Design, Synthesis, Spectral, Structural Analysis, and Biological Evaluation of Novel Pyrazole Derivatives as Anti-Tumor, Antimicrobial, and Anti-Biofilm Agents

TL;DR

This paper reports the design and evaluation of new pyrazole compounds with potential as anti-tumor, antibacterial, and anti-biofilm agents.

Contribution

The paper introduces novel pyrazole derivatives with iodine atoms and demonstrates their promising biological activities.

Findings

Compound 4c showed significant anti-tumor activity without cytotoxicity to normal cells.

Compounds 5a and 5c had the highest antibacterial activity against Staphylococcus aureus.

Several compounds exhibited strong anti-biofilm effects with a minimum biofilm inhibition concentration of 0.023 μg/mL.

Abstract

Objective: Based on our previous findings, we designed new molecules by extending functionalized pyrazole derivatives containing iodine atoms, which are linked via an amino bond to halogen-substituted phenyl groups. In addition, these newly developed pyrazole compounds exhibit anti-tumor, antibacterial, and anti-biofilm activities. Methods: Three new series of pyrazole compounds were designed. Fifteen novel pyrazole derivatives, distributed across three series (4a–d, 5a–d, and 6a–g), were synthesized and structurally characterized by 1H-NMR, 13C-NMR, FTIR, UV-Vis spectroscopy, and elemental analysis. Results: Among them, compound 4c, which exhibited notable anti-tumor activity, crystallized in a monoclinic system and was further analyzed via single-crystal X-ray diffraction. All synthesized compounds were evaluated in vitro on NCTC normal fibroblast cells and HEp-2 tumor epithelial cells. Compound 4c demonstrated significant anti-tumor activity while displaying no cytotoxic effects on normal cells. The antibacterial and anti-biofilm activities of the compounds were also assessed against four bacterial strains. Compounds 5a and 5c exhibited the highest antibacterial activity against Staphylococcus aureus ATCC 25923, both with a minimum inhibitory concentration (MIC) of 0.023 μg/mL. Additionally, compounds 4a, 5a, 6a, 6e, and 6f showed the strongest anti-biofilm effects, each presenting a minimum biofilm inhibition concentration (MBIC) of 0.023 μg/mL. ADME and ADMET in silico predictions indicated that all compounds exhibit generally favorable, drug-like physicochemical properties. Conclusions: The study reinforces the applicability of these compounds as promising anticancer, antibacterial, and anti-biofilm drugs.

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Full text

1. Introduction

Nitrogen-containing heterocyclic frameworks represent fundamental structural motifs in medicinal chemistry [1]. Among these, functionalized azole-type heterocycles, such as pyrazoles [2,3], triazoles [4], and tetrazoles [5], are extensively investigated for the development of new biologically active molecules aimed at combating a variety of diseases. Within this class, pyrazole derivatives stand out due to their broad spectrum of therapeutic activities, including anti-tumor, antibacterial, and antifungal effects [2,6,7,8], as well as anti-inflammatory [8,9], antidiabetic, antioxidant [8,10], antiviral [11], and local anesthetic properties [12,13].

Figure 1 shows small molecules of some azaheterocycles, which are successful drugs approved by the USFDA.

Clinically approved nonsteroidal anti-inflammatory drugs (NSAIDs), such as Tepoxalin, Celecoxib, and Phenylbutazone, as well as Epirizol and Lonazolac, the last two also being analgesic and antipyretic, all have a pyrazole ring in their structure, with two exceptions, and they contain fluorine and chlorine atoms.

Clinically approved drugs with analgesic and antipyretic activity, such as antipyrine and metamizole, are pyrazolones. Pyrazofurin (pyrazomycin), naturally produced by Streptomyces candidus, has anticancer and antiviral properties, while Crizotinib is used as an anticancer medication; both are clinically approved drugs. Sulfaphenazole is a bacteriostatic sulfonamide used to treat or prevent bacterial infections.

Rimonabant is an inverse agonist for the cannabinoid receptor CB1 and was first-in-class for clinical development and anti-obesity. Tartrazine is a synthetic lemon-yellow azo dye primarily used as a food coloring [14].

All these clinically approved drugs contain a pyrazole nucleus, especially fluorine or chlorine atoms and amino groups.

Pathogenic microorganisms are responsible for numerous infectious and potentially life-threatening diseases. Despite the availability of a wide range of clinical antibiotics, the incidence of bacterial and fungal infections continues to rise at an alarming rate. Consequently, the development of new antimicrobial agents remains a pressing need in modern therapeutics [15].

Pyrazole derivatives with antimicrobial properties act primarily by disrupting essential biological processes within bacterial and fungal cells [7].

In our previous work, we reported the synthesis of alkylaminopyrazoles that exhibited enhanced antimicrobial activity [16]. Building on these findings, we extended our research by designing a new series of pyrazole–benzimidazole hybrid molecules, which were subsequently evaluated for their antimicrobial effects against planktonic microbial cells. The synthesized compounds were tested against four bacterial strains, namely, the Gram-positive S. aureus and E. faecalis, as well as the Gram-negative P. aeruginosa and E. coli [16]. Structure–activity relationship (SAR) analysis indicated that the presence of a nitro substituent at position 4 of the 3,5-dimethylpyrazole moiety in the pyrazole–benzimidazole hybrid significantly enhanced antibacterial activity against the tested strains [17].

Globally, cancer remains one of the leading causes of mortality. The pyrazole scaffold is highly versatile and is frequently incorporated into anticancer agents due to its ability to modulate diverse biological pathways, including those involved in cell proliferation and apoptosis [1,18,19].

More recently, we have intensified our investigations on the 3,5-dimethylpyrazole scaffold bearing a nitro substituent at position 4, along with phenyl rings functionalized with halogen atoms and amino groups structural features associated with both anti-tumor and antimicrobial activities [20]. Within this series, compound 4b′ emerged as the most active derivative. It contains a chlorine atom at position 4 of the phenyl ring and exhibited a broad antimicrobial spectrum against all four tested bacterial strains (Figure 2) [20].

Compound 5d′, featuring an iodo substituent on the phenyl ring and crystallizing in a triclinic system, induced apoptosis in HEp-2 cervical carcinoma cells, achieving an apoptotic rate of 20.84% at a concentration of 50 μg/mL. Moreover, this compound demonstrated good biocompatibility when tested on normal fibroblasts across the entire concentration range. The Mannich base 5d′ exhibits the strongest anti-tumor activity within the series, likely due to the presence of iodine, which decreases the hydrophilicity of the molecule and enhances its interaction with the biological receptor (Figure 2) [20].

The pyrazole core is considered a versatile pharmacophore with diverse pharmacological activities. It is used in the design and development of medicines, especially for various ailments.

Structurally, drugs may include a pyrazole ring as such, or it may be linked to other heterocyclic nuclei, such as pyridinic, pyrimidinic, imidazole, benzimadazole, and triazole nuclei. These heterocyclic nuclei can have in their structure the key substituent fluorine atom, but also other halogen atoms, and an amino group attached to the pyrazole nucleus or on the other listed heterocyclic nuclei. These two structural peculiarities mentioned are crucial for the potential applications and therapeutic targets in drug development.

Based on our previous findings, we designed new molecules by extending functionalized pyrazole derivatives bearing iodine atoms, which are linked via an amino bond to halogen-substituted phenyl moieties. In addition, these newly developed pyrazole compounds exhibit anti-tumor, antibacterial, and anti-biofilm activities. Three series of new pyrazole compounds were designed.

Below, we explain the rationale for designing these three series of compounds, focusing on bromine and iodine.

The biological activity of some compounds containing halogen atoms differs; for example, the fluorine atom has modest activity, chlorine has good activity, bromine has maximum activity, and iodine has low activity.

These observations argue in favor of the bromine atom. It has a larger volume than chlorine and high polarizability, and therefore it has increased affinity for the biological target. The bromine atom increases lipophilicity and can facilitate cell membrane crossing and intracellular accumulation, all of which are very relevant in tumor cells.

The study of structure–activity relationships revealed a significant influence of the nature of the halogen on the phenyl ring on the anti-tumor activity of pyrazole compounds, with the brominated analogue exhibiting the highest biological potency. This result suggests that bromine provides a favorable steric and electronic balance for interaction with the biological target. However, given the potential toxicological concerns associated with brominated and iodinated compounds, they should be considered mainly as potency markers, while fluorinated and chlorinated analogues represent more suitable candidates for further development.

Drugs containing halogen atoms (F, Cl, Br, and I) often show improved pharmacological properties, such as increased lipophilicity, membrane permeability, and receptor binding. Strategic halogenation aims to optimize absorption, distribution, and binding affinity. However, halogens can also influence metabolic stability and toxicity, which must be considered.

