CA1338320C - Resorufin derivatives - Google Patents

Resorufin derivatives

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Publication number
CA1338320C
CA1338320C CA000514621A CA514621A CA1338320C CA 1338320 C CA1338320 C CA 1338320C CA 000514621 A CA000514621 A CA 000514621A CA 514621 A CA514621 A CA 514621A CA 1338320 C CA1338320 C CA 1338320C
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Prior art keywords
groups
resorufin
group
reactive
ligand
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CA000514621A
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French (fr)
Inventor
Christian Klein
Hans-Georg Batz
Rupert Herrmann
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Roche Diagnostics GmbH
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Boehringer Mannheim GmbH
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals
    • G01N33/533Production of labelled immunochemicals with fluorescent label
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D265/00Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/281,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/341,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
    • C07D265/38[b, e]-condensed with two six-membered rings
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/80Fluorescent dyes, e.g. rhodamine
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/802Chromogenic or luminescent peptides

Abstract

Resorufin derivatives of the general formula:

?

Description

~ 1 - 1 338320 The present invention is concerned with resorufin derivatives, which can be used as fluorescent-labelled low or high molecular weight compounds, as well as with a process for the preparation thereof.
S Fluorescing compounds are widely used as labelled compounds in chemical and biological processes, for example in clinical analyses but also in increasingly new fields, because of their ability to emit certain wavelengths after excitation. In biochemistry, fluor-escent coloured materials are increasingly used as highly sensitive labellings. There are thereby used not only compounds of fluorescent coloured materials with low molecular weights but also high molecular weight substances. As examples of the wide field of use of such fluorescent conjugates, there may be mentioned:
Fluorescence immunoassays for which there is used a hapten, antigen or a specific antibody labelled with a fluorescent coloured material. Due to the specific binding of the antibody to the hapten or antigen, the concentration of these materials or of the antibody can be determuned according to various processes.
In immunofluorescent microscopy, antigens, for example whole cells or proteins, are made visible under the microscope by fluorescent-labelled antibodies (see Wang et al., Meth. Enzymology, 85, 514 et seq.).
- 2 _ 1 33 83 20 The distribution of a hapten or antigen in a cell can also be observed directly when the compound in question is introduced into the cell in fluorescent labelled form and nitored under a microscope.
Fluorescent-labelled latex particle~ are used in the sorting of cells in a ~fluorescent activated cell sorterH (FACS).
Not least, -~ubstrates which carry a fluorescing labelling serve for the determination and measurement of the activity of enzymes.
Competitive immunoassays are based on the compet-ition between a ligand to be determine~ in a sample and a labelled ligand present in known concentration for a known but limited number of ligand binding points on antibodies which are ~pecific not only for the ligand to be determined but also for the labelled ligand.
The concentration of the ligand to be determined in the sample is decisive for how many labelled ligand molecules are bound to the antibodies. The concentrat-ion of complexes of antibodies and fluorescent-labelled ligands can be determined by spectroscop~c methods. It is inversely proportional to the ligand concentration to be determined present in the sample. As labelled ligands which are added in known concentration to the sample with the ligands to be determined, there were originally preponderantly used ligands labelled with radioisotopes. Because of the known disadvantages of a radioisotope labelling, labelling with fluorescent compounds is achieving ever more importance. Fluor-e~cing compound~ are hereby bound to the molecules of the substance to be determined. These conjugate~
can then, in principle, be used for the mo~t varied fluorescence ;mmlnsassays, for example fluorescence polarisation ;mm~lnoassay~ fluorescence quenching no~gsay and fluorescence enhAncement ;mmllno~ssay a~ labelling~, there can, in principle, be used 10 - all fluorescing coloured materials which possess a large extinction coefficient and a high quantum yield, as well as a ~ufficient stability under the test con-ditions. Hitherto, there were, therefore, used fluorescein or fluorescein derivatives (see J. Landon and R.S. Kamel in T~lnoassays 80s, Univ. Park Press, Baltimore, Md, 1980, pp. 91-112). However, high or low molecular weight compounds labelled with fluore~cein or derivative~ thereof have di~advantages. Absorption and emission maxima of the fluorescein-labelled substances lie in a wavelength range of from 490 to 520 nm. Since a considerable number of analytical methods and espec-ially fluorescent immunoassay processe~, is carried out in body fluids, such as serum, in the said ~pectral range, disturbance~ occur due to the inherent fluor-e~cence of biological materials in the sample~. Bili-rubin, which also absorbs light in the region of about 500 nm and emits fluorescence, i9 mainly responsible for this ~ - 4 - 1 3 3 8 3 ~

Some measurement arrangements require labelling substances with a Stokes ~hift which i8 as great as po~qible. In the caQe of fluore~cein derivatives, this shift i-~ at most 30 nm. This gives rise to light qcattering problems which impair the ~en~itivity of the fluore~cence measurements. For -~uch ca~es, com-pounds with a greater Stokes ~hift than that of the fluoreQcein derivativeQ would be desirable.

The present invention seeks to provide compounds which no longer display these disadvantages. This is achieved by the provi ion of the resorufin derivatives aceording to the present invention.
Thu~, according to the present invention, there are provided resorufin derivatives of the general formulae:

- A ~ z - A

(Ia) (Ib) wherein Rl, R2, R3, R and R , which can be the same or different, are hydrogen, halogen, carboxyl, carbox-amido, lower alkoxycarbonyl, cyano or nitro groups orlower alkyl or lower alkoxy radicals, whieh ean be substituted by carboxyl, carhox~mido~ lower alkoxy-- ~ 5 ~ 1 3 3 8 3 2 0 carbonyl, cyano or nitro groups, and wherein R4 and R5 can together also repre~ent an anellated aromatic residue, Z i8 a bridge member, A is the residue of a ligand and n is a whole number of from 1 to 200.
The lower alkyl and lower alkoxy radicals in the definitions of R , R , R , R and R5 contain hydrocarbon chain-q with up to 5 and preferably up to 3 carbon atoms, the methyl, ethyl, methoxy and ethoxy radicals being eqpecially preferred.
Halogen in the definitions of R , R2, R3, R and R5 means fluorine, chlorine, bromine or iodine, chlorine and bromine being especially preferred.
~ The anellated aromatic residue possibly formed by R4 and R5 is preferably h~7ene or naphthalene, benzene being especially preferred. The said aromatic residues can be unsubstituted or can each carry one or more sub-stituents selected from S03H, COOH and Cl-C5-alkoxy.
The bridge member Z is formed by conventional addition or condensation reactions between a reactive substituent Xl of the resorufin basic ~tructure and a reactive group x2 Of the ligand or ligand analogon, optionally with the insertion of a bifunctional compound X3-M-X , wherein X3 and X are reactive groups and M the remaining part of the molecule.
In the following Table 1, there are set out, by way of example, some possible meanings for such reactive substituents Xl and x2 aq well as the bridge members Z

- 6 - 1 3~83~

re~ulting in the ca~e of the reaction.

Table Some meaninqs of the reactive qroups Xl and x2 and the bridge members Z resulting therefrom xl x2 Z

-COOT * -NH2 -CONH--COOCO2T ** -NH2 -CONH--NCO -NH2 _N~CQ~-4N-~ -NH2 ~N--~

Cl - NH--HC=CH-CO- -SH -S-CH-CH2-CO--CHO -NH2 -CH=N--SO2Cl -NH2 2 15 -COCH2-halogen -SH -COCH2-S--COCH2-halogen -OH -COCH2-0-( 2)m 2 -COOH -(CH2)m-NH-CO_ --CO--N3 COOq! ~ --NH2 --CO--N3 CO--NE~--* T is an alkyl radical with up to 5 carbon atoms or an electronegatively-activated ester group, for example an N-hydroxysuccinimide ester group.
** Tl i8 an alkyl radical with up to 5 carbon atom~.
*** m i8 0 or a whole number of from 1 to 3.

Instead of primary amine~ such as are set out in Table 1, as reactive groups Xl and x2 there can also be used, in the same way, secondary amines with the formation of corresponding products.
A-q bifunctional compounds X3-M-X4, there may be mentioned diamines, dicarboxylic acids, a~ well as derivatives thereof, dialdehydes, aminocarboxylic acids and further compounds which are conventionally used for the production of such linkages. RYAmrle~ Of such cG..,~ounds include piperazine, 1,2-ethylenediamine, succinic acid, glutaric dialdehyde, glycine, sarcosine, ~-alanine and piperidine-4-carboxylic acid.
By a ligand in the definition of A, there are to be understood haptens, antigens, antibodies and sub-strates, as well as carriers and c~,.~ounds derivedtherefrom.
By a hapten, according to the present invention there is to be understood a substance with a low molecular weight which, as a rule, is not able to produce antibodies. Compounds with a molecular weight of from about 100 to about 2000 are to be regarded as being substances with a low molecular weight. Examples of such ~ubstances include physiologically-active sub-~tances which are present in the mamm~l;an or hl~-n organism, as well as metabolites thereof and pharma-ceutical substances which are administered to anim~ls and humans, as well as metabolites thereof. However, ~ 8 - 1 33832~

by the term hapten there can also be understood all further low molecular weight compounds insofar aq they only have a molecular weight in the above-mentioned range. Example~ of poq~ible hapten-~ include amines, ~teroid-~, hormones, carbohydrates, peptides, oligonucleotides, combinations thereof and the like.
Antigens are high molecular weight compounds which are uqually able to produce antibodies in organisms treated therewith. According to the present invention, high molecular weight compounds are those which have a molecular weight of at lea~t about 2000 but preferably, however, a molecular weight of at least about 5000. The molecular weight of such com-pounds cannot be upwardly limited. The value can amount to up to 20 million but can also be greater than that. Antigens which can be present as ligandQ in com-pounds of general formulae (Ia) and (Ib) include,~fôr example, proteins, nucleic acids, polysaccharides, combinations thereof and other high molecular weight substances. According to the present invention, the term antigen is to be understood to mean all high molecular weight compounds which have a minimum molecular weight of about 2000.
Antibodies are all those proteins or glycoproteins which react specifically with antigen~ or haptens, as well as with compounds derived therefrom, to form a complex. Accord-ing to the present invention, as ligandq in compoundq~

of general formulae (Ia) and (Ib), there can be used intact antibodies as well as fragments thereof.
According to the present invention, these fragments can also be called antibodies insofar as they are able to bind antigens and haptens, as well as compounds derived therefrom, By substrates there are to be understood compounds which undergo a detectable change in a chemical reaction.
For example, amongst these are to be understood all those compounds or materials derived therefrom upon which enzymes act, for example amino acids, peptides, proteins, glycosides, oligo- and polys~cch~rides, nucleotides, nucleic acids and combinations thereof and other enzymatically changeable substances.
Carriers can be naturally-occurring or synthetic, cross-linked or non-cross-linked materials with or without a definite shape. Hereunder are to be under-stood individual compounds or mixtures of compounds.
As carriers, there can be used, for example, compounds or mixtures of compounds, for example polysaccharides, nucleic acids, peptides, proteins and combinations thereof, as well as rubber, lignin, glass, charcoal, qynthetic addition and condensation polymers, for example, polystyrene, polyacrylics, vinyl compounds, polyesters, polyethers and polyamides, and also complex structures, such as latex particle~, vesicles, liposomes, cell wall parts or even whole cells.

~ .

i_ .
lO - 1 338320 A compound derived from a particular ligand i 8 referred to as a ligand analogue. A ligand analogue is to be understood to be a substance which structur-ally differR only slightly from the corresponding ligand but, with regard to itq properties, display-q no significant difference. The difference can be due, for example, to an additional substituent or to a missing part of the molecule.
In principle, all ligands can be uqed for the formation of the col~ounds (Ia) and (Ib) according to the preqent invention which carry free amino, hydroxyl, sulphhydryl or carboxyl groupq via which the ligands can be attached to the resorufin basic structure, posqibly with the insertion of a bridge member. Free amino or carboxyl groups are especially advantageous.
Free amino groups are to be understood to be not only ` primary but also secondary amino groups. If the ligands do not pos-qeqs suitable groups, then quch groups, for example amino or carboxyl groups, must be introduced by synthetic mean~. Furthermore, it i~ possible chemically to activate non-reactive functional groups of such ligands possibly present. Since the thus resulting substances are no longer completely identical with the original compounds, they are referred to a~ ligand analogue~.
The number n indicates how many resorufin molecules are attached to a ligand or a ligand analogue.

