WO2008030120A1 - A method for the fluorescent detection of nitroreductase activity using nitro-substituted aromatic compounds - Google Patents

A method for the fluorescent detection of nitroreductase activity using nitro-substituted aromatic compounds Download PDF

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WO2008030120A1
WO2008030120A1 PCT/NZ2007/000262 NZ2007000262W WO2008030120A1 WO 2008030120 A1 WO2008030120 A1 WO 2008030120A1 NZ 2007000262 W NZ2007000262 W NZ 2007000262W WO 2008030120 A1 WO2008030120 A1 WO 2008030120A1
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coor
nitroreductase
opo
nitro
conr
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PCT/NZ2007/000262
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French (fr)
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Jeffrey Bruce Smaill
Adam Vorn Patterson
Dean Craig Singleton
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Auckland Uniservices Limited
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Priority to US12/440,267 priority Critical patent/US20100173332A1/en
Publication of WO2008030120A1 publication Critical patent/WO2008030120A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/20—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
    • C07D219/06—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/88—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/94—Nitrogen atoms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • This invention relates generally to a method for the fluorescent detection of nitroreductase activity using at least one fluorogenic probe.
  • the method utilises one or more probes, which are non-fluorescent aromatic compounds containing at least one NO 2 group, that is reduced to NHOH or NH 2 by the action of a nitroreductase resulting in the production of a strongly fluorescent molecule.
  • the invention relates to a method of detection based on the use of a plurality of such probes in a common environment.
  • a novel class of nitro-substituted compounds is also provided.
  • nitroacridone derivatives are described in US20040191792A1 as dyes having characteristic fluorescence lifetimes. These acridone dyes do not require reduction by a nitroreductase to exhibit fluorescence. The acridone chromophore is inherently highly fluorescent.
  • Nitro quenched cyanine dyes are taught in US20030186348A1 as a way of enhancing the fluorescence output of the cyanine dyes for the detection of microbial nitroreductases with the emphasis on reporter gene applications. These compounds have considerable fluorescence in their quenched form in cell culture and upon the action of a nitroreductase increase in fluorescence by three to four-fold offering a ' limited dynamic range for reporter gene applications.
  • renilla luciferases may only be quantified in cultured mammalian cells following cell lysis (destruction) a procedure that is incompatible with many other assays.
  • Multiplexing unrelated reporter genes is usually problematic, or at best requires each to be assayed separately employing different chemistry and detection methods, with sequential measurements and iterative chemistry steps or sample replating.
  • nitroreductase detection is a nondestructive assay protocol that can be detected in a common environment, for example an individual well or a single cell, using the same detection method simultaneously (e.g. fluorescence emission), without the need to quench one before measuring another. Nitroreductases are thus ideal for multiplexing on sub-cellular imaging systems including microscopes, sub-cellular imagers and plate readers.
  • Another object of the invention is to provide a novel class of nitro-substituted aromatic compounds, or to at least provide a useful alternative.
  • the present invention provides a method for the fluorescent detection of nitroreductase activity using a fluorogenic probe suitable for use as a nitroreductase probe, the method including the step of applying a plurality of probes to a sample and monitoring for the presence of at least one nitroreductase enzyme in a common test environment.
  • the step of monitoring for the presence of at least one nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of the fluorogenic probe.
  • the reduced derivatives may be excited at predetermined wavelengths in the UV/visible range and the fluorescence emission determined.
  • the excitation wavelength will be between 200-700nm. More preferably, the excitation wavelength may be selected from 295, 340, 355, 405, 440 and 485 nm.
  • the fluorescence emission will be in the UV/visible/IR range.
  • the fluorescence emission wavelength will be between 300-800nm. More preferably, the fluorescence emission wavelength may be selected from 370, 460, 510, 535, 540 and 585nm.
  • the method further includes the step of quantifying the activity of at least one nitroreductase. More preferably, the step of quantifying the nitroreductase activity includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
  • the monitoring of a plurality of fluorogenic probes can be performed simultaneously.
  • the monitoring of a plurality of fluorogenic probes can be performed sequentially.
  • the present invention provides a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro-aromatic compounds of Formula I-V as defined herein, suitable for use as a nitroreductase probe, the method including the step of applying the one or more fluorogenic probes to a sample and monitoring for the presence of at least one nitroreductase.
  • the step of monitoring for the presence of nitroreductase activity includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula I-V.
  • the method further includes the step of quantifying the nitroreductase activity. More preferably, the step of quantifying the nitroreductase activity includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
  • the method uses a plurality of fluorogenic probes.
  • the monitoring can be performed in a common test environment.
  • the present invention provides a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- aromatic compounds of Formula I suitable for use as a nitroreductase probe
  • X represents N 1 NH, NR 6 , O or S
  • Z represents C, CH or N; wherein R 1 , if present, may be selected from H, R 7 , (CR 7 R 8 ) n COOH, (CR 7 R 8 ) n COOR 9 ,
  • R 2 may represent H, R 11 , (CR 11 R 12 J n COO(CR 11 R 12 J n NR 13 R 14 ,
  • CNNR 20 (CR 21 R 22 ) n OH, CNNR 20 (CR 21 R 22 J n NR 23 R 24 , CNNR 20 (CR 21 R 22 ) n OPO(OH) 2 , CONR 20 (CR 21 R 22 ) n OH, SONR 20 (CR 21 R 22 J n OH, SO 2 NR 20 (CR 21 R 22 ) n OH, CONR 20 (CR 21 R 22 J n COOR 23 , SONR 20 (CR 21 R 22 ) n COOR 23 , SO 2 NR 20 (CR 21 R 22 ) n COOR 23 , CONR 20 (CR 21 R 22 J n NR 23 R 24 , SONR 20 (CR 21 R 22 J n NR 23 R 24 , SO 2 NR 20 (CR 21 R 22 J n NR 23 R 24 , CONR 20 (CR 21 R 22 J n OPO(OH) 2 , SONR 20 (CR 21 R 22 J n OPO(OH) 2 , SO 2 NR 20
  • the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula I.
  • the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
  • X is O or NH
  • Z is CH or NH
  • R 3 is H.
  • the fluorogenic probe of Formula I is selected from
  • a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- substituted aromatic compounds of Formula Il
  • the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula II.
  • the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
  • the compound of Formula Il is selected from
  • a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- substituted aromatic compounds of Formula III
  • R 1 may represent COR 2 , SOR 2 , SO 2 R 2 , CO(CR 2 R 3 ) n OH, SO(CR 2 R 3 ) n OH, SO 2 (CR 2 R 3 ) n OH, CO(CR 2 R 3 ) n COOR 4 , SO(CR 2 R 3 ) n COOR 4 SO 2 (CR 2 R 3 ) n COOR 4 , CO(CR 2 R 3 ) n NR 4 R 5 , SO(CR 2 R 3 ) n NR 4 R 5 , SO 2 (CR 2 R 3 ) n NR 4 R 5 , CO(CR 2 R 3 ) n OPO(OH) 2 , SO(CR 2 R 3 ) n OPO(OH) 2 , SO 2 (CR 2 R 3 ) n OPO(OH) 2 , CONR 2 R 3 , SONR 2 R 3 , SO 2 NR 2 R 3 , CNNR 2 (CR 3 R 4 ) n COOR 5 , CNNR
  • the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula III.
  • the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
  • the compound of Formula III is selected from methyl 4- ⁇ [(5-nitro-1 -naphthyl)sulfonyl]amino ⁇ butanoate, methyl 4- ⁇ [(5-nitro-2-naphthyl)sulfonyl]amino ⁇ butanoate and methyl 4- ⁇ [(8-nitro-2-naphthyl)sulfonyl]amino ⁇ butanoate and any pharmaceutically acceptable salt thereof.
  • the compound of Formula III' is selected from methyl 4- ⁇ [(5-nitro-1-naphthyl)sulfonyl]amino ⁇ butanoate, methyl 4- ⁇ [(5-nitro-2-naphthyl)sulfonyl]amino ⁇ butanoate and methyl 4- ⁇ [(8-nitro-2-naphthyl)sulfonyl]amino ⁇ butanoate and any pharmaceutically acceptable salt thereof.
  • a seventh aspect there is provided a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- substituted aromatic compounds of Formula IV
  • the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula IV.
  • the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
  • R 1 is (CR 2 R 3 ) n NR 4 R 5 .
  • the compound of Formula I is selected from:
  • R 1 may represent H, R 4 , COR 4 , SOR 4 , SO 2 R 4 , CO(CR 4 R 5 ) n OH, SO(CR 4 R 5 ) ⁇ OH, SO 2 (CR 4 R 5 ) n OH, CO(CR 4 R 5 ) n COOR 6 , SO(CR 4 R 5 ) n COOR 6 SO 2 (CR 4 R 5 ) n COOR 6 , CO(CR 4 R 5 ) n NR 6 R 7 , SO(CR 4 R 5 J n NR 6 R 7 , SO 2 (CR 4 R 5 ) n NR 6 R 7 , CO(CR 4 R 5 ) n OPO(OH) 2 , SO(CR 4 R 5 ) ⁇ OPO(OH) 2 , SO 2 (CR 4 R 5 ) n OPO(OH) 2 , CONR 4 R 5 , SONR 4 R 5 , SO 2 NR 4 R 5 , CNNR 4 (CR 5 R 6 ) n COOR 7
  • the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula V.
  • the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
  • R 2 and R 3 together represent Ar.
  • the compound of Formula V is selected from 3-nitro-6H-benzo[c]chromen-6-one and any pharmaceutically acceptable salt thereof.
  • the one or more nitroreductase is a human oxidoreductase selected from known human enzymes classified as EC 1 in the EC number classification of enzymes. Oxidoreductases are classified into 22 subclasses of which 6 have known nitroreductase activities:
  • EC 1.1 includes oxidoreductases that act on the CH-OH group of donors e.g. Aldose reductase [ALDR1 ; E.C.1.1.1.21]; e.g. Aldehyde reductase [AKR1 B10; E.C. 1.1.1.2].
  • ALDR1 Aldose reductase
  • E.C.1.1.1.21 Aldehyde reductase
  • EC 1.2 includes oxidoreductases that act on the aldehyde or oxo group of donors
  • EC 1.3 includes oxidoreductases that act on the CH-CH group of donors
  • EC 1.4 includes oxidoreductases that act on the CH-NH 2 group of donors
  • EC 1.5 includes oxidoreductases that act on CH-NH group of donors
  • EC 1.6 includes oxidoreductases that act on NADH or NADPH e.g. DT-diaphorase [NQO1 ; E.C.1.6.99.2]; e.g. Cytochrome P450-reductase [CYPOR; E.C. 1.6.2.4]; e.g. Cytochrome B5 reductase [DIA1 ; E.C.1.6.2.2]; EC 1.7 includes oxidoreductases that act on other nitrogenous compounds as donors
  • EC 1.8 includes oxidoreductases that act on a sulfur group of donors e.g. Thioredoxin-disulfide reductase [TXNRD; E.C.1.8.1.9];
  • EC 1.9 includes oxidoreductases that act on a heme group of donors
  • EC 1.10 includes oxidoreductases that act on diphenols and related substances as donors
  • EC 1.11 includes oxidoreductases that act on peroxide as an acceptor (peroxidases)
  • EC 1.12 includes oxidoreductases that act on hydrogen as donors
  • EC 1.13 includes oxidoreductases that act on single donors with incorporation of molecular oxygen (oxygenases)
  • EC 1.14 includes oxidoreductases that act on paired donors with incorporation of molecular oxygen e.g. Inducible nitric oxide synthase [NOS2A; E. C.1.14.13.39];
  • EC 1.15 includes oxidoreductases that act on superoxide radicals as acceptors
  • EC 1.16 includes oxidoreductases that oxidize metal ions; e.g. Methionine synthase reductase [MTRR; E. C.1.16.1.8];
  • EC 1.17 includes oxidoreductases that act on CH or CH 2 groups; e.g. Xanthine oxidase [XO; E. C.1.17.3.2]; e.g. Xanthine dehydrogenase [XDH; E.C.1.17.1.4];
  • EC 1.18 includes oxidoreductases that act on iron-sulfur proteins as donors; e.g. Adrenodoxin oxidoreductase [FDXR; E.C.1.18.1.2]
  • EC 1.19 includes oxidoreductases that act on reduced flavodoxin as a donor
  • EC 1.21 includes oxidoreductases that act on X-H and Y-H to form an X-Y bond
  • EC 1.97 includes other oxidoreductases
  • the one or more nitroreductase is a microbial or fungal nitroreductase selected from type I nitroflavin reductase NfsA-like and NfsB-like superfamilies or the NQO1-like and YieF-like nitroreductase enzymes, or any putative nitroreductase gene showing evidence of significant sequence homology thereof.
  • a method for identifying the presence of cellular hypoxia by contacting in a first step an effective amount of a compound of Formula I to V as defined above to the one or more cellular samples, and in a second step monitoring for the formation of a fluorescent derivative arising from the reduction of the nitro group of the compound of Formula I to V by one or more nitroreductase present in the cellular sample.
  • the one or more nitroreductase is a human nitroreductase selected from
  • Cytochrome P450-reductase [CYPOR; E.C. 1.6.2.4];
  • Cytochrome B5 reductase [DIA1 ; E.C.1.6.2.2]; Xanthine oxidase [XO; E.C.1.17.3.2];
  • Adrenodoxin oxidoreductase [FDXR; E.C.1.18.1.2];
  • Methionine synthase reductase [MTRR; E.C.1.16.1.8];
  • Aldose reductase [ALDR1 ; E.C.1.1.1.21]; and Aldehyde reductase [AKR1 B10; E.C. 1.1.1.2]
  • the method further includes the analytical step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
  • an assay for the detection of nitroreductase including the steps of: (i) contacting an effective amount of a plurality of fluorogenic probes with a sample; (ii) monitoring for the formation of fluorescent derivatives.
  • the sample may be added to a common test environment containing a plurality of fluorogenic probes.
  • an assay for the detection of nitroreductase including the steps of: (iii) contacting an effective amount of at least one compounds of formula
  • an assay comprising at least one test environment containing a plurality of fluorogenic probes, wherein a sample may be added and the test environment monitored for the formation of fluorescent derivatives.
  • the fluorogenic probes are selected from compounds of formula I-V as defined in the second aspect.
  • test environment is compatible with sustained cell viability, permitting real time multiple analyses with synchronous detection.
  • a compound of formula I-V as defined in the second aspect wherein one or more nitro substituents is replaced by an amine or hydroxyl amine moiety.
  • Figure 1 shows the structural representations of representative nitro-substituted aromatic compounds 1 to 16 of the present invention.
  • Figure 2 shows representative fluorescent amino-substituted aromatic compounds 17 to 23 of the present invention.
  • Figure 3 shows the fluorescent intensity observed for compounds 1 to 15 when reduced in the presence of E.coli nitroreductase (nfsB).
  • Figure 4 shows the rate of fluorescence signal generation for compound 2 when reduced in the presence of E.coli nitroreductase (nfsB).
  • Figure 5 shows the fluorescent intensity observed for compounds 1 to 15 when reduced in the presence of human aerobic reductase NAD(P)H dehydrogenase quinone 1 (NQO1).
  • Figure 6 shows the rate of fluorescence signal generation for compound 1 when reduced in the presence of human aerobic reductase NAD(P)H dehydrogenase quinone 1 (NQO1).
  • Figure 7 shows the shows the fluorescent intensity observed for compounds 1 to 15 when reduced in the presence of the human anaerobic reductase NADPH Cytochrome P450 reductase (CYPOR).
  • Figure 8 shows the fluorescent intensity of compounds 1 and 13 when reduced in the presence of E.coli nitroreductase B (nfsB) expressing cells co-cultured in the presence of non-expressing cells. Cells were washed and media was replaced after 1 hour with fluorescence monitored over a 4 hour time frame.
  • nfsB E.coli nitroreductase B
  • Figure 9 shows the fluorescent intensity of compounds 4, 13, 14, and 15 when reduced in the presence of E.coli nitroreductase (nfsB) expressing cells (B) or non- expressing cells (A) for 1.5hr and imaged 6 hours after cells were washed free and fresh media was replaced. (NB The image has been rendered monochromatic for the purposes of publication quality.)
  • Figure 10 shows the superiority of compound 2 relative to methyl 7-nitrocoumarin carboxylate (methyl 7-nitro-2-oxo-2/-/-chromene-3-carboxylate) as described in US20020031795A1 as a nitroreductase fluorescent reporter.
  • Figure 11 shows the superior aqueous stability of compound 1 and 2 compared with the disclosed compounds methyl-7-nitrocoumarin carboxylate and 7-nitrocoumarin-3- carboxylic acid as described in US20020031795A1 and Letters in Applied Microbiology, (33) 403-8, 2001.
  • Figure 12 shows the superior aqueous stability under aerobic or anaerobic conditions of compounds 1 to 15 compared with the disclosed compounds methyl-7-nitrocoumarin carboxylate, 7-nitrocoumarin-3-carboxylic acid and 6-chloro-9-nitro-5H- benzo[a]phenoxazin-5-one (Molecular Probes Handbook, 10 th Edition, page 535).
  • Figure 13 shows 3 fluorescent amine reporter molecules of non-overlapping excitation/emission spectra suitable for multiplexed reporter gene applications.
