MXPA00003443A - Novel fluorescent reporter molecules and their applications including assays for caspases - Google Patents

Abstract

The present invention relates to novel fluorescent dyes, novel fluorogenic and fluorescent reporter molecules and new enzyme assay processes that can be used to detect the activity of caspases and other enzymes involved in apoptosis in whole cells, cell lines and tissue samples derived from any living organism or organ. The reporter molecules and assay processes can be used in drug screening procedures to identify compounds which act as inhibitors or inducers of the caspase cascade in whole cells or tissues. The reagents and assays described herein are also useful for determining the chemosensitivity of human cancer cells to treatment with chemotherapeutic drugs. The present invention also relates to novel fluorogenic and fluorescent reporter molecules and new enzyme assay processes that can be usedto detect the activity of type 2 methionine aminopeptidase, dipeptidyl peptidase IV, calpain, aminopeptidase, HIV protease, adenovirus protease, HSV-1 protease, HCMV protease and HCV protease.

Description

NEW FLUORESCENT REPORTER MOLECULES AND APPLICATIONS INCLUDING CASSETTE TESTS FIELD OF THE INVENTION This invention is in the field of intracellular enzyme detection using fluorogenic or fluorescent tests. The invention relates to new fluorescent dyes and the application of these dyes for the preparation of new amino acid derivatives or fluorescent or fluorogenic peptides which are substrates of proteases and peptidases. In particular, the invention relates to new fluorescent or fluorogenic peptide derivatives which are substrates of enzymes involved in apoptosis, such as caspases and Granzyme B serine protease derived from lymphocytes. The invention also relates to a process for measuring the activity of caspases and other enzymes involved in apoptosis in whole cells dead or alive, tissue samples or cell lines derived from any healthy, diseased, cancerous or infected organ or tissue. The invention also relates to the use of fluorogenic or fluorescent substrates in a new assay system to discover or detect inhibitors or inducers REF; 33113 of apoptosis in collections of compounds or genetics of compounds. In addition, the invention relates to the use of fluorogenic or fluorescent substrates to determine the sensitivity of cancer cells to treatment with chemotherapeutic drugs. The invention also relates to novel fluorescent or fluorogenic peptides which are substrates of exopeptidases such as aminopeptidase A and N, methionine aminopeptidase and dipeptidyl peptidase IV, endopeptidases such as calpain, proteases such as HIV proteases, HCMV protease , HSV protease, HCV protease and adenovirus protease.

BACKGROUND OF THE INVENTION Undesirable cells eliminated from organisms by a process variously known as regulated cell death, programmed cell death or apoptosis. Such cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26: 59-86 (1951); Glucksmann, A., Archives from Biologie 76: 419-437 (1965); Ellis et al., Dev. 112: 591-603 (1991); Vaux et al., Cell 16: 111-119 (1994)). The apoptosis regulates the cell number, facilitates morphogenesis, removes harmful or otherwise abnormal cells and eliminates cells that have already performed their function. Additionally, apoptosis occurs in response to various physiological stresses, such as hypoxia or ischemia (published PCT application O96 / 20721). There are a number of morphological changes distributed by cells experiencing regulated cell death, including blistering of the nuclear membrane and plasma, cell shrinkage (condensation of nucleoplasm and cytoplasm), compaction and relocation organelle, chromatin condensation and production of bodies apoptotic (particles enclosed with membrane containing intracellular material) (Orrenius, S., J. Internal Medicine 237: 529-536 (1995)). Apoptosis was achieved through an endogenous mechanism of cellular suicide (Yllie, A. H., in Cell Dea in Biology and Paology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activates its internally encoded suicide program as a result of internal or external signals. The suicide program is executed through the activation of a carefully regulated genetic program (Wylie et al., Int, Rev. Cyt. 68: 251 (1980); Ellis et al., Ann, Rev. Cell Bio., 7: 663 (1991)). The bodies and apoptotic cells are usually recognized and removed by the approach of cells or macrophages before lysis. Because of this clearance or clearance mechanism, inflammation is not induced despite the removal or clearance of large numbers of cells (Orrenius, S., J. Internal Medicine 237: 529-536 (1995)). Interleukin-LSS (IL-LSS) mammalian plays an important role in various pathologic processes, including chronic and acute inflammation and autoimmune diseases (Oppenheim, J. H. et al. Immunology Today, 7, 45-56 (1986)). IL-1β is synthesized as a cell-associated precursor polypeptide (pro-IL-1β) that is unable to bind IL-1 receptors and is biologically inactive (Mosley et al., J. Biol. Chem. 262: 2941-2944 (1987)).

By inhibiting the conversion of the IL-1β precursor to mature IL-1β, the activity of interleukin-1 can be inhibited. IL-1 is also a cytokine involved in mediating a wide range of biological responses including inflammation, septic shock, wound healing, hematopoiesis, and the growth of certain leukemias (Dinarello, CA, Blood 77: 1627-1652 (1991); DiGiovine et al. ., Immunology Today 11: 13 (1990)). The enzyme that converts interleukin-lβ (for its acronym in English, ICE) is a protease responsible for the activation of interleukin-lβ (IL-lß) (Thornberry, NA, et al., Na ture 356: 768 (1992); Yuan, J., et al., Cell 75: 641 (1993)). ICE . * _ »» **, is a substrate-specific cysteine protease that cleaves inactive prointerleucine-1 to produce mature IL-1. The genes coding for ICE and CPP32 are elements of the mammalian ICE / Ced-3 gene family, which presently include at least twelve elements: ICE, CPP32 / Yama / Apopain, mICE2, ICE4, ICH1, TX / 1CH- 2, MCH2, MCH3, MCH4, FLICE / MACH / MCH5, ICE-LAP6 and ICErelIII. The proteolytic activity of this family of cysteine proteases, whose active site cysteine residue is essential for apoptosis mediated by ICE, seems critical in the mediation of cell death (Miura et al., Cell 75: 653-660 (1993) )). This gene family has recently been named caspases (Alnernri, E. S. et al., Cell 87: 111 (1996)). A death activator, such as Tumor Necrosis Factor, FAS ligand, oxygen or nutrient deprivation, viruses, toxins, anticancer drugs, etc., can activate caspases within cells in a manner similar to cascade where caspases that are upstream in the cascade (for example FLICE / MACH / MCH5) can activate caspases further downstream in the cascade (for example CPP-32 / Yama / Apopain). Activation of the caspase cascade leads to cell death. A wealth of scientific evidence suggests that, in many diseases, caspase cascade is activated 'Jfa_ÉJfeü - itf is ¿^ í ^ when it should not be. This leads to excessive excessive cell suicide and organ failure. Diseases that involve the inappropriate activation of the caspase cascade and subsequent cellular suicide include myocardial infarction, congestive heart failure, autoimmune diseases, AIDS, viral infections, kidney failure, liver failure, rheumatoid arthritis, ischemic attack, diseases neurodegenerative, atherosclerosis, etc. Therefore, the discovery of new drugs that can block or inhibit the caspase cascade activation could have a wide impact on the variation in the treatment of degenerative diseases of more, if not all, of the organ systems of the human body. Caspases are also thought to be crucial in the development and treatment of cancer. There is mounting evidence that cancer cells, while containing caspases, lack parts of the molecular machinery that activates the caspase cascade (Los et al., Blood Vol. 90. No. 8: 3118-3129 (1997)). This causes the cancer cells to lose their capacity to suffer cell suicide and the cells become immortal-they themselves become cancerous. It has been shown that chemotherapy drugs (anticancer) can activate cancer cells to suffer suicide by reactivating the inactive caspase cascade. This may be a crucial aspect of the mode of action of more, if not all, known anti-cancer drugs (Los et al., Blood, Vol. 90. No 8: 3118-3129 (1997): Friesen et al., Na Med 2: 574 (1996) .Chemotherapeutic drugs may differ in their ability to activate the caspase system in different classes of cancers.In addition, it is likely that anticancer drugs differ in their ability to activate the caspase cascade in a given cancer (for example, lung cancer) and in different patients. In other words, there are differences from one patient to another in the chemosensitivity of, for example, lung cancer cells, to several anticancer drugs. In summary, excessive activation of the caspase cascade plays a crucial role in a wide variety of degenerative organ diseases, whereas a caspase system that does not work is a hallmark of cancer cells. New drugs that inhibit or stimulate caspase cascade are appropriate to revolutionize the treatment of numerous diseases in humans ranging from infections, cardiovascular, endocrine, kidney, liver and brain diseases to diseases of the immune system and cancer.

To find drugs that inhibit or stimulate the caspase cascade, it is necessary to develop high performance caspase activation assays (HTCA). These HTCA assays should be able to verify the activation or inhibition of the caspase cascade within living or complete cells. Ideally, HTCA assays should be versatile enough to measure the activity of the caspase cascade within any living or complete cell, without matter that its origin should be: Cancer cells, tumorigenic cells, immune cells, brain cells, cells endocrine system, cells or cell lines of different organ systems, biopsy samples, etc. In addition, such HTCA assays should be able to measure -in living or complete cells- the activation or inhibition of any of the caspase enzymes or any other enzymes that are involved in the caspase cascade. Developing such versatile HTCA assays represents a substantial advance in the field of drug selection. The commonly available HTCA assays do not allow internal cell selection for compounds that can activate or inhibit the caspase cascade. There are high-throughput, cell-free, screening assays available that can measure the activity of «™« ™ «individually isolated caspase enzymes, or assays that can measure the activity of caspases in dead cells which have been permeabilized by osmotic attack, for example (Los et al., Blood, Vol. 90. No 8: 3118 -3129 (1997)). But these enzyme assays can not predict the effect of a compound on the caspase cascade in living cells for the following reasons: 1. ) Cell-free assays, or assays using permeabilized, dead cells, can not predict the ability of compounds to penetrate the cell membrane. This is crucial because the caspase cascade resides inside the cells. To be active, a compound must not be able to modulate only the enzyme or enzymes of caspase, but it must also be capable of penetrating the intact cell membrane. Cell-free assays or assays using dead cells, therefore, are unable to determine whether or not a compound will be potentially useful as a drug. 2.) Caspases isolated in free cell assays are highly susceptible to oxidation and compounds that can cause oxidation of enzymes. This property of isolated caspases makes selection trials of cell-free caspase, highly susceptible to artifacts, and has avoided the successful use of these assays for the high-throughput selection of combination chemistry (or other) genetacs. Previous sizing or screening efforts, using cell-free caspase enzyme assays, have led to the discovery of numerous inhibitors which oxidize caspases, but not compounds that could be useful as a potential drug. Similar difficulties have been reported by others. 3.) Numerous cellular receptors, proteins, cellular constituents and cofactors - many of which are still unknown - can influence the caspase cascade in living cells. The cell-free caspase assay or assays using permeabilized dead cells do not take into account these cellular receptors and cofactors. Because of this, it is possible that a compound identified in a caspase assay free of cells or dead cells will not work or function in living cells. On the other hand, a compound that must inhibit or stimulate the caspase cascade indirectly through one of the cell receptors or cofactors might not occur completely in a cell-free or cell-free caspase assay.

It is highly probable that the caspase cascade functions differently in cells derived from different organs.There is growing evidence that the receptors and cofactors that influence the caspase cascade They differ between cell types Using cell-free or dead cell assays, it could be virtually impossible to identify specific organ modulators or cell types from the caspase cascade.

A potentially important application of an HTCA assay system for measuring intracellular caspase enzymes or any other enzymes involved in apoptosis is tested for chemical sensitivity of human cancers. It is known that there is a genetic difference in the susceptibility of human cancers to the anti-cancer drugs commonly sold. For example, lung cancer cells in a patient must be sensitive to Drug A, while another cancer of the patient's lung must be insensitive to Drug A, but sensitive to Drug B. This pharmacogenetic difference in the chemosensitivity of cancer cells in different individuals, it is a well-known phenomenon.

In the past, attempts have been made to determine the sensitivity to cancer cell chemicals taken from individual patients prior to the designation of a treatment regimen with one or more anticancer drugs sold. However, the chemical sensitivity test has not found widespread use, because the procedures involved have some inherent technical difficulties. The test is a long time consumer (six or more days per screen or selection) and requires the culture of the cells prior to selection. Cell culture leads to the clonal selection of cells and the cultured cells then are not too representative of cancer in the patient. An HTCA assay system for rapidly measuring intracellular caspase activity could be used to very quickly determine the chemical sensitivity profile of recently excised cancer cells. If the assay has high performance, it may be desirable to test the sensitivity to chemicals from multiple samples taken from the same patient, for example, from different metastases. This information could then be used to designate a treatment regimen using combinations of anticancer drugs sold for which the cells show much greater sensitivity.

It is clear that there is a need for HTCA assays and reagents for such assays that can be employed in cell discovery or diagnostic procedures to rapidly detect and measure the activity of the compounds that activate or inhibit the caspase cascade or other enzymes involved. in apoptosis inside whole cells alive or dead. A reagent for this type of test or cell test should ideally meet the conditions The following: a) should be a very large difference in fluorescent signal between the peptide-reporter molecule and reporter molecule after the amide bond in the peptide-reporter is cleaved by the caspases or other enzymes involved in apoptosis, preferably the The reporter-peptide molecule should not be fluorescent and more preferably the reporter-peptide molecule should not be fluorescent and discolored; b) the peptide-reporter molecule must be a permeable cell, so it must be of minimum numbers of hydrophilic groups in The molecule and the size of the molecule should preferably be smaller; c) the peptide-reporter molecule should preferably not be expanded outside the cell once it permeates the cell membrane; d) the reporter molecule should not expand preferably outside the cell once it is released from the peptide. The method of selecting inducers or inhibitors of apoptosis in whole cells against assay of cell-free enzymes can also be used for the selection of inhibitors of enzymes other than caspases. Traditionally, enzyme inhibitors were first identified by cell-free enzyme assays. The cell cultures were then used by the secondary assay to assess the activity of the active compounds in intact cells. A fluorescent or fluorogenic substrate permeable to cells will make it possible to select inhibitors of proteases and peptidases and other enzymes directly in whole living cells. There are several advantages in whole-cell assays against cell-free enzyme assay. One of the advantages is that in whole cell assays, the inhibitor will have to penetrate cells to be detected. Since many proteases in living cells are regulated by other proteins, receptors or genes, selection using live cells will allow the identification of small molecule compounds which interfere with cellular proteases binding to the active site, as well as compounds that modulate the Protease function by interference with transcription, translation, biosynthesis, assembly of subunits, cellular cofactors or mechanisms of signal translation (or virus entry into host cells, in the case of viral proteases). In addition, since there is an abundance of aminopeptidases in the cells, these aminopeptidases can be used in the design of fluorogenic or fluorescent substrates by whole cell assays that will not otherwise work in assays or tests of cell-free enzymes. Therefore, there is a need to develop high-throughput screening trials (for its acronym in English, HTS) and reagents for such assays in whole cells which can be used for drug discovery or diagnostic procedures. AGM-1470 (also known as TNP-470) is an inhibitor of angiogenesis in clinical trials for a variety of cancers. The mechanism of action of AGM-1470 was recently discovered by two independent search groups (Sin, N., et al., Proc.Nat.Acid.Sci.U.S.A. 54: 6099-6103 (1997); Griffith, E.C., et al., Chem. Biol. 4: 461-471 (1997)). They find that AGM-1470 and the like are inhibitors of methionine type 2 aminopeptidase (MetAP-2). The potency for the inhibition of endothelial cell proliferation and the inhibition of methionine aminopeptidase activity was determined by a series of AGM-1470 analogs and a significant correlation was found between the two activities. Since angiogenesis inhibitors are known to be capable of selectively killing cancer cells, a cell screening assay for MetAP-2 inhibitors may result in new anticancer drugs. Therefore, fluorescent or fluorogenic substrates permeable to cells by MetAP-2 can be used by the selection of MetAP-2 inhibitors in endothelial cells that could lead to new anti-cancer agents. Recently, inhibitors of HIV proteases such as ritonavir and viracept have been shown to be very effective in the treatment of patients infected with HIV. These inhibitors were designated based on the substrate structure of HIV proteases. The activities of these inhibitors were first determined against the HIV protease. Active compounds were then tested for the inhibition of HIV infection in cell cultures. A fluorescent or fluorogenic cell-permeable substrate for HIV protease can be used for the selection of HIV protease inhibitors in HIV-infected cells, which could accelerate the process for the discovery of new HIV protease inhibitors and lead to the new and better treatment for HIV infection. Since HIV proteases process viral precursor proteins in a preceding state in viral replicates, a fluorescent or fluorogenic substrate permeable to cells for HIV protease can also be used to select compounds which inhibit the transcription or translation of the gene, viral entry, or other key proteins in the earliest stage of HIV infection. Fluorescent or fluorogenic substrates could also be used for the diagnosis of HIV infection, which should be more sensitive than commonly available methods. Applying the same principle, fluorescent or fluorogenic cell permeable substrates for cathepsin B can be used for the selection of cathepsin B inhibitors. Fluorescent or fluorogenic cell permeable substrates for dipeptidyl peptidase IV can be used for the selection of inhibitors of dipeptidyl peptidase IV. Fluorescent or fluorogenic cell-permeable substrates for renin can be used for the selection of renin inhibitors and fluorescent or fluorogenic cell-permeable substrates for adenovirus proteases or other In addition, viral proteases can be used for the selection of adenovirus protease or other viral protease inhibitors. U.S. Patent Nos. 4,557,862 and 4,640,893 disclose Rhodamine 110 derivatives as fluorogenic substrates for proteinases of the formula: wherein Ri and R2, which are the same or different, are selected from the group consisting of amino acids, amino acid derivatives, blocked amino acids, blocked amino acid derivatives, and peptides. Examples of (AA) 2-Rhodamines and (peptide) 2-Rhodamines are (Z-Arg) 2-Rhodamine 110, (Arg) 2-Rhodamine 110, (Z-Ala-Arg) 2-Rhodamine 110, (Z-GlN) -Arg) 2-Rhodamine 110, (Z-Glu-Arg) 2-Rhodamine 110, (Z-Gly-Arg) 2-Rhodamine 110, (Z-Leu-Arg) 2-Rhodamine 110, (Z-Met-Arg) ) 2-Rhodamine 110, (Z-Phe-Arg) 2-Rhodamine 110, (Z-Pro-Arg) 2-Rhodamine 110, (Z-Trp-Arg) 2-Rhodamine 110, (Z-Val-Arg) 2-Rhodamine 110, (Z-Ile-Pro-Arg) 2-Rhodamine 110.

WO 96/36729 discloses compounds or their salts for testing the activity of an enzyme within a complete, etabolically active cell. This test group is to include a separation group and an indicator group. The separation group is selected from the group consisting of amino acids, peptides, saccharides, sulfates, phosphates, esters, phosphate esters, nucleotides, polynucleotides, nucleic acids, pyrimidines, purines, nucleosides, lipids and mixtures. The indicator group is selected from compounds having a first state when they are bound to the separation group, and a second state when the separation group is cleaved from the indicator group by the enzyme. Preferred indicator compounds are Rhodamina 110, rodol, and fluorescein and analogs of these compounds. The patent application lists many enzymes and enzyme substrates. US Patent 5,576,424 discloses haloalkyl derivatives of reporter molecules used to analyze the metabolic activity in cells of the formula: XR-SPACER-REPORTER-BLOCK where -BLOCK is a group selected to be removable by the action of a specific analyte, to give different spectral reporter properties from those of the substrate; -REPORTERA- is a molecule that, when not bound to the BLOCK by a BLOCK-REPORTER link, has spectral properties different from those of the substrate; -ESPACIADOR- is a covalent bond; and XR- is a haloalkyl portion that can covalently react with an intracellular thiol to form a thioether conjugate. Preferred reporter compounds are those which include Rhodamine-110, rodol, fluorescein and others.

BRIEF DESCRIPTION OF THE INVENTION The invention relates to fluorogenic or fluorescent reporter compounds of Formula I: x-y-z (I! or biologically acceptable salts or pro-parent molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein x and z is the same or different and is a peptide or amino acid or acyl group or other structure such as the compounds of Formula I, is a substrate for caspases, or a substrate for other proteases or peptidases or other enzymes; and wherein the cleavable link is only one or both links xy and yz in Formula I when x is the same as z, or where the cleavable link is only one of the links x and yz in Formula I when x is not the same as z, and it is a fluorogenic or fluorescent portion. Preferred compounds are represented by Formula II: Ri- (AA) n-Asp-y-Asp- (AA) n-R? (II) or biologically acceptable salts or pro-parent molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein Ri is an N-terminal protecting group such as t-butyloxycarbonyl, acyl, and benzyloxycarbonyl; each AA is independently a residue of any natural or unnatural α-amino acid or β-amino acid, or derivatives of an α-amino acid or β-amino acid, each n is independently 0-5, and y is a fluorogenic moiety or fluorescent. Preferably Y is a Rhodamine which includes Rhodamine 110, Rhodamine 116 and Rhodamine 19. More preferred and is Rhodamine 110. Especially preferred compounds are represented by Formula III: itr __tep »• * or biologically acceptable salts or pro-parent molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein Ri, AA, n are as previously defined in formula II. Ri is preferably t-butoxycarbonyl, acyl and benzyloxycarbonyl. The preferred values of n are 1-3. The invention also relates to a method for the preparation of a compound of the formula III, comprising (a) the condensation of Rhodamine together with N-fmoc-L-aspartic acid β-t-butyl ester to produce the (Fmoc) -Asp (Obu-t)) 2-Rhodamine; (b) removal of the Fmoc group to produce (Asp (Obu-t)) 2-Rhodamine; (c) the condensation of (Asp (Obu-t) 2-Rhodamine with Z- (AA) n to produce the (Z- (AA) n-Asp (Obu-t)) 2-Rhodamine, and (d) the elimination of the protective group Obu-t In a preferred embodiment, - (AA) n is WEH SEC ID: O: l, YVA SEQ ID: O: 2, LEH SEQ ID: O: 3, DET SEQ ID: O: 4 , DEV SEQ ID NO: 5, DEH SEQ ID NO: 6, VEH SE ID NO: 7, LET SEQ ID NO: 8, LEV SEQ ID NO: 9, SHV SEQ ID NO: 10, SEQ ID NO: 11, DGP SEQ ID NO: 12, DEP SEQ ID NO: 13, DGT SEQ ID NO: 14, DLN SEQ ID NO: 15, DEE SEQ ID NO: 16, DSL SEQ ID NO: 17, DVP SEQ ID NO: 18, DEA SEQ ID NO: 19, DSY SEQ ID NO: 20, ELP SEQ ID NO: 21, VED SEQ ID NO: 22, IEP SEQ ID NO: 23, or IET SEQ ID NO: 24, and amino acids containing carboxy are protected by a group OBu-t which is eliminated in the final stage.Another group of the preferred compounds that are within the scope of Formula I include the compounds where it is not the same as z.The preferred compounds of this group include those in where x is a peptide or other structure which makes the compound a substrate for caspases, or a substrate for other proteases or peptidases or other enzymes; and the x-y bond in Formula I is the cleavable bond under biological conditions, z is a blocking group and the y-z bond in Formula I is not a cleavable bond under biological conditions. Specifically, the novel fluorogenic or fluorescent reporter compounds of this invention are of Formula V: Ri- (AA) n-Asp-y-Re (V) , • ** •• «-. you - *. or biologically acceptable salts or pro-parent molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein: Ri, AA, n and y are as previously defined in formula II; and Rβ is a blocking group which is not an amino acid or a derivative of an amino acid. In particular, preferred embodiments of the compounds of Formula V are represented by Formula VII: or biologically acceptable salts or pro-parent molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein: Ri, Re, AA, and n are as previously defined in Formulas II and V: R2 and R3 are the same or different and are independently hydrogen, alkyl or aryl; R4 and Rs are the same or different and are independently hydrogen or alkyl. Another group of preferred embodiments of the compounds of Formula I are represented by Formula VIII: or biologically acceptable salts or pro-parent molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein: Ri, Re, AA, and n are as previously defined in the Formulas II and V; m is an integer from 0-3, R2 and R3 are the same or different and are independently hydrogen, alkyl or aryl; and R4 and R5 are the same or different and are independently hydrogen or alkyl. Yet another group of preferred embodiments of the compounds of Formula I are represented by the c__g_ _a_t. nuglü'djf aze¡ $ ¿? m *? & É ^ É ^ M Formula IX: or biologically acceptable salts or pro-parent molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein: Ri, Re, AA, and n are as previously defined in Formulas II and V; m is an integer from 0-3, R2 and R3 are the same or different and are independently hydrogen, alkyl or aryl; And R and Rs are the same or different and are independently hydrogen or alkyl. This invention also relates to a method for the preparation of a compound of Formula VII, which comprises (a) reacting Rhodamine with acetic anhydride to produce the N-acetyl-Rhodamine of Formula VI; (b) the condensation of the N-acetyl-Rhodamine of Formula VI together with the β-t-butyl ester of N-fmoc-L-aspartic acid to produce the N- (Fmoc-Asp (OBu-t)) -N'-acetyl-Rhodamine; (c) removal of the Fmoc group to produce N- (Asp (OBu-t)) -N'-acetyl-Rhodamine; (d) the condensation of N- (Asp (OBu-t)) -N'-acetyl-Rhodamine with Z- (AA) n to produce N- (Z- (AA) n-Asp (OBu-t) ) -N '-acetyl-Rhodamine; and (e) removing the OBU-t protecting group to produce the N- (Z- (AA) n-Asp) -N'-acetyl-Rhodamine; or alternatively (a) reacting Rhodamine with acetic anhydride to produce the N-acetyl Rhodamine of Formula VI; (b) the condensation of the N-acetyl-Rhodamine of Formula VI with Z- (AA) n-Asp (β-OBu-t) to produce the N- (Z- (AA) n-Asp (OBu-t )) -N'-acetyl-Rhodamine; and (c) the removal of the OBu-t protecting group to produce the N- (Z- (AA) n-Asp) -N'-acetyl-Rhodamine. In this embodiment, wherein (AA) n includes amino acids such as glutamic acid or aspartic acid, the carboxy group is protected with an OBu-t group which is inserted in the last stage. Thus, the invention also relates to novel fluorescent dyes of Formula VII which are derivatives of Rhodamine. These compounds are prepared by the introduction of a blocking group Re into one of the two amino groups of Rhodamine. The R2NH group in formula VI provides the attack point for the reaction with a potential enzyme substrate, such as the carboxyl group of an N-blocked peptide, to form a peptide amide bond. The reaction will convert the fluorescent molecule of formula VI into a reporter molecule of the non-fluorescent peptide of Formulas VII-IX which is a substrate for a protease or peptidase. The decomposition of reporter-peptide amide linkage cleavable in peptide-reporter by proteases or peptidases produces the compound of Formula VI or VI 'which is fluorescent. Specifically, the new fluorescent dyes of this invention are of Formula VI: or biologically acceptable salts wherein: R2 and R3 are the same or different and are independently hydrogen, alkyl or aryl; Re is a blocking group which is not an amino acid or derivative of an amino acid; R4 and Rs are the same or different and are independently hydrogen or alkyl. Preferably R2 and R3 are hydrogen, methyl or ethyl; Preferably R4 and Rs are hydrogen or methyl. The invention also relates to a process of using the reporter compounds represented by Formula I to measure the activity of caspases intracellular or other enzymes involved in apoptosis in tissues or whole cells alive or dead. The invention also relates to the methods of using the compounds represented by Formula I and the assay processes described herein to measure the activation or inhibition of any of the enzymes of caspase within any tissue or whole cell living or dead (normal or cancerous) by a test of substance or substances. The compounds represented by Formula I are permeable to cells, ie they are can enter tissue samples or whole cells. The compounds are fluorogenic or fluorescent and can be designated to be specific for any of the known caspases or for any other intracellular enzymes involved in the apoptosis.

Thornberry, N.A., et al., J. Biol. Chem. 272: 11901 (1997), describes the optimal sequences for various caspase substrates and for the Granzima B substrate. The optimal substrate sequences are shown in Table 1.

Table 1 * Enzymes are identified for both the new and the old nomenclature (in parentheses). ** Standard letter abbreviations for amino acids are used to indicate the sequences amino acids optimum.

Using the optimal sequences described by Thornberry et al. , fluorogenic or fluorescent substrates for specific caspases can be synthesize by the procedures described here.

^^^^^^^ Mm ?? It is also possible to designate other fluorogenic or fluorescent substrates for known or unknown caspases using potential or known decomposition site peptide sequences from potential or known natural substrates of caspase enzymes. Table 2 shows the peptide sequences corresponding to potential or known decomposition sites in proteins that can be natural substrates for caspases.

Table 2 ** Standard letter abbreviations for amino acids are used to indicate amino acid sequences.

Fluorogenic or fluorescent substrates can also be designated to measure more than one enzyme at the same time, by designating substrates that are recognized and inserted by more than one of the enzymes involved in the caspase cascade. The substrates Fluorogenic or fluorescent which are "promiscuous" for more than one caspase can be used using the assay process described here to measure the activity of caspases not yet known. When the caspase cascade is activated by a stimulus that induces the dead cell, the fluorogenic or fluorescent reporter molecules described herein are inserted and respond with a large increase in fluorescence emission. The change in fluorescence can be measured spectrofluorometrically. The molecules reporters can also be used to measure the activity of the layer at the baseline in cells that do not suffer from apoptosis. The method is easily adaptable to the tests of a screening of high performance or ultra high performance.

Yes *? ^ Lf __fcft.Ji The test system is very versatile. Examples of the extreme versatility of the test system are given below.

The assay can be used to select or sift a cell or tissue for the activity at the baseline of any caspase enzyme or any other enzyme involved in apoptosis. The assay can be used with equal ease to select or screen compounds that can either activate or inhibit the caspase cascade. This means that the assay can be used to select or screen drugs against degenerative diseases or for anti-cancer drugs. The assay can be used to select or screen the activation or inhibition of the caspase cascade in any of the cell lines or living or dead cells derived from any organic system in the body including, but not limited to, hair, brain, peripheral nervous system, eye, nose, mouth, tonsil, tooth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, system immune, liver, stomach, intestinal tract, pancreas, endocrine glands, and tissues, kidney, bladder, reproductive organs and glands, skin and bones. The assay can be used for the selection of drugs with potential use in any disease of any organic system in the body that includes malfunction of caspase cascade. 4. The assay can be used in the selection of drugs that can directly or indirectly modulate the caspase cascade, that is, by modulating caspase by itself or by modulating the cellular receptors and the co-factors that influence the cascade. of caspasa.

