Synthesis of Two Novel Homologous Polyphosphoesters Containing Aminophosphonate Units and Cytotoxicity of Some Low-Molecular and Polymeric Aminophosphonate Derivatives

Institute of Polymers, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University-Sofia, Sofia 1000, Bulgaria Institute of Biodiversity and Ecosystems Research, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Department of Pharmacognosy, Faculty of Pharmacy, Medical University-Sofia, Sofia 1000, Bulgaria


Introduction
e aminophosphonic acid derivatives are an interesting class of biologically active compounds, which possess diverse and useful properties, including herbicidal, antibacterial, antiviral, and antitumor activity [1][2][3].
e immobilization of aminophosphonate moieties to biodegradable polymer carriers is an effective way for the formation of new type of polymer prodrugs with improved properties [19,20].e polymer-drug conjugates seem to hold great potential to circumvent major disadvantages of the conventional drug therapy [21,22].
Polyphosphoesters comprise a class of biodegradable and biocompatible phosphorus-containing polymers [23].
ey degrade into nontoxic products through hydrolysis and enzymatic cleavage of their phosphoester bonds under physiological conditions [22,24].e poly(alkylene H-phosphonate)s contain reactive P-H groups, which allow for the chemical attachment of drug molecules to the polymer chain [22,23].
In the present study, we report on the synthesis and spectroscopic characterization of two novel homologous polyphosphoesters containing anthracene-and furanderived aminophosphonate units.One of them was evaluated for cytotoxicity against a panel of human leukemic cell lines, including HL-60, SKW-3, REH, and K-562 cells.
e polymer analogous reaction was carried out using an excess of the Schiff base and in the presence of CdI 2 as a catalyst.e polymer P-12 was obtained in a good yield (69%) by conventional heating of the reagents at 60 °C for 25 h.e preparation of the polymer P-13 was accomplished for a shorter reaction time (1 h) and in a higher yield (88%) with the assistance of microwave irradiation.e products P-12 and P-13 were obtained as viscous oils, soluble in common organic solvents (toluene, chloroform, methanol, and ethanol) and in water.
e structure of the polyphosphoesters P-12 and P-13 was verified by means of IR and NMR spectral analysis.e IR spectra displayed absorption bands characteristic for stretching vibration of NH, PH, P�O, and P-O-C groups. 1 H NMR spectra of the products showed doublet signals at 5.68 and 5.71 ppm (P-12, in CDCl 3 and in DMSO-d 6 , respectively) and 5.69 ppm (P-13, in CDCl 3 ) with 2 J PH about 23 Hz which can be assigned to the CHP proton from their units formed by addition reaction between the polymeric H-phosphonate and the Schiff base.e NH proton signal is masked in the spectra taken in CDCl 3 solution.In the spectrum of P-12 measured in DMSO-d 6 , the signal of NH proton appears at 4.60 ppm as a broad singlet.e signals for the anthracene protons (AnthH) of P-12 and P-13 are found in the expected region of the spectra [20,25]. 1 H NMR spectra of P-12 and P-13 reveal two different doublet signals with large coupling constants that are due to the PH proton from H-phosphonate repeating units and PH(O)OH end groups, respectively. 13C{ 1 H} NMR spectrum of P-12 shows the signal of CHP carbon atom as a doublet at 54.79 ppm with a large coupling constant ( 1 J PC � 159.7 Hz). e spectrum exhibits three distinct doublet signals which belong to the methylene carbon of the POCH 2 CH 2 fragment from H-phosphonate and aminophosphonate repeating units and of the POCH 2 group from H-phosphonate repeating units.
e carbon signals of the nonequivalent POCH 2 groups from aminophosphonate repeating units appear as two doublets.
