Synthesis of New Oxindoles and Determination of Their Antibacterial Properties

A versatile method for the synthesis of new oxindoles was developed by the reaction between substituted isatins and 5-aminopyrazoles. +e reaction was carried out at room temperature in ethanol using p-toluenesulfonic acid as the catalyst. +e products were obtained with acceptable to excellent yields (44–96%). Structures of the new compounds were unambiguously established by spectroscopic and analytical techniques. +e antibacterial activity was determined by microdilution assays. Compounds 3b, 3e, and 3g showed antistaphylococcal activity, particularly compound 3e displayed a potent activity against the vancomycin intermediate Staphylococcus aureus (VISA). Compounds 3i, 3j, and 3o inhibited Neisseria gonorrhoeae growth.

Recently, akur et al. described that isatin-based glucoconjugated oxindoles had potent activity against Plasmodium falciparum with a good selectivity index [32]. Pervez et al. reported some isatin-derived bis-Schiff bases as promising cytotoxic agents and urease inhibitors. Although in other research they evaluated several isatin-3-hydrazonothiazolines as urease inhibitors, despite some good results, substituents like 5-OCF 3 and 5-F over the isatin ring decreased the activity in various cases [33,34]. In a more recent research, Pervez et al. reported several thiosemicarbazone-isatin derivatives with important activity as a urease inhibitors with lower IC 50 (0.87 to 11.23 μM) than thiourea (IC 50 � 22.3 μM) [35][36][37]. According to the abovementioned background about this kind of products, we have designed a series of isatin-pyrazole hybrids that could be effective as antimicrobials. In this sense, we examined the potential inhibitory activity of these novel compounds against several relevant drug-sensitive and -resistant bacteria.

Results and Discussion
2.1. Chemistry. In this work, we focused on the synthesis of highly substituted 3-(4-pyrazolyl)oxindoles 3 bearing lipophilic groups like hexyl and benzyl over the nitrogen atom of isatin, owing to several studies about the relationship of the lipophilic character of compounds and their biological activities [38,39]. Another structural modification undertaken in order to improve the biological activity corresponded to the introduction of different halides such as chlorine and fluorine in the aryl ring, oriented to the known halogen bond effect [40][41][42], which is characterized for showing σ-hole interactions, that could increase the possibility for improving the activity of the studied compounds.
In a first attempt, we carried out the reaction between 3methyl-1-phenyl-1H-pyrazol-5-amine (1a) and isatin (2a) using p-toluenesulfonic acid (PTSA) as the catalyst in ethanol at room temperature (Scheme 1). e reaction was monitored by thin layer chromatography (TLC). After 30 min, a white solid formed, it was isolated by filtration under vacuum, and washed with ethanol. e product 3a, so obtained, did not require further purification with a yield of 69%. Spectroscopic and analytical data (NMR, FT-IR, EI-MS and elemental analysis) of compound 3a matched with the information supplied for the same structure in references [24][25][26][27]. e main 1 H-NMR signals for 3a correspond to the NH 2 protons at 5.28 ppm as a broad singlet, and the hydroxyl proton as a singlet at 6.61 ppm.
In order to evaluate the versatility and scope of this procedure, we performed the reaction, under the same reaction conditions, starting from substituted aminopyrazoles 1a-d and isatins 2a-i (Scheme 2), leading to the formation of diverse 3-(4-pyrazolyl) oxindole derivatives, 3a-p (Scheme 2, Table 1).
Despite the acceptable-to-good reaction yields (44-96%) obtained in our approach, in comparison with the procedure described by Yang et al. [27], remarkably, our methodology required less reaction time and less consumption of energy.
A possible mechanistic route for the described reaction is outlined in Scheme 3. e procedure should involve the protonation of isatin and formation of intermediate (I), and then the 5-aminopyrazole 1 performed the addition over C-3, affording the intermediate (II). Subsequently, a tautomeric process afforded the isolated products 3.  Table 2.

