Synthesis and in vitro antimicrobial activity screening of new pipemidic acid derivatives

Abstract This article describes the synthesis and antimicrobial activity evaluation of new pipemidic acid derivatives. New compounds were obtained on the basis of Mannich reaction of 4,5-disubstituted 1,2,4-triazole-3-thiones with pipemidic acid. Antimicrobial tests revealed high antibacterial activity of obtained derivatives. Gram-negative rods belonging to Enterobacteriaceae family were particularly most sensitive to new pipemidic acid derivatives. Synthesized compounds exhibited very strong activity towards Proteus mirabilis ATCC 12453, Salmonella typhimurium ATCC 14028 and Escherichia coli ATCC 25922. The minimum inhibitory concentrations of new pipemidic acid derivatives which inhibited the growth of these bacteria were 0.98–7.81 µg/ml, 0.98–7.81 µg/ml and 0.98–3.91 µg/ml, respectively. The antibacterial activity of newly synthesized pipemidic acid derivatives in many cases was far better than the activity of substances used as positive controls (nitrofurantoin, cefuroxime, ampicillin and pipemidic acid). Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s12272-018-1025-3) contains supplementary material, which is available to authorized users.


Introduction
Microorganisms play a key role in the functioning of the environment but they are also a real threat to human life and health. The widespread diseases and infections caused by different bacteria and fungi have encourage the need for scientific research and advances in medicinal chemistry (Coates et al. 2002;Spellberg et al. 2008;Llor and Bjerrum 2014). The unequivocal reports regarding the scale of pathogenic microbial resistance to commonly used drugs have forced the search for new antimicrobial agents (Coates et al. 2002;Spellberg et al. 2008;Llor and Bjerrum 2014).
Literature review prove that researchers around the world are looking for substances that will be better tolerated by patients, less toxic, and at the same time more effective in combating with microbes (Spellberg et al. 2008;Llor and Bjerrum 2014). Many medicinal chemists are concentrating their studies on compounds having heterocyclic systems with nitrogen atoms in their structure (Spellberg et al. 2008;Llor and Bjerrum 2014). Especially, 1,2,4-triazole-3-thiones focused attention of many researchers mainly due to their usefulness in different reactions e.g., Mannich reaction (Bala et al. 2014). Due to the simplicity of the Mannich reaction and favorable pharmacological effect of its products, this reaction is often used in the pharmaceutical industry (Bala et al. 2014).
Mannich bases display wide spectrum of biological activity, like: antibacterial (Pandeya et al. 2000;Ashok et al. 2007), antifungal (Pandeya et al. 2000;Singh et al. 2007), antitubercular (Mulla et al. 2011), antimalarial (Barlin and Jiravinya 1990), anti-HIV (Sriram et al. 2009), anti-inflammatory (Köksal et al. 2007;Ivanova et al. 2007), anti-cancer (Gul et al. 2000), anticonvulsant (Vashishtha et al. 2004), analgesic (Malinka et al. 2005;Köksal et al. 2007), antipsychotic activity (Scott et al. 1992) and activity against herpes virus (Edwards et al. 1983). Additionally, it is worth to mention that in our previous reports Mannich bases derived form 1,2,4-triazole-3-thiones displayed interesting antiproliferative and antimicrobial activity (Popiołek et al. 2014(Popiołek et al. , 2017. In our current research we focused our attention on both Mannich bases and pipemidic acid. Pipemidic acid is an antimicrobial agent from the quinolones group. Its mechanism of action involves inhibition of DNA synthesis in bacterial cells by blocking class II topoisomerases, namely DNA gyrase which is responsible for DNA stranding and spatial DNA isomer formation, and topoisomerase I responsible for DNA strand separation after replication process. Inhibition of the activity of these enzymes leads to the damage of bacterial DNA. Inhibition of DNA gyrase results in loosening of the structure and increasing of the space occupied by DNA in the bacterial cell (Shumitzu et al. 1975;Domagala 1994;Hawkey 2003;Andersson and MacGowan 2003). Pipemidic acid is therefore considered to be a bactericidal compound (Shumitzu et al. 1975). From the chemical point of view, pipemidic acid is a pyrimidine derivative active against Gram-negative bacteria, including Pseudomonas aeruginosa, as well as some Gram-positive bacteria. Its activity is generally greater than pyrimidine acid and nalidixic acid (Shumitzu et al. 1975). In addition to this, literature findings proved that the connection of 1,3,4-thiadiazoles or 1,2,4-triazoles with (fluoro)quinolones in one molecule may have beneficial influence on the antimicrobial activity of such hybrid compounds (Foroumadi et al. 2006;Plech et al. 2013).
In the view of above mentioned facts in this current research we decided to synthesize novel pipemidic acid derivatives with the use of Mannich reaction of 4,5-disubstituted 1,2,4-triazole-3-thione derivatives with pipemidic acid in believe to obtain compounds with interesting antimicrobial activity.

