© 2016 Taylor & Francis Group, LLC.The purpose of the present study was to investigate the antibacterial activity of novel alkyl esters of carboxylate phosphabetaine: β-(carboxyalkyl)ethyltributylphosphonium bromides 4–8. The in vitro microbiological activity of the synthesized phosphonium bromides against gram-positive, gram-negative bacteria and the yeast Candida albicans was determined in comparison to standard agents. Microbiological results indicate the synthesized phosphonium salts possess a broad spectrum of activity against the testedmicroorganisms. Every newly synthesized compound was characterized by elemental analyses, IR, 1H NMR, 31P NMR spectral studies

Aksunova A.

Bakhtiyarov D.

Bakhtiyarova Y.

Egorova S.

Galkin V.

Galkina I.

Pozdeev O.

Shulaeva M.

Phosphorus Sulfur and Silicon and the Related Elements

Antimicrobial evaluation of novel carboxylate phosphabetaines derivatives with long alkyl chain

Bakhtiyarova Yuliya Valerevna

Aksunova Alfiya Faritovna

Galkin Vladimir Ivanovich

Galkina Irina Vasilevna

Egorova Svetlana Nikalaevna

Pozdeev Oskar Kimovich

Shulaeva Marina Petrovna

Bahtiyarov Dmitrij Ilgizarovich

Kazan Federal University Digital Repository

Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=gpss20Download by: [178.205.215.228] Date: 30 August 2016, At: 06:26Phosphorus, Sulfur, and Silicon and the Related ElementsISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20Synthesis and Biological Evaluation of NovelCarboxylate Phosphabetaines Derivatives withLong Alkyl ChainsIrina V. Galkina, Alfiya F. Aksunova, Dmitriy I. Bakhtiyarov, Marina P.Shulaeva, Oskar K. Pozdeev, Svetlana N. Egorova, Yuliya V. Bakhtiyarova &Vladimir I. GalkinTo cite this article: Irina V. Galkina, Alfiya F. Aksunova, Dmitriy I. Bakhtiyarov, Marina P.Shulaeva, Oskar K. Pozdeev, Svetlana N. Egorova, Yuliya V. Bakhtiyarova & Vladimir I. Galkin(2016): Synthesis and Biological Evaluation of Novel Carboxylate Phosphabetaines Derivativeswith Long Alkyl Chains, Phosphorus, Sulfur, and Silicon and the Related Elements, DOI:10.1080/10426507.2016.1227821To link to this article:  http://dx.doi.org/10.1080/10426507.2016.1227821Accepted author version posted online: 29Aug 2016.Published online: 29 Aug 2016.Submit your article to this journal View related articles View Crossmark dataACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 1 Synthesis and Biological Evaluation of Novel Carboxylate Phosphabetaines Derivatives with Long Alkyl Chains Irina V. Galkina,1 Alfiya F. Aksunova,1 Dmitriy I. Bakhtiyarov,1 Marina P. Shulaeva,2  Oskar K. Pozdeev,2 Svetlana N. Egorova,2 Yuliya V. Bakhtiyarova,1 Vladimir I. Galkin1  1Kazan Federal University, Kazan, Russia.  2Kazan State Medical University, Kazan, Russia.   Address correspondence to name Irina V. Galkina e-mail vig54@mail.ru   GRAPHICAL ABSTRACT   Abstract The purpose of the present study was to investigate the antibacterial activity of novel alkyl esters of carboxylate phosphabetaine: β-(carboxyalkyl)ethyltributylphosphonium bromides 4-8. The in vitro microbiological activity of the synthesized phosphonium bromides against gram-positive, gram-negative bacteria and the yeast Candida albicans was determined in comparison to standard agents. Microbiological results indicate the synthesized phosphonium salts possess a broad spectrum of activity against the tested microorganisms. Every newly synthesized compound was characterized by elemental analyses, IR, 1H NMR, 31P NMR spectral studies. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2  Keywords Phosphabetaines; long alkyl chain; antimicrobial activity   ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 3  INTRODUCTION  Demand for new antimicrobial agents is high because more microorganisms develop resistance against drugs currently available on the market. Resistance of pathogenic bacteria to antibiotics is rapidly becoming a major problem in the medical community and hospital-based healthcare settings. The search for novel agents to combat resistant bacteria has become one of the most important areas of antibacterial research today1,2. Pharmaceutical and organic chemists are trying to synthesize new drugs with better pharmacokinetic and dynamic properties. We have earlier described the methods of preparation various phosphabetaines based on tertiary phosphines and unsaturated carboxylic acids and reported on their structure and reactivity3-7. In this study, we prepared tributyl-substituted phosphonium salts 4-8 on the basis of phosphabetaines (1) containing alkyl chains of various lengths. The synthesis of such phosphonium salts is more difficult in comparison with ammonium analogs8,9. Betaines easily react with alkyl halogenides with short alkyl chains to form the corresponding phosphonium salts without biological activity10,11. RESULTS AND DISCUSSION  Chemistry  In this paper we present the synthesis and biological activity of a series of nanosized (30 nm) quaternary phosphonium salts 4-8 with long alkyl chains (R = CnH2n+1; n = 10, 12, 14, 16 18; here n is the number of carbon atoms in alkyl groups) on the basis of phosphabetaine 1 and ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 4 higher alkyl halogenides. All the synthesized compounds were characterized by elemental analysis, IR, 1H NMR, and 31P NMR spectroscopy. The synthetic routes are given in Scheme 1. Scheme 1 Depicts the synthesis of β-(carboxyalkyl)ethyltributylphosphonium bromides (4-8). Treatment of acrylic acid with tributylphosphine at room temperature in chloroform during 6 hours yielded (87%) phosphabetaine 1. Alkylation of the starting phosphabetaine 1 – β-tributylphosphonium ethylcarboxylate with alkyl halogenides (reflux for 14 hours in CH3Cl) gave the corresponding phosphonium bromides 4-8 with long alkyl chains. The yield was 52%-72%.   Biological results  The synthesized compounds – a new class of bioactive nanomolecules – were screened for antibacterial and antifungal activity at 50µ/0.1mL concentration by using the cup-plate agar diffusion method, and standard drugs used were Chlorhexidine 9, Penicillin 10, and Griseofulvin 11. The novel synthesized compounds 4-8 with long alkyl chains (n = 10, 12, 14, 16, 18) show maximal activity against pathogenic microorganisms. Starting phosphabetaine 1 and all phosphonium salts with shot alkyl chains, synthesized earlier10,11, were not active at all. Compound 6 and 7 were highly significant against tested bacteria as well as fungi. Our results are reported in Table 1.  Such a high biological activity of cationic biocides 4-8 we explain by their ability to be integrated into the lipid layers of biomembranes of pathogenic microflora eventually leading to the destruction of this last. To confirm this idea we studied the interaction mechanism of β-(carboxyalkyl)ethyltriphenylphosphonium bromides – analogs of compounds 4-8 and phosphorus ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 5 analogs synthetic of biomembranes – with natural biological membranes (lecithin) using the model of Langmuir monolayers11. It was discovered that alkylated phosphabetaines interact with lecithin, by forming a pores, and thus deteriorating the membrane functions.    Many years we thought that it is impossible to grow single crystals of oil products with long alkyl chains suitable for X-ray diffraction, but after five years we have a real chance to obtain the crystalline structure of the β-(carboxyhexadecyl)ethyltriphenylphosphonium bromide, which gave good quality crystals3 (Figure 1). CONCLUSIONS In conclusion, carboxylate phosphabetaines derivatives with alkyl chains of various lengths were synthesized in good yield, characterized by different spectral studies, and their antimicrobial activity has been evaluated. Compounds 6 and 7 demonstrated good inhibitions against all the strains tested comparable to Chlorhexidine, Penicillin and Griseofulvin as positive standard. So, it may be concluded from our results that the synthesized compounds are potent nanoantimicrobial agents against pathogenic bacteria and fungi.  