Aminoferrocene-based prodrugs and their effects on human normal and cancer as well as bacterial cells

Transcription

1 S SUPPORTING INFORMATION Aminoferrocene-based prodrugs and their effects on human normal and cancer as well as bacterial cells Paul Marzenell, Helen Hagen, Leopold Sellner, Thorsten Zenz, Ruta Grinyte, Valeri Pavlov, Steffen Daum, Andriy Mokhir* Content Experiments with Escherichia coli bacteria: Protocol and Results Experiment with Streptococcus agalactiae bacteria: Protocol and Results Determination of logp-values Determination of membrane permeability of prodrugs p. S p. S p. S p. S5

2 S Experiments with Escherichia coli bacteria Protocol. Inoculate Mueller Hinton agar plate with E. coli. Dip a sterile cotton swab into the overnight culture of E. coli, and wipe off any excess on the inside of the tube. Inoculate a Mueller Hinton agar plate by streaking the entire surface of the plate with the swab, turning the plate 90, swabbing a second time, turning the plate 5 and swabbing a third time. Run the swab around the circumference of the plate. Be sure to cover the entire plate. Discard the swab.. Allow the plates to dry for 5 minutes before placing the samples disks on the plate surface.. Apply four samples disks to the agar surface of one plate using sterile forceps. To sterilize the forceps, dip them in alcohol, letting the excess drip into the beaker, and pass them through the Bunsen burner flame. Allow the alcohol to burn off. Be sure to space the disks at least 5 cm apart to prevent overlapping zones of growth inhibition. Press each disk gently with sterile forceps so that the disk makes good contact with the surface of the agar. Samples discs preparation Sterile disks with approximate diameter of 0mm (Sigma) were used. It was assumed that a sterile disk could absorb 0.08 ml of solutions. Were prepared the samples (a, e, and a,negative control Fc, ampicilin, control (without any compound)) of 0.ml the followed concentrations 0., 0., 0.9,.8 mm in %, %, 9%, 8% DMF, water and 0.0, 0.0, 0.09, 0.8 mm sodium ascorbat. Results No effect for bacteria of all the compound of different concentrations except antibiotic (ampicilin). In Figure (a and b) you can see the bacteria resistant for the 0.9 mm compound. a) b) 5 5 ) Ampicilin ) Prodrug a ) Prodrug e ) Prodrug a 5) Control sample ) Sample Negative control Figure S. Only around the disc of ampicilin you can see the zone of inhibition

3 S Experiment with Streptococcus agalactiae bacteria Protocol. Inoculate Blood agar +% sheep blood plate (I) or Mueller Hinton agar + % sheep blood plate (II) with Streptococcus agalactiae. Dip a sterile cotton swab into the overnight culture of Streptococcus agalactiae, and wipe off any excess on the inside of the tube. Inoculate a plate by streaking the entire surface of the plate with the swab, turning the plate 90, swabbing a second time, turning the plate 5 and swabbing a third time. Run the swab around the circumference of the plate. Be sure to cover the entire plate. Discard the swab.. Allow the plates to dry for 5 minutes before placing the samples disks on the plate surface.. Apply four samples disks to the agar surface of one plate using sterile forceps. To sterilize the forceps, dip them in alcohol, letting the excess drip into the beaker, and pass them through the Bunsen burner flame. Allow the alcohol to burn off. Be sure to space the disks at least 5 cm apart to prevent overlapping zones of growth inhibition. Press each disk gently with sterile forceps so that the disk makes good contact with the surface of the agar. Samples discs preparation Sterile disks with approximate diameter of 0mm (Sigma) were used. It was assumed that a sterile disk could absorb 0.08 ml of solutions. Were prepared the samples (a, e, and a,negative control Fc, ampicilin, control (without any compound)) of 0.ml the followed concentration 0.9 mm in 9% DMF, water and 0.09 mm sodium ascorbat. Results No effect for bacteria of all the compound of different concentrations except antibiotic (ampicilin). In Figure (I and II you can see the bacteria resistant for the 0.9 mm compound in different plates. I) II) 5 5 ) Ampicilin ) Prodrug a ) Prodrug e ) Prodrug a 5) Control sample ) Sample Negative control Figure S. Only around the disc of ampicilin you can see the zone of inhibition

