WO1985000605A1

WO1985000605A1 - Process for selective nitrile reduction

Info

Publication number: WO1985000605A1
Application number: PCT/US1984/001146
Authority: WO
Inventors: Wayne Manning; Aida Soto; Mario Castillo
Original assignee: American Hospital Supply Corporation
Priority date: 1983-07-27
Filing date: 1984-07-20
Publication date: 1985-02-14
Also published as: EP0154631A4; EP0154631A1

Abstract

Process for selective reduction of nitrile-functional groups on complex molecules without otherwise alteration of the complex molecule. In brief, the process of this invention involves the use of a reagent comprising cobalt chloride and a stoichiometric excess of an alkali metal borohydride. This reagent enables the selective reduction of a nitrile-functional group of a complex molecule without otherwise alteration of the molecule. The ability to selectively reduce the nitrile-functional group to the corresponding amine can be used to advantage in the cross-coupling of haptens to a variety of labels, such as enzymes and fluorophores. These labelled haptens are useful in immunoassay of biological samples.

Description

PR O C E S S F O R S E L E C T I V E N I T R I L E R E D U C T I O N BACKGROUND OF THE INVENTION

Field of the Invention

This invention is directed to a process and a composition of matter. More specifically, this invention concerns itself with the selective reduction of a nitrile-functional group on complex molecules and the subsequent interaction of these complex molecules via a cross-coupling reagent with a label such as an enzyme or a fluorophore.

Description of the Prior Art

The use of sodium borohydride as a reducing agent is well documented.

In general, it is a non-specific agent which reduces ketones, aldehydes, disulfides and other functional groups under certain stated conditions. This non-specific agent is not regarded as capable of reducing nitrile-functional groups. In the absence of catalysts, its reductive capability is generally limited to carbonyl, amino and hydroperoxide groups, R.C. Wade, J.

Molecular Catalysis, 18:273 (1983). The literature also discloses that sodium borohydride can effect reduction of barbituric acid derivatives. The products of such reduction are generally classifiable in four groups:

a. dihydrobarbiturates where either the carbonyl groups at positions 4 and 6 have been reduced to a secondary hydroxyl group; b. tetrahydrobarbiturates where both the carbonyl groups at positions 4 and 6 have been reduced to secondary hydroxyl groups; c. primary alcohols formed via the reductive cleavage of the barbiturate ring; and d. urea derivatives formed simultaneously with primary alcohols.

The sodium borohydride reduction of barbituric acid derivatives is more fully described in Ventron Alembic, Issue number 27, September 1982.

The combination of sodium borohydride with cobalt, nickel, copper and rhodium halides has been employed to reduce functional groups such as nitriles, amides and olefins which are inert to the sodium borohydride alone, Satoh, T. et al; Chern. Pharm. Bull. 19:817 (1971). The use of cobalt chloride in conjunction with sodium borohydride as a reagent for the selective reduction of nitriles has also been previously disclosed by Heinzman and Ganem; J. Amer. Chem. Soc. 104, 6801 (1982). The authors of this paper suggest that their experiments tend to support the proposition that the cobalt boride (which is formed by the interaction of the sodium borohydride and the cobalt chloride) is capable, by coordinating certain functional groups, to catalyze the heterogeneous reduction by the sodium borohydride. The specific nitrile compounds which were reduced in this fashion included benzonitrile, benzylnitrile and 5-cyano-3-methyl-2,4-pentadienoic acid methyl ester.

The authors of this paper fail to appreciate the specificity afforded by the cobalt chloride in the selective reduction of nitrile-functional groups on complex molecules which are sensitive to sodium borohydride reduction.