EXPERIMENTAL  Chemistry Materials and Methods All chemicals purchased from Sigma-Aldrich were reagent grade and used without purification. Analytical data were obtained from Perkin Elmer 2400 LS. And were found within ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 6 + 0.4% of the theoretical values. IR spectra were taken on a spectrophotometer Specord 251R in the range 400-3700 cm-1 from mulls in mineral oil between KBr plates. 1H NMR (D2O) and 31P NMR (DMSO-d6) spectra were determined on a Bruker Avance digital spectrometer 400.13 MHz. Chemical shifts were determined with respect to external reference, 85% H3PO4. The purity and thermal stability of crystal compounds were determined by simultaneous TG/DSC analysis on a NETZSCH STA 449C instrument (temperature range 20-400oC, heating rate 10 deg/min, argon atmosphere). Synthesis of tributyl-(3-alkoxy-3-oxopropyl)-phosphonium bromides (4-8)  General Procedure. A mixture of equimolar quantities of 3-(tributylphosphonio) propanoate 1 (0.01 mol) and appropriate alkyl halogenides (0.01 mol) was refluxed in dry chloroform (75 ml) for 1 h. Excess of solvent was removed under reduced pressure. The resulting salts 4-8 were obtained as yellow oils and purified by diethyl ether from starting reagents.  Preparation of tributyl-(3-decyloxy-3-oxopropyl)-phosphonium bromide (4). Yield 67%. IR (cm-1): 1720, 1125. 1H NMR (D2O 400.13 MHz): 0.78-0.93 (m, 12H, 4Me), 1.23-1.45 (m, 26H, 13CH2), 1.58-1.65 (m, 2H, OCCH2), 2.35-2.41 (m, 2H, PCCH2), 2.48-2.87 (m, 8H, PCH2), 4.13-4.23 (m, 2H, OCH2). 31P NMR (DMSO-d6, 161.9 MHz): 35.4. Anal (%) Calc. for C25H52BrO2P: C, 60.61; H 10.51. Found: C, 59.97; H, 10.73. (FW=494.9). Preparation of tributyl-(3-dodecyloxy-3-oxopropyl)-phosphonium bromide (5). Yield 60%. IR (cm-1): 1721, 1121. 1H NMR (D2O, 400.13 MHz): 0.75-0.97 (m, 12H, 4Me), 1.25-1.43 (m, 30H, 15CH2), 1.60-1.69 (m, 2H, OCCH2), 2.31-2.41 (m, 2H, PCCH2), 2.47-2.85 (m, 8H, PCH2), 4.11-4.25 (m, 2H, OCH2); 31P NMR (DMSO-d6, 161.9 MHz): 35.2. Anal (%) Calc. for C27H56BrO2P (FW=522.9): C 61.95; H 10.71. Found: C, 60.35; H, 10.39. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 7 Preparation of tributyl-(3-tetradecyloxy-3-oxopropyl)phosphonium bromide (6). Yield 52%. IR (cm-1): 1720, 1123. 1H NMR (D2O, 400.13 MHz): 0.81-0.91 (m, 12H, 4Me), 1.20-1.44 (m, 34H, 17CH2), 1.57-1.67 (m, 2H, OCCH2), 2.37-2.43 (m, 2H, PCCH2), 2.45-2.97 (m, 8H, PCH2), 4.13-4.23 (m, 2H, OCH2); 31P NMR (DMSO-d6, 161.9 MHz): 35.5. Anal (%) Calc. for C29H60BrO2P: C, 63.16, H; 10.89. Found: C, 63.70; H, 10.13. (FW=550.9). Preparation of tributyl-(3-hexadecyloxy-3-oxopropyl)phosphonium bromide (7). Yield 47%. IR (cm-1): 1719, 1120. 1H NMR (D2O, 400.13 MHz): 0.73-0.92 (m, 12H, 4Me), 1.13-1.45 (m, 38H, 19CH2), 1.52-1.61 (m, 2H, OCCH2), 2.31-2.38 (m, 2H, PCCH2), 2.43-2.93 (m, 8H, PCH2), 4.13-4.23 (m, 2H, OCH2); 31P NMR (DMSO-d6, 161.9 MHz): 35.0. Anal (%) Calc. for C31H64BrO2P (FW=579): C, 64.25; H, 11.05. Found: C, 65.11; H, 11.66. Preparation of tributyl-(3-octadecyloxy-3-oxopropyl)phosphonium bromide (8). Yield 72%. IR (cm-1): 1720, 1125. 1H NMR (D2O, 400.13 MHz): 0.77-0.97 (m, 12H, 4Me), 1.19-1.47 (m, 42H, 21CH2), 1.59-1.65 (m, 2H, OCCH2), 2.33-2.39 (m, 2H, PCCH2), 2.45-2.86 (m, 8H, PCH2), 4.13-4.23 (m, 2H, OCH2); 31P NMR (DMSO-d6, 161.9 MHz): 35.3. Anal (%) Calc. for C33H68BrO2P (FW=606.9): C, 65.24; H, 11.20; found C, 65.87; H, 11.11. Biological results The antimicrobial activity of the newly synthesized compounds was determined in vitro using the agar disk-diffusion method using Mueller-Hilton agar medium against a variety of pathogenic microorganisms: Staphylococcus aureus (ATCC 29213) (Gram-positive bacteria), Escherichia coli (ATCC 25922), Pseudomonas aeruginoza (ATCC 27853), Bacillus cereus (ATCC 11778) and Proteus mirabilis (ATCC 12453) (Gram-negative bacteria) and fungus Candida albicans (ATCC 885-653). The inhibition zones of the tested compounds were ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 8 measured after 24-48 h incubation at 37oC