Phosphorus-nitrogen compounds: Part 69—Unsymmetrical dispiro(N/N)cyclotriphosphazenes containing different pendant arms: syntheses, characterization, stereogenism, photophysical and bioactivity studies

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/cisN/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 the 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.


Introduction
Phosphazenes, especially cyclo, polymeric, dendrimeric phosphazene derivatives, continue to be an increasingly important area of interest for researchers [1][2][3][4][5][6][7]. Hexachlorocyclotriphosphazene, N 3 P 3 Cl 6 (HCCP, trimer), remains the focus of research on 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 substituents. For this, generally, mono and bidentate ligands containing OH/NH/SH functional group/groups have been reported in the literature [8][9][10][11][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][14][15][16][17][18][19][20][21][22][23][24][25]. 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, the 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 31 P NMR spectra [26][27][28].
Cyclophosphazene derivatives also have an important place in various applications such as catalysis [29], chemical [30] and biological imaging [31], and biomedical materials [32], multisite coordination ligands [33] and fluorescent dyes [34]. 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 [35]. 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) [36]. The anti-carcinogenic properties of anthraquinonedecorated cyclotriphosphazenes [37,38] and spermine-substituted cyclotriphosphazenes [39] were also investigated in different cell lines. Meanwhile, BODIPY-containing cyclotriphosphazene dendrimers were used for the first time under high-power laser irradiation in vivo phototherapy [40].
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 [41] and anticancer activity [42]. Some carbazole derivatives have been reported to have antioxidant activity [43].
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 1 3 Phosphorus-nitrogen compounds: Part 69-Unsymmetrical… spectroscopic, crystallographic and thermal properties were discussed in detail [44]. In addition, carbazole decorated cyclotriphosphazenes with intense luminescence properties have also been declared [45][46][47]. A literature survey revealed that there are quite a number of manuscripts on symmetrically substituted dispirocyclotriphosphazenes [15,17,23,24]. However, there are a few studies on unsymmetrical dispirocyclotriphosphazenes without pendant arms in the literature [48][49][50][51]. Moreover, to the best of our knowledge, there is only one report dealing with dispirocyclotriphosphazenes containing different spiro-rings with different unsymmetrical pendant arms [52].

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 B 254 sheets using toluene-tetrahydrofuran mixture as 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 1 H, 13 C and 31 P 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 [53] 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 H 2 SO 4 ), (λexc = 310 nm).
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 H 2 SO 4 ) [55] 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 α radiation (λ = 0.71073 Å) at T = 120 (2) K. No absorption correction was applied. The structure was solved by direct methods [56] and refined by full-matrix least-squares against F 2 using all data [57]. 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 [58]. 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.

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 (Et 3 N; 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. The first product was the monospirophosphazene (1

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 (Et 3 N; 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. The first product was monospirophosphazene (1)

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 (Et 3 N; 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 the mobile phase. The first product was the monospirophosphazene (2). Yield: 0.74 g, 1.63 mmol (28%). The transphosphazene (7a), the second white powder product, was obtained. Yield: 1.

Syntheses of 8a and 8b
The exact same process was 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 (Et 3 N; 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. The first product was the monospirophosphazene (2). Yield: 0.88 g, 1.94 mmol (29%). The trans-phosphazene (8a), the second product, was obtained as a white powder
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) (Scheme 1).
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.

