Design, synthesis, and antitumor activity of novel compounds based on 1,2,4-triazolophthalazine scaffold: Apoptosis-inductive and PCAF-inhibitory effects

Article abstract of DOI:10.1016/j.bioorg.2020.104019

The antitumor activity of newly synthesised triazolophthalazines (L-45 analogues) 10–32 was evaluated in human hepatocellular carcinoma (HePG-2), breast cancer (MCF-7), prostate cancer (PC3), and colorectal carcinoma (HCT-116) cells. Compounds 17, 18, 25, and 32 showed potent antitumor activity (IC₅₀, 2.83–13.97 μM), similar to doxorubicin (IC₅₀, 4.17–8.87 μM) and afatinib (IC₅₀, 5.4–11.4 μM). HePG2 was inhibited by compounds 10, 17, 18, 25, 26, and 32 (IC₅₀, 3.06–10.5 μM), similar to doxorubicin (IC₅₀, 4.50 μM) and afatinib (IC₅₀, 5.4 μM). HCT-116 and MCF-7 were susceptible to compounds 10, 17, 18, 25, and 32 (IC₅₀, 2.83–10.36 and 5.69–11.36 μM, respectively), similar to doxorubicin and afatinib (IC₅₀ = 5.23 and 4.17, and 11.4 and 7.1 μM, respectively). Compounds 17, 25, and 32 exerted potent activities against PC3 (IC₅₀, 7.56–12.28 μM) compared with doxorubicin (IC₅₀, 8.87 μM) and afatinib (IC₅₀ 7.7 μM). Compounds 17 and 32 were the strongest PCAF inhibitors (IC₅₀, 5.31 and 10.30 μM, respectively) and compounds 18 and 25 exhibited modest IC₅₀ values (17.09 and 32.96 μM, respectively) compared with bromosporine (IC₅₀, 5.00 μM). Compound 17 was cytotoxic to HePG2 cells (IC₅₀, 3.06 μM), inducing apoptosis in the pre-G phase and arresting the cell cycle in the G2/M phase. Molecular docking for the most active PCAF inhibitors (17 and 32) was performed.

Full text of DOI:10.1016/j.bioorg.2020.104019

Journal Pre-proofs
Design, synthesis, and antitumor activity of novel compounds based on 1,2,4-
triazolophthalazine scaffold: Apoptosis-inductive and PCAF-inhibitory effects
Abdallah Turky, Ashraf H. Bayoumi, Adel Ghiaty, Adel S. El-Azab, Alaa A.-
M. Abdel-Aziz, Hamada S. Abulkhair
PII:
S0045-2068(20)31316-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.104019
YBIOO 104019
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
13 April 2020
3 June 2020
12 June 2020
Please cite this article as: A. Turky, A.H. Bayoumi, A. Ghiaty, A.S. El-Azab, A. A.-M. Abdel-Aziz, H.S.
Abulkhair, Design, synthesis, and antitumor activity of novel compounds based on 1,2,4-triazolophthalazine
scaffold: Apoptosis-inductive and PCAF-inhibitory effects, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.104019
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Design, synthesis, and antitumor activity of novel compounds
based on 1,2,4-triazolophthalazine scaffold: Apoptosis-inductive
and PCAF-inhibitory effects
Abdallah Turkya, Ashraf H. Bayoumia, Adel Ghiatya, Adel S. El-Azabb,
Alaa A.-M. Abdel-Azizb,*, Hamada S. Abulkhaira,c,**
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Nasr City 11884,
Cairo, Egypt
bDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh
11451, Saudi Arabia
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Horus University - Egypt, International Costal
Road, New Damietta, Egypt
*Corresponding author:
E-mail address: almoenes@ksu.edu.sa (A.A.-M. Abdel-Aziz)
Address: Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O.
Box 2457, Riyadh 11451, Saudi Arabia
**Corresponding author:
E-mail address: hamadaorganic@azhar.edu.eg (H.S. Abulkhair)
Address: Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar
University, Nasr City 11884, Cairo, Egypt

2
Design, synthesis, and antitumor activity of novel compounds
based on 1,2,4-triazolophthalazine scaffold: Apoptosis-inductive
and PCAF-inhibitory effects
Abstract: The antitumor activity of newly synthesised triazolophthalazines (L-45
analogues) 10–32 was evaluated in human hepatocellular carcinoma (HePG-2), breast
cancer (MCF-7), prostate cancer (PC3), and colorectal carcinoma (HCT-116) cells.
Compounds 17, 18, 25, and 32 showed potent antitumor activity (IC50, 2.83–13.97 μM),
similar to doxorubicin (IC50, 4.17–8.87 μM) and afatinib (IC50, 5.4–11.4 μM). HePG2
was inhibited by compounds 10, 17, 18, 25, 26, and 32 (IC50, 3.06–10.5 μM), similar to
doxorubicin (IC50, 4.50 μM) and afatinib (IC50, 5.4 μM). HCT-116 and MCF-7 were
susceptible to compounds 10, 17, 18, 25, and 32 (IC50, 2.83–10.36 and 5.69–11.36 μM,
respectively), similar to doxorubicin and afatinib (IC50 = 5.23 and 4.17, and 11.4 and 7.1
μM, respectively). Compounds 17, 25, and 32 exerted potent activities against PC3 (IC50,
7.56–12.28 μM) compared with doxorubicin (IC50, 8.87 µM) and afatinib (IC50 7.7 μM).
Compounds 17 and 32 were the strongest PCAF inhibitors (IC50, 5.31 and 10.30 μM,
respectively) and compounds 18 and 25 exhibited modest IC50 values (17.09 and 32.96
μM, respectively) compared with bromosporine (IC50, 5.00 μM). Compound 17 was
cytotoxic to HePG2 cells (IC50, 3.06 μM), inducing apoptosis in the pre-G phase and
arresting the cell cycle in the G2/M phase. Molecular docking for the most active PCAF
inhibitors (17 and 32) was performed.
Keywords: Synthesis; 1,2,4-triazolophthalazine scaffold; antitumor activity; apoptosis
induction; PCAF-inhibition
1. Introduction
Cancer, which is abnormal growth of cells that invade adjacent normal cells, is the most
common cause of death worldwide1. Accordingly, the discovery of new target compounds for
the treatment of cancer is of great importance1-8. In addition, DNA methylation and histone
modification are the two accessible patterns of epigenetic processes9-10. The post-translational