Iodine and bromine are important in medicinal chemistry. Iodine is well absorbed, essential for metabolism, and used in supplements, drugs, disinfectants, wound care, and X-ray contrast media. Its broad-spectrum antimicrobial action—disrupting cell walls, denaturing proteins, and interfering with nucleic acids—makes iodine-based antiseptics highly effective in medical procedures [21,22].

Bromine is important in medicinal chemistry, used in synthesizing drugs for oxidative stress, cancer, and inflammation. Its derivatives are key in antiseptics, anesthetics, and anticancer agents. While effective against many pathogens with prolonged action, bromine can increase toxicity and accumulation in the body [23].

In conclusion, “bromine and iodine atoms are not intrinsically toxic, and potential adverse effects are dependent on the structural context and overall properties of the compound, the metabolism profile of the compound.”

2. Results and Discussion

2.1. Chemistry

Scheme 1 shows the intermediate pyrazole needed to synthesize the aminopyrazole derivatives of the three series (4a–d, 5a–d, and 6a–g).

Intermediate compound 1a was commercially available, while all the other compounds were synthesized by us.

The intermediate pyrazole derivatives 1b–1d were prepared following previously reported synthetic protocols, achieving good yields for pure compounds: 91%, 78%, and 76% (1b–1d) (Scheme 1) [24,25]. The methylol derivatives 2a (80%) and 2b (59%) were synthesized according to established literature procedures [26,27]. Notably, compounds 2c (63%) and 2d (83%) were synthesized for the first time by our group and have not been previously reported in the literature (Scheme 1) [28]. For the synthesized compounds 2a–2d, the reaction yields after purification are mentioned.

We aimed to synthesize three series of aminopyrazole derivatives, designated as 4a–d (Scheme 2), 5a–d (Scheme 3), and 6a–g (Scheme 4).

As in our previous work, the new compounds were prepared by a modified Mannich reaction, carried out in two steps.

In the first step, methylol derivatives 2a–d were obtained by condensation of pyrazoles 1a–d with a 37% formaldehyde solution (Scheme 1).

In the second step, the intermediate pyrazole compounds 2a–d were reacted with halogenated anilines (F, Cl, Br, and I) (3) in different molar ratios, yielding compounds 4a–d with a single pyrazole nucleus (Scheme 2) or compounds 5a–d with two pyrazole nuclei (Scheme 3), to study the influence of the pyrazole units on biological activity.

To synthesize compounds 6a–g, pyrazole intermediates 2a–d were reacted with anilines carrying chlorine substituents at the 2,4- or 2,6-positions of the phenyl ring, allowing us to compare how the functionalized pyrazole ring affects biological activity (Scheme 4).

The structures of both the intermediate and final compounds were confirmed by spectroscopic methods, and the corresponding retention factor (R_f_) values are provided in the experimental section.

Since the methylene group connecting the pyrazole and phenyl rings is extremely thermally labile, the purification of these compounds was carried out using a preparative column. The stationary phase was alumina (particle size: 50–200 µm), and the elution system consisted of petroleum ether/ethyl ether/methylene chloride/ethyl acetate in a 5:1:2:2 (v/v/v/v) ratio.

2.2. Spectroscopic Characterization of Compounds of Substituted Pyrazoles 4a–d, 5a–d, and 6a–g

2.2.1. FTIR Spectra

The molecular structures of the newly synthesized aminopyrazole derivatives 4a–d, 5a–d, and 6a–g were confirmed by FTIR spectroscopy using potassium bromide pellets.

For compounds 4a–d and 6a–g, the IR spectra indicated the presence of secondary amine functionalities, with ν_N-H_ stretching vibrations observed in the range 3484–3121 cm^−1^. Characteristic aromatic ν_C-N_ stretching vibrations were observed at 1306–1200 cm^−1^, while aliphatic ν_C-N_ stretching appeared at 1186–1050 cm^−1^. The pyrazole ring exhibited stretching vibrations in the ranges 1435–1381 cm^−1^ and 1360–1300 cm^−1^. Additionally, the asymmetric and symmetric stretching vibrations of the nitro group, ν(NO_2_)asym and ν(NO_2_)sym, were detected at 1565–1527 cm^−1^ and 1353–1352 cm^−1^, respectively.

In contrast, the FTIR spectra of compounds 5a–d confirmed the presence of tertiary amines, evidenced by the absence of ν_N-H_ stretching vibrations. Aromatic ν_C-N_ stretching was observed at 1268–1259 cm^−1^, while aliphatic ν_C-N_ stretching appeared consistently at 1222 cm^−1^. The pyrazole ring exhibited characteristic stretching vibrations at 1390–1361 cm^−1^ and 1175–1154 cm^−1^.

2.2.2. Electronic Spectra

The electronic spectra were recorded in ethanol, and the λmax values were in the range 237–761 nm. The absorption bands were assigned to π-π* transitions.

2.2.3. NMR Spectral Analysis

The NMR spectra of the new pyrazole derivatives 4a–c, 5a–d, and 6a–g confirmed their structures. Aminopyrazoles 4a–d and 6a–g presented in their structure one pyrazole ring, whereas 5a–d included two pyrazole residues.

In the ^1^H-NMR spectra of the pyrazoles 4a–d and 6a–g registered in deuterated chloroform, the proton of the NH group appears as a broad singlet or as a triplet. The triplet multiplicity of the NH proton is due to its coupling with the neighboring methylene protons. In the case of series 5a–d containing in their structures two pyrazole residues, the protons of the two methylene groups are magnetically equivalent, showing one set of signals with appropriate integrals.

The ^13^C-NMR spectra of the compounds from the series 4a–c, 5a–d, and 6a–g showed all the expected signals. The chemical shifts for the three carbon atoms in the pyrazole moieties are influenced by the nature of the substituents. The C-3 and C-5 from the pyrazole ring are strongly deshielded due to the vicinity of the nitrogen atoms. The variations in chemical shifts for C-4 in the compounds 4–6 are influenced by the electronic effects of the attached substituents. In the case of pyrazoles containing a hydrogen atom at C-4, the chemical shift is at ca. 106 ppm. The substitution of H-4 with an iodine atom shielded the signal of C-4 with ca. 64 ppm, whereas the substitution with nitro group deshielded C-4 with ca. 131 ppm compared to the hydrogen atom.

The NMR spectra recorded for the compounds are attached in the Supplementary Materials (Figures S1–S30).

2.2.4. X-Ray Crystallography

Given the exceptional anticancer activity of compound 4c, we investigated its structure using X-ray crystallography to analyze how its molecules are packed.

Compound 4c crystallizes in the monoclinic system, space group P2_1_/c (No. 14). The asymmetric unit is shown in Figure 3.

Figure 4 shows packing details for compound 4c, which exhibits hydrogen bonds between two adjacent molecules.

The crystal packing for compound 4c along the a, b, and c axes is depicted in Figure 5.

Compound 4c, which exhibits the best anti-tumor activity and the lowest cytotoxicity, is distinguished by an optimal combination of substituents, namely, the iodine atom located at position 4 of the pyrazole nucleus, which is bulky, highly polarizable, and lipophilic, favoring cell membrane permeation; the methyl groups at positions 3 and 5 of the pyrazole nucleus, which confer rigidity to the core, increase conformational stability, and protect the nucleus from metabolic degradation; and the bromine atom at position 4 of the phenyl nucleus, which is lipophilic and polarizable, strengthening hydrophobic interactions and hydrogen bonding. The amino group acting as a bridge between the two nuclei allows the formation of hydrogen bonds and is essential both for biological activity and for self-assembly in the crystal. These structural features provide an optimal balance between lipophilicity, rigidity, and molecular interaction capacity. The fact that this compound is the only one in the series to generate a single crystal suitable for X-ray diffraction analysis suggests high conformational stability and well-defined intermolecular interactions, characteristics that may be correlated with efficient molecular recognition at the biological level.

The details of the crystal parameters, data collection, and refinement for the compound 4c are listed in Table S1. A summary of selected bond lengths (Å) and angles (o) is given in Table S2. CCDC 2502445, including all supplementary crystallographic data for the compounds 4c and packaging details, is shown in Figures S31–S36.

2.3. Biological Activity

2.3.1. Evaluation of In Vitro Cytotoxicity of the Synthetized Compounds 4a–d, 5a–d, and 6a–g

The new compounds were tested in vitro to evaluate their cytotoxicity on normal NCTC fibroblasts and their anticancer activity on human HEp-2 epithelial tumor cells over a concentration range of 3.1–50 µg/mL. Cell viability was assessed using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide).

