Design and synthesis of new phthalazinone derivatives containing benzyl moiety with anticipated antitumor activity

Article abstract of DOI:10.1248/BPB.B15-00656

The acetohydrazide derivative reacted with carbon electrophiles such as acid chlorides, acetylacetone, ethyl acetoacetate and aromatic aldehydes to give some interesting heterocyclic compounds. The hydrazide derivative reacted with acetophenone which in turn underwent Vielsmeier-Haack reaction. Also, the phthalazinethione has been synthesized and its behavior towards hydrazine hydrate, oxidizing agent and ethyl chloroacetate has been investigated. The newly synthesized compounds were characterized by spectroscopic data. The antimicrobial, the cytotoxic, and the antioxidant activities of some of the synthesized products were evaluated. Some of the tested compounds showed very strong cytotoxic activity with respect to the standard.

Full text of DOI:10.1248/BPB.B15-00656

Vol. 39, No. 2
Biol. Pharm. Bull. 39, 239–251 (2016)
239
Regular Article
Design and Synthesis of New Phthalazinone Derivatives Containing
Benzyl Moiety with Anticipated Antitumor Activity
,a
Magda Ismail Marzouk,* Soheir Ahmad Shaker,a Aisha Ali Abdel Hafiz,a and
Khaled Zakaria El-Baghdadyb
a Department of Chemistry, Faculty of Science, Ain Shams University; Abassia, Cairo 11566, Egypt: and b Department
of Microbiology, Faculty of Science, Ain Shams University; Abassia, Cairo 11566, Egypt.
Received August 25, 2015; accepted November 9, 2015
The acetohydrazide derivative reacted with carbon electrophiles such as acid chlorides, acetylacetone,
ethyl acetoacetate and aromatic aldehydes to give some interesting heterocyclic compounds. The hydra-
zide derivative reacted with acetophenone which in turn underwent Vielsmeier–Haack reaction. Also, the
phthalazinethione has been synthesized and its behavior towards hydrazine hydrate, oxidizing agent and
ethyl chloroacetate has been investigated. The newly synthesized compounds were characterized by spec-
troscopic data. The antimicrobial, the cytotoxic, and the antioxidant activities of some of the synthesized
products were evaluated. Some of the tested compounds showed very strong cytotoxic activity with respect to
the standard.
Key words phthalazinone; acetohydrazide; antitumor activity; antibacterial activity
Heterocyclic compounds containing hydrazine have re- with IC50 of 71nM, also, it showed in vitro cytotoxic activity
ceived considerable attention because of their pharmacological against HCT116 colon cell line with IC50 of 5.44nM.
properties and clinical applications.1–3) The diverse biologi-
Tubulin binding molecules29) have gained much interest
cal activities of various functional derivatives of 4-substi- among cytotoxic agents due to its success in clinical oncol-
tuted alkyl-1-(2H)phthalazinones are well known. Some of the ogy. They differ from the other anticancer drugs in their mode
phthalazinone derivatives have found application in clinical of action because they target the mitiotic spindle and not the
medicine due to their pronounced antihypertensive proper- DNA, for example, E7010 (V).
ties,4) cardiotonic,5,6) anticonvulsant,7,8) antidiabetic,9,10) anti-
Oltipraz (VI) and related dithiolethione derivatives30) are an
thrombotic,11) antimicrobial,12,13) vasorelexant,14) antipyretic, important class of chemopreventive agents that enhance the
analgesic, antitumor,15–18) and cytotoxic,19) while others have expression of carcinogen detoxication and antioxidant agents.
shown interesting vasodilator,20) vascular endothelial growth Since, many synthetic compounds have been used to protect
factor receptor (VEGFR) II inhibitory activities,21) and tu- animals against a variety of cancer, hydrogen sulfide30) has
berculostatic activity allergic rhinitis.22) Phthalazin-1-(2H)- been shown to be a potent endogenous gaseous mediator in
ones bearing a substituent at 4-position represent key inter- many physiological and pathological processes. Hydrogen sul-
mediates in the synthesis of various compounds with high fide (H2S) in mammalian tissues is produced from L-cysteine,
interesting pharmacological properties such as the blood the sulfhydryl group (SH) be the essential species responsible
platelet aggregation inhibitor23) MV-5445 [1-(3-chloroanilino)- for the biological activities of these compounds (Fig. 1).
4-phenylphthalazine] which has been found to be a selec-
The observations outlined above, enthused us to develop
tive phosphodiesterase V inhibitor24) or the thromboxane A2 new phthalazinone derivatives and to investigate their activi-
synthetase inhibitor or bronchodilator, 2-[2-(1-imidazolyl)- ties as antimicrobial, anticancer and antioxidant agents.
ethyl]-4-[3-pyridyl]phthalazine-1-(2H)one.25)
During the last two decades there is a growing interest
in the synthesis of several phthalazines as promising drug
candidates for the treatment of cancer. The latter research
RESULTS AND DISCUSSION
In the present investigation, we have reported the syn-
efforts have led to the discovery of several leading phthal- thesis of some new phthalazinone derivatives. Some of the
azines with different cellular and enzymatic targets. Inhibitors newly synthesized compounds were tested for their biological
of poly(adenosine diphosphate-ribose)polymerases (PARPs) activity. A convenient four step procedure was used to syn-
family of proteins are currently being evaluated as potential thesize the target 2-(4-benzyl-1-oxophthalazin-2(1H)-yl)-
anticancer medicines at both preclinical and clinical levels.26) acetohydrazide 4 as outlined in Chart 1.
A series of 4-substituted-2H-phthalazin-1-ones have been
The commercially available phthalic anhydride was fused
investigated as potent orally bioavailable PARP inhibitor.27) with phenylacetic acid in the presence of fused sodium acetate
For example, Olaparib (I), MRU-868 (II) and KU0058958 in an oil bath at 180°C to afford the 3-benzylidenephthalide
(III) are the most interesting PARP inhibitors based on the 1, which was then reacted with hydrazine hydrate31) in boil-
4-substituted-2H-phthalazin-1-ones scaffold (Fig. 1).
ing ethanol to give the benzylphthalazinone derivative 2.
Interestingly, Prime et al.28) have reported a novel class of Oxidation of the phthalazinone derivative 2 with KMnO4 in
potent, selective, and orally bioavailable inhibitors of aurora- the presence of K2CO3 was failed to give 2,3-dihydrophthal-
A kinase based upon a 4-(pyrazole-3-ylamino)phenyl-2H- azine-1,4-dione A, this result indicated that the phthalazinone
phthalazin-1-one scaffold. Compound (IV) inhibits aurora-A derivative 2 exists in the tautomer form 2a not 2b (Chart 1).
*To whom correspondence should be addressed. e-mail: magda_marzouk@sci.asu.edu.eg
© 2016 The Pharmaceutical Society of Japan

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Biol. Pharm. Bull.