Antibacterial
N. gonorrhoeae and VISA have been identified by the World Health Organization as priority pathogens as resistance has been developed against the first-line treatment and other commonly used antibiotics [43]. ree compounds (3b, 3e, and 3f ) showed antistaphylococcal activity with MICs ranging from 3.9 μg/mL to 250 μg/mL. Compound 3e (the benzyl-chloro-hydroxyl indolin) induced substantial growth inhibition of the VISA strain.
is compound also showed discrete growth inhibition of the other two Staphylococcus strains (MRSA and MSSA). SAR analysis revealed that the 1-benzyl-5-chloro-substitution in the pyrazol-indolin seemed to confer to activity against the VISA strain whereas the 1-hexyl instead of the 1-benzyl improved growth inhibition of MSSA. Figure 1. VISA growth inhibition was improved 4 times in 3e compared with 3b analog suggesting that the activity is clearly enhanced by 5-chloro substitution at the isatin core. Two analogue compounds 3i (the benzyl-fluoro-hydroxyl  Figure 1. e 4-fluoro or 4-chloro substituent at phenyl moiety of the pyrazole ring and the C-5 of the benzyl or hexyl-isatin seemed to be important for gonococcal inhibition. e presence of electron withdrawing substituents at C-5 of the isatin core has been previously associated with biological activity here, as well as moieties at N-1 may also modulate the activity [29]. e ability of the biologically active isatin-pyrazole compounds 3 to induce hemolysis in human red blood cells ranged from 0 to 7%. None of these compounds seemed to be active against E. coli and drug-resistant K. pneumoniae or P. aeruginosa. e most active compounds are highlighted in bold. MIC values >1 mg/mL were considered as not relevant or not active.

Conclusions
In summary, we synthesized a series of 3-(4-pyrazolyl)oxindoles 3 by the reaction of 5-aminopyrazoles 1 with highly substituted isatins 2 using PTSA as catalyst in mild conditions, room temperature, short reaction times, and in acceptable-to-excellent yields (44-96%). Some compounds of this series showed antistaphylococcal and antigonococcal activity; the benzyl substitution at N-1 and chloro substit-

General Information.
Reagents and solvents used were obtained from commercial sources. e progress of the reactions was monitored by TLC with 0.2 μm precoated plates of silica gel 60GF254 (Merck). Melting points were measured using a Stuart SMP3 melting point apparatus. IR spectra were obtained with a Shimadzu IRAffinity-1 with ATR probe. e 1 H and 13 C-NMR spectra were recorded in a BRUKER DPX 400 spectrophotometer operating at 400 and 100 MHz, respectively, using dimethyl sulfoxide-d 6 as the solvent. e mass spectra were obtained on a SHIMADZU-GCMS-QP2010 spectrometer operating at 70 eV.

General Procedure for Preparation of 3-(4-Pyrazolyl)oxindoles 3a-p.
An equimolar mixture of 5-aminopyrazoles 1 and the corresponding isatin 2, in the presence of 10 mol% of p-toluenesulfonic acid in 1 mL of ethanol, was stirred at room temperature for 1 h. e solid formed was filtered under vacuum and washed with ethanol. Products 3 did not require further purification.                [44]. Stock solutions of the novel compounds were diluted in MHB containing 5% DMSO and 0.1% Tween 80 [46] and added to 90 μL of the bacterial inoculum. e microplates were incubated for 24 h at 35-37°C. MICs defined the lowest concentration with visible inhibition of bacterial growth [44] and/or detection using resazurin (125 μg/mL). Gentamicin and tetracycline (Sigma-Aldrich) were included as control of inhibition growth; MHB and DMSO were used as a negative control. Experiments were performed in duplicate. For N. gonorrhoeae, those compounds showing growth inhibition in the screening test were further tested for MIC on agar plates as described by the Centers for Disease Control and Prevention [47] and the Clinical and Laboratory Standards Institute [45] with modifications. Briefly, GC agar supplemented with 1% isovitalex was prepared containing increasing concentrations of the novel compounds and inoculated with 10 μL of the bacterial suspension (i.e., 1 × 10 4 CFU).

Hemolytic Activity. Human red blood cells (huRBC)
were adjusted to 5% hematocrit in phosphate buffered saline (PBS). 240 μL was placed into each well of 96-well plate and subsequently exposed against 200 μg/mL of each compound. Free hemoglobin was measured after 24 h incubation at 37°C, by spectrophotometry (420 nm Cytation 3M, Biotek). Nonspecific absorbance was subtracted from a blank. Determinations were performed by triplicate in at least two independent experiments [48].

Data Availability
e data used to support the findings of this study are provided in Section 4 and in the supplementary materials. Additional inquiries may be directed to the corresponding author.

Conflicts of Interest
e authors declare no conflicts of interest.