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inorganics Inorganics 2019，7，42of 15 Inorganics 2019， 7， 4； doi：10.3390/inorganics7010004www.mdpi.com/journal/inorganics Article Synthesis， Characterization， and BiologicalEvaluation of Red-Absorbing Fe(II)Polypyridine Complexes Johannes Karges 1D， Philippe Goldner D；) and Gilles Gasser 1，*D Laboratory for Inorganic Chemical Biology， Chimie Paris Tech， PSL University， 75005 Paris， France；johannes.karges@chimieparistech.psl.eu 2 Chimie ParisTech， PSL University， CNRS， Institut de Recherche de Chimie Paris， 75005 Paris，France；philippe.goldner@chimieparistech.psl.eu Correspondence： gilles.gasser@chimieparistech.psl.eu； Tel.：+33-1-44-27-56-02 Received： 7 December 2018； Accepted： 28 December 2018； Published： 7 January 2019 check forupdates Abstract： Cancer is known to be one of the major causes of death nowadays. Among others，chemotherapy with cisplatin is a commonly used treatment. Although widely employed， cisplatin isknown to cause severe side effects， such as nerve and kidney damage， nausea， vomiting，and bonemarrow suppression. Most importantly， a number of cancer tumors are acquiring resistance tocisplatin， limiting its clinical use. There is therefore a need for the discovery of novel anticancer agents.Complementary to chemotherapy， Photodynamic Therapy (PDT)has expanded the range of treatmentopportunities of numerous kinds of cancer. Nonetheless， the currently approved PDT photosensitizers(PSs) suffer from major drawbacks， which include poor water solubility or photobleaching， in additionto a slow clearance from the body that causes photosensitivity. Due to these limitations， there isa need for the development of new PDT PSs. To overcome these problems， a lot of research groupsaround the world are currently focusing their attention towards the development of new metalcomplexes as PDT PSs. However， most synthesized compounds reported so far show limited usedue to their poor absorption in the phototherap工eutic window. Herein， we report on the preparationand characterization of three Fe(II) polypyridine complexes (4-6) and evaluate their potential as bothanticancer agents and PDT PSs. Very importantly， these compounds are stable in human plasma，photostable upon continuous LED irradiation， and absorb in the red region of the spectrum. We coulddemonstrate that through additional sulfonic acid groups on the polypyridine ligand being used(bphen： 4，7-diphenyl-1，10-phenanthroline)， the water solubility of the complexes could be highlyimproved， whereas the photophysical properties did not significantly change. One of these complexes(4) shows interesting toxicity， with IC5o values in the low micromolar range in the dark as well assome phototoxicity upon irradiation at 480 and 540 nm against RPE-1 and HeLa cells. Keywords： anticancer； medicinal inorganic chemistry； metals in medicine； photodynamic therapy 1. Introduction Over the last decades， cancer has emerged as one of the deadliest diseases worldwide. Classicaltreatments are chemotherapy， surgery， radiotherapy， and immunotherapy. To efficiently treat tumors，a combination of these techniques is most commonly used [1，2]. Although cisplatin is still the mostcommonly used chemotherapeutic drug against cancer， it is responsible for a number of severeside-effects (e.g.， nerve and kidney damage， nausea， vomiting， and bone marrow suppression).Additionally， a number of cancer tumors are acquiring resistance to cisplatin， limiting its clinical use.There is therefore a need for the development of new chemotherapeutic drug candidates. To tackle this issue， the use of metal-based complexes is intensively studied due to their large structural varietyas well as the possibility of ligand exchange or biological redox reactions [3-5]. Most research so farhas been made on the metals Pt and Ru [6-13]. Complementary to chemotherapy， Photodynamic Therapy (PDT) has expanded the range of treatment opportunities of numerous kinds of cancer. This method is already approved to treat differentkinds of cancer as well as other bacterial， fungal， or viral infections. Typically， during a PDT treatment，a preferably non-toxic photosensitizer (PS) is selectively activated upon light irradiation to createreactive oxygen species (ROS). More specially， the excited state of the PS is able to transfer electrons orprotons from/to the biological environment to generate radicals or to transfer its energy to molecularoxygen generating singlet oxygen (O2). So far， most of the research has been performed on PSs witha tetrapyrrolic scaffold. However， based on their similar structures， these compounds suffer from similardrawbacks (e.g.poor water solubility， photobleaching or a slow clearance from the body that causesphotosensitivity) [14，15]. To overcome these limitations， metal complexes are currently investigated asalternatives to chlorins， phtalocyanines， or porphyrins due to their attractive physico-chemical properties(i.e.