Design and Synthesis of New Phthalazinone Derivatives Containing Benzyl Moiety with Anticipated Antitumor Activity

Abstract

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.

Heterocyclic compounds containing hydrazine have received considerable attention because of their pharmacological properties and clinical applications.^1–3) The diverse biological activities of various functional derivatives of 4-substituted alkyl-1-(2H)phthalazinones are well known. Some of the phthalazinone derivatives have found application in clinical medicine due to their pronounced antihypertensive properties,⁴⁾ cardiotonic,^5,6) anticonvulsant,^7,8) antidiabetic,^9,10) antithrombotic,¹¹⁾ antimicrobial,^12,13) vasorelexant,¹⁴⁾ antipyretic, analgesic, antitumor,^15–18) and cytotoxic,¹⁹⁾ while others have shown interesting vasodilator,²⁰⁾ vascular endothelial growth factor receptor (VEGFR) II inhibitory activities,²¹⁾ and tuberculostatic activity allergic rhinitis.²²⁾ Phthalazin-1-(2H)ones bearing a substituent at 4-position represent key intermediates in the synthesis of various compounds with high interesting pharmacological properties such as the blood platelet aggregation inhibitor²³⁾ MV-5445 [1-(3-chloroanilino)-4-phenylphthalazine] which has been found to be a selective phosphodiesterase V inhibitor²⁴⁾ or the thromboxane A₂ synthetase inhibitor or bronchodilator, 2-[2-(1-imidazolyl)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 efforts have led to the discovery of several leading phthalazines with different cellular and enzymatic targets. Inhibitors of poly(adenosine diphosphate-ribose)polymerases (PARPs) family of proteins are currently being evaluated as potential anticancer medicines at both preclinical and clinical levels.²⁶⁾ A series of 4-substituted-2H-phthalazin-1-ones have been investigated as potent orally bioavailable PARP inhibitor.²⁷⁾ For example, Olaparib (I), MRU-868 (II) and KU0058958 (III) are the most interesting PARP inhibitors based on the 4-substituted-2H-phthalazin-1-ones scaffold (Fig. 1).

Fig. 1. Structure of the Lead Anticancer Derivatives (I)–(VI) and the Designed Target Compounds 4, 12a, c, 15, 16 and 19

Interestingly, Prime et al.²⁸⁾ have reported a novel class of potent, selective, and orally bioavailable inhibitors of aurora-A kinase based upon a 4-(pyrazole-3-ylamino)phenyl-2H-phthalazin-1-one scaffold. Compound (IV) inhibits aurora-A with IC₅₀ of 71 nM, also, it showed in vitro cytotoxic activity against HCT116 colon cell line with IC₅₀ of 5.44 nM.

Tubulin binding molecules²⁹⁾ have gained much interest among cytotoxic agents due to its success in clinical oncology. They differ from the other anticancer drugs in their mode of action because they target the mitiotic spindle and not the DNA, for example, E7010 (V).

Oltipraz (VI) and related dithiolethione derivatives³⁰⁾ are an important class of chemopreventive agents that enhance the expression of carcinogen detoxication and antioxidant agents. Since, many synthetic compounds have been used to protect animals against a variety of cancer, hydrogen sulfide³⁰⁾ has been shown to be a potent endogenous gaseous mediator in many physiological and pathological processes. Hydrogen sulfide (H₂S) in mammalian tissues is produced from L-cysteine, the sulfhydryl group (SH) be the essential species responsible for the biological activities of these compounds (Fig. 1).

The observations outlined above, enthused us to develop new phthalazinone derivatives and to investigate their activities as antimicrobial, anticancer and antioxidant agents.

RESULTS AND DISCUSSION

In the present investigation, we have reported the synthesis of some new phthalazinone derivatives. Some of the newly synthesized compounds were tested for their biological activity. A convenient four step procedure was used to synthesize the target 2-(4-benzyl-1-oxophthalazin-2(1H)-yl)acetohydrazide 4 as outlined in Chart 1.

Chart 1. Synthesis of Compounds 1–5

The commercially available phthalic anhydride was fused with phenylacetic acid in the presence of fused sodium acetate in an oil bath at 180°C to afford the 3-benzylidenephthalide 1, which was then reacted with hydrazine hydrate³¹⁾ in boiling ethanol to give the benzylphthalazinone derivative 2. Oxidation of the phthalazinone derivative 2 with KMnO₄ in the presence of K₂CO₃ was failed to give 2,3-dihydrophthalazine-1,4-dione A, this result indicated that the phthalazinone derivative 2 exists in the tautomer form 2a not 2b (Chart 1).

Subsequent interaction of the benzylphthalazinone derivative 2a with ethyl chloroacetate in boiling acetone in the presence of anhydrous K₂CO₃ led to the oxophthalazinyl acetate derivative 3. The structure of compound 3 was inferred from spectroscopic data. The IR showed the presence of 2C=O bands at 1735 and 1665 cm⁻¹. The ¹H-NMR spectrum showed signals attributed to CH₃, CH₂, CH₂Ph, CH₂CO and aromatic protons at δ 1.22, 4.16, 4.33, 4.97, 7.09–8.48 ppm, respectively. The oxophthalazinyl acetate 3 was then reacted with hydrazine hydrate in boiling ethanol to give the desired acetohydrazide derivative 4. The structure of compound 4 was inferred from spectroscopic data. The IR showed the absence of the C=O band attributed to the ester group and the appearance of bands at 3290 and 3318 cm⁻¹ attributed to NH and NH₂ groups. The ¹H-NMR spectrum devoid of any signals attributed to protons of ethyl group; instead it showed signals attributed to NH and NH₂ protons at δ 4.56 and 9.31 ppm, respectively, as well as signals corresponding to CH₂Ph, CH₂CO and aromatic protons at δ 4.30, 4.75 and 7.29–8.27 ppm, respectively.

A chemical prove for the structure of compound 3 was gained from its reaction with benzaldehyde in the presence of piperidine that yielded the adduct 5, which was devoid of signal corresponding to CH₂CO protons in its ¹H-NMR spectrum.

Interaction of the acetohydrazide 4 with acetyl chloride and benzoyl chloride in the presence of dry benzene afforded the oxadiazol derivatives 6a and b (Chart 2). The structure of compounds 6a and b were confirmed from spectroscopic data. The reaction proceeded via nucleophilic attack followed by ring closure to afford the desired oxadiazol derivatives 6a and b.

