Tripodal Phosphine Oxide Ligand with Tetrazole Functionality

Tris[2-(3′-cyanopropoxy)phenyl]phosphine oxide has been obtained via the alkylation of tris(2hydroxyphenyl)phosphine oxide with 4-bromobutyronitrile in the presence of K2CO3. The product structure has been elucidated by means of X-ray diffraction analysis. The terminal cyano groups in the obtained phosphine oxide have been converted into tetrazole rings via the click-reaction with NH4N3 to give a new hybrid tripodal propeller ligand, tris{2-[3′-(tetrazol-5′′-yl)propoxy]phenyl}phosphine oxide. Palladium(II) complexes of the prepared ligand and its short-linker analog, tris[2-(tetrazol-5′-ylmethoxy)phenyl]phosphine oxide, have been synthesized. Cytotoxicity of the ligands and their Pd(II) complexes has been studied.

The use of triphenylphosphine oxide fragment as the core of a tripodal ligand can favor the complex formation with metal ions [25][26][27][28][29]. The combination of the P=O fragment and tetrazole functional groups in hybrid ligands affords the compounds promising as ligands and biologically active molecules.
This study aimed to synthesize hybrid ligand 1 containing the phosphine oxide core and tetrazole side groups and to investigate cytotoxicity of the ligand and its palladium complex toward tumor cells. It was also interesting to compare ligand 1 to its analog 2 with a shorter linker [Bykhovskaya, O.V. et al., Russ. J. Gen., Chem., 2019, vol. 89, no. 12] (Scheme 1).
Composition and structure of the obtained trinitrile 4 were confirmed by the data of elemental analysis as well as IR and NMR ( 1 Н, 13 С, and 31 Р) spectroscopy. IR spectrum of compound 4 contained the ν(С≡N) bands at 2241 cm -1 and the ν(P=O) bands at 1176 cm -1 . The 1 H NMR spectrum of the solution in CDCl 3 revealed the signals of the linker methylene protons as triplets or quintets at 1.6-4.1 ppm. The signals of the phenyl protons of the triphenylphosphine oxide core revealed chemical shifts and multiplicities typical of the related compounds [31] and appeared as doublets and triplets at 6.8-7.7 ppm. The 13 C NMR spectrum contained the singlet signals of the linker methylene groups at 13-66 ppm and the CN group signals at 119.3 ppm. The signals of the carbon atoms of the triphenylphosphine oxide core appeared as doublets at 111-161 ppm with typical spin-spin coupling constants of J PС 1.7 to 111.2 Hz. The 31 P NMR spectrum contained a singlet at 28.1 ppm, typical of triarylphosphine oxides.
Molecular structure of tris[2-(3′-cyanopropoxy)phenyl]phosphine oxide 4 was confirmed by the X-ray diffraction analysis method (Fig. 1). Selected bond lengths are collected in Table 1. The molecule of compound 4 was asymmetric, with two of three ortho-substituents located to the same side as the P=O group with respect to the carbon atoms of the POC 3 moiety and the third substituent was directed oppositely. The O 1 P 1 C 22 O 4 angle corresponding to the substituent located oppositely to the phosphine oxide group equaled -172.2(1)°, two other substituents forming the OPCO angles of 38.6(1) to 64.8(1)°. In the earlier described cases of tris(tetrazoles) and tris(alkanols) [31], the molecule conformation has been additionally stabilized by two C-H···O(P) bonds. Molecule of compound 4 also contained two O-H···O(P) intramolecular bonds formed by the substituents which were to the same side as the P=O group (Fig. 1). The bond lengths and angles were typical of the earlier described phosphoryltris(2,1-phenylene)oxyalkenes [27,29,[31][32][33].  (2) In the second stage, trinitrile 4 was introduced in the reaction with NH 4 N 3 prepared in situ from NH 4 Cl and NaN 3 , affording ligand 1 in 74.2% yield (Scheme 2). The product structure was elucidated by means of IR, Raman, and NMR ( 1 H, 13 C, and 31 P) spectroscopy. Tris[2-(tetrazol-5′-ylmethoxy)phenyl]phosphine oxide 2 has been prepared by us earlier [Bykhovskaya, O.V. et al., Russ. J. Gen., Chem., 2019, vol. 89, no. 12] using the same method from the corresponding cyano derivative [25].
