Bioorganic & Medicinal Chemistry 22 (2014) 2844–2854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Exploring 5-nitrofuran derivatives against nosocomial pathogens: Synthesis, antimicrobial activity and chemometric analysis Rodrigo Rocha Zorzi a,⇑, Salomão Dória Jorge a,⇑, Fanny Palace-Berl a, Kerly Fernanda Mesquita Pasqualoto b, Leandro de Sá Bortolozzo a, André Murillo de Castro Siqueira a, Leoberto Costa Tavares a a b
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmacy, University of São Paulo, Av. Prof Lineu Prestes, 580, São Paulo, SP 05508-900, Brazil Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP 05503-900, Brazil
a r t i c l e
i n f o
Article history: Received 28 January 2014 Revised 19 March 2014 Accepted 29 March 2014 Available online 8 April 2014 Keywords: 5-Nitrofuran derivatives Molecular modification Nosocomial infection Bacterial resistance Exploratory data analysis
a b s t r a c t The burden of nosocomial or health care-associated infection (HCAI) is increasing worldwide. According to the World Health Organization (WHO), it is several fold higher in low- and middle-income countries. Considering the multidrug-resistant infections, the development of new and more effective drugs is crucial. Herein, two series (I and II) of 5-nitrofuran derivatives were designed, synthesized and assayed against microorganisms, including Gram-positive and -negative bacteria, and fungi. The pathogens screened was directly related to either the most currently relevant HCAI, or to multidrug-resistant infection caused by MRSA/VRSA strains, for instance. The sets I and II were composed by substituted[N0 -(5-nitrofuran-2-yl)methylene]benzhydrazide and 3-acetyl-5-(substituted-phenyl)-2-(5-nitro-furan2-yl)-2,3-dihydro-1,3,4-oxadiazole compounds, respectively. The selection of the substituent groups was based upon physicochemical properties, such as hydrophobicity and electronic effect. The compounds have showed better activity against Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis. The findings from S. aureus strain, which was more susceptible, were used to investigate the intersamples and intervariables relationships by applying chemometric methods. It is noteworthy that the compound 4-butyl-[N0 -(5-nitrofuran-2-yl)methylene]benzhydrazide has showed similar MIC value to vancomycin, which is the reference drug for multidrug-resistant S. aureus infections. Taken the findings together, the 5-nitrofuran derivatives might be indeed considered as promising hits to develop novel antimicrobial drugs to fight against nosocomial infection. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Nosocomial or health care-associated infection (HCAI) caused by multidrug-resistant microorganisms comprises one of the most concerning problems pointed out by the World Health Organization (WHO). It is several fold higher in low- and middle-income than high income countries. It was estimated that around nine percent of hospitalized patients exhibits HCAI,1 resulting in 4 million hospitalizations and, at least, 140,000 deaths every year in the United States and Europe.2,3 The situation is getting even worse due to the acquired resistance by microorganisms to conventional therapy, being about 50–60% of total HCAI currently caused by multidrug-resistant microorganisms.1,4–6 The improper use and/or abuse of antibiotics can be pointed out as quite important to the development of resistance.1 ⇑ Corresponding authors. Tel.: +55 11 30913693; fax: +55 11 38156386. E-mail addresses: [email protected] (R.R. Zorzi), [email protected] (S.D. Jorge). http://dx.doi.org/10.1016/j.bmc.2014.03.044 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
The Gram-positive bacteria Staphylococcus aureus is commonly associated with HCAI, specially the MRSA (Methicillin-resistant S. aureus), VISA (Vancomycin-intermediate S. aureus), and VRSA (Vancomycin-resistant S. aureus) strains.1,4–7 The treatment for infections caused by these pathogens are extremely limited, since the strains are commonly non-susceptible to b-lactams, quinolones, tetracycline, and aminoglycosides. There are also several strains resistant to the new treatments, which have used Daptomycin and Linezolid, for instance.5–11 The therapeutic available options, then, would be Telavancin (Vancomycin derivative), Ceftaroline fosamil (5th generation cephalosporin), and Tigecycline (glycylcycline class).6,7 Other microorganisms which can be commonly associated to multidrug-resistant HCAI are Gram-negative bacteria, including ESBL-producing (Extended-Spectrum Beta-lactamase) strains. The available treatment for these strains is based upon carbapenems (e.g., Imipenem, Meropenem, and Doripenem).8 However, mainly Enterobacteriaceae, which is able to produce Klebsiella pneumoniae carbapenemase (KPC), can develop resistance to those antibiotics,
R. R. Zorzi et al. / Bioorg. Med. Chem. 22 (2014) 2844–2854
reducing the treatment options to the use of polimyxins (e.g., Colistin) and Tigecycline.8–21 The resistance of Gram-negative bacteria strains to Tigecycline has already been reported.9,10 In addition, cases of sepsis especially associated with fungal infections can also be a serious concern for healthcare systems. The number of fungal infections has been increased more than 200% in the last two decades, being the third most common cause of bloodstream infections.11,12 Candida spp. is responsible for 75% of all nosocomial infections caused by fungus, and Candida albicans causes 45% of the total cases primarily in immunosuppressed patients.13 The treatment available comprises polyenes (Amphoterecin B), azoles (Fluconazole and Itraconazole), and, more recently, echinocandins. However, similar to bacteria, several strains have already presented resistance to those drugs.14,15 In this regard, it