Article pubs.acs.org/bc
GSH Induced Controlled Release of Levofloxacin from a PurposeBuilt Prodrug: Luminescence Response for Probing the Drug Release in Escherichia coli and Staphylococcus aureus Suman Pal,† Vadde Ramu,† Nandaraj Taye,‡ Devraj G. Mogare,‡ Amar M. Yeware,† Dhiman Sarkar,*,† D. Srinivasa Reddy,*,† Samit Chattopadhyay,*,‡,¶ and Amitava Das*,†,# †
Organic Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India Chromatin and Disease Biology Lab, National Centre for Cell Science, Ganeshkhind, Pune 411007, India # Central Salt and Marine Chemical Research Institute, G.B. Marg, Bhavnagar 364002, India ‡
S Supporting Information *
ABSTRACT: Fluoroquinolones are third-generation broad spectrum bactericidal antibiotics and work against both Grampositive and Gram-negative bacteria. Levofloxacin (L), a fluoroquinolone, is widely used in anti-infective chemotherapy and treatment of urinary tract infection and pneumonia. The main pathogen for urinary tract infections is Escherichia coli, and Streptococcus pneumoniae is responsible for pneumonia, predominantly a lower respiratory tract infection. Poor permeability of L leads to the use of higher dose of this drug and excess drug in the outer cellular fluid leads to central nervous system (CNS) abnormality. One way to counter this is to improve the lipophilicity of the drug molecule, and accordingly, we have synthesized two new Levofloxacin derivatives, which participated in the spatiotemporal release of drug via disulfide bond cleavage induced by glutathione (GSH). Recent studies with Streptococcus mutants suggest that it is localized in epithelial lining fluid (ELF) of the normal lower respiratory tract and the effective [GSH] in ELF is ∼430 μM. E. coli typically cause urinary tract infections and the concentration of GSH in porcine bladder epithelium is reported as 0.6 mM for a healthy human. Thus, for the present study we have chosen two important bacteria (Gram + ve and Gram − ve), which are operational in regions having high extracellular GSH concentration. Interestingly, this supports our design of new lipophilic Levofloxacin based prodrugs, which released effective drug on reaction with GSH. Higher lipophilicity favored improved uptake of the prodrugs. Site specific release of the drug (L) could be achieved following a glutathione mediated biochemical transformation process through cleavage of a disulfide bond of these purpose-built prodrugs. Further, appropriate design helped us to demonstrate that it is possible also to control the kinetics of the drug release from respective prodrugs. Associated luminescence enhancement helps in probing the release of the drug from the prodrug in bacteria and helps in elucidating the mechanistic pathway of the transformation. Such an example is scarce in the contemporary literature.
■
INTRODUCTION Prodrugs are designer drug conjugates, which are inactive and undergo an intracellular biochemical transformation for the release of the active form of the drug. 1−8 Modified luminescence response on the explicit release of the active drug at a site could allow the real time monitoring by noninvasive imaging microscopy. Despite its immense significance, examples of such photoactive prodrugs in the contemporary literature are scarce.8 Limited reports reveal that the design aspect of such prodrugs basically involves © XXXX American Chemical Society
cleavage of amide or ester linkage either by caspase-3 or upon reaction with H2O2.9−14 Other approaches include use of low cellular pH,15−20 reducing species,21 and enzymatic process.22,23 However, such approaches have rarely offered the choice for the real time monitoring of the site specific drug release by noninvasive imaging technique. Received: June 20, 2016 Revised: August 6, 2016
A
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
normal human beings and this is much higher than [GSH] in plasma (∼3 μM).28−30 Reports also suggest that administration of acivicin, a γ-glutamyltransferases inhibitor, or purified reduced glutathione to the lung through aerosol inhalation could further enhance the GSH level in respiratory tract lining fluids.31,32 E. coli typically cause urinary tract infections,33 and in general, the concentration of GSH in porcine bladder epithelium is reported to be 0.6 mM for a healthy human.33 Thus, for the present study we have chosen two important bacteria (Gram + ve and Gram − ve), which are operational in regions having high extracellular GSH concentration. Literature reports reveal that increased extracellular GSH concentration is desired for enhancing GSH concentration for cells and microbes (bacteria). More recent literature reports suggest that esterified GSH in extracellular fluid helps to increase the intracellular GSH level.34,35 Most recently Vergauwen and co-workers have shown that GshT, a type-3 solute binding protein, can bind with GSH selectively and enhances the GSH level of Gram-positive bacteria in in vitro manner.36 Considering these, our results in achieving the bactericidal effect of these new prodrugs (PD1 and PD2) in the presence of exogenous GSH have significance. Thus, we could demonstrate a proof-of-concept for designing a theranostic antibiotic prodrug that would allow us to utilize a much simpler − S−S− bond cleavage by GSH and an eventual release of the drug that is effective against Staphylococcus aureus and Escherichia coli,37−45 which are bacteria species of clinical and microbiological relevance.
