Aminophenyl Esters of 5-Carboxyalkyl Barbituric Acids
Abstract
Aminoaryl esters and amino-lower alkyl amides of carboxy substituted barbiturates are linked via an amide linkage to carboxylated latex polymers to form reagents which are useful in a sensitive diagnostic test to detect the presence of barbiturates in body fluids. The large increase in the abuse of therapeutic agents, particularly the barbiturates, by the general population as well as military personnel, has brought with it a substantial need to improve analytical techniques for the determination of such agents in biological fluids. In many instances, medical treatment centers are faced with the immediate need for determining the identity of a barbiturate taken by a patient who is unable, being in a comatose condition, or unwilling to supply such information to the treating physician. Early procedures involved the identification of barbiturates by extraction and thin-layer, gas chromatographic and spectrophotometric methods. These techniques have the disadvantages of being relatively time-consuming, laborious and lacking great sensitivity. Recently, a rapid and sensitive immunoassay procedure involving the reaction between antibodies and barbiturate antigen was described by S. Spector in U.S. Pat. No. 3,766,162 and by S. Spector and E. J. Flynn in Science, 174, 1037 (1971). This procedure, however, requires sophisticated and expensive equipment, such as scintillation counters. Therefore, it would be desirable to develop a rapid and highly sensitive assay for detecting the presence of barbiturates in biological fluids which would not require sophisticated equipment and could be easily performed on site by laboratory technicians having a minimum of training. The present invention relates to a novel class of barbiturate derivatives, namely, aminoaryl esters and amino-lower alkyl amides of carboxy substituted barbiturates, which esters and amides may be covalently coupled via an amide linkage to a carboxylated latex polymer. The barbiturate thus linked to the latex polymer by means of the linking group can then be utilized as a reagent in a sensitive diagnostic assay for the presence of barbiturates in biological fluids. This assay method is dependent upon the well known binding of antigen to antibodies specific therefor, which is manifested by an insolubilization or agglutination followed by flocculation. When either the antigen or the antibody is linked to a suitable polymer such as a latex polymer, as hereinafter described, the detection of the antigen-antibody binding by means of agglutination is significantly enhanced by means of the latex so that such agglutination reaction is easily visualized by the naked eye. The general technique of utilizing latex particles as carriers for antigens or antibodies for easy visualization of the antigen-antibody reaction has been previously described in the literature, for example, U.S. Pat. No. 3,857,931. The starting materials which are used for the preparation of the latex reagents of the present invention are aminoaryl esters and amino-lower alkyl amides of carboxy substituted barbiturates. As used herein, the expression "lower alkyl" is meant to include straight and branched-chain saturated hydrocarbon radicals having from 2 to 8 carbon atoms, inclusive, such as ethyl, propyl, n-butyl, iso-butyl and the like. The term "aryl" denotes an aromatic radical derived from an unsubstituted or substituted arene and includes phenyl, naphthyl, halophenyl, tolyl, anisyl, nitrophenyl, hydroxyphenyl and the like. The term "halide" denotes iodide, bromide and chloride. The barbiturates useful for binding to latex polymers are those having free carboxylic acid groups. The barbiturate of particular preference in the practice of the present invention is 5-allyl-5-(1-carboxy-isopropyl)barbituric acid (allonalcarboxylic acid) since it has a carboxylic acid group in the sidechain and is readily obtainable. Thus, particularly preferred reagents are aminoaryl esters and amino-lower alkyl amides of the carboxylic acid group of allonalcarboxylic acid. However, the present assay, as hereinafter described, will detect barbiturates with or without free or functionalized carboxylic acid groups, such as barbital, phenobarbital, amobarbital, butabarbital, pentobarbital, etc. The aminoaryl esters and amino-lower alkyl amides of carboxy substituted barbiturates, as described above, are