Septeniber, lY(i4 Isolation and Characterization of Urinary Metabolites of Benzquinamide and Benzquinamide Alcohol B. KESKETH KOE ASD REX PINSON, JR. llfpdzcnl Rwrnrch Tdorntorirs, C'hns. Pjizpr R. Company, Tnc., Groton, Conn~cticirt Rereieed Aprd 17, 1864 Unchanged benzquinamide and six crystalline metabolites isolated from the urine of dogs treated with benz- quinaniide were characterized. Five other metabolites were recovered as (#rude preparations and related by chemical transformations to the crystalline met'abolites. Evidence for six more met,abolites of unknown structure was obtained. The chemical nature of the metabolites demonstrated that benzquinamide is metabolized by at least three pathways: (a) 0-deacetylation, (b) 0-dernethylation and conjugat,ion, and (c) N-dealkylation (one and two groups). The same series of 2-acetoxy and 2-hydroxy metabolites was formed from benzquinamide in both man and the dog. A corresponding series of 2-hydroxy metabolites was formed from benzquinamide alcohol in man. Conjugates in the urine were demonstrated by enzymatic hydrolysis or acid hydrolysis to 0-demethyl metabolites. In either the dimethoxy series or the conjugated 0-demethyl series, the K-deethyl compounds ap- peared to be the major metabolites in the urine. Benzquinamide,' 2-acetoxy-3-diethylcarbamoyl-9,10- dimethoxy-l,2,3,4,G,T-hexahydro-1lbH-benzo [alquin o- lizine (l), is a new berizoquinolizine conipound? of interest as a psychotherapeutic agent.3 Xs a part of a study of the metabolism of benzquin- amide,4 the chemical nature of the urinary metabolites formed in man and in the dog was determined. The human urinary metabolites of the pharniacologically active benzquinaniide alcohol (2) were also investi- gated. The exaniination of paper chromatograms of crude extracts of various uriiies collected from human sub- jects or from dogs treated with benzquinamide disclosed a number of zones, the intense fluorescence of which indicated that these substances were metabolites of benzquinaniide (Fig. 1-4, Table I). Earlier observa- tions from the chemical synthesis of benzquinaniide and related compounds2b had shown that the color of the characteristic fluorescence developed by these com- pounds on extended exposure of their paper chroma- tograms to ultraviolet light5 could be correlated with the functional group at C-2: yellow fluorescence for 2-acetoxy compounds and blue fluorescence (slowly turning yellow) for 2-hydroxy compounds. The paper chromatograms of extracts of benzquinaniide urines contained both yellow-fluorescent and blue-fluorescent zones, suggesting the presence of 2-acetoxy and 2- hydroxy metabolites, while extracts of benzquinaniide alcohol urines exhibited only blue-fluorescent (2- hydroxy) metabolite zones. Methanolysis (deacety- lation) and acetylation experiments carried out on crude extracts or on partially purified fractions (Table (1) Quantrilm. (2) (a) K. F. Finger, A. Weissman. and J. R. Tretter, The Pharmacologist, 3, 75 (1961); (b) J. R. Tretter, J. G. Lombardino, K. F. Finger. and A. Weissman, Abstracts of Papers, 140th National If eeting of American Chenii- cal Society, Chicago, Ill., Sept., 1961, p. 40: (c) A. Weissman and K. F. Finger, Biochem. Pharmacol., 11, 871 (1962): (d) .I, Scriabine, A. Weissman, I<. F. Finger, C. S. Delahunt, J. W. Constantine, and J. A. Srhneider, J. Am. .Wed. Assoc., 184, 276 (1963). J. E. Overall, L. E. Hollister. J. L. Bennett, Jr., J. Shelton, and E. 31. Caffey, Jr., Current Therap. Res., 6, 335 (1963). (4) E. H. Wiseman, E. C. Schreiber, and R. Pinson, Jr., T~P Pharmacolo- gist. 4, 156 (1962); Biochrm. Pharmacol., in press. (5) Subsequent photodecomposition products formed by the benzoquino- liaine zones on the paper chromatograms were responsible for the visible fluorescence color observed. Compounds 1, 2, 3, 4, 6, and 6 all exhibited the same type of fluorescence spectrum (uncorrected fluorescence A,,, 330 mp. activation Amax 280 mp in 0.1 HCI) as tetrabenazine [G. P. Quinn, P. A. Shore, and B. B. Brodie, J. Pharmacol. EzptZ. Therap., 127, 103 (1959)l. (3) M. E. Smith, Diseases A'eruous System, 24, 116 (1963): 11) followed by paper chromatographic comparison of the products and starting materials established the relationship between several pairs of yellow- and blue-fluorescent zones as the C-2 acetates and alcohols, respectively, of the sanie benzoquinolizine moieties. Similarly, methylation experiments with diazomethane followed by paper chromatographic comparison of the products with untreated samples related some phenolic zones (located in the chromatograms by their color reaction with p-nitrobenzenediazoniuni reagent) to certain fluorescent zones, demonstrating the occurrence of several pairs of metabolites differing in the degree of 0-methylation. The fractionation of the chloroform extract of alkalinized urine from benzquinamide- treated dogs by a combination of column chroma- tography and preparative paper chromatography led to the isolation of crystalline unchanged benzquinaniide (1) and six crystalline metabolites, 2, 3, 4, 5, 6, and 10. Rz CH ,O CON( I K3 OR I Characterization or identification of these compounds depended on elemental analysis (when possible), on ultraviolet and infrared absorption spectra, and on paper chromatographic comparisons with synthetic compounds. Five 0-deniethyl metabolites-7,8,9, 11, and 12-were not isolated in the pure state but could be readily detected in paper chromatograms and chemi- cally related to the corresponding dimethoxybenzo- quinolizines (A) by the methylation experiments nien- tioned previously. Three pairs of unidentified zones possessing the same type of fluorescence as the A com- pounds are considered to be drug-related metabolites, because methanolysis converted three yellow-fluorescent zones (a, c, e) to slower moving blue-fluorescent zones
Transcript
Septeniber, lY(i4
Isolation and Characterization of Urinary Metabolites of Benzquinamide and Benzquinamide Alcohol