Vol. 39, No. 2 (2016)
Fig. 1. Structure of the Lead Anticancer Derivatives (I)–(VI) and the Designed Target Compounds 4, 12a, c, 15, 16 and 19
Chart 1. Synthesis of Compounds 1–5
Subsequent interaction of the benzylphthalazinone deriva- signals attributed to CH3, CH2, CH2Ph, CH2CO and aromatic
tive 2a with ethyl chloroacetate in boiling acetone in the pres- protons at δ 1.22, 4.16, 4.33, 4.97, 7.09–8.48ppm, respectively.
ence of anhydrous K2CO3 led to the oxophthalazinyl acetate The oxophthalazinyl acetate 3 was then reacted with hydrazine
derivative 3. The structure of compound 3 was inferred from hydrate in boiling ethanol to give the desired acetohydrazide
spectroscopic data. The IR showed the presence of 2C=O derivative 4. The structure of compound 4 was inferred from
1
bands at 1735 and 1665cm−1. The H-NMR spectrum showed spectroscopic data. The IR showed the absence of the C=O

Vol. 39, No. 2 (2016)
Biol. Pharm. Bull.
241
Chart 2. Synthesis of Compounds 6–11
band attributed to the ester group and the appearance of bands corresponding to C=O group of the pyrazolone ring. The
at 3290 and 3318cm−1 attributed to NH and NH2 groups. The 1H-NMR spectrum showed a singlet signal for CH3 protons
1H-NMR spectrum devoid of any signals attributed to protons at δ 1.55ppm and a signal for CH2 protons group of the
of ethyl group; instead it showed signals attributed to NH and pyrazolone ring at δ 2.28ppm, beside signals corresponding
NH2 protons at δ 4.56 and 9.31ppm, respectively, as well as to CH2Ph, CH2CO and aromatic protons at δ 3.44, 4.29 and
signals corresponding to CH2Ph, CH2CO and aromatic protons 7.26–8.70ppm, respectively. The mass spectrum showed the
at δ 4.30, 4.75 and 7.29–8.27ppm, respectively. molecular ion peak at m/z 374 which is in agreement with the
A chemical prove for the structure of compound 3 was molecular weight.
gained from its reaction with benzaldehyde in the presence of
Furthermore, 4′-fluoro acetophenone in methanol contain-
piperidine that yielded the adduct 5, which was devoid of sig- ing a catalytic amount of glacial acetic acid was reacted with
1
nal corresponding to CH2CO protons in its H-NMR spectrum. compound 4 to give ethylidene acetohydrazide derivative 9.
Interaction of the acetohydrazide 4 with acetyl chloride The structure of compound 9 was confirmed by spectroscopic
and benzoyl chloride in the presence of dry benzene afforded data. The IR spectrum showed bands at 3214, 1714 (the high
the oxadiazol derivatives 6a and b (Chart 2). The structure value is due to mutual induction), 1655 and 1614cm−1 at-
of compounds 6a and b were confirmed from spectroscopic tributable to NH, 2C=O and C=N groups, respectively. The
data. The reaction proceeded via nucleophilic attack followed 1H-NMR spectrum showed two singlet signals for CH3 and
by ring closure to afford the desired oxadiazol derivatives 6a NH protons at δ 1.85 and 10.32ppm, respectively, in addi-
and b.
tion to signals corresponding to CH2Ph, CH2CO and aro-
Treatment of the acetohydrazide 4 with acetyl acetone matic protons at δ 4.30, 4.87 and 7.16–8.27ppm, respectively.
and ethyl acetoacetate afforded the pyrazol derivatives 7 and Further confirmation for the structure of compound 9 was
8, respectively. The structures of compounds 7 and 8 were achieved through its reaction with Vilsmeier–Haack reagent
1
corroborated by spectroscopic data. The H-NMR spectrum (N,N-dimethyl formamide (DMF)/POCl3) that yielded the
1
of compound 7 showed the presence of 2CH3 groups at δ pyrazole carbaldehyde derivative 10. The H-NMR spectrum
2.02ppm, in addition to a singlet signal at δ 5.63ppm for the of compound 10 was in a good agreement with the suggested
proton of the pyrazole ring. Also it showed signals at δ 4.33, structure (cf. Experimental); it showed the disappearance of
5.00 and 7.18–8.28ppm for CH2Ph, CH2CO and aromatic pro- the signals corresponding to the methyl and the NH protons.
tons respectively. The structure of compound 8 was supported The mass spectrum of compound 10 showed the molecular ion
from its IR spectrum which displayed a band at 1731cm−1 peak at m/z 466 which is coincident with the molecular weight

242
Biol. Pharm. Bull.
Vol. 39, No. 2 (2016)
Chart 3. Vilsmeier–Haack Reaction Mechanism
Chart 4. Synthesis of Compounds 12–15
(466) as supported the identity of the structure. A possible at m/z 437 which is in agreement with the molecular weight.
mechanism for the formylation of compound 9 is explained in
Interaction of the acetohydrazide 4 with the appropriate
Chart 3.
aldehydes namely benzaldehyde, 4-fluorobenzaldehyde, and
Treatment of the acetohydrazide 4 with isatin in ethanol and 4-methoxybenzaldehyde in boiling ethanol yielded the cor-
1
drops of acetic acid yielded oxoindolinylidene acetohydrazide responding Schiff bases 12a–c (Chart 4). The H-NMR spec-
derivative 11. Its IR spectrum showed carbonyl absorption tra of compounds 12a–c were devoid of any signals for NH2
bands in addition to the NH absorption band 1719, 1646 and group and showed signals attributed to CH2Ph, NH, CH2CO,
3147 cm−1, respectively. Further evidence was gained from =CH and aromatic protons in the range of δ 4.30–4.92,
1H-NMR spectrum as it exhibited signals for 2NH at 11.25 5.07–5.15, 5.31–5.53, 8.25–8.96 and 6.99–8.46ppm, respective-
and 12.67, beside signals corresponding to CH2Ph, CH2CO ly. Also compound 12c showed a singlet signal at δ 3.80ppm
and aromatic protons at δ 4.34, 5.47 and 6.93–8.30ppm, re- attributed to OCH3 protons. Further evidence were gained
spectively. The mass spectrum showed the molecular ion peak from mass spectra as they showed the correct molecular ion

Vol. 39, No. 2 (2016)
Biol. Pharm. Bull.
243
Chart 5. Reaction of Compound 4 with Methoxy Benzylidene
Chart 6. Synthesis of Compounds 16–20
peaks for compounds 12a and b and [M+−CH3, +3H] for
compound 12c beside some important peaks.