This number depends upon the number of reactive groups in the appropriate ligand or ligand analogue. The more reactive group~ the ligand or ligand analogue po-~qesse-q, the more resorufin molecules can be bound.
The number n iq usually from 1 to 200 and is especially preferably from 1 to 100.
The present invention also provides a proceq-q for the preparation of the compound~ according to the present invention of general formulae (Ia) and (Ib).
According to thi~ process, compounds of the general formulae:

R4 ~ ` ~ 1 = R ~ N ~ X
H0 ~ 0 ~ 0 0 ~ o ~ OH

(IIa) (IIb) wherein R , R , R , R and R have the same meanings as in general formulae (Ia) and (Ib) and Xl is a reactive group, are reacted a) with a ligand of the general formula:-X - A (III) wherein x2 iq a reactive group and A i~ the residue of the ligand: or b) with a bifunctional compound of the general formula:

X - M - X (IV) _ 12 - 1 3 3 8 3 2 0 in which X3 and X4 are reactive groups and M is the residue of the bifunctional compound, and with a ligand of the general formula:
x2 A (III) in which X and A have the same meanings as above, whereby possibly in the case of individual process steps, protective groups are introduced and subse-quently again -~plit off and also individual reactive groups X , X , X3 and X can be converted into other reactive groups.
By reactive groups in the definitions of Xl, X2, X3 and X there can, in principle, be understood all conventional reactive functional groups. Especially preferred functional groups include acid residues, for 15 example, carboxylic and sulphonic acids, as well as groups derived there_rom, for example, esters, amides, anhydrides, acid halides, and residue~ for example, primary or secondary amine, cyanate, isocyanate, thiocyanate, isothiocyanate, aldehyde, sulphhydryl, hydroxyl and a-ketohalide radicals and the like. Examples of reactive groups are set out above in Table 1 under X and X .
These recidues can, of course, be e~h~n~ed for one another and can also assume the meanings of the reactive groups X3 and X4.
By the residue M of the bifunctional compound (IV), there can, in principle, be understood any organic or inorganic residue. However, those residues are preferred ~_ --13 in which M iQ a straight-chained or branched, aliphatic, cycloaliphatic or aromatic reqidue or a combination of such residues. E~pecially preferred are aliphatic residues containing up to 10 and prefer-S ably up to 7 carbon atoms or those residues whichinclude both or only one of the two reactive groupQ
X3 and X4 in a cycloaliphatic residue.
Compounds of general formulae (IIa) and (IIb) are advantageously obtained from nitrosoresorcinol derivatives of the general formula:-R~NO (V) HO ~OH

. _ wherein R3, R4 and R5 have the meanings given ingeneral formulae (Ia) and (Ib), by react~on with resorcinol derivatives of the general formula:-R

H ~[~Xl (VI) R

wherein Rl and R2 have the meanings given in generalformulae (Ia) and (Ib) and Xl i8 a reactive group.
The reaction of compounds of general formula (V) with those of general formula (VI) preferably takes place in the presence of pyrolusite and sulphuric ~, .

_ 14 _ t 3 3 8 3 2 0 acid at a low temperature. Resazurin derivative~ are thereby first formed which can easily be converted into resorufin derivatives of general formulae (IIa) and (IIb).
The reaction of compounds of general formula (V) with compounds of general formula (VI) is usually carried out at a temperature of from -lO to +50C. and preferably of from 0 to 30C. The reaction takes place especially gently when the compound~ of genèral formulae (V) and (VI) are mixed at about 0 C. and the reaction mixture i~ subsequently allowed to warm up to ambient temperature; The concentration of the pyrolusite is preferably from 0.5 to 5 and more preferably from l to 2 mole/litre. The ~ulphuric acid concentration should lS be from 0.5 to 5 and preferably from l to 3 mole/litre.
The reduction of the initially formed resazurin - derivatives to re~orufins of general formulae (IIa) and (IIb) i-~ preferably carried out in ammoniacal solution with zinc dust (cf. Nietzki et al., Ber. Dtsch. Chem.
Ges., 22, 3020/1889) or with sodium borohydride. As solvent, there i-~ preferably u~ed a water-alcohol mixture and preferably a mixture of l part of water with 0 to 4 parts of methanol. Per mole of substance to be reduced, there are added l to 20 and preferably l to 5 mole of zinc dust or sodium borohydride. The temperature of the reaction Qolution i 8 thereby main-tained at -lO to +35C. and preferably at +5 to +10C.

-~ - 15 - 1 338320 The exact mainte~nc~ of the temperature range has proved to be necessary for a definite course of the reaction. Without cooling, the exothermal reaction gives rise to by-products which are difficult to separate.
Under the selected mild conditions, the reaction between the compounds of general formulae (V) and (VI) takes place unambiguously and with good yield. The selected synthesis route is capable of variation. This open-~ up numerous possibilities of synthesis, especially having regard to the preparation of asymmetrically substituted resorufin derivatives. ~ue to this prepar-ation process which is capable of many variations, a large number of resorufin-labelled compounds can be obtained, which cover a wide colour range due to their different substituents in various positions in the chromophore.
Before the reaction of the re~orufin derivatives of general formulae (IIa) and (IIb) with compounds of the general formula (III) or with compounds of the general formulae (IV) and (III), the former are prefer-ably converted into triacyldihydroresorufin derivatives of the general formula:-.

R5R ~N Rl R6 Jl~ o ~ o ~ 1 R6 (VIr) wherein Rl R2 R3 R4 RS and Xl have the meanings given in general formulae (IIa) and (IIb) and R is a lower alkyl, aryl or aralkyl radical.
Lower alkyl in the definition of R means an alkyl radical containing up to 5 and preferably up to 3 carbon atoms, the methyl and ethyl radical~ being e~pecially preferred. As aryl radical, the phenyl radical is especially preferred. The aralkyl radical preferably contains a phenyl radical as the aryl moiety and the alkyl moiety contains up to 5 and preferably up to 3 carbon atoms. The aralkyl radical is preferably a benzyl radical.
For the preparation of the triacyl derivatives of general formula (VII), the corresponding resorufin~
derivatives of general formulae (IIa) and (IIb) are fir~t reduced with a strong reducing agent, for example stannous chloride or chromic acetate, or electro-chemically. For the reduction, the re~orufin derivative is heated for from 10 to 60 minute~ with 2 to 10 and preferably with 2 to 6 equivalents of reducing agent ~ 17 - 1 338320 in an appropriate solvent, preferably with stannous chloride in 5 to 35% aqueous hydrochloric acid. Upon cooling, the dihydro compound precipitates out. The acylation takes place in the usual manner with an appropriate acylation agent, for example acetic anhydride, benzoyl chloride or the like. The compounds of general formula (VII) are preferably prepared in a one-pot proces~ by reductive acylation of the resorufin derivatives (IIa) and (IIb). For this purpose, the appropriate resorufin derivative is heated under reflux with 2 to 6 equivalents of reducing agent for 5 to 180 minutes in the presence of the acylation agent in an appropriate solvent or is stirred at ambient temperature for from 4 to 16 hours.
The reactive group X in the triacyldihydro-resorufin derivatives (VII) obtained can possibly be converted into another reactive group before the further reaction. Especially when Xl is a carboxyl group, it is preferable to convert this into a carboxylic acid chloride, carboxylic acid anhydride or reactive ester function. This can take place in a large variety of ways, for example with carbodiimides, alcohols, halides, N-hydroxysuccinimide or the like. Numerous processes are known from the literature. Especially preferred is the conversion of the carboxylic acid function into a carboxylic acid chloride, for ex~mr1e with thionyl chloride/dimethylformamide or oxalyl chloride/dimethyl-- 18 - 1 33832~

formamide, as well as into an activated ester, for example with a N-hydroxysuccinimide ester.
If the ligands or ligand analogues possess free amino, hydroxyl or sulphhydryl groups, then these can react as reactive groups X2. The ligands or ligand analogues X2-A can then be reacted directly with com-pounds (IIa) and (IIb), possibly after previous con-version into triacyldihydroresorufin derivatives and/or after activation of the group X with the formation of l~ amide, ester or thioester ~ol,.~ounds. Because of their stability, the formation of at least one amide bond is especially preferred, whereby, for the formation thereof, it is preferable to start from compounds of general formulae (IIa)/(IIb) or (VII), wherein X is a carboxylic acid halide group. The reaction thereof with an amino group-containing ligand or ligand analogue takes place according to conventional methods, for example in an organic ~olvent, for example, dichloro-methane, with the addition of a tertiary amine, for example, triethylamine, as base. Depending upon the size of the ligand or ligand analogue and thus consequently upon the number of its free amino groups and of the ~nount used of compounds (IIa)/(IIb), several chromo-phores can be bound per ligand or ligand analogue molecule.
If, for the preparation of the co..~ounds of general formulae (Ia) and (Ib) according to the present ,9 1 338320 invention, there are u~ed triacyl derivatives of general formula (VII), then, after the reaction, the acyl radical~ of the dihydroresorufin used as pro-tective groups must be selectively split off and the re-~ultant leuko coloured material residue oxidised to the chromophore of the compound (Ia)/(Ib).
The 0-acyl radicals of the dihydroresorufin moiety are -~p~it off especially advantageously by reaction with 2 to 10 mole and preferably with 2 to 4 mole of sodium sulphite in a mixture of water and a water-soluble solvent, for example 1,4-dioxan, methanol or ethanol and preferably water/1,4-dioxan (1:1 v/v).
The reaction temperature can be from 20 to 100C. and preferably from 80 to 100C. N-Acyldihydroresorufin derivatives can be prepared in high yields under these reaction conditions.
The oxidation of the dihydroresorufin to give compounds of general formulae (Ia) and (Ib) can be carried out with mild oxidation agents. It is preferred to use potassium ferricyanide which is employed in 2 to 6 and preferably in 2 to 4 molar excess with regard to the leuko coloured material in a mixture of water and a water-soluble solvent, for example 1,4-dioxan, methanol or ethanol and preferably in water/methanol (3:1 v/v). The react-ion is preferably carried out in the presence of an adjuvant ba~e, for e~A~rle sodium hydrogen carbonate or sodium carbonate. The reaction ~- , ~ - 20 - 1 3 3 ~3~

temperature is from 10 to 40C. and preferably ambient temperature.
The selective splitting off of the protective N-acyl radical and the oxidation of the dihydrore~orufin moiety can take place e~pecially advantageously in a one-pot reaction. For this purpose, the N-acylated dihydrore-~orufin in water/methanol (3:1 v/v) i-~ first mixed with 2 to 4 mole sodium hydrogen carbonate and an eguimolar amount of lN aqueous ~odium hydroxide solution and subsequently with a 2 to 4 mole excess of potassium ferricyanide. After a period of from about 10 to 120 minute-~ and preferably of 30 minutes at ambient temperature, the reaction is complete.

...