  • Figure 14 shows the multiplex use of compounds 4 and 16 to identify concurrently two nitroreductase expressing cell populations in a common environment.
  • Figure 15 shows the multiplex use of compounds 11 and 13 to identify concurrently two nitroreductase expressing cell populations in a common environment.
  • the invention broadly relates to a method for the detection of nitroreductase activity using at least one fluorogenic probe. More specifically, the invention relates to a method that may be adapted to detect and/or identify a plurality of nitroreductases sequentially or simultaneously in a common test environment. The ability to use a common environment for multiple determinations leads to advantages in assay systems for detection and/or diagnosis.
  • a single detection method can be used (e.g. fluorescence emission) without the need to quench between readings, permitting time- dependent monitoring using such noninvasive detection methods.
  • the method utilises one or more fluorogenic probes, which may be reduced by the action of one or more nitroreductase(s), resulting in one or more strongly fluorescent molecules.
  • the fluorescent output may be detected simultaneously or sequentially in a common test environment.
  • the fluorogenic probes of the invention are readily available and are stable in their non-fluorescent nitro form and as the fluorescent reduced amine or hydroxylamine derivatives. While multiple probes may co-exist in the fluorescent and non-fluorescent forms in a common test environment, the presence of individual fluorescent derivatives may be quickly and easily detected independently. The fluorescent derivatives may be independently detected either sequentially or simultaneously by monitoring their often characteristic fluorescence emission.
  • the inventors have employed singleton synthesis and substructure screening of in- house chemical libraries to collate a Fluorogenic Substrate Library (FSL) including of a range of nitro-substituted aromatic compounds that are likely to be fluorescent upon bioreduction.
  • FSL Fluorogenic Substrate Library
  • High-throughput fluorogenic cell-based screening assays have been developed and several fluorogenic probes have been identified for specific nitroreductases.
  • the nitroreductases that can be detected by this technology may be of microbial or human origin, for example the Escherichia coli oxygen-insensitive minor nitroreductase (NTR) [nfsB], or human DT-diaphorase (DTD) [NQO1 ; E.C.1.6.99.2] and human cytochrome P450-reductase (P450R) [CYPOR; E.C.1.6.2.4].
  • NTR Escherichia coli oxygen-insensitive minor nitroreductase
  • DTD human DT-diaphorase
  • P450R human cytochrome P450-reductase
  • Other human nitroreductases may include
  • Nitro reductases such as NTR and DTD have been shown to catalyse oxygen- insensitive two electron reduction of a nitro (NO 2 ) group to a hydroxylamine (NHOH) group (the four electron reduction product) which may be subsequently reduced to an amine group (the six electron reduction product), while nitroreductases such as P450R catalyse reduction that proceeds via an oxygen-sensitive one electron intermediate as shown in Scheme 1. In the presence of oxygen this one electron intermediate is back- oxidised to regenerate the starting material. In the absence of oxygen (hypoxia) further reduction to a hydroxylamine and amine can occur.
  • the nitro containing molecules (substrates) of interest are non- fluorescent dyes which upon metabolism by a nitroreductase yield stable fluorescent products (the hydroxylamine and amine containing compounds) that emit light upon excitation over a broad range of the spectrum that is proportional to their concentrations. Therefore, metabolic conversion of substrates yields products that are strongly fluorescent, reporting the presence of nitroreductase activity.
  • the stable fluorescent derivatives may be excited using light from the UV/visible spectrum and the fluorescent emission determined using any instrument adapted to detect and quantify light emissions, for example, a UV/vis spectrometer. Compounds of the invention will generally also emit in the UV/visible/IR range (200-800nm).
  • Any non-ubiquitous enzyme which does not occur naturally may be inserted into a cell of interest in such a way that expression of the enzyme is linked to the expression of a cellular gene of interest. For example, it may be placed under the control of an appropriate transcriptional or post-transcriptional control sequence.
  • a nitroreductase of microbial, fungal or mammalian origin utilised in this context is defined as a reporter gene.
  • the catalytic generation of a fluorescent signal from a non-fluorescent substrate correlates with the expression of the reporter gene, thus providing a quantitative and /or spatial measure of the activity of the regulatory sequence and expression of a gene of interest.
  • the fluorescent product may be entrapped within the cell of origin thereby identifying individual cells or tissue regions expressing the reporter gene at the time of compound exposure.
  • Uses of entrapped and freely-diffusing probes can include high-throughput cell based screening assays for compound discovery or identification of regional reporter gene expression within tissue regions of interest. This may include identification of nitroreductase delivered by exogenous vector systems, for example gene therapy, or expressed from tissue specific promoters, for example transgenic animals. Probe use may include identification of cells for subsequent nitroreductase-mediated ablation therapy.
  • the reporter enzyme may be coupled to an assay component of any binding assay such as an antibody/antigen in an immunoassay or a hormone/receptor in an affinity assay or a nucleic acid molecule in a nucleic acid hybridization assay (DNA/DNA, DNA/RNA, DNA/protein) or biotin/streptavidin or lectin/glycoprotein.
  • any binding assay such as an antibody/antigen in an immunoassay or a hormone/receptor in an affinity assay or a nucleic acid molecule in a nucleic acid hybridization assay (DNA/DNA, DNA/RNA, DNA/protein) or biotin/streptavidin or lectin/glycoprotein.
  • the various compounds of the invention incorporate a variety of chromophores and as such can be utilised to determine the presence of at least one nitroreductase in a single test environment.
  • the reduced derivatives of the compounds of the invention fluoresce at characteristic wavelengths. Subsequently, the detection of a characteristic emission signal indicates the presence of a particular reduced substrate. As many of the reduced substrates have unique characteristic emission signals, more than one reduced substrate can be detected in a single/common test environment.
  • Specific compounds of the invention may function as indicative probes for specific nitroreductases. Therefore, a specific nitroreductase may be identified on detection of one or more fluorescent probes. Furthermore, as more than one fluorescent probe can be detected in a common test environment, the method can be applied to identify multiple nitroreductase enzymes in a common test environment.
  • the fluorescent probes can be detected sequentially or simultaneously in the common test environment, as desired by the user. This provides process advantages over other similar assay type systems, which require separate reagents and/or detection methods for each probe used. The ability to obtain multiple results from a single test will allow much faster screening of samples resulting in improved efficiency in detection and/or diagnostic methods. No requirement is imposed for each reporter activity to be assayed separately, generally employing different chemistry and detection methods, with sequential measurements and iterative chemistry steps or sample separation.
  • the emission spectra of the florescent probes used in the multiplex environment will be sufficiently discrete to allow detection of the individual fluorescent derivatives.
  • Test environments can include high throughput small molecule of biological molecule screening platforms designed to establish the differential biological effects on one cell population over another, or effects on a specific signal transduction pathway relative the another in order to aid in the identification of agents that are active for a given utility.
  • This can include, but is not limited to the use of differential promoter assay to identify modulators of certain signal transduction pathways and mixed cell populations where an intended effect upon a subpopuiation is desired. Screening can be conducted in separate cell populations that can be subsequently mixed in a single test environment or multiple promoter activities within a single cell population.
  • Other platforms can include single cell fluorescent microscopy with high content image analysis for high throughput applications, including signal ratio calculations of multiplex signals to provide additional information relating to the differential activity of reporter gene nitroreductases in a common test environment.
  • signal ratio calculations of multiplex signals to provide additional information relating to the differential activity of reporter gene nitroreductases in a common test environment.
  • enzyme generated fluorescent signal detection including confocal microscopic detection of cell populations to monitor intracellular processes such as protein trafficking with the aid of split excitation and lasar photobleaching.
  • a sample can be applied to an assay test environment (eg an assay well) containing a plurality of fluorogenic probes.
  • an assay test environment eg an assay well
  • Light from the UV/visible spectrum can be used to excite any reduced derivatives in the test environment, which may fluoresce at a characteristic wavelength, thus indicating the presence of specific nitroreductase enzyme(s).
  • Nitroreductase activity is common, being found in the majority of organisms including obligate aerobic and anaerobic bacteria, fungi and eukaryotic parasites. Conversion of a non-fluorescent substrate to a fluorescent product provides a universal test for the presence of microorganisms in samples or cultures. Uses may include, but are not limited to, bioremediation, sterility tests, antibiotic susceptibility and quantification of organisms present in any sample.
  • the invention may be employed to demonstrate the presence of nitroreductase activity in any test sample containing one or more microorganisms of commercial value (e.g. food product, soil sample, aqueous sample) or medical interest (e.g. body fluids).
  • test sample containing one or more microorganisms of commercial value (e.g. food product, soil sample, aqueous sample) or medical interest (e.g. body fluids).
  • Compounds disclosed in this invention may be used in the detection and/or diagnostic tests for human nitroreductase activity.
  • certain obligate two- electron reductases for example NAD(P)H dehydrogenase quinone 1 (DT-Diaphorase; NQO1 , E. C.1.6.99.2)
  • DT-Diaphorase NQO1 , E. C.1.6.99.2
  • one-electron reductases for example NADPH cytochrome P450 reductase (CYPOR, E.C. 1.6.2.4
  • CYPOR E.C. 1.6.2.4
  • compounds may be employed to detect the total (composite) reductive activity of ubiquitous one-electron reductase activities. This has utility for predicting total reductive catalytic capacity of living tissue samples or preparations there of and may be of value in predicting reductive metabolism of therapeutic agents, for example hypoxic cytotoxins.
  • the conversion of a non-fluorescent substrate to a fluorescent product provides a measure of nitroreductase activity of interest and correlates with catalytic activity in the sample.
  • the fluorescent product may be entrapped within the cell of origin thereby identifying it as expressing the reductase of interest at the time of compound exposure. Signal retention may correlate with amplitude of enzyme catalysis providing a measure of the heterogeneity within a cell, tissue or analyte sample series.
  • Compounds disclosed in this invention may be used in the detection and/or diagnostic tests for tissue hypoxia ( ⁇ 1% O 2 ). Conversion of a non-fluorescent substrate to a fluorescent product by ubiquitous one-electron reductases, which occurs selectively in the absence of oxygen, provides a test for the relative absence of oxygen in a specific cell population or tissue region. In certain utilities, the fluorescent product may be entrapped within the cell of origin thereby identifying it as hypoxic at the time of compound exposure.
  • the conversion of a non-fluorescent substrate to a fluorescent product by an oxygen-inhibited reductase can provide a measure of hypoxia in any test system of interest.
  • the generation of fluorescence signal correlates with the concentration of oxygen in the sample.
  • the human breast cancer cell line (MDA ⁇ aO and a clonal derivative (MDA231 NTR ) engineered to express the reporter gene E.coli nitroreductase (nfsB) under the control of a constitutive promoter were seeded into 96-well plates at a density of 1x10 5 cells/well.
  • nfsB E.coli nitroreductase
  • Figure 1 compounds 1 to 15 were added to a final concentration of 100 ⁇ M for 4 hr.
  • Test groups were cell-free culture media alone (control), MDA231 m and MDA231 NTR .
  • the fluorescence signal was monitored at an excitation wavelength of 355 nm and emission wavelength of 460 nm (355/460) except for compounds 4 and 11 that were monitored at 405/585 and compounds 8 and 10 that were monitored at 355/585 and 355/535 respectively (figure 1). No fluorescence was observed in either the cell-free control or parental MDA231 WT containing cultures. Compounds 1 - 15 inclusive gave rise to a fluorescent signal specifically in the presence of E.coli nitroreductase (nfsB) expression.
  • nfsB E.coli nitroreductase
  • the human colorectal cancer cell line (HCT1 ' ⁇ 6 WT ) and a clonal derivative (HCT116 NTR ) engineered to express the reporter gene E.coli nitroreductase (nfsB) under the control of a constitutive promoter were suspended in stirred culture media at a density of 5x10 6 cells/ml.
  • nfsB E.coli nitroreductase
  • test groups were culture media alone (control), HCT116 ⁇ " and HCT116 NTR .
  • HCT116 NTR cells rapidly reduced compound (2) a process that approached completion by 9hrs. No detectable fluorescence was observed in either the control or parental HCT116 ⁇ containing cultures.
  • the human breast cancer cell line (MDA231 WT ) and a clonal derivative (MDA231 DTD ) engineered to express the human aerobic reductase, NAD(P)H dehydrogenase quinone 1 (DT-diaphorase; NQO1) under the control of a constitutive promoter were seeded into 96-well plates at a density of 1x10 5 cells/well. When samples were equilibrated to 37°C, 5%CO 2 for 2 hr and compounds 1 to 15 were added to a final concentration of 100 ⁇ M for 4 hr. Test groups were cell-free culture media alone (control), MDA231 m and MDA231 DTD .
  • the fluorescence signal was monitored at 355/460 except for compounds 4 and 11 that were monitored at 405/585 and compounds 8 and 10 that were monitored at 355/585 and 355/535 respectively (figure 5). No detectable fluorescence was observed in either the control or parental
  • Compounds 1 and 3 gave rise to a fluorescent signal specifically in the presence of human NQO1 expression.
  • the human breast cancer cell line (MDA231 wr ) and a clonal derivative (MDA231 DTD ) engineered to express the human NQO1 gene (DT- diaphorase) under the control of a constitutive promoter were seeded into 96-well tissue culture plates at 1x10 5 cells/well. Samples were equilibrated to 37 0 C, 5%CO 2 , compound (1) (6-nitro-4(1H)-quinolinone) was added to a final concentration of 300 ⁇ M.
  • Test groups were culture media alone (control), MDA231 WT cells and MDA231 DTD cells.
  • the rate of fluorescence signal generation at 355/460 was monitored as a function of time ( Figure 6).
  • MDA231 DTD cells reduced compound 1 and 3 (see Figure 5) to generate a fluorescent signal. No detectable fluorescence was observed in either the wells containing compound 1 alone (control) or parental MDA231 WT .
  • Test groups were cell-free culture media alone (control), MDA231 P450R under normoxic (air) and anoxic (N 2 ) conditions.
  • the fluorescence signal was monitored at 355/460 except for compounds 4 and 11 that were monitored at 405/585 and compounds 8 and 10 that were monitored at 355/585 and 355/535 respectively (figure 7).
  • No detectable fluorescence was observed in either the control or aerobic MDA231 P450R containing cultures.
  • Compounds 1 - 5 and 10 - 15 gave rise to a fluorescent signal specifically in the presence of human cytochrome P450 reductase expression when oxygen was absent.
  • Cellular Entrapment A particularly attractive property of a number of fluorogenic probes described herein is that of cellular entrapment of the fluorescent reporter molecule produced upon nitroreductase activity.
  • These compounds comprise of at least one NO 2 group and at least one of the groups R 1 , R 2 , R 3 , R 4 , R 5 of the molecule of formula I 1 R 1 , R 2 of formula II, R 1 of formula III, R 2 , R 3 , R 4 , R 5 of formula III', R 1 of formula IV, R 1 , R 2 , R 3 of formula V that provides for cell membrane permeabilising properties.
  • Membrane permeant compounds can generally be provided by masking hydrophilic groups. After entry into the cell the masking group can be designed to be cleaved to produce a hydrophilic fluorogenic substrate that provides a cell entrapped fluorescent report in the presence of reductase activity.
  • compounds comprising of at least one NO 2 group and at least one of the groups R 1 , R 2 , R 3 , R 4 , R 5 of the molecule of formula I, R 1 , R 2 of formula II, R 1 of formula III, R 2 , R 3 , R 4 , R 5 of formula III', R 1 of formula IV, R 1 , R 2 , R 3 of formula V that provides for increased DNA affinity can provide a nuclear localised cell entrapped fluorescent report in the presence of nitroreductase activity.
  • HCT116 m human colorectal cancer cell line
  • HCT116 NTR a clonal derivative engineered to express the reporter gene E.coli nitroreductase (nfsB) under the control of a constitutive promoter
  • nfsB E.coli nitroreductase
  • HCT116 m human colorectal cancer cell line
  • HCT116 NTR a clonal derivative engineered to express the reporter gene E.coli nitroreductase (nfsB) under the control of a constitutive promoter
  • the human breast cancer cell line (MDA231 WT ) and a clonal derivative (MDA231 NTR ) engineered to overexpress the E.coli nitroreductase (nfsB) under the control of a constitutive promoter were seeded into 96-well plates at a density of 2x10 4 cells/well.
  • nfsB E.coli nitroreductase
  • the fluorescence signal was monitored at 355/460 over a 7.5 hour time frame (figure 10).
  • no detectable fluorescence (above background) was observed in the control cell line MDA231 WT containing cultures, whereas a robust fluorescent signal was observed in the nitroreductase-expressing cell line MDA231 NTR giving rise to a 480-fold differential signal at 7.5 hours.
  • the methyl 7-nitrocoumarin carboxylate compound generated measurable fluorescence in the control cell line MDA231 WT containing cultures, which was only elevated 1.6-fold in the nitroreductase-expressing cell line MDA231 NTR . Therefore compound 2 is demonstrably superior to methyl 7- nitrocoumarin carboxylate as disclosed in US20020031795A1.
  • the fluorescence signal was monitored at 355/460 except for compounds 4 and 11 that were monitored at 405/585 and compounds 8, 10 and 6-CI-9-nitro-5-oxo-5H- benzo[a]phenoxazine that were monitored at 355/585, 355/535 and 530/585 respectively ( Figure 12).
