Optimization of Pyrazole Compounds as Antibiotic Adjuvants Active against Colistin- and Carbapenem-Resistant Acinetobacter baumannii

The diffusion of antibiotic-resistant, Gram-negative, opportunistic pathogens, an increasingly important global public health issue, causes a significant socioeconomic burden. Acinetobacter baumannii isolates, despite causing a lower number of infections than Enterobacterales, often show multidrug-resistant phenotypes. Carbapenem resistance is also rather common, prompting the WHO to include carbapenem-resistant A. baumannii as a “critical priority” for the discovery and development of new antibacterial agents. In a previous work, we identified several series of compounds showing either direct-acting or synergistic activity against relevant Gram-negative species, including A. baumannii. Among these, two pyrazole compounds, despite being devoid of any direct-acting activity, showed remarkable synergistic activity in the presence of a subinhibitory concentration of colistin on K. pneumoniae and A. baumannii and served as a starting point for the synthesis of new analogues. In this work, a new series of 47 pyrazole compounds was synthesized. Some compounds showed significant direct-acting antibacterial activity on Gram-positive organisms. Furthermore, an evaluation of their activity as potential antibiotic adjuvants allowed for the identification of two highly active compounds on MDR Acinetobacter baumannii, including colistin-resistant isolates. This work confirms the interest in pyrazole amides as a starting point for the optimization of synergistic antibacterial compounds active on antibiotic-resistant, Gram-negative pathogens.


Introduction
Antibacterial agents of both natural and synthetic origins represent invaluable therapeutic resources that not only have allowed for the effective treatment of once deadly bacterial infections, but also have paved the way for (and still is a pillar of) modern medicine (including organ transplantations, invasive surgery, etc.). However, the effectiveness of antibacterial drugs is undermined by the emergence and spread of resistant strains, which have developed resistance mechanisms to evade the activity of virtually all classes of antibacterial drugs [1,2]. Several other factors (the low financial attractiveness of antibiotics and costs of discovery and development, the evolution of clinically relevant species with multidrug resistance, and more stringent requirements for the approval of new drugs) have led to a significant shortage of new antibacterial drugs on the market in the last two decades and, consequently, to the increase in resistance to antibiotics, especially carbapenems, which are increasingly used to treat infections caused by multidrug-resistant

Chemistry
Considering the paucity of information available regarding the structural determi- Figure 1. Chemical structure of hit compounds identified during a previous antibacterial screening campaign of an unfocused library [12].

