Synthesis and spectroscopic properties of (N/O) mono- and dispirocyclotriphosphazene derivatives with benzyl pendant arms: study of biological activity

The Cl replacement reactions of hexachlorocyclotriphosphazene (trimer; N 3 P 3 Cl 6 ) with sodium (N-benzyl)- aminopropanoxides (1 and 2) produced monospiro- (3 and 4), cis-, and trans-dispirocyclotriphosphazenes (13–16). The monospiro tetrakis-aminocyclotriphosphazenes (5–12) were obtained by the Cl substitutions of 3 and 4 with different secondary amines. The cis- (13 and 14) and trans-dispirophosphazenes (15 and 16) possessed 2 chiral P centers, and they were able to present meso and racemic forms, respectively. Moreover, the structures of compounds 5 and 14 were designated using X-ray data. The absolute configuration of compound 14 was found as SR in the solid state. Analytical and spectroscopic data of the phosphazenes were consistent with their suggested structures. Antimicrobial activities of the benzyl-pendant-armed cyclotriphosphazenes were scrutinized against G(+) and G(−) bacteria and yeast strains. The bacterium most affected by the synthesized compounds was Pseudomonas aeruginosa . Minimum inhibitory concentrations and minimal bacterial concentrations were in the range of 125–500 μM. Interactions between the phosphazenes (3–12 and 15) and plasmid DNA were studied with agarose gel electrophoresis. The phosphazene- DNA interaction studies of the cyclotriphosphazenes revealed that phosphazenes 3, 4, and 15 had a substantial effect on supercoiled DNA by cleavage of the double helix.


Introduction
Phosphazenes refer to phosphorus-nitrogen compounds that occur by the sequential bonding of atoms to each other [1,2]. Hexachlorocyclotriphosphazene (N 3 P 3 Cl 6 , trimer) is versatile and has the ability to react easily with different mono-, di-, and multifunctional reagents [3][4][5]. When trimer is reacted with monofunctional agents, at least 6 different cyclotriphosphazene derivatives can be formed [6]. In addition, the Cl replacement reactions of trimer with one equimolar difunctional reagent may predominantly yield monospiro products when compared to other expected ansa-and bino-isomers [7,8]. Moreover, when two equimolar difunctional agents are used, dispirocyclotriphosphazenes with cis-and trans-geometrical and meso/racemic optical isomers can occur [9][10][11]. It is difficult to separate these isomers from each other; hence, very few optical and geometrical isomers of dispiro derivatives have been separated in the literature [9][10][11][12][13]. Geometric isomers of dispirocyclotriphosphazenes can be separated by column chromatography, while optical isomers may be determined using 31 P { 1 H}, circular dichroism, X-ray data, and/or high-pressure liquid chromatography [14][15][16][17]. Among these methods, X-ray data are important to define the absolute configurations of optically active phosphazenes.
In the literature, some cis-and trans-dispirophosphazene products have been presented, but dispirocyclotriphosphazene derivatives with pendant arms are less common [25,26]. The isomer distributions, chiralities, and structural and spectral features of dispiro products with pendant arms were determined in these studies. In order to compare with the reports in the literature, (N/O) mono-and dispirocyclotriphosphazene derivatives with benzyl pendant arms were prepared in this study. Furthermore, the mono-and dispirocyclotriphosphazene derivatives reported here were also prepared for the determinations of their chemical and biological aspects.
It has already been mentioned that phosphazenes 3-6 were presented in the literature [27]. According to the literature, phosphazenes 3 and 4 were synthesized in THF, while compounds 5 and 6 were synthesized in toluene. However, in the present study, the same compounds (3)(4)(5)(6) were synthesized in THF. The reaction yields of 3-6 previously reported in the literature were 44%, 55%, 34%, and 45%, respectively [27]. As understood, the reaction yields in this study were higher than those given in the literature. For comparison, the yields of compounds 3-6 can be seen in Section 3.3. Recently, the syntheses of some cis-and transspirocyclotriphosphazenes with 4-chloro/4-nitro/4-fluoro-benzyl pendant arms were reported in the literature Scheme. Replacement reactions of N 3 P 3 Cl 6 with the monodentate and bidentate amines. [28,29]. Although the trans-geometric isomers of these compounds were purely obtained, the cis-geometric isomers were not isolated from the reaction mixture using crystallization, column chromatography, or preparative TLC. Furthermore, the cis-and trans-isomers of dispirocyclotriphosphazenes with benzyl pendant arms were isolated by column chromatography in this study.
The cis-(13 and 14) and trans-phosphazenes (15 and 16) possessed 2 stereogenic P atoms, and they were probably present in the meso (RS/SR) and racemic (RR/SS) forms, respectively. As expected, cis-isomer 13 was present as a meso (SR) configuration.
The IR, APIES-MS, NMR, and microanalytical results were consistent with the suggested formulae of the mono-and dispirophosphazene derivatives. Protonated molecular ion peaks ([MH] + ) appeared for compounds 7-12, whereas molecular ion peaks ([MH] + emerged for compounds 13-16 in the mass spectra.

