Phthalazines and phthalazine hybrids as antimicrobial agents: Synthesis and biological evaluation

Abstract

Phthalazine and phthalazinone derivatives are important owing to their significant biological activities and pharmacological properties. Herein, a benzoic acid derivative (2), a benzoxazin-1-one derivative (3), and an oxophthalazin-2(1H)-yl)acetohydrazide (13) are utilized as precursors to construct a novel series of phthalazinones bearing various valuable functional groups in excellent yields via several simple and promising approaches. Finally, the antimicrobial activity of the newly synthesized phthalazines is screened against different microbial strains; namely, Gram-negative and Gram-positive bacteria utilizing Amoxicillin as a standard drug.

Keywords

benzoxazinone Friedel Craft’s reaction 1,3,4-oxadiazole phthalazine pyrazole

Introduction

Phthalazine, also known as benzo-orthodiazine, 2,3-diazonaphthalene, and benzo[d]pyridazine, is a compound in which pyridazine is fused with a benzene ring.

Although, N-containing heterocyclic compounds are widely distributed in nature, the presence of two adjacent nitrogen atoms in the phthalazine ring makes it rare and not very familiar in isolated extracts from living organisms, owing to the fact that it can be constructed from hydrazine which is an uncommon amine source in nature.

Phthalazines, as an important class of bicyclic N-heterocycles, have attracted sizable attention due to their valuable biological and pharmacological activities.^1,2

Phthalazines are popular pharmacophores as they are the core chemical motifs in many commercially available drugs such as Azelastin (antihistamine),³ Vatalanib (vascular endothelial growth factor receptor (VEGFR) inhibitor),⁴ and Hydralazine (antihypertensive agent).⁵ Phthalazine derivatives are also considered as p38MAP kinase inhibitors,⁶ selective binders of gamma-aminobutyric acid (GABA) receptors,⁷ cyclooxygenase-2 (COX-2) inhibitors,⁸ and high-affinity ligands of voltages gated calcium channels.⁹

Like many other isomeric benzodiazines, phthalazine derivatives reveal numerous pharmacological and biological activities such as antimicrobial,^10–12 antidiabetic,¹³ analgesic,^14,15 anticonvulsant,^16,17 antitumor,^18–20 antiproliferative,²⁰ antiepileptic,^21,22 anti-inflammatory,^23,24 and vasorelaxant.^25,26

In addition, phthalazines are known as serotonin reuptake inhibitors and are considered as anti-depression agents.²⁷ Also, they are crucial precursors in the synthesis of many compounds with interesting pharmacological properties like phosphodiesterase inhibitors and blood platelet aggregation inhibitors.^27–29

Encouraged by the above-mentioned facts, and in our endeavor to develop novel synthetic approaches to construct new valuable heterocycles for biological screening,^30,31 we report herein the synthesis of novel and diverse phthalazine, 1,3,4-oxadiazole, and pyrazole derivatives from readily available and inexpensive scaffolds utilizing short and straightforward synthetic routes.

Results and discussion

Owing to the aforementioned therapeutic properties of phthalazines, aroylation of cumene by phthalic anhydride exploiting Friedel Craft’s reaction conditions was achieved and 2-(4-isopropylbenzoyl)benzoic acid (2) was produced and subsequently used as a scaffold to construct our desired heterocyclic systems (Scheme 1).³²

Scheme 1.

Synthesis of o-benzoic acid derivative 2.

Having o-aroylbenzoic acid derivative 2 in hand encouraged us to study its behavior toward various nitrogen nucleophiles; for instance, hydroxylamine hydrochloride, hydrazine hydrate, thiosemicarbazide, and thiocarbonohydrazide.

Initially, cyclization of 2-(4-isopropylbenzoyl)benzoic acid (2) to the promising benzo[1,2]oxazinone derivative 3 was accomplished using hydroxylamine hydrochloride in pyridine (Scheme 2). The structure of compound 3 was established through its elemental analysis and spectral data. The infrared (IR) spectrum of 3 exhibited the disappearance of the hydroxy group broad absorption band at 3293–3455 cm⁻¹ and the appearance of a cyclic imine C=N absorption frequency at 1596 cm⁻¹. Also, the mass spectrum of 3 revealed the molecular ion peak at m/z = 265 (50%).

Scheme 2.

Compounds 2 and 3 as precursors for various phthalazines.

According to a reported procedure,³² phthalazin-1-one derivatives were readily accessible through the condensation reaction of o-benzoyl benzoic acid with hydrazine hydrate in boiling ethanol. Consequently, adopting this procedure and allowing 2-(4-isopropylbenzoyl)benzoic acid (2) to react with hydrazine hydrate gave 4-(4-isopropyl phenyl)phthalazin-1(2H)-one 4 (Scheme 2). Compound 4 was identified through its spectral and analytical data in which the IR spectrum showed the disappearance of the hydroxy group absorption and the appearance of a cyclic amide carbonyl absorption band at 1664 cm⁻¹ in addition to a characteristic signal for the NH group at 3155 cm⁻¹. In addition, the mass spectrum of 4 exhibited the molecular ion peak at m/z = 264 (99%), which was in accordance with the molecular formula C₁₇H₁₆N₂O.

It is noteworthy that 1-oxophthalazine derivatives 5 and 6 were obtained via the reaction of 2-(4-isopropylbenzoyl)benzoic acid (2) with thiosemicarbazide or thiocarbonohydrazide, respectively, in refluxing pyridine (Scheme 2). The structure of compound 5 was substantiated through the IR spectrum which was devoid of the characteristic hydroxy group absorption and revealed strong absorption bands at 3178 and 3367 cm⁻¹ characteristic for an NH₂ group, an intense absorption band at 1643 cm⁻¹ for the amide carbonyl group, and at 1284 cm⁻¹ due to the C=S group. The ¹H NMR spectrum exhibited the amino group signal at δ 6.90, while the mass spectrum revealed the molecular ion peak at m/z = 323 (5%), which was in accordance with the molecular formula of C₁₈H₁₇N₃OS. Likewise, the IR spectrum of compound 6 showed the disappearance of the hydroxy group band and the appearance of strong absorption bands at 3440 and 3155 cm⁻¹ due to NH and NH₂ groups, respectively, a sharp absorption band at 1666 cm⁻¹ for the amide carbonyl group, and an absorption band at 1423 cm⁻¹ for the C=S. The mass spectrum showed the molecular ion peak at m/z = 338 (10%) which was equivalent to the molecular formula C₁₈H₁₈N₄OS.

In the same context, investigation of the behavior of benzoxazinone 3 toward nitrogen nucleophiles was appealing. Interestingly, phthalazinone 4 was obtainable not only from o-aroylbenzoic acid derivative 2, as illustrated before, but also from benzoxazinone derivative 3 through its ammonolysis by fusing it with ammonium acetate at 150 °C (Scheme 2).^33,34 The formation of phthalazinone 4 was confirmed from its IR spectrum, mass spectrum, ¹H NMR, melting and mixed melting points.

Another convenient approach for the preparation of phthalazinone derivatives was achieved via the reaction of benzoxazinone derivative 3 with benzyl amine in ethanol to afford 2-benzyl-4-(4-isopropylphenyl)phthalazin-1(2H)-one (7) (Scheme 2).^35,36

Moreover, 1-oxophthalazine derivatives 8 and 9 were synthesized through the reactions of benzoxazinone derivative 3 with thiosemicarbazide or thiocarbonohydrazide, respectively, in refluxing pyridine (Scheme 2). The structure of 1-(4-(4-isopropylphenyl)-1-oxophthalazin-2(1H)-yl)thiourea (8) was substantiated via its spectroscopic data. The IR spectrum exhibited intense absorption bands at 3178 and 3367 cm⁻¹ due to the NH and NH₂ groups, respectively, along with an amide carbonyl group at 1643 cm⁻¹. Also, the ¹H NMR spectrum of compound 8 revealed signals at δ 4.50 and 8.63 due to NH₂ and NH, respectively. The mass spectrum showed the molecular ion peak at m/z = 338 (21%) equivalent to the molecular formula C₁₈H₁₈N₄OS. Similarly, the IR spectrum of N-(4-(4-isopropylphenyl)-1-oxophthalazin-2(1H)-yl)hydrazinecarbothioamide (9) revealed two absorption bands at 3212 and 3444 cm⁻¹ corresponding to 2NH and an NH₂ group, respectively. The mass spectrum showed the molecular ion peak at m/z = 353 (24%), which was equivalent to the molecular formula C₁₈H₁₉N₅OS.

