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Hydrolysis mechanism of anticancer drug lobaplatin in aqueous medium under neutral and acidic conditions: A DFT study
Chemical Physics Letters 663 (2016) 115–122
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Hydrolysis mechanism of anticancer drug lobaplatin in aqueous medium under neutral and acidic conditions: A DFT study Venkata P. Reddy B. a, Subhajit Mukherjee a, Ishani Mitra a, Sujay Mahata a, Wolfgang Linert b, Sankar Ch. Moi a,⇑ a b
Department of Chemistry, National Institute of Technology, Durgapur 713209, W.B., India Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt, 9/163-AC, 1060 Vienna, Austria
a r t i c l e
i n f o
Article history: Received 10 August 2016 In final form 3 October 2016 Available online 4 October 2016 Keywords: Lobaplatin Third generation anticancer drug DFT Aqueous medium Hydrolysis mechanism
a b s t r a c t We have studied the hydrolysis mechanism of lobaplatin in aqueous medium under neutral and acidic conditions using density functional theory combining with CPCM model. The stationary states located on potential energy surface were fully optimized and characterised. The rate limiting step in neutral conditions, ring opening reaction with an activation energy of 110.21 kJ mol1. The completely hydrolysed complex is expected to be the reactive species towards the DNA purine bases. In acidic conditions, ligand detachment is the rate limiting step with an activation energy of 113.82 kJ mol1. Consequently, monohydrated complex is expected to be the species reacting with DNA. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Platinum complexes has one of the greatest success stories of metals in medicine. The serendipitous discovery of antitumor properties of cis-diamminedichloroplatinum also known as cisplatin by Rosenberg [1] and his co-workers has led to a new age of metal based chemotherapy. Cisplatin based chemotherapy has become a fundamental treatment in some types of tumors that were significantly lethal prior to the introduction of this drug. Unfortunately, the therapeutic success of cisplatin is impaired by its serious side effects such as nausea, nephrotoxicity, ototoxicity and neurotoxicity [2–5]. Numerous mechanisms which bring about intrinsic or acquired resistance to cisplatin in vitro have been recognized [6]. Pathways that prevent the formation of platinumDNA adducts are reduced import, greater detoxification and enhanced efflux [7]. This has promoted the pursuit of novel metal-based compounds [8] that are able to overcome such inadequacies. Its successors with improved pharmacological properties such as oxaliplatin [9,10] carboplatin [11,12] and nedaplatin [13] have not yet exhibited remarkable advantages over cisplatin. The third generation anticancer drug lobaplatin is a 1:1 diastereomeric mixture of R,R,S and S,S,S configuration of platinum(II) complexes with 1,2-bis(aminomethyl)cyclobutane as the carrier ligand and ⇑ Corresponding author. E-mail address: [email protected] (S.Ch. Moi).
http://dx.doi.org/10.1016/j.cplett.2016.10.004 0009-2614/Ó 2016 Elsevier B.V. All rights reserved.
lactate as the leaving group. The structure of cisplatin and diastereomers of lobaplatin are shown in Scheme 1. Lobaplatin was developed by ASTA Pharma AG, and its advancement is undertaken by Zentaris AG. It is approved in China for the treatment of chronic myelogenous leukemia, small cell lung cancer and inoperable and metastatic breast cancer [14]. It has also completed phase II clinical trials in US, EU, Brazil, Australia and South Africa for the treatment of various cancers, including esophageal, breast, ovarian and lung cancers [15–18]. According to the claims of Zentaris its mechanism of action involves inhibition of DNA polymerase and RNA polymerase. The drug is heat-labile and does not require protection against normal daylight like cisplatin [19]. Lobaplatin showed significant activity against cisplatin-resistant testicular and human ovarian carcinoma xenografts in vivo [20] and a wide range of preclinical tumour models. It also has an improved therapeutic index as compared to cisplatin [21] and seems to overcome tumour resistance to cisplatin and carboplatin [15,22,23]. The antitumor activity of lobaplatin is thought to result from the formation of DNA-drug adducts, mainly as GG and AG intra-strand cross-links similar to cisplatin and oxaliplatin [24]. The cellular target of the classical platinum based anticancer drugs is the N7 atom of the purine bases in the DNA. It is generally accepted that these compounds induce apoptosis in tumour cells by conversion to their active hydrolysed forms which binds to nuclear DNA. Cisplatin undergoes a fast first aquation resulting in a monoaquated species which interacts with DNA, however for
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Scheme 1. Chemical structure of cisplatin and diastereomers of lobaplatin.