IR and Raman spectra of ligand 1 were compared to those earlier described for ligand 2 with a known structure ( Table 2). IR spectrum of compound 1 did not contain the bands of the starting trinitrile 4 at 2241 cm -1 [ν(С≡N)] and 1176 cm -1 [ν(P=O)]. Instead, the ν(P=O) at 1086 cm -1 appeared in the spectrum as in the case of ligand 2, corresponding to a phosphoryl group involved in a bifurcate hydrogen bond. The spectra of ligands 1 and 2 were similar in the range of the ν(NH) vibrations. It should be noted that the spectrum of a solution of ligand 1 in DMSO-d 6 contained the ν(P=O) band assigned to the phosphoryl group involved in hydrogen bond (1157 cm -1 ) and, in contrast to ligand 2, the band of the free P=O group (1185 cm -1 ). That feature could be due to longer linker providing higher mobility of the pendant group.
Assignment of the ring vibrational frequencies was based on the calculation of the frequency and shape of normal vibrations in 5-substituted tetrazoles [34]. The absorption bands at 1600-1400 cm -1 mainly related to the ν(C=N) and ν(N=N) vibrations overlapped with strong bands assigned to the phenoxy groups in the spectra of compounds 1 and 2 and thus could not be reliably detected. The band at 1480 cm -1 in the spectrum of ligand 1 ( Table 2) was assigned mainly to the ν(C-N) vibrations, the band at 1242 cm -1 corresponded to the N=N bonds vibrations mixed with ring deformations, whereas the bands at 1124 and 1110 cm -1 were assigned to the δ(NH) deformational vibrations. The band at 1040 cm -1 was due to the out-of-plane ρ(NH) vibrations mixed with the ring deformations, and the band at 990 cm -1 was assigned to the deformation of the cycle. Dissolution of ligand 1 in DMSO-d 6 led to the   (Table 2). 1 H NMR spectrum of ligand 1 contained the signals of the linker methylene groups as three broadened singlets at 1.7-3.9 ppm. The aryl protons appeared as multiplets at 6.8-7.3 and 7.4-7.7 ppm. The 1 H NMR spectrum did not contain the signals of the NH group, this feature is typical of the tetrazoles spectra. The 13 C NMR spectrum revealed three singlet signals of the linker methylene groups at 19-67 ppm, a singlet of the tetrazole carbon atom at 155.67 ppm, and several doublets of the aryl carbon atoms at 113-160 ppm. The 31 P NMR spectrum contained a singlet at 28 ppm, typical of triarylphosphine oxides.
Palladium complexes with different heterocyclic compounds have been recognized for the antitumor activity [35]. In view of that, we synthesized the 1 : 1 complexes of the new ligand 1 (n = 3) and the previously prepared ligand 2 (n = 1) with PdCl 2 : complexes 5 and 6, respectively (Scheme 3).
The complexes were isolated and characterized by means of elemental analysis as well as IR and NMR spectroscopy. According to the elemental analysis data, ligands 1 and 2 formed the 1 : 1 complexes with PdCl 2 .
