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

Abstract

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.

1. Introduction

The benzo[c]pyridazine nucleus, better-known as cinnoline, and its derivatives have received considerable interest due to their wide range of pharmacological profiles, e.g., antibacterial [1], antitumor [2,3], antifungal [4] and anti-inflammatory [5] activities. Certain compounds of the cinnoline series have antithrombocytic [6] and antituberculosis [7] properties, and also exhibit anesthetizing [8], and sedative [9] activity, in addition to their use as agrochemicals [10].

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].

Figure 1. Cinoxacine (1) and ICI-D-7569 (2).

Figure 1. Cinoxacine (1) and ICI-D-7569 (2).

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.

2. Results and Discussion

2.1. 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.

Scheme 1. The synthetic route for compounds 8–10.

In the ¹H-NMR (CDCl₃) 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 ¹³C-NMR spectra of compounds 8–10, the methyl (CH₃) 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.

2.2. Antibacterial and Antifungal Activity

2.2.1. 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 mg/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 10231and 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.

Table 1. Antifungal activity of compounds (8–10) at 25 mg/mL.

Compound (25 µg/mL)	8a	8b	8c	8d	9a	9b	Nystatin
C. albicans ATCC 10231	NA	NA	NA	NA	7 ± 0.2	7 ± 0.2	17 ± 1.5
C. glabirata ATCC 15126	8 ± 0.5	7 ± 0.1	7 ± 0.8	7 ± 0.5	NA	NA	20 ± 0.5
C. albicans Clinical isolate	7 ± 0.5	7 ± 0.1	7 ± 0.5	7 ± 0.1	8 ± 0.6	7 ± 0.2	15 ± 1
Compound (25 µg/mL)	9c	9d	10a	10b	10c	10d	Nystain
C. albicans ATCC 10231	NA	7 ± 0.2	8 ± 0.4	NA	NA	7 ± 0.5	17 ± 1.5
C. glabirata ATCC 15126	NA	NA	8 ± 0.5	8 ± 0.7	8 ± 0.4	9 ± 0.3	20 ± 0.5
C. albicans Clinical isolate	7 ± 0.6	7 ± 0.5	8 ± 0.3	7 ± 0.1	7 ± 0.1	8 ± 0.2	15 ± 1

The results are the mean ± SD (n = 3) in unit of mm. The well is 6 mm wide. NA: inactive at 25 mg/mL of the compound tested. Nystatin impregnated discs with 5 mm wide wells.

Table 1. Antifungal activity of compounds (8–10) at 25 mg/mL.

Compound (25 µg/mL)	8a	8b	8c	8d	9a	9b	Nystatin
C. albicans ATCC 10231	NA	NA	NA	NA	7 ± 0.2	7 ± 0.2	17 ± 1.5
C. glabirata ATCC 15126	8 ± 0.5	7 ± 0.1	7 ± 0.8	7 ± 0.5	NA	NA	20 ± 0.5
C. albicans Clinical isolate	7 ± 0.5	7 ± 0.1	7 ± 0.5	7 ± 0.1	8 ± 0.6	7 ± 0.2	15 ± 1
Compound (25 µg/mL)	9c	9d	10a	10b	10c	10d	Nystain
C. albicans ATCC 10231	NA	7 ± 0.2	8 ± 0.4	NA	NA	7 ± 0.5	17 ± 1.5
C. glabirata ATCC 15126	NA	NA	8 ± 0.5	8 ± 0.7	8 ± 0.4	9 ± 0.3	20 ± 0.5
C. albicans Clinical isolate	7 ± 0.6	7 ± 0.5	8 ± 0.3	7 ± 0.1	7 ± 0.1	8 ± 0.2	15 ± 1

The results are the mean ± SD (n = 3) in unit of mm. The well is 6 mm wide. NA: inactive at 25 mg/mL of the compound tested. Nystatin impregnated discs with 5 mm wide wells.

2.2.2. 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.

Table 2. The MIC and MFC in mg/mL for compounds (8a–d) against Candida species.

Entry	8a		8b		8c		8d		Nystatin
	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC
C. albicans clinical isolate	0.4	0.9	0.3	0.6	0.4	0.9	3.0	5.0	0.008	0.03
C.galibrata ATCC 15126	0.4	0.4	0.2	0.2	0.4	0.4	3.0	3.0	0.003	0.02

NA: inactive, MFC: minimum fungicidal concentration.

Table 2. The MIC and MFC in mg/mL for compounds (8a–d) against Candida species.

