Inhibitors of oplophorus luciferase-derived bioluminescent complexes

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 679,205, filed on 1 June 2018, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to compounds that can inhibit bioluminescent complexes derived from Oplophorus luciferase, particularly bioluminescent complexes of two or more non-luminescent peptides 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, a reporter molecule, e.g., a complementary amino acid chain from a bioluminescent protein or enzyme, e.g., a peptide or polypeptide, is fused to the colocalizing and / or interacting molecule. Reporter molecules are conventionally 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 possess 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 colocalizing / interacting molecules (e.g., proteins). When molecules colocalize and / or interact, non-luminescent peptide and / or polypeptide units associate to form bioluminescent complexes, which can generate luminescence signals indicating molecular colocalization / interaction in the presence of a substrate (e.g., coelenterazine or a coelenterazine derivative substrate). In certain applications, controlling the luminescence signal from Oplophorus luciferase-derived bioluminescent complexes may be even more advantageous. Selective inhibitors for such bioluminescent complexes are useful in luminescence assays. Luciferase inhibitors may 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 [Overview of the Initiative]

Means for Solving the Problem

[0005] In one aspect, the present disclosure relates to a compound of formula (I), or a salt thereof: TIFF2023178294000001.tif37170(wherein: R 1-10 , D , 1-10 , A , C , A , 1-10 , , 1-10 , C , 2 , 1-4 , 1-4 , <00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​A , -C 1-4 Alkilen-CO-R A , -CO-OR A , -C 1-4 Alkilen-CO-OR A ,-CO-NHR A , -C 1-4 Alkilen-CO-NHR A , -NR B R C , -C 1-4 Alkilen-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 groups substituted with -C≡CR A , or -C≡CC 1-4 Alkilen-OR A and or two R 2 However, they are combined Together with the carbon atoms in the TIFF2023178294000002.tif1315 portion, they form a 5- or 6-membered condensed ring; Each R 3 Independently, C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, -OR A , -C 1-4 Alkilen-OR A ,-CO-R A , -CO-OR A , or -CO-NHR A and or two R 3 However, they are combined Together with the carbon atoms in the TIFF2023178294000003.tif1720 portion, they form a 5- or 6-membered condensed ring; R 4 is H or C 1-4 It is alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R in each appearanceA H and C are independent of each other. 1-4 Alkyl, or C 1-4 It is a haloalkyl; R in each appearance B and R C H or C 1-4 Alkyl, or R B and R C These, together with the N atoms to which they are bonded, form a 5- or 6-membered heterocycle; R in each appearance D Independently, C 1-4 Alkyl, -OC 1-4 (It is alkyl, -CN, or halogen.) To provide.

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

[0007] In one embodiment, the present disclosure is a method for regulating the luminescence of an Oplophorus luciferase-derived bioluminescent complex in a sample, the method being: (a) Contacting the sample with a coelenterazine substrate and the compounds described herein, (b) Detecting light emission in the sample, Includes, The compound provides a method for causing a decrease in luminescence from a bioluminescent complex.

[0008] In one embodiment, the present disclosure is a method for detecting interactions or co-localization between a first molecule and a second molecule in a sample, the method being: (a) Contacting a sample with a coelenterazine substrate and a compound described herein, the sample is (i) A first fusion comprising a non-luminescent peptide of luciferase derived from Oplophorus and the first molecule, (ii) A second fusion comprising an oplochorus-derived luciferase nonluminescent polypeptide and a second molecule, wherein the nonluminescent polypeptide is capable of forming a bioluminescent complex with the nonluminescent peptide, Including contact, (b) Detecting light emission in the sample, Includes, The detection of luminescence provides a method for indicating an interaction or colocalization between a first molecule and a second molecule.

