Metabolic N-Oxidation of 3-Substituted Pyridines: Identification of Products by Mass Spectrometry David A. Cowan, Lyaquatali A. Damanit and John W. Gorrod Department of Pharmacy, Chelsea College, University of London, Manresa Road, London SW3 6LX, UK
The mass spectral characteristics of the N-oxides of a range of 3-substituted pyridines, and of quinolme and isoquinoline, are described. The molecular ion is the base peak in the majority of cases, provided that thermolysis is minimized when using the direct probe or gas chromatograph inlets. Chromatographic and mass spectral evidence is presented which indicates that biological oxidation of the heteroaromatic nitrogen of 3-substituted pyridines is a route of metabolism in viw and in vitro.
Pharmacological effect
INTRODUCTION c TN C O N H Z
Many naturally occurring compounds contain the aromatic heterocyclic amino group (for example, purines, pyrimidines and co-enzymes) as do many drug molecules (see examples below). A large number of the basic constituents of tobacco and tobacco smoke also contain a heteroaromatic amino group. Despite the extensive occurrence of this type of compound, the biological oxidation of the constituent nitrogen has not been widely observed.' The only in vitro reports of the N-oxidation of compounds containing a pyridyl nitrogen appear to be for nicotinamide (1)2 nikethamide (2)3and bromazepam (3); although the metabolism of compounds containing heteroaromatic nitrogen has been widely reported. The high polarity of the resulting N-oxides and their low extractability using conventional extraction techniques probably have prevented the detection of these compounds in previous metabolic studies. The use of more polar solvents for extraction, coupled with sensitive gas-liquid chromatographic methods has allowed the of these, often thermolabile, identification
Vitamin
1
Nicotinamide
2
Respiratory stimulant
3
Tranquilizer
Nikethamide
Br
Bromazepam
compound^.^ Thin-layer chromatographic examination of Noxides is hampered by the lack of a specific visualizing reagent.6 Identification of N-oxides is usually based on visualization under UV light using fluorescing TLC plates, or by reduction with titanous chloride to the parent amine and comparison of chromatographic properties with the appropriate reference N-oxide or amine. Similarly, gas chromatographic analysis of Noxides usually relies on reduction to the parent amine prior to chromatography.' Mass spectra of several aromatic amine N-oxides have been reported previ~usly*-'~ and the presence of a molecular ion peak and a [M-16]+ ion peak was considered to be of diagnostic value. It now seems evident that the latter ion is formed solely by ther~ molysis; for example, Duffield and B ~ c h a r d t 'report t Present address: University College Hospital Medical School, Laboratory of Toxicology and Pharmacokinetics, University Street, London WC1 655, UK.
Cardiac vasodilator
OCH,
Dimoxyline
Antiseptic
Aminacrine
CCC-0306-042X/78/0005-055 1$03.00 @ Heyden & Son Ltd, 1978
BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 9, 1978 551
D. A. COWAN, L. A. DAMANI, AND J. W. GORROD
that the variation in the intensity of the [M- 16]+ ion peaks of various quinoline and isoquinoline N-oxides is dependent upon the ion source temperature. We now report conditions which minimize this thermolysis and allow MS and GLCMS analysis of various 3-substituted pyridine-N-oxides. In this paper we also present chromatographic and mass spectral evidence that biological oxidation of heteroaromatic nitrogen of 3substituted pyridines is a route of metabolism in vivo and in vitro.