A structure–activity relationship (SAR) study was performed to highlight cell viability following the results obtained after testing the compounds on normal NCTC fibroblast cells and HEp-2 tumor epithelial cells.

We investigated the effect of halogen substituents (F, Cl, Br, and I) at position 4 of the phenyl ring on pyrazole nuclei, which vary in number and contain an iodine atom at position 4.

Testing of the compounds on normal NCTC fibroblast cells yielded the following results:

Series of compounds 4a–d:

• Compound 4c with the bromine atom in position 4 of the phenyl ring was biocompatible with normal cells, exhibiting a non-cytotoxic profile across the entire tested concentration range (3.1–50 µg/mL) at both 24 and 48 h, with cell viability values > 80%. Compound 4d with the iodine atom in position 4 of the phenyl ring showed a similar pattern, except at 50 µg/mL after 48 h, where viability decreased to 75.27%.
• Compound 4c (Br) at concentrations of 6.25–12.5 µg/mL appeared to stimulate cell proliferation, producing viability values ranging from 98.59% to 110.43%.
• Compound 4d (I) displayed a comparable effect over the same concentration range, with viability values between 100.54% and 104.02%.
• Compounds 4a with the fluorine atom and 4b with the chlorine atom in position 4 of the phenyl ring exhibited slight cytotoxicity at 24 h of the experiment (at 50 µg/mL, viability < 80%), while at 48 h their mild cytotoxic effects were observed starting at 25 µg/mL and became pronounced at 50 µg/mL (<60% viability).

Series of compounds 5a–d:

• Compounds 5b (Cl) and 5c (Br) were non-cytotoxic at 24 h across the entire concentration range (3.1–50 µg/mL), with cell viability values between 94.47% and 101.70%. However, at 48 h, both compounds exhibited increasing cytotoxicity starting from 12.5 µg/mL, reaching viability values below 35% at 50 µg/mL.

Conclusion for compounds of series 4 and 5:

• Compound 4c (Br) was the most biocompatible with normal NCTC fibroblast cells at both time intervals and across the entire concentration range, achieving a maximum cell viability of 110.43% at 12.5 µg/mL. It was closely followed by compound 4d (I), which showed comparable values.

Figure 6 and Figure 7 show the graphic representation of the cell viability induced by the compounds of the two series 4a–d and 5a–d for the determination of cytotoxicity in vitro on normal NCTC fibroblasts.

Testing on HEp-2 tumor epithelial cells: series of compounds 4a–d and 5a–d:

• Compound 4c (Br) exhibited anti-tumor activity on HEp-2 cells at 48 h, starting at 25 µg/mL (75.68% viability), with a more pronounced effect at 50 µg/mL, where cell viability decreased to 59.95%. Notably, compound 4c was non-cytotoxic to normal NCTC fibroblasts over the same concentration range (25–50 µg/mL), maintaining cell viability > 83%.
• Compound 4d (I) showed moderate anti-tumor activity at 48 h, with HEp-2 cell viability of 75.74% at 25 µg/mL and 62.47% at 50 µg/mL.
• Compound 5b (Cl) displayed anti-tumor activity at 24 h over 25–50 µg/mL, with HEp-2 cell viability of 76.42% and 63.44%, respectively, while remaining non-cytotoxic to normal NCTC cells at the same concentrations. At 48 h, although the anti-tumor effect of 5b (Cl) was stronger (HEp-2 viability: 36.06%), its cytotoxicity toward normal cells also increased, reducing NCTC cell viability to a minimum of 30.43%.

Figure 8 and Figure 9 show the graphic representation of the cell viability induced by the compounds of the two series 4a–d and 5a–d for the determination of cytotoxicity in vitro on tumor Hep-2 epithelial cells.

We analyze how the substituents on the pyrazole nucleus influence the effect of the dichloro substituents located at the 2,4- and 2,6-positions of the phenyl nucleus.

Series of compounds 6a–g:

• Compound 6c contains the iodine atom in position 4 of the pyrazole ring, and the phenyl ring contains chlorine atoms in positions 2,4. It was biocompatible with normal NCTC cells, exhibiting no cytotoxicity across the entire tested concentration range (3.1–50 µg/mL) at both 24 and 48 h, with cell viability values > 85%.
• Compounds 6a, 6b, 6c, and 6d have in common the chlorine atom substituents in positions 2,4 of the phenyl ring, and 6f has the same, but in positions 2,6 it differs in the nature of the substituents of the pyrazole ring. The compounds 6a, 6b, 6c, 6d, and 6f demonstrated high biocompatibility with normal cells over the concentration range of 3.1–25 µg/mL at 24 h, stimulating proliferation of normal fibroblasts, with viability predominantly > 100%. Maximum proliferation was observed for compounds 6a with the unsubstituted pyrazole nucleus (113–121%) and 6b with hydrogen in position 4 of the pyrazole ring and methyl groups in position 3,5 (115.38–117.43%).
• Compound 6b exhibited pronounced cytotoxicity at 50 µg/mL at both 24 and 48 h, with cell viability of 58.59% and 47.54%, respectively.

Testing on HEp-2 tumor epithelial cells: series of compounds 6a–g:

• Compound 6g with nitro in position 4 of the pyrazole ring exhibited a slight anti-tumor effect at 48 h at 25 µg/mL, with HEp-2 cell viability of 78.29%. At the same concentration, 6g was non-cytotoxic to normal NCTC cells, with 82.21% viability.
• Compound 6e with hydrogen in position 4 of the pyrazole ring and methyl groups in position 3,5 and chlorine atoms at the 2,6 positions of the phenyl ring was strongly cytotoxic at the maximum concentration of 50 µg/mL at both 24 and 48 h on both normal and tumor cell lines, reducing viability to 57.34% and 43.55%, respectively.
• Compounds 6a–c did not show any anti-tumor activity on HEp-2 cells over the tested concentration range. Compounds 6d–g exhibited anti-tumor effects only at the maximum concentration (50 µg/mL), predominantly at 48 h.

Figure 10 and Figure 11 show graphic representations of the cell viability induced by the compounds of the series 6a–g for the determination of cytotoxicity in vitro on normal NCTC fibroblasts and on tumor Hep-2 epithelial cells, respectively.

The results are presented in Tables S3 and S4, attached in the Supplementary Materials, and show the cytotoxicity and the anti-tumor activity of the compounds depending on the tested concentration, presented as means ± SDs (n = 3).

The IC_50_ values were calculated, and the results are presented in Table 1 to highlight the cell viability of the compounds from the three series 4a–d, 5a–d, and 6a–g following 48 h in vitro testing on normal NCTC fibroblasts and HEp-2 epithelial tumor cells.

ED_50_ Plus v1.0 software was used to calculate IC_50_ values.

The anti-tumor activity of the newly synthesized compounds was evaluated on HEp-2 human cervical carcinoma cells using the MTT assay after 48 h of treatment. All the compounds from series 5a–d and compound 6e exhibited moderate anti-tumor activity, with IC_50_ values ranging from 36.63 to 49.01 μg/mL. The other compounds showed IC_50_ values above 50 μg/mL and were therefore considered inactive under these conditions.

Dioscin, used as a reference, demonstrated strong anti-tumor activity with an IC_50_ of 14.65 μg/mL. Among the tested compounds, 5c, which contains a bromine atom at position 4 of the phenyl ring and two pyrazole rings, showed the highest anti-tumor activity (IC_50_ = 36.63 μg/mL) but remained moderate compared to dioscin.

At 48 h on normal NCTC fibroblasts, 5c was highly cytotoxic at 50 μg/mL (32.46% cell viability), whereas 4c maintained high biocompatibility (83.53% viability). Compound 4c also displayed significantly higher cytotoxicity toward HEp-2 tumor cells than toward normal cells, highlighting its potential as a promising anti-tumor agent. Consequently, compound 4c demonstrated superior selective anti-tumor activity compared to 5c (Table S3).

2.3.2. Antimicrobial Activity

All newly synthesized compounds from the three series 4a–d, 5a–d, and 6a–g were evaluated in vitro for their antibacterial activity against planktonic microbial cells using the agar diffusion method. The antibacterial activity was assessed against two Gram-positive bacterial strains, Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212, and two Gram-negative bacterial strains, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922. Erythromycin, a clinically used antibiotic for treating various bacterial infections, was employed as the reference drug.

Qualitative Antibacterial Analysis

For the qualitative assessment of antimicrobial activity, the disk diffusion method (Kirby–Bauer) was employed to determine the zone of inhibition, the circular area where bacterial growth is prevented. The qualitative results for the newly synthesized compounds 4a–d, 5a–d, and 6a–g demonstrated inhibitory effects on microbial growth, as evidenced by the formation of growth inhibition zones. These results are summarized in Table 2.