is important to adopt urgent measures to prevent the inappropriate impact caused by multidrug-resistant microorganisms on healthcare systems. Among this measures are the correct prescription of antibiotics; the development and implementation of protocols for cleaning and disinfecting patient rooms, surfaces, equipment, and common areas in hospitals environments; and, also the discovery of new drugs capable of treating the multidrug-resistant bacterial infections. Nitrofurans are a class of nitro compounds, which have been used to treat bacterial infections since 1940s.16 Several studies involving this chemical class have been carried out regarding different therapeutic uses, such as tuberculostatic,17,18 antileishmanial,19,20 trypanocidal activity,21 and anti-proliferative effect on cancer cells lines.22 Nifuroxazide (NF), for instance, is a nitro compound, which was widely used as antibacterial drug in 1970s and 80s. Recently, NF has been reported as a potential QuorumSensing inhibitor on Pseudomonas aerugionosa, even though it seems not be able to stop the bacterial growth.23 NF is also considered an excellent lead compound due to its chemical structure, which benefits molecular modifications by rational design strategy. One of these modifications is the synthesis of 3-acetyl2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole ring system by cyclization reaction of N-acylhydrazone compounds, which was reviewed by Rollas and Karakusß.24 Briefly, these structures were investigated as monoamine oxidase inhibitors,25 antifungals,26 anticonvulsants,27 anti-inflammatory agents,28 antibactericidals,29,30 and trypanocidal agents.29 The findings have emphasized the potential of these molecular structures for the development of novel drugs. Herein, two series of compounds structurally analogous of NF (Fig. 1A) were designed, synthesized, and experimentally tested. Series I presents a N-acylhydrazone structure (azomethine derivatives), and series II has a heterocyclic ring system 3-acetyl-2,5disubstituted-2,3-dihydro-1,3,4-oxadiazole (oxadiazoline series) (see Fig. 1B). Antimicrobial activity was evaluated against microorganisms reported as HCAI pathogens (Candida albicans,
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Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter clocae, Escherichia coli, Serratia marcescens, and Staphylococcus aureus). In addition, derivatives that have showed promising activity in S. aureus were evaluated against multidrug-resistant S. aureus VISA3. The molecular properties were investigated by applying exploratory data analysis, a chemometric procedure, which comprises the principal component analysis (PCA) and hierarchical cluster analysis (HCA).31,32 2. Results and discussion 2.1. Chemistry The designed compounds were obtained as show in Scheme 1. The compounds of series I (substituted-((5-nitrofuran-2-yl)methylene)benzohydrazide, 2a–v) were obtained from the reaction of substituted benzohydrazides (1a–v) with 5-nitrofuran-2-carbaldehyde.33 The compounds of series II (3-acetyl-5-(substituted-phenyl)-2-(5-nitro-furan-2-yl)-2,3-dihydro-1,3,4-oxadiazole, 3a–v) were obtained by cyclization reaction of 2a–v with acetic anhydride.29,34,35 The substituent groups attached at benzene moiety were chosen based on their physicochemical properties, such as hydrophobicity and electronic effect.36 In this study, 41 compounds (22 compounds of series I and 19 compounds of series II) were synthesized and identified. All compounds were synthesized in two steps, starting from the corresponding benzohydrazides. The first step was based on classical Schiff’s base formation, whose synthesis and mechanism of reaction have been well reported and discussed.29,37 Satisfactory yields (around 90%) were obtained in this step. The second step was performed by a cyclization reaction of Schiff’s base, and presented 66% yield.35 The compounds were structurally identified (see the Supplementary information section, p. S2–S49). Compounds 3h (R1 = SO2NH2), 3j (R1 = N(CH3)2, 3k (R1 = NH2), and the respective oxadiazole analogue of NF (R1 = OH) were not obtained probably due to a reaction of the substituent groups with acetic anhydride. The presence of unbound electrons seems to provide stronger nucleophiles, as the amidic nitrogen from Schiff’s base, for instance. This observation was confirmed by 1H NMR and 13C NMR spectra (Fig. 2), considering the chemical deviation (d) related to the internal standard reference (tetrametilsilane). Regarding the 1H NMR spectra of NF (Fig. 2A), it can be noticed the presence of a singlet signal around d 12 ppm, which is related to the proton of amidic nitrogen (H8). Also, a singlet signal at d 8.40 ppm indicated the azomethine hydrogen atom (H6). After a cyclization reaction, the absence of the signal corresponding to the amidic nitrogen and a singlet signal at d 7.35 ppm (H2), which indicates the 2,3-dihydro-1,3,4-oxadiazoline ring group formation, were observed in 1H NMR spectra of the product (Fig. 2B). Furthermore, a singlet with six protons integration at d 2.29 ppm was observed, confirming the three protons related to the acetyl group and to the acetoxylation of hydroxyl group. Analyzing the 13C NMR spectra (Fig. 2C), there are signals at 167.1 and 20.9 ppm, which indicate the presence of carbonyl (C20) and alkyl groups (C22), respectively. Also, the signals at 153.4 (C5) and 84.6 ppm (C2) are related to the oxadiazole ring system. The presence of the acetoxy group attached to benzene moiety can be observed through the signals at 168.5 and 20.7 ppm. 2.2. Biological activity
Figure 1. (A) Chemical structure of nifuroxazide (NF), pointing out the regions where molecular modifications were carried out on this study. (B) General chemical structures of series I and II.