， high water solubility， high chemical stability and photostability，high 102 production) [16-19].The most commonly studied transition metals in this field of research are Ru(II) [18，20-23]，Os(II) [24，25]，Rh(III) [26，27]， and Ir(III) [28-30] complexes. However， these metals are not abundant. It wouldtherefore be extremely interesting to develop a class of PDT PSs based on cheap， abundant metals.In this perspective， we have recently investigated the potential of easy-to-synthesize Cr(III) complexesas PDT PSs [31]. There have also been made some efforts in the design of Fe-based complexes.However， it is well established that Fe(II) polypyridine complexes are generally poorly emissivewith very short lifetimes. This is the case due to the fast depopulation of the photophysically activeMLCT state to lower lying 3MC and 5MC states [32，33]. Nonetheless， since Fe is a bioessentialmetal， it is anticipated to have a reduced metal-induced cellular toxicity [34，35] in comparison toother metal-based complexes. Chakravarty and co-workers have already reported several Fe(III)-basedcomplexes with a dipyrido[3，2-d：2'，3'-f]quinoxaline [35-37] or a dipicolylamine [34，38-45] moiety aseffective PSs. As an impressive example， the Fe(III) complexes 1 was prepared with a dipicolylamineligand combined with an anthracenyl moiety， which is able to intercalate in the DNA， and a catecholligand， which promotes an intense ligand-to-metal charge-transfer band in the near IR window(Figure 1). These complexes were shown to act as potent fluorophores and to have a nuclear uptake.Very importantly， they caused apoptotic cell death upon light irradiation [34]. Worthy of note， the samegroup also synthesized bimetallic Fe(III) complexes as PSs in view of DNA cleavage [46，47] and site1ihlicspecific protein cleavage [48]. Additionally， a recent publication by the same authors showed that theconjugation of a phototoxic Fe(III) complex to biotin allows for selective tumor delivery of the PDTagent [49]. In comparison to complexes with a +3 oxidation state， the studies of Fe complexes witha +2 oxidation state as a PS are scare. Roelfes and co-workers have， for example， designed a series ofFe(II)-N4Py complexes (N4Py=N，N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) as effective PSsfor DNA cleavage [50，51]. In addition， Fe(II)-terpyridine complexes with aromatic moieties， which werefound to be localizing in the nucleus， showed a remarkable phototoxicity upon visible light irradiationwhile remaining only slightly cytotoxic in the dark [52]. A recent study has also demonstrated theuse of pyridyldipyridophenazine substituted Fe(II) complexes as partial DNA intercalative binders.The complexes had phototoxicity upon visible light irradiation， in addition to a low dark toxicity [53]. Inspired from the works described above， we have decided to investigate the potential of Fe(II)polypyridyl complexes as chemotherapeutic agents and PDT PSs. For this， we capitalized on the workon Ru(II) polypyridine complexes and especially on the ones on 4，7-diphenyl-1，10-phenanthroline(bphen)-containing complexes [54-63]. With the work of Glazer and co-workers on the Ru(II) basedcomplexes 2 and 3 in mind (Figure 1)， we have designed the two analogous Fe(II) complexes(complexes 4 and 5， Figure 2). We have also prepared an isomer of complexe 5 (complex 6， Figure 2).As demonstrated for the Ru(II) complexes， the substituents that were introduced in complexes 5and 6 were able to drastically improve the water solubility of the complexes. At the same time， this derivatization did not influence their photophysical properties [54，64]. Worthy of note， 4 hasalready been reported in the literature as a catalyst for the autoxidation of cumene and cyclohexane，demonstrating that this complex is able to interact with oxygen [65]. Herein， we present the synthesis，in-depth characterization， and biological evaluation of three Fe(II) complexes. As shown below，we were able to unveil a compound with cytotoxicity in the low micro molar range in the dark andsome phototoxicity in RPE-1 and HeLa cells. Figure 1. Chemical structures of a Fe(III) complex (1) investigated by Chakravarty and co-workersand Ru(II) bphen complexes (2，3) investigated by Glazer and co-workers as Photodynamic Therapyphotosensitizers (PDT PSs). Figure 2. Chemical structures of the Fe(II) complexes investigated in this work. Complexes 4 wasisolated as a PF6 salt and the complexes 5 and 6 were isolated as Na salts. 2. Results and Discussion 2.1. Synthesis and Characterization The complexes that were synthesized in this work are visualized in Figure 2. The synthesis ofcomplex 4 has already been reported in the literature [65]， contrary to those of complexes 5 and 6. All ofthe complexes were synthesized by the complexation of bphen and its derivatives with FeCl·4H2O.The identity of all complexes was confirmed by 1H， 13C-NMR， and HRMS， and the purity by highperformance liquid chromatography (HPLC) and elemental analysis (Figures S1-S9). 