  • Figure 12 For compounds 1 -15 no detectable fluorescence (above background) was observed under either oxic or anoxic conditions.
  • the methyl 7-nitrocoumarin carboxylate compound, the 7-nitrocoumarin-3-carboylic acid compound and the 6-chloro-9-nitro-5H-benzo[a]phenoxazin-5-one compound generated measurable fluorescence in cell-free culture media, specifically under conditions of low oxygen, indicative of instability.
  • compounds 1-15 are demonstrably superior for detecting mammalian anaerobic reductase activities to methyl 7-nitrocoumarin carboxylate (methyl 7-nitro-2-oxo-2/-/-chromene-3-carboxylate) and 7-nitrocoumarin-3-carboylic acid as disclosed in US20020031795A1 , and 6-chloro- 9-nitro-5/-/-benzo[a]phenoxazin-5-one (also known as C22220, CNOB) as disclosed by Invitrogen (Molecular Probes Handbook, 10 th Edition, page 535).
  • Compounds 23, 18 and 22 (as representative fluorescent amino-substituted aromatic compounds of the present invention) were utilized to exemplify the capacity to specifically and independently monitor multiple fluorescent signal outputs from a mixture of compounds within a sample. Compounds 23, 18 and 22 were placed in
  • compounds 4 and 16 gave fluorescent output at Ex/Em wavelengths of 485/535 and 355/460 respectively; detecting the presence of Eshericia coli nfsB and human cytochrome P450 reductase (CYPOR) enzyme activity independently ( Figure 14a).
  • CYPOR cytochrome P450 reductase
  • MDA-231 cells expressing Eshericia coli nfsB and human cytochrome P450 reductase were mixed 1 :1 , seeded into a glass 96-well plate (15,000 cells/well in 0.1 mL ⁇ MEM + 10% FCS + P/S) and allowed to attach overnight.
  • Compound 16 and 4 were diluted into ⁇ MEM (from DMSO stock solutions) and 0.1 mL was added to achieve a final concentration of 300 ⁇ M and 10 ⁇ M respectively. Samples were incubated at 37°C for 45 minutes, washed three times in PBS and images were acquired on a Nikon TE-2000 inverted fluorescence microscope.
  • Figure 14b provides a co-registration image of compounds 16 and 4 (DAPI and FITC filter set respectively).
  • the fluorescent emissions of compounds 16 and 14 appear as blue and green marks on the co-registration image.
  • Figure 14c and 14d identify each individual cell population, with overlays of 16 and 4 respectively with the corresponding phase contrast image (note: figure 14c shows blue fluorescence emission, while figure 14d shows green fluorescence emission.
  • Figure 14b-d shows that two co-cultured cell populations can be readily identified and distinguished as mutually exclusive cell populations in a common environment through the co-application of two representative nitro-substituted aromatic compounds of the present invention.
  • Figure 14b-d This illustrates that multiple independent outputs can be recorded from a single sample providing appropriate excitation and emission wavelengths are utilized. The nature of the output may enable co-registration of independent signals within a single test environment which may be correlated to specific mammalian or microbial nitroreductase activities within the test sample of interest.
  • compounds 11 and 13 gave fluorescent output at Ex/Em wavelengths of 485/535 and 355/460 respectively; detecting the presence of Eshericia coli nfsB and human cytochrome P450 reductase (CYPOR) enzyme activities.
  • CYPOR cytochrome P450 reductase
  • MDA-231 wild-type cells or cells expressing Eshericia coli nfsB and human cytochrome P450 reductase were mixed 1 :1 :1 , seeded onto glass coverslips (15,000 cells/well in 0.1 ml. ⁇ MEM + 10% FCS + P/S) and allowed to attach overnight.
  • FIG. 15c provides a co-registration image of compounds 11 and 13 (FITC and DAPI filter set respectively).
  • Figure 15c identifies each individual cell population, with overlay of the corresponding phase contrast image; wild-type cells (W) or clones stably expressing either Eshericia coli nfsB (N) or human cytochrome P450 reductase (R).
  • Examples 7, 8 and 9 clearly demonstrates that a plurality of fluorescent probes can be detected and identified in a common test environment. Therefore a plurality of probes may be used to identify one or more nitroreductase(s) in a common environment. Thus, multiplex reporter output may be achieved.

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Abstract

A method utilising one or more fluorogenic probes, for the detection of nitroreductase activity. The non-fluorescent probes are reduced in the presence of nitroreductase to form fluorescent derivatives that may be detected using fluorescence spectroscopy. In particular, the method may be used to detect and/or identify a plurality of nitroreductase in a single test environment.

Description

A method for the fluorescent detection of nitroreductase activity using nitro-substituted aromatic compounds
This invention relates generally to a method for the fluorescent detection of nitroreductase activity using at least one fluorogenic probe. The method utilises one or more probes, which are non-fluorescent aromatic compounds containing at least one NO2 group, that is reduced to NHOH or NH2 by the action of a nitroreductase resulting in the production of a strongly fluorescent molecule. In particular, the invention relates to a method of detection based on the use of a plurality of such probes in a common environment. A novel class of nitro-substituted compounds is also provided.
Background
A small group of non-fluorescent 7-nitrocoumarins are described in US20020031795A1 that are reduced by nitroreductase into a fluorescent derivative for the fluorogenic detection of microbial infection. The present inventors have found that the 7-nitrocoumarins are activated in wild type neoplastic mammalian cells and tend to lack stability in solution over a period of time. The inventors have also established that the intensity of the fluorescent signal of the 7-nitrocoumarins can be variable making it very difficult to undertake quantitative diagnostic studies.
A 6-chloro-9-nitro-5H-benzo[a]phenoxazin-5-one (C-22220, CNOB) has been described as a fluorogenic probe for nitroreductase activity, but no specific information on particular reductases is given (Molecular Probes Handbook, 10th Edition, page 535). The inventors have established that this compound lacks stability in culture medium under conditions of low oxygen making it unsuitable as a probe for mammalian single- electron reductases which require anaerobic conditions for activity.
Some nitroacridone derivatives are described in US20040191792A1 as dyes having characteristic fluorescence lifetimes. These acridone dyes do not require reduction by a nitroreductase to exhibit fluorescence. The acridone chromophore is inherently highly fluorescent.
Nitro quenched cyanine dyes are taught in US20030186348A1 as a way of enhancing the fluorescence output of the cyanine dyes for the detection of microbial nitroreductases with the emphasis on reporter gene applications. These compounds have considerable fluorescence in their quenched form in cell culture and upon the action of a nitroreductase increase in fluorescence by three to four-fold offering a ' limited dynamic range for reporter gene applications.
The ability to assay for more than one reporter enzyme is particularly inefficient and laborious due to the current inability to identify multiple reporter enzymes in a common test environment. For example firefly or renilla luciferases may only be quantified in cultured mammalian cells following cell lysis (destruction) a procedure that is incompatible with many other assays. Multiplexing unrelated reporter genes is usually problematic, or at best requires each to be assayed separately employing different chemistry and detection methods, with sequential measurements and iterative chemistry steps or sample replating. In contrast, nitroreductase detection is a nondestructive assay protocol that can be detected in a common environment, for example an individual well or a single cell, using the same detection method simultaneously (e.g. fluorescence emission), without the need to quench one before measuring another. Nitroreductases are thus ideal for multiplexing on sub-cellular imaging systems including microscopes, sub-cellular imagers and plate readers.
It is an object of the invention to provide a method for the fluorescent detection of nitroreductase activity using nitro-substituted aromatic compounds as one or more fluorogenic probes to assay, qualify and/or quantify for diagnostic purposes, including the detection of microbial infection, the use in imaging applications, quantification of microorganisms in test samples, diagnostic testing for human reductase activity and detection in reporter gene assays, or to at least provide the public with a useful choice. Another object of the invention is to provide a novel class of nitro-substituted aromatic compounds, or to at least provide a useful alternative.
Summary of the Invention
In a first aspect, the present invention provides a method for the fluorescent detection of nitroreductase activity using a fluorogenic probe suitable for use as a nitroreductase probe, the method including the step of applying a plurality of probes to a sample and monitoring for the presence of at least one nitroreductase enzyme in a common test environment.
Preferably, the step of monitoring for the presence of at least one nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of the fluorogenic probe. Preferably, the reduced derivatives may be excited at predetermined wavelengths in the UV/visible range and the fluorescence emission determined.
Preferably, the excitation wavelength will be between 200-700nm. More preferably, the excitation wavelength may be selected from 295, 340, 355, 405, 440 and 485 nm.
Preferably, the fluorescence emission will be in the UV/visible/IR range.
Preferably, the fluorescence emission wavelength will be between 300-800nm. More preferably, the fluorescence emission wavelength may be selected from 370, 460, 510, 535, 540 and 585nm.
Preferably, the method further includes the step of quantifying the activity of at least one nitroreductase. More preferably, the step of quantifying the nitroreductase activity includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
Preferably, the monitoring of a plurality of fluorogenic probes can be performed simultaneously.
Preferably, the monitoring of a plurality of fluorogenic probes can be performed sequentially.
In a second aspect the present invention provides a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro-aromatic compounds of Formula I-V as defined herein, suitable for use as a nitroreductase probe, the method including the step of applying the one or more fluorogenic probes to a sample and monitoring for the presence of at least one nitroreductase.
Preferably, the step of monitoring for the presence of nitroreductase activity includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula I-V.
Preferably, the method further includes the step of quantifying the nitroreductase activity. More preferably, the step of quantifying the nitroreductase activity includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
Preferably, the method uses a plurality of fluorogenic probes.
Preferably, the monitoring can be performed in a common test environment.
In a third aspect the present invention provides a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- aromatic compounds of Formula I suitable for use as a nitroreductase probe
Figure imgf000005_0001
wherein X represents N1 NH, NR6, O or S;
Z represents C, CH or N; wherein R1, if present, may be selected from H, R7, (CR7R8)nCOOH, (CR7R8)nCOOR9,
(CR7R8JnCONH2, (CR7R8JnCONHR9, (CR7R8JnCONR9R10, (CR7R8JnOH, (CR7R8JnOR9,
(CR7R8JnOPO(OH)2, COOH, COOR7, (CR7R8JnNR9R10, (CR7R8)n-morpholinyl, (CR7R8Jn- piperazinyl; (CR7R8Jn-I -methylpiperazinyl; (CR7R8)n-piperidinyl; (CR7R8)n-pyrrolidinyl or
(CR7R8)n-imidazolyl; wherein R2 may represent H, R11, (CR11R12JnCOO(CR11R12JnNR13R14,
(CR11R12JnCONH(CR11R12JnNR13R14, wherein C and C" may be optionally and independently substituted with C1-C6 alkyl and/or OH;
R3 and R4 may independently represent H, R15, Ar, -(CH=CH)nAr; SO3H, CN wherein Ar may represent a substituted or unsubstituted phenyl, pyridyl, pyrimidinyl, thiazolyl, oxazoylyl, imidazolyl, furanyl, pyrrolyl, benzoxazolyl, benzthiazolyl, benzofuranyl, indolyl, indazolyl, benzimdazolyl, wherein each Ar is optionally substituted with NO2, R16, OH, OR16, SH, SR16, halogen, CF3, NH2, NHR16, NR16R17,
NHCOR16, NR16COR17, NHCOOR16, NR16COOR17, (CR16R17JnCOOH,
(CR16R17JnCOOR18, (CR16R17JnCONH2, (CR16R17JnCONHR18, (CR16R17JnCONR18R19, (CR16R17)nOH, (CR16R17)nOR18, (CR16R17)nOPO(OH)2, COOH, COOR16, CONH2, CONHR16, CONR16R17, COR16, CN, SOR16, SO2R16, SO2NR16R17, SO3H, or when Z represents C1 R3 and R4 may together form a fused aromatic ring optionally substituted at one or more of the available carbons with a Ci-C6 alkyl, halogen, SO3H or CN; wherein R5 may represent H, C1-C6 alkyl, halogen, CN, NO2, Ar, -(CH=CH)nAr, COR20, SOR20, SO2R20, CO(CR20R21 )nOH, SO(CR20R21 )nOH, SO2(CR20R21)nOH, CO(CR20R21 JnCOOR22, SO(CR20R21)nCOOR22 SO2(CR20R21)nCOOR22, CO(CR20R21JnNR22R23, SO(CR20R2^)nNR22R23, SO2(CR20R21JnNR22R23, CO(CR20R21JnOPO(OH)2, SO(CR20R21)nOPO(OH)2, SO2(CR20R21JnOPO(OH)2, CONR20R21, SONR20R21, SO2NR20R21, CNNR20(CR21R22)πCOOR23,
CNNR20(CR21R22)nOH, CNNR20(CR21 R22JnNR23R24, CNNR20(CR21R22)nOPO(OH)2, CONR20(CR21R22)nOH, SONR20(CR21 R22JnOH, SO2NR20(CR21R22)nOH, CONR20(CR21 R22JnCOOR23, SONR20(CR21R22)nCOOR23 , SO2NR20(CR21R22)nCOOR23 , CONR20(CR21 R22JnNR23R24, SONR20(CR21 R22JnNR23R24, SO2NR20(CR21 R22JnNR23R24, CONR20(CR21 R22JnOPO(OH)2, SONR20(CR21 R22JnOPO(OH)2, SO2NR20(CR21 R22JnOPO(OH)2; wherein n = O, 1 , 2, 3, 4, 5 or 6; and R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24 independently may represent H, C1-C6 alkyl, halogen, OH, (CR25R26)nCOOR27, (CR25R26JnNR27R28, (CR25R26JnOH, (CR25R26JnOPO(OH)2 and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl, wherein R25, R26, R27 and R28 may represent H, C1-C6 alkyl, halogen and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and any pharmaceutically acceptable salt thereof, the method including the step of applying the one or more fluorogenic probes to a sample and monitoring for the presence of at least one nitroreductase.
Preferably, the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula I.
Preferably, the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
Preferably, in the compound of Formula I X is O or NH, Z is CH or NH and R3 is H. Preferably the fluorogenic probe of Formula I is selected from
6-nitro-4(1 H)-quinolinone,
1 -methyl-6-nitro-4(1 H)-quinolinone,
2-methyl-6-nitro-4(1H)-quinolinone,
Λ/-[2-(dimethylamino)ethyl]-2-nitro-9-oxo-9,10-dihydro-4-acridinecarboxamide,
6-nitro-4(3H)-quinazolinone,
6-nitro-2-phenyl-4(3/-0-quinazolinone, methyl (6-nitro-4-oxo-1 (4H)-quinolinyl)acetate,
Λ/,Λ/-dimethyl-3-[(7-nitro-4-quinolinyl)oxy]-1-propanamine,
/v^/^-dimethyl-Λ/^y-nitro^-quinolinyO-i ,3-propaπediamine, and
ΛΛ/^-dimethyl-Λ/^δ-nitro^-quinazolinylJ-i ,3-propanediamine and any pharmaceutically acceptable salt thereof.
In a fourth aspect there is provided a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- substituted aromatic compounds of Formula Il
Figure imgf000007_0001
wherein X represents N, CH, O or S; Z represents C or N; wherein R1 and R2 if present, may independently represent H, Ci-C6 alkyl, Ar, -(CH=CH)nAr, (CR3R4JnNR5R6, (CR3R4)nCOOR5, (CR3R4JnOH, (CR3R4JnOPO(OH)2; wherein n = 0, 1 , 2, 3, 4, 5 or 6; wherein Ar may represent a substituted or unsubstituted phenyl, pyridyl, pyrimidinyl, thiazolyl, oxazoylyl, imidazolyl, furanyl, pyrrolyl, benzoxazolyl, benzthiazolyl, benzfuranyl, indolyl, indazolyl, benzimdazolyl, wherein each Ar is optionally substituted with one or more NO2, CN, R3, OH, OR3, SH, SR3, halogen, CF3, NH2, NHR3, NR3R4, NHCOR3, NR3COR4, NHCOOR3, NR3COOR4, (CR3R4JnCOOH, (CR3R4JnCOOR5, (CR3R4JnCONH2, (CR3R4JnCONHR5, (CR3R4JnCONR5R6, (CR3R4JnOH, (CR3R4JnOR5, (CR3R4JnOPO(OH)2, COOH, COOR3, CONH2, CONHR3, CONR3R4, COR3, SOR3, SO2R3, SO2NR3R4, SO3H; wherein R3, R4, R5 or R6 may independently represent H, C1-C6 alkyl, halogen, OH, (CR7R8)nNR9R10, (CR7R8JnOH, (CR7R8)πOPO(OH)2 and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R7, R8, R9 and R10 may represent H or C1-C6 alkyl and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and any pharmaceutically acceptable salts thereof the method including the step of applying the one or more fluorogenic probes to a sample and monitoring for the presence of at least nitroreductase.
Preferably, the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula II.
Preferably, the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
Preferably, in the compound of Formula Il X is N and Z is C, R1 = H and R2 = Ar.
Preferably the compound of Formula Il is selected from
5-nitro-2-phenyl-1 H-benzimidazole and any pharmaceutically acceptable salt thereof.
