Microbial metabolism of phenelzine and pheniprazine

Life Sciences, Vol. 42, pp. 285-292 Printed in the U.S.A.

MICROBIAL

METABOLISM

Pergamon Journals

OF PHENELZINE

B. C. Foster + , R. T. Coutts . I ,

AND P H E N I P R A Z I N E

F. M. Pasutto* and A. Mozayanl

*Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 and +Division of Pharmaceutical Chemistry Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario, Canada KIA OL2 (Received in final form November 16, 1987)

Summary Phenelzine and pheniprazine were used as substrates f o r metabolic studies with Gunninqhamellaechinulata and Mycobacterium smeqmatis. Metabolites were identified by means of g a s - l i q u i d chromatography and mass spectrometry. 1-Acetyl-2-(2-phenylethyl)hydrazine and 1-acetyl-2-(1-methyl-2-phenylethyl)hydrazinewere the major products of C. echinulata metabolism of phenelzine and pheniprazine, respectively. In addition, M. smeqmatis produced a second metabolite from each substrate; these metabolites were unequivocally identified as N-acetylphenylethylamine and N-acetylamphetamine from phenelzine and pheniprazine, r e s p e c t i v e l y . Phenelzine (la, Fig. I) and pheniprazine (2a) are monoamine Oxidase inhibitors structurally related to the phenylethylamine class of compounds. These inhibitors have been used clinically for the treatment of depression and hypertension [1, 2]. Little is known concerning t h e i r metabolic fate in either man or laboratory animals and several investigators have reported considerable a n a l y t i c a l d i f f i c u l t i e s with these compounds [3, 4]. Phenelzine i s known to be metabolized in the rat brain to phenylethylamine [5, 6] and by rat monoamine oxidase to phenylacetic acid [7, 8]. Phenelzine is polymorphically N-acetylated in the rabbit by N-acetyltransferase which is also involved in the polymorphic a c e t y l a t i o n of i s o n i a z i d , sulfamethazine, procainamide and 2-aminofluorene [9]. The N-acetylated metabolites of phenelzine or pheniprazine have not been characterized by either gas-liquid chromatography (GLC) or GLC-mass spectrometry (MS) and there is no unequivocal direct evidence for acetylated biotransformation products in vivo. In addition, N-hydroxylation of the terminal nitrogen has been proposed although no Nooxidation products have been detected [3]. The metabolic pathways involved in the transformation of pheniprazine are poorly understood. It has been reported that t h i s compound is inactivated by reaction with endogenous b i o l o g i c a l molecules [10] and by N-N bond cleavage to amphetamine [5]. I Author f o r r e p r i n t requests

0024--3025/88 $3.00 + ,00 Copyright (c) 1988 Pergamon Journals Ltd.

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Microbial Metabolism of Hydrazino MAOIs

