JP2001519368A - Novel fluorescent reporter molecules and their applications including caspase assays - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel fluorescent dyes, novel fluorogenic and fluorescent reporter molecules, and novel enzymatic assay processes, which can be used in whole cells, cell lines and tissue samples from any living organism or organ. Caspases and other enzymes involved in apoptosis can be used to detect activity. This reporter molecule and assay process can be used in drug screening procedures to identify compounds that 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 a chemotherapeutic drug. The invention also relates to novel fluorogenic and fluorescent reporter molecules and novel enzymatic assay processes, which include methionine aminopeptidase type 2, dipeptidyl peptidase IV, calpain, aminopeptidase, HIV protease, adenovirus protease, It can be used to detect the activity of HSV-1 protease, HCMV protease and HCV protease.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention is in the field of intracellular detection of enzymes using fluorogenic or fluorescent probes. The present invention involves novel fluorescent dyes and their application for the preparation of novel fluorogenic peptides or fluorescent peptides or amino acid derivatives that are substrates for proteases and peptidases. In particular, the present invention
It relates to novel fluorogenic peptides or derivatives of fluorescent peptides that are substrates for enzymes involved in apoptosis (eg, caspase and lymphocyte-derived serine protease granzyme B). The invention also relates to caspases and other enzymes involved in apoptosis in the survival or death of whole cells, cell lines, or tissue samples from any healthy, diseased, infected or cancerous organ or tissue. Related to a process for the determination of activity. The present invention also relates to the use of fluorogenic or fluorescent substrates in novel assay systems for the discovery or detection of inhibitors or inducers of apoptosis in a collection of compounds or a library of compounds. In addition, the present invention relates to the use of fluorogenic or fluorescent substrates in determining the susceptibility of cancer cells to treatment with a chemotherapeutic agent. The invention also relates to aminopeptidases A and N, methionine aminopeptidase and exopeptidases such as dipeptidyl peptidase IV, endopeptidases such as calpain, HIV protease, HCMV protease, HSV protease, HCV protease and adenovirus protease. It relates to novel fluorogenic peptides or derivatives of fluorescent peptides that are substrates for proteases.

Related Art [0002] Living organisms eliminate unwanted cells by processes known variously as regulated cell death, programmed cell death or apoptosis. Such cell death occurs as a normal aspect of animal development and in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cumb).
Ridge Philos. Soc. 26: 59-86 (1951); Gluc.
ksmann, A .; Archives de Biologie 76:41
9-437 (1965); Ellis et al., Dev. 112: 591-603 (1
991); Vaux et al., Cell 76: 777-779 (1994)). Apoptosis regulates cell number, promotes morphogenesis, removes harmful or otherwise abnormal cells, and eliminates cells that have already performed their function. In addition, apoptosis occurs in response to various physiological stresses such as hypoxia and ischemia (PCT published application WO 96/20721).

[0003] Regulated cell death, including plasma and nuclear blebbing, cell atrophy (nuclear and cytoplasmic aggregation), organelle rearrangement and condensation, chromatin aggregation and apoptotic bodies (membrane-surrounding particles containing intracellular material) There are many morphological changes that are shared by cells that have experienced the production of (Orrenius, SJ Internal).
Medicine 237: 529-536 (1995)).

[0004] Apoptosis is achieved via an endogenous mechanism of cell suicide (Wyll).
ie, A.I. H. in Cell Death in Biology and
Pathology, Bowen and Lockshin, Ed., Chapman
and Hall (1981), pp. 9-34). Cells activate their internally encoded suicide programs as a result of either internal or external signals. This suicide program is performed through the activation of a carefully regulated gene program (Wylie et al., Int. Rev. Cyt. 68: 251 (
1980); Ellis et al., Ann. Rev .. Cell Bio. 7: 663 (
1991)). Apoptotic cells and bodies are usually recognized and removed by neighboring cells or macrophages prior to lysis. Due to this removal mechanism, inflammation is not induced despite removal of many cells (
Orrenius, S.M. J. Internal Medicine 237: 5
29-536 (1995)).

[0005] Mammalian interleukin 1β (IL-1β) plays an important role in a variety of pathological processes including chronic and acute inflammatory and autoimmune diseases (Op
Penheim, J.M. H. Et al., Immunology Today, 7, 45.
-56 (1986)). IL-β is unable to bind to the IL-1 receptor and is associated with a biologically inactive cell-associated precursor polypeptide (pro-IL-1β
(Mosley et al., J. Biol. Chem. 262: 29).
41-2944 (1987)). By inhibiting the conversion of precursor IL-1β 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, sepsis, wound healing, hematopoiesis and the growth of certain leukemias (Dinarello, C.
. A. Blood 77: 1627-1652 (1991); diGiovin.
e et al., Immunology Today 11:13 (1990)). Interleukin 1β convertase (ICE) is interleukin 1β (IL-1β)
(Thornberry, NA, et al., Nat.
ure 356: 768 (1992)); Yuan, J. et al. Cell 75: 6
41 (1993)). ICE is a substrate-specific cysteine protease that cleaves inactive prointerleukin-1 to produce mature IL-1. IC
The genes encoding E and CPP32 currently have at least 12 members:
ICE, CPP32 / Yama / Apopain, mICE2, ICE4, IC
H1, TX / ICH-2, MCH2, MCH3, MCH4, FLICE / MAC
Mammalian ICs containing H / MCH5, ICE-LAP6 and ICEre1III
It is a member of the E / Ced-3 gene family. The proteolytic activity of this family of cysteine proteases, whose cysteine residues in the active site are essential for ICE-mediated apoptosis, appears to be important in mediating cell death (
Miura et al., Cell 75: 653-660 (1993)). This gene family has recently been termed caspases (Alnernri, ES
. Et al., Cell, 87: 171 (1996)). Triggers of death, such as tumor necrosis factor, FAS ligands, oxygen or nutrient deficiencies, viruses, toxins, anticancer drugs, etc., are caspases upstream of the cascade (eg, FLICE / MACH / MCH5).
Is a caspase downstream of the cascade (eg, CPP-32 / Yama / Ap
Caspases can be activated in cells in a cascade-like manner that can activate C. opain. Activation of the caspase cascade leads to cell death.

[0006] The abundance of scientific evidence suggests that (in many diseases) the caspase cascade
If it should not be activated, it will be activated. This leads to excessive cell suicide and organ failure. Diseases associated with inappropriate activation of the caspase cascade and subsequent cell suicide include myocardial infarction, congestive heart failure, autoimmune disease, AIDS, viral infection, renal failure, liver failure, rheumatoid arthritis, ischemic attack,
Including neurodegenerative diseases, atherosclerosis and the like. Thus, the discovery of new agents that can block or inhibit the activation of the caspase cascade has a wide range of effects on most (if not all) treatment of degenerative diseases of the human body organ system.

[0007] Caspases are also considered critical in the development and treatment of cancer. There is increasing evidence that cancer cells containing caspases lack part 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 ability to cause suicide of the cells, and the cells become immortal-they become cancerous.

[0008] Chemotherapy (anti-cancer) drugs have been found to induce cancer cells to commit suicide by reactivating the dormant caspase cascade. This may be a critical aspect of most (if not all) modes of action of known anticancer drugs (Lo
s et al., Blood, Vol. 90, no. 8: 3118-3129 (1997)
Friesen et al., Nat. Med. 2: 574 (1996)). Chemotherapeutic drugs can differ from their ability to activate the caspase system in different classes of cancer. In addition, anticancer drugs appear to differ in the ability to activate the caspase cascade in a given cancer (eg, lung cancer) and different patients. In other words, for example, the chemosensitivity of lung cancer cells to various anticancer drugs varies between patients.

[0009] In summary, excessive activation of the caspase cascade plays a critical role in a wide variety of degenerative organ diseases, whereas the non-functionalized caspase system is a hallmark of cancer cells. New drugs that inhibit or stimulate the caspase cascade seem to revolutionize the treatment of many human diseases ranging from infectious, cardiovascular, endocrine, renal, liver and brain diseases to immune system diseases and cancer is there.

[0010] In order to find drugs that inhibit or stimulate the caspase cascade, there is a need to develop high power caspase activity (HTCA) assays. These HTC
The A assay must be able to monitor the activation or inhibition of the caspase cascade in living or whole cells. Ideally, the HTCA assay is performed on any living cell or whole cell (even if its origin is: cancer cells, tumor cells, immune cells, brain cells, cells of the endocrine system, cells or cell lines from different organ systems, It should be sufficiently diverse to measure caspase cascade activity in biopsy samples (and so on). Furthermore, such an HTCA assay should be able to measure the activation or inhibition of any caspase enzyme or any other enzyme involved in the caspase cascade-in living cells or whole cells. The development of such diverse HTCA assays shows substantial advantages in the field of drug screening.

[0011] Currently, available HTCA assays do not allow intracellular screening for compounds that can activate or inhibit the caspase cascade. There are only cell-free high power screening assays available that can measure the activity of individually isolated caspase enzymes, or assays that can measure caspase activity in dead cells that have been permeabilized, for example, by osmotic shock. (Los et al., Blood, Vol. 90, No. 8: 3118-3129 (1997)). However, these enzyme assays cannot predict the effect of a compound on the caspase cascade in living cells for the following reasons: A) Cell-free assays or assays using dead and permeabilized cells do not predict the ability of the compound to penetrate cell membranes. What is unpredictable is crucial because the caspase cascade is inside the cell. To be active,
The compound must not only be able to regulate the caspase enzyme, but it must be able to penetrate the entire cell membrane. As such, cell-free assays or assays using dead cells cannot determine whether a compound is potentially useful as a drug. 2. C) Caspases isolated in cell-free assays are highly sensitive to oxidation and to compounds that can cause oxidation of the enzyme. This property of isolated caspases renders cell-free caspase screening assays highly sensitive to artifacts and the successful use of these assays for high power screening of combinatorial (or other) chemical libraries Was eliminated. Many previous screening efforts using cell-free caspase enzyme assays have led to the development of many inhibitors that oxidize caspases,
It did not lead to compounds that could be useful as potent drugs. Similar difficulties have been reported by others. 3. 2.) Many cell receptors, proteins, cellular components and cofactors, most of which are unknown, can affect the caspase cascade in living cells. Cell-free caspase assays or assays using permeabilized dead cells
Do not consider their cell receptors and cofactors. Because of this, compounds identified in cell-free or dead-cell caspase assays may not work in living cells. On the other hand, compounds that can inhibit or stimulate the caspase cascade indirectly through one of the cell receptors or cofactors,
It is completely missed in cell-free or dead cell caspase assays. 4. 2.) It is quite likely that the caspase cascade functions differently in cells from different organs. There is evidence that receptors and cofactors affecting the caspase cascade differ between cell types. Using cell-free or dead-cell assays, it is virtually impossible to identify cell type-specific or organ-specific modulators of the caspase cascade.

A potentially important application of the HTCA assay system for measuring intracellular caspase enzymes or any other enzymes involved in apoptosis is the chemosensitivity testing of human cancers. At present, it is known that there is a genetic difference in the sensitivity of human cancer to commercially available anticancer drugs. For example, one patient's lung cancer cells are sensitive to drug A, while another patient's cancer is insensitive to drug A but sensitive to drug B. This pharmacogenetic difference in chemosensitivity of cancer cells from different individuals is a well-known phenomenon.

In the past, attempts have been made to determine the chemosensitivity of cancer cells obtained from individual patients before designing a treatment regimen with one or more commercially available anticancer drugs. However, chemosensitivity testing did not find widespread use. Because the procedure involved has some inherent technical difficulties:
It is very time consuming (more than 6 days per screen) and requires culturing of cells before screening. This cell culture leads to clonal selection of the cells, and the cultured cells are no longer the proxy for the cancer in the patient. The HTCA assay system for rapidly measuring intracellular caspase activity can be used to very quickly determine the chemosensitivity profile of freshly excised cancer cells. If the assay is high power, many samples (eg, different metastases) obtained from the same patient may be tested for chemosensitivity. This information can then be used to design a treatment regimen using a combination of commercially available anti-cancer drugs on the cells with the greatest sensitivity.

[0014] Can be used in drug development or diagnostic procedures to rapidly detect and measure the activity of compounds that activate or inhibit the caspase cascade or other enzymes involved in apoptosis inside living or dead cell bodies. For such assays, the need for HTCA assays and the presence of reagents is apparent. Reagents for this type of cell assay should ideally meet the following conditions: a) After the amide bond in the peptide reporter has been cleaved by caspases or other enzymes involved in apoptosis, the peptide reporter molecule There should be a large difference in the fluorescent signal between the and the reporter molecule, preferably the peptide reporter molecule should be non-fluorescent, and most preferably the peptide reporter molecule should be non-fluorescent and colorless . b) The peptide reporter molecule should be cell permeable, so the number of hydrophilic groups in the molecule should be minimal, and the size of the molecule should preferably be small; Once it has penetrated the cell membrane, it should preferably not diffuse out of the cell; d) the reporter molecule should preferably not diffuse out of the cell once it is released from the peptide.

[0015] The methods for screening for apoptosis inhibitors or inducers in whole cell assays versus cell-free enzyme assays can also be used for screening for inhibitors of other enzymes of caspases. Traditionally, enzyme inhibitors were first identified by cell-free enzyme assays. The cell culture is then used for a secondary assay to assess the activity of the activating compound on intact cells. Cell permeable, fluorogenic or fluorescent substrates can be screened directly for inhibitors of proteases and peptidases and other enzymes in whole living cells. Whole cell assays have several advantages over cell-free enzyme assays. One advantage of whole cell assays is that inhibitors must penetrate cells in order to be detected. Many proteases in living cells are regulated by other proteins, receptors or genes,
Screening with live cells involves screening small molecule compounds that interfere with cellular proteases by binding to the active site, as well as transcription, translation, biosynthesis, subunit assembly, cell cofactors or signaling mechanisms (or, in the case of viral proteases, Allows the identification of compounds that modulate protease function by interfering with virus entry into host cells). In addition, due to the enrichment of aminopeptidases in the cells, these aminopeptidases can be used in designs for whole cell assays with fluorogenic or fluorescent substrates that otherwise do not work in cell-free enzyme assays. Thus, high power screening for such assays in whole cells that can be used in drug development or diagnostic procedures (
There is a need for the development of HTS) assays and reagents.

AGM-1470 (also known as TNP-470) is an inhibitor of angiogenesis in clinical trials for various cancers. The mechanism of action of AGM-1470 is currently discovered by two separate research groups (Sin, N. et al., Proc. Natl. Acad. USA 94: 6099-6103 (1).
997); Griffith, E .; C. Et al., Chem. Biol. 4: 461-
471 (1997)). They found that AGM-1470 and analogs were inhibitors of type 2 methionine aminopeptidase (MetAP-2). The titers of inhibition of endothelial cell proliferation and of methionine aminopeptidase activity were measured with a series of AGM-1470 analogs and a significant correlation between the two activities was found.

Since angiogenesis inhibitors are known to be able to selectively kill cancer cells,
Cell screening assays for inhibitors of MetAP-2 may lead to new anticancer drugs. Thus, cells permeable to a fluorogenic or fluorescent substrate for MetAP-2 can be used for screening for inhibitors of MetAP-2 in endothelial cells, which can result in novel anti-cancer drugs.

Recently, HIV protease inhibitors such as ritonavir and viraccept have been shown to be very effective in treating patients infected with HIV. These inhibitors were designed based on the structure of the HIV protease substrate. The activity of these inhibitors was first determined on HIV protease. The active compounds were then tested for inhibition of HIV infection in cell culture. Cells able to penetrate a fluorogenic or fluorescent substrate for HIV protease can speed up the process for the discovery of novel HIV protease inhibitors and provide a new and better treatment for HIV infection Can be used for screening for HIV protease inhibitors in HIV infected cells. HIV protease processes the viral precursor protein at a later stage of viral replication,
Cells permeable to fluorogenic or fluorescent substrates for proteases can also be used for screening for compounds that inhibit gene transcription or translation, viral entry, or other key proteins in the early stages of HIV infection. . Fluorogenic or fluorescent substrates can also be used for the diagnosis of HIV infection, which can be more sensitive than currently available methods.

By applying the same principle, cells permeable to a cathepsin B fluorogenic or fluorescent substrate can be used for screening for cathepsin B inhibitors. Cells that can penetrate the fluorogenic or fluorescent substrate of dipeptidyl-peptidase IV
It can be used for screening for dipeptidyl-peptidase IV inhibitors. Cells that are permeable to a fluorogenic or fluorescent substrate for renin can be used for screening for renin inhibitors, and a fluorogenic or fluorescent substrate for adenovirus or other viral proteases. Cells that are permeable to can be used for screening for adenovirus protease inhibitors or other viral protease inhibitors.

US Pat. Nos. 4,557,862 and 4,640,893 disclose rhodamine 110 derivatives as fluorogenic substrates for proteinases of the following formula:

[0021]

Embedded image Here, R ₁ and R ₂ (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. Typical (AA) ₂ -rhodamines and (peptide) ₂ -rhodamines are (Z-Arg) ₂ -rhodamine 110, (Ar
g) 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-Ar g) 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-Ar g) 2 - Rhodamine 110, and (Z -Ile-Pro-Arg) ₂ -rhodamine 110.

WO 96/36729 discloses compounds or their salts for assaying the activity of enzymes in metabolically active whole cells. The assay compound is said to contain a leaving group and an indicator group. Leaving groups include amino acids, peptides, sugars, sulfates, phosphates, esters, phosphates, nucleotides, polynucleotides, nucleic acids, pyrimidines, purines, nucleosides, lipids and mixtures. Selected from the group consisting of: The indicator group is selected from a compound having a first state when attached to the leaving group and a compound having a second state when the leaving group is cleaved from the indicator group by an enzyme. Preferred indicating compounds are said to be rhodamine 110, rhodol and fluorescein and analogs of these compounds. This patent application has listed a number of enzymes and enzyme substrates.

US Pat. No. 5,576,424 disclosed a haloalkyl derivative of a reporter molecule used to analyze metabolic activity in cells of the formula: XR-spacer-reporter-block, wherein: A block is a group that has been selected so as to be removable by the action of a specific analyte (to give the reporter different spectral properties than those of the substrate); the reporter is no longer blocked by the reporter bond Molecules that, when not attached to a block, have spectral properties different from those of the substrate;
A spacer is a covalent bond; XR- is a haloalkyl moiety that covalently reacts with an intracellular thiol to form a thioether conjugate. Preferred reporter compounds are said to include rhodamine-110, rhodal, fluorescein and others.

SUMMARY OF THE INVENTION The present invention involves a fluorogenic or fluorescent reporter compound of Formula I: xyz (I) or a biologically acceptable salt or proreporter molecule thereof. (For example,
(A methyl ester type of a carboxyl-containing amino acid residue). Where x and z
Are the same or different and whether the compound of formula I is a substrate for caspases,
Alternatively, it is a peptide or amino acid or an acyl group or other structure, such as being a substrate for another protease or peptidase or other enzyme; and wherein the scissile bond is of the formula I One or both of the xy and yz bonds. Or, here, the only cleavable bond is one of the xy or yz bond of Formula I where x is not the same as z. y is a fluorogenic or fluorescent moiety.

Preferred compounds are represented by the following formula II: R _1- (AA) _n -Asp-y-Asp- (AA) _n -R 1 (II) or a biologically acceptable salt thereof or Proreporter molecules (eg, the methyl ester form of a carboxyl-containing amino acid residue). Where R ₁ is N- 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 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. Preferred y is rhodamine, including rhodamine 110, rhodamine 116 and rhodamine 19. Most preferred y is rhodamine 110.

Particularly preferred compounds are represented by the following formula III:

[0027]

Embedded image Alternatively, it is a biologically acceptable salt or proreporter molecule (eg, a methyl ester form of a carboxyl-containing amino acid residue). Where R ₁ , AA, n are already defined in Formula II. Preferred R ₁ is t-butyloxycarbonyl, acyl and benzyloxycarbonyl. Preferred values for n are 1-3.

The present invention also relates to a process for the preparation of a compound of formula III, comprising: (a) N to obtain (Fmoc-Asp (OBu-t)) ₂ -rhodamine -Condensation of fmoc-L-aspartic acid β-t-butyl ester with rhodamine; (b) removal of Fmoc group to obtain (Asp (OBu-t)) ₂ -rhodamine; (c) (Z- (AA) _n -Asp (OBu-t)) _2- to obtain rhodamine,
Condensation of Z- (AA) _n with (Asp (OBu-t)) ₂ -rhodamine; and (d) removal of the OBu-t protecting group.

In a preferred embodiment,-(AA) _n is SEQ ID NO: 1 WEH, SEQ ID NO: 2 YVA, SEQ ID NO: 3 LEH, SEQ ID NO: 4 DET, SEQ ID NO: 5 D
EV, SEQ ID NO: 6 DEH, SEQ ID NO: 7 VEH, SEQ ID NO: 8 LET,
SEQ ID NO: 9 LEV, SEQ ID NO: 10 SHV, SEQ ID NO: 11 DEL, SEQ ID NO: 12 DGP, SEQ ID NO: 13 DEP, SEQ ID NO: 14 DGT, SEQ ID NO: 15 DLN, SEQ ID NO: 16 DEE, SEQ ID NO: : 17 DSL, SEQ ID NO: 18 DVP, SEQ ID NO: 19 DEA, SEQ ID NO: 20 DSY, SEQ ID NO: 21 ELP, SEQ ID NO: 22 VED, SEQ ID NO: 23 IEP, or SEQ ID NO: 24 IET, and Carboxy-containing amino acids are protected with an OBu-t group that is removed in a final step.

Another group of preferred compounds falling within the scope of Formula I include those wherein x is not the same as z. Preferred compounds of this group are those wherein x comprises a peptide or other structure that makes the compound a substrate for caspases, or a substrate for other proteases or peptidases or other enzymes; and xy in formula I A bond is a bond that breaks under biological conditions. z is a blocking group,
The yz bond of Formula I is a bond that is not fragile under biological conditions.

Specifically, the novel fluorogenic or fluorescent reporter compounds of the present invention are of Formula V: R _1- (AA) _n -Asp-y-R ₆ (V) or biologically An acceptable salt or proreporter molecule (eg, a methyl ester form of a carboxyl-containing amino acid residue). Wherein: R ₁ , AA, n and y are already defined in Formula II; and R ₆ is a blocking group that is not an amino acid or a derivative of an amino acid.

In particular, a preferred embodiment of the compound of formula V is represented by formula VII:

[0033]

Embedded image Alternatively, it is a biologically acceptable salt or proreporter molecule (eg, a methyl ester form of a carboxyl-containing amino acid residue). Where: R ₁ , R ₆ ,
AA and n are as previously defined in formulas II and V; and R ₂ and R ₃ are the same or different and are individually hydrogen, alkyl or aryl; and R ₄ and R ₅ are , The same or different, and individually, is hydrogen or alkyl.

Another group of preferred embodiments of the compounds of formula I is represented by formula VIII:

[0035]

Embedded image Alternatively, it is a biologically acceptable salt or proreporter molecule (eg, a methyl ester form of a carboxyl-containing amino acid residue). Where: R ₁ , R ₆ ,
AA and n are already defined in formulas II and V; m is 0-3
Is an integer. R ₂ and R ₃ are the same or different and are independently hydrogen, alkyl or aryl; and R ₄ and R ₅ are the same or different and are independently hydrogen or alkyl.

A further group of preferred embodiments of the compounds of formula I is represented by formula IX:

[0037]

Embedded image Alternatively, it is a biologically acceptable salt or proreporter molecule (eg, a methyl ester form of a carboxyl-containing amino acid residue). Where: R ₁ , R ₆ ,
AA and n are already defined in formulas II and V; m is 0-3
Is an integer. R ₂ and R ₃ are the same or different and are independently hydrogen, alkyl or aryl; and R ₄ and R ₅ are the same or different and are independently hydrogen or alkyl.