The minimal inhibitory concentration (MIC) was determined by broth microdilution method against the following strains: Candida albicans 537Y, Klebsiella pneumoniae ATCC 700603, Enterococcus faecalis ATCC 29212, Enterobacter clocae ATCC 23355, Escherichia coli ATCC 25922, Serratia marcescens ATCC 14576, and
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Scheme 1. Synthesis of series I and II compounds.
Staphylococcus aureus ATCC 29213. The derivatives with better activity profile in S. aureus were evaluated against multidrugresistant S. aureus VISA3.38,39 The activity of compounds was determined by the first wellvariant without turbidity, which was considered as the MIC value. The results are displayed in Tables 1 and 2. The compounds and the drugs used as reference were dissolved in DMSO. The range of DMSO concentration varied from 5.0% to 0.01%, and no effect on microbial growth was observed in the presence of up to 5.0% DMSO (v/v). All derivatives have presented MIC values derived from a DMSO concentration much lower than the compound concentration needed to kill the microorganism (see Tables 1 and 3). In this regard, the complete inhibition of bacterial and fungal growth was referred as due to an intrinsic activity of the investigated compounds, and any kind of synergism between compounds and DMSO was discarded. Regarding antifungal tests against C. albicans, compounds of series II were more active than compounds of series I, suggesting the improvement on activity could be due to the cyclization of N-acylhydrazone moiety. The findings were similar to previous data of our group.29 The most active compounds were 3p (R1 = OC2H5; MIC = 16.0–8.0 lM/5.5–2.7 lg/ml) and 3a (R1 = H; MIC = 16.0–8.0 lM/4.8–2.4 lg/ml). Although eighteen derivatives were non-active against E. coli strains at the concentrations tested, the compound 2k (R1 = NH2; MIC = 16.0–8.0 lM/4.3–2.1 lg/ml) has displayed 2-fold better activity than 2a (R1 = H; MIC = 32.0–16.0 lM/8.3–4.1 lg/ml). The lead compound (R1 = OH) has shown MIC value of 64.0–32.0 lM (17.6–8.8 lg/ml). Thus, the activity of compounds of series II was not quite interesting against E. coli strains. Compound 2k has also showed better activity against E. clocae strain (MIC = 8.0–4.0 lM/2.2–1.1 lg/ml). Otherwise, compounds 2t and 2u can be considered as weak inhibitors. The second most active compound was the lead compound (R1 = OH; MIC = 64.0–32.0 lM/ 17.6–8.8 lg/ml). The remaining compounds of both series did not present any activity, even at higher concentrations. The results found for the E. faecalis strain were quite similar to those for E. clocae. The compound 2k (R1 = NH2; MIC = 8.0–4.0 lM/2.2–1.1 lg/ml) and NF (R1 = OH; MIC = 16.0–8.0 lM/4.4–2.2 lg/ml) were the most active molecules. Then, there could be a positive influence on activity by the presence of hydrophilic and electron donating substituent groups attached at R1 position. In this regard, compounds of series II did not show any activity against those strains. Compounds of both series mostly did not display a suitable activity against the S. marcescens strains. Only eight compounds (2e, 2s, 2t, 2u, 3e, 3f, 3o, 3p) have presented some biological activity,
