Article pubs.acs.org/OPRD
Rapid Synthesis of Pharmaceutical Oxidation Products Using Electrochemistry: A Systematic Study of N‑Dealkylation Reactions of Fesoterodine Using a Commercially Available Synthesis Cell Susana Torres,*,† Roland Brown,† Roman Szucs,† Joel M. Hawkins,‡ Garry Scrivens,† Alan Pettman,† Debbie Kraus,† and Mark R. Taylor† †
Pfizer Worldwide R+D, Ramsgate Road, Sandwich, Kent CT139NJ, United Kingdom Pfizer Worldwide R+D, Eastern Point Road, Groton, Connecticut, United States
‡
ABSTRACT: A new method for the fast and convenient synthesis of pharmaceutical oxidation products is described. Two oxidation products of fesoterodine were electrochemically synthesized, isolated, and characterized. The influence of synthetic operating parameters such as pH, percentage of organic solvent in diluent, initial electrolyte concentration, and substrate concentration on the oxidation product profile was investigated. This synthetic procedure proved to be rapid, clean, and efficient compared to traditional synthetic methods and may be particularly useful for generating milligram quantities of reference samples of degradation products used as markers in chromatographic methods.
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INTRODUCTION The stability of a drug or drug product is its capability to maintain its physical, chemical, and therapeutic integrity during the storage time and usage by the patient. Maintaining the quality, safety, and efficacy of an approved drug product makes it necessary to identify drug degradants and establish their degradation mechanisms and pathways at an early stage of the development.1 The major mechanisms of chemical degradation of drugs include hydrolysis, oxidation, isomerization, rearrangements, condensation, decarboxylation, dimerization, polymerization, photolysis, and reactions with excipients/salt forms.2 Oxidative reactions are amongst the most commonly observed chemical degradation pathways for pharmaceuticals; stress methods for generating oxidative degradation typically involve the use of oxidising agents such as peroxide or iron(III) chloride and are often nonselective, yielding mixtures of oxidised products for further characterization.3,4 In recent years electrochemistry (EC) coupled to liquid chromatography (LC) and/or mass spectrometry (MS) has shown promise for supporting drug metabolism studies.5−7 Electrochemistry proved to be useful in the imitation of drug metabolism by cytochrome P450 (CYP) enzymes with a series of reactions being simulated electrochemically. Benzylic hydroxylation,8 hydroxylation of aromatic rings,8,9 allylic and aliphatic hydroxylation,10 N-dealkylation,11,12 N,S-oxidation,8,13 dehydrogenation,8,14 and O-dealkylation8,15 are the examples of reactions that can be simulated electrochemically. Here, we discuss the possibility of using electrochemistry as an oxidative stress condition and also as a means of synthesizing milligram quantities of oxidative compounds for subsequent NMR characterization, for use as markers to aid method development and to assist in stability and toxicity studies.
Figure 1. Chemical structure of fesoterodine (F) and of the electrochemically synthesized oxidative N-dealkylation products (1 and 2).16
electrochemistry in synthesizing two oxidative N-dealkylation products of the tertiary amine function of fesoterodine, which have been observed in long-term stability studies. Two oxidation products of fesoterodine were electrochemically synthesized at a constant potential of 950 mV using a three-electrode cell, controlled by a potentiostat (Figure 1). These two compounds are the result of the oxidative Ndealkylation of the tertiary amine function of fesoterodine. Masui et al. studied the oxidation of aliphatic tertiary amines by cyclic voltammetry and controlled potential electrolysis at a glassy-carbon electrode in aqueous alkaline solution,17 and their
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Special Issue: Oxidation and Oxidative Reactions
RESULTS AND DISCUSSION Fesoterodine fumarate (Pfizer WR+D) (Figure 1) was selected as a model compound in order to study the effectiveness of © XXXX American Chemical Society
Received: October 2, 2014
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Figure 2. Scanning voltammetry plot of fesoterodine. Current (Icell) vs potential (Ecell) for a single (half) scan cycle (solid line) and corresponding first derivative (dotted line) showing that at maximum gradient Ecell = 830 mV. Conditions: E1 = 0 V; E2 = 1.5 V; cycle: continuous; scan rate: 20 mV/s; flow rate: 50 μL/min; Fesoterodine fumarate concentration = 0.1 mg/mL in 5 mM aqueous ammonium acetate solution.
