CA1193194A - Immunogens obtained from unlabelled derivatives of butylamino propiophenone and a process for immunogen preparation - Google Patents

Abstract

ABSTRACT

An immunogen comprises a conjugate comprising a compound of formula:

Description

~3~

This invention relates to immunogens obtained from unlabelled derivatives of butylamirlo propiophenone and a process for immunogen preparation.
This application is a division of Canadian Patent Application S.N. 375,361, filed April 13, 1981.
The present invention is primarily directed to a radioimmunoassay for clinical or experimental testing -for the presence of and quantitatlon of bupropion L(t)-2-tert-butyl-amino-3l-chloropropiopherione]~ a pharmacologically active anti-depressant compound, in biological Fluids, including especially human sera or plasma.
The radioimmunoassay technique is findins increasing application for quantitation of drugs in biological fluids.
Monitoring of plasma concentrations of drugs enables more precise dose administration to ensure efficacy. In the hospital labo-ratory 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 biological fluids, especially human sera or plasma.
It is another purpose of the invention -to provide suitable radiolabelled agents for use in the radioimmunoassay.
In accordance with the present invention there is provided an immunogen comprising a conjugate comprising a compound oF formula (XX):

'3 ., . .~ ,,~

It is another pur~ose of the invention to provide anti-l~odies di~ected against bupropion ~lso referred to as bupropion-specific antisera and as anti-bupropion sP-ra. In this regard, it is a further purpose of the present inven-tion to provide suitable immunogens for usè to raise bupropion-specific antisera, and to provide rnethods suitable for raising such antisera.
The radioim~noassay of the present invPntion is based on campetition between bupropion ar~ a fixed quantity of a dis-tinguishable competitor of bupropion for a limited number of bind-ing sites on bupropion-specific antibodies. The distinguishable ccmpetitor is preferably a labeled c~mpound, .such as a ccmpound labeled with a radioactive isotope (~'radiolabele~").
m e drug and its radiolabeled competitor are added to the anti~odies and the reaction mixture is allowed to equilibrate.
When the antibody-~ound portion is separated fram the free drug arld free radiolabeled competitor, then the amount of radioactivity present in the antibody-bour~d portion will be inversely related to the quantity of unlabeled drug originally added to the reaction mixture. mus, the greater the amount of ~unlabeled drug added, the less radiolabeled competitor will be bound to the antibodies.
Conversely, the amount of radioactivity present in the reaction rnixture fram which the antibcdy-~ound portion has been removed will be directly related to the quantity of unlabeled drug originally added to the reaction mixture; i.e. the greater the arnount of unlabeled drug added, the rnore radiolabeled competitor will remain in the reaction r~ixture from which the antibody-bo-~d portion has been removed.
A standard curve can be produced hy employing a range -;r ~..

3~
. 3 _ of known col~centrations of unlabeled drug in a series of reaction mixtures while holding constc~n-t the amount of antibody and radio-labeled ccmpetitor employedO llhe standard curve a]lows the concen-tration of drug in an unknown sample to be interFolated fr~m the amount of radioactivity present in the antibody-bo~d portion of the equilibrated reaction mixt~re to which it WdS added.
Thus, it can be seen that the radioimmunoassay of the present i~vention requir~s ~o prinicipal reagents other than the u3iknown sample of biological fluid to be assayed. These are the radiolabeled competitor of the drug to be assayed, bupropion, and a bupropion-specific antiserum.

Bupropion-specific antisera Antisera to be used in the radioi~nunoassay of the present invention must have high specificit~ for bupropion, and poor recognition of (i.e., lcw cross-reaction with) other species which might be present in the biological fluid to be tested, e.g., metabolites of bupropion. According to one asp~ct of the present invention, suitable immunogens are described, which may be utilized to ;nduce formation of antibodies specific to bupropion and having low rec~gnition of other species in the medium.

Immunogens The immunogens of the present invention are utilized to induce formation of antib~dies specific to bupropion. The immuno-gens are presented, as by injection with suitable adjuvant, to the im~Nne response system of a host animal. Improve titers can be obtained by a series oE injections over a period of time. Suitable host animals include ma~als such as horses, goats, guinea pigs ar3d - r ;3~

and sheep. The preferrecl host c~nimals are rabbits.
m e mmunogens of khe presenk invention ccmprise suit-able compounds lir~ed via the aromatic ring or via khe side chain kekone, ko a suitable carrier material. Suitable carrier materials are known to those skilled in the art a~d include, for example, proteins; natural or synthetic polymeric ~ terials such as poly-peptides, e.g., polylysine and copolymers of amino acids; poly-~saccharides; and the like. Among the suitable proteins useful in the practice of the present invention are m~mmalian ser~m proteins such as, for example, human gamma globulin, human seru~ albumin, rabbit serum altumin, a~d bovine gamma globulin. '~ne prefe~-ed carrier material is bovine serum albumin (BS~).
~ hile not intending to be bound by theory, it is presently believed that metabolites of bupropion in m~n involve reduction of the side-chain ketone with or without hydro~ylation of the t-butyl group on t'ne nitrogen. Accordingly, the present invention provides methods of making novel bupropion variants ~lich can be linked to a suitable carrier material, preferably via tne aromatic ring, thus leaving the side chain available for recognition by the host animal imnLlne system.
According to another aspect of the present invention discussed below novel methods are provided for making ~he drug/carrier conjugate.
The new compounds provided by the present invention are of the formula I:

.~

33~

R_ Rl--~ C ~CH - CH3 Xl NH C(CH3)3 wherein (1) Xl is Cl, R2 is axygen and R is ~(~12~n~{)H
or -(OEI2)n-O-(CO)~(CH2)pCOZ
or -C-(CH2)pOOZ

( 2)p 11 or is a radioisotope or (2) Xl is Cl, Rl is H and R is =N O(CH2)qCOZ
or ~3) Xl is a radiolsotope, R2 is oxygen and is OH in the 4-position and wherein n is an integer of 1-5, m is 0 or 1, p is an integer of 1-4, q is an integer of 1-3~ Rll is a Cl~C3 alkyl group an~ Z
is OH or a group of formula:

~\/ 6

2 ~'~

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

~r"/~
,,.~

~D3~
~, ~2 Cl wherein eith~er ~ is oxygen and Rl is (CH2)n-0-(CO)m-(CH2)pCOOH
where n is an integer from O -to 5, m is O or 1, and p is an integer from 1 to 4; or ~ is hydrogen and R2 is N~O~(CH2)q~COOH
where q is an integer frcm 1 to 3. Included are each enantiomer and any mixture thereof.
Preferred conpolmds include:
a -t-butylamunc-3-chloropropiophenone-carboxymethyloxime, (comp~u~ l);
a-t-butylamino-3-chloro-4-carbomethoxy-propiophenone, (com~ound 2); ar,d 4-t -k~Utylaltli~3-ChlOrO-4 -y-hydrOXyprOpyl-propiophenone hemi~uccinate, (compound 3);
each of which has been found to be highly suit~lble for prepara-tion of Immun3gens according to the present invention for t,he purpose of bupropion-specific antisera preparation.
The preferred ccmpound a--t-kutylam m o-3-chloropropiophenone-carbo~me-thyloxime, camp3und lo N