ranging from 128.0 to 64.0 lM. Similarly, just two compounds (2t and 2u) were active against K. pneumoniae (128.0 to 64.0 lM). Otherwise, against S. aureus, both series have showed a promising inhibitory activity. Compounds of series I, specially, 2g (R1 = n-C4H9, MIC = 8.0–4.0 lM/2.5–1.3 lg/ml), 2u (R1 = OC3H7; MIC = 8.0–4.0 lM/2.5–1.3 lg/ml), 2t (R1 = C3H7; MIC = 8.0– 4.0 lM/2.4–1.2 lg/ml), 2m (R1 = I; MIC = 8.0–4.0 lM/3.1–1.5 lg/ml), and 2f (R1 = NO2; MIC = 8.0–4.0 lM/2.4–1.2 lg/ml), have displayed better activity than NF (MIC = 16.0–8.0 lM/4.4–2.2 lg/ml). The lowest activity was observed for the 2b derivative (R1 = CH3; MIC = 16.0–8.0 lM/4.4–2.2 lg/ml). Regarding series II, compound 3e (R1 = CN; MIC = 4.0–2.0 lM/1.3–0.6 lg/ml) has showed better activity, and the less active compounds were 3g (R1 = n-C4H9; MIC = 32.0–16.0 lM/11.4–5.7 lg/ml) and 3v (R1 = OC4H9; MIC = 32.0–16.0 lM/11.9–6.0 lg/ml). Compounds 3l (R1 = t-C4H9) and 3u (R1 = OC3H7) did not display any biological activity considering the concentrations tested. In order to explore the findings against S. aureus, the top ten most active compounds for the standard strain were assayed against the multidrug resistant VISA3 strain, isolated from a hospital in São Paulo/Brazil. The compounds’ selection, regarding each series, was based on the potency values (log 1/C values; see Table S3 in Supplementary material section). VISA3 strain is resistant to nineteen antimicrobial agents currently available in therapeutics, and moderately resistant to Vancomycin (MIC P4 lg/ ml).38,39 The MIC values are presented in Table 3, and the most promising derivative was 2g (R1 = n-C4H9; MIC = 4.0–2.0 lM/1.3– 0.6 lg/ml), which showed similar results for the standard strain. Of note, compounds 2u (R1 = OC3H7; MIC = 8.0–4.0 lM/2.6– 1.2 lg/ml) and 3d (R1 = Br; MIC = 16.0–8.0 lM/6.0–3.0 lg/ml) displayed lower activity values, which could be related to the inherent resistance mechanism of VISA and VRSA strains. The physical feature observed for those compounds was a significantly thicker cell wall in comparison to the standard strain.40 This feature can interfere directly on compound´s permeation across the bacterial cellular membrane. The MIC method is used to determine the lowest concentration of a drug that is able to inhibit a microorganism growth based on visualization of turbidity.41 Besides MIC method is widely used for evaluation of antimicrobial activity of several new compounds and drugs, the results are not highly qualified for qualitative or quantitative studies of structure–activity relationships, mainly because the accurate value of percentage of inhibition for each compound seems not well defined. Then, in this study, in addition to the visualization related to MIC determination, microbial growth responses were determined using absorbance readings at
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Figure 2. (A) 1H NMR spectra of NF and zoom out of spectrum ranged from 8.00 to 6.70 ppm, indicating the hydrogen atoms at benzene moiety (H11,H15 and H12,H14) and at furan system (H3 and H4). (B) 1H NMR spectra of 3-acetyl-5-(4-acetoxy-phenyl)-2-(5-nitro-furan-2-yl)-2,3-dihydro-1,3,4-oxadiazole and zoom out of spectrum ranged from 8.00 to 7.70 ppm, indicating the hydrogen atoms at benzene moiety (H15,H19 and H16,H18), at furan system (H7 and H8), and at 2,3-dihydro-1,3,4-oxadiazoline ring group (H2). (C) 13C NMR spectra of 3-acetyl-5-(4-acetoxy-phenyl)-2-(5-nitro-furan-2-yl)-2,3-dihydro-1,3,4-oxadiazole and zoom out of spectrum ranged from 22.5 to 19.5 ppm, indicating the carbons at alkyl group on acetyl (C22) and acetoxyl (Cb) groups, respectively. NMR attributions were carried out according to the numbered chemical structure.
