Bioorganic & Medicinal Chemistry 22 (2014) 874–882
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis, characterization, antitumor activity and safety testing of novel polyphosphoesters bearing anthracene-derived aminophosphonate units I. Kraicheva a,⇑, E. Vodenicharova a, S. Shenkov a, E. Tashev a, T. Tosheva a, I. Tsacheva a, A. Kril b, M. Topashka-Ancheva c, A. Georgieva b, I. Iliev b, I. Vladov b, Ts. Gerasimova c, K. Troev a a
Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 103A, 1113 Sofia, Bulgaria Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 25, 1113 Sofia, Bulgaria c Institute of Biodiversity and Ecosystems Research, Bulgarian Academy of Sciences, 2 Gagarin Str., 1113 Sofia, Bulgaria b
a r t i c l e
i n f o
Article history: Received 12 September 2013 Revised 11 November 2013 Accepted 2 December 2013 Available online 8 December 2013 Keywords: Polyphosphoesters Aminophosphonic acids NMR Antitumor activity Cytotoxicity Genotoxicity
a b s t r a c t Novel polyphosphoesters containing anthracene-derived aminophosphonate units, poly(oxyethylene aminophosphonate)s (4 and 5) and poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s (6 and 7), were synthesized via an addition of poly(oxyethylene H-phosphonate)s to 9-anthrylidene-p-toluidine. The IR, NMR (1H, 13C and 31P) and fluorescence emission spectral data of the polymers are presented. The copolymers 6 and 7 were tested for in vitro antitumor activity on a panel of seven human epithelial cancer cell lines. Safety testing was performed both in vitro (3T3 NRU test) and in vivo on ICR mice for genotoxicity and antiproliferative activity. The copolymer 7 showed excellent antiproliferative activity to HBL100, MDA-MB-231, MCF-7 and HepG2 cell lines. However, the in vitro safety testing revealed significant toxicity to Balb/c 3T3 mouse embryo cells. In contrast, the copolymer 6 showed complete absence of cytotoxicity to Balb/c 3T3 cells, but inhibited the growth of breast cancer cells, cervical carcinoma cells (HeLa) and hepatocellular carcinoma cell cultures after prolonged (72 h) exposure. The polymers (4–6) exhibited low (4 and 6) to moderate (5) clastogenicity in vivo and slightly inhibited bone marrow cell division, compared to Mitomycin C. The subcellular distribution of the copolymers 6 and 7 were studied in model cell culture systems. The tested polyphosphoesters are expected to act in vivo as prodrugs of aminophosphonates and could be valuable as a new class of biodegradable polymer drug carriers. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The polyphosphoesters comprise interesting groups of biodegradable and biocompatible phosphorus-containing polymers, like polyphosphates, polyphosphonates, polyphosphites and polyphosphoramides, which have found important biomedical applications, such as in drug and gene delivery systems, tissue engineering and biosensors.1–4 The polyphoshoesters possess hydrolitically unstable phosphoester linkages in the polymer backbones. These polymers degrade into nontoxic products through hydrolysis and enzymatic cleavage of their phosphoester bonds under physiological conditions.5,6 The pentavalency of the phosphorus atom from ⇑ Corresponding author. Tel.: +359 2 979 6633; fax: +359 2 870 0309. E-mail addresses: [email protected] (I. Kraicheva), [email protected] (E. Vodenicharova), [email protected] (S. Shenkov), [email protected] (E. Tashev), [email protected] (T. Tosheva), [email protected] (I. Tsacheva), [email protected] (A. Kril), [email protected] (M. Topashka-Ancheva), [email protected] (A. Georgieva), [email protected] (I. Iliev), [email protected] (I. Vladov), [email protected] (Ts. Gerasimova), ktroev@ polymer.bas.bg (K. Troev). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.12.001
