Molecular docking and biological assessment of substituted phthalazin-1(2H)-one derivatives

Article abstract of DOI:10.1002/jhet.3913

2, 4-Disubstituted phthalazin-1(2H)-one derivatives were synthesized via nucleophilic attack of N-2 of phthalazin-1(2H)-one derivatives on different alkyl halides. In addition, reactive phthalazinone acetohydrazide derivative was utilized as a scaffold to synthesize different heterocyclic systems. The assigned structures for all the newly synthesized derivatives were confirmed on the basis of elemental analyses and spectral data. Mechanistic illustrations for some of the synthesized compounds were also discussed. The synthesized compounds were evaluated for in vitro antimicrobial and antitumor activity. Most of the tested compounds exhibited significant potency as antimicrobial and antitumor agents. Moreover, MOE 2014.09 software was utilized to carry out computational studies to support the biological activity results.

Full text of DOI:10.1002/jhet.3913

Received: 27 September 2019
DOI: 10.1002/jhet.3913
Revised: 5 January 2020
Accepted: 20 January 2020
A R T I C L E
Molecular docking and biological assessment of substituted
phthalazin-1(2H)-one derivatives
Naglaa F. H. Mahmoud
| Galal A. Elsayed
Chemistry Department, Faculty of
Science, Ain Shams University, Cairo,
Egypt
Abstract
2, 4-Disubstituted phthalazin-1(2H)-one derivatives were synthesized via
nucleophilic attack of N-2 of phthalazin-1(2H)-one derivatives on different
alkyl halides. In addition, reactive phthalazinone acetohydrazide derivative
was utilized as a scaffold to synthesize different heterocyclic systems. The
assigned structures for all the newly synthesized derivatives were confirmed
on the basis of elemental analyses and spectral data. Mechanistic illustrations
for some of the synthesized compounds were also discussed. The synthesized
compounds were evaluated for in vitro antimicrobial and antitumor activity.
Most of the tested compounds exhibited significant potency as antimicrobial
and antitumor agents. Moreover, MOE 2014.09 software was utilized to carry
out computational studies to support the biological activity results.
Correspondence
Naglaa F. H. Mahmoud, Chemistry
Department, Faculty of Science, Ain
Shams University, Cairo 11566, Egypt.
Email: naglaa.fawzy@yahoo.com
1 | INTRODUCTION
the synthesized compounds has also been conducted in
order to appraise both of their antimicrobial and anti-
Phthalazines as N-heterocycles have received considerable
attention, owing to their various biological activities. In par-
ticular, Some natural products and their semi-synthetic
derivatives containing phtalazinone moiety have been
known for their potencies, especially as anticonvulsant,[1–7]
anti-tumor,[8] anti-hypertensive,[9] anti-thrombotic,[10] anti-
diabetic,[11] anti-trypanosomal,[12] anti-inflammatory,[13]
cardiotonic[14] and vasorelaxant activities.[15] Moreover,
different substituted and fused phthalazinone derivatives
have been clinically used for the treatment of fungal infec-
tions.[16] Some potent phthalazinone derivatives are
depicted in (Scheme 1). In order to have ample quantities
of phthalazinone derivatives for profound biological
assessment, different synthetic strategies have been
reported.[17–23] In view of these activities, great efforts
have been directed to develop efficient synthetic strate-
gies for phthalzinone derivatives. As an extension of our
study[24–26] which aims for the synthesis of different het-
erocyclic derivatives, herein, we report a convenient
synthetic methodology for construction of some new
heterocyclic derivatives containing phthalazine moiety.
In addition, in vitro biological assessment for some of
tumor cytotoxicities. Most of synthesized compounds
exhibited moderate to strong antimicrobial and antitumor
activities. Meanwhile, molecular docking study of those
phthalazinone derivatives was performed by Molecular
Operating Environment (MOE) 2014.09v to predict the
potential activity and the selectivity towards the carcino-
genic cells receptors. In the course of identifying various
chemical substances which may serve as leads for design-
ing novel antitumor agents, we are particularly interested
in the present work in phthalazine derivatives which may
serve as a new class of cancer chemotherapeutic agents.
2 | RESULTS AND DISCUSSION
The synthesis of 4-aryl 1-(2H)-phthalazinones 4a-c was
commenced with a Friedel-ring closure between aromatic
hydrocarbons and phthalic anhydride to afford 2-aroyl
benzoic acids 3a-c which in turn were treated with
hydrazine hydrate to afford 4a-c.[27]
On the other hand, the synthetic strategy to construct
2-(Prop-2-yn -1-yl) phthalazin-1(2H)-one derivatives 5a-c
J Heterocyclic Chem. 2020;1–18.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

2
MAHMOUD AND ELSAYED
SCHEME 1 Some potent phthalazinone derivatives
was emanated from treatment of 4a-c with propargyl bro-
mide in the presence of anhydrous potassium carbonate
(K2CO3) and dry acetone (Scheme 2). Meanwhile, utiliz-
ing allyl bromide as an alternative-alkylating agent under
the same condition afforded the 2-allyl-phthalazin-1(2H)-
one derivatives 6a-c where the alkylation reaction took
place via SN2-mechanism. All the IR spectra of com-
pounds 5a-c, 6a-c revealed the presence of bands at
1660-1652 cm-1 attributable to vCO of lactam ring which
unambiguously proved that the reaction took place via-
N-alkylation rather than o-alkylation of the
The structures of 10a-c were elucidated from the IR spec-
tra which lacked any NH bands and the existence of two
carbonyl bands in 1749-1658 cm-1 region corresponding
to ketonic and ester functional groups. When, an
ethanolic solution of 10b possessing the chlorine sub-
stiuent was treated with hydrazine hydrate under reflux
1
conditions, it afforded the hydrazide derivative 11b. H-
NMR of 11b exhibited two peaks corresponding to NH
and NH2 at δ= 10.24 and 4.19 ppm, respectively. Upon
treatment of 4a-c with 2-(bromo methoxy) ethyl acetate
and/or 4-bromo butyl acetate, the derivatives 12a-c and
13a-c were furnished, respectively (Scheme 2). Struc-
tural elucidations of 12a-c, 13a-c were ascertained from
the spectroscopic and elemental analyses data. For exam-
ple, all IR spectra exhibited two carbonyl groups, one
for lactam ring and the other represents the acetoxy
group which agreed well with the proposed structures.
Deacetylation of cyclo nucleosides 12a-c, 13a-c by treat-
ment with CH3OH/TEA and few drops of H2O afforded
the free acyclo nucleosides 14a-c, 15a-c. Assignments of
14a-c, 15a-c structures are based on the correct elemental
analyses, IR and 1H-NMR spectral data. The IR spectra of
14a-c, 15a-c revealed the presence of free hydroxyl
groups in 3445-3460 cm-1 region and the absence of
acetoxy carbonyl groups (Scheme 2).
Treatment of 11b with phthalic anhydride and/or
isatin afforded the N-phthalimido acetamide derivative 16
and indol-2-on-3-yl-acetylhydrazone derivative 17, respec-
tively (Scheme 3). Meanwhile, treatment of the hydra-
zide 11b with ethyl acetoacetate and/or acetylacetone
afforded the pyrazoloacetyl phtalzinone 18, pyrazolonoacetyl
phtalzinone 19, respectively. The structure of 18 was
proved by the existence of three ketonic peaks in the IR
range υ= 1713-1661cm-1. Furthermore 1H-NMR exhibited
a singlet peak at δ=3.03 ppm corresponding to CH2 of the
pyrazolone ring. When an ethanolic solution of 11b was
refluxed with phenyl isothiocyanate, the thiocarbamate
derivative 20 was afforded. Base-catalyzed cyclization
of thiocarbamate 20 using an aqueous solution of
sodium hydroxide under reflux condition afforded the
1
phthalazinone derivatives. Meanwhile, H-NMR spectra
revealed the lacking of signal corresponding to NH
group. In order to study the enhancement of biological
activity upon insertion of halogen moieties, compounds
6a-c were allowed to react with bromine in carbon tetra-
chloride as
a
solvent to give the anticipated
2,3-dibromopropyl-phthalazin-1(2H)-one derivatives 7a-
c. Treatment of phthalazin-1(2H)-one derivatives 4a-c
with epichlorohydrin in the presence of anhydrous potas-
sium carbonate (K2CO3) in dimethyl formamide (DMF)
afforded the 2-(Oxiran-2-ylmethyl) phthalazin-1(2H)-one
derivatives 8a-c. IR spectra of compounds 8a-c showed
bands in 1660-1650 cm-1 region which are characteristic
for vCO of lactam ring that indicated the formation of N-
alkyl products. The reaction took place via the ring
opening-ring closure of the oxirane nucleus[27] (Scheme
2). From biological aspects, it is well known that plethora
of halogen moieties as substiunents on the heterocyclic
frame could enhance the biological activity, therefore,
compounds 4a-c were treated with 1,3-dichloro-
2-propanol to furnish the 2-(3-chloro-2-hydroxypropyl)
phthalazin-1(2H)-one derivatives 9a-c. The structures of
9a-c were ascertained from IR spectra which revealed
bands in 1664-1651 and 3397-3345 cm-1 regions attributed
to υCO and υOH, respectively. On the other hand, when
phthalazin-1(2H)-one derivatives 4a-c were subjected to
react ethyl chloroacetate in the presence of-anhydrous
K2CO3 and dry dimethyl formamide (DMF), the
corresponding N-acyclo nucleosides 10a-c were afforded.