. The assay can be used to determine the site of action in which the modulator of caspase cascade interferes. That is, that the assay can help define or know the molecular mechanism of action of a novel modulator drug caspase cascade. The invention further relates to the use of fluorescent or fluorogenic substrates represented by formula I to find new compounds or new uses for known compounds in the reduction, prevention, or treatment of diseases in which apoptotic cell death is the causative factor or a result. Some examples of uses of the present invention include selection for compounds that can protect the nervous system that tracks focal ischemia as well as global ischemia; selection for compounds that treat degenerative disorders of neurons such as Alzheimer's disease, Huntington's disease, prion diseases, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia, telangiectasia, and atrophy spinobulbar; the selection for compounds that can treat heart disease includes myocardial infarction, congestive heart failure and cardiomyopathy; selection for compounds that can treat disorders of the autoimmune system include lupus erythematosus, rheumatoid arthritis, type I diabetes, Sjögren's syndrome and glomerulonephritis, selection for compounds that can treat polycystic kidney disease, and anemia / arithropoiesis; the selection for diseases that can treat disorders of the immune system, include AIDS and SCIDS; selection for compounds that reduce or prevent damage to cells, tissues and organs during transplantation (for example, grafting against disease in the procedures of jj sÉtfSs? áß & '.-Ci. : '*' 'transplant of the bone marrow); the selection for compounds that can reduce or prevent the death of the cell line in industrial biotechnology; the selection for compounds that reduce or prevent alopecia (hair loss); and the selection for compounds that reduce the premature death of skin cells. The present invention also relates to the use of fluorescent or fluorogenic substrates represented by formula I, in selection procedures wherein the genetcs of known combination medicaments, or other genetically composed compounds are selected for compounds having anticancer or antitumor activity. Cells or cancer cell lines can be derived from any internal or external organic system in the body that include but are not limited to: the hair, the brain, the peripheral nervous system, the eye, nose, mouth, amygdala, tooth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands, and tissues, kidney, bladder, glands and reproductive organs ( for example the prostate gland), joints, skin and bones.

The present invention further relates to the use of fluorescent or fluorogenic substrates represented by formula I in diagnostic procedures for the determination of chemical sensitivity or resistance of cancer cells taken from an animal or a human to be treated with drugs. for chemotherapy. Cells or cancer cell lines that can be derived from any cancer in any internal or external organic system in the body that includes but is not limited to: hair, brain, peripheral nervous system, eye, nose, mouth, tonsil, tooth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands, and tissues, kidney, bladder, glands and reproductive organs (for example the prostate gland), skin and bones. In particular, the invention relates to a method for the detection of an enzyme included in the apoptosis cascade in one or more cells, comprising (a) contacting one or more cells with a reporter compound according to the invention under conditions of which the reporter compound is taken in one or more cells, and (b) recording the fluorescence of one or more cells, wherein the relative change in fluorescence, either of magnitude (ie increase) or of wavelength within of one or more cells, compared to control cells which have not been contacted in this way or one that has been contacted with the reporter compound and a known competitive inhibitor of the enzyme, is an indication of the presence of the enzyme. The invention further relates to a method for measuring the activity of an enzyme involved in the apoptosis cascade in one or more cells, comprising (a) contacting one or more cells with the reporter compound according to the invention under conditions in the which the reporter compound is taken in one or more cells, and (b) recording the fluorescence of one or more cells, wherein the change in fluorescence, either of magnitude or wavelength, within one or more cells compared with the control cells which have not been contacted or have been contacted with the reporter compound and a known competitive inhibitor of the enzyme, is a measurement of the activity of the enzyme.

The invention further relates to a method for determining whether a test sance has an effect on an enzyme involved in the apostosis cascade in one or more test cells, comprising, (a) contacting one or more test cells with the test sance and reporter compound according to the invention under conditions in which the reporter compound is taken in one or more cells and the test sance is either interacted with an outer membrane receptor or is taken in said cells, and ( b) recording the fluorescence of the test cells, wherein the change in fluorescence, either of magnitude or wavelength, within the cells compared to the control cells which have only been put in contact with the compound reporter and not with the test sance, is an indication that the test sance has an effect either directly or indirectly on the apoptosis enzyme that is being ex me nothing. In practicing this aspect of the invention, the test cells may be in contact with said test sance before or after, or santially simultaneously with, the reporter compound according to the invention. The method can be used to detect whether the test sance stimulates or inhibits the activity of the enzyme. The invention also relates to an additional contact of the test cells with a second test sance or a mixture of the test sances in the presence of a first test sance. In a preferred embodiment, the test cell is a cancer cell or line of cells derived from a human in need of treatment with a chemotherapy drug and the test sance is a chemotherapy agent or a mixture of chemotherapy agents. The invention further relates to a method for determining the sensitivity of an animal to cancer to treatment with one or more chemotherapy agents, comprising (a) contacting one or more chemotherapeutic agents and the reporter compound according to the invention. invention, the cancer cells taken from said animal, under conditions in which the reporter compound is taken in cancer cells and one or more drugs either interacting with an outer membrane receptor or taken within said cell, ( b) recording the fluorescence of the cancer cells, wherein a change in fluorescence, either of magnitude or wavelength, in the cancer cells as compared to the control cells that have only been contacted with the reporter compound, is an indication that cancer cells are sensitive to the chemicals of one more agents and that the animal is sensitive to treatment. The invention also relates to a method for verifying the treatment of an animal with one or more chemotherapy drugs, comprising (a) administering one or more chemotherapy drugs to the animal, (b) contacting the cells taken from the animal afterwards. of the administration of the reporter compound according to the invention under conditions in which the reporter compound is taken up in the cells, and (c) recording the fluorescence of the cells contacted with the reporter compound, wherein a change in fluorescence, already whether of magnitude or wavelength, within cells taken from the animal after administration compared to control cells which have been taken from the animal prior to administration is an indication that the animal is sensitive to the chemotherapeutic agent . In this embodiment, the animal may suffer from a disease in which the death of the apoptotic cell is either a causative factor or a result.

»Srt < * - ¡- ** »..

The invention further relates to a method for determining whether a test sance inhibits or prevents cell death in one or more test cells, comprising (a) contacting the test cell with the test sance and reporter compound according to the invention under conditions in which the test sance either interacts with an outer membrane receptor or is placed in the cell and the reporter compound is placed in the cell, and (b) recording the fluorescence of the test cells, wherein a change in fluorescence, either in magnitude or in wavelength, within the test cells compared to the control cells that have only been put in contact with the reporter, it is an indication that the test substance inhibits or prevents the death of the cells. The invention also relates to a method for determining whether a test substance causes or increases the death of the cells in one or more test cells, comprising (a) contacting the test cells with the test substance and the test substance. reporter compound according to the invention under conditions in which the test substance interacts with an outer membrane receptor or is placed in the cells and the reporter compound is placed in the cells, (b) recording the fluorescence of the test cells, in where a change in fluorescence, either of magnitude or wavelength, within the test cells compared to the control cells that have only been contacted with the reporter compound is an indication that the test substance causes or it increases the death of the cells. The invention also relates to a process for the use of the reporter compounds represented by formula IX to measure the activity of intracellular peptidases and proteases in fully alive cells, including but not limited to, methionine-2-aminopeptidase in endothelial cells and HIV protease in HIV-infected cells. The invention also relates to methods for the use of compounds represented by the formula IX and the compound or to the assay processes described herein for measuring the inhibition or activation of enzymes within a fully living cell by a test compound or compounds . The reporter compounds represented by formula IX are permeable to cells, that is, they can be introduced into whole cells. The compounds are fluorogenic or fluorescent and can be designed to be specific for the known enzymes of interest, such as aminopeptidase methionine or the HIV protease. The invention also relates to a method for detecting a viral protease in one or more cells, comprising (a) contacting the cells with the reporter compound according to the invention under conditions in which the reporter compound is placed in the cells, and (b) recording the fluorescence of said cells, wherein a change or increase in fluorescence within the compared cells to control the cells that have not been contacted in this way, is an indication of the presence of the viral protease. . The invention also relates to a method for measuring the activity of a viral protease in one or more cells infected with the virus, comprising (a) contacting one or more cells infected with the virus with the reporter compound according to the invention under conditions in which the reporter compound is placed in one or more cells infected with the virus, and (b) recording the fluorescence of one or more cells, wherein a change or increase in fluorescence within one or more cells infected with the Compared viruses to control cells that have not been contacted, is a measure of the activity of the viral protease. The invention further relates to a method for determining whether a test substance has an effect on the activity of the viral protease in one or more cells infected with the virus, which comprises (a) contacting the infected test cells with the virus with the test substance and the reporter compound according to the invention under conditions in which said reporter compound is placed in the infected test cells, and (b) recording the fluorescence of the infected test cells compared to the infected control cells that have only been put in contact with the reporter compound, wherein a change or increase in fluorescence within the infected test cells compared to the infected control cells, is an indication that the test substance has an effect on the viral protease. In a preferred embodiment, the cells are cells infected with HIV, and the viral protease is the HIV protease. In 'Another preferred, the t cells are cells infected with the adenovirus and the viral protease is the adenovirus protease. In another preferred embodiment, the cells are cells infected with the VSH and The viral protease is the protease of the VSH. In another preferred embodiment, the cells are cells infected with the VMCH and the viral protease is the protease of the VMCH. In another preferred embodiment, the cells are cells infected with the HCV and the viral protease is the protease of the VCH. The invention also relates to a method for measuring the activity of the protease or peptidase in cells, which comprises (a) contacting the test cells with The reporter compound according to the invention under conditions in which the reporter compound is placed in the test cells, or the reporter compound is interacting with a protease or peptidase of the outer membrane of said cells, and 20 (b) recording the fluorescence of the cells, wherein a change or increase in fluorescence within the test cells compared to the control cells which has not been contacted is a measure of the activity of the protease or peptidase.

The invention also relates to a method for determining whether a test substance has an effect on the activity of the protease or peptidase in the test cells, comprising (a) ) contacting the test cells with the test substance and the reporter compound according to the invention under conditions in which the reporter compound is placed in the test cells, or the reporter compound is interacting with a membrane protease or peptidase of said cells, and (b) recording the fluorescence of the test cells compared to the test cells which have only been contacted with the reporter compound, where a change or increase in fluorescence within the cells of the test compared to the control cells which is an indication that the test substance has an effect on the protease or peptidase. In a preferred embodiment, the cells are endothelial cells and the peptidase is methionine aminopeptidase type 2. In another preferred embodiment, the cells are T cells and the peptidase is dipeptidyl peptidase-IV. In another preferred embodiment, the cells are cells of the neuron and the protease is calpain.

BRIEF DESCRIPTION OF THE FIGURES FIGS la-1F show photographs of HL-60 cells stained by N-octyloxycarbonyl-Rhodamine 110 (Fig. IA), N-decyloxycarbonyl-Rhodamine-110 (Fig. IB), N-dodecyloxycarbonyl-Rhodamine- 110 (Fig. 1C), hexyloxycarbonyl-Rhodamine-110 (Fig. ID), N- (ethylthio) carbonyl-Rhodamine 110 (Fig. 1E) and Rhodamine 10 (Fig. 1F). Figures 2A-2L show the bar graphs of the decomposition of caspase substrates of N-Z-VD-N'-ethoxycarbonyl-RllO, NZ-EVD-N '-ethoxycarbonyl-R110, NZ-DEVD- N'-ethoxycarbonyl-RllO SEQ ID NO: 5, N-Ac-DEVD-N'-ethoxycarbonyl-RllO SEQ ID NO: 5, N-Ac-DEVD-N '-octyloxycarbonyl-RUO SEQ ID NO: 5, N-Ac-DEVD-N '-hexyloxycarbonyl-RUO SEQ ID NO: 5, and NZ-DEVD-N'- (ethylthio) carbonyl-RUO SEQ ID NO: 5, by r-caspase-3 (Figures, 2A, 2B, 2D, 2G, and 2J) and lysates of the HL-60 cell treated with vlinbastine (Figures 2C, 2E, 2H and 2K) compared to the control lysates HL-60 (treated with DMSO) (Figures 2F, 21 and 2L). Figures 3A-3E show photographs of cells stained by incubation with N-Ac-DEVD-N '-octyloxycarbonyl-RUO SEQ ID NO: 5. Vinblastine (Figure 3A) and H-60 cells treated with DMSO (Figure 3D), the HL-60 cells treated with vinblastine with the N-Ac-DEVD-CHO SE ID NO: 5 are added in the assay stage (Figure 3C), antiFas (Figure 3D) and PBS (Figure 3E) ) treated with Jurkat cells. Figure 4 shows a graph demonstrating the results of a decomposition assay of N-Ac-DEVD-N '-octyloxycarbonyl-RUO SEQ ID NO: 5 by antiFas and PBS treated with Jurkat cells. Figure 5 shows a bar graph demonstrating the results of a decomposition test of N-Ac-LEVD-N'-ethoxycarbonyl-RllO SEQ ID NO: 5 by caspase-3, -6, -7 and -8 . Figures 7A-B show photographs of HL-60 cells treated with N-Z-G-N '-octyloxycarbonyl-R110 (A) and N-G-N' -octyloxycarbonyl-RUO (B).

DETAILED DESCRIPTION OF THE INVENTION The fluorogenic or fluorescent substrates of the present invention are compounds having the general Formula I: xyz (I) or biologically acceptable salts or pro-parent molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein xyz is the same or different and is a peptide or amino acid or acyl group or other structure such that Formula I is a substrate for caspases, or other proteases or peptidases or other enzymes; and wherein the cleavable link is only one or both links x and yz in Formula I when x is the same as z, or where the cleavable link is only one of the links x and yz in Formula I when x is not the same as z, and it is a fluorogenic or fluorescent portion. Preferred displacement compounds within the scope of Formula I include compounds wherein x is the same as z, and the first amino acid attached to y is an Asp. More preferably, x is mime as z and is an N-blocked tetrapeptide substrate of a caspase including WEHD SEQ ID NO: 1, YVAD SEQ ID NO: 2, LEHD SEQ ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID NO: 5, DEHD SEQ ID NO: 6, VEHD SEQ ID NO: 7, LETD SEQ ID NO: 8, LEHD SEQ ID NO: 3, SHVD SEQ ID NO: 10, DELD SEQ ID NO: 11 , DGPD SEQ ID NO: 12, DEPD SEQ ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 26, IETD SEQ ID NO: 24 or an N-blocked tetrapeptide substrate of granzyme B that includes IEPD SEQ ID NO: 23 and VEPD SEQ ID NO: 27; ox is the same as z and is an N-blocked peptide which corresponds to a carboxyterminal or amino terminal fragment consisting of 1, 2 or 3 amino acids of the tetrapeptide substrate of a caspase including WEHD SEQ ID NO: 1, YVAD SEQ ID N0: 2, LEHD SEQ ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID NO: 5, DEHD SEQ ID NO: 6, VEHD SEQ ID NO: 7, LETD SEQ ID NO: 8, LEHD SEQ ID NO : 3, SHVD SEQ ID NO: 10, DELD SEQ ID NO: 11, DGPD SEQ ID NO: 12, DEPD SEQ ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 23 and VEPD SEQ ID NO: 27 . Preferred displacement compounds within the scope of Formula I include compounds wherein y is Rhodamine 110. In particular, preferred embodiments of the compounds of Formula I are represented by Formula II: Ri- (AA) n-Asp-y-Asp- (AA) n-R? (II) or biologically acceptable salts or pro-reporter molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein Ri is an N-terminal protecting group that includes t-butoxycarbonyl, acetyl, benzyloxycarbonyl; each AA is independently a residue of any natural or unnatural α-amino acid or β-amino acid, or derivatives of an α-amino acid or β-amino acid; each n is independently 0-5; and y is a fluorogenic or fluorescent moiety. An example of a pre-reporter molecule is the methyl ester form of carboxyl-containing amino acid residues comprising the compounds of Formula II. Another example of a pre-reporter molecule is the acetoxymethyl ester (AM) form of carboxyl-containing amino acid residues of the compounds of Formula II. AM esters of carboxyl-containing compounds are known to be permeable to cells and can be hydrolyzed by esterases within cells. Once hydrolyzed, the carboxyl-containing compounds arrive to waterproof the cells and are entrapped within the cells (Adams et al., J. Am. Chem. Soc. 111: 7957-7968 (1989)). The AM esters can be prepared by the reaction of the corresponding carboxy-containing compounds with bromomethyl acetate.