Figure 3. UV chromatograms at 224 nm. (A) 0.25 mg/mL fesoterodine fumarate solution in 50 mM aqueous ammonium acetate (no potential was applied to the SynthesisCell). The observed peak “F” corresponds to fesoterodine. (B) Reaction mixture after 2 h (a constant potential of 950 mV was applied to the cell). A comparison of two chromatograms shows almost complete conversion of fesoterodine into the two oxidation compounds. (For the chemical structures, see Figure 1.) For both chromatograms, the peak at t = 0.2 min corresponds to fumarate and acetate molecules.19
grams of the drug were generated using the Roxy System equipped with a μ-PrepCell fitted with a glassy carbon working electrode (Figure 2). A solution of ammonium acetate (5 mM) in purified water was used as a supporting electrolyte. The working potential range was established by means of scanning voltammetry in flow injection conditions.18 A continuous full scan was applied to the cell from 0 to 1500 mV, and the first derivative of the measured cell current (Icell) was calculated in order to visualize the region where the oxidation takes place. A current peak occurred between 600 and 1000 mV indicating substrate oxidation (Figure 2). Considering these results, a voltage of 850 mV was selected as the working potential for the SynthesisCell. In a first approach, the two N-dealkylated oxidation products were
postulated mechanism for the oxidation of tertiary amines involves abstraction of an electron from the lone-pair of electrons on the amino-nitrogen, followed by rapid proton loss to form a neutral radical, which then loses an electron and is hydrolyzed to the products. In the case of the molecule under study there are two types of α-protons that can be lost in the second step of the proposed mechanism. If the N−CH proton is lost, that gives rise to the formation of oxidation product 1 (and 2-propanone). However, the loss of one of the CH2−N protons leads to the formation of oxidation product 2 (and diisopropylamine). In order to understand the electrochemical oxidative behavior of fesoterodine and to determine the potential range for the synthesis of the two oxidation compounds, voltammoB
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Figure 4. 1H NMR spectra for oxidation product 1 (a) and oxidation product 2 (b). Chemical shifts (δ) are given in ppm relative to (CD3)2SO and CDCl3 for oxidation product 1 and oxidation product 2, respectively. For 1H and 13C peak assignment, see the Experimental Section. *Unrelated impurity.
generated in the SynthesisCell at a constant potential (850 mV), using glassy carbon as the working electrode and 50 mM
aqueous ammonium acetate as the supporting electrolyte. The reaction was monitored over a 2 h period of time. Aliquots of C
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Table 1. Design of experiments study (DoE) run order
pH
initial concentration of fesoterodine (mg/mL)
concentration of electrolyte (mM)
amount of acetonitrile in diluent (%v/v)
1 2 3 4 5 6 7 8 9 10 low mid high
6.75 4.0 9.5 4.0 9.5 4.0 9.5 4.0 9.5 6.75 4 6.75 9.5
0.55 0.1 0.1 1 1 0.1 0.1 1 1 0.55 0.1 0.55 1
55 100 10 100 10 10 100 10 100 55 10 55 100
30 50 50 10 10 10 10 50 50 30 10 30 50
calculated. Conversion of substrate was calculated dividing the decrease on fesoterodine peak area (initial peak area (0 V) − final peak area (950 mV, t = 2 h) by the initial fesoterodine peak area. The selective formation of products 1 and 2 was calculated by dividing the final peak area of each of the oxidation products by the initial peak area of fesoterodine. The obtained results show good repeatability between replicates compared between run variability (Figure 5). Variance component analysis estimated at least 95% of the total variance in the results of the study was between experimental run settings with less than 5% made up of repeatability within a set of conditions. As can be seen from Figure 6, the obtained results indicate that there exists a good correlation between fesoterodine conversion and oxidation product 2 (aldehyde moiety) formation. Run 5 and 7 (circled points) showed lower results for both replicates than the linear relationship suggests. However, a much poorer correlation was obtained between fesoterodine conversion and oxidation product 1 (secondary amine moiety) formation. The obtained results also show that the formation of aldehyde moiety is favored when compared with the secondary amine moiety. According to Masui et al.,20 this can be explained by the fact that