4-nitrobenzyl) pendant arms: Structural and stereogenic properties and DNA interactions

Inorganica Chimica Acta 490 (2019) 179–189

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Research paper

Phosphorus-Nitrogen compounds part 47: The conventional and microwaveassisted syntheses of dispirocyclotriphosphazene derivatives with (4-fluoro/ 4-nitrobenzyl) pendant arms: Structural and stereogenic properties and DNA interactions

T

Güler İnci Tanrıkulua, Mehtap Yakut Özgürb, Aytuğ Okumuşb, , Zeynel Kılıçb, , Tuncer Hökelekc, Betül Aydınd, Leyla Açıkd ⁎

⁎

a

Department of Chemistry, Amasya University, 05100 Amasya, Turkey Department of Chemistry, Ankara University, 06100 Ankara, Turkey Department of Physics, Hacettepe University, 06800 Ankara, Turkey d Department of Biology, Gazi University, 06500 Ankara, Turkey b c

ARTICLE INFO

ABSTRACT

Keywords: Dispirocyclotriphosphazenes Crystallography Stereoisomers Spectroscopy

The Cl exchange reactions of hexachlorocyclotriphosphazene, N3P3Cl6, with two equimolar amounts of N-alkylN′-mono(4-fluorobenzyl)diamines (1–3), FC6H4CH2NH(CH2)nNHR1 (n = 2 and 3, R1 = CH3 or C2H5), and Nalkyl-N′-mono(4-nitrobenzyl)diamines (4 and 5), NO2C6H4CH2NH(CH2)nNHR1 (n = 2, R1 = CH3 or C2H5), led to the formation of the mono(4-fluorobenzyl) (1a-3a) and mono(4-nitrobenzyl) (4a and 5a) spirocyclotriphosphazenes as minor products, and trans-bis(4-fluorobenzyl) (1b-3b) and trans-bis(4-nitrobenzyl) (4b and 5b) spirocyclotriphosphazenes as major products. The bis(4-fluorobenzyl)spirocyclotriphosphazene (1b) reacted with excess pyrrolidine to give fully substituted (1c) phosphazene. The structures of the new compounds were elucidated by elemental analyses, ESI-MS, FTIR, 1H, 13C, and 31P NMR techniques. The molecular and crystal structures of 1a, 3b and 6 were identified by single crystal X-ray crystallography. The absolute configurations of 3b and 6 were unambiguously established as SS and R respectively, using X-ray crystallographic data. On the other hand, the interactions of 1b, 1c, 3b-5b and 6 with plasmid DNA indicated that compounds 3b, 4b, and 5b caused a decrease in the mobilities and intensities of form I and form II DNA. Compounds 1b, 1c and 6 caused a double strand break of plasmid DNA. All of the tested compounds inhibited enzyme cleavage indicating compound bindings to the specific G/G and A/A nucleotides.

1. Introduction Heterocyclic chlorophosphazene ring systems (NPCl2)n constitute the regular and homologous series, and the ring sizes vary remarkably [1]. Hexachlorocyclotriphosphazene, (NPCl2)3, is the renowned starting compound and used for the preparation of a large amount of stereogenic cyclotriphosphazene derivatives by nucleophilic Cl exchange reactions [2]. The sequential replacement reactions of Cl atoms in (NPCl2)3 by primary and secondary amines give the partly and fully substituted aminocyclotriphosphazenes [3]. However, the condensation reactions of the bidentate reagents with (NPCl2)3 afford regio- and/or stereo-selectively spiro, ansa-, dispiro-, trispiro-, spiroansa-cyclotriphosphazenes [4–7]. Some of these multiheterocyclic trimeric phosphazenes have geometrical (geminal/nongeminal cis–trans) and optical ⁎

(meso/racem) isomers [8,9]. In recent years, chirality in phosphazene chemistry has become a very interesting new research area for the evaluation of the stereogenic properties and absolute configurations of the chiral phosphazenes [10–12]. In addition, some of the stereochemical terms, eg. prochirality, pseudoasymmetric centers, homo, enantio- and diastereotopic atoms or substituents were involved in phosphazene chemistry [13,14,15]. Besides, the stereogenic properties of the cyclophosphazenes were investigated using 31P NMR spectroscopy, chiral HPLC and X-ray crystallography techniques [16–18]. On the other hand, some of the organocyclophosphazenes have also found chemical, technological and biological applications, for example as ligating agents and strong bases in coordination chemistry [19], in the preparation of phosphazene polymers [20], dendrimers [21], liquid crystalline materials [22], ionic liquids [23], and flame retardant

Corresponding authors. E-mail addresses: [email protected] (A. Okumuş), [email protected] (Z. Kılıç).

https://doi.org/10.1016/j.ica.2019.03.018 Received 25 January 2019; Received in revised form 13 March 2019; Accepted 13 March 2019 Available online 14 March 2019 0020-1693/ © 2019 Elsevier B.V. All rights reserved.

Inorganica Chimica Acta 490 (2019) 179–189

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Cl

Cl P N

N

P

) )

NHR2

2

XAr: X

3

1

4 6

5

X: F, NO2

Cl

Cl

Cl

P

X

Comp.

(CH2)2 CH3

F

(1)

(CH2)2 CH2CH3

F

(2)

(CH2)3 CH3

F

(3)

(CH2)2 CH3

NO2

(4)

(CH2)2 CH2CH3 NO2

(5)

P

X

Comp.

F

(1a)

F

(2a)

(CH2)3 CH3

F

(3a)

(CH2)2 CH3

NO2

(4a)

(CH2)2 CH2CH3 NO2

(5a)

(CH2)2 CH3 (CH2)2 CH2CH3

PCl2: Prochiral P centers

N

*P

XArCH2 N

N

N

N

R1

XArCH2

trans-bis(4-fluoro/nitrobenzyl) spirophosphazenes , C2 SS/RR R2 R1 comp. X

R1

N

P*

R2

*

N

N

*P

CH2ArX N

N

R1

)

mono(4-fluoro/nitrobenzyl) spirophosphazenes , Cs R2

P*

R2

XArCH2

R1

N

R2

)

R1

R1

N

Cl P

)

N

N

)

N

P

N

)

Cl

XArCH2 N

)

Cl

R2

N

Cl

Cl P

)

N

R2

R1

R1

XArCH2NH

Cl Cl

)

P

)

Cl

N

)

Cl

R2

cis-bis(4-fluorobenzyl) spirophosphazenes , Cs RS/SR R2 R1 comp. X

F

(1b)

(CH2)2 CH3

F

(CH2)2 CH2CH3

F

(2b)

F

(2b')

(CH2)3 CH3

(3b)

(CH2)2 CH2CH3

F

F

(CH2)2 CH3

NO2

(4b)

(CH2)3 CH3

(3b')

(CH2)2 CH2CH3

NO2

(5b)

(CH2)2 CH3

Cl: Homotopic Cl atoms

(1b')

* : Chiral P centers Cl: Diastereotopic Cl atoms

* : In cis-bis(4-fluorobenzyl)spirocyclotriphosphazenes, when one of the Cl atoms is replaced with a substituent, the P atom becomes the pseudo-asymmetric center (r or s).

