Novel Guanidine Compound against Multidrug-Resistant Cystic Fibrosis-Associated Bacterial Species

Chronic pulmonary infection is a hallmark of lung disease in cystic fibrosis (CF). Infections dominated by non-fermentative Gram-negative bacilli are particularly difficult to treat and highlight an urgent need for the development of new class of agents to combat these infections. In this work, a small library comprising thiourea and guanidine derivatives with low molecular weight was designed; these derivatives were studied as antimicrobial agents against Gram-positive, Gram-negative, and a panel of drug-resistant clinical isolates recovered from patients with CF. One novel compound, a guanidine derivative bearing adamantane-1-carbonyl and 2-bromo-4,6-difluouro-phenyl substituents (H-BDF), showed potent bactericidal activity against the strains tested, at levels generally higher than those exhibited by tobramycin, ceftazimide and meropenem. The role that different substituents exert in the antimicrobial activity has been determined, highlighting the importance of the halo-phenyl group in the guanidine moiety. The new compound displays low levels of cytotoxicity against THP-1 and A549 cells with a selective index (SI) > 8 (patent application PCT/IB2017/054870, August 2017). Taken together, our results indicate that H-BDF can be considered as a promising antimicrobial agent.


Introduction
In recent years, increasing infections due to antibiotic-resistant pathogens have made the formerly routine therapy of many infectious diseases challenging, and in many instances, extremely difficult or impossible to be eradicated [1][2][3]. Multidrug resistance is specially associated with respiratory tract infection in cystic fibrosis (CF) [4] where opportunistic pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia and species of the Burkholderia cepacia complex (Bcc) infect patient's lung and airways. Although for some patients the infection may occur only transiently, their acquisition most typically results in a chronic infection with acute debilitating exacerbations, causing a severe decline in respiratory function which contributes to disease progression and premature mortality [5,6]. In addition, they are important nosocomial pathogens affecting both immunocompetent and immunocompromised patients, and are responsible for a considerable proportion of infections in patients in Intensive Care Units (ICUs) worldwide [7]. Despite the emergence and dissemination of resistant bacteria and the need of more effective therapies, the development of new antimicrobial agents against these life-threatening infections is declining [8]. The impermeable nature of Gram-negative bacteria envelope, and the presence of multiple efflux pumps, in combination with other resistance mechanisms, has made the discovery of new effective antibacterial drugs very difficult [9].
Thioureas as well as guanidines represent two important groups of compounds due to their wide range of application as pharmaceutical agents. They possess a broad biological activity range including anti-inflammatory, anticancer, antiviral, antiparasitic, antifungal and antimicrobial properties [10,11]. Such a diverse range of biochemical behavior can be attributed to their flexible structure and the presence of nitrogen atoms in these molecules that make it possible to bear various substituents. For instance, it is well known that the 1-aroyl-3-(substituted-2-benzothiazolyl) thioureas exhibit potent antibacterial activity [12]. In addition, 1-(benzoyl)-3-(substituted) thioureas are antimicrobial agents [13] and the fluorinated analogues exhibit good antifungal activity [14]. Furthermore, due to efficient resonance stabilization of the charged protonated state, the guanidine groups have a relatively high acid dissociation constant which makes them stronger bases better suited for stable electrostatic interaction with the negative charged membranes of bacteria. This property improves the penetration of guanidine-bearing compounds through membranes and thus their biological activity [15,16]. On the other hand, the introduction of fluorine or appropriate fluorinated groups into organic compounds has advanced over recent decades in medicinal chemistry. The incorporation of fluorine atoms may contribute to increase metabolic stability, binding affinity and lipid solubility, thereby enhancing rates of absorption and transport of drugs in vivo [17,18]. Several studies further indicated that the incorporation of fluor and/or different electron withdrawing groups, such as bromo, chloro, acetyl, and nitro groups, on aromatic rings results in an improvement in antibacterial activity [16,[19][20][21].
Taking into account the aforesaid biological and synthetic significance of thioureas and guanidines on one hand, and the multifunctional value of the electron withdrawing groups in drug design on the other, the endeavor of the current work was to investigate the activity of newly synthesized halophenyl substituted thioureas and guanidines against drug-resistant clinical isolates recovered from patients with CF.

