Chem. Res. Toxicol. 2007, 20, 1093–1098
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Disproportionation of Clozapine Radical: A Link between One-Electron Oxidation of Clozapine and Formation of Its Nitrenium Cation Adam Sikora, Jan Adamus, and Andrzej Marcinek* Institute of Applied Radiation Chemistry, Technical UniVersity of Lodz, Zeromskiego 116, 90-924 Lodz, Poland ReceiVed April 24, 2007
The primary products of one-electron oxidation of clozapine and olanzapine, very effective atypical antipsychotic drugs, have been spectroscopically characterized. The oxidation process has been studied under glassy matrix conditions and by a pulse radiolysis technique in aqueous solution. The rate constants for the oxidation of clozapine with dibromide radical anion (k ) 2 × 109 M-1 s-1) and azide radical (k ) 2.3 × 109 M-1 s-1) in aqueous solution were measured. The computational DFT results support the identification of the transient species. The mechanistic aspects of reactivity of radical cations, radicals, and nitrenium cations have been investigated. A disproportionation reaction (k g 1 × 108 M-1 s-1) was proposed as a link between the products of one-electron oxidation and formation of the nitrenium cations of clozapine and olanzapine, products likely responsible for the pathogenesis of adverse drug reactions. The rate constants for the reactions of nitrenium cation of clozapine with glutathione (k ) 3.4 × 104 M-1 s-1) and cysteine (k ) 9.8 × 104 M-1 s-1) were determined. Introduction Clozapine [8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzodiazepine (CLZ)] 1 and olanzapine [2-methyl-4-(4-methyl1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine (OLZ)] are atypical antipsychotic drugs used in treatment of schizophrenia (1). The use of clozapine, which is more effective than standard neuroleptic drugs in the treatment of refractory schizophrenia, is, however, limited because of a relatively high incidence of drug-induced agranulocytosis (2–5). In addition, clozapine causes many other adverse reactions, including myocardity, cardiomyopathy, and hepatotoxicity (6, 7). On the other hand, a thiobenzodiazepine derivative, olanzapine, which was expected to be a safe alternative to clozapine also induces agranulocytosis (8). In contrast to the effects of clozapine, the adverse effects of olanzapine remain, however, less recognized since they occur at doses of the drug higher than those typically used in therapy (9, 10). The mechanisms of these CLZ- and OLZ-induced adverse reactions are still unknown, but it is believed that they may be due to chemically reactive metabolites and involve both toxic and immunological components (3, 6, 11, 12). It has been demonstrated that clozapine can be oxidized by activated neutrophils or by hypochlorous acid to its nitrenium cation, a reactive metabolite, that covalently binds to the neutrophils (13–15). This reactive intermediate can be trapped by glutathione, what results in the formation of the corresponding conjugates (Scheme 2) (14). Clozapine also undergoes bioactivation in hepatic and cardiac microsomal incubations and bone marrow cells. This has been demonstrated mainly in GSH trapping studies throughout the * To whom correspondence should be addressed. Telephone: +48-426313096. Fax: +48-42-6365008. E-mail:
[email protected]. 1 Abbreviations: CLZ, clozapine; OLZ, olanzapine; MCLZ, methylclozapine; MPO, myeloperoxidase; HRP, horseradish peroxidase; BMIM+PF6-, 1-butyl-3-methylimidazolium hexafluorophosphate; TD-DFT, time-dependent density functional theory.
Scheme 1. Structures of Clozapine and Olanzapine
Scheme 2. Formation of Glutathione Conjugates in Enzymatic Oxidation of Clozapine by a Horseradish Peroxidase (HRP)/H2O2 or Myeloperoxidase (MPO)/H2O2 System
formation of glutathione conjugates mentioned above (16). The same glutathione adducts were observed upon oxidation of clozapine by horseradish peroxidase (HRP)/H2O2 or myeloperoxidase (MPO)/H2O2 systems (17, 18). The formation of radical cations and radicals was postulated in those processes (16–18). In this paper, we present experimental results of one-electron oxidation of clozapine and olanzapine. Understanding the properties and reactivity of the primary oxidation products may be essential for elucidation of the mechanism of enzymatic
10.1021/tx700128c CCC: $37.00 2007 American Chemical Society Published on Web 07/13/2007
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bioactivation of the drugs. It seems especially important to recognize the possible mechanisms linking postulated reactive metabolites of clozapine and olanzapine, i.e., radical cations, radicals, and nitrenium cations. Recently, we have presented a novel method for spectroscopic characterization of nitrenium cations generated upon oneelectron oxidation of N-nitrosoamines (19). We have also studied the reactivity of the radical cations, radicals, and nitrenium cations generated from amines and N-nitrosoamines, under matrix conditions and by pulse radiolysis in solution, and we have found some important similarities in the reactivity of those species. 2 Therefore, in this paper, we explore the same experimental techniques of stabilization and spectral characterization of radical ions upon their generation in low-temperature matrices by an ionizing radiation (20, 21). In combination with pulse radiolysis, it allows us to monitor the reactivity of, unambiguously identified and spectroscopically characterized, transient species of interest.
