New Pyrazolyl Thioureas Active against the Staphylococcus Genus

Abstract

To meet the urgent need for new antibacterial molecules, a small library of pyrazolyl thioureas (PTUs) was designed, synthesized and tested against difficult-to-treat human pathogens. The prepared derivatives are characterized by a carboxyethyl functionality on C4 and different hydroxyalkyl chains on N1. Compounds 1a–o were first evaluated against a large panel of Gram-positive and Gram-negative pathogens. In particular, the majority of PTUs proved to be active against different species of the Staphylococcus genus, with MIC values ranging from 32 to 128 µg/mL on methicillin-resistant Staphylococcus strains, often responsible for severe pulmonary disease in cystic fibrosis patients. Time-killing experiments were also performed for the most active compounds, evidencing a bacteriostatic mechanism of action. For most active derivatives, cytotoxicity was evaluated in Vero cells, and at the tested concentrations and at the experimental exposure time of 24 h, none of the compounds analysed showed significant toxicity. In addition, favourable drug-like, pharmacokinetic and toxicity properties were predicted for all new synthesized derivatives. Overall, the collected data confirmed the PTU scaffold as a promising chemotype for the development of novel antibacterial agents active against Gram-positive multi-resistant strains frequently isolated from cystic fibrosis patients.

1. Introduction

The World Health Organization (WHO) estimates that there are 700,000 casualties per year worldwide due to drug-resistant infections with a projection of 10 million deaths by 2050 and a general cost for the global economy up to USD 100 trillion. Unfortunately, the WHO also noted that in 2020, none of the 43 antibiotics in clinical use had fully solved the problem of drug resistance [1]. In this regard, the bacteria included in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species) have been recognized by the Infectious Diseases Society of America (IDSA) as the most dangerous pathogens, due to their remarkable resistance to the most common conventional antibiotics. Additional concerns derived from the isolation of strains resistant to vancomycin and linezolid, considered the last line of defence against Gram-positive bacterial infections [2,3,4]. The clinical management of infections caused by these exceptional pathogens is often complex and problematic, especially in patients hospitalized or suffering from concomitant diseases.

In this regard, cystic fibrosis (CF) is a genetic disorder associated with the production of sticky and thick mucus that accumulates in different organs, including the lungs. This condition facilitates the adhesion and growth of pathogens in the respiratory epithelium and exposes CF patients to respiratory infections, mainly caused by methicillin-resistant Staphylococcus aureus (MRSA). Noteworthy, chronic lung infections with this Gram-positive multi-resistant pathogen have been associated with more severe pulmonary disease and increase the decline in lung function in CF patients [5,6]. Clinical studies on CF patients have shown that the incidence of methicillin-sensitive S. aureus (MSSA) is significantly lower than that of MRSA [7,8,9].

The pyrazole scaffold represents a privileged substructure in medicinal chemistry research and a number of pyrazole derivatives have been evaluated as effective compounds in different therapeutic areas [10,11,12,13,14,15,16], including infectious diseases [17,18] and also against MRSA [19]. In previous studies, pyrazole derivatives I (Figure 1) showed significative antibacterial activity against Gram-positive antibiotic-resistant strains [20]. The prepared molecules were characterized by a N1-hydroxy-2-phenylethyl chain, a carboxyethyl or tert-butyl substituents on C3 or C4 and a (thio)ureido moiety at position 5 of the pyrazole ring. The structure activity relationships (SARs) evidenced that a carboxyethyl group on C4 and a substituted thioureido function on C5 are key structural determinants for the antimicrobial activity of this series (derivatives II, Figure 1). Pyrazolyl thioureas (PTUs) II resulted inactive against all tested Gram-negative species but showed a good antibacterial potency against strains of the Staphylococcus genus resistant to methicillin and linezolid (MIC values between 32 and 64 µg/mL) and also against vancomycin-resistant Enterococcus strains (MIC values between 32 and 64 µg/mL).

The subject of this work is the design and synthesis of a novel small library of PTU molecules (compounds 1a–o) to confirm if this scaffold is a pharmaceutically relevant chemotype for the development of novel antibacterial agents, particularly active against Gram-positive species.

2. Results and Discussion

The novel pyrazole small library of PTUs (compounds 1a–o,

Table 1. Chemical structure, melting point and reaction yield of novel PTU derivatives 1a–o.

Compound	R	X	Melting Point (°C)	Yield (%)
1a	CH3	H	106–108 °C	64%
1b	CH3	o-F	Yellow oil	45%
1c	CH3	m-F	Yellow oil	35%
1d	CH3	p-F	131–133 °C	39%
1e	C2H5	H	Yellow oil	55%
1f	C2H5	o-F	Yellow oil	41%
1g	C2H5	m-F	Yellow oil	40%
1h	C2H5	p-F	120–122 °C	50%
1i	C3H7	H	126–128 °C	55%
1j	C3H7	o-F	Yellow oil	36%
1k	C3H7	m-F	Yellow oil	51%
1l	C3H7	p-F	134–136 °C	61%
1m	C4H9	H	108–110 °C	43%
1n	C4H9	m-F	Yellow oil	47%
1o	C4H9	p-F	114–116 °C	94%

) are characterized by: (1) a carboxyethyl function on C4 as previous derivatives II, (2) an unsubstituted or fluoro-substituted benzoyl thiourea segment on C5 and (3) various N1 hydroxyalkyl chains with different length, replacing the 2-hydroxy-2-phenylethyl chain of I and II. In detail, hydroxypropyl (compounds 1a–d), hydroxybutyl (compounds 1e–h), hydroxypentyl (compounds 1i–l) and finally hydroxyhexyl (compounds 1m–o) chains have been inserted on the N1 position of the pyrazole scaffold.

