CA1205089A - Unlabelled and radioactive labelled derivatives of butylamino propiophenone and their preparation - Google Patents

Abstract

ABSTRACT

Unlabelled and radioactive labelled compounds of formula:
wherein:
i) R10 is GH, R11 is a radioisotope and R12 is 0; or ii) R10 is H, R11 is C1 and R12 is a radical of formula:
wherein q is an integer from 1 to 3, and X1 is OH or a radical of formula:

Description

~L2~5C)~9 This invention relates to new unlabelled and radio-active labelle~ derivati~es o~ butylamino propiophenone and their preparation.
This application is a division of Canadian Patent Application S.N. 375,361, filed April 13, 1981.
The ?resent invention is primarily directed to a radioimmunoassay for clinical or experimental testing for the presence of and quantitation of bupropion [(+)-2-tert-butylamino-3'-chloropropiophenone], a pharmacologically active antidepressant compound, in biological fluids, including especially human sera or plasma.
The radioimmunoassay technique is finding increasing application for quantitation of drugs in biological fluids.
Monitoring of pl~sma concentrations of drugs enables more precise dose administration to ensure efficacy. In the hospital laboratory setting, specific radioimmunoassay methods can offer considerable advantages such as improved sensitivity and specificity and, particularly, greater speed and sample capacity, over the generally more laborious methods of gas chromatography or thin-layer chromatography.
Accordingly, it is a purpose oF the present invention to provide a radioimmunoassay procedure to determine the presence and concentration of bupropion in biiological fluids, especially human sera or plasma.
It is another purpose of the invention to provide suitable raJiolabelled agents for use in the raclioimmunoassay.

~2~i0~3 ,

- 2 -It is another purpose of the mvention ~o provide anki-bodies directed against bupropion also re~err~d to as bupropion-specific antisera and as anti-bupropion sera. In this reyard, it is a further purpose of the presen~ invention to provide suitable immunogens for use to raise bupropion-specific antisera, and to provide methods suitable for raising such antisera.
The radioimmunoassay of the present invention is based on cc~petition between bupropion and a fixed quantity of a dis-tinyuishable ccmpetitor of bupropion for a limited number of bind-ing sites on bupropion-specific antibodies. The distinguishable : competitor is preferably a labeled ccmpcu~d, such as a com~ound labeled with a radioactive isotope ("radiolabeled").
The drug and its radiolabeled competitor are added to the antibodies and the reaction mixture is allcwed to equilibrate.
When the antibody-kound portion is seFarated from the free drug and free radiolabeled competitor, then the amount of radioactivit~
present in the antibody-bound portion will be inversely related to the quantity of unlabeled drug originally added to the reaction muxtureO Thus, the greater the amount of unlabeled drug added, the less radiolabeled ccmpetitor will be boun~ to the antibodies.
Conversely, the amount of radioactivity present in ~he reaction mixture from which the antibody~bound portion has b en removed will be directly related to the quantity of unlabeled drug originally added to the reaction nuxture; i.e. the greater the amount of unlabeled drug added, the more radiolabeled ocmp~titor will remain in khe reaction mixture rom which the antibody-bound portion has ; been removed.

A standard curve can be prGduced by employing a range .~

s~l - ~ -of known concentrakions of unlabeled drug in a series of reaction 7~xkures while holding constant the amount of antikc~ly and radio--- labeled campetitor ~mployed~ ~he standard curve allo~7s the concen-tration of drug in an unknown sample to be interpolated frcm the amount of radioactivity present in the ankibody-bound portion of the equilibrated reaction mixture to which it was added.
Thus, it can be seen that the radioimmunoassay of the present invention requires t~70 prinicipal reagents other than the unkncwn s~mple of biological fluid to be assayed. ~hese are the radiolabeled ccmpetitor of the drug to be assayed, bupropion, and a bupropion-specific antiserum.

upropion-specific antisera Antisera to be used in the radioimmunoassay o-f the present invention must have high specificity for bupropion, and poor recognition of (i.e., low cross-reaction with) other species which might be present in the biological fluid to be tested, e.g., metabolites of bupropion. According to one aspect of the present invention, suitable immunogens are described, which may be utilized to induce formation of antikodies specific to bupropion and ha~ing low recognition of other species in the medium.

Immunogens The immunogens of the present invention æ e utilized to induce formation of antibodies specific to bu~ropion. The i~muno-gens are present~d, as by inj~ction with suitable adjuvant, to the im~une response system of a host an~mal. Improve titers can be obtained by a series of injections over a period of time. Suitable host animals include mammals such as horses, goats, guinea pigs and ~Z~5~~'3 and sheep. The prefe~red h~st animals are rabbits.
The immNnogens of the presenk invention comprise suit-able ccnpounds linked vLa the ar.cmatic ring or via ~he side chain ketone, to a suitable carrier material. Suitable carrier materials are known to those skilled in the art and include, for ex~mple, p~oteins; natural or synthetic polymeric materials such as poly pe~tides, e.g., p~lylysine and copolymers of aminD acids; poly-saccharides; and the like. Among the suitable proteins useful in the practice o the present invention are mammalian serum proteins such as, for e~ample, human ga~ma globulin, human serum aIbumin~
rabbit serum albumin, ~nd bcvine ga~ma globwlin. The preferred carrier material is bovine serum albumin (B5A).
While not intending to be bound ~y theory, it is presently believed ~hat metabolites of ~upropion in man involve reduction of the side-chain ketone with or without hydroxylation of the t-butyl grcup on the nitrogen. Accordingl~, the present invention provides meth~ds of maXing novel bupropion variants which can be linked to a suitable carrier ~aterial, preferably via the aromatic ring, thus leaving the side chain available for recognition by the host a mmal immune system.
According to another aspect of the present invention discussed below novel methods are provided for making the dnlg/carrier conjugate.
The new ccmpounds provided by the present invention are of the formula I:

I lR2 ~ CH - OE13 C (c~13)3 wherein (l) Xl is Cl, R2 is oxygen and R is ~(OE12)n~H
or -(OEI2)n ~-(oO~m(CH2)pCOZ
or -O-(CH2)pCOZ
or -O-(CH2)pCc~ 1 or is a radioisotope or (2) Xl is Cl, R is H and R is =N'O(CH2)qcOZ
or (3) Xl is a radioisotope, R2 is oxygen and : is OH in the 4-position and wherein n is an integer of 1~5, m is O or 1, p is an integer of 1-4, q is an integer of 1-3, ~ l is a Cl-C3 aIkyl group and Z
is OH or a group of formula:

where s is an integer of 1-4, R6 is a radioactive nuclide and is OH.
The compounds of the present invention for use in immuno-gen preparation include compounds of Formula IA.

~2 ~ - ~, f"l, IA

Cl wherein either R2 is oxygen and Rl is (CH2)n-0-(CO)m-(CH2)pCOOH
where n is an integer from 0 to 5, m is 0 or 1, and p is an int~ger from 1 to 4; or Rl is hydrogen and R2 is N-o-(GH2) -CCOH
where q i5 an integer from 1 ~o 3. Included are each enantio~er and any mixture thereof.
Preferred CCmpOllndS include:
a-t-butylamuno-3-chloropropi~phenone-carboxymethyloxlme, (comp~und l);
~=t-butylamino-3-chloro-4-carb~methaxy-propiophenone, (compound 2); ar~
o-t.-hltylam ~ 3-chloro-4-y-hydrc~ypropyl-propiophenone hemisuccinate, (compound 3);
each of which has been found to be highly suitable for preparation of i~nunogens according to the present invention for the purFose of bupropion-specific antisera preparation.
The preferred compoun~ a-t-butylamino-3-chloropropiophenone~
carkoxymethyloxime, compaund lo `~ 11 ' . ~

~æoso~

(and suitable analags and derivatives as would be obvious in view thereof) can be prepared by reaction of bupropion with carboxy-methoxylamlne hemihvdrochloride in the presence of anhydrou~
sodium acetate in suitable solvent, ~such as ethanol/water. Suitable variations in the method and alternate reayents will be obvious to one skilled in the art in view of the instant disclosure.
The preferred compound ~-t-but~1amino-3-chloro-4-carboxy-methoxypropiophenone, compound 2, Il C:~