conveniently prepared from carboxy substituted barbiturates. Using allonalcarboxylic acid as an example, one can conveniently introduce the requisite aminoaryl or the amino-lower alkyl moiety by methods well known in the art. Thus, for the preparation of aminoaryl esters of the carboxy substituted barbiturates, one can esterify allonalcarboxylic acid with, for example, p-nitrophenol to afford 5-allyl-5-(1-p-nitrophenyloxycarbonyl-isopropyl)barbituric acid which one can then reduce to 5-propyl-5-(1-p-aminophenyloxycarbonyl-isopropyl) barbituric acid. The esterification is performed in the presence of a condensing agent dissolved in an inert organic solvent. Suitable condensing agents include carbodiimides such as N,N'-diphenylcarbodiimides and N,N'-dicyclohexylcarbodiimides. Suitable inert organic solvents include polar aprotic solvents such as N,N-dimethylformamide, dimethylsulfoxide and hexamethylphosphoramide alone or admixed with non-polar aprotic solvents such as acetone, acetonitrile and ethyl acetate. A particularly preferred condensing agent is N,N'-dicyclohexylcarbodiimide and a particularly preferred organic solvent system is N,N-dimethylformamide-ethyl acetate. The temperature of the esterification reaction is not narrowly critical. The reaction may be carried out between about 0.degree. and 50.degree. C., most preferably at about 0.degree. to 25.degree. C. The reduction is performed by treating the nitroester, so obtained, dissolved in a suitable inert organic solvent, preferably an alkanol such as methanol, ethanol, or 2-propanol, with hydrogen in the presence of a suitable hydrogenation catalyst until the cessation of the uptake of hydrogen. Included among suitable hydrogenation catalysts are platinum, palladium, rhodium, ruthenium and nickel, unsupported or supported on carriers such as carbon, silica, alumina and the like. A particularly preferred hydrogenation catalyst is 10% palladium-on-carbon. While the temperature and pressure at which the hydrogenation is accomplished is not critical, it is preferred to carry out the reaction at about room temperature and about atmospheric pressure. For the preparation of amino-lower alkyl amides of carboxy substituted barbiturates, one can aminate allonalcarboxylic acid with, for example, 1,4-diaminobutane to afford 5-allyl-5-[1-(4-aminobutylcarbamoyl)-isopropyl]barbituric acid. The amination is performed in the presence of a condensing agent dissolved in an inert organic solvent. Suitable condensing agents include carbodiimides such as N,N'-diphenylcarbodiimide and N,N'-dicyclohexylcarbodiimide and carbonyldiimidazoles such as 1,1'-carbonyldiimidazole. Suitable inert organic solvents are ethereal solvents such as monoglyme, diglyme, dioxane and tetrahydrofuran. A particularly preferred condensing agent is 1,1'-carbonyldiimidazole. A particularly preferred inert organic solvent is tetrahydrofuran. The temperature at which the amination is carried out is not narrowly critical. A reaction temperature within the range of about 0.degree. to about the boiling point of the solvent is preferred, a reaction temperature of about 25.degree. C. being most preferred. The carboxy substituted barbiturates are also conveniently prepared by methods well known to a chemist of ordinary skill in the art. For example, the carboxy substituted barbiturates can be readily prepared by reductive alkylation of barbituric acid with an aldehydo- or keto-ester followed by alkylation and saponification of the ester group of the resulting 5,5-disubstituted-barbituric acid. The reductive alkylation is conducted by treating barbituric acid and an aldehydo- or keto-ester such as ethyl formylacetate, ethyl acetoacetate and ethyl levulinate, with hydrogen in the presence of a metal hydrogenation catalyst to afford a 5-monosubstituted barbituric acid derivative. Suitable metal catalysts are nickel and the nobel metals such as platinum, palladium, rhodium, ruthenium and so forth. The catalysts are normally employed in finely divided form and may be either unsupported or present on a suitable inert carrier such as carbon, aluminum, silica, calcium carbonate and the like. A particularly preferred catalyst is 10% palladium-on-carbon. As solvents for the reductive alkylation, there may be mentioned alcohols such as methanol, ethanol, 2-propanol and the like, and esters such as ethyl acetate and so forth. While the reductive alkylation