Novel PTU library 1 was evaluated for their antibacterial activity against several Gram-positive and Gram-negative species, using oxacillin as the reference compound. Time-killing experiments on MRSA strains and cytotoxicity evaluation on Vero cells were also performed for the most active compounds. Additionally, in silico prediction of pharmacokinetic properties, drug-likeness and toxicity (ADMET) of all novel pyrazole compounds was performed.

2.1. Chemistry

Novel pyrazole library was obtained following a consolidate procedure [20], following Scheme 1. Briefly, the condensation of the commercially available oxiranes 2a–d with hydrazine monohydrate led to the corresponding hydrazino-ethanols 3a–d, that were reacted with ethyl ethoxymethylene cyanoacetate in anhydrous toluene (3a) or absolute ethanol (3b–d) at 70–80 °C to give the suitable pyrazole intermediates 4a–d as yellow solids [21]. Finally, the thiourea moiety was introduced via a one-pot reaction in anhydrous THF for 12 h between the 5-amino-pyrazoles 4a–d and the proper benzoyl isothiocyanate 5a–d, commercially available or prepared according to the literature method [22]. Derivatives 1 were obtained in yields ranging from 35% to 94% (Table 1) as yellow oils or crystalline white solids.

2.2. Antibacterial Activity

The antibacterial potency of PTU 1a–o was evaluated against a panel of fifteen bacterial isolates (

Table 2. MIC values (expressed as µg/mL) of PTU 1 and reference compound oxacillin (oxa) against bacteria of the Gram-positive and Gram-negative species, obtained from experiments carried out at least in triplicate. * denotes resistant to methicillin; ** denotes resistant to methicillin and linezolid; ^# denotes resistance to vancomycin; NDM: New Delhi metallo-beta-lactamase producer; MDR: multidrug-resistant strain; CR carbapenem resistant. In bold are evidenced the most active compounds.

	MIC (µg/mL)
Strain	1a	1b	1c	1d	1e	1f	1g	1h	1i	1j	1k	1l	1m	1n	1o	oxa
Gram-positive
S. aureus 17 *	128	>128	64	128	128	>128	64	128	64	>128	64	>128	64	64	>128	256
S. aureus 18 *	128	>128	64	64	64	>128	128	128	64	>128	64	>128	64	64	>128	512
S. aureus 187 *	64	>128	32	32	64	>128	64	64	64	>128	32	>128	32	32	>128	512
S. aureus 195 *	64	>128	64	64	64	>128	64	64	64	>128	64	>128	32	64	>128	256
S. epidermidis 22 *	32	>128	64	32	32	>128	>128	64	32	>128	64	>128	32	64	>128	256
S. epidermidis 171 *	64	>128	128	32	64	>128	128	32	64	>128	64	>128	64	64	>128	128
S. epidermidis 181 **	64	>128	64	32	64	>128	128	32	64	>128	128	>128	64	128	>128	256
E. faecalis 365 #	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	-
E. faecalis 28	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	-
E. faecium 152 #	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	-
E. faecium 158	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	-
Gram-negative
E. coli 462 (NDM, CR)	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	-
E. coli 475 (CR)	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	-
P. aeruginosa 1V MDR	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	-
P. aeruginosa 6G MDR	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	>128	-

), representative of clinically relevant Gram-positive (eleven strains) and Gram-negative (four strains) species including four S. aureus strains (MRSA), three S. epidermidis isolates (two MRSE and one resistant to methicillin and linezolid), two E. faecalis strains (one vancomycin-sensitive and one vancomycin resistant, VRE), two E. faecium isolates (one vancomycin-sensitive and one VRE), two E. coli isolates resistant to carbapenem (one was a New Delhi metallo-β-lactamase (NDM)-producing isolate) and two P. aeruginosa (multidrug-resistant isolates, MDR) strains.

All tested compounds resulted inactive (MIC > 128 µg/mL) against the Gram-negative and Enterococcus genus. Conversely, the majority of derivatives (10 out of 15) showed a widespread activity against the most clinically relevant Staphylococcus species (i.e., S. aureus MRSA and S. epidermidis MRSE), with MIC values lower in some cases than those of oxacillin, used as a reference compound (32–128 mg/mL against 128–512 mg/mL). Interestingly, PTUs 1i and 1m were identified as the most active derivatives of the series because they showed MIC values in a close range (32–64 µg/mL) against seven of the considered Gram-positive isolates. Also compounds 1d, 1e, 1k and 1n resulted active against six Gram-positive strains.

To further define the antibacterial properties of the series, PTUs 1a,d,e,h,i,m were selected as representative examples of differently N1 substituted pyrazoles (1a and 1d, R = Me; 1e and 1h, R = Et; 1i, R = nPr; 1m, R = nBu) and tested against additional Staphylococcus species for a total of 14 isolates (

Table 3. MIC values of some PTUs 1 and reference compound oxacillin (oxa) against other Staphylococcus species, obtained from experiments carried out at least in triplicate, expressed as µg/mL. * denotes resistant to methicillin. In bold are evidenced the most active compounds.

	MIC (µg/mL)
Strains	1a	1d	1e	1h	1i	1m	oxa
S. saprophyticus 41	>128	>128	>128	>128	>128	>128	0.5
S. capitis 71 *	128	64	128	128	128	128	64
S. capitis 121	>128	128	>128	128	>128	>128	0.25
S. warneri 74 *	>128	>128	>128	>128	>128	>128	64
S. simulans 94 *	>128	>128	>128	>128	>128	>128	16
S. simulans 163 *	>128	>128	>128	>128	>128	>128	16
S. lugdunensis 96	32	32	32	32	32	32	1
S. lugdunensis 137 *	32	32	16	32	16	32	16
S. haemoliticus 115 *	128	64	64	64	64	128	64
S. haemoliticus 193 *	>128	128	128	128	>128	>128	16
S. haemoliticus 174	>128	>128	128	>128	>128	>128	0.25
S. hominis 124 *	128	128	128	64	128	>128	16
S. hominis 125 *	128	128	128	128	128	128	24
S. auricularis 136 *	32	32	16	32	16	16	16

). All analysed compounds proved to be ineffective against S. saprophyticus, S. warneri and S. simulans, but showed relevant antibacterial activity against S. lugdunensis and S. auricularis species (MIC value range = 16–32 µg/mL). Moreover, derivative 1d specifically inhibited the proliferation of methicillin-resistant S. capitis 71 strain (MIC = 64 µg/mL), whereas compound 1h affected the growth of S. hominis 124, without influencing the S. hominis 125. Finally, derivatives 1d,e,h,i showed similar activity against S. haemoliticus 115 isolate, resulting ineffective against the other two considered S. haemoliticus strains. Regarding S. auricularis, compounds 1e,i,m resulted the most potent, displaying MIC values of 16 µg/mL.