2.2. Photophysical Properties After full chemical characterization， the photophysical properties of the prepared complexes wereevaluated in view of potential applications as PDT PS. It is well established that the used wavelength ina PDT treatment has direct influence on the tissue light penetration depth and therefore the possibilityto treat deep tumors. It is important to mention that there is no ideal PS for PDT due to the variousapplications of this technique as well as the different depth of a tumor. For deep seated tumors，absorption in the near-infrared region (NIR) is desired to have an increased light penetration depth.However， if the tumor is seated only on the surface， a deep penetration would be undesirable due tothe possibility of damaging healthy underlying tissue. Worthy of note， PSs with very low extinctioncoefficients in the red region have already been shown to still be able to be activated [18，66，67].Based on this， a major aim in the development of new (metal-based) PSs is a red shift of the absorptiontowards the therapeutic window (600 to 900 nm) [16]. Keeping this in mind， the absorption profile ofall complexes (Figure 3) was measured in CH3CN. Importantly， the compounds showed an absorptionmaximum at 530 nm for complex 4 and 535 nm for complexes 5 and 6 with an absorption tail in thephoto-therapeutic window. For further investigation of the photophysical properties， the complexeswere excited at 450 nm and the emission measured in CHCN. The luminescence of all complexeswas found to be between 550 and 800 nm with an emission maximum at 610 nm for compound 4 and620 nm for compounds 5 and 6 (Figures S10-S12). Worthy of note， the complexes were found to be onlyweakly emissive and the emission could only be measured at the detection limit of our apparatus withluminescence quantum yields <0.01% in CH3CN. Additionally， the measurement of the excited statelifetimes in degassed and aerated CH3CN was not possible due to a necessary minimal delay betweenexcitation and detection with our apparatus， indicating that the compounds 4-6 have lifetimes <29 ns.These observations are fitting with other excited state studies， which confirm the low luminescenceand very short lifetime of Fe complexes [68，69]. The comparison between all investigated complexesshows very similar absorption and emission properties， indicating that the additional sulfonic acidgroup in complex 5 and 6 does not significantly change the photophysical properties when comparedto complex 4. Figure 3. Normalized UV /Vis spectra of the complexes 4-6 in CHCN. 2.3. Singlet Oxygen Generation Althoughthere can be radicals involved， for most approved PSs， the formation of singlet oxygen('O2) is the predominant species responsible for cell/bacteria death. Therefore， to assess the potentialof our PSs， we have investigated their ability to produce 102 upon light irradiation. To quantitativelyevaluate this， we used two methods： (1) a direct method by measurement of the phosphorescence of 102 and (2) an indirect method by measurement of the absorption changes of a reporter molecule [23，70].The results (Table 1) show that complexes 4-6 are poorly producing 102 with quantum yields from 2%to 4% in aerated CH3CN and <1% in aerated PBS at the detection limit of our used system. These lowvalues can be explained by the very short lifetime of the excited state. These short lifetimes are limitingthe necessary energy transfer from the excited MLCT state to molecular oxygen (32) to generate 102. Table 1. Singlet oxygen quantum yields (d(O2)) in CHgCN and aqueous solution determined bya direct and an indirect method upon excitation at 450 nm. Average of three independent measurements(±10%). Compounds CH3CN CH3CN D2O PBS Direct Method Indirect Method Direct Method Indirect Method 4 n.d. 4% n.d. <1% 5 n.d. 2% n.d. <1% 6 n.d. 2% n.d. <1% n.d. = not determinable. 2.4. Stability in Human Plasma In PDT， the stability of the PS is crucial. Therefore， the stability of our complexes under physiologicalconditions， more specifically in human plasma， was investigated. For this purpose， all of the complexeswere incubated in human plasma at 37°C in the dark for 48h. After extraction of the PS， its stabilitywas analysed via high performance liquid chromatography (HPLC). As an internal reference， coffeine，which has already been shown to be stable under these conditions [71]， was used. For analysis of thechromatogramms (Figures S13-S15)， the signal of the coffeine and of our complexes were integrated andthe ratios before and after incubation in human plasma were compared. Since the ratio did not changesignificantly， we concluded that there is no decomposition in the tested time interval， confirming hencethe stability of the compounds 4-6. 2.5. Photostability Next to the stability in an biological environment， the stability of our compounds upon lightexposure was investigated. The currently used PSs which are based on a tetrapyrrolic scaffold areknown to undergo structural physical modications (i.e.， photobleaching) upon light irradiation [72，73].To investigate this potential property of our metal complexes， they were exposed to a continous LEDirradiation for 120 min at 450 nm. The potential change of the absorbtion properties of the complexeswere monitored by UV/Vis spectroscopy. As a positive control，[Ru(bipy)3]Cl2 was used， whereasProtoporphyrin IX (PpIX) was employed as a negative control. The comparison of the temporalchange of the investigated complexes 4-6 shows only a small decrease in the absorption profile for ourthree complexes (Figures S16-S20)， in a similar fashion than [Ru(bipy)3]Cl2. This indicates that lightexposure has only a minimal effect on the stability of the compounds. 