Phosphorus–nitrogen compounds: Part 31. Syntheses, structural and stereogenic properties, in vitro cytotoxic and antimicrobial activities, and DNA interactions of bicyclotetraphosphazenes containing bulky side group

Accepted Manuscript Phosphorus-nitrogen compounds: Part 31. Syntheses, structural and stereogenic properties, in vitro cytotoxic and antimicrobial activities, and DNA interactions of bicyclotetraphosphazenes containing bulky side group Gürcü Mutlu, Gamze Elmas, Zeynel Kılı ç, Tuncer Hökelek, L. Yasemin Koç, Mustafa Türk, Leyla Aç ık, Betül Aydın, Hakan Dal PII: DOI: Reference:

S0020-1693(15)00361-8 http://dx.doi.org/10.1016/j.ica.2015.07.027 ICA 16620

To appear in:

Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

12 March 2015 14 July 2015 16 July 2015

Please cite this article as: G. Mutlu, G. Elmas, Z. Kılı ç, T. Hökelek, L. Yasemin Koç, M. Türk, L. Aç ık, B. Aydın, H. Dal, Phosphorus-nitrogen compounds: Part 31. Syntheses, structural and stereogenic properties, in vitro cytotoxic and antimicrobial activities, and DNA interactions of bicyclotetraphosphazenes containing bulky side group, Inorganica Chimica Acta (2015), doi: http://dx.doi.org/10.1016/j.ica.2015.07.027

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Phosphorus-nitrogen compounds : Part 31. Syntheses, structural and stereogenic properties, in

vitro

cytotoxic

and

antimicrobial

activities,

and

DNA

interactions

of

bicyclotetraphosphazenes containing bulky side group

Gürcü Mutlua, Gamze Elmasa, Zeynel Kılıça,*, Tuncer Hökelekb, L. Yasemin Koçc, Mustafa Türkd, Leyla Açıke, Betül Aydıne, Hakan Dalf

a

Department of Chemistry, Ankara University, 06100 Tandoğ an-Ankara, Turkey

b

Department of Physics, Hacettepe University, 06800 Beytepe-Ankara, Turkey

c

Department of Biology, Ankara University, 06100 Tandoğ an-Ankara, an Turkey

d

Department of Bioengineering, Kırıkkale University, 71450 Yahș ihan-Kırıkkale, ihan Turkey

e

Department of Biology, Gazi University, 06500 Teknikokullar-Ankara, Turkey

f

Department of Chemistry, Anadolu University, 26470 Yunus Emre Kampüsü-Eskiș ehir, Turkey

Abstract Hexachlorocyclotriphosphazene, N3P3Cl6, and octachlorocyclotetraphosphazene, N4P4Cl8, were reacted with K2N2O2 salt of symmetric tetradentate ligand to obtain spiro-bino-spiro [(sbs) (2)] and 2,6-spiro-ansa-spiro [(2,6-sas) (3)] phosphazenes, respectively. The sbs was obtained in a very poor yield, whereas, 2,6-sas was obtained in a moderate yield. The derivatives of 2,6-sas with mono- and diamines were synthesized. When the reactions were carried out, one equimolar amount of 2,6-sas with an excess pyrrolidine, piperidine, morpholine, 1,4-dioxa-8-azaspiro[4,5]decane (DASD), N-methylethane-1,2-diamine, N-ethylethane-1,2-diamine and N-methylpropane-1,3diamine, along with the fully substituted 2,6-sas-cyclotetraphosphazene derivatives (4a, 4b and 5a7a), were prepared. However, the excess morpholine and DASD with 2,6-sas yielded the geminal bis- (4c and 4e) and tris- (4d and 4f) cyclotetraphosphazenes, respectively. The Cl replacement reaction of 2,6-sas with one equimolar amount of 7 led to the formation of partly substituted 2,6sas (7b). The structures of the compounds were verified by elemental analyses, MS, FTIR, 1H,

13

C{1H},

31

P-NMR, HSQC, HMBC and X-ray crystallography (for 3 and 4a) techniques. All the

2,6-sas cyclotetraphosphazenes (except 3, 4a and 4b) have stereogenic P-atoms. All the compounds were screened for antibacterial and antifungal activities against bacteria and yeast strains. The interactions of the compounds with supercoiled plasmid pBR322 DNA were investigated. The evaluations for cytotoxic activity, and apoptotic and necrotic effects against A549 Lung cancer and L929 Fibroblast cell lines were introduced.

Keywords: Bicyclotetraphosphazenes, Spectroscopy, Stereogenism, Cytotoxicity, DNA binding, Crystallography * Corresponding author. Tel.:+90 0312 2126720-1043 E-mail address: [email protected] (Z. Kılıç). 1. Introduction

Phosphazene derivatives are hybrid molecules with an essentially linear and/or cyclic backbone of alternating phosphorous nitrogen atoms with the same and/or different organic side groups bonded to each phosphorous atom [1]. The chlorocyclophosphazenes, N3P3Cl6 and N4P4Cl8, are the best-known starting compounds and as such they have been extensively studied in the field of phosphazene chemistry [2, 3]. Although a large number of N4P4Cl8 derivatives have been synthesized with mono- and difunctional ligands [4-6], discussions of the substitution reaction patterns

with

polyfunctional

ligands

are

very

limited

in

the

literature

[7-9].

Hexachlorocyclotriphosphazene, N3P3Cl6, and octachlorocyclotetraphosphazene, N4P4Cl8, with bidentate and/or polydentate amines can produce spiro, ansa, dispiro (2,4- and 2,6-), trispiro, tetraspiro, spiro-ansa (2,4- and 2,6-), spiro-ansa-spiro (sas), bino, spiro-bino, and di(spiro-bino) products depending on the reaction conditions [10-12]. Up to now, two kinds of 2,6-bicyclo tetraphosphazene derivatives were obtained from the reactions of N4P4Cl8 with mono-functional amines (Fig. 1a) [13] and multi- functional reagents (Fig. 1b) [8]. Fig. 1 and Scheme 1. here about

A wide range of side groups (R1 and R2, Fig. 1a) may be bonded to this skeleton, leading to products with a similarly diverse range of physical and chemical properties [14]. Previously, to our knowledge there were only two papers about 2,4-sas and 2,6-sas bicyclophosphazenes in the literature [8, 9]. As part of our ongoing study of the reactions of N4P4Cl8 with multidendate ligands, we have concentrated primarily on the substituent exchange reactions of N4P4Cl8 with potassium {2,2'-[1,3-phenylenebis(methyleneiminomethylene)]diphenoxide, K2N2O2, (1a)} with the aim of obtaining bicyclotetraphosphazene derivatives and also exploring their biological activity. As known, mono- and polyamino substituted cyclotriphosphazene derivatives (e.g. aziridine, spermine and spermidine) have attracted a great deal of attention for their potential as anti-cancer agents [15, 16]. They exhibited cytotoxic activity against HT-29 (human colon adenocarcinoma), Hep2 (Human epidermoid carcinoma of the larynx), and Vero (African green monkey kidney) cells and stimulated apoptosis [17]. In addition, the antimicrobial activity of cyclotri- and tetraphosphazene derivatives was investigated against various bacteria and fungi [18, 9]. On the other hand, studies on the biological activity of cyclotetraphsphazene derivatives are very limited. It is known that octapyrrolidinocyclotetraphosphazene demonstrates significant anticancer activity [15]. The Cu(II) complex of a fully phenoxy-substituted star-branched cyclotetraphosphazene is active in the oxidative cleavage of DNA [19]. The present study focuses on the Cl replacement reactions of N4P4Cl8 with N2O2 tetradentate ligand (1) (Scheme 1) with the aim of obtaining the new 2,6-sas-bicyclotetraphosphazene derivatives, and to investigate the in vitro cytotoxic activity and apoptosis and necrosis effects against A549 Lung cancer and L929 Fibroblast cell lines. The evaluation of antimicrobial activity and DNA interactions of all the compounds were also presented.