In some cases, it is preferable not to attach the compoundQ (IIa)/(IIb) directly to the ligand or ligand analogue (III) but rather to introduce a spacing grouping X -M-X . As ~pacer, there can be used all compoundq with at least two reactive groups which are conventionally employed for this purpose, diamines and aminocarboxylic acids being especially preferred for this purpose. The choice depends upon the nature of the functional group~ X and X which are to be attached with the spacer.
The c~l"~ound-~ which result by the reaction of resorufin derivatives of the general formulae (IIa)/
(IIb) wnth bifunctional compounds of general formula (IV) in one or more steps, for example via compounds of general formula (VII), can be represented by the following general formulae: -R5 Rl R Rl R4 ~ N ~ X13-M-X4 = ~ N ~ X -M-X
H0 ~ ~ 2 . 0 0 ~ OH

(VIIIa) (VIIIb) wherein R , R , R , R and R have the same meanings as in general formulae (Ia) and (Ib), M and X4 have _ - - 22 ~ 1 3 3 8 3 20 the ~ame meanings as in general formula (IV) and X 3 is a functional group resulting from the reaction of xl and X3.
Functional groups X13 can be all conceivable groups resulting from the reaction with one another of the reactive residues X and X , preferred groups X13 including amides, thioethers, ethers, secondary and tertiary amines, as well as urea and thiourea groups.
If, in general formulae (IIa)/(IIb) and (VII), X is a carboxylic acid function or a reactive group derived therefrom and if the reactive groups x2 Of the ligand or ligand analogue are amino, hydroxyl or sulph-hydryl groups, then it is preferable to select a ~pacer lS X3-M-X4 in which X3 is an amino group and X4 is a carboxylic acid function or a reactive derivative thereof. The amino end can be attached to the carboxylic acid function of the compounds (IIa)/(IIb) or (VII) and the carboxyl end to the ligand or ligand ana~ogue X2-A.
If, however, not only X but also x2 are both carboxylic acid functions or activated derivatives derived there-from, then diamines have proved to be useful as spacers.
The reaction of resorufin derivatives of general fonmulae (IIa) and (IIb) with a bifunctional spacer 25 grouping (IV) and the ligand or ligand analogue (III) advantageously takes place in two step-~. First, the compounds (IIa)/(IIb), pos-~ibly after previous conversion into the active compounds (VII), are attached to the spacer (IV). The derivatives result-ing herefrom can be reacted in a second step with the ligands or ligand analogue-q (III). The opposite method of proceeding can, of course, also be used in which, in a first step, the spacer (IV) is attached to the ligand or ligand analogue (III) and the product obtained then reacted in a second step with the resorufin derivative (IIa)/(IIb) or the activated compound (VII).
A-~ aminocarboxylic acids, it is preferred to u~e amino acids, glycine, alanine, sarcosine and piperidine-4-carboxylic acid having proved to be especially useful.
The coupling of resorufin derivatives of general formulae (IIa)/(IIb) or (VII) with aminocarboxylic acids with the formation of an amide bond takes place by methods which are well known. For this purpose, it is especially advantageous to use the methyl or tert.-butyl esters of the appropriate amino acids. After amide formation has taken place, protective groups which have possibly been previously introduced are selectively split off according to known methods. If, for example, an activ-ated derivative (VII) is reacted with a carboxy-protected aminocarboxylic acid, then it is necessary selectively to hydrolyse the 0- and N-acyl groups, again to oxidise the leuko coloured material to the resorufin sy~tem and subsequently to split off the carboxyl protective group of the bound aminocarboxylic - _ - 24 - 1 33832~

acid under conventional conditions and preferably with trifluoroacetic acid. The free carboxyl group can then be activated for the conjugation of a ligand or ligand analogue (III) in an appropriate manner such as has been described for resorufin derivatives of general formulae (IIa) and (IIb). The carrying out of the conjugation it-qelf also takes place in a manner analogou-q to that which haq been described for the direct conjugation of ligands or ligand analogueq to resorufin derivatives of general formulae (IIa) and (IIb).
In the case of ligand or ligand analogues con-taining carboxyl groups, it is preferable to convert resorufin derivatives of general formulae (IIa)/(IIb) or (VII) into amino group-containing compounds which can then be reacted with the carboxylic acid group-containing ligands or ligand analogues. For this purpose, it has proved to be especially simple and advantageous to react compounds of general formulae (IIa)/(IIb) or (VII) with diamines (general formula (IV) in which X3 and X are amino groups). Preferred diamines in thiq sen~e include, for example, piperazine, 1,2-diaminoethane and 1,3-diaminopropane.
The conversion of re~orufin derivatives of general fonmulae (IIa)/(IIb) or (VII) with diamines into derivatives with free amino groupq takes place according to well known methodq. In order to achieve especially high yield~ of monosubstituted diamines, those diamines are preferably reacted which only have one reactive amino group, the second functional group being blocked by a protective group. In principle, all conventional amino protective group-~ can be used which can be split off again without impairment of the amide bonds, the use of tert.-butoxycarbonyl and benzoyloxycarbonyl protective groups having proved to be eQpecially advantageous.
After the reaction of mono-protected diamines with resorufin derivatives of general formulae (IIa)/
(IIb) or (VII), protective groups which have possibly been introduced are split off again and a leuko coloured material which is possibly formed a~ an inter-mediate is oxidised to the resorufin system. The splitting off of the amino protective groups of the bound diamine hereby takes place under conventional conditions and preferably with trifluoroacetic acid.
The amino group-containing resorufin derivatives thus obtained can be conjugated in conventional manner with ligands or ligand analogues (III) which, as reactive groups X , contain carboxyl groups. These carboxyl groups are advantageously activated. This can take place in the above-described manner. It is preferred to use ligands or ligand analogues of general formula (III) in which X is activated e~ter groups, ~-hydroxy~uccinimide e~ters having proved to be especially advantageous. The carrying out of the conjugation itself takes place, in principle, analogously to the procedure described above for the direct conjuqation of resorufin derivatives with ligand or ligand analogues, taking into account the changed roles of the functional groups.
The further reaction of the products which are obtained after the linking of resorufin derivatives (IIa)/(IIb) or (VII) with an intermediate member X3-M-X4 of general formula (IV) with low molecular weight ligands X2-A, for example, haptens, preferably takes place in a mixture of water or buffer and a water-soluble solvent, for example l,4-dioxan, methanol or ethanol, a mixture of O.lM potassium phosphate buffer (pH 8.5)/1,4-dioxan in a ratio of 1:1 v/v being preferred. It is possible to monitor the course of the reaction by thin layer chromatography.
The reaction period can be from 1 to 24 hours but the reaction is usually completely finished after 1 to 18 hours.

- 27 - 1 3 3 8 32 ~

For the reaction of resorufin derivatives (IIa)/(IIb), (VII) or (VIIIa)/(VIIIb) with high molecular weight ligands of general formula (III), N-hydroxysuccinimide esters prove to be especially advantageous. Thus, for example, in the case of the coupling of rabbit IgG, even 6 mole of resorufin derivative per mole of IgG suffice in order to achieve a degree of labelling of 3 molecules of resorufin per mole IgG. The previously used fluorescent coloured materials for this purpose, which display a maximum absorption wavelength of ~maX>470 nm, contain, as reactive group, an isothiocyanate or sulphonic acid chloride residue, for example fluorescein isothiocyanate or Texas Red. In these cases, for coupling to rabitt IgG, there must be used a substantially greater colouring material excess in order to achieve the same degree of labelling.
The conjugation of high molecular weight compounds of general formula (III), for example of proteins, preferably takes place in a buffer and especially advantageously in O.lM potassium phosphate buffer (pH 8.0 to 9.0) when the reactive group Xl or x2 is an N-hydroxysuccinimide ester. The reaction temperature can be from 10 to 35C., the reaction preferably being carried out at ambient temperature.

~. -_ 28 - 1 33~320 Because of their especially good spectral prop-erties, the present invention i-~ also concerned with the use of the compounds of general formulae (Ia) and (Ib) according to the preqent invention in analytical processes in which a fluorescent property of the com-pounds of general formulae (Ia)/(Ib) or of a reaction product thereof is measured.
In heterogeneou~ im~l~nQassays, a -qeparation of ligands bound to antibody and free ligands by precip-itation with appropriate ~ubstances or by the use ofantibodies bound to solid carriers is necessary before the concentration of free or bound ligands is determined.
In homogeneous ;m~lnoassays~ the inveqtigation of the antibody-ligand complex formation in the sample takes place without such a separation. The homogeneous immlno-assay methods include, for example, fluorescence quenching, fluorescence enhancement and fluorescence polarisation methods, in which fluorescing substances are used as labelling agents. Especially the last-mentioned method suffers from the disadvantagesmentioned initially of the fluorescence labelling conventionally used. Since the compounds of general formulae (Ia) and (Ib) according to the present invention possess absorption and emission maxima which lie far outside those of biological materialq in body fluids which disturb due to their inherent fluorescence, they are especially u~eful in fluore~cence polarisation -~ - 29 - ~ 3 3 8 3 2 ~

ir~l-noas~ays (FPIA). A further advantage of the com-pounds according to the present invention is that, in the case of appropriate substitution, they di~play an e-cpecially high Stokes shift of up to about 70 nm.
The FPIA processes are, in principle, based on the principle of conventional fluorescence ;mmllno_ a~says.
If appropriately fluorescence-labelled ligands are excited to fluoresce with linear polarised light, then, on the basis of the small time delay between excitation and emission, the molecule rotates before it emits radiation. In this way, the plane of the linear polarised light is also rotated through a definite angle. A number of molecules can, within this short period of time, lead to a certain depolar-isation of the fluorescence emission due to rotation diffusion. For the polarisation of the emitted fluor-escence, it is the greater the greater is the molecule and consequently the slower is the rotation. This association can be utilised for the measurement of the binding of ligands to antibodies since free, labelled ligands possess a smaller molecular volume than com-plexes of labelled ligands bound to antibodies. The polarisation is inversely proportional to the ligand concentration to be determined and present in the sample.
The concentration of the labelled ligand or ligand analogue and of the antibody necessary for such im~llno-~ ~ - 30 - 1 338~0 logical processes depends upon the measurement apparatus used, as well as upon the particular characteristic properties of the labelled ligand or ligand analogue used and of the antibody itself. In principle, these concentrations naturally also depend upon the concen-tration of the ligand to be determined and must, there-fore, be empirically ascertained. This ascertainment can be made by simple optimisation.
The ligand concentration which is to be determined generally varies from about 10 2 to about 10 13 molar.
For the measurement of a ligand concentration, it is especially advantageous to adjust in the sample a con-centration of from about 10 to about 10 12 molar and it is especially advantagous to adjust in the sample a concentration of from about 10 4 to about 10 10 molar. Higher ligand concen-trations can be measured after dilution of the original sample.

The measurement takes place at pàrticular pH
values which can extend from about 3 to 12. Usually, they lie in the range of from about 5 to about 10 and preferably in a pH range of from about 6 to about 9.
For the achievement and mainten~c~ of the pH value during the measurement, there can be used various buffers, for example borate, phosphate, carbonate or tris buffer. Which buffer is used is not decisive for the present invention. The choice depends, in the first place, upon the antibody u~ed and upon the ligand which is to be determined, as well as upon the fluor-e-~cence lAh~tling used.

The FPIA method is preferably carried out at a constant temperature Normally, the temperature can be selected from the range of about 0 to 50C. and preferably of from about 10 to about 40C.
The precise relationship between polarisation and concentration of a ligand or ligand analogue to be determined can be read off from a calibration curve.
These are obtained by measurement of the polarisation values of solutions of different but known concentrat-ions of appropriate substances. Unknown ligand concen-trations of a sample to be investigated can then be determined from such calibration curves from a knowledge of the polarisation.
A wide field of use of the compounds of general formulae (Ia) and (Ib) according to the present invention is to be seen in their general usefulness as fluorescent labellings. Thus, for example, in ;mmllnofluorescence microscopy, proteins as antigens or whole cells can be made visible by fluorescent-labelled antibodies. In a corresponding manner, the distribution of a hapten or antigen in a cell can also be directly observed and monitored, for example under a microscope, when the compound in question is introduced into the cell in fluorescent-labelled form. In contradistinction to the above-de~cribed homogeneous fluorescence ;m~llno-assay, the fluorescent properties of the resorufin derivative hereby do not change.

