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European Journal of Medicinal Chemistry 72 (2014) 127e136
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European Journal of Medicinal Chemistry
journal homepage: http: / /www.elsevier .com/locate/ejmech
Original article
Polyphosphoester conjugates of dinuclear platinum complex:Synthesis and evaluation of cytotoxic and the proapoptotic activity
Violeta Mitova a, Stoyanka Slavcheva a, Pavletta Shestakova b, Denitsa Momekova c,Nikolay Stoyanov d, Georgi Momekov e,*, Kolio Troev a, Neli Koseva a
a Institute of Polymers, Bulgarian Academy of Sciences, Acad. Georgi Bonchev Str. Bl.103, 1113 Sofia, Bulgariab Laboratory of Nuclear Magnetic Resonance, Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad.Georgi Bonchev Str. Bl. 9, 1113 Sofia, BulgariacDepartment of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgariad Laboratory of Hematopathology and Immunology, National Specialized Hospital for Active Treatment of Hematological Diseases, 6 Plovdivsko pole Str.,1756 Sofia, BulgariaeDepartment of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria
a r t i c l e i n f o
Article history:Received 22 July 2013Received in revised form22 October 2013Accepted 11 November 2013Available online 11 December 2013
Keywords:Dinuclear platinum complexPolyphosphoester conjugateDOSY NMRCytotoxicityProapoptotic activityRenal epithelial cellsNephrotoxicity
Abbreviations: DMPh, dimethyl H-phosphonate; DNMR spectroscopy.* Corresponding author. Tel.: þ359 2 9236 509; fax
E-mail addresses: [email protected] (V. Mitobg (S. Slavcheva), [email protected] (P. Shestakov(D. Momekova), [email protected] (N. Stoyan(G. Momekov), [email protected] (K. Troe(N. Koseva).
0223-5234/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2013.11.014
a b s t r a c t
Macromolecular conjugates of a dinuclear platinum complex with a spermidine bridge were synthesizedusing poly(oxyethylene H-phosphonate)s as precursor polymer. The complex species were attached tothe polymer chain via a phosphoramide bond resulting from the reaction between the H-phosphonategroups and the middle amino group of the spermidine moiety. 1H and 31P{H} DOSY NMR spectral datawere used to prove the conjugation reaction and to characterize the new species. The conjugatesexhibited profound cytotoxicity in a panel of five chemosensitive human tumor cell lines and onecisplatin-resistant model (HL-60/CDDP), and were found to induce apoptotic cell death. A flow cyto-metric analysis encountered a cisplatin-dissimilar modulation of the cell cycle progression in KG-1leukemic cells, following exposure to the dinuclear agents. Moreover, the novel compounds displayedless pronounced inhibitory activity against cultured murine renal epithelial cells, as compared tocisplatin.
� 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
New platinum drugs are continuously emerging with the aim toexpand the spectrum of activity of cisplatin, or to reduce the sideeffects and cell resistance. Farrell et al. [1] synthesized a series ofmultinuclear platinum(II) complexes, which formDNA adducts thatdiffer markedly in structure, sequence specificity, and formationkinetics from those generated by cisplatin and its mononuclearanalogs. The multinuclear platinum(II) complexes with amine li-gands are cationic in nature and show excellent solubility in water.
OSY NMR, diffusion ordered
: þ359 2 9879 874.va), [email protected]), [email protected]), [email protected]), [email protected]
son SAS. All rights reserved.
More importantly, they display high antitumor activity in vitro andin vivo against cisplatin sensitive and cisplatin-resistant tumor celllines [2]. Some of these complexes are active at nanomolar con-centrations. The increased cytotoxicity is explained with the abilityfor pre-covalent association and the formation of long-range ad-ducts with DNA [3,4]. However, clinical trials have shown that thetrinuclear complex BBR3464, and other multinuclear platinumdrugs, did not yield results substantially different from cisplatin,possibly due to their binding and degradation by human plasmaproteins [5,6]. Therefore research efforts have been directed toreduce the high irreversible plasma protein binding and improvethe chemical andmetabolic stability, as well as to decrease the toxiceffects that led to failure in clinical trials. Different approaches havebeen applied such as design of new derivatives with chloride li-gands replaced by alkylcarboxylates [7] encapsulation into lipo-somes (the cisplatin liposomal formulation Lipoplatin is underclinical trials [8]), prodrug delivery, i.e. application of less toxic andbetter tolerated derivatives of the active species that are subse-quently converted to the pharmacologically active agent [9,10].
V. Mitova et al. / European Journal of Medicinal Chemistry 72 (2014) 127e136128
An alternative approach is drug conjugation to a macromolec-ular carrier. It is a widely explored strategy especially in the field ofcancer therapy. A significant number of polymer-bound antitumoragents have been developed so far including taxol [11], doxorubicin[12], camptothecin [13], cisplatin [14,15] and oxaliplatin [16].Polymers such as poly(ethylene glycol) (PEG) [17], poly(-hydroxypropylmethacrylamide) [18], etc. have been used for drugconjugation. Poly(oxyethylene H-phosphonate)s present a family ofbiodegradable, hydrophilic, and nontoxic polymers, which mimicbiomacromolecules. These polymers contain PEG segments asbuilding blocks linked byH-phosphonate groupse reactive sites forconjugation of amino- or hydroxyl compounds under mild reactionconditions. All these valuable features make poly(oxyethyleneH-phosphonate)s promising candidates for immobilization tem-plates [19].