MAHMOUD AND ELSAYED
3
SCHEME 2 Synthesis of C-2 phthalazinone derivatives
corresponding 1,2,4-triazolo derivative 21 while under
acid-catalyzed cyclization of 20, the thiadizolo derivative
22 was afforded. Structural elucidations of both 21 and 22
afforded, respectively. The mechanistic pathways for 21
and 22 and 24 are illustrated in (Schemes 4 and 5).
1
were unambiguously proven from their H-NMR spectra
where 21 chart exhibited a peak at δ=12.86 ppm
corresponding to SH while H-NMR spectrum of 22 rev-
2.1 | The anti-microbial studies
1
ealed a singlet peak at δ=10.16 ppm corresponding to NH
group. Treatment of an ethanolic solution of hydrazide
derivative 11b with p-anisaldehyde under reflux condi-
tion, the arylidine hydrazide derivative 23 was afforded
(Scheme 3). Finally, when the arylidene hydrazide deriv-
ative 23 was subjected to react with different sulfur nucle-
ophiles such as thioglycollic acid and 2-amino thiophenol,
the corresponding thia-michael adducts 24 and 25 were
Screening of all the synthesized derivatives was done
using the agar diffusion assay.[28,29] This screening was
performed against Gram-positive bacteria, Gram-
negative bacteria as Staphylococcus aureus (Atcc 06538),
Escherechia coli (Atcc 10536), pseudomonas aeruginosa
(Atcc 6404) and the antifungal activity assessment was
performed against pathogenic fungi candida albicans
(Atcc 1023).

4
MAHMOUD AND ELSAYED
SCHEME 3 Utility of hydrazide 11b as a precursor to synthesize different heterocyclic systems
From table 1, it is shown that most of the compounds
exhibited moderate to strong antimicrobial activity. In
particular. compounds 5b, 7b, 9b, 14b, 19 and 25 showed
antimicrobial activities higher than the standard com-
pounds. As anticipated compound 9b showed the most
significant activity since it has two chlorine atoms. As a
conclusion all of the aforementioned compounds could
be considered as promising antimicrobial agents. The
results for anti-microbial evalution are shown in table 1
as follow.
drug under the same conditions using a colorimetric
MTT assay,[30,31] it was observed that the best results are
associated with compounds 5b, 7b, 8b and 13b. All the
results are depicted in Table 2. In addition, derivatives
8c, 9b and 11b have also showed strong cytotoxicity. On
the other hand, derivatives 4a, 4b, 5c, 6b and 7a showed
moderate cytotoxic activity while derivatives 4c, 5a, 6a,
9a, 9c, 12a, 13a, 17, 20 and 23 showed weak cytotoxicity
2.3 | Structure-Activity
Relationship (SAR)
2.2 | Cytotoxic activities of some
derivatives against human tumor cells
Investigations of the biological assessment results rev-
ealed that biological activities are dependent on the exis-
tence of certain structural features of the tested
compounds. For example derivatives with chlorine atoms
in ortho position of the aryl moiety have more in vitro
inhibitory activity than compounds having methyl group
at the position. This could be attributed to the electron
withdrawing power of the chlorine atom with –I
The in vitro growth inhibitory activities of the newly syn-
thesized compounds were investigated against three car-
cinoma cell lines named, mammary gland carcinoma cell
line (MCF-7), colorectal carcinoma cell line (HCT-116)
and human hepatocellular carcinoma cell line (HepG-2).
Comparing with Doxorubicin, the anticancer standard

MAHMOUD AND ELSAYED
5
SCHEME 4 Mechanistic illustration for 21 and 22 formation
SCHEME 5 Mechanistic illustration for 24 formation
inductive effect. Meanwhile, compounds with CH3 with +I
inductive effect at the same position when tested as anti-
tumor agents showed poor activity. The most cytotoxic
derivative was found to be 9b which has two chlorine atoms
in accordance with the previous conclusion. Therefore, we
can unequivocally conclude that derivatives with electron
withdrawing substituents exhibited remarkable activities as
anti-tumor candidates when compared with those having
electron donating substituents.
2.4 | Molecular Docking Studies
Molecular docking study of phthalazinone derivatives
was performed by Molecular Operating Environment
(MOE) 2014.09.[32–34] The Epidermal growth factor recep-
tor (EGFR) code (1M17) was downloaded from protein
data bank(http://www.rcsb.org/pdb) and prepared for
docking process. The co-crystalline ligands were re-
docked in the active pockets to validate the docking

6
MAHMOUD AND ELSAYED
TABLE 1 Anti-microbial activities of the synthesized derivatives
Synthesized Staph. Escherich Pseud.
Candida Synthesized Staph.
Pseud.
Candida
Compounds aureus coli aeruginosa albicans Compounds aureus Escherich coil aeruginosa albican
4a
18
19
17
13
20
17
28
21
22
20
15
30
16
0.0
0.0
20
23
20
9
21
22
21
12
20
21
18
17
16
18
21
27
19
0.0
0.0
15
16
14
15
21
20
21
14
16
16
12
22
13
0.0
22
12c
13b
13c
14b
15b
16
15
16
20
22
19
19
18
17
20
13
12
17
26
14
22
21
20
20
18
23
20
23
13
11
18
28
21
13
18
19
17
21
24
22
21
20
0.0
15
16
24
0.0
12
15
16
22
15
13
17
13
21
15
0.0
11
20
0.0
4b
4c
5a
5b
19
18
22
19
18
17
17
22
16
22
0.0
6b
7b
17
7c
18
8a
19
8b
20
9a
21
9b
22
12b
25
Ampicillin
Nystatin
Streptomycin 20
Note: Zone of Inhibition measured in mm. No activity (0.0), inhibition zone (<7 mm), weak activity (7-10), moderate activity (11-15 mm), strong activity
(>15 mm), solvent CDCl3 (6 mm).
TABLE 2 Cytotoxic activities of some derivatives against human tumor cells
In vitro Cytotoxicity IC50 (μM)
In vitro Cytotoxicity IC50 (μM)
Compounds
HePG2
HCT-116
5.23 0.3
50.90 2.0
25.16 3.9
>100
MCF-7
Compounds
HePG2
HCT-116
MCF-7
DOX
4a
4.50 0.2
41.31 1.8
38.81 3.1
93.34 5.3
78.56 4.5
12.34 1.2
49.34 2.2
23.62 1.2
32.69 2.5
11.35 1.0
4.17 0.2
57.43 2.5
33.12 3.2
84.25 5.1
71.36 4.4
10.71 0.9
61.34 2.1
27.09 1.6
42.28 3.0
7.28 0.6
8b
5.50 0.2
4.20 3.9
55.92 0.7
66.21 1.1
68.13 2.4
7.11 1.3
77.34 2.6
64.01 3.1
64.01 2.0
62.01 0.7
9.23 0.8
7.71 4.57
69.26 0.8
80.23 1.4
75.07 2.7
9.34 1.2
83.34 1.5
62.48 3.9
66.01 3.3
64.01 3.7
7.91 0.7
6.03 3.4
56.80 1.6
35.06 1.9
70.59 2.8
8.2 2.1
9b
4b
4c
10b
12a
13a
13b
13c
17
5a
81.14 4.7
9.06 0.6
53.34 3.2
14.28 1.5
38.55 2.1
10.45 0.4
5b
5c
72.34 1.9
76.03 3.3
79.01 1.9
66.01 2.8
6c
7a
20
7b
23
Note: IC50 (μM): 1 – 10 (very strong). 11 – 20 (strong). 21 – 50 (moderate). 51 – 100 (weak) and above 100 (non-cytotoxic).
Abbreviation: DOX, Doxorubicin.
protocol. The structures of the target compounds were
drawn in Chem Draw Ultra 14.0 (Chem Office package)
and the energy was minimized using the MMFF94x force
field until an RMSD (Root mean-square deviation) of
atomic position gradient of (0.01) Kcal mol-1A-1.
MMFF94x was reported as the efficient force field for
minimizing ligand-protein complexes. The docking Algo-
rithm was done by MOE-DOCK default which uses a
flexible technique for posing the molecule inside the
cavity. All rotatable bonds of ligands are allowed to
undergo free rotation to explore the conformational space
inside the rigid receptor binding site.
The MOE 2014.09 package software was used to ana-
lyze all binding energies and docking poses between com-
pounds 5b, 7b, 8b, 9b, 13b and the enzyme Epidermal
growth factor receptor (EGFR) code (1M17) to evaluate
the affinity of compounds according to its binding energy
with the enzyme. The total binding energy of compound

MAHMOUD AND ELSAYED
7
FIGURE 1 Two and three-dimensional representations for the interactions of compound 5b with the enzyme pocket amino acids
[Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 2 Two and three-dimensional representations for the interactions of compound 7b with the enzyme pocket amino acids
[Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 3 Two and three-dimensional representations for the interactions of compound 8b with the enzyme pocket amino acids
[Color figure can be viewed at wileyonlinelibrary.com]
5b equals -6.33 E (kcal/mol), showed good affinity with the
enzyme by forming one hydrogen donor interaction with
ASP 831 and one Pi-H interaction with LYS 721 amino acid
of the target enzyme (Figure 1), compound 7b equals -6.59
E (kcal/mol), showed good affinity with the enzyme by for-
ming one hydrogen donor interaction with ASP 831 and
three Pi-H interaction with LYS 721 and VAL702 amino
acid of the target enzyme (Figure 2), compound 8b equals

8
MAHMOUD AND ELSAYED
-6.75 E (kcal/mol), showed good affinity with the enzyme
by forming one hydrogen donor interaction with ASP(831)
and three Pi-H interaction with LYS 721 and VAL
702 amino acid of the target enzyme (Figure 3), compound
9b equals -6.52 E (kcal/mol), showed good affinity with the
enzyme by forming two hydrogen donor interaction with
FIGURE 4 Two and three-dimensional representations for the interactions of compound 9b with the enzyme pocket amino acids
[Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 5 Two and three-dimensional representations for the interactions of compound 13b with the enzyme pocket amino acids
[Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 6 Two and three-dimensional representations for the interactions of reference 1M17 with the enzyme pocket amino acids
[Color figure can be viewed at wileyonlinelibrary.com]

