A Dogma in Doubt: Hydrolysis of Equatorial Ligands of PtIV Complexes under Physiological Conditions

Abstract Due to their high kinetic inertness and consequently reduced side reactions with biomolecules, PtIV complexes are considered to define the future of anticancer platinum drugs. The aqueous stability of a series of biscarboxylato PtIV complexes was studied under physiologically relevant conditions. Unexpectedly and in contrast to the current chemical understanding, especially oxaliplatin and satraplatin complexes underwent fast hydrolysis in equatorial position (even in cell culture medium and serum). Notably, the resulting hydrolysis products strongly differ in their reduction kinetics, a crucial parameter for the activation of PtIV drugs, which also changes the anticancer potential of the compounds in cell culture. The discovery that intact PtIV complexes can hydrolyze at equatorial position contradicts the dogma on the general kinetic inertness of PtIV compounds and needs to be considered in the screening and design for novel platinum‐based anticancer drugs.

Dedicated to Professor Wolfgang Weigand on the occasion of his 60th birthday Abstract: Due to their high kinetic inertness and consequently reduced side reactions with biomolecules,P t IV complexes are considered to define the future of anticancer platinum drugs. The aqueous stability of as eries of biscarboxylato Pt IV complexes was studied under physiologically relevant conditions.U nexpectedly and in contrast to the current chemical understanding,e specially oxaliplatin and satraplatin complexes underwent fast hydrolysis in equatorial position (even in cell culture medium and serum). Notably,t he resulting hydrolysis products strongly differ in their reduction kinetics, acrucial parameter for the activation of Pt IV drugs,whichalso changes the anticancer potential of the compounds in cell culture.The discovery that intact Pt IV complexes can hydrolyze at equatorial position contradicts the dogma on the general kinetic inertness of Pt IV compounds and needs to be considered in the screening and design for novel platinum-based anticancer drugs.
Pt II complexes still play av ery important role in cancer treatment, [1] contributing to about 50 %o fa ll chemotherapies. [2] Cis-, carbo-, and oxaliplatin are widely used against various forms of cancer. [3] Furthermore,v ery recent clinical data show impressive synergistic effects of platinum drugs together with checkpoint inhibitor immunotherapy. [4] The mode of action of these metal complexes involves aquation, binding to DNA, and subsequently induced apoptosis. [5] However,P t II complexes bind to DNAn ot only in the tumor tissue,b ut also in healthy cells,r esulting in (severe) side effects like nephro-, neuro-, and gastrointestinal-tract toxicity. [6] To reduce these adverse effects,Pt IV complexes are attracting increasing interest. [7] Such low-spin d 6 octahedral complexes are considered to be kinetically more inert [8] and consequently far less reactive towards biomolecules. [9] Activation by reduction to the active Pt II complexes can occur via low-molecular-weight compounds like ascorbate and glutathione, [10] and/or high-molecular-weight proteins. [11] Another advantage of Pt IV complexes are the two additional axial ligands,w hich can be used to optimize chemical and biological properties,l ike lipophilicity and reduction potential. Furthermore,b ioactive substances or drug-targeting moieties can be used as axial ligands. [8b,12] Despite these benefits,n oP t IV drug has been clinically approved so far. [2] Nevertheless,s ome candidates have entered clinical trials. However,T etraplatin, ctc-[Pt(DACH)Cl 4 ]( DACH = (1R,2R)-(À)-1,2-diaminocyclohexane), was discontinued because of its neurotoxicity, [13] while Iproplatin, ctc-[Pt(IPA) 2 -(OH) 2 Cl 2 ]( IPA = isopropylamine), did not show superior anticancer activity. [14] Them ost prominent representative Satraplatin, ctc-[Pt(NH 3 )(CHA)(OAc) 2 Cl 2 ]( CHA = cyclohexylamine), ultimately failed to show improved overall survival in aphase III study. [2] Nevertheless,current research strongly focuses on Pt IV complexes,asthey are considered to be the next generation of platinum-based anticancer drugs. [7] Consequently,t he underlying chemical properties and reactivities are of high importance for the specific design of novel drugs with optimized biological properties.A sa lready mentioned above,P t IV complexes are considered to be kinetically inert. However,t here are af ew reports that the axial ligands can be hydrolyzed when electron-withdrawing ligands like dichloroacetate are present. [15] Concerning the equatorial ligands of Pt IV complexes,astudy using plasma from patients treated with Satraplatin showed not only the parent Pt II drug JM118, but unexpectedly also the mono-and dihydrated Pt IV species,where one or two equatorial chlorido ligands were exchanged with hydroxido ligands. [16] This is in strong contrast to the "dogma" of kinetic inertness of Pt IV drugs.A ss uch ah ydrolysis would strongly impact the chemical characteristics (e.g. reduction potential) and con-sequently also the biological properties,w ei nvestigated in this study the hydrolysis of the equatorial ligands for apanel of different Pt IV complexes in comparison to Satraplatin ( Figure 1).