The present invention also relates to a method of preparing a compound of formula VII, which includes: (a) reacting acetic anhydride with rhodamine to obtain N-acetyl-rhodamine of formula VI; VI N-acetyl-rhodamine and N-fmoc-L-aspartic acid β-t-butyl ester are condensed together to form N- (Fmoc-Asp (OBu-
t)) -N'-acetyl-rhodamine is obtained; (c) N- (Asp (OBu-t))-N'-acetyl-
(D) N- (Asp (OBu-t))-N'-acetyl-rhodamine and Z- (
AA) condensing _n to give N- (Z- (AA) _n -Asp (OBu-t))-N′-acetyl-rhodamine; and (e) removing N- (Z -(AA) _n -Asp) N'-acetyl-rhodamine; or alternatively (a) reaction of acetic anhydride with rhodamine to obtain N-acetyl-rhodamine of formula VI; (b) N- of formula VI Acetyl-rhodamine and Z- (AA) _n -Asp (β-O Bu-t) are condensed to give N- (Z- (AA) _n -Asp (OBu-t))-N′-acetyl-rhodamine. And (c) removing the OBu-t protecting group to give N- (Z- (AA) _n -Asp) N′-acetyl-rhodamine.

In this embodiment, (AA) _n comprises an amino acid such as glutamic acid or aspartic acid, and the carboxyl group is an OBu that is cleaved in a final step.
Protected as a -t group.

Thus, the present invention also relates to novel fluorescent dyes of the formula VI, which are derivatives of rhodamine. These compounds, the blocking group R ₆ is prepared by introducing into a 2 Tsunoa amino groups rhodamine.

The R ₂ HN group of Formula VI provides a linking site for reacting with a potential enzyme substrate (eg, the group of the carboxyl group of an N-blocked peptide) to form a peptide amide bond. . This reaction converts the fluorescent molecule of Formula VI to a non-fluorescent peptide reporter molecule of Formulas VII-IX that is a substrate for a protease or peptidase. Cleavage of the cleavable peptide-reporter amide bond in the peptide-reporter by a protease or peptidase yields a compound of formula VI or VI ′ that is fluorescent.

In particular, the novel fluorescent dyes of the present invention are of formula VI:

[0043]

Embedded image Or, a biologically acceptable salt thereof. Wherein: R ₂ and R ₃ are the same or different and are independently hydrogen, alkyl or aryl; R ₆ is a blocking group that is not an amino acid or an amino acid derivative; R ₄ and R ₅ are the same or Different and, separately, hydrogen or alkyl.

Preferred R ₂ and R ₃ are hydrogen, methyl or ethyl; preferred R ₄ and R ₅ are hydrogen or methyl.

The present invention also relates to the step of using a reporter compound of formula I to measure the activity of intracellular caspases or other enzymes involved in apoptosis in whole or dead cells or tissues. ing. The invention also relates to methods using the compounds of the formula I and to the determination or activation or inhibition of any caspase enzyme in any whole or dead cells or in tissues (normal or cancerous) by a test substance. To the assay process described herein. The compound represented by Formula I is cell permeable,
This can be introduced into whole cells or tissue samples. The compound is fluorogenic or fluorescent and can be designed to be specific for any known caspase or for any other intracellular enzyme involved in apoptosis.

Thornbury, N.M. A. J. et al. Biol. Chem. 272: 17
907 (1997) describe optimal sequences for various caspase substrates and granzyme B substrates. The optimal substrate sequence is shown in Table 1.

[0047]

[Table 1] ^* Enzymes are identified by both new and old nomenclature (in parentheses). ^** Standard amino acid abbreviations for amino acids are used to indicate optimal amino acids.

Using the optimal sequences described by Thornberry et al., A fluorogenic or fluorescent substrate for a particular caspase can be synthesized by the procedures described herein.

By utilizing the peptide sequence of a known or potential cleavage site from a known or potential natural substrate of a caspase enzyme, other fluorogenic for known or unknown caspases Or it is also possible to design a fluorescent substrate. Table 2 shows the peptide sequences corresponding to known or potential cleavage sites in proteins that may be natural substrates for caspases.

[0050]

[Table 2] ^{** The} amino acid sequence is shown using standard one letter abbreviations for amino acids.

[0051] Fluorogenic or fluorescent substrates can also be used at a time by designing substrates that are recognized and cleaved by two or more enzymes involved in the caspase cascade.
It can be designed to measure one or more enzymes. For two or more caspases,
A "promiscuous" fluorogenic or fluorescent substrate can be utilized using the assay process described herein to further measure the activity of unknown caspases.

When the caspase cascade is activated by a cell death-inducing stimulus, the fluorogenic or fluorescent reporter molecules described herein are cleaved and respond with a large increase in fluorescence excitation. The change in fluorescence can be measured spectrofluorometrically. This reporter molecule can also be used to measure reference caspase activity in cells that have not undergone apoptosis. This method is easily compatible with high power or ultra high power screening assays.

This assay system is very versatile. Examples of extreme versatility of this assay system include: This assay is used to screen cells or tissues for the basal activity of any caspase enzyme or any other enzyme involved in apoptosis.
Can be 2. This assay can be used as easily as screening for compounds that can either activate or inhibit the caspase cascade. This means that the assay can be used to screen drugs for degenerative diseases or drugs for cancer. 3. This assay is performed on hair, brain, peripheral nervous system, eyes, ears, nose, mouth, tonsils, teeth, esophagus, lungs, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach Any living or dead cell or cell line from any organ system in the body, including but not limited to, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands, joints, bones and skin Can be used to screen for activation or inhibition of the caspase cascade. This assay can be used to screen for drugs that have potential use in any disease of any organ system of the body that is involved in the multifunctionality of the caspase cascade. 4. This assay is used to screen for drugs that can directly or indirectly modulate the caspase cascade (ie, by modulating the caspase itself or by modulating cellular receptors and cofactors that affect the caspase cascade). obtain. 5. This assay can be used to determine the site of action that a caspase cascade modulator interferes with. Thus, this assay may help define the molecular mechanism of action of novel caspase cascade modulator drugs.

The present invention also relates to the discovery of new compounds or new uses of known compounds in the reduction, prevention or treatment of diseases in which apoptotic cell death is either a causative factor or a consequence. , Formula I, or the use of a fluorogenic or fluorescent substrate. Examples of uses of the invention include screening for compounds that can protect the nervous system after focal and systemic ischemia; Alzheimer's disease, Huntington's disease, prion disease, Parkinson's disease, multiple sclerosis, amyotrophic Screening of compounds capable of treating neurodegenerative disorders such as lateral sclerosis, ataxia, telangiectasia and spinal cord atrophy; compounds capable of treating heart diseases including myocardial infarction, congestive heart failure and cardiomyopathy Screening; screening for compounds that can treat retinal disorders; screening for compounds to treat autoimmune disorders including lupus erythematosus, rheumatoid arthritis, type I diabetes, Sjogren's syndrome and glomerulonephritis; polycystic nephropathy and anemia / Screening for compounds that treat erythropoiesis; AID
Screening for compounds that treat disorders of the immune system, including S and SCIDS; screening for compounds that reduce or prevent cell, tissue and organ damage during transplantation (eg, graft versus host disease in bone marrow transplant procedures); Screening for compounds that reduce or prevent cell line death in advanced biotechnology; screening for compounds that reduce or prevent alopecia (loss of hair); and screening for compounds that reduce premature death of skin cells.

The present invention has also been shown in Formula I in a screening procedure where a library of known drugs or a combinatorial library or a library of other compounds is screened for compounds having anti-tumor or anti-cancer activity. Related to the use of fluorogenic or fluorescent substrates. The cancer cells or cell lines include brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver Any cancer of any internal or external organ system in the body including, but not limited to, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands (eg, prostate), joints, bones and skin It can be of origin.

The present invention also relates to a fluorogenic agent of formula I in a diagnostic procedure for determining the chemosensitivity or resistance of cancer cells obtained from animals or humans for treatment with a chemotherapeutic agent. Or the use of fluorescent substrates. The cancer cells or cell lines include brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver Any cancer of any internal or external organ system in the body including, but not limited to, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands (eg prostate), joints, bones and skin From.

In particular, the present invention involves a method for determining an enzyme involved in the apoptotic cascade in one or more cells, including: (a) a reporter compound according to the present invention. Contacting the reporter compound with the one or more cells under conditions in which is taken up by the one or more cells,
And (b) recording the fluorescence of one or more cells described above. Here, the magnitude of fluorescence in the one or more cells as compared to control cells that have not been so contacted, or to those that have been contacted with a known competitive inhibitor of the reporter compound and enzyme (
Changes in either the wavelength (i.e., increase) or the wavelength are indicative of the presence of the enzyme.

The present invention also relates to a method for measuring the activity of an enzyme involved in the apoptosis cascade in one or more cells, comprising:

(A) contacting the reporter compound with the one or more cells under conditions in which the reporter compound is taken up by one or more cells according to the present invention; and (b) Recording of cell fluorescence. Here, the magnitude or wavelength of fluorescence in the one or more cells is compared to control cells that have not been so contacted, or those that have been contacted with a known competitive inhibitor of a reporter compound and an enzyme. Relative change is a measure of the activity of the enzyme.

The present invention also relates to a method for determining whether a test substance has an effect on an enzyme involved in the apoptotic cascade in one or more test cells,
This includes:

(A) In accordance with the present invention, under conditions in which the reporter compound is taken up by one or more cells and the test substance interacts with an external membrane receptor or is taken up by said cells, Contacting the one or more test cells with the test substance and the reporter compound; and (b) recording the fluorescence of the test cells. Here, the change in magnitude or wavelength in fluorescence in this cell, as compared to a control cell that was only contacted with the reporter compound and not the test substance, indicates that the test substance is being tested. It is an indicator that it has either a direct or indirect effect on apoptotic enzymes.

In the practice of this aspect of the invention, the test cell may be contacted with the test substance before, after, or substantially simultaneously with contacting the reporter compound according to the invention. This method can be used to detect whether a test substance stimulates or inhibits the activity of the enzyme.

The invention also relates to the further contacting of test cells with a secondary test substance or a mixture of test substances in the presence of a primary test substance.

In a preferred embodiment, the test cell is a cancer cell or cell line from a human in need of treatment with a chemotherapeutic agent, and the test substance is a chemotherapeutic agent or a mixture of chemotherapeutic agents.

The present invention also relates to a method for determining the susceptibility of an animal with cancer for treatment with one or more chemotherapeutic agents, comprising: (a) a reporter according to the present invention; Under conditions in which the compound is taken up by the cancer cells and one or more drugs interacts with the extramembrane receptor or is taken up by the cells described above, one of the cancer cells obtained from the animal is obtained. Contacting with the above chemotherapeutic agent and reporter compound; and (b) recording the fluorescence of the cancer cells. Here, a change in magnitude or wavelength in fluorescence in the cancer cell, as compared to a control cell contacted only with the reporter compound, indicates that the cancer cell is chemosensitive to one or more drugs. And that the animal is susceptible to treatment.

The present invention also relates to a method for monitoring treatment of an animal with one or more chemotherapeutic agents, including: (a) administering one or more chemotherapeutic agents to the animal. (B) contacting cells obtained from the animal after administration of the reporter compound under conditions in which the reporter compound is taken up by the cells according to the present invention; and (c) recording the fluorescence of the cells contacted with the reporter compound. thing. Here, a change in magnitude or wavelength in fluorescence in cells obtained from the animal after administration, as compared to control cells obtained from the animal before administration, indicates that the animal is susceptible to the chemotherapeutic agent. Is an indicator that In this embodiment, the animal may suffer from a disease in which apoptotic cell death is either a causative factor or a consequence.

The present invention also relates to a method for determining whether a test substance inhibits or prevents cell death in one or more test cells, including: (a) according to the present invention Contacting the test cell with the test substance and the reporter compound under conditions where the test substance interacts with or is taken up by the cell and the reporter compound is taken up by the cell; and (b) the test comprises: Record the fluorescence of the cells. Here, a change in the magnitude or wavelength in the fluorescence in the test cells compared to control cells that have been contacted only with the reporter compound is an indication that the test substance inhibits or prevents cell death.

The present invention also relates to a method for determining whether a test substance causes or enhances cell death in one or more test cells, comprising: According to the invention, contacting the test cell with the test substance and the reporter compound under conditions in which the test substance interacts with or is incorporated into the cell and the reporter compound is incorporated into the cell; and (b) ) Record the fluorescence of the test cells. Here, a change in the magnitude or wavelength in fluorescence in the test cell, as compared to a control cell contacted only with the reporter compound, is an indication that the test substance causes or enhances cell death. .

The present invention also relates to methionine aminopeptidase type 2 and H
Related to the process of using a reporter compound of formula IX to measure the activity of intracellular peptidases and proteases in whole living cells, including but not limited to HIV protease in IV infected cells.
The present invention also relates to methods of using the compounds of formula IX and the assay processes described herein for measuring the inhibition or activation of enzymes in whole cells of a living by a test compound or compounds. Related. The reporter compounds shown in Formula IX are cell permeable, that is, they can be introduced into whole cells. The compound can be designed to be fluorogenic or fluorescent and specific for a known enzyme of interest, such as methionine aminopeptidase or HIV protease.

The present invention also relates to a method for the detection of a viral protease in one or more cells, comprising: (a) in accordance with the present invention, under conditions wherein a reporter compound is incorporated into the cell, Contacting the cells with a reporter compound; and (b) recording the fluorescence of the cells. Here, a change or increase in fluorescence in this cell, as compared to a control cell that has not been so contacted, is an indication that a viral protease is present.

The present invention also relates to a method for measuring the activity of a viral protease in one or more virus-infected cells, comprising: (a) according to the present invention, a reporter compound comprising one or more reporter compounds; Contacting one or more virus-infected cells with a reporter compound under conditions that are incorporated into the virus-infected cells; and (b) recording the fluorescence of the one or more cells. Here, a change or increase in fluorescence in the one or more virus-infected cells, as compared to control cells that have not been so contacted, is indicative of the activity of the viral protease.

The present invention also relates to a method for determining whether a test substance has an effect on the activity of a viral protease in one or more virus-infected cells, comprising: According to the invention, contacting the virus-infected test cell with a test substance and the reporter compound under conditions in which the reporter compound is taken up by the infected test cell; and (b) determining the fluorescence of the infected test cell by the reporter compound. The change or increase in fluorescence in the infected test cells, compared to the infected control cells, compared to the infected control cells, only compared to the infected control cells contacted with It is an indicator that it has an effect on

[0073] In a preferred embodiment, the cell is an HIV infected cell and the viral protease is an HIV protease. In another preferred embodiment, the cell is an adenovirus infected cell and the viral protease is an adenovirus protease. In another preferred embodiment, the cells are HS
V-infected cells, and the viral protease is an HSV protease. In another preferred embodiment, the cell is an HCMV infected cell and the viral protease is an HCMV protease. In another preferred embodiment, the cell is an HCV infected cell and the viral protease is HCV
V protease.

The present invention also relates to a method for measuring the activity of a protease or peptidase in a cell, including: (a) according to the present invention, a reporter compound is incorporated into a test cell. Contacting the test cell with the reporter compound under conditions or conditions in which the reporter compound interacts with a membrane protease or peptidase outside of the cell, and (b) recording the fluorescence of the cell That is, where a change or increase in fluorescence in the test cells, as compared to control cells that have not been so contacted, is an indication of protease or peptidase activity.

The present invention also relates to a method for determining whether a test substance has an effect on the activity of a protease or peptidase in a test cell, comprising the following: According to the invention, contacting the test cell with the test substance and the reporter compound under conditions in which the reporter compound is taken up by the test cell or under conditions in which the reporter compound interacts with a membrane protease or peptidase outside the cell. And (b) recording the fluorescence of the test cells relative to control cells that have been contacted only with the reporter compound. Here, compared to the control cells,
A change or increase in fluorescence in the test cells is an indication that the test substance has an effect on proteases or peptidases.

In a preferred embodiment, the cell is an endothelial cell and the peptidase is type 2 methionine aminopeptidase. In another preferred embodiment,
The cell is a T cell and the peptidase is dipeptidyl peptidase IV. In another preferred embodiment, the cell is a neuronal cell,
And the protease is calpain.

Detailed Description of the Invention The fluorogenic or fluorescent substrate of the present invention is a compound having the following general formula I: xyz (I) or a biologically acceptable thereof Or a reporter molecule (eg, a methyl ester form of a carboxyl-containing amino acid residue). Where: x and z
Are the same or different and are peptides or amino acids or acyl groups or other structures such that Formula I is a caspase or other protease or peptidase or other enzyme substrate; and wherein x is z If the same as
The cleavable bond in Formula I is only one or both of the xy and yz bonds or, if x is not the same as z, in Formula I the cleavable bond is xy or yy Only one of the z bonds. y is a fluorogenic or fluorescent moiety.

Preferred compounds falling within the scope of Formula I include those where x is the same as z and the first amino acid attached to y is Asp. Most preferably,
x is the same as z and is the substrate for the N-blocked tetrapeptide of caspase, including: SEQ ID NO: 1 WEHD, SEQ ID NO: 2 YV AD, SEQ ID NO: 3 LEHD, SEQ ID NO: 4 DETD, SEQ ID NO: 5 DE
VD, SEQ ID NO: 6 DEHD, SEQ ID NO: 7 VEHD, SEQ ID NO: 8 LE
TD, SEQ ID NO: 3 LEHD, SEQ ID NO: 10 SHVD, SEQ ID NO: 11
DELD, SEQ ID NO: 12 DGPD, SEQ ID NO: 13 DEPD, SEQ ID NO:
14 DGTD, SEQ ID NO: 15 DLND, SEQ ID NO: 16 DEED, SEQ ID NO: 17 DSLD, SEQ ID NO: 18 DVPD, SEQ ID NO: 19 DEAD
, SEQ ID NO: 20 DSYD, SEQ ID NO: 21 ELPD, SEQ ID NO: 26 V
Granzyme B N-blocked tetrapeptide substrate comprising EID, SEQ ID NO: 24 IETD, or SEQ ID NO: 23 IEPD and SEQ ID NO: 27 VEPD; or x is the same as z and includes the tetras of caspase N-blocked peptide corresponding to a carboxyl- or amino-terminal fragment consisting of 1, 2 or 3 amino acids of a peptide substrate: SEQ ID NO:
1 WEHD, SEQ ID NO: 2 YVAD, SEQ ID NO: 3 LEHD, SEQ ID NO:
4 DETD, SEQ ID NO: 5 DEVD, SEQ ID NO: 6 DEHD, SEQ ID NO:
7 VEHD, SEQ ID NO: 8 LETD, SEQ ID NO: 3 LEHD, SEQ ID NO:
10 SHVD, SEQ ID NO: 11 DELD, SEQ ID NO: 12 DGPD, SEQ ID NO: 13 DEPD, SEQ ID NO: 14 DGTD, SEQ ID NO: 15 DLND
, SEQ ID NO: 16 DEED, SEQ ID NO: 17 DSLD, SEQ ID NO: 18 D
VPD, SEQ ID NO: 19 DEAD, SEQ ID NO: 20 DSYD, SEQ ID NO: 2
Granzyme B comprising 1 ELPD, SEQ ID NO: 26 VEID, SEQ ID NO: 24 IETD and SEQ ID NO: 23 IEPD and SEQ ID NO: 27 VEPD.

Preferred compounds falling within the scope of Formula I include those where y is rhodamine 110.

In particular, preferred embodiments of the compounds of formula I are shown in formula II. _{R 1 - (AA) n -Asp} -y-Asp- (AA) n -R1 (II) or a biologically acceptable salt or pro-reporter molecules (e.g., methyl ester of carboxyl-containing amino acid residues ). Wherein: R ₁ is an N-terminal protecting group including t-butyloxycarbonyl, acetyl, benzyloxycarbonyl; each AA is independently any natural or unnatural α-amino acid or is a β-amino acid residue, or a residue of an α-amino acid or β-amino acid derivative; n is independently 0-5; and y is a fluorogenic or fluorescent moiety. An example of a proreporter molecule is the methyl ester form of a carboxyl-containing amino acid residue comprising a compound of formula II. Another example of a proreporter molecule is the acetoxymethyl (AM) ester form of a carboxyl-containing amino acid residue of a compound of Formula II. AM esters of carboxyl-containing compounds are known to be cell permeable and can be hydrolyzed by intracellular esterases. Once hydrolyzed, the carboxyl-containing compound becomes cell impermeable and is trapped inside the cell (Adams et al., J. Am. Chem. S.
oc. 111: 7957-7968 (1989)). AM esters can be prepared by reaction of the corresponding carboxy-containing compound with bromomethylacetic acid.

A particularly preferred embodiment of the compounds of formula I is shown in formula III.

[0082]

Embedded image Alternatively, it is a biologically acceptable salt or proreporter molecule (eg, a methyl ester form of a carboxyl-containing amino acid residue). Where: R ₁ , AA, n have already been defined in Formula II. Desirable R ₁ is a t-butyloxycarbonyl group, an acetyl group or a benzyloxycarbonyl group. The preferred value of n is 1-3.

Another group of preferred compounds falling within the scope of Formula I include those wherein x is not the same as z. Preferred compounds of this group include those wherein x is a peptide or other structure that produces a substrate compound for caspases or other proteases or peptidases or other enzymes; and x in formula I
The -y bond is a bond that is fragile under biological conditions; z is a blocking group, and the yz bond of Formula I is a bond that is fragile under biological conditions.
Most preferably, x is an N-blocked tetrapeptide substrate of caspase comprising: SEQ ID NO: 1 WEHD, SEQ ID NO: 2 YVAD, SEQ ID NO: 3 LEHD, SEQ ID NO: 4 DETD, SEQ ID NO: : 5 DEVD, SEQ ID NO: 6 DEHD, SEQ ID NO: 7 VEHD, SEQ ID NO: 8 LETD, SEQ ID NO: 3 LEHD, SEQ ID NO: 10 SHVD, SEQ ID NO: 11 DELD, SEQ ID NO: 12 DGPD, SEQ ID NO: 13 DEPD, SEQ ID NO: 14 DGT
D, SEQ ID NO: 15 DLND, SEQ ID NO: 16 DEED, SEQ ID NO: 17
DSLD, SEQ ID NO: 18 DVPD, SEQ ID NO: 19 DEAD, SEQ ID NO:
20 DSYD, SEQ ID NO: 21 ELPD, SEQ ID NO: 26 VEID, SEQ ID NO: 24 IETD, or SEQ ID NO: 23 IEPD and SEQ ID NO: 27
N is a N-blocked tetrapeptide substrate of granzyme B containing VEPD; or x is an N-blocked peptide corresponding to a carboxyl- or amino-terminal fragment consisting of 1, 2, or 3 amino acids of a tetrapeptide substrate of caspase, including: Yes: SEQ ID NO: 1 WEHD, SEQ ID NO: 2 YVAD, SEQ ID NO: 3 LEHD, SEQ ID NO: 4 DETD, SEQ ID NO: 5 DEVD, SEQ ID NO: 6 DEHD, SEQ ID NO: 7 VEHD, SEQ ID NO: 8 LETD, SEQ ID NO: 3 LEHD, SEQ ID NO: 10 SHVD, SEQ ID NO: 11 DELD
, SEQ ID NO: 12 DGPD, SEQ ID NO: 13 DEPD, SEQ ID NO: 14 D
GTD, SEQ ID NO: 15 DLND, SEQ ID NO: 16 DEED, SEQ ID NO: 1
7 DSLD, SEQ ID NO: 18 DVPD, SEQ ID NO: 19 DEAD, SEQ ID NO: 20 DSYD, SEQ ID NO: 21 ELPD, SEQ ID NO: 26 VEID,
SEQ ID NO: 24 IETD or SEQ ID NO: 23 IEPD and SEQ ID NO: 2
Granzyme B with 7 VEPD; or x is an N-block peptide corresponding to a carboxyl- or amino-terminal fragment consisting of 1, 2, 3 or 4 amino acids of a tetrapeptide substrate of caspase, including: SEQ ID NO: 1
WEHD, SEQ ID NO: 2 YVAD, SEQ ID NO: 3 LEHD, SEQ ID NO: 4
DETD, SEQ ID NO: 5 DEVD, SEQ ID NO: 6 DEHD, SEQ ID NO: 7
VEHD, SEQ ID NO: 8 LETD, SEQ ID NO: 3 LEHD, SEQ ID NO: 10
SHVD, SEQ ID NO: 11 DELD, SEQ ID NO: 12 DGPD, SEQ ID NO: 13 DEPD, SEQ ID NO: 14 DGTD, SEQ ID NO: 15 DLND, SEQ ID NO: 16 DEED, SEQ ID NO: 17 DSLD, SEQ ID NO: 18 DVP
D, SEQ ID NO: 19 DEAD, SEQ ID NO: 20 DSYD, SEQ ID NO: 21
Granzyme B comprising ELPD, SEQ ID NO: 26 VEID, SEQ ID NO: 24 IETD and SEQ ID NO: 23 IEPD and SEQ ID NO: 27 VEPD,
G In addition, A, GA, P ₁ of caspase substrates including GG and AG - 1 to 2 amino acids corresponding to 'P _2' moiety.

