Phosphorus–nitrogen compounds: Part 19. Syntheses, structural and electrochemical investigations, biological activities, and DNA interactions of new spirocyclic monoferrocenylcyclotriphosphazenes

Polyhedron 29 (2010) 2933–2944

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Phosphorus–nitrogen compounds: Part 19. Syntheses, structural and electrochemical investigations, biological activities, and DNA interactions of new spirocyclic monoferrocenylcyclotriphosphazenes a,* _ Elif Ece Ilter , Nuran Asmafiliz b, Zeynel Kılıç b, Leyla Açık c, Makbule Yavuz c, E. Burcu Bali c, Ali Osman Solak b, Fevziye Büyükkaya b, Hakan Dal d, Tuncer Hökelek e _ TÜBITAK, Scientific and Technical Research Council of Turkey, 06540 Çankaya, Ankara, Turkey Department of Chemistry, Ankara University, 06100 Tandog˘an, Ankara, Turkey c Department of Biology, Gazi University, 06500 Teknikokullar, Ankara, Turkey d Department of Chemistry, Anadolu University, 26470 Eskisßehir, Turkey e Department of Physics, Hacettepe University, 06800 Beytepe, Ankara, Turkey a

b

a r t i c l e

i n f o

Article history: Received 19 April 2010 Accepted 17 July 2010 Available online 22 July 2010 Keywords: DNA interactions DNA cleavage Antituberculosis activity Ferrocenylphosphazenes Crystal structure Electrochemistry

a b s t r a c t The reactions of hexachlorocyclotriphosphazatriene, N3P3Cl6, with N-alkyl-N-ferrocenylmethylethylene diamines, FcCH2NH(CH2)2NHR1 [R1 = Me (1) and Et (2)], and sodium [3-(N-ferrocenylmethylamino)-1propanoxide] (3) produce spirocyclic monoferrocenyl tetrachlorophosphazenes (1a–3a). The tetrapyrrolidinophosphazenes (1b–3b) are prepared from the reactions of corresponding phosphazenes (1a–3a) with excess pyrrolidine. The reaction of 1a with excess morpholine affords geminal-morpholino phosphazene (1c), whilst the reactions of 2a and 3a give diethylaminotrimorpholino (2c) and fully substituted morpholino products (3c), respectively. The structural investigations of the compounds are examined by Fourier transform IR, MS, 1H, 13C, 31P NMR, DEPT, HETCOR, and HMBC techniques. The crystal structures of 3b and 3c are determined using X-ray crystallography. Cyclic voltammetric and chronoamperometric data show that compounds 1a–3a, 1b–3b, and 1c–3c exhibit electrochemically reversible one-electron oxidation of Fc redox centers which are hardly affected by the substituents on the phosphazene ring. The compounds 1b, 2b, 3b, and 3c are screened for antibacterial activities against Gram-positive and Gram-negative bacteria and for antifungal activities against yeast strains. In addition, the antituberculosis activities (in vitro) of these compounds are evaluated against INH-susceptible reference strain M. tuberculosis H37Rv, and six multi-drug resistant clinical M. tuberculosis isolates. Compound 2b is found to be the most active against the susceptible the reference strain. In addition, 1b, 2b, and 3c are active against all the multidrug-resistant clinical isolates at the highest concentrations. Gel electrophoresis data indicate that the compounds promote the formation of strand breaks in plasmid DNA. Almost all the concentrations lost of supercoiled DNA suggests that the compound 3b is very efficient plasmid-modifier. The compounds inhibit BamHI cleavage of pUC18 DNA while restricting HindIII. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Cyclophosphazenes, which cover a broad range of molecules, are an important family of inorganic ring systems, all of which contain phosphorus and nitrogen atoms linked by regularly unsaturated bonds [1–5]. The N3P3Cl6 is the best known and standard compound in the field of phosphazene chemistry. Most of the phosphazene derivatives are obtained by nucleophilic substitution reactions on N3P3Cl6 due to the ease of introducing a wide variety of different functional inorganic and organic groups onto P centers [6,7]. Cyclophosphazenes are used as building blocks for polyorg* Corresponding author. Tel.: +90 3124685300x2223; fax: +90 3124677298. _ E-mail address: [email protected] (E.E. Ilter). 0277-5387/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2010.07.017

anophosphazenes and dendrimers [3,8]. In addition, phosphazene derivatives are attracting increased interest in various areas of technological and medicinal importance [3,4]; such as the production of lubricants [9], inflammable textile fibers and elastomers [3,10], rechargeable lithium batteries [11,12], anticancer agents [13], antibacterial reagents [14], synthetic bones [15,16], and membranes [17]. Ferrocene (Fc) derivatives are also very important compounds for photochemistry [18,19] and organometallic chemistry [20,21], since they are good candidates for producing novel materials possessing interesting chemical, electrical, optical, and magnetic properties [20]. These compounds can be thought of as excellent electron-transfer mediators, redox-active probe materials for the modification of electrodes [22], and materials for non-linear optic

_ E.E. Ilter et al. / Polyhedron 29 (2010) 2933–2944

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culosis isolates; (vi) investigations of antibacterial and antifungal activities of 1b–3b and 3c; and (vii) interactions between these compounds and pUC18 plasmid DNA.

devices [23,24]. Additionally, the biological activities of ferrocenyldiamines against Mycobacterium tuberculosis H37Rv are known [25]. Throughout the world, approximately eight million people develop active tuberculosis every year and two millions die as a result of infection; this situation has only deterioted since the emergence of multidrug-resistant tuberculosis [26,27]. The increasing problem of multidrug-resistant tuberculosis has focused consideration on developing new drugs that are not only active against drug-resistant tuberculosis, but also shorten the length of therapy. Therefore, there is an immediate need and an important interest in developing new tuberculosis drugs [28]. The DNA binding abilities of compounds can be examined by agarose gel electrophoresis. The principle behind this method is that DNA molecules migrate in the agarose gel as functions of their masses and shapes, with supercoiled DNA (form I) migrating faster than open circular molecules (form II) of the same mass [29]. The geometry of supercoiled plasmid DNA molecule may be altered, which affects the intrinsic twisting of the DNA helix [30]. As far as we are aware, there are a limited number of reports on the reactions of monofunctional and difunctional reagents containing pendant Fc groups [31–35]. Thus, this study primarily focuses on the substitution reactions of N3P3Cl6 with ferrocenylamines with the aim of preparation of electron-rich reservoir complexes. We report here (i) the synthesis of new mono ferrocenylphosphazenes (1a–3a, 1b–3b, and 1c–3c) (Scheme 1), (ii) the determination of the structures of compounds by elemental analyses, mass spectrometry, Fourier transform (FTIR), one-dimensional (1D) 1H, 13C, and 31P NMR, distortionless enhancement by polarization transfer (DEPT), two-dimensional (2D) correlation spectroscopy (COSY), heteronuclear shift correlation (HETCOR), and heteronuclear multiple-bond correlation (HMBC) techniques; (iii) the solid-state structures of 3b and 3c established by X-ray diffraction techniques; (iv) the electrochemical behavior and structural correlations of ferrocenylphosphazene derivatives, (v) the biological activities of 1b–3b and 3c against Mycobacterium tuberculosis H37Rv (ATCC 27294) reference strain and multidrug-resistant clinical M. tuber-

2. Experimental 2.1. Reagents Hexachlorocyclotriphosphazatriene (Aldrich), ferrocenecarboxaldehyde (Aldrich), aliphatic amines (Fluka), pyrrolidine (Fluka), morpholine (Fluka) and 3-amino-1-propanol (Fluka) were purchased and used without further purification. THF was dried over 3 Å molecular sieves. All reactions were monitored using thin-layer chromatography in different solvents and chromatographed using silica gel. All experiments were carried out in an argon atmosphere. 2.2. Instruments The melting points were measured on a Gallenkamp apparatus using a capillary tube. 1H, 13C, and 31P NMR, DEPT, HETCOR, and HMBC spectra were recorded on a Bruker DPX FT-NMR (500 MHz) spectrometer (SiMe4 as internal and 85% H3PO4 as external standards). The spectrometer was equipped with a 5 mm PABBO BB inverse-gradient probe. Standard Bruker pulse programs [36] were used. The IR spectra were recorded on a Mattson 1000 FTIR spectrometer in KBr disks and were reported in cm1 units. APIES mass spectrometric analyses were performed on an Agilent 1100 MSD spectrometer. All electrochemical experiments were performed using a BAS-100B electrochemical analyzer (Bioanalytical System Inc., Lafayette, IL, USA) and Gamry Reference 600 workstation (Gamry, USA) both equipped with BAS C3 cell stands. Mycobacterial susceptibility testing was performed by the agar-well diffusion method [37] (Supplementary material, Section S1). Antituberculosis activity against the reference strain M. tuberculosis H37Rv (ATCC 27294) was carried out (Supplementary material, Section S2). The DNA binding abilities were examined using