2.6. Distribution Coefficient It is well known that the lipophilicity of a compound plays a crucial role in the extent of itscellular uptake and its cellular localisation [74，75]. Therefore， we investigated the lipophilicity ofall synthesised compounds by using the "shake-flask" method. The complex was dissolved andrepeatedly shaken between a PBS and octanol phase. After equilibration， the distribution betweenthe organic and water phase was determined. As expected， due to the large lipophilic bphen ligands，compound 4 was found in the organic phase (Table 2). In comparison， complexes 5 and 6 with theadditional sulfonic acid groups were found in the aqueous phase. Worthy of note， the Ru(II)-basedanalogous compounds were found to have very similar log P values [54]， indicating that the metalion does not significantly influence this value. Interestingly， compound 6 is more lipophilic than compound 5. This can be explained by the location of its sulfonic acid groups that are not decorated atthe outside of the complex. They are therefore a bit shielded and they do not interact with water asmuch as complex 5. Table 2. Distribution coefficients of the compound 4-6 between Phosphate-Buffered Saline (PBS) andoctanol. Average of three independent measurements. Compound log P 4 +1.7±0.1 5 -2.2±0.1 6 -1.9±0.1 2.7. Cytotoxicity and Photocytotoxicity After having asssessed the (photo-)physical properties of our complexes， we have investigatedthe influence of our compounds on the cell viability in the dark and upon light irradiation.For this purpose， compounds 4-6 were incubated in non-cancerous retinal pigment epithelium (RPE-1)and human cervical carcinoma (HeLa) cells in the dark and upon irradiation at 480 and 540 nm.After 48 h， cell viability was assessed by exposing the cells to resazurin. An overview of the IC50values is presented in Table 3. Interestingly， complex 4 was found to be toxic in the dark in the lowmicromolar range， as the anticancer drug cisplatin. Disapointingly， light exposure had only a smalleffect on cell viability. These results were expected due to the poor generation of 102 upon lightirradiation of our PSs. The phototoxic index (PI) is defined as the ratio between the IC50 value inthe dark and the IC50 value upon light exposure. Complex 4 was found to have a PI value of 1.2 at480 nm and 1.1 at 540 nm for RPE-1 cells and 1.3 at 480 nm and 1.4 at 540 nm in HeLa cells， respectively.These values are extremely low in comparison to established PDT PSs. In comparison， complexes 5and 6 have no measurable cytotoxic effect in both the dark or upon light irradition. Worthy of note，the dark cytotoxcities of 4 and 5 are in a very similar range than the Ru(II) analogous， which havebeen investigated by Glazer and co-workers in other cancer cell lines. This implies that the metalcore is not primary responsible for the observed dark toxicity [54]. The comparison between thenon-cancerous cell line RPE-1 and the human cervical carcinoma cell line HeLa shows no selectivityfor cancer. Overall， this study demonstrates that a small structural change， like additional sulfonicacid groups at the outer sphere of the complex， completely changes the effect that these compoundshave on the cells. Table 3. IC5o values in the dark and upon irradiation for the complexes 4-6 incubated in humancervical carcinoma (HeLa) and non-cancerous retinal pigment epithelium (RPE-1) cells. Average ofthree independent measurements. HeLa RPE-1 IC50/uM IC50/uMIIC50/uMPIC50/uMPI IC50/uMIPIC50/uMPI Dark 480 nm 540 nm Dark 480 nm 540 nm 4 9.3±0.8 7.2±0.6 1.3 6.6±0.1 1.4 10.8±1.0 9.3±0.5 1.2 9.8±0.5 1.1 5 >100 >100 n.d. >100 n.d. >100 >100 n.d. >100 n.d. 6 >100 >100 n.d. >100 n.d. >100 >100 n.d. >100 n.d. PpIX >100 2.5±0.1 >40 2.1±0.3 >48 >100 3.8±0.1 >26 3.1±0.1 >32 Cisplatin 10.5±0.8 - - - - 29.3±1.4 - - - n.d. = not determinable. 3. Materials and Methods 3.1. Instrumentation and Methods 1H- and 13C-NMR spectra were recorded on a 400 MHz NMR spectrometer (Bruker France，Wissembourg， France). Chemical shifts (8) are reported in parts per million (ppm)referenced to tetramethylsilane (8 0.00) ppm using the residual proton solvent peaks as internal standards. Couplingconstants (J) are reported in Hertz (Hz) and the multiplicity is abbreviated， as follows： s (singlet)，d (doublet)， and m (multiplet). Electrospray Ionization-Mass Spectrometry (ESI-MS) experiments werecarried out using a LTQ-Orbitrap XL from Thermo Scientific (Thermo Fisher Scientific， Courtaboeuf，France) and operated in positive ionization mode， with a spray voltage at 3.6 kV. No sheath andauxiliary gas was used. Applied voltages were 40 and 100 V for the ion transfer capillary and thetube lens， respectively. The ion transfer capillary was held at 275 °C. Detection was achieved in theOrbitrap with a resolution set to 100，000 (at m/z 400) and an m/z range between 150 and 2000 inprofile mode. Spectrum was analyzed using the acquisition software XCalibur 2.1 (Thermo FisherScientific， Courtaboeuf， France). The automatic gain control(AGC)allowed for the accumulation of upto 2 ×10 ions for Fourier Transform Mass Spectrometry (FTMS) scans， maximum injection time wasset to 300 ms and 1 us scan was acquired. 