Cl ~and suitable analogs and derivatives as w~uld be obvious in view thereof) can be prepared according to known methods by basic h~rolysis of a-t-butylamino-3-chloro-4-carbaxymethoxypropiophenone methyl ester hydrochloride, compound 4, (or suitable corresponding inter-; ~ mediate) such as by reaction with potassium hydro~ide.
Intermediate campound 4 can be prepared frGm the readilyavailable starting materials o-chlorophenol and propionyl chloride which react wlth heating to give o chlorophenyl propionate.
Treatment of the product with alumlnum chloride prcduces mixed reaction products in~luding 3-chloro-4-h~draxypropiophenone. To a mixture of 3-chloro-4-hydroxypropiophenone and sodium methoxide dissol~ed in suitable solvent is added ethyl bromoacetate and re-action at reflex gives ethyl 2-(2-chloro-4-propionylphenoxy~acetate.
Subse~uent ~rcmination in methanol gives methyl 2-[2-chloro-4-(2-~L2~S~

bromopropionyl)]acetate ~hich reacts with t-but~lamlne to yive compound 4. Mod.ifications of the method of synthesis such as selection of sui.table and alternate solvents, reaction conditions and reagents, is well within the skill of the art in view of the present disclosure ana such modifications do not bring the disclosed method outside the scope of the present invention.
Sîmilarly in view of the present disclosure, it w w ld be wit ~1 ~he skill of the art to modify the above method to produce other i~termdiates for ccmpaunds of Formula I. To the extent there is a preferred e~bodiment of one or more aspects of the method of making ccmp~und 4, each is incorporatea into the meth~d of Example I.
An alternate ~ethod of synthesis of compou~d 4 is diagra~matically illustrated in Syn~hesis Meth3d A.
m e preferr~d compound ~-t-butylamuno-3-chloro-4-~h~dro droxypropylpropiophenone hemisuccinate, oonpound 3, O

/~~
H2C(CH2)2C(CH2)3 ~1 ~and suitable analogs and derivatives as w~uld be obvious in view thereof) is prepared by reaction of ~-t-butylamino~3-chloro-4-(3-hydroxypropyl)-propiophenone h~drochloride three-~ou~ths hydrate, compou~d 5, g~

~L2~S~

g ~CC~IC1~3 . 3/4H20 5 2)3 ~I _--Cl with succ m ic anhydride in pyridine to ~orm the _-hemisuccinate.
Intermediate compound 5 iS suitably prepared from readily a~ail~ble starting materials~ Accordingly, 3-chloro~4imethyl-benzonitrile, N~bromosuccinimide and benzoyl peroxide react at r~1ux. with illumlnation to give 2 chloro-4-cyanobe$zyl bromide. The pro~uct compound can be reacted with diethyl malonate in a solution of sodium in ethanol t~ give diethyl2-(2-chloro-4-cyan~benzyl)~alonate which, upon subsequent treatment with sodium chloride and water in DMSO produces ethyl 3-(2-chloro-4-cyanophenyl)propanDate. Treatment with suitable base, such as potassium hydroxide and alcoh~lic sol-vent gives ~he corresponding propanoic acidl 3-(2-chloro-4-cyanc, phenyl)prDpRnoic acid which is then reduced tD the corresponding alcDhol, such as by treabment with B2H6 in tetrahydrofuran tTHF).
A mixture of the alcohol, 3-(2-chloro-4-cyanophenyl)pro~arol and ethyl magnesium hromide in dry ethyl ether is reacted at reflux to give 3-chloro-4-(3-hydroxypropyl)propiophenone which yields compDund 5 upon brcmination t~ a-bromo~3-chloro-4-(3-hydroxypropyl)propio-phenone and subsequent reaction of the brcm~ketone with an excess of t-kutylamine in suitable solvent, such as CH3CN.

In view of the present disclosure, it ~Duld be within the skill of the art -to modify the above method to produce other inter-mediates for comp~unds of Formula I and to modify it by selection ~L2C~ 8~

of alternate suitable solvents, reagents and re~ction con~itions, none o$ whi~h mcdifications take it outside th~ scope o~ the presen-t mventiDn. Tb the extent there is a preferred embcdiment of some aspects of this me~hod, each is incorporat~d into the method of Example II.
The application of the abcve described method of making conpound 3, to other ccmpounds within formula I haviny the formula o : I N

i Cl wherein ~ is (CH2) -O-CO(CH~ -C02H where n and p are as defined for formula I, w~uld be within the skill of the art. Thus such ccmpounds can be made by reactiny an intermediate compound of formula o HO(CH2) ¦ H

~ herein n is as defined for formula I, which IOCO(OEl~)pCol where m p is as defined for formula I.
The above described methNd of~making comF.ound 2 is readily applied to make other ccmFounds within formula I having the formula ~1 8~

o ¦ H
C~
wherein ~ O is O-(CH2)pCCC~1 where p i5 as defined abcve~. Accordingly such ccmpounds can be made by basic hydrolysis of an intermediate campnund of formula o RllOOC (CH2) ~

Cl ~here p is as defined above and ~ 1 is alkyl of 1 to 3 carbons~
Prefer~bly potassium or sodium hydroxide is used.
Immunogen Prep_ration m e coupling of a bupropion variant of the present invention to the Im~unoyenic carrier material can be readily accomplished utilizing techniques well known to the skilled in the art~ Thus, for example, one suitable technique ccmprises dissolving the b~propion variant, the carrier material anLI a suitable coupling agent in suitable lnert solvent to react. As discussed akove, for assay of human sera or plasma the bupropion variant should be coupled to the carrier material in ~such a way that regions believed to be affected by me W olic changes in man are presented for ready recognition by the host animal immune system. Thus, coupling is preferably vla the aromatic ring. Accoxdingly, as one asp~ct of the present invention, there are now disclosed preferred routes to conjugates of the bupropion v æ iants of the present invention such as campounds 2 and 3 disclosed above, linked vla the arcmatic ring to suitable carrier material. Such conjuyates present the bupropion side chain for recognition hy the host anImal immune system.
A first prefeLred conjugate ccmprises ccmpound 2 cou~led to B5A using a sui~able, water-soluble carbodiimide catalyst. Present understanding sugyests ~hat the c æ ~odiimide catalyzes the foYmation of peptide bon~s bet~een the free acid moiety of compound 2 and the e-amino group of lysyl residues of BSA. A preferred c æ ~odiimide is l-ethyl-3-(3-dimethylam mopropyl)-carbodiimide hydrochloride ("EDC"). In view of the present dis~
closure, it is wlt~tin the skill of the art to modify this method of forming the conjugate to apply it to other campounds within formula I or to employ alternate suit~ble catalyst, none of which modifications take the methnd outside the scope of the present invention~ To th~ extent there is a preferred em~odiment of some aspects of this method, it is incorporate~ into ~he method illustrated in Example III.
A second preferred conjugate comprises compound 3 coupled to B5A using the mixed an~tydride method. Thus, for e~ample, canFouutd 3 can be reacted with triethylamine and isobut~lchloro-formate in dimethylformamide to form the mixed ar~t~dri~e. Upon addition to BSA in sodiulm car~onate the desired conjugate is fonmed.
Ihis meth~d is illustrated in Examlple IV and in view of the present disclosure it is within the skill o-f the art to apply the met~tod of Example IV to other ComFoun~s of fo~nula I. Mbdifications such -, ~

as, for ex~nple, in the choice of reaction conditions ar~ reagents, will be obvious to the ~skilled in the art and are not outside the scope of the present invention, Tb the extent there is a preferred emkodim~lt of some aspects of this me~hod, it is incorporated into the me~h~d illustrated in Example IV.
The possibility i9 seen that ring-hydroxylated metakDlites of bupropion could exist in the biological fluid to be assa~ed.
The antisera raised utilizing the inmediately aboved described conjugates might to some extent crossreact with such metaholites.
While the efficacy of the radioimmunoassay of the present invention w~uld be maintained notwithstanding such possible cross-reaction, there ls also provided, as one aspect of the present invention, the preparation of conjugates CQmpriSing bupropion variant linked to sui~able carrier material, preferably BSA, via the side chain ketone. Utilizing such immunogen provides antisera which would not cross-re~ct substantially with ring-hydroxylated metabolites of bupropion. Such antisera could be expected to cross-react with side-chain alcoh~l metabolites.
~ccordingly, a third preferred conjugate comprises ccmpound 1 coupled to ~SA enploying a water soluble carbodiimide method.
This method is illustrated in Example V.
The radioimmunoassay of the present invention is based on the fin~ing that bupropion will successfully compete with radiolabe]ed bupropion, or a suitable radiolabeled competitor o~
k~lpropion according to the present invention, for binding to anti-sera raised by presenting a suitable bupropion variant-carrier material conjugate ~o the immune syst~m of host animals, such as rabbits.