Entry	8a		8b		8c		8d		Nystatin
	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC
C. albicans clinical isolate	0.4	0.9	0.3	0.6	0.4	0.9	3.0	5.0	0.008	0.03
C.galibrata ATCC 15126	0.4	0.4	0.2	0.2	0.4	0.4	3.0	3.0	0.003	0.02

NA: inactive, MFC: minimum fungicidal concentration.

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.

Table 3. The MIC and MFC in mg/mL for compounds (9a–d) against Candida species.

Entry	9a		9b		9c		9d		Nystatin
	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC
C .albicans ATCC 10231	0.6	1.0	0.6	1.0	NA	NA	0.8	2.0	0.006	0.03
C. albicans clinical isolate	0.2	0.3	0.6	1.0	0.08	0.2	0.4	0.8	0.008	0.03
C. galibrata ATCC 15126	0.3	0.3	0.6	1.0	0.16	0.3	0.4	0.4	0.003	0.02

NA: inactive.

Table 3. The MIC and MFC in mg/mL for compounds (9a–d) against Candida species.

Entry	9a		9b		9c		9d		Nystatin
	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC
C .albicans ATCC 10231	0.6	1.0	0.6	1.0	NA	NA	0.8	2.0	0.006	0.03
C. albicans clinical isolate	0.2	0.3	0.6	1.0	0.08	0.2	0.4	0.8	0.008	0.03
C. galibrata ATCC 15126	0.3	0.3	0.6	1.0	0.16	0.3	0.4	0.4	0.003	0.02

NA: 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.

2.3. 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.

Table 4. Percentage cell survival of MCF-7 following 72 h exposure to 50 µg/mL of 8–10.

Compound	8a	8b	8c	8d	9a	9b	9c	9d	10a	10b	10c	10d
MCF-7% survival	95	43	92	101	99	102	102	94	101	42	74	45
Standard deviation	±2.60	±3.53	±1.85	±1.10	±7.02	±3.21	±2.49	±0.71	±2.25	±1.48	±1.84	±2.31

Table 4. Percentage cell survival of MCF-7 following 72 h exposure to 50 µg/mL of 8–10.

Compound	8a	8b	8c	8d	9a	9b	9c	9d	10a	10b	10c	10d
MCF-7% survival	95	43	92	101	99	102	102	94	101	42	74	45
Standard deviation	±2.60	±3.53	±1.85	±1.10	±7.02	±3.21	±2.49	±0.71	±2.25	±1.48	±1.84	±2.31

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 IC₅₀ 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 IC₅₀ value of 5.56 μM. Compounds 10b and 10d have IC₅₀ values of 11.79 and 8.57, respectively.

Table 5. Effect of compounds 8b, 10b, and 10d on MCF-7.

Compound	8b	10b	10d	Doxorubicin
IC50 MCF-7 (μM)	5.56 ± 0.30	11.79 ± 2.05	8.57 ± 0.85	0.31 ± 0.01

Table 5. Effect of compounds 8b, 10b, and 10d on MCF-7.

Compound	8b	10b	10d	Doxorubicin
IC50 MCF-7 (μM)	5.56 ± 0.30	11.79 ± 2.05	8.57 ± 0.85	0.31 ± 0.01

3. Experimental

3.1. General

Melting points were recorded on SMP1 Stuart apparatus and are uncorrected. The ¹H- and ¹³C-NMR spectra were recorded on a Bruker DPX-300 spectrometer in CDCl₃ 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 CDCl₃, 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).

3.2. 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.

1-(2-(4-Fluorophenyl)hydrazono)-1-(4-phenylpiperazin-1-yl)propan-2-one (5b). Yield: 76%; mp = 123–125 °C. ¹H-NMR (CDCl₃) δ: 2.43 (3H, s, CH₃), 3.24 (4H, m, H₂-3'+H₂-5'), 3.26 (4H, m, H₂-2'+H₂-6'), 6.87–7.32 (9H, m, Ar), 9.15 (1H, s, N–H). ¹³C-NMR (CDCl₃) δ: 25.8 (CH₃), 48.1 (C-2'/C-6'), 50.3 (C-3'/C-5'), 115.4 (d, ³J_C-F = 7.5 Hz, C–H), 116.2 (d, ²J_C-F = 22.5 Hz, C–H), 116.4 (C–H), 120.3 (C–H), 129.3 (C–H), 139.0 (C), 143.3 (C), 151.5 (C), 158.6 (d, ¹J_C-F = 240.0 Hz, C), 195.1 (C=O). HRMS (ESI) m/z: 341.16797 (Calcd for C₁₉H₂₂FN₄O [M+H]⁺: 341.16994). Anal. Calcd for C₁₉H₂₁FN₄O: C, 67.04; H, 6.22; N, 16.46. Found: C, 66.88; H, 6.19; N, 16.33.

