Inhibitors of oplophorus luciferase-derived bioluminescent complexes

Abstract

To provide compounds that may selectively inhibit Oplophorus luciferase-derived bioluminescent complexes, e.g., NanoBiT(R) bioluminescent complex, as well as compositions and kits comprising the compounds, and methods of using the compounds.SOLUTION: The present invention provides compounds of formula (I) that may selectively inhibit NanoBiT(R) bioluminescent complex.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 679,205, filed on June 1, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure is directed to compounds that can inhibit a bioluminescent complex derived from Oplophorus luciferase, particularly a bioluminescent complex of two or more non - luminescent peptide and / or polypeptide units from Oplophorus - derived luciferase.

Background Art

[0003] Protein fragment complementation (PFC) or enzyme fragment complementation (EFC) systems are valuable tools for monitoring co-localization and / or intermolecular interactions. In such systems, a reporter molecule, e.g., a complementary amino acid chain from a bioluminescent protein or enzyme, e.g., a peptide or polypeptide, is fused to the co-localizing and / or interacting molecules. Reporter molecules are customarily used to monitor molecular events in the fields of biology, biochemistry, immunology, cell biology, and molecular biology. Luciferase based on luciferase secreted from the deep-sea shrimp Oplophorus gracilirostris can be used as a reporter molecule and has been shown to have advantageous features including broad substrate specificity, high activity, and high quantum yield. For example, non-luminescent peptide and / or polypeptide units of Oplophorus luciferase variants can be fused to co-localizing / interacting molecules (e.g., proteins). When the molecules co-localize and / or interact, the non-luminescent peptide and / or polypeptide units associate to form a bioluminescent complex, which can generate a luminescence signal indicating the co-localization / interaction of the molecules in the presence of a substrate (e.g., coelenterazine or a coelenterazine derivative substrate). For certain applications, it may be further advantageous to control the luminescence signal from the bioluminescent complex derived from Oplophorus luciferase. Selective inhibitors for such bioluminescent complexes are useful in luminescence assays. Luciferase inhibitors can be further derivatized to provide desired properties useful for studying enzyme activity and cellular processes.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Means for Solving the Problem

[0005] In one aspect, the present disclosure relates to a compound of formula (I), or a salt thereof: JPEG2025105624000001.jpg37170(wherein: R 1 is aryl, cycloalkyl, heteroaryl, heterocycle, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocycle thereof are optionally substituted with one or more R W each R W is independently C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A -C 1-10 alkylene-OR A -CO-R A -C 1-10 alkylene-CO-R A -CO-OR A -C 1-10 alkylene-CO-OR A -CO-NHR A -C 1-10 alkylene-CO-NHR A -NR B R C -C 1-10 alkylene-NR B R C -NH-CO-C 1-4 alkyl, -C 1-10 alkylene-NH-CO-C 1-4 alkyl, phenyl, and phenyl substituted with 1, 2, 3, or 4 R D groups; each R 2 is independently C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A -C 1-4 alkylene-OR A -CO-RA ,-C 1-4 alkylene-CO-R A ,-CO-OR A ,-C 1-4 alkylene-CO-OR A ,-CO-NHR A ,-C 1-4 alkylene-CO-NHR A ,-NR B R C ,-C 1-4 alkylene-NR B R C ,-NH-CO-C 1-4 alkyl,-C 1-4 alkylene-NH-CO-C 1-4 alkyl,phenyl,1,2,3,or 4 R D groups substituted phenyl,-C≡C-R A ,or-C≡C-C 1-4 alkylene-OR A and either two R 2 are such that they are attached to the carbon atoms of the 1315 part of JPEG2025105624000002.jpg to form a 5- or 6-membered fused ring; each R 3 is independently C 1-10 alkyl,C 1-10 haloalkyl,halogen,-CN,-OR A ,-C 1-4 alkylene-OR A ,-CO-R A ,-CO-OR A ,or-CO-NHR A and either two R 3 are such that they are attached to the carbon atoms of the 1720 part of JPEG2025105624000003.jpg to form a 5- or 6-membered fused ring; R 4 is H or C 1-4 alkyl; p is 0,1,2,3,or 4; q is 0,1,2,3,or 4; each occurrence of RA is, independently, H, C 1-4 alkyl, or C 1-4 haloalkyl; R at each occurrence B and R C is, independently, H or C 1-4 alkyl, or R B and R C together with the N atom to which they are attached form a 5- or 6-membered heterocycle; R at each occurrence D is, independently, C 1-4 alkyl, -OC 1-4 alkyl, -CN, or halogen) is provided.

[0006] In one aspect, the disclosure provides a method of inhibiting an Oplophorus luciferase-derived bioluminescent complex, the method comprising contacting the bioluminescent complex with a compound described herein.

[0007] In one aspect, the disclosure provides a method for modulating the luminescence of an Oplophorus luciferase-derived bioluminescent complex in a sample, the method comprising (a) contacting the sample with a coelenterazine substrate and a compound described herein, (b) detecting the luminescence in the sample, wherein the compound causes a decrease in luminescence from the bioluminescent complex. is provided.

[0008] In one aspect, the disclosure provides a method for detecting an interaction or co-localization between a first molecule and a second molecule in a sample, the method comprising (a) contacting the sample with a coelenterazine substrate and a compound described herein, wherein the sample (i) A first fusion, the first fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule, the first fusion; (ii) A second fusion, the second fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a second molecule, the non-luminescent polypeptide being capable of forming a bioluminescent complex with the non-luminescent peptide, the second fusion; comprising contacting; (b) Detecting luminescence in the sample; comprising; Detection of luminescence indicates an interaction or co-localization between the first molecule and the second molecule, providing a method.

[0009] In one aspect, the present disclosure is a method for detecting an interaction or co-localization between a first molecule and a second molecule in a sample, the method comprising: (a) Contacting the sample with a coelenterazine substrate and a compound described herein, the sample comprising: (i) A first polynucleotide encoding a first fusion, the first fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule, the first polynucleotide; (ii) A second polynucleotide encoding a second fusion, the second fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a second molecule, the non-luminescent polypeptide being capable of forming a bioluminescent complex with the non-luminescent peptide, the second polynucleotide; comprising contacting; (b) Detecting luminescence in the sample; comprising; Detection of luminescence indicates an interaction or co-localization between the first molecule and the second molecule, providing a method.

[0010] In one aspect, the present disclosure is a method for detecting an interaction or co-localization of a first molecule and a second molecule in a sample, the method comprising: (a) Contacting a sample with a non-luminescent polypeptide of Oplophorus-derived luciferase, a coelenterazine substrate, and a compound described herein, wherein the sample comprises (i) a first polynucleotide encoding a first fusion, the first fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule, the non-luminescent peptide being capable of forming a bioluminescent complex with the non-luminescent polypeptide, the first polynucleotide, and (ii) a second polynucleotide encoding a second fusion, the second fusion comprising a fluorescent acceptor molecule and a second molecule, the second polynucleotide, and including, contacting; (b) Detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of an interaction or co-localization of the first molecule and the second molecule; including, providing a method.

[0011] In one aspect, the present disclosure provides a method for detecting an interaction or co-localization of a first molecule and a second molecule in a sample, the method comprising: (a) Contacting a sample with a non-luminescent peptide of Oplophorus-derived luciferase, a coelenterazine substrate, and a compound described herein, wherein the sample comprises (i) a first polynucleotide encoding a first fusion, the first fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a first molecule, the non-luminescent polypeptide being capable of forming a bioluminescent complex with the non-luminescent peptide, the first polynucleotide, and (ii) a second polynucleotide encoding a second fusion, the second fusion comprising a fluorescent acceptor molecule and a second molecule, the second polynucleotide, and including, contacting; (b) Detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of an interaction or co-localization of the first molecule and the second molecule; Provide a method including

[0012] In one aspect, the present disclosure provides a method for detecting the interaction or co-localization of molecules in a sample, the method comprising: (a) contacting the sample with a coelenterazine substrate and a compound described herein, the sample comprising: (i) a first fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a first molecule; (ii) a second fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a second molecule, the non-luminescent peptide being capable of forming a bioluminescent complex with the non-luminescent polypeptide; (iii) a third fusion comprising a fluorescent acceptor molecule and a third molecule; and contacting; (b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of an interaction or co-localization between the first, second, and third molecules in the sample. Provide a method including

[0013] In one aspect, the present disclosure provides a method for detecting a target molecule in a sample, the method comprising: (a) contacting a sample comprising a target molecule fused to a non-luminescent peptide of Oplophorus-derived luciferase with: (i) a coelenterazine substrate; (ii) a compound described herein; and (iii) a non-luminescent polypeptide of Oplophorus-derived luciferase, the non-luminescent polypeptide being capable of forming a bioluminescent complex with the non-luminescent peptide; and contacting; (b) detecting luminescence in the sample; and the detection of luminescence indicates the formation of a bioluminescent complex between the non-luminescent peptide and the non-luminescent polypeptide.

[0014] In one aspect, the present disclosure is a method for detecting a target molecule in a sample, the method comprising: (a) contacting a sample containing a target molecule fused to a non-luminescent polypeptide of Oplophorus luciferase with (i) a coelenterazine substrate; (ii) a compound disclosed herein; and (iii) a non-luminescent peptide of Oplophorus luciferase, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; and (b) detecting luminescence in the sample, wherein detection of the luminescence indicates formation of a bioluminescent complex between the non-luminescent peptide and the non-luminescent polypeptide.

[0015] In one aspect, the present disclosure is a method for detecting a target molecule in a sample, the method comprising: (a) contacting a sample containing a target molecule fused to a non-luminescent peptide of Oplophorus luciferase with (i) a coelenterazine substrate; (ii) a compound described herein; and (iii) a fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a fluorescent moiety, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; and (b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of detection of the molecule.

[0016] In one aspect, the present disclosure is a method for detecting a target molecule in a sample, the method comprising: (a) A sample containing a molecule to be fused to a non-luminescent polypeptide of Oplophorus-derived luciferase is (i) coelenterazine substrate; (ii) a compound disclosed herein; and (iii) a fusion containing a non-luminescent peptide of Oplophorus-derived luciferase and a fluorescent moiety, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; contacted with;

[0017] (b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of the detection of the molecule; and provides a method.

[0018] In one aspect, the present disclosure is a bioluminescence resonance energy transfer (BRET) system comprising (a) a first fusion containing a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule; (b) a second fusion containing a non-luminescent polypeptide of Oplophorus-derived luciferase and a fluorescent moiety, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; (c) coelenterazine substrate; (d) a compound described herein; and provides a bioluminescence resonance energy transfer (BRET) system.

[0019] In one aspect, the present disclosure is a bioluminescence resonance energy transfer (BRET) system comprising (a) a first fusion containing a non-luminescent polypeptide of Oplophorus-derived luciferase and a first molecule; (b) a second fusion containing a non-luminescent peptide of Oplophorus-derived luciferase and a fluorescent moiety, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; (c) coelenterazine substrate; (d) a compound described herein, and provides a bioluminescence resonance energy transfer (BRET) system comprising the same.

[0020] In one aspect, the present disclosure provides a bioluminescence resonance energy transfer (BRET) system comprising: (a) a first fusion comprising a first molecule and a non-luminescent peptide of Oplophorus-derived luciferase; (b) a second fusion comprising a second molecule and a fluorescent acceptor molecule; (c) a non-luminescent polypeptide of Oplophorus-derived luciferase capable of forming a bioluminescent complex with a non-luminescent polypeptide of Oplophorus-derived luciferase; (d) a coelenterazine substrate; (e) a compound described herein; and provides a bioluminescence resonance energy transfer (BRET) system comprising the same.

[0021] In one aspect, the present disclosure provides a bioluminescence resonance energy transfer (BRET) system comprising: (a) a first fusion comprising a first molecule and a non-luminescent polypeptide of Oplophorus-derived luciferase; (b) a second fusion comprising a second molecule and a fluorescent acceptor molecule; (c) a non-luminescent peptide of Oplophorus-derived luciferase capable of forming a bioluminescent complex with a non-luminescent polypeptide of Oplophorus-derived luciferase; (d) a coelenterazine substrate; (e) a compound described herein; and provides a bioluminescence resonance energy transfer (BRET) system comprising the same.

[0022] In one aspect, the present disclosure provides (a) a compound described herein; (b) a first polynucleotide encoding a non-luminescent peptide of Oplophorus-derived luciferase; (c) A second polynucleotide encoding a non-luminescent polypeptide of Oplophorus-derived luciferase, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with a non-luminescent peptide, and A kit comprising

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0024] The disclosed compounds can selectively inhibit the bioluminescent complex derived from Oplophorus luciferase. For example, the disclosed compounds have (a) a peptide amino acid sequence having less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) and more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 2; and (b) a polypeptide amino acid sequence having less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) and more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 3. The bioluminescent complex derived from Oplophorus luciferase, which can exhibit detectable luminescence in the presence of the coelenterazine substrate, can be selectively inhibited by the disclosed compounds. In certain embodiments, the disclosed compounds have (a) a peptide comprising the peptide amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6; and (b) a polypeptide comprising the polypeptide amino acid sequence of SEQ ID NO: 5. The bioluminescent complex derived from Oplophorus luciferase, which can exhibit detectable luminescence in the presence of the coelenterazine substrate, can be selectively inhibited by the disclosed compounds. Exemplary bioluminescent complexes that can be inhibited using the disclosed compounds are described in U.S. Patent Nos. 9,797,889 and 9,797,890, the entire contents of which are incorporated herein by reference in their entirety. For example, the disclosed compounds can selectively inhibit the NanoBiT® HiBiT / LgBiT bioluminescent complex. As another example, the disclosed compounds can selectively inhibit the NanoBiT® LgBiT / SmBiT bioluminescent complex.

[0025] Due to their stability, potential for excretion from cells, and the presence of cell debris generated from cultured cells, in certain applications, it may be advantageous to use the selective inhibitors of the present invention to suppress luminescence from Oplophorus luciferase-derived bioluminescent complexes. For example, in applications involving time multiplexing of multiple luminescence systems, it may be beneficial to have a selective inhibitor for each system to enable measurement and / or detection of only one luminescence signal at a time. Also, in some plate-based assays, a predetermined amount of luciferase may be excreted from cells or present in the medium from cell debris. An extracellular inhibitor compound would be able to selectively suppress luminescence from luciferase in the medium. This can thus help improve the signal-to-background ratio in a given assay.

[0026] In certain embodiments, the light generated from the NanoBiT® bioluminescent complex can be selectively suppressed by the compounds disclosed herein. Advantageously, such selective inhibition can be used to enable time multiplexing of multiple bioluminescence systems such as NanoBiT and NanoLuc. Further, the disclosed compounds provide selective bioluminescence inhibition (e.g., intracellular or extracellular selectivity) to enable certain plate-based luminescence assays. The compounds can compete for binding to the coelenterazine substrate of luciferase and can be modified to produce cell-permeable and cell-impermeable inhibitors.

[0027] 1. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Although preferred methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The substances, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0028] The terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a", "and", and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0029] As used herein, the term "substituent" or "suitable substituent" is intended to mean a chemically acceptable functional group, e.g., a moiety that does not deactivate the activity of the compounds of the present invention. Exemplary examples of suitable substituents include halo group, perfluoroalkyl group, perfluoroalkoxy group, alkyl group, alkenyl group, alkynyl group, hydroxy group, halo group, oxo group, mercapto group, alkylthio group, alkoxy group, nitro group, azidoalkyl group, sulfonic acid group, aryl or heteroaryl group, aryloxy or heteroaryloxy group, aralkyl or heteroaralkyl group, aralkoxy or heteroaralkoxy group, HO-(C=O)- group, heterocyclic group, cycloalkyl group, amino group, alkyl- and dialkyl-amino groups, carbamoyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkoxycarbonyl group, alkylaminocarbonyl group, dialkylaminocarbonyl group, arylcarbonyl group, aryloxycarbonyl group, alkylsulfonyl group, arylsulfonyl group, etc., but are not limited thereto. Substituents may be substituted by additional substituents. Substituents may also be in the form of salts (e.g., a sulfonic acid group may be in the form of a sulfonate group).

[0030] The definitions of specific functional groups and chemical terms are described in detail below. For the purposes of the present disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., back cover, and specific functional groups are generally defined as described therein. Also, general principles of organic chemistry, and specific functionalities and reactivities are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March’s Advanced Organic Chemistry, 5 thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are hereby incorporated by reference.

[0031] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon chain containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl. The alkenyl groups of the present invention may be unsubstituted or substituted by one or more suitable substituents as defined above, preferably 1 to 3 suitable substituents.

[0032] As used herein, the term "alkoxy" refers to an alkyl group as defined herein attached to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0033] As used herein, the term "alkoxyalkoxy" refers to an alkoxy group as defined herein attached to a parent molecular moiety via another alkoxy group as defined herein. Representative examples of alkoxyalkoxy include, but are not limited to, tert-butoxymethoxy, 2-ethoxyethoxy, 2-methoxyethoxy, and methoxymethoxy.

[0034] As used herein, the term "alkoxyalkoxyalkyl" means an alkoxyalkoxy group as defined herein attached to a parent molecular moiety via an alkylene group as defined herein. Representative examples of alkoxyalkoxyalkyl include, but are not limited to, tert-butoxymethoxymethyl, ethoxymethoxymethyl, (2-methoxyethoxy)methyl, and 2-(2-methoxyethoxy)ethyl.

[0035] As used herein, the term "alkoxyalkyl" refers to an alkoxy group as defined herein attached to a parent molecular moiety via an alkyl group as defined herein. Representative examples of alkoxyalkyl include, but are not limited to, tert-butoxymethyl, 2-ethoxyethyl, 2-methoxyethyl, and methoxymethyl.

[0036] As used herein, the term "alkoxycarbonyl" refers to an alkoxy group as defined herein attached to a parent molecular moiety via a carbonyl group as defined herein. Representative examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, and tert-butoxycarbonyl.

[0037] The term "alkoxycarbonylalkyl", as used herein, refers to an alkoxycarbonyl group as defined herein attached to the parent molecular moiety via an alkylene group as defined herein. Representative examples of alkoxycarbonylalkyl include, but are not limited to, ethoxycarbonylmethyl, 3-methoxycarbonylpropyl, 4-ethoxycarbonylbutyl, and 2-tert-butoxycarbonylethyl.

[0038] As used herein, the term "alkyl" preferably refers to a linear or branched hydrocarbon group having from 1 to 30 carbon atoms, from 1 to 12 carbon atoms, from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. The term "C1-C8-alkyl" is defined to include alkyl groups having 1, 2, 3, 4, 5, 6, 7 or 8 carbons in a linear or branched arrangement. For example, "C1-C8-alkyl" particularly includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, pentyl (e.g., n-pentyl), hexyl (e.g., n-hexyl), heptyl (e.g., n-heptyl) and octyl (e.g., n-octyl). The term "C1-C6-alkyl" is defined to include alkyl groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement. For example, "C1-C6-alkyl" particularly includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, pentyl (e.g., n-pentyl), and hexyl (e.g., n-hexyl). The term "C1-C4-alkyl" is defined to include alkyl groups having 1, 2, 3, or 4 carbons in a linear or branched arrangement. For example, "C1-C4-alkyl" particularly includes methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, iso-butyl and tert-butyl. The alkyl groups of the present invention may be unsubstituted or may be substituted by one or more suitable substituents as defined above, for example, 1 to 3 suitable substituents. For example, the alkyl group may be substituted by one or more halo substituents to form a haloalkyl group, or by one or more hydroxy substituents to form a hydroxyalkyl group, or by one or more alkoxy groups to form an alkoxyalkyl group.

[0039] As used herein, the term "alkylamino" refers to an alkyl group as defined herein attached to the parent molecular moiety via an amino group as defined herein. Representative examples of alkylamino include, but are not limited to, methylamino, ethylamino, iso-propylamino, butyl-amino, and sec-butylamino.

[0040] As used herein, the term "alkylaminoalkyl" refers to an alkyl group as defined herein attached to the parent molecular moiety via an aminoalkyl group as defined herein. Representative examples of alkylaminoalkyl groups include, but are not limited to, methylaminoethyl and methylamino-2-propyl.

[0041] As used herein, the term "alkylcarbonyl" refers to an alkyl group as defined herein attached to the parent molecular moiety via a carbonyl group as defined herein. Representative examples of alkylcarbonyl include, but are not limited to, acetyl, 1-oxopropyl, 2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl.

[0042] As used herein, the term "alkylcarbonylalkyl" refers to an alkylcarbonyl group as defined herein attached to the parent molecular moiety via an alkyl group as defined herein.

[0043] As used herein, the term "alkylcarbonylalkylamide" refers to an alkylcarbonyl group as defined herein attached to the parent molecular moiety via an alkylamide group as defined herein.

[0044] The term "alkylene" means a divalent group derived from a saturated, straight-chain or branched-chain hydrocarbon having 1 to 10 carbon atoms. Representative examples of alkylene include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2C(CH3)2-, and -CH2CH(CH3)CH2-.

[0045] As used herein, the term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons and having one or more carbon-carbon triple bonds. The alkynyl groups of the present invention include, but are not limited to, ethynyl, propynyl, and butynyl. The alkynyl groups of the present invention may be unsubstituted or may be substituted by one or more suitable substituents as defined above, preferably 1 to 3 suitable substituents.

[0046] As used herein, the term "amide" refers to an amino group (i.e., -CONH2) added to a parent molecular moiety via a carbonyl group as defined herein. The term "alkylamide" as used herein refers to an alkylamino group or a dialkylamino group added to a parent molecular moiety via a carbonyl group as defined herein. Representative examples of alkylamides include, but are not limited to, methylaminocarbonyl, dimethylaminocarbonyl, ethylmethylaminocarbonyl, and n-hexylaminocarbonyl.

[0047] As used herein, the term "amino" refers to the -NH2 group.

[0048] As used herein, the term "aminoalkyl" refers to at least one amino group as defined herein attached to a parent molecular moiety via an alkyl group as defined herein. Representative examples of aminoalkyl include, but are not limited to, aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, and 6-aminohexyl.

[0049] As used herein, the term "aminoalkylamide" refers to at least one amino group as defined herein attached to a parent molecular moiety via an alkylamide group as defined herein.

[0050] As used herein, the term "amino protecting group" refers to a moiety that prevents chemical reactions from occurring on a nitrogen atom to which the protecting group is attached. The amino protecting group must also be removable by a chemical reaction. Such groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3 rd edition, John Wiley & Sons, 1999 (which is hereby incorporated by reference in its entirety). Suitable amino protecting groups include, but are not limited to, carbobenzyloxy (-NHCO-OCH2C6H5 or -NH-Cbz); t-butyloxycarbonyl (-NHCO-OC(CH3)3 or -NH-Boc); 9-fluorenylmethyloxycarbonyl (-NH-Fmoc), 2,2,2-trichloroethyloxycarbonyl (-NH-Troc), and allyloxycarbonyl (-NH-Alloc). In each of the above, -NH- represents the nitrogen from the protected amino group.

[0051] As used herein, the term "aminonorsiferin" refers to (4S)-2-(6-amino-1,3-benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid, or a substituted analog of this molecule.