,J'¢.,~ '' . .~ .. . .. --~s~

Radiolabeled Bupropion Competitor Radioactively labeled bupropion or the radioactively labeled bupropion competitors o~ the present .invention can be labeled in the manner well know.n to the art with any suitable radionuclide~ A listing of the radionuclides which æ e now con-ventionally in use in reagents and which may be used in this in-vention are listed in the index of radionuclides found on page 81 of the 1978 edition of the Catalogue of the New England NUC1 OE
Corporation, Boston, Massachusetts, U.S.A.. (New England Nuclear, 1977). Among radionuclides which are preferred in this invention the following may be mentioned: hydrogenr3 (tritiu~) and the radio isotopes of iodine (l23I~ 124I 125I 126I 128I 130I 131I an~
132I) with 125I and H being preferred from considerations of availability, half life and specific activity and/or the ability of these to be readily detected using a conventional gamma co~mter usually available in hospitals and sold by Packard Inst~uments or others. Alternate suitable labeled ccmpetitors include those labeled with any other detectable an~ distinguishing label such as for example, an electron spin resonance group. Okher suitable labels include chromophores, fluorophors, enzymes~ latex particles and the like. Preferably the label used is an isotope.
The radiolabeled competitors of the present invention are of Formula II: .

R' ~5 ¦ H

wherein R' is a suitable radioi.sotope as described abcve and is hvdrogen, R4 is Cl, and R5 is oxygen, or R' is h~drogen and either a) R3 is H and R4 is Cl and R5 is NO(CH2)~CO-X
where q is as defined for formula I and X is a group ha~ing formula III

-NH(CH2)S ~ R6 III

wherein R6 is a s~itable radioisotope as described abo~e, R7 is hydroxyl, and s is an integer from 1 to 4; or X is a group having iv formula III(~) 2 ( ~ ~ III¦~) wherein R6 is a suitable radioistope as described above, an~ AIk is a lower alkyl containing 1 to 4 carbons, b) R4 is Cl, R5 is oxygen, and R3 is (CH2)n-0-(CO)m-(CH2)p-CC-X where n, m, and p are as defined for Eormula I and X is as defined above; or c) R3 is hydroxy, R5 is oxygen, and R4 is a suitabl~
radioisotope as defined above.
Mbst preferabl~, the radiolabeled canpetitor is selected from a-t-Butylamino-3-chloropropiophenone-carboxymethyloxime-l25I-tyramine amide, (Compound 6) a-~ tylamino-3-chloro-4-carbometho~ypropiophenone-V85~

5I -tyramLne amide (compo~md 7) ~-t-: 3ut~1amiTx>-3-ch:lo,xo-4- y-hydro~ rop~71propio-phenone-h~nisuccinate-125I-tyramine amide (cQ~ound 8) 5I~ -Butylamino-3-iodo-4~hydroxypropiophe~one (ccmpound g) ~-t--Butylamino-3-c~oro-2-[ El]-propiophen3ne h~drochloride ( compound 10 ).

In view of the present disclosure, the radiolabeled compe~itors of the present invention ~an be made according to methods well known to th~se skilled in the art. Thus, for the preparation of ; 10 compound 6, c~mpound 1 can be coupled to tyram~ne using dicyclo hexylcarbodiimide to catalyze the formation of a peptide bond between the free ac.id of campound 1 and the.primary amino group of tyraTnine~ The product is s~ sequently iodin~t~d.in accordance wqth the Hunter-GreenwDod Chloramine-T Method, describ~d in W. M.
Hunter and F. C. Greenw~od, Nature, 194, 495 (1962).
Preferred radiolabeled cQmpound 7 is prepared frcm ocmpound 2 and tyramine using the muxed anhydride method. Similarly compound ~ can be prepared from compound 3 using the mixed anh~ride method. In each case, ~he starting reagent is reacted with TE~ and isobutyl chlorofc~ate in LMF to form the mixed anhydride. Ihe reaction mixture is then added to tyramine and the product icdinated as described abo~e.

The application of t~is mathod to other oom~oun~s of formula III w~uld be within the skill of the art. mus, compounds wi~hin formula III having the formula .

~z~ g ~ 17 -R - ~ ~ N

'' Cl 9 ( 2)n 0-CO(CH2)pCO-X where n, p and x are as defined above, can be ma~e by reactiny a canpound of formula 1 with trialkylamine and alkyl haloformate to form the mixed anhydride and subsequently adding the reaction mixture to HOC~H4(CM2)SNEI2 wherein s is as defined above. The product is subsequently iodinated preferably with 125I. The trialkylamine preferably com~rises an alkyl moiety of about 1 to 4 carbon and the alkyl moiety of alkyl haloformate preferably has 1 to 7 car~onO Txiethylamine and isobutyl chloroformate are most preferred.
In ~his Specification TEA means triethyla~ine, nMF mea~s dimethylfornE~Iide, DM50 means dimethylsulfoxide, ES~ means bovine serum albumen.

Similarly, compounds within form~la II having the formula O

N

Cl wherein R12 is 0-(CH2? CC-X where p is as defined above and X is III or III(a) ~ i ,...

-NH(CH2)S ~ R6 III

~ ~ ~ OH III~a~

where R6, ~ s and Alk are as defin0d above, can ke r~de by reacting a compound of for~ula 1 with triaIkylamine alkyl halofcmate, as those terms were used above, to form the mixed anhydrideO S~bsequently adding the reaction muxture to HOC6H4(CH2~SN~I2 w above or to HOC6H4OE12CH(Co2Alk)NH2 where Alk is as defined above and then iodinatin~ preferably with 125I gives tha desired radio-labeled oompetitor compound.
Compound 9 can be prepared by demethylation of R-t-butyl-an~n~-4-rnethoxypropiophenone i1 ' ~ ~ .

follcwed by iodination of the phenolic product~
Compound 10 is 3H labeled bupropion and can be prepared as shown herein.

I

~` ` .
......

~.--.,, .. . ~ .

m e Bupropion Radioimmunoassax ~rocedure PreEerably for high speciEicity, the antiser~n is in each case raised to a hlpropion variant which corresponds to the radiok~beled c y titor used in the assay. mus, radiolabeled c~npetitor canpound 6, is preferably used in conjunction with anti-(a~t-butyl~nino-3~chorGpropiophenone~carboxymethyloxime-B5~) sera, ("ant i ccmpound 1 sera"). Ccm~ound 7 and ccmpound ~ are each ; preferably used with antisera raised to cm immunogen wherein the bupropion variant is linked to the carrier material vla the aromatic ring. Thus, each is preferably used with either anti-camp~und 2 sera or with anti~compound 3 sera. Radiolabeled ccmpetitor compounds 9 and 10 also are each used preferably with antisera raised to immu~ogens linked via the ar~makic ring to the carrier material. Ccnpetition between the bupropion, if any, present in the biological fluid being assayed and the radiolabeled ccmpetitor proceeds in such a manner that equilibrium is achie~ed corresponding to the relative concentration of bupropion and radiolabeled comr petitor in the assay ~uxture.
The present invention is also based in part upon the discovery that once the competition of bupropion and radiolabeled competitor- has proceeded for khe desir~d time, preferably to equi-librium, khe antibody bound portion of the drug and competitor can be separated frcm the free portion of the drug and competitor.
Following such separation~ the amount of radioactivity in the anti-body bound portion gives a measure of the amount of unlabeled bupropion which was present in the test muxture.
According to the preferred radioimmunoassay o the present invention, a standard curve is constructed by running the .

