Antimicrobial and Antibiofilm Activities of 4,5-Dihydro-1H-pyrazole-1-carboximidamideHydrochlorideagainst Salmonella spp.

In the present study, the antimicrobial and antibioﬁlm activities of two 4,5-dihydro-1H-pyrazole-1-carboximidamide hydrochloride, (triﬂuoromethyl) phenyl-substituted (compound 1 ) and bromophenyl-substituted (compound 2 ), were evaluated against four Salmonella spp. serotypes through broth microdilution and bioﬁlm-forming activity. Further, the cytotoxicity of the compounds was evaluated by cell viability assays using cultures of HeLa and Vero cell lines, and the mutagenic potential was assessed by the Ames test. In the broth microdilution test, compound 1 inhibited 90% of the strains tested at the minimum inhibitory concentration of 62.5 μ gmL − 1 . Furthermore, both compounds prevented bioﬁlm formation, with a reduction of up to 5.2 log 10 . HeLa and Vero cells exhibited 100% viability in the presence of compound 1 . In contrast, low cell viability was observed in the presence of 15 µ gmL − 1 of compound 2 . Furthermore, no mutagenic potential was detected at any of the tested concentrations of compound 1 .


Introduction
Salmonellosis is an enteric infection caused by bacteria of the genus Salmonella and is among the primary causes of foodborne illness and is a major public health problem worldwide [1,2]. According to the Ministério da Saúde [3], salmonellosis symptoms include fever, abdominal pain, diarrhea, nausea, and vomiting while immunocompromised individuals present a rapid progression to septicemia that can lead to death [4]. Data from the Centers for Disease Control and Prevention (CDC) estimate that approximately 1.35 million illnesses and 420 deaths annually occur due to non-typhoidal Salmonella annually in the United States [5]. e costs of treating salmonellosis have a great economic impact on the medical, food, and agricultural industries. Millions of dollars are spent each year on food-borne infections, justifying research for control and prevention [6,7].
Studies have shown that Salmonella infection occurs following ingestion of contaminated protein-rich food, such as beef, pork, and poultry [4,8]. Another important factor in Salmonella transmission and infection is the ability of this microorganism to adhere and form biofilms on different surfaces [9]. ese biofilms become a source of Salmonella dissemination during food processing in industrial and domestic environments [10,11]. e presence of extracellular matrix confers biofilms with a high resistance to antimicrobial and sanitizing agents, hindering their removal and elimination [10].
In medicinal chemistry, compounds containing pyrazoline substructures and their derivatives have attracted interest due to their promising biological activities, such as antimicrobial, anti-inflammatory, antidepressant, and antitumor activities [12][13][14][15]. Different substituents of these moieties have been assayed in the search for more active and selective molecules. Halogenated substituents have been widely used to improve the antimicrobial activity of pyrazole compounds and have been studied by several researchers worldwide [14,16,17]. In this context, Sid et al. [18] reported that there is an improvement in lipophilicity with the insertion of bromo-substituted compounds and fluorine atoms and that the latter also provides greater permeability for cellular membranes, facilitating transport in biological systems.

Synthesis of Pyrazoline
Hydrochlorides. All the pyrazoline hydrochlorides were synthesized according to the procedure reported by us recently [29] with some modifications (Scheme 1). To a 50 mL vial, a mixture of 3-(hetero) aryl-1-(2-hydroxyphenyl)enones (1.0 mmol), aminoguanidine hydrochloride (0.22 g, 2.0 mmol), and potassium hydroxide (0.11 g, 2.0 mmol) in ethanol (15 mL) were added. e US probe was placed in the reaction vial, which was sonicated for 30 min. After the completion of the reaction, the excess of aminoguanidine hydrochloride was filtered off and in the filtrate 10% hydrogen chloride (15 mL) was added. e mixture was subsequently extracted with chloroform (3 × 30 mL) and organic phase was dried over MgSO 4 and filtered and the solvent was evaporated under reduced pressure. e residue was purified by recrystallization from ethyl acetate-methanol (10 : 1), resulting in the pyrazoline hydrochlorides as crystalline solids.

Minimum Inhibitory Concentration.
e antimicrobial activity of compounds 1 and 2 was determined by the broth microdilution test, following document M07-A9 of the Clinical and Laboratory Standards Institute with some modifications [30].
After cultivation in Trypticase Soy Agar (TSA) at 37°C for 24 h, the bacteria were standardized to 1.5 × 10 8 colonyforming units (CFU).mL −1 . A serial dilution (1 : 2) was performed from the concentration of 1.000 µg mL −1 to the concentration of 1.9 µg mL −1 . Chloramphenicol was used as reference, considering sensitive ≤8, intermediate 16, and resistant ≥32. A 0.1% triphenyl tetrazolium chloride (TTC) solution was used to interpret the results. e minimum inhibitory concentration (MIC) was defined as the lowest concentration of the compound capable of inhibiting bacterial growth. e MIC50 and MIC90 values were calculated defined as the concentration at which the compounds were able to inhibit 50% and 90% of the total isolates tested. e tests were performed in triplicate at two different time points.