Quantitative Antibacterial Analysis

Following the evaluation of antibacterial activity, the minimum inhibitory concentration (MIC) of the newly synthesized compounds was determined and expressed in μg/mL, as presented in Table 3. The objective was to compare the antibacterial efficacy of the pyrazole compounds with that of erythromycin.

The three series of synthesized compounds were systematically evaluated for their antibacterial activity.

Halogen atoms, particularly fluorine, play a crucial role in medicinal chemistry and drug design due to their interactions, which enhance drug affinity in biological systems, modulate enzymatic activity, and improve both binding and thermal stability [29]. As shown in Figure 12, both halogen substituents and the nitro group significantly affect the antimicrobial activity of the pyrazole and phenyl nuclei [7,30]. The antibacterial activity is largely determined by the substituents on the phenyl and pyrazole rings, with the presence of the pyrazole ring being essential (Figure 13).

Compounds in series 4a–d and 5a–d differ in the number of pyrazole rings while maintaining the same substituents, and all feature halogen atoms (fluorine, chlorine, bromine, and iodine) on the phenyl ring.

Among these, compounds 5a (fluorine at the 4-position of the phenyl ring) and 5c (bromine at the 4-position) exhibited the strongest antibacterial activity against Staphylococcus aureus ATCC 25923, with a minimum inhibitory concentration (MIC) of 0.023 μg/mL.

Compound 4a showed moderate activity against all four tested bacterial strains. Similarly, compounds 5a and 5c displayed moderate activity against Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922, with MIC values of 0.046 μg/mL.

The weakest antibacterial activity was observed for compounds 4b (chlorine) and 4d (iodine) at the 4-position of the phenyl ring, with MICs of 0.187 μg/mL, and for compound 5b (chlorine at the 4-position), with an MIC of 0.093 μg/mL.

Overall, these results indicate that both the type and position of the halogen substituent on the phenyl ring, as well as the presence of the pyrazole ring, strongly influence antibacterial potency.

This finding can be explained by the fact that the fluorine atom can modulate the electronic density of the amino group, favoring hydrogen bonds or interactions with bacterial enzymes, and can stabilize the molecule, reducing oxidative degradation, which can lead to better bioavailability, with better penetration into the bacterial cell wall. Fluorine, although less lipophilic, could interact more efficiently with intracellular targets. The bromine atom offers a balance between volume, polarizability, and stability, which may make it more active than iodine.

The chlorine atom is less polarizable than bromine or iodine; it can generate an unfavorable electronic distribution in the overall structure, affecting the binding to bacterial receptors.

Chlorine can be more easily deactivated metabolically, for example, by oxidative cleavage, which leads to a decrease in biological activity and can destabilize the compound in the biological environment.

The iodine atom is more active due to its greater lipophilicity and larger volume and therefore better Van der Waals interactions, but at the same time it is more easily oxidized and therefore unstable in biological environments, and more prone to the formation of reactive species and therefore more toxic; therefore, it is more rapidly eliminated by cells.

The compounds of the third series, 6a–g, exhibit structural diversity with respect to the substituents on both the pyrazole and phenyl rings. For instance, the phenyl ring bears chlorine atoms at positions 2, 4 or 2, 6, while the pyrazole ring may be unsubstituted or substituted with methyl groups at positions 3 and 5, and with hydrogen, iodine, or nitro groups at position 4.

The number and position of chlorine substituents appear to influence both antibacterial and anti-biofilm activities. Compounds 6e and 6f, which contain a pyrazole nucleus with different substituents and chlorine atoms at the 2, 6 positions of the phenyl ring, show moderate antibacterial activity, with a minimum inhibitory concentration (MIC) of 0.046 μg/mL against three bacterial strains: Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922.

Compound 6g, featuring a pyrazole nucleus with different substituents and chlorine atoms at the 2, 6 positions of the phenyl ring, exhibits the weakest antibacterial activity, with an MIC of 0.187 μg/mL against S. aureus ATCC 25923, P. aeruginosa ATCC 27853, and E. coli ATCC 25922, and 0.375 μg/mL against Enterococcus faecalis ATCC 29212.

Figure 14 shows the structures of the most active compounds from series 5a–d (compounds 5a and 5c) against S. aureus ATCC 25923, which demonstrated an MIC of 0.023 μg/mL.

The twenty antimicrobial activities of compounds 4a, 5a, 5c, 5d, 6e, and 6f against the four tested bacterial strains were superior to those of the reference drug.

Quantitative Anti-biofilm Analysis

The minimum biofilm inhibition concentrations of bacterial biofilms (MBICs) in μg/mL for the tested compounds in series 4a–d, 5a–d, and 6a–g were measured to determine their anti-biofilm activity (Table 4).

Compounds 4a and 5a, bearing a fluorine atom at the 4-position of the phenyl ring, showed the strongest activity against Staphylococcus aureus ATCC25923 (MBIC = 0.023 μg/mL) and moderate activity against Enterococcus faecalis ATCC29212 and Escherichia coli ATCC25922 (MBIC = 0.046 μg/mL).

Compounds 6a, 6b, and 6e also exhibited potent activity against S. aureus ATCC25923 (MBIC = 0.023 μg/mL), while compound 6f was highly active against both S. aureus ATCC25923 and E. faecalis ATCC29212 (MBIC = 0.023 μg/mL).

Furthermore, compounds 6e and 6f displayed moderate activity against E. faecalis ATCC29212, P. aeruginosa ATCC27853, and E. coli ATCC25922 (MBIC = 0.046 μg/mL).

Figure 15 and Figure 16 show the structures of the most active compounds from the three series against Staphylococcus aureus ATCC25923, with an MIC of 0.023 μg/mL.

Figure 17 shows the structures of the most active compound, 6f, against Staphylococcus aureus ATCC25923 and Enterococcus faecalis ATCC29212, with an MBIC of 0.023 μg/mL.

Eighteen anti-biofilm activities of compounds 4a, 5a, 6e, and 6f were also superior to the reference drug across the four tested bacterial strains.

2.4. Predicted ADME and ADMET Profiles

For all synthesized compounds and three clinically approved drugs selected for comparison, a predictive profile was generated. Physicochemical properties, pharmacokinetic parameters, and drug-likeness were assessed using ADME predictions [31,32]. Additional features, including QED, absorption, metabolism, and a broad range of toxicity aspects, were evaluated through ADMET predictions [33,34], with the two prediction platforms providing complementary information.

2.4.1. Evaluation of ADME Profiles

Favorable oral bioavailability falls within an acceptable range of physicochemical parameters: DIMENSION (molecular weight), INSATU (degree of unsaturation), FLEX (flexibility), POLAR (TPSA), LIPO (lipophilicity), and INSOLU (solubility). This is illustrated by the pink areas in the radar models (Figure 18).

The ideal range for all characteristics is indicated by the pink area (size: molecular weight between 150 and 500 g/mol, establishment: proportion of carbon atoms in sp3 hybridization not less than 0.25, flexibility: no more than 6 rotatable bonds, polarity: TPSA between 20 and 130 Å, lipophilicity: log Po/w below 5, and solubility: log S below 6). The Veber rule states that molecules with rotatable bonds and TPSAs equal to or less than 10 and 140 have superior oral bioavailability [35,36,37,38,39].

These characteristics are necessary for a molecule to have improved oral bioavailability.

As shown in Table 5 and Table 6, all the newly synthesized compounds in series 4a–d, 5a–d, and 6a–g fall within the ideal range of the bioavailability radar and all of them comply with the Veber rule, with one exception regarding the molecular weight, which is greater than 500 g/mol, for compounds in series 5a–d.

To further illustrate the ideal bioavailability characteristics of the synthesized compounds, we compared them with those of several clinically approved drugs containing a pyrazole ring (crizotinib, pyrazofurin, and sulfaphenazole). Among these three drugs, only pyrazofurin does not comply with the Veber rule (TPSA > 140 Å).

2.4.2. Evaluation of ADMET Profiles

The ADMET properties of the newly synthesized compounds, as well as those of selected clinically approved drugs, were evaluated using ADMETlab 3.0, an advanced in silico prediction framework designed to estimate pharmacokinetic behavior, toxicity, and safety-related attributes, thereby completing the spectrum of evaluated properties [34].

The ADMET parameter predictions are illustrated by the pink areas in the radar models (Figure 19) [40].

Table 7 and Table 8 present the ADMET properties of the newly synthesized compounds in comparison with three clinically approved drugs containing a pyrazole ring.

The estimates for QED, Synth, and Promiscuous compounds are consistent for all compounds, except QED for the comparator drugs.

Metabolism predictions indicate values within acceptable limits. Very good predictions can be observed for compounds 4a, 4b, and 6b, whereas compounds 6e–g show values close to the upper limit. Overall, the predictions align well with those of the comparator drugs.