MAHMOUD AND ELSAYED
9
TABLE 3 The docking binding free energies of the
synthesized compounds with TS, forming hydrogen bonding and
cationic-π interactions
3.1.1 | 4-Aryl phthalazin-1(2H)-one (4a-c)
A solution of 2-aroyl benzoic acids (0.01 mol) and hydra-
zine hydrate (0.015 mol, 0.75mL) in ethanol (30 mL) was
heated under reflux for 2 h. The precipitate formed after
cooling was filtered off and recrystallized from acetic acid.
Binding
EnergyΔG hydrogen No. of cationic-π
kcal/mol bonding interactions
No. of
Comp.
5b
-6.330
-6.596
-6.751
-6.522
-7.261
1 (ASP 831) 1 (LYS721)
7b
1(ASP 831) 3 (LYS 721 and VAL 702)
1(ASP 831) 3 (LYS 721 and VAL 702)
2( ASP 831) 1 ( VAL 702 )
3.1.2 | 4-(Phenyl) phthalazin-1(2H)-
one (4a)
8b
9b
Yield (87%), mp. 242-243oC. FT-IR (KBr) (cm-1): 3262 (NH),
13b
1(LYS 721) 2( LYS 721 and VAL 702)
1
reference -7.46
1M17
1(MET 769)
1(GLN 767)
—
1660 (CO). H-NMR (DMSO-d6): δH (ppm) 12.85 (br.s, 1H,
NH, D2O exchangeable), 8.36-7.35 (m, 9H, ArH). MS, m/z
(%): 222 (M.+, 100.0%). 13C-NMR (DMSO-d6) δ 159.4, 134.6,
132.6, 132.3, 131.5, 131.0, 130.4, 129.2 (2), 128.2 (2), 127.5,
126.0, 123.3. Anal. Calcd. for C14H10N2 O (222): C, 75.66; H,
4.54; N, 12.60; Found: C, 75.59; H, 4.77; N, 12.53.
ASP 831 and one Pi-H interaction with VAL 702 amino acid
of the target enzyme (Figure 4), the total binding energy of
compound 13b equals -7.26 E (kcal/mol), showed good
affinity with the enzyme by forming one hydrogen
acceptor interaction with LYS 721, two Pi-H interac-
tion with VAL 702 and one pi-cation with LYS
721 amino acid of the target enzyme (Figure 5). The
binding energy of reference (1M17) with the enzyme
pocket amino acids is shown (Figure 6) where it
shows -7.46 E (kcal/mol) and good affinity with the
enzyme by forming one hydrogen acceptor interaction
with MET 769 and one hydrogen donor interaction
with GLN 767 as shown in table 3. Both compounds
13b and reference 1M17 have close binding energies
-7.26 E (kcal/mol) and-7.46 E (kcal/mol), respectively
which is in accordance with the remarkable biological
potency of 13b.
3.1.3 | 4-(2-Chlorophenyl) phthalazin-1
(2H)-one (4b)
o
Yield (93 %), mp. 255-256 C. FT-IR (KBr) (cm-1): 3299
1
(NH), 1661 (CO). H-NMR (DMSO-d6): δH (ppm) 12.74
(br.s, 1H, NH, D2O exchangeable ), 8.34-7.65 (m, 8H,
ArH). MS, m/z (%): 256 (M.+, 100.0%), 258 (M.++2,
32%).13C-NMR (DMSO-d6) δ 159.4, 134.5, 133.1, 132.3(2),
131.5, 131.1, 130.9, 129.8, 129.2, 127.5, 126.5, 125.8, 123.1.
Anal. Calcd. for C14H9ClN2O (256): C, 65.51; H, 3.53; N,
10.91; O, 6.23. Found: C, 65.87; H, 3.70; N, 10.74.
3.1.4 | 4-(o-Tolyl) phthalazin-1(2H)-
one (4c)
3 | MATERIALS AND METHODS
3.1 | Experimental
Yield (93 %), mp. 271-272 oC. FT-IR (KBr) (cm-1): 3297 (NH),
1663 (CO). 1H-NMR (DMSO-d6): δH (ppm) 12.72 (br.s, 1H, NH,
D2O exchangeable), 8.35-7.62 (m, 8H, ArH), 2.39 (s, 3H, CH3).
MS, m/z (%): 236 (M.+, 100.0%). 13C-NMR (DMSO-d6) δ 159.7,
138.9, 134.2, 133.1, 132.3, 131.5, 131.1, 130.9, 129.2, 127.5, 126.5,
125.8, 123.3, 129.1, 19. Anal. Calcd. for C15H12N2O (236): C,
76.25; H, 5.12; N, 11.86; Found: C, 76.42; H, 4.95; N, 11.54.
All melting points were measured on a Gallenkamp elec-
tric melting point apparatus are uncorrected.-The infra-
red spectra were recorded in potassium bromide disks on
a pye Unicam SP-3-300 and Shimdazu FT IR 8101 PC
infrared spectrophotometers at the central laboratory of
1
faculty of science, Ain Shams University. The H-NMR
(300MHz) and 13C-NMR (75MHz) were measured on
Varian Gemini instrument with chemical shifts (δ)
expressed in ppm downfield from TMS. Mass spectra
were recorded on Schimadzu-GC-MS-QP 1000 EX instru-
ment operating at 70 e.v. Elemental analyses were carried
out at the Micro analytical center of Ain Shams Univer-
sity. All the reactions and the purity of the new com-
pounds were followed and checked by TLC.
3.1.5 | 2-Alkyl-4-Aryl phthalazin-1(2H)-
one (5a-c, 6a-c)
A solution of the appropriate alkyl halide namely,
propargle bromide and/or allyl bromide (0.01mol), com-
pounds 4a-c (0.01 mol) and anhydrous K2CO3 (0.01 mol)
in dry acetone (30 mL) was heated under reflux for 24 h.
The solvent was evaporated and the residue was diluted

10
MAHMOUD AND ELSAYED
with water, the solid obtained was filtered off, dried and
recrystallized from ethanol.
(m, 1H, CH=CH2), 5.14 (d, 2H, CH=CH2, J=5.5Hz ),
4.76 (m, 2H, NCH2). MS, m/z (%): 296 (M.+, 100.0%),
298 (M.++2, 32%). Anal. Calcd. for C17H13ClN2O
(296): C, 68.81; H, 4.42; N, 9.44; Found: C, 68.97; H,
3.97; N, 9.98.
3.1.6 | 4-Phenyl-2-(prop-2-yn-1-yl)
phthalazin-1(2H)-one (5a)
Yield (75 %), mp. 151-152 oC. FT-IR (KBr) (cm-1): 1656 (CO).
1H-NMR (DMSO-d6): δH (ppm) 7.98 – 7.56 (m, 9H, ArH),
4.81 (s, 2H, NCH2), 3.16 (s, 1H, acetylenic proton), MS, m/z
(%): 260 (M.+, 100.0%). Anal. Calcd. for C17H12N2O (260): C,
78.44; H, 4.65; N, 10.76; Found: C, 78.39; H, 4.69; N 10.81.
3.1.11 | 2-Allyl-4-(o -tolyl) phthalazin-1
(2H)-one (6c)
Yield (60 %), mp. 112-114oC. FT-IR (KBr) (cm-1): 1657
1
(CO). H-NMR (DMSO-d6): δH (ppm) 8.35-7.68 (m, 8H,
ArH), 5.98 (m, 1H, CH=CH2), 5.16 (d, 2H, CH=CH2,
J=5.5Hz), 4.78 (m, 2H, NCH2), 2.4 (s, 3H, CH3). MS,
m/z (%): 276 (M.+, 100.0%). Anal. Calcd. for C18H16N2O
(276): C, 78.24; H, 5.84; N, 10.14; Found: C, 77.97; H,
5.67; N, 10.48.
3.1.7 | 4-(2-Chlorophenyl)-2-(prop-2-yn-
1-yl) phthalazin-1(2H)-one (5b)
Yield (69 %), mp. 170-172oC. FT-IR (KBr) (cm-1): 1656
1
(CO). H-NMR (DMSO-d6): δH (ppm) 8.39-7.61 (m, 8H,
ArH). 4.98 (s, 2H, NCH2), 3.22 (s, 1H, acetylenic proton).
MS, m/z (%): 294 (M.+, 100.0%), 296 (M.++2, 31%). Anal.
Calcd. for C17H11ClN2O (294): C, 69.28; H, 3.76; N, 9.50;
Found: C, 68.97; H, 3.70; N, 10.04.
3.1.12 | 2-(2,3-Dibromopropyl)–4-aryl
phthalzin-1(2H)-one (7a-c)
A solution of 4a-c (0.01 mol) in carbon tetrachloride
(20 mL) and bromine (0.015 mol) in carbon tetrachlo-
ride (20 mL) were stirring at room temperature during
30 min. After complete addition, the stirring was con-
tinued for 3 h, the solid that formed was filtered off
dried and crystallized from ethanol.
3.1.8 | 2-(Prop-2-yn-1-yl)-4-(o-tolyl)
phthalazin-1(2H)-one (5c)
Yield (78 %), mp. 159-160oC. FT-IR (KBr) (cm-1): 1660
1
(CO). H-NMR (DMSO-d6): δH (ppm) 8.36-7.35 (m, 8H,
ArH), 4.97 (s, 2H, NCH2), 3.21 (s, 1H, acetylenic proton),
2.41 (s, 3H, CH3). MS, m/z (%): 274 (M.+, 100.0%). Anal.
Calcd. for C18H14N2O (274): C, 78.81; H, 5.14; N, 10.21;
Found: C, 78.42; H, 4.95; N,10.25.
3.1.13 | 2-(2,3-Dibromopropyl)–4-phenyl
phthalazin-1(2H)-one (7a)
Yield (58 %), mp. 155-156oC. FT-IR (KBr) (cm-1): 1660 (CO).
1H-NMR (DMSO-d6): δH (ppm) 8.34 –7.53 (m, 9H, ArH),
4.75 (m, 1H, CHBr), 4.71-4.61 (m, 2H, CH2Br), 4.12 (m, 2H,
NCH2). MS, m/z (%): 422 (M.+, 100.0%). Anal. Calcd. for
C17H14 Br2N2O (422): C, 48.37; H, 3.34; N, 6.64; Found:
C,48.43; H, 3.09; N 6.71.
3.1.9 | 2-Allyl-4-phenyl phthalazin-1(2H)-
one (6a)
Yield (62 %), mp. 125-126oC. FT-IR (KBr) (cm-1): 1658
1
(CO). H-NMR (DMSO-d6): δH (ppm) 8.34 – 7.53 (m, 9H,
ArH), 5.96 (m, 1H, CH=CH2), 5.14 (d, 2H, CH=CH2,
J=5.5Hz), 4.78 (m, 2H, NCH2). MS, m/z (%): 262 (M.+,
100.0%). Anal. Calcd. for C17H14N2O (262): C, 77.84; H,
5.38; N, 10.68; Found: C,78.19; H, 5.09; N 10.81.
3.1.14 | 4-(2-Chlorophenyl)-
2-(2, 3-dibromopropyl)–phthalazin-1(2H)-
one (7b)
o
Yield (60 %), mp. 152-154 C. FT-IR (KBr) (cm-1): 1659
1
(CO). H-NMR (DMSO-d6): δH (ppm) 8.41-7.62 (m, 8H,
3.1.10 | 2-Allyl-4-(2-chlorophenyl)
phthalazin-1(2H)-one (6b)
ArH), 4.88 (m, 1H, CHBr), 4.71-4.61 (m, 2H, CH2Br),
4.14 (m, 2H, NCH2). MS, m/z (%): 456 (M.+, 100.0%),
458 (M.++2, 32%). Anal. Calcd. for C17H13Br2ClN2O
(456.5): C, 44.72; H, 2.87; N, 6.14; Found: C, 44.76.; H,
2.94; N, 6.28.
Yield (60 %), mp. 102-104oC. FT-IR (KBr) (cm-1): 1656 (CO).
1H-NMR (DMSO-d6): δH (ppm) 8.33-7.59 (m, 8H, ArH), 5.98