the repeating P–O–C bonds allows numerous functional pendant groups to be introduced to the polymer main chain, that give much possibilities for further modifications of the polymers, including drug conjugation.1,7,8 The polyphosphoesters could serve as hydrophilic segments in amphiphilic block copolymers.9,10 Polyphosphoester-based copolymers are used as drug carriers for Doxorubicin and Paclitaxel delivery.9,11,12 The polyesters of the H-phosphonic acid, the poly(alkylene H-phosphonate)s, are one of the most promising drug carriers among this class of polymers.13 They contain highly reactive P–H groups in the repeating units, which can conjugate covalently low-molecular weight drugs in mild reaction conditions. The presence of highly polar repeating P@O groups enables for physical immobilization of drugs.14 Furthermore the poly(alkylene H-phosphonate)s are able to generate themselves new biologically active substances, like aminophosphonate fragments, connected to their polymer backbones.15,16 Using this approach, new type of polymer prodrugs, as well as new polymer drug carriers can be obtained. The aminophosphonic acid derivatives are considered to be phosphorus analogues of natural amino acids and are widely used as peptidomimetics, enzyme inhibitors,
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I. Kraicheva et al. / Bioorg. Med. Chem. 22 (2014) 874–882
haptens of catalytic antibodies, antitumor and antiviral agents.17–21 The introduction of aminophosphonate moieties, bearing DNA intercalating anthracene ring, to the biodegradable polymer carriers deserve particular attention in the development of new antitumor therapeuticals with improved properties. The anthracene-derived compounds exhibit a broad spectrum of antitumor activity and play a major role as chemotherapeutic agents in cancer therapy, act as inhibitors of tubulin polymerization and find valuable bioanalytical applications as chromophores for fluorescence measurements.22–25 Earlier, we described the synthesis, antitumor activity and genotoxicity of some poly(oxyethylene aminophosphonate)s.15,16,26 Here we report the synthesis, characterization, in vitro antitumor activity and safety, as well as the clastogenic and antimitotic potential in vivo of two novel poly(oxyethylene aminophosphonate)s and two poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s, containing anthracene rings. The fluorescent properties of the anthracene moieties have been used for studies on the subcellular distribution of the polymers in tumor and normal cells. The antitumor activity and safety testing of the low-molecular analogues of the polymers and their cellular distribution have been previously reported.27 2. Results and discussion 2.1. Chemistry Novel polyphosphoesters, namely poly(oxyethylene aminophosphonate)s 4 and 5 (Scheme 1) and poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s 6 and 7 (Scheme 2) have been synthesized via an addition reaction of poly(oxyethylene H-phosphonate)s 1 and 2 to the anthracene derived Schiff base 9-anthrylidene-p-toluidine 3. The polymer analogous reaction was carried out in the presence of catalytic amount of CdI2
O P
H3CO
H
O
O
O
O
O
x P H
O
n
HC
O H3CO
P
O
O
CHNH
CH3
O x P
1, 2
P x
H
OH
CH3 3
N
O O n
CHNH
O
CH3
P x
OH
CHNH
4, 5
CH3
x=13 (PEG 600) : 1, 4 x= 4 (PEG 200) : 2, 5 Scheme 1. Synthesis of poly(oxyethylene aminophosphonate)s 4 and 5. Reagents and conditions: 4, poly(oxyethylene H-phosphonate) (Mn = 8300 Da), 9-anthrylidene-p-toluidine, toluene, CdI2, 80 °C, 25 h; 5, poly(oxyethylene H-phosphonate) (Mn = 3200 Da), 9-anthrylidene-p-toluidine, benzene, CdI2, reflux, 23 h.
O
O
O
O O
x P
O
m CHNH
P x
H
l
CH3 6, 7
m+l=n
x=13 (PEG 600) : 6 x= 4 (PEG 200) : 7 Scheme 2. Poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s 6 and 7: repeating units. Reagents and conditions: 6, poly(oxyethylene H-phosphonate) (Mn = 8300 Da), 9-anthrylidene-p-toluidine, toluene, 60 °C, 26 h; 7, poly(oxyethylene H-phosphonate) (Mn = 3200 Da), 9-anthrylidene-p-toluidine, benzene, CdI2, reflux, 4 h.