EXPERIMENTAL Standards The reference N-oxides were synthesized according to published from the parent amines obtained from Aldrich Chemical Co., Gillingham, Kent, UK, or from Koch Light Laboratories, Colnbrook, UK. 1-(3-Pyridyl)ethanol was obtained by reduction of 3-acetylpyridine. l7 Cotinine was synthesized by a published method.I8 Full details of these procedures have been described elsewhere.: All compounds were checked chromatographically for purity using systems described previously.6The animals used were all male, as follows: albino Wistar rats (300400 g); albino Dunkin-Hartley guinea-pigs (400600 8); albino New Zealand White rabbits (2.5-3.0 kg); Syrian hamsters (80-100 g) and LACA albino mice (30-40 g). Microsomal fractions were prepared as described previously.*' Incubation of substrates Substrates (5 pmol), dissolved in distilled water (OSml), in 25ml Erhlenmeyer flasks with pH7.4 phosphate buffer (2 ml) containing NADP (2 pmol), magnesium chloride (25 pmol), glucose-6-phosphate (10 pmol) and glucose-6-phosphate dehydrogenase (1 unit) were mixed and pre-incubated at 37 "Cfor 5 min prior to the addition of hepatic or pulmonary microsoma1 preparations equivalent to 0.5 g of original liver or lung (in 1ml Tris/KCl buffer). The incubations were continued for 1 h using a shaking water bath (Gallenkamp). Control experiments containing heat inactivated (boiled) microsomal preparations or without co-factors or substrates were treated similarly to the active preparations. A further control consisting of the normal active enzyme/substrate system to which sodium hydroxide (0.5 ml, 1.0 M) was added at zero time (to terminate enzymic activity) was also used. Extraction of N-oxides After incubation, the flasks were chilled in an ice bath and sodium hydroxide (0.5m1, 1.OM) was added rapidly to terminate enzyme activity. The contents of each flask were transferred to a screw-capped tube (Sovirel, SVL, 10 ml) containing sodium chloride (1 g) and extracted with freshly distilled ether (2 x 5 ml). The ether extracts were discarded and double-distilled dichloromethane (3 x 5 ml) was used to extract the Noxides. The dichloromethane extracts were concentrated (to 10-20 p1) in tapered evaporating tubes21on a water bath at 45°C. This extraction procedure recovered more than 80% of each N-oxide." 552 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 9, 1978
Thin-layer chromatography Glass plates (20 x 20 cm) were spread to a thickness of 0.25 mm with silica gel GF254(type 60, Merck, Darmstadt). The coated plates were air dried and then activated at 105 "C for 1h. The concentrated dichloromethane extracts from each test or control flask and authentic reference N-oxide were applied to these plates at 1.5cm intervals. The plates were developed in either chloroform-ethanol-ammonia solution (S.G. 0.88) (100:8:0.5, by volume) or in acetone-ethanol-diethylamine (95 :5 : 1, by volume). After development, the spots were visua!ized under UV light from an Hanovia lamp (model CHI/294) at 254 nm. Compounds originating from metabolic extracts were tentatively identified as N-oxides if Rf values of these materials corresponded with Rfvalues of authentic N-oxides in both solvent systems, and if no material was observed from extracts from control flasks having identical Rf values. The N-oxide was extracted from the silica-gel using a suitable solvent and identity confirmed by the measurement of UV or IR spectra, or by reduction of N-oxide back to the arent pyridine, or by GLC, MS or combined GLCMS. "Details of the MS and GLCMS of some N-oxides formed in vitro using rabbit liver microsomes or isolated from urine of rabbits administered pyridine are described below.
Mass spectrometry A VG Micromass MM 12F single focusing mass spectrometer was used with an ion source temperature of 240 "C, an electron energy of 70 eV, a trap current of 100 pA, an accelerating voltage of 4 kV and a resolution of 800. The samples were introduced via the direct insertion probe and the spectra recorded on oscillographic paper as rapidly as possible after sample introduction without additional heating of the probe. The gas chromatographic inlet system was used for the analysis of the N-oxides of pyridine, 3-methylpyridine, 3-ethylpyridine, 3-fluoropyridine, 3-chloropyridine and 3-bromopyridine with a 1 m X 4 mm i d . glass column containing 2% w/w Carbowax 20 M and 5% w/w KOH on Chromosorb W HP (8O/lOO mesh) mounted in a Pye 104 oven maintained at 170 "C.The N-oxides of 3-cyanopyridine, 3-acetylpyridine, quinoline and isoquinoline were analysed using a 1 m x 4 mm i.d. glass column containing 3% w/w OV-17 on Gas Chrom Q (100/120 mesh). Helium was used as a carrier gas at a flow rate of 20 ml min-'. The glass-lined stainless steel tube connecting the GC column outlet to the single stage glass jet separator was maintained at 250°C. The samples were injected rapidly onto the column with a 7 cm syringe needle.