Cl (and sui-table analogs and deriva-tives as wDuld be obvious in view thereof) can be prepared by reaction of bupropion with carboxy-methoxylamine hemihydrochloride in t'ne presence of anhydrous sodium acetate in suitable solvent, such as ethanol/water. .Suitable variations in -the method and alternate reagents will be obvious to one skilled in the art in view of ~he instant disclosure, T'ne preferred com~ound ~-t-butylamino-3-chloro-4-carboxy-me-th~xypropiophenone, compound 2, o 1 ,' N
HO2CCH2O ¦ H
~1 (and suitable analogs and derivatives as would be obvious in view thereof) can be prepared according to known methods by basic hydrolysis of a -t-butylan~no-3-chloro-4-carboxymethoxypropiophenone methyl ester hydrochloride, compound 4, (or suitable corresFonding inter-mediate) such as by reaction with potassium hydroxide.
Intermediate compound 4 can be prepared frc~ the readily available starting materials o-chlorophe~ol and propionyl chloride which react with heating to give = chlorophenyl propionateO
Treatment of the product with aluminum chloride produces mixed reaction products including 3-chloro-4-hydroxypropiophenone. 1~ a mixture of 3-chloro-4-hydroxypropiophenone and sodium methoxide dissolved in suitable solvent is added ethyl bromoacetate and re-action at reflex gives ethyl 2-(2-chloro-4-,propionylphenQxy)acetate.
Subsecluent bromination in methanol gives methyl 2-[2-chloro-4-(2-brcmopropionyl)~acet~te which reacts with t--butylamine to give compo~u~d 4. M~difications of the methcd of ~ynthesis such as selection of suitable ar~ alternate solvents, reaction conditions and reagents, is well within the skil.l oE the art in view of the present disclosure and such modificatio.ns do not bring the disclosed method outside the scope of the present inverltion.
Sinilarly in view of the present di.sclosure, it ~uld be within the skill of the art to modi.fy the above method -to produce other inte~mediates for cc~pounds of Eorrnula I. Tb the extent there is a preferred er~odiment of one or more aspects of the method of making ccmpound 4, each is incor~orated^into the rnethod of EXample I.
An alternate method of synthesis of c~mpound 4 is diagramrnatically illustrated in Synthesis Method A.
The preferred compound a--t-butylaminc-3-chloro-4-~-.hydro droxypropylpropiophenone hemisuccinate, compound 3, O

~ I I
HO2C(C~2)2OCO(CH2)3 ¦ H

(and suitable analogs and derivatives as would be obvious in view thereof) is prepared by reaction of a-t-butylamino-3-chloro-4-13-hydroxypropyl)-propiophenone hydrochloride three-fourths hydrate, campound 5, 33~

o . l~/cchcH3 N~l. HCl ~ .3/4H20 5 HO lCH2 ) 3 1 __ __ C~ .
with succinic anhydride in pyridine to form the o-he~isuccinateO
Intermediate c~mpound 5 is suitably prepared from readily available starting materials. Accordingly, 3-chloro-Q-methyl-b~nzonitrile, N-bromosuccinimide and benzoyl peroxicle react at reflux with illumination to give 2-chloro-4-cyanobenzyl bromide. ~he product compound can be reacted with diethyl malonate :in a solution of sodium in ethanol to give diethyl2-(2-chloro-4-cyanobenzyl)malonate which, upon subsequent treatment with sodium chloride and water in DMSO produces ethyl 3-(2-chloro-4-cyanophenyl)propanoate. Treatment with suitable base, such as potassium hydroxide an~ alco~olic sol-vent gives the correspon~ing propanoic acid, 3-(2-chloro-4-cyano-phenyl)propanoic acid which is then reduced to the corresponding alcohol, such as by treatment with B2H6 in tetrahydrofuran (THF).
A mixture of the alcohol, 3-(2-chloro-4-cyanophenyl)propanol and ethyl magnesium bromide in dry ethyl ether is reacted at refl~lx to give 3-chloro-4-(3-hydroxypropyl)propiophenone which yields comFound 5 upon bro~lnation to Q-brom3-3-chloro-~-(3-hydroxypropyl)propio-phenone and subsequent reaction of the bromoketone with an excess ~ of t-butylamine in suitable solvent, such as OE13CN.
In view of the present disclo Æ e, it would be within the skill of the art to ~cdify the ab3ve method to pro~uce other inter-mediates for comF~unds oE Formula I and to modify it by selection ~33~
- ~.o - I
of alternate su;.t~able sol.vents, .reacJents and reaction cc>nditions, none of ~nich ~cdifications take it outside the scope of the present invention. To the extent there is a preferr~d embodiment of some aspects of this method, each is incorporated into the method of EXample II.
The applic~tion of the ci~ove descxi~3d met~d of making comFound 3, to other ccmpounds within formula I having the formula R,8 ~'` /
1~1 1 1 ~ N ~

wherein ~ is (CH2)n-0-CO(CH2) -C02H whexe n and p are as defined for formula I, would be within the skill of the art. ~nus such comFounds can be made by reacting an intermediate co~ound of formula o , y / ' HO(CH2)n ~ N
¦ H
C~

~ herein n is as defined for Eormula I, which C ~ ~ O where m p is as defined for formula I.

The above described method o~ making com~ound 2 is readily applied to make other ccmFounds within formula I having the formula , .~

Rlo ¦ H

wherein ~ O is O-(C~12)pCOOH where p is as defined abuve . ~ccor~ingly such c~npounds can be rnade by basic hydrolysis of an intermediate ccmpound of formNla o Rl100C (CH2)pO ~ ),f ~'1 where p is as defined above and ~ 1 is alkyl of 1 to 3 carkons~
Preferably potassium or sodium hydroxide lS used.

The coupling of a bupropion variant of the present invention to the imm~nogenic.carrier material can be readily acccmplished utilizing techniques well known to the skilled in the art. Thus, for example, one suitable technique comprises dissolving the bupropion variant, the carrier material and a suitable coupling agent in suitable inert solvent to react. As discussed above7 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 metabolic changes in man are presented for ready recognition by the host animal immune system. ThusJ coupling is preferably via the aranatic ring. Accor~ingly, as one aspect of the present invention, there are no~ disclosed preferred routes to conjugates of the bupropion variants of the present i~vention such as comcounds 2 and 3 disclosed abcve, li~ked via the aromatic ring to suitable carrier material. Such conjuyates present the bupropion side chain for recognition by the host animal immune system.
A first preferred con~ugate comprises compound 2 coupled to ~SA using a suitable, water-soluble carbodiimide catalyst. Present understarldiny suggests that the carbodiimide catalyzes the formation of peptide konds between the free acid moiety of compourd 2 and the ~-amino group of lysyl residues of BSA. A preferred carbodiimide is l-ethyl-3-(3-dimethylamunopropyl)-carkodiimide hydrochloride ("EDC"). In view of the present dis-closure, it is within the skill of the art to modify this method of formin~ the conjugate to apply i-t to other campounds within formula I or to employ alternate suitable catalyst, none of which modifications take the method outside the scope of the present invention. 1~ the extent there is a preferred embodiment of some aspects of this method, it is incorporated into the method illustrated in EXample III.
A secor,d preferred conjugate ccmprises comp~und 3 coupled to BSA using the mixed anhydride method. Thus, for example, compound 3 can be reacted with triethylamine and iso~utylchloro-formate in dimethylformamide to form the mixed anhy~ride. Upon addition to BSA in sodium carkonate the desired conjugate is formed.
This method is illustrated in Example IV and in view of the present disclosure it is within -the skill of the art to apply the method of Example IV to other cumpoun~s of formula I. Mbdific~tions such ~ ~ !