MAHMOUD AND ELSAYED
11
3.1.15 | 2-(2, 3-Dibromopropyl)-4-(o-tolyl)
phthalazin-1(2H)-one (7c)
3.1.19 | 2-(Oxiran-2-ylmethyl)-4-(o-tolyl)-
phthalazin-1(2H)-one (8c)
Yield (60 %), mp. 140-142oC. FT-IR (KBr) (cm-1): 1659
(CO). H-NMR (DMSO-d6): δH (ppm) 8.40-7.31 (m, 8H,
Yield (54 %), mp. 124-126oC. FT-IR (KBr) (cm-1): 1659
(CO). H-NMR (DMSO-d6): δH (ppm) 8.43-7.48 (m, 8H,
1
1
ArH), 4.91 (m, 1H, CHBr), 4.74- 4.62 (m, 2H, CH2Br),
4.11 (m, 2H, NCH2), 2.41 (s, 3H, CH3). MS, m/z (%):
436 (M.+, 100.0%). Anal. Calcd. for C18H16Br2N2O
(436): C, 49.57; H, 3.70; N, 6.42; Found: C, 49.17; H,
3.72; N, 6.48.
ArH), 4.68-4.42 (dd, 2H, NCH2, J=5.52), 4.32-4.25 (m, 1H
proton of oxirane ring), 4.15-4.05 (m, 2H, methylene pro-
tons of oxirane ring), 2.38 (s, 3H, CH3). MS, m/z (%):
292 (M.+, 100.0%). 13C-NMR (DMSO-d6) δ 158.4, 138.9,
134.7, 132.9, 132.3, 131.5, 131.0, 130.4, 129.2, 128.2, 127.5,
126.2, 123.3, 55.4, 47.4, 43.1, 19.2 Anal. Calcd. for
C18H16N2O2 (292): C, 73.95; H, 5.52; N, 9.58; Found: C,
73.71; H, 5.37; N, 9.65.
3.1.16 | 2-(Oxiran-2-ylmethyl)-4-aryl
phthalazin-1(2H)-one (8a-c), 4-aryl-
2-(3-chloro-2-hydroxy prop yl) phthalazin-1
(2H)-one (9a-c)
3.1.20 | 2-(3-Chloro-2-hydroxypropyl)-
4-phenyl phthalazin-1(2H)-one (9a)
A mixture of 4a-c (0.01 mol), epichlorohydrin and/or
1,3-dichloropropanol (0.01 mol) and anhydrous K2CO3
(0.01 mol) in dry DMF (30 mL) was heated on a water
bath for 15 h. The excess solvent was evaporated under
reduced pressure and the residue was diluted with water.
The solid that obtained was recrystallized from ethanol.
Yield (35%), mp. 112-114oC. FT-IR (KBr) (cm-1): 3397
(OH), 1660 (CO). 1H-NMR (DMSO-d6): δH (ppm)
8.38-7.35 (m, 9H, ArH), 4.81 (s, 1H, OH, exchangeable
with D2O), 4.62 (m, 1H, CHOH), 4.35-4.04 (m, 2H, meth-
ylene protons of CH2Cl), 3.43 (m, 2H, NCH2). MS, m/z
(%): 314 (M.+, 100.0%), 316 (M.++2, 30%). 13C-NMR
(DMSO-d6) δ 158.4, 134.7, 132.9, 132.3, 131.5, 131.0,
130.4, 129.2 (2), 128.2(2), 127.5, 126.2, 123.3, 71.2, 55.4,
48.4. Anal. Calcd. for C17H15ClN2 O2 (314): C, 64.87; H,
4.80; N, 8.90; Found: C, 64.94; H, 4.67; N, 8.75.
3.1.17 | 2-(Oxiran-2-ylmethyl)-
4-phenylphthalazin-1(2H)-one (8a)
o
Yield (53 %), mp. 142-143 C. FT-IR (KBr) (cm-1): 1660
1
(CO). H-NMR (DMSO-d6): δH (ppm) 8.31-7.55 (m, 9H,
ArH), 4.87-4.67 (dd, 2H, NCH2, J=5.52), 4.32-4.25 (m, 1H
proton of oxirane ring), 4.18-4.05 (m, 2H, methylene pro-
tons of oxirane ring), MS, m/z (%): 278 (M.+, 100.0%).
13C-NMR (DMSO-d6) δ 158.4, 134.7, 132.9, 132.3, 131.5,
131.0, 130.4, 129.2 (2), 128.2 (2), 127.5, 126.2, 123.3, 55.4,
47.4, 43.1. Anal. Calcd. for C17H14N2 O2 (278): C,
73.37; H, 5.07; N, 10.07; Found: C, 73.41; H,
5.37; N, 10.25.
3.1.21 | 2-(3-Chloro-2-hydroxypropyl)-
4-(2-chlorophenyl)-phthalazin-1(2H)-
one (9b)
Yield (33%), mp. 132-133oC. FT-IR (KBr) (cm-1): 3444
(OH), 1664 (CO). 1H-NMR (DMSO-d6): δH (ppm)
8.39-7.64 (m, 8H, ArH), 4.81 (s, 1H, OH, exchangeable
with D2O), 4.62 (m, 1H, CHOH), 4.26-4.16 (m, 2H, meth-
ylene protons of CH2Cl), 3.43 (m, 2H, NCH2). MS, m/z
(%): 348 (M.+, 100.0%), 351 (M.++2, 62%). 13C-NMR
(DMSO-d6) δ 158.4, 138.6, 134.7, 132.9, 132.3, 131.5,
131.0, 130.4, 129.2, 128.2, 127.5, 126.2, 123.3, 71.2, 55.4,
48.4, 19.2. Anal. Calcd. for C17H14Cl2N2 O2 (348): C,
58.47; H, 4.04; N, 8.02; Found: C, 58.17; H, 4.27; N, 8.35.
3.1.18 | 4-(2-Chlorophenyl)-2-(oxiran-
2-ylmethyl)-phthalazin-1(2H)-one (8b)
Yield (51 %), mp. 131-133oC. FT-IR (KBr) (cm-1): 1659
1
(CO). H-NMR (DMSO-d6): δH (ppm) 8.38-7.59 (m, 8H,
ArH), 4.77-4.64 (dd, 2H, NCH2, J=5.52), 4.30-4.27 (m, 1H
proton of oxirane ring), 4.22-4.15 (m, 2H, methylene pro-
tons of oxirane ring), MS, m/z (%): 312 (M.+, 100.0%),
314 (M.++2, 32%). 13C-NMR (DMSO-d6) δ 158.4, 134.7,
132.9, 132.3, 131.5, 131.0, 130.4, 129.2 (2), 128.2 (2), 127.5,
126.2, 123.3, 55.4, 47.4, 43.1. Anal. Calcd. for C17H13ClN2
O2 (312): C, 65.29; H, 4.19; N, 8.96; Found: C, 65.41; H,
4.37; N, 8.65.
3.1.22 | 2-(3-Chloro-2-hydroxypropyl)-
4-(o-tolyl)-phthalazin-1(2H)-one (9c)
Yield (36%), mp. 122-123oC. FT-IR (KBr) (cm-1): 3445
(OH), 1664 (CO). 1H-NMR (DMSO-d6): δH (ppm)
8.34-7.33 (m, 8H, ArH), 4.83 (s, 1H, OH, exchangeable
with D2O), 4.62 (m, 1H, CHOH), 4.27-4.16 (m, 2H,

