophores from different bioactive
substances [43]. This approach is expected to produce hybrid structures
with improved bioactivity when compared to individual parent drugs.
This strategy may also result in new scaffolds with dual modes of action
[44]. Here, inspired by the versatility of the [1,2,4]triazolo[3,4-a]
phthalazine pharmacophoric ring skeleton of L-45 and the hydrazone
moiety mentioned above, three novel series of hybrid structures were
designed and synthesized to evaluate their anticancer activities (Fig. 4).
Targeting critical regulators of apoptosis with the aim of encoura-
ging the programmed death in cancer cells is a successful and attractive
strategy for the discovery of new anticancer agents [31,32]. Numerous
phthalazine derivatives were reported to induce apoptotic-induced cell
death by elevating the level of cleaved caspase-3 expression [33]. The
cytotoxic effect observed in response to the [1,2,4]triazolo[3,4-a]
phthalazine derivative 4 is associated with the induction of apoptotic
Fig. 2. Structures of some reported hydrazone incorporating anticancer agents.
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H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
triazolophthalazine-hydrazone hybrids is illustrated in Scheme 1.
Phthalic anhydride reacted with hydrazine hydrate in acetic acid to
yield 2,3-dihydrophthalazine-1,4-dione (10). The latter reacted under
reflux with phosphorus oxychloride to yield 1,4-dichlorophthalazine
(11). Compound 11 was further reacted with hydrazine hydrate at
room temperature in ethanol to produce 1-chloro-4-hy-
drazinylphthalazine. Reaction of the hydrazinyl derivative 12 with
trifluoroacetic acid yielded 6-chloro-3-(trifluoromethyl)-[1,2,4]triazolo
[3,4-a]phthalazine (13a). Alternatively, treating 12 with 4-methyl-
benzoyl chloride or 4-chlorobenzoyl chloride in refluxing ethanol pro-
duced the triazolophthalazine derivatives 13b,c [25]. Reaction of the
latter with hydrazine hydrate in refluxing ethanol afforded the starting
materials 14a-c. Finally, all the synthesized compounds 15–29 were
readily obtained by refluxing 14a-c with the appropriate aromatic al-
dehydes in ethanol.
Fig. 3. The key interactions of L-45 with the active site of histone acetyl-
2.2. Evaluation of biological activity
transferase GCN5.
2.2.1. Cytotoxicity assay
Different substitution patterns were introduced to both the C-3 of
triazolophthalazine scaffolds and the terminal phenyl ring to in-
vestigate the effect on the cytotoxicity of the designed compounds. All
the synthesized compounds were evaluated for their in vitro cytotoxic
activities against four cancer cell lines namely; hepatocellular carci-
noma (HePG-2), mammary gland breast cancer (MCF-7), human pros-
tate cancer (PC3) and colorectal carcinoma (HCT-116). In addition, the
possible underlying mechanisms of the most potent compounds were
further investigated to measure its ability to induce apoptosis and to
inhibit the activity of p300/CBP-associated factor (PCAF). Moreover, a
molecular docking study for all the synthesized compounds was per-
formed to predict their binding affinity towards the active site of his-
tone acetyltransferase GCN5 as a proposed mode of their cytotoxic
activity. Finally, the structure–activity relationship of the synthesized
compounds was illustrated based on the results of cytotoxicity evalua-
tion against four human cancer cell lines (HePG2, MCF-7, PC3, and
HCT-116).
Cytotoxic activities of the synthesized compounds were evaluated
against four cancer cell lines namely; hepatocellular carcinoma (HePG-
2), mammary gland breast cancer (MCF-7), human prostate cancer
(PC3) and colorectal carcinoma (HCT-116) using MTT assay [45] and
Doxorubicin as a reference anticancer agent. The results of preliminary
antiproliferative evaluation are shown in Table 1. The results of the
cytotoxicity assay showed that most of the synthesized compounds
displayed moderate to good cytotoxic activities against the selected four
cancer cell lines. Concentrations of target compounds needed to inhibit
50% of tumor cell proliferation were as low as 4.28 µM. Compounds 16,
17, 21, 22, 26, and 27 were the most potent derivatives. Compound 16
showed an equal inhibitory effect with Doxorubicin against both he-
patocellular carcinoma (HePG2) and colorectal carcinoma (HCT-116)
cell lines with IC50 values of 5.70 and 4.25 µM respectively. Also,
compound 26 revealed comparable activity with the reference cyto-
toxic drug against both cell lines with IC50 values of 6.45 and 7.03 µM
respectively. Higher doses (up to 19.68 µM) of hydrazone derivatives
17, 21, 22, and 27 needed to Inhibit 50% of cell proliferation in-
dicating good to moderate activity of these derivatives. The electronic
effect of the substituents attached to the terminal phenyl ring had a
remarkable influence on the cytotoxic activity. Compound 16, 17, 21,
22, 26, and 27 with electron-donating groups attached to C-4 of the
terminal phenyl ring, all displayed significant inhibitory potencies with
IC50 values ranging from 4.25 to 19.68 µM. Lower activities were ob-
served for the remaining compounds with no electron-donating group
2. Results and discussion
2.1. Chemistry
The final target compounds of the present work were readily ob-
tained in six consecutive steps utilizing commercially available phthalic
anhydride. The general route for the synthesis of our target
Fig. 4. Molecular hybridization of triazolophthalazine scaffold with hydrazone moiety.
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H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
Scheme 1. Synthetic route of the designed new triazolophthalazine-hydrazone hybrids.
attached at the para position of the terminal phenyl ring. On the other
hand, the introduction of a phenyl group at C-3 of the triazolophtha-
lazine skeleton has a little negative impact on cytotoxic activity. The
derivatives of series A with trifluoromethyl substituents at C-3 were
found more potent than the analogous derivatives in series B and C.
These relative lower inhibitory effects of series B and C may attribute to
the presence of a bulky group at C-3, making derivatives with such a
bulky group relatively unable to accommodate the binding site of the
receptor.
Cytometric quantification analyses were performed to characterize the
mode of HePG2 cell death induced by these two compounds using
Annexin-V-FITC & propidium iodide and BD FACS Caliber reader [46].
After 12 and 24 h of treatment with 10 µM molar concentrations of each
compound, cells were labeled with the above two dyes, and the re-
sulting fluorescence was monitored by flow cytometry. As shown in
Figs. 5, 6, after treatment of cells with the test compounds for 12 h,
compound 16 caused an increase in the early apoptosis from 0.41% of
the control sample (DMSO) to 3.92%. After 24 h, compound 16
markedly increased the late apoptosis from 0.29% to 10.86%. After
treatment of the cells with compound 26 for 12 h, a significant increase
in the early apoptosis from 0.41 of the control sample (DMSO) to 8.40
has obtained. Upon treatment of the cells with compound 26 for 24 h,
the late apoptosis markedly increased from 0.29% to 15.20%.
2.2.2. Apoptosis assay
Encouraging apoptosis in cancer cells is a major way that most of
the cytotoxic agents kill tumor cells [31]. The two most active com-
pounds 16 and 26 were chosen to explore their action mechanisms.