The reaction of thiol to form a carbon–sulfur bond consti-
tutes a key reaction in biosynthesis as well as in the synthesis
The reaction of compound 4 with 2-(4-methoxybenzylidene)- of biologically active compounds.32) The schiff base 12b was
malononitrile afforded the methoxy benzylidene derivative submitted to react with thiophenol in benzene with stirring
12c not the pyrazolone derivative 12′c. The amino group in to yield the thia type adduct 14. The structure of compound
compound 4 underwent nucleophilic addition reaction to the 14 was corroborated by spectroscopic data. The IR spectrum
double bond of 2-(4-methoxybenzylidene)malononitrile via showed bands at 3298, 1734, 1653cm−1 attributed to NH and
Michael type addition reaction, by refluxing in ethanol con- 2C=O, the high value of νC=O is due to mutual induction.
taining few drops of piperidine to give a substance whose The 1H-NMR spectrum showed signals attributed to NH,
structure should be either the pyrazole derivative 12′c (route [CH2Ph, CH2CO], SCH, aromatic protons, at δ 1.85 4.24–4.31,
a) or the methoxy benzylidene derivative 12c (route b). The 5.01, 7.21–8.44ppm, respectively.
actual structure of the product was assigned as the methoxy
Reaction of acetohydrazide 4 with benzenesulfonyl chlo-
benzylidene derivative 12c based on the spectroscopic data. ride in pyridine yielded compound 15. The structure of the
The IR spectrum devoid any signal for C≡N group. The synthesized compound 15 was elucidated by studying its IR,
1
proton H-NMR spectrum showed signals attributed to NH 1H-NMR, mass spectra, and elemental analysis. The IR spec-
and=CH protons, and was devoid of NH2 signal which should trum showed bands at 1343 and 1170cm−1 attributed to SO2
have appeared if the reaction product was 12′c (Chart 5).
group beside bands for NH and C=O at 3220, 1703, 1635cm−1.
1
The Schiff bases 12a and b were allowed to react with The H-NMR spectrum showed signals attributed to CH2Ph,
thioglycolic acid in boiling benzene to yield thiazolidinyl CH2CO, aromatic and NH protons at δ 4.27, 4.71, 7.19–8.26,
acetamide derivatives 13a and b, respectively (Chart 4). The 9.74, 9.98ppm, respectively. The mass spectrum showed the
reaction takes place via thia addition type on azamethine molecular ion peak at m/z 448 which is in agreement with the
moiety followed by 5-exo-trig ring closure. The structures molecular weight.
of compounds 13a and b were corroborated by spectroscopic
Treatment of the phthalazinone derivative 2a with phos-
data. The 1H-NMR spectrum showed signals attributed to phorus pentasulfide in boiling dry toluene yielded the phthal-
CH2Ph, CH2CO, SCH2, aromatic and NH protons in the range azinethione derivative 16. The 1H-NMR spectrum showed
of δ 3.88–4.19, 4.26–4.32, 4.78–4.96, 5.33–5.80, 7.16–8.29, signals attributed to CH2Ph, aromatic and NH protons at δ
10.59–11.71ppm, respectively. The mass spectra of compounds 4.37, 7.24–8.91, 11.80ppm, respectively. The IR spectrum
13a and b showed the correct molecular ion peaks at m/z 470 showed bands at 3159 and 1242cm−1 attributed to NH, C=S
and 488, respectively.
and devoid any band of C=O. The mass spectrum showed the

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Biol. Pharm. Bull.
Vol. 39, No. 2 (2016)
molecular ion peak at m/z 252 which is in agreement with the pounds 12c, 17–19 (Fig. 3 and Table 1). For other compounds
molecular weight. no activity on Staphylococcus aureus was detected. For
The reaction of compound 16 with hydrazine hydrate in Gram-negative clinical isolates only compound 7 significantly
boiling ethanol yielded the hydrazine derivative 17 (Chart 6). inhibited both Escherichia coli and Pseudomonas aeruginosa,
The IR spectrum revealed NH2 and NH stretching bands at while compounds 12c and 19 inhibited only Escherichia coli.
1
3379 cm−1. While the H-NMR spectrum revealed the presence Both isolates were resistant to erythromycin.
of the NH2 and NH protons as a broad singlet at 4.78ppm, in
The mode of action of compounds 17 and 18 was differ-
addition to the other aromatic protons appearing as multiplet ent from compounds 7, 12c and 19, since they inhibited the
at 7.27–8.42ppm and to CH2Ph at 4.30ppm.
growth of Gram-positive clinical isolate, while, compound
Oxidation33) of the phthalazinethione 16 with concentrated 7 inhibited the growth of Gram-negative clinical isolates
HNO3 at room temperature afforded the sulfonic acid deriva- growth. Compounds 12c and 19 were able to inhibit both
tive 18. The structure of compound 18 was inferred from its isolates.
solubility in sodium bicarbonate solution and spectroscopic
Sulfones are competitive inhibitors of folic acid synthesis
1
data. The H-NMR spectrum revealed the presence of the OH and are used as bacteriostatic and in the treatment of Lep-
proton as a singlet signal at 13.25ppm beside CH2Ph and aro- rosy.34) Many of aryl sulfones are used as antifungal, antibac-
matic protons at δ 3.35 and 7.90–9.00ppm, respectively.
On the other hand, oxidation of the phthalazinethione 16
with chlorine gas in acetic acid gave the sulfonyl chloride
derivative 19. The analytical and the spectral data are in a
good agreement with the proposed structure. The 1H-NMR
spectrum revealed the presence of signals at δ 4.29 and
7.18–8.26ppm attributed to CH2Ph and aromatic protons. The
mass spectrum of compound 19 showed the correct molecular
ion peaks at m/z 318.
Alkylation of the phthalazinethione 16 with ethyl chloro-
acetate in the presence of anhydrous K2CO3 in boiling acetone
yielded the ethyl acetate derivative 20. The reaction takes
place via SN2 mechanism in which the lone pair of the sulfur
atom attacks the chloro moiety ester, while the function of
K2CO3 is to pull off the chloride ion. Here the authors offer
a speculation to explain the activities of the thioamide and
iminothiol equilibrium based on their thermodynamic and ki-
netic controlled formation under the experimental conditions.
Fig. 3. Diameter of Inhibition Zones for Effective Compounds on A)
Staphylococcus aureus, B) Escherichia coli and C) Pseudomonas aeru-
ginosa
Firstly, in the presence of anhydrous K2CO3 and acetone, the
conjugate base of iminothiol tautomer is more thermodynami-
cally stable than the conjugate base derived from thioamide
V: vancomycin; E: erythromycin and T: tetracyclin.
tautomer via the back donation involving the vacant D-orbital
of the sulfur atom. Therefore, the iminothiol tautomer is more
Table 1. The Antibacterial Activities of Some Compounds
predominant under such conditions. Secondly, the sulfur anion
is a stronger nucleophile than the nitrogen anion (nucleophilic-
ity is kinetic control). Thus, the iminothiol tautomer is more
thermodynamically and kinetically formed than thioamide
tautomer, which spells out the reactivity of the iminothiol tau-
tomer (Fig. 2). The structure of compound 20 was confirmed
from spectroscopic data. The IR showed band at 1737cm−1
Diameter of inhibition zones (cm)
Compd. no.
Staphylococcus
aureus
Escherichia
coli
Pseudomonas
aeruginosa
DMSO
0
0
0
3
0
0
0
1
attributed to C=O of the ester group. The H-NMR spectrum
4
0
0
0
revealed the presence of signals at δ 1.19, 4.12–4.17, 4.30, 4.69,
7.15–8.28ppm attributed to CH3, CH2, CH2Ph, CH2S and aro-
matic protons, respectively.