Interestingly, changing the solvent and performing the reaction of benzoxazinone derivative 3 with thiocarbonohydrazide in refluxing dioxane instead of refluxing pyridine gave access to a new fused tricyclic system containing a triazine moiety; namely, 7-(4-isopropylphenyl)-2H-[1,2,4,5]tetrazino[6,1-a]phthalazine-3(4H)-thione (10) (Scheme 2). The IR spectrum of compound 10 was devoid of any absorption band for a carbonyl group, but new frequencies at 1284 cm⁻¹ corresponding to C=S and at 3212 and 3270 cm⁻¹ corresponding to 2NH groups were recorded and the molecular ion peak equivalent to the molecular formula C₁₈H₁₇N₅S was detected in the mass spectrum at m/z = 335 (11%). Another piece of evidence was acquired from the ¹H NMR spectrum in which two new slightly broad singlet peaks were revealed at 7.68 and 8.91 ppm and were characteristic of two new NH groups.

In addition, dihydroisoquinoline derivative 11 was readily obtained through the reaction between benzoxazinone 3 and acetylacetone as a carbon nucleophile (Scheme 2).^36,37 The structure of compound 11 was confirmed by spectroscopic analysis. The IR spectrum exhibited strong absorption bands at 1735 and 1666 cm⁻¹ attributed to three carbonyl groups, and at 1600 cm⁻¹ corresponding to a cyclic imine C=N. Also, the ¹H NMR spectrum supported the proposed structure of compound 11 by revealing a new characteristic methyl group singlet signal integrating for six protons at δ 2.50. Finally, the mass spectrum showed the molecular ion peak at m/z = 347 (26%) equivalent to the molecular formula C₂₂H₂₁NO₃.

In our attempts to build up additional fused heterocycles, phthalazin-1-one derivative 4 was allowed to react with more diversified carbon nucleophiles such as ethyl bromoacetate and benzoyl chloride (Scheme 2). Accordingly, 4-(4-isopropylphenyl)phthalazin-1(2H)-one (4) was allowed to react with ethyl bromoacetate and anhydrous K₂CO₃ in dry acetone to afford the corresponding N-alkyl derivatives 12.

It is worthwhile to mention that no O-alkylation product was isolated and the reaction most probably took place via S_N² reaction mechanism and this may be attributed to the fact that the nitrogen atom is more nucleophilic than oxygen and therefore the N-alkylation product was the sole product. The structure of compound 12 was confirmed on the basis of its elemental analysis and spectral data. The IR spectrum showed characteristic absorption bands at 1742 cm⁻¹ corresponding to the C=O of the ester and at 1657 cm⁻¹ corresponding to the cyclic amide C=O, while it was devoid of any absorption bands for NH. Also, the ¹H NMR spectrum of 12 exhibited the distinctive ethyl group triplet–quartet splitting pattern at δ 1.20 and 4.15. Also, it confirmed the introduction of a new methylene group (CH₂CO) at 4.98. The mass spectrum of compound 12 revealed the molecular ion peak at m/z = 350 (0.02%) equivalent to the molecular formula C₂₁H₂₂N₂O₃.

In addition, the chemical structure of the latter compound 12 was confirmed chemically via its interaction with hydrazine hydrate to afford 2-(4-(4-isopropylphenyl)-1-oxophthalazin-2(1H)-yl)acetohydrazide (13). The IR spectrum of 13 revealed absorption bands at 1662 cm⁻¹ corresponding to the C=O groups, and at 3329 and 3444 cm⁻¹ corresponding to the NH and NH₂ groups, respectively, while ¹H NMR spectrum revealed NH₂ and NH signals at δ 4.28 and 9.29, respectively. The mass spectrum of compound 13 showed the molecular ion peak at m/z = 336 (15%) equivalent to the molecular formula C₁₉H₂₀N₄O₂.

Refluxing phthalazinone derivative 4 with benzoyl chloride on a steam bath gave access to 2-benzoyl-4-(4-isopropylphenyl)phthalazin-1(2H)-one (14). The IR spectrum of 14 was devoid of any NH absorption bands, but showed an intense absorption band at 1692 cm⁻¹ attributed to the two C=O groups. The mass spectrum showed a peak corresponding to the molecular ion at m/z = 368 (53%). Interestingly, hydrazinolysis of phthalazin-1-one derivative 14 with hydrazine hydrate in ethanol afforded a mixture of benzoyl hydrazine and phthalazinone derivative 4.

Driven by the high functionality of the side chain of oxophthalazine acetohydrazide derivative 13, the effect of various carbon nucleophiles was explored. For instance, reactions of compound 13 with aromatic aldehydes to obtain a new series of biologically active Schiff’s bases is described, in which the corresponding Schiff bases 15a–c were obtained through a condensation reaction between hydrazide 13 and different aromatic aldehydes; namely, benzaldehyde, p-chlorobenzaldehyde, and p-nitrobenzaldehyde in ethanol (Scheme 3). The IR spectra of compounds 15a–c showed stretching bands around 1579–1584 cm⁻¹ corresponding to the imine group C=N. The ¹H NMR spectrum of compound 15a, as a representative example, showed signals for NH at δ 4.95 and for CH at δ 8.72, while the mass spectrum revealed the molecular ion peak at m/z = 424 (100%) equivalent to the molecular formula C₂₆H₂₄N₄O₂. Subsequent cyclization of aryl methylidene hydrazide derivatives 15a–c to oxadiazole derivatives 16a–c was achieved through boiling in acetic anhydride (Scheme 3). The IR spectra of compounds 16a–c revealed absorption bands around 1661 cm⁻¹ corresponding to C=O groups and were devoid of any bands for NH groups. Also, the ¹H NMR spectrum of compound 16a, as a representative example, showed the oxadiazole singlet signal at δ 7.03 and the new methyl singlet signal at δ 2.04, while the mass spectrum showed the molecular ion peak at m/z = 466 (100%) equivalent to the molecular formula C₂₈H₂₆N₄O₃.

Scheme 3.

Reactions of acetic acid hydrazide derivative 13.

In addition, a cyclization reaction took place when acetic acid hydrazide derivative 13 was allowed to react with carbon disulfide in alcoholic potassium hydroxide to afford the corresponding oxadiazolo-2-thione derivative 17 (Scheme 3). The IR spectrum of compound 17 revealed the introduction of a C=S group at 1423 cm⁻¹. Also, the ¹H NMR spectrum of compound 17 showed a methylene bridge singlet signal at δ 5.51 and an NH group signal at 14.6, whereas the mass spectrum showed a peak corresponding to the molecular ion at m/z = 378 (4%) equivalent to the molecular formula C₂₀H₁₈N₄O₂S.

A new approach was adopted to synthesize more oxadiazole rings attached to the phthalazine moiety in which hydrazide derivative 13 was allowed to reflux with triethylorthoformate for 15 h to afford 2-((1,3,4-oxadiazol-2-yl)methyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (18) (Scheme 3). The IR spectrum of compound 18 was devoid of any absorption bands for NH groups, while the ¹H NMR spectrum of compound 18 exhibited the methylene bridge singlet signal at δ 4.88 and the oxadiazole CH singlet signal at δ 9.63. The mass spectrum showed the molecular ion peak at m/z = 346 (17%) equivalent to the molecular formula C₂₀H₁₈N₄O₂.

Likewise, an oxadiazole ring could be attached to our phthalazinone heterocycle by the reaction of hydrazide 13 with acetic acid in presence of phosphorus oxychloride to afford oxadiazolophthalazine derivative 19 (Scheme 3). The IR spectrum of compound 19 showed the disappearance of the NH and NH₂ absorption bands, while the ¹H NMR spectrum showed the presence of a new singlet signal integrating for three protons at δ 2.51, and the mass spectrum showed the molecular ion peak at m/z = 360 (32%).

Moreover, 2-{2-[4-(4-isopropylphenyl)-1-oxophthalazin-2(1H)-yl]acetyl}-N-phenylhydrazine-1-carbothioamide (20) was accessible through the reaction of hydrazide derivative 13 with phenyl isothiocyanate in pyridine (Scheme 3). The IR spectrum of compound 20 showed the presence of a new absorption band for the C=S group at 1427 cm⁻¹, while the mass spectrum showed the molecular ion peak at m/z = 471 (7%) equivalent to the molecular formula C₂₆H₂₅N₅O₂S.

Interestingly, a pyrazole ring could be attached to the phthalazine moiety through the reaction of the hydrazide derivative 13 with various activated reagents such as acetyl acetone, diethyl malonate, or malononitrile. Accordingly, refluxing the key starting compound 13 with the aforementioned reagents in boiling ethanol afforded the corresponding pyrazole derivatives 21–23. The ¹H NMR spectrum of compound 21 showed new singlet signals at δ 2.25 and 2.46 integrating to three protons each and attributed to the introduction of two new methyl groups, in addition to a singlet signal at δ 6.29 attributed to the CH of the pyrazole.