Chart 2. Synthesis of Compounds 6–11

Treatment of the acetohydrazide 4 with acetyl acetone and ethyl acetoacetate afforded the pyrazol derivatives 7 and 8, respectively. The structures of compounds 7 and 8 were corroborated by spectroscopic data. The ¹H-NMR spectrum of compound 7 showed the presence of 2CH₃ groups at δ 2.02 ppm, in addition to a singlet signal at δ 5.63 ppm for the proton of the pyrazole ring. Also it showed signals at δ 4.33, 5.00 and 7.18–8.28 ppm for CH₂Ph, CH₂CO and aromatic protons respectively. The structure of compound 8 was supported from its IR spectrum which displayed a band at 1731 cm⁻¹ corresponding to C=O group of the pyrazolone ring. The ¹H-NMR spectrum showed a singlet signal for CH₃ protons at δ 1.55 ppm and a signal for CH₂ protons group of the pyrazolone ring at δ 2.28 ppm, beside signals corresponding to CH₂Ph, CH₂CO and aromatic protons at δ 3.44, 4.29 and 7.26–8.70 ppm, respectively. The mass spectrum showed the molecular ion peak at m/z 374 which is in agreement with the molecular weight.

Furthermore, 4′-fluoro acetophenone in methanol containing a catalytic amount of glacial acetic acid was reacted with compound 4 to give ethylidene acetohydrazide derivative 9. The structure of compound 9 was confirmed by spectroscopic data. The IR spectrum showed bands at 3214, 1714 (the high value is due to mutual induction), 1655 and 1614 cm⁻¹ attributable to NH, 2C=O and C=N groups, respectively. The ¹H-NMR spectrum showed two singlet signals for CH₃ and NH protons at δ 1.85 and 10.32 ppm, respectively, in addition to signals corresponding to CH₂Ph, CH₂CO and aromatic protons at δ 4.30, 4.87 and 7.16–8.27 ppm, respectively. Further confirmation for the structure of compound 9 was achieved through its reaction with Vilsmeier–Haack reagent (N,N-dimethyl formamide (DMF)/POCl₃) that yielded the pyrazole carbaldehyde derivative 10. The ¹H-NMR spectrum of compound 10 was in a good agreement with the suggested structure (cf. Experimental); it showed the disappearance of the signals corresponding to the methyl and the NH protons. The mass spectrum of compound 10 showed the molecular ion peak at m/z 466 which is coincident with the molecular weight (466) as supported the identity of the structure. A possible mechanism for the formylation of compound 9 is explained in Chart 3.

Chart 3. Vilsmeier–Haack Reaction Mechanism

Treatment of the acetohydrazide 4 with isatin in ethanol and drops of acetic acid yielded oxoindolinylidene acetohydrazide derivative 11. Its IR spectrum showed carbonyl absorption bands in addition to the NH absorption band 1719, 1646 and 3147 cm⁻¹, respectively. Further evidence was gained from ¹H-NMR spectrum as it exhibited signals for 2NH at 11.25 and 12.67, beside signals corresponding to CH₂Ph, CH₂CO and aromatic protons at δ 4.34, 5.47 and 6.93–8.30 ppm, respectively. The mass spectrum showed the molecular ion peak at m/z 437 which is in agreement with the molecular weight.

Interaction of the acetohydrazide 4 with the appropriate aldehydes namely benzaldehyde, 4-fluorobenzaldehyde, and 4-methoxybenzaldehyde in boiling ethanol yielded the corresponding Schiff bases 12a–c (Chart 4). The ¹H-NMR spectra of compounds 12a–c were devoid of any signals for NH₂ group and showed signals attributed to CH₂Ph, NH, CH₂CO, =CH and aromatic protons in the range of δ 4.30–4.92, 5.07–5.15, 5.31–5.53, 8.25–8.96 and 6.99–8.46 ppm, respectively. Also compound 12c showed a singlet signal at δ 3.80 ppm attributed to OCH₃ protons. Further evidence were gained from mass spectra as they showed the correct molecular ion peaks for compounds 12a and b and [M⁺−CH₃, +3H] for compound 12c beside some important peaks.

Chart 4. Synthesis of Compounds 12–15

The reaction of compound 4 with 2-(4-methoxybenzylidene)malononitrile afforded the methoxy benzylidene derivative 12c not the pyrazolone derivative 12′c. The amino group in compound 4 underwent nucleophilic addition reaction to the double bond of 2-(4-methoxybenzylidene)malononitrile via Michael type addition reaction, by refluxing in ethanol containing few drops of piperidine to give a substance whose structure should be either the pyrazole derivative 12′c (route a) or the methoxy benzylidene derivative 12c (route b). The actual structure of the product was assigned as the methoxy benzylidene derivative 12c based on the spectroscopic data. The IR spectrum devoid any signal for C≡N group. The proton ¹H-NMR spectrum showed signals attributed to NH and=CH protons, and was devoid of NH₂ signal which should have appeared if the reaction product was 12′c (Chart 5).

Chart 5. Reaction of Compound 4 with Methoxy Benzylidene

The Schiff bases 12a and b were allowed to react with thioglycolic acid in boiling benzene to yield thiazolidinyl acetamide derivatives 13a and b, respectively (Chart 4). The reaction takes place via thia addition type on azamethine moiety followed by 5-exo-trig ring closure. The structures of compounds 13a and b were corroborated by spectroscopic data. The ¹H-NMR spectrum showed signals attributed to CH₂Ph, CH₂CO, SCH₂, aromatic and NH protons in the range of δ 3.88–4.19, 4.26–4.32, 4.78–4.96, 5.33–5.80, 7.16–8.29, 10.59–11.71 ppm, respectively. The mass spectra of compounds 13a and b showed the correct molecular ion peaks at m/z 470 and 488, respectively.

The reaction of thiol to form a carbon–sulfur bond constitutes a key reaction in biosynthesis as well as in the synthesis of biologically active compounds.³²⁾ The schiff base 12b was submitted to react with thiophenol in benzene with stirring to yield the thia type adduct 14. The structure of compound 14 was corroborated by spectroscopic data. The IR spectrum showed bands at 3298, 1734, 1653 cm⁻¹ attributed to NH and 2C=O, the high value of νC=O is due to mutual induction. The ¹H-NMR spectrum showed signals attributed to NH, [CH₂Ph, CH₂CO], SCH, aromatic protons, at δ 1.85 4.24–4.31, 5.01, 7.21–8.44 ppm, respectively.