10 uL was injected using a Thermo Finnigan Surveyor HPLCsystem (Thermo Fisher Scientific， Courtaboeuf， France) with a continuous infusion of methanol at100 uL·min-1. Elemental microanalyses were performed on a Thermo Flash 2000 elemental analyzer.For analytic HPLC， the following system has been used： 2×Agilent G1361 1260 Prep Pump system(Agilent Technologies， Massy， France) with Agilent G7115A 1260 DAD WR Detector equipped withan Agilent Pursuit XRs 5C18 (Analytic： 100 A， C18 5 um 250×4.6 mm， Preparative： 100 A， C185 um250 ×300 mm) Column and an Agilent G1364B 1260-FC fraction collector. The solvents (HPLC grade)were millipore water (0.1% TFA， solvent A) and acetonitrile (0.1% TFA， solvent B). The followingmethod was used： 0-3 min： isocratic 95% A (5%B)； 3-17 min： linear gradient from 95% A (5% B) to 0%A (100% B)； 17-23 min： isocratic 0% A (100% B). 3.2. Materials All of the chemicals were obtained from commercial sources and were used without furtherpurification. 4，7-Diphenyl-1，10-phenanthroline (98%) and Iron (II) chloride tetrahydrate (98%) wereobtained from Alfa Aesar (Schiltigheim， France). Disodium-4，7-Diphenyl-1，10-phenanthroline-4 4"-disulfonicacid hydrate (98%) and Disodium-4，7-Diphenyl-1，10-phenanthroline-3，8-disulfonic acid hydrate (98%)were obtained from abcr GmbH (Karlsruhe， Germany). Dulbecco’s Modified Eagles Medium (DMEM)，Dulbecco's Modified Eagles Medium supplemented with nutrient mixture F-12 (DMEM/F-12)， Fetal BovineSerum (FBS)， Gibco Penicillin-Streptomycin-Glutamine (Penstrep)， Dulbecco’s Phosphate-Buffered Saline(PBS) were purchased from Fisher Scientific (Illkirch， France)，and Resazurin from ACROS Organics (Noisyle Grand，France). 3.3. Synthesis Fe(4，7-Diphenyl-1，10-phenanthroline)3(PF6)2(1)： The synthesis of Fe(4，7-Diphenyl-1，10-phenanthroline)3(PF6)2is already published [65]. However， in this work， another synthetic route was employed. 4，7-Diphenyl-1，10-phenanthroline (750 mg， 2.26 mmol) and FeCl24H2O(128 mg， 0.65 mmol) were suspended in ethanol(50 mL) under nitrogen atmosphere and the mixture was refluxed for 16 h. After that， the solution was cooleddown and undissolved residue was removed via filtration. To the obtained solution， an aqueous saturatedsolution of NH4PF6(5mL) was added. The crude product， which precipitated as a PF6 salt， was collectedby centrifugation and washed with EtOH (20 mL)， HzO (50 mL)， and EtzO (50 mL). The crude productwas dissolved in dichloromethane (100 mL) and washed with a 5% LiC aqueous solution (3×100 mL)，brine (3×100 mL) and H2O(3×100 mL). The obtained solid was washed with pentane (100 mL) and driedunder vacuum. 794 mg of 1 (0.59 mmol， 91%) were yielded as a dark red solid. 1H-NMR (400 MHz， CD3CN，298 K)： 8.25 (s， 6H)， 7.94 (d，6H， J=5.4 Hz)， 7.67-7.60 (m， 30 H)；13C-NMR (100 MHz， CD3CN， 298 K)： 156.4，151.3， 150.6， 136.5， 130.7， 130.7，130.1， 129.5，127.1，126.9； ESI-HRMS： m/z 526.1640 [M]2+ (calcd C72H48N6Fe，526.1639)； Elemental analysis for C72H48F12FeN6P2+C5H12 (1427.12)： Calcd.： C 65.33； H4.28；N 5.94； Found：C 65.72；H4.15； N 5.94. Fe(4，7-Diphenyl-1，10-phenanthroline-4 ，4"-disulfonic acid)3Na6 (2)： Disodium-4，7-Diphenyl-1，10-phenanthroline-4 ，4"-disulfonic acid (600 mg， 1.08 mmol) and FeCl24H2O (61 mg， 0.31 mmol) weresuspended in ethanol (50 mL) under nitrogen atmosphere and the mixture was refluxed for 14 h.After that， the solution was cooled down and undissolved residue was removed via filtration. The crudeproduct was dissolved in a H2O/CHCN solution (50 mL)and fractionated precipitated by addingdropwise Et2O. The obtained solid was washed with CHCl3 (20 mL) and Et2O (50 mL). The productwas isolated after recrystallization from EtOH/Pentane. The obtained product was dried undervacuum. 211 mg of 2 (0.13 mmol， 42%) were yielded as a dark solid.H-NMR (400 MHz， CD3OD，298 K)： 8.34 (s， 6H)， 8.12-8.03 (m， 18H)， 7.80-7.70 (m， 18H)；13C-NMR (100 MHz， CD3OD， 298 K)：156.8， 151.8， 150.6， 147.6，137.0，132.7，130.0， 128.4，127.8， 127.7； ESI-HRMS m/z 381.5065 [M]4-(calcdC72H42N6O18S6Fe，381.5063)； Elemental analysis for C72H42FeNNa6O18S6(1665.30)： Calcd.： C 51.93；H 2.54； N 5.05； Found： C 51.86；H2.50；N 4.92. Fe(4，7-Diphenyl-1，10-phenanthroline-3，8-disulfonic acid)3Na6 (3)： Disodium-4，7-Diphenyl-1，10-phenanthroline-3，8-disulfonic acid (696 mg， 1.30 mmol) and FeCl2·4H2O (81 mg， 0.41 mmol) weresuspended in ethanol (50 mL) under nitrogen atmosphere and the mixture was refluxed for 16 h.After that， the solution was cooled down and undissolved residue was removed via filtration. The crudeproduct was dissolved in a H2O/CH3CN solution (50 mL) and fractionated precipitated by addingdropwise Et2O. The product was isolated after recrystallization from EtOH/Pentane. The obtainedproduct was dried under vacuum. 178 mg of 3 (0.11 mmol， 27%) were yielded as a dark solid. 