2. Experimental section

2.1. Materials and method All reactions were monitored using thin-layer chromatography (TLC) on Merck DC Alufolien Kiesegel 60 B254 sheets. Column chromatography was performed on Merck Kiesegel 60 (230-400 mesh ATSM) silica gel. The reactions were run out under argon atmosphere. Melting points were assessed with a Gallenkamp apparatus using a capillary tube. The Fourier transform infrared (FTIR) spectra were recorded on a Jasco FT/IR-430 spectrometer in KBr discs and reported in cm-1 units. One-dimensional (1D) 1H,

13

C and

31

P NMR and two-dimensional (2D)

heteronuclear single quantum coherence (HSQC), and heteronuclear multiple- bond correlation (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 5mm PABBO BB inverse gradient probe. Standard Bruker pulse programs [20] were utilized. APIES mass spectra were recorded on the AGILENT 1100 MSD spectrometer.

2.2. Synthesis of compounds 2,2'-[1,3-phenylenebis(methyleneiminomethylene)]diphenol (1) was synthesized according to the methods reported in the literature [21, 22].

2.2.1. Synthesis of sbs (2) K2CO3 (2.40 g, 18.00 mmol) was added to a stirred solution of (1) (1.04 g, 3.01 mmol) in dry THF (150 mL). The mixture was refluxed for 4 h and cooled to ambient temperature. Afterwards, a mixture of triethylamine (2.42 mL, 17.4 mmol) and N3P3Cl6 (1.05 g, 3.00 mmol) in dry THF (100 mL) was added dropwise to the stirred solution of (1) at -10 °C under Ar atmosphere. The mixture was stirred for over 5 h at the same temperature. After the mixture was allowed to warm to ambient temperature, it was stirred for 4 days. The precipitated amine hydrochloride and excess of K2CO3 were filtered off, and the solvent was evaporated. The product (2) was purified by column

chromatography with toluene, and it was crystallized from acetonitrile. In addition, the experiments were also repeated in toluene and acetonitrile. But, the product could not be obtained in acetonitrile. Yield: 0.12 g (5%) in THF, 0.09 g (3%) in toluene. mp: 175 °C. Anal. Calcd. for C22H20O2N8P6Cl8: C, 29.42; H, 2.21; N, 12.48. Found: C, 29.27; H, 2.31; N, 12.48. APIES-MS (fragments are based on

35

Cl, Ir %, Ir designates the fragment abundance percentage): m/z 895

([MH]+, 57). FTIR (KBr, cm-1): ν 3066 (asymm.) and 3041 (symm.) (C-H arom.), 2915; 2852 (C-H aliph.), 1587 (C=C), 1219 (asymm.) and 1182 (symm.) (P=N), 576 (asymm.) and 520 (symm.) (PCl).

2.2.2. Synthesis of tetrakischloro-2,6-sas (3) The work-up procedure was similar to that of compound 2, using 1 (1.78 g, 5.05 mmol), K2CO3 (4.30 g, 30.60 mmol), N4P4Cl8 (2.38 g, 5.05 mmol), and triethylamine (4.30 mL, 30.9 mmol). The product (3) was purified by column chromatography with toluene, and it was crystallized from acetonitrile. In addition, the experiments were also repeated in toluene and acetonitrile, but the product could not be obtained in toluene. Yield: 1.20 g (35%) in THF, 0.1 g (3%) in acetonitrile. mp: 213 °C. Anal. Calcd. for C22H20O2N6P4Cl4.CH3CN: C, 40.79; H, 3.44; N, 14.41. Found: C, 40.35; H, 3.39; N, 13.62. APIES-MS (fragments are based on 35Cl, Ir %): m/z 665 ([MH]+, 65). FTIR (KBr, cm-1): ν 3066 (asymm.) and 3033 (symm.), (C-H arom.), 2958;2852 (C-H aliph.), 1585 (C=C), 1298 (asymm.) and 1178 (symm.) (P=N), 551 (asymm.) and 491 (symm.) (PCl).

2.2.3. Synthesis of tetrakispyrrolidino-2,6-sas (4a)

A solution of pyrrolidine (0.40 mL, 4.80 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.33 mL, 2.40 mmol) and (3) (0.40 g, 0.60 mmol) in dry THF (100 mL) at room temperature. It was stirred for 4 days under Ar atmosphere. The product (4a) was purified by column chromatography with benzene-THF (4:1) and a white powder was crystallized from acetonitrile. Yield: 0.22 g (45%). mp: 199 °C. Anal. Calcd. for C38H52O2N10P4 : C, 56,72; H, 6,47; N, 17,41. Found: C, 56,78; H, 6,19; N, 17,76. APIES-MS (fragments are based on 35Cl, Ir %): m/z 806 ([M-2H]+, 100). FTIR (KBr, cm-1): ν 3066 (asymm.) and 3039 (symm.) (CH arom.), 2950; 2863 (C-H aliph.), 1583 (C=C), 1296 (asymm.) and 1192 (symm.) (P=N).

2.2.4. Synthesis of tetrakispiperidino-2,6-sas (4b) A solution of piperidine (0.60 mL, 6 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.33 mL, 2.40 mmol) and (3) (0.50 g, 0.80 mmol) in dry THF (100 mL) at room temperature. It was stirred for 4 days under Ar atmosphere. The product (4b) was purified by column chromatography with benzene and a white powder was crystallized from acetonitrile. Yield: 0.25 g (39%). mp: 155 °C. Anal. Calcd. for C42H60O2N10P4 : C, 58.56; H, 6.97; N, 16.27. Found: C, 58.60; H, 7.02; N, 16.27. APIES-MS (fragments are based on

35

Cl, Ir %): m/z 862

([M+2H]+, 100). FTIR (KBr, cm-1): ν 3072 (asymm.) and 3037 (symm.) (C-H arom.), 2929; 2848 (C-H aliph.), 1583 (C=C), 1279 (asymm.) and 1187 (symm.) (P=N).

2.2.5. Syntheses of geminal-bismorpholino-2,6-sas (4c) and trismorpholino-2,6-sas (4d) A solution of morpholine (0.42 mL, 4.80 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.33 ml, 2.40 mmol) and (3) (0, 40 g, 0.60 mmol) in dry THF (100 mL) at room temperature. It was stirred for 4 days under Ar atmosphere. The products 4c and 4d were purified by column chromatography with toluene-THF (6:1). Compound (4c) was crystallized from acetonitrile. Yield: 0. 11 g (24%). mp: 240 °C. Anal. Calcd. for C30H36O4N8P4Cl2: C, 46,92; H, 4.69; N, 14.60. Found: C, 47.44; H, 4.45; N, 14.89. APIES-MS

(fragments are based on 35Cl, Ir %): m/z 767 ([MH]+,100). FTIR (KBr, cm-1): ν 3053 (asymm.) and 3031 (symm.) (C-H arom.), 2954; 2840 (C-H aliph.), 1587 (C=C), 1255 (asymm.) and 1187 (symm.) (P=N), 576 (asymm.) and 549 (symm.) (PCl). Compound (4d) was crystallized from acetonitrile. Yield: 0. 18 g (37%). mp: 148 °C. Anal. Calcd. for C34H44O5N9P4Cl: C, 49.94; H, 5.39; N, 15.42. Found: C, 50.35; H, 5.50; N, 15.39. APIES-MS (fragments are based on 35Cl, Ir %): m/z 818 ([MH]+, 100). FTIR (KBr, cm-1): ν 3050 (asymm.) and 3032 (symm.) (C-H arom.), 2955; 2841 (C-H aliph.), 1587 (C=C), 1255 (asymm.) and 1162 (symm.) (P=N), 577 (PCl).