The need for such selectivity is particularly critical where such reduction of a nitrile group is attempted on a complex molecule having pharmaceutical properties. Even minor modifications of these compounds will generally change their pharmaceutical properties and thereby prevent their later accurate identification, by either conventional analytical procedures or highly specific immunochemical assays. The presence of a nitrile group on a pharmaceutical compound, either native to such compound or by its synthetic introduction, provides a convenient site for the formation of the corresponding amine which is generally a preferred functional group for the cross-coupling of the drug to a suitable label such a fluorophore or enzyme. Once the drug has been labeled in this fashion, it can be used in conjunction with other reagents for immunochemical analysis of the same drug in a patient sample.

Up to now, a variety of techniques have been previously disclosed for the labeling of drugs with an enzyme or a fluorophore. Typically, these procedures require an elaborate synthetic protocol and, in certain instances, may be unsuitable because the conditions required to effect such crosscoupling can also cause modification of the pharmaceutical agent. As noted hereinabove, even minor modifications of the pharmaceutical agent invariably changes its chemical and/or steric properties; and, thus renders the reagent unsuitable for later use in an analytical protocol. OBJECTS OF THE INVENTION

It is the object of this invention to remedy the above as well as related deficiencies in the prior art.

More specifically, it is the principal object to provide a process which is directive for the synthesis of amine-functional compounds which can be used in the synthesis of reagents for analysis of pharmaceutical agents.

It is yet another object of this invention to provide a process for the synthesis of enzyme-labeled drug conjugates which are suitable for use in immunoassay.

It is yet another object of this invention to provide a process for the synthesis of fluorophore-labeled drug conjugates which are suitable for use in immunoassay.

SUMMARY OF THE INVENTION

The above related objects are achieved by providing an improved process for the selective reduction/modification of a pharmaceutical agent so as to permit its use in the synthesis of an immunochemical agent for immunoassay. This process generally involves the selective reduction of a nitrile-functional group on such pharmaceutical agents to the corresponding amine with a reagent comprising cobalt chloride and a stoichiometric excess of an alkali metal borohydride. The pharmaceutical agent can then be further reacted through this amine-functional group with a bifunctional cross-coupling reagent. The remaining functional sites of the cross-coupling reagent are available for further interaction with a suitable label, such as a fluorophore or an enzyme. DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS

The process of this invention involves the selective reduction of a nitrile group on a complex molecule, such as a pharmaceutical agent, without otherwise alteration of the chemical and/or steric properties of the pharmaceutical agent. The conditions of the reductions are extremely mild and the nitrile group is only reduced to the corresponding amine. This aminefunctional group on the pharmaceutical agent can be the site of later cross-coupling of a suitable label to the pharmaceutically, active agent for the synthesis of a reagent (hereinafter "conjugate") which can be used in immunoassay.

While the basic process of this invention is to be later described in detail for quinidine, it is also applicable to any one of a number of other pharmaceutically active compounds which can tolerate introduction of either a nitrile or an amine functional group (primary or secondary) without alteration of the drug's pharmaceutical and immunochemical properties. For example, conjugates of quinidine can be prepared by initial introduction of a nitrile functional group followed by its selective reduction to the corresponding amine whereas, compounds such as primidone, phenobarbital, ethosuximide and carbamazepine already having an N-H function present can simply be cyanoethylated.

Pharmaceutical compounds containing moieties conducive to cyanoethylation are included in the application of this method. This group includes the barbiturates (possessing N-H groups); steroids such as cortisol and estriol (possessing OH groups); and, purine-based drugs such as theophilline and theobromine (through the N-H moiety).

Other classes of drugs which are amenable to the process of this invention include tricyclic antidepressants (such as protripyline and clomIpramine); opiates (morphine); anticonvulsants (such as methabarbital, phenytoIn and methylphenidate); and, many of the beta-blockers (propranolol, nadolol, oxprenolol, sotalol and timolol being representative). Some drugs must have certain groups protected before derivatization, which can later be deprotected. In pharmaceuticals where multiple reactive groups are present, prior chemical modification is necessary to furnish single derivatives. For example, in the eases of the B-aminoalcohols such as propranolol acetylation by well-known procedures (such as acetic anhydride/anhydrous sodium acetate) and alkaline work-up "protects" the N-H group but leaves the O-H in its reactive form. After cyanoethylation the amide can be hydrolyzed under acid conditions to restore the N-H gorup. Such "protection" and "deprotection" methods are well-established and constitute the present art. See, for example, Protective Group in Organic Synthesis, Theo W. Greene, Wiley & Sons (NYC 1981).

