fluconazole-pyridoxine bis-triazolium compounds with potent...

Research ArticleFluconazole-Pyridoxine Bis-Triazolium Compounds withPotent Activity against Pathogenic Bacteria and Fungi IncludingTheir Biofilm-Embedded Forms

Marsel R Garipov1 Roman S Pavelyev1 Svetlana A Lisovskaya2

Elena V Nikitina1 Airat R Kayumov1 Alina E Sabirova1 Oksana V Bondar1

Albina G Malanyeva1 Alexander M Aimaletdinov1 Alfia G Iksanova1

Konstantin V Balakin13 and Yurii G Shtyrlin1

1Kazan (Volga Region) Federal University Kremlyovskaya 18 Kazan 420008 Russia2Kazan Scientific and Research Institute of Epidemiology and Microbiology Bolshaya Krasnaya 67 Kazan 420015 Russia3IM Sechenov First Moscow State Medical University Trubetskaya 8 Bldg 2 Moscow 119991 Russia

Correspondence should be addressed to Yurii G Shtyrlin yuriishtyrlingmailcom

Received 4 September 2017 Accepted 21 November 2017 Published 27 December 2017

Academic Editor Daniela De Vita

Copyright copy 2017 Marsel R Garipov et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Two novel quaternary ammonium salts bis-triazolium derivatives of fluconazole and pyridoxine were synthesized by reactionof fluconazole with pyridoxine-based synthetic intermediates The leading compound demonstrated pronounced antimycoticand antibacterial in vitro activity comparable to or exceeding that of the reference antifungal (fluconazole terbinafine) andantibacterialantiseptic (miramistin benzalkonium chloride) agents In contrast to many antimicrobials the leading compoundwas also active against biofilm-embedded staphylococci and Escherichia coli While no biofilm structure destruction occurred allcompounds were able to diffuse into the matrix and reduce the number of colony-forming units by three orders of magnitude at16 times MBC The leading compound was significantly less toxic than miramistin and benzalkonium chloride and more toxic thanthe reference antifungal drugsThe obtained results make the described chemotype a promising starting point for the developmentof new broad-spectrum antimicrobial therapies with powerful effect on fungal and bacterial pathogens including their biofilm-embedded forms

1 Introduction

In the last decades there is drastic increase of diseasescaused bymicroscopic fungi Among them dermatomycetessuch as Trichophyton spp which are the main causativeagents for dermatomycoses in both immunocompetent andimmunocompromised patients become a serious clinicalproblem The widespread distribution of microscopic fungiin the natural environment results in multiple direct contactsof humans with the pathogens The anthropogenic factorsalso contribute to accumulation of hazardous fungal strainsin the environment and to an increased number of immun-odeficient people prone to secondarymycoses caused by con-ditionally pathogenic fungi including Aspergillus RhizopusFusarium Paecilomyces Candida and Rhodotorula [1]

Candida albicans is one of the most widely distributeddimorphic microscopic fungi growing as both yeast andfilamentous cells and causing deep and cutaneous forms ofcandidiasis in humans being responsible for 50ndash90 of allcases of candidiasis in humans [2] Systemic fungal infections(fungemias) including those which are caused by C albicanshave emerged as important causes ofmorbidity andmortalityin immunocompromised patients (eg patients with AIDSafter cancer chemotherapy and organ or bone marrowtransplantation) [3 4]

C albicanspossesseswell developedmechanisms of adap-tation which can significantly enhance their pathogenicityOne of such mechanisms is forming the biofilms on tissuesand abiotic surfaces and implants that dramatically increase

HindawiJournal of ChemistryVolume 2017 Article ID 4761650 15 pageshttpsdoiorg10115520174761650

2 Journal of Chemistry

the pathogenrsquos resistance to antifungal agents [5 6] Withinthe biofilm fungal cells are protected from the immunesystem of the host antimycotics and antiseptic agents Inbiofilms the specific fungal populations survive with themost resistant and virulent phenotypes formed due to aprolonged action of biological and chemical agents As aresult the cells released from biofilms often cause acute fun-gal infections highly resistant to the treatment Candidiasiscommonly occurs on mucous membranes of mouth vaginaand other organs where C albicans forms rigid biofilmswhich have been considered as one of the critical factorsproviding fungal resistance to traditional antimycotic drugsincluding azoles polyenes and echinocandins [7]

Microbial associations natural communities of severalmicroorganisms represent additional serious clinical prob-lem [8 9] Certain types of microorganisms involved into theassociation and resistant to antimicrobial agents enhance thevital activity and resistance of other species as a result of theinteraction As a result such associations are very resistantto various factors Many patients with chronic skin mycosesare often infected with various fungal strains belonging todifferent species genera and even classes Therefore theeffective antifungal drug should have a systemic action whichwarrants its efficient penetration into damaged organs andtissues and the fungal mono- and polymicrobial biofilms

There are a wide number of antimycotic drugs in currentclinical practice [10] One of the most popular azole com-pounds fluconazole is a first-generation fungistatic triazoleoften used for the treatment of cutaneous and other formsof candidiasis [11] Fluconazole inhibits ergosterol synthesisand thus alters membranes of fungal cell Fluconazole isactively used for the treatment of Candida infections andpossesses favorable safety profile and good pharmacokineticcharacteristics However its narrow antifungal spectrumin particular low activity against non-Candida fungi hasgreatly affected its therapeutic efficacy in clinic [12] Thereis also extensive documentation of intrinsic and developedresistance to azole antifungals among Candida species [13]In addition poor water solubility of fluconazole makes itproblematic to develop effective delivery forms for intra-venous administration These issues stimulated many stud-ies associated with structural modification of fluconazoleincluding esterification of hydroxyl group and modificationof 24-difluorobenzyl and triazolyl moiety aimed to improveits water solubility andor antimicrobial spectrum (eg [14ndash16]) A successful example was fosfluconazole a prodrugdeveloped for the treatment of infections caused by Candidaalbicans and Cryptococcus which exhibited broader antifun-gal activity spectrum than fluconazole [17]

In a more general context several studies have revealedthat the transformation of triazolyl ring into triazoliumcould result in enhanced antimicrobial action Triazoliumis able to form additional hydrogen bonds and produceelectrostatic interaction with biological targets and thesestructural features can affect the diffusion and interactionin bacterial tissues with the enhancement of water solubilityand membrane permeability [18] Thus quaternization ofN-4 position of triazole ring in fluconazole produced the

corresponding triazolium compounds which demonstratedpromising antibacterial potential [19]

In light of the described promising results it was reason-able to explore further structural modifications of flucona-zole In this work we have obtained two novel quaternaryammonium salts bis-triazolium derivatives of fluconazoleand pyridoxine The introduction of pyridoxine (vitaminB6) moiety into the developed hybrid structures can poten-tially enhance the transmembrane transport of the wholeconstructs via several possible mechanisms like either spe-cific pyridoxine transporters in cellular membranes [20] orrecognition of pyridoxine-modified molecules by the cell asendogenous [21] Another practical reason for introductionof pyridoxine-based cyclic acetals into the developed hybridsis the possibility of easily varying the acetal substituents inorder to modify lipophilicity steric parameters and otherphysicochemical properties of the obtained structures

2 Materials and Methods

21 Synthetic Procedures 1H and 13C NMR spectra wererecorded on a Bruker AVANCE 400 at operating frequencyof 400 and 10156MHz respectively Chemical shifts weremeasured with reference to the residual protons of thesolvent (DMSO-d

6 1H 250 ppm 13C 3952 ppm CDCl

3

1H 726 ppm 13C 7716 ppm) Coupling constants (J) aregiven in Hertz (Hz) The following abbreviations are usedto describe coupling s = singlet d = doublet t = tripletm = multiplet q = quartet br s = broad singlet qd =quartet of doublets AB = AB system Melting points weredetermined using a Stanford Research Systems MPA-100OptiMelt melting point apparatus and are uncorrected ForTLC analysis silica gel plates from Sorbfil (Krasnodar Rus-sia) were used with UV light (t 254 nm365 nm) or iron(III) chloride as developing agent Column chromatographywas performed on silica gel (60ndash200mesh) from Acros orreversed phase chromatography on chromatographPuriFlash450 (PF-15C18HP column Interchim) HRMS mass spectrawere obtained on a quadrupole time-of-flight (t qTOF) ABSciex Triple TOF 5600 mass spectrometer using turbo-ionspray source (nebulizer gas nitrogen a positive ionizationpolarity needle voltage 5500V) Recording of the spectrawas performed in ldquoTOF MSrdquo mode with collision energy10 eV declustering potential 100 eV and resolutionmore than30000 full-width half-maximum Samples with the analyteconcentration 5120583molL were prepared by dissolving the testcompounds in a mixture of methanol (HPLC-UV GradeLabScan) and water (LC-MS Grade Panreac) in 1 1 ratio[22]HPLC-MS analysis was performed usingAgilent ZorbaxEclipse Plus C18 column (21 times 100mm particle size 35120583m)with the flow rate of mobile phase 400 120583Lmin The HPLCpurity of compounds 4ab was gt98

Synthesis and analytical parameters of compounds2ab and 3a have been described in our previous paper[23] Fluconazole has been purchased from Sigma-Aldrich(USA)mdashSKU-Pack Size PHR1160-1G

6-(Bromomethyl)-8-methyl-2-propyl-4H-[13]dioxino[45-c]pyridin-5-yl)methanol (3b) To a solution of compound

Journal of Chemistry 3

2b (094 g 371mmol) in 100ml of dichloromethane wereadded triphenylphosphine (093 g 354mmol) and thenportionwise NBS (063 g 354mmol) and the reactionmixture was stirred at room temperature for 05 h Thenthe reaction mixture was concentrated under vacuumand purified by column chromatography (petroleumetherdiethyl ether = 1 4) Yield 509 (057 g) white solidmp 118ndash120∘C 1H NMR (CDCl

3) 120575 099 (t 3H 3JHH =

74Hz CH2CH2CH3) 150ndash159 (m 2H CH

2CH2CH3)

177ndash190 (m 2H CH2CH2CH3) 236 (s 3H CH

3Pyr)

459 463 (AB system 2H 2JHH = minus100Hz CH2) 459

467 (AB system 2H 2JHH = minus128Hz CH2) 498 (t 1H

3JHH = 52Hz CH) 501 (s 2H CH2) 13C NMR (CDCl

3)

120575 1399 (s CH2CH2CH3) 1702 (s CH

2CH2CH3) 1816 (s

CH3Pyr) 3238 (s CH

2CH2CH3) 3626 (s CH

2Br) 5771

(s CH2) 6461 (s CH

2) 9995 (s CH) 12867 (s CPyr)

12928 (s CPyr) 14518 (s CPyr) 14706 (s CPyr) 14826 (sCPyr) HRVS-ESI found [V +H]+ 3160543 C