Bacterial Cell Viability.
Based on the results of the antibacterial activity, a representative of each serotype (Braenderup, Enteritidis, Worthington, and Infantis), in addition to S. Enteritidis (ATCC 13076) and S. Typhimurium (ATCC 14028), were selected to evaluate the impact of compounds 1 and 2 on bacterial cell viability.
e compounds were tested at concentrations of 250, 125, and 62.5 μg mL −1 . After the 24 h of the broth microdilution test described above, serial dilutions of the compounds were prepared in saline solution (0.85%), and plating on Müeller Hinton agar using the drop plate technique [31]. e plates were incubated for 24 h at 37°C, the number of CFUs was counted, and the results were converted to log 10 CFU [32].
e experiment was carried out in triplicate.

Effect of Compounds 1 and 2 on Biofilm Formation.
Considering the antibacterial activity of the compounds 1 and 2, their effects were evaluated on biofilm formation according to the methodology of Costa et al. [33] with modifications. Salmonella strains were cultured in Tryptone Soy Broth (TSB) for 18 h at 37°C under 80 rpm. Subsequently, the cells were centrifuged at 3000 g for 10 min, washed three times with phosphate-buffered saline (PBS), and resuspended in TSB to a concentration of 1 × 10 8 CFU/ mL. In order to evaluate the effect of compounds on Salmonella strains biofilm formation, the compounds were added at the same time of Salmonella inoculum. For that, 100 µL of inoculum and 100 µL of compounds 1 and 2 at concentrations 250, 125, and 62.5 μg mL −1 were added to the wells of 96-well microplates. e microplates were incubated with orbital shaking at 80 rpm for 48 h. e wells were washed three times with PBS to remove weakly adhered cells and then were scraped using a micropipette tip to remove the biofilms. e resulting cell suspensions were homogenized by vortexing for 5 min to disaggregate the cells and serial dilutions were prepared, which were plated on Müller Hinton agar using the drop plate technique [31]. Plates were incubated at 37°C for 24 h. After incubation, the CFUs were counted, and results were converted into log 10 CFU. e experiment was conducted in triplicate at two different time points.

Cytotoxicity Assay.
e cytotoxicity of compounds 1 and 2 was evaluated following the methodology proposed by Capoci et al. [34] using HeLa and Vero cells. HeLa and Vero cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and in RPMI 1640 medium, respectively. Cell concentrations were adjusted to 2 × 10 5 cells.mL −1 , added to 96-well microplates, and incubated under 5% CO 2 at 37°C for 24 h. After the incubation period, adherent cells were washed and treated with the compounds at concentrations of 500, 150, and 15 μg mL −1 and then incubated under the same conditions. e culture media and the cell suspensions were used as the growth controls (blanks).
Cytotoxicity was evaluated by measuring the reduction of MTS (3-[4, 5- in DMEM without phenol red. After a 3 h incubation at 37°C, the formazan absorbance was measured at 490 nm using a microplate reader. e percentage of cell viability (CV%) was calculated using the following formula: where At and Ac correspond to the absorbance of the tested substance and the control (untreated cells), respectively [35]. e cytotoxicity of the compounds 1 and 2 was presented as the average of three independent experiments with three replicates.

Mutagenicity Assay.
Mutagenicity was assessed by the Ames test, a microsuspension method described by Kado et al. [36]. e Salmonella Typhimurium TA98 and TA100 strains were standardized to a concentration of 1 × 10 8 CFU.mL −1 . e assays were performed in the presence and absence of the S9 exogenous microsomal fraction.
To each tube, 50 µL of phosphate buffer or the S9 fraction, 5 μL of compound 1 at different concentrations (5000, 1500, 500, 150, and 50 μg.plate −1 ), and 50 μL of bacterial suspension were added. After the tubes were preincubated for 90 minutes at 37°C, 2 mL of top agar was immediately added, and the mixture was poured onto plates with minimal agar. e plates were incubated at 37°C for 48-66 hours, and the revertant colonies (His+) were counted. DMSO was used as a negative control, while 4nitrophenylenediamine (NPD) (10 μg.plate −1 ) was used as positive control for the TA98 strain and sodium azide (2.5 μg.plate −1 ) in the absence of S9 and 2-aminoanthracene in the presence of S9 were used as positive controls for the TA100 strain. e mutagenicity index (MI) was calculated according to the following formula: MI � no.of induced revertants no.of pontaneous revertants .
(2) e compound was considered to have mutagenic potential when the MI was equal to or greater than 2.0 for at least one of the concentrations tested and when there was a dose-response relationship between the concentrations and the number of induced revertants. Concentrations with an MI below 0.7 were considered cytotoxic [37].