In particular, the novel fluorogenic or fluorescent reporter compounds of the invention have the formula V: R _1- (AA) _n -Asp-y-R ₆ (V) or a biologically acceptable compound thereof. A possible salt or pro-reporter molecule (eg, a methyl ester form of a carboxyl-containing amino acid residue), wherein: R ₁ comprises t-butyloxycarbonyl, acetyl, octanoyl, and benzyloxycarbonyl; An N-terminal protecting group; each AA is independently any natural or unnatural α-amino acid or β
-An amino acid, or a residue that is an α-amino acid or β-amino acid derivative; n is 0-5; y is a fluorogenic or fluorescent moiety; and R ₆ is an amino acid or amino acid derivative. Not a blocking group.

In particular, the novel fluorogenic or fluorescent reporter molecules of the invention of formula VII-IX are derivatives of rhodamine, including rhodamine 110, rhodamine 116, and rhodamine 19. These novel fluorogenic specific or fluorescent reporter molecules, first a blocking group R _6, is prepared by introducing into one of the two amino groups of Rhodamine, the novel fluorescent dyes of Formula VI give. Remaining HNR ₂ group is used for reaction with a potential enzyme substrate, providing a fluorogenic specific substrate of Formula VII-IX. By blocking one of the two amino groups in rhodamine, the overall size of the substrate is reduced as compared to a bis-substituted rhodamine (eg, a bispeptide rhodamine). More importantly, the blocking group is a) stable under biological conditions such that amino acids are excluded, since the formed peptide bond can be cleaved by peptidases potentially present in the cell, and Preferably not too bulky (e.g., small) to reduce the overall size of the peptide-reporter molecule formed, so that it is not hydrolyzed; and so as to be a better enzyme substrate; c. ) In order to increase the cell permeability of the fluorogenic or fluorescent reporter molecule, it is preferably chosen to be essentially hydrophobic.

Preferred R ₆ blocking groups are C _2-12 alkyloxycarbonyl groups (eg, methoxycarbonyl, ethoxycarbonyl, hexyloxycarbonyl, octyloxycarbonyl, decyloxycarbonyl, and dodecyloxycarbonyl) ; C _2-12 (alkylthio) carbonyl group (e.g., (ethylthio) carbonyl group, (hexylthio) carbonyl group, (octylthio) carbonyl group); arylalkyloxycarbonyl group (e.g., benzyloxycarbonyl group); C _2-12 Acyl (alkanoyl) groups (eg, acetyl and octanoyl groups); carbamyl groups (eg, dimethylcarbamyl group, N-methyl-N
-Hexylcarbamyl group); and alkyl, haloalkyl, or aralkylsulfonyl groups (e.g., methanesulfonyl group).

Particularly preferred R₆The blocking group is CH_ThreeOCO-, CH_Three(CH_Two)_pOCO- (p = 1-11), Cbz, Cl_ThreeCCH_TwoOCO- and PhCH_TwoCH_TwoOC
O- (carbamate series); Me (OCH_TwoCH_Two)_qOCO- (q = 1-4), and CH_Three(CH_Two)_r(OCH_TwoCH_Two)_sOCO- (r = 0-5, s = 1-
4), (alkyloxyalkyl carbamate series); EtSCO-, CH _Three (CH_Two)_FiveSCO-, CH_Three(CH_Two)₇SCO-, CH_Three(CH_Two)₉SCO-, and CH_Three(CH_Two)_tSCO- (t = 0-11) (thiocarbamate series); Ts-, PhSO_Two-, MeSO_Two-, CH_Three(CH_Two)_uSO_Two− (U = 0-1
1), PhCH_TwoSO_Two− And CF_ThreeSO_Two-(Sulfonamide series); Me _Two NCO-, Et_TwoNCO-, and N-Me-N-CH_Three(CH_Two)_vNCO (v = 0-9) (urea series); and HCO-, CH_ThreeCO-, CH_Three(CH_Two )_wCO (w = 0-9), PhCH_TwoCO- and Ph-CO- (amide series
). Most preferred R₆Blocking groups are those that contain a hydrophobic group similar to membrane lipids and thus the cell permeability of a fluorogenic or fluorescent reporter molecule,
And cells after cleavage of a substrate by a targeted protease or peptidase
Increase the retention of fluorescent moieties within. These preferred R₆The blocking group is CH_Three(CH_Two)_pOCO- (p = 1-11) (carbamate series); Me (OCH_TwoCH_Two)_qOCO- (q = 1-4), and CH_Three(CH_Two)_r(OCH _Two CH_Two)_sOCO- (r = 0-5, s = 1-4), (alkyloxyalkyl carbamate series); EtSCO-, CH_Three(CH_Two)_FiveSCO-, CH_Three(CH _Two )₇SCO- and CH_Three(CH_Two)₉SCO- (thiocarbamate series); CH_Three(CH_Two)_tSO_Two-(T = 0-11), (sulfonamide series); N
-Me-N-CH_Three(CH_Two)_uNCO (u = 0-9) (urea series); and CH_Three(CH_Two)_wIncluding, but not limited to, CO (w = 0-9) (amide series).

The novel fluorogenic or fluorescent reporter molecule of Formula VII-IX is capable of combining the amino group NHR ₂ of the novel fluorescent dye of Formula VI with a potential enzyme substrate (eg, N-blocked peptide Carboxyl group) to form a peptide amide bond. The reaction converts a fluorescent molecule of Formula VI to a non-fluorescent peptide reporter molecule of Formula VII-IX, which is a substrate for a protease or peptidase. It is therefore very important that the blocking group R ₆ bond of formula VII should not be cleaved and that the peptide reporter amide bond be a bond that is susceptible to cleavage under biological conditions. Cleavage of the cleavable peptide reporter amide bond of formula VII-IX by a protease or peptidase yields a compound of formula VI or VI ′, which is fluorescent.

A particularly preferred embodiment of the compound of formula V is represented by formula VII:

[0090]

Embedded image Or a biologically acceptable salt or proreporter molecule thereof (eg, the methyl ester form of a carboxyl-containing amino acid residue), wherein: R ₂ and R ₃ are the same or different and are independently hydrogen, R ₆ is a blocking group which is not an amino acid or a derivative of an amino acid; R ₄ and R ₅ are the same or different and are independently hydrogen or alkyl; R ₁ is N Each AA is independently the 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; 0-5; and the cleavable bond is the Asp-N bond of Formula VII.

Preferred R ₂ and R ₃ are hydrogen, methyl, or ethyl; preferred R ₄ and R ₅ are hydrogen or methyl; preferred amino acids are natural amino acids (tyrosine, glycine, phenylalanine, methionine , Alanine, serine, isoleucine, leucine, threonine, valine, proline, lysine, histidine, glutamine, glutamic acid, tryptophan, arginine, aspartic acid, aspartic acid, and cysteine)
including. Unnatural amino acids include t-butylglycine and N, N-dimethylglutamine.

An example of a pro-reporter molecule is the methyl ester form of a carboxy-containing amino acid residue, including compounds of Formula VII. Another example of a pro-reporter molecule is the carboxy-containing amino acid residue acetoxymethyl (AM) of the compound of Formula VII.
It is an ester type.

Another group of preferred embodiments of the compounds of formula I is represented by formula VIII:

[0094]

Embedded image Or a biologically acceptable salt or pro - a reporter molecule (e.g., methyl ester of carboxyl-containing amino acid residues), wherein: R _1, R _6, AA and n are already formulas II and V M is an integer from 0-3; R ₂ and R ₃ are the same or different, and are independently hydrogen, alkyl or aryl; and R ₄ and R ₅ are the same as defined above. Or different, and is independently hydrogen or alkyl.

Compounds of formula VIII are novel fluorogenic or fluorescent substrates for caspases or other apoptosis-related enzymes. If m is 0, Asp
Cleavage of the amide bond between and rhodamine converts the fluorogenic substrate to a fluorochrome of formula VI. If m is not zero, cleavage of the amide bond between Asp and (AA) _m releases rhodamine bound to NH _2- (AA) _m . The remaining amino acids (AA) _m are then removed by aminopeptidase present in the cells, giving a fluorescent dye of formula VI. (AA) _m can be designed to correspond to the P ′ sequence of a cleavage site for a substrate for caspases or enzymes involved in apoptosis. Incorporation of the P 'sequence of a known substrate for caspases or enzymes involved in apoptosis is expected to increase the specificity and affinity of the fluorogenic substrate. Since aminopeptidases are widely present in cells, the (AA) _m sequence can be inserted in the design of substrates of formula VIII for whole cell assays. This is another advantage of whole cell assays over cell-free enzyme assays. For example, (AA
If _m is Gly, the substrate of formula VIII acts in a whole cell assay, but otherwise in a cell-free caspase assay. Because Asp-
This is because cleavage of the Gly amide bond releases Gly bound to rhodamine (which is not fluorescent).

Examples of pro-reporter molecules are the methyl or ethyl ester forms of carboxyl-containing amino acid residues, including compounds of formula VIII. Another example of a pro-reporter molecule is the acetoxymethyl (AM) ester form of the carboxy-containing amino acid residue of the compound of Formula VIII or pivaloyloxymethyl (PM)
It is an ester type. AM esters of carboxy-containing compounds are known to be cell permeable and can be hydrolyzed by esterases inside cells.
Once hydrolyzed, the carboxy-containing compound becomes cell impermeable and is trapped inside cells (Adams et al., J. Am. Chem. Soc. 111).
: 7957-7968 (1989)). AM esters can be prepared by reacting with a carboxy-containing compound having the corresponding bromomethyl acetate.

Yet another group of preferred embodiments of the compounds of Formula I is represented by Formula IX:

[0098]

Embedded image Or a biologically acceptable salt or pro-reporter molecule thereof (eg, the methyl ester form of a carboxyl-containing amino acid residue), wherein: R ₁ , R ₆ , AA and n are prior to formulas II and V. M is an integer from 0-3; R ₂ and R ₃ are the same or different and are independently hydrogen, alkyl or aryl; and R ₄ and R ₅ are the same as defined above. Or different, and is independently hydrogen or alkyl.

Preferred R₁Is t-butyloxycarbonyl, acetyl, octanoyl, dodecanoyl, and benzyloxycarbonyl. Preferred n is 1-4
is there. Preferred R_TwoAnd R_ThreeIs hydrogen, methyl or ethyl. Preferred R _Four And R_FiveIs hydrogen or methyl. Preferred R₆The blocking group is C_{2-1 Two} Alkyloxycarbonyl group (for example, methoxycarbonyl group, ethoxycarbonyl group, hexyloxycarbonyl group, octyloxycarbonyl group, decyl
An oxycarbonyl group and a dodecyloxycarbonyl group); C_2-12(Alkyl
Thio) carbonyl group (for example, (ethylthio) carbonyl group, (hexylthio)
Carbonyl group, (octylthio) carbonyl group; arylalkyloxycarbo
Nyl group (for example, benzyloxycarbonyl group); C_2-12Acyl (alkanoyl
) Groups (eg, acetyl and octanoyl groups; carbamyl groups (eg,
Butylcarbamyl group, N-methyl-N-hexylcarbamyl group);
, Haloalkyl, or aralkylsulfonyl groups (eg, methanesulfonyl
Groups), but are not limited thereto.

In Formula IX, (AA) _n is designed to be an amino acid or peptide that is recognized as a p-side sequence by a particular peptidase or protease, and is cleaved by the targeted peptidase or protease.
(AA) _m is designed to be an amino acid or peptide that is recognized as a P ′ sequence by a particular peptidase or protease, which can be removed by an aminopeptidase provided in the cell. When R ₁ is an N-terminal protecting group (eg, t-butyloxycarbonyl, Cbz, or acetyl),
The compound of formula IX is a substrate for an endopeptidase (eg, cathepsin D) or a protease (eg, HIV protease), and when R ₁ is H, the compound of formula IX is an exopeptidase (eg, methionine) Aminopeptidase).

In particular, the compound of formula IX is a methionine aminopeptidase type 2 (Me
It is designed to be a substrate for tAP-2). MetAP-2 has recently been identified by two research groups as a common target for the angiogenesis inhibitor AGM-1470, an anti-cancer drug currently in clinical trials (Griffith,
E. FIG. C. Et al., Chem. Biol. 4: 461-471 (1997) and Si
n, N. Proc. Natl. Acad. Sci. USA 94: 6099
-6103 (1997)). MetAP-2 is a bifunctional enzyme, eIF-2
It also regulates protein synthesis by affecting the phosphorylation state of the protein.
AGM-1470 has only been reported to inhibit 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 (1
997)). Since it is known that angiogenesis inhibitors such as AGM-1470 can selectively kill cancer cells, inhibitors of MetAP-2 have anti-angiogenic properties and are potent new anti-angiogenic agents. It is expected to be a cancer drug.

[0102] MetAP-2 is a cobalt-dependent enzyme that hydrolyzes the amino-terminal methionine of certain proteins. The preferred substrate is Met-XY.
X is an amino acid with a small and uncharged side chain, such as Gly, Ala, Ser, while Leu, Met, Arg and Tyr are known to produce inactive substrates. Y can be Ser, Met, Gly, or other amino acids (Li, X. and Chang Y.-H., Biochem. B
iophy. Res. Com. 227: 152-159 (1996)). Since rhodamine is a much larger molecule than amino acids, it is most likely that compounds with methionine directly attached to rhodamine are not substrates for MetAP-2. By exploiting the presence of aminopeptidases in whole cells, insertion of the (AA) _m sequence between methionine and rhodamine creates a good substrate for MetAP-2. This type of substrate is expected to work well in whole cell assays, but otherwise will not work in cell-free MetAP-2 enzyme assays.

For compounds of formula IX designed to be substrates for MetAP-2,
Preferred R ₁ is H, preferred (AA) _n is Met, and preferred (
AA) _m is Gly, Ala, Gly-Gly, Ala-Gly, or Gly-Ala. Methionine is cleaved by type 2 methionine aminopeptidase in endothelial cells to give rhodamine bound to (AA) _m . The aminopeptidase present in the cells then removes (AA) _m to give the fluorescent dye of formula VI. Compounds of formula IX have been used for screening for inhibitors of MetAP-2 in endothelial cells, which is expected to lead to the identification of new anticancer agents.

Compounds of formula IX can also be designed to be substrates for HIV protease. The HIV protease is an aspartic protease, which comprises g
It processes polypeptides transcribed from the ag and pol genes and is essential for the maturation of infectious virus. Thus, HIV protease has become one of the major targets for HIV chemotherapy intervention. Recently, some HIV protease inhibitors have been shown to be very potent in treating HIV,
It was approved for commercialization. Most of these HIV protease inhibitors were designed based on the structure of the protease substrate. Thus, these compounds are either peptides or peptidomimetics. The search for new and novel HIV protease inhibitors is expected to provide more effective drugs for combating this deadly disease.

Preferred substrates for HIV protease are peptides having a fragile hydrophobic-hydrophobic or aromatic-proline peptide bond between P ₁ -P ₁ ′ (Wes
t, M. L. And Fairlie, D .; P. , Trand. Pharm. S
ci. 16: 67-74 (1995)). Viral gag and gag-po
Nine different sites in the l protein were found to be cleaved by the protease (Martin, JA et al., Prog. Med. Chem. 3).
2: 239-287 (1995)). P ₄ -P ₃ 'sequences of these nine sites,
Ser-Gln-Asn-Tyr-Pro-Ile-Val SEQ ID NO: 28, A
la-Arg-Val-Leu-Ala-Glu-Ala SEQ ID NO: 29, Al
a-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-P
he-Ser-Phe-Pro-Gln-Ile SEQ ID NO: 33, Thr-Le
u-Asn-Phe-Pro-Ile-Ser SEQ ID NO: 34, Ala-Glu
-Thr-Phe-Tyr-Val-Asp SEQ ID NO: 35 and Arg-Ly
s-Val-Leu-Phe-Leu-Asp SEQ ID NO: 36. Many substrates of HIV protease, fluorogenic, radioactive, or chromogenic, are H
It has been prepared based on these natural substrates for IV protease activity assays. It is quenched fluorogenic specific substrates in the molecule, 2-aminobenzoyl -Thr-Ile-Nle- (4- NO 2 -Phe) -Gln-Arg-NH 2
SEQ ID NO: 141 (bond susceptible to cleavage here is _{Nle- (4-NO 2 -Phe)} ) , based on the hexapeptide substrate from p24 / p15 cleavage site was prepared (Toth, M.V. And Marshall, GR, Int.
Pept. Protein Res. 36: 544-550 (1990)). A fluorometric assay for HIV protease activity using HPLC using the substrate N-Dns-Ser-Gln-Asn-Tyr-Pro-Ile-Val SEQ ID NO: 28 is described by Tamburini et al. (Tamburini, P.P.
Et al., Anal. Biochem. 186: 363-368 (1990)), where Tyr-Pro was a cleavable bond. Many other HIV protease substrates have been developed that incorporate sequences from both the P and P 'sides of the cleavage site of the HIV protease substrate, and that
-Α-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide SEQ ID NO: 37 (this is the Phe recognized by HIV-1 protease
-Pro contains dipeptide bonds (Tyagi, SC and Carter, C
. A. , Anal Biochem. 200: 143-148 (1992)))
; Radiolabeled heptapeptide substrate [tyrosyl ^{-3,5- 3 H] Ac-Ser- Gln} -Asn-Tyr-Pro-Val-Val-NH 2 SEQ ID NO: 38 (this is the viral polyprotein substrate Pr55gag (Hyland , LJ et al.
Anal. Biochem. 188: 408-415 (1990)), based on the p17-p24 cleavage site Tyr-Pro); the peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Ph based on angiotensin I.
e-His-Leu-Leu-Glu-Glu-Ser SEQ ID NO: 39 (which produces angiotensin I (Ang I)) and Leu-Glu-Glu
-Ser SEQ ID NO: 40 (Evans, DB et al., Anal. Biochem.
. 206: 288-292 (1992)); intramolecular fluorescence resonance energy transfer (F
RET) substrate 4- (4-dimethylaminophenylazo) benzoic acid (DABCYL)
) -Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-5-
[(2-aminoethyl) amino] naphthalene-1 SEQ ID NO: 41 Sulfonic acid (E
DANS) (where Tyr-Pro is the cleavage site (Matayoshi, E
. D. Et al, Science 247: 954-958 (1990) )); and chromophores peptide substrate H-Ser-Gln-Asn- Leu-Phe (NO 2) -Leu-Asp-Gly-NH 2 SEQ ID NO: 42 and acetyl -Arg amide bond between the -L ys-Ile-Leu-Phe (NO 2) -Leu-Asp-Gly-NH 2 SEQ ID NO: 43 (where p- nitrophenyl alanyl and leucyl residues, binding to susceptible to cleavage Is included). Furthermore, a chromogenic substrate, Ly
s-Ala-Arg-Val-Leu-Phe (NO ₂ ) -Glu-Ala-Met SEQ ID NO: 44 (where Leu-Phe (NO ₂ ) is the cleavage site) was reported (Richards, A D. et al., J. Biol.
: 7733-7736 (1990))). Norleucine, SAR studies
Met, Phe, or substitution of Leu residues at P ₁ having a Tyr is different that when it is determined at pH values, the minimum to the kinetic parameters (K _cat and K _cat / K _m), has the effect, peptides containing Ile or Val in P _1, the hydrolysis was found to be extremely slow. By taking advantage of the presence of non-specific aminopeptidases in whole cells, the fluorogenic or fluorescent substrate of the HIV protease of Formula IX can be converted to P- and P'-sides of the HIV substrate for application in whole cell assays. It can be designed to incorporate amino acids from both. After the peptide sequence on the P side is cleaved by the HIV protease, the peptide sequence on the P 'side is expected to be removed by the aminopeptidase present in the cell.

For compounds of formula IX designed to be substrates for HIV protease, preferred R ₁ is acetyl or Cbz, and preferred (AA) _n is Thr-I
le-Nle, and preferred (AA) _m is Phe-Gln-Arg, Phe-Gln, or Phe; or preferred (AA) _n is Ser-Leu-Asn-Phe SEQ ID NO: 54 or Leu-Asn -Phe, and preferred (AA) _m is Pro-Ile-Val, Pro-Ile, or Pro; or preferred (AA) _n is Ser-Gln-Asn-Tyr SEQ ID NO: 45, or Gln-Asn -T
a yr, and preferred (AA) _m is, Pro-Ile-Val-Gln SEQ ID NO: 46, Pro-Ile-Val, Pro-Val-Val-NH 2, P ro-Val-NH 2, Pro-Ile, Or Pro; or preferred (AA) _n is Arg-Gly-Phe, and preferred (AA) _m is Pro;
Alternatively, (AA) _n is preferably Lys-Ala-Arg-Val-Leu SEQ ID NO: 47, Ala-Arg-Val-Leu SEQ ID NO: 48, or Arg-
Val-Leu, and preferred (AA) _m are Phe-Glu-Ala-Met SEQ ID NO: 49, Phe-Glu-Ala, Phe-Glu, or Phe
Or preferred (AA) _n is Pro-Phe-His-Leu SEQ ID NO: 50, or Phe-His-Leu, and preferred (AA) _m is Leu-Glu-Glu-Ser SEQ ID NO: 40, Leu -Glu-Glu, Le
u-Glu or Leu; or preferred (AA) _n is Ser-Gln-Asn-Leu-Phe SEQ ID NO: 140, Gln-Asn-Leu-P
he SEQ ID NO: 51, Asn-Leu-Phe, Arg-Lys-Ile-Le
u-Phe SEQ ID NO: 52, Lys-Ile-Leu-Phe SEQ ID NO: 53,
Or Ile-Leu-Phe and the preferred (AA) _m, is, Leu-As p-Gly- NH 2, Leu-Asp-NH 2, or a Leu-NH _2. More preferred (AA) _n is Ser-Leu-Asn-Phe SEQ ID NO: 54, or Leu-Asn-Phe, and more preferred (AA) _m is Pro-Ile-Val, Pro-Ile, or Pro; Or more preferred (AA) _n is Arg-Gly-Phe, and more preferred (AA) _m is Pro.

The substrates for the HIV protease of formula IX are expected to work in whole cell assays, but otherwise will not work in cell-free assays. Cleavage of the (AA) _n- (AA) _m amide bond by HIV protease in HIV-infected cells gives rhodamine bound to (AA) _m . The aminopeptidase present in the cells then removes (AA) _m to give a fluorescent dye of formula VI. Compounds of Formula IX are used for screening for inhibitors of HIV protease in HIV infected cells. This should accelerate the process of finding novel HIV protease inhibitors, especially non-peptide or non-peptide analog HIV protease inhibitors, that could lead to better anti-HIV agents than currently available drugs. Because HIV protease processes the viral precursor protein late in viral replication, the cell-permeable fluorogenic or fluorescent substrate of HIV protease may also be responsible for gene transcription or translation, virus entry, or early stage of HIV infection. Can be used to screen for compounds that inhibit other proteins that are key. Therefore, this method can lead to the identification of inhibition of HIV infection with a novel mechanism that cannot be identified in cell-free enzyme assays. In addition, since the HIV protease in HIV infected cells cleaves the cell permeable substrate of Formula IX to produce a fluorescent dye of Formula VI in the cell, the substrate of Formula V is also useful for diagnosis of HIV infection. Can be used.

The compounds of formula IX can also be designed to be substrates for adenovirus protease. Adenoviruses are sporadic which can lead to pneumonia
It is responsible for several diseases, including respiratory diseases and epidemic acute respiratory diseases. Adenovirus proteases are cysteine proteases that cleave some viral proteins and are required for viral maturation and infectivity (Webe
r, J. et al. M. , Curr. Top. Microbiol. Immunol. 19
9 / I: 227-235 (1995)). Preferred substrates for adenovirus protease include (M, L, I) XGX-G and (M, L, I) XGG-X.
Substrate specificity is primarily determined by the P ₂ and P ₄ amino acids (Diouri
, M .; J. et al. Biol. Chem. 271: 32511-32514 (199
6)). Met, hydrophobic amino acids such as Leu and Ile are good preferable in P _4. Small amino acid such as Gly is preferred in P _2. Small and hydrophobic amino acids such as Ala and Gly is also preferred in P ₁ and P ₁ '; whereas P ₃ may hardly applicable to any amino acid. These observations were supported by the recently determined crystal structure of the human adenovirus proteinase with its 11 amino acid cofactors, and substrate modeling based on the crystal structure (Ding, J. et al., EMBO J. 15: 1778). −1783 (
1996)). Taking advantage of the presence of the aminopeptidase in whole cells, the fluorogenic or fluorescent substrate of the adenovirus protease can be used for application in whole cell assays from only the P-side of the adenovirus protease substrate or from the P-side and It can be designed to incorporate amino acids from either of the P'sides.