RESULTS A N D DISCUSSION Direct inlet mass spectrometry The EI mass spectra of pyridine and pyridine-N-oxide are shown in Fig. 1. The molecular ion peak of the N-oxide is the most useful peak to distinguish the @ Heyden & Son Ltd, 1978
METABOLIC N-OXIDATION OF 3-SUBSTITUTED PYRIDINES
3-ethylpyridine (Fig. 4). The mass spectra of the other N-oxides examined are tabulated in Table 1. The majority of the compounds examined were surprisingly volatile compared with alkylamine-N-oxides. The polarity of pyridine-N-oxide is less than might be expected because of the various tautomeric forms possible, as shown.23
0- o-0- -0
-
N+
N
N
N
00-
0
0
0
I
70
This volatility resulted in a large concentration of compound in the ion source and in the appearance of large [M+l]+ ion peaks in the spectra. Reducing the sample size allowed spectra to be obtained with [M+ 11' ion peaks of the intensity expected considering the natural13C abundance. Thermolysis of the compound was minimized by recording the spectrum as soon as possible after sample insertion. A comparison of the relative abundance of the [MI+, [M-16]+ and [M17]+ ion peaks obtained in our laboratories with those published is shown in Table 2. In the majority of cases we observed less thermolysis than other workers, as indicated by the [M- 16]+ion intensity.
m/e
Figure 1. Mass spectra (a) pyridine and (b) pyridine Noxide.
former from the parent amine. The molecular ion usually produced the base peak in the spectrum of each N-oxide but the [M- 16]+/[M]f intensity ratio varied with the operating conditions. the mass spectra of the isomeric 2-, 3- and 4-hydroxypyridines (Fig. 2) were markedly different from that of pyridine-N-oxide in that no [M-16]+ ion peak was observed. Weak [MHI' and more intense [M - 27]+ ion peaks, which were absent in the spectrum of pyridine-N-oxide, were observed for the 3- and 4-hydroxypyridines. 2-Hydroxypyridine exhibited an intense [M- 28]+ ion peak which was less intense, but present, in the spectra of the 3- and 4- hydroxypyridines and absent in the N-oxide spectrum. A metastable ion peak was observed for the fragmentation of the molecular ion of 2-hydroxypyridine to form the (M- 28) ion peak, this probably being due to the loss of CO via the tautomeric 2pyridone. 22 The EI mass spectrum of the primary alcohol 3pyridyl carbinol [Fig. 3(a)] has a base peak corresponding to [M-HI' whilst the [M- 15]+ ion produces the most intense peak for the secondary alcohol l-(3-pyridyl) eth'anol (Fig. 3(b), thus readily distinguishing these compounds from the N-oxides of 3-methylpyridine and
Gas chromatography coupled with mass spectrometry The mass spectra obtained using the G C inlet system were substantially the same as those obtained via the direct insertion probe except that usually in the former the [M- 16]+peak was more intense. Nevertheless, in the majority of cases the molecular ion peak remained the base peak (see Table 2). The degree of thermolysis was minimized by a rapid injection of the sample directly onto the all-glass column. A G C peak due to the formation of the parent amine was observed when the sample was injected more slowly. Thermolysis was also observed on one occasion when the glass-lined stainless steel tube connecting the chromatograph to
100
ooH
00
-g ...? -
z
60
u) c W c
40
0
W
n
20
L I
i0
70
L 90
50
70
90
m/e
Figure 2. Mass spectra of 2-, 3- and Qhydroxypyridines.
@ Heyden & Son Ltd, 1978
BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 9, 1978 553
D. A. COWAN, L. A. DAMANI, AND J. W. GORROD
"1
I
Table 1. Major ion peaks in the direct inlet mass spectra of some pyridine-N-oxides
m/e (% relative intensity)
N
1
0
R=H 30
50
70
3-CH3
90 m/e
Rgure 3. Mass spectra of (a) 3-pyridylcarbinol 1-(3-pyridyl)ethanol.
and (b)
3-CzH5
3-F 3-CI
(a 1
3-Br QCH3
3-COCHa
4
0
3-NHz
3-NHCOCH,
m/e
Figure 4. Mass spectra of (a) 3-methylpyridine-Noxide and (b) 3-ethylpyridine-Noxide.
3-CN 3-CONHZ 3-COOH
the jet separator had been damaged, exposing the eluted compounds to the hot metal surface.