~ ~ ~3~'3~a as, for exarnple, in the choice of reaction conditions and reagents, will be obvious to the skilled in the art and are not outside the scope of the present invention. To the extent there is a preferxed embodiment of s~ne aspects of this method, it is incor~orated into the method illus-~-ated in EXample IV.
The possibility is seen tha-t ring-hydroxylated metabolites of bupropion could exjst in the biological fluid to be assayed.
Ihe antisera raised utilizing the immediately aboved described conjugates might to .~ne extent crossreact with such metabolites.
~Lile the efficacy of the radioimmunoassay of the present invention would be maintained notwithstanding such possible cross-reaction, there is also provided, as one aspect of the present invention~
the preparation of conjugates c~nprising bupropion variant linked to suitable carrier material, preferably BSA, via the side chain ketone. Utilizing such imnunogen provides antisera which would not cross-react substantially with ring-hydr~xylated met~bolites of bupropion. Such antisera could be expected to cross-react with side-chain alcohol metabolites.
Accordingly, a third preferred conjugate comprises compound 1 coupled to BSA employing a water soluble carbodiimide me-thod.
This method is illustrated in EXample V.
The radioi~munoassay of the present invention is based on the finding that bupropion will successfully ccmpete with radiolabeled bupropion, or a suitable radiolabeled competitor of bupropion according to the present invention, for bi~ing to anti-sera raised b~ presenting a suitable bupropion variant-carrier material conjugate to the imm~ne sys-tem of host animals, such as rabbits.

Radiolabeled B~lpropion Ccmpetitor _ Radioactively labeled bupropion or the radioactively labeled bupropion ccmpetitors of the present invention can be labeled in the manner well kncwn to the art with any suitable radionuclide. A listing of the radionuclides which are 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 Nuclear Corporation, 30ston, Massachusetts, U.S.A.. (New England Nuclear, 1977). Among radionuclides which are preferred in this invention the following may be mentioned: hydrogen-3 (tritium) and the radio isotopes of iodine (l23I~ 124I 125I 126I 128I 130I 131I a d 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 counter usually available in hospitals and sola by Packard Instrum~nts or others. Alternate suitable labeled competitors include those labeled with any other detectable and distinguishing label such as for example, an electron spin resonance group. Other suitable labels include chr~mophores, fluorophors, enzymes, latex particles and the like. Preferably the label used is an isotope.
I~e radiolabeled competitors of the present invention are o~ Formula II:

R' ~5 ~ ~

3~ N
¦ H

''~.

3~9~

wherein R' is a suitab]e radioi~sotope as described abcve and R3 is hydrogen, R4 is Cl, and R5 is oxygen, or R' is hy~rogen and either a) R3 is H and R4 is Cl and R5 is NO(CH2)qCO~X
where q is as defined for for~ula I and X is a group havin~ formula I .

-NH(CH2)s ~ R6 III

wherein P6 is a suitable radioisotope as described above, R7 is hydroxyl, and s is an integer from 1 to 4; or X is a group having lv formula III(à) C12~C ~ R6 --NH - CH - CH2 ~ - OH III(a) wherein R6 is a suitable radioistope as descr~bed abôve, and Alk is a lcwer aIkyl containing 1 to 4 carbons, b) R4 is Cl, R~ is oxygen, and ~ is (CH2)n-0-(CO)m-(CH2)p-CO-X where n, m, and p are as defined for formula I and X is as defined above; or c) R3 is hydroxy, R5 is oxygen, and R4 is a suitable radioisotope as defined above.
Most preferably, the radiolabeled ccmpetitor is selected 0 frcm a-t-Butylarnir~3-chloropropiophenone-car~r~xymethyloxime-125I-tyrc~mine arnide, (Com~ound 6) a -t-B~ltylanL~3-chloro-4-carb~rnethoxypropiophenone-5I -tyramine amide (~rnpou~d 7) Q-t-Butylamun~r3-ehloro-4-y-hy~roxypyropylpropio-phenone-hernisuccinate-l25I-tyramine arnide (cGmpound 8) 125I- _-~-ButylanlnK-3-iodo-4-hy~roxypropiophenone (compound 9) ~-t-Butylamino-3-chloro-2-[ H~-propiophenone h~rochloride ( compound 10 ).

In view of the present disclosure, the radiolabeled ccanpetitors of the present invention can be made according to rnethcds well known to those skilled in the art. Thus, for the preparation of ccmpouncl 6, cam~ouncl 1 can be coupled to tyramine using dicyclo-hexylcarbodiimide to catalyze the formation of a peptide bond between the free acid of c~mpound 1 and the primary amino group of tyramine. The product is subsequently iodi~ted in accordance with the Hunter-Greenwcod Chloramine-T Method, described in W. M.
Hunter and F. C. Gre~nwood, Nature, 194, 495 (1962)~
Preferred radioiabeled cornpound 7 is prepared frcm compound 2 and tyramine using the mixed anhydride method. Similarly coTnpound 8 can be prepared frcm c~npound 3 using the mixed ar~lydride methsd. In each case, the starting reagent is reacted with TEA and isobutyl chlorofarnate in nMF to form the mixed anhydride. The reaction mixture is then added to tyramine and the product iodinated as described above.
The applicatic~n of this method to other ccmpounds of form~la III would be wichin the skill of the art. Thus, cc~pounds within form~la III having the formula ~3 ,~ /
R ~

¦ H

C'l wherein Rg is (CH2)n-0-CO(CH2)pCo-X where n, p and x are as defined above, can be made by reacting a compound of formula 1 with trialkyla~une ar~ aIkyl haloformate to fonn the mixed anhydride and subsequently adding the réaction mixture to ~ICC~H41CH2)SNH2 wherein s is as defined above. The product is s~bsequently iodina-ted preferably with 125I. The trialkylr~nine preferably c~n~rises an alkyl moiety of about 1 to 4 car~on and the alkyl moiety of alkyl haloformate preferably has 1 to 7 carbon. Triethylarnine r~nd isobutyl chloroformate are most p~eferred.
In this Specification TE~ means triethylamine~ ~F mea~s dimethylformanide, DMSO means dimethylsulfoxide, BSA means bovine serum alkumen.
Similarly, comFounds wi-thin fornL~a II havir}g the formula -¦ H

Cl wherein ~ 2 is 0-(CH2! CO-X where p is as defined above and X is III or III(a) 3~
- 18 ~

( 2~s ~ III

I ~ R6 -NH - CH - CH2 OH III(a) where R6, ~ s and Alk are as definea above, can be made bv reactin~
a ccmpoun~ of form~la 1 with trialkylamine al~l halofanate, as those terms were used akove, to form the mixed anhydride. Subsequently -adding the reaction mixture to HOC6H4(CH2)SNH2 where s is as defined above or to HOC6H4CH2CH(CO ~ k)NH2 where Plk is as defined above and then iodinating preferably with 125I gives the desired radio-labeled ccmpetitor compoundO
Compound 9 can be prepared by demethylation of ~-t-butyl-amino~4~methoxypropiophenone o 1~' / N
~H30 H

followed by iodination of the..phenolic productO
Ccm~ound 10 is H la~eled kupropion an~ can be prepared as shown herein.