" ~ v~

assay on t~o or more, preferably fcur to 8 solutions, each having a different known bupropion concentration within a suitable con-centration range. In addition, ~m assay is run with only the radiolabeled competitor without the addition of bupropion, such ; that the maximlm possible binding can be quantified. The assay for each standard solution can be expressed as a percent of maxi-mum binding and plo~ted on a graph against the concentration of ; bupropion m the assayed sc~mple. The value obtained upon assay of the unknown biological sample expressed as percent of max~m binding can then be used to interpolate its bupropion concentration from the standard curve.
Preferably, duplicate measures of standard bupropion solutions, preferably in blank pla~ma, are prepared such as by pipetting equal amounts of each st~ndard solution into plastic tubes. In addition, duplicate "nonspecific binding tubes", that is tubes which will not receive antisera, and duplicate "maximum binding tubes", that is tubes which will not receive unlabeled bupropion, also each receive a like measure of blan~ plasma. While it is indicated that each sample is assayed in duplicate, this is merely preferred for greater accuracy and the average value for each pair is used. If desired, the assay mayeven be performed with triplicate samples. After being placed in an ice bath, all tubes receive a portion of radiolabeled competitor and s~bsequently all tubes, except n~n-specific binding tubes, receive anti-bupropion serum raised to an appropriate immunogen of the present invention.
Preferably, the radiolabeled competitor and antiserum are in suit-able assay buffer, such as phosphate~buffered isotonic saline containing EDrA and gelatin. The non-specific binding tubes receive '~

2~3~ï;0~43 blank assay buffer.
Foll~wing incubation, when the hupropion and the com-peting radiolaheled competitor :in the assay mixture have s~lh-; stantially reached equilibrium, antibody-bound radiolabeled ccm-petitor is separated from the free portion thereof by any suitable means such as are known to the s~illed in the art. In one preferred method a complex is formed between protein in the assay mixture and another added protein by incubation, for example, overnight at ambient temperature. The complex precipitates out and can be centrifuged to a pellet for measurement of radioactivity. Altern-ately, ammonium sulfate can be added to get a faster precipitation.
~ owever, a novel and useful feature of the radioim~unD-assay of the present invention is the ethanol precipitation of antib~dy-bound 125I-bupropion campetitor from the equilibrium assay mixture. In this novel and preferred methDd for quick precipitation, the assay mixture must be kept ice-cold, such as by immersion in an ice bath. Absolute ethanol is added to the ice-cold mixture and quickly precipitates all the protein in the assay mixture. m e precipitate can be pelletized and thus use of ethanol precipitation is significantly advantageous in allowing ~uantitation of 125I in the protein p~llets.
The quanti~ation of the antibcdy-bound radiolabeled competitor such as, for example, by beta or ga~ma radiation count of the antibody-b~und H or 2 I lakeled competitor respectively minus the average count obtained for the non-specific bir~ing tubes, is e~p~essed as a percentage of the quantitation of the maximum binding tubes (also minus the average count obtained for the non-specific binding tubes). Using the results obtained for ~2~3sa~

the tubes containing st~ndard bupropion solution samples, a standardcurve can be constructed as already described. The assa~v procedure will be 1Lrther explained by the illus~ration Ln the ex~nples.
The antisera raised to the above descriked ring function-alized imm~no~ens have been found to provide excellent specificity.
: Cross reaction with the bupropion metabolites believed to occur in man, has been found to be of a low level.
The novel immunogens and antibodies of the present invention may be utilized in conjunction with conventional additives, buffers, stabilizers, diluents, or in combination with other physio-logically active or inactive substances.
As one aspect of the present invention a kit is provided, such as for a mercantile unit, for practicing the radioimmunoassay of the present invention. Such kit comprises at least one container, such as, or example, a test bube, containing bupropion-specific ; antisera and bupropion competitor. In one emkodiment, the antisera, presented for ~xample, in freeze dried form, is adhered to a first portion of the container and bupropion ocmpetitor is adhered to a second, separate portion of the container. In such embodiment, any suitable adhesive means can be used, such as, for example, a water soluble adherent which will not interfere with the binding of bupropion or of the competitor to the antisera. Alternately, each reagent can be presente individually, each in one or more separate containers. m e kit can also comprise, in the same or different container(s), standard amount(s) of bupropion, antisera and com-petitor. This would be preferred for example, where a standard curve is to be constructed.

,'' :`

s~

In the accanpanying drawinys Fig. 1 is a s-tandard curve show.ing inhibition of the binding of radiolabele~ oanpetitor as a function of bupropion concentration and is explained in more detail in EXample 12 below;
Fig. 2 shows standard curves obtained using certain 1 iodinated bupropion ccmpetitors and antisera according to the present in~ention an~ is explained in more detail Ln Examples 13- `
17 belGw.
Whenever reference is made in this Specification to alkyl or a group ccmprising aIkyl, the alkyl moiety thereof contains 1 to 6 ccarbon atcms-unless otherwise specified and t-butylami~o means tert-butyL~mino.

.
The following Ex~mples ar~ given to illustrate the invention.

EXAMPIE 1 .

Preparation of ~-t-butylamino-3-chloro-4~methoxycar~onylmethoxy-propio~henone methYl ester hYdrochloride, compound 4 , l ~CH2COC CH3 CO-CH-NHC(CH3~3 A. o-Chlorophenyl propionate r ~ ~

IOH O-C(}C2H5 ~ t C2H5COCl > ~ 1 .

~' ~' " ~ S~

_-Chlorophenol (64 ~, 0.5 mole) and propionyl chloride (50 g, 0.55 mole) were mlxed at room temperature an~ then heated at 100C (steam bath) for 2 - 3 hrs. ~Iydrogen chloride gas was evolved. After 2 - 3 hrs. the reaction mixture wag distilled in vacuo and gave 78.8 g (86%) of o-Chlorophenyl propionate, bpllmm = 111C.
~nal for Cg H9 2 Cl M.W. 184.63 Calcd: C, 58.54; H, 4.91 Found: C, 58.31; H, 4.88 B. 3-chloro-4-hy _ oxpropiophenone ~ CC-C2H5 OH qH
g~C_~lC13 ~ ~clcl~c~c2}~5 Tb anhydrous aluminium chloride (37 g, 0.27 mole), o-ckloro-phenyl propionate 124.8 g, 0.13 ~ole) WdS added rapidly (vigourous reaction). The reaction mixture was heated at 120-130C (in a metal bath) for 30 - 45 min. After cooling, the aluminum chloride reaction mlxture was decomposed wi-th a mixture of ice and concentrated ~Cl.
The solid organic material was filtered off by suction, and was washed with much cold water giving 18~20 g of solids. ~is product was recrystallized se~eral times from ethyl ace~ate-hexane giving two pRrts: (1) 13-14 g insoluble in hexane and (2) 5-6 g from e~aporation of the ethyl acetate-hexane filtrates.
The 13-14 g of hexane insoluble material was recrystallized several times frQm mixbures of ethyl acetate and hexane. It was charcoaled while in the hot ethyl acetate solution and gave finally , .

~;~05[J~
- 2~ -

3-chloro-4-hydroxypropiophenone (12 g, 48.4~) mp 114-115C.
(D. Chakravarti and B. Majurndar., JO Indi~n Chem. Soc., 16, 151~159 (1939) re,ported a mp of 80C for this prcduct).
Anal for Cg Hg O~ Cl M.W. 184.63 Calcd: C, 58.54; H 4.91 Found: C, 58.41; H 4.76.
The material from the ethyl acetate-hexane soluble filtrate after several recrystallizations from hexane gave 2-hydroxy-3-chloropropiophenone (4.3 g, 17.3 %) mp 42-45Co Anal for Cg Hg 2 Cl MoW~ 184.63 Calcd: C, 58.54; H 4.91 Found: C, 58.42; H 4.76 C. Ethyl 2-(2-chloro-4-~ropionylphenoxy)acetate ~H ~ H2COOC2H5 + Br CH2COCC2Hs NaOCH3 > ~ 1 C(~C2H5 CC2~15 Sodium methoxide ~2.5 g, 0O045 mole) was dissolved in absolute eth-anol (40 ml) 3-Chloro-4-hydroxy-propiophenone ~7.4,g, 0.04 mole) was added and the mixture warmed for 1-2 minutes. Ethyl bromoacetate (7.3 g, 0.44 mole) was added and the reaction mixture was refluxed at 100C (steam bath) for 2~3 hrsO Mbst of the ethanol was evaForated, ice and water were added and the mixture was neutralized to pH 4-5 with dilute HCl. Cooling and scratching of the flask im tiated crystallization. The resulting solid was dissolved in excess ether, washed 3x with 5% NaOH, then with water. The ethereal solution was dried over sodium sulfate (anhydrous), ~iltered and evaporated and i~, gave 9-10 g of crude mater~al. Several recrys~allizations ~rcm ethyl acetate ~ave ethyl 2-(2-chloro-4-propionylphenoxy)acetate (8.3 g, 77%), mp 129-130C, Anal for C13 H15 4 Cl M.W. 7 Calcd: C, 57.67; H 5.59 Found: C, 57.67; H 5.43.
D. Methyl 2-[2-chloro-4-(2-bromopropionyl)phenoxy]acetate.