(polymers 4, 5 and 7) and without a catalyst (copolymer 6). Polymers 4–6 were obtained using an excess of the Schiff base, while in the case of 7 the reaction was conducted with an excess of the polymeric H-phosphonate. The details of the synthetic procedures are described in the Section 4. The products were obtained in good yields as viscous oils (4, 6 and 7) or a powder (5). All polymers (4–7) are soluble in common organic solvents: benzene, toluene, chloroform and ethanol. The copolymers 6 and 7 are water soluble. The structure of the polymers was elucidated by means of IR and NMR (1H, 13C and 31P) spectroscopy. The IR spectra showed the expected28,29 absorption bands for stretching vibrations of NH, P@O and PAOAC groups in polymers 4–7, as well as for stretching vibration of a P–H group in copolymers 6 and 7. In the 1 H NMR spectra of the polymers, a doublet signal appears at 6.40 (4), 6.38 (5), 6.40 (6) and 6.41 (7) ppm with a PH two-bond coupling of 27.4–31.8 Hz, which can be assigned to the proton from CHP fragment formed by addition reaction between the polymeric H-phosphonates and the Schiff base. The signals for the aromatic (ArH0 ) and anthracene (AnthH) protons of 4–7 are found in the expected regions of the spectra.27,30 1H NMR spectra of both copolymers 6 and 7 reveal a doublet signal at 6.97 ppm with 1JPH of 715.9 and 715.8 Hz, respectively, for the PH proton from H-phosphonate repeating units and a doublet at 6.84 (6) and 6.87 (7) ppm for the PH proton from PH(O)OH end groups. 13C{1H} NMR spectra of 4–7 exhibit doublets with large coupling constants of 154.3–156.2 Hz, which belong to the carbon atoms from CHP fragments in the polymer backbone. The signals of the three types of methylene carbons (OCH2CH2, POCH2 and POCH2CH2) from the aminophosphonate (4–7) and H-phosphonate (6 and 7) repeating units were observed in the spectra. The assignment of the anthracene carbons in the polymers 4–7 is based on the analysis of their HSQC spectra, and is in accordance with the data obtained for low-molecular anthracene-derived aminophosphonates.27 31P{1H} NMR spectra of the polymers display singlet signals in the regions of 24.55–24.63 and 9.34–9.47 ppm, which belong to the phosphorus atoms of CHP and PH groups from the aminophosphonate (4–7) and H-phosphonate (4, 6 and 7) repeating units, respectively (Fig. 1). Singlet signals for the phosphorus atom of CHP(O)OH (4, 5 and 6), PH(O)OCH3 (6 and 7) and PH(O)OH (4, 6 and 7) end groups appear in the spectra. In the case of 4, the signal at 9.35 ppm assigned to the PH phosphorus atom of the H-phosphonate repeating unit, is with very low intensity. Such signal was not observed in the spectrum of polymer 5. The monoester end groups CHP(O)OH are formed by the addition of the PH(O)OH end groups of the starting
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I. Kraicheva et al. / Bioorg. Med. Chem. 22 (2014) 874–882
Figure 1.
31
P{1H} NMR spectrum of poly(oxyethylene aminophosphonate) 4 in CDCl3.
polymers, to the azomethine. The addition of the PH group of phosphonic acid monoesters to Schiff bases has already been reported.16,31 The phosphorus signal of nonreacted PH(O)OH groups is found upfield in the spectra (6.10–6.94 ppm), which suggests that these groups possess a ionic structure. It is noteworthy that in the 31P{1H} NMR spectra of the polymers 4–7, the signals of the phosphorus from CHP diester end groups appear as two distinct singlets with nearly equal intensity and very close chemical shifts (the chemical shift differences are of 0.07 ppm). This indicates that in the polymers 4–7, more than one diastereomeric species exist. The average degree of polymerization of the synthesized polymers n (Scheme 1) and m + l (Scheme 2) and the degree of polymerization of the starting polymers n, estimated on the basis of the 31P{1H} NMR spectra32, is equal: n = 12 and m + l = 12. The content of the aminophosphonate units in the polymers 4–7, calculated from the 31P{1H} NMR spectra, is as follows: 90% (4), 100% (5), 43% (6) and 13% (7). The ratio between the aminophosphonate and the H-phosphonate units was controlled by varying the reaction conditions. Thus, the copolymers 6 and 7 were obtained with predominant content of hydrophilic H-phosphonate units, which increases their solubility in water. The fluorescence emission spectra of the polymers 4–7 were recorded at two excitation wavelengths of 375 and 395 nm and the emission maxima appear in the regions from 480 to 512 nm and from 485 to 515 nm, respectively, that is, in the blue—blue-green spectral range (Figs. S1 and S2). The mass spectra of the polymers 4–7 show that they undergo fragmentation processes. The break of the C–P bond of the anthracene-containing aminophosphonate fragment (b-bond towards the anthracene ring) lead to the ion C22H18N+, which forms the basic peak at m/z = 296.08–296.17 (C22H18N+, calcd 296.39). Many other peaks that differed with m/z = 44 (CH2CH2O+, calcd 44.05) have been observed in the range of m/z = 177.08–1753.95, due to fragmentation of the polymeric chains.
