Inhibitors of the bioluminescent complex derived from Oplophorus luciferase

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 to U.S. Provisional Patent Application No. 62 / 679,205, filed June 1, 2018, which is incorporated herein by reference in its entirety.

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

[0003] Protein fragment complementation (PFC) or enzyme fragment complementation (EFC) systems are valuable tools for monitoring colocalization and / or intermolecular interactions. In such systems, reporter molecules, such as complementary amino acid chains (e.g., peptides or polypeptides) from bioluminescent proteins or enzymes, are fused to colocalized and / or interacting molecules. Reporter molecules are routinely used to monitor molecular events in the fields of biology, biochemistry, immunology, cell biology, and molecular biology. Luciferases based on luciferase secreted from the deep-sea shrimp Oplophorus gracilirostris can be used as reporter molecules and have been shown to have advantageous characteristics, 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 colocalized / interacting molecules (e.g., proteins). When the molecules colocalize and / or interact, the non-luminescent peptide and / or polypeptide units associate to form a bioluminescent complex, which, in the presence of a substrate (e.g., a coelenterazine or coelenterazine derivative substrate), can generate a luminescent signal indicating the colocalization / interaction of the molecules. In certain applications, it may be advantageous to further control the luminescent signal from Oplophorus luciferase-derived bioluminescent complexes. Selective inhibitors for such bioluminescent complexes are useful in luminescence assays. Luciferase inhibitors can be further derivatized to provide desired properties useful for studying enzymatic activity and cellular processes. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 210294 [Patent Document 2] International Publication No. 2007 / 101225 [Patent Document 3] International Publication No. 2014 / 152895 Summary of the Invention [Means for solving the problem]

[0005] In one aspect, the present disclosure provides a compound of formula (I), or a salt thereof: JPEG0007815514000001.jpg37170 (in the formula: R 1 is an aryl, cycloalkyl, heteroaryl, heterocycle, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocycle may be optionally joined by one or more R W and each R W 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 1, 2, 3, or 4 R D phenyl substituted with a group; Each R 2 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 phenyl substituted with a group, -C≡CR A , or -C≡CC 1-4 Alkylene-OR A or two R 2 But they are combined JPEG0007815514000002.jpg1315 together with the carbon atoms of the moiety to form a 5- or 6-membered fused ring; Each R 3 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 R 3 But they are combined JPEG0007815514000003.jpg1720 together with the carbon atoms of the moiety to form a 5- or 6-membered fused ring; R 4 is H or C 1-4 is alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R in each occurrenceA are independently H, C 1-4 Alkyl, or C 1-4 is haloalkyl; R in each occurrence B and R C are independently H or C 1-4 alkyl, or R B and R C form a 5- or 6-membered heterocyclic ring together with the N atom to which they are attached; R in each occurrence D independently, C 1-4 Alkyl, -OC 1-4 alkyl, -CN, or halogen) to provide.

[0006] In one aspect, the disclosure provides a method of inhibiting a bioluminescent complex derived from Oplophorus luciferase, 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 luminescence in the sample; Including, The compound causes a decrease in luminescence from the bioluminescent complex, methods are provided.

[0008] In one aspect, the present 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 a sample with a coelenterazine substrate and a compound described herein, the sample comprising: (i) a first fusion, the first fusion comprising a non-luminescent peptide of Oplophorus luciferase and a first molecule; (ii) a second fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a second molecule, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; contacting the (b) detecting luminescence in the sample; Including, Detection of the emitted light provides a method that indicates an interaction or co-localization between the first and second molecules.

[0009] In one aspect, the present 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 a 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-light-emitting peptide of Oplophorus luciferase and a first molecule; (ii) a second polynucleotide encoding a second fusion, the second fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a second molecule, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; contacting the (b) detecting luminescence in the sample; Including, Detection of the emitted light provides a method that indicates an interaction or co-localization between the first and second molecules.

[0010] 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-light-emitting polypeptide of Oplophorus luciferase, a coelenterazine substrate, and a compound described herein, wherein the sample (i) a first polynucleotide encoding a first fusion, the first fusion comprising a non-luminescent peptide of Oplophorus luciferase and a first molecule, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; (ii) a second polynucleotide encoding a second fusion, the second fusion comprising a fluorescent acceptor molecule and a second molecule; and contacting the (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates an interaction or co-localization of the first molecule and the second molecule; The present invention provides a method comprising:

[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-light-emitting peptide of Oplophorus luciferase, a coelenterazine substrate, and a compound described herein, wherein the sample: (i) a first polynucleotide encoding a first fusion, the first fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a first molecule, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; (ii) a second polynucleotide encoding a second fusion, the second fusion comprising a fluorescent acceptor molecule and a second molecule; and contacting the (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates an interaction or co-localization of the first molecule and the second molecule; The present invention provides a method comprising:

[0012] In one aspect, the present disclosure provides a method for detecting molecular interactions or co-localization in a sample, the method comprising: (a) contacting a sample with a coelenterazine substrate and a compound described herein, the sample comprising: (i) a first fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a first molecule; (ii) a second fusion comprising a non-luminescent peptide of Oplophorus luciferase and a second molecule, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; (iii) a third fusion comprising a fluorescent receptor molecule and a third molecule; and contacting the (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates an interaction or co-localization between the first molecule, the second molecule, and the third molecule in the sample; The present invention provides a method comprising:

[0013] In one aspect, the present disclosure provides a method for detecting a molecule of interest in a sample, the method comprising: (a) A sample containing a molecule of interest fused to a non-luminescent peptide of Oplophorus luciferase is (i) Coelenterazine substrate; (ii) a compound described herein; and (iii) a non-luminescent polypeptide of Oplophorus luciferase, which is capable of forming a bioluminescent complex with a non-luminescent peptide; and contacting the (b) detecting luminescence in the sample; Including, Detection of luminescence provides a method that indicates the formation of a bioluminescent complex between the non-luminescent peptide and the non-luminescent polypeptide.

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

[0015] In one aspect, the present disclosure provides a method for detecting a molecule of interest in a sample, the method comprising: (a) A sample containing a molecule of interest fused to a non-luminescent peptide of Oplophorus luciferase is (i) 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 contacting the (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates detection of the molecule; The present invention provides a method comprising:

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

[0017] (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates detection of the molecule; The present invention provides a method comprising:

[0018] In one aspect, the present disclosure provides a bioluminescence resonance energy transfer (BRET) system, comprising: (a) a first fusion comprising a non-luminescent peptide of Oplophorus luciferase and a first molecule; (b) a second 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; (c) a coelenterazine substrate; (d) a compound described herein; and The present invention provides a bioluminescence resonance energy transfer (BRET) system comprising:

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

[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 luciferase; (b) a second fusion comprising a second molecule and a fluorescent receptor molecule; (c) a non-luminescent polypeptide of Oplophorus luciferase capable of forming a bioluminescent complex with a non-luminescent polypeptide of Oplophorus luciferase; (d) a coelenterazine substrate; (e) a compound described herein; and The present invention provides a bioluminescence resonance energy transfer (BRET) system comprising:

[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 luciferase; (b) a second fusion comprising a second molecule and a fluorescent receptor molecule; (c) a non-luminescent peptide of Oplophorus luciferase capable of forming a bioluminescent complex with a non-luminescent polypeptide of Oplophorus luciferase; (d) a coelenterazine substrate; (e) a compound described herein; and The present invention provides a bioluminescence resonance energy transfer (BRET) system comprising:

[0022] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) a compound described herein; and (b) a first polynucleotide encoding a non-luminescent peptide of Oplophorus luciferase; (c) a second polynucleotide encoding a non-luminescent polypeptide of Oplophorus luciferase, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; A kit is provided, comprising: [Brief explanation of the drawings]

[0023] [Figure 1] 1A and 1B show the inhibition of bioluminescent complexes by exemplary compounds of the invention. A shows the inhibition of the NanoBiT® HiBiT / LgBiT bioluminescent complex. B shows the inhibition of the NanoBiT® SmBiT / LgBiT bioluminescent complex. C is a bar graph showing calculated IC50 values ​​for exemplary compounds shown in FIGS. 1A and 1B. [Figure 2] The NANOLUC® (Nluc) inhibitory activity of exemplary compounds of the invention is compared to PBI-6096, a known Nluc inhibitor. JRW-1004, HL-0005, and HL-0010 show no appreciable inhibition of NanoLuc, but selective inhibition of the Oplophorus luciferase-derived bioluminescent complex. [Figure 3] Figure 1 shows the inhibition of intracellular bioluminescent complexes by exemplary compounds of the invention. (A) shows the inhibition of NanoBiT® HiBit / LgBit bioluminescent complexes by HL-0005 in a cellular context. (B) compares IC50 values ​​in lytic and non-lytic conditions, demonstrating that HL-0005 is largely cell permeable. DETAILED DESCRIPTION OF THE INVENTION

[0024] The disclosed compounds can selectively inhibit the bioluminescence complex derived from Oplophorus luciferase. For example, the disclosed compounds include (a) peptides comprising a peptide amino acid sequence having less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) sequence identity to SEQ ID NO:2 and more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity to SEQ ID NO:3; and (b) peptides comprising a peptide amino acid sequence having less than 100% (e.g., >99%, <95%, <90%, <80%) sequence identity to SEQ ID NO:3. and (b) a polypeptide comprising a polypeptide having a polypeptide amino acid sequence having at least 40% (e.g., <70%, <60%, <50%, etc.) sequence identity to a coelenterazine substrate, wherein the coelenterazine substrate exhibits detectable luminescence in the presence of a coelenterazine substrate. In certain embodiments, the disclosed compounds are capable of selectively inhibiting a bioluminescent complex derived from Oplophorus luciferase, comprising a polypeptide comprising a polypeptide amino acid sequence having at least 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity to a coelenterazine substrate. In certain embodiments, the disclosed compounds are capable of selectively inhibiting a bioluminescent complex derived from Oplophorus luciferase, comprising (a) a peptide comprising the peptide amino acid sequence SEQ ID NO:4 or SEQ ID NO:6; and (b) a polypeptide comprising a polypeptide amino acid sequence having SEQ ID NO:5, wherein the bioluminescent complex exhibits detectable luminescence in the presence of a coelenterazine substrate. 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 by reference in their entireties. 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 cellular excretion, and the presence of cellular debris resulting from cultured cells, it may be advantageous in certain applications to use selective inhibitors of the present invention to suppress luminescence from Oplophorus luciferase-derived bioluminescent complexes. For example, in applications involving the temporal multiplexing of multiple luminescent systems, it may be beneficial to have a selective inhibitor for each system to allow measurement and / or detection of only one luminescent signal at a time. Also, in some plate-based assays, a given amount of luciferase may be excreted from cells or present in the medium from cellular debris. An extracellular inhibitor compound would allow for selective suppression of luminescence from luciferase in the medium, which may help improve the signal-to-background ratio in certain assays.

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

[0027] 1.Definition 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 document, including definitions, will control. Preferred methods and materials are described below, although 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 materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0028] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude 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," and "consist essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[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 invalidate the activity of the compound of the present invention. Illustrative examples of suitable substituents include, but are not limited to, halo groups, perfluoroalkyl groups, perfluoroalkoxy groups, alkyl groups, alkenyl groups, alkynyl groups, hydroxy groups, halo groups, oxo groups, mercapto groups, alkylthio groups, alkoxy groups, nitro groups, azidoalkyl groups, sulfonic acid groups, aryl or heteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl or heteroaralkyl groups, aralkoxy or heteroaralkoxy groups, HO-(C=O)- groups, heterocyclic groups, cycloalkyl groups, amino groups, alkyl- and dialkyl-amino groups, carbamoyl groups, alkylcarbonyl groups, alkylcarbonyloxy groups, alkoxycarbonyl groups, alkylaminocarbonyl groups, dialkylaminocarbonyl groups, arylcarbonyl groups, aryloxycarbonyl groups, alkylsulfonyl groups, arylsulfonyl groups, etc. The substituents may be substituted with additional substituents. The substituents may also be in the form of a salt (e.g., a sulfonic acid group may be in the form of a sulfonate group).