B. KESKETH KOE ASD REX PINSON, JR.
l l fpdzcnl Rwrnrch Tdorntorirs , C'hns. Pjizpr R. Company, Tnc., Groton, Conn~cticirt
Rereieed A p r d 17, 1864
Unchanged benzquinamide and six crystalline metabolites isolated from the urine of dogs treated with benz- quinaniide were characterized. Five other metabolites were recovered as (#rude preparations and related by chemical transformations to the crystalline met'abolites. Evidence for six more met,abolites of unknown structure was obtained. The chemical nature of the metabolites demonstrated that benzquinamide is metabolized by a t least three pathways: (a) 0-deacetylation, (b) 0-dernethylation and conjugat,ion, and (c ) N-dealkylation (one and two groups). The same series of 2-acetoxy and 2-hydroxy metabolites was formed from benzquinamide in both man and the dog. A corresponding series of 2-hydroxy metabolites was formed from benzquinamide alcohol in man. Conjugates in the urine were demonstrated by enzymatic hydrolysis or acid hydrolysis to 0-demethyl metabolites. In either the dimethoxy series or the conjugated 0-demethyl series, the K-deethyl compounds ap- peared to be the major metabolites in the urine.
Benzquinamide,' 2-acetoxy-3-diethylcarbamoyl-9,10- dimethoxy-l,2,3,4,G,T-hexahydro-1lbH-benzo [alqui n o- lizine (l) , is a new berizoquinolizine conipound? of interest as a psychotherapeutic agent.3 Xs a part of a study of the metabolism of benzquin- amide,4 the chemical nature of the urinary metabolites formed in man and in the dog was determined. The human urinary metabolites of the pharniacologically active benzquinaniide alcohol (2) were also investi- gated.
The exaniination of paper chromatograms of crude extracts of various uriiies collected from human sub- jects or from dogs treated with benzquinamide disclosed a number of zones, the intense fluorescence of which indicated that these substances were metabolites of benzquinaniide (Fig. 1-4, Table I). Earlier observa- tions from the chemical synthesis of benzquinaniide and related compounds2b had shown that the color of the characteristic fluorescence developed by these com- pounds on extended exposure of their paper chroma- tograms to ultraviolet light5 could be correlated with the functional group a t C-2: yellow fluorescence for 2-acetoxy compounds and blue fluorescence (slowly turning yellow) for 2-hydroxy compounds. The paper chromatograms of extracts of benzquinaniide urines contained both yellow-fluorescent and blue-fluorescent zones, suggesting the presence of 2-acetoxy and 2- hydroxy metabolites, while extracts of benzquinaniide alcohol urines exhibited only blue-fluorescent (2- hydroxy) metabolite zones. Methanolysis (deacety- lation) and acetylation experiments carried out on crude extracts or on partially purified fractions (Table
(1) Quantrilm. (2) (a) K. F. Finger, A . Weissman. and J. R. Tretter, T h e Pharmacologist,
3, 75 (1961); (b) J. R. Tretter, J. G. Lombardino, K. F. Finger. and A. Weissman, Abstracts of Papers, 140th National If eeting of American Chenii- cal Society, Chicago, Ill., Sept., 1961, p. 4 0 : (c) A . Weissman and K. F. Finger, Biochem. Pharmacol., 11, 871 (1962): (d) .I, Scriabine, A. Weissman, I<. F. Finger, C. S. Delahunt, J. W. Constantine, and J. A. Srhneider, J . A m . .Wed. Assoc., 184, 276 (1963).