Especially preferred embodiments of the compounds of Formula I are represented by Formula III: ,, - ^ or biologically acceptable salts or pre-reporter molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein Ri, AA, n are as previously defined in Formula II. Preferred Ri is t-butoxycarbonyl, acetyl and benzyloxycarbonyl. Also the preferred values for n are 1-3. Another group of preferred displacement compounds within the scope of Formula I includes compounds wherein x is not the same as z. Preferred compounds of this group include those wherein x is a peptide or other structure which makes the compound a substrate for caspases, or other proteases or peptidases or other enzymes; and the bond x-y in Formula I is the cleavable bond under biological conditions; z is a blocking group and the y-z bond in Formula I is not a cleavable bond under biological conditions. More preferably, x is an N-blocked tetrapeptide substrate of a caspase including WEHD SEQ ID NO: 1, YVAD SEQ ID NO: 2, LEHD SEQ ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID NO. : 5, DEHD SEQ ID NO: 6, VEHD SE ID N0: 7, LETD SEQ ID NO: 8, LEHD SEQ ID NO: 3, SHVD SEQ ID NO: 10, DELD SEQ ID NO: 11, DGPD SEQ ID NO: 12, DEPD SEQ ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19 , DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 26, IETD SEQ ID NO: 24 or an N-blocked tetrapeptide substrate of granzyme B including IEPD SEQ ID NO: 23 and VEPD SEQ ID NO: 27; ox is an N-blocked peptide which corresponds to a carboxy-terminal or amino-terminal fragment consisting of 1, 2 or 3 amino acids of the tetrapeptide substrates of a caspase including WEHD SEQ ID NO: 1, YVAD SEQ ID NO: 2, LEHD SEC ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID NO: 5, DEHD SEQ ID NO: 6, VEHD SE ID NO: 7, LETD SEQ ID NO: 8, LEHD SE ID NO: 3, SHVD SEQ ID NO: 10, DELD SEQ ID NO: ll, DGPD SEQ ID NO: 12, DEPD SEQ ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO : 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 26, IETD SEQ ID NO: 24 or granzima B including IEPD SEQ ID NO: 23 and VEPD SEQ ID NO: 27; ox is an N-blocked peptide which corresponds to a carboxy-terminal or amino-terminal fragment consisting of 1, 2, 3 or 4 amino acids of the tetrapeptide substrate of a caspase including WEHD SEQ ID NO: 1, YVAD SEQ ID NO: 2, LEHD SEQ ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID NO: 5, DEHD SEQ ID NO: 6, VEHD SEQ ID NO: 7, LETD SEQ ID NO: 8, LEHD SEQ ID NO: 3, SHVD SEC ID NO: 10, DELD SEQ ID NO: 11, DGPD SEQ ID NO: 12, DEPD SEQ ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 26, IETD SEQ ID NO: 24 and granzima B including IEPD SEQ ID NO: 23 and VEPD SEQ ID NO: 27, amino acids 1-2 positive correspond to the portion P? '- P27 of the substrate of a caspase including G, A, GA, GG and AG. Specifically, the novel fluorogenic or fluorescent reporter compounds of this invention are of Formula V: Ri- (AA) n-Asp ^ y-Re (V) or biologically acceptable salts or reporter molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein: Ri is an N-terminal protecting group that includes t-butoxycarbonyl, acetyl, octanoyl and benzyloxycarbonyl; each AA is independently a residue of any natural or unnatural α-amino acid or β-amino acid, or a derivative of an α-amino acid or β-amino acid; n is 0-5; and is a fluorogenic or fluorescent moiety; and Re is a blocking group which is not an amino acid or a derivative of an amino acid. In particular, the new fluorogenic or fluorescent reporter molecules of this invention of Formula VII-IX are derivatives of Rhodamines which include Rhodamine 110, Rhodamine 116 and Rhodamine 19. These new fluorogenic or fluorescent reporter molecules are prepared by initially introducing a group Re-blocking in one of the two amino groups of a Rhodamine to produce new fluorescent dyes of Formula VI. The remaining group HNR2 is used for the reaction with a substrate of potential enzyme to produce a fluorogenic substrate of Formulas VII-IX. Blocking one of the two amino groups in a Rhodamine, the total size of the substrate is reduced compared to a bis-substituted Rhodamine, such as a bis-peptide-Rhodamine. More importantly, the blocking group is selected such that a) is stable and will not hydrolyze under biological conditions, thus the amino acids are excluded because the bond of the formed peptide can be potentially divided by peptidases which are present in the cells; b) it is preferably not too bulky (for example it is small) to reduce the total size of the peptide reporter molecule so much that this will be a better enzyme substrate; c) is preferably hydrophobic in nature to increase the cellular permeability of the fluorogenic or fluorescent reporter molecule. Preferred blocking groups Re include, but are not limited to, an alkoxycarbonyl group of 2 to 12 carbon atoms such as methoxycarbonyl, ethoxycarbonyl, hexyloxycarbonyl, octyloxycarbonyl, decyloxycarbonyl and dodecyloxycarbonyl; a (alkylthio) carbonyl group of 2 to 12 carbon atoms such as (ethylthio) carbonyl, (hexylthio) carbonyl, (octylthio) carbonyl; an arylalkyloxycarbonyl group such as benzyloxycarbonyl, an acyl (alkanoyl) group of 2 to 12 carbon atoms such as acetyl and octanoyl, a carbamyl group such as dimethylcarbamyl, N-methyl-N-hexylcarbamyl, and an alkyl, haloalkyl or aralkylsulfonyl group such as methanesulfonyl. Particularly preferred Rβ blocking groups are CH3OCO-, CH3 (CH2) pOCO- (p = 1-11), Cbz, Cl3CCH2OCO- and PhCH2CH2OCO- (carbamate series); Me (OCH2CH2) qOCO- (q = 1-4), and CH3 (CH2) r (OCH2CH2) sOCO- (r = 0- ! _ $ _ & - * ___. , s = 1-4), (alkyloxyalkylcarbamate series); EtSCO-, CH3 (CH2) 5SCO-, CH3 (CH2) 7SCO-, CH3 (CH2) gSCO- and CH3 (CH2) tSCO- (t = 0-11) (thiocarbamate series); Ts-, PhS02-, MeS02-, CH3 (CH2) uS02- (u = 0-11), PhCH2S02- and CF3S02- (series of 5 sulfonamide); Me2NCO-, Et2NCO-, and N-Me-N-CH3 (CH2) vNCO (v = 0-9) (urea series); and HCO-, CH3CO-, CH3 (CH2) "CO (w = 0-9), PhCH2CO- and PhCO- (amide series). The most preferred blocking groups R are those which contain a hydrophobic group similar to the membrane lipid, or thereby increase the cellular permeability of the fluorogenic or fluorescent reporter molecules, as well as the retention of the fluorescent portion in the cells after the Substrate division by the target protease or peptidase. These preferred R 5 blocking groups include, but are not limited to CH 3 (CH 2) pOCO- (p = 1-11) (carbamate series): Me (OCH 2 CH 2) qOCO- (q = 1-4), and CH 3 ( CH2) r (OCH2CH2) sOCO- (r = 0-5, s = 1-4), (alkyloxyalkylcarbamate series); EtSCO-, CH3 (CH2) 5SCO-, CH3 (CH2) 7SCO-, and CH3 (CH2) 9SCO- (thiocarbamate series); CH3 (CH2) tS02- (t = 0-11), (sulfonamide series); N-Me-N- CH3 (CH2) uNCO (u = 0-9) (urea series); and CH3 (CH2) "CO (w = 0-9) (amide series). The new fluorogenic or fluorescent reporter molecules of Formula VII-IX are prepared by the reaction of the amino group NHR2 of the new dyes "X" * fluorescers of Formula VI with a substrate of pootential enzyme, such as the carboxyl group of an N-blocked peptide, to form a peptide amide bond.The reaction converts the fluorescent molecule of Formula VI into a reporter molecule of the non-fluorescent peptide of Formulas VII-IX which is a substrate for a protease or peptidase However it is very important that the Re-N bond of the blocking group of Formula VII could not be divided and that the linkage of the peptide reporter amide could be the cleavable bond under biological conditions.The division of the cleavable link of the reporter amide of the peptide of Formulas VII-IX by the proteases or peptidases produces a compound of Formula VI or VI 'which is fluorescent The preferred modalities specifically of the compounds of Formula V are represented by Formula VII: or biologically acceptable salts or pre-reporter molecules (such as the methyl ester form of the S & amp; carboxyl-containing amino acid residues thereof, wherein: R2 and R3 are the same or different and are independently hydrogen, alkyl or aryl; Rβ is a blocking group which is not an amino acid or a derivative of an amino acid; R and Rs are the same or different and are independently hydrogen or alkyl, Ri is an N-terminal protecting group; each AA is independently a residue of any natural or unnatural α-amino acid or β-amino acid, or a derivative of an α-amino acid or β-amino acid; n is 0-5; and the cleavable link is the Asp-N link in the Formula VII. Preferred R2 and R3 are hydrogen, methyl or ethyl; Preferred R4 and R5 are hydrogen or methyl. Preferred amino acids include the natural amino acids including tyrosine, glycine, phenylalanine, methionine, alanine, serine, isoleucine, leucine, threonine, valine, proline, lysine, histidine, glutamine, glutamic acid, tryptophan, arginine, aspartic acid, aspargin, and cysteine Unnatural mainoacids include t-butylglycine and N, N-dimethylglutamine. An example of a reporter molecule is the methyl ester form of carboxyl-containing amino acid residues comprising the compounds of the Formula VII. Another example of a pro-reporter molecule is the acetoxymethyl ester (AM) form of the carboxyl-containing amino acid residues of the compounds of Formula VII. Another group of preferred embodiments of the compounds of Formula I are represented by Formula VIII: or biologically acceptable salts or pro-reporter molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein Ri, R, AA and n are as previously defined in Formulas II and V; m is an integer of 0-3. R2 and R3 are the same or different and are independently hydrogen, alkyl or aryl; and R and Rs are the same or different and are independently hydrogen or alkyl. The compounds of Formula VIII are new fluorogenic or fluorescent substrates for caspases or other enzymes related to apoptosis. When m is 0, the cleavage of the amide bond between Asp and Rhodamine will convert the fluorogenic substrate into the fluorescent dye of Formula VI. When m is not 0, the division of the amide bond between Asp and (AA) m will separate the Rhodamine bound to NH2- (AA) m. The remaining amino acids (AA) m will then be removed by the aminopeptidases present in the cells to provide the fluorescent dye of Formula VI. (AA) m may be designated to correspond to the sequence P 'of the cleavage site of caspase substrates or enzymes related to apoptosis. The incorporation of the P 'sequence of known substrates of caspases or enzymes related to apoptosis is expected to increase the specificity and affinity of the fluorogenic substrates. Since aminopeptidases are widely present in cells, one can insert a sequence of (AA) m into the design of substrates of Formula VIII for whole cell assays. This is another advantage of whole-cell assays on cell-free enzyme assays. For example, when (AA) m is Gly, a substrate of formula VIII will work in whole cell assays but otherwise it will not work in the cell-free caspase assay because the division of the amide bond Asp-Gly It will divide the Gly junction to Rhodamine, which is not fluorescent. An example of a pro-reporter molecule are the methyl or ethyl ester forms of the carboxyl-containing amino acid residues comprising the compounds of Formula VIII. Another example of a pro-parent molecule is the acetoxymethyl ester (AM) or pivaloyloxymethyl ester (PM) form of the carboxyl-containing amino acid residues of the compounds of Formula VIII. AM esters of carboxyl-containing compounds are known to be permeable to cells and can be hydrolyzed by esterases within cells. Once hydrolyzed, the carboxyl-containing compounds become impermeable to the cells and are trapped within the cells (Adams et al., J. Am. Chem. Soc. 111: 7957-7968 (1989)). The AM esters can be prepared by the reaction of the corresponding carboxy-containing compounds with bromomethyl acetate. Yet another group of preferred embodiments of the compounds of Formula I are represented by Formula IX: IX or biologically acceptable salts or pro-exporting molecules (such as the methyl ester form of carboxyl-containing amino acid residues) thereof, wherein Ri, Re, AA and are not as previously defined in Formula II and V; m is an integer from 0-3, R2 and R3 are the same or different and are independently hydrogen, alkyl or aryl; and R4 and Rs are the same or different and are independently hydrogen or alkyl. Ri is preferably t-butyloxycarbonyl, acetyl, octanoyl, dodecanoyl and benzyloxycarbonyl. Preferably n is 1-4. Preferred R2 and R3 are hydrogen, methyl or ethyl. The R and R5 are hydrogen or methyl. Preferred R-blocking groups include, but are not limited to, an alkoxycarbonyl group of 2 to 12 carbon atoms such as methoxycarbonyl, ethoxycarbonyl, hexyloxycarbonyl, ethoxycarbonyl, hexyloxycarbonyl, octyloxycarbonyl, decyloxycarbonyl and dodecyloxycarbonyl; a (alkylthio) carbonyl group of 2 to 12 carbon atoms such as (ethylthio) carbonyl, (hexylthio) carbonyl, (octylthio) carbonyl; an arylalkyloxycarbonyl group such as benzyloxycarbonyl, an acyl (alkanoyl) group of 2 to 12 carbon atoms such as acetyl and octanoyl, a carbamyl group such as dimethylcarbamyl, N-methyl-N-hexylcarbamyl, and an alkyl, haloalkyl or aralkylsulfonyl group such as methanesulfonyl. In Formula IX, (AA) n is designated to be an amino acid or a peptide which is recognized by a specific protease or peptidase as the sequence on the p-side and will be divided by the target protease or peptidase. (AA) m is designated as an amino acid or peptide which is recognized by a specific protease or peptidase as the sequence on the P 'side, and which can be removed by the aminopeptidases present in the cells. When Ri is an N-terminal protecting group such as a terbutoxycarbonyl, Cbz or acetyl, the compounds of Formula IX are substrates for endopeptidases such as cathepsin D or protease such as HIV protease; when Ri is H, the compounds of Formula IX are substrates for exopeptidases such as methionine aminopeptidase. Specifically, the compounds of Formula IX are designated as being methionine aminopeptidase substrates of type 2 (MetAP-2). MetAP-2 was recently identified by two search groups (Griffith, EC, et al., Chem. Biol. 4: 461-471 (1997) and Sin, N., et al., Proc. Na ti. Acad. Sci. USA 94: 6099-6103 (1997)) is the common target of the AGM-1470 inhibitor of angiogenesis, an anti-cancer drug presently undergoes clinical trials. MetAP-2 is a bifunctional enzyme which also regulates the synthesis of the protein by affecting the phosphorylation state of eIF-2. AGM-1470 is reported to inhibit only the aminopeptidase activity of MetAP-2 and has no effect on the regulatory activity of MetAP-2 (Griffith, E.C., et al., Chem. Biol. 4: 461 -471 (1997)). Since the antiogenesis inhibitor such as AGM-1470 is known to be capable of screening for killer cancer cells, inhibitors of MetAP-2 are expected to have anti-giogenic properties and to be potential new anticancer agents. MetAP-2 is a cobalt-dependent enzyme that hydrolyzes amino-teminal methionine of certain proteins. Their preferred substrates are Met-X-Y. X is an amino acid with small and discharged side groups, such as Gly, Ala, Ser, while Leu, Met, Arg and Tyr are known to result in inactive substrates. And it can be Ser, Met, Gly or other amino acids (Li, X, &Chang Y.-H., Biochem. Biophy, Res. Com. 227: 152-159 (1996)). Since Rhodamine is much larger than an amino acid, a compound with methionine directly bound to Rhodamine will most likely not be a substrate for MetAP-2. Taking advantage of the presence of aminopeptidase in whole cells, the insertion of a sequence (AA) n between methionine and Rhodamine will make a good substrate for MetAP-2. This type of substrate is expected to work well in a whole-cell assay but otherwise it will not work in a cell-free MetAP-2 enzyme assay. For compounds of Formula IX designated to be MetAP-2 substrates, Ri is H, (AA) n Preferred is Met, and (AA) m is preferred Gly, Ala, Gly-Gly, Ala-Gly or Gly-Ala . The methionine will be cleaved by the methionine aminopeptidase of type 2 in endothelial cells to give the Rhodamine bound to (AA) m. The aminopeptidases present within the cells will then remove the (AA) to give the fluorescent dye of Formula VI. The compounds of Formula IX will be used for the selection of MetAP-2 inhibitors in endothelial cells, which are expected to lead to the identification of new anticancer drugs. The compounds of Formula IX can also be designated to be HIV protease substrates. The HIV protease is an aspartic protease which processes polypeptides transcribed from the gag and pol genes and is essential for the maturation of the infectious virus. Therefore, HIV protease has been one of the main targets or targets of HIV chemotherapeutic intervention. Recently, several HIV protease inhibitors have shown great potential in the treatment of HIV and have been taken advantage of for m-0 r $ & amp; amp; amp; amp;; -o. More of these HIV protease inhibitors were designated based on the structure of the protease substrates. Therefore, these compounds are peptides or peptidomimetics. The search for new and recent HIV protease inhibitors is expected to provide more effective drugs to combat this deadly disease. Preferred substrates of HIV proteases are peptides with a proline-aromatic or hydrophobic-hydrophobic peptide linkage cleavable between Pi-Pi '(West, ML, and Fairlie, DP, Trand, Pharm. Sci. 16: 67-74 ( nineteen ninety five) ) . Nine different sites in the gag and viral gag-pol proteins have been found to be cleaved by the protease (Martin, J.A., et al., Prog. Med. Chem. 32: 239-287 (1995)). The P4-P3 'sequences of these nine sites are Ser-Gln-Asn-Tyr-Pro-Ile-Val SEQ ID NO: 28, Ala-Arg-Val-Leu-Ala-Glu-Ala SEQ ID NO: 29, Ala -Thr-Ile-Met-Met-Gln-Arg SEQ ID NO: 30, Arg-Gln-Ala-Asn-Phe-Leu-Gly SEQ ID NO: 31, Pro-Gly-Asn-Phe-Leu-Gln-Ser SEQ ID NO: 32, Ser-Phe-Ser-Phe-Pro-Gln-Ile SEQ ID NO: 33, Thr-Leu-Asn-Phe-Pro-Ile-Ser SEQ ID NO: 34, Ala-Glu-Thr- Phe-Tyr-Val-Asp SEQ ID NO: 35 and Arg-Lys-Val-Leu-Phe-Leu-Asp SEQ ID NO: 36. Many fluorogenic substrates, radioactive ^^^ P-fromosenic HIV protease have been prepared based on these natural substrates for assays of HIV protease activity. An intramolecularly cooled fluorogenic substrate, 2-aminobenzoyl-Thr-Ile-Nle- (4-N02-Phe) -Gln-Arg-NH2 SEQ ID NO: 141, wherein the cleavable link is Nie- (4-N02-Phe) ), was prepared based on the hexapeptide substrate derived from the cleavage site p24 / pl5 (Toth, MV, and Marshall, GR, Int. J. Pept. Protein Res. 36: 544-550 (1990)). A fluorometric assay for HIV protease activity using HPLC with the substrate N-Dns-Ser-Gln-Asn-Tyr-Pro-Ile-Val SEQ ID NO: 28 was reported by Tamburini et al. (Tamburini, P.P., et al., Anal. Biochem. 186: 363-368 (1990)), where Tyr-Pro is the cleavable link. Many other HIV protease substrates that incorporate sequences from both the P side and the P 'side of the cleavage sites of HIV protease substrates have been developed, and these include N-alpha-benzoyl-Arg-Gly-Phe -Pro-MeO-beta-naphthylamide SEQ ID NO: 37, fluorogenic, containing the dipeptide linkage Phe-Pro recognized by the HIV-1 protease (Tyagí, SC, and Cárter, CA, Anal. Biochem. 200: 143- 148 (1992)); the radiolabelled heptapeptide substrate, [tyrosyl-3, 5-3H] Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 SEQ ID NO: 38, which is based on the cleavage site Tyr-Pro pl7- p24 found in the viral pigment protein substrate Pr55g | ÉK fyland, LJ, et al. , Anal. Biochem. 188: 408-415 (1990)); the angiotensin I-based peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser SEQ ID NO: 39, which produces angiotensin I (Ang I) and Leuko- Glu-Glu-Ser SEQ ID NO: 40 (Evans, DB, et al., Anal. Biochem. 206: 288-292 (1992)); 4- (4-dimethylaminophenylazo) benzoic acid of the intramolecular fluorescence resonance energy transfer substrate (FRET), (DABCYL) -Ser-Gln-Asn-Tyr-pro-Ile-Val-Gln -5- [(2-aminoethyl) amino] naphthalene-1 SEQ ID NO: 41, sulfonic acid (EDANS), wherein Tyr-Pro is the cleavage site (Matayoshi, ED, et al., Science 247: 954- 958 (1990)); and substrates of chromophoric peptide H-Ser-Gln-Asn-Leu-Phe (N02) -Leu-Asp-Gly-NH2 SEQ ID NO: 42 and acetyl-Arg-Lys-Ile-Leu-Phe (N02) -Leu -Asp-Gly-NH2 SEQ ID NO: 43, wherein the amide bond between the p-nitrophenylalanyl and leucyl residues is the cleavable bond. In addition, the chromogenic substrate, Lys-Ala-Arg-Val-Leu-Phe (N02) -Glu-Ala-Met SEQ ID NO: 44, where Leu-Phe (N02) is the cleavage site, was reported ( Richards, AD, et al., J. Biol. Chem. 265: 1133-1136 (1990)). SAR studies find that the substitution of Leu residue in Pi with norleucine, Met, Phe, or Tyr had minimal effects on the kinetic parameters (Kcat and KCat / Km), as determined in the values jjggj ^ H _JQá of different pH, while the peptides containing lie or Val in Pi were found to hydrolyze extremely slowly. Taking advantage of the presence of non-specific aminopeptidases in whole cells, fluorogenic or fluorescent substrates of HIV protease of Formula IX can be designated incorporating amino acids from the P side and P 'side of the HIV substrate for application in assays of whole cells. It is expected that after the peptide sequence on the P side is cleaved by the HIV protease, the sequence of the peptide in the P 'laso will be removed by aminopeptidases present in the cells. For compounds of Formula IX designated to be HIV protease substrates, Ri is preferred acetyl or Cbz, (AA) n preferred is Thr-Ile-Nle, and (AA) m preferred is Phe-Gln-Arg, Phe-Gln , or Phe; or (AA) n preferred is Ser-Leu-Asn-Phe SEQ ID NO: 54, or Leu-Asn-Phe, and (AA) m preferred is Pro-Ile-Val, Pro-lie, or Pro; or (AA) n preferred is Ser-Gln-Asn-Tyr SEQ ID NO: 45, or Gln-Asn-Tyr, and (AA) m preferred is Pro-Ile-Val-Gln SEQ ID NO: 46, Pro-Ile -Val, Pro-Val-Val-NH2, Pro-Val-NH2, Pro-lie, or Pro; or (AA) n preferred is Arg-Gly-Phe, and (AA) m preferred is Pro; or (AA) n preferred is Lys-Ala-Arg-Val-Leu SEQ ID NO: 47, Ala-Arg-Val-Leu SEQ ID NO: 48, or Arg-Val-Leu, and (AA) m preferred is Phe -Glu-Ala-Met SEQ ID NO: 49, Phe-Glu-Ala, Phe-Glu, or Phe; (AA) n preferred 2 < H¡ @ - pro-Phe-His-Leu SEQ ID NO: 50, or Phe-His-Leu, and (AA m preferred is Leu-Glu-Glu-Ser SEQ ID NO: 40, Leu-Glu-Glu, Leu -Glu, or Leu, or (AA) n preferred is Ser-Gln-Asn-Leu-Phe SEQ ID NO: 140, Gln-Asn-Leu-Phe SEQ ID NO: 51, Asn-Leu-Phe, Arg-Lys -Ile-Leu-Phe SEQ ID NO: 52, Lys-Ile-Leu-Phe SEQ ID NO: 53, or Ile-Leu-Phe, and (AA) m preferred is Leu-Asp-Gly-NH2, Leu-Asp -NH2, or Leu-NH2. (AA) n most preferred is Ser-Leu-Asn-Phe SE ID NO: 54, or Leu-Asn-Phe, and (AA) m most preferred is Pro-Ile-Val, Pro -lie, or Pro; or (AA) n is most preferred Arg-Gly-Phe, and (AA) m more preferred is Pro. The HIV protease substrates of Formula IX are expected to function in whole cell assays but otherwise, it will not work in cell-free enzyme assays Cleavage of the amide bond (AA) n- (AA) m by HIV protease in HIV-infected cells will provide Rhodamina bound to (AA) M. The aminopeptidases present inside the cells will then remove the (AA) m to give the dye fl uorescent of Formula IV. The compounds of Formula IX will be used for the selection of HIV protease inhibitors in HIV-infected cells. This should accelerate the process for the discovery of new HIV protease inhibitors, especially the discovery of non-peptidomimetic or non-peptide HIV protease inhibitors, which should lead to better anti-HIV agents than t 7 drugs commonly Since HIV protease processes viral precursor proteins in an earlier state in viral replication, a fluorogenic or fluorescent substrate permeable to cells by an HIV protease can also be used to select compounds which inhibit the transcription or translation of genes , viral entry, or other key proteins in the earliest stage of HIV infection. Therefore, this method can lead to the identification of inhibitors of HIV infections with a new mechanism, which could not be identified in a cell-free enzyme assay. In addition, since the HIV protease in HIV-infected cells will cleave the cell-permeable substrates of Formula IX to produce the fluorescent dye of Formula VI within the cells, the substrates of Formula V can also be used for diagnosis of HIV infection. The compounds of Formula IX can also be referred to as adenovirus protease substrates. Adenoviruses are the cause of several diseases including sporopathic respiratory disease and acute respiratory disease that can lead to pneumonia. Adenovirus protease is a cysteine protease that cleaves several viral proteins and is required for virus maturation and infectious quality (Weber, J.M., Curr. Top. MJ _? - »c *" b, iol Immunol 199/1: 221-235 (1995).) Preferred substrates of adenovirus protease include (M, L, I) XGX-G and (M, L, I) XGG-X The specificity of the substrates is determined primarily by amino acids P2 and P (Diouri, M., et al., J. Biol. Chem. 271: 32511-32514 (1996).) Hydrophobic amino acids such as Met, Leu e lie are preferred at P4, Minor amino acids such as Gly are preferred at P2, A minor hydrophobic amino acid is also preferred for Pi and Pi ', such as Ala and Gly.; whereas P3 can accommodate almost any amino acid. These observations are supported by the newly determined crystal structure of human adenovirus proteinase with its 11 amino acid cofactor and substrate that is modeled based on the crystal structure (Ding, J., et al., EMBO J. 15: 1118- 1183 (1996)). Taking advantage of the presence of aminopeptidase in whole cells, the fluorogenic or fluorescent substrates of adenovirus protease can be designated to incorporate amino acids either on the P side only, or both on the P side and on the P 'side of the adenovirus protease substrate for the application in whole cell assays. For compounds of Formula IX designated as adenovirus protease substrates, Ri is preferred acetyl or Cbz, (AA) n preferred is Leu-Arg-Gly-Gly SEQ ID NO: 55, Met-Arg-Gly-Gly SEQ ID NO: 55 NO: 56, Ile-Arg-Gly-Gly SEQ ID NO: 57, Leu-Val-Gly-Gly SEQ ID NO: 58, Met-Val-Gly-Gly SEQ ID NO: 59 or Ile-Val-Gly-Gly SEQ ID NO: 60, and (AA) m is preferred Gly, Ala, om = 0. When m is 0, the cleavage of the amide bond of (AA) n-Rhodamine by the adenovirus protease will produce a fluorescent dye of Formula VI. When m is not 0, the cleavage of the amide bond (AA) n- (AA) by adenovirus protease in the cells will provide the Rhodamine bound to (AA) m. The aminopeptidases present within the cells will then remove (AA) m to give the fluorescent dye of Formula VI. The compounds of Formula IX will be used for the selection of adenovirus protease inhibitors in cells infected with adenovirus. The compounds of Formula IX can also be designated as protease substrates of herpes simplex virus type 1 (HSV-1). The simple human herpes virus type 1 is responsible for herpes labialis (cold ulcers). The HSV-1 protease is a serine protease and is responsible for proteolytic processing by itself and ICP35 for the assembly of viral capsid (Gao, M., et al., J. Virol. 68: 3102-3112 (1994)). Two proteolytic sites have been identified as Ala247 and Ser248 and Ala610 and Ser611 within the protease (Dilanni, C.L., et al., J. Biol. Chem. 268: 25449-25454 (1993)). of eight amino acids that is cleaved as efficiently as peptide from the 20-mer maturation site, and the sequence from P to Pi was defined as the minimum substrate recognition domain for the HSV-1 protease (O'Boyle, DR , et al., Virology 236: 338-341 (1997)). It has also been reported that the specificity of the HSV-1 protease resides within the P-P region? of cleavage sites (McCann, P.J., et al., J. Virol 68: 526-529 (1994)). Taking advantage of the presence of aminopeptidase in whole cells, fluorescent or fluorogenic substrates of HSV-1 protease are designated to incorporate amino acids of P-P? only, or both of P4-P? and the P 'side of the HSV-1 protease substrate for application in whole cell assays. For compounds of Formula IX designated to be HSV-1 protease substrates, Ri is preferred acetyl or Cbz, (AA) n preferred is Leu-Val-Leu-Ala SEQ ID NO: 62, and (AA) m preferred is Ser, Ser-Ser, om = 0. When m is 0, cleavage of the (AA) n-Rhodamine bond by the HSV-1 protease will produce the fluorescent dye of Formula VI. When it is not 0, cleavage of the amine bond (AA) n- (AA) m by HSV-1 in the cells will provide the Rhodamine linked to (AA) m. The aminopeptidases present within the cells then reagent (AA) m to provide the fluorescent dye of Formula VI. The compounds of Formula IX will be used for the selection of HSV-1 protease inhibitors in cells infected with HSV-1. The compounds of Formula IX can also be designated as human cytomegalovirus protease substrates (HCMV). HCMV can cause life-threatening infections in congenitally infected children, immunocompromised individuals, and transplant or immunosuppressed patients. The human cytomegalovirus (HCMV) encodes a protease that it cleaves by itself and the HCMV congregation protein and is essential for the replication of the virus, therefore, it is a potential target for therapeutic intervention. The HCMV protease is a serine protease and two proteolytic processing sites within the protease were identified in Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). (Sztevens, J.T., et al., Eur. J. Biochem. 226: 361-361 (1994)). A fluorogenic substrate, DABCYL-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser-Arg-Leu-Ala-EDANS SEQ ID NO: 63 was synthesized and found to be efficiently cleaved by the CMV protease in the peptide binding Ala-Ser (Holskin, BP, et al., Anal. Biochem. .. • 227: 148-155 (1995)). Rec ± | É | t & it was reported that the replacement of the Ire Val-Val sequence that corresponds to the P-P2 residues of the enzyme's maturation site by the optimized Tbg-Tbg-Asn (NMe2) (Tbg, t-butylglycine) sequence significantly increases the affinity of the substrate for the protease. An AMC fluorogenic substrate was prepared with the lateral peptide P sequence including these improved amino acids (Bonneau, P.R., et al., Anal.Biochem 255: 59-65 (1998)). Taking advantage of the presence of aminopeptidase in whole cells, fluorescent or fluorogenic substrates of HCMV protease are designated to incorporate amino acids either from the P side only, or both from the P side and from the P 'side of the HCMV protease substrate for the application in whole cell assays. For compounds of Formula IX designated to be HCMV protease substrates, Ri is preferred acetyl or Cbz, (AA) n preferred is Val-Val-Asn-Ala SEQ ID NO: 64, Tbg-Tbg-Asn-Ala SEQ ID NO: 65, and (AA) m preferred is Ser, Ser-Ser, om = 0. When m is 0, the cleavage of the amide bond (AA) n-Rhodamine by the HCMV protease will produce the fluorescent dye of the Formula SAW. When it is not 0, cleavage of the amide bond of (AA) n- (AA) m by HCMV in the cells will provide the Rhodamine bound to (AA) m. The aminopeptidases present inside the cells will remove mtía * ~ j v ^ & 7 then the fluorescent dye of Formula VI. The compounds of Formula IX will be used for the selection of HCMV protease inhibitors in cells infected with HCMV. The compounds of Formula IX can also be designated to be substrates of the hepatitis C virus protease (HCV). HCV is the main causative agent of both sporadic and parenterally transmitted hepatitis A and non B, which infects an estimated 50 million people worldwide. The NS3 protease of HCV and its activator of protein NS4A participates in the processing of the viral polyprotein, thus the NS3 / 4A protease complex is an attractive target for antiviral therapy against HCV. The HCV protease is a serine protease and Cys-Ser has been identified as a cleavage site. A sequence of the substrate is Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Tyr SEQ ID NO: 66, and Pi Cys and P3 Val were found to be critical (Zhang, R., et al. , J. Virol. 71: 6208-6213 (1997)). Taking advantage of the presence of aminopeptidase in whole cells, fluorogenic or fluorescent substrates of HCV protease are designated to incorporate amino acids from both the P side and the P 'side of the HCV protease substrate for application in whole cell assays.

For compounds of Formula IX designated to be HCV protease substrates, Ri is preferred acetyl or Cbz, (AA) n preferred is Asp-Asp-Ile-Val-Pro-Cys SEQ ID NO: 67, Asp-Ile-Val -Pro-Cys SEQ ID NO: 68, or Ile-Val-Pro-Cys SEQ ID NO: 69 and (AA) m preferred is Ser-Met-Ser-Tyr SEQ ID NO: 70, Ser-Met-Ser, Ser -Met, Ser, om = 0. When m is 0, the cleavage of the amide bond (AA) n-Rhodamine by the HCV protease will produce the fluorescent dye of Formula VI. When m is not 0, the cleavage of the amide bond (AA) n- (AA) by HCV in the cells will give the Rhodamine bound to (AA) m. The aminopeptidases present within the cells will then remove the (AA) m to give the fluorescent dye of Formula VI. The compounds of Formula IX will be used for the selection of HCV protease inhibitors in cells infected with HCV. The invention also relates to novel compounds of Formula VI which are derivatives of a Rhodamine and are obtained by introducing a blocking group R into one of the two amino groups in a Rhodamine. The R2HN group in Formula VI provides the binding site for the reaction with a potential enzyme substrate, such as the carboxyl group of an N-blocked peptide, to form an amide bond of the peptide. The reaction converts the fluorescent molecule of Formula VI into a non-fluorescent molecule of Formulas VII-IX which produces a repo-a-peptide molecule that functions as a substrate for a pro-ase or peptidase. The reporter peptide-amide bond in Formulas VII-IX is the cleavable bond under biological conditions. Cleavage of the reporter peptide-amide bond cleavable in the reporter peptide by proteases or peptidases, produces a compound of Formula VI or VI 'which is fluorescent. More importantly, the blocking group can incorporate a hydrophobic group. The hydrophobic group is designed to increase the permeability of the membrane of the substrates, and results in an accumulation of the substrate within the cells, as well as to increase the retention of the fluorescent portion within the cells after their cleavage by protease or white peptidase. The new fluorescent dyes of this invention are of Formula VI: or biologically acceptable salts wherein R2-Rβ are defined above with respect to Formula VII. Preferred R2 and R3 are hydrogen, methyl or ethyl; Preferred R and Rs are hydrogen or methyl.

The compounds of Formula VI of the present invention can exist in tautomeric forms, particularly the ring opening form of Formula VI '. The invention includes all tautomeric forms including VI and VI '.

Fluorogenic or fluorescent substrates of the present invention are compounds having the Formula II and include, but are not limited to: (Z-WEHD) 2-Rhodamine 110, SEQ ID NO: 1 (Z-YVAD) 2-Rhodamine 110, SEQ ID NO: 2 (Z-DETD) 2-Rhodamine 110, SEQ ID NO: 4 (Z-DEVD) 2-Rhodamine 110, SEQ ID NO: 5 (Z-DEHD) 2-Rhodamine 110, SEQ ID NO: 6 (Z-VEHD) 2-Rhodamine 110, SEQ ID NO: 7 (Z-LETD) 2-Rhodamine 110, SEQ ID NO: 8 (Z-LEHD) 2-Rhodamine 110, SEQ ID NO: 3 20 (Z-LEVD) 2-Rhodamine 110, SEQ ID NO: 9 (Z- IEPD) 2-Rhodamine 110, SEQ ID NO: 23 (Z-VEPD) 2-Rhodamine 110, SEQ ID NO: 27 (Z-SHVD) 2-Rhodamine 110, SEQ ID NO: 10 (Z-DELD) 2-Rhodamine 110, SEQ ID NO: 11 m msiB (Z-DGPD) 2-Rhodamine 110, SEQ ID NO: 12 (Z-DEPD) 2-Rhodamine 110, SEQ ID NO: 13 (Z-DGTD) 2-Rhodamine 110, SEQ ID NO: 14 (Z- DLND) 2-Rhodamine 110, SEQ ID NO: 15 (Z-DEED) 2-Rhodamine 110, SEQ ID NO: 16 (Z-DSLD) 2-Rhodamine 110, SEQ ID NO: 17 (Z-DVPD) 2-Rhodamine 110, SEQ ID NO: 18 (Z-DEAD) 2-Rhodamine 110, SEQ ID NO: 19 (Z-DSYD) 2-Rhodamine 110, SEQ ID NO: 20 (Z-ELPD) 2-Rhodamine 110, SEQ ID NO : 21 (Z-VEID) 2-Rhodamine 110, SEQ ID NO: 26 (Z-IETD) 2-Rhodamine 110, SEQ ID NO: 24 (Z-VD) 2-Rhodamine 110, (Z-TD) 2-Rhodamine 110, (Z-AD) 2-Rhodamine 110, (Z-VAD) 2-Rhodamine 110, (Boc-WEHD) 2-Rhodamine 110, SEQ ID NO: 1 (Boc-YVAD) 2-Rhodamine 110, SEQ ID NO. : 2 (Boc-DETD) 2-Rhodamine 110, SEQ ID NO: 4 (Boc-DEVD) 2-Rhodamine 110, SEQ ID NO: 5 (Boc-DEHD) 2-Rhodamine 110, SEQ ID NO: 6 (Boc-DED) VEHD) 2-Rhodamine 110, SEQ ID NO: 7 (Ac-YVAD) 2-Rhodamine 110, SEQ ID NO: 2 (Ac-LETD) 2-Rhodamine 110, SEQ ID NO: 8 (Ac-LEHD) 2-Rhodamine 110, SEQ ID NO: 3 (Ac-DEVD) 2-Rhodamine 110, SEQ ID NO: 5 (Ac-LEVD) 2-Rhodamine 110, SEQ ID NO: 9 (Ac-IEPD) 2-Rhodamine 110, SEQ ID NO: 23 (Ac-VEPD) 2-Rhodamine 110, SEQ ID NO: 27 5 (Ac-VD) 2-Rhodamine 110, (Ac-TD) 2 -Rhodamine 110, (Ac-AD) 2-Rhodamine 110, (Ac-VAD) 2-Rhodamine 110, (Z-YVAD) 2-Rhodamine 116, SEQ ID NO: 2 10 (Z-LEHD) 2-Rhodamine 116, SEQ ID NO: 3 (Z-DETD) 2-Rhodamine 116, SEQ ID NO: 4 (Z-DEVD) 2-Rhodamine 116, SEQ ID NO: 5 (Z-YVAD) 2-Rhodamine 19, SEQ ID NO: 2 (Z-LEHD) 2-Rhodamine 19, SEQ ID NO: 3 15 (Z-DETD) 2-Rhodamine 19, SEQ ID NO: 4 (Z-DEVD) 2-Rhodamine 19, SEQ ID NO: 5 (Z-YVAD) (OAM)) 2-Rhodamine 110, SEQ ID NO: 2 (Z-LE (OAM) HD (OAM)) 2-Rhodamine 110, SEQ ID NO: 3 (ZD (OAM) E (OAM) TD (OAM)) 2-Rhodamine 110, SEQ ID NO: 4 20 (ZD (OAM) E (OAM) VD (OAM)) 2-Rhodamine 110, SEQ ID NO: 5 (ZD (OMe) E (OMe) VD (ODAM)) 2 -Rhodamine 110, and SEQ ID NO: 5 (ZD (OMe) E (OMe) VD) 2-Rhodamine 110, SEQ ID NO: 5 Fluorogenic or fluorescent substrates of the present invention are compounds having Formula 25 VII and include , but are not limited to: £ • "£ £ £ £? N- (Z-WEHD -N'-acetyl -Rhodamine 110, SEQ ID NO: l N- (Z-YVAD-N '-acetyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-LEHD - N '-acetyl-Rhodamine 110, SEQ ID NO: 3 N- (Z-LEVD-N' -acetyl-Rhodamine 110, SEQ ID NO: 9 N- (Z-DETD-N '-acetyl-Rhodamine 110, SEQ ID NO: 4 N- (Z-DEVD-N '-acetyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-DEHD-N' -acetyl-Rhodamine 110, SEQ ID NO: 6 N- ( Z- • VEHD -N'-acetyl -Rhodamine 110, SEQ ID NO: 7 N- (Z-LETD -N'-acetyl -Rhodamine 110, SEQ ID NO: 8 N- (Z-IEPD -N '- acetyl -Rhodamine 110, SEQ ID NO: 23 N- (Z- • VEPD-N '-acetyl-Rhodamine 110, SEQ ID NO: 27 N- (Z- • SHVD-N' -acetyl-Rhodamine 110, SEQ ID NO. : 10 N- (Z- • DELD -N '-acetyl -Rhodamine 110, SEQ ID NO: ll N- (Z- DGPD -N' -acetyl '-Rhodamine 110, SEQ ID NO: 12 N- (Z- • DEPD -N '-acetyl-Rhodamine 110, SEQ ID NO: 13 N- (Z- DGTD-N' -acetyl-Rhodamine 110, SEQ ID NO: 14 N- (Z- DLND -N '-acetyl-- Rhodamine 110, SEQ ID NO: 15 N- (Z- DEED-N'-acetyl-Rhodamine 110, SEQ ID NO: 16 N- (Z- DSLD-N '-acetyl-Rhodamine 110, SEQ ID NO: 17 N- (Z- DVPD-N '-acetyl-Rhodamine 110, SEQ ID NO: 18 N- (Z-DEAD-N' -acetyl-Rhodamine 110, SEQ ID NO: 19 N - (Z- DSYD-N'-acetyl-Rhodamine 110, SEQ ID NO: 20 N- (Z- ELPD-N'-acetyl-Rhodamine 110, SEQ ID NO: 21 N- (Z-VEID -N ' acetyl-Rhodamine 110, SEQ ID NO: 26 N- (Z-IETD-N'-acetyl-Rhodamine 110, SEQ ID NO: 24 N- ((Z-VD) -N '-acetyl-Rhodamine 110, N- ((Z-TD) -N'-acetyl-Rhodamine 110, N- ((Z-AD) -N '-acetyl-Rhodamine 110, N- ((Z-VAD) -N' -acetyl-Rhodamine 110 , N- ((Boc-WEHD) -N '-acetyl-Rhodamine 110, SEQ ID NO: l N- ((Boc-YVAD) -N' -acetyl-Rhodamine 110, SEQ ID NO: 2 N- ((Ac -ELD) -N'-acetyl-Rhodamine 110, SEQ ID NO: 8 N- ((Ac-LEHD) -N '-acetyl-Rhodamine 110, SEQ ID NO: 3 N- ((Z-DEVD) -N' -methoxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- ((Z-YVAD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- ((Z-LEVD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- ((Z-LEHD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 3 N- ((Ac-WEHD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: l N- ((Ac-YVAD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- ((Ac-DEVD) -N '-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- ((Ac-DEHD) -N '-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 6 N- ((Ac-DETD) -N '-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 4 N- ((Ac-LEVD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- ((Ac-LEHD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 3 N- ((Ac-LETD) -N '-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 8 N- ((Ac-VEHD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 7 N- ((Ac-IEPD) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 23 N- ((Z-WEHD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: l N- ((Z-YVAD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- ((Z-DEVD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N'-ethoxycarbonyl-Rhodamine 110 , SEQ ID NO: 5 N- (Ac-DEHD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N ' -ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: l N- (Z-YVAD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-) DEVD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: N- (Ac-WEHD) -N'-hexyloxycarbonyl-Rhodamine 110, SEC ID NO: l N- (Ac-YVAD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Ac- DEHD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N'-hexyloxycarbonyl- Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N '-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) - N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 1 N- (Z-YVAD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: l N- ( Ac-YVAD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N - (Ac-DEHD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N '-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N '-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N'-octyloxycarbonyl-Rhodamine 110, SEC ID NO: 8 N- (Ac-VEHD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z -WEHD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: l N- (Z-YVAD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N'-decyloxycarbonyl -Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Ac-DEHD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) - N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: l N- (Z-YVAD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- ( Ac-WEHD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N '- dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Ac-DEHD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO : 4 N- (Ac-LEVD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) ) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N'-dodecyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 7 N- (Ac-IEPD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-DEVD) -N'- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO. : N- (Z-YVAD) -N'- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-LEVD) -N'- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Z-LEHD) -N'- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-WEHD) -N'- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N'- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N'- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Ac -DEHD) -N '- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N' - (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD ) -N '- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N' - (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) - N '- (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N' - (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N ' - (methylthio) carbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Z-YVAD) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N - (Z-DEVD) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- ( Ac-WEHD) -N '- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N' - (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac- DEVD) -N '- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Ac-DEHD) -N' - (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N '- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N' - (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N '- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N' - (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N '- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N '- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) -N'- (hexylthio ) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Z-YVAD) -N'- (hexylthi o) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N'- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N '- • (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N' - (hexylthio) carbonyl -Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N '- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Ac-DEHD) -N' - (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N '- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N' - (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N '- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N' - (hexylthio) carbonyl-Rhodamine 110, SEQ ID. NO: 8 N- (Ac-VEHD) -N '- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N' - (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO. : 23 N- (Z-WEHD) -N'- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 1 N- (Z-YVAD) -N'- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N'- (octylthio) carboni l-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N '- (octylthio) carbonyl-Rhodamine 110, SEC ID NO: l N- (Ac-YVAD) -N '- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N' - (octylthio) carbonyl-Rhodamine 110, SEQ ID NO. : N- (Ac-DEHD) -N '- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N' - (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N '- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N' - (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N '- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 8 N- (AC-VEHD) -N' - (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N '- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Z-YVAD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Ac-DEHD) -N'- (decylthio) carbonyl-Rhodamma 110, SEQ ID NO: 6 N- (Ac-DETD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N '- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Z-YVAD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N' - (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5 N- (Ac-DEHD) -N' - (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N' - (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) -N' - (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 1 N- (Z YVAD) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N' - (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD ) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N' - (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 1 N- (Ac-YVAD) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N' - (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 5 N- (Ac-DEHD) -N '- (dimethylcarbamyl) ) -Rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N' - (dimethylcarbamyl) -Rhodamine 110 , SEQ ID NO: 9 N- (Ac-LEHD) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 6 N- (Ac-LETD) -N' - (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO. : 8 N- (Ac-VEHD) -N '- (d imethylcarbamyl) -Rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 23 N- (Z-WEHD) -N'- (N-hexyl -N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: l N- (Z-YVAD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: 2 N- (Z-DEVD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: 5 N- (Z-LEVD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: 9 N- (Ac-WEHD) -N '- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: l N- (Ac-YVAD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamina 110, SEC ID NO: 2 N- (Ac-DEVD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEC ID NO: 5 N- (Ac-DEHD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEC ID NO: 6 N- (Ac-DETD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEC ID NO: 4 N- (Ac-LEVD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD) -N'- (N-hexyl-N -metilcarbamil) -Rhodamina 110, SEC ID NO: 3 N- (Ac-LETD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEC ID NO: 8 N- (Ac-VEHD) -N '- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEC ID NO: 7 N- (Ac-IEPD) -N'- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEC ID NO: 23 N (Z-DEVD) -N '-methansulfonyl-Rhodamine 110, SEQ ID NO: 5 N (Z-YVAD) -N' -methansulfonyl-Rhodamine 110, SEQ ID NO: 2 N (Z-DEVD) -N'-acetyl-Rhodamine 116, SEQ ID NO: 5 N (Z-YVAD) -N'-methanesulfonyl-Rhodamine 116, SEQ ID NO: 2 N (Z-DEVD) -N'-acetyl-Rhodamine 19, SEC ID NO: 5 N (Z-YVAD) -N '-metanesulfonyl-Rhodamine 19, SEQ ID NO: 2 N (Z-YVAD (OAM)) -N' -acetyl-Rhodamine 110, SEQ ID NO: 2 N- ( Z-LE (0AM) HD (0AM)) -N'-acetyl-Rhodamine 110, SEQ ID NO: 3 N- (ZD (OAM) E (OAM) TD (OAM)) -N'-acetyl-Rhodamine 110, SEQ ID NO: 4 N- (ZD (OAM) E (OAM) VD (OAM) )) -N'-acetyl-Rhodamine 110, SEQ ID NO: 5 N- (ZD (OMe) E (OMe) VD (ODAM)) -N'-acetyl-Rhodamine 110, and SEQ ID NO: 5 N- ( ZD (OMe) E (OMe) VD) -N'-acetyl-Rhodamine 110, SEQ ID NO: 5 N- (Z-VD (OAM)) -N'-acetyl-Rhodamine 110, and N- (ZE (OAM) ) VD (OAM)) -N'-acetyl-Rhodamine 110. Other preferred fluorogenic or fluorescent substrates of the present invention are compounds having the Formula VIII and include, but are not limited to: N- (Z-WEHDG) -N '-acetyl-Rhodamine 110, SEQ ID NO: 71 N- (Z-YVADG) -N' -acetyl-Rhodamine 110, SEQ ID NO: 72 N- (Z-LEHDG) -N '-acetyl-Rhodamine 110,' SEQ ID NO: 73 N- (Z-LEVDG) -N '-acetyl-Rhodamine 110, SEQ ID NO: 74 N- (Z-DETDG) -N' -acetyl-Rhodamine 110, SEQ ID NO: 75 N- ( Z-DEVDG) -N'-acetyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-LETDG) -N '-acetyl-Rhodamine 110, SEQ ID NO: 77 N- (Ac-LEHDG) -N' - acetyl-Rhodamine 110, SEQ ID NO: 73 N- (Ac-WEHDG) -N'-methoxycarbonyl-Rho damina 110, SEQ ID NO: 71 N- (Ac-YVADG) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Z-WEHDG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 79 N- (Z-YVADG) - N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Z-DEVDG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Z-LEVDG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 74 N- (Ac-WEHDG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 72 N- ( Ac-DEVDG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N '-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N' -hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N '-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N '-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N '-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N '-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N' -octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 72 NO: 76 N- (Ac-DEHDG) -N '-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N'- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N'- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N'- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N'- (hexylthio) carbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N'- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N'- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N'- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N'- (octylthio) carbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 142 N- (Ac-DEVDG) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N'- (decylthio) carbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID N0: 71 N- (Ac-YVADG) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N' - (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N '- (dodecylthio) carbonyl-Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N'- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N '- (dimethylcarbamyl) -Rhodamine 110, SEQ ID NO: 78 N- (Ac-WEHDG) -N '- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: 71 N- (Ac-YVADG) -N '- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: 72 N- (Ac-DEVDG) -N '- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: 76 N- (Ac-DEHDG) -N '- (N-hexyl-N-methylcarbamyl) -Rhodamine 110, SEQ ID NO: 78 N (Z-DEVDG) -N'-methanesulfonyl-Rhodamine 110, SEC ID NO: 76 N (Z-YVADG) -N'-methanesulfonyl-Rhodamine 110, SEQ ID NO: 72 N (Z-DEVDG) -N'-acetyl-Rhodamine 116, SEQ ID NO: 76 N (Z-YVADG) -N'-methanesulfonyl-Rhodamine 116, SEQ ID NO: 72 N (Z-DEVDG) -N '- acetyl-Rhodamine 19, and SEQ ID NO: 76 N (Z-YVADG) -N'-methanesulfonyl-Rhodamine 19 SEQ ID NO: 72. Other preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula IX and include , but are not limited to: N- (GP) -N'-octyloxycarbonyl-Rhodamine 110, N- (GPG) -N '-octyloxycarbonyl-Rhodamine 110, N- (GP) -N'-ethoxycarbonyl-Rhodamine 110, N - (GPG) -N'-ethoxycarbonyl-Rhodamine 110, N- (GPA) -N'-ethoxycarbonyl-Rhodamine 110, N- (GP) -N'-hexyloxycarbonyl-Rhodamine 110, N- (GPG) -N '- hexyloxycarbonyl-Rhodamine 110, N- (GP) -N '- (ethylthio) carbonyl-Rhodamine 110, N- (GPG) -N' - (ethylthio) carbonyl-Rhodamine 110, N- (MG) -N '-octyloxycarbonyl- Rhodamina 110, N- (MA) -N'-octyloxycarbonyl-Rhodamine 110, N- (MGG) -N '-octyloxycarbonyl-Rhodamine 110, N- (MGA) -N' -octyloxycarbonyl-Rhodamine 110, N- (MAG) -N '-octyloxycarbonyl-Rhodamine 110, NGN' -octyloxycarbonyl-Rhodamine 110, N- (MG) -N'-ethoxycarbonyl-Rhodamine 110, N- (MA) -N '-ethoxycarbonyl-Rhodamine 110, NGN' -ethoxycarbonyl-Rhodamine 110 , N- (MG) -N '-hexyloxycarbonyl-Rhodamine 110, N- (MA) -N' -hexyloxycarbonyl-Rhodamine 110, NGN '-hexyloxycarbonyl-Rhodamine 110, N- (MG) -N' - (ethylthio) carbonyl -Rhodamine 110, NGN '- (ethylthio) carbonyl-Rhodamine 110, N- (Boc-LM) -N' -octyloxycarbonyl-Rhodamine 110, N- (Ac-LM) -N '-octyloxycarbonyl-Rhodamine 110, N- ( Boc-LM) -N'-ethoxycarbonyl-Rhodamine 110, N- (Ac-LM) -N'-ethoxycarbonyl-Rhodamine 110, N- (Boc-LM) -N '-hexyloxycarbonyl-Rhodamine 110, N- (Ac- LM) -N '-hexyloxycarbonyl-Rhodamine 110, N- (Boc-LM) -N "- (ethylthio) carbonyl-Rhodamine 110, N- (Ac-LM) -N'- (ethylthio) carbonyl-Rhodamine 110, N - (Ac-SLNFPIV) -N'-octyloxycarbo nil-Rhodamine 110, SEQ ID NO: 80 N- (Ac-SLNFPI) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 81 N- (Ac-SLNFP) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 82 N- (Ac-LNFPIV) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 83 N- (Ac-LNFPI) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 84 02 N- (Ac-LNFP) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 85 N- Ac-RGFP) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 37 5 N- Z-LNFPIV) -N'- octyloxycarbonyl-Rhodamine 110, SEQ ID NO 83 N- Z-LNFPI) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 84 N- Z-LNFP) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 85 N- Z-RGFP) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 37 N- Z-RQANFLG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 31 N- Z-RQANFL) -N'- octyloxycarbonyl-Rhodamine 110, SEQ ID NO 86 15 N-Z-RQANF) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 87 N- Z-RKVLFLD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 36 N- Z-RKVLFL) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 88 N- Z-RKVLF) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 89 N- Z-ARVLFLG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 90 _ ^ fe £ _fe N- (Z-ARVLFL) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 91 N- Z-ARVLF) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 92 N- Z-SQNYFLG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 93 N- Z-SQNYFL) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 94 N- Z-SQNYF) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO. 95 N-Ac-SLNFPIV) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO 80 N-Ac-SLNFPI) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 81 N-Ac-SLNFP) -N'- ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 82 N-Ac-RGFP) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 37 N-Ac-SLNFPIV) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 37 NO 80 N-Ac-SLNFPI) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO 81 N- Ac-SLNFP) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO 82 N- Ac- RGFP) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO 37 N-Ac-MRGGG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO 96 N- (Ac-IRGGG) -N'-octyloxycarbonyl- Rhodamina 110, SEC ID NO: 97 N- (Ac-LVGGG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 98 N- (Ac-MVGGG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 99 N- (Ac-IVGGG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 100 N- (Ac-LRGGG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 101 N- (Ac-LRGGA) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 102 N- (Ac-LRGG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 55 N- (Z-LRGGG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO.101 N- (Z-LRGGA) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO.