13H19BrNO

3

calculated [V + H]+ 3160543

111015840-(2-(24-Difluorophenyl)-2-hydroxypropane-13-diyl)bis(4-((5-(hydroxymethyl)-228-trimethyl-4H-[13]dioxino[45-c]pyridin-6-yl)methyl)-1H-124-triazol-4-ium) Bromide (41)Compound 3a (15 g 496mmol) and fluconazole (076 g248mmol) were dissolved in acetonitrile (50mL) and thereaction mixture was stirred at 70∘b for 1 week Then thesolvent was removed in vacuo and the residue was dissolvedin ethanol (5mL) Diethyl ether (30mL) was added and theformed precipitate was collected by filtration The productwas purified by column chromatography on C18-reversedphase silica gel with gradient elution (isopropanolwater0ndash35) Yield 47 (106 g) white solid mp 147∘C (decomp)1H NMR (DMSO-d

6) 120575 149 (s 12H 4CH

3) 222 (s 6H

2CH3Pyr) 443ndash450 (br m 4H 2CH

2OH) 499 (s 4H

2CH2) 506 (br s 4H 2CH

2) 520ndash531 (brm 2H 2CH

2OH)

567 (br s 4H 2CH2) 681ndash685 (m 1H CHAr) 705 (d 1H

5JHF = 17Hz OH) 711ndash717 (m 1H CHAr) 729ndash735 (m1H CHAr) 910 (br s 1H CHAr) 911 (br s 1H CHAr)1013ndash1015 (m 2H 2CHAr) 13C NMR (DMSO-d

6) 120575 1829

(s CH3Pyr) 2443 (s CH

3) 2447 (s CH

3) 4889 (s CH

2)

5506 (s CH2) 5698 (s CH

2) 5845 (s CH

2) 7284 (d

3JCF = 39Hz C-OH) 9957 (s C(CH3)2) 10442 (dd 2JCF

= 266Hz 2JCF = 268Hz CAr) 11136 (d 2JCF = 211 HzCAr) 12116 (dd 2JCF = 128Hz 4JCF = 31 Hz CAr) 12664(s CAr) 12847 (s CAr) 12976 (dd 3JCF = 86Hz 3JCF =42Hz CAr) 14033 (s CAr) 14447 (s CAr) 14504 (s CAr)14526 (s CAr) 15909 (dd 1JCF = 2482Hz 3JCF = 124HzCAr) 16255 (dd 1JCF = 2480Hz 3JCF = 127Hz CAr)HRVS-ESI found [V minus 2Br]2+ 3751644 C

37H44F2N8O7

calculated [V minus 2Br]2+ 3751645

111015840-(2-(24-Difluorophenyl)-2-hydroxypropane-13-diyl)bis(4-((5-(hydroxymethyl)-8-methyl-2-propyl-4H-[13]dioxino[45-c]pyridin-6-yl)methyl)-1H-124-triazol-4-ium) Bromide (4b)Compound 3b (054 g 171mmol) and fluconazole (020 g065mmol) were dissolved in acetonitrile (50mL) andthe reaction mixture was stirred at 70∘b for 1 week Thesolvent was removed in vacuo and then a diethyl etherwater

mixture (5 3 40mL) was added to the residue The aqueousand organic phases were separated and the aqueous layerwas concentrated to a volume of 1mL Ethanol (1mL)was added and the dissolved product was purified bycolumn chromatography on C18-reversed phase silica gelwith gradient elution (isopropanolwater 0ndash30) Yield64 (039 g) yellow solid mp 120∘C (decomp) 1H NMR(CDCl

3) 120575 097 (t 12H 3JHH = 73Hz 4CH

2CH2CH3)

147ndash156 (m 8H 4CH2CH2CH3) 173ndash186 (m 8H

4CH2CH2CH3) 228 (s 6H 2CH

3Pyr) 228 (s 6H

2CH3Pyr) 453 458 (AB system 8H 2JHH = minus120Hz

4CH2) 481ndash501 (m 16H 4CH

2 4CH

2-OH 4CH) 504 514

(AB system 8H 2JHH = minus140Hz 4CH2) 567ndash582 (m 4H

4CH2-OH) 658ndash669 (br m 2H 2CHAr) 678ndash683 (m 2H

2CHAr) 686 (br s 2H 2OH) 727ndash733 (m 2H 2CHAr)866 (s 2H 2CHAr) 867 (s 2H 2CHAr) 1055 (br s 4H4CHAr) 13C NMR (CDCl

3) 120575 1402 (s CH

2CH2CH3) 1704

(s CH2CH2CH3) 1845 (s CH

3Pyr) 3624 (s CH

2CH2CH3)

5060 (s CH2) 5601 (s CH

2) 5730 (s CH

2) 5733 (s CH

2)

6469 (s CH2) 7334 (d 3JCF = 23Hz C-OH) 9981 (s

CH) 10437 (dd 2JCF = minus267Hz 2JCF = minus271Hz CAr)11212 (dd 2JCF = minus203Hz 4JCF = minus45Hz CAr) 12034(dd 2JCF = minus94Hz 4JCF = minus35Hz CAr) 12846 (s CAr)12898 (s CAr) 13039 (dd 3JCF = 97Hz 3JCF = 51HzCAr) 14040 (s CAr) 14044 (s CAr) 14415 (s CAr)14424 (s CAr) 14452 (s CAr) 14455 (s CAr) 14718 (sCAr) 14720 (s CAr) 14809 (s CAr) 14811 (s CAr) 15923(dd 1JCF = 2480Hz 3JCF = 128Hz CAr) 16341 (dd1JCF = 2516Hz 3JCF = 132Hz CAr) HRVS-ESI found[Vminus2Br]2+ 3891801 C

39H48F2N8O7 calculated [Vminus2Br]2+

3891801

22 In Vitro Studies

221 Antimycotic Activity Antimycotic activity of theobtained compounds 4ab and the reference drugsfluconazole and terbinafine (terbinafine was purchasedfrom Sigma-Aldrich (USA)mdashSKU-Pack Size PHR1298-1G)was evaluated on several fungal strains causing cutaneousand systemic mycoses The following strains were obtainedfrom a collection of clinical isolates of the Kazan Institute ofMicrobiology and Epidemiology (Kazan Russia) Candidaalbicans K-1663 (clinical strain isolated from pharynxmucosa) Aspergillus fumigatus 1320-13 (clinical strainisolated from nasal cavity) and Trichophyton rubrum P-1(clinical strain isolated from skin) Rhizopus nigricans 600was taken from the All-Russian collection ofmicroorganisms(etalon strain)

For the inoculum preparation the pure 2- and 5-daycultures of yeasts and filamentous fungi respectively grownon Sabouraud dense nutrient medium were used Yeastcultures of C albicans were prepared by flushing the culturefrom the surface of the solid agar medium Cultures ofmycelial fungi T rubrum R nigricans A fumigatus werepreground in a mortar A suspension of microorganisms wasprepared in a sterile isotonic solution of sodium chlorideThecell concentrations were (1 divide 5) times 103 for yeast fungi and (04divide 5) times 104 for mycelial fungi


The study of the antifungal activity of substances invitro was carried out in a liquid nutrient medium (glucoseSabouraud broth) in biological test tubes by 2-fold serialdilutions approach Test compounds were prepared at con-centrations ranging from 400 to 038 120583gmL A test tube inthe absence of test compounds served as a control To eachtube 50 120583L of inoculumwas addedThe tubes were incubatedfor 2ndash7 days at 30∘C To the end of this period the resultswere assessed by visual analysis of optical density of themedium The following MIC endpoints were determined 0= clear solution no growth 1 = weak growth (25 control)2 = significant inhibition of growth (50 control) 3 =insignificant growth inhibition (75 control) 4 = no growthinhibition All experiments were carried out in duplicate

222 Activity against the Clinical Strains of C albicans inBiofilms The ability of fluconazole and 4b to inhibit thebiofilm formation was evaluated on the following C albicansstrains C albicans P-1663 (isolated from pharynx mucosa)C albicans P-4467 (isolated from skin) These strains wereobtained from a collection of clinical isolates of the KazanInstitute of Microbiology and Epidemiology (Kazan Russia)

To detect the inhibitory effect of the studied compoundson C albicans biofilm formation a semiquantitative deter-mination of biofilm formation was performed in microtiterplates as described earlier [24 25] The cell culture seededin Sabouraud liquid medium was incubated in orbital shaker(180 rpm) for 24 h at 30∘C Then the culture was washedtwice with a sterile phosphate buffer and resuspended inSabouraud liquid medium to achieve the final cell density 10times 106 cellsmL A 100120583L cell suspension was added to 96-wellflat-bottom polystyrene microsheets (CorningCostar USA)and incubated for 24 hours at 37∘C After biofilm formationthe plates were washed three times with sterile phosphatebuffer and the biofilm formed at this point was taken as thebiofilm level before the treatment Then 125120583L aliquots oftest compounds were added in various concentrations andthe plates were incubated for 48 hours After incubationthe plates were washed three times with sterile phosphatebuffer dried for 30 minutes at 37∘C and stained with 125 120583Lof a 1 aqueous solution of crystal violet After staining for20 minutes at 37∘C the plates were washed 125120583L of 95ethanol was added to dissolve the stained dye and the opticaldensity at 620 nm was measured using a spectrophotometerto assess the amount of adhered and stained cells Allexperiments were carried out in triplicate

223 Antibacterial Activity Antibacterial activity of com-pounds 4ab was evaluated on a number of Gram-positive(Staphylococcus aureus ATCC 29213 Bacillus subtilis 168Staphylococcus epidermidis (clinical isolate) andMicrococcusluteus (clinical isolate)) and Gram-negative (Escherichia coli0fbb 25922 Pseudomonas aeruginosa 0fbb 27853Salmonella typhimurium TA100 K pneumoniae (clinical iso-late)) bacteria Clinical isolates of Staphylococcus epidermidisMicrococcus luteus and Klebsiella pneumoniae were obtainedfrom the Kazan Institute of Epidemiology and Microbiology(Kazan Russia)

MICs were determined by using the broth microdilutionmethod in 96-well plates (Eppendorf) according to theEUCAST rules for antimicrobial susceptibility testing [26]with some modifications Briefly the 108 cellsml bacterialsuspensions were subsequently diluted 100-fold with TSBbroth to obtain a 1 times 106 cellsmL suspension and variousconcentrations of antimicrobials were added in microw-ell plates up to final concentrations of 05ndash64 120583gmL Thecultures were incubated at 37∘C for 24 h The minimuminhibitory concentration was defined as the lowest concen-tration of compound at which no visible growth could beseen after 24 h of cultivation at 37∘C To determine minimumbactericidal concentration 5 120583L of culture liquid from wellswithout visible growth was plated on solid LB medium andincubated for 24 h at 37∘C MBC was assumed at antimicro-bials concentrations where no viable planktonic cells wereobserved All experiments were performed in triplicate

For investigation of the antimicrobial mechanism ofcompound 4b MICs were also determined in the presenceof 01M CaCl

2in nutrient broth on six Gram-positive and

Gram-negative strains from the same bacterial panel

224 Activity against Bacterial Biofilms Bacterial biofilmswere grown under static conditions in BM for 72 h at37∘C Subsequently the supernatants were carefully removedand the biofilms were washed with fresh sterile BM brothAliquots (500 120583L) of antimicrobials solutionswith concentra-tions between 1 and 16MBC in fresh BMbuffer were added tothe wells and cultivation was continued for 24 hours at 37∘CTo evaluate the viability of biofilm-embedded cells the wellswere washed several times with sterile phosphate-bufferedsaline (PBS) to remove nonadherent and detached cells Thewashed biofilms were suspended in PBS by scratching thewell bottoms with following treatment in a sonicator bathfor 2min at 20 kHz to favor the disintegration of bacterialclumps and viable cells were counted by a drop platemethod with minor modifications [27 28] The serial 10-fold dilutions of each well were prepared and a 5 120583L aliquotof the suspension was dropped onto LB agar plates CFUswere counted from those drops containing 5ndash10 colonies andpresented as CFU per mL

