Phosphorus-nitrogen compounds: Part 69. Unsymmetrical Dispiro(N/N)cyclotriphosphazenes Containing Different Pendant Arms: Syntheses, Characterization, Stereogenism, Photophysical and Bioactivity Studies

doi:10.21203/rs.3.rs-2442654/v1

In this research, the starting compounds tetrachloromonospirocyclotriphosphazenes, (BzSpiro-5)R¹(N₃P₃)Cl₄ [Bz: Benzyl; R¹: Me (1) and R¹: Et (2)], were synthesized from the reactions of hexachlorocyclotriphosphazene, N₃P₃Cl₆ (HCCP, trimer) with N/N-donor type benzyldiamines. Both phosphazenes were reacted with 9-ethyl-N-methyl-3-carbazolyldiamines (3 and 4) to form dichloro trans/cis N/N-dispirocyclotriphosphazenes, [(BzSpiro-5)R¹(N₃P₃)(CzSpiro-n)R²]Cl₂ (Cz: Carbazolyl; R¹, R²: Me or Et; n= 5 or 6; (5a-8a and 5b-8b), containing unsymmetrical diamino precursors. The structure of the cis-5b was clarified by single crystal X-ray crystallography. The space group of the chiral cis-5b isomer is P -1, which is centrosymmetric, and both enantiomers must be present in the unit cell. Since the absolute configuration of an enantiomer is found as SR' according to the X-ray crystallography, the other must be RS'. The chiralities of the compounds were also confirmed by ³¹P NMR spectroscopy recorded upon addition of chiral solvating agent [(S)-(+)-2,2,2-trifluoro-1-(9’-anthryl)ethanol; CSA] (for 5a, 5b and 8b) and the circular dichroism (CD) (for 5a, 5b, 6a and 6b) spectra. All N/N-dispirocyclotriphosphazenes (5a-8b) bearing carbazole units exhibited a fluorescence profile with a lifetime of about 4.9-6.5 ns and a quantum yield in the range of 0.11-0.20. On the other hand, P. aeruginosa was sensitive to compounds 5b, 7a, 8a and 8b, while E. coli was sensitive to most of the phosphazenes. The cytotoxic activities of phosphazenes against Vero cells (healthy) and MDA-MB-231 breast cancer cells were determined. In addition, compounds 5a and 5b appear to have very high antioxidant activity with respect to radical scavenging activities of 55.1% and 68.3%, respectively.

Unsymmetrical dispirocyclotriphosphazene

photophysical properties

crystal structure

chirality

antimicrobial activity

cytotoxicity

Phosphazenes, especially cyclo, polymeric, dendrimeric phosphazene derivatives, continue to be an increasingly important area of interest for researchers [1-7]. Hexachlorocyclotriphosphazene, N₃P₃Cl₆ (HCCP, trimer), remains on the focus of research of cyclophosphazenes due to its thermal stability, easy functionality, biocompatibility and versatile chemistry. Chlorocyclotriphosphazenes are robust cores suitable for nucleophilic substitution reactions due to their spatial orientations and significantly reactive P-Cl bonds. Thus, HCCP has the feature of functionalization with different substituents and gaining different properties depending on the types of the substituents. For this, generally, mono and bidentate ligands containing OH/NH/SH functional group/groups have been reported in the literature [8-12]. There are fewer articles on the reactions of HCCP with bidentate ligands than with monodentate reagents. Nucleophilic substitution reactions of trimer with bidentate reagents produce compounds of different architectures such as spiro, ansa, bino, dispiro and trispiro [13-24]. Considering the reported articles in the scientific literature, it is also clear that the number of monospirocyclotriphosphazenes is larger than those of dispiro or trispiro compounds. On the other hand, chirality of phosphazenes is one of the very interesting topics that has been studied continuously for the last two decades. This topic is investigated by X-ray crystallography, high-pressure liquid chromatography (HPLC), circular dichroism (CD) and/or chiral solvating agent (CSA)-added ³¹P NMR spectra [25-27].

Cyclophosphazene derivatives also have an important place in various applications such as catalysis [28], chemical [29] and biological imaging [30], and biomedical materials [31], multisite coordination ligands [32] and fluorescent dyes [33]. On the other hand, the supramolecular systems, containing cyclophosphazenes as a main platform, have been employed in the systems such as host-guest complexes, liquid crystals and nanostructures [34]. Many cyclic phosphazenes exhibit significant biological properties as well. Recently, cytotoxic activity studies of paraben-decorated monospiro-cyclotriphosphazenes have been performed on cancer cell lines (human breast adenocarcinoma, MCF-7 and colorectal adenocarcinoma, DLD-1) [35]. Meanwhile, BODIPY-containing cyclotriphosphazene dendrimers were used for the first time under high-power laser irradiation in vivo phototherapy [36].

As it is well known, carbazole is an intriguing tricyclic structure in chemistry. Carbazole and its derivatives serve as an important platform in bioactive natural products and synthetic compounds that achieve broad biological properties, including antifungal, antibacterial [37] and anticancer activity [38]. Some carbazole derivatives have been reported to have antioxidant activity [39].

In recent years, studies have been carried out on the binding and integration of carbazole and its derivatives to trimeric phosphazene. In a recent study, trimeric phosphazene functionalized with carbazolyldiamines was synthesized and its spectroscopic, crystallographic and thermal properties were discussed in detail [40]. In addition, carbazole decorated cyclotriphosphazenes with intense luminescence properties have also been declared [41-43]. A literature survey revealed that there are quite a number of manuscripts on symmetrically substituted dispirocyclotriphosphazenes [14,16,22,23]. However, there are a few studies on unsymmetrical dispirocyclotriphosphazenes without pendant arms in the literature [44-47]. Moreover, to the best of our knowledge, there is not any report dealing with dispirocyclotriphosphazenes containing different spiro-rings with different unsymmetrical pendant arms.

In the current study, primarily the syntheses, spectroscopic, stereogenic, photophysical and crystallographic properties of dispirocyclotriphosphazenes with unsymmetrical bulky benzyl and carbazolyl pendant arms, [(BzSpiro-5)R¹(N₃P₃)(CzSpiro-n)R²]Cl₂ (Bz: Benzyl, Cz: Carbazolyl; n, designates the number of members of the spiro-rings; R¹=R²: Me, n=5 trans-5a and cis-5b; R¹=R²: Me, n=6 trans-6a and cis-6b; R¹: Et, R²= Me, n=5 trans-7a and cis-7b; R¹: Et, R²= Me, n=6 trans-8a and cis-8b), are investigated, where the obtained compounds are the second examples of unsymmetrical cis/trans (N/N) dispirocyclotriphosphazenes with different bulky side groups [48]. In addition, the antimicrobial, anticancer and antioxidant effects of these phosphazenes and their interactions with plasmid DNA are considerably investigated in detail.