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Table 1 Biological activity of 4-substituted-((5-nitrofuran-2-yl)methylene)benzohydrazide derivatives (2a–v) against several microorganisms related to nosocomial infections R1
R2
R3
K. pneumoniae ATCC 700603 MICa lM
S. aureus ATCC 29213 MIC lM
E. faecalis ATCC 29212 MIC lM
E. clocae ATCC 23355 MIC lM
E. coli ATCC 25922 MIC lM
S. marcescens ATCC 14576 MIC lM
C. albicans ATCC 537Y MIC lM
2a H H 2b CH3 H H 2c OCH3 2d Br H 2e CN H 2f NO2 H 2g n-C4H9 H 2h SO2NH2 H 2i Cl H 2j N(CH3)2 H 2k NH2 H 2l t-C4H9 H 2m I H 2n Cl Cl 2o Cl H 2p OC2H5 H 2q C2H5 H 2r CF3 H 2s F H H 2t C3H7 2u OC3H7 H 2v OC4H9 H Nifuroxaide Vancomycin Nitrofurantoin Levofloxacin Ampicillin Chloramphenicol Sulfamethoxazol Itraconazole DMSO (%)
H H H H H H H H H H H H H H Cl H H H H H H H
n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 128.0–64.0 128.0–64.0 n.a. n.a. — n.a.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Exploring 5-nitrofuran derivatives against nosocomial pathogens: Synthesis, antimicrobial activity and chemometric analysis Rodrigo Rocha Zorzi a,⇑, Salomão Dória Jorge a,⇑, Fanny Palace-Berl a, Kerly Fernanda Mesquita Pasqualoto b, Leandro de Sá Bortolozzo a, André Murillo de Castro Siqueira a, Leoberto Costa Tavares a a b
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmacy, University of São Paulo, Av. Prof Lineu Prestes, 580, São Paulo, SP 05508-900, Brazil Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP 05503-900, Brazil
a r t i c l e
i n f o
Article history: Received 28 January 2014 Revised 19 March 2014 Accepted 29 March 2014 Available online 8 April 2014 Keywords: 5-Nitrofuran derivatives Molecular modification Nosocomial infection Bacterial resistance Exploratory data analysis
a b s t r a c t The burden of nosocomial or health care-associated infection (HCAI) is increasing worldwide. According to the World Health Organization (WHO), it is several fold higher in low- and middle-income countries. Considering the multidrug-resistant infections, the development of new and more effective drugs is crucial. Herein, two series (I and II) of 5-nitrofuran derivatives were designed, synthesized and assayed against microorganisms, including Gram-positive and -negative bacteria, and fungi. The pathogens screened was directly related to either the most currently relevant HCAI, or to multidrug-resistant infection caused by MRSA/VRSA strains, for instance. The sets I and II were composed by substituted[N0 -(5-nitrofuran-2-yl)methylene]benzhydrazide and 3-acetyl-5-(substituted-phenyl)-2-(5-nitro-furan2-yl)-2,3-dihydro-1,3,4-oxadiazole compounds, respectively. The selection of the substituent groups was based upon physicochemical properties, such as hydrophobicity and electronic effect. The compounds have showed better activity against Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis. The findings from S. aureus strain, which was more susceptible, were used to investigate the intersamples and intervariables relationships by applying chemometric methods. It is noteworthy that the compound 4-butyl-[N0 -(5-nitrofuran-2-yl)methylene]benzhydrazide has showed similar MIC value to vancomycin, which is the reference drug for multidrug-resistant S. aureus infections. Taken the findings together, the 5-nitrofuran derivatives might be indeed considered as promising hits to develop novel antimicrobial drugs to fight against nosocomial infection. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Nosocomial or health care-associated infection (HCAI) caused by multidrug-resistant microorganisms comprises one of the most concerning problems pointed out by the World Health Organization (WHO). It is several fold higher in low- and middle-income than high income countries. It was estimated that around nine percent of hospitalized patients exhibits HCAI,1 resulting in 4 million hospitalizations and, at least, 140,000 deaths every year in the United States and Europe.2,3 The situation is getting even worse due to the acquired resistance by microorganisms to conventional therapy, being about 50–60% of total HCAI currently caused by multidrug-resistant microorganisms.1,4–6 The improper use and/or abuse of antibiotics can be pointed out as quite important to the development of resistance.1 ⇑ Corresponding authors. Tel.: +55 11 30913693; fax: +55 11 38156386. E-mail addresses: [email protected] (R.R. Zorzi), [email protected] (S.D. Jorge). http://dx.doi.org/10.1016/j.bmc.2014.03.044 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
The Gram-positive bacteria Staphylococcus aureus is commonly associated with HCAI, specially the MRSA (Methicillin-resistant S. aureus), VISA (Vancomycin-intermediate S. aureus), and VRSA (Vancomycin-resistant S. aureus) strains.1,4–7 The treatment for infections caused by these pathogens are extremely limited, since the strains are commonly non-susceptible to b-lactams, quinolones, tetracycline, and aminoglycosides. There are also several strains resistant to the new treatments, which have used Daptomycin and Linezolid, for instance.5–11 The therapeutic available options, then, would be Telavancin (Vancomycin derivative), Ceftaroline fosamil (5th generation cephalosporin), and Tigecycline (glycylcycline class).6,7 Other microorganisms which can be commonly associated to multidrug-resistant HCAI are Gram-negative bacteria, including ESBL-producing (Extended-Spectrum Beta-lactamase) strains. The available treatment for these strains is based upon carbapenems (e.g., Imipenem, Meropenem, and Doripenem).8 However, mainly Enterobacteriaceae, which is able to produce Klebsiella pneumoniae carbapenemase (KPC), can develop resistance to those antibiotics,
R. R. Zorzi et al. / Bioorg. Med. Chem. 22 (2014) 2844–2854