3
acetylation of histone proteins contributes to epigenesis, leading to changes in protein
conformation and protein-protein interactions9. Therefore, the regulation of histone acetyl
transfer is a desirable feature for novel antitumor agents9-10. Consequently, histone
acetyltransferases (HATs) have emerged as interesting prime targets in antitumor
chemotherapy9-10. Similarly, the bromodomain family of protein interaction modules has
attracted special interest as unprecedented targets for pharmacological intervention11. Protein
dysfunction and the irregular expression of bromodomain-containing proteins have been
related to tumourigenesis11. New avenues for developing antitumor drugs have been
highlighted by the observed potent antitumor activity of selective bromodomain inhibitors12.
The HATs paralogues p300/CBP-associated factor (PCAF) bromodomain and GCN5
bromodomain-containing lysine acetyl transferase, which is highly homogeneous to PCAF, are
members of subfamily I of the phylogenetic bromodomain tree13,14. The dysregulation of PCAF
has been linked to a variety of diseases including HIV infection15, neuro-inflammation16, and
cancer17, and dysregulation of PCAF signalling pathway leads to its over-expression in
different types of cancer cells, including hepatocellular17, mammary gland18, colorectal17, and
prostate carcinoma19. In addition, targeting critical apoptosis regulators with the objective of
inducing apoptosis in tumor cells is a successful strategy for the discovery and the
development of new antitumor agents20. Recently, 1,2,4-triazolopthalazines were categorised
into a structural class comprising potent bromodomain and extra-terminal motif inhibitors14,21
.
1,2,4-Triazolopthalazines are considered a promising class of PCAF inhibitors that inhibit
histone acetyl transferases such as bromosporine (1), and L-45 (2) (Figure 1)14,22. Furthermore,
1,2,4-triazolophthalazine is considered an important pharmacophore ring system incorporated
in several antitumor agents, such as derivatives 3–5 (Figure 1)23. In the past decade, many
synthetic 1,2,4-triazolophthalazines have been reported to exhibit good antitumor activities
compared to sorafenib and 5-fluorouracil23. The antitumor effects that were induced by the

4
1,2,4-triazolophthalazine derivative 5 (Figure 1) were associated with the induction of
apoptotic cell death23. Recently, phthalazine derivatives were reported to induce apoptotic cell
death through elevation of cleaved caspase-3 expression23,24. Moreover, molecular modelling is
an important technique to study and build a structure model that incorporates all the structural
features required for the biological activities of the designed target molecules25-27. Docking is a
tool used to study the theoretical drug receptor interaction and predict the binding mode of the
ligand molecules inside the putative binding site of the target receptor or enzyme28-30
.
Based on the aforementioned rationale, we have reported the following herein: (i) The
synthesis of novel derivatives based on the 1,2,4-triazolophthalazine scaffold. The target
molecules were designed based on the structure of L-45 (2) with two main bioisosteric
modifications. Firstly, the 1,2,4-triazolophthalazine core structure was linked at C-6 with a
different amine, aminoalcohol, or hydrazine derivatives. Secondly, the methyl group at C-3 in
L-45 (2) was replaced with either trifluoromethyl or 4-substituted phenyl moieties. (ii) The
investigation of the in vitro antitumor activities of the synthesised molecules against four types
of human cancer cell: liver cancer (HePG2 cell line), colon cancer (HCT-116 cell line), breast
cancer (MCF-7 cell line), and prostate cancer (PC3 cell line). (iii) An investigation of the
structure-activity relationships (SAR) depending on in vitro antitumor activity and a
comparison of the activity of 1,2,4-triazolophthalazines incorporating aminoalcohol or
hydrazine to those of substituted amines. (iv) An assay of the induction of apoptotic cell death
of the most active derivative using the HePG2 cell line. (v) An elucidation of the inhibitory
effects on the PCAF enzyme. (vi) A molecular docking study to elucidate the virtual binding
mode of the synthesised compounds.

5
2. Results and Discussion
2.1. Chemistry
6-Chloro-[1,2,4]triazolo[3,4-a]phthalazines 9a–c were obtained from phthalic anhydride via a
sequence of reactions (Scheme 1). Phthalic anhydride was heated with hydrazine hydrate in
acetic acid, to yield 2,3-dihydrophthalazine-1,4-dione (6), which was refluxed with phosphorus
oxychloride to produce 1,4-dichlorophthalazine (7). Compound 7 was reacted with hydrazine
hydrate in ethanol to give 1-chloro-4-hydrazinylphthalazine (8), which was reacted with an
appropriate acid/acid chloride to yield the starting 6-chloro-[1,2,4]triazolo[3,4-a]phthalazines
9a–c23b,31 as key intermediates. 3,6-Disubstituted-[1,2,4]triazolo[3,4-a]phthalazines 10–32
were obtained by reacting compounds 9a–c with appropriate amines in ethanol containing
potassium carbonate (Scheme 1). Different spectroscopic analyses were performed to confirm
the structures of the newly synthesised target compounds, 10–32.
2.2. Biological evaluation
2.2.1. In vitro antitumor evaluation and SAR
The antitumor activity of compounds 10–32 was evaluated using the standard MTT assay32 in
four human tumor cell lines: hepatocellular carcinoma (HePG-2), breast cancer (MCF-7),
prostate cancer (PC3), and colorectal carcinoma (HCT-116). The results for the tested
compounds 10–32 and the reference drugs, doxorubicin and afatinib, are listed in Table 1.
Compounds 10, 17, 18, 25, 26, and 32 were the most active antitumor agents among the tested
derivatives, with IC50 value range of 2.83–18.7 μM. Compounds incorporating
triazolophthalazine bearing hydrazino fragments, such as compounds 17, 25, and 32, possessed
remarkable activity and displayed potent antitumor activity against the tested cell lines (IC50
values: 2.83–12.28 μM) compared with the reference drugs doxorubicin (IC50 values: 4.17–

6
8.87 μM), and afatinib (IC50 values: 5.4–11.4 μM). The replacement of the hydrazino moiety in
compounds 17, 25, and 32 with an aminoalcohol fragment, such as in compounds 10, 18, and
26, maintained the potent antitumor activity (IC50 values: 7.88–18.7 μM) against all tested
cancer cell lines compared with the reference drugs. Moreover, the replacement of the
aminoalcohol moiety of compound 10 into N-alkyl fragments of the same scaffold, as in
compounds 11–16, resulted in a sharp decrease in antitumor activity against all the tested
cancer cell lines, with IC50 values of 34.03–79.43 μM. Moreover, compounds 19 and 20
exhibited moderate antitumor activity (IC50 values: 26.34–35.87 μM) against certain cancer
cell lines compared with compound 18 (IC50 values: 7.88–13.97 μM). In addition, substitution
of an aminoalcohol fragment of compound 26 with a derivative bearing the N-propyl moiety,
as in compound 27, led to slight decrease in antitumor activity (IC50 values, 18.13–25.53 μM);
in contrast, weak activity was observed for the scaffold incorporating N-alkyl derivatives, such
as compounds 28–31, with IC50 values of 44.93–87.59 μM.
In the analysis of each individual cell line, HePG2 was shown to be potently inhibited by
compounds 10, 17, 18, 25, 26, and 32, with IC50 values of 9.13, 3.06, 7.88, 6.48, 10.5, and 4.89
μM, respectively, compared with reference drugs (doxorubicin IC50 = 4.50 M and afatinib
IC50= 5.4 μM). Whereas, compounds 10, 17, 18, 25, and 32 effectively inhibited MCF-7, with
IC50 values of 11.36, 5.69, 9.28, 8.63, and 6.18 μM, respectively, compared with reference
drugs (doxorubicin IC50 = 4.17 M; afatinib IC50= 7.1 μM). PC3 cell line was highly
susceptible to compounds 17, 25, and 32, with IC50 values of 7.56, 12.28, and 9.71 μM,
respectively, compared with reference drugs (doxorubicin IC50 = 8.87 M; afatinib IC50 = 7.7
μM). HCT-116 cell line was strongly inhibited by compounds 10, 17, 18, 25, and 32, with IC50
values of 10.36, 2.83, 8.29, 7.13, and 3.76 μM, respectively, compared with the reference drugs
(doxorubicin IC50 = 5.23 M; afatinib IC50 = 11.4 μM).