Known coloured materials which have previously been used as fluorescent labellings include, for example, fluoresceins, such as fluorescein isothiocyanate, and rhodamine dyes, such as ~exa~ Red. How-ever, as previously stated, fluoresceins have the dis-advantage that they fluoresce at relatively low wave-lengths. Furthermore, as is known from experience, the yield of the coupling reaction to the carrier in question is mostly small and, in addition, the colour stability of the coupling products is poor. This also applies to rhodamine dyes Precisely with regard to this point, the compounds of general formulae (Ia) and (Ib) according to the present invention display distinct advantages over the fluorescent labellings known from the - 15 prior art. mey fluoresce at long wavelengths with a good colour stability and can be prepared in good yields from compounds of general formulae (IIa) and (IIb) and appropriate coupling components.
The compounds of general formulae (IIa) and (IIb) according to the present invention can, of course, also be used for labelling substances in processes other than the above-mentioned fluorescence immunological processes.
Thus, the labelling of a component of another complex-forming system is possible with these reactive resorufin compounds. By complex-forming systems are hereby to be understood all those combinations of substances which, on the basis of specific interaction forces, are able ~ 33 ~ 1 338320 to form complexes. Known combinations include, for example, hormone/specific receptor, biotin/avidin, carbohydrate derivative/lectin and the like. For example, proteins labelled with biotin can be determined by means of a coupling product from avidin and a reactive compound of general formula (IIa) or (IIb) according to the present invention. A further advantageous field of uqe of compounds of general formulae (Ia) and (Ib) according to the preqent invention is in the determin-ation of a component of the lectin/carbohydrate derivative-system.
Fluorescent-labelled latex particles find use in the sorting of cells in a "fluorescent activated cell sorter". Such particles can al-qo be readily fluorescent labelled, for example by the reaction of latex particles containing hydroxyl, sulphhydryl, amino or also carboxyl or qulphonic acid groups with reactive resorufin com-pounds of general formulae (IIa) and (IIb).
Compounds of general formulae tIa) and (Ib) can also be advantageously used for the determination of enzymes. For this purpose, a resorufin derivative of general formula (IIa) or (IIb) can be bound to a sub-strate which can be split by the enzyme to be deter-mined. This coupling product is also a compound of general formula (Ia) or (Ib) according to the present invention. After reaction of the resorufin-labelled substrate with the enzyme and separation of the fission products and unreacted substrate, the activity of the enzyme can be determined. For example,a reactive resorufin derivative of general formula (IIa) or (IIb) .

can be bound to a glycopeptide and the coupling product hereby obtained used as a substrate for the detection of endoglucosidase activity. Labelling with dansyl compounds is known for the determination S of endoglucosidases (see Iwase et al., Anal. Biochem., 113, 93-95/1981). In comparison with such compounds, resorufin derivatives are characterised by an especially high sensitivity.
The following Examples, which are given for the purpose of illustrating the present invention, show the fundamental possibility of labelling low and high molecular compounds and the use thereof.
Example 1.
Resorufin-4-carboxylic acid 3-(1-diphenylhydantoinyl-ethylcarbonyl)-piperazide.
a) Resorufin-4-carboxylic acid.
16 g. Nitrosoresorcinol, 15.5 g. 2,6-dihydroxy-benzoic acid and 8.6 g. pyrolusite are suspended in 200 ml. methanol and cooled to 0C. 10.6 ml. concen-trated sulphuric acid are then added dropwise thereto and the reaction mixture is further stirred for 2 hours at ambient temperature. The precipitated red resazurin-- 4-carboxylic acid is filtered off, washed with methanol and dried, The resazurin derivative is taken up in 200 ml.

water and 50 ml. 25% aqueous ammonia ~olution and filtered. 50 g. zinc dust are added portionwise to the L~

_ - 35 -1 33832~
blue filtrate, with ice cooling, and the reaction mixture i~ then allowed to warm up to ambient temper-ature. The course of the reduction can easily be monitored by thin layer chromatography (elution agent:
methanol/ethyl acetate 1:1 v/v, silica gel TLC plates).
The reaction solution is filtered and the filtrate i~
then acidified with glacial acetic acid and a little concentrated hydrochloric acid. The precipitated resorufin-4-carboxylic acid is filtered off and dried in a vacuum over phosphorus pentoxide. The yield is 16.33 g.
Rf (silica gel elution agent: n-butanol/glacial acetic acid/water 4:1:1 v/v/v) = 0.4.
b) N,0,0-Triacetyldihydroresorufin-4-carboxylic acid.
12.9 g. Resorufin-4-carboxylic acid are taken up in 30 ml. glacial acetic acid and 30 ml. acetic anhydride, mixed with 27.6 g. stannous chloride and stirred for 1 hour at 80C. The reaction mixture is then poured on to 600 ml. ice water, stirred for 1 hour and the precipitate is filtered off. After drying, the solid material is taken up in 500 ml. acetone. It is then filtered with suction and the filtrate is evaporated to give, after drying, 11.3 g. of product.
lH-NMR (D6-DMSO): ~ = 2.24, 2.26 and 2.29 (each s, 9H):
6.94 (dd, J = 8.5 and 2.2 Hz, lH) 6.98 (d, J = 2.2 Hz, lH); 7.04 (d, J = 9 Hz, lH) 7.61 (d, J =

8.5 Hz, lH); 7.67 ppm (d, J - 9 Hz, lH) .

Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.46.
c) N,O,O-Triacetyldihydroresorufin-4-carboxylic acid chloride.
38.5 g. of the triacetate described in Example lb) are mixed with 54 ml. oxalyl chloride and cooled to 0C.
A drop of dimethylformamide is added thereto and the reaction mixture is allowed to warm up to ambient temp-erature. The educt thereby dissolves with the evolution of gas. The reaction mixture is evaporated to dryness in a vacuum, taken up three times with, in each case, 200 ml. amounts of dry methylene chloride and again evaporated to dryness. The yield is 41 g d) N-BOC-piperazine.
12.61 g. N-Benzhydrylpiperazine (EMKA Chemie) are taken up in 100 ml. 1,4-dioxan/water (3:1 v/v) and 12.0 g. di-tert.-butyl dicarbonate dissolved in 50 ml.
1,4-dioxan are added dropwise thereto. After stirring for 30 minutes, 50 ml. water are added dropwise thereto, filtered and the precipitate dried. Yield: 16.2 g. N-BOC-N'-benzhydrylpiperazine.
Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.92.

7 g. N-BOC-N'-benzhydrylpiperazine are taken up in 100 ml. ethyl acetate and 5 ml. glacial acetic acid.

_ .

It is hydrogenated in the presence of 0.3 g. palladium on active charcoal, thereafter filtered off from the catalyst and the filtrate evaporated to dryness. The residue is mixed with 100 ml. water and 20 ml. lN
hydrochloric acid and filtered. The filtrate is extracted twice with ethyl acetate and the aquéous phase then rendered basic with aqueous sodium hydroxide solution. The oily product which separates out is extracted with dichloromethane. After drying with anhydrous sodium sulphate and evaporating, there are obtained 3.2 g. N-BOC-piperazine -in $he form of an oil which, after a few days, crystallises completely.
Rf (silica gel: elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v)= 0.05 becomes blue with ninhydrin.
e) N,O,O-Triacetyldihydroresorufin-4-carboxylic acid N'-BOC-piperazide.
A solution of 13.8 g. N-BOC-piperazide in 50 ml.
dichloromethane is added dropwise at 0 C. to 25 g. of the acid chloride described in Example 1 c) and 17.3 ml.
triethylamine in 450 ml. dichloromethane. The reaction mixture is stirred for 1 hour without cooling, then shaken out three times with water and the organic phase is evaporated. The yield is 36.0 g.
Rf (silica gel: elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.64.

` - 38 _ 1 3 3 8 3 2 0 f) N-Acetyldihydroresorufin-4-carboxylic acid N'-BOC-piperazide.
34.3 g. of the triacetate described in Example 1 e) and 17.1 g. sodium sulphite are stirred for 1 hour at 60C. in 500 ml. 1,4-dioxan/water (1:1 v/v).
The reaction mixture is subsequently evaporated and the residue is taken up in ethyl acetate, filtered off from inqoluble salts and chromatographed on 2 litre~
of silica gel (elution agent: ethyl acetate/dichloro-methane 4:1 v/v: as soon as the product is eluted, change over to pure ethyl acetate). The yield is 14 g.
Rf (silica gel: elution agent: ethyl acetate/dichloro-methane 4:1 v/v) = 0.28.
g) Resorufin-4-carboxylic acid N'-BOC-piperazide.
5 g. of the N-acetyl compound~obtained in ~Y~rl e 1 f) are di~solved in 200 ml. methanol and 600 ml.
water. 1.8 g. Sodium hydrogen carbonate and 10.7 ml.
lN aqueous sodium hydroxide solution are added thereto, followed by 14 g. potassium ferricyanide. After stirr-ing for 30 minutes at ambient temperature, the pH is adjusted to 5. The product precipitates out and is filtered off with suction. The yield is 2.72 g.
Rf (silica gel elution agent: chloroform/methanol/
glacial acetic acid 9:1:0,1 v/v/v) = 0.28.
h) Resorufin-4-carboxylic acid piperazide trifluoro-acetate.
1 g. of the BOC derivative obtained in Example 1 3~83~

1 g) is left to stand for 15 minutes in 20 ml. tri-fluoroacetic acid. The reaction mixture is then evaporated, the residue is digested with diethyl ether and the product is filtered off. Yield 0.96 g.
Rf (silica gel: elution agent: chloroform/methanol/
glacial acetlc acid 9:1:0.1 v/v/v) = 0.02.
i) Couplinq of resorufin-4-carboxylic acid piperazide with 3-(1-diphenylhydantoinyl)-propionic acid N-hydroxysuccinimide ester.
191 mg. of the piperazide trifluoroacetate obtained in Example 1 h) and 210 mg. 3-(1-diphenyl-hydantoinyl)-propionic acid N-hydroxy~uccinimide ester (prepared from diphenylhydantoin sodium salt and ethyl 3-bromopropionate analogously to Cook et al., Res.
~ommlln;cations in Chemical Pathology and Pharmacology, 5, 767/1973) are stirred for 15 hours in 20 ml. dioxan and 20 ml. O.lM potassium phosphate buffer (pH 8.5).
The precipitated product is filtered off and the filtrate is evaporated and chromatographed on silica gel RP18 (elution agent: isopropanol), additional product thereby being obtained. The product obtained is crystallised from ethyl acetate/ methanol to give a total of 250 mg. of coupling product.
Rf (silica gel, elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.61.
H-NMR (D6-DMS0): S = 2.6-2.8 (m, 2H), 3.0-3.8 (m, lOH) 6.74 (d, J = 2.2 Hz, lH) 6.82 - 40 - 1 3 3~3~

(d, J = 9.5 Hz, lH); 6~91 (dd, J z 9.5 and 2.2 HZ) 7.25-7.33 (m, lOH);
7.55 and 7.66 ppm (each d, J = 9.5 Hz, 2H).
W /VIS (O.lM potassium phosphate buffer, pH 7.5) =
~ max = 576.8 nm Fluorescence emission: ~max = 592 nm.
Example 2.
Couplinq of resorufin-4-carboxylic acid piperazide with 3-(1-diphenylhydantoinyl)-acetic acid N-hydroxy-succinimide ester.
Analogously to Example 1 i), from 365 mg.
resorufin-4-carboxylic acid piperazide trifluoroacetate and 339 mg. 3-(1-diphenylhydantoinyl)-acetic acid N-hydroxysuccinimide ester, there are obtained 210 mg.
of the desired product.
Rf (silica gel: elution agent: n-butanol/glacial acetic acid/water 4:1:1 v/v/v) = 0.82.
H-NMR (D6-DMSO): ~ = 3.2-4.5 (m, lOH); 6.60 (d, J =
2.4 Hz, lH), 6.71 (d, J = 9.5 Hz, lH), 6.80 (dd, J = 9.05 and 2.4 Hz, lH), 7.39 ("s", lOH), 7.52 (d, J =
9.5 Hz, lH); 7.61 (d, J = 9.0 HZ, lH), 9.65 ppm (s, lH).
W /VIS (O.lM potassium phosphate buffer, pH 8.0):

~ max = 575.4 nm.
fluorescence emission: ~max = 592 nm.
-1 33~3~
Example 3.
Couplinq of reqorufin-4-carboxylic acid piperazide with N-BOC-L-thyroxine-N-hydroxyquccinimide ester.
Analogously to Example 1 i), from 212 mg.
resorufin-4-carboxylic acid piperazide trifluoroacetate and 419 mg. N-BOC-L-thyroxine-N-hydroxysuccinimide ester, there are obtained 320 mg. of the desired product.
Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.58.
The N-BOC-L-thyroxine-N-hydroxysuccinimide ester is obtained in the following manner:
a) N-BOC-thyroxine.
A solution of 10 g. (12.5 mMole) L-thyroxine sodium salt mQ~Qhydrate in a mixture of 300 ml. dioxan/
water (2:1 v/v) and 15 ml. 1~ aqueous sodium hydroxide solution is mixed with 3 g. (13 75 mMole) di-tert.-butyl dicarbonate ((BOC)20) and stirred for 2 hours at ambient temperature with the exclusion of light. The pH is adjusted to 2 with 2M potassium hydrogen sulphate solution, the solution is extracted with ethyl acetate and the ethyl acetate extract is washed with water, dried over anhydrous sodium sulphate and evaporated.
The solid residue is triturated with petroleum ether, filtered off with quction and dried in a desiccator.
Yield 9.45 g. (86% of theory).
Rf (silica gel; elution agent: chlorofonm/ligroin/