Scheme 1. Phosphazene derivatives (1a-5b) obtained from the reactions of N3P3Cl6 with the ligands (1–5). The cis compounds (1b'-3b') were not isolated.

additives [24]. Furthermore, the interactions of DNA with phosphazene derivatives were also investigated to understand the chemotherapeutic effect of the molecules and their potential application as antimicrobial and anti-cancer agents [25–28]. The monospirocyclotriphosphazenes with (4-fluoro/nitrobenzyl) pendant arms (1a- 5a) were obtained and published earlier by our group [29,30]. As a particular attention in our ongoing studies in this area, this extended study primarily focuses on the Cl exchange reactions of (NPCl2)3 with N-alkyl-N′-mono(4-fluorobenzyl) (1–3) and N-alkyl-N′mono(4-nitrobenzyl)diamines (4 and 5) (Scheme 1). At the same time, the main goals of this study are the investigations of the geometrical (cis/trans) and optical isomers of the new dispirocyclotriphosphazenes (1b- 5b and 1c) and the interactions of DNA with the compounds (1b, 1c, 3b-5b and 6), as well.

data were determined using the Leco CHNS-932 instrument. Fourier transform infrared (FTIR) spectra were recorded on a Perkin Elmer 100 spectrometer in KBr discs and reported in cm−1 units. Electrospray Ionization Mass Spectrometry (ESI-MS) of the phosphazenes were enrolled with a Waters 2695 Alliance Micromass ZQ spectrometer. The 1 H, 13C{1H} and 31P{1H} NMR spectra were recorded on a Varian Mercury (400 MHz) FT spectrometer (SiMe4 as an internal standard for 1 H and 85% H3PO4 as an external standard for 31P NMR), operating at 400.13, 100.62, and 161.97 MHz. The spectrometer is equipped with a 5-mm PABBO BB inverse-gradient probe, and standard Bruker pulse programs [31] were used. Crystallographic data were recorded on a Bruker Kappa APEXII CCD area-detector diffractometer using Mo Kα radiation (λ = 0.71073 Å) at T = 296(2) K. Microwave-assisted experiments have been performed with a Mars 5 Digeston Microwave system using a weflonTM magnet. The atom numbering scheme of protons and carbons of the 4-fluoro/4-nitrophenyl rings is given in Scheme 1 for the interpretations of 1H and 13C{1H} NMR spectra.

2. Experimental Part 2.1. Materials and method

2.2. Materials used in the syntheses

All the condensation reactions were carried out under Ar(g) atmosphere, and pursued by thin-layer chromatography (TLC) on Merck DC Alufolien Kieselgel 60 B254 sheets in suitable solvents. The column chromatography was made on Merck Kieselgel 60 (230–400 mesh ATSM) and preparative TLC on silica gel by Merck (PF254-366 nm). The melting points of the new phosphazene derivatives were determined on a Gallenkamp apparatus using a capillary tube. The microanalytical

The Cl replacement reactions were performed under argon atmosphere. The solvents were dried and purified using standard methods. N3P3Cl6 (Aldrich and recrystallized from hot n-hexane), 4-fluorobenzaldehyde, 4-nitrobenzaldehyde, N-methylethylenediamine, Nmethyl-1,3-propanediamine, N-ethylethylenediamine (Merck) and pyrrolidine (Fluka) were purchased. 180

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z 594 ([M]+, 100). FTIR (KBr, cm−1): ν 3028 (CeH arom.), 2938, 2841 (CeH aliph.), 1227 (asymm.), 1191 (symm.) (P]N), 1048 (C-F), 562 (asymm.), 539 (symm.) (P-Cl). NMR δH (CDCl3, ppm): 7.36 (d, 4H, 3 JHH = 8.8 Hz, 4JFH = 5.6 H3 and H5), 7.01 (d, 4H, 3JHH 8.8, 3JFH 8.4, H2 and H6), 4.13 (d, 2H, 3JPH = 14.8 Hz, ArCH2N), 4.04 (d, 2H, 3 JPH = 14.4 Hz, ArCH2N), 3.12 (m, 4H, NCH2), 3.01 (m, 4H, CH2NR), 2.57 (d, 6H, 3JPH = 13.6 Hz, NCH3), 1.76 (m, 4H, NCH2CH2),; NMR δC (CDCl3, ppm): 162.3 (1JFC = 245.4 Hz, C1), 134.2 (3JPC = 5.9 Hz, 4 JFC = 2.8 Hz C4), 130.1 (3JFC = 8.5 Hz, C3 and C5), 115.4 (2JFC = 21.5 Hz, C2 and C6), 50.5 (ArCH2N), 50.2 (NCH2), 46.0 (CH2NR), 36.1 (NCH3), 24.1 (NCH2CH2). Syntheses of monospiro- (4a) and dispiro- (4b): N3P3Cl6 (0.35 g, 1.0 mmol) in dry toluene (150 mL) was added into N-methyl-N'-(4-nitrobenzyl)-1,2-ethandiamine (0.83 g, 4.0 mmol). The mixture was refluxed for over 12 h. The precipitated salt was filtered off, and the solvent was evaporated. The products, 4a and 4b, were separated by column chromatography using toluene-THF (4:1) as an eluent. Compound 4a was crystallized from n-hexane. Yield: 0.32 g (65%) and M.p.: 136 °C (Lit. M.p.: 135 °C). The white powder product 4b was crystallized from n-hexane. Yield: 0.12 g (21%). M.p.: 213 °C. Anal. Calcd. for P3N9Cl2O2C20H26: C, 38.73; H, 4.23; N, 20.32. Found: C, 38.81; H, 4.36; N, 19.34. ESI-MS (fragments are based on 35Cl, Ir %): m/ z 621 ([MH]+, 100). FTIR (KBr, cm−1): 3062 (CeH arom.), 2943, 2853 (CeH aliph.), 1521, 1350 (C–NO2 arom.), 1235 (asymm.), 1172 (symm.) (P]N), 596 (asymm.), 567 (symm.) (PCl). NMR δH (CDCl3, ppm): 8.22 (d, 4H, 3JHH = 8.4 Hz, H2 and H6), 7.65 (d, 4H, 3 JHH = 8.4 Hz, H3 and H5), 4.22 (d, 2H, 3JPH = 8.5 Hz, ArCH2N), 4.19 (d, 2H, 3JPH = 8.5 Hz, ArCH2N), 3.20 (m, 4H, NCH2), 3.09 (m, 4H, CH2NR), 2.51 (t, 6H, 3JPH = 5.8 Hz, NCH3); NMR δC (CDCl3, ppm): 147.8 (C1), 144.8 (3JPC = 7.3 Hz C4), 128.9 (C3 and C5), 124.2 (C2 and C6), 48.7 (ArCH2N), 47.2 (2JPC = 12.3 Hz, NCH2), 44.7 (2JPC = 14.6 Hz, CH2NR), 31.3 (NCH3). Syntheses of monospiro- (5a) and dispiro- (5b): The procedure used for 4a and 4b was followed for the syntheses of 5a and 5b using Nethyl-N'-(4-nitrobenzyl)-1,2-ethandiamine (0.89 g, 4.0 mmol). Compound 5a was purified by column chromatography using benzene as eluent and afterwards recrystallized from toluene. Yield: 0.28 g (53%) and M.p.: 103 °C (Lit. M.p.: 104 °C). The white powder product 5b was crystallized from n-hexane. Yield: 0.18 g (30%). M.p.: 175 °C. Anal. Calcd. for P3N9Cl2O4C22H30: C, 40.76; H, 4.66; N, 19.44. Found: C, 40.76; H, 4.63; N, 19.07. ESI-MS (fragments are based on 35Cl, Ir %): m/z 648 ([M]+, 100). FTIR(KBr,cm−1): 3062 (CeH arom.), 2922, 2850 (CeH aliph.), 1542, 1345 (C–NO2 arom.), 1285 (asymm.), 1196 (symm.), (P]N), 574 (asymm.),515 (symm.) (PCl); NMR δH (CDCl3, ppm): 8.22 (d, 4H, 3JHH = 8.8 Hz, H2 and H6), 7.64 (d, 4H, 3 JHH = 8.8 Hz, H3 and H5), 4.17 (d, 2H, 3JPH = 8.8 Hz, ArCH2N), 4.15 (d, 2H, 3JPH = 8.8 Hz, ArCH2N), 3.19 (m, 4H, NCH2), 3.05 (m, 4H, CH2NR), 2.83 (m, 4H, NCH2CH3), 1.08 (t, 6H, 3JHH = 7.2 Hz, NCH2CH3); NMR δC (CDCl3, ppm): 147.5 (C1), 145.5 (3JPC = 6.9 Hz C4), 128.6 (C3 and C5), 123.8 (C2 and C6), 48.3 (ArCH2N), 44.7 (2JPC = 13.8 Hz, NCH2), 43.6 (2JPC = 12.9 Hz, CH2NR), 39.1 (2JPC = 3.4 Hz NCH2CH3), 13.7 (3JPC = 4.4 Hz NCH2CH3). Synthesis of dipyrolidino-bisspiro (1c): A solution of 1b (0.50 g, 0.83 mmol) in dry toluene (100 mL) was slowly added into a solution of pyrrolidine (1.00 mL, 8.30 mmol) in dry toluene (50 mL) with stirring and refluxing for 26 h. The precipitate was filtered off, and the solvent was evaporated. The oily product was purified by column chromatography with THF as eluent. The white powder product (1c) was crystallized from n-hexane. Yield: 0.26 g (51%) M.p.: 148 °C. Anal. Calcd. for P3N9F2C28H42: C, 52.91; H, 6.66; N, 19.83. Found: C, 53.05; H, 6.77; N, 19.75. ESI-MS (Ir %): m/z 636 ([MH]+, 100). FTIR(KBr,cm−1): ν 3034 (CeH arom.), 2964, 2914 (CeH aliph.), 1221 (asymm.), 1188 (symm.) (P]N), 1081 (C-F), NMR δH (CDCl3, ppm): 7.41 (dd, 4H, 3 JHH = 8.8 Hz, 4JFH = 5.6 Hz, H3 and H5), 6.97 (dd, 4H, 3JHH = 8.8 Hz, 3 JFH = 8.6 Hz H2 and H6), 4.01 (d, 4H, 3JPH = 6.4 Hz, ArCH2N), 3.20 (m, 8H, p: NCH2), 3.13 (m, 8H p: NCH2), 3.06 (m, 4H, CH2NR), 2.97 (m,