7
The SAR of the antitumor activity for the synthesised compounds revealed several findings. (i)
Generally, compounds 10–17, incorporating a trifluoromethyl substituent group at C-3,
compounds 18–25, incorporating 4-tolyl substituent group at C-3, and compounds 26–32,
incorporating 4-cholorophenyl substituent group at C-3 possessed equipotent antitumor activity
regardless of the substituent at C-6. The nature of the substituent group at C-3 had no impact
on the activity against the tested cancer cell lines. Exceptionally, 3-(4-chlorophenyl)-N-propyl-
[1,2,4]triazolo[3,4-a]phthalazin-6-amine (27) is approximately three-fold more potent (IC50 =
18.13–25.53 µM) against the tested cancer cell lines than the corresponding N-propyl-3-
(trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (11), which had IC50 values of
49.35–64.7 µM. In addition, compound 27 exhibited almost two-fold stronger antitumor
activity than the corresponding N-propyl-3-(p-tolyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine
(19), which had IC50 values of 31.48–46.87 M against all tested cancer cell lines. (ii)
Compounds possessing an aminoalcohol substituent at the C-6 of the triazolophthalazine core
structure, such as derivatives 10, 18, and 26, exhibited remarkable and potent antitumor
activity (IC50 = 7.88–18.70 M) compared with similar compounds bearing morpholine and
piperazine moieties at C-6, such as derivatives 15, 16, 23, 24, and 31 (IC50 values = 42.67–
89.92 M). (iii) Replacement of the aminoalcohol fragment, such as in compounds 10, 18, and
26, with a hydrazine moiety, such as in compounds 17, 25, and 32, resulted in an increase of
the antitumor activity by two- to three-fold, as indicated by the IC50 values of 2.83–12.28 µM.
(iv) Replacement of the hydrazino or amino alcohol fragments, such as in compounds 18, 25,
26, and 32, with the corresponding N-propyl or N-butyl led to compounds with moderate
antitumor activity, such as compounds 19 (IC50 = 31.48–46.87 µM), 20 (IC50 = 26.34–32.31
M), and 27 (IC50 = 18.13–25.53 M).

8
2.2.2. Apoptosis assay
2.2.2.1. Annexin V-FITC apoptosis assay
The induction of apoptosis in cancer cells is the major method through which most cytotoxic
agents kill tumor cells20. During apoptosis, the cellular changes taking place are the
translocation of phosphatidylserine (PS) through the plasma membrane from the inner to the
outer side. Annexin-V, which binds to phosphatidylserine, is used as sensitive probe for PS in
the outer side of the plasma membrane33. Cytometric quantification analysis was performed to
differentiate between the apoptosis and necrosis mode of HePG2 cell death induced by the
most active compound, compound 17, by using PI and annexin-V using the BD FACSCalibur33
(Table 2). The annexin-V/PI staining methodology was performed in HePG2 cells at mixed
molar concentration of 10 µM of with compound 17 for 24 h (Table 2). As shown in Figures 2,
and 3 and Table 2, the treatment of HePG2 cells with compound 17 for 24 h resulted in an
increase in the early apoptosis ratio (lower right quadrant of the cytogram), from 0.41% in the
control sample (DMSO) to 8.41%, and a significant increase in proportion of cells in late
apoptosis (higher right quadrant of the cytogram), from 0.29% to 15.19%. These results
indicate that compound 17 may trigger the apoptotic mechanism of programmed cell death,
rather than the necrotic pathway.
2.2.2.2 Cell cycle analysis.
Cancer cell growth inhibitors invariably cause an alteration in cell cycle distribution,
preferentially resulting in G2/M blockade34,35. The mechanism responsible for the most potent
antitumor agent, compound 17 (IC50 = 3.06 μM), was examined by the effect of this compound
on cell cycle progression of the HePG2 cell line (Figures 4, and 5 and Table 3). The cell cycle
of HePG-2 cells was assessed after treatment with compound 17 for 24 h by using propidium
iodide staining and flow cytometric analysis. As seen in Figures 4, and 5 and Table 3,

9
compared with that in control cells, compound 17 caused aggregation of cells in both the G2/M
and Pre-G1 phases, with a 20.8-fold increase in the percentage of cells in the Pre-G1 phase,
which could be indicative of apoptosis, and a 3.3-fold increase in the percentage of cells in the
G2/M phase. These increases were concomitantly accompanied by a decrease in the count of
cells in the other two phases of the cell cycle. The Pre-G1 and G2/M phase results indicated
that compound 17 induced apoptosis and arrest the cell cycle at the G2/M phase.
2.2.3. PCAF inhibition
The dysregulation of the PCAF signalling pathway leads to the over-expression in several
types of cancer cells, including liver cancer, breast cancer, colon cancer, and prostate cancer17-
19. Accordingly, the most potent antitumor compounds, 17, 18, 25, and 32, were selected for
further assessment of enzyme inhibition effect on PCAF36 (Table 4). The inhibitory effects of
the selected compounds against PCAF are presented in Table 4. The results indicated that the
selected compounds were promising PCAF-inhibitory candidates. Compound 17 possessed the
highest inhibitory effect, with an IC50 value of 5.31 μM, similar to the reference bromosporine
(IC50 value = 5.00 μM); in addition, compound 32 exhibited a powerful PCAF-inhibitory
effect, with an IC50 value of 10.30. Finally, compounds 18 and 25 showed moderate PCAF
inhibition, with IC50 values of 17.09 and 32.96 μM, respectively.
2.3. Molecular modelling study
Molecular modelling is an important tool to study theoretical SARs and the interactions of
bioactive molecules with the enzyme of interest or putative binding sites of the receptor25-30
.
The binding mode of triazolophthalazine toward the HAT GCN5 (highly homologous, with
64% identity, to PCAF)14 was performed using molecular docking implemented in the MOE
2008.10 program (Chemical Computing Group Inc., Montreal, QC, Canada)37. The docking
analysis studied the mode of interaction between the most active compounds, 17 and 32, and

10
the PfGCN5 binding site (Figure 6). The crystallographic binding site on the HAT GCN5 along
with co-crystallised L-45 ligand was derived from Protein Data Bank (PDB code: 5TPX)
(Figure 6, upper panels)14b. To validate the docking procedure, the co-crystallised L-45 ligand
was submitted for one-ligand run docking calculation. The interaction of the co-crystallised L-
45 ligand inside the putative binding pocket of PfGCN5 was governed by three types of forces:
the salt bridge among the Glu1389 residue (conserved in PCAF as Glu756) and the
dimethylamino fragment of the L-45 ligand (2.71 and 3.32 Å); the hydrogen bonding
interaction between the Asn1436 residue (conserved in PCAF as Asn803) and the triazole part
of triazolophthalazine (3.71 Å); and a combination of three interactions, the pi-pi stacking
interaction between Tyr1442 residues (conserved in PCAF as Tyr809) and the pyridazine
fragment of the triazolophthalazine core structure, the CH---pi interaction between the
phthalazine ring and amino acid residue Ala1390, and the CH---O interaction of the
dimethylamino moiety with amino acid residues Pro1384 (3.38 Å) and Glu1389 (3.23 Å)
(Figure 4, upper panels).
The result of the docking calculation of compound 17 are shown in Figure 6 (lower left panel),
which exhibited a binding mode almost similar to that of the co-crystallised L-45 ligand. The
triazole fragment formed a hydrogen bond with the Asn1436 residue (2.94 Å), whereas the
pyridazine ring adopted hydrophobic recognition at the binding cavity through pi-pi stacking,
with Tyr1442 similar to the bound inhibitor L-45. Moreover, the hydrazino moiety at C-6 of
the triazolophthalazine core structure was adapted to interact with the amino acid residue
Glu1389 by hydrogen bond formation (3.09 Å). In addition, the trifluoromethyl moiety adopted
nonclassical hydrogen bonds with the amino acid residue Phe1381 (3.06, and 3.26 Å) (Figure 6,
lower left panel).
In contrast, compound 32 showed binding mode in a different manner than compound 17
(Figure 6, lower right panel). The result of the docking calculation of compound 32 revealed

