Microbiological Screening of 5-Functionalized Pyrazoles for the Future Development of Optimized Pyrazole-Based Delivery Systems

The pyrazole ring represents a widely applied chemical scaffold in medicinal chemistry research and we have observed that the physicochemical and biological features of highly substituted pyrazoles can be successfully improved by their encapsulation in dendrimer nanoparticles (NPs). For the future development of new optimized antibacterial delivery systems, we report the synthesis and biological evaluation of 5-amino functionalized pyrazole library (compounds 2–7). In detail, new derivatives 2–7 were differently decorated in C3, C4 and C5 positions. An in silico study predicted pyrazoles 2–7 to exert good drug-like and pharmacokinetic properties. Compounds 3c and 4b were endowed with moderate, but nanotechnologically improvable activity against multidrug-resistant (MDR) clinical isolates of Gram-positive species, especially of the Staphylococcus genus (MICs = 32–64 µg/mL). In addition, derivatives 3c and 4a showed moderate activities against Mycobacterium tuberculosis and 4a evidenced activity also against MDR strains. Overall, the collected evidence supported that, upon nano-formulation with proper polymer matrices, the new synthesized compounds could provide new pyrazole-based drug delivery systems with an enhanced and enlarged-spectrum of antibacterial activity.


Introduction
The five-membered heterocycle pyrazole ring represents a widely applied chemical scaffold in medicinal chemistry research. Several pyrazole derivatives show a broad spectrum of biological properties, such as antimicrobial [1], analgesic [2], antifungal [3], anti-inflammatory [4], and antioxidant activities [5]. Collectively, the pyrazole nucleus possesses almost all types of pharmacological activities, and throughout the years, many researchers have studied its skeleton both chemically and biologically. Currently, the pyrazole nucleus is present in numerous pharmacological agents belonging to different therapeutic categories (Figure 1), as anti-inflammatory/analgesic compounds (Celecoxib, Tepoxalin, Betazole), anticancer (Crizotimib), antiobesity (Surinabant, Difenamizole), antidepressant and tranquillizer (Fezolamine, Mepiprazole) and many others, confirming the pharmacological potential of this heterocyclic ring [6]. Particularly, 5-pyrazolyl-ureas (5-PUs) (Figure 1) are reported and studied for the treatment of different microbial infections caused by bacteria or protozoa of the genera Plasmodium, Toxoplasma, and others [7] or to treat cancer (e.g., Figure 1. Chemical structure of some drugs endowed with the pyrazole structure Crizotinib, Surinabant, Fezolamine) and 5-PUs, BBB4, CB1H and I previously anticancer/anti-inflammatory, antibacterial and anti-Mycobacterium compounds, respec In the current scenario of bacterial infections, those caused by bacteria of t (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Ac baumannii, Pseudomonas aeruginosa, Enterobacter) family, which are rec dangerous superbugs by the Infectious Diseases Society of America (IDSA), ar a global concern because they are almost untreatable [6]. Unfortunately, ESKA are endowed with an increasing tendency to develop MDR to the bactericid conventional antibiotics, which are no longer effective and need substitution efficient new antibacterial agents suitable for clinical application [6]. To mee urgency of new antimicrobial compounds, the aim of this work is to inve antibacterial/anti-Mycobacterium activity of a small library of 5-amino fun pyrazoles (compounds 2-7, see the workflow of the whole study in Figure 2), s related to previously reported BBB4, CB1H, 5-PUs and I. To reduce the lipoph therefore increase water solubility) and preserve antimicrobial activity, the ne derivatives do not bear the 3,5-diphenyl groups but share with the parent com Additionally, we have observed that the water solubility and antibacterial effects of two differently functionalized pyrazoles synthetized by us (BBB4 and CB1H) (Figure 1) were remarkably improved by their encapsulation in polyester-based dendrimer nanoparticles (NPs) and in styrene-based copolymer NPs [6,14]. In particular, the obtained BBB4-G4K NPs and CB1H-P7 NPs were able to contrast multidrug-resistant (MDR) pathogenic species, also including clinical isolates particularly difficult to treat, due to the onset of resistance versus the most potent antibiotics, such as carbapenems and colistin [15,16]. Additionally, the developed encapsulated formulations showed lower cytotoxicity in human cells and higher selectivity indices than the free pyrazole compounds [15,16]. On the other hand, other 5-pyrrolyl-pyrazoles I (Figure 1), characterized as our previous 5-PUs by an N1hydroxy-2-phenylethyl chain, evidenced some anti-Mycobacterium activity [13].