The predicted Caco-2 and PAMPA permeability profiles were highly consistent across all compounds, indicating good passive membrane permeability. These results were supported by high human intestinal absorption (HIA) probabilities, suggesting favorable oral absorption potential for the entire series and the comparator drugs.

The predicted toxicity profiles revealed distinct trends across the 4-, 5-, and 6-series compounds, as well as the comparator drugs. Overall, hERG blockade probabilities were low to moderate for most derivatives, indicating a limited potential for cardiotoxicity, with crizotinib exhibiting the highest value.

Compounds from the 4- and 5-series consistently displayed low DILI probabilities. In contrast, compounds from the 6-series—particularly 6a, 6b, 6d, 6e, and 6g—as well as crizotinib and pyrazofurin, showed markedly higher values, with sulfaphenazole exhibiting the highest values, indicating an increased potential for hepatotoxicity potentially associated with this scaffold.

AMES mutagenicity predictions were generally low to moderate across the entire dataset. Compounds 4d, 5b, 5c, and 5d exhibited particularly low AMES scores, whereas slightly higher values were observed for certain 6-series compounds, particularly 6d, 6g, and pyrazofurin, indicating a modest increase in mutagenicity risk. Sulfaphenazole exhibited the highest score.

Assessment of rat oral acute toxicity (ROA) indicated moderate systemic toxicity for most compounds. Slightly higher probabilities were observed for compounds 4a, 5a, 6f, and 6g, whereas lower values were seen for 5d, 6a, 6b, and pyrazofurin, suggesting potentially improved acute tolerability for these compounds.

Carcinogenicity predictions were consistently moderate to high, exhibiting limited variability across the dataset.

Predictions for drug-induced nefrotoxicity were generally low to moderate. The lowest values were observed for 4c, 5b, 5c, and 5d.

Similarly, predictions for A549 cytotoxicity indicated the lowest values for compound 4c and for the entire 5a–d series.

Predictions for hematotoxicity were similar across the series, with the lowest values observed for compounds 4b, 4c, 4d, 5c, and 5d.

All evaluated compounds were considered compliant, indicating generally favorable drug-like ADMET properties. None of the structures were flagged as pan-assay interference compounds (PAINS), reducing the likelihood of assay-related false positives.

Based on the combined evaluation of low DILI and AMES probabilities, moderate hERG liability, and acceptable rat oral acute toxicity, the compounds that best satisfy these criteria are 5b and 5c. Compound 5b exhibits low predicted hepatotoxicity and mutagenicity, along with moderate hERG blockade risk and balanced acute oral toxicity. Similarly, 5c combines minimal DILI and AMES liabilities with moderate predicted cardiotoxicity and systemic toxicity.

In addition, compounds 4d and 5d exhibit very low DILI and AMES scores.

Among all the synthesized compounds and the selected clinically approved drugs, crizotinib exhibits the highest hERG probability. This indicates a potentially higher cardiotoxic risk for this drug, making it less optimal.

Overall, the ADMET properties of the synthesized compounds were found to be consistent, showing optimal profiles in terms of QED, absorption, and metabolism, while toxicity-related parameters fell within acceptable to moderate ranges.

3. Materials and Methods

3.1. Chemistry

The purity of the synthesized compounds was evidenced by determining the technical melting temperature, using TLC, Merck plates with silica gel, an elution system of n-butanol/acetic acid/water = 4:1:5 v/v/v, and spot detection with a UV lamp (λ = 254 nm and 365 nm), by elemental analysis (Perkin Elmer 2400 Series II CHNS/O; Perkin Elmer Waltham, Waltham, MA, USA), the experimental error being within ±0.4%. FT-IR spectra were recorded with a Varian Resolutions spectrometer, and electronic ones were recorded with a VSU-2P Zeiss-Jena spectrophotometer (Analytik Jena GmbH, Jena, Germany). ^1^H-NMR and ^13^C-NMR spectra were recorded with a Varian Gemini 300BB spectrometer (Systems, Palo Alto, CA, USA) at 300 MHz for ^1^H and 75 MHz for ^13^C in CDCl3 with TMS as an internal standard.

X-ray crystallography. The crystal data were collected on a Rigaku R-AXIS RAPID II diffractometer using graphite monochromated Mo-Kα radiation (λ = 0.71075 A) and the ω-ϕ scan technique, at room temperature. The single crystals were mounted on glass fiber. The data were collected with the Crystal Clear program. The structures were solved by direct methods [41] and refined anisotropically using a full-matrix least-squares method based on F2. The atoms were anisotropically refined, calculations were performed using Olex2 [42], and the crystal data for 4c were solved with the olex2.solve [42] structure solution program using Charge Flipping and refined with the olex2.refine [43] refinement package using Gauss-Newton minimization.

3.2. Synthesis and Characterization

3.2.1. General Method for the Synthesis of Series Compounds 4a–4d, 5a–5d, and 6a–6g

Synthesis of compound 4a

A 10 mL solution of 4-fluoroaniline (8.3 mmol) in methylene chloride was added dropwise to a solution of the corresponding 1-(hydroxymethyl)-3,5-dimethyl-4-iodopyrazole 3 (8.3 mmol) in 40 mL methylene chloride. The crystallized crude product was obtained after stirring the reaction mixture for 30 h and removing the solvent under vacuum, monitoring the progress of the reaction by thin-layer chromatography.

Synthesis of N-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-4-fluoroaniline (4a)

Yield 88%; mp 144–145 °C; R_f_ 0.85; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.11, 2.16 (2s, 6H, 2CH_3_), 5.25 (s, 2H, NCH_2_N), 5.35 (s, 1H, NH), 6.64–6.82 (m, 4H, FC_6_H_4_).

^13^C-NMR (75 MHz, CDCl_3_) δ: 12.2, 14.1 (2CH_3_); 60.6 (NCH_2_N); 66.2 (C-4), 115.8, 116.1, 124.5 (4C, C-2′, C-3′, C-5′ C-6′), 140.2, 140.6, 146.2 (C-1′, C-3, C-5), 155.9 (C-4′).

IR (KBr, cm^−1^) ν 3277 w (NH), 1089 i (Caliphatic-N), 1204 vi (Caromatic-N), 1381 w, 1301 w (pyrazole ring); UV-Vis. λmax (log ε) 292 (4.000), 3.66 (2.937), 445 (1.664), 536 (1.217), 761 (0.745) nm. Anal. calcd. for C_12_H_13_FIN_3_ (345.16): C 41.76; H 3.80; N 12.17 Found: C 41.36; H 3.50; N 12.52.

Synthesis of N-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-4-chloroaniline (4b)

Compound 4b was synthesized according to the general procedure described for 4a.

Yield 70%; mp 156–157 °C; R_f_ 0.83; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.10, 2.20 (2s, 6H, 2CH_3_), 4.40 (bs, 1H, NH), 5.26 (s, 2H, NCH_2_N), 6.62 (d, 2H, J = 8.2 Hz, H-2′, H-6′), 7.03 (d, 2H, J = 8.2 Hz, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 12.3, 14.2 (2CH_3_); 59.6 (NCH_2_N); 65.3 (C-4), 115.5, 129.2 (4C, C-2′, C-3′, C-5′, C-6′), 124.3 (C-4′), 140.4 (C-1′), 143.9, 149.7 (C-3, C-5).

IR (KBr, cm^−1^) ν 3280 m (NH), 1058 i (Caliphatic-N), 1258 vi (Caromatic-N), 1430 w, 1311 w (pyrazole ring); UV-Vis. λmax (log ε) 283 (4.000), 367 (2.937), 445 (1.667), 536 (1.223), 761 (0.735) nm. Anal. calcd. for C_12_H_13_ClIN_3_ (361.61): C 39.86; H 3.62; N 11.62 Found: C 40.25; H 3.43; N 11.95.

Synthesis of N-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-4-bromoaniline (4c)

Compound 4c was synthesized according to the general procedure described for 4a.

Yield 92%; mp 123–124 °C; R_f_ 0.81; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.12, 2.24 (2s, 6H, 2CH_3_), 3.60 (bs, 1H, NH), 5.29 (d, 2H, NCH_2_N), 6.45 (d, 2H, J = 8.8 Hz, H-2′, H-6′), 7.42 (d, 2H, J = 8.8 Hz, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 12.6, 14.2 (2CH_3_); 59.3 (NCH_2_N), 64.8 (C-4), 81.0 (C-4′), 116.4, 138.1 (4C, C-2′, C-3′, C-5′, C-6′), 140.3 (C-1′), 145.0, 150.5 (C-3, C-5).