Chemistry
Considering the paucity of information available regarding the structural determinants for synergistic antibacterial activity with the colistin of hit compounds 1 and 2, we kept the pyrazole ring as the critical structural element and undertook a wide exploration of the chemical space around this ring to obtain, in the end, a library of 47 pyrazole derivatives. Based on the hit compound 1, a first group of 20 pyrazole 4-carboxamide derivatives ( Figure 2) was synthesized by varying substituents R1, R2, and R3, whereas a wider investigation of chemical substitution at every position of the pyrazole ring, starting from the hit compound 2, led to a second family of derivatives characterized by the presence of a carboxamide substituent at position 3 ( Figure 3). No particular attention was paid to the design of compounds with optimal physicochemical properties, favoring, in the first instance, the exploration of chemical space, and postponing the structural optimization of the most promising compounds to a later stage. Compounds 3-22, featuring a carboxamide group at position 4, were prepared as outlined in Schemes 1-3, whereas the preparation of compounds 23-49, featuring a carboxamide substituent at position 3 of the pyrazole nucleus, as shown by the hit compound 2, is described in Schemes 4 and 5. The reaction between the appropriate hydrazines and diethyl ethoxymethylenemalonate (Scheme 1) yielded the 5-aminopyrazole derivatives 50-53, of which 50-52 were converted into the pyrrol-1-yl derivatives 54-56 by using the Clauson-Kaas procedure [13]. Compound 56 was subjected to a Suzuki-Miyaura reaction with p-tolylboronic acid to produce compound 57. Esters 54, 55, and 57 were, in turn, hydrolyzed [14] to the corresponding acids 58-60, which were subjected to an amidation reaction, which was conducted by following previously published procedures [15], to give the final amides 3-14.
To investigate whether the particular antibacterial properties of compound 40 (vide infra) might depend on the acidity of its carboxyl group or on the position of the same on the benzene ring, a small family of compounds was synthesized (Scheme 5) in which the carboxyl group was shifted to the meta-or ortho-position (compounds 42 and 44), or replaced by other functional groups with different pK a values, namely, a phenolic hydroxyl (45) or a sulfonamide (46), acylsulfonamide (47), or sulfoximine group (49). Acid 68 was amidated with methyl 3-aminobenzoate using EDC/HOBt as coupling reagents to give the intermediate 41, which was then hydrolyzed (NaOH/MeOH) to obtain the free carboxylic acid 42. Similarly, 68 was also converted to 43 using 2-aminobenzoate methyl ester, which was then hydrolyzed to 44, whereas for the preparation of 45, 4-aminophenol PyBOP/HOBt carboxyl group was shifted to the meta-or ortho-position (compounds 42 and 44), o placed by other functional groups with different pKa values, namely, a phenolic hydr (45) or a sulfonamide (46), acylsulfonamide (47), or sulfoximine group (49). Acid 68 amidated with methyl 3-aminobenzoate using EDC/HOBt as coupling reagents to giv intermediate 41, which was then hydrolyzed (NaOH/MeOH) to obtain the free carbo acid 42. Similarly, 68 was also converted to 43 using 2-aminobenzoate methyl ester, w was then hydrolyzed to 44, whereas for the preparation of 45, 4-aminophenol BOP/HOBt in dry DMF was used. The activation of 68 to the corresponding acyl chlo followed by a reaction with sulfanilamide led to 46, which was, subsequently, tr formed into the acylsulfonamide 47 via a treatment with methyl chloroformate. The tion between acid 68 and 4-methylthioaniline, as described for the synthesis of 41 an gave the amide 48 which, when treated with (diacetoxyiodo)benzene in the presen ammonium hydroxide as a nitrogen source, gave the sulfoximine derivative 49.

Biological Evaluation of Pyrazole-4-and Pyrazole-3-Carboxamide Derivatives
Compounds 3-40, 42, 44-47, and 49 (Schemes 1-5) were subjected to a detailed in tigation of their direct and synergistic antibacterial activity and potential cytotoxicit using several methods. The direct antibacterial activity of these compounds was d mined using both agar diffusion and broth microdilution methods. The former was tially used to identify potentially active compounds and was tested on eight refer strains, with representatives of both Gram-positive and Gram-negative clinically rele

