The Metabolic Formation of y - (3-Pyridyl) -y ...reaction and then with successive 500-ml portions of methanol- ether: 5, 10, and 15% methanol by volume. The eluate (0 to 15% methanol)

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 239, No. 11, November 1964

Printed in U.S.A.

The Metabolic Formation of y - (3-Pyridyl) -y -hydroxybutyric

Acid and Its Possible Intermediary Role in

the Mammalian Metabolism of Nicotine*

HERBERT MCKENNIS, JR., SORELL L. ScHwARTz,t LENNOX B. TURNBULL, EINOSUKE TAMAKIJ AND EDWARD R. BOWMAN

From the Department of Pharmacology, Medical College of Virginia, Richmond 19, Virginia

(Received for publication, February 20, 1964)

In recent studies (1) on the mammalian degradation of the pyrrolidine ring of (-)-nicotine, y-(3-pyridyl)-y-oxobutyric acid was isolated, after chemical conversion to the methyl ester, from the urine of the rat and the dog after administration of (-)- cotinine, an intermediate in the metabolism of (-)-nicotine. Concurrently in the rat, the hydrolysis of y-(3-pyridyl)-y-oxo-N- methylbutyramide, another metabolite of ( -)-nicotine (2, 3)) was demonstrated.

These findings, preliminary experiments in which the metabolism of y-(3-pyridyl)-y-oxobutyric to y-3-pyridyl-y-hydroxybutyric acid was shown in the rabbit (4), and the early experiments of Knoop (5) on y-phenyl-y-oxobutyric acid led to the suggestion that y-3-pyridyl-y-hydroxybutyric acid arose as an intermediate in the metabolism of ( -)-nicotine to 3-pyridylacetic acid (1). These data and the recent demonstration (6) that (-)-demethylcotinine, a metabolite of (-)-nicotine and (-)- nornicotine, is converted by the rat to y-(3.pyridyl)-y-oxobutyric acid and y-(3-pyridyl)-y-hydroxybutyric acid have given rise to a more detailed investigation of the metabolism of y-(3-pyridyl)- y-oxobutyric acid.

EXPERIMENTAL PROCEDURE

Chromatography-Paper chromatograms were prepared by the descending method on Whatman No. 1 paper, unless otherwise noted, at ambient temperature. The solvent mixtures employed were the systems previously designated (7) B (ammonia-ethanol-n-butyl alcohol) and C (90% formic acid-set-butyl alcohol- water). The Koenig-positive areas were disclosed with p-amino- benzoic acid and cyanogen bromide as previously described (2). The Dowex resins were 50 to 100 mesh.

Elementary Analyses-Elementary analyses were performed by Spang Microanalytical Laboratory and by Weiler and Strauss.

Melting Points-All melting points, unless otherwise noted, are corrected micromelting points.

Ultraviolet Absorption Spectra-All ultraviolet absorption spectra were obtained in 95% ethanol with a Cary model 1lPM recording spectrophotometer.

* A preliminary report of this work was presented at the 49th annual meeting of the Federation of American Societies for Experi- mental Biology in Philadelphia, Pennsylvania, April 15, 1958.

t Present,address, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Marvland.

$ Present address, Central Research institute, Japan Monopoly Corporation, Tokyo, Japan.

Animals-Rats were obtained from Albino Farms, Red Bank, New Jersey. Rabbits were obtained from J. C. Heath, Ashland, Virginia. Mongrel dogs were obtained from pooled pound facilities.

y-(S-Pgridyl)-y-oxobutyric Acid-To a suspension of sodium hydride (18 g, 0.75 mole, 33 g of a 55.4% dispersion in mineral oil) in 200 ml of refluxing benzene were added 227 g (1.5 moles) of freshly distilled ethyl nicotinate. After the addition of 5 ml of absolute ethanol, freshly distilled diethyl succinate (131 g, 0.75 mole) was introduced dropwise over a period of 30 minutes into the stirred refluxing mixture. After an additional lBmin- ute period of stirring, the reaction mixture was well cooled by immersion into an ice water bath, and 500 ml of 5 N hydrochloric acid were added slowly with stirring, which was continued for 10 minutes after completion of the addition. The lower aqueous phase was collected, extracted with a portion of benzene, and then adjusted to approximately pH 9 by addition of cold concentrated ammonium hydroxide. The alkaline aqueous solution was exhaustively extracted with three portions of chloroform (500 ml each). After drying over sodium sulfate, the filtered chloroform solution was concentrated under diminished pressure to obtain a residue of crude diethyl cr-nicotinylsuccinate and ethyl nicotinate as a dark brown oil.

By rapid distillation through a Claisen flask with a pot temperature of llO-115”, 120 g of ethyl nicotinate (b.p. 7%84”, 1 mm of Hg) were recovered from the foregoing mixture. The pot temperature was increased to 190” to obtain 120 g of diethyl a-nicotinylsuccinate (b.p. 171-172”, 1 mm of Hg). The yield of diethyl Lu-nicotinylsuccinate was 57.8% of the amount calculated on the basis of diethyl succinate employed.

This procedure affords a generally higher and more consistent yield of diethyl ar-nicotinylsuccinate than the previously pub- lished condensation procedures on which it is based (8, 9).

A mixture of 100 g of diethyl cr-nicotinylsuccinate and 250 ml of 5 N sulfuric acid was heated under reflux for 36 hours. The cooled solution was adjusted to pH 4 to 5, until no additional precipitation occurred, by addition of sodium hydroxide. The precipitate was collected, washed with small portions of water, and then dried to obtain 49 g (79 y0 based on the nicotinyl ester) of y-(3-pyridyl)-y-oxobutyric acid, m.p. 162.5-163.5” (undepressed by admixture with an authentic sample (9)). The ultraviolet spectrum of the keto acid, X,,, 228 (e 8757), X,,, 267 (E 2436), and X, in 250 (E 1740) in ethanol, was in good agree- ment with previously reported values (8).