2.2.6. Syntheses of geminal-bis(1,4-dioxa-8-azaspiro[4,5]deca)-2,6-sas (4e) and tris(1,4-dioxa -8azaspiro [4,5]deca)-2,6-sas (4f and 4f') A solution of DASD (0.62 mL, 4.80 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.33 mL, 2.40 mmol) and (3) (0, 40 g, 0.60 mmol) in dry THF (100 mL) at room temperature. It was stirred for 4 days under Ar atmosphere. The products 4e and 4f were purified by column chromatography with toluene-THF (6:1). Compound (4e) was crystallized from acetonitrile. Yield: 0. 12 g (23%). mp: 216 °C. Anal. Calcd. for C36H44O6N8P4Cl2: C, 46,15; H, 5.01; N, 12.74. Found: C, 49.53; H, 5.43; N, 12.52. APIES-MS (fragments are based on 35Cl, Ir %): m/z 879 ([MH]+,100). FTIR (KBr, cm-1): ν 3052 (asymm.) and 3023 (symm.) (C-H arom.), 2969; 2850 (C-H aliph.), 1583 (C=C), 1279 (asymm.) and 1186 (symm.) (P=N), 549 (asymm.) and 482 (symm.) (PCl). Compounds (4f and 4f') were crystallized from acetonitrile. Yield: 0. 11 g (19%). mp: 136 °C. Anal. Calcd. for C43H56O8N9P4Cl: C, 52.32; H, 5.68; N, 12.79. Found: C, 51.42; H, 5.54; N, 12.17. APIES-MS (fragments are based on 35Cl, Ir %): m/z 986 ([MH]+, 98). FTIR (KBr, cm-1): ν 3057 (asymm.) and 3026 (symm.) (C-H arom.), 2956; 2877 (C-H aliph.), 1583 (C=C), 1286 (asymm.) and 1180 (symm.) (P=N), 570 (PCl).

2.2.7. Synthesis of geminal-bis(N-methylethane-1,2-diamino)-2,6-sas (5a) A solution of N-methylethane-1,2-diamine (0.30 mL, 3 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.33 mL, 2.40 mmol) and (3) (0.40 g, 0.60 mmol) in dry THF (100 mL) at room temperature. It was stirred for 4 days under Ar atmosphere. The product (5a) was purified by column chromatography with toluene/THF (2:1) and a white powder was crystallized from acetonitrile. Yield: 0.17 g (42%). mp: 86 °C. Anal. Calcd. for C28H36O2N10P4.(C7H8)1/2: C, 52.94; H, 5.60; N, 19.61. Found: C, 52.80; H, 6.02; N, 18.93. APIESMS (fragments are based on 35Cl, Ir %): m/z 668 ([M]+, 100). FTIR (KBr, cm-1): ν 3057 (asymm.) and 3039 (symm.) (C-H arom.), 2927; 2870 (C-H aliph.), 1581 (C=C), 1278 (asymm.) and 1184 (symm.) (P=N).

2.2.8. Synthesis of geminal-bis(N-ethylethane-1,2-diamino)-2,6-sas (6a) A solution of N-ethylethane-1,2-diamine (0.30 mL, 2.8 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.33 mL, 2.40 mmol) and (3) (0.40 g, 0.60 mmol) in dry THF (100 mL) at room temperature. It was stirred for 4 days under Ar atmosphere. The product (6a) was purified by column chromatography with toluene/THF (2:1) and a white powder was crystallized from acetonitrile. Yield: 0.14 g (33%). mp: 77 °C. Anal. Calcd. for C30H40O2N10P4.(C7H8)1/2: C, 54.18; H, 5.93; N, 18.87. Found: C, 53.87; H, 5.88; N, 18.17. APIESMS (fragments are based on 35Cl, Ir %): m/z 697 ([MH]+, 100). FTIR (KBr, cm-1): ν 3057 (asymm.) and 3039 (symm.) (C-H arom.), 2962; 2852 (C-H aliph.), 1583 (C=C), 1280 (asymm.) and 1186 (symm.) (P=N).

2.2.9. Synthesis of geminal-bis(N-methylpropane-1,3-diamino)-2,6-sas (7a) A solution of N-methylpropane-1,3-diamine (0.30 mL, 2.9 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.33 mL, 2.40 mmol) and (3) (0.40 g, 0.60 mmol) in dry THF (100 mL) at room temperature. It was stirred for 4 days under Ar atmosphere.

The product (7a) was purified by column chromatography with toluene/THF (2:1) and a white powder was crystallized from acetonitrile. Yield: 0.15 g (37%). mp: 207 °C. Anal. Calcd. for C30H40O2N10P4.(C7H8)1/2:

C, 54.18; H, 5.93; N, 18.87. Found: C, 54.33; H, 6.19; N, 17.89.

APIES-MS (fragments are based on 35Cl, Ir %): m/z 697 ([MH]+, 100). FTIR (KBr, cm-1): ν 3056 (asymm.) and 3039 (symm.) (C-H arom.), 2938; 2869 (C-H aliph.), 1581 (C=C), 1280 (asymm.) and 1187 (symm.) (P=N).

2.2.10. Synthesis of mono(N-methylpropane-1,3-diamino)-2,6-sas (7b) A solution of N-methylpropane-1,3-diamine (0.13 mL, 1.3 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.33 mL, 2.40 mmol) and (3) (0.42 g, 0.63 mmol) in dry THF (100 mL) at room temperature. It was stirred for 3 days under Ar atmosphere. The product (7b) was purified by column chromatography with toluene/THF (2:1) and a white powder was crystallized from acetonitrile. Yield: 0.06 g (14%). mp: 103 °C. Anal. Calcd. for C26H30O2N8P4Cl2.(C7H8)1/2: C, 48.69; H, 4.68; N, 15.41. Found: C, 48.00; H, 5.24; N, 14.93. APIES-MS (fragments are based on

35

Cl, Ir %): m/z 680 ([M]+, 100). FTIR (KBr, cm-1): ν 3080

(asymm.) and 3033 (symm.) (C-H arom.), 2958; 2854 (C-H aliph.), 1585 (C=C), 1297 (asymm.) and 1178 (symm.) (P=N), 553 (asymm.) and 516 (symm.) (PCl).

2.3. X-ray crystallography The colorless crystals of compounds 3 and 4a were crystallized from acetonitrile at room temperature. The crystallographic data were given in Table 1, selected bond lengths and angles were listed in Table S1, S designates Supplementary Data, and hydrogen bond data were presented in Table S2. Crystallographic data were recorded on a Bruker Kappa APEXII CCD area-detector diffractometer using Mo Kα radiation (λ=0.71073 Å) at T=100(2) K. Absorption corrections by multi-scan [23] were applied. Structures were solved by direct methods and refined by full-matrix least squares against F2 using all data [24]. All non-H atoms were refined anisotropically.

Table 1 here about

3. Results and discussion

3.1. Synthesis The reaction of N3P3Cl6 with an equimolar amount of K2N2O2 (1a) in THF and toluene gave only sbs (2) product in a poor yield. When the reaction was carried out in acetonitrile, no product was isolated. However, the reaction of N4P4Cl8 with an equimolar amount of 1a in THF produces the partly (2,6-dispiro-bicyclo) substituted 2,6-sas compound (3) with 35% yield. The yield of 3 in acetonitrile was 3%, but the same compound was not obtained in toluene. The other expected products e.g. 2,4-sas, 2,4,6-asa and sbs were not detected in the reaction mixture. However, gummy products were left in the column during the column chromatography. The compound 3 has four susceptible Cl atoms at the 4,8-positions. These atoms can be replaced with mono- and difunctional reagents (Scheme 1). The condensation reactions of 3 with excess pyrrolidine and piperidine produced the fully substituted tetrapyrrolidino (4a) and tetrapiperidino (4b) cyclotetraphosphazenes. When the reactions were carried out with excess morpholine and DASD, the geminal di- (4c and 4e) and tri- (4d and 4f) cyclotetraphosphazenes were isolated, but the expected fully substituted compounds were not obtained, possibly due to the steric interactions of the bulky substituents. Furthermore, compound 3 with excess diamines (5-7) gave stereoselectively dispiro cyclotetraphosphazenes (5a-7a) with moderate yields (ca. 37%). The

31

P NMR spectra of

these reaction mixtures disclosed that the monospiro-2,6-sas cyclotetraphosphazenes (5b-7b) also occur as byproducts. Fig. 2 illustrates that compounds 5a and 5b are present together in the reaction mixture. In addition, compound 7b was obtained directly from the reaction of one equimolar amount of 3 with one equimolar amount of 7 with a poor yield (15%). In all these reactions, gummy and sticky products, possibly oligomeric or polymeric phosphazenes, were observed.