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H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
Table 1
In vitro anticancer activity of the designed new triazolophthalazine-hydrazone hybrids.
Cpd.
R1
R2
R3
R4
IC50 (µM)*
HePG2
MCF-7
PC3
HCT-116
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
CF3
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
Cl
37.28
5.70
2.34
32.52
8.04
2.23
42.58
11.15
15.79
36.48
94.02
> 100
13.97
18.70
26.98
55.96
46.12
12.28
19.68
> 100
> 100
8.87
2.32
1.05
1.31
2.25
5.46
47.46
4.25
2.47
CF3
OH
OCH3
Cl
0.41
0.71
0.75
0.29
CF3
9.13
11.36
29.72
79.16
81.49
9.28
0.90
2.12
4.27
4.66
10.36
41.38
65.26
90.02
8.29
0.95
2.12
3.60
4.90
CF3
32.75
61.56
89.40
7.88
2.05
CF3
H
3.50
4.78
4-CH3C6H4
4-CH3C6H4
4-CH3C6H4
4-CH3C6H4
4-CH3C6H4
4-Cl-C6H4
4-Cl-C6H4
4-Cl-C6H4
4-Cl-C6H4
4-Cl-C6H4
Doxorubicin
H
OH
OCH3
Cl
0.53
0.82
1.12
1.43
1.49
2.90
2.54
1.18
1.51
0.67
10.50
19.49
45.08
41.93
6.45
1.01
0.85
2.90
2.62
14.90
22.45
39.01
36.91
8.63
1.24
1.02
2.53
2.48
13.50
21.89
57.26
49.80
7.03
1.10
1.03
2.74
2.58
H
H
OH
OCH3
Cl
0.42
0.71
0.41
13.46
90.09
88.47
4.50
1.16
4.90
4.51
15.37
83.57
79.76
4.17
1.39
4.80
4.32
17.68
92.46
87.92
5.23
1.30
5.51
4.76
H
0.27
0.20
0.66
0.30
* Cytotoxicity was assayed by treating cells with the test compound for 72 h and 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.
2.2.3. Cell cycle analysis
Growth inhibitors of cancer cells invariably cause an alteration of
cell cycle distribution, with preferential G2/M blockade [47]. There-
fore, we decided to examine the effects of compounds 16 and 26 on cell
cycle progression of the HePG2 cell line (Figs. 7, 8). HePG2 cells were
treated with 10 µM molar concentration of both compounds for 12 h
and 24 h. Compounds 16 and 26 caused aggregation of cells in both
G2/M and Pre G1 phases and decrease in the count of cells in the other
two phases. Specifically, the percentages of cells in G2/M and pre G1
phase increased from 12.81% and 1.27% to 32.68 and 17.22 respec-
tively for compound 16. The percentages of cells in G2/M and pre G1
phase increased to 42.26% and 26.51% respectively for compound 26
in comparison to 12.81% and 1.27% of control.
Fig. 5. The apoptotic effects of compounds 16 and 26 on the cell cycle dis-
tribution of HePG2 cell line.
Fig. 6. Annexin V-FITC/PI double staining for detection of apoptosis in HePG2 cells treated with compounds 16 and 26.
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H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
Fig. 7. Bar diagram of cell cycle phase distribution in HePG2 cells treated with
compounds 16 and 26.
2.2.4. PCAF inhibitory effects
Fig. 9. IC50 of the highest potent triazolophthalazine-hydrazone hybrids and
Hepatocellular carcinoma comprises 6% of all incident cancer cases
worldwide and the third driving cause of cancer mortality worldwide.
Mis-regulation of PCAF in hepatocellular cancer cells is well known
[48]. In this article, compounds that showed the highest potent cyto-
toxicity effects against hepatocellular carcinoma cell line 16, 17, 21,
22, 26, and 27 were selected for further assessment of enzyme in-
hibitory effects on P300/CBP-associated factor (PCAF). As presented in
Fig. 9, the results indicated that the selected compounds are promising
PCAF inhibiting candidates. Compound 26 showed the highest effect
with an IC50 value of 5.30 μM which is nearly equal to the IC50 of the
reference PCAF inhibitor, bromosporine. Compound 16 showed an IC50
value of 8.13 μM.
bromosporine (BSP) on PCAF.
Table 2
The binding free energies (ΔG, kcal/mol) of the target triazolophthalazine-hy-
drazone hybrids and co-crystallized ligand (L-45) with the binding site of his-
tone acetyltransferase GCN5.
Compound
ΔG (kcal/mol)
Compound
ΔG (kcal/mol)
15.
16.
17.
18.
19.
20.
21.
22.
−11.04
−11.63
−11.25
−10.56
−10.79
−11.42
−11.35
−10.43
23.
24.
25.
26.
27.
28.
29.
L-45
−10.92
−10.46
−10.79
−11.76
−11.55
−10.73
−10.55
−13.16
2.3. Molecular docking studies
In the present work, molecular docking studies were conducted to
give guidance of molecular binding modes of the target compounds
inside the pocket of histone acetyltransferase GCN5. Docking studies
were conducted using MOE2014 to determine the free energy and
binding mode. The selection of the most promising molecules depended
on the perfect binding mode and the binding free energy. Free energies
of binding of all compounds together with that of the reference ligand
are summarized in Table 2. The binding mode of the co-crystallized
ligand, L-45 with the pocket of histone acetyltransferase GCN5, ex-
hibited a binding energy of −13.16 kcal/mol. There are three main key
interactions between the ligand and the binding site of histone acetyl-
transferase GCN5 a) salt bridge between Glu1389 residues and the di-
methylamino motif of L-45; b) a π-π stacking interaction between
Tyr1442 and the pyridazine ring of the triazolophthalazine motif; c) a
hydrogen bond between the triazole ring and Asn1436 residue.
16 formed a hydrogen bond with Asn1436 residue. The pyridazine ring
formed a π-π stacking with Tyr1442 while the NH of hydrazone moiety
at C-6 interacted via hydrogen bond with Glu1389. The terminal ben-
zene ring of compound 16 showed additional arene-H interaction with
Trp1379 (Fig. 10).
Similarly, compound 17, as a representative example of hydrazone
derivatives with a methoxy group at the para position of the terminal
benzene ring (17, 22, and 27), showed a binding mode almost like that
of L-45, with affinity value of −11.15 kcal/mol. The N-1 of the triazole
ring formed a hydrogen bond with Asn1436 residue. The pyridazine
ring formed a π-π stacking with Tyr1442 and the NH of the hydrazone
moiety at C-6 interacted with Glu1389 by a hydrogen bond (Fig. 10).
Independently, compound 23, as a representative example of hy-
drazone derivatives with electron-withdrawing group at the para posi-
tion of the terminal benzene, exhibited different virtual binding mode
with that of L-45 (Fig. 10). Compound 23 revealed an affinity value of
−10.93 kcal/mol and showed four different interactions with the
binding site of histone acetyltransferase GCN5. These interactions
The binding mode of compound 16, as a representative example of
hydrazones with OH substituent at the para position of the terminal
benzene ring (16, 21, and 26), exhibited an affinity value of
−11.43 kcal/mol. Obeying the same interaction pattern of L-45 with
the binding site of histone acetyltransferase GCN5, the triazole rings of
Fig. 8. Flow cytometry analysis of cell cycle phase distribution in HePG2 cells treated with compounds 16 and 26.