Reaction of acetohydrazide 4 with benzenesulfonyl chloride in pyridine yielded compound 15. The structure of the synthesized compound 15 was elucidated by studying its IR, ¹H-NMR, mass spectra, and elemental analysis. The IR spectrum showed bands at 1343 and 1170 cm⁻¹ attributed to SO₂ group beside bands for NH and C=O at 3220, 1703, 1635 cm⁻¹. The ¹H-NMR spectrum showed signals attributed to CH₂Ph, CH₂CO, aromatic and NH protons at δ 4.27, 4.71, 7.19–8.26, 9.74, 9.98 ppm, respectively. The mass spectrum showed the molecular ion peak at m/z 448 which is in agreement with the molecular weight.

Treatment of the phthalazinone derivative 2a with phosphorus pentasulfide in boiling dry toluene yielded the phthalazinethione derivative 16. The ¹H-NMR spectrum showed signals attributed to CH₂Ph, aromatic and NH protons at δ 4.37, 7.24–8.91, 11.80 ppm, respectively. The IR spectrum showed bands at 3159 and 1242 cm⁻¹ attributed to NH, C=S and devoid any band of C=O. The mass spectrum showed the molecular ion peak at m/z 252 which is in agreement with the molecular weight.

The reaction of compound 16 with hydrazine hydrate in boiling ethanol yielded the hydrazine derivative 17 (Chart 6). The IR spectrum revealed NH₂ and NH stretching bands at 3379 cm⁻¹. While the ¹H-NMR spectrum revealed the presence of the NH₂ and NH protons as a broad singlet at 4.78 ppm, in addition to the other aromatic protons appearing as multiplet at 7.27–8.42 ppm and to CH₂Ph at 4.30 ppm.

Chart 6. Synthesis of Compounds 16–20

Oxidation³³⁾ of the phthalazinethione 16 with concentrated HNO₃ at room temperature afforded the sulfonic acid derivative 18. The structure of compound 18 was inferred from its solubility in sodium bicarbonate solution and spectroscopic data. The ¹H-NMR spectrum revealed the presence of the OH proton as a singlet signal at 13.25 ppm beside CH₂Ph and aromatic protons at δ 3.35 and 7.90–9.00 ppm, 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 7.18–8.26 ppm 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 chloroacetate in the presence of anhydrous K₂CO₃ in boiling acetone yielded the ethyl acetate derivative 20. The reaction takes place via S_N2 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 kinetic controlled formation under the experimental conditions. Firstly, in the presence of anhydrous K₂CO₃ and acetone, the conjugate base of iminothiol tautomer is more thermodynamically stable than the conjugate base derived from thioamide tautomer via the back donation involving the vacant D-orbital of the sulfur atom. Therefore, the iminothiol tautomer is more predominant under such conditions. Secondly, the sulfur anion is a stronger nucleophile than the nitrogen anion (nucleophilicity is kinetic control). Thus, the iminothiol tautomer is more thermodynamically and kinetically formed than thioamide tautomer, which spells out the reactivity of the iminothiol tautomer (Fig. 2). The structure of compound 20 was confirmed from spectroscopic data. The IR showed band at 1737 cm⁻¹ attributed to C=O of the ester group. The ¹H-NMR spectrum revealed the presence of signals at δ 1.19, 4.12–4.17, 4.30, 4.69, 7.15–8.28 ppm attributed to CH₃, CH₂, CH₂Ph, CH₂S and aromatic protons, respectively.

Fig. 2. Thioamide–Iminothiol Tautomerism

Pharmacology

Antibacterial Activity

Staphylococcus aureus was significantly inhibited by compounds 12c, 17–19 (Fig. 3 and Table 1). For other compounds no activity on Staphylococcus aureus was detected. For Gram-negative clinical isolates only compound 7 significantly inhibited both Escherichia coli and Pseudomonas aeruginosa, while compounds 12c and 19 inhibited only Escherichia coli. Both isolates were resistant to erythromycin.

Fig. 3. Diameter of Inhibition Zones for Effective Compounds on A) Staphylococcus aureus, B) Escherichia coli and C) Pseudomonas aeruginosa

V: vancomycin; E: erythromycin and T: tetracyclin.

Table 1. The Antibacterial Activities of Some Compounds

Compd. no.	Diameter of inhibition zones (cm)
	Staphylococcus aureus	Escherichia coli	Pseudomonas aeruginosa
DMSO	0	0	0
3	0	0	0
4	0	0	0
6a	0	0	0
6b	0	0	0
7	0	1.7±0.05	1.9±0.05
12a	0	0	0
12b	0	0	0
12c	2.4±0.01	0.37±0.01	0
13a	0	0	0
16	0	0	0
17	1.7±0.05	0	0
18	2.4±0.1	0	0
19	3.0	3.12	0
20	0	0	0
Vancomycin	2±0.05	0	0
Erythromycin	3±0.1	0	0
Tetracycline	1.8±0.05	1.1±0.05	0

The mode of action of compounds 17 and 18 was different from compounds 7, 12c and 19, since they inhibited the growth of Gram-positive clinical isolate, while, compound 7 inhibited the growth of Gram-negative clinical isolates growth. Compounds 12c and 19 were able to inhibit both isolates.

Sulfones are competitive inhibitors of folic acid synthesis and are used as bacteriostatic and in the treatment of Leprosy.³⁴⁾ Many of aryl sulfones are used as antifungal, antibacterial or antitumor agents³⁵⁾ and inhibitors for several³⁶⁾ cyclic oxygenase-2 (COX-2). Compounds containing C=N may act as a Michael acceptor for nucleophiles containing NH₂ or sulfhydryl SH groups. In case of enzymes they are converted from the active form to the inactive form by these compounds. Some of these enzymes are responsible for building cell walls of bacteria and /or fungi; which explain the activities of compounds 7, 12c, 17–19.