Based on our experience in synthesis and application of poly(-oxyalkylene H-phosphonate)s as drug carriers [20e22] we under-went a study on conjugation of a dinuclear platinum complex withspermidine acting as a bridge between the platinum centers.Among the “non-classical” multinuclear platinum(II) complexes,the dinuclear platinum complexes containing a charged polyaminelinker are exceptionally potent compounds as they show high ac-tivity against tumor cell lines resistant to cisplatin [2]. On the otherside, citotoxicity in the nano/micromolar range might result in arelatively narrow therapeutic index that could limit the clinicalapplication of these compounds. Less toxic low molecular pro-drugs were synthesized via introduction of cleavable carbamateor amide groups at the middle nitrogen of the spermidine linker[23,24].
Herewe report the preparation ofmacromolecular conjugates ofthe dinuclear platinum complex with a spermidine bridge andpoly(oxyethylene H-phosphonate)s. The aim is to obtain a macro-molecular pro-drug form where the polymer component has adouble function e through conjugation to decrease the toxicity ofthe bioactive complex and secondly, to protect it against bindingand degradation by human plasma proteins. The complex specieswere attached to the polymer chain via a phosphoramide bondresulting from the reaction between the H-phosphonate groups
Fig. 1. Synthesis of low molecular conjugate e complexes
and the middle amino group of the spermidine moiety. The newconjugates were characterized by multinuclear and diffusion or-dered NMR spectroscopy. The conjugates exhibited profoundcytotoxicity, albeit inferior than that of the low molecular weightprototype, indicative for their pro-drug properties. Noteworthy, thenovel compounds proved to bypass the resistance mechanisms in acisplatin-resistant cell line. A flow cytometric analysis of KG-1 cellsconfirmed the cisplatin-dissimilar mode of action of the dinuclearagent and its conjugates conditioned by their distinct chemicalstructure. Moreover, the presented compounds displayed lowernephrotoxic potential in an in vitro test system, as compared to thereference anticancer agent.
2. Results and discussion
2.1. Chemistry
The 1,1/t,t-spermidine dinuclear platinum complex, [{trans-PtCl(NH3)2}2{m-spermidine-N1,N8}]Cl3, was prepared by knownsynthetic pathways [25]. This complex is soluble in water and notsoluble in polar organic solvents. The conjugation reaction with H-phosphonates requires a water free-medium. To increase the sol-ubility of the spermidine bridged platinum complex in polar sol-vents such as DMF, DMSO, etc. the chloride counterions werereplaced with nitrate ones. The compound is assigned as complex(1).
Diesters of the H-phosphonic acid react with amines in thepresence of tetrachloromethane (CCl4) and triethylamine (Et3N) atroom temperature known as the AthertoneTodd reaction [26,27].The model reaction between 1 and the dimethyl H-phosphonate(DMPh) in mole ratio 1:2, respectively, was performed in anhy-drous DMF and with the addition of NaOH (dissolved in methanol)in an equimolar ratio to 1 to convert the middle amino group of thespermidine chain from a salt to basic form (Fig. 1). The drugconjugation via the formation of a phosphoramide bond wasconfirmed by the signal at 14.96 ppm in the 31P NMR spectrum. Thesecond signal at 3.73 ppm which appeared as septet in the samespectrum corresponds to a phosphate anion (CH3O)2P(O)O�
2 and polyphosphorester-drug conjugates 4a and 4b.
Fig. 2. 31P{H} DOSY NMR spectrum of 2.
V. Mitova et al. / European Journal of Medicinal Chemistry 72 (2014) 127e136 129
obtained due to NaOH and the excess of DMPh added to the reac-tion mixture. The integrated intensities ratio of the two signals is1:1 which implies completion of the conjugation reaction(Suppl. 1). The assignment of the chemical shifts correspond to thedata in literature [28,29]. The phosphate anion was isolated as acounterion of the targeted reaction product.
The conjugation between 1 and DMPh was additionally provenby 31P DOSY NMR (Fig. 2). The spectrum shows two peaks withdifferent diffusion coefficients that imply the presence of twospecies containing phosphorus in the solution. The diffusion coef-ficient of the signal at 14.96 ppm, indicative for the formation of PeN bond, is 3.80� 10�10 m2/s. This value is lower as compared to thediffusion coefficients measured in the individual solutions of thedinuclear platinum(II) complex 1 andDMPh. The solvated dimethylphosphate anion (the signal at 3.73 ppm) displays a diffusion co-efficient of 6.60 � 10�10 m2/s. The preparation of complex 2 can beregarded as a reaction pathway to a lowmolecular weight pro-drugform and also as a model reaction to obtaining drug-polymerconjugates of 1.
The poly(oxyethylene H-phosphonate)s were obtained via pol-ytransesterification of dimethyl H-phosphonate and poly(ethyleneglycol)s with number average molecular weight 200 g/mol (PEG200) (3a) and 600 g/mol (PEG 600) (3b) following a proceduredescribed elsewhere [20].
The structure of the two polymers (Fig. 3) was confirmed by 1H,13C and 31P NMR spectra. The average degree of polymerization (n)was estimated from 1H and 31P NMR spectra of the polymers. Thenumber average molecular weight was calculated: 3500 g/mol for3a (n¼ 13) and 7100 g/mol for 3b (n¼ 10). The polydispersity of thesynthesized polyphosphoesters 3a and 3b was 1.3.