[0009] In one embodiment, the present disclosure is a method for detecting interactions or colocalization between a first molecule and a second molecule in a sample, the method being: (a) Contacting a sample with a coelenterazine substrate and a compound described herein, the sample is (i) A first polynucleotide encoding the first fusion, wherein the first fusion comprises a non-luminescent peptide of luciferase derived from Oplophorus and the first molecule, (ii) A second polynucleotide encoding a second fusion, the second fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and a second molecule, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide and the second polynucleotide, Including contact, (b) Detecting light emission in the sample, Includes, The detection of luminescence provides a method for indicating an interaction or colocalization between a first molecule and a second molecule.

[0010] In one embodiment, the present disclosure is a method for detecting interactions or colocalization of a first molecule and a second molecule in a sample, the method being: (a) Contacting a sample with a non-luminescent polypeptide of luciferase derived from Oplophorus, a coelenterazine substrate, and a compound described herein, wherein the sample is (i) A first polynucleotide encoding a first fusion, the first fusion comprising a non-luminescent peptide of Oplophorus-derived luciferase and a first molecule, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with a non-luminescent polypeptide and the first polynucleotide, (ii) A second polynucleotide encoding the second fusion, wherein the second fusion comprises a fluorescent receptor molecule and the second molecule, Including contact, (b) Detecting bioluminescent resonance energy transfer (BRET) in a sample that shows interaction or co-localization of the first and second molecules, This provides a method that includes [something].

[0011] In one embodiment, the present disclosure is a method for detecting interactions or colocalization of a first molecule and a second molecule in a sample, the method being: (a) Contacting a sample with a non-luminescent peptide of luciferase derived from Oplophorus, a coelenterazine substrate, and a compound described herein, wherein the sample is (i) A first polynucleotide encoding the first fusion, the first fusion comprising a non-luminescent polypeptide of Oplophorus-derived luciferase and the first molecule, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide and the first polynucleotide, (ii) A second polynucleotide encoding the second fusion, wherein the second fusion comprises a fluorescent receptor molecule and the second molecule, Including contact, (b) Detecting bioluminescent resonance energy transfer (BRET) in a sample that shows interaction or co-localization of the first and second molecules, This provides a method that includes [something].

[0012] In one embodiment, the present disclosure is a method for detecting interactions or colocalization of molecules in a sample, the method being (a) Contacting a sample with a coelenterazine substrate and a compound described herein, the sample is (i) A first fusion comprising a non-luminescent polypeptide of luciferase derived from Oplophorus and the first molecule, (ii) A second fusion comprising a non-luminescent peptide of luciferase derived from Oplophorus 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, Including contact, (b) Detecting bioluminescent resonance energy transfer (BRET) in the sample that indicates interactions or co-localization between the first, second, and third molecules in the sample, This provides a method that includes [something].

[0013] In one embodiment, the present disclosure relates to a method for detecting a target molecule in a sample, the method being: (a) A sample containing the target molecule fused to a non-luminescent peptide of luciferase derived from Oplophorus, (i) Celenterazine substrate; (ii) Compounds described herein; and (iii) A non-luminescent polypeptide of luciferase derived from Oplophorus, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with a non-luminescent peptide; To bring it into contact with, (b) Detecting light emission in the sample, Includes, The detection of luminescence provides a method for demonstrating the formation of a bioluminescent complex between a non-luminescent peptide and a non-luminescent polypeptide.

[0014] In one embodiment, the present disclosure relates to a method for detecting a target molecule in a sample, the method being: (a) A sample containing the target molecule fused to a non-luminescent polypeptide of luciferase derived from Oplophorus, (i) Celenterazine substrate; (ii) Compounds disclosed herein; and (iii) A non-luminescent peptide of luciferase derived from Oplophorus, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with a non-luminescent polypeptide; To bring it into contact with, (b) Detecting light emission in the sample, Includes, The detection of luminescence provides a method for demonstrating the formation of a bioluminescent complex between a non-luminescent peptide and a non-luminescent polypeptide.