reducing the treatment options to the use of polimyxins (e.g., Colistin) and Tigecycline.8–21 The resistance of Gram-negative bacteria strains to Tigecycline has already been reported.9,10 In addition, cases of sepsis especially associated with fungal infections can also be a serious concern for healthcare systems. The number of fungal infections has been increased more than 200% in the last two decades, being the third most common cause of bloodstream infections.11,12 Candida spp. is responsible for 75% of all nosocomial infections caused by fungus, and Candida albicans causes 45% of the total cases primarily in immunosuppressed patients.13 The treatment available comprises polyenes (Amphoterecin B), azoles (Fluconazole and Itraconazole), and, more recently, echinocandins. However, similar to bacteria, several strains have already presented resistance to those drugs.14,15 In this regard, it is important to adopt urgent measures to prevent the inappropriate impact caused by multidrug-resistant microorganisms on healthcare systems. Among this measures are the correct prescription of antibiotics; the development and implementation of protocols for cleaning and disinfecting patient rooms, surfaces, equipment, and common areas in hospitals environments; and, also the discovery of new drugs capable of treating the multidrug-resistant bacterial infections. Nitrofurans are a class of nitro compounds, which have been used to treat bacterial infections since 1940s.16 Several studies involving this chemical class have been carried out regarding different therapeutic uses, such as tuberculostatic,17,18 antileishmanial,19,20 trypanocidal activity,21 and anti-proliferative effect on cancer cells lines.22 Nifuroxazide (NF), for instance, is a nitro compound, which was widely used as antibacterial drug in 1970s and 80s. Recently, NF has been reported as a potential QuorumSensing inhibitor on Pseudomonas aerugionosa, even though it seems not be able to stop the bacterial growth.23 NF is also considered an excellent lead compound due to its chemical structure, which benefits molecular modifications by rational design strategy. One of these modifications is the synthesis of 3-acetyl2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole ring system by cyclization reaction of N-acylhydrazone compounds, which was reviewed by Rollas and Karakusß.24 Briefly, these structures were investigated as monoamine oxidase inhibitors,25 antifungals,26 anticonvulsants,27 anti-inflammatory agents,28 antibactericidals,29,30 and trypanocidal agents.29 The findings have emphasized the potential of these molecular structures for the development of novel drugs. Herein, two series of compounds structurally analogous of NF (Fig. 1A) were designed, synthesized, and experimentally tested. Series I presents a N-acylhydrazone structure (azomethine derivatives), and series II has a heterocyclic ring system 3-acetyl-2,5disubstituted-2,3-dihydro-1,3,4-oxadiazole (oxadiazoline series) (see Fig. 1B). Antimicrobial activity was evaluated against microorganisms reported as HCAI pathogens (Candida albicans,
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Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter clocae, Escherichia coli, Serratia marcescens, and Staphylococcus aureus). In addition, derivatives that have showed promising activity in S. aureus were evaluated against multidrug-resistant S. aureus VISA3. The molecular properties were investigated by applying exploratory data analysis, a chemometric procedure, which comprises the principal component analysis (PCA) and hierarchical cluster analysis (HCA).31,32 2. Results and discussion 2.1. Chemistry The designed compounds were obtained as show in Scheme 1. The compounds of series I (substituted-((5-nitrofuran-2-yl)methylene)benzohydrazide, 2a–v) were obtained from the reaction of substituted benzohydrazides (1a–v) with 5-nitrofuran-2-carbaldehyde.33 The compounds of series II (3-acetyl-5-(substituted-phenyl)-2-(5-nitro-furan-2-yl)-2,3-dihydro-1,3,4-oxadiazole, 3a–v) were obtained by cyclization reaction of 2a–v with acetic anhydride.29,34,35 The substituent groups attached at benzene moiety were chosen based on their physicochemical properties, such as hydrophobicity and electronic effect.36 In this study, 41 compounds (22 compounds of series I and 19 compounds of series II) were synthesized and identified. All compounds were synthesized in two steps, starting from the corresponding benzohydrazides. The first step was based on classical Schiff’s base formation, whose synthesis and mechanism of reaction have been well reported and discussed.29,37 Satisfactory yields (around 90%) were obtained in this step. The second step was performed by a cyclization reaction of Schiff’s base, and presented 66% yield.35 The compounds were structurally identified (see the Supplementary information section, p. S2–S49). Compounds 3h (R1 = SO2NH2), 3j (R1 = N(CH3)2, 3k (R1 = NH2), and the respective oxadiazole analogue of NF (R1 = OH) were not obtained probably due to a reaction of the substituent groups with acetic anhydride. The presence of unbound electrons seems to provide stronger nucleophiles, as the amidic nitrogen from Schiff’s base, for instance. This observation was confirmed by 1H NMR and 13C NMR spectra (Fig. 2), considering the chemical deviation (d) related to the internal standard reference (tetrametilsilane). Regarding the 1H NMR spectra of NF (Fig. 2A), it can be noticed the presence of a singlet signal around d 12 ppm, which is related to the proton of amidic nitrogen (H8). Also, a singlet signal at d 8.40 ppm indicated the azomethine hydrogen atom (H6). After a cyclization reaction, the absence of the signal corresponding to the amidic nitrogen and a singlet signal at d 7.35 ppm (H2), which indicates the 2,3-dihydro-1,3,4-oxadiazoline ring group formation, were observed in 1H NMR spectra of the product (Fig. 2B). Furthermore, a singlet with six protons integration at d 2.29 ppm was observed, confirming the three protons related to the acetyl group and to the acetoxylation of hydroxyl group. Analyzing the 13C NMR spectra (Fig. 2C), there are signals at 167.1 and 20.9 ppm, which indicate the presence of carbonyl (C20) and alkyl groups (C22), respectively. Also, the signals at 153.4 (C5) and 84.6 ppm (C2) are related to the oxadiazole ring system. The presence of the acetoxy group attached to benzene moiety can be observed through the signals at 168.5 and 20.7 ppm. 2.2. Biological activity
Figure 1. (A) Chemical structure of nifuroxazide (NF), pointing out the regions where molecular modifications were carried out on this study. (B) General chemical structures of series I and II.