Once the pharmaceutically active compound has been reduced to the corresponding amine and reacted with the appropriate bifunctional reagent, the resultant intermediate can thereafter be readily cross-coupled to an appropriate label. The preferred labels for cross-coupling to this reactive intermediate compound can preferably be either a fluorophore or an enzyme. Fluorophores which are suitable for cross-coupling with such reactive intermediates include (but are not limited to): bimane; 4-methylumbelliferyl derivatives; fluorescein and its derivatives, in particular dichlorotriazinylaminoflorescein (DTAF); rhodamine and its derivatives; dansylchloride and its derivatives; rare earth chelates; 2-methoxy-24-diphenyl-3(2H)-furanone (MDPF), and acridine and its derivatives. Enzyme labels which are suitable for cross-coupling to such reactive intermediates include (but are not limited to): horseradish peroxidase, glucose oxidase and Beta-galactosidase. For the purposes of exemplification of the novel process of this invention, two reaction schemes have been provided to illustrate the preparation of quinidine immunogens and quinidine conjugates.

Reaction Schemes I and ll involve the synthesis of reagents suitable in an immunoassay, such as an enzyme immunoassay. The pharmaceutical agent exemplified in these reaction schemes is quinidine. Quinidine is a pharmaceutical agent generally prescribed for regulation of arrhythmic heartbeat and, thus, its concentration in a patient's blood is critical and it is carefully monitored during its administration. Reaction Scheme I (illustrated in the following equations) describes the synthesis of an immunogen; an agent used for the production of antibodies specific for immunochemical recognition of quinidine and conjugates of quinidine. The synthesis of this immunogen initially involves demethylation of the quinidine according to established laboratory procedures (see for example Small et al, J. Med. Chem. 22, 1014 (1979), which is hereby incorporated by reference in its entirety). Once the quinidine is demethylated, it can then be alkylated with methyl 5-bromovalerate. The alkylated product is thereafter subject to saponif ication followed by reaction with bovine serum albumin. The resultant product can be used to raise antibodies to quinidine by simple injection thereof into a host animal followed by isolation of appropriate protein fragments (antibodies). Quinidine alone is incapable of antibody stimulation in the host because of its relatively low molecular weight; thus, the need for the foregoing procedure.

SCHEME I

SYNATHESIS OF IMMUNOGEN

1 DEMETHYLATION OF QUINDINE.

2. ALKYLATION OF DEMETHYLATED QUINIDINE WITH

METHYL 5-BROMOVALERATE .

3. SAPONIFICATION OF ESTER DERIVATIVE

COUPLING TO BSA BY MIXED ANHYDRIDE PROCEDURE

In Reaction Scheme ll (illustrated in the following equations), an enzyme label is prepared by initial thiolation of alkaline phosphatase according to well established procedures (see Carlsson et al, J. Biochem, 273, 723 (1978) which is hereby incorporated by reference in its entirety).