A new pyrazole ring attached to the phthalazine ring was obtained through the reaction of hydrazide derivative 13 with acrylonitrile to yield 2-[2-(5-amino-2,3-dihydro-1H-pyrazol-1-yl)-2-oxoethyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (24) (Scheme 3). The IR spectrum of compound 24 showed the presence of the two C=O groups with an absorption band at 1673 cm⁻¹ and NH and NH₂ absorptions at 3266 and 3440 cm⁻¹, respectively. The mass spectrum showed the molecular ion peak at m/z = 389 (91%), equivalent to the molecular formula C₂₂H₂₃N₅O₂.

Furthermore, acetic acid hydrazide derivative 13 reacted with phthalic anhydride, as a dicarbonyl compound in a cyclic structure, to afford N-(1,3-dioxoisoindolin-2-yl)-2-[4-(4-isopropylphenyl)-1-oxophthalazin-2(1H)-yl]acetamide (25). The IR spectrum of compound 25 exhibited absorption bands for the four C=O groups at 1662, 1737, and 1792 cm⁻¹, while the ¹H NMR spectrum showed a single signal for the methylene bridge at δ 5.11 and revealed an NH signal at δ 11.05. Finally, the mass spectrum showed the molecular ion peak at m/z = 466 (0.05%) equivalent to the molecular formula C₂₇H₂₂N₄O₄.

Antimicrobial assay

Phthalazine and phthalazine hybrid structures, in which a phthalazine moiety is attached to another heterocycle, are considered to be of immense importance owing to their wide spectrum of biological activities.^1,2,10 Accordingly, the in vitro antimicrobial activity of the newly synthesized heterocycles was appraised against two Gram-positive bacteria, namely, Bacillus subtilis and Staphylococcus aureus, in addition to two Gram-negative bacteria, namely, Pseudomonas aeruginosa and Escherichia coli.

In the current in vitro antimicrobial assay, Amoxicillin was employed as a standard and the antibacterial activities of the compounds under investigation were recorded as inhibition diameter zones (IZ) in millimeters (mm) (Table 1).

Table 1.

In vitro antimicrobial activity of the synthesized compounds.

Entry	Compound	Gram-positive bacteria				Gram-negative bacteria
		Bacillus subtilis		Staphylococcus aureus		Pseudomonas aeruginosa		Escherichia coli
		IZ ± SD^a	Activity index (%)	IZ ± SD^a	Activity index (%)	IZ ± SD^a	Activity index (%)	IZ ± SD^a	Activity index (%)
1	2	5 ± 0.29	62.5	9 ± 0.15	112.5	12 ± 0.29	60.0	8 ± 0.50	80.0
2	3	3 ± 0.29	37.5	NA	–	NA	–	6 ± 0.78	60.0
3	4	NA	–	NA	–	NA	–	3 ± 0.76	30.0
4	5	5 ± 0.15	62.5	13 ± 0.87	162.5	12 ± 0.36	60.0	9 ± 0.12	90.0
5	6	4 ± 0.50	50.0	6 ± 0.20	75.0	12 ± 0.29	60.0	6 ± 0.20	60.0
6	7	3 ± 0.76	37.5	3 ± 0.29	37.5	8 ± 0.20	40.0	NA	–
7	8	5 ± 0.36	62.5	NA	–	14 ± 0.58	70.0	NA	–
8	9	NA	–	8 ± 0.50	100	6 ± 0.15	30.0	NA	–
9	10	5 ± 0.20	62.5	9 ± 0.15	112.5	15 ± 0.12	75.0	4 ± 0.20	40.0
10	11	4 ± 0.15	50.0	2 ± 0.5	25.0	NA	–	NA	–
11	12	4 ± 0.12	50.0	3±0.20	37.5	10 ± 0.29	50.0	3 ± 0.29	30.0
12	13	2 ± 0.58	25.0	6 ± 0.29	75.0	12 ± 0.58	60.0	6 ± 0.12	60.0
13	14	9 ± 0.20	112.5	8 ± 0.36	100	14 ± 0.36	70.0	8 ± 0.29	80.0
14	15a	NA	–	15 ± 0.87	187.5	2 ± 0.87	10.0	3 ± 0.20	30.0
15	15b	2.5 ± 0.36	31.3	2 ± 0.29	25.0	5 ± 0.50	25.0	NA	–
16	15c	NA	–	6 ± 0.12	75.5	NA	–	2 ± 0.50	20.0
17	16a	6 ± 0.12	75.0	9 ± 0.12	112.5	13 ± 0.15	65.5	5 ± 0.58	50.0
18	16b	6 ± 0.29	75.0	2 ± 0.50	25.0	12 ± 0.20	60.0	3 ± 0.29	30.0
19	16c	NA	–	5 ± 0.20	50.0	13 ± 0.12	65.5	3 ± 0.12	30.0
20	17	6 ± 0.20	75.0	10 ± 0.87	125.0	4 ± 0.76	20.0	6 ± 0.20	60.0
21	18	4 ± 0.50	50.0	4 ± 0.36	50.0	10 ± 0.58	50.0	7 ± 0.15	70.0
22	19	7 ± 0.36	87.5	NA	–	15 ± 0.15	75.0	NA	–
23	20	5 ± 0.12	62.5	4 ± 0.20	50.0	11 ± 0.36	55.0	NA	–
24	21	NA	–	5 ± 0.12	62.5	14 ± 0.20	70.0	NA	–
25	22	6 ± 0.15	75.0	4 ± 0.29	50.0	9 ± 0.36	45.0	NA	–
26	23	3 ± 0.50	37.5	11 ± 0.15	137.5	5 ± 0.76	25.0	NA	–
27	24	7 ± 0.20	87.5	2 ± 0.50	25.0	10 ± 0.36	50.0	NA	–
28	25	4 ± 0.50	50.0	7 ± 0.12	87.5	6 ± 0.29	30.0	NA	–
29	Amoxicillin	8	100	8	100	20	100	10	100

NA: No antimicrobial activity detected; SD: standard deviation.

Inhibition diameter zones expressed in millimeters (mm).

As shown in Table 1, the compounds under investigation revealed a wide range of variation in their biological activities.

Compound 4 was inactive against the two tested Gram-positive bacterial strains while compound 11 was inactive against the two Gram-negative bacteria used in this test (Table 1, entries 3 and 10). Moreover, of all the tested compounds, 14 showed the highest activity against B. subtilis (Table 1, entry 13). Likewise, compound 15a exhibited very promising inhibition effects against S. aureus bacteria (Table 1, entry 14) followed by compounds 5, 23, and 17 (Table 1, entries 4, 26, and 20). In addition, compounds 2, 10, and 16a showed exactly the same high activity value against S. aureus (Table 1, entries 1, 9, and 17).However, and with respect to Gram-negative bacteria, the highest activity index in the case of Pseudomonas aeruginosa was observed for compounds 10 and 19 (Table 1, entries 9 and 22). Contrarily, compounds 3, 4, and 15c were inactive against the above-mentioned pathogen. Finally, the highest activity against E. coli was recorded in the case of tested compound 5 (Table 1, entry 4). However, compounds 7–9, 15b, and 19–25 were totally inactive against E. coli (Table 1, entries 6–8, 15, and 22–28).

Conclusion

In summary, o-aroylbenzoic acid derivative 2, benzoxazinone derivative 3, and acetic acid hydrazide derivative 13 were utilized as precursors to construct novel and valuable phthalazine derivatives via simple and straightforward synthetic routes. Subsequently, carbon and nitrogen nucleophiles were exploited to give access to plentiful and diverse 1,3,4-oxadiazole and pyrazole rings attached to the phthalazine moiety. Finally, the in vitro antimicrobial activity of the newly synthesized heterocyclic compounds was tested against assorted pathological strains. Interestingly, many of the products revealed excellent inhibition efficiency against the tested pathogens.

Experiment

General

Melting points were determined with an electrothermal melting point apparatus and are uncorrected. The reaction times were determined using thin-layer chromatography (TLC) which was performed with fluorescent silica gel plates HF₂₄₅ (Merck) and the plates were viewed with iodine. Merck silica gel (230–400 mesh) was used for flash chromatography separations. The Microanalytical Center of Cairo University performed the microanalyses. IR (KBr) spectra were recorded on a Pye-Unicam IR spectrophotometer SP 2000 (Faculty of Science, Fayoum University). The mass spectra were run using a Shimadzu-GC-MS-GP 1000 EX instrument by employing the direct inlet system. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury 300-MHz spectrometer using tetramethylsilane (TMS) as the internal standard at the National Research Center. Chemical shifts (δ) and coupling constants (J) are recorded in parts per million (ppm) and Hertz (Hz), respectively.