Reagents and Equipment
1-adamantane carboxylic acid, thionyl chloride, triethylamine, potassium thiocyanate, mercury(II) chloride and substituted anilines were commercial products (Sigma-Aldrich, St. Louis, MO, USA) and were used as received. Analytical grade (Merck, Kenilworth, NJ, USA) acetone and dimethyl formamide, DMF, were dried and freshly distilled prior to use.
Melting points were recorded using a digital Gallenkamp (SANYO, Moriguchi, Japan) model MPD.BM 3.5 apparatus and are uncorrected. 1 H and 13 C NMR spectra were determined in CDCl 3 at 300 MHz and 75.4 MHz, respectively, using a Bruker spectrophotometer (Billerica, Middlesex, MA, USA). FTIR spectra were acquired by a FTS 3000 MX spectrometer. Elemental analyses were conducted using a LECO-183 CHNS analyzer (LECO Corporation, MI, USA). Thin layer chromatography (TLC) was carried out on 0.25 mm silica gel plates (60 F254, Merck, Darmstadt, Germany). Visualization was achieved by ultraviolet light.

Synthesis of Compounds
Thirteen compounds were synthesized and their structures were confirmed by a combination of elemental analysis, infrared and nuclear magnetic resonance spectroscopy. 1-(Adamantane-1-carbonyl)-3-substituted thiourea compounds were prepared by the addition reaction between adamantyl isothiocyanate with a variety of suitably substituted anilines [22][23][24][25]. The starting material 1-adamantane carbonyl chloride was obtained via the reaction of 1-adamantane carboxylic acid with thionyl chloride. A solution of adamantane-1-carbonyl chloride in dry acetone was treated with an equimolar quantity of potassium thiocyanate in dry acetone to yield the adamantane-1-carbonyl-isothiocyanate as intermediate ( Figure 1). A treatment of the latter with an equimolar quantity of cyclohexylamine (for compound 1, Table 1) and a variety of substituted anilines (compounds 2-7, Table 1) in acetone produced the thiourea derivatives. In a typical procedure, a freshly distilled solution of adamantane-1-carbonyl chloride (10 mmol) in dry acetone (50 mL) was added dropwise to a suspension of potassium thiocyanate (10 mmol) in acetone (30 mL) and the reaction mixture was refluxed for 30 min under nitrogen. After cooling to room temperature, a solution of the substituted aniline (10 mmol) in acetone (10 mL) was added and the resulting mixture refluxed for 2-4 h. The reaction mixture was poured into cold water and the precipitated thioureas were recrystallized from suitable solvents. acid with thionyl chloride. A solution of adamantane-1-carbonyl chloride in dry acetone was treated with an equimolar quantity of potassium thiocyanate in dry acetone to yield the adamantane-1-carbonyl-isothiocyanate as intermediate ( Figure 1). A treatment of the latter with an equimolar quantity of cyclohexylamine (for compound 1, Table 1) and a variety of substituted anilines (compounds 2-7, Table 1) in acetone produced the thiourea derivatives. In a typical procedure, a freshly distilled solution of adamantane-1-carbonyl chloride (10 mmol) in dry acetone (50 mL) was added dropwise to a suspension of potassium thiocyanate (10 mmol) in acetone (30 mL) and the reaction mixture was refluxed for 30 min under nitrogen. After cooling to room temperature, a solution of the substituted aniline (10 mmol) in acetone (10 mL) was added and the resulting mixture refluxed for 2-4 h. The reaction mixture was poured into cold water and the precipitated thioureas were recrystallized from suitable solvents. Three 1-acyl-3-(2-bromo-4,6-difluoro-phenyl)thioureas (compounds 8-10, Table 1) were synthesized in a similar way by treating the corresponding acyl chloride derivatives (1-naphthoyl chloride, 2,4-dichloro-benzoyl chloride and 4-methyl-benzoyl chloride, respectively) with potassium thiocyanate in dry acetone followed by the addition of 2-bromo-4,6-difluoro-aniline.