Experiments were carried out in biological triplicates (ienewly prepared cultures and medium) with 3 independentrepeats in each one Since the drop plate assay results wereassessed from 10-fold dilutions where the number of colonieswas typically countable only in the two latter dilutions toassess the statistical significance we compared 10log

10(119888)

where 119888 is the obtained cell number using Pearsonrsquos chi-squared homogeneity test

225 Cytotoxic Activity The human fibroblast cells (HFC)or embryonic kidney 293 cells (HEK-293) were cultured in120572-MEM supplemented with 10 FBS 2mM L-glutamine100 120583gmL penicillin and 100UmL streptomycin The cellswere seeded in 96-well plates at the density of 1000 cellsper well and then allowed to attach overnight Cells werecultured in the presence of the tested compoundrsquos solutions


(015ndash1mgmL) for 72 h at 37∘C and 5 CO2 After incuba-

tion the medium with the tested compoundrsquos solutions wasremoved by aspiration and replaced with 80 120583L of fresh 120572-MEMmedium MTT solution (20120583L of 5mgmL MTT) wasadded to each well and the plates were incubated for 35hours at 37∘C in culture hood After the incubation periodthe medium with MTT solution was removed and 100120583Lof DMSO was added to each well to dissolve the resultingformazan crystals The colored product of MTT reductionby viable cells was detected on Infinite 200 PRO analyzer at530 nm All measurements were performed in triplicate

226 Genotoxicity The SOS-chromotest was performed byusing the Salmonella typhimurium TA1535pSK1002 Brieflyaliquots of 05mL of an overnight culture of the tester strainswere diluted in 5mL of LB medium and then incubatedwith rigorous agitation in presence of the ficin substancesMitomycinC (Sigma) at concentration of 1120583gmLwas used asa positive control in SOS-chromotest After 4 h of incubationthe cell density (A600) and the 120573-galactosidase activitywere measured by Millerrsquos protocol [29] with modifications[30] Cells were harvested from 05ndash15ml of culture liquidand resuspended in 800 120583l of Z-buffer (60mM Na

2HPO4times

7H2O 40mM NaH

2PO4times H2O 10mM KCl and 1mM

MgSO4times 7H2O (pH 70)) containing additionally 0005

cetyl trimethylammonium bromide (CTAB) and 50mM 120573-mercaptoethanol was added After preincubation at 30∘C for5min the reaction was started by adding 200120583L of 4mgmLo-nitrophenyl-120573-D-galactopyranoside in Z-buffer When theyellow color appeared the reaction was stopped by 500120583Lof 1M Na

2CO3 For the blank solution the Na

2CO3was

added prior the incubation The 120573-galactosidase activity wasmeasured at A420 nm To calculate the Miller units we usedthe following formula [A420(A600 of 1 10 dilution of cellstimes time of incubation)] times 1000

S typhimurium strains TA98 TA100 TA102 TA1535 andTA1537 were used for the Ames test Briefly S typhimuriumstrains [31] were grown overnight in 5mL of LB mediumand diluted 4-times by prewarmed LB and then incubationwas continued for 2 h Cells were harvested washed onceby 1x salt base solution (sodium citrate times 3X

2 ndash 05 gL

P2X`3times 3X

2 ndash 14 gL PX

2`3ndash 6 gL (NH

4)2SO4

ndash 1 gL MgSO4times 7X

2 ndash 05 gL) and resuspended in

6mL of 1x salt base About 100 120583L of bacterial suspensionwas mixed with top agar (05 agar 05 NaCl 5mM L-histidine 5mM biotin pH 74 42∘C) in a final volume of3mL and with the substance to be tested Each mixturewas then seeded onto the minimal agar plates (15 agarin the 1x salt base supplemented with 05 glucose andampicillin 10 120583gmL) Then the plates were incubated at37∘C for 72 hours and colonies were counted Sodium azidewas used as a positive control for S typhimurium TA100(5 120583gplate) and S typhimurium TA102 (20 120583gplate) andTA1535 (5120583gplate) 9-aminoacridine was used as a positivecontrol for S typhimurium TA1537 (5 120583gplate) 4-nitro-o-phenylenediamine was used as a positive control for Styphimurium TA98 (25 120583gplate)

3 Results and Discussion

31 Results

311 Synthesis The synthetic way to the studied compoundsis shown in Scheme 1 The key intermediates 2ab have beenobtained from initial pyridoxine hydrochloride 1 accord-ing to procedure described previously [23] Reaction of2ab with triphenylphosphine and N-bromosuccinimide indichloromethane gave the corresponding bromides 3ab asthe main products Reaction of 3ab with fluconazole inacetonitrile led to the target compounds 4ab The synthesisis convenient and well reproducible at a laboratory scaleCompounds 4ab were purified using flash chromatographyon C18 silica gel Synthesis and analytical parameters ofcompounds 2ab and 3a have been described previously [23]while compounds 3b and 4ab are described in this work forthe first time

312 Antimycotic Activity Antimycotic activity of com-pounds 4ab was studied on a panel of fungal pathogens(Table 1) in comparison with fluconazole and terbinafineas reference drugs Compound 4b exhibited a pronouncedantimycotic activity against all the tested fungi with min-imum inhibitory concentrations (MIC) 15 625 312 and312 120583gmL against C albicans No 1663 T rubrum Afumigatus and R nigricans respectively which were 15ndash30-fold lower than those of fluconazole and similar with those ofterbinafine In the case of A fumigatus 4b exhibited higheractivity than terbinafine (MICs 312120583gmL and 125 120583gmLrespectively) Compound 4a demonstrated amoderate fungi-cidal effect against the clinical strain of C albicans (MIC25 120583gmL) but was less active against the mycelial fungiwith MICs of 200120583gmL Interestingly the direct analog ofthe obtained compounds fluconazole was inactive in thisexperiment against the investigated mycelial fungi and onlymoderately active against the clinical strain of C albicans(MIC 50 120583gmL)

313 Prevention of C albicans Biofilm Growth Formation ofbiofilms is one of the important mechanisms of C albicanssurvival We tested whether fluconazole and the leadingcompound 4b are able to inhibit the biofilm formation ofclinical isolates of C albicans in vitro (Figure 1)The 24-hour-old biofilm of C albicans was prepared in 96-well platesIn control wells the biofilm was stained with crystal violetand taken as biofilm level before treatment (green line)In experimental wells various concentrations of fluconazoleand 4b in Sabouraud broth were then added After 48 h ofincubation the biofilms were evaluated using a crystal violetassay

Figure 1 demonstrates that fluconazole inhibits thebiofilm biomass growth only at concentrations higher than200120583gmL At lower concentrations it increases the biofilmformation by C albicans clinical isolates leading to 2ndash25-fold increase of total biofilm mass in comparison withuntreated control this effect is maximally pronounced atconcentrations between 25 and 50 120583gmL The effect of


1

OH

N

NF

F

N

N

N

N

HOO

O

O

O

OH

N

OHHO

HO

N

OH

Br

O

O

Fluconazole

N

OHO

O

OH

PPh3NBSCH2Cl2

R1

R2

R2

R2

R1

R1

R1

R2

2a R1= R2

= CH3[22]

b R1= H R2

= C3H7[22]

3a R1= R2

= CH3[22]

3b R1= H R2

= C3H7

4a R1= R2

= CH3

4b R1= H R2

= C3H7

N+Brminus

N+Brminus

CH3CN 70∘C(038ndash05 equiv)

Scheme 1 Synthesis of compounds studied in this work

Table 1 Activity of compounds 4ab on a panel of fungal pathogens

Compound Strains MIC 120583gmL400 200 100 50 25 125 625 312 15 075 038

Fluconazole

C albicans 0 0 0 0 1 1 3 4 4 4 4T rubrum 2 4 4 4 4 4 4 4 4 4 4A fumigatus 3 4 4 4 4 4 4 4 4 4 4R nigricans 4 4 4 4 4 4 4 4 4 4 4

Terbinafine


4a


4b


Note 0 = clear solution no growth 1 = weak growth (25 of control) 2 = significant inhibition of growth (50 of control) 3 = insignificant growth inhibition(75 of control) 4 = no growth inhibition (100 of control)


C albicans P-1663C albicans P-4467

Biofilm before treatmentC albicans P-4467C albicans P-1663Fluconazole

4b

00

01

02

03

Biofi

lm O

D570

1256231 25 50 100 200 400 800 16000Concentration xMBC

Figure 1 The effect of fluconazole and 4b on C albicans biofilmformation The 24-h biofilms (green line) were treated with31ndash1600 120583gmL of fluconazole (lines) or 4b (bars) for 48 h andquantifiedwith crystal violet assay Data are present as averages fromfive independent experiments with standard deviations C albicansP-1663 isolated from pharynxmucosa (blue) andC albicansP-4467isolated from skin (red) were used

escape of Candida from inhibition by antifungal agents atconcentrations above the MIC (paradoxical or Eagle effect)was previously reported Thus a growth of some C albicansstrains in vitro [32 33] in vivo [34] and in biofilms [35] wasobserved for caspofungin It was also shown that fluconazolecan induce the growth of planktonic C krusei at sub-MICs[36] Although the molecular mechanisms of such effectsof antifungal agents remain largely unknown it has beenargued that the salvage pathways associated with changes incell morphology and cell wall rearrangements [37] play aprincipal role

In contrast to fluconazole compound 4b effectively sup-pressed the biofilm growth even at minimal concentrationsas compared to untreated sample (Figure 1) Of note thetreatment with fluconazole and 4b did not lead to destructionof preformed biofilm

314 Antibacterial Activity Theantibacterial activity of com-pounds 4ab was evaluated on various Gram-positive andGram-negative bacteria Table 2 shows MICs of compoundsin comparison with the reference antifungal drugs (flucona-zole and terbinafine) and antibacterial drugs (benzalkoniumchloride and miramistin) Compound 4b demonstrated highantibacterial activity withMICs in the range of 05ndash32 120583gmLfor all the studied pathogens Its activity against the Gram-positive strains in this test (MICs 05ndash8 120583gmL) was compa-rable with that of the reference antibacterial drugs At thesame time it was active against all the four Gram-negativestrains (MICs 8ndash32 120583gmL) while benzalkonium chloridewas inactive against K pneumoniae Compound 4a showeda moderate activity against two Gram-positive strains (MIC32 120583gmL) while both antifungal drugs were inactive in this

experiment For all the studied strains the MBCMIC ratioof 4b was found to be 2ndash4 suggesting its biocidal properties

Antibacterial activity of compound 4b has also beentested on six bacterial strains from the same panel ofpathogens in the presence of CaCl