New polyphosphoester pro-drugs 4a and 4b were synthesizedby conjugating the dinuclear platinum(II) complex 1 with poly(-oxyethylene H-phosphonate)s 3a and 3b under the conditions usedfor the model reaction (Fig. 1). The conjugates were characterized
Fig. 3. Poly(oxyethylene H-phosphonate)s: 3a and 3b.
by multinuclear 1H, 13C and 31P NMR spectroscopy. The 1H and 13CNMR spectra (Suppl. 2) displayed signals of the two components ofthe conjugates e the spermidine bridged platinum complex andthe polymer. The conjugates formation was also evidenced by the31P NMR spectral data e the signal at 12.2 ppmwhich is assigned tothe formation of PeN bond, i.e. the complex was bound to thepolymer chain through a covalent PeN bond (see the ExperimentalSection, as well as Suppl. 3 and Suppl. 4). The integral intensities ofthe signals for the P atoms in the phosphoramide groups (in thespectral region 14-12 ppm) and those in the region of the phos-phate groups (at 2.5 ppm for methyl phosphate groups and at 1.2e1.3 ppm for hydroxyphosphate groups) were used for determina-tion of the compositions of the conjugates. The composition of eachpolymer conjugate was expressed via the mole fractions (in %) ofthe three structural units (Fig. 1) in the polymer chain obtained as aresult of the conjugation reaction. It is seen that 4a contains 44 mol% reactive centers bound to platinum(II) complex, while for theother conjugate - 30 mol% of the phophorous centers of 4b bearspermidine bridged complex. The experimental data from theelemental analysis confirmed the composition of the conjugates 4aand 4b (Suppl. 5).
The 1H and 31P DOSY NMR spectroscopy was also used to provethe conjugation reaction between the polymer carriers (3a and 3b)and the bioactive agent (1) as well as to determine the size and themobility of the obtained new entities. Further, assuming sphericalshape approximation the apparent hydrodynamic radius, Rh, of thepolymer particles can be estimated using the StokeseEinsteinequation and the obtained value of the diffusion coefficient. Asinitial step DOSY spectra of solutions of 3a and 3b poly-phosphoesters in D2O were measured (see Suppl. 6). The values ofthe diffusion coefficients determined for 3a and 3b in their indi-vidual solutions were: D(3a) ¼ 9.33 � 10�11 m2/s andD(3b) ¼ 2.65 � 10�11 m2/s. According to StokeseEinstein equationthese values correspond to particles with apparent hydrodynamicradius, Rh of 1.84 nm and 6.47 nm.
Figs. 4 and 5 show the DOSY spectra of the polyphosphoester-drug conjugates 4a and 4b, respectively. The conjugation of thedrug to themacromolecular carrier is confirmed by the fact that thesignals of the drug and the polymer chains display the samediffusion coefficients indicating that they belong to the same entity.The values for the diffusion coefficients obtained from the DOSYspectrawere used for the calculation of the apparent hydrodynamicradii of the obtained conjugates: Rh(4a) ¼ 5.46 nm andRh(4b) ¼ 12.00 nm. The Rh values for the conjugates given presentan average values obtained from the corresponding 1H and 31PDOSY spectral data.
1H and 31P NMR measurement were done for estimation of thehydrolytic stability of the conjugates 4a and 4b. First, solutions of4a and 4b in aqueous medium (D2O) at 22 �C were measured over aperiod of 7 days and no changes in the structure of the conjugateswere detected. Then measurements were performed in twodifferent media e phosphate buffer solution at pH 7.5 and citricbuffer solution at pH 4.5 and heating at 37 �C for 5 days. Againno changes in the conjugate structure were observed (see Suppl. 7and 8).
2.2. Pharmacological data
The cytotoxicity of the conjugate 2 and the polyphosphoesteranalogs 4a and 4b were evaluated in comparison to the non-modified spermidine bridged dinuclear platinum complex 1 in apanel of human tumor cell lines, namely HL-60 and KG-1 (acutemyeloid leukemia), HL-60/CDDP (cisplatin-resistant clone), BV-173(chronic myeloid leukemia), SKW-3 (T-cell leukemia), and MDA-MB-231 (estrogen receptor-negative breast cancer). Cisplatin was
Fig. 4. DOSY 1H and 31P{H} DOSY NMR spectra of polyphosphoester-drug conjugate 4a.
V. Mitova et al. / European Journal of Medicinal Chemistry 72 (2014) 127e136130
used as a reference anticancer compound. The IC50 values calcu-lated from the concentrationeresponse curves are summarized inTable 1.
Evident from the results obtained the prototypic dinuclearcompound 1 proved to exert superior cytotoxicity compared tocisplatin against all leukemic cell lines with the exception of thebreast cancer derived MDA-MB-231 cell line. Noteworthy thedinuclear agent 1 proved to be far more active against the cisplatin-resistant cell line HL-60/CDDP, displaying a 6 fold lower resistanceindex as compared to the reference anticancer drug (Fig. 6). The lowmolecular weight prodrug 2 albeit less potent than 1 still retainedcytotoxic activity causing 50% cell growth inhibition of leukemiccell lines at lowmicromolar levels and accordingly proved to be farless active against the solid tumor model. Both polyphosphoesteranalogs exhibited profound cytotoxicity, superior to that of conju-gate 2. On the basis of the IC50 values obtained the highermolecularweight polyphosphoester compound 4bwas generally more activeand its cytotoxicity against chemosensitive leukemic models wascomparable to that of cisplatin. A noteworthy finding of the cyto-toxicity screening bioassay was the well-established ability of the
Fig. 5. 1O and 31P{H} DOSY NMR spectra o
conjugates to bypass the resistance mechanisms in HL-60/CDDP,whereby the resistance indices in all conjugates were even lowerthan those of the prototype dinuclear agent 1 (Fig. 6, Table 1).