3-OH 3-N(CH3)z 3-CHZOH 3-CON(CZH&
Metabolism of 3-substituted pyridines 3-CONHCZHs
The in vitro metabolism of several 3-substituted pyridines, and of cotinine and quinoline was studied using hepatic and pulmonary microsomal preparations from various animal species. Pyridines in which the 3-substituent was -H, -CH3, -CzHs, -F, -el, -Br, -COCH3, -CN and -CON(CzHs)z were all converted to the corresponding N-oxides by fortified hepatic microsomal preparations from hamsters, guinea-pig, rabbit, rat and mouse, and by pulmonary preparations from guineapig and rabbit (see Table 3). This is the first report of in vifro metabolic N-oxidation of these pyridines, except for a single report3 on the metabolic N-oxidation of N, N-diethylnicotinamide (nikethamide) using rat liver homogenates. The in vitro metabolic N-oxidation of 3-acetylpyridine has not been described previously, although the N-oxide of 1-(3-pyridyl)ethanol has been identified in the urine'of ratsz4 as a metabolite of 3-acetylpyridine; the N-oxide isolated in vitro, in the present investigation, was that of 3-acetylpyridine itself. The primary metabolite in vivo of 3-acetylpyridine may be its N-oxide, which is metabolized further to form the N-oxide of 1-(3-pyridyl)ethanol as the secondary 554 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 9, 1978
Nicotine-1-oxide Cotinine-N-oxide 2-CH3 4-CH3
4-Cd-k Quinoline N-oxide lsoquinoline N-oxide
79(12) 95(100) 39(59) 109(100) 93(13) 65(22) W27) 123(100) 107(18) 81(42) 65(20) 39(50) 40(53) 113(100) 97(25) 70(21) 58(25) 131(33) 129(100) 78(22) 73129) 40(69) 39(54) 175(100) 173( 100) 66(50) 78(27) 39(59) 137(100) 122(35) 51(19) 53(22) 94(31) 110(100) 55(22) 66(15) 39(28) 136(19) 152(25) 67(18) 93(18) 39(23) 120(100) 104(20) 76( 16) 77( 16) 138(100) 122(18) 39(21) 78( 15) 139( 100) 123(96) 78(41) 51(28) 95(16) 111(100) 138(100) 124(20) 42(75) 95( 17) 125(100) 109(17) 63(22) 78(42) 194(18) 178(12) 78(42) 106(100) 150(36) 166(43) 78(66) 106( 100) 39(24) 162(16) 178(30) 121(15) 119(32) 192(100) 176(3) 42(10) 93(41) 109(100) 65(96) 66(21) 109(100) 93( 10) 39(20) 123(59) 108(100) 39(20) 145(100) 129(17) 89(311 W54) 145(100) 129(10) 90(78) 89(62) 39(19)
78(9)
40(20)
92(7) 5363) 106116) 53(47)
80(25) 39(75) 92(26) 51(21)
966) 57(53) 113(12) 63(23)
86(10) 102(11) 50(27)
157(13) 63(37)
146(12) 51(37)
121(7) 43(51) 81(11) 5442)
78(42)
11O(24) 43(62)
94(100) 40(24)
103(8) 65(W 120(9)
93(13) 6430) 9411)
106(26) 39(28) 55131) 122(12) 39(60) 108(15) 39(87) 177(28) 51(18) 149(30) 51(40)
67(17) 40(26)
105(43) 39(27) 121(37) 80(37) 122(42) 39(12) 122(41) 44011
161(30) 84(100) 175(6)
133(28) 40(50) 98(99)
92(93) 40W) 65(10)
78(13) 39(30) 53(24)
91(19)
65(12)
128(10)
117(27)
128(8) 63(32)
118(45)
metabolite. In addition, 1-(3-pyridyl)ethanol was produced in vitro as a reduction product of 3-acetylpyridine. The same compound was formed, but by pC-oxidation, on incubation of 3-ethylpyridine. @ Heyden & Son Ltd, 1978
METABOLIC N-OXIDATION OF 3-SUBSTITUTED PYRIDINES
Table 2. Mass spectral characteristics of some pyridine-N-oxides. The values in parentheses are for combined GLCMS % Relative intensity (literature values)
% Relative intensity Compound
[ M - 161t
[MI:
[MI!
