Urinary Metabolites of 7-Chloro-1-(2-diethylaminoethyl)-5-(2

J. Med. Chem. , 1968, 11 (4), pp 770–774. DOI: 10.1021/jm00310a608. Publication Date: July 1968. ACS Legacy Archive. Cite this:J. Med. Chem. 11, 4, ...
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July 1968

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OF BN r \ T 1 - 1 ) I E T H Y L A R 1 I N O E T H Y L I ) E N Z O ~ I A ~ E ~ l ~ ~

(at a final concentrat,ion of 1% v/v), then readjusted to pH 7.0, and reextracted with two equal volumes of EtOAc. This EtOAc extract (treated as above and brought t’o 1 ml with E t O H ) was designated “EtAc A.G.” and contained those metabolites which were originally present in the urine as glucosuronic acid and/or sulfate conjugates. The extracted metabolites were separated by tlc on a fluorescent silica gel (Camag Kieselgel DF-5). The following solvent syst.ems were used: 1, EtOAc-EtOH-concentrated NH3 (90: 10:0.3); 2, heptane-CHC13-EtOH-concentrated NH3 (50:50: 2 5 : 1); 2a, heptane-CHCl3-Et0H-concentrated NH3 (50: 50:20: 1); 3, i-PrOH-concentrated KH3 (100: 1); 4, ?.Ie2CO-CHCI3concentrated NH3 (50:50: 1);5 , EtOAc-Et’OH-concentrated NH3 (90: 10: 1 ) ; 6, heptane->Ie&O-EtOH-concent,rat,ed PI”, (50: 50:25: 1);7, CHC13-31e2CO-concentrated NH3 (80:60: 1);and 8, C6H6-EtOhc-EtOH-concentrated XHa (80:20: 20: 2). Solvent, system 1 was the initial system used to separate the met,abolites. The migration of I and of related compounds is shown in Table I. Detection of I and metabolites was accomplished by spraying t,he plate with a modified Dragendorff reagent7 which was prepared by mixing equal volumes of a solution of 1.7% (w ’v) bismuth nitrate in 20% AcOH with 40%, (w/v) aqueous solution of KI and diluting the mixture with 4 vol of 10% H2S04. For isolation purposes, greater aliquots were streaked across large ‘LO X 20 cm chromatoplat,es and after development’ in system 1 a narrow vertical channel on each plate was sprayed with the Dragendorff reagent. Those bands seen under short,-wave uv light which were Dragendorff positive were scraped off, eluted with EtOH (5-10 ml twice), and concentrated. The tlc (in different, solvent systems) and elution of Dragendorff-positive bands was then repeated twice with each concentrate. Mass Spectrometry.-The preparation of samples for introduction into the Consolidated Electrodynamics Corp. 21-110 mass spectrometer and a brief description of how the high-resolution mass spectral data were obtained have been reported.4

Results and Discussion Human Urinary Metabolite.-Tlc in system 1 of the ether, EtAc B.G., and EtAc A.G. extracts of pre- and posttreatment urine gave one clear-cut indication of a metabolite: the presence of IV in the EtAc A.G. extracts of both subjects. This ethyl acetate extract of the posttreatment urine of subject B was used for isolation and further identification of this metabolite. Tlc systems 2 , 4 , and 5 were used serially for the purification of Dragendorff-positive metabolite which in each system migrated as authentic IV. High-resolution mass +pectral analysis of this metabolite (Table IV, see last column labeled Human) yielded a molecular composition (C17H14FClS202)which differed from that of I by -C4H& 0 and indicated that the diethylamino group in the side chain had been replaced by a hydroxyl group ; the loss of CH,OH to form the m / e 301 fragment and loss of CZHdO to form m/e 285 supported this conclusion. Finally, the mass spectrum of this metabolite was identical with that obtained with IV and on two-dimensional tlc (system 6 followed by system 3 ) ; the metabolite and IV did not separate but migrated as one compound. It was therefore concluded that Ia xas metabolized to IV which was excreted as a glucosuroriic acid and /or sulfate conjugate. An aliquot of EtAc A.G. extract of subject A posttreatment urine was chromatographed in system 1 alongside graded amounts of IV. Visual comparison of the intensity of the Drageridorff reaction of metabolite and btaridards led to the estimation that roughly

+

(7) This reagent, developed I)y R . Colarusso, E. Heylweil, a n d B. Z. Jenkowski, Analytical Research Laboratory, Hoffmann-La Roche I n c . , becomes more sensitive as it ages. .ifter 3-4 weeks it detected 1-2 ~g of I a n d analogs a s rust red spots o n a light yellox. hackground.