[0030] Definitions of certain functional groups and chemical terms are described in detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th The general principles of organic chemistry and specific functional moieties and reactivities are identified in accordance with the back cover of "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and 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 incorporated herein by reference.

[0031] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing 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 can be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 3 suitable substituents, as defined above.

[0032] The term "alkoxy," as used herein, refers to an alkyl group, as defined herein, appended 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] The term "alkoxyalkoxy," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through 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] The term "alkoxyalkoxyalkyl," as used herein, means an alkoxyalkoxy group, as defined herein, appended to the parent molecular moiety through 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] The term "alkoxyalkyl," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through 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] The term "alkoxycarbonyl," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through 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, appended to the parent molecular moiety through 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" refers to a linear or branched hydrocarbon group, preferably having 1 to 30 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. The term "C-C-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, "C-C-alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, 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 "C-C-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" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, 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" specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, isobutyl, and tert-butyl. Alkyl groups of the present invention can be unsubstituted or substituted with one or more suitable substituents, for example, 1 to 3 suitable substituents, as defined above. For example, alkyl groups can be substituted with one or more halo substituents to form a haloalkyl group, or one or more hydroxy substituents to form a hydroxyalkyl group, or one or more alkoxy groups to form an alkoxyalkyl group.

[0039] The term "alkylamino," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through 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] The term "alkylaminoalkyl," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an aminoalkyl group, as defined herein. Representative examples of alkylaminoalkyl groups include, but are not limited to, methylaminoethyl and methylamino-2-propyl.

[0041] The term "alkylcarbonyl," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through 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] The term "alkylcarbonylalkyl," as used herein, refers to an alkylcarbonyl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0043] The term "alkylcarbonylalkylamido," as used herein, refers to an alkylcarbonyl group, as defined herein, appended to the parent molecular moiety through an alkylamido group, as defined herein.

[0044] The term "alkylene" means a divalent group derived from a saturated, straight or branched chain hydrocarbon of from 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 hydrocarbon group having 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons and having one or more carbon-carbon triple bonds. Alkynyl groups of the present invention include, but are not limited to, ethynyl, propynyl, and butynyl. Alkynyl groups of the present invention can be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 3 suitable substituents, as defined above.

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

[0047] As used herein, the term "amino" refers to the group --NH.sub.2.

[0048] The term "aminoalkyl," as used herein, refers to at least one amino group, as defined herein, appended to the parent molecular moiety through 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 "aminoalkylamido" refers to at least one amino group, as defined herein, appended to the parent molecular moiety through an alkylamido group, as defined herein.

[0050] As used herein, the term "amino protecting group" refers to a moiety that prevents a chemical reaction from occurring on the nitrogen atom to which the protecting group is attached. The amino protecting group must also be removable by chemical reaction. Such groups are well known in the art and are described in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M.Wuts, 3 rd edition, John Wiley & Sons, 1999, which is incorporated herein by reference in its entirety. Suitable amino-protecting groups include, but are not limited to, carbobenzyloxy (-NHCO-OCHCH or -NH-Cbz); t-butyloxycarbonyl (-NHCO-OC(CH) 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 "aminoluciferin" 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" refers to 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 be optionally substituted with one or more suitable substituents, preferably 1 to 5 suitable substituents, as defined above. As used herein, aryl is appended to a parent molecular moiety and includes a phenyl fused to a cycloalkyl group (e.g., indanyl or 5,6,7,8-tetrahydronaphthalen-2-yl), a phenyl fused to a phenyl group (i.e., naphthyl), or a phenyl fused to a non-aromatic heterocycle (e.g., benzo[d][1,3]dioxol-5-yl).

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

[0054] The term "arylcarbonyl," as used herein, refers to an aryl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein.

[0055] As used herein, the term "carbonyl" or "(C=O)" (as used in phrases such as alkylcarbonyl, alkyl-(C=O)-, or alkoxycarbonyl) refers to the connection of a >C=O moiety to a second moiety, such as an alkyl or amino group (i.e., an amide group). Alkoxycarbonylamino (i.e., alkoxy(C=O)-NH-) refers to an alkylcarbamate group. A carbonyl group is also defined herein equivalently as (C=O). Alkylcarbonylamino refers to groups such as acetamido.

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

[0057] The term "carboxyalkyl," as used herein, refers to a carboxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0058] The term "carboxyalkylamido," as used herein, refers to a carboxyalkyl group, as defined herein, appended to the parent molecular moiety through 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, bicyclo[5.2.0]nonanyl, and the like), optionally containing one or two double bonds. The cycloalkynyl groups of the present invention can be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 5 suitable substituents, as defined above.

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

[0061] The term "cycloalkylamido," as used herein, refers to a cycloalkyl group, as defined herein, appended to the parent molecular moiety through an amide group, as defined herein.

[0062] The term "dialkylamino," as used herein, refers to two independently selected alkyl groups, as defined herein, appended to the parent molecular moiety through 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] The term "dialkylaminoalkyl," as used herein, refers to a dialkylamino group, as defined herein, appended to the parent molecular moiety through 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] The term "dialkylaminoalkylamido," as used herein, refers to a dialkylamino group, as defined herein, appended to the parent molecular moiety through an alkylamido 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 a monocyclic heteroaryl or a bicyclic heteroaryl. A monocyclic heteroaryl is a 5- or 6-membered ring. A 5-membered ring contains two double bonds. A 5-membered ring may contain one heteroatom selected from O or S; or 1, 2, 3, or 4 nitrogen atoms and optionally one oxygen or sulfur atom. A 6-membered ring may contain three 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 a monocyclic heteroaryl fused to a phenyl, or a monocyclic heteroaryl fused to a monocyclic cycloalkyl, or a monocyclic heteroaryl fused to a monocyclic cycloalkenyl, or a monocyclic heteroaryl fused to a monocyclic heteroaryl, or a monocyclic heteroaryl fused to a 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 groups of the present invention can be unsubstituted or substituted with 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 the parent molecular moiety through an alkyl group, as defined herein.Representative examples of heteroarylalkyl include, but are not limited to, furan-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 term "heterocycle" or "heterocyclyl" refers to a monocyclic heterocycle, bicyclic heterocycle, or tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 7- and 8-membered ring contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. Representative examples of monocyclic heterocycles include 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, phosphinane, piperazinyl, piperidinyl, pyrazolinyl, and the like. Examples of thiomorpholinyl include, but are not limited to, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, trithianyl, and 2,5-dioxo-pyrrolidinyl.A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a bridged monocyclic heterocyclic ring system in which 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 bicyclic heterocycles 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. Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which 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 tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-admantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7Heterocyclic groups of the present invention may be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 3 suitable substituents, as defined above. Heterocyclic groups of the present invention may contain one or more oxo (=O) or thioxo (=S) groups attached to the ring.

[0071] As used herein, the term "heterocyclylalkyl" refers to a heterocyclyl group, as defined herein, appended to the parent molecular moiety through 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] The term "heterocyclylamide," as used herein, refers to a heterocyclyl group, as defined herein, appended to the parent molecular moiety through 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 with 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 with 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] The term "hydroxyalkylamido," as used herein, refers to a hydroxyalkyl group attached to an amide group, e.g., -amido-alkyl-OH.

[0077] The term "hydroxycarbonyl," as used herein, refers to a hydroxy group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein.

[0078] The term "methylenedioxy," as used herein, refers to a -OCHO- group, in which the oxygen atoms of the methylenedioxy are attached to the parent molecular moiety through two adjacent carbon atoms.

[0079] As used herein, the term "oxo" refers to a double-bonded oxygen (=O) group where the bond 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 joined through a backbone by peptide amide bonds (-C(O)NH-). The term "peptide" typically refers to short amino acid polymers (e.g., chains having fewer than 25 amino acids), while the term "polypeptide" typically refers to longer amino acid polymers (e.g., chains having more than 25 amino acids).

[0081] A prefix attached to a multi-component substituent applies only to the first component that it precedes. To illustrate, the term "cycloalkylalkyl" contains two components: alkyl and cycloalkyl. Thus, C1- 10 Cycloalkylalkyl C1- 10 The prefix C1-C6- denotes that the alkyl portion of the cycloalkylalkyl contains 1 to 6 carbon atoms; the prefix C1-C6- does not describe the cycloalkyl portion. 10 The term haloalkyl refers to halomethyl, haloethyl, halopropyl, halobutyl, halopentyl, and halohexyl. To further illustrate, the prefix "halo" in 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 instead be described as "alkoxyhaloalkyl."

[0082] A substituent is "substitutable" if it contains at least one carbon or nitrogen atom that is bonded to one or more hydrogen atoms. Thus, for example, hydrogen, halogen, and cyano are not included in this definition. Also, sulfur atoms in heterocyclyls containing such atoms can be substituted with one or two oxo substituents.

[0083] When a substituent is described as "substituted", a non-hydrogen group exists in place of a hydrogen group on the 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 exists in place of a hydrogen group on the alkyl substituent.For example, monofluoroalkyl is an alkyl substituted with a fluoro group, and difluoroalkyl is an alkyl substituted with two fluoro groups.When multiple substitutions exist on a substituent, it should be recognized that each non-hydrogen group can be the same or different (unless otherwise stated).

[0084] When a substituent is described as "unsubstituted" and not "substituted" or "optionally substituted," it means that the substituent does not have any substituents. When a substituent is described as "optionally substituted," the substituent can be (1) unsubstituted or (2) substituted. When a substituent is described as being optionally substituted with up to a specified number of non-hydrogen groups, the substituent can be (1) unsubstituted or (2) substituted with up to the specified number of non-hydrogen groups or up to the maximum number of substitutable positions on the substituent, whichever is less. Thus, for example, if a substituent is described as a heteroaryl optionally substituted with up to three non-hydrogen groups, then any heteroaryl having fewer than three substitutable positions would be optionally substituted only with up to the same number of non-hydrogen groups as the heteroaryl has substitutable positions. To illustrate, tetrazolyl (having only one substitutable position) would be optionally substituted with up to one non-hydrogen group. To further illustrate, if it is said that an amino nitrogen is optionally substituted with up to two non-hydrogen groups, then a primary amino nitrogen is optionally substituted with up to two non-hydrogen groups, while a secondary amino nitrogen is optionally substituted with up to only one non-hydrogen group.

[0085] When substituents are described as being "independently selected" from a group, each substituent is selected independently of the others, and therefore each substituent can be the same or different from the other substituent(s).

[0086] As used herein, the term "bioluminescence" or "luminescence" can refer to light produced as a result of a reaction between a light-producing enzyme and a substrate. Examples of such enzymes (bioluminescent enzymes) include Oplophorus luciferase, e.g., Oplophorus gracilirostris, firefly luciferase, e.g., Photinus pyralis or Photuris pennsylvanica, click beetle luciferase, Renilla luciferase, Cypridina 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 one another. In one aspect, "contact" or, more specifically, "direct contact" means that two or more molecules are in close enough proximity that attractive non-covalent interactions, such as van der Waals forces, hydrogen bonding, ionic and hydrophobic interactions, etc., dominate the interaction of the molecules. In such an aspect, a complex of molecules (e.g., peptides and polypeptides) forms under assay conditions such that the complex is thermodynamically favored (e.g., compared to the unaggregated, or uncomplexed, states of its component molecules). As used herein, the term "complex," unless otherwise specified, refers to a population of two or more molecules (e.g., peptides, polypeptides, or combinations thereof).