m e Buproplon Radioim ~noas ~ Procedl~e Preferably for high specificity, the antiserum is in each case raisad to a bupropion variant which corresponds to the radiolabeled competitor used in the assay. Thus, radiolabeled competitor compo~md 6, is preferably used in conjunction with anti~ t-butylamino-3-choropropiophenone-carboxymethyloxime-~SA) sera, ("anti-cc~pound l sera"). Cb~our~ 7 and c~n~x~und 8 are each preferably used with antisera raised to an immunogen wherein the bupropion variant is linked to the carrier material via the aromatic ring. Thus, each is preferably used with either anti-com~Dund 2 sera or with anti-c~mpound 3 sera. Radiolabeled cc~petitor co~pounds 9 and lO also are each used preferably wi-th antisera raised to im~unogens linked via the aromatic ring to the-carrier material. Competition between the bupropion, if any, present in the biological fluid being assayed and the radiolabeled c~mpetitor proceeds in such a manner that equilibrium is achieved corresponding to the relative concentration of bupropion and radiolabeled c~m-petitor in the assay mixture.
The present invention is also based in part upon the discovery that once the ccmpetition of bupropion and radiolabeled c~mpetitor- has proceeded for the desired time, preferably to equi-librium, the antibody bound Fortion of the drug and competitor can be separated from the free portion of the drug and competitor Following such separation, the amount of radioactivity in the anti-body bound Fortion gives a measure of the amount oE unlabeled bupropion which was present in the test mixture.
According to the preferred radioim~L~oassay of the present inven-tion, a standard curve is constructed by r mning the . ,, ,, 3~
- 20 ~
assay on tw~ or more, preferably four to 8 solutions, each having a different known bupropion concen~ration wit~lin a suitable con-centration range. In addition, an assay is run with on1y the radiolabeled competitor without the addition of bupropion, such that the T~xImum possible binding can be quantified. I~e assay for each standard solution can be expressed as a percent of ~LYi-TT~m binding and plotted on a graph againLst the concentration of bupropion in the assayed sample. The value obtained upon assay of the unknown biological sample expressed as p~rcent of n~LYiT~m binding can then be used to interpolate its bupropion concentration frcn the standard curve.
Prefexably, duplicate measures of standard bupropion soluticns, prefexably in blank plasma, are prepRred such as by pipetting equal amounts of each standard solution into plastic tubes. ~n addition, duplicate "nonspecific binding tubes", that is tubes which will not receive antisexa, and duplicate "T~xim~Tm binding tubes", that is tubes which will not receive unlabeled bupropion, also each receive a like measure of bla ~ plasma. While it is indicated that each sample is assayed in duplicate, this is merely preferred for greater accuracy and the avexage value for each pair is used. If desire~, the assay mayeJen be performed with triplicate samples. After being placed in an ice bath, all tubes receive a portion of radiolabeled competitor and subsequently all tubes, except non-specific binding tubes, receive anti-bupropion serum raised to an appropriate immunogen of the present invention.
Preferably, the radiolabeled ccmpetitor and antiserum are in suit~
able assay buffer, such as phosphate-buffered isotonic saline containing EYrA and gelatin. The non-specific binding tubes receive 3~

blank assay buffer.
Follawing incubation, when the bupropion and the ccm-peting radiolakeled competitor in the assay m~xture have sub-stantially reached equilibriu~, anti~xdy-kound radiolabeled com-petitor is separated from the free portion thereof by any suitable means such as are known to the skilled in the art. In one preferred methcd a ccmplex 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 measure~ent of radioactivity. Altern-ately, am~onium sulfate can be added to get a faster precipitation.
However, a novel and useful feature of the radioimmunc-assay of the present invention is the ethanol precipitation of antibody-bound 1 5I-bupropion competitor frcm the equilibrium assay mixture. In this novel and preferred method 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. The precipitate can be pelletized and thus use of ethanol precipitation is significantly advantageous in allowing quantitation of I in the protein pellets.
The quantitation of the antibody-bound radiolabeled competitor such as, for example, by beta or gamma radiation count of the antibody-bound H or 125I labeled competitor respectively minus the average count obtained for the non-specific binding tubes, is expressed as a percentage of the quantitation of the maximum bind~g tubes (also minus the average count obtained -Eor -the non-specific binding tubes). Using the results obtained for 3:~9~

the tubes con~aining standard bupropion solution s~mples, a standard curve can be cons-tructed as ~lreadv described. The assay proce~ure will be further explained by the illustration in the ex~mples me antisera raised to -~he ab~ve descri~xd ring f~mction-alized il~mogens have been found to provide excellent specificity.
Cross reaction wi~h the bupropion metabolites belie~7ed to occur in man, has been found to be of a low level.
m e novel immunogens and antibodies of the present invention may be utilized in conjunction with conventional additives, buffers, stabilizers, diluents, or in canbination 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 radioin~noassay of the present invention. Such kit cc~prises at least one container, such as, for example, a test tube, containing bupropion-specific antisera and bupropion competitor. In one el~kodiment, the antisera, presented for example, in free~e dried fo~m, is adhered to a fi~st portion of t~e container and bupropion c~npetitor is adhered to a second, se,~arate rortion of the container. In such em~odiment, any suitable adhesive means can be used, such as, for exampIe, a water soluble adherent which will not interfere with the binding of bupropion or of the canpetitor to the antisera. Alternately, each reagent can be presente~ individually, each in one or re separate containers. m e kit can also comprise, in the same or different container(s), standard amount(s) of bupropion, antisera and com~
petitor. 1~is w~uld be preferred for ex~nple, whele a standard curve is -to be constructed.

J

In the accompanying drawings Fig. 1 is a standard c~ve sh~w,iny i~ihibition of the binding of radiolabel~d c~npetitor as a function of bupropion concentration and is explained in more ae~ail in Example 12 below;
Fig. 2 shows standar-d curves obtaine~ using certain 125I
iodinated bupropion competitoxs ana antisera according to the present invention and is explained in more detail in EXamples 13-17 below.
Whenever reference is made in this Specification to alkyl or a group comprising alkyl, the alkyl moiety thereof contains 1 to 6 carbon atoms unless otherwise specified and t-butylamino means tert-butylanLino.
The following EXamples are given to illustrate the invention.
EX~IE 1 .

Preparation of ~-t-butylaminc-3-chloro-4-methoxycarbonylmethoxy-propiophenone methyl ester hydrochloride, compound 4 (~CH2C~H3 ~5H3 CO-CH-~HC(CH3)3 A. o-Chlorophenyl propionate ~ C2H5COCl 1.~,, :~LY`~3~
- 2~ -_-Chlorophenol (64 g, 0.5 mole) and propionyl chloride (50 g, 0.55 mole) were mixed at room te~perature and the~ heated at 100C (steam bath) for 2 - 3 hrs. Hydrogen chloride gas was evolve~. After 2 - 3 hrs. the reaction mixture was distilled in vacuo and gave 78.8 g (86~) of o-Chlorophenyl propionate, bpllmm = 111C.
Anal for Cg Hg 2 Cl M.W. 184.63 CalcdO C, 58054; H, 4.91 Four~: C, 58.31; H, 4.88 B. 3-chloro-4-hydroxpropiophe~one CO-C2H5 OH O~l ~/ 1 ~,Cl Cl~,CO-C2H5 ' - . C~C2H5 Tb anhydrous alumunium chloride t37 g, 0.27 mole), o-chl~rc-phe~yl propionate (24.8 g, 0.13 mole) was added rapidly (viyourous reaction). ~he reaction mixture was heated at 120-130UC (in a metal bath) for 30 - 45 min. After cooling, the aluminum chloride reaction mixture was decomposed with a mixture of ice an~ concentrated HCl.
m e solid organic material was filtered off by suction, and was washed with much cold water giving 18-20 g of solids. ~his product was recrystallized several times frcm ethyl acetate-hexane giving tw~ parts: (1) 13-14 g insol~ble in hexane and (2) 5-6 g frcm evaForation of the ethyl acetate-hexane filt~ates.
The 13-14 g of hexane insoluble material was recrystallized several times from mixtures of ethyl acetate and hexane. It was charcoaled while in the hot ethyl acetate solution an~ gave finally ~''.