Materials and Methods Compounds. Clozapine was isolated from tablets (10 × 100 mg tablets, Klozapol, Anpharm, Poland). The tablets were crushed and extracted with chloroform (50 mL). The solution was centrifuged and filtered to remove insoluble material. Then solvent was removed under reduced pressure. The residue was dissolved in 0.2 M HCl, and then the solution was filtered. After addition of a 1 M NaHCO3 solution, the clozapine precipitate was filtered out and washed with (hot) water. For further purification, clozapine was crystallized from an acetone/hexane mixture: yellow crystals; mp 184–184.5 °C. To obtain olanzapine, Zolafren (Adamed, Poland) tablets (30 × 10 mg of active substance) were crushed in a mortar and extracted with 30 mL of a 0.2 M HCl solution. The suspension was filtered through paper, and the filtrate was basified to pH 8.5 with NaHCO3 and extracted with ethyl ether (2 × 20 mL). The ether solution was dried with anhydrous MgSO4, the solvent evaporated on a rotary evaporator, and the residue crystallized from 3 mL of anhydrous acetonitrile to give 0.276 g of olanzapine: light-orange crystals; mp 196 °C dec. Other chemicals were commercially available from SigmaAldrich. All aqueous solutions were prepared using water purified by a Millipore Milli-Q system. Cryogenic Measurements. Glassy samples of a 1-butyl-3methylimidazolium hexafluorophosphate/chloroform mixture [1:1 (v/v) BMIM+PF6-:CHCl3 ratio] were prepared by immersing room-temperature solutions in liquid nitrogen in special cryogenic cuvettes. The samples were 0.5–3 mm thick and were placed in a liquid nitrogen-cooled cryostat (Oxford Instruments) where any temperature between 77 and 150 K can be maintained by controlled heating. The optical absorption spectra were measured on a Cary 5 (Varian) spectrophotometer. The samples mounted in a cryostat were irradiated with 4 µs electron pulses from an ELU-6 linear accelerator. In the paper, the difference steady-state transient absorption spectra are presented (after minus before irradiation) for the direct comparison with the spectra obtained in the pulse radiolysis experiments. Pulse Radiolysis. Pulse radiolysis experiments were carried out with high-energy (8 MeV) 17 ns electron pulse generated from an ELU-6 linear electron accelerator. The dose absorbed per pulse was determined with a N2O-saturated aqueous solution of KSCN (0.01 M), assuming G[(SCN)2•-] ) 6.2 × 10-7 mol/J and ε[(SCN)2•-] ) 7600 M-1 cm-1 (G represents the radiation chemical yield, and ε is the molar absorption coefficient at 475 nm) (22). The dose delivered per pulse was within the range of 5–80 Gy. Low-dose pulses were used for the determination of the 2
Unpublished results of A. Sikora, A. Marcinek, et al.
Sikora et al. rate constants of the observed reactions. Details of the pulse radiolysis system are given elsewhere (23, 24). To identify the products of one-electron oxidation of the investigated drugs, we studied their reactions with strong oxidants like N3• radicals or dibromide radical anions Br2•-. We have found that Br2•- [E°(Br2•-/2Br-) ) 1.63 V, vs NHE] which is a stronger oxidant than N3• [E°(N3•/N3-) ) 1.33 V, vs NHE] (25, 26) is more suitable for monitoring and visualization of the oxidation process as it allows observation of the Br2•- decay and product formation (radical cations of the drugs). Dibromide radical anions, Br2•-, were generated by the pulse radiolysis carried out in the solution of KBr saturated with N2O so that the •OH radicals react with bromide anions to form oxidizing species (25, 26).
Br- + •OH f •Br + OH•
Br + Br- f Br2•-
(1) (2)
The solution was saturated with N2O to convert eaq- into hydroxyl radicals (25).
eaq- + N2O f •OH + OH- + N2
(3)
In some cases, to avoid interference of dibromide radical anion absorption with the absorption of the products, N3• radicals were used as oxidizing species. Pulse radiolysis of aqueous solution of NaN3 leads to the formation of N3• radicals in the reaction of •OH radicals with N3-.