Fluoroquinolones are third-generation broad spectrum bactericidal antibiotics. Furthermore, literature reports confirm that fluoroquinolones work against both Gram-positive and Gram-negative bacteria.24 Levofloxacin is extensively prescribed for anti-infective chemotherapy, urinary tract infection, and pneumonia.25 The main pathogen for urinary tract infections is Escherichia coli (E. coli, a Gram-negative bacteria), whereas Streptococcus pneumoniae (a Gram-positive bacteria) is predominantly a lower respiratory tract infection, responsible for pneumonia.25 However, overdose and frequent use of Levofloxacin cause some adverse effects on the gastrointestinal as well as the central nervous system (CNS). The adverse effects of levofloxacin on CNS cause headache, restlessness, tremor, insomnia, hallucination, convulsion, anxiety, and so forth.26 Among the regular patients on Levofloxacin, around 0.9−11% people are suffering with CNS abnormality and 3− 17% people are suffering from gastrointestinal problems.27 Poor permeability of Levofloxacin is attributed to usage of a higher dose of the drug by the patients. The extra Levofloxacin that exists in the outer cellular fluid binds with the cationic γaminobutyric acid (GABA) receptor channel instead of GABA, leading to CNS abnormality.26,27 It has been argued that the effective dose for such a drug could be reduced by enhancing its bacterial membrane permeability, thereby improving the effective uptake. One of the ways to achieve this goal is to improve the lipophilicity of the drug molecule. Accordingly, we have attached lipophilic appendages to the Levofloxacin through a disulfide linker in such a way that, on internalization of these Levofloxacin derivatives, spatiotemporal release of drug could be achieved through glutathione (GSH) induced disulfide bond cleavage. Studies have also revealed that these newly synthesized Levofloxacin derivatives are less toxic than Levofloxacin itself. Importantly, for two new prodrugs (PD1 and PD2), acid functionality is converted to the corresponding ester group and this nullifies any possibility of the respective Levofloxacin fragments (as the part of these prodrugs) of PD1 and PD2 to exist in anionic form in extracellular fluid. This essentially is expected to reduce the option for the prodrugs to be able to attach to the receptor of GABA.26,27 Thus, this approach should help us in reducing the CNS abnormality, which is one of the major side effects of Levofloxacin due to the higher dose of the drug (Scheme 1). A report on GSH estimation in the epithelial lining fluid (ELF) of the normal lower respiratory tract, where it is thought to play a crucial role in providing antioxidant protection to the epithelial cells and killing the bacterial pathogens responsible for pneumonia, reveals that effective [GSH] is ∼430 μM in
■
RESULT AND DISCUSSION Fluorescence Quenching in PD1 and PD2. Spectroscopic data for Levofloxacin, LSSTr, and LSSDMTr were recorded in an essentially aqueous medium (H2O:DMSO of 99:1, v/v) and these spectra were solely dominated by transitions associated with Levofloxacin. Deconvolution of the spectra of Levofloxacin (Figure S4), LSSTr, and LSSDMTr revealed three distinct absorption bands at around 295, 325, and 335 nms (Figure S4). On excitation at 299 nm, an emission band with maximum at 455 nm was observed (relative quantum yield (Φ) of 0.25 (ΦL) for Levofloxacin.46 Absorption band maximum at 299 nm was attributed to the π → π* and/or n → π* transitions. Interestingly, Φ for LSSTr (ΦLSSTr = 0.0051) and LSSDMTr (ΦLSSDMTr = 0.00495) were found to be much less. Presumably, folded conformation (vide infra) of LSSTr and LSSDMTr favored an efficient nonradiative deactivation of Levofloxacinbased excited state and accounted for the observed luminescence quenching. 1D and 2D 1H NMR spectra for Levofloxacin, LSSTr, and LSSDMTr with complete assignment are provided in the Supporting Information. A close look at the rotating-frame overhauser spectra (ROESY) for LSSTr (Figure 1) reveals a distinct interaction between Hj and Hd and He, while a weak interaction is also evident between Hi/Hg−Hf (Figure S13). Such observations agree well with the proposed folded conformation for LSSTr in DMSO-d6 solution. It may not be unreasonable to presume that such a conformation also prevails for LSSDMTr under similar experimental conditions. Such interactions are generally operational over a shorter distance,47 which presumably favored the CT transition between the trityl and Levofloxacin moieties and accounted for an efficient luminescence quenching of the Levofloxacinbased excited states in LSSTr/LSSDMTr. For all luminescence
Scheme 1. Synthetic Methodology for the Preparation of LSS-Tr and LSS-DMTr
B
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
levofloxacin (I), led to the release of levofloxacin through an intramolecular cyclization reaction (vide infra). Scheme 2. Proposed Disulfide Bond Cleavage in LSSTr and LSSDMTr
Attempts were made for isolation of each component by column chromatography. The crude of reaction mixture was divided into two parts. First, half of this reaction mixture was subjected to solvent extraction with dichloromethane (20 mL × 3) and the organic part was evaporated under reduced pressure to isolate the crude solid. This was subjected to column chromatography for separation of two of the four reaction products with Rf value of 0.75 (15 mg) and Rf value of 0.45 (∼10 mg) in its pure form. The major component with Rf value of 0.75 was identified as trityl alcohol based on results of the ESI-MS, 1H NMR, D2O exchange in 1H NMR, 13C NMR and DEPT studies (Figures S19−23). The 13C NMR spectrum, recorded for this fraction with Rf value of 0.75 (15% methanol in dichloromethane), showed a signal at 82 ppm and this confirmed the presence of the quaternary C atom of trityl fragment. As anticipated, the signal of a quaternary C atom disappeared in the DEPT spectrum. Also, a signal for the HOH in the trityl alcohol appeared at 2.7 ppm and this disappeared when treated with D2O. These evidences confirmed the generation of the trityl alcohol, as proposed in Scheme 2. The absence of any 13C signal at 62 ppm nullified any possibility for the formation of triphenyl methane, which could have been generated through the cleavage of the O−CTriphenyl bond in LSSTr, instead of O−CC2H4. A second component with Rf value of 0.45 was identified as unreacted LSSTr (10 mg) based on the results of the 1H, 13C, and ESI-MS spectral studies. The other half of reaction mixture was also subjected to solvent extraction with dichloromethane (20 mL × 3) and a crude reaction product was isolated on evaporation of the organic fraction under vacuum. This crude was purified via preparative TLC (using PLC Silica gel 60 F254, 2 mm plate). Two components with Rf values of 0.25 (3 mg) and 0.12 (2 mg) were isolated. HRMS data (Figure S25) confirmed that the product with Rf value of 0.12 was the thiol ethyl ester of levofloxacin (4) with a distinct signal at 421 corresponding (Figures S24−S25) to m+/ z for C20H24F N3O4S; while the product with Rf value of 0.25 was identified as Levofloxacin (m+/z of 362 for C18H21FN3O4) (Figures S26−S27). 1H NMR spectrum for each of these two components was also consistent with these interpretations (SI). Based on these results, the above-mentioned reaction pathway
Figure 1. Changes in luminescence spectral pattern for (A) [LSSTr] (7.2 × 10−5 M) or (B) LSSDMTr (8.0 × 10−5 M) on reaction with varying [GSH] ((0−7.2) × 10−4 M for LSSTR and (0−36.0) × 10−4 M for LSSDMTr), λExt = 299 nm, and aq. buffer−DMSO medium (99:1, v:v; pH 6.5) solution were used for the studies. (C) Partial 2D ROSEY spectrum (400 MHz) of LSSTr in DMSO-d6 revealing appreciable interactions between Hj[L] and Hd and He[ethylene] protons. Cartoon representation of the prodrug LSSTr depicting the folded conformation, as suggested by 1H NMR studies.