6a
0
0
0
6b
0
0
0
7
0
1.7 0.05
1.9 0.05
12a
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Pharmacology
Antibacterial Activity
Staphylococcus aureus was significantly inhibited by com-
12b
0
0
12c
2.4 0.01
0.37 0.01
13a
0
0
16
0
0
17
18
1.7 0.05
2.4 0.1
3.0
0
0
19
3.12
20
0
0
Vancomycin
Erythromycin
Tetracycline
2
3
0.05
0.1
0
0
1.8 0.05
1.1 0.05
Fig. 2. Thioamide–Iminothiol Tautomerism

Vol. 39, No. 2 (2016)
Biol. Pharm. Bull.
245
Fig. 4. Cytotoxic Activity of the Tested Compounds on Different Cell Lines
Table 2. Cytotoxicity (IC50) of the Tested Compounds on Different Cell Lines
Compd. no.
IC50 (µg/mL)a)
HePG2
HCT-116
PC3
MCF-7
4
12a
12c
15
29.9 1.33
85.9 5.71
53.8 4.32
7.8 0.23
5.6 0.22
93.7 5.97
95.1 6.42
7.9 0.12
12.9 0.96
96.5 6.65
62.9 2.97
8.5 0.31
7.7 0.25
93.6 6.28
>100
26.9 1.57
95.4 5.30
66.6 4.36
8.9 0.51
8.6 0.34
>100
19.2 1.64
94.9 5.18
41.2 2.96
8.9 0.62
5.0 0.41
>100
16
19
20
>100
8.3 0.25
>100
5.4 0.21
5-FU
5.3 0.14
a) IC50 (µg/mL): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), above 100 (non-cytotoxic).
terial or antitumor agents35) and inhibitors for several36) cyclic (12.9 0.69µg/mL), and MCF-7 cell (19.2 1.64 µg/mL), also it
oxygenase-2 (COX-2). Compounds containing C=N may act showed moderate activity towards HePG-2 cell (29.9 1.33 µg/
as a Michael acceptor for nucleophiles containing NH2 or mL) and PC3 cell (26.9 1.57 µg/mL). While compounds 12a,
sulfhydryl SH groups. In case of enzymes they are converted c, 19 and 20 observed activity ranged from moderate to non-
from the active form to the inactive form by these compounds. cytotoxic with IC50 41.2 2.96 to >100. [The relative viability
Some of these enzymes are responsible for building cell walls of cells (%) for the tested compounds is depicted in Tables S1
of bacteria and /or fungi; which explain the activities of com- and S2 (Supplementary materials)].
pounds 7, 12c, 17–19.
Antioxidant Activity Using 2.2′-Azino-bis(3-ethylbenz-
Antitumor Activity Using in Vitro Ehrlich Ascites Assay thiazoline-6-sulfonic (ABTS) Acid Inhibition Seven com-
We assessed the cytotoxic action of the compounds listed in pounds were tested for antioxidant activity as reflected in
Table 2 against four human tumor cell lines namely; hepa- the ability to inhibit oxidation in rat brain and kidney ho-
tocellular carcinoma (liver) HePG2, colon cancer HCT-116, mogenates Table 3. Compounds 16 and 15 showed high %
human prostate cancer cell line PC3, and mammary gland inhibition 85.3, 78.4%, respectively. While the rest of the
breast MCF-7. In general, activity was observed by all of compounds 4, 12a, c, 19 and 20 exhibited moderate to weak
these molecules ranged from very strong to non-cytotoxic. antioxidant activity ranged from 53.9–30.1%.
The optimal results were observed for compounds 15 and
Bleomycin-Dependent DNA Damage The bleomycin is
16 (very strong activity) with IC50 7.8 0.23, 5.6 0.22µg/ a family of glycopeptides antibiotics that are used routinely
mL for HePG-2 cell, 8.5 0.31, 7.7 0.25µg/mL for HCT-116 as antitumor agents. The bleomycin assay has been adopted
cell, 8.9 0.51, 8.6 0.34µg/mL for PC3 cell and 8.9 0.62, for assessing the pro-oxidant effect of food antioxidants. The
5.0 0.41 µg/mL for MCF-7 cell, respectively. Compound 16 antitumor antibiotic bleomycin binds iron ions and DNA. The
exhibited stronger activity (IC50 5.6 0.22, 0 0.41 µg/mL) than bleomycin–iron complex degrades DNA when heated with
the 5-fluorouracil (5-FU) as a standard (7.9 0.12, 5.4 0.21 µg/ thiobarbituric acid (TBA) to yield a pink chromogenic. Upon
mL) for HePG2 and MCF-7, respectively. Also it showed ap- the addition of suitable reducing agents antioxidant competes
proximately equal activity to the 5-FU as a standard for PC3 with DNA and diminishes chromogenic formation.37)
cell line with IC50 8.6 0.34 and 8.3 0.25µg/mL, respectively.
To show the mechanism of action of the tested compounds
Compound 15 showed approximately equal activity to the 4, 12a, c, 15, 16, 19 and 20, their protective activity against
ascorbic acid as a standard for HePG2 and PC3 cells with IC50 DNA damage induced by the bleomycin–iron complex were
7.8 0.23, 8.9 0.51 and 7.9 0.12, 8.3 0.25µg/mL, respective- examined. The results in Table 3 showed that compounds 4,
ly. Compound 4 showed strong activity towards HCT-116 cell 12a, c, 15, 16, 19 and 20 have the ability to protect DNA from

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Biol. Pharm. Bull.
Vol. 39, No. 2 (2016)
Table 3. Antioxidant Activity and Bleomycin Dependent DNA Damage
for the Tested Compounds
duces the cytotoxic activity against all cell line may be due
to the lack of the sulfhydryl group.
Antioxidant activity (ABTS method)
Bleomycin dependent
CONCLUSION
Compd. no.
DNA damage
Absorbance
Inhibition (%)
The objective of the present study was to synthesize the
phthalazinone based compounds scafold and study their
antibacterial, cytotoxicity, antioxidant and bleomycin depen-
dent DNA damage activities. Staphylococcus aureus was
significantly inhibited by compounds 12c, 17–19. For other
compounds no activity on Staphylococcus aureus was de-
tected. For Gram-negative clinical isolates only compound 7
significantly inhibited both Escherichia coli and Pseudomo-
nas aeruginosa, while compounds 12c and 19 inhibited only
Escherichia coli. Both isolates were resistant to erythromycin.
The tested compounds showed very strong to non-cytotoxic
activity against four anticancer cell lines. The best results
4
0.222
0.337
0.268
0.104
0.071
0.300
0.302
0.053
53.9
30.1
44.4
78.4
85.3
37.7
37.3
89.0
0.096
0.137
0.088
0.072
0.096
0.130
0.142
0.073
12a
12c
15
16
19
20
Ascorbic acid
All experiments were performed three times. The data are expressed as the
mean standard error of the mean (S.E.M.).
the induced damage by bleomycin. Compound 15 showed were observed for compounds 15 and 16 (very strong activ-
very high protection (0.072) against DNA damage induced by ity). Compound 15 showed approximately equal activity to the
the bleomycin–iron complex which is approximately equal to 5-FU as a standard against HePG2 and PC3 cells. Compound
ascorbic acid as a standard (0.073). Compounds 4, 12c and 16 16 exhibited stronger activity than the 5-FU as a standard
showed high ability. On the other hand, the rest of the com- against HePG2 and MCF-7.
pounds 12a, 19 and 20 exhibited moderate activities. Thus,
all the tested compounds diminish the chromogenic formation
between the damage DNA and TBA.