and cervix. The copolymers exerted concentration-dependent antiproliferative effects after 24 and 72 h exposure which enabled the construction of concentration–response curves (not shown) and the calculation of the corresponding IC50 values. As evident from the cytotoxicity data the polymer 7 proved to be more potent than the polymer 6 after 24 h treatment and appeared to be the most active cytotoxic agent towards colostrum-derived myoepithelial cell line HBL-100. In addition, the cytotoxic potential of the same polymer was comparable to that of the positive control substance Doxorubicin, used in the experiments, when tested on cell lines MDA-MB-231 (highly metastatic carcinoma of the breast) and HepG2 (hepatocellular carcinoma). In comparison to the activity of the positive control drug, the effect of 7 on tumor cell lines 647-V, MCF-7 and HeLa was two, three and four times lower, respectively. The colon carcinoma cells (HT-29 line) were found to be resistant to the antitumor activity of both tested polymers (Table 1). The prolongation of the incubation time for 72 h resulted in an enhancement of the in vitro antitumor activity of both tested substances. The cytotoxicity of 6 towards cell lines MCF-7, MDA-MB231 and HeLa was approximately two times higher and for the cell line HepG2 three times higher than values obtained after 24 h treatment. No alterations of IC50 values were noted after treatment
Table 1 Comparative cytotoxic activity of copolymers 6 and 7 versus referent substance doxorubicin in a panel of human tumor cell lines after 24 h treatment (MTT-dye reduction assay)
MCF-7 MDA-MB-231 HBL-100 HepG2 HT-29 647-V HeLa BALB/c 3T3
2.2. In vitro antitumor activity The copolymers 6 and 7 were tested for cytotoxicity on a panel of seven human cancer cell lines representative of important solid epithelial tumors of the breast, liver, colon, urinary bladder
Mean IC50 valuesa (mg/ml)
Cell lines
a
Compound 6
Compound 7
Doxorubicin
>1.000 >1.000 >1.000 >1.000 >1.000 >1.000 >1.000 >1.000
0.199 ± 0.020 0.089 ± 0.001 0.118 ± 0.005 0.094 ± 0.003 >1.000 0.114 ± 0.022 0.286 ± 0.003 0.179 ± 0.005
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis, characterization, antitumor activity and safety testing of novel polyphosphoesters bearing anthracene-derived aminophosphonate units I. Kraicheva a,⇑, E. Vodenicharova a, S. Shenkov a, E. Tashev a, T. Tosheva a, I. Tsacheva a, A. Kril b, M. Topashka-Ancheva c, A. Georgieva b, I. Iliev b, I. Vladov b, Ts. Gerasimova c, K. Troev a a
Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 103A, 1113 Sofia, Bulgaria Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 25, 1113 Sofia, Bulgaria c Institute of Biodiversity and Ecosystems Research, Bulgarian Academy of Sciences, 2 Gagarin Str., 1113 Sofia, Bulgaria b
a r t i c l e
i n f o
Article history: Received 12 September 2013 Revised 11 November 2013 Accepted 2 December 2013 Available online 8 December 2013 Keywords: Polyphosphoesters Aminophosphonic acids NMR Antitumor activity Cytotoxicity Genotoxicity
a b s t r a c t Novel polyphosphoesters containing anthracene-derived aminophosphonate units, poly(oxyethylene aminophosphonate)s (4 and 5) and poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s (6 and 7), were synthesized via an addition of poly(oxyethylene H-phosphonate)s to 9-anthrylidene-p-toluidine. The IR, NMR (1H, 13C and 31P) and fluorescence emission spectral data of the polymers are presented. The copolymers 6 and 7 were tested for in vitro antitumor activity on a panel of seven human epithelial cancer cell lines. Safety testing was performed both in vitro (3T3 NRU test) and in vivo on ICR mice for genotoxicity and antiproliferative activity. The copolymer 7 showed excellent antiproliferative activity to HBL100, MDA-MB-231, MCF-7 and HepG2 cell lines. However, the in vitro safety testing revealed significant toxicity to Balb/c 3T3 mouse embryo cells. In contrast, the copolymer 6 showed complete absence of cytotoxicity to Balb/c 3T3 cells, but inhibited the growth of breast cancer cells, cervical carcinoma cells (HeLa) and hepatocellular carcinoma cell cultures after prolonged (72 h) exposure. The polymers (4–6) exhibited low (4 and 6) to moderate (5) clastogenicity in vivo and slightly inhibited bone marrow cell division, compared to Mitomycin C. The subcellular distribution of the copolymers 6 and 7 were studied in model cell culture systems. The tested polyphosphoesters are expected to act in vivo as prodrugs of aminophosphonates and could be valuable as a new class of biodegradable polymer drug carriers. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The polyphosphoesters comprise interesting groups of biodegradable and biocompatible phosphorus-containing polymers, like polyphosphates, polyphosphonates, polyphosphites and polyphosphoramides, which have found important biomedical applications, such as in drug and gene delivery systems, tissue engineering and biosensors.1–4 The polyphoshoesters possess hydrolitically unstable phosphoester linkages in the polymer backbones. These polymers degrade into nontoxic products through hydrolysis and enzymatic cleavage of their phosphoester bonds under physiological conditions.5,6 The pentavalency of the phosphorus atom from ⇑ Corresponding author. Tel.: +359 2 979 6633; fax: +359 2 870 0309. E-mail addresses: [email protected] (I. Kraicheva), [email protected] (E. Vodenicharova), [email protected] (S. Shenkov), [email protected] (E. Tashev), [email protected] (T. Tosheva), [email protected] (I. Tsacheva), [email protected] (A. Kril), [email protected] (M. Topashka-Ancheva), [email protected] (A. Georgieva), [email protected] (I. Iliev), [email protected] (I. Vladov), [email protected] (Ts. Gerasimova), ktroev@ polymer.bas.bg (K. Troev). 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.12.001
the repeating P–O–C bonds allows numerous functional pendant groups to be introduced to the polymer main chain, that give much possibilities for further modifications of the polymers, including drug conjugation.1,7,8 The polyphosphoesters could serve as hydrophilic segments in amphiphilic block copolymers.9,10 Polyphosphoester-based copolymers are used as drug carriers for Doxorubicin and Paclitaxel delivery.9,11,12 The polyesters of the H-phosphonic acid, the poly(alkylene H-phosphonate)s, are one of the most promising drug carriers among this class of polymers.13 They contain highly reactive P–H groups in the repeating units, which can conjugate covalently low-molecular weight drugs in mild reaction conditions. The presence of highly polar repeating P@O groups enables for physical immobilization of drugs.14 Furthermore the poly(alkylene H-phosphonate)s are able to generate themselves new biologically active substances, like aminophosphonate fragments, connected to their polymer backbones.15,16 Using this approach, new type of polymer prodrugs, as well as new polymer drug carriers can be obtained. The aminophosphonic acid derivatives are considered to be phosphorus analogues of natural amino acids and are widely used as peptidomimetics, enzyme inhibitors,
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I. Kraicheva et al. / Bioorg. Med. Chem. 22 (2014) 874–882
haptens of catalytic antibodies, antitumor and antiviral agents.17–21 The introduction of aminophosphonate moieties, bearing DNA intercalating anthracene ring, to the biodegradable polymer carriers deserve particular attention in the development of new antitumor therapeuticals with improved properties. The anthracene-derived compounds exhibit a broad spectrum of antitumor activity and play a major role as chemotherapeutic agents in cancer therapy, act as inhibitors of tubulin polymerization and find valuable bioanalytical applications as chromophores for fluorescence measurements.22–25 Earlier, we described the synthesis, antitumor activity and genotoxicity of some poly(oxyethylene aminophosphonate)s.15,16,26 Here we report the synthesis, characterization, in vitro antitumor activity and safety, as well as the clastogenic and antimitotic potential in vivo of two novel poly(oxyethylene aminophosphonate)s and two poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s, containing anthracene rings. The fluorescent properties of the anthracene moieties have been used for studies on the subcellular distribution of the polymers in tumor and normal cells. The antitumor activity and safety testing of the low-molecular analogues of the polymers and their cellular distribution have been previously reported.27 2. Results and discussion 2.1. Chemistry Novel polyphosphoesters, namely poly(oxyethylene aminophosphonate)s 4 and 5 (Scheme 1) and poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s 6 and 7 (Scheme 2) have been synthesized via an addition reaction of poly(oxyethylene H-phosphonate)s 1 and 2 to the anthracene derived Schiff base 9-anthrylidene-p-toluidine 3. The polymer analogous reaction was carried out in the presence of catalytic amount of CdI2
O P
H3CO
H
O
O
O
O
O
x P H
O
n
HC
O H3CO
P
O
O
CHNH
CH3
O x P
1, 2
P x
H
OH
CH3 3
N
O O n
CHNH
O
CH3
P x
OH
CHNH
4, 5
CH3
x=13 (PEG 600) : 1, 4 x= 4 (PEG 200) : 2, 5 Scheme 1. Synthesis of poly(oxyethylene aminophosphonate)s 4 and 5. Reagents and conditions: 4, poly(oxyethylene H-phosphonate) (Mn = 8300 Da), 9-anthrylidene-p-toluidine, toluene, CdI2, 80 °C, 25 h; 5, poly(oxyethylene H-phosphonate) (Mn = 3200 Da), 9-anthrylidene-p-toluidine, benzene, CdI2, reflux, 23 h.