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3982 Metabolic Formation of y-(SPyridyl)-y-hydroxybutyric Acid Vol. 239, No. 11

A picrate was obtained by treating the keto acid (40 mg, 0.22 mmole) with 50 mg (0.22 mmole) of picric acid (10% water) as a saturated solution in ethanol. The yellow precipitate was recrystallized from absolute ethanol, m.p. 139-142” (with decomposition). For analysis, the sample was dried at 75” and 1 mm of Hg over KOH.

CISHI&~OK, (408.13)

Calculated: C 44.10, H 2.96, N 13.72 Found : C 44.23, H 2.93, N 13.76

(A)-T-(S-Pyridyl).y-hydroxybutyric Acid-Potassium hydroxide pellets were slowly added with stirring to a mixture of 10 g (0.056 mole) of y-(3-pyridyl)-y-oxobutyric acid and 150 ml of water until the pH of the resultant clear solution was approximately 11. Sodium borohydride (1.65 g, 0.044 mole) in 25 ml of water was added dropwise to the stirred solution. After addition was complete, the solution was stirred for an additional 2 hours and then was concentrated to dryness, under diminished pressure. The residue was treated at room temperature with 250 ml of ethanol and then filtered. The residue from concentration of the filtrate was treated with 100 ml of absolute ethanol and then filtered. On concentration under diminished pressure, the filtrate yielded a light yellow oil (10 g, 0.055 mole), which showed on paper chromatography in Solvent C a single Koenig- positive zone at Rr 0.36. The product slowly solidified on cooling in the refrigerator. In preliminary attempts, a satisfac- tory recrystallization of the material, which liquified on exposure to room air, was not achieved.

A reduction of the keto acid to y-(3-pyridyl)-y-hydroxybutyric acid was also effected when a molar equivalent of aqueous sodium borohydride was added to a mixture of T-(3-pyridyl)-y-oxobutyric acid and water which was initially at pH 5 to 6. The paper chromatograms of the reaction mixture indicated, however, the presence of unchanged keto acid (Rr 0.65, Solvent C) and an unidentified Koenig-positive component (RF 0.57, Solvent C), possibly a diol which formed from the reduction of the lactone of Y-(3-pyridyl)-y-hydroxybutyric acid. Wolfrom and Wood (10) have noted the reduction of n-gluco-n-guloheptano- y-lactone with sodium borohydride to the diol, n-gluco-n-gulo- heptose.

(A)-5-(S-PyridyZ)tetrahydrofuranone-%-To a mixture of 1 liter of methanol and 30 ml of concentrated sulfuric acid were added 40 g of Y-(3-pyridyl)-y-hydroxybutyric acid obtained from reductions with sodium borohydride (above). The mixture was heated under reflux for 12 hours and then, after cooling, neutralized with ammonium hydroxide. The solution was then concentrated to dryness under diminished pressure. The residue was treated at room temperature with approximately 400 ml of ethanol, and the mixture was filtered. The filtrate was concentrated under diminished pressure with removal of successive crops of ammonium sulfate until no further precipitation occurred. The oily residue from evaporation of the filtrate showed, on paper chromatography with Solvent C, three Koenig-positive zones: Rp 0.46, 0.30, and 0.25. This mixture was placed on a column (5 x 21 cm) of alumina. The column was treated with ether until the eluate no longer gave a positive Koenig reaction and then with successive 500-ml portions of methanol- ether: 5, 10, and 15% methanol by volume.

The eluate (0 to 15% methanol) that contained only a single Koenig-positive component at RF 0.46 was concentrated to ob-

tain 5-(3-pyridyl)tetrahydrofuranone-2 (12 g) as a light brown oil (Fraction A).

(+)-5-(3-Pyridyl)tetrahydrofuranone-2 picrate formed readily when the oil (227 mg, 1.70 mmoles) was treated with picric acid (400 mg, 1.74 mmoles) as a saturated solution in ethanol. The crystalline picrate recrystallized readily from ethanol or water. For analysis, the sample was recrystallized from ethanol, m.p. 133.5-135.5” with decomposition (153 mg). The compound was dried at 100” and 0.5 mm of Hg over KOH.

‘.L~HxzN~O~ (392.27)


The lactone was also formed from (+)-y-3-pyridyl-y-hydroxybutyric acid by heating the acid at 150” in an atmosphere of nitrogen for 10 to 30 minutes, or by treatment with pyridine- acetic anhydride at room temperature overnight. The resultant crude lactone mixture readily afforded the foregoing (=I=-5- (3-pyridyl)tetrahydrofuranone-2 picrate on treatment with a saturated aqueous solution of picric acid.

(-)-5-(S-Pyridyl)tetrahydrofuranone-2 by Resolution of Syn- thetic (&)-La&one-To a hot saturated solution of 10 g (0.061 mole) of the (=t)-la&one (Fraction A, above) in isopropyl alcohol were added 9.25 g (0.061 mole) of a saturated solut.ion of L-( +)-tartaric acid in isopropyl alcohol. After this was cooled and the container was scratched, a mixture of oil and crystals was obtained. A hot saturated solution of the mixture in methanol deposited a white crystalline tartrate (1.67 g) on cooling. After additional recrystallizations from methanol, the resultant product, m.p. 136.5-138.5” (with decomposition), weighed 300 mg and had a constant specific rotation: [o(]:tal -5.95” (3.52% in methanol). An additional crop (43 mg), m.p. 136.5-138.5” (with decomposition), was obtained from the mother liquors.

The combined tartrate (343 mg) was dissolved in approximately 2 ml of isopropyl alcohol containing enough methanol to effect a clear solution. The mixture was placed on a column (0.8 x 8.5 cm) of alumina. The column was treated with methanol-ether (30% methanol by volume) until the eluate was no longer Koenig-positive. Concentration of the solvent afforded 215 mg of (-)-5.(3-pyridyl)tetrahydrofuranone-2 as a colorless oil, which gave a single Koenig-positive zone on paper chromatography (Rp 0.42, Solvent C), [a],““,,, -7.4” (2.84% in methanol) .

The levorotatory lactone on treatment with a molar equivalent of picric acid as a saturated solution in ethanol readily yielded a crystalline picrate. The air-dried product which was recrystallized from ethanol melted at 143.5-145.5” (with decomposition), [o~]:“,h - 12.3” (3.65y0 in methyl Cellosolve). For analysis, the product was dried at 100” and 0.5 mm of Hg over KOH for 3 hours; m.p. 143.5-145.5”, [(Y]::$ -18.5” (2.71% in methyl Cellosolve).