carboplatin and oxaliplatin, the first aquation step was found to be slower, followed by a second rapid step where the monodentate leaving group is completely substituted to give the diaqua complex as the active species [25,26]. Lobaplatin which is generally administered as an intravenous bolus injection, also undergoes aqueous degradation in a biphasic process before interacting with DNA. Recent studies on pharmacokinetics between the two diastereoisomers of lobaplatin proves that there was no apparent stereospecificity [27]. To acquire a better understanding of the mode of action, it is imperative to identify the active species of drug reaching the DNA and to get an insight into the hydrolysis mechanism of platinum compounds which is an essential step in triggering cell death. The present study describes the degradation pathways of LB-D1 (R, R,S-diastereomer) in aqueous medium under neutral and acidic conditions using density functional theory (DFT) with conductor like polar continuum model (CPCM) calculations. With the rapid development of computational chemistry, DFT methods have been successfully applied to the hydrolysis processes of metal-based anticancer drugs [28–34]. To the best of our knowledge, no attempt has been made to study in detail the hydrolysis mechanism of lobaplatin either experimentally or theoretically. Lobaplatin undergoes hydrolysis in two consecutive steps: addition of the first water molecule simultaneous with the ring-opening, followed by addition of a secondwater leading to the loss of lactic acid.
31++G(2df,2pd) for more accurate energies. Thermal contribution of the energetic properties was also considered at the standard state, namely, at 298.15 K and 1 atm. Potential energy surface profiles were estimated from total electronic energies calculated at B3LYP/6-31++G(2df,2pd)/LANL2DZ level of theory adding zeropoint and enthalpy corrected energies.
2. Computational details
3.1.1. Underneutral conditions Lobaplatin is expected to undergo water degradation in a biphasic process, first step is ring-opening by the addition of water molecule followed by the release of the lactic acid upon reaction with second water molecule. The proposed mechanism of hydrolysis of lobaplatin in neutral conditions along path A and path B is depicted in Scheme 2. The detachment of ligand may occur by rupture of the bond that involves the oxygen a to the carbonyl group (path A) or by breaking the other PtAO bond (path B). The optimized stationary states (TS, RI, PI along path A and TS0 , RI0 , PI0 along path B) on potential energy surface for first and second step aquation process along path A, are shown in Figs. 2 and 3 respectively. In the first step, the product obtained has shown a proton transfer from entered water molecule to the carbonyl oxygen of the leaving group. At physiological pH and temperature, the hydroxo complexes available, even if in the presence of macromolecules the local pH could be altered, affecting hydrolysis rates [48,49]. The PtAO (oxygen of ligand) bond distance increases as water molecule approaches the metal centre, until transition geometry is reached. From the optimized structures of the reactive intermediates, we have observed that entering water molecule is initially hydrogen bonded with amine groups of the carrier ligand, resulting in stabilisation. The imaginary frequencies were observed at 168.78i cm1 and 397.03i cm1 for TS1 and TS10 respectively. Both the transition states show penta-coordinate geometry. The animation of these vibrational modes clearly indicates the rupture of the PtAO (ligand) and formation of PtAO (water) bond. Along path A, the entering water molecule approaches the platinum centre with a
All calculations were performed with Gaussian 09 [35] programme. For optimisation of stationary states on potential energy surface, we have used gradient-corrected density functional theory of three parameter fit of exchange and correlation functionals of Becke (B3LYP) [36], and correlation functional of Lee, Yang and Parr (LYP) [37] by combining with conductor like polar continuum model (CPCM) [38–41]. The relativistic effective core potential (ECP) and associated valence double n (zeta) basis set of Hay and Wadt [42,43] (LANL2DZ) were employed for Pt. This comprises of electrons in the 6s, 6p, and 5d orbitals. The standard split valence basis set 6-31G(d) [44,45], which was described by the quasirelativistic Stuttgart–Dresden pseudo potential was applied for carbon, oxygen, nitrogen and hydrogen. The geometry of the studied diastereomer was optimized at the same level of theory. Vibrational frequency analysis was done on the basis of analytical second derivatives of the Hamiltonian, in order to confirm proper convergence to equilibrium and transition state geometries at the same level of theory. Geometries of fully optimized stationary points located on the PES were characterized as minima or first order transition state (one imaginary frequency). The intrinsic reaction coordinates (IRC) paths [46,47] were traced in order to check the energy profiles connecting each transition state to two associated minima of the proposed mechanism. For every transition state, the corresponding geometries were confirmed by the IRC method and frequency calculation. Single point energy calculations were also carried out on the optimized geometries with diffused and polarisable functions containing the larger basis set 6-
3. Results and discussion 3.1. Hydrolysis mechanism The hydrolysis reactions of the classical platinum(II) based anticancer drugs belong to the class of second-order nucleophilic substitution (SN2) reactions. These reactions for square-planar complexes proceed via a collision between the reactant with two consecutive nucleophilic species attacking the metal centre to release the ligand. Transition states in which the entering and the leaving groups around the metal centre are in weakly bonded compared to substrate complex, suggesting that the associative mechanism is proposed. The equatorial plane of the fivecoordinated transition state structure plays an important role in determining the hydrolysis behaviour. The optimized geometry and important structural parameters of studied diastereomer of lobaplatin with configuration R,R,S is shown in Fig. 1.