[0088] The terms "bioluminescent complex" or "Oplophorus luciferase-derived bioluminescent complex," used interchangeably herein, refer to an assembled complex of two or more non-luminescent peptides and / or polypeptides. The bioluminescent complex catalyzes or enables the conversion of the substrate of the bioluminescent complex to an unstable form; the substrate then emits light. When uncomplexed, the two non-luminescent components that form a bioluminescent complex may be referred to as a "non-luminescent pair." When a bioluminescent complex is formed by three or more non-luminescent peptides and / or polypeptides, the uncomplexed components of the bioluminescent complex may be referred to as a "non-luminescent group." The Oplophorus luciferase-derived bioluminescent conjugates include (a) a peptide comprising a peptide amino acid sequence having less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity to SEQ ID NO:2; and and (b) a polypeptide comprising a polypeptide amino acid sequence having less than 100% identity (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity to SEQ ID NO:3, wherein the bioluminescent conjugate exhibits detectable luminescence. In certain embodiments, the invention provides a bioluminescent conjugate comprising: (a) a peptide comprising a peptide amino acid sequence having SEQ ID NO:4 or 6; and (b) a polypeptide comprising a polypeptide amino acid sequence having SEQ ID NO:5, wherein the bioluminescent conjugate exhibits detectable luminescence. Exemplary Oplophorus luciferase-derived bioluminescent conjugates include NanoBiT® technology, including 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 detectable amounts 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 exhibits no 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 exhibits, for example, 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10) luminescence compared to a complex of the NLpoly with its non-luminescent complementary peptide. 3 times 1×10 4 times 1×10 5 times 1×10 6 times 1×10 7 In some embodiments, an entity is "non-luminescent" if any light emission is minimal enough 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) refer to a non-luminescent peptide that emits substantially no light (e.g., in the presence of a substrate), or that is below noise or that does not emit a significant signal (e.g., a luminescent conjugate) by 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10) under standard conditions (e.g., physiological conditions, assay conditions, etc.) using a typical instrument (e.g., a luminometer, etc.). 3 times 1×10 4 times 1×10 5 times 1×10 6 times 1×10 7 "non-luminescent element" refers to peptides and polypeptides that exhibit an amount less than (e.g., 1-fold) the amount of non-luminescent peptides and polypeptides present in the bioluminescent complex. 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" refers to a non-luminescent peptide or polypeptide.

[0091] As used herein, the term "interacting element" or "interacting molecule" refers to a moiety that helps bring together a pair of non-luminescent elements or groups to form a bioluminescent complex. In a typical embodiment, a pair of interacting elements (also known as an "interacting pair") binds to a pair of non-luminescent elements (e.g., a non-luminescent peptide / polypeptide pair), and an attractive interaction between the two interacting elements promotes the formation of a bioluminescent complex; however, the present invention is not limited to such a mechanism, and an understanding of the mechanism is not required for the practice of the present invention. An interacting element may promote the formation of a bioluminescent complex by any suitable mechanism (e.g., bringing the non-luminescent pair / group into close proximity, positioning the non-luminescent pair / group in a suitable conformation for stable interaction, reducing the activation energy for complex formation, a combination thereof, etc.). An interacting element can be a protein, polypeptide, peptide, small molecule, cofactor, nucleic acid, lipid, carbohydrate, antibody, etc. An interacting pair can be composed of two of the same interacting elements (i.e., a homopair) or two different interacting elements (i.e., a heteropair). In the case of heteropairs, the interacting elements can be the same type of moiety (e.g., a polypeptide) or two different types of moieties (e.g., a polypeptide and a small molecule). In some embodiments in which complex formation by an interacting pair is studied, the interacting pair can be referred to as a "target pair" or "pair of interest," and the individual interacting elements are 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 of interest (e.g., a target sequence, etc.) linked 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 the first polypeptide or protein and the second peptide, polypeptide, or protein are fused end-to-end (e.g., C-terminus to N-terminus or N-terminus to C-terminus).

[0093] As used herein, the terms "coelenterazine," "coelenterazine substrate," "coelenterazine derivative," or "coelenterazine derivative substrate" refer to a class of reporter molecules that emit light when acted upon by various bioluminescent proteins, such as luciferases (e.g., marine luciferases). 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 naturally occurring analogs thereof, including 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-methylcoelenterazine, 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).

[0094] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples can include blood products such as plasma and serum. Samples can also refer to cell lysates or purified forms 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. Samples can also include cell-free expression systems. Environmental samples include environmental materials such as surface materials, soil, water, crystals, and industrial samples. However, such examples should not be construed as limiting the types of samples applicable to the present invention.

[0095] The term "energy acceptor" or "acceptor 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 acceptor is a fluorophore or other detectable chromophore.Suitable fluorophores include 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.), prene 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., porphine, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA Examples of suitable dyes include, but are not limited to, 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," used interchangeably herein, refer to a class of oxidative enzymes used in bioluminescence, in which the enzyme generates and emits light when provided with a substrate. The luciferase can be a naturally occurring luciferase, a recombinant luciferase, or a mutant luciferase that uses a luciferase substrate. The luciferase substrate can be luciferin, a luciferin derivative or analog, a pre-luciferin derivative or analog, coelenterazine, or a coelenterazine derivative or analog. If the luminescent enzyme is naturally occurring, or is a recombinant or mutant luminescent enzyme, for example, one that retains activity in the luciferase-coelenterazine or luciferase-luciferin reaction of a naturally occurring luminescent enzyme, it can be easily obtained from cultures of bacteria, yeast, mammalian cells, insect cells, plant cells, and the like that have been transformed to express a nucleic acid encoding the luminescent enzyme. Additionally, recombinant or mutant luminescent enzymes can be derived from in vitro cell-free systems using nucleic acids encoding luciferase.Suitable luminescent enzymes include luciferases from bioluminescent decapod crustaceans, such as luciferases from Oplophoroidea (e.g., Oplophorus luciferase), beetle luciferases (e.g., Photinus pyralis, Photuris pennsylvanica, etc.), marine organisms such as Cnidarians (e.g., Renilla luciferase), decapod families Aristeidae, Solenoceridae, Luciferidae, Sergestidae, Pasipheidae, and Thalassocarididae, Caenorhabditis luciferases, such as Gaussia luciferases, e.g., Gaussia princeps luciferase, Metridia luciferases, e.g., Metridia longa and Metridia pacifica luciferase, Vargula luciferases, e.g., Vargula hilgendorfii luciferase, Pleuromamma luciferases, and the like. xiphias luciferase, and photoproteins such as aequorin, as well as variants, recombinants, and mutants thereof.

[0097] A "luminescent reaction mixture" contains materials that enable a 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 materials needed to generate a luminescent signal, as well as the specific concentrations and / or amounts, will vary depending on the luminescent enzyme used and the type of assay being performed. Often, other materials are added to the solution, including buffers to maintain the reaction at the appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) to help maintain enzyme activity, reducing agents, detergents, etc.

[0098] As used herein, the terms "Oplophorus luciferase" and "Oplophorus-derived luciferase" are used interchangeably and refer to the luciferase secreted from the deep-sea shrimp Oplophorus gracilirostris (e.g., SEQ ID NO: 1), including wild-type, variant, and mutant forms thereof. For example, suitable Oplophorus luciferase variants are described in U.S. Patent Nos. 8,557,970 and 8,669,103, each of which is incorporated 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 share the same biophysical characteristics and can be classified into families, such as acidic (e.g., aspartate, glutamic acid), 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). "Percent sequence identity" (or "percent sequence similarity") 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 specified window); (2) determining the number of positions containing identical (or similar) monomers (e.g., the same amino acid is present in both sequences, the similar amino acid is present in both sequences) to obtain the number of matched positions; (3) dividing the number of matched 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 specified window); and (4) multiplying the result by 100 to obtain the percent sequence identity or sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but one position, then peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., both are acidic), then peptide A and peptide B would 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 in the peptide C portion, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity over the optimal comparison window of peptide C.For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is treated as a mismatch at that position.

[0100] As used herein, the term "reporter moiety" may refer to a moiety that generates a detectable signal directly or indirectly under appropriate conditions. Exemplary reporter moieties include, but are not limited to, fluorophores, luminescent molecules, dyes, radioactive labels, and substrates for enzymes such as luciferase. In some embodiments, a reporter moiety may indirectly generate a detectable signal, for example, when the reporter moiety is a substrate for an enzyme. Reaction of the enzyme with the substrate then generates a detectable signal, such as fluorescence or luminescence. As used herein, the term "bioluminescent reporter moiety" may refer to a moiety that is a substrate for luciferase. For example, a bioluminescent reporter moiety can be luciferin, a luciferin derivative, such as pre-luciferin, aminoluciferin, chionorylluciferin, napthylluciferin, fluoroluciferin, chloroluciferin, a precursor of a luciferin derivative, coelenterazine, or a coelenterazine derivative or analog, such as furimazine. The generated luminescent 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, silixanthene, or carbopyronine. Fluorescence can be detected using a fluorometer.

[0101] For the description of numerical ranges herein, each intervening number therebetween, to the same degree of precision, is expressly 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 expressly contemplated.

[0102] 2.Compound Provided herein are compounds that can selectively inhibit Oplophorus luciferase-derived bioluminescent complexes, e.g., inhibit the luciferase activity of the bioluminescent complexes. In one embodiment, a compound of formula (I), or a salt thereof: JPEG0007815514000004.jpg43170 (in the formula: R 1 is an aryl, cycloalkyl, heteroaryl, heterocycle, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocycle may be optionally joined by one or more R W and each R W 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 1, 2, 3, or 4 R D phenyl substituted with a group; Each R 2 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 phenyl substituted with a group, -C≡CR A , or -C≡CC 1-4 Alkylene-OR A or two R 2 But they are combined JPEG0007815514000005.jpg1315 together with the carbon atoms of the moiety to form a 5- or 6-membered fused ring; Each R 3 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 R 3 But they are combined JPEG0007815514000006.jpg1720 together with the carbon atoms of the moiety to form a 5- or 6-membered fused ring; R 4 is H or C 1-4 is alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R in each occurrenceA are independently H, C 1-4 Alkyl, or C 1-4 is haloalkyl; R in each occurrence B and R C are independently H or C 1-4 alkyl, or R B and R C form a 5- or 6-membered heterocyclic ring together with the N atom to which they are attached; R in each occurrence D 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 an aryl, cycloalkyl, heteroaryl, or heterocycle, and the aryl, cycloalkyl, heteroaryl, and heterocycle may be joined by one or more R W and each R W 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 1-4alkyl, phenyl, and 1, 2, 3, or 4 R D phenyl substituted with a group; Each R 2 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, 1, 2, 3, or 4 R D phenyl substituted with a group, -C≡CR A , or -C≡CC 1-4 Alkylene-OR A or two R 2 But they are combined JPEG0007815514000007.jpg1315 together with the carbon atoms of the moiety to form a 5- or 6-membered fused ring; Each R 3 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 R 3 But they are combined JPEG0007815514000008.jpg1720 together with the carbon atoms of the moiety to form a 5- or 6-membered fused ring; R 4 is H or C 1-4 is alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R in each occurrence A are independently H, C 1-4 Alkyl, or C 1-4 is haloalkyl; R in each occurrence B and R C are independently H or C 1-4 alkyl, or R B and R C form a 5- or 6-membered heterocyclic ring together with the N atom to which they are attached; R in each occurrence D independently, C 1-4 Alkyl, -OC 1-4 It is alkyl, —CN, or halogen.

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

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

[0106] In some embodiments, R 1 is an aryl (C 6-20 aryl) or heteroaryl (5- to 12-membered heteroaryl), and the aryl and heteroaryl are each independently one or more R W In some embodiments, R 1 is C 6-20 aryl, wherein the aryl is selected from one or more R WIn some embodiments, R 1 is one or more R W In some embodiments, R is a 5- to 12-membered heteroaryl optionally substituted with 1 is one or more R W C optionally substituted with 5-10 In some embodiments, R 1 is one or more R W is a 5- to 12-membered heterocycle optionally substituted with

[0107] In some embodiments, R 1 teeth, JPEG0007815514000009.jpg22170, each containing one or more R W In some embodiments, R 1 teeth, JPEG0007815514000010.jpg22170, each of which is unsubstituted. In some embodiments, R 1 teeth, JPEG0007815514000011.jpg22170, and each of these is C 1-4 Alkyl or C 1-4 It is substituted with haloalkyl.

