Synthesis and Biological Activity of Some 3-(4-(Substituted)-piperazin-1-yl)cinnolines

A new series of 6-substituted-4-methyl-3-(4-arylpiperazin-1-yl)cinnolines 8–10 were synthesized as potential antifungal agents via intramolecular cyclization of the respective 1-(2-arylhydrazono)-1-(4-arylpiperazin-1-yl)propan-2-ones 5–7, mediated by polyphosphoric acid (PPA). The amidrazones themselves were synthesized via direct interaction of the appropriate hydrazonoyl chlorides 4a–d with the corresponding N-substituted piperazine in the presence of triethylamine. The structures of the new prepared compounds were confirmed by elemental analyses, 1H-NMR, 13C-NMR, and ESI-HRMS spectral data. The antitumor, antibacterial, and antifungal activity of the newly synthesized compounds was evaluated.

Significant commercial interest in the development of benzopyridazine derivatives, particularly pharmaceutical uses of pyridazines and cinnolines, is shown by the large number of patents filed in this area [11]. Their ring system is an isosteric relative to either quinoline or isoquinoline, therefore, in many cases the synthesized compounds were designed as analogs of previously obtained quinoline or isoquinoline derivatives; for example cinoxacin (1) is a cinnoline analogue of the quinoline antibacterials used for urinary tract infection [12] and ICI-D-7569 (2) is an anxiolytic agent [13] (Figure 1). Meanwhile, attention has been paid to the synthesis of heterocyclic compounds bearing a cinnoline moiety; an excellent review on the synthesis and characteristics of cinnolines has been published by Haider and colleagues [14]. In view of the interest in the activity spectrum and profile of cinnolines, and in continuation of our work on the synthesis of new compounds of pharmacological and biological interest [15][16][17], we describe herein the preparation and spectroscopic characterization of some new 3-(4-(substituted)piperazin-1-yl)cinnolines (shown in Scheme 1), together with their antitumor, and antifungal activities.

Chemistry
The synthesis of 3-piperazinyl cinnolines 8-10 was carried out via interamolecular cyclization of the piperazinyl amidrazones 5-7 using PPA as a cyclizing agent as shown in Scheme 1. Syntheses of the respective amidrazones 5-7 in good yield were achieved according to a modified procedure [15] which involved treatment of the appropriate hydrazonyl chloride 4a-d with N-substituted piperazine in the presence of triethylamine. Compounds 4a-d were prepared by coupling of the respective arenediazonium salts with 3-chloro-2,4-pentanedione via the Japp-Klingemann reaction [18][19][20], according to reported procedures [21,22]. Scheme 1. The synthetic route for compounds 8-10.
In the 1 H-NMR (CDCl 3 ) spectra of cinnoline derivatives, a singlet peak appears in the range δ 2.50-2.95 ppm corresponding to the methyl protons. The methylene protons of the piperazine moiety appear as two broad singlets or multiplet peaks in the range δ 3.32-3.48 ppm and δ 2.68-3.61 ppm. The aromatic protons signals resonate around δ 6.78-8.69 ppm. In the 13 C-NMR spectra of compounds 8-10, the methyl (CH 3 ) carbon, resonates upfield between δ 12.6-17.7 ppm, which is indicative of the formation of cyclized product through acylation of the benzene ring; the methylene carbons of the piperazine moiety appear around δ 49.5-50.9 and 50.7-53.5 ppm, while the aromatic carbons resonate in the range δ 105.5-164.6 ppm.

Compound Susceptibility Testing by Kirby Bauer Method
The newly synthesized compounds 8-10 were screened for their antibacterial activity against Gram negative (Escherechia coli ATCC 8739) and Gram-positive (Staphylococcus aureus ATCC 25923) microorganisms at 25 g/mL. In-vitro antibacterial screening of the compounds showed that they were inactive against both organisms. In addition, these compounds were also inactive against Candida glabirata clinical neonatal isolates 1 and 2.
In addition, whereas compounds 8a-d and 9a-d showed no activity against C. albicans ATCC 10231 and C. glabirata ATCC 15126, respectively, fairly good activity was found when tested against C. albicans clinical isolates (compounds 8a-d, 9c, 10b and 10c) with a percentage of inhibition zone of 40%-55% when compared to nystain. These results are shown in Table 1. The results are the mean ± SD (n = 3) in unit of mm. The well is 6 mm wide. NA: inactive at 25 g/mL of the compound tested. Nystatin impregnated discs with 5 mm wide wells.