In order to elucidate the involvement of proapoptotic mecha-nism for the cytotoxicity mode of action of the newly synthesizedcompounds we evaluated their ability to trigger oligonucleosomalDNA-fragmentation after 24 h exposure to equitoxic concentrationof the tested agents. In the chemosensitive models KG-1 and HL-60the series of dinuclear agents and cisplatin evoked profound DNA-fragmentation, implicative for the induction of apoptosis. Albeitcisplatin and 1 were superior in this respect the novel conjugatesalso displayed concentration-dependent proapoptotic activity, andcould be ranged in the following order of increasing potency2 < 4a < 4b, in corroboration to the MTT-bioassay data. In thecisplatin-resistant cell line HL-60/CDDP however the proapoptoticactivity of cisplatin was far less prominent as compared to thatevoked by 1 and its conjugates (Fig. 7).
The co-incubation of cells with the pancaspase inhibitor Boc-Asp(OMe)-fluoromethyl ketone (PCI) totally abrogated the abilityof platinum agents to trigger oligonucleosomal DNA-fragmen
f polyphosphoester-drug conjugate 4b.
Table 1IC50 values of the spermidine bridged dinuclear complex 1 and its conjugates 2, 4a, 4b vs. cisplatin in a panel of five chemosensitive and one cisplatin-resistant human tumorcell lines after 72 h exposure (MTT-dye reduction assay).
Exp. Series IC50 value (mmol/L)
HL-60 HL-60/CDDP KG-1 BV-173 SKW-3 MDA-MB-231
1 4.6* � 0.7 14.9* a � 1.3 10.4 � 1.1 3.8* � 0.90 4.3* � 1.0 20.2* � 1.22 15.1* � 1.9 27.1* b � 3.1 29.2* � 2.4 25.5 � 2.7 50.1* � 3.1 168.4* � 7.94a 9.3* � 1.2 20.8* c � 1.9 17.3* � 1.9 18.8* � 1.4 47.1* � 3.2 97.4* � 8.84b 8.2* � 0.9 18.7* d � 2.4 12.3 � 2.0 10.8 � 1.1 14.7* � 1.1 79.7* � 2.4Cisplatin 7.0 � 1.2 135.2e � 9.3 11.5 � 1.5 9.3 � 1.7 10.4 � 0.9 8.8 � 1.2
*Significantly different (p � 0.05) vs. cisplatin (t-test).a Resistance indices (IC50 HL-60/CDDP/IC50 HL-60): 3.2.b Resistance indices (IC50 HL-60/CDDP/IC50 HL-60): 1.8.c Resistance indices (IC50 HL-60/CDDP/IC50 HL-60): 2.2.d Resistance indices (IC50 HL-60/CDDP/IC50 HL-60): 2.3.e Resistance indices (IC50 HL-60/CDDP/IC50 HL-60): 19.3.
V. Mitova et al. / European Journal of Medicinal Chemistry 72 (2014) 127e136 131
tation, which firmly implies that the induction of programmed celldeath is dependent on recruitment of caspase-dependent apoptoticsignaling pathways (Fig. 7).
A flow cytometric analysis (FCA) of KG-1 cells was carried out tofurther explore the ability of the tested agents to induce apoptosisand to characterize their influence on cell cycle progression. After24 h exposure mimicking the DNA-fragmentation protocol emer-gence of the characteristic sub-G1 population in the FCA histogramsconfirmed the ability of cisplatin and the dinuclear agents 1, 4a, 4bto induce apoptosis. In this assay the conjugate 2 was devoid ofdetectable proapoptotic activity (Table 2, Fig. 8).
The cell cycle progression of non-apoptotic cells however dis-played quite distinct patterns following exposure to cisplatin andthe series of free or conjugated dinuclear agents (Table 2, Fig. 8).The reference drug caused concentration-dependent G1 arrestconcomitant with drastic decrease of the S phase population,indicative for inhibition of the cell cycle progression. In a dissimilarfashion the dinuclear agent 1 caused only marginal modulation ofthe S-phase fraction, whereas its conjugates (especially 2 and 4b)even evoked relative increase of the percentage of S-phase popu-lation. These findings together with the aforementioned bypassingof the resistance mechanisms in HL-60/CDDP further justify for thecisplatin-dissimilar mode of action of the dinuclear agent 1 and itsconjugates conditioned by their distinct chemical structure and thepresence of a second metal center.
The in vitro nephrotoxic potential of the novel compounds wasevaluated in comparison to cisplatin against cultured murine kid-ney epithelial cells. While the reference drug caused significantconcentration-dependent decrease in cellular viability, the testeddinuclear agents and their poly-phosphoester conjugates proved to
Fig. 6. Concentration-response curves of the spermidine bridged dinuclear platinum compleits cisplatin-resistant variant HL-60/CDDP after 72 h treatment (MTT-dye reduction assay).
exert far less prominent detrimental effects upon this non-malignant cellular population (Table 3). Considering the profoundinhibitory effects of the novel species against tumor cell lines thelower potential to inhibit the viability of kidney epithelium, in-dicates superior therapeutic index, considering the fact that thekidneys are a key target organ for cisplatin toxicity.
3. Conclusion
New conjugates of a platinium (II) dinuclear complex withspermidine bridge were obtained via covalent bonding to dimethylH-phosphonate and poly(oxyethylene H-phosphonate)s applyingthe AthertoneTodd reaction. The conjugation reaction between thepolymer carrier and the bioactive agent was proved by NMRspectroscopy. The results from the DOSY spectra showed that thenew polymeric conjugates form particles with average size from10 nm to 24 nm depending on the molecular mass and the lengthsof the PEG segment of the polyphosphoester carrier used. Theircytotoxic activities were determined in a panel of five chemo-sensitive human tumor cell lines and one cisplatin-resistant model(HL-60/CDDP), whereby albeit less active than the prototypedinuclear agent the conjugates retained its ability to decrease thecellular viability and to induce apoptosis at micromolar concen-trations. All dinuclear agents displayed superior activity against HL-60/CDDP as compared to cisplatin and proved to evoke cisplatin-dissimilar modulation of cell cycle progression in KG-1 leukemiccells. Moreover, the novel compounds displayed less pronouncedinhibitory activity against cultured murine renal epithelial cells, ascompared to cisplatin.