NO: 102 N- (Z-LRGG) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 55 N- (Ac-LRGGG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 101 N- (Ac-LRGGA) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 102 N- (Ac-LRGG) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 55 N- (Ac-LRGGG) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 101 N- (Ac-LRGGA) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 102 N- (Ac-LRGG) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 55 N- (Ac-LVLASSS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 103 N- (Ac-LVLASS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 104 N- (Ac-LVLAS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 105 N- (Ac-LVLA) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 62 N- (Z-LVLASSS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 103 N- (Z-LVLASS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 104 N- (Z-LVLAS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 105 N- (Z-LVLA) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 62 N- (Ac-LVLASS) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 104 N- (Ac-LVLAS) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 105 N- (Ac-LVLA) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 62 N- (Ac-LVLASS) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID N- (Ac-LVLAS) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 105 N- (Ac-LVLA) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 62 N- (Ac-VVNASS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 106 N- (Ac-VVNAS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 107 N- (Ac-VVNA) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 64 N- (Ac-Tbg-Tbg-NASS) -N'-octyloxycarbonyl-Rhodamine 110, SEC ID NO: 108 N- (Ac-Tbg-Tbg-NAS) -N'-octyloxycarbonyl-Rhodamine 110, SEC ID NO: 109 N- (Ac-Tbg-Tbg-NA) -N'-octyloxycarbonyl-Rhodamine 110, SEC ID NO: 110 N- (Z-Tbg-Tbg-NASS) -N'-octyloxycarbonyl-Rhodamine 110, SEC ID NO: 108 N- (Z-Tbg-Tbg-NAS) -N '-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 109 N- (Z-Tbg-Tbg-NA) -N' -octyloxycarbonyl-Rhodamine 110, SEC ID NO.110 N- (Ac-Tbg-Tbg-NASS) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 108 X & amp;? ^^ & N- (Ac-Tbg-Tbg-NAS) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 109 N- (Ac-Tbg-Tbg-NA) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 110 N- (Ac-Tbg-Tbg-NASS) -N '- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 108 N- (Ac-Tbg-Tbg-NAS) -N'- (ethylthio) carbonyl-Rhodamine 110, SEC ID NO: 109 N- (Ac-Tbg-Tbg-NA) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 110 N- (Ac-DDIVPCSMST) -N'-octyloxycarbonyl-Rhodamine 110, SEC ID NO.lll N- (Ac-DIVPCSMST) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 112 N- (Ac-IVPCSMST) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 113 N- (Ac-IVPCSMS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 114 N- (Ac-IVPCSM) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 115 N- (Ac-IVPCS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 116 N- (Ac-IVPC) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 69 N- (Z-IVPCSMST) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 113 N- (Z-IVPCSMS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 114 N- (Z-IVPCSM) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 115 N- (Z-IVPCS) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 116 N- (Ac-IVPCSMS) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 114 N- (Ac-IVPCSM) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 115 N- (Ac-IVPCS) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 116 N- (Ac-IVPCSMS) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 114 N- (Ac-IVPCSM) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 115 N- (Ac-IVPCS) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 116 wherein Z is benzyloxycarbonyl, BOC is tert-butoxycarbonyl, Ac is acetyl, Tbg is t-butylglycine, and AM is acetoxymethyl. The new preferred fluorescent dyes of the present invention are the compounds having the Formula VI and include, but are not limited to: N-formyl-Rhodamine 110, N-acetyl-Rhodamine 110, N-hexanoyl-Rhodamine 110, N-octanoyl-Rhodamine 110, N-decanoyl-Rhodamine 110, N-dodecanoyl-Rhodamine 110, N-methoxycarbonyl-Rhodamine 110, N -ethoxycarbonyl-Rhodamine 110, N-butoxycarbonyl-Rhodamine 110, N-hexyloxycarbonyl-Rhodamine 110, N-octyloxycarbonyl-Rhodamine 110, N-decyloxycarbonyl-Rhodamine 110, N-dodecyloxycarbonyl-Rhodamine 110, N-benzyloxycarbonyl-Rhodamine 110, N- (2-butoxyethoxycarbonyl) -Rhodamine 110, N- (2, 5, 8-trioxadecyloxycarbonyl) -Rhodamine 110, N- (methylthio) carbonyl-Rhodamine 110, N- (ethylthio) carbonyl-Rhodamine 110, N- (butylthio) carbonyl-Rhodamine 110, N- (hexylthio) carbonyl-Rhodamine 110, N- (octylthio) carbonyl-Rhodamine 110, N- (decylthio) carbonyl-Rhodamine 110, N- (dodecylthio) carbonyl-Rhodamine 110, N-methanesulfonyl-Rhodamine 110, N-ethanesulfonyl-Rhodamine 110, N-hexansulfonyl-Rhodamine 110, N-octansulfonyl-Rhodamine 110, N- decansulfonyl-Rhodamine 110, N-dodecansulfonyl-Rhodamine 110, 5 N-trifluoromethanesulfonyl-Rhodamine 110, N-dimethylcarbamyl-Rhodamine 110, N-diethylcarbamyl-Rhodamine 110, N- (N-methyl-N-hexylcarbamyl) -Rhodamine 110, N - (N-methyl-N-octylcarbamyl) -Rhodamine 110, N- (N-methyl-N-decylcarbamyl) -Rhodamine 110, N-acetyl-Rhodamine 116, N-methoxycarbonyl-Rhodamine 116, N-ethoxycarbonyl-Rhodamine 116, N-octyloxycarbonyl-Rhodamine 116, N-hexyloxycarbonyl-Rhodamine 116, N-benzyloxycarbonyl-Rhodamine 116, N-methanesulfonyl-Rhodamine 116, N-trifluoromethanesulfonyl-Rhodamine 116, N-octansulfonyl-Rhodamine 116, N-acetyl-Rhodamine 19, N-ethoxycarbonyl-Rhodamine 19, N-octyloxycarbonyl-Rhodamine 19, N-methoxycarbonyl-Rhodamine 19, and N-methanesulfonyl-Rhodamine 19. 25 . * iáxás, *} . ' S ^ c f »2 &&&&&&Typical aryl groups are aryl groups of 6 to 10 carbon atoms including phenyl, naphthyl, fluorenyl and the like, any of which can be subsituated with the halo or alkyl groups. Typical alkyl groups are alkyl groups of 1 to 10 carbon atoms including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and branched chain isomers thereof. Typical acyl (alkanoyl) groups are alkanoyl groups of 2 to 10 carbon atoms such as acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and the like as well as the branched chain isomers thereof. Typical biologically acceptable salts of the compounds of the invention include sodium, potassium, ammonium, TRIS and the like. Some of the compounds of the present invention may be in tautomeric forms, particularly in the y-portion of Formula I. The invention includes all those tautomers. The invention also includes the stereoisomers, the racemic mixtures of such stereoisomers as well as the individual enantiomers that can be separated according to the invention by methods that are well known to those of ordinary skill in the art.

The compounds of this invention can be prepared using methods known to those skilled in the art. Specifically, compounds with Formulas I-III can be prepared as illustrated by the example reactions in Schemes 1-5. Scheme 1 is the least preferred method since deprotection with HBr / HOAc leads to the elimination of both the t-butoxy groups such as benzyloxycarbonyl (Z), which makes the next coupling reaction complicated. Thus, where the t-butoxy group is desired, it can be reintroduced. When an N- (9-fluorenylmethoxycarbonyl) (fmoc) group is employed as the N-blocking group (Scheme 2), it can be selectively removed with morpholine, piperidine or other amine base without removal of the protective groups t- butoxy, thus allowing the initial introduction of additional amino acids or Z-blocked peptides (see Schemes 2-4). The final Z-blocked compounds can be selectively deprotected with trifluoroacetic acid (TFA) to remove the t-butoxy group without removal of the Z group.

Scheme 1 , s ** fcí Scheme 2 "^^^^ - ^^^^^^ Scheme 3 HClNH2-Asp (0Bu-t)} -NH, HC1 Z -Val -Ala, EDC / Piri «fln-i. DMF Í? SM? i Scheme 4 Z-Tyr-Val-Ala, EDC / plridine, DMF Scheme 5 Z-Asp (? Bu-t) -Glu (0Bu-t) -Val-Asp < OBu-t) EDC / pyridine DMF < Z-Asp (0Bu-t) -Glu (0Bu-t) -Val-Asp (0Bu-t)), - Rhodamine TFA Accordingly, the invention also relates to a method for the preparation of a compound of Formula III, comprising (a) the condensation of Rhodamine 110 together with the β-t-butyl ester of N-fmoc-L- acid aspartic to produce (Fmoc-Asp (OBu-t)) 2-Rhodamine 110; (b) removal of the Fmoc group to produce (Asp (OBu-t)) 2-Rhodamine 110; (c) condensation of (Asp (OBu-t)) 2-Rhodamine 110 with Z- (AA) n to produce (Z- (AA) n-Asp (OBu-t)) 2-Rhodamine 110; and (d) the removal of the OBu-t protecting group. In a preferred embodiment, - (AA) n is EH, YVA, LEH, DET, DEV, DEH, VEH, LET, SHV, DEL, DGP, DEP, DGT, DLN, DEE, DSL, DVP, DEA, DSY, ELP , VED, IEP or IET. Where the amino acid is replaced by a carboxy group, it is protected with a protective OBu-t group which is eliminated in the final stage. The condensation reaction can be performed using any conventional condensing agent that is used for the synthesis of the peptide. In a preferred embodiment, the condensing agent is l- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), or 2-ethoxy-l-ethoxycarbonyl-1,2-dihydroguinoline (EEDQ). The solvent for the reaction may be pyridine or dimethylformamide (DMF). The reaction is generally carried out at room temperature. The proportion of the condensation agent for Rhodamine may be about 10: 1 and the proportion of the protecting peptide or amino acid for Rhodamine or (Asp (OBu-t)) 2-Rhodamine 110 may also be about 10: 1.

^^ - ^ - - - * - »• '-« - »The Fmoc group is generally removed by treatment with morpholine, piperidine or other amine base, in a polar aprotic solvent such as DMF. In general, the morpholine is added in excess, and the reaction is carried out at room temperature. The α, α-α?-Methyl-3,5-dimethoxybenzyloxycarbonyl (ddz) is another group of N-blocking which can be used in place of the Fmoc. Accordingly, the ß-t-butyl ester of N-Ddz-L-aspartic acid can be used in place of the β-t-butyl ester of N-fmoc-L-aspartic acid. The Ddz can be selectively cleaved in the presence of the t-butoxy group by 1% TFA in methylene chloride. The OBu-t group is removed with trifluoroacetic acid in an aprotic solvent such as methylene chloride at room temperature. Compounds with Formula VI can be prepared as illustrated by the example reaction in Scheme 6.

Scheme 6 The compounds with Formulas VII-IX can be prepared as illustrated by the example reactions in Schemes 7-10.

Scheme 7 • ^ - ». ^ * Scheme 8 Z-Tyr-Val-Ala, EDC / plridlna, QMF Scheme 9 Z-As (OB-) -Glu (OBu-t) -Val-Asp (OBu-t) EDC / pyridine, DMF Scheme 10 Accordingly, the invention also relates to a method for the preparation of a compound of Formula VII, which comprises (a) reacting Rhodamine with acetic anhydride to give N-acetyl-Rhodamine; (b) condensing the N-acetyl-Rhodamine together with the β-t-butyl ester of N-fmoc-L-aspartic acid to give N- (Fmoc-Asp (OBu-t)) -N'-acetyl-Rhodamine; (c) removing the Fmoc group to give N- (Asp (OBu-t)) -N'-acetyl-Rhodamine; (d) condense N- (Asp (OBu-t)) -N'-acetyl-Rhodamine with Z- (AA) n to give N- (Z- (AA) n-Asp (OBu-t)) -N ' acetyl-Rhodamine; and (e) removing the protecting group OBu-t to provide N- (Z- (AA) n-Asp) -N'-acetyl-Rhodamine; or alternatively (a) reacting the Rhodamine with acetic anhydride to give N-acetyl-Rhodamine; (b) condensing N-acetyl-Rhodamine with Z- (AA) n-Asp (OBu-t) to give N- (Z- (AA) n-Asp (OBu-t)) -N'-acetyl-Rhodamine; and (c) removing the OBU-t protecting group to provide N- (Z- (AA) N-Asp) -N'-acetyl-Rhodamine.

In a preferred embodiment, - (AA) n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SHV, DEL, DGP, DEP, DGT, DLN, DEE, DSL, DVP, DEA, DSY, ELP , VED, IEP or EIT. The compounds of Formula VII can also be prepared using an acyl (alkanoyl) chloride in place of acetic anhydride, such as acetyl chloride, hexanoyl chloride, octanoyl chloride and decanoyl chloride. Other reagents may be used in place of included acetic anhydride, but not limited to carbamyl chloride such as dimethylcarbamyl chloride, diethylcarbamyl chloride and N-methyl-N-hexylcarbamyl chloride; chloroformate such as methyl chloroformate, ethyl chloroformate, octyl chloroformate, 2-butoxyethyl chloroformate and 2,5,8-trioxadecyl chloroformate; chlorothiolformate such as methyl chlorothiolformate, ethyl chlorothiolformate, octyl chlorothiolformate; alkyl, haloalkyl and aralkyl sulfonyl halides such as methanesulfonyl chloride, octansulfonyl chloride, trifluoromethanesulfonyl chloride and tosyl chloride. The reaction is carried out in the presence of a base, such as (Et) 3 N, (i-Pr) 2-NEt or pyridine. The preferred solvent is DMF. The reaction is generally carried out at room temperature. The ratio of anhydride or acyl chloride to Rhodamine is about 1: 1.

The condensation reaction can be performed using any conventional condensing agent that is used for peptide synthesis. In a preferred embodiment, the condensing agent is EDC or EEDQ, and the solvent for the reaction is pyridine or dimethylformamide (DMF). The reaction is generally carried out at room temperature. The ratio or ratio of the condensing agent to N-acetyl-Rhodamine is about 3: 1 and the ratio of the protected amino acid or peptide to N-acetyl-Rhodamine or N- (Asp (OBu-t)) -N '-acetyl -Rhodamine is approximately 3: 1. The condensation of N-acetyl-Rhodamine with a peptide such as Z- (AA) n-Asp (OBu-t) to provide N- (Z- (AA) n-Asp (OBu-t)) -N '-acetyl -Rhodamine in a one-step reaction is a preferred procedure. A) Yes, the compounds of Formula VI provide fluorescent dyes which can be condensed with any peptide or other structure for the preparation of fluorogenic or fluorescent compounds which are substrates for proteases or peptidases. Initially, the compounds of Formula VII can also be prepared first by condensation of a peptide with a Rhodamine to give N-peptide-Rhodana, then reacting the N-peptide-Rhodamine with acetyl anhydride or other acylation reagent for »&» a > flBMfci - '? j; give, for example, N-acetyl-N '-peptide-Rhodamine. However, a) the peptides are generally much more expensive than the acyl chlorides or anhydrides, b) the condensation reaction between the peptide and Rhodamine is not an efficient reaction. For these reasons it is preferred to bind the peptide a to N-acetyl-Rhodamine rather than to bind the acyl group to N-peptide-Rhodamine. In one aspect, the invention relates to a method for determining whether a test substrate has an effect on an enzyme involved in the apoptosis cascade in a test cell, comprising (a) contacting the test cell with the test substance and the reporter compound according to the invention under conditions whereby the test substance interacts with an outer membrane receptor of the cell or is taken into the cell and the reporter compound is taken into the cell, and (b) ) record the fluorescence of the test cells, where a change in fluorescence, either of magnitude or wavelength, within the tested cell compared to the control cell which has contacted only the reporter compound and not with the test substance, it is an indication that the test substance has an effect on the enzyme.

The results obtained by this method can be compared with the results obtained within the test compounds which are known to affect enzymes involved in the apoptosis cascade in cells, to generate a measure of the relative effectiveness of the test substance. The compounds which may be used include known activators and inhibitors of enzymes involved in the apoptosis cascade. Activators, either by direct or indirect mechanisms, of enzymes involved in the apoptosis cascade, include but are not limited to known chemotherapeutic agents, such as etoposide (Yoon HJ, Choi IY, Kang MR, Kim SS, Muller MT, Spitzner JR, Chung IK (1998), Biochim Biophys Acta 1395: 110-120) and doxorubicin (Gamen S, Anel A, Lasierra P. Alava MA, Martinez-Lorenzo MJ, Pineiro A, Naval J (1997), FEBS Lett 417 : 360-364) which are inhibitors of topoisomerase II; cisplatin (Maldonado V, Melendez-Zajgla J. Ortega A (1997), M ta t Res 381: 61-15); chlorambucil (Hickman JA. (1992) Cancer Metastasis Rev. 11: 121-139) which is an alkylating agent; and fluorouracil, an RNA / DNA antimetabolite (Hickman JA. (1992), Cancer Metastasis Rev. 11: 121-139). These apoptosis activators can also be used to induce apoptosis when inhibitor of apoptosis is selected in whole cells. Activators, either direct or indirect, of enzymes involved in the apoptosis cascade include but are not limited to endogenous proteins including Bcl-2 (Joensuu H, Pylkkanen L, Toikkanen S (1994). Am, J. Pathol. 5: 1191-1198), the viral produced p35 agent (Miller LK (1997), J. Cell Physiol. 173: 178-182) and the synthetic caspase inhibitor Z-Vad-FMK (An S, Knox KA (1996) , FEBS LETT 386: 115-122). In particular, the invention relates to the use of reporter compounds having Formulas I-III, V, VII and VIII in whole cell assays, using whole cells or tissue samples that have been included to undergo apoptosis, to select compounds that inhibit either directly or indirectly an enzyme or enzymes involved in apoptosis (programmed cell death). These screening assays using compounds having Formulas I-III, V, VII and VIII are expected to lead to the discovery of new drugs or new uses for known drugs that kill cells en bloc or slowly in a variety of clinical conditions in the which occurs the loss of cells, tissues or complete organs. The reporter compounds having Formulas I-III, V, VII and VIII and the screening assays of the present invention can be used to identify drugs that reduce or prevent cell death in the nervous system (brain, spinal cord, and peripheral nervous system) under various conditions of ischemia and excitotoxicity, including, but not limited to, focal ischemia due to apoplectic stroke and global ischemia due to cardiac arrest. Selection tests can also be used to identify compounds that reduce or prevent cell death in the nervous system due to traumatic damage (such as head trauma or damage to the spinal cord), nerve cell death induced by radiation or infection viral (for example, as a side effect of cancer radiotherapy) or environmental toxicity (for example, by certain halogenated hydrocarbons). Selection tests can also be used to identify inhibitors of cell death which are useful for reducing or preventing cell death in a range of neurodegenerative disorders, including but not limited to Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, and spinobulbar atrophy. The screening assays of this invention can be used to identify compounds that prevent cell death under any conditions that potentially result in cardiac muscle death.

This includes myocardial infection, congestive heart failure and cardiomyopathy. A particular application of the screening assay is to identify compounds which reduce or prevent cell death to the myocardium that occurs in certain viral heart infections. Selection tests of the invention can be used to identify compounds that prevent retinal neuron cell death that occur in disorders associated with increased intraocular pressure (such as glaucoma) or retinal disorders associated with the aging process (such as macular degeneration related to age). The assays can also be used to identify compounds which treat hereditary degenerative disorders of the retina, such as retinitis pigmentosa. Selection tests of the invention can be used to identify inhibitors of cell death that can be used to reduce or prevent premature death of cells in the immune system, and are particularly useful for identifying inhibitors which are useful for treating deficiency disorders. immune, such as acquired deficiency immune syndrome (AIDS), various combined immune deficiency syndromes (SCIDS) and related diseases. Selection tests can also be used to identify inhibitors of cell death that can be used to treat radiation-induced immunosuppression. Selection tests of the invention can also be used to identify drugs useful in organ transplantation procedures. The transplantation of human organs and tissues is a common treatment for organ failure. However, during the transplant process, the donor organ or tissue at risk of cell death is deprived of its normal blood supply prior to being implanted in the host. This ischemic state can be treated with inhibitors of cell death by infusion into the donor organ or tissue, or by direct addition of the cell death inhibitors to the organ / tissue storage medium. Inhibitors of cell death can be identified using the screening assays described in this invention. Inhibitors of cell death can also be used to reduce or prevent cell death in the donor organ / tissue after it has been transplanted to protect it from the effects of host immune cells which exterminate their targets by the activation of apoptosis. The screening assays described in this invention can be used to identify cell death inhibitors useful for protecting transplanted rejection organs. The cytoprotective effects of cell death inhibitors can also be used to prevent the death of human or animal sperm and eggs used in in vitro fertilization procedures. These inhibitors can be used during the collection process and can also be included in the storage medium. Cell death inhibitors useful for application in fertilization procedures can be identified using the screening assay methods described in this invention.

Yeast cells and mammalian cell lines are commonly used to produce large amounts of recombinant proteins (such as antibodies, enzymes or hormones) for industrial or medical use. The lifespan of some of these cell lines is limited due to growth conditions, the nature of the recombinant molecule that is expressed (some are toxic) and other unknown factors. The lifespan of cell lines in the industry can be extended including inhibitors of cell death in the growth medium. Inhibitors of cell death useful for extending the lifespan of cell lines can be identified using the screening assay procedures described in this invention.