4 logp S Determination of logp-values (Table S, Figures S, S) LogP-values were determined by using reversed phase - thin layer chromatography (RP-TLC) on TLC plates from Macherey-Nagel (Germany):, Alugram RP-8W/UV5, stationary phase thickness: 0.5 mm. The spots of prodrugs and reference compounds were initially monitored by using UV-imaging and permanently stained either by aqueous KMnO solution (KMnO ( g), K CO (5 g) in water (00 ml)) or by vanillin - sulfuric acid mixture (vanillin (0.5 g), H SO /EtOH (/, v/v, 00 ml). A set of reference compounds was used, for which logp-values were reported: benzylalcohol (.), 8-hydroxyquinoline (.9),,,5-trichlorophenol (.7) 5 and anthracene (.5). A calibration plot of R f versus logp, which was obtained based on these data, was used to determine logp values of new prodrugs (Figure S). Table S. Octanol-water partition coefficients (logp) and membrane permeabilities of aminoferrocenebased prodrugs. Number Prodrug Permeability logp a b c d e a b i i 9 a b. +.5 i Spots of these compounds on TLC were broad, which precluded accurate determination of R f. logp = -,588 * R f +, ,5 Retention factor (R f ) Figure S. A calibration plot (retentions factor (R f ) versus octanol-water partition coefficient (logp)).

5 Permeability S5 Determination of membrane permeability of prodrugs (Table S, Figure S) HL-0-cells grown in RPMI 0 medium supplemented with 0% FCS, % glutamine, and % penicillin/streptomycin were centrifuged, and the medium was replaced with RPMI 0 medium (5% FCS, % L-glutamine, % penicillin/streptomycin) to obtain suspensions containing 0 cells/ml. Solutions of prodrugs (5 μl, solvent DMF containing % water and % sodium ascorbate) were added to the suspensions (500 μl) and incubated for h. The final concentration of the prodrugs in the suspensions was 00 μm. Then the cells were washed three times with PBS buffer ( 500 μl) and treated with concentrated H O solution (00 μl, M) for 0 min, and all volatiles were removed by lyophilization. Dry, lysed cells were washed with water (00 μl), and aqueous solution obtained was acidified with HCl (00 μl, 0. M). Then this solution was extracted with -ethyl-,-hexanediol (00 μl, 0% in CHCl, v/v), and a portion of the organic phase obtained (0 μl) was mixed with H SO /CH CO H (00 μl, /, v/v). Curcumin solution in methyl isobutyl ketone (500 μl, mg in ml of the solvent) was added and allowed to react for h. The reaction was quenched by addition of water ( ml). Light absorbance at 550 and 780 nm of the organic phase was measured. The value A(550 nm) A(780 nm) was proportional to the concentration of boron in the mixture. The amount of boron released from prodrug a was taken as a reference. All other values were determined relative to this reference (Table S, Figure S). b d 0 a b a c,8 logp e a Figure S. Dependence of membrane permeability (OY axis) from octanol-water partition coefficient (LogP, OX axis); for a series of aminoferrocene-prodrugs, which release iron in cancer cells (a, b, c and d, indicated with red color), permeability correlates with logp; for other prodrugs (N-benzylsubstituted (green colored) and bis-substituted (blue colored)) no obvious correlation between these values was observed. References:. Biagi, G. L.; Barbaro, A. M.; Guerra, M. C.; Gamba, M. F. Partition data of penicillins determined by means of reversed-phase thin-layer chromatography. J. Chromatography, 99,, 7-79.

6 S. Biagi, G. L.; Barbaro, A. M.; Guerra, M. C.; Gamba, M. F. Partition data of cephalosporins determined by means of reversed-phase thin-layer chromatography J. Chromatography, 99,, Sangster, J. Phys. Chem. Ref. Data, 989, 8, htm; Pesticide Properties Database (PPDB)