Fig. 2 here about The plausible reaction pathways, SN2 (P) and/or SN1 (P) are shown in Fig. S1 according to the isolated product distributions. It appears that the dominant reaction pathway of N4P4Cl8 with 1a, at least for the first stage of Cl replacement, is likely to be the SN2 (P) type, involving the formation of the five-coordinated intermediate. The geminal reaction pathway occurs with SN1 (P) route in the second stage of the Cl replacement reaction to produce a spiro ring. But it is also possible to form the 2,6-ansa ring via SN1 (P) pathway. On the other hand, the reactions of 3 with secondary amines and diamines gave di-, tri- and tetra- substituted products in THF. All the geminal and spiro products are likely to be formed according to the SN1 (P) route. However, the occurrence of geminal products does not prove SN1 (P). In this case, the spirocyclic products are most likely the result of minimizing ring strain. The tri- and tetra- substituted derivatives at least may occur competitively via SN1 (P) or SN2 (P) pathways. The 2,6-sas compound (3) is the first example of cyclotetraphosphazene derivatives with the bulky N2O2 donor-type aminopodand. Data obtained from the microanalyses FTIR, APIES-MS and 1H,

13

C{1H} and

31

P NMR,

HSQC and HMBC are in agreement with the proposed structures of the phosphazene derivatives. The mass spectra of all the compounds show the molecular (M)+, protonated molecular (MH)+ and diprotonated molecular (M+2H)+ ion peaks. The crystal structures of 3 and 4a were also determined by X-ray crystallography.

3.2. NMR and FTIR spectroscopy The spin systems of the tetrameric phosphazenes show that only one kind of tetrameric phosphazene derivatives, namely 2,6-sas (3, 4a-4f, 5a-7a and 5b-7b), were formed (Fig. 3). Fig. 3 here about

As anticipated, the 1H-decoupled 31P NMR spectra of 2, 3 and the fully substituted derivatives (4a, 4b and 5a-7a) show AX2, A2B2 and A2X2 type spectra, respectively, because of the two different phosphorus environments within the molecules (Table 2). The chemical shifts of spiro and PCl2 values of 3 are -11.81 and -9.52 ppm. The δ OPN and δ NPN shifts of fully substituted (4a and 4b) and dispiro-2,6-sas (5a-7a) cyclotetraphosphazenes are significantly larger than those of the starting compound 3 due to the substitution of Cl atoms with secondary amines and diamines. The geminal disubstituted 2,6-sas compounds (4c, 4e and 7b) having three different phosphorus environments (Fig. 3), display the A2MX (for 4c and 4e) and A2BX (for 7b) spin systems consisting of three multiplets at the 1 H-decoupled 31P NMR spectra. As examples, the 1Hdecoupled and coupled 31P NMR spectra of 4e are depicted in Figs. 4a and 4b, respectively. All the phosphorus atoms were unambiguously distinguished from the 1H-coupled

31

P NMR spectrum of

4e, showing three sets of multiplets corresponding to the P (DASD)2 (PX), P spiro (PA) and PCl2 (PM) groups (Fig. 4b). The δ P and the coupling constant (2JPP) values of the other geminal phosphazenes were also assigned as 4e. Fig. 4 and Table 2 here about

The chirality of the cyclotri- and cyclotetraphosphazenes is an interesting subject that has been investigated more frequently during the last decade [9, 25]. In these phosphazene derivatives, when the tetracoordinated pentavalent phosphorus atoms have two different substituents, they are likely to be stereogenic centres. The investigations reported the stereogenic properties of cyclotriphosphazene derivatives, including those with one, two equivalent, two different and three different stereogenic centres [26]. The chiralty in cyclotriphosphazenes was evaluated by spectroscopic methods such as X-ray crystallography, CSA added NMR spectroscopy and/or chiral HPLC in the literature [27-29]. To date, the studies about chiral cyclotetraphosphazenes are very limited in the literature [9]. In this study, the chiral cyclotetraphosphazene derivatives were synthesized using dipotassium salt (1a) of the tetradentate symmetric ligand (1). The choice of the ligand (1) is very important to this study, because this ligand causes restricted conformations of the

cyclotetraphosphazene derivatives. Therefore, optical isomers occur in a certain number. Theoretical stereoisomer distributions and expected geometrical isomers of the compounds 4c-4f, 5a-7a and 5b-7b are given in Table S3. The stereoisomer distributions can be rationalized with Fig. 5, a stick diagram (Fig. 6), the X-ray crystallographic data and NMR results. The starting compound 3 certainly has a meso form, according to the results obtained by X-ray and from NMR data. The findings showed that 2,6 sas cyclotetraphosphazenes (4c and 4e) with two equivalent chiral

centres

give

only

meso

(RS/SR)

compounds

(Fig.

6a).

In

contrast,

the

cyclotetraphosphazenes (4d, 4f and 5b-7b) that have one different-two equivalent chiral centres give two racemic isomers (RSR'/RSS' and SRR'/SRS') (Figs. 6b and 6d). However, the compounds (5a-7a) with one different-two equivalent chiral centres only produce two products (RSS' and SRS' diastereoisomer), according to Fig. 6c. The reason for this is that if two phosphorus atoms have the same substituents and one P atom has different substituents in a cyclotetraphosphazene ring, the fourth P atom is not chiral. Therefore, the cyclotetraphosphazenes such as compounds 5a-7a have one different-two equivalent chiral centres. The tri-substituted 2,6-sas compounds (4d, 4f and 4f') have A2MX spin systems possessing three sets of triplets corresponding to the Pspiro (PA), PNCl (PM) and PNN (PX) groups. The NMR data for the reaction mixtures of 4f and 4f' show that both of the compounds are likely to be diastereoisomers (Table S3). In contrast, the

31

P NMR data for the reaction mixtures of the other

cyclotetraphosphazenes indicate that the diastereoisomers were not present. However, 4f and 4f' were not separated using column chromatography. Fig. 5 and Fig. 6 here about The 1 H and

13

C{1H} NMR signals of the phosphazene derivatives were interpreted in the light of

chemical shifts, multiplicities and coupling constants (Tables S1 and S2). The assignments were established by HSQC using values corresponding to

1

J(CH) and by HMBC using values

corresponding to 2J(CH), 3J(CH) and 4J(CH) between the protons and carbons. The HSQC and HMBC spectra of 4b are given in Figs. S2a and S2b. In light of the 1 H and 13C{1H} NMR data, the sbs (2) and 2,6-sas (3, 4a-4c and 4e) compounds appear to have symmetric structures in solution.