O
O
O
O O
x P
O
m CHNH
P x
H
l
CH3 6, 7
m+l=n
x=13 (PEG 600) : 6 x= 4 (PEG 200) : 7 Scheme 2. Poly[oxyethylene(aminophosphonate-co-H-phosphonate)]s 6 and 7: repeating units. Reagents and conditions: 6, poly(oxyethylene H-phosphonate) (Mn = 8300 Da), 9-anthrylidene-p-toluidine, toluene, 60 °C, 26 h; 7, poly(oxyethylene H-phosphonate) (Mn = 3200 Da), 9-anthrylidene-p-toluidine, benzene, CdI2, reflux, 4 h.
(polymers 4, 5 and 7) and without a catalyst (copolymer 6). Polymers 4–6 were obtained using an excess of the Schiff base, while in the case of 7 the reaction was conducted with an excess of the polymeric H-phosphonate. The details of the synthetic procedures are described in the Section 4. The products were obtained in good yields as viscous oils (4, 6 and 7) or a powder (5). All polymers (4–7) are soluble in common organic solvents: benzene, toluene, chloroform and ethanol. The copolymers 6 and 7 are water soluble. The structure of the polymers was elucidated by means of IR and NMR (1H, 13C and 31P) spectroscopy. The IR spectra showed the expected28,29 absorption bands for stretching vibrations of NH, P@O and PAOAC groups in polymers 4–7, as well as for stretching vibration of a P–H group in copolymers 6 and 7. In the 1 H NMR spectra of the polymers, a doublet signal appears at 6.40 (4), 6.38 (5), 6.40 (6) and 6.41 (7) ppm with a PH two-bond coupling of 27.4–31.8 Hz, which can be assigned to the proton from CHP fragment formed by addition reaction between the polymeric H-phosphonates and the Schiff base. The signals for the aromatic (ArH0 ) and anthracene (AnthH) protons of 4–7 are found in the expected regions of the spectra.27,30 1H NMR spectra of both copolymers 6 and 7 reveal a doublet signal at 6.97 ppm with 1JPH of 715.9 and 715.8 Hz, respectively, for the PH proton from H-phosphonate repeating units and a doublet at 6.84 (6) and 6.87 (7) ppm for the PH proton from PH(O)OH end groups. 13C{1H} NMR spectra of 4–7 exhibit doublets with large coupling constants of 154.3–156.2 Hz, which belong to the carbon atoms from CHP fragments in the polymer backbone. The signals of the three types of methylene carbons (OCH2CH2, POCH2 and POCH2CH2) from the aminophosphonate (4–7) and H-phosphonate (6 and 7) repeating units were observed in the spectra. The assignment of the anthracene carbons in the polymers 4–7 is based on the analysis of their HSQC spectra, and is in accordance with the data obtained for low-molecular anthracene-derived aminophosphonates.27 31P{1H} NMR spectra of the polymers display singlet signals in the regions of 24.55–24.63 and 9.34–9.47 ppm, which belong to the phosphorus atoms of CHP and PH groups from the aminophosphonate (4–7) and H-phosphonate (4, 6 and 7) repeating units, respectively (Fig. 1). Singlet signals for the phosphorus atom of CHP(O)OH (4, 5 and 6), PH(O)OCH3 (6 and 7) and PH(O)OH (4, 6 and 7) end groups appear in the spectra. In the case of 4, the signal at 9.35 ppm assigned to the PH phosphorus atom of the H-phosphonate repeating unit, is with very low intensity. Such signal was not observed in the spectrum of polymer 5. The monoester end groups CHP(O)OH are formed by the addition of the PH(O)OH end groups of the starting
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I. Kraicheva et al. / Bioorg. Med. Chem. 22 (2014) 874–882
Figure 1.