2-(4-Isopropylbenzoyl)benzoic acid (2)

In a 1000 mL three-necked flask equipped with a condenser, a mixture of phthalic anhydride (14.8 g, 0.1 mol) and cumene (15 mL) was treated with then anhydrous aluminum chloride (20.0 g, 0.15 mol) gradually during stirring over a period of half an hour.³² The reaction mixture was refluxed in a water bath for 2 h and then the mixture was allowed to stand at room temperature overnight. Ice-HCl was added to the mixture and the excess cumene was removed by steam distillation. The collected solid was filtered off, washed well with water, and dried. Recrystallization from ethanol afforded 2 in 65% yield as beige crystals; m.p. 230–231 °C, IR (cm⁻¹): 1656, 1709 (2CO), 2955 (CH aliphatic), 3293–3455 (OH). Anal. calcd for C₁₇H₁₆O₃: C, 76.10; H, 6.01; found: C, 76.06; H, 5.97%.

4-(4-Isopropylphenyl)-1 H -benzo[d][1,2]oxazin-1-one (3)

A mixture of compound 2 (2.6 g, 0.01 mol) and hydroxylamine hydrochloride (0.01 mol) in dry pyridine (15 mL) was re-fluxed for 15 h. On cooling, the reaction mixture was poured onto ice-cold water acidified with HCl and the produced solid was filtered off, washed with water, and recrystallized from ethanol as colorless crystals in 71% yield; m.p. 110–111 °C, IR (KBr): 1596 cm⁻¹ (C=N), 1731 cm⁻¹ (CO), 2962 cm⁻¹ (CH aliphatic).¹H NMR (dimethyl sulphoxide (DMSO)-d₆, 300 MHz, ppm) δ 1.24 (d, J = 6.4 Hz, 6H, 2CH₃), 2.90–3.03 (m, 1H, CH), 7.33–8.20 (m, 8H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 125.7, 126.6, 126.9, 127.6, 128.1, 129.7, 130.4, 133.0, 134.8, 148.4, 152.3, 158.6. Anal. calcd for C₁₇H₁₅NO₂: C, 76.96; H, 5.70; N, 5.28; found: C, 76.93; H, 5.68; N, 5.30%. MS (70 eV) m/z (%): 265 (50) (M⁺), 106 (100).

4-(4-Isopropyl phenyl)phthalazin-1(2 H )-one (4)

Method A: To a solution of 2 (2.6 g, 0.01 mol) in absolute ethanol (15 mL), 98% hydrazine hydrate (0.3 mL) was added.³² The reaction mixture was refluxed for 4 h and after cooling the obtained solid was filtered off and recrystallized from ethanol to give 4 in 57% yield as colorless crystals.

Method B: A mixture of benzoxazine 3 (2.65 g, 0.01 mol) and ammonium acetate (0.77 g, 0.01 mol) was fused for 2 h in an oil bath at 150 °C. After cooling, the obtained solid was washed with water, filtered off, and recrystallized from ethanol to give 4 in 57% yield as colorless crystals; m.p. 260–261 °C, IR (KBr): 1606 cm⁻¹ (C=N), 1664 cm⁻¹ (CO), 2956 (CH aliphatic), 3155 cm⁻¹ (NH). Anal. calcd for C₁₇H₁₆N₂O: C, 77.25; H, 6.10; N, 10.60; found: C, 77.20; H, 6.12; N, 10.58%. MS (70 eV) m/z (%): 264 (99) (M⁺), 249 (100).

4-(4-Isopropylphenyl)-1-oxophthalazine-2(1 H )-carbothioamide (5)

A mixture of 2 (2.6 g, 0.01 mol) and thiosemicarbazide (0.01 mol) was refluxed in pyridine (20 mL) for 8 h. On cooling, the reaction mixture was poured onto ice-cold water acidified with HCl and the solid produced was filtered off, washed with water, and crystallized from ethanol to give 5 in 69% yield as colorless crystals; m.p. 278–279 °C, IR (KBr): 1284 cm⁻¹ (C=S), 1619 cm⁻¹ (C=N), 1643 cm⁻¹ (CO), 3178 and 3367 cm⁻¹ (NH₂). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.04 (d, J = 6.3 Hz, 6H, 2CH₃), 3.18–3.21 (m, 1H, CH), 6.90 (s, 2H, NH₂), 7.10 (d, J = 7.5 Hz, 2H, ArH), 7.17 (d, J = 7.5 Hz, 2H, ArH), and 7.26–8.11 (m, 4H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 125.8, 126.3, 126.9, 127.5, 127.8, 128.6, 129.9, 132.6, 134.3, 149.6, 167.1, 168.35, 172.1. Anal. calcd for C₁₈H₁₇N₃OS: C, 66.85; H, 5.30; N, 12.99; S, 9.91; found: C, 66.81; H, 5.27; N, 12.86; S, 9.89%. MS (70 eV) m/z (%): 323 (5) (M⁺), 205 (11), 77 (100).

4-(4-Isopropylphenyl)-1-oxophthalazine-2(1 H )-carbothiohydrazide (6)

A mixture of 2 (2.6 g, 0.01 mol) and thiocarbonohydrazide (1.06 g, 0.01 mol) was refluxed in pyridine (20 mL) for 8 h. On cooling, the reaction mixture was poured onto ice-cold water acidified with HCl and the produced solid was filtered off, washed with water, and recrystallized from ethanol to give 6 in 63% yield as colorless crystals; m.p. >300 °C, IR (KBr): 1423 cm⁻¹ (C=S), 1604 cm⁻¹ (C=N), 1666 cm⁻¹ (CO), 2958 cm⁻¹ (CH aliphatic), 3155 and 3440 cm⁻¹ (NH) and (NH₂), respectively. ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.4 Hz, 6H, 2CH₃), 3.12–3.31 (m, 1H, CH), 4.65 (s, 2H, NH₂), 7.22 (d, J = 7.5 Hz, 2H, ArH), 7.61 (d, J = 7.5 Hz, 2H, ArH), 7.75–7.92 (m, 4H, ArH), 8.21 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.3, 34.2, 126.1, 126.5, 126.8, 127.5, 128.1, 129.1, 129.9, 132.7, 134.5, 150.6, 168.8, 170.7, 172.1. Anal. calcd for C₁₈H₁₈N₄OS: C, 63.88; H, 5.36; N, 16.56; S, 9.47; found: C, 63.86; H, 5.26; N, 16.50; S, 9.50%. MS (70 eV) m/z (%): 338 (10) (M⁺), 307 (8), 265 (100).

2-Benzyl-4-(4-isopropylphenyl)phthalazin-1(2H)-one (7)

A mixture of benzooxazine 3 (2.65 g, 0.01 mol) and benzyl amine (1.10 mL, 0.01 mol) in ethanol (20 mL) was heated at reflux temperature for 20 h and left to cool. The solid product was collected, dried, and recrystallized from ethanol to afford 7 in 63% yield as colorless crystals; m.p. >300 °C, IR (KBr): 1581 cm⁻¹ (C=N), 1662 cm⁻¹ (CO), 2954 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.29 (d, J = 6.4 Hz, 6H, 2CH₃), 2.98–3.19 (m, 1H, CH), 4.84 (s, 2H, CH₂), 7.10–7.54 (m, 5H, ArH), 7.62 (d, J = 7.5 Hz, 2H, ArH), 7.78 (m, 2H, ArH), 7.93–8.02 (m, 4H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 53.3, 126.3, 126.5, 126.7, 127.6, 127.8, 127.9, 128.6, 128.9, 129.3, 130.6, 132.7, 134.3, 135.8, 143.9, 150.5, 158.2. Anal. calcd for C₂₄H₂₂N₂O: C, 81.33; H, 6.26; N, 7.90; found: C, 81.31; H, 6.21; N: 7.88%. MS (70 eV) m/z (%): 354 (1) (M⁺), 275 (10), 250 (100).