The factors that govern hair growth and loss are largely unknown. Nevertheless, there is some evidence, that the regression of the hair follicle (referred to as catageno) may be due to the lower partially apoptosis. Therefore, it is possible that cell death inhibitors can be used to treat hair loss that occurs due to various conditions, including but not limited to baldness as a man's sampler, hair loss induced by chemotherapy or induced by radiation, and hair loss due to emotional stress. There is also evidence that apoptosis can play a role in the loss of hair color. Therefore, it is possible that inhibitors of cell death can also be used in the treatment of cases in which the hair becomes gray. Inhibitors of cell death useful in the treatment or prevention of hair loss or hair turning white can be identified using the screening assay procedures described in this invention. Skin epithelial cell death can occur after exposure to high levels of radiation, heat or chemicals. It is possible that cell death inhibitors can be used to reduce or prevent this type of skin damage. In a particular application, cell death inhibitors can be applied in an ointment to treat acute overexposure to the sun and prevent the formation of blisters and peeling of the skin. Inhibitors of cell death useful in the treatment or prevention of skin cell death can be identified using the screening assay procedures described in this invention.

Another important aspect of the present invention is to use the reporter compounds having Formulas I-III, V, VII and VIII in whole cell assays using live or dead complete cells or tissue samples to select the compounds they stimulate, either directly or indirectly, an enzyme or enzymes involved in apoptosis. Therefore, these screening assays using compounds having Formulas I-III, V, VII and VIII are expected to lead to the discovery of new drugs or new uses for known drugs that act as anti-cancer agents in diseases such as cancers, tumors and cell hyperplasias, etc. The compounds that can be found using screening or screening assays and reagents described herein are useful for the treatment of cancers, tumors or tissue hyperplasias including but not limited to cancers or tumors of the brain, peripheral nervous system, eyes, ears , nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, gallbladder , reproductive organs and glands, joints, bones and skin. Another important aspect of the present invention is the use of reporter compounds having Formulas I-III, V, VII and VIII in whole-cell assays using yeast and other fungi, and bacteria to select genetically composed compounds for antifungal or antibacterial drugs that they act by inducing, either directly or indirectly, the caspase cascade or other enzymes involved in apoptosis in these cells. Another important aspect of the invention is to use reporter compounds having Formulas I-III, V, VII and VIII to verify the therapeutic effects of therapeutic agents or treatment provided to patients in order to reduce, prevent or treat diseases in which apoptotic cell death is a cause or a result. Another important aspect of the present invention is to use reporter compounds having Formulas IX to select HIV protease inhibitors in HIV infected cells, comprising (a) contacting the test cell with the test substance and the test substance. reporter compound according to the invention, under conditions whereby the test substance interacts with an outer membrane receptor of the cell or enters the cell and the reporter compound enters the cell, and (b) records the fluorescence of the test cell, wherein a change in fluorescence, either of magnitude or wavelength, within the tested cell compared to the control cell which has contacted only the reporter compound and not the substance of the test cell. test, is an indication that the test substance has an inhibitory effect on the HIV protease. Yet another important aspect of the present invention is to use reporter compounds having Formulas IX for the diagnosis of HIV infection, comprising (a) contacting a test cell from an individual suspected of having an infection due to HIV infection. HIV with the reporter compound according to the invention, under conditions whereby the reporter compound is put into the cell, and (b) recording the fluorescence of the test cell, wherein a change in fluorescence, either of magnitude or of wavelength, within the tested cell compared to the control cell which has contacted the reporter compound, is an indication that the test cell has been infected by the HIV virus and that the individual has become infected with HIV. By applying the same procedure for the selection of HIV protease inhibitors in HIV infected cells, the reporter com pounds having the Formula IX of the present invention can be used to select the adenovirus protease inhibitors in adenovirus infected cells. Reporter compounds having the Formula IX of the present invention can also be used to select protease inhibitors of herpes simplex virus type-1 (for its acronym in English, HSV-1) in cells infected by HSV-1. Reporter compounds can also be used to select inhibitors of human cytomegalovirus (HCMV) proteases in cells infected with HCMV; for selecting protease inhibitors of hepatitis C virus (HCV) in cells in cells infected with HCV; to select DPP-IV inhibitors in T cells; as well as to select inhibitors of type 2 methionine aminopeptidase (MetAp-2) in endothelial cells.

Additionally, using the same procedure for the diagnosis of HIV infection, the reporter compounds having the Formula IX of the present invention can also be used for the diagnosis of adenovirus, simple herpes virus type 1, human cytomegalovirus and hepatitis virus. C. Compositions within the scope of this invention include all compositions wherein the fluorogenic or fluorescent compounds of the present invention are contained in an amount which is effective to achieve the intended purpose. While the quantities may vary from test to test, the determination of the optimum ranges of the effective amounts of each component is within the skill of the art. Typically, fluorogenic or fluorescent substrate compounds can be applied to cellular or mammalian cell lines, eg, human, u - * - ** JL other animals incubating the cells or tissues containing the cells with the fluorogenic or fluorescent substrate at a concentration of about 0.01 nanomolar to approximately 1 molar, or an equivalent amount of a salt or pro-reporter molecule thereof in a physiologically absorber compatible. Such buffers include cell growth media, an example for cancer cells derived from leukemia which is RPMI-1640 with or without 10% fetal bovine serum. Other known cell incubation buffers could involve isotonic solutions buffered with either phosphate or HEPES. One of ordinary skill in the art can identify other suitable shock absorbers with no more than routine experimentation. Cells can be derived from any organ or organ system for which it is desirable to find -by means of screening assays- drugs that could be useful in the treatment of disorders mediated by apoptosis, for example, neuronal cell death, disease of the heart, liver disease, diseases of the retinas, kidney, diseases of joints and bones, disorders of the immune system, cancers, tumors and tissue hyperplasias, etc. Suitable solubilizers can be used to present the fluorogenic or fluorescent compounds of the present invention to tissues, cells or cell lines. Suitable solubilizers include aqueous solutions of the active compounds in the water soluble form, for example, water soluble salts and alkaline solutions. In addition, suspensions of the compounds as appropriate oily suspensions may be presented to the cells or tissues. Suitable lipophilic solvents or carriers include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400) or dimethyl sulfoxide (DMSO) ) or other suitable solubilizers. Optionally, the suspension or solution may also contain stabilizers. Optionally, electroporation or presentation of the reporter molecules in liposomes or detergents can be used to improve the cellular permeability of the fluorogenic or fluorescent reporter molecules. Typically, the cells are contacted with the reporter compounds of the invention and the test substance for about 30 minutes at about 5 hours, more preferably, about 1 hour. The invention also relates to the pro-reporter derivatives of the compounds of the invention. Such pro-reporter derivatives include compounds that are cleaved in situ by endogenous enzymes to give the compounds of Formulas I-III, V, and VII-IX. Such pro-reporter derivatives include lower alkyl esters of carboxyl-containing amino acid residues such as Asp and Glu. Especially the preferred pro-reporter derivatives include the methyl esters and acetoxymethyl esters (AM) of compounds containing Asp and Glu. The following examples demonstrate the utility of the invention to measure the activity of caspases and other enzymes involved in apoptosis in cells or tissues. The examples also demonstrate the utility of the invention in drug screening assays that can be used to find enhancers or inhibitors of apoptosis. These examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in in vitro assays, drug selection procedures or diagnostic procedures which are obvious to those skilled in the art are within the spirit and scope of the invention.

EXAMPLE 1 [Fmoc-Asp (OBu-t)] 2-Rhodamine 110 To a solution of the acid β-t-butyl ester Fmoc-L-aspartic (4.9 g, 11.91 mmol) dissolved in a 1: 1 anhydrous mixture of dimethylformamide and pyridine (30 mL) at 0 ° C was added EDC (2.28 g, 11.91 mmol). The solution was stirred for 45 min, then a solution of Rhodamine 110 HCl (0.44 g, 1.19 mmol) in the same solvent was added. The reaction mixture was stirred at room temperature for 60 h and concentrated in vacuo to about 10 mL. Then the residue was diluted with 100 mL of water and extracted with ethyl acetate (3 x 50 mL). The organic phase was washed with IN HCl (2 x 50 mL) and water (2 x 50 mL). The solution was dried over Na2SO and concentrated to give the crude product which was purified by column chromatography (CH2Cl2 / EtOAc 10: 1), affording 0.89 g (67%) of the title compound as a colorless solid, mp 156-158. ° C. XH NMR (CDC13): d 8.75 (bs, 2H), 8.02 (d, J = 9.3 Hz, 2H), 7.80-6.70 (m, 26H), 6.12 (bs, 2H), 4.64 (bs, 2H), 4.46 (d, J = 6.8 Hz, 4H), 4.22 (t, J = 6.8 Hz, 2H), 2.82 (m, 4H), 1.45 (s, 18H).

AXIS 2 [Asp (OBu-t)] 2-Rhodamine 110 2HC1 A cooled solution of DMF / morpholine (3 mL, 1: 1) was added dropwise in a stirred solution of [Fmoc-Asp (OBu-t)] 2-Rhodamine 110 (150 mg, 0.13 mmol) in dimethylformamide (3 mL). The solution was stirred for 20 minutes and poured into ice water (100 mL) and extracted with ethyl acetate (2 x 100 mL). The organic phase was washed with water (3 x 100 mL) and dried over Na 2 SO 4. To the solution was added IN HCl in ether (0.39 mL) and concentrated to give a red solid. The red solid was collected and dissolved in methanol (1 mL), and precipitated with ether (50 mL) to yield the title compound (64 mg, 77%) as a red solid, melting point 200 ° C (dec. ).

EXAMPLE 3 [Z-Ala-Asp (OBu-t)] 2-Rhodamine 110 From benzyloxycarbonyl-L-alanine (376 mg, 1.69 mmol), EDC (258 mg, 1.35 mmol) and [Asp (OBu-t)] 2-Rhodamine 110 2HC1 (50 mg, 0.072 mmol) in 1: 1 anhydrous DMF / pyridine (10 f) was obtained 63 mg (86%) of the title compound as a solid, mp 124-126 ° C. XH NMR (CDCl 3) d 8.85 '(d, 2H) , 7.90 (m, 2H), 7.60 (m, 4H), 7.32 (m, 10H), 7.10 (m, 2H), 6.68 (m, 2H), 5.20 (s, 2H), 5.10 (d, 4H), 4.90 (s, 2H), 4.18 (m, 2H), 2.82 (m, 4H), 1.42 (m, 24H).

EXAMPLE 4 (Z-Ala-Asp) 2-Rhodamine 110 To a cooled (0 ° C) solution of [Z-Ala-Asp (OBu-t)] 2-Rhodamine 110 (41 mg, 0.038 mmol) in methylene chloride (5 mL) was added 50% trifluoroacetic acid in chloride of methylene (16 mL). The solution turned orange and was stirred at room temperature for 3 hours. The solvent was removed and the crude product was purified by flash column chromatography (EtOAc / CF3C02H = 20: 0.5) to yield 34 mg (91%) of the title compound. XH NMR (CD3OD) d 8.02 (d, J = 8.1 Hz, 1H), 7.85 (s, 2H), 7.73 (t, J = 7.5 Hz, 2H), 7.34-6.60 (m, 15H), 5.08 (d, 4H), 4.05 (m, 2H), 2.95 (m, 4H), 1.38 (d, J = 6.4 Hz, 6H).

EXAMPLE 5 [Z-Asp (OBu-t)] 2-Rhodamine 110 From the ß-t-butyl ester of benzyloxycarbonyl-L-aspartic acid (4.41 mg, 13.63 mmol), EDC (2.61 mg, 13.63 mmol) and Rhodamine 110 HCl (0.50 g, 1.36 mmol) in 1: 1 anhydrous DMF / pyridine (40 mL) at 0 ° C gave 1.09 g (82%) of the title compound as a colorless solid. , mp 127-129 ° C. ? R NMR (CDC13) d 8.75 (bs, 2H), 8.06 (d, J = 6.3 Hz, 2H), 7.68-7.56 (m, 4H), 7.40-6.60 (m, 15H), 6.12 (bs, 2H), 5.16 (s, 4H), 4.62 (bs, 2H), 2.97 (dd, Ji = 17.1 Hz, J2 = 3.3 Hz, 2H), 2.69 (dd, J? = 17.3 Hz, J2 = 6.9 Hz, 2H), 1.44 (s, 18H).

EXAMPLE 6 (Asp) 2-Rhodamine 110 2HBr A cooled solution of 30% HBr in acetic acid (5 mL) was added dropwise to a stirred solution of [Z-Asp (OBu-t)] 2-Rhodamine 110 (200 mg, 0.21 mmol) in acetic acid (2). mL). The solution was stirred at room temperature for 1 hour and was then concentrated in vacuo. The residue was added 100 mL of anhydrous ether to give a red precipitate which was isolated after centrifugation to give 118 mg (78%) of a solid, melting point 130 ° C. XH NMR (DMSO-de) d 10.81 (s, 2H), 8.35 (bs, 6H), 8.05-6.79 (m, 10H), 4.25 (bs, 2H), 2.96 (m, 4H).

EXAMPLE 7 (Z-Val-Asp) 2-Rhodamine 110 From benzyloxycarbonyl-L-valine (628 mg, 2.5 mmol), EDC (383 mg, 2 mmol) and (Asp) 2-Rhodamine 110 2 HBr (72 mg, 0.1 mmol) in 1: 1 anhydrous DMF / pyridine ( 12 mL) at 0 ° C was obtained 38 mg (37%) of the title compound, melting point 169-171 ° C. XH NMR (DMSO) d 8.70 (d, 2H), 8. 04 (d, J = 7.5 Hz, 1H), 7.80 (, 2H), 7.48-7.20 (m, 1H), 7. 05 (m, 2H), 5.02 (bs, 4H), 4.70 (m, 2H), 3.85 (t, 2H), 3.20 (m, 2H), 2.60 (m, 2H), 2.05 (bs, 2H), 0.85 (t, 12 H).

EXAMPLE 8 -S-íS-. [Z-Val-Ala-Asp (OBu-t)} 2-Rhodamine 110 From benzyloxycarbonyl-L-Val-L-Ala (200 mg, 0.62 punol), EDC (110 mg, 0.57 mmol) and [Asp (OBu-t)] 2-Rhodamine 110 2HC1 (31 mg, 0.043 mmol) in 1: 1 anhydrous DMF / pyridine at 0 ° C there was obtained 45 mg (85%) of the title compound, melting point 85-87 ° C. XH NMR (CDC13) d 9.10 (bs, 1H), 8.90 (bs, 1H), 8.80 (d, 1H), 7.80-6.60 (m, 19H), 5.42 (bs, 2H), 5.10 (bs, 4H), 4.90 (s, 2H), 4.00 (d, 2H), 2.90 (m, 4H), 2.12 (m, 2H), 1.35 (s, 18H), 1.30 (d, J = 6.4 Hz, 6H), 0.92 (m , 12H).

EXAMPLE 9 (Z-Val-Ala-Asp) 2-Rhodamine 110 From the [Z-Val-Ala-Asp (OBu-t)] 2-Rhodamine 110 (28 mg, 0.022 mmol) and trifluoroacetic acid in methylene chloride (30 mL) at 0 ° C, 23 mg (88 mg) was obtained. %) of the title compound. XH NMR (CD3OD) δ 8.00 (s, 1H), 7.92 (bs, 1H), 7.86 (bs, 1H), 7.70 (m, 2H), 7.40-7.21 (m, 13H), 6.70 (s, 1H), 6.68 (s, 1H), 5.08 (d, 4H), 4.22 (m, 2H), 3.90 (m, 2H), 2.92 (m, 4H), 2.06 (m, 2H), 1.34 (d, J = 6.4 Hz , 6H), 0.95 (m, 12H).

EXAMPLE 10 [Z-Tyr-Val-Ala-Asp (OBu-t)] 2-Rhodamine 110 SEQ ID NO: 2 From benzyloxycarbonyl-L-Tyr-Val-L-Ala 10 (339 mg, 0.70 mmol), EDC (122 mg, 0.64 mmol) and [Asp (OB-t)] 2-Rhodamine 110 2HC1 (39 mg, 0.058 mmol) in 1: 1 anhydrous DMF / pyridine at 0 ° C afforded 61 mg (65%) of the title compound, melting point 155-157 ° C. XH NMR (CD3OD) d 8.02-6.60 (m, 28H), 5.02 (bs, 4H), 4.92 (t, 2H), 4.38 (m, 15 2H), 4.22 (m, 2H), 3.05-2.62 (m, 8H), 2.02 (m, 2H), 1.42 (s, 18H), 1.32 (d, J = 6.6 Hz, 6H), 0.92 (m, 12H).

EXAMPLE 11 (Z-Tyr-Val-Ala-Asp) 2-Rhodamine 110 SEQ ID NO: 2 From [Z-Tyr-Val-Ala-Asp (OBu-t)] 2- Rhodamine 110 SEQ ID NO: 2 (47 mg, 0.029 mmol) and acid trifluoroacetic acid in methylene chloride (30 mL) at 0 ° C obtained 36 mg (82%) of the title compound, melting point 115 ° C. 1 NM NMR (CD3OD) δ 8.02 (bs, 2H), 7.82 (bs, 2H), 7.70 (m, 2H), 7.40-6.60 (m, 22H), 5.02 (bs, 4H), 4.80 (m, 2H), 4.38 (m, 2H), 4.22 (m, 2H), 4.10 (m, 2H), 3.10-2.60 (m, 6H), 2.06 (m, 2H), 1.36 (d, J = 7.2 Hz, 6H), 0.91 (d, J = 7.5 Hz, 12H).

EXAMPLE 12 N-Acetyl-Rhodamine 110 To a solution of Rhodamine 110 (500 mg, 1.36 mmol) dissolved in DMF (20 ml) at 0 ° C was added N, N-diisopropylethylamine (176 mg, 1.36 mmol), then acetic anhydride was added dropwise to the previous solution. (167 mg, 1.64 mmol). The reaction solution was stirred at room temperature for 24 hours, and then diluted with 100 mL of water and extracted with ethyl acetate (3 x 50 L). The organic phase was washed with water (2 x 100 mL), dried over Na 2 SO and concentrated to yield the crude product which was purified by column chromatography (Hexane / EtOAc 1: 1) to give 210 mg (41%) of the title compound as a colorless solid, mp 179 ° C (dec). Rf = 0.36 (EtOAc / CH2Cl2 = 1: 1). ? H NMR (CDC13) d 8.00 (d, J = 6.7 Hz, 1H), 7.78-7.52 (m, 4H), 7.14 (d, J = 6.7 Hz, 1H), 6.93 (d, J = 6.7 Hz, 1H ), 6.64 (d, J = 7.9 Hz, 1H), 6.54-6.28 (m, 3H), 3.86 (bs, 2H), 2.15 (s, 3H).

EXAMPLE 13 N- [Fmoc-Asp (OBu-t)] -N '-acetyl-Rhodamine 110 From the t-butyl ester of L-aspartic acid (739 mg, 1.79 mmol), EDC (302 mg, 1.57 mmol) and N-acetyl-Rhodamine 110 (160 mg, 0.43 mmol) in 1: 1 anhydrous DMF / pyridine (8 mL) at 0 ° C, 276 mg (84%) of the title compound was obtained as a colorless solid. Rf = 0.75 (EtOAc / CH2Cl2 = 1: 1). XH NMR (CDCl 3): d 8.72 (bs, 1H), 8.02 (d, J = 6.3 Hz, 1H), 7.80-6.68 (m, 17H), 6.12 (bs, 1H), 4.63 (bs, 1H), 4.47. (d, J = 5.5 Hz, 2H), 4.22 (t, J = 7.2 Hz, 1H), 2.96 (m, 1H), 2.68 (, 1H), 2.20 (s, 3H), 1.46 (s, 9H).

EXAMPLE 14 N- [Asp (OBu-t)] -N'-acetyl-Rhodamine 110 From a cooled solution of DMF / morpholine (3 mL, 1: 1) and N- [Fmoc-Asp (OBu-t)] - N "-acetyl-Rhodamine 110 (100 mg, 0.13 mmol) gives the title compound (67 mg, 95%) as a solid, mp 131-133 ° C. H NMR (CDCl 3): d 9.73 (s, 1H ), 8.01 (d, J = 7.2 Hz, 1H), 7.72 (s, 1H), 7.70-6.65 (m, 9H), 3.80 (m, 1H), 2.88 (d, J = 16.5 Hz, 1H), 2.70 (m, 1H), 2.17 (s, 3H), 1.90 (bs, 2H), 1.44 (s, 9H).

EXAMPLE 15 N- [Z-Ala-Asp (OBu-t)] -N '-acetyl-Rhodamine 110 From benzyloxycarbonyl-L-alanine (43 mg, 0.19 mmol), EDC (37 mg, 0.19 mmol) and N-Asp (OBu-t) -N'-acetyl-Rhodamine 110 (30 mg,, 0.055 mol) in anhydrous DMF / pyridine 1: 1 at 0 ° C, 38 mg (92%) of the title compound is obtained as a solid, mp 138-140 ° C. Rf = 0.42 (EtOAc / hexane 4: 1). XH NMR (CDCl 3): d 8.82 (bs, 1H), 8.01 (d, J = 6.6 Hz, 1H), 7.70-6.67 (m, 14H), 5.20 (, 1H), 5.16 (bs, 2H), 4.90 ( m, 1H), 4.20 (m, 1H), 3.12 (m, 1H), 2.61 (m, 1H), 2.19 (s, 3H), 1.56 (bs, 3H), 1.43 (s, 9H). 25 EXAMPLE 16 N- (Z-Ala-Asp) -N '-acetyl-Rhodamine 110 From N- [Z-Ala-Asp (OBu-t)] - N '-acetyl-Rhodamine 110 (38 mg, 0.052 mmol) and 50% trifluoroacetic acid in methylene chloride (3 mL) at 0 ° C 34 (96%) of the title compound are obtained. Rf = 0.6 (10 mL EtOAc with 5 drops of CF3C02H). XH NMR (CD3OD): d 8.02 (d, J = 7.5 Hz, 1H), 7.80-6.69 (m, 14H), 5.10 (bs, 2H), 2.92 (m, 2H), 2.14 (s, 3H), 1.37 (d, J = 7.5 Hz, 3H).

EXAMPLE 17 N- [Z-Val-Asp (OBu-t)] -N '-acetyl-Rhodamine 110 From benzyloxycarbonyl-L-valine (49 mg, 0.19 mmol), EDC (37 mg, 0.19 mmol) and N-Asp (OBu-t) -N '-acetyl-Rhodamine 110 (30 mg,, 0.055 mmol) in Anhydrous DMF / pyridine 1: 1 (4 L) at 0 ° C gives 40 mg (94%) of the title compound as a solid, mp 155-157 ° C. Rf = 0.5 (EtOAc / hexane 4: 1). XH NMR (CDC13): d 8.87 (s, 1H), 8.01 (d, J = 7.5 Hz, 1H), 7.64-6.66 (m, 14H), 5.32 (s, 1H), 5.11 (d, J = 3.3 Hz , 2H), 4.92 (m, 1H), 4.02 (m, 1H), 3.06 (d, J = 15.6 Hz, 1H), 2.62 (m, J = 15.8 Hz, 1H), 2.20 (s, 3H), 1.43 (s, 9H), 1.26 (bs, 1H), 0.96 (m, 6H).

EXAMPLE 18 N- (Z-Val-Asp) -N '-acetyl-Rhodamine 110 From N- [Z-Val-Asp (OBu-t)] - N '-acetyl-Rhodamine 110 (40 mg, 0.051 mmol) and 50% trifluoroacetic acid in methylene chloride (3 mL) at 0 ° C 37 mg (99%) of the title compound are obtained. Rf = 0.6 (10 mL EtOAc with 5 drops of CF3C02H). H NMR (CD3OD): d 8.01 (d, J = 7.5 Hz, 1H), 7.78-6.60 (m, 14H), 5.08 (d, J = 6.5 Hz, 2H), 4.83 (m, 1H), 3.89 (d, J = 5.7 Hz, 1H), 3.02 (m, 1H ), 2.85 (m, 1H), 2.14 (s, 3H), 1.25 (m, 1H), 0.97 (bs, 6H).

EXAMPLE 19 N- [Z-Val-Ala-Asp (OBu-t)] -N'-acetyl-Rhodamine 110 From benzyloxycarbonyl-L-valine-L-alanine (63 mg, 0.019 mmol), EDC (37 mg, 0.019 mmol) and N-Asp (OBu-t) -N '-acetyl-Rhodamine 110 (30 mg, 0.052 mmol) in anhydrous DMF / pyridine 1: 1 to 0 ° C 40 mg (97%) of the title compound is obtained, mp 101-103 ° C. Rf = 0.35 (EtOAc / hexane 6: 1). XH NMR (CDCl 3): d 7.99 (d, J = 8.4 Hz), 7.80-6.62 (m, 14H), 5.42 (m, 1H), 5.11 (bs, 2H), 4.90 (m, 1H), 4.40 (bs) , 1H), 4.08 (m, 1H), 3.68 (bs, 2H), 3.50 (m, 1H), 2.90 (m, 2H), 2.16 (bs, 3H), 1.41 (s, 9H), 1.26 (d, J = 6.3 Hz, 3H), 1.25 (m, 1H), 0.93 (m, 6H).

EXAMPLE 20 N- (Z-Val-Ala-Asp) -N '-acetyl-Rhodamine 110 From N- [Z-Val-Ala-Asp (OBu-t)] - N '-acetyl-Rhodamine 110 (36 mg, 0.043 mmol) and 50% trifluoroacetic acid in methylene chloride (4 mL) at 0 ° C 35 mg (100%) of the title compound is obtained. Rf = 0.4 (10 mL EtOAc with 4 drops of CF3C02H). XH NMR (CD3OD): d 8.02 (d, J = 5.7 Hz, 1H), 7.95-7.12 (m, 12H), 6.71 (s, 1H), 6.68 (s, 1H), 5.09 (bs, 2H), 4.82 (m, 1H), 4.25 (m, 1H), 3.88 (m, 1H), 3.64 (bs, 1H), 2.94 (m, 2H), 2.14 (s, 3H), 1.30 (d, J = 6.0 Hz, 3H), 0.95 (d, J = 6.9 Hz, 6H).

EXAMPLE 21 SS ..IÍ ...? N- [Z-Tyr-Val-Ala-Asp (OBu-t)] -N'-acetyl-Rhodamine 110 SEQ ID NO: 2 From benzyloxycarbonyl-L-tyrosine-L-valine-L-alanine (119 mg, 0.25 mmol), EDC (47 mg, 0.25 mmol) and N-Asp (OBu-t) -N '-acetyl-Rhodamine 110 ( 30 mg, 0.055 mmol) in anhydrous DMF / pyridine 1: 1 at 0 ° C gives 50 mg (95%) of the title compound. Rf = 0.5 (EtOAc). XH NMR (CD3OD): d 8.02 (d, J = 7.8 Hz, 1H), 7.82-6.58 (m, 18H), 4.90 (m, 2H), 4.35 (m, 1H), 4.24 (m, 1H), 4.08 (m, 1H), 2.93 (m, 1H), 2.73 (m, 1H), 2.13 (s, 3H), 1.43 (s, 9H), 1.37 (d, J = 7.2 Hz, 3H), 1.29 (bs, 1H), 0.91 (m, 6H).

EXAMPLE 22 N- [Z-Tyr-Val-Ala-Asp) -N '-acetyl-Rhodamine 110 SEQ ID NO: 2 From N- [Z-Tyr-Val-Ala-Asp (OBu-t)] -N'-acetyl-Rhodamine 110 SEQ ID NO: 2 (49 mg, 0.049 mmol), and 50% trifluoroacetic acid in chloride of methylene (4 mL) at 0 ° C gives 42 mg (89%) of the title compound.

Rf = 0.62 (10 mL EtOAc with 5 drops of CF3C02H). 1H NMR (CD3OD): d 8.00 (d, J = 7.2 Hz, 1H), 7.84-6.56 (m, 18H), 4.99 (bs, 2H), 4.80 (m, 1H), 4.32 (m, 1H), 4.23 (m , 1 HOUR) , ¿_4 * á? Tf? "* * - > - ^" "* 4.10 (m, 1H), 2.97 (m, 2H), 2.13 (s, 3H), 1.37 (d, J = 6.9 Hz, 3H), 1.23 (, 1H), 0.90 (m, 6H).

EXAMPLE 23 N- [Z-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N '-acetyl-Rhodamine 110 SEQ ID NO: 5 From benzyloxycarbonyl-L-Asp (OBu-t) -L-Glu (OBu-t) -L-Val-L-Asp (OBu-t) (262 mg, 0.34 mmol), EDC (65 mg, 0.34 mmol ) and N-acetyl-Rhodamine 110 (30 mg, 0.08 miriol) in anhydrous DMF / pyridine 1: 1 (4 mL) at 0 ° C yields 73 mg (81%) of the title compound, mp 127-129 ° C. Rf = 0.69 (EtOAc / CH2Cl2 = 1: 1). H NMR (CD3OD): d 9.05 (s, 1H), 8.79 (d, J = 10.8 Hz, 1H), 8.38 (bs, 1H), 8.01-6.65 (m, 15H), 6.10 (m, 1H), 5.14 (m, 2H), 4.92 (bs, 1H), 4.52 (m, 1H), 4.42 (m, 1H), 4.18 (m, 1H), 3.92 (m, 1H), 3.10-2.64 (m, 4H), 2.48 (m, 2H), 2.17 (bs, 3H), 1.40 (m, 27H), 0.99 (bs, 6H).

EXAMPLE 24 N- [Z-Asp-Glu-Val-Asp) -N '-acetyl-Rhodamine 110 SEQ ID NO: 5 From N- [Z-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N '-acetyl-Rhodamine 110 SEQ ID NO: 5 (49 mg, 0.043 mol ), and 50% trifluoroacetic acid in methylene chloride (5 mL) at 0 ° C gives 38 mg (92%) of the title compound. Rf = 0.52 (10 mL EtOAc with 5 drops of CF3C02H). XH NMR (CD3OD): d 8.04-6.63 (m, 15H), 5.05 (m, 2H), 4.50 (, 1H), 4.40 (m, 1H), 4.05 (m, 1H), 3.93 (d, J = 7.9 Hz, 1H), 3.10-2.61 (, 4H), 2.37 (m, 2H), 2.13 (s, 3H), 0.97 (m, 6H).

EXAMPLE 25 N-Ethoxycarbonyl-Rhodamine 110 To a solution of Rhodamine 110 (3.00 g, 8.18 mmol) was dissolved in dimethylformamide (140 mL) at -50 ° C, N, N-diisopropylethylamine (1.27 g, 1.2 mmol) was added, then it was added dropwise to the previous solution ethyl chloroformate (1.07 g, 9.81 mmol). The reaction solution was slowly warmed up to room temperature and kept stirring for 5 h. It was then diluted with 200 mL of ice water and extracted with ethyl acetate (3 x 50 mL). The organic phase was washed with brine water (3 x 100 mL), dried over Na 2 SO and concentrated to give the crude product which was purified by column chromatography (hexane / EtOAc 3: 1) to give 1.31 g (40% ) of the title compound as a colorless solid. Rf = 0.4 (EtOAc / Hexane = 2: 1). XH NMR (CDCl 3): d 8.00 (d, J = 6.9 Hz, 1H), 7.65-7.50 (m, 4H), 7.15 (d, J = 7.2 Hz, 1H), 6.88-6.32 (m, 8H), 4.24 (q, J = 7.2 Hz, 2H), 3.92 (bs, 2H), 1.33 (t, J = 7.2 Hz, 3H).