However, 3 and 4a do not have symmetric structures in the solid state, according to the X-ray crystallographic data. The 1 H NMR spectra of the phosphazene derivatives are given in Table S4. The chemical shifts of diastereotopic benzylic Ar-CH2 and O-Ar-CH2 protons are separated from each other and can be clearly distinguished. The benzylic Ar-CH2 peak groups are in the range of δ 4.59-4.22 ppm and the benzylic O-Ar-CH2 protons are in the range of δ 4.41-4.18 ppm. The benzylic protons of 2, 3, 4a, 4b, 5a and 6a give rise to the doublets, while the benzylic protons of 4c-4f (its diastereomer 4f'), 7a and 7b give rise to an ABX spin system, due to the geminal proton-proton coupling and vicinal coupling with the phosphorus-31 nucleus. Thus, the peaks of benzylic protons are observed as doublets of doublets for 4c-4f (4f'), 7a and 7b. The average 2JHH and 3JPH coupling constants of these compounds, which have ABX spin systems, are 4.0 and 15.2 Hz for benzylic Ar-CH2 and 5.3 and 4.0 Hz for benzylic O-Ar-CH2. As expected, the δOCH2 shifts of the geminal (4c and 4e) and trisubstituted (4d, 4f and 4f') cyclotetraphosphazenes are observed separately because of two different -OCH2 environments within the geminal substituents (Fig. 3). The

13

C{1H} NMR data for all the phosphazene derivatives are listed in Table S5. The two

bond-coupling constants, 2JPNC for the CH3-N-CH2 carbons of the five-membered spiro rings of compounds 5a and 6a are larger than those of 7a and 7b, which contain the six membered spiro rings. The 2JPNC values of aromatic C6 carbons of the cyclotetraphosphazenes are in the range of 6.0-6.9 Hz. The average value is 6.4 Hz. In addition, the 3JPNCC values of aromatic C1 and C5 carbons are in the ranges of 8.4-9.3 Hz and 8.5-9.9 Hz. The average values of C1 and C5 carbons are estimated as 8.8 Hz and 9.0 Hz, respectively. On the other hand, the 3JPNCC values of bicyclo phenyl carbons (C1' and C5') for all the phosphazanes, excluding sbs compound (2), are not observed. This situation may depend on the interactions of the π electrons of the tetrameric and phenyl rings. In other words, the ring currents of the tetrameric ring may affect the electron delocalizations of the bridged phenyl ring. The characteristic νN-H band of aminopodand (1) at 3277 cm-1 is not present in the FTIR spectra of the phosphazenes. The sbs (2) phosphazene displays strong νP=N absorption frequencies

in the range of 1219-1182 cm-1. All of the cyclotetraphosphazenes exhibit strong absorption frequencies in the ranges of 1298-1279 cm-1 (asymm.) and 1192-1162 cm-1 (symm.), which are ascribed to νP=N bands of the phosphazene ring [30, 9], and two medium-intensity absorption signals between 3080-3050 cm-1 and between 3041-3032 cm-1 attributed to the asymmetric and symmetric stretching vibrations of the Ar-H bonds.

3.3. X-ray structures of 3 and 4a The molecular and solid-state structures of 3 and 4a, along with the atom-numbering schemes, are illustrated in Figs. 7 and 8. The torsion angles of the ring bonds display the conformation of the phosphazene rings (Fig. S3). Compounds 3 and 4a consist of the noncentrosymmetric non-planar macrocyclic tetrameric phosphazene rings with the tetradentate ligand (1) bonded to the P atoms in a spiro-ansa-spiro (2,6-sas) fashion. Both of the tetrameric rings [(P1/N1/P2/N2/P3/N3/P4/N4)] have boat conformations in 3 [Fig. S4a; QT= 0.669(2) Å [31], ϕ2= 65.28(1)°, θ2= 84.66(1)°] and 4a [Fig. S5a; QT= 0.877(1) Å, ϕ2= -140.82(0.1)°, θ2= 83.01(0.09)°]. In 3 and 4a, the bicyclic parts (Figs. S4b and S5b) are made up of eight-membered tetrameric N4P4 and ansa (N1/P1/N5/C13/C14/C19/C18/C20/N6/P3/N2/P2) rings fused by the PNPNP fragments. The six-membered spiro rings [(P1/N5/C6/C7/C12/O1) and (P3/N6/C21/C22/C27/O2)] of 3 [Fig. S4c; QT= 0.640(2) Å, ϕ2= 116.29(0.3)°, θ2= 41.62(0.18)°] and 4a [Fig. S5c; QT= 0.889(5) Å, ϕ2= -32.49(0.26)°, θ2= 93.43(0.26)°] are in twisted-boat conformations. The endocyclic P—N bond lengths of 3 and 4a are in the ranges of 1.544(2) to1.590(2) Å and 1.559(2) to 1.596(2) Å, respectively. The average endocyclic P—N bond lengths in tetrameric phosphazene rings are 1.565(2) and 1.574(2) Å, respectively, which are shorter than the average exocyclic P—N bond lengths of 1.643(2) Å and 1.655(2) Å for 3 and 4a, respectively. Moreover, in compounds 3 and 4a, P-O, P-Cl, exocylic P-N and endocylic P-N bonds separately seem to be almost equal to each other. It is known that the PN single and double bonds in the phosphazenes vary in the ranges of 1.628 to 1.691 Å and 1.571 to 1.604 Å [32].

In compound 3, the opposite two P—N—P angles [P1—N1—P2; 132.88(13)° and P3—N3— P4; 133.04(12)°] are much smaller than those of the other endocylic P—N—P [P1—N4—P4; 142.91(13)° and P2—N2—P3; 144.92(12)°] angles. However, as can be seen from Table S1, the two opposite endocylic N—P—N angles are almost the same. The variations in the endocyclic N— P—N and P—N—P bond angles are quite large, ranging from 117.46(10)° to 123.88(10)°, and from 132.88(13) to 144.92(12), respectively. In compound 4a, the endocyclic P—N—P angles [P1—N1—P2; 130.88(14)° and P3—N3—P4; 130.49(13)°] are nearly the same, while the [P2— N2—P3; 141.98(14)°] bond angle is much smaller than [P1—N4—P4; 148.03(14)°], probably due to the intermolecular C19—H19···N2 hydrogen bonding (Table S2). The endocylic P—N—P bond angles in compounds 3 and 4a are greatly affected by the N2O2 donor-type tetradentate ligand 1, bonded to the P atoms, while the endocylic N—P—N and exocylic O—P—N bond angles are affected to a lesser extent as compared to the corresponding angles in the standard compound, N4P4Cl8. In N4P4Cl8, the endocyclic N—P—N, P—N—P and exocylic Cl—P—Cl bond angles are 121.2º, 131.3º and 102.8º, respectively [33]. Recently, the formation of the Pπ-Nπ bonds of the cyclophosphazenes have been explained by negative hyperconjugation [34-36]. Eventually, the shortening of the P-N bonds and variations of bond angles in 3 and 4a could be attributed to the presence of negative hyperconjugation. Compounds 3 and 4a, have intramolecular C—H—N bonds. In addition, there is an intermolecular C-H-N hydrogen bond between 3 and an acetonitrile molecule (Fig. 7). In both compounds, the C—H···л contacts may further stabilize the packing of the crystal structures.

Fig. 7 and Fig. 8 here about

3.4. Cytotoxicity screenings of the compounds Cytotoxicity was determined via a WST-1 test and experimental details are given in Section S1. Compounds 3 and 4f were evaluated for their cytotoxic activity against L929 Fibroblast and A549 lung cancer cell lines. Approximately 5000 cells (L929 and A549) in their exponential

growth phase were seeded in a flat-bottomed 96-well plate and were then incubated for 24 h at 37 °C in a 5% CO2 incubator. A series of dilutions (6.25-100 µg/mL) of 3 and 4f in the medium were added to the plate in hexaplets. The results obtained from the cytotoxic activity of 3 and 4f are given in Table S6 and Figs. 9a and 9b. The experiments were conducted using different concentrations of 3 and 4f to cause cytotoxicity on L929 Fibroblast and A549 lung cancer cell lines. The data show that more than 78% fibroblast cells were viable after incubation with 3 at concentrations of 100 µg/mL. In addition, a series of decreasing concentrations of 3 shows excellent cell viability. There was a marginal cytotoxic effect of 3 on A549 lung cell lines more than the fibroblast cells and demonstrated low cytotoxic activity with decreasing concentrations of 3. The viability of cell cancer varies between 24-85% (Table S6). After treatments of 3 and 4f, L929 fibroblast (22%) and (25%) show, respectively, cytotoxicity even up to a concentration of 100 µg/mL and have lower toxicity with decreasing concentrations of 3 and 4f. In contrast, 4f exhibits 92-101% viability of A549 lung cancer cell lines at different concentrations. Compound 3 shows 24±0.05% relative cell viability at 100 µg/mL. It is important to note that 3 and 4f displayed moderate cytotoxic activity against fibroblast cell lines at low concentrations, but 4f exhibited a strong cytotoxic effect against A549 lung cancer cell lines at 12.5 µg/mL concentrations. Fig. 9 here about