spectral studies, solution pH of 6.5 was maintained using 0.5 mmol HEPES-HCl buffer, unless mentioned otherwise.48 Prodrugs (LSSTr and LSSDMTr) were treated with varying [GSH] in aq. buffer-DMSO medium (99:1, v/v; pH 6.5). A sharp increase in the intensity at ∼455 nm (Figure 1) was observed for both experiments and emission spectra recorded after 3 h of incubation with GSH, matched closely with the emission spectra for Levofloxacin (Figure 1A,B). These results tended to suggest that reaction of GSH with LSSTr or LSSDMTr led to the eventual release of Levofloxacin. This was confirmed from results of various spectroscopic (ESI-Ms, HRMS, 1H, 13C, and DEPT NMR) as well as the results of the HPLC studies. Fragmentation Mechanism Using Reverse Phase HPLC and Kinetics Studies. To have a clear idea about the products, LSSTr (50 mg, 0.0675 mM) was treated with GSH (5 equiv) in DMSO water system (1:10, v:v) and the reaction mixture was allowed to stir for 3 h at RT. The resultant reaction mixture was subjected to a TLC study (using PLC Silica gel 60 F254, 2 mm plate, and 15% methanol in CH2Cl2 as eluent) and this showed four major spots apart from the one for LSSTr (Rf = 0.45). Other spots were identified as (vide infra) trityl alcohol (Rf = 0.75), reaction intermediate (I) thiol ethyl ester of levofloxacin (Rf = 0.12), and released Levofloxacin drug Rf = 0.25 (Scheme 2). Reaction intermediate, thiol ethyl ester of C
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
where m = LSSTrk1 or LSSDMtrk1) of the pseudo-first-order rate constants (LSSTRkobs and LSSDMTrkobs) on [GSH] was observed for two prodrugs (Figure 2A,B) and this clearly suggested that the rate-determining step for these two reactions involved GSH. Also, intercepts for both plots were negligible and were ignored, which also suggested a negligible side reaction that could contribute to these observed luminescence changes. This helped us in evaluating absolute rate constant for respective prodrugs (LSSTr and LSSDMTr): LSSTrk1 = (20.28 ± 0.5) s−1 or LSSDMtrk1 = (2.4 ± 0.3) s−1 (at 37 °C in 10 mmol phosphatebuffered saline (PBS) buffer medium of pH 6.5). The linear dependency of kobs on [GSH] and the negligible intercept suggested that observed changes in fluorescence at 455 nm were primarily due to the reaction shown in step 1 of Scheme 2, which led to the generation of the intermediate 4. Rate constant for the generation of L from 4 (step 2 of Scheme 2) was evaluated based on the results of the kinetic studies performed with HPLC (Figure S18) technique for an essentially aq. buffer solution (aq. HEPES-buffer−methanol; 99:1; v/v; pH 6.5) of isolated pure intermediate 4 (5 μM) using Kromasil column RP-18 (250 × 4.6 mm). Details about the experimental procedures are provided in the SI. Under this specified condition, a peak for 4 appeared at retention time of 5.26 min. This solution of 4 was allowed to stir at 23 °C and subsequent chromatograms (Figure S18) recorded with increasing time intervals revealed a steady decrease in [4] with concomitant increase in peak intensity with retention time of 8.06 min. Control experiment with pure L under the identical experimental condition revealed that the signal at 8.06 min was solely for L. This confirmed that with time intermediate 4 was getting converted to L. Another control experiment with externally added GSH (50 μM) revealed that GSH failed to alter either the rate of the reaction or the extent of conversion of 4 to L. These data revealed that GSH had no role in the conversion shown in step 2 of Scheme 2. The good linear fit for decrease in [4] vs time or increase in [L] vs time (Figure 2C,D) confirmed the zero-order reaction. This further confirmed the proposed intramolecular cyclization reaction for intermediate 4 (step 2 of Scheme 2) and establised the fact that GSH had no role in influencing step 2 of this reaction. The rate constant for this step 2 was evaluated either by determining the rate of disappearance of I (−Ik2 = (2.57 ± 0.09) × 10−8 M L−1 s−1) or by the rate of formation of L (+Lk2 = (2.46 ± 0.07) × 10−8 M L−1 s−1) (Figure 2C,D). It is worth mentioning here that the generic drug L is reported to be most soluble in the pH range 6.5−6.7 and this is the primary reason for performing reaction kinetics at pH 6.5.48 These kinetic data for Step 1 (Scheme 2) also revealed that the cleavage process was more efficient for LSSTr than that of LSSDMTr (Figure 2A,B) as reflected in the rate constants for the respective reaction. This demonstrated the fact that perhaps one could achieve the slower release of the effective drug through appropriate design of the prodrug. Kinetic data also revealed that the extent of disulfide bond cleavage induced by Cys or Hcy was negligible compared to those observed for GSH (Figure 2A,B) and these led us to perform detailed studies only with GSH. Unique Reaction Pathway for S−S Bond Cleavage. To the best of our knowledge, there are only limited reports that had utilized the GSH induced disulfide bond cleavage reaction for drug release from a prodrug. All such examples revealed formation of a bis-carbamate/bis-amide derivative of a thiol moiety as an intermediate, which underwent an intramolecular cyclization reaction for eventual release of the drug along with
is proposed for LSSTr (Scheme 2). It is not unreasonable to presume that LSSDMTr will also undergo a similar transformation. The same reaction mixture of LSSTr (50 mg, 0.0675 mmol) and GSH (5 mol equiv) in the DMSO−H2O system (1:10, v/v at RT for 3 h) was analyzed by reverse phase HPLC (Kromasil RP-18 (150 × 4.6 mm) column) to further validate our results (Figure S17). Interestingly, the HPLC results also revealed that [4] was reduced over time with concomitant increase in [L] in the absence of any external agents, which also corroborated our presumption about the intramolecular cyclization reaction of 4 that eventually led to the release of L. Isolation of the intermediate 4 during the reaction of GSH with LSSTr suggested that the release of drug L happened in two steps (Scheme-2): the first step being the immediate cleavage of disulfide bond induced by the nucleophilic attack of GSH, which led to the generation of the thioester intermediate (4) and the second step being the intramolecular nucleophilic substitution reaction involving the free sulfhydryl group of 4, which eventually led to the release of L and the thiirane moiety. Fluorescence quantum yield for 4 (ΦI = 0.2) was found to be comparable to L (ΦL = 0.25). Thus, the observed enhancement in emission intensities at ∼455 nm for reactions of GSH with LSSTr/LSSDMTr could be accounted either for Step I (generation of 4) or for the concerted process involving Steps I and II (Scheme 2). To unravel this, systematic kinetic studies were performed by monitoring luminescence changes at 455 nm (Figure 2). Pseudo-first-order rate constants (kobs) for reaction with LSSTR or LSSDMTr were plotted against respective [GSH] used. Linear dependency (kobs = m[GSH],
