Abstract
The emerging resistance to antimicrobial drugs demands the synthesis of new remedies for microbial infections. Attempts have been made to prepare new compounds by modifications in the quinolone structure. An important method for the synthesis of new quinolone is using Vilsmeier approach but has its own limitations. The present work aimed to synthesize novel norfloxacin analogues using modified Vilsmeier approach and conduct preliminary investigations for the evaluation of their physicochemical properties, photochemical probe, and antimicrobial effects. In an effort to synthesize norfloxacin analogues, only 7-bromo-6-N-benzyl piperazinyl-4-oxoquinoline-3-carboxylic acid was isolated using Vilsmeier approach at high temperature, where -bis-(4-fluoro-3-nitrophenyl)-oxalamide and -bis-(3-chloro-4-fluorophenyl)-malonamide were obtained at low temperature. Correlation results showed that lipophilicity, molecular mass, and electronic factors might influence the activity. The synthesized compounds were evaluated for their antimicrobial effects against important pathogens, for their potential use in the inhibition of vitiligo.
1. Introduction
The structure activity relationship (SAR) for the quinolone skeleton 1-alkyl-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid studies revealed that the 6-halogen atom, especially the 6-fluorine, is responsible for the potency as represented by the binding capacity with DNA gyrase and topoisomerase IV [1]. It is clear that chemical modifications at C-7 are suitable to control the pharmacokinetic properties and, hence, changes in the cell permeability of these antibiotics. -piperazinyl derivatives of fluoroquinolones were introduced and demonstrated for various biological activities that possess broad-spectrum activity [2β6]. Furthermore, it is clear that the neutral species of fluoroquinolones are more lipophilic than the Zwitterionic form. Therefore, factors that can affect -protonation like steric and electronic effect or charge density can also affect lipophilicity [7β9].
Procopiou et al. [10] prepared a series of asymmetrical 1,4-disubstituted piperazines as a novel class of non-brain-penetrant histamine H3 receptor antagonists. In addition, Foroumadi et al. [11] synthesized a modified norfloxacin via heteroarylation of norfloxacin on -piperazinyl position (Scheme 1). The antibacterial activity of these modified norfloxacin depends not only on the bicyclic heteroaromatic pharmacophore but also on the nature of the peripheral substitutions and their spatial relationship, such as solubility, thermal stability, hydrolysis, and a possibility to form a Zwitter ion. Meth-Cohn and Taylor [12] reported an important method for the synthesis of quinolones using reverse Vilsmeier approach but has its own limitations, like uncompleted cyclisation to the target quinolone.
In the light of these observations, the aim of this work was to synthesize novel norfloxacin analogues using modified Vilsmeier approach and conduct preliminary investigations for the evaluation of their physicochemical properties, photochemical probe, and antimicrobial effects.
2. Materials and Methods
2.1. Equipment Used for the Characterization of the Produced Compounds
Electrothermal 9100 (fisher Scientific, US) was used to determine melting points or ranges. Infrared (IR) spectra were recorded on a Unicam Research Series 2000 FTIR. NMR spectra were recorded in DMSO or CDCl3 on a Bruker AVANCE 300 at 300βMHz. Mass spectrometry was performed on an Esquire 3000 plus, or Bruker ApexII, for low and high resolution. Elemental analysis was performed on an Exeter Analytical CE-440; GCMS was performed on Shimadzu GC-17A and QP-5000 Mass Spectrometer.
2.2. Materials Used for Microbiological Assay
Nutrient Agar, MacConkey Agar, Sabouraud Dextrose Agar, and dimethylformamide (DMF) were obtained from Sigma; Nalidixic acid (30βΞΌg/disk, Bioanalize, Egypt) and Nystain (manufactured by Pasteur Lab., Egypt, NS 100 units (100βΞΌg/disk) were used as reference antibiotics.
2.3. Synthesis of Norfloxacin Analogues
We used a solid phase via Merrifield resin through reactions of substituted piperazine with 3-bromo-4-fluoronitrobenzene. In the synthetic sequence, the Merrifield resin (1) was first suspended in dry DMF, and to this suspension was added an excess of piperazine (2-3 equivalents) in pyridine or anhydrous K2CO3 (6β8 equivalents). The reaction mixture was continued at 40Β°C for 24 hours then piperazine resin (2) was obtained, filtered, washed with CH2Cl2, and dried. Compound 2 was resuspended in DMF and reacted with 3-bromo-4-fluoronitrobenzene (3) to give the 4-piperazine resin-supported-3-bromo-1-nitrobenzene (4) (not the expected 3-piperazine resin-supported-4-fluoro-1-nitrobenzene), (Scheme 2), which on reduction with SnCl2-EtOH yielded the 3-bromo-4-(4β²-resin-supported benzyl piperazinyl)-1-aniline (5) and then by treatment with an excess of formic acid at room temperature for 12 hours produced the corresponded 3-bromo-4-(4β²-resin-supported benzyl piperazinyl)-1-formanild (6). The dry resin-supported formanilide 6, when reacted with Phosphorus oxychloride or Oxalyl chloride and methyl malonyl chloride (7) under reverse Vilsmeier conditions, mainly gave the resin-supported quinolone, 6-fluoro-7-piperazino-4-oxo-3-quinolone carboxylic acid, (8) (Scheme 3). The procedure, in general, yielded a mixture of by-products in low quantities, and TLC and GCMS were used for the assessment of the recovered cleavage products.