2.3. Syntheses of the compounds The starting compounds, N-methyl-N'-(4-fluorobenzyl)-1,2-ethandiamine, N-ethyl-N'-(4-fluorobenzyl)-1,2-ethandiamine and N-methylN'-(4-fluorobenzyl)-1,3-propanediamine were synthesized from the reactions of 4-fluorobenzaldehyde with N-methylethylendiamine, Nethylethylendiamine, and N-methyl-1,3-propanediamine in ethanol, respectively, according to the published procedure [30]. In addition, as the starting materials, N-methyl-N'-(4-nitrobenzyl)-1,2-ethandiamine and N-ethyl-N'-(4-nitrobenzyl)-1,2-ethandiamine were prepared from the reactions of 4-nitrobenzaldehyde with the corresponding diamines in ethanol according to the literature method [29]. 2.3.1. Conventional syntheses of cyclotriphosphazenes Syntheses of monospiro- (1a) and dispiro- (1b): N3P3Cl6 (0.35 g, 1.0 mmol) in dry toluene (150 mL) was added into N-methyl-N'-(4fluorobenzyl)-1,2-ethandiamine (0.73 g, 4.0 mmol). The mixture was refluxed for over 12 h. The precipitated salt was filtered off, and the solvent was evaporated. The crude products, 1a and 1b, were separated by preparative thin layer chromatography (TLC) using toluene-THF (4:1) as an eluent. The white powder product (1a) was crystallized from n-hexane. Yield: 0.17 g (39%) and M.p.: 111 °C (Lit. M.p.:112 °C). Compound 1b was crystallized from n-hexane. Yield: 0.11 g (20%). M.p.: 133 °C. Anal. Calcd. for P3N7Cl2F2C20H26: C, 42.42; H, 4.63; N, 17.31. Found: C, 42.23; H, 4.66; N, 17.38. ESI-MS (fragments are based on 35Cl, Ir %): m/z 566 ([M]+, 100). FTIR (KBr, cm−1): ν 3062 (CeH arom.), 2923, 2864 (CeH aliph.), 1221 (asymm.), 1171 (symm.) (P] N.), 1082 (C-F), 595 (asymm.), 562 (symm.) (P-Cl). NMR δH (CDCl3, ppm): 7.43 (dd, 4H, 3JHH = 8.8 Hz, 4JFH = 5.6 Hz, H3 and H5), 7.03 (dd, 4H, 3JHH = 8.8 Hz, 3JFH = 8.6 Hz H2 and H6), 4.09 (d, 2H, 3 JPH = 8.5 Hz, ArCH2N), 4.05 (d, 2H, 3JPH = 8.5 Hz, ArCH2N), 3.17 (m, 4H, NCH2), 3.04 (m, 4H, CH2NR), 2.53 (d, 6H, 3JPH = 11.6 Hz, NCH3); NMR δC (CDCl3, ppm): 162.4 (1JFC = 245.3 Hz, C1), 133.5 (C4), 129.9 (3JFC = 8.5 Hz, C3 and C5), 115.6 (2JFC = 21.5 Hz, C2 and C6), 48.1 (ArCH2N), 47.4 (2JPC = 13.1 Hz, NCH2), 44.2 (2JPC = 14.6 Hz, CH2NR), 31.4 (NCH3). Syntheses of monospiro- (2a), and dispiro- (2b): The procedure used for 1a and 1b was followed for the synthesis of 2a and 2b, using Nethyl-N'-(4-fluorobenzyl)-1,2-ethandiamine (0.78 g, 4.0 mmol). The crude products, 2a and 2b, were separated by TLC using toluene-THF (4:1) as an eluent. Compund 2a was crystallized from n-hexane. Yield: 0.15 g (32%) and M.p.: 59 °C (Lit. M.p.: 60 °C). Compound 2b was crystallized from hexane. Yield: 0.12 g (20%). M.p.: 82 °C. Anal. Calcd. for P3N7Cl2F2C22H30: C, 44.46; H, 5.09; N, 16.50. Found: C, 44.23; H, 4.98; N, 16.53. ESI-MS (fragments are based on 35Cl, Ir %): m/z 595 ([MH]+, 100). FTIR (KBr, cm−1): ν 3052 (CeH arom.), 2917, 2871 (CeH aliph.), 1221 (asymm.), 1154 (symm.) (P]N), 1048 (C-F), 592 (asymm.), 564 (symm.) (P-Cl). NMR δH (CDCl3, ppm): 7.38 (dd, 4H, 3 JHH = 8.4 Hz, 4JFH = 5.2 Hz, H3 and H5), 6.99 (dd, 4H, 3JFH = 8.6 Hz, 3 JHH = 8.4 Hz H2 and H6), 3.99 (d, 4H, 3JPH = 8.4 Hz, ArCH2N), 3.35 (m, 4H, NCH2), 3.19 (m, 4H, CH2NR), 3.03 (m, 4H, NCH2CH3), 1.14 (t, 6H, 3JHH = 7.2 Hz, NCH2CH3); NMR δC (CDCl3, ppm): 162.2 (1JFC = 245.3 Hz, C1), 133.3 (3JPC = 11.2 Hz, 4JFC = 3.5 Hz, C4), 129.5 (3JFC = 8.5 Hz, C3 and C5), 115.4 (2JFC = 21.4 Hz, C2 and C6), 47.6 (ArCH2N), 44.0 (2JPC = 15.4 Hz, NCH2), 43.5 (2JPC = 14.5 Hz, CH2NR), 39.0 (2JPC = 2.3 Hz, NCH2CH3), 13.9 (3JPC = 3.4 Hz, NCH2CH3). Syntheses of monospiro- (3a), and dispiro- (3b): The procedure used for 1a and 1b was followed for the syntheses of 3a and 3b, using Nmethyl-N'-(4-fluorobenzyl)-1,3-propanediamine (0.78 g, 4.0 mmol). The crude products, 3a and 3b, were separated by TLC using tolueneTHF (4:1) as an eluent. Compound 3a was crystallized from n-hexane. Yield: 0.18 g (39%) and M.p.: 119 °C (Lit. M.p.:120 °C). Compound 3b was crystallized from hexane. Yield: 0.15 g (26%). M.p.: 91 °C. Anal. Calcd. for P3N7Cl2F2C22H30: C, 44.46; H, 5.09; N, 16.50. Found: C, 44.25; H, 5.10; N, 16.62. ESI-MS (fragments are based on 35Cl, Ir %): m/ 181