12
MAHMOUD AND ELSAYED
methylene protons of -CH2Cl), 3.45 (m, 2H, NCH2), 2.39
(s, 3H, CH3). MS, m/z (%): 328 (M.+, 100.0%), 341 (M.
++2, 31%). 13C-NMR (DMSO-d6) δ 158.4, 138.6, 134.7,
132.9, 132.3, 131.5, 131.0, 130.4, 129.2, 128.2, 127.5, 126.2,
123.3, 71.2, 55.4, 48.4, 19.2. Anal. Calcd. for
C18H17ClN2O2 (328): C, 65.75; H, 5.21; N, 8.52; Found: C,
65.70; H, 5.27; N, 8.35.
322 (M.+, 100.0%).13C-NMR (DMSO-d6) δ: 169.5, 158.4,
138.3, 134.5, 133.7, 132.3, 131.1, 131.0, 130.8, 130.6, 129.1,
127.6, 126.3, 125.9, 123.4, 61.0, 52 6, 19.2, 14.1. Anal.
Calcd. for C19H18N2O3 (322): C, 70.79; H, 5.63; N, 8.69;
Found: C, 71.02; H, 5.75; N, 8.54.
3.1.27 | 2-(4-(2-Chlorophenyl)-
1-oxophthalazin-2(1H)-yl)
acetohydrazide (11b)
3.1.23 | Ethyl 2-(4-aryl-1-oxophthalazin-2
(1H)-yl) acetate (10a-c)
A mixture of 10b (0.01 mol, 3.42 g) and hydrazine hydrate
(2 mL) in boiling ethanol (50 mL) was refluxed 3 h then
cooled at room temperature. The solid that formed was
filtered off, dried and recrystlised from ethanol.
A mixture of 4a-c (0.01 mol), ethyl bromoacetate (0.01
mol, 2.5 mL) and anhydrous K2CO3 (0.03 mol, 4.1 g) in
dry DMF (30 mL) was heated under reflux for 24 h. the
solvent was evaporated and the residue was diluted with
water, the solid afforded was filtered off, dried and rec-
rystlised from proper solvent.
o
Yield (88%), mp. 232-234 C. FT-IR (KBr) (cm-1): 3417,
1
3325 (NH2), 3299 (NH), 1662, 1656 (CO). H-NMR (DMSO-
d6): δH (ppm) 10.24 (br.s, 1H, NH, D2O exchangeable),
8.14-7.35 (m, 8H, ArH), 4.19 (br.s, 2H, NH2, D2O exchange-
able), 3.44 (s, 2H, NCH2). MS, m/z (%): 328 (M.+, 100.0%),
331 (M.++2, 32%). 13C-NMR (DMSO-d6) δ: 170.0, 159.1, 134.6,
132.8, 132.6, 132.4, 131.5, 131.1, 130.9, 130.4, 130.1, 127.5, 126.5,
125.8, 123.1, 57.3 Anal. Calcd. for C16H13ClN4O2 (328): C,
58.46; H, 3.99; N, 17.04; Found: C, 58.37; H, 3.79; N, 16.94.
3.1.24 | Ethyl 2-(1-oxo-4-phenyl
phthalazin-2(1H)-yl)acetate (10a)
Yield (75%), mp. 132-134oC. FT-IR (KBr) (cm-1): 1751,
1
1658 (CO). H-NMR (DMSO-d6): δH (ppm) 8.28-7.33 (m,
9H, ArH), 4.94 (s, 2H, NCH2), 4.14 (q, 2H, CH2CH3), 1.18
(t, 3H, CH2CH3). MS, m/z (%): 308 (M.+, 100.0%). 13C-
NMR (DMSO-d6) δ 169.3, 158.4, 134.7, 132.9, 132.4,
131.1, 131.0, 130.4, 129.2 (2), 128.2(2), 127.5, 126.2, 123.7,
61.2, 53.4, 14.4. Anal. Calcd. for C18H16N2O3 (308): C,
70.12; H, 5.23; N, 9.09; Found: C, 69.94; H, 4.97; N, 9.15
3.1.28 | 2-((1-Oxo-4-arylphthalazin-2(1H)-
yl)methoxy) ethyl acetate (12a-c) and
4-(1-Oxo-4-arylphthalazin-2(1H)-yl) butyl
acetate (13a-c)
A mixture of 4a-c (0.01 mol), 4-bromobutylacetate and/or
2-(bromomethoxy) ethyl acetate (0.01 mol) and anhy-
drous K2CO3 (0.01 mol) in dry DMF (30 mL) was stirred
at room temperature for 24 h. The excess solvent was
evaporated under reduced pressure, and the reaction
mixture was diluted with water. The solid that obtained
was recrystallized from ethanol.
3.1.25 | Ethyl 2-(4-(2-chlorophenyl)-
1-oxophthalazin-2(1H)-yl)acetate (10b)
Yield (93 %), mp. 205-207 oC. FT-IR (KBr) (cm-1): 1749, 1661
(CO). 1H-NMR (DMSO-d6): δH (ppm) 8.34-7.65 (m, 8H,
ArH), 4.94 (s, 2H, NCH2), 4.14 (q, 2H, CH2CH3), 1.18(t, 3H,
CH2CH3). MS, m/z (%): 342 (M.+, 100.0%), 344 (M.++2,
32%).13C-NMR (DMSO-d6) δ: 169.5, 159.1, 136.3, 134.5, 133.1,
132.3, 131.1, 131.0, 130.8, 130.6, 129.1, 128.7 (2), 126.5, 123.1,
61.4, 52 5, 14.1. Anal. Calcd. for C18H15ClN2O3 (342): C,
63.07; H, 4.41; N, 8.17; Found: C, 65.87; H, 3.70; N, 10.74.
3.1.29 | 2-((1-Oxo-4-phenylphthalazin-2
(1H)-yl)methoxy)ethyl acetate (12a)
Yield (60%), mp 86-88 oC. FT-IR (KBr) (cm-1): 1738
1
(CO ester), 1675 (CO amide). H-NMR (DMSO-d6): δH
(ppm) 8.35-7.52 (m, 9H, ArH), 4.43(s, 2H, NCH2O), 4.24
(t, 2H, CH2OCO, J = 5.5Hz), 3.75 (t, 2H, OCH2, J= 5.5
Hz), 1.95 (s, 3H, CH3CO). MS, m/z (%): 338 (M.+,
100.0%). 13C-NMR (DMSO-d6) δ: 170.0, 158.4, 134.7,
132.9, 132.3, 131.5, 131.0, 130.4, 129.2 (2), 128.2(2), 127.5,
126.2, 123.3, 87.4, 66.1, 63.7, 20.5. Anal. Calcd. for
C19H18N2 O4 (338): C, 67.45; H, 5.36; N, 8.28; Found: C,
67.54; H, 5.38; N 8.30.
3.1.26 | Ethyl 2-(1-oxo-4-(o-tolyl)
phthalazin-2(1H)-yl)acetate (10c)
o
Yield (84 %), mp. 110-111 C. FT-IR (KBr) (cm-1): 1745,
1
1660 (CO). H-NMR (DMSO-d6): δH (ppm) 8.34-7.65 (m,
8H, ArH), 4.94 (s, 2H, NCH2), 4.14 (q, 2H, CH2CH3), 2.41
(s, 3H, ArCH3), 1.18(t, 3H, CH2CH3). MS, m/z (%):