the relative amount of dealkylation in unsymmetrical amines is predominantly governed by the acidity and the number of α-protons. In the case of the molecule under study there are two types of αprotons that can be abstracted from the molecule. If the N−CH proton is lost, that gives rise to the formation of oxidation product 1. However, the loss of one of the CH2−N protons leads to the formation of oxidation product 2. In the case of the molecule under study, the explanation for the fact that we form more aldehyde than secondary amine is more likely related to steric effects at the isopropyl group because the molecule of fesoterodine has two CH2−N α-protons and two N−CH αprotons (two isopropyl groups). The abstraction of the proton in the isopropyl group is likely more difficult than the abstraction of one of the CH2−N protons which may contribute to the higher formation of aldehyde when compared to the secondary amine dealkylated product. The assessment of the effects of the experimental parameters has been done with the aid of graphical methods. Half-normal plots for fesoterodine and the two N-dealkylated products were constructed (Figure 7). The half-normal plots allowed us to determine the statistically significance of the experimental parameters. These plots are useful for separating potential real effects from those who are indistinguishable from noise. If none
the reacting solution were taken at given time points and analyzed using high-performance liquid chromatography with UV and mass detection. After turning on the cell voltage, a decrease in fesoterodine peak area was observed with concomitant formation of the two N-dealkylated oxidation products. These experimental conditions generated an 89% conversion of fesoterodine into the two N-dealkylated oxidation products over a 2 h period of time (data not shown). However, an increment of the cell voltage to 950 mV led to almost complete conversion in the same period of time (Figure 3). Based on these results, the potential of 950 mV was chosen as the working potential for further studies. The two oxidation products were purified by reverse-phase preparative high-performance liquid chromatography and subsequently characterized by NMR. 1H NMR spectra for the two oxidation products can be seen in Figure 4, and 1H and 13C peak assignments can be found in the Experimental Section. Chemical shifts (δ) are given in ppm relative to CDCl3 (1H, δ = 7.27; 13C, δ = 77.00) or (CD3)2SO (1H, δ = 2.50; 13C, δ = 39.51). 1H, 13C, COSY, HMQC, and HMBC NMR data were obtained for each compound. The next step of this study was to optimize the conditions for the synthesis of the two oxidation products. To do so, a design of experiments (DoE) study was performed in order to understand the effect of various parameters on the synthesis of oxidation compounds. The effect of pH, electrolyte concentration, substrate concentration, and percentage of organic (acetonitrile) in the sample diluent was studied. All of the experiments were performed using a constant working potential of 950 mV. The effect of the four factors was studied by means of an 8-run fractional factorial 24−1 IV design with a repeated center-point run (Table 1). The experimental results were analysed using the software Statgraphics Plus. In this study, 8 runs were performed in order to obtain information on how the four factors influence the conversion of fesoterodine into the two oxidation products. These 8 runs (2− 9) were done in duplicate with a repeteated centre point run (1 and 10). The synthesis of the two oxidation products was done using the experimental conditions listed in Table 1, and the working potential was set to 950 mV. A sample of the reaction mixture was taken 2 h after turning on the cell voltage and injected in the HPLC instrument with UV detection. The peaks areas corresponding to fesoterodine and to the two Ndealkylated oxidation products were determined by HPLCUV. The percentage of fesoterodine conversion and the percentage of formation of the two oxidation products were D
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Figure 7. Half-normal plots for fesoterodine (a) and the two oxidation products (b = oxidation product 2, c = oxidation product 1) showing the importance of the four studied experimental parameters (A: pH, B: initial concentration of fesoterodine, C: concentration of electrolyte and D: percentage of acetonitrile in the diluent) and their interactions on the fesoterodine conversion and oxidation products formation.