Biological Evaluation of Pyrazole-4-and Pyrazole-3-Carboxamide Derivatives
Compounds 3-40, 42, 44-47, and 49 (Schemes 1-5) were subjected to a detailed investigation of their direct and synergistic antibacterial activity and potential cytotoxicity by using several methods. The direct antibacterial activity of these compounds was determined using both agar diffusion and broth microdilution methods. The former was initially used to identify potentially active compounds and was tested on eight reference strains, with representatives of both Gram-positive and Gram-negative clinically relevant bacterial species or genera ( Figure 4).  As somewhat expected, considering that the parent compounds were selected for their synergistic rather than direct-acting antibacterial activity [12], limited growth inhibition was observed for these compounds as the best compounds showed a growth inhibition zone with a diameter of a max. of 7 mm. Interestingly, some compounds, specifically 15, 16, 17, 19, and 42, showed a broad-spectrum, though moderate, direct-acting antibacterial activity. All these compounds, which are analogues of parent compound 1, but where no direct antibacterial activity was observed [12], are characterized by the presence of free carboxylic acid on the carboxamide substituent at position 4 of the pyrazole heterocycle. However, none of these compounds exhibited MIC values lower than 512 µ g/mL when tested using a broth microdilution method. As somewhat expected, considering that the parent compounds were selected for their synergistic rather than direct-acting antibacterial activity [12], limited growth inhibition was observed for these compounds as the best compounds showed a growth inhibition zone with a diameter of a max. of 7 mm. Interestingly, some compounds, specifically 15, 16, 17, 19, and 42, showed a broad-spectrum, though moderate, direct-acting antibacterial activity. All these compounds, which are analogues of parent compound 1, but where no direct antibacterial activity was observed [12], are characterized by the presence of free carboxylic acid on the carboxamide substituent at position 4 of the pyrazole heterocycle. However, none of these compounds exhibited MIC values lower than 512 µg/mL when tested using a broth microdilution method.
Notable exceptions were represented by 40, 42, 44, and 47, which showed a remarkable antibacterial activity on Gram-positive organisms (although slightly lower on Enterococcus faecalis), with growth inhibition zones having diameters of up to 18 mm. This activity was confirmed in broth microdilution assays and translated in MIC values ranging from 32 µg/mL for Bacillus subtilis, Enterococcus faecalis, and Staphylococcus aureus, to 16 µg/mL for Streptococcus pyogenes. From a structural standpoint, these four appear to be related to parent compound 2, as they feature a 3-carboxamide (rather than a 4-carboxamide) substituent. Interestingly, the 3-carboxamide substituent also contains a free carboxylic group in three of these compounds, although the nature of the linker varies from that of the analogs of 1, being made up of a phenyl rather than an aliphatic chain. Overall, these data suggest that the introduction of a free carboxyl group into parent compounds 1 and 2 leads to an improvement, albeit modest overall, in their direct-acting antibacterial activity against Gram-positive organisms, which is maintained when the acid group is moved to other positions on the benzene ring or replaced by a functional group, such an acylsulfonamide, with a similar pK a value. Although potentially interesting, none of these compounds exhibited sufficient activity on Gram-negative bacteria, which nowadays, represent a far more relevant target for the discovery of novel antibacterial scaffolds, and were not further studied in this work.
Second, we established a rapid screening method to identify compounds showing synergistic activity with antibiotics on representative Gram-negative species. This method was based on the detection of growth inhibition in liquid medium (Mueller-Hinton II culture medium) in the presence of a single concentration of the compound (64 µg/mL) and subinhibitory concentrations (0.5 × MIC) of colistin (see the Materials and Methods section for details), as this antibiotic appears to be the best potentiator of parent compounds [12]. From this analysis, 11 compounds (8, 9, 13, 20, 22, 27, 28, 31, 39, 45, and 49) were identified that showed synergistic activity on at least one organism (out of the four representative Gram-negative bacteria) ( Table 1). a Compounds tested at 64 µg/mL, 1% DMSO was used as the negative control (no growth inhibition observed); b colistin concentration was 0.12 µg/mL (Eco and Kpn) or 0.25 µg/mL (Pae and Aba), corresponding to 0.5 × MIC; c -, not determined due to limited solubility of the compound in these conditions. Interestingly, these compounds were structurally different from those showing directacting activity, and were analogues of both parent compounds 1 and 2. Compound 39 showed synergistic activity with colistin on Aba only. A broader spectrum of activity was observed with all other compounds, most notably, 9, 28, 31, 45, and 49, which inhibited the growth of all tested bacteria except P. aeruginosa. None of the compounds actually proved active on that organism. This synergistic activity was further investigated by determining the MIC values of these compounds in the presence of a subinhibitory concentration of colistin and provided additional data regarding their antibacterial spectrum and potency ( Table 2).
Compounds 9, 45, and 49 emerged as the most promising pyrazolo-carboxamide derivatives, with MAC values ranging from 4 to 8 µg/mL on both Aba and Enterobacterales, interestingly showing that both scaffolds (pyrazole-4-and pyrazole-3-carboxamides) allowed for the identification of potent compounds showing synergistic activity with colistin. A similarly potent synergistic activity was also observed with compound 8 on Aba. Compounds 8 and 9 are analogues of parent compound 1, with modifications of the nature of the 4-carboxamide substituent (e.g., adamantanyl). Other modifications of compound 1, including the introduction of butanoate ester on the 4-carboxamide substituent (13) or the substitution of the 5-pyrrole ring with a tert-butoxycarbonylamino moiety (20 and 22), did not translate into a similar increase in antibacterial synergistic activity. Regarding pyrazole-3-carboxamide derivatives, some new analogues of compound 2 (27, 28, 31, and 39) did not prove to be significantly better than the parent compound. However, compounds 45 and 49 did provide some form of significant improvement in their synergistic activity, when com-pared to that of parent compound 2. Compounds 8, 9, 45, and 49 showed the best overall activity, including on Aba, which we consider a more relevant bacterial target in our drug discovery programs than Enterobacterales, in relation to the paucity of antibiotics currently in clinical