Ciprofloxacin conjugated to diphenyltin(IV); A novel formulation with enhanced antimicrobial activity.

The metalloantibiotic of formula Ph 2 Sn(CIP) 2 ( CIPTIN) ( HCIP = ciprofloxacin) was synthesized by reacting ciprofloxacin hydrocloride ( HCIP·HCl ) (an antibiotic in clinical use) with the diphenyltin dichloride (Ph 2 SnCl 2 DPTD ). The complex was characterized in solid state by melting point, FT-IR, X-ray Powder Diffraction (XRPD) analysis, 119 Sn Mössbauer spectroscopy, X-ray Fluorescence (XRF) spectroscopy, Thermogravimetry/Differential Thermal Analysis (TG-DTA) and in solution by UV-Vis, 1 H-NMR spectroscopic techniques and Electrospray Ionisation Mass Spectrometry (ESI-MS). The crystal structure of CIPTIN and its processor HCIP were also determined by X-ray crystallography. The antibacterial activity of CIPTIN , HCIP·HCl , HCIP and DPTD was evaluated against the bacterial species Pseudomonas aeruginosa ( P. aeruginosa ), Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ) and Staphylococcus epidermidis ( S. epidermidis ), by the means of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Inhibition Zones (IZs). CIPTIN shows lower MIC values than those of HCIP·HCl (up to 4.2-folds), HCIP (up to 2.7-folds) or DPTD (>135-folds), towards the tested microbes. CIPTIN is classified to bactericidal agents according to MBC/MIC values. The developing IZs are 40.8±1.5, 34.0±0.8, 36.0±1.1 and 42.7±0.8 mm, respectively which classify the microbe’s P. aeruginosa , E. coli , S. aureus and S. epidermidis to susceptible ones to CIPTIN . These IZ’s are greater to the corresponding ones of the HCIP·HCl by 1.1 to 1.5-folds against both Gram negative and positive bacteria tested. CIPTIN eradicates the biofilm of P. aeruginosa and S. aureus more efficiently than HCIP·HCl and HCIP . The in vitro toxicity and genotoxicity of CIPTIN were tested against human skin keratinocyte cells (HaCaT) (IC 50 = 2.33 μΜ). CIPTIN exhibits 2 to 9-folds lower MIC values against than its IC 50 against HaCaT, while its genotoxic effect determined by micronucleus assay is equivalent to the corresponding ones of HCIP·HCl or HCIP . Melting points measured in a Stuart and uncorrected. IR spectra in the region of cm -1 were obtained on Cary 670 FTIR spectrometer, Agilent Technologies. A UV-1600 PC series spectrophotometer of VWR was used to obtain electronic absorption spectra. The 1 H-NMR spectra were recorded on a Bruker AC 250, 400 MHFT-NMR instrument in DMSO- d 6 . ESI-MS spectra were measured with an Agilent 1100/LC−MS system. 119 Sn Mössbauer spectra were collected at various sample temperatures (85 K), with a constant-acceleration spectrometer equipped with a CaSnO 3 source kept at room temperature. The calibration of the spectrometer was carried out with a 57 Co source and an Fe absorber at room temperature. The line widths were very close to the natural width (0.28 mm/s). The content of the tin samples was calculated to be approximately 4 mg Sn in the 2-cm 2 area of the sample holder. XRF measurements were carried out using an Am-241 radio isotopic source (exciting radiation 59.5 keV). Thermal Gravimetric– Differential out on a Seiko SII Graphical Abstract Ciprofloxacin conjugated The steroisomer Δ- cis -[Ph 2 Sn(CIP) 2 ] ( CIPTIN) was obtained from ciprofloxacin and DPTC. CIPTIN exhibits stronger activity than ciprofloxacin against Gram positive or negative bacteria


Introduction
Ciprofloxacin, a second-generation fluoroquinolone, is effective against both Gram-positive and Gram-negative pathogenic bacteria [1] . It exhibits good bioavailability or tissue permeability, low incidence of toxic effects and bacteriostatic activity [2] . Generally, the bacteriocidal agents are preferred than bacteriostatic ones, against infections, since they limit the development of resistance towards bacteria [3] . However, microbes such as P. aeruginosa, E. coli, S. aureus and S. epidermidis are reported to have developed resistance towards ciprofloxacin [4] . Hence efforts were made in recent years to modify ciprofloxacin with metal ions, in order to increase its antibacterial activity against resistant strains [5][6][7][8] .
The conjugation of metals with drugs (CoMeDs), is a new research area being investigated for discovery of new synergistic therapeutic modalities. The term "conjugation" is used for polymeric drug formulations, to emphasize the synergistic effect of a metal with a drug [9][10] .
CoMeDs combine metals with specific classes of drugs aiming to create new agents possessing altogether new properties than the drugs from which they were prepared [9][10] . From this point of view, organotin(IV) compounds which contain active drugs as ligands are expected to exhibit enhance effectiveness due to the synergy which is developing between the R n Sn(IV) (R= alkyl or aryl group; n=1 to 3) moiety and the drug. Besides, organotin compounds are commercially used as agricultural biocides [11][12] . Recently, Pokharia et.al have shown that triorganotin(IV) complexes of ciprofloxacin (R 3 Sn(CIP) (R= Me-, Cy-)) exhibit better antibacterial effect than free ciprofloxacin against E. faecalis, S. aureus, K. pneumoniae, E. coli, P. aeruginosa, and P. mirabilis [13] .
With the aim to develop new antimicrobial agents with enhanced activity capable of overcoming bacterial resistance [9,10,[14][15][16][17] , the metalloantibiotic of ciprofloxacin (HCIP, (Scheme 1)), having the formula Ph 2 Sn(CIP) 2 (CIPTIN) was synthesized, characterized and evaluated against bacterial strains P. aeruginosa, E. coli, S. aureus and S. epidermidis. Moreover, the combination of an antibiotic, ciprofloxacin, which possesses pharmacological significance and a 4 metal with known biological activity, tin(IV), are expected to throw light on principles that govern the synergy between metals and drugs in these metallodrugs.