To investigate whether PTUs act as bacteriostatic or bactericidal, time-killing experiments were carried out on MRSA isolates, very relevant in the daily clinical practice of CF patients. Compounds 1c,d,n,h were selected as representative examples of the chemical diversity of the series and tested against four different MRSA strains (i.e., S. aureus 17, 18, 187 and 195). The experiments were carried out at concentrations four times the MIC values. As exemplified in Figure 2, the four tested compounds proved to act as a bacteriostatic agent, because they were all able to maintain virtually unchanged (10⁵ CFU/mL) the concentration of the initial bacterial inocula for all 24 h of this study. Similar trends were obtained for all the analysed MRSA strains.

2.3. Cytotoxicity Evaluation

To verify if PTUs here reported are characterized by a cytotoxicity activity, selected compounds 1c (R = methyl) and 1n (R = butyl), chosen among the most active ones and as representative examples of the chemical diversity of the series, were tested on Vero cells at the most representative MIC values obtained (32 µg/mL and 64 µg/mL for both compounds, Figure 3).

2.4. Pharmacokinetic Properties, Drug-Likeness and Toxicity (ADMET) Prediction

To further characterize the pharmaceutical potentials of PTUs 1, the drug-likeness and pharmacokinetic properties of the series were calculated using the SwissADME program [23]. Derivatives IIa,b (Figure 1) were used as reference molecules (Table 4). Collectively, this in silico evaluation predicted for PTUs 1 favourable physiochemical and DMPK properties that, in some case, would result better than those of previously described compounds II.

In detail, the replacement of the II phenyl group with aliphatic, linear chains (namely, methyl, ethyl, propyl or butyl R substituents, Table 1) led to an increase in the Csp₃ fraction with an improvement of the predicted bioavailability. Respect to previous II, in most cases the number of rotatable bonds of H bond acceptors and H bond donors are the same of previous II. Moreover, 1 and II would display the similar polarity, as indicated by the topological polar surface area (TPSA) descriptor (137.57 Ų). This descriptor has proved to be indispensable for predicting the permeability of a molecule towards biological membranes. It has in fact been demonstrated that when the TPSA value is greater than 140 Ų, the molecules have difficulty permeating the barriers; on the contrary, when it is less than 140 Ų, the passage through the lipophilic barriers is easier.

The different N1 substituents of the pyrazole ring would also affect the lipophilicity of the compounds, being the logPvalues within the desired range (logP between −0.7 and +5.0) for all analysed PTUs. Except for 1m–o (N1 hydroxyhexyl derivatives), all compounds were predicted to be water soluble rather than moderately soluble as compounds II.

Compounds 1a–i,1m would be highly absorbed in the gastrointestinal tract, whereas derivatives 1j–l and 1n–o, characterized by a fluoro-substituted phenyl ring on thioureido function and a more embedded chain on N1 (hydroxypentyl and hydroxyexyl) would be poorly absorbed. As derivatives II, the novel compounds would not be able to pass the blood–brain barrier (BBB) and enter in the central nervous system. Furthermore, unlike the previous II, derivatives 1 would be substrates of the P-gp efflux pump.

The CYP inhibition properties of compounds 1 would be different from that predicted for derivatives II. Thus, 1A2 and 2D6 isoforms would not be affected by PTUs 1, while 2C19, 2C9 and 3A4 enzymes would be inhibited by derivatives 1e–o.

As derivatives II, no violations of the Lipinski rules have been identified for PTUs 1 that would not display any pan-assay interference compounds (PAINS) alerts. According to the Brenk filters, the thiourea thiocarbonyl group on the C5 position was spotted as a problematic fragment [24].

Table 4. Predicted pharmacokinetics and drug-like properties of compounds 1a–o in comparison with previously synthesized derivatives IIa,b.

	1a	1b–d	1e	1f–h	1i	1j–l	1m	1n–o	IIa,b
Physicochemical Property
MW (g/mol)	376.43	394.42	390.46	408.45	404.48	422.47	418.51	436.50	456.49
Fraction Csp3	0.29	0.29	0.33	0.33	0.37	0.37	0.40	0.40	0.18
Rotatable bonds	10	10	11	11	12	12	13	13	11
H-bond acceptors	5	6	5	6	5	6	5	6	6
H-bond donors	3	3	3	3	3	3	3	3	3
TPSA a (Å2)	137.57	137.57	137.57	137.57	137.57	137.57	137.57	137.57	137.57
Lipophilicity
LogP b	2.06	2.16	2.59	2.69	2.95	3.05	3.49	3.59	3.26
Water solubility
Solubility (mg/mL) c	0.282	0.203	0.133	0.095	0.080	0.057	0.037	0.026	0.018
Solubility class d	S	S	S	S	S	S	MS	MS	MS
Pharmacokinetics
GI absorption	high	high	high	high	high	low	high	low	low
BBB permeant	no	no	no	no	no	no	no	no	no
P-gp substrate	yes	yes	yes	yes	yes	yes	yes	yes	no
CYP1A2 inhibitor	no	no	no	no	no	no	no	no	no
CYP2C19 inhibitor	no	no	yes	yes	yes	yes	yes	yes	yes
CYP2C9 inhibitor	no	no	yes	yes	yes	yes	yes	yes	yes
CYP2D6 inhibitor	no	no	no	no	no	no	no	no	yes
CYP3A4 inhibitor	no	no	yes	yes	yes	yes	yes	yes	yes
Drug-likeness
Lipinski violations	0	0	0	0	0	0	0	0	0
Medicinal chemistry
PAINS alerts	0	0	0	0	0	0	0	0	0
Brenk alerts	1	1	1	1	1	1	1	1	1

^a Topological polar surface area. ^b Predicted according to the XLOGP3 program. ^c Values predicted by the ESOL method [25]. ^d S = soluble; MS = moderately soluble.