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2H, NCH2), 2.51 (d, 6H, 3JPH = 11.6 Hz, NCH3); NMR δC (CDCl3, ppm): 162.0 (1JFC = 244.5 Hz, C1), 134.7 (4JFC = 3.5 Hz C4), 129.5 (3JFC = 7.7 Hz, C3 and C5), 115.0 (2JFC = 21.3 Hz, C2 and C6), 48.5 (ArCH2N), 47.1 (2JPC = 11.6 Hz, NCH2), 46.2 (2JPC = 3.8 Hz, p: NCH2), 44.2 (2JPC = 11.6 Hz, CH2NR), 31.9 (NCH3), 26.3 (p:NCH2CH2), 26.2 (3JPC = 9.1 Hz, p:NCH2CH2).

T = 296(2) K. Absorption corrections by multi-scan [32] were applied. The structures of the compounds were solved by direct methods and refined by full-matrix least-squares against F2 using all data [33,34]. All non-H atoms were anisotropically refined. The molecular and packing diagrams together with the ring conformations of 1a, 3b and 6 were drawn by the ORTEP-3 and PLATON programs incorporated into the WinGX package [35]. The H atom positions were calculated geometrically at distances of 0.93 Å (for aromatic CH), 0.98 Å (for methine CH), 0.97 Å (for methylene) and 0.96 Å (for methyl) from the parent C atoms; during the refinement process a riding model was used, and the Uiso(H) values were constrained to be 1.5 Ueq(for methyl carrier atoms) and 1.2 Ueq(for other carrier atoms). The Cl atoms [(Cl1A, Cl1B and Cl2A, Cl2B) in compound 3b] and one of the F atoms [(F2A and F2B) in compound 6] were disordered over two sites with occupancy ratios of 0.50:0.50 and 0.70:0.30, respectively, (in compound 3b) and 0.80:0.20 (in compound 6).Table 3

2.3.2. Microwave-assisted syntheses of cyclotriphosphazenes Synthesis of dispiro- (1b): N3P3Cl6 (0.35 g, 1.0 mmol) in dry toluene (50 mL) was added into N-methyl-N'-(4-fluorobenzyl)-1,2-ethandiamine (0.73 g, 4.0 mmol). The mixture was refluxed for over 4 h in microwave oven. The precipitated salt was filtered off, and the solvent was evaporated. The by-product 1a did not occur, but the compound 1b was purified by column chromatography using toluene-THF (4:1) as an eluent. It is crystallized from n-hexane. Yield: 0.22 g (39%). M.p.: 133 °C. Syntheses of monospiro (2a) and dispiro- (2b): The microwave assisted procedure used for 1b was followed for the syntheses of 2a and 2b using N-ethyl-N'-(4-fluorobenzyl)-1,2-ethandiamine (0.78 g, 4.0 mmol). The products 2a and 2b were separated by column chromatography using toluene-THF (4:1). The obtained white powder product 2a was crystallized from n-hexane. Yield: 0.03 g (6%). M.p.: 60 °C (Lit. M.p.: 60 °C). Compound 2b was also crystallized from n-hexane. Yield: 0.20 g (34%). M.p.: 82 °C. Syntheses of mono-spiro (3a) and bis-spiro- (3b) cyclotriphosphazenes: The microwave assisted procedure used for 1b was followed for the syntheses of 3a and 3b using N-ethyl-N'-(4-fluorobenzyl)-1,2-ethandiamine (0.78 g, 4.0 mmol). The products, 2a and 2b, were separated by column chromatography using toluene-THF (4:1). The white powder product 3a was crystallized from n-hexane. Yield: 0.07 g (15%). M.p.: 119 °C (Lit. M.p.: 120 °C). Compound 3b was also crystallized from nhexane. Yield: 0.18 g (31%). M.p.: 91 °C.

2.5. Determination of the DNA interactions with the compounds The interactions between the phosphazenes (1b, 1c, 3b, 4b, 5b and 6) and plasmid DNA were examined by agarose gel electrophoresis [36]. The stock solutions of the compounds in DMSO were prepared and used within 1 h, and they were incubated with plasmid DNA in an incubator at 310 K for 24 h and 48 h in the dark. The compound-DNA mixtures were loaded onto the 1% agarose gel with a loading buffer (0.1% bromophenol blue, 0.1% xylene cyanol). The agarose gel electrophoresis was made in TAE buffer (0.05 M Tris base, 0.05 M glacial acetic acid and 1 mM EDTA, pH = 8.0) for 2 h at 70 V. Then, the gel was stained with ethidium bromide (0.5 μg/mL) and visualized under UV light using a transilluminator (BioDoc Analyzer, Biometra). The image was photographed with a video camera and saved as a TIFF file. The cleavage and binding efficiency were measured by determining the ability of the complex to convert the supercoiled (Form I) DNA to nicked circular form (Form II) and linear form (Form III).

