HK1143189A - Caspase imaging probes - Google Patents

Description

Caspase imaging probes

The present invention relates to molecular probes (substrates) for observing the catalytic activity of a single proteolytic enzyme or a group of proteolytic enzymes in an in vitro assay, in a cell or a multicellular organism. The invention also relates to methods of synthesizing and designing such probes (substrates).

Background

Proteolytic enzymes (proteases) cleave or degrade other enzymes or polypeptides either inside or outside of living cells. Proteases are involved in many critical processes, many of which are critical processes in cell signaling and tissue homeostasis. Abnormal or enhanced protease activity has been implicated in a variety of diseases, including cancer, osteoarthritis, atherosclerosis, inflammation, and the like. Since proteolytic activity must be strictly controlled in living systems, many proteases are expressed as inactive precursor proteins (zymogens) and then activated after proteolytic cleavage under control. Other controls for proteolytic activity come from endogenous inhibitors that bind to and inactivate the catalytically active form of the proteolytic enzyme. Because of this tight control, it is desirable to monitor protease activity rather than just protease expression when studying protease function in a cellular or physiological event. Thus, reports have also been made in the literature on various activity-based chemical probes. Typical protease probes generate a detectable signal by one of two pathways: (i) the spectral properties of the reporter system are altered by cleavage of peptide bonds, or (ii) by covalent attachment of a mechanistic inhibitor to the subject protease of interest. The search for localization and quantification of the activity and inhibition of a particular protease or group of proteases (e.g. in cellular assays or whole animal imaging experiments) requires the development of imaging probes that are capable of (i) reaching physiologically relevant sites of protease action (e.g. specific organs in the cytosol or whole animal imaging) and (ii) being selective for the desired protease or group of proteases. The preparation of protease-selective probes poses a great challenge to this field. The present invention relates to (i) novel selective probes for cysteine proteases, preferably for the caspase subfamily, (ii) the use of these probes in vitro assays, in cells or multicellular organisms (e.g. by molecular imaging) and (iii) methods of synthesizing and designing such probes.

In recent years, several molecular imaging techniques (optical and non-optical) have become increasingly important for the non-invasive visualization of specific molecular targets and pathways in vivo. Since the information content of any image signal is initially a function of internal contrast, the development of internally quenched imaging probes that can be activated in enzymatic reactions (e.g., cleavage of peptide bonds) has been commonly used in the imaging and localization of catalytically active proteases. Probes that are selective for individual proteases and that are capable of reaching the in vivo site of action of the protease are rarely obtained by conventional methods. Pharmaceutical chemists in the pharmaceutical industry are also faced with related challenges in developing drugs with appropriate pharmacokinetic profiles and appropriate selectivity for a given target. In the present invention, we have invented a new route to prepare probes with selective activity against cysteine proteases, which we have applied to proteases of the caspase subfamily.

Cysteine proteases are characterized by having a cysteine residue at the active site that acts as a nucleophilic group in the catalytic process. The catalytic cysteine is typically hydrogen bonded to an appropriate adjacent group to enable formation of a thiolate ion. When the protease recognizes the substrate, a cleavable peptide bond is placed in the vicinity of the catalytic cysteine which attacks the carbonyl carbon atom to form an oxoanion intermediate. The amide bond is then cleaved, releasing the C-terminal peptide in the amine form. The N-terminal portion of the scissile peptide remains in the covalent acylase intermediate, which is subsequently cleaved with water to regenerate the enzyme. The N-terminal cleavage product of the substrate is released in the form of a carboxylic acid.

Caspases are a family of caspase proteases. The human genome encodes 11 caspases. Of these 8 (caspase-2, 3, 6, 7, 8, 9, 10 and 14) play a role in apoptosis or programmed cell death. They act through a highly regulated signal transduction cascade. In rank order, some caspases initiate cleavage of enzymes (caspase-2, 8, 9 and 10) and activate caspase effectors (caspase-3, 6 and 7). These caspases are associated with cancer, autoimmune diseases, degenerative disorders and stroke. The functions of the other three caspases (caspase-1, 4 and 5) are completely different: inflammation is mediated by activation of a subset of inflammatory cytokines.

Caspase-1, also known as interleukin-1 beta-converting enzyme (ICE), is predominantly present in monocytes. The protease is mainly responsible for the production of proinflammatory cytokines interleukin-1 beta and interleukin-18. It has been shown that inhibition of caspase-1 may produce beneficial effects in human models of inflammatory disease, including rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, and asthma.

Caspase-3 is responsible for proteolytic cleavage of a variety of essential proteins including cytoskeletal proteins, kinases, and DNA repair enzymes. In neurons, caspase-3 is a key mediator of apoptosis. Inhibition of caspase-3 has been shown to have beneficial effects in models such as stroke, traumatic cerebrospinal injury, hypoxic brain injury, myocardial ischemia, and reperfusion injury.

Caspase-8 is an apoptotic caspase initiator, located downstream of the death receptors of the TNF superfamily. The substrates include caspase effectors and family members of the pro-apoptotic factor Bcl-2 involved in apoptosis. It is known that apoptosis resistance of cancer is associated with low caspase-8 expression levels, and that inhibition of caspase-8 may potentiate resistance to apoptosis-inducing stressors such as chemotherapy and radiation therapy. Caspase-8 is therefore an attractive target for the treatment of tumors and metastatic lesions. Gene knockout studies have also found that caspase-8 has many other potential effects unrelated to apoptosis. For example, caspase-8 knock-out is followed by defects in leukocyte differentiation, proliferation and immune response.

In the case of proteolytic enzymes, it is their activity rather than just the expression level that determines their functional role in cell physiology and pathology. Thus, molecules that inhibit caspase activity can be used as therapeutics for treating diseases and for developing specific imaging biomarkers that can visualize proteolytic activity as well as inhibition thereof by candidate drugs, which can accelerate target validation, Drug Discovery and even clinical trials (h. pien et al, Drug Discovery Today, 2005, 10, 259-266). Specific proteins or protein families can be readily detected in complex protein mixtures, intact cells, and even in vivo using activity-based imaging reagents. In addition, enzyme class specific probes can be used in screening methods for developing small molecule inhibitors for functional experiments (d.a. jeffery, m.bogyo curr. opp.biotech.2003, 14, 87-95).

To date, activity-based imaging probes incorporating peptide substrates have been developed for monitoring and labeling caspase-1 (W.Nishii et al, FEBS Letters 2002, 518, 149-. In addition, a near infrared fluorescent probe has been reported which can be used to detect caspase-1 activity in living animals (S. Messerli et al, Neoplasia 2004, 6, 95-105).

The enzymatic mechanism of caspases is well studied and found to be highly conserved. Based on research and screening data for cleavable peptides, electrophilic substrate analogs that react only with this conserved active site were developed. The electrophilic center in such probes is usually part of a so-called "warhead", which is a molecular entity whose electrophilic properties and geometrical arrangement are optimized so that it fits well with the active site of a caspase and reacts with a catalytic cysteine residue at that site. A wide variety of such electrophilic substrates have been described as mechanistic cysteine protease inhibitors, including but not limited to: diazomethyl ketone, fluoromethyl ketone, acyloxymethylketone, O-acylhydroxylamine, vinyl sulfone and epoxysuccinic acid derivatives (S.Verhelst, M.Bogyo QSAR comb.Sci.2005, 24, 261-269).