development targeting this specific pathogen (see Introduction), and were selected for further investigation. A chequerboard analysis, to further assess whether they would exhibit true synergistic activity or rather a simple additive effect with colistin, was carried out (Table 3, Figure 5). All new tested compounds (8,9,45, and 49) were confirmed to be synergistic with colistin on antibiotic-susceptible reference strains of E. coli, K. pneumoniae, and Aba. Compound 8 was slightly inferior to 9, as expected from the data reported in Table 2. A different picture emerged when these compounds were tested on clinical isolates showing a multidrug-or pandrug-resistant phenotype, including to polymyxin antibiotics. On one hand, synergistic activity could not be observed with the pan-resistant K. pneumoniae isolate, whose resistance to colistin was acquired through the insertional inactivation of mgrB (a negative regulator of PhoP/PhoQ) [17]. On the other hand, the potentiation effect was stronger on a colistin-resistant Aba strain when compared to that of the antibiotic-susceptible reference strains, as the resulting average FIC index values were lower when measured on the colistin-resistant Aba strain ( Figure 5). Table 2. MAC (minimum antibacterial concentration) values of selected compounds on representative Gram-negative bacteria (Eco, Escherichia coli CCUG T ; Kpn, Klebsiella pneumoniae ATCC 13833; Aba, Acinetobacter baumannii ATCC 17978), when tested in the presence of 0.5 × MIC colistin. The minimal ratio between the MIC of the compound alone and the MAC in the presence of colistin is given between parentheses and used to quantify the improvement of the antibacterial activity of the tested compounds when tested in the presence of colistin. Data for parent compounds 1 and 2 are provided for comparison [12]. These data confirmed that the new analogues 8 and 9 were promising potentiators of polymyxin antibiotics, especially on colistin-resistant Aba. Encouraged by these results, we wanted to understand whether any of these two active compounds would show sufficient selectivity, and thus, validate the scaffold as a potentially useful new series of antibiotic adjuvants. The potential cytotoxic activity of both compounds was assessed using several methods. First, a simple membrane integrity assay carried out on HeLa cells showed that none of the compounds induced an LDH release at concentrations of up to 256 µg/mL after 24 h of exposure (Table 4). This is in agreement with the fact that no hemolytic activity could be observed, not only for compounds 8 and 9, but also for compounds 1, 2, 45, and 49. Considering that these compounds would show potent antibacterial activity in the presence of colistin (a membrane-interacting antibiotic known for its suboptimal safety profile [19,20]), we also wanted to investigate whether the cytotoxicity of our compounds would be affected by the presence of this antibiotic. Colistin, when tested alone in a similar assay, showed an IC 50 value of 1240 µg/mL, confirming its membrane-damaging activity in eukaryotic cells at higher concentrations. Based on this result, the cytotoxicity of our compounds was measured in the presence of a subtoxic concentration of colistin (512 µg/mL). In this case, compounds 8 and 9 showed a significantly different behavior, as compound 8 appeared to enhance the membrane-damaging activity of colistin at rather low concentrations (IC 50 , 16.4 µg/mL). Interestingly, this was not observed with compound 9, which was devoid of any membrane-damaging activity both in the absence and in the presence of 512 µg/mL of colistin. Subsequently, the cytotoxicity of compound 9 was further assessed using cell proliferation/viability assays (see the Materials and \methods section for details). In these assays, the proliferation of HeLa cells was not significantly altered in the presence of 16 µg/mL of compound for up to 72 h. Moreover, similar results were obtained when this experiment was performed in the presence of 512 µg/mL of colistin in the medium.  These data confirmed that the new analogues 8 and 9 were promising potentiators of polymyxin antibiotics, especially on colistin-resistant Aba. Encouraged by these results, we wanted to understand whether any of these two active compounds would show sufficient selectivity, and thus, validate the scaffold as a potentially useful new series of antibiotic adjuvants. The potential cytotoxic activity of both compounds was assessed using several methods. First, a simple membrane integrity assay carried out on HeLa cells showed that none of the compounds induced an LDH release at concentrations of up to  Finally, and considering the promising selectivity showed by compound 9, its antibacterial activity with colistin on the colistin-resistant Aba N50 strain was further characterized using time-kill curves ( Figure 6). The presence of colistin alone (tested at 2 µg/mL, i.e., the susceptibility breakpoint), although showing an initial moderate impact on bacterial survival, was not expectedly able to show any bactericidal effect (defined as the capacity of a compound to decrease the initial bacterial population by at least 3 log 10 [21]). However, a combination of 2 µg/mL of colistin and 8 µg/mL of 9 did exhibit a significantly faster and better bacterial killing, with a 4 log 10 reduction achieved within 5 h. Unfortunately, complete eradication could not achieved, leading to the recovery of the bacterial growth. This was confirmed by the determination of the MBC, which yielded values >64 µg/mL for compounds 8, 9, 45, and 49 when tested in the presence of 2 µg/mL of colistin.  Finally, and considering the promising selectivity showed by compound 9, its antibacterial activity with colistin on the colistin-resistant Aba N50 strain was further characterized using time-kill curves ( Figure 6). The presence of colistin alone (tested at 2 µ g/mL, i.e., the susceptibility breakpoint), although showing an initial moderate impact on bacterial survival, was not expectedly able to show any bactericidal effect (defined as the capacity of a compound to decrease the initial bacterial population by at least 3 log10 [21]). However, a combination of 2 µ g/mL of colistin and 8 µ g/mL of 9 did exhibit a significantly faster and better bacterial killing, with a 4 log10 reduction achieved within 5 h. Unfortunately, complete eradication could not achieved, leading to the recovery of the bacterial growth. This was confirmed by the determination of the MBC, which yielded values >64 µ g/mL for compounds 8, 9, 45, and 49 when tested in the presence of 2 µ g/mL of colistin. Figure 6. Time-dependent kill-curve analysis of the MDR, colistin-resistant A. baumannii clinical isolate (N50) in the presence 2 µ g/mL of colistin (squares), 2 µ g/mL of colistin, and 8 µ g/mL of 9 (circles). The growth control is shown as triangles (similar results were obtained in the presence of 8 µ g/mL of 9, as this compound alone does not exhibit antibacterial activity).