[0052] As used herein, the term "aryl" means a monocyclic, bicyclic, or tricyclic aromatic group. Representative examples of aryl groups include, but are not limited to, phenyl, dihydroindenyl, indenyl, naphthyl, dihydronaphthalenyl, and tetrahydronaphthalenyl. The aryl groups of the present invention may optionally be substituted by one or more suitable substituents, preferably 1 to 5 suitable substituents, as defined above. Aryl, as used herein, includes phenyl attached to the parent molecular moiety and condensed to a cycloalkyl group (e.g., indanyl or 5,6,7,8 - tetrahydronaphthalen - 2 - yl), phenyl condensed to a phenyl group (i.e., naphthyl), or phenyl condensed to a non - aromatic heterocyclic ring (e.g., benzo[d][1,3]dioxol - 5 - yl).

[0053] As used herein, the term "arylalkyl" refers to an aryl group as defined herein attached to the parent molecular moiety via an alkyl group as defined herein. Representative examples of arylalkyl include, but are not limited to, phenylmethyl (i.e., benzyl) and phenylethyl.

[0054] As used herein, the term "arylcarbonyl" refers to an aryl group as defined herein attached to the parent molecular moiety via a carbonyl group as defined herein.

[0055] As used herein, the term "carbonyl" or "(C = O)" (when used in phrases such as alkylcarbonyl, alkyl - (C = O)- or alkoxycarbonyl) refers to the linker of the >C = O moiety to a second moiety, e.g., an alkyl or amino group (i.e., an amide group). Alkoxycarbonylamino (i.e., alkoxy(C = O)-NH -) refers to an alkyl carbamate group. The carbonyl group is also defined herein as equivalent to (C = O). Alkylcarbonylamino refers to groups such as acetamide.

[0056] As used herein, the term "carboxy" refers to a -C(O)OH group.

[0057] As used herein, the term "carboxyalkyl" refers to a carboxy group as defined herein attached to the parent molecular moiety via an alkyl group as defined herein.

[0058] As used herein, the term "carboxyalkylamide" refers to a carboxyalkyl group as defined herein attached to the parent molecular moiety via an amide group as defined herein.

[0059] As used herein, the term "cycloalkyl" refers to a monocyclic, bicyclic or tricyclic carbocyclic group (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclopentenyl, cyclohexenyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl and bicyclo[5.2.0]nonanyl, etc.) which optionally contains one or two double bonds. The cycloalkyl group of the present invention may be unsubstituted or may be substituted by one or more suitable substituents as defined above, preferably 1 to 5 suitable substituents.

[0060] As used herein, the term "cycloalkylalkyl" refers to a cycloalkyl group as defined herein attached to the parent molecular moiety via an alkyl group as defined herein. Representative examples of cycloalkylalkyl include, but are not limited to, cyclohexylmethyl.

[0061] As used herein, the term "cycloalkylamide" refers to a cycloalkyl group as defined herein attached to the parent molecular moiety via an amide group as defined herein.

[0062] As used herein, the term "dialkylamino" refers to two independently selected alkyl groups as defined herein attached to the parent molecular moiety via an amino group as defined herein. Representative examples of dialkylamino include, but are not limited to, N,N-dimethylamino, N-ethyl-N-methylamino, and N-isopropyl-N-methylamino.

[0063] As used herein, the term "dialkylaminoalkyl" refers to a dialkylamino group as defined herein attached to the parent molecular moiety via an alkyl group as defined herein. Representative examples of dialkylaminoalkyl include, but are not limited to, N,N-dimethylaminoethyl and N,N-methyl(2-propyl)aminoethyl.

[0064] As used herein, the term "dialkylaminoalkylamide" refers to a dialkylamino group as defined herein attached to the parent molecular moiety via an alkylamide group as defined herein.

[0065] As used herein, the term "halogen" or "halo" refers to a fluoro, chloro, bromo or iodo group.

[0066] As used herein, the term "haloalkoxy" refers to an alkoxy group as defined herein substituted by one, two, three or four halogen atoms. Representative examples of haloalkoxy include, but are not limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, and pentafluoroethoxy.

[0067] As used herein, the term "haloalkyl" refers to an alkyl group as defined herein substituted by one, two, three or four halogen atoms. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and 4,4,4-trifluorobutyl.

[0068] As used herein, the term "heteroaryl" refers to monocyclic heteroaryl or bicyclic heteroaryl. Monocyclic heteroaryl is a 5- or 6-membered ring. The 5-membered ring contains 2 double bonds. The 5-membered ring may contain 1 heteroatom selected from O or S; or 1, 2, 3 or 4 nitrogen atoms and optionally 1 oxygen or sulfur atom. The 6-membered ring may contain 3 double bonds and 1, 2, 3 or 4 nitrogen atoms. Representative examples of monocyclic heteroaryl include, but are not limited to, furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1,3-oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, 1,3-thiazolyl, thienyl, triazolyl, and triazinyl. Bicyclic heteroaryl includes monocyclic heteroaryl fused to phenyl, or monocyclic heteroaryl fused to monocyclic cycloalkyl, or monocyclic heteroaryl fused to monocyclic cycloalkenyl, or monocyclic heteroaryl fused to monocyclic heteroaryl, or monocyclic heteroaryl fused to monocyclic heterocycle. Representative examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, 6,7-dihydro-1,3-benzothiazolyl, imidazo[1,2-a]pyridinyl, indazolyl, indolyl, isoindolyl, isoquinolinyl, naphthyridinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, thiazolo[5,4-b]pyridin-2-yl, thiazolo[5,4-d]pyrimidin-2-yl, 5,6,7,8-tetrahydroquinolin-5-yl, cyclopenta[b]thiophen-2-yl, and 4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl. The heteroaryl group of the present invention may be unsubstituted or may be substituted by one or more suitable substituents, preferably 1 to 5 suitable substituents, as defined above.

[0069] As used herein, the term "heteroarylalkyl" refers to a heteroaryl group as defined herein appended to a parent molecular moiety via an alkyl group as defined herein. Representative examples of heteroarylalkyl include, but are not limited to, fur-3-ylmethyl, 1H-imidazol-2-ylmethyl, 1H-imidazol-4-ylmethyl, 1-(pyridin-4-yl)ethyl, pyridin-3-ylmethyl, 6-chloropyridin-3-ylmethyl, pyridin-4-ylmethyl, (6-(trifluoromethyl)pyridin-3-yl)methyl, (6-(cyano)pyridin-3-yl)methyl, (2-(cyano)pyridin-4-yl)methyl, (5-(cyano)pyridin-2-yl)methyl, (2-(chloro)pyridin-4-yl)methyl, pyrimidin-5-ylmethyl, 2-(pyrimidin-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl.

[0070] As used herein, the terms "heterocyclic ring" or "heterocyclyl" refer to a monocyclic heterocyclic ring, a bicyclic heterocyclic ring, or a tricyclic heterocyclic ring. A monocyclic heterocyclic ring is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least 1 heteroatom independently selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 3- or 4-membered ring contains 0 or 1 double bond and 1 heteroatom selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 5-membered ring contains 0 or 1 double bond and 1, 2, or 3 heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 6-membered ring contains 0, 1, or 2 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 7- and 8-membered ring contains 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. Representative examples of monocyclic heterocyclic rings include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, phosphinan, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, trithianyl, and 2,5-dioxo-pyrrolidinyl.The bicyclic heterocyclic ring is a monocyclic heterocyclic ring condensed with a phenyl group, or a monocyclic heterocyclic ring condensed with a monocyclic cycloalkyl, or a monocyclic heterocyclic ring condensed with a monocyclic cycloalkenyl, or a monocyclic heterocyclic ring condensed with a monocyclic heterocyclic ring, or a bridged monocyclic heterocyclic ring system, wherein two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of the bicyclic heterocyclic ring include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, 9-phosphabicyclo[3.3.1]nonane, 8-phosphabicyclo[3.2.1]octane, and tetrahydroisoquinolinyl. The tricyclic heterocyclic ring is exemplified by a bicyclic heterocyclic ring condensed with a phenyl group, or a bicyclic heterocyclic ring condensed with a monocyclic cycloalkyl, or a bicyclic heterocyclic ring condensed with a monocyclic cycloalkenyl, or a bicyclic heterocyclic ring condensed with a monocyclic heterocyclic ring, or a bicyclic heterocyclic ring, wherein two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of the tricyclic heterocyclic ring include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 decane), oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7), and 2,4,6-trioxa-8-phosphatricyclo[3.3.1.13,7]decane, but are not limited thereto. The heterocyclic group of the present invention may be unsubstituted or may be substituted by one or more suitable substituents as defined above, preferably 1 to 3 suitable substituents. The heterocyclic group of the present invention may contain one or more oxo groups (=O) or thioxo groups (=S) bonded to the ring.

[0071] As used herein, the term "heterocyclylalkyl" refers to a heterocyclyl group as defined herein attached to the parent molecular moiety via an alkyl group as defined herein. Representative examples of heterocyclylalkyl include, but are not limited to, piperidin-4-ylmethyl, piperazin-1-ylmethyl, 3-methyl-1-pyrrolidin-1-ylbutyl, (1R)-3-methyl-1-pyrrolidin-1-ylbutyl, (1S)-3-methyl-1-pyrrolidin-1-ylbutyl, and 3-morpholinopropyl.

[0072] As used herein, the term "heterocyclylamide" refers to a heterocyclyl group as defined herein attached to the parent molecular moiety via an amide group as defined herein.

[0073] As used herein, the term "hydroxy" refers to an -OH group.

[0074] As used herein, the term "hydroxyalkoxy" refers to an alkoxy group as defined herein substituted by at least one hydroxy group. Representative examples of hydroxyalkoxy include, but are not limited to, hydroxyethoxy and 2-hydroxypropoxy.

[0075] As used herein, the term "hydroxyalkyl" refers to an alkyl group as defined herein substituted by at least one hydroxy group. Representative examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 2,3-dihydroxypentyl, 4-hydroxybutyl, 2-ethyl-4-hydroxyheptyl, 3,4-dihydroxybutyl, and 5-hydroxypentyl.

[0076] As used herein, the term "hydroxyalkylamide" refers to a hydroxyalkyl group attached to an amide group, e.g., -amide-alkyl-OH.

[0077] As used herein, the term "hydroxycarbonyl" refers to a hydroxy group as defined herein attached to a parent molecular moiety via a carbonyl group as defined herein.

[0078] As used herein, the term "methylenedioxy" refers to an -OCH2O- group in which the oxygen atoms of the methylenedioxy are attached to the parent molecular moiety via two adjacent carbon atoms.

[0079] As used herein, the term "oxo" refers to a double-bonded oxygen (=O) group in which the bonding partner is a carbon atom. Such a group may also be considered a carbonyl group.

[0080] As used herein, unless otherwise specified, the terms "peptide" and "polypeptide" refer to a polymeric compound of two or more amino acids linked through a peptide amide bond (-C(O)NH-) along the backbone. The term "peptide" typically refers to a short amino acid polymer (e.g., a chain having less than 25 amino acids), while the term "polypeptide" typically refers to a longer amino acid polymer (e.g., a chain having more than 25 amino acids).

[0081] The prefix attached to a multi-component substituent applies only to the first component that it precedes. By way of illustration, the term "cycloalkylalkyl" contains two components: alkyl and cycloalkyl. Thus, C1- 10 C1- of cycloalkylalkyl 10 the prefix means that the alkyl component of cycloalkylalkyl contains from 1 to 6 carbon atoms; the C1-C6- prefix does not describe the cycloalkyl component. By further illustration, C1- 10 the term "haloalkyl" refers to halomethyl, haloethyl, halopropyl, halobutyl, halopentyl, and halohexyl. By further illustration, the prefix "halo" of haloalkoxyalkyl indicates that only the alkoxy component of the alkoxyalkyl substituent is substituted with one or more halogen groups. If halogen substitution can occur only on the alkyl component, the substituent would be described instead as "alkoxyhaloalkyl".

[0082] A substituent is "substitutable" if it contains at least one carbon or nitrogen atom bonded to one or more hydrogen atoms. Thus, for example, hydrogen, halogen, and cyano do not fall within this definition. Also, a sulfur atom in a heterocyclyl containing such an atom is substitutable with one or two oxo substituents.

[0083] When a substituent is described as "substituted", a non-hydrogen group is present in place of a hydrogen group on a carbon or nitrogen of the substituent. Thus, for example, a substituted alkyl substituent is an alkyl substituent in which at least one non-hydrogen group is present in place of a hydrogen group on the alkyl substituent. By way of illustration, monofluoroalkyl is alkyl substituted with a fluoro group, and difluoroalkyl is alkyl substituted with two fluoro groups. It should be recognized that when multiple substitutions are present on a substituent, each non-hydrogen group can be the same or different (unless otherwise described).

[0084] When a substituent is referred to as "unsubstituted" and not referred to as "substituted" or "optionally substituted", it means that the substituent has no substituents. When a substituent is described as "optionally substituted", the substituent may be (1) unsubstituted or (2) substituted. When a substituent is described as optionally substituted with a maximum number of non-hydrogen groups, the substituent may be (1) unsubstituted or (2) substituted by up to the lesser of the maximum number of such non-hydrogen groups or the maximum number of substitutable positions on the substituent. Thus, for example, when a heteroaryl is described as optionally substituted with up to 3 non-hydrogen groups, any heteroaryl having less than 3 substitutable positions will be optionally substituted by up to the same number of non-hydrogen groups as the heteroaryl has substitutable positions. By way of illustration, tetrazolyl (which has only 1 substitutable position) will be optionally substituted with up to 1 non-hydrogen group. Further by way of illustration, when an amino nitrogen is described as optionally substituted with up to 2 non-hydrogen groups, a primary amino nitrogen will be optionally substituted with up to 2 non-hydrogen groups while a secondary amino nitrogen will be optionally substituted with up to only 1 non-hydrogen group.

[0085] When a substituent is described as "independently selected" from a group, each substituent is selected independently of the others. Thus, each substituent may be the same as or different from the other substituent(s).

[0086] As used herein, the terms "bioluminescence" or "luminescence" may refer to light produced as a result of a reaction between an enzyme and a substrate that produces light. Examples of such enzymes (bioluminescent enzymes) include Oplophorus luciferase, such as Oplophorous gracilirostris, firefly luciferase, such as Photinus pyralis or Photuris pennsylvanica, glowworm luciferase, Renilla luciferase, sea pansy luciferase, aequorin photoprotein, obelin photoprotein, and the like.

[0087] As used herein, the term "complex" refers to a population or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with each other. In one aspect, "contact" or more specifically, "direct contact" means that two or more molecules are in proximity such that attractive non-covalent interactions, such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, are sufficient to govern the interaction of the molecules. In such an aspect, a complex of molecules (e.g., peptides and polypeptides) is formed under assay conditions such that the complex is thermodynamically favored (e.g., as compared to the non-aggregated, or non-complexed state of its component molecules). As used herein, the term "complex" refers to a population of two or more molecules (e.g., peptides, polypeptides, or combinations thereof), unless otherwise specified.

[0088] The terms "bioluminescent complex" or "bioluminescent complex derived from Oplophorus luciferase", which are used interchangeably herein, refer to a complex assembled from two or more non-luminescent peptides and / or non-luminescent polypeptides. The bioluminescent complex catalyzes or enables the conversion of the substrate of the bioluminescent complex into an unstable form; the substrate then emits light. When not complexed, the two non-luminescent elements that form the bioluminescent complex can be referred to as a "non-luminescent pair". When the bioluminescent complex is formed by three or more non-luminescent peptides and / or non-luminescent polypeptides, the non-complexed components of the bioluminescent complex can be referred to as a "non-luminescent group". A bioluminescent complex derived from Oplophorus luciferase may include: (a) a peptide comprising a peptide amino acid sequence having an identity of less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) with SEQ ID NO: 2 and more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%); and (b) a polypeptide comprising a polypeptide amino acid sequence having an identity of less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) with SEQ ID NO: 3 and more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%), and the bioluminescent complex exhibits detectable luminescence. In certain embodiments, the invention provides a bioluminescent complex comprising: (a) a peptide comprising the peptide amino acid sequence of SEQ ID NO: 4 or 6; and (b) a polypeptide comprising the polypeptide amino acid sequence of SEQ ID NO: 5, wherein the bioluminescent complex exhibits detectable luminescence. Exemplary bioluminescent complexes derived from Oplophorus luciferase include the NanoBiT® technology, which includes the SmBiT non-luminescent peptide (SEQ ID NO: 6), the NanoBiT® HiBiT non-luminescent peptide (SEQ ID NO: 4), and / or the NanoBiT® LgBiT non-luminescent polypeptide (SEQ ID NO: 5).

[0089] As used herein, the term "non-luminescent" refers to an entity (e.g., a peptide, polypeptide, complex, protein, etc.) that exhibits the characteristic of not emitting a detectable amount of light in the visible spectrum (e.g., in the presence of a substrate). For example, an entity may be referred to as non-luminescent if it does not exhibit detectable luminescence in a given assay. As used herein, the term "non-luminescent" is synonymous with the term "substantially non-luminescent". For example, a non-luminescent polypeptide (NLpoly) is substantially non-luminescent, and compared to a complex of NLpoly having its non-luminescent complementary peptide, luminescence exhibits a decrease of, for example, 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10 3 times, 1×10 4 times, 1×10 5 times, 1×10 6 times, 1×10 7 times, etc.). In some embodiments, an entity is "non-luminescent" if any light emission is sufficiently minimal so as not to create an interfering background for a particular assay.

[0090] As used herein, the terms "non-luminescent peptide" (e.g., NLpep) and "non-luminescent polypeptide" (e.g., NLpoly) are peptides and polypeptides that are substantially non-luminescent (e.g., in the presence of a substrate), or below noise, or when compared to a significant signal (e.g., a luminescent complex) using a typical instrument (e.g., a luminometer, etc.) under standard conditions (e.g., physiological conditions, assay conditions, etc.), exhibit an amount that is 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10 3 times, 1×10 4 times, 1×10 5 times, 1×10 6 times, 1×10 7 times, etc.) lower. In some embodiments, such non-luminescent peptides and polypeptides assemble according to the criteria described herein to form a bioluminescent complex. As used herein, a "non-luminescent element" is a non-luminescent peptide or non-luminescent polypeptide.

[0091] As used herein, the term "interaction element" or "interaction molecule" refers to a moiety that aids in bringing together a pair of non-luminescent elements or non-luminescent groups to form a bioluminescent complex. In typical embodiments, a pair of interaction elements (also known as an "interaction pair") binds to a pair of non-luminescent elements (e.g., a non-luminescent peptide / polypeptide pair), and the attractive interaction between the two interaction elements promotes the formation of the bioluminescent complex, although the invention is not limited to such a mechanism and understanding of the mechanism is not required for the practice of the invention. The interaction elements can promote the formation of the bioluminescent complex by any suitable mechanism (e.g., bringing non-luminescent pairs / groups into proximity, arranging non-luminescent pairs / groups in a suitable conformation for stable interaction, reducing the activation energy for complex formation, combinations thereof, etc.). The interaction elements can be proteins, polypeptides, peptides, small molecules, cofactors, nucleic acids, lipids, carbohydrates, antibodies, etc. The interaction pair can be composed of two of the same interaction element (i.e., a homodimer) or two different interaction elements (i.e., a heterodimer). In the case of a heterodimer, the interaction elements can be of the same type of moiety (e.g., polypeptide) or of two different types of moieties (e.g., polypeptide and small molecule). In some embodiments where complex formation by an interaction pair is being studied, the interaction pair can be referred to as a "target pair" or "pair of interest", and the individual interaction elements can be referred to as "target elements" (e.g., "target peptide", "target polypeptide", etc.) or "elements of interest" (e.g., "peptide of interest", "polypeptide of interest", etc.).

[0092] As used herein, the terms "fusion", "fusion polypeptide", and "fusion protein" refer to a chimeric protein containing a first protein or polypeptide (e.g., a target sequence, etc.) of interest conjugated to a second, different peptide, polypeptide, or protein (e.g., a detectable sequence, an isolatable sequence, a tag, etc.). The term "conventional fusion" refers to a fusion in which a first polypeptide or protein and a second peptide, polypeptide, or protein are fused end-to-end (e.g., from the C-terminus to the N-terminus, or from the N-terminus to the C-terminus).

[0093] As used herein, the terms "coelenterazine", "coelenterazine substrate", "coelenterazine derivative", or "coelenterazine derivative substrate" refer to a classification of reporter molecules that emit light when acted upon by various bioluminescent proteins such as luciferase (e.g., marine luciferase). As used herein, the terms "coelenterazine", "coelenterazine substrate", "coelenterazine derivative", or "coelenterazine derivative substrate" refer to naturally occurring ("native") coelenterazine. As used herein, the terms "coelenterazine", "coelenterazine substrate", "coelenterazine derivative", or "coelenterazine derivative substrate" refer to native coelenterazine as well as synthetic, e.g., derivatives or variants, and natural analogs thereof, including, in addition to those disclosed in WO2003 / 040100; U.S. Application No. 12 / 056,073 (paragraph

[0086] ); and U.S. Patent No. 8,669,103 (the disclosures of which are incorporated herein by reference in their entireties), coelenterazine-h, coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine.

[0094] As used herein, the term "sample" is used in its broadest sense. In one sense, it means including not only biological samples and environmental samples, but also samples or cultures obtained from any source. Biological samples are obtained from animals (including humans) and can include fluids, solids, tissues, and gases. Biological samples can include blood products such as plasma and serum. A sample can also refer to a cell lysate or purified form of the peptides and / or polypeptides described herein. Cell lysates can include cells lysed with a lysing agent or lysates such as rabbit reticulocyte or wheat germ lysates. A sample can also include a cell-free expression system. Environmental samples include environmental substances such as surface materials, soil, water, crystals, and industrial samples. However, such examples are not to be construed as limiting the types of samples applicable to the present invention.

[0095] The term "energy receptor" or "receptor molecule" refers to any small molecule (e.g., chromophore), macromolecule (e.g., autofluorescent protein, phycobiliprotein, nanoparticle, surface, etc.), or molecular complex that generates a readily detectable signal in response to energy absorption (e.g., resonance energy transfer). In certain embodiments, the energy receptor is a fluorophore or other detectable chromophore.Suitable fluorophores include, but are not limited to, xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), perylene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile Red, Nile Blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, phycobilisome) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, etc. In some embodiments, the fluorophore is a rhodamine analog (e.g., a carboxyrhodamine analog). In certain embodiments, energy acceptors include, but are not limited to, small molecule fluorescent dyes such as NCT, quenchers, fluorescent particles such as quantum dots, luminescent metal complexes, and any other known energy acceptors.

[0096] The terms "luminescent enzyme", "bioluminescent enzyme", or "luciferase", which are used interchangeably herein, refer to a classification of oxidizing enzymes used in bioluminescence that generate and emit light when the enzyme is provided with a substrate. Luciferase can be a naturally occurring luciferase, a recombinant luciferase, or a mutant luciferase that uses a luciferase substrate. Luciferase substrates can be luciferin, luciferin derivatives or analogs, pre-luciferin derivatives or analogs, coelenterazine, or coelenterazine derivatives or analogs. Luminescent enzymes, when naturally occurring, can be readily obtained from organisms by those skilled in the art. Whether the luminescent enzyme is a naturally occurring one, a recombinant or mutant luminescent enzyme, e.g., one that retains activity in the luciferase-coelenterazine or luciferase-luciferin reaction of a naturally occurring luminescent enzyme, it can be readily obtained from cultures of bacteria, yeast, mammalian cells, insect cells, plant cells, etc., that have been transformed to express a nucleic acid encoding the luminescent enzyme. Furthermore, recombinant or mutant luminescent enzymes can be derived from in vitro cell-free systems using nucleic acids encoding luciferase.Suitable luminescent enzymes include luciferases derived from bioluminescent decapod crustaceans such as Oplophoroidea (e.g., luciferase derived from Oplophorus), firefly luciferases (e.g., Photinus pyralis, Photuris pennsylvanica, etc.), marine organisms such as cnidarians (e.g., Renilla luciferase), Aristeidae, Solenoceridae, Luciferidae, Sergestidae, Pasipheidae, and Thalassocarididae decapod families, krill luciferases such as Gaussia luciferase (e.g., Gaussia princeps luciferase), Metridia luciferase (e.g., Metridia longa and Metridia pacifica luciferase), Vargula luciferase (e.g., Vargula hilgendorfii luciferase), Pleuromamma xiphias luciferase, and photoproteins such as aequorin, as well as variants, recombinants, and mutants thereof.