11
that the triazole fragment formed a hydrogen bond with the Tyr1442 residue (2.92 Å), whereas
the pyridazine ring adapted to a hydrophobic recognition at the binding cavity through pi-pi
stacking with Tyr1442. Meanwhile, the hydrazino moiety at the C-6 of the triazolophthalazine
core structure formed hydrogen bonds with the amino acid residue Pro1380 (3.02 Å). In
addition, the phthalazine moiety adapted a nonclassical CH---O hydrogen bond with the amino
acid residue Asn1436 (3.83 Å) (Figure 6, lower right panel).
Conclusion
The antitumor activities of new synthesised triazolophthalazines derivatives 10–32 against four
human cancer cell lines (HePG2, MCF-7, PC3, and HCT-116) was evaluated by using a
standard MTT assay and compared with the reference drug doxorubicin and afatinib.
Compounds 10, 17, 18, 25, 26, and 32 exerted the most potent antitumor activities against
human cancer cell lines (HePG2, MCF-7, PC3, and HCT-116) with IC50 values of 2.83–18.7
μM, compared with doxorubicin (IC50 values of 4.17–8.87 μM) and afatinib (IC50 values of
5.4–11.4 μM). The hydrazinyl-triazolophthalazine derivatives 17, 25, and 32 displayed the
most potent antitumor activity against the four tested cell lines (IC50 values of 2.83–12.28 μM),
which was comparable with that of doxorubicin and afatinib. Triazolophthalazines
incorporating aminoalcohol in compounds 10, 18, and 26 exhibited potent antitumor activity
(IC50 values of 7.88–18.7 μM) against the tested cancer cell lines, compared with doxorubicin
and afatinib. In contrast, the N-alkyl-triazolophthalazines 11–16 demonstrated a sharp
reduction in antitumor activity against the tested cancer cell lines, with IC50 values of 34.03–
79.43 μM. Compounds 17, 18, 25, and 32, showed the most potent antitumor activity, and were
selected for further assessment of their PCAF-inhibitory effects. Compounds 17 and 32
possessed the highest PCAF-inhibitory effect, with IC50 values of 5.31 and 10.30 μM,
respectively, compared with bromosporine (IC50 = 5.00 μM). The strength of the PCAF-
inhibitory effects indicated the importance of the halogen atoms in compounds 17 and 32. In

12
contrast, compounds 18 and 25 exhibited an adequate PCAF-inhibitory effect, with IC50 values
of 17.09 and 32.96 μM, respectively, compared with bromosporine. Compound 17 increased
the percentage of cells in early apoptosis from 0.41% to 8.41% and significantly increased the
percentage of cells in late apoptosis from 0.29% to 15.19%. Cell cycle analysis indicated that
the percentage of apoptotic cells in the Pre-G1 phase was increased from 1.27% to 26.51% and
that the percentage of cells in the G2/M phase was increased from 12.81% to 42.26%. The
results of the docking experiments supported the superior activities of compounds 17 and 32 by
indicating potential binding interactions with the active site of HAT GCN5 (highly
homologous to PCAF) and exhibited similar binding characteristics to that of the bound
inhibitor L-45.
3. Materials and methods
3.1. General experimental procedures
Melting points were measured by using an electrothermal apparatus (Stuart SMP30) apparatus
and were uncorrected. IR spectra were recorded on a Pye Unicam SP 1000 IR
spectrophotometer at the Pharmaceutical Analytical Unit, Faculty of Pharmacy, Al-Azhar
University. 1H NMR spectra were recorded 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
and CDCl3 at 400 MHz on a Joel ECA spectrometer at NMR Lab, Microanalytical Unit,
Faculty of Pharmacy, Mansoura University. 13C NMR spectra were determined at 100 MHz in
DMSO-d6 on a Varian Mercury NMR spectrometer. Chemical shifts were reported relative to
that of the solvent, with TMS as an internal standard. Mass spectra and microanalyses were
performed at The Regional Center for Mycology Biotechnology, Al-Azhar University, Cairo,
Egypt. The progress of the reactions was monitored by TLC using TLC sheets precoated with a
UV fluorescent silica gel Merck 60 F254 plates and was visualised using a UV lamp, with

13
different solvents as the mobile phases. The starting reagents, phthalic anhydride, hydrazine
hydrate, phosphorus oxychloride, acetic anhydride, and acid chloride derivatives, were
purchased from Aldrich chemical company and were used as received. Compounds 9a–c were
synthesised in accordance with reported procedures23b,31
.
3.2. Synthesis procedures
3.2.1. General procedure for synthesis of 3,6-disubstituted-[1,2,4]triazolo[3,4-
a]phthalazines 10–32
A suitable 6-chloro-3-substituted-[1,2,4]triazolo[3,4-a]phthalazine derivative 9a–c (0.001 mol)
was dissolved in ethanol (20 mL) containing the appropriate amine (0.001 mol) or hydrazine (5
mL) and potassium carbonate (0.5 g). The mixture was then refluxed for 6–10 h. After
completion reaction (as monitored by TLC), the mixture was allowed to cool; water (200 mL)
was added, and the mixture allowed to stand overnight. The produced solid was collected by
filtration and recrystallised from dichloromethane to afford the corresponding final target
compounds (10–32).
3.2.1.1. 2-((3-(Trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-yl)amino)ethan-1-ol (10).
White solid (0.21 g, 73%); melting point [MP] = 262°C–264°C; IR (KBr) νmax cm-1 3305 (NH),
1
3194 (OH); H NMR (DMSO-d6) δ: 8.49 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.05
(brs, 1H), 8.01 (t, J = 7.5 Hz, 1H), 7.95 (t, J = 7.7 Hz, 1H), 4.83 (t, J = 5.6 Hz, 1H), 3.73 (q, J =
5.9 Hz, 2H), 3.50 (q, J = 5.8 Hz, 2H); 13C NMR (DMSO-d6) δ: 153.0, 144.2, 138.8, 133.8,
132.0, 124.8, 123.6, 122.5, 119.3, 117.8, 58.5, 44.7; MS (m/z, %): 297 (M+, 100%), 247
(C8H8F3 N5O, 41.12%). Anal. Calc. for: (C12H10F3N5O) (M.W. = 297): C, 48.49%; H, 3.39%;
N, 23.56%; Found: C, 48.21%; H, 3.62%; N, 23.34%.
3.2.1.2. N-Propyl-3-(trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (11). White