Discussion and Conclusions
In this study, we investigated the direct-acting and synergistic antibacterial activity of 44 new analogues of pyrazole-3-carboxamides and pyrazole-4-carboxamides identified in a previous work [12]. A single compound showed significant direct-acting activity primarily on Gram-positive organisms, a result that was somewhat expected considering that the parent compounds were previously identified and characterized for their synergistic activity with colistin. Interestingly, nine new analogues of both parent compounds 1 and 2 maintained a synergistic activity on Gram-negative bacteria, with the exception of P. aeruginosa. Two compounds, 8 and 9, were significantly more active than their parent

Discussion and Conclusions
In this study, we investigated the direct-acting and synergistic antibacterial activity of 44 new analogues of pyrazole-3-carboxamides and pyrazole-4-carboxamides identified in a previous work [12]. A single compound showed significant direct-acting activity primarily on Gram-positive organisms, a result that was somewhat expected considering that the parent compounds were previously identified and characterized for their synergistic activity with colistin. Interestingly, nine new analogues of both parent compounds 1 and 2 maintained a synergistic activity on Gram-negative bacteria, with the exception of P. aeruginosa. Two compounds, 8 and 9, were significantly more active than their parent compound 1, and were characterized by a different substituent on the 4-carboxamide moiety. Apparently, this structural difference largely affects the properties of compound 8, which not only exhibited a narrower spectrum of synergistic activity, but also a more apparent enhancement of the membrane-damaging activity of colistin on eukaryotic cells. More strikingly, compound 9 showed synergistic activity on both colistin-susceptible Enterobacterales and Aba, but also proved, in combination with a subinhibitory concentration of colistin, to significantly enhance the rate of killing of a colistin-resistant Aba clinical isolate at concentrations as low as 8 µg/mL while showing a promising selectivity. Furthermore, compounds 45 and 49, which are pyrazole-3-carboxamide derivatives, also proved to have a better synergistic activity than their parent 2, although the improvement on Aba was less remarkable. These data, overall, further highlight the potential of pyrazole-4-and pyrazolo-3-carboxamide analogues to obtain selective and synergistic compounds to be further optimized, and hopefully represent a promising source of new therapeutic solutions that are able to restore the activity of the last-resort polymyxins for the treatment of infections caused by extensively drug-or pandrug-resistant clinical isolates of Acinetobacter baumannii. Furthermore, the synergistic activity exhibited by these compounds in the presence of colistin may rely on the permeabilizing effect of the latter, and provides hope that the further optimization of these compounds, particularly regarding their rate of diffusion through bacterial membranes, may allow for the identification of potent and direct-acting compounds.