Materials and methods

Benzaldehyde (Sigma), N-ethyl-3-carbazolecarboxaldehyde (Acros Organics), trimer (TCI) and aliphatic diamines (Fluka) were purchased. The solvents used in this study were dried and distilled by standard methods. All reactions were monitored by thin layer chromatography (TLC) on Merck DC Alufolien Kieselgel 60 B₂₅₄ sheets using toluene-tetrahydrofuran mixture as an eluent. The column chromatography was performed on Merck Kieselgel 60 (230−400 mesh ATSM) silica gel. Melting points were measured with a Gallenkamp apparatus using a capillary tube. Microanalyses (C, H, N) were performed using a Leco CHNS-932 elemental analyzer at the Central Instrumental Analysis Laboratory in the Faculty of Pharmacy at Ankara University. The ¹H, ¹³C and ³¹P NMR spectra were recorded on a Bruker DPX FT-NMR (500 MHz) spectrometer. The spectrometer was equipped with a 5 mm PABBO BB inverse-gradient probe. Standard Bruker pulse programs [49] were used. The IR spectra were recorded on a Mattson 1000 FTIR spectrometer in KBr disks and were reported in cm^-1 units. Electrospray ionization (ESI) mass spectrometric analyses were performed on a Waters 2695 Alliance Micromass ZQ spectrometer for the other monoferrocenylphosphazenes.

Ultraviolet (UV) absorption and CD spectra of phosphazenes were obtained with a Jasco J720 spectropolarimeter, flushed with N2, and scanned at 10 nm/min with a spectral bandwidth of 1 nm and a data resolution of 0.2 nm. The solvent was MeCN and cells were strained free with path lengths of 1 cm and 0.2 cm. CD spectra were presented relative to a normalized absorbance in a 1 cm cell, A(265.6 nm) = 1. UV-Vis absorption spectra were recorded on a Shimadzu, UV-1800 UV-Vis spectrophotometer. Photoluminescence (PL) characterizations were performed with a Varian Eclipse spectrofluorometer. Time-correlated single photon counting measurements were made with the Horriba-Jobin-Yvon-SPEX Fluorolog 3-2iHR device with a 310 nm pulsed laser diode and the Fluoro Hub-B Single Photon Counting Controller. PL quantum yields in dichloromethane (DCM) were calculated by comparing the fluorescence of the Quinine sulfate standard (ΦPL = 0.54 in 0.10 N H₂SO₄), (λexc = 310 nm).

The parameters for fluorescence quantum yields

The fluorescent quantum yields (ΦF) of cis/trans-dispirocyclotriphosphazenes 5-12 were determined according to the comparative method (Eq. 1) [50].

Where F and F_Std are the areas under the fluorescence emission curves of the dispirocyclotriphosphazenes and the standard, respectively. A and A_Std are the respective absorbances of dispirocyclotriphosphazenes and the standard at the excitation wavelengths, respectively. The refractive indices (n) of the solvents were used to calculate the fluorescent quantum yields in different solvents. Quinine sulfate (Φ_PL= 0.54 in 0.10 N H₂SO₄) [51] was used as a standard.

X-ray crystallography

The suitable single crystals of compound 5b were obtained from acetonitrile at ambient temperature. The crystallographic data were recorded on a Bruker APEX II QUAZAR three-circle diffractometer using Mo K_aradiation (l=0.71073 Å) at T=120 (2) K. No absorption correction was applied. Structure was solved by direct methods [52] and refined by full-matrix least-squares against F² using all data [53]. All of the non-H atoms were refined anisotropically. The molecular and packing diagrams together with the ring conformations were drawn by ORTEP-3 program incorporated in the WinGX package [54]. The C-bound H atoms were positioned geometrically with C-H = 0.93, 0.97 and 0.96 Å, for aromatic, methylene and methyl H atoms, respectively, and constrained to ride on their parent atoms, with Uiso(H) = k X Ueq(C), where k = 1.5 for methyl H atoms and k = 1.2 for aromatic and methylene H-atoms.

Preparations of compounds

Tetrachloromonospirophosphazene derivatives (1 and 2) and diamines (3 and 4) were synthesized according to published procedures [15,40].

Syntheses of 5a and 5b

Tetrachloromonospirophosphazene (1) (3.24 g, 7.38 mmol) in THF (150 mL) was added to the solution of carbazolyldiamine (3) (2.08 g, 7.38 mmol) in THF and triethylamine (Et₃N; 14.76 mmol, 0.76 g/mL, 2.06 mL) at room temperature. After the mixture was stirred for 72 h, the precipitated triethylamine hydrochloride was filtered out. The solvent was evaporated, and then a monospiro and two dispiro phosphazene products were purified by column chromatography using toluene-THF (5:1) as eluent. First product was the monospirophosphazene (1). Yield: 1.96 g (30%). The trans-phosphazene (5a), white powder second product, was crystallized from acetonitrile at room temperature. Yield: 1.73 g (36%), mp: 115 °C. Rf= 0.46 {in toluene-THF (5:1)}. Anal. Calc. for C₂₈H₃₅Cl₂N₈P₃.1/2H₂O (%): C, 51.23; H, 5.53; N, 17.31. Found (%): C, 51.62; H, 5.59; N, 17.07. APIES-MS (fragments are based on ³⁵Cl, Ir %): m/z Calc. 647.17 ([MH]⁺, 100.0). Found: 647.94 ([MH]⁺, 100.0). FTIR: ν 3057 (C-H arom.),2976 (asymm.), 2860 (symm.) (C-H aliph.), 1170 (asymm.) (P=N), 576 (asymm.), 517 (symm.) (P-Cl aliph.). The cis-phosphazene (5b), white powder third product, was crystallized from acetonitrile at room temperature. Yield: 1.47 g (31%), mp: 143 °C. Rf= 0.34 {in toluene-THF (5:1)}. Anal. Calc. for C₂₈H₃₅Cl₂N₈P₃.1/2H₂O (%): C, 51.23; H, 5.53; N, 17.07. Found (%): C, 51.15; H, 5.37; N, 16.84. APIES-MS (fragments are based on ³⁵Cl, Ir %): m/z Calc. 647.17 ([MH]⁺, 100.0). Found: 647.94 ([MH]⁺, 100.0). FTIR: ν 3057 (C-H arom.),2971 (asymm.), 2871 (symm.) (C-H aliph.), 1157 (asymm.) (P=N), 572 (asymm.), 513 (symm.) (P-Cl aliph.).