In the current scenario of bacterial infections, those caused by bacteria of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter) family, which are recognized as dangerous superbugs by the Infectious Diseases Society of America (IDSA), are becoming a global concern because they are almost untreatable [6]. Unfortunately, ESKAPE bacteria are endowed with an increasing tendency to develop MDR to the bactericidal effect of conventional antibiotics, which are no longer effective and need substitution with more efficient new antibacterial agents suitable for clinical application [6]. To meet the global urgency of new antimicrobial compounds, the aim of this work is to investigate the antibacterial/anti-Mycobacterium activity of a small library of 5-amino functionalized pyrazoles (compounds 2-7, see the workflow of the whole study in Figure 2), structurally related to previously reported BBB4, CB1H, 5-PUs and I. To reduce the lipophilicity (and therefore increase water solubility) and preserve antimicrobial activity, the new pyrazole derivatives do not bear the 3,5-diphenyl groups but share with the parent compounds the N1 2-hydroxy-2-phenylethyl chain identified in previous studies as a relevant pharmacophoric portion [11][12][13]. Particularly, compounds 2 and 3 (Table 1) are characterized by an ureido moiety in the 5 position and a carboxyethyl (derivatives 2a-d) or a tert-butyl (derivatives 3a-d) substituent at C4 and C3, respectively. Compounds 4-6 (Table 1) share with derivatives 2 the 4-carboxyethyl substituent but bear at C5 a benzoyl-thiourea (derivatives 4a-d), an amide (derivatives 5a-d) or a sulfonamide function (derivative 6). Finally, compound 7 is a positional isomer of derivative 4c characterized by a carboxyethyl substituent moved from position 4 to position 3. Within each series of compounds, the substituent at position 5 was varied to define structure-activity relationships (SAR) on the effect of this functionalization on antibacterial/anti-Mycobacterium activity. In fact, except for compounds 5a, all compounds bear at different positions (ortho, meta, para) of the phenyl ring in the functional group at position 5 a fluorine atom or a trifluoromethyl group to increase lipophilicity.
x FOR PEER REVIEW 3 of 15 characterized by an ureido moiety in the 5 position and a carboxyethyl (derivatives 2a-d) or a tert-butyl (derivatives 3a-d) substituent at C4 and C3, respectively. Compounds 4-6 (Table 1) share with derivatives 2 the 4-carboxyethyl substituent but bear at C5 a benzoylthiourea (derivatives 4a-d), an amide (derivatives 5a-d) or a sulfonamide function (derivative 6). Finally, compound 7 is a positional isomer of derivative 4c characterized by a carboxyethyl substituent moved from position 4 to position 3. Within each series of compounds, the substituent at position 5 was varied to define structure-activity relationships (SAR) on the effect of this functionalization on antibacterial/anti-Mycobacterium activity. In fact, except for compounds 5a, all compounds bear at different positions (ortho, meta, para) of the phenyl ring in the functional group at position 5 a fluorine atom or a trifluoromethyl group to increase lipophilicity.       Figure 2. Workflow of the applied strategies leading to the design, biological evaluation and future development of novel pyrazole compounds 2-7.  [11] [11] 2b COOEt H Figure 2. Workflow of the applied strategies leading to the design, biological evaluation and future development of novel pyrazole compounds 2-7.  [11] [11] 2c COOEt H Figure 2. Workflow of the applied strategies leading to the design, biological evaluation and future development of novel pyrazole compounds 2-7.  [11] [11] 2d COOEt H Figure 2. Workflow of the applied strategies leading to the design, biological evaluation and future development of novel pyrazole compounds 2-7.