2[38] The MIC val-

ues of 4b in the presence of Ca2+ ions were significantlyincreased for both Gram-positive and Gram-negative strains(gt64 120583gmL as compared to 1ndash32120583gmL under Ca2+-freeconditions) This observation suggests that the cell walldamage could be associated with the Ca2+ removal andmightrepresent the possible mechanisms of antibacterial action of4b

315 Activity against Bacterial Biofilms Activity of 4bagainst the biofilm-embedded cells was evaluated on fourcommon human resident Gram-positive (S aureus S epi-dermidis) and Gram-negative (E coli P aeruginosa) bacterialstrains causing nosocomial infections and forming rigidbiofilms on tissues and abiotic surfaces Similar toC albicansthe 24-h bacterial biofilms were established on 96-well plateswashed and incubated with different concentrations of 4bmiramistin and benzalkonium chloride in BM broth for thenext 24 h The untreated 24-hour-old biofilm was taken asbiofilm level before the treatmentThen thewells were stainedwith crystal violet and quantified All the antimicrobialsinsignificantly increased the biofilm mass at concentrations(05ndash4) times MBCs while (8ndash16) times MBCs almost completelyrepressed the biofilm growth (Figure 2)

Since no biofilm eradication occurred at all concentra-tions of 4b tested its antimicrobial activity against biofilm-embedded cells was evaluated by counting of viable cells(colony-forming units CFUs) inside the biofilm (Figure 3)The activity of 4b in these experiments was comparable withthat of other ammoniumquaternary salts likemiramistin andbenzalkonium chloride as judged with Pearsonrsquos chi-squaredhomogeneity test Similar to reference antimicrobials 4bwasactive against the biofilm-embedded staphylococci (Figures3(a) and 3(b)) and E coli (Figure 3(c)) reducing the CFUsamount by 2-3 orders of magnitude at 16 timesMBC By contrastall compounds were almost inactive against P aeruginosaeven at 16 timesMBC (Figure 3(d))

316 Genotoxicity In order to evaluate the genotoxicityof 4b the Ames test [31] was performed using five Styphimurium TA98 TA100 TA102 TA1535 and TA1537strains The positive controls for each strain are describedin the Methods section The samples were taken in concen-trations of 02 04 08 and 16 120583gmL since higher concen-trations were toxic for S typhimurium In all the studiedstrains no increase in the number of revertant colonieswas detected as well as no dose-dependence was observed(Table 3) suggesting the absence of mutagenic potential of4b

Genotoxicity of compound 4b was also evaluated usingSOS-chromotest in S typhimuriumTA1535pSK1002 strain aspreviously described [39]MitomycinCwas used as a positive


Table2In

vitro

antib

acteria

lactivity

oftheo

btainedcompo

unds

4ab

andther

eference

drugs(MICs120583gmL)lowast

Com

poun

ds

Gram

(+)

Gram

(minus)

Saureus

0fbb

29213

Bsubtilis

168

Sepidermidis

Mluteus

Ecoli

0fbb

25922

Kpn

eumoniae

Paeruginosa

0fbb

27853

Styphim

urium

TA100

4agt64

3232

nm

gt64

gt64

gt64

nm

4b05

28

416

3232

84b

+Ca

Cl2

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Flucon

azole

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Terbinafine

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Benzalkonium

chlorid

e4

28

416

gt64

328

Mira

mistin

21

44

8nm

644

lowastn mno

tmeasured


S aureus

Benzalkonium chlorideMiramistin

Biofilm before treatment

4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570

Concentration xMBC025 050 1 2 4 8 16




00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570

Figure 2The effect of 4b miramistin and benzalkonium chloride on bacterial biofilm formationThe 24-h biofilms (green line) were treatedwith 025ndash16 timesMBCs of antimicrobials or 4b (bars) for 24 h and then were quantified with crystal violet assay Data are present as averagesfrom five independent experiments with standard deviations Arrows indicate no differences with the initial biofilm mass

control in SOS-chromotest The optical density at 420 nm(OD420) was measured and 120573-galactosidase activity wasnormalized to the amount of cells estimated from the OD600values SOS induction factor was calculated as a ratio of 120573-galactosidase activity in the presence of compounds and thesolvent control (Table 4)The concentrations of 4bwere in therange of 075ndash150 120583gmLThe tested compound as well as thereference biocides miramistin and benzalkonium chlorideled to dose-dependent increase of 120573-galactosidase activitysuggesting the development of SOS-response in cells at highconcentrationsDNA-damaging activity of compound4bwassimilar to that of benzalkonium chloride and significantlylower as compared to that of miramistin under the testedconcentrations

317 Cytotoxicity Cytotoxicity of compounds 4ab and thereference antifungal and antibacterial drugs was evaluated inhuman fibroblast cells (HFC) and human embryonal kidney(HEK-293) cells (Table 5) Fluconazole demonstrated thelowest cytotoxicity among the studiedmolecules Compound4a was more toxic than fluconazole but less toxic thanall other compounds The leading compound 4b was moretoxic than 4a and fluconazole slightly more toxic thanterbinafine and significantly less toxic than miramistin andbenzalkonium chloride The latter were the most cytotoxicin the studied group For deeper characterization of com-pounds cytotoxicity their CC

50MBC ratios were calculated

with minimal and maximal MBC values established for thebacteria analyzed (Table 5)


S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

Concentration xMBC1x0 2x 4x 8x 16x

(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)

Figure 3 Antimicrobial effect of 4b on biofilm-embedded bacteria

32 Discussion

321 Antimycotic Activity Fluconazole is a drug widely usedfor the treatment of various fungal infections Despite certainside effects it has low toxicity and is generally well toler-ated and the recommended therapeutic regimens are veryappealing to the patient However many fungal pathogensincluding various Candida species develop resistance tofluconazole In addition the data reported in literature andobtained in this work indicate that this drug can be ineffectivefor the biofilms-associated infections In this relation thedevelopment of more effective therapeutic agents for thetreatment of various forms of candidiasis represents a highlyactual task

To overcome the problems associated with fluconazole-based therapies in this work we have attempted to design anovel hybrid construction based on quaternary ammoniumderivatives of fluconazole and pyridoxine Our aim was

to increase antimycotic activity including activity againstbiofilm-forming fungi and reduce ability of fungal pathogensto develop resistance

Since the 1930s quaternary ammonium compounds(QACs) are widely used for the control of bacterial andfungal growth Broad-spectrum antimicrobial activity [4041] has made many QACs such as benzalkonium chlo-ride miramistin and cetylpyridinium chloride the usefulhygienic adjuncts in disinfectant formulations and theyhave also been used in therapy of patients with localpyoinflammatory processes QACs can also be active againstthe main pathogenic fungi such as Candida albicans [42]Cryptococcus neoformans [43] Saccharomyces cerevisiae [44]and Aspergillus flavus [45]

The underlying idea for introduction of pyridoxine(vitamin B6) moiety into the developed hybrid structuresis that the presence of pyridoxine moiety can enhancetransmembrane transport of the obtained constructs viaseveral possible mechanisms First of all many cells have


Table 3 Mutagenicity of 4b in the Ames test (ratio fold increase over the solvent control)

S typhimurium strain Positive control Concentration 120583gmL02 04 08 16

TA98 342 plusmn 34 07 plusmn 03 12 plusmn 05 09 plusmn 02 06 plusmn 02





Table 4 DNA-damage activity of 4b in SOS-chromotest (ratio fold increase over the solvent control)

Compounds Concentration 120583gmL150 75 15 75 15 075

4b 24 plusmn 05 32 plusmn 06 20 plusmn 13 18 plusmn 14 09 plusmn 06 10 plusmn 04

Benzalkonium chloride 50 plusmn 11 20 plusmn 03 18 plusmn 12 15 plusmn 02 12 plusmn 06 12 plusmn 08

Miramistin 59 plusmn 11 48 plusmn 07 29 plusmn 06 21 plusmn 09 18 plusmn 08 15 plusmn 10

Mitomycin C - - - - - 117 plusmn 25

Table 5 Cytotoxicity of 4ab and the reference drugs (mean plusmn SD)

Compounds HFCCC50 120583gmL

HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438

4b 168 plusmn 20 05ndash2 331 plusmn 113 2ndash4Fluconazole gt2000 gt2000Terbinafine 411 plusmn 190 630 plusmn 122

Miramistin 41 plusmn 04 1-2 41 plusmn 08 1-2Benzalkonium chloride 21 plusmn 01 05ndash2 20 plusmn 09 05ndash2

specific pyridoxine transporters in their membranes (eg[20]) Furthermore vitamin B6 is a cofactor for more than140 essential enzymatic reactions and pyridoxine-modifiedmolecules are often recognized by many pathogenic cellsand microorganisms as endogenous As a result they haveincreased cellular permeability and increased bioactivity Forexample the development of pyridoxine-modified nanopar-ticles for efficient intracellular delivery of doxorubicin (DOX-B6-SA-NP) was reported [21] It was demonstrated thatthe treatment with DOX-B6-SA-NP kept higher doxoru-bicin accumulation inside the cells than conventional lipidnanoparticlesThe positive charge of nanoparticles facilitatedthe endosomal escape and promoted the nuclear accumu-lation of the drug In vitro studies confirmed the enhancedefficacy of DOX-B6-SA-NP in comparison to free doxoru-bicin and lipid nanoparticles Intravenous pharmacokineticsand biodistribution studies demonstrated that pyridoxine-modified nanoparticles can significantly prolong the bloodcirculation time of doxorubicin in the biological systemand increase the drug accumulation in the tumor tissue Ascompared to free drug DOX-B6-SA-NP exhibited increasedtherapeutic efficacy and lower toxicity in animal models Ingeneral the obtained results suggest that the pyridoxine-modified nanoparticles represent a prospective platform foranticancer drugs delivery

Another practical reason for introduction of pyridoxine-based cyclic acetals into the developed hybrids is the possibil-ity of easily varying the acetal substituents in order to modifylipophilicity steric parameters and other physicochemicalproperties of the obtained structures

The obtained results demonstrate that the developedstructures have potent antimycotic activity against severalfungal pathogens including C albicans T rubrum Afumigatus and R nigricans with MICs in the range of15ndash625 120583gmL for the leading compound 4b The latter alsoinhibited the growth of C albicans biofilms Interestinglyfluconazole the direct analog of the obtained compoundswas almost inactive in this experiment against the mycelialfungi and only moderately active against the clinical strain ofC albicans (MIC 50 120583gmL)

It is well known that C albicans biofilms are highlyresistant to the action ofmany clinically important antifungaland antimicrobial agents including fluconazole [46 47]C albicans biofilm formation proceeds via at least threedevelopmental phases (i) early phase (0 to 11 h) involvingadhesion of fungal cells to the substrate (ii) intermediatephase (sim12 to 30 h) during which the blastospores coaggre-gate and proliferate forming communities while producinga carbohydrate-rich extracellular matrix (ECM) and (iii)maturation phase (sim31 to 72 h) in which the fungal cells


are completely encased in a thick ECM [46] Acquisition ofantifungal resistance by C albicans biofilms correlates withthe developmental phases of these biofilms It was reportedthat at later developmental phases (12 and 48 h) biofilmsformed by C albicans typically displayed complete resistanceto fluconazole [48]