RI

Vol. 42, No. 3, 1988

R1

R

OCH2CH2NNHR20CH2~HNNHR2

~CH2CHNHCOCH ] 3

CH3 _2 RI

R2

a

H

H

b c d

H COCH3 COCH~

COCH3 H COCH3

3_

RI

R2

R

a

H

H

a

H

b c

H COCH3

COCH3 COCH3

b

CH3

FIG. I S t r u c t u r e s of Substrates and Metabolites

Initial drug metabolism studies are generally conducted in laboratory animals although there are problems associated with these systems when extrapolating the results to man [11, 12]. Alternatively, the application and advantages of microbial models of mammalian drug metabolizing systems for studying the biotransformation of medicinally important compounds have been reported by several investigators [13-15]. This study describes the extraction and identification of phenelzine and pheniprazine metabolites from microbial i n c u b a t i o n broths. Materials and Methods Substrates and Reference Compounds Phenelzine sulfate was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.), and (_+)-pheniprazine h y d r o c h l o r i d e was a g i f t from Merrell Pharmaceuticals (Concord, Ontario, Canada). 1-Acetyl-2-(2-phenylethyl)hydrazine ( I b ) , m . p . 70-71°C, was synthesized from phenylacetaldehyde and acetylhydrazine by a reported method [16]; 1-acetyl-2-(1-methyl-2-phenylethyl)hydrazine (2b), m.p. 77-79°C, was similarly prepared from 1-phenyl-2-propanone and acetylhydrazine by the literature procedure [16]. 1-Acetyl-1-(2-phenylethyl)hydrazine (Ic), m.p. 67-69°C, 1,2-diacetyl-1-(1-methyl-2-phenylethyl)hydrazine (2c), m.p. 83-85°C, and 1,2-diacetyl-1-(2-phenylethyl)hydrazine (]d), m.p. 61-63°C, were prepared from phenelzine, pheniprazine and the monoacetate (Ib), respectively, by reaction with an aqueous alkaline (NaHCO:~; pH 8) solution of acetic anhydride [cf. ref. 17]. N-Acetylamphetamine (3b-)__, m.p. 93-95°C, and N - a c e t y l - 2 - p h e n y l e t h y l a m i n e (3a), m.p. 47-48°C, were synthesized from amphetamine and 2-phenylethylamine r e s p e c t i v e l y , by a previously reported procedure [18]. All acetates were colorless compounds and were characterized by elemental analysis, mass spectrometry and proton magnetic resonance spectroscopy. Further details on their syntheses and properties will be published elsewhere.

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287

Instrumentation Combined GLC/electron-impact MS analyses were performed on a HewlettPackard Model 5710A gas chromatograph interfaced with a Hewlett-Packard Model 5980A mass spectrometer and Model 5934A data system. The injection port and transfer line temperature was 300°C. The operating conditions for the mass spectrometer were: electron energy, 70 eV; emission current, 35 ma; source temperature 180°C. A 15 m DB-5 fused silica GLC column (J & W Scientific, Palo Alto, CA, U.S.A.), 0.32 mm i.d., was employed. The carrier gas (helium) flow rate was 2 mL/min, and the oven temperature was programmed from 130°-290°C (8°C/min). Retention times reported in Table I were recorded under these conditions. Samplinq Protocol All incubation broths were analyzed by withdrawing 2 mL aliquots at various times up to 168 h. With some substrates, two d i f f e r e n t analytical methods were necessary in order to assay the incubation broths for all possible products. In such cases, each aliquot was divided into two equal I mL portions and treated as follows. Method A: a I mL sample was made alkaline (solid potassium carbonate) and extracted with three times five volumes of a diethyl ether/methylene chloride mixture (55:45 v/v). The solvents were f r e s h l y distilled prior to use. The extracts were combined and concentrated at 50°C to approximately 50 uL. Method B: a I mL sample was made acidic (conc. HCI) and extracted as outlined above. The combined extracts were evaporated to dryness and derivatized with 50 uL of chloroethanol/HCl solution at 50°C for 15 min. Method C: a portion of the extract obtained by Method A was reacted with an equal volume of acetic anhydride as described p r e v i o u s l y [17]. The reaction mixtures obtained in Methods B and C were reduced to dryness under a stream of nitrogen and the residue r e d i s s o l v e d in the d i e t h y l ether/methylene chloride mixture to a final volume of 50 uL. The final extracts obtained by Methods A, B, or C were then analyzed by GLC-MS. Incubation Protocol C. echinulata ATCC 9244 was maintained on oatmeal agar slants at 4°C. Spores from the slants were used to inoculate medium containing trypticase soy broth (BBL), 30 g/L; yeast extract, 7 g/L and dextrose, 16 g/L. Dextrose was autoclaved separately and added aseptically. Thick, three day culture was homogenized and added as a 10% inoculum to 25 mL of fresh medium in 125 mL Erlenmeyer flasks. Filter sterilized substrates (7 mg/flask) were then added aseptically to 48 h cultures. Cultures were incubated at 28°C on a New Brunswick Scientific Model G-tO gyratory shaker equipped with 45 ° angle brackets operated at 250 rpm. M_. smeqmatis ATCC 14468 was maintained in nutrient broth (Difco) at 4°C. Cultures were started by transferring 0.5 mL of stock into 125 mL Erlenmeyer flasks containing 25 mL of nutrient broth (Difco), 8 g/L and dextrose, 16 g/L. Dextrose was added aseptically after autoclaving. Fresh medium was inoculated with I mL of overnight culture and f i l t e r sterilized solutions of substrates (0.2 mg/flask) were added aseptically after a f u r t h e r incubation of 6 h.