Figure 2. Plot of kobs vs [GSH] at 37 °C in 0.5 mM HEPES buffer medium having 1% DMSO (v/v) for prodrugs (A) LSSTr and (B) LSSDMtr, respectively, using [LSSTr] or [LSSDMTr] = 5.0 × 10−5 M and λEmsMon = 445 nm. Good linear fit of the zero-order kinetic plots for the conversion of intermediate 4 to L by monitoring (C) the decrease in [4] with time and (D) the increase in [L] with time by monitoring changes in signal at RT of 5.25 and 8.06 min, respectively, in HPLC chromatogram performed with aq. buffer solution (aq. HEPES-buffer−methanol; 99:1, v/v; pH 6.5) using isolated pure intermediate I (5 μM). D
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry 1,3-oxathiolan-2-one.39,45 In the present study, we introduced a unique synthon involving an ester derivative of a thiol moiety, which underwent an intramolecular cyclization reaction for elimination of thiirane and the eventual release of levofloxacin (Scheme 2). Elimination of thiirane owing to an internal cyclization reaction was reported earlier.2,37−45 Recent studies with Streptococcus mutants suggest that GSH accumulates in Gram-positive bacteria. These mutants make its own GSH as well as imports GSH to create intracellular concentrations that are significantly higher than those attained from de novo synthesis.36 This report further corroborates our proposed proof of concept in releasing the effective drug Levofloxacin from prodrugs PD1 and PD2 upon reaction with GSH. Antibacterial Activity of these Prodrugs. Preliminary antimicrobial studies with newly synthesized Levofloxacin derivatives (PD1 and PD2) using E. coli and Staphylococcus aureus revealed that, in the presence of exogenous GSH, these reagents were as effective as Levofloxacin, which was reflected in their minimum inhibitory concentration (MIC) values for respective drugs presented in the SI. For these studies, these bacterial stains, after prior incubation with GSH, were further exposed to PD1 and PD2. Such bacterial stains were viewed under CLSM. CLSM images shown in Figure 3 clearly reveal that accumulation of the released Levofloxacin from PD1 and PD2 in these bacterial strains are much better than that of pure Levofloxacin. It has been argued by many researchers that lipophilicity of the reagent is one of the most essential criteria for achieving improved cell membrane permeability.49,50 Higher partition coefficients (logP) for these two prodrugs (logP = 1.63 for
LSSTr and logP = 1.67 for LSSDMTr) than that for L (logP = 1.3) were also expected to improve the lipophilicity and the cell membrane permeability. Enhanced lipophilicity could be attributed to better penetration or uptake of prodrugs (PD1 and PD2) and the subsequent release of Levofloxacin accredited to GSH induced disulfide bond cleavage. Solution studies had clearly revealed that Φ values for PD1 and PD2 are substantially lower than that of Levofloxacin (Figure 1A,B and Scheme 1). PD2 being more lipophilic than PD1, effective accumulation of Levofloxacin in both Gram + ve and Gram − ve bacteria was higher (in short-term) for PD2. Furthermore, CLSM images also reveal that intake of PD1 and PD2 is greater in Gram − ve E. coli than that of Gram + ve bacteria Staphylococcus aureus, which is not an unusual phenomena considering the difference in the intrinsic nature of the cell wall for Gram + ve and Gram − ve bacteria (Figure S2).51 Minimum inhibitory concentration (MIC)52−57 study reveals that PD1 or PD2, in combination with GSH, could be used as
Figure 4. (A) Antibacterial activity plots: Plot of log10CFU/mL [CFU: colony formation unit] or values after 12 h of incubation for control and for strains of Staphylococcus aureus in the presence of different drugs and prodrugs at their MIC concentration (GSH of 5 equiv was used as indicated). B. Plot of percentage of growth inhibition of bacterial colonies of S. aureus at different GSH concentration for optimizing the GSH concentration that is required for the release of Levofloxacin from PD1 and PD2 (Levofloxacine, PD1, and PD2 used as their MIC conc). Figure 3. Kinetic study of intake prodrugs and concomitant release of drugs by GSH which is also incubated before prodrug incubation in E. coli. First set of 4 showing confocal images using Hoechst channel (λExt = 352 nm and λEms = 460 nm) of original E. coli bacterial pictures incubated with only GSH, GSH-Levofloxacin (conc 1 μM where GSH used 5 equiv respectively), GSH-PD1, GSH-PD2 (for PD1 and PD2MIC conc have been used with 5 equiv GSH); second set of 4 showing their intensity plot only.
prodrug for both Gram + ve and Gram − ve bacteria. Colony formation unit (CFU) studies were also performed for optimizing the GSH concentration,58−60 which confirmed that either PD1 or PD2 could inhibit (Figures 4 and 5 and Figure S1) the bacterial growth 99% in the presence of 5 equiv GSH. E
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Bioconjugate Chemistry
■
Article
CONCLUSIONS In brief, we have designed two new Levofloxacin derivatives (LSSTr (PD1) and LSSDMTr (PD2)), which could act as antibiotic prodrugs. Both of these prodrugs produced Levofloxacin upon specific reaction with GSH. This was primarily achieved through the disulfide bond cleavage reaction induced by the sulfhydryl functionality. Different intermediates and final product as Levofloxacin were isolated based on the reverse phase chromatography. These were also confirmed based on other spectroscopic studies. This enabled us to study the kinetics and establish the mechanistic pathway for this reaction. Studies also reveal that GSH induced release of Levofloxacin happens in Gram + ve and Gram − ve bacteria in a slightly controlled manner. Higher lipophilicity of LSSDMTr (PD2) as compared to the drug Levofloxacin further facilitate higher accumulation of these two prodrugs as well as the subsequent release of Levofloxacin in these bacterial strains. MIC study reveals that PD1-GSH or PD2-GSH combinations can be used as a drug in lower concentration (μM). Results of the CFU study confirmed that this PD1-GSH or PD2-GSH combination can inhibit bacterial growth within 12 h with more than 99% efficiency. MTT assay suggest that PD1 and PD2 are less toxic than the original drug Levofloxacin. Higher permeability due to higher lipophilicity could perhaps help in lowering the usage of the effective concentration of the drug for bacterial growth inhibition. The present example also demonstrates a new reaction pathway for the release of the drug from a prodrug through a cleavage of the disulfide bond by GSH. Results of the kinetic studies reveal that disulfide bond cleavage (first step) is faster for PD1 than for PD2. However, higher lipophilicity favors the better uptake of the prodrug PD2 in bacterial strains and Hct116 cells.
Figure 5. Antiproliferative influence of Levofloxacin (Control), PD1, and PD2. Snapshot of the time dependent growth of bacterial colonies (Staphylococcus aureus): (A) only bacteria, (B) bacteria with Levofloxacin, (C) bacteria with PD1 and GSH, and (D) bacteria with PD2 and GSH.