Bacterial Strains
The antibacterial activity of the compounds was tested against the reference strains Escherichia coli ATCC25922, Bordetella bronchiseptica 9.73H+ [27], Pseudomonas aeruginosa ATCC15692, Burkholderia cenocepacia J2315, Pandorea apista DSM16535, Staphyloccocus aureus ATCC6538, Bacillus cereus ATCC10876. A total of forty non-fermenting Gram-negative bacilli and two Methicillin-Resistant Staphylococcus aureus (MRSA) clinical isolates collected from sputum samples of patients with CF attended at different hospitals and CF Centers in the period 2004 to 2017 were used in this study. They were selected from the collection of microorganisms CAMPA (Colección Argentina de Microorganismos Patógenos y Ambientales) of CINDEFI, at the Faculty of Exact Sciences in La Plata University [28]. All Bcc isolates were identified by PCR-recA technology (amplification, PCR-recA RFLP HaeIII, and sequencing). Additionally hisA, gyrB, or other gene from the current multilocus sequence typing (MLST) scheme were sequenced when the identification remained ambiguous [29,30]. The isolates were maintained both as lyophilized and frozen at −80 • C in Trypticase-soy broth with 10% (v/v) glycerol until further analysis.

Antimicrobial Activity Assays
The in vitro susceptibility tests (Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) tests) were determined using the micro-dilution method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI) [31]. Briefly, serial two-fold dilutions of each compound were prepared (final volume of 50 µL) in 96-well polypropylene microtiter plates (Sarstedt, Nümbrecht, Germany) with Mueller Hinton (MH) broth. Each dilution series included control wells without any compound and control wells without bacteria. Then, a total of 50 µL of the adjusted inoculum (approximately 5 × 10 5 cells/mL) in MH broth was added to each well. The MIC was taken as the lowest concentration of antimicrobial compound resulting in the complete inhibition of visible growth after 18 h of incubation at 37 • C. Minimal bactericidal concentration (MBC) assay was performed following MIC assay. After reporting the MIC assay value, 10 µL aliquots of the medium were taken from wells with no visible bacterial growth. These were plated on LB agar and incubated for 24 h to allow colony growth. The lowest concentration of the compound at which no growth occurred on LB plates was denoted as the MBC. Results are mean values of at least two independent determinations.

Checkerboard Assay
The activity of compound 11 in combination with meropenem, tobramycin and ciprofloxcin was analyzed using the checkerboard broth dilution method [32]

Cytotoxicity Assays
A trypan blue exclusion assay [34] was performed to check the cytotoxicity of compound 11 against THP-1 human monocytic leukemia cells (ATCC, TIB-202, Manassas, VA, USA) and A549 alveolar epithelial cells (ATCC, CCL185, Rockville, MD, USA). Cells were routinely maintained in Complete Medium RPMI-1640 and Dulbecco's Modified Eagle's medium (DMEM), respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS). For the cytotoxicity assay, cells were seeded at a density of 5 × 10 4 per well in a 96 well plate and were incubated with serial dilutions of compound 11 to a total of 200 µL, at 37 • C in a humidified atmosphere of 5% CO 2 for 24 h. Two negative controls were included: cells in drug-free culture media and cells treated for 24 h with the maximum concentration of the drug solvent used in the experiment (4% dimethyl sulfoxide).
Cells were subsequently stained with 0.2% trypan blue and incubated for 3 min at room temperature. The number of dye-excluding cells was counted by microscopy. A minimum of 200 cells were counted and the percent viability was calculated in comparison to the control. The IC50 value was defined as the highest drug concentration at which 50% of the cells are viable relative to the control. Results are mean values of at least five independent determinations. The selectivity index (SI) was calculated as the ratio of IC50 and the MIC [35].