[0015] In one embodiment, the present disclosure relates to a method for detecting a target molecule in a sample, the method being: (a) A sample containing the target molecule fused to a non-luminescent peptide of luciferase derived from Oplophorus, (i) Celenterazine substrate; (ii) Compounds described herein; and (iii) A fusion comprising a non-luminescent polypeptide and a fluorescent moiety of luciferase derived from Oplophorus, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide; To bring it into contact with, (b) Detecting bioluminescent resonance energy transfer (BRET) in a sample that indicates the detection of molecules, This provides a method that includes [something].

[0016] In one embodiment, the present disclosure relates to a method for detecting a target molecule in a sample, the method being: (a) A sample containing the target molecule fused to a non-luminescent polypeptide of luciferase derived from Oplophorus, (i) Celenterazine substrate; (ii) Compounds disclosed herein; and (iii) A fusion comprising a non-luminescent peptide and a fluorescent moiety of luciferase derived from Oplophorus, wherein the non-luminescent peptide is capable of forming a bioluminescent complex with a non-luminescent polypeptide; To bring it into contact with,

[0017] (b) Detecting bioluminescent resonance energy transfer (BRET) in a sample that indicates the detection of molecules, This provides a method that includes [something].

[0018] In one aspect, the present disclosure relates to a bioluminescent resonance energy transfer (BRET) system, (a) A first fusion comprising a non-luminescent peptide of luciferase derived from Oplophorus and the first molecule, (b) A second fusion comprising a non-luminescent polypeptide and a fluorescent moiety of luciferase derived from Oplophorus, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with the non-luminescent peptide, (c) Coelenterazine substrate and (d) Compounds described herein, We provide a bioluminescent resonance energy transfer (BRET) system that includes [the specified component].

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

[0022] In one aspect, the present disclosure provides (a) a compound described herein, (b) a first polynucleotide encoding a non-luminescent peptide of Oplophorus-derived luciferase, (c) A second polynucleotide encoding a non-luminescent polypeptide of luciferase derived from Oplophorus, wherein the non-luminescent polypeptide is capable of forming a bioluminescent complex with a non-luminescent peptide, We provide a kit that includes this. [Brief explanation of the drawing]

[0023] [Figure 1] This shows the inhibition of bioluminescent complexes by exemplary compounds of the present 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 the calculated IC50 values ​​of the exemplary compounds shown in Figures 1A and 1B. [Figure 2] The NANOLUC® (Nluc) inhibitory activity of exemplary compounds of the present invention is compared with that of PBI-6096, a known Nluc inhibitor. JRW-1004, HL-0005, and HL-0010 show no sensible inhibition of NanoLuc but exhibit selective inhibition of the Oplophorus luciferase-derived bioluminescent complex. [Figure 3] This shows the inhibition of intracellular bioluminescent complexes by exemplary compounds of the present invention. A shows the inhibition of NanoBiT® HiBit / LgBit bioluminescent complexes by HL-0005 under cellular conditions. B compares IC50 values ​​under soluble and insoluble conditions, showing that HL-0005 is generally cell-permeable. [Modes for carrying out the invention]