_ _ 42 -acetic acid 6:3:1 v/v/v) = 0.6.
b) N-BOC-Thyroxine-N-hydroxysuccinimide ester.
1.2 g. (9.5 mMole) N-hydroxysuccinimide is added to a solution of 8.8 g. L-BOC-thyroxine in 200 ml.
ethyleneglycol dimethyl ether. The solution is cooled to 10C. and mixed dropwise with a solution of 2.3 g.
(9.9 mMole) dicyclohexylcarbodiimide in 40 ml. ethylene-glycol dimethyl ether. After stirring for 2 hour~ at ambient temperature, the precipitated dicyclohexylurea lo is filtered off with suction and the filtrate evaporated in a vacuum at 40C. The residue is triturated with isopropanol and filtered off with suction. The product is dried at ambient temperature in-a desiccator. Yield 9.19 g. (94% of theory) (total yield referred to thyroxine = 81%).
Rf (HPTLC-RP 18, elution agent: nitromethane/ethanol 9:1 v/v) = 0.8, or (HPTLC-RP 18, elution agent:
acetonitrile/water 8:2 v/v) = 0.6.
lH-NMR (D6-DMSO): ~ = 1.36 (s, 9H): 2.81 (s, 4H), 2.9-3.2 (m, 2H), 4.5-4.9 (m, lH), 7.03 (s, 2H) 7.63 (d, J = 9 Hz, lH), 7.90 (s, 2H), 9.2 (s, lH), Example 4.
CouPlinq of resorufin-4-carboxylic acid Piperazide with 3-0- r 3-(N-succinimidoxycarbonyl)-propyll-oestradiol.
Analogously to Example 1 i), from 212 mg.
f 1 33832~
re~orufin-4-carboxylic acid piperazide trifluoroacetate and 220 mg. 3-0-[3-(N-su~cinimidyloxycarbonyl)-propyl]-oestradiol, there are obtained 295 mg. of the desired product.
Rf (silica gel, elution agent- chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.58.
3-0-[3-(N-succinimidoxycarbonyl)-propyl]-oestradiol is obtained in the usual manner from 3-0-carboxypropyloestradiol (obtained from oestradiol and bromobutyric acid analogously to Lubke et al., in Tmm~lnQlogische Teste fur niedermolekulare Wirkstoffe, pub. G. Thieme Verlag, Stuttgart, p. 94) and N-hydroxy-succinimide in the presence of dicyclohexylcarbodiimide.
EXample 5.
Couplinq of resorufin-4-carboxylic acid piperazide with N-r3-(N-succinimidoxycarbonyl)-propyll-pheno-barbital.
Analogously to Example 1 i), from 212 mg.
resorufin-4-carboxylic acid piperazide trifluoroacetate and 205 mg. N-[3-(N-succinimidoxycarbonyl)-propyl]-phenobarbital, there are obtained 220 mg. of the desired - product.
Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.45 N-[3-(N-succinimidoxycarbonyl)-propyl]-pheno-barbital is obtained in the usual ~ner from pheno-barbital-l-butyric acid (T. Nistikawa et al., Clin.

-- - 1 338~

Chim. Acta, 91, 59/1979) and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide.
Example 6.
Couplinq of resorufin-4-carboxylic acid piperazide with theophylline-7-propionic acid N-hydroxy-succinimide ester.
Analogously to Example 1 i), from 212 mg.
resorufin-4-carboxylic acid piperazide trifluoroacetate and 175 mg. theophylline-7-propionic acid N-hydroxy-succinimide e~ter, there are obtained 200 mg. of thedesired product.
Rf (silica gel: elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.44.
Theophylline-7-propionic acid N-hydroxysuccinimide ester is obtained in the usual manner from theophylline-7-propionic acid (T. Nistikawa et al., Chem. Pharm.
` Bull, 27, 893/1979) and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide.
Example 7.
Couplinq of N-(4-resorufincarbonyl)-sarcosine-N'-hydroxysuccinimide ester with l-(2-aminoethyl)-diphenylhydantoin.
a) N,0,0-Triacetyldihydroresorufin-4-carboxylic acid (tert.-butoxycarbonylmethyl)-methylamide.
10 g. of the acid chloride described in Example 1 c) are reacted with sarcosine tert.-butyl ester analogously to Example 1 e). Yield 7.5 g.

- 45 ~ 1 3 3 8 3 2 a Rf (silica gel elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.77.
b) N-Acetyldihydroresorufin-4-carboxylic acid (tert.-butoxycarbonylmethyl)-methylamide.
7.5 g. of the product according to Example 7 a) are deacetylated analogously to Example 1 f). Yield 5.2 g.
Rf (silica gel elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.56.
c) Resorufin-4-carboxylic acid (tert.-butoxycarbonyl-methyl)-methylamide.
4.5 g. of the product according to Example 7 b) - are reacted analogously to Example 1 g). Yield 2.6 g.
Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.64.
d) Resorufin-4-carboxylic acid(carboxymethyl)methYlamide.
0.55 g. of the product according to Example 7 c) is left to stand for 1 hour at ambient temperature in 6 ml. trifluoroacetic acid. The reaction mixture is then evaporated to dryness and the residue is triturated with diethyl ether and filtered. Yield 0.45 g.
Rf (silica gel elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.11.
e) N-(4-Resorufinylcarbonyl)-sarcosine-N'-hydroxy-succinimide ester.
- 200 mg. of the product according to Example 7 d) are stirred for 14 hours in 40 ml. tetrahydrofuran with - 46 - 1 3 3 8 3 2 ~

72 mg. N-hydroxysuccinimide and 138 mg. dicyclohexyl-carbodiimide. The precipitated urea is filtered off, the filtrate is evaporated and the residue is chromato-graphed on silica gel RP 18 (elution agent: nitromethane/
ethanol 4:1 v/v). Yield 150 mg.
Rf (silica gel RP 18, elution agent: nitromethane/
ethanol 4:1 v/v) = 0.79.
f) Couplinq of N-(4-resorufinylcarbonyl)-sarcosine N'-hydroxysuccinimide ester with 1-(2-aminoethyl)-diphenylhydantoin.
125 mg. of the N-hydroxysuccinimide ester obtained in Example 7 e) are stirred with 90 mg. 1-(2-aminoethyl)-diphenylhydantoin in 40 ml. dioxan/potassium phosphate buffer (pH 8.5) (1:1 v/v) for 1 hour. The dioxan is then evaporated off, ammonia is added until the colour change is complete and then filtered and the product is precipitated from the filtrate with hydrochloric acid. Yield 110 mg.
Rf (silica gel, elution agent: n-butanoljglacial acetic acid/water 4:1:1 v/v/v) = 0.78.
W/VIS (0.1 M pota-~sium phosphate buffer, pH 8.0):

~ max = 575 nm.
fluorescence emission: ~ max = 592 nm-Example 8.
Resorufin-4-carboxylic acid 2-(1-diphenylhydantoinyl)-- ethylamide.

- 4~ -~ 1 338320 a) N,0,0-Triacetyldihydroresorufin-4-carboxylic acid 2-tl-diphenylhydantoinyl)-ethylamide.
Analogously to Example 1 e), 1.37 g. 1-(2-amino-ethyl)-diphenylhydantoin is reacted with 1.2 g. N,0,0-triacetyldihydroresorufin-carboxylic acid chloride, 1.9 g. of product being obtained as a slightly coloured foam.
b) Resorufin-4-carboxylic acid 2-(1-diphenylhydantoinyl)-ethylamide.
The product obtained in Example 8 a) is deacetyl-ated analogously to Examples 1 f) and 1 g). From 1.9 g.
of educt, there are obtained 600 mg. of product.
Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.68.
lH-NMR (D6-DMSO): ~ = 3.2-3.6 (m, 4H) 6.73 (d, J =
2.2 Hz, lH) 6.84 (d, J = 9.5 Hz, 1H); 6.86 (dd, J = 9.5 and 2.2 HZ, lH); 7.2-7.4 (m, lOH); 7.62 and 7.66 (each d, J = 9.5 Hz, 2H), 8.66 (t, wide, J = 5 Hz, lH);
9.58 ppm (s, lH).
W /VIS (O.lM potassium phosphate buffer, pH 8.0) A max =
575 nm fluorescence emission: A max = 591 nm.

Example 9.
Couplinq of 6-methylresorufin-4-carboxylic acid piperazide with 2-(1-diphenylhydantoinyl)-acetic acid N-hydroxysuccinimide ester.

~_ _ 48 - 1 33 8 32~

a) 2-Methyl-4-nitrosoresorcinol.
19.8 g. 2-Methylresorcinol and 13.4 g. potassium hydroxide are dissolved in 120 ml. ethanol and cooled to 5C. 24 ml. Isopentyl nitrite are added dropwise S thereto, the reaction mixture is stirred for 3 hours and the precipitate is filtered off with suction. The yellow solid material is stirred into 200 ml. SN
sulphuric acid, the bright yellow product thereby precipitating out. Yield 22 g.
Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.53.
b) 6-Methylresazurin-4-carboxylic acid.
15.3 g. 2-Methyl-4-nitrosoresorcinol, 15,4 g.
2,6-dihydroxybenzoic acid, 8.8 g. pyrolusite and 11 ml.
concentrated sulphur-ic acid are reacted anàlogously to Example 1 a). Yield 28.7 g.
Rf (silica gel: elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.15.
c) N,0,0-Triacetyl-6-methyldihydroresorufin-4-carboxylic acid.
Analogously to Example 1 b), from 10 g. 6-methyl-resazurin-4-carboxylic acid, 19.8 g. stannous chloride, 20 ml. acetic anhydride and 150 ml. glacial acetic acid, the triacetylated leuko compound is obtained directly.
The crude product is purified by boiling out with acetone. Yield 7.3 g.
Rf (silica gel; elution agent: chloroform/methanol/

_ 49 - 1 338320 glacial acetic acid 9:1:0.1 v/v/v) = 0.51.
H-NM~ (D6-DMSO): ~ = 2.10, 2.25, 2.29, 2.33 (each s, 12H) 7.00, 7.09, 7.50 and 7.74 ppm (each d, J = 8.8 Hz, 4H).
d) N,O,O-Triacetyl-6-methyldihydroresorufin-4-carboxylic acid N'-BOC-piperazide.
Analogously to Example~ 1 d) and 1 e), from 5 g.
N,O,O-triacetyl-6-methyldihydroresorufin-4-carboxylic acid, 10.7 ml. oxalyl chloride and 2 g. N-BOC-piperazine, there are obtained 3 g. of the desired product.
Rf (silica gel, elution agent: ethyl acetate) = 0.57.
e) 6-Methylresorufin-4-carboxylic acid piperazide trifluoroacetate.
1 g. of the triacetyl derivatives obtained accord-ing to Example 9 d) is reacted analogously to Examples 1 g) and 1 h). Yield 0.43 g.
f) Couplinq of 6-methylresorufin-4-carboxylic acid piperazide with 2-(1-diphenylhydantoinyl)-acetic acid N-hydroxysuccinimide ester.
222 mg. of the compound obtained inlExample 9 e) are reacted with 200 mg. 2-(1-diphenylhydantoinyl)-acetic acid N-hydroxysuccinimide ester. Yield 250 mg.
W/VI~ (0.lM potassium phosphate buffer, pH 8.0):

~ max = 584 nm.
fluorescence emission: ~ max = 600 nm.

_ 50 - 1 3 3 8 3 2 0 Example 10.
Couplinq of 9-hydroxy-5-benzoralphenoxazone-8-carboxylic acid piperazide with 2-(1-diphenyl-hydantoinyl)-acetic acid N-hydroxysuccinimide ester.
a) 9-Hydroxy-5-benzoralphenoxazone-8-carboxylic acid 12-oxide.
2.84 g. 1~3-dihydroxy-4-nitrosonaphthalene~ 2.31 g, 2,6-dihydroxybenzoic acid, 1.29 g. pyrolusite and 1.6 ml.
concentrated sulphuric acid are reacted analogously to Example 1 a). Yield 2.8 g.
Rf (silica gel; elution agent: n-butanol/glacial acetic acid/water 4:1:1 v/v/v) = 0.63 b) 12-Acetyl-5,9-diacetoxybenzoralphenoxazone-8-carboxylic acid.
1~ Analogously to Example 9 c), from 2.4 g. 9-hydroxy-5-benzo[a]phenoxazone-8-carboxylic acid 12-oxide, there is obtained 1.8 g. of the triacetylated dihydroxy compound.
Rf (silica gel, elution agent: chloroform/methanol/
2~ glacial acetic acid 9:1:0,1 v/v/v) = 0.31.
c:) 12-Acetyl-5,9-diacetoxybenzoralphenoxazine-8-carboxylic acid N'-BOC-piperazide.
1.6 g. of the triacetyl compound obtained accord-ing to Example 10 b) is reacted with oxalyl chloride and N-BOC-piperazine analogously to Example 9 d).
Yield 1.2 g.