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Fig. 1. Optimized geometries and important structural parameters of lobaplatin diastereomers.
Scheme 2. Degradation path ways of lobaplatinin neutral conditions where first step is ring-opening followed by the loss of ligand.
distance of 3.59 Å which becomes 2.49 Å in the transition state and 2.05 Å in the product. The angle of entering group and leaving oxygen of ligand at Pt centre is found to be 60.80°. The optimized stationary states for first aquation step along path B is given in Fig. S1. Along path B, the entering water molecule approaches the platinum centre with a distance of 3.71 Å which becomes 2.45 Å in the transition state and 2.04 Å in the product. The angle of entering group and leaving oxygen of ligand at Pt centre in TS10 is found 58.12° and the bond lengths being broken and formed are more or less same as observed along path A. Our calculation shows that first aquation step must overcome an energy barrier of 113.62 kJ mol1 along path A and 119.73 kJ mol1 along path B. Hence the activation energy along path A (rupture of the bond that involves the oxygen a to the carbonyl group) is less than path B. The reaction is endothermic by 26.25 kJ mol1 and 34.02 kJ mol1 along path A and path B respectively. For the second aquation step which leads to the loss of lactic acid can also occur in two different ways. The fully optimized stationary states along path A are given in Fig. 3 and along path B are shown in Fig. S2. In the intermediate structure along path A, the water molecule approaches the platinum centre with a distance of 3.40 Å that becomes 2.43 Å in TS and 2.04 Å in product. In the intermediate structure along path B, the water molecule approaches the platinum centre with a distance of 3.74 Å that becomes 2.55 Å in TS and 2.12 Å in product. The imaginary frequencies were observed at 323.03i cm1 and 213.29i cm1 for TS2 and TS20 , corresponding to the change of PtAO (ligand) and formation of PtAO (water) bond respectively.
According to the calculated potential energy diagram shown in Fig. 4. The most energetically favoured path is Path A with an activation energy of 110.21 kJ mol1 and 108.14 kJ mol1 for first and second aquation step respectively. For both the steps proton transfer has been observed from entering water molecule to the leaving group. In neutral conditions, it has been already established that cisplatin in its monohydrated form interacts with DNA [32]. While we expect that lobaplatin undergoes complete hydrolysis and the dihydrated complex is suggested to be the reactive species with the DNA purine bases.
3.1.2. Underacidic conditions Lobaplatin degradation pathways has also been investigated in acidic conditions. We have taken into account that the lactate ligand is protonated under such a condition. The main purpose ofthis study isto know the effect of acidification onthe rate of degradation. It has been previously reported that rate of hydrolysis reaction [28,34,50] of some currently used anticancer drugs increases under acidic conditions. The investigated reaction paths are depicted in Scheme 3. The ring opening of ligand by addition of water molecule in the first aquation step can occur in two different ways. In path A, the detachment of ligand may occur by rupture of the bond that involves the oxygen a to the carbonyl group and in path B, by breaking the PtAO hydroxyl bond. The optimized stationary states and selected structural parameters along A has been shown in Fig. 5 and along path B is in Fig. S3.
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Fig. 2. Fully optimized stationary states of first step aquation along path A under neutral conditions.
Fig. 3. Fully optimized stationary states of second step aquation along path A under neutral conditions.
The entering water molecule approaches the platinum centre with a distance of 3.62 Å which becomes 2.68 Å in the transition state and 2.12 Å in the product. The angle of entering group and
leaving oxygen of ligand at Pt centre is found to be 62.32°. The imaginary frequency for the transition state (TS3) is 143.80i cm1 and the animation of this corresponds to the breaking of PtAO
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Fig. 4. Enthalpy of activation energy profile diagram of hydrolysis of lobaplatin under neutral conditions along path A and path B.
Scheme 3. Degradation path ways of lobaplatin in acidic conditions where first step is ring-opening followed by the loss of ligand.
(ligand) and formation of PtAO (water) bond. Along path B, the proton transfer has been observed from entering water molecule to the detaching ligand. The potential energy profile in acidic conditions is depicted in Fig. 7. The most favoured path for the ring opening reaction is path A i.e. the addition of water molecule on the same side of protonated carbonyl group where the proton transfer has not been observed from entering water molecule to the detaching ligand. Therefore, for the first aquation step which leads to ring opening due to rupture of the bond involving the oxygen a to the protonated carbonyl group has an activation energy of 64.13 kJ mol1. Ring opening in acidic conditions is exothermic by 6.87 kJ mol1. According to the results obtained, the activation barrier is lower than that for neutral conditions by 46.08 kJ mol1. For the second aquation step which leads to the loss of lactic acid, path B is more favourable with an activation energy of 52.67 kJ mol1. Along path A, the activation energy of second aquation is 113.82 kJ mol1. According to activation energy profile, the most stable path for first aquation step is path A and the product formed in this step reacts with second water molecule leading to loss of lactic acid. Acidification results in lowering of activation energy and induces a spontaneous proton transfer from entering water molecule to a negatively charged oxygen atom of the leaving lactate group. The optimized stationary states along path A are shown in Fig. 6 and along path B are shown in Fig. S4.