Syntheses of 6a and 6b

The procedure performed in the syntheses of 5a and 5b was followed for the syntheses of 6a and 6b. Compound (1) (3.27 g, 7.45 mmol) in THF (150 mL) was added to the solution of carbazolyldiamine (4) (2.20 g, 7.45 mmol) in THF and triethylamine (Et₃N; 14.90 mmol, 0.76 g/mL, 2.08 mL) at room temperature. After the mixture was stirred for 72 h, the precipitated triethylamine hydrochloride was filtered out. The solvent was evaporated, and then a monospiro and two dispiro phosphazene products were purified by column chromatography using toluene-THF (5:1) as eluent. First product was the monospirophosphazene (1). Yield: 1.05 g (32%). The second white powder product (6a), trans-phosphazene, was isolated from column. Yield: 1.74 g (35%), mp: 126 °C. Rf= 0.57 {in toluene-THF (5:1)}. Anal. Calc. for C₂₉H₃₇Cl₂N₈P₃ (%): C, 52.66; H, 5.64; N, 16.94. Found (%): C, 52.42; H, 5.60; N, 16.80. APIES-MS (fragments C₂₉H₃₇Cl₂N₈P₃are based on ³⁵Cl, Ir %): m/z Calc. 661.18 ([MH]⁺, 100.0). Found: 661.65 ([MH]⁺, 100.0). FTIR (KBr, cm^-1): ν 3057 (C-H arom.),2973 (asymm.), 2859 (symm.) (C-H aliph.), 1167 (asymm.) (P=N), 575 (asymm.), 516 (symm.) (P-Cl aliph.). The white powder third product (6b), cis-phosphazene, was obtained. Yield: 1.49 g (30%), mp: 148 °C. Rf= 0.43 {in toluene-THF (5:1)}. Anal. Calc. for C₂₉H₃₇Cl₂N₈P₃ (%): C, 52.66; H, 5.64; N, 16.94. Found (%): C, 52.31; H, 5.66; N, 16.92. APIES-MS (fragments are based on ³⁵Cl, Ir %): m/z Calc. 661.18 ([MH]⁺, 100.0). Found: 661.67 ([MH]⁺, 100.0). FTIR (KBr, cm^-1): ν 3060 (C-H arom.),2973 (asymm.), 2859 (symm.) (C-H aliph.), 1167 (asymm.) (P=N), 577 (asymm.), 517 (symm.) (P-Cl aliph.).

Syntheses of 7a and 7b

The same procedure was followed for the syntheses of 7a and 7b. Compound (2) (2.64 g, 5.82 mmol) in THF (150 mL) was added to the solution of carbazolyldiamine (3) (1.64 g, 5.82 mmol) in THF and triethylamine (Et₃N; 11.64 mmol, 0.76 g/mL, 1.62 mL) at room temperature. The mixture was stirred for 72 h, then the precipitated triethylamine hydrochloride was filtered off. After the solvent was evaporated, a monospiro and two dispiro phosphazene products were purified by column chromatography using toluene-THF (5:1) as mobile phase. First product was the monospirophosphazene (2). Yield: 0.74 g (28%). The trans-phosphazene (7a), second white powder product, was obtained. Yield: 1.39 g (36%), mp: 114 °C. Rf= 0.50 {in toluene-THF (5:1)}. Anal. Calc. for C₂₉H₃₇Cl₂N₈P₃.1/2H₂O. (%): C, 51.95; H, 5.71; N, 16.71. Found (%): C, 52.06; H, 5.72; N, 16.73. APIES-MS (fragments C₂₉H₃₇Cl₂N₈P₃are based on ³⁵Cl, Ir %): m/z Calc. 661.18 ([MH]⁺, 100.0). Found: 661.96 ([MH]⁺, 100.0). FTIR (KBr, cm^-1): ν 3054 (C-H arom.),2970 (asymm.), 2849 (symm.) (C-H aliph.), 1169 (asymm.) (P=N), 575 (asymm.), 520 (symm.) (P-Cl aliph.). The white powder third product (7b), cis-phosphazene, was produced. Yield: 1.20 g (31%), mp: 127 °C. Rf= 0.42 {in toluene-THF (5:1)}. Anal. Calc. for C₂₉H₃₇Cl₂N₈P₃ (%): C, 52.66; H, 5.64; N, 16.94. Found (%): C, 52.84; H, 5.93; N, 16.71. APIES-MS (fragments are based on ³⁵Cl, Ir %): m/z Calc. 661.18 ([MH]⁺, 100.0). Found: 661.90 ([MH]⁺, 100.0). FTIR (KBr, cm^-1): ν 3057 (C-H arom.),2972 (asymm.), 2871 (symm.) (C-H aliph.), 1160 (asymm.) (P=N), 575 (asymm.), 517 (symm.) (P-Cl aliph.).

Syntheses of 8a and 8b

The exact same process used in the syntheses of 8a and 8b. Compound (2) (3.04 g, 6.71 mmol) in THF (150 mL) was added to the solution of carbazolyldiamine (4) (1.98 g, 6.71 mmol) in THF and triethylamine (Et₃N; 13.42 mmol, 0.76 g/mL, 1.87 mL) at room temperature. After the mixture was stirred for 72 h, the precipitated triethylamine hydrochloride was filtered out. The solvent was evaporated, and then a monospiro and two dispiro phosphazene products were purified by column chromatography using toluene-THF (5:1) as eluent. First product was the monospirophosphazene (2). Yield: 0.88 g (29%). The trans-phosphazene (8a), second product, was obtained as a white powder. Yield: 1.54 g (34%), mp: 95 °C. Rf= 0.61 {in toluene-THF (5:1)}. Anal. Calc. for C₃₀H₃₉Cl₂N₈P₃ (%): C, 53.34; H, 5.82; N, 16.59. Found (%): C, 53.14; H, 5.99; N, 16.38. APIES-MS (fragments C₃₀H₃₉Cl₂N₈P₃are based on ³⁵Cl, Ir %): m/z Calc. 675.20 ([MH]⁺, 100.0). Found: 675.21 ([MH]⁺, 100.0). FTIR (KBr, cm^-1): ν 3060 (C-H arom.),2934 (asymm.), 2859 (symm.) (C-H aliph.), 1170 (asymm.) (P=N), 575 (asymm.), 517 (symm.) (P-Cl aliph.). The cis-phosphazene, third product (8b), was isolated as a white powder. Yield: 1.46 g (32%), mp: 112 °C. Rf= 0.55 {in toluene-THF (5:1)}. Anal. Calc. for C₃₀H₃₉Cl₂N₈P₃ (%): C, 53.34; H, 5.82; N, 16.59. Found (%): C, 53.08; H, 5.72; N, 16.46. APIES-MS (fragments are based on ³⁵Cl, Ir %): m/z Calc. 675.20 ([MH]⁺, 100.0). Found: 675.21 ([MH]⁺, 100.0). FTIR (KBr, cm^-1): ν 3060 (C-H arom.),2926 (asymm.), 2853 (symm.) (C-H aliph.), 1165 (asymm.) (P=N), 539 (asymm.), 518 (symm.) (P-Cl aliph.).

On the other hand, methods for the determinations of antimicrobial and antioxidant activities, MIC and MBC/MFC values, cytotoxicity assay, DNA-compound interactions and BamHI and HindIII digestion were given in Supplementary Information (SI).