Statistical Analysis.
e results of the biofilm and bacterial cell viability tests were analyzed by GraphPad Prism ® 5.01, using analysis of variance (ANOVA), and the means were compared by Tukey's test, with a p value < 0.05 considered significant. e Ames test data were analyzed by ANOVA using the statistics program Salanal ® 1.0, with a p value < 0.05 considered significant.

Results and Discussion
A series of amidino pyrazolines were synthesized and were subjected to a screening of biological activities. Two of these compounds showed promising results and were selected to continue further biological tests. e selected compounds were named as follows: compound 1: 3-(2-hydroxyphenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazole-1carboximidamide hydrochloride and compound 2: 5-(4bromophenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboximidamide hydrochloride (Scheme 1). e synthesis was accomplished by cyclocondensation reactions between facile prepared chalcones (3-(hetero) aryl-1-(2-hydroxyphenyl)enones) and commercially available aminoguanidine hydrochloride, using ethanol as solvent and potassium hydroxide as base, promoted by ultrasonic irradiation. Compounds 1 and 2 were isolated in 63 and 68% of yields, respectively, after liquid-liquid extraction and purification by recrystallization. e success of the synthesis is evidenced by the results from nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (EMAR) analyses. For compound 1, the 1 H NMR spectrum shows a broad singlet at 9.93 ppm attributed to the OH hydrogen and another broad singlet at 8.04 ppm due to the four equivalent hydrogens attached to the nitrogens. , respectively. e identity of compound 1 is also confirmed by the 13 C NMR spectrum that shows 16 signals at the expected chemical shifts for the 16 nonequivalent carbons present in the structure. e 13 C NMR spectrum also shows three quadruplets due to the C-F couplings. Unequivocally, the HRMS analyses confirm the identity of the proposed elemental composition for compound 2 because the comparison between the theoretical exact m/z for [C 17  Compound 1 and 2 were tested against Salmonella spp., and both exhibited antimicrobial activity. For compound 1, MIC50 and MIC90 against all Salmonella species were 62.5 µg mL −1 . For compound 2, MIC50 and MIC90 against all Salmonella species tested were 125 µg mL −1 , with the exception of S. Infantis, which was more sensitive to the compound, with MIC50 of 62.5 µg mL −1 . With regard to internal control, all Salmonella spp. tested resulted in being sensitive to chloramphenicol, with MIC of 4 µg mL −1 . e greater antimicrobial activity of compound 1 was attributed to the presence of fluorine atoms in the molecule that promote increased biological activity due to the intrinsic properties of fluorine, small atomic radius, high electronegativity, and stability [12,[38][39][40][41]. e high electronegativity of fluorine may enhance the electrophilicity of neighboring groups in the molecule and the substitution of a methyl group by a trifluoromethyl group, resulting in increased lipophilicity.
is lipophilicity provides greater permeability in cell membranes and facilitates the absorption and transport of these molecules within biological systems, improving its properties [42]. Furthermore, literature data describe that the antimicrobial effect of fluorine is associated with the inhibition of intracellular acid, inhibition of the transformation and use of some nutrients, affecting the synthesis and storage of polysaccharides, and inhibition of enzymes related to bacterial pathogenicity, such as catalase, phosphatase, and others [43].
In addition, the effect of the compounds on the number of viable bacterial cells was evaluated and the results showed a significant reduction against all the evaluated serotypes. At a concentration of 62.5 µg.mL-1, compound 1 showed reduction ranging from 3, 3-6, 6 log 10 CFU, while at a concentration of 250 µg.mL-1 reduction ranging from 7, 7-11, 39 log 10 CFU. Regarding compound 2, the reduction ranged from 0, 5-3 log 10 CFU at a concentration of 62.5 µg.mL-1, and from 5, 3-8 log 10 CFU at a concentration of 250 µg.mL-1, which corroborate the MIC50 and MIC90 values (Figures 1(a) and 1(b)). As biofilms are more resistant than bacteria in planktonic form, the effect of the compounds on biofilm formation was also evaluated, and compounds 1 and 2 reduced the number of cells present in the biofilm of Salmonella spp. in all tested concentrations. At the highest concentration (250 µg.mL-1), compound 1 was able to elicit decrease 2.1-4.9 log10 in the formation of biofilms, while compound 2 at the same concentration decreased 3.8-5.2 log10 (Figures 2(a) and 2(b)). e antibiofilm activity of the compounds can be attributed to halogenated substituents, which generate changes in relative binding affinities to the bacterial surface. Halogenated substitutes reduce