3-c}-loro-4-hyaroxypropiophenone (12 y, 48.4%) mp 114-115C~
(D. Chakravarti and B. Maj~x~ar., J. Indian Chem. Soc., 16J 151-159 ~1939) reported a mp of 80C for this product).
Anal fc,r Cg H9 2 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 yave 2-h~droxy-3-chloropropiophenone (4.3 g, 17~3 %) mp 42-45Co Anal for Cg Hg 2 Cl M.W. 184.63 Calcd: C, 58.54; H 4.91 Found: C, 58.42; H 4.76 C~ Ethyl 2-l2-chloro-4-propionylphenoxy)acetate lH SJCH2COOC2H5 ~ t sr CH2COOC2H5 NaOCH3 > ~ 1 .

Sodium methoxlde (2.5 g,~ 0O045 mole) was dissolved in absolute eth-anol (40 ml) 3-Chloro-4-hydroxy-propiophenone ~7.4 g, 0.0~ mole) ~as added an~ the mixture warmed for 1-2 minutes. Ethyl bramoacetate (7.3 g, 0.44 le) was added and the reaction mix-ture was refluxed at 100C (steam bath) for 2-3 hrs. M~st of the ethanol was evaporated, ice and water were added and the mixture was neutralized to p~ 4-5 with dilute HCl. Cooling and scratching of the flask initiated 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), filtered and evaporated and ~ ;.

gave 9-10 g oE crude m~terial. Scrveral recrystallizations from eth~l acetate gave ethyl 2-(2-chloro-4-propionylphenoxy)acetate (8.3 g, 77%), mp 129-130C.
Aral for C13 H15 O4 Cl M-W- 270-Calod: C, 57.67; H 5.59 Found: C, 57.67; H 5~43.
D. _ethyl 2-[2-chloro-4-(2-bromopropionyl)phenoxy]acetate._ O-cH2cooc2H5CCH2COOCH3 2 HCl ~O-C2~5 C~-CH-Br Ethyl -2-(2-chloro-4-propionylphenoxy)acetate (4 g)~s dissolved in methanol (40-5~). HCl (2 drops, conc) was added, followed b~ the dropwise addition over 5-10 minutes of bromine (2.62 g, 0.016 mole). After the bronine addition was ccmplete the mixture was heated on a steam bath for 15-30 minutes allowing much of the solvent to evaporate~ The residue was washed with cold water and the re~aining solid recrystallized several times from hexane to give methyl 2-[2-chloro-4-(2-bromopropionyl)ph~noxy]
acetate (4.6 g, 90%) mp 63-65C.

Calcd: C, 42.93; H 3.60 Found: C, 43.83; H 3.45.

'~-33.~L~4 E. Methyl 2- 2-chl ro-4-(2-t-butylami_ p o ionyl)p~ noxyl~acetate _yd hloride CCH2COCCH3 . OC~2CCOCH3 t- 2 ~CH3)3CNH2 ~ j ~ Cl ~ 3 ~13 CO-CH-Br CO-CII-N~IC(CH3)3 'rO methyl 2- L 2-chloro-4-(2-brcmopropionyl)phenoxy] acetate (3.3 g, 0.01 mole) in acetonitrile (20 ml) t-butylamine (3.3 g, 0.044 mole) was added, and -the reaction muxture was left overnight at 40C (near a warm steam bath). In the morning the mixture was heated on a steam bath, allowing solvent and excess amine to evap-orate(to dryness). Dhe residue was treated with cold water and NaOH

to pH 11 .In~ extracted quickly with ether. m e ethereal solution was dried over anhydrous sodium sulfate, filtered, and evap~rated to remove ether and any traces of t-butylamine. The residue was redissolved in ether and precipitated as the hydrochloride salt by the addition of a little alcoholic H~l. The white crystalline solid was recrystallized tw~ times from mixtures of methanol-ethyl acetate-ether to give methyl 2-[2-chloro-4-(2-t-butylaminopropionyl)phenoxy~
acetate hydrochloride (2.5 g, 68.5%), mp 213-215C.

Calcd: C, 52.76; H, 6.36; N, 3.85; O, 17.57; Cl, 19.47 2Q Found: C, 52.48; H, 6.42; ~, 3.71; C1, 19.36 ,`~1 ., 33~

EXAMPLE II
Preparation of a ~-t~~tylamino-3-chloro-4-y-hydroxypropylpropio-phenone hemisuccinate, ccmpound 3 A. 2-Chloro-4-cyanobenzyl br ~ide ~ J 4 ~ j Cl benzoyl peroxide Cl A mixbLre of 3-chloro-4-m4thyl-benzo~itrile (30.0 g, 0.2 mol~, N-br~nosuccinimide (3600 g, 0.21 mol~ and benzoyl peroxide ~100 mg) in 600 mL of CC14 was refluxed with ill~nination for 72 h. N-Brcmo-succinimide (5.0 g) was added and the r~action was continued for an additional 24 h. The m~xtur2 was cooled, filtered and evaporated.
Recrystallization fram neptane gave pure 2-chloro-4-cyanobenzyl bromide: yield 24~5 g; mp 83-85C. Anal. calcd for C8H5 BrCIN; C, 41.69; H, 2.19; N, 6.07. Found: C, 41.48; H, 1.80; N, 5.76.

B. Diethyl 2-(2-chloro-4-cyanobenzyl)malonate.
~ CN

BrCH
Cl Na/EtOH
, CH2(~)2~)2 ~" " ~ / CN
l O
(Et2C)2 Cl .~

3~

T~ sodium (2.0 g, 0~09 mol) dissolved in ethanol was added diethyl-ma]onate (15a7 g~ 0~1 mol) followed by 2-chloro-4-cyanobeJnzyl bronide (20.0 g, 0.09 mo]). The mixture was refluxed for 3.5 h.
The solvent was evaporated and 120 mL of water and 3.6 mL of concentrated hydrochloric acid was added to the residue. Separation and distillation gave die-thyl 2-(2-chloro-4-cyanobenzyl)malonate as an oil: 12.5 g; bp 140-142C (0.1 mm Hg). Anal. calcd for C15H16CINO~; C, 58.16; ~1, 5.21; N, 4.52. Found: C, 58.07; H, 5.22;
N, 4.52.

C. Ethyl 3-(2-chloro-4-cyanolphenyl)propanoate _.

~0 ~ ~ NaCl, ~ MSO ~f Et,02CCH2CH2 (EtO2C)2CHCH2 Cl Cl A muxture of diethyl 2-(2-cnloro-4-cyanobenzyl)malonate ~9.0 g, 0.03 mol), sodium chloride (2.12 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 170~C over a 3 h period. The mixture was diluted with water and extracted with 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 CIN02: C, 60.64; H, 5.09; N, 5.89. Found: C, 60.48; H, 5.08; N, 5.84.

D. 3-(2-chloro-4-cyanophenyl)propanoic acid ~C~

C:l 3.~

A m~ture 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 warmed at 40C for 1 h, The JILi~ture was neutralized and evaporated to dryness. Iqle residue was dissolLved in 5% NaHC()3 ar~ washed with EtOAc. Acidification and filtratlon gave crystalline 3-(2-chloro-

4-cyanophenyl) propanoic acid: 18~4 g; IT~ 127-219C.