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H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
Fig. 10. 3D interactions of compounds 16 (upper left panel), 17 (upper right panel), 23 (lower left panel), and 25 (lower right panel) with the active site of histone
acetyltransferase GCN5.
involve three arene-H interactions between the pyridazine, pyrazole,
and the terminal benzene rings in the target compounds with Glu1389,
Ala1390, and Trp1379 residues respectively. The last interaction is a π-
π stacking between the aryl ring at C-3 of triazolophthalazine with
Tyr1442 residue.
cytotoxic agents is a major objective of the present work. Comparing
the cytotoxic activity of compounds 15–19 incorporating tri-
fluoromethyl substituent group at C-3 (Series A) with that of com-
pounds 20–24 incorporating p-tolyl substituent group at C-3 (Series B),
indicated that there is a mild decrease in the activity upon increasing
the bulkiness of substituent at C-3. The same result is also clear if series
C compared with series A (Fig. 11). The relative lower cytotoxic in-
hibitory effects of series B and C may be due to the inability of such
compounds to accommodate the binding site of the receptor. The
electronic nature of the substituent group attached to the terminal
phenyl ring has a great impact on the cytotoxicity against the four se-
lected cell lines. Comparing the cytotoxic activities of the individual
derivatives in each series revealed that compounds with electron-do-
nating group at C-4 of the terminal phenyl ring (16, 17, 21, 22, 26, and
27) are more potent than all other derivatives. Finally, compound (19,
24, and 29) with electron-withdrawing groups at C-2 and C-6 of the
terminal phenyl ring all displayed the least inhibitory potency against
The absence of electron-donating group at the C-4 position of the
terminal benzene ring as in compounds 20 and 25 has an undesirable
effect on the affinity for the receptor (Table 2). The obtained result for
compound 20 is virtually the same as that of compound 25. In addition,
the presence of a bulky substituent at C-3 may decrease the affinity of
the last two compounds due to the inability of these compounds to
accommodate the binding site of the receptor (Fig. 10). With
−10.77 kcal/mol free energy of binding, the binding mode of 25 in-
volves a) a hydrogen bond between the triazole ring and Asn1436 re-
sidue b) a π-π stacking of the pyridazine ring with Tyr1442 c) a hy-
drogen bond between the NH of the hydrazone moiety at C-6 with
Glu1389.
Collectively, the obtained results of molecular docking studies
showed that all the studied compounds have almost similar position
and orientation inside the binding site of histone acetyltransferase
GCN5. As well, the distribution of binding free energies calculated from
MOE software showed preferentially that the two most active deriva-
tives; 16 and 26 are highly correlated with the biological activity,
Moreover, the presence of electron donating group attached at the C-4
of terminal benzene ring played an important role in the binding affi-
nity and may be responsible for the higher activity of compounds 16,
17, 21, 22, 26, and 27.
2.4. Structure-activity relationship study
Fig. 11. Summary of SAR study of the synthesized triazolophthalazine-hy-
drazone hybrids.
As outlined in the rationale ligand-based drug design, studying SAR
of the newly synthesized triazolophthalazine-hydrazone hybrids as
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H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
selected cancer cell lines with IC50 values ranging from 45.08 up to over
100 µM.
4.2.1. 6-(2-Benzylidenehydrazinyl)-3-(trifluoromethyl)-[1,2,4]triazolo
[3,4-a]phthalazine (15)
White solid (0.28 g, 80%); mp = 253–255 °C; IR (KBr) νmax cm−1
:
3363 (NH), 3066 (CH aromatic), 2935 (CH aliphatic), 1573 (C]C); 1H
NMR (DMSO‑d6) δ: 11.55 (brs, 1H), 8.6 (t, J = 7.6 Hz, 2H), 8.53 (s,
1H), 8.07 (m, 2H), 7.79 (d, J = 7.3 Hz, 2H), 7.49 (m, 3H); 13C NMR
(DMSO‑d6) δ: 150.30, (phthalazine C-1), 146.98, (triazole C-3), 144.58,
(CH), 139.29, (phthalazine C-4), 134.82, (phenyl C-1), 132.21,
(phthalazine C-6), 130.45, (phthalazine C-7), 129.37, (phenyl C-4),
127.31, (phenyl C-2, C-6), 125, (phenyl C-3, C-5), 123.8, (phthalazine
C-5), 122.95, (phthalazine C-8a), 120.39, (phthalazine C-8), 118.29,
(phthalazine C-4a), 117.71, (CF3); MS (m/z, %): 356 (M+, 10.11%),
279 (C17H11F3N6, 10.10%), 130 (C8H6N2, 2.24%), 77 (C6H5, 100%).
Anal. Calc. for C17H11F3N6 (M.W. = 356): C, 57.31; H, 3.11; N, 23.59%;
Found: C, 57.49; H, 3.29; N, 23.45%.
3. Conclusion
In conclusion, we are here reporting the design, synthesis, in vitro
anticancer evaluation, in silico docking studies of three novel series of
triazolophthalazine derivatives bearing hydrazone moiety as potent
inhibitors of P300/CBP-associated factor (PCAF). The target com-
pounds were designed based on molecular hybridization of the triazo-
lophthalazine scaffold of L-45 with hydrazone moiety. Different sub-
stitution patterns were introduced to both the C-3 of
triazolophthalazine scaffolds and the terminal phenyl rings to in-
vestigate the effect on cytotoxic activity. Six of our designed hybrid
derivatives showed good cytotoxicity effects against four human cancer
cell lines (HePG2, MCF-7, PC3, and HCT-116). Compound 16 showed
more inhibitory effects than the reference antitumor Doxorubicin
against HCT-116, and almost equipotent against HePG2. Also, com-
pound 26 revealed comparable activity with Doxorubicin against
HePG2, PC3, and HCT-116 cell lines. Additionally, apoptosis study re-
vealed that both compounds 16 and 26 significantly induce the early
and late apoptosis. Moreover, cell cycle analysis and PCAF inhibitory
studies confirmed the spectacular cytotoxic and apoptotic effects of
both compounds. Finally, molecular docking and SAR studies were
conducted. Results of docking experiments assisted the superior activ-
ities of compounds 16 and 26 by anticipating potential binding inter-
actions with the active site of histone acetyltransferase GCN5. The most
active candidates in the quest for effective cytotoxic agents will serve as
useful leads and merit further investigations.