TLCMIGR.ITION OF I

.\NU

TABLE I RELATED CUMIWVNL)~ IN SYSTEM 1 Kr

Compounda

’i-Chloro-l-(2-diethylaminoethyl )-5- (2-fluoropheny1)1,3-dihydro-PH- 1,4-benzodiaxepin-2-one (I)

0 5O

CH CH.KH-C,H,

7-Chloro-1,3-dihydro-l-(%ethylaminoethyl)-b-(2fluorophenyl)-2H-1,4-benzodiazepin-Bone (11)

1 -

6;

a N - Y C H 2 CI

0 OD

7H.CH.X”.

l-(2-A1ninoethyl)-’i-~hluro1,3-dihydro-5- (2-fluuropheny1)-2H-l,4benzodiazepin-2-one (111)

0.09

@ FH,CHIOH

i-Chloro-l-(2-(hydroxyethyl)5-(2-fluorophenyl)-1,3-dihydro-2H- 1,bbenzodiazepin-2-one (IV)

0 il

7-Chloro-5-(2-fluorophenyl)1,3-dihydro-2H- 1,Pbenxodiaxepin-2-one (V)

0 hS

a -4s reference compounds for tlc the dihydrochlorides of I, 11, and I11 and the monohydrochloride of IV were used. Ia represents the dihydrochloride of I.

25% of the 60-mg dose of Ia first day as conjugated IV.

wab

excreted during the

Urinary Metabolites of the Chronically Treated Dog.--Aliquots of the ether, E t h c B.G., arid EtXc A.G. extracts prepared from 50 ml of urine from a control dog and one treated for 6 months with Ia I\ ere chromatographed in system 1. As seen in Table I1 there were four definite metabolite bands, two in the ether extract and two in the EtAc A.G. extract. Band 3 and band 4 components of the EtXc A.G. extract were each separated into a number of Dragendorff-positive components (Table 111). Band 3 yielded 3A, 3B, and 3C; and 3C migrated as IT’. Band 4 yielded 4A1 (which migrated us 111),4Az, 3B (which migrated ax 11),arid 4C. T.ABLE I1 I ) I ~ . ~ ~ ~ : . ~ ~ o I L F F - ~SUBST~.INCES ’ o Y I T I v E I N 1Sx:~11.1~12 OF UI~INE FROM A 1)OG CHROKICALLY TREATED \YITH 1.4 WHICH WERE ABSENTFROM COKTROL DOGURINEEXTR.ACTS Crine extracta

Ether

Dragendorff-positive bands on tlc in system 1 Designation Rt

Band 1 O..il Band 2 0 . 15 EtAc B.G. None E t A c A.G. Band 3 0.i6 Band 4 0.16 a Preparation of extracts is described in the Experinient,al Section.

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r17io ('wxitial data obtaiiied from high-reiolutiori of the\c isolated fraction5 arc - ] ) c v m l l :ln:ll) ~ ) i * c w ~ i t iei di Tahlc V, 3-1 uid 313 had the \aii~(' iiiolcc~ri1:ir coinposition (at 771 e 304) which indicated that t tic critirc S-1 side cliairi of I had been replaced h;

lll:l\h

:L hydrogen :uid one oxygen had been added t o t h r rcni:uiider of the molecule. However, the fragmentation Imtteriis of Xi arid 3B differed. \JTitli r c i l m t to 3-1,t h e prcsence of the m c 24s fragment ( i c c footnottl c of Table IY) and tlie -\I - CHJ tr:igmciit (nhicah iiiclicatey the retention of botli 11) d i v g ( ~ ~ati ~('-:3 of 1,4-he1izodinzepiiie~~~~) togcthcr witli the a h ~ e i i c eof \ I - H L O strongl). sliggested :I 1 ) I l C ~ l l O l l Cstrllctul~c.

VI1

E'rom Table I11 i t I - evi(hit that 4,12 was isolutc(1 t roiii 4.1, the ma,jor ronipoiwiit , whj rh in otily migrated A- 111 brit \v:i? clearly scp:ir:ttetl from N'. Tht- suggested t1i:it I7I1 m g l i t he fornietl during t l i c t lc purification 1)). tlehytlratiori of 111. This spec.\ iiiorcb cicfiiiitc .tructurc n:i- oht:iiiird lor :{I3 ulation was supportcd by further tlc experiments; I11 twc:iuw the preaeiicc of the m , e 232 u r d -\I - HrO fragments indicated the oxygen was added at C-3. as the free base, arid to u much lesser extent as the This compound, 7-chloro-5-(%-fluorophe1iyl)-3-h~~-dihydrochloride salt which was used as reference compound (Table I), yirlde adily detectable amounts of droxq-1-1,3-dihydro-2HH-l ,4-benzodiazepin-2-one (VI), tciiis used for purificatioii of \\:is suhsequeiitly i> iitheiisetl' a t i d t l i v in: T 3 1 on tlc i i i tlic. solveii band 4. Therefore. VI1 is much more likely to have :$l3 and VI were idelltical. heen an artifact produced from 111 than :t metatbolitc T h c 1 1:ist conil)orient, :{C, exhibitrd :i 1iio1ecul:ir ioii origindlj presciit iii 1):iiid 4. : t i i d fr:igiuciit:itioti p i t t w i i idciitiwl with that oi t l i c s