14
solid (0.22 g, 77%); MP = 269°C–271°C; IR (KBr) νmax cm-1 3317 (NH); 1H NMR (DMSO-d6)
δ: 8.47 (d, J = 8.0 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.02 (brs, 1H), 7.98 (t, J = 7.5 Hz, 1H),
7.92 (t, J = 8.0 Hz, 1H), 3.35 (t, J = 8.0 Hz, 2H), 1.7 (h, J = 7.3 Hz, 2H), 0.95 (t, J = 7.4 Hz,
3H); 13C NMR (DMSO-d6) δ: 152.8, 144.1, 138.8, 138.4, 133.7, 132.0, 124.76, 123.6, 122.5,
117.8, 43.6, 21.3, 12.0; MS (m/z, %): 295 (M+, 36.07%), 252 (C10H5F3N5, 28.77%), 169
(C9H5N4, 69.4%), 107 (C7H9N, 100%). Anal. Calc. for: (C13H12F3N5) (M.W. = 295): C,
52.88%; H, 4.1%; N, 23.72%; Found: C, 53.14%; H, 4.29%; N, 23.56%.
3.2.1.3. N-Butyl-3-(trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (12). White
solid (0.22 g, 72%); MP = 263ºC–265°C; IR (KBr) νmax cm-1 3305 (NH); 1H NMR (DMSO-d6)
δ: 8.47 (d, J = 7.6 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.07–7.95 (m, 2H), 7.93 (t, J = 7.8 Hz,
1H), 3.46–3.37 (m, 2H), 1.7 (p, J = 7.2 Hz, 2H), 1.39 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.3 Hz,
3H); 13C NMR (DMSO-d6) δ: 152.9, 144.1, 138.8, 133.7, 132.0, 124.7, 122.5, 120.5, 119.3,
117.8, 41.4, 30.1, 20.2, 14.1; MS (m/z, %): 309 (M+, 100%), 246 (C9H11F3N5, 10.47%). Anal.
Calc. for: (C14H14F3N5) (M.W. = 309): C, 54.37%; H, 4.56%; N, 22.64%; Found: C, 54.25%;
H, 4.78%; N, 22.81%.
3.2.1.4. N-Cyclohexyl-3-(trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (13).
1
White solid (0.26 g, 78%); MP = 269ºC–271°C; IR (KBr) νmax cm-1 3309 (NH); H NMR
(DMSO-d6) δ: 8.55–8.46 (m, 2H), 8.01 (t, J = 8.0 Hz, 1H), 7.94 (t, J = 8.0 Hz, 1H), 7.64 (d, J =
8.0 Hz, 1H), 3.83 (m, 1H), 2.08 (d, J = 12.0 Hz, 2H), 1.80 (t, J = 12.0 Hz, 2H), 1.65 (d, J =
12.5 Hz, 1H), 1.48 – 1.26 (m, 4H), 1.30 – 1.14 (m, 1H); 13C NMR (DMSO-d6) δ: 152.0, 144.1,
138.8, 133.8, 131.9, 125.1, 123.6, 122.6, 119.3, 117.8, 51.1, 31.8, 25.9, 25.4; MS (m/z, %): 335
(M+, 40.75%), 252 (C10H5F3N5, 28.77%), 129 (C8H5N2, 8.04%), 84 (C6H11, 10.86%), 55
(C2H3N2, 100%). Anal. Calc. for: (C16H16F3N5) (M.W. = 335): C, 57.31%; H, 4.81%; N,
20.89%; Found: C, 57.31%; H, 4.97%; N, 20.61%.

15
3.2.1.5. N,N-Dimethyl-3-(trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (14).
1
White solid (0.22 g, 79%); MP = 267ºC–269°C; H NMR (DMSO-d6) δ: 8.55 (d, J = 8.0 Hz,
1H), 8.27 (d, J = 8.0 Hz, 1H), 8.05 (t, J = 7.6 Hz, 1H), 7.96 (t, J = 7.3 Hz, 1H), 3.11 (s, 6H);
13C NMR (DMSO-d6) δ: 159.3, 144.8, 139.0, 138.7, 134.0, 131.9, 128.1, 123.9, 120.7, 117.7,
42.7; MS (m/z, %): 281 (M+, 78.57%), 232 (C8H9F3N5, 100%). Anal. Calc. for: (C12H10F3N5)
(M.W. = 281): C, 51.25%; H, 3.58%; N, 24.9%; Found: C, 51.49%; H, 3.67%; N, 24.78%.
3.2.1.6. 6-Morpholin-4-yl-3-(trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazine (15). White
1
solid (0.23 g, 72%); MP = 265ºC–267°C; H NMR (DMSO-d6) δ: 8.57 (d, J = 7.6, Hz, 1H),
8.23 (d, J = 8.0 Hz, 1H), 8.07 (t, J = 7.6 Hz, 1H), 7.97 (t, J = 7.6 Hz, 1H), 3.88 (t, J = 4.0 Hz,
4H), 3.39 (t, J = 4.0 Hz, 4H); 13C NMR (DMSO-d6) δ: 159.0, 144.9, 138.9, 134.4, 132.2, 127.6,
124.0, 123.7, 120.5, 117.6, 66.0, 51.7; MS (m/z, %): 323 (M+, 100%), 280 (C12H9F3N5, 3.52%).
Anal. Calc. for: (C14H12F3N5O) (M.W. = 323): C, 52.02%; H, 3.74%; N, 21.66%; Found: C,
52.58%; H, 3.86%; N, 21.84%.
3.2.1.7. 6-(Piperazin-1-yl)-3-(trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazine (16). White
solid (0.24 g, 76%); MP = 258ºC–260°C; IR (KBr) νmax cm-1 3259 (NH); 1H NMR (DMSO-d6)
δ: 8.57 (d, J = 7.8 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.05 (t, J = 7.5 Hz, 1H), 7.96 (t, J = 7.5
Hz, 1H), 4.16 (s, 1H), 2.97 (t, J = 7.8, 4H), 2.51 (t, J = 7.8, 4H); 13C NMR (DMSO-d6) δ:
159.3, 144.8, 139.3, 138.9, 134.1, 132.1, 127.5, 123.9, 123.6, 117.6, 52.6, 45.4; MS (m/z, %):
322 (M+, 5.52%), 239 (C10H6F3N4, 12.04%), 56 (C3H6N, 100%). Anal. Calc. for: (C14H13F3N6)
(M.W. = 322): C, 52.17; H, 4.07; N, 26.08%; Found: C, 52.41%; H, 4.28%; N, 25.85%.
3.2.1.8. 6-Hydrazinyl-3-(trifluoromethyl)-[1,2,4]triazolo[3,4-a]phthalazine (17). White solid
1
(0.19 g, 69%); MP = 157ºC–159°C; IR (KBr) νmax cm-1 3294, 3109, (NH); H NMR (DMSO-
d6) δ: 9.40 (brs, 1H), 8.48 (d, J = 7.8 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H), 7.99 (t, J = 7.6 Hz, 1H),
13
7.90 (t, J = 7.7 Hz, 1H), 4.50 (s, 2H); C NMR (DMSO-d6) δ: 154.4, 144.4, 138.9, 133.7,