4.2.2. 6-(2-(4-Hydroxybenzylidene)hydrazinyl)-3-(trifluoromethyl)-
[1,2,4]triazolo[3,4-a]phthalazine (16)
Yellowish white solid (0.27 g, 74%); mp = 259–261 °C; IR (KBr)
νmax cm−1: 3433 (OH), 3317 (NH), 3012 (CH aromatic), 2962 (CH
aliphatic), 1593 (C]C); 1H NMR (DMSO‑d6) δ: 11.31 (brs, 1H), 9.96 (s,
1H), 8.576.9 (m, 9H); 13C NMR (DMSO‑d6) δ: 159.75, (phthalazine C-
1), 150.14, (phenyl C-4), 147.46, (triazole C-3), 144.43, (CH), 139.19,
(phthalazine C-4), 133.87, (phthalazine C-6), 131.91, (phthalazine C-
7), 129.12, (phenyl C-2, C-6), 125.9, (phthalazine C-5), 124.88, (phenyl
C-1), 123.63, (phthalazine C-8a), 122.82, (phthalazine C-8), 120.42,
(phthalazine C-4a), 118.26, (phenyl C-3, C-5), 117.74, (CF3); MS (m/z,
%): 372 (M+, 100%), 77 (C6H5, 22.24%). Anal. Calc. for C17H11F3N6O
(M.W. = 372): C, 54.84; H, 2.98; N, 22.57; O, 4.3%; Found: C, 55.08;
H, 3.17; N, 22.38%.
4. Experimental section
4.1. General
4.2.3. 6-(2-(4-Methoxybenzylidene)hydrazinyl)-3-(trifluoromethyl)-
[1,2,4]triazolo[3,4-a]phthalazine (17)
Melting points were taken on electrothermal (stuart SMP30) appa-
ratus and were uncorrected. IR spectra were recorded on Pye Unicam
SP 1000 IR spectrophotometer at the Pharmaceutical Analytical Unit,
Faculty of Pharmacy, Al-Azhar University. 1H NMR spectra were re-
corded in: 1) DMSO‑d6 at 300 MHz on a Varian Mercury VXR-300 NMR
spectrometer at NMR Lab, Faculty of Science, Cairo University, 2)
DMSO‑d6 at 400 MHz on Joel ECA spectrometer at NMR Lab,
Microanalytical Unit, Faculty of Pharmacy, Mansoura University, 13C
spectra were determined at 100 MHz in deuterated dimethyl sulfoxide
(DMSO‑d6) on a Varian Mercury NMR spectrometer. Chemical shifts
were related to that of the solvent, TMS was used as an internal stan-
dard. Mass spectra and microanalyses were carried out at the Regional
Center for Mycology Biotechnology, Al-Azhar University, Cairo, Egypt.
The progress of reactions was monitored with Merck silica gel IB2-F
plates (0.25 mm thickness) and was visualized using a UV lamp using
different solvents as mobile phases. Starting reagents, phthalic anhy-
dride, hydrazine hydrate, phosphorus oxychloride, trifluoroacetic acid,
and acid chloride derivatives were purchased from Aldrich chemical
company and were used as received. Compounds 14a-c were synthe-
sized following the directions of reported procedures [22].
Yellowish white solid (0.30 g, 79%); mp = 264–266 °C; IR (KBr)
νmax cm−1: 3541 (NH), 3012 (CH aromatic), 2970 (CH aliphatic), 1593
(C]C); 1H NMR (DMSO‑d6) δ: 11.39 (brs, 1H), 8.57 (t, J = 6.7 Hz, 2H),
8.46 (s, 1H), 8.09–8 (m, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.06 (d,
J = 8.4 Hz, 2H), 3.82 (s, 3H); 13C NMR (DMSO‑d6) δ: 161.26,
(phthalazine C-1), 150.3, (phenyl C-4), 150, (triazole C-3), 147.07,
(CH), 144.63, (phthalazine C-4), 134.24, (phthalazine C-6), 132.28,
(phthalazine C-7), 128.97, (phenyl C-2, C-6), 127.40, (phthalazine C-5),
124.98, (phenyl C-1), 123.85, (phthalazine C-8a), 122.94, (phthalazine
C-8), 118.39, (phthalazine C-4a), 114.92, (phenyl C-3, C-5), 114.92,
(CF3), 55.78, (CH3); MS (m/z, %): 386 (M+, 100%), 253
(C14H13F3N4O, 6.12%). Anal. Calc. for C18H13F3N6O (M.W. = 386): C,
55.96; H, 3.39; N, 21.75; O, 4.14%; Found: C, 56.13; H, 3.62; N,
22.01%.
4.2.4. 6-(2-(4-Chlorobenzylidene)hydrazinyl)-3-(trifluoromethyl)-[1,2,4]
triazolo[3,4-a]phthalazine (18)
White solid (0.26 g, 67%); mp = 252–253 °C; IR (KBr) νmax cm−1
:
3363 (NH), 3068 (CH aromatic), 2937 (CH aliphatic), 1560 (C]C); 1H
NMR (DMSO‑d6) δ: 11.61 (brs, 1H), 8.63 (t, J = 7.6 Hz, 2H), 8.53 (s,
1H), 8.09 (m, 2H), 7.79 (d, J = 7.3 Hz, 2H), 7.49 (m, 2H); 13C NMR
(DMSO‑d6) δ: 150.40, (phthalazine C-1), 146.99, (triazole C-3), 144.67,
(CH), 139.39, (phthalazine C-4), 134.92, (phenyl C-1), 132.31,
(phthalazine C-6), 130.55, (phthalazine C-7), 129.47, (phenyl C-4),
127.41, (phenyl C-2, C-6), 125,10 (phenyl C-3, C-5), 123.90, (phtha-
lazine C-5), 123.05, (phthalazine C-8a), 120.49, (phthalazine C-8),
118.39, (phthalazine C-4a), 117.81, (CF3); MS (m/z, %): 392 (M++2,
7.02%) 390 (M+ 12.15%), 102 (C7H4N, 100%); Anal. Calc. for
4.2. General procedures for synthesis of 3-(trifluoromethyl/aryl)-6-(2-
(benzylidene)hydrazinyl)-[1,2,4]triazolo[3,4-a]phthalazines (15–29)
The hydrazine derivatives 14a-c (0.001 mol) were added to a so-
lution of the appropriate aldehyde (0.001 mol) in ethanol (25 mL)
containing few drops of glacial acetic acid. The mixture was heated to
120 °C for 4 h. After the reaction was complete (monitored by TLC), the
reaction mixture allowed to stand overnight. The produced solid pro-
ducts were collected by filtration, recrystallized from ethanol to obtain
the corresponding final compounds (15–29).
C17H10ClF3N6 (M.W. = 390): C, 52.25; H, 2.58; N, 21.51%; Found: C,
52.31; H, 2.62; N, 21.58%.