2.4. Single crystal X-ray structure determinations of 1a, 3b and 6

2.6. HindIII and BamHI digestion of the compounds-plasmid DNA

The compounds (1a, 3b and 6) were crystallized from n-hexane at room temperature. The crystallographic data, and the selected bond lengths and angles were given in Tables 1 and 2, respectively. The crystallographic data were recorded on a Bruker Kappa APEXII CCD area-detector diffractometer using Mo Kα radiation (λ = 0.71073 Å) at

The affinity evaluations between the compounds (1b, 1c, 3b, 4b, 5b and 6) and guanine-guanine (G/G) and/or adenine-adenine (A/A) regions of DNA were performed through restriction endonuclease analyses of the compound- plasmid DNA adducts by HindIII and BamHI enzymes. The compound- plasmid DNA mixtures were incubated for 24 h, and after that restricted with BamHI or HindIII enzyme at 310 K in order to the test compounds binding to DNA. BamHI enzyme binds at the sequence 5′-G/GATCC-3′ and since plasmid DNA contains a single sequence of that, cleaves this sequence. Afterward, BamHI converts supercoiled Form I and open circular Form II to Form III. HindIII recognizes the sequence 5′-A/AGCTT-3′ and cleaves this sequence. Consequently, HindIII converts Form I and Form II to Form III similarly to BamHI. The restricted DNA was subjected to 1% agarose gel electrophoresis in 1 h at 70 V in TAE buffer [37] . The gel was stained with ethidium bromide (0.5 µg/mL), and then the gel was viewed with a transilluminator. The image was photographed with a video-camera and saved as a TIFF file.

Table 1 Crystallographic details for 1a, 3b and 6.

Empirical Formula Fw Crystal System Space Group a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å 3) Z μ (cm−1) ρ (calcd) (g cm−3) Number of Reflections Total Number of Reflections Unique Rint 2θmax (°) Tmin / Tmax Number of Parameters R [F2 > 2σ(F2)] wR

1a

3b

6

C10H13Cl4FN5P3 456.96 monoclinic P21/n 19.5744(4) 8.2801(3) 23.2624(5) 90.00 96.990(3) 90.00 3742.3(2) 8 0.71073 (Mo Kα) 1.622 49,347

C22H30Cl2F2N7P3 594.34 triclinic P-1 9.5803(3) 9.7713(3) 16.2166(4) 74.821(2) 78.651(3) 78.036(7) 1416.86(7) 2 0.71073 (Mo Kα) 1.393 66,629

C18H20F2N2 302.36 orthorhombic Pna21 15.1581(3) 5.5478(2) 19.1654(3) 90.00 90.00 90.00 1611.70(7) 4 0.71073 (Mo Kα) 1.246 26,712

6612

7011

3142

0.0571 50.06 0.687/0.749 417 0.0462 0.1179

0.0396 56.08 0.925/0.945 345 0.0644 0.1449

0.0429 51.98 0.660/0.746 209 0.0546 0.1346

3. Results and discussion 3.1. Synthesis The conventional condensation reactions of N3P3Cl6 with four equimolar amounts of (4-fluorobenzyl)diamines, FC6H4CH2NHR1NHR2 (1–3), and (4-nitrobenzyl)diamines, NO2C6H4CH2NHR1NHR2 (4 and 5), gave two kinds of products, namely, (4-fluorobenzyl)monospiro (1a-3a) and (4-nitrobenzyl)monospiro (4a and 5a) [28,29] and trans-bis(4fluorobenzyl) (1b-3b), cis-bis(4-fluorobenzyl) (1b'-3b') and trans-bis(4nitrobenzyl) (4b and 5b) cyclotriphosphazenes, respectively. The 182

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Table 2 The Selected Bond Lengths (Å) and Angles (deg) for compounds 1a, 3b and 6.

P1-N1 P1-N3 P2-N1 P2-N2 P3-N2 P3-N3 P1-N4 P1-N5 P4-N6 P4-N8 P6-N8 P6-N7 P5-N7 P5-N6 P4-N9 P4-N10 P2-N6 P2-N7 C2-N1 C2-C3 C3-N4 C5-N4 C5-N1

1a

3b

6

1.607(3) 1.601(3) 1.557(3) 1.570(3) 1.578(3) 1.555(3) 1.623(3) 1.629(3) 1.607(3) 1.600(3) 1.556(3) 1.580(3) 1.572(3) 1.557(3) 1.620(3) 1.624(3) – – – – – – –

1.599(3) 1.607(2) 1.576(3) 1.630(2) 1.558(3) 1.566(7) 1.658(2) 1.659(3) – – – – – – – – 1.659(3) 1.505(2) – – – – –

– – – – – – – – – – – – – – – – – – 1.472(4) 1.509(4) 1.456(4) 1.459(3) 1.463(3)

N1-P1-N3 P1-N1-P2 N1-P2-N2 P2-N2-P3 N2-P3-N3 P3-N3-P1 N5-P1-N4 N6-P4-N8 P4-N8-P6 N8-P6-N7 P6-N7-P5 N7-P5-N6 P5-N6-P4 N9-P4-N10 N7-P2-N6 C5-N1-C2 N1-C2-C3 C2-C3-N4 C3-N4-C5 N4-C5-N1

D-H∙∙∙A

D-H

H∙∙∙A

D∙∙∙A

1a

C10-H10B···Cg4i C20-H20C···Cg1ii C20-H20B···F2iii C8-H8B···Cg1iv C3-H3B···F1v C12-H12···F2Avi C2-H2B···Cg3vii

0.96 0.96 0.97 0.97 0.97 0.93 0.97

2.67 2.96 2.53 2.80 2.51 2.42 2.94

3.561 3.733 3.394 3.690 3.299 3.269 3.839

3b 6

D-H∙∙∙A (4) (5) (6) (3) (4) (10) (3)

3b

6

111.28(14) 124.14(18) 120.58(15) 117.78(18) 119.69(15) 125.57(18) 95.46(15) 111.01(15) 125.49(20) 119.84(16) 117.58(19) 120.35(15) 124.42(19) 94.97(17) – – – – – –

115.71(12) 125.31(15) 113.90(13) 121.31(16) 122.01(14) 119.74(16) 104.03(13) – – – – – – – 102.75(13) – – – – –