In this work we measured the biofilm biomass growthat the maturation phase (48 h) Compound 4b demonstratedexpressed ability to inhibit growth of biofilm biomass inall concentrations studied as compared to untreated sample(Figure 2) The inhibition was maximal at concentrationsmore than 400 120583gmL while in the range of 31ndash25 120583gmLthe effect was less expressed In agreement with the literaturedata the biofilms formed by the studied clinical isolates of Calbicanswere resistant to fluconazole at the same experimen-tal conditions (Figure 1) Moreover in concentrations below100 120583gmL fluconazole clearly stimulated biofilm growthThe effects of fluconazole stimulated growth of biofilmsformed by Candida species were reported in literature (eg[36 49]) The obtained results indicate that the leadingcompound 4b represents a useful candidate for the treatmentof candidiasis caused by C albicans biofilms

322 Antibacterial Activity Another interesting and usefulproperty of compound 4b is ability to inhibit growth ofbacterial pathogens Recently we described a wide seriesof phosphonium and ammonium derivatives of pyridoxine[22 23 50 51] Some of the described compounds pos-sessed potent antibacterial activity with minimum inhibitoryconcentrations (MICs) in the range of 05ndash64120583gmL Theseresults encouraged us to test the quaternary ammoniumderivatives 4ab in vitro for their ability to inhibit growthof a number of bacterial pathogens We have observed thatthe antibacterial activity of 4b was comparable to that of thetwo widely used quaternary ammonium salts benzalkoniumchloride andmiramistin In general compound 4b efficientlyinhibited the growth of Gram-positive bacteria while exhibit-ing less efficiency against Gram-negative ones suggesting adifferent mechanism of antimicrobial activity against thesetwo groups of microorganisms In most cases the MBCMICratio was found to be 2ndash4 suggesting that 4b exhibits biocidalrather than biostatic properties

According to literature data [52] QACs generally actby disrupting the cytoplasmic and outer membrane lipidbilayers through association of the positively charged qua-ternary nitrogen with the anionic head groups of acidicphospholipids and interaction of the lipophilic tail withthe hydrophobic membrane core As a result QACs formmixed-micelle aggregates with hydrophobic membrane com-ponents leading thereby to membrane solubility and cell lysisbecause of generalized and progressive leakage of cytoplasmicmaterials At the same time other biomolecular complexeswithin the bacterial and fungal cells are potential targets foraction of cationic surfactants For example a correlation ofantifungal activity with fungal phospholipase inhibition hasbeen described for a series of bis-quaternary ammonium salts[43]

To gain insight into possible mechanism of action ofcompound 4b its activity has been tested on six bacterialstrains from the same panel of pathogens in the presenceof CaCl

2[38] The mechanism of action of the membrane

damaging drugs is often related to removal of Ca2+ fromthe cellular membranes Ca2+ ions stabilize membranes bycross-linking of the negatively charged head groups of lipidsand this effect plays an important structural role in theintegrity of the outer lipopolysaccharide layer and the cellwalls of bacterial cells Therefore possible modification ofantibacterial activity of the tested compound by Ca2+ ionsmay indicate that it exerts its antimicrobial activity by causingcell wall damage

In accordance with this hypothesis we have observedthat the MIC values of 4b in the presence of Ca2+ ions weresignificantly increased for both Gram-positive and Gram-negative strains (gt64 120583gmL as compared to 1ndash32120583gmLunder Ca2+-free conditions) (Table 2) The observed activitydecrease is probably related to the membrane-stabilizingeffect The increased concentration of Ca2+ ions in theextracellular space prevents their removal from the cell uponthe action of the tested compounds Therefore it can besuggested that the cell wall damage associated with theremoval of Ca2+ ions is one of the possible mechanisms oftheir antibacterial activity Similar observations have beenreported in our recent paper [53] for a series of quaternarybis-phosphonium salts of pyridine derivatives which exhib-ited broad-spectrum antibacterial activity against Gram-positive pathogens including methicillin-resistant strains ofS aureus

So far no specific target has been identified for mostQACs it is assumed that the effect is rather generalized thanspecific to one target However as discussed in literature[52] there should be some target specificities for exampleas shown for the bis-quaternary bis-naphthalimide MT02[54] because the activity of QACs toward different bacterialspecies varies substantially and cannot be explained simplyby the structure of cationic and hydrophobic portions [55]It is therefore possible that compound 4b exerts membranedamage leading to disruption of the cell envelope and arrest-ing intracellular activity by binding targets in the cytoplasmThe above described antibacterial effects in the presence ofCa2+ ions suggest that 4b interacts with bacterial membranesHowever the role of intracellular targets in its antibacte-rial action remains unclear Pyridoxine molecule is a well-established cofactor formany enzymesTherefore pyridoxinederivatives can participate in many intracellular interactionsthus leading to enhanced or more specific antibacterialaction The observed effect of 4b in the SOS-chromotest onS typhimurium may suggest some specific interaction withDNA however this hypothesis requires further experimentalinvestigation

It was also observed that Gram-positive bacteria aregenerally more sensitive to 4b than Gram-negative bacteriawhich is in agreement with literature data on QACs [52]

According to our recent report quaternary ammoniumpyridoxine derivatives are able to penetrate the bacterialbiofilms and efficiently eradicate them [28] Therefore it was


interesting to evaluate capability of 4b to target the biofilm-embedded bacterial cells To address this issue four commonhuman resident Gram-positive (S aureus S epidermidis) andGram-negative (E coli and P aeruginosa) bacteria causingnosocomial infections and forming rigid biofilms on tissuesand abiotic surfaces were chosen as model objects Thebacterial strains were grown in basal medium (BM) broth in24-well plates for 72 hours to obtain rigid biofilms Then thewells were washed filled with fresh BM containing differentconcentrations of antimicrobials and incubated for the next24 hours The amount of colony-forming units (CFUs) inthe biofilm was quantified using a drop plate approach Bothbenzalkonium chloride and miramistin were active againstthe biofilm-embedded staphylococci (Figures 3(a) and 3(b))reducing the CFUs amount by 3 orders of magnitude at (4ndash6)times MBC (minimum bactericidal concentration) At the sametime both drugswere less effective againstE coli (Figure 3(c))and almost inactive against P aeruginosa (Figure 3(d)) Theactivity of 4b in these experiments was not so expressed(Figure 3) Thus it was inactive against S epidermidis andP aeruginosa strains even at 10 timesMBC and only moderatelyactive against S aureus and E coli At the same time although4b was not so efficient in eradicating the studied biofilmsthe observed activity against S aureus and E coli suggestsits ability to penetrate through the biofilm wall Taking intoaccount the fact thatMIC ofmany antibiotics against biofilm-embedded bacteria increases up to 1000-fold as compared totheir planktonic forms it can also be suggested that furtherstructural modification can increase the activity

323 Safety Issues The results of the Ames test suggest theabsence of mutagenic potential of 4b At the same time thetested compound as well as the reference biocides miramistinand benzalkonium chloride led to dose-dependent increaseof 120573-galactosidase activity suggesting the development ofSOS-response in cells at high concentrationsDNA-damagingactivity of compound 4bwas similar to that of benzalkoniumchloride and significantly lower as compared to that ofmiramistin

The cytotoxicity studies on human fibroblast cells andhuman embryonal kidney cells demonstrate that compound4b was more toxic than the reference antifungal drugs(fluconazole and terbinafine) but significantly less toxicthan miramistin and benzalkonium chloride the effectiveantiseptics for the local treatment of infected wounds withdeclared low side effects [56 57] In particular CC

50value

of benzalkonium chloride for the normal human fibroblastswas reported to be 67120583gmL with CC

50MBC ratio of 005

[58] In our studies CC50

of benzalkonium chloride wasfound a bit less (21 120583gmL) while the CC

50MBC for both

human fibroblasts and 2 HEK-293 cells was in range of 05ndash2(Table 5) For 4b the CC

50MBC ratio was also found in

a range of 05ndash2 for human fibroblasts and 2ndash4 for HEK-293 cells suggesting that it has at least similar therapeuticindex with benzalkonium chloride which is widely used asa biocide for outer treatment [56 57 59]

In general comparative evaluation of activity and safetyparameters for compound 4b and the reference antifungal

and antibacterial drugs suggest promising potential of theobtained chemotype in the design of novel broad-spectrumantimicrobial agents

4 Conclusion

In this work we have synthesized two novel quaternaryammonium salts 4ab bis-triazolium derivatives of flucona-zole and pyridoxine and studied their antimycotic andantibacterial activity cytotoxicity and genotoxicityThe lead-ing compound 4b demonstrated potent antimycotic activityagainst several fungal pathogens including C albicans Trubrum A fumigatus and R nigricans with MICs in therange of 15ndash625120583gmL It also inhibited the growth of Calbicans biofilms Under the same experimental conditionsfluconazole was inactive or moderately active against thestudied fungal pathogens In addition 4b demonstratedhigh antibacterial activity on a panel of Gram-positive andGram-negative bacterial strains with MICs in the range of1ndash32 120583gmL which was comparable or better than that of thereference antibacterial drugs benzalkonium chloride andmiramistin Antibacterial activity studies in the presence ofCaCl2suggested that the cell wall damage associated with

the removal of Ca2+ ions from the bacterial membrane isone of the possible mechanisms of antibacterial activity Incontrast to many antimicrobials 4b was also active againstbiofilm-embedded staphylococci and Escherichia coli Whileno biofilm structure destruction occurred 4b was able todiffuse into the matrix and reduce the number of colony-forming units by three orders of magnitude at 16 timesMBCTheAmes test in S typhimurium showed the lack ofDNA-damageactivity for 4b at the same time it showed some muta-genic potential in the SOS-chromotest comparable to thatof benzalkonium chloride Cytotoxicity studies on humanskin fibroblasts and embryonic kidney cells demonstratedthat 4b was more toxic than 4a and fluconazole slightlymore toxic than terbinafine and significantly less toxic thanmiramistin and benzalkonium chloride The obtained resultsmake the described chemotype a promising starting point forthe development of new antimicrobial therapies with a broadspectrum of antifungal and antibacterial activity and abilityto inhibit biofilm growth

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this manuscript

Acknowledgments

This work was supported by the Russian Science FoundationGrant no 15-14-00046 and by the Programs of CompetitiveGrowth of Kazan Federal University and IM Sechenov FirstMoscow State Medical University


References

[1] M V Castelli M G Derita and S N Lopez ldquoNovel antifungalagents a patent review (2013-present)rdquo Expert Opinion onTherapeutic Patents vol 27 no 4 pp 415ndash426 2017

[2] R L Harvey and J P Myers ldquoNosocomial fungemia in a largecommunity teaching hospitalrdquo JAMA Internal Medicine vol147 no 12 pp 2117ndash2120 1987

[3] Y Tong and J Tang ldquoCandida albicans infection and intestinalimmunityrdquoMicrobiological Research vol 198 pp 27ndash35 2017

[4] M Ruhnke ldquoMucosal and systemic fungal infections in patientswithAIDS prophylaxis and treatmentrdquoDrugs vol 64 no 11 pp1163ndash1180 2004