Cl

Cl P N

N

P

P

Cl Cl

Cl Cl

N

Fc CH 2

THF

NH

XR1

X NH NH

R1 Me Et

OH

-

n 0 0 1

Compound 1 2 3

( ) N

XR1

N

N

P

P

HN Cl

Cl N

X

R1

-

1 5 CH2 2

4

Fe

n

Fc CH2 N

P

Cl

Fc-CH2 : 3

( )

n Fc CH2

2

3 ( ) n

O

or

P

HN R3

THF Cl n 0 0 1

N

N

P

P

R4 Compound 1a 2a 3a

XR1

N

R2 R2

X N N

R1 Me Et

R2 C4H8N C 4H8N

R3 C4H8N C4H8N

R4 C4H8N C4H8N

n 0 0

Compound 1b 2b

O N N

-

Me Et

C4H8N OC4H8N OC 4H8N

C4H8N Cl OC4H8N

C4H8N Cl N(C2H5)2

1 0 0

3b 1c 2c

OC4H8N

OC4H8N

OC4H8N

1

3c

O

-

Scheme 1. The chloride replacement reaction pathway of N3P3Cl6 with amines.

_ E.E. Ilter et al. / Polyhedron 29 (2010) 2933–2944

agarose gel electrophoresis (Supplementary material, Section S3) [38,39]. 2.3. Preparation of compounds 3-(N-ferrocenylmethylamino)-1-propanol (3) was obtained from the reaction of ferrocenecarboxaldehyde with 3-amino-1propanol according to the methods reported in the literature [42]. 2.3.1. N-(1-ferrocenylmethyl)-N-methyl-ethylenediamine (1) A solution of ferrocenecarboxaldehyde (3.00 g, 14.02 mmol) in methanol (100 mL) was added to 1.04 g of N-methyl-ethylenediamine (14.02 mmol) in methanol (50 mL). The mixture was refluxed for 3 h and then the excess sodium borohydride (1.59 g, 42.06 mmol) was added. The crude product was extracted with dichloromethane (150 ml, three times). Yield: 3.52 g (92%) and m.p.: 76–77 °C. Anal. Calc. for C14H20N2Fe: 61.78; H, 7.41; N, 10.29. Found: C, 61.53; H, 7.72; N, 10.17%. FTIR (KBr, cm1): m 3374; 3224 (N–H), 3091; 3065 (C–H arom.), 2939; 2852 (C–H aliph.). 2.3.2. N-(1-ferrocenylmethyl)-N-ethyl-ethylenediamine (2) A solution of ferrocenecarboxaldehyde (3.00 g, 14.02 mmol) in methanol (100 mL) was added to 1.47 g of N-ethyl-ethylenediamine (14.02 mmol) in methanol (50 mL). The mixture was refluxed for 3 h and then the excess sodium borohydride (1.59 g, 42.06 mmol) was added. The crude product was extracted with dichloromethane (150 ml, three times). Yield: 3.75 g (93%). M.p.: 95 °C. Anal. Calc. for C15H22N2Fe: C, 62.95; H, 7.75; N, 9.79. Found: C, 62.53; H, 7.72; N, 9.47%. FTIR (KBr, cm1): m 3311; 3224 (N–H), 3093; 3078 (C–H arom.), 2966; 2806 (C–H aliph.). 2.3.3. Spiro(ethane-1,2-diamino)[N-(1-ferrocenylmethyl)-N-methyl]4,4,6,6-tetrachlorocyclotriphosphazatriene (1a) A solution of 1 (3.80 g, 14.00 mmol) in THF (150 mL) and triethylamine (3.94 mL) was added to a stirred solution of N3P3Cl6 (4.86 g, 14.00 mmol) in THF (50 mL) at room temperature. The mixture was stirred for 25 h and the precipitated triethylaminehydrochloride was filtered off. The solvent was evaporated and the product purified by column chromatography with toluene. An orange powder was crystallized from n-heptane. Yield: 4.75 g (62%) and m.p.: 142 °C. Anal. Calc. for C14H18N5FeP3Cl4: C, 30.75; H, 3.32; N, 12.81. Found: C, 30.70; H, 3.45; N, 12.67%. APIES-MS (fragments are based on 35Cl and 56Fe, Ir%): m/z 545 ([M]+, 85.0). FTIR (KBr, cm1): 3094; 3070 (C–H arom.), 2975; 2858 (C–H aliph.), 1225; 1178 (P@N), 564;527 (PCl). 2.3.4. Spiro(ethane-1,2-diamino)[N-(1-ferrocenylmethyl)-N-ethyl]4,4,6,6-tetrachlorocyclotriphosphazatriene (2a) The work-up procedure was similar to that of compound 1a, using 2 (2.42 g, 8.45 mmol) and N3P3Cl6 (2.94 g, 8.45 mmol) and triethylamine (2.38 mL). The product was purified by column chromatography using benzene and crystallized from n-hexane. Yield: 3.00 g (63%) and m.p.: 125 °C. Anal. Calc. for C15H20N5FeP3Cl4: C, 32.12; H, 3.59; N, 12.49. Found: C, 32.15; H, 3.54; N, 12.50%. APIES-MS (fragments are based on 35Cl and 56Fe, Ir%): m/z 559 ([M]+, 8.5). FTIR (KBr, cm1): m 3092; 3073 (C–H arom.), 2974; 2857 (C–H aliph.), 1228; 1187 (P@N), 563; 528 (PCl). 2.3.5. Spiro[3-(N-ferrocenylmethylamino)-1-propanoxy]-4,4,6,6tetrachlorocyclotriphosphazatriene (3a) A total of 2.00 g of N3P3Cl6 (5.74 mmol) in THF (150 mL) was added sodium (3-amino-1-propanoxide) (1.70 g, 5.74 mmol) and triethylamine (0.81 mL) at room temperature. The mixture was stirred for 28 h, and the precipitated triethylaminehydrochloride and sodium chloride were filtered off. The solvent was evaporated