X-ray Powder Diffraction (XRPD) analysis:
The XRPD spectrum of the powder of CIPTIN is identical to the simulated one which is derived by using single crystal XRD data ( Figure S2). This 119 Sn Mössbauer spectroscopy: 119 Sn Mössbauer spectrum at 77 K of CIPTIN is shown in Figure 4.
The broth dilution method was used for the determination of the MIC values of CIPTIN, HCIP·HCl, HCIP and DPTD, upon incubation of Gram-negative (P. aeruginosa and E. coli) or Gram-positive (S. aureus and S. epidermidis) bacteria for 20 h (Table 1). Table 1 The which is 4.2-fold higher than that of HCIP·HCl and 2.7 fold than that of HCIP. Moreover CIPTIN activity rises up to 135-folds than DPTD stronger activity against these bacteria. Therefore, CIPTIN exerts superior activity than both its ingredients (antibiotic and DPTD).
Microbes are classified to susceptible (MIC< 50 μM) or resistant (MIC> 100 μM) towards an antimicrobial agent by their MIC values [8,10] . Thus, all tested strains here are considered as susceptible to CIPTIN.  Table 1 Minimum bactericidal concentration (MBC): The MBC values for CIPTIN were also evaluated.
The  Table 1, Figures S14-S15). In the case of DPTD, the MBC values are higher than 100 μΜ ( Figure S16). Thus, CIPTIN exhibits higher bactericidal activity than those of free drugs which rises up to 3.6-fold in respect of HCIP·HCl or up to 2.8-fold for HCIP. DPTD has no bactericidal activity.
Since the MBC/MIC values of CIPTIN lie between 1.08-2.62, against the tested microbial strains, (Table 1) the CIPTIN is classified as bactericidal agent. This is because when the MBC/MIC value is ≤2, an agent is considered as bactericidal one, while when MBC/MIC value is ≥4, as bacteriostatic one [8,10,[14][15][16][17] . The MBC/MIC values of HCIP·HCl and HCIP varied between 1.22-2.82 and they are bactericidal drugs.

Inhibition zone (IZ):
The IZ was evaluated with agar disk-diffusion method [8,10,[14][15][16][17] . The calculated diameter of IZ is used to determine the effectiveness of an antibiotic. Therefore, the bacterial strains might be classified as susceptible to an agent based on the IZ developed [8,10,[14][15][16][17] . Thus, Shungu et.al [25] classified the microbes in three categories according to the size of IZ caused by antibiotics.
aeruginosa is 656 μΜ and for S. aureus is 752 μΜ (Figure 6). We have recently determined the BEC value of HCIP·HCl against P. aeruginosa (670 μΜ) and S. aureus (952 μΜ) [8] . The corresponding BEC values of HCIP are: 2140 μΜ (P. aeruginosa) and 2463 μΜ (S. aureus) ( Figure S19). CIPTIN eliminates the biofilm of P. aeruginosa and S. aureus more efficiently than the neutral drug HCIP, while its BEC value is comparable with the corresponding one of HCIP·HCl. The therapeutic window of an agent is defined by the selectivity index (SI) [8,26] : Evaluation of in vitro genotoxicity by micronucleus assay: The micronucleus assay is frequently used to evaluate the mutagenic, genotoxic or teratogenic effect of metallodrugs [27] . In the presence of a genotoxic factor, micronuclei (MN) appear as small membrane-bound DNA fragments in the cytoplasm of interphase cells and they are formed during the metaphase-anaphase transition of the mitotic cycle [28] . The micronucleus assay has been widely used in monitoring genetic damage in order to avoid the screening of drugs on animals.