IM-161t
16(20) 5
The mass spectral characteristics of the N-oxides of a range of 3-substituted pyridines, and of quinolme and isoquinoline, are described. The molecular ion is the base peak in the majority of cases, provided that thermolysis is minimized when using the direct probe or gas chromatograph inlets. Chromatographic and mass spectral evidence is presented which indicates that biological oxidation of the heteroaromatic nitrogen of 3-substituted pyridines is a route of metabolism in viw and in vitro.
Pharmacological effect
INTRODUCTION c TN C O N H Z
Many naturally occurring compounds contain the aromatic heterocyclic amino group (for example, purines, pyrimidines and co-enzymes) as do many drug molecules (see examples below). A large number of the basic constituents of tobacco and tobacco smoke also contain a heteroaromatic amino group. Despite the extensive occurrence of this type of compound, the biological oxidation of the constituent nitrogen has not been widely observed.' The only in vitro reports of the N-oxidation of compounds containing a pyridyl nitrogen appear to be for nicotinamide (1)2 nikethamide (2)3and bromazepam (3); although the metabolism of compounds containing heteroaromatic nitrogen has been widely reported. The high polarity of the resulting N-oxides and their low extractability using conventional extraction techniques probably have prevented the detection of these compounds in previous metabolic studies. The use of more polar solvents for extraction, coupled with sensitive gas-liquid chromatographic methods has allowed the of these, often thermolabile, identification
Vitamin
1
Nicotinamide
2
Respiratory stimulant
3
Tranquilizer
Nikethamide
Br
Bromazepam
compound^.^ Thin-layer chromatographic examination of Noxides is hampered by the lack of a specific visualizing reagent.6 Identification of N-oxides is usually based on visualization under UV light using fluorescing TLC plates, or by reduction with titanous chloride to the parent amine and comparison of chromatographic properties with the appropriate reference N-oxide or amine. Similarly, gas chromatographic analysis of Noxides usually relies on reduction to the parent amine prior to chromatography.' Mass spectra of several aromatic amine N-oxides have been reported previ~usly*-'~ and the presence of a molecular ion peak and a [M-16]+ ion peak was considered to be of diagnostic value. It now seems evident that the latter ion is formed solely by ther~ molysis; for example, Duffield and B ~ c h a r d t 'report t Present address: University College Hospital Medical School, Laboratory of Toxicology and Pharmacokinetics, University Street, London WC1 655, UK.
Cardiac vasodilator
OCH,
Dimoxyline
Antiseptic
Aminacrine
CCC-0306-042X/78/0005-055 1$03.00 @ Heyden & Son Ltd, 1978
BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 9, 1978 551
D. A. COWAN, L. A. DAMANI, AND J. W. GORROD
that the variation in the intensity of the [M- 16]+ ion peaks of various quinoline and isoquinoline N-oxides is dependent upon the ion source temperature. We now report conditions which minimize this thermolysis and allow MS and GLCMS analysis of various 3-substituted pyridine-N-oxides. In this paper we also present chromatographic and mass spectral evidence that biological oxidation of heteroaromatic nitrogen of 3substituted pyridines is a route of metabolism in vivo and in vitro.