2935

completely, and the oily residue was purified by column chromatography with toluene. The powder product was crystallized from n-heptane. Yield: 2.30 g (73%) and m.p.: 152 °C. Anal. Calc. for C14H17N4OFeP3Cl4: C, 30.69; H, 3.12; N, 10.23. Found: C, 31.03; H, 3.28; N, 10.29%. APIES-MS (fragments are based on 35Cl and 56Fe, Ir%): m/z 546 ([M]+, 1.3). FTIR (KBr, cm1): m 3092; 3072 (C–H arom.), 2947; 2851 (C–H aliph.), 1242; 1192 (P@N), 571; 527 (PCl). 2.3.6. Spiro(ethane-1,2-diamino)[N-(1-ferrocenylmethyl)-N-methyl]4,4,6,6-tetrapyrrolidinocyclotriphosphazatriene (1b) A solution of compound 1a (1.20 g, 2.19 mmol) in dry THF (150 mL) was added slowly to a solution of pyrrolidine (2.16 mL, 26.00 mmol) with stirring and refluxing for 30 h. After excess triethylamine (1.23 mL) was added to the solution, the mixture was refluxed for 4 h. The oily product was purified by column chromatography using benzene-THF (3:2) as eluent and was crystallized from n-heptane. Yield: 1.93 g (62%) and m.p.: 203 °C. Anal. Calc. for C30H50N9FeP3: C, 52.55; H, 7.30; N, 18.39. Found: C, 52.47; H, 7.21; N, 18.20%. APIES-MS (fragments are based on 56Fe, Ir%): m/z 686 ([M]+, 100.0). FTIR (KBr, cm1): 3091; 3073 (C–H arom.), 2951; 2839 (C–H aliph.), 1224; 1183 (P@N). 2.3.7. Spiro(ethane-1,2-diamino)[N-(1-ferrocenylmethyl)-N-ethyl]4,4,6,6-tetrapyrrolidinocyclotriphosphazatriene (2b) The work-up procedure was similar to that of compound 1b, using 2a (1.00 g, 1.80 mmol), pyrrolidine (1.77 mL, 22.00 mmol) and triethylamine (1.00 mL). Yield: 0.83 g (66%) and m.p.: 211 °C. Anal. Calc. for C31H52N9FeP3: C, 53.22; H, 7.49; N, 18.02. Found: C, 53.28; H, 7.14; N, 17.86%. APIES-MS (fragments were based on 56 Fe, Ir%): m/z 700 ([M]+, 100.0). FTIR (KBr, cm1): m 3092; 3073 (C–H arom.), 2961; 2840 (C–H aliph.), 1218; 1189 (P@N). 2.3.8. Spiro[3-(N-ferrocenylmethylamino)-1-propanoxy]-4,4,6,6tetrapyrrolidinocyclotriphosphazatriene (3b) The mixture of 3a (0.54 g, 0.99 mmol) and pyrrolidine (0.97 mL, 12.00 mmol) in THF (150 mL) was refluxed for 30 h. After the excess of triethylamine (0.55 mL) was added to the solution, the mixture was refluxed for another 4 h. The product was purified by column chromatography with benzene-THF (3:1) and was crystallized from n-heptane. Yield: 0.42 g (62%) and m.p.: 151–153 °C. Anal. Calc. for C30H49N8OFeP3: C, 52.48; H, 7.19; N, 16.32. Found: C, 52.67; H, 7.00; N, 16.17%. APIES-MS (fragments were based on 56 Fe, Ir%): m/z 687 ([M]+, 100.0). FTIR (KBr, cm1): m 3091; 3073 (C–H arom.), 2961; 2850 (C–H aliph.), 1204; 1187 (P@N). 2.3.9. Spiro(ethane-1,2-diamino)[N-(1-ferrocenylmethyl)-N-methyl]4,4-dichloro-6,6-dimorpholinocyclotriphosphazatriene (1c) A solution of compound 1a (1.18 g, 2.16 mmol) in dry THF (150 mL) was added slowly to a solution of morpholine (2.26 mL, 26.0 mmol) and was then stirred and refluxed for 30 h. After excess triethylamine (1.21 mL) was added to the solution, the mixture was refluxed for 4 h. The product was purified through column chromatography using benzene-THF (3:1) as an eluent and was crystallized from n-heptane. Yield: 0.63 g (45%) and m.p.: 147 °C. Anal. Calc. for C22H34N7O2FeP3Cl2: C, 40.77; H, 5.29; N, 15.13. Found: C, 41.10; H, 4.99; N, 15.16%. APIES-MS (fragments were based on 35Cl and 56Fe, Ir%): m/z 648 ([M]+, 100.0). FTIR (KBr, cm1): m 3091; 3070 (C–H arom.), 2957; 2848 (C–H aliph.), 1221; 1171 (P@N), 569; 522 (PCl). 2.3.10. Spiro(ethane-1,2-diamino)[N-(1-ferrocenyl-methyl)-N-ethyl]4-diethylamino-4,6,6-trimorpholinocyclotriphosphazatriene (2c) The work-up procedure was similar to that of compound 1c, using 2a (1.20 g, 2.14 mmol), morpholine (2.24 mL, 26.00 mmol) and triethylamine (1.20 mL). Yield: 0.87 g (54%) and m.p.: 237 °C. Anal. Calc. for C31H54N9O3FeP31/2C6H6: C, 51.78; H, 7.28; N,

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15.98. Found: C, 51.61; H, 6.93; N, 15.84%. APIES-MS (fragments were based on 56Fe, Ir%): m/z 749 ([M]+, 34.0). FTIR (KBr, cm1): m 3093; 3074 (C–H arom.), 2963; 2842 (C–H aliph.), 1221; 1183 (P@N). 2.3.11. Spiro[3-(N-ferrocenylmethylamino)-1-propanoxy]-4,4,6,6tetramorpholinocyclotriphosphazatriene (3c) The mixture of 3a (1.00 g, 1.82 mmol) and morpholine (0.90 mL, 24.00 mmol) in THF (150 mL) was refluxed for 30 h. After the excess triethylamine (1.02 mL) was added to the solution, the mixture was refluxed for another 4 h. After the solvent was evaporated, the crude product was purified through column chromatography with benzene-THF (3:1) and was crystallized from nheptane. Yield: 0.81 g (59%) and m.p.: 246 °C. Anal. Calc. for C30H49N8O5FeP3: C, 48.01; H, 6.58; N, 14.93. Found: C, 48.48; H, 6.42; N, 14.56%. APIES-MS (fragments were based on 56Fe, Ir%): m/z 751 ([M]+, 100.0). FTIR (KBr, cm1): m 3097; 3073 (C–H arom.), 2954; 2838 (C–H aliph.), 1255; 1187 (P@N). 2.4. X-ray crystallography The suitable crystals of compounds 3b and 3c were crystallized from n-heptane at room temperature. The crystallographic data are Table 1 Crystallographic data for 3b and 3c.

Empirical formula Formula weight Crystal system Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) V (Å3) Z l (cm1) q (calcd) (g cm1) Number of reflections total Number of reflections unique Rint 2hmax (°) Tmin/Tmax Number of parameters R [F2 > 2r(F2)] wR

3b

3c

C30H49FeN8OP3 686.53 monoclinic P21 10.6888(3) 10.6938(3) 14.9834(5) 90 99.617(2) 90 1688.59(9) 2 0.626 (Mo Ka) 1.350 16337 7968 0.0437 56.84 0.780/0.898 388 0.0414 0.0972

C30H49FeN8O5P3 750.53 orthorhombic Pbca 10.5219(2) 21.5811(3) 30.9634(5) 90 90 90 7031.00(2) 8 0.616 (Mo Ka) 1.418 34837 8614 0.0563 56.56 0.829/0.855 424 0.0553 0.1411

Table 2 Selected bond lengths (Å) and angles (°) for 3b and 3c.

P1–N1 P1–N3 P2–N1 P2–N2 P3–N2 P3–N3 P3–N4 P3–O1 N1–P1–N3 N7–P2–N8 N1–P2–N2 N5–P1–N6 N2–P3–N3 N4–P3–O1 P1–N1–P2 P3–N2–P2 P1–N3–P3

3b

3c

1.596(2) 1.599(3) 1.599(2) 1.598(3) 1.592(2) 1.578(2) 1.659(2) 1.601(2) 116.2(1) 101.3(1) 115.7(1) 101.0(1) 116.7(1) 101.7(1) 123.7(2) 123.0(1) 122.5(1)

1.591(3) 1.598(3) 1.598(3) 1.589(3) 1.599(2) 1.579(3) 1.653(3) 1.593(2) 116.6(1) 102.4(1) 116.6(1) 101.4(1) 113.1(1) 117.3(2) 123.4(2) 121.3(2) 122.0(2)

Table 3 Hydrogen-bond geometries (Å), (°) for 3b and 3c. D–H  A

D–H

H  A

D  A

D–H  A

3b C4-H4A  N3 C27–H27B  N1 C8–H8  O1i C12–H12  N3i C10–H10  N2ii C2–H2A  Cg2iii 3c C1–H1A  O2iv C4–H4B  N3 C8–H8  O1v C11–H11  N3v C18––H18B  N1 C19–H19B  N3 C22-H22A  N5 C23––H23A  N2 C30–H30A  N1 C2–H2B  Cg2vi

0.97 0.97 0.93 0.93 0.93 0.97 0.97 0.97 0.93 0.93 0.97 0.97 0.97 0.97 0.97 0.97

2.74 2.59 2.5 2.5 2.52 2.76 2.58 2.69 2.5 2.6 2.44 2.49 2.6 2.53 2.45 2.82

3.187(4) 3.053(5) 3.361(3) 3.384(3) 3.414(4) 3.425(3) 3.438(5) 3.155(4) 3.362(4) 3.503(4) 2.975(5) 2.967(4) 3.047(5) 2.957(5) 2.976(4) 3.480(4)

109 109 153 158 161 126 147 110 154 165 114 110 108 107 114 126

Symmetry codes: (i) 2  x, ½ + y, z, (ii) 1  x, ½ + y, z, (iii) x, y  1, z, (iv) 1 + x, y, z, (v) x  ½, ½  y, z, (vi) 1 + x, y, z. Ring centroids: Cg2 (for 3b) and Cg2 (for 3c) are centroids of rings (C10–C14) (for 3b) and (C10–C14) (for 3c), respectively.