Another tool for monitoring protease activity is in bioluminescent assays. The method utilizes an amino-modified luciferin precursor (begles pro-luciferase) (Caged luciferin) or a carboxy-terminal derivative thereof linked to a protease substrate. The first proteolytic cleavage explains the release of luciferin which is subsequently converted by luciferase, and the luminescent signal can be detected. The secondary assay has a similar range of applications as fluorescent probes, with the additional advantage of high signal to noise ratio.

Protease inhibitors not only need to have high potency but also must have highly selective binding to specific proteases in order to be effective biological tools. The development of small molecule inhibitors against specific proteases often begins with peptide substrates. Although peptides have a variety of biological properties, their use as pharmaceuticals is limited by instability and poor oral bioavailability. For it to be effective as a drug, it is desirable that the peptide-like character of the protease inhibitor obtained should be weak, highly stable to non-selective proteolytic degradation, highly selective for a given protease, and have good bioavailability at the site of protease action. In accordance with these requirements, caspase inhibitors a-B have been developed, where a is a chemical backbone covalently linked to an electrophilic warhead B. In the presence of caspases, B reacts covalently with a catalytic cysteine, a mechanistic inhibitor. In many cases, the selectivity and pharmacokinetic properties of such inhibitors have been successfully optimized in biomedical research. In order to be able to initiate an efficient electrophilic attack against the catalytic cysteine, the electrophilic center of such inhibitors must be precisely located within the active site of the enzyme. The particular arrangement of catalytic cysteine to electrophilic carbon atoms in the warhead corresponds to the spatial arrangement of the peptide carbonyl groups in the catalytic cysteine and the cleavable peptide substrate. Under the guidance of this comparison, we thought that it should be possible to "redesign" the optimized covalent inhibitor (with chemical backbone a and electrophilic warhead B) as a cleavable substrate. Since chemical backbone a can be considered a determinant of inhibitor selectivity, our approach can translate the selectivity or partial selectivity of an optimized inhibitor into an activity-based chemical probe. We refer to this process as "reverse engineering" of activity-based selective probes from selective caspase inhibitors.

The present invention relates to molecular probes against cysteine proteases of formula (I)

{L1-R1-L}_n-A-CO-NH-R2-L2 (I)

Wherein

A is a group recognizable by caspase;

r1 is a linking group;

r2 is a bond or a linking group;

l is a bond or a group capable of allowing the L1 group to bind easily;

l1 and L2 are independently of each other at least one labeling group optionally attached to a solid support; and is

n is 1;

or

R2 is a bond;

l2 is a substrate suitable for coupled bioluminescent assays; and is

n is 0.

The compounds of formula (I) are activity-based probes (substrates) for cysteine proteases, preferably cysteine proteases of the caspase family.

In its most basic form, the chemical probe consists of four functional elements, namely, a) an amide group-CO-NH-as a reactive group, which can be cleaved by the action of a protease, b) a backbone a, which defines the selectivity for a given protease target, c) linker groups R1 and R2 linking the various subunits, and d) a set of label groups L1 and L2 for detection.

Preferably the a group is a determinant of specificity for a given caspase or group of caspases, preferably caspase-1, 3 and 8, as shown for example in compounds 1-43 in tables 1, 2, 3. The activity-based probes of the invention have a selectivity factor for a given caspase of 1000: 1, preferably 10: 1, where selectivity is defined by the relative turnover (turnover of enzyme 1: turnover of enzyme 2) at the preferred substrate concentration. The relative turnover number for each pair of enzymes is determined by dividing the turnover number for the enzyme of interest (enzyme 1) by the turnover number for the other enzyme for which selectivity is desired (enzyme 2). For in vivo applications, it is desirable to have high selectivity at lower substrate concentrations (e.g., micromolar or submicromolar).

Scheme 1 shows the reaction of protease P, wherein A is a specificity determining group and P is a protease, the reactive cysteine of which contains a thiol group S, with a substrate^-：

(scheme 1)

The reaction rate depends on the substrate structure.

The linking groups R1 or R2 are preferably flexible linking groups which are linked to a marker group L1 or L2, respectively, or to a plurality of identical or different marker groups L2 or L1. The choice of linker group is made according to the envisaged application, i.e. according to the activity-based imaging probe for the specific protease. The linking group may also increase the solubility of the substrate in a suitable solvent. The linking group used is chemically stable under the conditions of practical use. The linker does not interfere with the reaction of the selected protease target, nor with the detection of the marker groups L1 and/or L2, but may be constructed with consideration that the linker may be cleaved at some point in time. In particular, the linking group R1 or R2 is a linear or branched alkylene group containing from 1 to 300 carbon atoms, wherein optionally,

(a) one or more carbon atoms are replaced by oxygen, particularly wherein every third carbon atom is replaced by oxygen, for example a polyoxyethylene group containing from 1 to 100 oxyethylene units; and/or

(b) One or more carbon atoms are replaced by nitrogen bearing a hydrogen atom and the adjacent carbon atom is replaced by an oxo group, forming an amide function-NH-CO-; and/or

(c) One or more carbon atoms are replaced by an ester function-O-CO-;

(d) the bond between two adjacent carbon atoms is a double or triple bond; and/or

(e) Two adjacent carbon atoms are replaced by disulfide bonds.

The labeling groups L1 and L2 of the substrate can be selected by the person skilled in the art according to the intended use of the probe.

The labeling groups L1 and L2 are, independently of one another, spectroscopic probes such as fluorophores; a quencher group (quencher) or a chromophore; a magnetic probe; a contrast agent; a molecule that is part of a specific binding pair that specifically binds to a ligand; a molecule that is a substrate for an enzyme; molecules covalently attached to a polymer support, a dendrimer, a glass slide, a microtiter plate known to those skilled in the art; or a molecule having a combination of any of the above properties.

A preferred embodiment of the invention is the use of a modified aminoluciferin or a carboxy-terminally protected derivative thereof as a reporter group which, when cleaved from the central scaffold A, is converted by luciferase to generate a luminescent signal. Thus, the labeling group L2 may also be a substrate suitable for coupled bioluminescent assays, characterized by having a modified aminofluorescein or its carboxy-terminally protected derivative as reporter group.

US7148030 discloses examples of bioluminescent protease assays comprising a peptide as a caspase substrate linked to a modified aminofluorescein.

Preferred probes contain an intramolecular quenched fluorescent probe comprising a polymeric backbone (backbone) and a plurality of fluorophores covalently linked to the backbone through backbone A at a density sufficient to cause fluorescence quenching.