x 1, its conjugates 2, 4a, 4b, and cisplatin against the chemosensitive HL-60 cell line and
Fig. 7. Apoptotic DNA-fragmentation induced by the spermidine bridged dinuclearplatinum complex 1, its conjugates 2, 4a, 4b, and cisplatin after 24 h treatment of HL-60, HL-60/CDDP and KG-1 cells at cytotoxic concentrations (½ IC50 and IC50), appliedalone or in combination with 100 mmol/l pancaspase inhibitor (PCI). The cytosolicenrichment factor is proportional to the cellular levels of histone associated mono- andoligonucleosomal DNA fragments, determined by ‘Cell death detection ELISA’ kit. Eachcolumn is representative for the arithmetic mean (�sd) of three independentexperiments.
V. Mitova et al. / European Journal of Medicinal Chemistry 72 (2014) 127e136132
4. Experimental
4.1. General
Spermidine (4-azaoctane-1,8-diamine) and anhydrous LiCl werepurchased from Fluka. trans-[PtCl2(NH3)2] (TDDP) was purchasedfrom Alfa Aesar. Ethyl trifluoroacetate, di-tert-butyl dicarbonate[(BOC)2O], anhydrous DMF and AgNO3 were purchased fromAldrich and used without further purification. Acetonitrile anddichloromethane were purchased from SigmaeAldrich, dried overP2O5 and distilled prior to use. Dimethyl H-phosphonate and trie-thylamine were Fluka products and distilled prior to use. Poly-ethylene glycols with molecular weight 200 (PEG200) and 600(PEG 600) was obtained from Fluka and dried by azeotropicdistillation with toluene. Carbon tetrachloride, methanol and
toluene were dried and freshly distilled prior to use. All otherssolvents were of p.a. grade and used without further purification.1H, 13S{H}, 31R, 19F, 195Pt NMR spectra were recorded on BrukerAvance IIþ600 MHz spectrometer in CDCl3 or D2O using tetrame-thylsilane or 85% H3PO4 as external standarts. 1O and 31R DOSYNMR spectra were measured on Bruker Avance IIþ600 MHz spec-trometer using 5mm direct detection dual broadband probe, with agradient coil delivering maximum gradient strength of 63 G/cm.The experiments were performed at a temperature of 293 K. 1HNMR spectra were acquired with 32 K time domain points, spec-trum width of 9600 Hz and 128 scans. The DOSY measurementswere performed with samples dissolved in D2O at concentration of12 mg/ml.
4.2. Synthesis
4.2.1. Synthesis and characterization of the platinum dinuclearcomplex 1,1/t,t-spermidine dinuclear platinum complex, [{trans-PtCl(NH3)2}2{m-spermidine-N1,N8}](NO3)3, (1)
The [{trans-PtCl(NH3)2}2{m-spermidine-N1,N8}]Cl3 was syn-thesized following a known synthetic pathways [25]. Productsobtained at each step of the synthesis were isolated, purified andanalyzed by NMR spectroscopy to verify their composition andstructure. [{Trans-PtCl(NH3)2}2{m-spermidine-N1,N8}]Cl3 (1.000 g,0.42 mmol) was dissolved in 60 ml of distilled water. A solution ofAgNO3 (0.655 g, 3.84 mmol) in 14 ml water was added dropwiseunder stirring. After 10e15 min the precipitated AgCl was filteredoff. The filtrate was evaporated and the solid obtained waswashed with acetone:ether (1:1) and dried. Yield 80%. 1H NMR(D2O), d (ppm): 2.97 (2t, Ha,a`); 2.66 (2t, Hbb`); 1.97 (q, Hc); 1.63 (m,Hd,e).
4.2.2. Model reaction between 1,1/t,t-spermidine complex (1) andDMPh yielding complex 2
Complex (1) (0.124 g, 1.4 � 10�4 mol) was dissolved in 10 ml ofanhydrous DMF under gentle flow of dry argon. To the solution thefollowing reagents were added: NaOH (5.8 mg, 1.4 � 10�4 mol) in0.15 ml methanol, 0.5 ml of tetrachloromethane, triethylamine(34 mg, 3.4 � 10�4 mol) in 1 ml of DMF and DMPh (0.0318 g,2.89 � 10�4 mol) in 0.1 ml of DMF. The latter was added dropwiseand solution cooled to 10 �C for 1 h. Stirring continued for 24 h atroom temperature. The product 2 was isolated by precipitation inacetone. The precipitate was washed two times with chloroformand dried. Yield 86%. 31P NMR (D2O, d, ppm): 14.96 (nonet;(CH3O)2P(O)N<); 3.73 (septet, (CH3O)2P(O)O�).
4.2.3. Synthesis and characterization of the poly(oxyethylene H-phosphonate)s (3a) and (3b)
The poly(oxyethylene H-phosphonate)s were obtained via pol-ytransesterification of dimethyl H-phosphonate and poly(ethyleneglycol)s with number average molecular weight 200 g/mol (PEG200) and 600 g/mol (PEG 600) respectively products (3a) and (3b)following a procedure described [20]. The two products 3a and 3bwere analyzed by 1H, 13C and 31P NMR spectroscopy. Their spectradisplayed signals with the same chemical shifts and different valuesof the integral intensities.
Poly(oxyethylene H-phosphonate): 1H NMR (CDCl3, d, ppm):6.86 (d, 1J(P,H) ¼ 716.2 Hz, 1H, P-H repeating unit), 6.79 (d,
Table 2The effects of 1, 2, 4a, and 4b treatment on cell cycle progression and apoptosis rates of KG-1 cells as determined by flow cytometric analysis. Cisplatin was used a sa referenceanticancer drug (mean � sd; n ¼ 3).