Chemistry
Compounds 2-7 were synthesized by a divergent synthetic protocol starting from the pyrazole key intermediates 1a-c (Scheme 1) [12,17] by functionalization of the 5-amino group with different electrophilic reagents as isocyanates, benzoylisocyanates, benzoyl chlorides and 4-fluorobenzenesulfonyl chloride. In general, reaction yields are good for compounds 2-4 (from 42% to 51%), and lower for compounds 5 and 6 (from 23% to 61%). In general, compounds derived from pyrazole 1a (substituted in C4 position) were obtained with yields higher than compounds obtained from pyrazoles 1b and 1c (substituted at the C3 position), as demonstrated comparing the reaction yields of compound 4c with its isomer 7 (51% versus 37%), evidencing a different reactivity versus electrophilic reagents of amino group at the 5 position of pyrazole 1a withrespect to pyrazoles 1b,c. As previously reported [11,12], compounds 2a-d and 3a-d, characterized by a urea moiety at the 5 position of the pyrazole nucleus, were synthesized by refluxing in anhydrous toluene 1a or 1c with a small excess (1.1. equivalents) of suitable phenyl isocyanate for 6 h. Compounds 4a-c and 7, in which the urea moiety was transformed into thiourea, were obtained by reacting pyrazoles 1a,b with the suitable benzoyl isothiocyanate, previously prepared modifying a method from the literature [18], in anhydrous THF at reflux for 12 h. Finally, amides 5a-e and the sulphonamide 6 were obtained by condensing 1 with benzoyl chlorides (for derivatives 5) or 4-fluorobenzenesulfonyl chloride (for derivative 6) in anhydrous THF in the presence of triethylamine (TEA) at reflux for 18 h (compounds 5) or 3 days (compound 6).

Pharmacokinetic Properties and Druglikeness Prediction
To evaluate the pharmaceutical relevance of this 5-amino functionalized library pharmacokinetics properties, as well as the druglikeness of compounds 2-7 were ca

Pharmacokinetic Properties and Druglikeness Prediction
To evaluate the pharmaceutical relevance of this 5-amino functionalized library, the pharmacokinetics properties, as well as the druglikeness of compounds 2-7 were calculated by SwissADME [19]. Collectively, an in silico study predicted these compounds to exert good drug-like and pharmacokinetic properties, particularly regarding physicochemical properties, lipophilicity and water solubility. Interestingly, no violations of the Lipinski rules were detected, as well as any pan assay interference compound (PAINS) alerts were reported. Exclusively for compounds 4 and 7, the presence of the thiocarbonyl functionality at the C5 position was spotted as problematic fragment(s) according to the Brenk filters [20]. Overall, the collected data support the pharmaceutical potentials of derivatives 3 and 4.

Antimicrobial Activity
Supported by the promising pharmacokinetic and druglikeness properties predicted for derivatives 2-7 and based on the structural similarities with the previous compounds BBB4, CB1H and I, the antibacterial activity of all compounds was preliminarily evaluated. Out of this initial screening, which established the inactivity of all compounds against bacteria of the Gram-negative species, compounds 3a-c and 4a-c, characterized by a urea moiety (3) or thiourea function (4) at the C5 position, evidenced in some cases promising antibacterial profiles. Based on these results, they have been selected for further evaluation against twenty-one clinical and MDR isolates of the genera Staphylococcus and Enterococcus (Table 4). Compounds were considered inactive against a specific strain when MIC values higher than 128 µg/mL were observed. Thus, compound 3c showed widespread activity (MICs = 32-64 µg/mL) on selected isolates, emerging ineffective only against E. faecalis 365, and less clinically relevant enterococci and staphylococci, including E. gallinarum 150, E. casseliflavus 184, S. simulans 163 and S. haemolyticus 193 strains. Interestingly, the substitution pattern of the urea phenyl ring deeply affects the antibacterial properties of the compounds. Specifically, 5-PUs 3a (ortho-F substituted) and 3b (meta-F substituted) showed reduced activity in comparison with their 4-fluorophenyl analogue 3c, still capable of affecting the duplication of strains not influenced by 3c (namely, E. gallinarum 150 and E. casseliflavus 184). Collectively, compound 3c demonstrated significant antibacterial activity against practically all the isolates considered, regardless of their resistance patterns. Particularly, as reported in Table 4, compound 3c displayed MICs = 32-64 µg/mL against all staphylococci considered in this study, except for one isolate, and MICs = 64 µg/mL against all E. faecium isolates  129 (4a)). Overall, compound 3c and, although with a very narrow spectrum on staphylococci, compound 4b resulted as the most promising molecules between compounds 3a-c and 4a-c for future nano-formulation studies. To further define their potency, the antibacterial effects of 3c and 4b were compared with those of commonly used antibiotics (Table 5). Both compounds showed similar or improved activities in comparison to the reference antibiotics (i.e., ampicillin, ciprofloxacin and oxacillin) against sensitive isolates, thus supporting the pharmaceutical potentials of these pyrazole derivatives as novel antibacterial agents. Compounds 3c (para-F substituted) and 4b (meta-F substituted), characterized by a urea or thiourea function at C5 of the pyrazole nucleus, respectively, were found as the most active derivatives. On the contrary, the presence in this position of an amide (compounds 5) or sulfonamide (compounds 6) moiety negatively affected the antibacterial properties. Furthermore, relevant structural features for antibacterial activity include: (i) the carboxyethyl group at C4 and not at C3 (compare 4a with 7, Table 4); (ii) a tertbutyl substituent at C3 (as in compounds 3). However, the simultaneous presence of the carboxyethyl substituent at C4 and of a urea function at C5 (as in compounds 2) caused a reduction in biological activity. Finally, compounds in which the aromatic ring at the 5 position is not substituted (as in 5a) or presented a trifluoromethyl group (as in 3d and 5d) showed decreased activity with respect to fluoro-substituted analogues.
Data reported in Table 5 established that, both compounds 3c and 4b show promise in the development of new drug delivery systems to specifically counteract infections sustained by staphylococci, where both ciprofloxacin and most beta-lactam drugs are no longer active. Additionally, compound 3c also proved to be promising against enterococci isolates that are difficult to inhibit with ampicillin.