[0024] The disclosed compounds can selectively inhibit the bioluminescent complex derived from Oplophorus luciferase. For example, the disclosed compounds include (a) peptides containing a peptide amino acid sequence having less than 100% (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) and more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 3; and (b) peptides containing a peptide amino acid sequence having less than 100% (e.g., >99%, <95%, <90%, <80%) sequence identity with SEQ ID NO: 3. A bioluminescent complex derived from Oplophorus luciferase comprising a polypeptide containing a polypeptide amino acid sequence having sequence identity of <70%, <60%, <50%, etc. and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%), wherein the bioluminescent complex can selectively inhibit a bioluminescent complex that exhibits detectable luminescence in the presence of a coelenterazine substrate. In a particular embodiment, the disclosed compound is a bioluminescent complex derived from Oplophorus luciferase comprising (a) a peptide containing the peptide amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 6; and (b) a polypeptide containing the polypeptide amino acid sequence having SEQ ID NO: 5, wherein the bioluminescent complex can selectively inhibit a bioluminescent complex that 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. Patents 9,797,889 and 9,797,890, the entirety of which is incorporated by reference. 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 efflux from cells, and the presence of cellular debris from cultured cells, it may be advantageous in certain applications to suppress luminescence from the Oplophorus luciferase-derived bioluminescent complex using the selective inhibitors of the present invention. For example, in applications involving the temporal multiplexing of multiple luminescence systems, it may be beneficial to have a selective inhibitor for each system to enable the measurement and / or detection of only one luminescence signal at a time. Also, in some plate-based assays, a certain amount of luciferase may be efflux from cells or present in the culture medium from cellular debris. Extracellular inhibitor compounds would allow for the selective suppression of luminescence from luciferase in the culture medium. This may help improve the signal-to-background ratio in a given assay.

[0026] In certain embodiments, the light generated from NanoBiT® bioluminescent complexes can be selectively suppressed by 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 suppression (e.g., intracellular or extracellular selectivity) to enable a given plate-based luminescence assay. The compounds may compete for luciferase binding to coelenterazine substrates and may be modified to produce cell permeability and cell impermeability inhibitors.

[0027] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, the definitions in this document shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in the practice or testing of the present invention. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative and not intended to limit the scope of this invention.

[0028] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and their variations are intended, when used herein, to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The singular forms “a,” “and,” and “the” include multiple references unless the context otherwise explicitly indicates. This disclosure also contemplates other embodiments that “include,” “consist of,” and “essentially consist of,” the embodiments or elements presented herein, whether expressly or otherwise.

[0029] As used herein, the terms “substituent” or “preferred substituent” are intended to mean a chemically acceptable functional group, for example, a portion that does not inactivate the activity of the compound of the present invention. Exemplary examples of preferred substituents include, but are not limited to, halo groups, perfluoroalkyl groups, perfluoroalkoxy groups, alkyl groups, alkenyl groups, alkynyl groups, hydroxyl 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, and arylsulfonyl groups. The substituent may be substituted by additional substituents. The substituent may also be in the form of a salt (for example, a sulfonic acid group may be in the form of a sulfonate group).

[0030] Definitions of specific functional groups and chemical terms are described in detail below. For the purposes of this disclosure, chemical elements are as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Identified according to the back cover of the ed., specific functional groups are generally defined as described therein. Also, general principles of organic chemistry, as well as specific functional parts and reactivity, are as follows: Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5 thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd This information is contained in Edition, Cambridge University Press, Cambridge, 1987, and the entirety of each of these contents is incorporated herein by reference.

[0031] As used herein, the term “alkenyl” refers to a linear or branched hydrocarbon chain containing 2 to 10 carbon atoms and at least one carbon-carbon double bond formed by the removal of two hydrogen atoms. Typical examples of alkenyls 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 group of the present invention may be unsubstituted or may be substituted with one or more suitable substituents as defined above, preferably 1 to 3 suitable substituents.

[0032] As used herein, the term “alkoxy” refers to an alkyl group as defined herein, which is added to the parent molecule via an oxygen atom. Typical examples of alkoxys include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