Figure 5. Plot of the fesoterodine (substrate) conversion and oxidation products formation versus run (10 runs; run 2−9 were done in duplicate).
of the factors or interactions are real, i.e., the variability in the data is just due to random noise, we would expect all of the points in the half-Normal plot to fall on a line through (0,0). Values that fall to the right of the line point to effects that are larger than due to chance alone and are likely to be a real effect of a factor (or an interaction between two factors). These plots indicated that the formation of oxidation product 2 (aldehyde moiety) was strongly affected by the pH of the solution, by the percentage of acetonitrile used in the diluent and by the interaction between these two factors (Figure 7b). The half-normal plots also suggest that the solution pH is an important factor on fesoterodine conversion as well as the interaction between pH and percentage of acetonitrile (Figure 7a). As the N-dealkylated amine moiety is concerned, it is unclear which factors are having an effect on the formation of this compound, although pH is estimated to be the largest factor effect (Figure 7c).
Figure 6. Plot showing the correlation between fesoterodine conversion and formation of oxidation products (oxidation product 1 = ×, oxidation product 2 = □) after 2 h of electrochemistry using the synthesis cell with applied voltage of 950 mV. For run 2−9 the points plotted correspond to the average of the duplicates.
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Figure 8. Interaction plots for fesoterodine (a) and N-dealkylated aldehyde (b) showing the interaction between pH and percentage of organic in the diluent on the substrate conversion and oxidation product 2 formation. Each point on the plot corresponds to the mean response for 2 runs.
Interaction plots for the substrate conversion and oxidation product 2 formation were also constructed (Figure 8). As can be seen in the figure, the interaction plot between factor A (pH) and factor D (% of acetonitrile in the diluent) consists of two lines, each of which joins two points. The four points on the plot are the mean responses for runs at (i) low level of A and low level of D; (ii) low level of A and high level of D; (iii) high level of A and low level of D; (iv) high level of A and high level of D. The first line joins points (i) and (ii), and the second line joins points (iii) and (iv). If the lines are not parallel, then an interaction may be present, meaning that the effect of factor A will be different depending on the level of factor D. The obtained data showed that the higher pH and higher percentage of acetonitrile generated higher levels of Ndealkylated aldehyde formation and complete fesoterodine conversion. The initial concentration of electrolyte and fesoterodine do not have an impact on the fesoterodine conversion and aldehyde formation. The fact that the pH of the solution plays such a significant effect is likely due to the fact that at the higher pH the electrons from the lone-pair of electrons on the amino nitrogen are more readily accessible for abstraction than they are at acidic conditions, which then favors the formation of the oxidation products. The percentage of acetonitrile can be important in this particular reaction because it can prevent the adsorption of the generated product molecules on the electrode and assist solubility. Consequently, conversion of high quantities of fesoterodine is possible which is advantageous for productivity. From the analysis of the half-normal plot for oxidation product 1 (secondary amine moiety), it is unclear which factors are affecting the formation of this oxidation product, and Figure 9 shows that the centre-point runs give the highest oxidation product formation, which suggests that the relationship is nonlinear with the highest oxidation product formation occurring at mid-pH range. In order to understand the reason for this behavior, another run was performed (pH = 8, 50% of acetonitrile in the diluent, 55 mM of ammonium acetate, and 0.55 mg/mL of fesoterodine). This additional run suggests that the relationship is nonlinear with the highest results occurring at the centre-point conditions. Some degree of oxidation of product 1 (secondary amine) to the primary amine and to the aldehyde (oxidation product 2) could explain this behavior. In order to evaluate this hypothesis, secondary amine compound obtained after purification by preparative HPLC was oxidized at 950 mV using the SynthesisCell at high pH conditions. The reaction was monitored over time using UV and mass HPLC,
Figure 9. Scatter plots of the percentage of N-dealkylated compound formed versus pH for different percentages of organic (□ = 10% organic, × = 30% organic, ○ = 50% organic). The plotted values correspond to the percentage of oxidation compound 1 at 120 min. For run 2−9 the points plotted correspond to the average of the duplicates. This plot includes the extra run at pH 8.