31
P{1H} NMR spectrum of poly(oxyethylene aminophosphonate) 4 in CDCl3.
polymers, to the azomethine. The addition of the PH group of phosphonic acid monoesters to Schiff bases has already been reported.16,31 The phosphorus signal of nonreacted PH(O)OH groups is found upfield in the spectra (6.10–6.94 ppm), which suggests that these groups possess a ionic structure. It is noteworthy that in the 31P{1H} NMR spectra of the polymers 4–7, the signals of the phosphorus from CHP diester end groups appear as two distinct singlets with nearly equal intensity and very close chemical shifts (the chemical shift differences are of 0.07 ppm). This indicates that in the polymers 4–7, more than one diastereomeric species exist. The average degree of polymerization of the synthesized polymers n (Scheme 1) and m + l (Scheme 2) and the degree of polymerization of the starting polymers n, estimated on the basis of the 31P{1H} NMR spectra32, is equal: n = 12 and m + l = 12. The content of the aminophosphonate units in the polymers 4–7, calculated from the 31P{1H} NMR spectra, is as follows: 90% (4), 100% (5), 43% (6) and 13% (7). The ratio between the aminophosphonate and the H-phosphonate units was controlled by varying the reaction conditions. Thus, the copolymers 6 and 7 were obtained with predominant content of hydrophilic H-phosphonate units, which increases their solubility in water. The fluorescence emission spectra of the polymers 4–7 were recorded at two excitation wavelengths of 375 and 395 nm and the emission maxima appear in the regions from 480 to 512 nm and from 485 to 515 nm, respectively, that is, in the blue—blue-green spectral range (Figs. S1 and S2). The mass spectra of the polymers 4–7 show that they undergo fragmentation processes. The break of the C–P bond of the anthracene-containing aminophosphonate fragment (b-bond towards the anthracene ring) lead to the ion C22H18N+, which forms the basic peak at m/z = 296.08–296.17 (C22H18N+, calcd 296.39). Many other peaks that differed with m/z = 44 (CH2CH2O+, calcd 44.05) have been observed in the range of m/z = 177.08–1753.95, due to fragmentation of the polymeric chains.
and cervix. The copolymers exerted concentration-dependent antiproliferative effects after 24 and 72 h exposure which enabled the construction of concentration–response curves (not shown) and the calculation of the corresponding IC50 values. As evident from the cytotoxicity data the polymer 7 proved to be more potent than the polymer 6 after 24 h treatment and appeared to be the most active cytotoxic agent towards colostrum-derived myoepithelial cell line HBL-100. In addition, the cytotoxic potential of the same polymer was comparable to that of the positive control substance Doxorubicin, used in the experiments, when tested on cell lines MDA-MB-231 (highly metastatic carcinoma of the breast) and HepG2 (hepatocellular carcinoma). In comparison to the activity of the positive control drug, the effect of 7 on tumor cell lines 647-V, MCF-7 and HeLa was two, three and four times lower, respectively. The colon carcinoma cells (HT-29 line) were found to be resistant to the antitumor activity of both tested polymers (Table 1). The prolongation of the incubation time for 72 h resulted in an enhancement of the in vitro antitumor activity of both tested substances. The cytotoxicity of 6 towards cell lines MCF-7, MDA-MB231 and HeLa was approximately two times higher and for the cell line HepG2 three times higher than values obtained after 24 h treatment. No alterations of IC50 values were noted after treatment
Table 1 Comparative cytotoxic activity of copolymers 6 and 7 versus referent substance doxorubicin in a panel of human tumor cell lines after 24 h treatment (MTT-dye reduction assay)
MCF-7 MDA-MB-231 HBL-100 HepG2 HT-29 647-V HeLa BALB/c 3T3
2.2. In vitro antitumor activity The copolymers 6 and 7 were tested for cytotoxicity on a panel of seven human cancer cell lines representative of important solid epithelial tumors of the breast, liver, colon, urinary bladder
Mean IC50 valuesa (mg/ml)
Cell lines
a
Compound 6
Compound 7
Doxorubicin
>1.000 >1.000 >1.000 >1.000 >1.000 >1.000 >1.000 >1.000
0.199 ± 0.020 0.089 ± 0.001 0.118 ± 0.005 0.094 ± 0.003 >1.000 0.114 ± 0.022 0.286 ± 0.003 0.179 ± 0.005
