Preparation of Metabolites by Chemical Reaction: Conversion of Antipsychotic Phenothiazines to Their Sulfoxides and Tertiary

396 Journal of Health Science, 50(4) 396–406 (2004) Preparation of Metabolites N-oxides from parent drug could be confirmed when the collisional energy was decreased to 10 eV. 1H- and by Chemical Reaction: 13C-NMR spectral data confirmed the structure of the Conversion of Antipsychotic prepared mianserin N-oxide to be 2-oxide. In conclu- sion, a simple and rapid preparation method for oxide Phenothiazines to their metabolites of phenothiazines and tertiary amino cy- Sulfoxides and Tertiary clic antidepressants available as analytical standards was established. Amino Cyclic Key words —–— metabolite, chemical preparation, ox- Antidepressants ide, phenothiazine, cyclic antidepressants, mass spectros- to their N-Oxide with copy Hydrogen Peroxide Using Titanosilicate INTRODUCTION Catalyst The identification and quantification of drugs Akira Ogamo* and Mariko Fukumoto ingested into human bodies require comprehensive information covering the target drugs themselves and School of Pharmaceutical Sciences, Kitasato University, their metabolites. To carry out a quantitative analy- Shirokane 5–9–1, Minato-ku, Tokyo 108–8641, Japan sis, the standard substances of metabolites are es- (Received March 5, 2004; Accepted April 11, 2004) sential. Some important metabolites can be obtained as commercial products, but many metabolites, es- The aim of this study was to establish a prepara- pecially those of new drugs, are difficult to obtain tion method needed to analyze the metabolites of an as standard samples. These problems have been analytical procedure for antipsychotic phenothiazines solved by using experimental animals or isolated and tertiary amino cyclic antidepressants by chemical enzyme systems originating from biological mate- reaction. These drugs were oxidized to their sulfoxide rials. But defects such as low yield, high cost, and and N-oxide, respectively, with hydrogen peroxide us- lengthy preparation time have necessitated the search ing titanosilicate as the catalyst. An acidic medium for another effective method. Preparing metabolites (pH 3.0) containing 20% methanol was found to be optimal for the preparation of phenothiazine sulfoxide, by applying a chemical reaction to target drugs may and an alkaline medium (pH 10.5) containing 50% be effective, if the reaction is specific, rapid, simple, methanol was optimal for the preparation of the N- and applicable to a broad range of organic com- oxide of tertiary amino cyclic antidepressants. Each pounds. preparative scale reaction (2 mmol) was carried out Drugs acting on the central nervous system are and the crystallized product of each oxide was ob- the main targets of analysis in emergency medicine tained. The amount of hydrogen peroxide needed to and forensic analytical toxicology. The major me- obtain the best yields of sulfoxide was 1.3–2.0 times tabolites of phenothiazine drugs in blood and urine the molar equivalent of the phenothiazines. The puri- are their corresponding sulfoxides together with the ties of the prepared crystallized product of the phe- hydroxylated form.1) Although the sulfoxides gen- nothiazine sulfoxides were good except for periciazine erally lack antipsychotic activity, they may be re- sulfoxide. The amounts of hydrogen peroxide needed sponsible for the adverse effects (e.g. cardiotoxic to obtain the best yields of N-oxide were 4–5 times the activity2,3)) associated with drug intake. Conventional molar equivalent of tertiary amino cyclic antidepres- preparation methods for phenothiazine sulfoxide sants. The purities of the crystals of each N-oxide were have been reported.4–9) The yields of these methods 99.5–100%. The characteristic mass fragment ions of are not sufficient, and the phenothiazine derivatives each prepared oxide that were distinguishable as the reported have been limited to simple structural phenothiazine derivatives and drugs produced at an early *To whom correspondence should be addressed: School of Phar- stage of development. maceutical Sciences, Kitasato University, Shirokane 5–9–1, Most of the cyclic antidepressants used clinically Minato-ku, Tokyo 108–8641, Japan. Tel.: +81-3-5791-6332; Fax: include a ternary amino group in their structures. +81-3-3442-1946; E-mail: [email protected] Major metabolites of these cyclic antidepressants are No. 4 397 the N-demethylated and hydroxylated forms.1) Al- The absence of any band or signal in the range of though N-oxide is a minor metabolite,1,10–12) it retains 310–320 nm indicates the absence of an extra-frame- its original structure and is useful for identification. work octahedral TiO2 phase in the TS-1 sample. Conventional preparation methods for the N-oxide Small-Scale Reaction of Antipsychotic Phenothi- of general ternary amino compounds are oxidation azines with Hydrogen Peroxide Using Titano- by hydrogen peroxide or organic peroxide.13–15) The silicate as Catalyst —–— Each phenothiazine so- reaction rate with hydrogen peroxide is slow and the lution (0.1 mmol) was mixed