J. E. Overall, L. E. Hollister. J. L. Bennett, J r . , J. Shelton, and E. 31. Caffey, Jr., Current T h e r a p . Res., 6 , 335 (1963).
(4) E. H. Wiseman, E. C. Schreiber, and R. Pinson, J r . , T ~ P Pharmacolo- gist. 4, 156 (1962); Biochrm. Pharmacol., in press.
( 5 ) Subsequent photodecomposition products formed by the benzoquino- liaine zones on the paper chromatograms were responsible for the visible fluorescence color observed. Compounds 1, 2 , 3, 4 , 6, and 6 all exhibited the same type of fluorescence spectrum (uncorrected fluorescence A,,, 330 m p . activation Amax 280 mp in 0.1 HCI) as tetrabenazine [G. P. Quinn, P. A . Shore, and B. B. Brodie, J . Pharmacol. EzptZ. Therap., 127, 103 (1959)l.
(3) M. E. Smith, Diseases A'eruous System, 24, 116 (1963):
11) followed by paper chromatographic comparison of the products and starting materials established the relationship between several pairs of yellow- and blue-fluorescent zones as the C-2 acetates and alcohols, respectively, of the sanie benzoquinolizine moieties. Similarly, methylation experiments with diazomethane followed by paper chromatographic comparison of the products with untreated samples related some phenolic zones (located in the chromatograms by their color reaction with p-nitrobenzenediazoniuni reagent) to certain fluorescent zones, demonstrating the occurrence of several pairs of metabolites differing in the degree of 0-methylation. The fractionation of the chloroform extract of alkalinized urine from benzquinamide- treated dogs by a combination of column chroma- tography and preparative paper chromatography led to the isolation of crystalline unchanged benzquinaniide (1) and six crystalline metabolites, 2, 3, 4, 5, 6, and 10.
Rz CH ,O
CON( I K3
OR I
Characterization or identification of these compounds depended on elemental analysis (when possible), on ultraviolet and infrared absorption spectra, and on paper chromatographic comparisons with synthetic compounds. Five 0-deniethyl metabolites-7,8,9, 11, and 12-were not isolated in the pure state but could be readily detected in paper chromatograms and chemi- cally related to the corresponding dimethoxybenzo- quinolizines (A) by the methylation experiments nien- tioned previously. Three pairs of unidentified zones possessing the same type of fluorescence as the A com- pounds are considered to be drug-related metabolites, because methanolysis converted three yellow-fluorescent zones (a, c, e) to slower moving blue-fluorescent zones
6:36 E. KENNETH KOE ANI) REX PINSON, JR. Vol. 7
d
ismide metabolitcs i n syslr i i i I i'i p:isscs). ( ~ l h r c ~ f ~ m ~ ~ s d u l i o n applied at A, extract l , , , , , , : , , , ~ l ~ ~ , , ~ ~ i , ~ i , , : ~ , ~ ~ i , l ~ urine: at I $ cxtrart nf human-benzquinninirie ; ~ I v c h l urine; a t C, est,rnct of dog- benzquinamide urine; rel:ibive R r vnlues: a, 1.00; 1, 0.61; 2, 0.42; 7, 0.14.
(b, d, f, respectively), a transformation associated with deacetylation at C-2. The major metabolites observed in extracts of human- and dog-benzquinamide urines were essentially the same except for differences in the minor unidentified fluorescent zones.
a hensquinamide ( 1 ) - benzquinamide alrohol (2)
h/ 1 s cl'<
7( f - 4 8
The metabolites identified in the urine deliionstrated that benzquinamide is metabolized by a t least three pathways: (a) deacetylation of the 2-acetoxy group, (b) demethylation of the 9- or 10-niethoxy group and conjugation, arid (c) deethylation of the 3-diethyl- carbamoyl group.6 The N-deethyl compouuds ap- peared to he the major urinary metabolites with the benzoquinolizine structure in either the dimethoxy series (A) or in the conjugated 0-demethyl series (B).' (6) A number of urinary metaholitea and oonjuwtes have been reported
for tetrabenazine by A. Pletaoher, A. Rrorsi, and K. F. Gey [Intern. RPU. NeuroUol.. 1, 275 (1962)l. O ~ l y two rnetaholitea. the two epimeric aloo- hols from reduotion of the 2-keto protip, have been identified by eom~srison with synthetic compounds. EEq-cH2cH(cH,,)2 0
tetrabenazine (7) Approximately 3% (range, 1.5-6.6%) of sn oral dose (100 to 300 mg.)
of benrquinamide in mania found sa unohanged d m e in the urine In. Cahn, L. S. Brahen, E. H. Wiaeman, and R. Pinson, J r . , Current Thcmp. Rea.. 6 . 301 (1963)l. The amount of met%b&te S excreted in the urine is estimated to he shout 3-6 timea that of unchanged drug.