To get afirst overview,weanalyzed the hydrolytic stability of the model complexes 1-3 and satraplatin 4 ( Figure 1) under physiologically relevant conditions.A ll complexes possess two acetato ligands in axial position and an equatorial core consisting of cisplatin (1), carboplatin (2), oxaliplatin (3), or cis-amminedichlorido(cyclohexylamine) (4). Thec omplexes were incubated in phosphate buffer (PB) at 37 8 8Cand pH 7.4 for 24 ha nd monitored using HPLC-MS.I ndeed, after 24 h most of the satraplatin (72 %hydroxido and 3% dihydroxido species) and the oxaliplatin-based complex 3 (43 %hydroxido and 14 %d ihydroxido;F igure 2) was already hydrolyzed. In contrast, the cisplatin analogue 1 was widely stable (5 % hydroxido species) and the carboplatin derivative remained completely unchanged ( Figure S1). To exclude as ignificant impact of the column on the hydrolysis process,the incubated solutions were also directly injected into the mass spectrometer. This revealed the same hydrolyzed products as observed with HPLC-MS (data not shown).
Notably,r epetition of these experiments at pH values of 8.0 and 9.0 even resulted in distinctly accelerated hydrolysis (Table 1). At pH 9.0 the parental oxaliplatin derivative 3 completely disappeared with exclusive formation of the dihydroxido species.A lso satraplatin was fully converted into its hydrolyzed species (25 %h ydroxido/75 %d ihydroxido). In contrast, in the case of the cisplatin analogue 1,s till % 70 %ofthe intact complex could be observed together with % 20 %h ydroxido and 5% dihydroxido species.C arboplatin was again completely unaffected. These findings indicated ad istinct pH-dependencyo ft he reaction, which could be exemplarily proven for 3,w hich displayed ap erfect linear correlation between pH and the reaction rate ( Figure S2).
To rule out the involvement of phosphate in the hydrolysis,o ther buffer systems,t hat is,H EPES and ammonium carbonate,were investigated for 3 yielding comparable results ( Figure S3). Next, 3 was investigated in cell culture medium (RPMI-1640), and similar ratios of hydroxido/dihydroxido complex formation were observed as in pure PB solution ( Figure S3). Notably,t he pH value of fresh cell culture medium was about 7.4 but increased up to pH 8-9 within 24 h. Consequently,t he cell culture medium was phosphate-buffered (150 mm), generating stable pH values over > 24 h. Finally,t he hydrolysis of 3 was also investigated in mouse serum (also buffered with 150 mm phosphate), again yielding similar results ( Figure S3). This clearly indicates that all the components of cell culture medium or serum (amino acids, proteins,i norganic salts,a nd vitamins) have no influence on the hydrolysis process of the Pt IV complex. Instead this reaction is solely dependent on the presence of water as asolvent and the appropriate pH value.
Notably,t he hydrolysis of oxaliplatin with % 10 % [17] is distinctly slower than that of 3 with % 50 %a fter 24 ha t   pH 7.4. Al ikely explanation is that the Pt II complex with am onodentate oxalate ligand possesses ap K a of 7.23 [18] and therefore an aqua ligand is partially present. This enables af ast ring-closing reverse reaction to the bidentate-bound oxalate and reformation of oxaliplatin. Consequently,t he release of oxalate is suppressed. In contrast, the pK a of the Pt IV complex 3a is much lower at 3.5 (see below). Therefore ah ydroxido ligand is still present at pH 7.4 and the reverse reaction back to 3 is hindered. This results in as hift of the equilibrium towards the dihydroxido species and as ac onsequence the hydrolysis is faster than in the case of Pt II .