The entering water molecule approaches the platinum centre with a distance of 3.30 Å which becomes 2.40 Å in the transition state and 2.04 Å in the product. The imaginary frequency observed for the transition state is 249.72i cm1. The animation of frequency corresponds to the breaking of PtAO (ligand) and formation of PtAO (water) bond, while the proton transfer occurred from entering water molecule to the oxygen of the detaching ligand. The angle of entering and leaving group at Pt centre is found 58.84° in the transition state. From Fig. 7, it can be concluded that second hydrolysis step is unfavourable and can be considered as rate limiting step. The obtained final product is [Pt(1,2-bis(aminomethyl)cyclobutane)(H + 2O)(OH)] by the loss of ligand. Being second aquation step being the rate limiting step in acidic conditions, we expect that monohydrated form of lobaplatin probably reach DNA. Due to the presence of the large monodentate lactate group in monohydrated form of lobaplatin, it can promote stereochemical hindrance in reaction with DNA. 3.2. Comparison of activation energies For a better understanding of how the platinum containing anticancer drugs reach DNA, several computational studies have been done [28–34] but it is not still established whether the mono-aqua
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Fig. 5. Fully optimized stationary states of first step aquation along path A under acidic conditions.
Fig. 6. Fully optimized stationary states of second step aquation along path A under acidic conditions.
or di-aqua complex acts as a platinating species on DNA. Herein, we have compared the results obtained in this work with previ-
ously reported hydrolysis of Pt-anticancer drugs which is shown in Fig. 8. Second and third generation Pt-drugs show slower
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Fig. 7. Enthalpy of activation energy profile diagram of hydrolysis of lobaplatin under acidic conditions along path A and path B.
Fig. 8. Comparison of calculated activation energies of lobaplatin under neutral and acidic conditions with cisplatin, carboplatin, oxaliplatin, nedaplatin.
hydrolysis rates compared with cisplatin due to the introduction of the kinetically less labile ligands and presence of large carrier amine groups. In neutral conditions, activation energies of hydrolysis for carboplatin, oxaliplatin, nedaplatinand lobaplatin reveal that the ring opening reaction is therate limiting step. In case of lobaplatin, path A shows similar activation energy for the first and second aquation step process but along path B, first aquation step has higher energy barrier than second one. Consequently in neutral conditions, lobaplatin undergo complete hydrolysis. Whereasin acidic conditions, ligand detachment step is the rate limiting step for carboplatin, oxaliplatin, nedaplatin and lobaplatin with lower activation energy. 4. Conclusions In summary, we have investigated the hydrolysis mechanism of third generation anticancer drug lobaplatin in aqueous medium both in neutral and acidic conditions using desity functional theory (DFT) by combining with conductor like polar continuum model (CPCM). The stationary states located on potential energy surface including penta-coordinated transition states (TS), reactive intermediates (RI) and product intermediates (PI) were fully optimized
and confirmed by frequency analysis. Reaction paths were confirmed by IRC calculations. Hydrolysis proceeds through two steps: first, ring opening and followed by the detachment of ligand. The computed potential energy surface reveals that the rate limiting step in neutral conditions is the first hydrolysis process with an activation energy of 110.21 kJ mol1. The final product formed in neutral conditions is a dihydroxo Pt-complex with the loss of lactic acid. Proton transfer from entering water molecule to the oxygen of leaving group has been observed for both the substitution processes. Lobaplatin undergoes complete hydrolysis in neutral conditions and the dihydrated complex is belived to be the species reacting with the DNA purine bases. Whereas in acidic conditions, ligand detachment step is the rate limiting process with an activation energy of 113.82 kJ mol1. For the second aquation step, proton transfer is observed from entering water molecule to detaching ligand and the product formed is [Pt (1,2-bis(aminomethyl)cyclobutane)(H2O)(OH)]+. We can therefore hypothesize that lobaplatin reaches DNA in its monohydrated form. A comparison between lobaplatin with other currently used Pt(II)containing anticancer drugs in the medicalprotocols, reveals some general trends. The hydrolysis reaction of lobaplatin in neutral and acidic conditions is in analogy with the other second generation Pt-containing drugs. This study will provide a better insight into the hydrolysis controlling mechanisms which is essential to identify the active form of drug, which reacts with DNA.
Acknowledgements The authors V.P. Reddy B. and S.C. Moi are pleased to thank National Institute of Technology Durgapur-713209 for necessary assistance and also Department of Science and Technology, Government of India for providing DST project fund (Project No: SB/EMEQ-028/2013), for carrying out this work. IM and SM are also thankful to DST Inspire, Govt. of India for research fellowship.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cplett.2016.10. 004.