Table 4. Predicted pharmacokinetics and drug-like properties of compounds 1a–o in comparison with previously synthesized derivatives IIa,b.

	1a	1b–d	1e	1f–h	1i	1j–l	1m	1n–o	IIa,b
Physicochemical Property
MW (g/mol)	376.43	394.42	390.46	408.45	404.48	422.47	418.51	436.50	456.49
Fraction Csp3	0.29	0.29	0.33	0.33	0.37	0.37	0.40	0.40	0.18
Rotatable bonds	10	10	11	11	12	12	13	13	11
H-bond acceptors	5	6	5	6	5	6	5	6	6
H-bond donors	3	3	3	3	3	3	3	3	3
TPSA a (Å2)	137.57	137.57	137.57	137.57	137.57	137.57	137.57	137.57	137.57
Lipophilicity
LogP b	2.06	2.16	2.59	2.69	2.95	3.05	3.49	3.59	3.26
Water solubility
Solubility (mg/mL) c	0.282	0.203	0.133	0.095	0.080	0.057	0.037	0.026	0.018
Solubility class d	S	S	S	S	S	S	MS	MS	MS
Pharmacokinetics
GI absorption	high	high	high	high	high	low	high	low	low
BBB permeant	no	no	no	no	no	no	no	no	no
P-gp substrate	yes	yes	yes	yes	yes	yes	yes	yes	no
CYP1A2 inhibitor	no	no	no	no	no	no	no	no	no
CYP2C19 inhibitor	no	no	yes	yes	yes	yes	yes	yes	yes
CYP2C9 inhibitor	no	no	yes	yes	yes	yes	yes	yes	yes
CYP2D6 inhibitor	no	no	no	no	no	no	no	no	yes
CYP3A4 inhibitor	no	no	yes	yes	yes	yes	yes	yes	yes
Drug-likeness
Lipinski violations	0	0	0	0	0	0	0	0	0
Medicinal chemistry
PAINS alerts	0	0	0	0	0	0	0	0	0
Brenk alerts	1	1	1	1	1	1	1	1	1

^a Topological polar surface area. ^b Predicted according to the XLOGP3 program. ^c Values predicted by the ESOL method [25]. ^d S = soluble; MS = moderately soluble.

Collectively, this in silico evaluation predicted for this new PTU library good physicochemical, lipophilicity and water solubility properties; in some cases, better than previous II.

In addition, also predicted acute toxicity (lethal dose, LD₅₀) for rats after oral administration (

Table 5. Predicted toxicity properties of compounds 1a–o.

Cpd	LD50 (mg/kg)	Reliability (R.I.)
1a	2600	0.45
1b–d	1600	0.39
1e	2800	0.45
1f–h	1700	0.39
1i	3000	0.45
1j–l	1900	0.40
1m	3300	0.45
1n–o	2000	0.40

) was calculated using the Advanced Chemistry Development (ACD) Percepta platform (ACD/Percepta Platform. Advanced Chemistry Development, Inc.; Toronto, ON, Canada: 2015).

Reliability index values are shown as R.I. (values higher than 0.30 are ranked as reliable by the software). The software prediction is performed based on the software implemented training libraries, which include experimentally determined pharmacokinetic and safety properties for different series of compounds.

Notably, the newly developed compounds 1a–o were predicted to have LD₅₀ values in the 1600–3300 mg/kg range.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Information

All solvents and reagents were purchased from Chiminord s.r.l. (Milan, Italy) and Merk (Aldrich Chemical, Milan, Italy). Solvents were reagent grade. All commercial reagents were used without further purification. For thin-layer chromatography (TLC), aluminium-backed silica gel plates (Merck DC-Alufolien Kieselgel 60 F254, Darmstad, Germany) were used. For chromatography, Merck silica gel, 230–400 mesh, was used. Flash chromatography was performed using the Isolera One instrument (Biotage, Uppsala, Sweden) using a silica gel column. Melting points were not “corrected” and were obtained with a Buchi M-560 instrument (Buchi instruments, Flawil, Switzerland). NMR spectra were recorded on a JEOL JNM ECZ-400S/L1 (400 MHz, Tokyo, Japan).

Elemental analysis was determined with an elemental analyser EA 1110 (Fison-Instruments, Milan, Italy); compounds have been considered pure when the difference between calculated and found values is ± 0.4 (Table S1 in Supporting Material). Hydrazines 3 and pyrazole intermediates 4a–c were prepared according to the already published procedures [21,26]. Benzoyl isothiocyanates 5b–d were prepared according to the literature method [20,22].

3.1.2. Synthesis of Ethyl 5-amino-1-(2-hydroxyhexyl)-1H-pyrazole-4-carboxylate 4d

A mixture of ethyl ethoxymethylene acetate (3.38 g, 20 mmol) and 2-hydrazinohexane-2-ol 3d (2.64 g, 20 mmol) [26] in absolute ethanol (40 mL) was heated at 70–80 °C for 8 h. After cooling at room temperature, the mixture was evaporated under vacuum; 6N HCl (30 mL) was added and the acid solution was washed with diethyl ether (20 mL), then alkalized with 4M NaOH to obtain a yellow solid that was filtered and recrystallized from a mixture of diethyl ether/ligroin (b.p. 40–60 °C) (1:1).