1-[4-(4-Isopropylphenyl)-1-oxophthalazin-2(1 H )-yl]thiourea (8)

A mixture of benzooxazine 3 (2.65 g, 0.01 mol) and thiosemicarbazide (0.91 g, 0.01 mol) was refluxed in pyridine (20 mL) for 8 h. On cooling, the reaction mixture was poured onto ice-cold water acidified with HCl and the solid product was filtered off, washed with water, and recrystallized from ethanol to afford 8 in 69% yield as beige crystals; m.p. 168–169 °C, IR (KBr): 1284 cm⁻¹ (C=S), 1619 cm⁻¹ (C=N), 1643 cm⁻¹ (CO), 2966 cm⁻¹ (CH aliphatic), 3178 and 3367 cm⁻¹ (NH) and (NH₂), respectively. ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.3 Hz, 6H, 2CH₃), 2.95–3.03 (m, 1H, CH), 4.50 (s, 2H, NH₂), 7.19–7.56 (m, 8H, ArH), 8.63 (s, 1H, NH);¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.1, 126.3, 126.7, 127.2, 127.6, 129.2, 129.8, 130.1, 132.5, 134.5, 146.4, 150.5, 161.4, 179.3. Anal. calcd for C₁₈H₁₈N₄OS: C, 63.88; H, 5.36; N, 16.56; S, 9.47; found: C, 63.85; H, 5.32; N, 16.50; S, 9.43%. MS (70 eV) m/z (%): 338 (21) (M⁺), 279 (11), 236 (13), 57 (100).

N-[4-(4-isopropylphenyl)-1-oxophthalazin-2(1 H )-yl]hydrazinecarbothioamide (9)

A mixture of benzooxazine 3 (2.65 g, 0.01 mol) and thiocarbonohydrazide (1.06 g, 0.01 mol) was refluxed in pyridine (20 mL) for 8 h. On cooling, the reaction mixture was poured onto ice-cold water acidified with HCl and the solid product was filtered off, washed with water, and crystallized from ethanol to furnish 9 in 63% yield as beige crystal; m.p. 296–297 °C, IR (KBr): 1425 cm⁻¹ (C=S), 1604 cm⁻¹ (C=N), 1668 cm⁻¹ (CO), 2960 cm⁻¹ (CH aliphatic), 3212 and 3444 cm⁻¹ (2NH) and (NH₂), respectively. ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.26 (d, J = 6.4 Hz, 6H, 2CH₃), 3.00–3.22 (m, 1H, CH), 4.86 (s, 2H, NH₂), 7.24 (d, J = 7.5 Hz, 2H, ArH), 7.38 (d, J = 7.4 Hz, 2H, ArH), 7.89–8.14 (m, 4H, ArH), 8.43 (s,2H, 2NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 126.5, 126.7, 127.2, 127.7, 129.1, 129.8, 130.2, 132.3, 134.5, 146.2, 150.6, 160.9, 179.3. Anal. calcd for C₁₈H₁₉N₅OS: C, 61.17; H, 5.42; N, 19.82; S: 9.07; found: C, 61.15; H, 5.40; N: 19.79; S, 9.04%. MS (70 eV) m/z (%): 353 (24) (M⁺), 322 (7), 264 (52), 76 (100).

7-(4-Isopropylphenyl)-2H-[1,2,4,5]tetrazino[6,1-a]phthalazine-3(4H)-thione (10)

A mixture of benzooxazine 3 (2.65 g, 0.01 mol) and thiocarbonohydrazide (1.06 g, 0.01 mol) in dioxane (20 mL) was heated at reflux for 8 h and then left to cool. The precipitated solid was collected, dried, and recrystallized from ethanol to afford 10 in 69% yield as yellow crystals; m.p. 160–161 °C, IR (KBr): 1284 cm⁻¹ (C=S), 1639 cm⁻¹ (C=N), 2958 cm⁻¹ (CH aliphatic), 3212 and 3270 cm⁻¹ (2NH). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.30 (d, J = 6.4 Hz, 6H, 2CH₃), 2.98–3.21 (m, 1H, CH), 7.38 (d, J = 7.5 Hz, 2H, ArH), 7.49 (d, J = 7.5 Hz, 2H, ArH), 7.68 (s, 1H, NH), 7.87–8.25 (m, 4H, ArH), 8.91 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 120.7, 126.3, 126.5, 127.1, 127.4, 127.9, 128.5, 129.1, 129.9, 134.5, 150.5, 148.8, 167.8. Anal. calcd for C₁₈H₁₇N₅S: C, 64.45; H, 5.11; N, 20.88; S, 9.56; found: C, 64.41; H, 4.81; N, 20.68; S, 9.53%. MS (70 eV) m/z (%): 335 (11) (M⁺), 320 (23), 287 (40), 261 (100).

1,1′-[1-(4-Isopropylphenyl)-4-oxo-3,4-dihydroisoquinoline-3,3-diyl]bis(ethan-1-one) (11)

To a solution of benzooxazine 3 (2.65 g, 0.01 mol) in ethanol (20 mL) and a few drops of piperidine, acetylacetone (1.0 mL, 0.01 mol) was added and the mixture was refluxed for 30 h and left to cool. The precipitated solid was collected, dried, and recrystallized from ethanol to give 11 in 62% yield as yellow crystals; m.p. 140–141 °C, IR (KBr): 1600 cm⁻¹ (C=N), 1666 cm⁻¹, 1735 cm⁻¹ (3CO), 2958 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.3 Hz, 6H, 2CH₃), 2.50 (s, 6H, 2CH₃), 2.92–3.04 (m, 1H, CH), 7.36 (d, J = 7.5 Hz, 2H, ArH), 7.48 (d, J = 7.5 Hz, 2H, ArH), 7.53–8.37 (m, 4H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.1, 29.6, 34.2, 125.4, 126.3, 126.5, 128.9, 130.2, 131.1, 132.3, 134.8, 135.7, 151.3, 162.2, 176.2, 182.5, 187.4. Anal. calcd for C₂₂H₂₁NO₃: C, 76.06; H, 6.09; N, 4.03; found: C, 75.96; H, 6.03; N, 3.83%. MS (70 eV) m/z (%): 347 (26) (M⁺), 262 (11), 234 (9), 76 (100).

Ethyl 2-[4-(4-isopropylphenyl)-1-oxophthalazin-2(1H)-yl]acetate (12)

A mixture of 4 (2.6 g, 0.01 mol), ethyl bromoacetate (0.03 mol), and potassium carbonate (4.1 g, 0.03 mol) in dry acetone (30 mL) was heated under reflux for 30 h, cooled to room temperature, and poured into ice-cold water. The obtained solid was filtered off and recrystallized from ethanol to give 12 in 85% yield as colorless crystals; m.p. 162–163 °C, IR (KBr): 1606 cm⁻¹ (C=N), 1657 cm⁻¹ (amide CO), 1742 cm⁻¹ (ester CO), 2959 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.20 (t, J = 6Hz, 3H, CH₃), 1.26 (d, J = 6.4 Hz, 6H, 2CH₃), 2.95–3.02 (m, 1H, CH), 4.15 (q, J = 6Hz, 2H, CH₂), 4.98 (s, 2H, CH₂CO), 7.42 (d, J = 7.5 Hz, 2H, ArH), 7.51 (d, J = 7.5 Hz, 2H, ArH), 7.74–8.38 (m, 4H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 14.8, 23.2, 34.2, 48.6, 60.9, 126.3, 126.5, 127.2, 127.7, 127.9, 129.1, 130.9, 132.5, 134.6, 146.3, 150.5, 159.3, 167.1. Anal. calcd for C₂₁H₂₂N₂O₃: C, 71.98; H, 6.33; N, 7.99; found: C, 71.93; H, 6.31; N, 8.03%. MS (70 eV) m/z (%): 350 (0.02) (M⁺), 263 (0.33), 71 (100).

2-[4-(4-Isopropylphenyl)-1-oxophthalazin-2(1H)-yl]acetohydrazide (13)

A mixture of 12 (3.5 g, 0.01 mol) and hydrazine hydrate (2 mL) in absolute ethanol (30 mL) was refluxed for 6 h. The reaction mixture was allowed to cool down and the separated solid product was filtered off and dried. Recrystallization of the crude product with ethanol afforded 13 in 78% yield as colorless crystals; m.p. 158–159 °C, IR (KBr): 1662 cm⁻¹ (2CO), 2955 cm⁻¹ (CH aliphatic), 3329 and 3444 cm⁻¹ (NH) and (NH₂ ), respectively. ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.27 (d, J = 6.4 Hz, 6H, 2CH₃), 2.97–3.04 (m, 1H, CH), 4.28 (s, 2H, NH₂), 4.77 (s, 2H, CH₂CO), 7.43 (d, J = 7.5 Hz, 2H, ArH), 7.51 (d, J = 7.5 Hz, 2H, ArH), 7.74–8.37 (m, 4H, ArH), 9.29 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.1, 53.9, 126.3, 126.7, 126.9, 127.4, 127.7, 129.1, 130.8, 132.7, 134.5, 145.8, 150.5, 159.6, 172.6. Anal. calcd for C₁₉H₂₀N₄O₂: C, 67.84; H, 5.99; N, 16.66; found: C, 67.79; H, 6.02; N, 16.67%. MS (70 eV) m/z (%):336 (15) (M⁺), 264 (13), 249 (100).