may be performed over a wide range of temperatures and pressures from, for example, about room temperature to about 150.degree. C. and about atmospheric pressure to about 1000 psi, it is preferable to employ a reaction temperature of about 90.degree. to about 100.degree. C. and a pressure of about 700 psi. The alkylation step is accomplished by treating the monosubstituted barbituric acid derivative with an alkylating agent such as methyl halide, propyl halide, allyl halide, hexyl halide, cyclohexenyl halide and the like, in a suitable inert solvent in the presence of a base to afford a 5,5-disubstituted barbituric acid derivative. Suitable solvents include, among others, alcohols such as methanol, ethanol, 2-propanol and the like, water and mixtures of water and alcohols. Suitable bases are alkali metal and alkaline earth hydroxides such as sodium and potassium hydroxide and calcium hydroxide, and alkali metal alkoxides such as sodium methoxide, potassium ethoxide and potassium tertiary-butoxide. A particularly preferred base and solvent system is about 20% aqueous sodium hydroxide. The alkylation may be conducted over a temperature range of from about 10.degree. to about 80.degree. C., most preferably between about 25.degree. to 60.degree. C. The saponification step is conducted by treating the disubstituted barbituric acid derivative with an aqueous acid such as hydrochloric acid, hydrobromic acid, dilute sulfuric acid and the like, at an elevated temperature. For this conversion, aqueous hydrochloric acid having a normality of about 1 and a reaction temperature of about the reflux temperature of the reaction mixture is preferred. In order to prepare the diagnostic reagent useful for the practice of the present invention, aminoaryl esters and amino-lower alkyl amides of carboxy substituted barbiturates are covalently bonded by means of an amide linkage to a latex polymer containing carboxyl groups. Suitable latex polymers for this purpose are carboxylated styrene butadienes, carboxylated polystyrenes, acrylic acid polymers and the like. Among the commercial latex polymers which are included in the aforementioned classes are Dow 421, Dow 816, Dow 620, Fluka 241 and Dow 241. Dow batch 1721, a latex polymer of the polystyrene type having a particle size of about 0.2 to about 0.3 microns, percent solid composition of about 8 to about 12% and a specific gravity of about 1.02, is also suitable. Particularly preferred polymers are carboxylated styrene butadiene copolymers, preferably Fluka 241 or Dow 241. Suitable latex carrier particles are generally supplied commercially as an aqueous latex suspension, usually in concentrations of about 5 to about 60% solids. These polymers are water insoluble, have a particle size in the range from about 0.01 to about 0.9 microns, preferably between about 0.1 and about 0.3 microns, and a specific gravity near that of water enabling them to remain in aqueous suspension. The particles should have sufficient surface charge density so that when coupled to the aminoaryl esters and amino-lower alkyl amides of carboxy substituted barbiturates, their repulsive forces are enough to prevent aggregation. The aminoaryl esters and amino-lower alkyl amides of carboxy substituted barbiturates are coupled to the carboxylated latex polymers by means of an amide linkage initiated in the presence of a water soluble carbodiimide condensing agent. The degree of coupling is dependent upon the density of the reactive groups in the polymer. The density of the reactive groups is not critical to the operability of this invention, as long as a sufficient number of reactive groups are present to provide coupling of a sufficient amount of barbiturate moiety to be useful in a diagnostic test. However, a suitable density would be in the range of from about 1 to about 5%, preferably about 3%, by weight. The coupling reaction with carbodiimides is described in detail in U.S. Pat. No. 3,857,931. Once the latex coupled product is formed, it can be utilized in specific diagnostic tests for the detection of barbiturates. It can be used in any convenient concentration, depending upon the specific test and samples involved. However, concentrations of from about 0.1 to about 2% by weight of latex solids are suitable and the preferred concentrations are from about 0.3 to about 1.5% by weight. In a typical test, a measured amount of antiserum against barbiturates