NMR and IR spectroscopies
The data obtained from the 31 P { 1 H } spectra of the mono-(3-12) and cis-and trans-dispirophosphazenes (13-16) are given in Table 1. The partly and fully substituted monospiro- (3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and dispirocyclotriphosphazenes (13)(14)(15)(16) had AX 2 spin systems. A triplet for 1 P atom and a doublet for 2 P atoms were determined in the spectra of the phosphazenes with AX 2 spin systems. Moreover, the cis-and trans-dispiro products (13)(14)(15)(16) had a triplet for 1 P atom and a doublet for 2 P (spiro) atoms. The chemical shifts and 2 J P P values of benzylamino monospirophosphazenes containing 5-membered spiro precursors were larger than those of the other phosphazenes bearing 6-membered spiro-rings. Moreover, the average 2 J P P constants were 48.4 and 46.2 Hz for the products, including the 5-and 6-membered spiro rings. These findings were inconsistent with the literature data [28,29]. The shifts, multiplicities, and coupling constants of the phosphazenes in the 13 C and 1 H spectra were evaluated (Tables S1 and S2). The expected carbon peaks were interpreted using the 13 C spectra. The carbon peaks of the phenyl rings, C1-C4, were assigned between 125.27 and 138.97 ppm (Table S1). The peaks of the O C H 2 carbons of the spiro rings were between 68.23 and 63.00 ppm. In addition, the signals of the benzylic carbons (Ph C H 2 ) of the products, including the 6-membered spiro rings (4, 6, 8, 10, 12, 14, and 16), were shifted further downfield than those of the 5-membered rings (3, 5, 7, 9, 11, 13, and 15), as observed previously [28,29]. The same phosphorus atoms, to which 2 heterocyclic groups had bonded, showed 2 groups of N C H 2 , NCH 2 C H 2 , NCH 2 CH 2 C H 2 , and O C O peaks, with small separations in the 13 C spectra.
Additionally, coupling constants between the C1 and P atoms ( 3 J P C ) emerged in all of the phosphazenes, and the average 3 J P C value was 8.6 Hz. Coupling constants ( 2 J P C ) of the NCH 2 spiro-groups of the products, including the 5-membered spiro rings (3, 5, 7, 9, 11, 13, and 15), were considerably large. The average 2 J P C value was 12.3 Hz.
The expected proton signals of the phosphazenes were determined from the 1 H spectra (Table S2) Asymmetric ν PN vibrations of the trimeric cyclophosphazenes were assigned in the range of 1200-1172 cm −1 in the IR spectra [30]. The aromatic C-H bands were between 3040 cm −1 and 3100 cm −1 . Moreover, the asymmetric ν PCl 2 vibrations of the partly substituted tetra (3 and 4) and dichloro phosphazenes (13)(14)(15)(16) were determined in the range of 562-576 cm −1 . These peaks were not observed in the IR spectra of the tetrakis-substituted monospirophosphazene derivatives (5-12).