Exp. Series Dose Apoptotic cells (%)a Fold increasevs. control
Cell cycle progression (%)b
G1-phase G2-phase S-phase
Control e 1.18 � 0.19 e 33.51 � 2.17 19.04 � 1.09 47.48 � 4.101 ½ IC50 2.30* � 0.14 1.95 39.54* � 2.10 13.85* � 1.10 46.61 � 2.29
IC50 7.76* � 1.14 6.58 41.42* � 2.31 17.84 � 1.78 40.74* � 1.332 ½ IC50 0.19 � 0.11 0.16 19.01* � 1.09 9.63* � 2.14 71.36* � 4.22
IC50 2.03* � 0.12 1.72 25.13* � 3.17 0.00 74.87* � 2.984a ½ IC50 1.32* � 0.47 1.12 25.17* � 3.94 20.91 � 1.92 53.92* � 2.22
IC50 9.81* � 2.34 8.31 30.81* � 2.78 21.41 � 2.11 47.78 � 3.924b ½ IC50 5.86* � 0.90 4.97 16.87* � 2.21 27.04* � 2.01 56.09* � 4.25
IC50 7.16* � 1.02 6.06 19.34* � 1.54 18.68 � 1.09 61.98* � 3.73Cisplatin ½ IC50 24.98* � 2.51 21.17 71.45* � 3.27 5.32* � 0.19 23.23* � 2.96
IC50 30.40* � 1.59 25.76 90.87* � 2.51 9.13* � 2.06 0.00
*Significantly different (p � 0.05) vs. untreated control (t-test).a Percentage of all cellular events (>10 000 per treatment group).b Percentage of cycling, non-apoptotic cells.
V. Mitova et al. / European Journal of Medicinal Chemistry 72 (2014) 127e136 133
1J(P,H) ¼ 708.8 Hz, 1H, P(H)OCH3 end group), 6.74 (d,1J(P,H) ¼ 690.3 Hz, 1H, P(H)OH end group), 4.19e4.07 (m, 4H,CH2OP(O)OCH2), 3.62e3.55 (m, 50H, CH2OCH2); 13C{H} NMR(CDCl3, d, ppm): 70.43 (CH2OCH2), 70.03 (d, 3J(P,C) ¼ 5.8 Hz,POCH2CH2), 64.57 (d, 2J(P,C)¼ 6.2 Hz, POCH2CH2); 31P NMR (CDCl3, d,ppm): 11.17 (d of sextet, 1J(P,H) ¼ 708.8 Hz, 3J(P,H) ¼ 10.5 Hz), 10.47 (d
Fig. 8. Effects of the spermidine bridged dinuclear platinum complex 1, its conjugates 2,Exponentially growing cells were cultured in RPMI 1640 supplemented with 10% FBS, andexposure, cells were collected, washed with PBS, digested with RNase and then cellular DNThereafter cell cycle distribution was examined by flow cytometric analysis.
of quintet, 1J(P,H) ¼ 716.2 Hz, 3J(P,H) ¼ 9.9 Hz), 8.37 (d of t,1J(P,H) ¼ 690.3 Hz, 3J(P,H) ¼ 10.97 Hz).
4.2.4. Polyphosphorester-drug conjugation e products 4a and 4b4.2.4.1. Polyphosphorester prodrug 4a. Complex 1 (0.200 g,2.3 � 10�4 mol) was dissolved in 16 ml of anhydrous DMF under
4a, 4b, and cisplatin on the apoptotic rate and cell cycle progression in KG-1 cells.treated with tested agents at equitoxic concentrations (½ IC50 and IC50). After 24 hA was stained with propidium iodide as described in Materials and methods section.
Table 3Nephrotoxicity of 1, 2, 4a, and 4b vs. cisplatin on murine kidney epithelium cells after 72 h treatment as assessed by MTT dye reduction assay.
Concentration (mM) Survival fraction (% of untreated control)
Cisplatin 1 2 4a 4b
0 100 � 2.7 e e e e
10 49.3* � 7.8 67.0*x � 3.4 85.4*x � 7.2 83.7*x � 5.3 90.1*x � 4.920 35.7* � 4.4 48.3*x � 3.7 67.1*x � 5.6 58.9*x � 9.4 55.7*x � 6.140 28.9* � 7.1 35.9* � 7.3 37.3* � 9.2 31.2* � 7.8 34.1* � 5.8
*P < 0.05 as compared to control.xP < 0.05 as compared to cisplatin treatment at equal concentrations (paired t-test).