Antitumor Activity Using in Vitro Ehrlich Ascites Assay

We assessed the cytotoxic action of the compounds listed in Table 2 against four human tumor cell lines namely; hepatocellular carcinoma (liver) HePG2, colon cancer HCT-116, human prostate cancer cell line PC3, and mammary gland breast MCF-7. In general, activity was observed by all of these molecules ranged from very strong to non-cytotoxic. The optimal results were observed for compounds 15 and 16 (very strong activity) with IC₅₀ 7.8±0.23, 5.6±0.22 µg/mL for HePG-2 cell, 8.5±0.31, 7.7±0.25 µg/mL for HCT-116 cell, 8.9±0.51, 8.6±0.34 µg/mL for PC3 cell and 8.9±0.62, 5.0±0.41 µg/mL for MCF-7 cell, respectively. Compound 16 exhibited stronger activity (IC₅₀ 5.6±0.22, 0±0.41 µg/mL) than the 5-fluorouracil (5-FU) as a standard (7.9±0.12, 5.4±0.21 µg/mL) for HePG2 and MCF-7, respectively. Also it showed approximately equal activity to the 5-FU as a standard for PC3 cell line with IC₅₀ 8.6±0.34 and 8.3±0.25 µg/mL, respectively. Compound 15 showed approximately equal activity to the ascorbic acid as a standard for HePG2 and PC3 cells with IC₅₀ 7.8±0.23, 8.9±0.51 and 7.9±0.12, 8.3±0.25 µg/mL, respectively. Compound 4 showed strong activity towards HCT-116 cell (12.9±0.69 µg/mL), and MCF-7 cell (19.2±1.64 µg/mL), also it showed moderate activity towards HePG-2 cell (29.9±1.33 µg/mL) and PC3 cell (26.9±1.57 µg/mL). While compounds 12a, c, 19 and 20 observed activity ranged from moderate to non-cytotoxic with IC₅₀ 41.2±2.96 to >100. [The relative viability of cells (%) for the tested compounds is depicted in Tables S1 and S2 (Supplementary materials)].

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	29.9±1.33	12.9±0.96	26.9±1.57	19.2±1.64
12a	85.9±5.71	96.5±6.65	95.4±5.30	94.9±5.18
12c	53.8±4.32	62.9±2.97	66.6±4.36	41.2±2.96
15	7.8±0.23	8.5±0.31	8.9±0.51	8.9±0.62
16	5.6±0.22	7.7±0.25	8.6±0.34	5.0±0.41
19	93.7±5.97	93.6±6.28	>100	>100
20	95.1±6.42	>100	>100	>100
5-FU	7.9±0.12	5.3±0.14	8.3±0.25	5.4±0.21

a) IC₅₀ (µg/mL): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), above 100 (non-cytotoxic).

Fig. 4. Cytotoxic Activity of the Tested Compounds on Different Cell Lines

Antioxidant Activity Using 2.2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic (ABTS) Acid Inhibition

Seven compounds were tested for antioxidant activity as reflected in the ability to inhibit oxidation in rat brain and kidney homogenates Table 3. Compounds 16 and 15 showed high % inhibition 85.3, 78.4%, respectively. While the rest of the compounds 4, 12a, c, 19 and 20 exhibited moderate to weak antioxidant activity ranged from 53.9–30.1%.

Table 3. Antioxidant Activity and Bleomycin Dependent DNA Damage for the Tested Compounds

Compd. no.	Antioxidant activity (ABTS method)		Bleomycin dependent DNA damage
	Absorbance	Inhibition (%)
4	0.222	53.9	0.096
12a	0.337	30.1	0.137
12c	0.268	44.4	0.088
15	0.104	78.4	0.072
16	0.071	85.3	0.096
19	0.300	37.7	0.130
20	0.302	37.3	0.142
Ascorbic acid	0.053	89.0	0.073

All experiments were performed three times. The data are expressed as the mean±standard error of the mean (S.E.M.).

Bleomycin-Dependent DNA Damage

The bleomycin is a family of glycopeptides antibiotics that are used routinely as antitumor agents. The bleomycin assay has been adopted for assessing the pro-oxidant effect of food antioxidants. The antitumor antibiotic bleomycin binds iron ions and DNA. The bleomycin–iron complex degrades DNA when heated with thiobarbituric acid (TBA) to yield a pink chromogenic. Upon the addition of suitable reducing agents antioxidant competes with DNA and diminishes chromogenic formation.³⁷⁾

To show the mechanism of action of the tested compounds 4, 12a, c, 15, 16, 19 and 20, their protective activity against DNA damage induced by the bleomycin–iron complex were examined. The results in Table 3 showed that compounds 4, 12a, c, 15, 16, 19 and 20 have the ability to protect DNA from the induced damage by bleomycin. Compound 15 showed very high protection (0.072) against DNA damage induced by the bleomycin–iron complex which is approximately equal to ascorbic acid as a standard (0.073). Compounds 4, 12c and 16 showed high ability. On the other hand, the rest of the compounds 12a, 19 and 20 exhibited moderate activities. Thus, all the tested compounds diminish the chromogenic formation between the damage DNA and TBA.

Structure–Activity Relationship (SAR)

DNA is made of chemical building blocks called nucleotides. The four types of nitrogen bases found in nucleotides are: adenine (A), thymine (T), guanine (G) and cytosine (C). The base adenine always pairs with thymine, while guanine always pairs with cytosine through hydrogen bond. The cytotoxic activity towards different cell lines depends on two factors: (i) The formation of intermolecular hydrogen bond with DNA bases (ii) the positive charge on the tested compounds attracted to the negative charge on the cell wall. By comparing the experimental cytotoxicity of the compounds reported in this study to their structures, the following SAR were postulated.

CONCLUSION

The objective of the present study was to synthesize the phthalazinone based compounds scafold and study their antibacterial, cytotoxicity, antioxidant and bleomycin dependent DNA damage activities. Staphylococcus aureus was significantly inhibited by compounds 12c, 17–19. For other compounds no activity on Staphylococcus aureus was detected. For Gram-negative clinical isolates only compound 7 significantly inhibited both Escherichia coli and Pseudomonas 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 were observed for compounds 15 and 16 (very strong activity). Compound 15 showed approximately equal activity to the 5-FU as a standard against HePG2 and PC3 cells. Compound 16 exhibited stronger activity than the 5-FU as a standard against HePG2 and MCF-7.

EXPERIMENTAL

Chemistry

All melting points were uncorrected. IR spectra were recorded in KBr on FTIR Mattson Spectrometers. ¹H-NMR spectra were measured on Varian Gemini 300 MHz instrument with chemical shift (δ) expressed in ppm downfield from TMS. MS were recorded on Shimadzu GC-MS-QP 1000 Ex and MS_5988 instruments at 70 eV. All the spectral measurements as well as elemental analyses were carried out at the Micro analytical Center of Cairo University, Egypt and at Faculty of Pharmacy, Ain Shams University. Compound 2 was prepared according to the literature procedure.³¹⁾ All products were characterized by IR; ¹H-NMR; mass spectroscopy and elemental analyses.