Figure 6
In

Conclusions
The design and the development of antimicrobial agents are of great importance. The new metalloantibiotic CIPTIN, has been synthesized and characterized within this work. The crystal structure reveals that the geometry around the metal center is octahedral ( Figure 1B). The stereoselective synthesis results to the Δ-cis-[Ph 2 Sn(CIP) 2 ] isomer only ( Figure 1B). 2D layers supramolecular assemblies are established based on strong hydrogen bonding interactions ( Figure   3). Four molecules form squares planes with their tins atoms to occupy its acnes of each square.
CIPTIN exhibits stronger activity than ciprofloxacin against both Gram positive and negative bacteria tested as well as the [Me 3 Sn(CIP)], [Cy 3 Sn(CIP)] [13] suggesting that the presence of the tin(IV) ion enhances ciprofloxacin's effectiveness even more (Table 1, Figure 8). While DPTC's activity is significant lower than that of CIPTIN, this should be attributed to the synergistic effect of both the organotin moiety and ciprofloxacin. CIPTIN exerts superior activity than penicillin against Gram negative, as expected since penicillin is inactive against Gram negative Among the compounds of ciprofloxacin, CIPTIN exhibits higher activity from CIPAG against S. epidermidis, which however is more effective against S. aureus and P. aeruginosa (Figure 8).
Therefore, the antibiotic activity is differentiated by the type of the metal ion which is coordinating on the drug. Moreover, among organotins, the [Me 3 Sn(CIP)] and the [Cy 3 Sn(CIP)] [13] show stronger activity than CIPTIN against P. aeruginosa and S. aureus (Table 1). However this should be assigned to the higher toxicity of tri-than di-organotin moieties. Although PenAg shows better activity against S. aureus, CIPTIN, however is more drastic against S. epidermidis and P.
aeruginosa where ciprofloxacin itself shows better activity than free penicillin (Figure 8). Therefore the activity of the metallodrug is following the corresponding efficiency of the antibiotic, regardless the type of the metal ion Sn(IV) or Ag(I). Moreover CIPTIN shows superior activity than silver(I) nitrate against all microbes tested (Figure 8) confirming the conclusion which is already withdrawn for the synergy of the metal ion with the drug on the activity of the metallodrug. CIPTIN eliminates the biofilm in a similar manner with the drug ( Table 1). The in vitro toxicity of the CIPTIN against HaCaT cells shows a selectivity index (SI) higher than 1 suggesting wide therapeutic window, while its in vitro genotoxicity is even lower than that of HCIP·HCl or HCIP.
In conclusion, the coordination of a drug on a metal ion strongly enhances its activity. The antimicrobial information which has been stored in a drug, affects the activity of the metallodrug and it is affected by the coordination demands stored in the metal ion, creating a combination of a new entity with enhance and novel biological properties.  immediately filtered off. Crystals of CIPTIN suitable for X-ray analysis were grown from slow evaporation of the solution after one day. X-ray Structure Determination: Intensity data for the crystals of CIPTIN and HCIP were collected on an Oxford Diffraction CCD instrument, using graphite monochromated Mo radiation (λ=0.71073 Å). Cell parameters were determined by least-squares refinement of the diffraction data from 25 reflections. All data were corrected for Lorentz-polarization effects and absorption [29] . The structures were solved with direct methods with SHELXS97 [30] and refined by full-matrix leastsquares procedures on F 2 with SHELXL97 [31] . All non-hydrogen atoms were refined anisotropically, hydrogen atoms were located at calculated positions and refined via the "riding model" with isotropic thermal parameters fixed at 1.

Minimum inhibitory concentration (MIC) testing:
This study was performed according standard procedure, as described previously [8][9][10][14][15][16][17] . Briefly, the bacterial strains were streaked onto in trypticase soy agar plates, which were incubated for 18-24 h at 37 o C. Then, three to five isolated colonies were selected of the same morphological appearance from the fresh agar plate using a sterile loop and transferred into a tube containing 2 mL of sterile saline solution. The optical density at 620 nm is 0.1, which corresponds in to 10 8 cfu/mL. The final inoculum size for broth dilution is occurs when no colony formation is observed. The lowest concentration at which the tested compounds could give complete inhibition of microbes' growth was defined as MBC value [8][9][10][14][15][16][17] .
Effects on biofilm formation: Bacterial strains of P. aeruginosa or S. aureus with a density of 6.7×10 6 cfu/mL were inoculated into LB medium (total volume= 1500 μL) and cultured for 24 h at

20
Excess stain was rinsed off with 1 mL methanol and 2 mL ddH 2 O. The tubes are left to dry for 24 hand the bounded crystal violet was released by adding 30% glacial acetic acid (2 mL) and after 3 mL ddH 2 O. The optical density of the solution yielded is measured at 550 nm, to give the biofilm biomass [8][9][10][14][15][16][17] .
In vitro toxicity evaluation Sulforhodamine B Assay: These studies were performed in accordance with the previous reported method [10,15] . Evaluation of genotoxicity by micronucleus assay: The micronucleus assay was carried out as previously described [27] . Briefly HaCaT cells were seeded (at a density of 240000 cells/well) in glass cover slips which were afterwards placed in six-well plates, with 3 mL of cell culture medium and incubate for 24 h. HaCaT cells exposed with compounds in IC 50 values for a period of 48 h.