[0097] A "luminescent reaction mixture" contains substances that enable the luminescent enzyme to generate a light signal, i.e., luminescence. The mixture may also contain an enzyme, e.g., a luciferase enzyme or luciferase. The substances required to generate the luminescence signal, as well as the specific concentrations and / or amounts, vary depending on the luminescent enzyme used and the type of assay being performed. Often, other substances are added to the solution, such as a buffer to maintain the reaction at an appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) to assist in maintaining enzyme activity, reducing agents, detergents, etc.

[0098] As used herein, the terms “Oplophorus luciferase” and “Oplophorus-derived luciferase” are used interchangeably and refer to luciferase secreted from the deep-sea shrimp Oplophorus gracilirostris (e.g., SEQ ID NO: 1), including its wild-type, variants, and mutants. For example, suitable Oplophorus luciferase variants are described in U.S. Pat. Nos. 8,557,970 and 8,669,103, each of which is incorporated herein by reference in its entirety.

[0099] As used herein, the term "sequence identity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequence composition of monomer subunits. The term "sequence similarity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be classified into families, such as acidic (e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The "sequence identity rate" (or "sequence similarity rate") is calculated by (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specific window), (2) determining the number of positions that contain identical (or similar) monomers (e.g., the same amino acid is present in both sequences, similar amino acids are present in both sequences) to obtain the number of matching positions, (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specific window), and (4) multiplying the result by 100 to obtain the sequence identity rate or sequence similarity rate. For example, if both Peptides A and B are 20 amino acids in length and have the same amino acid at all positions except one, then Peptides A and B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., both are acidic), then Peptides A and B will have 100% sequence similarity. As another example, if Peptide C is 20 amino acids long and Peptide D is 15 amino acids long, and 14 of the 15 amino acids in Peptide D are identical to those of a portion of Peptide C, then Peptides C and D have 70% sequence identity, but Peptide D has 93.3% sequence identity relative to the optimal comparison window of Peptide C.For the purpose of calculating the "sequence identity" (or "sequence similarity") in this specification, any gap in the aligned sequences is treated as a mismatch at that position.

[0100] As used herein, the term "reporter moiety" can refer to a moiety that directly or indirectly generates a detectable signal under appropriate conditions. Exemplary reporter moieties include, but are not limited to, fluorophores, luminescent molecules, dyes, radiolabels, and substrates of enzymes such as luciferase. In some embodiments, the reporter moiety can indirectly generate a detectable signal, for example, when the reporter moiety is a substrate of an enzyme. Then, a detectable signal such as fluorescence or luminescence is generated by the reaction of the enzyme and the substrate. As used herein, the term "bioluminescent reporter moiety" can refer to a moiety that is a substrate of luciferase. For example, the bioluminescent reporter moiety can be luciferin, a luciferin derivative, such as pre-luciferin, aminoluciferin, quinolyl luciferin, naphthyl luciferin, fluoroluciferin, chloroluciferin, a precursor of a luciferin derivative, coelenterazine, or a coelenterazine derivative or analog, such as furimazine. The generated luminescence signal can be detected using a luminometer. As used herein, the term "fluorescent reporter moiety" can refer to a moiety that emits fluorescence. For example, the fluorescent reporter moiety can be a fluorophore such as coumarin, R110, fluorescein, DDAO, resorufin, cresyl violet, xanthene, or carbopyronin. Fluorescence can be detected using a fluorometer.

[0101] Regarding the description of numerical ranges in this specification, each intervening number having the same degree of precision is explicitly contemplated. For example, in the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0102] 2. Compound This specification provides compounds that can selectively inhibit bioluminescent complexes derived from Oplophorus luciferase, for example, compounds that can inhibit the luciferase activity of bioluminescent complexes. In one aspect, a compound of formula (I), or a salt thereof: JPEG2025105624000004.jpg43170(wherein: R 1 is aryl, cycloalkyl, heteroaryl, heterocycle, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocycle thereof are optionally substituted with one or more R W ; each R W is independently C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-10 alkylene-OR A , -CO-R A , -C 1-10 alkylene-CO-R A , -CO-OR A , -C 1-10 alkylene-CO-OR A , -CO-NHR A , -C 1-10 alkylene-CO-NHR A , -NR B R C , -C 1-10 alkylene-NR B R C , -NH-CO-C 1-4 alkyl, -C 1-10 alkylene-NH-CO-C 1-4 alkyl, phenyl, and phenyl substituted with 1, 2, 3, or 4 R D groups; and is selected from the group consisting of; each R 2 is independently C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-4 alkylene-OR A , -CO-RA 、 -C 1-4 alkylene - CO - R A 、 -CO - OR A 、 -C 1-4 alkylene - CO - OR A 、 -CO - NHR A 、 -C 1-4 alkylene - CO - NHR A 、 -NR B R C 、 -C 1-4 alkylene - NR B R C 、 -NH - CO - C 1-4 alkyl, -C 1-4 alkylene - NH - CO - C 1-4 alkyl, phenyl, phenyl substituted with 1, 2, 3, or 4 R D groups, -C≡C - R A 、 or -C≡C - C 1-4 alkylene - OR A and either two Rs 2 are such that they combine with the carbon atoms of the 1315 part of JPEG2025105624000005.jpg to form a 5 - or 6 - membered fused ring; each R 3 is independently C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A 、 -C 1-4 alkylene - OR A 、 -CO - R A 、 -CO - OR A 、 or -CO - NHR A and either two Rs 3 are such that they combine with the carbon atoms of the 1720 part of JPEG2025105624000006.jpg to form a 5 - or 6 - membered fused ring; R 4 is H or C 1-4 alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; each occurrence of RA is, independently, H, C 1-4 alkyl, or C 1-4 haloalkyl; R in each occurrence B and R C is, independently, H or C 1-4 alkyl, or R B and R C together with the N atom to which they are attached form a 5- or 6-membered heterocycle; R in each occurrence D is, independently, C 1-4 alkyl, -OC 1-4 alkyl, -CN, or halogen) is disclosed.

[0103] In some embodiments, in the compound of formula (I): R 1 is aryl, cycloalkyl, heteroaryl, or heterocycle, and the aryl, cycloalkyl, heteroaryl, and heterocycle are optionally substituted with one or more R W s, and each R W is, independently, C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A -, -C 1-10 alkylene-OR A -, -CO-R A -, -C 1-10 alkylene-CO-R A -, -CO-OR A -, -C 1-10 alkylene-CO-OR A -, -CO-NHR A -, -C 1-10 alkylene-CO-NHR A -, -NR B R C -, -C 1-10 alkylene-NR B R C -, -NH-CO-C 1-4 alkyl, -C 1-10 alkylene-NH-CO-C 1-4Selected from the group consisting of alkyl, phenyl, and phenyl substituted with 1, 2, 3, or 4 R D groups; Each R 2 is, independently, C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-4 alkylene-OR A , -CO-R A , -C 1-4 alkylene-CO-R A , -CO-OR A , -C 1-4 alkylene-CO-OR A , -CO-NHR A , -C 1-4 alkylene-CO-NHR A , -NR B R C , -C 1-4 alkylene-NR B R C , -NH-CO-C 1-4 alkyl, -C 1-4 alkylene-NH-CO-C 1-4 alkyl, phenyl, phenyl substituted with 1, 2, 3, or 4 R D groups, -C≡C-R A , or -C≡C-C 1-4 alkylene-OR A , or two Rs 2 are taken together with the carbon atoms of the JPEG2025105624000007.jpg1315 moiety to form a 5- or 6-membered fused ring; Each R 3 is, independently, C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-4 alkylene-OR A , -CO-R A , -CO-OR A , or -CO-NHR A , or two Rs 3 are taken together with the carbon atoms to which they are attached to form a 5- or 6-membered fused ring; combines with 1720 carbon atoms to form a 5- or 6-membered fused ring; R 4 is H or C 1-4 alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R in each occurrence A is independently H, C 1-4 alkyl, or C 1-4 haloalkyl; R in each occurrence B and R C are independently H or C 1-4 alkyl, or R B and R C combine with the N atom to which they are attached to form a 5- or 6-membered heterocyclic ring; R in each occurrence D is independently C 1-4 alkyl, -OC 1-4 alkyl, -CN, or halogen.

[0104] In some embodiments, R 1 is unsubstituted aryl, unsubstituted cycloalkyl, unsubstituted heteroaryl, unsubstituted heterocyclic, or unsubstituted arylalkyl.

[0105] In some embodiments, R 1 is unsubstituted aryl, unsubstituted cycloalkyl, unsubstituted heteroaryl, or unsubstituted heterocyclic.

[0106] In some embodiments, R 1 is aryl (such as C 6-20 aryl) or heteroaryl (such as 5- to 12-membered heteroaryl), and the aryl and heteroaryl are optionally substituted with one or more R W s. In some embodiments, R 1 is C 6-20 aryl, and the aryl is substituted with one or more R Wis optionally substituted. In some embodiments, R 1 is a 5- to 12-membered heteroaryl optionally substituted with one or more R W . In some embodiments, R 1 is a C W cycloalkyl optionally substituted with one or more R 5-10 . In some embodiments, R 1 is a 5- to 12-membered heterocyclic ring optionally substituted with one or more R W .

[0107] In some embodiments, R 1 is JPEG2025105624000009.jpg22170, each of which is optionally substituted with one or more R W . In some embodiments, R 1 is JPEG2025105624000010.jpg22170, each of which is unsubstituted. In some embodiments, R 1 is JPEG2025105624000011.jpg22170, each of which is substituted with C 1-4 alkyl or C 1-4 haloalkyl.

[0108] In some embodiments, R 1 is JPEG2025105624000012.jpg22170, each of which is optionally substituted with one or more R W . In some embodiments, R 1 is JPEG2025105624000013.jpg22170. In some embodiments, each R W is independently selected from the group consisting of C 1-10 alkyl, C 1-10 haloalkyl, halogen, or -CN. In some embodiments, R W is -OR A , -C 1-4Alkylene-OR A , -CO-R A , -C 1-4 Alkylene-CO-R A , -CO-OR A , -C 1-4 Alkylene-CO-OR A , -CO-NHR A , or -C 1-4 Alkylene-CO-NHR A In some embodiments, R W -NR B R C , -C 1-4 Alkylene-NR B R C , -NH-CO-C 1-4 Alkyl, or -C 1-4 Alkylene-NH-CO-C 1-4 In some embodiments, R W is phenyl or 1, 2, 3, or 4 R D In some embodiments, R W is C 1-10 Alkyl, C 1-10 haloalkyl, halogen, or -CN. In some embodiments, R W is C, such as methyl, ethyl, propyl, or butyl. 1-4 It is an alkyl.

[0109] In some embodiments, R W is C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, -OR A , -C 1-10 Alkylene-OR A , -CO-OR A , -C 1-10 Alkylene-CO-OR A In some embodiments, each R A is independently selected from hydrogen, methyl, and ethyl.

[0110] In some embodiments, R 1is one or more R W and is an arylalkyl (such as benzyl) optionally substituted with W .

[0111] In some embodiments, R 1 is aryl, cycloalkyl, heteroaryl, heterocycle, or arylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocycle are each substituted with one R W where R W is C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-10 alkylene-OR A , -CO-OR A , -C 1-10 alkylene-CO-OR A , and phenyl, and each R A is independently selected from hydrogen, methyl, and ethyl.

[0112] In some embodiments, R 1 is JPEG2025105624000014.jpg43170, each of which is unsubstituted or substituted with one R W where R W is C 1-10 alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, n-pentyl, n-hexyl, or n-octyl), C 1-10 haloalkyl (e.g., -(CH2)4Br), halogen (e.g., fluoro, chloro, or bromo), -CN, -OR A (e.g., -OCH3), -C 1-10 alkylene-OR A (e.g., -(CH2)4-OH or -(CH2)6-OH), -CO-OR A (e.g., -COOH, -COOCH3, or -COOCH2CH3), -C 1-10 alkylene-CO-OR AIt is selected from the group consisting of, for example, -CH2COOH, -CH2COOCH2CH3, -(CH2)5-COOH, or -(CH2)5-COOCH3, and phenyl.

[0113] In some embodiments, p is 0, 1, or 2. In some embodiments, p is 0. In some embodiments, p is 1.

[0114] In some embodiments, R 2 is C 1-10 alkyl, halogen, C 1-4 haloalkyl, -OH, C 1-4 alkylene-OH, -OC 1-4 alkyl, -NH 2、 phenyl, or -CO-OC 1-4 alkyl. In some embodiments, R 2 is C 1-10 alkyl. In some embodiments, R 2 is C 1-4 alkyl such as methyl, ethyl, propyl, or butyl. In some embodiments, R 2 is methyl. In some embodiments, R 2 is halogen, C 1-4 haloalkyl, -OH, C 1-4 alkylene-OH, -OC 1-4 alkyl, or -NH2.

[0115] In some embodiments, R 2 is C 1-10 alkyl, C 1-10 haloalkyl, halogen, -OR A , -C 1-4 alkylene-OR A , -CO-R A , -CO-OR A , -C 1-4 alkylene-CO-OR A , -CO-NHR A , -NR B R C , -NH-CO-C 1-4Alkyl, phenyl, and -C≡C-C 1-4 Alkylene-OR A selected from, each R A is independently selected from hydrogen, methyl, ethyl, and trifluoromethyl.

[0116] In some embodiments, R 2 is C 1-10 alkyl (e.g., C such as methyl, ethyl, isopropyl, n-propyl, n-butyl, n-pentyl, or n-hexyl 1-6 alkyl), C 1-10 haloalkyl (e.g., C such as trifluoromethyl 1-4 haloalkyl), halogen (e.g., fluoro, chloro, or bromo), -OR A (e.g., -OH, -OCH3, -O(CH2)3CH3, or -OCF3), -C 1-4 alkylene-OR A (e.g., -CH2OH or -(CH2)3-OH), -CO-R A (e.g., -COH), -CO-OR A (e.g., -COOH or -COOCH3), -C 1-4 alkylene-CO-OR A (e.g., -(CH2)2COOCH3, -CO-NHR A (e.g., -CONH(CH2)3CH3, -NR B R C (e.g., -NH2 or -NH(CH2)3CH3, -NH-CO-C 1-4 alkyl (e.g., -NH-CO-CH3), phenyl, -C≡C-R A (e.g., -C≡C-(CH2)3CH3) and -C≡C-C 1-4 alkylene-OR A (e.g., -C≡C-CH2-OH) selected from. In some embodiments, each R A is independently selected from hydrogen, methyl, ethyl, n-propyl, and n-butyl.

[0117] In some embodiments, p is 2. In some embodiments, two Rs2 combine with the carbon atoms of the 1315 moiety thereof to form a 5- or 6-membered fused ring. For example, two Rs JPEG2025105624000015.jpg1315 moiety to form 2 combine with the carbon atoms of the 1315 moiety thereof to form JPEG2025105624000016.jpg1315 moiety to form JPEG2025105624000017.jpg1326.

[0118] In some embodiments, q is 0, 1, or 2. In some embodiments, q is 0. In some embodiments, q is 1.

[0119] In some embodiments, R 3 is C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, and -OR A selected from the group consisting of, and R A is selected from hydrogen and C 1-4 alkyl. In some embodiments, R 3 is C 1-10 alkyl (such as C 1-4 alkyl like methyl, ethyl, isopropyl, n-propyl, or n-butyl), C 1-10 haloalkyl (such as C 1-4 haloalkyl like trifluoromethyl), halogen (such as fluoro, chloro, or bromo), -CN, and -OR A (such as -OH or -OCH3) selected from the group consisting of. In some embodiments, R 3 is C 1-4 alkyl, halogen, -CN, -OH, or -OC 1-4 alkyl. In some embodiments, R 3 is halogen. In some embodiments, R 2 is C 1-4 alkyl (such as methyl), and R 3 is C 1-4 alkyl, halogen, -CN, -OH, or -OC 1-4It is alkyl.

[0120] In some embodiments, q is 2. In some embodiments, the two Rs 3 together with the carbon atoms of the portion to which they are attached form a 5- or 6-membered fused ring. For example, the two Rs 3 together with the portion to which they are attached may form JPEG2025105624000020.jpg2622.

[0121] In some embodiments, p is 1 and q is 0. In some embodiments, p is 1 and q is 1. In some embodiments, R 2 is C 1-4 alkyl (such as methyl), p is 1, and q is 0. In some embodiments, R 2 is C 1-4 alkyl (such as methyl), R 3 is C 1-4 alkyl, halogen, -CN, -OH, or -OC 1-4 alkyl, p is 1, and q is 1. In some embodiments, R 2 is methyl, R 3 is -CH3, halogen (such as F), -OCH3, or -CN, p is 1, and q is 1.

[0122] In some embodiments, R 4 is H. In some embodiments, R 4 is C 1-4 alkyl. In some embodiments, R 4 is ethyl.

[0123] Each occurrence of R A (R 1 , R 2 , or R 3As substituents in), independently, are H, C 1-4 alkyl, or C 1-4 haloalkyl. In some embodiments, R A is H. In some embodiments, R A is C 1-4 alkyl. In some embodiments, R A is methyl. In some embodiments, R A is C 1-4 haloalkyl such as -CF3.

[0124] In each occurrence, R B and R C (as substituents in R 1 or R 2 ) are independently H or C 1-4 alkyl, or R B and R C together with the N atom to which they are attached form a 5- or 6-membered heterocycle. In some embodiments, R B and R C are both H. In some embodiments, R B is H and R C is C 1-4 alkyl. In some embodiments, R B is C 1-4 alkyl and R C is C 1-4 alkyl. In some embodiments, R B and R C together with the N atom to which they are attached form a 5- or 6-membered heterocycle.

[0125] In some embodiments, R D is C 1-4 alkyl such as methyl. In some embodiments, R D is -OC 1-4 alkyl such as -OCH3. In some embodiments, R D is -CN or a halogen.

[0126] In some embodiments, R 1 is JPEG2025105624000021.jpg22170, each of which is C 1-4 alkyl or C 1-4 haloalkyl substituted, or R 1 is JPEG2025105624000022.jpg22170, R 2 is 1-4 alkyl, halogen, C 1-4 haloalkyl, -OH, C 1-4 alkylene-OH, -OC 1-4 alkyl, or -NH2, and R 3 is 1-4 alkyl, halogen, -CN, -OH, or -OC 1-4 alkyl, p is 1, and q is 0 or 1.

[0127] In some embodiments, when p is 0, then R 1 is substituted with one or more R W (e.g., one R W ). In some embodiments, when p is 0 and R 1 is phenyl, then R 1 is substituted with one or more R W (e.g., one R W ). In some embodiments, when R 1 is unsubstituted, then p is 1, 2, 3, or 4 (e.g., p is 1 or 2, or p is 1). In some embodiments, when R 1 is unsubstituted phenyl, then p is 1, 2, 3, or 4 (e.g., p is 1 or 2, or p is 1). In some embodiments, the compound is not N-phenyl-2-(phenylsulfonamide)benzamide.

[0128] In some embodiments, the compound of formula (I) is a compound of formula (I-a), or a salt thereof, JPEG2025105624000023.jpg37170 (wherein R 1 is aryl or 5- to 12-membered heteroaryl, and the aryl and heteroaryl are each optionally substituted with one or more R 6-20 ; W p is 0, 1, or 2; q is 0 or 1; R 3 is C 1-4 alkyl, halogen, -CN or -OR A ; A R is H or C 1-4 alkyl; 2 R and R W are as defined herein) 1 ). is.

[0129] In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 6-20 is unsubstituted C 1 aryl or unsubstituted 5- to 12-membered heteroaryl) is disclosed.

[0130] In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R W is JPEG2025105624000024.jpg22170, each optionally substituted with one or more R 1 ). In some embodiments, R of formula (I-a) 1 is JPEG2025105624000025.jpg22170.

[0131] In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 1-4 is C 1-4 alkyl or C 1 haloalkyl) is disclosed. For example, R of formula (I-a) 1-4 is It can be JPEG2025105624000026.jpg22170, each being C 1-4 alkyl or C 1-4 is substituted with haloalkyl.

[0132] In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 1 is JPEG2025105624000027.jpg22170, each of which is optionally substituted with one or more R W is disclosed. In some embodiments, R 1 is JPEG2025105624000028.jpg22170.

[0133] In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 1 is JPEG2025105624000029.jpg43170, each of which is unsubstituted or substituted with one R W , where R W is C 1-10 alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, n-butyl, n-pentyl, n-hexyl, or n-octyl), C 1-10 haloalkyl (e.g., -(CH2)4Br), halogen (e.g., fluoro, chloro, or bromo), -CN, -OR A (e.g., -OCH3), -C 1-10 alkylene-OR A (e.g., -(CH2)4-OH or -(CH2)6-OH), -CO-OR A (e.g., -COOH, -COOCH3, or -COOCH2CH3), -C 1-10 alkylene-CO-OR A (e.g., -CH2COOH, -CH2COOCH2CH3, -(CH2)5-COOH, or -(CH2)5-COOCH3), and is selected from the group consisting of phenyl) is disclosed.

[0134] In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 2 is C 1-4 alkyl (such as methyl, ethyl, propyl, or butyl), halogen, C 1-4 haloalkyl, -OH, C 1-4 alkylene-OH, -OC 1-4 alkyl, -NH 2、 phenyl, or -CO-OC 1-4 alkyl, or R 2 is such that they are attached to the carbon atoms of the JPEG2025105624000030.jpg1315 moiety to form a 5- or 6-membered fused ring) is disclosed. For example, two Rs of formula (I-a) 2 is such that it is attached to the JPEG2025105624000031.jpg1315 moiety to form JPEG2025105624000032.jpg1326. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 2 is C 1-4 alkyl (such as methyl)) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 2 is -OH, -C 1-4 alkylene-OH (such as -CH2OH), -OC 1-4 alkyl (such as -OCH3), or -NH2) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein p is 1 and q is 0) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 2 is C 1-4 alkyl (such as methyl), -OH, -C 1-4 alkylene-OH (such as -CH2OH), -OC 1-4is alkyl (such as -OCH3) or -NH2, p is 1, and q is 0) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein p is 1 and q is 1) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 2 is methyl and R 3 is -CH3, halogen (such as F), -OCH3, or -CN, p is 1, and q is 1) is disclosed.

[0135] In some embodiments, a compound of formula (I-a), or a salt thereof (wherein p is 0 and when R 1 is phenyl, then R 1 is substituted with one or more R W is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein when R 1 is unsubstituted phenyl, then p is 1, 2, 3, or 4 (for example, p is 1 or 2, or p is 1)) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein when p is 0, then R 1 is substituted with one or more R W (for example, with one R W ) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein p is 0 and when R 1 is phenyl, then R 1 is substituted with one or more R W (for example, with one R W ) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein when R 1 is unsubstituted, then p is 1, 2, 3, or 4 (for example, p is 1 or 2, or p is 1)) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof (wherein R 1When it is unsubstituted phenyl, then p is 1, 2, 3, or 4 (for example, p is 1 or 2, or p is 1) is disclosed. In some embodiments, a compound of formula (I-a), or a salt thereof, wherein the compound is not N-phenyl-2-(phenylsulfonamido)benzamide is disclosed.