Synthesis of Ethyl 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)acetate (3)

A solution of 2 (2.36 g, 0.01 mol), ethyl chloroacetate (4.4 g, 0.04 mol) and anhydrous potassium carbonate (5.5 g, 0.04 mol) in acetone (60 mL) was heated on a water bath for 20 h. The excess solvent was evaporated and the reaction mixture was poured on water. The separated solid was filtered off and recrystallized from petroleum ether 60–80°C to give compound 3. Violet crystals; yield (2.1 g, 66%); mp 128–130°C (Lit.,³⁶⁾ mp 76–78°C); ¹H-NMR (CHCl₃) δ: 1.22 (3H, t, J=6.0 Hz), 4.16 (2H, q, J=6.0 Hz), 4.33 (2H, s), 4.97 (2H, s), 7.09–8.48 (9H, m). IR (KBr) cm⁻¹: 1735, 1665. MS m/z: 322 (M⁺), 249, 91. Anal. Calcd for C₁₉H₁₈N₂O₃: C, 70.79; H, 5.63; N, 8.69. Found: C, 70.80; H, 5.58; N, 8.64.

Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)acetohydrazide (4)

A mixture of 3 (3.22 g, 0.01 mol) and hydrazine hydrate (0.05 mL, 0.01 mol) in ethanol (30 mL) was refluxed for 3 h. The separated solid was filtered off and recrystallized from ethanol to give compound 4. White crystals; yield (2.6 g, 85%); mp 184–186°C (Lit.,³¹⁾ mp 130–132°C); ¹H-NMR (DMSO-d₆) δ: 4.30 (2H, s), 4.56 (2H, s), 4.75 (2H, s), 7.26–8.27 (9H, m), 9.31 (1H, s). IR (KBr) cm⁻¹: 3290, 3318, 1661, 1615. MS m/z: 308 (M⁺), 277, 91.05. Anal. Calcd for C₁₇H₁₆N₄O₂: C, 66.22; H, 5.23; N, 18.17. Found: C, 66.36; H, 5.27; N, 18.20.

Synthesis of Ethyl 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-3-phenylacrylate (5)

A mixture of 3 (3.22 g, 0.01 mol), benzaldehyde (1 mL, 0.01 mol) and piperidine (1 mL) in ethanol (30 mL) was refluxed for 3 h. The reaction mixture was cooled and the separated solid was filtered off and recrystallized from petroleum ether to give compound 5. White crystals; yield (2.6 g, 64%); mp 174–176°C; ¹H-NMR (DMSO-d₆) δ: 1.22 (3H, t, J=6.0 Hz), 4.15–4.23 (2H, q, J=6.0 Hz), 4.91 (2H, s), 7.09–8.43 (15H, m). IR (KBr) cm⁻¹: 3100, 1735, 1656. MS m/z: 396 (M⁺−CH₃, +H^·), 294, 250, 249, 207, 176, 178, 130. Anal. Calcd for C₂₆H₂₂N₂O₃: C, 76.08; H, 5.40; N, 6.82. Found: C, 76.11; H, 5.43; N, 6.86.

General Procedure for the Synthesis of 6a, b

A solution of 4 (3.08 g, 0.01 mol), acetyl chloride or benzoyl chloride (0.01 mol) in benzene (30 mL) was refluxed for 4 h. The separated solid was filtered off dried and recrystallized from the suitable solvent to give compounds 6a and b.

4-Benzyl-2-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)phthalazin-1(2H)-one (6a)

Brown crystals; yield (2.87 g, 82%); mp 222–224°C; ethanol; ¹H-NMR (DMSO-d₆) δ: 2.49 (3H, s), 4.31 (2H, s), 5.50 (2H, s), 7.16–8.30 (9H, m). IR (KBr) cm⁻¹: 3136, 2946, 1634. MS m/z: 318 (M⁺−CH₃, +H), 290, 250, 249, 222, 130. Anal. Calcd for C₁₉H₁₆N₄O₂: C, 68.66; H, 4.85; N, 16.86. Found: C, 68.17; H, 5.10; N, 16.80.

4-Benzyl-2-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)phthalazin-1(2H)-one (6b)

White crystals; yield (3.2 g, 80%); mp 228–230°C; toluene; ¹H-NMR (DMSO-d₆) δ: 4.33 (2H, s), 4.96 (1H, s), 7.19–8.30 (14H, m). IR (KBr) cm⁻¹: 3192, 1650, 1604. MS m/z: 394 (M⁺), 383, 381, 120, 92. Anal. Calcd for C₂₄H₁₈N₄O₃: C, 73.08; H, 4.60; N, 14.20. Found: C; 73.20, H; 4.53, N; 14.25.

Synthesis of 4-Benzyl-2-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-2-oxoethyl)phthalazin-1(2H)-one (7)

A solution of 4 (3.08 g, 0.01 mol) and acetyl acetone (1 mL, 0.01 mol) in ethanol (30 mL) was refluxed for 3 h. The separated solid was filtered off and recrystallized from toluene to give compound 7. Orange crystals; yield (2.73 g, 70%); mp 206–208°C; ¹H-NMR (DMSO-d₆) δ: 2.02 (6H, s), 4.33 (2H, s), 5.00 (2H, s), 5.63 (1H, s), 7.18–8.28 (9H, m). IR (KBr) cm⁻¹: 3026, 1684, 1652. MS m/z: 372 (M⁺−H₂O), 277, 250, 249, 237, 222, 195, 193, 130, 107, 102, 91. Anal. Calcd for C₂₂H₂₀N₄O₂: C, 70.95; H, 5.41; N, 15.04: Found: C, 71.00; H, 5.69; N, 15.14.

Synthesis of 4-Benzyl-2-(2-(3-methyl-5-oxo-4,5-dihydropyrazol-1-yl)-2-oxoethyl)phthalazin-1(2H)-one (8)

A solution of compound 4 (3.08 g, 0.01 mol) and ethyl acetoacetate (1.3 mL, 0.01 mol) in ethanol (30 mL) was refluxed for 3 h. The separated solid was filtered off and recrystallized from ethanol to give compound 8. White crystals; yield (2.94 g, 70%); mp 138–136°C; ¹H-NMR (CDCl₃) δ: 1.55 (3H, s), 2.28 (2H, s,), 3.44 (2H, s), 4.29 (2H, s), 7.26–8.70 (9H, m,). IR (KBr) cm⁻¹: 1731, 1689, 1654. MS m/z: 374 (M⁺), 368, 286, 188, 186, 185, 131, 117, 71, 58, 57, 56. Anal. Calcd for C₂₁ H₁₈ N₄ O₃: C, 67.37; H, 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′-(1-(4-fluorophenyl)ethylidene)acetohydrazide (9)

A mixture of compound 4 (3.08 g, 0.01 mol) and 4′-fluoro acetophenone (1.38 g, 0.01 mol) in glacial acetic acid (20 mL) was refluxed for 5 h, cooled to room temperature, and then poured over crushed ice and allowed to stand overnight. The separated solid was collected by filtration, dried and recrystallized from ethanol to give 9. Brown crystals; yield (3.4 g, 80%); mp 220–222°C; ¹H-NMR (DMSO-d₆) δ: 1.85 (3H, s), 4.30 (2H, s), 4.87 (2H, s), 7.16–8.27 (13H, m), 10.32 (1H, s). IR (KBr) cm⁻¹: 3214, 1714, 1655, 1614. MS m/z: 428 (M⁺, 0.00), 249, 91. Anal. Calcd for C₂₅H₂₁FN₄O₂: C, 70.08; H, 4.94; N, 13.08. Found: C, 70.36; H, 4.96; N, 13.12.