3.5. Apoptosis and necrosis Determination of apoptosis and necrosis with using a double-staining method are given in Section S1. The percentage of apoptotic cells displayed at 16 ± 2.0% of L929 fibroblast cells and 18 ± 3.0% of A549 when treated with the highest concentration of 3 (100 ߤg/mL), while the highest percentage of the necrotic cells were found to be 18.0 ± 1.0% of L929 fibroblast cells and 52 ± 5.0% on A549 when treated with 100 ߤg/mL of 3 (Table S7). The apoptotic and necrotic cells seem to remain moderate by reducing the concentrations. The increase in the percentage of apoptotic cells when treated with 4f are similar to the results when treated with 3. When A549 cells

are treated with 4f, the necrotic cells appear to increase more than L929 fibroblast cells at 15.0 ± 3.0% with 100 ߤg/mL (Fig. 10). Therefore, 3 and 4f have the capability to induce apoptosis and necrosis on cancer cell lines, dose dependently. In addition, using compound 3 on L929 fibroblast cells, especially A549 cancer cells, is more effective than using 4f. Fig. 10 here about 3.6. Antimicrobial activity The antimicrobial activity of a compound can depend on a combination of steric, electronic and pharmacokinetic factors. The action of the compound may involve the formation of a hydrogen bond through the active centers of the cell constituents with the hydrogen donor/acceptor atoms, resulting in interference with normal cell processes. All the compounds are screened for antimicrobial activity against 7 types of bacteria and 3 yeast strains, with the experimental details explained Section S1. The results of the antimicrobial activity of the cyclotetraphosphazenes are given in Table S8a. Moreover, aminopodand 1, diamines 5-7, the starting compounds (N3P3Cl6 and N4P4Cl8), pyrrolidine, morpholine, piperidine and DASD are all checked against the same bacteria and fungi. These findings are presented in Table S8b and S8c for comparison with the cyclotetraphosphazene derivatives. The antimicrobial activity of the cyclotetraphosphazenes may have arisen from the whole structure of these compounds. Most of the bacteria and all of the yeast strains demonstrated some degree of sensitivity to the compounds. However, the most effective compounds were aminopodand 1 and diamines 5-7. In general, the microorganisms show similar susceptibilities to all the compounds, with minimum inhibitory concentrations (MIC) ranging from 312 to 5000 µM (Table S9). The aminopodand 1, diamines 5-7, and 5a appear preferentially more active than the other compounds, exerting greater inhibitory activity against E. coli. The microbotoxicity of these compounds may be ascribed to the formation of hydrogen bonds with the active centers of the bacterial cell constituents.

3.7. Interactions of DNA with the compounds

The interaction of a compound with DNA may depend on the binding/cleavage properties with the nucleobases of DNA, resulting in changes to the DNA conformation. These changes in DNA conformation can play an important role in the rate of migration of DNA in an electric field [38]. Interactions between the starting compounds (N3P3Cl6 and N4P4Cl8), cyclotetraphosphazene derivatives and pBR322 plasmid DNA are studied using agarose gel electrophoresis. These experimental details are described Section S1. [39, 40]. Fig. 11 shows the electrophoretograms applied to incubated mixtures of pBR322 DNA at varying concentrations (5000 µM to 312 µM) of compounds. Lane 1 applies to the untreated pBR322 plasmid DNA (control DNA), showing the major supercoiled circular form I and minor singly nicked relaxed circular form II of the plasmid DNA. Lanes 2-6 apply to pBR322 plasmid DNA incubated with compounds ranging from 5000 µM to 312 µM. Further evaluation of the compounds with plasmid DNA has demonstrated that aminopodand 1 and diamines 5-7 have a similar effect on DNA, which slightly decreased the mobility of supercoiled circular plasmid DNA as piperidine and DASD. No change was observed in the mobilities of 4c, 4d, 4e and 4f on supercoiled plasmid DNA as pyrrolidine and morpholine. However, the mobilities of 5a, 6a, 7a and 7b on supercoiled circular plasmid DNA were slightly increased. Compounds 2 and 3 caused a decrease in mobility of form I and form II, indicating intercalation, which involves the insertion of a compound between DNA base pairs, resulting in a decreased DNA helixal twist and lengthening of DNA as N3P3Cl6 and N4P4Cl8. Fig. 11 here about

3.8. BamHI and HindIII digestion of compounds-pBR322 plasmid DNA In order to assess whether the compounds show an affinity towards guanine-guanine (GG) and/or adenine-adenine (AA) regions of DNA, the restriction analyses of the compound-pBR322 plasmid DNA adducts by HindIII and BamHI enzymes were carried out (Section S1). HindIII and BamHI enzymes bind at the recognition sequences 5′-A/AGCTT-3′ and 5′-G/GATCC-3′ cleave these sequences just after the 5′-adenine and 5′-guanine sites, respectively, and as a result they

convert supercoiled plasmid (Form I) and relaxed circular form DNA (Form II) to linear DNA (Form III) [41]. Fig. 12a and 12b present the electrophoretograms applied to incubate the mixtures of pBR322 plasmid DNA and compounds for 24h at 37°C, followed by HindIII (a) and BamHI (b) digestions for a further 1 h at the same temperature. It was found that digestion with BamHI and HindIII was prevented for the compounds indicating that the GG and AA adducts with DNA were formed. Fig. 12 here about

4. Conclusions

The Cl replacement reactions of N4P4Cl8 with the dipotassium salt, K2N2O2 (1a) of ligand (1) gave 2,6-sas-cyclotetraphosphazene (3) in THF. When the reactions were made one equimolar amount of 3 with an excess mono- and diamines, along with the fully substituted 2,6-sas cyclotetraphosphazenes (4a, 4b and 5a-7a) were prepared. Although the excess morpholine and DASD were used in the reactions, the geminal bis- (4c and 4e) and tris- (4d and 4f) cyclotetraphosphazenes occurred. In addition, the reactions of one equimolar amount of 3 with two equimolar amounts of diamines (5-7) resulted in di- and monospiro 2,6-sas compounds (5a-7a and 5b-7b). The cyclotetraphosphazenes were fully characterized using one- and two-dimensional NMR techniques, where the molecular and solid state structures of 3 and 4a were established crystallographically. The results exhibit that the controlled stereogenic phosphorus centers are likely to be generated using tetradentate symmetric ligand (1) in a certain number. Furthermore, all the compounds seem to be slightly active against the tested microorganisms. The interactions of the compounds with supercoiled pBR322 DNA were investigated using agarose gel electrophoresis. It was observed that compounds 2 and 3 had better cleavage abilities than those of other compounds. Digestion with HindIII and BamHI was prevented, indicating that the GG and AA adducts with DNA comprised. The compounds 3 and 4f exhibit cytotoxic effects against L929 fibroblast cells, and have lower toxicity with decreasing concentrations of 3 and 4f. Compound 4f

also displays strong cytotoxicity against A549 lung cancer cell lines at 12.5 µg/mL concentration. In addition, 3 and 4f have the capability to induce apoptosis and necrosis on cancer cell lines depending on the dose amount.

Acknowledgements The authors thank the “Scientific and Technical Research Council of Turkey” (Grant No. 211T019), and Z. K. thanks Turkish Academy of Sciences (TÜBA) for partial support.