For compounds of formula IX designed to be substrates for adenovirus protease, preferred R ₁ is acetyl or Cbz, and preferred (AA) _n is Leu
-Arg-Gly-Gly SEQ ID NO: 55, Met-Arg-Gly-Gly- SEQ ID NO: 56, Ile-Arg-Gly-Gly SEQ ID NO: 57, Leu-V
al-Gly-Gly SEQ ID NO: 58, Met-Val-Gly-Gly SEQ ID NO: 59, or Ile-Val-Gly-Gly SEQ ID NO: 60, and preferred (AA) _m is Gly, Ala, or m = 0 It is. When m is 0, cleavage of the (AA) _n -rhodamine amide bond by adenovirus protease produces a fluorescent dye of formula VI. If m is not 0, cleavage of the (AA) _n- (AA) _m amide bond by adenovirus protease in the cell will result in (AA
) Give rhodamine bound to _m . The aminopeptidase present in the cells then removes (AA) _m to give a fluorescent dye of formula VI. The compound of formula IX is used for screening for inhibitors of adenovirus protease in adenovirus infected cells.

The compounds of formula IX can also be designed to be substrates for the herpes simplex virus type 1 (HSV-1) protease. Human herpes simplex virus type 1
Herpes simplex). HSV-1 protease is a serine protease, and itself and ICP3 for viral capsid assembly.
5 are responsible for proteolytic processing (Gao, M. et al., J. Vir.
ol. 68: 3702-3712 (1994)). The two proteolytic sites inside the protease are Ala247 and Ser248, and Ala610.
And Ser611 (Dilani, CL et al., JB
iol. Chem. 268: 25449-25454 (1993)). Recently, 8
One of the amino acid consensus peptide LVLASSSF SEQ ID NO: 61, 20 to be similarly efficient cleavage and maturation site peptide mer found, and minimal substrate recognition of sequences of P ₁ is HSV-1 protease from P ₄ (O'Boyle, DR, et al., Virology 236: 3).
38-347 (1997)). Specificity of HSV-1 protease, it is also reported to exist in P ₄ -P ₁ 'region of the cleavage site (McCann, P.J.
J. et al. Virol. 68: 526-529 (1994)). Taking advantage of the presence of aminopeptidases in whole cells, the fluorogenic or fluorescent substrate of the HSV-1 protease can be used for application in whole cell assays, where the HSV-1 protease substrate P ₄ -P ₁ alone or P 4 ₄ is designed from one of both -P ₁ and P 'side to capture an amino acid.

For compounds of formula IX designed to be substrates for HSV-1 protease, preferred R ₁ is acetyl or Cbz, and preferred (AA) _n is Leu-V
al-Leu-Ala SEQ ID NO: 62, and preferred (AA) _m is Ser, Ser-Ser, or m = 0. When m is 0, cleavage of the (AA) _n -rhodamine amide bond by the HSV-1 protease produces a fluorescent dye of formula VI. If m is not 0, cleavage of the (AA) _n- (AA) _m amide bond by HSV-1 in the cell gives rhodamine bound to (AA) _m . The aminopeptidase present in the cells then removes (AA) _m to give a fluorescent dye of formula VI. Compounds of formula IX are useful for HSV-
Used for screening for inhibitors of one protease.

The compound of formula IX can also be designed to be a substrate for human cytomegalovirus (HCMV) protease. HCMV can cause life-threatening infections in congenitally infected infants, immunocompromised individuals, and immunosuppressed cancer and transplant patients. Human cytomegalovirus (HCMV)
Encodes a protease that cleaves itself and the HCMV assembly protein and is essential for viral replication. Therefore, it is a potential target for therapeutic intervention. HCMV protease is a serine protease, and two proteolytic processing sites inside the protease are Ala256
-Ser257 (release site), and Ala643-Ser644 (maturation site)
(Sztevens, JT, et al., Eur. J. Bioch).
em. 226: 361-367 (1994)). DABC is a fluorogenic substrate
YL-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser-A
rg-Leu-Ala-EDANS SEQ ID NO: 63 was synthesized and Ala-
It was found to be efficiently cleaved by CMV protease at the Ser peptide bond (Holskin, BP et al., Anal. Biochem. 2).
27: 148-155 (1995)). Recently, the Val-Val-Asn sequence corresponding to the P ₄ -P ₂ residue of the mature part of the enzyme, optimized Tbg-Tbg-A
sn replaced by (NMe ₂₎ (Tbg is t- butyl glycine) sequence, to increase significantly the substrate affinity for the protease was reported. AMC fluorogenic substrates were prepared using P-side peptide sequences containing these improved amino acids (Bonneau, PR et al., Anal. Biochem).
. 255: 59-65 (1998)). Taking advantage of the presence of aminopeptidases in whole cells, the fluorogenic or fluorescent substrate of HCMV protease can be used for application in whole cell assays, with only the P-side, or both the P- and P'-sides of the HCMV protease substrate. Designed to incorporate amino acids from any of

For compounds of formula IX designed to be substrates for HCMV protease, preferred R ₁ is acetyl or Cbz, and preferred (AA) _n is Val-Va
l-Asn-Ala SEQ ID NO: 64, Tbg-Tbg-Asn-Ala SEQ ID NO: 65, and preferred (AA) _m is Ser, Ser-Ser, or m = 0. When m is 0, cleavage of the (AA) _n- rhodaminamide bond by HCMV protease produces a fluorescent dye of formula VI. If m is not zero, cleavage of the (AA) _n- (AA) _m amide bond by HCMV in the cell will result in (A
A) Give rhodamine bound to _m . The aminopeptidase present in the cells then removes (AA) _m to give a fluorescent dye of formula VI. Compounds of formula IX are used for screening for inhibitors of HCMV protease in HCMV infected cells.

The compounds of formula IX can also be designed to be substrates for hepatitis C virus (HCV) protease. HCV is a major causative agent of non-A and non-B hepatitis, both parenterally infected and sporadic, and is estimated to affect 50 million people worldwide. The HCV protease NS3 and its protein activator NS4A are involved in the processing of viral polyproteins, and thus the NS3 / 4A protease complex is an attractive target for antiviral therapy against HCV. HCV protease is a serine protease, and Cys-Ser has been identified as a cleavage site. One of the sequences of the substrate is Asp-
Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Tyr
SEQ ID NO: 66, and P ₁ Cys and P ₃ Val were found to be important (Zhang, R. et al., J. Virol. 71: 6208-6213 (1
997)). Taking advantage of the presence of aminopeptidases in whole cells, fluorogenic or fluorescent substrates of HCV protease can be used for applications in whole cell assays.
It is designed to incorporate amino acids from both the P and P 'sides of the HCV protease substrate.

Compounds of formula IX designed to be substrates for HCV protease
And preferred R₁Is acetyl or Cbz, preferably (AA)_nIs Asp-Asp
-Ile-Val-Pro-Cys SEQ ID NO: 67, Asp-Ile-Val-
Pro-Cys SEQ ID NO: 68, or Ile-Val-Pro-Cys sequence
No. 69 and preferred (AA)_mIs Ser-Met-Ser-Tyr SEQ ID NO: 70, Ser-Met-Ser, Ser-Met, Ser, or
m = 0. When m is 0, (AA) by HCV protease_n-Cleavage of the rhodamine amide bond yields a fluorescent dye of formula VI. When m is not 0
(AA) by HCV in cells_n-(AA)_mCleavage of the amide bond is performed by (AA) _m To give rhodamine conjugated. The aminopeptidase present in the cell is then (AA)_mTo give a fluorescent dye of formula VI. Compounds of formula IX can be used to screen for inhibitors of HCV protease in HCV infected cells.
Used for

The present invention also relates to novel compounds of formula VI which are derivatives of rhodamine and are obtained by introducing a blocking group R ₆ into one of the two amino groups of rhodamine. R ₂ HN group of formula VI are, N- blocking peptides such as carboxyl groups, to provide attachment points for the reaction using an enzyme substrate that can form a peptide amide bond. This reaction converts the fluorescent molecule of Formula VI to a non-fluorescent molecule of Formula VII-IX, and produces a peptide reporter molecule that functions as a substrate for a protease or peptidase. Formula VII
The peptide-reporteramide bond in -IX is a bond that is susceptible to cleavage under biological conditions. Cleavage of the cleavable peptide-reporter amide bond in the peptide-reporter by a protease or peptidase yields a compound of formula VI or VI ′ that is fluorescent. More importantly, the blocking group may incorporate a hydrophobic group. Hydrophobic groups are designed to increase the membrane permeability of the substrate and to result in accumulation of the substrate inside the cell, as well as to increase retention of the fluorescent moiety inside the cell after its cleavage by a targeted protease or peptidase Designed to be.

The novel fluorescent dye of the present invention has the following chemical formula VI:

[0118]

Embedded image Or a biologically acceptable salt, wherein R ₂ -R ₆ are defined above with respect to Formula VII.

Preferred R2 and R3 are hydrogen, methyl, or ethyl; preferred R4 and R5 are hydrogen or methyl.

The compounds of formula VI of the invention may exist in tautomeric forms, in particular in the ring-opened form of formula VI ′. The present invention includes all tautomeric forms, including VI and VI '.

[0121]

Embedded image A preferred fluorogenic or fluorescent substrate of the present invention is a compound having Formula II and includes, but is 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 (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-S VD) ₂ - Rhodamine 110, SEQ ID NO: 10 (Z-DELD) ₂ - Rhodamine 110, SEQ ID NO: 11 (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-VEHD) ₂ - rhodamine 110, SEQ ID NO: 7 (Ac-YVAD) ₂ - rhodamine 110, SEQ ID NO: ₂ (Ac-LETD) 2 - rhodamine 110, SEQ No. 8 (Ac-LEHD) ₂ -rhodamine 110, SEQ ID NO: 3 (Ac-DEVD) ₂ -B -Damine 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 (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 (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 (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 (Z -D (OAM) E (OAM) TD (OAM)) ₂ -Rhodamine 110, SEQ ID NO: 4 (ZD (OAM) E (OAM) VD (OAM)) ₂ -Rhodamine 110, SEQ ID NO: 5 (ZD (OMe) E (OMe) VD (OAM)) ₂ -rhodamine 110, and SEQ ID NO: 5 (ZD (OMe) E (OMe) VD) ₂ -rhodamine 110, SEQ ID NO: 5.

A preferred fluorogenic or fluorescent substrate of the present invention is a compound having formula VII and includes, but is not limited to: N- (Z-WEHD) -N′-acetyl-rhodamine 110 SEQ ID NO: 1 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- ETD) -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, sequence No. 27 N- (Z-SHVD) -N'-acetyl-rhodamine 110, SEQ ID NO: 10 N- (Z-DELD) -N'-acetyl-rhodamine 110, SEQ ID NO: 11 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 1 0, N- (Boc-WEHD) -N'-acetyl-rhodamine 110, SEQ ID NO: 1 N- (Boc-YVAD) -N'-acetyl-rhodamine 110, SEQ ID NO: 2 N- (Ac-LETD) -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'-methoxy Carbonyl-rhodamine 110, SEQ ID NO: 3 N- (Ac-WEHD) -N'-methoxycarbonyl-rhodamine 110, SEQ ID NO: 1 N- (A -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-rhodami 110, SEQ ID NO: 23 N- (Z-WEHD) -N′-ethoxycarbonyl-rhodamine 110, SEQ ID NO: 1 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: 1 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'-eth Cycarbonyl-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: 3N -(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: 1 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: 9 N- (Ac-WEHD) -N′-hexyloxycarbonyl-rhodamine 110
SEQ ID NO: 1 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: 1 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
SEQ 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: 1 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'-decyl Oxycarbonyl-rhodamine 110,
SEQ ID NO: 1 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: 1 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: 1 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 110
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: 5 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: 1 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: 1 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: 1 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: 1 N- (Z-YVAD) -N '-(hexylthio) 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 11
0, SEQ ID NO: 1 N- (Ac-YVAD) -N '-(hexylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 2 N- (Ac-DEVD) -N ′-(hexylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 5 N- (Ac-DEHD) -N ′-(hexylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 6 N- (Ac-DETD) -N ′-(hexylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 4 N- (Ac-LEVD) -N ′-(hexylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 9 N- (Ac-LEHD) -N ′-(hexylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 3 N- (Ac-LETD) -N ′-(hexylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 8 N- (Ac-VEHD) -N ′-(hexylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 7 N- (Ac-IEPD) -N ′-(hexylthio) carbonyl-rhodamine 11
0, 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) carbonyl-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 11
0, SEQ ID NO: 1 N- (Ac-YVAD) -N '-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 2 N- (Ac-DEVD) -N ′-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 5 N- (Ac-DEHD) -N ′-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 6 N- (Ac-DETD) -N ′-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 4 N- (Ac-LEVD) -N ′-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 9 N- (Ac-LEHD) -N ′-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 3 N- (Ac-LETD) -N ′-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 8 N- (Ac-VEHD) -N ′-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 7 N- (Ac-IEPD) -N ′-(octylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 23 N- (Z-WEHD) -N ′-(decylthio) carbonyl-rhodamine 110,
SEQ ID NO: 1 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: 1 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-rhodamine 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: 1 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 11
0, SEQ ID NO: 1 N- (Ac-YVAD) -N '-(dodecylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 2 N- (Ac-DEVD) -N ′-(dodecylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 5 N- (Ac-DEHD) -N ′-(dodecylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 6 N- (Ac-DETD) -N ′-(dodecylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 4 N- (Ac-LEVD) -N ′-(dodecylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 9 N- (Ac-LEHD) -N ′-(dodecylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 3 N- (Ac-LETD) -N ′-(dodecylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 8 N- (Ac-VEHD) -N ′-(dodecylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 7 N- (Ac-IEPD) -N ′-(dodecylthio) carbonyl-rhodamine 11
0, 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 ′-(dimethylcarbamyl) -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: 1 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: 1 N- (Ac-YVAD) -N '-(N-hexyl-N-methylcarbamyl) -rhodamine 110, SEQ ID NO: 2 N- (Ac-DEVD) -N'-(N-hexyl-N-methylcarbamyl)- Rhodami 110, SEQ ID NO: 5 N- (Ac-DEHD) -N '-(N-hexyl-N-methylcarbamyl) -rhodamine 110, SEQ ID NO: 6 N- (Ac-DETD) -N'-(N-hexyl- N-methylcarbamyl) -rhodamine 110, SEQ ID NO: 4 N- (Ac-LEVD) -N '-(N-hexyl-N-methylcarbamyl) -rhodamine 110, SEQ ID NO: 9 N- (Ac-LEHD)- N ′-(N-hexyl-N-methylcarbamyl) -rhodamine 110, SEQ ID NO: 3 N- (Ac-LETD) -N ′-(N-hexyl-N-methylcarbamyl) -rhodamine 110, SEQ ID NO: 8 N- (Ac-VEHD) -N '-(N-hexyl-N-methylcarbamyl) -rhodamine 110, SEQ ID NO: 7 N- (Ac-IEPD) -N'-(N-hexyl-N-methylcarbamic ) -Rhodamine 110, SEQ ID NO: 23 N- (Z-DEVD) -N'-methanesulfonyl-rhodamine 110, SEQ ID NO: 5 N- (Z-YVAD) -N'-methanesulfonyl-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, SEQ ID NO: 5 N- (Z-YVAD) -N'-methanesulfonyl-rhodamine 19, SEQ ID NO: 2 N- (Z-YVAD (OAM))-N'-acetyl-rhodamine 110, SEQ ID NO: 2 N- (Z-LE (OAM) HD (OAM))-N'-acetyl-rhodamine 11
0, 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 (OAM))-N'-acetyl-rhodamine 110, SEQ ID NO: 5 N- (Z- D (OMe) E (OMe) VD) -N'-acetyl-rhodamine 11
0, SEQ ID NO: 5 N- (Z-VD (OAM))-N'-acetyl-rhodamine 110, and N- (ZE (OAM) VD (OAM))-N'-acetyl-rhodamine 110
.

Another preferred fluorogenic or fluorescent substrate of the present invention is a compound having the formula VIII, including but 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-LEDG) -N'-acetyl-rhodamine 110, SEQ ID NO: 77 N- (Ac-LEHDG) -N'-acetyl-rhoda 110, SEQ ID NO: 73 N- (Ac-WEHDG) -N'-methoxycarbonyl-rhodamine 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-WEHDGG) -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: 7 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 11
0, SEQ ID NO: 71 N- (Ac-YVADG) -N'-hexyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 72 N- (Ac-DEVDG) -N'-hexyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 76 N- (Ac-DEHDG) -N'-hexyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 78 N- (Ac-WEHDG) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 71 N- (Ac-YVADG) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 72 N- (Ac-DEVDG) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 76 N- (Ac-DEHDG) -N'-octyloxycarbonyl-rhodamine 11
0, 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 11
0, SEQ ID NO: 71 N- (Ac-YVADG) -N'-dodecyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 72 N- (Ac-DEVDG) -N'-dodecyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 76 N- (Ac-DEHDG) -N'-dodecyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 78 N- (Ac-WEHDG) -N ′-(ethylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 71 N- (Ac-YVADG) -N '-(ethylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 72 N- (Ac-DEVDG) -N '-(ethylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 76 N- (Ac-DEHDG) -N '-(ethylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 78 N- (Ac-WEHDG) -N ′-(hexylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 71 N- (Ac-YVADG) -N '-(hexylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 72 N- (Ac-DEVDG) -N '-(hexylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 76 N- (Ac-DEHDG) -N '-(hexylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 78 N- (Ac-WEHDG) -N '-(octylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 71 N- (Ac-YVADG) -N '-(octylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 72 N- (Ac-DEVDG) -N '-(octylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 76 N- (Ac-DEHDG) -N '-(octylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 78 N- (Ac-WEHDG) -N ′-(decylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 71 N- (Ac-YVADGGG) -N '-(decylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 142 N- (Ac-DEVDG) -N '-(decylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 76 N- (Ac-DEHDG) -N ′-(decylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 78 N- (Ac-WEHDG) -N '-(dodecylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 71 N- (Ac-YVADG) -N '-(dodecylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 72 N- (Ac-DEVDG) -N '-(dodecylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 76 N- (Ac-DEHDG) -N '-(dodecylthio) carbonyl-rhodamine 1
10, 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, SEQ ID NO: 76 N- (Z-YVADG) -N'-methanesulfonyl-rhodamine 110, SEQ ID NO: 72 N- (Z-DEVDG) -N'-acetyl-rhodamine 116, SEQ ID NO: 76N -(Z-YVADG) -N'-methanesulfonyl-rhodamine 116, SEQ ID NO: 72 N- (Z-DEVDG) -N'-acetyl -Rhodamine 19, SEQ ID NO: 76 N- (Z-YVADG) -N'-methanesulfonyl-rhodamine 19, SEQ ID NO: 72.

Another preferred fluorogenic or fluorescent substrate of the present invention is a compound having the formula IX, including but 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-rhodamine 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-N'-octyloxycarbonyl-rhodamine 110, N- ( MG) -N'-ethoxycarbonyl-rhodamine 110, N- (MA) -N'-ethoxycarbonyl-rhodamine 110, NGN-N'-ethoxycarbonyl-rhodamine 110, N- (MG) -N'-hexyloxy Carbonyl-rhodamine 110, N- (MA) -N'-hexyloxycarboni Le-rhodamine 110, NGN-hexyloxycarbonyl-rhodamine 110, N- (MG) -N '-(ethylthio) carbonyl-rhodamine 110, NGN-N'-(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- Damine 110, N- (Ac-LM) -N '-(ethylthio) carbonyl-rhodamine 110, N- (Ac-SLNFPIV) -N'-octyloxycarbonyl-rhodamine 110, SEQ ID NO: 80 N- (Ac-SLNFPI) -N'-octyloxycarbonyl-rhodamine 1
10, SEQ ID NO: 81 N- (Ac-SLNFP) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 82 N- (Ac-LNFPIV) -N'-octyloxycarbonyl-rhodamine 1
10, SEQ ID NO: 83 N- (Ac-LNFPI) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 84 N- (Ac-LNFP) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 85 N- (Ac-RGFP) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 37 N- (Z-LNFPIV) -N'-octyloxycarbonyl-rhodamine 11
0, 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 1
10, SEQ ID NO: 31 N- (Z-RQANFLG) -N'-octyloxycarbonyl-rhodamine 1
10, SEQ ID NO: 86 N- (Z-RQANF) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 87 N- (Z-RKVLFLD) -N'-octyloxycarbonyl-rhodamine 1
10, SEQ ID NO: 36 N- (Z-RKVLFL) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 88 N- (Z-RKVLF) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 89 N- (Z-ARVLFLG) -N'-octyloxycarbonyl-rhodamine 1
10, SEQ ID NO: 90 N- (Z-ARVLFL) -N′-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 91 N- (Z-ARVLF) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 92 N- (Z-SQNYFLG) -N'-octyloxycarbonyl-rhodamine 1
10, SEQ ID NO: 93 N- (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: 80 N- (Ac-SLNFPI) -N'-(ethylthio) carbonyl-rhodamine 1
10, SEQ ID NO: 81 N- (Ac-SLNFP) -N ′-(ethylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 82 N- (Ac-RGFP) -N '-(ethylthio) carbonyl-rhodamine 110
SEQ ID NO: 37 N- (Ac-MRGGGG) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 96 N- (Ac-IRGGGG) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 97 N- (Ac-LVGGG) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 98 N- (Ac-MVGGGG) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 99 N- (Ac-IVGGGG) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 100 N- (Ac-LRGGG) -N′-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 101 N- (Ac-LRGGA) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 102 N- (Ac-LRGG) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 55 N- (Z-LRGGGG) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 101 N- (Z-LRGGA) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID 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 11
0, SEQ ID NO: 101 N- (Ac-LRGGA) -N '-(ethylthio) carbonyl-rhodamine 11
0, 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 1
10, SEQ ID NO: 104 N- (Ac-LVLAS) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 105 N- (Ac-LVLA) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 62 N- (Z-LVLASSS) -N'-octyloxycarbonyl-rhodamine 1
10, SEQ ID NO: 103 N- (Z-LVLASS) -N'-octyloxycarbonyl-rhodamine 11
0, 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′-octyloxycarbonyl-rhodamine 1
10, 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 1
10, SEQ ID NO: 104 N- (Ac-LVLAS) -N ′-(ethylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 105 N- (Ac-LVLA) -N ′-(ethylthio) carbonyl-rhodamine 110
SEQ ID NO: 62 N- (Ac-VVNASS) -N'-octyloxycarbonyl-rhodamine 1
10, SEQ ID NO: 106 N- (Ac-VVNAS) -N'-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 107 N- (Ac-VVNA) -N'-octyloxycarbonyl-rhodamine 110
SEQ ID NO: 64 N- (Ac-Tbg-Tbg-NASS) -N'-octyloxycarbonyl-
Rhodamine 110, SEQ ID NO: 108 N- (Ac-Tbg-Tbg-NAS) -N'-octyloxycarbonyl-rhodamine 110, SEQ ID NO: 109 N- (Ac-Tbg-Tbg-NA) -N'-octyloxycarbonyl- Rhodamine 110, SEQ ID NO: 110 N- (Z-Tbg-Tbg-NASS) -N'-octyloxycarbonyl-rhodamine 110, SEQ 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, SEQ ID NO: 110 N- (Ac-Tbg-Tbg-NASS) -N'-ethoxycarbonyl-rhodamine 110, SEQ ID NO: 108 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, SEQ ID NO: 109 N- (Ac-Tbg-Tbg-NA) -N'-(ethylthio) Carbonyl-rhodamine 110, SEQ ID NO: 110 N- (Ac-DDIVPCSMST) -N'-octyloxycarbonyl-rhodamine 110, SEQ ID NO: 111 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'-octyloxy Carbonyl - rhodamine 1
10, SEQ ID NO: 115 N- (Ac-IVPCS) -N'-octyloxycarbonyl-rhodamine 11
0, 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 1
10, SEQ ID NO: 114 N- (Z-IVPCSM) -N'-octyloxycarbonyl-rhodamine 11
0, 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 1
10, SEQ ID NO: 115 N- (Ac-IVPCS) -N ′-(ethylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 116, wherein Z is benzyloxycarbonyl, and BOC is tert. -Butoxycarbonyl, Ac is acetyl, Tbg is t-butylglycine, and AM is acetoxymethyl.