1-(4-Benzylpiperazin-1-yl)-1-(2-phenylhydrazono)propan-2-one (6a). Yield: 76%; mp = 102–105 °C. ¹H-NMR (CDCl₃) δ: 2.41 (3H, s, CH₃), 2.54 (4H, m, H₂-3'+H₂-5'), 3.06 (4H, m, H₂-2'+H₂-6'), 3.57 (2H, s, CH₂Ph), 6.97 (1H, t, J = 7.3, H-4), 7.17–7.37 (10H, m, Ar), 9.13 (1H, s, N–H). ¹³C-NMR (CDCl₃) δ: 25.7 (CH₃), 47.8 (C-2'/C-6'), 53.8 (C-3'/C-5'), 63.2 (CH₂Ph), 114.1 (C–H), 122.0 (C–H), 127.1 (C–H), 128.3 (C–H), 129.1 (C–H), 129.4 (C–H), 138.0 (C), 142.5 (C), 143.5 (C), 195.0 (C=O). HRMS (ESI) m/z: 337.20029 (Calcd for C₂₀H₂₅N₄O [M+H]⁺: 337.19501). Anal. Calcd for C₂₀H₂₄N₄O: C, 71.40; H, 7.19; N, 16.65. Found: C, 71.48; H, 7.17; N, 16.57.

1-(4-Benzylpiperazin-1-yl)-1-(2-(4-fluorophenyl)hydrazono)propan-2-one (6b). Yield: 76%; mp = 121–124 °C. ¹H-NMR (CDCl₃) δ: 2.39 (3H, s, CH₃), 2.53 (4H, m, H₂-3'+H₂-5'), 3.06 (4H, m, H₂-2'+H₂-6'), 3.57 (2H, s, CH₂Ph), 6.99–7.04 (2H, m, C₆H₄F), 7.11–7.14 (2H, m, C₆H₄F) 7.25–7.37 (5H, m, Ar), 9.08 (1H, s, N–H). ¹³C-NMR (CDCl₃) δ: 25.7 (CH₃), 47.8 (C-2'/C-6'), 53.8 (C-3'/C-5'), 63.2 (CH₂Ph), 115.1 (d, ³J_C-F = 7.5 Hz, C–H), 116.1 (d, ²J_C-F = 22.5 Hz, C–H), 127.1 (C–H), 128.3 (C–H), 129.1 (C–H), 138.0 (C), 138.9 (C), 143.5 (C), 158.3 (d, ¹J_C-F = 240.0 Hz, C), 194.8 (C=O). HRMS (ESI) m/z: 355.18287 (Calcd for C₂₀H₂₄FN₄O [M+H]⁺: 355.18559). Anal. Calcd for C₂₀H₂₃FN₄O: C, 67.78; H, 6.54; N, 15.81. Found: C, 67.87; H, 6.50; N, 15.72.

1-(4-Benzylpiperazin-1-yl)-1-(2-(4-chlorophenyl)hydrazono)propan-2-one (6c). Yield: 63%; mp = 115–117 °C. ¹H-NMR (CDCl₃) δ: 2.41 (3H, s, CH₃), 2.54 (4H, m, H₂-3'+H₂-5'), 3.06 (4H, m, H₂-2'+H₂-6'), 3.57 (2H, s, CH₂Ph), 6.92–6.95 (1H, m, Ar), 7.01–7.04 (1H, m, Ar), 7.20–7.37 (m, 7H, Ar), 9.08 (1H, s, N–H). ¹³C-NMR (CDCl₃) δ: 25.8 (CH₃), 47.8 (C-2'/C-6'), 53.8 (C-3'/C-5'), 63.1 (CH₂Ph), 114.2 (C–H), 127.1 (C–H), 128.3 (C–H), 129.0 (C–H), 130.4 (C–H), 135.2 (C), 138.0 (C), 143.8 (C), 144.2 (C), 195.0 (C=O). HRMS (ESI) m/z: 371.15332 (Calcd for C₂₀H₂₄ClN₄O [M+H]⁺: 371.15604).Anal. Calcd for C₂₀H₂₃ClN₄O: C, 64.77; H, 6.25; N, 15.11. Found: C, 64.56; H, 6.27; N, 15.06.