Syntheses of dispirocyclotriphosphazenes

Monospirocyclotriphosphazenes, (BzSpiro-5)R¹(N₃P₃)Cl₄ [R¹: Me (1) and R¹: Et (2)], were synthesized using HCCP and benzyldiamines according to the published procedure [15]. Condensation reactions of N-ethyl-3-carbazolecarboxaldehyde with N-methyl-1,2-diaminoethane and N-methyl-1,3-diaminopropane yielded 9-ethyl-N-methyl-3-carbazolyl-1,2-diaminoethane (3) and 9-ethyl-N-methyl-3-carbazolyl-1,3-diaminopropane (4), respectively, using the published procedure [40]. On the other hand, nucleophilic substitution reactions of equimolar amounts of monospirocyclotriphosphazenes (1 and 2) with equimolar amounts of N-ethyl-3-carbazolyl diamines (3 and 4) afforded unsymmetrical target cis/trans dispirocyclotriphosphazenes with carbazolyl and benzyl pendant arms, [(BzSpiro-5)R¹(N₃P₃)(CzSpiro-n)R²]Cl₂ (R¹=R²: Me, n=5 trans-5a and cis-5b; R¹=R²: Me, n=6 trans-6a and cis-6b; R¹: Et, R²= Me, n=5 trans-7a and cis-7b; R¹: Et, R²= Me, n=6 trans-8a and cis-8b). in THF. According to the methyl or ethyl groups bonded to the spiro rings, cis and trans isomers arose. As seen from the ³¹P {1H} NMR spectrum of the reaction mixture in Fig. 1, besides monospirocyclotriphosphazene (1), cis-6a and trans-6b isomers are also present in the mixture. The relative yields of compound (1) and trans/cis isomers (6a and 6b) were calculated as 63 and 37%, respectively. Geometrical isomers were detected by thin layer chromatography (TLC) and purified by column chromatography using silica gel as the stationary phase. The yields of all the trans and cis isomers were calculated to be in the ranges of 34-36% and 32-30%, respectively. However, it can be argued that the yields of the cis isomers are less than the yields of the trans isomers, possibly due to the steric hindrances between the bulky substituents.

The NMR, MS and microanalytical data prove that the proposed structures of cyclotriphosphazenes are correct. Protonated molecular ion peaks ([MH]⁺) appear in the mass spectra of all compounds.

Table 1 The ³¹P NMR (decoupled) spectral data of three phosphazenes and the effect of CSA on ³¹P NMR chemical shifts^a

NMR and IR spectroscopies

The structures of all novel compounds were confirmed by IR, MS, ³¹P, ¹H and ¹³C NMR and elemental analysis data. On the other hand, trans (5a, 6a, 7a and 8a) and cis (5b, 6b, 7b and 8b) dispirophosphazenes have two stereogenic P atoms. Hence, they are expected to exist as optical isomer mixtures (RR'/SS' and RS'/SR’), respectively (Fig. 2). To examine the stereogenic properties of dispirophosphazenes, CSA-added ³¹P NMR (for 5a, 5b and 8b) and CD (for 5a, 5b, 6a and 6b) spectra were recorded and visualized in Figs. 3 and 4, respectively. The effects of the addition of CSA on the ³¹P NMR spectra of 5a, 5b and 8b were evaluated for the mole ratio of compound to CSA at 15:1 (Fig. 3). The effects of CSA on the ³¹P NMR chemical shifts are listed in Table 1. It is observed that the addition of CSA has significant effects on the chemical shifts and coupling constants in the ³¹P NMR spectra of compounds 5a and 8b. All ³¹P NMR signals of 5a and 8b were observed to split into two lines, suggesting that it exists as a racemic mixture. However, it is a different case that the signals of 5b do not split in the CSA-added spectrum. As a result of this observation, it is considered that compound 5b behaves like a meso compound. Therefore, compound 5b can be stated as a pseudo-meso configuration [55]. Moreover, UV and CD spectra of 5a, 5b, 6a and 6b are recorded and given in Fig. 4. For these four compounds, “positive cotton effects” and “negative cotton effects’’ were observed in CD spectra. The figures also indicate that these compounds are racemic mixtures in solution. These findings have been encountered in the literature before [56]. Also, X-ray crystallography findings (see crystal structure solution section 3.4) confirmed that both enantiomers of cis-5b isomer were present in the crystal lattice. As a result, it can be stated that CSA-added ³¹P NMR, CD and X-ray crystallography data are compatible with each other.

The ³¹P (¹H) NMR data of the dispirophosphazenes are listed in Table 2. When ³¹P NMR spectroscopic data are evaluated, it can be stated that cis/trans dispirophosphazenes have A₂X (for 5b), AMX (for 6a and 6b) and ABX (for 5a, 7a, 7b, 8a and 8b) spin systems. These compounds have three different phosphorus environments. Thus, the signals of all phosphorus atoms, except cis-5b, appeared as doublets of doublets. It is worth emphasizing that the P(spiro/carbazolyl) signals shift downwards in the five-membered spiro-rings relative to the P(spiro/carbazolyl) atoms containing the six-membered spiro-rings. As explained before, in cis-5b, P(spiro/carbazolyl) and P(spiro/benzyl) signals seem to be overlapping. Thereof, compound 5b has a triplet and a doublet signals. Also, the spin-spin coupling constants (²J_PP) of the five-membered carbazolyl-spiro ring compounds are larger than those of the six-membered ones. Average ²J_PP constants are 50.5 Hz and 44.8 Hz for dispirophosphazenes containing five and six-membered carbazolyl-spiro rings, respectively.

The characteristic carbon and proton signals of the dispirophosphazenes were determined by evaluating the ¹³C and ¹H NMR spectra. Chemical shifts, coupling constants and multiplicities are presented in Tables S1 and S2). According to the ¹³C spectral data of the dispirophosphazenes (Table S1), the signals appearing in the ranges of 48.54-49.06 ppm and 48.98-50.93 ppm belong to the PhCH₂ and CzCH₂ carbons, respectively. The most significant signals in the compounds are the ipso-carbon peaks of the benzene (C1) and carbazole (C1') rings. It was observed that the C1 and C1' carbons resonate in the ranges of 137.58-137.88 ppm and 127.80-128.14 ppm, respectively. However, chemical shift values of other aromatic carbons were observed between 108.22-140.21 ppm. Another important signals are the peaks belonging to the N-CH₂-CH₃carbons bonded to the nitrogen of the carbazole rings appearing between 13.80-13.84 ppm. The N-CH₂-CH₃ carbons of the carbazole rings resonate in the range of 37.52-37.59 ppm. On the other hand, the average ³J_PC1’ coupling constant (9.8 Hz) of all compounds containing six-membered spiro-rings is greater than that of the five-membered ones (7.2 Hz). In contrast, the average ³J_PC between CH₂-carbazolyl-carbons and P atoms in phosphazenes with the six-membered spiro-rings (2.2 Hz) is smaller than in the five-membered ones (4.9 Hz). However, the average coupling constant of ³J_PC1 in all compounds was calculated as 6.9 Hz. Moreover, the coupling constants of ²J_PC between the phosphorus atoms and the N-CH₂ spiro-carbons of unsymmetrical dispirophosphazenes containing the five-membered spiro-rings are considerably larger than those of the six-membered ones, as observed previously [14,25,57].

The expected proton signals were determined from the ¹H spectra of the dispirophosphazenes (Table S2). It is quite important to point out a few characteristic peaks. Some of these are benzylic (PhCH₂) and carbazolyl (CzCH₂) protons, and their chemical shifts were found to be in the ranges of 3.98-4.33 ppm for PhCH₂ and 3.96-4.22 ppm for CzCH₂. These protons can differentiate from each other according to their chemical environments, and they are called diastereotopic. Accordingly, the PhCH₂ and CzCH₂ protons of all dispirophosphazenes are expected to be both diastereotopic. These diastereotopic protons emerge as doublets of doublets due to the geminal (²J_HH) and vicinal (³J_PH) couplings (for AMX spin system). Other characteristic peaks belong to methly (NCH₃) protons. The NCH₃ protons of all compounds were determined to be between 2.54 and 2.70 ppm, and the average coupling constant with the P-atom (³J_PH) was calculated as 12.0 Hz. In addition, aromatic hydrogens occur in the range of 7.14-8.15 ppm.