EXPERIMENTAL Standards The reference N-oxides were synthesized according to published from the parent amines obtained from Aldrich Chemical Co., Gillingham, Kent, UK, or from Koch Light Laboratories, Colnbrook, UK. 1-(3-Pyridyl)ethanol was obtained by reduction of 3-acetylpyridine. l7 Cotinine was synthesized by a published method.I8 Full details of these procedures have been described elsewhere.: All compounds were checked chromatographically for purity using systems described previously.6The animals used were all male, as follows: albino Wistar rats (300400 g); albino Dunkin-Hartley guinea-pigs (400600 8); albino New Zealand White rabbits (2.5-3.0 kg); Syrian hamsters (80-100 g) and LACA albino mice (30-40 g). Microsomal fractions were prepared as described previously.*' Incubation of substrates Substrates (5 pmol), dissolved in distilled water (OSml), in 25ml Erhlenmeyer flasks with pH7.4 phosphate buffer (2 ml) containing NADP (2 pmol), magnesium chloride (25 pmol), glucose-6-phosphate (10 pmol) and glucose-6-phosphate dehydrogenase (1 unit) were mixed and pre-incubated at 37 "Cfor 5 min prior to the addition of hepatic or pulmonary microsoma1 preparations equivalent to 0.5 g of original liver or lung (in 1ml Tris/KCl buffer). The incubations were continued for 1 h using a shaking water bath (Gallenkamp). Control experiments containing heat inactivated (boiled) microsomal preparations or without co-factors or substrates were treated similarly to the active preparations. A further control consisting of the normal active enzyme/substrate system to which sodium hydroxide (0.5 ml, 1.0 M) was added at zero time (to terminate enzymic activity) was also used. Extraction of N-oxides After incubation, the flasks were chilled in an ice bath and sodium hydroxide (0.5m1, 1.OM) was added rapidly to terminate enzyme activity. The contents of each flask were transferred to a screw-capped tube (Sovirel, SVL, 10 ml) containing sodium chloride (1 g) and extracted with freshly distilled ether (2 x 5 ml). The ether extracts were discarded and double-distilled dichloromethane (3 x 5 ml) was used to extract the Noxides. The dichloromethane extracts were concentrated (to 10-20 p1) in tapered evaporating tubes21on a water bath at 45°C. This extraction procedure recovered more than 80% of each N-oxide." 552 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 9, 1978
Thin-layer chromatography Glass plates (20 x 20 cm) were spread to a thickness of 0.25 mm with silica gel GF254(type 60, Merck, Darmstadt). The coated plates were air dried and then activated at 105 "C for 1h. The concentrated dichloromethane extracts from each test or control flask and authentic reference N-oxide were applied to these plates at 1.5cm intervals. The plates were developed in either chloroform-ethanol-ammonia solution (S.G. 0.88) (100:8:0.5, by volume) or in acetone-ethanol-diethylamine (95 :5 : 1, by volume). After development, the spots were visua!ized under UV light from an Hanovia lamp (model CHI/294) at 254 nm. Compounds originating from metabolic extracts were tentatively identified as N-oxides if Rf values of these materials corresponded with Rfvalues of authentic N-oxides in both solvent systems, and if no material was observed from extracts from control flasks having identical Rf values. The N-oxide was extracted from the silica-gel using a suitable solvent and identity confirmed by the measurement of UV or IR spectra, or by reduction of N-oxide back to the arent pyridine, or by GLC, MS or combined GLCMS. "Details of the MS and GLCMS of some N-oxides formed in vitro using rabbit liver microsomes or isolated from urine of rabbits administered pyridine are described below.
Mass spectrometry A VG Micromass MM 12F single focusing mass spectrometer was used with an ion source temperature of 240 "C, an electron energy of 70 eV, a trap current of 100 pA, an accelerating voltage of 4 kV and a resolution of 800. The samples were introduced via the direct insertion probe and the spectra recorded on oscillographic paper as rapidly as possible after sample introduction without additional heating of the probe. The gas chromatographic inlet system was used for the analysis of the N-oxides of pyridine, 3-methylpyridine, 3-ethylpyridine, 3-fluoropyridine, 3-chloropyridine and 3-bromopyridine with a 1 m X 4 mm i d . glass column containing 2% w/w Carbowax 20 M and 5% w/w KOH on Chromosorb W HP (8O/lOO mesh) mounted in a Pye 104 oven maintained at 170 "C.The N-oxides of 3-cyanopyridine, 3-acetylpyridine, quinoline and isoquinoline were analysed using a 1 m x 4 mm i.d. glass column containing 3% w/w OV-17 on Gas Chrom Q (100/120 mesh). Helium was used as a carrier gas at a flow rate of 20 ml min-'. The glass-lined stainless steel tube connecting the GC column outlet to the single stage glass jet separator was maintained at 250°C. The samples were injected rapidly onto the column with a 7 cm syringe needle.