– – – – – – – – – – – – – – – 106.08(20) 105.34(22) 103.94(23) 104.31(19) 102.08(18)

differences are likely to be depending on the fact that the compounds have different pendant arms. Besides, the bis(4-fluoro/nitrobenzyl)spirocyclotriphosphazenes (1b-5b and 1c) had two equivalent chiral P-centers. The trans isomers were expected to be in the racemic (RR/SS) mixtures. The chiral P atoms are indicated in Scheme 1, together with the stereochemical terms, such as prochirality, homo, and diastereotopic atoms. In cis- bis (4-fluorobenzyl)spirocyclotriphosphazenes, when one of the Cl atoms is replaced with a substituent, the P atom becomes the pseudo-asymmetric center (r or s). Then, the two meso diastereomeric forms; meso 1 (R,r,S) and meso 2 (S,s,R) are likely to be expected, according to the literature 15, indicating that both of the Cl atoms are diastereotopic. In addition, the crystal structure of 3b was determined using X-ray crystallography (see crystallographic part) indicating that compound 3b was in a trans-configuration. Furthermore, in the reaction of 2 with N3P3Cl6, it was noticed that the compound 6, which is an organic substance as by-product, occurred in addition to the expected compound 2b. Thus, the formation of 6 is explained in Scheme 2. According to this reaction, it is understood that in the synthesis of (4-fluorobenzyl)diamine (2), 4-fluorobenzaldehyde remains in the reaction mixture and reacts with 2 to form 6. According to this finding, an excess of the diamine, NH2(CH2)nNHR1 (n = 2 and 3, R1 = CH3 or C2H5), was used in the reaction to ensure that the 4fluorobenzaldehyde did not remain in the mixture. The suggested structures of the new compounds were enlightened using the microanalyses, mass spectrometry (ESI-MS), Fourier transform infrared (FTIR), 1H, 13C and 31P{1H} NMR results. The ESI-MS spectra of the phosphazenes exhibit the protonated molecular [MH]+ ion peaks. The molecular and solid state crystal structures of 1a, 3b and 6 were also determined by X-ray crystallography.

Table 3 Hydrogen-bond geometries (Å, o) for compounds 1a, 3b and 6. Compound

1a

156 138 149 152 138 152 155

Symmetry codes: [(i) ×, y + 1, z; (ii) ×, y − 1, z; (iii) −x + 1, −y + 1, −z + 1; (iv) −x + 2, −y + 1, −z; (v) −x + 3/2, y + 1/2, z − 1/2; (vi) −x + 2, −y + 1, z + 1/2 ; (vii) −x − 1/2, y + 1/2, z + 1/2]. Cg1, Cg3 and Cg4 are the centroids of the (C1—C6), (C15—C20) and (C11—C16) rings, respectively.

expected cis-bis(4-nitrobenzyl)cyclotriphosphazenes were not obtained. The reaction mixtures were subjected to TLC, the monospiro (1a-3a) and trans (1b-3b) products were purely separated. Whereas, the cis products (1b' and 3b') were not isolated purely. The presence of the cis isomers was determined using the 31P NMR spectra of the reaction mixtures (Fig. 1.). The yields of trans-bis phosphazenes were smaller than those of the corresponding mono phosphazene derivatives. On the other hand, the microwave-assisted reactions were carried out in the same reaction solvent. The yields of the bis products increased and the reaction time was reduced with respect to the conventional synthesis (Scheme 1). In addition, the bis pyrrolidino dispiro phosphazene (1c) was prepared from the reaction of the trans compound (1b) with excess pyrrolidine (see Table 4). Recently, our group published an article on cis/ trans dispirocyclotriphosphazenes [38]. The difference of the present paper from that article is that the pendant arms are different. In the previous article, pendant arms were bulky ferrocenyl groups, compared with 4-fluoro/nitrobenzyl groups of the synthesized cyclotriphosphazenes. Ferrocenyl dispiro compounds were prepared only by the conventional method in a polar solvent (THF) and the cis/trans isomers were separated by column chromatography. In the present study, the syntheses of the compounds were carried out in toluene with both of the conventional and microwave methods. However, cis/trans isomers of bis(4-fluorobenzyl)spirocyclotriphosphazenes could not be separated by column chromatography and TLC methods. Consequently, these

3.2. Characterizations of reaction products by NMR spectroscopy The 31P{1H} NMR data of the cyclotriphosphazenes were listed in Table 4. The spin systems of the compounds 1a-5a, 1c and 1b-5b are found to be as AX2 and A2X, respectively. The 31P spectra of the mixtures of trans 1b and cis 1b', and pure 1b are given in Fig. 1, as an example. The δP-shifts and the coupling constants, 2JPP, are written on the spectra. The 31P spectra of the other trimeric phosphazenes are interpreted by taking into account Fig. 1. The δP-shifts of the dispiro compounds are larger than those of the mono spiro ones. The coupling 183

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Fig. 1.

31

P {1H} NMR spectra of a) the mixtures of 1b and 1b', and b) pure 1b.

phosphazene (3b) was observed at 36.08 ppm. As expected, the 2JPC values of the NCH2 carbons of five membered rings in compounds (1b, 2b, 4b and 5b) are very large. The average 2JPC value was 13.9 Hz, which was in agreement with the published data of the trimeric phosphazene derivatives containing the five membered rings [39]. On the other hand, second order effects were observed in the spectra of the phosphazenes containing the five membered spiro rings. As examples, the spectra of compounds (1b, 4b and 1c) were given in Fig. 2, for indicating the second-order effects. The second-order effects have been known for a long time in the 13CNMR spectra of symmetrically substituted cyclotriphosphazenes [40–42]. The origin of this phenomenon was explained by physical properties and mathematical equations in a review [43]. These effects essentially emerge from the long range interactions of 31P-13C nuclei in the symmetric cyclotriphosphazene ring. The subject is also addressed in a nice paper published by Vicente et al [44]. The coupling constants, 2JPC, which occurred depending on

constants and the δP-shifts of the trans and cis compounds are very close to each other. When the Cl atoms are exchanged with pyrrolidine, the δPA shift of the compound (1c) decreases by 10.47 ppm, while the δP spiro shifts increase by 3.84 ppm. The 13C {1H} NMR signals of all the phosphazenes were interpreted on the basis of chemical shifts, multiplicities and coupling constants, e.g. 1JFC, 2JFC, 3JFC,4JFC and 3JPC (ipso C4) values for aromatic and 2JPC, 3 JPC values for aliphatic carbons. The results of the bis(4-fluoro/nitrobenzyl)spirocyclotriphosphazenes were given in the “Experimental Part”. The expected carbon atoms of the suggested structures, δC shifts and coupling constants were assigned. The δC shifts and the coupling constants of C1, C2/C6, C3/C5 and C4 carbons of the (4-fluoro/nitrobenzyl) pendant armed phosphazenes are in agreement with literature values [26,29,30]. The average δC-shift of NCH3 carbons of phosphazenes containing the five membered spiro rings (1b, 4b and 1c) was 31.55 ppm. Whereas, the δC shift of NCH3 carbon of six membered 184

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Table 4 31 P NMR spectral data of the cyclotriphosphazenesa.