Table 2 The ³¹P NMR (decoupled) spectral data of the phosphazenes. [Chemical shifts (δ) are reported in ppm and J values in Hz]^a.[d: doublet, t: triplet and dd: doublet of doublets]

Compound	Spin system	PCl₂	P(NN)_benzyl	P(NN)_carbazolyl	²J_PP
5a	ABX	29.30(dd)	24.77(dd)	24.35(dd)	²J_AB= 55.7 ²J_AX= 52.6 ²J_BX = 43.5
5b	A₂X	29.40(t)	24.37(d)	24.37(d)	²J_AX= 54.7
6a	AMX	28.14(dd)	23.69(dd)	20.08(dd)	²J_AM= 48.6 ²J_AX= 46.2 ²J_MX = 41.3
6b	AMX	28.31(dd)	23.35(dd)	20.10(dd)	²J_AM= 48.6 ²J_AX= 46.2 ²J_MX = 41.3
7a	ABX	28.99(dd)	24.44(dd)	23.57(dd)	²J_AB= 55.8 ²J_AX= 53.4 ²J_BX = 53.4
7b	ABX	29.10(dd)	24.36(dd)	23.39(dd)	²J_AB= 48.6 ²J_AX= 46.2 ²J_BX = 41.3
8a	ABX	27.93(dd)	22.83(dd)	20.33(dd)	²J_AB= 47.4 ²J_AX= 41.3 ²J_BX = 41.3
8b	ABX	28.21(dd)	23.37(dd)	20.09(dd)	²J_AB= 48.6 ²J_AX= 46.6 ²J_BX = 40.5
^{a 31}P NMR measurements in CDCl₃ solutions at 293 K

The characteristic peaks expected from the dispirophosphazenes were determined in the IR spectra. The asymmetric νPN and νPCl stretching bands of all dispirophosphazenes emerged in the ranges of 1157–1170 cm⁻¹and 539–577 cm⁻¹, respectively. On the other hand, the characteristic aromatic νCH vibrations bands were observed between 3054 cm⁻¹and 3060 cm^-1.

Photophysical properties

Optical properties of trans (5a, 6a, 7a and 8a) and cis phosphazene stereoisomers (5b, 6b, 7b and 8b) were evaluated by UV-Vis and fluorescence spectroscopies. Primarily, the electronic absorption and fluorescent emission properties of trans-5a and cis-5b were studied in various polar and nonpolar solvents at room temperature (Fig. S1A/B and S1C/D). Among these solvents, DCM has been found to be suitable for both solubility and photophysical characteristics. Therefore, further studies were performed in DCM and the results were summarized in Table 3. Three main absorption bands were observed for the trans (5a, 6a, 7a and 8a) and cis (5b, 6b, 7b and 8b) phosphazenes in the ranges of 260–270 nm, 290–300 nm, and 290–300 nm (Fig. 5A/B). The absorption bands between 290-300 nm are mainly due to the π−π* electronic transitions in these molecules. Moreover, the absorptions of these compounds in the 330-350 nm range are mainly attributed to the n–π* electronic transitions.

On the other hand, all trans (5a, 6a, 7a and 8a) and cis (5b, 6b, 7b and 8b) derivatives give rise to maximum doublet emissions at 360 and 370 nm when excited at 290 nm (Fig. 5C/D). According to these UV-Vis absorption and fluorescent emission spectra, all trans and cis isomers are almost identical to those of the carbazole unit [58] due to the optical inertness of the cyclophosphazene core [41, 42, 59]. This means that the carbazole groups do not have an effective ground state interaction. Although the absorption and emission patterns of all isomers remain almost the same in DCM (Fig. S2A-H), the corresponding quantum yield and fluorescence lifetime values show slight decreases from trans to cis isomers (Table 3). Fluorescent quantum yields (Φ_F) of all dispirophosphazenes were measured in DCM and a solution of quinine sulfate in 0.10 N H₂SO₄ used as a standard. As an example, the fluorescence quantum yields of 5a and 5b were calculated as 0.16 and 0.11, respectively. The differences in fluorescence lifetime values were found to be compatible with fluorescence quantum yields. The fluorescence lifetimes (τ_F) of the isomers of the dispirophosphazenes (5a-8b) were determined using the time-correlated single photon count (TCSPC) technique in DCM. According to the lifetime spectra in Figs. 6A-D, the fluorescence lifetimes of all isomers were measured by bi-exponential calculation, which may result from various π-system conjugations in carbazole and NN-phenyl-decorated cyclotriphosphazenes [60]. While the lifetime values for all isomers varied around 5 and/or 6 ns, the trans isomers exhibited longer decay profiles than the cis isomers and the data were presented in Table 3.

Table 3 Photophysical properties of compounds 5a-8b

Compound	5a	5b	6a	6b	7a	7b	8a	8b
*λ_ab,* nm^a	265;296;334; 350	266;297;334; 349	266;297;337; 349	266;297;337; 350	266;297;336; 350	266;297;335; 350	266;297;336; 350	266;298;335; 350
*λ_em*, nm^a	360;371	358;373	361;371	360;373	360;371	360;371	360;373	361;371
τ_F(ns)^b	5.282	4.890	6.451	5.342	5.090	4.891	5.121	4.941
Φ_F^c	0.164	0.110	0.197	0.186	0.189	0.124	0.192	0.160

^a DCM. ^b Lifetime, ^cFluorescence quantum yield.

X-Ray structure of 5b

The crystal structure of 5b was determined by single crystal X-ray crystallography. The ORTEP diagram of cis-5b with atom-numbering scheme is shown in Fig. 7 and experimental details are listed in Table 4. The N₃P₃ ring A (P1/N1/P2/N2/P3/N3) [Fig. S3a;j₂ = 97.8(5)°, q₂= 62.1(4)°] is in twisted-boat conformation with the total puckering amplitude, Q_T, of 0.200(1) Å [61]. This conformation determined according to the j₂and q₂ values was found to be compatible with the literature data [21]. Moreover, the five-membered rings B (P1/N6/N7/C13/C14) and C (P3/N4/N5/C1/C2) are in half chair conformations [Fig. S3b; φ₂ = 307.9(4)° (for ring B) and φ₂ = 119.2(3)° (for ring C)].