e methylene CH 2 Fur carbon gives a doublet signal at 44.36 ppm.e HSQC spectrum shows the connection between this doublet and the proton signals at 3.66 and 3.42 ppm.ese two signals are overlapped with the complex multiplet attributed to the OCH 2 CH 2 , POCH 2 CH 2 , and OCH 3 proton signals of P-12.e signals for the furan protons (P-12 and P-13) and for the furan carbons (P-12) are also observed in the 1 H and 13 C NMR spectra, respectively.e assignment of the anthracene carbons of P-12 is based on the analysis of the HSQC spectrum and is in accordance with the data of similar aminophosphonate derivatives [20,25]. 31P{ 1 H} NMR spectra of the products, taken in CDCl 3 (P-12 and P-13) and DMSO-d 6 (P-12) solutions, display a singlet signal about 25 ppm and another   [20].e signal of the nonreacted PH(O)OH groups is found up field (5.01-2.42ppm) in the spectra.In the coupled spectra, this signal appears as a doublet with coupling constant ( 1 J PH ) 647-610 Hz, which indicates that these groups possess an ionic structure.e phosphorus atom from CHP diester end groups gives two distinct singlets with very close chemical shifts because of the presence of more than one diastereomeric species in the molecules of the products.On the basis of the 31 P{ 1 H} NMR spectra is estimated the average degree of polymerization of the starting polyphosphoester 1 (n � 6) and of the synthesized homologous polyphosphoesters P-12 (m + l � 5.4) and P-13 (m + l � 6).From the 31 P{ 1 H} NMR spectral data is calculated the content of the aminophosphonate units in the products: 54% (P-12) and 81% (P-13).
e fluorescence spectra of the synthesized homologues show that the emission maxima appear in the region 402-446 nm. e mass spectra of the polyphosphoesters P-12 and P-13 are performed using electron impact mass spectrometry of the products in acetonitrile/water solutions.e analysis of the spectra reveals that they undergo strong fragmentation processes, which cause cleavages of their polymer backbones.e most intense peak (basic peak) in both spectra observed at m/z � 286.1218 (P-12) and 286.1216 (P-13) can be assigned to the ion C 20 H 16 N + O (calcd 286.1226).
e structure of this cation [C 14 H 9 CH�N + HCH 2 C 4 H 3 O] corresponds to the protonated form of the starting Schiff base S-2: 9anthrylidene-furfurylamine cation.e mass spectra of the polyphosphoesters P-8-P-11, studied previously [20], showed base peaks for an ion (C 22 H 18 N + ) at about 296 Da which structure was identical with the structure of the protonated synthetic precursor (protonated Schiff base S-1) of these polymers.In both cases, an electron elimination at the nitrogen atom and a break of the C-P bond of the anthracene-containing aminophosphonate moiety lead to the formation of the protonated forms of the starting Schiff bases S-2 and S-1, respectively.Many other peaks are observed in the mass spectra of P-12 and P-13 due to the fragmentation of the polymeric chains.
Recently, we described the synthesis and the in vitro antitumor activity against a panel of human epithelial cancer cell lines and the in vitro and in vivo safety evaluation of anthracene-derived low-molecular and polymeric aminophosphonates and their precursors [20,[25][26][27][28]: Schiff bases S-1 and S-2, α-aminophosphonates A-3-A-6, bis-aminophosphonate B-6 (R, S diastereomer), and polyphosphoesters P-8-P-11, containing aminophosphonate units (Figure 1).e α-aminophosphonates A-3 and A-4 [25] and the polyphosphoesters P-8-P-11 [20] have been synthesized from the anthracene-derived Schiff base S-1, while the compounds A-5 [25] and A-6 [28] have been prepared using the anthracene-and furan-containing Schiff base S-2.e racemic compound A-5 has been separated into its two enantiomers A-5a and A-5b by HPLC technique [28].Among the studied compounds A-3-A-5, aminophosphonate A-5, bearing anthracene and furan ring, showed the best antitumor effects against all tested human epithelial cancer cell lines.is result provokes us to incorporate such aminophosphonate fragments, which contain anthracene and furan units, into polymer backbone to obtain novel polyphosphoesters P-12 and P-13.
e polyphosphoesters P-8-P-13 have been synthesized on the basis of biodegradable polymer carriers, which possess repeating hydrolytically unstable phosphorus ester linkages in their backbone [22].e polymers P-8 and P-10 are built only of aminophosphonate repeating units, while the copolymers P-9 and P-11-P-13 contain aminophosphonate and hydrophilic H-phosphonate links.

Pharmacology.