and the analysis of the obtained chromatograms shows that the secondary amine, previously obtained from the oxidation of the tertiary amine, is oxidized to the primary amine and to the aldehyde (oxidation product 2). These findings suggest that the fact we do not observe a linear relationship between the substrate conversion and the oxidation product 1 formation (secondary amine moiety) is likely due to the fact the secondary amine is subsequently oxidized, forming the corresponding primary amine and the oxidation product 2 (aldehyde moiety), which is also formed in the first oxidation. The obtained results indicate that running the reaction at high pH and high percentage of acetonitrile in the diluent favors the substrate conversion and the formation of oxidation product 2 (aldehyde moiety). Using these experimental conditions, the percentage of formation of oxidation product 1 is less than what is expected if a linear relationship was being followed, because this compound likely participates in a second oxidation, forming the primary amine and more aldehyde (oxidation product 2). With this particular molecule, we should also take into consideration that both the substrate and the oxidation products have an ester linkage which is hydrolysed under basic conditions. Even if the results suggest that this hydrolysis reaction is slower than the oxidation reaction, the formation of hydrolysed products is observed. The obtained results suggest that a much cleaner reaction mixture is obtained F
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performed in a thin-layer three-electrode electrochemical cell (μ-PrepCell, Antec, Zoeterwoude, The Netherlands) using glassy carbon (GC) as the working electrode. Analytical high-performance liquid chromatography was performed using either an Agilent 1200 Series instrument equipped with a UV detector or a Hewlett-Packard 1100 instrument equipped with UV and a mass detector (Waters Micromass ZQ). Preparative HPLC was performed using an Agilent 1200 series liquid chromatograph equipped with a UV detector. Fractions obtained after preparative HPLC were concentrated using a Genevac EZ2 centrifugal evaporator. 1H, 13 C, COSY, HMQC, and HMBC NMR spectra were run on a Bruker 400 NMR spectrometer, operating at 400 and 100 MHz for 1H and 13C, respectively. Scanning Voltammetry with Electrochemical Detection. A 0.1 mg/mL solution of fesoterodine fumarate dissolved in 5 mM aqueous ammonium acetate was pumped through the thin-layer cell, fitted with a glassy carbon electrode at a flow rate of 50 μL/min. The potential was scanned from 0 to 1.5 V at a scan rate of 20 mV/s in a continuous cycle. Using the Dialogue software, the current intensity (Icell) was measured as a function of time or as a function of the voltage applied to the cell (Ecell). Electrochemical Synthesis. A solution of fesoterodine fumarate (0.25 mg/mL) in 50 mM ammonium acetate (80 mL) was transferred to the glass reaction vessel. The synthesis cell was assembled and connected via the electric cables to the ROXY potentiostat. A constant cell voltage of 950 mV was applied to the cell, and the progress of the reaction was monitored over time by taking an aliquot of the synthesis cell solution and analyzing it by HPLC with UV and MS detection. Design of Experiments Study (DoE). For each run the right amount of fesoterodine fumarate (8, 44, or 80 mg) was dissolved in 80 mL of a mixture of ammonium acetate and acetonitrile. Different percentages of organic (10, 30, and 50%) were used. Ammonium acetate solutions of different concentrations (10, 55, 100 mM) and pH (4.00, 6.75 and 9.50) were prepared. For each concentration of ammonium acetate, the pH of the solution was adjusted either by adding ammonium hydroxide or acetic acid. For detailed information on the experimental conditions for each run, see Table 1. Analytical and Preparative HPLC. For all analytical HPLC analysis a reverse-phase column (Agilent Extend C18, 3. 0 × 50 mm, 1.8 μm) was used. Aqueous ammonium hydroxide (0.1% NH4OH) and acetonitrile (ACN) were used as eluting solvents, and separation was achieved by means of a gradient profile, described in Table 2. The flow rate was set to 1.2 mL/
using the centre-point experimental conditions, even if the substrate conversion is not complete.