The next step in Reaction Scheme π involves the preparation of the quinidine enzyme-labeled conjugate. This is achieved through the use of a bifunctional reagent which is capable of cross-coupling the quinidine to the thiolated alkaline phosphatase. Quinidine is demethylated in the same fashion as described hereinabove in the preparation of the immunogen. Subsequent to demethylation of the quinidine, it is alkylated with bromobutyronitrile; thereby introducing a nitrile group onto the quinidine. The nitrile-functional quinidine is thereafter reduced, in an alcoholic medium, with a reagent comprising cobalt chloride and a stoichiometric excess of sodium borohydride or other suitable alkali metal borohydride. The reaction conditions are very mild (ambient laboratory conditions) and thus reduction is limited to conversion of a nitrile to an amine, while the remainder of the compound remains unaffected. The procedures used in the reduction of the nitrile to the amine generally follow those described in the literature of T. Satoh et al, Tet. Lett. 455 (1969) (which is hereby incorporated by reference in its entirety). Following reduction of the nitrile-functional quinidine to the corresponding amine, it is further reacted with a bifunctional cross-coupling agent. Any compatible heterofunctional reagent can be used, as well as other coupling reagents, e.g., carbodiimides, glutaraldehyde, dimethyl suberimidate and dimethyl adipimidate. Meta-maleimidobenzoyl-N-hydroxysuccinimide ester (also known as MBS) is used in a preferred embodiment of this invention, and coupled to the quinidine, according to the procedures described in the literature (see Kitagawa et al, J. Biochem. 79, 233 (1976) which is hereby incorporated by reference in its entirety). The MBS reacts with the amine-functional group of the quinidine. The resultant compound is further reacted with the thiolated enzyme through other functional groups on the MBS. This cross-coupling of the quinidine to the alkaline phosphatase in the above manner produces an enzyme-labeled conjugate which is suitable as a reagent in an immunochemical assay for quinidine.

Typically, an immunoassay will involve the competitive binding of the conjugate and quinidine contained in the patient's sample with antibodies which have been raised to the immunogen prepared as previously described. In a solid phase immunoassay system, the antibody, which is specific for the conjugate and the quinidine in the patient's sample, is immobilized on/within a solid support. Following a suitable incubation period during which time the conjugate and the quinidine in the patient's sample compete for available binding sites on the antibody, the unbound materials are separated from the solid phase and the enzyme activity of either the solid phase or the fluid fraction measured. The level of enzyme activity within the solid phase indirectly correlates to the level of quinidine within the patient's sample. The enzyme activity can be measured through the addition of a chromogenic or fluorogenie substrate for which the enzyme is specific. The enzymatic action on the substrate produces a fluorophore or chromophore which can be monitored spectrophotometrically.

Employing analogous procedures, the process of this invention can effect cross-coupling of pharmaceutically active compounds to antibodies. Such conjugates can be used in the type of classical competitive heterogeneous assay of the type described in U.S. Patent 3,850,752 (which is hereby incorporated by reference in its entirety) or in a sandwich immunoassay of the type described by Grubb in U.S. Patent 4,168,146 (which is hereby incorporated by reference in its entirety).

The Examples which follow further define, describe and illustrate a number of preferred embodiments of this invention. Parts and percentages appearing in such Examples are by weight unless otherwise stipulated.

Apparatus and techniques used in both the synthesis and evaluation of the products of such synthesis are standard or as hereinbefore described. Example I

SYNTHESIS OF QUINIDINE-AMINE DERIVATIVE

6'-Hvdroxycinchonine. A 4.82 g portion (13.8 mmol) of quinidine (Boehringer- Mannheim, Lot # 426999, Serial #13193) was demethylated according to a published procedure (Small, et al, J. Med. Chem. 22:1014 (1979)). The recovered product, 6'-hydroxycinchonine, exhibited an Rf of 0.30 in ethyl EtOAc:iPrOH:NH₄OH:H₂O = 11:7:2:2 and had a mp of 200ºC with decomposition.

6'-(4-Cyanobutyl)oxycinchonine. A 250 mg portion (0.71 mmol) of 6'- hydroxycinchonine was dissolved in 4 ml DMF (Aldrich, Lot #102547). Two eq (119 mg, 1.44 mmol) of K₂CO₃ (Mallinckrodt, Lot #ES2) were added to the above DMF solution followed by 10 eq (0.72 ml, 7.2 mmol) of 4- bromobutyronitrile (Aldrich, Lot #1226 EH). The mixture was stirred at room temperature for 36 hours at which time the DMF solution was pipetted into a separatory funnel. Twenty-five ml of deionized water were added and the pH was adjusted to 9-9.5 with 10% w/v NaOH. This solution was washed five times with 10% v/v MeOH in CH₂Cl₂ (Baker, Lot #119818 & Aldrich, Lot

#TC 111687, respectively). The washings were discarded. After evaporating the DMF-H2O layer in vacuo, MeOH and Et₂O were added successively to the recovered residue to precipitate out the remaining inorganic salts. These were filtered off and discarded. The filtrate was evaporated in vacuo to afford a light-beige powder which was recrystallized from MeOH-Et₂O (93% yield, mp 182°C (dec)).