'r.4Br.E I B E N Z Q U N A M I D E AND METABOr.rTES
((llms,ntogm,,l,ic: No. rolllpolln.i systems
I Benzquinamide I, 3 passes'; IT, 20 hr. 2 Benzquinamide aleohol I, 3 passes; IV, 20 hr. :3 N-neethylhensquinamide 11; V, 2.5 hr. 4 N-l)eethylbenaquinamide alcohol 11; V, 2.5 hr. 5 ~',?;-Risdeetlrylbenntluinamide 111, 3 passes; TI, 2.5
b Alcohol of a (2-hydroxy) I, 2 passes c Unidentified metabolite (2-are- V, 2.5 hr.
d Alcohol of c (2-hyroxy) V, 2.5 hr. e Unidentified metabolite (2- 111, 3 passes; VI, 2.5
aeetoxy) hr. f Alcohol of e (2-hydroxy) 1-1, 18 hr.
toxy)
" Systems, using formamide-acetone (2:3) treated paper: I, hexsn~bbenzenediethylsmine (27:9:4), formamide-saturated; 11, benzenediethylamine (9: l ) , formamidesaturated; 111, benzene-chloroform-diethylamine (13: 6: l), formamide-satu- rated; cf. D. Waldi, A x h . Pharn., 292, 206 (1959). Relative Rr values are given in Fig. 14. The following order of decreas- ing rate of migration wa8 observed in the diethylamine systems (I, 11,111): a, b, 1, 2, 7, 8, 3, 4, 9, 5, e, IO, 6, 11, and 12. Supple- mentary formamide-saturated systems, using pH 8 buffered paper: IV, hexane, 20 hr. (relative Rt values: 1, 1.00; 2.0.65); V, benzene, 2.5-4 hr. for 3 to 4 region (relative Rr values for 2.5- hr. Nn: 3, 1.00; 4, 0.35; 9, 0.15; 5, 0.11); VI, benzene- chloroform (1: l ) , 2.5-18 hr. for 5 to 12 region (relative Rr valulues for2.5-hr. run: 5, 1.00; 9,0.83; e, 0.46; 10 ,024; 6, 0.17).
One pass is defined a8 the time required for the descending sol- vent front to reach 20 mm. above the lower edge of the BUS- pended sheet, which was 40-cm. long.
Dealkylation from a carboxamide nitrogen is well documented in the special instance of imide nitrogen such as the dealkylation of K-alkylbarbiturates or the demethylation of 5-ethyl-3-methyl-5-phenylhy- dantoin.R Metabolic removal of alkyl groups from aliphatic tertiary amides have been described more recently; e.g., the demethylation in the rat of (-)- cotinine to (-)-demethylcotinines* and the monode- alkylation of N,N-dialkylcarbamate esters by rat-liver microsomes. gh The metabolic dealkylation sequence of tertiary amide + secondary amide -+ primary amide, as in the formation of 5 and 6 from benzquinamide, is not an unexpected series of transforoiations in view of the deethylation of N-ethylbenzilamide to benzil- amide in the dog.1°
The structures assigned to the X-deethyl metabolites, (8) R. T. Williams, "Detoxication Meohaniama," 2nd Ed., John Wiley and
(9) (a) H. MoKennis, Jr.. L. B. Turnbull, S. L. Sehwartr. E. Tsmaki, and (h) E. Hodgaon and J. E.
(10) J. R. Albert, 0. C. Borill, sod J. H. Weikel, Jr.. J . Pharmocol.
Sons, Ino.. New York, N. Y.. 1859, p p . 581, 604405.
E. R. Bowman, .I. B i d . Chew., S W , 541 (1962): Casida. Biochen. P h o m e o l . , 8 , 179 (1961).
ELIXI. Themp.. 1S1, 85 (1961).
TABLE 11 CHROMATOGRAPHIC FRACTIONS OF IhG-URINE HENEQUINAMll>E
METABOI.I.LE MIXTURE" voi- Mets-
Yrao- u*ne, Residue bolite ti"" 101. neveloper r t .? P. rontPot"
I 125 Sll%o CHCI, 11.09, oil iL
I1 42.5 hO% CHCls 0.74. oil 1, 2. :I. 1) . . 111 37.5 5&7.5% CHCl, 11.19; g. oil 1, 2 IV 500 75% CHCh 1.26, cryst. 3 Y 275 75% CHCI, 0.58 , oil :I, 4, i, S, c YI 1675 75% CHCla-lllO/, 0.77, oil 4, 7, s, (.
YII 2110 10%~ M ~ O H 0 7 5 , amorph. 5, 6, 0, 111, e VI11 250 15qZ MeOH 0.05, oil 5, 6, !J, 111.