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CONCLUSIONS The synthesis of analytical standards of API oxidation products is an important process for quality assurance and control, product development, method validation, and toxicity testing. The synthesis of oxidative standards by wet chemistry procedures can be a time-consuming and expensive approach. Here, we showed how electrochemistry can be used as an alternative approach for the synthesis of oxidative API compounds. This method proved to be fast, efficient, and environmentally friendly. Of great importance was its selectivity, repeatability, and tuneability for the oxidation products of interest. Such selective oxidation performance would be difficult or impossible to achieve using traditional synthetic procedures. By using this electrochemical oxidation approach with the commercial synthesis cell, sufficient amounts (>10 mg quantities) of the two oxidation products were generated over a 2 h time period without need to use oxidizing agents. The oxidation compounds were subsequently purified by HPLC and characterized by NMR. Moreover, the present study gave us some insights on how different experimental conditions affect the conversion of fesoterodine and the formation of the two N-dealkylated oxidation products. In this particular reaction, elevated pH and percentage of acetonitrile favored the conversion of fesoterodine and the formation of one of the oxidation products (aldehyde moiety), with the initial concentration of electrolyte and fesoterodine having no particular influence. For the other oxidation product (secondary amine moiety), it is unclear from the half normal plot which factors have significant effect on its formation, and data suggest that the secondary amine participates in a second oxidation forming the aldehyde oxidation product and the primary amine. The high pH and high percentage of organic in the diluent ensures high fesoterodine conversion and formation of oxidation products, but it also favors the hydrolysis of the ester linkage which means that for the synthesis of the two oxidation products the centre-point conditions give rise to a much cleaner reaction mixture which makes it easier to purify. Studies are being conducted in order to understand the kinetics of this oxidation reaction.
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EXPERIMENTAL SECTION General. Fesoterodine fumarate was obtained from Pfizer Worldwide R+D, Sandwich, Kent. Ammonium acetate (Optima LC-MS grade) was purchased from Fisher Scientific. Ammonium hydroxide solution (>25% in H2O) was obtained from Fluka Analytical, and acetic acid (>99.99%) was purchased from Sigma-Aldrich. Formic acid 99+% was obtained from Thermo Scientific. Acetonitrile HPLC grade was obtained from Fisher Chemical. Water used for sample preparation and HPLC mobile phases was purified with a Millipore Water purification system. All chemicals were used in the highest quality available. Instrumentation. The electrochemical synthesis was performed in a three-electrode cell (SynthesisCell, Antec, Zoeterwoude, The Netherlands) connected to a ROXY potentiostat controlled using Dialogue software. The synthesis cell is composed of a working electrode (tubular reticulated glassy carbon, RGC), a reference electrode (Pd/H2, HyREF), and an auxiliary electrode (coiled platinum wire). Initial experiments to determine the optimal potential range were
Table 2. Gradient profile used for analytical HPLC separation time/min ACN (%)
0 5
1 5
9 100
11.5 100
11.6 5
12 5
