Processing and synthetic intermediates for preparing...

Details Processing and synthetic intermediates for preparing... Processing and synthetic intermediates for preparing...

2001-01-25

2002-06-25

Aulakh, Charanjit S. (Department: 1625)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C546S023000, C546S103000, C546S104000

Reexamination Certificate

active

06410732

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to synthetic processes and intermediates useful for preparing N-arylacridancarboxylic acid derivatives. The N-arylacridancarboxylic acid derivatives are useful in methods to produce chemiluminescence, for example by reaction with a peroxide and a peroxidase. The chemiluminescent reaction is useful in methods of analysis for detecting peroxidase enzymes or hydrogen peroxide. It is also useful in methods to detect and quantify various biological molecules wherein a peroxidase is used as a label and in methods to detect oxidase enzymes which generate hydrogen peroxide.
BACKGROUND OF THE INVENTION
The detection and quantitation of biological molecules has been accomplished historically with excellent sensitivity by the use of radiolabeled reporter molecules. Recently numerous non-radioactive methods have been developed to avoid the hazards and inconvenience posed by these materials. Methods based on enzyme-linked analytes offer the best sensitivity since the ability to catalytically turn over substrate to produce a detectable change achieves an amplification. Substrates which generate color, fluorescence or chemiluminescence have been developed, the latter achieving the best sensitivity.
Further increases in assay sensitivity will expand the range of utility of chemiluminescence-based methods by permitting the detection of analytes present in smaller quantities or reducing the amount of time and/or reagents required to perform the assay. A way to increase the speed and sensitivity of detection in an enzymatic chemiluminescent assay is through the use of substrates which generate light with a higher efficiency or for a greater length of time.
Among the enzymes used in enzyme-linked detection methods such as immunoassays, detection of oligonucleotides and nucleic acid hybridization techniques, the most extensively used to date has been horseradish peroxidase. Chemiluminescent reagents known in the art do not permit full advantage to be taken of the beneficial properties of this enzyme in analysis mainly due to sensitivity limitations. A reagent which permits the detection of lower amounts of enzyme is needed to enable the use of peroxidase conjugates in applications requiring ultrasensitive detection. Specifically, reagents are required which generate higher levels of chemiluminescence without an accompanying increase in the background or non-specific chemiluminescence. The increased chemiluminescence can be accomplished via either a higher maximum intensity or a longer duration than compounds known in the art.
a. Enzymatic oxidation of N-alkylacridancarboxylic acid derivatives. Applicants' U.S. Pat. Nos. 5,491,072, 5,523,212, 5,593,845, 5,670,644, 5,723,295 and 5,750,698 disclose the use of a peroxidase enzyme to oxidize substituted and unsubstituted N-alkylacridancarboxylic acid derivatives to generate chemiluminescence. In the presence of a peroxidase enzyme and a peroxide, N-alkylacridancarboxylic acid derivatives are efficiently oxidized to produce the N-alkylacridone and blue chemiluminescence. N-aryl-substituted acridan-carboxylic acid derivatives are not disclosed.
U.S. Pat. No. 6,030,803 discloses a group of acridancarboxylic acid derivatives having a substituted alkoxy or alkylthio leaving group but not aryloxy or arylthio leaving groups as chemiluminescent substrate for peroxidase enzymes. N-aryl acridan compounds are claimed but no examples of N-aryl compounds are provided. All exemplary compounds contain a methyl group as the substituent on the acridan ring nitrogen atom. U.S. Pat. No. 6,162,610 discloses a group of acridancarboxylic acid derivatives having a substituted alkoxy, alkylthio or amide leaving group as chemiluminescent substrate for peroxidase enzymes. The claimed compounds bear a group designated —OX alleged to be a triggering group. No examples of N-aryl compounds are provided nor is a basis for the alleged triggering effect.
N-arylacridancarboxylic acid derivatives having a heteroaromatic group bound to the nitrogen atom are not taught or suggested in this or any of the cited publications nor in any other publication prior to the present invention.
OBJECTS
It is therefore an object of the present invention to provide processes for the synthesis of N-arylacridancarboxylic acid derivatives for use in generating chemiluminescence. It is another object of the present invention to provide synthetic intermediates useful in methods for preparing N-arylacridancarboxylic acid derivatives. It is still another object of the present invention to provide N-arylacridancarboxylic acid derivatives for use in generating chemiluminescence. It is also an object of the present invention to provide N-arylacridancarboxylic acid derivatives for use in methods of analysis and detection. It is a further object to provide chemiluminescent methods for the detection of biological materials and compounds. It is also an object of the present invention to provide a chemiluminescent method for detecting peroxidase enzymes and enzyme-conjugates. Additionally, it is an object of the present invention to provide improved methods for use in solution or on surfaces in nucleic acid assays, protein-binding assays, Western blots, Southern blots and other DNA and RNA hybridization assays and for detection of haptens, proteins and antibodies in enzyme immunoassays.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
The term “substituted” when used to describe an organic moiety such as a chain or ring group refers to the replacement of one or more hydrogen atoms on the chain or ring with another atom or group. Exemplary groups include halogen, trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl, carboxyl, carboxamide, carboalkoxy, formyl (—CHO), keto, amino, substituted amino, imino, amido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl, alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate, phosphonium, phosphate and phosphonate groups.
The term “leaving group ability” as used herein refers to the propensity for a group when attached to the carbonyl group of the acridancarboxylic acid derivative to be displaced in the nucleophilic reaction of the invention involving a peroxide or hydroperoxide or its anion.
The present invention relates to N-arylacridancarboxylic acid derivatives of the formula:
wherein R
1
is selected from alkyl, substituted alkyl, heteroalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl and heteroaryl groups, R
2
to R
9
are independently selected from substituents which contain from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms, wherein Ar is an aryl, substituted aryl or heteroaryl group and Z is selected from O and S atoms or the group ZR
1
is an —NR
10
R
11
group wherein R
10
and R
11
are independently selected from alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkylsulfonyl and arylsulfonyl groups, and wherein R
10
and R
11
can be combined with N into a heterocycle with leaving group ability including pyrazole, imidazole, benzimidazole, triazole, benzotriazole, tetrazole, oxazole, benzoxazole, thiazole and benzothiazole.
When R
10
or R
11
is alkylsulfonyl or arylsulfonyl it is preferably selected from methanesulfonyl, trifluoromethanesulfonyl, benzenesulfonyl or substituted benzenesulfonyl and the other of R
10
or R
11
is preferably an alkyl, phenyl or substituted phenyl group. When R
1
is a substituted alkyl or substituted aryl group the group is preferably substituted with one or more electron withdrawing groups, preferably halogen atoms and most preferably with fluorine.
Compounds having Formula I which contain an N-aryl group are useful in methods for producing chemiluminescence. These compounds distinguish from known prior art compounds which all have an alkyl group substituted on the ring nitrogen atom, usually a methyl group. In contrast, the compounds of the present invention bear an aromatic or heteroaromatic ring group on the ring nitrogen. Representative aryl groups include phenyl, naphthyl, biphenyl, anthryl, pyrenyl, pyridyl, quinolyl, acridinyl

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