[5] M A Al-Fattani and L J Douglas ldquoBiofilm matrix of Candidaalbicans and Candida tropicalis chemical composition and rolein drug resistancerdquo Journal of Medical Microbiology vol 55 no8 pp 999ndash1008 2006

[6] S Tobudic C Kratzer A Lassnigg and E Presterl ldquoAntifungalsusceptibility of Candida albicans in biofilmsrdquoMycoses vol 55no 3 pp 199ndash204 2012

[7] H T Taff K FMitchell J A Edward andD R Andes ldquoMecha-nisms of Candida biofilm drug resistancerdquo Future Microbiologyvol 8 no 10 pp 1325ndash1337 2013

[8] M S Tuttle E Mostow P Mukherjee et al ldquoCharacterizationof bacterial communities in venous insufficiency wounds byuse of conventional culture andmolecular diagnostic methodsrdquoJournal of Clinical Microbiology vol 49 no 11 pp 3812ndash38192011

[9] F Z Hu and G D Ehrlich ldquoPopulation-level virulence factorsamongst pathogenic bacteria relation to infection outcomerdquoFuture Microbiology vol 3 no 1 pp 31ndash42 2008

[10] M K Kathiravan A B Salake A S Chothe et al ldquoThe biologyand chemistry of antifungal agents a reviewrdquo Bioorganic ampMedicinal Chemistry vol 20 no 19 pp 5678ndash5698 2012

[11] L R Peyton S Gallagher and M Hashemzadeh ldquoTriazoleantifungals a reviewrdquo Drugs of Today vol 51 no 12 pp 705ndash718 2015

[12] X Che C Sheng W Wang et al ldquoNew azoles with potentantifungal activity design synthesis and molecular dockingrdquoEuropean Journal of Medicinal Chemistry vol 44 no 10 pp4218ndash4226 2009

[13] S G Whaley E L Berkow J M Rybak A T Nishimoto K SBarker andPD Rogers ldquoAzole antifungal resistance inCandidaalbicans and emerging non-albicansCandida Speciesrdquo Frontiersin Microbiology vol 7 article 2173 2017

[14] S Sobue K Tan L Shaw G Layton and R Hust ldquoComparisonof the pharmacokmetics of fosfluconazole and fluconazoleafter single intravenous administration of fosfluconazole inhealthy Japanese and Caucasian volunteersrdquo European Journalof Clinical Pharmacology vol 60 no 4 pp 247ndash253 2004

[15] N-H Nam S Sardari M Selecky and K Parang ldquoCarboxylicacid and phosphate ester derivatives of fluconazole synthesisand antifungal activitiesrdquo Bioorganic amp Medicinal Chemistryvol 12 no 23 pp 6255ndash6269 2004

[16] G-P Yu L-Z Xu X Yi W-Z Bi Q Zhu and Z-W ZhaildquoSynthesis and fungicidal evaluation of 2-arylphenyl ether-3-(1H-124-triazol-1-yl)propan-2-ol derivativesrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 11 pp 4854ndash4860 2009

[17] A Bentley M Butters S P Green et al ldquoThe discoveryand process development of a commercial route to the watersoluble prodrug fosfluconazolerdquo Organic Process Research ampDevelopment vol 6 no 2 pp 109ndash112 2002

[18] KMHindi T J Siciliano S Durmus et al ldquoSynthesis stabilityand antimicrobial studies of electronically tuned silver acetateN-heterocyclic carbenesrdquo Journal of Medicinal Chemistry vol51 no 6 pp 1577ndash1583 2008

[19] Y-Y Zhang J-LMi C-H Zhou and X-D Zhou ldquoSynthesis ofnovel fluconazoliums and their evaluation for antibacterial andantifungal activitiesrdquo European Journal of Medicinal Chemistryvol 46 no 9 pp 4391ndash4402 2011

[20] N Szydlowski L Burkle L Pourcel M Moulin J Stolz and TB Fitzpatrick ldquoRecycling of pyridoxine (vitamin B6) by PUP1in ArabidopsisrdquoThePlant Journal vol 75 no 1 pp 40ndash52 2013

[21] S Sharma A Verma J Singh et al ldquoVitamin B6 tetheredendosomal PH responsive lipid nanoparticles for triggeredintracellular release of doxorubicinrdquo ACS Applied Materials ampInterfaces vol 8 no 44 pp 30407ndash30421 2016

[22] S V Sapozhnikov N V Shtyrlin A R Kayumov et al ldquoNewquaternary ammonium pyridoxine derivatives synthesis andantibacterial activityrdquoMedicinal Chemistry Research vol 26 no12 pp 3188ndash3202 2017

[23] M V Pugachev N V Shtyrlin S V Sapozhnikov et al ldquoBis-phosphonium salts of pyridoxine the relationship betweenstructure and antibacterial activityrdquo Bioorganic and MedicinalChemistry vol 21 no 23 pp 7329ndash7341 2013

[24] G A OrsquoToole and R Kolter ldquoInitiation of biofilm formationin Pseudomonas fluorescens WCS365 proceeds via multipleconvergent signalling pathways a genetic analysisrdquo MolecularMicrobiology vol 28 no 3 pp 449ndash461 1998

[25] G Ramage K VandeWalle B L Wickes and J L Lopez-RibotldquoCharacteristics of biofilm formation by Candida albicansrdquoRevista Iberoamericana de Micologıa vol 18 no 4 pp 163ndash1702001

[26] R Leclercq R Canton D F J Brown et al ldquoEUCAST expertrules in antimicrobial susceptibility testingrdquo Clinical Microbiol-ogy and Infection vol 19 no 2 pp 141ndash160 2013

[27] B Herigstad M Hamilton and J Heersink ldquoHow to optimizethe drop plate method for enumerating bacteriardquo Journal ofMicrobiological Methods vol 44 no 2 pp 121ndash129 2001

[28] A R Kayumov A A Nureeva E Y Trizna et al ldquoNewderivatives of pyridoxine exhibit high antibacterial activ-ity against biofilm-embedded staphylococcus cellsrdquo BioMedResearch International vol 2015 Article ID 890968 10 pages2015

[29] J H Miller In Experiments in Molecular Genetics Cold SpringHarbor Laboratory Press New York Ny USA 1972

[30] K Fedorova A Kayumov K Woyda O Ilinskaja and KForchhammer ldquoTranscription factor TnrA inhibits the biosyn-thetic activity of glutamine synthetase in Bacillus subtilisrdquo FEBSLetters vol 587 no 9 pp 1293ndash1298 2013

[31] J McCann and B N Ames ldquoA simple method for detectingenvironmental carcinogens as mutagensrdquo Annals of the NewYork Academy of Sciences vol 271 pp 5ndash13 1976

[32] D A Stevens T C White D S Perlin and C P SelitrennikoffldquoStudies of the paradoxical effect of caspofungin at high drugconcentrationsrdquoDiagnosticMicrobiology and Infectious Diseasevol 51 no 3 pp 173ndash178 2005

[33] G Chamilos R E Lewis N Albert and D P KontoyiannisldquoParadoxical effect of echinocandins across Candida species invitro evidence for Echinocandin-Specific and Candida species-related differencesrdquo Antimicrobial Agents and Chemotherapyvol 51 no 6 pp 2257ndash2259 2007


[34] K V Clemons M Espiritu R Parmar and D A StevensldquoAssessment of the paradoxical effect of caspofungin in therapyof candidiasisrdquoAntimicrobial Agents and Chemotherapy vol 50no 4 pp 1293ndash1297 2006

[35] C J Walraven S M Bernardo N P Wiederhold and S A LeeldquoParadoxical antifungal activity and structural observations inbiofilms formed by echinocandin-resistant Candida albicansclinical isolatesrdquo Medical Mycology vol 52 no 2 pp 131ndash1392014

[36] EMMaiolo U F Tafin O Borens and A Trampuz ldquoActivitiesof fluconazole caspofungin anidulafungin and amphotericinB on planktonic and biofilm candida species determined bymicrocalorimetryrdquo Antimicrobial Agents and Chemotherapyvol 58 no 5 pp 2709ndash2717 2014

[37] F C Bizerra A S A Melo E Katchburian et al ldquoChangesin cell wall synthesis and ultrastructure during paradoxicalgrowth effect of caspofungin on four different candida speciesrdquoAntimicrobial Agents and Chemotherapy vol 55 no 1 pp 302ndash310 2011

[38] T Das S Sehar L Koop et al ldquoInfluence of calcium inextracellular DNA mediated bacterial aggregation and biofilmformationrdquo PLoS ONE vol 9 no 3 Article ID e91935 2014

[39] Y Oda S-I Nakamura I Oki T Kato and H ShinagawaldquoEvaluation of the new system (umu-test) for the detection ofenvironmental mutagens and carcinogensrdquoMutation Researchvol 147 no 5 pp 219ndash229 1985

[40] K P CMinbioleM C Jennings L E Ator et al ldquoFrom antimi-crobial activity to mechanism of resistance the multifacetedrole of simple quaternary ammonium compounds in bacterialeradicationrdquo Tetrahedron vol 72 no 25 pp 3559ndash3566 2016

[41] E Obłak A Piecuch A Krasowska and J Łuczynski ldquoAnti-fungal activity of gemini quaternary ammonium saltsrdquo Micro-biological Research vol 168 no 10 pp 630ndash638 2013

[42] N Lincopan and A M Carmona-Ribeiro ldquoLipid-covered drugparticles Combined action of dioctadecyldimethylammoniumbromide and amphotericin B ormiconazolerdquo Journal of Antimi-crobial Chemotherapy vol 58 no 1 pp 66ndash75 2006

[43] C K L Ng D Obando F Widmer L C Wright T CSorrell andKA Jolliffe ldquoCorrelation of antifungal activitywithfungal phospholipase inhibition using a series of bisquaternaryammonium saltsrdquo Journal of Medicinal Chemistry vol 49 no 2pp 811ndash816 2006

[44] E Obłak A Gamian R Adamski and S UłaszewskildquoThe physiological and morphological phenotype of a yeastmutant resistant to the quaternary ammonium salt N-(dodecyloxycarboxymethyl)-NNN-trimethyl ammoniumchloriderdquo Cellular amp Molecular Biology Letters vol 15 no 2pp 215ndash233 2010

[45] M H El-Newehy H El-Hamshary S S Al-Deyab and AAbdel-Megeed ldquoSynthesis of quaternized amine-terminatedpolyacrylonitrile and their antimicrobial assessmentrdquo Journalof Macromolecular Science Part A Pure and Applied Chemistryvol 51 no 6 pp 527ndash537 2014

[46] J Chandra D M Kuhn P K Mukherjee L L Hoyer TMcCormick and M A Ghannoum ldquoBiofilm formation by thefungal pathogen Candida albicans development architectureand drug resistancerdquo Journal of Bacteriology vol 183 no 18 pp5385ndash5394 2001

[47] J Morschhauser ldquoThe development of fluconazole resistance inCandida albicans ndash an example of microevolution of a fungalpathogenrdquo Journal of Microbiology vol 54 no 3 pp 192ndash2012016

[48] P KMukherjee J ChandraDMKuhn andMAGhannoumldquoMechanism of fluconazole resistance in Candida albicansbiofilms phase-specific role of efflux pumps and membranesterolsrdquo Infection and Immunity vol 71 no 8 pp 4333ndash43402003