-~ 13r2 CO~C2H5 -Br Ethyl -2-(2-chloro,4-propionylpheno~y)ace~ate (4 g)~as dissolv~d in methanol (40-5Qml). HCl ~2 drop5, conc) was added, followed by the dropwise addition over 5-10 minutes of bromlne (2.62 g, 0.016 mole). After the bromine addition was complete the muxture was heated on a steam ~ath for 15-30 minutes allowing much of the solvent to evaporate. m e residue was washed with cold - water and the remaining solid recrystallized several times from hexane to give methyl 2-[2-chloro-4-(2-~romopropionyl)phen~xy]
acetate (4.6 g, 90%) mp 63-65C.
A~al for C12 H12 4 1 Calod: C, 42.93; H 3.60 Found: C, 43.83; H 3.46.

~ 71 ~l2~S~

E. Methyl 2-[2-chloro-4-(2-t-buty~l~a ___ propion~l)pheno~ ~acetate hydrochloride OCH2COOCH3 ~12COOCH3 t 2 ~C33)3C~12 ~

Br Cc-cH-l~Hc(c~13)3 Tb methyl 2~~2-chloro-4-(2-bromopropionyl)phenoxy] acetate (3.3 g, 0.01 mole) in acetonitrile (20 ml) t-butylanine (3.3 g, 0.044 mole) was added, and the reaction miK-ture was left overnight at 40C (near a waxm steam bath). In the morning the mixture was heated on a steam bath, allowing solvent and excess amine ~o evap-orate(to dryn~ss). m e residue was treated with cold water and NaOH

~o pH 11 and extracted quickly with ether. The ethereal solution was dried over anhydrous sodium sulfate, filtered, an~ evaporated to remove ether and any traces of t-butylamlne. The residue was redissolved in ether and precipitated as the hydrochloride salt by the addition of a little alooholic HCl. The white crys-talline solid was recrystallized two times fron mixtures of methanol-ethyl acetate-ether to give methyl 2-[2-chloro 4-(2-t-butylamlnopropion~l)phenoxy~
acetate hydrochloride (2.5 g, 68.5%), mp 213 215C.
Anal for C16 H23 NO4 C12 M~W. 364.29 Calcd: Ct 52.76; H, 6.36; N, 3.85; O, 17.57; Cl, 19.47 Found: C, 52.48; H, 6.42; N, 3.71; Cl, 19.36 -` ~2~

EXAMPLE II
. .
~repa-ration of a ~-t-butykanino-3-chloro-4-y-h~drox}F~ pi~-.
phenone hemisuccinate, c~~~ound 3 _ A. 2-Chloro-4-~yanobenzyl bromide ~,h~ BrCH2/ C f Cl benzoyl peroxide Cl A mixture of 3-chloro-4-~eth~l-benzonitrile (30.0 g, 0~2 m~
N-bromosuccinimide (36.0 g~ 0.21 mol) and b_nzoyl peroxide (100 mg) in 600 mL of CC14 was reLluxed with illumination for 72 h. N-Bromo-succinimide (5.0 g) was added and the reaction was continued for an additio~al 24 h. The mixture was cooled, filtered and eNaporated.
Recrystallization from neptane gave pure 2-chloro-4-cyanobenzyl bromide: yield 24.5 g; mp 83-85C. Anal. calod for C8H5 ~rCIN; C, 41.69; H, 2.19; N, 6.07~ Found: C, 41.48; H, 1.80; N, 5.76.

B. Di~ yl 2-(2-c oro-4~y~n benzyl)malonate.

~ CN
. ~
BrCH2 Cl Na/EtOH
! ~ ~H2(CO2Et)2 CN

tO C) E 2 2~ n2 Cl i.,.,~

~ 29 -Tb ~sodium (2.0 gr 0.09 mol) dissolved in e~hanol was added die~hyl-malonate ~15.7 g, 0.1 mol) ~ollowed by 2-chloro-4-cyanobenzyl brcmide (20.0 y, 0.09 mol). The mLxture was refluxed for 3.5 h.
The so~vent was evaporated and 120 mL of water and 3.6 mL of concentrated hydrochloric acid was added to the residue. Separation and distillation gave diethyl 2-(2-chloro-4-c~a~obenzyl)malonate as an oil: 12.5 g; bp 140-142C (0.1 mm Hg). Anal. calcd for C15H16CIN04; C, 58.16; H, 5.21; N, 4.52. Found: C, 58.07; H, 5.22;
~, ~.52.

C. Eth~l 3-(2-chloro-4-c~anolphenyl)propanoate ~ ~ < NaCl~ H2O~ DMSO

EtO2CCH2CH2 (EtO2C) 2(~ICH2 Cl Cl A muxture of diethyl 2-(2-chloro-4-cyano~enzyl)malonate (9.0 g, 0.03 mol), sodium chloride (2012 g, 0.04 mol), and water (0.86 g, 0.05 mol) in 30 mL of DMSO was heated to 135C and then the temp-erature was gradually raised to 170C over a 3 h period. The mixture was diluted with water and extracted wi~h ether. Evaporation and distillation gave ethyl 3-~2-chloro-4-cyanophenyl)propanoate as an oil: 5.8 g; bp 138-140C (0.1 mm Hg) Anal. calced for C12H12 CIN32: C, 60.64; H, 5~09; N, 5089. Found: C, 60.48; H, 5.08; N, 5.84.

D. 3-(2-chloxo-4-c~anophen~l)propanoic acid ~ CN

E~OOC~I2CH2 `~
Cl ~sa~

A rLLKtlIre of ethyl 3-(chloro~4-cyanophenyl)propanoate (33.0 g, 0.14 mol), KOH (6.27 g, 0.11 mol) and 95% ethanol (109 mL) was wanmed at 40C for 1 h. The mux~ure was neutralized and evaporated to dryness. The residue was dissolved in 5% Na~CO3 and washed wlth EtQ~c. Acidification an~ filtration gave crystalline 3-(2-chloro-

4-c~anophenyl) propanoic acid: 18.4 g; mp 127-219C.

E 3-(2-Chloro~4-cyanophenyl)propanol - ~ CN

HCCH2CH2cH2 1' Cl To 3-(2-chloro-4-cyanophenyl)-propan~ic acid (14.3 g, 0.068 mol~

in 70 mL of dry ~ at -18C was added B2H6 (72 ml, 0.068 mol) in THF. The mixture was stirred at ro~n ternperature overnight. After addition of water, the layers were separated and the aqueous phase was extracted with ether. Evaporation and distillation gave 3-(2-chloro-4-cyanophenyl)propanol as an oil: 11.0 g; bp 55-60C
(0.005 mm Hg). Anal. calcd for CloHloCINO: C, 61.39; H, 5.15; N, 7.16. Found: C, 61.02; H, 5~19; N, 6.76.

F. 3-Chloro-4-(3-hydrox~propyl)propiophenone .

,~/CCH2CH3 HO(~12C~12C~i~

~s~

A mixture of 3-(2-chloro-4-c~anop~enyl)propanol (18.0 g, 0.09 mol) and ethyl magnesium brc~Lide (36.8 g, 0.276 mol) in dry ethyl ether was refluxed for 4 h. After quenching the reaction with 150 mL of 15% HCl, the layers were separated and the aqueous layer was extracted with ethyl acetate. Evap~ration an~ recrystallization from eth~l ether-pentane gave 3-chloro-4 (3-hydroxpropyl)propio-phenone: 7.9 g; mp 46-48C.