G~H~xN~O~ (392.27)


(+)-5-(S-PyridyZ)tetrahydrofuranone-,%-The tartrate mother liquors from the isolation of the levorotatory lactone tartrate (above) and subsequent resolutions, representing a total of 15 g


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of synthetic racemic lactone, were concentrated to dryness under diminished pressure. The residue was dissolved in water and adjusted to pH 9 by addition of ammonium hydroxide. The solution was exhaustively extracted with chloroform. After drying over sodium sulfate and filtration, the chloroform solution was concentrated to obtain 7.55 g of a light amber oil,

blL f3.01” (4.64% in methanol). To a hot saturated solution of 7.1 g of the oil in isopropyl

alcohol were added 6.95 g of D( -)-tartaric acid as a saturated solution in isopropyl alcohol. Diligent scratching and careful cooling promoted the formation of a crystalline precipitate (1.47 g). After six recrystallizations from methanol, the colorless crystalline tartrate (300 mg) melted at 135.5-137.5” and had a constant specific rotation: [o(]::,, +5” (5.2% in methanol).

The tartrate (250 mg) was dissolved in methanol-ether (20% methanol by volume) and placed on a column (1 X 6 cm) of alumina. Elution was effected with the same solvent mixture until the eluate no longer gave a positive Koenig reaction. The solution was concentrated under diminished pressure on the water bath to obtain 182 mg of (+)-5-(3-pyridyl)tetrahydrofuranone-2 (Fraction B) asa light yellow oil, [or]ii$ + 8.3” (6.05% in methanol).

The dextrorotatory lactone (Fraction B), 60 mg, was treated with 85 mg of picric acid as a saturated ethanolic solution. The resultant crystalline picrate was recrystallized from ethanol, m.p. 143.5-145.5” (with decomposition), and dried at 100” and 0.5 mm of Hg over KOH to obtain the analytical sample with constant specific rotation: [a],““,,, +17.11” (2.5% in methyl Cellosolve).

C~~HVZN~O~ (392.27)

Calculated: C 45.92, H 3.08 Found : C 45.93, H 2.88

A solution of 122 mg of (+)-5-(3-pyridyl)tetrahydrofuranone-2 (Fraction B) was dissolved in water and adjusted to pH 9 by addition of ammonium hydroxide. The solution was placed on a column (1 X 10 cm) of Dowex 21K-OH- and allowed to stand overnight at room temperature. The effluent and water washings from the column at this time showed no positive Koenig reaction. (+)=y-(3..Pyridyl)-y-hydroxybutyric acid was then obtained by treating the column with 1 N acetic acid until the eluate no longer gave a strong positive Koenig reaction. The product was obtained as a light yellow oil (82 mg) by concentration of the acidic solution at room temperature under diminished pressure. A solution of the oil in ethanol cochromatographed at RF 0.35 (Solvent C) with a synthetic sample of ( +)---(3-pyridyl)-y-hydroxybutyric acid and showed an optical rotation: [cY]::~~ +22.84” (2.32% in 50% ethanol).

Metabolism of y-(3Pyridyl)-y-oxobutyric Acid in Rats--Six

male albino (Wistar strain) rats (385 to 525 g each) received a total of 8.55 g of y-(3-pyridyl)-y-oxobutyric acid by oral intuba- tion in aqueous solution as the sodium salt (250 mg of acid per ml, 500 mg of acid per kg of body weight in single doses on each of 6 successive days). The animals were allowed food (Purina rat chow) and water ad libitum. Urine was collected as daily runoff from metabolism cages into toluene and sodium fluoride until 48 hours after the last dose. The combined urine, which was stored frozen until processing, was filtered with Celite and adjusted to pH 5 with hydrochloric acid for placement on a column (2.5 X 28 cm) of Dowex 50W-Hf. The initial effluent and water washings obtained by treating the column with water

until the effluent was neutral were not investigated. The column was then treated with 1 N ammonium hydroxide until the effluent no longer gave a positive Koenig reaction. The ammoniacal solution was exhaustively extracted with chloroform.

The aqueous phase which remained from the chloroform extraction was concentrated to dryness on the steam bath under diminished pressure. The residue was heated under reflux with 1.5 liters of ethanol for 4 hours. After cooling to room temperature, the mixture was filtered. The residue (13 g) from evaporation of the filtrate was dissolved in water and after adjustment to pH 8 with ammonium hydroxide was placed on a column (2.5 X 28 cm) of Dowex 21K-OH-. The effluent, which gave a positive Koenig reaction, and a water wash were combined and saved for possible future investigation. The column was then treated with 1 N acetic acid until the eluate was Koenig- negative. Concentration of the acidic solution under diminished pressure afforded a brown oil (6.53 g) which showed, on paper chromatography with Solvent C, Koenig-positive zones at Rp 0.15, 0.19, 0.30, 0.40, and 0.62 (Table I) as well as a series of faint Koenig-positive zones between the origin and the zones at RF 0.15. The oil was heated under reflux with 175 ml of absolute methanol and 6 ml of concentrated sulfuric acid for 16 hours. After cooling and neutralization with ammonium hydroxide, the mixture was concentrated to dryness under diminished pressure. A solution of the residue in approximately 25 ml of water was adjusted to approximately pH 9 by addition of ammonium hydroxide and then extracted exhaustively with

TABLE I Chromatographic data on Koenig-positive componentsa of urine

ajter administration of r-(S-pyridyl)-r-oxobutyric acid

Male albino rat (oral)

Male mongrel dog (intravenous)

RF

0.15 0.19 0.30d 0.40 0.33

0.42e 0.620

0.49h 0.60il i 0.7gk

RF

0.15 0.16 0.20 0.30d 0.29

0.418 0.630 0.46

0.50h 0.56-0.60is i 0.79k

Female albino rabbit (oral)