E 3- (2-Chloro-4-cyanophenyl) propanol .

CN

HCCH2CH2C~/
Cl To 3-(2-chloro-~ cyanophenyl)-propanoic acid (14.3 g, 0.068 mol) in 70 nL of dry THF at -18C was added B2H6 (72 ml, 0.068 n~l) in ~rHF. The m~xture was stirred at roan temperature 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.Q05 mm Hg). Anal. calcd for CloHloCl~ C, 61.39; H, 5.15; N, 7.16. Found: C, 61.02; H, 5.19; N, 6.76.

F. 3-Chloro-4- (3 hydroxypropyl) propiophenone o ~/ 2 3 HCCH2(~2C~2 Cl ~3~
~ 31 -A muxture of 3-(2-chloro-4--cyanop~enyl)propanol (18,0 g, 0.09 m~l) aJ~d ethyl ma~nesium brcmlde (36.8 g, 0.276 ~1~ m dry ethyl ether was refluxed for 4 h. After quenching the reaction with 150 ~L of 15% HCl, ~he layers were separa-ted and the aqueous layer was extracted with ethyl acet~te. Evaporation and recrystallization from ethyl e-ther-pentane gave 3-c~oro-4-(3-hydroxpropyl)propio-phenone: 7.9 g; mp 46-48C.

G. Q-t-sutylamuno-3-chloro-4--(3-hydroxpropyl)propiophenone hydrochloride three-fourths hydrate _ ~ 2 3 1) ~r2MeoH

~ ~ 2) t- BuN~CH3cN

Cl o ~ J l~ ICl HO (CH2)3 ~ l .3/4H~O

Cl CH3- - - CH3 A mixture of 3-chloro-4-(3-hydroxypropyl)propiophenone ~7./ g, 0.03 mol) and bromine (5.7 g, 0.04 mol) in dry methanol was stirred at r room temperature overnight. Evaporation and distillation gave a-brcmo-3-chloro-4-(3-hydroxypropyl)propiophenone: 9.6 y; bp 102-105~C
(0.005 mm ~g). The brc~oketone was stirred overnight with an excess of tert-butylamine in CH3C~ at room temperature. The crude ~-t-butylamino-3-chloro-4-(3-hydrc~y-propyl)propiopherlone was isolated and converted to the hydroc~oride salt; yield 1.25 g; mp 203-205C

~;

-- 32 ~
ded. Anal. C~lcd. for C16H24ClN02HC13/4H20; C, 55.22; H, 7.68; N, 4.02. Found: C, 55.14; H, 7.39; N, 4.16.

H. ~-t-Butylall~3-chloro-4- (3-hydro~yproE~yl)propiophenone hernisuccinate (car~?ow~ 3) Q-t-butylarnino-3-chloro-4- (3-hydroxypropyl ) propiophenone _, _ (100 mg; 0.288 mrnole) and succinic anhydride (31.7 rng) were dissolved in pyridine (2.5 rnl); the resulting solution was allowed to stand at roorn temperature overnight. Benzene (lO ml) was added then evaE~orated under reduced pressure; this step WdS repeated four rnore times! giving a brown oil. The oil was dissolved in rnethanol (0.5 rnl), and the solution was applied to two 0.5 r~n silica gel plates (20 cm x 20 cm). The plates were developed using chloroforrn/methanol/aqueous amnonia (80/20/1) as the mobile phase. The silica gel strip containing the product (located by uv) ~as scraped off and the product extracted with chloroforn~/methanol (60/40; 2 x lO0 rnl). Ille canbined extract was filtered and the solve solvent removed under reduced pressure. Ethyl acetate (30 ml) was added and the solution filtered. Evaporation of solvent gave ~-t-butylamino-3-chloro-4-(3-hydroxypropyl)propiophenone hemi-succinate (25.4 mg~ as a yellow oil.Analysis: l) thin layer chromatography~ 0.15 on silica gel (0.2~) plates developed in chloroform~methanol/aqueous amnonia (80/20/1); 2) uv (in iso-tonic saline): ~max = 261 nrn,~ Tin = 231 nm; nmr ar~l mass spectometry data were consistent with the assigned stnlcture.

" ( 33~
- 33 -~

EX~MPIE IIl ~n~oGEN PREPAR rION

t-Butylamunc~3-chloro-4-carboxymethoxypropiophenone-Bs~ (C~npound 2-BS~) Ccmpound 2 (25.4 mg; 0.088 mmole) (obtained by basic hydrolysis of the corresponding methyl ester obtained in Example 1) was reacted with BSA (40 mg; 0.00057 mmole) and 1-ethyl-3-(3-di~
methylaminopropyl)-carbodiimide ~EDC) (25 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 ultrafi]tration and the immunogen lyophilized overnight.
EXAMPIE IV
~-t-Butylamino-3-chloro-4-y-hydroxypropylpropiophenone hemisuccinate __ _ -sSA ( CXxnpound 3-BS~) Compound 3 (25.5 mg; 0.064 mmole) reacted wi-th triethyl-amine (TEA, 9.8 mg; 0.097 mmole) and isobutylchloroforma-te (9.8 mg;
0.072 n~ole) in 1 ml dimethylformamide (DMF) at 4C for 30 minu,es to form the mixed anhydride. This reaction mixture was slowly added to a well-stirred solution of B~ (30 mg; 0.00043 mmole) in 4 ml of 0.1 M sodium carbonate at 4C and allowed to react overnight.
The reaction mixture was pressure dialyzed and lyophilized as in Example III.
E~AMPLE V
~-t-~utylamino-3-chloropropiophenone-carboxyrnethyloxime(CQmpound 1) -Bupropion (free base, 500 mg; 2.085 m mole), carbo~y methoxylamine hemihydrochloride (1~3~ g; 6.255 m mole) and anhydrous sodium acetate (684 mg; 8.34 m mole) in 50-r~ aqueous ethanol (10 ml) were allowed to react at 25C for 5 days. The pH was adjusted to 33~

2.0 with 0.1 N HCl, and the mL~ture extrac-ted with chloroform (3 x 30 ml)~ The co~bined extract was evaporated under reduced pressure, redissolved in chloroform (25 rnl), evaporated under reduced pressure and redissolved in chloroform (2 ml). This sol-ution was applied to 8 2 mm silica gel plates (20 cm x 20 cm) which were then developed with chloroform/methanol/aqueous am~onia (80/20/1). The band at Rf 0.2-0.3 was scraped off and extracted with ethyl acetate/methanol 90/10 (5 x 100 ml). The combined extract was filtered, evaporated under reduced pressure and dis-solved in ethyl acetate (5 ml). This was filtered and evaporated under reAùced pressure to give ~-t-butylanino-3-chloropropiophen-one-carboxymethyloxime as an oil (135 mg). Analysis: uv (in iso-tonic saline): ~max = 250 nm, ~ min = 230 r~m; r~r was consistent with the assigned structure.
B. ~-t-Butylamino-3-chloropropionphenone-carboxyme-thyloxime-BSA, _ (Compound l-BSA) CGmpound 1 was coupled to bovine serum albl~nin (BSA) using a water soluble carbodiimide to catalyze the formation of peptide konds between the free acid moiety of II and the ~-amino groups of lysyl residues of BSA. Ccm~ound 1 (25 mg; 0.080 ~ole) was reacted with BSA (40 mg; 0.00057 mmole) and 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimlde (~DC, 25 mg) in 15 ml 10~ dioxane/water at pH 5.7 overnight at 25C. The reaction mixture was press~re dialyzed vs deionized water, concentrated by ultra-filtration and the immunogen lyophilized overnight.
EXAMPIE VI
R-t-Butylamino-3-chloropropiophenone-carboxymethyloxime-125I-tyra-m ne amide, (CGmFound 6) 7~