with 0.2 M acetate yield is low, while the latter method represented by buffer (pH 3.0) and methanol (final concentration: m-chloroperbenzoic acid requires the maintenance 20%) in a test tube with a screw plug, and stirred of a low reaction temperature and purification of the with the indicated amounts of 30% hydrogen per- column chromatography system. oxide and titanosilicate in a water bath at 60°C for Hydrogen peroxide is a very attractive oxidant 1 hr. The total volume of the reaction mixtures was because it is relatively cheap and can oxidize or- 0.5 ml, although that of periciazine was increased ganic compounds with the generation of water as to 1 ml by increasing the amounts of methanol and the only theoretical by-product. Recently, many cata- buffer in order to improve its solubility. As control lysts have been developed to enhance the efficiency tubes, the drug solutions were incubated in the same of the oxidation ability of hydrogen peroxide. We acetate buffer and methanol without titanosilicate selected titanosilicate as the catalyst for oxidation and hydrogen peroxide under the same conditions. by hydrogen peroxide since the effective catalytic Prochlorperazine maleate was converted into the free ability of titanosilicate to form sulfoxide from a form to improve the solubility. An equal molar simple structural sulfide (thioether) in the presence amount of 35% hydrogen chloride (0.1 mmol) was of hydrogen peroxide has been reported.16–18) added to the reaction mixtures containing free base Titanosilicate has been used in the field of chemical type phenothiazine (pericyazine, perphenazine and industry as a catalyst for hydrogen peroxide.19) prochlorperazine). This article deals with the search for the optimal After the reactions, the reaction mixture was di- reaction conditions of reaction to form the oxide of luted with cold milli-Q water (1.5 ml) and the pH of each drug using hydrogen peroxide and titanosilicate the solution was adjusted to 10.0 with 1 M sodium in a small scale reaction (0.1 mmol). Larger amounts carbonate. The reaction mixture was vigorously of drugs (2 mmol) were then oxidized on the basis stirred with chloroform twice (1.5 ml each) for of the optimal conditions obtained, and purification 3 min. After centrifugation, the chloroform phase of the oxides formed was carried out using crystalli- was removed using a pasteur pipette, and the com- zation. bined extract was dried under a stream of N2 gas at 40°C. The residue was dissolved in methanol (HPLC grade, 1 ml), and 0.1 ml of this methanol solution MATERIALS AND METHODS was diluted to 100-fold with HPLC eluting solution, and then analyzed by HPLC. Materials —–— Chlorpromazine hydrochloride, Small-Scale Reaction of Cyclic Antidepressants promethazine hydrochloride, periciazine (synonym: with Hydrogen Peroxide Using Titanosilicate as pericyazine, propericiazine), imipramine hydrochlo- Catalyst —–— Each methanol solution of the HCl ride, and 30% hydrogen peroxide (9.88 mmol/ml) salts of cyclic antidepressants (0.1 mmol) was mixed were purchased from Wako Pure Chemical Indus- with 1 M carbonate buffer (pH 10.5) and methanol tries (Osaka, Japan). Promazine hydrochloride, (final volume: 50%) and stirred with the indicated fluphenazine dihydrochloride, trifluoperazine amounts of 30% hydrogen peroxide and titanosilicate dihydrochloride, perphenazine, prochlorperazine in a water bath at 60°C for 1 hr (total volume: dimaleate, clomipramine hydrochloride, amitrip- 0.5 ml). As control tubes, the drug solutions were tyline hydrochloride, and mianserin hydrochloride incubated in the indicated buffer and methanol with- were purchased from Sigma Chemical Co. (St. Louis, out titanosilicate and hydrogen peroxide under the MO, U.S.A.). Titanosilicate catalyst, TS-1, was same conditions. After the reaction, the reaction kindly supplied by Süd-Chemie Catalysts Japan Inc. mixture was diluted with cold milli-Q water (1.5 ml) (Tokyo, Japan). The Si/Ti molar ratio in the TS-1 and extracted with chloroform twice (1.5 ml each). used was 70, and average particle size as determined The rest of the procedure was the same as that for by scanning electron micrographs was 100–200 nm. phenothiazines described above. 398 Vol. 50 (2004) Preparation of Phenothiazine Sulfoxide —–— silane column (Mightysil RP-18, 4.6 × 250 mm, Each solid phenothiazine (2 mmol) was transferred 5 µm, Kanto Chemicals Co., Inc., Tokyo, Japan) with to an Erlenmeyer flask, and dissolved in 2 ml of the eluting solution of 67 mM phosphate buffer 20) methanol and a calculated amount (8 ml – volume (pH 3.0) - CH3CN (91 : 49, v/v) at a flow rate of of hydrogen peroxide) of 0.2 M sodium acetate 1.2 ml/min. The wavelengths of the UV detector for buffer (pH 3.0). Titanosilicate (100 mg) and the in- phenothiazine were set at 247–260 nm depending dicated amount of 30% hydrogen peroxide were on each drug as described in the Results. The wave- added. The reaction mixture (10 ml) was stirred in a length setting for the cyclic antidepressants was water bath at 60°C for 1 hr.

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