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Cultures were incubated at 37°C in a New Brunswick Scientific Model G-25 gyratory shaker equipped with 45 ° angle brackets operated at 250 rpm. Culture controls were prepared by incubating the microorganism in the absence of substrate. Samples of each culture were plated on nutrient and Sabouraud dextrose agar (Difco). No contamination was observed. Substrate c o n t r o l s were prepared by incubating substrates in s t e r i l e medium. Results Extracts of control and incubation broths were examined by GLC and GLC-MS. Identification of metabolites was based on comparison of t h e i r GLC retention times and mass spectral fragmentation patterns with those of a u t h e n t i c reference compounds (Table I). Different extraction procedures were generally required in view of the dissimilar solubility characteristics of the possible metabolites. All incubation broths were make alkaline prior to e x t r a c t i o n (Method A) in order to recover neutral and basic metabolites and unmetabolized substrate. These extracts were analyzed by GLC-MS to confirm that GLC peaks of metabolites and substrates were free from i n t e r f e r i n g peaks. After removal of neutral and basic constituents, broths were acidified and re-extracted to remove potential carboxylic acid metabolites (Method B). Extracts were then esterified with a chloroethanol/HCl solution to facilitate GLC-MS analysis. The aqueous l a y e r was not examined f u r t h e r . TABLE I Gas-liquid Chromatographic Retention Times (Rt) and Mass Spectrometric Fragmentation Data of Metabolites and D e r i v a t i v e s

Compound

Rt (min) I

l__bb

15.0

Ic

14.6

l_._dd

18.7

2___b_b

15.0

2c

18.6

3.__~a

12.9

3b

13.1

MS Data 2 (% Relative Abundance)

178(2), I05(i0), 91(20), 87(100), 45(43), 43(12) 178(12), 136(8), 105(11), 104(11), 91(38), 87(31), 74(49), 45(100), 43(35) 220(I), 178(6), 116(22), 105(9), 91(19), 87(100), 74910), 45(17), 43(20) 101(100), 91(40), 83(22), 59(29), 43(26), 42(27) 143(18), 118(12), 117(15), 101(100), 91(34), 83(11), 59(14), 43(38), 42(13) 163(20), 104(100), 91(45), 72(20), 65(37), 43(79), 30(80) 177(abs), 118(12), 91(17), 86(23), 65(11), 44(100) 43(24)

I GLC c o n d i t i o n s are described in Materials and Methods 2 Major fragmentation ions

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Incubation broths containing M. smeqmatis and phenelzine were extracted at day 5 (Method A) and found to contain two metabolites and trace amounts of substrate. The f i r s t GLC metabolite peak was unequivocally i d e n t i f i e d as N-acetyl-2-phenylethylamine (3a, Fig. I) by direct comparison of i t s GLC and MS properties with those of a synthetic sample of 3a. The mass spectrum of the second metabolite contained diagnostic fragment ions (m/z 105, 91, 87, 45 and 43) consistent with those expected for a mono-N-acetyl d e r i v a t i v e of phenelzine (Fig. 2). This was confirmed when a portion of the original extract of the M. smeqmatis incubation broth was reacted with acetic anhydride (Method C) and re-examined by GLC. The peaks ascribed to phenelzine and its N-acetyl derivative were now absent from the GLC trace, having been replaced by a single new peak which ~as found to have GLC-MS properties identical to those of authentic N', NC-diacetylphenelzine (I(I). However, the p o s i t i o n of the N-acetyl group in the mono-N-acetyl metabolite was not readily discernible from the MS fragmentation data. Two structures were possible (Ib and l c ) , and further characterization of the metabolite was required. Both isomers ( I b and Lc) were synthesized and found to possess d i f f e r e n t GLC and MS properties (Table I). The metabolite produced by M_. smeqmatis d i s p l a y e d p r o p e r t i e s i d e n t i c a l to those of l - a c e t y l - 2 - ( 2 - p h e n y l e t h y l ) h y d r a z i n e ( I b ) . PhCH2CH2N-NH2~ J

or

PhCH2CH2NHNHCOCH3~

COCH3

1._cc

m/z 178

L PhCH2CH2+ m/z 105 + ---'- CH2=N-NH2

or

11)