MAHMOUD AND ELSAYED
13
3.1.30 | 2-((4-(2-Chlorophenyl)-
1-oxophthalazin-2(1H)-yl) methoxy) ethyl
acetate (12b)
372 (M.++2, 32%). 13C-NMR (DMSO-d6) δ: 170.0, 158.4,
138.1, 134.7, 133.1, 132.9, 132.3, 131.5, 131.0, 130.4, 129.2,
128.2, 127.5, 126.2, 123.3, 64.1, 41.1, 26.7, 21.7, 20.5. Anal.
Calcd. for C20H19ClN2O3 (370): C, 64.78; H, 5.16; N, 7.55;
Found: C, 64.82; H, 5.05; N 7.51.
Yield (55%), mp. 96-97 oC FT-IR (KBr) (cm-1): 1740 (CO ester),
1
1662 (CO amide). H-NMR (DMSO-d6): δH (ppm) 8.37-7.61
(m, 8H, ArH), 4.43(s, 2H, NCH2O), 4.25 ( t, 2H, CH2OCO,
J = 6.6Hz), 3.78 (t, 2H, OCH2, J= 6.9 Hz), 1.91 (s, 3H,
CH3CO). MS, m/z (%): 372 (M.+, 100.0%), 374 (M.++2, 32%).
13C-NMR (DMSO-d6) δ 170.0, 158.4, 138.2, 134.7, 133.5, 132.9,
132.3, 131.5, 131.0, 130.4, 129.2, 128.2, 127.5, 126.2, 123.3, 87.4,
66.1, 63.7, 20.5. Anal. Calcd. for C19H17ClN2 O4 (372): C,
61.21; H, 4.60; N, 7.51; Found: C, 61.54; H, 4.56; N, 7.59.
3.1.34 | 4-(1-Oxo-4-(o-tolyl) phthalazin-2
(1H)-yl) butyl acetate (13c)
Yield (98%), mp. 92-94oC. FT-IR (KBr) (cm-1): 1731 (CO ester),
1
1656 (CO amide). H-NMR (DMSO-d6): δH (ppm) 8.38-7.69
(m, 8H, ArH), 4.22 (t, 2H, CH2O, J = 6.6), 4.03 (t, 2H, NCH2,
J=6.6Hz), 1.96 (s, 3H, CH3CO), 1.79 (m, 2H, CH2), 1.58 (m,
2H, CH2). MS, m/z (%): 350 (M.+, 100.0%). 13C-NMR (DMSO-
d6) δ: 170.0, 158.4, 138.1, 134.7, 133.1, 132.9, 132.3, 131.5,
131.0, 130.4, 129.2, 128.2, 127.5, 126.2, 123.3, 64.1, 41.1, 26.7,
21.7, 20.5, 19.2. Anal. Calcd. for C21H22N2O3 (350): C, 71.98; H,
6.33; N, 7.99; Found: C, 71.82; H, 6.45; N 7.71.
3.1.31 | 2-((1-Oxo-4-(o-tolyl) phthalazin-2
(1H)-yl)methoxy)ethyl acetate (12c)
Yield (61%), mp. 90-92oC. FT-IR (KBr) (cm-1): 1737
(CO ester), 1656 (CO amide). 1H-NMR (DMSO-d6): δH
(ppm) 8.38-7.66 (m, 8H, ArH), 4.43(s, 2H, NCH2O), 4.24 (t,
2H, CH2OCO, J = 6.6Hz), 3.75 (t, 2H, OCH2, J= 6.9 Hz),
2.36 (s, 3H, ArCH3), 1.90 (s, 3H, CH3CO). MS, m/z (%):
352 (M.+, 100.0%). 13C-NMR (DMSO-d6) δ: 170.0, 158.4,
138.2, 134.7, 132.9, 132.3, 131.5, 131.0, 130.4, 129.2, 128.2,
127.5, 126.2, 123.3, 87.4, 66.1, 63.7, 20.5, 19.1. Anal. Calcd.
for C20H20N2 O4 (352): C, 68.17; H, 5.72; N, 7.95; Found: C,
68.78; H, 5.70; N, 7.89.
3.1.35 | 4-Aryl 2-((2-hydroxyethoxy)
methyl)-pthalazin-1(2H)-one (14a-c) and
4-Aryl-2-(4-hydroxybutyl)-phthalazin-1
(2H)-one (15a-c)
A mixture of 10a-c and/or 11a-c (0.01 mol), triethyl amine
(1 mL), and few drops of water in methanol (30 mL) was
stirred at room temperature for15 h and then left over night.
The excess solvent was evaporated under reduced pressure
and the formed residue was recrystallized from ethanol.
3.1.32 | 4-(1-Oxo-4-phenyl phthalazin-2
(1H)-yl) butyl acetate (13a)
Yield (98%), mp. 90-92oC. FT-IR (KBr) (cm-1): 1730 (CO ester),
1665 (CO amide). H-NMR (DMSO-d6): δH (ppm) 8.31-7.55
3.1.36 | 2-((2-Hydroxyethoxy)methyl)-
4-phenylphthalazin-1(2H)-one (14a)
1
(m, 9H, ArH), 4.21 (t, 2H, CH2O, J = 6.6), 4.01 (t, 2H, NCH2,
J=6.6Hz), 1.96 (s, 3H, CH3CO), 1.82 (m, 2H, CH2), 1.61 (m,
2H, CH2). MS, m/z (%): 338 (M.+, 100.0%). 13C-NMR (DMSO-
d6) δ: 170.0, 158.4, 134.7, 132.9, 132.3, 131.5, 131.0, 130.4, 129.2
(2), 128.2(2), 127.5, 126.2, 123.3, 64.1, 41.1, 26.7, 21.7, 20.5.
Anal. Calcd. for C20H20N2O3 (336): C, 71.41; H, 5.99; N, 8.33;
Found: C, 71.46; H, 5.95; N 8.51.
Yield (80%), mp. 112-114oC. FT-IR (KBr) (cm-1): 3445 (OH),
1656 (CO amide). H-NMR (DMSO-d6): δH (ppm) 8.35-7.52
(m, 9H, ArH), 4.81 (t, 1H, OH, J=6.6Hz, exchangeable in
D2O), 4.43(s, 2H, NCH2O), 3.61(t, 2H, CH2OCO, J = 5.5Hz),
3.34 (t, 2H, OCH2, J= 5.5 Hz). MS, m/z (%): 296 (M.+, 100.0%).
Anal. Calcd. for C17H16N2O3 (296): C, 68.91; H, 5.44; N, 9.45;
Found: C, 68.97; H, 5.50; N 9.50.
1
3.1.33 | 4-(2-Chlorophenyl)-
1-oxophthalazin-2(1H)-yl) butyl
acetate (13b)
3.1.37 | 4-(2-Chlorophenyl)-
2-((2-hydroxyethoxy)methyl)phthalazin-1
(2H)-one (14b)
Yield (96%), mp. 97-99 oC. FT-IR (KBr) (cm-1): 1729
(CO ester), 1664 (CO amide). 1H-NMR (DMSO-d6): δH (ppm)
8.37-7.65 (m, 8H, ArH), 4.21 (t, 2H, CH2O, J = 6.6), 4.02
(t, 2H, NCH2, J=6.6Hz), 1.95 (s, 3H, CH3CO), 1.84 (m, 2H,
CH2), 1.60 (m, 2H, CH2). MS, m/z (%): 370 (M.+, 100.0%),
o
Yield (75%), mp. 117-119 C. FT-IR (KBr) (cm-1): 3460
1
(OH), 1656 (CO amide). H-NMR (DMSO-d6): δH (ppm)
8.53-7.63 (m, 8H, ArH), 4.83 (t, 1H, OH, J=5.5Hz,
exchangeable in D2O), 5.32 (s, 2H, NCH2O), 3.62 (t, 2H,