EXPERIMENTAL
Structure–Activity Relationship (SAR) DNA is made of
Chemistry All melting points were uncorrected. IR spec-
chemical building blocks called nucleotides. The four types of tra were recorded in KBr on FTIR Mattson Spectrometers.
nitrogen bases found in nucleotides are: adenine (A), thymine 1H-NMR spectra were measured on Varian Gemini 300MHz
(T), guanine (G) and cytosine (C). The base adenine always instrument with chemical shift (δ) expressed in ppm downfield
pairs with thymine, while guanine always pairs with cytosine from TMS. MS were recorded on Shimadzu GC-MS-QP 1000
through hydrogen bond. The cytotoxic activity towards dif- Ex and MS_5988 instruments at 70eV. All the spectral mea-
ferent cell lines depends on two factors: (i) The formation surements as well as elemental analyses were carried out at
of intermolecular hydrogen bond with DNA bases (ii) the the Micro analytical Center of Cairo University, Egypt and at
positive charge on the tested compounds attracted to the nega- Faculty of Pharmacy, Ain Shams University. Compound 2 was
tive charge on the cell wall. By comparing the experimental prepared according to the literature procedure.31) All products
1
cytotoxicity of the compounds reported in this study to their were characterized by IR; H-NMR; mass spectroscopy and
structures, the following SAR were postulated.
elemental analyses.
・Compound 16 showed very strong activity, this is due to
Synthesis of Ethyl 2-(4-Benzyl-1-oxophthalazin-2(1H)-
the presence of the sulfhydryl group (SH) and NH groups yl)acetate (3) A solution of 2 (2.36g, 0.01mol), ethyl chlo-
which may be added to any unsaturated moiety in DNA roacetate (4.4g, 0.04mol) and anhydrous potassium carbonate
(thia or aza Michael addition) or forming hydrogen bond (5.5g, 0.04mol) in acetone (60mL) was heated on a water bath
with either one of the nucleobases of the DNA and causes it for 20h. The excess solvent was evaporated and the reaction
damage.
mixture was poured on water. The separated solid was filtered
・Compound 15 showed very strong activity, this is due to the off and recrystallized from petroleum ether 60–80°C to give
presence of 2NH groups available to form hydrogen bond compound 3. Violet crystals; yield (2.1g, 66%); mp 128–130°C
with either one of the nucleobases of the DNA and causes it (Lit.,36) mp 76–78°C); 1H-NMR (CHCl3) δ: 1.22 (3H, t,
damage. Also the presence of the SO2Ph group as a strong J=6.0Hz), 4.16 (2H, q, J=6.0Hz), 4.33 (2H, s), 4.97 (2H, s),
electron attracting group rendered the molecule positively 7.09–8.48 (9H, m). IR (KBr) cm−1: 1735, 1665. MS m/z: 322
charged forming electrostatic attraction with the DNA (M+), 249, 91. Anal. Calcd for C19H18N2O3: C, 70.79; H, 5.63;
nucleobases. Moreover, the SO2 group acts on the mitotic N, 8.69. Found: C, 70.80; H, 5.58; N, 8.64.
spindle.29)
Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)ace-
・Compound 4 contains NH and NH2 groups forming hydro- tohydrazide (4) A mixture of 3 (3.22g, 0.01mol) and hy-
gen bond with either one of the nucleobases of the DNA drazine hydrate (0.05mL, 0.01mol) in ethanol (30mL) was
and causes it damage.
refluxed for 3h. The separated solid was filtered off and re-
・Incorporation of –N=CHAr moiety has rendered com- crystallized from ethanol to give compound 4. White crystals;
pounds 12a and c with moderate to weak cytotoxicity ac- yield (2.6g, 85%); mp 184–186°C (Lit.,31) mp 130–132°C);
tivities against the four tumor cell types.
1H-NMR (DMSO-d6) δ: 4.30 (2H, s), 4.56 (2H, s), 4.75 (2H,
・Compounds 12a and c contain only 1NH groups forming s), 7.26–8.27 (9H, m), 9.31 (1H, s). IR (KBr) cm−1: 3290, 3318,
hydrogen bond with either one of the nucleobases of the 1661, 1615. MS m/z: 308 (M+), 277, 91.05. Anal. Calcd for
DNA and causes it damage but to a little effect.
C17H16N4O2: C, 66.22; H, 5.23; N, 18.17. Found: C, 66.36; H,
・Conversion of compound 16 to compounds 19 and 20 re- 5.27; N, 18.20.

Vol. 39, No. 2 (2016)
Biol. Pharm. Bull.
247
Synthesis of Ethyl 2-(4-Benzyl-1-oxophthalazin-2(1H)- from ethanol to give 9. Brown crystals; yield (3.4g, 80%); mp
1
yl)-3-phenylacrylate (5) A mixture of 3 (3.22g, 0.01mol), 220–222°C; H-NMR (DMSO-d6) δ: 1.85 (3H, s), 4.30 (2H, s),
benzaldehyde (1mL, 0.01mol) and piperidine (1mL) in etha- 4.87 (2H, s), 7.16–8.27 (13H, m), 10.32 (1H, s). IR (KBr) cm−1:
nol (30mL) was refluxed for 3h. The reaction mixture was 3214, 1714, 1655, 1614. MS m/z: 428 (M+, 0.00), 249, 91. Anal.
cooled and the separated solid was filtered off and recrystal- Calcd for C25H21FN4O2: C, 70.08; H, 4.94; N, 13.08. Found: C,
lized from petroleum ether to give compound 5. White crys- 70.36; H, 4.96; N, 13.12.
1
tals; yield (2.6g, 64%); mp 174–176°C; H-NMR (DMSO-d6)
Synthesis of 1-(2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-
δ: 1.22 (3H, t, J=6.0Hz), 4.15–4.23 (2H, q, J=6.0Hz), 4.91 acetyl)-3-(4-fluorophenyl)-1H-pyrazole-4-carbaldehyde
(2H, s), 7.09–8.43 (15H, m). IR (KBr) cm−1: 3100, 1735, 1656. (10) POCl3 (0.612g, 0.004mol) was added to cold DMF
MS m/z: 396 (M+−CH3, +H·), 294, 250, 249, 207, 176, 178, (0.117g, 0.0016mol) at 0°C, and a solution of compound 9
130. Anal. Calcd for C26H22N2O3: C, 76.08; H, 5.40; N, 6.82. (0.25g, 0.0006mol) in DMF (10mL) was added dropwise, then
Found: C, 76.11; H, 5.43; N, 6.86.
the reaction mixture was heated on water bath at 65–70°C
General Procedure for the Synthesis of 6a, b A solu- for 4h; the reaction mixture was cooled and then poured into
tion of 4 (3.08g, 0.01mol), acetyl chloride or benzoyl chloride cold water; the separated solid was filtered off, washed with
(0.01mol) in benzene (30mL) was refluxed for 4h. The sepa- water, dried and recrystallized from benzene/petroleum ether
rated solid was filtered off dried and recrystallized from the 60–80°C (2:1) to give 10. White crystals; yield (1.8g, 40%);
1
suitable solvent to give compounds 6a and b.
mp 110–113°C; H-NMR (DMSO-d6) δ: 3.69 (2H, s), 5.06 (2H,
4-Benzyl-2-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)- s), 7.18 (1H, s), 7.20–8.26 (13H, m), 8.28 (1H, s). IR (KBr)
phthalazin-1(2H)-one (6a)
cm−1: 1738, 1637. MS m/z: 466 (M+), 237, 128. Anal. Calcd for
Brown crystals; yield (2.87g, 82%); mp 222–224°C; etha- C27H19FN4O3: C, 69.52; H, 4.11; N, 12.01. Found: C, 69.43; H,
1
nol; H-NMR (DMSO-d6) δ: 2.49 (3H, s), 4.31 (2H, s), 5.50 4.33; N, 12.24.