N3- + •OH f N3• + OH-
(4)
•-
The radiation chemical yield of Br2 was determined on the basis of its molar absorption coefficient at 360 nm [Br2•-, ε360 ≈ 8 × 103 M-1 cm-1 (27)]. In the case of azide radical, a dosimetry with (SCN)2•- was used for the yield estimation. The pH of solutions was adjusted with perchloric acid, sodium hydroxide, or phosphate buffer and measured with the ORION 420A pH meter (Orion Research). The pH values of very basic solutions (>12) were calculated from the concentrations of sodium hydroxide. Kinetic analysis was done with Levenberg–Marquardt algorithm. The first- or second-order rate constants were evaluated from the plot of ∆A versus time. For the reaction of the transient products with glutathione (GSH) and cysteine (Cys), the bimolecular rate constants were determined from the slope of the linear plot of pseudo-first-order rate constants of the decay of transient products versus the concentration of GSH and Cys, respectively. Quantum Chemical Calculations. The geometries of all species were optimized by the B3LYP density functional method (28, 29) as implemented in the Gaussian 03 suite of programs (30) using the 6-31G* basis set. Electronic transitions were predicted by density functional-based time-dependent response theory (31). We used the formulation of TD-DFT implemented in the Gaussian 03 program (30), together with the same functional and Gaussian basis sets as mentioned above.
Results and Discussion In the paper, we use the term radical cation to name the primary products of one-electron oxidation of clozapine and olanzapine. This in fact may not be completely correct since on the basis of the pKa values found for clozapine [pKa1 ) 3.70; pKa2 ) 7.60 (32)], in some cases one-electron oxidation products should be considered as radical di- or trications. However, since the protonation takes place on nitrogen atoms [piperazine nitrogen atoms and/or the N(10) atom in the central ring] other than that in the oxidation process center of the molecule [N(5) atom], it is more convenient to leave out the aspects of protonation; moreover, pKa values for appropriate radical cations, radicals, and nitrenium cations remain unknown.
Disproportionation of Clozapine Radical
Figure 1. Electronic absorption spectra of clozapine (A) and olanzapine (B) nitrenium cations obtained on oxidation with 50 µM (BrPh)3N•+SbCl5-. The samples were 1 cm thick. Vertical bars are the TD-DFT/B3LYP-631G* predictions of the electronic transitions of clozapine and olanzapine nitrenium cations.
It is also worth mentioning at the beginning that the three most important transient species identified in this paper, i.e., radical cations, radicals, and nitrenium cations, are characterized by very similar absorption spectra (strong band around 300 nm, weak band around 400 nm, and weak, broad band at 500– 600 nm). 3 Therefore, very careful spectroscopic characterization supported by time-dependent DFT calculations is required to distinguish these elusive species. The main target of this investigation was spectroscopic characterization of the primary products of one-electron oxidation of CLZ and OLZ and mechanistic aspects of their reactivity leading to the appropriate nitrenium cations. These latter final transient products, nitrenium cations of clozapine (CLZ+) and olanzapine (OLZ+), can be obtained by a very simple, chemical oxidation. Figure 1 presents spectra of the species obtained upon
two-electron oxidation of the compounds of interest in reaction with radical cation of hexachloroantimonate tris(p-bromophenyl)amine (BrPh)3N•+SbCl6- [“magic blue”; λmax ) 725 nm, CH2Cl2; log ε ) 4.51 (33)]. The transient products generated by this method remained stable for several minutes to hours which allowed for their steady-state, spectroscopic characterization. The vertical bars seen in Figure 1 represent the results of the TD-DFT predictions of the spectra of CLZ+ and OLZ+. The experimentally observed spectra are in good accord with the results of the TD-DFT method which predicts correctly all strongest absorption bands in both cases (λmax ) 310, 380, and 480 nm and 350, 430, and 550 nm for CLZ+ and OLZ+, respectively). The observed spectra remain also in agreement with the spectra obtained previously in reactions of CLZ and OLZ with HOCl (13–15), and therefore, they can be used for spectral confirmation of the formation of such products in the processes of interest. 3 All presented spectra are colored according to their assignment to different transient species: radical cations (blue), radicals (black), and nitrenium cations (red).
Chem. Res. Toxicol., Vol. 20, No. 8, 2007 1095
Figure 2. Electronic absorption spectra of clozapine (A) and olanzapine (B) radical cations obtained upon radiolysis of 2 mM clozapine (A) and 2 mM olanzapine (B) embedded in low-temperature glasses (77 K) of a BMIMPF6/CHCl3 mixture (1:1, v/v). The sample was 3 mm thick and received a radiation dose of 5 kGy. Vertical bars are the TD-DFT/B3LYP-631G* predictions of the electronic transitions of clozapine and olanzapine radical cations.
