Synthesis and evaluation of antimicrobial activity of some new 3-(pyrrol-4-yl)acrylamide derivatives

.


Introduction
It is reported by WHS that a microbial resistance is one of the most challenging global threats to public health. 1As a bacterial multi-resistance grows in various strains of gram-positive bacteria including the methicillin-resistant bacteria of the genus Staphylococcus, penicillin-resistant bacteria of the genus Streptococcus, and vancomycin-resistant Enterococcus, 2 the search for new effective antibacterial compounds becomes more and more topical.Besides such bacterial resistance, a fungal resistance also grows rapidly in many fungal pathogens, which brings new challenges for the health protection system. 3That is why, the construction of new anti-germ compounds, including those based on the heterocyclic molecules, keeps its topicality.
In the framework of the development of the highly efficient antimicrobial agents based on the polyfunctional and condensed pyrrole systems, [4][5][6] this work represents the results of the investigation of some previously unknown amides of acrylic acid consisting a functionalized pyrrole fragment in the alkenylic part of the molecule.Even though the information related to this type of compounds is very scarce, [7][8][9] special attention should be given to the recently published method of synthesis of such compounds that is based on the Ru(0)-catalyzed alkenization in the β-position of α-iminofunctionalized pyrroles 10 .2][13][14][15] On the other hand, another type of benzoanalogs of the above unknown amides, derivatives of indolyl-3-acrylamides are known as pharmaceutically active chemicals.For instance, they can be used as the inhibitors of hepatitis C virus (HCV), 16 human immune deficit virus (HIV), 17 human deacylglycerol acyltransferase-2 (DGAT-2), 18 and also as antiproliferation agents. 19,20nce the pyrrole scaffold is important for the construction of various bioactive compounds, [21][22][23] the synthetically potent formyl and ester groups, and a chlorine atom as a pharmacophoric substitute should be present in the basic pyrrole substrates to be transformed into further pharmaceutical agents.It should be emphasized that the pharmaceutical effectiveness of organic compounds increases if they consist of chlorine. 24,25This effect is based on the improved absorption, 26 and allocation of the biotargets in the hydrophobic 'pockets' 27 .A series of esters of 5-chloro-4-formylpyrrol-3-carboxylic acids 1a-e recently synthesized by us 28 and the authors of 29 completely satisfies the above requirements and that is why these compounds were used for the synthesis of the target pyrrolacrylamides.As shown before, the compounds of class 1 can be effectively used as synthetic units in construction of some pyrrol[2,3-b]quinoline 30 and pyrrol [3,4-b;3',4'-d]pyridine 31 systems.

Synthesis and spectra characteristics
We have developed a preparative simple method of synthesis in which the esters of 5-chloro-4-formylpyrrol-3-carboxylic acids 1a-e are transformed in the corresponding acrylamides (Fig. 1).At the first stage, the compounds 1a-e interact with malonic acid by the Knoevenagel reaction and form the respective 3-(pyrrol-4-yl)acrylic acids 2a-e which yields 86-93 %.Their IR spectra reveal the medium intensity absorbance bands corresponding to the groups С=С (1639-1645 sm -1 ), carboxylic С=О (1700-1710 sm -1 ), carboxylatic C=O (1720-1730 sm -1 ), and wider absorbance bands of the carboxylic О-Н (2524-2832 sm -1 ).The peaks related to the pyrrole-like derivatives, the carboxylic group singlets at 12.15-12.25ppm, and the doublets of С 2 Н= and С 3 Н= protons in the ranges 6.38-6.50 and 7.98-8.04ppm respectively were found in the 1 Н-NMR spectra of the above compounds recorded for J = 16.0Hz.It is a proof of the trans-configuration of a pyrrole fragment and a carboxylic group in the regard of a double bond.
At the second stage, chloroanhydrides of the acids 2a-e were synthesized by a 4 h long boiling of the respective acids in a benzene solution of excessive thionyl chloride.Further, the raw uncleaned chloroanhydrides reacted with aromatic or aliphatic amines in a boiling acetonitrile solution of triethylamine.This process lasted during 3 h.As a result the amides 3a-l of 3-(pyrrol-4-yl)acrylic acid were obtained with yields 72-94% (Table 1).Even though a pyrrole fragment of the intermediate chloroanhydrides consists of an electrophilic ester group, the reactions with substituted anilines and more basic aliphatic amines remain regioselective and involve only chlorocarbonyl function.AcONa, Py, Δ. 12h   Structural composition of the amides 3a-l was confirmed by their spectral characteristics.For instance, the absorption bands of the valent asymmetric oscillations of bonds C=C (1634-1649 sm -1 ), amide C=O (1665-1676 sm -1 ), and ester C=O (1720-1728 sm -1 ) were found in their IR spectra.The NH absorption band of monosubstituted amides 3a, b, d, e, h-k was registered at 3240-3254 sm -1 .The doublet peaks of C 2 H= and C 3 H= protons were registered in the 1 H-NMR spectra of synthesized compounds 3a-l at 6.73-7.19 and 7.94-8.23ppm for J = 16.0Hz.Thus, the introduction of aromatic amines in the structure of compounds 3 does not cause any changes in the shift of the С 3 Н= protons, and leads to a weak-field shift of the С 2 Н= protons on average by 0.3 ppm on average as compared to acids 2. On the contrary, the similar effect caused by the introduction of cycloalkylamino groups (compounds 3с, f, g) is more significant, and it causes a shift of the abovementioned protons towards a weaker field by 0.6-0.7 ppm.