(2H, s), 7.16–8.30 (9H, m). IR (KBr) cm−1: 3136, 2946, 1634.
Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N′-
MS m/z: 318 (M+−CH3, +H), 290, 250, 249, 222, 130. Anal. (2-oxoindolin-3-ylidene)acetohydrazide (11) The aceto-
Calcd for C19H16N4O2: C, 68.66; H, 4.85; N, 16.86. Found: C, hydrazide derivative 4 (3.08g, 0.01mol) was condensed with
68.17; H, 5.10; N, 16.80.
4-Benzyl-2-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)- of few drops of acetic acid on a water bath for 3h. The solvent
phthalazin-1(2H)-one (6b) was evaporated and the reaction mixture was poured into
isatin (1.47g, 0.01mol) in ethyl alcohol (25mL) in the presence
White crystals; yield (3.2g, 80%); mp 228–230°C; toluene; crushed ice. The separated solid was filtered off, dried and
1H-NMR (DMSO-d6) δ: 4.33 (2H, s), 4.96 (1H, s), 7.19–8.30 recrystallized from ethanol to give 11. Brown crystals; yield
1
(14H, m). IR (KBr) cm−1: 3192, 1650, 1604. MS m/z: 394 (M+), (4.0g, 92%); mp 254°C (dec); H-NMR (DMSO-d6) δ: 4.34
383, 381, 120, 92. Anal. Calcd for C24H18N4O3: C, 73.08; H, (2H, s), 5.47 (2H, s), 6.93–8.30 (13H, m), 11.25 (1H, s), 12.67
4.60; N, 14.20. Found: C; 73.20, H; 4.53, N; 14.25.
(1H, s). IR (KBr) cm−1: 3147, 1719, 1696, 1654, 1623. MS m/z:
Synthesis of 4-Benzyl-2-(2-(3,5-dimethyl-1H-pyrazol- 437 (M+), 277, 91. Anal. Calcd for C25H19N5O3: C, 68.64; H,
1-yl)-2-oxoethyl)phthalazin-1(2H)-one (7) A solution of 4 4.38; N, 16.01. Found: C, 68.83; H, 4.26; N, 16.10.
(3.08g, 0.01mol) and acetyl acetone (1mL, 0.01mol) in etha-
nol (30mL) was refluxed for 3h. The separated solid was fil-
General Procedure for the Synthesis of 12a–c
Method (A)
tered off and recrystallized from toluene to give compound 7.
A mixture of compound 4 (3.08g, 0.01mol) and aromatic
1
Orange crystals; yield (2.73g, 70%); mp 206–208°C; H-NMR aldehydes namely benzaldehyde, 4-fluoro benzaldehyde, and
(DMSO-d6) δ: 2.02 (6H, s), 4.33 (2H, s), 5.00 (2H, s), 5.63 (1H, 4-methoxy benzaldehyde (2mL) in boiling ethanol (30mL)
s), 7.18–8.28 (9H, m). IR (KBr) cm−1: 3026, 1684, 1652. MS was refluxed for 3h. The separated solid was filtered off and
m/z: 372 (M+−H2O), 277, 250, 249, 237, 222, 195, 193, 130, recrystallized from the suitable solvent to give compounds
107, 102, 91. Anal. Calcd for C22H20N4O2: C, 70.95; H, 5.41; N, 12a–c.
15.04: Found: C, 71.00; H, 5.69; N, 15.14.
2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-Nꢀ-benzylidene-
Synthesis of 4-Benzyl-2-(2-(3-methyl-5-oxo-4,5-dihydro- acetohydrazide (12a)
pyrazol-1-yl)-2-oxoethyl)phthalazin-1(2H)-one (8) A solu-
White crystals; yield (2.76g, 71%); mp 202–204°C; ethanol;
tion of compound 4 (3.08g, 0.01mol) and ethyl acetoacetate 1H-NMR (CDCl3) δ: 4.32 (2H, s), 5.07 (1H, s), 5.53 (2H, s),
(1.3mL, 0.01mol) in ethanol (30mL) was refluxed for 3h. The 7.26–8.46 (14H, m), 8.96 (1H, s). IR (KBr) cm−1: 3192, 1688,
separated solid was filtered off and recrystallized from ethanol 1646. MS m/z: 396 (M+), 278, 250, 249, 222, 90, 77. Anal.
to give compound 8. White crystals; yield (2.94g, 70%); mp Calcd for C24H20 N4O2: C, 72.71; H, 5.08; N, 14.13. Found: C,
1
138–136°C; H-NMR (CDCl3) δ: 1.55 (3H, s), 2.28 (2H, s,), 3.44 72.64; H, 5.12; N, 14.20.
(2H, s), 4.29 (2H, s), 7.26–8.70 (9H, m,). IR (KBr) cm−1: 1731,
Nꢀ-(4-Fluorobenzylidene)-2-(4-benzyl-1-oxophthalazin-2(1H)-
1689, 1654. MS m/z: 374 (M+), 368, 286, 188, 186, 185, 131, yl)acetohydrazide (12b)
117, 71, 58, 57, 56. Anal. Calcd for C21 H18 N4 O3: C, 67.37; H,
White crystals; yield (3.1g, 75%); mp 208–210°C; ethanol;
1H-NMR (CDCl3) δ: 4.30 (2H, s), 5.15 (1H, s), 5.50 (2H, s),
4.85; N, 14.96. Found: C, 67.45; H, 4.93; N, 14.87.
Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N′- 7.30–8.33 (13H, m), 8.25 (1H, s). IR (KBr, cm−1): 3196, 1686,
(1-(4-fluorophenyl)ethylidene)acetohydrazide (9)
A
mix- 1645. MS m/z: 414 (M+), 278, 277, 250, 249, 222, 108, 91.
ture of compound 4 (3.08g, 0.01mol) and 4′-fluoro aceto- Anal. Calcd for C24H19FN4O2: C, 69.55; H, 4.62; N, 13.52.
phenone (1.38g, 0.01mol) in glacial acetic acid (20mL) was Found: C, 69.74; H, 4.60; N, 13.58.
refluxed for 5h, cooled to room temperature, and then poured
over crushed ice and allowed to stand overnight. The sepa- yl)acetohydrazide (12c)
rated solid was collected by filtration, dried and recrystallized White crystals; yield (2.8g, 68%); mp 198–200°C; ethanol;
Nꢀ-(4-Methoxybenzylidene)-2-(4-benzyl-1-oxophthalazin-2(1H)-

248
Biol. Pharm. Bull.