Synthesis of 1-(2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)acetyl)-3-(4-fluorophenyl)-1H-pyrazole-4-carbaldehyde (10)

POCl₃ (0.612 g, 0.004 mol) was added to cold DMF (0.117 g, 0.0016 mol) at 0°C, and a solution of compound 9 (0.25 g, 0.0006 mol) in DMF (10 mL) was added dropwise, then the reaction mixture was heated on water bath at 65–70°C for 4 h; the reaction mixture was cooled and then poured into cold water; the separated solid was filtered off, washed with water, dried and recrystallized from benzene/petroleum ether 60–80°C (2 : 1) to give 10. White crystals; yield (1.8 g, 40%); mp 110–113°C; ¹H-NMR (DMSO-d₆) δ: 3.69 (2H, s), 5.06 (2H, s), 7.18 (1H, s), 7.20–8.26 (13H, m), 8.28 (1H, s). IR (KBr) cm⁻¹: 1738, 1637. MS m/z: 466 (M⁺), 237, 128. Anal. Calcd for C₂₇H₁₉FN₄O₃: C, 69.52; H, 4.11; N, 12.01. Found: C, 69.43; H, 4.33; N, 12.24.

Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N′-(2-oxoindolin-3-ylidene)acetohydrazide (11)

The acetohydrazide derivative 4 (3.08 g, 0.01 mol) was condensed with isatin (1.47 g, 0.01 mol) in ethyl alcohol (25 mL) in the presence of few drops of acetic acid on a water bath for 3 h. The solvent was evaporated and the reaction mixture was poured into crushed ice. The separated solid was filtered off, dried and recrystallized from ethanol to give 11. Brown crystals; yield (4.0 g, 92%); mp 254°C (dec); ¹H-NMR (DMSO-d₆) δ: 4.34 (2H, s), 5.47 (2H, s), 6.93–8.30 (13H, m), 11.25 (1H, s), 12.67 (1H, s). IR (KBr) cm⁻¹: 3147, 1719, 1696, 1654, 1623. MS m/z: 437 (M⁺), 277, 91. Anal. Calcd for C₂₅H₁₉N₅O₃: C, 68.64; H, 4.38; N, 16.01. Found: C, 68.83; H, 4.26; N, 16.10.

General Procedure for the Synthesis of 12a–c

Method (A)

A mixture of compound 4 (3.08 g, 0.01 mol) and aromatic aldehydes namely benzaldehyde, 4-fluoro benzaldehyde, and 4-methoxy benzaldehyde (2 mL) in boiling ethanol (30 mL) was refluxed for 3 h. The separated solid was filtered off and recrystallized from the suitable solvent to give compounds 12a–c.

2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N′-benzylideneacetohydrazide (12a)

White crystals; yield (2.76 g, 71%); mp 202–204°C; ethanol; ¹H-NMR (CDCl₃) δ: 4.32 (2H, s), 5.07 (1H, s), 5.53 (2H, s), 7.26–8.46 (14H, m), 8.96 (1H, s). IR (KBr) cm⁻¹: 3192, 1688, 1646. MS m/z: 396 (M⁺), 278, 250, 249, 222, 90, 77. Anal. Calcd for C₂₄H₂₀ N₄O₂: C, 72.71; H, 5.08; N, 14.13. Found: C, 72.64; H, 5.12; N, 14.20.

N′-(4-Fluorobenzylidene)-2-(4-benzyl-1-oxophthalazin-2(1H)-yl)acetohydrazide (12b)

White crystals; yield (3.1 g, 75%); mp 208–210°C; ethanol; ¹H-NMR (CDCl₃) δ: 4.30 (2H, s), 5.15 (1H, s), 5.50 (2H, s), 7.30–8.33 (13H, m), 8.25 (1H, s). IR (KBr, cm⁻¹): 3196, 1686, 1645. MS m/z: 414 (M⁺), 278, 277, 250, 249, 222, 108, 91. Anal. Calcd for C₂₄H₁₉FN₄O₂: C, 69.55; H, 4.62; N, 13.52. Found: C, 69.74; H, 4.60; N, 13.58.

N′-(4-Methoxybenzylidene)-2-(4-benzyl-1-oxophthalazin-2(1H)-yl)acetohydrazide (12c)

White crystals; yield (2.8 g, 68%); mp 198–200°C; ethanol; ¹H-NMR (DMSO-d₆) δ: 3.80 (3H, s), 4.32 (2H, s), 4.91 (1H, s), 5.30 (2H, s), 6.97–8.27 (13H, m), 8.30 (1H, s). IR (KBr) cm⁻¹: 3149, 3141, 1698, 1633. MS m/z (%): 414 (M⁺−CH₃, +3H), 250, 249, 91, 89, 77, 59. Anal. Calcd for C₂₅H₂₂N₄O₃: C, 70.41; H, 5.20; N, 13.14. Found: C, 70.48; H, 5.14; N, 13.17.

Method (B)

The acetohydrazide derivative 4 (3.08 g, 0.01 mol) was refluxed with 2-(4-methoxybenzylidene)malononitrile (1.84 g, 0.01 mol) in ethyl alcohol (30 mL) in the presence of few drops of piperidine for 6 h. The separated solid after cooling was filtered off, dried and recrystallized from the ethanol to give compound 12c.

General Procedure for the Synthesis of 13a and b

A solution of compounds 12a and b (0.01 mol) and thioglycolic acid (0.7 mL, 0.01 mol) in dry benzene (30 mL) was refluxed for 6 h. The separated solid was filtered off and recrystallized from the suitable solvent to give compounds 13a and b.