G. Q-t-Butylamuno-3-chloro-4-(3-hydroxpropyl)propiophenone hy~rochloride three-fourths hydrate ~ CX~I2C~13 1) Br2MeOH
/~J ;~) t-BuNH2~cH3cN

Cl ~ J I .~ICl HO~CH2)3 ~ ~ .3/4H2O

Cl 3 CH3 A mixture of 3-chlorc-4-(3-hydroxyproFyl)propiophenone (7.7 g, 0.03 mol) and bromine (5.7 g, 0.04 mol) in dry methanol was stirred at r roQ~ temperature overnight. EvaForation and distillation gave ~-bromo-3-chloro-4-(3-hydroxypropyl)propiophenone: 9.6 g; bp 102-105C
(0.005 mm Hg). The bromoketone was st~rred overnight with an excess of tert-butylamine in CH3CN at room temp~rature. me crude ~-t-butylamunc-3-chlorc~4-(3-hydrQxy-propyl)propiophenone was isolated and converted to the hydrochloride salt; yield 1.25 g; mp 203-205C

'';~

~L2~;3S~

ded. Anal. Calcd. for Cl6H24CINO2~C13/4H2 ;
4.02. Found: C, 55.14; H, 7.39; N, 4.16.

H. ~-t-Buty a~no-3-chloro-4-(3~h ~ ox~pro~y1)propiophenone hemisucc mate (compound 3) ~ -t-butylamuno,3-chloro-4-(3-hydroxypropyl)propiophenone (100 mg; 0.288 mmole) and succinic anhydride (31.7 mg) were dissolved in pyridine (2.5 ml); the resulting solution was allowed to s~an~ at roam temperature overnight. Benzene (10 ml) WrdS added then evaporated under reduced pressure; this step WdS repeated four more times, giving a brown oil.The oil was dissolved in methanol (0.5 ml), and the solution was applied to tw~ 0.5 mm silica gel plates (20 cm x 20 cm). The plates were developed us ms chloroform/methanol/aqueous ammonia (80/20~1) as the mobile phase. The silica ~el strip containing the product (located by uv) was scraped off and the product extracted with chloroform/methanol (60/40; 2 x 100 ml). m e combined extract was filtered and the solve solvent removed un~er r~duced pressure. Ethyl acetate (30 ml) was added and the solution filtered. Evaporation of solvent ga~e ~-~-butylanino-3-chloro-4-(3-hydroxypropyl)propiophenone hemi-succinate (25.4 mg) as a yellow oiL Analysis: 1) thin layer chromatograp~ly: Rf = 0.15 on silica gel (0.25mm) plates develop~d in chloroform/methanol/aqueous ammo~ia (80/20/1); 2) uv (in iso-tonic saline): ~ max = 261 nm,~ min = 231 nm; nmr and mass spectometry data were consistent with the assigned structure.

s~
- 33 ~

EXAMPLE III

IM~UNOGEN PREP~RAI'ION

~-t-Butylaminor3-chloro-4-carkoxymethcxypropiophenone-BsA (Ccmpound 2-B~SA) .
Compound 2 (25.4 mg; 0.088 mm~le) (obtained by basic hydrolysis of the corresponding methyl est~r obtained in Example 1) was reacted with BSA (40 mg; 0.00057 mmole) and l~ethyl-3-(3-di-met~ylaminopropyl)-carbodiimide (~DC) t25 mg) in 15 ml 10% dioxane/
water at pH 5.7 overnight at 25C. The reaction mixture was pressure dialyzed vs. deionized water, concentrated by ultrafiltration and the im~unogen lyophil~zed overnight.
EX~LE IV
-t-Butylamino-3-chloro-4-y-hydroxyp opylpropiophe~one h~misuccinate -BSA ( ComFound 3-BSA) .
Compound 3 (25.5 mg; 0.064 mm~le) reacted with triethyl-amlne (TE~, 908 mg; 0.097 le) and isobutylchloroformate (9.8 mg;
0.072 mmole) in 1 ml dimethylformamide (nMF) at 4~C for 30 minutes to form the mixed anhydride. This reaction mixture was slowly added to a well-stirred solution of BSA (30 mg; 0.00043 mmole) in 4 ml of 0.1 M sodium carbonate at 4~C and allowed to react overnight.
The reaction muxture was pressure dialyzed and ly~philized as in EXample III.

EX~qPLE V
A ~-t-But~lamir.o-3-chloropropiophenone-carboxymethyloxime(Compound 1) Bupropion (free base, 500 mg; 2.085 m mole), carboxy meth~xylamune hemihydrochlOride ~1.37 g; 6.255 m mole) and anhy~rous sodium acetate! (684 m~; 8.34 m mole) in 50% aqueous ethanol (10 ml) were allowed to react at 25C for 5 days. The pH was adjusted to ; `

~Z6~

2.0 with 0.1 N ~ICl, and the mixture extractecl with chloroforrn t3 x 30 ml)~ The combined extract wa~ evaporated u~der reduced press~e, redissolved in chloroEorm (25 ml), evaporated under reduced pres~ure and redissolved m chloroform (2 ml). This sol-ution was applied to 8 2 mm silica gel plates (20 cm x 20 ~n) which were then developed with chloroforrq/methanol/aqueous ammonia (80/20/l). The kand at Rf 0.2-0.3 was scrap~d off and extracted with ethyl acetate/methanol 90/lO (5 x lO0 ml). The ccmbined extract was filtered, evaporated under reduced pressure and dis-solved in ethyl acetate (5 ml). This was filtered and evaporated under reduced pressure to give ~-t-butylamino-3-chloropropiophen-one-carkoxymethyloxime as an oil (135 mg). Analysis: uv (in iso-tonic saline): ~m~x = 250 nm, ~ min = 230 nm; nmr was consistent with the assigned structure.
B. ~-t-Butylamino-3-chloropropionphenone-carbox~methyloxime-BS~, (Com ound l-BSA) Ckmpaund l was coupled to bovine serum albumin (B5A) using a water soluble carbcdiimide to catalyze the formation of peptide konds between the free acid m~iety of II and the ~-aminG groups of ly.syl residues of BS~. Compound 1 (25 mg; 0.080 mmole) was reacted with BS~ (40 mg; 0.00057 ~mole) and l-ethyl-3-(3-dimethyl-aminopropyl)-car~odiimide (EDC, 25 mg) in 15 ml 10% diQxane/water at pH 5.7 overnight at 25C. m e reaction mixture was pressure dialyzed vs deionized water, concentrated by ultra-filtration and the immuno~en lyophilized o~ernight.
E~æIE VI
R-t-Butylamin~3-chloropropiophenon ~ carkox~nethyloxime 125I-tyra-_ mine amide~ (CcmFound 6~

~ 1 3L;~ 9 Cbmpaun~ 1 was coupled to tyramine using dicyclohexyl-carbcdiimide to catalyze the formation of a peptide bond bet~een the free acid of II and the primary amLno group of tyr~mine.
Campound 1 (15 mg; 0.048 mmole) react~d with tyramine (free base, 13 mg; 00096 mmole) and dicyclohexylcarbcdiimide (DCC; 19 mg) in 2 ml dry dioxane at pH 5 overnight at 25C. The product was ~rified by TLC, characterized spectrally and subsequently iodinated (Hu~-ter-GreeN~Dod Chloramine-T Method).
EX~MPLE VII
~-t-ButylanLuno-3-chloro-4-car~ome-th~x~propiophenone-125I-tyramine amide, (Compound 7) Co~pound 2 was coupled to tyramine usin~ ~Jhe mixed anhyd-ride method. Compound 2 (25 mg; 0.076 m mole) was reacted with TE~
(9~8 mg; 0.097 m mole) and isobutyl chloroformate (9.8 mg; 0.072 mmole) in 1 ml DMF at 4C for 30 minutes to form the mixed anhyd-ride. The reaction mixture was slo-~ly added to a well-stirred solution of tyramine (free base, 10 mg; 0.073 mmole) in 1 ml ~MF
at 4C and allowed to react overnight to yield the phenolic product.
The product was purified by TIC, characteriz~d spectrally and subseq~ently iodinated as in ExaTnple VI.
EXAMPLE VIII
~-t-Butylam mo-3-chloro-4-Y-hydroxypropylpropiophenone-he~isucc ate-125I-tyramine amide,(compound 8) Compound 3 was coupled to tyramine usiny ~he mixed anhyd-ride method of Example VII. This product wa5 purified by TLC and subsequently iodinated as described in Example VI.