~3~

Ccmpound 1 was coupled to tyramine using dicyclohexyl-carbodiimide to catalyze the formation of a peptide bond between the free acid of II and the primary amino group of tyramine.
Co~poun 1 (15 mg; 0.048 mmole) reacted with tyramine (free base, 13 mg; 0.096 mmole) and dicyclohexylcarbcdiimi-de (DCC; 19 mg) in 2 ml dry dioxane at p~ 5 overnight at 25~C. The product was purified by TLC, characterized spectrally and subseq~lently iodinated (Hun-ter-Greenw~od Chloramine-T Method).
EXAMPLE VII
10 a-t-Butyla~T ~ 3-chlOro-4-carb~methoxypropiophenone-125I-tyramine amide, (Compound 7) Compound 2 was coupled to tyramine using the nLixed anhyd-ride methcd. Compound 2 (25 mg; 0.076 m mole) was reacted with TEh (9.8 m~; 0.097 m mole) and isobutyl chloroformate (9.8 mg; 0.072 nmcle) in 1 ml DMF at 4C for 30 minutes to form the mixed anhyd-ride. The reaction mixture was slowly added to a well-stirred solution of tyramine (free base, 10 mg; 0.073 mmole) in 1 ml DMF
at 4C and allowed to react overnight to yiela the phenolic product~
The product was purified by TLC, characterized spectrally and 20 subsequently iodinated as in Example VI.
EXAMPLE VIII
~-t-Butylamino 3-chloro-4-y-hydroxypropylpropiophenone-hemisuccin-ate-l 5I tyramine amide,(compoun~
Compound 3 was coupled to tyramine using the mixed anl~yd-ride method of Example VII. This product was purified by ~C an~
subsequently iodinated as described in Example VI.

, ~

~3~

EXAMPLE IX
t-sutylanulo-3-iodo-4-hydrQxy~ropiophenone~(comp~und 9) __ __ Q-t-butylami~c-4-methoxypropiophenone hydrochloride (20.6 mg; 0.076 mmole) was suspended in 1 ml dry methylene chloride and ccoled to -80C in an acetone-dry ice bath. Boron tribromide (41.9 mg; 0.167 mmole) was added, the reaction mixture was protected from moisture with a calcium chloride drying tube, and the reaction was allowed to proceed overnight, during which time the temp~rature rose to 25C. The reaction mixture was shaken with one volume methylene chloride (containung 0.02~ wrdter impurity~

to liberate the product. The product was purified by TIC and subsequently iodinated as described in Example VI.
EX~MPLE X

Prepa~ation of ~-t-butylamino-3-chloro-2-tritiopropiophenone hydrochloride ~ H buprcpion) (Compound 10) A. 2-(3-Chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline 2-(3-Chlorophenyl3-4,4-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-amino-2-methylpropanol to give the amide and cyclization of the amide with thionyl 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 r~) was cooled to -78C and tert-butyllithium (2~0 m moles) in pentane (1.12 ml) was added under nitrogen with stirring. Stirring was continuod a-t ~78C. for 4 1/2 hrs. Solvent-free T2O (~.5 ml, 9o% isotopic purity) was added and the solution was allowed to slowly warm to rocm temF~rature and stirred overnight. m e solution was dried over anhydrous magnesium sulfate and iltered, and the solvent was removed in vacuo to give an orange oil. Purification was achieved by column chrfJI~tography on silica (12.6 g) packed as a chlorofonn slurry. The crude reaction product was added to the column as a chloroform solution. Elution was with chloroform-ethyl acetate (3:1 V~V). F~actions containing the radiolabeled oxazoline were cr~mbined. Solvent remcval 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 was about 90% of theoretical.
B. 2-(3-Chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline N-Methyl-uorosulfonate To 2-(3-chloro-2-tritiophenyl)-4,4-dimethyl 2-oxazoline (670~7 mg) in dry b~nzene (7 ml) under nitrogen was added methane fluorosulfonate ~1.4 g). The mixture was stirred overnight at rocm temperature under nitrogen. The white precipitate was collected under nitrogen, washed with b~nzene (1 x 10 ml) and ether (2 ~ 10 ml) and dried to give 2-(3-chloro-2-tritiophenyl)-4,4-dime-thyl-2-oxazoline N-methylfluorosulfonate in greater than 90~ yieldO
C. 2-(3-Chloro-2-tritiophenyl)-2-ethyl-4,4-dimethyl-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 ethylmaynesium chloride (8.0 m moles~ in ether (2.7 ml). I'he mixture was stirred for 1 hour at ro~m t.emperature and then poured into an ice-water mixture (75 ml) with stirring. m e resulting suspension was extracted with ether .;

~t~

(1 x 100 ml followecl by 1 x 50 ml). rme ctmbined ether extrac-ts were washed with water (1 x 50 ml) and dried over anhydrous mag-nesi~n sulfate. Filtration and removal of solvent ln vacuo gave 2-(3-chloro-2-tri-tiophenyl)-2-ethyl-4,4-dimethyl~N-me~hyloxa zolidine (83% yield) as a yellow oil.
D. 3-Chloro-3-tritiopropiophenone _ . _ A mixture of the crude oxazolidine frtm step C ~670.5 mg) ar}d oxalic acid (475~6 mg) in water (10 ml) was heated a-t reflex for 1 hour and then cooled to room temperature. Additional wa-ter (15 ml) was added, and the product was extrac-ted with ether (2 x 15 ml). The ether layer was washed with 5% potassium bicarbonate solution (2 x 15 ml) and dried over anhydrous magnesium sulfate.
Solvent was removed in vacuo -to give 3-chloro-2-tritiopLopiophenone (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 ~-bra~o 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 Bupropion Samples of six standard solutions of bupropion in blank plasma having concentrations of 0.39 -to 50 ng/~1 are pipetted (0.1 ml each) in duplicate into 12 x 75 mm plas-tic tubes. ~uplica-te non-specific binding (no an-tibcdy) and ma~irr~n binding -tubes (no unlabeled bupropion) receive 0.1 ml blank plasma. Spiked plasma controls at several concen-trations of (e.g., 1.0, 10.0, and 100 "~, 3~

ng/ml) and plaqma s~mples for assay (diluted into the standard range with blank plasma if necessary) are assayed in duplicate.
Pfter being placed in an ice bath, all tubes receive (in order) 0.2 ~1 H bupropion (416 p~. contai m ~g approximately 10 cFm) an~ 0.7 ml anti-bupropion serum (raised to a ring-functionalized immunogen accordin~ to the present invention) (at pre-determ-ined dilutions such that approximately 40% of ~he H bupropion added is bound), both in assay buffer (0.05 M phosphate-buffered iso-tonic saline, containing 0.01 M EDTA and 0.1~ gelatin, p~ 7.0).

