Bis(2-furanylmethyl)monospiro(N/N)cyclotriphosphazenes: synthesis, structural characterization, antiproliferative, and antimigratory activity studies

Abstract In this study, the condensation reaction of hexachlorocyclotriphosphazene, N3P3Cl6 (HCCP) with N 1,N3 -bis(furan-2-ylmethyl)-1,3-diamino-propane (1) yielded tetrachlorobis(2-furanyl-methyl)monospiro(N/N)cyclotriphosphazene (2). Fully substituted tetraaminobis(2-furanylmethyl)-spiro(N/N)cyclotriphosphazenes (2a and 2b) were obtained by Cl replacement reactions with excess pyrrolidine and piperidine, respectively, using dry THF under reflux of 2. The structures of the phosphazenes were confirmed by data obtained by elemental analysis, FT-IR, ESI-MS, 1H, 13C, and 31P NMR. Moreover, the molecular structure of 2b was elucidated by X-ray crystallography. The antiproliferative effects of phosphazenes (2, 2a, and 2b) on prostate (PC-3) and colon cancer (HT-29) cell lines were determined by cell viability analysis using MTT assay. Antimigratory effects were also investigated by scratch analysis. In addition, the effects of compounds against genes involved in apoptosis and cell migration were evaluated by gene expression analysis. As a result, it was found that compound 2a was more effective than compound 2b in both prostate and colon cancer cell lines. GRAPHICAL ABSTRACT