2.4. Preparation of 3-bromo-4-fluoronitrobenzene (3)
Equimolar mixture of nitric acid and sulphuric acid (1β:β1, 25βmLβ:β25βmL) was stirred at ~5Β°C. A solution of 2-fluorobromobenzene (25βg, 0.143βmoL) in methanol (30βmL) was added to the mixture with gradual stirring over a period of 20β30 minutes. After complete addition, the temperature was raised gradually to 70Β°C for 1βh. After cooling, the reaction mixture was poured into cold water (20βmL), and the immediate cream solid precipitate was collected by filtration. Crystallization with CHCl3 gave a cream shiny crystals (29.23βg, 93% yield), mp 60β62Β°C (lit. [13] mp 58-59Β°C); /cmβ1 1535 and 1342 (NO2); (300βMHz; CDCl3) 7.29 (1H, t, βHz, H-5), 8.24 (1H, m, H-6), 8.50 (1H, dd, and 4.3βHz, H-2); (75βMHz; CDCl3) 110.1 (d, βHz, C-3), 117.1 (d, βHz, C-5), 123.3 (d, βHz, C-6), 129.6 (C-2), 144.4 (C-1), 162.9 (d, βHz, C-4); (MHz;CDCl3)-74.22 (s); 221(M+, 44%), 219 (M+, 46%), 203 (3), 189 (17), 173 (38), 161 (14), 94 (M-Br-NO2, 100), 68 (25), 61 (7), 50 (38).
2.5. Preparation of 4-(4β²-benzylpiperazin-1β²-yl)-3-bromo-1-nitrobenzene (9)
Under dry conditions, 3-bromo-4-fluoronitrobenzene (3) (5.1βg, 23βmmoL) was dissolved in dry acetonitrile (2βmL), then anhydrous K2CO3 (9.6βg, 69.2βmmoL) was added followed by addition of N-benzylpiperazine (8βg, 46βmmoL) to the suspension mixture using a syringe; the temperature gradually raised to reflux for 12βh (or until the complete disappearance of the starting material). The reaction was monitored by TLC (CHCl3: petroleum ether (40β60), 50%). The acetonitrile was removed under vacuo, and the resulting solid was stirred in cold water (200βmL) for 20 minutes. The pale brown solid formed was recrystallized from CHCl3 to give bright yellow needle-like crystals of 9 (5.8 g, 81% yield), mp 123-124Β°C; [C17H18BrN3O2 Calc. C, 54.3; H, 4.8; N, 11.2. Found C: 54.5; H, 4.8; N, 11.1]; /cmβ1 1580, and 1339 (NO2); (300βMHz; CDCl3), 2.57 (4H, m, H-3β², and H-5β²), 3.17 (4H, m, H-2β² and H-6β²), 3.56 (2H, s, Ph-CH2), 7.12 (1H, d, βHz, H-5), 7.24 (5H, m, Ph), 8.08 (1H, dd, and 9.0βHz, H-6), 8.26 (1H, d, βHz, H-2); (75βMHz; CDCl3) 51.1 (C-3β² and C-5β²), 52.8 (C-2β² and C-6β²), 62.4 (CH2-Ph), 116.9 (C-3), 121.1 (C-5), 124.7 (C-6), 127.5 (C-2), 129.5 (Ph), 142.3 (C-1), 156.6 (C-4); (M+373/375).
2.6. Preparation of 4-(4β²-benzylpiperazin-1β²-yl)-3-bromo-4-phenylamine (10) [14]
A pale yellow oil (2.7βg, 60% yield); cmβ1 3150 (NH2); (300βMHz; CDCl3) 2.68 (4H, s, CH2-3β² and 5β²) and 3.01 (4H, s, CH2-2β² and 6β²), 3.57 (2H, s, Ph-CH2), 6.62 (1H, dd, and 4.2βHz, H-6), 6.94 (1H, d, βHz, H-5), 6.97 (1H, d, βHz, H-2), 7.37 (5H, m, Ph); (75βMHz; CDCl3) 52.2 (C-3β² and C-5β²), 53.6 (C-2β² and C-6β²), 63.3 (Ph-CH2), 114.9 (C-3), 120.1 (C-6), 121.1 (C-5), 121.8 (C-2), 128.4 (Ph), 142.3 (C-1), 143.4 (C-4); HRMS (ESI). Found: MH+, 346.0908. Calc. for C17H20BrN3: MH+ = 346.0919.