Chemistry
The reagents and solvents used in this research were obtained from Merck Co. (Darmstadt, Germany) and Sigma-Aldrich (Munich, Germany). Melting points were determined with the use of Fisher-Johns blocks melting point apparatus (Fisher Scientific, Germany) and presented without correction. The 1 H NMR and 13 C NMR spectra were recorded with the use of Bruker Avance 300 apparatus (Bruker BioSpin GmbH, Germany). The DMSO-d 6 was used as solvent and TMS as the internal standard. Chemical shifts in this article are reported in ppm (δ). The coupling constants (J) are presented in Hertz. The purity of obtained compounds and the progress of the reaction were determined by thin-layer chromatography (TLC) with the use of pre-coated aluminum sheet 60 F254 plates (Merck Co. USA), and CHCl 3 /C 2 H 5 OH (10:1, v/v) solvent system. The spots were identified by the exposure to the UV light at 254 nm. The elemental analysis of obtained compounds was carried out with the use of AMZ 851 CHX analyser (PG, Gdańsk, Poland). The results of elemental analysis (C, H, N) were within±0.4% of the calculated values.

Synthesis of thiosemicarbazide derivatives (3-18)
0.002 Mole of appropriate carboxylic acid hydrazide (3methoxybenzhydrazide-1 or 4-tert-butylbenzhydrazide-2, respectively) was dissolved in 10 ml of ethanol (96%). Then 0.0022 mol of appropriate isothiocyanate was added and heated under reflux for 3 h. Subsequently obtained solution was put to the refrigerator for 24 h. After that formed precipitate was filtered off and re-crystallized from ethanol. The procedure of this synthesis was based on our previous article (Popiołek et al. 2013a).
Detailed physicochemical data of thiosemicarbazide derivatives (3-18) is presented in Supplementary Materials.

Synthesis of new pipemidic acid derivatives (35-50)
In order to obtained new pipemidic acid derivatives we applied the Mannich reaction and the procedure reported by our group earlier (Popiołek et al. 2014(Popiołek et al. , 2017. 0.003 Mole of appropriated 4,5-disubstituted 1,2,4-triazole-3thione derivative (19-34) was added to the conical flask and dissolved with stirring in 5 ml of ethanol (96%). After that 200 µl of formaldehyde and 0.0033 mol of pipemidic acid was added to the flask. The content of the flask was stirred by magnetic stirrer for 1 h. Subsequently, 15 ml of distilled water was added to the flask. The precipitate which formed was filtered off and re-crystallized from methanol.
Detailed physicochemical data of new pipemidic acid derivatives (35-50) is presented in Supplementary Materials.

Microbiology
The examined compounds 3-50 were screened in vitro for antibacterial and antifungal activities using the broth microdilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST discussion document E. Dis 5.1 2003) and Clinical and Laboratory Standards Institute guidelines (Reference method for broth dilution antifungal susceptibility testing of yeasts. M27-S4 2012) against a panel of reference and clinical or saprophytic strains of microorganisms. Ciprofloxacin, nitrofurantoin, cefuroxime, ampicillin and pipemidic acid (Sigma-Aldrich, Munich, Germany) were used as a reference antibacterial compounds. Nystatin (Sigma-Aldrich, Munich, Germany) was used as reference antifungal positive control. Detailed procedure of antimicrobial screening is presented in Supplementary Materials. The statistical analysis of obtained results is presented in the Tables 1S, 2S, and 3S in Supplementary Materials.
The 1 H NMR spectra of new pipemidic acid derivatives (35-50) showed characteristic singlet signal for CH 2 group in the range of δ 5.18-5.36 ppm, what confirmed common aminomethylation reaction of 1,2,4-triazole-3-thiones. Signals for other aliphatic and aromatic fragments of synthesized compounds on 1 H NMR and 13 C NMR were shown at expected shift range.