2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N-(4-oxo-2-phenylthiazolidin-3-yl)acetamide (13a)

White crystals; yield (3.0 g, 64%); mp 184–186°C; ethanol; ¹H-NMR (DMSO-d₆) δ: 3.88 (2H, s), 4.26 (2H, s), 4.78 (2H, s), 5.80 (1H, s), 7.18–8.27 (14H, m), 10.59 (1H, s). IR (KBr, cm⁻¹): 3203, 1730, 1697, 1631. MS m/z: 470 (M⁺), 277, 250, 249, 222, 178, 91, 77. Anal. Calcd for C₂₆H₂₂N₄O₃S: C, 66.37; H, 4.71; N, 11.91; S, 6.81. Found: C, 66.69; H, 4.64; N, 11.97; S, 6.89.

2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)acetamide (13b)

White crystals; yield (3.5 g, 73%); mp 178–180°C; ethanol; ¹H-NMR (DMSO-d₆) δ: 4.19 (2H, s), 4.32 (2H, s), 4.96 (2H, s), 5.33 (1H, s), 7.16–8.29 (13H, m), 11.71 (1H, s). IR (KB, cm⁻¹): 3206, 1729, 1699. MS m/z: 488 (M⁺), 489 (M+1), 250, 249, 165, 91. Anal. Calcd for C₂₆H₂₁FN₄O₃S: C, 63.92; H, 4.33; N, 11.47; S, 6.56. Found: C, 64.23; H, 4.27; N, 11.45; S, 6.58.

Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N′-((4-fluorophenyl)(phenylthio)methyl)acetohydrazide (14)

A solution of compound 12b (4.14 g, 0.01 mol) and thiophenol (1.1 mL, 0.01 mol) in dry benzene (30 mL) was refluxed for 3 h with stirring. The separated solid was filtered off and recrystallized from petroleum ether 60–80°C to give compound 14.Yellowish white crystals; yield (3.7 g, 73%); mp 118–120°C; ¹H-NMR (CDCl₃) δ: 1.85 (2H, br s), 4.24–4.31 (4H, s), 5.01 (1H, s), 7.21–8.44 (18H, m). IR (KBr) cm⁻¹: 3298, 1734, 1653. MS m/z: 522 (M⁺−2), 278, 277, 249, 110, 92, 58. Anal. Calcd for C₃₀H₂₅FN₄O₂S: C, 68.68; H, 4.80; N, 10.68; S, 6.11. Found: C, 68.73; H, 4.76; N, 10.57; S 6.14.

Synthesis of 2-(4-Benzyl-1-oxophthalazin-2(1H)-yl)-N′-(phenylsulfonyl)acetohydrazide (15)

Benzenesulfonyl chloride (0.176 g, 0.001 mol) was added to the solution of acetohydrazide derivative 4 (0.308 g, 0.001 mol) in pyridine (10 mL) at 0°C. The resulting mixture was stirred at room temperature for 5 h. At the end of this period, the reaction mixture was poured into ice water. The precipitate was filtered off, dried, and crystallized from ethanol to give compound 15. Off white crystals; yield (3.3 g, 75%); mp 234–236°C; ¹H-NMR (300 MHz, DMSO-d₆) δ: 4.27 (2H, s), 4.71 (2H, s), 7.19–8.26 (14H, m), 9.74 (1H, s), 9.98 (1H, s). IR (KBr) cm⁻¹: 3220, 1703, 1635, 1343, 1170. MS m/z: 448 (M⁺), 277, 91. Anal. Calcd for C₂₃H₂₀N₄O₄S: C, 61.59; H, 4.49; N, 12.49; S, 7.15. Found: C, 61.46; H, 4.46; N, 12.50; S, 7.12.

4-Benzylphthalazine-1(2H)-thione (16)

A solution of compound 2 (2.36 g, 0.01 mol) and P₂S₅ (8 g, 0.02 mol) in dry toluene (50 mL) was refluxed for 2 h. The reaction mixture was filtered off while hot and concentrated. The separated solid on cooling was filtered off and recrystallized from toluene to give compound 16. Yellow crystals; yield (1.25 g, 50%); mp 160–162°C; ¹H-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 (%): 252 (M⁺), 251, 91, 69. Anal. Calcd for C₁₅H₁₂N₂S: C, 71.40; H, 4.79; N, 11.10; S, 12.71. Found: C, 71.47; H, 4.71; N, 11.04; S, 12.80.

Synthesis of 1-(4-Benzylphthalazin-1-yl)hydrazine (17)

A solution of compound 16 (2.5 g, 0.01 mol) and hydrazine hydrate (0.5 mL, 0.01 mol) in ethanol (30 mL) was refluxed for 3 h. The separated solid was filtered off and crystallized from benzene to give compound 17. Orange crystals; yield (1.6 g, 65%); mp 228–230°C; ¹H-NMR (DMSO-d₆) δ: 4.30 (2H, s), 4.78 (3H, s), 7.27–8.42 (9H, m). IR (KBr) cm⁻¹: 3379, 1616. MS m/z: 234 (M⁺−NH₂), 129, 102, 92, 91, 76. Anal. Calcd for C₁₅H₁₄N₄: C, 71.98; H, 5.64; N, 22.38. Found: C, 72.11; H, 5.60; N, 22.22.

Synthesis of 4-Benzylphthalazine-1-sulfonic Acid (18)

Nitric acid (0.01 mol, 60–68%) was added to compound 16 (2.52 g, 0.01 mol) with stirring for 3 d, then poured on water, the separated solid was filtered off, washed with water and recrystallized from benzene to give compound 18. Yellow crystals; yield (1.5 g, 50%); mp 208–210°C; ¹H-NMR (DMSO-d₆) δ: 3.35 (2H, s), 7.90–9.00 (9H, m), 13.25 (1H, br s). IR (KBr) cm⁻¹: 3431, 3170, 1602, 1200, 1050, 650. MS m/z (%): 270 (M⁺−O₂, +2H^·), 220, 178, 176, 165, 150, 149, 130, 104, 92, 77. Anal. Calcd for C₁₅H₁₂N₂O₃S: C, 59.99; H, 4.03; N, 9.33; S, 10.68. Found: C, 60.20; H, 3.97; N, 9.41; S, 10.61.