1-(4-Benzylpiperazin-1-yl)-1-(2-(4-bromophenyl)hydrazono)propan-2-one (6d). Yield: 96%; mp = 106–108 °C. ¹H-NMR (CDCl₃) δ: 2.39 (3H, s, CH₃), 2.53 (4H, m, H₂-3'+H₂-5'), 3.04 (4H, m, H₂-2'+H₂-6'), 3.56 (2H, s, CH₂Ph), 7.06 (2H, d, J = 8.7, Ar), 7.40 (2H, d, J = 8.7, Ar), 7.25–7.34 (5H, m, Ar), 9.08 (1H, s, N–H). ¹³C-NMR (CDCl₃) δ: 25.7 (CH₃), 47.8 (C-2'/C-6'), 53.7 (C-3'/C-5'), 63.1 (CH₂Ph), 115.6 (C–H), 127.1(C–H), 128.2 (C–H), 129.1(C–H), 132.2 (C–H), 138.0 (C), 141.3 (C), 141.7 (C), 143.9 (C), 194.9 (C=O). HRMS (ESI) m/z: 415.10680 (Calcd for C₂₀H₂₄BrN₄O [M+H]⁺: 415.10552). Anal. Calcd for C₂₀H₂₃BrN₄O: C, 57.84; H, 5.58; N, 13.49. Found: C, 57.72; H, 5.60; N, 13.36.

1-(2-(4-Fluorophenyl)hydrazono)-1-(4-(4-fluorophenyl)piperazin-1-yl)propan-2-one (7b). Yield: 72%; mp = 157–159 °C. ¹H-NMR (CDCl₃) δ: 2.42 (3H, s, CH₃), 3.20 (8H, br m, H₂-3'+H₂-5'/H₂-2'+H₂-6'), 6.88–7.17 (8H, m, Ar), 9.13 (1H, s, N–H). ¹³C-NMR (CDCl₃) δ: 25.7 (CH₃), 48.0 (C-2'/C-6'), 51.1 (C-3'/C-5'), 115.2 (d, ³J_C-F = 7.5 Hz, C–H), 115.6 (d, ²J_C-F = 22.5 Hz, C–H), 116.1 (d, ²J_C-F = 22.5 Hz, C–H), 118.2 (d, ³J_C-F = 7.5 Hz, C–H), 138.8 (C), 143.1 (C), 148.0 (C), 157.3 (d, ¹J_C-F = 233.0 Hz, C), 158.4 (d, ¹J_C-F = 240.0 Hz, C), 194.90 (C=O). HRMS (ESI) m/z: 359.15970 (Calcd for C₁₉H₂₁F₂N₄O [M+H]⁺: 359.16052). Anal. Calcd for C₁₉H₂₀F₂N₄O: C, 63.68; H, 5.62; N, 15.63. Found: C, 63.58; H, 5.66; N, 15.70.

3.3. 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.

4-Methyl-3-(4-phenylpiperazin-1-yl)cinnoline (8a). Yield: 75%; mp = 164–166 °C. ¹H-NMR (CDCl₃) δ: 2.64 (3H, s, CH₃), 3.42 (4H, m, H₂-3'+H₂-5'), 3.56 (4H, m, H₂-2'+H₂-6'), 6.89 (1H, t, J = 7.2 Hz, Ar), 7.02 (2H, d, J = 8.0 Hz, Ar), 7.30 (2H, t, J = 8.0, Ar), 7.62–7.65 (2H, m, Ar), 7.89–7.92 (1H, m, Ar), 8.36–8.39 (1H, m, Ar). ¹³C-NMR (CDCl₃) δ: 12.6 (CH₃), 49.5 (C-2'/C-6'), 50.9 (C-3'/C-5'), 116.2 (C–H), 119.1 (C–H), 122.2 (C), 122.8 (C–H), 127.9 (C–H), 128.2 (C), 129.1 (C–H), 130.1 (C–H), 130.3 (C–H), 148.0 (C), 151.4 (C), 159.2 (C). HRMS (ESI) m/z: 305.17107 (Calcd for C₁₉H₂₁N₄ [M+H]⁺: 305.16880). Anal. Calcd for C₁₉H₂₀N₄: C, 74.97; H, 6.62; N, 18.41. Found: C, 74.88; H, 6.57; N, 18.22.