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

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

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

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

[0037] The term "alkoxycarbonylalkyl," as used herein, refers to an alkoxycarbonyl group, as defined herein, attached to a parent molecule via an alkylene group, as defined herein. Typical examples of alkoxycarbonylalkyls 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 “C1-C8-alkyl” is defined to include alkyl groups having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms in a linear or branched arrangement. For example, “C1-C8-alkyl” particularly includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, pentyl (e.g., n-pentyl), hexyl (e.g., n-hexyl), heptyl (e.g., n-heptyl), and octyl (e.g., n-octyl). The term “C1-C6-alkyl” is defined to include alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement. For example, "C1-C6-alkyl" particularly includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, pentyl (e.g., n-pentyl), and hexyl (e.g., n-hexyl). The term "C1-C4-alkyl" is defined as including alkyl groups having 1, 2, 3, or 4 carbon atoms in a linear or branched arrangement. For example, "C1-C4-alkyl" particularly includes methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, iso-butyl, and tert-butyl. The alkyl groups of the present invention may be unsubstituted or may be substituted with one or more preferred substituents as defined above, for example, 1 to 3 preferred substituents. For example, an alkyl group may 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] As used herein, the term “alkylamino” refers to an alkyl group as defined herein, which is added to the parent molecule via an amino group as defined herein. Typical examples of alkylaminos include, but are not limited to, methylamino, ethylamino, isopropylamino, butylamino, and sec-butylamino.

[0040] As used herein, the term “alkylaminoalkyl” refers to an alkyl group as defined herein, which is added to the parent molecule via an aminoalkyl group as defined herein. Typical examples of alkylaminoalkyl groups include, but are not limited to, methylaminoethyl and methylamino-2-propyl.

[0041] As used herein, the term “alkylcarbonyl” refers to an alkyl group as defined herein that is added to the parent molecule via a carbonyl group as defined herein. Typical examples of alkylcarbonyls include, but are not limited to, acetyl, 1-oxopropyl, 2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl.

[0042] As used herein, the term “alkylcarbonylalkyl” refers to an alkylcarbonyl group, as defined herein, attached to the parent molecule via an alkyl group, as defined herein.

[0043] As used herein, the term “alkylcarbonylalkylamide” refers to an alkylcarbonyl group, as defined herein, attached to the parent molecule via an alkylamide group, as defined herein.

[0044] The term "alkylene" refers to a divalent group derived from saturated, linear, or branched hydrocarbons containing 1 to 10 carbon atoms. Typical examples of alkylenes 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 linear or branched hydrocarbon group having 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms and one or more carbon-carbon triple bonds. The alkynyl groups of the present invention include, but are not limited to, ethynyl, propynyl, and butynyl. The alkynyl groups of the present invention may be unsubstituted or may be substituted with one or more preferred substituents, preferably 1 to 3 preferred substituents, as defined above.

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

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

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

[0049] As used herein, the term “aminoalkylamide” means at least one amino group, as defined herein, attached to the parent molecule via an alkylamide group, as defined herein.

[0050] As used herein, the term “amino protecting group” refers to a portion 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, as seen in *Protecting Groups in Organic Synthesis*, TW Greene and PGMWuts, 3. rd This includes those described in detail in 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-OCH2C6H5 or -NH-Cbz); t-butyloxycarbonyl (-NHCO-OC(CH3)3 or -NH-Boc); 9-fluorenylmethyloxycarbonyl (-NH-Fmoc), 2,2,2-trichloroethyloxycarbonyl (-NH-Troc), and allyloxycarbonyl (-NH-Alloc). In each of the above, -NH- represents the nitrogen from the protected amino group.

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

[0052] As used herein, the term “aryl” means a monocyclic, bicyclic, or tricyclic aromatic group. Typical 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, as defined above. As used herein, aryls include phenyl (e.g., indanyl or 5,6,7,8-tetrahydronaphthalen-2-yl) attached to the parent molecule and condensed to a cycloalkyl group, phenyl (i.e., naphthyl) condensed to a phenyl group, or phenyl (e.g., benzo[d][1,3]dioxol-5-yl) condensed to a non-aromatic heterocycle.

[0053] As used herein, the term “arylalkyl” refers to an aryl group, as defined herein, attached to a parent molecule via an alkyl group, as defined herein. Typical examples of arylalkyls include, but are not limited to, phenylmethyl (i.e., benzyl) and phenylethyl.

[0054] As used herein, the term “arylcarbonyl” refers to an aryl group as defined herein that is attached to the parent molecule via a carbonyl group as defined herein.

