United States Patent 9} Wagner et al, [54] {751 (731 [22) an (521 (1) 158) (56) 3,754,021 METHADONE ANALOG COMPOUNDS Inventors: Daniel Wagner, Palo Alto; Edwin F, ‘Ullman, Atherton, both of Calif. ‘Assignee: Syva Company, Palo Alto, Calif. Filed: Sept. 5, 1972 Appl. No.: 286,519 US. Ch oss 260/404, 260/112 R, 260/112 B, 260/112.5, 260/293.76, 260/326.47, 260/463, 260/570.8 K, 424/12 Cite 3/00 260/404 Int. Cl. Field of Search... References Cited UNITED STATES PATENTS. 8/1973 Shen et al 260/518 R un 3,843,696 145] Oct. 22, 1974 Primary Examinet—Lorraine A. Weinberger Assistant Examiner—L. A. Thaxton 7) ABSTRACT Methadone derivatives are provided for use in prepar- ing reagents for immunoassays and for preparing anti- bodies to methadone. A carboxy group is bonded through an aliphatic chain to a group which conforms to a substantial portion of methadone. Accurate and selective immunoassays are obtained by employing a modified methadone derivatized detector with the an- tibodies prepared to the methadone analog. 4 Claims, No Drawings3,843,696 1 METHADONE ANALOG COMPOUNDS BACKGROUND OF THE INVENTION |. Field of the Invention Immunoassays have been finding wide application for 5 assaying for physiologically active materials. By em- ploying naturally occurring receptors, one is frequently capable of assaying for a class of compounds, a small group of compounds, and in many instances, a single compound, where a number of other compounds may '0 be present of similar and/or dissimilar structure. ‘Among the most popular naturally occurring receptors for immunoassays are antibodies. Since a large number of compounds which are of interest for assaying are not antigenic, but rather haptenic, itis usually necessary to modify the compound of interest, so as to be able to bond the compound to an antigenic protein. The hap- ten modified protein may then be introduced into an animal for production of antibodies to the hapten. In modifying the hapten to introduce an active fune- tionality, one must consider a wide variety of potential problems. The modification of the hapten must occur in such a way that antibodies which are formed will rec- ognize the hapten itself. In addition, it may or may not be desirable that the antibody recognize one or more metabolites of the hapten. Also, where the assay is per- formed by causing a competition for antibody sites be- tween the naturally occurring hapten and a hapten bonded to a detector, itis essential that the bridging group between the hapten and the detector allow for binding to the antibody in competition with the natu- rally occurring hapten. ‘The group introduced must permit formation of anti- bodies to the free hapten. The bridging group should be capable of being activated, so as to be active with both proteins and, where employed, the detector. It is often preferable to employ the same linking group for bond- ing to the protein antigen, that is employed for forma- tion of the antibody, as is employed for bonding to the detector. ‘Also, the modified hapten when joined to a detector should provide a product which is soluble in the assay medium at the concentrations employed. Because aqueous solvents are employed and the haptens and/or the detectors are frequently hydrophobic, the bridging group usually should be devised so as not to greatly en- hance the lipophilicity of the product. 2. Description of the Prior Art Copending application Ser. No. 143,609, filed May 14, 1971 now abandoned, describes a method for im- munoassays employing enzymes as the detector. Co- pending application Ser. No. 141,516 now abandoned, filed May 10, 1971, describes the method of immuno- assays employing stable free radicals as the detector. M.M. Baiser, Bull. of Narcotics, 1953, 32, in an article entitled Methadone Chemistry, describes the method of synthesis for methadone. SUMMARY OF THE INVENTION 2,2-Diphenyl-4-dimethylaminopentanoyl substituted aliphatic carboxylic acids are used in the preparation of | derivatives for preparing antibodies and for joining with detector systems to be used in immunoassays for methadone. Detecting systems of special interest are enzymes and stable free radicals. 