8

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BioorganicChemistry100(2020)103899
4.2.5. 6-(2-(2,6-Dichlorobenzylidene)hydrazinyl)-3-(trifluoromethyl)-
[1,2,4]triazolo[3,4-a]phthalazine (19)
C-4),139.82, (phthalazine C-6), 133.81, (phthalazine C-7), 131.01,
(phenyltriazole C-4), 129.67, (phenyl C-2, C-6), 128.96, (phenyltriazole
C-3, C-5), 127.36, (phenyltriazole C-1), 126.32, (phthalazine C-4),
124.81, (phenyl C-1), 124.77, (phenyltriazole C-2, C-6), 124.15,
(phthalazine C-8a), 123.39, (phthalazine C-8), 117.62, (phthalazine C-
4a), 116.38 , (phenyl C-3, C-5), 55.84 (OCH3), 21.63, (CH3); MS (m/z,
%): 408 (M+, 62.42%), 377 (C23H17N6, 25.32%), 327 (C19H15N6,
White solid (0.28 g, 67%); mp = 269–271 °C; IR (KBr) νmax cm−1
:
3182 (NH), 3008 (CH aromatic), 2924 (CH aliphatic), 1566 (C]C); 1H
NMR (DMSO‑d6) δ: 11.83 (brs, 1H), 8.78 (s, 1H), 8.64 (d, J = 7.6 Hz,
1H), 8.60 (d, J = 7.2 Hz, 1H), 8.07 (m, 2H), 7.59 (d, J = 7.6 Hz, 2H),
7.44 (t, J = 8.1 Hz, 1H); 13C NMR (DMSO‑d6) δ: 150.53, (phthalazine
C-1), 144.68, (triazole C-3), 141.84, (CH), 134.42, (phthalazine C-4),
134.37, (phenyl C-2, C-6), 132.2, (phthalazine C-6), 131.36, (phthala-
zine C-7), 130.36, (phenyl C-1), 129.9,(phenyl C-4), 125.36, (phenyl C-
3, C-5), 123.81, (phthalazine C-5), 123.1, (phthalazine C-8a), 120.34,
(phthalazine C-8), 118.4, (phthalazine C-4a), 117.67, (CF3); MS (m/z,
%): 428 (M++2, 1.57%) 426 (M+, 7.07%), 279 (C11H6F3N6, 1.71%),
171 (C9H7N4, 4.34%), 102 (C7H4N, 100%). Anal. Calc. for
27.42%), 113 (C6H13N2, 100%). Anal. Calc. for C24
H N6O
20
(M.W. = 408): C, 70.57; H, 4.94; N, 20.58; Found: C, 70.38; H, 5.12; N,
20.69%.
4.2.9. 6-(2-(4-Chlorobenzylidene)hydrazinyl)-3-(p-tolyl)-[1,2,4]triazolo
[3,4-a]phthalazine (23)
White solid (0.32 g, 79%); mp = 267–269 °C; IR (KBr) νmax cm−1
:
C
17H9Cl2F3N6 (M.W. = 425): C, 48.02; H, 2.13; N, 19.77%; Found: C,
3213 (NH), 3032 (CH aromatic), 2912 (CH aliphatic), 1597 (C]C); 1H
NMR (DMSO‑d6) δ: 11.66 (brs, 1H), 8.6 – 8.56 (m, 5H), 8.06 (t,
J = 7.5 Hz, 1H), 7.98 (t, J = 7.7 Hz, 1H), 7.86 (d, J = 8.3 Hz, 2H),
7.64 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 2.46 (s, 3H); 13C
NMR (DMSO‑d6) δ: 159.79, (phthalazine C-1), 149.17, (phenyl C-4),
147.62, (triazole C-3), 146.43, (CH), 143.11, (phthalazine C-4), 139.95,
(phthalazine C-6), 133.84, (phthalazine C-7), 131.14, (phenyltriazole
C-4), 129.80, (phenyl C-2, C-6), 129.09, (phenyltriazole C-3, C-5),
127.49, (phenyltriazole C-1), 126.45, (phthalazine C-4), 124.94,
(phenyl C-1), 124.89, (phenyltriazole C-2, C-6), 124.28, (phthalazine C-
8a), 123.52, (phthalazine C-8), 117.75, (phthalazine C-4a), 116.51 ,
(phenyl C-3, C-5), 21.73, (CH3); MS (m/z, %): 414 (M++2, 24.35%)
412 (M+, 24.57%), 363 (C22H15N6, 28.79%), 144 (C8H6N3, 100%),
125 (C7H6Cl, 23.87%). Anal. Calc. for C23H17ClN6 (M.W. = 412): C,
66.91; H, 4.15; N, 20.36%; Found: C, 66.85; H, 4.34; N, 20.18%.
48.3; H, 2.41; N, 19.54%.
4.2.6. 6-(2-Benzylidenehydrazinyl)-3-(p-tolyl)-[1,2,4]triazolo[3,4-a]
phthalazine (20)
White solid (0.26 g, 70%); mp = 242–244 °C; IR (KBr) νmax cm−1
:
3221 (NH), 3028 (CH aromatic), 2924 (CH aliphatic), 1624 (C]N),
1597 (C]C); 1H NMR (DMSO‑d6) δ: 11.45 (s, 1H), 8.61 (d, J = 7.7 Hz,
2H), 8.55 – 8.50 (m, 3H), 8.01 (t, J = 7.7 Hz, 1H), 7.95 (t, J = 7.8 Hz,
1H), 7.85 (d, J = 7.2 Hz, 2H), 7.55 (t, J = 7.5 Hz, 2H), 7.48 (d,
J = 7.3 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H), 2.44 (s, 3H); 13C NMR
(DMSO‑d6) δ: 159.68, (phthalazine C-1), 149.06, (phenyl C-4), 147.51,
(triazole C-3), 146.32, (CH), 143.00, (phthalazine C-4),139.84,
(phthalazine C-6), 133.83, (phthalazine C-7), 131.03, (phenyltriazole
C-4), 129.69, (phenyl C-2, C-6), 128.98, (phenyltriazole C-3, C-5),
127.38, (phenyltriazole C-1), 126.34, (phthalazine C-4), 124.83,
(phenyl C-1), 124.78, (phenyltriazole C-2, C-6), 124.17, (phthalazine C-
8a), 123.41, (phthalazine C-8), 117.64, (phthalazine C-4a), 116.40 ,
(phenyl C-3, C-5), 21.62, (CH3); MS (m/z, %): 378 (M+, 40.78%), 249
(C15H13N4, 100%), 172 (C9H7N4, 21.08%), 77 (C6H5, 58.36 Anal. Calc.
for C23H18N6 (M.W. = 378): C, 73; H, 4.79; N, 22.21%; Found: C,
73.24; H, 4.95; N, 22.43%.