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July 196s

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‘Uie et her-ex trac t able I>ragendorfY-poait i ve component>, bands 1 and 2 (Table 11),were characterized with little difficult>.. Rand 1 nhich migrated :i\ intact I (compare Rfwith that of I 111 Table I) was found to ~clcwticnlwith I b:, further tlc imd b:, high-reqolutioii n i m h spectrometr> (Table I\r). Band 2 . 011 purificatioii by tlc, yielded :t pattern of metabolite. (and artifact) which \vas almost i t i i exact duplicate of that obtuincti n ith bniitl 1. Component. 2Al. 2.1?. 9 3 , a t i d 2C :itialogou> to the t i a i i d 4 conipoiieiiti (Table 111) \rere obtained arid each component \\as found tiy high-reholution mass spectromctr?. (T:iblc 117)to he itlentic:il with the itnalogouh band 4 comporienl. F’urthcrmore, components :ind 2C \yere more re:dily identified :is TI1 becau+e the:,. (lid tiot cont:iiri ttic (v&meouL: uz/ e 302 fragment. I t i h evideiit from the ahovc that rioncorijugatetl 11, 111, :11ic1 intact drug (I) \\ere excreted in the urine. h

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Acknowledgments.-We are itidcbted t o I l r . I{. 1.: Bagdon for hupplying u, \\ ith the dog uriric u-ed 111 t he\(> studies and to I)r. H. I’occliiiki) and Ilr. \T. ,J. I lor for the huniati urine. We :ire :d.o iiidebtetl to 1Ir.. A . Goetz for operatioil of the mas. ypectroiiicter.

Quinazolines and 1,l-Benzodiazepines. XL.

The Synthesis of Metabolites of ?-Chloro-l-(2-diethylaminoethyl)-5-(2-fluorophenyl) 1,3-dihydro-2H-l,4-benzodiazepin-2-one

-

The syiihe-is of :t n i u i i h of compuunds related to the hypnotic., 7-(~hlo~o-I-~‘L-dicth~lariliIloethJ.l)-~-(~-flllori)~~tieiiyl)-l,3-dihydro-~R-1,4-benzodiaaepin-%-one (2), is reported. These compoiinds were prepared as potential metabolites and many were found to he identical with the metabolites isolated and discussed in t,he preceding paper.2 It1 cotitiectioii with the metabolic studies of the hypnotic, i-chloro- 1-(2-diethylaminoet hyl) -3- (2-fluorophen:,l)-1,3-dihydro-2H-l,4-benzodiazepin-%-otie (2).’ diqcwsed in the preceding paper,2 we have syrit heaized a number of related compounds designed a s possible 111 vivo and/or in vitro metabolite.. B:,mean\ of direct comparisori or by a comparison of mabs bpectra and the use of tlc techniques, many of these derivatives n-ere showti b:, Schwartz, Vane, :tnd l’ostma2 to be identical with the metabolites of 2. Some of these compounds were synthesized after their initial tentative identification by an interpretation of mass spectral data while others were prepared hascd oii our knowledge of the metabolism of other l,~-beiizodi:tzepiries(e.!/., diazepam is known to yield a 3-11)tlroxy derivat h e 4 ) . Tlic .\ nthesia of tlic riiorioet1iyl;imino cierivative carried o u t b:, a voti Braun degradation of the iitle chain of 2. Thus treatment of 2 with cyanogen I I j I’auer S S S I S : .\I. 1%:. Ueriea, it. 1. Fryer, a n d L. ti. Sternbacii. J . . V e d . Chem., 11, ‘312 (1868). ( 2 ) A l . .I. Sclinartz, Y. Vane, a n d E. P u s h a , ibid., 11, i T O (1968). (: lene oxide. Another c o m p o u d .yiithesized as a possiblv n i t s tabolite wa5 the aminoeth:, 1 derivative 8. A2g:tintlic unalkylated compound 1 \va> used a> t h e startiiig material arid \\-as treated firbt with .odium mc~thoxidc iiiid the11 with cttrbohenzox:, bromocthy1;iinitie to givcL 7. Compound 7 wab then treated with a +elution ot HBr in g1:icial acetic acid to give the free ;tiiiii~o derivative 8. The dehydration product 16 fornicd t)> heating 8 uritler reflux in ethanol’ was found :is :111 artifact of 8 in the metabolic studies carried out h> Schwartz and l’oqtma.? T h e %hydroxy eonipourid (12) was piepared 111 t h e coiiventiorial niatiner from 10 by a Polonovdii rv:trrangement of the S-oxide to give 11 which ~ i i . \ubeeyuently hydrolyzed to 12. (’ompouiid 10 way \jilthesized from 1 in two bteps. In the fir.t itc’p. 1 \vas oxidized with peracetic acid to g i w the riitrotirb 9, ; l i d in the wcoiid z t i y 9 \\as alkylated uia tlic -od~o