In contrast to the lanthanum complex with deprotonated ligand 2 described by us earlier [Bykhovskaya, O.V. et al., Russ. J. Gen., Chem., 2019, vol. 89, no. 12], the amino groups capable of hydrogen bonding were retained in complex 6, which could affect the coordination properties of the ligand. The ν(NH) bands of the palladium complex were changed in comparison with the free ligand spectrum: the maximum at 2700 cm -1 became weaker, and the second maximum appeared at ~3100 cm -1 , which could be assigned to weaker bound NH groups and the ν(ОH) vibrations of bound water. The ν(Р=О) band, as in the ligand spectrum, appeared at about 1090 cm -1 , evidencing preservation of the intramolecular hydrogen bons of the phosphoryl oxygen atom with two tetrazole rings, the ring vibrational bends being changed ( Table 2). The Raman spectrum of complex 6 contained a band at 300 cm -1 , which could be assigned to the ν s (Pd-Cl) symmetric vibrations in the trans complex [36], hence palladium evidently coordinated two tetrazole rings in the trans configuration. In view of the 1 : 1 complex composition, it could be suggested that those tetrazole rings belonged to different molecules, and the dimers with one of the tetrazole rings acting as bridge were formed. Moreover, a water molecule could be bound to proton as well as nitrogen atom, linking the complexes.
The ν(Р=О) band in the spectrum of complex 5, as in the case of complex 6, was not shifted in comparison with the free ligand spectrum, hence it could be suggested that the node with the bifurcate hydrogen bond was retained. Position of the ring bands was slightly changed ( Table 2). The ν s (Pd-Cl) band at 299 cm -1 in the Raman spectrum was stronger than that in the spectrum of complex 6 and was also typical of trans geometry of the coordination plane. Overall, comparison of the vibrational spectra  revealed structural similarity of the obtained palladium complexes.
Hence, the analysis of the vibrational spectra suggested the trans structure of complexes 5 and 6 with coordination of tetrazole rings of two ligands and preservation of the bifurcate intramolecular hydrogen bond with the phosphoryl group.
Complexes 5 and 6 were slowly dissolved in DMSO, seemingly without decomposition (as per the IR spectroscopy data).
1 Н NMR spectrum of the solution of complex 5 contained a broad singlet of the central methylene group of the linker at 1.74 ppm and a triplet of the CH 2 N group protons at 2.63 ppm, whereas the signal of the CН 2 О group protons was masked with the water signal ( Table 3). The aryl protons of complex 5 were observed as triplets at 6.98 (Н 5 ), 7.53 ppm (Н 4 ) and a doublet of doublets at 7.08 (Н 3 ), 7.47 ppm (Н 6 ).
The 1 Н NMR spectrum of complex 6 contained a broad singlet of the CН 2 О group at 5.38 ppm and the signals of the aromatic protons as triplets at 6.96 (Н 5 ), 7.58 ppm (Н 4 ), triplet of doublets at 7.34 ppm (Н 3 ), and doublet of doublets at 7.26 ppm (Н 6 ). Comparison of the signals of protons for the ligands 1, 2 and complexes 5, 6 ( Table 3) revealed that the complex formation with PdCl 2 did not significantly change the 1 Н NMR spectra as well as the 31 P NMR spectra: the signal of the phosphorus atom was shifted downfield by 0.1-2.5 ppm. Those data evidenced no coordination with the P=O group during the complex formation.
The tested compounds revealed weaker activity in comparison with the reference (Camptothecin). The palladium complex 5 was inactive towards the used cell cultures, and other compounds were inactive toward the НCT116 cells (colon carcinoma). Ligands 1 and 2 revealed almost equal cytotoxicity against the А549, HeLa, and MCF7 cells. At the same time, complex 6 revealed twice higher cytotoxicity against the А549, HeLa, and MCF7 in comparison with the starting ligand 2. Overall, the Pd(II) complex of ligand 2 with the short linker was the most active of the tested compounds.