1H-NMR(400 MHz， CD3OD， 298 K)： 8.35 (s， 6H)， 8.12-8.01 (m， 18H)， 7.81-7.65 (m， 18H)；13C-NMR (100MHz，CD3OD，298K)：156.8，151.8，150.6，147.6，137.1，132.7，130.0，128.4，127.7，125.4.ESI-HRMS m/z 381.5063[M]4-(calcd C72H42N6O18S6Fe， 381.5063)； Elemental analysis for C72H42FeN6Na6O18S6 (1665.30)：Calcd.： C 51.93；H2.54；N5.05； Found： C 51.80；H 2.47； N 4.93. 3.4. Spectroscopic Measurements The absorption of the samples has been measured with a SpectraMax M2 Spectrometer (MolecularDevices， San Jose， CA， USA). The emission was measured by irradiation of the sample in fluorescencequartz cuvettes (width 1 cm) using a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla，Vilnius， Lithuania) at 450 nm. Luminescence was focused and collected at a right angle to the excitationpathway and it was directed to a Princeton Instruments Acton SP-2300i monochromator. As a detectora PI-Max 4 CCD camera (Princeton Instruments， Trenton，NJ，USA) has been used. 3.5. Luminescence Ouantum Yield Measurements For the determination of the luminescence quantum yield， the samples were prepared in a CHCNsolution with an absorbance of 0.1 at 450 nm. This solution was irradiated in fluorescence quartz cuvettes(width 1 cm) using a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla， Vilnius， Lithuania)at 450 nm. The emission signal was focused and collected at a right angle to the excitation pathway andit was directed to a Princeton Instruments Acton SP-2300i monochromator. As a detector， a PI-Max 4CCD camera (Princeton Instruments， Trenton， NJ， USA) has been used. The luminescence quantumyields were determined by comparison with the reference [Ru(bipy)3]Cl2 in CH3CN (Dem=5.9%) [76]applying the following formula： Pem =luminescence quantum yield， F= fraction of light absorbed， I = integrated emission intensities，n = refractive index，A=absorbance of the sample at irradiation wavelength. 3.6. Lifetime Measurements For the determination of the lifetimes， the samples were prepared in an air saturated and ina degassed CH3CN solution with an absorbance of 0.1 at 355 nm. This solution was irradiated influorescence quartz cuvettes (width 1 cm) using a NT342B Nd-YAG pumped optical parametricoscillator (Ekspla， Vilnius， Lithuania) at 450 nm. The emission signal was focused and collectedat right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300imonochromator. As a detector， a R928 photomultiplier tube (Hamamatsu， Hamamatsu， Japan) hasbeen used. 3.6.1. Singlet Oxygen Measurements-Direct evaluation The samples were prepared in an air saturated CHCN or D2O solution with an absorbanceof 0.2 at 450 nm. This solution was irradiated in fluorescence quartz cuvettes (width 1 cm) usinga mounted M450LP1 LED (Thorlabs， Dachau， Germany)， whose irradiation， centered at 450 nm， has beenfocused with aspheric condenser lenses. The intensity of the irradiation has been varied using a T-CubeLED Driver (Thorlabs， Dachau， Germany) and measured with an optical power and energy meter.The emission signal was focused and collected at right angle to the excitation pathway and directed toa Princeton Instruments Acton SP-2300i monochromator. A longpass glass filter was placed in front ofthe monochromator entrance slit to cut off light at wavelengths shorter than 850 nm. As a detector anEO-817L IR-sensitive liquid nitrogen cooled germanium diode detector (North Coast Scientific Corp.，Santa Rosa， CA，USA) has been used. The singlet oxygen luminesce at 1270 nm was measured byrecording spectra from 1100 to 1400 nm. For the data analysis， the singlet oxygen luminescence peaks atdifferent irradiation intensities were integrated. The resulting areas were plotted against the percentageof the irradiation intensity and the slope of the linear regression was calculated. The absorbance ofthe sample was corrected with an absorbance correction factor. As reference for the measurement inan CHgCN solution phenalenone (phenaleone =95%) [77] and for the measurement in a D2O solution[Ru(bipy)3]Clz(PRu(bipy)3Cl2=22%) [78] was used and the singlet oxygen quantum yields were calculatedwhile using the following formula： D=singlet oxygen quantum yield， S = slope of the linear regression of the plot of the areas of the singletoxygen luminescence peaks against the irradiation intensity， I = absorbance correction factor， Io=lightintensity of the irradiation source， and A =absorbance of the sample at irradiation wavelength. 3.6.2. Singlet Oxygen Measurements - Indirect evaluation For the measurement in CH3CN： The samples were prepared in an air-saturated CH3CN solutioncontaining the complex with an absorbance of 0.2 at the irradiation wavelength， N，N-dimethyl-4-nitrosoaniline aniline (RNO， 24 uM) and imidazole (12mM). For the measurement in PBS buffer：The samples were prepared in an air-saturated PBS solution containing the complex with an absorbanceof 0.1 at the irradiation wavelength，N，N-dimethyl-4-nitrosoaniline aniline (RNO， 20 uM)， and histidine(10 mM). The samples were irradiated on 96 well plates with an Atlas Photonics LUMOS BIOirradiator (Atlas Photonics， Fribourg， Switzerland) for different times. The absorbance of the sampleswas measured during these time intervals with a SpectraMax M2 Microplate Reader (MolecularDevices， San Jose， CA， USA). The difference in absorbance (A0-A) at 420 nm for the CHCNsolution or at 440 nm a PBS buffer solution was calculated and plotted against the irradiation times.From the plot， the slope of the linear regression was calculated as well as the absorbance correctionfactor determined. The singlet oxygen quantum yields were calculated using the same formulas asused for the direct evaluation. 