Acidic fractionb

Ester fractionC

RF

0.18 0.19 0.24 0.31d 0.40 0.34-0.38 0.44f 0.406 0.640 0.46

0.4gh 0.5Bisi 0.74k

a Data obtained from chromatograms developed in Solvent C. 6 Alcohol-soluble, not extracted from alkaline aqueous media

with chloroform (see text). c Acidic fraction after refluxing with methanol-sulfuric acid

and extraction with chloroform (see text). d Cochromatographed with 3-pyridylacetic acid and r-(3.pyri-

dyl)-7.hydroxybutyric acid. e Corresponded in RF value to 5-@pyridyl)tetrahydrofura-

none-2. f Corresponded in RF value to r-(3.pyridyl)butyric acid. Q Corresponded in RF value to r-(3-pyridyl)--y-oxobutyric acid. h Corresponded in RF value to methyl 3-pyridylacetate. i Corresponded in RF value to methyl r-3-(pyridyl)butyrate. i Very faint zone in all cases. k Corresponded in RF value to methyl r-(3-pyridyl)-y-oxobu-

tyrate.


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3984 Metabolic Formation of y-(S-Pyridyl)-y-hydroxybutyric Acid Vol. 239, No. 11

chloroform. The chloroform solution on evaporation yielded a reddish brown oil (4.7 g). A sample of this ester fraction showed a number of Koenig-positive zones on paper chromatography (Table I).

The crude oily ester fraction was dissolved in benzene and placed upon a column (2.5 x 32 cm) of Florisil. The column was treated with benzene until the effluent was Koenig-positive. An additional 100 ml of benzene, followed by loo-ml portions of benzene containing, respectively, 5, 15, 20, 30, 40, 50, 70, and 90% acetone (v/v) and finally 100% acetone, were passed through the column and collected in 11-ml fractions. Paper chromatography of the effluent in Solvent C showed Koenig- positive components, which were obtained from the column in the following sequence: RF 0.78, 0.60, 0.49, 0.27, and 0.33.

The fractions with Koenig-positive components at RF 0.49, 0.60, and 0.78 were combined and concentrated to an oil (3.0 g), which solidified on cooling. The crystalline mass was recrystallized from isopropyl alcohol to obtain 1.1 g of methyl y-(3- pyridyl)-y-oxobutyrate as colorless crystals, m.p. 66-67.5”, undepressed by admixture with an authentic sample (1). The mother liquors yielded crude methyl y-(3-pyridyl)-y-oxobutyrate (RF 0.76) which on paper chromatography in Solvent C showed evidence of possible contamination with methyl 3-pyridylacetate (RF 0.49) and methyl y-(3-pyridyl)butyrate (RF 0.60).

The fractions from the Florisil column that showed material at RF 0.42 as the only Koenig-positive component were combined and concentrated to obtain a brown oil (140 mg). To this were added 150 mg of picric acid (15% water) as a saturated solution in ethanol. The resulting picrate (16 mg) was recrystallized from ethanol, m.p. 144-146.5”. Admixture with an authentic sample of ( -)-5-(3-pyridyl)tetrahydrofuranone-2 picrate produced no melting point depression. The sample for analysis was dried at 60” and 1 mm of Hg over KOH.

‘&H~N409 (392.27)

Calculated: C 45.92, H 3.08, N 14.28 Found: C -16.13, H 3.08, N 14.00

iMetabolism of y-(S-Pyridyl)-y-oxobutyric Acid in a Dog-A male mongrel dog (17.5 kg) under pentobarbital anesthesia received, during an g-hour period, by continuous intravenous infusion into a femoral vein, 4.0 g of y-(3-pyridyl)-y-oxobutyric acid (28.5 mg per kg per hour) as the sodium salt in 480 ml of 0.02 M sodium chloride. On completion of this infusion, 0.9% sodium chloride (150 ml) was infused over a period of 1 hour. Urine was collected via an indwelling bladder catheter into a flask packed in carbon dioxide-acetone during the infusions and a subsequent period, for a total of 24 hours.

Upon thawing, the urine was adjusted to pH 9 with ammonium hydroxide and then filtered for exhaustive extraction with chloroform. The extracted urine was concentrated under diminished pressure to a brown residue. The residue was treated for 2 hours with ethanol (1 liter) under reflux. After removal of the ethanol by filtration, the remaining solid residue was extracted in a Soxhlet apparatus with ethanol for 16 hours. The combined ethanol solutions gave 36.5 g of brown, semisolid material on concentration under diminished pressure An aqueous solution of this residue was adjusted to pH 5 with hydrochloric acid and then placed on a column (4 x 28 cm) of Dowex 5OW-H+. The column was washed with water until the effluent was neutral and then was treated with 1 N ammonium hydroxide until the

Koenig reaction of the eluate was no longer positive. The ammonical solution was placed on a column (4 x 16 cm) of Dowex 21K-OH-. The effluent, which contained Koenig-positive material at Rp 0.12 (major), RF 0.07 (minor), and RF 0.18 (minor), with Solvent C and a wash that was obtained by treating the column with water until the effluent was neutral were not investigated in detail. The column was then treated with 1 N acetic acid until the eluate was no longer Koenig-positive. The acidic solution was concentrated under diminished pressure to a dark brown oil (5.1 g), which showed on paper chromatography in Solvent C a variety of Koenig-positive material as indicated in Table I. The oil was heated under reflux for 18 hours in a mixture of methanol (350 ml) and sulfuric acid (10.5 ml). A chloroform solution of the resultant esters was obtained essentially as described under the experiment on rats (above). The residue (2.8 g) from evaporation of the chloroform, a dark brown oil, showed Koenig-positive zones as indicated in Table I.

The mixture of oily esters was dissolved in benzene and placed upon a column (2.5 X 36 cm) of Florisil. The column was treated with benzene, benzene-acetone, and finally acetone, by the procedure essentially as described above for the rat. Koe- nig-positive components emerged from the column, as deter- mined by paper chromatography in Solvent C: RF 0.79, 0.56, 0.46, 0.42, 0.60, 0.50, 0.29, and 0.16.