11, 12, e IX 500 2040'% MeOH 0.05, cryst. 9, 10, 1 1 ,
12, origin
*Column (length, 46.5 cm.; diameter, 5.2 cm.): neutral Woelm, grade 111, alumina (395 g.); charge: benzene snlut.im (160 ml.) of crude metabolite gum (8 g.); developers: 0.5-1. volumes of 5, 10, and 25% chloroform-benzene; 1-1. volumes of 50 and 75% chloroform-benzene; 1 I. of chloroform; 0.25.1. volumes of 1,2, 10, 15,20, and 40% (twice) methanol-ohh,roforrn.
Reeidue weight after evaporation of the fraction and not the weight of metabolites in residue (except for 3). 0 The rdnnm cuts were examined for metabolite content by paper rhromatog- raphy in system 11, with additional resolution in other systems when necessary. The eluate up to 50% benzene-chloroform con- tained no fluwescent metahdites, while the remaining eluates (2.5ml. ruts) were rombined into fractions I to I X .
3 and 4, 011 the basis of elenrental analysis, niethoxyl group determination, and spectral data werc verified by deacetylating 3 to 4 and establishing the identity of the latter compound with totally synthetic N- deethylhenzquinamide alcohol.*b Corresponding phys- ical and chemical data support the structure, S,S- hisdeethylbenaquinatnide, for 5. Since 5 gave 6 on deacetylation, the latter is N,K-hisdeethylbenzquina- mide alcohol. The structure assignments for 7-10 based on the results of methylation and deacetylation experi- ments of the type mentioned earlier" were strengthened by the isolation of several crystalline methylation products, which were identical with authentic samples: 1 from 7 (fraction V), 3 from 9, 4 from 10. The phe- nolic metabolites are considered to he 0-inonodemethyl compounds for three reasons. (1) Dihydmxy deriva- tives, like the catecholamines, would be unstahle under the alkaline conditions used for extraction and paper chromatography. (2 ) The shift in Lx (280 to 295 mp region) in going from acid to alkaline solu- tion for 10 (shift = 7 mp; e in HCI: c in XaOH = 0.93) resembled that of 3-hydroxy4-inethoxyphenylethyl- amine (shift = 8 nip; e in HCI:c in NaOH = 0.91) hut not that of dopamine (shift = 11.5 nip; e in HCI: c in NaOH = 0.68). (3) A paper chromatographic comparison showed that 8 (enriched sample prepared by subjecting fraction V to niethanolysis) was the same as one of the two 0-monodeniethylhenzquinamide alcohols derived chemically from authentic bena- quinamide alcohol.'2 The detection of 0-demethyl compounds in extracts of untreated urines suggested
( I l l Since I (fraction VI - 1 and 8 -1 on methylation. while 1 - 8 and 1 - 1 on deacetylation. I and 8 *re 0-dsmBthylben.quinamids and 0- demethylbenvsuinsmide aloohal, respectively. Sirnilsrly, 9 - 3 and 10 - 1 on methylation, while 9 - 10 and 3 - 1 on deacetylation. Since atruotura of 3 and 6 have been determined, 0 and 10 me N-dFethyl-O-demethylbsn.- ouinamide and N - d ~ t h r l - o - d ~ m e t h y i b ~ ~ ~ q " i " ~ m i d ~ alcohol, respectively.
MeOH
Fig. 2.- I ~ N P S I . P I I I jmper ~ , l , ~ , , , , , : * ~ , , ~ ~ : , , , ~ Iwv.rluin;tmide Inetaholiles i i i s>sleir 1 1 . Si~iiie s,IIulioiis as iii Fig. 1 : Ilrvahtes: 2, ( ] . T i ; 7, l l , ( i~ l : 3,O..il: 4, Il.:N; 9, I l . IT : 5, 11.11: 6, 0.05.
the occurrencc of cotijugated nietabolites, the presence of which was coriaborated by the formation of B by either acid hydrolysis or enzyinatic hydrolysis of urines pie-extracted exhaustively with chloroforni at pH 10. The absence of heirzquinaniide alcohol in similar acid hydrolysatcs iiidicated that conjugation did not occur at the 2-hydroxy group. The intact conjugates of 9 and 10 werc dctccted in paper chroniatograins of con- centrated huiiiaii-betiz~~uinaiiiide urine hy acid hy- drolysis of the eluted zones to 10.