[0108] In some embodiments, R 1 teeth, JPEG0007815514000012.jpg22170, each of which contains one or more R W In some embodiments, R 1 teeth, JPEG0007815514000013.jpg22170. In some embodiments, each R W independently, C 1-10 Alkyl, C 1-10 In some embodiments, R is selected from the group consisting of haloalkyl, halogen, or —CN. W -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 is -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 is phenyl substituted with a phenyl group. W is C 1-10 Alkyl, C 1-10 haloalkyl, halogen, or -CN. In some embodiments, R W is a C such as methyl, ethyl, propyl, or butyl. 1-4 It is 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 and phenyl. 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 optionally substituted arylalkyl (such as benzyl).

[0111] In some embodiments, R 1 is an aryl, cycloalkyl, heteroaryl, heterocycle, or arylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocycle are each independently selected from one R W is substituted with 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, each R A is independently selected from hydrogen, methyl, and ethyl.

[0112] In some embodiments, R 1 teeth, JPEG0007815514000014.jpg43170, and each of these is non-permutation or one R W is substituted with 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 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 In some embodiments, R 2 is C 1-10 In some embodiments, R 2 is a C such as methyl, ethyl, propyl, or butyl. 1-4 In some embodiments, R 2 is methyl. In some embodiments, R 2 is a 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≡CC 1-4 Alkylene-OR A are selected from, and 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≡CR A (e.g., -C≡C-(CH2)3CH3) and -C≡CC 1-4 Alkylene-OR A (e.g., —C≡C—CH—OH). 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 R2 are combined JPEG0007815514000015.jpg1315 carbon atoms together to form a 5- or 6-membered fused ring. For example, two R 2 are combined JPEG0007815514000016.jpg1315 together JPEG0007815514000017.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 R A is hydrogen and C 1-4 In some embodiments, R 3 is C 1-10 Alkyl (e.g., C such as methyl, ethyl, isopropyl, n-propyl, or n-butyl) 1-4 alkyl), C 1-10 Haloalkyl (e.g., C such as trifluoromethyl) 1-4 haloalkyl), halogen (e.g., fluoro, chloro, or bromo), —CN, and —OR A (e.g., —OH or —OCH). In some embodiments, R 3 is C 1-4 Alkyl, halogen, -CN, -OH, or -OC 1-4 In some embodiments, R 3 is halogen. In some embodiments, R 2 is C 1-4 alkyl (e.g., 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, two R 3 are combined JPEG0007815514000018.jpg1720 together with the carbon atoms of the moiety to form a 5- or 6-membered fused ring. For example, two R 3 are combined JPEG0007815514000019.jpg1720 together JPEG0007815514000020.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 (e.g., methyl), and 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 and R 3 is —CH 3 , halogen (such as F), —OCH 3 , 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 In some embodiments, R 4 is ethyl.

[0123] R in each occurrence A (R 1 , R 2 , or R 3(as a substituent in 1-4 Alkyl, or C 1-4 In some embodiments, R A is H. In some embodiments, R A is C 1-4 In some embodiments, R A is methyl. In some embodiments, R A is C such as -CF3 1-4 It is haloalkyl.

[0124] R in each occurrence B and R C (R 1 or R 2 (as a substituent in 1-4 alkyl, or R B and R C are taken together with the N atom to which they are attached to 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 In some embodiments, R B is C 1-4 alkyl, and R C is C 1-4 In some embodiments, R B and R C together with the N atom to which they are attached form a 5- or 6-membered heterocyclic ring.

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

[0126] In some embodiments, R 1 teeth, JPEG0007815514000021.jpg22170, and each of these is C 1-4 Alkyl or C 1-4 substituted with haloalkyl, or R 1 teeth JPEG0007815514000022.jpg22170, R 2 is C 1-4 Alkyl, halogen, C 1-4 Haloalkyl, -OH, C 1-4 Alkylene -OH, -OC 1-4 alkyl, or -NH2, and R 3 is C 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 one or more R W (e.g., one R W In some embodiments, p is 0 and R 1 If is phenyl, then R 1 is one or more R W (For example, one R W In some embodiments, 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, 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-(phenylsulfonamido)benzamide.

[0128] In some embodiments, the compound of Formula (I) is a compound of Formula (Ia), or a salt thereof: JPEG0007815514000023.jpg37170 (in the formula, R 1 is C 6-20 aryl or 5- to 12-membered heteroaryl, and the aryl and heteroaryl are each independently one or more R W optionally substituted with; p is 0, 1, or 2; q is 0 or 1; R 3 is C 1-4 Alkyl, halogen, -CN or -OR A and; R A is H or C 1-4 is alkyl; R 2 and R W are as defined herein) is.

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

[0130] In some embodiments, a compound of formula (Ia), or a salt thereof, wherein R 1 teeth, JPEG0007815514000024.jpg22170, each containing one or more R W In some embodiments, R of formula (Ia) is optionally substituted with 1 teeth, JPEG0007815514000025.jpg22170.

[0131] In some embodiments, a compound of formula (Ia), or a salt thereof, wherein R 1 is C 1-4 Alkyl or C 1-4 For example, R in formula (Ia) is haloalkyl. 1 teeth, JPEG0007815514000026.jpg22170, each of which is C 1-4 Alkyl or C 1-4 It is substituted with haloalkyl.

[0132] In some embodiments, a compound of formula (Ia), or a salt thereof, wherein R 1 teeth, JPEG0007815514000027.jpg22170, and each of these contains one or more R W In some embodiments, R 1 teeth, JPEG0007815514000028.jpg22170.

[0133] In some embodiments, a compound of formula (Ia), or a salt thereof, wherein R 1 teeth, JPEG0007815514000029.jpg43170, and each of these is non-permutation or one R W is substituted with 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., selected from the group consisting of -CH2COOH, -CH2COOCH2CH3, -(CH2)5-COOH, or -(CH2)5-COOCH3), and phenyl).

[0134] In some embodiments, a compound of formula (Ia), 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 are combined 1315) form a 5- or 6-membered fused ring together with the carbon atoms of the moiety. For example, two R 2 It is binding JPEG0007815514000031.jpg1315 together In some embodiments, a compound of formula (Ia), or a salt thereof, wherein R 2 is C 1-4 In some embodiments, compounds of formula (Ia), or a salt thereof, wherein R 2 -OH, -C 1-4 Alkylene -OH (such as -CH2OH), -OC 1-4 In some embodiments, compounds of formula (Ia), or a salt thereof, are disclosed, wherein p is 1 and q is 0. In some embodiments, compounds of formula (Ia), or a salt thereof, wherein R 2 is C 1-4 Alkyl (e.g., methyl), -OH, -C 1-4 Alkylene -OH (such as -CH2OH), -OC 1-4In some embodiments, compounds of formula (Ia), or salts thereof, are disclosed, wherein R is alkyl (such as -OCH3), or -NH2, and p is 1 and q is 0. ... 2 is methyl and R 3 is -CH3, halogen (such as F), -OCH3, or -CN, p is 1, and q is 1) are disclosed.

[0135] In some embodiments, a compound of Formula (Ia), or a salt thereof, wherein p is 0 and R 1 If is phenyl, then R 1 is one or more R W In some embodiments, compounds of formula (Ia), or salts thereof, wherein 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, compounds of formula (Ia), or a salt thereof, are disclosed, wherein when p is 0, then R 1 is one or more R W (e.g., one R W In some embodiments, compounds of formula (Ia), or a salt thereof, wherein p is 0 and R 1 If is phenyl, then R 1 is one or more R W (e.g., one R W In some embodiments, compounds of formula (Ia), or salts thereof, wherein 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, compounds of formula (Ia), or salts thereof, wherein R 1is unsubstituted phenyl, then p is 1, 2, 3, or 4 (e.g., p is 1 or 2, or p is 1) are disclosed. In some embodiments, compounds of formula (Ia), or a salt thereof, are disclosed, wherein the compound is not N-phenyl-2-(phenylsulfonamido)benzamide.

[0136] In some embodiments, the compound of Formula (Ia) is a compound of Formula (Ia-1), or a salt thereof: JPEG0007815514000033.jpg43170 (in the formula: R 1 teeth, JPEG0007815514000034.jpg22170, and each is a halogen, C 1-4 Alkyl, C 1-4 optionally substituted with haloalkyl, or phenyl; or R 1 teeth, JPEG0007815514000035.jpg20150; R 2 is a 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 is as defined in formula (Ia). is.

[0137] In some embodiments, a compound of formula (Ia-1), or a salt thereof, wherein R 1 teeth, In some embodiments, a compound of formula (Ia-1), or a salt thereof, wherein R 1 teeth, JPEG0007815514000037.jpg22170, each of which is a halogen, C 1-4 Alkyl, C 1-4In some embodiments, compounds of formula (Ia-1), or a salt thereof, wherein R 1 teeth, JPEG0007815514000038.jpg22170) is disclosed.

[0138] In some embodiments, a compound of formula (Ia-1), or a salt thereof, wherein R 2 is C 1-4 Alkyl (e.g., methyl), halogen (e.g., Br), or C 1-4 In some embodiments, compounds of formula (Ia-1), or a salt thereof, wherein R 2 is C 1-4 In some embodiments, compounds of formula (Ia-1), or a salt thereof, wherein R 2 -OH, -C 1-4 Alkylene -OH (such as -CH2OH), -OC 1-4 In some embodiments, compounds of formula (Ia-1), or salts thereof, wherein R 2 is C 1-4 Alkyl (e.g., methyl), halogen (e.g., Br), -OH, C 1-4 Alkylene -OH (-CH2-OH, etc.), -OC 1-4 In some embodiments, compounds of formula (Ia-1), or a salt thereof, wherein R 2 is C 1-4 and q is 0. In some embodiments, compounds of formula (Ia-1), or a salt thereof, are disclosed, wherein R 2 is C 1-4 alkyl (e.g., methyl), and R 3 is —CH 3 , halogen (such as F), —OCH 3 , or —CN, and q is 1).

[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.

[0140] (1) Salt form The thienopyrrole compounds described herein can be in the form of salts.The neutral form of the compound can be regenerated by contacting the salt with a base or acid, and isolating the parent compound by conventional methods.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 becomes -COO - (which may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, + and K. + Alkali metal ions such as Ca 2+ and Mg 2+ Examples of suitable organic cations include, but are not limited to, alkaline earth cations such as ammonium ions (i.e., NH + ) and substituted ammonium ions (e.g., NH3R1 + , NH2R2 + , NHR3 + , NR4 + Examples of some suitable substituted ammonium ions include, but are not limited to, 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 becomes -NH3+ (which may be:) then salts may be formed with suitable anions. 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-acetyoxybenzoic 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, carboxymethyl cellulose.

[0144] Unless otherwise specified, a reference herein to a particular thienopyrrole compound also includes its salt forms.

[0145] (2) Isomers A given thienopyrrole compound may exist in one or more particular 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, hereinafter collectively referred to as "isomers" (or "isomeric forms").

[0146] In some embodiments, the compounds described herein may be enantiomerically enriched isomers of the stereoisomers described herein. For example, the compounds may 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 compounds disclosed herein are enriched for an isomer of the compound having a selected stereochemistry, e.g., R or S, corresponding to a selected stereocenter. For example, the compound has a purity corresponding to at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the compound having the selected stereochemistry at the selected stereocenter.

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

[0149] An "enriched preparation," as used herein, is enriched for a selected configuration of one, two, three, or more selected stereocenters in a subject compound. Exemplary selected stereocenters and their exemplary configurations can be selected from those provided herein, for example, 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, this is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Enriched refers to a level of the subject molecule(s) and does not imply a process limitation unless otherwise specified.