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Research paper
Hydrolysis mechanism of anticancer drug lobaplatin in aqueous medium under neutral and acidic conditions: A DFT study Venkata P. Reddy B. a, Subhajit Mukherjee a, Ishani Mitra a, Sujay Mahata a, Wolfgang Linert b, Sankar Ch. Moi a,⇑ a b
Department of Chemistry, National Institute of Technology, Durgapur 713209, W.B., India Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt, 9/163-AC, 1060 Vienna, Austria
a r t i c l e
i n f o
Article history: Received 10 August 2016 In final form 3 October 2016 Available online 4 October 2016 Keywords: Lobaplatin Third generation anticancer drug DFT Aqueous medium Hydrolysis mechanism
a b s t r a c t We have studied the hydrolysis mechanism of lobaplatin in aqueous medium under neutral and acidic conditions using density functional theory combining with CPCM model. The stationary states located on potential energy surface were fully optimized and characterised. The rate limiting step in neutral conditions, ring opening reaction with an activation energy of 110.21 kJ mol1. The completely hydrolysed complex is expected to be the reactive species towards the DNA purine bases. In acidic conditions, ligand detachment is the rate limiting step with an activation energy of 113.82 kJ mol1. Consequently, monohydrated complex is expected to be the species reacting with DNA. Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction Platinum complexes has one of the greatest success stories of metals in medicine. The serendipitous discovery of antitumor properties of cis-diamminedichloroplatinum also known as cisplatin by Rosenberg [1] and his co-workers has led to a new age of metal based chemotherapy. Cisplatin based chemotherapy has become a fundamental treatment in some types of tumors that were significantly lethal prior to the introduction of this drug. Unfortunately, the therapeutic success of cisplatin is impaired by its serious side effects such as nausea, nephrotoxicity, ototoxicity and neurotoxicity [2–5]. Numerous mechanisms which bring about intrinsic or acquired resistance to cisplatin in vitro have been recognized [6]. Pathways that prevent the formation of platinumDNA adducts are reduced import, greater detoxification and enhanced efflux [7]. This has promoted the pursuit of novel metal-based compounds [8] that are able to overcome such inadequacies. Its successors with improved pharmacological properties such as oxaliplatin [9,10] carboplatin [11,12] and nedaplatin [13] have not yet exhibited remarkable advantages over cisplatin. The third generation anticancer drug lobaplatin is a 1:1 diastereomeric mixture of R,R,S and S,S,S configuration of platinum(II) complexes with 1,2-bis(aminomethyl)cyclobutane as the carrier ligand and ⇑ Corresponding author. E-mail address: [email protected] (S.Ch. Moi).
http://dx.doi.org/10.1016/j.cplett.2016.10.004 0009-2614/Ó 2016 Elsevier B.V. All rights reserved.
lactate as the leaving group. The structure of cisplatin and diastereomers of lobaplatin are shown in Scheme 1. Lobaplatin was developed by ASTA Pharma AG, and its advancement is undertaken by Zentaris AG. It is approved in China for the treatment of chronic myelogenous leukemia, small cell lung cancer and inoperable and metastatic breast cancer [14]. It has also completed phase II clinical trials in US, EU, Brazil, Australia and South Africa for the treatment of various cancers, including esophageal, breast, ovarian and lung cancers [15–18]. According to the claims of Zentaris its mechanism of action involves inhibition of DNA polymerase and RNA polymerase. The drug is heat-labile and does not require protection against normal daylight like cisplatin [19]. Lobaplatin showed significant activity against cisplatin-resistant testicular and human ovarian carcinoma xenografts in vivo [20] and a wide range of preclinical tumour models. It also has an improved therapeutic index as compared to cisplatin [21] and seems to overcome tumour resistance to cisplatin and carboplatin [15,22,23]. The antitumor activity of lobaplatin is thought to result from the formation of DNA-drug adducts, mainly as GG and AG intra-strand cross-links similar to cisplatin and oxaliplatin [24]. The cellular target of the classical platinum based anticancer drugs is the N7 atom of the purine bases in the DNA. It is generally accepted that these compounds induce apoptosis in tumour cells by conversion to their active hydrolysed forms which binds to nuclear DNA. Cisplatin undergoes a fast first aquation resulting in a monoaquated species which interacts with DNA, however for
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Scheme 1. Chemical structure of cisplatin and diastereomers of lobaplatin.