IR (KBr, cm^−1^) ν 3281 i (NH), 1053 vi (Caliphatic-N), 1257 vi (Caromatic-N), 1466 m, 1322 w (pyrazole ring); UV-Vis. λmax (log ε) 292 (4.000), 366 (2.978), 536 (1.189) nm. Anal. calcd. for C_12_H_13_BrIN_3_ (406.06): C 35.49; H 3.23; N 10.35 Found: C 35.11; H 2.85; N 10.62.

Synthesis of N-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-4-iodoaniline (4d)

Compound 4d was synthesized according to the general procedure described for 4a.

Yield 72%; mp154–155 °C; R_f_ 0.65; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.13, 2.23 (2s, 6H, 2CH_3_), 3.80 (bs, 1H, NH), 5.29 (s, 2H, NCH_2_N), 6.58 (d, 2H, J = 8.5 Hz, H-2′, H-6′), 7.18 (d, 2H, J = 8.8 Hz, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 12.3, 14.3 (2CH_3_); 59.5 (NCH_2_N); 64.3 (C-4), 100.1 (C-4′), 116.0, 132.2 (4C, C-2′, C-3′, C-5′, C-6′), 140.3 (C-1′), 144.4, 149.9 (C-3, C-5).

IR (KBr, cm^−1^) ν 3283 i (NH), 1054 i (Caliphatic-N), 1257 vi (Caromatic-N), 1465 m, 1300 w (pyrazole ring); UV-Vis. λmax (log ε) 288 (4.000), 366 (2.937), 4.45 (1.624), 536 (1.179) nm. Anal. calcd. for C_12_H_13_I_2_N_3_ (453.06): C 31.81; H 2.89; N 9.27 Found: C 31.44; H 3.21; N 9,65.

Synthesis of compound 5d

A 10 mL solution of 4-iodoaniline (5.95 mmol) in methylene chloride was dropped over a corresponding chloromethylene solution (40 mL) of 1-(hydroxymethyl)-3,5-dimethyl-4-iodo pyrazole 3 (11.9 mmol). The crude product, 5d, nicely crystallized after stirring the reaction mixture for 30 h and removing the solvent under vacuum, monitoring the progress of the reaction by thin-layer chromatography.

Synthesis of N, N-bis-[(3,5-dimetyl-4-iodo-1H-pyrazol−1-yl)-methyl]-4-fluoroaniline (5a)

Compound 5a was synthesized according to the general procedure described for 5d.

Yield 91%; mp 82–85 °C; R_f_ 0.78; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.13, 2.29 (2s, 6H, 2Me), 5.37 (s, 2NCH_2_N), 6.80–6.87 (m, 4H, H-2′, H-3′, H-5′, H-6′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 12.2, 13.1 (2CH_3_); 64.2 (C-4), 66.2 (2NCH_2_N); 116.1 (C-3′, C-5′), 124.5, 124.6 (d, ^3^JC-F = 8.3 Hz, C-2′, C-6′), 141.2, 142.3, 150.0 (C-1′, C-4′, C-3, C-5).

IR (KBr, cm^−1^) ν 1222 vi (Caliphatic-N), 1262 vi (Caromatic-N), 1361 m, 1155 m (pyrazole ring); UV-Vis. λmax (log ε) 240 (4.000), 299 (3.027), 390 (2.942) nm. Anal. calcd. for C_18_H_20_FI_2_N_5_ (579.20): C 37.33; H 3.48; N 12.09; Found: C 37.72; H 3.19; N 12.45.

Synthesis of N, N-bis-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-4-chloroaniline (5b)

Compound 5b was synthesized according to the general procedure described for 5d.

Yield 84%; mp 101–102 °C; R_f_ 0.55; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.16, 2.25 (2s, 6H, 2Me), 5.42 (s, 4H, 2NCH_2_N), 6.65 (d, 2H, J = 8.7 Hz, H-2′, H-6′), 7.06 (d, 2H, J = 8.7 Hz, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 12.3, 14.2 (2CH_3_); 59.5 (2NCH_2_N); 64.3 (C-4), 115.3, 129.5 (4C, C-2′, C-3′, C-5′, C-6′), 124.1, 140.4 (C-1′, C-4′), 144.0, 149.8 (C-3, C-5).

IR (KBr, cm^−1^) ν 1222 m (Caliphatic-N), 1268 vi (Caromatic-N), 1322 w, 1175 w (pyrazole ring); UV-Vis. λmax (log ε) 245 (4.103), 290 (3.019), 366 (2.912) nm. Anal. calcd. for C_18_H_20_ClI_2_N_5_ (595.65): C 36.30; H 3.38; N 11.76 Found: C 36.28; H 3.40; N 11.75.

Synthesis of N, N-bis-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-4-bromoaniline (5c)

Compound 5c was synthesized according to the general procedure described for 5d.

Yield 86%; mp 113–114 °C; R_f_ 0.63; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.11, 2.29 (2s, 6H, 2Me), 5.26 (d, 4H, NCH_2_N), 6.57 (d, 2H, J = 8.8 Hz, H-2′, H-6′), 7.17 (d, 2H, J = 8.8 Hz, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 11.7, 14.1 (2CH_3_); 43.5 (NCH_2_N); 59.3 (C-4), 111.5 (C-4′), 115.9, 132.2 (4C, C-2′, C-3′, C-5′, C-6′), 140.4 (C-1′), 144.4, 149.8 (C-3, C-5).

IR (KBr, cm^−1^) ν 1222 i (Caliphatic-N), 1259 vi (Caromatic-N), 1390 w, 1155 w (pyrazole ring); UV-Vis. λmax (log ε) 242 (4.105), 299 (3.018) nm. Anal. calcd. For C_18_H_20_BrI_2_N_5_ (640.10): C 33.78; H 3.15; N 10.94; Found: C 34.05; H 3.36; N 11.17.

Synthesis of N,N-bis-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-4-iodoaniline (5d)

Yield 79%; mp 99–100 °C; R_f_ 0.24; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.14, 2.32 (2s, 6H, 2Me), 5.41 (NCH_2_N), 6.52 (d, 2H, J = 8.5 Hz, H-2′, H-6′), 7.40 (d, 2H, J = 8.8 Hz, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 12.3, 14.2 (2CH_3_); 59.2 (NCH_2_N); 64.3 (C-4), 80.9 (C-4′; 116.5, 138.2 (4C, C-2′, C-3′, C-5′, C-6′), 140.4 (C-1′), 145.1, 149.9 (C-3, C-5).

IR (KBr, cm^−1^) ν 1222 i (Caliphatic-N), 1265 vi (Caromatic-N), 1367 w, 1154 w (pyrazole ring); UV-Vis. λmax (log ε) 237 (4.103), 304 (3.027) nm. Anal. calcd. for C_18_H_20_I_3_N_5_ (687.10): C 31.47; H 2.93; N 10.19; Found: C 31.85; H 3.08; N 10.48.

Synthesis of compound 6c

A 10 mL solution of 2,4-dichloroaniline (6.1 mmol) in methylene chloride was added dropwise to a solution of the corresponding 1-(hydroxymethyl)-3,5-dimethyl-4-iodopyrazole 3 (6.1 mmol) in 30 mL methylene chloride. The crystallized crude product was obtained after stirring the reaction mixture for 30 h and removing the solvent under vacuum, monitoring the progress of the reaction by thin-layer chromatography.

Synthesis of N-[(1H-pyrazol-1-yl)-methyl]-2,4-dichloroaniline (6a)

Compound 6a was synthesized according to the general procedure described for 6c.

Yield 68%; mp 103–105 °C; R_f_ 0.84; ^1^H-NMR (300 MHz, CDCl_3_) δ: 4.78 (bs, 1H, NH), 5.58 (s, 2H, NCH_2_N), 6.27 (t, 1H, J = 1.9 Hz, H-3), 6.82 (d, 1H, J = 8.8 Hz, H-6′), 7.09 (dd, 1H, J = 8.8, 2.4 Hz, H-5′), 7.52–7.54 (m, 2H, H-3, H-5).

^13^C-NMR (75 MHz, CDCl_3_) δ: 60.1 (NCH_2_N); 106.5 (C-4), 113.2, 128.0, 129.1, (3C, C-3′, C-5′, C-6′), 120.2, 123.8, 140.3 (3C, C-1′, C-2′, C-4′), 139.8, 145.7 (C-3, C-5).

IR (KBr, cm^−1^) ν 3306 m (NH), 1090 i (Caliphatic-N), 1268 i (Caromatic-N), 1434 w, 1360 w (pyrazole ring); UV-Vis. λmax (log ε) 284 (4.000), 367 (3.024), 446 (1.675), 536 (1.231) nm. Anal. calcd. for C_10_H_9_Cl_2_N_3_ (242.11): C 49.61; H 3,75; N 17.36; Found: C 49.93; H 3.51; N 17.54.