(

R1

PX

XP

Cl Cl

(1a-5a)

Compound

1a* 2a* 3a* 4a* 5a* 1b 1b' 2b 2b' 3b 3b' 4b 5b 1c

Spin System

AX2 AX2 AX2 AX2 AX2 A2 X A2 X A2 X A2 X A2 X A2 X A2 X A2 X AX2

CH3; CH2CH3

R : F; NO2

P X

RArCH2 N N

R1

N

N A

PA

R2

R1: (CH2)2 ; (CH2)3

Cl

N (1b-5b)

P

R2:

R2

CH3; CH2CH3

R : F; NO2

N

N

R1

FArCH2 N N CH3

RArCH2

N

N

N

)

N

N

Cl

)

Cl

R2:

P A

N Cl

R1: (CH2)2 ; (CH2)3

NR2

N

)

CH2

)

(

R

P A

PX N (1c)

N XP

CH3 N N CH2ArF

2

δ(ppm) PA

PX

19.22 17.72 14.34 19.35 17.81 24.40 24.31 23.41 23.36 19.98 20.90 20.53 23.75 18.94

24.17 23.78 22.71 24.40 23.94 29.41 29.47 28.86 28.81 25.85 25.70 26.89 29.14 28.24

2

JPP (Hz)

JAX: JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2 JAX: 2

42.2 42.0 36.4 43.0 42.4 54.7 54.7 53.4 54.7 37.3 38.9 46.7 53.6 49.8

a31

P NMR data for the compounds in CDCl3 solutions at 293 K. *The values of compounds (1a–5a) were taken from the literature [29,30] for comparison. PA designates for spiro and PX for the geminal P atoms.

the second-order effects, were calculated using the frequencies of the external transitions of triplets in the 13C spectra [44,45]. On the other hand, the 1H NMR data of the bis(4-fluoro/nitrobenzyl)spirocyclotriphosphazenes were presented in the “Experimental Part”. The 3JFH, 4JFH, 3JHH and δ shifts of H2/H6 and H3/H5 protons of the FPh groups of the phosphazenes (1b-3b and 1c) are clearly interpreted. The average 3JFH, 4JFH and 3JHH values of H2/ H6 and H3/H5 protons of the FPh groups are found to be 3JFH = 8.6 Hz, 4 JFH = 5.5 Hz and 3JHH = 8.7 Hz, respectively. These data are in agreement with the literature values [30]. The 1H NMR spectra of the pendant armed dispirophosphazenes are highly complex, since all the protons are diastereotopic. In addition, the protons H2/H6 and H3/H5 are observed at ca. 8.22 and 7.65 ppm, as two groups of doublets. The average 3JHH value is 8.6 Hz. The NCH3 protons of the trans-dispirophosphazenes (1b, 1c, 3b and 4b) are observed at ca. 2.52 ppm and gave rise to doublets. The average coupling constant, 3JPH, is 12.6 Hz. This value is very large regardless of the spiro rings. The NCH2CH3 protons of 2b and 5b arise at ca. 1.10 ppm, as triplets, as well. Besides, the characteristic νP=N stretching bands are observed in the ranges of 1227–1221 cm−1 and 1191–1154 cm−1, respectively, indicating the frequencies of the P]N bonds of the P3N3 rings [46]. The average νC-F band is observed at 1065 cm−1. This value is in agreement with the literature data of the trimeric phosphazenes containing Ph-F substituents [47].

3.3. X-ray structures of 1a, 3b and 6 The molecular and crystal structures of compounds (1a, 3b and 6) were established using their crystallographic data. Compound 1a was synthesized previously, but its molecular and crystal structures were not reported in the literature [30]. Nevertheless, the structure of 1a was also given in this manuscript and compared with the structure of 3b. The ORTEP drawings with the atom-numbering schemes of 1a, 3b and 6 were illustrated in Figs. 3, 4 and 5, respectively. The conformations of the trimeric phosphazene rings of 1a and 3b were presented by the torsion angles of the ring bonds (Fig. S1, S designates Supplementary Material), indicating a pseudo-twofold axis running through the P3….N1 and P6….N6 (for 1a), P3…N1 (for 3b) atoms. Compound 1a has two independent molecules in the asymmetric unit. In compounds (1a and 3b), the six-membered phosphazene [(P1/N1/P2/N2/P3/N3) and (P4/N6/P5/N7/P8/N8), (P1/N1/P2/N2/P3/N3)] rings are in nearly planar [Fig. S2; the total puckering amplitudes, QT [48], 0.090(2) Å, φ2 = 52.86(1.87)°, θ2 = 45.74(1.44) ° and QT = 0.285(8) Å, φ2 = 24.21(2.30) °, θ2 = 114.2(9) ° (for 1a), Fig. S3a; QT = 0.132(2) Å, φ 2 = -123.13(1.12) °, θ2 = 132.8(8) ° (for 3b)], respectively. These findings are in agreement with the QT values found for the twisted and flattened conformations observed for some mono and bis amino cyclotriphosphazenes [29,30,49]. While, the six membered spiro rings of 3b, (P1/N4/C8/C9/C10/N5) and (P2/N7/C19/C20/C21/N6), are in Scheme 2. The synthesis of compound 6.

185

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Fig. 2. The second order effects in

13

C {1H} NMR spectra of 1b, 1c and 4b.

Fig. 5. An ORTEP-3 [34] drawing of 6 with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level.

chair conformations, respectively [Fig. S3b; QT = 0.9289(7) Å, φ 2 = 156.5(5) °, θ2 = 60.2(2) ° and QT = 0.6452(4) Å, φ 2 = 37.1(3) °, θ2 = 91.7(4) ° (for 3b)]. The average exocyclic PeN bond lengths [1.626(3) Å and (1.622(3) Å) (for 1a) and 1.657(3) Å (for 3b)] are considerably longer than the average endocyclic PeN bond lengths [1.578(3) Å and 1.578(3) Å (for 1a) and 1.589(3) Å (for 3b)], implying single and double bond characters of the exocyclic and endocyclic PeN bonds [50]. In addition, the endocyclic N1P1N3 (α) and N6P4N8 (α) angles of 1a are significantly narrowed, and whereas the endocyclic P1N3P3 (β) and P4N6P5 (β) bond angles are enlarged. Meanwhile, the endocyclic P2N2P3 (δ) and P5N7P6 (δ) angles are also narrowed according to the corresponding angles [α:118.3(2) °, β:121.4(3) °, δ: 121.4(3) °] of the starting compound N3P3Cl6 [51]. In compound 3b, the N1P1N3 and N1P2N2 angles are considerably narrowed due to the steric and electronic properties of spiro rings, but the P1N1P2 angle is enlarged. In the pendant armed mono and bis cyclotriphosphazenes, the similar variations of the bond lengths and bond angles are also observed. These changes are consistent with the literature data [29,30,49]. Moreover, the most important finding in this paper is the absolute configuration of 3b using the X-ray crystallographic data. The space group of 3b is P −1, and it is among the chiral Sohncke space groups [52,53]. Compounds having this space group may be chiral or achiral. The absolute configurations of P1 and P2 atoms are found to be SS. Compound 3b should be conformationally-locked, and hence it is

Fig. 3. An ORTEP-3 [34] drawing of 1a with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. Hydrogen atoms have been omitted for clarity.

Fig. 4. An ORTEP-3 [34] drawing of 3b with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. Hydrogen atoms have been omitted for clarity.