In a fifth aspect there is provided a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- substituted aromatic compounds of Formula III
Figure imgf000008_0001
wherein R1 may represent COR2, SOR2, SO2R2, CO(CR2R3)nOH, SO(CR2R3)nOH, SO2(CR2R3)nOH, CO(CR2R3)nCOOR4, SO(CR2R3)nCOOR4 SO2(CR2R3)nCOOR4, CO(CR2R3)nNR4R5, SO(CR2R3)nNR4R5, SO2(CR2R3)nNR4R5, CO(CR2R3)nOPO(OH)2, SO(CR2R3)nOPO(OH)2, SO2(CR2R3)nOPO(OH)2, CONR2R3, SONR2R3, SO2NR2R3, CNNR2(CR3R4)nCOOR5, CNNR2(CR3R4)nOH, CNNR2(CR3R4)nNR5R6, CNNR2(CR3R4)nOPO(OH)2, CONR2(CR3R4)nOH, SONR2(CR3R4)πOH, SO2NR2(CR3R4)πOH, CONR2(CR3R4)πCOOR5, SONR2(CR3R4)nCOOR5, SO2NR2(CR3R4)πCOOR5 CONR2(CR3R4)nNR5R6, SONR2(CR3R4)nNR5R6, SO2NR2(CR3R4)nNR5R6, CONR2(CR3R4)nOPO(OH)2, SONR2(CR3R4)nOPO(OH)2, SO2NR2(CR3R4)nOPO(OH)2; wherein n = 0, 1 , 2, 3, 4, 5 or 6; and R2, R3, R4, R5, R6 independently may represent H, Ci-C6 alkyl, halogen, OH, (CR7R8JnNR9R10, (CR7R8)nOH, (CR7R8JnOPO(OH)2 and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R7, R8, R9 and R10 may represent H, C1-C6 alkyl, halogen and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and any pharmaceutically acceptable salt thereof the method including the step of applying the one or more fluorogenic probes to a sample and monitoring for the presence of at least one nitroreductase.
Preferably, the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula III.
Preferably, the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
Preferably, in the compound of Formula III R1 is SO2NR2(CR3CR4)nCOOR5, wherein R2, R3, R4 and R5, may represent H, C1-C6 alkyl, halogen or OH and wherein n = 0, 1 , 2, 3, 4, 5 or 6.
Preferably the compound of Formula III is selected from methyl 4-{[(5-nitro-1 -naphthyl)sulfonyl]amino}butanoate, methyl 4-{[(5-nitro-2-naphthyl)sulfonyl]amino}butanoate and methyl 4-{[(8-nitro-2-naphthyl)sulfonyl]amino}butanoate and any pharmaceutically acceptable salt thereof.
In sixth aspect there is provided a class of nitroaromatic compounds of formula III'
Figure imgf000010_0001
M,< wherein R2, R3, R4, R5 independently may represent H, C1-C6 alkyl, halogen, OH, (CR6R7)πNR8R9, (CR6R7)nOH, (CR6R7)nOPO(OH)2 and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R6, R7, R8 and R9 may represent H, Ci-C6 alkyl, halogen and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and wherein n = 0, 1 , 2, 3, 4, 5 or 6; and any pharmaceutically acceptable salt thereof
Preferably the compound of Formula III' is selected from methyl 4-{[(5-nitro-1-naphthyl)sulfonyl]amino}butanoate, methyl 4-{[(5-nitro-2-naphthyl)sulfonyl]amino}butanoate and methyl 4-{[(8-nitro-2-naphthyl)sulfonyl]amino}butanoate and any pharmaceutically acceptable salt thereof.
In a seventh aspect there is provided a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- substituted aromatic compounds of Formula IV
Figure imgf000010_0002
wherein R1, may be selected from H, R2, (CR2R3)nCOOH, (CR2R3)nCOOR4, (CR2R3JnCONH2, (CR2R3JnCONHR4, (CR2R3JnCONR4R5, (CR2R3JnOH, (CR2R3JnOR4, (CR2R3JnOPO(OH)2, (CR2R3JnNR4R5, (CR2R3)n-morpholinyl, (CR2R3)n-piperazinyl; (CR2R3)n-1-methylpiperazinyl; (CR2R3)n-piperidinyl; (CR2R3)n-pyrrolidinyl or (CR2R3)n- imidazolyl; wherein n = 0, 1 , 2, 3, 4, 5 or 6; and R2, R3, R4, R5 independently may represent H, C1-C6 alkyl, halogen, OH, (CR6R7)nNR8R9, (CR6R7)nOH, (CR6R7JnOPO(OH)2 and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R6, R7, R8 and R9 may represent H, Ci-C6 alkyl, halogen and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and any pharmaceutically acceptable salt thereof the method including the step of applying the one or more fluorogenic probes to a sample and monitoring for the presence of at least one nitroreductase.
Preferably, the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula IV.
Preferably, the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
Preferably, in the compound of Formula IV R1 is (CR2R3)nNR4R5.
Preferably the compound of Formula I is selected from
2-[4-(dimethylamino)butyl]-5-nitro-1H-benzo[de]isoquinoline-1 ,3(2f/)-dione and any pharmaceutically acceptable salt thereof.
In a eighth aspect there is provided a method for the fluorescent detection of nitroreductase activity using as one or more fluorogenic probes a class of nitro- substituted aromatic compounds of Formula V
Figure imgf000011_0001
wherein R1 may represent H, R4, COR4, SOR4, SO2R4, CO(CR4R5)nOH, SO(CR4R5)πOH, SO2(CR4R5)nOH, CO(CR4R5)nCOOR6, SO(CR4R5)nCOOR6 SO2(CR4R5)nCOOR6, CO(CR4R5)nNR6R7, SO(CR4R5JnNR6R7, SO2(CR4R5)nNR6R7, CO(CR4R5)nOPO(OH)2, SO(CR4R5)πOPO(OH)2, SO2(CR4R5)nOPO(OH)2, CONR4R5, SONR4R5, SO2NR4R5, CNNR4(CR5R6)nCOOR7, CNNR4(CR5R6)πOH,
CNNR4(CR5R6)nNR7R8, CNNR4(CR5R6)nOPO(OH)2, CONR4(CR5R6)nOH, SONR4(CR5R6)nOH, SO2NR4(CR5R6)nOH, CONR4(CR5R6)nCOOR7, SONR4(CR5R6)nCOOR7 , SO2NR4(CR5R6)nCOOR7 , CONR4(CR5R6)nNR7R8, SONR4(CR5R6)nNR7R8, SO2NR4(CR5R6)nNR7R8, CONR4(CR5R6)nOPO(OH)2, SONR4(CR5R6)nOPO(OH)2, SO2NR4(CR5R6)nOPO(OH)2;
R2 and R3 may independently represent H, R9, (CR9R10JnCOOH, (CR9R10JnCOOR11, (CR9R10JnCONH2, (CR9R10JnCONHR11, (CR9R10JnCONR11R12, (CR9R10JnOH, (CR9R10JnOR11, (CR9R10JnOPO(OH)2, COOH, COOR9, CONH2, CONHR9, CONR9R10, COR9, CN, SOR9, SO2R9, SO2NR9R10, or R2 and R3 may together form a fused aromatic ring optionally substituted at one or more of the available carbons with a C1- C6 alkyl, halogen, SO3H or CN; wherein n = O, 1 , 2, 3, 4, 5 or 6; and R4, R5, R6, R7, R8, R9, R10, R11, R12 independently may represent H, C1-C6 alkyl, halogen, OH, (CR13R14JnNR15R16, (CR13R14JnOH, (CR13R14JnOPO(OH)2 and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R13, R14, R15 and R16 may represent H, C1- C6 alkyl, halogen and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and any pharmaceutically acceptable salt thereof the method including the step of applying the one or more fluorogenic probes to a sample and monitoring for the presence of at least one nitroreductase.
Preferably, the step of monitoring for the presence of nitroreductase includes the step of monitoring for the presence of a reduced fluorescent derivative of a compound of Formula V.
Preferably, the method further includes the step of quantifying the nitroreductase. More preferably, the step of quantifying the nitroreductase includes the step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
Preferably, in the compound of Formula V R2 and R3 together represent Ar. Preferably the compound of Formula V is selected from 3-nitro-6H-benzo[c]chromen-6-one and any pharmaceutically acceptable salt thereof.
Preferably in the method aspects defined above the one or more nitroreductase is a human oxidoreductase selected from known human enzymes classified as EC 1 in the EC number classification of enzymes. Oxidoreductases are classified into 22 subclasses of which 6 have known nitroreductase activities:
EC 1.1 includes oxidoreductases that act on the CH-OH group of donors e.g. Aldose reductase [ALDR1 ; E.C.1.1.1.21]; e.g. Aldehyde reductase [AKR1 B10; E.C. 1.1.1.2].
EC 1.2 includes oxidoreductases that act on the aldehyde or oxo group of donors
EC 1.3 includes oxidoreductases that act on the CH-CH group of donors
EC 1.4 includes oxidoreductases that act on the CH-NH2 group of donors EC 1.5 includes oxidoreductases that act on CH-NH group of donors
EC 1.6 includes oxidoreductases that act on NADH or NADPH e.g. DT-diaphorase [NQO1 ; E.C.1.6.99.2]; e.g. Cytochrome P450-reductase [CYPOR; E.C. 1.6.2.4]; e.g. Cytochrome B5 reductase [DIA1 ; E.C.1.6.2.2]; EC 1.7 includes oxidoreductases that act on other nitrogenous compounds as donors
EC 1.8 includes oxidoreductases that act on a sulfur group of donors e.g. Thioredoxin-disulfide reductase [TXNRD; E.C.1.8.1.9];
EC 1.9 includes oxidoreductases that act on a heme group of donors
EC 1.10 includes oxidoreductases that act on diphenols and related substances as donors
EC 1.11 includes oxidoreductases that act on peroxide as an acceptor (peroxidases)
EC 1.12 includes oxidoreductases that act on hydrogen as donors
EC 1.13 includes oxidoreductases that act on single donors with incorporation of molecular oxygen (oxygenases) EC 1.14 includes oxidoreductases that act on paired donors with incorporation of molecular oxygen e.g. Inducible nitric oxide synthase [NOS2A; E. C.1.14.13.39];
EC 1.15 includes oxidoreductases that act on superoxide radicals as acceptors
EC 1.16 includes oxidoreductases that oxidize metal ions; e.g. Methionine synthase reductase [MTRR; E. C.1.16.1.8];
EC 1.17 includes oxidoreductases that act on CH or CH2 groups; e.g. Xanthine oxidase [XO; E. C.1.17.3.2]; e.g. Xanthine dehydrogenase [XDH; E.C.1.17.1.4];
EC 1.18 includes oxidoreductases that act on iron-sulfur proteins as donors; e.g. Adrenodoxin oxidoreductase [FDXR; E.C.1.18.1.2] EC 1.19 includes oxidoreductases that act on reduced flavodoxin as a donor EC 1.21 includes oxidoreductases that act on X-H and Y-H to form an X-Y bond EC 1.97 includes other oxidoreductases
In the alternative, the one or more nitroreductase is a microbial or fungal nitroreductase selected from type I nitroflavin reductase NfsA-like and NfsB-like superfamilies or the NQO1-like and YieF-like nitroreductase enzymes, or any putative nitroreductase gene showing evidence of significant sequence homology thereof.
In the ninth aspect of the invention there is provided a method for identifying the presence of cellular hypoxia by contacting in a first step an effective amount of a compound of Formula I to V as defined above to the one or more cellular samples, and in a second step monitoring for the formation of a fluorescent derivative arising from the reduction of the nitro group of the compound of Formula I to V by one or more nitroreductase present in the cellular sample.
Preferably the one or more nitroreductase is a human nitroreductase selected from
DT-diaphorase [NQO1 ; E.C.1.6.99.2];
Cytochrome P450-reductase [CYPOR; E.C. 1.6.2.4];
Inducible nitric oxide synthase [NOS2A; E. C.1.14.13.39];
Cytochrome B5 reductase [DIA1 ; E.C.1.6.2.2]; Xanthine oxidase [XO; E.C.1.17.3.2];
Xanthine dehydrogenase [XDH; E.C.1.17.1.4];
Adrenodoxin oxidoreductase [FDXR; E.C.1.18.1.2];
Methionine synthase reductase [MTRR; E.C.1.16.1.8];
Aldose reductase [ALDR1 ; E.C.1.1.1.21]; and Aldehyde reductase [AKR1 B10; E.C. 1.1.1.2]
Thioredoxin reductase [TXNRD; E.C.1.8.1.9]
Preferably the method further includes the analytical step of quantifying the formation of the fluorescent derivative from the fluorescence emission intensity.
In a tenth aspect of the invention, there is provided an assay for the detection of nitroreductase including the steps of: (i) contacting an effective amount of a plurality of fluorogenic probes with a sample; (ii) monitoring for the formation of fluorescent derivatives.
Preferably, the sample may be added to a common test environment containing a plurality of fluorogenic probes.
In an eleventh aspect of the invention, there is provided an assay for the detection of nitroreductase including the steps of: (iii) contacting an effective amount of at least one compounds of formula
I-V as defined in the second aspect, with a sample; (iv) monitoring for the formation of fluorescent derivatives.
In a twelfth aspect of the invention, there is provided an assay comprising at least one test environment containing a plurality of fluorogenic probes, wherein a sample may be added and the test environment monitored for the formation of fluorescent derivatives.
Preferably, the fluorogenic probes are selected from compounds of formula I-V as defined in the second aspect.
Preferably the test environment is compatible with sustained cell viability, permitting real time multiple analyses with synchronous detection.
In a thirteenth aspect of the invention, there is provided a compound of formula I-V as defined in the second aspect, wherein one or more nitro substituents is replaced by an amine or hydroxyl amine moiety.
Further aspects of the present invention will become apparent from the following
Figures and Examples which are given by way of example only:
Brief Description of Drawings
Figure 1 shows the structural representations of representative nitro-substituted aromatic compounds 1 to 16 of the present invention.
Figure 2 shows representative fluorescent amino-substituted aromatic compounds 17 to 23 of the present invention.
Figure 3 shows the fluorescent intensity observed for compounds 1 to 15 when reduced in the presence of E.coli nitroreductase (nfsB). Figure 4 shows the rate of fluorescence signal generation for compound 2 when reduced in the presence of E.coli nitroreductase (nfsB). Figure 5 shows the fluorescent intensity observed for compounds 1 to 15 when reduced in the presence of human aerobic reductase NAD(P)H dehydrogenase quinone 1 (NQO1).
Figure 6 shows the rate of fluorescence signal generation for compound 1 when reduced in the presence of human aerobic reductase NAD(P)H dehydrogenase quinone 1 (NQO1). Figure 7 shows the shows the fluorescent intensity observed for compounds 1 to 15 when reduced in the presence of the human anaerobic reductase NADPH Cytochrome P450 reductase (CYPOR).
Figure 8 shows the fluorescent intensity of compounds 1 and 13 when reduced in the presence of E.coli nitroreductase B (nfsB) expressing cells co-cultured in the presence of non-expressing cells. Cells were washed and media was replaced after 1 hour with fluorescence monitored over a 4 hour time frame.
Figure 9 shows the fluorescent intensity of compounds 4, 13, 14, and 15 when reduced in the presence of E.coli nitroreductase (nfsB) expressing cells (B) or non- expressing cells (A) for 1.5hr and imaged 6 hours after cells were washed free and fresh media was replaced. (NB The image has been rendered monochromatic for the purposes of publication quality.)
Figure 10 shows the superiority of compound 2 relative to methyl 7-nitrocoumarin carboxylate (methyl 7-nitro-2-oxo-2/-/-chromene-3-carboxylate) as described in US20020031795A1 as a nitroreductase fluorescent reporter. Figure 11 shows the superior aqueous stability of compound 1 and 2 compared with the disclosed compounds methyl-7-nitrocoumarin carboxylate and 7-nitrocoumarin-3- carboxylic acid as described in US20020031795A1 and Letters in Applied Microbiology, (33) 403-8, 2001.
Figure 12 shows the superior aqueous stability under aerobic or anaerobic conditions of compounds 1 to 15 compared with the disclosed compounds methyl-7-nitrocoumarin carboxylate, 7-nitrocoumarin-3-carboxylic acid and 6-chloro-9-nitro-5H- benzo[a]phenoxazin-5-one (Molecular Probes Handbook, 10th Edition, page 535). Figure 13 shows 3 fluorescent amine reporter molecules of non-overlapping excitation/emission spectra suitable for multiplexed reporter gene applications. Figure 14 shows the multiplex use of compounds 4 and 16 to identify concurrently two nitroreductase expressing cell populations in a common environment.
Figure 15 shows the multiplex use of compounds 11 and 13 to identify concurrently two nitroreductase expressing cell populations in a common environment. Detailed Description of the Invention
The invention broadly relates to a method for the detection of nitroreductase activity using at least one fluorogenic probe. More specifically, the invention relates to a method that may be adapted to detect and/or identify a plurality of nitroreductases sequentially or simultaneously in a common test environment. The ability to use a common environment for multiple determinations leads to advantages in assay systems for detection and/or diagnosis. A single detection method can be used (e.g. fluorescence emission) without the need to quench between readings, permitting time- dependent monitoring using such noninvasive detection methods. When multiplexed on sub-cellular imaging systems including; microscopes, sub-cellular imagers and plate readers, relationships between multiple reporter gene cell populations, or single cell populations harboring multiple nitroreductase reporter genes can be quantified as a function of time. This avoids the pitfalls of cell lysis, synchronizes detection, and allows for specific and direct comparison of two or more promoter-regulated nitroreductase activities in a multiplex format.