2-Benzoyl-4-(4-isopropylphenyl)phthalazin-1(2H)-one (14)

A mixture of phthalazine 4 (2.60 g, 0.01 mol) and excess benzoyl chloride (5 mL) was refluxed for 3 h on a steam bath. The reaction mixture was poured into ice-cold water and the separated solid was filtered off, washed well with water, and dried. Recrystallization from ethanol afforded 14 in 58% yield as colorless crystals; m.p. 134–135 °C, IR (KBr): 1591 cm⁻¹ (C=N), 1692 cm⁻¹ (2CO), 2935 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.4 Hz, 6H, 2CH₃), 3.00–3.19 (m, 1H, CH), 7.23–7.47 (m, 7H, ArH), 7.69–8.30 (m, 6H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 126.2, 126.6, 127.4, 127.7, 127.8, 128.8, 129.2, 129.5, 129.9, 132.5, 132.8, 132.9, 134.4, 150.5, 154.9, 156.8, 163.3. Anal. calcd for C₂₄H₂₀N₂O₂: C, 78.24; H, 5.47; N, 7.60; found: C, 78.31; H, 5.40; N, 7.57%. MS (70 eV) m/z (%): 368 (53) (M⁺), 305 (100), 263 (6).

Reactions of phthalazine hydrazide derivative 13 with aromatic aldehydes 15a–c

General procedure

A mixture of acetic acid hydrazide 13 (0.30 g, 0.001 mol), the appropriate aromatic aldehyde; namely, benzaldehyde, p-chlorobenzaldehyde, or p-nitrobenzaldehyde (0.001 mol) and a few drops of piperidine was refluxed in boiling ethanol (20 mL) for 6 h. After cooling, the separated solid was collected by filtration, dried, and recrystallized from ethanol to give aryl methylidene hydrazide derivatives 15a–c.

N′-Benzylidene-2-[4-(4-isopropylphenyl)-1-oxophthalazin-2(1H)-yl]acetohydrazide (15a)

Colorless crystals were isolated in 76% yield; m.p. 228–229 °C, IR (KBr): 1579 cm⁻¹ (C=N), 1659 cm⁻¹ (2CO), 2958 cm⁻¹ (CH aliphatic), 3217 cm⁻¹ (NH). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.27 (d, J = 6.4 Hz, 6H, 2CH₃), 2.98–3.02 (m, 1H, CH), 4.95 (s, 1H, NH), 5.35 (s, 2H, CH₂), 7.44 (d, J = 7.5 Hz, 2H, ArH), 7.51 (d, J = 7.5 Hz, 2H, ArH), 7.70–8.40 (m, 9H, ArH), 8.72 (s, 1H, CH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.1, 54.1, 126.3, 126.6, 127.5, 127.7, 128.9, 129.1, 129.3, 130.9, 132.6, 134.5, 134.8, 145.7, 146.8, 150.5, 152.9, 155.4, 158.6, 163.3. Anal. calcd for C₂₆H₂₄N₄O₂: C, 73.56; H, 5.70; N, 13.20; found: C, 73.50; H, 5.73; N, 13.24%. MS (70 eV) m/z (%): 424 (100) (M⁺), 305 (94), 76 (2).

N′-(4-Chlorobenzylidene)-2-(4-(4-isopropylphenyl)-1-oxopht-halazin-2(1H)-yl)acetohydrazide (15b)

Colorless crystals were isolated in 82% yield; m.p. 270–271 °C, IR (KBr): 1580 cm⁻¹ (C=N), 1676 cm⁻¹ (2CO), 2958 cm⁻¹ (CH aliphatic), 3068 cm⁻¹ (CH aromatic), 3205 cm⁻¹ (NH). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.4 Hz, 6H, 2CH₃), 3.03–3.21 (m, 1H, CH), 5.41 (s, 2H, CH₂), 7.22–7.35 (m, 4H, ArH), 7.42–8.05 (m, 8H, ArH), 8.21 (s,1H, NH), 8.79 (s, 1H, CH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.37, 34.20, 54.2, 126.3, 126.5, 127.2, 127.6, 127.9, 128.9, 129.2, 129.7, 130.2, 132.4, 134.3, 134.7, 135.1, 144.5, 146.8, 150.5, 159.1, 164.1. Anal. calcd for C₂₆H₂₃ClN₄O₂: C, 68.04; H, 5.05; N, 12.21; found: C, 68.08; H, 5.01; N, 12.23%. MS (70 eV) m/z (%): 458 (82) (M⁺), 305 (100).

2-[4-(4-Isopropylphenyl)-1-oxophthalazin-2(1H)-yl]-N′-(4-nitrobenzylidene)aceto-hydrazide (15c)

Beige crystals were isolated in 72% yield; m.p. 298–299 °C, IR (KBr): 1584 cm⁻¹ (C=N), 1694 cm⁻¹ (2CO), 2959 cm⁻¹ (CH aliphatic), 3068 cm⁻¹ (CH aromatic), 3446 cm⁻¹ (NH). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.4 Hz, 6H, 2CH₃), 2.99–3.05 (m, 1H, CH), 5.70 (s, 2H, CH₂), 7.30–7.48 (m, 5H, ArH), 7.60 (s, 1H, NH), 7.65–8.12 (m, 7H, ArH), 8.61 (s,1H, CH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 54.2, 124.6, 126.3, 126.6, 127.1, 127.6, 127.9, 128.2, 129.1, 131.2, 132.7, 134.5, 138.9, 145.6, 147.9, 148.7, 150.5, 159.3, 164.2. Anal. calcd for C₂₆H₂₃N₅O₄: C, 66.51; H, 4.94; N, 14.9; found: C, 66.00; H, 5.10; N, 14.6%. MS (70 eV) m/z (%): 469 (2) (M⁺), 305 (100).

Cyclization of Schiff’s bases 15a–c: general procedure

Compounds 15a–c (0.002 mol) and acetic anhydride (2 mL) were heated under reflux for 5 h. The reaction mixture was cooled, poured onto water, and allowed to stand at room temperature for 3 h. The solid product formed was collected and recrystallized from ethanol to afford 16a–c.

2-[(4-Acetyl-5-phenyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (16a)

Colorless crystals were isolated in 73% yield; m.p. 130–131 °C, IR (KBr): 1581 cm⁻¹ (C=N), 1662 cm⁻¹ (2 CO), 2958 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.22 (d, J = 6.4 Hz, 6H, 2CH₃), 2.04 (s, 3H, CH₃), 2.98–3.02 (m, 1H, CH), 5.01 (s, 2H, CH₂), 7.03 (s, 1H, CH oxadiazole), 7.21–7.51 (m, 5H, ArH), 7.52–8.38 (m, 8H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.1, 23.5, 34.2, 46.4, 98.9, 126.3, 126.6, 126.9, 127.1, 127.6, 127.8, 128.9, 129.3, 130.1, 130.9, 132.7, 134.3, 134.6, 145.7, 150.5, 153.7, 157.7, 159.3. Anal. calcd for C₂₈H₂₆N₄O₃: C, 72.09; H, 5.62; N, 12.01; found: C, 72.05; H, 5.60; N, 12.05%. MS (70 eV) m/z (%): 466 (100) (M⁺), 424 (91), 277 (3).

2-[(4-Acetyl-5-(4-chlorophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (16b)

Colorless crystals were isolated in 79% yield; m.p. 140–141 °C, IR (KBr): 1583 cm⁻¹ (C=N), 1661 cm⁻¹ (2 CO), 2959 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.25 (d, J = 6.4 Hz, 6H, 2CH₃), 2.07 (s, 3H, CH₃), 2.98–3.05 (m, 1H, CH), 5.28 (s, 2H, CH₂), 7.11 (s, 1H, CH oxadiazole), 7.27 (d, J = 7.5 Hz, 2H, ArH), 7.32 (d, J = 7.5 Hz, 2H, ArH), 7.49–7.73 (m, 4H, ArH), 7.98–8.38 (m, 4H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 23.4, 34.2, 46.3, 99.3, 126.3, 126.5, 127.3, 127.5, 127.8, 128.8, 129.2, 129.6, 130.1, 132.7, 134.0, 134.5, 136.4, 146.2, 150.5, 154.1, 158.2, 159.6. Anal. calcd for C₂₈H₂₅ ClN₄O₃: C, 67.13; H, 5.03; N, 11.18; Cl, 7.08; found: C, 67.15; H, 5.00; N, 11.22; Cl, 6.88%. MS (70 eV) m/z (%): 500 (3) (M⁺), 73 (100).