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

[0151] Specifically excluded from the term "isomer" as used herein are structural (or conformational) isomers (i.e., isomers that differ solely by the bonds between atoms and not simply by the position of the atoms in space), excluding tautomeric forms as discussed below. For example, a reference to a methoxy group, i.e., -OCH, should not be construed as a reference to its structural isomer, a hydroxymethyl group, i.e., -CHOH. Similarly, a reference to ortho-chlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, reference to a structural class may fully include structural isomeric forms that fall within that class (e.g., C-alkyl or propyl includes n-propyl and iso-propyl; C-alkyl or butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

[0152] The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-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.

[0153] In particular, it should be noted that the term "isomer" includes compounds with one or more isotopic substitutions. For example, H is 1 H, 2 H(D), and 3 H(T) can be in any isotopic form; C can be in any isotopic form, including 12 C. 13 C, and 14 C can be any isotopic form, including 16 O and 18 It can be any isotopic form containing O, etc.

[0154] 3. Bioluminescent complex derived from Oplophorus luciferase The disclosed compounds can be used to inhibit Oplophorus luciferase-derived bioluminescent complexes. The disclosed compounds can inhibit the luciferase activity of Oplophorus luciferase-derived bioluminescent complexes. 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] Oplophorus luciferase-derived bioluminescent conjugates can be an assembly of two or more non-luminescent peptide and / or polypeptide units (e.g., a non-luminescent pair). Oplophorus luciferase-derived bioluminescent conjugates can include (a) a peptide comprising a peptide amino acid sequence having less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity to SEQ ID NO:2; and (b) SEQ ID NO:3. and (b) a polypeptide comprising a polypeptide amino acid sequence having SEQ ID NO:5, wherein the bioluminescent conjugate exhibits detectable luminescence in the presence of a coelenterazine substrate. In certain embodiments, the invention provides a bioluminescent conjugate comprising: (a) a peptide comprising a peptide amino acid sequence having SEQ ID NO:4 or SEQ ID NO:6; and (b) a polypeptide comprising a polypeptide amino acid sequence having SEQ ID NO:5, wherein the bioluminescent conjugate exhibits detectable luminescence in the presence of a coelenterazine substrate. Exemplary Oplophorus luciferase-derived bioluminescent conjugates include NanoBiT® technology, which includes the NanoBiT® SmBiT nonluminescent peptide (SEQ ID NO: 6), the NanoBiT® HiBiT nonluminescent peptide (SEQ ID NO: 4), and / or the NanoBiT® LgBiT nonluminescent polypeptide (SEQ ID NO: 5). Oplophorus luciferase-derived bioluminescent conjugates are described in U.S. Patent 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 competing with or interfering with coelenterazine or coelenterazine derivative substrates from binding to luciferase. Coelenterazine substrates are a class of reporter molecules that emit light upon the action of luciferase and other bioluminescent proteins. Examples of coelenterazine substrates include coelenterazine; those disclosed in WO 2003 / 040100, U.S. Patent Publication No. 2008 / 0248511, and U.S. Patent Publication No. US 2012 / 0117667; as well as coelenterazine derivatives and / or analogs such as 2-furanylmethyl-deoxy-coelenterazine (furimazine), coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, and coelenterazine. Coelenterazine substrates include, but are not limited to, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl-coelenterazine; pro-coelenterazines (i.e., compounds that are not substrates for non-luminescent enzymes that convert the compound into a luciferase substrate), quinone-masked coelenterazines, etc. Further examples of coelenterazine substrates are described, for example, in U.S. Publication No. 2012 / 0107849, U.S. Publication No. 2013 / 0130289, U.S. Patent Application No. 14 / 608,910, and U.S. Patent Application No. 14 / 609,372, each of which is incorporated herein by reference.

[0157] 5. Methods for inhibiting the activity of bioluminescent complexes derived from Oplophorus luciferase The disclosed compounds can be used in methods for inhibiting Oplophorus luciferase-derived bioluminescent complexes, e.g., for inhibiting the luciferase activity of a bioluminescent complex. The methods can include contacting a compound disclosed herein with a cell expressing or containing an Oplophorus luciferase-derived bioluminescent complex or with a non-luminescent peptide and / or polypeptide of an Oplophorus luciferase-derived bioluminescent complex, where the disclosed compound can selectively inhibit the Oplophorus luciferase-derived bioluminescent complex. The methods can include contacting a compound disclosed herein with a non-luminescent peptide and / or polypeptide of an Oplophorus luciferase-derived bioluminescent complex, where the disclosed compound inhibits the Oplophorus luciferase-derived bioluminescent complex when the complex assembles. The disclosed compounds can be used to selectively inhibit signal from an Oplophorus luciferase-derived bioluminescent complex in assays used to detect the presence or activity of an enzyme that uses an Oplophorus luciferase-derived bioluminescent complex. For example, the disclosed compounds can be used in bioluminescence methods employing an Oplophorus luciferase-derived bioluminescent complex and a coelenterazine or coelenterazine derivative substrate to detect one or more molecules in a sample, such as a protein of interest (e.g., an enzyme, binding partner, ligand, etc.), a cofactor for an enzymatic reaction, an enzyme substrate, an enzyme inhibitor, an enzyme activator, or an OH group, or one or more conditions, such as a redox condition. While the coelenterazine substrate acts as a substrate for the Oplophorus luciferase-derived bioluminescent complex, the disclosed compounds can act to inhibit the luciferase-derived bioluminescent complex, selectively suppressing a luminescent signal in embodiments, such as may be desired in applications involving, for example, temporal multiplexing of multiple bioluminescent 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 Oplophorus luciferase-derived bioluminescent complexes.

[0158] (1) Protein complementation assay In accordance with the above, the disclosed compounds can be used to inhibit Oplophorus luciferase-derived bioluminescent complexes when used in other methods for detecting ligand-protein and protein-protein interactions or proximity or colocalization, 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, e.g., biomolecules or polypeptides. PCA utilizes two fragments of the same protein, e.g., an enzyme, fused to a polypeptide of interest, which produces light only when the two fragments are brought into proximity with each other through the binding interaction of their fusion partners, e.g., the polypeptide of interest, and reassemble into a functionally active protein, e.g., an enzyme. For example, NANOBIT® technology (Promega Corporation) can be used to detect molecular proximity by the reassembly of luminescent enzymes through the binding interaction of the enzymatic units. The NanoBiT® system can include two or more non-luminescent peptide and / or polypeptide units that can be expressed as fusions with a target molecule of interest. In some embodiments, the two units can include a NanoBiT® LgBiT non-luminescent polypeptide (NLpoly) and a NanoBiT® SmBiT non-luminescent peptide (NLpep). In some embodiments, the two units can include a NanoBiT® LgBiT non-luminescent polypeptide (NLpoly) and a NanoBiT® HiBiT non-luminescent peptide (NLpep). Oplophorus luciferase-derived bioluminescent complexes are 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 into two units, e.g., non-luminescent peptides or polypeptides, e.g., in a moiety or moieties tolerant to separation, and each unit, e.g., a non-luminescent peptide or polypeptide, can be fused to one of a pair of polypeptides of interest that are thought to interact, e.g., FKBP and FRB. If the two polypeptides of interest interact, the non-luminescent units, e.g., then reassemble into a bioluminescent complex in close 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 complementation system similar to lac-Z (Langley et al., PNAS 72:1254-1257 (1975)) or ribonuclease S (Levitt 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 cells or cell lysates containing fragment B. In some embodiments, the source of unit B can be the same cell (e.g., if the gene for unit B is integrated into the cell's genome or contained on a separate plasmid in the cell), or it can be a lysate or purified protein 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 is capable of binding to a solid support. In some embodiments, luminescence can be used to demonstrate successful capture or to quantify the amount of captured material.

[0160] (2) Molecular detection assays In accordance with the above, the disclosed compounds can be used to inhibit Oplophorus luciferase-derived bioluminescent complexes when such bioluminescent complexes are used in other methods to detect molecules of interest. For example, NANOBIT® technology (Promega Corporation) can be used to detect molecules of interest. 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. The two units can include a NanoBiT® LgBiT non-luminescent polypeptide (NLpoly) and a NanoBiT® HiBiT non-luminescent peptide (NLpep). In some embodiments, the NanoBiT® HiBiT NLpep can be fused to a molecule of interest. A sample containing the NanoBiT® HiBiT NLpep fused to a molecule of interest can be contacted with the NanoBiT® LgBiT NLpoly. For example, NanoBiT® LgBiT NLpoly can be added to a detection reagent containing a coelenterazine substrate. The resulting bioluminescence can be detected, measured, or inhibited with a compound disclosed herein. In some embodiments, NanoBiT® LgBiT NLpoly can be fused to a molecule of interest. A sample containing NanoBiT® LgBiT NLpoly fused to a molecule of interest can be contacted with NanoBiT® HiBiT NLpep. For example, NanoBiT® HiBiT NLpep can be added to a detection reagent containing a coelenterazine substrate.

[0161] (3) Use of cell-impermeable compounds In certain embodiments, the methods disclosed herein involve contacting a sample (e.g., cells) 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 establish initial luminosity in a high-throughput screening assay format and then selectively inhibit any Oplophorus luciferase-derived bioluminescent complexes that may be exported from the cell, thereby selectively inhibiting luminescence that may 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., cells) 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 cells and selectively inhibit Oplophorus luciferase-derived bioluminescent complexes therein. Such methods can be advantageous in multiplexed assays involving the use of two or more luciferases, allowing for the inhibition of luminescence from Oplophorus luciferase-derived bioluminescent complexes so that luminescence from alternative luciferases within the cells can be selectively viewed.

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

[0164] (6) Multiplexing The disclosed compounds can be used to inhibit Oplophorus luciferase-derived bioluminescent complexes when applied to temporal multiplexing with other luciferases and assays. In some embodiments, Oplophorus luciferase-derived bioluminescent complexes can be multiplexed with another enzyme (e.g., luciferase) that emits light at a different wavelength, such as green firefly luciferase, e.g., Photinus pyralis (e.g., Luc2; Promega Corp.) or red click beetle luciferase (CHROMA-LUC™ luciferase; Promega Corp.). For example, when an 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 nonspecific 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 the red CHROMA-LUC (approximately 610 nm) can be easily resolved using a luminometer with wavelength-discriminating 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 the other luciferase can be selectively viewed.

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

[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., 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 Oplophorus luciferase-derived bioluminescent conjugates are used to detect ligand-protein and / or protein-protein interactions. In various embodiments, Oplophorus luciferase-derived bioluminescent conjugates can be used to transfer energy to an energy acceptor. One such method is bioluminescence resonance energy transfer (BRET). With BRET, energy transfer from a bioluminescent donor to a fluorescent acceptor results in a shift in the spectral distribution of light emission. This energy transfer can enable real-time monitoring of protein-protein or ligand-protein interactions in vitro or in vivo.

[0168] In some embodiments, Oplophorus luciferase-derived bioluminescent complexes used in BRET assays can be used to determine whether two molecules are capable of binding to each other or colocalize within a cell. For example, Oplophorus luciferase-derived bioluminescent complexes can be used as bioluminescent donor molecules, in which case one of the non-luminescent units is combined with a molecule or protein of interest to generate a first fusion protein. In some embodiments, a non-luminescent peptide can be combined with a molecule or protein of interest to generate a first fusion protein. In other embodiments, a non-luminescent polypeptide can be combined with a molecule or protein of interest to generate a first fusion protein. In various embodiments, a first fusion protein containing a non-luminescent unit (e.g., a non-luminescent peptide or polypeptide) of an Oplophorus luciferase-derived bioluminescent complex 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 complementary non-luminescent unit (e.g., a non-luminescent polypeptide or peptide) of a second protein of interest or bioluminescent complex. For example, a non-luminescent polypeptide of an Oplophorus luciferase-derived bioluminescent complex can be fused to HALOTAG, expressed in a cell or animal, and labeled with a fluorescent HALOTAG® ligand, such as the HALOTAG® TMR ligand. The fusion can then be excited to fluoresce in the presence of a cell-permeable luminescent enzyme substrate. As another example, a non-luminescent peptide of an Oplophorus luciferase-derived bioluminescent complex can be fused to HaloTag, expressed in a cell or animal, and labeled with a fluorescent HaloTag® ligand, such as the 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 may be fused to a second protein of interest and a complementary non-luminescent unit (e.g., a non-luminescent polypeptide or peptide) of an Oplophorus luciferase-derived bioluminescent complex added via a detection reagent. In some embodiments, BRET may be performed using an Oplophorus luciferase-derived bioluminescent complex in combination with a fluorescent protein, including, but not limited to, green fluorescent protein (GFP) or red fluorescent protein (RFP), or a fluorescent label including fluorescein, rhodamine green, Oregon green, or Alexa488, to name a few non-limiting examples.