[49] C F Rodrigues and M Henriques ldquoOral mucositis causedby Candida glabrata biofilms failure of the concomitant useof fluconazole and ascorbic acidrdquo Therapeutic Advances inInfectious Disease vol 4 no 1 pp 10ndash17 2017

[50] MV PugachevNV Shtyrlin L P Sysoeva et al ldquoSynthesis andantibacterial activity of novel phosphonium salts on the basis ofpyridoxinerdquo Bioorganic amp Medicinal Chemistry vol 21 no 14pp 4388ndash4395 2013

[51] N V Shtyrlin S V Sapozhnikov S A Koshkin et al ldquoSynthesisand antibacterial activity of novel quaternary ammonium pyri-doxine derivativesrdquoMedicinal Chemistry vol 11 no 7 pp 656ndash665 2015

[52] M Tischer G Pradel K Ohlsen and U Holzgrabe ldquoQuater-nary ammonium salts and their antimicrobial potential targetsor nonspecific interactionsrdquo ChemMedChem vol 7 no 1 pp22ndash31 2012

[53] E V Nikitina M I Zeldi M V Pugachev et al ldquoAntibacterialeffects of quaternary bis-phosphonium and ammonium saltsof pyridoxine on Staphylococcus aureus cells a single basehitting two distinct targetsrdquoWorld Journal of Microbiology andBiotechnology vol 32 no 1 article 5 pp 1ndash7 2016

[54] L Gonzalez-Bulnes and J Gallego ldquoIndirect effects modulatingthe interaction between DNA and a cytotoxic bisnaphthalimidereveal a two-step binding processrdquo Journal of the AmericanChemical Society vol 131 no 22 pp 7781ndash7791 2009

[55] H H Locher D Ritz P Pfaff et al ldquoDimers of nostocarbolinewith potent antibacterial activityrdquo Chemotherapy vol 56 no 4pp 318ndash324 2010

[56] I L Bernstein ldquoIs the use of benzalkonium chloride as a preser-vative for nasal formulations a safety concern A cautionarynote based on compromisedmucociliary transportrdquoTheJournalof Allergy and Clinical Immunology vol 105 no 1 I pp 39ndash442000

[57] C Fromm-Dornieden J-D Rembe N Schafer J Bohm and EK Stuermer ldquoCetylpyridinium chloride andmiramistin as anti-septic substances in chronic wound managementmdashprospectsand limitationsrdquo Journal of Medical Microbiology vol 64 no 4pp 407ndash414 2015

[58] O Damour S Zhi Hua F Lasne M Villain P Rousselleand C Collombel ldquoCytotoxicity evaluation of antiseptics andantibiotics on cultured human fibroblasts and keratinocytesrdquoBurns vol 18 no 6 pp 479ndash485 1992

[59] N Akimitsu H Hamamoto R-I Inoue et al ldquoIncrease inresistance of methicillin-resistant Staphylococcus aureus tobeta-lactams caused by mutations conferring resistance tobenzalkonium chloride a disinfectant widely used in hospitalsrdquoAntimicrobial Agents and Chemotherapy vol 43 no 12 pp3042-3043 1999

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal ofInternational Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


the pathogenrsquos resistance to antifungal agents [5 6] Withinthe biofilm fungal cells are protected from the immunesystem of the host antimycotics and antiseptic agents Inbiofilms the specific fungal populations survive with themost resistant and virulent phenotypes formed due to aprolonged action of biological and chemical agents As aresult the cells released from biofilms often cause acute fun-gal infections highly resistant to the treatment Candidiasiscommonly occurs on mucous membranes of mouth vaginaand other organs where C albicans forms rigid biofilmswhich have been considered as one of the critical factorsproviding fungal resistance to traditional antimycotic drugsincluding azoles polyenes and echinocandins [7]

Microbial associations natural communities of severalmicroorganisms represent additional serious clinical prob-lem [8 9] Certain types of microorganisms involved into theassociation and resistant to antimicrobial agents enhance thevital activity and resistance of other species as a result of theinteraction As a result such associations are very resistantto various factors Many patients with chronic skin mycosesare often infected with various fungal strains belonging todifferent species genera and even classes Therefore theeffective antifungal drug should have a systemic action whichwarrants its efficient penetration into damaged organs andtissues and the fungal mono- and polymicrobial biofilms

There are a wide number of antimycotic drugs in currentclinical practice [10] One of the most popular azole com-pounds fluconazole is a first-generation fungistatic triazoleoften used for the treatment of cutaneous and other formsof candidiasis [11] Fluconazole inhibits ergosterol synthesisand thus alters membranes of fungal cell Fluconazole isactively used for the treatment of Candida infections andpossesses favorable safety profile and good pharmacokineticcharacteristics However its narrow antifungal spectrumin particular low activity against non-Candida fungi hasgreatly affected its therapeutic efficacy in clinic [12] Thereis also extensive documentation of intrinsic and developedresistance to azole antifungals among Candida species [13]In addition poor water solubility of fluconazole makes itproblematic to develop effective delivery forms for intra-venous administration These issues stimulated many stud-ies associated with structural modification of fluconazoleincluding esterification of hydroxyl group and modificationof 24-difluorobenzyl and triazolyl moiety aimed to improveits water solubility andor antimicrobial spectrum (eg [14ndash16]) A successful example was fosfluconazole a prodrugdeveloped for the treatment of infections caused by Candidaalbicans and Cryptococcus which exhibited broader antifun-gal activity spectrum than fluconazole [17]

In a more general context several studies have revealedthat the transformation of triazolyl ring into triazoliumcould result in enhanced antimicrobial action Triazoliumis able to form additional hydrogen bonds and produceelectrostatic interaction with biological targets and thesestructural features can affect the diffusion and interactionin bacterial tissues with the enhancement of water solubilityand membrane permeability [18] Thus quaternization ofN-4 position of triazole ring in fluconazole produced the

corresponding triazolium compounds which demonstratedpromising antibacterial potential [19]

In light of the described promising results it was reason-able to explore further structural modifications of flucona-zole In this work we have obtained two novel quaternaryammonium salts bis-triazolium derivatives of fluconazoleand pyridoxine The introduction of pyridoxine (vitaminB6) moiety into the developed hybrid structures can poten-tially enhance the transmembrane transport of the wholeconstructs via several possible mechanisms like either spe-cific pyridoxine transporters in cellular membranes [20] orrecognition of pyridoxine-modified molecules by the cell asendogenous [21] Another practical reason for introductionof pyridoxine-based cyclic acetals into the developed hybridsis the possibility of easily varying the acetal substituents inorder to modify lipophilicity steric parameters and otherphysicochemical properties of the obtained structures

2 Materials and Methods

21 Synthetic Procedures 1H and 13C NMR spectra wererecorded on a Bruker AVANCE 400 at operating frequencyof 400 and 10156MHz respectively Chemical shifts weremeasured with reference to the residual protons of thesolvent (DMSO-d

6 1H 250 ppm 13C 3952 ppm CDCl

3

1H 726 ppm 13C 7716 ppm) Coupling constants (J) aregiven in Hertz (Hz) The following abbreviations are usedto describe coupling s = singlet d = doublet t = tripletm = multiplet q = quartet br s = broad singlet qd =quartet of doublets AB = AB system Melting points weredetermined using a Stanford Research Systems MPA-100OptiMelt melting point apparatus and are uncorrected ForTLC analysis silica gel plates from Sorbfil (Krasnodar Rus-sia) were used with UV light (t 254 nm365 nm) or iron(III) chloride as developing agent Column chromatographywas performed on silica gel (60ndash200mesh) from Acros orreversed phase chromatography on chromatographPuriFlash450 (PF-15C18HP column Interchim) HRMS mass spectrawere obtained on a quadrupole time-of-flight (t qTOF) ABSciex Triple TOF 5600 mass spectrometer using turbo-ionspray source (nebulizer gas nitrogen a positive ionizationpolarity needle voltage 5500V) Recording of the spectrawas performed in ldquoTOF MSrdquo mode with collision energy10 eV declustering potential 100 eV and resolutionmore than30000 full-width half-maximum Samples with the analyteconcentration 5120583molL were prepared by dissolving the testcompounds in a mixture of methanol (HPLC-UV GradeLabScan) and water (LC-MS Grade Panreac) in 1 1 ratio[22]HPLC-MS analysis was performed usingAgilent ZorbaxEclipse Plus C18 column (21 times 100mm particle size 35120583m)with the flow rate of mobile phase 400 120583Lmin The HPLCpurity of compounds 4ab was gt98

Synthesis and analytical parameters of compounds2ab and 3a have been described in our previous paper[23] Fluconazole has been purchased from Sigma-Aldrich(USA)mdashSKU-Pack Size PHR1160-1G

6-(Bromomethyl)-8-methyl-2-propyl-4H-[13]dioxino[45-c]pyridin-5-yl)methanol (3b) To a solution of compound



3) 120575 099 (t 3H 3JHH =


2CH2CH3)


3Pyr)




3)

120575 1399 (s CH2CH2CH3) 1702 (s CH

2CH2CH3) 1816 (s

CH3Pyr) 3238 (s CH

2CH2CH3) 3626 (s CH

2Br) 5771

(s CH2) 6461 (s CH

2) 9995 (s CH) 12867 (s CPyr)


13H19BrNO

3



6) 120575 149 (s 12H 4CH

3) 222 (s 6H


2OH) 499 (s 4H

2CH2) 506 (br s 4H 2CH


2OH)



6) 120575 1829


3) 2447 (s CH

3) 4889 (s CH

2)

5506 (s CH2) 5698 (s CH

2) 5845 (s CH

2) 7284 (d



37H44F2N8O7




3) 120575 097 (t 12H 3JHH = 73Hz 4CH

2CH2CH3)



3Pyr) 228 (s 6H


4CH2) 481ndash501 (m 16H 4CH

2 4CH

2-OH 4CH) 504 514




3) 120575 1402 (s CH

2CH2CH3) 1704


3Pyr) 3624 (s CH

2CH2CH3)

5060 (s CH2) 5601 (s CH

2) 5730 (s CH

2) 5733 (s CH

2)

6469 (s CH2) 7334 (d 3JCF = 23Hz C-OH) 9981 (s



3891801

22 In Vitro Studies














10(119888)







2HPO4times

7H2O 40mM NaH





2CO3was



2 ndash 05 gL

P2X`3times 3X

2 ndash 14 gL PX

2`3ndash 6 gL (NH

4)2SO4





31 Results






1

OH

N

NF

F

N

N

N

N

HOO

O

O

O

OH

N

OHHO

HO

N

OH

Br

O

O

Fluconazole

N

OHO

O

OH

PPh3NBSCH2Cl2

R1

R2

R2

R2

R1

R1

R1

R2

2a R1= R2

= CH3[22]

b R1= H R2

= C3H7[22]

3a R1= R2

= CH3[22]

3b R1= H R2

= C3H7

4a R1= R2

= CH3

4b R1= H R2

= C3H7

N+Brminus

N+Brminus





Fluconazole


Terbinafine


4a


4b






4b

00

01

02

03

Biofi

lm O

D570








2[38] The MIC val-







Table2In

vitro

antib

acteria

lactivity

oftheo

btainedcompo

unds

4ab

andther

eference


Com

poun

ds

Gram

(+)