2.7. Preparation of 4-(4β²-benzylpiperazin-1β²-yl)-3-bromoformamide (11)
Formic acid (5βmL, 0.13βmoL) was added to 4-(4β²-benzylpiperazin-1β²-yl)-3-bromo-4-phenylamine (12) (5βg, 14.4βmmoL), and the resulting clear solution was refluxed for 2βh. After cooling to room temperature, the reaction mixture was poured into ice water (10βmL), then NaHCO3ββsolution (10% w/v, 20βmL) was added gradually until no more effervescence (formation of neutral to slightly basic solution) was observed and the solution extracted with CH2Cl2. (3 Γ 20βmL). The organic layers were combined, washed with NaHCO3 solution (10%, 20βmL), and dried over MgSO4. The solvent was removed in vacuo until complete dryness to give 11 as a brown solid which was purified by column chromatography on silica, eluted with CHCl3 to give a white solid (2.94βg, 54%), mp 73-74Β°C; [C18H20BrN3O Calc. C, 57.76; H, 5.39; N, 11.23. Found: C, 57.79; H, 5.41; N, 11.23]; /cmβ1 3320 (br, NH), 1716 (NCHO); (300βMHz; CDCl3) 2.68 (4H, br s, CH2-3β² and 5β²), 3.06 (4H, br s, CH2-2β² and 6β²), 3.62 (2H, s, Ph-CH2), 7.34 (6H, m, Ph+H-5), 7.48 (1H, dd, and 4.2βHz, H-6 ), 7.81 (1H, d, , H-2 ), 8.34 (1H, s, CHO), 8.58 (1H, s, NH); (75βMHz; CDCl3) 51.7 (C-3β² and C-5β²), 53.2 (C-2β² and C-6β²), 63.2 (Ph-CH2), 119.3 (C-3), 120.1 (C-5), 121.0 (C-6), 125.5 (C-2), 129.4 (C-Ph), 132.5 and 132.8 (C-1), 147.7 and 148.5 (C-4), 158.9 and 162.5 (-CHO).
2.8. Vilsmeier Reaction of 4-(4β²-benzylpiperazin-1β²-yl)-3-bromoformanilide (9) and Formation of Compound 12
In dry atmosphere, a solution of 4-(4β²-benzylpiperazin-1β²-yl)-3-bromoformamide (11) (1βg, 2.7βmmoL) in POCl3 (5βmL) was stirred for 15 minutes at 25Β°C. A solution of methyl malonyl chloride (1.12βg, 8.5βmmoL) in POCl3 (2βmL) was gradually added to the reaction mixture through a syringe. After addition was complete, the oil bath temperature was gradually raised to 130β140Β°C, and the reaction was continued for 12βh. The excess POCl3 was removed in vacuo, and the cooled black residue was dissolved in diethyl ether (20βmL), poured into ice (50βmL), and vigorously stirred for 2βh. The resulting mixture was made basic by the addition of aq. NaOH solution (30%, 10βmL), refluxed for 2βh, and cooled for 12βh in fridge (<5Β°C). Column chromatography on the resulting black gum (CHCl3β:βMeOH, 90β:β10) gave 6-(4β²-benzylpiperazin-1β²-yl)-7-bromo-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (12). It was recrystallized from EtOH to produce a yellow solid as (0.1βg, 5% yield); mp 285-286Β°C; /cmβ1 3525 (carboxylic OH), 1699 (carboxylic C=O), 1611 (COOβ st as), 1462 (COOβ st sy); (600βMHz; DMSO-) 3.13 (8H, br s, piperazine), 4.19 (2H, s, Ph-CH2), 7.45 (5H, m, Ph), 7.83 (1H, s, H-8), 8.15 (1H, s, H-5), 8.87 (1H, s, H-2), 15.21 (1H, br s, NH); 51.21 (piperazine), 60.0 (CH2), 107.4 (C-3), 115.0 (C-8), 124.4 (C-5), 124.6 (C-7), 126.1 (C-10), 128.6 and 130.6 (Ph), 136.1 (C-9), 147.3 (C-2), 166.1 (C-6), 177.3 (CO2H), 206.5 (C=O); HRMS (ESI). Found: MH+, 442.0764. Calc. for C17H20BrN3β:βMH+ = 442.0761.
2.9. Solid-Phase Synthesis with 4-fluoro-3-bromo-1-Nitrobenzene
2.9.1. Loading the Piperazine to Merrifield Resin
General Resin Preparation
The Merrifield resin (1) (5βg) was a suspension in dry DMF (20βmL) for 6β12βh. The resin had a gel-like appearance double its original volume.
To the resin suspension, a molar excess of free piperazine (5βg), pyridine (2βmL), or K2CO3 (3βg), stirred at 80Β°C for 24βh. The cold resin was then filtered and washed with water (2 Γ 20βmL), MeOH (2 Γ 20βmL), and CH2Cl2 (2 Γ 10βmL), then dried in vacuo for a minimum of 24βh or until a constant weight was achieved (5.6βg); /cmβ1 3441 (NH); (Found: C, 85.1; H, 10.4; N, 2.9%).
2.9.2. Preparation of 3-bromo-4-(Resin-Supported benzylpiperazine)-1-nitrobenzene (4)
3-Bromo-4-fluoro-1-nitrobenzene (3) (2βg) was stirred in dry DMF (10βmL), and anhydrous K2CO3 (3βg) was added to the suspended piperazine-Merrifield resin (2) (3βg), and the reaction was continued at 50Β°C for 24βh. The cold resin was filtered, then washed with water (2 Γ 20βmL), MeOH (4 Γ 10βmL), and finally with CH2Cl2 (2 Γ 10βmL). The solid was dried under vacuo for 24βh or until constant weight (4.6βg); /cmβ1 1509 and 1339 (NO2).
