Synthesis and Biological Screening of 4-Benzyl-2H-phthalazine Derivatives

Preparation of 4-benzyl-2-substituted phthalazin-1-one derivatives 2-8 is reported. Condensation of 4-benzyl-1-chlorophthalazine (9) with a series of different nucleophiles gave 4-benzylphthalazin-1-ylamino derivatives (10-13 and 16) and 4-amino-2-[N′-(4-benzylphthalazin-1-yl)-hydrazino]-6-arylpyrimidine-5-carbonitriles (14a,b). Interaction of 9 with ambident anions was also studied. 5-Benzyl-6,6a,12-triazobenzo[a]-anthracen-7-one (15) is obtained from 9 and anthranilic acid derivatives. Treatment of 16 with (EtO)3CH/Ac2O under reflux afforded the corresponding ethoxymethylene derivative 17, while aqueous ammonium hydroxide treatment afforded carboxamide derivative 18. The structures of the newly synthesized derivatives were confirmed by their elemental analysis, IR, 1H NMR, 13C NMR and mass spectral studies. Antimicrobial activities of some selected compounds were also studied and some of these were found to exhibit promising effects against Gram-positive and Gram-negative bacteria and fungi.


Introduction
The phthalazine derivative azelastine (A, Figure 1) is an antihistamine used in the treatment of allergic rhinitis [1]. Newer agents are more selective inhibitors of the cGMP-inhibited phosphor diesterase (PDE) and casn be exemplified by phthalazine derivatives like MY5445 (B, Figure 1) [2][3][4][5]. Zopolrestat (C, Figure 1) is a phthalazinone derivative that has been in clinical trials; it inhibits aldose reductase and has potential use in the prevention of retinopathy, neuropathy, and cataract formation in diabetes [6]. The chemiluminescence reactions of luminol (D, Figure 1) and related phthalazines have found analytical applications, particularly in biological systems where the inherent signal strength and low signal noise ratio contribute to sensitivity. The hydrogen peroxide/luminol system has been used for the on-line determination by chemiluminescence of nitric oxide in isolated organ perfusates [7][8][9][10].  Phthalazine derivatives have been widely applied as therapeutic agents due to their anticonvulsant, cardiotonic, vasorelaxant and anti-inflammatory properties [11][12][13][14][15][16][17] in addition to having antimicrobial activity [18].

General
Melting points were determined on a Stuart melting point apparatus and are uncorrected. IR spectra were recorded in KBr using a FT-IR 5300 spectrometer and Perkin Elmer spectrum RXIFT-IR system (ν, cm −1 ). The 1 H NMR at (300 MHz) and 13 C NMR spectra (75 MHz) were recorded in CDCl 3 or DMSO-d 6 on a Varian Mercury VX-300 NMR spectrometer. Chemical shifts (δ) are related to that of the solvent. Mass spectra were measured on a Shimadzu GMMS-QP-1000 EX mass spectrometer at 70 eV. The elemental analyses were performed at the Microanalytical Center, Cairo University. Cairo (Egypt). (2). A mixture of compound 1 (0.23 g, 1.0 mmol) and formaldehyde solution 38% (0.8 mL) in ethanol (30 mL) was refluxed for 3 hours. The solvent was evaporated under vacuum, and then water (25 mL) was added. The solid obtained was filtered off and recrystallized from ethanol. Colourless crystals, 90%, 0. Ethyl (4-benzyl-1-oxo-1H-phthalazin-2-yl)acetate (6). A mixture of compound 1 (0.23 g, 10 mmol) and ethyl chloroacetate (2 mL) and anhydrous K 2 CO 3 (0.13 g, 1.0 mmol) was refluxed for 4 hours. The solvent was evaporated under vacuum, then water (50 mL) was added. The solid obtained was filtered off and recrystallized from pet. ether and chloroform, respectively. Violet crystals, 65%, 0.

General Procedure for the Synthesis of Benzylphthalazin-1-ylamino Derivatives 10a-h
A mixture of compound 9 (0.25g, 10 mmol) and aromatic amine (10 mmol) in ethanol (30 mL) was refluxed for 3 hours. The solvent was evaporated under vacuum. The solid obtained was filtered off to give crude products

Antibacterial Activities
The screening results indicate that compounds 2-8 and 11 show weaker inhibitory activity than the standard drugs, while compounds 15-18 are moderately inhibitory, compared to the standard drugs .  Compounds 10a-j, 12, 13 and 14a,b showed nearly the same inhibition activity asn antibacterial activity (Table 1). It clear that decomposition of chloride atom at C-1 of 4-benzyl-1-chlorophthalazine with N-nucleophiles is responsible for the antimicrobial activities.

Conclusions
The results from this screening demonstrated that replacement of the hydrogen atom attached to the phthalazine nucleus at N-1 with amino derivatives (compounds 10a-j), diimino derivatives (compounds 12a,b-13) and pyrimidine derivatives (14a,b) resulted in spectrum if moderate antibacterial activity against all tested Gram positive and Gram negative fungi.