Preferred novel fluorescent dyes of the present invention are 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-hexyloxy Carbonyl-rhodamine 110, N-octyloxycarbonyl-rhodamine 110, N-decyloxycarbonyl-rhodamine 110, N-dodecyloxycarbonyl-rhodamine 110, N-benzoyloxyca Bonyl-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-hexanesulfonyl-rhodamine 110, N-octanesulfonyl-rhodamine 1 0, N-decanesulfonyl-rhodamine 110, N-dodecanesulfonyl-rhodamine 110, 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-benzoyloxycarbonyl-rhoda Min 116, N-methanesulfonyl-rhodamine 116, N-trifluoromethanesulfonyl-rhodamine 116, N-octanesulfonyl-rhodamine 116, N-acetyl-rhodamine 19, N-ethoxycarbonyl-rhodamine 19, N-octyloxycarbonyl-rhodamine 19, N-methoxycarbonyl-rhodamine 19, and N-methanesulfonyl-rhodamine 19.

Exemplary aryl groups are C _6-10 aryl groups, including phenyl, naphthyl, fluorenyl, and the like, any of which may be substituted with halo or alkyl groups.

Representative alkyl groups are C _1-10 alkyl groups including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and its branched isomers.

Representative acyl (alkanoyl) groups include acetyl, propionyl, butanoyl
, Pentanoyl, hexanoyl, and the like, and their branched isomers _2-10 Alkanoyl group.

Representative biologically acceptable salts of the compounds of the present invention include sodium, potassium, ammonium, TRIS and the like.

Certain compounds of the present invention may exist in tautomeric forms, especially in the y-portion of Formula I. The present invention includes all such tautomers. The present invention also includes stereoisomers, racemic mixtures of such stereoisomers, as well as individual enantiomers that can be separated according to methods well known to those skilled in the art.

The compounds of the present invention can be prepared using methods known to those skilled in the art. For details,
Compounds having Formulas I-III can be prepared as illustrated by the exemplary reactions in Schemes 1-5.

Deprotection using HBr / HOAc leads to the removal of both the t-butoxy and benzyloxycarbonyl (Z) groups, which complicates the subsequent coupling reaction, so that at least Scheme 1 is the preferred method. It is. Therefore, if a t-butoxy group is desired, it must be reintroduced. When the N- (9-fluorenylmethoxycarbonyl) (fmoc) group is used as an N-blocking group (Scheme 2), it can be converted to morpholine, piperidine or other amine without removing the t-butoxy protecting group. It can be selectively removed with a base, thus allowing the immediate introduction of additional Z-blocked amino acids or peptides (see Schemes 2-4). The final Z block compound is selectively deprotected with trifluoroacetic acid (TFA) to remove the t-butoxy group without removing the Z group.

[0133]

(Equation 1)

[0134]

(Equation 2)

[0135]

(Equation 3)

[0136]

(Equation 4)

[0137]

(Equation 5) Accordingly, the present invention also relates to a process for the preparation of a compound of formula III, comprising: (a) condensing rhodamine 110 with N-fmoc-L-aspartic acid β-t-butyl ester and (Fmoc- (B) a step of providing Asp (OBu-t) ₂ -rhodamine 110; (b) removing an Fmoc group to give (Asp (OBu-t)) ₂ -rhodamine 110; (c) (Asp (OBu-t) Condensing ₂ -rhodamine 110 with Z- (AA) _n to give (Z- (AA) _n -Asp (OBu-t)) ₂ -rhodamine 110; and (d) removing the OBu-t protecting group Step.

In a preferred embodiment,-(AA) _n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SHV, DEL, DGP, DEP, DG
T, DLN, DEE, DSL, DVP, DEA, DSY, ELP, VED, IE
P or IET. If this amino acid is replaced with a carboxyl group, it is protected with an OBu-t protecting group which is removed in the last step.

The condensation reaction can be performed using any conventional condensing agent used for peptide synthesis. In a preferred embodiment, the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC) or 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ).
The solvent for this reaction can be pyridine or dimethylformamide (DMF). This reaction is generally performed at room temperature. The ratio of condensing agent to rhodamine can be about 10: 1, and the ratio of protected amino acid or peptide to rhodamine or (Asp (OBu-t)) ₂ -rhodamine 110 can also be
It may be about 10: 1.

The Fmoc group is generally removed by treatment with morpholine, piperidine or other amine base in a polar aprotic solvent (eg, DMF).
Generally, morpholine is added in excess and the reaction is performed at room temperature. α,
α-dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz)
Another N-blocking group that can be used in place of moc. Therefore, ND
dz-L-Aspartic acid β-t-butyl ester can be used instead of N-fmoc-L-aspartic acid β-t-butyl ester. Ddz can be selectively cleaved by 1% TFA in methylene chloride in the presence of a t-butoxy group.

[0141] The OBu-t group is removed with trifluoroacetic acid at room temperature in an aprotic solvent such as methylene chloride.

Compounds having formula VI can be prepared as shown by the exemplary reactions in Scheme 6.

[0143]

(Equation 6) Compounds having Formula VII-IX may be prepared as illustrated by the exemplary reactions in Schemes 7-10.

[0144]

(Equation 7)

[0145]

(Equation 8)

[0146]

(Equation 9)

[0147]

(Equation 10) Accordingly, the present invention also relates to a process for the preparation of a compound of formula VII, comprising: (a) reacting acetic anhydride with rhodamine to give N-acetyl rhodamine; (b) converting N-acetyl-rhodamine to N -Fmoc-L-aspartic acid β-
Condensed with t-butyl ester, N- (Fmoc-Asp (OBu-t))-
(C) removing the Fmoc group to give N- (Asp (OBu-t))-N'-acetyl-rhodamine; (d) N- (Asp (OBu- t)) -N'-acetyl-rhodamine is converted to Z- (
AA) condensing with _n to give N- (Z- (AA) n-Asp (OBu-t))-N'-acetyl-rhodamine; and (e) removing the OBu-t protecting group, -(Z- (AA) _n -Asp) -N'-acetyl-rhodamine; alternatively, (a) reacting rhodamine with acetic anhydride to give N-acetyl-rhodamine; (b ) N-acetyl-rhodamine is condensed 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 give 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 IET.

The compounds of formula VII also provide acyl (alkanoyl) (
For example, acetyl chloride, hexanoyl chloride, octanoyl chloride and decanoyl chloride). Other reagents that can be used in place of acetic anhydride include carbamyl chloride (eg, dimethylcarbamyl chloride, diethylcarbamyl chloride, and N-methyl-N-hexylcarbamyl chloride); chloroformate (eg, methylchloroformyl) Acid, ethylchloroformylic acid, octylchloroformylic acid, 2-butoxyethylchloroformylic acid and 2,5,8-trioxadecylchloroformylic acid); Alkyl, haloalkyl, and aralkylsulfonyl halides (eg, methanesulfonyl chloride, octanesulfonyl chloride, trifluoromethanesulfonyl chloride, and tosyl chloride); But it is not limited to. This reaction is carried out in the presence of _{(Et) 3 N, (i} -Pr) 2 -NEt or bases, such as pyridine. The preferred solvent is DMF. This reaction is generally performed at room temperature. The ratio of anhydride or acyl chloride to rhodamine is about 1: 1.

[0149] The condensation reaction can be performed using any conventional condensing agent 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
). This reaction is generally performed at room temperature. The ratio of condensing agent to N-acetyl-rhodamine is about 3: 1 and the ratio of protected amino acid or peptide to N-acetyl-rhodamine or N- (Asp (OBu-t))-N'-acetyl-rhodamine. Is about 3: 1.

In a one-step reaction, a peptide such as Z- (AA) _n -Asp (OBu-t) is condensed with N-acetyl-rhodamine to form N- (Z- (AA) _n -Asp (OBu- t)) Obtaining -N'-acetyl-rhodamine is a preferred procedure. Thus, the compound of Formula VI provides a fluorochrome that can be condensed with any peptide or other structure for the preparation of a fluorogenic compound or a fluorescent compound that is a substrate for a protease or peptidase.

In principle, the compounds of formula VII can also be prepared by first condensing a peptide with rhodamine to give N-peptide-rhodamine, then reacting N-peptide-rhodamine with acetic anhydride or another acylating agent. Thus, for example, by providing N-acetyl-N'-peptide-rhodamine. However, a) the peptide is generally much more expensive than the acyl chloride or anhydride, b) the condensation reaction between the peptide and rhodamine is not an efficient reaction. For this reason, it is preferred to attach the peptide to N-acetyl-rhodamine rather than the acyl group to N-peptide-rhodamine.

In one aspect, the invention relates to a method for determining whether a test substance has an effect on an enzyme involved in the apoptotic cascade in a test cell, comprising: (a) the test substance comprising an outer membrane of the cell; Contacting a test cell with a test substance and a reporter compound according to the present invention under conditions that either interact with the receptor or are taken up by the cell, and the reporter compound is taken up by the cell; and (B) recording the fluorescence of the test cells, wherein the fluorescence of either size or wavelength in the test cells is compared with the control cells that have been contacted only with the reporter compound and not with the test substance. The change is an indicator that the test substance has an effect on the enzyme.

The results obtained by this method can be compared to the results obtained with test compounds known to affect enzymes involved in the apoptotic cascade in cells where the relative potency of the test substance can be measured. Compounds that can be used include known activators and inhibitors of enzymes involved in the apoptotic cascade. Activators by either direct or indirect mechanisms of enzymes involved in the apoptotic cascade include known chemotherapeutic agents (eg, etoposide (Yoon HJ,
Choi IY, Kang MR, Kim SS, Muller MT, Spi
tzner JR, Chung IK (1998), Biochem Biop
hys Acta 1395: 110-120) and doxorbin (Game)
n S, Anel A, Lasierra P, Alava MA, Marti
nes-Lorenzo MJ, Pineiro A, Naval J (199
7), FEBS Lett 417: 360-364), which are topoisomerase II inhibitors; cisplatin (Maldonado V, Me
lendez-Zajgla J, Ortega A (1997), Mutat
Res 381: 67-75); chlorambucil (Hickman JA.
1992), Cancer Metastasis Rev. 11: 121-1
39) This is an alkylating agent; and the RNA / DNA antimetabolite fluorouracil (Hickman JA. (1992), Cancer Met).
astasis Rev. 11: 121-139), but is not limited thereto. These activators of apoptosis can also be used to induce apoptosis when screening for inhibitors of apoptosis in whole cells. Non-activators by either direct or indirect mechanisms of enzymes involved in the apoptotic cascade include endogenous proteins including Bcl-2 (Joensuu H, Pylkkanen L, Toikkanen L
S (1994), Am. J. Pathol. 5: 1191-1198), the viral producer p35 (Miller LK (1997), J. Cell Phys.
iol. 173: 178-182) and synthetic caspase inhibitor ZV
AD-FMK (AnS, Knox KA (1996), FEBS Lett.
386: 115-122), but is not limited thereto.

In particular, the present invention relates to Formulas I-III, V, VII and V in whole cell assays.
The use of reporter compounds having III induces apoptosis to screen for compounds that either directly or indirectly inhibit the enzyme (s) associated with apoptosis (programmed cell death) Use the prepared whole cell or tissue sample. Formulas I-III, V, VII
These screening assays using compounds having
It is expected to lead to the discovery of new drugs or new uses for known drugs that delay or block cell death in various clinical conditions where loss of cells, tissues or organs occurs.

The reporter compounds having Formulas I-III, V, VII and VIII of the present invention and screening assays can be used under various conditions of ischemia and excitotoxicity (focal ischemia due to stroke and systemic due to cardiac arrest due to cardiac arrest). Can be used to identify drugs that reduce or prevent cell death in the nervous system (brain, spinal cord and peripheral nervous system) in, but not limited to, ischemia. This screening assay may also include traumatic injury (eg, head or spinal injury), viral infection or radiation-induced neuronal death (eg, as a side effect of cancer treatment) or environmental toxicity (eg, certain halogenated hydrocarbons). ) Can be used to identify compounds that reduce or prevent cell death in the nervous system. The screening assay may also include 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 spinal medulla atrophy. Can be used to identify cell death inhibitors useful for reducing or preventing cell death in E. coli.

The screening assays of the invention can be used to identify compounds that prevent cell death in any condition that potentially results in myocardial death. This includes myocardial infarction, congestive heart failure and cardiomyopathy. One particular application of this screening assay is to identify compounds that reduce or prevent myocardial cell death resulting from a particular viral infection of the heart.

The screening assays of the present invention prevent retinal neuronal cell death that occurs in disorders associated with increased intraocular pressure (eg, glaucoma) or aging processes associated with the aging process (eg, age-related macular degeneration). Can be used to identify compounds. This assay can also be used to identify compounds that treat a genetic degenerative disorder of the retina, such as retinitis pigmentosa.

The screening assays of the present invention can also be used to identify cell death inhibitors that can be used to reduce or prevent immature death of cells in the immune system, and include immunodeficiency disorders such as acquired The present invention is particularly useful for identifying inhibitors that are useful in the treatment of experimental immune syndrome (AIDS), severe combined immune syndrome (SCIDS), and related diseases.This screening assay is also used to treat radiation-induced immunosuppression. Can be used to identify cell death inhibitors that can be used.

The screening assays of the present invention can also be used to identify drugs useful in organ transplant procedures. Transplantation of human organs and tissues is a common treatment for organ failure. However, during the transplantation process, the donor organ or tissue is at risk for cell death. Because it is deprived of this normal blood supply before it is transplanted in the host. The ischemic condition can be treated with a cell death inhibitor by injection into the organ or tissue of the donor or by direct addition of the cell death inhibitor to the organ / tissue storage medium. Such cell death inhibitors can be identified using the screening assays described in the present invention. Cell death inhibitors are also used to reduce or prevent cell death in a donor organ / tissue after it has been transplanted and protect it from the effects of host immune cells that kill their targets by causing apoptosis. obtain. The screening assays described in the present invention can be used to identify cell death inhibitors useful in protecting transplanted organs from rejection. The cytoprotective effects of cell death inhibitors can also be used to prevent human or animal sperm and egg death used in in vitro fertilization procedures. These inhibitors can be used during the harvesting 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 the present invention.

[0160] Mammalian cell lines and yeast cells are commonly used to produce large amounts of recombinant proteins (eg, antibodies, enzymes and hormones) for industrial or medical use. The life span of some of these cell lines is limited due to growth conditions, the nature of the recombinant molecule being expressed (some are cytotoxic), and other unknown factors. The lifespan of industrial cell lines can be extended by including a cell death inhibitor in the growth medium. Cell death inhibitors useful in extending the lifespan of a cell line may be identified using the screening assay procedures described in the present invention.

The factors that govern hair growth and loss are largely unknown. However, there is some evidence that hair follicle degeneration (referred to as catagen) may be at least partially due to apoptosis. Thus, cell death inhibitors may be caused by a variety of conditions, including but not limited to male pattern baldness, radiation-induced or chemotherapy-induced hair loss, and hair loss due to emotional stress. Can be used to treat loss. There is also evidence that apoptosis may play a role in hair color loss. Thus, cell death inhibitors could also be used in the treatment of immature gray hair. Cell death inhibitors useful in treating or preventing hair loss or gray hair may be identified using the screening assay procedures described in the present invention.

[0162] Death of skin epithelial cells can occur after exposure to high levels of radiation, heat, or chemicals. Cell death inhibitors can be used to reduce or prevent this type of skin damage. In one particular application, cell death inhibitors may be applied to ointments to treat acute overexposure to the sun and prevent blistering and exfoliation of the skin. Cell death inhibitors useful in treating or preventing skin cell death can be identified using the screening assay procedures described in the present invention.

Another important aspect of the invention is the enzyme (s) associated with apoptosis
Of reporter compounds having Formulas I-III, VII and VIII in whole cell assays using live or dead whole cell or tissue samples to screen for compounds that promote either directly or indirectly It is. Therefore, these screening assays using compounds having Formulas I-III, VII and VIII may provide new drugs or new uses for known drugs that act as anti-cancer agents in diseases such as cancer, tumors and cell hyperplasia. It is expected to lead a discovery. Compounds that can be found using the screening methods and reagents described herein include cancer, tumor or tissue hyperplasia (brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus) Cancer of the lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and gonads, joints, bone and skin Or, including but not limited to, tumors).

Another important aspect of the present invention is that antifungal or antibacterial agents that act by inducing directly or indirectly, in these cells, the caspase cascade or other enzymes associated with apoptosis. Use of reporter compounds having Formulas I-III, VII and VIII in whole cell assays using yeast and other fungi and bacteria to screen compound libraries for agents.

[0165] Another important aspect of the invention is the therapeutic effect of a therapeutic agent or treatment given to a patient for the purpose of reducing, preventing or treating a disease caused or caused by apoptotic cell death. Formulas I-III, VII and VI to monitor
Is to use a reporter compound having II.

Another important aspect of the present invention is the use of a reporter compound having Formula IX to screen for HIV protease inhibitors in HIV infected cells, comprising: Contacting a test cell with a test substance and a reporter compound according to the invention under conditions such that they either interact or are taken up by said cell, and said reporter compound is taken up into the cell;
And (b) recording the fluorescence of the test cells, wherein the magnitude or wavelength of the fluorescence in the test cells compared to control cells that were only contacted with the reporter compound and not contacted with the test substance Any change is an indicator that the test substance has an inhibitory effect on HIV protease.

Yet another important aspect of the present invention is the use of a reporter compound having formula IX for diagnosing HIV infection, comprising: (a) subjecting the reporter compound under conditions in which the reporter compound is taken up by cells Contacting a test cell from an individual suspected of having an HIV interaction with a reporter compound according to the invention; (b) recording the fluorescence of the test cell, wherein said step is compared to control cells contacted with said reporter compound. A change in either the magnitude or wavelength of the fluorescence in the test cells is an indication that the test cells have been infected by the HIV virus and that the individual has been infected with HIV.

Applying the same procedure for screening for HIV protease inhibitors in HIV infected cells, reporter compounds having Formula IX of the present invention can be used to screen for adenovirus protease inhibitors in adenovirus infected cells. . The reporter compound having Formula IX of the present invention also provides for herpes simplex virus type 1 (HSH-1) in HSV-1 infected cells.
V-1) Can be used to screen for protease inhibitors. The reporter compound may also be used to screen for human cytomegalovirus (HCMV) protease inhibitors in HCMV infected cells; to screen for hepatitis C virus (HCV) protease inhibitors in HCV infected cells; DPP-IV in T cells It can be used to screen for inhibitors; as well as to screen for type 2 methionine aminopeptidase (MetAP-2) inhibitors in endothelial cells.

In addition, using the same procedure for the diagnosis of HIV infection, the reporter compound having formula IX of the present invention also contains adenovirus, herpes simplex virus type 1
, Human cytomegalovirus and hepatitis C virus.

Compositions within the scope of the present invention include all compositions wherein the fluorogenic or fluorescent compounds of the present invention are contained in an amount effective to achieve its intended purpose. While amounts can vary from assay to assay, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typically, the fluorogenic or fluorescent substrate compound is present in a physiologically compatible buffer at a concentration of about 0.01 nanomolar to about 1 molar or an equivalent of a salt thereof or a proreporter molecule thereof and a cell or a fluorogenic or fluorescent substance. It can be applied to cells or cell lines from mammals (eg, humans), or other animals, by incubating the tissue containing the cells. Such buffers include RPMI-1640 with or without 10% bovine eradication serum.
And a cell growth medium which is an example for leukemia derived from cancer cells. Other known cell incubation buffers may include isotonic solutions buffered with either phosphate or HEPES. One of skill in the art can identify other suitable buffers without going beyond routine experimentation. The cells can be useful in the treatment of apoptosis-mediated disorders such as neuronal cell death, heart disease, liver disease, retinal disease, kidney, joint and bone disease, immune system disorders, cancer, tumor and tissue hyperplasia, and the like. The drug can be from any organ or organ system for which it is desired to find the drug by a screening assay.

Suitable stabilizers can be used for the fluorogenic or fluorescent compounds of the present invention that are present on a tissue, cell or cell line. Suitable stabilizers include aqueous solutions of the active compounds in 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 cells or tissues. Suitable lipophilic solvents or vehicles include fatty oils (e.g., sesame oil) or synthetic fatty acid esters (e.g., ethyl oleate or triglyceride) or polyethylene glycol-400 (the compound is PEG-40
0) or dimethyl sulfoxide (DMSO) or another suitable stabilizer. If necessary, the suspension or solution may also contain stabilizers. If desired, presentation of a reporter molecule or surfactant in electroporation or liposomes can be used to increase the cell permeability of the fluorogenic reporter molecule or the fluorescent reporter molecule.

Typically, the cells are treated with a reporter compound of the invention and a test substance for about 30 minutes.
The contact is for minutes to about 5 hours, most preferably for about 1 hour.

The present invention also relates to proreporter derivatives of the compounds of the present invention. Such a proreporter derivative is cleaved in situ by an endogenous enzyme and has the formula I
-III, V and VII-IX. Such proreporter derivatives include lower alkyl esters of carboxy-containing amino acid residues such as Asp and Glu. In particular, preferred proreporter derivatives are methyl esters and acetylmethyl (AM) of Asp and Glu containing compounds.
) Esters.

The following examples demonstrate the utility of the present invention in measuring the activity of caspases and other enzymes associated with apoptosis in cells and tissues. This example also demonstrates the utility of the present invention in drug screening assays that can be utilized to find enhancers or inhibitors of apoptosis. These examples are illustrative, but not limiting, of the methods and compositions of the present invention. Other suitable modifications and adaptations of the various conditions and parameters usually encountered in in vitro assays, drug screening procedures or diagnostic procedures apparent to those of skill in the art include:
It is the spirit and scope of the present invention.

Example 1 [Fmoc-Asp (OBu-t)] ₂ -rhodamine 110 Fmoc-L-aspartic acid β-t- dissolved in an anhydrous 1: 1 mixture of dimethylformamide and pyridine (30 mL). Butyl ester (4.9 g, 11
. EDC (2.28 g, 11.91 mmol) was added to the solution at 0 ° C. The solution was stirred for 45 minutes and then rhodamine 110 hydrochloric acid (0.44 g
, 1.19 mmol) in the same solvent (2 mL). The reaction mixture was stirred at room temperature for 60 hours and concentrated under reduced pressure to about 10 mL. The residue was then diluted with 100 mL of water and extracted with ethyl acetate (3 × 50 mL). The organic layer was washed with 1N hydrochloric acid (2 × 50 mL) and water (2 × 50 mL). The solution was dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by column chromatography (CH ₂ Cl ₂ / EtOAc 10: 1) to give 0.89 g (67%) as a colorless solid ) Was obtained. Melting point 156-15
8 ° C., ¹ H NMR (CDCl ₃ ): δ 8.75 (bs, 2H), 8.02 (d,
J = 9.3 Hz, 2H), 7.80-6.70 (m, 26H), 6.12 (b
s, 2H), 4.64 (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).

Example 2 [Asp (OBu-t)] ₂ -rhodamine 110 2HCl) A cooled solution of DMF / morpholine (3 mL, 1: 1) was added to dimethylformamide (
To a stirred solution of [Fmoc-Asp (OBu-t)] ₂ -rhodamine 110 (150 mg, 0.13 mmol) in 3 mL) was added. The solution was stirred for 20 minutes and it was poured into ice water (100 mL) and ethyl acetate (2 × 100 m
L). The organic layer was washed with water (3 × 100 mL) and dried over Na ₂ SO ₄ . To this solution was added 1N hydrochloric acid in ether (0.39 mL) and concentrated to give a red solid. The red solid was collected and added to methanol (1 m
L) and precipitated with ether (50 mL) to give the title compound (65 mg).
, 77%) as a red solid (melting point 200 ° C. (dec)).