carboplatin and oxaliplatin, the first aquation step was found to be slower, followed by a second rapid step where the monodentate leaving group is completely substituted to give the diaqua complex as the active species [25,26]. Lobaplatin which is generally administered as an intravenous bolus injection, also undergoes aqueous degradation in a biphasic process before interacting with DNA. Recent studies on pharmacokinetics between the two diastereoisomers of lobaplatin proves that there was no apparent stereospecificity [27]. To acquire a better understanding of the mode of action, it is imperative to identify the active species of drug reaching the DNA and to get an insight into the hydrolysis mechanism of platinum compounds which is an essential step in triggering cell death. The present study describes the degradation pathways of LB-D1 (R, R,S-diastereomer) in aqueous medium under neutral and acidic conditions using density functional theory (DFT) with conductor like polar continuum model (CPCM) calculations. With the rapid development of computational chemistry, DFT methods have been successfully applied to the hydrolysis processes of metal-based anticancer drugs [28–34]. To the best of our knowledge, no attempt has been made to study in detail the hydrolysis mechanism of lobaplatin either experimentally or theoretically. Lobaplatin undergoes hydrolysis in two consecutive steps: addition of the first water molecule simultaneous with the ring-opening, followed by addition of a secondwater leading to the loss of lactic acid.
31++G(2df,2pd) for more accurate energies. Thermal contribution of the energetic properties was also considered at the standard state, namely, at 298.15 K and 1 atm. Potential energy surface profiles were estimated from total electronic energies calculated at B3LYP/6-31++G(2df,2pd)/LANL2DZ level of theory adding zeropoint and enthalpy corrected energies.
2. Computational details
3.1.1. Underneutral conditions Lobaplatin is expected to undergo water degradation in a biphasic process, first step is ring-opening by the addition of water molecule followed by the release of the lactic acid upon reaction with second water molecule. The proposed mechanism of hydrolysis of lobaplatin in neutral conditions along path A and path B is depicted in Scheme 2. The detachment of ligand may occur by rupture of the bond that involves the oxygen a to the carbonyl group (path A) or by breaking the other PtAO bond (path B). The optimized stationary states (TS, RI, PI along path A and TS0 , RI0 , PI0 along path B) on potential energy surface for first and second step aquation process along path A, are shown in Figs. 2 and 3 respectively. In the first step, the product obtained has shown a proton transfer from entered water molecule to the carbonyl oxygen of the leaving group. At physiological pH and temperature, the hydroxo complexes available, even if in the presence of macromolecules the local pH could be altered, affecting hydrolysis rates [48,49]. The PtAO (oxygen of ligand) bond distance increases as water molecule approaches the metal centre, until transition geometry is reached. From the optimized structures of the reactive intermediates, we have observed that entering water molecule is initially hydrogen bonded with amine groups of the carrier ligand, resulting in stabilisation. The imaginary frequencies were observed at 168.78i cm1 and 397.03i cm1 for TS1 and TS10 respectively. Both the transition states show penta-coordinate geometry. The animation of these vibrational modes clearly indicates the rupture of the PtAO (ligand) and formation of PtAO (water) bond. Along path A, the entering water molecule approaches the platinum centre with a
All calculations were performed with Gaussian 09 [35] programme. For optimisation of stationary states on potential energy surface, we have used gradient-corrected density functional theory of three parameter fit of exchange and correlation functionals of Becke (B3LYP) [36], and correlation functional of Lee, Yang and Parr (LYP) [37] by combining with conductor like polar continuum model (CPCM) [38–41]. The relativistic effective core potential (ECP) and associated valence double n (zeta) basis set of Hay and Wadt [42,43] (LANL2DZ) were employed for Pt. This comprises of electrons in the 6s, 6p, and 5d orbitals. The standard split valence basis set 6-31G(d) [44,45], which was described by the quasirelativistic Stuttgart–Dresden pseudo potential was applied for carbon, oxygen, nitrogen and hydrogen. The geometry of the studied diastereomer was optimized at the same level of theory. Vibrational frequency analysis was done on the basis of analytical second derivatives of the Hamiltonian, in order to confirm proper convergence to equilibrium and transition state geometries at the same level of theory. Geometries of fully optimized stationary points located on the PES were characterized as minima or first order transition state (one imaginary frequency). The intrinsic reaction coordinates (IRC) paths [46,47] were traced in order to check the energy profiles connecting each transition state to two associated minima of the proposed mechanism. For every transition state, the corresponding geometries were confirmed by the IRC method and frequency calculation. Single point energy calculations were also carried out on the optimized geometries with diffused and polarisable functions containing the larger basis set 6-
3. Results and discussion 3.1. Hydrolysis mechanism The hydrolysis reactions of the classical platinum(II) based anticancer drugs belong to the class of second-order nucleophilic substitution (SN2) reactions. These reactions for square-planar complexes proceed via a collision between the reactant with two consecutive nucleophilic species attacking the metal centre to release the ligand. Transition states in which the entering and the leaving groups around the metal centre are in weakly bonded compared to substrate complex, suggesting that the associative mechanism is proposed. The equatorial plane of the fivecoordinated transition state structure plays an important role in determining the hydrolysis behaviour. The optimized geometry and important structural parameters of studied diastereomer of lobaplatin with configuration R,R,S is shown in Fig. 1.