' ~ ' ~z~s~
. --EX~PIE ~, t-But~lamino-3-iodo-~ r~ prop~ enone,(campound 9) ~ -t-butylam m o-4-methoxy~ropiophenone hydrochloride (20.6 mg; 0.076 mmole) was suspended in 1 ml dry meth~lene chloride and cooled to -80C in an acetone-dry ice bath. Boron tribromide (41.9 mg; 0.167 mmole) was added, the reaction mixture was protected fxon moisture with a calcium chloride drying tube, and the reaction was allowed to proceed overnight, during which time -the temperature rose to 25C. The reaction mixture was shaken with one volume methylene chloride (containing 0.02% water impurity) to liberate the product. The product was purified by TIC and subsequently iodinated as described in Example VI.
EXAMPLE X
Preparation of ~-t-butylamino-3-chloro-2-kritio~ropiophenone hydrochloride _( H bupro~ion) (Compound 10) A. 2-(3-Chloro~2-tritiophenyl)-4,4-dimethyl-2-oxazoline 2-~3-Chlorophenyl)-4,4-dimethyl-2-oxazoline was prepared from 3-chlorobenzoic acid by reaction with thionyl chloride to give 3-chlorobenzoylchloride, reaction of this acid chloride with 2-aminD-2-methylpropanol to give the amide and cy~lization of the amide with thionyl chloride followed by neutralization with dilute sodium hydroxide -to give 2-(3-chlorophenyl)-4,4-dfmeth~1-2-oxa-zoline.
m e oxaæoline ~419~2 mg) in dry ether (15 ml) was cooled to -78~C and tert-butyllithium (2.0 m moles) in pentane (1.12 ml) was added under nitrogen with stirring. Stirring was continued at -78C. for 4 1/2 hrs. Solvent-free T2O (0.5 ml, 90% isotopic purity) was added and the solution was allow~d to slowly wanm to ro~n t~mperature and stirred overnight. m e solution ~7as dried over anhy~rous magnesium sulfa~e and filtered, and the solvenk was removed :Ln v uo to give an orange oil. Purification was achieved by column chrcmatography on silica ~12.6 g) packed as a chlor~form slurry. The crude reaction product was added to the column as a chloroform solution. Elution was with chloroform-ethyl acetate (3:1 V~V). Fractions containing the radiolabeled oxazoline were combined. Solvent r~moval in vacuo gave 2-(3-chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline (334 mg, 79~) as a yellow oil. N~r and mass spectral date confirmed the structure; the radio-label ~7as akout 90~ of theoretical.
B. 2-(3-Chloro-2-kritiophenyl)-4,4-dimethyl 2-oxazoline N-Mekhyl-fluorosulfonate Tb 2 (3-chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline (670.7 mg) in dry benzene (7 ml) under nitrogen was added methane fluorosulfonate (1.4 g). The mixture ~7as stirred overnight at room temperature under nitrogen. The white precipitate was collected under nitrogen, washed with kenzene (1 x 10 ml) and ether (2 x 10 ml) and dried to give 2-(3-chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline N-methylfluorosulfonate in ~reater than 90% yield.
C. 2-(3-Chloro-2-tritiophenyl)-2-ethyl-4,4-dime~h~l-N-methyloxa-zolidine _ To the dry 2-(3-chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline N-methylfluorosulfonate from step C in dry THF (15 ml) under nitrogen was rapidly added ethylma~nesium chloride (8.0 m moles) in ether (2.7 ml). The mixture was stirred for 1 hour at roon temperature and then poured into an ice-water mixture (75 ml) with stirring. The resulting suspension was extracted with ether .

s~

(1 x 100 ml followed by 1 x 50 ml). ~he combined ether extracts w~re washed with water (1 x 50 ml) arrl dried aver c~nhydrous may-nesium sulfate. ~iltration and removcal of solvent in vauo gave 2-(3-chloro-2-tritiophenyl)-2-e~hyl-4,4-dimethyl-N-methyloxa-zolidine (B3% yield) as a yellow oil.
D. 3-Chloro-3-tritiopropiophenone A nL~blre of the crude oxazolidine from step C (670.5 mg) and oxalic acid (475.6 mg) in water (10 ml) was heated at reflex for 1 hour and then cooled to ro~m temperature. Additional water (15 ml) was added, and the product was extracted with ether (2 x 15 ml). The ether layer was washed with 5~ potassium bicarkonate solution (2 x 15 ml) and dried over anhydrous magnesium sulfate.
Solvent was removed _ vacuo to give 3-chloro-2-tritiopropiophenone (405 mg).
E. ~-t-Butylamino-3-chloro-2 tritiopropiophenone hydrochloride Following the procedure of U.S. Patent No. 3,819,706 3-chloro-2-tritiopropiophenone was brominated and the ~-bromo product treated with tert-butylamine to give~-t-butylamino-3-chloro-2-tritiopropiophenone which was isolated as the hydro-chloride ~43~ yield).
EXAMPLF XI
RIA of Bupropion in Plasma Using H 3upropion Samples of six standard solutions of bupropion in blank plasma having concentrations of 0.39 to 50 ng/ml are pipetted (0~1 ml each) in duplicate into 12 x 75 ~Im plastic tubes. Duplicate non-specific binding (no antibody) and maxim~m binding tubes (no unlabeled bupropion) recei~e 0.1 ml blank plasma. Spiked plasma controls at several concentrations of (e.gO, 1.0, 10.0, and 100 503~

~ 39 ~

ng/ml) and plasma samples Eor assay ~diluted into the st~ndard range with blank plasma if necessary) are assayed in duplicate.
After being placed in an ice bath, all tube~ receive ~in order) 0~2 ml 3H bupropion (416 pg. containing approxlmately 104 ~pn) and 0.7 ml anti-bupropion serum (raised to a ring-functionalized immunogen according to the present invention) (at pre-determine dilutions such tbat approximately 40% of the 3H bupropion added is kound), both in assay buffer (0.05 M phosphate-buffered iso-tonic saline, containing 0.01 M EDTA and 0.1% gelatin, pH 7.0).
Non-specific bindimg tubes receive 0.7 ml assay buffer.) Following incukation either at 25C for 2 hours or overnight at 4C, bound and free 3H bupropion are separated by incubation for 10 mun. at 0C with 0.5 ml ice-cold dextran-coated charcoal solution (0.25%
dextran, 5mg/ml charcoal, in assay buffer) followel by centrifug-ation for lO minutes at 4C (S000 rFm) to pellet the charcoal.
The supernatants, con~aining anti~ody-bound 3H bupropion are care-fully decanted into liquid scintillation cocktail for quantitation of 3H. After counts (minus average non-specific binding c~m) are expressed as percentages of average maximum b~nding cFm, bupropion concentrations in plasma .samples and spiked con~rols are read from a standard curve (log concentration vs. percent average maxumum binding)~
EXAMPLE XII
Ihe procedure of Exanple XI has been applied to a study of bupropion (supplied as Wellbutrin*, produced by Burroughs Wellcome Co.) pharmacokinetics in plasma after oral administration of 200 mg bupropion-HCl (in tWD 100 mg tablets) to four normal male subjects.
Venous blood samples were collected at various times following *trade mar~

-- ~o --dosin~, and concentrations in plasma samples dete~mined as des-cribed. The assay standard curve obtain0d by this method (~inter-assay standard deviation, N=8) is shown in Figure 1. Spiked controls ( at 1, 10 and 100 ng/ml) run with 8 separate assays gave inter-assay values oE 0.89 + 0.11 (S.D.), 10.35 + 0.60, and 92.72 +

