Synthesis and Antimicrobial Activity of New 4-Heteroarylamino Coumarin Derivatives Containing Nitrogen and Sulfur as Heteroatoms

ynthesis, spectral analysis and bioactivity of new coumarin derivatives are described in this paper. Eight new coumarin derivatives were synthesized in moderate to good yields by condensation of 4-chloro-3-nitrocoumarin and the corresponding heteroarylamine. The synthesized compounds were tested for their in vitro antimicrobial activity, in a standard disk diffusion assay, against thirteen strains of bacteria and three fungal strains. They have shown a wide range of activity - from one completely inactive compound to medium active ones.


Introduction
Coumarin derivatives have been shown to possess a remarkably broad spectrum of biological activity including antibacterial [1,2], antifungal [3][4][5], anticoagulant [6], anti-inflammatory [7], antitumor [8,9] and anti-HIV [10] activity. In addition, these compounds are used as additives in food and cosmetics [11], dispersed fluorescent brightening agents and as dyes for tuning lasers [12]. Main representatives of the class are the hydroxyl derivatives, 4-and 7-hydroxycoumarins, also biologically active and very important for the synthesis of other coumarin derivatives.
On the other hand, the nitrogen and sulfur heterocyclic system families are very interesting due to their physicochemical properties, especially in the sense of design of new drugs and new materials. The chemistry and pharmacology of thiazole derivatives has been of great interest to medicinal chemists lately [13]. The pyrazole ring is a prominent structural moiety found in numerous pharmacologically active compounds. Pyrazole-based derivatives have been regarded as anxiolytics [14], GABA receptor antagonists and insecticides [15], potential PET ligands for CB1 receptors [16], anti-inflammatory, antimicrobial [17], and growth inhibition agents [18].
In continuation of our ongoing interest in synthesis of the new coumarin derivatives [20][21][22][23][24], and having in mind the above considerations, we have been prompted to synthesize new, possibly more potent, pharmacologically active compounds. We decided to combine the coumarinic system with the above named groups of compounds in hope that the resulting novel heterocycles would be biologically active. Additionally, a recent QSAR study of the antimicrobial activity of some 3-nitrocoumarins has put forward some new arguments in this direction [19]. In connection with our previous work [20][21][22][23][24], in the present paper we report on the synthesis of novel 4-heteroarylamino-3-nitrocoumarin derivatives and the screening of their in vitro antimicrobial activity.
The synthesized compounds 5a-h were screened for their in vitro antimicrobial activity against thirteen strains of bacteria and three fungi strains using a disk diffusion assay [20,25]. The compounds were tested at the dose of 500 μg per disk [50 μL of the samples solutions (10 mg/mL) in DMSO]. Measured susceptibility zones to the nearest mm were the clear zones around the disk inhibiting the microbial growth. The obtained results are listed in Table 2. As it can be seen, the prepared compounds possess a wide range of activities -from a completely inactive compound 5e to medium active ones. The synthesized compounds 5a-h showed no special selectivity towards any particular microorganism, although the bacterial strains showed greater susceptibility. Also, the activity of the synthesized compounds was slightly higher against Gram-positive compared to Gram-negative bacteria. The largest inhibition zones were noted for S. aureus and S. lutea . On the other hand, the most resistant strain was S. cerevisiae, being almost completely unsusceptible to the tested compounds. Considering the antimicrobial effect towards the different strains of the same microorganism, in the case of E. coli ATCC 8739 and E. coli ATCC 25922, the latter was less resistant to all compounds. Two different strains of P. aeruginosa exhibited similar susceptibility to the tested compounds. The most active compounds were 5c, 5g and 5h, showing reduction of bacterial and fungal growth comparable with the one exhibited by the standards used as positive control (tetracycline and nystatine), especially against medically important pathogens, though in a much larger dose. The lack of susceptibility of all tested microorganisms toward 5e was probably the consequence of sterically-hindered nitrogen atoms of the pyrazole ring. In order to make the discussion more easy to follow and the conclusions statistically supported, we performed agglomerative hierarchical clustering (AHC) on the mentioned samples (Table 2), using the Excel program plug-in XLSTAT version 2008.6.07. The method was applied utilizing the values of diameters of growth inhibition zones as original variables without any recalculation. The results of AHC are presented in Figure 1. AHC was performed using Pearson dissimilarity (as aggregation criteria simple linkage, unweighted pair-group average and complete linkage were used) and Euclidean distance (aggregation criterion: weighted pair-group average, unweighted pair-group average and Ward's method). The definition of the groups was based on Pearson correlation, using complete linkage and unweighted pair-group average method. AHC analysis has clearly indicated the existence of four groups of compounds under study (designations of the compounds were given in Scheme 1). Compound 5e showed no activity at all towards the tested microorganisms, and was not included in the AHC analysis. A single compound from the first group, 5h, is distinguished from the rest of the compounds. Structurally speaking, in 5h the indazole substituent was connected to the coumarin moiety in such a way that the larger distance of the indazole nitrogen atoms from the nitro-group of coumarin moiety might be responsible for the observed activity. No correlation between the structure of the compounds and the observed antimicrobial activity can be drawn out for the compounds placed in the three remaining groups. The marked antimicrobial activity of the synthesized compounds makes the 4-arylamino-3-nitrocoumarin derivatives, with the nitrogen and sulfur as heteroatoms, interesting for further investigation and shows that they are a good basis for the synthesis of new, potentially more physiologically active compounds.