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

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

[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 acids, or carbohydrate polymers, or lipid membrane complexes). The disclosed compounds can generally be non-toxic to viable cells and other biological components within the range of concentrations used.

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

[0173] 7. Kit Kits for determining the presence or activity of an Oplophorus luciferase-derived bioluminescent complex are disclosed. The kits may include one or more of the following: a compound or composition of the invention capable of inhibiting an Oplophorus luciferase-derived bioluminescent complex, a coelenterazine or coelenterazine derivative substrate, and a polynucleotide for expression of an Oplophorus luciferase-derived bioluminescent complex, e.g., a non-luminescent peptide and / or polypeptide of an Oplophorus luciferase-derived bioluminescent complex, instructions for conducting a luminescent assay, and reaction buffer(s). The reaction buffers may be present in separate formulations for the non-luciferase and luminescent enzymatic reactions, or in a single formulation for a single-step assay. The reaction buffer may contain a non-luminescent unit of the Oplophorus luciferase-derived bioluminescent complex, e.g., a non-luminescent peptide. The kit may also contain other inhibitors, activators, and / or enhancers for the non-luciferase enzyme(s). The kit may also contain positive and / or negative controls for the assay. [Example]

[0174] 8. Working Example Example 1 Compound synthesis General Procedure A: Sulfonamide Bond Formation.

[0175] To a solution of the aniline derivative (1 equiv.) in pyridine was added substituted benzenesulfonyl chloride (1.1 equiv.). The solution was stirred at room temperature for 4-18 h. The mixture was diluted with dichloromethane and washed with HCl (2 M). 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 the methyl or ethyl ester (1 equiv.) in dioxane was added sodium hydroxide (2 M, 2 equiv.). 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 crude in the next step.

[0178] General Procedure C1: Acid Chloride-Mediated Amide Bond Formation.

[0179] To a solution of the acid chloride derivative (1 equiv.) in dichloroethane was added pyridine (3-5 equiv.) and an amine (1 equiv.). The reaction was stirred at room temperature for 2-18 h. The mixture was diluted with DCM and 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: Carboxylate-mediated Amide Bond Formation.

[0181] To a solution of the carboxylic acid derivative (1 equiv.) in DMF was added an amine (1.2 equiv.), HBTU (2 equiv.), and diisopropylethylamine (3 equiv.). The reaction was heated to 60-85 °C for 2-18 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.

[0182] General Procedure D: Nitro Reduction.

[0183] To a solution of the nitro derivative (1 equiv.) in ethyl acetate was added tin chloride hydrate (3-5 equiv.). 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, and 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 JPEG0007815514000039.jpg53128

[0185] Example 2 N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-0998) JPEG0007815514000040.jpg2659Step 1. N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-nitrobenzamide (JRW-0994) JPEG0007815514000041.jpg2744

[0186] Following 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) JPEG0007815514000042.jpg2543

[0188] Following 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 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] Step 3. N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-0998) JPEG0007815514000043.jpg2661

[0190] Following 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) JPEG0007815514000044.jpg2661Step 1. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-nitrobenzamide (JRW-1000) JPEG0007815514000045.jpg2847

[0192] Following 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) JPEG0007815514000046.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) was added ammonium chloride (474 ​​mg, 8.8 mmol) and iron filings (100 mg, 1.8 mmol). The suspension was heated to 60 °C for 18 h. The reaction was filtered, and 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) JPEG0007815514000047.jpg3268

[0196] Following 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) JPEG0007815514000048.jpg2654Step 1. N-(3-cyanothiophen-2-yl)-2-nitrobenzamide (JRW-1001). JPEG0007815514000049.jpg2134

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

[0199] Step 2. 2-Amino-N-(3-cyanothiophen-2-yl)benzamide (JRW-1003). JPEG0007815514000050.jpg2134

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

[0201] Step 3. N-(3-cyanothiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1006). JPEG0007815514000051.jpg2654

[0202] Following 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 give the desired product (97 mg, 66%) as a pale brown solid. ESI MS m / z 398 [M+H] +

[0203] Example 5 N-(2-cyanophenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1008) JPEG0007815514000052.jpg2656Step 1. N-(2-cyanophenyl)-2-nitrobenzamide (JRW-1005). JPEG0007815514000053.jpg2639

[0204] Following 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). JPEG0007815514000054.jpg2540

[0206] Following general procedure D, N-(2-cyanophenyl)-2-nitrobenzamide (200 mg, 0.75 mmol) was reacted with tin chloride hydrate (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). JPEG0007815514000055.jpg2656

[0208] Following 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) JPEG0007815514000056.jpg2656Step 1. 2-Nitro-N-phenylbenzamide (JRW-1009) JPEG0007815514000057.jpg2639

[0210] Following 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). JPEG0007815514000058.jpg2538

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

[0213] Step 3. 2-((4-methylphenyl)sulfonamido)-N-phenylbenzamide (JRW-1011). JPEG0007815514000059.jpg3063

[0214] Following 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) Following 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 pale 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) JPEG0007815514000061.jpg2664Step 1. N-(benzo[b]thiophen-2-yl)-2-nitrobenzamide (HL-0028). JPEG0007815514000062.jpg2847

[0217] Following 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). JPEG0007815514000063.jpg2747

[0219] Following general procedure D, N-(benzo[b]thiophen-2-yl)-2-nitrobenzamide (100 mg, 0.33 mmol) was reacted with tin 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] Step 3. N-(benzo[b]thiophen-2-yl)-2-((4-formylphenyl)sulfonamido)benzamide (HL-0038). JPEG0007815514000064.jpg2662

[0221] Following 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) Following 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 pale 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) Following 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) JPEG0007815514000067.jpg267To 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 40°C was added lithium borohydride (4 mg, 0.19 mmol). The reaction was allowed to warm 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) JPEG0007815514000068.jpg2969Step 1. 2-Nitro-N-(p-tolyl)benzamide (JRW-1243). JPEG0007815514000069.jpg2143

[0226] Following 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 pale brown solid. ESI MS m / z 257 [M+H] + .

[0227] Step 2. 2-Amino-N-(p-tolyl)benzamide (JRW-1247). JPEG0007815514000070.jpg2549

[0228] Following general procedure D, 2-nitro-N-(p-tolyl)benzamide (4.6 g, 18.0 mmol) was reacted with tin chloride hydrate (11.2 mg, 54.0 mmol) to give 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). JPEG0007815514000071.jpg2869

[0230] Following 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 give 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) Following 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 give 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) Following 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 give 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) Following 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 give 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) JPEG0007815514000075.jpg2970 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 to 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 give 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) To a mixture of 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoic acid (54 mg, 0.13 mmol) in DCM (5 mL) was added 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 give the desired product (29 mg, 55%) as a white solid. ESI MS m / z 397[M+H] + .

[0236] Example 18 2-((3-(butylcarbamoyl)phenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1263) Following 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 give the desired product (43 mg, 76%) as a white solid. ESI MS m / z 466 [M+H] + .

[0237] Example 19 2-((3-bromophenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1267) Following 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 give the desired product (620 mg, 90%) as a pale brown solid. ESI MS m / z 446 [M+H] + .

[0238] Example 20 2-((3-(butylamino)phenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1269) JPEG0007815514000079.jpg2988 To 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, after which sodium triacetoxyborohydride (55 mg, 0.26 mmol) was added. The reaction was stirred at room temperature for 5 hours, quenched with a saturated solution of NaHCO3, 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 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) To a solution of 2-((3-bromophenyl)sulfonamido)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in DMF (5 mL) was added 1-hexyne (36 mg, 0.45 mmol), triethylamine (68 mg, 0.67 mmol), triphenylphosphine (6 mg, 0.022 mmol), and PdCl(PPh) (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 h. The reaction was diluted with ethyl acetate and water, extracted with ethyl acetate, and 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) JPEG0007815514000081.jpg2990 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 was stirred at 40 psi of hydrogen at room temperature for 2 hours. 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) To a solution of 2-((3-bromophenyl)sulfonamido)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in DMF (5 mL) was added propargyl alcohol (25 mg, 0.45 mmol), triethylamine (68 mg, 0.67 mmol), triphenylphosphine (6 mg, 0.022 mmol), and PdCl(PPh) (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 85 °C for 48 h. The reaction was diluted with ethyl acetate and water, extracted with ethyl acetate, and 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) JPEG0007815514000083.jpg2981 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) was added palladium on carbon (5 mg). The reaction was stirred at room temperature under 40 psi of hydrogen for 1 hour. 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) Following 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) Following 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) Following 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) JPEG0007815514000087.jpg2669 To a solution of 2-((4-bromophenyl)sulfonamido)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in dioxane (5 mL) was 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 to 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) Following 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) Following 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 carboxylates JPEG0007815514000090.jpg60153 Example 31 N-(benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamido)benzamide (HL-0005) JPEG0007815514000091.jpg2661Step 1. Methyl 2-((4-methylphenyl)sulfonamido)benzoate (HL-0001). JPEG0007815514000092.jpg2651

[0250] Following 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). JPEG0007815514000093.jpg2646

[0252] Following 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). JPEG0007815514000094.jpg2661

[0254] Following 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) Following 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) Following general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with naphthalen-2-amine (29 mg, 0.21 mmol) to give the desired product (40 mg, 54%) as a pale 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) Following general procedure C2, 2-((4-methylphenyl)sulfonamido)benzoic acid (50 mg, 0.17 mmol) was reacted with 5,6,7,8-tetrahydronaphthalen-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) Following 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) Following 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) Following 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) Following 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) Following 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)benzamido)benzoate (HL-0026) Following 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)benzamido)phenyl)acetic acid (HL-0030) Following general procedure B, ethyl 2-(4-(2-((4-methylphenyl)sulfonamido)benzamido)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)benzamido)benzoic acid (HL-0031) Following 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) Following 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) JPEG0007815514000107.jpg3765Step 1. Methyl 3-((4-methylphenyl)sulfonamido)-2-naphthoate (HL-0049). JPEG0007815514000108.jpg3657

[0268] Following 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). JPEG0007815514000109.jpg3346

[0270] Following 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). JPEG0007815514000110.jpg3766

[0272] Following 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 pale brown solid. ESI MS m / z 473 [M+H] + .

[0273] Example 45 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide (HL-0059) Following 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) JPEG0007815514000112.jpg3867Step 1. Ethyl 5-methyl-2-((4-methylphenyl)sulfonamido)benzoate (HL-0053). JPEG0007815514000113.jpg3761

[0275] Following 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). JPEG0007815514000114.jpg3446

[0277] Following 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). JPEG0007815514000115.jpg3461

[0279] Following 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) Following 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) JPEG0007815514000117.jpg3866Step 1. Methyl 5-cyano-2-((4-methylphenyl)sulfonamido)benzoate (HL-0056). JPEG0007815514000118.jpg3351

[0282] Following 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). JPEG0007815514000119.jpg3751

[0284] Following 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] Step 3. N-(benzo[b]thiophen-2-yl)-5-cyano-2-((4-methylphenyl)sulfonamido)benzamide (HL-0071). JPEG0007815514000120.jpg3361

[0286] Following 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 pale brown solid. ESI MS m / z 448 [M+H] + .