5.02, respectively. Plots of elapsed time vs plasma bupropion concentration for each subject were made. Using these data, various pharmacokinetic parameters were calculated using the C-strip an~
NO~LIN comput~r pro~rams. The mean elimination half-life, maximum plasma bupropion concentration and time to reach this maximum concentration w~e 2.9 + 0.4(S.D.) hours, 217+ 37 ng/ml, a~d 1.5 + 0.4 hours, respectively.
EXAMPLES XIII~XVII
RIA of Bupropion using I-labeled Bupropion Competitors RIA's have been run accordïng to the method of EXample X utilizing 1 I labeled bupropion ccmpetitors of the presen~
invention in conjunction with antisera obtained following immuni-zation with immQnogens of the present invention. CGmpound 6 was ; used in conjunction with anti-compound 1 sera. Compounds 7, 8 and 9 were each used with anti-compound 2 sera, and with anti-comFound 3 sera. Following the ethanol precipation of antibody-bound 25I-bupropion competitor at equilibrium, 1 I in the protein pellets was quantifie~. Figure 2 shows standard curves obtained with the various iodinated ocmpetitors.
It can be seen that co~pound 9, in conjunction with either antisera produces RI~'s approximately ten times more sensitive than the compound 10 (3H bupropion) assay s~stem. Substitution of 7 or 8 yields sensitivi~y approximately equal to that obtained with the 5~

compound 10 system. The anti-ccmpound 1 sera and compound 6, which preferably are used Eor RJA only in conjunction with each other, produced the l~ast sensltive R~ k~w~ver, by virtue of derivatization via the ketone, these antisera are believed to ; exhibit much less cross reaction with rin~ hydroxylated metabolites, - albeit at the expense of specificity for changes in structure at the ketone.
EXAMPLE XVIII
Preparation of R-t-butylamino~3-chloro-2-tritiopropiophenone hydro c oride (ccmpound 10) A. 2-(3-Chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline 2~(3-Chlorophenyl)-4r4-dimethyl-2-oxazoline was prepared from 3-chlorobenzoic acid by reaction wi~h thionyl chloride to give 3-chlorobenzoylchloride~ reaction of this acid chloride with 2-aminc-2-methylpropanol to give the amide and cyclization of the amide with thion~l chloride followed by neutralization with dilute sodium hydroxide to give 2-(3-chlorophenyl)-4,4-dimethyl-2-oxa-zoline.
The oxazoline (419.2 mg) in dry ether (15 ml~ was cooled to -78C and tert-~utyllithium (2.~m m3les) in pentane (1.12 ml) was added under nitrogen with stirring. Stirring was continued at -78C for 4 1/2 hours. Solvent-free T20 (0.5 ml, 90% isotopic purity) was added, and the solution was allowed to slowly warm - to roQm temperature and stirred overnight~ The solution was dried over anhydrous magnesi~lm sulfate and filtered, and the sol-vent was removed in vacuo to give an orange oil. Purification was achie~ed b~ column chromatography on silica (12.6 g) packed as a chloroform slurry~ The crude reaction product was added to the 8 2(~;08~
- ~2 -column as a chloroform solution. Elution was with chloroform~
ethyl acetate (3:1 V~V). Fractions containing -~he radiolabeled oxazoline were combined. So~venk removal in vacuo yave 2-(3--chloro-2-tritiophenyl)~4,4-dimethyl-2-oxazoline (334 mg, 79%~ as a a yellow oil. Nmr and mass spectral data confi~med the structure;
the radiolabel was about 90% of theoretical.
B. 2-(3-Chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline N-Methyl-fluorosllfonate Ib 2-(3-chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline (670.7 mg) in dry benzene (7 ml) under nitrogen was added methane fluorosulfonate (1.4 g). m e mixture was stirred overnight at room te~perature under nitrogen. The white precipitate was coll ected lmder nitrogen, washed with benzene (1 x 10 ml) and ether ( 2 x 10 ml) an~ dried to give 2-(3-chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline N-methylfluorosulfonate in greater than 90%
yieldO
C. 2-(3-Chloro-2-tri_iophenyl)-2-ethyl-4~4-dimethyl-N-me oxazoli ine To the dry 2-(3-chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline N-methylfluorosulfonate from step C in dry T~F (15 ml) under nitrogen ~as rapidly added ethylmagnesium chloride (8.0 m moles) in eth~r (2~7 ml)~ The mixture was stirred for 1 hour at room temperature and then poured into an ice-water mi~ture (75 ml) with stirring. m e resulting suspension wa~ extracted with ether (1 x 100 ml follow~l by 1 x 50 ml)~ The combined ether extracts were washed with water (1 x 50 nLl) and dried over anhydrous mag-nesium sulfate. Filtration and removal of solvent in vacuo gave 2-(3-chloro-2-tritiophenyl)-2 ethyl-4,4-dimethyl-N-methyloxazol-:, .~ 6)8~

- ~3 ~
idine (83% yield) as a yellow oil.
D. 3-~hloro-2-t itio~ oE~
A m:ixture of the cr~e oxazolidin~ ~rom step C (670.5 mg) and oxalic acid ~475.6 n~) in water (10 ~1) was heated at reflex for 1 h~ux and ~hen cooled to roQm t~mperatura. ~dditional water ( 15 ml) was added, and the product was extracted with ether (2 x 15 ml). m e ether layer was washed with 5% potassium bicarbonate solution (2 x 15 ml) and dried over anh~drous magnesium sulfate.
Solvent was removed in vacuo to give 3-chloro-2~tritiopropiophen-one ~405 mg)~
E. ~-t-Butylaminc-3-chloro-2-tritiopropiophenone hydrochloride Following the procedure of U.S.Patent No~ 3,819,706, 3-chloro-2-tritiopropiophenone was hrominated and the a-brQmo product treated with tert-butylamine to give a-t-butylaminc-3-chloro-2-tritiopropiophenone which was isolated as the hydro-chloride ~A3~ yield).

'~ '-~i~

SYNliHESIS ME~OD A

+ Br CH2C2C2H5 NaCCH~ ) ~

l S02C12 Cll2 3 O~H2COOC2H5 , Cl ~ ~Cl CO-OEI-B~ CO C2H5 1 ) (CH3 ) 3CNH2 2) ~Cl ~12COO CH3 ~1 C~-NH-C (C~13 ) 3 ,~1 .

Claims (16)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound of formula:

wherein:
i) R10 is OH, R11 is a radioisotope and R12 is O; or ii) R10 is H, R11 is C1 and R12 is a radical of formula:
=N-O(CH2)qCO-X1 wherein q is an integer from 1 to 3, and X1 is OH
or a radical of formula:

wherein R6 is a radioactive nuclide, R7 is hydroxyl and s is an integer from 1 to 4.

2. A compound of the formula wherein R4 is a radioisotope,

3. A compound according to claim 2, wherein R4 is selected from 3H and 125I.

4. A compound according to claim 3, wherein R4 is 125I.

5. A compound according to claim 2, wherein R4 is 3H.

6. A compound of the formula:
where q is an integer from 1 to 3.

7. A compound according to claim 6, in which q is 1.

8. The method of making a compound of claim 6 comprising reacting bupropion with carboxyalkoxylamine hemihydrochloride, wherein the alkyl moiety of carboxyalkoxylamine comprises one to three carbons.

9. The method of claim 8, for making the compound of claim 7 comprising reacting bupropion with carboxymethoxylamine hemihydrochloride.

10. A compound of the formula:

wherein R5 is NO(CH2)qCO-X where q is an integer of from 1 to 3 and X is a group of formula:
wherein R6 is a radioactive nuclide, R7 is hydroxyl and s is an integer from 1 to 4.

11. A compound according to claim 10, wherein said radioactive nuclide is selected from 3H and 125I.

12. A compound according to claim 10, wherein said radio-active nuclide is 125I.

13. A compound according to claim 10, which is ?-t-butylamino-3-chloropropiophenone-carboxymethyloximme-125I-tyramine amide.

14. A method of making a compound of the formula:
wherein R5 is NO(CH2)qCO-X where q is an integer of from 1 to 3 and X is a group of formula:

wherein R6 is 125I, R7 is hydroxyl and s is an integer of from 1 to 4, comprising reacting a compound of formula:
where q is an integer of from 1 to 3, with a compound of formula:
HOC6H4(CH2)sNH2 wherein s is an integer of from 1 to 4 and subsequently iodinating the product.

15. A method according to claim 14, wherein dicyclohexyl-carbodiimide is used to catalyze the reaction.

16. A method according to claim 14 or 15, wherein q is l and said compound is reacted with tyramine.