Example 3 [Z-Ala-Asp (OBu-t)] ₂ -rhodamine 110) Benzyloxycarbonyl-L in 1: 1 anhydrous DMF / pyridine (10 mL)
-Alanine (376 mg, 1.69 mmol), EDC (258 mg, 1.35)
mmol) and [Asp (OBu-t)] ₂ -rhodamine 110 2HCl (50 mg, 0.072 mmol) at 63C (86%) at 0 <0> C to give the title compound as a solid (mp 124-126 <0> C). ¹ H NMR (CDCl ₃ ): δ8
. 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 [Z-Ala-Asp (OBu-t)] ₂ -rhodamine 110 (41 mg, 0.038 mmol) in methylene chloride (5 mL) To a cooled solution of (0 ° C.) was added 50% trifluoroacetic acid in methylene chloride (16 mL). The solution turned orange and was stirred at room temperature for 3 hours. The solvent is removed and the crude product is purified by flash column chromatography (EtOAc / CF ₃ CO ₂ H = 20:
0.5) to give 34 mg (91%) of the title compound. ¹ H NMR (CD ₃ OD): δ 8.02 (d, J = 8.1 Hz, 1H), 7.85 (s, 2H), 7.73 (t, J = 7.5 Hz, 2H), 7.34 to 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 Benzyloxycarbonyl-L in 1: 1 anhydrous DMF / pyridine (40 mL)
-Aspartic acid β-t-butyl ester (4.41 g, 13.63 mmol
), EDC (2.61 g, 13.63 mmol) and Rhodamine 110 HC
1 (0.50 g, 1.36 mmol) at 0 ° C. to give 1.09 g (82%) of the title compound as a colorless solid (mp 127-129 ° C.). _{^{1 H NMR (CDC l 3)}} : δ8.75 (bs, 2H), 8.06 (d, J = 6.3Hz, 2H), 7.68~7.56 (m, 4H), 7.40~ 6.60 (m, 15H), 6.1
2 (bs, 2H), 5.16 (s, 4H), 4.62 (bs, 2H), 2.97
_{(Dd, J 1 = 17.1Hz,} J 2 = 3.3Hz, 2H), 2.69 (dd, J 1 = 17.3Hz, J 2 = 6.9Hz, 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 to [ZA in acetic acid (2 mL).
sp (OBu-t)] ₂ -rhodamine 110 (200 mg, 0.21 mmol) was added by titration to a stirred solution. The solution was stirred at room temperature for 1 hour and then concentrated under reduced pressure. 100 mL of anhydrous ether was added to the residue to give a red precipitate. It was isolated after centrifugation and 118 mg (78%) solid (mp 130
° C). ¹ H NMR (DMSO-d ₆ ): δ 10.81 (s, 2H),
8.35 (bs, 6H), 8.05 to 6.79 (m, 10H), 4.25 (bs
, 2H), 2.96 (m, 4H).

Example 7 (Z-Val-Asp) ₂ -Rhodamine 110 At 0 ° C., benzyloxycarbonyl-L-valine (628 mg, 2.5 mmol) in 1: 1 anhydrous DMF / pyridine (12 mL). , EDC (383 mg, 2 m
mol) and (Asp) ₂ -rhodamine 110 2HBr (72 mg, 0.1 mmol) gave 38 mg (37%) of the title compound, mp 169-171 ° C.

[0182]

[Equation 11] (Example 8: [Z-Val-Ala-Asp (OBu-t)] ₂ -rhodamine 110) Benzyloxycarbonyl-LV in 1: 1 anhydrous DMF / pyridine at 0 ° C.
al-L-Ala (200 mg, 0.62 mmol), EDC (110 mg, 0
. 57 mmol) and [Asp (OBu-t)] ₂ -rhodamine 1102 HCl (31 mg, 0.043 mmol) gave 45 mg (85%) of the title compound, mp 85-87 ° C.

[0183]

(Equation 12) (Example 9: (Z-Val-Ala-Asp) ₂ -rhodamine 110) At 0 ° C, [(Z-Val-Ala-Asp (O) in methylene chloride (30 mL) was used.
(Bu-t)] ₂ -rhodamine 110 (28 mg, 0.022 mmol) and 50% trifluoroacetic acid to give 23 mg (88%) of the title compound.

[0184]

(Equation 13) (Example 10: [(Z-Tyr-Val-Ala-Asp (OBu-t)]] _Two -Rhodamine 110, SEQ ID NO: 2) Benzyloxycarbonyl-LT in 1: 1 anhydrous DMF / pyridine at 0 ° C
yr-L-Val-L-Ala (339 mg, 0.70 mmol), EDC (1
22 mg, 0.64 mmol) and [Asp (OBu-t)]_Two-From rhodamine 110 2HCl (39 mg, 0.058 mmol), 61 mg (65%
)), Mp 155-157 ° C.

[0185]

[Equation 14] (Example 11: (Z-Tyr-Val-Ala-Asp) ₂ -rhodamine 110, SEQ ID NO: 2) [Z-Tyr-Val-Ala-As in methylene chloride (30 mL) at 0 ° C.
p (OBu-t)] ₂ -rhodamine 110, SEQ ID NO: 2 (47 mg, 0.029 mmol) and 50% trifluoroacetic acid gave 36 mg (82%) of the title compound, mp 115 ° C.

[0186]

(Equation 15) (Example 12: N-acetyl-rhodamine 110) Rhodamine 110 (500 mg, 1 mg) dissolved in DMF (20 mL) at 0 ° C.
. 36 mmol) in a solution of N, N-diisopropylethylamine (176 mg).
, 1.36 mmol) and then acetic anhydride (167 mg, 1.64 mmol).
) Was added dropwise to the above solution. The reaction solution was stirred at room temperature for 24 hours, then it was diluted with 100 ml of water and extracted with ethyl acetate (3 × 50 m
L). The organic phase was washed with water (2 × 100 mL), dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by column chromatography (hexane / Et.
Purification by OAc 1: 1) afforded 210 mg (41%) of the title compound as a colorless solid, mp 179 ° C (dec).

[0187]

(Equation 16) Example 13: N- [Fmoc-Asp (OBu-t)]-N'-acetyl-rhodamine 110) Fmoc-L-aspartic acid t- in 1: 1 anhydrous DMF / pyridine (8 mL) at 0 ° C. Butyl ester (739 mg, 1.79 mmol), EDC (302
mg, 1.57 mmol) and N-acetyl-rhodamine 110 (160 m
g, 0.43 mmol) to give 276 mg (84%) of the title compound as a colorless solid.

[0188]

[Equation 17] (Example 14: N- [Asp (OBu-t)]-N'-acetyl-rhodamine 110) Cold DMF / morpholine solution (3 mL, 1: 1) and N- [Fmoc-As
p (OBu-t)]-N'-acetyl-rhodamine 110 (100 mg, 0.1
3 mmol) to give the title compound (67 mg, 95%) as a solid, mp 131-13.
Obtained as 3 ° C. p.

[0189]

(Equation 18) (Example 15: N- [Z-Ala-Asp (OBu-t)]-N'-acetyl-rhodamine 110) Benzyloxycarbonyl-L-alanine (43 mg) in 1: 1 anhydrous DMF / pyridine at 0 ° C. , 0.19 mmol), EDC (37 mg, 0.19 mmol)
) And N-Asp (OBu-t) -N'-acetyl-rhodamine 110 (30
mg, 0.055 mmol) to give 38 mg (92%) of the title compound as a solid, mp 138-140 ° C.

[0190]

[Equation 19] (Example 16: N- (Z-Ala-Asp) -N'-acetyl-rhodamine 110) N- [Z-Ala-Asp (OBu-t) in methylene chloride (3 mL) at 0 ° C.
)]-N'-acetyl-rhodamine 110 (38 mg, 0.052 mmol) and 50% trifluoroacetic acid gave 34 mg (96%) of the title compound.

[0191]

(Equation 20) Example 17: N- [Z-Val-Asp (OBu-t)]-N'-acetyl-rhodamine 110) Benzyloxycarbonyl-L- in 1: 1 anhydrous DMF / pyridine (4 mL) at 0 ° C. Valine (49 mg, 0.19 mmol), EDC (37 mg, 0.19 mmol)
mmol) and N-Asp (OBu-t) -N'-acetyl-rhodamine 11
0 (30 mg, 0.055 mmol) gave 40 mg (94%) of the title compound as a solid, mp 155-157 ° C.

[0192]

(Equation 21) (Example 18: N- (Z-Val-Asp) -N'-acetyl-rhodamine 110) N- [Z-Val-Asp (OBu-t) in methylene chloride (3 mL) at 0 ° C.
)]-N'-acetyl-rhodamine 110 (40 mg, 0.051 mmol) and 50% trifluoroacetic acid gave 37 mg (99%) of the title compound.

[0193]

(Equation 22) (Example 19: N- [Z-Val-Ala-Asp (OBu-t)]-N '
-Acetyl-rhodamine 110) Benzyloxycarbonyl-L-valine-L-alanine (63 mg, 0.019 mmol) in 1: 1 anhydrous DMF / pyridine at 0 ° C, EDC (37 mg, 0
. (019 mmol) and N-Asp (OBu-t) -N′-acetyl-rhodamine 110 (30 mg, 0.052 mmol) gave 40 mg (97%) of the title compound as a solid, mp 101-103 ° C.

[0194]

(Equation 23) (Example 20: N- (Z-Val-Ala-Asp) -N'-acetyl-rhodamine 110) N- [Z-Val-Ala-Asp (O) in methylene chloride (4 mL) at 0 ° C.
Bu-t)]-N'-acetyl-rhodamine 110 (36 mg, 0.043 mm)
ol) and 50% trifluoroacetic acid to give 35 mg (100%) of the title compound.

[0195]

(Equation 24) (Example 21: N- [Z-Tyr-Val-Ala-Asp (OBu-t)
] -N'-acetyl-rhodamine 110, SEQ ID NO: 2) Benzyloxycarbonyl-L-tyrosine-L-valine-L-alanine (119 mg, 0.25 mmol) in 1: 1 anhydrous DMF / pyridine at 0 ° C. EDC
(47 mg, 0.25 mmol) and N-Asp (OBu-t) -N′-acetyl-rhodamine 110 (30 mg, 0.055 mmol) from 50 mg (9
(5%) of the title compound.

[0196]

(Equation 25) (Example 22: N- (Z-Tyr-Val-Ala-Asp) -N'-acetyl-rhodamine 110, SEQ ID NO: 2) N- [Z-Tyr-Val in methylene chloride (4 mL) at 0 ° C. -Ala-A
sp (OBu-t)]-N'-acetyl-rhodamine 110, SEQ ID NO: 2 (49
mg, 0.049 mmol) and 50% trifluoroacetic acid to give 42 mg (8
9%) of the title compound were obtained.

[0197]

(Equation 26) (Example 23: N- [Z-Asp (OBu-t) -Glu (OBu-t)-
Val-Asp (OBu-t)]-N'-acetyl-rhodamine 110, SEQ ID NO: 5) Benzyloxycarbonyl-L-Asp (OBu-t) in 0: 1 1: 1 anhydrous DMF / pyridine (4 mL). -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, 0.0
8 mmol), 73 mg (81%) of the title compound, mp = 127-129 ° C.
I got

[0198]

[Equation 27] (Example 24: N- (Z-Asp-Glu-Val-Asp) -N'-acetyl-rhodamine 110, SEQ ID NO: 5) N- [Z-Asp (OBu) in methylene chloride (5 mL) at 0 ° C. -T) -Gl
From u (OBu-t) -Val-Asp (OBu-t)]-N'-acetyl-rhodamine 110, SEQ ID NO: 5 (49 mg, 0.043 mmol) and 50% trifluoroacetic acid, 38 mg (92%) title. The compound was obtained.

[0199]

[Equation 28] (Example 25: N-ethoxycarbonyl-rhodamine 110) Rhodamine 1 dissolved in dimethylformamide (140 mL) at -50 ° C
To a solution of 10 (3.00 mg, 8.18 mmol) was added N, N-diisopropylethylamine (1.27 g, 1.2 mmol) and then ethyl chloroformate (1.07 g, 9.81 mmol) was added to the above solution. Added dropwise. Then
The reaction solution was slowly warmed to room temperature and kept stirring for 5 hours. It was then diluted with 200 ml of ice water and extracted with ethyl acetate (3 × 50
mL). The organic phase was washed with brine (3 × 100 mL), dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by column chromatography (hexane / EtOAc 3: 1) 1.31 g (40%) of the title compound were obtained as a colorless solid.

[0200]

(Equation 29) (Example 26: N- [Cbz-Val-Asp (OBu-t)]-N'-ethoxycarbonyl-rhodamine 110) 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) provided 47 mg (50%) of the title compound as a solid (as described in Example 1).

[0201]

[Equation 30] Example 27: N- (Cbz-Val-Asp) -N'-ethoxycarbonyl-rhodamine 110 N- [Z-Val- in 50% trifluoroacetic acid in methylene chloride (2 mL)
Asp (OBu-t)]-N'-ethoxycarbonyl-rhodamine 110 (20
mg, 0.025 mmol) to give 15 mg (80%) of the title compound.

[0202]

(Equation 31) (Example 28: N- [Cbz-Asp (OBu-t) -Glu (OBu-t
) -Val-Asp (OBu-t)]-N'-ethoxycarbonyl-rhodamine 110, SEQ ID NO: 5) Cbz-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (O
Bu-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) to 81 m
g (58%) of the title compound were obtained as a solid.

[0203]

(Equation 32) (Example 29: N- (Cbz-Asp-Glu-Val-Asp) -N '
-Ethoxycarbonyl-rhodamine 110, SEQ ID NO: 5) N- [Z-Asp (OBu-t) -Glu (OB) in methylene chloride (3 mL)
ut) -Val-Asp (OBu-t)]-N′-ethoxycarbonyl-rhodamine 110, SEQ ID NO: 5 (100 mg, 0.086 mmol) and 50% trifluoroacetic acid (as described in Example 4) ), 85 mg (99%) of the title compound.

[0204]

[Equation 33] (Example 30: N- [Ac-Asp (OBu-t) -Glu (OBu-t)
-Val-Asp (OBu-t)]-N'-ethoxycarbonyl-rhodamine 1
10, SEQ ID NO: 5) Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OB
ut), SEQ ID NO: 5 (307.6 mg, 0.45 mmol), EDC (86 m
g, 0.45 mmol) and N-ethoxycarbonyl-rhodamine 110 (6
0 mg, 0.15 mmol) (as described in Example 1) to 128 mg
(80%) of the title compound was obtained as a solid.

[0205]

(Equation 34) (Example 31: N- (Ac-Asp-Glu-Val-Asp) -N'-)
Ethoxycarbonyl-rhodamine 110, SEQ ID NO: 5) N- [Ac-Asp (OBu-t) -Glu (O) in methylene chloride (2 mL)
Bu-t) -Val-Asp (OBu-t)]-N'-ethoxycarbonyl-rhodamine 110, 65 mg (86%) of the title compound from SEQ ID NO: 5 (90 mg, 0.084 mmol) and 50% trifluoroacetic acid. I got

[0206]

(Equation 35) (Example 32: N-octyloxycarbonyl-rhodamine 110) Rhodamine 110 (500 mg, 1.36 mmol); From N-diisopropylethylamine (351.6 mg, 2.76 mmol) and octyl chloroformate (316 mg, 1.64 mmol) (as described in Example 25)
182 mg (28%) of the title compound as a colorless solid.

[0207]

[Equation 36] (Example 33: N- [Ac-Asp (OBu-t) -Glu (OBu-t)
-Val-Asp (OBu-t)]-N'-octyloxycarbonyl-rhodamine 110, SEQ ID NO: 5) Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OB
ut), 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 were obtained as a solid.

[0208]

(37) (Example 34: N- (Ac-Asp-Glu-Val-Asp) -N'-
Octyloxycarbonyl-rhodamine 110, SEQ ID NO: 5) N- [Z-Asp (OBu-t) -Glu (OB) in methylene chloride (2 mL)
ut) -Val-Asp (OBu-t)]-N'-octyloxycarbonyl-rhodamine 110, SEQ ID NO: 5 and 50% trifluoroacetic acid, from 16.5
mg (100%) of the title compound were obtained.

[0209]

(38) (Example 35: N-methoxycarbonyl-rhodamine 110) Rhodamine 110 (600 mg, 1.64 mmol); From N-diisopropylethylamine (254 mg, 1.96 mmol) and methyl chloroformate (201 mg, 2.13 mmol) (as described in Example 25), 28
mg (4.4%) of the title compound were obtained as a colorless solid.

[0210]

[Equation 39] (Example 36: N-methylsulfonyl-rhodamine 110) Rhodamine 110 (500 mg, 1.36 mmol); From N-diisopropylethylamine (211 mg, 1.64 mmol) and methylsulfonyl chloride (187 mg, 1.64 mmol) (as described in Example 25),
2.1 mg (9.4%) of the title compound were obtained.

[0211]

(Equation 40) (Example 37: N-acetyl-rhodamine 116) Rhodamine 116 (458.8 mg, 1.0 mmol); N-diisopropylethylamine (129.3 mg, 1.0 mmol) and acetic anhydride (122
mg, 1.2 mmol) to 141 mg (as described in Example 25).
4%) of the title compound as a colorless solid.

[0212]

[Equation 41] (Example 38: N-dimethoxycarbamyl-rhodamine 110) Rhodamine 110 (1.0 mg, 2.73 mmol); From N-diisopropylethylamine (0.42 g, 3.27 mmol) and dimethylcarbamyl chloride (0.35 mg, 3.27 mmol) (as described in Example 25).
To give 10 mg (1%) of the title compound as a solid.

[0213]

(Equation 42) (Example 39: N-hexyloxycarbonyl-rhodamine 110) Rhodamine 110, diisopropylethylamine (0.24 mL, 2.52 m)
mol) and hexyl chloroformate (0.27 mL, 1.64 mmol)
From (as described in Example 25) gave 80 mg (13%) of the title compound as an orange solid.

[0214]

[Equation 43] Example 40: N-decyloxycarbonyl-rhodamine 110 The title compound was prepared as described in Example 25.

[0215]

[Equation 44] Example 41: N-dodecyloxycarbonyl-rhodamine 110 The title compound was prepared as described in Example 25.

[0216]

[Equation 45] (Example 42: N- [Ac-Asp (OBu-t) -Glu (OBu-t)
-Val-Asp (OBu-t)]-N'-hexyloxycarbonyl-rhodamine 110, SEQ ID NO: 5) Ac-Asp (OBu-t) -Glu (OBu-t) -Val-Asp (OB
ut) -OH, SEQ ID NO: 5 (263 mg, 0.383 mmol), EDC (7
4 mg, 0.39 mmol) and N-hexyloxycarbonyl-rhodamine 110 (44 mg, 0.096 mmol) (according to Example 1) gave the title compound as a white solid (30 mg, 28%).

[0217]

[Equation 46] (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.

[0218]

[Equation 47] (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.

[0219]

[Equation 48] (Example 45: N- (Ac-Asp-Glu-Val-Asp) -N'-hexyloxycarbonyl-rhodamine 110, SEQ ID NO: 5) 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) gave the title compound as a yellow solid (22 mg, 85%). .

[0220]

[Equation 49] Example 46: N- (Ac-Asp-Glu-Val-Asp) -N'-decyloxycarbonyl-rhodamine 110, SEQ ID NO: 5 The title compound was prepared as described in Example 45.

[0221]

[Equation 50] 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.

[0222]

(Equation 51) Example 48: N- (ethylthio) carbonyl-rhodamine 110) Rhodamine 11 dissolved in dimethylformamide (12 mL) at -61 ° C.
0 (500 mg, 1.36 mmol), N, N-diisopropylethylamine (264 mg, 2.64 mmol) was added, followed by acetic anhydride (204 mg).
, 1.64 mmol) was added dropwise to the above solution. Then, the reaction solution is
Warmed slowly to room temperature and maintained stirring for 1 hour. Then add 100ml
Diluted with ice water and extracted with ethyl acetate (3 × 30 mL). The organic phase was washed with brine (2 × 50 mL), dried over Na ₂ SO ₄ and concentrated to give the crude product, which was purified by chromatography (hexane / EtOAc 2: 1), 238 mg (42%) of the title compound were obtained as a solid.

[0223]

(Equation 52) (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.

[0224]

(Equation 53) (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.

[0225]

(Equation 54) Example 51: 2,5,8-trioxadecyl chloroformate Triethylene glycol monomethyl ether (2 g) in ether (15 ml)
, 12.2 mg) was added dropwise over 20 minutes to a stirred, ice-cold solution of 20% phosphogen in toluene (11.36 mL, 21.92 mmol). The reaction mixture was allowed to warm to room temperature and stirring was continued for 15 hours. Evaporation of the solvent gives 2.63 g (95 g) of the title compound.

[0226]

[Equation 55] Example 52: N- (2,5,8-trioxadecylcarbonyl) -rhodamine 110 Rhodamine 110 (500 mg, 1.36 mmol), N.D. From N-diisopropylethylamine (317 mg, 2.45 mmol) and 2,5,8-trioxadecyl chloroformate (371 mg, 1.64 mmol), 261 mg
(37%) of the title compound was obtained as a solid.

[0227]

[Equation 56] (Example 53: N- [Ac-Leu-Glu (OBu-t) -Val-As)
p (OBu-t)]-N'-ethoxycarbonyl-rhodamine 110, SEQ ID NO: 9) Ac-Leu-Glu (OBu-t) -Val-Asp (OBu-t) (18
9 mg, 0.3 mmol) SEQ ID NO: 9, EDC (57.5 mg, 0.3 mmol)
) And N-ethoxycarbonyl-rhodamine 110 (40 mg, 0.1 mmol)
From 1) (according to Example 1), 48 mg (47%) of the title compound were obtained as a solid.

[0228]

[Equation 57] 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.

[0229]

[Equation 58] (Example 55: N- (Cbz-Gly-Pro) -N'-ethoxycarbonyl-rhodamine 110) 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) to 68 mg (98%
) Was obtained as a solid.

[0230]

[Equation 59] (Example 56: N- (Gly-Pro) -N'-ethoxycarbonyl-rhodamine 110. HBr)

[0231]

[Equation 60] Example 57: 1-hexylchlorothiol formate 1-hexanethiol (3.72 g, 31.5 mm) in ether (15 ml)
ol) was added to a stirred ice-cold solution of 20% phosphogen in toluene (25 ml).
L, 47 mmol) over 20 minutes. The reaction mixture was allowed to warm to room temperature and stirring was continued for 15 hours. Evaporation of the solvent gave 6.1 g (98%) of the title compound.

[0232]

[Equation 61] Example 58: N- (hexylthio) carbonyl-rhodamine 110 The title compound was prepared according to Example 25.

[0233]

(Equation 62) Example 59: 2-butoxyethyl chloroformate From a solution of 2-butoxyethanol (3.72 g, 31.5 mmol) and 20% phosphogen (25 mL, 47 mmol) in toluene, 4.51 g (7
9%) of the title compound were obtained.

[0234]

[Equation 63] Example 60: N- (2-butoxyethoxycarbonyl) -rhodamine 11
0) The title compound was prepared according to Example 25.

[0235]

[Equation 64] (Example 61: N- [Cbz-Asp (OEt) -Glu (OEt) -Va
l-Asp (OEt)]-N'-ethoxycarbonyl-rhodamine 110, SEQ ID NO: 5) Cbz-Asp (OEt) -Glu (OEt) -Val-Asp (OEt),
SEQ ID NO: 5 (181 mg, 0.3 mmol), EDC (57.5 mg, 0.3 m
mol) and N-ethoxycarbonyl-rhodamine 110 (40 mg, 0.1
mmol) (according to Example 1) to give 71 mg (72%) of the title compound as a solid.

[0236]

[Equation 65] (Example 62: N- [Ac-Leu-Glu (OBu-t) -Val-As)
p (OBu-t)]-N'-octyloxycarbonyl-rhodamine 110, SEQ ID NO: 9) The title compound was prepared according to Example 1.

[0237]

[Equation 66] (Example 63: N- (Z-Gly) -N'-ethoxycarbonyl-rhodamine 110, SEQ ID NO: 9) Z-glycine (284 mg, 1.356 mmol), EDC (260 mg, 1
. 356 mmol) and N-ethoxycarbonyl-rhodamine 110 (58 m
g, 0.135 mmol) (according to Example 1) to give the title compound as a solid (70 mg, 83%).