16
132.0, 124.1, 123.5, 122.1, 118.3, 117.7; MS (m/z, %): 268 (M+, 100%), 291 (C6H6F3N6,
10.21%). Anal. Calc. for: (C10H7F3N6) (M.W. = 268): C, 44.78%; H, 2.63%; N, 31.34%;
Found: C, 45.86%; H, 2.87%; N, 31.08%.
3.2.1.9. 2-((3-(p-Tolyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-yl)amino)ethan-1-ol (18). White
solid (0.21 g, 65%); MP = 251ºC–253°C; IR (KBr) νmax cm-1 3337 (NH), 3198 (OH); 1H NMR
(DMSO-d6) δ: 8.48 (d, J = 7.6 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.35 (d, J = 7.7 Hz, 2H), 7.96
(t, J = 7.6 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H), 7.7 (brs, 1H), 7.40 (d, J = 7.7 Hz, 2H), 4.84 (t, J =
5.6 Hz, 1H), 3.77 (q, J = 5.9 Hz, 2H), 3.57 (q, J = 5.8 Hz, 2H), 2.41 (s, 3H); 13C NMR
(DMSO-d6) δ: 152.0, 147.3, 142.6, 139.4, 133.3, 130.7, 129.6, 127.2, 124.8, 124.6, 123.7,
123.1, 118.6, 58.9, 44.9, 21.4; MS (m/z, %): 319 (M+, 95.65%), 251 (C14H13N5, 100%). Anal.
Calc. for: (C18H17N5O) (M.W. = 319): C, 67.70%; H, 5.37%; N, 21.93%; Found: C, 67.49%;
H, 5.6%; N, 21.68%.
3.2.1.10. N-Propyl-3-(p-tolyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (19). White solid
1
(0.24 g, 77%); MP = 251ºC–253°C; IR (KBr) νmax cm-1 3263 (NH); H NMR (DMSO-d6) δ:
8.47 (d, J = 7.8 Hz, 1H), 8.37 (d, J = 7.9 Hz, 2H), 7.95 (t, J = 7.6 Hz, 1H), 7.86 (t, J = 7.6 Hz,
1H), 7.82 (brs, 1H), 7.38 (d, J = 7.9 Hz, 2H), 3.44 (q, J= 6.6, 2H), 2.4 (s, 3H), 1.79 (h, J = 7.4
13
Hz, 2H), 1.01 (t, J = 7.4 Hz, 3H); C NMR (DMSO-d6) δ: 151.8, 147.2, 142.6, 139.4, 133.2,
130.7, 129.5, 127.1, 124.9, 124.5, 123.7, 123.1, 118.6, 43.9, 21.4, 12.2; MS (m/z, %): 317 (M+,
100%), 260 (C15H10N5, 19.21%). Anal. Calc. for: (C19H19N5) (M.W. = 317): C, 71.9%; H,
6.03%; N, 22.07%; Found: C, 71.76%; H, 6.21%; N, 21.89%.
3.2.1.11. N-Butyl-3-(p-tolyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (20). White solid (0.26
g, 80%); MP = 264ºC–266°C; IR (KBr) νmax cm-1 3317 (NH); 1H NMR (DMSO-d6) δ: 8.47 (d,
J = 7.8 Hz, 1H), 8.37 (t, J = 7.2 Hz, 3H), 7.96 (t, J = 7.6 Hz, 1H), 7.86 (t, J = 7.8 Hz, 1H), 7.8
(brs, 1H), 7.38 (d, J = 7.9 Hz, 2H), 3.48 (q, J = 6.7 Hz, 2H), 2.4 (s, 3H), 1.76 (p, J = 7.4 Hz,

17
13
2H), 1.74 (h, J = 7.5 Hz, 2H), 0.9 (t, J= 7.4 Hz, 3H); C NMR (DMSO-d6) δ: 151.8, 147.2,
142.6, 139.5, 133.3, 130.8, 129.5, 127.1, 124.9, 124.6, 123.7, 123.1, 118.6, 41.8, 30.2, 21.4,
20.5, 14.2; MS (m/z, %): 331 (M+, 100%), 259 (C15H9N5, 31.92%). Anal. Calc. for: (C20H21N5)
(M.W. = 331): C, 72.48%; H, 6.39%; N, 21.13%; Found: C, 72.71%; H, 6.53%; N, 21.05%.
3.2.1.12. N-Cyclohexyl-3-(p-tolyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (21). White solid
(0.28 g, 79%); MP = 252ºC–254°C; 1H NMR (DMSO-d6) δ: 8.47 (d, J = 8.1 Hz, 2H), 8.40 (d, J
= 8.0 Hz, 2H), 7.96 (t, J = 7.6 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.44 (d, J = 7.0 Hz, 1H), 7.39
(d, J = 8.0 Hz, 2H), 3.88 (s, 1H), 2.41 (s, 3H), 2.18 (s, 2H), 1.86 (s, 2H), 1.73 (d, J = 10.8 Hz,
1H), 1.45 (q, J = 10.8 Hz, 4H), 1.26 (s, 1H); 13C NMR (DMSO-d6) δ: 150.9, 147.1, 142.5,
139.4, 133.2, 130.6, 129.5, 127.0, 124.9, 124.8, 123.8, 123.0, 118.5, 51.6, 32.1, 26.0, 25.6,
21.4; MS (m/z, %): 357 (M+, 100%), 129 (C8H5N2, 28.06%). Anal. Calc. for: (C22H23N/)
(M.W. = 357): C, 73.92%; H, 6.49%; N, 19.59%; Found: C, 73.68%; H, 6.63%; N, 19.75%.
3.2.1.13. N,N-Dimethyl-3-(p-tolyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (22). White solid
(0.21 g, 69%); MP = 247ºC–249°C; 1H NMR (DMSO-d6) δ: 8.54 (d, J = 4.0 Hz, 1H), 8.33 (d,
J = 8.0 Hz, 2H), 8.22 (d, J = 8.0 Hz, 1H), 8 (t, J = 8.0 Hz, 1H), 7.89 (t, J = 8.0 Hz, 1H), 7.42
13
(d, J = 8.0 Hz, 2H), 3.1 (s, 6H), 2.41 (s, 3H); C NMR (DMSO-d6) δ: 158.2, 147.7, 143.1,
139.8, 133.5, 130.7, 129.7, 127.6, 127.4, 124.6, 124.3, 123.4, 119.9, 42.9, 21.4; MS (m/z, %):
303 (M+, 100%), 143 (C8H5N3, 11.88%). Anal. Calc. for: (C18H17N5) (M.W. = 303): C,
71.27%; H, 5.65%; N, 23.09%; Found: C, 71.5%; H, 5.89%; N, 22.86%.
3.2.1.14. 4-(3-(p-Tolyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-yl)morpholine (23). White solid
(0.25 g, 70%); MP = 258ºC–260°C; 1H NMR (DMSO-d6) δ: 8.54 (d, J = 7.8 Hz, 1H), 8.29 (d, J
= 8.0 Hz, 2H), 8.20 (d, J = 8.1 Hz, 1H), 8.02 (t, J = 7.6 Hz, 1H), 7.89 (t, J = 7.7 Hz, 1H), 7.42
13
(d, J = 8.0 Hz, 2H), 3.91 (t, J = 4.5 Hz, 4H), 3.39 (t, J = 4.5 Hz, 4H), 2.41 (s, 3H); C NMR
(DMSO-d6) δ: 157.6, 148.0, 143.1, 139.9, 133.8, 131.0, 129.7, 127.4, 127.1, 124.6, 124.1,