Figure 3. Transient absorption spectra obtained by pulse radiolysis of clozapine (100 µM) in a N2O-saturated aqueous solution containing 0.1 M KBr and 0.01 M HClO4. The spectra were collected (squares) 200 ns and (circles) 30 µs after the electron pulse. The inset depicts changes in absorbance at 310 and 360 nm. The sample was 1 cm thick and received a radiation dose of 50 Gy.
Despite the poor solubility of clozapine and olanzapine, their radical cations can be spectroscopically characterized under organic matrix conditions (glassy BMIM+PF6-/CHCl3 mixture) or upon reaction with one-electron oxidants such as dibromide radical anions or azide radicals in aqueous solutions. Figure 2 presents the spectra of the radical cations, CLZ•+ and OLZ•+, obtained under cryogenic, matrix conditions. Similar spectra were also observed in aqueous solutions by using the complementary time-resolved technique, pulse radiolysis (see Figure 3). The kinetics of oxidation of CLZ and OLZ by dibromide radical anions could be monitored both by the observation of the decay of Br2•- [λmax ) 360 nm (27)] and by the formation of the strongest absorption band of these radical cations (λmax ) 310 nm). The second-order rate constant of the oxidation of CLZ by dibromide radical anion [kox(Br2•-) ) 2 × 109 M-1 s-1] was estimated from the analysis of the kinetic traces (see Figure 3) and the known concentration of clozapine in its saturated solution. The fast formation of radical cations was followed by their slow decay. Assuming that the molar absorption coefficients for the radical cations can be estimated by a comparison with the molar absorption coefficient of Br2•- (CLZ•+, ε310 g 8 × 103 M-1 cm-1; OLZ•+, ε310 g 5 × 103 M-1 cm-1), the secondorder rate constant for the decay of radical cations (kd) can be estimated to be >1 × 108 M-1 s-1 for both drugs.
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Figure 4. Transient absorption spectrum obtained by pulse radiolysis of clozapine (80 µM) in a N2O-saturated aqueous solution containing 0.1 M NaN3 and 0.1 M NaOH. The spectrum was collected (b) 10 µs after the electron pulse. The sample was 1 cm thick and received a radiation dose of 50 Gy. Vertical bars are TD-DFT/B3LYP-631G* predictions of the electronic transitions of clozapine radical.
The experiments in aqueous solutions were conducted under acidic conditions to avoid deprotonation of the radical cations and the formation of neutral radicals. These latter species were characterized in aqueous solutions (see Figure 4) on one-electron oxidation of the clozapine by the reaction with the azide radicals, N3•, under basic conditions (in the case of olanzapine, Br2•was used as an oxidant because of the fast reaction of secondary products with azide anions; see further discussion in the paper). The rate constant for the oxidation reaction of clozapine with azide radical was found also to be close to the diffusioncontrolled limit [kox(N3•) ) (2.3 ( 0.3) × 109 M-1 s-1]. The high degree of similarity of the spectra of radical cations and radicals in aqueous solution did not allow an unambiguous determination of the pKa of these radical cations. The decay of the radical absorptions follows also the secondorder kinetics 2k/εl ) (5.0 ( 0.2) × 104 s-1. The rate of the process is dose-dependent (thus dependent on the concentration of the radical generated) which further confirms the secondorder nature of this reaction. Upon evaluation of the molar absorption coefficient for CLZ•, similar to that of CLZ•+ which is on the order of 1 × 104 M-1 cm-1, the rate constant for the observed second-order reaction [kd(CLZ•)] can be estimated to be 1 × 108 M-1 s-1. Comparison of the spectra of the products of radical cation and neutral radical decays (see Figure 5 for the products observed at neutral or slightly basic pH) with the spectra of the appropriate nitrenium cations leaves no doubt that these species are the final products of the reactivity of clozapine and olanzapine upon their one-electron oxidation. The second-order nature of the observed reactions suggests that the disproportionation reaction can be responsible for the formation of the nitrenium cation product (Scheme 3). In the case of clozapine, its nitrenium cation only very slowly reacts with azide anion. Pseudo-first-order decays of the absorption bands of CLZ+ in the presence of different concentrations of NaN3 (0.01–0.08 M) showed that the rate constant for this reaction [kCLZ(N3-) ) 20 ( 1 M-1 s-1] is very low. In contrast, olanzapine nitrenium cation reacts 3 orders of magnitude faster [kOLZ(N3-) ) (5.9 ( 0.4) × 103 M-1 s-1]. Fast generation of CLZ+ in the reaction of clozapine with azide radicals and at the same time its slow reaction with azide anion allowed us to investigate the reaction of CLZ+ with cysteine (Cys) and glutathione (GSH). Although both these thiols also react with azide radicals [kGSH(N3•) ) 9.5 × 106 M-1 s-1 (34) and kCys(N3•) ) 1.4 × 107 M-1 s-1 (35) for GSH and Cys, respectively], their rate constants are 2–3 orders of magnitude lower than those of the CLZ and OLZ oxidation reactions. The course of the reaction of clozapine nitrenium
Sikora et al.