14
MAHMOUD AND ELSAYED
CH2OCO, J = 5.5Hz), 3.44 (t, 2H, OCH2, J= 5.5 Hz). MS,
m/z (%): 330 (M.+, 100.0%). Anal. Calcd. for
C17H15ClN2O3 (330): C, 61.73; H, 4.57; N, 8.47; Found: C,
61.77; H, 4.53; N 8.50.
Anal. Calcd. for C19H20N2O2 (308): C, 74.00; H, 6.54; N,
9.08; Found: C, 74.12; H, 6.59; N 9.38.
3.1.42 | 2-(4-(2-Chlorophenyl)-
1-oxophthalazin-2(1H)-yl)-N-
(1,3-dioxoisoindolin-2-yl) acetamide (16)
3.1.38 | 2-((2-Hydroxyethoxy)methyl)-
4-(o-tolyl)phthalazin-1(2H)-one (14c)
Amixture of hydrazide 11b (0.01 mol, 3.28 g), phthalic anhy-
dride (0.01mol, 1.4 g) was fused in an oil bath at 180 oC for
1 h. The fused mixture was treated with ethanol and filtered.
The crude product was recrystlised from dioxane.
Yield (7%), mp. 114-115 oC. FT-IR (KBr) (cm-1): 3445
(OH), 1662 (CO amide). H-NMR (DMSO-d6): δH (ppm)
1
8.53-7.63 (m, 8H, ArH), 4.89 (t, 1H, OH, J=6.9Hz,
exchangeable in D2O), 5.34 (s, 2H, NCH2O), 3.52 ( t, 2H,
CH2OCO, J = 5.5Hz), 3.34 (t, 2H, OCH2, J= 5.5 Hz), 2.23
(s, 3H, ArCH3) MS, m/z (%): 310 (M.+, 100.0%). Anal.
Calcd. for C18H18N2O3 (310): C, 69.66; H, 5.85; N, 9.03;
Found: C, 69.70; H, 5.73; N 9.10.
o
Yield (90%), mp.268-270 C. FT-IR (KBr) (cm-1): 3235
1
(NH), 1790, 1735, 1690, 1656 (4CO). H-NMR (DMSO-d6):
δH (ppm) 10.78 (br.s, 1H, NH, D2O exchangeable), 8.10-7.35
(m, 12H, ArH), 3.84 (s, 2H, NCH2). MS, m/z (%): 459 (M.+,
100.0%), 461 (M.++2, 33.0%),.13C-NMR (DMSO-d6) δ 170.1,
164.4(2), 158.6, 132.8, 132.5, 132.4, 132.2(3), 132.0(2), 130.5,
130.3, 130.1, 127.5, 126.5, 125.8, 123.7, 123.3(3), 123.1, 57.5,
Anal. Calcd. for C24H15ClN4O4 (459): C, 62.82; H, 3.30; N,
12.21; Found: C, 63.12; H, 3.39; N 12.38.
3.1.39 | 2-(4-Hydroxybutyl)-4-phenyl
phthalazin-1(2H)-one (15a)
Yield (91%), mp.112-113oC. FT-IR (KBr) (cm-1): υ 3445 (OH),
1
1661 (CO amide). H-NMR (DMSO-d6): δH (ppm) 8.39-7.54
3.1.43 | 2-(4-(2-Chlorophenyl)-
1-oxophthalazin-2(1H)-yl)-N'-(2-oxoindolin-
3-ylidene) acetohydrazide (17)
(m, 9H, ArH), 4.39 (t, 1H, J =5.5, OH, exchangeable with
D2O), 4.22 (t, 2H, CH2O, J = 6.6), 3.42 (t, 2H, NCH2,
J=6.6Hz), 1.79 (m, 2H, CH2), 1.43 (m, 2H, CH2). MS, m/z (%):
294 (M.+, 100.0%). Anal. Calcd. for C18H18N2 O2 (294): C,
73.45; H, 6.16; N, 9.52; Found: C, 73.50; H, 6.20; N 9.53.
A mixture of hydrazide 11b (0.01 mol, 3.28 g), isatin (0.01
mol, 1.4 g) and few drops of acetic acid in ethanol
(20 mL) was refluxed 10 h. After cooling, the obtained
solid was collected, filtered and recrystlised from ethanol.
Yield (92%), mp.218-220 oC. FT-IR (KBr) (cm-1): 3220,
3.1.40 | 4-(2-Chlorophenyl)-
2-(4-hydroxybutyl) phthalazin-1(2H)-
one (15b)
1
3170 (2NH), 1705, 1670, 1660 (3CO). H-NMR (DMSO-
d6): δH (ppm) 10.78 (br.s, 1H, NH, D2O exchangeable),
9.68 (br.s, 1H, NH, D2O exchangeable), 8.25-7.39 (m,
12H, ArH), 3.84 (s, 2H, NCH2). MS, m/z (%): 458 (M.+,
100.0%), 460 (M.++2, 33.0%).13C-NMR (DMSO-d6)
δ:170.9, 168.4, 158.5, 141.0, 134.7, 134.4, 132.8, 132.5,
132.4, 132.2, 131.2(2), 130.5, 130.3, 130.1, 129.3, 127.5,
126.8, 126.2, 124.2, 123.7, 119.1, 117.4, 57.5. Anal. Calcd.
for C24H16ClN5O3 (458): C, 62.96; H, 3.52; N, 15.30;
Found: C, 63.12; H, 3.59; N 15.38.
Yield (90%), mp.127-128 oC. FT-IR (KBr) (cm-1): 3450 (OH),
1
1657 (CO amide). H-NMR (DMSO-d6): δH (ppm) 8.38-7.56
(m, 8H, ArH), 4.38 (t, 1H, J =5.2, OH, exchangeable with
D2O), 4.19 (t, 2H, CH2O, J = 6.6), 3.43 (t, 2H, NCH2,
J=6.6Hz), 1.81 (m, 2H, CH2), 1.42 (m, 2H, CH2). MS, m/z (%):
294 (M.+, 100.0%). Anal. Calcd. for C18H17ClN2O2 (328): C,
65.75; H, 5.21; N, 8.52; Found: C, 65.79; H, 5.21; N 8.59.
3.1.41 | 2-(4-Hydroxybutyl)-4-(o-tolyl)
phthalazin-1(2H)-one (15c)
3.1.44 | 4-Aryl-2-(2-(3-methyl-5-oxo-
4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)
phthalazin-1(2H)-one (18) and 4-Aryl-
2-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-
2-oxoethyl)phthalazin-1(2H)-one (19)
Yield (95%), mp.121-123oC. FT-IR (KBr) (cm-1): 3465
1
(OH), 1661 (CO amide). H-NMR (DMSO-d6): δH (ppm)
8.38-7.35 (m, 8H, ArH), 4.41 (t, 1H, J =5.2, OH,
exchangeable with D2O), 4.16 (t, 2H, CH2O, J = 6.6), 3.41
(t, 2H, NCH2, J=6.6Hz), 2.10 (s, 3H, ArCH3), 1.79 (m, 2H,
CH2), 1.45 (m, 2H, CH2). MS, m/z (%): 308 (M.+, 100.0%).
A mixture of hydrazide 11b (0.01 mol, 3.28 g), ethyl
acetoacetate and/or acetylacetone (0.01 mol) and few
drops of acetic acid in ethanol (20 mL) was refluxed for

MAHMOUD AND ELSAYED
15
8 h. After cooling, the obtained solid was collected, fil-
tered and recrystlised from ethanol.
3.1.48 | 4-(2-Chlorophenyl)-
2-((5-mercapto-4-phenyl-4H-1,2,4-triazol-
3-yl) methyl)phthalazin-1(2H)-one (21)
3.1.45 | 4-(2-Chlorophenyl)-
2-(2-(3-methyl-5-oxo-4,5-dihydro-1H-
pyrazol-1-yl)-2-oxoethyl) phthalazin-1(2H)-
one (18)
A mixture of 20 (0.01 mol, 4.6 g) and aguous sodium
hydroxide 20% (20 mL) was refluxed 3 h, after cooling the
reaction mixture neutralized with dil. HCl. The solid that
formed was filtered off, dried and recrystlized from petro-
leum ether(80-100 oC).
Yield (75%), mp.170-172 oC. FT-IR (KBr) (cm-1): 1713,
1675, 1661 (3CO). 1H-NMR (DMSO-d6): δH (ppm)
8.15-7.36 (m, 8H, ArH), 4.04 (s, 2H, NCH2). 3.03 (s, 2H,
CH2 pyrazolone), 2.21 (s, 3H, CH3). MS, m/z (%): 394 (M.
+, 100.0%), 396 (M.++2, 33.0%).13C-NMR (DMSO-d6) δ
170.3, 163.4, 159.2, 158.3, 134.7, 132.8, 132.6, 132.4, 132.2,
131.2, 130.5, 130.3, 130.1, 127.5, 126.8, 126.2, 123.7, 55.5,
42.1, 16.2. Anal. Calcd. for C20H15ClN4O3 (394): C,
60.84; H, 3.83; N, 14.19; Found: C, 61.12; H, 3.59; N 14.38.
Yield (95%), mp.153-154 C. FT-IR (KBr) (cm-1):1659
o
(CO). 1H-NMR (DMSO-d6): δH (ppm) 12.86 (br.s, 1H, SH,
D2O exchangeable), 8.15-7.38 (m, 13H, ArH), 4.40 (s, 2H,
NCH2). MS, m/z (%): 445 (M.+, 100.0%), 447 (M.++2,
32.0%). Anal. Calcd. for C23H16ClN5OS (445): C, 61.95; H,
3.62; N, 15.71; Found: C, 62.11; H, 3.73; N 15.78.
3.1.49 | 4-(2-Chlorophenyl)-
2-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)
methyl) phthalazin-1(2H)-one (22)
3.1.46 | 4-(2-Chlorophenyl)-
2-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-
2-oxoethyl)phthalazin-1(2H)-one (19)
A mixture of 20 (0.01 mol, 4.6 g) and concentrated sul-
phric acid 70% (5 mL) was allowed to stand at room tem-
perature overnigh. The reaction mixture poured onto ice/
H2O. The solid that formed was filtered off, dried and
recrystlized from ethanol.
Yield (73%), mp.178-180 oC. FT-IR (KBr) (cm-1): 1675,
1661 (2CO). 1H-NMR (DMSO-d6): δH (ppm) 8.15-7.36 (m,
8H, ArH), 6.20 (s, 1H, pyrazole), 4.34 (s, 2H, NCH2). 2.50
(s, 3H, CH3 ), 2.21 (s, 3H, CH3). MS, m/z (%): 392 (M.+,
100.0%), 394 (M.++2, 32.0%).13C-NMR (DMSO-d6) δ:
168.1, 158.2, 152.3, 134.3, 132.8, 132.5, 132.4, 132.2, 131.2,
130.5, 130.3, 130.1, 127.5, 126.4, 126.2, 123.7, 110.0, 52.5,
12.9, 13.2. Anal. Calcd. for C21H17ClN4O2 (392): C,
64.21; H, 4.36; N, 14.26; Found: C, 64.12; H, 4.53;
N 14.38.
o
Yield (63%), mp.134-135 C. FT-IR (KBr) (cm-1): 3265
1
(NH), 1659 (CO). H-NMR (DMSO-d6): δH (ppm) 10.16
(br.s, 1H, NH, D2O exchangeable), 8.15-7.38 (m, 13H,
ArH), 4.20 (s, 2H, NCH2). MS, m/z (%): 445 (M.+, 100.0%),
447 (M.++2, 32.0%). Anal. Calcd. for C23H16ClN5OS
(445): C, 61.95; H, 3.62; N, 15.71; Found: C, 62.11; H, 3.73;
N 15.78.
3.1.50 | 2-(4-(2-Chlorophenyl)-
1-oxophthalazin-2(1H)-yl)-N'-
(4-methoxybenzylidene)
acetohydrazide (23)
3.1.47 | 2-(2-(4-(2-Chlorophenyl)-
1-oxophthalazin-2(1H)-yl)acetyl)-N-phenyl
hydrazine-1-carbothioamide (20)
A mixture of hydrazide 11b (0.01 mol, 3.28 g), phenyl iso-
thiocyanate (0.02 mol, 2.70 g) in absolute ethanol (30 mL)
was refluxed for 5 h. After cooling, the obtained solid was
collected, filtered and recrystlised from ethanol.
A mixture of hydrazide 11b (0.01 mol, 3.28 g), p-
anisaldehyde (0.01 mol,1.36 g) was refluxed in absolute
ethanol (30 mL) for 9 h. After cooling, the separated solid
was filtered, dried and recrystlised from ethanol.
o
o
Yield (83%), mp.196-198 C. FT-IR (KBr) (cm-1): 3320,
Yield (88%), mp.224-226 C. FT-IR (KBr) (cm-1): 3275
3275, 3195 (3NH), 1673, 1659 (2CO), 1243 (C=S). 1H-NMR
(DMSO-d6): δH (ppm) 11.30 (br.s, 1H, NH, D2O exchange-
able ), 10.78 (br.s, 1H, NH, D2O exchangeable ), 7.80 (br.s,
1H, NH, D2O exchangeable ), 8.15-7.10 (m, 13H, ArH),
3.98 (s, 2H, NCH2). MS, m/z (%): 463 (M.+, 100.0%),
465 (M.++2, 34.0%). Anal. Calcd. for C23H18ClN5O2S
(463): C, 59.54; H, 3.91; N, 15.10; Found: C, 59.51; H, 4.03;
N 15.38.
(NH), 1685, 1659 (CO). H-NMR (DMSO-d6): δH (ppm)
1
11.16 (br.s, 1H, NH, D2O exchangeable ), 8.49 (s, 1H,
Ar'CH=), 8.15-7.18 (m, 12H, ArH), 4.11 (s, 2H, NCH2),
3.86 (s, 3H, OCH3). MS, m/z (%): 446 (M.+, 100.0%),
448 (M.++2, 32.0%) 13C-NMR (DMSO-d6) δ 170.8, 162.4,
159.1, 144.3, 134.6, 132.8, 132.6, 132.4, 131.5, 131.1(2),
130.9, 130.4, 130.1(2), 127.5, 126.5, 126.2, 125.8, 123.1,
114.2(2), 57.3, 55.4. Anal. Calcd. for C24H19ClN4O3

