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CN-116217656-B - Activity-based probe compounds, compositions, and methods of use thereof

CN116217656BCN 116217656 BCN116217656 BCN 116217656BCN-116217656-B

Abstract

The present invention provides activity-based probe compounds for labeling cysteine proteases. The compounds target proteases through specific target elements. The compounds also include a detectable element, such as a fluorescent label, radiolabel, or chelator. In some cases, the compound further includes a quenching element that is released upon reaction with the protease. The invention also provides compositions comprising the compounds, and methods of using the compounds, such as for labeling proteases in an animal and visualizing tumors in an animal.

Inventors

  • Matthew, S, Bo Jiao
  • Matikin Veldos

Assignees

  • 里兰斯坦福初级大学理事会

Dates

Publication Date
20260508
Application Date
20171223
Priority Date
20161223

Claims (17)

  1. 1. A compound for labelling a protease having the formula (II): wherein D comprises a benzindole dye substituted with a sulfonyl or carbonate group; l 1 is a linking group; AA 1 is an amino acid side chain; U is O, NH or S; R 1 is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, or a protecting group, and is optionally substituted with 1 to 3A groups; Each a is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkanylamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkylamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkylamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkylamino, hydroxy, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidino, ureido, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamino, or azido; l 3 is a linking group, and Q comprises a quencher.
  2. 2. The compound of claim 1, wherein the benzindole dye has the structure:
  3. 3. the compound of claim 1, wherein the benzindole dye has the structure:
  4. 4. The compound of claim 1, wherein L 1 is an optionally substituted alkyl linking group, wherein each carbon atom is optionally replaced with a heteroatom.
  5. 5. The compound of claim 1, wherein AA 1 is an aralkylamino acid side chain, optionally substituted with 1 to 3 a groups.
  6. 6. The compound of claim 1, wherein U is O.
  7. 7. The compound of claim 1, wherein L 3 is an optionally substituted alkyl linking group, wherein each carbon atom is optionally replaced with a heteroatom.
  8. 8. The compound of claim 1, wherein L 3 -Q is Wherein R comprises a QSY quencher or QC-1 quencher, and N is an integer from 1 to 8.
  9. 9. The compound of claim 8, wherein the QSY quencher is a hydrophilic QSY quencher.
  10. 10. The compound of claim 9, wherein the hydrophilic QSY quencher is a sulfo-QSY quencher.
  11. 11. A compound according to claim 8, wherein the QC-1 quencher has the structure:
  12. 12. The compound of claim 1, having formula (III): Wherein R comprises a QSY quencher or a QC-1 quencher, and m and n are independently integers from 1 to 8.
  13. 13. The compound of claim 12, wherein R is: And D is:
  14. 14. a compound having the structure:
  15. 15. The compound of claim 14, wherein the compound has a structure according to the formula:
  16. 16. A composition for labeling a protease in an animal comprising a compound of any one of claims 1 to 15 and a pharmaceutically acceptable carrier.
  17. 17. A method of labeling a protease in an animal comprising the step of administering the composition of claim 16 to the animal.

Description

Activity-based probe compounds, compositions, and methods of use thereof Technical Field Cross Reference to Related Applications The present application claims priority from U.S. provisional application No.62/438,959 filed on date 2016, 12, 23, the disclosure of which is incorporated herein by reference in its entirety. Government support description The present invention was made under the project of government supported contract EB005011 (issued by the national institutes of health). The government has certain rights in this invention. Background Various techniques are currently being developed for use in the fields of molecular imaging and disease monitoring. In particular, optical fluorescence imaging is a method that begins to present potential as a clinical tool due to its sensitivity, specificity and non-invasiveness. The specificity of fluorescent optical probes may in some cases be provided by their biological targets. For example, optical probes recognized by enzyme targets in biological samples tend to produce very specific signals if the fluorescence of the probe is only released in the case of an enzymatic reaction. Ideally, the fluorescent moiety of the probe remains attached to its enzyme target even in the case where the fluorescent signal has been activated by an enzymatic reaction. The use of such fluorescence activity-based probes (ABPs) for protease targets has been disclosed. Blum et al, (2009) PLoS One 4:e6374; doi:10.1371/journ.pone.0006374. ABP can be distinguished from simple fluorogenic substrates by the permanent covalent bond created by the reaction between ABP and the active site catalytic residues of the enzyme. While signal amplification due to catalytic inversion by the target enzyme may appear to be advantageous for fluorogenic substrates, APB has been found to exhibit increased tissue uptake kinetics and prolonged probe retention in target tissue due to covalent modification of the target enzyme. Among the target enzymes of interest for use with fluorescence-based optical probes are proteases, particularly cysteine proteases. Cysteine cathepsins are a family of proteases that play an important role in health and disease. Reiser et al, (2010) J.Clin.Invest.120:3421-31. Although their function has been described primarily as limited to the endosomal pathway, there is growing evidence that they are major mediators of matrix degradation, suggesting that they also function extracellularly.&Wilson(2011)Role of Cysteine Cathepsins in Extracellular Proteolysis.Biology of Extracellular Matrix Volume 2 23-51. Furthermore, it has been shown that members of the cysteine cathepsin family are major participants .Mohamed&Sloane(2006)Nat.Rev.Cancer(2006)6:764-75;Palermo&Joyce(2008)Trends Pharmacol.Sci.29:22-8. in the development and progression of several types of cancer and, in addition, alterations in the expression of endogenous inhibitors of cysteine cathepsins (cysteine protease inhibitors) have been observed in cancer. Cox (2009) CYSTATINS AND cancer front biosci.14:463-74. These observations, in combination with the potential changes in the intracellular and extracellular environments, underscore the importance of tools that allow the direct assessment of the activity of these proteases in the case of natural tumor microenvironments. Several ABPs have been synthesized for the cysteine cathepsin family. Edgington et al, (2011) Curr.Opin.chem.biol.15:798-805. In particular, fluorescence quenching ABP (qABP) has proven to be a powerful tool for non-invasive optical imaging of cancer and subsequent characterization of target cathepsins at the tissue, cellular and protein levels. Blum et al, (2007) Nat.chem.biol.3:668-77; verdones et al (2012) chem.biol.19:619-28. Inhibitors of the active-based dipeptidyl peptidase I based on 2,3,5, 6-tetrafluorophenoxy aryl methyl ketone reactive groups have been reported (Deu et al, (2010) Chem biol. 17:808-819), but these inhibitors are non-peptide and do not include a detectable group. Quenched activity-based peptide inhibitors for fluorescence imaging of cells containing active proteases (e.g., cathepsins) are also reported. See, for example, U.S. patent application publication No.2007/0036725. These probes employ an ester-linked acyloxymethyl ketone reactive group to bind to the protease active site. In some cases, the activity-based fluorescent probe is a non-peptide probe. See, for example, PCT international publication No. wo 2012/118715. In some cases, activity-based probes are used to radiolabel their target enzymes. See, for example, PCT international publication No. wo 2009/124265. PCT International publication No. WO 2012/021800, U.S. patent application publication No.2002/0052323, U.S. patent application publication No.2002/0028774, PCT International publication No. WO 96/41638 and European patent application publication No. EP 0272671 report other activity-based caspases and other cysteine protease inhibitors. However, there remains a nee