4.2.10. 6-(2-(2,6-Dichlorobenzylidene)hydrazinyl)-3-(p-tolyl)-[1,2,4]
triazolo[3,4-a]phthalazine (24)
Yellowish white solid (0.33 g, 74%); mp = 263–265 °C; IR (KBr)
νmax cm−1: 3417 (NH), 3032 (CH aromatic), 2912 (CH aliphatic), 1512
(C]C); 1H NMR (DMSO‑d6) δ: 11.70 (brs, 1H), 8.86 (s, 1H), 8.63 – 8.53
(m, 4H), 8.05 (t, J = 7.6 Hz, 1H), 7.96 (t, J = 7.6 Hz, 1H), 7.64 (d,
J = 8.1 Hz, 2H), 7.45 (t, J = 8.1 Hz, 1H), 7.35 (d, J = 8 Hz, 2H), 2.41
(s, 3H); 13C NMR (DMSO‑d6) δ: 159.65, (phthalazine C-1), 149.03,
(phenyl C-4), 147.48, (triazole C-3), 146.29, (CH), 142.97, (phthalazine
C-4),139.81, (phthalazine C-6), 133.80, (phthalazine C-7), 131.00,
(phenyltriazole C-4), 129.66, (phenyl C-2, C-6), 128.95, (phenyltriazole
C-3, C-5), 127.35, (phenyltriazole C-1), 126.31, (phthalazine C-4),
124.80, (phenyl C-1), 124.75, (phenyltriazole C-2, C-6), 124.14,
(phthalazine C-8a), 123.38, (phthalazine C-8), 117.61, (phthalazine C-
4a), 116.37 , (phenyl C-3, C-5), 21.59, (CH3); MS (m/z, %): 449 (M+
+2, 8.88%) 447 (M+, 10.1%), 344 (C16H12Cl2N5, 100%), 212
(C10H8N6, 9.45%), 146 (C8H6N3, 14.82%). Anal. Calc. for C23H16Cl2N6
(M.W. = 447): C, 61.76; H, 3.61; N, 18.79%; Found: C, 61.59; H, 3.87;
N, 19.02%.
4.2.7. 6-(2-(4-Hydroxybenzylidene)hydrazinyl)-3-(p-tolyl)-[1,2,4]
triazolo[3,4-a]phthalazine (21)
White solid (0.30 g, 77%); mp = 245–247 °C; IR (KBr) νmax cm−1
:
3441 (OH), 3313 (NH), 3008 (CH aromatic), 2974 (CH aliphatic), 1581
(C]C); 1H NMR (DMSO‑d6) δ: 11.27 (brs, 1H), 9.95 (s, 1H), 8.62 (d,
J = 8.1 Hz, 2H), 8.58 – 8.47 (m, 2H), 8.42 (s, 1H), 8.02 (t, J = 7.6 Hz,
1H), 7.94 (t, J = 7.5 Hz, 1H), 7.7 (d, J = 8.4 Hz, 2H), 7.44 (d,
J = 8.0 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 2.44 (s, 3H); 13C NMR
(DMSO‑d6) δ: 159.63, (phthalazine C-1), 149.01, (phenyl C-4), 147.46,
(triazole C-3), 146.27, (CH), 142.95, (phthalazine C-4), 139.79,
(phthalazine C-6), 133.78, (phthalazine C-7), 130.98, (phenyltriazole
C-4), 129.64, (phenyl C-2, C-6), 128.93, (phenyltriazole C-3, C-5),
127.33, (phenyltriazole C-1), 126.29, (phthalazine C-4), 124.78,
(phenyl C-1), 124.74, (phenyltriazole C-2, C-6), 124.12, (phthalazine C-
8a), 123.36, (phthalazine C-8), 117.59, (phthalazine C-4a), 116.35 ,
(phenyl C-3, C-5), 21.57, (CH3); MS (m/z, %): 394 (M+, 44.97%), 379
(C22H15N6O, 26.67%), 245 (C15H9N4, 50.6%), 204 (C11H16N4, 100%).
Anal. Calc. for C23H18N6O (M.W. = 394): C, 70.04; H, 4.6; N, 21.31; O,
4.06%; Found: C, 70.31; H, 4.83; N, 21.47%.
4.2.11. 6-(2-Benzylidenehydrazinyl)-3-(4-chlorophenyl)-[1,2,4]triazolo
[3,4-a]phthalazine (25)
White solid (0.28 g, 72%); mp = 255–257 °C; IR (KBr) νmax cm−1
:
3263 (NH), 3021 (CH aromatic), 2893 (CH aliphatic), 1593 (C]C); 1H
NMR (DMSO‑d6) δ: 11.52 (brs, 1H), 8.74 (d, J = 8.8 Hz, 2H), 8.58 –
8.53 (m, 3H), 8.06 (t, J = 7.6 Hz, 1H), 7.98 (t, J = 7.6 Hz, 1H), 7.86 (d,
J = 7.2 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.57 (t, J = 7.6 Hz, 2H),
7.47 (t, J = 7.2 Hz, 1H); MS (m/z, %):400 (M++2, 14%), 398 (M+,
100%), 279 (C15H8 ClN4, 4.57%). Anal. Calc. for C22H15ClN6
(M.W. = 398): C, 66.25; H, 3.79; N, 21.07%; Found: C, 66.43; H, 4.01;
N, 20.91%.
4.2.8. 6-(2-(4-Methoxybenzylidene)hydrazinyl)-3-(p-tolyl)-[1,2,4]
triazolo[3,4-a]phthalazine (22)
White solid (0.31 g, 77%); mp = 248–250 °C; IR (KBr) νmax cm−1
:
3417 (NH), 3005 (CH aromatic), 2912 (CH aliphatic), 1566 (C]C); 1H
NMR (DMSO‑d6) δ: 11.57 (brs, 1H), 8.64–8.58 (m, 4H), 8.53 (s, 1H),
8.09 (t, J = 7.4 Hz, 1H), 8.02 (t, J = 7.6 Hz, 1H), 7.81 (d, J = 8.8 Hz,
2H), 7.49 (d, J = 8 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 3.86 (s, 3H), 2.47
(s, 3H); 13C NMR (DMSO‑d6) δ: 159.71, (phthalazine C-1), 149.04,
(phenyl C-4), 147.49, (triazole C-3), 146.30, (CH), 142.98, (phthalazine
4.2.12. 3-(4-Chlorophenyl)-6-(2-(4-hydroxybenzylidene)hydrazinyl)-
[1,2,4]triazolo[3,4-a]phthalazine (26)
Yellowish white solid (0.29 g, 72%); mp = 269–271 °C; IR (KBr)
νmax cm−1: 3363 (OH), 3224 (NH), 3021 (CH aromatic), 2893 (CH
9

H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
aliphatic), 1597 (C]C); 1H NMR (DMSO‑d6) δ: 11.27 (brs, 1H), 9.93 (s,
1H), 8.71 (d, J = 8.6 Hz, 2H), 8.50 (m, 2H), 8.41 (s, 1H), 8.01 (t,
J = 7.5 Hz, 1H), 7.93 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 8.3 Hz, 4H),
6.93 (d, J = 8.4 Hz, 2H); 13C NMR (DMSO‑d6) δ: 159.69, (phthalazine
C-1), 149.22, (phenyl C-4), 146.75, (triazole C-3), 146.43, (CH),
143.11, (phthalazine C-4), 134.85, (phenyltriazole C-4), 134.75,
(phthalazine C-6), 133.86, (phthalazine C-7), 131.32, (phenyl C-2, C-6),
130.68, (phenyltriazole C-3, C-5), 129.14, (phenyltriazole C-2, C-6),
129.03, (phenyltriazole C-1), 126.13, (phthalazine C-5), 124.69,
(phenyl C-1), 123.64, (phthalazine C-8a), 123.46, (phthalazine C-8),
117.64, (phthalazine C-4a), 116.63, (phenyl C-3, C-5); MS (m/z, %):
416 (M++2, 58.3%), 414 (M+, 76.97%) 321 (C16H10ClN6, 27.03%),
170 (C9H6N4, 100%). Anal. Calc. for C22H15ClN6O (M.W. = 414): C,
63.7; H, 3.64; N, 20.26; Found: C, 63.54; H, 8.23; N, 20.43%.