[0055] As used herein, the terms “carbonyl” or “(C=O)” (as used in phrases such as alkylcarbonyl, alkyl-(C=O)-, or alkoxycarbonyl) refer to the conjugate of the >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. The carbonyl group is also defined herein as equivalent to (C=O). Alkylcarbonylamino refers to groups such as acetamide.

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

[0057] As used herein, the term "carboxyalkyl" refers to a carboxyl group as defined herein that is attached to the parent molecule via an alkyl group as defined herein.

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

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

[0060] As used herein, the term "cycloalkylalkyl" refers to a cycloalkyl group as defined herein, which is added to the parent molecule via an alkyl group as defined herein. Typical examples of cycloalkylalkyls include, but are not limited to, cyclohexylmethyl.

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

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

[0063] As used herein, the term “dialkylaminoalkyl” refers to a dialkylamino group as defined herein, which is added to the parent molecule via an alkyl group as defined herein. Typical examples of dialkylaminoalkyls include, but are not limited to, N,N-dimethylaminoethyl and N,N-methyl(2-propyl)aminoethyl.

[0064] As used herein, the term “dialkylaminoalkylamide” refers to a dialkylamino group as defined herein that is attached to the parent molecule via an alkylamide group as defined herein.

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

[0066] As used herein, the term “haloalkoxy” refers to an alkoxy group as defined herein, substituted with one, two, three, or four halogen atoms. Typical examples of haloalkoxys 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 with one, two, three, or four halogen atoms. Typical examples of haloalkyls include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and 4,4,4,-trifluorobutyl.

[0068] As used herein, the term “heteroaryl” refers to monocyclic heteroaryl or bicyclic heteroaryl. Monocyclic heteroaryls are five- or six-membered rings. A five-membered ring contains two double bonds. A five-membered ring may contain one heteroatom selected from O or S; or one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom. A six-membered ring may contain three double bonds and one, two, three or four nitrogen atoms. Representative examples of monocyclic heteroaryls include, but are not limited to, furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1,3-oxazolyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, 1,3-thiazolyl, thienyl, triazolyl, and triazinyl. Bicyclic heteroaryls include monocyclic heteroaryls condensed with phenyl, monocyclic heteroaryls condensed with monocyclic cycloalkyls, monocyclic heteroaryls condensed with monocyclic cycloalkenyls, monocyclic heteroaryls condensed with monocyclic heteroaryls, and monocyclic heteroaryls condensed with monocyclic heterocyclics. Typical 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, naphthilidinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, thiazolo[5,4-b]pyridinyl-2-yl, thiazolo[5,4-d]pyrimidine-2-yl, 5,6,7,8-tetrahydroquinoline-5-yl, cyclopenta[b]thiophene-2-yl, and 4,5,6,7-tetrahydrobenzo[b]thiophene-2-yl. The heteroaryl groups of the present invention may 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 that is added to the parent molecule via an alkyl group as defined herein. Typical examples of heteroarylalkyls include, but are not limited to, flu-3-ylmethyl, 1H-imidazole-2-ylmethyl, 1H-imidazole-4-ylmethyl, 1-(pyridine-4-yl)ethyl, pyridine-3-ylmethyl, 6-chloropyridine-3-ylmethyl, pyridine-4-ylmethyl, (6-(trifluoromethyl)pyridine-3-yl)methyl, (6-(cyano)pyridine-3-yl)methyl, (2-(cyano)pyridine-4-yl)methyl, (5-(cyano)pyridine-2-yl)methyl, (2-(chloro)pyridine-4-yl)methyl, pyrimidine-5-ylmethyl, 2-(pyrimidine-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl.