Another preferred embodiment of the invention is the use of dendrimers to which two or more fluorophores are covalently attached via backbone A at a density sufficient to cause fluorescence quenching. The advantage of using polymeric probes is that the probes can be delivered locally (targeted), which have a prolonged circulation time in the blood of animals or humans. Polymer conjugation alters the biodistribution of low molecular weight species, enabling tumor-specific targeting (via enhanced permeation and retention effects (EPR effects), reducing the amount of access to toxic sites, conjugation of conjugated polymers with low molecular weight imaging probes is the most preferred embodiment of the invention for imaging multicellular organisms (including mammals such as mice, rats, etc.) the polymer backbone may be composed of any biocompatible polymer, and may contain polypeptides, polysaccharides, nucleic acids, or synthetic polymers Other copolymers of methoxypolyethylene glycol and ethylene glycol.

Probes of the invention may also contain targeting groups such as antibodies, antibody fragments, receptor binding ligands, peptide fragments, or synthetic protein inhibitors.

The marker groups L1 and L2 may also be positively charged linear or branched polymers. It is known to those skilled in the art that such polymers can facilitate the transport of molecules linked therein across living cell membranes. The polymers are more preferred for substances that are less permeable or virtually impermeable to living cell membranes. A cell-impermeable chemical probe will be able to penetrate the cell membrane after conjugation to such L1 or L2 groups. Such cell membrane transport promoting groups L1 and L2 include, for example, linear poly (arginine) s of D-and/or L-arginine containing 6-15 arginine residues, linear polymers containing 6-15 subunits, each of which carries a guanidinium group, oligomers or short chain polymers containing 6 to up to 50 subunits, a portion of which carries a guanidinium group and/or a portion of the HIV-tat protein sequence, for example, the Tat49-Tat57 subunit (one letter amino acid is encoded as RKKRRQRRR.) if L1 is one member of two interacting spectroscopic probes L1/L2 and L2 is the other member, for example in the case of FRET pairs, then linear poly (arginine) of D-and/or L-arginine containing 6-15 arginine residues is preferably used as the polymer label.

Most preferred L1 and/or L2 labeling groups are spectroscopic probes. The most preferred L2 labeling groups are molecules that are part of a spectral pair that forms an interaction with L1, and labeling groups that are capable of specifically binding to ligands and molecules covalently attached to a solid support.

Among the particularly preferred labeling groups, L1 is one member of two interacting spectroscopic probes L1/L2 and L2 is the other member thereof, wherein energy can be transferred between the donor and acceptor (quencher) in a non-radiative manner by dynamic or static quenching. The label pair L1/L2 undergoes a change in spectroscopic properties after the corresponding caspase reaction/cleavage. An example of such a label pair L1/L2 is FRET (FRET: (R) (R))Resonance energy transfer) pairs, such as fluorescent probe precursors (pro-fluorescent probes), which are covalently labeled with a donor (reporter group) at one end (e.g., L1) and an acceptor (quencher group) at the other end (L2), or vice versa.

Specifically, L1 is a donor (reporter) and L2 is an acceptor (quencher), or L1 is a quencher and L2 is a reporter. When this probe is used, the reaction of cysteine protease with the probe causes a change in fluorescence. When the double-labeled substrate reacts with protease, the distance between the reporter group and the quenching group in the double-labeled substrate is changed, so that the reporter group and the quenching group are spatially separated, and fluorescence is emitted or the emission wavelength is changed. A variety of reporter groups may be used as the L1 or L2 marker groups, respectively, including, for example, near infrared light emitting fluorophores. The substrate containing the reporter and quencher groups remains dark until the reaction with the protease, and when the reaction is initiated, the reaction mixture is "lit" and fluorophore emission is initiated due to the spatial separation of the reporter and quencher groups. Fluorescence quenching and energy transfer can be measured by the emission of either the quencher or the energy donating label. When energy transfer occurs, the label group receiving the energy also fluoresces, and the fluorescence of this acceptor label group can also be measured. Of the two interacting labeling groups, the donor labeling group may be selected from chemiluminescent donor probes, thus eliminating the need for an excitation lamp and reducing the level of acceptor background fluorescence. The specific method described using this double-labeled substrate can be used to determine the reaction kinetics based on fluorescence time measurements, and can be applied in vivo or in vitro.

Alternatively, the labelling group L2 may be, or otherwise attached to, a solid support, or attached or attachable to a polymer/solid support. For the L1/L2 FRET pair, linear poly (arginine) of D-and/or L-arginine containing 6-15 arginine residues is preferably used as the polymer label.

A particularly preferred combination is two different affinity labelling groups, in particular a pair of interacting spectroscopic labelling groups L1/L2, such as a FRET pair. The definition of affinity tag group refers to a molecule that is part of a specific binding pair capable of specifically binding to a ligand. Specific binding pairs refer to, for example, biotin and avidin or streptavidin, and in addition methotrexate, which is a tight binding inhibitor of dihydrofolate reductase (DHFR).

One skilled in the art can select the appropriate reporter and quencher pair. Typically, the reporter and quencher are fluorescent dyes with high spectral overlap, e.g., fluorescein as the reporter and rhodamine as the quencher. Other quenching groups are gold clusters and metal cryptates.

The second type of quencher used in the present invention is a "dark quencher", i.e. a dye that is not fluorescent by itself, whose absorption spectrum overlaps with the emission spectrum of a common reporter dye to induce maximum FRET quenching. Furthermore, the dye pairs may be selected such that the absorption bands overlap in order to promote a resonant dipole-dipole interaction mechanism (static quenching) in the ground-state complex.

Specific fluorophores and quenching groups are: alexa dyes including Alexa350, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 635 and Alexa 647(US5696157, US6130101, US 6716979); dimethylaminocoumarin (e.g., 7-dimethylaminocoumarin-4-acetate succinimidyl ester, product D374 from Invitrogen, CA92008, USA); quenching groups QSY 35, QSY 9 and QSY 21(Invitrogen, CA92008, USA); cyanine-3 (Cy 3), cyanine 5(Cy 5), and cyanine 5.5(Cy 5.5) (Amersham-GE Healthcare, Solingen, Germany); BHQ-1, BHQ-2 and BHQ-3(Biosearch Technologies, Inc., Novato, CA 94949, Black Hole Quencher, USA Inc.)^TM) (ii) a Fluorophores ATTO 488, ATTO532, ATTO 600 and ATTO 655 and quencher groups ATTO 540Q and ATTO 612Q (ATTO-Tec, D57076 Siegen, Germany); fluorophores DY-505, DY-547, DY-632, and DY-647(Dyomics, Jena, Germany); 5/6-carboxyfluorescein, tetramethylrhodamine, 4-dimethylaminoazobenzene-4 '-sulfonyl derivative (Dabsyl) and 4-dimethylaminoazobenzene-4' -carbonyl derivative (Dabcyl). These substances can advantageously be combined as follows:

fluorophores Quenching group

● Alexa350, dimethylaminocoumarin, 5/6- ● Dabsyl, Dabcyl,

Carboxyfluorescein, Alexa 488, ATTO 488, BHQ 1, QSY 35

DY-505

● 5/6-carboxyfluorescein, Alexa 488, Alexa ● BHQ2, QSY 9,

532、Alexa 546、Alexa 555、ATTO ATTO 540Q

488. ATTO532, tetramethyl rhodamine, Cy3,

DY-505、DY-547

●Alexa 635、Alexa 647、ATTO 600、 ●BHQ 3、ATTO

ATTO 655、 612Q、QSY 21

DY-632、Cy 5、DY-647 Cy 5.5

The light generated by bioluminescence analysis associated with enzymatic reactions is related to the immediate rate of the catalytic reaction. The method comprises amino-modified amino-luciferin or a derivative protected by a carboxyl terminal thereof, wherein an amino group in the amino-luciferin is connected with a central skeleton A through an amido bond, and an obtained substrate is identified and cracked by caspase. The enzymatic activity of caspases cleaves the peptide bond linking the aminoluciferin to backbone a releasing the substrate aminoluciferin of luciferase. Subsequent reaction of the luciferase with its substrate produces a detectable signal (luminescence). Thus, the method correlates caspase activity with a second enzymatic reaction, producing a readable light signal. Such assays require the development of a "luciferin precursor" ("Caged luciferin") that is recognized by the luciferase as a substrate only after being converted to luciferin by a previous enzymatic reaction, such as proteolytic cleavage. Thus, the light signal is directly dependent on the previous enzymatic reaction. Therefore, another embodiment of the present invention is directed to a probe for detecting the proteolytic activity of caspases by luminescent means.

In a particular embodiment, the method involves a substrate wherein L2 is a solid support or is attached to a solid support carrying one member of a reporter/quencher pair, or wherein L2 is a combination of a solid support and one member of a reporter/quencher pair and L1 is the other member of the pair. Thus, a dark solid support will fluoresce when reacted with an appropriate protease.

The solid support may be a glass slide, a microtiter plate or any polymer known to those skilled in the art, such as a functionalized polymer (preferably in bead form), a chemically modified oxidized surface such as silica, tantalum pentoxide or titanium dioxide, or a chemically modified metal surface such as a noble metal surface such as a gold or silver surface. Solid supports may also be suitable sensing elements.

Preferably, the compounds of formula (I) contain an A group which is a caspase-1 inhibitor. Methods for the preparation of scaffold a having caspase-1 inhibitory activity are described in, for example, US 5670494; WO 9526958; WO 9722619; WO 9816504; WO 0190063; WO 03106460; WO 03104231; WO 03103677; harter, bioorg, med, chem, lett, 2004, 14, 809-812; shahripour et al, bioorg.Med.chem.Lett.2001, 11, 2779-; shahripour et al, bioorg.med.chem.2002, 10, 31-40; M.C. Laufersweeler et al, bioorg.Med.chem.Lett.2005, 15, 4322-4326; chapman, bioorg, med, chem, lett, 1992, 2, 613-; dolle et al, J.Med.chem.1997, 40, 1941-1946; soper et al, bioorg.Med.chem.Lett.2006, 16, 4233-; soper et al, bioorg.Med.chem.2006, 14, 7880-; D.J.Lauffer et al, bioorg.Med.chem.Lett.2002, 12, 1225-; and C.D.Ellis et al, bioorg.Med.chem.Lett.2006, 16, 4728-. More preferably, the compound of formula (I) is a probe for caspase-1, characterized by the following compounds (table 1) containing the preferred backbone a:

TABLE 1 examples of Selective probes (I) for caspase-1

Wherein the variables in groups 1 to 28 are as defined in the respective compound neighborhood; x is-CONH-R2-L2; y is-L-R1-L1; and R1, R2, L, L1 and L2 are as described above.

It is further preferred that the compounds of formula (I) contain an A group which is a caspase-3 inhibitor. Methods for the preparation of scaffold a having caspase-3 inhibitory activity are described, for example, in WO 0032620; WO 0055127; WO 0105772; WO 03024955; WO 2008/008264; tawa et al, Cell Death and Differentiation 2004, 11, 439-; micale et al, J.Med.chem.2004, 47, 6455-6458; and Berger et al, Molecular Cell, 2006, 23, 509-. More preferably, the compound of formula (I) is a probe for caspase-3, characterized by the following compounds (table 2) containing the preferred backbone a:

TABLE 2-Selective probes (I) against caspase-3

Wherein the variables in groups 29 to 42 are as defined for the corresponding compounds; x is-CONH-R2-L2; y is-L-R1-L1; and R1, R2, L, L1 and L2 are as described above.

It is furthermore preferred that the compounds of formula (I) contain an A group as caspase-8 inhibitor. Methods for the preparation of scaffold A having caspase-8 inhibitory activity are described, for example, in Berger et al, Molecular Cell, 2006, 23, 509-521; and Garcia-Calvo, J.biol.chem.1998, 273(49), 32608-32613. More preferably, the compound of formula (I) is a probe for caspase-8, characterized by a compound containing the following preferred backbone A (Table 3):

TABLE 3 examples of Selective probes (I) against caspase-8

Wherein X is-CONH-R2-L2; y is-L-R1-L1; and R1, R2, L, L1 and L2 are as described above.

Unless otherwise stated, the following definitions apply.

Alkyl refers to a straight or branched chain hydrocarbon group having 1 to 6 carbon atoms. (C)₁-C₆) Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, isobutyl, butyl, tert-butyl, sec-butyl, pentyl and hexyl.

Acyl is defined as-C (═ O) alkyl.

Aryl is defined as an aromatic hydrocarbon having 6 to 10 carbon atoms. Examples of aryl groups include phenyl and naphthyl.

Heteroaryl is defined as an aryl group in which one or more of the arene carbon atoms are replaced with a heteroatom, where "heteroatom" includes oxygen, nitrogen, sulfur and phosphorus. Heteroaryl groups include furan, thiophene, benzothiophene, pyrrole, thiazole, pyridine, pyrimidine, pyrazine, benzofuran, indole, coumarin, quinoline, isoquinoline and naphthyridine.

Cycloalkyl refers to cyclic alkyl groups having 3 to 10 carbon atoms. Examples of cycloalkyl groups include cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

Heterocycle or heterocyclyl refers to cycloalkyl groups in which one or more carbon atoms are replaced by a heteroatom. Examples of heterocyclic groups include piperazine, morpholine, and piperidine.

The aryl, heteroaryl or cycloalkyl groups may be substituted by one or more identical or different substituents. Examples of suitable substituents include alkyl, alkoxy, thioalkoxy, hydroxy, halo, trifluoromethyl, amino, alkylamino, dialkylamino, NO₂、CN、CO₂H、CO₂Alkyl, SO₃H. CHO, C (═ O) alkyl, CONH₂CONH-alkyl, CONHR_qC (═ O) N (alkyl)₂、(CH₂)_nNH₂、OH、CF₃、O(C₁-C₆) Alkyl group, (CH)₂)_nNH-alkyl, NHR_q、NHCOR_qPhenyl, wherein n is 1 to5 and R_qIs hydrogen or (C)₁-C₆) An alkyl group.