(phenyltriazole C-2, C-6), 126.09, (phenyltriazole C-1), 124.78, (phenyl
C-3, C-5), 123.91, (phthalazine C-5), 123.35, (phthalazine C-8a),
123.35, (phthalazine C-8), 117.34, (phthalazine C-4a); MS (m/z, %):
470 (M++2, 45.25%), 468 (M+, 100%), 321 (C16H10ClN6, 41.32%).
Anal. Calc. for C22H13Cl3N6) (M.W. = 467): C, 56.49; H, 2.8; N,
17.97%; Found: C, 56.73; H, 3.07; N, 18.23%.
4.3. Biological evaluation
4.3.1. In vitro cytotoxic activity
Four human tumor cell lines namely; Hepatocellular carcinoma
(HePG-2), Mammary gland breast cancer (MCF-7), Human prostate
cancer (PC3), and Colorectal carcinoma (HCT-116), were obtained from
VACSERA, Cairo, Egypt. The reagents RPMI-1640 medium, MTT and
DMSO, Fetal Bovine Serum (GIBCO, UK). Doxorubicin was used as a
standard anticancer 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 [45]. Cells were cultured in
RPMI-1640 medium with 10% fetal bovine serum. Penicillin (100 µg/
ml) and streptomycin (100 units/ml) antibiotics were added at 37 °C in
a 5% CO2 incubator. The cells were then seeded in a 96-well plate at a
density of 1.0x104 cells/well at 37 °C for 48 h under 5% CO2. After
incubation, the cells were treated with different concentrations of
compounds and incubated for 24 h. After 24 h of drug treatment, 20 µl
of MTT solution at 5 mg/ml was added and incubated for 4 h. 100 µl
DMSO is added into each well to dissolve the purple formazan formed.
The colorimetric assay is measured and recorded at absorbance of
570 nm using a plate reader (EXL 800, USA). The relative cell viability
in percentage was calculated. Individual IC50 values of the final target
compounds were summarized in Table 1.
4.2.13. 3-(4-Chlorophenyl)-6-(2-(4-methoxybenzylidene)hydrazinyl)-
[1,2,4]triazolo[3,4-a]phthalazine (27)
Yellowish white solid (0.31 g, 73%); mp = 274–276 °C; IR (KBr)
νmax cm−1: 3294 (NH), 3032 (CH aromatic), 2997 (CH aliphatic), 1593
(C]C); 1H NMR (DMSO‑d6) δ: 11.34 (brs, 1H), 8.74 (d, J = 8.6 Hz,
2H), 8.54 (m, 2H), 8.47 (s, 1H), 8.04 (t, J = 7.6 Hz, 1H), 7.96 (t,
J = 7.7 Hz, 1H), 7.80 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H),
7.14 (d, J = 8.6 Hz, 2H), 3.86 (s, 3H); 13C NMR (DMSO‑d6) δ: 161.15,
(phthalazine C-1), 149.12, (phenyl C-4), 146.51, (triazole C-3), 133.38,
(CH), 131.19, (phthalazine C-4), 129.16, (phenyltriazole C-4), 128.94,
(phthalazine C-6), 128.82, (phthalazine C-7), 127.62, (phenyl C-2, C-6),
127.62, (phenyltriazole C-3, C-5), 126.26, (phenyltriazole C-2, C-6),
126.26, (phenyltriazole C-1), 124.64, (phthalazine C-5), 124.64,
(phenyl C-1), 123.86, (phthalazine C-8a), 123.41, (phthalazine C-8),
117.54, (phthalazine C-4a), 115, (phenyl C-3, C-5), 55.81, (CH3); MS
(m/z, %): 430 (M++2, 22.63%), 428 (M+, 43.53%), 350 (C18H15ClN6,
22.36%), 243 (C15H7N4, 100%). Anal. Calc. for C23
H
ClN6O
4.3.2. Apoptosis assay
17
(M.W. = 428): C, 64.41; H, 4; N, 19.6; Found: C, 64.19; H, 4.18; N,
The effects of the two most potent cytotoxic compounds 16 and 26
as apoptosis inducers were analyzed using Annexin V-FITC/PI apoptosis
detection kit. HepG-2 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
HepG-2 cells were trypsinized, washed with phosphate-buffered saline
(PBS), and finally stained for 15 min at 37 °C in the dark. FACS Caliber
flow cytometer was used in analysis process [46].
19.84%.
4.2.14. 6-(2-(4-Chlorobenzylidene)hydrazinyl)-3-(4-chlorophenyl)-
[1,2,4]triazolo[3,4-a]phthalazine (28)
White solid (0.28 g, 65%); mp = 275–277 °C; IR (KBr) νmax cm−1
:
3309 (NH), 3035 (CH aromatic), 2897 (CH aliphatic), 1597 (C]C); 1H
NMR (DMSO‑d6) δ: 11.54 (brs, 1H), 8.71 (d, J = 8.4 Hz, 2H), 8.56–8.48
(m, 3H), 8.04 (t, J = 7.6 Hz, 1H), 7.96 (t, J = 7.7 Hz, 1H), 7.85 (d,
J = 8.2 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H); 13
C
4.3.3. Cell cycle analysis
NMR (DMSO‑d6) δ: 144.53, (phthalazine C-1), 143.17, (triazole C-3),
135.05, (CH), 134.8, (phthalazine C-4), 134.61, (phenyl C-4), 133.98,
(phenyl C-1), 133.92, (phenyltriazole C-4), 131.13, (phthalazine C-6),
130.08, (phthalazine C-7), 129.51, (phenyl C-2, C-6), 129.13, (phe-
nyltriazole C-3, C-5), 128.86, (phenyltriazole C-2, C-6), 128.68, (phenyl
C-3, C-5), 128.20, (phenyltriazole C-1), 126.13, (phthalazine C-5),
124.84, (phthalazine C-8a), 123.36, (phthalazine C-8), 117.81,
(phthalazine C-4a); MS (m/z, %): 435 (M++2, 36.22%), 433 (M+,
42.76%), 381 (C18H11 Cl2N6, 5.56%), 231 (C11H8ClN4, 33.89%), 215
(C10H11N6, 100%). Anal. Calc. for C22H14Cl2N6 (M.W. = 433): C,
60.98; H, 3.26; N, 19.4%; Found: C, 60.79; H, 3.45; N, 19.26%.
Hepatocellular carcinoma cells (HepG-2) were seeded at density of
2 X 105 cells/well and incubated for 24 h in six-well plates. Fetal bovine
serum (10%) was added, then, cells were incubated at 37 °C in 5%
carbon dioxide. The medium was replaced with (DMSO 1% v/v) con-
taining 2.5 µM of the test compound, then incubated for 48 h, washed
with phosphate buffered saline, fixed with 70% ethanol, rinsed with
phosphate buffered saline then stained with the DNA fluorochrome PI,
kept for 15 min at 37 °C. Then samples were analyzed with a FACS
Caliber flow cytometer [49].