min, the injection volume was 2, 25, or 50 μL, and the temperature of the column was maintained at 50 °C. Compounds were detected either by UV at a wavelength of 224 nm or by mass spectrometry. For the preparative HPLC a reverse-phase column (Phenomenex Luna C18 (2), 5 μ, 100 A, 150 × 21.20 mm) was used. The crude solution (80 mL) was pumped at a flow rate of 1 mL/min through the preparative column, using a JASCO PU-1580 Intelligent HPLC pump. The flow rate for purification was set to 20.00 mL/min; the column was kept at G
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(5) Karst, U. Angew. Chem., Int. Ed. 2004, 43, 2476−2478. (6) Baumann, A.; Karst, U. Expert Opin. Drug Metab. Toxicol. 2010, 6 (6), 715−731. (7) Jahn, S.; Karst, U. J. Chromatogr., A 2012, 1259, 16−49. (8) Johansson, T.; Weidolf, L.; Jurva, U. Rapid Commun. Mass Spectrom. 2007, 21, 2323−2331. (9) van Leeuwen, S. M.; Hayen, H.; Karst, U. Anal. Bioanal. Chem. 2004, 378, 917−925. (10) Baumann, A.; Lohmann, W.; Schubert, B.; Oberacher, H.; Karst, U. J. Chromatogr., A 2009, 1216, 3192−3198. (11) van Leeuwen, S. M.; Blankert, B.; Kauffmann, J.-M.; Karst, U. Anal. Bioanal. Chem. 2005, 382, 742−750. (12) Lohmann, W.; Karst, U. Anal. Chem. 2007, 79, 6831−6839. (13) Blankert, B.; Hayen, H.; van Leeuwen, S. M.; Karst, U.; Bodoki, E.; Lotrean, S.; Sandulescu, R.; Diez, N. M.; Dominguez, O.; Arcos, J.; Kauffmann, J.-M. Electroanalysis 2005, 17 (17), 1501−1510. (14) Lohmann, W.; Karst, U. Anal. Bioanal. Chem. 2006, 386, 1701− 1708. (15) Lohmann, W.; Karst, U. Anal. Bioanal. Chem. 2009, 394, 1341− 1348. (16) Using electrochemistry as a synthetic approach for the synthesis of the two oxidation products, we are able to obtain approximately 100 mg of both oxidation products in 24 h. Using the traditional synthetic methods, the two compounds are obtained using two different synthetic routes. To obtain 85 mg of the oxidation product 1 (amine moiety), a two-step synthetic approach was used, and a very complex reaction mixture was obtained. Starting with 800 mg of the resultant reaction mixture, 85 mg of pure oxidation product was obtained after purification by preparative HPLC. It took 3 days to obtain 85 mg the desired product. The other oxidation product (aldehyde moiety) was obtained after a more time-consuming synthetic procedure. This particular oxidation product was obtained following a seven-step synthetic approach taking one week to complete with an overall yield of 10% of the desired oxidation product. In addition this electrochemical synthetic approach is also environmentally friendly as the consumption of organic reagents and solvents is significantly reduced. (17) Masui, M.; Sayo, H.; Tsuda, Y. J. Chem. Soc. B 1968, 973−976. (18) Antec Manual 204.0018, Edition 2nd ed.; Antec: Zoeterwoude, The Netherlands, 2012. (19) The initial studies conducted to understand the electrochemical oxidative behavior of fesoterodine were performed using a flowthrough electrochemical cell. Those studies showed that the optimal voltage for the conversion of fesoterodine into the two oxidation products was around 850 mV. When using this result in the synthesis cell (large surface-area working electrode immersed in an actively stirred bulk solution), we observed that higher conversion was obtained using a cell voltage of 950 mV. We think that this observation can be related with the fact that the electrochemical cells used are quite different, and the potential of 850 mV (optimal for the flow-through cell) is not optimal for the synthesis cell. (20) Masui, M.; Sayo, H. J. Chem. Soc. B 1971, 1953−1956.