[0238]

[Equation 67] (Example 64: N-Gly-N'-ethoxycarbonyl-rhodamine 110
. HBr) The title compound was prepared according to Example 6.

[0239]

[Equation 68] (Example 65: N- (Z-Gly-Pro-Gly) -N'-ethoxycarbonyl-rhodamine 110) 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) to give the title compound as a pale yellow solid (70 mg, 96%). .

[0240]

[Equation 69] (Example 66: N- (Gly-Pro-Gly) -N'-ethoxycarbonyl-rhodamine 110.HBr)

[0241]

[Equation 70] Example 67 N-hexyl-methyl carbamyl chloride To a solution of 0.35 ml of (i-Pr) ₂ NEt in 10 ml of diethyl ether at 0 ° C. was added phosphogen (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 stirred at 25 ° C for a further 14 hours. The mixture was filtered and the solvent was removed under reduced pressure. This product was used without further purification for the next step reaction.

[0242]

[Equation 71] Example 68: N- (N-hexyl-N-methylcarbamyl) -rhodamine 110) Rhodamine 110 (0.5 g, 1.36) in DMF (15 mL) at -61 ° C.
mmol) in N, N-diisopropylethylamine (0.25 ml) and N-hexyl-N-methylcarbamyl chloride (2.05 mmol) in DMF.
l) was added. The reaction mixture was stirred at −61 ° C. for 1 hour and then slowly warmed to room temperature. The reaction mixture was stirred for a further 14 hours and then partitioned between water and ethyl acetate saturated with NH ₄ Cl solution (2 × 50 ml). The organic phase was washed with brine (100 mL) and dried over Na ₂ SO ₄ . The solvent was removed and the crude product was flash chromatographed (hexane: EtOAc
= 1: 1). The title compound was obtained as a solid. (115 mg, 1
8%).

[0243]

[Equation 72] Example 69: N- (octylthio) carbonyl-rhodamine 110 The title compound was prepared according to Example 25.

[0244]

[Equation 73] Example 70: N- [Z-Gly] -N'-octylthiocarbonyl-rhodamine 110 The title compound was prepared according to Example 1.

[0245]

[Equation 74] (Example 71: N-Gly-N'-octylthiocarbonyl-rhodamine 1
10 HBr) The title compound was prepared according to Example 6.

[0246]

[Equation 75] (Example 72: N-acetyl-rhodamine 1 compared to rhodamine 110)
10 Fluorescence and Stability) The activity of rhodamine 110 and N-acetyl rhodamine 110 as fluorescent moieties for synthetic substrates was measured in a fluorometric assay. The fluorescent signal is read in a spectrofluorimeter or fluorimetric microtiter plate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Using this assay, the relative values of the fluorescence were determined for the two fluorescent moieties.

Fluorescence was measured using the following buffer conditions: 10% sucrose, 1% CHAP
S, 100 mM with 5 mM glutathione and 1-200 nM test compound
mM HEPES pH 7.5. Assays for stability are typically 37
C. for 2 days.

The ratio of the fluorescent signal was 10.2 at time zero (rhodamine 110 / N-acetyl rhodamine 110) and 1 after 2 days incubation at 37 ° C.
0.1. This result, similar to rhodamine 110, shows that N-acetyl-rhodamine 110 is stable and an efficient fluorescent indicator.

Example 73 Fluorescence of Modified Rhodamine Dye The modified rhodamine dye was evaluated using both conventional and spectrofluorimetric measurements. For both types of analysis, the dye was methanol or 50 mM Tri.
s at any final dye concentration ranging from 10 nM to 100 μM. 2
The absorbance spectrum from a wavelength of 00 nm to 700 nm was measured by Beckman DU-7.
Each dye was measured using a 000 spectrometer. The pigments are all about 470
It had an absorption peak at 480 nm. This wavelength was selected as the fluorescence excitation wavelength, and all fluorescence emission spectra were determined using a Hitachi F-2000 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: Fluorescence of modified rhodamine dye)

[0250]

[Table 3] Example 74 Uptake and Retention of Modified Rhodamine Dye by HL-60 Cells HL-60 cells were treated with 5 ml of 10 μM or 50 μM of N-octyloxycarbonyl rhodamine 110, N-decyloxycarbonyl rhodamine 110,
N-dodecyloxycarbonyl rhodamine 110, N-hexyloxycarbonyl rhodamine 110, N- (ethylthio) carbonyl rhodamine 110 or Iscove's medium containing rhodamine 110 (no serum or phenol red). The cells were incubated for various times at 37 ° C. in a CO ₂ incubator, harvested by centrifugation, and washed in 50 mL of ice-cold medium. The cells were recentrifuged and the final pellet was resuspended in 50 μL of fresh medium. Aliquots of each cell suspension were placed on μ slides and viewed on a Nikon inverted microscope equipped with epifluorescent illumination. As shown in FIGS. 1A-1F, N-octylcarbonyl-rhodamine 110 (FIG. 1A), N-decyloxycarbonyl-rhodamine 110 (FIG. 1B), and N-dodecyloxycarbonyl-rhodamine 110 (FIG. 1C) are HL -60 cells stained intensely and there was little dye leakage into the medium. N-hexyloxycarbonyl-
Rhodamine 110 (FIG. 1D) did not stain HL-60 cells very strongly, but still retained it well. N- (ethylthio) carbonyl-rhodamine 110
(FIG. 1E) gave moderate, but still easily detectable staining, but there was slight leakage. Rhodamine 110 (FIG. 1F) stained cells rapidly, but the dye quickly leaked out of the cells, resulting in low intensity cell staining and high fluorescence in the medium containing cells. Thus, the modified rhodamine dye is rhodamine 11
Better than 0. Because they are readily taken up by HL-60 cells and retained within the cells for at least 30 minutes.

Example 75 Enzyme Activity of Substrate N- (Z-VD) as a synthetic substrate for recombinant CPP32 and ICE
-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, activity of SEQ ID NO: 2 Was measured in a fluorescent colorimetric enzyme assay. Recombinant CPP32 and ICE proteins were prepared by expressing DNA clones encoding these enzymes in insect host cells (sf9 cells) using baculovirus as a vector. Webb, N.M. R. Et al., "Expr
session of protein using recombinant
Baculovirus "Technologies 2: 173-188 (199
See 0). Cleavage of the synthetic substrate by this enzyme results in a fluorescent signal that is read on a spectrofluorometer or fluorimetric microtiter plate reader. Using this assay, the K _m and V _max values determined for each substrate using either CPP32 or ICE.

CPP32 and ICE-dependent substrate cleavage was measured using the following buffer conditions: 10% sucrose, 1% CHAPS, 5 mM glutathione, and 1-1.
100 mM HEPES pH 7.5 with 00 μM test substrate. The non-specific enzymatic cleavage is carried out with the sequence As, each having an aldehyde group conjugated at the C-terminus.
Determined by the use of specific CPP32 and ICE inhibitors consisting of p-Glu-Val-Asp or oligomers having the sequence Tyr-Val-Ala-Asp. Assays for enzyme activity were typically performed at 37 ° C. for 60 minutes.

Table 4 shows that N- (Z-VD)-as a substrate for CPP32 and ICE.
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 listed K _m and V _max values.

Table 4: Substrate cleavage by CPP32 and ICE

[0255]

[Table 4] No activity is observed when NA = 1-100 μM substrate, incubation at 37 ° C. for 3 hours.

The results shown in Table 4 indicate that N- (Z-DEVD) -N′-acetyl-rhodamine 11
0, indicating that SEQ ID NO: 5 is an efficient substrate for both ICE and CPP32. Also shown is that N- (Z-VD) -N'-acetyl-rhodamine 110 is an efficient substrate for CPP32 but not for ICE, and N- (Z-VAD) -N ' -Acetyl-rhodamine 110, N-
(Z-YVAD) -N'-acetyl-rhodamine 110, SEQ ID NO: 2, (ZV
AD) ₂ -rhodamine 110 and (Z-YVAD) ₂ -rhodamine 110;
SEQ ID NO: 2 is an efficient substrate for ICE, but not for CPP32.

Example 76 Cleavage of Caspase-3 Substrate by Lysates of Recombinant Human Caspase-3 and Apoptotic HL-60 Cells Caspase substrates were assayed by lysates prepared from recombinant caspase-3 and apoptotic HL-60 cells. . The assay was performed at 37 ° C. in a 96-well plate with 30 μL of caspase 3 preparation or cell lysate, 10 μM
Or 50 μM substrate, and caspase assay buffer (40 mM 1,4-
Piperazine bis (ethanesulfonic acid) (PIPES, Aldrich Chem)
(Ical Company) pH 7.2, 100 mM NaCl; 10% sucrose; 0.1% CHAPS; 1 mM EDTA; 10 mM DTT) in 100 μl incubations. At the end of the incubation period, fluorescence was measured on a Bio-Tek FL500 fluorescence microplate reader using excitation and emission wavelengths of 485 nm and 530 nm, respectively. Two different controls: 1) an enzyme blank consisting of a sample containing the substrate but no enzyme or cell lysate; 2) caspase inhibitor A
An inhibitor control consisting of a sample containing c-DEVD-CHO (final concentration, 10 μM) was performed. Table 5 is a summary of the results obtained from these substrates.

(Table 5. Cleavage of substrate by caspase 3 and lysate)

[0259]

[Table 5] As shown in FIG. 2A, cleavage of the dipeptide substrate NZ-VD-N'-ethoxycarbonyl-R110 resulted in very high concentrations of recombinant caspase 3 (the amount of enzyme required for tripeptide and tetrapeptide substrates). 50 times). Even with this large amount of enzyme, the signal was low. In contrast, the tripeptide substrate NZ-EVD-N'-ethoxycarbonyl-R110 (FIG. 2A)
And all tetrapeptide substrates (FIGS. 2B-2L) were efficiently cleaved by caspase 3 and apoptotic lysates.

Example 77: Staining of apoptotic HL-60 cells with caspase 3 substrate, N-Ac-DEVD-N'-octyloxycarbonyl R110 N-Ac-DEVD-N'-octyloxycarbonyl-R110 SEQ ID NO: 5 detect the ability to detect caspase activation in intact cells.
Tested using L-60 and Jurkat cells. These whole cell assays were performed in two stages: 1) induction of apoptosis; 2) incubation with substrate. For HL-60 cells, apoptosis was induced by treatment with 10 μg / ml vinblastine for 4 hours. D for control sample
Treated with MSO. For Jurkat cells, 500n apoptosis
Induction was achieved by treatment with g / ml of agonistic anti-Fas antibody for 2 hours.
Control samples were treated with PBS. Following induction of apoptosis, the cells were combined with 50 μM of N-Ac-DEVD-N′-octyloxycarbonyl-R110 SEQ ID NO: 5 in caspase assay buffer (40 mM P
IPES, pH 7.4, 100 mM NaCl; 10% sucrose; 1 mM
(EDTA; 10 mM DTT). The cells were then transferred to glass microslides and epifluorescence irradiation was viewed on a Nikon inverted microscope. As shown in FIG. 3A, vinblastine-treated HL-60 cells were N-
Ac-DEVD-N'-octyloxycarbonyl-R110 Intensely stained with SEQ ID NO: 5. DMSO treated cells also showed some staining (
FIG. 3B), signal intensity was significantly lower than vinblastine treated cells.
HL-60 cells treated with 50 μM Ac-DEVD-CHO SEQ ID NO: 5 during the assay phase (FIG. 3C) showed little fluorescent signal.
This indicates that the staining observed in vinblastine-treated cells is almost entirely due to caspase-mediated cleavage. Jurkat cells induced to undergo apoptosis by anti-Fas (FIG. 3D) also show N-Ac
-DEVD-N'-octyloxycarbonyl-R110 showed intense staining with SEQ ID NO: 5, whereas control cells showed only mild staining (Figure 3F).
. These experiments were performed with N-Ac-DEVD-N'-octyloxycarbonyl-R
110 SEQ ID NO: 5 can be used to measure apoptosis in intact cells, and the signal obtained from N-Ac-DEVD-N'-octyloxycarbonyl-R110 SEQ ID NO: 5 is caspase-dependent Demonstrate that.

Example 78 Cleavage of Caspase 3 Substrate, N-Ac-DEVD-N'-octyloxycarbonyl-R110 SEQ ID NO: 5, by Whole Apoptotic Jurkat Cells N-Ac-DEVD-N'-octyloxy To quantify the whole-cell cleavage of carbonyl-R110 SEQ ID NO: 5, an assay was performed in which the fluorescent signal generated from this substrate by apoptotic Jurkat cells was measured on a spectrofluorescent plate reader. Jurkat cells were incubated with 500 ng / ml anti-Fas antibody in 96-well plates for different times to induce apoptosis. Control cells were incubated with PBS. At the end of the treatment period, the cells were harvested, centrifuged in 1.5 ml tubes and resuspended in 25 μL of medium containing 1% FBS. 50 μM N
-Ac-DEVD-N'-octyloxycarbonyl-R110 25 μL of caspase buffer containing SEQ ID NO: 5 was added and the cells were incubated for 1 hour. At the end of the incubation period, three 5 μL aliquots from each time point were placed in a 96-well plate and the fluorescence was set to 485/530 nm.
At the excitation / emission wavelength of FIG. 4 shows that cells treated with PBS produced a small fluorescent signal that did not increase over the treatment time used.
However, cells treated with anti-Fas produced a detectable fluorescent signal approximately 1 hour after apoptosis induction, and this signal had a signal to background ratio of approximately 7 and was not detected until 2 hours. Continued to increase. In this experiment, N-Ac-
DEVD-N'-octyloxycarbonyl-R110 SEQ ID NO: 5 produces a strong signal throughout apoptotic cells, and thus N-Ac-DEVD-N
It demonstrates that '-octyloxycarbonyl-R110 SEQ ID NO: 5 can be used for quantitative measurement of caspase-mediated apoptosis in a cell-based assay.

Example 79: Caspase 8 substrate, N-Ac-LEVD-N'-ethoxycarbonyl R110 SEQ ID NO: 9, recombinant human caspases 3, 6, 7, and 8
Cleavage by) N-Ac-LEVD-N'-ethoxycarbonyl R110 SEQ ID NO: 9 was assayed by recombinant human caspases 3, 6, 7, and 8. The assay was performed at 37 ° C. in 96-well plates with recombinant human caspase, 10 μM.
N-Ac-LEVD-N'-ethoxycarbonyl R110 SEQ ID NO: 9, and caspase assay buffer (40 mM PIPES, pH 7.2, 100 mM
NaCl; 10% sucrose; 0.1% CHAPS; 1 mM EDTA;
Performed in a 100 μL incubation containing 10 mM DTT). At the end of the incubation period, fluorescence was measured on a Bio-Tek FL500 fluorescence microplate reader using excitation and emission wavelengths of 485 nm and 530 nm, respectively. To correct for endogenous fluorescence of the uncleaved substrate, a control consisting of a sample containing 10 μM N-Ac-LEVD-N′-ethoxycarbonyl R110 SEQ ID NO: 9 without enzyme (“enzyme blank”) was used. Ran. A further control is the caspase inhibitor Ac-
A sample containing DEVD-CHO SEQ ID NO: 5 was included. As shown in FIG. 5, caspase 6 and caspase 8 cleave N-Ac-LEVD-N′-ethoxycarbonyl R110 SEQ ID NO: 9 to produce a readily measured fluorescent signal (about 13 fold for caspase 6). , Signal to background ratio of about 26 for caspase 8). Caspase 3 is N-Ac-LEVD-N
'-Ethoxycarbonyl R110 SEQ ID NO: 9 was cleaved less efficiently, resulting in a signal that was approximately 5-fold above the value of the enzyme blank. Caspase 7 produced virtually no signal. These experiments show that N-Ac-LEVD-N'-ethoxycarbonyl R110 SEQ ID NO: 9 can be cleaved by members of the caspase 8 subfamily, and that N-Ac-LEVD-N'-ethoxycarbonyl R110
110 shows that SEQ ID NO: 9 can be used to report the activity of this type of caspase.

Example 80 Aminopeptidase substrate NG with HL-60 cell lysate
Cleavage of -N'-octyloxycarbonyl-R110 Aminopeptidases are present in many cells and continuously remove unblocked amino acid residues from peptides starting at the N-terminus. Peptides with blocked amino termini are not cleaved. HL-60 lysates were prepared by homogenizing HL-60 cells in caspase buffer, and these lysates were NZGN-octyloxycarbonyl-R110 and NGN
The ability to cleave '-octyloxycarbonyl-R110 was tested in a microtiter plate assay. FIG. 6 shows that HL-60 cell lysates readily cleaved NGN′-octyloxycarbonyl-R110 and that the size of the signal was dependent on the concentration of the substrate. In contrast, no signal was produced from NZGN'-octyloxycarbonyl-R110 by HL-60 cell lysate.

Example 81 Use of a Fluorescent Assay in the Screening of Drugs That Stimulate the Caspase Cascade The drugs that stimulate the caspase cascade in the absence of Fas ligand are:
For example, it may be useful as an anti-cancer chemotherapeutic agent. Similar conditions as in Example 78, except that the Fas ligand reagent is replaced with a known or unknown compound having known or unknown anti-cancer or anti-tumor activity using the assay described in Example 78. By performing the assays below, one can screen for drugs that stimulate the caspase cascade.

Example 82 Use of Fluorescent Assays in Screening for Drugs that Inhibit or Enhance the Caspase Cascade Stimulated with Fas Ligand or Another Apoptosis Inducer Drugs that inhibit the caspase cascade include It may be useful in the treatment of degenerative and other diseases caused by or associated with inappropriate activation. Drugs that enhance the action of other caspase stimulating factors (eg, Fas ligand or anti-cancer drug or anti-cancer agent) treat cancer or tumors caused by or associated with improper functioning of the caspase cascade. May be appropriate for The assays and reagents described in the present invention can be used to screen for drugs that either inhibit or enhance the caspase cascade in cells. This screen inhibits the assay described in Example 78 using Fas ligand or any other agent that stimulates the caspase cascade or other apoptotic pathway by inhibiting the action of an inducer of the first apoptotic cascade or caspase cascade. Or in the presence of a test substance that acts, enhances or acts synergistically.

Example 83 Use of Fluorescence Assay in Testing Sample Cancer Cells from Patients for Chemosensitivity to Anticancer Drugs The same cancer in different patients shows significant individual differences to treatment with anticancer drugs. The indications are well known. Thus, it is very difficult to predict whether a cancer in a patient will be treatable with a particular anticancer drug before the start of treatment. The fluorescent assay according to the present invention makes it possible to test chemosensitivity or drug resistance of cancer or tumor cells or tissue samples taken from individual cancer or tumor patients. To perform a chemosensitivity test, a fluorescence assay using cancer cell or tissue samples taken from a patient may be performed as described in Example 78. Using this approach, different drugs with known or unknown chemotherapeutic activity can be tested for their ability to stimulate the caspase cascade. The results of this assay provide information that can be used to design the optimal chemotherapeutic drug treatment regime for the patient.

Example 84: Uptake and cleavage of aminopeptidase substrate NGN'-octyloxycarbonyl-R110 by HL-60 cells. HL-60 cells are treated with 5 ml of Iscove's medium (without serum or phenol red). ) Wherein 10 μM of NGN′-octyloxycarbonyl-R11
It was placed in a medium containing 0 or 10 μM NZGN-octyloxycarbonyl-R110. Three million HL-60 cells were incubated for 3 hours at 37 ° C. in a CO ₂ incubator, harvested by centrifugation, and
Washed in μL ice-cold medium. The cells were centrifuged again and the final pellet was resuspended in 50 μL of fresh medium. An aliquot of each cell suspension was placed in a microtiter 96-well plate and an excitation wavelength of 485 nm,
Reading was performed on a Wallac 1420 microplate reader using an emission wavelength of 525 nm. Aliquots of each cell suspension were also placed on microslides and viewed on a Nikon inverted microscope using epi-fluorescence irradiation. As shown in Table 6, only cells incubated with 10 μM NGN′-octyloxycarbonyl-R110 showed a signal. NZGN '
There was no signal from cells incubated with -octyloxycarbonyl-R110. Similarly, NGN'-octyloxycarbonyl-
Only cells incubated with R110 show fluorescence under the microscope,
And no fluorescent signal was observed from the cells incubated with NZGN'-octyloxycarbonyl-R110 (FIGS. 7A-B).

(Table 6, NGN'-octyloxycarbonyl- by HL-60 cells
R110 cutting)

[0269]

[Table 6] Having fully described the invention, those skilled in the art will appreciate that they do not impair the scope of the invention or any of its embodiments, but within the broad and equivalent scope of conditions, formulations and other parameters. It is understood that the present invention can be implemented. All patents, patent applications, and publications cited herein are fully incorporated by reference in their entirety.

[Sequence list]

[Brief description of the drawings]

1A-1F show N-octyloxycarbonyl-rhodamine 110 (FIG. 1A).
), N-decyloxycarbonyl-rhodamine 110 (FIG. 1B), N-dodecyloxycarbonyl-rhodamine 110 (FIG. 1C), N-hexyloxycarbonyl-rhodamine 110 (FIG. 1D), N- (ethylthio) carbonyl-rhodamine 110 (FIG. 1E), and HL- stained with rhodamine 110 (FIG. 1F).
A photograph of 60 cells is shown.

FIGS. 2A-2L show caspase substrates, NZ-VD-N′-ethoxycarbonyl-R110, NZ-EVD-N′-ethoxycarbonyl-R110, N
-Z-DEVD-N'-ethoxycarbonyl-R110 SEQ ID NO: 5, N-Ac
-DEVD-N'-ethoxycarbonyl-R110 SEQ ID NO: 5, N-Ac-D
EVD-N'-octyloxycarbonyl-R110 SEQ ID NO: 5, N-Ac-
DEVD-N'-hexyloxycarbonyl-R110 SEQ ID NO: 5, and N
-Z-DEVD-N '-(ethylthio) carbonyl-R110 of SEQ ID NO: 5,
Cleavage by r-caspase 3 (FIGS. 2A, 2B, 2D, 2G, and 2J) and vinblastine-treated HL-60 cell lysates (FIGS. 2C, 2E, 2H, and 2K) was compared with HL-60 control (DMSO treatment). Figure 4 shows a bar graph compared to lysates (Figures 2F, 21 and 2L).

FIGS. 3A-3E show N-Ac-DEVD-N′-octyloxycarbonyl-R.
110 shows a photograph of cells stained by incubation with SEQ ID NO: 5. HL-60 cells treated with vinblastine (FIG. 3A) and DMSO (FIG. 3B), HL-60 cells treated with vinblastine and added N-Ac-DEVD-CHO SEQ ID NO: 5 (FIG. 3C), anti-Fas ( 3D) and PBS (FIG. 3E).
) Treated Jurkat cells.

FIG. 4 shows N-Ac-DEVD-N′-octyloxycarbonyl-R110.
Figure 7 shows a graph showing the results of a cleavage assay with Jurkat cells treated with anti-Fas of SEQ ID NO: 5 and PBS.

FIG. 5 shows a bar graph showing the results of a cleavage assay with N-Ac-LEVD-N′-ethoxycarbonyl-R110 SEQ ID NO: 5 by caspases 3, 6, 7, and 8.

FIG. 6 shows NZGN′-octyloxycarbonyl-R110 and N-
Figure 5 shows a bar graph showing the results of a cleavage assay of GN'-octyloxycarbonyl-R110 with HL-60 cell lysates.

FIGS. 7A-B show NZGN′-octyloxycarbonyl-R110 (A
) And HL-60 cells treated with NGN'-octyloxycarbonyl-R110 (B).

[Procedure amendment]

[Submission date] October 13, 2000 (2000.10.13)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Embedded image A compound having the formula:

Embedded image Alternatively, a biologically acceptable salt or proreporter molecule thereof, wherein R ₂ and R ₃ are independently hydrogen, methyl or ethyl, and R ₄ and R ₅ are independently Or a compound or a biologically acceptable salt or proreporter molecule thereof.

Embedded image Alternatively, a biologically acceptable salt or proreporter molecule thereof, wherein R ₁ is hydrogen or an N-terminal protecting group; R ₆ is a blocking group that is not an amino acid or a derivative of an amino acid ; each AA is an independently residues of α-amino acids or β amino acids or α-amino acids or β amino acid derivative,; n is an integer from 0 to 5; m is an integer of 0 to 3; R ₂ And R ₃ is independently hydrogen, methyl or ethyl, and R ₄ and R ₅ are independently hydrogen or methyl, or a biologically acceptable salt or proreporter molecule thereof.