given in Table 1, selected bond lengths and angles are listed in Table 2, and hydrogen bond data are given in Table 3. Crystallographic data were recorded on a Bruker Kappa APEXII CCD areadetector diffractometer using Mo Ka radiation (k = 0.71073 Å) at T = 100(2) K. Absorption corrections by multi-scan [40] were applied. Structures were solved by direct methods and refined by full-matrix least squares against F2 using all data [41]. All non-H atoms were refined anisotropically. In compounds 3b and 3c, H atom positions were calculated geometrically at distances of 0.93 Å (CH) and 0.97 Å (CH2) from the parent C atoms; a riding model was used during the refinement process and the Uiso(H) values were constrained to 1.2 Ueq(carrier atom). 3. Results and discussion 3.1. Synthesis The starting monoferrocenylamines (1–3) were obtained from the reactions of appropriate amines and ferrocenecarboxaldehyde. The reactions of N3P3Cl6 with new N-alkyl-N-ferrocenylmethylethylene diamines, FcCH2NH(CH2)2NHR1 [R1 = Me (1) and Et (2)], and sodium [3-(N-ferrocenylmethylamino)-1-propanoxide] (3) create spirocyclic monoferrocenyl phosphazenes (1a–3a) in THF at room temperature. Scheme 1 depicts the reaction pathway of N3P3Cl6 with the ligands for providing a better understanding of the nucleophilic replacement reactions. The reactions of N3P3Cl6 with the corresponding amines (1–3) seem to be regioselective because only the spiro arrangement is favored. The tetrapyrrolidinophosphazenes (1b–3b) were prepared from the reactions of corresponding partly substituted phosphazenes (1a–3a) with excess pyrrolidine in boiling THF. On the other hand, compound 1a affords only the geminal product (1c) with excess morpholine in boiling THF or toluene. The geminal structure of 1c is evidenced by a 1Hcoupled 31P NMR spectrum (Fig. S1). The reactions of 2a and 3a with excess morpholine resulted in the unexpected dietilaminotrimorpholino (2c, in boiling toluene) and fully substituted morpholino products (3c, in boiling THF), respectively. Compound 2c appears to have been formed from the reaction of chlorotrimorpholino monoferrocenylphosphazene, which was present as an impurity in the reaction mixture with diethylamine. Diethylamine may have come from the triethylamine used as an HCl acceptor (see Section 2). Data from the microanalyses, FTIR, APIES-MS, and NMR were consistent with the proposed structures of the compounds. The mass spectra of 1a–3c showed the molecular (M+) ion peaks.

_ E.E. Ilter et al. / Polyhedron 29 (2010) 2933–2944

3.2. FTIR and NMR spectroscopy The FTIR spectra of the monoferrocenylphosphazene derivatives showed strong stretching absorption bands between 1255 and 1171 cm1 attributed to mP@N vibrations of the phosphazene ring [43,44]. Asymmetric and symmetric stretching bands of mAr–H were found at 3097–3091 cm1 and 3078–3070 cm1. In addition, mPCl2(asym) and mPCl2(sym) absorption peaks arose for the tetrachloro derivatives (1a–3a) in the ranges of 571–563 cm1 and 528– 522 cm1, respectively. In the FTIR spectra of 3b and 3c mCH. . .N (hydrogen bonds) stretching bands were observed at 3419– 3306 cm1 and the X-ray structural data indicated the existence of intramolecular C–H  N hydrogen bonds for 3b and 3c (Table 3). The 1H-decoupled 31P NMR data of the phosphazenes are given in Table 4, and indicate that all of the compounds have spiro architectures. The spin systems are interpreted as simple AX2, ABX, AMX, and AB2. The compounds show a typical five-lines resonance pattern consisting of a triplet for one P(spiro) atom and a doublet for two other phosphorus atoms, except for 1c and 2c. The 31P NMR of 1c illustrates a 12-line resonance pattern consisting of a doublet of doublets for all of the P atoms, indicating that it has a geminal structure. A similar resonance pattern was also observed for 2c. In addition, one P atom in 1c and two P atoms in 2c are expected to be stereogenic centers and they may be in the mixture of enantiomers (R and S for 1c and RR, RS, SR, SS for 2c). The stereogenic properties of the phosphazenes can be determined by 31P NMR spectroscopy with the addition of a chiral solvating agent (CSA) [35,45]. The signals of P(spiro) atoms of tetrapyrrolidino (1b–3b) and tetramorpholino ferrocenylphosphazenes (3c) are downfieldshifted with respect to the corresponding P(spiro) phosphorus

2937

atoms of tetrachloro derivatives (1a–3a). In the case of oxygen atoms (12.54 ppm for 3b and 11.13 ppm for 3c) and the NEt group (9.41 ppm for 2b) in the spiro-ring, this downfield shiftings is larger than that of NMe (3.59 ppm for 1b). The average of the coupling constants of the compounds is ca. 43.9 Hz. The 1H and 13C NMR signals are assigned on the basis of chemical shifts, multiplicities and coupling constants of all the phosphazenes (Tables 5 and 6). Additionally, the assignments are made clear by the DEPT, COSY, HETCOR, and HMBC spectra (Fig. S2). The spectra of 2b are illustrated in Supplementary Fig. S2, as examples, all of the 1H and 13C NMR assignments are written on the spectra. The NMR data of 1c and 2c show complex 1H NMR spectra since all of the aliphatic protons are diastereotopic. The geminal FcCH2N protons of 1c and 2c give rise to an ABX spin system because of the geminal proton–proton coupling and the vicinal coupling with the P-31 nucleus. The protons of cyclopentadienyl rings are distinguished using HETCOR and HMBC, unambigously (Fig. S2). The 3JPH values of FcCH2N protons are between 10.3 and 10.6 Hz for tetrachloro (1a–3a) and; 5.1–6.7 Hz for tetrapyrrolidinophosphazenes (1b–3b). In the 1H NMR spectra of tetrapyrrolidinophosphazenes (1b–3b), and geminal- (1c), tri- (2c) and tetramorpholinophosphazenes (3c), the two substituents bonded to the same phosphorus atom show two groups of NCH2 and NCH2CH2 signals with small separations (Table 6, Fig. S2). The expected carbon signals are interpreted from the 13C NMR spectra of the compounds; the FcCH2, NCH2 (spiro) and NCH2 (in the pyrrolidino and morpholino rings) carbon signals were assigned by the HETCOR and HMBC experiments. The two groups of carbon peaks for NCH2 and NCH2CH2 (in the geminal pyrrolidino and morpholino rings) are easily distinguishable for 1b–3c

Table 4 31 P NMR (decoupled) spectral data of the compounds. [Chemical shifts (d) reported in ppm and J values in Hz]a.

R1= Me or Et, X= N or O, Y=Cl or pyrr

( )n Fc CH2

N

( )

n

Fc CH2

XR1

N

P A N

P A Y

Y Y

N

N

P X

(B)

P N

X

Y

(B)

R3 R4

1a-3a, 1b-3b and 3c

Compound

Spin system

PCl2

XR1

P X

N

N

P B

R2 R2

(M)

1c and 2c

d(ppm)

P(mor)2

P(mor)3(Et2N)

2

41.1 41.0 50.4 41.9 42.2 41.8 54.0 39.8 44.4 46.6 42.7 41.7 43.2

JPP

P(NR)2 P(pyrr)2 1a 2a 3a 1b 2b 3b 1c

AX2 AX2 AX2 AX2 AX2 AX2 AMX

23.93 23.58 23.09 – –– – 26.30

2c

ABX

3c

AB2

a 31

(d) (d) (d)

18.55 (t) 17.19 (t) 8.49 (t) 22.14 (t) 26.60 (t) 21.03 (t) 22.32 (dd)

– – – 13.87 (d) 18.30 (d) 19.00 (d) –

– – – – – – 18.25 (dd)

– – – – – – –

–

26.97 (dd)

–

22.25 (dd)

20.08 (dd)

–

19.62 (t)

–

21.50 (d)

–

(dd)

P NMR measurements in CDCl3 solutions at 293 K.