Interestingly, results of the MTT assay (SI Table S3 and Figure S3) revealed that LSSTr and LSSDMTr were less toxic
■
EXPERIMENTAL SECTION General Experimental Information. Starting materials like Levofloxacin, trityl chloride, dimethoxy trityl chloride, diisopropyl-ethyl-amine, bis(2-hydroxyethyl)-disulfide, and solvents like dichloromethane, pyridine, hexane, and ethyl acetate have been procured from Sigma-Aldrich, Alfa Aesar, and Ranchem Company. For recording spectra, spectroscopy-grade solvent have been used. For purification of the products column chromatography has been employed using Merck 100−200 silica gel. For separation of the fragments, preparative TLC plates (using PLC Silica gel 60 F254, 2 mm plate) have been employed. 1H, DEPT, and other NMR spectra were recorded in Bruker 500, 200 MHz, Jeol 400 MHz spectrometer. Mass and HRMS spectra were recorded in JEOL JM AX 505 HA mass spectrometer. Absorption and emission spectra of those compounds has been recorded using Cary 500 and Edinburgh instrument Xe-900 spectrometer, while confocal cell images were recorded in Olympus Fluoview microscope instrument. HPLC separation of fragments from the reaction mixture is performed by using Merck Hitachi instrument (PUMP L-7100, UV Detector L-7400) using reverse-phase Kromasil column RP-18 (250 × 4.6 mm). Experimental Details. Trityl-SS-OH (2) and Dimethoxy Trityl-SS-OH are prepared according to the reported method.64,65 1 H NMR of trityl-SS-OH: 1H NMR (200 MHz, CDCl3, ppm), δ 7.56−7.5 (m, 6H), 7.5−7.23 (m, 9H), 3.78 (t, J = 6.0 Hz, 2H), 3.39 (t, J = 6.5 Hz, 2H), 2.88 (t, J = 6.5, 2H), 2.7 (t, J = 6.0 Hz, 2H), 1.89 (s, 1H).
Figure 6. CLSM images for colocalization studies with ER Tracker-1 Green and Levofloxacin released from LSSDMTr on reaction with intracellular GSH in HCT116 cells after incubation with LSSDMTr (100 μM) for 1 h.
than the original drug L toward live Hct116 (colon cancer cells). The presence of Levofloxacin moiety as an ester derivative in PD1 and PD2 presumably nullifies the possibility of these prodrugs to be able to attach with the receptor of GABA and could accounted for the lower toxicity. Colocalization studies were performed with ER-TrackerGreen (BODIPY FL Glibenclamide)61−63 and LSSDMTr. Merged image (having Pearson’s coefficient 0.78 with overlap of 0.93) (Figure 6 and SI Figure S28) clearly revealed that the Levofloxacin produced from the lipophilic prodrug PD2 (LSSDMTr) was localized solely in the lipid dense regions (cytoplasm and ER region) of cells and not in the nucleus. It is not unreasonable to presume that LSSTr will also behave similarly. This could have also favored the observed lower toxicity of these two prodrugs. F
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry Prodrug 1 (LSSTr) (3a). Levofloxacin (100 mg, 0.27 mmol, 1 equiv), (2-tritylethyl, 2′-hydroxyethyl)-disulfide (210 mg, 0.54 mmol, 2 equiv), and O-(1H-benzotriazole-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate (HBTU) (150 mg, 0.405 mmol, 1.5 equiv) were taken in 10 mL dry dichloromethane in inert dinitrogen atmosphere followed by the addition of triethylamine (150 μL, 1.08 equiv). The reaction mixture was allowed to stir at room temperature for 72 h in inert atmosphere. The reaction mixture washed with water thoroughly and the organic part was evaporated to dryness. The crude was separated by column chromatography (eluent: 5% methanol in dichloromethane). 120 mg pure product was obtained with 60% yield. 1H NMR (500 MHz, CDCl3, ppm), δ 8.31 (s, 1H), 7.58 (d, J = 12.5 Hz, 1H), 7.46 (d, J = 12.5, 6H), 7.31 (t, J = 7.5, 6H), 7.23 (t, J = 7.25 Hz, 3H), 4.54−4.48 (m, 2H), 4.34−4.28 (m, 3H), 3.4 (t, J = 6.25 Hz, 6H), 2.95 (t, J = 6.75 Hz, 2H), 2.91 (t, J = 6.25 Hz, 2H), 2.70 (s, 4H), 2.47 (s, 3H), 1.48 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, ppm) δ 173.1, 164.9, 156.7−154.8 (d, J = 238 Hz), 145.5, 143.9, 139.8, 131.5, 131.4, 129.6, 128.7, 127.8, 127.1, 123.7, 123.3, 108.9, 106.1, 105.6−105.4 (d, J = 25 Hz), 86.9, 68.1, 62.6, 62.2, 60.4, 55.6, 54.8, 50.1, 46.1, 41.4, 39.6, 37.1, 18.2. ε at 290 nm 1568 M−1, 330 nm 7527 M−1. Observed HRMS peak at 740.2600 attributed to C41H43O5N3FS2 for (M + H+) and 762.2400 for C41H42O5N3FS2Na (M + Na+). Prodrug 2 (LSSDMTr) (3b). Levofloxacin (100 mg, 0.27 mmol, 1 equiv), (2-dimethoxytritylethyl, 2′-hydroxyethyl)disulfide64,65 (250 mg, 0.54 mmol, 2 equiv), and O-(1Hbenzotriazole-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) (150 mg, 0.405 mmol, 1.5 equiv) were taken in 10 mL dry dichloromethane in inert dinitrogen atmosphere followed by the addition of triethylamine (150 μL, 1.08 equiv). The reaction mixture was allowed to stir at room temperature for 72 h in inert atmosphere. The reaction mixture washed with water thoroughly and the organic part was evaporated to dryness. The crude was separated by column chromatography (eluent: 5% methanol in dichloromethane). 130 mg pure product was obtained with 60% yield. 1H NMR (500 MHz, CDCl3, ppm) δ 8.35 (s, 1H), 7.61 (d, J = 12.5 Hz, 1H), 7.46 (d, J = 7.0 Hz, 2H), 7.34 (d, J = 9.0 Hz, 4H), 7.28 (t, J = 8.0 Hz, 2H), 7.2 (t, J = 7.25 Hz, 1H), 6.82 (dd, J1 = 1.5 Hz, J2 = 9 Hz, 4H), 4.5 (t, J = 6.5 Hz, 2H), 4.35−4.25 (m,3H), 3.38 (m, 6H), 2.96−2.91 (m, 4H), 2.7 (s, 4H), 2.46 (s, 3H), 1.5 (d, J = 6.5, 3H) and 1.43 (d, J = 7.0 Hz, 6H). 13C NMR (125 MHz, CDCl3, ppm) δ 173.2, 164.7, 158.5, 156.8−154.8 (d, J = 250 Hz), 145.7, 144.8, 139.8, 136.1, 131.6, 130, 128.2, 127.8, 126.8, 123.8, 113.1, 108.7, 105.3, 86.3, 68.2, 62.6, 62, 55.6, 55.2, 54.9, 54.3, 50.1, 46.1, 42.5, 39.7, 37, 18.2, 12.5. ε at 290 nm 19221 M−1 and at 330 nm 9234 M−1. Observed HRMS at 800.2828 is attributed to C43H47O7N3FS2 (M+H+). 1 H and 13C NMR of hydroxy trityl fragment 7. 1H NMR (200 MHz, CDCl3, ppm) δ 7.21 (m, 15H), 2.7 (s, 1H). 13C NMR (100 MHz, CDCl3, ppm) δ 146.8, 127.9, 127.3, 82. Observed HRMS peak at 243.1169 is attributed to C19H15. 1 H NMR of hydroxy ethyl ester of Levofloxacin (intermediate [4]). 1H NMR (200 MHz, MeOD, ppm) δ 8.97 (s, 1H), 7.66 (d, J = 12.5 Hz, 1H), 4.6 (m, 3H), 3.43 (m, 8H), 2.7 (m, 4H), 2.44 (s, 3H), 1.57 (d, J = 6.8 Hz, 3H). Observed HRMS peak at 421.1455 is attributed to C20H24O4N3FS (intermediate 1+H). 1 H NMR of Levofloxacin fragment 6. 1H NMR (200 MHz, CD3OD, ppm) δ 8.7 (s, 1H), 7.53 (d, J = 12, 1H), 4.47−4.43 (m, 3H), 3.33 (m, 4H), 2.57 (m, 4H), 2.31 (s, 3H), 1.45 (d, J =
6, 3H). Observed HRMS peak at 362.1505 is attributed to C18H21O4N3F (M+H+).