The minimal inhibitory concentration (MIC) was determined by broth microdilution method against the following strains: Candida albicans 537Y, Klebsiella pneumoniae ATCC 700603, Enterococcus faecalis ATCC 29212, Enterobacter clocae ATCC 23355, Escherichia coli ATCC 25922, Serratia marcescens ATCC 14576, and
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Scheme 1. Synthesis of series I and II compounds.
Staphylococcus aureus ATCC 29213. The derivatives with better activity profile in S. aureus were evaluated against multidrugresistant S. aureus VISA3.38,39 The activity of compounds was determined by the first wellvariant without turbidity, which was considered as the MIC value. The results are displayed in Tables 1 and 2. The compounds and the drugs used as reference were dissolved in DMSO. The range of DMSO concentration varied from 5.0% to 0.01%, and no effect on microbial growth was observed in the presence of up to 5.0% DMSO (v/v). All derivatives have presented MIC values derived from a DMSO concentration much lower than the compound concentration needed to kill the microorganism (see Tables 1 and 3). In this regard, the complete inhibition of bacterial and fungal growth was referred as due to an intrinsic activity of the investigated compounds, and any kind of synergism between compounds and DMSO was discarded. Regarding antifungal tests against C. albicans, compounds of series II were more active than compounds of series I, suggesting the improvement on activity could be due to the cyclization of N-acylhydrazone moiety. The findings were similar to previous data of our group.29 The most active compounds were 3p (R1 = OC2H5; MIC = 16.0–8.0 lM/5.5–2.7 lg/ml) and 3a (R1 = H; MIC = 16.0–8.0 lM/4.8–2.4 lg/ml). Although eighteen derivatives were non-active against E. coli strains at the concentrations tested, the compound 2k (R1 = NH2; MIC = 16.0–8.0 lM/4.3–2.1 lg/ml) has displayed 2-fold better activity than 2a (R1 = H; MIC = 32.0–16.0 lM/8.3–4.1 lg/ml). The lead compound (R1 = OH) has shown MIC value of 64.0–32.0 lM (17.6–8.8 lg/ml). Thus, the activity of compounds of series II was not quite interesting against E. coli strains. Compound 2k has also showed better activity against E. clocae strain (MIC = 8.0–4.0 lM/2.2–1.1 lg/ml). Otherwise, compounds 2t and 2u can be considered as weak inhibitors. The second most active compound was the lead compound (R1 = OH; MIC = 64.0–32.0 lM/ 17.6–8.8 lg/ml). The remaining compounds of both series did not present any activity, even at higher concentrations. The results found for the E. faecalis strain were quite similar to those for E. clocae. The compound 2k (R1 = NH2; MIC = 8.0–4.0 lM/2.2–1.1 lg/ml) and NF (R1 = OH; MIC = 16.0–8.0 lM/4.4–2.2 lg/ml) were the most active molecules. Then, there could be a positive influence on activity by the presence of hydrophilic and electron donating substituent groups attached at R1 position. In this regard, compounds of series II did not show any activity against those strains. Compounds of both series mostly did not display a suitable activity against the S. marcescens strains. Only eight compounds (2e, 2s, 2t, 2u, 3e, 3f, 3o, 3p) have presented some biological activity,
ranging from 128.0 to 64.0 lM. Similarly, just two compounds (2t and 2u) were active against K. pneumoniae (128.0 to 64.0 lM). Otherwise, against S. aureus, both series have showed a promising inhibitory activity. Compounds of series I, specially, 2g (R1 = n-C4H9, MIC = 8.0–4.0 lM/2.5–1.3 lg/ml), 2u (R1 = OC3H7; MIC = 8.0–4.0 lM/2.5–1.3 lg/ml), 2t (R1 = C3H7; MIC = 8.0– 4.0 lM/2.4–1.2 lg/ml), 2m (R1 = I; MIC = 8.0–4.0 lM/3.1–1.5 lg/ml), and 2f (R1 = NO2; MIC = 8.0–4.0 lM/2.4–1.2 lg/ml), have displayed better activity than NF (MIC = 16.0–8.0 lM/4.4–2.2 lg/ml). The lowest activity was observed for the 2b derivative (R1 = CH3; MIC = 16.0–8.0 lM/4.4–2.2 lg/ml). Regarding series II, compound 3e (R1 = CN; MIC = 