EXAMPLE 26 N- [Cbz-Val-Asp (OBu-t)] -N'-ethoxycarbonyl-Rhodamine 110 From Cbz-Val-Asp (OBu-t) (197 mg, 0.47 mmol), EDC (89.53 mg, 0.47 mmol) and N-ethoxycarbonyl-Rhodamine 110 (47 mg, 0.12 mmol) (as described in Example 1) 47 mg (50%) of the title compound is obtained as a solid. XH NMR (CDC13): d 9.04 (bs, 1H), 8.00 (d, J = 6.9 Hz, 1H), 7.82-6.62 (m, 15H), 5.30 (bs, 1H), 5.20-5.11 (m, 2H) , 4.98 (m, 1H), 4.23 (q, J = 6.9 Hz, 2H), 3.74 (t, 1H), 3.15 (d, J = 16.5 Hz, 1H), 2.59 (m, 1H), 2.10 (m, 1H), 1.45 (s, 9H), 1.32 (t, J = 6.9 Hz, 3H), 1.01 (m, 6H).

EXAMPLE 27 N- (Cbz-Val-Asp) -N'-Ethoxycarbonyl-Rhodamine 110 Starting from N- [Z-Val-Asp (OBu-t)] -N'-ethoxycarbonyl Rhodamine 110 (20 mg, 0.025 mmol) in 50% trifluoroacetic acid of methylene chloride (2 mL) gives 15 mg ( 80%) of the title compound. XH NMR (CD3OD): d 8.02 (d, J = 6.6 Hz, 1H), 7.86-6.59 (m, 14H), 5.10 (m, 2H), 4.97 (m, 1H), 4.20 (q, J = 6.9 Hz , 2H), 3.77 (d, J = 8.1 Hz, 1H), 3.04 (m, 1H), 2.76 (m, 1H), 2.00 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 1.02 (m, 6H).

EXAMPLE 28 n- [Cbz-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N'-ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From Cbz-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t) SEQ ID NO: 5 (374 mg, 0.48 mmol), EDC (92 mg, 0.48 mmol) and N Ethoxycarbonyl-Rhodamine 110 (48.28 mg, 0.12 mmol) (as described in Example 1) affords 81 mg (58%) of the title compound as a solid. 1H NMR (CDC13): d 9.02 (bs, 1H), 8.80 (d, 1H), 8.00-6.78 (m, 15H), 6.18-6.02 (m, 1H), 5.13 (bs, 2H), 4.94 (bs, 1H), 4.60 (bs, 1H), 4.44 (bs, 1H), 4.22 (m, 2H), 3.89 (m, 1H), 3.15-2.00 (m, 8H), 1.46-1.31 (m, 27H), 1.26 (m, 1H), 1.05-0.98 (m, 9H).

EXAMPLE 29 N- (Cbz-Asp-Glu-Val-Asp) -N'-ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From N- [Z-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] - N '-ethoxycarbonyl Rhodamine 110 SEQ ID NO: 5 (100 mg, 0.086 mmol) and 50% trifluoroacetic acid methylene chloride (3 mL) (as described in Example 4) affords 85 mg (99%) of the title compound. Rf = 0.5 (10 mL of EtOAc with 5 drops of CF3C02H). XH NMR (CD3OD): d 8.02-6.63 (m, 15H), 5.08-5.04 (d, 2H), 4.48-3.92 (m, 6H), 3.10-1.95 (m, 8H), 1.31 (t, J = 7.2 Hz, 3H), 1.05-0.96 (m, 9H).

EXAMPLE 30 N- [Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N * ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t) SEQ ID NO: 5 (307.6 mg, 0.45 mmol), EDC (86 mg, 0.45 mmol) and N Ethoxycarbonyl-Rhodamine 110 (60 mg, 0.15 mmol) (as described in Example 1) gives 128 mg (80%) of the title compound as a solid. Rf = 0.35 (EtOAc / CH2Cl2 = 1: 1). XH NMR (CDCl 3): d 9.00 (d, J = 7.5 Hz), 8.80-8.54 (m, 2H), 8.05-6.90 (m, 9H), 6.72 (s, 1H), 6.69 (s, 1H), 4.93 -4.02 (m, 6H), 3.09-2.30 (m, 6H), 2.10 (s, 3H), 2.05 (m, 2H), 1.48-1.30 (m, 29H), 1.06-0.96 (m, 6H).

EXAMPLE 31 N- [Ac-Asp-Glu-Val-Asp) -N'-ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From N- [Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N "-ethoxycarbonyl Rhodamine 110 SEQ ID NO: 5 (90 mg, 0.084 mmol) and 50% trifluoroacetic acid in methylene chloride (2 mL) gives 65 mg (86%) of the title compound XH NMR (CD3OD): d 8.12-7.26 (m, 9H), 7.21 (d, J = 7.2 Hz, 1H), 7.08-7.05 (m, 1H), 6.79-6.64 (m, 2H), 4.67 (m, 1H), 4.40 (, 1H), 4.20 (q, J = 6.9 Hz, 2H), 4.07- 3.52 (m, 2H), 3.07-2.68 (m, 4H), 2.38 (m, 2H), 2.13 (, 2H), 1.98-1.94 (t, 3H), 1.32 (t, J = 6.9 Hz, 3H), 1.04-0.95 (m, 6H).

EXAMPLE 32 N-Octyloxycarbonyl-Rhodamine 110 From Rhodamine 110 (500 mg, 1.36 mmol), N, N-diisopropylethylamine (351.6 mg, 2.76 mmol), and octyl chloroformate (316 mg, 1.64 mmol) (as described in Example 25) is obtained. mg (28%) of the title compound as a solid. Rf = 0.7 (EtOAc / hexane = 1: 1). XH NMR (CDCl 3): d 7.99 (d, J = 7.2 Hz, 1H), 7.65-7.56 (m, 2H), 7.52 (bs, 1H), 7.15 (d, J = 7.5 Hz, 1H), 6.88-6.32 (m, 6H), 4.17 (t, J = 6.6 Hz, 2H), 3.9 (2H), 1.68 (m, 2H), 1.42-1.26 (m, 8H), 0.89 (t, J = 6.3 Hz, 3H) .

EXAMPLE 33 N- [Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N "octyloxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t) SEQ ID NO: 5 (123.7 mg, 0.18 mmol), EDC (34.5 mg, 0.18 mmol) and N -octyloxycarbonyl-Rhodamine 110 (29 mg, 0.06 mmol) (as described in Example 1) 43 mg (62%) of the title compound is obtained as a solid. H NMR (CDC13): d 9.01-8.53 (m, 3H), 8.07-7.99 (, 1H), 7.77-6.78 (m, 9H), 6.72 (s, 1H), 6.69 (s, 1H), 4.93 (m , 1H), 4.76-4.64 (m, 1H), 4.39 (m, 1H), 4.16 (m, 2H), 4.06 (m, 1H), 3.08-2.02 (m, 9H), 2.10 (s, 3H), 1.47-1.29 (m, 39H), 1.05-0.96 (m, 6H), 0.88 (t, J = 5.7 Hz, 3H).

EXAMPLE 34 N- (Ac-Asp-Glu-Val-Asp) -N "-octyloxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From N- [Z-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] - N '-octyloxycarbonyl R-110 SEQ ID NO: 5 and 50% trilofluoroacetic acid in methylene chloride (2 mL) gives 16.5 mg (100%) of the title compound. Rf = 0.46 (10 mL of EtOAc with 5 drops of CF3C02H). XH NMR (CD3OD): d 8.02-7.04 (m, 8H), 6.79-6fß4 (, 2H), 4.67 (m, 1H), 4.40 (m, 1H), 4.15 (t, J = 6.6 Hz, 2H), 4.15-3.94 (m, 2H), 3. 10-2.10 (m, 6H), 2.01-1.94 (m, 3H), 1.69 (m, 2H), 1.29 (bs, 10H), 1.05-0.86 (m, 9H).

EXAMPLE 35 N-Methoxycarbonyl-Rhodamine 110 From Rhodamine 110 (600 mg, 1.64 mmol), N, N-diisopropylethylamine (254 mg, 1.96 mmol) and methyl chloroformate (201 mg, 2.13 mmol) (as described in Example 25) yield 28 mg (4.4%) of the title compound as a colorless solid. Rf = 0.77 (EtOAc / hexane = 3: 1). 1R NMR (CDC13): d 8.10-6.53 (m, 9H), 6.35 (d, J = 7.2 Hz, 1H), 3.90 (bs, 2H), 3.80 (s, 3H).

EXAMPLE 36 N-Methylsulfonyl-Rhodamine 110 From Rhodamine 110 (500 mg, 1.36 p-mol), N, N-diisopropylethylamine (211 mg, 1.64 mmol) and methylsulfonyl chloride (187 mg, 1.64 mmol) (as described in Example 25) is obtained 42.1 mg (9.4%) of the title compound. 1H NMR (CDC13): d 8.02 (d, J = 7.5 Hz, 1H), 7.71-7.62 (m, 3H), 7.24-6.36 (m, 7H), 3.95 (bs, 2H), 3.18 (s, 3H) .

EXAMPLE 37 N-Acetyl-Rhodamine 116 From Rhodamine 116 (458.8 mg, 1.0 mmol), N, N-diisopropylethylamine (129.3 mg, 1.0 mmol) and acetic anhydride (122 mg, 1.2 mmol) (as described in Example 25) there is obtained 141 mg ( 9.4%) of the title compound as a colorless solid. Rf = 0.64 (EtOAc / CH2Cl2 = 2: 1). XH NMR (CDCl 3): d 8.01 (d, J = 7.5 Hz, 1H), 7.69-7.62 (m, 3H), 7.24-6.36 (, 7H), 3.95 (bs, 2H), 3.28 (s, 3H), 2.87 (s, 3H), 1.95 (bs, 3H).

EXAMPLE 38 N-Dimethylcarbamyl-Rhodamine 110 From Rhodamine 110 (1.0 g, 2.73 mmol), N, N-diisopropylethylamine (0.42 g, 3.27 mmol) and dimethylcarbamyl chloride (0.35 g, 3.27 mmol) (as described in Example 25), 10 mg (1%) of the title compound is obtained as a solid. Rf = 0.3 (EtOAc). ? ti NMR (CD3OD): d 8. 00 (d, J = 7.2 Hz, 1H), 7.80-7.64 (m, 3H), 7.55 (d, J = 2. 1 Hz, 1H), 7.21-6.40 (m, 6H), 3.03 (s, 6H).

EXAMPLE 39 N-Hexyloxycarbonyl-Rhodamine 110 From Rhodamine 110, diisopropylethylamine (0.24 mL, 2.52 mmol) and hexyl chloroformate (0.27 mL, 1.64 mmol) (as described in Example 25) the title compound is obtained as an orange solid (80 mg, 13%). XH NMR (CDCl 3): d 7.99 (d, J = 6. 6, 1H), 7.67-7.56 (m, 2H), 7.51 (s, 1H), 7.14 (d, J = 7.5, 1H), 6.87 (dd, J = 1.8, 8.7, 1H), 6.79 (s, 1H) ), 6.66 (d, J = 8.7, 1H), 6.54-6.50 (m, 2H), 6.33 (dd, J = 2.1, 8. 7, 1H), 4.17 (t, J = 6.6, 2H), 3.92 (s, 2H), 1.68 (m, 2H), 1.34 (brs, 6H), 0.91 (t, J = 6.0, 3H).

EXAMPLE 40 N-Decyloxycarbonyl-Rhodamine 110 The title compound was prepared as described in Example 25.? U NMR (CDCI3): d 7.98 (d, J = 7. 2, 1H), 7.67-7.56 (m, 2H), 7.51 (s, 1H), 7.14 (d, J = 7. 5, 1H), 6.89-6.87 (m, 2H), 6.66 (d, J ~ 8.4, 1H), 6.53- 6.49 (m, 2H), 6.32 (d, J = 8.4, 1H), 4.17 (t, J = 6.6, 2H), 3.93 (s, 2H), 1.67 (m, 2H), 1.27 (brs, 14H), 0.89 (t, J = 6.9, 3H).

EXAMPLE 41 N-Dodecyloxycarbonyl-Rhodamine 110 The title compound was prepared as described in Example 25. 1 H NMR (CDCl 3): d 8.00 (d, J = 6.9, 1H), 7.68-7.57 (m, 2H), 7.51 (s, 1H), 7.16 ( d, J = 6.9, 1H), 6.86 (d, J = 8.4, 1H), 6.89-6.53 (m, 4H), 6.35 (d, J = 7.1, 1H), 4.17 (t, J = 6.3, 2H) , 3.89 (s, 2H), 1.67 (m, 2H), 1.27 (brs, 18H), 0.89 (t, J = 5.7, 3H).

EXAMPLE 42 N- [Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N '-hexyloxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t) -OH SEQ ID NO: 5 (263 mg, 0.383 mmol), EDC (74 mg, 0.39 mmol) and N-hexyloxycarbonyl-Rhodamine 110 (44 mg, 0.096 mmol) (according to Example 1) the title compound is obtained as a white solid (30 mg, 28%). H NMR (CDC13): d 8.99 (d, J = 6.9, 1H), 8.78 (d, J = 11.7, 1H), 8.52 (s, 1H), 8.06-6.69 (m, 13H), 4.97-4.01 (m , 6H), 3.08-2.04 (m, 12H), 1.70-1.34 (m, 39H), 1.04-0.89 (, 9H).

EXAMPLE 43 N- [Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N * -decyloxycarbonyl-Rhodamine 110 SEQ ID NO: 5 The title compound was prepared as described in Example 1. 1 H NMR (CDCl 3): d 9.01-8.76 (m, 3H), 8.51 (s, 1H), 8.02-6.69 (m, 13H), 4.97-4.01 (m, 6H), 3. 08-2.04 (m, 12H), 1.70-1.34 (m, 47H), 1.04-0.88 (m, 9H).

EXAMPLE 44 N- [Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N'-dodecyloxycarbonyl-Rhodamine 110 SEQ ID NO: 5 The title compound was prepared as described in Example 1. XH NMR (CDC13): d 9.01-8.76 (m, 3H), 8.51 (s, 1H), 8.02-6.69 (, 13H), 4.97-4.01 (m, 6H), 3. 08-2.04 (, 12H), 1.70-1.34 (m, 51H), 1.04-0.88 (m, 9H).

EXAMPLE 45 N- [Ac-Asp-Glu-Val-Asp) -N '-hexyloxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From N- [Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N '-hexyloxycarbonyl-Rhodamine 110 SEQ ID NO: 5 (30 mg, 0.027 mmol ) and trifluoroacetic acid (2 mL) the title compound is obtained as a yellow solid (22 mg, 85%). XH NMR (CD3OD): d 8.13-732 (, 8H), 7.26 (d, J = 6.6, 1H), 7.12 (d, J = 8.7, 1H), 6.83-6.69 (m, 2H), 4.72 (, 1H) ), 4.44-3.99 (m, 5H), 3.11-2.77 (m, 3H), 2.43 (m, 2H), 2.17 (m, 2H), 2.03-1.98 (m, 5H), 1.73 (m, 2H), 1.49-1.34 (m, 8H), 1.04-0.97 (m, 9H).

EXAMPLE 46 N- (Ac-Asp-Glu-Val-Asp) -N "-decyloxycarbonyl-Rhodamma 110 SEQ ID NO: 5 The title compound was prepared as described in Example 45. XH NMR (CD3OD): d 8.13-7.32 (m, 8H), 7.26 (d, J = 7.8, 1H), 7.12 (d, J = 8.1, 1H ), 6.83-6.69 (m, 2H), 4.72 (m, 1H), 4.44-3.99 (m, 5H), 3.11-2.77 (m, 3H), 2.45 (m, 2H), 2.22-2.10 (m, 2H) ), 2.05-1.98 (m, 5H), 1.73 (m, 2H), 1.49-1.26 (m, 14H), 1.06-0.92 (m, 9H).

EXAMPLE 47 N- (Ac-Asp-Glu-Val-Asp) -N'-dodecyloxycarbonyl-Rhodamine 110 SEQ ID NO: 5 The title compound was prepared as described in Example 45. XH NMR (CD3OD): d 8.07-7.08 (m, 10H), 6.81-6.68 (m, 2H), 4.73 (m, 1H), 4.44-3.35 (m, 5H), 3.26-2.02 (m, 12H), 1.73 (m, 2H), 1.49-1.29 (m, 18H), 1. 06-0.92 (m, 9H).

EXAMPLE 48 N- (Ethylthio) carbonyl-Rhodamine 110 The solution of Rhodamine 110 (500 mg, 1.36 mmol) was dissolved in dimethylformamide (12 mL) at -61 ° C, N, N-diisopropylethylamine (264 mg, 2.04 mmol) was added, then chloroformate was added dropwise to the previous solution. of ethyl (204 mg, 1.64 mmol). The reaction solution was then slowly heated to room temperature and kept stirring for 1 hour. It was then diluted with 100 mL of ice water and extracted with ethyl acetate (3 x 30 mL). The organic phase was washed with brine (2 x 50 mL) and dried over Na 2 SO and concentrated to give the crude product which was purified by chromatography (Hexane / EtOAc 2: 1), giving 238 mg (42%) of the compound of the title as a solid. Rf = 0.6 (EtOAc / Hexane = 1: 1). 1H NMR (CDC13): d 8.01 (d, J = 6.9 Hz, 1H), 7.68-7.57 (m, 3H), 7.21 (bs, 1H), 7.14 (d, J - 7.2 Hz, 1H), 6.88 (dd) , Ji = 8.4 Hz, J2 = 2.1 Hz, 1H), 6.68 (d, 1H), 6.56-6.50 (m, 2H), 6.34 (dd, Ji = 8.4 Hz, J2 = 2.1 Hz, 1H), 3.91 (s) , 2H), 3.00 (q, J = 7.5 Hz, 2H), 1.36 (t, J = 7.5 Hz, 3H).

EXAMPLE 49 N- [Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OBu-t)] -N '- (ethylthio) carbonyl-Rhodamine 110 SEQ ID NO: 5 The title compound was prepared as described in Example 1. Rf = 0.56 (EtOAc / CH2Cl2 = 2: 1). 1u NMR (CDCl 3): d 9.04-8.46 (m, 3H), 8.01-6.68 (m, 11H), 4.93-3.86 (m, 4H), 3.15-1.85 (m, 8H), 2.14 (d, 3H), 1.48-1.33 (m, 29H), 1.06-0.98 (m, 6H).

EXAMPLE 50 N- (Ac-Asp-Glu-Val-Asp) -N '- (ethylthio) carbonyl-Rhodamine 110 SEQ ID NO: 5 The title compound was prepared as described in Example 45. 1 H NMR (CD3OD): d 8.12-7.09 (m, 11H), 6.79-6.66 (m, 2H), 4.67-3.93 (m, 4H), 3.08- 2.68 (m, 6H), 2.38 (m, 2H), 2.12 (m, 2H), 1.99-1.94 (t, 3H), 1.32 (t, J = 7.5 Hz, 3H), 1.04-0.94 (m, 6H) .

EXAMPLE 51 2, 5, 8-Trioxadecyl Chloroformate A solution of triethylene glycol monomethyl ether (2 g, 12.2 mmol) in ether (15 mL) was added dropwise to a stirred solution cooled with ice of 20% phosgene in toluene (11.36 mL, 21.92 mmol) over 20 min. The reaction mixture was allowed to warm to room temperature and stirring was continued for 15 h. evaporation of the solvent gave 2.63 g (95%) of the title compound. XH NMR (CDC13): d 4.44 (m, 2H), 3.37 (m, 2H), 3.68-3.63 (m, 6H), 3.55 (m, 2H), 3.38 (s, 3H).

EXAMPLE 52 N- (2,5, 8-Trioxadecyloxycarbonyl) -Rhodamine 110 From Rhodamine 110 (500 mg, 1.36 mmol), N, N-diisopropylethylamine (317 mg, 2.45 mmol) and 2, 5, 8, 8-trioxadecyl chloroformate (371 mg, 1.64 mmol) yields 261 mg (37%) ) of the title compound as a solid. Rf = 0.52 (EtOAc / Hexane = 4: 1). H NMR (CDCl 3): d 8.00 (d, J = 6.6 Hz, 1H), 7.66-7.58 (m, 3H), 7.49 (s, 1H), 7.26 (bs, 1H), 6.88 (d, J = 9.0 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 6.54-6.31 (m, 3H), 4.33 (t, J = 4.2 Hz, 2H), 3.93 (bs, 2H), 3.74 (t, J = 3.3 Hz, 2H), 3.70-3.64 (m, 6H), 3.55 (m, 2H), 3.37 (bs, 3H).

EXAMPLE 53 N- [Ac-Leu-Glu (OBu-t) -Val-Asp (OBu-t)] -N'-ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 9 From Ac-Leu-Glu (OBu-t) -Val-Asp (OBu-t) (189 mg, 0.3 mmol) SEQ ID NO: 9, EDC (57.5 mg, 0.03 mmol) and N-ethoxycarbonyl-Rhodamine 110 (40 mg, 0.1 mmol) (according to Example 1) there is obtained 48 mg (47%) of the title compound as a solid. Rf = 0.35 (EtOAc / CH2Cl2 = 1: 1); XH NMR (CD3OD): d 8.02-7.04 (m, 8H), 6.79-6.93 (m, 2H), 4.35 (m, 2H), 4.20 (q, J = 6.9 Hz, 2H), 4.11 (m, 1H) , 3.94 (d, J = 6.3 Hz), 3.00-2.65 (m, 2H), 2.30 (bs, 2H), 2.00 (m, 2H), 1.98-1.95 (d, 3H), 1.62-1.28 (m, 25H) ), 1.04-0.82 (m, 12H).

EXAMPLE 54 N- (Ac-Leu-Glu-Val-Asp) -N'-ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 9 The title compound was prepared as described in Example 45. H NMR (CD3OD): d 8.40-7.05 (m, 9H), 6.79-6.63 (m, 2H), 4.37 (m, 1H), 4.31 (m, 1H), 4.20 (q, J = 6.9 Hz, 2H), 4.11 (m, 1H), 3.94 ( m, 1H), 3.07-2.68 (m, 2H), 2.38 (m, 2H), 2.08 (m, 2H), 1.99-1.95 (t, 3H), 1.68-1.48 (m, 3H), 1.31 (t, J = 6.9 Hz, 3H), 1.04-0.80 (m, 12H).

EXAMPLE 55 N- [Cbz-Gly-Pro] -N'-ethoxycarbonyl-Rhodamine 110 From Cbz-Gly-Pro (91.8 mg, 0.3 mmol), EDC (57.5 mg, 0.3 mmol) and N-ethoxycarbonyl-Rhodamine 110 (40.2 mg, 0.1 mmol) (according to Example 1) gave 68 mg (98%) of the title compound as a solid. Rf = 0.6 (EtOAc / CH2Cl2 = 4: 1). XH NMR (CDCl 3): d 9.45 (s, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.61-6.91 (m, 8H), 6.68-6.63 (m, 2H), 5.69 (bs, 1H), 5.11 (s, 2H), 4.72 (d, 1H), 4.22 (q, J = 6. 9 Hz, 2H), 4.03 (bs, 2H), 3.58 (1H), 3.43 (m, 1H), 2.49- 1.90 (m, 4H), 2.42 (bs, 1H), 2.18-1.95 (m, 3H), 1.31 (t, J = 6.9 Hz, 3H).

H? ' EXAMPLE 56 N- (Gly-Pro) -N'-ethoxycarbonyl-Rhodamine 110. HBr 1H NMR (CD3OD): d 8.11 (d, J = 7.8 Hz, 1H), 8.00 (bs, 1H), 7.84-7.72 (m, 3H), 7.32-7.15 (m, 3H), 6.95-6.87 (m, 2H), 4.64 (m, 1H), 4.23 (q, J = 6.9 Hz, 2H), 3.96 (s, 2H), 3.68 (m, 2H), 2.35-2.05 (m, 4H), 1.33 (t, J = 6.9 Hz, 3H).

EXAMPLE 57 1-Hexyl Chlorotiolformate A solution of 1-hexanet? Ol (3.72 g, 31.5 mmol) in ether (15 mL) was added dropwise to an ice-cooled solution of 20% phosgene in toluene (25 mL, 47 mmol) stirred for 20 minutes. The reaction mixture was allowed to warm to room temperature and stirring was continuous for 15 hours. Evaporation of the solvent gave 6.1 g (98%). xti NMR (CDCI3): d 2.96 (t, J = 7.2 Hz, 2H), 1.64 (m, 8H), 0.91 (t, J = 6.3 Hz, 3H).

EXAMPLE 58 N- (Hexylthio) carbonyl-Rhodamine 110 The title compound was prepared according to Example 25. Rf = 0.8 (EtOAc / Hexane = 1: 1). XH NMR (CDCl 3): d 8.00 (d, J = 7.2 Hz, 1H), 7.65-7.58 (m, 3H), 7.24 (bs, 1H), 7.14 (d, 1H), 6.87 (dd, 1H), 6.68 -6.32 (m, 4H), 3.92 (bs, 2H), 2.99 (t, J = 6.9 Hz, 2H), 1.68 (m, 2H), 1.42 (m, 2H), 1.42 (m, 2H), 1.34- 1.26 (m, 4H), 0.90 (t, J = 7.2 Hz, 3H).

EXAMPLE 59 2-Butoxyethyl Chloroformate From 2-butoxyethanol (3.72 g, 31.5 mmol) and 20% phosgene in toluene (25 mL, 47 mmol), 4.51 g (79%) of the title compound was obtained. 1 H NMR (CDCl 3): d 4.46 (t, 2 H), 3.70 (m, 2 H), 3.51 (t, J = 6.6 Hz, 2 H), 1.56 (m, 2 H), 1.40 (m, 2 H), 0.94 (t , J = 6.9 Hz, 3H).

EXAMPLE 60 N- (2-Butoxyethoxycarbonyl) -Rhodamine 110 The title compound was prepared according to Example 25. Rf = 0.58 (EtOAc / Hexane = 1: 1). XH NMR (CDCl 3): d 8.00 (d, J = 7.5 Hz, 1H), 7.67-7.49 (m, 3H), 7.15 (d, J = 7.8 Hz, 1H), 6.89-6.32 (m, 6H), 4.34 (m, 2H), 3.91 (bs, 2H), 3.68 (m, 2H), 3.50 (t, J = 6.6 Hz), 1.59 (m, 2H), 1.39 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H).

EXAMPLE 61 N- [Cbz-Asp (OEt) -Glu (OEt) -Val-Asp (OEt)] - N "-ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 5 From Cbz-Asp (OEt) -Glu (OEt) -Val-Asp (OEt) SEQ ID NO: 5 (181 mg, 0.3 mmol), EDC (57.5 mg, 0.3 mmol) and N-ethoxycarbonyl-Rhodamine 110 ( 40 mg, 0.1 mmol) (according to Example 1) 71 mg (72%) of the title compound was obtained as a solid. Rf = 0.3 (EtOAc / CH2Cl2 = 4: 1). XH NMR (CDCl 3): d 9.00 (bs, 1H), 8.76 (d, 1H), 8.43 (bs, 1H), 8.14-6.90 (m, 10H), 6.80-6.62 (, 2H), 5.1-3.8 (m , 6H), 3.2-2.0 (m, 11H), 1.35-1.22 (m, 12 H), 1.12-0.84 (m, 7H).

EXAMPLE 62 N- [Ac-Leu-Glu (OBu-t) -Val-Asp (OBu-t)) -N '-octyloxycarbonyl-Rhodamine 110 SEQ ID NO: 9 The title compound was prepared according to Example 1. Rf = 0.65 (EtOAc / CH2Cl2 1: 1), XH NMR (CD3OD): d 8.02-7.04 (m, 8H), 6.79-6.93 (m, 2H), 6.20 ( bs, 1H), 4.95 (bs, 1H), 4.52-4.20 (, 5H), 3.15-2.00 (m, 9H), 1.68 (m, 2H), 1.48 (s, 9H), 1.45 (s, 9H), 1.41-1.29 (m, 12H), 1.18-0.88 (m, 15H).

EXAMPLE 63 N- (Z-Gly) -N'-ethoxycarbonyl-Rhodamine 110 From Z-Glycine (284 mg, 1356 mmol), EDC (260 mg, 1356 mmol), and N-ethoxycarbonyl Rhodamine 110 (58 mg, 0.135 mmol) (according to Example 1) was obtained the title compound (70 mg, 83%) as a solid. XH NMR (CDCl 3): d 8.95 (bs, 1H), 7.98 (s, 2H), 7.53 (m, 8H), 7.30 (s, 5H), 7.05 (m, 2H), 6.63 (dd, 2H, J = 8.4, 11.4 Hz), 5.09 (s, 2H), 4.19 (q, 2H, J = 7.5 Hz), 1.27 (t, 3H, J = 7.5 Hz).

EXAMPLE 64 N-Gly-N '-ethoxycarbonyl-Rhodamine 110 HBr The title compound was prepared according to Example 6. XH NMR (CDC13): d 9.41 (bs, 1H), 9.20 (s, 1H), 8.04 (m, 1H), 7.62 (m, 3H), 7.33 (m, 2H), 7.12 (m, 2H), 6.97 (, 2H), 6.74 (m, 2H), 4.25 (q, 2H, J = 6.9), 1.33 (t, 3H, J = 6.9 Hz). 10 Example 65 N- (Z-Gly-Pro-Gly) -N'-ethoxycarbonyl-Rhodamine 110 15 From Z-glycine-proline (315 mg, 1.03 mmol), EDC (197 mg, 1.03 mmol) and N-Gly-N '-ethoxycarbonyl- Rhodamine 110 (50 mg, 0.103 mmol) (according to Example 1) the title compound (70 mg, 96%) was obtained as a solid pale yellow. XH NMR (CDCl 3): d 8.97 (bs, 1H9, 7.94 (d, 1H, J = 7.8 Hz), 7.77 (d, 1H, J = 15 Hz), 7.52 (m, 4H), 7.32 (s, 5H) , 7.22 (m, 2H), 7.15 (m, 2H), 6.64 (m, 1H), 5.92 (bs, 2H), 5.78 (bs, 1H), 5.57 (bs, 1H), 5.09 (s, 2H), 4.95 (m, 2H), 4.74 (m, 2H), 4.16 (m, 2H), 3.62 (m, 1H), 3.46 (m, 1H), 1.98 (m, 4H), 1.26 (m, 3H). £ ____ * »* .. !! * tt____» _._. _-_- A_ _ff EXAMPLE 66 N- (Gly-Pro-Gly) -N'-ethoxycarbonyl-Rhodamine 110 HBr XH NMR (CD3OD): d 8.02 (d, 1H, J = 7.8 Hz), 7.71 (m, 3H), 7.28 (m, 2H), 7.19 (m, 1H), 7.01 (m, 1H), 6.71 (m , 2H), 4.48 (m, 1H), 4.19 (q, 2H, J = 7.2 Hz), 4.05 (m, 2H), 3.94 (m, 2H), 3.61 (m, 2H), 2.12 (m, 4H) , 1.30 (t, 3H, J = 7.2 Hz).