6-Fluoro-4-methyl-3-(4-phenylpiperazin-1-yl)cinnoline (8b). Yield: 78%; mp = 196–198 °C. ¹H-NMR (CDCl₃) δ: 2.58 (3H, s, CH₃), 3.43 (4H, m, H₂-3'+H₂-5'), 3.57 (4H, m, H₂-2'+H₂-6'), 6.89 (1H, t, J = 7.2 Hz, Ar), 7.02 (2H, d, J = 8.1 Hz, Ar), 7.28–7.33 (2H, m, Ar), 7.37–7.47 (2H, m, Ar), 8.37–8.43 (1H, m, Ar). ¹³C-NMR (CDCl₃) δ: 13.0 (CH₃), 49.6 (C-2'/C-6'), 50.9 (C-3'/C-5'), 105.9 (d, ²J_C-F = 22.5 Hz, C–H), 116.3 (C–H), 119.3 (d, ²J_C-F = 26.0 Hz, C–H), 120.0 (C–H), 121.2 (C), 129.3 (C–H), 129.8 (C), 133.7 (d, ³J_C-F = 11.0 Hz, C–H), 145.9 (C), 151.5 (C), 159.2 (C), 162.9 (d, ¹J_C-F = 255.0 Hz, C). HRMS (ESI) m/z: 323.16065 (Calcd for C₁₉H₂₀FN₄ [M+H]⁺: 323.15937). Anal. Calcd for C₁₉H₁₉FN₄: C, 70.79; H, 5.94; N, 17.38. Found: C, 70.63; H, 6.01; N, 17.27.

6-Chloro-4-methyl-3-(4-phenylpiperazin-1-yl)cinnoline (8c). Yield: 43%; mp = 156–160 °C. ¹H-NMR (CDCl₃) δ: 2.95 (3H, s, CH₃), 3.42 (4H, m, H₂-3'+H₂-5'), 3.57 (4H, m, H₂-2'+H₂-6'), 6.78–6.97 (1H, m, Ar), 7.02 (2H, d, J = 7.9, Ar), 7.26–7.33 (2H, m, Ar), 7.46–7.58 (1H, m, Ar), 7.67 (1H, d, J = 7.2, Ar), 8.30–8.36 (1H, m, H-8). ¹³C-NMR (CDCl₃) δ: 17.7 (CH₃), 49.5 (C-2'/C-6'), 50.7 (C-3'/C-5'), 116.2 (C–H), 119.9 (C–H), 124.6 (C), 127.3 (C–H), 128.5 (C), 129.1 (C–H), 130.2 (C–H), 131.5 (C), 132.5 (C–H), 149.1 (C), 151.3 (C), 160.7 (C). HRMS (ESI) m/z: 361.11905 (Calcd for C₁₉H₁₉ClN₄Na [M+Na]⁺: 361.11959). Anal. Calcd for C₁₉H₁₉ClN₄: C, 67.35; H, 5.65; N, 16.54. Found: C, 67.39; H, 5.54; N, 16.43.

6-Bromo-4-methyl-3-(4-phenylpiperazin-1-yl)cinnoline (8d). Yield: 20%; mp = 194–196 °C. ¹H-NMR (CDCl₃) δ: 2.59 (3H, s, CH₃), 3.43 (4H, m, H₂-3'+H₂-5'), 3.56 (4H, m, H₂-2'+H₂-6'), 6.89 (1H, t, J = 7.2, Ar), 7.02 (2H, d, J = 8.0, Ar), 7.25–7.33 (2H, m, Ar), 7.69 (1H, d, J = 9.0, Ar), 8.10 (1H, s, Ar), 8.24 (1H, d, J = 9.0, Ar). ¹³C-NMR (CDCl₃) δ: 12.8 (CH₃), 49.5 (C-2'/C-6'), 50.9 (C-3'/C-5'), 116.2 (C–H), 120.0 (C–H), 120.5 (C), 125.3 (C–H), 125.6 (C–H), 129.2 (C–H), 131.6 (C–H), 132.0 (C–H), 132.7 (C), 146.4 (C), 151.4 (C), 159.5 (C). HRMS (ESI) m/z: 405.06853 (Calcd for C₁₉H₁₉BrN₄Na [M+Na]⁺: 405.06908). Anal. Calcd for C₁₉H₁₉BrN₄: C, 59.54; H, 5.00; N, 14.62. Found: C, 59.38; H, 4.93; N, 14.71.

3-(4-Benzylpiperazin-1-yl)-4-methylcinnoline (9a). Yield: 30%; mp = 162–164 °C. ¹H-NMR (CDCl₃) δ: 2.58 (3H, s, CH₃), 2.71 (4H, m, H₂-3'+H₂-5'), 3.42 (4H, m, H₂-2'+H₂-6'), 3.63 (2H, s, CH₂Ph), 7.24–7.40 (5H, m, Ar), 7.58–7.64 (2H, m, Ar), 7.84–7.89 (1H, m, Ar), 8.32–8.37 (1H, m, Ar). ¹³C-NMR (CDCl₃) δ: 12.8 (CH₃), 50.9 (C-2'/C-6'), 53.5 (C-3'/C-5'), 63.3 (CH₂Ph), 121.8 (C), 123.0 (C–H), 127.2 (C–H), 127.8 (C–H), 128.4 (C–H), 129.3 (C–H), 130.3 (C–H), 132.9 (C), 138.1 (C), 148.0 (C), 159.5 (C). HRMS (ESI) m/z: 319.18272 (Calcd for C₂₀H₂₃N₄ [M+H]⁺: 319.18445). Anal. Calcd for C₂₀H₂₂N₄: C, 75.44; H, 6.96; N, 17.60. Found: C, 75.23; H, 6.91; N, 17.52.