20 as 30 35 40 4s so Cy 6s 2 DESCRIPTION OF THE SPECIFIC EMBODIMENTS The subject compositions are 2,2-diphenyl-4-dim: thylaminopentanoyl substituted aliphatic carboxylic acids and derivatives of such carboxylic acids, such as the mixed anhydride with carbonates, esters and a wide variety of amides. Included among the amides are those derived from synthetic or naturally occurring polype tides. The subject compounds are useful for derivati2- ing detector molecules having amino groups, such as enzymes and nitroxide amines. ‘The amides, depending upon the group bonded to the amide nitrogen atom, find use in preparation of ant 5 bodies to methadone, and as reagents in the carrying cout of immunoassays for the determination of metha- done. In preparing antibodies to methadone, an acti- vated derivative of the aforementioned carboxylic acid is combined with an antigenic substance, normally a polypeptide or protein, to provide at least one metha- done derivative bound to the antigenic substance. The resulting product is then introduced into an animal to initiate antibody formation to methadone. By following known techniques the antibodies or y-globulins are harvested and may be subjected to further treatment, as required. The compounds of this invention and derivatives thereof will for the most part have the following for- mula: ( wherein R is a divalent aliphatic group of from 4 to 10 carbon atoms and 0 to I heteroatoms (O, N, with N being bonded solely to carbon) other than terminal, usually aliphatic hydrocarbon (hereinafter referred to as “‘aliylene”) of from 4 to 6 carbon atoms, which may be straight chain or branched chain, usually straight chain, or if branched having not more than two branches, usually methyl, and has from 0-1 site of ali phatic unsaturation, usually ethylenic, there being at least four carbon atoms between the two carbonyl groups; and Z is hydroxyl, nitrophengl, alkyl carbonate (OCO,R! wherein R! is alkyl of from 1 to 6 carbon atoms, more usually 1 to 4 carbon atoms), —Y, wherein Y is a poly. Peptide residue (including polypeptide subunits of pro- teins) and —NH—X, wherein X is a stable free radical ‘group, usually nitroxide, and more usually cyclic ni: troxide. n is one except when Z is —Y, when 1 will be equal to the number of acyl groups bonded to the amino groups of Z and will be at least 1 and not greater than the number of amino functional groups available for bonding, usually not more than the molecular ‘weight of Z divided by 500, more usually not more than the molecular weight divided by 1,500; m is 0 or 1; and E is an anion of a mineral acid, e'g., CI", Br-; HSO.-, ete, The carboxylic acid (Z=OH) will for the most part have the following formula: ° ty COHCHO—CRcomt coum gf3,843,696 3 wherein R? is a divalent aliphatic group having 0 to I heteroatoms (O,N) of from 4 to 10 carbon atoms, ‘more usually aliylene of from 4 to 6 carbon atoms, hav- ing from 0-1 site of aliphatic unsaturation, usually eth- ylenic, and may be straight chain or branched chain, preferably straight chain, there being at least 4 carbon atoms between the two carbonyl groups, Usually a het- eroatom will be separated from a carbonyl group by at least 2 carbon atoms, Illustrative groups for R* are tetramethylene, penta- methylene, hexamethylene, _octamethylene, 3,3-dimethylpentylene, 2-methythexylene, 3+ oxapentylene, N-methyl 3-azapentylene, etc. Usually the two valences of R® will be alpha-omega, the terminal carbon atoms being primary. ‘The mixed anhydride which finds use in this inven- tion will have the following formula: M—R*—CO,CO,R® wherein M has the formula: cHoHcHc—6— cua R? is as defined previously; and Ris alkyl of from 1-6 carbon atoms, more usually of from 2-4 carbon atoms, e. ethyl, isopropyl, butyl and hexyl. Of particular interest are compounds where the cat- boxyl group is bonded to an amino group, which is part of a polypeptide or protein structure. One group of polypeptides and proteins is antigenic, so that by bond- ing the carboxyl modified methadone to the polypep- tide or protein, antibodies can be formed to metha- done. A narrower class of proteins, which also can be used as antigens, but will not normally be used as such, are enzymes which are employed as the detector in an immunoassay system. As antigens, inactive enzymes can be used. Polypeptides usually encompass from about 2 to 100 ‘amino acid units (usually less than about 12,000 molec- ular weight). Larger polypeptides are arbitrarily called proteins. Proteins are usually composed of from 1 to 20 polypeptide chains, called subunits, which