[0287] Example 49 N-(4-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1076) Following 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 pale 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) JPEG0007815514000122.jpg2661Step 1. Methyl 2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamido)benzoate (HL-0068). JPEG0007815514000123.jpg2649

[0289] Following 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 give 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) JPEG0007815514000124.jpg2646

[0291] Following 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 give 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). JPEG0007815514000125.jpg2661

[0293] Following 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 give 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) Following 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) Following 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) Following 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) Following 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) JPEG0007815514000130.jpg3083Step 1. Methyl 2-((4-butylphenyl)sulfonamido)benzoate (JRW-1111) JPEG0007815514000131.jpg3068

[0299] Following 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) JPEG0007815514000132.jpg2864

[0301] Step 3. N-(benzo[b]thiophen-2-yl)-2-((4-butylphenyl)sulfonamido)benzamide (JRW-1114) JPEG0007815514000133.jpg2881

[0302] Following 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) Following 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) JPEG0007815514000135.jpg2679Step 1. Methyl 2-((4-pentylphenyl)sulfonamido)benzoate (JRW-1115) JPEG0007815514000136.jpg2667

[0305] Following 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) JPEG0007815514000137.jpg2664

[0307] Following 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) JPEG0007815514000138.jpg2679

[0309] Following 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) JPEG0007815514000139.jpg5070 Step 1. 5-Butyl-2-((4-methylphenyl)sulfonamido)benzoic acid (JRW-1142) JPEG0007815514000140.jpg4652

[0311] Following 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] Step 2. N-(benzo[b]thiophen-2-yl)-5-butyl-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1146) JPEG0007815514000141.jpg4667

[0313] Following 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) Following 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) JPEG0007815514000143.jpg2679To a solution of N-(4-(4-hydroxybutyl)phenyl)-2-((4-methylphenyl)sulfonamido)benzamide (40 mg, 0.091 mmol) in DCM (5 mL) was added carbon tetrabromide (60 mg, 0.18 mmol) and triphenylphosphine (47 mg, 0.18 mmol). The reaction was stirred overnight at room temperature. 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 (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) JPEG0007815514000144.jpg3769Step 1. Methyl 5-methoxy-2-((4-methylphenyl)sulfonamido)benzoate (JRW-1159) JPEG0007815514000145.jpg3860

[0317] Following 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 give 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) JPEG0007815514000146.jpg3852

[0319] Following 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 give 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) JPEG0007815514000147.jpg3768

[0321] Following 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 give 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) Following 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 give the desired product (34 mg, 24%) as a pale brown solid. ESI MS m / z 453 [M+H] + .

[0323] Example 63 4-Methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1202) JPEG0007815514000149.jpg3267Step 1. Methyl 4-methoxy-2-((4-methylphenyl)sulfonamido)benzoate (JRW-1195) JPEG0007815514000150.jpg3356

[0324] Following 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) JPEG0007815514000151.jpg3351

[0326] Following 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) JPEG0007815514000152.jpg3370

[0328] Following 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) Following 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) JPEG0007815514000154.jpg2973Step 1. Methyl 2-((3-methoxyphenyl)sulfonamido)benzoate (JRW-1196) JPEG0007815514000155.jpg2960

[0331] Following 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) JPEG0007815514000156.jpg2953

[0333] Following 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] Step 3. 2-((3-Methoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1205) JPEG0007815514000157.jpg2667

[0335] Following general procedure C2, 2-((3-methoxyphenyl)sulfonamido)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 pale brown solid. ESI MS m / z 397 [M+H] + .

[0336] Example 66 N-(benzo[b]thiophen-2-yl)-2-((3-methoxyphenyl)sulfonamido)benzamide (JRW-1230) Following general procedure C2, 2-((3-methoxyphenyl)sulfonamido)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)sulfonamido)-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 a saturated solution of NaHCO. 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 give 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 a saturated solution of NaHCO. 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 give the desired product (140 mg, 76%) as a pale brown solid. ESI MS m / z 383 [M+H] + .

[0339] Example 69 2-((3-butoxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (JRW-1266) JPEG0007815514000161.jpg2988 To a solution of 2-((3-hydroxyphenyl)sulfonamido)-N-(p-tolyl)benzamide (40 mg, 0.10 mmol) in THF (3 mL) was added 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 hours. 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) Following 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) Following 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) Following 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) Following 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 pale 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) JPEG0007815514000166.jpg2974 To a solution of N-(3-bromophenyl)-2-((4-methylphenyl)sulfonamido)benzamide (110 mg, 0.24 mmol) in dioxane (5 mL) was added 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 to 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) Following 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) Following 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 pale brown solid. ESI MS m / z 397 [M+H] + .

[0347] Example 77 2-((N-ethyl-4-methylphenyl)sulfonamido)-N-(4-methoxyphenyl)benzamide (JRW-1325) JPEG0007815514000169.jpg2871 To a solution of N-(4-methoxyphenyl)-2-((4-methylphenyl)sulfonamido)benzamide (50 mg, 0.12 mmol) in DMF (3 mL) was added diisopropylethylamine (49 mg, 0.38 mmol) and ethyl iodide (0.5 mL). The solution was stirred for 18 h at 60 °C. 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 (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) JPEG0007815514000170.jpg2961Step 1. Methyl 4-fluoro-2-((4-methylphenyl)sulfonamido)benzoate (JRW-1342). JPEG0007815514000171.jpg3357

[0349] Following 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 give 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). JPEG0007815514000172.jpg3151

[0351] Following 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 give 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). JPEG0007815514000173.jpg3266

[0353] Following 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 give 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) JPEG0007815514000174.jpg3766Step 1. Methyl 5-fluoro-2-((4-methylphenyl)sulfonamido)benzoate (JRW-1343). JPEG0007815514000175.jpg3351

[0355] Following 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 give the desired product (1.9 g, 99%) as a white solid. ESI MS m / z 324 [M+H] + .

[0356] Step 2. 5-Fluoro-2-((4-methylphenyl)sulfonamido)benzoic acid (JRW-1345). JPEG0007815514000176.jpg3752

[0357] Following 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). JPEG0007815514000177.jpg3868

[0359] Following 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 pale brown solid. ESI MS m / z 441 [M+H] + .

[0360] Example 80 N-benzyl-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1383) Following 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 pale brown solid. ESI MS m / z 381 [M+H] + .

[0361] Example 81 N-(4-Methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide (JRW-1384) Following 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) JPEG0007815514000180.jpg3362Step 1. Methyl 2-((4-methylphenyl)sulfonamido)-5-(trifluoromethyl)benzoate (JRW-1382) JPEG0007815514000181.jpg3350

[0363] Following 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) JPEG0007815514000182.jpg3346

[0365] Following 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) JPEG0007815514000183.jpg3362

[0367] Following 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 pale brown solid. ESI MS m / z 491 [M+H] + .

[0368] Example 83 Selective inhibition of bioluminescent complexes The following examples provide 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 exemplary compounds. NanoBiT® HiBiT nonluminescent peptide and NanoBiT® LgBiT nonluminescent polypeptide were each diluted in PBS + 0.01% BSA (0.1 nM and 100 nM final concentrations, respectively) and incubated with serially diluted concentrations of the indicated compounds in the presence of RPMI medium (2.5% FBS final) for 2 hours at room temperature. Samples were analyzed after the addition of furimazine (10 μM final concentration) using a GloMax®-Multi+ plate reader. Each sample was normalized to a "no inhibitor" control. The IC 50 Values ​​were determined using GraphPad Prism (log [inhibitor] vs. normalized response). Figure 1B shows the inhibition of the NanoBiT® SmBiT / LgBiT bioluminescent complex by exemplary compounds of the invention. NanoBiT® SmBiT-annexin fusion and NanoBiT® LgBiT-annexin fusion were diluted in PBS / 0.01% BSA to final concentrations of 60 nM and 30 nM, respectively, and incubated for 2 hours at room temperature with serially diluted concentrations of the indicated compounds in the presence of K562 cell lysate diluted in RPMI medium (2.5% FBS final). Samples were analyzed after the addition of furimazine (10 μM final concentration) using a GloMax®-Multi+ plate reader. Each sample was normalized to a "no inhibitor" control. IC 50 Values ​​were determined using GraphPad Prism (log[inhibitor] vs. normalized response). Figure 1C shows the calculated IC between NanoBiT® HiBiT / LgBiT and SmBiT / LgBiT complexes for the exemplary compounds shown in Figures 1A and 1B. 50 It is a bar chart comparing values.

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

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

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

[0372] Example 85 IC of inhibitor 50 Decision The following examples illustrate the IC of the compounds disclosed herein. 50 Values ​​are provided. The results are shown in Table 1. NanoBiT® HiBiT non-luminescent peptide and LgBiT non-luminescent non-polypeptide were diluted to 0.1 nM and 1 nM, respectively, in TBS buffer with 0.01% BSA to make the detection reagent. A 3-fold dilution series of each inhibitor was then made in the detection reagent. A "no inhibitor" control was also made for each sample. 50 ul 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 determined using GraphPad Prism (log[inhibitor] vs. normalized response). Table 1 JPEG0007815514000184.jpg203143 JPEG0007815514000185.jpg203143 JPEG0007815514000186.jpg62131 No NA activity NT - Not tested

[0373] It is understood that the foregoing detailed description and accompanying examples are illustrative only and are not to be taken as limitations on the scope of the invention, which is defined solely 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, with respect to the chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof. Annex

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

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

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

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

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

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

[0381] SEQ ID NO:7 - NanoLuc MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLFRVTINGVTGWRLCERILA Another aspect of the present invention may be as follows. [1] A compound of formula (I) or a salt thereof: JPEG0007815514000187.jpg40170 (In the formula: R 1 is an aryl, cycloalkyl, heteroaryl, or heterocycle, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocycle may be optionally joined by one or more R W and each R W independently, C1-10 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 1, 2, 3, or 4 R D phenyl substituted with a group; 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, 1, 2, 3, or 4 R D phenyl substituted with a group, -C≡CR A , or -C≡CC 1-4 Alkylene-OR A or two R 2 However, the above-mentioned JPEG0007815514000188.jpg1316 together with said carbon atoms of the moiety form 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 or two R 3 However, the above-mentioned JPEG0007815514000189.jpg1818 together with said carbon atoms of the moiety form a 5- or 6-membered fused ring; R 4 is H or C 1-4 is alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R in each occurrence A are independently H, C 1-4 Alkyl, or C 1-4 is haloalkyl; R in each occurrenceB and R C are independently H or C 1-4 alkyl, or R B and R C form a 5- or 6-membered heterocyclic ring together with the N atom to which they are attached; R in each occurrence D independently, C 1-4 Alkyl, -OC 1-4 alkyl, -CN, or halogen; The compound is not N-phenyl-2-(phenylsulfonamido)benzamide). 〔2〕R 1 is aryl or heteroaryl, and the aryl and heteroaryl are each independently selected from one or more R W The compound according to [1] above, or a salt thereof, optionally substituted with 〔3〕R 1 is one or more R W C optionally substituted with 6-20 The compound according to any one of the above [1] to [2], or a salt thereof, which is aryl. 〔4〕R 1 is one or more R W The compound according to any one of the above [1] and [2], or a salt thereof, wherein R is 5 to 12-membered heteroaryl optionally substituted with R. [5] The compound according to any one of the above [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 [1] to [5] above, or a salt thereof, [7] The compound or salt thereof according to any one of the above [1] to [6], 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 The compound according to any one of the above [1] to [7], or a salt thereof, wherein the compound is alkyl. [9] The compound has the formula (Ia): JPEG0007815514000190.jpg40170 (In the formula: R 1 is C 6-20 aryl or 5- to 12-membered heteroaryl, and the aryl and heteroaryl are each independently one or more R W optionally substituted with; p is 0, 1, or 2; q is 0 or 1; R 3 is C 1-4 Alkyl, halogen, -CN or -OR A and; R A is H or C 1-4 is alkyl; R 2 and R W is as defined in [1] above) The compound according to [1] above, or a salt thereof, having the following structure: 〔10〕R 1 teeth, JPEG0007815514000191.jpg22150 and each of them contains one or more R W The compound according to [9] above, or a salt thereof, optionally substituted with 〔11〕R 1 is unsubstituted, or R 1 can contain one, two, or three R W are substituted with R W are independently halogen, C 1-4 Alkyl, C 1-4 The compound according to any one of the above [9] to

[10] , or a salt thereof, which is haloalkyl or phenyl. 〔12〕R 1 teeth, JPEG0007815514000192.jpg24170 The compound according to any one of [9] above, or a salt thereof,

[13] p is 1 or 2, and each R 2 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 1-4alkyl, or two R 2 However, the above-mentioned JPEG0007815514000193.jpg1316 Together with the parts JPEG0007815514000194.jpg1326 The compound according to any one of [9] to

[12] above, or a salt thereof, which forms:

[14] The compound has the formula (Ia-1): JPEG0007815514000195.jpg40170 (In the formula: R 1 teeth, JPEG0007815514000196.jpg24170 and each is a halogen, C 1-4 Alkyl, C 1-4 optionally substituted with haloalkyl, or phenyl; or R 1 teeth, JPEG0007815514000197.jpg24170 and; R 2 is a halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, -OH, -C 1-4 Alkylene -OH, -OC 1-4 Alkyl, or -NH 2 is) The compound according to [9] above, or a salt thereof, having the formula:

[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)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 The compound according to [1] above, or a salt thereof, selected from the group consisting of:

[16] A method for inhibiting a bioluminescent complex derived from Oplophorus luciferase, the method comprising contacting the bioluminescent complex with a compound described in any one of [1] to

[15] above.