2.9.3. Preparation of 3-bromo-4-(4β²-Resin-Supported benzylpiperazino)-1-aniline (5)
3-Bromo-4-(4β²-resin-supported benzylpiperazine)-1-nitrobenzene 4 (2βg) was suspended in dry DMF (10βmL) for 12βh. An excess of stannous chloride (5βg) and EtOH (5βmL) was added to the resin. The resulting reaction mixture was stirred at 50Β°C for 8βh. At this time, the resin color changed from yellow to pale yellow. The cold resin was filtered and washed with water (4 Γ 20βmL). The resin was stirred in a solution of NaHCO3 (20% w/v, 20βmL), filtered, washed several times with water (2 Γ 20βmL), NaHCO3 solution (2 Γ 20βmL), MeOH (2 Γ 20βmL), and finally with CH2Cl2 (2 Γ 20βmL), and dried to give a yellow resin (1.8βg); /cmβ1 3360 (NH2).
2.9.4. Preparation of 3-bromo-4-(4β²-Resin-Supported benzylpiperazino)-1-formamide (6)
The resin-supported amine 5 (1βg) was suspended in dry DMF (10βmL) for 12βh before the addition of formic acid (5βmL). The reaction suspension was stirred and heated at 50Β°C for 2βh. the cooled reaction mixture was filtered and washed with water (4 Γ 10βmL) to remove the excess of formic acid. The resin was washed with NaHCO3 solution (30% w/v, 20βmL), MeOH (2 Γ 10βmL), and finally with CH2Cl2 (2 Γ 10βmL) to give derivatized resin 6 (1.2βg); /cmβ1 3362 cmβ1 (NH), 1721 cmβ1 (C=O).
2.9.5. Preparation of Resin-Supported 7-bromo-6-piperazino-4-oxo-3-quinolone Carboxylic Acid (7)
3-Bromo-4-(4β²-resin-supported benzylpiperazino)-1-formamide (6) (1βg) was suspended in dry DMF (10βmL) for 12βh. Phosphorus oxychloride (POCl3, 5βmL) was added to the suspended resin, and the mixture was stirred for 30 minutes at 25Β°C. A solution of methyl malonyl chloride (1.32βg, 9.6βmmoL) in POCl3 (2βmL) was gradually added to the reaction mixture. When the addition was completed, the temperature was gradually raised to 100Β°C for 24βh. After cooling, the reaction mixture was added gradually and carefully to ice (20βmL) then stirred for a further 20 minutes. The solution was basified using NaOH (10% w/v, 5βmL) and refluxed for a further 30 minutes. The resin was filtered and washed with water (2 Γ 10βmL), MeOH (2 Γ 10βmL), and finally with CH2Cl2 (2 Γ 10βmL) and dried in vacuo to constant weight (1.2βg); /cmβ1 1719 cmβ1 (C=O).
2.10. Cleavage from the Resin
2.10.1. Using the Hydrogenator
General Method
Resin-supported compound 4β7 (0.3βg) was placed in a hydrogenator vessel and suspended in dry CH2Cl2 (5βmL). Pd/C (0.05βg) was added to the resin suspension and the hydrogenation system was securely sealed. The reaction was carried out under 2βatm of hydrogen for 24βh. The reaction mixture was filtered, and the resin was washed several times with MeOH (4 Γ 5βmL); the resulting filtrates combined and the solvent was removed in vacuo to give a black residue (0.05βg). TLC showed a mixture of several spots, while the 1H NMR spectrum gave a complicated and noncharacterizable spectrum.
2.10.2. Cleavage by Catalytic Transfer Hydrogenation (Hydrogenolysis)
General Method
The resin-supported compound 4β7 (0.3βg) was suspended in dry MeOH (10βmL). Cyclohexene (5βmL) and 20% Pd(OH)2 on carbon (1β:β3 catalyst substrate by weight) was added. The suspended mixture was stirred under dry nitrogen at reflux for 12β48βh; extra cyclohexene (10βmL) was added in two portions during this reaction time, and the reaction was monitored by TLC (CHCl3β:βMeOH, 90β:β10). The reaction mixture was filtered through celite and washed with MeOH (3 Γ 10βmL). The combined filtrates were collected, dried over MgSO4, and concentrated to give a residue for characterization. None of the compounds 4β7 gave an acceptable cleavage product.
2.10.3. Cleavage by Formation of a Solid-Supported Tertiary Amine Using Alkyl Halide
General Method
The compound on resin support 4β7 (0.3βg) was swollen with a mixture of DMF (5βmL), and an excess of MeI or EtI (3-4βmL) was added; the mixture was refluxed with slow stirring for 60βh. The resin was cross-washed with MeOH (5 Γ 10βmL), CH2Cl2 (5 Γ 10βmL), and diethyl ether (10βmL). The dry resin was swollen again with morpholine (4βmL) and heated at 110Β°C for 20β40βh and then washed with MeOH (2 Γ 3βmL), and the filtrate was evaporated. The resulting solid was partitioned between CH2Cl2 (5βmL) and aqueous sodium carbonate (10%, 5βmL). Organic layers were collected, dried, and concentrated. None of the expected cleavage products was obtained.
2.10.4. Cleavage by Formation of a Solid-Supported Tertiary Amine Using Ξ±-Chloroethyl Chloroformate (ACE-Cl)
General Method
Compounds on the resin support (0.5βg) were first suspended in 1,2-dichloropropane (5βmL), followed by the addition of an excess of Ξ±-chloroethyl chloroformate (10βmL). The resulting suspension was stirred at room temperature for 48βh. The resin was filtered through a bed of silica gel, and the filtrate was then concentrated in vacuo until dryness. The residue dissolved in methanol and refluxed for 3βh. The solvent was removed to yield the secondary amines as their HCl salts.