To investigate whether the hydrolysis of 3 takes place via an attack on the electrophilic carboxylic group of the oxalate ligand or on platinum itself,w ea nalyzed the hydrolysis process in H 2 18 O( buffered with 50 mm phosphate). This measurement resulted in an exact mass of m/z = 488 for ctc-[Pt(DACH)(OAc) 2 ( 18 OH) 2 ] + Na + ,w hich proves that the platinum core is directly attacked and not the oxalato ligand ( Figure S4). This is in line with H 2 18 Ostudies of the hydrolysis of the axial dichloroacetate ligands of mitaplatin. [15] To gain more insight on the exact impact of the different equatorial ligands,t he additional Pt IV model complexes 5-7 ( Figure 1) were synthesized. Table 2presents an overview on the speed of hydrolysis of all complexes 1-7 after incubation in PB at pH 7.4 and 37 8 8Cfor 24 h. Thefollowing trends could be observed:1 )Whent he derivatives with ac hlorido ligand as aleaving group are compared, the oxaliplatin-like complex (DACH; 5)h as the fastest hydrolysis with complete disappearance of the parental compound after 24 h, followed by satraplatin (NH 3 /CHA; 4), the ethylenediamine complex (en; 6), and the cisplatin derivative (NH 3 ; 1)w ith just 5% hydrolysis.2 )Comparison of the complexes with two NH 3 equatorial ligands shows generally very slow hydrolysis rates with the cisplatin derivative 1 as the fastest (5 %hydrolysis), followed by the completely stable oxalate-bearing complex 7 and the carboplatin analogue 2.3)When complexes with the same amine ligand but chlorido vs.o xalato leaving groups (e.g. 5 vs. 3)are compared, the complex with chlorido ligands hydrolyzes faster (81 %hydroxido for 5)than the one with the bidentate oxalate ligand (41 %f or 3). However,w hen the DACH ligand in complexes 5 and 3 is exchanged for two NH 3 moieties,t he hydrolytic stability dramatically increases,w ith only 5% hydroxide species for 1 and no hydrolysis for 7.This impressively shows that the influence of the leaving group (chlorido vs.oxalate) has much less impact on the hydrolysis compared to the stable amine ligand (DACHv s. NH 3 ). Notably,i ncubation of 1-7 in cell culture medium (RPMI-1640) resulted in similar amounts of hydrolysis products.A s RPMI-1640 contains % 100 mm NaCl, this also confirms that the presence of chloride ions cannot significantly change the rate of hydrolysis (this could be also supported by comparison of 4, 5,and 6 in 50 mm PB vs.50mm PBS with 150 mm NaCl).
In order to obtain deeper insight into the underlying binding energies (BE) and heights of the activation barriers, as et of quantum-chemical data was calculated. TheB E obtained from energy decomposition of the individual reactants (Table S1) are in good agreement with the trends mentioned above.F or example:comparison of the BE of the leaving chlorido ligands resulted also in the order 5 % 4 > 6 > 1 (in the case of 4 the weaker-bound chlorido ligand was considered). Furthermore,B Eo ft he ammine ligands in complex 1, 2,a nd 7 were in line with the experimental data, although the BE of the acetato ligands are quite different. These results were also verified by extended transition state combined with natural orbitals for chemical valence (ETS-NOCV) [19] analysis and average local ionization potential (ALIP) maps. [20] Additionally,t he calculated heights of activation barrier of the hydrolysis (Scheme S1) of the equatorial ligands correlate very well with the HPLC-MS experiments ( Table 1). As summarized in Table 3, the activation energies for the hydrolysis in the case of 3 and 4 are distinctly lower than those for 1 and 2.T his confirms that the higher s-donor properties of asecondary amine compared to simple NH 3 ligands are important for an accelerated reaction, which explains the differences observed in the HPLC-MS experiments. [21] Furthermore,a lso the second hydrolysis step DE(TS2) of 3,w here the monodentate-bound oxalate ligand is finally released, possesses ad istinctly lower activation barrier than that of 4 with two chlorido ligands;this is in exact agreement with the HPLC-MS studies (Table 1). Thec alculations in the case of 4 further revealed ahigher trans-effect of the CHA ligand compared to NH 3 explaining the two peaks observed with HPLC-MS.T his is in accordance with areport on the respective Pt II complex JM118. [22] To test how the hydrolysis alters the chemical and biological properties,t he two derivatives of 3 were synthesized. This was achieved through incubation of 3 at pH 8-9 and 37 8 8Cand subsequent purification via preparative HPLC. Theh ydroxido (3a)a nd the dihydroxido (3b)s pecies were characterized by 1 Ha nd 13 CN MR, mass spectrometry,a nd

Angewandte Chemie
Communications elemental analysis.Furthermore,the pK a values of 3a and 3b were determined via 1 HN MR analysis and found to be 3.5 and 4.0, respectively ( Figure 3). This is in line with data of 3a after % 3hof incubation at pH of 2.5, which resulted in ac omplete transformation back to its parental complex 3.A ccording to the pK a value,t he hydroxido ligand gets protonated at such low pH values and the thereby generated aqua ligand can be released under reformation of the bidentate oxalato complex 3.I nc ontrast, 3a is stable at pH 5.5, which again fits with the pK a value of 3.5.