Chemistry
Reagents were purchased from commercial suppliers and used without further purification. Anhydrous reactions were run under a positive pressure of dry N 2 . Merck silica gel 60 was used for flash chromatography (23-400 mesh). 1 H NMR and 13 C NMR were recorded at 400 and 100 MHz, respectively, on a Bruker Advance DPX400. Chemical shifts are reported relative to tetramethylsilane at 0.00 ppm. Mass spectral (MS) data were obtained using the Agilent 1100 LC/MSD VL system (G1946C) with a 0.4 mL/min flow rate and using a binary solvent system of 95:5 methanol/water. UV detection was monitored at 254 nm. Mass spectra were acquired either in positive or in negative mode by scanning over the mass range of 105-1500. Melting points were determined on a Gallenkamp apparatus and are uncorrected. Elemental analyses were performed on a PerkinElmer PE 2004 elemental analyzer and the data for C, H, and N are within 0.4% of the theoretical values. The chemical purity of most of the target compounds was determined using an ACQUITY Waters UPLC-MS system with a Waters BEH C18 reversed-phase column (2.1 mm × 50 mm, 1.7 µm); the method was carried out under the following conditions: gradient elution, solvent A (0.1% formic acid in water), solvent B (0.1% formic acid in acetonitrile) 90:10 to 0:100 over 2.9 min, flow rate of 0.5 mL/min, UV detector, and 254 nm. The chemical purity of compounds 10, 14, and 19 was determined using an Agilent 1260 Infinity instrument constituted of a binary pump, an autosampler, an UV-DAD, and an ESI-MS detector. The chromatographic separation was realized with a Symmetry ® C18 column (4.6 × 75 mm, 3.5 µm). Analysis was carried out with methanol as the mobile phase at a flow rate of 0.5 mL/min. UV detection was monitored at 254 nm. The purity of each compound was ≥ 95% in either analysis, with the exception of compounds 3, 4, 6, 7, and 20, whose purity was in the range of 90-94%; accordingly, elemental analyses were not performed on these compounds.

General Procedure for the Synthesis of Ethyl 5-Pyrrol-1-yl-1H-pyrazole-4carboxylates 54-56
Compounds 54 and 55 were prepared according to known procedures [15,25]. As an example, the preparation of the new compound 56 is reported below.
A mixture of 57 (200 mg, 0.54 mmol) in EtOH (4 mL) and NaOH (216 mg, 5.4 mmol) in water (4 mL) was heated at reflux for 1 h, then cooled in an ice bath and acidified with conc. HCl. The precipitate was extracted with EtOAc and the combined organic layer was dried over sodium sulfate and evaporated under reduced pressure. The solid residue was triturated with PE/Et 2 O to give the title compound 60 (81% yield) as a white solid. Mp 240-243 • C. 1

