Synthesis and biological evaluation of О -acyloximes of 5-chloro-4-formyl- 1 Н -pyrrol-3-carboxylates as antimicrobial agents

Aim. Elaboration of effective methods for the synthesis of the polysubstituted oximes with the O -acyloxime groups and investigation of their antibacterial and antifungal activity. Methods. Organic synthesis, analytical and spectral methods, pharmaceutical screening. Results. A series of new O -acyloximes of 4-formylpyrroles has been synthesized, and the screening of their antibacterial and antifungal activity was performed. It was found that the synthesized compounds exhibit antimicrobial activity, and their minimum inhibitory concentration (MIC) is ranged between 7.81 and 125 μg/mL. A comparatively high antibacterial activity has been registered for some synthesized compounds against the gram-negative bacteria of the genus Proteus (МIC=7.8-62.5 μg/mL). Conclusions. The most active antibacterial О -acyloximes were identified among the array of the synthesized substances, and the MIC of the compound 10 consisting of an m -nitrobenzoilic fragment against the bacterial test strains Proteus aeruginosa АТСС 27853 and Proteus mirabilis АТСС 410 was 15.625 μg/mL. In the case of the latter strain, this value is close to the MIC value of the control drug. The MIC of the compound 9 against the bacterial strain Proteus mirabilis АТСС 410 was 7.81 μg/mL, which is greater than the corresponding control MIC.

cally active agents [1,2] and as the target compounds exhibiting a distinct biologic activity. In the latter context, the following agents can be mentioned: inhibitors of the N-end kinases [3], selective covalent inhibitors of serine hydrolase [4], the antioxidant [5], antiinflammatory [6] and herbicide [7] agents. Special attention is also paid to the О-acyloximes exhibiting high antibacterial and antifungal activity. For instance, it was found that the antibacterial activity of aroyloxime I against the bacteria Е. coli and P. aeruginosa was close to the activity of streptomycin [5], whereas its fungicide efficiency against A. flavus was higher than that of the antifungal medication fluconazole. The antimicrobial screening of the acyloxime of benzaldehyde II regarding the strains of S. аureus and E.coli proved that its activity was close to the acti vity of the test drug bromheramin [8]. The bacteriostatic activity of the О-aroyloxime III concerning Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli was sufficient to stop their proliferation, and its MIC was 1.562 μg/mL that is close to the efficiency of penicillin [9]. As seen from the published scientific papers, only the derivatives of simple pyrrole-2and 3-carbaldehydes with an O-acylated oxime fragment were investigated previously. Among them, there are the inhibitors of the lipoprotein-associated phospholipase А2 [10], the BODIPY-based photoelectronic platforms for the transportation of valproic acid (VPA) known as an inhibitor of histone diacetylase and an inductor of the tumor cell apoptosis [11]. Many О-acyloximes are used also as synthetic blocks in the designing of the condensed pyrrole-containing systems [12,13].
However, further structural modification of functionalized pyrroles by the acyloxime fragment seems reasonable. This assumption is based on the fact that such pyrroles are the key components of a wide array of important natural and synthetic compounds used as a promising scaffold for further transformation into various bioactive agents [14][15][16].
A wide-scale proliferation of tuberculosis bacteria, Staphylococcus aureus, and non-fermenting gram-positive germs of multidrug resistance is one of the most acute problems in today's public health system [17][18][19]. Besides, the lack of efficacious antifungal drugs and the rising resistance of the fungal pathogens also require intense counter-efforts [20][21][22]. That is why, the research and development of new highly active wide-range antimicrobial/antifungal agents seem a promising direction to address the problem of germ multidrug resistance.
Therefore, our investigation was planned in the framework of the search for new antimicrobial agents. It dealt with the elaboration of the effective methods of synthesis of the polysubstituted pyrroles with the O-acyloxime group and the study of their antibacterial and antifungal properties.