Compound Susceptibility Testing by Microbroth Dilution Method
As has been mentioned earlier, compounds 8a-d did not have any activity against C. albicans ATCC 10231 strain and C. galibrata clinical isolates 1 and 2; but they showed fungicidal rather than fungistatic activity in the range 0.2-3.0 mg/mL against C. galibrata ATCC 15126 strain. The minimum inhibitory concentration (MIC) of these compounds ranged from 0.3-5.0 mg/mL against C. albicans clinical isolates as displayed in Table 2. Results in Table 2 reveal that compounds 8a, 8b and 8c are more effective, with no significant difference against C. albicans clinical isolate and C. galibrata ATCC 15126 strains when compared to 8d, which has an MIC value of 3.0 mg/mL against the same strains. The fungicidal concentration values (MFC) in Table 2 reveal that compounds 8a-d displayed fungicidal activity against C. galibrata ATCC 15126 in the concentration range 0.2-3.0 mg/mL. On the other hand, the MFCs of the same compounds against C. albicans clinical isolate were in the 0.9-5.0 mg/mL range. We conclude that compounds 8a-d MFC corresponds to about ¼-½ lesser concentration against C. galibrata ATCC 15126 when compared to C. albicans clinical isolate. Shown in Table 3 is the antifungal activity of compounds 9a-d. The results reveal that these compounds have no antifungal activity against C. galibrata clinical isolates (1 and 2), and C. albicans ATCC 10231; the results also show that 9a and 9d display fungicidal activity against C. galibrata ATCC 15126 strain. The results in Table 3 indicate that compounds 9a, 9b, and 9d, exhibit moderate antifungal activity against C. albicans strains (ATCC 10231 and clinical isolate) and C. galibrata ATCC 15126 strain only, while 9c is inactive. In conclusion, the prepared compounds included in this study have no antibacterial effect. In addition, the tested compounds have no activity against C. galibrata clinical isolates (1 and 2) but some antifungal activity against C. albicans clinical isolate. Some of the tested compounds such as 10c, 10d and 8a-d have fungicidal activity rather than fungistatic effects. However, only 9a had bactericidal activity against E. coli strain with MBC of 1.0 mg/mL. These analyses emphasize the possible diversity in mechanisms that result in a phenotype of compounds resistance and selectivity amongst bacterial and fungal strains. These compounds should not be considered at this stage as potent therapeutic agents in mycosis especially when compared to nystatin. However selective compounds with antifungal activity could be potential agents in industrial mycology and microbiology especially if they prove to have low cytotoxicity in humans and animals. In addition; we emphasize the necessity for further work to modify the structures of the compounds to increase their activity firstly and secondly to decrease their cytotoxity in humans and animals.

Antitumor Activity
The antitumor activity of compounds 8-10 was characterized by conducting cell viability assays using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cultures of MCF-7 breast cancer cells were treated first at a concentration of 50 µg/mL and the results are shown in Table 4. Compounds 10b, 10d, and 8b showed potential anti-MCF-7 activity and were able to reduce the viability after 72 h to less than 50% ( Table 4). The anti-leukemic effect of these compounds was next tested against the K562 cell line, but none has shown any activity at ≤100 µg/mL. Furthermore, we determined the IC 50 values for compounds 8b, 10b and 10d on the MCF-7. Results, which are shown in Table 5, clearly reveal that compound 8b was the most potent against MCF-7 cells, scoring an IC 50 value of 5.56 μM. Compounds 10b and 10d have IC 50 values of 11.79 and 8.57, respectively.