For the synthesis of activity-based probes of the invention, the central backbone A can be chemically constructed using appropriate protecting groups known to those skilled in the art and linked to one of the two linking groups and the labeling group L1 or L2 via the L group and the-C (O) -NH-group. Suitable building blocks and FRET pairs such as cyanine dyes (e.g. Cy 3B, Cy 5.5, Cy 7) are commercially available (e.g. Sigma-Aldrich, GE-Healthcare). Solid phase synthesis Methods are particularly useful for the subset of probes described herein (B.J. Merrifield, Methods in Enzymology 1997, 289, 3-13). Depending on the synthetic requirements, attachment of the linker, quencher or fluorophore can be accomplished on a solid support or by solution phase chemistry.

In general, the reactive side-chain groups and optional L groups on the central backbone a will be protected and then subsequently released for further modification with L1R1 and L2R2 subunits, respectively. The combination of these subunits can be accomplished according to methods known in the art of chemical synthesis. It is particularly useful to carry out a reaction between the dye active ester and the primary amine groups of backbone A such that the two units are linked by an amide linkage. Intermediate and final product probe molecules can be purified by High Performance Liquid Chromatography (HPLC) and identified by mass spectrometry and analytical HPLC prior to use in labeling and imaging experiments.

The following paragraphs illustrate the invention by way of some non-limiting examples.

In a preferred embodiment, the probe of formula (I) contains a backbone a derived from a tetrapeptide caspase-1 inhibitor (table 1, compound 2), which carries chromophores at the C-and N-termini. The chromophore is suitably selected so that its spectral properties are suitable for Fluorescence Resonance Energy Transfer (FRET). The chromophore may or may not emit fluorescent light. In principle, a wide variety of chromophores can be used in the present invention, provided that the basic requirement is met that the spectrum changes after proteolytic cleavage of peptide bonds. The linkage of such interacting chromophores to the central backbone can optionally be via a linker unit.

Preferably the fluorophore is selected from a xanthene dye or a cyanine dye. More preferably a cyanine dye selected from the group consisting of carbocyanines, thiacyanines, oxacyanines and azacyanines. Cyanine dyes suitable for use in the present invention are disclosed in US5268468 and US 5627027. Dyes with the trade names Cy3, Cy 3B, Cy 3.5.5, Cy 5, Cy 5.5, Cy 7 and Cy 7.5 are included (Amersham, ge healthcare).

Preferred quenching units are non-fluorescent chromophores including 2, 4-dinitrophenyl, 4- (4-dimethylaminophenyl) azobenzoic acid (DABCYL), 7-methoxycoumarin-4-yl) acetyl and non-fluorescent cyanine dyes as disclosed in WO 9964519.

In preferred embodiments, the quencher does not emit significantly, more preferably the quencher is a non-fluorescent chromophore. In this embodiment, the imaging agent contains a fluorophore and a non-fluorescent (dark) acceptor chromophore.

More preferred are probes of formula (I) based on a tetrapeptide backbone (table 1, compound 2) carrying a QSY 21-quencher at the N-terminus and a CY 5.5 fluorophore at the C-terminus (scheme 2):

(scheme 2).

Another preferred embodiment comprises the same backbone, carrying a dark quencher BHQ 3 at the N-terminus and a Cy 7 fluorophore at the C-terminus (scheme 3):

(scheme 3).

In a preferred embodiment, fluorescein and tetramethylrhodamine are selected as the interacting FRET pair, and the tetramethylrhodamine is located on the N-terminal side of the backbone, while fluorescein is attached to the C-terminus, as shown in (scheme 4):

(scheme 4).

In another preferred embodiment, one member of the interacting FRET pair contains a nanoparticle. More preferred of the invention are CdSe nanoparticles (e.g., Quantum dots), lanthanide ion-doped oxide nanoparticles (e.g., Y)_0.6Eu_0.4VO₄) And iron oxide nanoparticles (e.g., aminospack 680 and aminospack 750 available from VisEn Medical, inc., MA 01801, USA). If such a nanoparticle is to act as a donor in a FRET pair, the excitation wavelength is much shorter than the acceptor absorption wavelength, so that direct acceptor excitation can be minimized. Moreover, the emission wavelength range of the donor is narrow and does not overlap with the emission band of the acceptor. Furthermore, such nanoparticles have proven to be optically more stable than organic dyes that can undergo rapid photobleaching. Activated quantum dots for chemical conjugation are commercially available (Invitrogen, CA92008, USA) and the emission wavelength can be selected from a variety of products.

Schemes 5 and 6 show quantum dot based probes of formula (I) specific for caspase-1. Thus, in further preferred probes of formula (I), quantum dots (e.g. QD605 supplied by Invitrogen, CA92008, USA) may be attached to the N-terminus of the caspase-1 probe (scheme 5) or to the C-terminus of caspase-1 (scheme 6) via a suitable linking group.

(scheme 5);

(scheme 6).

Quantum dots are represented by black circles and suitable acceptor molecules are represented by cyanine dye CY 7.

In another preferred embodiment, the quantum dots in the probe of formula (I) are linked to the gold nanoparticles by proteolytically cleavable subunits (scheme 7):

(scheme 7).

The quantum dots and gold nanoparticles are represented by black circles.

Gold nanoparticles (aunps) have been shown to be efficient quenching groups for organic fluorescent dyes and quantum dots. The combined use of quantum dots and aunps is disclosed in, for example, WO 2006126570.

In another preferred embodiment, the probe of formula (I) consists of a multiple FRET system, wherein two specific protease probes are covalently linked together (scheme 8):

(scheme 8).

In this configuration, excitation at a single wavelength and the use of different emission ratios as unique FRET signals is possible (K.E Sapsford et al, Angew. chem. int. Ed.2006, 45, 4562-. The probe combines two unique special structures in one molecule, namely a framework of caspase-1 and a framework of caspase-3.

In another preferred embodiment, the probe of formula (I) is designed to have a long cycle time, a high tumor accumulation and contains a quenched fluorescent label that fluoresces in the near infrared spectrum upon enzyme activation. These probes are based on synthetic graft copolymers [ poly-L-lysine partially modified with methoxypoly (ethylene glycol) ] in which multiple NIR fluorophores are attached to free polylysine residues. The fluorescence of these macromolecules is greatly attenuated due to internal quenching caused by the high density and close proximity of the NIR chromophore.

For example, scheme 9 shows a polymer-based caspase-1 probe, where the attachment of a to the polylysine backbone of D-and/or L-lysine is done through a C-terminal linking group, while the NIR chromophore Cy 5.5 is attached through an N-terminal linking group:

(scheme 9).

The opposite case is given in scheme 10, where the attachment of a to the poly-amino acid backbone of D-and/or L-lysine is done via an N-terminal linking group, and the NIR chromophore Cy 5.5 is attached via a C-terminal linking group:

(scheme 10).