RESULTS A N D DISCUSSION Direct inlet mass spectrometry The EI mass spectra of pyridine and pyridine-N-oxide are shown in Fig. 1. The molecular ion peak of the N-oxide is the most useful peak to distinguish the @ Heyden & Son Ltd, 1978
METABOLIC N-OXIDATION OF 3-SUBSTITUTED PYRIDINES
3-ethylpyridine (Fig. 4). The mass spectra of the other N-oxides examined are tabulated in Table 1. The majority of the compounds examined were surprisingly volatile compared with alkylamine-N-oxides. The polarity of pyridine-N-oxide is less than might be expected because of the various tautomeric forms possible, as shown.23
0- o-0- -0
-
N+
N
N
N
00-
0
0
0
I
70
This volatility resulted in a large concentration of compound in the ion source and in the appearance of large [M+l]+ ion peaks in the spectra. Reducing the sample size allowed spectra to be obtained with [M+ 11' ion peaks of the intensity expected considering the natural13C abundance. Thermolysis of the compound was minimized by recording the spectrum as soon as possible after sample insertion. A comparison of the relative abundance of the [MI+, [M-16]+ and [M17]+ ion peaks obtained in our laboratories with those published is shown in Table 2. In the majority of cases we observed less thermolysis than other workers, as indicated by the [M- 16]+ion intensity.
m/e
Figure 1. Mass spectra (a) pyridine and (b) pyridine Noxide.
former from the parent amine. The molecular ion usually produced the base peak in the spectrum of each N-oxide but the [M- 16]+/[M]f intensity ratio varied with the operating conditions. the mass spectra of the isomeric 2-, 3- and 4-hydroxypyridines (Fig. 2) were markedly different from that of pyridine-N-oxide in that no [M-16]+ ion peak was observed. Weak [MHI' and more intense [M - 27]+ ion peaks, which were absent in the spectrum of pyridine-N-oxide, were observed for the 3- and 4-hydroxypyridines. 2-Hydroxypyridine exhibited an intense [M- 28]+ ion peak which was less intense, but present, in the spectra of the 3- and 4- hydroxypyridines and absent in the N-oxide spectrum. A metastable ion peak was observed for the fragmentation of the molecular ion of 2-hydroxypyridine to form the (M- 28) ion peak, this probably being due to the loss of CO via the tautomeric 2pyridone. 22 The EI mass spectrum of the primary alcohol 3pyridyl carbinol [Fig. 3(a)] has a base peak corresponding to [M-HI' whilst the [M- 15]+ ion produces the most intense peak for the secondary alcohol l-(3-pyridyl) eth'anol (Fig. 3(b), thus readily distinguishing these compounds from the N-oxides of 3-methylpyridine and
Gas chromatography coupled with mass spectrometry The mass spectra obtained using the G C inlet system were substantially the same as those obtained via the direct insertion probe except that usually in the former the [M- 16]+peak was more intense. Nevertheless, in the majority of cases the molecular ion peak remained the base peak (see Table 2). The degree of thermolysis was minimized by a rapid injection of the sample directly onto the all-glass column. A G C peak due to the formation of the parent amine was observed when the sample was injected more slowly. Thermolysis was also observed on one occasion when the glass-lined stainless steel tube connecting the chromatograph to
100
ooH
00
-g ...? -
z
60
u) c W c
40
0
W
n
20
L I
i0
70
L 90
50
70
90
m/e
Figure 2. Mass spectra of 2-, 3- and Qhydroxypyridines.
@ Heyden & Son Ltd, 1978
BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 9, 1978 553
D. A. COWAN, L. A. DAMANI, AND J. W. GORROD
"1
I
Table 1. Major ion peaks in the direct inlet mass spectra of some pyridine-N-oxides
m/e (% relative intensity)
N
1
0
R=H 30
50
70
3-CH3
90 m/e
Rgure 3. Mass spectra of (a) 3-pyridylcarbinol 1-(3-pyridyl)ethanol.
and (b)
3-CzH5
3-F 3-CI
(a 1
3-Br QCH3
3-COCHa
4
0
3-NHz
3-NHCOCH,
m/e
Figure 4. Mass spectra of (a) 3-methylpyridine-Noxide and (b) 3-ethylpyridine-Noxide.
3-CN 3-CONHZ 3-COOH
the jet separator had been damaged, exposing the eluted compounds to the hot metal surface.