The rate of enzyiiiatic dealkylation of aromatic methyl ethers'3h is apparently increased by para electron-attractitig groups (-It, -I effects), which can enhance the clectroir-i,epelliiig effect (+R) of the aromatic incthoxy gro~ps. '~b Such a correlation would account for the greater rate of eiizyinatic dealkylation of p-0-methylcatechols, like 3-hydroxy-4-methoxy- acetophenolie, 3-hydroxy-4-inethoxyphenylmethylcar- hinol, paranephrine, and norparaiiephrine, as compared to the corresponding m-O-niethylcatechols,'Se as well as the iiietabolic dealkylation of the 6-methoxy rather than the 4-methoxy group in griseofuIvin.lad Both 0-niononiethyldopamines are demethylated in o i t m a t the same rate (ahout one-half that of paranephrine or norparaiiephrine),13c suggesting that the aniino- ethyl group has little influence. In contrast, attaching a p-aminomethyl group ( -I effect) to anisole increased the demethylation rate about e i g h t f ~ l d . ' ~ ~ Since the 9-methoxy group in the benzoquinolizine A series is part of a similar benzylamine system and would he
(12) J . R. Tretter. unpablishsd synthesis. The location of the phenolic OH in the two 0-monodernethylbenrquinamide alcohol8 (at G D or C-10) hsa not been established. For synthetic 6 the shift in bar from seid toalkaline solution is 8 r n ~ (e in HCI: c in NaOH = 0.8s).
(bl Theaubstituent ef- fect on thia oxidative 0-demethylation by liver miorosomes has a wsi i tat ive similarity to the effect of SubatituentB on nuoleophilie aromatio eubstitation (c. K. Ingold. "Structure and Mechanism in Or~anio Cherniatry," Cornell University Prea. Ithaca, N. Y.. 1913. pp. 802-8111. The reaultant partial poaitive charge on the ether oxygen could be signifioant in the meehaniem of dealkylstion by 0-demethylase I d . R. E. MoMahon. H. W. Culp, J . Milla. and F. J. Marahall, J . Mad. Chem.. 6, 343 (196.311. (01 J. W. Dab. J. Axelrod. and B. Witkop. J . B;oL Chem.. X35, 1115 (18601. (d) M. S. Barnes and 11. Ibothroyd, Biochem. J.. '76, 41 (19611.
(13) (a) S. Axelrod, Biocham. J.. 63, 634 (1866).
September, 1964 METABOLITES OF BESZQUIKAMIDE G30
2,3,4,6, 7-hexahydro-1 1 bH-benzo [a] quinolizine ( m. p. 148- 150 O ) prepared by Tretter.zb
B. From Urine&-Crystalline 4 was isolated: (1) from frac- tion V by preparative paper chromatography (system 11, 2 passes), 10-mg. yield, m.p. 146-149"; and ( 2 ) from human-benz- quinamide alcohol urine (Fig. 3) by the same method described in Table 11, 3-mg. yield from 21 l., m.p. 146-147". Both products proved to be identical with 4 from part -4 by paper chromatographic comparison, by comparison of infrared spectra, and by acetylation (acetic anhydride, concentrated H&Oa catalyst) to 3 (latter demonstrated by resolution of t,he reaction product on paper).
N,N-Bisdeethylbenzquinamide (5).-Metabolit,e 5 was present in human- and dog-benzquinamide urines but absent in benz- quinamide alcohol urine (Fig. 4). Fraction VI1 (260 mg.) was chromatographed in system 111 ( 3 passes) using 5-10 mg. per sheet of paper. The yellow-fluorescent 5 band was c u t out and stirred 20 min. with 0.01 S HCl (650 ml.). The eluate was extracted twice with 0.5 volumes of chloroform a t pH 10. The dried extract on evaporation gave a crystalline residue, which was recrystallized from ether; yield, 71 mg. For analysis a sample was recrystallized from acetone, m.p. 193-195" dec.;
284.j nip ( t 3380); vmbX (cni.-l): 3448, 3289, 3165 ( S H , OH), 1724 (acetoxy C=O), 1681 (amide C=o).
N,N-Bisdeethylbenzquinamide Alcohol (6j.-Crystalline 6 was prepared by treating 5 (10 mg.) with 1 AT methanolic NaOH ( 5 ml.) as described for 4; yield, 4 mg.; m.p. 182-185" dec.; Amax 285 mp ( e 3430); vmnx (cni.-l): 3448, 3333, 3183 (XH, OH), I667 (amide C=O). This deacetylation product of 5 was iden- tical paper rshromatographically with a blue-fluorescent meta- bolite present in extracts of benzquinamide and benzquinamide alcohol urines (Fig. 4).
0-Demethylbenzquinamide (7).-A solution of fraction V (350 mg.) in methanol (1 nil.) treated with ethereal diazomethane (0.24 J I , 90 nil.) was refrigerated overnight, kept 4 hr. at room temperature, and evaporated. The residue on paper chroma- tography (8 sheets, system I, 3 passes) exhibited zones cor- responding to benzquinamide and benzquinamide alcohol. Crystalline 1 was recovered from the former zone by eluting with 0.01 HCL; yield, 4 mg.; m.p. 125-128'; infrared spectrum identical with that of authentic benzquinamide. Spraying the paper chromatograms (system I1 j of unt,reated fraction V (1 and 2 absent,) with p-nitrobenzenediazonium solution and dilute Kaz- C08 revealed two brownish violet zones, 7 (major one), migrating slightly faster than 3, and 8, having approximately the same Rr as 3 (Fig. 2). Both phenolic zones were absent in the chroma- tograms of the diazomethane-treated solution, indicating that 7 and 8 are 0-demethylbenzquinaniide and O-demethylbenz- quinamide alcohol, respertively.