[0136] In some embodiments, the compound of formula (I-a) is a compound of formula (I-a-1), or a salt thereof, JPEG2025105624000033.jpg43170(wherein: R 1 is JPEG2025105624000034.jpg22170, each of which is optionally substituted with halogen, C 1-4 alkyl, C 1-4 haloalkyl, or phenyl; or R 1 is JPEG2025105624000035.jpg20150; R 2 is halogen, C 1-4 alkyl, C 1-4 haloalkyl, -OH, -C 1-4 alkylene-OH, -OC 1-4 alkyl, or -NH2; R 3 and q are as defined in formula (I-a)) is.

[0137] In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 1 is JPEG2025105624000036.jpg22170) is disclosed. In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 1 is JPEG2025105624000037.jpg22170, each of these being halogen, C 1-4 alkyl, C 1-4Halogenoalkyl, or phenyl-substituted, is disclosed. In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 1 is JPEG2025105624000038.jpg22170) is disclosed.

[0138] In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 2 is C 1-4 alkyl (such as methyl), halogen (such as Br), or C 1-4 halogenoalkyl (such as -CF3)) is disclosed. In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 2 is C 1-4 alkyl (such as methyl)) is disclosed. In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 2 is -OH, -C 1-4 alkylene-OH (such as -CH2OH), -OC 1-4 alkyl (such as -OCH3), or -NH2) is disclosed. In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 2 is C 1-4 alkyl (such as methyl), halogen (such as Br), -OH, C 1-4 alkylene-OH (such as -CH2-OH), -OC 1-4 alkyl (such as -OCH3), or -NH2, and q is 0) is disclosed. In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 2 is C 1-4 alkyl (such as methyl) and q is 0) is disclosed. In some embodiments, a compound of formula (I-a-1), or a salt thereof (wherein R 2 is C 1-4 alkyl (such as methyl), R 3 is -CH3, halogen (such as F), -OCH3, or -CN, and q is 1) is disclosed.

[0139] Suitable compounds include: N-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-Cyanothiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(2-Cyanophenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 2-((4-Methylphenyl)sulfonamido)-N-phenylbenzamide; N-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((4-formylphenyl)sulfonamido)benzamide; Methyl 3-(4-(N-(2-(benzo[b]thiophen-2-ylcarbamoyl)phenyl)sulfamoyl)phenyl)propanoate; N-(Benzo[b]thiophen-2-yl)-2-((3-methylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((4-(3-hydroxypropyl)phenyl)sulfonamido)benzamide; 2-([1,1'-Biphenyl]-3-sulfonamido)-N-(p-tolyl)benzamide; Methyl 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoate; 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoic acid; 2-((3-Acetamidophenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-Aminophenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(Hydroxymethyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(Butylcarbamoyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-Bromophenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(Butylamino)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(Hex-1-yn-1-yl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-Hexylphenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(3-Hydroxyprop-1-yn-1-yl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(3-Hydroxypropyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; N-(p-Tolyl)-2-((4-(trifluoromethyl)phenyl)sulfonamido)benzamide; 2-((4-Methoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((4-Bromophenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-([1,1'-Biphenyl]-4-sulfonamido)-N-(p-tolyl)benzamide; N-(p-Tolyl)-2-((3-(trifluoromethyl)phenyl)sulfonamido)benzamide; N-(p-Tolyl)-2-((3-(trifluoromethoxy)phenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-Cyclohexyl-2-((4-methylphenyl)sulfonamido)benzamide; 2-((4-Methylphenyl)sulfonamido)-N-(naphthalen-2-yl)benzamide; 2-((4-Methylphenyl)sulfonamido)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzamide; Methyl trans-4-(2-((4-methylphenyl)sulfonamido)benzamido)cyclohexane-1-carboxylate; Trans-4-(2-((4-methylphenyl)sulfonamido)benzamido)cyclohexane-1-carboxylic acid; 2-((4-Methylphenyl)sulfonamido)-N-(p-tolyl)benzamide; Ethyl 2-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)acetate; N-(3-Isopropylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; Ethyl 3-(2-((4-methylphenyl)sulfonamido)benzamido)benzoate; 2-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)acetic acid; 3-(2-((4-methylphenyl)sulfonamido)benzamido)benzoic acid; 2-((4-Methylphenyl)sulfonamido)-N-(m-tolyl)benzamide; N-(Benzo[b]thiophen-2-yl)-3-((4-methylphenyl)sulfonamido)-2-naphthamide; 2-((4-Methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(Benzo[b]thiophen-2-yl)-5-methyl-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-Butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-5-cyano-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-Butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzamide; N-(4-Hexylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 2-((4-Methylphenyl)sulfonamido)-N-(4-octylphenyl)benzamide; Methyl 6-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)hexanoate; 6-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)hexanoic acid; N-(Benzo[b]thiophen-2-yl)-2-((4-butylphenyl)sulfonamido)benzamide; N-(4-(6-Hydroxyhexyl)phenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((4-pentylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-5-butyl-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-(4-Hydroxybutyl)phenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-(4-Bromobutyl)phenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 5-Methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide; N-(Benzo[b]thiophen-2-yl)-5-methoxy-2-((4-methylphenyl)sulfonamido)benzamide; 4-Methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide; N-(Benzo[b]thiophen-2-yl)-4-methoxy-2-((4-methylphenyl)sulfonamido)benzamide; 2-((3-Methoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((3-methoxyphenyl)sulfonamido)benzamide; 5-Hydroxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-Hydroxyphenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-Butoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide; N-(2-Bromophenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-Bromophenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-([1,1'-Biphenyl]-4-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(2-Methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-([1,1'-Biphenyl]-3-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-Methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-Methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 2-((N-Ethyl-4-methylphenyl)sulfonamido)-N-(4-methoxyphenyl)benzamide; N-(Benzo[b]thiophen-2-yl)-4-fluoro-2-((4-methylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-5-fluoro-2-((4-methylphenyl)sulfonamido)benzamide; N-Benzyl-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-Methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide; and N-(Benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzamide, or a salt thereof is included.

[0140] (1) Salt form The thienopyrrole compounds described herein can be in salt form. The neutral form of the compound can be regenerated by contacting the salt with a base or an acid and isolating the parent compound by conventional techniques. The parent form of the compound differs from the various salt forms in certain physical properties such as solubility in polar solvents, but otherwise, the salts are equivalent to the parent form of the compound for the purposes of this disclosure.

[0141] For example, if the compound is anionic or has a functional group that can become anionic (e.g., -COOH can become -COO - ), then the salt can be formed with a suitable cation. Examples of suitable inorganic cations include alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and others. Examples of suitable organic cations include ammonium ions (i.e., NH4 + ) and substituted ammonium ions (e.g., NH3R1 + , NH2R2 + , NHR3 + , NR4 + ), but are not limited thereto. Examples of some suitable substituted ammonium ions are ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and those derived from amino acids such as lysine and arginine.

[0142] If the compound is cationic or has a functional group that can become cationic (e.g., -NH2 can become -NH3+ and when that is the case, the salt can be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0143] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose.

[0144] Unless otherwise specified, references to specific thienopyrrole compounds herein include their salt forms as well.

[0145] (2) Isomers The specified thienopyrrole compounds can exist in one or more specific geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including, but not limited to, cis and trans forms, E and Z forms, c, t, and r forms; endo and exo forms; R, S, and meso forms; D and L forms; d and l forms; (+) and (-) forms; keto, enol, and enolate forms; syn and anti forms; synclinal and anticlinal forms; a and β forms; axial and equatorial forms; boat, chair, twist, envelope, and half-chair forms; and combinations thereof, collectively referred to herein as "isomers" (or "isomeric forms").

[0146] In some embodiments, the compounds described herein can be enantiomerically enriched isomers of the stereoisomers described herein. For example, the compounds can have an enantiomeric excess of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Enantiomers, as used herein, refer to either of a pair of chemical compounds whose molecular structures are mirror images of each other.

[0147] In some embodiments, preparations of the compounds disclosed herein are enriched for isomers of the compounds having a selected stereochemistry corresponding to a selected stereocenter, e.g., R or S. For example, the compounds have a purity corresponding to at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the compounds having the selected stereochemistry of the selected stereocenter.

[0148] In some embodiments, the compositions described herein include preparations of the compounds disclosed herein that are enriched for one or more structures having a selected stereochemistry, e.g., R or S, at a selected stereocenter. Exemplary R / S configurations can be those provided in the examples described herein.

[0149] "Enriched preparation", as used herein, is enriched for a selected stereoconfiguration of one, two, three or more selected stereocenters within the compound of interest. Exemplary selected stereocenters and their exemplary stereoconfigurations can be selected herein, for example, from those provided in the examples described herein. Enriched means that, for example, at least 60% of the molecules of the compound in the preparation have the selected stereochemistry of the selected stereocenter. In embodiments, it is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Enriched refers to the level of the molecule(s) of interest and does not imply process limitations unless otherwise specified.

[0150] The compounds can be prepared in racemic form or as individual enantiomers or diastereomers by either stereospecific synthesis or resolution. The compounds can be resolved into their enantiomers or diastereomers by standard techniques such as, for example, formation of stereoisomeric pairs by salt formation with an optically active base followed by fractional crystallization and regeneration of the free acid. The compounds can also be resolved by formation of esters or amides of the stereoisomers followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds can be resolved using a chiral HPLC column. Enantiomers can also be obtained from the kinetic resolution of a racemic compound of the corresponding ester using a lipase enzyme.

[0151] Except for tautomers, as discussed below, structural (or constitutional) isomers (i.e., isomers that differ not merely in the position of atoms in space but in the bonds between atoms) are specifically excluded from the term “isomer” as used herein. For example, reference to a methoxy group, i.e., -OCH3, should not be construed as a reference to its structural isomer, a hydroxymethyl group, i.e., -CH2OH. Similarly, reference to ortho-chlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, reference to a classification of structures may fully include structural isomeric forms that fall within that classification (e.g., C3-alkyl or propyl includes n-propyl and iso-propyl; C4-alkyl or butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

[0152] The above exclusion does not relate to tautomeric forms, such as the following tautomeric pairs: keto / enol, imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, N-nitroso / hydroxyazo, and nitro / aci-nitro, and keto-, enol-, and enolate-forms.

[0153] In particular, it should be noted that the term “isomer” includes compounds having one or more isotope substitutions. For example, H can be any isotopic form including 1 H, 2 H(D), and 3 H(T); C can be any isotopic form including 12 C, 13 C, and 14 C; O can be any isotopic form including 16 O and 18 O, and so on.

[0154] 3. Bioluminescent complex derived from Oplophorus luciferase The disclosed compounds can be used to inhibit an Oplophorus luciferase-derived bioluminescent complex. The disclosed compounds can inhibit the luciferase activity of an Oplophorus luciferase-derived bioluminescent complex. The Oplophorus luciferase can be a wild-type Oplophorus luciferase or a variant of Oplophorus luciferase. The Oplophorus luciferase can be a variant of the luciferase of SEQ ID NO: 7. Variants of Oplophorus luciferase are described in U.S. Patent Nos. 8,557,970 and 8,669,103, each of which is incorporated herein by reference in its entirety.

[0155] The bioluminescent complex derived from Oplophorus luciferase can be an aggregate of two or more non-luminescent peptides and / or polypeptide units (e.g., non-luminescent pairs). The bioluminescent complex derived from Oplophorus luciferase comprises: (a) a peptide comprising a peptide amino acid sequence having a sequence identity of less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) with SEQ ID NO: 2 and more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%); and (b) a polypeptide comprising a polypeptide amino acid sequence having a sequence identity of less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) with SEQ ID NO: 3 and more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%), and the bioluminescent complex exhibits detectable luminescence in the presence of the coelenterazine substrate. In certain embodiments, the invention provides a bioluminescent complex comprising: (a) a peptide comprising the peptide amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6; and (b) a polypeptide comprising the polypeptide amino acid sequence of SEQ ID NO: 5, wherein the bioluminescent complex exhibits detectable luminescence in the presence of the coelenterazine substrate. Exemplary bioluminescent complexes derived from Oplophorus luciferase include the NanoBiT® technology comprising the NanoBiT® SmBiT non-luminescent peptide (SEQ ID NO: 6), the NanoBiT® HiBiT non-luminescent peptide (SEQ ID NO: 4), and / or the NanoBiT® LgBiT non-luminescent polypeptide (SEQ ID NO: 5). Bioluminescent complexes derived from Oplophorus luciferase are described in U.S. Pat. Nos. 9,797,889 and 9,797,890, each of which is incorporated herein by reference in its entirety.

[0156] 4. Coelenterazine Substrate The disclosed compounds of the present invention can be used to inhibit luciferase activity by binding to luciferase and thus competing with or interfering with the coelenterazine or coelenterazine derivative substrates. Coelenterazine substrates are a class of reporter molecules that emit light when acted upon by luciferase and other bioluminescent proteins. Examples of coelenterazine substrates include coelenterazine; in addition to those disclosed in WO2003 / 040100, US Patent Publication No. 2008 / 0248511, and US Patent Publication No. US2012 / 0117667, coelenterazine derivatives and / or analogs such as 2-furanylmethyl-deoxy-coelenterazine (furimazine), coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl-coelenterazine; pro-coelenterazine (i.e., a compound that is not a substrate of a non-luminescent enzyme that converts the compound to a substrate of luciferase), quinone-masked coelenterazine, and the like, but are not limited thereto. Further examples of coelenterazine substrates are described, for example, in US Publication No. 2012 / 0107849, US Publication No. 2013 / 0130289, US Patent Application No. 14 / 608,910, and US Patent Application No. 14 / 609,372, each of which is incorporated herein by reference.

[0157] 5.Method for inhibiting the bioluminescence complex activity derived from Oplophorus luciferase The disclosed compounds inhibit the bioluminescent complex derived from Oplophorus luciferase and can be used, for example, in a method for inhibiting the luciferase activity of the bioluminescent complex. The method can include contacting the compounds disclosed herein with cells that express or contain the bioluminescent complex derived from Oplophorus luciferase or with non-luminescent peptides and / or polypeptides of the bioluminescent complex derived from Oplophorus luciferase, and the disclosed compounds can selectively inhibit the bioluminescent complex derived from Oplophorus luciferase. The method can include contacting the compounds disclosed herein with non-luminescent peptides and / or polypeptides of the bioluminescent complex derived from Oplophorus luciferase, and the disclosed compounds inhibit the bioluminescent complex derived from Oplophorus luciferase when the complex assembles. The disclosed compounds can be used to selectively inhibit the signal from the bioluminescent complex derived from Oplophorus luciferase in an assay used to detect the presence or activity of an enzyme that uses the bioluminescent complex derived from Oplophorus luciferase. For example, the disclosed compounds can be used in a bioluminescence method that employs the bioluminescent complex derived from Oplophorus luciferase and coelenterazine or a coelenterazine derivative substrate to detect one or more molecules in a sample, such as a target protein (e.g., an enzyme, a binding partner, a ligand, etc.), a cofactor of an enzyme reaction, an enzyme substrate, an enzyme inhibitor, an enzyme activator, or an OH group, or one or more conditions, such as redox conditions. While coelenterazine substrate acts as a substrate for the bioluminescent complex derived from Oplophorus luciferase, the disclosed compounds can act to inhibit the luciferase-derived bioluminescent complex and selectively suppress the luminescence signal in embodiments, where such suppression can be desired, for example, in applications involving temporal multiplexing of multiple bioluminescence systems or in some plate-based luminescence assays. For example, the disclosed compounds can be used to inhibit the activity of intracellular and / or extracellular bioluminescent complexes derived from Oplophorus luciferase.

[0158] (1) Protein complementation assay According to the above, the disclosed compounds can be used to inhibit such a bioluminescent complex when the Oplophorus luciferase-derived bioluminescent complex is used in other methods for detecting ligand-protein and protein-protein interactions or proximity or co-localization, such as protein complementation assays (PCA) or enzyme fragment complementation (EFC) assays. PCA and EFC assays provide a means for detecting the interaction of two interacting elements, such as biomolecules, polypeptides. PCA utilizes two fragments of the same protein that generate light only when they are brought into proximity to each other via the binding interaction of their fusion partners, such as the polypeptide of interest, and reassembled into a functionally active protein, such as an enzyme. For example, the NANOBIT® technology (Promega Corporation) can be used to detect molecular proximity by the reassembly of a luminescent enzyme via the binding interaction of enzyme units. The NanoBiT® system can include two or more non-luminescent peptides and / or polypeptide units that can be expressed as fusions with the target molecule of interest. In some embodiments, the two units can include the NanoBiT® LgBiT non-luminescent polypeptide (NLpoly) and the NanoBiT® SmBiT non-luminescent peptide (NLpep). In some embodiments, the two units can include the NanoBiT® LgBiT non-luminescent polypeptide (NLpoly) and the NanoBiT® HiBiT non-luminescent peptide (NLpep). The Oplophorus luciferase-derived bioluminescent complex is described in U.S. Patent Nos. 9,797,889 and 9,797,890, each of which is incorporated herein by reference in its entirety.

[0159] For example, Oplophorus luciferase or a variant of Oplophorus luciferase can be separated, for example, in a portion (s) tolerant to separation, into two units, for example, into a non - luminescent peptide or polypeptide, and each unit, for example, the non - luminescent peptide or polypeptide, can be fused to one of a pair of polypeptides that are intended to interact, for example, one of FKBP and FRB. When the two target polypeptides interact, the non - luminescent units then, for example, reassemble into a bioluminescent complex in proximity to each other. In some embodiments, the activity of the bioluminescent complex can then be detected and measured. In some embodiments, the bioluminescent complex can be used in a more general complementary system similar to lac - Z (Langley et al., PNAS 72:1254 - 1257 (1975)) or ribonuclease S (Levit and Berger, J. Biol. Chem. 251:1333 - 1339 (1976)). In some embodiments, a luminescent enzyme unit (designated "A") known to be complementary to another luminescent enzyme unit ("B") can be fused to a target protein, and the resulting fusion can be monitored via luminescence in a cell or cell lysate containing fragment B. In some embodiments, the source of unit B can be the same cell (for example, if the gene for unit B is integrated into the cell's genome or contained in another plasmid within the cell), or it can be a lysate or purified protein derived from another cell. In some embodiments, this same fusion protein (unit A) can be captured or immobilized using a fusion between unit B and a polypeptide such as HaloTag that can bind to a solid support. In some embodiments, luminescence can be used to demonstrate successful capture or to quantify the amount of the captured substance.

[0160] (2) Molecular detection assay Accordingly, the disclosed compounds can be used to inhibit an Oplophorus luciferase-derived bioluminescent complex when such a bioluminescent complex is used in other methods for detecting a target molecule. For example, the NANOBIT® technology (Promega Corporation) can be used to detect a target molecule. The NanoBiT® system can include two or more non-luminescent peptide and / or polypeptide units. One or more of the non-luminescent peptide and / or polypeptide units can be fused to a target molecule. The two units can include the NanoBiT® LgBiT non-luminescent polypeptide (NLpoly) and the NanoBiT® HiBiT non-luminescent peptide (NLpep). In some embodiments, the NanoBiT® HiBiT NLpep can be fused to a target molecule. A sample containing the NanoBiT® HiBiT NLpep fused to a target molecule can be contacted with the NanoBiT® LgBiT NLpoly. For example, the NanoBiT® LgBiT NLpoly can be added to a detection reagent containing the coelenterazine substrate. The resulting bioluminescence can be detected, measured, and inhibited with the compounds disclosed herein. In some embodiments, the NanoBiT® LgBiT NLpoly can be fused to a target molecule. A sample containing the NanoBiT® LgBiT NLpoly fused to a target molecule can be contacted with the NanoBiT® HiBiT NLpep. For example, the NanoBiT® HiBiT NLpep can be added to a detection reagent containing the coelenterazine substrate.

[0161] (3) Use of cell-impermeable compounds In certain embodiments, the methods disclosed herein include contacting a sample (e.g., a cell) with a mixture of a cell-permeable coelenterazine substrate and a compound described herein that has been modified to be cell-impermeable. In such embodiments, the disclosed compounds and methods can be used to construct an initial luminescence in a high-throughput screening assay format and then selectively inhibit any Oplophorus luciferase-derived bioluminescent complex that can be excreted from the cell, to selectively inhibit luminescence that can occur outside the cell. Such methods can provide a more selective signal within the cell.

[0162] (4) Use of cell-permeable compounds In certain embodiments, the methods disclosed herein include contacting a sample (e.g., a cell) with a mixture of a cell-permeable coelenterazine substrate and a cell-permeable compound described herein. In such embodiments, the disclosed compounds can enter the cell and selectively inhibit an Oplophorus luciferase-derived bioluminescent complex therein. Such methods can be advantageous in multiplex assays that involve the use of two or more luciferases, and can allow for the inhibition of luminescence from an Oplophorus luciferase-derived bioluminescent complex so that luminescence from another luciferase within the cell can be selectively observed.

[0163] (5) Use with transcriptional reporters The disclosed compounds can be used with gene transcription reporter systems. In certain embodiments, a method for measuring the activity of a promoter in a sample, wherein the promoter is operably linked to a gene encoding a non-luminescent unit of a bioluminescent complex derived from Oplophorus luciferase, e.g., a non-luminescent polypeptide, is provided. The method comprises (a) contacting a sample expressing a non-luminescent polypeptide of a bioluminescent complex derived from Oplophorus luciferase that is capable of forming a bioluminescent complex with a non-luminescent peptide of the bioluminescent complex derived from Oplophorus luciferase fused to the promoter, with coelenterazine substrate and a non-luminescent peptide of the bioluminescent complex derived from Oplophorus luciferase that is capable of forming a bioluminescent complex with the non-luminescent polypeptide of the bioluminescent complex derived from Oplophorus luciferase; and (b) determining the activity of the promoter by measuring the luminescence of the sample. The method may further comprise contacting the sample with a compound described herein to selectively inhibit the luminescence produced from the bioluminescent complex. The promoter may be operably linked to the gene via a translational or transcriptional fusion. The biological pathway of interest may be examined, for example, by treating cells containing a promoter operably linked to a gene encoding a non-luminescent unit of a bioluminescent complex derived from Oplophorus luciferase, e.g., a non-luminescent polypeptide, with an agent that induces the pathway. The activity of this promoter is then measured and monitored to study any correlation between the activity of the promoter and the pathway of interest, and kinetic measurements related to gene expression (e.g., induction, repression, and activation) may be obtained. The compounds described herein can be used to selectively inhibit the luminescence from a bioluminescent complex derived from Oplophorus luciferase.

[0164] (6) Multiplexing The disclosed compounds can be used to inhibit Oplophorus luciferase-derived bioluminescent complexes when applied to time multiplexing with other luciferases and assays. In some embodiments, the Oplophorus luciferase-derived bioluminescent complex can be multiplexed with another enzyme (e.g., luciferase) that emits light at a different wavelength, such as green firefly luciferase, such as Photinus pyralis (e.g., Luc2; Promega Corp) or red rice weevil luciferase (CHROMA-LUC™ luciferase; Promega Corp.). For example, when the Oplophorus luciferase-derived bioluminescent complex is used as a functional reporter, then green firefly luciferase or red CHROMA-LUC™ luciferase can be used to control for non-specific effects on gene regulation or to normalize for transfection efficiency. In some embodiments, the luminescence generated from the Oplophorus luciferase-derived bioluminescent complex (approximately 460 nm) and red CHROMA-LUC (approximately 610 nm) can be readily resolved using a luminometer having wavelength discrimination filters that allow measurement of both signals from the same sample. In such embodiments, the compounds described herein can be used to selectively inhibit the Oplophorus luciferase-derived bioluminescent complex so that the signal from other luciferases can be selectively viewed.