On the other hand, X-ray crystallographic data reveal that phosphazene 5b is in the cis configuration with respect to its methyl groups. In addition, according to the crystallographic results, the absolute configurations of P1 and P3 atoms of cis-5b were determined to be S and R', respectively. However, cis-5b has two different stereogenic P-centers and is expected to exist as a racemic mixture. Compound cis-5b crystallizes in the P -1 (Z’=2) space group and does not belong to Sohncke space groups [62] since it has the inversion symmetry operation (centrosymmetric). For such a racemic structure crystallizing in the P-1 space group, both enantiomers must be present in the crystal lattice. In pseudo-meso cis-5b, it is clear that the absolute configuration of one enantiomer should be SR' and the other enantiomer RS' in the unit cell. The N₃P₃ ring of cis-5b also has no pseudocentrosymmetry with respect to torsion angles (Fig. S4). The endocyclic and exocyclic P–N bond lengths in the trimer ring of cis-5b range from 1.5653(16)-1.6246(17)A and 1.6391(17)-1.6526(17)A, respectively (Table 5). In addition, the average endocyclic P–N bond length [1.5949(16) Å] was calculated to be considerably shorter than the average exocyclic P–N bond length [1.6449(17) Å], consistent with literature data [15,63]. In this unsymmetrical dispirocyclotriphosphazene, there are significantly regular changes in the distances to P1: P1–N1 ≈ P3–N2 > P1–N3 ≈ P3–N3 > P2–N1 ≈ P2–N2. On the other hand, the endocyclic N1-P1-N3 and N2-P3-N3 (γ) angles of N₃P₃ rings [112.74(8)° and 113.27(8)°] (Table 5) are significantly narrowed with respect to the corresponding ones in the starting compound, N₃P₃Cl_6, [α, α’, β and δ: 118.3(2)°, 101.2(1)°, 121.4(1)° and 121.4(1)° [64]. The exocyclic NPN (γ’) bond angles of 5b are 93.95(8)° and 93.92(8)°, while the endocyclic PNP (β and δ) bond angles are 119.86(10)°, 120.24(10)° and 128.69(10)° (Table 5). The large PNP bond angle (δ), 128.69(10)°, indicates that the repulsions between the bulky substituents are very strong. Consequently, as observed previously, these changes in bond lengths and angles can be attributed to bulky substituents, conformations of phosphazene and spiro rings and negative hyperconjugation [65,66]. The planar rings, D (C5—C10), E (C15—C20), F (N8/C17/C18/C21/C26) and G (C21—C26), are oriented at dihedral angles of D/E = 53.91(7)°, D/F = 53.15(8)°, D/G = 51.23(7)°, E/F = 1.40(8)°, E/G = 2.77(8)° and F/G = 1.98(8)°. So, the E, F and G ring system is almost coplanar.

Table 4 Experimental details for 5b.

5b
Empirical Formula	C₂₈H₃₅Cl₂N₈P₃	m (cm^-1) (Mo K_a)	0.41
Fw	647.45	r (Calc.) (g cm^-1)	1.425
Crystal System	Triclinic	Number of Reflections Total	18862
Space Group	P -1	Total Number of Reflections Unique	6921
a (Å)	9.0355(3)	R_int	0.031
b (Å)	12.6247(5)	2q_max (°)	54.97
c (Å)	14.6643(5)	T_min/ T_max	0.943/0.956
a (°)	68.434(2)	Number of Parameters	373
b (°)	75.880(3)	R [F²>2s(F²)]	0.038
g (°)	84.133(3)	wR	0.099
V ( ³)	1508.51(10)	S	1.05
Z	2

In crystal structure, intermolecular C—H···Cl hydrogen bonds (Table 6) link the molecules into infinite chains along the a-axis direcrion (Fig. S5). A weak C—H···π interaction (Table 6) is also observed.

Table 5 Selected bond lenghts (Å) and angles (deg) for cis-5b
	5b
P1– N1	1.6241(16)	N1– P1– N3 (γ)	112.74(8)
P1– N3	1.5969(15)	N1– P2– N2 (α)	121.18(9)
P2– N1	1.5675(16)	N2– P3– N3 (γ)	113.27(8)
P2– N2	1.5653(16)	P1– N1– P2 (β)	119.86(10)
P3– N2	1.6246(17)	P2– N2– P3 (β)	120.24(10)
P3– N3	1.5907(15)	P1– N3– P3 (δ)	128.69(10)
P1– N6	1.6391(17)	N6– P1– N7 (γ’)	93.92(8)
P1– N7	1.6434(16)	N4– P3– N5 (γ’)	93.95(8)
P3– N4	1.6443(16)	Cl1– P2– Cl2 (α’)	98.85(3)
P3– N5	1.6526(17)

Table 6 Hydrogen-bond geometry (Å, º)

D—H···A	D—H	H···A	D···A	D—H···A
C1—H1B···Cl1ⁱ	0.97	2.81	3.511 (2)	130
C2—H2B···Cg7ⁱⁱ	0.97	2.62	3.552 (3)	162

Symmetry codes: (i) x+1, y, z; (ii) −x+2, −y, −z. Cg7 is the centroid of ring G (C21—C26).

Cell Viability Assay

The cytotoxicities of 5a, 5b, 7b and 8a were determined on breast cancer cells (MDA-MB-231) and healthy cells (Vero) using the MTT method. The results are shown in Fig. 8. According to MTT assay, none of compounds were as effective as the cis-Diammineplatinum(II) dichloride. Compound 5b was showed almost 50% inhibition effect at 250 µM concentration on Vero cells. However, same compounds did not show inhibition effect on cancer cells. As a result, the compounds tested were more effective against epithelial cells than breast cancer cells. Other values of concentration did not form a regular inhibition curve on both of cells.

Antimicrobial activity

In this study, agar well diffusion method was used to determine the antimicrobial activity of the microorganism based on the effect of chemical compounds. The agar plate surfaces were inoculated to allow bacterial and fungal microorganism to spread over the entire agar in this procedure (Supplementary Information, SI). Compounds were determined on Mueller Hinton Agar for bacteria strains and Sabouraud Dextrose Agar for yeast. The diameter of the inhibition zones were defined on Table S3. Zones of inhibition ranged from 10 millimeters to 20 millimeters. The antimicrobial effects of the compounds against both bacteria and fungi were then defined. According to the results obtained, all the compounds tested (except 6a and 6b) have very weak antimicrobial activity against tested microorganisms. Compounds 6a and 6b were not effective on all microorganisms. The minimum inhibitory concentration (MIC) is defined as the lowest concentration that prevent the growth of microorganisms. MIC determination was performed by serial dilution method in 96-well microplate. MIC values were found between 156.3 µM and 2500 µM. E. coli and B. subtilis were susceptible to most of the phosphazenes. However, P. aeruginosa was sensitive to compounds 5b, 7a, 8a and 8b (Table 7).