Synthesis of N-[(3,5-dimetyl-1H-pyrazol-1-yl)-methyl]-2,4-dichloroaniline (6b)

Compound 6b was synthesized according to the general procedure described for 6c

Yield 91%; mp 108–110 °C; R_f_ 0.69; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.21, 2.29 (2s, 6H, 2Me), 5.17 (t, 1H, J = 6.4 Hz, NH), 5.40 (d, 2H, J = 6.4Hz, NCH_2_N), 5.80 (s, 1H, H-4), 7.10 (m, 2H, H-5′, H-6′), 7.23–7.24 (m, 1H, H-3′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 11.3, 13.6 (2CH_3_), 58.5 (NCH_2_N), 106.5 (C-4), 114.0, 127.9, 128.8 (3C, C-3′, C-5′, C-6′), 120.2, 123.8, 140.5 (3C, C-1′, C-2′, C-4′), 138.8, 147.7 (C-3, C-5).

IR (KBr, cm^−1^) ν 3382 m (NH), 1162 i (Caliphatic-N), 1275 i (Caromatic-N), 1415 w, 1313 w (pyrazole ring); UV-Vis. λmax (log ε) 229 (4.000), 366 (2.832), 444 (1.607), 536 (1.164) nm. Anal. calcd. for C_12_H_13_Cl_2_N_3_ (270.16): C 53.35; H 4,85; N 15.55; Found: C 53.16; H 5.03; N 15.26.

Synthesis of N-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-2,4-dichloroaniline (6c)

Yield 70%; mp 78–80 °C; R_f_ 0.85; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.24, 2.37 (2s, 6H, 2Me), 5.24 (bs, 1H, NH), 5.47 (d, 2H, J = 4.7 Hz, NCH_2_N), 7.13 (m, 2H, H-5′, H-6′), 7.28 (m, 1H, H-3′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 12.3, 14.2 (2CH_3_), 59.2 (NCH_2_N), 64.8 (C-4), 113.9, 128.0, 128.9 (3C, C-3′, C-5′, C-6′), 120.2, 123.7, 140.2 (3C, C-1′, C-2′, C-4′), 140.4, 149.6 (C-3, C-5).

IR (KBr, cm^−1^) ν 3397 m (NH), 1095 vi (Caliphatic-N), 1216 vi (Caromatic-N), 1411 w, 1309 w (pyrazole ring); UV-Vis. λmax (log ε) 284 (4.000), 368 (2.937), 536 (1.241) nm. Anal. calcd. for C_12_H_12_Cl_2_IN_3_ (396.06): C 36.39; H 3.05; N 10.61; Found: C 36.05; H 3.41; N 10.76.

Synthesis of N-[(3,5-dimetyl-4-nitro-1H-pyrazol-1-yl)-methyl]-2,4-dichloroaniline (6d)

Compound 6d was synthesized according to the general procedure described for 6c

Yield 93%; mp 136–138 °C; R_f_ 0.88; ^1^H-NMR (300 MHz, CDCl_3_δ: 2.50, 2.72 (2s, 6H, 2Me), 5.37 (bs, 1H, NH), 5.52 (d, 2H, J = 6.0 Hz, NCH_2_N), 7.08–7.16 (m, 2H, H-5′, H-6′), 7.25 (bs, 1H, H-3′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 11.8, 14.3 (2CH_3_), 59.4 (NCH_2_N), 131.9 (C-4), 114.0, 128.0, 129.1 (3C, C-3′, C-5′, C-6′), 120.5, 124.3, 140.3 (4C, C-5, C-1′, C-2′, C-4′), 139.7, 145.9 (C-3, C-5).

IR (KBr, cm^−1^) ν 3423 m (NH), 1094 i (Caliphatic-N), 1299 i (Caromatic-N), 1565 i (NO_2_asym), 1353 vi (NO_2_sym), 1401 w, 1304 w (pyrazole ring); UV-Vis. λmax (log ε) 294 (3.306), 366 (2.860), 444 (1.603), 536 (1.172) nm. Anal. calcd. for C_12_H_12_Cl_2_N_4_O_2_ (315.16): C 45.73; H 3.84; N 17.78; Found: C 46.07; H 4.11; N 17.59.

Synthesis of N-[(3,5-dimetyl-1H-pyrazol-1-yl)-methyl]-2,6-dichloroaniline (6e)

Compound 6e was synthesized according to the general procedure described for 6c

Yield 35%; mp 108–110 °C; R_f_ 0.79; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.22, 2.33 (2s, 6H, 2Me), 4.80 (bs, NH), 5.40 (s, NCH_2_N), 5.83 (s, 1H, H-4) 6.85–6.88 (m, 1H, H-4′), 7.20–7.23 (m, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 10.8, 13.2 (2CH_3_); 70.2 (NCH_2_N); 106.3 (C-4), 124.2, 128.7, (3C, C-3′, C-4′, C-5′), 129.1, 140.0 (C-1′, C-2′, C-6′), 146.8, 148.6 (C-3, C-5).

IR (KBr, cm^−1^) ν 3121 w (NH), 1073 vi (Caliphatic-N), 1306 i (Caromatic-N), 1412 w, 1310 w (pyrazole ring); UV-Vis. λmax (log ε) 281 (3.376), 366 (2.864), 446 (1.616), 536 (1.179) nm. Anal. calcd. for C_12_H_13_Cl_2_N_3_ (270.16): C 53.35; H 4.85; N 15.55; Found: C 53.08; H 5.01; N 15.83.

Synthesis of N-[(3,5-dimetyl-4-iodo-1H-pyrazol-1-yl)-methyl]-2,6-dichloroaniline (6f)

Compound 6f was synthesized according to the general procedure described for 6c

Yield 51%; mp 81–82 °C; R_f_ 0.76; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.19, 2.37 (2s, 6H, 2Me), 4.80 (bs, NH), 5.49 (d, 2H, J = 4.7 Hz, NCH_2_N), 6.70–6.92 (m, 1H, H-4′), 7.11–7.26 (m, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 11.6, 13.9 (2CH_3_); 60.7 (NCH_2_N); 64.7 (C-4), 124.3, 128.7 (3C, C-3′, C-4′, C-5′), 139.1, 140.4 (C-1′, C-2′, C-6′), 141.6, 150.2 (C-3, C-5).

IR (KBr, cm^−1^) ν 3348 w (NH), 1050 vi (Caliphatic-N), 1219 i (Caromatic-N), 1427 w, 1311 w (pyrazole ring); UV-Vis. λmax (log ε) 284 (3.285), 366 (2.836), 445 (1.608), 536 (1.173) nm. Anal. calcd. for C_12_H_13_Cl_2_IN_3_ (396.06): C 36.39; H 3.05; N 10.61; Found: C 36.15; H 3.39; N 10.42.

Synthesis of N-[(3,5-dimetyl-4-nitro-1H-pyrazol-1-yl)-methyl]-2,6-dichloroaniline (6g)

Compound 6g was synthesized according to the general procedure described for 6c.

Yield 86%; mp 112–113 °C; R_f_ 0.85; ^1^H-NMR (300 MHz, CDCl_3_) δ: 2.29, 2.52 (2s, 6H, 2Me), 3.47 (bs, NH), 5.50 (NCH_2_N), 6.57–6.61 (m, 1H, H-4′), 7.05–7.40 (m, H-3′, H-5′).

^13^C-NMR (75 MHz, CDCl_3_) δ: 11.2, 14.2 (2CH_3_); 64.8 (NCH_2_N); 131.7 (C-4), 127.8, 129.5 (3C, C-3′, C-4′, C-5′), 135.7, 140.3 (C-1′, C-2′, C-6′), 139.3, 146.6 (C-3, C-5).

IR (KBr, cm^−1^) ν 3484 w (NH), 1186 i (Caliphatic-N), 1200 i (Caromatic-N), 1527 i

(NO_2_asym), 1352 vi (NO_2_sym), 1435 w, 1310 w (pyrazole ring); UV-Vis. λmax (log ε) 295 (4.000), 366 (2.937), 536 (1.168) nm. Anal. calcd. for C_12_H_12_Cl_2_N_4_O_2_ (315.16): C 45.73; H 3.84; N 17.78; Found: C 45.92; H 4.02; N 17.67.

3.3. Biological Assay

3.3.1. Cytotoxicity of Samples

The biocompatibility of the compounds was assessed using the NCTC normal fibroblast cell line (clone 929), while their anticancer activity was evaluated on the HEp-2 human epithelial tumor cell line. Cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, and 500 µg/mL neomycin). In the cytotoxicity assays, dioscin (Merck-Darmstadt, Germany) was used as a reference control and tested under the same conditions as the studied compounds.