_ E.E. Ilter et al. / Polyhedron 29 (2010) 2933–2944

2938

Table 5 13 C NMR (decoupled) spectral data for 1a–3a, 1b–3b and 1c–3c. [Chemical shifts (d) reported in ppm and J values in Hz].[mor: morpholine, pyrr: pyrrolidine]. Compound [d(ppm), J(Hz)] 1a

2a

3a

1b

2b

3b

1c

2c

3c

N–CH2–CH3

–

13.39 3 JPC = 4.3

–

–

14.22 3 JPC = 6.4

–

–

–

N–CH3

30.85 2 JPC = 4.0 –

–

–

–

–

–

26.34 JPC = 9.0 26.45 3 JPC = 9.6 39.33 2 JPC = 4.3

26.38 3 JPC = 9.4 26.52 3 JPC = 9.4

31.52 2 JPC = 4.7 –

–

–

31.76 2 JPC = 4.0 26.39 3 JPC = 10.5 26.29 3 JPC = 10.1 –

14.12 3 JPC = 7.0 (spiro) 26.52 3 JPC = 9.4 (Et2N–) – –

–

39.37 2 JPC = 4.2 (spiro) 46.70 2 JPC = 4.0 (Et2N–) –

–

N–CH2–CH2 (pyrr)

N–CH2–CH3

–

39.21 JPC = 4.7

–

25.73 3 JPC = 5.6 –

2

O–CH2–CH2

–

–

CH3–N–CH2

43.80 2 JPC = 5.8 –

–

C2H5–N–CH2

–

47.10 2 JPC = 13.6 –

43.97 JPC = 3.5 44.06 2 JPC = 14.3 –

44.10 2 JPC = 5.8 –

43.83 2 JPC = 5.5 –

47.25 2 JPC = 3.2 68.08 2 JPC = 7.2

43.97 2 JPC = 10.7 –

2

N–CH2 N–CH2 (pyrr or mor) a

C5

O–CH2

47.05 JPC = 11.9 46.08 46.19 45.14 2 JPC = 5.2 – 2

–

– 44.10 2 JPC = 9.7 43.55 2 JPC = 11.9 46.12 46.35 45.28 2 JPC = 6.7 –

–

26.53 3 JPC = 4.9 – – 45.39 46.07 46.37 47.72

43.61 2 JPC = 13.7 – 47.16 JPC = 14.5 44.44 44.54 44.44

65.78 2 JPC = 6.8

67.06 3 JPC = 6.8 67.12 3 JPC = 7.3

68.54 68.15 68.43 69.15 69.69 83.21 3 JPC = 9.1

68.60 68.45

68.59 68.45

68.68 68.48

67.61 68.18

68.24 67.80

68.29 67.89

C2a

69.55

69.58

69.91

69.80

69.71

70.43

82.45 3 JPC = 5.7

82.44 3JPC = 6.3

3

81.76 JPC = 9.1

3

84.72 JPC = 11.7

3

84.91 JPC = 13.4

84.01 3 JPC = 13.6

C1

–

2

C4a C3a

a

a

45.11

3

– 44.20 2 JPC = 11.3 43.53 2 JPC = 12.1 44.83 44.91 45.49 2 JPC = 4.0 67.43

26.38 3 JPC = 3.4 – – 45.33 44.66 44.92 47.90 66.17 (spiro) JPC = 6.9 67.28 (mor) 3 JPC = 8.2 67.36 (mor) 3 JPC = 8.6 68.41 68.12 2

68.38 68.06 67.95 69.28 69.53 84.31 3 JPC = 11.9

70.13 83.28 3 JPC = 13.1

Numberings of protons are given in Scheme 1.

(Fig. S2). The three-bond couplings of 1b–3b and 3c (for pyrrolidine and morpholine) give rise to triplets in the case of doublets, as illustrated in Fig. S3. This situation may be due to second-order effects, which have previously been observed [35] and the 3JPC coupling constants have been estimated using the external transitions of the triplets [46]. The 3JPC between the ipso-C atoms of the Fc (C1) and P atoms observed for all the phosphazene derivatives. The 3JPC values of the tetrachloro compounds (1a–3a) are smaller than those of tetrapyrrolidinophosphazenes (1b–3b), geminal- (1c), tri- (2c) and tetramorpholinophosphazenes (3c). On the other hand, the 3JPC values of the compounds containing six-membered spiro-rings (3a–3c) are larger than the five-membered ones (1a–2c). In addition, the signals of nonprotonated carbon atoms disappear in the DEPT spectra, as compared with the 1H-decoupled 13C NMR spectra. 3.3. X-ray structures of 3b and 3c The molecular and solid-state structure determinations of 3b and 3c are verified by the assignments of their structures from spectroscopic data. The molecular structures of 3b and 3c along, with the atom-numbering schemes, are illustrated in Figs. 1 and 2, respectively. The phosphazene rings of 3b and 3c are in flattened-boat forms (Supplementary material, Fig. S4a; u2 = 69.9(1.3)°, h2 = 142.5(0.9)°, Fig. S5a; u2 = 139.6(0.8)°, h2 = 53.8(0.8)°) having total

puckering amplitudes QT of 0.130(2) (for 3b) and 0.166(2) (for 3c) [47]. In 3b and 3c, the six-membered rings (P3/N4/O1/C1–C3) are in chair conformations (Supplementary material, Fig. S4b; QT = 0.636(3) Å, u2 = 27.0(0.2)°, h2 = 97.0.(0.3)°, Fig. S5b; QT = 0.691(3) Å, u2 = 150.2(0.3)°, h2 = 90.1(0.3)°). The average P–N bond lengths in phosphazene rings are 1.594(2) and 1.592(3) Å which are shorter than the exocyclic (spiro-ring) P–N bonds of 1.659(2) and 1.653(3) Å for 3b and 3c, respectively. In the phosphazene rings, the endocyclic P–N bond lengths are in the ranges 1.578(2)–1.599(3) Å (for 3b) and 1.579(3)–1.599(2) Å (for 3c) (Table 2). In the phosphazenes the P–N single and double bonds are generally in the ranges 1.628– 1.691 Å and 1.571–1.604 Å, respectively [48]. They are known to be the most intriguing bonds in chemistry. In recent years, natural bond orbital (NBO) and topological electron density analyses have been used to investigate the electronic structure of phosphazenes [49]. There are two phosphazene bonding alternatives, namely, negative hyperconjugation and ionic bonding, which are evaluated using NBO. The calculations indicate that the ionic component is the dominant feature. But, they are not mutually exclusive alternatives and in fact, two phosphazenes bonding alternatives are both important. Moreover, the presence of the negative hyperconjugation strongly contributed to the multiple-bond character, and the electron withdrawing substituents bonding to the P atoms increase the negative hyperconjugation of the exocyclic P–N bonds. Thus,

Table 6 1 H NMR spectral data for 1–3, 1a–3a, 1b–3b, and 1c–3c.[mor: morpholine, pyrr: pyrrolidine, s: singlet, d: doublet, t: triplet, m: multiplet, and bp: broad peak]. Compound [d(ppm), J(Hz)] 2

1a

2a

3a

1b

2b

3b

1c

2c

3c

–

1.16 (t, 3H) 3 JHH = 7.1

–

1.20 (t, 3H) 3 JHH = 7.2

–

–

1.13 (t, 3H) 3 JHH = 7.2

–

–

–

N–CH3

–

2.57 (d, 3H) 3 JPH = 12.5 –

–

–

–

–

–

–

2.71 (m, 2H) 3 JHH = 7.1

–

–

–

2.92 (spiro) (m, 2H) 2.98–3.30 (Et2N–) (m, 4H)

–

NH

2.38

–

–

–

–

–

–

–

–

O–CH2–CH2

2.86 2.88 –

2.94 (m, 2H) 3 JPH = 11.6 3 JHH = 7.2 –

1.78 (m, 16H) 1.89 (m, 16H) –

–

N–CH2–CH3

1.80 (m, 16H) 1.87 (m, 16H) 2.91 (m, 2H)3JHH = 7.2

2.54 (d, 3H) 3 JPH = 11.9 –

–

–

2.53 (d, 3H) 3 JPH = 12.1 1.79 (m, 16H) 1.85 (m, 16H) –

–

N–CH2–CH2 (pyrr)

2.50 (s, 3H) –

1.15 (spiro) (t, 3H) 1.91 (Et2N–) (t, 6H) –

–

–

1.79 (m, 2H) 3 JHH = 7.4

CH3–N–CH2

3.05 (m, 2H)

–

–

3.13 (m, 2H) JPH = 9.4 3 JHH = 5.1 –

–

1.92 (m, 2H) 3 JHH = 6.2 3 JHH = 5.4 –

3

C2H5–N–CH2

–

2.81 (m, 2H)

N–CH2

3.05 (m, 2H)

2.81 (m, 2H)

2.94 (m, 2H) JPH = 10.1 3 JHH = 6.1 –

–

–

3.00–3.30 (m, 2H)

–

–

–

–

2.98–3.30 (m, 2H)

–

3.02 (m, 2H) JPH = 10.1 3 JHH = 6.1 3.17 (m, 16H) 3.24 (m, 16H) 3.75 (d) 3 JPH = 5.2