Appendix A. Supplementary data Listings of the selected bond lengths (Å) and angles (deg) for 3 and 4a (Table S1), hydrogenbond geometries (Table S2), the tentative reaction pathways of N4P4Cl8 with the N2O2 donor type aminopodand (1), pyrrolidine, piperidine, morpholine, DASD, 5, 6 and 7 in THF (Fig. S1), theoretical

stereoisomer

distributions

and

expected

geometrical

cyclotetraphosphazene derivatives 3, 4a-4f, 5a-7a and 5b-7b (Table S3),

13

isomers

of

the

C{1H} and 1H NMR

spectral data of the compounds (Tables S4 and S5), HSQC and HMBC spectra of 4b (Fig. S2), the shapes of the phosphazene rings in 3 and 4a with torsion angles (deg) given (Fig. S3), crystal ring conformations of 3 and 4a (Figs. S4 and S5), cell viability (%) of A549 and L929 fibroblast cell treated with 3 and 4f (Table S6), apoptotic and necrotic effects of 3 and 4f complexes on A549 cell line in vitro (Table S7), antimicrobial activities of (a) the compounds 2b, 2c, 3b, 4b, 5a,5b, (b) aminopodand 1 and diamines 5-7 and (c) starting compound N4P4Cl8, N3P3Cl6, pyrrolidine, morpholine, piperidine, DASD (Table S8), the in vitro antimicrobial activities of compounds 2, 3, 4a, 4b, 4d-4f, 5a-7a, 1 and 5-7 (Minimum Inhibitory ConcentrationValues, µM) (Table S9), determination of cytotoxicity with WST-1 and apoptosis/necrosis with double staining method, antimicrobial activity, DNA binding studies and restriction enzyme digestion (Section S1. Experimental Part of Biological Studies), and X-ray crystallographic file in CIF format for 3 and 4a are available free of charge at http://dx.doi.org/... Crystallographic data for the structure

reported herein have been deposited with the Cambridge Crystallographic Data Centre as Supporting Information, CCDC Nos.823467 for 3 and 823466 for 4a. Copies of the data can be obtained through application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. (fax: +44 1223 336033 or e-mail: [email protected] or at http://www.ccdc.cam.ac.uk).

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Figure Legend Fig. 1 (a) General structures of bicyclophosphazenes and (b) 2,6-sas bicyclophosphazene. Scheme 1. The Cl replacement reaction pathway of cyclophosphazenes with aminopodand, mono and diamines. Fig. 2 1H-decoupled

31

P NMR spectrum of the reaction mixture of 3 with 5 indicating that mono-

(5b) and dispiro- (5a) cyclotetraphosphazenes were formed. Fig. 3 Spatial views of all of the compounds based on the ORTEP diagrams of 3 and 4a. Fig. 4 1H-decoupled (a) and coupled (b) 31P NMR spectrum of 4e. Fig. 5 The diastereoisomers of compounds 4d, 4f, 5a-7a and 5b-7b (NCH2ArCH2N groups are bonded 2,6-cis fashion). Fig. 6 The isomers and racemic forms of compounds 3, 4a-4f, 5a-7a and 5b-7b via stick diagrams. Fig. 7 An ORTEP-3 [37] drawing of 3 with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. Fig. 8 An ORTEP-3 [37] drawing of 4a with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. Fig. 9 Cell viability (%) of L929 fibroblast (a) A549 lung cancer (b) cell treated with3 and 4f. Fig. 10 Apoptotic and necrotic effects of 3 and 4f complexes on L929 Fibroblast (a) A549 cell (b) line in vitro. Fig. 11 Gel electrophoretic mobility of pBR322 plasmid DNA, when incubated with different concentrations of starting compound (N4P4Cl8), N3P3Cl6, pyrrolidine, morpholine, piperidine, DASD, 1, 5, 6, 7 and cyclotetraphosphazene derivatives 2, 3, 4a-4f, 5a-7a, 7b. Concentrations (in µM) are as follows: lanes 1 untreated pBR322 plasmid DNA; (lanes 2-6 compound treated DNA) lanes 2: 5000, lanes 3: 2500, lanes 4: 1250, lanes 5: 625, lanes 6: 312. Fig. 12 Electrophoretograms for the BamHI (a) and HindIII (b) digested mixtures of pBR322 plasmid DNA after their treatment with cyclotetraphosphazene derivatives 2, 3, 4a-4f, 5a-7a and

7b, and amines 1, 5, 6 and 7. P applies to untreated pBR322 plasmid DNA. P/H and P/B apply untreated DNA digested with HindIII and BamHI, respectively.

R1

N

P

N

R2 N

N

P

N

P

Cl

R1

N

N

P

Cl

N

NH

R1

N

N

P

N

R N

(

R1

P

(

R1

P

R1

P

Cl Cl

(a)

(b) Fig. 1

Cl OH

OK

HO

NH

KO

NH

HN

Cl

HN

K 2C O 3

Cl

N 4 P 4C l8

Cl P

N

N

P

P

O

Cl

TH F

,

Cl

P

N 3 P 3C l6 , T H F

N

N

N

N

N

P

P

O

Cl Cl

N

(1a )

(1 )

spiro -b in o-spiro (sb s) (2)

(

X P

X

N

N

P

4 eq uim olar M on oa m ine s

N

N N

P

N

P

Cl

N

N

Y

O

Z

N

P

1 e qu im o lar D ia m in es

N

N

P

N

R1 NH2 NHR2

(

P

R1

O

Cl

P

Cl

R1

R2

(C H 2 ) 3 C H 3

Y

X

Z

N

N

N

N

Cl

O N

O

4b

Cl

4c

Cl

4d

O

Cl

4e

Cl

4f

H

N

R2

NHR2 C o m po u n ds

(C H 2 ) 2

CH3

5

(C H 2 ) 2

C 2H 5

6

(C H 2 ) 3

CH3

7

P

N

P

N

P

O

O O N

R1

R2

C om pound

(C H 2 ) 2

CH3

5b

(C H 2 ) 2

C 2H 5

6b

(C H 2 ) 3

CH3

7b

NR2

(N

P

H

N

O

P

N

O

P

N

N N

R1

(

N

NR2

P HN

R1

R2

C o m po u n ds

(C H 2 ) 2

CH3

5a

(C H 2 ) 2

C 2H 5

6a

(C H 2 ) 3

CH3

7a

Cl

7

(

O

N

Cl

N

C o m p o un d

O

N

(

Cl

N

N

R1

N

O

R1

NH2

2 eq uim olar D ia m in e

P

R1

(3)

(

O

4a

(

N

N

N

2 ,6-sp ir o-an sa -spir o (2,6 -sas)

C o m p o u nd s

NR 2

(N O

(

O

Cl

O

Scheme 1. The Cl replacement reaction pathway of cyclophosphazenes with aminopodand, mono and diamines.

Fig. 2

X

X

O

N

)

2,6-sas (3, 4a-4f)

N

N

O

N

)

N

N

di-spiro-2,6-sas (5a-7a)

Fig. 3

O

N

)

N

N

O

N

Cl

Cl

)

Y

N

))

) Z

O

))

N

))

) O

mono-spiro-2,6-sas (5b-7b)

Fig. 4

X

NR2

(

O N

N N

P

N

X

O

Cl

N

( O

N

P

N

N

P

P

N

N HN

N NR2 R1

(

H

P

Cl

P

N

P

H

N X

N

N

O

P

P

N

N

O N

H N

P

R1

R2N

O

O Cl

N

N

Cl

O

(

(N

N

O

(N

Cl

NR2

R1

P

P

(

P

X

N

P

N

P

N

N

P

P

N

N H N

R1

R2N

Fig. 5

Cl

P

N

N

(

O

N

P

O

P

P

N

N N

P

N HN

NR2 R1

(

N

P

(

X

N

R1

(

P

O

(

X

( (racemic)1

Cl

X

O

X

(N

(O

X

O

X

N

SRR'

NH N

NH

O

( (

NH O

Cl

NH

RSS'

NR O

N

Cl

( O

=

(N

X

O

X

N

Cl X

SRS'

O

Cl Cl

N

SRR'

(

(5b-7b)#

(X: pyr, pip, mor, DASD; R: CH3, C2H5) *: The stereogenic centre. #: Two equivalent chiral centre which give two racemic forms.