After the incubation of 3b in MeOH/Et 2 Oaf ew single crystals could be obtained and were analyzed by X-ray diffraction. Thestructure reveals an octahedral geometry with two axial acetato ligands and the equatorial DACH moiety. However,t he two hydroxido groups were exchanged by methoxido ligands (Figure 4; for bond lengths and angles see Tables S5 and S6) Notably,t he crystals contain both the R,R and S,S isomers as ar acemic mixture.T his can be explained by the % 2%S,S isomer present in the commercially available DACH compound and the often observed preference of compounds to crystallize as ar acemate and not as the pure isomers. [23] As anext step,the reactivity of 3b with different organic solvents was investigated. In contrast to aqueous cell culture medium or serum, incubation of 3b with, for example, DMSO,a cetonitrile,M eOH, or EtOH for 1h resulted in the exchange of one hydroxido ligand, which could be proven by mass spectrometry and an altered HPLC retention time ( Figure S5). This indicates that at very high excess,t he hydroxido ligands indeed can be substituted, which could also be used as anew synthetic pathway for introducing equatorial ligands into already existing Pt IV complexes.
As anext step,the thermodynamic reduction properties of 3, 3a,a nd 3b were compared using cyclic voltammetry.A ll three complexes showed irreversible reduction peaks with decreasing potentials the more hydroxido ligands are present in the molecule (3: À630 mV vs.N HE; 3a: À670 mV vs. NHE; 3b: À920 mV vs.NHE). This trend is in line with data from similar Pt IV complexes,h owever,w ith one or two axial hydroxido groups. [24] Thekinetic reduction rates of 3, 3a, and 3bwere investigated by HPLC after incubation with 10 equiv. of l-ascorbic acid at 20 8 8C. While 3 was completely stable over 6h,3a and 3b were reduced much faster and fully converted to the respective Pt II species already after 3-4 h( Figure 5). Consequently,these hydroxide species are even more rapidly reduced than the cisplatin complex 1,which is well-known to be much more sensitive than oxaliplatin or carboplatin derivatives. [25] Thus,a lthough the thermodynamic reduction potential decreases with the increasing number of OH groups, the reduction rate accelerates dramatically.A lthough this   seems to be unexpected, these data are in line with astudy of Gibson et al. [24] using axial mono-and dihydroxido derivatives of complex 3 and support the importance of the Pt IV reduction kinetics.
To evaluate whether the changed chemical properties of the hydrolysis products result in differences in biological activity,the anticancer activity of 3, 3a,and 3b against three cancer cell lines (HCT116, RKO, and CT-26) was evaluated. These experiments revealed that 3b had asignificantly lower IC 50 value (up to 2-fold more active) than the parental species 3 or the monohydroxido species 3a (Figure 6a nd Figure S6; Table 4).
An explanation for this could be that after reduction of 3a, the hydroxido group in the respective Pt II complex is protonated (pK a = 7.23). [18] This aqua ligand represents ag ood leaving group and facilitates ring closure to the bidentate-bound oxalate and reformation of oxaliplatin. Consequently, 3 and 3a can be expected to have av ery similar cytotoxic activity.I nc ontrast, in the case of 3b, oxaliplatin cannot be regenerated, and instead the Pt II complex [Pt(DACH)(H 2 O)OH] + is formed, which is able to directly interact with biological targets.I na ddition, cellular uptake of 3, 3a,a nd 3b was studied on HCT-116 and CT-26 cells after 3hincubation. Notably,n os ignificant differences in the uptake could be observed, with the parental complex 3 showing the highest cellular platinum levels ( Figure S7).
Taken together,o ur data indicates that we have to reconsider the current understanding of anticancer Pt IV complexes and doubt the dogma of the generally very high hydrolytic stability of Pt IV complexes.Depending on the exact equatorial coordination sphere,there are massive differences in their stability at physiological pH, and the resulting hydrolyzed Pt IV complexes possess vastly altered physicochemical properties.E specially their rate of reduction, the crucial factor in the activation of Pt IV prodrugs,i ss trongly accelerated upon prior hydrolysis.These observations are also important for, for example,s imple physiologically buffered solutions of oxaliplatin(IV) complexes,where more than 50 % hydrolysis can be expected within 24 h. Furthermore,not the leaving group itself,b ut the substituents at the equatorial amine ligands have the major impact on the rate of hydrolysis. In addition, it is important to mention that (cancer) cells possess different pH values in their organelles,f or example, up to pH 8i nm itochondria. [26] Consequently,e ven higher levels of hydrolysis can be expected, as this reaction is further accelerated at more alkaline pH values.U nder such conditions also cisplatin derivatives start to hydrolyze to as ignificant degree.
Thus,i tis essential to carefully select the equatorial core of Pt IV prodrugs not only according to the biological activity of the active Pt II analogue,but also to the hydrolytic stability of the Pt IV prodrugs (satraplatin < oxaliplatin ! cisplatin < carboplatin) and the rate of reduction (satraplatin > cisplatin @ oxaliplatin % carboplatin). All these parameters influence the biological properties and have to be considered in the future design of novel Pt IV anticancer prodrugs.