3-(4-Benzylpiperazin-1-yl)-6-fluoro-4-methylcinnoline (9b). Yield: 20%; mp = 113–117 °C. ¹H-NMR (CDCl₃) δ: 2.50 (3H, s, CH₃), 2.70 (4H, m, H₂-3'+H₂-5'), 3.42 (4H, m, H₂-2'+H₂-6'), 3.62 (2H, s, CH₂Ph), 7.25–7.37 (7H, m, Ar), 8.33–8.38 (1H, m, Ar). ¹³C-NMR (CDCl₃) δ: 13.0 (CH₃), 50.6 (C-2'/C-6'), 53.3 (C-3'/C-5'), 63.2 (CH₂Ph), 105.7 (d, ²J_C-F = 22.5 Hz, C–H), 118.9 (d, ²J_C-F = 26.0 Hz, C–H), 120.9 (C), 127.2 (C–H), 128.3 (C–H), 129.2 (C–H), 129.8 (C), 133.5 (d, ³J_C-F = 7.5 Hz, C–H), 137.9 (C), 145.7 (C), 159.3 (C), 162.7 (d, ¹J_C-F = 255.0 Hz, C). HRMS (ESI) m/z: 337.17230 (Calcd for C₂₀H₂₂FN₄ [M+H]⁺: 337.17502). Anal. Calcd for C₂₀H₂₁FN₄: C, 71.41; H, 6.29; N, 16.65. Found: C, 71.38; H, 6.27; N, 16.54.

3-(4-Benzylpiperazin-1-yl)-6-chloro-4-methylcinnoline (9c). Yield: 60%; mp = 150–152 °C. ¹H-NMR (CDCl₃) δ: 2.53 (3H, s, CH₃), 2.69 (4H, m, H₂-3'+H₂-5'), 3.43 (4H, m, H₂-2'+H₂-6'), 3.62 (2H, s, CH₂Ph), 7.25–7.39 (5H, m, Ar), 7.51 (1H, dd, J = 1.9, 9.2, Ar), 7.84 (1H, d, J = 1.9, Ar), 8.28 (2H, d, J = 9.0, Ar). ¹³C-NMR (CDCl₃) δ: 12.9 (CH₃), 50.7 (C-2'/C-6'), 53.3 (C-3'/C-5'), 63.2 (CH₂Ph), 120.3 (C), 121.7 (C–H), 127.1 (C–H), 128.3 (C–H), 128.9 (C–H), 129.0 (C), 129.2 (C–H), 132.0 (C–H), 136.6 (C), 138.0 (C), 146.1 (C), 159.6 (C). HRMS (ESI) m/z: 353.14475 (Calcd for C₂₀H₂₂ClN₄ [M+H]⁺: 353.14547). Anal. Calcd for C₂₀H₂₁ClN₄: C, 68.08; H, 6.00; N, 15.88. Found: C, 67.95; H, 5.87; N, 15.73.

3-(4-Benzylpiperazin-1-yl)-6-bromo-4-methylcinnoline (9d). Yield: 45%; mp = 153–155 °C. ¹H-NMR (CDCl₃) δ: 2.52 (3H, s, CH₃), 2.70 (4H, m, H₂-3'+H₂-5'), 3.42 (4H, m, H₂-2'+H₂-6'), 3.62 (2H, s, CH₂Ph), 7.24–7.39 (5H, m, Ar), 7.64 (1H, dd, J = 1.9, 9.0, Ar), 8.03 (1H, d, J = 1.9, Ar), 8.20 (1H, d, J = 9.0, Ar). ¹³C-NMR (CDCl₃) δ: 13.0 (CH₃), 50.8 (C-2'/C-6'), 53.4 (C-3'/C-5'), 63.3 (CH₂Ph), 120.2 (C), 125.3 (C–H), 125.5 (C), 127.2 (C–H), 128.4 (C–H), 129.3 (C–H), 129.5 (C), 131.4 (C–H), 132.0 (C–H), 138.0 (C), 146.3 (C), 159.7 (C). HRMS (ESI) m/z: 419.08418 (Calcd for C₂₀H₂₁BrN₄Na [M+Na]⁺: 419.08473). Anal. Calcd for C₂₀H₂₁BrN₄: C, 60.46; H, 5.33; N, 14.10. Found: C, 60.29; H, 5.27; N, 13.99.