Fig. 6

=

(N

(racemic)2

NR O

(N ( NH

O

NH O

NH

NR N

NR

(

SRR'

(5a-7a)#

(

(racemic)1

( (

RSR'

(O

Cl

X

NR

(

O

RSS'

N

=

X

SR

(racemic)2

(N (NR N

NR

NR O

NH N

Cl

(N

Cl

(4c and 4e) (meso)

(

=

O

(

NR N

(O ( NH

N

=

O

(4d and 4f)#

(racemic)1

(

N

X

RSS'

N

NR

NH

RSR'

(d)

RS

( ( (

(

NH N

(O ( NR O

(O

Cl

X

Cl Cl

O

=

(N

SRS'

NH O

Cl

NR N

Cl

(

RSR'

=

O

(

Cl

X

O

(

(

X O

N

Cl

( (

X

N

SR

(

(c)

(O

X

( (

X N

(

N

N

X

(

(O

N

X

(

(b)

X

(4a and 4b) (meso)

( N

O

( ( (

(

RS

=

X

X

(

O

O

(N

SR

(

X

X

(

N

(3) (cis-meso)

(

(a)

X

Cl

(

( N

Cl N

Cl

(

Cl

RS

(O

(N

=

(

Cl O

(

(O

Cl O

O

Cl

(

N

Cl

N

(racemic)2

SRS'

Fig. 7

34

Fig. 8

35

(a)

(b)

Fig. 9

36

(a)

(b)

Fig. 10

37

Fig. 11

38

Fig. 12

39

Table 1. Crystallographic data for 3 and 4a. 3

4a

Empirical Formula Fw CrystalSystem Space Group o a( A ) b ( Ao )

C22H20Cl4N6O2P4.C2H3N 707.17 monoclinic P 2 1/c 9.6773(2) 24.2038(6)

C38H52N10O2P4 804.78 triclinic P -1 9.0581(3) 12.2960(4)

c (A ) α (°) β (°) γ (°) V ( Ao 3) Z µ(MoKα) (cm-1) ρ(calcd) (g cm-3) Number of Reflections Total Number of ReflectionsUnique Rint 2θmax (°) Tmin/ T max Number of Parameters R [F2 >2σ(F2)] wR

12.6327(3) 90 99.297(1) 90 2920.06(12) 4 0.664 1.609 27529 7298 0.0433 56.84 0.775/0.830 371 0.0365 0.0992

18.3897(5) 84.048(3) 76.732(2) 77.255(3) 1941.39(11) 2 0.244 1.377 34947 9824 0.0467 57.10 0.887/0.948 487 0.0548 0.1516

o

40

Table 2. 31P{1H}NMR Spectral Data of the Compounds. Y= Cl, pyrr, mor, pip, DASD, 5, 6 or 7 Y Y O N

(B) X P N

A P

N

N

P A

N

O

Y

N

Y

Y P X Y (B)

A 2X 2 (4a, 4b and 5a-7a) A 2B2 (3)

X P

N

N O N

P A

A P

O

Y

N

Y

A P

N

N

N N

X P

Cl P M Y

O N

A 2MX (4d, 4f (4f') and 4f'(4f))

P A

O Y

N

N N

Cl P M Cl (B)

X P

N

Y

N

N

P A

A2MX (4c and 4e) A2BX (5b-7b)

Y

X P

N O

Y

P Y X

Y

N

O N

N

P A

N

Y P X Y

AX 2 (2)

2

JPP (Hz)

2

JAX: 56.9

−

2

JAB: 52.6

−

−

2

JAX: 54.2

-1.82

−

−

2

JAX: 50.2

-9.54

4.21

-14.03

−

2

JAM: 49.8 JAX: 50.9

A2MX

-7.02

0.81

−

- 1.21

4e

A2MX

-10.19

4.55

-14.54

−

4f (4f')

A2MX

5.45

18.54

−

-1.75

4f'(4f)

A2MX

1.19

16.29

−

-7.51

5a

A2X2

-3.74

15.52

−

−

5b

A2BX

13.2

17.4

-3.2

−

6a

A2X2

-4.23

15.91

−

−

6b

A2BX

11.3

16.9

-4.1

−

7a

A2X2

-6.34

2.11

−

−

2

JAX: 51.5

−

2

JAB: 52.1 JAX: 47.9

Compound

Spin System

OPN

NPN

δ(ppm) PCl2

2

AX2

5.60

−

24.00

3

A2B2

-9.52

−

-11.81

4a

A2X2

-8.85

-1.76

4b

A2X2

-8.13

4c

A2MX

4d

7b

A2BX

0.84

6.17

-4.62 41

NPCl −

2

2 2

2 2

2 2

2 2

2

JAM: 53.9 JAX: 54.5 JAM: 47.8 JAX: 50.8 JAX: 45.3 JAM: 50.8 JAX: 50.4 JAM: 52.9 JAX: 46.8

2

JAB: 49.1 JAX: 46.8 2 JAX: 45.7

2

2 2

2

JAB: 46.8 JAX: 45.7

a 31

P NMR measurements for the compounds in CDCl 3 solutions at 293 K. [Chemical shifts (δ) are reported in ppm, J values in Hz].

42

Phosphorus-nitrogen compounds : Part 31. Syntheses, structural and stereogenic properties, in

vitro

cytotoxic

and

antimicrobial

activities,

and

DNA

interactions

of

bicyclotetraphosphazenes containing bulky side group

Gürcü Mutlua, Gamze Elmasa, Zeynel Kılıça,*, Tuncer Hökelekb, L. Yasemin Koçc, Mustafa Türkd, Leyla Açıke, Betül Aydıne, Hakan Dalf

Graphical Abstract (synopsis) N4P4Cl8 was reacted with aminopodand to obtain 2,6-sas-phosphazene. The mono- and diamino2,6-sas phosphazenes were prepared, and their characterizations were made. The interactions between the phosphazenes and pBR322 plasmid DNA, the evaluations for cytotoxic activity, and apoptotic and necrotic effects against A549 Lung cancer and L929 Fibroblast cell lines were studied.

43

Phosphorus-nitrogen compounds : Part 31. Syntheses, structural and stereogenic properties, in

vitro

cytotoxic

and

antimicrobial

activities,

and

DNA

interactions

of

bicyclotetraphosphazenes containing bulky sidegroup

Gürcü Mutlua, Gamze Elmasa, Zeynel Kılıça,*, Tuncer Hökelekb, L. Yasemin Koçc, Mustafa Türkd, Leyla Açıke, Betül Aydıne, Hakan Dalf

X

X

N

N

O

N

O

Y

N

O

N

N

N

O

N

N

Cl

Cl

mono-spiro-2,6-sas (5b-7b)

)

di-spiro-2,6-sas (5a-7a)

X, Y, Z: Cl, Pyr, Pip, Mor, DASD

)

N

)

2,6-sas (3, 4a-4f)

N

))

)

N

Z

O

)

O

))

)

))

)

Graphical Abstract (figure)

N

N: Diamines (5-7)

44

Phosphorus-nitrogen compounds : Part 31. Syntheses, structural and stereogenic properties, in

vitro

cytotoxic

and

antimicrobial

activities,

and

DNA

interactions

of

bicyclotetraphosphazenes containing bulky side group

Gürcü Mutlua, Gamze Elmasa, Zeynel Kılıça,*, Tuncer Hökelekb, L. Yasemin Koçc, Mustafa Türkd, Leyla Açıke, Betül Aydıne, Hakan Dalf

Highlights The partly and fully substituted spiro-ansa-spiro-cyclotetraphosphazenes were obtained. The compounds were tested against A549 Lung cancer and L929 Fibroblast cell lines. All of the phosphazenes were screened G(+) and G(-) bacteria and yeast strains. Interactions between the conjugates and pBR322 plasmid DNA were scrutinized. The crystallographic, stereogenic and spectral data of the compounds were presented.

45