4.3.4. In vitro PCAF inhibition assay
The effects of the highest potent antitumor compounds as PCAF
bromodomain inhibitors were assessed a well-established fluorescence-
based method with a fluorescence plate reader [50]. Bromosporine, a
potent PCAF inhibitor belonging to 1,2,4-triazolophthalazines, was
used as a positive control.
4.2.15. 3-(4-Chlorophenyl)-6-(2-(2,6-dichlorobenzylidene)hydrazinyl)-
[1,2,4]triazolo[3,4-a]phthalazine (29)
White solid (0.31 g, 68%); mp = 277–279 °C; IR (KBr) νmax cm−1
:
3329 (NH), 3008 (CH aromatic), 2970 (CH aliphatic), 1577 (C]C); 1H
NMR (DMSO‑d6) δ: 11.67 (brs, 1H), 8.83 (s, 1H), 8.70 (d, J = 8.4 Hz,
2H), 8.55 (t, J = 7.3 Hz, 2H), 8.04 (t, J = 7.5 Hz, 1H), 7.95 (t,
J = 7.6 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H),
7.44 (t, J = 8.1 Hz, 1H); 13C NMR (DMSO‑d6) δ: 148.84, (phthalazine
C-1), 146.5, (triazole C-3), 143.28, (CH), 141.34, (phthalazine C-4),
134.64, (phenyltriazole C-4), 134.31, (phenyl C-2, C-6), 134.01,
(phthalazine C-6), 131.1, (phthalazine C-7), 130.12, (phenyl C-1),
129.88, (phenyltriazole C-3, C-5), 129.05, (phenyl C-4), 128.92,
4.4. Docking studies
In the present work, all docking experiments were performed for all
the final target hybrid structures using Molecular Operating
Environment software (MOE2014, https://www.chemcomp.com/
Products.htm) to evaluate the free energy of binding and to explore
the binding mode toward histone acetyltransferase GCN5. Each
10

H.S. Abulkhair, et al.
BioorganicChemistry100(2020)103899
experiment used histone acetyltransferase GCN5 retrieved from the
Brookhaven Protein Databank (PDB: 5TPX, Resolution: 2.1 Å, https://
www.rcsb.org/structure/5TPX) and considered as a target for docking
simulation [23]. Firstly, the crystal structure of the protein was pre-
pared by removing water molecules and retaining the essential chain
and the ligand, (1S,2S)-N1,N1-dimethyl-N2-(3-methyl-[1,2,4]triazolo
[3,4-a]phthalazin-6-yl)-1-phenylpropane-1,2-diamine (L-45). After
that, protein was protonated, the energy was minimized, and the
binding pocket of the protein was defined. The 3D structures of the
target compounds were sketched using Chem3D 15.0 and saved in MDL
molfile format after minimizing the energy. Molecular docking of the
final target compounds was performed using a default protocol against
the target receptor. In each case, 20 docked structures were generated
using genetic algorithm searches, Affinity dG & London dG were used
for scoring 1 and Scoring 2 respectively. The London dG scoring func-
tion predicts the free energy of binding of the ligand from a given pose.
The functional form is a sum of terms:
https://doi.org/10.1021/cb500996u.
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03.011.
G = c + Eflex
+
CHB FHB
+
CM FM
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Di
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h
bond
m
lig
atomi
where C is the average gain or loss of rotational and translational en-
tropy; Eflex represents the energy upon loss of flexibility of the ligand;
CHB and FHB are the energy of an ideal hydrogen bond and the geo-
metric imperfections of hydrogen bonds respectively; CM and FM re-
present the energy of an ideal metal ligation and the measure of geo-
metric imperfections of metal ligations respectively; Di is the
desolvation energy of atom i. The most abundant low energy clusters
were selected for analysis. The protein–ligand interaction plots were
generated, using MOE module ligand-target interaction.
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Declaration of Competing Interest
[19] H. Almahli, E. Hadchity, M.Y. Jaballah, R. Daher, H.A. Ghabbour, M.M. Kabil,
N.S. Al-shakliah, W.M. Eldehna, Development of novel synthesized phthalazinone-
based PARP-1 inhibitors with apoptosis inducing mechanism in lung cancer, Bioorg.
Chem. 77 (2018) 443–456, https://doi.org/10.1016/j.bioorg.2018.01.034.
[20] O. Fedorov, H. Lingard, C. Wells, O.P. Monteiro, S. Picaud, T. Keates, C. Yapp,
M. Philpott, S.J. Martin, I. Felletar, B.D. Marsden, P. Filippakopoulos, S. Müller,
S. Knapp, P.E. Brennan, [1,2,4]Triazolo[4,3- a ]phthalazines: inhibitors of diverse
bromodomains, J. Med. Chem. 57 (2014) 462–476, https://doi.org/10.1021/
jm401568s.
The authors declared that there is no conflict of interest.
Acknowledgement
The authors would like to express their appreciation to Dr. Shaimaa
Hassanin, lecturer of English language at Horus University in Egypt for
here valuable contribution throughout proofreading the article and the
revision stages. Thanks, and profound gratitude are also paid to Heba F.
Ashour, teaching assistant of Pharmaceutical Chemistry at Horus
University in Egypt, for her great help and sincere guidance in the
molecular modeling study.
[21] D.-Q. Xue, X.-Y. Zhang, C.-J. Wang, L.-Y. Ma, N. Zhu, P. He, K.-P. Shao, P.-J. Chen,
Y.-F. Gu, X.-S. Zhang, C.-F. Wang, C.-H. Ji, Q.-R. Zhang, H.-M. Liu, Synthesis and
anticancer activities of novel 1,2,4-triazolo[3,4-a]phthalazine derivatives, Eur. J.
Med. Chem. 85 (2014) 235–244, https://doi.org/10.1016/j.ejmech.2014.07.031.
[22] A.-G.A. El-Helby, H. Sakr, R.R.A. Ayyad, K. El-Adl, M.M. Ali, F. Khedr, Design,
Synthesis, In Vitro Anti-cancer Activity, ADMET Profile and Molecular Docking of
Novel Triazolo[3,4-a]phthalazine Derivatives Targeting VEGFR-2 Enzyme,
Anticancer. Agents Med. Chem. 18 (2018) 1184–1196, https://doi.org/10.2174/
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Appendix A. Supplementary material
[23] M. Moustakim, P.G.K. Clark, L. Trulli, A.L. Fuentes de Arriba, M.T. Ehebauer,
A. Chaikuad, E.J. Murphy, J. Mendez-Johnson, D. Daniels, C.F.D. Hou, Y.H. Lin,
J.R. Walker, R. Hui, H. Yang, L. Dorrell, C.M. Rogers, O.P. Monteiro, O. Fedorov,
K.V.M. Huber, S. Knapp, J. Heer, D.J. Dixon, P.E. Brennan, Discovery of a PCAF
bromodomain chemical probe, Angew. Chemie – Int. Ed. 56 (2017) 827–831,
https://doi.org/10.1002/anie.201610816.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103899.
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