X-ray structures of compounds 5 and 14
The crystal structures of compounds 5 and 14 were assigned crystallographically. The crystallographic data of the new products are tabulated in Table 2, and ORTEP diagrams with suitable atom numbering are illustrated in Figures 1 and 2 indicating total puckering amplitudes Q T of 0.120(2) Å (for 5) and 0.145(2) Å (for 14) [31]. As expected, the 5-membered spiro-ring of 5 was in envelope conformation ( Figure S3). Both of the spiro-rings of 14 were in chair conformations ( Figure S4). Compound 14 crystallized in the P bca space group. The absolute configurations of the P1 and P2 atoms of 14 were determined as S and R, respectively. The shapes of the phosphazene skeletons in 5 and 14 with torsion angles are given in Figure S5, displaying the pseudo mirror plane running from the P1-N1 atoms of the cyclotriphosphazene ring. Table 3  . The exocyclic PN bonds were longer than the endocyclic PN bonds. Furthermore, the endocyclic PN bonds were shorter than the PN single bond reported in the literature [32], indicating that the phosphazene ring had nearly double the bond character.
Furthermore, regular variations of the bonds in 5 and 14 were also observed, with the distances from P3: P3-N3 The endocyclic NPN ( α) angles of tetrakis-pyrrolidino monospiro (5) and cis-dispirophosphazenes (14) [116.27 (12) (Table 3). Additionally, it was concluded that the were considerably narrow when compared with N 3 P 3 Cl 6 . All of the variations in the bond angles and lengths may be attributed to the steric interactions between the bulky groups and the negative hyperconjugation [34].
When compared with the literature findings, it was clear that the variations in the bond angles and length variations of 5 and 14 were in good agreement with the previously reported data [35].

Antimicrobial activity of the compounds
Experiments were performed to determine their MICs and MBCs against strains of S. typhimurium, P. vulgaris, S. aureus, P. aeruginosa, E. hirae, B. cereus, C. tropicalis, and C. krusei (Tables 4 and 5). The MICs and MBCs were in the range of 125-500 µ M. Table 4. MIC values of compounds 3, 4, and 6-12 on different bacterial and fungal species (in µ M) (Amp: ampicillin, C: chloramphenicol (antibacterial), and Keto: ketoconazole (antifungal) used as a control; "-": the compounds did not cause any growth inhibition; NS: not studied).  Consequently, the antibacterial and antifungal activity results observed in this study were comparable with the literature data. In the literature, pyrrolidino-and DASD-substituted cyclotriphosphazenes containing ferrocenyl/arylspirocyclic pendant arms also had activity against some G( +) and G(-) bacteria and fungi [8,25,26,28,36]. In the present study, the chloro-, pyrrolidino-, morpholino-, piperidino-, and DASD-substituted cyclotriphosphazenes had different antibacterial and fungal activities, as listed in Tables 4 and 5. The antibacterial and antifungal activities of the mono-and dispirocyclotriphosphazene derivatives with benzyl pendant arms could have been due to the formation of hydrogen bonding, dipole-dipole interactions between the DNA of the bacteria and fungi, or the phosphazene derivatives used in this study. On the other hand, the substituents (chloro, pyrrolidino, DASD, piperidino, and morpholino), the number of members, and the conformations of the spiro rings may also have played an important role in the activity.

Interactions of DNA with compounds 3-12 and 15
In the present study, 2 DNA bands, namely Forms I and II, appeared for both untreated and treated pBR322 with decreasing concentrations of compounds 6-15 (except for compounds 3, 4, and 15) (Figure 3). In compound 15, a faint, almost coalesced band was observed at 2500 µ M (Lane 1), and when the concentrations of the products decreased, the mobility of the bands of Forms I and II increased. The change was less significant with compound 3 at three concentrations, but Form I disappeared at the highest concentration. With compound 4, Forms I and II disappeared at three high concentrations. This was the most damaging compound for the DNA when compared to compound 15. With 15, the separations between the bands were the smallest at 2500-1250 µ M (Lanes 1 and 2), below which the separations were larger (Lanes 3 and 4). The presence of a coalesced band suggested a change in the conformation of the DNA of Form I, from the supercoiled Form I to the negative form and from the negative 1 to the positive form [37]. In all of the other compounds, two bands corresponding to Forms I and II were observed in all of the lanes, and a single faint band could be seen in