3-OH 3-N(CH3)z 3-CHZOH 3-CON(CZH&
Metabolism of 3-substituted pyridines 3-CONHCZHs
The in vitro metabolism of several 3-substituted pyridines, and of cotinine and quinoline was studied using hepatic and pulmonary microsomal preparations from various animal species. Pyridines in which the 3-substituent was -H, -CH3, -CzHs, -F, -el, -Br, -COCH3, -CN and -CON(CzHs)z were all converted to the corresponding N-oxides by fortified hepatic microsomal preparations from hamsters, guinea-pig, rabbit, rat and mouse, and by pulmonary preparations from guineapig and rabbit (see Table 3). This is the first report of in vifro metabolic N-oxidation of these pyridines, except for a single report3 on the metabolic N-oxidation of N, N-diethylnicotinamide (nikethamide) using rat liver homogenates. The in vitro metabolic N-oxidation of 3-acetylpyridine has not been described previously, although the N-oxide of 1-(3-pyridyl)ethanol has been identified in the urine'of ratsz4 as a metabolite of 3-acetylpyridine; the N-oxide isolated in vitro, in the present investigation, was that of 3-acetylpyridine itself. The primary metabolite in vivo of 3-acetylpyridine may be its N-oxide, which is metabolized further to form the N-oxide of 1-(3-pyridyl)ethanol as the secondary 554 BIOMEDICAL MASS SPECTROMETRY, VOL. 5, NO. 9, 1978
Nicotine-1-oxide Cotinine-N-oxide 2-CH3 4-CH3
4-Cd-k Quinoline N-oxide lsoquinoline N-oxide
79(12) 95(100) 39(59) 109(100) 93(13) 65(22) W27) 123(100) 107(18) 81(42) 65(20) 39(50) 40(53) 113(100) 97(25) 70(21) 58(25) 131(33) 129(100) 78(22) 73129) 40(69) 39(54) 175(100) 173( 100) 66(50) 78(27) 39(59) 137(100) 122(35) 51(19) 53(22) 94(31) 110(100) 55(22) 66(15) 39(28) 136(19) 152(25) 67(18) 93(18) 39(23) 120(100) 104(20) 76( 16) 77( 16) 138(100) 122(18) 39(21) 78( 15) 139( 100) 123(96) 78(41) 51(28) 95(16) 111(100) 138(100) 124(20) 42(75) 95( 17) 125(100) 109(17) 63(22) 78(42) 194(18) 178(12) 78(42) 106(100) 150(36) 166(43) 78(66) 106( 100) 39(24) 162(16) 178(30) 121(15) 119(32) 192(100) 176(3) 42(10) 93(41) 109(100) 65(96) 66(21) 109(100) 93( 10) 39(20) 123(59) 108(100) 39(20) 145(100) 129(17) 89(311 W54) 145(100) 129(10) 90(78) 89(62) 39(19)
78(9)
40(20)
92(7) 5363) 106116) 53(47)
80(25) 39(75) 92(26) 51(21)
966) 57(53) 113(12) 63(23)
86(10) 102(11) 50(27)
157(13) 63(37)
146(12) 51(37)
121(7) 43(51) 81(11) 5442)
78(42)
11O(24) 43(62)
94(100) 40(24)
103(8) 65(W 120(9)
93(13) 6430) 9411)
106(26) 39(28) 55131) 122(12) 39(60) 108(15) 39(87) 177(28) 51(18) 149(30) 51(40)
67(17) 40(26)
105(43) 39(27) 121(37) 80(37) 122(42) 39(12) 122(41) 44011
161(30) 84(100) 175(6)
133(28) 40(50) 98(99)
92(93) 40W) 65(10)
78(13) 39(30) 53(24)
91(19)
65(12)
128(10)
117(27)
128(8) 63(32)
118(45)
metabolite. In addition, 1-(3-pyridyl)ethanol was produced in vitro as a reduction product of 3-acetylpyridine. The same compound was formed, but by pC-oxidation, on incubation of 3-ethylpyridine. @ Heyden & Son Ltd, 1978
METABOLIC N-OXIDATION OF 3-SUBSTITUTED PYRIDINES
Table 2. Mass spectral characteristics of some pyridine-N-oxides. The values in parentheses are for combined GLCMS % Relative intensity (literature values)
% Relative intensity Compound
[ M - 161t
[MI:
[MI!
IM-161t
16(20) 5