Non-specific ~indi~g tubes receive 0.7 ml assay buffer.) Following inc~ibation either at 25C for 2 hours or overnight a~ 4C, bouna and free 3H bupropion are separated by incubation for 10 min. at 0C with 0.5 ml ice-cold dextran-coated charcoal solution (0.25~
dextran, 5mg/ml charcoal, in assay buffer) followed by centrifug-ation for 10 minutes at 4C (5000 rFm~ to pellet the charcoal.
The supernatants, containing antibody-bound 3H bupropion are care-fully decanted into liguid scintillation cocktail for quantitation of 3H. After counts (minus average non-specific binai~g cpm) are expressea as percentages of average maximum binding c~m, bupropion concentrations in plasma samples ana spiked controls are read from a standara curve ~log concentration vs. percent average maximum binding) .
EXAMPLE XII
m e procedure of Example XI has been applied to a study of bupropion (supplied as Wellbutrin*, produced by Burroughs W~llcome Co.) pharmacokinetics in pla~na after oral administration of 200 mg bupropion-HCl (in two 100 mg tablets) to four normal male subjects.
Venous blor~d samples were collected at various times following *trade mark --dosing, and concentrations in plasma samples determined as des-cribed. The as~a~ standard curve obtained by this me~d (+inter-assay standard deviation, N-8) is shown in Figure 1. Spiked controls ( at 1, 10 and 100 ng/~l) run with 8 separate assays gave inter-assay values of 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 and ~NLIN computer programs. The mean elimination half-life, maximum plasma bupropion concentration and time to reach this maximum concentration were 2.9 + 0.4(S.D.) h~urs, ?17~ 37 ng/ml, and 1.5 + 0.4 hours~ respectively~
EXAMPLES XIIl-XV I
RIA of Bu~ropion using I-labeled ~upropion Competitors RI~'s have been run according to the met~d of Example X utilizing 125I labeled kupropion cQmpetitors of the present invention in conjunction with antisera obtained following imn~ni-zation with immunogens of the present invention. Cbmpound 6 was used in conjunction with anti-ccmpound 1 sera. Compounds 7, 8 and 9 were each used with anti-cQ~npound 2 sera, and with anti-compound 3 sera. Following the etnanol precipation of antibody-bour~ I-bupropion competitor at equilibrium, I in the protein pellets was quantified. Figure 2 shcws standard curves obtained with the various iodinated competitors.
It can be seen that compound 9! in conjunction with either antisera produces RIA's appro~imately ten times more sensitive than the ccmpound 10 (3H bupropion) assay system. Substitu-tion of 7 cr 8 yields sensitivity approximately equal to that obtained with the :

fil -compound 10 system. The anti-canpol~nd 1 sexa and cornpour~ 6, which preferably are used for RIA only in conjunction with each other, produced the least ser~itive RI~. However, ky vir~ue of derivatiz~tion via the ketone, these antisera are believed to exhibit much less cross reaction with ring hydroxylated metabolites, albeit at the expense of specificity for changes in struc~ure at the ketone~
- EXAMPLE XVIII
Preparation of ~-t-butylamino-3-chloro-2-tritiopropiophenone hydro-chloride (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-amino-2-methylpropanol to give the amide and cyclization of the arnide with thionyl chloride followed by neutralization with dilute sodium hydroxide to give 2-(3-chlorophenyl)-4,4-dimeth~1-2-oxa-zoline.
I~e oxazoline (419.2 mg) in dry ether (15 ml) was cooled to -78C and tert-butyllithium (2.Gm moles) in pentane (1.12 ml) was added under nitrogen with stirring. Stirring was continued at ~78C for 4 1/2 hours. Solvent-free T2O (0.5 ml, 90% isotopic purity~ was added, and ~he solution was allowed to slowly warm to room temperature and stirred overnight. The solution was dried over anhy~rous magnesium sulfate and filtered, and the sol-vent was removed in vacuo to give an orange oil. Purification was achieved by col mn chr~natography on silica (12.6 g) packed as a chloroform slurry. Ihe crude reaction product was added to the .~

~3~
- ~2 -column as a chloroform solution. Elution was wi-th c~oroform-ethyl acetate (3:1 V~V). Fractions contain~g the radiolakeled oxazoline were combined. ~Solvent removal Ln vacuo gave 2-(3-chloro-2-tritiop~enyl)-4,4-dirnethyl-2-Oxazoline (334 mg, 79~) as a a yellow oil. Nmr and mass spectral data confirme-~ the structure;
the radiolabel WdS about 90% of theoretical.
B. 2-(3-C}~oro-2-tritjopheny~)-4l4-dimethyl-2-oxazoline N-Methyl-fluorosulfonate ~ =
Io 2-(3-chloro-2-tritiophenyl)--4,4-dimethyl-2-oxazoline (670.7 mg) in dry benzene (7 nLl) under nitrogen was added methane fluorosulfonate (1.4 g). The mixture was stirred overnight at roam temperature under nitrogen. The white precipitate ~as co~l-ected under nitrogen, washed with benzene (1 x 10 ml3 and ether ( 2 x 10 ml) and dried -to give 2-(3~chloro-2-tritiophenyl)-4,4-dimethyl-2-oxazoline N-methylfluorosulfonate in greater than 90%
yield.
C. 2-(3-chloro--2-tritiophenyl)-2-ethyl-4~4-dimethyl-N-meth oxazolidine ~b 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 ethylmagnesium chloride (8.0 m moles) in ether (2.7 ml). The mi~ture was stirred for 1 hour at room temperature and then ~oured into an ice-water mixture (75 ml) with stirring. The resulting suspension was ex-tracted with ether (1 x 100 ml followed by 1 x 50 ml). The comhined ether extracts were washed with water (1 ~ 50 ml) and dried over anhydrous mag-nesium sulfate. Filtration and removal of solvent Ln vacuo gave 2-(3-chloro-2-tritiophenyl)-2-ethyl-4,4-dimethyl-N-methyloxazol-~, 31~

idine (83% yield) as a yellow oil.
D. 3-Chloro-2-tritiopropiophenone A mixture of the crude o~azolidine frcm step C (670.5 mg) and oxalic acid 1475.6 mg) in water (10 ml) was heated at reflex for 1 hour and then cooled to roorn temp~rature. Additional wate,r ( 15 ml) was added, and the product was extracted with ether (2 x 15 ml). The ether layer was washed with 5% po-tassiurn bicarb3nate solution (2 x 15 ml) and drie,d over anhydrous magnesiurn sulfate.

Solvent was removed ir. vacuo to give 3-chloro-2-tritiopropiophen-one (405 mg).
E. ~-t-Butylamino-3-chloro-2-tritiopropiophenone hydrochloride Following the procedure of ~.S.Patent No. 3,819,706, 3-chloro-2-tritiopropiophenone was brominated and the a-bro~3 product treate,d with tert-butylamine to give a-t-khltylar~ino-3-chloro-2-tritiopropiophenone which ~s isolated as the hydro-chloride (43% yield).

't3~

qq _ SYNTHESIS ME~IOD A
_ _ . _ _ _ H CO C H NaOCH
t Br C 2 2 2 5 I SO,~C12 ~CH CH30~ ~
CO-CH-Br CO C2H5 1) (CH3) 3C~2 2) HCl ~1 Cl C~H3 CO-C!H-NH-C (CH3 ) 3

Claims (6)

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

1. An immunogen comprising a conjugate comprising a compound of formula (XX):

(XX) wherein q is an integer of from 1 to 3, coupled to a suitable immunogenic carrier material.

2. An immunogen according to claim 1, wherein the immunogenic carrier material is bovine serum albumin.

3. An immunogen according to claim 1 or 2, in which q is 1.

4. A method of making an immunogen according to claim 1, comprising reacting said compound of formula (XX), as defined in claim 1, with an immunogenic carrier material and a suitable coupling agent.

5. A method according to claim 4, wherein said carrier material is bovine serum albumin.

6. A method according to claim 4 or 5, in which q is 1.