As evidenced by the data presented in Table 1, in all of the screened leukemic lines calculated IC 50 values of the tested compounds vary substantially both within and between the series.Comparable cytotoxicity was noted only for the precursors (anthracene-based Schiff bases S-1 and S-2) with the highest sensitivity of the ALL (REH) cells.e encountered inhibitory concentrations of both compounds are similar to cisplatin's, whereas HL-60, SKW-3, and K-562 cells retained a far greater proliferative capacity under the same conditions.α-Aminophosphonates (A3-A6) showed lesser consistency in the produced growth inhibitory effect against the tested leukemic cells.Highest cytotoxic potential in all in vitro tumor models was encountered with α-aminophosphonate A-4 whose IC 50 estimates correlate with those of the referent drug.e other anthracene-derivative carrying an identical aminophosphonate residue, that is, A-3, produced an in-between inhibitory effect inferior to cisplatin's in HL-60, SKW-3, and REH test systems, whereby the estimated equieffective concentration for the K-562 cells was 10-to 15fold that of the referent drug.e racemic compound A-5, obtained from the anthracene-and furan-containing Schiff base S-2, appeared least effective in terms of growth inhibition, whereas both corresponding enantiomers (A-5a and A-5b) achieved markedly lower IC 50 values, especially in the leukemic subtypes of lymphoid origin (SKW-3 and REH cells).e other anthracene-and furan-carrying α-aminophosphonate congener, that is, A-6, delivered a moderate anticancer efficacy ranging between that of A-5 and both chiral isomers A-5a and A-5b.e cytostatic activity of the same compound is evidenced to be essentially invariable in all of the tested cell lines (closely comparable IC 50 values).

Advances in Materials Science and Engineering
In line with the A-4 toxicity profile, the polymeric derivatives, that is, polyphosphoesters P-8 and P-9, yielded distinctly lower equally effective concentrations in all four tumor lines as compared to the newly synthesized P-12.Cytotoxicity of both P-8 and P-9 is largely surpassing that of cisplatin in HL-60 and SKW-3 and 2-fold higher (P-8) or equal (P-9) to the referent in REH cells, whereas the chronic myeloid cell line K-562 displayed 2-fold lower chemosensitivity towards P-8 as compared to the referent drug.e same leukemic line showed a slightly lower responsiveness towards the P-9 analogue, however estimated IC 50 value remaining in the range of cisplatin's.e other two polyphosphoesters, namely, P-10 and P-11, bearing the same aminophosphonate residue and a shorter length of the PEG (4 units) chain demonstrated poor cytostatic activity, whereby the K-562 cell line was rated again as the least sensitive one.e newly synthesized P-12 showed consistently lower cytotoxicity than cisplatin that was oddly most pronounced in the CML K-562 cells, followed by the SKW-3, REH, and HL-60 myeloproliferative cell lines.
Overall tumor-inhibiting activity of the studied anthracene derivatives varies by compound and cell line, albeit some correlation was observed between cancer chemosensitivity and number and type of polymer units.e promyelocytic and T-cell leukemic cell lines (HL-60 and SKW-3 cells, respectively) were generally more sensitive to the α-aminophosphonates A-3 and A-4 than to the Schiff bases, whereas the latter exerted substantially stronger cytotoxic effects against the B-cell lymphoma cell line (REH).e most potent low-molecular analogue in the tested panel of human leukemic cell lines was the α-aminophosphonate A-4, whereas highest cytotoxicity among the polymeric species was determined for the corresponding polyphosphoester P-8.In the K-562 cell line, the newly synthesized polyphosphoesters P-12 demonstrated cytotoxicity matching that of the leader polymers P-8 and P-9 as well as the reference drug, while a fewfold variability in tumor cell response was established for the rest of the leukemic lines.e data for P-12, given in Table 1   show that the immobilization of anthracene-and furancontaining aminophosphonate units into the polymer chain results in a higher cytotoxicity of the polymer against the tested human leukemic cell lines compared to that of its low-molecular analogues A-5 and A-6.However, polymer P-12 exhibits moderate cytotoxicity compared to polymers P-8 and P-9. e novel polyphosphoesters P-12 and P-13 will be of interest for future investigation on human epithelial cancer cell lines, based on the results of their lowmolecular analogue (A-5) obtained from its study with such cancer cell lines [25].Among the all tested compounds, we considered that aminophosphonate A-4 and, especially, polymers P-8 and P-9 appear very promising new materials for development of anticancer drugs.