Mp: 103–105 °C. Yield: 57%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.80 (t, J = 7.0, 3H, CH₃), 1.07–1.34 (m, 9H, CH₃ + 3CH₂), 3.73–3.82 (m, 3H, CHOH + NCH₂), 4.11 (q, J = 7.0, 2H, CH₂O), 4.91 (d, 1H, OH, exchangeable with D₂O), 6.05 (br s, 2H, NH₂ exchangeable with D₂O), 7.41 (s, 1H, H-3). ¹³C-NMR (101 MHz, DMSO-d₆): δ 160.71, 149.11, 143.44, 98.61, 69.38, 55.67, 53.06, 31.80, 27.42, 23.44, 16.27. Anal. (C₁₂H₂₁N₃O₃) calcd for C, H, N.

3.1.3. General Synthesis of 5-Thioureido Pyrazoles 1a–o

The proper 5-amino-pyrazoles 4a–d (1 mmol) and the suitable benzoyl isothiocyanate 5a–d (1 mmol), commercially available or previously prepared modifying the literature method [22], in anhydrous THF (10 mL) was refluxed for 12 h. After cooling to room temperature, the solution was concentrated under reduced pressure and the crude was dissolved in ethyl acetate (20 mL); the organic phase was washed with 6N HCl (10 mL), then with NaHCO₃ saturated solution (10 mL) and water (10 mL), dried (MgSO₄) and concentrated under reduced pressure. The crude was purified by flash chromatography using a mixture of diethyl ether/ligroin (b.p. 40–60 °C) 3/1 as the eluent.

Ethyl 5-(3-benzoylthioureido)-1-(2-hydroxypropyl)-1H-pyrazole-4-carboxylate 1a. White solid. M.p.: 106–108 °C. Yield: 64%. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.07 (d, J = 6.0, 3H, CH₃), 1.18 (t, J = 7.0, 3H, CH₃), 3.91–4.05 (m, 3H, CHOH + CH₂N), 4.13 (q, J = 7.0, 2H, CH₂O), 5.05 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.37–8.03 (m, 6H, 5 Ar + H-3), 12.10 (br s, 1H, NH, exchangeable with D₂O), 12.20 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 180.47, 165.88, 161.44, 140.22, 139.93, 134.94, 132.03, 128.50, 128.09, 103.44, 66.87, 60.12, 57.55, 20.94, 14.28. Anal. (C₁₇H₂₀N₄O₄S) calcd for C, H, N, S.

Ethyl 5-(3-(2-fluorobenzoyl)thioureido)-1-(2-hydroxypropyl)-1H-pyrazole-4-carboxylate 1b. Yellow oil. Yield: 45%. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.05 (d, J = 6.0, 3H, CH₃), 1.18 (t, J = 7.0, 3H, CH₃), 3.90–4.09 (m, 3H, CHOH + CH₂N), 4.12 (q, J = 7.0, 2H, CH₂O), 5.05 (br s, 1H, OH exchangeable with D₂O), 7.29–7.87 (m, 4H, Ar), 7.89 (s, 1H, H-3), 11.89 (br s, 1H, NH, exchangeable with D₂O), 12.16 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 176.97, 164.60, 161.87, 158.29, 140.22, 131.76, 129.99, 122.75, 122.22, 115.82, 104.44, 67.63, 59.28, 55.77, 23.81, 16.39. Anal. (C₁₇H₁₉N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-(3-fluorobenzoyl)thioureido)-1-(2-hydroxypropyl)-1H-pyrazole-4-carboxylate 1c. Yellow oil. Yield: 35%. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.07 (d, J = 6.0, 3H, CH₃), 1.22 (t, J = 7.0, 3H, CH₃), 3.90–4.04 (m, 3H, CHOH + CH₂N), 4.13 (q, J = 7.0, 2H, CH₂O), 5.05 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.52–7.87 (m, 4H, Ar), 7.92 (s, 1H, H-3), 12.04 (br s, 1H, NH, exchangeable with D₂O), 12.16 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 178.28, 165.68, 162.48, 160.01, 158.56, 140.22, 140.09, 139.93, 130.66, 122.97, 115.03, 114.47, 104.20, 68.20, 60.08, 57.55, 21.16, 13.60. Anal. (C₁₇H₁₉N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-(4-fluorobenzoyl)thioureido)-1-(2-hydroxypropyl)-1H-pyrazole-4-carboxylate 1d. White solid. M.p.: 131–133 °C. Yield: 39%. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.16 (t, J = 6.0, 3H, CH₃), 1.42 (t, J = 7.0, 3H, CH₃), 4.09–4.30 (m, 3H, CHOH + CH₂N), 4.32 (q, J = 7.0, 2H, CH₂O), 5.05 (br s, 1H, OH exchangeable with D₂O), 7.34–7.71 (m, 4H, Ar), 7.84 (s, 1H, H-3), 11.86 (br s, 1H, NH, exchangeable with D₂O), 11.98 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 175.35, 166.01, 163.54, 161.44, 139.81, 130.17, 129.67, 115.99, 101.92, 68.20, 60.12, 57.55, 16.27, 9.97. Anal. (C₁₇H₁₉N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-benzoylthioureido)-1-(2-hydroxybutyl)-1H-pyrazole-4-carboxylate 1e. Yellow oil. Yield: 55%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 6.0, 3H, CH₃), 1.18 (t, J = 7.0, 3H, CH₃), 1.22–1.47 (m, 2H, CH₂), 3.72–4.04 (m, 3H, CHOH + CH₂N), 4.14 (q, J = 7.0, 2H, CH₂O), 5.02 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.45–8.03 (m, 6H, 5Ar + H-3), 12.10 (br s, 1H, NH, exchangeable with D₂O), 12.20 (br s, 1H, NH, exchangeable with D₂O). ¹³C -NMR (101 MHz, DMSO-d₆): δ 174.97, 167.07, 158.96, 141.86, 140.22, 136.90, 132.41, 128.50, 128.09, 101.68, 74.10, 60.95, 53.62, 30.22, 16.59, 7.13. Anal. (C₁₈H₂₂N₄O₄S) calcd for C, H, N, S.