18
123.4, 119.6, 66.3, 51.8, 21.4; MS (m/z, %): 345 (M+, 100%), 330 (C19H16N5, 5.56%). Anal.
Calc. for: (C20H19N5O) (M.W. = 345): C, 69.55%; H, 5.54%; N, 20.28%; Found: C, 69.76%;
H, 5.78%; N, 20.51%.
3.2.1.15. 6-(Piperazin-1-yl)-3-(p-tolyl)-[1,2,4]triazolo[3,4-a]phthalazine (24). Yellowish
1
white solid (0.24 g, 73%); MP = 258ºC–260°C; IR (KBr) νmax cm-1 3305 (NH); H NMR
(DMSO-d6) δ: 8.53 (d, J = 7.8 Hz, 1H), 8.30 (d, J = 8.0 Hz, 2H), 8.15 (d, J = 8.0 Hz, 1H), 8 (t,
J = 7.6 Hz, 1H), 7.89 (t, J = 7.7 Hz, 1H), 7.42 (d, J = 7.9 Hz, 2H), 3.33 (s, 4H), 3.02 (t, J = 4.7
Hz, 4H), 2.41 (s, 3H); 13C NMR (DMSO-d6) δ: 158.1, 147.9, 143.1, 139.9, 133.6, 130.9, 129.6,
127.4, 127.1, 124.5, 124.1, 123.4, 119.8, 52.6, 45.6, 21.4; MS (m/z, %): 344 (M+, 100%), 259
(C16H11N4, 1.81%). Anal. Calc. for: (C20H20N6) (M.W. = 344): C, 69.75%; H, 5.85%; N,
24.4%; Found: C, 69.61%; H, 6.09%; N, 24.17%.
3.2.1.16. 6-Hydrazinyl-3-(p-tolyl)-[1,2,4]triazolo[3,4-a]phthalazine (25). White solid (0.27 g,
78%); MP = 158ºC–160°C; 1H NMR (DMSO-d6) δ: 9.09 (s, 1H), 8.48 (d, J = 7.6 Hz, 2H), 8.44
(d, J = 7.6 Hz, 1H), 8.31 (d, J = 8.2 Hz, 1H), 7.96 (t, J = 7.4 Hz, 1H), 7.83 (t, J = 7.4 Hz, 1H),
7.38 (d, J = 8.0 Hz, 2H), 4.53 (brs, 2H), 2.41 (s, 3H); 13C NMR (DMSO-d6) δ: 154.1, 147.7,
142.9, 139.6, 133.5, 131.0, 129.7, 127.6, 124.5, 124.1, 123.4, 123.2, 117.8, 21.4; Anal. Calc.
for: (C16H14N6) (M.W. = 290): C, 66.19%; H, 4.86%; N, 28.95%; Found: C, 66.25%; H,
4.78%; N, 29.04%.
3.2.1.17. 2-((3-(4-Chlorophenyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-yl)amino)ethan-1-ol (26).
White solid (0.28 g, 67%); MP = 270ºC–272°C; IR (KBr) νmax cm-1 3329 (NH), 3201 (OH); 1H
NMR (DMSO-d6) δ: 8.49 (d, J = 8.0 Hz, 3H), 8.41 (d, J = 7.8 Hz, 1H), 7.97 (t, J = 7.6 Hz, 1H),
7.88 (t, J = 7.4 Hz, 1H), 7.83 (brs, 1H), 7.66 (d, J = 8.1 Hz, 2H), 4.83 (t, J = 5.6 Hz, 1H), 3.78
13
(q, J = 5.9 Hz, 2H), 3.58 (q, J = 5.8 Hz, 2H); C NMR (DMSO-d6) δ: 152.2, 146.3, 142.9,
134.4, 133.4, 131.0, 129.2, 128.8, 126.4, 124.6, 123.5, 123.2, 118.6, 58.8, 44.9; MS (m/z, %):

19
341 (M++2, 36.01%), 339 (M+, 100%). Anal. Calc. for: (C17H14ClN5O) (M.W. = 339): C,
60.09%; H, 4.15%; N, 20.61%: C, 59.82%; H, 4.36%; N, 20.49%.
3.2.1.18. 3-(4-Chlorophenyl)-N-propyl-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (27). White
solid (0.27 g, 80%); MP = 267ºC–269°C; IR (KBr) νmax cm-1 3263 (NH); 1H NMR (DMSO-d6)
δ: 8.51 (d, J = 8.7 Hz, 2H), 8.49 (d, J = 10.0 Hz, 1H), 8.40 (d, J = 7.96 Hz, 1H), 7.98 (t, J = 7.6
Hz, 1H), 7.93–7.88 (m, 2H), 7.66 (d, J = 8.4 Hz, 2H), 3.45 (q, J = 6.6 Hz, 2H), 1.78 (h, J = 7.5
Hz, 2H), 1.02 (t, J = 7.4 Hz, 3H); MS (m/z, %): 339 (M++2, 49.15%), 337 (M+, 72.17%,), 294
(C15H9ClN5, 5.07%), 279 (C15H8ClN4, 15.59%), 102 (C8H6, 100%). Anal. Calc. for:
(C18H16ClN5) (M.W. = 337): C, 64%; H, 4.77%; N, 20.73%; Found: C, 63.83%; H, 4.91%; N,
20.89%.
3.2.1.19. N-Benzyl-3-(4-chlorophenyl)-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (28). White
solid (0.27 g, 70%); MP = 271ºC–273°C; IR (KBr) νmax cm-1 3348 (NH); 1H NMR (DMSO-d6)
δ: 8.56 (brs, 1H), 8.45 (d, J = 8.0 Hz, 2H), 8.19 (d, J = 8.4 Hz, 2H), 7.95 (t, J = 7.6 Hz, 1H),
7.87 (t, J = 7.5 Hz, 1H), 7.48 (dd, J = 10.5, 8.1 Hz, 4H), 7.36 (t, J = 7.5 Hz, 2H), 7.24 (t, J =
7.4 Hz, 1H), 4.66 (d, J = 5.4 Hz, 2H); MS (m/z, %): 387 (M++2, 13.66%), 385 (M+, 41.32%),
347 (C19H14ClN5, 100%). Anal. Calc. for: (C22H16ClN5) (M.W. = 385): C, 68.48%; H, 4.18%;
N, 18.15%; Found: C, 68.31%; H, 4.42%; N, 17.98%.
3.2.1.20. 3-(4-Chlorophenyl)-N-cyclohexyl-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (29).
1
White solid (0.28 g, 74%); MP = 261ºC–263°C; IR (KBr) νmax cm-1 3348 (NH); H NMR
(DMSO-d6) δ: 8.50 (dd, J = 8.6, 8.2 Hz, 4H), 7.97 (t, J = 7.6 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H),
7.65 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 6.4 Hz, 1H), 3.88 (s, 1H), 2.18 (s, 2H), 1.83 (s, 2H), 1.71
(d, J = 12.9 Hz, 1H), 1.43 (m, 4H), 1.25 (s, 1H); 13C NMR (DMSO-d6) δ: 151.1, 146.1, 142.8,
134.5, 133.4, 130.9, 129.0, 128.7, 126.4, 124.8, 123.6, 120.2, 118.6, 51.6, 32.0, 26.0, 25.5; MS
(m/z, %): 379 (M++2, 34.28%), 377 (M+, 100%). Anal. Calc. for: (C21H20ClN5) (M.W. = 377):