Physical measurements
Microanalytical data were obtained using a LECO CHNS-932 analyzer (St. Joseph, MI, USA). The NMR spectra were supplied on a Bruker DPX FT-NMR (500 MHz) spectrometer (Billerica, MA, USA) (SiMe 4 as internal and 85% H 3 PO 4 as external standards). The spectrometer was equipped with a 5-mm PABBO BB inverse-gradient probe. Standard Bruker pulse programs [38] were used. The IR spectra were obtained on a Mattson 1000 IR spectrometer in KBr disks and were given as cm −1 . APIES mass analyses were made on a Waters 2695 Alliance Micromass ZQ spectrometer (Milford, MA, USA).

Preparations of the compounds
Compounds 1-6 were synthesized according to the literature [27], whereas compounds 1 and 2 were synthesized in ethanol and phosphazenes 3-6 were synthesized in THF. Information on synthesis of the fully substituted monospirocyclotriphosphazenes (7-12) is given in Table  6.

16)
The mixtures of sodium (N-benzyl)aminopropanoxide and Et 3 N in THF were added to a stirred solution of N 3 P 3 Cl 6 in THF (50 mL), and stirred and refluxed for 30 h. After the precipitated triethylaminehydrochloride was filtered off, the solvent was evaporated. The three products were purified by column chromatography with toluene and THF (15:1). The first product was the tetrachloro monospirophosphazene derivative. The second product was the cis-dispirocyclophosphazene. The last product was the trans-dispirocyclic compound (15). The compounds were crystallized from petroleum ether (60-80 • C) at room temperature. Information on synthesis of the dispirocyclotriphosphazenes (13-16) is given in Table 7.
The IR, APIES-MS, and microanalytical data of the products (7-16) are tabulated in Table 8.

X-ray crystallography
Single crystals of compounds 5 and 14 were grown in acetonitrile at 296 K, and their crystallographic data were collected on a Bruker APEXII CCD area-detector diffractometer by Mo K α . The multiscan absorption correction [39] applied data were processed by SHELX program packages [40,41] for solving and refining the structures, and the ORTEP-3 program [42] was used for the drawings. H atom positions were calculated geometrically at distances of 0.93 and 0.97 Å for methine and methylene, respectively, and refined using a riding model by applying the constraint of 1.2 U eq (carrier atom) for the U iso (H) values.
Determinations of the antimicrobial activities and MIC/MBC/MFC values of the phosphazenes as well as DNA interactions with the compounds were performed according to the method in the literature [43].

Conclusion
The present study focused on the syntheses of monospiro (3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and dispirocyclotriphosphazenes (13-16) with benzyl pendant arms. One of the most important aspects of this study was to separate the cis (13 and 14) and trans (15 and 16) isomers in pure form. The structures of 5 and 14 were determined using X-ray data. The spectral data ( 1 H, 13 C, and 31 P) showed that the structures of all of the compounds were in good agreement with the proposed formulae. The structures of 5 and 14 were symmetric in the solid state with respect to X-ray crystallography. The data showed that the absolute configuration of 14 was SR. In addition, the monospiro (3 and 4), cis (meso) (13 and 14), and trans (racemic) (15 and 16) cyclotriphosphazenes possessed prochiral, diastereotopic, and homotopic atoms. Additionally, most of the compounds were found to inhibit most of the antibacterial activities of bacteria, except for P. aeruginosa, S. aureus, and B. cereus. The antimicrobial activities and MIC values revealed that the most effective compounds were 6-8 and 11, and the bacterium most affected by the compounds was P. aeruginosa. The MBC and MFC values of compounds 3, 4, and 6-12 ranged from 250 to 1000 µ M. DNA interaction studies of the cyclotriphosphazenes revealed that compounds 3, 4, and 15 had a strong effect on supercoiled DNA by cleavage of the double helix.
In conclusion, the partly tetrachloro monospiro (3 and 4) and dichloro dispirocyclotriphosphazenes (13-16) could be useful starting compounds for the synthesis of the new organocyclotriphosphazenes as a chiral

Supplementary data
Listings of the 13 C (decoupled) and 1 H data of the phosphazenes (Tables S1 and S2)