General
Melting points were determined on a Kofler hot-plate apparatus and are uncorrected. HRMS(EI) spectra were recorded on a Finnigan-MAT 8230 BE mass spectrometer. The IR measurements (ATRattenuated total reflectance) were carried out with a Thermo Nicolet model 6700 FTIR instrument. The NMR spectra were recorded on a Varian Gemini 200 spectrometer( 1 H-NMR at 200 MHz, 13 C-NMR at 50 MHz), using DMSO-d 6 as the solvent. Chemical shifts are expressed in δ (ppm) using TMS (Me 4 Si) as the internal standard. For TLC, silica gel plates (Kiesel 60 F 254 , Merck) were used. Visualization was affected by spraying the plates with 1:1 aqueous sulfuric acid and then heating. All the reagents and solvents were obtained from commercial sources (Aldrich, USA; Merck, Germany; Fluka, Germany) and used as received, except that the solvents were purified by distillation.

Synthesis of 4-chloro-3-nitrocoumarin 3
According to the previously published procedure [26], 4-hydroxycoumarin (1) was nitrated in glacial AcOH with 72% HNO 3 to afford 4-hydroxy-3-nitrocoumarin (2). Starting compound 3 was prepared from 4-hydroxy-3-nitrocoumarin (2) following the method of Kaljaj et a l. [27]. The preparation was carried out in the following manner: N,N-dimethylformamide (DMF, 2 mL, 26 mmol) was cooled to 10 ºC in an ice bath. With stirring, POCl 3 (4 g, 26 mmol) was added dropwise, and the obtained mixture was stirred for an additional 15 min. Then, the ice bath was removed and the reaction was left to proceed at room temperature for a further 15 min. Finally, the solution of 4-hydroxy-3nitrocoumarin (2, 5.4 g; 26 mmol) in DMF (12.5 mL) was added dropwise. After 15 minutes of stirring, the reaction was stopped by adding cold water (15 mL). The precipitated solid was collected by filtration and washed with saturated sodium-bicarbonate solution and water. Recrystallisation from the mixture of benzene-hexane (1:1 volume ratio) yielded yellow crystals of 3 (5.1 g; 22.6 mmol) in 87% yield, mp 162-163 ºC. The procedure was repeated twice.

General procedure for the synthesis of 4-heteroarylamino-3-nitrocoumarins 5a-h
A solution of 4-chloro-3-nitrocoumarin (3, 1 g, 4.4 mmol) and the appropriate heteroarylamine 4ah (4.4 mmol) in ethyl acetate (10 mL) was refluxed in the presence of triethylamine (1 mL, 7.2 mmol) for 3-6 h. After cooling, the precipitated solid was filtered off, washed with ethyl acetate and water. The purity of the synthesized compounds was checked by TLC.     and Virology ''Torlak", 30 μg of the active component, diameter 6 mm) were used individually as positive controls, while the disks imbued with 50 μL of pure DMSO were used as a negative control. The diameters of the inhibition zones were measured in millimeters (to the nearest mm) using a ''Fisher-Lilly Antibiotic Zone Reader" (Fisher Scientific Co., USA). Each test was performed in quintuplicate. In order to evaluate statistically any significant differences among mean values, a oneway ANOVA test was used. In all tests the significance level at which we evaluated critical values differences was 5%.

Conclusions
In summary, this paper describes the synthesis, spectral characterization and screening of antimicrobial activity of some new 4-arylamino-3-nitrocoumarin derivatives. 4-Chloro-3-nitrocoumarin proved to be a good electrophilic substrate in these reactions. The synthesized compounds showed a wide range of potentially promising antimicrobial activities. The notable antimicrobial effect of certain compounds confirms that these are a good basis for the production of a number of new, possibly physiologically active coumarin derivatives.