3-Bromo-4-(4β²-resin-supported benzylpiperazine)-1-nitrobenzene (4) (0.5βg) was swollen in 1,2-dichloropropane (5βmL) for 12βh, and ACE-Cl (10βmL) was then added. The resulting suspension was stirred at room temperature for 48βh and then treated as for the general method. The resulting black residue (0.3βg) was refluxed in ethanol for 3βh, and reaction was monitored by TLC. (CHCl3:petroleum ether (40β60), 60β:β40). The TLC showed a complicated mixture of spots; the major product at was separated by preparative thin layer chromatography to give 3-bromo-4-ethoxy-1-nitrobenzene.
2.11. Preparation of N-(2-fluoro-5-nitrophenyl) piperazine (13) [15] the N,N-bis-(2-chloroethyl)ammonium chloride is very toxic and must be handled with care only in fuming hood
A mixture of 2-fluoro-5-nitroaniline (1βg, 6.4βmmoL) and N,N-bis-(2-chloroethyl)ammonium chloride (1.3βg, 7.0βmmoL) in diethylene glycol monomethyl ether (1βmL) was heated under dry nitrogen at 150Β°C for 24βh. The reaction was monitored by TLC (ethyl acetateβ:βCHCl3, 80β:β20), product , the dark solid of -(2-fluoro-5-nitrophenyl) piperazine 13 (0.87βg, 60%); mp 216-217Β°C; /cmβ1 3386 (NH), 1522 and 1346 (NO2); (300βMHz; DMSO-d6) [16] 3.26 (4H, m, CH2-3β² and CH2-5β²), 3.40 (4H, m, CH2-2β² and CH2-6β²), 7.49 (1H, dd, βHz and 12βHz, H-3), 7.85 (1H, dd, and 9βHz, H-6), 7.94 (1H, m, H-4), 9.59 (1H, br s, NH); (75βHz; DMSO-) 43.0 (C-3β² and C-5β², 47.0 (C-2β² and C-6β²), 115.2 (d, βHz, C-6), 117.8 (d, βHz, C-3), 119.2 (d, βHz, C-4), 139.9 (d, βHz, C-1), 144.9 (C-5), 158.8 (d, βHz, C-2).
2.12. Solid Phase Reaction Using p-nitrophenyl Carbonate Wang Resin 14
2.12.1. Reactions of N-(2-fluoro-5-nitrophenyl) piperazine with p-nitrophenyl Carbonate Wang Resin (14)
-Nitrophenyl carbonate Wang resin 14 (1βg, loading: 0.60β1.20βmmoL/g resin) was first suspended in dry DMF (5βmL) for 5βh, and -(2-fluoro-5-nitrophenyl) piperazine 13 (1.6βg, 7.1βmmoL), and dry pyridine (2βmL) were then added to resin. The resulting suspension was then stirred and heated to 35Β°C for 24βh. After cooling to room temperature, the resin was filtered and washed with water (2 Γ 10βmL), methanol (3 Γ 10βmL) and CH2Cl2 (3 Γ 10βmL). The resin was dried under vacuo to give a brown resin (2.3βg). The residual product was verified by the complete disappearance of the characteristic carbonate resin band at 1760βcmβ1; /cmβ1 1555 and 1316 (NO2), 1669 (C=O).
2.12.2. Resin Cleavage [17]
The nitrocarbonate resin 15 (0.2βg) was suspended in trifluoroacetic acid (2βmL), dichloromethane (2βmL) and stirred at room temperature for 3βh. The cleavage reaction was monitored by TLC [(CH2Cl2β:βMeOH, 80β:β20) on the solution, product ]. The resin was filtered and washed with CH2Cl2 (4 Γ 20βmL), and the filtrate was collected and then extracted with NaHCO3 (10%, 4 Γ 20βmL). The CH2Cl2 layers were collected, washed with brine (2 Γ 20βmL), and dried over MgSO4. The solvent was removed under vacuo to give a yellow crystal of 13 (0.1βg).
2.12.3. Reduction of N-(2-fluoro-5-nitrophenyl)piperazine-carbonate Wang Resin (15)
N-(2-fluoro-5-nitrophenyl)piperazine-carbonate Wang resin 15 (0.3βg) was suspended in anhydrous DMF (5βmL) and Et3N (2βmL). Anhydrous stannous chloride (1βg) and absolute ethanol (5βmL) were then added to the resin, and the reaction mixture was stirred at room temperature for 24βh (the color changed from deep yellow to light grey). The resin was filtered washed with methanol (20βmL), water (3 Γ 20βmL), methanol (3 Γ 10βmL), and CH2Cl2 (3 Γ 10βmL). The resin was dried to give the resin supported amine (0.34βg); /cmβ1 3401 and 3385 (NH2), 1672 (OC=O).
2.12.4. Reaction of N-(2-fluoro-5-aminophenyl) piperazine-carbonate Wang Resin 16 with Ethyl Formate
-(2-Fluoroaniline) piperazine-carbonate Wang resin (0.3βg) was suspended in dry DMF (5βmL) (the resin doubled in volume), under a positive flow of dry nitrogen, and ethyl formate was added (5βmL). The resulting mixture was stirred at 30Β°C for 24βh and, after cooling to room temperature, the resin was filtered off. TLC of the filtrate showed a spot at (CHCl3β:βMeOH, 96β:β4). The resin was washed with water (3 Γ 10βmL), methanol (3 Γ 10βmL), and CH2Cl2 (2 Γ 10βmL) to give, after drying, the corresponding formamide resin 16 (0.21βg); /cmβ1 3406 (NH) and 1685 (-C=O), 1662 (OC=O).