2.94 (m, 2H) 3 JPH = 10.9 3 JHH = 6.2 3.04 (m, 2H) 3 JPH = 10.1 3 JHH = 6.4 3.16 (m,16H) 3.27 (m, 16H) 3.76 (d, 2H) 3 JPH = 5.1

2.94 (m, 2H) 3 JPH = 12.2 3 JHH = 6.1 3.15 (m, 16H) 3.28 (m, 16H) 3.82 (d, 2H) 3 JPH = 6.7

3.00–3.30 (m, 2H)

2.98–3.30 (m, 2H)

3.00–3.30 (m, 8H) 3.87 (dd, 2H) 3 JPH = 7.2 2 JHH = 14.1 3.69 (m, 4H) 3.75 (m, 4H)

2.98–3.30 (m, 12H) 3.60–3.85 (m, 2H)

2.97 (m, 2H) 3 JPH = 10.8 3 JHH = 7.4 3.05–3.27 (m, 16H) 3.66–3.78 (m, 2H)

3

–

3.13 (m, 2H) JPH = 11.1 3 JHH = 5.1 –

3.11 (m, 2H) 3 JPH = 11.2 3 JHH = 5.6 3.15 (m, 2H) 3 JPH = 11.7 3 JHH = 5.6 –

3.08 (m, 2H) 3 JPH = 12.5 3 JHH = 6.2 –

3

1.78 (m, 2H) 3 JHH = 6.1

3

N–CH2 (pyrr or mor) H5

–

–

3.63 (s, 2H)

3.56 (s, 2H)

3.87 (d, 2H) 3 JPH = 10.5

3.85 (d, 2H) 3 JPH = 10.3

3.87 (d, 2H) 3 JPH = 10.6

O–CH2

–

–

–

–

4.35 (m, 2H) 3 JPH = 10.7 3 JHH = 5.4

–

–

4.25 (m, 2H)

H4 H3

4.16 (bp, 5H) 4.16 (bp, 2H)

4.15 (bp, 5H) 4.12 (bp, 2H)

4.15 (bp, 5H) 4.15 (bp, 2H)

4.14 (bp, 5H) 4.14 (bp, 2H)

4.13 (bp, 5H) 4.15 (t, 2H)) JHH = 3.6 4.28 (t, 2H) 3 JHH = 3.6

4.07 (bp, 5H) 4.07 (bp, 2H)

4.08 (bp, 5H) 4.08 (bp, 2H)

4.07 (bp, 5H) 4.09 (bp, 2H)

4.14 (bp, 5H) 4.14 (bp, 2H)

4.14 (bp, 5H) 4.14 (bp, 2H)

4.23 (m) (spiro) (bp, 2H) 3.66–3.78 (mor) (m, 16H) 4.14 (bp, 5H) 4.14 (bp, 2H)

4.18 (t, 2H) JHH = 3.3

4.19 (t, 2H) JHH = 3.3

4.23 (m, 2H)

4.36 (bp, 2H)

4.21 (bp, 2H)

4.23 (bp, 2H))

3

3.60–3.85 (m, 12H)

_ E.E. Ilter et al. / Polyhedron 29 (2010) 2933–2944

1 N–CH2–CH3

3

H2

4.28 (bp, 2H)

4.21(bp, 2H)

4.27 (t, 2H) JHH = 3.2

3

4.27 (bp, 2H))

3

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_ E.E. Ilter et al. / Polyhedron 29 (2010) 2933–2944

molecules into a three-dimensional network. The p  p contacts between the Fc rings, Cg1  Cg2 [where Cg1 and Cg2 are centroids of the rings (C5–C9) and (C10–C14) of 3b and 3c, respectively] may further stabilize the structures, with centroid–centroid distances of 3.296(2) Å for 3b and 3.282(2) Å for 3c. There are also C–H  p interactions (Table 3). 3.4. Electrochemistry of spirocyclic monoferrocenylphosphazenes

Fig. 1. ORTEP-3 [52] drawing of 3b with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level.

Fig. 2. ORTEP-3 [52] drawing of 3c with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level.

the shortening of the P–N bond with electronegative substituents could be explained, qualitatively, using more efficient pN–r*PR2 overlapping [49,50]. As can be seen from Table 2, in 3b and 3c the endocylic N2–P3– N3 (a) and N3–P1–N1 (c) angles are narrowed, while the P3–N3– P1 (b) and P1–N1–P2 (d) angles are noticeably expanded with respect to the corresponding values in the ‘‘standard” compound, N3P3Cl6. In N3P3Cl6, the a, b, c and d angles are 118.3(2)°, 121.4(3)°, 118.3(2)°, and 121.4(3)°, respectively [51]. The variations in the endocyclic and exocyclic angles found in phosphazenes could be explained by the substituent-dependent charge at the P centers and negative hyperconjugation. The values of this charge separation between the P and N atoms in the phosphazene rings vary significantly depending on the electron-withdrawing capacities of the substituents bonded to the P atoms. It is particularly pronounced in the electronegative substituents such as F, CF3, NC2H4 that the PNP angles are expanded [49]. In addition, electronic factors and steric interactions between the bulky substituents play an important role on the endocyclic and exocyclic angles [45]. In compounds 3b and 3c, intramolecular C–H  N and intermolecular C–H  O and C–H  N hydrogen bonds (Table 3) link the

The electrochemistry of Fc derivatives is an important research area in material science [53], organometallics [19,20], catalysis [53], and photoluminescent systems [24]. The cyclic voltammograms (CVs) of the spirocyclic tetrachloro (Fig. 3a), tetrapyrrolidino (Fig. 3b) and morpholino monoferrocenylphosphazenes (Fig. 3c) are acquired in acetonitrile (0.1 M TBATFB) at the glassy carbon electrode to investigate the electrochemical behaviors and the effects of the structural differences on the electron transfer kinetics of pendant Fc group. The electrochemical data of the compounds are given in Table 7, and all groups of compounds have reversible CVs with one-electron anodic and cathodic peaks corresponding to the redox-responsible moiety of Fc. As Fig. 3 shows, the reversibility of the CV waves and the position of the peak potentials and peak currents are not significantly influenced by the structural changes in the phosphazene rings of 1a–3a and 1b–3b. This means that Fc redox centers are well isolated from the phosphazene ring. The presence of the pyrrolidino and morpholino groups results in significant changes in the peaks currents which can be attributed to either changes in diffusion coefficients or differences in the solubilities of compounds on the electrode surfaces. For instance, diffusion coefficients decrease from 1.71  105 cm2 s1 (for 1a and 2a) to 0.96  105 cm2 s1 (for 1b and 2b) when the Cl replacement reactions with pyrrolidine groups occur (Table 7). It is interesting to note that diffusion coefficients decrease in the order of 2a, 2b, and 2c. This decrease can be explained in light of the molecular weight of the stated order. As expected, the diffusion coefficient decreases as the molecular weight increases. The length of the alkyl chain [(CH2)n, where n = 0, 1] and the substituent on it (methyl or ethyl) have no readily discernible effect on the redox potentials of each group of compounds. Redox potentials were approximately equal to the half-wave potentials, assuming reversible oxidation and identical diffusion coefficients of oxidized and reduced forms of the ferrocenylphosphazenes. Also the oxygen insertion into the spiro-ring as in 3a, 3b, and 3c does not significantly affect the electrochemical behavior. Another important result that comes from the CVs is the identity of the number of electrons transferred for the compounds having different substituents in the phosphazene and spiro-ring. The values of cathodic-to-anodic peak current ratios are very close to one for all groups of compounds, indicating that there are no solubility differences between compounds and their oxidized forms [32] and the high electron-transfer rate of Fc centers [54]. The values of the slope of the log ipa versus log v plots, which are about 0.5, also verify that voltammograms are not influenced by the adsorption of either the compounds or their oxidized forms. 3.5. Antibacterial activity The results of antimicrobial activity of the compounds on Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Bacillus cereus NRRL-B-3711, and Enterobacter fecalis ATCC 292112, Candida albicans ATCC 10231, and Candida tropicalis ATCC 13803 are listed in Table 8. The compounds 1b, 2b, 3b, and 3c had antimicrobial effects on several of the bacterial species. Of all tested bacteria, S. aureus, P. aeruginosa, B. subtilis and B. cereus were the most sensitive to all phosphazene compounds. Compound 1b was active

_ E.E. Ilter et al. / Polyhedron 29 (2010) 2933–2944

2941

Fig. 3. Cyclic voltammograms of ferrocenylphosphazene derivatives (1 mM each) of (a) 1a–3a, (b) 1b–3b, (c) 1c–3c in acetonitrile (containing 0.1 M TBATFB) at the glassy carbon electrode versus Ag/AgNO3 (0.01 M). The scan rate is 100 mV/s.