Chemistry
Melting points were measured on a Kofler melting point-device and are uncorrected. IR spectra were recorded on Bruker Vertex 70 FT-IR spectrometer for samples in KBr pellets. 1 H NMR spectra were acquired in pulsed Fourier transform mode on a Varian VXR-400 spectrometer (400 MHz), whereas 13 CNMR spectra were acquired on a Bruker Avance DRX-500 spectrometer (125 MHz), using DMSO-d 6 as a solvent. Mass spectra were recorded on an Agilent LC/MSD SL chromatograph equipped with Zorbax SB-C 18 column (4.6x15mm), particle size 1.8 µm (PN 82(c)75-932), solvent DMSO, electrospray ionization at atmospheric pressure. Elemental analysis was performed on a PerkinElmer 2400 CHN Analyzer. The individuality of the obtained compounds was monitored by TLC on Silutol UV-254 plates.
Common experimental methods (Method 1). A mixture of 5 mmol of the corresponding carboxylic acid, 0.81 g (5 mmol) of 1,1-carbodiimidazole (CDI) and 100 mg of 4-dimethylenaminepyridine (DMAP) was added to a solution of 5 mmol of the oxime 1 in 10 mL of methylene chloride. The system was stirred at room temperature for 10 h, then the solvent was vacuum evaporated, and 10 mL of water were added to the remaining solid residue. The sediment was filtered out, dried and recrystallized from the 70% ethanol.
(Method 2). 0.89 g (7.5 mmol) of thionyl chloride were added to a solution of 5 mmol of the corresponding carboxylic acid in 10 mL of toluene, and then the mixture was boiled for 1 h. The solvent was vacuum evaporated, and 10 mL of acetonitrile, 5 mmol of the oxime 1 and 0.51 g (5 mmol) of triethylamine were added to the solid residue. The mixture was boiled for 1 h, and then the solvent was vacuum evaporated. Finally, 10 mL of water were added to the residue, and then the sediment was filtered out, dried and recrystallized from the 70% ethanol. The 1000 μg/ml DMSO solutions of all the compounds to be researched were prepared and then involved in experiments according to the serial dilutions micromethod. All the experiments were repeated three times until the relevant and not-contradictory data were obtained. DMSO was used as a reference system, whereas Decasan was used as a control drug in the investigation of antimicrobial efficiency.

Chemistry
This investigation was aimed at the elaboration of effective methods of the synthesis of new O-acyloximes of pyrrol-4-carbaldehydes with an additional functionalization by the etoxycarbonile group in the 3 rd position and by chlorine -in the 5 th position. Besides, all synthesized compounds were checked for their possible application as antimicrobial agents. Two preparatively convenient methods of acylation of the previously synthesized oximes of 4-formylpyrroles 1 [24] were applied to obtain the target compounds. The first of them implied an acylation by carboxylic acids in the presence of carbodiimydazole (CDI) and 4-dimethylaminopyridine (DMAP). It was shown that the  O-acyloximes 2, 5, 6, 8, 10, 11 were synthesized with the yields of 86-94 % after the 10hour stirring of corresponding reactants in dichloromethane at room temperature. According to the second method, the acylation was performed in situ by chloranhydrides of carboxylic acids interacting in boiling toluene with the oximes 1 in the presence of triethylamine acting as a base and leading to the O-acyloximes 3, 4, 7, 9, 12 with the yields of 85-92 %.
The structures of the synthesized compounds 2-12 were confirmed by NMR 1 Н, 13 С and chromato-mass spectra. In particular, the singlets of the azomethine group protons are present in the NMR spectra in the range 8.68-9.05 m.n. along with the specific signals of the substitutes R 1 and R 2 .

Antimicrobial activity
As seen from the results of bioscreening of the O-acyloximes 2-12, they exhibit some antimicrobial activity, and their MIC is ranged between 7.81-125 μg/mL (Table 1). It is also clear that their antifungal activity is lesser than the antibacterial efficiency, and this activity does not depend on the structure of acylic oxime fragment (МІC=31.25-62.5 μg/mL). Unlike the antifungal activity of the compounds, their antibacterial effect differs significantly depending on the type of germs and against the test strain Proteus mirabilis АТСС 410 was 7.81 mg/mL, which is higher than the control value. Taking into consideration the noticeable antibiotic resistance of these strains [25], the abovementioned results seem important for further detailed investigations in this direction.

Conclusions
The preparatively convenient methods for the synthesis of О-acyloximes of pyrrol-4-carbaldehydes 2-12 with an additional functionalization in the 3 rd position by an etoxycarbonile group and in the 5 th position -by chlorine are proposed and discussed. As seen from the bioscreening data, the target compounds show quite high antimicrobial activity against the gram-negative bacteria of the genus Proteus (МIC=7.8-62.5 mg/mL). It has been found that the MIC of the compound 10, consisting of an m-nitrobenzoilic fragment, against the test strain of bacteria Proteus aeruginosa АТСС and 27853 Proteus mirabilis АТСС 410 was 15.625 mg/mL. For the latter strain, this value is close to the activity of the control drug. * proliferation of bacteria detected ** Decasan (a solution consisting of 0.2 mg/mL of decamethoxin) made by "Yuria-Pharm" was used as the control drugs