For the experiments, the compounds were first dissolved in DMSO (3 mg/mL), and stock solutions at 50 µg/mL in culture medium were prepared. The concentrations tested were 3.1, 6.25, 12.5, 25, and 50 µg/mL. Normal NCTC cells were seeded in 96-well culture plates at a density of 4 × 10^4^ cells/mL, while HEp-2 tumor cells were seeded at 5 × 10^4^ cells/mL. Plates were incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO_2_. After 24 h, the compounds were added in triplicate at the specified concentrations, and the plates were maintained under standard conditions.

Cell viability was determined at 24 and 48 h using the MTT assay. In this assay, the tetrazolium salt reacted with mitochondrial proteins of living cells to form formazan crystals, which were subsequently solubilized with isopropanol. The absorbance of the resulting colored solutions in the wells was measured at 570 nm using a Berthold Technologies LB 940 microplate reader (Bad Wildbad, Germany). In the MTT assay, the optical density of the tested culture solution is directly proportional to the number of viable cells and is expressed as a percentage relative to the optical density of the control culture, which was considered 100%.

3.3.2. Statistical Analysis

Statistical analysis of data was performed using the Student’s t test. Data were expressed as means ± SDs for three independent samples (n = 3). Differences were considered statistically significant at p < 0.05.

3.3.3. Qualitative Evaluation of the Antimicrobial Activity

A 0.5 Standard McFarland suspension was used, which was inoculated onto Mueller–Hinton agar plates; 5 µL of each compound was spotted and incubated overnight at 37 °C. Antimicrobial activity was observed as zones of inhibition formed around the spots.

3.3.4. Quantitative Testing of Antimicrobial Activity on Bacterial Strains

For the quantitative determination of the minimum inhibitory concentration (MIC) (the minimum amount of chemical compound capable of inhibiting the growth of microbial cells), the method of serial microdilutions in liquid TSB (Tryptone Soy Broth) medium was used, using 96-well plates. In a volume of 90 µL of culture medium, serial binary dilutions of the compound stock solution prepared in DMSO (10 µg/mL) were performed. Subsequently, 90 µL of liquid culture medium and 90 µL of chemical compound were pipetted into the first well. Then, 90 µL was transferred from the first well to the second, 90 µL was transferred from the second well to the third, and so on, until the last well, from which 90 µL was discarded. Subsequently, the wells were seeded with 10 µL of microbial suspension with a density of 10^6^ CFU (Colony Forming Units). Microbial suspensions in SPW (sterile physiological water) from cultures obtained on plain agar for 24 h were used. Each test was performed with a microbial culture control and a medium sterility control. After incubation of the plates at 37 °C for 24 h, they were examined macroscopically, reading the absorbance at 620 nm. The mandatory sterility control did not show any bacterial growth; the liquid content remained clear and transparent. The concentration of the chemical compound corresponding to the last well in which no growth of the culture was observed was the minimum inhibitory concentration (MIC) value (mg/mL) for that compound.

3.3.5. Anti-Biofilm Assay

Microbial cells were cultured in 96-well plates with nutrient broth in the presence of the test compounds, then incubated at 37 °C for 24 h. The plates were emptied and washed three times with SPW (sterile physiological water). Then the adherent cells were treated with 110 μL of methanol for 5 min, after which the methanol solution was removed by inversion. The adherent cells were stained with a 1% alkaline crystal violet solution (110 μL/well) for 15 min. When the staining of the plates was observed, the solution was removed, and then the plates were washed under running water. The formed microbial biofilms were resuspended in 33% acetic acid, and the intensity of the stained suspension was assessed by reading the absorbance at 492 nm.

Standard reference compounds, erythromycin and dioscin, were included as positive controls in all biological assays. The choice of these compounds as reference agents is supported by previous studies [44,45].

3.4. ADME and ADMET Predictions

3.4.1. ADME Prediction

SwissADME provides reliable predictive models for estimating physicochemical properties, pharmacokinetic parameters, drug similarity, and the medicinal chemistry compatibility of molecules. The platform incorporates patented methods, which facilitate rapid decision-making by visualizing the oral bioavailability of molecules [32].

3.4.2. ADMET Prediction

The ADMETlab3.0 platform enables probabilistic and categorical predictions covering critical areas of drug distribution and toxicity. It facilitates the large-scale assessment of physicochemical properties of molecules, medicinal chemistry parameters, absorption, metabolism, and toxicity. Pharmacokinetics and toxicity are considered to be undesired factors that contribute to drug development failures. ADMETlab 3.0 has significantly expanded its capabilities, accelerating the drug development process [34].

4. Conclusions

The structures of the 15 compounds in series 4a–d, 5a–d, and 6a–g were confirmed through NMR, FTIR, UV–Vis spectroscopy, and elemental analysis following their synthesis and physicochemical characterization. Compound 4c (Br) was the only member of the series to form single crystals and was further characterized by X-ray diffraction. These structural features provide an optimal balance between lipophilicity, rigidity, and molecular interaction capacity.

The biocompatibility of the newly synthesized compounds was evaluated using the normal fibroblast NCTC (clone 929) cell line, while their anticancer activity was assessed on the human epithelial tumor cell line HEp-2. Cell viability was determined at 24 and 48 h using the MTT assay at concentrations of 3.1, 6.25, 12.5, 25, and 50 µg/mL.

Among the compounds tested, 4c exhibited the highest biocompatibility with normal NCTC fibroblasts at both time points across the full range of concentrations, reaching a maximum cell viability of 110.43% at 12.5 µg/mL. Compound 4d (I) showed comparable results with slightly lower values. Compound 6c (Cl and l) was also highly biocompatible, remaining non-cytotoxic over the entire tested concentration range (3.1–50 µg/mL) at both 24 and 48 h, with cell viability exceeding 85%.

Compound 4c exhibited the strongest anti-tumor activity against HEp-2 cells at 48 h, starting at a concentration of 25 µg/mL (cell viability 75.68%) and reaching a maximum effect at 50 µg/mL, reducing viability to 59.95%. Notably, at the same concentration range (25–50 µg/mL), 4c remained non-cytotoxic toward normal NCTC fibroblasts, maintaining cell viability above 83%.

The enhanced anti-tumor activity of 4c, which contains a bromine atom at the 4-position of the phenyl ring and crystallizes in the monoclinic system, is attributed to the presence of bromine. This substitution reduces the hydrophilic character of the molecule, facilitating improved interaction with the target receptor.

All synthesized compounds were evaluated for antibacterial activity. Among them, compounds 5a (bearing a fluorine atom) and 5c (bearing a bromine atom) at the 4-position of the phenyl ring showed the strongest activity against Staphylococcus aureus ATCC25923, with a minimum inhibitory concentration (MIC) of 0.023 μg/mL, and moderate activity (MIC = 0.046 μg/mL) against the other tested bacterial strains.

Compounds 4a and 5a, containing fluorine, exhibit higher polarity, enhancing their interaction with hydrophilic bacterial receptors and contributing to stronger antimicrobial activity. Furthermore, all synthesized compounds were tested against the same bacterial strains to evaluate their anti-biofilm properties.

Five compounds (4a, 5a, 6a, 6e, and 6f) exhibited the strongest anti-biofilm activity against Staphylococcus aureus ATCC25923, with a minimum biofilm inhibition concentration (MBIC) of 0.023 μg/mL. Notably, compound 6f also showed an MBIC of 0.023 μg/mL against both S. aureus ATCC25923 and Enterococcus faecalis ATCC29212.

The twenty antimicrobial activities of compounds 4a, 5a, 5c, 5d, 6e, and 6f against the four tested bacterial strains were superior to those of the reference drug, and eighteen anti-biofilm activities of compounds 4a, 5a, 6e, and 6f were also superior to the reference drug across the four tested bacterial strains.

In conclusion, compound 4c demonstrated the most potent anti-tumor activity while remaining non-cytotoxic, compounds 4a and 5a displayed strong antimicrobial activity, and five compounds (4a, 5a, 6a, 6e, and 6f) effectively inhibited biofilm formation. These results highlight their potential as promising candidates for therapeutic applications.

Following the evaluation of ADME predictions, all newly synthesized compounds in series 4a–d, 5a–d, and 6a–g have the ideal bioavailability radar range, all obeying Veber’s rule, with one exception regarding molecular weight, with a weight greater than 500g/mol, for compounds in series 5a–d.

ADMET in silico predictions indicated that all the compounds have generally favorable drug-like properties in terms of medicinal chemistry, absorption, metabolism, and toxicity. The study reinforces the applicability of these compounds as promising anticancer, antibacterial, and anti-biofilm drugs.