is mixed with a barbiturate free body fluid, for example, serum, saliva or urine. Then, a measured amount of aminoaryl or amino-lower alkyl barbiturate coupled latex is added and the mixture is allowed to incubate at a slightly elevated temperature, e.g., 37.degree. C., for a period of time, for example, from about 1 to about 3 hours, preferably for about 2 hours. The pH of the text mixture is suitably in the range of from about pH 5.0 to 8.5, most preferably about 6.5 to 7.5. After the incubation, flocculation or agglutination of the latex particles is noted. The concentration and quantity of both the antiserum and the latex complex are adjusted to produce a strong flocculation, and the minimum concentrations of both reagents which produce a strong flocculation are determined. The mixture of antiserum against barbiturate and barbiturate free body fluid may be incubated at a slightly elevated temperature, e.g., 37.degree. C., prior to the addition of the aminoaryl or amino-lower alkyl barbiturate coupled latex. The antisera which may be used in the present diagnostic test are antisera specific for barbiturates, such as secobarbital and pentobarbital. The preparation of such antisera is described in U.S. Pat. No. 3,766,162 and in Science, 174, 1037 (1971). After the control system is set up, as described above, various amounts of barbiturates, e.g., secobarbital, pentobarbital, butabarbital, amobarbital, phenobarbital and barbital are dissolved in barbiturate free body fluid. The minimum amount of barbiturate required to inhibit the flocculation is noted. This quantity will depend both upon the concentration and the amount of body fluid added, as well as upon the concentration and the strength of the antiserum utilized in the test. In a preferred test, the quantities and concentrations are adjusted so that approximately 400-500 microliters of serum or urine containing between about 100 and 200 nanograms of barbiturate per milliliter (total of between 40-50 and 80-100 nanograms of barbiturate) will be just sufficient to inhibit flocculation. Once the test has been standardized with one type of body fluid, for example, urine, another type of body fluid, for example, serum, should not be substituted, and a separate standard must be set up for this. Since the presence of flocculation is easily visualized by the naked eye, the present test serves as an extremely sensitive assay method for the detection of barbiturates, such as secobarbital, pentobarbital, butabarbital, amobarbital, phenobarbital and barbital. Thus, once the test has been standardized as mentioned above, the presence of nanogram quantities of these barbiturates in body fluids can easily be detected by noting the inhibition of flocculation caused by the presence of such barbiturates in the body fluid, as compared with the flocculation resulting when barbiturate free body fluid is employed. The test can be standardized so that a medically and statistically meaningful cut-off point is established. Thus, quantities of barbiturates in body fluid greater than this amount will cause inhibition of flocculation (a positive test for the presence of such drug in the body fluid) and quantities less than this amount will not inhibit flocculation (a negative test). The above described reagents can be conveniently packaged for commercial purposes, e.g., in a diagnostic reagent kit containing two separate containers: one with the antiserum against barbiturates and the other with the aminoaryl esters or amino-lower alkyl amides of carboxy substituted barbiturates bonded via an amide linkage to latex particles containing carboxyl groups, most preferably in aqueous suspension. The aminoaryl esters and amino-lower alkyl amides of carboxy substituted barbiturates can also be linked to immunogenic carrier materials such as proteins or polypeptides by means of an amide linkage to afford antigens which are useful for the elicitation of antibodies specific for barbiturates. The method of linkage to immunogenic carrier materials, as well as the elicitation of antibodies, are generally described in U.S. Pat. No. 3,766,162. The invention is further explained and illustrated in the following examples. All temperatures are in degrees Centigrade.
Metadata
Assignee
- Hoffmann-La Roche Inc.
Inventors
- Antonino Focella
- John Edward Heveran
- Sidney Teitel
- Manfred Weigele
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