PhCH2+

CH3CO+

m/z 91

m/z 43

+ CH2=NHNHCOCH 3

_ CH2=C=O ~

+ CH2=NHNH 2

I

COCH3

m/z 87

m/z 45

FIG. 2 Diagnostic fragmentation ions in the electron-impact mass spectrum of metabolically produced mono-N-acetylated phenelzine (see Table I). Samples of the M. smegmatis incubation broth containing phenelzine were also e x t r a c t e d under acidic c o n d i t i o n s (Method B). The l e v e l s of the chloroethyl derivative of phenylacetic acid were not s i g n i f i c a n t l y different from c o n t r o l s . Following procedures similar to those outlined above, extracts (Method A) from 5 day incubations of M. smeqmatis and pheniprazine were found to contain three compounds, viz, substrate, N-acetylamphetamine (3b) and 1-acetyl-2(1-methyl-2-phenylethyl)hydrazine (21), Table I). I d e n t i t i e s were confirmed by comparison of GLC-MS properties with those of authentic synthetic standards. The metabolite 21) was further acetylated (Method C) and in t h i s way converted to 1 , 2 - d i a c e t y l - 1 - ( 1 - m e t h y l - 2 - p h e n y l e t h y l ) h y d r a z i n e(2c). I t s GLC-MS properties were also i d e n t i c a l to those of an a u t h e n t i c 2c reference standard.

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In a parallel study to determine the metabolic transformations of phenelzine and pheniprazine by the filamentous fungus C. echinulata, i t was found that only the N-acetylation products (]b) and (21)) were formed, respectively. The levels of recovered metabolites were not sufficient to account for the total amount of added substrates. I n s t a b i l i t y of these substrates in alkaline solution, their reaction with media and cellular constituents, the possibility of additional metabolic pathways and autooxidation [19] may account for the portion not detected by the extraction/derivatization procedures employed. Discussion In this report we have described the isolation and identification of four novel metabolites of phenelzine and pheniprazine; ( ] b ) , (21)), (3a), and (3a). Two of these metabolites (]b) and (2b) have not been unequivocally identified as mammalian metabolites although they are reportedly products of phenotypic acetylation [I, 3, 9, 20, 21] and are important in determining the variability of the therapeutic response and incidence of drug t o x i c i t y to these substances [22, 23]. The N-acetyl metabolites (3a) and (3b) are conjugates of the products of N-N bond cleavage of phenelzine and pheniprazine, i.e. phenylethylamine and amphetamine r e s p e c t i v e l y . This study shows that in addition to the classical mechanisms for the detoxification of hydrazine-containing compounds, N-acetylation and oxidative dehydrazination [10, 24], metabolic N-N bond cleavage also occurs in the rat and produces pharmacologically active metabolites (phenylethylamine and amphetamine). To date, however, there is no evidence that N-N bond cleavage of phenelzine or pheniprazine occurs in man. The exact mechanism of N-N bond cleavage is not clear; at least two possible routes of transformation are possible. In previous studies with M. smeqmatis [25] we observed that N-oxidation was a major metabolic route for the phenylethylamine class of compounds. In view of the phenylethylamine character of phenelzine and pheniprazine, i t would be reasonable to suggest that this microorganism is able to N-oxidize the terminal nitrogen of these substrates. The resulting primary hydroxylaminewould be unstable and liable to decompose to hydroxylamine and the corresponding primary amine (Route a, Fig. 3). The

R-NHNH2

F HLR-NH-NH_]

H-N:O R-NH2

FIG. 3 Possible Mechanisms for the Metabolic N-N Bond Cleavage of Phenelzine and Pheniprazine by M_. smeqmatis