1 33832~
_ - 51 -- H-NMR (CDC13): ~= 1.49 (s, 9H): 2.12, 2.27, 2.46 (each s, 12H), 3.0-3.9 (m, 8H) 7,03 (d, J = 9 Hz, lH) 7.16-7.94 ppm (m, 6H).
d) 9-Hydroxy-5-benzoralphenoxazone-8-carboxylic acid N'-BOC-piperazide.
Analogously to Example 1 f), from 0.93 g. of the triacetyl compound of Example 10 c), there is obtained 0.51 g. of the desired product.
Rf (silica gel elution agent: chlorofonm/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.69.
e) 9-Hydroxy-5-benzoralphenoxazone-8-carboxylic acid piperazide trifluoroacetate.
From 0.5 g. of the BOC-protected compound des-cribed in Example 10 d), there is obtained 0.5 g. ofthe desired product analogously to Example 1 h).
Rf (silica gel elution agent: chlorofonm/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.02.
f) Couplinq of 9-hydroxy-5-benzoralphenoxazone-8-carboxylic acid piperazide wnth 2-(1-diphenyl-hydantoinyl)-acetic acid N-hydroxysuccinimide ester.
From 50 mg. of the piperazide prepared according to Example 10 e) and 150 mg. 2-(1-diphenylhydantoinyl)-acetic acid N-hydroxysuccinimide ester, there are obtained 70 mg. of the desired product analogously to Example 1 i).
Rf (silica gel elution agent: chloroform/methanol/

~~ - 52 - 1 33 ~ 3 2~

glacial acetic acid 9:1:0.1 v/v/v) = 0.57.
W/VIS (0.1 M potassium phosphate buffer, pH 8.0):
~ max = 560 nm.
fluorescence emission: ~max = 6-51 nm-H-NMR (D6-DMSO): ~ = 3.0-4.5 (m, lOH); 6.37 (s, lH) 6.80 (d, J = 8 Hz, lH) i.2-7.35 (m, lOH): 7.35-8.0 (m, 3H); 8.10 (dd, J = 8 and 2 Hz, lH) 8.56 (dd, J = 8 and 2 Hz, lH); 9.60 ppm (s, lH).
Example 11.
8-Ethylresorufin-4-carboxylic acid (l-diphenyl-hydantoinylmethylcarbonyl)-piperazide.
a) 6-Ethyl-4-nitrosoresorcinol.
Analogously to Example 9 a), from 7.5 g. ethyl- -resorc-inol, 4.5 g. potas~ium hydroxide and 8 ml. iso-pentyl nitrite, there is obtained 6-ethyl-4-nitroso-resorcinol as a yellow solid material. Yield 7.5 g.
Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.37.
b) 8-Ethylresorufin-4-carboxylic acid.
Analogously to Example 1 a), from 7.4 g. 6-ethyl-4-nitrosoresorcinol, 6.8 g. 2,6-dihydroxybenzoic acid, 3.9 g. manganese dioxide and 5 ml. concentrated sulphuric acid, there are obtained, after reduction wqth 8 g. zinc dust, 9.5 g. of the desired product.
Rf (silica gel elution agent: chloroform/methanol/

_ 53 _ 1 3 3 83 20 glacial acetic acid 9:1:0,1 v/v/v) = 0.05.
c) N,O,O-Triacetyl-8-ethyldihydroresorufin-4-carboxylic acid N'-BOC-piperazide.
Analogou~ly to Example 1 b), from 7.7 g. 8-ethyl-resorufin-4-carboxylic acid, 15.4 g. stannous chloride, 30 ml. glacial acetic acid and 15.3 ml. acetic anhydride, there is obtained the desired crude product which is directly further worked up analogously to Example 1 c) to give the acid chloride and this is further directly worked up analogously to Example 1 e) to give the BOC-piperazide. Yield 4 g.
Rf (silica gel: elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.86.
d) 8-Ethylresorufin-4-carboxylic acid N'-BOC-piperazide.
From 4 g. N,O,O-triacetyl-8-ethyldihydroresorufin-4-carboxylic acid N'-BOC-piperazide there is obtained, analogously to Examples 1 f) and 1 g), the corresponding carboxylic acid N'-BOC-piperazide. Yield 0.5 g.
Rf (silica gel, elution agent: chloroform/methanol 4:1 v/v) = 0.67.
e) 8-Ethylresorufin-4-carboxylic acid piperazide trifluoroacetate.
330 mg. of the appropriate BOC-piperazide are left to stand for 1.5 hours in 35 ml. dichloromethane/
trifluoroacetic acid. After evaporation, the residue is digested with diethyl ether, filtered off with suction and dried. Yield 350 mg.

~' _ 54 _ 1 3 3 8 3 2 0 Rf (~ilica gel: elution agent: butanol/glacial acetic acid/water 4:1:1 v/v/v) = 0.33.
f) Reaction with 2-(1-diphenylhydantoinyl)-acetic acid N-hydroxYsuccinimide e~ter.
Analogously to Example 1 i), from 325 mg. 8-ethyl-resorufin-4-carboxylic acid piperazide trifluoroacetate and 435 mg. 2-(1-diphenylhydantoinyl)-acetic acid N-hydroxysuccinimide ester, there are obtained 120 mg. of the desired product.
Rf (silica gel elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.43.
H-NMR (D6-DMSO): ~ = 1.15 (t, J = 7.2 Hz, 3H) 2.52 (q, J = 7.2 Hz, 2H) 3.1-4.0 (m, 8H): 4.25-4.45 (m, 2H): 6.42 (s, broad, lH) 6.94 (d, J = 9.0 Hz, - lH) 7.3-7.5 (m, llH) 7.68 (d, J = 9.0 Hz, lH): 9.54 (s, lH) 11.2 ppm ( 9, broad, lH).
W/VIS (O.lM potassium phosphate buffer, pH 8.0):
'~max = 575 nm.
fluorescence emission: ~max = 598 nm.
Example 12.
8-Chlororesorufin-4-carboxylic acid (l-diphenyl-hydantoinylmethylcarbonyl)-piperazide.
2S a) 8-Chlororesazurin-4-carboxylic acid.
~ rom 17.3 g. 4-chloro-6-nitrosoresorcinol (pre-pared according to Plampin and Cain, J. Med. Chem., 6, .

_ 55 _ 1 33832~

247/1963), 15.4 g. 2,6-dihydroxybenzoic acid, 8.6 g.
pyrolusite and 10.7 ml. concentrated sulphuric acid, there is obtained, analogously to Example 1 a), 8-chlororesorufin-4-carboxylic acid. Yield 17.1 g.
Rf (silica gel; elution agent: butanol/glacial acetic acid/water 4:1:1 v/v/v) = 0.58.
b) N,O,O-Triacetyl-8-chlorodihydroresorufin carboxylic acid.
16.3 g. 8-Chlororesazurin-4-carboxylic acid and 18.9 g. stannous chloride are heated to 80C. for 30 minutes in 100 ml. glacial acetic acid/acetic anhydride ( 1 1 v!v ) and then poured into 500 ml. ice water. The mixture is stirred for 2 hours, filtered off from precipitate and dried over Sicapent. The solid material obtained is taken up-in 500 ml. acetone and filtered off from undissolved residues. The filtrate is evaporated and, after drying, there are obtained 12.3 g. of the desired product.
Rf (silica gel, elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v) = 0.39.
H-~LR (D6-DMSO): ~ = 2.25 (s, 3H), 2.33 ("s", 6H), 7.16 (d, J = 8.8 Hz, lH), 7.30 (s, 1H), 7.76 (d, J = 8.8 Hz, lH), 7.90 ppm (s, lH) c) 8-Chlororesorufin-4-carboxylic acid N'-BOC-piperazide.
Analogously to Examples 1 b), 1 c) and 1 e), from ~ 56 - 1 338320 5 g. N,O,O-triacetyl-8-chlorodihydroresorufin-4-carboxylic acid, there is obtained 0.8 g. of the desired product.
Rf (silica gel: elution agent: chloroform/methanol/
5 glacial acetic acid 9:1:0.1 v/v/v) = 0.7.
d) 8-Chlororesorufin-4-carboxylic acid piperazide trifluoroacetate.
From 0.8 g. of the BOC-protected compound of Example 12 c), there is obtained, analogously to Example 1 h), 0.81 g. of the desired product.
Rf (silica gel; elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.07.
e) Couplinq of 8-chlororesorufin-4-carboxylic acid piperazide with 2-(1-diphenylhydantoinyl)-acetic acid N-hydroxysuccinimide ester.
Analogously to Example 1 i), from 400 mg. 8-chlororesorufin-4-carboxylic acid piperazide trifluoro-acetate and 410 mg. 2-(1-diphenylhydantoinyl)-acetic acid N-hydroxysuccinimide ester, there is obtained the desired product. Yield 150 mg.
Rf (silica gel elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.38.
W/VIS (0.1 M potassium phosphate buffer, pH 8.0):
~ = 581 nm fluorescence emi~sion: ~ = 597 nm.
max Bxample 13.
Couplinq of 8-chlororesorufin-1-carboxylic acid piperazide with theophylline-7-propionic acid 2-aminoethylamide.
a) 8-Chlororesorufin-4-carboxylic acid.
8.7 g. 4-Chloro-6-nitrosoresorcinol and 7.71 g.
3,5-dihydroxybenzoic acid are dissolved in 200 ml.
methanol, 4.8 g. of pyrolusite are added thereto at 0C. and portionwise 5.3 ml. concentrated sulphuric acid. The reaction mixture is stirred for 2 hours at ambient temperature, filtered and ammonia added thereto up to the~colour change to blue and 200 ml. water. The solution is filtered, the filtrate is mixed with 25 ml.
concentrated aqueous ammonia solution and 20 g. zinc dust, while cooling with ice, and thereafter, without further cooling, stirred for about 15 minutes. 200 mg.
active charcoal are added thereto, filtered, the filtrate i8 acidified to pH 2 and the precipitated resorufin derivative is then centrifuged off. Yield 3.9 g-Rf (silica gel: elution agent: n-butanol/glacial acetic acid/water 4:1:1 v/v/v) = 0.88.
b) N,0,0-Triacetyl-8-chlorodihydroresorufin-1-carboxylic acid.
Analogously to Example 1 b), from 3.5 g. 8-chloro-resorufin-l-carboxylic acid there are obtained 3.2 g.
of the deqired product.
-Rf (silica gel: elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.43.
-) 8-Chlorore~orufin-l-carboxylic acid piperazide trifluoroacetate.
From 3 g. of the triacetyl compound prepared according to Example 11 b), there is obtained 1.4 g. of the desired product analogously to Examples 1 c) to 1 h). -Rf (~ilica gel: elution agent: chloroform/methanol/
glacial acetic acid 9:1:0.1 v/v/v) = 0.08.
d) Couplinq of 8-chlororesorufin-1-carboxylic acid piperazide with theophylline-7-propionic acid 2-aminoethylamide.
Analogously to Example 8, from 420 mg. N,0,0-triacetyl-8-chlorodihydroresorufin-1-carboxylic acid piperazide and 300 mg. theophylline-7-propionic acid 2-aminoethylamide, there are obtained 190 mg. of the desired product.
Example 14.
Labellinq of ;mmllnoqlobulin G with N-(4-resorufinyl-carbonyl)-sarcosine N'-hydroxYsuccinimide ester.
100 mg. Human IgG are dissolved in 10 ml. O.lM
potassium phosphate buffer (pH 8.0) and mixed with 5 mg. N-(4-resorufinylcarbonyl)-sarcosine ~'-hydroxy-succinimide ester (cf. Example 7 e)). The reaction 2~ mixture is left to stand for 12 hours at ambient temp-erature and then chromatographed on Ultrogèl ACA 202 (LKB). The labelled protein is thereby eluted before trade mark the free low molecular weight resorufin. The degree of labelling is determined by mean~ of extinction measurement. It is 3, i.e. per molecule of IgG, there are bound 3 molecules of the resorufin derivative.
Example 15.
Labellinq of ;mmtlnoqlobulin G with N-(4-resorufinyl-carbonyl)-piperidine-4-carboxylic acid N'-hydroxy-succinimide ester.
a) N-(4-Resorufinylcarbonyl)-piperidine-4-carboxylic acid.
2.0 g. of the N,0,0-triacetyldihydrore~orufin-4-carboxylic acid chloride de~cribed in Example 1 c) are reacted analogously to Example 1 e) with 0.9 g. methyl piperidine-4-carboxylate hydrochloride, deacetylated analogously to Examples 1 f) and 1 g) and oxidised and then saponified with an aqueous solution of sodium hydroxide to give N-(4-resorufincarbonyl)-piperidine-4-carboxylic acid. Yield 0.9 g.
Rf (silica gel RP-18: elution agent: nitromethane/
ethanol 4:1 v/v) = 0.44.
W/VIS (0.1 M potassium phosphate buffer, pH 8.5):
~ max = 576.2 nm.
b) N-(4-Resorufinylcarbonyl)-piperidine-4-carboxylic acid N'-hydroxysuccinimide ester.
Analogously to Example 7 e) from 200 mg. N-(4-resorufinylcarbonyl)-piperidine-4-carboxylic acid, 240 mg. N-hydroxysuccinimide and 468 mg. dicyclocarbo-diimide, there are obtained 190 mg. of the desired product.
Rf (silica gel RP-18 elution agent: nitromethane/
ethanol 4:1 v/v) = 0.7.
IR (KBr pressed body): 3415 (m, broad), 1814 (w), 1773 (m), 1734 (s), 1626 (m), 1214 (m) cm 1 c) Labellinq of rabbit IqG with N-(4-resorufinYl-carbonyl)-piperidine-4-carboxylic acid N'-hydroxy-succinimide ester.
10 mg. Rabbit IgG, dissolved in 1 ml. O.lM
potassium phosphate buffer (pH 8.5), are mixed with 100!,1. of a solution of 1.9 mg. N-(4-resorufinyl-carbonyl)-piperidine-4-carboxylic acid N-hydroxy-succinimide ester in 1 ml. 1,4-dioxan and left to stand for 2 hours at ambient temperature. This corres-ponds to a ratio of 6.4 mole of resorufin derivative per 1 mole of rabbit IgG.
After chromatography on ACA 202 (eluent: 0.1 M
potassium phosphate buffer, pH 8.5), there is obtained a protein fraction which has an absorption ratio A578/
A280 = 0.97, corresponding to a degree of loading of 3.4 mole resorufin per mole of IgG.
~hen, in an analogous experiment, 10 mg. rabbit IgG are mixed with 20 ~1. of a solution of the activated resorufin, there is obtained, in the case of 1.05 mole of available coloured material per mole of igG, a -1 338~20 degree of loading of 0.8.
The absorption maximum of the resorufin-labelled IgG is 578 nm. The solution fluoresces strongly with a bright red colour.
If a solution of the resorufin-labelled IgG is exposed to daylight for a month, the fluorescence intensity drops to 59yO of the original value, whereas an analogously prepared IgG labelled with fluorescein isothiocyanate decreases to 16% and an IgG labelled with Texas Red decreases to 12%.
Example 16.
Diphenylhydantoin determination in human serum by means of FPIA.
1950 ~1. O,lM sodium phosphate buffer (pH 7.8) are mixed with 5 ~1. of sample (1), 25 ~1. of antibody solution (2) and 25 ~1. diphenylhydantoin-resorufin solution (3). After incubation for 5 minutes at 37 C., the fluorescence polarisation is measured (excitation wavelngth: 578 nm, emission wavelength 594 nm, measure-ment apparatus: fluorescence spectrometer 650-lOS, Hitachi).
1) Sample: human donor serum made up with a known amount of diphenylhydantoin. For the production of a calibration curve, there is used human donor serum which contains diphenylhydantoin in concentrations of:
a) 2.5 ~g./ml.
b) 5 ~g./ml.