V. Mitova et al. / European Journal of Medicinal Chemistry 72 (2014) 127e136134
gentle flow of dry argon. To the solution the following reagentswere added: NaOH (9.3 mg, 2.3� 10�4 mol) in 0.24 ml of methanol,0.5 ml of CCl4, Et3N (56 mg, 5.6 � 10�4 mol) in 1.6 ml of anhydrousDMF. The solution was cooled to 10e13 �S and the 3a (0.115 g,4.6 � 10�4 mol repeating unit) in 3.5 ml of DMF was added drop-wise. Stirring continued for 24 h at room temperature and inertatmosphere. The product 4awere isolated by evaporation almost todryness, the solid obtained was washed with acetone, dried anddialyzed in water at 10 �S for 24 h (MWCO 1000). The water wasevaporated and the solid obtained was washed twice with acetoneand dried in vacuo. Yield 70%. 31P NMR (D2O, d, ppm): 13.63(CH3O)(eCH2O)P (O)N<, 13.38 (HO)(eCH2O)P(O)N<, 12.27 (eCH2O)2P (O)N<, 2.49 (eCH2O)2P(O)OCH3, 1.23 (eCH2O)2P(O)O�
4.2.4.2. Polyphosphorester prodrug 4b. Complex 1 (0.200 g,2.3 � 10�4 mol) was dissolved in 16 ml of anhydrous DMF undergentle flow of dry argon. To the solution the following reagentswere added: NaOH (9.3 mg, 2.3� 10�4 mol) in 0.24 ml of methanol,0.5 ml of CCl4, Et3N (56 mg, 5.5 � 10�4 mol) in 1.6 ml of anhydrousDMF. The solution was cooled to 10e13 �S and the 3b (0.30 g,4.6 � 10�4 mol repeating unit) in 3.5 ml of DMF was added drop-wise. Stirring continued for 24 h at room temperature and inertatmosphere. The product 4bwere isolated by evaporation almost todryness, the solid obtained was washed with acetone, dried anddialyzed in water at 10 �S for 24 h (MWCO 1000). The water wasevaporated and the solid obtained was washed twice with acetoneand dried in vacuo. Yield 77%. 31P NMR (D2O, d, ppm): 12.24 (eCH2O)2P (O)N<, 2.54 (eCH2O)2P(O)OCH3, 1.31 (eCH2O)2P(O)O�.
4.3. DOSY measurements
All NMR measurements were performed on a Bruker AvanceIIþ600 NMR spectrometer, equipped with 5 mm dual 1H/31P Diff30probe and a 40 A gradient amplifier, providing maximum gradientstrength of 11.8 T/m. 1H NMR spectra were acquired with 32 K timedomain points, spectrum width of 7200 Hz and 128 scans. The 31Pspectra were measured with 64 K time domain data points, spec-trum width of 50 kHz, relaxation delay of 15 s and 128 scans. Theinverse-gated pulse sequence was used in order to ensure quanti-tative signal area measurement.
The 31P DOSY spectra were acquired with the Diff suite inte-grated in Topspin package using double stimulated echo pulsesequence [30], to eliminate possible convection during the exper-iments. An inverse-gated decoupling scheme was implemented inthe standard convection-compensated DOSY pulse sequence inorder to increase the sensitivity by eliminating the 1He31P spincoupling over two and three bonds. Monopolar sine shapedgradient pulses, a longitudinal eddy current delay of 20 ms andthree spoiling gradients were used. Diffusion delay (D) of 100 msand gradient pulse length (d) of 1 ms were used. The gradientstrength G was varied in 32 linear steps from 35.4 to 283.6 G/cm toensure complete signal attenuation. A gradient recovery delay (s) of100 ms and an eddy current delay (te) of 20 ms were used. All
spectra were recorded with 16 K time domain data points in the t2dimension, 128 transients for each gradient increment, and arelaxation delay of 5 s. Themeasurements were carried out withoutsample spinning. To minimize the effects of external temperaturevariations, the air conditioning in the laboratory and the air flow tothe probe were switched off, with only the water cooling system ofthe Diff30 probe was used to regulate the sample temperature.
The spectra were processed with an exponential windowfunction (line broadening factor 1), 64 K data points in F2 dimen-sion and 1 K data points in the diffusion dimension, using the fittingroutine integrated in Topspin3.1 package. The evaluation of thediffusion coefficients was performed by fitting the sum of the col-umns along the chemical shift of each signal in the DOSY spectrumwith the Gaussian distribution curve.
The StokeseEinstein equation was used to estimate theapparent hydrodynamic radius, Rh, of the solutes (the complexes,the polymer carriers and the conjugates):
Rh ¼ kT6phD
where ke Boltzmann constant, Te temperature (K) and he solventviscosity. In the present experiment: h(D2O)¼ 1.2518 � 10�3 Pa s at293 K (NIST, USA). D is the diffusion coefficient obtained from theDODY spectral data.
4.4. Biological assay
4.4.. 1Cell lines and culture conditionsThe cytotoxic activity of the tested platinum agents was
assessed against a panel of human tumor cell lines, namely HL-60(acute myelocyte leukemia), its cisplatin-resistant sub-line HL-60/CDDP, KG-1 (acute myelocyte leukemia), SKW-3 (T-cell leukemia),BV-173 (chronic myeloid leukemia) and MDA-MB-231 (breastcancer). The cisplatin-resistant sub-line HL-60/CDDP has beendeveloped at the Lab of Experimental Chemotherapy (Faculty ofPharmacy, MU-Sofia). It was established by continuous selection inmedium with gradually increasing concentrations of cisplatin. HL-60, KG-1, BV-173, SKW-3 and MDA-MB-231 were purchasedfrom the German Collection of Microorganisms and Cell Cultures(DSMZ GmbH, Braunschweig, Germany). The cells were grown incontrolled environmente cell culture flasks at 37 �C in an incubator‘BB 16-Function Line’ Heraeus (Kendro, Hanau, Germany) withhumidified atmosphere and 5% CO2. The growth medium was 90%RPMI-1640 þ 10% FBS. HL-60/CDDP cells were cultivated in thepresence of 25 mmol/l cisplatin in order to maintain their drugresistance phenotype.