room temperature, and 0.1% aqueous formic acid and acetonitrile were used as eluting solvents. The gradient profile used for the isolation and separation is described in Table 3. Compounds were detected by UV at a wavelength of 224 nm. Fractions were collected, and those corresponding to the compounds of interest were pooled together and concentrated. Table 3. Gradient profile used for preparative HPLC separation time/min ACN (%)
0 5
1.0 5
1.10 15
16 75
16.10 95
20 95
20.10 5
1
H NMR and 13C Peak Assignment. Oxidation product 1 (secondary amine moiety): 1H NMR (400 MHz, (CD3)2SO): δ (ppm) = 1.04 and 1.06 (2 × d, J = 2.2 Hz, 2 × 3H, NHCH(CH3)2); 1.22 and 1.26 (2 × d, J = 6.9 Hz, 2 × 3H, C(O)CH(CH3)2); 2.15−2.29 (m, 2H, CH2CH2NH); 2.54− 2.63 (m, 2H, CH2CH2NH); 2.86 (spt, J = 7.0 Hz, 1H, C(O)CH(CH3)2); 2.97 (spt, J = 6.4 Hz, 1H, C(O)CH(CH3)2); 4.13 (t, J = 7.7 Hz, 1H, CHCH2CH2); 4.47 (s, 1H, CH2OH), 6.96 (d, J = 8.2 Hz, 1H aromatic, OCCH); 7.15−7.30 (m, 6H, aromatic protons); 7.39 (d, J = 1.9 Hz, 1 H aromatic, OCCCH). 13C NMR (100 MHz, (CD3)2SO): δ (ppm) = 18.61 and 18.75 (C(O)CH(CH3)2); 20.16 and 20.22 (NHCH(CH3)2; 32.52 (CHCH2CH2); 33.31 (C(O)CH(CH3)2); 40.81 (CHCH2CH2); 43.24 (CHCH2CH2); 48.28 (NHCH(CH3)2); 62.48 (CH2OH); 122.34, 125.35, 125.82, 126.83, 127.52, 128.35 (CH aromatic); 135.30, 140.35, 143.29, 146.92 (C aromatic); 174.91 ((COO) ester). Oxidation product 2 (aldehyde moiety): 1H NMR (400 MHz, CDCl3): δ (ppm) = 1.28 and 1.32 (2 × d, J = 6.9 Hz, 2 × 3H, CH(CH3)2; 2.80 (spt, J = 6.9 Hz, 1H, CH(CH3)2; 3.07 (ddd, J = 17.1, 6.9, and 1.7 Hz, 1H, CH2CHO); 3.20 (ddd, J = 17.1, 8.3, and 1.9 Hz, 1 H, CH2CHO); 4.64 (s, 2H, CH2OH); 4.80 (t, J = 7.6 Hz, 1H, CHCH2CHO); 7.03 (d, J = 8.2 Hz, 1H aromatic, OCCH); 7.18−7.32 (m, 7 H, aromatic protons); 9.73 (t, J = 1.7 Hz, 1H, CHO). 13C NMR (100 MHz, CDCl3): δ (ppm) = 18.87 and 18.98 CH(CH3)2; 34.19 CH(CH3)2; 38.18 (CHCH2CHO); 48.70 (CHCH2CHO); 64.77 (CH2OH); 123.00 (OCHCH); 126.33, 126.79, 127.01, 127.70, 128.68 (CH aromatic); 135.29, 138.84, 141.80, 147.77 (C aromatic); 175.33 ((COO) ester); 200.45 (CHO).
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +44 1304643936. E-mail: susana.dasilvatorres@ pfizer.com. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Li, M. Organic Chemistry of Drug Degradation; RSC Publishing: London, 2012. (2) Baertschi, S. W.; Alsante, K. M.; Santafianos, D. Stress testing: The chemistry of drug degradation. In Pharmaceutical Stress Testing: Predicting Drug Degradation, Vol. 153; Taylor & Francis Group: London, 2005. (3) Baertschi, S. W; Jansen, P. J.; Alsante, K. M. Stress testing: A predictive tool. In Pharmaceutical Stress Testing: Predicting Drug Degradation, Vol. 153; Taylor & Francis Group: London, 2005. (4) Harmon, P.; Boccardi, G. Oxidative susceptibility testing. In Pharmaceutical Stress Testing: Predicting Drug Degradation, Vol. 153; Taylor & Francis Group: London, 2005. H
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