Additional Routes in the Metabolism of Nicotine to 3-Pyridylacetate THE METABOLISM OF DIHYDROMETANICOTINE”

The metabolism of dihydrometanicotine was investigated in the rat and dog. In preliminary studies, dihydrometanicotine difumarate was administered to rats. The RF values and retention times of methyl esters prepared from acidic Koenig-positive metabolites in the urine suggested the presence of 3-pyridylacetate and 4-(3-pyridyl)butyrate. Following administration of dihydrometanicotine difumarate to dogs, the pattern of excretion of acidic metabolites in the urine was similar to that found in the rat. Chemical conversion of the acidic metabolites to their methyl esters for separation by preparative gas chromatography afforded methyl 3-pyridylacetate and methyl 4-(3-pyridyl)butyrate. Methyl 3-pyridylacetate was identified by melting point, elemental analysis, and the infrared spectrum of its picric acid salt. Methyl 4-(3-pyridyl)butyrate was identified by mass spectroscopy and by the melting point, elemental analysis, and the infrared spectrum of the methyl ester picrate. the formation of 3-pyridylacetate from (-)nicotine via either (-)cotinine


JR.
From the Department of Pharmacology, JIerlical College of Virginia, Richmond, Virginia %%19

SUMMARY
The metabolism of dihydrometanicotine was investigated in the rat and dog. In preliminary studies, dihydrometanicotine difumarate was administered to rats.
The RF values and retention times of methyl esters prepared from acidic Koenig-positive metabolites in the urine suggested the presence of 3-pyridylacetate and 4-(3-pyridyl)butyrate. Following administration of dihydrometanicotine difumarate to dogs, the pattern of excretion of acidic metabolites in the urine was similar to that found in the rat.
Chemical conversion of the acidic metabolites to their methyl esters for separation by preparative gas chromatography afforded methyl 3-pyridylacetate and methyl 4-(3-pyridyl)butyrate. Methyl 3-pyridylacetate was identified by melting point, elemental analysis, and the infrared spectrum of its picric acid salt.
Methyl 4-(3-pyridyl)butyrate was identified by mass spectroscopy and by the melting point, elemental analysis, and the infrared spectrum of the methyl ester picrate.
In view of the report (DE CLERCQ, M., AND TRUHAUT, R. (1962) Bull. Sot. Chim. Biol., 44, 227) that dihydrometanicotine is a metabolite of nicotine in the rat, the present findings in the rat and dog suggest a third alternate route (via dihydrometanicotine) to 3-pyridylacetate.
Truhaut and de Clercq (1) administered nicotine to rats and obtained evidence for the formation of dihydrometanicotine (3. [4-(methylamino) their studies provide little or no data which would indicate formation of dihydrometanicotine. In addition to providing the first direct evidence for the natural occurrence of dihydrometanicotine, and a possible conjugate with phenylalanine, de Clercq and Truhaut (4) noted that dihydrometanicotine is more active than the parent nicotine in stimulating the synthesis of diphosphopyridine nucleotide from the precursors tryptophan, nicotinamide, and nicotinic acid, which were supplied to t'he experimental animals.
Evidence for the presence of dihydro-m&nicotine in tobacco smoke has been supplied by other investigators (5).
One distinguishing feature in the general mammalian metabolism of nicotine is access (6) to two distinct routes, one via cotinine and the other via demethylcotinine, which provide alternate pathways to the production of 3-pyridylacetic acid. The reported production of dihydrometanicotine as a nicotine met'abolite would provide on theoretical grounds a third alternate route to 3-pyridylacetic acid: via 4-(3.pyridyl)butyric acid and subsequent p oxidation.
These and other considerations led to our exploration of the metabolism of dihydrometanicotine in the rat and the dog. As part of the initial study, primary attention was directed to characterization and identification of pyridylcarboxylic acids that might arise from the oxidative metabolism of dihydrometanicotine.
Analytical and preparative gas chromatography were performed with an Aerograph Autoprep 700, fitted with an aluminum column (10 feet x Q inch OD and packed with 30% SE-30 on 60/80 mesh firebrick) at 221"; helium carrier, 100 ml per min; injection port at 243"; and thermal conductivity detector at 225". SpeclraP.\ll ultraviolet absorption spectra were determined in 95yG ethanol with a Cary niodel 11 I'11 recording spectroI)hotometer.
The acetyl chloride solut,ion n-as cooled in the ice bath, and nicotine n'as slowly added.
The mixture n-as allowed to come to room temperature and theri xvas reflused for 18 hours ulider protection of a ralciuni chloride tube. While still warm and free flowing, the mixture was treated lvilh 100 ml of 10% hydrochloric acid and vigorously shaken in a separatory funnel. The acidic aqueous layer MU removed and later conlbined with three subsequent acidic extractions (50 ml each).