16
MAHMOUD AND ELSAYED
(446): C, 64.50; H, 4.29; N, 12.54; Found: C, 64.56; H,
4.13; N 12.68.
3.2 | Experimental for Antimicrobial
activity
The samples were prepared by dissolving 2mg in 2mL of
DMSO and 100μl (containing 100μg) was used in this test.
The antimicrobial activity of different samples was inves-
tigated by the agar cup plate method. Four different test
microbes namely: Gram-positive bacteria, Gram-negative
bacteria and antifungal activity. Staphylococcus aureus
(Atcc 06538), Escherechia coli (Atcc 10536), pseudomonas
aeruginosa (Atcc 6404) and pathogenic fungi candida
albicans (Atcc 1023) were used. Nutrient agar plates were
heavily seeded uniformly with 1ml of 105-106 cells/ml in
case of bacteria and yeast. A Czapek-Dox agar plate
seeded by the fungus was used to evaluate the antifungal
activities. Then a hole was made in media by gel cutter
(Cork borer no.4) in sterile condition. Then one drop of
melted agar was poured into hole and allowed to solidify
to make a base layer. After that specific amount of cul-
ture filtrate (0.1 ml) was poured into the hole. Then
plates were kept at low temperature (4ꢀC) for 2-4 hours
to allow maximum diffusion. The plates were then incu-
bated at 37ꢀC for 24 hours for bacteria and at 30oC for
48 hours in upright position to allow maximum growth
of the organisms. The antimicrobial activity of the test
agent was determined by measuring the diameter of zone
of inhibition expressed in millimeter (mm). The experi-
ment was carried out more than once and mean of read-
ing was recorded.[28,29]
3.1.51 | 2-(4-(2-Chlorophenyl)-
1-oxophthalazin-2(1H)-yl)-N-
(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)
acetamide (24)
A mixture of 23 (0.01 mol, 4.46 g), and thioglycolic acid
(0.01 mol, 0.165 mL) was treated with few drops of piperi-
dine and heated on awater bath for 8 h. The reaction mix-
ture washed with petroleum ether (40-60 oC), the
separated solid was filtered, dried and recrystlised from
ethanol.
Yield (60%), mp.300-302 oC. FT-IR (KBr) (cm-1):
1
3275 (NH), 1705, 1676, 1659 (3CO). H-NMR (DMSO-
d6): δH (ppm) 11.16 (br.s, 1H, NH, D2O exchangeable ),
8.15-6.80 (m, 12H, ArH), 5.75 (s, 1H, S-CH), 4.11 (s,
2H, NCH2), 3.86 (s, 3H, OCH3), 3.45 (s, 2H, S-CH2) MS,
m/z (%): 520 (M.+, 100.0%), 522 (M.++2, 31.0%) 13C-
NMR (DMSO-d6) δ 170.4, 168.4, 159.1, 158.2, 144.3,
134.6, 132.8, 132.6, 132.4, 131.5, 131.1, 130.9, 130.4,
130.1(2), 129.4, 127.5, 126.5, 126.2, 125.8, 123.1, 114.2,
64.1, 57.3, 55.4, 35.2. Anal. Calcd. for C26H21ClN4O4S
(520): C, 59.94; H, 4.06; N, 10.75; Found: C, 60.10; H,
4.13; N 10.68.
3.1.52 | N'-(((2-Aminophenyl) thio)
(4-methoxyphenyl) methyl)-
2-(4-(2-chlorophenyl)-1-oxophth-alazin-2
(1H)-yl) acetohydrazide (25)
4 | CYTOTOXICITY ASSAY
4.1 | Materials and methods
A mixture of 23 (0.01 mol, 4.46 g), and 2-amino
thiophenol (0.015 mol, 0.5 mL) was treated with few
drops of piperidine and heated on awater bath for
8 h. The reaction mixture washed with petroleum ether
(40-60 oC), the separated solid was filtered, dried and rec-
rystlised from ethanol.
Cell line Hepatocellular carcinoma (HEPG-2), Mammary
gland (MCF-7) and Colorectal carcinoma (HCT-116). The
cell lines were obtained from ATCC via Holding com-
pany for biological products and vaccines (VACSERA),
Cairo, Egypt.
Yield (80%), mp.280-282 oC. FT-IR (KBr) (cm-1): 3424,
3339 (NH2), 3298, 3275 (NH), 1685, 1661 (2CO). 1H-NMR
(DMSO-d6): δH (ppm) 9.50 (br.s, 1H, NH, D2O exchange-
able), 8.87 (br.s, 1H, NH, D2O exchangeable), 8.15-6.88
(m, 16H, ArH), 5.50 (s, 1H, S-CH), 5.08 (br.s, 2H, NH2,
D2O exchangeable ), 4.18 (s, 2H, NCH2), 3.86 (s, 3H,
OCH3). MS, m/z (%): 571 (M.+, 100.0%), 573 (M.++2,
30.0%) 13C-NMR (DMSO-d6) δ 170.8, 160.4, 158.1, 148.0,
144.3, 135.6, 134.6, 134.2, 132.6, 132.4, 131.5, 130.9, 130.4,
130.1, 128.4 (2), 127.5, 126.5, 126.2, 125.8, 123.1, 119.6,
118.5 (2), 114.9, 114.2 (2), 71.1, 57.3, 55.4. Anal. Calcd. for
C30H26ClN5O3S(571): C, 62.99; H, 4.58; N, 12.24;
Found: C, 62.69; H, 4.63; N 12.18.
Doxorubicin was used as a standard anticancer drug
for comparison. Chemical reagents
The reagents RPMI-1640 medium, MTT and DMSO
(sigma co., St. Louis, USA), Fetal Bovine serum (GIBCO, UK).
4.2 | MTT assay
The cell lines mentioned above were used to determine
the inhibitory effects of compounds on cell growth using
the MTT assay. This colorimetric assay is based on the
conversion of the yellow tetrazolium bromide (MTT) to a
purple formazan derivative by mitochondrial succinate

MAHMOUD AND ELSAYED
17
dehydrogenase in viable cells. Cell lines were cultured in
RPMI-1640 medium with 10% fetal bovine serum. Anti-
biotics added were 100 units/ml penicillin and
100μg/ml streptomycin at 37oC in a 5% Co2 incubator.
The cell lines were seedes 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
concentration of compounds and incubated for 24 h.
After 24 h of drug treatment, 20 μl of MTT solution at
5mg/ml was added and incubated for 4 h. Dimethyl
sulfoxide (DMSO) in volume of 100 μl is added into
each well to dissolve the purple formazan formed. The
colorimetric assay is measured and recorded at absor-
bance of 570 nm using a plate reader (EXL 800, USA).
The relative cell viability in percentage was calculated
as (A570 of treated samples/A570 of untreated sample)
X 100.[30,31]
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
ORCID
Naglaa F. H. Mahmoud https://orcid.org/0000-0002-
6655-5741
Galal A. Elsayed https://orcid.org/0000-0002-5542-
667X
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5 | CONCLUSION
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A group of 2,4-disubstituted phthalazin-1(2H)-one
derivatives was synthesized via nucleophilic attack of
N-2 of phthalazin-1(2H)-one derivatives on different
alkyl halides. Furthermore, a reactive phthalazinone
acetohydrazide derivative was utilized as a scaffold to
synthesize different heterocyclic systems. Antibacterial
and antifungal assessment has been done on some of
the synthesized compounds. In addition, an evaluation
as antitumor agents for some of the synthesized was
run against three carcinoma cell lines named, mam-
mary gland carcinoma cell line (MCF-7), colorectal
carcinoma cell line (HCT-116) and human hepatocel-
lular carcinoma cell line (HepG-2). Some of the tested
compounds showed promising activities compared
with the anticancer standard drug (Doxorubicin).
Exclusively, the tested compounds 5b, 7b, 8b, 9b, 13b
exhibited remarkable activities. In addition, a molecu-
lar docking study of those phthalazinone derivatives
was performed by Molecular Operating Environment
(MOE) 2014.09 to investigate the binding affinity of
the tested compounds compared with the reference
1M17. These novel compounds may serve as leads
for the development of less toxic drugs with verstile
structural feature from those of the currently utilized
antifungal, antibacterial, and antitumor agents. The
structure-activity relationships discussed above can
provide useful information for further design and syn-
thesis of compounds with optimized bioactivity profile.
Further studies to optimize the structural feature as
well as to determine the mechanism of action of this
novel class of compounds is planned to start in near
future by our group.
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