IR spectra of solid compounds 1, 5, and 6 were obtained using a Bruker Tensor 37 IR spectrometer (resolution 2 cm -1 , range 4000-400 cm -1 , KBr pellets or Nujol suspension). IR spectra of the solutions in DMSO-d 6 (c 0.04 M, CaF 2 cuvettes with pathlength of 0.06 mm) were recorded over the 4000-1100 cm -1 range. Raman spectra of the powders were recorded over the 3500-100 cm -1 range using a Jobin-Ivon LabRAM 300 spectrometer equipped with a microscope and laser CCD detector. A 632.8 nm He-Ne laser (power not exceeding 2 mW) was used as the excitation source. 1 Н, 13 С{ 1 Н}, and 31 P{ 1 Н} NMR spectra of the solutions in CDCl 3 and DMSO-d 6 were recorded using a Bruker Avance 400 instrument operating at 400.13, 100.61, and 161.98 MHz ( 1 Н, 13 С, and 31 P, respectively). The signals of residual protons and carbon atoms were used as internal references in the 1 Н and 13 С NMR spectra, the accuracy of the chemical shifts determination being better than ±0.01 and ±0.03 ppm, respectively. The chemical shifts in the 31 P NMR spectra were determined relative to 85% H 3 PO 4 as external reference. The signals were assigned by comparison with the spectra of similar compounds [31]. Melting points were determined in a melting point apparatus using short Anschutz thermometers. The С, H, and N content was determined using a Carlo Erba 1106 instrument, the content of P was determined as described elsewhere [38].
Tris[2-(3′-cyanopropoxy)phenyl]phosphine oxide (4). A mixture of 0.74 g (0.005 mol) of 4-bromobutyronitrile, 2.1 g (0.015 mol) of freshly calcined K 2 CO 3 , and 0.49 g (0.0015 mol) of tris(2-hydroxyphenyl)phosphine oxide 3 in 20 mL of anhydrous DMF was heated at 95°C with stirring during 4 h. The solvent was removed, and 15 mL of CH 2 Cl 2 was added to the dry residue; the obtained mixture was washed with water (4×15 mL). The organic solution was dried with Na 2 SO 4 , and the solvent was removed. Light yellow oil crystallizing during keeping was obtained. The product was recrystallized from a CH 2 Cl 2 -Et 2 O mixture to obtain white crystalline substance. Yield 0.76 g (96.2%), mp 159°С. The crystals suitable for X-ray diffraction analysis were prepared via recrystallization from ethanol. IR spectrum ( [39] and refined via full-matrix least squares method over F 2 . The non-hydrogen atoms were refined under anisotropic approximation, the positions of the hydrogen atoms were calculated, and the hydrogen atoms were included in the refinement using the rider model with U iso (H) = 1.2U eq (C). The calculations were performed using SHELXL2014 [40] and OLEX2 [41] software package. CCDC 2085004 contains additional crystallographic information relevant to this paper. These data are available free of charge via http://www.ccdc.cam. ac.uk/structures/. МТТ-test). The А549 and НСТ116 human cells were cultivated in DMEM medium, and MCF7 and HeLa human cells were cultivated in ЕМЕМ medium with addition of 10% fetal calf serum, 2 mM of glutamine, and 1% gentamicin as antibiotic at 37°С under humid СО 2 atmosphere (5%). The cells of adhesion cultures А549, НСТ116, MCF7, and HeLa were seeded at concentration of 1×10 4 cells/200 μL in a 96-well plate and cultivated at 37°С under humid atmosphere with 5% of СО 2 . Upon 24 h of incubation, 100 to 1.56 рМ of the studied compounds was added to the cell cultures, and they were cultivated for further 72 h under the same conditions. The experiments were run in triplicate at each concentration. The compounds were dissolved in DMSO, final concentration of which in the well did not exceed 1% and was not toxic for the cells. Pure solvent (final concentration 1%) was added to the reference cells. Upon the incubation, 20 μL of МТТ (5 mg/mL) was added to each well, and the plate was incubated during 2 h. The medium was then removed from the wells, and 100 μL of DMSO was added to each well for dissolution of the formed formazan crystals. Absorbance at 536 nm was measured using a Cytation3 plate scanner (BioTeklnstruments, Inc). The value of the concentration inducing 50% inhibition of the cells growth (IC50, μM) was determined from the dose-dependent curves using OriginPro 9.0 software.