Ethyl 5-(3-(2-fluorobenzoyl)thioureido)-1-(2-hydroxybutyl)-1H-pyrazole-4-carboxylate 1f. Yellow oil. Yield: 41%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 6.0, 3H, CH₃), 1.03–1.46 (m, 5H, CH₃ + CH₂), 3.74–4.04 (m, 3H, CHOH + CH₂N), 4.14 (q, J = 7.0, 2H, CH₂O), 5.05 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.26–7.90 (m, 4H, Ar), 7.92 (s, 1H, H-3), 11.92 (br s, 1H, NH, exchangeable with D₂O), 12.15 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 176.97, 164.60, 161.47, 158.99, 140.22, 132.22, 129.99, 125.06, 123.62, 116.34, 103.44, 71.50, 60.12, 54.73, 27.50, 14.28, 9.79. Anal. (C₁₈H₂₁N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-(3-fluorobenzoyl)thioureido)-1-(2-hydroxybutyl)-1H-pyrazole-4-carboxylate 1g. Yellow oil. Yield: 40%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.88 (t, J = 6.0, 3H, CH₃), 1.16–1.43 (m, 5H, CH₃ + CH₂), 3.74–4.04 (m, 3H, CHOH + CH₂N), 4.14 (q, J = 7.0, 2H, CH₂O), 5.02 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.38–7.89 (m, 4H, Ar), 7.92 (s, 1H, H-3), 12.07 (br s, 1H, NH, exchangeable with D₂O), 12.16 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 177.08, 166.55, 162.79, 161.44, 160.33, 140.22, 137.37, 130.66, 123.42, 118.99, 115.19, 105.38, 74.10, 63.07, 54.64, 30.54, 15.56, 11.31. Anal. (C₁₈H₂₁N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-(4-fluorobenzoyl)thioureido)-1-(2-hydroxybutyl)-1H-pyrazole-4-carboxylate 1h. White solid. M.p.: 120–122 °C. Yield: 50%. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.21 (t, J = 6.0, 3H, CH₃), 1.60 (t, J = 7.0, 3H, CH₃), 1.63–1.79 (m, 2H, CH₂CH₃), 4.13–4.28 (m, 3H, CHOH + CH₂N), 4.52 (q, J = 7.0, 2H, CH₂O), 5.05 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.33–7.38 and 7.65–7.72 (2m, 4H, Ar), 7.88 (s, 1H, H-3), 11.72 (br s, 1H, NH, exchangeable with D₂O), 11.82 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 177.08, 166.01, 165.26, 163.54, 159.75, 139.50, 130.17, 118.10, 101.44, 71.50, 54.17, 47.55, 24.23, 10.29, 5.80. Anal. (C₁₈H₂₁N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-benzoylthioureido)-1-(2-hydroxypentyl)-1H-pyrazole-4-carboxylate 1i. White solid. M.p.: 126–128 C. Yield: 55%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.83 (t, J = 6.0, 3H, CH₃), 1.18 (t, J = 7.0, 3H, CH₃), 1.25–1.43 (m, 4H, 2CH₂), 3.81–4.03 (m, 3H, CHOH + CH₂N), 4.14 (q, J = 7.0, 2H, CH₂O), 5.00 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.55–8.02 (m, 6H, 5Ar + H-3), 12.10 (br s, 1H, NH, exchangeable with D₂O), 12.19 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 177.06, 165.63, 161.55, 140.22, 140.08, 135.30, 132.14, 128.66, 128.50, 128.09, 128.01, 103.44, 70.17, 60.12, 55.34, 37.09, 18.44, 14.28, 14.12. Anal. (C₁₉H₂₄N₄O₄S) calcd for C, H, N, S.

Ethyl 5-(3-(2-fluorobenzoyl)thioureido)-1-(2-hydroxypentyl)-1H-pyrazole-4-carboxylate 1j. Yellow oil. Yield: 36%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.83 (t, J = 6.0, 3H, CH₃), 1.15–1.42 (m, 7H, CH₃ +2CH₂), 3.82–4.05 (m, 3H, CHOH + CH₂N), 4.15 (q, J = 7.0, 2H, CH₂O), 5.02 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.34–7.90 (m, 4H, Ar), 7.92 (s, 1H, H-3), 11.92 (br s, 1H, NH, exchangeable with D₂O), 12.10 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 175.74, 164.60, 161.47, 158.99, 140.22, 132.22, 132.14, 125.06, 123.62, 116.34, 106.17, 73.08, 60.12, 55.34, 37.09, 20.30, 11.86. Anal. (C₁₉H₂₃N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-(3-fluorobenzoyl)thioureido)-1-(2-hydroxypentyl)-1H-pyrazole-4-carboxylate 1k. Yellow oil. Yield: 51%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.84 (t, J = 6.0, 3H, CH₃), 1.20–1.42 (m, 7H, CH₃ +2CH₂), 3.82–4.02 (m, 3H, CHOH + CH₂N), 4.14 (q, J = 7.0, 2H, CH₂O), 4.99 (br s, 1H, OH exchangeable with D₂O), 7.52–7.87 (m, 4H, Ar), 7.91 (s, 1H, H-3), 12.11 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 178.12, 167.65, 162.79, 161.44, 160.33, 140.22, 135.37, 130.66, 123.42, 119.36, 103.44, 69.14, 59.92, 54.41, 35.50, 18.44, 14.12. Anal. (C₁₉H₂₃N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-(4-fluorobenzoyl)thioureido)-1-(2-hydroxypentyl)-1H-pyrazole-4-carboxylate 1l. White solid. M.p.: 134–136 °C. Yield: 61%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.84 (t, J = 6.0, 3H, CH₃), 1.10–1.40 (m, 7H, CH₃ +2CH₂), 3.82–3.99 (m, 3H, CHOH + CH₂N), 4.13 (q, J = 7.0, 2H, CH₂O), 5.00 (br s, 1H, OH exchangeable with D₂O), 7.38–7.44-(m, 2H, Ar), 7.92 (s, 1H, H-3), 8.05–8.12 (m, 2H, Ar), 12.14 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 174.73, 166.01, 163.54, 161.44, 140.22, 130.26, 115.99, 105.38, 70.72, 60.71, 55.43, 37.09, 18.44, 13.99, 13.20. Anal. (C₁₉H₂₃N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-benzoylthioureido)-1-(2-hydroxyhexyl)-1H-pyrazole-4-carboxylate 1m. White solid. M.p.: 108–110 °C. Yield: 43%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.80 (t, J = 6.0, 3H, CH₃), 1.15 (t, J = 7.0, 3H, CH₃), 1.20–1.38 (m, 6H, 3CH₂), 3.76–4.07 (m, 3H, CHOH + CH₂N), 4.12 (q, J = 7.0, 2H, CH₂O), 4.96 (br s, 1H, OH exchangeable with D₂O), 7.41–7.98 (m, 6H, 5Ar + H-3), 12.06 (br s, 1H, NH, exchangeable with D₂O), 12.18 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 174.73, 166.52, 159.98, 140.22, 140.08, 137.13, 133.20, 128.50, 128.09, 127.68, 102.23, 71.74, 63.63, 57.32, 36.52, 25.26, 21.32, 14.46, 13.20. Anal. (C₂₀H₂₆N₄O₄S) calcd for C, H, N, S.