6'-(4-Aminobutypoxycinchonine. The procedure for the reduction of the cyano group in 6'-(4-cyanobutyl)oxycinchonine was adapted from Satoh et al,

Tet. Lett., 4555 (1969) as follows: a 100 mg portion (0.265 mmol) of 6'-(4-cyanobutyl)oxycinchonine along with 2.1 eq (132 mg) of CoCl₂.6H₂O

(Mallinckrodt, Lot #KHJB) were dissolved in 1.5 ml MeOH. Ten and four-ltenths eq (104 mg) of NaBH₄ (Eastman-Kodak, Lot #A8B) were added slowly and with rapid stirring since this mixing is exothermic. The reaction was complete after 30 minutes at room temperature. Deionized water was then added and the resulting suspension transferred to a separatory funnel. The aqueous layer was washed twice with 10% v/v MeOH-CHCl₃. Insoluble materials were filtered off before in vacuo evaporation of this aqueous phase. The recovered material contained some inorganic borate salts. R_f 0.10 in EtOAc:iPrOH:NH₄OH:H₂O = 11:7:2:2 (ninhydrin positive).

6'-(N-m-Maleimidobenzoyl-4-aminobutypoxycinchonine. The reduced quinidine-amine derivative was coupled to m -maleimidobenzoyl-N-hydroxy= succiniinide ester to yield the named adduct (hapten) using the procedure described in Kitagawa et al, J. Biochem. 79, 233 (1976).

Aklaline phosphatase-quinidine conjugate. Alkaline phosphatase was thiolated according to the procedure described in Carlsson et al, J. Biochem. 173, 726 (1978). The hapten was coupled to the thiolated enzyme following the method of Kitagawa et al, J. Biochem. 79, 233 (1976).

(Abbreviations used in Example I) Me = methyl Et = ethyl iPr = isopropyl DMF = dimethylformamide

OAc = acetate

Example ll

PRIMIDONE CYANOETHYLATION

A 250 mg (1.15 mmole) quantity of primidone was dissolved in 7 ml of dimethylformamide (DMF) (purified by passage through basic alumina) by mild heating in a 60C water bath. To this heated solution was added 0.2 ml 1 N NaOH. A solution of 60 mg (1.15 mmole) of acrylonitrile (Aldrich, Lot #EE- 531-2CE) in 3 ml of DMF was added in quarter portions to the primidone solution over a period of four (4) minutes. The reaction was aUowed to heat for another ten (10) minutes. Then the volatiles were removed by rotary evaporation at reduced pressure, yielding a clear oil. Acetone (8 ml) was added to the oil and the mixture warmed and stirred for ten (10) minutes. Filtration of the mixture furnished a clear filtrate that was applied to a silica gel preparative thin-layer chromatography plate (Analtech, Silica Gel GF, 2000u x 20 cm x 20 cm). After the plate was thoroughly dried, it was developed twice in CHCl₃:Acetone (6:1). The major UV absorbing band (middle Rf value) was scraped and eluted with acetone to give 124 mg (40% yield) of monocyanoethylated primidone, mp. 177-178C.

The cyanoethylated product prepared as described above is selectively reduced to the corresponding amine as described in Example I.

The amine-functional derivative of primidone is thereafter cross-coupled to alkaline phosphatase with the same bifunctional reaction and procedures of Example I.