Anti-Mycobacterium Activity
All compounds were preliminary screened at 50 µg/mL for growth inhibition of replicating cultures of Mycobacterium tuberculosis using the Microplate Alamar Blue Assay (MABA). This concentration was selected according to literature data [13] and is normally used in screening protocols to test compounds against Mycobacterium tubercolosis. Addi-tionally, pyrazoles 2-7 were tested against low oxygen-adapted M. tuberculosis containing a luxABCDE reporter under hypoxia using the low oxygen recovery assay (LORA) to assess compound activity against non-replicating cultures [22]. Most compounds 2-7 proved to be inactive, showing a percentage of inhibition concentration lower than 50% in the MABA test. Conversely, selected derivatives 3 and 4 evidenced a moderate activity (Table 6). Particularly, compounds 3c and 4a, characterized by a fluorophenyl substituent in para (3c) or ortho (4a) position, evidenced the most promising results, being able to block the duplication of M. tuberculosis at 50 µg/mL concentration. The derived MIC values for 3c and 4a were 35.8 and 39.3 µg/mL, respectively (Table 6). Unfortunately, none of the tested derivatives showed significant inhibition in the LORA test. Finally, compounds 2-7 underwent extensive preliminary screening to assess their potential to inhibit the growth of MDR M. tuberculosis strains, responsible for a form of tuberculosis resistant to at least two first-line drugs for tuberculosis (Lilly TB Drug Discovery Initiative) [23]. From this large screening, only compound 4a evidenced weak activity at 20 µM concentration (31% of inhibition), representing a good starting point to develop new agents able to counteract MDR M. tuberculosis.
Ethyl  Table 7). To a mixture of 5-amino-pyrazole 1a [17] (1.37 g, 5 mmol) in anhydrous THF (10 mL), TEA (1 mL) and the 4-fluorobenzenesulfonyl chloride (1.17 g, 6 mmol) were slowly added at 0 • C; then, the mixture was refluxed for 3 days. After cooling to room temperature, the solvent was removed under reduced pressure and the crude was solved in DCM (15 mL Table 7). A mixture of 5-amino-pyrazole 1b [11] (0.275 g, 1 mmol) and the 4-fluorobenzoyl isothiocyanate (0.181 g, 1 mmol), previously prepared following a method from the literature [18], in anhydrous THF (10 mL) was refluxed for 12 h. After cooling to room temperature, the white solid obtained was filtered and purified by flash chromatography using diethyl ether as the eluent. White solid.  Table 7).