2.13. Preparation of 1-(benzoylpiperazinyl)-2-fluoro-5-nitrobenzene (19 )
-(2-Fluoro-5-nitrophenyl)piperazine (13) (3βg, 13.3βmmoL) was dissolved in CHCl3 (20βmL), K2CO3 (5.5βg, 40βmml) and H2O (20βmL) were added to the above solution, and benzoyl chloride (3.73βg, 26.6βmmoL) was added gradually over 20 minutes. The reaction continued at 35Β°C for 1 hour. The organic layer was separated, washed with water (3 Γ 20βmL) and brine (30βmL), dried over MgSO4, and concentrated in vacuo to give a yellow shiny crystal of 19 (4βg, 92%); mp 108-109Β°C; (Calc. for C18H18FN3O2: C, 66.0; H, 5.5; N, 12.8. Found: C, 66.1; H, 5.5; N, 12.7); /cmβ1 1694 (CO-N), 1508 (NO2), 1347 (NO2); (300βMHz; DMSO-) 2.94 (4H, s, CH22β², CH2-6β²), 3.49 (2H, s, CH2-3β²), 3.71 (2H, s, CH2-5β²), 7.46 (6H, m, Ph + H-3), 7.81 (1H, dd, and 7.5βHz, H-6), 7.92 (1H, m, H-4); (75βMHz,; DMSO-) 41.3 (C-5β²), 46.9 (C-3β²), 49.6 (C-2β² and C-6β²), 115.2 (d, βHz, C-6), 117.7 (d, βHz, C-3), 118.8 (d, βHz, C-4), 128.9 (Ph), 135.9 (C-8), 140.7 (d, βHz, C-1), 144.9 (C-5), 158.9 (d, βHz, C-2), 169.6 (C=O).
2.14. Preparation of 1-(benzoylpiperazinyl)-2-fluoro-aniline (20) [14]
1-(benzoylpiperazinyl)-2-fluoroaniline (20) (2.14βg, 78%); mp 89-90Β°C; (Calc. for C17H18FN3Oβ:βC, 68.2; H, 6.1; N, 14.0. Found: C, 68.2; H, 6.05; N, 14.0); /cmβ1 3450 and 3350 (NH2), 1724 (CO-N); (300βMHz; DMSO-) 2.94 (4H, s, CH2-2β² and CH2-6β²), 3.48 (2H, s, CH2-3β²), 3.73 (2H, s, CH2-5β²), 4.84 (2H, br s, NH2), 6.13 (1H, m, H-4), 6.25 (1H, dd, and 7.5βHz, H-6), 6.77 (1H, dd, and 12.6βHz, H-3), 7.45 (5H, m, Ph); (75βMHz; DMSO-) 50.17 (2CH2), 50.22 (2CH2), 105.5 (C-6), 107.6 (d, βHz, C-4), 116.3 (d, βHz, C-3), 128.9 (Ph), 140.0 (d, βHz, C-1), 145.9 (C-5), 147.7 (d, βHz, C-2), 169.5 (C=O).
2.15. Preparation of 1-(benzoylpiperazinyl)-2-fluoro-formanilide (21)
Formic acid (5βmL) was added to 1-(benzoylpiperazin-1-yl)-2-fluoroaniline (20) (1βg, 3.34βmmoL); the resulting solution was heated at 70Β°C for 2βh. The cooled reaction mixture was added to cooled water (100βmL), extracted with CHCl3 (4 Γ 20βmL) and brine (30βmL), and dried over MgSO4. The resulting white solid was purified by column chromatography (CHCl3β:βMeOH, 97β:β3) to give white crystals of 21 (0.62βg, 56%); mp 189β191Β°C; (Calc. for C18H18FN3O2: C, 66.0; H, 5.5; N, 12.8. Found: C, 66.0; H, 5.5; N, 12.8); /cmβ1 3080 (NH) 1684 (NH-CHO), 1620 (CO-Ph); (300βMHz; DMSO-) 3.02 (4H, s, H-2β², H-6β²), 3.55 (2H, s, H-3β²), 3.74 (2H, s, H-5β²), 7.11 (1H, dd, and βHz, H-3), 7.19 (1H, ddd, , 2.7 and 1.5βHz, H-4), 7.35 (1H, dd, and βHz, H-6), 7.46 (5H, m, CO-Ph), 8.25 (1H, d, βHz, CHO), 10.12 (1H, br s, NH); (75βMHz; DMSO-) 50.2 and 50.22 (piperazine-C), 111.1 (C-6), 113.7 (d, βHz, C-4), 116.5 (d, βHz, C-3), 128.9 (Ph), 136.3 (C-5), 140.0 (d, βHz, C-1), 153.1 (d, βHz, C-2), 159.9 (CHO), 169.6 (C=O).
2.16. Vilsmeier Reaction of 3-nitro-4-fluoroformanilide
Preparation of , -Bis-(4-fluoro-3-nitrophenyl)oxala-mide 23.