Vol. 39, No. 2 (2016)
1H-NMR (DMSO-d6) δ: 3.80 (3H, s), 4.32 (2H, s), 4.91 (1H, s), toluene (50mL) was refluxed for 2h. The reaction mixture was
5.30 (2H, s), 6.97–8.27 (13H, m), 8.30 (1H, s). IR (KBr) cm−1: filtered off while hot and concentrated. The separated solid
3149, 3141, 1698, 1633. MS m/z (%): 414 (M+−CH3, +3H), on cooling was filtered off and recrystallized from toluene to
250, 249, 91, 89, 77, 59. Anal. Calcd for C25H22N4O3: C, 70.41; give compound 16. Yellow crystals; yield (1.25g, 50%); mp
1
H, 5.20; N, 13.14. Found: C, 70.48; H, 5.14; N, 13.17.
Method (B)
160–162°C; H-NMR (CDCl3) δ: 4.37 (2H, s), 7.24–8.91 (9H,
m), 11.80 (1H, s). IR (KBr) cm−1: 3159, 1242. MS m/z (%):
The acetohydrazide derivative 4 (3.08g, 0.01mol) was re- 252 (M+), 251, 91, 69. Anal. Calcd for C15H12N2S: C, 71.40; H,
fluxed with 2-(4-methoxybenzylidene)malononitrile (1.84g, 4.79; N, 11.10; S, 12.71. Found: C, 71.47; H, 4.71; N, 11.04; S,
0.01mol) in ethyl alcohol (30mL) in the presence of few drops 12.80.
of piperidine for 6h. The separated solid after cooling was
Synthesis of 1-(4-Benzylphthalazin-1-yl)hydrazine (17)
filtered off, dried and recrystallized from the ethanol to give A solution of compound 16 (2.5g, 0.01mol) and hydrazine
compound 12c.
hydrate (0.5mL, 0.01mol) in ethanol (30mL) was refluxed for
3h. The separated solid was filtered off and crystallized from
General Procedure for the Synthesis of 13a and b
A
solution of compounds 12a and b (0.01mol) and thioglycolic benzene to give compound 17. Orange crystals; yield (1.6g,
1
acid (0.7mL, 0.01mol) in dry benzene (30mL) was refluxed 65%); mp 228–230°C; H-NMR (DMSO-d6) δ: 4.30 (2H, s),
for 6h. The separated solid was filtered off and recrystallized 4.78 (3H, s), 7.27–8.42 (9H, m). IR (KBr) cm−1: 3379, 1616.
from the suitable solvent to give compounds 13a and b.
2-(4-Benzyl-1-oxophthalazin-2(1H )-yl)-N-(4-oxo-2- for C15H14N4: C, 71.98; H, 5.64; N, 22.38. Found: C, 72.11; H,
MS m/z: 234 (M+−NH2), 129, 102, 92, 91, 76. Anal. Calcd
phenylthiazolidin-3-yl)acetamide (13a)
5.60; N, 22.22.
White crystals; yield (3.0g, 64%); mp 184–186°C; ethanol;
Synthesis of 4-Benzylphthalazine-1-sulfonic Acid (18)
1H-NMR (DMSO-d6) δ: 3.88 (2H, s), 4.26 (2H, s), 4.78 (2H, s), Nitric acid (0.01mol, 60–68%) was added to compound 16
5.80 (1H, s), 7.18–8.27 (14H, m), 10.59 (1H, s). IR (KBr, cm−1): (2.52g, 0.01mol) with stirring for 3d, then poured on water,
3203, 1730, 1697, 1631. MS m/z: 470 (M+), 277, 250, 249, 222, the separated solid was filtered off, washed with water and re-
178, 91, 77. Anal. Calcd for C26H22N4O3S: C, 66.37; H, 4.71; N, crystallized from benzene to give compound 18. Yellow crys-
1
11.91; S, 6.81. Found: C, 66.69; H, 4.64; N, 11.97; S, 6.89.
tals; yield (1.5g, 50%); mp 208–210°C; H-NMR (DMSO-d6)
2-(4-Benzyl-1-oxophthalazin-2(1H )-yl)-N-(2-(4- δ: 3.35 (2H, s), 7.90–9.00 (9H, m), 13.25 (1H, brs). IR (KBr)
fluorophenyl)-4-oxothiazolidin-3-yl)acetamide (13b)
cm−1: 3431, 3170, 1602, 1200, 1050, 650. MS m/z (%): 270
White crystals; yield (3.5g, 73%); mp 178–180°C; ethanol; (M+−O2, +2H·), 220, 178, 176, 165, 150, 149, 130, 104, 92,
1H-NMR (DMSO-d6) δ: 4.19 (2H, s), 4.32 (2H, s), 4.96 (2H, s), 77. Anal. Calcd for C15H12N2O3S: C, 59.99; H, 4.03; N, 9.33; S,
5.33 (1H, s), 7.16–8.29 (13H, m), 11.71 (1H, s). IR (KB, cm−1): 10.68. Found: C, 60.20; H, 3.97; N, 9.41; S, 10.61.
3206, 1729, 1699. MS m/z: 488 (M+), 489 (M+1), 250, 249,
Synthesis of 4-Benzylphthalazine-1-sulfonyl Chloride
165, 91. Anal. Calcd for C26H21FN4O3S: C, 63.92; H, 4.33; N, (19) Molecular chlorine was bubbled through a solution of
11.47; S, 6.56. Found: C, 64.23; H, 4.27; N, 11.45; S, 6.58. compound 16 (2.52g, 0.01mol) in glacial acetic acid (25mL)
Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N′- and water (3mL) at 30°C for 20min, upon completion of
((4-fluorophenyl)(phenylthio)methyl)acetohydrazide (14) the chlorination, water (30mL) was added and the solution
A solution of compound 12b (4.14g, 0.01mol) and thiophenol was washed with chloroform (50mL aliquots), the combined
(1.1mL, 0.01mol) in dry benzene (30mL) was refluxed for 3h chloroform layers were washed with 2.5% sodium hydrox-
with stirring. The separated solid was filtered off and recrys- ide (2×50mL aliquots), the chloroform was then dried over
tallized from petroleum ether 60–80°C to give compound 14. MgSO4, filtered off, and left for slow evaporation. The sepa-
Yellowish white crystals; yield (3.7g, 73%); mp 118–120°C; rated solid was filtered off and recrystallized from petroleum
1H-NMR (CDCl3) δ: 1.85 (2H, brs), 4.24–4.31 (4H, s), 5.01 ether 40–60°C to give compound 19. Yellow crystals; yield
1
(1H, s), 7.21–8.44 (18H, m). IR (KBr) cm−1: 3298, 1734, 1653. (1.7g, 55%); mp 136–138°C; H-NMR spectrum (DMSO-d6)
MS m/z: 522 (M+−2), 278, 277, 249, 110, 92, 58. Anal. Calcd δ: 4.29 (2H, s), 7.18–8.26 (9H, m). IR (KBr) cm−1: 1602, 1346,
for C30H25FN4O2S: C, 68.68; H, 4.80; N, 10.68; S, 6.11. Found: 1180. MS m/z: 318 (M+), 237, 236, 235, 102, 78, 77. Anal.