EXAMPLE 67 N-hexyl-N-methylcarbamyl chloride To a solution of 0.35 ml of (i-Pr) 2NET in 10 ml of diethyl ether at 0 ° C was added phosgene (1.06 ml, 1.93 M solution in toluene) and N-hexylmethylamine (0.31 ml, 2.05 mm). The reaction mixture was allowed to warm to 25 ° C, and was further stirred at 25 ° C for 14 hours. The mixture was filtered, and the solvent was removed under vacuum. The product was used for the next reaction step without further purification. '"' H NMR (CDCl 3): d 3.42 (m, 2H), 3.07 (s, 3H), 1.59 (m, 2H), 1.31 (m, 6H), 0.90 (m, 3H).

EXAMPLE 68 N- (N-Hexyl-N-methylcarbamyl) -Rhodamine 110 To a solution of Rhodamine 110 (0.5 g, 1.36 mmol) in DMF (15 ml) at -61 ° C was added N, N-diisopropylethylamine (0.25 ml) and N-hexyl-N-methylcarbamyl chloride in DMF (2.05 mmol ). The reaction mixture was stirred at -61 ° C for 1 hour, then allowed to warm to room temperature. The reaction mixture was further stirred at room temperature for 14 hours, and then partitioned between saturated aqueous NH 4 Cl solution and ethyl acetate (2 X 50 ML). The organic solution was washed with brine (100 ml), and dried over Na2SO4. The solvent was removed and the crude product was purified by flash chromatography (hexane: EtOAc, 1: 1). The title compound (115 mg, 18%) was obtained as a solid. NMR (CDC13): d 7.97 (d, 1H, J = 7.2 Hz), 7.59 (m, 3H), 7.12 (d, 1H, J = 7.5 Hz), 6.85 (dd, 1H, J = 8.4, 2.1 Hz), 6.60 (dd, 2H, J = 5.1, 3.3 Hz), 6.48 (dd, 2H, J = 8.4, 2.1 Hz), 6.31 (dd, 1H, J = 8.4, 2.4 Hz), 3.93 (bs, 2H ), 3.33 (t, 2H, J = 6.9 Hz), 2.79 (s, 3H), 1.56 (m, 2H), 1.30 (m, 6H), 0.88 (t, 3H, J = 6.6 Hz).

EXAMPLE 69 N- (Octylthio) carbonyl Rhodamine 110 The title compound was prepared according to Example 25. X H NMR (CDCl 3): d 7.99 (d, 1 H, J = 7.5 Hz), 7. 59 (m, 3H), 7.11 (dd, 1H, J = 6.9, 0.9 Hz), 6.89 (dd, 1H, J = 8.4, 2.1 Hz), 6.64 (d, 1H, J = 8.4 Hz), 6.48 (dd, 2H, J = 8.4, 2.1 Hz), 6.31 (dd, 1H, J = 8.4, 2.4 Hz), 3.97 (bs, 2H), 2.95 (m, 2H), 1.67 (m, 2H), 1.29 (m, 10H), 0.88 (t, 3H, J = 6.6 Hz).

EXAMPLE 70 N- [Z-Gly] -N '-octyloxycarbonyl-Rhodamine 110 The title compound was prepared according to Example 1. 1 H NMR (CDCl 3): d 8.22 (s, 1 H), 7.11 (d, J = 7.5, 1 H), 7.04-6.97 (m, 2 H), 6.85 (s, 1 H), 6.70 (dd, J = 7. 5, 7.5, 2H), 5.57 (s, 1H), 5.16 (s, 1H), 5.16 (s, 2H), 4.18 (t, J = 6.6, 2H), 4.01 (d, J = 5.7, 1H), 1.68 (q, J = 6. 6, 2H), 1.29 (brs, 10H), 0.89 (t, J = 6.9, 3H).

EXAMPLE 71 N-Gly-N '-octyloxycarbonyl-Rhodamine 110 HBr The title compound was prepared according to Example 6. XH NMR (CD3OD): d 8.10 (d, J = 6.9, 1H), 7.93 8d, J = 2.4, 1H), 7.86-7.75 (m, 3H), 7.28-7.24 (m, 2H), 7. 11 (dd, J = 2.4, 9.0, 1H), 6.85 (d, J = 9.0, 1H), 6.78 (d, J = 9.0, 1H), 4.20 (t, J = 6.6, 2H), 3.94 (s, 2H), 1. 74 (m, 2H), 1.50-1.30 (br m, 10H), 0.94 (t, J = 6.6, 3H).

EXAMPLE 72 Fluorescence and Stability of N-Acetyl-Rhodamine 110 Compared with Rhodamine 110 The acties of Rhodamina 110 and N-acetyl-Rhodamine 110 as fluorescent portions for the synthetic substrates were measured in a fluorometric assay. The fluorescent signal is read on a spectrofluorometer or on a fluorometric microtiter plate reader at the excitation wavelength of 485 and emission 530. Using this assay or «• -'si" f- '- "?? ÍSL-Í test, the rela fluorescent values were determined for the two fluorescent portions. Fluorescence was measured using the following buffer conditions: 100 mM HEPES pH 7.5, with 10% sucrose, 1% CHAPS, 5 mM glutathione, and 1-200 nM of the test compound. The stability test was typically performed at 37 ° C for 2 days. The ratio or proportion of the fluorescent signal was 10.2 (Rhodamina 110 / N-acetyl Rhodamine 110) at time zero and 10.1 after two days of incubation at 37 ° C. The results show that similar to Rhodamine 110, N-acetyl-Rhodamine 110 is a stable and efficient fluorescent indicator.

EXAMPLE 73 Fluorescence of Modified Rhodamine Dye Modified Rhodamine dyes were evaluated using both spectrometry and conventional spectrofluorometry. For both types of analysis the dyes were dissolved in either methanol or 50 mM Tris at final dye concentrations ranging from 10 nM to 100 μM. An absorbance spectrum of wavelengths from 200 nm to 700 nm was determined for each dye using a DU-7000 spectrophotometer from Beckman. The dyes had peaks of absorbance at approximately 470 to 480 nm. This wavelength was chosen as the fluorescence excitation wavelength and a total fluorescence emission spectrum was determined using a Hitachi F-200 spectrofluorometer. For each dye, the emission peak was approximately 520 nm and the fluorescence output was measured under the conditions tested (see Table 1).

Table 3, Modified Rhodamine Dye Fluorescence EXAMPLE 74 Recovery and Retention of Modified Rhodamine Dyes by HL-60 Cells The HL-60 cells were placed in 5 ml of Iscove's medium (without serum or phenol-red) containing 10 μM or 50 μM of N-octyloxycarbonyl-Rhodamine 110, N-decyloxycarbonyl-Rhodamine 110, N-dodecyloxycarbonyl-Rhodamine 110 , M-hexyloxycarbonyl-Rhodamine 110, N- (ethylthio) carbonyl-Rhodamine 110 or Rhodamine 110. Cells were incubated for varying periods at 37 ° C in a CO 2 incubator, recovered by centrifugation, and washed in 50 mL of half cooled with ice. The cells were recentrifuged and the final pellet was resuspended in 50 μL of fresh or fresh medium. Aliquots of each cell suspension were placed in microslips and observed in a Nikon inverted microscope with epifluorescent illumination. As shown in Figures 1A-1F, HL-60 cells stained with N-octyloxycarbonyl-Rhodamine 110 (Figure IA), N-decyloxycarbonyl-Rhodamine 110 (Figure IB), and N-dodecyloxycarbonyl-Rhodamine 110 (Figure C) intensively and almost no leakage of the dye the middle. HL-60 cells stained with N-hexyloxycarbonyl-Rhodamine 110 (Figure ID) less intensively, but still stained well. N- (Ethylthio) carbonyl-Rhodamine 110 (Figure 1E) gave moderate, but still spotting easily detectable, although there was no slight leak, cells stained rapidly with Rhodamine 110 (Fig. 1F), but the dye leaks rapidly from the cells, resulting in a lower intensity of cell staining and a high degree of fluorescence in the medium containing the cells. Therefore, the modified Rhodamine dyes are superior to Rhodamine 110 since they are readily absorbed by HL-60 cells and maintained within the cells for at least 30 minutes.

EXAMPLE 75 Enzymatic Activity of Substrates The activities of N- (Z-VD) -N "-acetyl-Rhodamine 110, N- (Z-VAD) -N '-acetyl-Rhodamine 110, N- (Z-DEVD) -N' -acetyl-Rhodamine 110 SEQ ID NO: 5, N- (Z-YVAD) -N-acetyl-Rhodamine 110 SEQ ID NO: 2, (Z-VAD) 2-Rhodamine 110 and (Z-YVAD) 2-Rhodamine 110 SEQ ID NO: 2 as synthetic substrates for recombinant CPP32 and ICE, were measured in a fluorometric enzyme assay The recombinant CPP32 protein and the ICE protein were prepared by expressing DNA clones encoding these enzymes in an insect host cell (sf9 cells) using baculovirus as the vector See, Webb, NR et al., "Expression of proteins using recombinant Baculovirus", Techniques 2: 173-188 (1990) .Scission of synthetic substrates by the enzyme results in a fluorescent signal that is read in a spectrofluorometer or in a fluorometric microtiter plate reader.Using this assay, the Km and Vma? values were determined for each substrate with either CPP32 or ICE.

Cleavage of the substrate dependent on CPP32 and ICE was measured using the following buffer conditions: 100 mM HEPES pH 7.5, with 10% sucrose, 1% CHAPS, 5 mM glutathione, and 1-100 μM test substrate . Nonspecific enzymatic cleavage was determined with the use of specific CPP32 and ICE inhibitors consisting of an oligomer with the sequence Asp-Glu-Val-Asp or Tyr-Val-Ala-Asp, respectively, with an aldehyde group conjugated to C -terminal. The assay for enzymatic activity was typically performed at 37 ° C for 60 minutes.

Table 4 lists the values of Km and Vmax for N- (Z-VD) -N '-acetyl-Rhodamine 110, N- (Z-VAD) -N' -acetyl-Rhodamine 110, N- (Z-DEVD) -N'-acetyl-Rhodamine 110 SEQ ID NO: 5, N- (Z-YVAD) -N "-acetyl-Rhodamine 110 SEQ ID NO: 2, (Z-VAD) 2-Rhodamine 110 and (Z-YVAD) 2-Rhodamine 110 SEQ ID NO: 2 as substrates for CPP32 and ICE.

Table . Cleavage of the Substrate by CPP32 and ICE Enzyme CPP32 ICE Rn Vmax Rm Vmax (μM) (nmol / min) (μM) (ntnol / min) N- (Z-VD) -N '-Ac-Rhcdamine 110 60 11 NA N- (Z-VAD) -N '-Ac-Rhoda ina 110 NA 70 N- (Z-DEVD) -N' -Ac-Rhodamine 110 154 160 12 SEQ ID NO: 5 N- (Z-YVAD) -N '-Ac-Rhodamine 110 NA 32 96 SEQ ID NO: 2 (Z-VAD) 2-Rhodamine 110 NA 21 9 (Z-YVAD) 2-Rhodamine 110 NA 6 14 SEC ID NO: 2 Na = no activity observed at 1-100 UM of substrate, 37 ° C, 3 h of incubation The results shown in Table 4 show that N- (Z-DEVD) -N'-acetyl-Rhodamine 110 SEC I D NO: 5 is an efficient substrate for both ICE and CPP32. It is also shown that N- (Z-VD) -N'-acetyl-Rhodamine 110 is an efficient substrate for CPP32 and not for ICE and that N- (Z-VAD) -N "-acetyl-Rhodamine 110, n- ( Z-YVAD) -N'-acetyl-Rhodamine 110 SEQ ID NO: 2, (Z-VAD) 2-Rhodamine 110 and (Z-YVAD) 2-Rhodamine 110 SEQ ID NO: 2 are efficient substrates for ICE and not for CPP32.

EXAMPLE 76 Excision of Caspase-3 Substrates by Recombinant Human Caspase-3 and by Lysaties of Apoptotic Cells HL-60 The caspase substrates were tested by recombinant caspase-3 and by lysates prepared from apoptotic HL-60 cells. Assays or tests were performed at 37 ° C in 96-well plates in a 100 μL incubation containing 30 μL of caspase-3 preparation or cell lysate, 10 μM or 50 μM of the substrate, and the test buffer of caspase (40 mM 1,4-piperazinbis (ethanesulfonic acid) (PIPES, Aldrich Chemical Company) pH 7.2, 100 mM NaCl, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, 10 mM DTT). At the end of the incubation period, the fluorescence was measured in a fluorescent Bio-Tek FL500 microplate reader using excitation and emission wavelengths of 485 and 530 nm, respectively. Two different controls were put into operation: 1) an enzyme blank consisting of samples containing substrate, but without enzyme or cell lysate; 2) an inhibitor control, consisting of samples containing the caspase inhibitor, Ac-DEVD-CHO (f.c, 10 μM). Table 5 is a summary of the results obtained with these substrates.

Table 5. Splitting of the Substrate by Caspasa-3 and Lisatos As shown in Figure 2A, the cleavage of the dipeptide substrate, NZ-VD-N'-ethoxycarbonyl-RUO, requires extremely high concentrations of recombinant caspase-3 (a greater than 50-fold amount of enzyme needed for tripartite substrates). and tetrapeptides).

Even with this large amount of enzyme, the signal was low. In contrast, the tripeptide substrate, N-Z-EVD-N'-Ethoxycarbonyl-RllO (Fig. 2A), and all tetrapeptide substrates (Fig. 2B-2L) were efficiently cleaved by caspase-3 and apoptotic lysates.

EXAMPLE 77 Staining of Apoptotic Cells HL-60 by Caspase-3 Substrate, N-Ac-DEVD-N '-octyloxycarbonyl RllO The ability of N-Ac-DEVD-N '-octyloxycarbonyl-R110 SEQ ID NO: 5 to detect the activation of caspase in intact cells was tested using apoptotic HL-60 and Jurkat cells. These whole cell assays were performed in two stages: 1) induction of apoptosis; 2) incubation with the substrate. For HL-60 cells, apoptosis was induced by treatment with 10 μg / ml vinblastine for 4 hours. The control samples were treated with DMSO. For Jurkat cells, apoptosis was induced by treatment with 500 ng / ml of agonistic anti-FAS antibody for 2 hours. The control samples were treated with PBS. Following the induction of apoptosis, the cells were incubated with 50 μM N-Ac-DEVD-N '-octyloxycarbonyl-RllO SEQ ID NO: 5 in caspase assay buffer (40 mM PIPES, pH 7.4, 100 mM NaCl; 10% sucrose, 1 M EDTA, 10 mM DTT). The cells were then transferred to a glass microscope and observed by epifluorescent illumination on a Nikon inverted microscope. As shown in Figure 3A, the HL-60 cells treated with vinblastine were strongly stained by N-Ac-DEVD-N '-octyloxycarbonyl RllO SEQ ID NO: 5. DMSO treated cells also show some staining or staining ( Fig. 3B), although the intensity of the signal was significantly lower than that of the cells treated with vinblastine. HL-60 cells treated with 50 μM of Ac-DEVD-CHO SEQ ID NO: 5 during the test or test state (Fig. 3C) show the almost non-fluorescent signal, indicating that the staining or staining observed in cells treated with Vinblastine is almost complete due to caspase-mediated excision. Jurkat cells induced to undergo apoptosis by antiFas (Fig. 3D) also show intense staining by N-Ac-DEVD-N '-octyloxycarbonyl RllO SEQ ID NO: 5, whereas control cells show only light staining (Fig. 3F). These experiments demonstrate that N-Ac-DEVD-N '-octyloxycarbonyl RllO SEQ ID NO: 5 can be used to measure apoptosis in intact cells and that the signal obtained from N-Ac-DEVD-N' -octyloxycarbonyl-RllO SEQ ID NO: 5 is dependent on caspasa.

EXAMPLE 78 Excision of the Caspase-3 Substrate, N-Ac-DEVD-N'-octyloxycarbonyl RllO SEQ ID NO: 5, by Complete Apoptotic Jurkat Cells To quantitate the cleavage of N-Ac-DEVD-N '-octyloxycarbonyl-RllO SEQ ID NO: 5 by whole cells, an assay was performed in which the fluorescent signal generated from this substrate by apoptotic Jurkat cells was measured in a spectrofluorometric plate reader. Jurkat cells were incubated for several periods in 96-well plates with 500 ng / ml anti-FAS antibody to induce apoptosis. The control cells were incubated with PBS. At the end of the treatment period, the cells were harvested, centrifuged in 1.5 ml tubes and turned into suspension in 25 μL of the medium containing 1% FBS. 25 μL of caspase buffer containing 50 μM of N-Ac-DEVD-N '-octyloxycarbonyl RllO SEQ ID NO: 5 was added, and the cells were incubated for one hour. At the end of the period of gsj i -_ ____ incubation, three aliquots of 5 μL from each time point were placed in 96-well plates and the fluorescence was measured at the excitation / emission wavelength of 485/530 nm. Figure 4 shows that cells treated with PBS give a small fluorescence signal which does not increase over the treatment time used. Nevertheless, cells treated with antiFas give a detectable fluorescence signal at about one hour after the induction of apoptosis, and the signal continued to increase until 2 o'clock, with a signal for the basic ratio of approximately 7. This experiment demonstrates that N-Ac-DEVD-N '-octyloxycarbonyl RllO SEQ ID NO: 5 produces a robust signal in complete apoptotic cells and can therefore be used to quantitatively measure caspase-mediated apoptosis in a cell-based assay.

EXAMPLE 79 Substrate cleavage of Caspase-8 Ac-LEVD-N-N '- ethoxycarbonyl RllO SEQ ID NO: 9 Caspase-3, 6, 7, and Recombinant Human Ac-LEVD-N-N was tested' -ethoxycarbonyl SEQ ID RllO NO: 9 by caspase-3, 6, 7, and 8 recombinant human. Tests or assays were performed at 37 ° C in 96 well plates in 100 uL incubation caspase containing recombinant human 10 uM of N-Ac-LEVD-N'-ethoxycarbonyl RllO SEQ ID NO: 9, and cushion caspase assay (40 mM PIPES, pH 7.2, 100 mM NaCl, 10% sucrose, 0.1% CHAPS, 1 mM EDTA, 10 mM DTT). At the end of the incubation period, the fluorescence was determined in a Bio-Tek FL500 microplate or fluorescent microplate reader using excitation and emission wavelengths of 485 and 530 nm, respectively. To correct for endogenous fluorescence of uncleaved substrate, they were operated controls which consist of samples containing 10 uM of N-Ac-LEVD-N'-ethoxycarbonyl RllO SEQ ID NO: 9 without enzyme ( "white enzyme"). Samples include additional controls containing the caspase inhibitor DEVD-CHO Ac-SEQ ID NO:. 5. As shown in Fig 5, caspase-6 and caspase-8 cleaves Ac-LEVD-N-N'-ethoxycarbonyl RllO SEQ ID NO: 9 to give an easily measured fluorescent signal (signal for base ratios of about 13 by caspase-6 to about 26 by caspase-8). Caspase-3 cleaves N-Ac-LEVD-N '-ethoxycarbonyl RllO SEQ ID NO: 9 less efficiently, produces a signal that was 1 approximately 5 times higher than the value of the enzyme blank. Caspase-7 does not produce a virtual signal. These experiments show that N-Ac-LEVD-N '-ethoxycarbonyl RllO SEQ ID NO: 9 can be cleaved by elements of the caspase-8 subfamily and that it can be used to report the activity of this caspase type.

EXAMPLE 80 Excision of Aminopeptidase Substrate N-G-N "- octyloxycarbonyl RllO by Lyses of HL-60 Cells Aminopeptidases are present in many cells and consecutively unblocked amino acid residues of peptides are removed, starting from the N-termini. Peptides with blocked amino termini are not cleaved. Lysates of HL-60 were prepared by homogenizing HL-60 cells in buffer caspase, and the ability of these lysates for cleaving N-Z-G-N'-octiloxicarbonil RllO and N-G-N * -octiloxicarbonil-RllO tested in an assay microtiter plates. Figure 6 shows that the lysates of HL-60 cells readily cleaved N-G-N'-octyloxycarbonyl RllO, and the size of the signal was dependent on the concentration of the substrate. In contrast, no signal was generated by lysates of HL-60 cells of N-Z-G-N'-octyloxycarbonyl-RUO.

EXAMPLE 81 Use of fluorescence assay in the selection of drugs that stimulate caspase cascade Drugs that stimulate the caspase cascade in the absence of Fas ligand may be useful, for example, as anti-cancer chemotherapeutic agents. The essays. The tests described in Example 78 can be used to screen for drugs that stimulate the caspase cascade by performing the assay or test under similar conditions as in Example 78 except that a known or unknown compound with antitumorígena or anticancer activity known or unknown replace the Fas ligand reagent.

EXAMPLE 82 Use of fluorescent assay in the selection for drugs that inhibit or potentiate the caspase cascade stimulated with Fas ligand or other apoptosis inducer.

Drugs that inhibit the caspase cascade may be useful for the degenerative treatment and other diseases caused by or associated with an inappropriate activation of the caspase cascade. Drugs that enhance the action of another caspase stimulator, such as, for example, Fas ligand or a drug or anticancer agent, may be suitable for treating cancers or tumors caused by or associated with an inappropriate function of the caspase cascade. The assays and reagents described in this invention can be used to select drugs that either inhibit or potentiate the caspase cascade in cells, performing the assay or test as described in Example 78, using the Fas ligand or any other agent that stimulates the caspase cascade and other forms of apoptosis in the presence of a test substance that inhibits or potentiates or acts synergistically with the action of the first inducer of apoptosis or caspase cascade.

EXAMPLE 83 Use of fluorescence assay to test cancer cell samples from patients by chemosensitivity to anticancer drugs It is well known that the same cancer in different patients shows a great variability to treatment with anticancer drugs. Therefore, it is very difficult to predict whether a cancer in a patient is treatable with a particular anticancer drug before treatment has begun. The fluorescence tests described in this invention allow to test the chemosensitivity or drug resistance of tissue samples or tumorigenic or cancerous cells taken from patients with individual tumor or cancer. To perform the chemosensitivity test, a fluorescence assay using a tissue sample or cancer cell taken from a patient can be conducted as described in Example 78. Using this approach, different drugs with known or unknown chemotherapeutic activity can be tested for its ability to stimulate caspase cascade. The results of this trial provide information that can be used to design an optimal treatment regimen with chemotherapeutic drugs for the patient.

EXAMPLE 84 Absorption and Excision of the Aminopeptidase Substrate N-G- N '-octyloxycarbonyl-RllO by HL-60 Cells The HL-60 cells were placed in 5 ml of Iscove's medium (without serum or phenol-red) containing 10 μM of N-G-N'-octyloxycarbonyl-RllO or 10 μM of N-Z-G-N-octyloxycarbonyl-RUO. Three million HL-60 cells were incubated for 3 hours at 37 ° C in a C02 incubator, recovered by centrifugation, and washed in 50 μL of ice-cooled medium. The cells were recentrifuged and the final pellet was resuspended in 50 μL of the fresh medium. Aliquots of each cell suspension were placed in a 96-well microtitre plate and read on a Wallac 1420 microplate reader with excitation wavelength at 485 nm, emission wavelength at 525 nm. Aliquots of each cell suspension were also placed in micro-slides and were observed in a Nikon inverted microscope with epifluorescent illumination. As shown in Table 6, only cells incubated with 10 μM of N-G-N '-octyloxycarbonyl-RllO showed signal. There is no signal from the cells incubated with 10 μM of N-Z-G-N'-octyloxycarbonyl-RllO. Similarly, only cells incubated with N-G-N '-octyloxycarbonyl-RllO showed fluorescence under a microscope, and no fluorescence signal was observed from cells incubated with N-Z-G-N'-octyloxycarbonyl-RllO (Figures 7A-B).

Table 6, Cleavage of N-G-N "-octyloxycarbonyl-RllO by HL-60 Cells Having now fully described this invention, it will be understood by those of ordinary skill in the art that it may be performed within a broad and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any modality thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property Sequence Listing < 110 > Cytovia, Inc. < 120 > Novel Fluorescent or Fluorescent Reporting Molecules, and their Applications for Selective Classification Tests by Fluorescence in Whole Cells, for Caspases and other Enzymes, and the use of them < 130 > 1735.029PC02 < 140 > < 141 > < 150 > US 60 / 061,582 < 151 > 1997-10-10 < 150 > US 09 / 033,661 < 151 > 1998-03-03 < 160 > 142 < 171 > Patentln Ver. 2.0 < 210 > 1 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 1 Trp Glu His Asp 1 < 210 > 2 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 2 Tyr Val Wing Asp 1 < 210 > 3 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 3 Leu Glu His Asp 1 < 210 > 4 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 4 Asp Glu Thr Asp 1 < 210 > 5 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 5 Asp Glu Val Asp 1 < 210 > 6 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 6 Asp Glu His Asp 1 < 210 > 7 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 7 Val Glu His Asp 1 < 210 > 8 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 8 Leu Glu Thr Asp 1 < 210 > 9 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 9 Leu Glu Val Asp 1 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 10 Ser His Val Asp 1 < 210 > 11 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 11 Asp Glu Leu Asp 1 < 210 > 12 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 220 > < 400 > 12 Asp Gly Pro Asp 1 < 210 > 13 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 13 Asp Glu Pro Asp 1 < 210 > 14 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 14 Asp Gly Pro Asp 1 < 210 > 15 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 15 Asp Leu Asn Asp 1 < 210 > 16 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 16 Asp Glu Glu Asp 1 < 210 > 17 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 17 Asp Ser Leu Asp 1 < 210 > 18 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 18 Asp Val Pro Asp 1 < 210 > 19 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 19 Asp Glu Ala Asp 1 < 210 > 20 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 20 Asp Ser Tyr Asp 1 < 210 > 21 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 21 Glu Leu Pro Asp 1 < 210 > 22 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 22 Val Glu Asp Asp 1 < 210 > 23 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 23 lie Glu Pro Asp 1 < 210 > 24 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > He Glu Thr Asp 1 < 210 > 25 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 221 > INSECURE < 222 > (1) < 223 > The Amino Acid can be Tryptophan or Leucine < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 25 Xaa Glu His Asp 1 < 210 > 26 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 26 Val Glu He Asp 1 < 210 > 27 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 27 Val Glu Pro Asp 1 < 210 > 28 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 28 Ser Gln Asn Tyr Pro He Val 1 5 < 210 > 29 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 28 Ala Arg Val Leu Ala Glu Ala 1 5 < 210 > 30 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 30 Wing Thr He Met Met Gln Arg 1 5 < 210 > 31 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 31 Arg Gln Ala Asn Phe Leu Gly 1 5 < 210 > 32 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 28 Pro Gly Asn Phe Leu Gln Ser 1 5 < 210 > 33 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 33 Ser Phe Ser Phe Pro Gln He 1 5 < 210 > 34 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 34 Thr Leu Asn Phe Pro He Ser 1 5 < 210 > 35 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 35 Ala Glu Thr Phe Tyr Val Asp 1 5 < 210 > 36 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 236 Ala Lys Val Leu Phe Leu Asp 1 5 < 210 > 37 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 37 Arg Gly Phe Pro 1 < 210 > 38 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 38 Ser Gln Asn Tyr Pro Val Val 1 5 < 210 > 39 < 211 > 14 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 39 Asp Arg Val Tyr He His Pro Phe His Leu Glu Glu Ser 1 5 10 < 210 > 40 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 40 Leu Glu Glu Ser 1 < 210 > 41 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 41 Ser Gln Asn Tyr Pro He Val Gln 1 5 < 210 > 42 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > <; 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 42 Ser Gln Asn Leu Phe Leu Asp Gly 1 5 < 210 > 43 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 43 Arg Lys He Leu Phe Asp Gly 1 5 < 210 > 44 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 44 Lys Ala Arg Val Leu Phe Glu Ala Met 1 5 < 210 > 45 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 45 Ser Gln Asn Tyr 1 < 210 > 46 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence __ £ (_ &«<223> Description of the Artificial Sequence: Synthetic Peptide <400> 46 Pro He Val Gln 1 <210> 47 <211> 5 <212> PRT < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Synthetic Peptide < 400 > 47 Lys Ala Arg Val Leu 1 5 < 210 &48 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Synthetic Peptide < 400 > 48 Ala Arg Val Leu 1 < 210 > 49 < 211 > 4 <212> PRT <213> Artificial Sequence <220> <223> Description of the Artificial Sequence: Synthetic Peptide <400> 49 Phe Glu Ala Met 1 <210 > 50 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Pé Synthetic ptido < 400 > 50 Pro Phe His leu 1 < 210 > 51 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 51 Gln Asn Leu Phe 1 < 210 > 52 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 52 Arg Lys He Leu Phe 1 5 < 210 > 53 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 53 Lys He Leu Phe 1 < 210 > 54 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 54 Ser Leu Asn Phe 1 < 210 > 55 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 55 Leu Arg Gly Gly 1 < 210 > 56 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 56 Met Arg Gly Gly < 210 > 57 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 57 He Arg Gly Gly 1 < 210 > 58 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 58 Leu Val Gly Gly 1 < 210 > 59 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 59 Met Val Gly Gly 1 < 210 > 60 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 60 He Val Gly Gly 1 < 210 > 61 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 61 Leu Val Leu Ala Ser Ser Ser Phe 1 5 < 210 > 62 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 62 Leu Val Leu Ala 1 < 210 > 63 < 211 > 11 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 63 Arg Gly Val Val Asn Ala Ser Ser Arg Leu Ala 1 5 10 < 210 > 64 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 64 Val Val Asn Ala 1 < 210 > 65 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 221 > SITE < 222 > (1) ... (2) < 223 > t-butylglycine < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 65 Gly Gly Asn Ala 1 < 210 > 66 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 66 Asp Asp He Val Pro Cys Ser Met Ser Tyr 1 5 10 < 210 > 67 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the .?? AfcS & c, ~ - - * ms8 ¿aM < 210 > 68 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 68 Asp He Val Pro Cys 1 5 < 210 > 69 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 69 He Val Pro Cys 1 < 210 > 70 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 70 Ser Met Ser Tyr 1 < 210 > 71 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 71 Trp Glu His Asp Gly 1 5 < 210 > 72 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 72 Tyr Val Wing Asp Gly 1 5 < 210 > 73 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 73 Leu Glu His Asp Gly 1 5 < 210 > 74 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 75 Asp Glu Thr Asp Gly 1 5 < 210 > 76 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 76 Asp Glu Val Asp Gly 1 5 < 210 > 77 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 77 Leu Glu Thr Asp Gly 1 5 < 210 > 78 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 78 Asp Glu His Asp Gly 1 5 < 210 > 79 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 79 Trp Glu His Asp Gly Gly 1 5 < 210 > 80 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 80 Ser Leu Asn Phe Pro He Val 1 5 < 210 > 81 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 81 Ser Leu Asn Phe Pro ile 1 5 < 210 > 82 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 82 Ser Leu Asn Phe Pro 1 5 < 210 > 83 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 83 Leu Asn Phe Pro He Val 1 5 < 210 > 84 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 84 Leu Asn Phe Pro He 1 5 < 210 > 85 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 85 Leu Asn Phe Phe 1 < 210 > 86 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 86 Arg Gln Ala Asn Phe Leu 1 5 < 210 > 87 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 87 Arg Gln Ala Asn Phe 1 5 < 210 > 88 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence <; 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 88 Arg Lys Val Leu Phe Leu 1 5 < 210 > 89 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 89 Arg Lys Val Leu Phe 1 5 < 210 > 90 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 90 Ala Arg Val Leu Phe Leu Gly 1 5 < 210 > 91 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 91 Ala Arg Val Leu Phe Leu 1 5 < 210 > 92 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 92 Ala Arg Val Leu Phe 1 5 < 210 > 93 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 93 Ser Gln Asn Tyr Phe Leu Gly 1 5 < 210 > 94 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 94 Ser Gln Asn Tyr Phe Leu 1 5 < 210 > 95 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 95 Ser Gln Asn Tyr Phe 1 5 < 210 > 96 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 96 Met Arg Gly Gly Gly 1 5 < 210 > 97 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 97 He Arg Gly Gly Gly 1 5 < 210 > 98 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 98 Leu Val Gly Gly Gly 1 5 < 210 > 99 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 99 Met Val Gly Gly Gly 1 5 < 210 > 100 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 100 He Val Gly Gly Gly 1 5 < 210 > 101 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 101 Leu Arg Gly Gly Gly 1 5 you? < 210 > 102 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 102 Leu Arg Gly Gly Ala 1 5 < 210 > 103 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 103 He Val Leu Ala Ser Ser Ser 1 5 < 210 > 104 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 104 Leu Val Leu Ala Ser Ser 1 5 < 210 > 105 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 105 He Val Leu Ala Ser 1 5 < 210 > 106 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 106 He Val Leu Ala Ser Ser 1 5 < 210 > 107 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 107 Val Val Asn Ala Ser 1 5 < 210 > 108 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 221 > SITE < 222 > (1) ... (2) < 223 > t-butylglycine < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 108 Gly Gly Asn Ala Ser Ser 1 5 < 210 > 109 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 221 > SITE < 222 > (1) ... (2) < 223 > t-butylglycine < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 109 Gly Gly Asn Ala Ser 1 5 < 210 > 110 < 211 > 4 < 212 > PRT < 213 > Artificial Sequence < 220 > < 221 > SITE < 222 > (1) ... (2) < 223 > t-butylglycine < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 110 Gly Gly Asn Ala 1 < 210 > 111 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 111 Asp Asp He Val Pro Cys Ser Met Ser Thr 1 5 10 < 210 > 112 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 112 Asp He Val Pro Cys Ser Met Ser Thr 1 5 < 210 > 113 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 113 He Val Pro Cys Ser Met Ser Thr 1 5 < 210 > 114 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 114 He Val Pro Cys Ser Met Ser 1 5 < 210 > 115 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 115 He Val Pro Cys Ser Met 1 5 < 210 > 116 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 >; < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 116 He Val Pro Cys Ser 1 5 < 210 > 117 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 117 Ser Gln Asn Tyr Pro He 1 5 < 210 > 118 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 118 Ala Arg Val Leu Ala Glu 1 5 < 210 > 119 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic peptide 5 < 400 > 119 Ala Thr He Met Met Gln 1 5 < 210 > 120 10 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > 15 < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 120 Arg Gln Ala Asn Phe Leu 20 1 5 < 210 > 121 < 211 > 6 < 212 > PRT 25 < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide 30 < 400 > 121 Pro Gly Asn Phe Leu Gln 1 5 35 < 210 > 122 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence 40 < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 122 45 Ser Phe Ser Phe Pro Gln 1 5 < 210 > 123 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 123 Thr Leu Asn Phe Pro He 1 5 < 210 > 124 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 124 Ala Glu Thr Phe Tyr Val 1 5 < 210 > 125 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 125 Arg Lys Val Leu Phe Leu 1 5 < 210 > 126 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide ? - t-J-i < 400 > 126 Ser Gln Asn Tyr Pro 1 5 5 < 210 > 127 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence 10 < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 127 15 Ala Arg Val Leu Ala 1 5 < 210 > 128 < 211 > 5 20 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 128 Ala Thr He Met Met 1 5 30 < 210 > 129 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence 35 < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide 40 < 400 > 129 Arg Gln Ala Asn Phe 1 5 < 210 > 130 45 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 130 Pro Gly Asn Phe Leu 1 5 < 210 > 131 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 131 Ser Phe Ser Phe Pro 1 5 < 210 > 132 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 132 Thr Leu Asn Phe Pro 1 5 < 210 > 133 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 133 Ala Glu Thr Phe Tyr 1 5 < 210 > 134 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 134 Arg Lys Val Leu Phe 1 5 < 210 > 135 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 135 Met Arg Gly Gly Ala 1 5 < 210 > 136 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 136 He Arg Gly Gly Ala 1 5 < 210 > 137 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 137 Leu Val Gly Gly Ala 1 5 < 210 > 138 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 138 Met Val and Gly Ala 1 5 < 210 > 139 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 139 He Val Gly Gly Ala 1 5 < 210 > 140 < 211 > 5 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 140 Ser Gln Asn Leu Phe 1 5 < 210 > 141 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 400 > 141 Thr He Asn Phe Gln Arg 1 5 < 210 > 142 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Synthetic Peptide < 40 > 142 Tyr Val Wing Asp Gly Gly 1 5