2-[(4-Acetyl-5-(4-nitrophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (16c)

Beige crystals were isolated in 72% yield; m.p. 216–217 °C, IR (KBr): 1581 cm⁻¹ (C=N), 1662 cm⁻¹ (2 CO), 2962 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.26 (d, J = 6.4 Hz, 6H, 2CH₃), 2.05 (s, 3H, CH₃), 2.98–3.05 (m, 1H, CH), 5.34 (s, 2H, CH₂), 7.08 (s, 1H, CH oxadiazole), 7.22 (d, J = 7.5 Hz, 2H, ArH), 7.30 (d, J = 7.5 Hz, 2H, ArH), 7.41–7.68 (m, 4H, ArH), 7.88–8.27 (m, 4H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.3, 23.4, 34.2, 46.4, 99.2, 124.6, 126.4, 126.7, 126.9, 127.2, 127.5, 127.7, 129.3, 130.8, 132.6, 134.5, 142.1, 145.8, 148.1, 150.5, 154.0, 157.9, 159.7. Anal. calcd for C₂₈H₂₅ N₅O₅: C, 65.74; H, 4.93; N, 13.69; found: C, 65.71; H, 4.97; N, 13.65%. MS (70 eV) m/z (%): 511 (52) (M⁺), 469 (100), 277 (1).

4-(4-Isopropylphenyl)-2-[(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]phthalazin-1(2H)-one (17)

A mixture of the acetic acid hydrazide 13 (0.6 g, 0.002 mol) was added to potassium hydroxide (0.28 g, 0.005 mol) solution in absolute ethanol (40 mL) and then carbon disulfide (6 mL) was added portionwise and the reaction mixture was refluxed until no odor of hydrogen sulfide evolved (18 h). The reaction mixture was poured onto ice-cold water and rendered acidic with HCl. The precipitated solid was filtered off, dried, and recrystallized from ethanol to give 17 in 54% yield as yellow crystals; m.p. 210–211 °C, IR (KBr): 1423 cm⁻¹ (C=S), 1581 cm⁻¹ (C=N), 1663 cm⁻¹ (CO), 2958 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.27 (d, J = 6.4 Hz, 6H, 2CH₃), 2.98–3.02 (m, 1H, CH), 5.51 (s, 2H, CH₂), 7.43 (d, J = 7.5 Hz, 2H, ArH), 7.52 (d, J = 7.5 Hz, 2H, ArH), 7.76–8.4 (m, 4H, ArH), 14.6 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 46.4, 126.3, 126.5, 127.3, 127.6, 127.8, 129.1, 131.0, 132.7, 134.5, 146.4, 150.5, 155.7, 159.4, 178.9. Anal. calcd for C₂₀H₁₈N₄O₂S: C, 63.47; H, 4.79; N, 14.80; S, 8.47; found: C, 63.43; H, 4.77; N, 14.86; S: 8.45%. MS (70 eV) m/z (%): 378 (4) (M⁺), 304 (100), 277 (69).

2-[(1,3,4-Oxadiazol-2-yl)methyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (18)

A mixture of acetic acid hydrazide 13 (0.6 g, 0.002 mol) and triethylorthoformate (5mL) was heated under reflux for 15 h. After cooling, the formed solid was collected, dried, and recrystallized from ethanol to yield 18 in 73% yield as colorless crystals; m.p. 292–293 °C, IR (KBr): 1582 cm⁻¹ (C=N), 1663 cm⁻¹ (CO), 2959 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.26 (d, J = 6.4 Hz, 6H, 2CH₃), 2.97–3.01 (m, 1H, CH), 4.88 (s, 2H, CH₂), 7.41 (d, J = 7.5 Hz, 2H, ArH), 7.50 (d, J = 7.5 Hz, 2H, ArH), 7.70–8.37 (m, 4H, ArH), 9.63 (s, 1H, CH oxadiazole); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.3, 34.2, 45.7, 126.3, 126.6, 127.2, 127.5, 127.7, 129.2, 131.1, 132.7, 134.5, 145.9, 150.5, 158.7, 160.4, 162.8. Anal. calcd for C₂₀H₁₈N₄O₂: C, 69.35; H, 5.24; N, 16.17; found: C, 69.30; H, 5.28; N, 16.19%. MS (70 eV) m/z (%): 346 (17) (M⁺), 277 (26), 76 (9), 48 (100).

4-(4-Isopropylphenyl)-2-[(5-methyl-1,3,4-oxadiazol-2-yl)methyl]phthalazin-1(2H)-one (19)

A mixture of acetic acid hydrazide 13 (0.6 g, 0.002 mol) and acetic acid (0.12 mL, 0.002 mol) in phosphorous oxychloride (1 mL) was heated under reflux for 6 h. After cooling, the excess phosphorous oxychloride was removed under reduced pressure. The obtained solid was poured on ice-cold water (50 mL) and neutralized with saturated sodium bicarbonate, The obtained solid was collected by filtration and then recrystallized from ethyl acetate to give 19 in 70% yield as beige crystals; m.p. 220–221 °C, IR (KBr): 1581 cm⁻¹ (C=N), 1658 cm⁻¹ (CO), 2958 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.26 (d, J = 6.4 Hz, 6H, 2CH₃), 2.51 (s, 3H, CH₃), 2.97–3.02 (m, 1H, CH), 5.32 (s, 2H, CH₂), 7.39–7.35 (m, 5H, ArH), 7.72–8.35 (m, 3H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 11.2, 23.1, 34.2, 45.6, 126.4, 126.7, 127.3, 127.5, 127.8, 129.4, 131.2, 132.7, 134.5, 145.6, 150.5, 156.7, 157.2, 158.6. Anal. calcd for C₂₁H₂₀N₄O₂: C, 69.98; H, 5.59; N, 15.55; found: C, 70.01; H, 5.60; N, 15.50%. MS (70 eV) m/z (%): 360 (32) (M⁺), 345 (8), 277 (22), 249 (100).

2-{2-[4-(4-Isopropylphenyl)-1-oxophthalazin-2(1H)-yl]acetyl}-N-phenylhydrazine-1-carbothioamide (20)

A mixture of acetic acid hydrazide 13 (0.6 g, 0.002 mol) and phenyl isothiocyanate (0.2 mL, 0.002 mol) in pyridine (20 mL) was refluxed for 8 h. After cooling, the reaction mixture was poured on ice-cold water acidified with HCl. The precipitated solid was collected by filtration, dried, and crystallized from ethanol to afford 20 in 72% yield as colorless crystals; m.p. 200–201 °C, IR (KBr): 1427 cm⁻¹ (C=S), 1576 cm⁻¹ (C=N), 1656 cm⁻¹ (2CO), 2959 cm⁻¹ (CH aliphatic), 3258 cm⁻¹ (NH). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.4 Hz, 6H, 2CH₃), 3.05–3.18 (m, 1H, CH), 5.62 (s, 2H, CH₂), 7.10–7.32 (m, 5H, ArH), 7.41 (d, J = 7.5 Hz, 2H, ArH), 7.58 (d, J = 7.5 Hz, 2H, ArH), 7.73 (m, 2H, ArH), 7.91 (s, 1H, NH), 8.16 (s, 1H, NH), 8.22–8.39 (m, 2H, ArH), 8.64 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 53.4, 120.7, 124.6, 126.3, 126.6, 127.3, 127.5, 127.9, 128.8, 129.3, 131.1, 132.6, 134.5, 138.7, 146.2, 150.6, 158.5, 168.9, 176.4. Anal. calcd for C₂₆H₂₅N₅O₂S: C, 66.22; H, 5.34; N, 14.85; S, 6.80; found: C, 66.25; H, 5.33; N, 14.83; S, 6.83%. MS (70 eV) m/z (%): 471 (7) (M⁺), 379 (34), 305 (78), 277 (100).

2-[2-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-oxoethyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (21)

A mixture of acetic acid hydrazide 13 (0.6 g, 0.002 mol) and acetyl acetone (0.2 mL, 0.002 mol) in ethanol (20 mL) was refluxed for 10 h. The resulting solid was filtered and crystallized from ethanol to afford 21 in 82% yield as colorless crystals; m.p. 180–181 °C, IR (KBr): 1581 cm⁻¹ (C=N), 1659 cm⁻¹ (2CO), 2957 cm⁻¹ (CH aliphatic). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.25 (d, J = 6.4 Hz, 6H, 2CH₃), 2.25 (s, 3H, CH₃), 2.46 (s, 3H, CH₃), 2.95–3.03 (m, 1H, CH), 5.69 (s, 2H, CH₂), 6.29 (s, 1H, CH pyrazole), 7.41 (d, J = 7.5 Hz, 2H, ArH), 7.51 (d, J = 7.5 Hz, 2H, ArH), 7.70–8.39 (m, 4H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 13.4, 14.7, 23.2, 34.1, 52.8, 112.6, 126.5, 126.8, 127.2, 127.49, 127.7, 129.3, 130.8, 132.6, 134.5, 140.7, 145.6, 150.5, 151.8, 158.3, 167.9. Anal. calcd for C₂₄H₂₄N₄O₂: C, 71.98; H, 6.04; N, 13.99; found: C, 71.95; H, 6.08; N, 14.02%. MS (70 eV) m/z (%): 396 (0.2) (M⁺–4), 305 (0.05), 263 (2), 73 (100).