Figure 5. Transient absorption spectra obtained by pulse radiolysis of (A) clozapine (80 µM) in a N2O-saturated aqueous solution containing 0.1 M NaN3. The spectra were collected (squares) 10 and (circles) 450 µs after the electron pulse. The inset depicts the changes in absorbance at 310 nm (black) and the second-order kinetic fitting (red). The sample was 1 cm thick and received a radiation dose of 60 Gy. Transient absorption spectra obtained by pulse radiolysis of (B) olanzapine (80 µM) in a N2O-saturated aqueous solution containing 0.1 M KBr. The spectrum was collected 0.01 s after the electron pulse. The sample was 1 cm thick and received a radiation dose of 55 Gy.
Scheme 3. Disproportionation of CLZ Radicals and Radical Cations Leading to Nitrenium Cations
cation was monitored by the decay of its absorption in the aqueous solutions (saturated with N2O) of CLZ (0.8 mM), NaN3 (0.04 M), and glutathione or cysteine (0–0.6 mM). The rate constants for the reactions of nitrenium cation of clozapine with glutathione [kCLZ(GSH) ) (3.4 ( 0.1) × 104 M-1 s-1] and with cysteine [kCLZ(Cys) ) (9.8 ( 0.2) × 104 M-1 s-1] were found from the linear dependence of the rate of CLZ+ decay on the concentration of GSH and Cys, respectively. Similar results were obtained for methylclozapine [4-(8chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)-1,1-dimethylpiperazin-1-ium (MCLZ); kMCLZ(GSH) ) (5.1 ( 0.1) × 104 M-1 s-1 and kMCLZ(Cys) ) (1.1 ( 0.1) × 105 M-1 s-1] which show that the positive charge on the piperazine ring has no effect on the reactivity of nitrenium cation. Higher concentrations of glutathione did not seem to affect the formation of MCLZ+ through the reaction of GSH with radical cation or methylclozapine radical. The reactivity of the nitrenium cation of MCLZ with GSH is relatively more effective than the reactivity of its precursors, i.e., radical cations or radicals (Scheme 4). The results of the DFT calculations presented in Scheme 5 show that significant changes which accompany the oxidation of clozapine to its nitrenium cation occur mainly
Disproportionation of Clozapine Radical Scheme 4. Structure of Methylclozapine
Scheme 5. Comparison of the Structures of CLZ and Its Nitrenium Cation, CLZ+
in the central and side chlorophenyl rings, while the second phenyl ring and piperazine moiety remain almost unchanged. The structure of CLZ+ adopts an o-diiminecyclohexadiene character which explains the substitution of GSH at the C(6) and C(9) positions of the chlorophenyl ring, in agreement with the previous findings (14, 16–18). On the basis of computational DFT results, analogical conclusions can be drawn for olanzapine, but with one important difference. In contrast to the case for clozapine, part of the positive charge in the olanzapine nitrenium cation is localized on the side (thiophene) ring, which to some extent may affect the reactivity of the species.
Conclusions In this paper, we show a novel method of generation of nitrenium cations of clozapine and olanzapine, the two very important atypical antipsychotic drugs used in therapy of schizophrenia, which allows for their spectroscopic characterization and convenient investigations of the mechanistic aspects of their reactivity. These important very reactive transient products can be formed in the processes initiated by the one-electron oxidation of the drugs. The results link the reactivity of radical cations and neutral radicals of clozapine and olanzapine with the formation of their nitrenium cations. In light of our findings, the previously reported evidence for the formation of free radicals in the process of the formation of GSH conjugates remains in no contradiction to the assignment of nitrenium cations as major reactive metabolites leading to these products in reaction with glutathione (14, 16–18). The disproportionation reaction presented above seems to be a missing link between investigated transient species. It seems very likely that one-electron oxidation products of clozapine and olanzapine can exert their toxic effects through the formation of nitrenium cations. Acknowledgment. This work was supported by a grant from the Polish Ministry of Science and Higher Education (N205 003 32/0258).
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