In another preferred embodiment, the probe of formula (I) is designed with the goal of being used in an in-phase enzyme-linked luminescence assay. The following scheme gives a general view of the mechanism of action described above. Luciferin is a substrate for luciferase, and the second enzymatic reaction generates a light signal:

the following scheme shows the mechanism of action described above, where fluorescein is pyridazinodiazepineDerivative masking which releases by proteolytic activity of the caspase-1:

the invention also relates to a method for designing molecular probes for in vitro analysis, for observing the catalytic activity of a single proteolytic enzyme or of a group of proteolytic enzymes in a cell or a multicellular organism, characterized in that an inhibitor of a single proteolytic enzyme or of a group of proteolytic enzymes is converted into a selective imaging probe for said single proteolytic enzyme or group of proteolytic enzymes, preferably caspases. To this end, we replaced the electrophilic groups in certain known caspase inhibitors with a cleavable amide bond. Preferably, the compounds are synthesized in such a way that the enzymatic activity (e.g., proteolytic activity) of a particular target produces a detectable signal. In particular, preferred probes contain an internally quenched fluorophore (e.g., an appropriate FRET pair) linked to (i) the specificity determining group A at the N-terminus of the scissile bond and (ii) the C-terminus of the scissile bond. The present invention can transform the desired and previously optimized properties of known inhibitors into new activity-based probes.

The caspase inhibitors described in the prior art use electrophilic warheads at position P1. The activity-based probes of the invention utilize the known backbone and are modified in two places, first by conversion of the electrophilic warhead to a cleavable amide group, and second by placement of an interactive label pair or property modifier on both sides of the cleavable amide group.

In vitro, the reaction of the protease with the substrate of the invention can generally be carried out in a cell extract or with a purified or enriched form of the protease. For in vivo applications, the emission wavelength of the reporter group is preferably in the Near Infrared (NIR) band, since this band is not interfered with by bioluminescence. Known cyanine NIR dyes meeting these requirements are preferably incorporated into the substrates of the present invention.

The molecular architecture of the compounds of formula (I) consists of a central skeleton a bearing an amide function and two subunits L1R1 and L2R 2. As shown in formula (I), L2R2 is always linked to backbone a through an amide bond, since the amide bond can be cleaved by caspase enzymes. Those skilled in the art can select suitable functional groups for linking the subunit L1R1 to the backbone a, some examples of which are given below. The specific functional group L' in the precursor compound may be located on the backbone A so as to link the appropriate L1R1 subunit to form the L group within the compound of formula (I), which is limited only by the requirements of the synthetic strategy and the end use of such a substrate as an activity-based imaging agent. The choice of these groups will depend on the particular reagents chosen to construct the desired substrate. Examples of the functional group L' connecting A to the subunit L1R1 on the skeleton A include fluorine, chlorine, bromine, cyano group, nitro group, amino group, azide group, alkylcarbonylamino group, carboxyl group, carbamoyl group, alkoxycarbonyl group, aryloxycarbonyl group, aldehyde group, hydroxyl group, alkoxy group, aryloxy group, alkylcarbonyloxy group, arylcarbonyloxy group, carbon-carbon double bond, carbon-carbon triple bond and the like. Most preferred examples include amino, azido, hydroxyl, cyano, carboxyl, carbamoyl, aldehyde, carbon-carbon double or triple bonds. Therefore, L is preferably a bond or is selected from- (NRx) -, -O-, -C ═ N-, -C (═ O) -NH-, -NH-C (═ O) -, -C (═ O) H, -CRx ═ CRy-, -C ≡ C-and phenyl, where Rx and Ry are independently H or (C ≡ O) C-, and phenyl₁-C₆) An alkyl group.

In particular, a preferred method of synthesis of the compounds of formula (I) utilizes orthogonally protected functional groups. This choice of protecting groups allows for independent deprotection, whereby each released functional group can then be further chemically treated to attach the corresponding subunit to backbone a. Suitable protecting Groups can be selected by those skilled in the art for the envisaged functional Groups, for a general overview of protecting Groups see, for example, T.W.Greene and P.G.M.Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, New York 1991.

The compounds of formula L' -a-CO-OH (backbone) can be prepared according to standard methods in the art, for example international patent application US 5670494; WO 9526958; WO 9722619; WO 9816504; WO 0032620; WO 0055127; WO 0105772; WO 0190063; WO 03024955; WO 03106460; WO 03104231; WO 03103677; harter, bioorg, med, chem, lett, 2004, 14, 809-812; shahripour et al, bioorg.Med.chem.Lett.2001, 11, 2779-; shahripour et al, bioorg.med.chem.2002, 10, 31-40; M.C. Laufersweeler et al, bioorg.Med.chem.Lett.2005, 15, 4322-4326; chapman, bioorg, med, chem, lett, 1992, 2, 613-; dolle et al, J.Med.chem.1997, 40, 1941-1946; soper et al, bioorg.Med.chem.Lett.2006, 16, 4233-; soper et al, bioorg.Med.chem.2006, 14, 7880-; D.J.Lauffer et al, bioorg.Med.chem.Lett.2002, 12, 1225-; C.D.Ellis et al, bioorg.Med.chem.Lett.2006, 16, 4728-; tawa et al, Cell Death and Differentiation 2004, 11, 439-; micale et al, J.Med.chem.2004, 47, 6455-6458; berger et al, Molecular Cell, 2006, 23, 509-; and Garcia-Calvo, J.biol.chem.1998, 273(49), 32608-32613.

The invention also relates to a process for the preparation of compounds of formula (I), characterized in that, if n is 1:

(a) reacting a compound of formula (II)

L’-A-CO-OH (II)

Wherein A has the meaning defined above in general and in preferred meanings, L' is fluorine, chlorine, bromine, cyano, nitro, amino, azido, alkylcarbonylamino, carboxyl, carbamoyl, alkoxycarbonyl, aryloxycarbonyl, aldehyde, hydroxyl, alkoxy, aryloxy, alkylcarbonyloxy, arylcarbonyloxy, a carbon-carbon double bond, a carbon-carbon triple bond, preferably amino, azido, hydroxyl, cyano, carboxyl, carbamoyl, aldehyde, a carbon-carbon double bond or a carbon-carbon triple bond, more preferably amino,

with a compound of the formula L1-R1-H, wherein L1 has the definitions in the general and preferred meanings indicated above, under conditions known to those skilled in the art to give a compound of the formula (III),

L1-R1-L-A-CO-OH (III)

(b) reacting a compound of formula (III) with a compound H₂N-R2-L2 to give the compound of formula (I).

Optionally, the synthesis of compounds of formula (I) utilizes orthogonally protected functional groups. This choice of protecting groups allows for independent deprotection, whereby each released functional group can subsequently be further chemically treated to attach a label group or to introduce further extension groups on the linker groups R1 and/or R2. Suitable protecting Groups can be selected by those skilled in the art for the envisaged functional Groups, for a general description of protecting Groups see, for example, T.W.Greene and P.G.M.Wuts in "Protective Groups in organic Synthesis", John Wiley & Sons, New York 1991.

In another method for preparing the probe of formula (I), n is 1, which comprises

(a1) Reacting a compound of formula (II) with a compound of formula (IV) under conditions known to those skilled in the art

H₂N-L2-PG2 (IV)

To produce a compound of formula (V)

L’-A-CO-NH-R2-PG2 (V)

(b) Then reacting the compound of formula (V) with a compound of formula (VI)

PG1-R1-L” (VI)

Under the conditions required for each group known to those skilled in the art to form the compound

PG1-R1-L-A-CO-NH-R2-PG2 (VI)

Wherein PG1 and PG2 are independently of each other a protecting group, preferably an orthogonal protecting group, L 'is a linking group for L', or a bond, which needs to be selected by a person skilled in the art

(c1) Cleaving the PG2 group of compound (VI) and reacting the resulting compound with a labelling group L2, then cleaving the protecting group PG1 and reacting the resulting compound with a labelling group L1 to give a compound of formula (I), or

(c2) The PG1 group of compound (VI) is cleaved and the resulting compound is reacted with a labeling group L1, followed by cleavage of the protecting group PG2 and reaction of the resulting compound with a labeling group L2 to give the compound of formula (I).