[0165] In another example, a bioluminescent complex derived from Oplophorus luciferase can be used as a transcriptional reporter and paired with a luciferase that emits light at a different wavelength and is contained in an assay reagent. In another example, a bioluminescent complex derived from Oplophorus luciferase can be used with one or more additional luciferases, and the luminescence of each luciferase and the bioluminescent complex can be measured separately by use of a selective enzyme inhibitor. For example, the luminescence of a bioluminescent complex derived from Oplophorus luciferase can be measured by addition of an appropriate substrate and buffer, followed by measurement of a second luciferase by subsequent addition of an appropriate substrate and buffer and one or more of the compounds described herein that are selective for the bioluminescent complex derived from Oplophorus luciferase.

[0166] In some embodiments, the Oplophorus luciferase-derived bioluminescent complex can be multiplexed with another enzyme (e.g., luciferase) that emits light at the same wavelength. For example, NANOBIT™ technology (Promega Corporation) can be multiplexed with NANOLUC. The NanoBiT™ system can include two or more non-luminescent peptide and / or polypeptide units. One or more of the non-luminescent peptide and / or polypeptide units can be fused to a molecule of interest. In some embodiments, the NanoBiT™ LgBiT non-luminescent polypeptide and / or the NanoBiT™ HiBiT non-luminescent peptide can be fused to a molecule of interest. For example, the NanoBiT™ LgBiT non-luminescent polypeptide can be added to a detection reagent containing furimazine as a means to detect and quantify a protein of interest fused to the NanoBiT™ HiBiT non-luminescent peptide. As another example, the NanoBiT™ HiBiT non-luminescent peptide can be added to a detection reagent containing furimazine as a means to detect and quantify a protein of interest fused to the NanoBiT™ LgBiT non-luminescent polypeptide. The disclosed compounds can be used to inhibit the luminescence of the resulting bioluminescent complex (e.g., the HiBiT / LgBiT complex) without inhibiting the luminescence from NanoLuc.

[0167] (7) Bioluminescence Resonance Energy Transfer (BRET) The disclosed compounds can be used in any method in which a bioluminescent complex derived from Oplophorus luciferase is used to detect ligand-protein and / or protein-protein interactions. In various embodiments, the bioluminescent complex derived from Oplophorus luciferase can be used to transfer energy to an energy acceptor. One such method is bioluminescence resonance energy transfer (BRET). For BRET, energy transfer from a bioluminescent donor to a fluorescent acceptor results in a shift in the spectral distribution of the emitted light. This energy transfer can enable real-time monitoring of protein-protein or ligand-protein interactions in vitro or in vivo.

[0168] In some embodiments, the bioluminescent complex derived from Oplophorus luciferase used in BRET assays can be used to determine whether two molecules can bind to each other or co-localize within a cell. For example, the bioluminescent complex derived from Oplophorus luciferase can be used as a bioluminescent donor molecule, in which case one of the non-luminescent moieties is combined with a molecule or protein of interest to produce a first fusion protein. In some embodiments, a non-luminescent peptide can be combined with a molecule or protein of interest to produce a first fusion protein. In other embodiments, a non-luminescent polypeptide can be combined with a molecule or protein of interest to produce a first fusion protein. In various embodiments, the first fusion protein containing the non-luminescent moiety (e.g., non-luminescent peptide or non-luminescent polypeptide) of the bioluminescent complex derived from Oplophorus luciferase can be used in BRET assays to detect protein / protein interactions in systems including, but not limited to, cell lysates, intact cells, and live animals. In various embodiments, HALOTAG can be used as a fluorescent acceptor molecule. In some embodiments, HALOTAG can be fused to a second protein of interest or a complementary non-luminescent moiety (e.g., non-luminescent polypeptide or non-luminescent peptide) of the bioluminescent complex. For example, the non-luminescent polypeptide of the bioluminescent complex derived from Oplophorus luciferase can be fused to HALOTAG, expressed in cells or animals, and labeled with a fluorescent HALOTAG® ligand such as HALOTAG® TMR ligand. The fusion can then be excited to fluoresce in the presence of a cell-permeable luminescent enzyme substrate. As another example, the non-luminescent peptide of the bioluminescent complex derived from Oplophorus luciferase can be fused to HaloTag, expressed in cells or animals, and labeled with a fluorescent HaloTag® ligand such as HaloTag® TMR ligand. The fusion can then be excited to fluoresce in the presence of a cell-permeable luminescent enzyme substrate.In some embodiments, HALOTAG can be fused to a second target protein and a complementary non-luminescent unit of a bioluminescent complex derived from Oplophorus luciferase added via a detection reagent (e.g., a non-luminescent polypeptide or a non-luminescent peptide). In some embodiments, BRET can be carried out using a bioluminescent complex derived from Oplophorus luciferase in combination with a fluorescent protein including, but not limited to, a fluorescent label including green fluorescent protein (GFP) or red fluorescent protein (RFP) or fluorescein, rhodamine green, Oregon green, or Alexa488.

[0169] In some embodiments, quenching the signal from a bioluminescent complex derived from Oplophorus luciferase can improve the signal-to-background ratio when using a BRET-based plate assay.

[0170] In certain embodiments, a cell-permeable compound can be used to inhibit intracellular BRET. In certain embodiments, a cell-impermeable compound can be used to inhibit extracellular BRET. In certain embodiments, a cell-impermeable compound can be used in a target engagement model.

[0171] 6. Sample The disclosed compounds can be used with samples containing biological components. The sample can include cells. The sample can include a heterogeneous mixture of components (including intact cells, cell extracts, cell lysates, bacteria, viruses, organelles, exosomes, and mixtures thereof) or a single component or a homogeneous group of components (e.g., natural or synthetic amino acids, nucleic acid or carbohydrate polymers, or lipid membrane complexes). The disclosed compounds can generally be non-toxic to living cells and other biological components within the range of use concentrations.

[0172] The sample can include an animal (e.g., a vertebrate), a plant, a fungus, a physiological fluid (e.g., blood, plasma, urine, mucus secretion, etc.), a cell, a cell lysate, a cell supernatant, or a purified cell fraction (e.g., an intracellular fraction). In certain embodiments, the sample can be a cell. In some embodiments, the sample can be a living cell. The cell can be a eukaryotic cell, such as a yeast, avian, plant, insect or mammalian cell, including a human, ape, mouse, dog, cow, horse, cat, sheep, goat or pig cell, or a prokaryotic cell, or cells from two or more different organisms, or cell lysates or supernatants thereof, but not limited thereto. The cell can be one that has not been genetically modified by recombinant techniques (non-recombinant cell), or a recombinant cell transiently transfected with recombinant DNA, and / or a recombinant cell whose genome has been stably augmented with recombinant DNA, or a recombinant cell whose genome has been modified to disrupt a gene, e.g., to disrupt a promoter, intron or open reading frame, or to replace one DNA fragment with another. The recombinant DNA or replacement DNA fragment can encode a molecule that is detected by the methods of the invention, a moiety that changes the level or activity of the detected molecule, and / or a gene product that is unrelated to the molecule or moiety that changes the level or activity of the molecule. The cell may or may not express luciferase. The cell can be one that has been genetically modified by recombinant techniques.

[0173] 7. Kit A kit is disclosed for determining the presence or activity of a bioluminescent complex derived from Oplophorus luciferase. The kit may include one or more of the following: a compound or composition of the present invention that can inhibit a bioluminescent complex derived from Oplophorus luciferase, coelenterazine or a coelenterazine derivative substrate, and a polynucleotide for the expression of a non-luminescent peptide and / or polypeptide of a bioluminescent complex derived from Oplophorus luciferase, instructions for performing a luminescence assay, and reaction buffer(s). The reaction buffer may be present in individual formulations for non-luciferase enzyme reactions and luminescent enzyme reactions, or in a single formulation for a single-step assay. The reaction buffer may contain a non-luminescent unit of a bioluminescent complex derived from Oplophorus luciferase, such as a non-luminescent peptide. The kit may also contain other inhibitors, activators, and / or enhancers for non-luciferase enzyme(s). The kit may also contain positive and / or negative controls for the assay.

Example

[0174] 8. Example Example 1 Synthesis of Compounds General Procedure A: Formation of Sulfonamide Bond.

[0175] Substituted benzenesulfonyl chloride (1.1 equivalents) was added to a solution of aniline derivative (1 equivalent) in pyridine. The solution was stirred at room temperature for 4 - 18 hours. The mixture was diluted with dichloromethane and washed with HCl (2M). The organic layer was dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography.

[0176] General Procedure B: Saponification.

[0177] To a solution of methyl or ethyl ester (1 eq) in dioxane, sodium hydroxide (2 M, 2 eq) was added. The solution was stirred at 60 °C for 2 - 18 h. The solution was acidified with HCl (2 M), diluted with ethyl acetate and water, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and used as the crude product in the next step.

[0178] General procedure C1: Formation of amide bond via acid chloride.

[0179] To a solution of acid chloride derivative (1 eq) in dichloroethane, pyridine (3 - 5 eq) and amine (1 eq) were added. The reaction was stirred at room temperature for 2 - 18 h. The mixture was diluted with DCM, washed with water and HCl (2 M). The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography.

[0180] General procedure C2: Formation of amide bond via carboxylate.

[0181] To a solution of carboxylic acid derivative (1 eq) in DMF, amine (1.2 eq), HBTU (2 eq), and diisopropylethylamine (3 eq) were added. The reaction was heated at 60 - 85 °C for 2 - 18 h. The mixture was diluted with ethyl acetate, washed with water and brine. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography.

[0182] General procedure D: Nitro reduction.

[0183] To a solution of nitro derivative (1 eq) in ethyl acetate, tin(II) chloride hydrate (3 - 5 eq) was added. The mixture was heated to reflux for 24 - 48 h. Saturated potassium carbonate was added and stirred at room temperature for 1 h. The layers were separated, the organic layer was washed with water, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography.

[0184] Exemplary compounds can be prepared according to the representative synthetic methods of Scheme 1 or Scheme 2. Scheme 1. Representative synthesis of amide analogs via acid chlorides JPEG2025105624000039.jpg53128

[0185] Example 2 N-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-0998) JPEG2025105624000040.jpg2659 Step 1. N-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-nitrobenzamide (JRW-0994) JPEG2025105624000041.jpg2744

[0186] After General Procedure C1, 2-nitrobenzoyl chloride (113 mg, 0.61 mmol) was reacted with 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (100 mg, 0.61 mmol) to give the desired product (160 mg, 84%) as a yellow solid. ESI MS m / z 314 [M+H] + .

[0187] Step 2. 2-Amino-N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)benzamide (JRW-0996) JPEG2025105624000042.jpg2543

[0188] After General Procedure D, N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-nitrobenzamide (160 mg, 0.51 mmol) was reacted with tin(II) chloride hydrate (318 mg, 1.5 mmol) to give the desired product (80 mg, 55%) as a white solid. ESI MS m / z 284 [M+H] + .

[0189] Project 3. N-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-0998) JPEG2025105624000043.jpg2661

[0190] After General Procedure A, 2-amino-N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)benzamide (80 mg, 0.28 mmol) was reacted with 4-methylbenzenesulfonyl chloride (54 mg, 0.28 mmol) to give the desired product (75 mg, 61%) as a pale yellow solid. ESI MS m / z 438 [M+H] + .

[0191] Example 3 N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1004) JPEG2025105624000044.jpg2661 Project 1. N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-nitrobenzamide (JRW-1000) JPEG2025105624000045.jpg2847

[0192] After General Procedure C1, 2-nitrobenzoyl chloride (187 mg, 1.0 mmol) was reacted with 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (180 mg, 1.0 mmol) to give the desired product (290 mg, 87%) as a yellow solid. ESI MS m / z 328 [M+H] + .

[0193] Step 2. 2-Amino-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide (JRW-1002) JPEG2025105624000046.jpg2847

[0194] To a solution of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-nitrobenzamide (290 mg, 0.88 mmol) in ethanol / water (8 / 2 mL) were added ammonium chloride (474 mg, 8.8 mmol) and iron filings (100 mg, 1.8 mmol). The suspension was heated at 60 °C for 18 h. The reaction mixture was filtered, the filtrate was added to celite, concentrated, and purified by silica gel chromatography to give the desired product (57 mg, 21%) as a pale brown solid. ESI MS m / z 298 [M+H]+.

[0195] Step 3. N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1004) JPEG2025105624000047.jpg3268

[0196] After general procedure A, 2-Amino-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)benzamide (57 mg, 0.19 mmol) was reacted with 4-methylbenzenesulfonyl chloride (36 mg, 0.19 mmol) to give the desired product (35 mg, 40%) as a white solid. ESI MS m / z 452 [M+H] + 。

[0197] Example 4 N-(3-Cyanothiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1006) JPEG2025105624000048.jpg2654 Step 1. N-(3-Cyanothiophen-2-yl)-2-nitrobenzamide (JRW-1001). JPEG2025105624000049.jpg2134

[0198] After general procedure C1, 2-nitrobenzoyl chloride (171 mg, 0.93 mmol) was reacted with 2-aminothiophene-3-carbonitrile (115 mg, 0.93 mmol) to afford the desired product (190 mg, 75%) as a light brown solid. ESI MS m / z 274 [M+H] + 。

[0199] Step 2. 2-Amino-N-(3-cyanothiophen-2-yl)benzamide (JRW-1003). JPEG2025105624000050.jpg2134

[0200] After general procedure D, N-(3-cyanothiophen-2-yl)-2-nitrobenzamide (190 mg, 0.69 mmol) was reacted with tin(II) chloride hydrate (433 mg, 2.1 mmol) to afford the desired product (90 mg, 53%) as a light brown solid. ESI MS m / z 244 [M+H] + 。

[0201] Step 3. N-(3-cyanothiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1006). JPEG2025105624000051.jpg2654

[0202] After general procedure A, 2-amino-N-(3-cyanothiophen-2-yl)benzamide (90 mg, 0.37 mmol) was reacted with 4-methylbenzenesulfonyl chloride (85 mg, 0.44 mmol) to afford the desired product (97 mg, 66%) as a light brown solid. ESI MS m / z 398 [M+H] +

[0203] Example 5 N-(2-Cyanophenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1008) JPEG2025105624000052.jpg2656 Step 1. N-(2-Cyanophenyl)-2-nitrobenzamide (JRW-1005). JPEG2025105624000053.jpg2639

[0204] After the general procedure C1, 2-nitrobenzoyl chloride (189 mg, 1.0 mmol) was reacted with 2-aminobenzonitrile (120 mg, 1.0 mmol) to give the desired product (208 mg, 77%) as a white solid. ESI MS m / z 268 [M+H] + 。

[0205] Step 2. 2-Amino-N-(2-cyanophenyl)benzamide (JRW-1007). JPEG2025105624000054.jpg2540

[0206] After the general procedure D, N-(2-cyanophenyl)-2-nitrobenzamide (200 mg, 0.75 mmol) was reacted with tin(II) chloride dihydrate (466 mg, 2.2 mmol) to give the desired product (80 mg, 45%) as a white solid. ESI MS m / z 238 [M+H] + 。

[0207] Step 3. N-(2-Cyanophenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1008). JPEG2025105624000055.jpg2656

[0208] After the general procedure A, 2-amino-N-(2-cyanophenyl)benzamide (80 mg, 0.34 mmol) was reacted with 4-methylbenzenesulfonyl chloride (128 mg, 0.67 mmol) to give the desired product (91 mg, 68%) as a white solid. ESI MS m / z 392 [M+H]+.

[0209] Example 6 2-((4-Methylphenyl)sulfonamido)-N-phenylbenzamide (JRW-1011) JPEG2025105624000056.jpg2656 Step 1. 2-Nitro-N-phenylbenzamide (JRW-1009) JPEG2025105624000057.jpg2639

[0210] After general procedure C1, 2-nitrobenzoyl chloride (239 mg, 1.3 mmol) was reacted with aniline (120 mg, 1.3 mmol) to give the crude product (350 mg) as a white solid. ESI MS m / z 243 [M+H] + 。

[0211] Step 2. 2-Amino-N-phenylbenzamide (JRW-1010). JPEG2025105624000058.jpg2538

[0212] After general procedure D, 2-nitro-N-phenylbenzamide (1.3 mmol) was reacted with tin(II) chloride dihydrate (803 mg, 3.9 mmol) to give the desired product (200 mg, 73% over 2 steps) as a white solid. ESI MS m / z 213 [M+H] + 。

[0213] Step 3. 2-((4-Methylphenyl)sulfonamido)-N-phenylbenzamide (JRW-1011). JPEG2025105624000059.jpg3063

[0214] After general procedure A, 2-amino-N-phenylbenzamide (200 mg, 0.94 mmol) was reacted with 4-methylbenzenesulfonyl chloride (359 mg, 1.9 mmol) to give the desired product (340 mg, 98%) as a white solid. ESI MS m / z 367 [M+H] + 。

[0215] Example 7 N-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)benzamide (HL-0010) After the general procedure A, 2-amino-N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)benzamide (40 mg, 0.14 mmol) was reacted with benzenesulfonyl chloride (30 mg, 0.17 mmol) to give the desired product (17 mg, 26%) as a light brown solid. ESI MS m / z 424 [M+H] + .

[0216] Example 8 N-(Benzo[b]thiophen-2-yl)-2-((4-formylphenyl)sulfonamido)benzamide (HL-0038) JPEG2025105624000061.jpg2664Step 1. N-(Benzo[b]thiophen-2-yl)-2-nitrobenzamide (HL-0028). JPEG2025105624000062.jpg2847

[0217] After the general procedure C1, 2-nitrobenzoyl chloride (373 mg, 2.0 mmol) was reacted with benzo[b]thiophen-2-amine (300 mg, 2.0 mmol) to give the desired product (50 mg, 8%) as a solid. ESI MS m / z 299 [M+H] + .

[0218] Step 2. 2-Amino-N-(benzo[b]thiophen-2-yl)benzamide (HL-0036). JPEG2025105624000063.jpg2747

[0219] After the general procedure D, N-(benzo[b]thiophen-2-yl)-2-nitrobenzamide (100 mg, 0.33 mmol) was reacted with tin(II) chloride hydrate (209 mg, 1.0 mmol) to give the desired product (27 mg, 30%) as a solid. ESI MS m / z 269 [M+H] + .

[0220] Project 3. N-(Benzo[b]thiophen-2-yl)-2-((4-formylphenyl)sulfonamido)benzamide (HL-0038). JPEG2025105624000064.jpg2662

[0221] After General Procedure A, 2-amino-N-(benzo[b]thiophen-2-yl)benzamide (10 mg, 0.036 mmol) was reacted with 4-formylbenzenesulfonyl chloride (7 mg, 0.037 mmol) to give the desired product (5 mg, 31%) as a white solid. ESI MS m / z 437 [M+H] + .

[0222] Example 9 Methyl 3-(4-(N-(2-(benzo[b]thiophen-2-ylcarbamoyl)phenyl)sulfamoyl)phenyl)propanoate (HL-0040) JPEG2025105624000065.jpg2674 After General Procedure A, 2-amino-N-(benzo[b]thiophen-2-yl)benzamide (10 mg, 0.037 mmol) was reacted with methyl 3-(4-(chlorosulfonyl)phenyl)propanoate (10 mg, 0.037 mmol) to give the desired product (12 mg, 63%) as a light brown solid. ESI MS m / z 495 [M+H] + .

[0223] Example 10 N-(Benzo[b]thiophen-2-yl)-2-((3-methylphenyl)sulfonamido)benzamide (HL-0041) JPEG2025105624000066.jpg2661 After General Procedure A, 2-amino-N-(benzo[b]thiophen-2-yl)benzamide (10 mg, 0.032 mmol) was reacted with 3-methylbenzenesulfonyl chloride (6 mg, 0.032 mmol) to give the desired product (7 mg, 54%) as a white solid. ESI MS m / z 423 [M+H] + .

[0224] Example 11 N-(Benzo[b]thiophen-2-yl)-2-((4-(3-hydroxypropyl)phenyl)sulfonamido)benzamide (HL-0062) JPEG2025105624000067.jpgTo a solution of methyl 3-(4-(N-(2-(benzo[b]thiophen-2-ylcarbamoyl)phenyl)sulfamoyl)phenyl)propanoate (31 mg, 0.063 mmol) in DMF (2 mL) at 26740 °C was added lithium borohydride (4 mg, 0.19 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The mixture was diluted with ethyl acetate and water, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (17 mg, 60%) as a white solid. ESI MS m / z 467 [M+H] + 。

[0225] Example 12 2-([1,1'-Biphenyl]-3-sulfonamido)-N-(p-tolyl)benzamide (JRW-1248) JPEG2025105624000068.jpgStep 1. 2-Nitro-N-(p-tolyl)benzamide (JRW-1243). JPEG2025105624000069.jpg2143

[0226] After General Procedure C1, 2-nitrobenzoyl chloride (3.46 g, 18.6 mmol) was reacted with p-toluidine (2.0 g, 18.6 mmol) to give the desired product (4.6 g, 96%) as a light brown solid. ESI MS m / z 257 [M+H] + 。

[0227] Step 2. 2-Amino-N-(p-tolyl)benzamide (JRW-1247). JPEG2025105624000070.jpg2549

[0228] After general procedure D, 2-nitro-N-(p-tolyl)benzamide (4.6 g, 18.0 mmol) was reacted with tin(II) chloride hydrate (11.2 mg, 54.0 mmol) to afford the desired product (3.3 g, 81%) as a white solid. ESI MS m / z 227 [M+H] + .

[0229] Step 3. 2-([1,1'-Biphenyl]-3-sulfonamido)-N-(p-tolyl)benzamide (JRW-1248). JPEG2025105624000071.jpg2869

[0230] After general procedure A, 2-amino-N-(p-tolyl)benzamide (75 mg, 0.33 mmol) was reacted with [1,1'-biphenyl]-3-sulfonyl chloride (92 mg, 0.36 mmol) to afford the desired product (85 mg, 58%) as a white solid. ESI MS m / z 443 [M+H] + .

[0231] Example 13 Methyl 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoate (JRW-1250) JPEG2025105624000072.jpg2672 After general procedure A, 2-amino-N-(p-tolyl)benzamide (400 mg, 1.78 mmol) was reacted with methyl 3-(chlorosulfonyl)benzoate (498 mg, 2.1 mmol) to afford the desired product (670 mg, 89%) as a white foam. ESI MS m / z 425 [M+H] + .

[0232] Example 14 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoic acid (JRW-1251) After the general procedure B, methyl 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoate (650 mg, 1.5 mmol) was reacted with LiOH (110 mg, 4.6 mmol) to afford the desired product (580 mg, 92%) as a white solid. ESI MS m / z 411 [M+H] + .

[0233] Example 15 2-((3-Acetamidophenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1253) After the general procedure A, 2-amino-N-(p-tolyl)benzamide (400 mg, 1.78 mmol) was reacted with 3-acetamidobenzenesulfonyl chloride (495 mg, 2.1 mmol) to afford the desired product (690 mg, 92%) as a white foam. ESI MS m / z 424 [M+H] + .