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alternative mechanism takes into account the observation that o x i d a t i o n reduction reactions are important in the transformation of some aryl and alkyl hydrazines [26]. The N-N bond may be reduced by nucleotide electron donors to the respective amine (Route b, Fig. 3). The inability to detect the possible oxidative dehydrazination products of phenelzine (phenylacetic acid) and pheniprazine (1-phenyl-2-propanone, 1-phenyl-2-propanol, and benzoic acid) was not unexpected. The ultimate oxidation products phenylacetic and benzoic acids are common metabolic intermediates which can be rapidly incorporated into cellular constituents [27, 28]. We have observed (unpublished results) that oxidative deamination products of ring halogenated phenylethylamine analogues are not assimilated but accumulate in the incubation broth; unfortunately, halogenated analogues of phenelzine and pheniprazine were not available for further evaluation of possible o x i d a t i v e pathways. Since pheniprazine is a chiral drug it would be of interest to determine whether the (+)- and (-)-isomers are metabolized s t e r e o s e l e c t i v e l y . Both organisms produce the N-acetyl conjugate (2b) while _M. smegmatis also gives N-acetylamphetamine (31)). We have previously reported the C. echinulatamediated regioselective aromatic para-hydroxylation of racemic 1-isopropylamino-3-phenoxy-propan-2-ol [291; stereoselective oxidation was not observed. It is appropriate to note, however, that the metabolic oxidation is cytochrome P-450-mediated while acetylase enzymes are l i k e l y involved in the present study. Further experiments are warranted to c l a r i f y whether these enzymatic processes are s t e r e o s e l e c t i v e l y c o n t r o l l e d . Acknowledqements Financial assistance from the Alberta Heritage Foundation for Medical Research (B. C. Foster), the Medical Research Council of Canada (Grant MA-5728 to R. T. Coutts and F. M. Pasutto) and from the Alberta Provincial Mental Health Advisory Council is gratefully acknowledged. Useful discussions with Drs. G. B. Baker and M. Daneshtalab are also acknowledged. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10.

D.A.P. EVANS, K. DAVISON, and R.T.C. PRATT, Clin. Pharmacol. Ther. 6 430-435 (1965). V. CLINESCHMIDT and A. HORITA, Br. J. Pharmacol. Chemother. 30 67-77 (1967). B. CADDY, A.H. STEAD and E.C. JOHNSTONE, Br. J. Clin. Pharmacol. 6_ 185-188 (1978). B. CADDY, W.J. TILSTONE and E.C. JOHNSTONE, Br. J. Clin. Pharmacol. 3_ 633-637 (1976). A.A. BOULTON, S.R. PHILIPS, D.A. DURDEN, B.A. DAVIS, T.J. DANIELSON and G.B. BAKER, Proc. 21 Can. Spec. Confer. pp 40-48 (1975). L.E. DYCK, D.A. DURDEN and A.A. BOULTON, Proc. 8 Meet. I n t . Soc. Neurochem. pp 289 (1981). B.V. CLINESCHMIDT and A. HORITA, Biochem. Pharmacol. L8 1011-1020 (1969). B.V. CLINESCHMIDT and A. HORITA, Biochem. Pharmacol. 18 1021-1028 (1969). D.W. HEIN and W.W. WEBER, Drug Metab. Dispo. 10 225-229 (1982). M.R. JUCHAU and A. HORITA, Drug Metab. Rev. ! 71-100 (1972).

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11. A.H. BECKETT and D.A. COWLEY, Druq Metabolism Ln Man, J.W. Gorrod and A.H. Beckett (eds), pp 237-258, Taylor and Francis Ltd., London (1978). 12. R.L. SMITH, Drua Metabolism _ iD_nMan, J.W. Gorrod and A.H. Beckett (Eds), pp 97-106, Taylor and Francis Ltd., London (1978). 13. J.P. ROSAZZA and R.V. SMITH, Adv. Appl. Microbiol. 25 169-208 (1979). 14. A.M. CLARK, J.D. McCHESNEY and C.D. HUFFORD, Med. Res. Rev. 5 231-253 15.

(1985). J.B. R E I G H A R D

and J.E. KNAPP,

Pharmacy

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16.

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H.D. HALLER and R.K. FINN, Eur. J. Appl. Microbiol. 8_ 191-205 (1979). F.M. PASUTTO, N.N. SINGH, F. JAMALI, R.T. COUTTS, and S. ABUZAR, J. Pharm. Sci. 76 177-179 (1987).