In step (b), preferred combinations of L 'and L' and reaction types (in parentheses) are as follows:

when L 'is fluorine, chlorine, bromine, iodine, L' is amino (R-NH)₂) Hydroxyl (R-OH), triple bond (Sonogashira reaction), double bond (Heck reaction), alkyl borane (Suzuki reaction);

when L 'is cyano, L' is amino (R-NH)₂) Hydroxyl (R-OH), mercapto (R-SH);

when L' is amino, L "is an activated carboxylic acid (NHS ester.), aldehyde, fluorine, chlorine, bromine, iodine;

when L 'is azide, L' is a triple bond, phosphine group (Staudinger ligation);

when L' is carboxy, L "is amino, hydroxy, hydrazide;

when L' is alkoxycarbonyl, L "is amino, hydroxy, hydrazide;

when L' is aryloxycarbonyl, L "is amino, hydroxy, hydrazide;

when L' is hydroxy, L "is fluoro, chloro, bromo, iodo, hydroxy (Mitsunobu reaction), carboxy;

when L' is an aldehyde, L "is amino, hydrazine;

when L' is a carbon-carbon double bond, L "is bromine, chlorine, iodine (Heck reaction), alkylborane (Suzuki reaction);

when L 'is a carbon-carbon triple bond, L' is bromine, chlorine, iodine (Sonogashira reaction), azide.

Compounds of formula (I) wherein n is 0 can be prepared by reacting a compound of formula A-CO-OH (IV) with a compound H₂N-R2-L2 to prepare the probe of formula (I).

Cysteine protease substrates functionalized with different labeling groups are preferably synthesized on a solid support.

For the synthesis of caspase probes of formula (I) with peptidomimetic structures, non-peptide building blocks can be used in solid phase synthesis. The synthesis of the building blocks is further described in example 8.

Construction of the basic element (VII) it is preferred to use for the synthesis of caspase-1 probes, such as the compounds of examples 1 and 2,

the probe of the present invention is preferably a caspase-1, caspase-3 or caspase-8 probe.

The probes of the invention are useful for molecular imaging, including screening and whole animal imaging, in vitro, in cell culture assays, ex vivo assays, or in the context of living organisms (in vivo). Most preferred are imaging applications such as optical imaging and Magnetic Resonance Imaging (MRI).

The probes of the invention are intended for use in diagnostic imaging of protease activity. Most preferably, there is provided the use of a method of monitoring the effect of a drug or drug-like substance on a target protease. Administration of such drugs or drug-like substances should have a measurable effect on the signal emitted by the probes of the invention.

Another most preferred aspect of the probe of the invention is its use as an imaging agent for guiding surgery and monitoring the efficacy of drug therapy. Surgical guidance includes detection of tumor margins and detection of tumor metastasis progression.

Accordingly, another aspect of the invention is a method of imaging a living organism comprising:

(a) administering to the organism a probe of formula (I),

(b) exposing the organism to electromagnetic radiation that excites the non-quenched fluorophore to produce a detectable signal, and

(c) the signal is detected, thereby creating an image.

Alternatively, a method of imaging a living organism comprises:

(a) administering to the organism a probe of formula (I),

(b) exposing the organism to electromagnetic radiation that excites the fluorophore to produce a detectable signal, and

(c) the signal is detected, thereby creating an image.

The "living organism" may be any living cell or whole organism containing the cysteine protease to be detected, preferably the living organism is a mammal, such as a mouse or rat.

The probes of the invention have a high selectivity, so that the risk of false positives can be avoided.

Abbreviations:

DMF ═ dimethylformamide

DMSO ═ dimethyl sulfoxide

DCM ═ dichloromethane

equiv

sat. (saturated)

THF ═ tetrahydrofuran

DIPEA ═ diisopropylethylamine

HOAt ═ 1-hydroxy-7-azabenzotriazole

HATU ═ O- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate

NHS ═ N-hydroxysuccinimide ester

General procedure for solid phase peptide synthesis:

the following probes were synthesized according to standard solid phase peptide synthesis methods. 2-chlorotrityl resin was used as a solid support. When the resin was loaded, 2 equivalents of the Fmoc-protected amino acid and 3 equivalents of DIPEA were dissolved in DCM and the reaction mixture was added to the resin (loading 1.4 mmol/g). The reaction mixture was shaken at room temperature overnight. The resin was washed with DCM and DMF. For Fmoc deprotection, the resin was treated twice with 30% piperidine/DMF for 15 min. For solid phase peptide synthesis, standard protocols were used: 4 equivalents of Fmoc-protected amino acid, 4 equivalents of HATU, 4 equivalents of HOAt and 8 equivalents of DIPEA were dissolved in a DCM/DMF (1/1) mixture. The reaction mixture was stirred at room temperature for 20 minutes and then added to the resin. The reaction mixture was shaken for 2 hours, longer if the Fmoc-protected amino acid was sterically hindered. When cleavage from the solid phase was performed, the resin was treated twice with 5% TFA in DCM for 15 min. The solvent and toluene were co-distilled off under reduced pressure and the end product was purified by preparative HPLC (gradient: H)₂O + 0.05% TFA; 5 to 95% CH₃CN)。

Example 1: caspase-1 probes

According to aThe compounds were prepared on solid supports and purified by HPLC (H)₂O+0.05％TFA；4-95％CH₃CN). Calculated values: [ M + H ]]⁺1569.70, found: [ M + H ]]⁺1569.45. Yield: 54 percent.

Example 2: caspase-1 probes

The compound was prepared according to the general procedure on a solid support and purified by HPLC (H)₂O+0.05％TFA；4-95％CH₃CN) calculated value: [ M + H ]]⁺1583.73 found: [ M + H ]]⁺1583.2. Yield: 72 percent.

Example 3: caspase-1 probes

The compound was prepared according to the general procedure on a solid support and purified by HPLC (H)₂O+0.05％TFA；4-95％CH₃CN) calculated value: [ M + H ]]⁺1591.19 found: [ M + H ]]⁺1591.50. Yield: 66 percent.

Example 4: caspase-3 probes

The compound was prepared according to the general procedure on a solid support and purified by HPLC (H)₂O+0.05％TFA；4-95％CH₃CN). Calculated values: [ M +H]⁺1517.66 found: [ M + H ]]⁺1517.55. Yield: 59 percent.

Example 5: caspase-3 probes

The compound was prepared according to the general procedure on a solid support and purified by HPLC (H)₂O+0.05％TFA；4-95％CH₃CN) calculated value: [ M + H ]]⁺1546.79 found: [ M + H ]]⁺1546.35. Yield: 61 percent.