[0234] Example 16 2-((3-Aminophenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1255) To a solution of 2-((3-acetamidophenyl)sulfonamido)-N-(p-tolyl)benzamide (690 mg, 1.6 mmol) in methanol (20 mL) was added sodium hydroxide (5 mL, 2 M). The mixture was heated at 85 °C for 5 h. The reaction was cooled, acidified to pH 5, diluted with DCM and water, and the aqueous layer was extracted with DCM. The organic layers were combined, dried over sodium sulfate, filtered, concentrated and purified by silica gel chromatography to afford the desired product (62 mg, 10%) as a white solid. ESI MS m / z 382 [M+H]+.

[0235] Example 17 2-((3-(Hydroxymethyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1261) A mixture of 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoic acid (54 mg, 0.13 mmol) in DCM (5 mL) was added with HOBt (20 mg, 0.13 mmol) and EDC (25 mg, 0.13 mmol). The mixture was stirred at room temperature for 30 minutes. The solution was concentrated to a white foam, and then the solid was dissolved in THF (10 mL) and water (0.5 mL). The solution was cooled and sodium borohydride (10 mg, 0.26 mmol) was added. The reaction was stirred at room temperature for 18 hours, quenched with HCl, and diluted with ethyl acetate and water. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain the desired product (29 mg, 55%) as a white solid. ESI MS m / z 397 [M+H] + 。

[0236] Example 18 2-((3-(Butylcarbamoyl)phenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1263) After General Procedure C2, 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoic acid (50 mg, 0.12 mmol) was reacted with butylamine (17 mg, 0.21 mmol) to obtain the desired product (43 mg, 76%) as a white solid. ESI MS m / z 466 [M+H] + 。

[0237] Example 19 2-((3-Bromophenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1267) After General Procedure A, 2-amino-N-(p-tolyl)benzamide (350 mg, 1.5 mmol) was reacted with 3-bromobenzenesulfonyl chloride (474 mg, 1.8 mmol) to obtain the desired product (620 mg, 90%) as a light brown solid. ESI MS m / z 446 [M+H] + 。

[0238] Example 20 2-((3-(Butylamino)phenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1269) JPEG2025105624000079.jpgTo a solution of 2-((3-aminophenyl)sulfonamido)-N-(p-tolyl)benzamide (50 mg, 0.13 mmol) in THF (5 mL) was added butyraldehyde (14 mg, 0.20 mmol). The mixture was stirred at room temperature for 30 minutes, then sodium triacetoxyborohydride (55 mg, 0.26 mmol) was added. The reaction was stirred at room temperature for 5 hours, quenched with saturated NaHCO3 solution, diluted with ethyl acetate and water. The organic layers were combined, dried over sodium sulfate, filtered, concentrated and purified by silica gel chromatography to give the desired product (27 mg, 47%) as a white solid. ESI MS m / z 438 [M+H] + 。

[0239] Example 21 2-((3-(Hex-1-yn-1-yl)phenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1270) JPEG2025105624000080.jpgTo a solution of 2-((3-bromophenyl)sulfonamido)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in DMF (5 mL) were added 1-hexyne (36 mg, 0.45 mmol), triethylamine (68 mg, 0.67 mmol), triphenylphosphine (6 mg, 0.022 mmol), and PdCl2(PPh3)2 (8 mg, 0.011 mmol). The suspension was purged with nitrogen. Copper iodide (4 mg, 0.022 mmol) was added and the reaction was stirred at 60 °C for 18 hours. The reaction was diluted with ethyl acetate and water, extracted with ethyl acetate, the organic layers were combined, dried over sodium sulfate, filtered, concentrated and purified by silica gel chromatography to give the desired product (50 mg, 50%) as an orange oil. ESI MS m / z 447 [M+H] + 。

[0240] Example 22 2-((3-Hexylphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1271) JPEG2025105624000081.jpg Palladium on carbon (5 mg) was added to a solution of 2-((3-(hex-1-yn-1-yl)phenyl)sulfonamido)-N-(p-tolyl)benzamide (40 mg, 0.090 mmol) in ethanol (10 mL). The reaction mixture was stirred with hydrogen at 40 psi for 2 hours at room temperature. The mixture was filtered through Celite, concentrated, and purified by silica gel chromatography to give the desired product (25 mg, 62%) as a white solid. ESI MS m / z 451 [M+H] + .

[0241] Example 23 2-((3-(3-Hydroxyprop-1-yn-1-yl)phenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1272) JPEG2025105624000082.jpg Propargyl alcohol (25 mg, 0.45 mmol), triethylamine (68 mg, 0.67 mmol), triphenylphosphine (6 mg, 0.022 mmol), and PdCl2(PPh3)2 (8 mg, 0.011 mmol) were added to a solution of 2-((3-bromophenyl)sulfonamido)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in DMF (5 mL). The suspension was purged with nitrogen. Copper(I) iodide (4 mg, 0.022 mmol) was added and the reaction mixture was stirred at 85 °C for 48 hours. The reaction mixture was diluted with ethyl acetate and water, extracted with ethyl acetate, the organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (30 mg, 31%) as a pale yellow gum. ESI MS m / z 421 [M+H] + .

[0242] Example 24 2-((3-(3-Hydroxypropyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1275) JPEG2025105624000083.jpg2981 Palladium on carbon (5 mg) was added to a solution of 2-((3-(3-hydroxyprop-1-yn-1-yl)phenyl)sulfonamido)-N-(p-tolyl)benzamide (23 mg, 0.055 mmol) in ethanol (10 mL). The reaction was stirred at room temperature for 1 hour with hydrogen at 40 psi. The mixture was filtered through celite, concentrated, and purified by silica gel chromatography to give the desired product (17 mg, 74%) as a white solid. ESI MS m / z 425 [M+H] + .

[0243] Example 25 N-(p-Tolyl)-2-((4-(trifluoromethyl)phenyl)sulfonamido)benzamide (JRW-1284) JPEG2025105624000084.jpg2867 After the general procedure A, 2-amino-N-(p-tolyl)benzamide (50 mg, 0.22 mmol) was reacted with 4-(trifluoromethyl)benzenesulfonyl chloride (64 mg, 0.26 mmol) to give the desired product (86 mg, 89%) as a white solid. ESI MS m / z 434 [M+H] + .

[0244] Example 26 2-((4-Methoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1285) JPEG2025105624000085.jpg2973 After the general procedure A, 2-amino-N-(p-tolyl)benzamide (50 mg, 0.22 mmol) was reacted with 4-methoxybenzenesulfonyl chloride (55 mg, 0.26 mmol) to give the desired product (82 mg, 94%) as a white solid. ESI MS m / z 397 [M+H] + .

[0245] Example 27 2-((4-Bromophenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1287) JPEG2025105624000086.jpg2965After general procedure A, 2-amino-N-(p-tolyl)benzamide (190 mg, 0.84 mmol) was reacted with 4-bromobenzenesulfonyl chloride (257 mg, 1.0 mmol) to give the desired product (310 mg, 83%) as a white solid. ESI MS m / z 446 [M+H] + 。

[0246] Example 28 2-([1,1'-Biphenyl]-4-sulfonamido)-N-(p-tolyl)benzamide (JRW-1293) JPEG2025105624000087.jpg2669To a solution of 2-((4-bromophenyl)sulfonamido)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in dioxane (5 mL) were added phenylboronic acid (32 mg, 0.27 mmol) and Pd(dppf)Cl2 (18 mg, 0.022 mmol). The mixture was purged with nitrogen and then aqueous Cs2CO3 (0.67 mL, 1 M) was added. The reaction was heated at 80 °C for 2 h. The mixture was diluted with ethyl acetate and washed with water and brine. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to give the desired product (64 mg, 64%) as a white solid. ESI MS m / z 443 [M+H] + 。

[0247] Example 29 N-(p-tolyl)-2-((3-(trifluoromethyl)phenyl)sulfonamido)benzamide (JRW-1327) JPEG2025105624000088.jpg2969After general procedure A, 2-amino-N-(p-tolyl)benzamide (55 mg, 0.24 mmol) was reacted with 3-(trifluoromethyl)benzenesulfonyl chloride (71 mg, 0.29 mmol) to give the desired product (13 mg, 12%) as a white solid. ESI MS m / z 435 [M+H]+ .

[0248] Example 30 N-(p-Tolyl)-2-((3-(trifluoromethoxy)phenyl)sulfonamido)benzamide (JRW-1328) JPEG2025105624000089.jpg2971After general procedure A, 2-amino-N-(p-tolyl)benzamide (55 mg, 0.24 mmol) was reacted with 3-(trifluoromethoxy)benzenesulfonyl chloride (76 mg, 0.29 mmol) to give the desired product (42 mg, 38%) as a white solid. ESI MS m / z 451 [M+H] + .

[0249] Scheme 2. Representative synthesis of amide analogs via carboxylate JPEG2025105624000090.jpg60153Example 31 N-(Benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (HL-0005) JPEG2025105624000091.jpg2661Step 1. Methyl 2-((4-methylphenyl)sulfonamido)benzoate (HL-0001). JPEG2025105624000092.jpg2651

[0250] After general procedure A, methyl 2-aminobenzoate (1.0 g, 6.6 mmol) was reacted with 4-methylbenzenesulfonyl chloride (1.5 g, 7.9 mmol) to give the desired product (1.25 g, 62%) as a white solid. ESI MS m / z 306 [M+H] + .

[0251] Step 2. 2-((4-Methylphenyl)sulfonamido)benzoic acid (HL-0003). JPEG2025105624000093.jpg2646

[0252] After general procedure B, methyl 2-((4-methylphenyl)sulfonamido)benzoate (1.2 g, 4.1 mmol) was reacted with LiOH (294 mg, 12.3 mmol) to give the desired product (1.0 g, 86%) as a white solid. ESI MS m / z 292 [M+H]+.

[0253] Step 3. N-(Benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (HL-0005). JPEG2025105624000094.jpg2661

[0254] After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with benzo[b]thiophen-2-amine (31 mg, 0.21 mmol) to give the desired product (44 mg, 96%) as a brown solid. ESI MS m / z 423 [M+H] + 。

[0255] Example 32 N-Cyclohexyl-2-((4-methylphenyl)sulfonamido)benzamide (HL-0006) JPEG2025105624000095.jpg2656 After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with cyclohexylamine (20 mg, 0.21 mmol) to give the desired product (14 mg, 22%) as a pale yellow solid. ESI MS m / z 373 [M+H] + 。

[0256] Example 33 2-((4-Methylphenyl)sulfonamido)-N-(naphthalen-2-yl)benzamide (HL-0007) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with naphthalene-2-amine (29 mg, 0.21 mmol) to give the desired product (40 mg, 54%) as a light brown solid. ESI MS m / z 417 [M+H] + .

[0257] Example 34 2-((4-methylphenyl)sulfonamido)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzamide (HL-0008) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 5,6,7,8-tetrahydronaphthalene-2-amine (30 mg, 0.21 mmol) to give the desired product (33 mg, 44%) as a white solid. ESI MS m / z 421 [M+H] + .

[0258] Example 35 Methyl trans-4-(2-((4-methylphenyl)sulfonamido)benzamido)cyclohexane-1-carboxylate (HL-0009) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with methyl trans-4-aminocyclohexane-1-carboxylate HCl (40 mg, 0.21 mmol) to give the desired product (41 mg, 55%) as a solid. ESI MS m / z 431 [M+H] + .

[0259] Example 36 Trans-4-(2-((4-methylphenyl)sulfonamido)benzamido)cyclohexane-1-carboxylic acid (HL-0012) After general procedure B, methyl trans-4-(2-((4-methylphenyl)sulfonamido)benzamido)cyclohexane-1-carboxylate (33 mg, 0.077 mmol) was reacted with LiOH (4 mg, 0.16 mmol) to give the desired product (29 mg, 84%) as a pale brown solid. ESI MS m / z 417 [M+H]+.

[0260] Example 37 2-((4-Methylphenyl)sulfonamido)-N-(p-tolyl)benzamide (HL-0019) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 4-methyl-aniline (22 mg, 0.21 mmol) to give the desired product (34 mg, 52%) as a pale brown solid. ESI MS m / z 381 [M+H] + 。

[0261] Example 38 Ethyl 2-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)acetate (HL-0023) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with ethyl 2-(4-aminophenyl)acetate (37 mg, 0.21 mmol) to give the desired product (32 mg, 42%) as a solid. ESI MS m / z 453 [M+H] + 。

[0262] Example 39 N-(3-Isopropylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (HL-0025) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 3-isopropylaniline (28 mg, 0.21 mmol) to give the desired product (49 mg, 71%) as a white solid. ESI MS m / z 409 [M+H] + .

[0263] Example 40 Ethyl 3-(2-((4-methylphenyl)sulfonamido)benzamide)benzoate (HL-0026) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with ethyl 3-aminobenzoate (34 mg, 0.21 mmol) to give the desired product (50 mg, 67%) as a solid. ESI MS m / z 439 [M+H] + .

[0264] Example 41 2-(4-(2-((4-methylphenyl)sulfonamido)benzamide)phenyl)acetic acid (HL-0030) After general procedure B, ethyl 2-(4-(2-((4-methylphenyl)sulfonamido)benzamide)phenyl)acetate (23 mg, 0.052 mmol) was reacted with LiOH (3 mg, 0.11 mmol) to give the desired product (17 mg, 79%) as a brown solid. ESI MS m / z 425 [M+H] + .

[0265] Example 42 3-(2-((4-methylphenyl)sulfonamido)benzamide)benzoic acid (HL-0031) After general procedure B, ethyl 3-(2-((4-methylphenyl)sulfonamido)benzamido)benzoate (23 mg, 0.054 mmol) was reacted with LiOH (3 mg, 0.11 mmol) to give the desired product (12 mg, 54%) as a pale yellow solid. ESI MS m / z 411 [M+H] + .

[0266] Example 43 2-((4-methylphenyl)sulfonamido)-N-(m-tolyl)benzamide (HL-0044) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with m-toluidine (22 mg, 0.21 mmol) to give the desired product (57 mg, 87%) as a white solid. ESI MS m / z 381 [M+H] + .

[0267] Example 44 N-(benzo[b]thiophen-2-yl)-3-((4-methylphenyl)sulfonamido)-2-naphthamide (HL-0057) JPEG2025105624000107.jpg3765Step 1. Methyl 3-((4-methylphenyl)sulfonamido)-2-naphthoate (HL-0049). JPEG2025105624000108.jpg3657

[0268] After general procedure A, methyl 3-amino-2-naphthoate (300 mg, 1.5 mmol) was reacted with 4-methylbenzenesulfonyl chloride (341 mg, 1.8 mmol) to give the desired product (340 mg, 64%) as a solid. ESI MS m / z 356 [M+H]+.

[0269] Step 2. 3-((4-methylphenyl)sulfonamido)-2-naphthoic acid (HL-0052). JPEG2025105624000109.jpg3346

[0270] After general procedure B, methyl 3-((4-methylphenyl)sulfonamido)-2-naphthoate (340 mg, 0.96 mmol) was reacted with LiOH (69 mg, 2.9 mmol) to give the desired product (231 mg, 71%) as a solid. ESI MS m / z 342 [M+H] + 。

[0271] Step 3. N-(Benzo[b]thiophen-2-yl)-3-((4-methylphenyl)sulfonamido)-2-naphthamide (HL-0057). JPEG2025105624000110.jpg3766

[0272] After general procedure C2, 3-((4-methylphenyl)sulfonamido)-2-naphthoic acid (50 mg, 0.15 mmol) was reacted with benzo[b]thiophen-2-amine (22 mg, 0.15 mmol) to give the desired product (28 mg, 40%) as a light brown solid. ESI MS m / z 473 [M+H] + 。

[0273] Example 45 2-((4-Methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide (HL-0059) JPEG2025105624000111.jpg3864 After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 2-propylaniline (23 mg, 0.17 mmol) to give the desired product (28 mg, 40%) as a white solid. ESI MS m / z 409 [M+H] + 。

[0274] Example 46 N-(Benzo[b]thiophen-2-yl)-5-methyl-2-((4-methylphenyl)sulfonamido)benzamide (HL-0061) JPEG2025105624000112.jpg3867Project 1. Ethyl 5-methyl-2-((4-methylphenyl)sulfonamido)benzoate (HL-0053). JPEG2025105624000113.jpg3761

[0275] After general procedure A, ethyl 2-amino-5-methylbenzoate (300 mg, 1.7 mmol) was reacted with 4-methylbenzenesulfonyl chloride (638 mg, 3.4 mmol) to give the desired product (404 mg, 72%) as a solid. ESI MS m / z 334 [M+H] + 。

[0276] Step 2. 5-Methyl-2-((4-methylphenyl)sulfonamido)benzoic acid (HL-0058). JPEG2025105624000114.jpg3446

[0277] After general procedure B, ethyl 5-methyl-2-((4-methylphenyl)sulfonamido)benzoate (404 mg, 1.2 mmol) was reacted with LiOH (87 mg, 3.6 mmol) to give the desired product (262 mg, 71%) as a solid. ESI MS m / z 306 [M+H] + 。

[0278] Step 3. N-(Benzo[b]thiophen-2-yl)-5-methyl-2-((4-methylphenyl)sulfonamido)benzamide (HL-0061). JPEG2025105624000115.jpg3461

[0279] After general procedure C2, 5-methyl-2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.16 mmol) was reacted with benzo[b]thiophen-2-amine (24 mg, 0.16 mmol) to give the desired product (68 mg, 95%) as a pale yellow solid. ESI MS m / z 437 [M+H] + 。

[0280] Example 47 N-(3-Butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (HL-0070) JPEG2025105624000116.jpg2984After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 3-butylaniline (25 mg, 0.17 mmol) to give the desired product (18 mg, 25%) as a white solid. ESI MS m / z 423 [M+H] + 。

[0281] Example 48 N-(Benzo[b]thiophen-2-yl)-5-cyano-2-((4-methylphenyl)sulfonamido)benzamide (HL-0071) JPEG2025105624000117.jpg3866Step 1. Methyl 5-cyano-2-((4-methylphenyl)sulfonamido)benzoate (HL-0056). JPEG2025105624000118.jpg3351

[0282] After general procedure A, methyl 2-amino-5-cyanobenzoate (300 mg, 1.7 mmol) was reacted with 4-methylbenzenesulfonyl chloride (649 mg, 3.4 mmol) to give the desired product (360 mg, 64%) as a solid. ESI MS m / z 331 [M+H] + 。

[0283] Step 2. 5-Cyano-2-((4-methylphenyl)sulfonamido)benzoic acid (HL-0060). JPEG2025105624000119.jpg3751

[0284] After general procedure B, methyl 5-cyano-2-((4-methylphenyl)sulfonamido)benzoate (360 mg, 1.1 mmol) was reacted with LiOH (78 mg, 3.3 mmol) to give the desired product (140 mg, 41%) as a solid. ESI MS m / z 317 [M+H] + 。

[0285] Engineering 3. N-(Benzo[b]thiophen-2-yl)-5-cyano-2-((4-methylphenyl)sulfonamido)benzamide (HL-0071). JPEG2025105624000120.jpg3361

[0286] After General Procedure C2, 5-cyano-2-((4-methylphenyl)sulfonamido)benzoic acid (92 mg, 0.29 mmol) was reacted with benzo[b]thiophen-2-amine (43 mg, 0.29 mmol) to give the desired product (47 mg, 36%) as a light brown solid. ESI MS m / z 448 [M+H] + .

[0287] Example 49 N-(4-Butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1076) JPEG2025105624000121.jpg2679 After General Procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (55 mg, 0.19 mmol) was reacted with 4-butylaniline (42 mg, 0.28 mmol) to give the desired product (68 mg, 86%) as a light brown oil. ESI MS m / z 423 [M+H] + .

[0288] Example 50 N-(Benzo[b]thiophen-2-yl)-2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzamide (JRW-1077) JPEG2025105624000122.jpg2661 Step 1. Methyl 2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzoate (HL-0068). JPEG2025105624000123.jpg2649

[0289] After general procedure A, methyl 2-aminobenzoate (200 mg, 1.7 mmol) was reacted with 5,6,7,8-tetrahydronaphthalene-2-sulfonyl chloride (366 mg, 1.6 mmol) to afford the desired product (289 mg, 63%) as a solid. ESI MS m / z 346 [M+H] + 。

[0290] Step 2. 2-((5,6,7,8-Tetrahydronaphthalene)-2-sulfonamido)benzoic acid (HL-0072) JPEG2025105624000124.jpg2646

[0291] After general procedure B, methyl 2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzoate (289 mg, 0.84 mmol) was reacted with LiOH (60 mg, 2.5 mmol) to afford the desired product (264 mg, 95%) as a solid. ESI MS m / z 332 [M+H] + 。

[0292] Step 3. N-(Benzo[b]thiophen-2-yl)-2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzamide (JRW-1077). JPEG2025105624000125.jpg2661

[0293] After general procedure C2, 2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzoic acid (50 mg, 0.15 mmol) was reacted with benzo[b]thiophen-2-amine (27 mg, 0.18 mmol) to afford the desired product (18 mg, 26%) as a brown oil. ESI MS m / z 463 [M+H] + 。

[0294] Example 51 N-(4-Hexylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1090) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 4-hexylaniline (36 mg, 0.21 mmol) to give the desired product (61 mg, 79%) as a white solid. ESI MS m / z 451 [M+H] + .

[0295] Example 52 2-((4-methylphenyl)sulfonamido)-N-(4-octylphenyl)benzamide (JRW-1091) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 4-octylaniline (42 mg, 0.21 mmol) to give the desired product (67 mg, 81%) as a white solid. ESI MS m / z 479 [M+H] + .

[0296] Example 53 Methyl 6-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)hexanoate (JRW-1107) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with methyl 6-(4-aminophenyl)hexanoate (45 mg, 0.21 mmol) to give the desired product (65 mg, 76%) as an orange oil. ESI MS m / z 479 [M+H] + .

[0297] Example 54 6-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)hexanoic acid (JRW-1110) After the general procedure B, methyl 6-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)hexanoate (55 mg, 0.11 mmol) was reacted with LiOH (8 mg, 0.33 mmol) to give the desired product (50 mg, 94%) as a pale brown solid. ESI MS m / z 481 [M+H] + .

[0298] Example 55 N-(Benzo[b]thiophen-2-yl)-2-((4-butylphenyl)sulfonamido)benzamide (JRW-1114) JPEG2025105624000130.jpg3083 Step 1. Methyl 2-((4-butylphenyl)sulfonamido)benzoate (JRW-1111) JPEG2025105624000131.jpg3068

[0299] After the general procedure A, methyl 2-aminobenzoate (1.0 g, 6.6 mmol) was reacted with 4-butylbenzenesulfonyl chloride (1.7 g, 7.3 mmol) to give the crude product as a pale brown oil. ESI MS m / z 348 [M+H] + .