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00324. Detailed experimental procedure about biological experiments with necessary plots, lipophilicity measurement, HPLC analysis, fragment separation through HPLC with histograms, reference quantum yield measurement, ROSEY correlation, deconvolution of UV−vis spectra of prodrugs and Levofloxacin,and detailed characterization of compounds and fragments PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: *E-mail: *E-mail: *E-mail:
[email protected].
[email protected].
[email protected].
[email protected].
Present Address ¶
Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata-00032, India
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS A.D. acknowledges SERB (India) Grant (SB/S1/IC-23/2013) and CSIR-CSMCRI network project (CSC 0134) for funding. S.P. is thankful to BSC0124 and SERB YSS/2015/1081 (GAP 312526) for financial support and D.S.R. is thankful to CSIR, New Delhi for the financial support through BSC0124. V.R., N.T., and A.Y. are thankful to CSIR, New Delhi for their fellowships.
■
REFERENCES
(1) Wu, X. M., Sun, X. R., Guo, Z. Q., Tang, J. B., Shen, Y. Q., James, T. D., Tian, H., and Zhu, W. H. (2014) In vivo and in situ tracking cancer chemotherapy by highly photostable NIR fluorescent theranostic prodrug. J. Am. Chem. Soc. 136, 3579−3588. (2) Santra, S., Kaittanis, C., Santiesteban, O. J., and Perez, J. M. (2011) Cell-specific, activatable, and theranostic prodrug for dualtargeted cancer imaging and therapy. J. Am. Chem. Soc. 133, 16680− 16688. (3) Li, C. M., Chen, T., Ocsoy, I., Zhu, G. Z., Yasun, E. M., You, X., Wu, C. C., Zheng, J., Song, E., Huang, C. Z., and Tan, W. H. (2014) Gold-Coated Fe3O4 Nanoroses with Five Unique Functions for Cancer Cell Targeting, Imaging and Therapy. Adv. Funct. Mater. 24, 1772−1780. (4) Melancon, M. P., Zhou, M., and Li, C. (2011) Cancer theranostics with near-infrared light-activatable multimodal nanoparticles. Acc. Chem. Res. 44, 947−956. (5) Patra, C. R., Bhattacharya, R., Wang, E., Katarya, A., Lau, J. S., Dutta, S., Muders, M., Wang, S., Buhrow, S. A., Safgren, S. L., et al. (2008) Targeted delivery of gemcitabine to pancreatic adenocarcinoma using cetuximab as a targeting agent. Cancer Res. 68, 1970−1978. (6) Redy, O., and Shabat, D. (2012) Modular theranostic prodrug based on a FRET-activated self-immolative linker. J. Controlled Release 164, 276−282. (7) Cao, Y., Pan, R., Xuan, W., Wei, Y., Liu, K., Zhou, J., and Wang, W. (2015) Photo-triggered fluorescent theranostic prodrugs as DNA
G
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry alkylating agents for mechlorethamine release and spatiotemporal monitoring. Org. Biomol. Chem. 13, 6742−48. (8) Kuang, Y., Balakrishnan, K., Gandhi, V., and Peng, X. (2011) Hydrogen peroxide inducible DNA cross-linking agents: targeted anticancer prodrugs. J. Am. Chem. Soc. 133, 19278−19281. (9) Shi, H., Kwok, R. T. K., Liu, J., Xing, B., Tang, B. Z., and Liu, B. (2012) Real-Time Monitoring of Cell Apoptosis and Drug Screening Using Fluorescent Light-Up Probe with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc. 134, 17972−17981. (10) Kumar, R., Han, J., Lim, H. J., Ren, W. X., Lim, J. Y., Kim, J. H., and Kim, J. S. (2014) Mitochondrial induced and self-monitored intrinsic apoptosis by antitumor theranostic prodrug: in vivo imaging and precise cancer treatment. J. Am. Chem. Soc. 136, 17836−17843. (11) Kim, E. J., Bhuniya, S., Lee, H., Kim, H. M., Cheong, C., Maiti, S., Hong, K. S., and Kim, J. S. (2014) An activatable prodrug for the treatment of metastatic tumors. J. Am. Chem. Soc. 136, 13888−13894. (12) Liu, P., Xu, J., Yan, D., Zhang, P., Zeng, F., Li, B., and Wu, S. (2015) A DT-diaphorase responsive theranostic prodrug for diagnosis, drug release monitoring and therapy. Chem. Commun. 51, 9567−9570. (13) Wang, J. Q., Sun, X. R., Mao, W. W., Sun, W. L., Tang, J. B., Sui, M. H., Shen, Y. Q., and Gu, Z. W. (2013) Tumor redox heterogeneityresponsive prodrug nanocapsules for cancer chemotherapy. Adv. Mater. 25, 3670−3676. (14) Redy-Keisar, O., Ferber, S. K., Satchi-Fainaro, R., and Shabat, D. (2015) NIR Fluorogenic Dye as a Modular Platform for Prodrug Assembly: Real-Time in vivo Monitoring of Drug Release. ChemMedChem 10, 999−1007. (15) Li, S. Y., Liu, L. H., Jia, H. Z., Qiu, W. X., Rong, L., Cheng, H., and Zhang, X. Z. (2014) A pH-responsive prodrug for real-time drug release monitoring and targeted cancer therapy. Chem. Commun. 50, 11852−11855. (16) Yang, X. Z., Du, X. J., Liu, Y., Zhu, Y. H., Liu, Y. Z., Li, Y. P., and Wang, J. (2014) Rational design of polyion complex nanoparticles to overcome cisplatin resistance in cancer therapy. Adv. Mater. 