Example 6: caspase-8 probes

The compound was prepared according to the general procedure on a solid support and purified by HPLC (H)₂O+0.05％TFA；4-95％CH₃CN) calculated value: [ M + H ]]⁺1523.45 found: [ M + H ]]⁺1523.25. Yield: and 55 percent.

Example 7

The building block (VII) was prepared according to the general method described in WO 9722619.

Example 8: caspase-1 bioluminescent probes

The compound was prepared on a solid support according to the general procedure starting from 6-Fmoc-amino-D-fluorescein and purified by HPLC (H)₂O+0.05％TFA；4-95％CH₃CN). Calculated values: [ M + H ]]⁺772.82, found: [ M + H ]]⁺773.15. Yield: 13 percent.

Claims (18)

1. Molecular probes against cysteine proteases of formula (I)

{L1-R1-L}_n-A-CO-NH-R2-L2 (I)

Wherein

A is a group recognizable by caspase;

r1 is a linking group;

r2 is a bond or a linking group;

l is a bond or a group capable of allowing the L1 group to bind easily;

l1 and L2 are independently of each other at least one labeling group optionally attached to a solid support; and is

n is 1;

or

R2 is a bond;

l2 is a substrate suitable for coupled bioluminescent assays; and is

n is 0.

2. The probe of claim 1, wherein the caspase is caspase-1, caspase-3, or caspase-8.

3. The probe of any one of claims 1 or 2, wherein L is a bond or is selected from the group consisting of- (NRx) -, -O-, -C ═ N-, -C (═ O) -NH-, -NH-C (═ O) -, -C (═ O) H, -CRx ═ CRy-, -C ≡ C-and phenyl, where Rx and Ry are independently H or (C ≡ O) C-, and phenyl₁-C₆) An alkyl group.

4. The probe of any one of claims 1 to 3, wherein R1 or R2 is a linear or branched alkylene group containing 1 to 300 carbon atoms, wherein optionally,

(a) one or more carbon atoms are replaced by oxygen, particularly wherein every third carbon atom is replaced by oxygen, for example a polyoxyethylene group containing from 1 to 100 oxyethylene units; and/or

(b) One or more carbon atoms are replaced by nitrogen bearing a hydrogen atom and the adjacent carbon atom is replaced by an oxo group, forming an amide function-NH-CO-; and/or

(c) One or more carbon atoms are replaced by an ester function-O-CO-;

(d) the bond between two adjacent carbon atoms is a double or triple bond; and/or

(e) Two adjacent carbon atoms are replaced by disulfide bonds.

5. The probe according to any one of claims 1 to 4, wherein the labeling groups L1 and L2 independently of each other are spectroscopic probes such as fluorophores; a quencher or chromophore; a magnetic probe; a contrast agent; a molecule that is part of a specific binding pair that specifically binds to a ligand; a molecule covalently attached to a solid support, wherein the support can be a glass slide, a microtiter plate, or any polymer known to those skilled in the art; biomolecules with desirable enzymatic, chemical or physical properties; or a molecule having a combination of any of the above properties; or a positively charged linear or branched polymer.

6. The probe of claim 5, wherein the labeling groups L1 and L2 are independently from each other linked to a positively charged linear or branched polymer.

7. The probe of claim 6, wherein one of the labeling groups L1 and L2 is a linear poly (arginine) of D-and/or L-arginine containing 6-15 arginine residues.

8. The probe of any one of claims 5 to 7, wherein L1 is one member of two interacting spectroscopic probes L1/L2 and L2 is the other member thereof.

9. The probe of claim 8, wherein L1/L2 is a FRET pair.

10. The probe of claim 9, wherein one L1/L2 is a fluorophore selected from the group consisting of Alexa350, dimethylaminocoumarin, 5/6-carboxyfluorescein, Alexa 488, ATTO 488, DY-505, 5/6-carboxyfluorescein, Alexa 488, Alexa 532, Alexa 546, Alexa 555, ATTO 488, ATTO532, tetramethylrhodamine, Cy3, DY-505, DY-547, Alexa 635, Alexa 647, ATTO 600, ATTO 655, DY-632, Cy 5, DY-647Cy 5.5, and the other L1/L2 marker group is a quencher selected from the group consisting of Dabsyl, Dabcyl, BHQ 1, QSY 35, BHQ2, QSY 9, ATTO 540Q, BHQ 3, ATTO 612Q, QSY 21.

11. A probe according to any of claims 1 to 4 wherein n is 0, R2 is a bond and L2 is a substrate suitable for coupled bioluminescent assays, characterised in that a modified aminoluciferin or a carboxy-terminally protected derivative thereof is used as a reporter group which, when cleaved from the central scaffold A, is converted by luciferase to generate a luminescent signal.

12. The selective caspase-1 probe according to any one of claims 1 to 11, characterized by compounds 1-28 of the following table:

13. the selective caspase-3 probe of any one of claims 1 to 11, characterized by compounds 29-42 of the following table:

14. the selective caspase-8 probe according to any one of claims 1 to 11, characterized by compounds 43-44 of the following table:

15. method for preparing probes of formula (I) according to claims 1 to 14, characterized in that if n is 1:

(a) reacting a compound of formula (II)

L’-A-CO-OH (II)

With a compound of formula L1-R1-H to give a compound of formula (III),

L1-R1-L-A-CO-OH (III)

(b) reacting a compound of formula (III) with a compound H₂N-R2-L2 to give a probe of formula (I) wherein L' is fluorine, chlorine, bromine, cyano, nitro, amino, azide, alkylcarbonylamino, carboxyl, carbamoyl, alkoxycarbonyl, aryloxycarbonyl, aldehyde, hydroxyl, alkoxy, aryloxy, alkylcarbonyloxy, arylcarbonyloxy, a carbon-carbon double bond, a carbon-carbon triple bond, preferably amino, azide, hydroxyl, cyano, carboxyl, carbamoyl, aldehyde, a carbon-carbon double bond or a carbon-carbon triple bond, more preferably amino, and

r1 and/or R2 may be protected by suitable orthogonal protecting groups and cleaved sequentially during the preparation of the compounds of formula (I); and is

If n is 0:

reacting a compound of the formula A-CO-OH (IV) with a compound H₂N-R2-L2 to prepare the probe of formula (I).

16. Use of a probe of formula (I) according to claims 1 to 14 for molecular imaging in vitro, in cell culture experiments, ex vivo experiments or in vivo organisms.

17. Use of a probe of formula (I) according to claims 1 to 14 for molecular imaging of living organisms, comprising:

(a) administering to the organism a probe of formula (I),

(b) exposing the organism to electromagnetic radiation that excites the non-quenched fluorophore to produce a detectable signal, and

(c) the signal is detected, thereby creating an image.

18. Use of a probe of formula (I) according to claims 1 to 14 for molecular imaging of living organisms, comprising:

(a) administering to the organism a probe of formula (I),

(b) exposing the organism to electromagnetic radiation that excites the fluorophore to produce a detectable signal, and

(c) the signal is detected, thereby creating an image.