0-Demethylbenzquinamide Alcohol (8).-The content of 8 in fraction V was enriched by subjecating the latter to deacetyla- tion ( 7 +. 8) as described under 4. Treating a part of the meth- anolysis product with diazoniethane by the same method given in the preceding section demonstrated the conversion of 8 to 2. Paper chromatographic comparison with the two authentic 0- monodemethylbenzquinamide which differed in Rf value and the color produced with the p-nitrobenzenediazoniurn reagent (brownish violet ccilor for the faster zone, brown color for the slower zone), showed that, 8 was the same as the faster moving isomer.I7 Relative Rr values (system I, 16 hr.) are: metabolite 8 (violet zone), 1.00; synthetic 8 (O-demethylbenz- quinaniide alcohol, violet zone), 1.00; 0-demethylbenzyuinamide alcohol (brown zone), 0.60; 3, 0.88; 4, 0.20.
N-Deethyl-0-demethylbenzquinamide (9).--Spraying the Ixiper chromatograms (system 111, :3 passes) of fraction VI1 or of crude extracts of benzquinamide urines with the p-nitrobenzene- diazonium reagent disclosed two brownish violet phenolic zones, 9 (major one) and 10 (Fig. 3 and 4). Crude 9 isolated from Fraction VI1 ( 3 Y O nig.) by preparative paper chromatography (system 111, 3 passes) was treated with diazomethane, and rrys-
(17) Synthetic 8l2: m.p. 176-180°; A,,,,, 286 m p ( e 3840) in 0.01 .Y HCl- Xlnar 294 inp ( a 1060) in 0.01 .V NaOH-inetlianol; vmRX (cii i .-I): inethanol:
3378 (OH) , 1610 (amide C=O).
talline 3 (4 mg.) was recovered from the chromatograms (system 11) of the methylation product and identified by its infrared spectrum. For confirmation, part of the sample ( 2 mg.) was deacetylated to give 0.4 mg. of crystals, whose infrared spectrum was identical with that of 4. The formation of 3 from 9 indi- cated that 9 ita S-deethyl-0-demethylbenzquinamide. Meth- anolysis as described under 4 converted 9 to 10, as shown by resolution of the reaction product on paper. N-Deethyl-0-demethylbenzquinamide Alcohol (10). A. Acid
Hydrolysis of Urines.-The spent aqueous phase from the chloro- form extraction (pH 10) of dog-benzquinamide urine wm con- centrated via freeze drying t o 1.5 l., adjusted to pH 0-0.3 (con- centrated HClj, and heated a t 100" for 1 hr. These hydrolytic rwnditions deacet,ylated the 2-acetoxy compounds, but neither 2 nor 4 was demethylated by similar treatment with 0.6 AT to 1.2 AY HC1. The hydrolysate was washed with chloroform (1 volume), adjusted to pH 10, and extracted with chloroform (1 volume). The concentrated extract (10 nil. j, shown by paper chromatography to contain 8, 10 (predominant zone), and 12, was developed on a column of alumina (50 g.) with 100-ml. volumes of chloroform and 0.5%, 1 % (twice), 2vh, 5%, and 10% methanol-chlorofc,rm.'8 The 1 % methanol-chloroform eluate on concentrating to 5 nil. gave crystalline 10; yield, 54 mg.; m.p. 110-120" dec.; A,,, 287 mp ( e 3,580) in 0.01 HC1-meth- a n d ; A,,, 294 mp ( e 3870) in 0.01 .Y SaOH-methanol; vmax (cm.-l): 3521, 3333, 3279 ( S H , OH), 1637 (amide C=Oj. .4 second crop of 10 (90 mg.) was recovered from the mother liquor. In a similar manner 10 was also isolated from human- henzquinamide urine ( I 6 mg. from 22 1.) and from hunian- benzquinamide alcohol urine (7 mg from 21 1.).
Conversion to 4.-A solution of 10 (20 mg.) in methanol (0.5 ml.) treated with ethereal diazomethane (0.37 'If, 50 ml.) was chromatographed on paper (system 11). Eluting the band corresponding to 4 with 0.01 S HC1 and recrystallizing the prod- uct from acetone yielded 2 mg., m.p. 147-148"; infrared spec- trum identical with that of 4.