[0070] As used herein, the terms “heterocyclic” refer to monocyclic, bicyclic, or tricyclic heterocyclic rings. A monocyclic heterocyclic ring 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 0 or 1 double bond and 1 heteroatom selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 5-membered ring contains 0 or 1 double bond and 1, 2, or 3 heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 6-membered ring contains 0, 1, or 2 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. A 7 or 8-membered ring contains 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus, and sulfur. Representative examples of monocyclic heterocycles include azetidinyl, azepanyl, azilidinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolinyl, phosfinanyl, piperadinyl, piperidinyl, pyrazolinyl. This includes, but is not limited to, nyl, pyrazolinil, pyrazolidinil, pyrrolinil, pyrrolidinil, tetrahydrofuranil, tetrahydropyranil, tetrahydropyridinil, tetrahydropyrimidinil, tetrahydrothienyl, thiadiazolinil, thiadiazolidinil, thiazolinil, thiazolidinil, thiomorpholinil, 1,1-dioxidethiomorpholinil (thiomorpholine sulfone), thiopyranil, trithianil, and 2,5-dioxo-pyrrolidinil.A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl group, 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 group, or bicyclic heterocycles fused to a monocyclic cycloalkenyl group, or bicyclic heterocycles fused to a monocyclic heterocycle, where 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-admantan(1-azatricyclo[3.3.1.1. 3,7 ]decane), oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7This includes, but is not limited to, 2,4,6-trioxa-8-phosphatricyclo[3.3.1.13,7]decane. The heterocyclic group of the present invention may be unsubstituted or substituted with one or more preferred substituents, preferably 1 to 3 preferred substituents, as defined above. The heterocyclic group of the present invention may contain one or more oxo groups (=O) or thioxo groups (=S) bonded to the ring.

[0071] As used herein, the term “heterocyclylalkyl” refers to a heterocyclyl group as defined herein, added to the parent molecule via an alkyl group as defined herein. Typical examples of heterocyclylalkyls include, but are not limited to, piperidine-4-ylmethyl, piperazine-1-ylmethyl, 3-methyl-1-pyrrolidine-1-ylbutyl, (1R)-3-methyl-1-pyrrolidine-1-ylbutyl, (1S)-3-methyl-1-pyrrolidine-1-ylbutyl, and 3-morpholinopropyl.

[0072] As used herein, the term “heterocyclilamide” refers to a heterocyclyl group as defined herein that is attached to the parent molecule via an amide group as defined herein.

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

[0074] As used herein, the term “hydroxyalkoxy” refers to an alkoxy group as defined herein, substituted with at least one hydroxyl group. Typical examples of hydroxyalkoxys 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 hydroxyl group. Typical examples of hydroxyalkyls include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 2,3-dihydroxypentyl, 4-hydroxybutyl, 2-ethyl-4-hydroxyheptyl, 3,4-dihydroxybutyl, and 5-hydroxypentyl.

[0076] As used herein, the term "hydroxyalkylamide" refers to a hydroxyalkyl group bonded to an amide group, such as -amide-alkyl-OH.

[0077] As used herein, the term “hydroxycarbonyl” refers to a hydroxyl group as defined herein that is attached to the parent molecule via a carbonyl group as defined herein.

[0078] As used herein, the term "methylenedioxy" refers to an -OCH2O- group in which the oxygen atom of methylenedioxy is bonded to the parent molecule via two adjacent carbon atoms.

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

[0080] As used herein, unless otherwise specified, the terms “peptide” and “polypeptide” refer to high molecular weight compounds of two or more amino acids linked together via a peptide amide bond (-C(O)NH-) in their main chain. The term “peptide” typically refers to short amino acid polymers (e.g., chains with fewer than 25 amino acids), while the term “polypeptide” typically refers to longer amino acid polymers (e.g., chains with more than 25 amino acids).