Chemistry
A series of 11 novel closely related compounds belonging to the thiourea family (compounds 1-10) and a guanidine derivative (11) was prepared ( Figure 1 and Table 1). Primary amines substituted with different electron withdrawing groups were subjected to the addition reaction with isothiocyanates in order to be transformed into the corresponding thioureas by using the general method originally proposed by Douglas and Dains [22] (Figure 1). The substitution on both nitrogen positions (1 and 3) of the thiourea group was varied in order to better understand the role of different substituents in the biological activity. To rationalize this aspect, a series of closely related 1-(adamantane-1-carbonyl)-3-mono substituted thioureas was firstly prepared by taking into account the well-known capacity of the adamantyl group to enhance antibacterial activity [36][37][38][39][40]. Thus, several thioureas were prepared bearing the adamantyl group in R 1 (compounds 1-7, Table 1).
Moreover, taking into account the improvement in antibacterial activity exerted by the presence of phenyl groups substituted with electron withdrawing groups [16,[19][20][21], a second group of thioureas (compounds 7-10, Table 1) was substituted in R 2 with the 2-bromo-4,6-difluoro-phenyl group. Finally, the effect of replacing the thiocarbonyl (C=S) with aryl-guanidino functionality (Ar-N=C) was evaluated in compound 7, in which N-3 of the guanidine was substituted with the 2-bromo-4,6-difluoro-phenyl group. To this end, the procedure proposed by Vencato et al. [26] was applied and the acyl thiourea derivatives were treated with mercury(II) chloride under basic conditions in the presence of 2-bromo-4,6-difluoroaniline to produce the corresponding guanidine derivative (compound 11, Figure 1 and Table 1) [26,41].
The guanidine derivative 11 was characterized by two typical NH absorptions at ca. 3400 and 3240 cm −1 , the C=O stretching at around 1670 cm −1 and the absence of thiocarbonyl stretching when the FTIR spectra are compared with the corresponding thiourea reagent. The characteristic C=N stretching modes of the guanidine group are observed as an intense absorption at ca. 1575 cm −1 . In 1 H-NMR, two broad NH singlets appeared besides the aromatic protons. The carbonyl carbons are observed at 178-179 ppm in the 13 C-NMR spectrum, while the (C=N-Ar) appeared upfield at 174 ppm compared to the thiocarbonyl carbon.