Those fractions that contained the mat,erial at RF 0.42, and also the minor components at Rr 0.76 and Rp 0.46, were combined and concentrated to a light brown oil (614 mg). The mixture was dissolved in benzene and placed on a column (2 x 12 cm) of Florisil. An elution with benzene (32 ml), followed by benzene-acetone (32..ml portions, containing 1 y0 acetone and acetone sequentially increased to 12%, in increments of approximately 1 sl,), and finally 100% acetone produced Koenig- positive fractions. The terminal fractions that contained Koe- nig-positive material, RF 0.42 in Solvent C, were combined and concentrated to an oil (336 mg). To this were added 460 mg of picric acid (15% water) as a saturated solution in ethanol. The resulting picrate of ( -)-5-(3-pyridyl)tetrahydrofuranone-2 (708 mg) was recrystallized from ethanol, m.p. 143.5-145” (with decomposition), undepressed by admixture with an authentic sample. The sample for analysis was dried at 100” and 0.5 mm of Hg over KOH, [a!]:“,tl -21.3O (4.6% in methyl Cellosolve).

C~E,H,~N~O~ (392.27)


Metabolism of y-(S-Pyridyl)-y-oxobutyric Acid after Oral Ad- ministration to a Dog-A male mongrel dog received in a single oral dose 1.85 g of y-(3-pyridyl)-y-oxobutyric acid (100 mg per kg) as the sodium salt in aqueous solution. The 48-hour urine, which was collected daily as run-off from a metabolism cage into sodium fluoride, was processed on Dowex 50-H+ in a man- ner similar to that described for the rat (above). The ammoniacal eluate was adjusted to pH 2 by addition of sulfuric acid and then extracted continuously with ether for 20 hours. The aqueous solution remaining from the extraction was adjusted to pH 7 by addition of ammonium hydroxide. The residue from evaporation of the aqueous solution was extracted with hot chloroform. The chloroform filtrate and the ether solution (above) were combined and evaporated. The residue was dissolved in 60% aqueous methanol and on cooling deposited 100


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mg of y-(3-pyridyl)-y-oxobutyric acid, m.p. 162.5-163.5” with decomposition, undepressed by admixture with an authentic sample. The recovered acid chromatographed on paper to give a single Koenig-positive zone, RF 0.24 (Solvent B), and RF 0.65 (Solvent C) .

The insoluble residue from the foregoing chloroform extraction was treated with 220 ml of hot methanol. After removal of insoluble material by filtration, the methanolic solution was evaporated to dryness. The resultant residue was extracted with successive portions of hot absolute ethanol (40 ml, 20 ml, and 20 ml). The combined extracts were concentrated to a volume of 16 ml. The ethanolic solution was applied to large sheets of Whatman No. 1 paper for descending chromatography (Solvent C). The quenching zones in ultraviolet light corresponding in RF value to authentic y-(3-pyridyl)-y-oxobutyric acid were excised, cut into small pieces, and extracted with hot methanol. The residue from evaporation of the solvent was treated with 60% aqueous methanol. An additional 300 mg of y-(3- pyridyl)-y-oxobutyric acid, m.p. 162.5-163.5”, undepressed by admixture with an authentic synthetic sample, were obtained by careful concentrations of the solution and recrystallization from 60% aqueous methanol.

The ultraviolet quenching zones in the vicinity of R,w 0.36, corresponding approximately in value to authentic y-(3-pyridyl)- y-hydroxybutyric acid, were excised, cut into small pieces, and extracted with hot methanol. The methanolic solutions were combined and then concentrated to a volume of approximately 2 ml. After treatment with decolorizing carbon, the solution was concentrated to dryness. The residue was dissolved in water and placed on a column of Dowex l-OH-. After a water wash until the effluent was neutral, the column was treated with 1 N acetic acid until the eluate no longer gave a positive Koenig reaction. The acidic solution was concentrated to a light yellow oil which was heated to 150” in an atmosphere of nitrogen for lactonization. The product at room temperature was treated with 5 ml of water. The aqueous solution was extracted with 4 portions of chloroform (5 ml each). On concentration the combined chloroform solutions yielded an oil (210 mg). This afforded, on treatment with aqueous picric acid, a yellow picrate, m.p. 143.5-145.5”, undepressed on admixture with a sample of the synthetic levorotatory picrate of 5-(3-pyridyl)tetrahydrofuranone-2.

C&H12N409 (392.27)


iMetabolism of y-(S-Pyridylj-y-oxobutyric Acid by Rabbits- Experiment A: To four New Zealand white female rabbits (1.76 to 1.95 kg) was administered a total of 2.22 g of y-(3-pyridyl)- y-oxobutyric acid (150 mg per kg of body weight in single oral doses on each of 2 successive days as the sodium salt in aqueous solution containing 30 mg of acid per ml). The animals were allowed food and water ad libitum and urine was collected as run-off from metabolism cages until 48 hours after administration of the last dose. The combined urine, initially preserved with sodium fluoride, and final bladder urine, which was obtained by aspiration, were stored in the frozen state until processing. The residual dark brown oil obtained by concentrating the filtered urine under diminished pressure was heated under reflux with 1.5 liters of ethanol for 3 hours. The mixture was

allowed to cool to room temperature, and the remaining solid was separated by filtration. The solid was extracted with ethanol for 3 hours in a Soxhlet apparatus. The ethanolic solutions were combined and concentrated under diminished pressure to an oily residue (30 g). An aqueous solution of the residue was placed on a column (4 x 17 cm) of Dowex 5OW-H+. After a water wash, the column was treated with 1 N ammonium hydroxide until the eluat,e no longer gave a positive Koenig reaction. The ammoniacal solution was placed on a column (4 x 17 cm) of Dowex 21K-OH-. After a water wash, the column was treated with 1 N acetic acid until the eluate no longer gave a positive Koenig reaction. The brown oil (3.0 g) obtained from concentration of the acidic solution was heated under reflux with methanol (200 ml) and sulfuric acid (6 ml) for 9 hours. A solution of the resultant esters in chloroform was obtained essentially as described under the experiments on the rat (above). The residue (1.38 g) from evaporation of the chloroform showed Koenig-positive zones as indicated in Table I. This residue was chromatographed on Florisil (1.5 x 26 cm) essentially as described in the experiments with the rat. The collected fractions showed Koenig-positive zones on paper chromatography in Solvent C: RF 0.74, 0.56, and 0.48; RF 0.40; RF 0.46 and 0.38; RF 0.34 and 0.24; and RF 0.18.