2-[2-(3,5-Diamino-1H-pyrazol-1-yl)-2-oxoethyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (22)

A mixture of acetic acid hydrazide 13 (0.6 g, 0.002 mol) and malononitrile (0.002 mol) in ethanol (20 mL) was refluxed for 8 h. On cooling, the separated solid was filtered off and recrystallized from ethanol to give 22 in 75% yield as beige crystals; m.p. 246–247 °C, IR (KBr): 1583 cm⁻¹ (C=N), 1666 cm⁻¹ (2CO), 2958 cm⁻¹ (CH aliphatic), 3158 and 3442 cm⁻¹ (2NH₂). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.4 Hz, 6H, 2CH₃), 3.00–3.17 (m, 1H, CH), 4.92 (s, 1H, CH pyrazole), 5.65 (s, 2H, CH₂), 5.98 (s, 2H, NH₂), 6.30 (s, 2H, NH₂), 7.28 (d, J = 7.5 Hz, 2H, ArH), 7.54 (d, J = 7.5 Hz, 2H, ArH), 7.78–8.32 (m, 4H, ArH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.2, 51.9, 87.6, 126.3, 126.5, 127.1, 127.7, 127.9, 129.3, 131.2, 132.7, 134.4, 138.6, 145.8, 150.5, 158.7, 163.6, 167.4. Anal. calcd for C₂₂H₂₂N₆O₂: C, 65.66; H, 5.51; N: 20.88; found: C, 65.60; H, 5.53; N, 20.85%. MS (70 eV) m/z (%):402 (3) (M⁺), 305 (75), 277 (100).

1-{2-[4-(4-Isopropylphenyl)-1-oxophthalazin-2(1H)-yl]acetyl}pyrazolidine-3,5-dione (23)

A mixture of acetic acid hydrazide 13 (0.6 g, 0.002 mol) and diethyl malonate (0.002 mol) was refluxed in ethanol (20 mL) for 10 h. On cooling, the separated solid was filtered off and crystallized from ethanol gave 23 in 73% yield as colorless crystals; m.p. >300 °C, IR of (KBr): 1581 (C=N), 1631 (4CO), 2923 (CH aliphatic), 3421 cm⁻¹ (NH). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.29 (d, J = 6.4 Hz, 6H, 2CH₃), 3.03–3.15 (m, 1H, CH), 4.51 (s, 2H, CH₂ pyrazole), 5.70 (s, 2H, CH₂), 7.37 (d, J = 7.5 Hz, 2H, ArH), 7.58 (d, J = 7.5 Hz, 2H, ArH), 7.68–8.35 (m, 4H, ArH), 8.97 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.2, 34.1, 37.5, 51.3, 126.3, 126.5, 127.1, 127.5, 127.7, 129.2, 131.1, 132.6, 134.5, 146.0, 150.5, 158.5, 164.2, 171.2, 174.7. Anal. calcd for C₂₂H₂₀N₄O₄: C, 65.34; H, 4.98; N, 13.85; found: C, 65.30; H, 5.01; N, 13.88%. MS (70 eV) m/z (%): 404 (15) (M⁺), 305 (5),263 (11), 57 (100).

2-[2-(5-Amino-2,3-dihydro-1H-pyrazol-1-yl)-2-oxoethyl]-4-(4-isopropylphenyl)phthalazin-1(2H)-one (24)

A mixture of acetic acid hydrazide 13 (0.3 g, 0.001 mol) and acrylonitrile (0.001 mol) in boiling pyridine (20 mL) was refluxed for 10 h. On cooling, the reaction mixture was poured into ice-cold water acidified with HCl and the solid produced was filtered off, washed several times with water, and crystallized from ethanol to afford 24 in 76% yield as colorless crystals; m.p. 290–291 °C, IR (KBr): 1581 cm⁻¹ (C=N), 1673 cm⁻¹ (2CO), 2958 cm⁻¹ (CH aliphatic), 3266 cm⁻¹ (NH), 3440 cm⁻¹ (NH₂). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.25 (d, J = 6.4 Hz, 6H, 2CH₃), 2.98–3.12 (m, 1H, CH), 3.95 (d, J = 6.2 Hz, 2H, CH₂ pyrazole), 4.57 (t, J = 6.2 Hz, 1H, CH pyrazole), 5.58 (s, 2H, CH₂), 6.36 (s, 2H, NH₂), 7.35 (d, J = 7.5 Hz, 2H, ArH), 7.58 (d, J = 7.5 Hz, 2H, ArH), 7.72–8.29 (m, 4H, ArH), 8.61 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.1, 34.2, 53.4, 54.6, 93.8, 126.4, 126.7, 127.3, 127.6, 127.9, 129.2, 131.2, 132.6, 134.5, 144.3, 145.9, 150.5, 158.7, 169.8. Anal. calcd for C₂₂H₂₃N₅O₂: C, 67.85; H, 5.95; N, 17.98; found: C, 67.81; H, 5.91; N, 18.00%. MS (70 eV) m/z (%): 389 (91) (M⁺), 388 (100), 305 (5).

N-(1,3-Dioxoisoindolin-2-yl)-2-[4-(4-isopropylphenyl)-1-oxophthalazin-2(1H)-yl]acetamide (25)

A mixture of acetic acid hydrazide 13 (0.6 g, 0.002 mol) and phthalic anhydride (0.3 g, 0.002 mol) was heated under reflux in acetic acid (15mL) for 10 h. After cooling the obtained solid was filtered off, dried, and recrystallized from acetic acid to give 25 in 80% yield as beige crystals; m.p. 210–211 °C, IR (KBr): 1580 cm⁻¹ (C=N), 1662, 1737, 1792 cm⁻¹ (4CO), 2958 cm⁻¹ (CH aliphatic), 3210 cm⁻¹ (NH). ¹H NMR (DMSO-d₆, 300 MHz, ppm) δ 1.28 (d, J = 6.4 Hz, 6H, 2CH₃), 3.00–3.03 (m, 1H, CH), 5.11 (s, 2H, CH₂), 7.44 (d, J = 7.5 Hz, 2H, ArH), 7.51 (d, J = 7.5 Hz, 2H, ArH), 7.56–8.04 (m, 8H, ArH), 11.05 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 75 MHz, ppm) δ 23.1, 34.2, 52.2, 125.8, 126.4, 126.7, 127.3, 127.5, 127.7, 129.4, 131.2, 131.9, 132.6, 132.8, 134.5, 146.1, 150.5, 159.4, 161.7, 168.9. Anal. calcd for C₂₇H₂₂N₄O₄: C, 69.52; H, 4.75; N, 12.01; found: C, 69.55; H, 4.72; N, 12.05%. MS (70 eV) m/z (%): 466 (0.05) (M⁺), 305 (5), 277 (64), 235 (100).

Antimicrobial activity assay

The in vitro antimicrobial activity of the newly synthesized compounds was measured against specific microbial strains using the disk diffusion technique in which sterile Whatman No. 5 filter paper (11 mm diameter) disks were utilized.³⁸ Ethanol was selected to dissolve the tested compounds then the 11-mm disks were loaded with 10 mg mL⁻¹ of the test compound under investigation (50 µL). Subsequently, the disks were left with caution under hot air streams to reach complete dryness.

Test plates were made ready via pouring freshly prepared Muller–Hinton agar medium (10 mL) seeded with selected microbes. The disks were placed on the surface of agar plates then incubation for 1 h at 5 °C took place to secure good diffusion. Thereafter, all the plates were incubated at 37 °C for 24 h. Finally, the outgrowth of the microorganisms was recorded and the average IZ were measured in millimeters and used as criteria for the antimicrobial activity. Importantly, the average growth of microbes was calculated after the plates were done in triplicate. The observed clear zone size is directly proportional to the inhibitory effect of the investigated compound. In each experiment, a negative control was incorporated by using a pure solvent disk. For comparison, a disk with Amoxicillin (standard drug) was screened for antimicrobial activity using the same conditions.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors would like to extend their profound appreciation to Fayoum University for the financial support.

ORCID iD

Asmaa Kamal Mourad

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