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Fig. 1. Optimized geometries and important structural parameters of lobaplatin diastereomers.
Scheme 2. Degradation path ways of lobaplatinin neutral conditions where first step is ring-opening followed by the loss of ligand.
distance of 3.59 Å which becomes 2.49 Å in the transition state and 2.05 Å in the product. The angle of entering group and leaving oxygen of ligand at Pt centre is found to be 60.80°. The optimized stationary states for first aquation step along path B is given in Fig. S1. Along path B, the entering water molecule approaches the platinum centre with a distance of 3.71 Å which becomes 2.45 Å in the transition state and 2.04 Å in the product. The angle of entering group and leaving oxygen of ligand at Pt centre in TS10 is found 58.12° and the bond lengths being broken and formed are more or less same as observed along path A. Our calculation shows that first aquation step must overcome an energy barrier of 113.62 kJ mol1 along path A and 119.73 kJ mol1 along path B. Hence the activation energy along path A (rupture of the bond that involves the oxygen a to the carbonyl group) is less than path B. The reaction is endothermic by 26.25 kJ mol1 and 34.02 kJ mol1 along path A and path B respectively. For the second aquation step which leads to the loss of lactic acid can also occur in two different ways. The fully optimized stationary states along path A are given in Fig. 3 and along path B are shown in Fig. S2. In the intermediate structure along path A, the water molecule approaches the platinum centre with a distance of 3.40 Å that becomes 2.43 Å in TS and 2.04 Å in product. In the intermediate structure along path B, the water molecule approaches the platinum centre with a distance of 3.74 Å that becomes 2.55 Å in TS and 2.12 Å in product. The imaginary frequencies were observed at 323.03i cm1 and 213.29i cm1 for TS2 and TS20 , corresponding to the change of PtAO (ligand) and formation of PtAO (water) bond respectively.
According to the calculated potential energy diagram shown in Fig. 4. The most energetically favoured path is Path A with an activation energy of 110.21 kJ mol1 and 108.14 kJ mol1 for first and second aquation step respectively. For both the steps proton transfer has been observed from entering water molecule to the leaving group. In neutral conditions, it has been already established that cisplatin in its monohydrated form interacts with DNA [32]. While we expect that lobaplatin undergoes complete hydrolysis and the dihydrated complex is suggested to be the reactive species with the DNA purine bases.
3.1.2. Underacidic conditions Lobaplatin degradation pathways has also been investigated in acidic conditions. We have taken into account that the lactate ligand is protonated under such a condition. The main purpose ofthis study isto know the effect of acidification onthe rate of degradation. It has been previously reported that rate of hydrolysis reaction [28,34,50] of some currently used anticancer drugs increases under acidic conditions. The investigated reaction paths are depicted in Scheme 3. The ring opening of ligand by addition of water molecule in the first aquation step can occur in two different ways. In path A, the detachment of ligand may occur by rupture of the bond that involves the oxygen a to the carbonyl group and in path B, by breaking the PtAO hydroxyl bond. The optimized stationary states and selected structural parameters along A has been shown in Fig. 5 and along path B is in Fig. S3.
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Fig. 2. Fully optimized stationary states of first step aquation along path A under neutral conditions.
Fig. 3. Fully optimized stationary states of second step aquation along path A under neutral conditions.
The entering water molecule approaches the platinum centre with a distance of 3.62 Å which becomes 2.68 Å in the transition state and 2.12 Å in the product. The angle of entering group and
leaving oxygen of ligand at Pt centre is found to be 62.32°. The imaginary frequency for the transition state (TS3) is 143.80i cm1 and the animation of this corresponds to the breaking of PtAO
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Fig. 4. Enthalpy of activation energy profile diagram of hydrolysis of lobaplatin under neutral conditions along path A and path B.
Scheme 3. Degradation path ways of lobaplatin in acidic conditions where first step is ring-opening followed by the loss of ligand.
(ligand) and formation of PtAO (water) bond. Along path B, the proton transfer has been observed from entering water molecule to the detaching ligand. The potential energy profile in acidic conditions is depicted in Fig. 7. The most favoured path for the ring opening reaction is path A i.e. the addition of water molecule on the same side of protonated carbonyl group where the proton transfer has not been observed from entering water molecule to the detaching ligand. Therefore, for the first aquation step which leads to ring opening due to rupture of the bond involving the oxygen a to the protonated carbonyl group has an activation energy of 64.13 kJ mol1. Ring opening in acidic conditions is exothermic by 6.87 kJ mol1. According to the results obtained, the activation barrier is lower than that for neutral conditions by 46.08 kJ mol1. For the second aquation step which leads to the loss of lactic acid, path B is more favourable with an activation energy of 52.67 kJ mol1. Along path A, the activation energy of second aquation is 113.82 kJ mol1. According to activation energy profile, the most stable path for first aquation step is path A and the product formed in this step reacts with second water molecule leading to loss of lactic acid. Acidification results in lowering of activation energy and induces a spontaneous proton transfer from entering water molecule to a negatively charged oxygen atom of the leaving lactate group. The optimized stationary states along path A are shown in Fig. 6 and along path B are shown in Fig. S4.