The method utilises one or more fluorogenic probes, which may be reduced by the action of one or more nitroreductase(s), resulting in one or more strongly fluorescent molecules. Wherein the fluorescent output may be detected simultaneously or sequentially in a common test environment.
The fluorogenic probes of the invention are readily available and are stable in their non-fluorescent nitro form and as the fluorescent reduced amine or hydroxylamine derivatives. While multiple probes may co-exist in the fluorescent and non-fluorescent forms in a common test environment, the presence of individual fluorescent derivatives may be quickly and easily detected independently. The fluorescent derivatives may be independently detected either sequentially or simultaneously by monitoring their often characteristic fluorescence emission.
The inventors have employed singleton synthesis and substructure screening of in- house chemical libraries to collate a Fluorogenic Substrate Library (FSL) including of a range of nitro-substituted aromatic compounds that are likely to be fluorescent upon bioreduction. High-throughput fluorogenic cell-based screening assays have been developed and several fluorogenic probes have been identified for specific nitroreductases. The nitroreductases that can be detected by this technology may be of microbial or human origin, for example the Escherichia coli oxygen-insensitive minor nitroreductase (NTR) [nfsB], or human DT-diaphorase (DTD) [NQO1 ; E.C.1.6.99.2] and human cytochrome P450-reductase (P450R) [CYPOR; E.C.1.6.2.4]. Other human nitroreductases may include
Inducible nitric oxide synthase [NOS2A; E. C.1.14.13.39], Cytochrome B5 reductase [DIA1 ; E.C.1.6.2.2]; Xanthine oxidase [XO; E.C.1.17.3.2]; Xanthine dehydrogenase [XDH; E.C.1.17.1.4]; Adrenodoxin oxidoreductase [FDXR; E.C.1.18.1.2]; Methionine synthase reductase [MTRR; E.C.1.16.1.8]; Aldose reductase [ALDR1 ; E. C.1.1.1.21]; Aldehyde reductase [AKR1 B10; E.C. 1.1.1.2] and Thioredoxin reductase [TXNRD; E. C.1.8.1.9] or any appropriate human oxidoreductase from enzyme class EC 1.1.
Nitro reductases such as NTR and DTD have been shown to catalyse oxygen- insensitive two electron reduction of a nitro (NO2) group to a hydroxylamine (NHOH) group (the four electron reduction product) which may be subsequently reduced to an amine group (the six electron reduction product), while nitroreductases such as P450R catalyse reduction that proceeds via an oxygen-sensitive one electron intermediate as shown in Scheme 1. In the presence of oxygen this one electron intermediate is back- oxidised to regenerate the starting material. In the absence of oxygen (hypoxia) further reduction to a hydroxylamine and amine can occur. two electron reductases
(oxygen insensitive) _■ _. _. _-• , .
_ /^\ kl _, , further reduction D_A_Mι,
further reduction one electron reductases
Figure imgf000018_0001
back oxidation (in the presence of oxygen)
Scheme 1
In the present invention the nitro containing molecules (substrates) of interest are non- fluorescent dyes which upon metabolism by a nitroreductase yield stable fluorescent products (the hydroxylamine and amine containing compounds) that emit light upon excitation over a broad range of the spectrum that is proportional to their concentrations. Therefore, metabolic conversion of substrates yields products that are strongly fluorescent, reporting the presence of nitroreductase activity. The stable fluorescent derivatives may be excited using light from the UV/visible spectrum and the fluorescent emission determined using any instrument adapted to detect and quantify light emissions, for example, a UV/vis spectrometer. Compounds of the invention will generally also emit in the UV/visible/IR range (200-800nm).
Applications:
Use and detection of microbial or fungal nitroreductases in enzyme-reporter assays Any non-ubiquitous enzyme which does not occur naturally may be inserted into a cell of interest in such a way that expression of the enzyme is linked to the expression of a cellular gene of interest. For example, it may be placed under the control of an appropriate transcriptional or post-transcriptional control sequence. A nitroreductase of microbial, fungal or mammalian origin utilised in this context is defined as a reporter gene.
The catalytic generation of a fluorescent signal from a non-fluorescent substrate correlates with the expression of the reporter gene, thus providing a quantitative and /or spatial measure of the activity of the regulatory sequence and expression of a gene of interest. In certain utilities, the fluorescent product may be entrapped within the cell of origin thereby identifying individual cells or tissue regions expressing the reporter gene at the time of compound exposure. Uses of entrapped and freely-diffusing probes can include high-throughput cell based screening assays for compound discovery or identification of regional reporter gene expression within tissue regions of interest. This may include identification of nitroreductase delivered by exogenous vector systems, for example gene therapy, or expressed from tissue specific promoters, for example transgenic animals. Probe use may include identification of cells for subsequent nitroreductase-mediated ablation therapy.
The reporter enzyme may be coupled to an assay component of any binding assay such as an antibody/antigen in an immunoassay or a hormone/receptor in an affinity assay or a nucleic acid molecule in a nucleic acid hybridization assay (DNA/DNA, DNA/RNA, DNA/protein) or biotin/streptavidin or lectin/glycoprotein. The conversion of a non-fluorescent substrate to a fluorescent product provides a measure of bound nitroreductase activity and correlates with the amount of analyte in the assay. Multiplex detection of nitroreductase enzyme activity
The various compounds of the invention incorporate a variety of chromophores and as such can be utilised to determine the presence of at least one nitroreductase in a single test environment. The reduced derivatives of the compounds of the invention fluoresce at characteristic wavelengths. Subsequently, the detection of a characteristic emission signal indicates the presence of a particular reduced substrate. As many of the reduced substrates have unique characteristic emission signals, more than one reduced substrate can be detected in a single/common test environment.
Specific compounds of the invention may function as indicative probes for specific nitroreductases. Therefore, a specific nitroreductase may be identified on detection of one or more fluorescent probes. Furthermore, as more than one fluorescent probe can be detected in a common test environment, the method can be applied to identify multiple nitroreductase enzymes in a common test environment.
The fluorescent probes can be detected sequentially or simultaneously in the common test environment, as desired by the user. This provides process advantages over other similar assay type systems, which require separate reagents and/or detection methods for each probe used. The ability to obtain multiple results from a single test will allow much faster screening of samples resulting in improved efficiency in detection and/or diagnostic methods. No requirement is imposed for each reporter activity to be assayed separately, generally employing different chemistry and detection methods, with sequential measurements and iterative chemistry steps or sample separation.
In a preferred embodiment of the invention, the emission spectra of the florescent probes used in the multiplex environment will be sufficiently discrete to allow detection of the individual fluorescent derivatives.
Persons skilled in the art will appreciate the type of common test environment assay that may be used to perform the invention. Test environments can include high throughput small molecule of biological molecule screening platforms designed to establish the differential biological effects on one cell population over another, or effects on a specific signal transduction pathway relative the another in order to aid in the identification of agents that are active for a given utility. This can include, but is not limited to the use of differential promoter assay to identify modulators of certain signal transduction pathways and mixed cell populations where an intended effect upon a subpopuiation is desired. Screening can be conducted in separate cell populations that can be subsequently mixed in a single test environment or multiple promoter activities within a single cell population. Other platforms can include single cell fluorescent microscopy with high content image analysis for high throughput applications, including signal ratio calculations of multiplex signals to provide additional information relating to the differential activity of reporter gene nitroreductases in a common test environment. Those skilled in the art of cellular physiology and microscopy will appreciate the many other applications of enzyme generated fluorescent signal detection, including confocal microscopic detection of cell populations to monitor intracellular processes such as protein trafficking with the aid of split excitation and lasar photobleaching..
By way of example, a sample can be applied to an assay test environment (eg an assay well) containing a plurality of fluorogenic probes. Light from the UV/visible spectrum can be used to excite any reduced derivatives in the test environment, which may fluoresce at a characteristic wavelength, thus indicating the presence of specific nitroreductase enzyme(s).
Detection of microbial nitroreductase enzyme activity
Compounds disclosed in this invention may be used in the detection and/or diagnostic tests for microorganisms. Nitroreductase activity is common, being found in the majority of organisms including obligate aerobic and anaerobic bacteria, fungi and eukaryotic parasites. Conversion of a non-fluorescent substrate to a fluorescent product provides a universal test for the presence of microorganisms in samples or cultures. Uses may include, but are not limited to, bioremediation, sterility tests, antibiotic susceptibility and quantification of organisms present in any sample.
The invention may be employed to demonstrate the presence of nitroreductase activity in any test sample containing one or more microorganisms of commercial value (e.g. food product, soil sample, aqueous sample) or medical interest (e.g. body fluids).
Detection of mammalian nitroreductase enzyme activity
Compounds disclosed in this invention may be used in the detection and/or diagnostic tests for human nitroreductase activity. Under aerobic conditions certain obligate two- electron reductases, for example NAD(P)H dehydrogenase quinone 1 (DT-Diaphorase; NQO1 , E. C.1.6.99.2), can be detected in living tissue samples or preparations there of. Alternately, in the absence of oxygen (hypoxia) certain one-electron reductases, for example NADPH cytochrome P450 reductase (CYPOR, E.C. 1.6.2.4), can be detected in living tissue samples or preparations there of. Alternatively, compounds may be employed to detect the total (composite) reductive activity of ubiquitous one-electron reductase activities. This has utility for predicting total reductive catalytic capacity of living tissue samples or preparations there of and may be of value in predicting reductive metabolism of therapeutic agents, for example hypoxic cytotoxins.
In all cases the conversion of a non-fluorescent substrate to a fluorescent product provides a measure of nitroreductase activity of interest and correlates with catalytic activity in the sample. In certain utilities, the fluorescent product may be entrapped within the cell of origin thereby identifying it as expressing the reductase of interest at the time of compound exposure. Signal retention may correlate with amplitude of enzyme catalysis providing a measure of the heterogeneity within a cell, tissue or analyte sample series.
Detection of cellular hypoxia
Compounds disclosed in this invention may be used in the detection and/or diagnostic tests for tissue hypoxia (< 1% O2). Conversion of a non-fluorescent substrate to a fluorescent product by ubiquitous one-electron reductases, which occurs selectively in the absence of oxygen, provides a test for the relative absence of oxygen in a specific cell population or tissue region. In certain utilities, the fluorescent product may be entrapped within the cell of origin thereby identifying it as hypoxic at the time of compound exposure.
Additionally, the conversion of a non-fluorescent substrate to a fluorescent product by an oxygen-inhibited reductase can provide a measure of hypoxia in any test system of interest. The generation of fluorescence signal correlates with the concentration of oxygen in the sample.
EXAMPLES The present invention will now be described in more detail by referring to the following examples, but is not deemed to be limited thereto.
EXAMPLE 1
Method of detecting E.coli nitroreductase (nfsB) reporter gene activity in intact cells
The human breast cancer cell line (MDA∑aO and a clonal derivative (MDA231NTR) engineered to express the reporter gene E.coli nitroreductase (nfsB) under the control of a constitutive promoter were seeded into 96-well plates at a density of 1x105 cells/well. When samples were equilibrated to 370C, 5%CO2 for 2 hr and compounds 1 to 15 (Figure 1 ) were added to a final concentration of 100 μM for 4 hr. Test groups were cell-free culture media alone (control), MDA231m and MDA231NTR. The fluorescence signal was monitored at an excitation wavelength of 355 nm and emission wavelength of 460 nm (355/460) except for compounds 4 and 11 that were monitored at 405/585 and compounds 8 and 10 that were monitored at 355/585 and 355/535 respectively (figure 1). No fluorescence was observed in either the cell-free control or parental MDA231WT containing cultures. Compounds 1 - 15 inclusive gave rise to a fluorescent signal specifically in the presence of E.coli nitroreductase (nfsB) expression.
In a further exemplification, the human colorectal cancer cell line (HCT1 '\6WT) and a clonal derivative (HCT116NTR) engineered to express the reporter gene E.coli nitroreductase (nfsB) under the control of a constitutive promoter were suspended in stirred culture media at a density of 5x106 cells/ml. When samples were equilibrated to 37°C, 5%CO2, compound (2) (1-methyl-6-nitro-4(1H)-quinolinone) was added to a final concentration of 300 μM. Test groups were culture media alone (control), HCT116^" and HCT116NTR. The rate of fluorescence signal generation at 355/460 was monitored as a function of time (figure 4). HCT116NTR cells rapidly reduced compound (2) a process that approached completion by 9hrs. No detectable fluorescence was observed in either the control or parental HCT116^ containing cultures.
EXAMPLE 2 Method of detecting human aerobic nitroreductase activity
The human breast cancer cell line (MDA231WT) and a clonal derivative (MDA231DTD) engineered to express the human aerobic reductase, NAD(P)H dehydrogenase quinone 1 (DT-diaphorase; NQO1) under the control of a constitutive promoter were seeded into 96-well plates at a density of 1x105 cells/well. When samples were equilibrated to 37°C, 5%CO2 for 2 hr and compounds 1 to 15 were added to a final concentration of 100 μM for 4 hr. Test groups were cell-free culture media alone (control), MDA231m and MDA231 DTD. The fluorescence signal was monitored at 355/460 except for compounds 4 and 11 that were monitored at 405/585 and compounds 8 and 10 that were monitored at 355/585 and 355/535 respectively (figure 5). No detectable fluorescence was observed in either the control or parental
MDA231m containing cultures. Compounds 1 and 3 gave rise to a fluorescent signal specifically in the presence of human NQO1 expression. In a further exemplification, the human breast cancer cell line (MDA231wr) and a clonal derivative (MDA231DTD) engineered to express the human NQO1 gene (DT- diaphorase) under the control of a constitutive promoter were seeded into 96-well tissue culture plates at 1x105 cells/well. Samples were equilibrated to 370C, 5%CO2, compound (1) (6-nitro-4(1H)-quinolinone) was added to a final concentration of 300 μM. Test groups were culture media alone (control), MDA231WT cells and MDA231DTD cells. The rate of fluorescence signal generation at 355/460 was monitored as a function of time (Figure 6). MDA231 DTD cells reduced compound 1 and 3 (see Figure 5) to generate a fluorescent signal. No detectable fluorescence was observed in either the wells containing compound 1 alone (control) or parental MDA231WT.
EXAMPLE 3 :
Method of detecting human nitroreductase activity under anoxia A clonal derivative of the human breast cancer cell line (MDA231P450R), engineered to overexpress the human anaerobic reductase, NADPH cytochrome P450 reductase (CYPOR) under the control of a constitutive promoter were seeded into 96-well plates at a density of 1x105 cells/well. When samples were equilibrated to 37°C, 5%CO2 for 2 hr under 95% N2 and compounds 1 to 15 were added to a final concentration of 100 μM for 4 hr. Test groups were cell-free culture media alone (control), MDA231 P450R under normoxic (air) and anoxic (N2) conditions. The fluorescence signal was monitored at 355/460 except for compounds 4 and 11 that were monitored at 405/585 and compounds 8 and 10 that were monitored at 355/585 and 355/535 respectively (figure 7). No detectable fluorescence was observed in either the control or aerobic MDA231P450R containing cultures. Compounds 1 - 5 and 10 - 15 gave rise to a fluorescent signal specifically in the presence of human cytochrome P450 reductase expression when oxygen was absent.
Cellular Entrapment: A particularly attractive property of a number of fluorogenic probes described herein is that of cellular entrapment of the fluorescent reporter molecule produced upon nitroreductase activity.
These compounds comprise of at least one NO2 group and at least one of the groups R1, R2, R3, R4, R5 of the molecule of formula I1 R1, R2 of formula II, R1 of formula III, R2, R3, R4, R5 of formula III', R1 of formula IV, R1, R2, R3 of formula V that provides for cell membrane permeabilising properties. Membrane permeant compounds can generally be provided by masking hydrophilic groups. After entry into the cell the masking group can be designed to be cleaved to produce a hydrophilic fluorogenic substrate that provides a cell entrapped fluorescent report in the presence of reductase activity. Alternately, compounds comprising of at least one NO2 group and at least one of the groups R1, R2, R3, R4, R5 of the molecule of formula I, R1, R2 of formula II, R1 of formula III, R2, R3, R4, R5 of formula III', R1 of formula IV, R1, R2, R3 of formula V that provides for increased DNA affinity can provide a nuclear localised cell entrapped fluorescent report in the presence of nitroreductase activity.
EXAMPLE 4
Entrapment of fluorescent products within nitroreductase expressing cells
The human colorectal cancer cell line (HCT116m) and a clonal derivative (HCT116NTR) engineered to express the reporter gene E.coli nitroreductase (nfsB) under the control of a constitutive promoter were seeded onto glass coverslips at an equal density (50:50). Cells were equilibrated to 37°C, 5%CO2, and compound 1 and 13 was added to a final concentration of 100 μM for 1 hr. Cells were washed free of compound after 1 hour and fluorescence was monitored as a function of time (figure 8). By 4 hours, cells were no longer fluorescent following exposure to compound 1, whereas compound 13, bearing an ester group subject to intracellular hydrolysis, was still retained within the nitroreductase expressing (but not co-cultured parental) cells. This demonstrates that durable and specific retention of fluorescence is possible with nitroreductase positive cells even when co-cultured in the presence of non-expressing cells.