Antimicrobial Evaluation of Newly Synthesized Compounds
All obtained compounds were tested in vitro for their MIC and MBC against two reference Gram-negative non-fermentative bacilli strains, Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia J2315. These species play a critical role in morbidity and mortality associated with CF and they were selected on the basis of their high level of resistance to a variety of antimicrobial substances [44][45][46][47]. The results of antimicrobial activity are summarized in Table 1. The MIC and MBC values of meropenem, tobramycin and ceftazidime, three commonly used antibiotics for the treatment of chronic pulmonary bacterial infections [48], were analyzed in parallel. It is apparent from the results that only the guanidine derivative 11, namely H-BDF, showed a MIC value less than 2 µg/mL, and comparable or superior activity than standard drugs. Interestingly, this compound has the lowest MIC and MBC against B. cenocepacia J2315, a strain particularly resistant to meropenem [44].
A first look into structural activity relationship (SAR) indicates that, independent of the halogens introduced in the phenyl group, thiourea derivatives have poor or no antimicrobial activity. However, the replacement of thiourea in compound 7 for the guanidine group (compound 11) greatly improves antimicrobial activity. We next evaluated the impact of introducing changes in the phenyl ring of compound 11 in the biological activity. To this end, the guanidine derivatives 12 and 13 ( Figure 2) were synthesized and characterized. Compound 13 was designed to evaluate the effect of changing the substitution of the halophenyl groups by the incorporation of another electron withdrawing group (nitro) in N-2 and N-3, whereas compound 12 evaluates the effect of introducing a small change in N-3 by the substitution of bromine by fluorine in position 6.
The antimicrobial activity of the new compounds was tested against P. aeruginosa PAO1 and B. cenocepacia J2315 as well as other Gram-negative and Gram-positive reference strains. As shown in Table 2, when the phenyl group substituent of compound 11 was altered by the introduction of a nitro group at the meta position (compound 13), the guanidine derivate completely lost its inhibition potency, suggesting that not only the guanidine group but also the identity and/or position of the phenyl substitutions are decisive for the antibacterial activity. Moreover, whereas compound 11 exhibited very good inhibitory and bactericidal activity against all tested strains, compound 12, in which the 2-bromo-4,6-difluoro-phenyl group in N-2 was substituted by 2,6-dibromo-4-fluoro-phenyl ring, showed only moderate microbicidal activity, suggesting that the presence of fluorine atom in position 6 of the phenyl group in N-2 is critical to ensure high inhibition and bactericidal potency.

Cytotoxic Evaluation of H-BDF
As limited human cellular toxicity is an important feature for an antibiotic compound, the toxicity of H-BDF was evaluated using the human monocytic leukemia cell line THP-1 and the human lung epithelial cell line A549, commonly employed in toxicity evaluation of new compounds for pulmonary application [49,50]. The IC50 for compound 11 was 38.4 ± 5.4 µg/mL for A549 and 15.5 ± 3.1 µg/mL for THP-1 cells. On the basis of the MIC and IC50 values, the selectivity indices were calculated for standard strains (Table 3). It is generally considered that the ratio for a good therapeutic index for a drug should be >10, which is a cut-off point ensuring that overdose does not put the life of the patient in danger [35]. Good SI values were obtained with compound 11 suggesting that H-BDF can be considered as a promising antibacterial agent.

Synergistic Effects between H-BDF and Conventional Antibiotics
Developments of alternate antibacterial strategies to potentiate the antimicrobial activity of conventional antibiotics have become increasingly important due to the emerging threat of multi-drug resistant infection [51]. As many clinical isolates exhibit resistance to meropenem, ciprofloxacin and tobramycin, three of the different classes of antibiotics commonly used to treat CF pulmonary exacerbations [52], we next studied the ability of H-BDF to potentiate the antimicrobial activity of these antibiotics toward the multidrug-resistant strain B. cenocepacia J2315. To this end, the relationship between H-BDF and meropenem, tobramycin, and ciprofloxacin was assessed via a standard checkerboard assay [29]. Treatment with H-BDF reduced the minimum inhibitory concentration of ciprofloxacin and meropenem below their clinical sensitivity breakpoints (≤4 µg/mL and ≤1 µg/mL, respectively). Fractional inhibitory concentration calculations revealed that H-BDF exhibited a synergistic interaction with meropenem and ciprofloxacin with FICIs values of 0.3 and 0.4, respectively, and an additive interaction with tobramycin with a FICI value of 0.75. This preliminary study suggests that in addition to being used as antimicrobial agent alone, H-BDF has the potential to be used in combination with other antibiotics.