Gram

(minus)

Saureus

0fbb

29213

Bsubtilis

168

Sepidermidis

Mluteus

Ecoli

0fbb

25922

Kpn

eumoniae

Paeruginosa

0fbb

27853

Styphim

urium

TA100

4agt64

3232

nm

gt64

gt64

gt64

nm

4b05

28

416

3232

84b

+Ca

Cl2

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Flucon

azole

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Terbinafine

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Benzalkonium

chlorid

e4

28

416

gt64

328

Mira

mistin

21

44

8nm

644

lowastn mno

tmeasured


S aureus



4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570





00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570







S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



3) 120575 099 (t 3H 3JHH =


2CH2CH3)


3Pyr)




3)

120575 1399 (s CH2CH2CH3) 1702 (s CH

2CH2CH3) 1816 (s

CH3Pyr) 3238 (s CH

2CH2CH3) 3626 (s CH

2Br) 5771

(s CH2) 6461 (s CH

2) 9995 (s CH) 12867 (s CPyr)


13H19BrNO

3



6) 120575 149 (s 12H 4CH

3) 222 (s 6H


2OH) 499 (s 4H

2CH2) 506 (br s 4H 2CH


2OH)



6) 120575 1829


3) 2447 (s CH

3) 4889 (s CH

2)

5506 (s CH2) 5698 (s CH

2) 5845 (s CH

2) 7284 (d



37H44F2N8O7




3) 120575 097 (t 12H 3JHH = 73Hz 4CH

2CH2CH3)



3Pyr) 228 (s 6H


4CH2) 481ndash501 (m 16H 4CH

2 4CH

2-OH 4CH) 504 514




3) 120575 1402 (s CH

2CH2CH3) 1704


3Pyr) 3624 (s CH

2CH2CH3)

5060 (s CH2) 5601 (s CH

2) 5730 (s CH

2) 5733 (s CH

2)

6469 (s CH2) 7334 (d 3JCF = 23Hz C-OH) 9981 (s



3891801

22 In Vitro Studies














10(119888)







2HPO4times

7H2O 40mM NaH





2CO3was



2 ndash 05 gL

P2X`3times 3X

2 ndash 14 gL PX

2`3ndash 6 gL (NH

4)2SO4





31 Results






1

OH

N

NF

F

N

N

N

N

HOO

O

O

O

OH

N

OHHO

HO

N

OH

Br

O

O

Fluconazole

N

OHO

O

OH

PPh3NBSCH2Cl2

R1

R2

R2

R2

R1

R1

R1

R2

2a R1= R2

= CH3[22]

b R1= H R2

= C3H7[22]

3a R1= R2

= CH3[22]

3b R1= H R2

= C3H7

4a R1= R2

= CH3

4b R1= H R2

= C3H7

N+Brminus

N+Brminus





Fluconazole


Terbinafine


4a


4b






4b

00

01

02

03

Biofi

lm O

D570








2[38] The MIC val-







Table2In

vitro

antib

acteria

lactivity

oftheo

btainedcompo

unds

4ab

andther

eference


Com

poun

ds

Gram

(+)

Gram

(minus)

Saureus

0fbb

29213

Bsubtilis

168

Sepidermidis

Mluteus

Ecoli

0fbb

25922

Kpn

eumoniae

Paeruginosa

0fbb

27853

Styphim

urium

TA100

4agt64

3232

nm

gt64

gt64

gt64

nm

4b05

28

416

3232

84b

+Ca

Cl2

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Flucon

azole

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Terbinafine

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Benzalkonium

chlorid

e4

28

416

gt64

328

Mira

mistin

21

44

8nm

644

lowastn mno

tmeasured


S aureus



4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570





00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570







S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of












10(119888)







2HPO4times

7H2O 40mM NaH





2CO3was



2 ndash 05 gL

P2X`3times 3X

2 ndash 14 gL PX

2`3ndash 6 gL (NH

4)2SO4





31 Results






1

OH

N

NF

F

N

N

N

N

HOO

O

O

O

OH

N

OHHO

HO

N

OH

Br

O

O

Fluconazole

N

OHO

O

OH

PPh3NBSCH2Cl2

R1

R2

R2

R2

R1

R1

R1

R2

2a R1= R2

= CH3[22]

b R1= H R2

= C3H7[22]

3a R1= R2

= CH3[22]

3b R1= H R2

= C3H7

4a R1= R2

= CH3

4b R1= H R2

= C3H7

N+Brminus

N+Brminus





Fluconazole


Terbinafine


4a


4b






4b

00

01

02

03

Biofi

lm O

D570








2[38] The MIC val-







Table2In

vitro

antib

acteria

lactivity

oftheo

btainedcompo

unds

4ab

andther

eference


Com

poun

ds

Gram

(+)

Gram

(minus)

Saureus

0fbb

29213

Bsubtilis

168

Sepidermidis

Mluteus

Ecoli

0fbb

25922

Kpn

eumoniae

Paeruginosa

0fbb

27853

Styphim

urium

TA100

4agt64

3232

nm

gt64

gt64

gt64

nm

4b05

28

416

3232

84b

+Ca

Cl2

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Flucon

azole

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Terbinafine

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Benzalkonium

chlorid

e4

28

416

gt64

328

Mira

mistin

21

44

8nm

644

lowastn mno

tmeasured


S aureus



4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570





00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570







S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





2HPO4times

7H2O 40mM NaH





2CO3was



2 ndash 05 gL

P2X`3times 3X

2 ndash 14 gL PX

2`3ndash 6 gL (NH

4)2SO4





31 Results






1

OH

N

NF

F

N

N

N

N

HOO

O

O

O

OH

N

OHHO

HO

N

OH

Br

O

O

Fluconazole

N

OHO

O

OH

PPh3NBSCH2Cl2

R1

R2

R2

R2

R1

R1

R1

R2

2a R1= R2

= CH3[22]

b R1= H R2

= C3H7[22]

3a R1= R2

= CH3[22]

3b R1= H R2

= C3H7

4a R1= R2

= CH3

4b R1= H R2

= C3H7

N+Brminus

N+Brminus





Fluconazole


Terbinafine


4a


4b






4b

00

01

02

03

Biofi

lm O

D570








2[38] The MIC val-







Table2In

vitro

antib

acteria

lactivity

oftheo

btainedcompo

unds

4ab

andther

eference


Com

poun

ds

Gram

(+)

Gram

(minus)

Saureus

0fbb

29213

Bsubtilis

168

Sepidermidis

Mluteus

Ecoli

0fbb

25922

Kpn

eumoniae

Paeruginosa

0fbb

27853

Styphim

urium

TA100

4agt64

3232

nm

gt64

gt64

gt64

nm

4b05

28

416

3232

84b

+Ca

Cl2

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Flucon

azole

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Terbinafine

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Benzalkonium

chlorid

e4

28

416

gt64

328

Mira

mistin

21

44

8nm

644

lowastn mno

tmeasured


S aureus



4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570





00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570







S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


1

OH

N

NF

F

N

N

N

N

HOO

O

O

O

OH

N

OHHO

HO

N

OH

Br

O

O

Fluconazole

N

OHO

O

OH

PPh3NBSCH2Cl2

R1

R2

R2

R2

R1

R1

R1

R2

2a R1= R2

= CH3[22]

b R1= H R2

= C3H7[22]

3a R1= R2

= CH3[22]

3b R1= H R2

= C3H7

4a R1= R2

= CH3

4b R1= H R2

= C3H7

N+Brminus

N+Brminus





Fluconazole


Terbinafine


4a


4b






4b

00

01

02

03

Biofi

lm O

D570








2[38] The MIC val-







Table2In

vitro

antib

acteria

lactivity

oftheo

btainedcompo

unds

4ab

andther

eference


Com

poun

ds

Gram

(+)

Gram

(minus)

Saureus

0fbb

29213

Bsubtilis

168

Sepidermidis

Mluteus

Ecoli

0fbb

25922

Kpn

eumoniae

Paeruginosa

0fbb

27853

Styphim

urium

TA100

4agt64

3232

nm

gt64

gt64

gt64

nm

4b05

28

416

3232

84b

+Ca

Cl2

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Flucon

azole

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Terbinafine

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Benzalkonium

chlorid

e4

28

416

gt64

328

Mira

mistin

21

44

8nm

644

lowastn mno

tmeasured


S aureus



4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570





00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570







S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




4b

00

01

02

03

Biofi

lm O

D570








2[38] The MIC val-







Table2In

vitro

antib

acteria

lactivity

oftheo

btainedcompo

unds

4ab

andther

eference


Com

poun

ds

Gram

(+)

Gram

(minus)

Saureus

0fbb

29213

Bsubtilis

168

Sepidermidis

Mluteus

Ecoli

0fbb

25922

Kpn

eumoniae

Paeruginosa

0fbb

27853

Styphim

urium

TA100

4agt64

3232

nm

gt64

gt64

gt64

nm

4b05

28

416

3232

84b

+Ca

Cl2

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Flucon

azole

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Terbinafine

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Benzalkonium

chlorid

e4

28

416

gt64

328

Mira

mistin

21

44

8nm

644

lowastn mno

tmeasured


S aureus



4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570





00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570







S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Table2In

vitro

antib

acteria

lactivity

oftheo

btainedcompo

unds

4ab

andther

eference


Com

poun

ds

Gram

(+)

Gram

(minus)

Saureus

0fbb

29213

Bsubtilis

168

Sepidermidis

Mluteus

Ecoli

0fbb

25922

Kpn

eumoniae

Paeruginosa

0fbb

27853

Styphim

urium

TA100

4agt64

3232

nm

gt64

gt64

gt64

nm

4b05

28

416

3232

84b

+Ca

Cl2

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Flucon

azole

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Terbinafine

gt64

gt64

gt64

nm

gt64

gt64

gt64

nm

Benzalkonium

chlorid

e4

28

416

gt64

328

Mira

mistin

21

44

8nm

644

lowastn mno

tmeasured


S aureus



4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570





00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570







S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


S aureus



4b





4b



4b

S epidermidis

E coli

4b

P aeruginosa

00

01

02

03

04

05Bi

ofilm

OD

570





00

02

04

06

08

10

12

Biofi

lm O

D570

00

02

04

06

08

10

Biofi

lm O

D570

00

02

04

06

08

10Bi

ofilm

OD

570







S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


S aureus

Concentration xMBC


4b

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)

1x0 2x 4x 8x 16x

(a)


4b

S epidermidis

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


(b)

Ecoli

100

101

102

103

104

105

106

107

108

109


4b


Viab

le ce

lls (C

FUm

l)

(c)

P aeruginosa

100

101

102

103

104

105

106

107

108

109

Viab

le ce

lls (C

FUm

l)


4b


(d)


32 Discussion






















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















HFCCC50MBC

HEK-293CC50 120583gmL

HEK-293CC50MBC

4a 274 plusmn 124 1073 plusmn 438























50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

















50value


50MBC ratio of 005



50MBC for both






4 Conclusion





Acknowledgments



References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


References






































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

fluconazole-pyridoxine bis-triazolium compounds with potent...

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