All of the fractions containing material showing a Koenig- positive component only at RF 0.40 were combined and concentrated. The resultant light brown oil (97.0 mg) was treated with 125 mg of picric acid (15% water) as a saturated solution in ethanol. The resultant crystalline picrate (51 mg) was recrystallized from ethanol, m.p. 132.5-135.5”, undepressed by admixture with a sample of synthetic (h)-5-(3-pyridyl)tetra- hydrofuranonel2 picrate. The sample for analysis was dried at 100” and 0.5 mm of Hg over KOH, [o~]io4bq 0” (4.60% in methyl Cellosolve).

GsH~~N~O~ (392.27)


Experiment B: The combined 48-hour acidified urine from two New Zealand white female rabbits that received orally a total of 810 mg (150 mg per kg) of y-(3-pyridyl)-y-oxobutyric acid essentially as described above was placed on a column of Dowex 50-H+. After a water wash, the column was treated with 1 N ammonium hydroxide. The eluate was collected in 25-ml fractions. The first fraction (I) contained Koenig-positive material corresponding in R, value to authentic y-(3- pyridyl)-y-hydroxybutyric acid (RF 0.33, Solvent C) and three other Koenig-positive components (RF 0.16, 0.42, and 0.68, Solvent C). The three subsequent fractions, which showed on paper chromatography Koenig-positive material at RF 0.68 only, corresponding in value to y-(3-pyridyl)-y-oxobutyric acid, were combined and designated Fraction II.

Fraction I was adjusted to approximately pH 9 by addition of 1 N ammonium hydroxide and placed on a column (1 x 10 cm) of Dowex l-OH-. After a water wash, the column was treated with 0.5 N acetic acid and 11 subfractions (10 ml each) were collected. The first 5 subfractions (Sl to S5) showed a Koenig-positive component, RF 0.33 (Solvent C) corresponding to y-(3-pyridyl)-y-hydroxybutyric acid and minor Koenig- positive components (RF 0.16 and RF 0.42). The 6th subfraction contained Koenig-positive material at RR 0.68, corresponding to the 0x0 acid, and the two minor components.


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3986 Metabolic Formation of y-(S-Pyridyl)-r-hydroxybutyric Acid Vol. 239, No. 11

The subsequent subfractions, which showed evidence of the keto acid as a major component, were combined with Fraction II (above) and concentrated to dryness under diminished pressure. The residue was dissolved in methanol and treated with decolorizing carbon. The methanolic filtrate was concentrated to a volume of approximately 10 ml and diluted with an equal volume of water. On cooling, the solution deposited crystals of y-(3-pyridyl)-y-oxobutyric acid. After recrystallization from 60% methanol, the yield was 141 mg (17.4’% of the administered dose), m.p. 163-163.5” (with decomposition), undepressed on admixture with an authentic sample.

Subfractions Sl through S5 were combined and concentrated under diminished pressure. The residue was heated at 150” in an atmosphere of nitrogen for 15 minutes and after cooling dissolved in 3 ml of water. The aqueous solution was extracted with 10 portions of chloroform (5 ml each). The combined chloroform extracts were dried over sodium sulfate and then concentrated to a brown oil (66 mg). The oil was dissolved in chloroform and placed on a column of acid-washed alumina. Koenig-positive material (Rp 0.42, Solvent C) was obtained by treating the column with ether containing 5% methanol by volume. A light colored oil (66 mg) remained on evaporation

of the solvent. On addition of saturated aqueous picric acid, a crystalline picrate formed. After recrystallization from water, the product melted at 132.5-135.5” and did not depress the melting point of an authentic sample of (At)-5-(3-pyridyl)tetrahydrofuranone-2 picrate. The infrared spectrum (KBr pellet) showed (Fig. 1) no essential difference from an authentic synthetic sample.

In another experiment in which y-(3-pyridyl)-y-oxobutyric acid was administered orally to two rabbits, y-(3-pyridyl)-y- hydroxybutyric acid was also isolated from urine in the form of the lactone, 5-(3-pyridyl)tetrahydrofuranone-2. The ammoniacal solution which was obtained from the column of Dowex 50-H+ was concentrated to dryness. The residue was treated overnight with an excess of pyridine-acetic anhydride (1: 1 by volume). After removal of the volatile reagents under diminished pressure, the residue was dissolved in water and extracted with chloroform. The residual oil from evaporation of the chloroform was dissolved in methanol for chromatography in Solvent C on large sheets of S and S No. 589 green ribbon paper. The area at RF 0.42, corresponding to authentic lactone, was located as a quenching zone in ultraviolet light and then extracted with methanol. A brown oil, which formed a poorly

FIG. 1. Infrared absorption spectra of synthetic and metabolic 5-(3-pyridyl)tetrahydrofuranone-2 picrates in potassium bromide pellets.


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crystalline picrate, was obtained from evaporation of the solvent. The picrate was decomposed with 5 ml of 4 N hydrochloric acid. After an ether extraction to remove picric acid, the solution was made alkaline with ammonium hydroxide and then extracted with chloroform. The oily residue from evaporation of the chloroform was treated with an excess of picric acid. The crystalline picrate after three recrystallizations from water melted at 133-137”. Admixture with authentic (At)-lactone picrate did not depress the melting point.

C,sH,2N409 (392.27)


---- __- _. --. - *-- The aqueous solution remaining from the chloroform extrac-

tion of the pyridine-acetic anhydride treatment above was treated with decolorizing carbon. The filtrate was concentrated under diminished pressure. The residual mass on recrystallization from water afforded 34 mg of y-(3-pyridyl)y-oxobutyric acid, m.p. 163.5-164.5” (with decomposition), undepressed on admixture with an authentic sample.