26, 931− 936. (17) Fan, J. Q., Fang, G., Zeng, F., Wang, X. D., and Wu, S. Z. (2013) Water-dispersible fullerene aggregates as a targeted anticancer prodrug with both chemo- and photodynamic therapeutic actions. Small 9, 613−21. (18) Fan, J. Q., Zeng, F., Wu, S. Z., and Wang, X. D. (2012) Polymer Micelle with pH-Triggered Hydrophobic−Hydrophilic Transition and De-Cross-Linking Process in the Core and Its Application for Targeted Anticancer Drug Delivery. Biomacromolecules 13, 4126− 4137. (19) Chang, G. T., Yu, L., Ding, J. D., and Ci, T. Y. (2011) Enhancement of the fraction of the active form of an antitumor drug topotecan via an injectable hydrogel. J. Controlled Release 156, 21−27. (20) Liu, J., Huang, Y. R., Kumar, A., Tan, A., Jin, S. B., Mozhi, A., and Liang, X. J. (2014) pH-sensitive nano-systems for drug delivery in cancer therapy. Biotechnol. Adv. 32, 693−710. (21) Yuan, Y. Y., Chen, Y. L., Tang, B. Z., and Liu, B. (2014) A targeted theranostic platinum(IV) prodrug containing a luminogen with aggregation-induced emission (AIE) characteristics for in situ monitoring of drug activation. Chem. Commun. 50, 3868−3870. (22) Weinstain, R., Segal, E., Satchi-Fainaro, R., and Shabat, D. (2010) Real-time monitoring of drug release. Chem. Commun. 46, 553−555. (23) Sharma, K., Iyer, A., Sengupta, K., and Chakrapani, H. (2013) INDQ/NO, a bioreductively activated nitric oxide prodrug. Org. Lett. 15, 2636−2639. (24) Wispelwey, B., and Schafer, K. R. (2010) Fluoroquinolones in the management of community-acquired pneumonia in primary care. Expert Rev. Anti-Infect. Ther. 8, 1259−1271. (25) Dasaraju, P. V., and Liu, C. (1996) Infections of the Respiratory System, In Medical Microbiology, 4th ed. (Baron, S., Ed.) Chapter 93, University of Texas Medical Branch at Galveston. (26) Moorthy, N., Raghavendra, N., and Venkatarathnamma, P. N. (2008) Levofloxacin-induced acute psychosis. Indian. Indian J. Psychiatry 50, 57−58.
(27) Ashby, J. A., McGonigle, I. V., Price, K. L., Cohen, N., Comitani, F., Dougherty, D. A., Molteni, C., and Lummis, S. C. R. (2012) GABA Binding to an Insect GABA Receptor: A Molecular Dynamics and Mutagenesis. Study. Biophys. J. 103, 2071−2081. (28) Schock, B. C., Koostra, J., Kwack, S., Hackman, R. M., van der Vliet, A., and Cross, C. E. (2004) Ascorbic acid in nasal and tracheobronchial airway lining fluids. Free Radical Biol. Med. 37, 1393− 1401. (29) Cantin, A. M., North, S. L., Hubbard, R. C., and Crystal, R. G. (1987) Normal alveolar epithelial lining fluid contains high levels of glutathione. J. Appl. Physiol. 63, 152−157. (30) Cantin, A. M., Hubbard, R. C., and Crystal, R. G. (1989) Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis. Am. Rev. Respir. Dis. 139, 370−372. (31) Halliwell, B., and Gutteridge, J. M. C. (2015) Free Radical in Biology and Medicine, Oxford University Press. (32) Buhl, R., Vogelmeier, C., Critenden, M., Hubbard, R. C., Hoyt, R. F., Jr., Wilson, E. M., Cantin, A. M., and Crystal, R. G. (1990) Augmentation of glutathione in the fluid lining the epithelium of the lower respiratory tract by directly administering glutathione aerosol. Proc. Natl. Acad. Sci. U. S. A. 87, 4063−4067. (33) Mimata, H., Tanigawa, T., Ogata, J., and Takeshita, M. (1988) Regulation of prostaglandin synthesis by reduced glutathione in urinary bladder epithelium. J. Urol. 139, 616−620. (34) Martensson, J., Jain, A., and Meister, A. (1990) Glutathione is required for intestinal function. Proc. Natl. Acad. Sci. U. S. A. 87, 1715− 1719. (35) Aoyama, K., Watabe, M., and Nakaki, T. (2008) Regulation of neuronal glutathione synthesis. J. Pharmacol. Sci. 108, 227−238. (36) Vergauwen, B., Verstraete, K., Senadheera, D. B., Dansercoer, A., Cvitkovitch, D. G., Guédon, E., and Savvides, S. N. (2013) Molecular and structural basis of glutathione import in Gram-positive bacteria via GshT and the cystine ABC importer TcyBC of Streptococcus mutans. Mol. Microbiol. 89, 288−303. (37) Al-Hiari, Y. M., Al-Mazari, I. S., Shakya, A. K., Darwish, R. M., and Abu-Dahab, R. (2007) Synthesis and antibacterial properties of new 8-Nitrofluoroquinolone derivatives. Molecules 12, 1240−1258. (38) Lee, M. H., Kim, J. Y., Han, J. H., Bhuniya, S., Sessler, J. L., Kang, C., and Kim, J. S. (2012) Direct fluorescence monitoring of the delivery and cellular uptake of a cancer-targeted RGD peptideappended naphthalimide theragnostic prodrug. J. Am. Chem. Soc. 134, 12668−12674. (39) Maiti, S., Park, N., Han, J. H., Jeon, H. M., Lee, J. H., Bhuniya, S., Kang, C., and Kim, J. S. (2013) Gemcitabine-coumarin-biotin conjugates: a target specific theranostic anticancer prodrug. J. Am. Chem. Soc. 135, 4567−72. (40) Wu, X. M., Sun, X. R., Guo, Z. Q., Tang, J. B., Shen, Y. Q., James, T. D., Tian, H., and Zhu, W. H. (2014) In vivo and in situ tracking cancer chemotherapy by highly photostable NIR fluorescent Theranostic Prodrug. J. Am. Chem. Soc. 136, 3579−3588. (41) Sun, H. L., Guo, B. N., Cheng, R., Meng, F. H., Liu, H. Y., and Zhong, Y. (2009) Biodegradable micelles with sheddable polyethylene glycol shells for triggered intracellular release of doxorubicin. Biomaterials 30, 6358−6366. (42) Hu, X. L., Hu, J. M., Tian, J., Ge, Z. S., Zhang, G. Y., Luo, K. F., and Liu, S. Y. (2013) Polyprodrug amphiphiles: hierarchical assemblies for shape-regulated cellular internalization, trafficking, and drug delivery. J. Am. Chem. Soc. 135, 17617−17629. (43) Wang, H., Tang, L., Tu, C. L., Song, Z. Y., Yin, L. C., Zhang, Z. H., Cheng, J. J., and Yin, Q. (2013) Redox-responsive, core-crosslinked micelles capable of on-demand, concurrent drug release and structure disassembly. Biomacromolecules 14, 3706−3712. (44) Bhuniya, S., Lee, M. H., Jeon, H. M., Han, H., Lee, J. H., Park, N., Maiti, S., Kang, C., and Kim, J. S. (2013) A fluorescence off-on reporter for real time monitoring of gemcitabine delivery to the cancer cells. Chem. Commun. 49, 7141−7143. (45) Zhang, Y., Yin, Q., Yen, J., Li, J., Ying, H., Wang, H., Hua, Y., Chaney, E. J., Boppart, S. A., and Cheng, J. (2015) Non-invasive, realH