Introduction
6][7][8][9] Depending on the nucleophiles and reaction conditions, the Cl atoms in the N 3 P 3 Cl 6 ring can be easily replaced with the same or different substituents.[12][13] On the other hand, it is well known that these substituents bonded to ring phosphorus significantly affect the physical, chemical, and bioactivity of phosphazene derivatives.Inorganic-organic hybrid heterocyclic molecules were successfully designed using the trimeric phosphazene ring.Therefore, cyclotriphosphazenes are used to design different molecules for target applications, such as anticancer and antimicrobial agents, [14][15][16] fluorescent chemosensors, [17,18] metal-mediated biomimetic sensing/recognition and catalysis compounds, [19] thin films, [20] fire safety/flame retardants, [21][22][23] lithium-ion batteries, [24] and organic light-emitting diodes. [25,26][29][30][31][32] The lack of toxic effects of products such as ammonia and phosphate formed by the breakdown of the phosphazene skeleton also causes an increase in the research of these structures, and it is thought that this may provide important advantages for their use in the field of chemotherapy.
Nowadays, many researchers are working on the design and synthesis of new effective chemical molecules for the prevention or treatment of cancer, which is a leading cause of death worldwide. [13,30,33]With a wide variety of cancer types (colon, larynx, cervix, prostate, lung, etc.), however, there is still a great need to discover and create novel chemical molecules with therapeutic potential.There are very few studies on the antiproliferative and antimigratory activities of phosphazenes in the literature. [15,27,28]Recently, the potential of phosphazene derivatives as therapeutics for a wide range of diseases and conditions was the subject of intensive study and proposed as promising anticancer agents. [13,15,34,35]In addition, it has been reported that phosphazenes inhibit the growth of various cancer cells by triggering their programmed death mechanism, apoptosis. [36]It is thought that determining the antiproliferative and antimigratory effects of these compounds can greatly help in the treatment of cancer. [13,15,37,38]n this study, the antiproliferative and antimigratory properties of tetrachlorobis(2-furanylmethyl)spiro(N/N) cyclotriphosphazene (2) and their tetrapyrrolidino (2a) and tetrapiperidino (2b) derivatives in cancer progression were investigated (Scheme 1).Additionally, the article sheds light on the properties of these compounds from a spectroscopic and crystallographic perspective.A future development of such specific derivatives of cyclotriphosphazene could also be aided by the results of this study.
Chemical shifts, multiplicities, and coupling constants were determined from the 1 H and 13 C spectra of bis(2-furanylmethyl)spirocyclotriphosphazenes (2, 2a, and 2b) and clearly interpreted.It is the carbon signals of pyrrolidino and piperidino substituents that provide the most reliable evidence that all Cl atoms in 2 have been substituted.The N-CH 2 carbons of the pyrrolidino (2a) and piperidino (2b) phosphazenes appeared at 46.36 and 45.64 ppm, respectively.The average chemical shift of the C5 carbons of 2, 2a, and 2b was calculated to be 46.51ppm.However, d-shifts of the ipso-C4 furanyl carbons were observed as doublets.The coupling constants ( 3 J PC ) of the 2, 2a, and 2b ipso-C4 carbons were calculated as 7.4, 13.3, and 13.3 Hz, respectively.The coupling constant ( 2 J PC ) of the NCH 2 (spiro) carbon in the six-membered spiro ring was found to be 4.8 Hz (for 2) and an average of 2.5 Hz was found to be for 2a and 2b.Also, in the 13 C spectra of pyrrolidino (2a) and piperidino (2b) phosphazenes, the N-CH 2 -CH 2 carbons were clearly seen in triplets at about 26.5 ppm due to the second-order effects.In the 13 C-spectra of 2a and 2b visualized in Figures S8 and  S9, the 3 J PNCC values are calculated by the external transitions of the triple peaks [39] and are given in the spectra.][42] In a review, the source of the second-order effects is discussed by physical properties and mathematical equations. [43]n the other hand, the d-shifts of the furanyl protons (H1-H3) appear in the range of 7.38-6.20 ppm.Both H5 protons of cyclotriphosphazenes appear in the spectra as doublets, indicating that these protons are equivalent to each other.While the coupling constant ( 3 J PH ) was 11.3 Hz (for 2), this value was calculated as an average of 6.4 Hz for 2a and 2b.
31 P data of tetrachloro and tetraaminobis(2-furanylmethyl)spirocyclotriphosphazenes were listed (Table 1).The 31 P-spectra of cyclotriphosphazenes are also visualized in Figure 1.With the exception of 2b, the spin system of the products was determined as AX 2 .The spin system of 2b is AB 2 (Dt/ 2 J PP : 5.5).Cyclotriphosphazenes (2 and 2a) have a triplet for a P(spiro) atom and a doublet for two PCl 2 or two P(amino) phosphorus atoms.The 31 P NMR values of the analogous compounds (4, 4a, and 4b) taken from the literature [36] are added to Table 1 for comparison.The dP shift of tetrachloro-bis(2-furanylmethyl)spirocyclotriphosphazene (2) is smaller than tetrachloro-mono(2-furanylmethyl)spirocyclotriphosphazene (4).Whereas, the coupling constant, 2 J PP , of 2 is larger than that of 4. The dP shift and 2 J PP values of 2a, 2b, 4a, and 4b are almost the same.
Also, the distinctive characteristic FTIR vibration of products was given in the "Experimental Part".In addition, the FT-IR spectra of all cyclotriphosphazenes are presented in Figures S10-S12.t P-Cl stretching absorption bands (asymmetric and symmetrical) of 2 appeared at 567 and 505 cm À1 , respectively.As expected, these bands disappear in the FT-IR spectra of the tetraaminocyclotriphosphazenes (2a and 2b).The symmetrical and asymmetrical stretching vibrations of the P ¼ N bonds of the phosphazene ring appear as strong and intense absorption bands in all cyclotriphosphazenes in the range of 1251-1215 cm À1 and 1185-1169 cm À1 , respectively.Also, these data are very consistent with the literature findings. [12,16,44]ray structure of 2b Evaluation of the molecular structure of 2b was performed by X-ray crystallography.An ORTEP diagram with the atom-numbering scheme of 2b is visualized in Figure 2. Table 2 lists the experimental data.The N 3 P 3 ring (P1/N1/P2/N2/P3/N3) of 2b [Figure S13a; u 2 ¼ 74.89(1.32), h 2 ¼ 101.04 (1.33) ] is in flattened-boat conformation with the total puckering amplitude, Q T , of 0.0601(15) Å.It has also been observed in the literature [50] that trimeric phosphazene rings, N 3 P 3 , are mostly in planar or twisted conformations when the total puckering amplitude (QT) values are in the range of (0.026-0.200)Å.The spiro ring (P1/N4/N5/C6/C7/C8) of 2b [Figure S13(b); u 2 ¼ À36.22(0.20), h 2 ¼ 122.08 (0.11) ] is in chair conformation with the Q T of 1.1790 (44) Å.In N 3 P 3 ring, atoms N1, N2, and N3 are above or below the plane of the P-atoms (P1/ P2/P3) with the distances of 0.0555(18) Å, À0.0270(19) Å, and À0.0566(19) Å.It was also determined by the torsional angles of the N 3 P 3 ring that the phosphazene ring of 2b has a pseudo-mirror plane passing through the midpoints between the P1, N3, and P2, N2 atoms (Figure S14).Apart from these, it was found that the endocyclic P-N bond lengths of 2b were in the range of (1.583-1.598)Å and were almost close to each other (Table 3).The average value of the exocyclic P-N bond lengths of 2b was calculated as 1.667(2) Å, significantly longer than the endocyclic P-N bonds.This result is in good agreement with the literature findings.[53] In 2b, angles c 1 and c 3 contracted, while angles b 1 , b 2 , and d 1 expanded significantly (Table 3) with respect to the corresponding values in the standard compound HCCP.In HCCP, the a 1 , a 2 , b 1 , c 1 , c 2 , and d 1 angles are 118.3(2), 101.2(1) , 121.4(3) , 118.3(2) , 101.2(1) , and 121.4(3) , respectively. [51]These data imply that 2b has electron delocalization in the N 3 P 3 ring.In the structure, the molecules Table 1. 31 P NMR spectral data of tetrachloro(2) and tetraaminobis(2-furanylmethyl)spiro(N/N)-cyclotriphosphazenes (2a and 2b).