Antimicrobial activity
The 10 μg/ml solutions of synthesized amides 3a-l have been evaluated for antimicrobial activity on some archive strains and clinical isolates of antibiotic-sensitive (MS) and antibiotic-resistant (MR) microorganisms including gram-positive Staphylococcus aureus MR and MS, Staphylococcus haemolyticus MR, Staphylococcus epidermidis MS, Bacillus subtilis, Streptococcus pyogenes, Streptococcus oralis, Streptococcus gordonii; gram-negative Klebsiella pneumonniae and Escherichia coli ATCC 35218, and fungi Candida albicans and Candida tropicalis.Chlorhexidine 32 and Decamethoxin (Dekasan) 33 were used as the control antiseptic and disinfection agents revealing activity against bacteria and fungi.
It was found that the gram-negative bacteria Klebsiella pneumonniae and Escherichia coli are not sensitive to the studied amides 3a-l.In contrary, the gram-positive bacteria were found sensitive to the amides 3с, e, f, g, i (Table 2).In particular, Staphylococcus aureus MS, Streptococcus pyogenes and Streptococcus gordonii are sensitive to the amides 3g and 3f, Staphylococcus aureus MR, Staphylococcus haemolyticus MR and Staphylococcus epidermidis MS -to the amides 3с, f, g, Bacillus subtilis -to the amides 3f, g, i, and Streptococcus oralis -to the amides 3 e, f, g.Some antifungal activity has been found in the amides 3с, f, g, in particular, the amide 3с retards the proliferation of Candida albicans, and the compounds 3 с, f, g -Candida tropicalis.
Basing on these results, we can emphasize the antistaphylococcus activity of the amide 3f against Staphylococcus aureus MR and Staphylococcus epidermidis MS, for which the retardation area diameter was 14.65 mm, which is greater than that for the control agents.Such a notable antibacterial effect can be caused by the strong electrondonoring dimethylamino group of the amide fragment, which increases a lypophility of the molecule 34 .Taking into account the natural resistance of staphylococcus bacteria against many drugs, a deeper study of the amide 3f will be taken.

Conclusions
It can be concluded that an efficient method of synthesis of new antimicrobial active 3-(pyrrol-4-yl)acrylamides has been developed.These products consist of biophoric chlorine atoms and an easily modifiable ester group in the pyrrole cycle.This method is very promising as it involves readily available reactants, easy procedures of synthesis, and ensures high yields of intermediate and final compounds.Structural composition of the synthesized products was confirmed by IR-, 1 Н ( 13 С)-NMR-spectroscopy, mass spectrometry, and elemental analysis.As seen from the evaluation of antimicrobial properties of the synthesized amides, the compounds 3c, e, f, g, i are highly efficient against gram-positive bacteria.Special attention should be given to the compound 3f because its activity against Staphylococcus aureus MR and Staphylococcus epidermidis MS is greater than that of the control drugs.As seen from the antimicrobial activity investigation, the obtained 3-(pyrrol-4-yl)acrylamides deserve further study as prospective antigerm agents.

Materials and Methods
All chemicals were of analytical grade and commercially available.When performing the synthetic part of the work, the reagents of the company Merck (Germany) and Sigma-Aldrich (USA) were used.All reagents and solvents were used without further purification and drying.All the melting points were determined in an open capillary and left uncorrected.IR spectra were recorded on Bruker Vertex 70 FT-IR spectrometer for samples in KBr pellets. 1 H-NMR spectra were acquired in pulse Fourier transform mode on a Varian VXR-400 spectrometer (400 MHz) in DMSO-d6 (compounds 2 a-e) or CDCl3 (compounds 3 a-l), while 13 C-NMR spectra of all compounds were recorded on a Bruker Avance DRX-500 spectrometer.The solvent signal (DMSO-d6: 2.49 ppm for 1Н nuclei, 39.5 ppm for 13 С nuclei; CDCl3: 7.26 ppm for 1 Н nuclei) served as the internal standard.Mass spectra were recorded on an Agilent LC/MSD SL mass spectrometer; column: Zorbax SB-C18, 4.6 × 15 mm, 1.8 μm (PN 82 (c)75-932); DMSO solvent, atmospheric pressure electrospray ionization.Elemental analysis was performed on a Perkin Elmer 2400 CHN-analyzer.Melting points were determined on a Kofler bench and left uncorrected.

Antimicrobial activity
A micromethod of diffusion into agar was used to evaluate an antimicrobial activity of the synthesized compounds.Nutritional agar was placed in the Petri bowls, and a series of 4.0 mm holes was made in it.Then the agar was uniformly populated with the standard suspensions of test-cultures with the concentration 1×10 7 CFU/mL.20 μL of a 10 mg/mL solution of the studied compounds in a mixture DMSO/ethanol/ water (1:2:1) were added to each hole to evaluate their antimicrobial activity.To do that, a diameter of the bacterial colony's retardation area was measured 24 h after the addition of a compound by taking digital images of the colonies followed by their analysis performed by the software UTHSCSA ImageTool 3.0 (The University of Texas Health Science Center in San Antonio, © 1995-2002).Then the results were additionally processed using variational statistics methods.No retardation of the colony's growth was found in the control holes treated with a pure mixture DMSO/ethanol/ water (1:2:1).
In the above study, some archival strains and clinical isolates of the antibiotic sensitive and resistant microbes were involved.They were identified by their morphological, cultural features, and using the biochemical tests «STAPHYtest 16», «ENTEROtest 24», «STREPTOTtest 16» (by Lachema, Czechia), test-systems VITEK 2 GP and VITEK 2 YST (by Biomerieux, France) on the analyzer VITEK 2 Compact.

Table 1 .
Structure of synthesized compounds