Synthesis, Physiochemical Properties, Photochemical Probe, and Antimicrobial Effects of Novel Norfloxacin Analogues

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 N, N′-bis-(4-fluoro-3-nitrophenyl)-oxalamide and N, N′-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.


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. Npiperazinyl derivatives of fluoroquinolones were introduced and demonstrated for various biological activities that possess broad-spectrum activity [2][3][4][5][6]. Furthermore, it is clear that the neutral species of fluoroquinolones are more lipophilic than the Zwitterionic form. Therefore, factors that can affect N-protonation like steric and electronic effect or charge density can also affect lipophilicity [7][8][9].
Procopiou et al. [10] prepared a series of asymmetrical 1,4-disubstituted piperazines as a novel class of non-brainpenetrant histamine H3 receptor antagonists. In addition, Foroumadi et al. [11] synthesized a modified norfloxacin via heteroarylation of norfloxacin on N-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.

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 CDCl 3 on a Bruker AVANCE 300 at 300 MHz. 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.

Using the Hydrogenator
General Method. Resin-supported compound 4-7 (0.3 g) was placed in a hydrogenator vessel and suspended in dry CH 2 Cl 2 (5 mL). Pd/C (0.05 g) was added to the resin suspension and the hydrogenation system was securely sealed. The reaction was carried out under 2 atm of hydrogen for 24 h. The reaction mixture was filtered, and the resin was washed several times with MeOH (4 × 5 mL); the resulting filtrates combined and the solvent was removed in vacuo to give a black residue (0.05 g). TLC showed a mixture of several spots, while the 1 H NMR spectrum gave a complicated and noncharacterizable spectrum.

Cleavage by Catalytic Transfer Hydrogenation (Hydrogenolysis)
General Method. The resin-supported compound 4-7 (0.3 g) was suspended in dry MeOH (10 mL). Cyclohexene (5 mL) and 20% Pd(OH) 2 on carbon (1 : 3 catalyst substrate by weight) was added. The suspended mixture was stirred under dry nitrogen at reflux for 12-48 h; extra cyclohexene (10 mL) was added in two portions during this reaction time, and the reaction was monitored by TLC (CHCl 3 : MeOH, 90 : 10). The reaction mixture was filtered through celite and washed with MeOH (3 × 10 mL). The combined filtrates were collected, dried over MgSO 4 , and concentrated to give a residue for characterization. None of the compounds 4-7 gave an acceptable cleavage product.

Cleavage by Formation of a Solid-Supported Tertiary Amine Using Alkyl Halide
General Method. The compound on resin support 4-7 (0.3 g) was swollen with a mixture of DMF (5 mL), and an excess of MeI or EtI (3-4 mL) was added; the mixture was refluxed with slow stirring for 60 h. The resin was crosswashed with MeOH (5 × 10 mL), CH 2 Cl 2 (5 × 10 mL), and diethyl ether (10 mL). The dry resin was swollen again with morpholine (4 mL) and heated at 110 • C for 20-40 h and then washed with MeOH (2 × 3 mL), and the filtrate was evaporated. The resulting solid was partitioned between CH 2 Cl 2 (5 mL) and aqueous sodium carbonate (10%, 5 mL). Organic layers were collected, dried, and concentrated. None of the expected cleavage products was obtained.

Cleavage by Formation of a Solid-Supported Tertiary Amine Using α-Chloroethyl Chloroformate (ACE-Cl)
General Method. Compounds on the resin support (0.5 g) were first suspended in 1,2-dichloropropane (5 mL), followed by the addition of an excess of α-chloroethyl chloroformate (10 mL). The resulting suspension was stirred at room temperature for 48 h. 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 3 h. The solvent was removed to yield the secondary amines as their HCl salts. 3-Bromo-4-(4 -resin-supported benzylpiperazine)-1nitrobenzene (4) (0.5 g) was swollen in 1,2-dichloropropane (5 mL) for 12 h, and ACE-Cl (10 mL) was then added. The resulting suspension was stirred at room temperature for 48 h and then treated as for the general method. The resulting black residue (0.3 g) was refluxed in ethanol for 3 h, and reaction was monitored by TLC. (CHCl 3 :petroleum ether (40-60), 60 : 40). The TLC showed a complicated mixture of spots; the major product at R f = 0.34 was separated by preparative thin layer chromatography to give 3-bromo-4-ethoxy-1-nitrobenzene. (13) [15]  1 mmoL), and dry pyridine (2 mL) were then added to resin. The resulting suspension was then stirred and heated to 35 • C for 24 h. After cooling to room temperature, the resin was filtered and washed with water (2 × 10 mL), methanol (3 × 10 mL) and CH 2 Cl 2 (3 × 10 mL). The resin was dried under vacuo to give a brown resin (2.3 g). The residual product was verified by the complete disappearance of the characteristic carbonate resin band at 1760 cm −1 ; ν max /cm −1 1555 and 1316 (NO 2 ), 1669 (C=O).

Vilsmeier Reaction on 3-chloro-4-fluoroformanilide and Preparation of N,N -Bis-(3-chloro-4-fluorophenyl)malonamide (24)
. Under anhydrous conditions, 3-chloro-4fluoroformanilide (2 g, 12.98 mmoL) was dissolved in CHCl 3 (20 mL). Oxalyl chloride (2 mL) was added gradually over 30 min. (vigorous reaction). The resulting reaction mixture was heated to 40 • C for 30 minutes. Methyl malonyl chloride (2.13 g, 15.57 mmoL) was added gradually to the cooled reaction mixture over 30 minutes. When addition was complete, the reaction was continued at 40 • C for 3 h until TLC showed a complete consumption of the starting formanilide with formation of a new product above the starting compound [(CHCl 3 : MeOH, 97 : 3) R f = 0.53]. The reaction mixture was concentrated in vacuo; cold water (20 mL) 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 (CHCl 3 ). The solid was recrystallized from

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 • C (Figures 3  and 4). The highly bioactive pure tested compounds were also determined like melting point, water solubility and pKa values. The presterilized filter paper disks (6 mm diameter) were impregnated with 30, 40, and 50 μg 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 37 • C for bacteria and for 7 days at 28 • 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 (λ366 nm) for 3 hours before the incubation.

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 N,Nbis-(4-fluoro-3-nitrophenyl)-oxalamide and N,N -bis-(3chloro-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 N, N-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 N, N-bis-aryl derivative was reported as an HIV-1 integrase inhibition [19].

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. N-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).

Fourier Transforms Infrared Spectroscopy.
Generally, Fourier transforms infrared spectroscopy (FT-IR) studies of the obtained compounds in both the solid and solution (CHCl 3 ) 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. (c) Pka. Pure tested compounds at pH 5.7 and 9 at 24 • C showed different types of protons, in quinolone the -COOH and NH, while in the formylamino derivative, -COOH, -CHO, 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.

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.