Table 7 CVs data of ferrocenylphosphazene derivatives (1 mM each) obtained in acetonitrile (0.1 M TBATFB) at the glassy carbon electrode versus Ag/AgNO3 (0.01 M). The scan rate is 100 mV s1.

a b

Compound

Epa, Epc, E°0 (E1/2)a,b

DEp (Epa  Epc)

ipab (lA)

ipc/ipa

Slope of log(ia)  log(v)

Db cm2 s1

1a 2a 3a 1b 2b 3b 1c 2c 3c

203, 203, 216, 177, 165, 162, 197, 176, 181,

70 68 74 87 71 70 81 67 69

24.86 25.09 24.40 18.92 19.55 21.13 25.40 15.03 20.54

0.97 0.95 0.97 1.01 0.93 0.97 0.98 0.96 0.93

0.49 0.52 0.51 0.51 0.50 0.49 0.49 0.50 0.49

1.71  10–5 1.71  105 1.71  105 0.96  105 0.96  105 1.01  105 1.71  105 0.67  105 0.96  105

133, 168 135, 169 142, 179 90, 134 94, 130 92, 127 116, 157 109, 143 112, 147

E1/2 is calculated from the equation E1/2 = [(Epa  Epc)/2] + Epc assuming reversible oxidation of Fc groups [54]. The pooled standard deviation is 16 mV for the peak potential measurements, 0.16 lA for peak current measurements and 0.23  105 cm2 s1 for diffusion coefficients.

Table 8 Antimicrobial activities of 1b, 2b, 3b and 3c (Antibiotics; Amp = Ampicilin and C = Chloramfenicol, Antifungal; Keto = Ketoconozole). Test bacteria/compounds

Inhibition zone diameter (mm) Agar-well diffusion

E. fecalis ATCC 292112 E. coli ATCC 25922 E. coli ATCC 35218 S. aureus ATCC 25923 P. aeruginosa ATCC 27853 B. cereus NRRL-B-3711 B. subtilis ATCC 6633 P. vulgaris ATCC 8427 C. albicans ATCC 10231 C. tropicalis ATCC 13803

Antibiotics

Antifungal

1b

2b

3b

3c

Dioxan

Amp

C

Keto

16.6 ± 1.5a 11.0 ± 0.5b 10.6 ± 0.5b 18.9 ± 0.5c 12.0 ± 0.0d 16.6 ± 0.5e 18.0 ± 1.0f 10.3 ± 0.5g 11.0 ± 0.0h 12.0 ± 0.0d

10.3 ± 1.5a – 10.5 ± 0.7a 18.6 ± 0.5b 12.5 ± 0.7c 13.0 ± 0.0c 16.0 ± 1.0d 12.0 ± 1.0c 18.0 ± 0.0e 19.0 ± 0.0f

14.6 ± 0.5a 11.0 ± 1.0b 13.3 ± 0.5c 19.5 ± 1.2d 17.0 ± 2.0e 25.6 ± 1.1f 22.0 ± 0.0g 13.3 ± 1.1c 15.5 ± 1.2a 23.6 ± 1.0g

11.6 ± 0.5a 15.0 ± 1.0b 14.3 ± 0.5b 12.3 ± 0.5a 20.0 ± 0.0c 10.0 ± 0.0d 13.0 ± 1.0b 14.6 ± 0.5b 10.5 ± 1.2d 10.0 ± 0.8d

– – 10.6 ± 0.5a 10.0 ± 0.0a 10.3 ± 0.5a – 10.1 ± 0.7a 11.0 ± 1.0a 10.0 ± 0.0a 10.5 ± 0.5a

30.0 ± 0.0 20.3 ± 0.5 R 35.0 ± 0.0 R 10.0 ± 1.0 30.3 ± 0.5 R NS NS

29.0 ± 1.0 26.6 ± 1.1 10.5 ± 0.7 29.6 ± 0.5 R 23.4 ± 0.7 31.0 ± 1.0 30.3 ± 0.5 NS NS

NS NS NS NS NS NS NS NS 30.0 ± 0.0 29.0 ± 0.5

Values represent averages ± standard deviations for triplicate experiments. Values in the same column with different superscripts are significantly (p < 0.05) different. NS: not studied, R: resistant.

against E. fecalis, S. aureus, and B. subtilis, and 2b was active against S. aureus, C. albicans, and C. tropicalis. Compound 3c was active against E. coli and P. Aeruginosa, while, compound 3b was the most active compound against most of the tested bacteria and yeast strains.

3.6. Susceptibility testing: M. tuberculosis H37Rv and multi-drug resistant clinical M. tuberculosis isolates The compounds 1b, 2b, 3b, and 3c were evaluated for tuberculosis inhibition against the M. tuberculosis H37Rv reference strain.

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Susceptibility testing shows that the reference strain is susceptible to high concentrations of 1b, 3b, and 3c, but is resistant to dioxan. However, the M. tuberculosis strain was sensitive to all concentrations of 2b. The susceptibility results of compounds for reference strain are given in Table 9. To further investigate the antituberculosis activity, the compounds were tested against six clinical multidrug-resistant M. tuberculosis isolates. The antibiogram of the

Table 9 Susceptibility results of compounds for reference strain: M. tuberculosis H37Rv. R: resistant S: sensitive.

reference strain and multidrug-resistant clinical M. tuberculosis isolates are given in Table 10. Compounds 1b and 2b were active against all the multidrug-resistant clinical isolates at concentrations of 10 000 and 5000 lM (Table 11). Consequently, to the best of our knowledge, there is only one paper about anti-microbial/anti-bacterial activity of mono and bisferrocenylphosphazenes [35], published by our group. As a matter of fact, compounds 1b, 2b, 3b, and 3c exhibit significantly improved antibacterial, antifungal, and antituberculosis activity when compared to the tetrapyrrolidino ferrocenylphosphazenes, given in the literature 35.

Dilution rates and susceptibility results Compounds

10 000 lM

5.000 lM

2.500 lM

1.250 lM

625 lM

1b 2b 3b 3c Dioxan

S S S S R

S S S S R

S S R R R

R S R R R

R S R R R

Table 10 Antibiogram of reference strain and multi-drug resistant clinical isolates. Reference strain and clinical isolates name

Streptomycin

Isoniazid

Rifampin

Etambutol

H37Rv Clinical M. isolates Clinical M. isolates Clinical M. isolates Clinical M. isolates Clinical M. isolates Clinical M. isolates

S S

S R

S R

S R

S

R

R

S

R

R

R

R

S

R

R

S

S

R

R

S

S

R

R

R

tuberculosis 1 tuberculosis 2 tuberculosis 3 tuberculosis 4 tuberculosis 5 tuberculosis 6

3.7. Interactions of DNA with the compounds Interactions of 1b, 2b, 3b, and 3c with supercoiled pUC18 plasmid DNA were investigated. The compounds were incubated at a range of concentrations with supercoiled pUC18 plasmid DNA in the dark at 37 °C for 24 h. Fig. 4a depicts the electrophoretograms applying to the interaction of pUC18 plasmid DNA with the compounds 1b and 2b at concentrations of compounds ranging from 312 lM to 10 000 lM. Lane 1 applies to the untreated pUC18 plasmid DNA (control DNA), showing the major supercoiled (form I) and minor nicked (form II) forms of the plasmid DNA. Lanes 2–7 apply to pUC18 plasmid DNA incubated with compounds ranging from 10 000 lM to 312 lM. The pUC18 plasmid DNA interacted with decreasing concentrations of 1b, the mobility of form I slightly decreased in all the concentration tested. The plasmid DNA interaction with 2b leads to the complete digestion of the DNA and no form I or form II bands were detected for concentrations greater than 10 000 lM (Lane 9). Form I also diminished at the concentration of 5000 lM (Lane 10), while form II was observed from 5000 lM to 312 lM concentrations of 2b. The mobility of form I was slightly decreased for all concentrations. Fig. 4b and c show how the electrophoretograms apply to the interactions of pUC18 plasmid DNA with 3b and 3c at concentrations of compounds ranging from 312 lM to 10 000 lM, respectively. In the

Table 11 Susceptibility results of compounds for multi-drug resistant clinical M. tuberculosis isolates. R: resistant S: sensitive I: intermediate.

a

Compounds

10 000 lM

5.000 lM

2.500 lM

1.250 lM

1000 lM

Clinical M. tuberculosis isolates 1

1b 2b 3b 3c

S S S S

S S S I

S I I I

R R I R

R R R R

Clinical M. tuberculosis isolates 2

1b 2b 3b 3c

S S S S

S S I I

S S I R

R I R R

R I R R

Clinical M. tuberculosis isolates 3

1b 2b 3b 3c

S S S S

S S S I

S I S R

R R I R

R R R R

Clinical M. tuberculosis isolates 4

1b 2b 3b 3c

S S S R

S S S R

S R S R

R R R R

R R R R

Clinical M. tuberculosis isolates 5

1b 2b 3b 3c

S S S R

S S S R

S S R R

R I R R

R I R R

Clinical M. tuberculosis isolates 6

1b 2b 3b 3c Dioxana

S S S R R

S S R R R

I S R R R

R R R R R

R R R R R

Six multi-drug resistant clinical isolates are resistant to dioxan.