In further screening (figure 9), the human colorectal cancer cell line (HCT116m) and a clonal derivative (HCT116NTR) engineered to express the reporter gene E.coli nitroreductase (nfsB) under the control of a constitutive promoter were seeded separately into a 96-well glass plate. Cells were equilibrated to 370C, 5%CO2, and compounds were added to a final concentration of 10 μM for 1.5hr. Cells were washed free of compound and fluorescence was monitored 6 hours post-exposure. Compounds 4, 13, 14 and 15 are identified as possessing cellular retention properties, with fluorescence being observed within the nitroreductase expressing (B) but not parental (A) HCT116 cells. This demonstrates that durable and specific retention of fluorescence is possible with nitroreductase positive cells. Parental non-expressing cells failed to provide a signal. The image has been rendered monochromatic for the purposes of publication quality. EXAMPLE 5
Superior nitroreductase specificity of compound 2 over 7-nitrocoumarins
The human breast cancer cell line (MDA231WT) and a clonal derivative (MDA231NTR) engineered to overexpress the E.coli nitroreductase (nfsB) under the control of a constitutive promoter were seeded into 96-well plates at a density of 2x104 cells/well. When samples were equilibrated to 370C, 95% N2, 5%CO2 for 2 hr and compound 2 or methyl 7-nitrocoumarin carboxylate (methyl 7-nitro-2-oxo-2/-/-chromene-3-carboxylate) were added to a final concentration of 300 μM. The fluorescence signal was monitored at 355/460 over a 7.5 hour time frame (figure 10). For compound 2 no detectable fluorescence (above background) was observed in the control cell line MDA231WT containing cultures, whereas a robust fluorescent signal was observed in the nitroreductase-expressing cell line MDA231NTR giving rise to a 480-fold differential signal at 7.5 hours. In contrast, the methyl 7-nitrocoumarin carboxylate compound generated measurable fluorescence in the control cell line MDA231WT containing cultures, which was only elevated 1.6-fold in the nitroreductase-expressing cell line MDA231NTR. Therefore compound 2 is demonstrably superior to methyl 7- nitrocoumarin carboxylate as disclosed in US20020031795A1.
In a further experiment, the aqueous stability of compounds 1 and 2 were compared with the compounds methyl-7-nitrocoumarin carboxylate and 7-nitrocoumarin-3- carboxylic acid in the absence of cell culture. Minimal essential media was equilibrated to 37°C, 5%CO2 for 2 hr and compounds were added to a final concentration of 300 μM. The fluorescence signal was monitored at 355/460 over a 7.5 hour time frame (figure 11). Both the methyl-7-nitrocoumarin carboxylate and 7-nitrocoumarin-3- carboxylic acid compounds exhibited instability in culture media with a 4.2-fold and 5.3- fold increase in background florescence over 7.8 hours. In contrast, compounds 1 and 2 were significantly more stable over this time period, with a 1.4-fold increase in background florescence over 7.8 hours.
EXAMPLE 6
Superior anaerobic stability of compounds 1-15 over the 7-nitrocoumarins and 6- chloro-9-nitro-5W-benzo[a]phenoxazin-5-one.
Compounds 1 - 15 or methyl 7-nitrocoumarin carboxylate (methyl 7-nitro-2-oxo-2H- chromene-3-carboxylate) or 7-nitrocoumarin-3-carboylic acid or 6-chloro-9-nitro-5/-/- benzo[a]phenoxazin-5-one were added to a final concentration of 100 μM in cell-free culture media and were held at 37°C, 5%CO2 for 4 hr under either air or 95% N2. The fluorescence signal was monitored at 355/460 except for compounds 4 and 11 that were monitored at 405/585 and compounds 8, 10 and 6-CI-9-nitro-5-oxo-5H- benzo[a]phenoxazine that were monitored at 355/585, 355/535 and 530/585 respectively (Figure 12). For compounds 1 -15 no detectable fluorescence (above background) was observed under either oxic or anoxic conditions. In contrast, the methyl 7-nitrocoumarin carboxylate compound, the 7-nitrocoumarin-3-carboylic acid compound and the 6-chloro-9-nitro-5H-benzo[a]phenoxazin-5-one compound generated measurable fluorescence in cell-free culture media, specifically under conditions of low oxygen, indicative of instability. Therefore compounds 1-15 are demonstrably superior for detecting mammalian anaerobic reductase activities to methyl 7-nitrocoumarin carboxylate (methyl 7-nitro-2-oxo-2/-/-chromene-3-carboxylate) and 7-nitrocoumarin-3-carboylic acid as disclosed in US20020031795A1 , and 6-chloro- 9-nitro-5/-/-benzo[a]phenoxazin-5-one (also known as C22220, CNOB) as disclosed by Invitrogen (Molecular Probes Handbook, 10th Edition, page 535).
EXAMPLE 7
Suitability of compounds for use as multiplexed fluorescent reporter molecules
Compounds 23, 18 and 22 (as representative fluorescent amino-substituted aromatic compounds of the present invention) were utilized to exemplify the capacity to specifically and independently monitor multiple fluorescent signal outputs from a mixture of compounds within a sample. Compounds 23, 18 and 22 were placed in
10OmM phosphate buffer (pH 7.0) at 10 μM and subjected to excitation at wavelengths 295, 340 and 440 nm. Fluorescent emission was recorded from 300 to 750 nm for each excitation range (figure 13). When the data were collated it was evident that the fluorescent emission maxima of each compound could be independently quantified without interference from the other fluorescent molecules. Specifically, compounds 23, 18 and 22 gave fluorescent output at Ex/Em wavelengths of 295/370 = 683.3, 0.78, and 0.0 fluorescent units, respectively; at Ex/Em wavelengths of 340/510 = 1.2, 12.0 and 461.8 fluorescent units, respectively; at Ex/Em wavelengths of 440/540 = 0.05, 347.9 and 0.75 fluorescent units, respectively. This demonstrated that multiple independent outputs can be recorded from a single sample providing appropriate excitation and emission wavelengths are utilized. The nature of the output may enable co-registration of independent signals within a single test environment which may be correlated to specific mammalian or microbial nitroreductase activities within the test sample of interest. EXAMPLE 8
Use of compounds for multiplexed reporter gene applications
Compounds 4 and 16 (as representative nitro-substituted aromatic compounds of the present invention) were utilized to exemplify the ability to detect individual cell populations. Firstly, to demonstrate selectivity in a 96-well format, fluorescent platereader analyses were performed using MDA-231 wild-type cells (WT) or clones stably expressing either Eshericia coli nfsB (NfsB) or human cytochrome P450 reductase (CYPOR). 30,000 cells were seeded into a plastic 96-well plate in a volume of 0.1 mL After 2 h attachment samples were incubated with either 200μM 16 or 100μM 4 by dilution of DMSO stock solutions into culture medium and addition of 0.1 mL directly into wells. Plates were incubated for 4 h before being read on a fluorescent platereader at Ex/Em 355/460 and 485/535. Values represent the fluorescence in the presence of cells - blank (fluorescence. in the absence of cells). When the data were collated it was evident that the fluorescent output of each cell population could be identified with minimal interference from the other reductases-generated fluorescent molecules. Specifically, compounds 4 and 16 gave fluorescent output at Ex/Em wavelengths of 485/535 and 355/460 respectively; detecting the presence of Eshericia coli nfsB and human cytochrome P450 reductase (CYPOR) enzyme activity independently (Figure 14a). To establish the single cell specificity of compounds 4 and 16 for detection of activities in a mixed (co-cultured) cell population, cells were examined by fluorescent microscopy following concurrent exposure to compounds 4 and 16. MDA-231 cells expressing Eshericia coli nfsB and human cytochrome P450 reductase were mixed 1 :1 , seeded into a glass 96-well plate (15,000 cells/well in 0.1 mL αMEM + 10% FCS + P/S) and allowed to attach overnight. Compound 16 and 4 were diluted into αMEM (from DMSO stock solutions) and 0.1 mL was added to achieve a final concentration of 300μM and 10μM respectively. Samples were incubated at 37°C for 45 minutes, washed three times in PBS and images were acquired on a Nikon TE-2000 inverted fluorescence microscope. Figure 14b provides a co-registration image of compounds 16 and 4 (DAPI and FITC filter set respectively). The fluorescent emissions of compounds 16 and 14 appear as blue and green marks on the co-registration image. Figure 14c and 14d identify each individual cell population, with overlays of 16 and 4 respectively with the corresponding phase contrast image (note: figure 14c shows blue fluorescence emission, while figure 14d shows green fluorescence emission. This demonstrates that two co-cultured cell populations can be readily identified and distinguished as mutually exclusive cell populations in a common environment through the co-application of two representative nitro-substituted aromatic compounds of the present invention (Figure 14b-d). This illustrates that multiple independent outputs can be recorded from a single sample providing appropriate excitation and emission wavelengths are utilized. The nature of the output may enable co-registration of independent signals within a single test environment which may be correlated to specific mammalian or microbial nitroreductase activities within the test sample of interest.
EXAMPLE 9
Use of compounds for multiplexed reporter gene applications
Compounds 11 and 13 (as representative nitro-substituted aromatic compounds of the present invention) were utilized to exemplify the ability to detect individual cell populations. Firstly, to demonstrate selectivity, fluorescent microscope images were captured for each of compound 11 (Figure 15a) and 13 (Figure 15b) using appropriate filter settings following exposure of either MDA-231 wild-type cells (WT) or clones stably expressing either Eshericia coli nfsB (NfsB) or human cytochrome P450 reductase (CYPOR). When the data were collated it was evident that the fluorescent output of each cell population could be identified with minimal interference from the other reductases-generated fluorescent molecules. Specifically, compounds 11 and 13 gave fluorescent output at Ex/Em wavelengths of 485/535 and 355/460 respectively; detecting the presence of Eshericia coli nfsB and human cytochrome P450 reductase (CYPOR) enzyme activities. To establish the single cell specificity of compounds 11 and 13 for detection of activities in a mixed (co-cultured) cell population, cells were examined by fluorescent microscopy following concurrent exposure to compounds 11 and 13. MDA-231 wild-type cells or cells expressing Eshericia coli nfsB and human cytochrome P450 reductase were mixed 1 :1 :1 , seeded onto glass coverslips (15,000 cells/well in 0.1 ml. αMEM + 10% FCS + P/S) and allowed to attach overnight.
Compound 11 and 13 were diluted into αMEM (from DMSO stock solutions) and 0.1 mL was added to achieve a final concentration of 10μM and 100μM respectively. Samples were incubated at 370C for 45 minutes, washed three times in PBS and images were acquired on a Nikon TE-2000 inverted fluorescence microscope. Figure 15c provides a co-registration image of compounds 11 and 13 (FITC and DAPI filter set respectively). Figure 15c identifies each individual cell population, with overlay of the corresponding phase contrast image; wild-type cells (W) or clones stably expressing either Eshericia coli nfsB (N) or human cytochrome P450 reductase (R). This demonstrates that three co-cultured cell populations can be readily identified and distinguished as mutually exclusive cell populations in a common environment through the co-application of two representative nitro-substituted aromatic compounds of the present invention (Figure 15a-c). This illustrates that multiple independent outputs can be recorded from a single sample providing appropriate excitation and emission wavelengths are utilized. The nature of the output may enable co-registration of independent signals within a single test environment which may be correlated to specific mammalian or microbial nitroreductase activities within the test sample of interest.
Examples 7, 8 and 9 clearly demonstrates that a plurality of fluorescent probes can be detected and identified in a common test environment. Therefore a plurality of probes may be used to identify one or more nitroreductase(s) in a common environment. Thus, multiplex reporter output may be achieved.
Compounds
3-Nitro-6H-benzo[c]chromen-6-one (12) and 6-aminoindazole (23) were purchased from Aldrich Chemical Company.
The following compounds were prepared according to published procedures:
6-Nitro-4(1/-/)-quinolinone (1) [Ruche man, A. L.; Kerrigan, J. E.; Li1 T-K.; Zhou, N.; Liu, A.; Liu, L F.; LaVoie, E. J. Nitro and amino substitution within the A-ring of 5H-8.9- dimethoxy-5-(2-Λ/,Λ/-dimethylaminoethyl)dibenzo[c,ή][1 ,6]-naphthyridin-6-ones: influence on topoisomerase l-targeting activity and cytotoxicity. Bioorg. & Med. Chem. 2004, 12(13), 3731-42.]
1-Methyl-6-nitro-4(1/-/)-quinolinone (2) [Denny, W. A.; Atwell, G. J.; Baguley, B. C; Cain, B. F. Potential Antitumor Agents. 29. Quantitative Structure-Activity Relationships for the Antileukemic Bisquaternary Ammonium Heterocycles. J. Med. Chem. 1979, 22(2), 134-50.]
2-Methyl-6-nitro-4(1H)-quinolinone (3) [Chen, B.; Huang, X.; Wang, J. A versatile synthesis of 2-alkyl and 2-aryl-4-quinolones. Synthesis, 1987, 5, 482-83.]
Λ/-[2-(Dimethylamino)ethyl]-2-nitro-9-oxo-9, 10-dihydro-4-acridinecarboxamide (4) [Chen, Q.; Deady, L. W.; Baguley, B. C; Denny, W. A. Electron-Deficient DNA- Intercalating Agents as Antitumor Drugs: Aza Analogues of the Experimental Clinical Agent N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide. J. Med. Chem. 1994, 37(5), 593-97.] 6-Nitro-4(3H)-quinazolinone (5) [Morley, J. S.; Simpson, J. C. E. The chemistry of simple heterocyclic systems. Part 1. Reactions of 6- and 7-nitro-4-hydroxyquinazoline and their derivatives. J. Chem. Soc. 1948, 360-66.]
6-Nitro-2-phenyl-4(3/-/)-quinazolinone (6) [Goerdeler, J.; Sappelt, R. lmidazoline-4,5- diones. I. Chem.Ber. 1967, 100(6), 2064-76.]
5-Nitro-2-phenyl-1H-benzimidazole (7) [Tandon, V. K.; Kumar, M. BF3-Et2O promoted one-pot expeditious and convenient synthesis of 2-substituted benzimidazoles and 1 ,3,5-benzoxadiazepines. Tet. Lett. 2004, 45(21), 4185-87 (and references cited therein).]
2-[4-(Dimethylamino)butyl]-5-nitro-1/-/-benzo[de]isoquinoline-1 ,3(2H)-dione (11) was prepared according to the procedure described for the 2- and 3-carbon homologues [Zee-Cheng, R. K. Y.; Cheng, C. C; N-(Aminoalkyl)imide antineoplastic agents. J. Med. Chem. 1985, 28(9), 1216-22.]
Methyl (6-nitro-4-oxo-1(4H)-quinolinyl)acetate (13) [Hutt, M. P.; MacKellar, F. A.; Identification of quinoline nitration products by NOE. J. Het. Chem. 1984, 21(2), 349- 52.]
Λ/^Λ/^Dimethyl-Λ^^-nitro^-quinolinyO-I .S-propanediamine (15) [Denny, W. A.; Atwell, G. J.; Roberts, P. B.; Anderson, R. F.; Boyd, M.; Lock, C. J. L.; Wilson, W. R. Hypoxia-Selective Antitumor Agents. 6. 4-(Alkylamino)nitroquinolines: A New Class of Hypoxia-Selective Cytotoxins. J. Med. Chem. 1992, 35(26), 4832-41.]