[17] The method of

[16] , wherein the bioluminescent complex comprises two or more substantially non-luminescent peptide and / or polypeptide units, and the association of the two or more non-luminescent peptide and / or polypeptide units generates a detectable bioluminescent signal in the presence of a coelenterazine substrate.

[18] The method of

[17] , wherein the association of the two or more substantially non-luminescent peptide and / or polypeptide units produces a bioluminescent complex capable of binding the coelenterazine substrate.

[19] The bioluminescent complex comprises: a) a peptide comprising an amino acid sequence having less than 100% but more than 40% identity to SEQ ID NO:2; and b) a polypeptide comprising an amino acid sequence having less than 100% but more than 40% identity with SEQ ID NO: 3 Including, The method of claim 16, wherein the bioluminescent conjugate exhibits detectable luminescence in the presence of a coelenterazine substrate.

[20] The method described in

[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.

[21] 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 the compound according to any one of [1] to

[15] ; (b) detecting luminescence in the sample; Including, The method, wherein the compound according to any one of [1] to

[15] above causes a decrease in the luminescence from the bioluminescent complex.

[22] A method for detecting an interaction or colocalization between a first molecule and a second molecule in a sample, the method comprising: (a) contacting a sample with a coelenterazine substrate and the compound according to any one of [1] to

[15] above, wherein the sample is (i) a first fusion comprising a non-light-emitting peptide of Oplophorus luciferase and a first molecule; (ii) a second fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a second molecule, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; said contacting comprising: (b) detecting luminescence in the sample; Including, The method, wherein detecting said luminescence indicates an interaction or co-localization between said first protein and said second protein.

[23] A method for detecting an interaction or colocalization between a first molecule and a second molecule in a sample, the method comprising: (a) contacting a sample with a coelenterazine substrate and the compound according to any one of [1] to

[15] above, wherein the sample is (i) a first polynucleotide encoding a first fusion, the first fusion comprising a non-light-emitting peptide of Oplophorus luciferase and a first molecule; (ii) a second polynucleotide encoding a second fusion, the second fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a second molecule, the non-luminescent polypeptide being capable of forming a bioluminescent complex with the non-luminescent peptide; said contacting comprising: (b) detecting luminescence in the sample; Including, The method, wherein detecting the luminescence indicates an interaction or co-localization between the first molecule and the second molecule.

[24] 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 luciferase, a coelenterazine substrate, and the compound according to any one of [1] to

[15] above, wherein the sample is (i) a first polynucleotide encoding a first fusion, the first fusion comprising a non-luminescent peptide of Oplophorus luciferase and a first molecule, the non-luminescent peptide being capable of forming a bioluminescent complex with the non-luminescent polypeptide; (ii) a second polynucleotide encoding a second fusion, the second fusion comprising a fluorescent acceptor molecule and a second molecule; and said contacting comprising: (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates an interaction or co-localization of the first molecule and the second molecule; The method comprising:

[25] 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 luciferase, a coelenterazine substrate, and the compound according to any one of [1] to

[15] above, wherein the sample is (i) a first polynucleotide encoding a first fusion, the first fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a first molecule, the non-luminescent polypeptide being capable of forming a bioluminescent complex with the non-luminescent peptide; (ii) a second polynucleotide encoding a second fusion, the second fusion comprising a fluorescent acceptor molecule and a second molecule; and said contacting comprising: (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates an interaction or co-localization of the first molecule and the second molecule; The method comprising:

[26] A method for detecting molecular interactions or colocalization in a sample, the method comprising: (a) contacting a sample with a coelenterazine substrate and the compound according to any one of [1] to

[15] above, wherein the sample is (i) a first fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a first molecule; (ii) a second fusion comprising a non-luminescent peptide of Oplophorus luciferase and a second molecule, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; (iii) a third fusion comprising a fluorescent receptor molecule and a third molecule; and said contacting comprising: (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates an interaction or co-localization between the first molecule, the second molecule, and the third molecule in the sample; The method comprising:

[27] A method for detecting a molecule of interest in a sample, the method comprising: (a) a sample containing the molecule of interest fused to a non-luminescent peptide of Oplophorus luciferase; (i) Coelenterazine substrate; (ii) the compound according to any one of the above [1] to

[15] ; and (iii) a non-luminescent polypeptide of Oplophorus luciferase, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; and contacting the (b) detecting luminescence in the sample; Including, The method, wherein detection of luminescence indicates the formation of a bioluminescent complex between the non-luminescent peptide and the non-luminescent polypeptide.

[28] A method for detecting a molecule of interest in a sample, the method comprising: (a) a sample containing the molecule of interest fused to a non-luminescent polypeptide of Oplophorus luciferase; (i) Coelenterazine substrate; (ii) the compound according to any one of the above [1] to

[15] ; 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 contacting the (b) detecting luminescence in the sample; Including, The method, wherein detection of luminescence indicates the formation of a bioluminescent complex between the non-luminescent peptide and the non-luminescent polypeptide.

[29] A method for detecting a molecule of interest in a sample, the method comprising: (a) a sample containing the molecule of interest fused to a non-luminescent peptide of Oplophorus luciferase; (i) Coelenterazine substrate; (ii) the compound according to any one of the above [1] to

[15] ; 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 contacting the (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, indicative of detection of the molecule; The method comprising:

[30] A method for detecting a molecule of interest in a sample, the method comprising: (a) a sample containing the molecule of interest fused to a non-luminescent polypeptide of Oplophorus luciferase; (i) Coelenterazine substrate; (ii) the compound according to any one of the above [1] to

[15] ; and (iii) a fusion comprising a non-luminescent peptide and a fluorescent moiety of Oplophorus luciferase, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; and contacting the (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, indicative of detection of the molecule; The method comprising:

[31] The method according to any one of

[21] to

[30] , comprising contacting the sample with the coelenterazine substrate before contacting the sample with the compound according to any one of [1] to

[15] .

[32] The method according to any one of

[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 bioluminescent signal in the presence of a coelenterazine substrate.

[33] The method according to any one of

[21] to

[32] above, wherein the sample contains cells.

[34] The method according to any one of

[21] to

[33] above, wherein the coelenterazine substrate is coelenterazine, a coelenterazine derivative, a coelenterazine analog, pro-coelenterazine, or a quinone-masked coelenterazine.

[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 method according to any one of

[21] to

[34] above, wherein 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.

[36] A bioluminescence resonance energy transfer (BRET) system, (a) a first fusion comprising a non-luminescent peptide of Oplophorus luciferase and a first molecule; (b) a second 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; (c) a coelenterazine substrate; (d) a compound according to any one of the above [1] to

[15] ; The bioluminescence resonance energy transfer (BRET) system comprises:

[37] A bioluminescence resonance energy transfer (BRET) system, (a) a first fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a first molecule; (b) a second fusion comprising a non-luminescent peptide of Oplophorus luciferase and a fluorescent moiety, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with the non-luminescent polypeptide; (c) a coelenterazine substrate; (d) a compound according to any one of the above [1] to

[15] ; The bioluminescence resonance energy transfer (BRET) system comprises:

[38] A bioluminescence resonance energy transfer (BRET) system, (a) a first fusion comprising a first molecule and a non-luminescent peptide of Oplophorus luciferase; (b) a second fusion comprising a second molecule and a fluorescent receptor molecule; (c) a non-luminescent polypeptide of Oplophorus luciferase capable of forming a bioluminescent complex with said non-luminescent peptide of Oplophorus luciferase; (d) a coelenterazine substrate; (e) a compound according to any one of the above [1] to

[15] ; The bioluminescence resonance energy transfer (BRET) system comprises:

[39] A bioluminescence resonance energy transfer (BRET) system, comprising: (a) a first fusion comprising a first molecule and a non-luminescent polypeptide of Oplophorus luciferase; (b) a second fusion comprising a second molecule and a fluorescent receptor molecule; (c) a non-light-emitting peptide of Oplophorus luciferase capable of forming a bioluminescent complex with said non-light-emitting polypeptide of Oplophorus luciferase; (d) a coelenterazine substrate; (e) a compound according to any one of the above [1] to

[15] ; The bioluminescence resonance energy transfer (BRET) system comprises:

[40] 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 bioluminescent resonance energy transfer system according to any one of

[36] to

[39] above. 〔41〕 (a) a compound according to any one of the above [1] to

[15] ; (b) a bioluminescent complex derived from Oplophorus luciferase; Includes a kit.

[42] The Oplophorus luciferase-derived bioluminescent complex comprises: (a) a non-luminescent peptide comprising an amino acid sequence having less than 100% and more than 40% sequence identity with SEQ ID NO: 2; (b) a non-luminescent polypeptide comprising an amino acid sequence having less than 100% and more than 40% sequence identity with SEQ ID NO: 3; The kit according to

[41] , comprising:

[43] (a) a compound according to any one of the above [1] to

[15] ; (b) a first polynucleotide encoding a non-luminescent peptide of Oplophorus luciferase; (c) a second polynucleotide encoding a non-luminescent polypeptide of Oplophorus luciferase, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; and Includes a kit.

[44] The kit described in

[43] , wherein the non-luminescent peptide comprises an amino acid sequence having less than 100% but more than 40% sequence identity with SEQ ID NO: 2, and the non-luminescent polypeptide comprises an amino acid sequence having less than 100% but more than 40% sequence identity with SEQ ID NO: 3.

[45] The kit according to any one of

[41] to

[44] above, further comprising a coelenterazine substrate.

Claims

[Claim 1] N-(2-cyanophenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 2-([1,1'-biphenyl]-3-sulfonamido)-N-(p-tolyl)benzamide; Methyl 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoate; 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; 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; 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; Ethyl 2-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)acetate; N-(3-isopropylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; 2-(4-(2-((4-methylphenyl)sulfonamido)benzamido)phenyl)acetic acid; 3-(2-((4-methylphenyl)sulfonamido)benzamido)benzoic acid; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)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-(4-(6-hydroxyhexyl)phenyl)-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; 4-Methoxy-2-((4-methylphenyl)sulfonamido)-N-(p-tolyl)benzamide; 2-((3-methoxyphenyl)sulfonamido)-N-(p-tolyl)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-([1,1'-biphenyl]-4-yl)-2-((4-methylphenyl)sulfonamido)benzamide; N-([1,1'-biphenyl]-3-yl)-2-((4-methylphenyl)sulfonamido)benzamide; and 2-((N-ethyl-4-methylphenyl)sulfonamido)-N-(4-methoxyphenyl)benzamide A compound selected from the group consisting of:

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