Table 7 Minimum inhibitory concentrations (MIC) of the compounds and positive controls against test strains (in µM). (Amp: Ampicillin, C: Chloramphenicol and Keto: Ketoconazole)

Test microorganisms	Compounds								Positive Controls
	5a	5b	6a	6b	7a	7b	8a	8b	Amp	C	Keto
*E. coli* ATCC 35218 *G(-)*	625	1250	625	1250	625	625	625	625	2500	625	-
*E. coli* ATCC 25922 *G(-)*	312.5	312.5	625	625	312.5	312.5	312.5	312.5	<19.5	78.1	-
*B. cereus* NRRL B-3711 *G(+)*	312.5	312.5	1250	1250	312.5	312.5	1250	1250	156.3	156.3	-
*B. subtilis* ATCC 6633 *G(+)*	1250	1250	1250	1250	1250	1250	1250	1250	<19.5	78.1	-
*S. aureus* ATCC 25923 *G(+)*	625	1250	1250	1250	1250	1250	625	625	<19.5	156.3	-
*E. faecalis* ATCC 29212 *G(+)*	1250	1250	1250	1250	1250	1250	625	625	<19.5	312.5	-
*P. aeruginosa* ATCC 27853 *G(-)*	625	156.3	625	625	156.3	625	312.5	156.3	>2500	>2500	-
*K. pneumaniae* ATCC 13883 *G(-)*	625	625	625	625	625	625	625	625	1250	625	-
*S. typhimurium* ATCC 14028 *G(-)*	625	625	625	1250	625	625	625	312.5	<19.5	156.3	-
*E. hirae* ATCC 9790 *G(+)*	1250	1250	1250	1250	1250	1250	625	625	19.5	156.3	-
*P. vulgaris* RSKK 96029 *G(-)*	625	1250	625	625	625	625	625	312.5	1250	1250	-
*C. albicans* ATCC 10231	625	625	625	625	625	625	156.3	156.3	-	-	312.5
*C. krusei* ATCC 6258	156.3	156.3	625	625	156.3	156.3	156.3	78.1	-	-	<19.5
*C. tropicalis* Y-12968	156.3	625	625	625	625	156.3	312.5	156.3	-	-	78.1

The Minimum Bactericidal and Fungicidal Concentrations (MBC and MFC) define the minimum antimicrobial agent concentration that reduces the viability of the initial microorganism numbers by 99.9%. According to the MBC and MFC results (Table 8), the compounds used in this study are not sufficiently effective on pathogenic bacteria as E. coli, B. subtilis, S. aureus, E. faecalis, K. pneumaniae, S.typhimurium, E. hirae and P. vulgaris. However, these compounds were effective on fungi such as C. albicans, C. krusei and C. tropicalis. The MFC of the compounds ranged from >1250 µM to 156.3 µM. The lowest concentration of compounds 5b and 8b for P. aeruginosa and C. krusei was determined as 156.3 µM. P. aeruginosa was more sussceptible to all compounds tested compared to positive controls (Ampicillin, Chloramphenicol and Ketoconazole).

Table 8 Minimum bactericidal and fungicidal concentrations (MBC and MFC) of the compounds and positive controls against test strains (in µM)

Test microorganisms	Compounds								Positive Controls
	5a	5b	6a	6b	7a	7b	8a	8b	Amp	C	Keto
*E. coli* ATCC 35218 *G(-)*	1250	>1250	1250	>1250	1250	1250	1250	1250	2500	625	-
*E. coli* ATCC 25922 *G(-)*	625	625	1250	1250	625	625	625	625	<19.5	78.1	-
*B. cereus* NRRL B-3711 *G(+)*	625	625	>1250	>1250	625	625	>1250	>1250	156.3	156.3	-
*B. subtilis* ATCC 6633 *G(+)*	>1250	>1250	>1250	>1250	>1250	>1250	>1250	>1250	<19.5	78.1	-
*S. aureus* ATCC 25923 *G(+)*	1250	>1250	>1250	>1250	>1250	>1250	1250	625	<19.5	156.3	-
*E. faecalis* ATCC 29212 *G(+)*	>1250	>1250	>1250	>1250	>1250	>1250	1250	1250	<19.5	312.5	-
*P. aeruginosa* ATCC 27853 *G(-)*	1250	156.3	1250	1250	312.5	1250	625	312.5	>2500	>2500	-
*K. pneumaniae* ATCC 13883 *G(-)*	1250	1250	1250	1250	1250	1250	1250	1250	1250	625	-
*S. typhimurium* ATCC 14028 *G(-)*	1250	1250	1250	>1250	1250	1250	625	625	<19.5	156.3	-
*E. hirae* ATCC 9790 *G(+)*	>1250	>1250	>1250	>1250	>1250	>1250	1250	1250	19.5	156.3	-
*P. vulgaris* RSKK 96029 *G(-)*	1250	1250	1250	1250	1250	1250	1250	625	1250	1250	-
*C. albicans* ATCC 10231	1250	1250	1250	1250	1250	1250	312.5	312.5	-	-	312.5
*C. krusei* ATCC 6258	312.5	312.5	1250	1250	312.5	312.5	312.5	156.3	-	-	<19.5
*C. tropicalis* Y-12968	312.5	1250	1250	1250	1250	312.5	625	312.5	-	-	78.1

Antioxidant activities of phosphazenes 5a-8b

The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical scavenging activity method is the most widely used method to determine the antioxidant activity of natural or synthetic substances [67]. Characterized by having one or more unpaired electrons, free radicals are reactive molecules produced during metabolic reactions in cells. Reactive radicals have the potential to induce cancer by altering the redox system, damaging cellular structures, activate pro-carcinogens, cause various chronic diseases and cancers [68]. Natural or synthetic substances that can inhibit oxidation are called antioxidants. These molecules transfer their electrons to radicals to scavenge free radicals and minimize the damage done by free radicals to cells [69]. When there are not enough antioxidant molecules in the body, the balance between pro-oxidant and anti-oxidant species is corrupted. As a result of this lack of cellular balance, oxidative stress occurs in the body [70,71]. Oxidative stress is seen as the cause of many health problems such as cancer, diabetes, neurodegeneration, cardiovascular diseases, rheumatoid arthritis, kidney disease, eye disease, radiation-induced lung injury [70,71]. Therefore, there is increasing interest in the protective roles of antioxidants in the body and in pathological processes mediated by oxidative stress. Calculation of the antioxidant activities of the synthesized molecules is an important indicator in determining the use of these molecules in medical applications [72,73].

To determine the antioxidant activity of phophazenes 5a-8b and butylated hydroxytoluene (BHT), the scavenging effect of the compounds on DPPH free radicals was investigated. The experiment was repeated three times. Absorbance values at 517 nm were measured for the control group and compounds at concentrations in the range of 2500-156.3 µM. Radical scavenging activities (%) of these compounds were listed in Table 9. Compound 5b has the highest antioxidant capacity at a radical scavenging activity value of 68.34% among the other compounds. It is understood that the cis/trans configurations, the five-membered or six-membered spiro rings, and the methyl and/or ethyl substituents in the spiro rings are very effective in the radical scavenging activities of the phosphazenes. For instance, cis-5b is more active than trans-5a. Whereas, cis-5b with two N-methyl groups in both spiro rings is much more active than cis-7b with an N-ethyl group. Generally, the activity decreases in compounds with six-membered spiro rings. However, the antioxidant activities of all compounds are lower than the synthetic antioxidant BHT used as a positive control.