c) 10 ~g./ml.
d) 20 ~g./ml.
e) 40 ~g./ml.
2) Antibody solution: 450 ~g. antibody/ml. 0.1M sodium phosphate buffer (pH 7.8).
The antibodies are obtained in a conventional manner by ;~l~n;sing sheep with diphenylhydantoin which is bound to bovine serum albumin via glutar-dialdehyde. The antiserum is purified by ammonium sulphate precipitation and chromatography on DEAE-cellulose.
3) Diphenylhydantoin-resorufin solution (10 M):
diphenylhydantoin-resorufin conjugate of Example 1 i) in 0.1M sodium phosphate buffer (pH 7.8).
The measurement results, which are obtained with diphenylhydantoin solutions la) - le), are illustrated in Fig. 1 of the accompanying drawings in which the diphenylhydantoin concentrations of the samples ( ~ g./
ml.) are plotted against the measured polarisation values (mP), With the help of such a calibration curve, there can also be determined the diphenylhydantoin concent-ration in samples with an unknown content of diphenyl-hydantoin.
A comparable calibration curve is also obtained when, instead of the above-used diphenylhydantoin con-jugate from Example 1 i), there is, in each case, used - 63 - 1 33 83~

the diphenylhydantoin conjugate of Examples 2, 7, 8 or lOf).
Example 17.
Determination of an endoqlycosidase activity with resorufin-hiqh mannose qlycopeptide.
a) Labellinq of hiqh mannose qlycopeptide with N-(4-resorufinylcarbonyl)-sarcosine N'-hydroxy-succinimide ester.
50 mg. High mannose glycopeptide (prepared accord-ing to Huang et al., Carbohydrate Res., 13, 127-137/
1970) are mixed with 10 ml. O.lM pota-~sium phosphate buffer (pH 8.0), the solution subsequently being adjusted to a pH of 8Ø 25 mg. N-(4-resorufinyl-carbonyl)-sarcosine N'-hydroxysuccinimide ester, dis-solved in 3 ml. dioxan, are added thereto and, after 1 hour, the same amount of coloured material-N-hydroxy-succinimide ester in 3 ml. dioxan are added thereto.
The reaction mixture is stirred for 14 hours at ambient temperature, the dioxan is then evaporated off in a vacuum and the residue diluted with water to 70 ml. and thereafter with buffer A ~0.02M tris HCl, 2mM magnesium chloride, 2mM manganese chloride, 2mM calcium chloride;
pH 7.2) to 140 ml. The pH is adjusted to 7.2 with aqueous ammonia solution. Precipitate thereb~ formed is centrifuged off. The supernatant is applied to a Con A-Sepharos~ column (1 x 15 cm.) and the free coloured material washed out with buffer A. As soon trade mark `_ 1 338320 as the flow-through is no longer red, a first fraction of resorufin-high mannose glycopeptide is eluted with 2~/o methylmannoside in buffer A as eluent (about 100 ml.).
Thereafter, a second fraction is eluted with 2% aqueous 5 methylm~nnoside~ Both fractions are dialysed against water and lyophilised. Both fractions can be used for the determination of the endoglycosidase activity des-cribed hereinafter under b).
b) Determination of endoqlycosidase activity.
Resorufin-high mannose glycopeptide is incubated in an appropriate buffer with endoglycosidase, for example endoglycosidase H, and citrate buffer (pH 5. 5 ) (sample 1). Parallel thereto, a sample i~ used which does not contain endoglycosidase but which is otherwise identical (sample-2). After incubation, both samples are mixed with Con A-Sepharose and shaken in order to bind resorufin-high mannose glycopeptide. Resorufin-labelled peptide, in which the sugar part is split off by the enzyme activity, is not bound. After 15 minutes, 20 the Con A-Sepharose is centrifuged off, the supernatant is adjusted to pH 7. 5 and the fluoresence is measured (excitation, for example, 550 nm, emission A max =
595 nm). The di~ference between sample 1 and the blank (sample 2) gives the amount of split resorufin-high 25 mannose glycopeptide and is thus a measure of the enzyme activity.

-

Claims (18)

1. Resorufin derivatives of the general formula:

? (Ia) (Ib) wherein R1, R2, R3, R4 and R5, which can be the same or different, are hydrogen, halogen, carboxyl, carboxamido, lower alkoxycarbonyl, cyano or nitro groups or lower alkyl or lower alkoxy radicals, which are unsubstituted or substituted by carboxyl, carboxamide, lower alkoxycarbonyl, cyano or nitro groups, and wherein R4 and R5 can together also represent an anellated benzene or naphthalene, said benzene and naphthalene being unsubstituted or substituted one or more times by a substituent selected from -SO3H, -COOH and C1-C5-alkoxy, Z is a bridge member, A is the residue of a ligand and n is a whole number of from 1 to 200.
2. Resorufin derivatives according to claim 1, wherein the residue A is a hapten, antigen, antibody, substrate or carrier.
3. Resorufin derivatives according to claim 1 or 2, wherein R4 and R5 together represent an anellated benzene or naphthalene, said benzene and naphthalene being unsubstituted or substituted one or more times by a substituent selected from -SO3H, -COOH
and C1-C5-alkoxy.
4. Resorufin derivatives according to claim 1 or 2, wherein Z is formed by an addition or condensation reaction between a reactive substituent of the resorufin basic structure and a reactive group of the ligand or a ligand analogen, said reactive group and substituent being selected from a carboxylic or sulphonic acid radical or an ester-, amide-, anhydride-, acid-halide-, primary or secondary amino-, cyanate-, isocyanate-, thiocyanate-, isothiocyanate-, aldehyde-, sulphhydryl-, hydroxyl- or .alpha.-ketohalide-radical or the radical .
5. Resorufin derivatives according to claim 1 or 2, wherein Z is formed by an addition or condensation reaction between a reactive substituent of the resorufin basic structure, a bifunctional compound X3 - M - X4 and a reactive group of the ligand or a ligand analogen, said reactive group and substituent and X3 and X4 being selected from a carboxylic or sulphonic acid radical or an ester-, amide-, anhydride-, acid-halide-, primary or secondary amino-, cyanate-, isocyanate-, thiocyanate-, isothiocyanate-, aldehyde-, sulphhydryl-, hydroxyl- or .alpha.-ketohalide-radical or the radical , and M is a straight-chained or branched aliphatic, cycloaliphatic or aromatic residue, the residues containing up to 10 carbon atoms, or combinations of such residues.
6. A process for the preparation of a resoru-fin derivative of the formula:

? (Ia) (Ib) in which R1, R2, R3, R4 and R5, which can be the same or different, are hydrogen, halogen, carboxyl, carbox-am do, lower alkoxycarbonyl, cyano or nitro groups or lower alkyl or lower alkoxy radicals, which are un-substituted or substituted by carboxyl, carboxamido, lower alkoxycarbonyl, cyano or nitro groups, and in which R4 and R5 can together also represent an anellated benzene or naphthalene, said benzene and naphthalene being unsubstituted or substituted one or more times by a substituent selected from -SO3H, -COOH and C1-C5-alkoxy, Z is a bridge member, A is the residue of a ligand and n is a whole number of from 1 to 200, wherein a compound of the formula - ? (IIa) (IIb) in which R1, R2, R3, R4 and R5 have the same meanings as above and X1 is a group reactive with X2, is reacted a) with a ligand of the general formula:-X2 - A (III) in which X2 is a group reactive with X1 and A is the residue of the ligand; or b) with a bifunctional compound of the formula -X3-M-X4 (IV) in which X3 and X4 are groups reactive with X1 and x2 and M is a residue selected from the group of straight-chained or branched aliphatic, cycloaliphatic or aromatic residues, the residues containing up to 10 carbon atoms, or combinations of such residues, said compound (IV) being selected from diamines, dicar-boxylic acids and derivatives thereof, dialdehydes and aminocarboxylic acids, and with a ligand of formula (III);
wherein, in individual process steps, protective groups are split off, and individual reactive groups X1, X2, X3 and X4 are if desired, converted into other reactive groups, wherein one of X1 and X2 is selected from -COOH, -COOT in which T is alkyl of up to 5 carbon atoms or an elecronegatively-activated ester group, -COOCO2T1 in which T1 is alkyl of up to 5 carbon atoms, -NCO, -NCS, -HC=CH-CO-, -CHO, -SO2Cl, -COCH2Hl in which Hl is halogen, -(CH2)m-NH2 in which m is 0 or a whole number from 1 to 3, in which T is as defined hereinbefore, or and the other of X1 and X2 is selected from -NH2, -SH, ?NH, -OH and -COOH, provided that X1 and X2 are selected such that X1 is reactive with X2.
7. A process according to claim 6 b), wherein each of X3 and X4 is an amino-, carboxyl-, ester-, amido-, anhydride- or acid halide group or X3 is an amino group and X4 is a carboxyl group.
8. A process according to claim 7, wherein the bifunctional compound is piperazine, 1,2-ethylene-diamine, glycine, sarcosine, .beta.-alanine or piperidine-4-carboxylic acid.
9. In an analytical process in which a fluorescent property of a fluorescing component is measured, the improvement wherein the fluorescing component is a resorufin derivative of formula (Ia) or (Ib), as defined in claim 1 or 2.
10. In an analytical process in which a fluorescent property of a fluorescing component is measured, the improvement wherein the fluorescing component is a resorufin derivative of formula (Ia) or (Ib), as defined in claim 5.
11. In an immunoassay in which a biological entity is labeled with a fluorescing component and a fluorescent property of the labeled entity is measured, the improvement wherein the fluorescing component is a resorufin derivative of formula (Ia) or (Ib), as defined in claim 1 or 2.
12. Resorufin derivatives of the formula:

? (IIa) (IIb) wherein R1, R2, R3, R4 and R5 have the same mean-ings as in claim 1, and X1 is a reactive group selected from the group comprising of carboxylic acid radicals, sulphonic acid radicals, ester groups, amide groups, anhydride groups, acid halide groups, primary amino, secondary amino, cyanate, isocyanate, thiocyanate, isothiocyanate, aldehyde, sulphhydryl, hydroxyl, ?-keto-halide radicals, sulphoxy chloride, -HC=CH-CO- and -COCH2-Hl where Hl is halogen.
13. Resorufin derivatives of the formula:

? (VIIIa) (VIIIb) wherein R1, R2, R3, R4 and R5, which can be the same or different, are hydrogen, halogen, carboxyl, carboxamido, lower alkoxycarbonyl, cyano or nitro groups or lower alkyl or lower alkoxy radicals, which are unsubstituted or substituted by carboxyl, carboxamide, lower alkoxycarbonyl, cyano or nitro groups, and wherein R4 and R5 can together also represent an annellated benzene or naphthalene, said benzene and naphthalene being unsubstituted or substituted one or more times by a substituent selected from -SO3H, -COOH and C1-C5-alkoxy, M is a residue selected from the group of straight-chained or branched aliphatic, cycloaliphatic or aromatic residues, the residues containing up to 10 carbon atoms, or combinations of such residues, X13 is a functional group formed by the reaction of a reactive group X1 and a reactive group X3, which reactive groups X1 and X3 are each selected from the group consisting of carboxylic acid radicals, sulphonic acid radicals, ester groups, amide groups, anhydride groups, acid halide groups primary amino, secondary amino, cyanate, isocyanate, thiocyanate, isothiocyanate, aldehyde, sulphhydryl, hydroxyl, .alpha.-keto-halide radicals, sulphoxychloride, -HC=CH-CO- and -COCH2Hl where Hl is halogen, and X4 is a group derived from a compound (IV) X3-M-X4 (IV) in which M is as defined above and X3 and X4 are reactive groups, said compound (IV) being selected from diamines, dicarboxylic acids and derivatives thereof, dialdehydes and aminocarboxylic acids.
14. Resorufin derivatives of the formula:

? (Xa) (Xb) wherein R1, R2, R3, R4 and R5, which can be the same or different, are hydrogen, halogen, carboxyl, carboxamido, lower alkoxycarbonyl, cyano or nitro groups or lower alkyl or lower alkoxy radicals, which are unsubstituted or substituted by carboxyl, carboxamide, lower alkoxycarbonyl, cyano or nitro groups, and wherein R4 and R5 can together also represent an annellated benzene or naphthalene, said benzene and naphthalene being unsubstituted or substituted one or more times by a substituent selected from -SO3H, -COOH and C1-C5-alkoxy, and X14 is a reactive group selected from the group consisting of carboxylic acid radicals, sulphonic acid radicals, ester groups, amide groups, anhydride groups, acid halide groups, primary amino, secondary amino, cyanate, isocyanate, thiocyanate, isothiocyanate, aldehyde, sulphhydryl, hydroxyl, .alpha.-keto-halide radicals, sulphoxy chloride, -HC=CH-CO- and -COCH2Hl where Hl is halogen, or a group -X13-M-X4, wherein M
is a residue selected from the group of straight-chained or branched aliphatic, cycloaliphatic or aromatic residues, the residues containing up to 10 carbon atoms, or combinations of such residues, and X13 is a functional group formed by the reaction of a reactive group X1 and a reactive group X3, which reactive groups X1 and X3 are each selected from the group consisting of carboxylic acid radicals, sulphonic acid radicals, ester groups, amide groups, anhydride groups, acid halide groups, primary amino, secondary amino, cyanate, isocyanate, thiocyanate, isothiocyanate, aldehyde, sulphhydryl, hydroxyl, .alpha.-keto-halide radicals, sulphoxy chloride -HC=CH-CO- and -COCH2Hl where Hl is halogen, and X4 is a group derived from a compound (IV):
X3-M-X4 (IV) in which M and X3 are as defined above, said compound (IV) being selected from diamines, dicarboxylic acids and derivatives thereof, dialdehydes and aminocarboxylic acids.
15. Use of a resorufin derivative according to claim 1 or 2, as a fluorescing component in an immunoassay.
16. Use of a resorufin derivative according to claim 3, as a fluorescing compound in an immunoassay.
17. Use of a resorufin derivative according to claim 4, as a fluorescing component in an immuno-assay.
18. Use of a resorufin derivative according to claim 5, as a fluorescing component in an immuno-assay.
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Families Citing this family (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0319620A1 (en) * 1987-12-10 1989-06-14 Viomedics Inc. Novel oxazines-ureas and thiazine urea chromophors
DE3526565A1 (en) * 1985-07-25 1987-02-05 Boehringer Mannheim Gmbh RESORUFIN DERIVATIVES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE IN FLUORESCENT IMMUNOASSAYS
DE3644401A1 (en) * 1986-12-24 1988-07-07 Boehringer Mannheim Gmbh NEW HYDROLASE SUBSTRATES
JPH01211595A (en) * 1988-02-18 1989-08-24 Kikkoman Corp Novel n-acetyl-beta-d-glucosamine derivative, production thereof and utilization thereof to reagent for measuring n-acetyl-beta-d-glucosamidase activity
CA2227448C (en) * 1995-07-26 2008-02-05 Universite De Montreal Elisa serodiagnosis of pig pleuropneumonia serotypes 5a and 5b
ATE345501T1 (en) 1997-04-08 2006-12-15 Univ Montreal ELISA SERODIAGNOSIS OF SWINE PLEUROPNEUMONIA SEROTYPE 2
JP3810035B2 (en) 1997-09-08 2006-08-16 日本精機株式会社 Easy-open packaging bag and manufacturing apparatus thereof
US6514687B1 (en) 1998-12-14 2003-02-04 Vertex Pharmaceuticals (San Diego), Llc Optical molecular sensors for cytochrome P450 activity
CA2352631A1 (en) * 1998-12-14 2000-06-22 Aurora Biosciences Corporation Optical molecular sensors for cytochrome p450 activity
US6420130B1 (en) 1998-12-14 2002-07-16 Aurora Biosciences Corporation Optical molecular sensors for cytochrome P450 activity
US6143492A (en) * 1998-12-14 2000-11-07 Aurora Biosciences Corporation Optical molecular sensors for cytochrome P450 activity
GB9902068D0 (en) * 1999-01-29 1999-03-24 Smithkline Beecham Plc Compounds
US6727356B1 (en) 1999-12-08 2004-04-27 Epoch Pharmaceuticals, Inc. Fluorescent quenching detection reagents and methods
US20040081959A9 (en) * 1999-12-08 2004-04-29 Epoch Biosciences, Inc. Fluorescent quenching detection reagents and methods
US8569516B2 (en) * 2001-09-07 2013-10-29 Elitech Holding B.V. Compounds and methods for fluorescent labeling
US6972339B2 (en) 2001-09-07 2005-12-06 Epoch Biosciences, Inc. Compounds and methods for fluorescent labeling
WO2004026804A1 (en) * 2002-09-20 2004-04-01 Integrated Dna Technologies, Inc. Anthraquinone quencher dyes, their methods of preparation and use
WO2005040357A2 (en) * 2003-10-24 2005-05-06 Epoch Biosciences, Inc. Compounds and methods for fluorescent labeling
EP2298312B1 (en) 2003-10-31 2018-09-26 Molecular Probes Inc. Fluorinated resorufin compounds and their application in detecting hydrogen peroxide
US7439341B2 (en) * 2003-11-14 2008-10-21 Integrated Dna Technologies, Inc. Fluorescence quenching azo dyes, their methods of preparation and use
JP5214967B2 (en) 2004-08-13 2013-06-19 エポック バイオサイエンシズ インコーポレーティッド Phosphonic acid fluorescent dyes and complexes
JP2008545659A (en) * 2005-05-20 2008-12-18 インテグレイテッド ディーエヌエイ テクノロジーズ インコーポレイテッド Compounds and methods for labeling oligonucleotides
WO2011120049A1 (en) 2010-03-26 2011-09-29 Integrated Dna Technologies, Inc. Methods for enhancing nucleic acid hybridization
US9506057B2 (en) 2010-03-26 2016-11-29 Integrated Dna Technologies, Inc. Modifications for antisense compounds
US8735575B2 (en) 2010-07-30 2014-05-27 Washington University Phenoxazine derivatives and methods of use thereof
EP2614149B1 (en) 2010-09-07 2015-04-08 Integrated Dna Technologies, Inc. Modifications for antisense compounds
WO2013048583A2 (en) 2011-05-24 2013-04-04 Elitech Holding B.V. Detection of methicillin-resistant staphylococcus aureus
EP2755019B1 (en) * 2011-09-09 2018-01-03 Konica Minolta, Inc. Biological substance detection method
US9642858B2 (en) 2012-10-30 2017-05-09 University of Pittsburgh—of the Commonwealth System of Higher Education Use of resazurin, or analogs thereof, for antibacterial therapy
US20140255928A1 (en) 2013-03-11 2014-09-11 Elitech Holding B.V. Methods for true isothermal strand displacement amplification
EP2997161B1 (en) 2013-05-13 2017-09-27 Elitechgroup B.V. Droplet digital pcr with short minor groove probes
EP3230468B1 (en) 2014-12-12 2020-09-16 ELITechGroup, Inc. Methods and kits for detecting antibiotic resistant bacteria
WO2016094162A1 (en) 2014-12-12 2016-06-16 Elitechgroup B.V. Methods and compositions for detecting antibiotic resistant bacteria
WO2021080629A1 (en) 2019-10-23 2021-04-29 Elitechgroup, Inc. Methods for true isothermal strand displacement amplification
EP3932995A1 (en) 2020-07-04 2022-01-05 Emulseo SAS Novel fluorescent substrates and uses thereof in microfluidics

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB807687A (en) * 1955-10-10 1959-01-21 Bayer Ag Phenoxazone-dicarboxylic acid derivatives
US4141688A (en) * 1977-08-11 1979-02-27 Miles Laboratories, Inc. Composition, device and method for determining reducing agents
IT1140209B (en) * 1981-09-25 1986-09-24 Anic Spa IMMUNOLUORESCENCE REAGENTS AND METHOD FOR THEIR PREPARATION
US4667032A (en) * 1983-01-21 1987-05-19 Merck Frosst Canada, Inc. Phenothiazone derivatives and analogs
US4859667A (en) * 1983-01-21 1989-08-22 Merck Frosst Canada, Inc. Pharmaceutical compositions of phenothiazone derivatives and analogs
US4714763A (en) * 1985-07-11 1987-12-22 Viomedics Inc. Novel oxazine-ureas and thiazine urea chromophors as fluorescent labels
DE3526566A1 (en) * 1985-07-25 1987-02-05 Boehringer Mannheim Gmbh N-ACYL-DIHYDRORESORUFIN DERIVATIVES, METHOD FOR THE PRODUCTION THEREOF AND THE USE THEREOF FOR DETERMINING HYDROGEN PEROXIDE, PEROXIDATICALLY ACTIVE COMPOUNDS OR PEROXIDASE
DE3526565A1 (en) * 1985-07-25 1987-02-05 Boehringer Mannheim Gmbh RESORUFIN DERIVATIVES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE IN FLUORESCENT IMMUNOASSAYS
DE3644401A1 (en) * 1986-12-24 1988-07-07 Boehringer Mannheim Gmbh NEW HYDROLASE SUBSTRATES
US5242805A (en) * 1991-08-23 1993-09-07 Molecular Probes, Inc. Long wavelength lipophilic fluorogenic glycosidase substrates

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