4.4.2. Cytotoxicity assessment (MTT-dye reduction assay)The cytotoxicity of tested compounds was assessed using the
MTT-dye reduction assay, as previously described [31], with minormodifications [32]. In brief, exponentially proliferating cells wereseeded in 96-well flat-bottomed microplates (100 ml/well; at a
V. Mitova et al. / European Journal of Medicinal Chemistry 72 (2014) 127e136 135
density of 1 � 105 cells/ml for the leukemic cells and 2 � 104 forMDA-MB-231) and incubated for 24 h at 37 �C, in an incubator.Thereafter the cells were exposed to various concentrations of thetested compounds for 72 h. For each concentration a set of at least 8wells were used. After the treatment period 10 ml MTT solution (at aconcentration of 10 mg/ml in PBS) aliquots were added to eachwell. The microplates were further incubated for 4 h at 37 �C andthe MTT-formazan crystals formed were dissolved by addition of100 ml/well 5% HCOOH-acidified 2-propanol. The MTT-formazanabsorption was measured using a multimode microplate reader(Beckman Coulter DTX-880) at 580 nm. The cell survival data werenormalized as percentage of the untreated control (set as 100%viability), were fitted to sigmoidal dose response curves and thecorresponding IC50 values (concentrations inducing half-maximalsuppression of cellular viability) were calculated using non-linearregression analysis (GraphPad Prizm Software for PC). In additionthe resistance indices as a relativemerit for the level of resistance inHL-60/CDDP were determined as the ratio between the IC50 in themulti-drug resistant HL-60/CDDP and the corresponding IC50 in thesensitive parent line HL-60. All tests were run in triplicate.
4.4.3. Apoptotic DNA fragmentation assayThe oligonucleosomal DNA fragmentation, a key hallmark
feature of apoptosis, was examined using a commercially available‘Cell-death detection’ ELISA kit (Roche Applied Science). The assayallows for semi-quantitative determination of the apoptotichistone-associated mono- and oligonucleosomal DNA-fragmentation using ‘sandwitch’ ELISA. Exponentially growing cells wereexposed to cytotoxic concentrations of the tested compounds (1, 2,4a, 4b and cisplatin) and thereafter cytosolic fractions of 1 � 104
cells per group (treated or untreated) served as antigen source in asandwich ELISA, utilizing primary anti-histone antibody-coatedmicroplate and a secondary peroxidase-conjugated anti-DNAantibody. In order to evaluate the involvement of the caspaseactivation in the apoptotic process the cellular treatment wasperformed with or without co-incubation with a commerciallyavailable non-selective pan-caspase inhibitor Boc-Asp(OMe)-fluoromethyl ketone (PCI). The photometric immunoassay forhistone-associated DNA fragments was executed according to themanufacturers’ instructions at 405 nm, using a multimode micro-plate reader (Beckman Coulter DTX-880). The results are expressedas the oligonucleosome enrichment factor (representing a ratiobetween the absorption in the treated vs. the untreated controlsamples).
4.4.4. Flow cytometric analysis of apoptosis and cell cycleprogression
The pro-apoptotic and cell cycle modulatory effects of the testedconjugates 2, 4a, 4b, and the starting dinuclear complex 1 wereassessed in KG-1 cells by flow-cytometric analysis (FCM) asdescribed elsewhere [33]. In brief, control or treated cells werepelleted, washed with cold PBS, and resuspended in a mixture of100 ml PBS and 300 ml 96% ethanol. The cells were kept at �20 �C.Before the FCM measurements the cells were centrifuged andresuspended in 500 ml PBS, containing 20 mg/ml RNAase and 20 mg/ml propidium iodide (PI) at room temperature. The test tubes wereincubated at 4 �C for 1 h, protected from light, and the red fluo-rescence emitted from the PIeDNA complex was analyzed afterlaser excitation of the fluorescent dye at 488 nm by FACS Canto IIflow cytometer (B-D). DNAQC particles (B-D) and FACS Diva (B-D)were used to set instrument photomultiplier tube voltages andamplifier gains, check instrument resolution and linearity, andverify instrument alignment. At least 20 000 events were collectedfor each sample at a resolution of 262 144 linear channels usinglinear amplification of all signals. The population of apoptotic cells
(cells with fractional DNA content; sub-G0 cells) were defined onhistograms and expressed as percentages, by means of ModFit LTver3.0 software. Cisplatin was used as a reference anticancer drug.All tests were run in triplicate.
4.4.5. Murine renal epithelial cell culture and in vitronephrotoxicity testing
The procedure was carried out as described elsewhere [34] withminor modifications [35]. In brief, kidneys from 10 adult micewhere dissected out aseptically and minced until formation of tis-sue particles approximating 1 mm3. They were then washed in PBSseveral times and allowed to sediment between the washing pro-cedures and finally the supernatant (containing cellular debris andblood cells) was discarded. A sufficient amount of warm enzymaticsolution (0.1% collagenase/0.1% trypsin) was then added to thetissue mass and it was incubated for 100min in awater bath shakerat 37 �C. After the digestion completion the enzymatic solutionwasreplaced with FCS supplemented RPMI-1640 medium (withoutglucose). The tissue suspension yielded was turned in single cellsuspension by vigorously driving it through a Pasteur pipette.Finally the isolated cells were transferred in cell culture flasks at adensity of 1 � 105/cm2 and incubated at 37 �C for several days toallow the adhesion of the cells to the wall of the flask. Thereafterthe cells were harvested via hot trypsination, counted and trans-ferred in 96-well microplates (100 ml per well) at a density of 1.5e2 � 105 cells/ml. Following 24 h incubation in the microplates thekidney epithelial cells were exposed to various concentrations ofcisplatin and compounds 1, 2, 4a and 4b for 72 h. The cell viabilitywas thereafter assessed using the MTT-dye reduction assay.
4.4.6. Bioassay data processing and statisticsThe statistical processing of biological data included a two sided
Student’s t-test whereby values of p � 0.05 were considered asstatistically significant.
Acknowledgments
The support by the NSF of Bulgaria (Grants: DO 02-198/2008and DRNF 02-13/2009) is highly acknowledged.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2013.11.014
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