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Fig. 4. Electrophoretic results of the incubation of pUC18 plasmid DNA with varying concentrations of (a) 1b, (b) 2b, (c) 3b, (d) 3c (lane 1: pUC18 DNA, lane 2–7: pUC18 DNA modified by compounds 10 000 lM, 5.000 lM, 2.500 lM, 1.250 lM, 625 lM, and 312 lM. The roman numerals I and II indicate form I and form II pUC18 plasmid DNAs, respectively.

case of 3b, forms I and II of plasmid DNA diminished in three high concentrations. The form I band was lost in all concentrations tested. The mobility of the forms I and II bands changed such that the two bands co-migrated for the concentrations of 3b ranging from 1250 lM to 312 lM. When the two forms co-migrate, form I is fully relaxed [55]. If restriction occurs on one strand (nicking) of the supercoiled plasmid DNA, the supercoil will relax to generate a slower-moving open circular form (form II) [56]. If both strands are cut, a linear form (form III) will be generated that migrates between forms I and II (Fig. 4b). The conformational changes in form I of DNA were caused by the covalent binding of phosphazene derivatives with nucleotides in DNA. The co-migration in 3b indicated that the binding of a compound causes a greater unwinding of the supercoiled form I of DNA than all other compounds tested. In the case of 3c, binding of the compound to plasmid DNA was less effective than with 3b. The binding of 3c resulted in slightly decreased mobilities of the bands of forms I and II. There was no form I band at the highest concentration of the compound however; the intensity of form I of DNA increased after 5000 lM concentrations of 3c (Fig. 4c). The intensity of the band II was very high at two high concentrations of 3c and then started to decrease. Therefore, 3c changes the conformation of plasmid DNA from negatively supercoiled form I to a large amount of open circular form II. 3.8. BamHI and HindIII digestion of compounds-pUC18 plasmid DNA In order to assess whether the phosphazene compounds show affinity toward guanine–guanine (GG) and/or adenine–adenine (AA) regions of DNA, the restriction analyses of compound-

pUC18 plasmid DNA adducts by BamHI and HindIII enzymes were carried out (Fig. 5). BamHI and HindIII enzymes bind at the recognition sequences 50 -G/GATCC-30 and 50 -A/AGCTT-30 and cleave these sequences just after the 50 -guanine and 50 -adenine sites, respectively, and as a result, convert form I and form II to linear form III of DNA [39]. Only two concentrations were used for restriction analysis. It was observed that digestion with BamHI was prevented for all compounds, however, HindIII digestion was observed. 4. Conclusions NN (1 and 2) and N/O (3) donor-type monoferrocenylamines have, regioselectively, led to the formation of spirocyclic monoferrocenylphosphazenes (1a–3a) via the condensation reactions of N3P3Cl6. The fully substituted phosphazenes (1b–3b and 3c) were, respectively prepared from the reactions of compounds 1a, 2a, and 3a with pyrrolidine and morpholine. On the other hand, the reaction of 1a with excess morpholine afforded geminal-morpholino phosphazene (1c), whilst 2a produce dietilaminotrimorpholino (2c). The solid-state structures of 3b and 3c revealed the intramolecular C–H  N hydrogen bondings. In addition, all of the new phosphazenes possessing electrochemically active Fc groups, can be thought of as good candidates for electron-transfer mediators and materials for electronic devices. Compounds (1b, 2b, 3b, and 3c) have potential antibacterial activities with almost all Gramnegative and Gram-positive bacteria tested. In addition, the compounds 2b and 3b have very strong antifungal activities against C. albicans ATCC 10231 and C. tropicalis ATCC 13803. Compounds 1b, 2b, 3b, and 3c were evaluated for antituberculosis activities

Fig. 5. Electrophoretograms applying to incubated mixtures of pUC18 plasmid DNA and compounds 1b, 2b, 3c, and 3b followed by their restriction with BamHI (A) and HindIII (B) for a period of 1 h at 37 °C, at concentrations of compounds 1250 lM and 625 lM. Lane1 applies to pUC18 plasmid DNA only. Lanes 2–3 apply to plasmid DNA and 1b. Lanes 4–5 apply to plasmid DNA and 2b. Lanes 6–7 apply to plasmid DNA and 3c. Lanes 8–9 apply to plasmid DNA and 3b.

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(in vitro) against M. tuberculosis H37Rv and six multidrug-resistant clinical M. tuberculosis isolates. The compounds 1b, 2b, and 3c exhibited antitubercular activities at the highest concentrations (10 000 and 5000 lM). It is noteworthy that, they were good candidates for developing new drugs against tuberculosis. On the other hand, the compounds were able to perform a cleavage of DNA. They were very effective in changing the mobility of the pUC18 plasmid DNA; however, 3b had a stronger effect on forms I and II of plasmid DNA than the other compounds. Moreover, restriction analyses indicate that phosphazene derivatives bind to G/G of the DNA. Acknowledgements The authors acknowledge the ‘‘Scientific and Technical Research Council of Turkey” (Grant 108T892). The authors also thank the ‘‘Medicinal Plants and Medicine Research Center of Anadolu University, Eskisßehir” for the use of their X-ray and NMR facilities. T.H. is indepted to ‘‘Hacettepe University, Scientific Research Unit” (Grant 02 02 602 002) for their financial support. Appendix A. Supplementary data CCDC 760529 and 760530 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected]. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.poly.2010.07.017. References [1] V. Chandrasekhar, P. Thilagar, B.M. Pandian, Coord. Chem. Rev. 251 (2007) 1045. [2] M. Rajeswara Rao, G. Gayatri, A. Kumar, G. Narahari Sastry, M. Ravikanth, Chem. Eur. J. 15 (2009) 3488. [3] H.R. Allcock, Curr. Opin. Solid State Mater. Sci. 10 (2006) 231. [4] H.R. Allcock, Chemistry and Applications of Polyphosphazenes, John Wiley and Sons, 2003. [5] M. Gleria, R. De Jaeger, Inorg. Organomet. Polym. 11 (2001) 1. [6] S.R. Contractor, Z. Kılıç, R.A. Shaw, J. Chem. Soc., Dalton Trans. (1987) 2023. [7] J.E. Marck, H.R. Allcock, R. West, Inorganic Polymers, second ed., Oxford University Press, New York, 2005. [8] B. Çosut, F. Hacıveliog˘lu, M. Durmusß, A. Kılıç, S. Yesßilot, Polyhedron 28 (2009) 2510. [9] J. Zhu, W. Liu, R. Chu, X. Meng, Tribology Int. 40 (1) (2007) 10. [10] H.R. Allcock, M.E. Napierala, C.G. Cameron, S.J.M. O’Connor, Macromolecules 29 (1996) 1951. [11] H.R. Allcock, E.C. Kellam, R.V. Morford, D.A. Conner, D.T. Welna, Y. Chang, H.R. Allcock, Macromolecules 40 (2007) 322. [12] R. Klein, D.T. Welna, A. Weikel, H.R. Allcock, J. Runt, Macromolecules 40 (2007) 3990. [13] K. Brandt, T.J. Bartczak, R. Kruszynski, I. Porwolik-Czomperlik, Inorg. Chim. Acta 322 (2001) 138. _ Öztürk, N.S. Vanlı, S. Kırbag˘, M. Arslan, Bioorg. Chem. 30 [14] Ö. Yılmaz, F. Aslan, A.I. (2002) 303.

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