General Procedure for the Synthesis of Amide Derivatives 3-14
To a solution of the carboxylic acids 58-60 (1 mmol) in DCM (50 mL), HOBt (135 mg, 1 mmol), EDC (383 mg, 1.2 mmol), and the appropriate amine (1.5 mmol) were added successively. After stirring at room temperature for 12 h, the solution was washed with 1 N HCl, 10% NaHCO 3 solution, and brine. After drying on sodium sulfate, the solvent was removed under reduced pressure and the residue was purified by recrystallization from MeOH or flash chromatography on a silica gel using the reported eluent system.   1 C), 164.1, 139.0, 137.9,  136.9, 136.5, 129.2, 128.9, 128.1, 127.7, 126.9, 123.1, 122.1, 112.7, 112.1 (1 C), 111.3, 60.6 A solution of the amino derivatives 51 and 53 (3.6 mmol), DMAP (44 mg, 0.36 mmol), and di-tert-butyl dicarbonate (1.57 g, 7.2 mmol) in dry DCM (40 mL) was stirred at room temperature for 2-4 h. Afterward, it was washed with 1 N HCl, then brine, and finally, dried over anhydrous sodium sulfate. The removal of the solvent left an oily residue, which was purified by using flash chromatography on a silica gel eluted with PE/EtOAc (4:1) to produce the pure compounds 61 and 62. To a solution of the esters 61 and 62 (4 mmol) in 50 mL of EtOH, 6 N KOH (27 mL, 0.16 mol) was added and the mixture was refluxed for 1 h. After cooling, the dark-orange solution was acidified with conc. HCl and the precipitate was extracted with DCM. The organic layer was washed with brine, dried (Na 2 SO4), and evaporated to leave a solid, which was sufficiently pure to be used in the next step. bacterial inoculum of 5 × 10 5 CFU/mL, as recommended by the CLSI [26]. The minimum antibacterial concentration (MAC) of the compounds was determined in the presence of a fixed and subinhibitory concentration of colistin (as mentioned above) [21].

Chequerboard Analysis and FIC Determinations
The chequerboard analysis is a two-dimensional array in which individual microplate wells contain a unique combination of the concentrations of the tested compound and colistin. Tested concentrations of the compound ranged from 64 to 0.06 µg/mL along the xaxis, whereas polymyxin E1 concentrations ranged from 2 to 0.03 µg/mL (or 32-0.5 µg/mL when polymyxin E1-resistant clinical isolates were used) and varied along the y-axis. Determining the fractional inhibitory concentration (FIC) of both compounds and computing the average FIC index allowed us to determine whether a synergistic, additive, or antagonistic activity between the tested compounds could be observed. A synergistic activity occurred when the average FIC index was equal to or less than 0.5 [21,27].

Kill-Curve Analysis
The time-dependent bacterial killing of colistin, compound 9, or a combination thereof was investigated using established methods [25]. Briefly, a culture medium containing a starting inoculum of 5.0 × 10 6 CFU/mL of Acinetobacter baumannii N50 clinical isolate was incubated aerobically at 35 • C under orbital agitation (200 rpm) in Mueller-Hinton broth, in the absence (growth control) or presence of either 2 µg/mL of colistin (a concentration equivalent to the susceptibility breakpoint), 8 µg/mL of compound 9, or both. The bacterial count (expressed in CFU/mL) in the control and antibiotic-containing media was determined every hour for up to 6 h of incubation via serial 10-fold dilution and direct plating of 0.1 mL of the sample on the Mueller-Hinton Agar medium (without antibiotic), which was incubated for 24 h at 35 • C.

Membrane Integrity Assays
The potential cytotoxic activity of compounds was evaluated using the commercially available integrity assay (CytoTox 96 ® non-radioactive cytotoxicity assay, Promega). The compounds were tested for their ability to induce the lysis of HeLa cells by measuring the release of lactate dehydrogenase (LDH) after incubating the HeLa cell cultures (20,000 cells/well) for 24 h (37 • C, 5% CO 2 ) in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum, 4.5 mg/mL glucose, and 2 mM L-glutamine in the absence and presence of varying concentrations of the compounds (up to 256 µg/mL). Further controls included samples containing the medium only or in which cell lysis was induced by the addition of 9% Triton X-100 (maximum LDH release control). The percentage of cytotoxicity was computed as 100 × (sample LDH release)/(maximum LDH release). The variation of the percentage of cytotoxicity allowed us to compute an IC 50 value (compound concentration inducing 50% cytotoxicity).

Cell Viability and Proliferation Assays
The cytotoxicity of the compounds was also evaluated by measuring the number of viable cells in the culture with respect to the control culture (cells treated with DMSO only) using the RealTime-Glo™ MT Cell Viability Assay in the presence of varying concentrations of the compound. The assay is a nonlytic homogeneous, bioluminescent method that can be used to measure cell viability/proliferation in real time using NanoLuc ® luciferase and