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry time reporting drug release in vitro and in vivo. Chem. Commun. 51, 6948−6951. (46) Viola, G., Facciolo, L., Canton, M., Vedaldi, D., Dall'Acqua, F. D., Aloisi, G. G., Amelia, M., Barbafina, A., Elisei, F., and Latterini, L. (2004) Photophysical and phototoxic properties of the antibacterial fluoroquinolones levofloxacin and moxifloxacin. Chem. Biodiversity 1, 782−801. (47) Desiraju, G. R. (2002) Hydrogen bridges in crystal engineering: interactions without Borders. Acc. Chem. Res. 35, 565−573. (48) North, D. S., Pharm, D., Fish, D. N., Pharm, D., and Redington, J. (1998) Levofloxacin, a second-generation fluoroquinolones. Pharmacotherapy 18, 915−935. (49) Puckett, C. A., Ernst, R. J., and Barton, J. K. (2010) Exploring the cellular accumulation of metal complexes. Dalton Trans. 39, 1159− 1170. (50) Ramu, V., Gill, M. R., Jarman, P. J., Turton, D., Thomas, J. A., Das, A., and Smythe, C. (2015) A cytostatic ruthenium(II)platinum(II) bis(terpyridyl) anticancer complex that blocks entry into S phase by up-regulating p27. Chem. - Eur. J. 21, 9185−9197. (51) Scheffers, D. J., and Pinho, M. G. (2005) Bacterial cell wall synthesis: new insights from localization studies. Scheffers DJ1, Pinho MG. Microbiol. Mol. Biol. Rev. 69, 585−607. (52) Jabbour, A., Steinberg, D., Dembitsky, V. M., Moussaieff, A., Zaks, B., and Srebnik (2004) M. Synthesis and evaluation of oxazaborolidines for antibacterial activity against Streptococcus mutans. J. Med. Chem. 47, 2409−2510. (53) Villain-Guillot, P., Gualtieri, M., Bastide, L., Roquet, F., Martinez, J., Amblard, M., Pugniere, M., and Leonetti, J. P. (2007) Structure-activity relationships of phenyl-furanyl-rhodanines as inhibitors of RNA polymerase with antibacterial activity on biofilms. J. Med. Chem. 50, 4195−4204. (54) Wiles, J. A., Hashimoto, A., Thanassi, J. A., Cheng, J., Incarvito, C. D., Deshpande, M., Pucci, M. J., and Bradbury, B. J. (2006) Isothiazolopyridones: synthesis, structure, and biological activity of a new class of antibacterial agents. J. Med. Chem. 49, 39−42. (55) Seetharamsingh, B., Ramesh, R., Dange, S. S., Khairnar, P. V., Singhal, S., Upadhyay, D., Veeraraghavan, S., Viswanadha, S., Vakkalanka, S., and Reddy, D. S. (2015) Design, synthesis, and identification of silicon incorporated oxazolidinone antibiotics with improved brain exposure. ACS Med. Chem. Lett. 6, 1105−1110. (56) Pore, V. S., Divse, J. M., Charolkar, C. R., Nawale, L. U., Khedkar, V. M., and Sarkar, D. (2015) Design and synthesis of 11αsubstituted bile acid derivatives as potential anti-tuberculosis agents. Bioorg. Med. Chem. Lett. 25, 4185−90. (57) Shaikh, M. H., Subhedar, D. D., Arkile, M., Khedkar, V. M., Jadhav, N., Sarkar, D., and Shingate, B. B. (2016) Synthesis and bioactivity of novel triazole incorporated benzothiazinone derivatives as antitubercular and antioxidant agent. Bioorg. Med. Chem. Lett. 26, 561−569. (58) Haritova, A. M., Rusenova, N. V., Parvanov, P. R., Lashev, L. D., and Fink-Gremmels, J. F. (2006) Integration of pharmacokinetic and pharmacodynamic indices of marbofloxacin in Turkeys. Antimicrob. Agents Chemother. 50, 3779−3785. (59) Quelemes, P. V., Araruna, F. B., de Faria, B. E., Kuckelhaus, S. A. S., da Silva, D. A., Mendonça, R. Z., Eiras, C., Soares, M. J. S., and Leite, J. R. S. A. (2013) Development and antibacterial activity of cashew gum-based silver nanoparticles. Int. J. Mol. Sci. 14, 4969. (60) Akhtar, S., Khan, A., Sohaskey, C. D., Jagannath, C., and Sarkar, D. (2013) Nitrite reductase NirBD Is induced and plays an important role during in vitro dormancy of mycobacterium tuberculosis. J. Bacteriol. 195, 4592−4599. (61) Ali, F., Anila, H. A., Taye, N., Gonnade, R. G., Chattopadhyay, S., and Das, A. (2015) A fluorescent probe for specific detection of cysteine in the lipid dense region of cells. Chem. Commun. 51, 16932− 16935. (62) Anila, H. A., Reddy, U. G., Ali, F., Taye, N., Chattopadhyay, S., and Das, A. (2015) A reagent for specific recognition of cysteine in aqueous buffer and in natural milk: imaging studies, enzymatic reaction and analysis of whey protein. Chem. Commun. 51, 15592−15595.
(63) Ramu, V., Ali, F., Taye, N., Garai, B., Alam, A., Chattopadhyay, S., and Das, A. (2015) New imaging reagents for lipid dense regions in live cells and the nucleus in fixed MCF-7 cells. J. Mater. Chem. B 3, 7177−7185. (64) Jain, A. K., Gund, M. G., Desai, D. C., Borhade, N., Senthilkumar, S. P., Dhiman, M., Mangu, N. K., Mali, S. V., Dubash, N. P., Halder, S., et al. (2013) Mutual prodrugs containing biocleavable and drug releasable disulfide linkers. Bioorg. Chem. 49, 40− 48. (65) Kumar, A. (1993) A New Solid Phase Method for the Synthesis of Oligonucleotides with Terminal −3′-Phosphate. Nucleosides Nucleotides 12, 441−447.
I
DOI: 10.1021/acs.bioconjchem.6b00324 Bioconjugate Chem. XXXX, XXX, XXX−XXX