[0300] Step 2. 2-((4-Butylphenyl)sulfonamido)benzoic acid (JRW-1112) JPEG2025105624000132.jpg2864

[0301] Step 3. N-(Benzo[b]thiophen-2-yl)-2-((4-butylphenyl)sulfonamido)benzamide (JRW-1114) JPEG2025105624000133.jpg2881

[0302] After General Procedure C2, 2-((4-butylphenyl)sulfonamido)benzoic acid (120 mg, 0.36 mmol) was reacted with benzo[b]thiophen-2-amine (54 mg, 0.36 mmol) to give the desired product (25 mg, 15%) as an off-white solid. ESI MS m / z 465 [M+H] + 。

[0303] Example 56 N-(4-(6-Hydroxyhexyl)phenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1120) JPEG2025105624000134.jpg2690 After General Procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (150 mg, 0.51 mmol) was reacted with 6-(4-aminophenyl)hexan-1-ol (100 mg, 0.51 mmol) to give the desired product (60 mg, 25%) as a white foam. ESI MS m / z 467 [M+H] + 。

[0304] Example 57 N-(Benzo[b]thiophen-2-yl)-2-((4-pentylphenyl)sulfonamido)benzamide (JRW-1121) JPEG2025105624000135.jpg2679 Step 1. Methyl 2-((4-pentylphenyl)sulfonamido)benzoate (JRW-1115) JPEG2025105624000136.jpg2667

[0305] After General Procedure A, methyl 2-aminobenzoate (1.0 g, 6.6 mmol) was reacted with 4-pentylbenzenesulfonyl chloride (1.8 g, 7.3 mmol) to give the crude product (2.3 g) as an orange oil. ESI MS m / z 362 [M+H] + 。

[0306] Step 2. 2-((4-Pentylphenyl)sulfonamido)benzoic acid (JRW-1116) JPEG2025105624000137.jpg2664

[0307] After general procedure B, methyl 2-((4-pentylphenyl)sulfonamido)benzoate (2.3 g, 6.4 mmol) was reacted with NaOH (6.4 mL, 2 M, 12.7 mmol) to give the crude product (2.2 g) as a pale pink solid. ESI MS m / z 348 [M+H] + 。

[0308] Step 3. N-(Benzo[b]thiophen-2-yl)-2-((4-pentylphenyl)sulfonamido)benzamide (JRW-1121) JPEG2025105624000138.jpg2679

[0309] After general procedure C2, 2-((4-pentylphenyl)sulfonamido)benzoic acid (100 mg, 0.29 mmol) was reacted with benzo[b]thiophen-2-amine (34 mg, 0.23 mmol) to give the desired product (57 mg, 41%) as an orange foam. ESI MS m / z 479 [M+H] + 。

[0310] Example 58 N-(Benzo[b]thiophen-2-yl)-5-butyl-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1146) JPEG2025105624000139.jpg5070 Step 1. 5-Butyl-2-((4-methylphenyl)sulfonamido)benzoic acid (JRW-1142) JPEG2025105624000140.jpg4652

[0311] After general procedure A, 2-amino-5-butylbenzoic acid (220 mg, 1.1 mmol) was reacted with 4-methylbenzenesulfonyl chloride (325 mg, 1.7 mmol) to give the desired product (275 mg, 69%) as a brown solid. ESI MS m / z 348 [M+H] + 。

[0312] Engineering 2.N-(Benzo[b]thiophen-2-yl)-5-butyl-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1146) JPEG2025105624000141.jpg4667

[0313] After General Procedure C2, 5-butyl-2-((4-methylphenyl)sulfonamido)benzoic acid (275 mg, 0.79 mmol) was reacted with benzo[b]thiophen-2-amine (118 mg, 0.79 mmol) to give the desired product (30 mg, 8%) as a brown solid. ESI MS m / z 479 [M+H] + 。

[0314] Example 59 N-(4-(4-Hydroxybutyl)phenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1150) JPEG2025105624000142.jpg2682 After General Procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (250 mg, 0.86 mmol) was reacted with 4-(4-aminophenyl)butan-1-ol (170 mg, 1.0 mmol) to give the desired product (140 mg, 37%) as an oil. ESI MS m / z 439 [M+H] + 。

[0315] Example 60 N-(4-(4-Bromobutyl)phenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1152) A solution of N-(4-(4-hydroxybutyl)phenyl)-2-((4-methylphenyl)sulfonamido)benzamide (40 mg, 0.091 mmol) in DCM (5 mL) was added with carbon tetrabromide (60 mg, 0.18 mmol) and triphenylphosphine (47 mg, 0.18 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with ethyl acetate and water, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain the desired product (18 mg, 40%) as a clear oil. ESI MS m / z 502 [M+H] + .

[0316] Example 61 5-Methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1166) JPEG2025105624000144.jpg3769 Step 1. Methyl 5-methoxy-2-((4-methylphenyl)sulfonamido)benzoate (JRW-1159) JPEG2025105624000145.jpg3860

[0317] After General Procedure A, methyl 2-amino-5-methoxybenzoate (5.0 g, 27.6 mmol) was reacted with 4-methylbenzenesulfonyl chloride (5.8 g, 30.3 mmol) to obtain the crude product as a purple solid. ESI MS m / z 336 [M+H] + .

[0318] Step 2. 5-Methoxy-2-((4-methylphenyl)sulfonamido)benzoic acid (JRW-1161) JPEG2025105624000146.jpg3852

[0319] After general procedure B, methyl 5-methoxy-2-((4-methylphenyl)sulfonamido)benzoate (27.6 mmol) was reacted with NaOH (27.6 mL, 2 M, 55.2 mmol) to afford the desired product (8.4 g, 94%) as a pale purple solid. ESI MS m / z 322 [M+H] + .

[0320] Step 3. 5-Methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1166) JPEG2025105624000147.jpg3768

[0321] After general procedure C2, 5-methoxy-2-((4-methylphenyl)sulfonamido)benzoic acid (500 mg, 1.6 mmol) was reacted with p-toluidine (200 mg, 1.9 mmol) to afford the desired product (348 mg, 54%) as a white foam. ESI MS m / z 411 [M+H] + .

[0322] Example 62 N-(Benzo[b]thiophen-2-yl)-5-methoxy-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1167) JPEG2025105624000148.jpg3866 After general procedure C2, 5-methoxy-2-((4-methylphenyl)sulfonamido)benzoic acid (100 mg, 0.31 mmol) was reacted with benzo[b]thiophen-2-amine (51 mg, 0.34 mmol) to afford the desired product (34 mg, 24%) as a light brown solid. ESI MS m / z 453 [M+H] + .

[0323] Example 63 4-Methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1202) JPEG2025105624000149.jpg Project 3267 1. Methyl 4-methoxy-2-((4-methylphenyl)sulfonamido)benzoate (JRW-1195) JPEG2025105624000150.jpg 3356

[0324] After General Procedure A, methyl 2-amino-4-methoxybenzoate (1.0 g, 5.5 mmol) was reacted with 4-methylbenzenesulfonyl chloride (1.2 g, 6.1 mmol) to give the crude product (1.9 g) as a white foam. ESI MS m / z 336 [M+H] + .

[0325] Step 2. 4-Methoxy-2-((4-methylphenyl)sulfonamido)benzoic acid (JRW-1198) JPEG2025105624000151.jpg 3351

[0326] After General Procedure B, methyl 4-methoxy-2-((4-methylphenyl)sulfonamido)benzoate (5.5 mmol) was reacted with NaOH (5.6 mL, 2 M, 11.3 mmol) to give the desired product (1.7 g, 94%) as a pale yellow solid. ESI MS m / z 322 [M+H] + .

[0327] Step 3. 4-Methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1202) JPEG2025105624000152.jpg 3370

[0328] After General Procedure C2, 4-methoxy-2-((4-methylphenyl)sulfonamido)benzoic acid (510 mg, 1.6 mmol) was reacted with p-toluidine (200 mg, 1.9 mmol) to give the desired product (460 mg, 70%) as a white foam. ESI MS m / z 411 [M+H] + .

[0329] Example 64 N-(Benzo[b]thiophen-2-yl)-4-methoxy-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1203) JPEG2025105624000153.jpg3467After general procedure C2, 4-methoxy-2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.16 mmol) was reacted with benzo[b]thiophen-2-amine (27 mg, 1.8 mmol) to give the desired product (30 mg, 42%) as an orange solid. ESI MS m / z 453 [M+H] + 。

[0330] Example 65 2-((3-Methoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1205) JPEG2025105624000154.jpg2973Step 1. Methyl 2-((3-methoxyphenyl)sulfonamido)benzoate (JRW-1196) JPEG2025105624000155.jpg2960

[0331] After general procedure A, methyl 2-aminobenzoate (700 mg, 4.6 mmol) was reacted with 3-methoxybenzenesulfonyl chloride (1.05 g, 5.1 mmol) to give the desired product (1.3 g, 87%) as a white solid. ESI MS m / z 322 [M+H] + 。

[0332] Step 2. 2-((3-Methoxyphenyl)sulfonamido)benzoic acid (JRW-1199) JPEG2025105624000156.jpg2953

[0333] After general procedure B, methyl 2-((3-methoxyphenyl)sulfonamido)benzoate (1.3 g, 4.1 mmol) was reacted with NaOH (4.0 mL, 2 M, 8.0 mmol) to give the crude product (1.5 g) as a white solid. ESI MS m / z 308 [M+H] + 。

[0334] Engineering 3.2 - ((3 - Methoxyphenyl)sulfonamide)-N-(p - Tolyl)benzamide (JRW - 1205) JPEG2025105624000157.jpg2667

[0335] After general procedure C2, 2 - ((3 - Methoxyphenyl)sulfonamide)benzoic acid (455 mg, 1.5 mmol) was reacted with p - toluidine (190 mg, 1.8 mmol) to give the desired product (335 mg, 57%) as a light brown solid. ESI MS m / z 397 [M + H] + 。

[0336] Example 66 N - (Benzo[b]thiophen - 2 - yl)-2 - ((3 - Methoxyphenyl)sulfonamide)benzamide (JRW - 1230) JPEG2025105624000158.jpg3069 After general procedure C2, 2 - ((3 - Methoxyphenyl)sulfonamide)benzoic acid (60 mg, 0.19 mmol) was reacted with benzo[b]thiophen - 2 - amine (35 mg, 0.23 mmol) to give the desired product (30 mg, 35%) as a reddish - brown solid. ESI MS m / z 439 [M + H] + 。

[0337] Example 67 5 - Hydroxy - 2 - ((4 - Methylphenyl)sulfonamide)-N-(p - Tolyl)benzamide (JRW - 1232) To a solution of 5-methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide (260 mg, 0.63 mmol) in DCM (10 mL) at 0 °C was added boron tribromide (1.6 mL, 1.0 M, 1.6 mmol). The reaction was warmed to room temperature, stirred for 18 h, and quenched with saturated NaHCO3 solution. The mixture was diluted with DCM and water, and the aqueous layer was extracted with DCM. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to afford the desired product (29 mg, 11%) as a brown solid. ESI MS m / z 397 [M+H]+.

[0338] Example 68 2-((3-Hydroxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1236) To a solution of 2-((3-methoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (190 mg, 0.48 mmol) in DCM (10 mL) at 0 °C was added boron tribromide (0.96 mL, 1.0 M, 0.96 mmol). The reaction was warmed to room temperature, stirred for 18 h, and quenched with saturated NaHCO3 solution. The mixture was diluted with DCM and water, and the aqueous layer was extracted with DCM. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to afford the desired product (140 mg, 76%) as a light brown solid. ESI MS m / z 383 [M+H] + 。

[0339] Example 69 2-((3-Butoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1266) A solution of 2-((3-hydroxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (40 mg, 0.10 mmol) in 2988 THF (3 mL) was added with n-butanol (15 mg, 0.20 mmol), triphenylphosphine (30 mg, 0.12 mmol) and DIAD (46 mg, 0.23 mmol). The mixture was stirred at room temperature for 18 h. The mixture was diluted with ethyl acetate and water, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, filtered, concentrated and purified by silica gel chromatography to give the desired product (30 mg, 65%) as a white solid. ESI MS m / z 439 [M+H] + 。

[0340] Example 70 N-(2-Bromophenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1282) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (200 mg, 0.68 mmol) was reacted with 2-bromoaniline (141 mg, 0.82 mmol) to give the desired product (48 mg, 15%) as a white solid. ESI MS m / z 446 [M+H] + 。

[0341] Example 71 N-(3-Bromophenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1283) After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (200 mg, 0.68 mmol) was reacted with 3-bromoaniline (141 mg, 0.82 mmol) to give the desired product (122 mg, 40%) as a white solid. ESI MS m / z 446 [M+H] + 。

[0342] Example 72 N-([1,1'-Biphenyl]-4-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1288) JPEG2025105624000164.jpg3375After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with [1,1'-biphenyl]-4-amine (34 mg, 0.20 mmol) to give the desired product (42 mg, 55%) as a white solid. ESI MS m / z 443 [M+H] + 。

[0343] Example 73 N-(2-Methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1292) JPEG2025105624000165.jpg2961After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (190 mg, 0.65 mmol) was reacted with 2-methoxyaniline (96 mg, 0.78 mmol) to give the desired product (155 mg, 60%) as a light brown solid. ESI MS m / z 397 [M+H] + 。

[0344] Example 74 N-([1,1'-Biphenyl]-3-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1297) A solution of N-(3-bromophenyl)-2-((4-methylphenyl)sulfonamido)benzamide (110 mg, 0.24 mmol) in dioxane (5 mL) was added with phenylboronic acid (36 mg, 0.29 mmol) and Pd(dppf)Cl2 (20 mg, 0.024 mmol). The mixture was purged with nitrogen and then aqueous Cs2CO3 (0.74 mL, 1 M) was added. The reaction was heated at 80 °C for 2 h. The mixture was diluted with ethyl acetate and washed with water and brine. The organic layers were combined, dried over sodium sulfate, filtered, concentrated and purified by silica gel chromatography to give the desired product (105 mg, 96%) as a white solid. ESI MS m / z 443 [M+H] + 。

[0345] Example 75 N-(3-methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1299) After General Procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (190 mg, 0.65 mmol) was reacted with 3-methoxyaniline (96 mg, 0.78 mmol) to give the desired product (165 mg, 64%) as a white solid. ESI MS m / z 397 [M+H] + 。

[0346] Example 76 N-(4-methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1300) After General Procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (190 mg, 0.65 mmol) was reacted with 4-methoxyaniline (96 mg, 0.78 mmol) to give the desired product (156 mg, 60%) as a light brown solid. ESI MS m / z 397 [M+H] + 。

[0347] Example 77 2-((N-Ethyl-4-methylphenyl)sulfonamido)-N-(4-methoxyphenyl)benzamide (JRW-1325) JPEG2025105624000169.jpgTo a solution of N-(4-methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (50 mg, 0.12 mmol) in DMF (3 mL) were added diisopropylethylamine (49 mg, 0.38 mmol) and ethyl iodide (0.5 mL). The solution was stirred at 60 °C for 18 h. The mixture was diluted with ethyl acetate and washed with water and brine. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to afford the desired product (50 mg, 94%) as a pale yellow foam. ESI MS m / z 425 [M+H] + .

[0348] Example 78 N-(Benzo[b]thiophen-2-yl)-4-fluoro-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1346) JPEG2025105624000170.jpgStep 1. Methyl 4-fluoro-2-((4-methylphenyl)sulfonamido)benzoate (JRW-1342). JPEG2025105624000171.jpg3357

[0349] After General Procedure A, methyl 2-amino-4-fluorobenzoate (1.0 g, 5.9 mmol) was reacted with 4-methylbenzenesulfonyl chloride (1.2 g, 6.5 mmol) to afford the desired product (1.52 g, 79%) as a pale yellow solid. ESI MS m / z 324 [M+H] + .

[0350] Step 2. 4-Fluoro-2-((4-methylphenyl)sulfonamido)benzoic acid (JRW-1344). JPEG2025105624000172.jpg3151

[0351] After general procedure B, methyl 4-fluoro-2-((4-methylphenyl)sulfonamido)benzoate (1.5 g, 4.6 mmol) was reacted with NaOH (7 mL, 2 M, 14 mmol) to afford the crude product (1.6 g) as a pale yellow solid. ESI MS m / z 310 [M+H] + .

[0352] Step 3. N-(Benzo[b]thiophen-2-yl)-4-fluoro-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1346). JPEG2025105624000173.jpg3266

[0353] After general procedure C2, 4-fluoro-2-((4-methylphenyl)sulfonamido)benzoic acid (100 mg, 0.32 mmol) was reacted with benzo[b]thiophen-2-amine (58 mg, 0.39 mmol) to afford the desired product (93 mg, 65%) as a pale pink solid. ESI MS m / z 441 [M+H] + .

[0354] Example 79 N-(Benzo[b]thiophen-2-yl)-5-fluoro-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1347) JPEG2025105624000174.jpg3766 Step 1. Methyl 5-fluoro-2-((4-methylphenyl)sulfonamido)benzoate (JRW-1343). JPEG2025105624000175.jpg3351

[0355] After general procedure A, methyl 2-amino-5-fluorobenzoate (1.0 g, 5.9 mmol) was reacted with 4-methylbenzenesulfonyl chloride (1.2 g, 6.5 mmol) to afford the desired product (1.9 g, 99%) as a white solid. ESI MS m / z 324 [M+H] + .

[0356] Engineering 2.5-Fluoro-2-((4-methylphenyl)sulfonamido)benzoic acid (JRW-1345). JPEG2025105624000176.jpg3752

[0357] After general procedure B, methyl 5-fluoro-2-((4-methylphenyl)sulfonamido)benzoate (1.9 g, 5.9 mmol) was reacted with NaOH (9 mL, 2 M, 18 mmol) to give the crude product (1.8 g) as a white solid. ESI MS m / z 310 [M+H] + .

[0358] Step 3. N-(Benzo[b]thiophen-2-yl)-5-fluoro-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1347). JPEG2025105624000177.jpg3868

[0359] After general procedure C2, 5-fluoro-2-((4-methylphenyl)sulfonamido)benzoic acid (100 mg, 0.32 mmol) was reacted with benzo[b]thiophen-2-amine (58 mg, 0.39 mmol) to give the desired product (38 mg, 26%) as a light brown solid. ESI MS m / z 441 [M+H] + .

[0360] Example 80 N-Benzyl-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1383) JPEG2025105624000178.jpg2660 After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with benzylamine (31 mg, 0.21 mmol) to give the desired product (6 mg, 9%) as a light brown solid. ESI MS m / z 381 [M+H] + .

[0361] Example 81 N-(4-Methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1384) JPEG2025105624000179.jpg2872After general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 4-methoxy-benzylamine (31 mg, 0.21 mmol) to give the desired product (25 mg, 35%) as a pale brown solid. ESI MS m / z 411 [M+H] + 。

[0362] Example 82 N-(Benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzamide (JRW-1388) JPEG2025105624000180.jpg3362Step 1. Methyl 2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzoate (JRW-1382) JPEG2025105624000181.jpg3350

[0363] After general procedure A, methyl 2-amino-5-(trifluoromethyl)benzoate (1.0 g, 4.6 mmol) was reacted with 4-methylbenzenesulfonyl chloride (0.96 g, 5.0 mmol) to give the desired product (1.4 g, 81%) as a white solid. ESI MS m / z 374 [M+H] + 。

[0364] Step 2. 2-((4-Methylphenyl)sulfonamido)-5-(trifluoromethyl)benzoic acid (JRW-1386) JPEG2025105624000182.jpg3346

[0365] After general procedure B, methyl 2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzoate (1.4 g, 3.7 mmol) was reacted with NaOH (3.7 mL, 2 M, 7.4 mmol) to give the crude product (1.3 g) as a white solid. ESI MS m / z 360 [M+H] + 。

[0366] Step 3. N-(Benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzamide (JRW-1388) JPEG2025105624000183.jpg3362

[0367] After general procedure C2, 2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzoic acid (110 mg, 0.31 mmol) was reacted with benzo[b]thiophen-2-amine (55 mg, 0.37 mmol) to give the desired product (140 mg, 93%) as a light brown solid. ESI MS m / z 491 [M+H] + 。

[0368] Example 83 Selective inhibition of bioluminescent complexes The following examples provide for the use of the disclosed inhibitors to selectively inhibit various bioluminescent complexes, such as bioluminescent complexes derived from Oplophorus luciferase, without inhibiting NanoLuc® luciferase. Figure 1A shows the inhibition of the NanoBiT® HiBiT / LgBiT bioluminescent complex by an exemplary compound. The NanoBiT® HiBiT non-luminescent peptide and the NanoBiT® LgBiT non-luminescent polypeptide were each diluted in PBS + 0.01% BSA (final concentrations of 0.1 nM and 100 nM, respectively) and incubated at room temperature for 2 hours with the indicated compounds at sequentially diluted concentrations in the presence of RPMI medium (final 2.5% FBS). Samples were analyzed using a GloMax®-Multi+ plate reader after the addition of furimazine (final concentration of 10 μM). Each sample was normalized to the "no inhibitor" control. The IC 50 values were then determined using GraphPad Prism (log[inhibitor] vs. normalized response). Figure 1B shows the inhibition of the NanoBiT® SmBiT / LgBiT bioluminescent complex by an exemplary compound of the invention. The NanoBiT® SmBiT-annexin fusion and the NanoBiT® LgBiT-annexin fusion were each diluted in PBS / 0.01% BSA to final concentrations of 60 nM and 30 nM, respectively, and incubated at room temperature for 2 hours with the indicated compounds at sequentially diluted concentrations in the presence of K562 cell lysate diluted in RPMI medium (final 2.5% FBS). Samples were analyzed using a GloMax®-Multi+ plate reader after the addition of furimazine (final concentration of 10 μM). Each sample was normalized to the "no inhibitor" control. The IC 50 values were then determined using GraphPad Prism (log[inhibitor] vs. normalized response). Figure 1C is a bar graph comparing the calculated IC 50 values between the NanoBiT® HiBiT / LgBiT and SmBiT / LgBiT complexes for the exemplary compounds shown in Figures 1A and 1B.

[0369] Figure 2 compares the NANOLUC® (Nluc) inhibitory activity of exemplary compounds of the present invention to PBI-6096, known as an Nluc inhibitor. The inhibitor was diluted in CO2-independent medium with 10% FBS. Nluc was diluted to 2 ng / ml in NanoGlo® buffer with 100 μM furimazine. A series of dilutions of the inhibitor were added to the NanoLuc / furimazine / NanoGlo® solution and the samples were analyzed immediately using a GloMax®-Multi+ plate reader. As shown in Figure 2, JRW-1004, HL-0005, and HL-0010 showed no appreciable inhibition against NanoLuc, demonstrating the selectivity of the compounds against the bioluminescent complex derived from Oplophorus luciferase.

[0370] Example 84 Inhibition of Bioluminescent Complex in Cells Figures 3A - 3B show the inhibition of the bioluminescent complex in cells. In Figure 3A, HEK293 cells were transfected with DNA encoding an intracellular NanoBiT® HiBiT fusion protein, plated at 20,000 cells / 100 μL growth medium, and incubated for 24 hours. After 24 hours of expression, the cells were lysed with 50 ug / mL (final) digitonin in OptiMEM and treated with a series of dilutions of purified NanoBiT® LgBiT non-luminescent polypeptide and HL-0005. After incubation for 2 hours at room temperature, furimazine was added (final concentration of 10 μM) and luminescence was measured using a GloMax®-Multi+ plate reader.

[0371] In Figure 3B, DNA encoding an intracellular NanoBiT® HiBiT fusion protein and DNA encoding an intracellular NanoBiT® LgBiT fusion protein were transfected into HEK293 cells such that the fusion proteins were co-expressed intracellularly. The transfected cells were plated at 20,000 cells / 100 μL of growth medium and incubated for 24 hours. After 24 hours of expression, the cells were optionally lysed with 50 μg / mL digitonin in OptiMEM and treated with a series of dilutions of HL-0005. After incubation for 2 hours at room temperature, furimazine was added (final concentration of 10 μM), and luminescence was measured with a GloMax® Multi+ plate reader. Figure 3A shows the inhibition of the NanoBiT® HiBit / LgBit bioluminescent complex by HL-0005 in a cellular context. Figure 3B compares the IC 50 values and shows that HL-0005 is generally cell-permeable.