Compound
Spin system dPNN (spiro) (ppm) P A dPCl 2 (ppm) P X dPNN (ppm) P X (P B ) a The 31 P NMR values of compounds 4, 4a, and 4b were taken from the literature. [36]re elongated along the c-axis and stacked along the a-axis directions (Figure S15).

Antiproliferative and antimigrative activities of the cyclotriphosphazenes on cancer cell lines
The antiproliferative activity of phosphazenes (2, 2a, and 2b) against prostate (PC-3) and colon cancer (HT-29) cell lines was examined by the MTT assay.In this study, only cells treated with cell nutrient medium were used as negative control and 5-fluorouracil (5-Fu), which is widely used in conventional chemotherapy, was preferred as positive control.Percent survival of cells was calculated after application of phosphazenes (2, 2a, and 2b) at various concentrations (3.12-50) mM.The IC 50 values of phosphazenes were determined for PC-3 and HT-29 cells using GraphPad Prism version 9.0.0 software (Graphpad Software Inc., La Jolla, CA) and are given in Table S2.The most effective dose and incubation times of the compounds in inhibiting the proliferation of cancer cells were determined with the data obtained during the evaluation.It was determined that phosphazenes, whose possible antiproliferative effects on cancer cells were evaluated, suppressed the proliferation of prostate cancer cells more effectively and especially 2 and 2a had a negative effect on cell viability at all studied concentrations.The results obtained were statistically significant when compared with the negative control.It was understood that 2b suppressed cell proliferation in prostate cancer cells only at the highest dose studied (50 mM), and the compound lost its effect as the dose used decreased.When the viability analysis results of the compounds with HT-29 cells were examined, it was observed that compounds 2 and 2a were effective in both 24 and 48 h at concentrations in the range of (3.12-50) mM.Compound 2b suppressed cell viability at only the highest concentration in prostate cancer as well as colon cancer cell line.The effect of the compound disappeared as the dose used decrease.In this study, the antimigratory properties of phosphazenes (2, 2a, and 2b) were examined by in vitro scratch analysis.In this analysis, the IC50 concentrations found by MTT analysis of the compounds were used.However, no inhibition value could be calculated as the viability of cells treated with compound 2b was higher than that of the negative control.Therefore, compound 2b was applied at the highest concentration for scratch analysis.Thus, as a result of the analysis, the possible effects of the compounds against prostate and colon cancer cell metastasis and invasion were determined as preliminary.
As seen in Figures S16 and S17, all compounds were found to have antimigratory effects in prostate and colon cancer cells compared with the negative control.Compound Figure 2.An ORTEP-3 [48,54] illustration of 2b with the atom-numbering scheme.Displacement ellipsoids are drawn at the 30% probability level.Hydrogen atoms have been omitted for clarity.(symmetry code: 2a is the compound with the highest antimigratory effect in prostate cancer.Compounds 2 and 2a were also found to be quite effective in colon cancer cells.When comparing their antimigratory effects against cancer cells, the phosphazenes exhibited the excellent ability to suppress migration in colon cancer cells.

Investigation of antiproliferative and antimigratory effects at molecular level by gene expression analysis
The antiproliferative and antimigratory effects of phosphazenes (2, 2a, and 2b) have been demonstrated in the previous sections.It was decided to examine the expression levels of some gene regions (Bcl-2, Bax, and p53).Because it is thought that the antiproliferative effects of related compounds may result from triggering apoptosis, which is defined as a mechanism of programmed cell death, these gene regions may be important in the mechanism of apoptosis (Figure S18).
For gene expression analysis, cancer cells were incubated with compounds for 48 h at IC50 concentrations determined in previous experimental steps.As mentioned in the previous sections, only compounds 2 and 2a were used in gene expression analysis, since the IC50 value of 2b could not be calculated in colon and prostate cancer cells.Quantitative real-time PCR analysis indicated increased expression of p53 and Bax genes in both HT-29 and PC-3, while expression of Bcl-2 gene was suppressed.These expression changes highlighted that apoptosis was induced at the molecular level.These results clearly demonstrated that 2 and 2a suppress cell proliferation in prostate and colon cancers at both cellular and molecular levels.Cell migration and invasion is an important process in cancer metastasis.Within the scope of this study, the possible effects of phosphazenes in the relevant process were primarily evaluated by in vitro scratch analysis.Then, it was decided to conduct an investigation at the molecular level.Therefore, changes in the expression levels of MMP2, MMP9, VEGF, and HIF-1a, which are important gene regions that play an important role in this process, were determined by quantitative real-time PCR analysis.As seen in Figure S19, it was determined that compounds 2 and 2a suppressed the expression of genes related to cancer cell migration (MMP2, MMP9, VEGF, and HIF-1a) in HT-29 and PC-3 cells.When the results obtained were evaluated statistically, it was seen that the difference between the control and experimental groups was distinctive.As a result, it was understood that all the findings were compatible with the data obtained by in vitro scratch analysis.