Microbiology
Our antimicrobial activity screening results indicated, that most of synthesized compounds among thiosemicarbazide derivatives (3-18), especially compounds: 4-10, 12, 13, 15, 18, had no activity against all reference microorganisms (Table 1). Among remaining substances the compound 17 indicated the highest activity with strong and very strong bacteriostatic effect against Micrococcus luteus ATCC 10240 and both of Bacillus spp. ATCC strains. The minimum concentrations of 17, which inhibited the growth of these bacteria were 3.91-31.25 and 500 to[1000 µg/ml, respectively. The activity of the compound 17, on the basis of minimal inhibitory concentration (MIC) values, was 32 times better against M. luteus ATCC 10240 (MIC=3.91 µg/ ml) and two times better against B. cereus ATCC 10876 (MIC=7.81 µg/ml) in comparison with the activity of pipemidic acid (MIC=125 µg/ml and MIC=15.62 µg/ml, respectively), used as positive control (Table 1). The compound 17 also showed good bacteriostatic activity towards Staphylococcus aureus ATCC 6538 (MIC= 125 µg/ml and MBC[1000 µg/ml) and moderate or mild effect against other reference staphylococci and some The standard compounds used as positive controls: ciprofloxacin (CIP), nitrofurantoin (NIT), cefuroxime (CFX), ampicillin (APC) and pipemidic acid (PA) for bacteria and nystatin (NY*) for fungi. Compounds with bactericidal effect (MBC/MIC≤4) are marked in bold. All the experiments were repeated three times (n=3) and representative data is presented nd not determined, na not applicable, "-" no activity  (Table 1). Besides this, the compounds 11 and 17 indicated moderate or mild activity towards fungi belonging to all reference yeasts. The minimum concentrations of these substances, which inhibited the growth of Candida spp. were from 250 to 1000 µg/ml. The minimal fungicidal concentrations were similar 500 to [1000 µg/ml. The activity of the compound 3 against yeasts was assessed as mild (MIC=1000 µg/ml and MFC≥1000 µg/ml) (Table 1).
In addition, the compounds 19, 20 and 21 showed fungicidal activity. The most sensitive to these substances were Candida albicans ATCC 2091 and Candida albicans ATCC 10231. The minimum inhibitory concentrations of 19, 20 and 21, which inhibited their growth were 250-500 µg/ml, while MFC≥1000 µg/ml. These compounds indicated slightly lower effect against other reference species of Candida (MIC=500-1000 µg/ml and MFC≥ 1000 µg/ml). The remaining substances 22-33 were inactive towards bacteria and fungi from ATCC (Table 2).
In comparison to the activity of pipemidic used as positive control (MIC=62.5 µg/ml), the activity of the compounds 36 and 50 (MIC=15.62 µg/ml) was four times better and for the compounds 35, 37, 38, 44, 45, and 46 (MIC=31.25 µg/ml) was two times better against S. aureus ATCC 6538 on the basis of MIC values. The compound 50 showed two times lower MIC values (MIC=7.81 µg/ml) with bactericidal effect and the compounds 36 and 46 showed two times lower MBC values (MBC=31.25 µg/ml) with bactericidal effect against S. epidermidis ATCC 12228 than pipemidic acid (MIC=15.62 µg/ml, MBC=62.5 µg/ ml). The MBC values for the derivative 36 towards B. subtilis ATCC 6633 (MBC=3.91 µg/ml) were two times lower than for pipemidic acid (MBC=7.81 µg/ml). In the case of the activity against B. cereus ATCC 10876 the MIC values for the substance 37 were two times lower (MIC= 7.81 µg/ml) than for pipemidic acid (MIC=15.62 µg/ml) ( Table 3). High activity of synthesized compounds (35-50) against B. cereus ATCC 10876 is especially important due to the fact that this bacterium is responsible for an increasing number of foodborne diseases in industrial countries as well as postoperative and posttraumatic wound infections (Kotiranta et al. 2000;Bottone 2010).
Summarizing, in this research we synthesized and evaluated for in vitro antimicrobial activity a series of new pipemidic acid derivatives obtained by the Mannich reaction of appropriate 4,5-disubstituted 1,2,4-triazole-3thiones with pipemidic acid. Antimicrobial activity screening of synthesized compounds revealed interesting antibacterial properties of obtained derivatives. Our antimicrobial assays results indicated that newly synthesized pipemidic acid derivatives showed very high antimicrobial activity, especially against Gram-negative bacteria.

Compliance with ethical standards
Conflict of interest Authors declare no conflict of interest.
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