Table 9 DPPH radical scavenging activities of the compounds at

a concentration of 2500 µM

Compounds	Radical Scavenging Activity %
5a	55.09 ± 2.36
5b	68.34 ± 1.56
6a	1.46 ± 0.65
6b	18.61 ± 5.80
7a	26.22 ± 7.03
7b	29.40 ± 3.47
8a	2.18 ± 0.59
8b	24.18 ± 4.25
BHT	83.31 ± 2.05

Interactions of pBR322 DNA with the phosphazene derivatives

The interaction of pBR322 plasmid DNA with phosphazene derivatives was investigated by agarose gel electrophoresis. While there are two bands in the untreated plasmid DNA, form I and form II, the form III band is formed by the effect of DNA damage. The appearance of linear DNA band formation on the agarose gel is evidence that phosphazene derivatives cause DNA damage (double strand cleavage). Fig. 9 gives the electrophoretograms applying to the interaction of pBR322 plasmid DNA with decreasing concentrations of phosphazenes 5a–8b at concentrations ranging from 2500 µM to 156.2 µM. Phosphazenes 5a, 5b, 7a and 7b do not have any DNA bands in the presence of 2500 µM. This is because the compounds cause massive DNA damage at a concentration of 2500 µM. For compounds 6a, 6b and 7a at a concentration of 1250 µM, phosphazenes showed the effect of partially cleaving double-stranded DNA, resulting in Form III (linear form) (Fig. 9 ). Form I, which converted from super-stranded DNA to linear DNA, is believed to result from partial cleavage of the double-strand break by binding of the compound.

Restriction endonuclease reaction with BamHI and HindIII enzyme

BamHI and HindIII are restriction endonuclease enzymes that hydrolyze phosphodiester bonds in DNA. The recognition sites are the sequences 5'-G/GATCC-3', 5'-A/AGCTT-3' and are cut from 5'-guanine for BamHI and 5' adenine for the enzyme HindIII. The pBR322 plasmid DNA has a single restriction site that converts from supercoiled form I and circular form II to linear form III DNA for the two enzymes. Linear form III was generated when plasmid DNA was restricted by restriction endonucleases without compounds. Fig. 10 shows the electrophoretograms for the incubated mixtures of pBR322 plasmid DNA and the compounds (5a-8b), followed by BamHI and HindIII digestion As a result, BamHI enzyme partly restricted plasmid DNA interacts with compounds 5a, 6a, 6b, 7b and 8a, whereas phosphazenes 5a-8b partially inhibited restricition of plasmid DNA interacted with compounds (Fig. 10). In case of compound 7a, HindIII restriction enzyme was not inhibited by the compound.

The unsymmetrical inorganic-organic hybrid multi-heterocyclic cis/trans cyclotriphosphazenes containing different pendant arms were synthesized. The main purposes in obtaining these compounds were to demonstrate their photophysical and stereogenic properties, anticancer, antioxidant and antimicrobial activities and their interactions with DNA. The chiralities of these phosphazenes were determined by X-ray technique, CD and CSA-added ³¹P NMR spectra. CD spectra of phosphazenes, 6a and 6b, were recorded. In addition to the CD spectra of 5a and 5b, the CSA-added ³¹P NMR spectra were also examined. According to the X-ray results, the absolute configuration of one enantiomer of cis-5b was found to be SR'. All these data prove that unsymmetrical cis/trans-dispirocyclotriphosphazenes exist as racemic mixtures in solution and solid states. Photophysical properties of all dispirophosphazenes were interpreted using UV/vis and both steady-state and time-resolved fluorescence measurements. The dispirophosphazenes that were chemically stable under light excitation exhibited strong three absorption band regions at approximately 260-270 nm, 290-300 nm and 330-350 nm. Moreover, all derivatives displayed maximum double emission at 360 and 370 nm when excited at 290 nm. Although the absorption and emission profiles of the isomers are almost the same, the corresponding quantum yield and fluorescence lifetime values show slight decreases in the cis isomers relative to the trans isomers. This tendency can be attributed to carbazole-phenyl interactions in the crystal structures of the cis isomers. In cell vialibity assay, it was found that the compounds 5a, 5b, 7b and 8a did not show as much cytotoxic effect as the positive control. On the other hand, the compounds have partial DNA cleaving activity and DNA binding activity to A/A and G/G nucleotides. Of these phosphazenes, compounds 5b, 7a, 8a and 8b exhibited a better inhibitory effect on the pathogenic bacteria (especially P. aeruginosa). Phosphazenes 8a and 8b are highly susceptible to C. albicans ATCC 10231 and C. krusei ATCC 6258. In addition, compounds 5a and 5b were found to have the highest antioxidant capacity at radical scavenging activity values of 55.09% and 68.34%, respectively. As a result, the radical scavenging activities of the cis derivatives are found to be considerably greater than the trans phosphazenes.

Author contributions Reşit Cemaloğlu: Formal analysis, Investigation, Writing–original draft, Visualization. Nuran Asmafiliz: Conceptualization, Methodology, Formal analysis, Resources, Writing–original draft, Writing –review & editing, Project administration. Bünyemin Çoşut: Investigation, Formal analysis, Writing –original draft, Writing –review & editing. Zeynel Kılıç: Conceptualization, Methodology, Writing–original draft, Writing–review & editing, Project administration. Büşra Nur Sabah: Investigation, Writing–original draft. Leyla Açık: Investigation, Writing–original draft, Writing–review & editing. Hatice Mergen: Investigation, Writing–original draft. Tuncer Hökelek: Investigation, Formal analysis, Writing–original draft, Writing –review & editing.

Acknowledgements Z. K. thanks the Turkish Academy of Sciences (TÜBA) for partial support of this work.

Funding T. H. is grateful to Hacettepe University Scientific Research Project Unit (Grant No. 013 D04 602 004).

Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval Not applicable.

Availability of data and materials Not applicable.

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

SupplementaryMaterial.docx
Electronic supplementary Listings of ¹³C (decoupled) and ¹H NMR spectral data of the phosphazenes (Tables S1 and S2); antimicrobial activities of 5a-8b and positive controls against different bacterial and fungal strains¸(Table S3); optical absorption and fluorescence emission spectra of 5a and 5b in various polar and nonpolar solvents at room temperature (Fig. S1); comparison of optical absorption and fluorescence emission spectra of the trans and cis phosphazenes (Fig. S2); the ring conformation and the packing diagram of 5b (Fig. S3); the shape of the phosphazene ring in 5b with torsional angles (Fig. S4); a partial packing diagram of cis-5b (Fig. S5); determination of antimicrobial activities, MIC and MBC/MFC values, antioxidant activities, MTT cell viability, DNA-compound interactions and BamHI and HindIII digestion Crystallographic data for the structures reported herein have been deposited with the Cambridge Crystallographic Data Centre as Supporting Information, CCDC No. 2210972 (for 5b). 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).
scheme1.png
Scheme 1 The replacement reactions of tetrachlorobenzyl-(N/N)spirocyclotriphosphazenes with the carbazolyldiamines

Phosphorus-nitrogen compounds: Part 69. Unsymmetrical Dispiro(N/N)cyclotriphosphazenes Containing Different Pendant Arms: Syntheses, Characterization, Stereogenism, Photophysical and Bioactivity Studies

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Abstract

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Introduction

Experimental

Results And Discussion

Conclusion

Declarations

References

Scheme

Additional Declarations

Supplementary Files

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