[0372] Example 85 Determination of the IC 50 of Inhibitors The following examples provide the IC 50 values of the compounds disclosed herein. The results are shown in Table 1. NanoBiT® HiBiT non-luminescent peptide and LgBiT non-luminescent non-polypeptide were each diluted to 0.1 nM and 1 nM in TBS buffer with 0.01% BSA to prepare detection reagents. Next, a 3-fold dilution series of each inhibitor was prepared in the detection reagent. A "no inhibitor" control was also prepared for each sample. 50 μL of each inhibitor dilution was mixed with 6 μM (final) furimazine, and luminescence was measured. Each sample was normalized to the "no inhibitor" control. The IC 50 values were then determined using GraphPad Prism (log[inhibitor] vs. normalized response). Table 1 JPEG2025105624000184.jpg203143 JPEG2025105624000185.jpg203143 JPEG2025105624000186.jpg62131 NA - No activity NT - Not tested

[0373] It is understood that the foregoing detailed description and the accompanying examples are merely illustrative and should not be construed as limitations on the scope of the invention, which is defined only by the appended claims and their equivalents.

[0374] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those related to the chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof. Appendix

[0375] SEQ ID NO: 1 - Amino acid sequence of native growth Oplophorus luciferase FTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFRVTINGVTGWRLCENILA

[0376] SEQ ID NO: 2 - Wild - type NLpep MGVTGWRLCERILA

[0377] SEQ ID NO: 3 - Wild - type NLpoly MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINV

[0378] Amino acid sequence of SEQ ID NO: 4-HiBiT VSGWRLFKKIS

[0379] Amino acid sequence of SEQ ID NO: 5-LgBiT MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS

[0380] Amino acid sequence of SEQ ID NO: 6-SmBiT VTGYRLFEEIL

[0381] SEQ ID NO: 7-NanoLuc MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

Claims

1. A compound of formula (I), or a salt thereof: (Wherein: R 1 is aryl, cycloalkyl, heteroaryl, or heterocycle, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, wherein said aryl, cycloalkyl, heteroaryl, and heterocycle are optionally substituted with one or more R W groups, and each R W is independently selected from the group consisting of C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-10 alkylene-OR A , -CO-R A , -C 1-10 alkylene-CO-R A , -CO-OR A , -C 1-10 alkylene-CO-OR A , -CO-NHR A , -C 1-10 alkylene-CO-NHR A , -NR B R C , -C 1-10 alkylene-NR B R C , -NH-CO-C 1-4 alkyl, -C 1-10 alkylene-NH-CO-C 1-4 alkyl, phenyl, and phenyl substituted with 1, 2, 3, or 4 R D groups; R 2 is C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-4 alkylene-OR A , -CO-R A , -C 1-4 alkylene-CO-R A , -CO-OR A , -C 1-4 alkylene-CO-OR A , -CO-NHR A , -C 1-4 alkylene-CO-NHR A , -NR B R C , -C 1-4 alkylene-NR B R C , -NH-CO-C 1-4 alkyl, -C 1-4 alkylene-NH-CO-C 1-4 alkyl, phenyl, phenyl substituted with 1, 2, 3, or 4 R D groups, -C≡C-R A , or -C≡C-C 1-4 alkylene-OR A and either two R 2 are such that when they are attached to the said Together with the carbon atom of the moiety forms a 5- or 6-membered fused ring; R 3 is C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-4 alkylene-OR A , -CO-R A , -CO-OR A , or -CO-NHR A and alternatively two Rs 3 are such that when they are attached to the said Together with the carbon atom of the moiety forms a 5- or 6-membered fused ring; R 4 is H or C 1-4 alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R at each occurrence A is, independently, H, C 1-4 alkyl, or C 1-4 haloalkyl; R at each occurrence B and R C are, independently, H or C 1-4 alkyl, or R B and R C together with the N atom to which they are attached form a 5- or 6-membered heterocycle; R at each occurrence D is, independently, C 1-4 alkyl, -OC 1-4 alkyl, -CN, or halogen, and The compound is not N-phenyl-2-(phenylsulfonamido)benzamide).

2. R 1 is aryl or heteroaryl, and the aryl and heteroaryl are optionally substituted with one or more R W groups, the compound according to claim 1, or a salt thereof.

3. R 1 is C optionally substituted with one or more R W and is aryl, the compound according to any one of claims 1 to 2, or a salt thereof. 6-20 ​

4. R 1 is a 5- to 12-membered heteroaryl optionally substituted with one or more R W The compound according to any one of claims 1 to 2, or a salt thereof.

5. The compound according to any one of Claims 1 to 4, or a salt thereof, wherein p is 0, 1, or 2.

6. p is 1, and R 2 is C 1-4 alkyl, halogen, C 1-4 haloalkyl, -OH, -C 1-4 alkylene-OH, -OC 1-4 alkyl, or -NH 2 The compound according to any one of claims 1 to 5, or a salt thereof.

7. The compound according to any one of Claims 1 to 6, or a salt thereof, wherein q is 0 or 1.

8. q is 1, and R 3 is C 1-4 alkyl, halogen, -CN, -OH, or -OC 1-4 alkyl, and the compound according to any one of claims 1 to 7, or a salt thereof.

9. The compound is of formula (I-a): (Wherein: R 1 is C 6-20 aryl or 5- to 12-membered heteroaryl, and the aryl and heteroaryl are optionally substituted with one or more R W ; p is 0, 1, or 2; q is 0 or 1; R 3 is C 1-4 alkyl, halogen, -CN or -OR A ; R A is H or C 1-4 and is alkyl; R 2 and R W are as defined in claim 1) The compound according to Claim 1, or a salt thereof).

10. R 1 is and each of which is one or more R W The compound according to claim 9, or a salt thereof, which is optionally substituted with

11. R 1 is unsubstituted or R 1 is substituted with one, two or three R W each R W is independently halogen, C 1-4 alkyl, C 1-4 haloalkyl, or phenyl, a compound according to any one of claims 9 to 10, or a salt thereof.

12. R 1 is The compound according to any one of Claim 9, or a salt thereof.

13. p is 1 or 2, and each R 2 is, independently, C 1-4 alkyl, halogen, C 1-4 haloalkyl, -OH, -C 1-4 alkylene-OH, -OC 1-4 alkyl, -NH 2、 phenyl, or -CO-OC 1-4 alkyl, or two Rs 2 are such that when they are attached to the said Together with the moiety To form the compound according to any one of Claims 9 to 12, or a salt thereof.

14. The compound is of formula (I-a-1): (Wherein: R 1 is and each is optionally substituted with halogen, C 1-4 alkyl, C 1-4 haloalkyl, or phenyl; or R 1 is Is; R 2 is halogen, C 1-4 alkyl, C 1-4 haloalkyl, -OH, -C 1-4 alkylene-OH, -OC 1-4 alkyl, or -NH 2 (wherein) The compound according to Claim 9, or a salt thereof).

15. N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-cyanothiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(2-cyanophenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 2-((4-methylphenyl)sulfonamido)-N-phenylbenzamide; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)benzamide; N-(benzo[b]thiophen-2-yl)-2-((4-formylphenyl)sulfonamido)benzamide; Methyl 3-(4-(N-(2-(benzo[b]thiophen-2-ylcarbamoyl)phenyl)sulfamoyl)phenyl)propanoate; N-(Benzo[b]thiophen-2-yl)-2-((3-methylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((4-(3-hydroxypropyl)phenyl)sulfonamido)benzamide; 2-([1,1'-Biphenyl]-3-sulfonamido)-N-(p-tolyl)benzamide; Methyl 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoate; 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoic acid; 2-((3-Acetamidophenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-Aminophenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(Hydroxymethyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(Butylcarbamoyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-Bromophenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(Butylamino)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(Hex-1-yn-1-yl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-Hexylphenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(3-Hydroxyprop-1-yn-1-yl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-(3-Hydroxypropyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide; N-(p-Tolyl)-2-((4-(trifluoromethyl)phenyl)sulfonamido)benzamide; 2-((4-Methoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((4-Bromophenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-([1,1'-Biphenyl]-4-sulfonamido)-N-(p-tolyl)benzamide; N-(p-Tolyl)-2-((3-(trifluoromethyl)phenyl)sulfonamido)benzamide; N-(p-Tolyl)-2-((3-(trifluoromethoxy)phenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-Cyclohexyl-2-((4-methylphenyl)sulfonamido)benzamide; 2-((4-methylphenyl)sulfonamido)-N-(naphthalen-2-yl)benzamide; 2-((4-methylphenyl)sulfonamido)-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzamide; Methyl trans-4-(2-((4-methylphenyl)sulfonamido)benzamido)cyclohexane-1-carboxylate; Trans-4-(2-((4-methylphenyl)sulfonamido)benzamido)cyclohexane-1-carboxylic acid; 2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide; Ethyl 2-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)acetate; N-(3-Isopropylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; Ethyl 3-(2-((4-methylphenyl)sulfonamido)benzamido)benzoate; 2-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)acetic acid; 3-(2-((4-methylphenyl)sulfonamido)benzamido)benzoic acid; 2-((4-methylphenyl)sulfonamido)-N-(m-tolyl)benzamide; N-(Benzo[b]thiophen-2-yl)-3-((4-methylphenyl)sulfonamido)-2-naphthamide; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(Benzo[b]thiophen-2-yl)-5-methyl-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-Butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-5-cyano-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-Butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(Benzo[b]thiophen-2-yl)-2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzamide; N-(4-Hexylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 2-(((4-methylphenyl)sulfonyl)amino)-N-(4-octylphenyl)benzamide; Methyl 6-(4-(2-(((4-methylphenyl)sulfonyl)amino)benzamide)phenyl)hexanoate; 6-(4-(2-(((4-methylphenyl)sulfonyl)amino)benzamide)phenyl)hexanoic acid; N-(benzo[b]thiophen-2-yl)-2-(((4-butylphenyl)sulfonyl)amino)benzamide; N-(4-(6-hydroxyhexyl)phenyl)-2-(((4-methylphenyl)sulfonyl)amino)benzamide; N-(benzo[b]thiophen-2-yl)-2-(((4-pentylphenyl)sulfonyl)amino)benzamide; N-(benzo[b]thiophen-2-yl)-5-butyl-2-(((4-methylphenyl)sulfonyl)amino)benzamide; N-(4-(4-hydroxybutyl)phenyl)-2-(((4-methylphenyl)sulfonyl)amino)benzamide; N-(4-(4-bromobutyl)phenyl)-2-(((4-methylphenyl)sulfonyl)amino)benzamide; 5-methoxy-2-(((4-methylphenyl)sulfonyl)amino)-N-(p-tolyl)benzamide; N-(benzo[b]thiophen-2-yl)-5-methoxy-2-(((4-methylphenyl)sulfonyl)amino)benzamide; 4-methoxy-2-(((4-methylphenyl)sulfonyl)amino)-N-(p-tolyl)benzamide; N-(benzo[b]thiophen-2-yl)-4-methoxy-2-(((4-methylphenyl)sulfonyl)amino)benzamide; 2-(((3-methoxyphenyl)sulfonyl)amino)-N-(p-tolyl)benzamide; N-(benzo[b]thiophen-2-yl)-2-(((3-methoxyphenyl)sulfonyl)amino)benzamide; 5-hydroxy-2-(((4-methylphenyl)sulfonyl)amino)-N-(p-tolyl)benzamide; 2-(((3-hydroxyphenyl)sulfonyl)amino)-N-(p-tolyl)benzamide; 2-(((3-butoxyphenyl)sulfonyl)amino)-N-(p-tolyl)benzamide; N-(2-bromophenyl)-2-(((4-methylphenyl)sulfonyl)amino)benzamide; N-(3-bromophenyl)-2-(((4-methylphenyl)sulfonyl)amino)benzamide; N-( [1,1'-biphenyl]-4-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(2-methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-( [1,1'-biphenyl]-3-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(3-methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 2-((N-ethyl-4-methylphenyl)sulfonamido)-N-(4-methoxyphenyl)benzamide; N-(benzo[b]thiophen-2-yl)-4-fluoro-2-((4-methylphenyl)sulfonamido)benzamide; N-(benzo[b]thiophen-2-yl)-5-fluoro-2-((4-methylphenyl)sulfonamido)benzamide; N-benzyl-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide; and N-(benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzamide The compound according to claim 1, or a salt thereof, selected from the group consisting of.

16. A method for inhibiting an Oplophorus luciferase-derived bioluminescent complex, said method comprising contacting said bioluminescent complex with a compound according to any one of claims 1 to 15, said method.

17. The bioluminescent complex comprises two or more substantially non-luminescent peptide and / or polypeptide units, and the association of said two or more non-luminescent peptide and / or polypeptide units produces a detectable bioluminescence signal in the presence of a coelenterazine substrate. The method according to claim 16.

18. The association of said two or more substantially non-luminescent peptide and / or polypeptide units produces a bioluminescent complex capable of binding said coelenterazine substrate. The method according to claim 17.

19. The bioluminescent complex is a) a peptide comprising an amino acid sequence having an identity of less than 100% and more than 40% to SEQ ID NO: 2; and b) a polypeptide comprising an amino acid sequence having an identity of less than 100% and more than 40% with SEQ ID NO: 3 comprising The method according to claim 16, wherein the bioluminescent complex exhibits detectable luminescence in the presence of a coelenterazine substrate. **Claim 20** The method according to claim 19, wherein the bioluminescent complex comprises a polypeptide having the amino acid sequence of SEQ ID NO: 5 and a peptide having the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:

6. **Claim 21** A method for regulating the luminescence of an Oplophorus luciferase-derived bioluminescent complex in a sample, the method comprising: a) contacting the sample with a coelenterazine substrate and a compound according to any one of claims 1 to 15; b) detecting the luminescence in the sample; comprising The method according to any one of claims 1 to 15, wherein the compound causes a decrease in the luminescence from the bioluminescent complex. **Claim 22** A method for detecting an interaction or co-localization between a first molecule and a second molecule in a sample, the method comprising: a) contacting the sample with a coelenterazine substrate and a compound according to any one of claims 1 to 15, wherein the sample comprises: i) a first fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule; ii) a second fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a second molecule, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; comprising the contacting; b) detecting the luminescence in the sample; comprising The method, wherein the detection of the luminescence indicates an interaction or co-localization between the first protein and the second protein. **Claim 23** A method for detecting an interaction or co-localization between a first molecule and a second molecule in a sample, the method comprising: a) contacting the sample with a coelenterazine substrate and a compound according to any one of claims 1 to 15, wherein the sample comprises: (i)A first polynucleotide encoding a first fusion, wherein the first fusion comprises a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule, said first polynucleotide, and (ii)A second polynucleotide encoding a second fusion, wherein the second fusion comprises a non-luminescent polypeptide of Oplophorus-derived luciferase and a second molecule, and the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide, said second polynucleotide, and comprising said contacting, (b)Detecting luminescence in said sample, comprising, said method, wherein said detection of luminescence indicates an interaction or co-localization between said first molecule and said second molecule.

24. A method for detecting an interaction or co-localization of a first molecule and a second molecule in a sample, said method comprising: (a)Contacting a sample with a non-luminescent polypeptide of Oplophorus-derived luciferase, a coelenterazine substrate, and a compound according to any one of claims 1 to 15, wherein the sample comprises: (i)A first polynucleotide encoding a first fusion, wherein the first fusion comprises a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule, and the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide, said first polynucleotide, and (ii)A second polynucleotide encoding a second fusion, wherein the second fusion comprises a fluorescent acceptor molecule and a second molecule, said second polynucleotide, and comprising said contacting, (b)Detecting bioluminescence resonance energy transfer (BRET) in said sample indicating an interaction or co-localization between said first molecule and said second molecule, comprising said method.

25. A method for detecting an interaction or co-localization of a first molecule and a second molecule in a sample, said method comprising: (a)Contacting a sample with a non-luminescent peptide of Oplophorus-derived luciferase, a coelenterazine substrate, and a compound according to any one of claims 1 to 15, wherein the sample comprises: (i)A first polynucleotide encoding a first fusion, wherein the first fusion comprises a non-luminescent polypeptide of Oplophorus-derived luciferase and a first molecule, and the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide, the first polynucleotide; (ii)A second polynucleotide encoding a second fusion, wherein the second fusion comprises a fluorescent acceptor molecule and a second molecule, the second polynucleotide; Including the contacting; (b)Detecting bioluminescence resonance energy transfer (BRET) in the sample indicating interaction or co-localization of the first molecule and the second molecule; Including the method.

26. A method for detecting interaction or co-localization of molecules in a sample, the method comprising: (a)Contacting a sample with a coelenterazine substrate and a compound according to any one of claims 1 to 15, wherein the sample comprises: (i)A first fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a first molecule; (ii)A second fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a second molecule, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide, the second fusion; (iii)A third fusion comprising a fluorescent acceptor molecule and a third molecule; Including the contacting; (b)Detecting bioluminescence resonance energy transfer (BRET) in the sample indicating interaction or co-localization among the first molecule, the second molecule, and the third molecule in the sample; Including the method.

27. A method for detecting a target molecule in a sample, the method comprising: (a)A sample comprising the target molecule fused to a non-luminescent peptide of Oplophorus-derived luciferase is contacted with: (i)A coelenterazine substrate; (ii)A compound according to any one of claims 1 to 15; and (iii)A non-luminescent polypeptide of Oplophorus-derived luciferase, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; Contacting; (b)Detecting luminescence in the sample; Including The method, wherein detection of luminescence indicates formation of a bioluminescent complex between the non-luminescent peptide and the non-luminescent polypeptide.

28. A method for detecting a target molecule in a sample, the method comprising: (a) contacting a sample containing the target molecule fused to a non-luminescent polypeptide of Oplophorus-derived luciferase with (i) a coelenterazine substrate; (ii) a compound according to any one of claims 1 to 15; and (iii) a non-luminescent peptide of Oplophorus-derived luciferase, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; and (b) detecting luminescence in the sample, wherein detection of luminescence indicates formation of a bioluminescent complex between the non-luminescent peptide and the non-luminescent polypeptide.

29. A method for detecting a target molecule in a sample, the method comprising: (a) contacting a sample containing the target molecule fused to a non-luminescent polypeptide of Oplophorus-derived luciferase with (i) a coelenterazine substrate; (ii) a compound according to any one of claims 1 to 15; and (iii) a fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a fluorescent moiety, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; and (b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of detection of the molecule.

30. A method for detecting a target molecule in a sample, the method comprising: (a) contacting a sample containing the target molecule fused to a non-luminescent polypeptide of Oplophorus-derived luciferase with (i) a coelenterazine substrate; (ii) a compound according to any one of claims 1 to 15; and (iii) a fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a fluorescent moiety, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; and (b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of detection of the molecule.

31. A method for detecting a target molecule in a sample, the method comprising: (a) contacting a sample containing the target molecule fused to a non-luminescent polypeptide of Oplophorus-derived luciferase with (i) a coelenterazine substrate; (ii) a compound according to any one of claims 1 to 15; and (iii) a fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a fluorescent moiety, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; and (b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of detection of the molecule.

31. The method according to any one of claims 21 to 30, comprising contacting the sample with the selentrazine substrate before contacting the sample with the compound according to any one of claims 1 to 15.

32. The method according to any one of claims 21 to 26, wherein when the first molecule and the second molecule interact or co-localize, the non-luminescent peptide and the non-luminescent polypeptide associate to form a bioluminescent complex capable of generating a bioluminescence signal in the presence of a selentrazine substrate.

33. The method according to any one of claims 21 to 32, wherein the sample comprises cells.

34. The method according to any one of claims 21 to 33, wherein the selentrazine substrate is selentrazine, a selentrazine derivative, a selentrazine analog, pro-selentrazine, or quinone-masked selentrazine.

35. a) the non-luminescent peptide comprises an amino acid sequence having less than 100% and more than 40% sequence identity with SEQ ID NO: 2, b) the non-luminescent polypeptide comprises an amino acid sequence having less than 100% and more than 40% sequence identity with SEQ ID NO: 3, the method according to any one of claims 21 to 34.

36. A bioluminescence resonance energy transfer (BRET) system, comprising: a) a first fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule; b) a second fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a fluorescent moiety, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; c) a selentrazine substrate; d) a compound according to any one of claims 1 to 15. The bioluminescence resonance energy transfer (BRET) system.

37. A bioluminescence resonance energy transfer (BRET) system, comprising: a) a first fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a first molecule; b) a second fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a fluorescent moiety, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; c) a selentrazine substrate; (d) A compound according to any one of claims 1 to 15, and The bioluminescence resonance energy transfer (BRET) system comprising. **Claim 38** A bioluminescence resonance energy transfer (BRET) system, comprising: (a) A first fusion comprising a first molecule and a non-luminescent peptide of Oplophorus-derived luciferase; (b) A second fusion comprising a second molecule and a fluorescent acceptor molecule; (c) A non-luminescent polypeptide of Oplophorus-derived luciferase capable of forming a bioluminescent complex with the non-luminescent peptide of Oplophorus-derived luciferase; (d) A coelenterazine substrate; (e) A compound according to any one of claims 1 to 15, and The bioluminescence resonance energy transfer (BRET) system comprising. **Claim 39** A bioluminescence resonance energy transfer (BRET) system, comprising: (a) A first fusion comprising a first molecule and a non-luminescent polypeptide of Oplophorus-derived luciferase; (b) A second fusion comprising a second molecule and a fluorescent acceptor molecule; (c) A non-luminescent peptide of Oplophorus-derived luciferase capable of forming a bioluminescent complex with the non-luminescent polypeptide of Oplophorus-derived luciferase; (d) A coelenterazine substrate; (e) A compound according to any one of claims 1 to 15, and The bioluminescence resonance energy transfer (BRET) system comprising. **Claim 40** a) The non-luminescent peptide comprises an amino acid sequence having a sequence identity of less than 100% and more than 40% with SEQ ID NO: 2; b) The non-luminescent polypeptide comprises an amino acid sequence having a sequence identity of less than 100% and more than 40% with SEQ ID NO: 3; The bioluminescence resonance energy transfer system according to any one of claims 36 to 39. **Claim 41** (a) A compound according to any one of claims 1 to 15, and (b) An Oplophorus luciferase-derived bioluminescent complex, A kit comprising. **Claim 42** The Oplophorus luciferase-derived bioluminescent complex comprises: (a) A non-luminescent peptide comprising an amino acid sequence having a sequence identity of less than 100% and more than 40% with SEQ ID NO: 2; (b) A non-luminescent polypeptide comprising an amino acid sequence having a sequence identity of less than 100% and more than 40% with SEQ ID NO: 3; The kit according to claim 41. **Claim 43** (a) A compound according to any one of claims 1 to 15, and (b) A first polynucleotide encoding a non-luminescent peptide of Oplophorus-derived luciferase, and (c) A second polynucleotide encoding a non-luminescent polypeptide of Oplophorus-derived luciferase, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide, the second polynucleotide, A kit comprising.

44. The non-luminescent peptide comprises an amino acid sequence having a sequence identity of less than 100% and more than 40% with SEQ ID NO: 2, and the non-luminescent polypeptide comprises an amino acid sequence having a sequence identity of less than 100% and more than 40% with SEQ ID NO:

3. The kit according to claim 43.

45. The kit according to any one of claims 41 to 44, further comprising a coelenterazine substrate.

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