affinity to components of the bacterial structure, such as lipopolysaccharide, and this may facilitate the compounds' ability to cross the outer membrane, enhancing their antimicrobial activity [44,45]. Additionally, they are known to control biofouling and to be effective in penetration and disinfection of biofilms [46]. Compound 2 showed more highly significant effect in reducing biofilm formation by Salmonella, which can be related to the effect of the bromine atom, which possibly promoted the breakdown of the extracellular matrix secreted during the biofilm formation process to allow increased permeability of this compound to these strongly adherent cells. e presence of bromine as an important factor for antibiofilm activity was also reported by Galarraga-Vinueza et al. and Campana et al. [47,48]. In our study, we observed that fluoro-and bromo-substituted pyrazoline compounds were effective against Salmonella spp. in different in vitro analyses. e microdilution test enabled the determination of the minimum concentration that inhibited the multiplication of the bacterial cells, while the bacterial cell viability test showed the decrease in CFUs. To the best of our knowledge, there are not results describing regarding the action of such compounds against biofilm formation in the literature, advocating an innovative application of pyrazoline compounds for this purpose.
Due to the antibacterial and antibiofilm activity of our compounds, their cytotoxic potential was tested to examine the potential toxic effects. Compound 1 showed 100% viability for HeLa and Vero cells after 24 h of treatment in all concentrations, indicating that it does not present cytotoxicity in vitro. Compound 2, however, at the lowest concentration reduced viability to 23% and 19% for HeLa and Vero cells, respectively (Figure 3).
In a study by Altintop et al. [14] p-bromophenyl compounds derived from thiazolyl-pyrazolines showed high cytotoxicity, whereas the fluoro-substituted compounds had low cytotoxicity. ese data indicate that cytotoxicity is not related to the pyrazoline moiety.
Since it has no cytotoxic potential, compound 1 was subjected to the Ames test to evaluate its mutagenic potential.
e Salmonella Typhimurium TA98 and TA100 strains were tested and the assays were performed in the presence and absence of the S9 exogenous microsomal fraction (Table 1). is experiment indicated that there was no significant (p < 0.05) increase in the number of revertant colonies of S. typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation at the concentrations tested (50-5000 μg.plate −1 ) when compared to the negative control. e concentrations tested also did not present MI ≥ 2, indicating that compound 1 has no potential to cause gene base-pair substitution and frameshift mutations. However, at 5000 μg.plate −1 cytotoxicity was observed for TA98 in an assay without metabolic activation and for TA100 in the presence and absence of metabolic activation. Altintop et al. [14] also investigated the mutagenicity of substituted thiazolyl-pyrazoline derivatives against S. Typhimurium TA 98 and TA100, with and without metabolic activation, and did not observe any mutagenic potential. Furthermore, for both compounds, we performed an in silico prediction of the pharmacokinetics properties [49]. For this calculation, we used the open-source software Molinspiration [50] and OSIRIS Property Explorer [51].
Lipinski's rule of 5 [52] predicts that for a potentially drug candidate to present a good pharmacokinetic profile it has to meet some of the following requirements, such as MW (molecular weight) ≤ 500, ClogP ≤5.0 or MLogP ≤4.15, HBA (hydrogen bond acceptors) ≤ 10, and HBD (hydrogen bond donors) ≤ 5. Veber and coworkers [53] introduced other two important criteria for this type of prediction, that is, to have TPSA (topological polar surface area) ≤ 140Å 2 and 10 or fewer rotatable bonds. Compound 1 and compound 2 satisfied Lipinski and Veber criteria (Table 2). e results from in silico prediction showed that both compounds may present good absorption and permeation after oral administration, turning them good drug candidate.

Conclusions
e (trifluoromethyl)phenyl-substituted (1) and bromophenyl-substituted (2) 4,5-dihydro-1H-pyrazole-1-carboximidamide hydrochlorides assayed in this study exhibited promising antimicrobial activity. Both produced log reductions and were able to reduce biofilm formation. Besides, to these biological activities, compound 1 has displayed no cytotoxic or mutagenic potential. Taking into account challenges and difficulties in design novel and efficient drugs able to control the action of pathogenic microorganisms, these bioactive compounds could be used as alternatives for treatment, disinfection, and sanitizing agents in the food industry. In this sense, future in vivo studies will be necessary to confirm this potential and assess their risk to the human health.