CgHgN03 (179.17)


RESULTS AND DISCUSSION

As previously indicated (I), the study of the mammalian metabolism of y-(3-pyridyl)-y-oxobutyric acid retains a special interest since this keto acid appears to occupy a point of con- vergence for many possible routes in the metabolism of (-)- nicotine. Initial studies leading to the isolation of y-(3-pyridyl)- y-oxobutyric acid were performed with bacterial cultures (11). The precursor of the keto acid was considered to be 3-pyridyl y-(methylamino)propyl ketone. A variety of evidence points to the possible participation of intestinal flora and fauna in the metabolic disposal of nicotine by mammals (12, 13).

However, other routes to the keto acid appear to have an established role in mammalian metabolism. In a previous report (I), it was noted that administration of y-(3-pyridyl)-y-oxo- N-methylbutyramide, a mammalian metabolite of (-)-nicotine and (-)-cotinine, gives rise to the urinary excretion of y-(3- pyridyl)-y-oxobutyric acid.

The need for additional studies on the metabolism of y-(3- pyridyl)oxobutyric acid prompted our re-examination of synthetic methods for obtaining the compound. Although several methods (14,15) are available for its synthesis, the condensation of ethyl nicotinate and diethyl succinate (8, 9), to give the intermediate nicotinylsuccinate ester, which is readily hydrolyzed to the keto acid, was especially attractive for further study. The two starting materials are generally commercially available. By operating with a large excess of ethyl nicotinate (16), with sodium hydride as the condensing agent, self-condensation of diethyl succinate, which was previously noted as a limiting factor, was minimized, and diethyl a-nicotinylsuccinate was prepared in good yield. y-(3-Pyridyl)-y-oxobutyric acid was obtained by the conventional (8, 9) hydrolysis and decarboxylation of the diethyl cr-nicotinylsuccinate.

Synthetic +y-(3-pyridyl)-y-hydroxybutyric acid was obtained by reduction of y-(3-pyridyl)-y-oxobutyric acid under a variety of conditions. Virtually quantitative yields were obtained by

an alkaline reduction with sodium borohydride. Regrettably, preliminary attempts to resolve the hydroxy acid directly were unsuccessful and recourse was made to a resolution via its lactone, 5-(3-pyridyl)tetrahydrofuranone-2.

A reaction of L( +)-tartaric acid with ( -)-5-(3-pyridyl)tetrahydrofuranone-2 afforded a crystalline tartrate which was recrystallized to obtain a ( -)-5-(3-pyridyl)tetrahydrofuranone-2 (+)-tartrate in low yield. The free (-)-lactone, which was obtained as an oil from decomposition of the salt, was readily characterized as a levorotatory picrate.

Mother liquors from isolation of the levorotatory lactone tartrate were alkalinized to obtain a dextrorotatory-rich 5-(3- pyridyl)tetrahydrofuranone-2 as an oil. This oil, on treatment with D( -)-tartaric acid, yielded (+)-5-(3-pyridyl)tetrahydrofuranone-2 (-)-tartrate in low yield. The free (+)-lactone, which was obtained by decomposition of the salt, was characterized as a dextrorotatory picrate. The lower relative rotation of this enantiomorph indicated a lesser purity than that achieved in the isolation of the levorotatory form. The racemic and optically active forms served as reference compounds in the identification of the metabolic products.

After the oral administration of y-@pyridyl)-y-oxobutyric acid to male albino rats, the collected urine was processed with the aid of Dowex 50-H+ to obtain samples suitable for the paper chromatographic examination. An ammoniacal solution of acid components of the urine was thoroughly extracted with chloroform and subsequently chromatographed on paper (Table I). The RF values of the Koenig-positive components suggested, in addition to various unknown compounds, the presence of 3- pyridylacetic acid, Y-@pyridyl)-y-hydroxybutyric acid, y-(3- pyridyl)butyric acid, and unchanged y-(3-pyridyl)-y-oxobutyric acid. Administration of y-(3-pyridyl)-y-oxobutyric acid to the dog by the intravenous route led (Table I) to a similar type of excretion of Koenig-positive acidic components. The area on the chromatograms corresponding to 3-pyridylacetic acid and y-(3-pyridyl)butyric acid was, however, so low in Koenig-positive material or so contaminated with interfering material as to give a negative Koenig test.

Solutions of the partially purified acid fractions from both the dog and rat urine were treated with methanol-sulfuric acid. A paper chromatographic examination of the concentrated esterification mixture again indicated the presence of a variety of compounds. All of the pyridyl acids putatively present in the pre- esterification mixture, including y-(3-pyridyl)butyric acid in the case of the dog and rat, again evinced themselves, as adjudged by cochromatography with corresponding authentic methyl esters, except for y-(3-pyridyl)-y-hydroxybutyric acid, which lactonizes under the esterification conditions. The presence of the lactone was indicated by a Koenig-positive zone corresponding in RIF value to the authentic compound.

At the dosage levels employed throughout the animal experiments (including rabbits, rats, and dogs), there appeared, on the basis of intensity of colors observed in the various chromatograms, to be a high excretion of “unchanged” y-3-pyridyl-y-oxobutyric acid. The necessity for preliminary purification of urine and the possible chemical reaction by solvent systems employed in the chromatography could presumably have destroyed labile conjugates. This precludes reference to “unchanged” or “free” y-(3-pyridyl)-y-oxobutyric acid without some qualification. Similar limitations apply to Y-(3-pyridyl)-y-hydroxybutyric acid.


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3988 Metabolic Formation of -y-(%Pyridyl)-y-hydroxybutyric Acid Vol. 239, No. 11

The isolation and purification procedure employed on rat urine after administration of 8.55 g of y-(3-pyridyl)-y-oxobutyric acid led initially to the isolation of 3.0 g of an oil which appeared to be predominately methyl y+pyridyl)-y-oxobutyrate. On the basis of the crystalline methyl ester which was isolated from the oil and identified in comparison with an authentic sample, it is estimated that approximately 25% of the keto acid administered was excreted unchanged. In an experiment on the rabbit, in which the harsh conditions of the esterification procedure were avoided, the recovered urinary y-(3-pyridyl)-y-oxobutyric acid of analytical quality accounted for 17.5% of the administered keto compound.

is required for a quantitative interpretation. Urinary pH may significantly affect both the form and quantity of excretion with high acidity favoring production of the lactone form.