20
C, 66.75%; H, 5.34%; N, 18.53%; Found: C, 66.59%; H, 5.47%; N, 18.61%.
3.2.1.21. 3-(4-Chlorophenyl)-N,N-dimethyl-[1,2,4]triazolo[3,4-a]phthalazin-6-amine (30).
1
White solid (0.21 g, 65%); MP = 275ºC–277°C; H NMR (DMSO-d6) δ: 8.52 (d, J = 7.6 Hz,
1H), 8.45 (d, J = 8.8 Hz, 2H), 8.22 (d, J = 8.0 Hz, 1H), 7.99 (t, J = 7.6 Hz, 1H), 7.89 (t, J = 7.2
13
Hz, 1H), 7.67 (d, J = 8.8 Hz, 2H), 3.11 (s, 6H); C NMR (DMSO-d6) δ: 158.4, 146.8, 143.5,
134.8, 133.7, 131.0, 129.4, 129.1, 127.8, 125.9, 124.5, 123.5, 120.0, 42.9; MS (m/z, %): 325
(M++2, 45.35%), 323 (M+ 100%). Anal. Calc. for: (C17H14ClN5) (M.W. = 323): C, 63.06%; H,
,
4.36%; N, 21.63%; Found: C, 62.97%; H, 4.53%; N, 21.8%.
3.2.1.22. 6-Morpholin-4-yl-3-(4-chlorophenyl)-[1,2,4]triazolo[3,4-a]phthalazine (31). White
1
solid (0.25 g, 70%); MP = 263ºC–265°C; H NMR (DMSO-d6) δ: 8.55 (d, J = 7.2 Hz, 1H),
8.43 (d, J = 8.4 Hz, 2H), 8.21 (d, J = 8.0 Hz, 1H), 8.03 (t, J = 7.7 Hz, 1H), 7.93 (t, J = 8.4 Hz,
1H), 7.69 (d, J = 8.4 Hz, 2H), 3.9 (t, J = 4.5 Hz, 4H), 3.45 (t, J = 5.0 Hz, 4H); 13C NMR
(DMSO-d6) δ: 157.9, 147.1, 143.5, 135.0, 134.1, 131.3, 129.4, 129.3, 127.3, 125.8, 124.5,
123.6, 119.8, 66.3, 51.8; MS (m/z, %): 367 (M++2, 27.25%), 365 (M+, 100%). Anal. Calc. for:
(C19H16ClN5O) (M.W. = 365): C, 62.38%; H, 4.41%; N, 19.14%; Found: C, 62.51%; H,
4.53%; N, 19.4%.
3.2.1.23. 3-(4-Chlorophenyl)-6-hydrazinyl-[1,2,4]triazolo[3,4-a]phthalazine (32). White solid
1
(0.23 g, 73%); MP = 151ºC–153°C; IR (KBr) νmax cm-1 3275 (NH), 3101, 3070 (NH2); H
NMR (DMSO-d6) δ: 9.15 (brs, 1H), 8.59 (d, J = 8.6 Hz, 2H), 8.47 (d, J = 7.8 Hz, 1H), 8.31 (d,
J = 8.0 Hz, 1H), 7.96 (t, J = 7.6 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 4.53
(brs, 2H); 13C NMR (DMSO-d6) δ: 154.1, 146.4, 143.2, 134.4, 133.4, 131.0, 129.1, 129.1,
126.2, 124.1, 123.3, 123.1, 117.7; MS (m/z, %): 312 (M++2, 2.43%), 310 (M+, 8.12%), 102
(C7H4N, 100%). Anal. Calc. for: (C15H11ClN6) (M.W. = 310): C, 57.98%; H, 3.57%; N,
27.05%; Found: C, 58.14%; H, 3.78%; N, 26.89%.

21
3.3. Biological evaluation
3.3.1. In vitro antitumor activity
The antitumor activity was evaluated by using the tetrazolium salt 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in accordance with a reported
method32.
3.3.2. Apoptosis assay
The induction of apoptosis was evaluated by using the well-established method of the Annexin
5-FITC/PI apoptosis detection kit. Hepatocellular carcinoma cells (HepG-2) cells were used for
this assay and the FACSCalibur flow cytometer was used for analysis33.
3.3.3. Cell cycle analysis
Hepatocellular carcinoma cells (HepG-2) were seeded at a density of 2 × 105 cells/well and
incubated for 24 h in six-well plates. Foetal bovine serum (10%) was added and the cells were
incubated at 37°C in an atmosphere of 5% CO2. The medium was replaced with 1% (v/v)
DMSO containing 10.0 µM compound 17; the cells were then incubated for 48 h, washed with
phosphate-buffered saline, fixed with 70% ethanol, rinsed with phosphate-buffered saline, and
then stained with the DNA fluorochrome PI for 15 min at 37°C. Then samples were analysed
by using a FACSCalibur flow cytometer34,35
.
3.3.4. In vitro PCAF inhibition assay
The effect of the most potent antitumor compounds as PCAF bromodomain inhibitors was
assessed by using a well-established fluorescence-based method with a fluorescence plate
reader36.

22
3.4. Docking methodology
The molecular docking analysis was performed by using MOE 2008.10 (Chemical Computing
Group. Inc.)37 in accordance with previously established methods25-30
.

Acknowledgement
The authors acknowledge financial support from the Researchers Supporting Project number
(RSP-2019/41), King Saud University, Riyadh, Saudi Arabia.
Declaration of interest
Authors declare that there is no conflict of interest.
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28
N
N
N
N
N
N
N
H
CH3
H3C
O
N
CH3
N
N
N
N
O
N
HN
HN
NH
HN
CH3
O
O
S
O
H3C
N
N
CH3
H
CH3
CH3
bromosporine (1)
L-45 (2)
3
N
N
N
N
N
N
N
N
N
N
N
N
F
H
H
N
N
O
O
Cl
4
5
Structure based design
N
N
N
N
N
N
F
N
N
F
F
F
C
C
C
F
F
F
C
N
F
N
N
N
N
F
F
F
F
N
N
N
HN
HO
N
N
X
HN
HN
NH2
R
N
N
N
N
N
N
N
N
N
N
N
N
N
X
X
X
N
X
N
HN
HO
N
HN
HN
NH2
R
X
10-32
Designed compounds
Fig. 1. The reported antitumor and PCAF inhibitors (1-5) and the designed 1,2,4-triazolophthalazine derivatives
(10-32).

29
Fig. 2. Effect of DMSO (Left panel), and 17 (Right panel) on the percentage of annexin V-FITC-positive
staining in HepG2 cells.
Fig. 3. Bar diagram of the apoptotic effect of compound 17 on the cell cycle distribution of HePG2 cell line.