for bacteria and after 5 days of incubation at 28oC for fungi. Penicillin (Sigma-Aldrich) and Chlorhexidine (Sigma-Aldrich) were used as reference drug for bacteria, whereas Griseofulvin (Sigma-Aldrich) was used as reference drug for fungi. Every experiment in the antibacterial and antifungal assay was replicated twice. For the antibacterial and antifungal activity, the compounds were dissolved in dimethylsulfoxide (DMSO). The antifungal activity was carried out by using cup-plate method using cup-plate method using Sabouraud’s agar medium. Fungal strains used were Candida albicans (ATCC 885-653) with incubation period of 48 hours at temperature 28oC. The standard drug used was Griseofulvin (50 µg/0.1 mL) and the test compounds at concentration of 50 µg/0.1 mL by using dimethyl sulfoxide (DMSO). ACKNOWLEDGEMENTS This work was funded by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities.   ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 9  REFERENCES  1. Aysel, G.; Taylan, I.; Nalan, T.; and Gulten, O. Turkish Journal of Pharmaceutical Sciences, 2005, 2 (1), 134-136. 2. Costerton, J.W.; Cheng, K.-J. Journal of Antimicrob Chemother, 1975, 1, 363-377. 3. Galkina, I.; Bakhtiyarova, Yu.; Shulaeva, M.; Pozdeev, O.; Egorova, S.; Myrzakhmetova, N.; Cherkasov, R.; Galkin, V. Journal of Chemistry, 2013, ID 302937. 4. Galkin, V.I.; Bakhtiyarova, Yu.V.; Polezhaeva, N.A.; Shaikhutdinova, R.A.; Klochkov, V.V. Zhurnal Obshchei Khimii (Russian Journal of General Chemistry), 1998, 68, 1104-1108. 5. Galkin, V.I.; Bakhtiyarova, Yu.V.; Polezhaeva, N.A.; Cherkasov, R.A.; Krivolapov, D.B.; Gubaidullin, A.T.; Litvinov, I.A. Phosphorus, Sulfur Silicon Relat. Elem., 1999, 144-146, 918-920. 6. Galkin, V.I.; Bakhtiyarova, Yu.V.; Zaripova, A.N.; Galkina, I.V.; Cherkasov, R.A.; Krivolapov, D.B.; Gubaidullin, A.T.; Litvinov I.A.; Pudovik, A.N. Phosphorus, Sulfur, and Silicon and Related Elements, 2002, 177, 2063-2065. 7. Galkin, V.I.; Bakhtiyarova, Yu.V.; Sagdieva, R.I.; Galkina, I.V.; Cherkasov, R.A. Heteroatom Chemistry, 2006, 17(6), 557-565. 8. Kourai, H.; Horie, T.;Takeichi, K. Journal of Antibacterial and Antifungal Agents, 1980, 8, 9-17. 9. Kanazava, A.; Ikeda, T.; Endo, T. Antimicrobial Agents and Chemotherapy, 1994, 38 (5), 945-952. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 10 10. Galkin, V.I.; Bakhtiyarova, Yu.V.; Polezhaeva, N.A.; Galkina, I.V.; Cherkasov, R.A.; Krivolapov, D.B;. Gubaidullin, A.T.; Litvinov. I.A. Zhurnal Obshchei Khimii (Russian Journal of General Chemistry), 2002, 72, 404-411. 11. Galkina, I.V.; Melnikova, N.B.; Bakhtiyarova, Yu.V.; Stakheev, V.V.; Galkin, V.I.; Zhiltsova, O.Ye.; Zhukova, O.V.; Egorova. S.N. Pharmaciya (Russian Journal of Farmacy), 2010, 3, 47-49.   ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 11   Table 1. Antimicrobial activity of the newly synthesized compounds and the control drugs  (50 μg/0.1 mL) Zone of inhibition (mm) n Compound Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Bacillus cereus Proteus mirabilis Candida albicans 4 Bu3PCH2CH2COOC10H21Br  16 17 9 14 13 25 5 Bu3PCH2CH2COOC12H25Br  21 11.5 14 11 15 23.5 6 Bu3PCH2CH2COOC14H29Br  26 20 15 15 17.5 31 7 Bu3PCH2CH2COOC16H33Br  27.5 21 17 17.5 21 37 8 Bu3PCH2CH2COOC18H37Br  14 13 11 8 14 20.5 9 Chlorhexidine 16 15 13 13 15 14 10 Penicillin 20 17 11 7 10 - 11 Griseofulvin - - - - - 20  n: Compound number All tests were performed in triplicate. Zone of inhibition 22 to 31: highly significant, between 15 to 21 mm: less significant, below 14 mm: poor active.    ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 12  Figura 1. Molecular structure of the asymmetric unit of the Ph3PCH2CH2C(O)OC16H33 in crystal.   ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 13 4-8R = C10H21    (4)R = C12H25    (5)R = C14H29    (6)CH2=CHCOOHBu3P, CHCl320oC, 6 hBu3PCH2CH2COOCnH2n+1 Br, CHCl3reflux, 14 hBu3PCH2CH2COORBrR = C16H33    (7)R = C18H37    (8)1  Scheme 1. Synthetic routes of β-(carboxyalkyl)ethyltributylphosphonium bromides 4-8; reagents and conditions.   