3-(4-(4-Fluorophenyl)piperazin-1-yl)-4-methylcinnoline (10a). Yield: 35%; mp = 183–185 °C. ¹H-NMR (CDCl₃) δ: 2.64 (3H, s, CH₃), 3.35 (4H, m, H₂-3'+H₂-5'), 3.56 (4H, m, H₂-2'+H₂-6'), 6.95–7.03 (3H, m, Ar), 7.63–7.66 (2H, m, Ar), 7.89–7.92 (1H, m, Ar), 8.36–8.40 (1H, m, Ar). ¹³C-NMR (CDCl₃) δ: 12.6 (CH₃), 50.5, (C-2'/C-6'), 51.0 (C-3'/C-5'), 115.6 (d, ²J_C-F = 22.5 Hz, C–H), 118.0 (d, ³J_C-F = 7.5 Hz, C–H), 122.1 (C), 122.9 (C–H), 127.9 (C–H), 128.2 (C), 130.2 (C–H), 130.3 (C–H), 148.0 (C), 148.1 (C), 157.3 (d, ¹J_C-F = 235.0 Hz, C), 159.1 (C). HRMS (ESI) m/z: 323.16065 (Calcd for C₁₉H₂₀FN₄ [M+H]⁺: 323.15937). Anal. Calcd for C₁₉H₁₉FN₄: C, 70.79; H, 5.94; N, 17.38. Found: C, 70.71; H, 5.89; N, 17.35.

6-Fluoro-3-(4-(4-fluorophenyl)piperazin-1-yl)-4-methylcinnoline (10b). Yield: 25%; mp = 173–176 °C. ¹H-NMR (CDCl₃) δ: 2.57 (3H, s, CH₃), 3.34 (4H, m, H₂-3'+H₂-5'), 3.57 (4H, m, H₂-2'+H₂-6'), 6.93–7.03 (4H, m, Ar), 7.37–7.46 (2H, m, Ar), 8.40 (1H, dd, J = 5.7, 9.2, Ar). ¹³C-NMR (CDCl₃) δ: 13.1 (CH₃), 50.6 (C-2'/C-6'), 50.9 (C-3'/C-5'), 105.7 (d, ²J_C-F = 22.5 Hz, C–H), 115.5 (d, ²J_C-F = 22.5 Hz, C–H), 118.1 (d, ³J_C-F = 7.5 Hz, C–H), 119.4 (d, ²J_C-F = 26.0 Hz, C–H), 121.4 (C), 133.6 (d, ³J_C-F = 7.5 Hz, C–H), 145.9 (C), 148.1 (C), 153.7 (C), 157.4 (d, ¹J_C-F = 240.0 Hz, C), 159.2 (C), 162.0 (d, ¹J_C-F = 248.0 Hz, C). HRMS (ESI) m/z: 341.15123 (Calcd for C₁₉H₁₉F₂N₄ [M+H]⁺: 341.14995). Anal. Calcd for C₁₉H₁₈F₂N₄: C, 67.05; H, 5.33; N, 16.46. Found: C, 66.98; H, 5.38; N, 16.38.

6-Chloro-3-(4-(4-fluorophenyl)piperazin-1-yl)-4-methylcinnoline (10c). Yield: 75%; mp = 156–158 °C. ¹H-NMR (CDCl3) δ: 2.59 (3H, s, CH₃), 3.35 (4H, m, H₂-3'+H₂-5'), 3.56 (4H, m, H₂-2'+H₂-6'), 6.91–7.03 (4H, m, Ar), 7.69 (1H, d, J = 9.0, Ar), 8.08 (1H, s, Ar), 8.69 (1H, d, J = 9.1, Ar). ¹³C-NMR (CDCl₃) δ: 12.9 (CH₃), 50.6 (C-2'/C-6'), 50.9 (C-3'/C-5'), 115.6 (d, ²J_C-F = 21.0 Hz, C–H), 118.1 (d, ³J_C-F = 7.5 Hz, C–H), 120.3 (C), 121.8 (C–H), 129.0 (C), 129.4 (C–H), 131.8 (C–H), 138.1 (C), 146.1 (C), 147.5 (C), 157.4 (d, ¹J_C-F = 235.0 Hz, C), 159.8 (C). HRMS (ESI) m/z: 357.12010 (Calcd for C₁₉H₁₉ClFN₄ [M+H]⁺: 357.12040). Anal. Calcd for C₁₉H₁₈ClFN₄: C, 63.95; H, 5.08; N, 15.70. Found: C, 63.82; H, 5.02; N, 15.53.