e reaction was carried out by conventional heating (P-12) and under microwave irradiation (P-13) of the reagents.It was conducted using an excess of the Schiff base and in the presence of a catalytic amount of CdI 2 .e structure of the polyphosphoesters was studied by means of spectral methods: IR, NMR ( 1 H, 13 C, and 31 P), fluorescence, and mass spectra.e average degree of polymerization of the starting polyphosphoester 1 and of the synthesized polyphosphoesters P-12 and P-13 and the ratio between the aminophosphonate and the H-phosphonate units in the products were estimated from their 31 P{ 1 H} NMR spectral data.
e novel polyphosphoester P-12 and some anthracenederived compounds, among them, the α-aminophosphonates A-3-A-6, their precursors the Schiff bases S-1 and S-2, the bis-aminophosphonate B-6, the two enantiomers A-5a and A-5b of the racemic A-5, and the polyphosphoesters P-8-P-11, containing aminophosphonate units, have been tested for cytotoxicity against a panel of human tumor cell lines: HL-60, SKW-3, REH, and K-562.Both precursors S-1 and S-2 presented similar cytotoxicity profiles that are cisplatin-like only in the REH lymphoid cell line.Leader compound of the α-aminophosphonates A-3-A-6 is A-4 with cell deathinducing properties fully equaling those of the referent drug.Some of the polymeric analogues elicited moderate (P-10 and P-12) to low (P-11) cytotoxic activity, whereas the polyphosphoesters P-8 and P-9 produced in vitro antitumor effects largely surpassing cisplatin's potency.ree of the tested compounds, namely, P-8, P-9, and A-4, appear quite promising as new materials for the development of drug in chemotherapy of malignant leukemic diseases.

Synthesis. Dimethyl H-phosphonate (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany) was purified by vacuum distillation.Poly(ethylene glycol) with average molecular weight 600 Da (PEG 600) was purchased from Fluka and dried prior to use.All solvents were freshly distilled before use.IR spectra were taken on an IRAffinity-1 spectrophotometer. 1 H, 13 C{ 1 H}, and HSQC NMR spectra of polymer P-12 in CDCl 3 were recorded on an Avance 600 MHz spectrometer at room temperature and tetramethylsilane (TMS) was used as an internal standard. 1H NMR spectra of P-12 in DMSO-d 6 and P-13 in CDCl 3 were taken on a Bruker DRX-250 250 MHz spectrometer at room temperature and TMS was used as an internal standard. 31P NMR spectra of P-12 in CDCl 3 and DMSO-d 6 solutions and of P-13 in CDCl 3 were registered on an Avance 600 MHz (P-12) and on a Bruker DRX-250 250 MHz (P-13) spectrometers, using 85% H 3 PO 4 as an external standard.Fluorescence spectra were recorded on a JascoFp 6600 fluorometer (220-750 nm excitation range/220-1010 nm emission range).Microwave-assisted reaction was carried out in Microwave Reactor ROTOSYNTH Rotativ Solid Phase purchased from Milestone, Bergamo, Italy.
e mass spectra were taken on ermo Scientific Q Exactive Plus (Bremen, Germany) equipped with heated electrospray probe HESI-II in positive mode.e instrument operated in fullscan mode in m/z range 200-3000 at resolution 280000 (at m/z 200).e instrument parameters were as follows: spray voltage 3.5 kV, sheath gas 36 psi, auxiliary gas 11 a.u., ion transfer tube temperature 320 °C, and HESI-II vaporizer temperature 320 °C.Data acquisition and processing was done using ermo Scientific Xcalibur ver. 3.All solvents were of LC-MS grade and were purchased from ermo Fisher Scientific (Waltham, USA).

Enantiomers A-5a and A-5b.
e enantioseparation of the racemic α-aminophosphonate A-5 into its enantiomers A-5a and A-5b was reported in a previous paper [28] A-5a, e polymers P-8-P-11 were obtained through an addition reaction of poly(oxyethylene H-phosphonate)s to the Schiff base S-1 using synthetic procedures reported previously [20].
e polymers P-8 and P-9 were synthesized from polymeric H-phosphonate based on PEG 600 and polymers P-10 and P-11 based on PEG 200.