4.0–2.0 lM/1.3–0.6 lg/ml) has showed better activity, and the less active compounds were 3g (R1 = n-C4H9; MIC = 32.0–16.0 lM/11.4–5.7 lg/ml) and 3v (R1 = OC4H9; MIC = 32.0–16.0 lM/11.9–6.0 lg/ml). Compounds 3l (R1 = t-C4H9) and 3u (R1 = OC3H7) did not display any biological activity considering the concentrations tested. In order to explore the findings against S. aureus, the top ten most active compounds for the standard strain were assayed against the multidrug resistant VISA3 strain, isolated from a hospital in São Paulo/Brazil. The compounds’ selection, regarding each series, was based on the potency values (log 1/C values; see Table S3 in Supplementary material section). VISA3 strain is resistant to nineteen antimicrobial agents currently available in therapeutics, and moderately resistant to Vancomycin (MIC P4 lg/ ml).38,39 The MIC values are presented in Table 3, and the most promising derivative was 2g (R1 = n-C4H9; MIC = 4.0–2.0 lM/1.3– 0.6 lg/ml), which showed similar results for the standard strain. Of note, compounds 2u (R1 = OC3H7; MIC = 8.0–4.0 lM/2.6– 1.2 lg/ml) and 3d (R1 = Br; MIC = 16.0–8.0 lM/6.0–3.0 lg/ml) displayed lower activity values, which could be related to the inherent resistance mechanism of VISA and VRSA strains. The physical feature observed for those compounds was a significantly thicker cell wall in comparison to the standard strain.40 This feature can interfere directly on compound´s permeation across the bacterial cellular membrane. The MIC method is used to determine the lowest concentration of a drug that is able to inhibit a microorganism growth based on visualization of turbidity.41 Besides MIC method is widely used for evaluation of antimicrobial activity of several new compounds and drugs, the results are not highly qualified for qualitative or quantitative studies of structure–activity relationships, mainly because the accurate value of percentage of inhibition for each compound seems not well defined. Then, in this study, in addition to the visualization related to MIC determination, microbial growth responses were determined using absorbance readings at
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Figure 2. (A) 1H NMR spectra of NF and zoom out of spectrum ranged from 8.00 to 6.70 ppm, indicating the hydrogen atoms at benzene moiety (H11,H15 and H12,H14) and at furan system (H3 and H4). (B) 1H NMR spectra of 3-acetyl-5-(4-acetoxy-phenyl)-2-(5-nitro-furan-2-yl)-2,3-dihydro-1,3,4-oxadiazole and zoom out of spectrum ranged from 8.00 to 7.70 ppm, indicating the hydrogen atoms at benzene moiety (H15,H19 and H16,H18), at furan system (H7 and H8), and at 2,3-dihydro-1,3,4-oxadiazoline ring group (H2). (C) 13C NMR spectra of 3-acetyl-5-(4-acetoxy-phenyl)-2-(5-nitro-furan-2-yl)-2,3-dihydro-1,3,4-oxadiazole and zoom out of spectrum ranged from 22.5 to 19.5 ppm, indicating the carbons at alkyl group on acetyl (C22) and acetoxyl (Cb) groups, respectively. NMR attributions were carried out according to the numbered chemical structure.
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Table 1 Biological activity of 4-substituted-((5-nitrofuran-2-yl)methylene)benzohydrazide derivatives (2a–v) against several microorganisms related to nosocomial infections R1
R2
R3
K. pneumoniae ATCC 700603 MICa lM
S. aureus ATCC 29213 MIC lM
E. faecalis ATCC 29212 MIC lM
E. clocae ATCC 23355 MIC lM
E. coli ATCC 25922 MIC lM
S. marcescens ATCC 14576 MIC lM
C. albicans ATCC 537Y MIC lM
2a H H 2b CH3 H H 2c OCH3 2d Br H 2e CN H 2f NO2 H 2g n-C4H9 H 2h SO2NH2 H 2i Cl H 2j N(CH3)2 H 2k NH2 H 2l t-C4H9 H 2m I H 2n Cl Cl 2o Cl H 2p OC2H5 H 2q C2H5 H 2r CF3 H 2s F H H 2t C3H7 2u OC3H7 H 2v OC4H9 H Nifuroxaide Vancomycin Nitrofurantoin Levofloxacin Ampicillin Chloramphenicol Sulfamethoxazol Itraconazole DMSO (%)
H H H H H H H H H H H H H H Cl H H H H H H H
n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 128.0–64.0 128.0–64.0 n.a. n.a. — n.a.