Synthesis and biological evalution of novel carboxylate phosphabetaines derivatives with long alkyl chains

Phosphorus, Sulfur and Silicon and the Related Elements 2016 vol.191 N11-12, pages 1676-1678Synthesis and biological evaluation of novel carboxylatephosphabetaines derivatives with long alkyl chainsGalkina I., Aksunova A., Bakhtiyarov D., Shulaeva M., Pozdeev O., Egorova S., Bakhtiyarova Y.,Galkin V.Kazan Federal University, 420008, Kremlevskaya 18, Kazan, RussiaAbstract© 2016 Taylor & Francis Group, LLC.The purpose of the present study was to investigate theantibacterial  activity  of  novel  alkyl  esters  of  carboxylate  phosphabetaine:  β-(carboxyalkyl)ethyltributylphosphonium bromides 4–8. The in vitro microbiological activity of thesynthesized  phosphonium bromides  against  gram-positive,  gram-negative  bacteria  and  theyeast  Candida  albicans  was  determined in  comparison to  standard  agents.  Microbiologicalresults indicate the synthesized phosphonium salts possess a broad spectrum of activity againstthe testedmicroorganisms. Every newly synthesized compound was characterized by elementalanalyses, IR, 1H NMR, 31P NMR spectral studies.http://dx.doi.org/10.1080/10426507.2016.1227821Keywordsantimicrobial activity, long alkyl chain, Phosphabetaines