Claims (90)

1. A reporter compound that has the general Formula II: Ri- (AA) n-Asp-y-Asp- (AA) n-R? (ID or a biologically acceptable salt or a pro-parent molecule thereof, characterized in that Ri is an N-terminal protecting group; each AA is independently a residue of an α-amino acid or β-amino acid, or a derivative of an α-amino acid or β-amino acid; each n is independently 0-5; and y is a fluorogenic or fluorescent moiety.

2. The compound according to claim 1, characterized in that R? - (AA) n-Asp is a? -blocked tetrapeptide which is a substrate for a caspase enzyme.

3. The compound according to claim 2, characterized in that said tetrapeptide is WEHD SEQ ID NO: 1, YVAD SEQ ID NO: 2, LEHD SEQ ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID NO: 5, DEHD SEQ ID NO: 6, VEHD SEQ ID NO: 7, LETD SEQ ID NO: 8, LEHD SEQ ID NO: 3, SHVD SEQ ID NO: 10, DELD SEQ ID NO: 11, DGPD SEQ ID NO: 12, DEPD SEC ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 26 or IETD SEQ ID NO: 24.

4. The compound according to claim 1, characterized in that R? - (AA) n-Asp is an N-blocked tetrapeptide which is a substrate for granzyme B.

5. The compound according to claim 4, characterized in that R? - (AA) n-Asp is IEPD or VEPD.

6. The compound according to claim 1, characterized in that R? - (AA) n-Asp is an N-blocked peptide consisting of C-terminal Asp and 1.2 or 3 amino acids of a peptide chain selected from the group which consists of WEHD SEQ ID NO: 1, YVAD SEQ ID NO: 2, LEHD SEQ ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID NO: 5, DEHD SEQ ID NO: 6, VEHD SEQ ID NO: 7, LETD SEQ ID NO: 8, LEHD SEQ ID NO: 3, SHVD SEQ ID NO: 10, DELD SEQ ID NO: 11, DGPD SEQ ID NO: 12, DEPD SEQ ID NO: 13, DGTD SEQ ID NO: 14 , DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 26, IETD SEQ ID NO: 24, IEPD SEQ ID NO: 23 and VEPD SEQ ID NO: 27.

7. The compound according to claim 1, characterized in that y is a Rhodamine 110; and the reporter molecule is a lower alkyl ester or an acetoxymethyl ester (AM) of a compound containing Asp- or Glu-.

8. The compound according to claim 1, characterized in that it has the formula III: x- (AA) NH-Asp- (AA) ,, - R! (III

9. The compound according to claim 8, characterized in that Ri is t-butoxycarbonyl, acetyl, hexanoyl, octanoyl benzyloxycarbonyl.

10. The compound according to claim 8, characterized in that - (AA) n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SHV, DEL, DGP, DEP, DGT, DLN, DEE, DSL, DVP , DEA, DSY, ELP, VED, IEP or EIT.

11. The compound according to claim 1, characterized in that it is selected from the group consisting of (Z-YVAD) 2-Rhodamine 110, SEQ ID NO: 2; (Z-DEVD) 2-Rhodamine 110, SEQ ID NO: 5; (Z-YVAD) 2-Rhodamine 110; (Z-YVAD (OAM)) 2-Rhodamine 110, SEQ ID NO: 2; (Z-LE (OAM) HD (OAM)) 2-Rhodamine 110, SEQ ID NO: 3; (Z-D (OAM) E (OAM) TD (OAM)) 2-Rhodamine 110, SEQ ID NO: 4; (Z-D (OAM) E (OAM) VD (OAM)) 2-Rhodamine 110, SEQ ID NO: 5; (Z-D (OMe) E (OMe) VD (ODAM)) 2-Rhodamine 110, SEQ ID NO: 5; and (Z-D (OMe) E (OMe) VD) 2-Rhodamine 110, SEQ ID NO: 5. 22 k, *? T-

12. A method for the preparation of a compound according to claim 8, characterized in that it comprises (a) the condensation of Rhodamine 110 together with N-fmoc-L-t-butyl ester of N-fmoc-L - aspartic to produce (Fmoc-Asp (OBu-t)) 2-Rhodamine 110; (b) removal of the Fmoc group to produce (Asp (OBu-t)) 2-Rhodamine 110; (c) condensation of (Asp (OBu-t) 2-Rhodamine with Z- (AA) n to produce the (Z- (AA) n-Asp (OBu-t)) 2-Rhodamine 110, and (d) the removal of the OBU-t protecting group.

13. The method according to claim 12, characterized in that - (AA) n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SHV, DEL, DGP, DEP, DGT, DL ?, DEE, DSL, DVP, DEA, DSY, ELP, VED, IEP or EIT.

14. A reporter compound that has the general formula V: Ri- (AA) n-Asp-y-Re (V) or a biologically acceptable salt or pro-parent molecule thereof, characterized in that Ri is an N-terminal protecting group; R is a blocking group which is not an amino acid or a derivative of an amino acid; each AA is independently a residue of an α-amino acid or β-amino acid, or a derivative of an α-amino acid or β-amino acid; each n is 0-5; and y is a fluorogenic or fluorescent moiety.

15. The compound according to claim 14, characterized in that R? - (AA) n-Asp is a? -blocked tetrapeptide which is a substrate for a caspase enzyme.

16. The compound according to claim 15, characterized in that said tetrapeptide is WEHD SEQ ID NO: 1, YVAD SEQ ID NO: 2, LEHD SEQ ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID? O: 5, DEHD SEC ID? O: 6, VEHD SEC ID? O: 7, LETD SEC ID? O: 8, LEHD SEC ID? O: 3, SHVD SEC ID? O: 10, DELD SEC ID? O: ll, DGPD SEQ ID: O: 12, DEPD SEQ ID: O: 13, DGTD SEQ ID: O: 14, DL? D SEQ ID: O: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 26 or IETD SEQ ID NO: 24.

17. The compound according to claim 14, characterized in that R? - (AA) n-Asp is an N-blocked tetrapeptide which is a substrate for granzyme B.

18. The compound according to claim 17, characterized in that R? - (AA) n-Asp is N-blocked IEPD or VEPD.

19. The compound according to claim 14, characterized in that R? - (AA) n-Asp is an N-blocked peptide consisting of C-terminal Asp and 1.2 or 3 amino acids of a peptide chain selected from the group which consists of WEHD SEQ ID NO: 1, YVAD SEQ ID NO: 2, LEHD SEQ ID NO: 3, DETD SEQ ID NO: 4, DEVD SEQ ID NO: 5, DEHD SEQ ID NO: 6, VEHD SEQ ID NO: 7, LETD SEQ ID NO: 8, LEHD SEQ ID NO: 3, LEVD SEQ ID NO: 9, SHVD SEQ ID NO: 10, DELD SEQ ID NO: 11, DGPD SEQ ID NO: 12, DEPD SEQ ID NO: 13 , DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEC ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEC ID NO: 26 or IETD SEQ ID NO: 24, IEPD SEQ ID NO: 23 and VEPD SEQ ID NO: 27.

20. The compound according to claim 14, characterized in that y is a Rhodamine 110;

21. The compound according to claim 14, characterized in that Rβ is CH3OCO-, Cbz, Cl3CCH2OCO-, phCH2CH2OCO-, or CH3 (CH2) P-, where p is 1-11.

22. The compound according to claim 14, characterized in that Rβ is Me2NCO-, Et2NCO-, or N-Me-N-CH3 (CH2) vNCO- where v is 0-9.

23. The compound according to claim 14, characterized in that Rβ is Ts-, PhS02-, MeS02, PhCH2S02-, CF3S02- or CH3 (CH2) US02-, where u is 0-11.

24. The compound according to claim 14, characterized in that Rβ is CH3SCO-, or CH3 (CH2) tSCO-, where t is 0-11.

25. The compound according to claim 14, characterized in that R is HCO-, CH3CO-, PhCH2CO-, PhCO- or CH3 (CH2) wCO-, where w is 0-11.

26. The compound according to claim 14, characterized in that R is CH3 (OCH2CH2) qOCO-, or CH3 (CH2) r (OCH2CH2) s-OCO-, where q is 1-4, r is 0-5 and s is 1-4.

27. The compound according to claim 14, having the formula VII: VII or a biologically acceptable salt or a pro-exporter molecule thereof, characterized in that R2 and R3 are independently hydrogen, methyl or ethyl; and R and R5 are independently hydrogen or methyl.

28. The compound according to claim 27, characterized in that Ri is t-butoxycarbonyl, ectyl, hexanoyl, octanoyl or benzyloxycarbonyl; and R2, R3, R. and R5 are hydrogen.

29. The compound according to claim 27, characterized in that - (AA) n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SHV, DEL, DGP, DEP, DGT, DLN, DEE, DSL, DVP, DEA, DSY, ELP, VED, IEP or EIT.

30. The compound according to claim 27, characterized in that it is selected from the group consisting of N- (Z-YVAD) -N '-acetyl-Rhodamine 110, SEQ ID NO: 2; N- (Z-DEVD) -N '-acetyl-Rhodamine 110, SEQ ID NO: 5; N- (Z-VD) -N'-acetyl-Rhodamine 110; N- (Z-AD) -N '-acetyl-Rhodamine 110; -? * «. • < «**», H ^ 28 N- (Z-VAD) -N '-acetyl-Rhodamine 110; N- (Z-DEVD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 5; N- (Ac-DEVD) -N'-ethoxycarbonyl-Rhodamine 110, SEQ ID NO: 5; N- (Ac-DEVD) -N'-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5; N- (Ac-DEVD) -N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5; N- (Ac-DEVD) -N'-decyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5; N- (Ac-DEVD) -N'-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO: 5; and N- (Ac-DEVD) -N'- (ethylthio) carbonyl-Rhodamine 110, SEQ ID NO: 5

31. A method for the preparation of a compound according to claim 27, characterized in that it comprises (a) reacting Rhodamine with acetic anhydride to produce N-acetyl-Rhodamine; (b) the condensation of N-acetyl-Rhodamine together with the β-t-butyl ester of N-fmoc-L-aspartic acid to produce N- (Fmoc-Asp (OBu-t)) -N'-acetyl -Rhodamine; (c) the elimination of the Fmoc group to produce * e ~ *? - *, £ • - and? ~ T N- (Asp (OBu-t)) -N'-acetyl-Rhodamine; (d) the condensation of N- (Asp (OBu-t)) -N'-acetyl-Rhodamine with Z- (AA) n to produce N- (Z- (AA) n-Asp (OBu-t) ) -N '-acetyl-Rhodamine; and (e) removing the OBU-t protecting group to produce the N- (Z- (AA) n-Asp) -N'-acetyl-Rhodamine;

32. A method for the preparation of a compound according to claim 27, characterized in that it comprises (a) reacting Rhodamine with acetic anhydride to produce N-acetyl-Rhodamine; (b) the condensation of N-acetyl-Rhodamine with Z- (AA) n-Asp (OBu-t) to produce the N- (Z- (AA) n-Asp (OBu-t)) -N ' acetyl-Rhodamine; and (c) the removal of the OBu-t protecting group to produce the N- (Z- (AA) n-Asp) -N'-acetyl-Rhodamine.

33. The method according to claim 31 or 32, characterized in that - (AA) n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SHV, DEL, DGP, DEP, DGT, DL ?, DEE, DSL, DVP, DEA, DSY, ELP, VED, IEP or EIT.

34. The compound according to claim 1, having the formula IX: or a biologically acceptable salt or a pro-reporter molecule thereof, characterized in that Ri is hydrogen or an N-terminal protecting group; Rβ is a blocking group which is not an amino acid or derivative of an amino acid; each AA is independently a residue of an α-amino acid or β-amino acid, or a derivative of an α-amino acid or β-amino acid; and n is an integer of 0-5; m is an integer of 0-3; R2 and R3 are independently hydrogen, methyl or ethyl; and R4 and R5 are independently hydrogen or methyl.

35. The compound according to claim 34, characterized in that R2, R3, R4 and Rs are c.te? *! í ?. hydrogen.

36. The compound according to claim 34, characterized in that Ri is H; n = l; - (AA) n is M; m is an integer from 1-2; and (AA) m is selected from the group consisting of GG, GA, AG, G, and A.

37. The compound according to claim 34, characterized in that the compound is selected from the group consisting of N- (GP) -N'-ethoxycarbonyl-Rhodamine 110, N- (GPG) -N'-ethoxycarbonyl-Rhodamine 110, and NGN '-octyloxycarbonyl-Rhodamine 110.

38. The compound according to claim 34, characterized in that R is an N-terminal protecting group selected from the group consisting of t-butyloxycarbonyl, acetyl, hexanoyl, octanoyl, dodecanoyl and benzyloxycarbonyl.

39, The compound in accordance with s # * - i claim 34, characterized in that (AA) n- (AA) m is selected from the group consisting of SQNY-PIV SEQ ID NO: 28, ARLV-AEA SEQ ID NO: 29, ATIM-MQR SEC ID NO: 30, RQAN-FLG SEQ ID NO: 31, PGNF-LQS SEQ ID NO: 32, SFSF-PQI SEQ ID NO: 33, TLNF-PIS SEQ ID NO: 34, AETF-YVD SEQ ID NO: 35 or RKVL-FLD SEQ ID NO: 36; SQNY-PI SEQ ID NO: 117 ARLV-AE SEQ ID NO: 118, ATIM-MQ SEQ ID NO: 119, RQAN-FL SEQ ID NO: 120, PGNF-LQ SEQ ID NO: 121, SFSF-PQ SEQ ID NO : 122, TLNF-PI SEQ ID NO: 123, AETF-YV SEQ ID NO: 124 or RKVL-FL SEQ ID NO: 125; SQNY-P SEQ ID NO: 126, ARLV-A SEQ ID NO: 127, ATIM-M SEQ ID NO: 128, RQAN-F SEQ ID NO: 129, PGNF-L SEQ ID NO: 130, SFSF-P SEQ ID NO: 131, TLNF-P SEQ ID NO: 132, AETF-Y SEQ ID NO: 133 and RKVL-F SEQ ID NO: 134.

40. The compound according to claim 34, characterized in that (AA) n- (AA) m is selected from the group consisting of LRGGG SEQ ID NO: 101, LRGGA SEQ ID NO: 102, MRGGG SEQ ID NO: 96, MRGGA SEQ ID NO: 135, IRGGG SEQ ID NO: 97, IRGGA SEQ ID NO: 136, LVGGG SEQ ID NO: 98, LVGGA SEQ ID NO: 137, MVGGG SEQ ID NO: 99, MVGGA SEQ ID NO: 138, IVGGG SEQ ID NO: 100, IVGGA SEQ ID NO: 139, LRGG SEQ ID NO: 55, MRGG SEQ ID NO: 56, IRGG SE ID NO: 57, LVGG SEQ ID NO: 58, MVGG SEQ ID NO: 59 and IVGG SEC ID NO: 60

41. A compound that has the general formula SAW: or a biologically acceptable salt, characterized in that R2 and R3 are independently hydrogen, methyl or ethyl; R4 and R5 are independently hydrogen or methyl; R is a blocking group which is not an amino acid or derivative of an amino acid.

42. The compound according to claim 41, characterized in that R2, R3, R4 and Rs are hydrogen.

43. The compound according to claim 41, characterized in that R2 and R3 are methyl; R4 and Rs are hydrogen.

44 The comflifeinium according to claim 41, characterized in that R2 and R3 are ethyl R4 and R5 are methyl.

45. The compound according to claim 41, characterized in that it is selected from the group consisting of N-methoxycarbonyl-Rhodamine 110, N-ethoxycarbonyl-Rhodamine 110, N-hexyloxycarbonyl-Rhodamine 110, N-octyloxycarbonyl-Rhodamine 110, N- decyloxycarbonyl-Rhodamine 110, and N-dodecyloxycarbonyl-Rhodamine 110.

46. The compound according to claim 41, characterized in that it is selected from the group consisting of N-dimethylcarbamyl-Rhodamine 110, N- (N-methyl-N-hexylcarbamyl) -Rhodamine 110, N-methanesulfonyl-Rhodamine 110, N - (ethylthio) carbonyl-Rhodamine 110, N- (hexylthio) carbonyl-Rhodamine 110, N- (octylthio) carbonyl-Rhodamine 110, N-acetyl-Rhodailthes-110, N-acetyl-Rhodamine 116, N- (2, 5 , 8-trioxadeyloxycarbonyl) -Rhodamine 110 and N- (2-butoxyethoxycarbonyl) -Rhodamine 110.

47. A method for the detection of an enzyme included in the apoptosis cascade in one or more cells, characterized in that it comprises (a) contacting one or more cells with a reporter compound according to claim 1 or 14 under conditions in which the compound reporter is taken in one or more cells, and (b) record the fluorescence of one or more cells, where the relative change in fluorescence within one or more cells, compared to the control cells which have not been of this Contact form is an indication of the presence of the enzyme.

48. The method according to claim 47, characterized in that said enzyme is an intracellular caspase.

49. One method, to measure the activity of an enzyme involved in the apoptosis cascade in one or more cells, characterized in that it comprises (a) contacting one or more cells with the reporter compound according to claim 1 or 14 under conditions in the which the reporter compound is taken in one or more cells, and (b) recording the fluorescence of one or more cells, wherein the change in fluorescence, within one or more cells compared to the control cells which have not been Contacting is a measurement of the activity of the enzyme.

50. The method according to claim 49, characterized in that said enzyme is an intracellular caspase.

51. A method for determining whether a test substance has an effect on an enzyme involved in the apostosis cascade in one or more test cells, characterized in that it comprises (a) contacting one or more test cells with the test substance and the reporter compound according to claim 1 or 14 under conditions in which the test substance is either interacted with an external receptor or is taken in one or more cells, and the reporter compound is taken in one or more cells, and (b) ) record the fluorescence of the test cells, compared to the control cells which have only been contacted with the reporter compound, where a change in fluorescence within one or more test cells compared to the control cells it is an indication that the test substance has an effect on said enzyme.

52. The method according to claim 51, characterized in that said enzyme is an intracellular caspase.

53. The method according to claim 51, characterized in that one or more test cells are brought into contact with the test substance prior to the contact of the cells with the reporter compound.

54. The method according to claim 51, characterized in that one or more test cells are contacted with the test substance after contact with the reporter compound.

55. The method according to claim 51, characterized in that one or more test cells are substantially in contact with each other. 10 simultaneous with the test substance and the reporter compound.

56. The method according to claim 51, characterized in that the method is to determine if the test substance stimulates the activity of the enzyme.

57. The method according to claim 51, characterized in that the method is to determine if the test substance inhibits the activity of the enzyme. 25 _b- £ ^

58. The method according to claim 51, characterized in that the additional contacting step includes contacting one or more test cells with at least one second test substance in the presence of the first test substance. proof.

59. The method according to claim 51, characterized in that one or more test cells are derived from an organism of a single cell.

60. The method according to claim 51, characterized in that one or more test cells are derived from a multicellular organism.

61. The method according to claim 60, characterized in that said multicellular organism is selected from the group consisting of a mammal, an invertebrate animal, an insect and a plant. fc * í »a A» * "Ai» tfi4

62. The method according to claim 51, characterized in that one or more test cells and control cells are derived from the group consisting of hair, brain, peripheral nervous system, eye, ear, nose, mouth, amygdala, tooth, esophagus, lung, sinuses, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, glands and reproductive organs, joints , skin and bones of said multicellular organism.

63. The method according to claim 51, characterized in that one or more test cells are cancerous.

64. The method according to claim 63, characterized in that one or more cancerous test cells are derived from the group consisting of the brain, peripheral nervous system, eye, ear, nose, mouth, amygdala, tooth, esophagus, lung, sinuses, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, tract intestinal, pancreas, endocrine glands or tissues, kidney, bladder, glands or reproductive organs, joints, skin and bones of said multicellular organism.

65. The method according to claim 63, characterized in that one or more cancerous test cells are derived from a human in need of treatment with a chemotherapy drug and the test substance is a chemotherapy agent.

66. The method according to claim 51, characterized in that the test substance is a chemotherapy agent.

67. The method according to claim 51, characterized in that the test substance is a mixture of chemotherapy agents.

68. A method to determine the sensitivity of 2 an animal with cancer fifflBBÉlBl. treatment with one or more chemotherapy agents, characterized in that it comprises (a) contacting one or more chemotherapy agents and the reporter compound according to claim 1 or 14, the cancer cells taken from said animal, under conditions in which one or more agents either interact with an external receptor or taken within said cell, and (b) register the fluorescence of the cancer cells, compared with the control cells that have only been contacted with the reporter compound, where a change in fluorescence, in cancer cells compared to control cells is an indication that cancer cells are sensitive to the one more chemotherapy agents and that the animal is sensitive to treatment.

69. The method according to claim 68, characterized in that the animal is a human.

70. A method for verifying the treatment of an animal for treating it with one or more chemotherapy drugs, characterized in that it comprises (a) administering one or more chemotherapy agents to the animal, (b) contacting the cells taken from the animal after administration with the reporter compound according to claim 1 or 14 under conditions in which the reporter compound is taken in the cells, and (c) recording the fluorescence of the cells contacted with the reporter compound, compared to the control cells which have been taken from the animal prior to administration wherein a change in the fluorescence of the cells taken from the animal compared to the control cells is an indication that the animal is sensitive to the chemotherapeutic agents.

71. The method according to claim 70, characterized in that the animal suffers from a disease related to apoptotic cell death.

72. A method for determining whether a test substance inhibits or prevents cell death in one or more test cells, characterized in that it comprises (a) contacting one or more test cells with the test substance and reporter compound according to the claim 1 or 14 under conditions in which the reporter compound is placed in one or more cells, and (b) recording the fluorescence of one or more test cells, compared to control cells that have only been contacted with the compound reporter, where a decrease in fluorescence within one or more of the test compared to the control cells is an indication that the test substance inhibits or prevents the death of the cells.

73. The method according to claim 72, characterized in that one or more test cells are nerve cells.

74. The method according to claim 72, characterized in that one or more test cells are selected from the group consisting of myocardial cells, immune cells, cells of an organ to be transplanted, sperm, eggs, cell lines which produce a recombinant protein , hair cells, skin cells and nerve cells.

75. A method for determining whether a test substance causes or increases the death of the cells in one or more test cells, characterized in that it comprises (a) contacting one or more test cells with the test substance and reporter compound according to claim 1 or 14 ba or conditions in which the reporter compound is placed in one or more cells, and (b) record the fluorescence of one or more test cells, compared to the control cells which have only been placed in contact with the reporter compound, wherein an increase in fluorescence, within one or more test cells compared to the control cells is an indication that the test substance causes or increases the death of the cells.

76. The method according to claim 75, characterized in that one or more test cells are cancer cells, yeasts, fungi or bacteria.

77. A method for detecting a viral protease in one or more cells, characterized in that it comprises (a) contacting the cells with the reporter compound according to claim 34 under conditions in which the reporter compound is placed in the cells, and (b) recording the fluorescence of said cells, wherein a change or increase in fluorescence within the cells compared to control cells that have not been contacted in this way, is an indication of the presence of the viral protease.

78. A method for measuring the activity of a viral protease in one or more cells infected with the virus, characterized in that it comprises (a) contacting one or more cells infected with the virus with the reporter compound according to the invention. claim 34 under conditions in which the reporter compound is placed in one or more cells infected with the virus, and (b) register the fluorescence of one or more cells, wherein a change or increase in fluorescence within one or more cells infected with the virus compared to control cells that have not been contacted, is a measure of the activity of the viral protease.

79. A method for determining whether a test substance has an effect on the activity of the viral protease in one or more cells infected with the virus, characterized in that it comprises (a) contacting the test cells infected with the virus with the substance of the virus. test and the reporter compound according to claim 34 under conditions in which said reporter compound is placed in the infected test cells, and (b) recording the fluorescence of the infected test cells compared to the infected control cells which have only been tested. placed in contact with the reporter compound, wherein a change or increase in fluorescence within the infected test cells compared to the infected control cells, is an indication that the test substance has an effect on the viral protease.

80. The method according to any of claims 77-79, characterized in that the cells are cells infected with HIV and the viral protease is the HIV protease.

81. The method according to any of claims 77-79, characterized in that the cells are cells infected with the adenovirus and the viral protease is the adenovirus protease.

82. The method according to any of claims 77-79, characterized in that the cells are cells infected with the VSH and the viral protease is the protease VSH.

83. The method according to any of claims 77-79, characterized in that the cells are cells infected with the VMCH and the viral protease is the VMCH protease.

84. The method according to any of claims 77-79, characterized in that the cells are cells infected with the HCV and the protease Viral is the VCH protease.

85. A method for measuring the activity of the protease or peptidase in cells, characterized in that it comprises (a) contacting the test cells with the reporter compound according to claim 34 under conditions in which the reporter compound is placed in the test cells, or the reporter compound is 20 interacting with a protease or peptidase from the outer membrane of said cells, and (b) recording the fluorescence of the cells, wherein a change or increase in fluorescence within the test cells compared to the cells of ^: ^. ^ x¡ control which have not been contacted is a measure of the activity of the protease or peptidase.

86. A method for determining whether a test substance has an effect on the activity of the protease or peptidase in the test cells, characterized in that it comprises (a) contacting the test cells with the test substance and the reporter compound according to the claim 34 under conditions in which the reporter compound is placed in the test cells, or the reporter compound is interacting with a protease or peptidase of the outer membrane of said cells, and (b) registering the fluorescence of the test cells compared with the test cells which have only been contacted with the reporter compound, where a change or increase in fluorescence within the test cells compared to the control cells which is an indication that the test substance it has an effect on the protease or peptidase.

87. The method of compliance with any of 21 claims 85-86, characterized in that the cells are endotteliales cells and the peptidase is the methionine type 2 aminopeptidase.

88. The method according to any of claims 85-86, characterized in that the cells are T cells and the peptidase is dipeptidyl peptidase-IV.

89. The method according to any of claims 85-86, characterized in that the cells are neuronal cells and the protease is calpain.

90. The method according to any of claims 85-86, characterized in that the peptidase is the aminopeptidase.