Λ/1,Λ/1-dimethyl-Λ/3-(5-nitro-4-quinazolinyl)-1 ,3-propanediamine (16) [Denny, W. A.; Atwell, G. J.; Roberts, P. B.; Anderson, R. F.; Boyd, M.; Lock, C. J. L.; Wilson, W. R. Hypoxia-Selective Antitumor Agents. 6. 4-(Alkylamino)nitroquinolines: A New Class of Hypoxia-Selective Cytotoxins. J. Med. Chem. 1992, 35(26), 4832-41.] Preparation of methyl 4-{f(5-nitro-1-naphthyl)sulfoπyl]amino>butanoate (8), methyl 4- (f(5-nitro-2-naphthyl)sulfonyllamino}butanoate (9) and methyl 4-{f(8-nitro-2- naphthyl)sulfonvπamino}butanoate (10)
To a stirred solution of 1-nitronapthalene (2.1 g, 12.1 mmol) in dichloromethane (50 mL) at 0 0C was added chlorosulfonic acid (5 mL) dropwise. The resulting red solution was allowed to warm to room temperature over 18 hours and then poured onto ice water. The aqueous layer was extracted with ethyl acetate (3 times) and the combined organic extracts were washed with brine then dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude mixture of sulfonyl chloride isomers that was used directly. The residue was dissolved in tetrahydrofuran to which methyl 4-aminobutyrate hydrochloride (3.73 g, 24.2 mmol) and excess triethylamine (10 mL) were added. After stirring at room temperature for 1 hour the reaction was diluted with water and acidified with 1 N hydrochloric acid. The aqueous layer was extracted with ethyl acetate (3 times) and the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure and chromatographed on silica gel, eluting with hexane/ethyl acetate (2:1), to give at highest Rf, methyl 4-ff(5-nitro-1-naphthyl)sulfonvπamino}butanoate (8) (0.61 q, 14%) as a white solid, m.p. 94-96 0C (Et2O/hexane); 1H NMR [(CD3)2SO] δ 9.02 (d, J = 8.7 Hz, 1 H), 8.51 (d, J = 8.7 Hz, 1 H), 8.40 (dd, J = 7.6, 0.9 Hz, 1 H), 8.29 (dd, J = 7.4, 1.0 Hz, 1 H), 8.21 (br s, 1 H), 7.92 (m, 2H), 3.48 (s, 3H), 2.85 (t, J = 6.9 Hz, 2H), 2.20 (t, J = 7.3, 2H), 1.55 (m, 2H); MS found: 353 (M+H), 321 (M+H-MeOH); followed at intermediate Rf by methyl 4-W5-nitro-2-naphthyl)sulfonvπamino}butanoate (9) (0.24 g. 6%) as an off-white solid, m.p. 102-104 0C (Et2O/hexane); 1H NMR [(CD3)2SO] δ 8.66 (d, J = 1.9 Hz, 1 H), 8.62 (d, J = 8.3 Hz, 1 H), 8.57 (d, J = 9.2 Hz, 1 H), 8.49 (dd, J = 7.7, 1.1 Hz, 1 H), 8.08 (dd, J = 9.2, 2.0 Hz, 1 H), 7.91 (br s, 1 H), 7.87 (t, J = 7.9 Hz, 1 H), 3.51 (s, 3H), 2.84 (t, J = 7.0 Hz, 2H), 2.29 (t, J = 7.3, 2H), 1.63 (m, 2H); MS found: 353 (M+H), 321 (M+H-MeOH); followed at lowest Rf by methyl 4-(f(8-nitro-2- naphthyPsulfonyliaminolbutanoate (10) (70 mq. 2%) as an off-white solid, m.p. 109- 111 0C (Et2O/hexane); 1H NMR [(CD3)2SO] δ 8.89 (d, J = 1.7 Hz, 1 H)1 8.50 (m, 2H), 8.40 (d, J = 8.7 Hz, 1 H), 8.01 (dd, J = 8.7, 1.8 Hz, 1 H), 7.94 (br s, 1 H), 7.90 (t, J = 7.8 Hz, 1 H), 3.50 (s, 3H), 2.84 (t, J = 7.0 Hz, 2H), 2.28 (t, J = 7.3, 2H), 1.61 (m, 2H); MS found: 353 (M+H), 321 (M+H-MeOH). Preparation of /V.Λ/-dimethyl-3-f(7-nitro-4-quinolinyl)oxyl-1-propanamine (14)
To a solution of 7-nitro-4-quinolone [Ruchelman, A. L.; Kerrigan, J. E.; Li, T-K.; Zhou, N.; Liu, A.; Liu, L. F.; LaVoie, E. J. Nitro and amino substitution within the A-ring of 5H- 8,9-dimethoxy-5-(2-Λ/,Λ/-dimethylaminoethyl)dibenzo[c,/7][1 ,6]-naphthyridin-6-ones: influence on topoisomerase l-targeting activity and cytotoxicity. Bioorg. & Med. Chem. 2004, 12(13), 3731-42.] (0.30 g, 1.58 mmol) in dimethylformamide (10 mL) was added solid potassium carbonate (0.87 g, 6.32 mmol) followed by Λ/-(3-chloropropyl)-Λ/,Λ/- dimethylamine hydrochloride (0.37 g, 2.37 mmol). The resulting suspension was heated at 80 0C under nitrogen with stirring for 3 hours after which time the temperature was reduced to 50 0C for 18 hours. The reaction was diluted with brine and extracted with ethyl acetate (3 times). The combined organic extracts were washed with brine before being dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was then suspended in diethyl ether/hexane (1 :1 , 80 mL) and the undissolved solid (unreacted starting material) was removed by filtration. The filtrate was then concentrated in vacuo, re-dissolved in methanol (10 mL) and converted to the HCI salt by the addition of 1M anhydrous HCI in methanol (1 mL). Diethyl ether was added to precipitate the HCI salt which was collected by filtration. Reverse phase preparative HPLC (CH3CN/H2O/TFA) and concentration of the chromatography fractions under reduced pressure then gave Λ/,Λ/-dimethyl-3-[(7-nitro- 4-quinolinyl)oxyl-1-propanamine trifluoroacetate (14) (156 mg, 25%) as a white powder, m.p. 146-149 0C. 1H NMR [(CD3)2SO] δ 9.73 (br s, 1 H), 8.96 (d, J = 5.3 Hz, 1 H), 8.75 (d, J = 2.2 Hz, 1 H), 8.45 (d, J = 9.2 Hz, 1 H), 8.28 (dd, J = 2.2, 9.2 Hz, 1 H), 7.25 (d, J = 5.3 Hz, 1 H), 4.40 (t, J = 5.9 Hz, 3H), 3.36 (m, 3H), 2.87 (s, 6H), 2.28 (m, 3H). Found: C, 48.08; H, 4.62; N, 10.16. C16H18F3N3O5-SMH2O requires C, 47.71 ; H, 4.88; N, 10.43. Followed by 1-[3-(dimethylamino)propyl]-7-nitro-4(1/-/)-quinolinone trifluoroacetate (150 mg, 24%) as a yellow powder, m.p. 188-190 0C. 1H NMR [(CDs)2SO] δ 9.61 (br s, 1 H), 8.54 (d, J = 2.0 Hz, 1 H), 8.41 (d, J = 8.8 Hz, 1 H), 8.15- 8.11 (m, 2H), 6.25 (d, J = 7.8 Hz, 1 H), 4.42 (t, J = 7.2 Hz, 3H), 3.15 (m, 3H), 2.77 (s, 6H), 2.15 (m, 3H). Found: C, 49.06; H, 4.54; N, 10.75. C16H18F3N3O5 requires C, 49.36; H, 4.66; N, 10.79.
General procedure for the preparation of amines of Figure 2.
To a solution of the nitro precursor in methanol/ethyl acetate (1 :1) was added a catalytic amount of 5% Pd-C. The resulting suspension was stirred vigorously over an atmosphere of hydrogen (60 psi) for 2 hours and then filtered through celite and concentrated under reduced pressure. Trituration from diethyl ether/hexane then gave:
6-Amino-1-methyl-4(1H)-αuinolinone (17), m.p. 212-214 0C. 1H NMR [(CD3)2SO] δ 7.73 (d, J = 7.5 Hz, 1 H), 7.39 (d, J = 9.0 Hz, 1 H), 7.30 (d, J = 2.7 Hz, 1 H), 7.06 (dd, J = 9.0, 2.8 Hz, 1 H), 5.83 (d, J = 7.5 Hz, 1 H), 5.30 (s, 2H), 3.73 (s, 3H). MS found: 175 (M+H).
2-Amino-/V-f2-(dimethylamino)ethyl]-9-oxo-9,10-dihvdro-4-acridinecarboxamide (18), m.p. 198-202 0C. 1H NMR [(CD3)2SO] δ 11.73 (br s, 1 H), 8.75 (t, J = 5.0 Hz, 1H)1 8.18 (br d, J = 8.5 Hz, 1 H), 7.67-7.62 (m, 3H), 7.52 (d, J = 2.7 Hz, 1 H), 7.21-7.17 (m, 1 H)1 5.30 (s, 2H), 3.45 (q, J = 6.6 Hz, 2H), 2.49 (obsc t, J ~ 7 Hz, 2H), 2.23 (s, 6H). MS found: 325 (M+H).
5-Amino-2-[4-(dimethylamino)butyll-1H-benzo[de]isoquinoline-1.3(2H)-dione (19), m.p. 99-101 0C. 1H NMR [(CDa)2SO] δ 8.08 (dd, J = 7.2, 1.0 Hz, 1 H), 8.02 (dd, J = 8.4, 0.7 Hz, 1 H), 7.97 (d, J = 2.3 Hz, 1 H)1 7.61 (dd, J = 7.3, 8.2 Hz, 1 H)1 7.28 (d, J = 2.3 Hz1 1 H), 5.97 (s, 2H), 4.02 (t, J = 7.3 Hz, 2H), 2.22 (t, J = 7.1 Hz, 2H), 2.09 (s, 6H), 1.65- 1.58 (m, 2H)1 1.47-1.40 (m, 2H). MS found: 312 (M+H).
Methyl 4-(r(5-amino-1 -naphthynsulfonyliaminolbutanoate (20), m.p. 90-92 0C. 1H NMR [(CDs)2SO] δ 8.37 (d, J = 8.5 Hz, 1 H)1 8.01 (dd, J = 7.3, 1.0 Hz, 1 H), 7.71 (d, J = 8.5 Hz, 1 H), 7.77 (br s, 1 H), 7.45 (dd, J = 7.4, 8.5 Hz, 1 H)1 7.36 (dd, J = 7.7, 8.4 Hz, 1 H)1 6.79 (dd, J = 7.6, 0.7 Hz, 1H)1 5.93 (s, 2H), 3.50 (s, 3H), 2.76 (t, J = 6.9 Hz, 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.58-1.51 (m, 2H). MS found: 323 (M+H).
Methyl 4-(r(5-amino-2-naphthvnsulfonvnamino)butanoate (21), m.p. 171-175 0C. 1H NMR [(CDa)2SO] δ 8.25 (d, J = 8.9 Hz1 1H), 8.18 (d, J = 1.9 Hz, 1H), 7.64 (obsc br s, 1 H), 7.62 (dd, J = 8.9, 1.9 Hz, 1 H), 7.34 (t, J = 7.9 Hz, 1 H), 7.26 (d, J = 8.1 Hz, 1 H), 6.82 (dd, J = 7.5, 1.1 Hz, 1 H), 5.91 (s, 2H), 3.51 (s, 3H)1 2.78 (t, J = 7.0 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.65-1.58 (m, 2H). MS found: 323 (M+H).
Methyl 44f(8-amino-2-naphthyl)sulfonvHamino)butanoate (22), m.p. 162-167 0C. 1H NMR [(CDa)2SO] δ 8.53 (br s, 1 H), 7.89 (d, J = 8.7 Hz, 1 H), 7.67 (dd, J = 8.7, 1.8 Hz1 1 H), 7.56 (br s, 1 H), 7.36 (t, J = 7.8 Hz, 1 H), 7.15 (d, J = 8.1 Hz, 1 H), 6.79 (dd, J = 7.6, 0.9 Hz1 1 H)1 6.02 (s, 2H), 3.52 (s, 3H), 2.79 (t, J = 7.0 Hz, 2H)1 2.29 (t, J = 7.4 Hz, 2H), 1.64-1.58 (m, 2H). MS found: 323 (M+H). It is appreciated that the compounds of the invention may occur in different geometric and enantiomeric forms, and that both pure forms and mixtures of these compounds are included.
Wherein the foregoing description reference has been made to integers having known equivalents thereof, those equivalents are herein incorporated as if individually set forth.
It is to be appreciated that variations and modifications may be made to the invention as described without departing from the spirit and scope of the invention.
Throughout this specification, unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to".
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge.

Claims

What we claim is:
A method for the fluorescent detection of nitroreductase activity using a fluorogenic probe suitable for use as a nitroreductase probe, the method including the step of applying a plurality of probes to a sample and monitoring for the presence of at least one nitroreductase in a common test environment.
A method for the fluorescent detection of nitroreductase activity using at least one fluorogenic probe selected from compounds of formula I-V defined below:
Figure imgf000036_0001
wherein X represents N, NH, NR6, O or S;
Z represents C, CH or N; wherein R1, if present, may be selected from H, R7, (CR7R8)nCOOH,
(CR7R8)nCOOR9, (CR7R8JnCONH2, (CR7R8)nCONHR9, (CR7R8)nCONR9R10,
(CR7R8JnOH, (CR7R8JnOR9, (CR7R8)nOPO(OH)2, COOH, COOR7,
(CR7R8)nNR9R10, (CR7R8)n-morpholinyl, (CR7R8)n-piperazinyl; (CR7R8)n-1- methylpiperazinyl; (CR7R8)n-piperidinyl; (CR7R8)n-pyrrolidinyl or (CR7R8Jn- imidazolyl; wherein R2 may represent H, R11, (C'R11R12)nCOO(C"R11R12)nNR13R14,
(C'R11R12)nCONH(C"R11R12)nNR13R14, wherein C and C" may be optionally and independently substituted with C1-C6 alkyl and/or OH;
R3 and R4 may independently represent H, R15, Ar, -(CH=CH)nAr; SO3H, CN wherein Ar may represent a substituted or unsubstituted phenyl, pyridyl, pyrimidinyl, thiazolyl, oxazoylyl, imidazolyl, furanyl, pyrrolyl, benzoxazolyl, benzthiazolyl, benzofuranyl, indolyl, indazolyl, benzimdazolyl, wherein each Ar is optionally substituted with NO2, R18, OH, OR16, SH, SR16, halogen, CF3, NH2,
NHR16, NR16R17, NHCOR16, NR16COR17, NHCOOR16, NR16COOR17,
(CR16R17)nCOOH, (CR16R17)nCOOR18, (CR16R17)nCONH2, (CR16R17)nCONHR18, (CR16R17)nCONR18R19, (CR16R17)nOH, (CR16R17)nOR18, (CR16R17JnOPO(OH)2, COOH, COOR16, CONH2, CONHR16, CONR16R17, COR16, CN1 SOR16, SO2R16, SO2NR16R17,- SO3H, or when Z represents C, R3 and R4 may together form a fused aromatic ring optionally substituted at one or more of the available carbons with a C1-C6 alkyl, halogen, SO3H or CN; wherein R5 may represent H, C1-C6 alkyl, halogen, CN, NO2, Ar, -(CH=CH)nAr, COR20, SOR20, SO2R20, CO(CR20R21JnOH, SO(CR20R21)nOH, SO2(CR20R21)nOH, CO(CR20R21)nCOOR22, SO(CR20R21)nCOOR22 SO2(CR20R21)nCOOR22, CO(CR20R21JnNR22R23, SO(CR20R21JnNR22R23, SO2(CR20R21JnNR22R23, CO(CR20R21JnOPO(OH)2, SO(CR20R21JnOPO(OH)2, SO2(CR20R21)nOPO(OH)2,
CONR20R21, SONR20R21, SO2NR20R21, CNNR20(CR21 R22JnCOOR23, CNNR20(CR21R22)nOH, CNNR20(CR21 R22JnNR23R24,
CNNR20(CR21R22)nOPO(OH)2, CONR20(CR21R22)nOH, SONR20(CR21 R22JnOH, SO2NR20(CR21 R22JnOH, CON R20(CR21 R22JnCOOR23, SONR20(CR21 R22JnCOOR23, SO2NR20(CR21 R22JnCOOR23,
CONR20(CR21 R22JnNR23R24, SONR20(CR21 R22JnNR23R24, SO2NR20(CR21R22)nNR23R24, CONR20(CR21R22)nOPO(OH)2, SONR20(CR21R22)nOPO(OH)2, SO2NR20(CR21R22)πOPO(OH)2; wherein n = O, 1 , 2, 3, 4, 5 or 6; and R6, R7,,R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22,
R23, R24 independently may represent H, C1-C6 alkyl, halogen, OH, (CR25R26JnCOOR27, (CR2IR26JnNRi7R28, (CR25R26JnOH, (CR25R26JnOPO(OH)2 and may together form a ring selected from morpholinyl, piperazinyl, 1- methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl, wherein R25, R26, R27 and R28 may represent H, C1-C6 alkyl, halogen and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpjperazinyl, piperidinyl, pyrrolidinyl, imidazolyl, and any pharmaceutically acceptable salt thereof;
Figure imgf000037_0001
wherein X represents N, CH, O or S; Z represents C or N, wherein R1 and R2 if present, may independently represent H, C1-C6 alkyl, Ar, -(CH=CH)nAr, (CR3R4)nNR5R6, (CR3R4)nCOOR5, (CR3R4)nOH, (CR3R4)nOPO(OH)2; wherein n = 0, 1 , 2, 3, 4, 5 or 6; wherein Ar may represent a substituted or unsubstituted phenyl, pyridyl, pyrimidiny^thiazolyl, oxazoylyl, imidazolyl, furanyl, pyrrolyl, benzoxazolyl, benzthiazolyl, benzfuranyl, indolyl, indazolyl,-benzimdazolyl, wherein each Ar is optionally substituted with one or more NO2, CN, R3, OH, OR3, SH, SR3, halogen, CF3, NH2, NHR3, NR3R4, NHCOR3, NR3COR4, NHCOOR3, NR3COOR4, (CR3R4JnCOOH1 (CR3R4JnCOOR5T (CR3R4JnCONH2, (CR3R4JnCONHR5, (CR3R4JnCONR5R6, (CR3R4JnOH, (CR3R4JnOR5,
XCR3R4JnOPO(OH)2, COOH, COOR3, CONH2, CONHR3, CONR3R4, COR3, SOR3, SO2R3, SO2NR3R4, SO3H; ' wherein R3, R4, R5 or R6 may independently represent H, C1-C6 alkyl, halogen, OH, (CR7R8JnNR9R10, (CR7R8JnOH, (CR7R8JnOPO(OH)2 and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpιperazιnyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R7, R8, R9 and R10 may represent H or C1-C6 alkyl and may together form a ring selected from morpholinyl, piperazinyl, 1- methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and any pharmaceutically acceptable salts thereof;
Figure imgf000038_0001
" wherein R1 may represent COR2, SOR2, SO2R2,~CO(CR2R3)~ nOH, SO(CR2R3)nOH, SO2(CR2R3JnOH, CO(CR2R3JnCOOR4, SO(CR2R3JnCOOR4 SO2(CR2R3JnCOOR4, CO(CR2R3)nNR4R5, SO(CR2R3)nNR4R5, SO2(CR2R3)nNR4R5, CO(CR2R3)nOPO(OH)2, SO(CR2R3)nOPO(OH)2,
SO2(CR2R3)nOPO(OH)2, CONR2R3, SONR2R3, SO2NR2R3, CNNR2(CR3R4)nCOOR5, CNNR2(CR3R4)nOH, CNNR2(CR3R4)nNR5R6, CNNR2(CR3R4)nOPO(OH)2, CONR2(CR3R4)nOH, SONR2(CR3R4)nOH, SO2NR2(CR3R4)nOH, CONR2(CR3R4)nCOOR5, SONR2(CR3R4)nCOOR5 SO2NR2(CR3R4)nCOOR5 CONR2(CR3R4)nNR5Rβ, SONR2(CR3R4)nNR5R6,