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Fig. 6. Results of the electrophoresis after the incubation of plasmid DNA with the compounds for 24 h. The first Lane (P) indicates control untreated DNA.

effectively chiral as it is not converted to it mirror image [52,53]. However, compound 3b (the trans isomer) indicates that it is likely to be a racemic mixture. According to the literature (52), it may be claimed that the conformationally-locked compound 3b represents an example of a spontaneous resolution of a racemic mixture, indicating the crystallization of a single enantiomer (SS). Compound 6 has a five-membered heterocyclic ring with two bulky

substituents. The conformation of the five- membered ring is in a cover open-envelope. It is a chiral compound and the absolute configuration of the C5 atom is R. In compound 6, the average values of the endocyclic and exocyclic CeN bond lengths are 1.463(4) Å and 1.451(4) Å, respectively. They are consistent with the CeN bond lengths of organic compounds given in the literature [50]. In compound 1a, the molecules are stacked along the a-axis (Fig.

Fig. 7. Results of the electrophoresis after the incubation of plasmid DNA with the compounds for 48 h. The first Lane (P) indicates control untreated DNA. 187

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Fig. 8. Electrophoretogram for the incubated mixtures of plasmid DNA followed by digestion with BamHI (a) and HindIII (b). Lane P applies to untreated DNA, PB and PH, untreated but digested plasmid DNA.

S4). The weak CeH···π interactions (Table 3) and a π ···π interaction between the benzene rings [Cg1···Cg4i = 3.881(2) Å; symmetry code: (i) 1 – x, -y, -z, where Cg1 and Cg4 are the centroids of the A (C1—C6) and D (C11—C16) rings, respectively] may be effective in the stabilization of the crystal structure. In compound 3b, the intermolecular C—Hspiroring···F hydrogen bonds (Table 3) link the molecules into centrosymmetric dimers forming R22(20) ring motifs (Fig. S5). A weak CeH···π interaction (Table 3) may further stabilize the crystal structure. In compound 6, the intermolecular C—Hfmr···F and C—HPh···F [fmr = five membered A (N1/N4/C2/C3/C5) ring and Ph = phenyl B (C8—C13) ring] hydrogen bonds (Table 3) link the molecules into a network structure enclosing R44(34) ring motifs (Fig. S6). A weak CeH···π interaction (Table 3) may further stabilize the crystal structure.

site for the enzyme, the linear form will be produced, because both strands are cleaved. These restricted, not restricted, and single nicked DNA (if only one strand is cleaved), three plasmid DNA conformations were distinguishable when subjected to agarose gel electrophoresis. In order to determine the cleavage activity of the compounds, the incubated mixtures of plasmid DNA and compounds were cut with restriction enzyme BamHI and HindIII. Fig. 8a and 8b show the representative electrophoretograms from the cleavage of DNA by the compounds. While the BamHI and HindIII enzyme recognize and cut DNA at the 5′-G/GATCC-3′ and 5′-A/AGCTT-3′ sequence respectively, and Form I and Form II convert to linear Form III DNA. Untreated plasmid DNA is applied to Lane P. In the absence of the compounds, when untreated plasmid DNA is digested with the enzymes, only the linear form III band is observed (Lane PB for BamHI digestion; Lane PH for HindIII digestion). All of the tested compounds inhibited enzyme cleavage indicating compound binding the specific G/G and A/A nucleotides. The results indicate that the binding of the compounds to plasmid DNA has been able to prevent BamHI and HindIII digestion at the highest concentrations. The reason for inbition of cleavage is due to the binding of the compounds with the DNA to A/A and G/G nucleotides. The binding of the phosphazenes can be ascribed to the hydrogen bonds through the substituents bonded to the P atoms of the strong phosphazene bases with the active centers of plasmid DNA nucleotides. In addition, it is emphasized that the biological activities of the conjugates (4b and 5b) were expected due to the possible oxidative DNA damage by the redox active nitro groups [29]. The dipole–dipole and van der Waals interactions are also very important for the binding of the compounds to plasmid DNA.

3.4. Interactions of DNA with the compounds The interactions between DNA and the phosphazenes were examined by agarose gel electrophoresis. The binding was observed through changes in the mobility of the DNA. Figs. 6 and 7 show the interactions between plasmid DNA and decreasing concentrations of the compounds ranging from 2000 to 125 µM (Lane 1, 2000 μM; Lane 2, 1000 μM; Lane 3, 500 μM; Lane 4, 250 μM; Lane 5, 125 μM) for 24 h and 48 h, respectively. The plasmid DNA is found to be in three different forms; supercoiled Form I, singly nicked relaxed circular Form II and linear Form III. Typically, the gel electrophoresis shows two main bands (Form I and Form II) for the untreated plasmid (Lane P). Form III is observed in the middle when plasmid DNA is restricted by an enzyme or double strand break. Compounds (1b, 1c, 3b-5b and 6) incubated with plasmid DNA showed a certain degree of DNA cleavage of supercoiled plasmid DNA. All of the compounds caused a decrease in the mobilities and intensities of form I and form II DNA. There were small fragments in all the concentrations of 1b, 1c and 6 and the lowest concentration of 3b. Compounds (3b, 4b and 5b) caused the disappearance of supercoiled DNA at the highest concentrations of the compounds, the intensity and mobility decreased with the incubation. Fig. 6 also shows a representative gel from the concentration dependent cleavage of the DNA by the compounds (6, 3b, 4b, 5b, 1b and 1c). For all of these compounds linear form III DNA was observed. Compounds (6, 3b, 4b, 5b, 1b and 1c) showed significant DNA cleavage activity in a concentration dependent manner. The incubation time caused very little effect on mobility and the intensity of the DNA (Fig. 7).

4. Conclusions In this study, the nucleophilic Cl exchange reactions of N3P3Cl6, with two equimolar alkyl(4-fluorobenzyl) and (4-nitrobenzyl)diamines (1–5), were investigated. The trans-bis(4-fluorobenzyl) (1b-3b) and trans-bis(4-nitrobenzyl) (4b and 5b) spirocyclotriphosphazenes have been obtained. The structures of compounds (1b-5b and 1c) were determined by elemental analyses, ESI-MS, FTIR, 1H, 13C, and 31P NMR techniques. The molecular and crystal structures of 1a, 3b and 6 were established by X-ray crystallography. In this paper, the most important finding is that the absolute configurations of 3b and 6 were unambiguously determined as SS and R respectively, using X-ray crystallographic data. The interactions of compounds (1b, 1c, 3b-5b and 6) with plasmid DNA indicated that compounds (4b and 5b) caused the loss of form I, and other compounds (1b, 1c, 3b and 6) caused the decrease in the mobilities and intensities of form I and form II DNA and

3.5. HindIII and BamHI digestions of the compounds- plasmid DNA In the presence of a DNA restriction enzyme, if there is a restriction 188

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cleavage of DNA. The compounds caused changes in DNA conformation and also DNA damage. The changes in DNA conformation are believed to be due to DNA double strand breakage. Furthermore, the compounds inhibited enzymatic cleavage, showing the compounds binding to DNA at specific G/G and A/A sequences.

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