Synthesis of 4-Benzylphthalazine-1-sulfonyl Chloride (19)

Molecular chlorine was bubbled through a solution of compound 16 (2.52 g, 0.01 mol) in glacial acetic acid (25 mL) and water (3 mL) at 30°C for 20 min, upon completion of the chlorination, water (30 mL) was added and the solution was washed with chloroform (50 mL aliquots), the combined chloroform layers were washed with 2.5% sodium hydroxide (2×50 mL aliquots), the chloroform was then dried over MgSO₄, filtered off, and left for slow evaporation. The separated solid was filtered off and recrystallized from petroleum ether 40–60°C to give compound 19. Yellow crystals; yield (1.7 g, 55%); mp 136–138°C; ¹H-NMR spectrum (DMSO-d₆) δ: 4.29 (2H, s), 7.18–8.26 (9H, m). IR (KBr) cm⁻¹: 1602, 1346, 1180. MS m/z: 318 (M⁺), 237, 236, 235, 102, 78, 77. Anal. Calcd for C₁₅H₁₁ClN₂O₂S: C, 56.52; H, 3.48; Cl, 11.12; N, 8.79; S, 10.06. Found: C, 56.63; H, 3.43; Cl, 11.20; N, 8.93; S, 10.12.

Synthesis of Ethyl 2-(4-Benzylphthalazin-1-ylthio)acetate (20)

A solution of compound 16 (2.52 g, 0.01 mol), ethyl chloroacetate (4.4 g, 0.04 mol) and anhydrous potassium carbonate (5.5 g, 0.04 mol) in acetone (60 mL) was heated on water bath for 20 h. The excess solvent was evaporated and the reaction mixture was poured on water. The separated solid was filtered off and recrystallized from petroleum ether 60–80°C to give compound 20. Yellowish white crystals; yield (1.7 g, 54%); mp 74–76°C; ¹H-NMR (DMSO-d₆) δ: 1.19 (3H, t, J=6.0 Hz), 4.12–4.17 (2H, q, J=6.0 Hz), 4.30 (2H, s), 4.63 (2H, s), 7.15–8.28 (9H, m). IR (KBr) cm⁻¹: 3061, 3030, 2978, 2923, 1737. MS m/z: 338 (M⁺), 294, 293, 266, 265, 252, 248, 203, 91 (100). Anal. Calcd for C₁₉H₁₈N₂O₂S: C, 67.43; H, 5.36; N, 8.28; S, 9.67. Found: C, 67.25; H, 5.53; N, 8.18; S, 9.60.

MATERIALS AND METHODS

Biological Activities

Antibacterial Activity

Bacterial isolates were grown in nutrient broth medium (Oxoid) in an orbital shaking incubator for 24 h at 37°C. The bacterial growth centrifuged, washed twice with saline solution (0.9%) and then standardized to approximately 10⁶ colony forming unit (CFU)/mL using nutrient broth medium, during the assay.

Bacterial Sensitivity Test

Standard well agar diffusion method was carried out to detect the activity of the compounds against the clinical bacterial isolates according to Cheesbrough³⁸⁾ Standard antibiotic discs (vancomycin, erythromycin and tetracycline) were placed uniformly on the surface of nutrient agar plates seeded with 100 µL of 24 h bacterial culture prepared as mentioned above. For antibacterial activities of the compounds wells were made in these plates then from each compound 100 µL (100 µg) was placed in each well. The plates were left in refrigerator for 2 h. After that were incubated at 37°C for 24 h. Diameter of inhibition zones around the wells were measured. All microbiological statistical analyses in this study were carried out using Microsoft Excel 2003 (Microsoft Corporation). All data were calculated from at least 2 replicates and the standard errors for each datum were plotted on the graph.

Cytotoxicity Assay

The cytotoxic activity of seven compounds was tested against four human tumor cell lines namely; hepatocellular carcinoma (liver) HePG-2, colon cancer HCT-116, human (prostate) cancer cell line PC3, and mammary gland (breast) MCF-7. The cell lines were obtained from ATC C via Holding Company for biological products and vaccines (VACSERA), Cairo, Egypt. 5-Fluorouracil was used as a standard anticancer drug for comparison.

Chemical Reagents

The reagents are RPMI-1640 medium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO) and 5-fluorouracil (Sigma Co., St. Louis, MO, U.S.A.), Fetal Bovine serum (GIBCO, U.K.).

MTT Assay

The different cell lines^39,40) mentioned above was 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 MTT to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells. The cells were cultured in RPMI-1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/mL penicillin and 100 µg/mL streptomycin at 37°C in a 5% CO₂ incubator. The cells were seeded⁴¹⁾ in a 96-well plate at a density of 1.0×10⁴ cells/well at 37°C for 48 h under 5% CO₂ incubator. 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 5 mg/mL was added and incubated for 4 h. 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 absorbance of 570 nm using a plate reader (EXL 800 U.S.A.). The relative cell viability in percentage was calculated as (A₅₇₀ of treated samples/A₅₇₀ of untreated sample)×100.

Antioxidant Assay

ABTS Method

For each of the investigated compounds^42–44) (2 mL) of ABTS solution (60 µM) was added to 3 mL MnO₂ suspension (25 mg/mL), all prepared in (5 mL) aqueous phosphate buffer solution (pH 7, 0.1 M). The mixture was shaken, centrifuged, filtered and the absorbance of the resulting green blue solution (ABTS radical solution) at 734 nm was adjusted to approx. ca. 0.5. Then, 50 µL of (2 mM) solution of the tested compound in spectroscopic grade MeOH/phosphate buffer (1 : 1) was added. The absorbance was measured and the reduction in color intensity was expressed as inhibition percentage. L-Ascorbic acid was used as standard antioxidant (positive control). Blank sample was run without ABTS and using MeOH/phosphate buffer (1 : 1) instead of the tested compounds. Negative control was run with ABTS and MeOH/phosphate buffer (1 : 1) only.

Bleomycin-Dependent DNA Damage Assay

To the reaction mixtures^45,46) in a final volume of 1.0 mL, the following reagents were added: DNA (0.2 mg/mL), bleomycin sulfate (0.05 mg/mL), FeCl₃ (0.025 mM), magnesium chloride (5 mM), KH₂PO₄–KOH buffer pH 7.0 (30 mM), and ascorbic acid (0.24 mM) or the test fractions diluted in MeOH to give a concentration of (0.1 mg/mL). The reaction mixtures were incubated in a water bath at 37°C for 1 h. At the end of the incubation period, 0.1 mL of ethylenediaminetetraacetic acid (EDTA) (0.1 M) was added to stop the reaction (the iron-EDTA complex is unreactive in the bleomycin assay). DNA damage was assessed by adding 1 mL 1% (w/v) thiobarbituric acid (TBA) and 1 mL of 25% (v/v) hydrochloric acid followed by heating in a water-bath maintained at 80°C for 15 min. The chromogen formed was extracted into 1-butanol, and the absorbance was measured at 532 nm.

Acknowledgment

Technical support from Department of Chemistry, Faculty of Science, Ain Shams University is gratefully acknowledged.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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