Microbiology Bacterial Species Considered in This Study
Since in preliminary experiments none of the compounds reported in this study were active on bacteria of the Gram-negative species, in the following experiments several clinical isolates of Gram-positive species were tested for a total of 21 strains of Enterococcus and Staphylococcus genus. All bacteria belonged to a collection obtained from the School of Medicine and Pharmacy of University of Genoa (Italy). Their identification was carried out as described in our previous works [24,25]. Particularly, 9 strains were of the Enterococcus genus, including 3 vancomycin-resistant (VRE) E. faecalis strains, 3 VRE E. faecium isolates, 1 VRE E. durans, 1 VRE E. gallinarum and 1 VRE E. casseliflavus. The other clinical isolates were staphylococci. Particularly, 3 isolates were methicillin-resistant S. aureus (MRSA), 3 were methicillin-resistant S. epidermidis (MRSE), while 6 were strains belongonging to other species of the genus Stafilococcus, of which 3 resistant to methicillin (1 S. hominis, 1 S. simulans and 1 S. haemolyticus) and 3 susceptible to methicillin (including 1 S. saprophyticus, 1 S. warneri and 1 S. lugdunensis).

Determination of the Minimal Inhibitory Concentrations (MICs)
To assess the antimicrobial activity of compounds 3a-c and 4a-c their MICs were determined following the microdilution procedures detailed by the European Committee on Antimicrobial Susceptibility Testing EUCAST [26], as also reported in our previous studies [24,25]. Here, serial 2-fold dilutions of solutions of all the six samples (DMSO), ranging from 1 to 256 µg/mL, were used, using DMSO as control to verify the absence of antibacterial activity of the solvent used for the experiments. All MICs were obtained at least in triplicate, and results were expressed reporting the modal value, that is the value that has been observed most frequently. In the case of equivocal or unclear results, more than three determinations of MICs were carried out.

MABA and LORA Tests
MICs against replicating and non-replicating cultures of M. tuberculosis were determined using the Microplate Alamar Blue Assay (MABA) and the Low Oxygen Recovery Assay (LORA) [22].

Conclusions
To further extend the SARs of BBB4 and CB1H, 5-PUs and I derivatives, the synthesis, characterization, and microbiological evaluation of a small library of trisubstituted pyrazoles have been here reported. In addition, the compounds demonstrating more promise for future development of new drug delivery systems by nanoencapsulation have been found. Structurally, compounds 2-7 shared the N1 2-hydroxy-2-phenylethyl chain, previously identified as a relevant structural determinant for biological activity [8][9][10][11][12][13]. The compounds were prepared by a divergent synthetic protocol starting from the key intermediates 1a-c and were tested on a panel of twenty-one MDR clinical isolates of the Staphylococcus and Enterococcus genus. Compounds 3c and 4b, characterized by a urea or thiourea function at C5 of the pyrazole nucleus, respectively, were found to be the most active derivatives as antibacterial agents, whereas derivatives 3c and 4a show significant, albeit moderate, activity against M. tuberculosis, with 4a being weakly active against an MDR TB strain. Collectively, the presence of an amide (compounds 5) or sulfonamide (compounds 6) moiety at the C5 position negatively affected the biological properties. In silico study predicted these compounds to exert good drug-like and pharmacokinetic properties.
Overall, the collected data support the pharmaceutical potentials of derivatives 3 and 4 as novel antibacterial agents. In fact, the biological results, particularly against clinical isolates of enterococci and staphylococci, confirm that the N1 2-hydroxy-2-phenylethyl chain induces an improvement in antimicrobial activity with respect to previously reported BBB4 and CB1H, which were completely inactive when not encapsulated in proper nanosized macromolecules. Based on the excellent antibacterial effects obtained encapsulating the inactive BBB4 and CB1H in NPs, pyrazoles 3c and 4b here reported represent even more promising candidates for future development of pyrazole-based nano-delivery systems with further enhanced antibacterial properties in terms of potency and spectrum of activity.
In conclusion, the pyrazole-based library here developed contains at least two compounds, namely 3c and 4b which, upon nano-formulation with proper polymer matrices, could provide new pyrazole-based drug delivery systems with enhanced and broaderspectrum antibacterial activity.