are associ- ated by covalent of non-covalent bonds. Subunits are normally of from about 100 to 300 amino acid groups (or 10,000 to 35,000 molecular weight). For the pur- poses of this invention, polypeptide is intended to in- clude individual polypeptide units and polypeptides which are subunits of proteins, whether composed solely of polypeptide units or polypeptide units in com- bination with other functional groups, such as porphy- ris, as in haemoglobin or cytochrome oxidase ‘The number of methadone groups will vary depend- ing on whether the polypeptide is an enzyme or anti- gen. The maximum number of groups will be limited by the effect of substitution on solubility, activity and the like. For the formation of antibodies, a sufficient num- ber of methadone groups should be present, so as to provide a satisfactory harvest of antibodies to metha- done. Otherwise, the proportion of antibodies to meth- adone as compared to other protein may be undesir- ably low. 0 25 35 40 45 30 35 60 4 The first group of protein materials or polypeptides which will be considered are the antigenic polypep- tides. These may be joined to the non-oxocarbonyl group of the carboxy modified methadone through an amino group. The amide product can be used for the formation of antibodies to methadone. The protein ma- terials which may be used will vary widely, and will nor- mally be from 1,000 to 10 million molecular weight, more usually 25,000 to 500,000 molecular w: With the antigens, there will be no more than one methadone group per 500, more usually 1,000 molecu- lar weight, generally no more than one methadone group per 2,000 molecular weight and not less than one methadone group per $00,000 molecular weight, usu- ally not less than one methadone group per 50,000 mo- lecular weight. With intermediate molecular weight an- tigens, those having molecular weights in the range of 0,000 to 1,000,000 the number of methadone groups will generally be from about 2 to 250, usually from 4 to 100. Low molecular weight antigens (1,000 to 5,000 molecular weight) may have 1 to 10, usually 2 to 5 methadone groups so that there may frequently be as many as one methadone group per 500 molecular weight. Enzymes will normally be of molecular weights in the range of about 10,000 to 600,000, usually in the range of about 12,000 to 150,000, and more usually in the range of 12,000 to 80,000. Some enzymes will have a plurality of enzyme subunits. Itis intended when speak- ing of enzyme molecular weights to refer to the entire enzyme. There will be on the average at least about one methadone per enzyme, usually at least about two methadones per enzyme, when the labelling is not lim- ited to a specific amino group, and rarely more than 40 methadones per enzyme, usually not more than 35 methadones per enzyme. For example, with lysozyme the average number of methadone groups will be in the range of about 2 to 4, With malate dehydrogenase, the average number of methadones will be from 4 to 35, Specific labelling techniques can be used to introduce ‘one methadone per enzyme. While the methadone may be bonded through the carboxyl group to hydroxy! or mercapto groups, which are present in the proteins, for the most part the bond- ing will be to amino. Therefore, the compounds are de- scribed as amides, although esters and thioesters may also be present. Amino acids present in proteins which have free amino groups for bonding to the carboxy modified methadone include lysine, arginine, ornithine, ete. The hydroxyl and mercaptan containing amino ‘acids in- clude serine, cysteine, and threonine Various protein and polypeptide types may be em- ployed as the antigenic material. These types include albumins, enzymes, serum proteins, e.g. globulins, ocu- lar lens proteins, lipoproteins, etc. Mlustrative proteins include bovine serum albumin, keyhole limpet hemocy- anin, egg albumin, bovine gamma-globulin, etc. Small neutral polypeptides which are immunogenic such as gramicidins may also be employed. Various synthetic polypeptides may be employed, such as polymers of ly- sine, glutamic acid, phenylalanine, tyrosine, etc, either by themselves or in combination, Of particular interest is polylysine or a combination of lysine and glutamic acid. Any synthetic polypeptide must contain a suffi- cient number of free amino groups, as for example, provided by lysine.