Under anhydrous conditions, 3-chloro-4-fluoroforma-nilide (2βg, 10.86βmmoL) was dissolved in dry CHCl3 (20βmL), then (COCl)2 (2βmL) was added gradually over 30 minutes, (a vigorous reaction was observed). The resulting reaction mixture was heated to 40Β°C for 30 minutes. The reaction flask was removed from the oil bath; methyl malonyl chloride (1.78βg, 13.03βmmoL) in CHCl3 (2βmL) was added gradually to the Vilsmeier reagent over 30 min. The reaction was continued at 40Β°C for 3βh until the TLC of the reaction showed a complete consumption of the starting formanilide, with the formation of a new product above the starting compound, [(CHCl3β:βMeOH, 95β:β5) ]. The reaction mixture was concentrated in vacuo, followed by the addition of cooled water (20βmL), and stirred for 30 minutes. The resulting yellow solid was collected by filtration, washed with water, and recrystallized from CHCl3 to give yellow crystals of , -Bis-(4-fluoro-3-nitrophenyl)oxalamide (23) (0.62βg, 16%); mp 109β111Β°C; (Calc. for C14H8F2N4O6: C, 45.9; H, 2.2; N, 15.3. Found: C, 45.9; H, 2.2; N, 15.3); /cmβ1 3275 (NH), 1673 (NCO); 1H NMR (300βMHz; DMSO-) 7.64 (2H, t, βHz, H-5 and H-5β²), 8.23 (2H, m, H-6 and H-6β²), 8.80 (2H, dd, and 1.5βHz, H-2 and H-2β²), 11.40 (2H, s, 2NH); (75βMHz; CDCl3) 120.1 (C-2 and C-2β²), 121.7 (d, βHz, C-5 and C-5β²), 130.9 (d, βHz, C-6 and C-6β²), 137.2 (C-1 and C-1β²), 139.1 (d, βHz, C-3 and C-3β²), 154.1 (d, βHz, C-4 and C-4β²), 161.2 (C=O).
2.17. Vilsmeier Reaction on 3-chloro-4-fluoroformanilide and Preparation of ,-Bis-(3-chloro-4-fluorophenyl)malon-amide (24)
Under anhydrous conditions, 3-chloro-4-fluoroformanilide (2βg, 12.98βmmoL) was dissolved in CHCl3 (20βmL). Oxalyl chloride (2βmL) was added gradually over 30 min. (vigorous reaction). The resulting reaction mixture was heated to 40Β°C for 30 minutes. Methyl malonyl chloride (2.13βg, 15.57βmmoL) was added gradually to the cooled reaction mixture over 30 minutes. When addition was complete, the reaction was continued at 40Β°C for 3βh until TLC showed a complete consumption of the starting formanilide with formation of a new product above the starting compound [(CHCl3β:βMeOH, 97β:β3) ]. The reaction mixture was concentrated in vacuo; cold water (20βmL) was then added and the mixture stirred for 30 minutes. The resulting yellow solid was collected by filtration, washed with water, and purified by column chromatography (CHCl3). The solid was recrystallized from CHCl3 to give shiny needle-like crystals of compound 24ββ, -Bis-(3-chloro-4-fluorophenyl)malon-amide (0.72βg, 18%); mp 201-202Β°C; (Calc. for C15H10Cl2 F2N2O2: C, 50.2; H, 2.8; N, 7.8. Found: C, 50.2; H, 2.8; N, 7.8); /cmβ1 3281 (br, NH), 1677 (C=O), 1497 (NH), 811 (Cl-C=O); (300βMHz; DMSO-) 7.38 (2H, t, βHz, H-5 and H-5β²), 7.48 (2H, ddd, , 4.5 and 9.0βHz, H-6 and H-6β²), 7.93 (2H, dd, and 2.7βHz, H-2 and H-2β²), 10.39 (2H, s, 2NH); (75βMHz,; DMSO-) 46.3 (CH2), 117.5 (d, βHz, C-5 and C-5β²), 119.7 (d, βHz, C-3 and C-3β²), 119.9 (d, βHz, C-6 and C-6β²), 121.0 (C-2 and C-2β²), 136.6 (C-1 and C-1β²), 153.7 (d, βHz, C-4 and C-4β²), 165.9 (C=O).
2.18. Physicochemical Studies
The physicochemical studies include the lipophilicity, Fourier transforms infrared spectroscopy, and the thermal stability of highly bioactive compounds. The thermal behaviors for the bioactive compound 12 was investigated by thermogravimetric technique and indicated by the TGD peaks at 177 and 270 (Figures 3 and 4). The highly bioactive pure tested compounds were also determined like melting point, water solubility and pKa values.
2.19. Antimicrobial Assay
Some synthesized compounds, 19, 20, 21, 9, 11, 23, and 12, were evaluated for their antimicrobial effects by Agar diffusion disk method [18] using Nutrient Agar, MacConkey Agar, and Sabouraud Dextrose Agar. The potentialities of these compounds were estimated against some important and representative microbes like Gram + ve: Bacillus Subtilis (B.S.); Staphylococcus Aureus (S.A.), Gram βve: Escherichia Coli (E.C.); Klebsiella Pneumonia (K.P.), and Fungi: Candida Albicans (C.A.); Aspergillus Funigates (A.F.). The presterilized filter paper disks (6βmm diameter) were impregnated with 30, 40, and 50βΞΌg of the compound and dissolved in DMF as solvent, which has no effect on either bacteria or fungi. These disks were implanted on different sets of agar plates containing the microbes. The agar plates were then incubated for 24 hours at 37Β°C for bacteria and for 7 days at 28Β°C for fungi. Nalidixic acid and nystain were used as reference antibiotics.