6-Bromo-3-(4-(4-fluorophenyl)piperazin-1-yl)-4-methylcinnoline (10d). Yield: 62%; mp = 208–210 °C. ¹H-NMR (CDCl₃) δ: 2.57 (3H, s, CH₃), 3.34 (4H, m, H₂-3'+H₂-5'), 3.56 (4H, m, H₂-2'+H₂-6'), 6.92–7.02 (4H, m, Ar), 7.68 (1H, dd, J = 1.9, 9.0, Ar), 8.06 (1H, d, J = 1.8, Ar), 8.23 (1H, d, J = 9.0, Ar). ¹³C-NMR (CDCl₃) δ: 12.8 (CH₃), 50.5 (C-2'/C-6'), 50.8 (C-3'/C-5'), 115.6 (d, ²J_C-F = 22.5 Hz, C–H), 118.0 (d, ³J_C-F = 7.5 Hz, C–H), 120.5 (C), 125.2 (C–H), 125.6 (C), 129.4 (C), 131.6 (C–H), 131.9 (C–H), 146.4 (C), 148.0 (C), 157.3 (d, ¹J_C-F = 240.0 Hz, C), 159.4 (C). HRMS (ESI) m/z: 401.07116 (Calcd for C₁₉H₁₉BrFN₄ [M+H]⁺: 401.06989). Anal. Calcd for C₁₉H₁₈BrFN₄: C, 56.87; H, 4.52; N, 13.96. Found: C, 56.80; H, 4.54; N, 13.85.

3.4. Biological Activity Test Procedures

3.4.1. 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.

3.4.2. 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 mg/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.

3.4.3. Serial Dilution Method (Broth Microdilution Assay)

According to the National Committee for Clinical Laboratories Standards (NCCLS), a broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) [25,26]. The MIC of a compound was defined as the lowest concentration of the compound that resulted in complete inhibition of visible bacterial/fungal growth at 24 h. The inocula of the bacterial strains or Candida were prepared from overnight broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. A serial doubling dilution of the compounds was prepared in a 96/well microtiter plate. A double strength of Mueller Hinton broth/Sabouraud Dextrose broth was used as a diluent. The concentrations were in range of 25–0.02 mg/mL. Bacterial strains and Candida were inoculated in Mueller Hinton broth and Sabouraud dextrose broth respectively and inoculated into wells (the final concentration in each well adjusted to 2.0 × 10⁶ CFU/mL for bacteria and 2.0 × 10⁵ of Candida strains). The plate was incubated for 24 h at 37 °C. A control well containing the growth medium and the bacteria or Candida was set-up. Gentamicin and nystatin served as positive controls, while the solvent (DMSO) was used as a negative control. MIC was defined as the lowest concentration of compound that resulted in complete inhibition of visible (turbidity in the broth) bacterial/fungal growth at 24 h. To determine MBC/MFC broth was taken from each well and inoculated in Mueller Hinton agar for 24 h at 37 °C for bacteria or in Sabouraud dextrose agar for Candida strains, respectively.

The MBC/MFC were defined as the lowest concentration of the compound that kills 99.9% of the original inoculum in 24 h. Tables (1, 2 and 3) show the MIC and MBC/MFC to the corresponding compounds and the organisms used.

3.4.4. Statistical Analyses

Analysis of variance (ANOVA) was used to determine the significance (p ≤ 0.05) of the data obtained in all experiments. All results were determined to be within the 95% confidence level for reproducibility.

3.5. Cell Lines and Cell Culture

3.5.1. Materials and Methods

The K562 leukemia cell line was obtained from Dr. Mona Hassona (The University of Jordan, Department of Biology) and was cultured in RPMI; MCF-7 breast cancer cells were obtained from ATCC and were cultured in DMEM. All media were supplemented with 2 mM glutamine and 10% Fetal Bovine Serum (FBS, Gibco Life Technologies) and cells were maintained under standard cell culture conditions at 37 °C in a water-saturated atmosphere of 5% CO₂ in air.

3.5.2. Cell Proliferation Assay

MCF-7 and K562 cells were seeded at a density of 1 × 10⁴ and 4 × 10⁴ per well in 96-well plates in appropriate medium. For anti-MCF-7 screening, the cells were treated with 50 µM concentrations of the tested compounds. For the IC₅₀ determination the cells were treated with increasing concentrations of the tested compound (1.56–100 µM). In all assays, the drugs were dissolved in DMSO immediately 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-2-yl)-2,5-diphenyltetrazolium bromide (MTT), obtained from Sigma (Dorset, UK). IC₅₀ 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.