Single crystal X-ray structure determinations
The colorless suitable single crystal of 2b was obtained from n-hexane at ambient temperature.Crystallographic data were recorded on a Bruker APEX II QUAZAR three-circle diffractometer using Mo Ka radiation (k ¼ 0.71073 Å) at T ¼ 173 K. Absorption corrections by multi-scan [45] were applied.Structures were solved by direct methods [46] and refined by full-matrix least squares against F 2 using all data. [47]All of the non-H atoms were refined anisotropically.
The molecular and packing diagrams together with the ring conformations were drawn by the ORTEP-3 program incorporated in the WinGX package. [48]The H atoms were positioned geometrically at distances of 0.95 Å (CH) and 0.99 Å (CH 2 ) from the parent C atoms.A riding model was used during the refinement process.The Uiso(H) values were constrained to be 1.2 Ueq (carrier atom).
Crystallographic data for the structure reported in this article has been deposited with the Cambridge Crystallographic Data Center [deposition numbers: CCDC 2241465 (for 2b)].Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [e-mail: deposit@ccdc.cam.ac.uk].

Determination of the in vitro antiproliferative and antimigratory activities
Cancer cell lines HT-29 (HTB-38) and PC-3 (CRL-1435) used to investigate the antiproliferative and antimigratory effects of cyclophosphazene derivatives (2, 2a, and 2b) on cancer cells are provided from ATCC (American Type Culture Collection, Manassas, VA).Cell culture media and all other chemical reagents used in this study were commercially obtained.In addition, details of the method of in vitro antiproliferative and antimigratory activities are provided in the SM.

Investigation of antiproliferative and antimigratory effects at molecular level by gene expression analysis
The effects of phosphazene derivative compounds (2, 2a, and 2b) on genes involved in apoptosis and cell migration were analyzed by gene expression at the mRNA level.Details of the analysis are provided in the SM.

Conclusions
As it is well known, the replacement of Cl atoms in the cyclotriphosphazene core with different organic/inorganic mono, di, and polydentate substituents is very useful in the synthesis of new organic-inorganic hybrid heterocyclic cyclotriphosphazene derivatives.In this study, new hybrid tetrachloro-(2), tetrapyrrolidino-(2a), and tetrapiperidino-(2b) monospiro(N/N)cyclotriphosphazenes with bis(2-furanylmethyl) pendant arms were synthesized to examine their structures, antiproliferative and antimigratory activities.Meanwhile, reaction yields in THF are found to be very high (62-82%), and microanalytical, spectral (HRMS, FTIR, and NMR), and crystallographic data (for 2b) confirm the structures of the cyclotriphosphazenes.The 31 P NMR values of bis(2-furanylmethyl)phosphazenes (2, 2a, and 2b) were observed to be considerably different from those of mono(2furanylmethyl)phosphazenes (4, 4a, and 4b).In addition, the antiproliferative and antimigratory activity of bis(2-furanylmethyl)spirocyclotriphosphazenes (2, 2a, and 2b) against prostate cancer (PC-3) and colon cancer (HT-29) cells was investigated.According to the findings, it was determined that 2a was significantly effective against both cancer cell lines.In conclusion, it is concluded that these findings provide important preliminary data for the use of these compounds in cancer researches.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
We would like to thank the Tokat Gaziosmanpas ¸a University Research Fund (TOGU-BAP: 2019/44) for financial support.T. H. is grateful to Hacettepe University Scientific Research Unit (Grant No. 013 D04 602 004).
max , Dq min (e Å À3 ) 1.04, À0.41 efficacy in the inhibition of cancer cell migration.In addition, the result obtained in this study may have filled an important gap in the ability of phosphazenes to inhibit cancer cell migration.As a result, more research is needed to fully understand the mechanisms and potential use of these compounds in cancer treatment.
All spectra were analyzed using Jasco Spectra Manager version 2.14.05 software, JASCO Co., Tokyo, Japan.ESI-Mass spectra (HRMS) were recorded by LC-MS TOF electrospray ionization technique (Agilent Technologies 6230-A, Santa Clara, CA).The theoretical values of isotopic distributions were generated with the molecular mass calculator from MSTools (EPFL, Lausanne, Switzerland).