General
Melting points were recorded on SMP1 Stuart apparatus and are uncorrected. The 1 H-and 13 C-NMR spectra were recorded on a Bruker DPX-300 spectrometer in CDCl 3 with TMS as an internal standard. The chemical shifts are reported in parts per million (ppm) expressed in δ units; coupling constant (J) values are given in Hertz (Hz). High resolution mass spectra (HRMS) were acquired using electrospray ionization (ESI) technique on a Bruker APEX-4 instrument. The samples were dissolved in CDCl 3 , diluted in spray solution (methanol/water 1:1 v/v + 0.1% formic acid) and infused using a syringe pump with a flow rate of 2 µL/min. External calibration was conducted using arginine cluster in a mass range m/z 175-871. Elemental analyses were performed on a Euro Vector Elemental Analyzer (EA 3000 A). The following chemicals were used as received without further purification: Substituted anilines and polyphosphoric acid (Fluka), 3-chloro-2,4-pentanedione, 1-(4-fluorophenyl)piperazine, 1-phenylpiperazine, 1-benzylpiperazine (Acros). The reactions were monitored by thin layer chromatography (TLC), carried out on silica gel plates (60 F-254, Scharlau). Plates were visualized under UV light (where appropriate). Preparative thick layer chromatography was performed on 0.5 mm silica gel glass plates (60 F-254, Scharlau).

General Procedure for the Synthesis of Substituted Piperazin-1-yl amidrazones 5-7
To a stirred solution of 1-chloro-1-(4-subsitituted) phenylhydrazono)propan-2-one 4a-d (10 mmol) and triethylamine (3 mL) in THF (10 mL) was added the appropriate piperazine (25 mmol), and the resulting mixture was stirred at room temperature for 6-8 h. The reaction mixture was then diluted with water (60 mL) and extracted with diethyl ether (3 × 50 mL). The combined organic extracts were dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by recrystalization from ethanol.

General Procedure for the Synthesis of 4-methyl-3-[(4-substituted)piperazin-1-yl]cinnolines 8-10
A solution of the appropriate piperazinyl amidrazone 5-7 (1.5 mmol) in PPA (5.0 g) was stirred at 110-120 °C for 8-10 h. The reaction mixture was then cooled to room temperature, treated with crushed ice (10 g), and neutralized with 10% aqueous ammonium hydroxide. The reaction mixture was then extracted with ethyl acetate (3 × 50 mL) and the combined organic extracts were evaporated under reduced pressure to afford crude residue of the respective title compound which was recrystallized from ethanol.

Candida Cultures
Compounds 8-10 were tested for their activity against Candida (fungi or yeast) strains using laboratory controls from American Type Culture Collection (ATCC) (Rockville, MD, USA) and clinical isolates which were a gift from Basem Jaber (The University of Jordan, Department of Biological Sciences): Candida glabirata ATCC 15126, Candida albicans clinical isolate (urinary tract infection), Candida glabirata clinical isolate 1 and 2 (neonate infections). Candida strains were cultured overnight at 37 °C in Sabouraud Dextrose broth.

Compound Susceptibility Testing Disk Diffusion Method/(Kirby Bauer method)
The synthetic compounds 8-10 were tested in vitro for their antibacterial activity against Gram positive S. aureus ATCC 25923 and Gram negative E. coli ATCC 8739, and Candida at 25 g/mL by modified Kirby-Bauer agar diffusion method [23,24].
The National Committee for Clinical Laboratory Standards (NCCLS) guidelines recommends using Mueller-Hinton agar medium for bacteria and Sabouraud dextrose agar medium for Candida [24,25]. Wells were punched in the agar plates (6 mm wide) and inoculated with different bacteria and Candida. The wells were filled with 100 μL of the tested compound and the plates were incubated at 37 °C for 24 h. The diameters of the inhibition zones were measured in millimeters (mm). Each antimicrobial assay was performed in triplicates and mean values were reported. Standard antibiotics, gentamicin (10 μg/disc), and nystatin (25 μg/disc) served as positive controls for antimicrobial and Candida activity, respectively. Solvent control wells of dimethyl sulfoxide (100 μL of DMSO) were used to aid in solubilizing Nystatin and they were used as negative control. The inhibition zone diameters were measured. The organisms used and zone of inhibition to the corresponding compounds are shown in Table 1.
before the addition to cell cultures and equal amounts of the solvent were added to control cells. Cell viability was assessed, after 3 days of treatment, with tetrazolium dye 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), obtained from Sigma (Dorset, UK). IC 50 concentrations were obtained from the dose-response curves using Graph Pad Prism Software 5 (GraphPad Software, Inc. San Diego, CA, USA) [27], and doxorubicin as positive control.