Ethyl 5-(3-(3-fluorobenzoyl)thioureido)-1-(2-hydroxyhexyl)-1H-pyrazole-4-carboxylate 1n. Yellow oil. Yield: 47%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.85–0.96 (m, 6H, 2CH₃), 1.27–1.31 (m, 6H, 3CH₂), 3.95–4.25 (m, 3H, CHOH + CH₂N), 4.30 (q, J = 7.0, 2H, CH₂O), 7.35–7.92 (m, 4H, Ar), 7.98 (s, 1H, H-3), 9.49 (br s, 1H, NH, exchangeable with D₂O), 12.09 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 174.95, 168.91, 162.24, 161.44, 159.77, 140.29, 136.15, 130.66, 123.42, 118.99, 114.95, 103.44, 70.23, 60.12, 55.30, 35.74, 27.42, 22.70, 14.28, 12.41. Anal. (C₂₀H₂₅N₄O₄SF) calcd for C, H, N, S.

Ethyl 5-(3-(4-fluorobenzoyl)thioureido)-1-(2-hydroxyhexyl)-1H-pyrazole-4-carboxylate 1o. White solid. M.p.: 114–116 °C. Yield: 94%. ¹H-NMR (400 MHz, DMSO-d₆): δ 0.84 (t, J = 6.0, 3H, CH₃), 1.07–1.39 (m, 9H, CH₃ +3CH₂), 3.80–4.03 (m, 3H, CHOH + CH₂N), 4.13 (q, J = 7.0, 2H, CH₂O), 5.00 (d, J = 4.8, 1H, OH exchangeable with D₂O), 7.38–7.44 (m, 2H, Ar), 7.92 (s, 1H, H-3), 8.07–8.12 (m, 2H, Ar), 12.14 (br s, 1H, NH, exchangeable with D₂O). ¹³C-NMR (101 MHz, DMSO-d₆): δ 175.68, 167.91, 167.00, 165.45, 163.13, 139.81, 138.95, 132.11, 130.17, 117.86, 115.76, 102.23, 67.88, 59.53, 53.30, 33.61, 26.67, 22.26, 11.23, 10.92. Anal. (C₂₀H₂₅N₄O₄SF) calcd for C, H, N, S.

3.2. Microbiological Evaluation

3.2.1. Bacterial Species Considered in This Study

A total of 29 clinical strains were used in this study, all belonging to a collection obtained from the School of Medicine and Pharmacy of University of Genoa (Italy), identified by VITEK^® 2 (Biomerieux, Firenze, Italy) or the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric technique (Biomerieux, Firenze, Italy).

Particularly, fifteen strains were used to preliminary test the 15 new PTU derivatives, which were eleven Gram-positive strains (four MRSA Staphylococcus aureus strains), threeStaphylococcus epidermidis isolates (two MRSE and one resistant to methicillin and linezolid), two Enterococcus faecalis strains (one vancomycin-sensitive and one vancomycin-resistant (VRE)), two Enterococcus faecium isolates (one vancomycin-sensitive and one (VRE)) and four Gram-negative isolates (two Escherichia coli isolates, one was a New Delhi metallo-β-lactamase (NDM)-producing isolate and both carbapenem resistant, and two Pseudomonas aeruginosa MDR isolates).

In addition, fourteen isolates of the Staphylococcus genus were also evaluated, including one S. saprophyticus, two S. capitis (one was resistant to methicillin), one S. warneri (resistant to methicillin), two S. simulans (both resistant to methicillin), two S. lugdunensis (one was resistant to methicillin), three S. haemoliticus (two were resistant to methicillin), two S. hominis (both resistant to methicillin) and one S. auricularis (resistant to methicillin).