C, 68.73; H, 4.76; N, 10.57; S 6.14.
Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N′- S, 10.06. Found: C, 56.63; H, 3.43; Cl, 11.20; N, 8.93; S, 10.12.
(phenylsulfonyl)acetohydrazide (15) Benzenesulfonyl chlo- Synthesis of Ethyl 2-(4-Benzylphthalazin-1-ylthio)acetate
Calcd for C15H11ClN2O2S: C, 56.52; H, 3.48; Cl, 11.12; N, 8.79;
ride (0.176g, 0.001mol) was added to the solution of acetohy- (20) A solution of compound 16 (2.52g, 0.01mol), ethyl
drazide derivative 4 (0.308g, 0.001mol) in pyridine (10mL) at chloroacetate (4.4g, 0.04mol) and anhydrous potassium car-
0°C. The resulting mixture was stirred at room temperature bonate (5.5g, 0.04mol) in acetone (60mL) was heated on
for 5h. At the end of this period, the reaction mixture was water bath for 20h. The excess solvent was evaporated and
poured into ice water. The precipitate was filtered off, dried, the reaction mixture was poured on water. The separated
and crystallized from ethanol to give compound 15. Off solid was filtered off and recrystallized from petroleum ether
white crystals; yield (3.3g, 75%); mp 234–236°C; 1H-NMR 60–80°C to give compound 20. Yellowish white crystals; yield
1
(300MHz, DMSO-d6) δ: 4.27 (2H, s), 4.71 (2H, s), 7.19–8.26 (1.7g, 54%); mp 74–76°C; H-NMR (DMSO-d6) δ: 1.19 (3H, t,
(14H, m), 9.74 (1H, s), 9.98 (1H, s). IR (KBr) cm−1: 3220, 1703, J=6.0Hz), 4.12–4.17 (2H, q, J=6.0Hz), 4.30 (2H, s), 4.63 (2H,
1635, 1343, 1170. MS m/z: 448 (M+), 277, 91. Anal. Calcd for s), 7.15–8.28 (9H, m). IR (KBr) cm−1: 3061, 3030, 2978, 2923,
C23H20N4O4S: C, 61.59; H, 4.49; N, 12.49; S, 7.15. Found: C, 1737. MS m/z: 338 (M+), 294, 293, 266, 265, 252, 248, 203, 91
61.46; H, 4.46; N, 12.50; S, 7.12.
(100). Anal. Calcd for C19H18N2O2S: C, 67.43; H, 5.36; N, 8.28;
4-Benzylphthalazine-1(2H)-thione (16)
A
solution of S, 9.67. Found: C, 67.25; H, 5.53; N, 8.18; S, 9.60.
compound 2 (2.36g, 0.01mol) and P2S5 (8g, 0.02mol) in dry

Vol. 39, No. 2 (2016)
Biol. Pharm. Bull.
249
ABTS solution (60µM) was added to 3mL MnO2 suspension
(25mg/mL), all prepared in (5mL) aqueous phosphate buffer
solution (pH 7, 0.1M). The mixture was shaken, centrifuged,
filtered and the absorbance of the resulting green blue solution
MATERIALS AND METHODS
Biological Activities
Antibacterial Activity
Bacterial isolates were grown in nutrient broth medium (ABTS radical solution) at 734nm was adjusted to approx. ca.
(Oxoid) in an orbital shaking incubator for 24h at 37°C. The 0.5. Then, 50µL of (2mM) solution of the tested compound in
bacterial growth centrifuged, washed twice with saline solu- spectroscopic grade MeOH/phosphate buffer (1:1) was added.
tion (0.9%) and then standardized to approximately 106 colony The absorbance was measured and the reduction in color
forming unit (CFU)/mL using nutrient broth medium, during intensity was expressed as inhibition percentage. L-Ascorbic
the assay.
acid was used as standard antioxidant (positive control). Blank
Bacterial Sensitivity Test Standard well agar diffu- sample was run without ABTS and using MeOH/phosphate
sion method was carried out to detect the activity of the buffer (1:1) instead of the tested compounds. Negative control
compounds against the clinical bacterial isolates according was run with ABTS and MeOH/phosphate buffer (1:1) only.
to Cheesbrough38) Standard antibiotic discs (vancomycin,
Bleomycin-Dependent DNA Damage Assay To the reac-
erythromycin and tetracycline) were placed uniformly on the tion mixtures45,46) in a final volume of 1.0mL, the following
surface of nutrient agar plates seeded with 100µL of 24h bac- reagents were added: DNA (0.2mg/mL), bleomycin sulfate
terial culture prepared as mentioned above. For antibacterial (0.05mg/mL), FeCl3 (0.025mM), magnesium chloride (5mM),
activities of the compounds wells were made in these plates KH2PO4–KOH buffer pH 7.0 (30mM), and ascorbic acid
then from each compound 100µL (100µg) was placed in each (0.24 mM) or the test fractions diluted in MeOH to give a con-
well. The plates were left in refrigerator for 2h. After that centration of (0.1mg/mL). The reaction mixtures were incu-
were incubated at 37°C for 24h. Diameter of inhibition zones bated in a water bath at 37°C for 1h. At the end of the incuba-
around the wells were measured. All microbiological statisti- tion period, 0.1mL of ethylenediaminetetraacetic acid (EDTA)
cal analyses in this study were carried out using Microsoft (0.1 M) was added to stop the reaction (the iron-EDTA complex
Excel 2003 (Microsoft Corporation). All data were calculated is unreactive in the bleomycin assay). DNA damage was as-
from at least 2 replicates and the standard errors for each sessed by adding 1mL 1% (w/v) thiobarbituric acid (TBA) and
datum were plotted on the graph.
1mL of 25% (v/v) hydrochloric acid followed by heating in
Cytotoxicity Assay The cytotoxic activity of seven a water-bath maintained at 80°C for 15min. The chromogen
compounds was tested against four human tumor cell lines formed was extracted into 1-butanol, and the absorbance was
namely; hepatocellular carcinoma (liver) HePG-2, colon measured at 532nm.
cancer HCT-116, human (prostate) cancer cell line PC3, and
mammary gland (breast) MCF-7. The cell lines were obtained
Acknowledgment Technical support from Department of
from ATCC via Holding Company for biological products and Chemistry, Faculty of Science, Ain Shams University is grate-
vaccines (VACSERA), Cairo, Egypt. 5-Fluorouracil was used fully acknowledged.
as a standard anticancer drug for comparison.
Chemical Reagents
Conflict of Interest The authors declare no conflict of
The reagents are RPMI-1640 medium, 3-(4,5-dimethylthiazol- interest.
2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulf-
oxide (DMSO) and 5-fluorouracil (Sigma Co., St. Louis, MO,
U.S.A.), Fetal Bovine serum (GIBCO, U.K.).
Supplementary Materials The online version of this ar-
ticle contains supplementary materials.
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