Embedded image Or a biologically acceptable salt thereof, wherein R ₂ and R ₃ are independently hydrogen, methyl or ethyl; R ₄ and R ₅ are independently hydrogen or methyl A compound, or a biologically acceptable salt thereof, wherein R ₆ is a blocking group that is not an amino acid or a derivative of an amino acid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/15 G01N 33/15 Z 33/50 33/50 Z 33/574 33/574 A (81) Designation Country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG) , CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, D K, E, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, U Z, VN, YU, ZW (72) Inventor Cana, John F. W. Oregon 07405, Eugene, Onyx Street 3854 (72) Inventor Drew, John A. United States of America 92627, Costa Mesa, Pacific Ave. Irvine, Parkview Lane number 336 3800

Claims (90)

[Claims] 1. A reporter compound having the general formula II: R _1- (AA) _n -Asp-y-Asp- (AA) _n -R ₁ (II), or a biologically acceptable compound thereof. A salt or proreporter molecule, wherein R ₁ 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; And each n is independently 0-5; and y is a fluorogenic or fluorescent moiety, or a compound, or a biologically acceptable salt or proreporter molecule thereof. 2. The compound of claim 1, wherein R _1- (AA) _n -Asp is an N-blocked tetrapeptide that is a substrate for a caspase enzyme.
Compound. 3. The compound of claim 2, wherein 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, VE
HD 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
A compound which is SEQ ID NO: 26 or IETD SEQ ID NO: 24. 4. The compound of claim 1, wherein R _1- (AA) _n -Asp is an N-blocked tetrapeptide that is a substrate for granzyme B.
Compound. 5. The compound according to claim 4, wherein R _1- (AA) _n -Asp is IEPD or VEPD. 6. The compound of claim 1, wherein R _1- (AA) _n -Asp is a C-terminal Asp and 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, LEH
D 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
A compound which is an N-blocked peptide consisting of one, two or three amino acids of a peptide chain selected from the group consisting of SEQ ID NO: 23 and VEPD SEQ ID NO: 27. 7. The compound of claim 1, wherein y is rhodamine 110; and wherein the proreporter molecule is a low alkyl or acetoxymethyl (AM) ester of an Asp or Glu containing compound. A compound. 8. The compound according to claim 1, wherein the compound is of the formula III: A compound having the formula: 9. The compound according to claim 8, wherein R ₁ is t-butyloxycarbonyl, acetyl, hexanoyl, octanoyl or benzyloxycarbonyl. 10. The compound of claim 8, wherein-(AA) _n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SH.
V, DEL, DGP, DEP, DGT, DLN, DEE, DSL, DVP, DE
A, A, DSY, ELP, VED, IEP or IET. 11. The compound according to claim 1, wherein the compound comprises: (Z-YVAD) ₂ -rhodamine 110, SEQ ID NO: 2; (Z-DEVD) ₂ -rhodamine 110, SEQ ID NO: 5; (Z-VAD) 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; (D (OAM) E (OAM) TD (OAM)) ₂ -rhodamine 110, SEQ ID NO: 4; (ZD (OAM) E (OAM) VD (OAM)) ₂ -rhodamine 110, SEQ ID NO: 5; ZD (OMe) E (OMe) VD (OAM)) ₂ -rhodamine 110, SEQ ID NO: 5; and (ZD (OMe) E (OMe) VD) ₂ -rhodamine 110, SEQ ID NO: 5 Group Selected the compound. 12. A method for preparing a compound according to claim 8, comprising the following steps: (a) condensing rhodamine 110 with N-fmoc-L-aspartic acid β-t-butyl ester; (Fmoc-Asp (OBu-t )) 2 - step generates a Rodami emissions 110; (b) removing the Fmoc group (Asp (OBu-t)) 2 - step generates the rhodamine 110; (c) ( Asp (OBu-t)) ₂ -rhodamine is condensed with Z- (AA) _n to give (
Producing Z- (AA) _n -Asp (OBu-t)) ₂ -rhodamine 110;
And (d) removing the OBu-t protecting group. 13. The method of claim 12, wherein-(AA) _n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SH.
V, DEL, DGP, DEP, DGT, DLN, DEE, DSL, DVP, DE
A, A, DSY, ELP, VED, IEP or IET. 14. A reporter compound having the general formula V: R _1- (AA) _n -Asp-y-R ₆ (V), or a biologically acceptable salt or proreporter molecule thereof. Wherein, R ₁ is an N-terminal protecting group; R ₆ is a blocking group that 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 α And n is 0-5; and y is a fluorogenic or fluorescent moiety. Or a biologically acceptable salt or proreporter molecule thereof. 15. The compound according to claim 14, wherein R _1- (AA) _n -Asp is an N-blocked tetrapeptide which is a substrate for a caspase enzyme. 16. The compound of claim 15, wherein the 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, SHV
D SEQ ID NO: 10, DELD SEQ ID NO: 11, DGPD SEQ ID NO: 12, DEP
D SEQ ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEE
D SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEA
D SEQ ID NO: 19, DSYD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEI
D The compound which is SEQ ID NO: 26 or IETD SEQ ID NO: 24. 17. The compound of claim 14, wherein R _1- (AA) _n -Asp is an N-blocked tetrapeptide that is a substrate for granzyme B. 18. The compound according to claim 17, wherein R _1- (AA) _n -Asp is N-terminal protected IEPD or VEPD. 19. The compound according to claim 14, wherein R _1- (AA) _n -Asp is a C-terminal Asp, and 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, DE
LD SEQ ID NO: 11, DGPD SEQ ID NO: 12, DEPD SEQ ID NO: 13, DG
TD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DS
LD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DS
Consisting of 1, 2 or 3 amino acids of a peptide chain selected from the group consisting of YD SEQ ID NO: 20, ELPD SEQ ID NO: 21, VEID SEQ ID NO: 26, or IETD SEQ ID NO: 24, IEPD SEQ ID NO: 23, and VEPD SEQ ID NO: 27; A compound that is an N-blocked peptide. 20. The compound of claim 14, wherein y is rhodamine 110. 21. The compound according to claim 14, wherein R ₆ is CH ₃ OCO—, Cbz, Cl ₃ CCH ₂ OCO—, PhCH ₂ CH ₂ OCO—, or CH ₃ ( CH ₂₎ a _p OCO-, is wherein p is 1 to 11, compounds. 22. The compound according to claim 14, wherein R ₆ is Me ₂ NCO—, Et ₂ NCO—, or N-Me—N—CH ₃ (CH ₂ ) _v NCO—. Wherein v is 0-9. 23. The compound according to claim 14, wherein R ₆ is T s-, PhSO _2- , MeSO ₂ , PhCH ₂ SO _2- , CF ₃ SO ₂ -or CH ₃ (CH _{3 2} ) compounds wherein _u SO ₂ —, wherein u is 0-11 24. A compound according to claim 14, wherein, R ₆ is a C H ₃ SCO-or _{CH 3 (CH 2) t SCO-} , wherein t is at 0-11 There is a compound. 25. The compound according to claim 14, wherein R ₆ is H CO—, CH ₃ CO—, PhCH ₂ CO—, PhCO— or CH ₃ (CH ₂ ) _w CO—. Wherein w is 0-11. 26. The compound according to claim 14, wherein R ₆ is CH ₃ (OCH ₂ CH ₂ ) _q OCO— or CH ₃ (CH ₂ ) _r (OCH ₂ CH ₂ ). _s OC
O-, wherein q is 1-4, r is 0-5, and s is 1-4. 27. The compound according to claim 14, which has the following formula VII: Alternatively, a biologically acceptable salt or proreporter molecule thereof, wherein R ₂ and R ₃ are independently hydrogen, methyl or ethyl, and R ₄ and R ₅ are independently Or a compound or a biologically acceptable salt or proreporter molecule thereof. 28. The compound according to claim 27, wherein R ₁ is t-butyloxycarbonyl, acetyl, hexanoyl, octanoyl or benzyloxycarbonyl; and R ₂ , R ₃ , R ₄ And R ₅ is hydrogen. 29. The compound of claim 27, wherein-(AA) _n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, LE.
V, SHV, DEL, DGP, DEP, DGT, DLN, DEE, DSL, DV
A compound which is P, DEA, DSY, ELP, VED, IEP or IET. 30. The compound according to claim 14, wherein the compound is: N- (Z-YVAD) -N'-acetyl-rhodamine 110, SEQ ID NO: 2; N- (Z-DEVD) N- (Z-VD) -N'-acetyl-rhodamine 110; N- (Z-AD) -N'-acetyl-rhodamine 110; 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, sequence No. 5; N- (Ac-DEVD) -N′-hexyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 5; N- (Ac-DEVD) -N′-octyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 5; N- (Ac-DEVD) -N'-decyloxycarbonyl-rhodamine 110
N- (Ac-DEVD) -N'-dodecyloxycarbonyl-rhodamine 11
0, SEQ ID NO: 5; and N- (Ac-DEVD) -N '-(ethylthio) carbonyl-rhodamine 11
0, SEQ ID NO: 5, a compound selected from the group consisting of: 31. A method for preparing the compound of claim 27, comprising the following steps: (a) reacting rhodamine with acetic anhydride to produce N-acetyl-rhodamine; (b) N-acetyl-rhodamine is converted to N-fmoc-L-aspartic acid β-t.
-N- (Fmoc-Asp (OBu-t))
A step of producing -N'-acetyl-rhodamine; (c) removing the Fmoc group to give N- (Asp (OBu-t))-N'-acetyl-
(D) N- (Asp (OBu-t))-N′-acetyl-rhodamine is converted to Z- (A
A) condensing with _n to produce N- (Z- (AA) _n -Asp (OBu-t))-N′-acetyl-rhodamine; and (e) removing the OBu-t protecting group Producing N- (Z- (AA) _n -Asp) -N′-acetyl-rhodamine. 32. A method for preparing a compound according to claim 27, comprising the following steps: (a) reacting rhodamine with acetic anhydride to produce N-acetyl-rhodamine; (b) N-acetyl-rhodamine is condensed with Z- (AA) _n -Asp (OBu-t) to give N- (Z- (AA) _n -Asp (OBu-t))-N′-acetyl- Producing rhodamine; and (c) removing the OBu-t protecting group to produce N- (Z- (AA) _n- Asp) -N'-acetyl-rhodamine. 33. The method according to claim 31 or 32, wherein-
(AA) _n is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, LEV, SHV, DEL, DGP, DEP, DGT, DLN, DEE, D
SL, DVP, DEA, DSY, ELP, VED, IEP or IET,
Method. 34. A compound according to claim 1 having the following formula IX: Alternatively, a biologically acceptable salt or proreporter molecule thereof, wherein R ₁ is hydrogen or an N-terminal protecting group; R ₆ is a blocking group that is not an amino acid or a derivative of an amino acid ; each AA is an independently residues of α-amino acids or β amino acids or α-amino acids or β amino acid derivative,; n is an integer from 0 to 5; m is an integer of 0 to 3; R ₂ And R ₃ is independently hydrogen, methyl or ethyl, and R ₄ and R ₅ are independently hydrogen or methyl, or a biologically acceptable salt or proreporter molecule thereof. 35. The compound according to claim 34, wherein R ₂ , R ₃ ,
The compound wherein R ₄ and R ₅ are hydrogen. 36. The compound of claim 34, wherein R ₁ is H; n = 1; (AA) _m is M; m is an integer of 1-2. And (AA) _n is selected from the group consisting of GG, GA, AG, G, and A. 37. The compound of claim 34, wherein said compound is:
Hereinafter, a group consisting of N- (GP) -N'-ethoxycarbonyl-rhodamine 110, N- (GPG) -N'-ethoxycarbonyl-rhodamine 110, and N-GN'-octyloxycarbonyl-rhodamine 110 A compound selected from: 38. The compound of claim 34, wherein R ₇ is an N-terminal protecting group selected from the group consisting of t-butyloxycarbonyl, acetyl, hexanoyl, octanoyl, dodecanoyl, and benzyloxycarbonyl. A compound. 39. The compound according to claim 34, wherein (AA) _n- (AA) _m is SQNY-PIV SEQ ID NO: 28, ARVL-AEA SEQ ID NO: 29, ATIM-MQR SEQ 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-F
LD SEQ ID NO: 36; SQNY-PI SEQ ID NO: 117, ARVL-AE SEQ ID NO: 118, ATIM-MQ SEQ ID NO: 119, RQAN-FL SEQ ID NO: 12
0, PGNF-LQ SEQ ID NO: 121, SFSF-PQ SEQ ID NO: 122, TL
NF-PI SEQ ID NO: 123, AETF-YV SEQ ID NO: 124 or RKVL
-FL SEQ ID NO: 125; SQNY-P SEQ ID NO: 126, ARVL-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
A compound selected from the group consisting of SEQ ID NO: 132, AETF-Y SEQ ID NO: 133 and RKVL-F SEQ ID NO: 134. 40. The compound according to claim 34, wherein (AA) _n- (AA) _m is 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,
A compound selected from the group consisting of IVGGGG SEQ ID NO: 100, IVGGA SEQ ID NO: 139, LRGG SEQ ID NO: 55, MRGG SEQ ID NO: 56, IRGG SEQ ID NO: 57, LVGG SEQ ID NO: 58, MVGG SEQ ID NO: 59 and IVGG SEQ ID NO: 60. 41. A compound having the following general formula VI: Or a biologically acceptable salt thereof, wherein R ₂ and R ₃ are independently hydrogen, methyl or ethyl; R ₄ and R ₅ are independently hydrogen or methyl A compound, or a biologically acceptable salt thereof, wherein R ₆ is a blocking group that is not an amino acid or a derivative of an amino acid. 42. The compound according to claim 41, wherein R ₂ , R ₃ ,
The compound wherein R ₄ and R ₅ are hydrogen. 43. The compound of claim 41, wherein R ₂ and R ₃ are methyl; and R ₄ and R ₅ are hydrogen. 44. The compound of claim 41, wherein R ₂ and R ₃ are ethyl; and R ₄ and R ₅ are methyl. 45. The compound according to claim 41, wherein the compound comprises: N-methoxycarbonyl-rhodamine 110, N-ethoxycarbonyl-rhodamine 110, N-hexyloxycarbonyl-rhodamine 110, A compound selected from the group consisting of N-octyloxycarbonyl-rhodamine 110, N-decyloxycarbonyl-rhodamine 110, and N-dodecyloxycarbonyl-rhodamine 110. 46. The compound according to claim 41, wherein the compound comprises: 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-rhodamine 110, N-acetyl- Rhodamine 116, N- (2,5,8-trioxadecyloxycarbonyl) -rhodamine 110
And N- (2-butoxyethoxycarbonyl) -rhodamine 110, a compound selected from the group consisting of: 47. A method for detecting an enzyme involved in the apoptosis cascade in one or more cells, comprising the steps of: (a) converting the one or more cells into a reporter according to claim 1 or 14; A compound;
Contacting the reporter compound under conditions that allow it to be taken up by the one or more cells; and (b) recording the fluorescence of the one or more cells, wherein the contact is not so performed. Wherein a change in fluorescence in the one or more cells compared to control cells is indicative of the presence of the enzyme. 48. The method of claim 47, wherein said enzyme is an intracellular caspase. 49. A method for measuring the activity of an enzyme involved in the apoptosis cascade in one or more cells, comprising the steps of: (a) transferring the one or more cells according to claim 1 or 14; A reporter compound;
Contacting the reporter compound under conditions that allow it to be taken up by the one or more cells; and (b) recording the fluorescence of the one or more cells, wherein the contact is not so performed. Wherein the relative change in fluorescence in the one or more cells as compared to control cells is a measure of the activity of the enzyme. 50. The method of claim 49, wherein said enzyme is an intracellular caspase. 51. A method for determining whether a test substance has an effect on an enzyme involved in an apoptosis cascade in one or more test cells, comprising: (a) the one or more test cells And the reporter compound according to claim 1 or 14, and the test substance either interacts with an external receptor or is taken up by the one or more cells, and the reporter compound Contacting the compound under conditions that allow the compound to be taken up by the one or more cells; and (b) recording the fluorescence of the one or more test cells compared to control cells contacted with the reporter compound alone. Here, a change in fluorescence in the one or more test cells as compared to the control cells indicates that the test substance has an effect on the enzyme. It encompasses a target, process, and methods. 52. The method of claim 51, wherein said enzyme is an intracellular caspase. 53. The method of claim 51, wherein said one or more test cells are contacted with said test substance prior to contacting said test cells with said reporter compound. 54. The method of claim 51, wherein said one or more test cells are contacted with said reporter compound prior to contacting said test substance. 55. The method of claim 51, wherein the one or more test cells are contacted with the test substance and the reporter compound substantially simultaneously. 56. The method of claim 51, wherein said method determines whether said test substance stimulates the activity of said enzyme. 57. The method of claim 51, wherein said method determines whether said test substance inhibits the activity of said enzyme. 58. The method of claim 51, wherein said contacting causes said one or more test cells to undergo at least one second test cell in the presence of said first test substance. The method further comprising contacting the test substance with a test substance. 59. The method of claim 51, wherein said one or more test cells are from a single cell organism. 60. The method of claim 51, wherein said one or more test cells are from a multicellular organism. 61. The method of claim 60, wherein said multicellular organism is selected from the group consisting of mammals, invertebrates, insects and plants. 62. The method according to claim 51, wherein said one or more test cells and control cells comprise the hair, brain, peripheral nervous system of said multicellular organism.
Eyes, ears, nose, mouth, tonsils, teeth, esophagus, lungs, chest, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and endocrine tissues,
A method derived from the group consisting of kidney, bladder, reproductive organs or gonads, joints, bone and skin. 63. The method of claim 51, wherein said one or more test cells are cancerous. 64. The method of claim 63, wherein the one or more cancerous test cells are the brain, peripheral nervous system, eye, ear, nose, mouth, tonsil of the multicellular organism. , Teeth, esophagus, lung, breast, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine and endocrine tissues, kidney, bladder, reproductive organs or gonads, A method derived from the group consisting of joints, bones and skin. 65. The method of claim 63, wherein the one or more cancerous test cells are from a human in need of treatment with a chemotherapeutic drug, and the test substance is a chemical. A method that is a therapeutic agent. 66. The method of claim 51, wherein said test substance is a chemotherapeutic agent. 67. The method of claim 51, wherein the test substance is a mixture of chemotherapeutic agents. 68. A method for determining the susceptibility of an animal having a cancer to treatment with one or more chemotherapeutic agents, comprising the steps of: (a) removing cancer cells from the animal; The one or more chemotherapeutic agents and the reporter compound of claim 1 or 14 and the conditions under which the one or more agents interact with external receptors or are taken up by the cancer cells. Contacting, and (b) recording the fluorescence of the cancer cells as compared to control cells contacted only with the reporter compound, wherein the fluorescence was compared to the control cells.
Wherein a change in the fluorescence of the cancer cells is an indication that the cancer cells are chemosensitive to the one or more chemotherapeutic agents and that the animal is susceptible to the treatment. A method comprising: 69. The method of claim 68, wherein said animal is a human. 70. A method of monitoring treatment of an animal for treatment with one or more chemotherapeutic agents, wherein the method comprises the steps of: (a) replacing the one or more chemotherapeutic agents with: (B) contacting cells collected from the animal after the administration with the reporter compound according to claim 1 or 14 under conditions in which the reporter compound is incorporated into the cells; And (c) recording the fluorescence of the cells contacted with the reporter compound, as compared to control cells taken from the animal prior to the administration, wherein the animals are compared to the control cells. Wherein a change in the fluorescence of the cells collected from the animal is indicative of the susceptibility of the animal to the chemotherapeutic agent. 71. The method of claim 70, wherein said animal comprises:
Suffering from a disease associated with apoptotic cell death. 72. A method for determining whether a test substance inhibits or prevents cell death in one or more test cells, the method comprising the steps of: (a) the one or more test cells Is contacted with the test substance and the reporter compound according to claim 1 or 14 under conditions in which the reporter compound is taken up by the one or more cells, and (b) a control contacting only with the reporter compound. Recording the fluorescence of the one or more test cells as compared to a cell, wherein a decrease in the fluorescence of the one or more test cells as compared to the control cell indicates that the test substance is a cell. A step that is an indicator of inhibiting or preventing death. 73. The method of claim 72, wherein said one or more test cells are neurons. 74. The method of claim 72, wherein said one or more test cells produces cardiomyocytes, immune cells, cells of an organ to be transplanted, sperm, eggs, recombinant protein. A cell line, a hair cell, a skin cell, and a nerve cell. 75. A method for determining whether a test substance causes or enhances cell death in one or more test cells, the method comprising the steps of: (a) replacing the one or more test cells with: Contacting the test substance with the reporter compound according to claim 1 or 14 under conditions in which the reporter compound is taken up by the one or more cells; and (b) a control cell contacted only with the reporter compound. Recording the fluorescence of the one or more test cells in comparison, wherein the increase in fluorescence in the one or more test cells as compared to the control cells is indicative of the cell death of the test substance. A step that is an indicator of causing or enhancing the method. 76. The method of claim 75, wherein said one or more test cells is a cancer cell, yeast, fungus or bacterium. 77. A method for detecting viral protease in one or more cells, comprising: (a) treating the cells with the reporter compound of claim 34; Contacting under conditions that are taken up by the cells, and (b) recording the fluorescence of the cells, wherein the fluorescence in the cells relative to a control cell not so contacted is determined. Wherein the alteration is indicative of the presence of a viral protease. 78. A method for measuring the activity of a viral protease in one or more virus-infected cells, the method comprising the following steps: (a) isolating the one or more virus-infected cells according to claim 34. Contacting the reporter compound with the reporter compound under conditions in which the reporter compound is taken up by the one or more virus-infected cells, and (b) recording the fluorescence of the one or more cells, wherein: Wherein the change in fluorescence in the one or more virus-infected cells as compared to control cells that have not been so contacted is a measure of the viral protease activity. 79. A method for determining whether a test substance has an effect on the activity of a viral protease in one or more virus-infected cells, the method comprising the following steps: Contacting the test substance and the reporter compound according to claim 34 under conditions that allow the reporter compound to be taken up by the infected test cells, and (b) comparing the infected control cells with the reporter compound alone. Recording the fluorescence of the infected test cells, wherein a change in fluorescence in the infected test cells compared to the infected control cells is an indicator that the test substance has an effect on a viral protease. A method comprising the steps of: 80. The method according to any one of claims 77 to 79, wherein
Wherein the cells are HIV infected cells and the viral protease is an HIV protease. 81. The method according to any one of claims 77 to 79, wherein
Wherein the cells are adenovirus infected cells and the viral protease is an adenovirus protease. 82. The method according to any one of claims 77 to 79, wherein
Wherein the cell is an HSV-infected cell and the viral protease is an HSV protease. 83. The method according to any one of claims 77 to 79, wherein
Wherein the cells are HCMV-infected cells and the viral protease is HCMV protease. 84. The method according to any one of claims 77 to 79, wherein
Wherein the cells are HCV-infected cells and the viral protease is an HCV protease. 85. A method for measuring the activity of a protease or peptidase in a cell, comprising the steps of: (a) testing a test cell with the reporter compound according to claim 34; Contacting under conditions that are taken up by the cell or under conditions where the reporter compound interacts with a membrane protease or peptidase outside the cell, and (b) recording the fluorescence of the cell, Wherein the change in fluorescence in the test cell compared to a control cell not so 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 a protease or peptidase in a test cell, the method comprising the following steps: (a) replacing the test cell with the test substance and Contacting the reporter compound of claim 34 with a condition under which the reporter compound is taken up by the test cell or under a condition in which the reporter compound interacts with a membrane protease or peptidase outside the cell; and (B) recording the fluorescence of the test cell compared to a control cell contacted only with the reporter compound, wherein the change in fluorescence in the test cell compared to the control cell is A step which is an indicator that the substance has an effect on the protease or peptidase. 87. The method according to any one of claims 85 to 86, wherein
Wherein the cell is an endothelial cell and the peptidase is type 2 methionine aminopeptidase. 88. The method according to any one of claims 85 to 86, wherein
Wherein the cell is a T cell and the peptidase is dipeptidyl peptidase IV. 89. The method according to any one of claims 85 to 86, wherein
Wherein the cell is a neuron and the protease is calpain. 90. The method according to any one of claims 85 to 86, wherein
Wherein the peptidase is an aminopeptidase.