3.2.2. Determination of the Minimal Inhibitory Concentrations (MICs)

MIC values were determined following the microdilution procedures detailed by the European Committee on Antimicrobial Susceptibility Testing EUCAST [27], as also reported in our previous studies [20,28]. Briefly, serial two-fold dilutions in Mueller–Hinton (MH) broth (Merck, Darmstadt, Germany) of all the fifteen samples (dissolved in DMSO), ranging from 128 to 1 µg/mL, were used. DMSO was also tested as a control to verify the absence of antibacterial activity of the solvent used for the experiments. Cultures of all the selected bacteria, after overnight incubation, were diluted to yield a standardized inoculum of 1.5 × 10⁸ CFU/mL. Appropriate aliquots of each suspension were added to 96-well microplates containing dilutions of compounds to be tested to yield a final concentration of about 5 × 10⁵ cells/mL. After 24 h of incubation at 37 °C, the lowest concentration of sample that prevented visible growth was recorded as the MIC. 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 case of equivocal or not clear results, more than three determinations of MICs were carried out.

3.2.3. Killing Curves

Killing curve assays for most interesting compounds (1a, 1e, 1i and 1m) were performed on the four MRSA isolates selected for this study as previously reported [20,28].

A mid logarithmic phase bacterial culture was diluted in MH broth (10 mL) containing 4× MIC of all the compounds to give a final inoculum of 3.0 × 10⁵ CFU/mL. The same inoculum was added to MH broth, as a growth control. Tubes were incubated at 37 °C with constant shaking for 24 h. Samples of 0.20 mL from each tube were removed at 0, 2, 4, 6 and 24 h, diluted appropriately with a 0.9% sodium chloride solution to avoid carryover of compounds being tested, plated onto MH plates and incubated for 24 h at 37 °C. Growth controls were run in parallel. The percentage of surviving bacterial cells was determined for each sampling time by comparing colony counts with those of standard dilutions of the growth control. Results have been expressed as log₁₀ of viable cell numbers (CFU/mL) of of surviving bacterial cells over a 24 h period. All time-kill curve experiments were performed in triplicate.

3.3. Maintenance of Cell Cultures

Vero cell line, isolated from kidney epithelia cells extracted from Cercopithecus aethiops, was certified by STR DNA profile analysis by Biological Bank, a Core Facility of the IRCCS San Martino University Hospital-IST National Institute for Cancer Research (Genoa, Italy). Vero cells were routinely cultured al 37 °C under 5% CO₂ in a DMEM medium (Euroclone, Milan, Italy) plus 10% heat inactivated FBS serum (Euroclone, Milan, Italy). No antibiotic or anti-fungine solutions were added to the standard or experimental medium in order to avoid any potential interference of these drugs with the experimental conditions. The medium was changed every 2 to 3 days and cells were sub-cultured by TripLE^TM Express (Invitrogen, Life Technologies, Carlsbad, CA, USA) treatment when the original flask was approximately 75% confluent. All cell cultures were found to be mycoplasma-free during regular checks with the Reagent Set Mycoplasma Euroclone (Euroclone, Milan, Italy).

3.3.1. Cell Viability Index

MTT Test

At the end of each experimental treatment, the cell viability was assessed by thiazolyl blue tetrazolium dye reduction assay (MTT) (Euroclone, Milan, Italy) [29]. The optical densities (OD) of the dissolved formazan crystals (for the MTT test) were determined spectrophotometrically at 570 nm.

A chemical compound was considered toxic if the cell viability was reduced by 15% compared to untreated cultures, according to the ECVAM’s guidelines testing any cytotoxic effects of the compounds and in parallel for measuring cell proliferation, according to manufacturer’s instructions.

4. Conclusions

The obtained results pointed at the PTU scaffold as a pharmaceutically relevant chemotype for the development of novel antibacterial agents active against Gram-positive species. In fact, novel PTUs here reported proved to be equally or more active that previous derivatives I. Compounds 1a–o evidenced interesting activity towards Gram-positive resistant pathogen, often associated with severe pulmonary disease in CF patients [30,31,32,33,34]; in fact, several novel PTUs proved to be effective against different species of the Staphylococcus genus, with MIC values ranging from 32 to 128 µg/mL on MRSA and MRSE strains. In addition, time-killing experiments confirm the bacteriostatic actions of this class of compounds.

For all the synthesized compounds, favourable pharmacokinetic properties were calculated, evidencing for a major part of PTUs’ good ADMET properties. Finally, considering the preliminary cytotoxicity results obtained on Vero cells, it is reasonable to assume that the PTU library here reported have low toxicity.

Collected data allow us to draw the following SAR considerations regarding N1 and thiourea moiety substitution (Figure 4): (1) the replacement of hydroxy-2-phenylethyl chain with hydroxyalkyl substituent at N1 improves the antimicrobial activity of the compounds, but make PTUs here reported P-gp efflux pump substrates; (2) the presence of the N1 hydroxyexyl chain at N1 reduced solubility (compounds 1m–o); (3) the ortho-fluoro substitution of the thiourea benzoyl ring is detrimental for activity (as in derivatives 1b,f,j), whereas para and meta fluoro-substituted compounds evidenced a potency comparable to their non-substituted analogues (1a,e,i,m); (4) the simultaneous presence of the fluorine atom and longer hydroxyalkyl chain (hydroxypentyl 1j–l or hydroxyexyl 1n,1o) increases the Csp3 fraction with an improvement of the predicted bioavailability; (5) the carboxyethyl function at C4 position resulted relevant for biological activity, as previously evidenced for derivatives IIa,b.

Additional chemical modifications on the C4 and C3 pyrazole nucleus (as in previous derivatives I [20]) will be performed to extend the SAR consideration about the PTU chemotype.

As in our previous studies on pyrazole derivatives I and II [20], these collected results supported that, upon nano-formulation with proper polymer matrices, the new synthesized compounds could provide novel pyrazole-based drug delivery systems with enhanced and enlarged-spectrum of antibacterial activity, particularly against Gram-positive MRSA and MRSE species.

In conclusion, PTUs here reported could represent a new starting point for the development of new antibacterial pyrazole-based agents [35,36,37,38] able to counteract strains resistant to common antibiotics and consequently a new therapeutic approach for CF patients [39,40,41]. Additional studies will be necessary to identify the mechanism of action of this class of molecules.