The entering water molecule approaches the platinum centre with a distance of 3.30 Å which becomes 2.40 Å in the transition state and 2.04 Å in the product. The imaginary frequency observed for the transition state is 249.72i cm1. The animation of frequency corresponds to the breaking of PtAO (ligand) and formation of PtAO (water) bond, while the proton transfer occurred from entering water molecule to the oxygen of the detaching ligand. The angle of entering and leaving group at Pt centre is found 58.84° in the transition state. From Fig. 7, it can be concluded that second hydrolysis step is unfavourable and can be considered as rate limiting step. The obtained final product is [Pt(1,2-bis(aminomethyl)cyclobutane)(H + 2O)(OH)] by the loss of ligand. Being second aquation step being the rate limiting step in acidic conditions, we expect that monohydrated form of lobaplatin probably reach DNA. Due to the presence of the large monodentate lactate group in monohydrated form of lobaplatin, it can promote stereochemical hindrance in reaction with DNA. 3.2. Comparison of activation energies For a better understanding of how the platinum containing anticancer drugs reach DNA, several computational studies have been done [28–34] but it is not still established whether the mono-aqua
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Fig. 5. Fully optimized stationary states of first step aquation along path A under acidic conditions.
Fig. 6. Fully optimized stationary states of second step aquation along path A under acidic conditions.
or di-aqua complex acts as a platinating species on DNA. Herein, we have compared the results obtained in this work with previ-
ously reported hydrolysis of Pt-anticancer drugs which is shown in Fig. 8. Second and third generation Pt-drugs show slower
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Fig. 7. Enthalpy of activation energy profile diagram of hydrolysis of lobaplatin under acidic conditions along path A and path B.
Fig. 8. Comparison of calculated activation energies of lobaplatin under neutral and acidic conditions with cisplatin, carboplatin, oxaliplatin, nedaplatin.
hydrolysis rates compared with cisplatin due to the introduction of the kinetically less labile ligands and presence of large carrier amine groups. In neutral conditions, activation energies of hydrolysis for carboplatin, oxaliplatin, nedaplatinand lobaplatin reveal that the ring opening reaction is therate limiting step. In case of lobaplatin, path A shows similar activation energy for the first and second aquation step process but along path B, first aquation step has higher energy barrier than second one. Consequently in neutral conditions, lobaplatin undergo complete hydrolysis. Whereasin acidic conditions, ligand detachment step is the rate limiting step for carboplatin, oxaliplatin, nedaplatin and lobaplatin with lower activation energy. 4. Conclusions In summary, we have investigated the hydrolysis mechanism of third generation anticancer drug lobaplatin in aqueous medium both in neutral and acidic conditions using desity functional theory (DFT) by combining with conductor like polar continuum model (CPCM). The stationary states located on potential energy surface including penta-coordinated transition states (TS), reactive intermediates (RI) and product intermediates (PI) were fully optimized
and confirmed by frequency analysis. Reaction paths were confirmed by IRC calculations. Hydrolysis proceeds through two steps: first, ring opening and followed by the detachment of ligand. The computed potential energy surface reveals that the rate limiting step in neutral conditions is the first hydrolysis process with an activation energy of 110.21 kJ mol1. The final product formed in neutral conditions is a dihydroxo Pt-complex with the loss of lactic acid. Proton transfer from entering water molecule to the oxygen of leaving group has been observed for both the substitution processes. Lobaplatin undergoes complete hydrolysis in neutral conditions and the dihydrated complex is belived to be the species reacting with the DNA purine bases. Whereas in acidic conditions, ligand detachment step is the rate limiting process with an activation energy of 113.82 kJ mol1. For the second aquation step, proton transfer is observed from entering water molecule to detaching ligand and the product formed is [Pt (1,2-bis(aminomethyl)cyclobutane)(H2O)(OH)]+. We can therefore hypothesize that lobaplatin reaches DNA in its monohydrated form. A comparison between lobaplatin with other currently used Pt(II)containing anticancer drugs in the medicalprotocols, reveals some general trends. The hydrolysis reaction of lobaplatin in neutral and acidic conditions is in analogy with the other second generation Pt-containing drugs. This study will provide a better insight into the hydrolysis controlling mechanisms which is essential to identify the active form of drug, which reacts with DNA.
Acknowledgements The authors V.P. Reddy B. and S.C. Moi are pleased to thank National Institute of Technology Durgapur-713209 for necessary assistance and also Department of Science and Technology, Government of India for providing DST project fund (Project No: SB/EMEQ-028/2013), for carrying out this work. IM and SM are also thankful to DST Inspire, Govt. of India for research fellowship.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cplett.2016.10. 004.
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