The csombined aqueous layers were cooled and adjusted t,o pH 9 to 10 (pHydrion paper) by addition of 20% potassium hydroxide.
The alkaline solution was then continuously extracted for 48 hours with n-hexane, which removed unreacted nicotine (RF 0.93,solvent H), and a small amount of acetylmetanicotine (n, 0.82, solvent II). The aqueous phase was then continuously extracted with ether for 48 hours. The ether layer contained a major Koenig-positive component (RF 0.80,solvent II,corresponding in RF value to authentic aretylmetanicotine) and several minor unident#ified Koenig-positive components (RF 0.55 and 0.48, solvent II).
The ethereal solution was evaporated under reduced pressure to obtaiii crude acetylmetanicotine (127 g, 447;) as a brown oily residue. For l)urifi(xtion and identification, a sample of the crude a~etylmetallicotille (20 g) iii 40 ml of chloroforni was placed on a column (3 x 34 cm) of acid-washed alumina.
Al sample of this base (52 mg) was treated with 68 mg of picric acid (15% water) as a saturated solution in 95 % ethanol.
The yellow rrystalline precipitate (69 mg), which formed at room temperature, u-as recrystallized from absolute alcohol without change of melting point (139-139.5") and then dried for analysis at 64" and 1 mm of Hg over KOH. Acetyldihydrometanicotine-ii solution of 13 g of acetylmetanicotine in 50 ml of absolute alcohol was stirred at room temperature with 3.3 g of 10% palladium-charcoal and hydrogen at atmospheric pressure and room temperature for 2 hours, or until the calculated amount of hydrogen had been consumed.
After removal of the catalyst, the solution which contained txo Koenigpositive zones (RF 0.67 and IiF 0.13, solvent I) was evaporated under diminished pressure to a light brown oil.
The solvent was removed under nitrogen, and the crystalline lxoduct xv&s recrystallized frorn acetone; m.p. 1322134", 130 mg. For analysis, the salt was recrystallized from acetone without change of melting point, and was dried at 86" and 1 mm of Hg over KOH. Acetyldihydrometanicotine was previously prepared by I Iromatka (la), who acetylated dihydrometanicotine with acetic anhydride and then distilled the product (b.p. 213" at 15 mm of Hg).
Dihydrometanicotine-Acetyldihydrometanicotine (7 g) w'as heated under reflux with 50 ml of 12 x sulfuric acid for 27 hours. The hydrolysis mixture, which contained Koenig-positive zones (Solvent I) at RF 0.23 (major) and RF 0.63 (minor, and corresponding in RF value to unhydrolyzed acetyldihydrometanicotine), was adjusted to $1 9 to 10 (pHydrion paper) by addition of conrentrated ammonium hydroxide. The mixture was then extracted three times with methylene chloride (loo-ml portions).
A sample of the crude dihydrometanicotine (2.02 g) was treated with 3.01 g of fumaric acid in 40 ml of hot absolute ethanol and then concentrated to approximately 20 ml. The solution was cooled in an ice bath, and ether was added dropwise until no further increase in turbidity resulted. The product (4.5 g, representing a 92% yield based upon the acetyldihydrometanicotine employed in the hydrolysis) rnelted at 112-115". For analysis, the difumarate was dissolved in a minimal amount of warm 2-propanol and reprecipitated by dropwise addition of ethyl acetate. The sample was dried at 44" and 1 mm of Hg over KOH. For further identification, dihydrometanicotine base (91 mg) was treated with 0.4 ml of 40% HBr.
After removal of the solvent by evaporation at 140" in an atmosphere of nitrogen, the dihydrobromide salt was dissolved in 1 ml of ethanol-acetone (1: 1 by volume).
Upon cooling and scratching the wall of the container, the salt was precipitated.
After recrystallization from absolute ethanol, the product was dried at 40" and 1 mm of Hg over KOH for 3 hours, mp. 127-130" (capillary). The methylene chloride extract (B), which showed a single chromatographic examination (Fig. 1). Peak A corresponded The aqueous phase (A), remaining from extraction with methylene chloride, was placed on a column (6 X 18.5 cm) of Dowex 21-K (OH-).
After a water wash, material was eluted from the column with 2 N acetic acid until the eluate was no During the 43 hours of infusion and a subsequent lo-hour period, bladder urine was collect,ed by an indwelling catheter leading to a bottle immersed in dry ice-acetone and kept frozen until processing.