In addition, similar antimicrobial assay was performed for the biologically highly active compounds 20, 21, 11, 23, and 12 after exposure of the Petridishes containing microorganisms and the test compounds to UV light (Ξ»366βnm) for 3 hours before the incubation.
3. Results and Discussion
3.1. Synthesis of Novel Norfloxacin Analogues
In the present study, novel norfloxacin analogues were synthesized using basically the Vilsmeier method with some modifications. The 7-bromo-6-N-benzyl piperazinyl-4-oxoquinoline-3-carboxylic acid (12) was isolated at high temperature (mention the temperature). On the other hand, bis-compounds -bis-(4-fluoro-3-nitrophenyl)-oxalamide and -bis-(3-chloro-4-fluorophenyl)- malonamide (22) and (23) were obtained under reveres Vilsmeier approach using the modified method of commercially available Merrifield resin 14, which was modified by introduction of spacer with free hydroxyl group to enhance the activity of the substrates bound to the polymer. Besides the determination of their physiochemical properties, these compounds were evaluated for use in vitiligo and as antimicrobial agents.
Isolation of two novel , -bis-(aryl) compounds 23, 24 instead of norfloxacin analogue targets could be due to a type of interaction between oxalyl chloride with methyl malonyl chloride followed by monoacylation of anilidimide which hinders the formation of norfloxacin analogues via a second interaction with other anilidimide molecule (Scheme 4). Recently, nonfluorinated , -bis-aryl derivative was reported as an HIV-1 integrase inhibition [19].
3.2. Physiochemical Properties
3.2.1. Lipophilicity
The lipophilic and Zwitterionic form of the obtained compounds, as well as steric and electronic effects or charge density, plays an important role for chemical and biocidal activities. -Mannich base functional group can increase the lipophilicity of the tested compounds, for example, 12 at physicobiological pH values by decreasing their protonation resulting in the enhancement of absorption through biomembranes. It is clear that the neutral species of haloquinolones are more lipophilic than Zwitter ionic form. In addition, steric and electronic effects or molecular charge density can affect lipophilicity (Scheme 5).
3.2.2. Fourier Transforms Infrared Spectroscopy
Generally, Fourier transforms infrared spectroscopy (FT-IR) studies of the obtained compounds in both the solid and solution (CHCl3) states showed lack of some characteristic bands in the solution state, for example, compound 12 (Figure 2). This effect may be due to a type of intramolecular and/ or intermolecular H-bonding between functional group of the tested compounds and a functional group in the solvent used, which possibly act similarly to the functional groups of the organisms leading to inhibition of their vital activities and death. The results of the Fourier transform infrared spectroscopy are given in Figure 1.
(a) Solid state
(b) Solution state
3.2.3. Other Physicochemical Properties of Highly Bioactive Compounds
The physicochemical properties of highly bioactive pure tested compounds are demonstrated as follows. (a)Melting Points. They differ according to the type of solvent from which crystals are obtained, for example, compound 20 had approximately 87Β°C for pure crystallized from cyclohexane, and 90Β°C from chloroform.(b)Solubility in Water. Pure compound 20, for example, gave approximately, 200βΞΌg/L while compound 23 showed 350βΞΌg/L at 20Β°C.(c)Pka. Pure tested compounds at pH 5.7 and 9 at 24Β°C showed different types of protons, in quinolone the βCOOH and NH, while in the formylamino derivative, βCOOH, βCHO, and NH. This data indicated that tested compounds 20, 21, 11, 23, and 12 have a very low rate of hydrolysis because of its stability in suspension concentration under normal conditions Table 4.
3.3. Antimicrobial Assay
The potentialities of the tested compounds 19, 20, 21, 9, 11, 23, and 12 are given in Tables 1, 2 and 3.
3.4. Photochemical Probe Agents
Vitiligo is an acquired disorder characterized by patchy progressive depigmentation of the skin. It affects about 2% of world population. Vitiligo occurs equally in both sexes and has no age limits. It may be presented as a single path, which may be progressing or static for a long time and suddenly starts progressing or multiple patches, which are slowly progressing or stationary indefinitely. These depigmented molecules sometimes spontaneously pigment and depigment again and are often symmetrical and are called as vitiligo vulgarize. The etiology of nonsymmetrical Vitiligo, namely, segment vitiligo, is entirely different from symmetrical vitiligo. Often the exposed areas of the skin and areas around orifices of the body are depigmented rather than other areas [20]. The melanocytes successfully treated vitiligo patients by PUVA therapy [21]. Increasing use of PUVA-8MP could be responsible for a type of skin cancer [22]. Thus, some antibiotics like nalidixic acid and Nystatin are now used to control the vitiligo symptoms.
Acknowledgments
The authors would like to thank Professor O. Meth-Cohen, Professor P. Groundwater, Professor R. Anderson, and Dr. Amal Alkordy from Sunderland University, UK. Thanks are extended to Professor Zenib M. ElBaza and coworkers, Department of Pharmaceutical Microbiology, National Center for Radiation Research and Technology, Nasr City, Egypt. The authors are also grateful to the Deanship Research, King Abdul-Aziz University, Jeddah, Saudi Arabia, for the financial support.