0-Demethyl Metabolites 11 and 12.-The combined 5 and 10% methanol-chloroform eluates from the column for 10 were evap- orated, and an aliquot of the residue was treated with diazo- methane. Examination of the paper chromatogram (system 111, 3 passes) of the product showed the formation of a new zone corresponding to 5 not present in an untreated aliquot and a more intense blue-fluorescent zone for 6. The untreated sample contained a strong phenolic zone (unresolved mixture of 11 and 12), which on the basis of the diazomethane reaction are X,X- bisdeethyl-0-demethylbenzqclinamide and S,K-bisdeethyl-0- demethylbenzquinaniide alcohol, respectively. lg Relative Rf values (system 111, 3 passes) are: 9, 1.00; 6, 0.23; 11 and 12, 0.13. The phenolic zone corresponding to the 11 and 12 mixture was also detected in the same manner in fraction VI11 (Table IT).
Conjugates of 0-Demethyl Metabolites. A. Enzymatic Hydrolysis of Urines.-The substrate was prepared by extracting human-benzquinamide urine (concentrated 4-fold) four times with equal volumes of chloroform a t pH 10 to remove free 0-demethyl metabolites, adjusting the aqueous phase to pH 5.2 with glacial acetic acid, adding penicillin G (1.2 mg./ml. as a preservative), and diluting to the original urine volume with pH 5.2 0.1 Af acetate buffer. Aliquots (50 ml.) were incubated a t 37' for 42 hr. with 0.6 ml. of Glusulase (Endo Laboratories, 100,000 units/ml. of glucuronidase and 50,000 units/ml. of sul- fatase) or 400 mg. of 0-glucuronidase (hlann Research Labora- tories, 60.000 units/g.) and extracted with chloroform (1 volume) a t pH 10. The residue from the evaporation of the dried ex- tract was chromatographed in system I1 and in system I11 (3 passes) with appropriate controls, including the extract of sub- strate incubated without enzyme. The fcirmatirin of 7-10 by enzymatic. hydrolysis was shown clearly on the paper chronia- tograms. As expected, diazomethane treatment of the crude rnisture of 0-demethyl metabolites liberated by enzymatic hydrolysis formed the six A compounds, as demonstrated by resolution of the niethylation product in systems I, 11, and 111. Metabolite 10 was also detected following enzymatic treatment of human-benzquinamide alcohol urine in the same manner.
B.
(18) The combined chloroform and 0..5% methanol-chloroform eluates froin this column on evaporation yielded a gum (80 ma.) containing 8.
(19) Acid hydrolysis of concentrated urines did not completely deacetylate the 2-acetoxy metabolites, accounting for some 11 in the untreated aliquot; the latter a h o contained a trace of 6.
Acknowledgment.-\Ye arc' giatefui to 111.. . \ I . . I . IAyiivh for devising paper chromatographic I h . I<. L. IVagiier. J r . \ mid 1 iiieasurenieiit s a i d rnicroaiia Jr. . fo r espelrt t ec+hiiicd as l'retter for samples uf syiithetir beiizotliiiiioliziiie t '(JIl1-
pouiids and helpful tlixcussioiis.
. . - .- . . . ..
Synthesis and Biological Evaluation of Water- Soluble 2-Boronoethylthio Compounds
toxicity, \ \e i ( ' iouiid aiiioiig those borouic acids Iiaviiig a high water-lipid solwiit partition cocf1cient.3' I )
Thus a low lipid solubility sccined to he ail iiiipoitaiit rquirciiieiit for a t~orui i coiiipound. -1s cliiiical iii- forination Ixcaiiic available. it was apparcwt that thr probleiii was iiiorc iiivolvcd than the nicrc attniiiiiioiit of a high tuiiior braiii horoii ratio. H t prcparatioii and clvaluatioii of a series of hi wlublc horoiiic acids pi'ewiit \\ol-k oii t hr. ha
Sy lit hc tic i i t hod-, h for the gciicbi a1 pi (>paratioil of alkylboronic acids voii-
taiiiiiig hydrophilic substituc.nts. Thc facilc radicsl- catalyzed additioii of iiieimptans to the double hoiid of dibutyl cthyleiiehoroiiate4 has been cxploitcd iii this present work t o synthcsizc. ~ w e r a l horoiiic acids con-
iwtioiial group-, iiiaiiy of wliicli l ~ a v o cd I\ atcr ~olubility. of it Icrc:tptoacc.t ic i~ i id $ - i t i ( > i rapt ( I -
piopioniv :wit1 t o dibutyl c.thyleiic.boi.oiiat(~ \\ i'i i'
y awoiiiplidid. I5ithcr ultraviolet light oi ' a z o - otlutyroiiiti ilc could b(> uscd to initiate th(. ic\ac+ioii
but itli additioiinl cxpc.ric.iicc, it appearrd that thc, axonitrilo gavv 11101 o c o n stoiit and better cciiitrol1c.d iesults. Tho initially fo led liquid adducts dwoiii- posed oii attempted dis llation and were therefore- hydrolyzed dirrctly l o tlw corresponding lmroiiic acids, ~--horoiiorth~ltliioacetic ( la) , and X-(2-boroiio