Processing of Dog Urine-Celite (Johns-Manville) was added to the urine (3.7 liters) from 13 dogs which had received a total of 8.0 g of dihydrometanicotine, and the urine was filtered through Whatman No. 1 paper. Data obtained by paper chromatographic examination of the urine are shown in Table I. The filtered urine was adjusted to pH 9 to 10 (pHydrion paper) by addition of concentrated ammonium hydroxide.
This aqueous solution (A) was then continuously extracted with methylene chloride for 48 hours.
The methylene chloride solution (B) was evaporated to a dark brown residue (1.2 g). The residue was dissolved in distilled water and placed on a column (2 x 7.5 cm) of Dower; 21-K (OH-).
The effluent and a water wash were collected until Koenig negative.
The combined effluent and water wash were made acidic (pH 5 to 6 to pHydrion paper) with 10% hydrochloric acid and then placed on a column (2.5 x 4.5 cm) of Dowex 50-W (II+).
The effluent and water wash were discarded. The column was then treated with 1 liter of 2 N ammonium hydroxide and then with 1 liter of 5 N ammonium hydroxide. The combined ammonium hydroxide eluates were continuously extracted with methylene chloride for 24 hours. The methylene chloride was evaporated to a brown residue (399 mg). The residue wets dissolved in absolute ethanol and treated with decolorizing carbon (Norit A). After filtration through Celite, the mixture was concentrated on the st'eam bath with a stream of nitrogen to a brown residue (371 mg). An aliquot (70.1 mg) was treated with 113.9 mg of fumaric acid dissolved in hot ethanol.
After removal of the solvent on the steam bath under nitrogen, the residue was dissolved in 1 ml of 2-propanol.
The mixture was treated dropwise with ethyl acetate until no more turbidity developed.
After standing overnight at room temperature, the crystals (94.2 mg) were collected and dried with a stream of nitrogen.
A solution of the product in hot ethanol was treated wit.h Norit A and then filtered through Whatman No. 1 filter paper with the aid of Celite. Upon cooling and scratching, white crystals were deposited, m.p. 112-113"; on admixture with authentic dihydrometanicotine difumarate, the melting point was unchanged.
The metabolic product showed a single Koenig-positive zone at R,w 0.08 (solvent I), corresponding in RF value to the zone obtained from authentic dihydrometanicotine difumarate. Isolation and Identi$cation of Acidic Metabolites in Dog Urine-The aqueous phase (A) remaining from ext.raction with methylene chloride was filtered and then placed on a column (8 X 82 cm) of Dowex 21-K (OH).
The effluent. and a water wash contained Koenig-positive material and were saved for later st,udy. The column was treated with 2 IG acetic acid until the eluate was Koenig negative.
The effluent and water wash were discarded.
The column was treated with 2 N ammonium hydroxide until the eluate was Koenig negative.
The ammoniacal solution (C) showed six Koenig-positive zones on chromatography with solvent B (RF 0.40, 0.26, 0.53, 0.18, 0.10, and 0.02). The material at RF 0.40 corresponded in RF value to authentic 4-(3-pyridyl)butyric acid, and that at RF 0.26 to 3-pyridylacetic acid. Identiscation of 4-(S-Pyridyl)butyric Acid and S-Pyridyhcetic Acid as Methyl Esters-The ammoniacal solution (C) was concentrated over a steam bath and under reduced pressure to a brown oily residue. The residue was triturated with 100 ml of hot methanol and filtered.
The residue (10.2 g) from evaporation of the solvent was dissolved in 250 ml of fresh methanol and 9.4 ml of sulfuric acid. The mixture was heated under reflux for 16 hours. The esterification mixture was concentrated to one-half volume and then treated with an equal volume of crushed ice. The pH was adjusted to 9 to 10 with 10% SaOH, and the mixture was extracted with four portions of chloroform (200 ml each). The combined chloroform extracts were treat,ed with anhydrous magnesium sulfate and filtered; the filtrate was evaporated to dryness. The residue contained four Koenigpositive zones (Table I) on chromatography with solvent C (RF 0.65,0.55,0.44,and 0.20). The zone at RF 0.65 corresponded in RF value to methyl 4-(3-pyridyl)butyrate, and that at RF 0.55 to methyl 3-pyridylacetate.
The methyl esters described above were dissolved in benzene to a final volume of 4 ml. A 50.~1 sample, when subjected to quantitative gas chromatographic analysis, showed four peaks, two of which were identified as methyl 3-pyridylacetate and methyl 4-(3-pyridyl)butyrate, plus a solvent peak. The height of the methyl 3-pyridylacetate peak corresponded by calculation to 954 mg of methyl 3-pyridylacetate, or 12.9 molar percentage of the administered dihydrometanicotine.