In view of the possibilities, as previously discussed (I), that the metabolism of y-(3-pyridyl)-y-oxobutyric acid proceeds by a route, keto acid + Y-(3-pyridyl)-y-hydroxybutyric acid, 4-(3-pyridyl)-3-butenoic acid + y-(3-pyridyl)butyric acid -+ 3-(pyridyl)acetic acid, in analogy to the metabolism of y-phenyl- y-oxobutyric acid (5, 17), major emphasis was given to procedures which led to an isolation and characterization of y-(3- pyridyl)-y-hydroxybutyric acid, the first intermediate in the series.

Restrictions must apply also to an interpretive discussion of the isolation of ( -)-5-(3-pyridyl)tetrahydrofuranone-2 from the urine of the rat and the dog in contrast to the isolation of (h)-5- (3-pyridyl)tetrahydrofuranone-2 from the urine of rabbits after administration of y-(3-pyridyl)-y-oxobutyric acid. In all cases, the urine may contain a mixture of racemic and levorotatory Y-(3-pyridyl)-y-hydroxybutyric acid, with a high preponderance of racemic material in the rabbit leading to the isolation of the racemic picric acid salt. Conversely, a preponderance of the levorotatory isomer and the greater insolubility of the picrate of the latter would lead to the isolation of the optically active material from rat and dog urine.

The administration of a total of 8.55 g of keto acid to a group of six rats resulted in the urinary excretion of y-(3-pyridyl)-y- hydroxybutyric acid that was obtained in the form of 140 mg of crude lactone. The crude la&one was processed to obtain 16 mg of purified picric acid salt of (-)-5-(3-pyridyl)tetrahydrofuranone-2. The administration of y-(3-pyridyl)-y-oxobutyric acid to the dog was similarly followed by urinary excretion of optically active Y-@pyridyl)-y-hydroxybutyric acid, which was characterized in the form of the picric acid salt of the (-)-lactone. The analytical sample of this salt accounted for 8.1% of the administered keto acid. A levorotatory lactone was also obtained after oral administration of the keto acid to the dog. Identification of the lactone was accomplished by the physical constants of the picrate, which had an infrared spectrum (Fig. 1) which compared favorably with the synthetic compound.

To the foregoing must be added the fact that there are no systematic studies on the racemization of y-(3-pyridyl)-y-hydroxybutyric acid. In this connection, it should be noted that the methanol-sulfuric acid reflux conditions in one of the rabbit studies are comparable, except on the basis of time, to those in an experiment with the dog. The severity of the conditions in all cases may, be conducive to racemization; there is some evidence on the basis of analogy with other lactones (18) that the lactonization may in part proceed with inversion of configuration. Since the methanol-sulfuric acid treatment of the acid fractions from urine was performed over a period of 9 hours for the rabbit and extended to 16 to 18 hours for the rat and the dog, it may be tentatively concluded that, even in the presence of possible racemization, the proportion of levorotatory y-(3-pyridyl)-y-hydroxybutyric acid to the racemic form is greater in the dog and rat than in the rabbit. The presence of nonartifactual racemic form in rabbit urine is thus also indicated.

SUMMARY

Administration of T-(3-pyridyl))y-oxobutyric acid to the rabbit led to the urinary excretion of unchanged y-(3-pyridyl)-y- oxobutyric acid. Paper chromatographic evidence suggested the presence of y-(3-pyridyl)-y-hydroxybutyric acid, 3-pyridylacetic acid, and y-(3-pyridyl)butyric acid. In one experiment, in which y-(3-pyridyl)-y-oxobutyric acid was isolated in analytical purity from the urine, the total recovery amounted to 17.4~~ of the administered dose. In three separate experiments, y- (3.pyridyl)-y-hydroxybutyric acid was isolated in the form of the lactone picrate. In one case in which conversion of hydroxy acid to lactone was accomplished by the methanol-sulfuric acid method the purified picrate acid salt of the lactone accounted for 10.4% of the administered keto acid. In all cases, the isolated picrate showed no optical rotation and corresponded by melting point and mixture melting point to the racemic synthetic compound. The infrared spectrum (Fig. 1) compared well with the synthetic compound.

y-(3-Pyridyl)-y-oxobutyric acid, an intermediate in the bacterial and mammalian metabolism of (-)-nicotine, gives rise to Y-(3-pyridyl))y-hydroxybutyric acid after administration to the rat, the dog, and the rabbit. The hydroxy acid, which was prepared chemically by reduction of the keto acid with sodium borohydride, was converted to the lactone, 5-(3-pyridyl)tetrahydrofuranone-2, by methanol-sulfuric acid and then resolved in the form of (-)- and ( +)-tartrate salts. The resulting enanti- omorphs were characterized as the corresponding picrates. The synthetic levorotatory picrate corresponded to the product that was isolated from rat and dog urine. In contrast, the urine of rabbits yielded a racemic picrate of 5-(3-pyridyl)tetrahydrofuranone-2 that was identified in comparison with the synthetic compound. The evidence suggests that the ratio of optically active Y-(3-pyridyl)-y-hydroxybutyric acid to racemic acid in the urine of the rat and the dog exceeds that in the urine of the rabbit under the conditions of the study.

Although it would be attractive to assume that y-(3-pyridyl)- y-hydroxybutyric acid is excreted predominantly in the acid rather than the lactone form, a systematic study of the effect of pH on the equilibrium reaction

AcknowZedgments-We are grateful to The American Tobacco Company and the Tobacco Industry Research Committee for generous support of these investigations. The infrared data were obtained through the kindness of Mr. J. Scott Osborne, Jr., of The American Tobacco Company.

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and Edward R. BowmanHerbert McKennis, Jr., Sorell L. Schwartz, Lennox B. Turnbull, Einosuke Tamaki

Possible Intermediary Role in the Mammalian Metabolism of Nicotine-hydroxybutyric Acid and Itsγ-(3-Pyridyl)-γThe Metabolic Formation of

1964, 239:3981-3989.J. Biol. Chem.

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The Metabolic Formation of y - (3-Pyridyl) -y ...reaction and then with successive 500-ml portions of methanol- ether: 5, 10, and 15% methanol by volume. The eluate (0 to 15% methanol)