SO2NR2(CR3R4)nNR5R6, CONR2(CR3R4)nOPO(OH)2, SONR2(CR3R4)nOPO(OH)2, SO2NR2(CR3R4)nOPO(OH)2; wherein n = 0, 1, 2, 3, 4, 5 or 6r and R2, R3, R4, R5, R6 independently may represent H, Ci-C6 alkyl, halogen, OH, (CR7R8)nNR9R10, (CR7R8JnOH, (CR7R8JnOPO(OH)2 and may together form a ring selected fronvmorphplinyl, piperazinyl, 1-methylpιperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R7rR8, R9_and R10_may represent H, C1-C6 alkyl- halogen and may togetherJorm a ring selected from morpholinyl, piperazinyl, 1-methylpiperazinyl,_pιperidιnyl,_pyrrolidinyl, imidazolyl; and any -pharmaceutically acceptable salt thereof;
Figure imgf000039_0001
wherein R1, may be selected from H, R2, (CR2R3JnCOOH, (CR2R3JnCOOR4, (CR2R3JnCONH2, (CR2R3JnCONHR4, (CR2R3JnCONR4R5, (CR2R3JnOH, (CR2R3JnOR4, (CR2R3JnOPO(OH)2, (CR2R3JnNR4R5, (CR2R3)n-morpholinyl, (CR2R3)n-pιperazinyl; (CR-2R3Jn- 1 -methylpiperazinyl; (CR2R3)n-piperidinyl; (CR2R3)n-pyrrolidinyl or (CR2R3)n-jmidazolyl; wherein n = O, 1, 2, 3, 4, 5 or 6; and R2, R3, R4, R5 independently may represent H, CrC6 alkyl, halogen, OH, (CR6R7JnNR8R9, (CR6R7JnOH, (CR6R7JnOPO(OH)2 and mayJogether form a ring selected from morpholinyl, piperazinyl, 1 -methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R6, R7, R8 and R9 may represent H, Ci-C6 alkyl, halogen and may together form a ring selected from morpholinyl, piperazinyl, 1- methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and any pharmaceutically acceptable salt thereof,
Figure imgf000039_0002
wherein R1 may represent H, R4, COR4, SOR4, SO2R4, CO(CR4R5)nOH, SO(CR4R5)nOH, SO2(CR4R5JnOH1 CO(CR4R5JnCOOR6, SO(CR4R5)nCOOR6 SO2(CR4R5JnCOOR6, CO(CR4R5)nNR6R7, SO(CR4R5JnNR6R7,- SO2(CR4R5)nNR6R7, CO(CR4R5JnOPO(OH)2, SO(CR4R5)nOPO(OH)2, - SO2(CR4R5)nOPO(OH)£ CONR4R5, SONR4R5, SO2NR4R5, ' .-
CNNR4(CR5R6)nCOOR7, CNNR4(CR5R6)nOH, CNNR4(CR5R6)nNR7R8, CNNR4(CR5R6)nOPO(OH)2,-CONR4(CR5-R6)nOH, SONR4(CR5R6)nOH, SO2NR4(CR5R6)nOH, CONR4(CR5R6)nCOOR7, SONR4(CR5R6)nCOOR7, SO2NR4(CR5R6)nCOOR7 CONR4(CR5R6)nNR7R8, SONR4(CR5R6)nNR7R8, SO2NR4(CR5R6)nNR7R8, CONR4(CR5R6)nOPO(OH)2,
SONR4(CR5R6)nOPO(OH)2) SO2NR4(CR5R6)nOPO(OH)2; R2 and R3 may independently represent H, R9, (CR9R10JnCOOH, (CR9R10JnCOOR11 , (CR9R10JnCONH21-(CR9R10OnCONHR11 , (CR9R10JnCONR11R12, (CR9R10JnOH, (CR9R10JnOR11, (CR9R10JnOPO(OH)2, COOH, COOR9, CONH2, CONHR9, CONR9R10, COR9, CN, SOR9, SO2R9,
SO2NR9R10, or R2 and R3_may together form a fused aromatic ring optionally substituted at one or more of the available carbons with a Ci-C6 alkyl, halogen, SO3H or CN; wherein n = O, 1 ,
2, 3, 4, 5 or 6; and R4; R5, R6, R7, R8, R9~ R10, R11, R12"independently may represent H, C1-C6 alkyl, halogen, OH, (CR13R14JnNR15R16, (CR13R14JnOH, (CR13R14JnOPO(OH)2 and may together form a ring" selected from morpholinyl, piperazinyl, 1- methylpiperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; wherein R13, Ru, R15 and R16 may represent H, C1-C6 alkyl, halogen and may together form a ring selected from morpholinyl, piperazinyl, 1-methylpfperazinyl, piperidinyl, pyrrolidinyl, imidazolyl; and any pharmaceutically acceptable salt thereof the method including the step of applying at least one fluorescent probe to a sample and monitoring for the presence of at least one nitroreductase.
3. A method according to claim 2, wherein the method of detection uses a plurality of fluorogenic probes.
4. A method as defined in claim 1-3, wherein the fluorogenic probe(s) are selected from compounds of formula I as defined in claim 2.
5. A method according to claim 4, wherein X is O or NH, Z is CH or NH and R3 is H.
6. "A "method according to claim 4 of 5, wherein the compound of formula I is selected from: -
6-nitro-4(1 H)~-quinolinone; 1 -methylϊ6-nitro=4(.1H)-:quinolinone; :
2~-methyl-6-nitro"-4(-1 Λ/)-quinolinone; ~
Λ/^[2-(dirnethylamino)ethyl]-2fnitro-9:oxb-9r10-dihydro-4"-acridinecarboxamide;
6-nitro-4(3H)-quinazolinone; ^- - „=
6-nitro-2-phenyl-4(3H)-quinazolinone; methyl (6-nitrό^4-oxo-1 (4H)-quiήoliήyl)acetate;
A/.W-dimethyl-S-^-'nitro^quinolinyOoxyl-i-propanamine;
Λ/1,Λ/1-dimethykΛ/3r(7τnitro-4rquinolinyl)-1V3-pr6panediamine;
"/Vl IΛ/1-dimethyl-Λ/3-(5-πitro-4J :qutna2blinyl)-i73-propanediamine; or any pharmaceutically acceptable salt thereof.
7. A method as defined in claim 1-3;-wherein the fluorogenic probe(s) are selected from com pounds of formula Il as defined in claim 2.
8. A method according to claim 7, wherein X is N and Z is C, R1 = H and R2 = Ar.
9. A method according to claim 7 or 8, wherein the compound of formula Il is 5- nitro-2-phenyl-1/-/-benzimidazble or any pharmaceutically acceptable salt thereof.
10. -A method as defined in claim 1-3; wherein the fluorogenic probe(s) are selected _ from compounds of formula IfI as defined in claim 2.
11. - A method according to claim 10, wherein R1 is SO2NR2(CR3CR4)f,GOOR5, wherein R2, R3, R4 and R5, may represent H, C1-C6 alkyl, halogen or OH and wherein n = 0,-1 ,-2, 3, 4-5 or 6. -
12. A method according to claim 10 or 11 , wherein the compound of formula III is selected from: methyl 4-{[(5-nitro-1 -naphthyl)sulfonyl]amino}butanoate; methyl 4-{[(5-nitro-2-naphthyl)sulfonyl]amino}butanoate; methyl 4-{[(8-nitro-2-naphthyl)sulfonyl]amino}butanoate; or any pharmaceutically acceptable salt thereof.
13. A method as-defined-inxlaim 1^3,-wherein the-fluorogenic probe(s) are selected from compounds of formula IV as defined in claim -2.
14. A- method according toxlaim 13, wherein R1 is (CR2R3JnNR4R5.
15. A method according-to claim 13 or 14p wherein the compoundOf formula IV is =2-[4-(dimethylamino)butyl]-5-nitro-1 /-/-benzo[de]isoquinoline-1 ,3(2H)-dione or any pharmaceutically acceptable salt thereof.
16." A method as defined in claim -1-3, wherein the fluorogenic probe(s) are selected from compounds of formula-V as defined in claim-2.
17. A method according to^claim 16, wherein R^ and Rd together represent Ar.
18. A method according to claim 16 or 17; wherein the compound of formula V is 3- nitro-6H-benzo[c]chromen-6-one or any pharmaceutically acceptable salt thereof.
19. A method as defined in claims 1-18, wherein the at least one nitroreductase is a human oxidoreductase.
20. - A'method as defined in claim 19, wherein the human oxidoreductase is selected from known human enzymes classified as EC1.
21. A method as defined in claims 19 or-20, wherein the nitroreductase(s) is selected from:
E(M . Vr which includes oxidoreductases that act on the CH-OH group of donors e.g. Aldose reductase [ALDR1 ; E.C.V.1.1.21]; e.g-Aldehydeτeductase.[AKR1B10; E.C. 1.1.1.2].
EC 1.2: which includes oxidoreductases that act on the aldehyde or oxo group of donors -
EC 1.3: which includes oxidoreductases that act on the CH-CH group of donors
EC 1.4: which includes oxidoreductases-that act on-the CH-NH2 group of donors
EC 1.5: which includes oxidoreductases that act on CH-NH group of donors
EC 1.6: which includes oxidoreductases that act on NADH or NADPH e.g. DT-diaphorase [NQO1 ; E.C.1.6.99.2]; e.g. -Cytochrome P450-reductase [C-YPΘR;-E.G.~1?6.2r4]; ~ erg. Cytochrome B5 reductase [DIA1; E.G.1.6.2.2];
EC 1.7: which includes oxidoreductases that act on other nitrogenous compounds as donors
EC 1.8: which includes oxidoreductases that act on a sulfur group of donors e.gi Thioredoxin-disulfide-reductase [TXNRD; Ε.C.1τ8.1:9];'
EC 1.9: which includes oxidoreductases that act on a heme group of donors ' EC 1.10: which includes oxidoreductases that act on diphenols and related substances as donors
EC 1.11 : which includes oxidoreductases that act on peroxide as an acceptor
(peroxidases)
EC 1.12: which includes oxidoreductases that act on hydrogen as donors
EC 1.13:. which includes oxidoreductases that act on single donors with incorporation of molecular oxygen (oxygenases)
EC 1.14: which includes oxidoreductases that act on paired donors with
- incorporation of molecular oxygen -
■ e.g. Inducible nitric oxide synthase [NOS2A; E.C.1.14.13.39];
EC 1.15: which includes oxidoreductases that act on superoxide radicals as acceptors
EC 1.16: which includes oxidoreductases that oxidize metal ions; e.g. Methionine synthase reductase [MTRR; E.C.1.16.1.8];
- EC 1.17: which includes oxidoreductases that act on CH or CH2 groups; - e.g.-Xanthine-oxidase [XO;- E.C.1 /17.3.2]; e.g.- Xanthine dehydrogenase [XDH; E.C.1.17.1.4];
-EC -1τ18: which includes oxidoreductases that-act on-iron-sulfur proteins as donors; e.g. Adrenodoxin oxidoreductase [FDXR; -E.C.1.18r1.2]"-
EC 1.19: whichϊncludes oxidoreductases, that act on reduced flavodoxin as a donor -
EC 1.21 : which includes oxidoreductases that act on X-H-and Y-H to form an X-
Y bond
EC 1.97: which includes other oxidoreductases .
22. A method as defined in claims 1-18, wherein the at least one nitroreductase is a microbial or fungal nitroreductase.
23. A method as defined in claim 22, wherein the at least one nitroreductase is selected frorrktype I nitroflavin reductase NfsA and NfsB superfamilies or the NQOI-like and YieF-like nitroreductase enzymes, or any putative nitroreductase genejshowing-jevidence of significant sequence homology thereof.
24. .., A-method-foMdentifying the-preseήce of-cellular-hypoxia, trie method including:
(i) f contacting an effective amount of af least one compound otformula I-
.V as defined in claim.2, to at least one cellular sample; and - (ii) monitoring for the formation of at least one fluorescent derivative
: ■" ; arising from- reduction byat least one nitroreductase.
25. A method for- identifying the presence of cellular hypoxia, the method including: (i) - contacting-anaeffective amount of a;plurality of compounds of formula .. - I-V as defined in claim 2, to at least one cellular sample; and
(ii)- monitoring-for-the formation of at- least-one fluorescent derivative arising. from reduction by at least.one nitroreductase.
26. The method according to claim 24 or 25, wherein the nitroreductase is a human .nitroreductase.
27. The method according to claims 24-26,' wherein the nitroreductase is selected from:-
■DT-diaphorase [NQO1 ; E.C.1.6.99.2];- Cytochrome P450,reductase [CYPΘR; E.C.-1.6.2.4];
Jnducible-nitric oxide synthase_[NQS2Af E.G.1.14.13.39];
Cytochrome B5 reductase [DIA1 ; E.C.1.6.2.2];
Xanthinezoxidase [XOrE.C.,1 J 7.3.2]; -- .;- ,
Xanthine-dehydrogenase-[XDH; E.C.1.17.1.4]; , Adrenodoxin oxidoreductase [FDXR; E. C.1.18.1.2];
"Methionine synthase-reductase [MTRR-; E.C.1.-16.1.8]~
-Aldose reductase [ALDR1 ; E.C.1.1.1.21];
Aldehydej-eductase [AKR1B.10; B.C.-t1.1 r1.2].and
Thioredoxin reductase [TXNRD; E.C.-1.8.1.9]. -
28. A method as defined in claim 1-27, wherein the.step of monitoring -for the presence otnitroreductase includes-the step-of monitoring-forthe presence of a reduced fluorescent derivative ofat least one compound of formula I-V.
5 29. ~ A-method as defined in claim 28, wherein the presence of the reduced luorescent derivative is determined from the fluorescence emission.
30. - A method according to claim 29;\.wherein the reduced.derivative(s) may be excited at predetermined wavelengths in the UV/visible range, -t 10
31. A method according to clainrr30,* wherein the excitation wavelength is between 200-700nm
32. A method accordingio claims 30 or 31..wherein the excitation wavelength is 15 selected from 295, 340, 355, 405, 440 and 485 nm. ~
33. A method according to claim 29; wherein the fluorescence emission(s) is in the UV/visible/IR range.
20 34. A method according to claim 33, wherein the fluorescence emission wavelength is between 300-800nm
35. - A method according to claims 33 or 34, wherein the excitation wavelength is
- selected from-370, 460, 510, 535,' 540 and 585 nm. 25
36. A method as
Figure imgf000045_0001
wherein the method further includes the
- step of quantifying the one or more nitroreductase.
37. - - A method as defined in claim :36,~ wherein the quantification of nitroreductase -30 Jncludes.the step of quantifying the formation of the fluorescent derivative from the fluorescence emission-intensity.
38. A method^according to-claims 2-37, wherein the monitoring can be performed in a common test environment.
35
39. An assay for the detection of one or more nitroreductase including the steps of: (i) contacting an effective amount of a plurality of fluorogenic probes with
-a sample; (ii) monitoring for the formation of fluorescent derivatives.
—
40. An assay for detection of one.or more nitroreductase including the steps of:
(i) contacting an effective amount of at least one compound of formula I-
V-as defined in-claim 2-, with a sample; - (ιi) monitoring for; the formation of fluorescent derivatives
41. An assay according to claims 39 or 40, wherein the sample may be added to a common test environment containing aφlurahty of fluorogenic probes.
42. An assay according to claim 39^41, wherein the assay -further includes the analytical step of quantifying the formation of at least one fluorescent " derivative(s) from the fluorescence emission intensity.
43. An assayxomprising at least one test environment containing a plurality of fluorogenic probes, wherein a sample may be added and the test environment monitored for the formation of fluorescent derivative(s).
44. An assay according to claim 43, wherein the fluorogenic probes may be selected from compounds of formula I-V as defined in claim 2.
45. ^A compound of formula III as defined in claim 2,, wherein formula III represents one of the following: methyl.A-{[(5-nitro-1 -naphthyl)sulfonyl]amino}butanoate, - methyh4-{[(5-nitro-2-naphthyl)sulfonyl]amino}butanoate; methyl 4-{[(8-nitro-2-naphthyl)sulfonyl]amino}butanoate; or any-pharmaceutically acceptable salt thereof.
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