With the exit port at 180" and loo-p1 aliquots, preparative separation of five fractions plus initial solvent peaks was achieved.
The initial solvent peaks (Fraction I) which contained a small amount of Koenig-positive material were discarded and the subsequent fractions (II to V) were collected, with some mechanical loss, in glass bottles chilled in solid carbon dioxide-acetone: II (retention time 103 mm, primary major), III (retention time lS+ min, primary minor), IV (retention time 22+ min, secondary major), and V (retention time 36s mm, secondary minor).
Fractions III and V were not identified.
An aliquot (48 mg) of Fraction II material (465 mg; RF 0.56, solvent C) was dissolved in 0.5 ml of methanol and t.reated dropwise with approximately 90 mg of hydrous picric acid (15% water) as a saturated methanolic solution.
The sample, which showed a single Koenig- The infrared spectrum of the isolated compound showed no essential difference from an authentic sample (Fig. 2).
The mass spectrum of the free methyl ester (Fraction IV), obtained in an Hitachi RMU 6 H instrument,

AND DISCUSSION
A number of synthetic sources (14, 15), in addition to natural sources (4), have been described for dihydrometanicotine.
In the present study, attention was directed to the preparation of dihydrometanicotine via acetylmetanicotine, since there is some possibility (16) that the latter may be formed in vivo by a reaction analogous to the Pinner-Retard reaction.
Acetylmetanico-tine, which has been previously prepared (17) from acetic anhydride and nicotine, was conveniently obtained in our studies from acetyl chloride and nicotine.
Hydrogenation of acetylmetanicotine in the presence of palladium-charcoal afforded a 92% yield of acetyldihydrometanicotine.
Dihydrometanicotine, from the acidic hydrolysis of acetyldihydrometanicotine, was conveniently stored as the stable fumaric acid salt.
Preliminary studies were conducted on the metabolism of dihydrometanicotine in the rat, since it is in this species alone that dihydrometanicotine and the possible phenylalanine conjugate (3-[N-phenylalanyl-4-(n~ethylamino)butyl]pyridine) have been implicated (1) in the metabolism of nicotine. After the intraperitoneal administration of dihydrometanicotine, the urine of the animals provided evidence by paper chromatography for the presence of 3-pyridylacetate and 4-(3-pyridyl)butyrate. After removal of unchanged dihydrometanicotine by extraction with methylene chloride, the urine was processed and the acidic fraction was subjected to esterification with methanol-sulfuric acid. The resultant methyl ester fraction was examined by both paper and gas chromatography.
The RR values of Koenigpositive materials and the retention times of Koenig-positive materials corresponded to authentic methyl 3-pyridylacetate and methyl 4-(3-pyridyl)butyrate.
Experimental work was then extended to the dog to facilitate the use of larger quantities of dihydrometanicotine.
The dogs under pentobarbital anesthesia and bladder urine was firmation of identify was achieved through favorable comparison collected by means of an indwelling catheter. The methylene of the infrared spectra, melting point, and analysis of the picric chloride-soluble basic fraction of the urine contained unchanged acid salt. The isolation of 4-(3.pyridyl)butyrate in this indihydrometanicotine and three other Koenig-positive substances stance affords the first direct evidence for the participation of which were not ident,ified.
A preliminary chromatographic 4-(3.pyridyl)butyrate in the mammalian metabolism of nicoexamination of the acidic components of the urine pointed to the tine, if one assumes that dihydrometanicotine participates in the possible presence of 3-pyridylacetate and 4-(3.pyridyl)butyrate. metabolism of nicotine in the dog along the lines indicated by Confirmation of the presence of Y-pyridylacetate was achieved Truhaut and de Clercq (1) for metabolisrn of nicotine in the rat. by gas chromatographic separation of sufficient quantities of the From Knoop's early studies (18) on the metabolism of 4-phenylmethyl ester of B~pyridylacet~ate for preparation of the picric butyric acid to phenylacetic acid in the dog, it can be anticipated acid salt. The analytical sarnple was identified by melting that 4-(3-pyridyl)butyric acid is a precursor of 3-pyridylacetic point, mixed melt,ing point, and comparison of infrared spectra acid, which has been shown to arise from the metabolism of with authentic material.
nicotine in a variety of mammalian species. The gas chromatographic fraction which corresponded in retention time to authentic methyl 4-(3.pyridyl)butgrate showed a principal mass spectral peak m/e 179, corresponding to authentic methyl 4-(3-pyridyl)but'yrate (13). Additional con-Evidence for the metabolism of nicotine to 3-pyridylacetate was first arhiered (19)