3.7. Stability in Human Plasma The stability of the complexes was evaluated with caffeine as an internal standard， which hasalready been shown to be suitable for these experiments [71]. The pooled human plasma was obtainedfrom Biowest and caffeine from TCI Chemicals. Stock solutions of the compounds (20 uM) and caffeine(40 uM) were prepared in DMSO.One aliquot of the solutions was added to 975 uL of human plasmato a total volume of 1000 uL. Final concentrations of the compounds of 0.25 uM and caffeine of 0.5 uMwere achieved. The resulting solution was incubated for 48 h at 37 °C with continuous gentle shaking(ca.300 rpm). The reaction was stopped after the incubation time by the addition of 3 mL of methanol.The mixture was centrifuged for 60 min at 3000 rpm at 4°C. The methanolic solution was filteredthrough a 0.2 um membrane filter. The solvent was evaporated under reduced pressure and the residuewas dissolved in 1：1 (u/u) CHCN/HzO 0.1% TFA solution. The solution was filtered through a 0.2 ummembrane filter and analyzed using a HPLC System (Agilent Technologies， Massy， France). 3.8. Photostability The samples were prepared in an air saturated CHgCN solution with an absorbance of about 0.5 at450 nm. To measure the photostability， the samples were irradiated at 450 nm in 96 well plates with anAtlas Photonics LUMOS BIO irradiator (Atlas Photonics， Fribourg， Switzerland) during time intervalsfrom 0 to 120 min. The absorbance spectrum from 350 to 700 nm was recorded with a SpectraMax M2Microplate Reader (Molecular Devices， San Jose， CA， USA) after each time interval and then compared.As a positive control [Ru(bipy)3]Cl2 and as a negative control Protoporphyrin IX has been used. 3.9. Distribution Coefficient The lipophilicity of a compound was determined by measuring its distribution coefficient betweenthe PBS and Octanol phase by using the "shake-flask" method. For this technique， the used phaseswere previously saturated in each other. The compound was dissolved in the phase (A) with its majorpresence with an absorbance of about 0.5 at 450 nm. This solution was then mixed with an equalvolume of the other phase (B) at 80 rpm for 8 h with an Invitrogen sample mixer and then equilibratedovernight. The phase A was then carefully separated from phase B. The amount of the compound beforeand after the sample mixing was determined by UV/Vis spectroscopy at 450 nm using a SpectraMaxM2 Microplate Reader (Molecular Devices， San Jose， CA，USA). The evaluation of the complexes wasrepeated three times and the ratio between the organic and aqueous phase calculated. 3.10. Cell Culture Human cervical carcinoma (HeLa) cells were cultured using DMEM media and retinal pigmentepithelium (RPE-1) cells using DMEM/F-12 with addition of 10% FBS and 1% penstrep. The cellswere cultivated and maintained at 37 °C in a cell culture incubator at 37 °C with 5%CO2 atmosphere.Before an experiment， the cells were passaged three times. 3.11. Cytotoxicity and Photocytotoxicity The cytotoxicity of the compounds was accessed by measuring the cell viability using a fluorometricresazurin assay. The cultivated cells were seeded in triplicates in 96 well plates with a density of 4000cells per well in 100 uL of media. After 24 h， the medium was removed and the cells were treated withincreasing concentrations of the compound diluted in cell media achieving a total volume of 200 uL.The cells were incubated with the compound for 4 h. After this time， the media was removed andreplaced with 200 uL of fresh medium. For the phototoxicity studies， the cells were exposed to lightwith an Atlas Photonics LUMOS BIO irradiator (Atlas Photonics，Fribourg， Switzerland). Each well wasconstantly illuminated with either a 480 nm or 510 nm irradiation. During this time， the temperaturewas maintained constantly at 37 C. The cells were grown in the incubator for an additional 44 h. For thedetermination of the dark cytotoxicity， the cells were not irradiated and after the medium exchange directly incubated for 44 h. After this time， the medium was replaced with fresh medium containingresazurin with a final concentration of 0.2 mg/mL. After 4 h incubation，the amount of the fluorescentproduct resorufin was determined upon excitation at 540 nm and measurement of its emission at 590 nmusing a SpectraMax M2 Microplate Reader (Molecular Devices， San Jose， CA， USA). The obtained datawas analyzed with the GraphPad Prism software. 