Activity of Compound H-BDF against Multidrug-Resistant Clinical Isolates Recovered from Respiratory Samples of CF Patients
Respiratory infections with opportunistic pathogens with intrinsic antibiotic resistance to most clinically available antimicrobials are life-threatening in patients with CF [53][54][55]. Although P. aeruginosa and S. aureus remain the most common pathogens in CF lung infections, other bacteria such as species within the Bcc, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans, have emerged as significant opportunistic human pathogens in the last decades [56][57][58][59]. To investigate whether the guanidine derivative H-BDF would have clinical utility against current multidrug resistant bacteria, we determined the MIC and MBC of compound 11 against thirty eight Bcc clinical isolates, one Achromobacter xylosoxidans, one Stenotrophomonas maltophilia and two MRSA recovered from sputum samples of CF patients and selected on the basis of their high level of resistance to a variety of antimicrobial substances [55] (Table 4). MIC values of compound H-BDF were generally lower than those of meropenem, ceftazimide and tobramycin. In total, 69% of Bcc clinical isolates had H-BDF MIC values less than or equal to 4 µg/mL whereas only 41% of isolates were classified as susceptible to meropenem (MIC values ≤4 µg/mL), 49% were classified as susceptible to ceftazimide (MIC values ≤8 µg/mL), and 2.6% of isolates were classified as susceptible to tobramycin (MIC values ≤4 µg/mL). The activity of compound H-BDF against B. cenocepacia strains was impressive, with 92% susceptible at 4 µg/mL compared with only 31% susceptible to meropenem at 4 µg/mL, and 69% susceptible to ceftazimide at 8 µg/mL (Table 4). Interestingly, some clinical isolates were resistant to more than 16 antibiotics, such as B. seminalis CBC040 [55] had H-BDF MIC values ≤ 4 µg/mL. Indeed, H-BDF was active against two methicillin-resistant S. aureus clinical isolates with MIC values varying from 1 to 2 µg/mL. In conclusion, compound H-BDF was active in vitro against a significant number of multi-resistant clinical isolates recovered from CF patients.  Isolates recovered from patients with cystic fibrosis whose complete antibiotic susceptibilities to 17 antimicrobial agents were previously reported (reference [55]).

Conclusions
We have reported the synthesis and preliminary evaluation of the antimicrobial activity of 13 novel thiourea and guanidine derivatives. The results evidenced that H-BDF, a guanidine derivative bearing adamantane-1-carbonyl and two 2-bromo-4,6-di-fluoro-phenyl groups, can be considered as a promising antimicrobial agent, since it exhibited higher in vitro antibacterial potency against Gram-positive and Gram-negative reference strains than previously reported guanidine compounds [10,11,15]. Moreover, the novel compound was active in vitro against a panel of multidrug-resistant clinical isolates recovered from sputum samples of patients with CF. Preliminary studies further suggest that H-BDF was able to significantly potentiate antibacterial synergy with meropenem and ciprofloxacin. From the structure activity relationship, it can be concluded that the antimicrobial activity depends mainly on the presence of a guanidine group. It has been proposed that most of the biological properties of guanidine derivatives are related to their strong basicity due to efficient resonance stabilization of the charged protonated state. The pKa of H-BDF was not determined; however, it is expected that under physiological conditions, the guanidine group exists mainly in its protonated form [60]. We can hypothesize that under this state, the guanidine moiety may alter bacterial outer membrane permeability by binding to a negatively charged site in the lipopolysaccharide layer, causing cell death. This mechanism of action have been proposed for several guanidine derivatives with antibacterial activity [61]. Alternatively, the protonated forms may interact with the active site of proteins and enzymes altering its function [11]. By analyzing the role that different substituents exert in the antimicrobial activity, the importance of the halo-phenyl group in the guanidine moiety was also demonstrated. The substituted fluorine in position 6 of the phenyl group in N-2 may contribute to increase binding affinity and/or lipid solubility [18]. Also, the electron-withdrawing group may activate the guanidine binding moiety to enhance its interaction with amine groups present in the cell membrane. Future studies will be directed towards elucidating the targets of H-BDF and the mechanisms of action.
Importantly, this compound displays low levels of cytotoxicity against THP-1 and A549 cell lines. Future research will be performed to evaluate its efficacy and safety in animal models of infection in order to validate its development as a novel antimicrobial.