Example III

Immunogens for the quinidine and primidone are prepared as previously described. Antibodies are raised to their respective immunogens in the conventional manner, isolated and subsequently immobilized on a solid phase. The resultant immobilized antibodies and their corresponding conjugates are used in an immunoassay of a patient sample in accordance with procedures described in U.S. Patent 3,850,752.

Claims

WHAT IS CLAIMED IS

1. A process for the preparation of a coupling product of a polycyclic compound having nitrile or amino functional groups, comprising:

(a) combining cobalt chloride with a stoichiometric excess of an alkali metal borohydride under conditions favoring formation of cobalt boride;

(b) contacting a polycyclic compound, having both a nitrile-functional group and moieties sensitive to sodium borohydride reduction, with the reagent prepared in accordance with step (a) in a lower alkyl alcohol under ambient laboratory conditions so as to selectively reduce the nitrile group to the corresponding amine; and

(c) reacting the product of the reduction of step (b) with a bifunctional cross-coupling reagent through the amine group of said product.

2- A process for the synthesis of conjugates for immunoassay from pharmaceutically active compounds said process comprising:

(a) providing a pharmaceutically active compound having a nitrile functional group;

(b) selectively reducing said nitrile-functional group on said pharmaceutically active compound to the corresponding amine with a reagent comprising cobalt chloride and a stoichiometric excess of alkali metal borohydride;

(c) contacting the reduction product of step (b) with a bifunctional cross-coupling reagent so as to effect reaction of said cross- coupling reagent with the reduction product through the amine- functional group of said pharmaceutically active compound; and

(d) coupling the product of step (c) with a label through the bifunctional cross-coupling reagent.

3. The process of claim 2, wherein the label is an enzyme, and said enzyme is thiolated prior to its interaction with product of step (c).

4. The process of claim 2, wherein the label is a fluorophore, and said fluorophore is thiolated, if such a group is lacking prior to its interaction with the product of step (c).

5. The process of claim 2, wherein said pharmaceutically active compound is selected from the group consisting of: theophylline, theobromine, primidone, ethosuximide, carbamazepine, phenpbarbital and the other barbiturates.

6. The process of claim 2, wherein said pharmaceutically active compounds or steroids are selected from the group consisting of: propranolol, nadolol, oxprenolol, sotalol, timolol, cortisol, estriol, protripyline, chlomipramine, opiates (morphine), methabarbital, phenytoin and methyl phenidate and said compound or steroid is initially modified without alteration of its chemical or steric properties by the introduction of a nitrile functional group.

7. The process of claim 2 wherein the pharmaceutically active compound is initially subjected to modification by cyanoethylation so as to introduce a nitrile functional group onto said compound without modification of its pharmaceutical and/or steric properties.

8. A process for the preparation of quinidine/alkaline phosphatase conjugates, comprising:

(a) demethylating quinidine with boron tribromide;

(b) alkylation of the demethylated product of step (a) with 4- bromobutyronitrile;

(c) selective reduction of the nitrile-functional group of the alkylated product of step (b) to the corresponding amine with a reagent comprising cobalt chloride and a stoichiometric excess of alkali metal borohydride; (d) contacting the reduction product of step (c) with a bifunctional cross-coupling reagent so as to effect reaction of said cross- coupling reagent with the reduction product through the amino functional group; and

(e) coupling the product of step (d) with a thiolated ester of alkaline phosphatase through the bifunctional cross-coupling reagent.

A process for the preparation of primidone/alkaline phosphatase conjugates, comprising:

(a) cyanoethylation of primidone in the acrylonitrile;

(b) selective reduction of the nitrile-functional group of the eyanoethylated product of step (a) to the corresponding amine with a reagent comprising cobalt chloride and a stoichiometric excess of alkalin metal borohydride;

(c) contacting the reduction product of step (b) with a bifunctional cross-coupling reagent so as to effect reaction of said cross- coupling reagent with the reduction product through the amino functional group; and

(d) coupling the product of step (c) with a thiolated ester of alkaline phosphatase through the bifunctional cross-coupling reagent.