4. Conclusions In this work， we report on the characterization and biological properties of three Fe(Il)complexes containing different derivatives of 4，7-diphenyl-1，10-phenanthroline (bphen) (complexes 4-6).The complexes were found to absorb strongly in the red region， which is a desired and importantrequirement for PDT treatment of deep cancers. Stability studies in human plasma as well as photostabilitystudies showed that the investigated complexes are stable in physiological conditions and importantly donot have a photobleaching effect as some of the currently approved PDT PSs. Photophysical studies ofthese compounds revealed that they are weakly luminescent， have a very short lived excited state， and arepoorly producing singlet oxygen. Interestingly， the addition of sulfonic acid groups to the bphen ligand togive complexes 5 and 6 do not significantly change these properties. However， as expected， these groupsare able to strongly increase the water solubility of the complexes. Complexes 5 and 6 were shown to haveno cytotoxic effect in the dark as well as upon light exposure contrary to compound 4 that had a darktoxicity in the low micromolar range. Light exposure was shown to have only a small effect on cell viability.We assume that this is due to the poor photophysical properties (i.e.， short luminescent lifetimes and poor-O2 production) of the complexes. We are currently investigating other options to prepare Fe(II) complexeswith longer luminescent lifetimes， which will be more appropriate for PDT applications. Supplementary Materials： The following are available online at http：//www.mdpi.com/2304-6740/7/1/4/s1，Figure S1.1H-NMR spectrum of 4 in CD3CN， 400 MHz， Figure S2. 13C-NMR spectrum of 4 in CD3CN， 100 MHz，Figure S3. ESI-HRMS spectrum of 4 (positive detection mode)， Figure S4. 1H-NMR spectrum of 5 in CD，OD，400 MHz， Figure S5. 13C-NMR spectrum of 5 in CD3OD， 100 MHz， Figure S6. ESI-HRMS spectrum of 5 (negativedetection mode)， Figure S7. 1H-NMR spectrum of 6 in CD3OD， 400 MHz， Figure S8. 13C-NMR spectrum of 6in CD3OD， 100 MHz， Figure S9. ESI-HRMS spectrum of 6 (negative detection mode)， Figure S10. NormalisedEmission Spectrum of 4 in CH3CN， Figure S11. Normalised Emission Spectrum of 5 in CHCN， Figure S12.Normalised Emission Spectrum of 6 in CH3CN， Figure S13. HPLC chromatogram of Caffeine (internal standard)and 4 after 0 and 48 h incubation in human pooled plasma， Figure S14. HPLC chromatogram of Caffeine (internalstandard) and 5 after 0 and 48 h incubation in human pooled plasma， Figure S15. HPLC chromatogram of Caffeine(internal standard) and 6 after 0 and 48 h incubation in human pooled plasma， Figure S16. Temporal change of theUV /Vis spectra of [Ru(bipy)3]Cl2 by irradiation at 450 nm in CH3CN， Figure S17. Temporal change of the UV/Visspectra of 4 by irradiation at 450 nm in CHCN， Figure S18. Temporal change of the UV/Vis spectra of 5 byirradiation at 450 nm in CH3CN， Figure S19. Temporal change of the UV/Vis spectra of 6 by irradiation at 450 nmin CHCN， Figure S20. Temporal change of the UV/Vis spectra of PpIX by irradiation at 450 nm in CHCN. 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Acta 1996，79，1222-1238.[C ro s sRef] ) 2019 by the authors. Licensee MDPI， Basel， Switzerland. This article is an open accesscc article distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http：//creativecommons.org/licenses/by/4.0/). Cancer is known to be one of the major causes of death nowadays. Among others,chemotherapy with cisplatin is a commonly used treatment. Although widely employed, cisplatin is known to cause severe side effects, such as nerve and kidney damage, nausea, vomiting, and bone marrow suppression. Most importantly, a number of cancer tumors are acquiring resistance to cisplatin, limiting its clinical use. There is therefore a need for the discovery of novel anticancer agents. Complementary to chemotherapy, Photodynamic Therapy (PDT) has expanded the range of treatment opportunities of numerous kinds of cancer. Nonetheless, the currently approved PDT photosensitizers (PSs) suffer from major drawbacks, which include poor water solubility or photobleaching, in addition to a slow clearance from the body that causes photosensitivity. Due to these limitations, there is a need for the development of new PDT PSs. To overcome these problems, a lot of research groupsaround the world are currently focusing their attention towards the development of new metalcomplexes as PDT PSs. However, most synthesized compounds reported so far show limited use due to their poor absorption in the phototherapeutic window. Herein, we report on the preparation and characterization of three Fe(II) polypyridine complexes (4–6) and evaluate their potential as both anticancer agents and PDT PSs. Very importantly, these compounds are stable in human plasma, photostable upon continuous LED irradiation, and absorb in the red region of the spectrum. We could demonstrate that through additional sulfonic acid groups on the polypyridine ligand being used (bphen: 4,7-diphenyl-1,10-phenanthroline), the water solubility of the complexes could be highly improved, whereas the photophysical properties did not significantly change. One of these complexes (4) shows interesting toxicity, with IC50 values in the low micromolar range in the dark as well as some phototoxicity upon irradiation at 480 and 540 nm against RPE-1 and HeLa cells.