NMR and IR spectroscopies
The structures of all novel compounds were confirmed by IR, MS, 31 P, 1 H and 13 C 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 Phosphorus-nitrogen compounds: Part 69-Unsymmetrical… dispirophosphazenes, CSA-added 31 P 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 31 P NMR spectra of 5a, 5b and 8b were  (1) and c pure trans (6a) and cis (6b) isomers evaluated for the mole ratio of compound to CSA at 15:1 (Fig. 3). The effects of CSA on the 31 P 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 31 P NMR spectra of compounds 5a and 8b. All 31 P 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 in 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 [59]. 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 [60]. Also, X-ray crystallography findings (see crystal structure solution) confirmed that both enantiomers of cis-5b isomer were present in the crystal lattice. As a result, it can be stated that CSA-added 31 P NMR, CD and X-ray crystallography data are compatible with each other. The 31 P ( 1 H) NMR data of the dispirophosphazenes are listed in Table 2. When 31 P NMR spectroscopic data are evaluated, it can be stated that cis/trans dispirophosphazenes have A 2 X (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 ( 2 J PP ) of the five-membered carbazolyl-spiro ring compounds are larger than those of the six-membered ones. Average 2 J 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 13 C and 1 H NMR spectra. Chemical shifts, coupling constants and multiplicities are presented in Tables S1 and S2). According to the 13 C 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 PhCH 2 and CzCH 2 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 and 140.21 ppm. Another important signals are the peaks belonging to the N-CH 2 -CH 3 carbons bonded to the nitrogen of the carbazole rings appearing between 13.80 and 13.84 ppm. The N-CH 2 -CH 3 carbons of the carbazole rings resonate in the range of 37.52-37.59 ppm. On the other hand, the average 3 J PC1′ coupling constant (9.8 Hz) of all compounds containing sixmembered spiro-rings is greater than that of the five-membered ones (7.2 Hz). In contrast, the average 3 J PC between CH 2 -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 3 J PC1 in all compounds was calculated as 6.9 Hz. Moreover, the coupling constants of 2 J PC between the phosphorus atoms and the N-CH 2 spiro-carbons of unsymmetrical dispirophosphazenes containing the five-membered spiro-rings are considerably larger than those of the six-membered ones, as observed previously [15,26,61].
The expected proton signals were determined from the 1 H spectra of the dispirophosphazenes (Table S2). It is quite important to point out a few characteristic peaks. Some of these are benzylic (PhCH 2 ) and carbazolyl (CzCH 2 ) protons, and their chemical shifts were found to be in the ranges of 3.98-4.33 ppm for PhCH 2 and 3.96-4.22 ppm for CzCH 2 . These protons can differentiate from each other according to their chemical environments, and they are called diastereotopic. Accordingly, the PhCH 2 and CzCH 2 protons of all dispirophosphazenes are expected to be both diastereotopic. These diastereotopic protons emerge as doublets of doublets due to the geminal ( 2 J HH ) and vicinal ( 3 J PH ) couplings (for the AMX spin system). Other characteristic peaks belong to methyl (NCH 3 ) protons. The NCH 3 protons of all compounds were determined to be between 2.54 and 2.70 ppm, and the average coupling constant with the P-atom ( 3 J PH ) was calculated as 12.0 Hz. In addition, aromatic hydrogens occur in the range of 7.14-8.15 ppm.
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 −1 and 539-577 cm −1 , respectively. On the other hand, the characteristic aromatic νCH vibrations bands were observed between 3054 and 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 and 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 [62] due to the optical inertness of the cyclophosphazene core [45,46,63]. 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 H 2 SO 4 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 Fig 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 [64]. 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.

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 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 [66] 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 N 3 P 3 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)  In crystal structure, intermolecular C-H···Cl hydrogen bonds (Table 6) link the molecules into infinite chains along the a-axis direction (Fig. S5). A weak C-H···π interaction ( Table 6) is also observed.

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 the MTT assay, none of the compounds were as effective as the cis-Diammineplatinum(II) dichloride. Compound 5b showed almost 50% inhibition effect at 250 µM concentration on Vero cells. However, the same compounds did not show an inhibition effect on cancer cells. As a result, the compounds

3
Phosphorus-nitrogen compounds: Part 69-Unsymmetrical… tested were more effective against epithelial cells than breast cancer cells. Other values of concentration did not form a regular inhibition curve on both cells.

Antimicrobial activity
In this study, the 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 the bacterial and fungal microorganisms 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 diameters of the inhibition zones were defined in Table S3. Zones of inhibition ranged from 10 to 20 mm. 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 prevents the growth of microorganisms. MIC determination was performed by serial dilution method in a 96-well microplate. MIC values were found between 156.3 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).  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 such 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 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 susceptible to all compounds tested compared to positive controls (Ampicillin, Chloramphenicol and Ketoconazole).

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 [71]. 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, activating pro-carcinogens, cause various chronic diseases and cancers [72]. 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 [73]. 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 [74,75]. Oxidative stress is seen as the cause of many health problems such as cancer, diabetes, neurodegeneration, cardiovascular diseases, rheumatoid arthritis, kidney disease, eye disease and radiation-induced lung injury [74,75]. 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 [76,77].
To determine the antioxidant activity of phosphazenes 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.

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). Figure 9 gives the electrophoretograms applying to the interaction of pBR322 plasmid DNA with decreasing concentrations of phosphazenes 5a-8b at concentrations ranging from 2500 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′ and 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 a 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. Figure 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 restriction of plasmid DNA interacted with compounds ( Fig. 10). In case of compound 7a, HindIII restriction enzyme was not inhibited by the compound.

Conclusion
The unsymmetrical inorganic-organic hybrid multi-heterocyclic cis/trans cyclotriphosphazenes containing different pendant arms were synthesized. The main purposes of 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 31 P NMR spectra. CD spectra of phosphazenes, 6a and 6b, were recorded. In addition to the CD spectra of 5a and 5b, the CSA-added 31 P 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 the cell viability 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 10,231 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.

Fig. 10
Electrophoretograms applying to the incubated mixtures of pBR322 plasmid DNA and compound followed by digestion with BamHI and HindIII. Lane P1 applied to the untreated pBR322 plasmid DNA and undigested with enzyme, lane P/B and P/H applied to untreated but digestion with BamHI and HindIII, respectively