[0081] The prefix attached to a multicomponent substituent applies only to the first component it precedes. For example, the term "cycloalkylalkyl" contains two components: alkyl and cycloalkyl. Therefore, C1- 10 Cycloalkylalkyl C1- 10 The prefix indicates that the alkyl component of cycloalkylalkyl contains 1 to 6 carbon atoms; the C1-C6- prefix does not describe the cycloalkyl component. For further example, C1- 10 The term haloalkyl refers to halomethyl, haloethyl, halopropyl, halobutyl, halopentyl, and halohexyl. For further illustration, 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 bonded to one or more hydrogen atoms. Therefore, for example, hydrogen, halogens, and cyanos do not fall under this definition. Furthermore, sulfur atoms in heterocyclines containing such atoms are substituted with one or two oxo substituents.

[0083] When a substituent is described as "substituted," a non-hydrogen group is present in place of a hydrogen group on the carbon or nitrogen of the substituent. Therefore, for example, a substituted alkyl substituent is an alkyl substituent in which at least one non-hydrogen group is present in place of a hydrogen group on the alkyl substituent. For illustrative purposes, a monofluoroalkyl is an alkyl substituted with a fluoro group, and a difluoroalkyl is an alkyl substituted with two fluoro groups. When multiple substitutions are present on a substituent, it should be recognized that each non-hydrogen group may be the same or different (unless otherwise stated).

[0084] If a substituent is described as “unsubstituted” and not as “substituted” or “optionally substituted,” it means that the substituent has no substituents. If a substituent is described as “optionally substituted,” the substituent may be (1) unsubstituted or (2) substituted. If a substituent is described as being optionally substituted with a maximum of a specific number of nonhydrogen groups, the substituent may be (1) unsubstituted or (2) substituted with a maximum of that specific number of nonhydrogen groups or up to the maximum number of substituted positions on the substituent, whichever is less. For example, if a substituent is described as a heteroaryl optionally substituted with a maximum of three nonhydrogen groups, then any heteroaryl having fewer than three substituted positions will be optionally substituted with a maximum of the same number of nonhydrogen groups as the heteroaryl has substituted positions. For illustrative purposes, tetrazolyl (having only one substituted position) will be optionally substituted with a maximum of one nonhydrogen group. To illustrate further, if it is stated that an amino nitrogen is optionally substituted with up to two non-hydrogen groups, then the primary amino nitrogen is optionally substituted with up to two non-hydrogen groups, while the secondary amino nitrogen is optionally substituted with up to one non-hydrogen group.

[0085] When it is stated that substituents are "independently selected" from the group, each substituent is selected independently of the others. Therefore, each substituent can be identical or different from the other substituents.

[0086] As used herein, the terms “bioluminescence” or “luminescence” may 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 Opophorus luciferase, e.g., Opophorus gracilirostris; firefly luciferase, e.g., Photinus pyralis or Photuris pennsylvanica; click mushroom luciferase; Renilla luciferase; sea firefly luciferase; aequorin photoprotein; oberin photoprotein; and others.

[0087] As used herein, the term “complex” refers to a group or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with one another. In one embodiment, “contact” or more specifically, “direct contact” means that two or more molecules are close enough together that attractive non-covalent interactions, such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, dominate the interaction of the molecules. In such embodiments, the complex of molecules (e.g., peptides and polypeptides) is formed under assay conditions such that the complex is thermodynamically favorable (e.g., compared to the non-aggregated or non-complexed state of its component molecules). As used herein, the term “complex” refers to a group of two or more molecules (e.g., peptides, polypeptides, or combinations thereof) unless otherwise stated.

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

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

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

[0091] As used herein, the terms “interacting element” or “interacting molecule” refer to a portion that assists in bringing together a pair of non-luminescent elements or groups of non-luminescent elements 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 the attractive interaction between the two interacting elements promotes the formation of the bioluminescent complex. However, the present invention is not limited to such mechanisms, and an understanding of the mechanism is not required to carry out the present invention. Interacting elements may promote the formation of the bioluminescent complex by any preferred mechanism (e.g., bringing non-luminescent pairs / groups into proximity, arranging non-luminescent pairs / groups in a suitable conformation for stable interaction, reducing the activation energy for complex formation, or a combination thereof). Interacting elements may be proteins, polypeptides, peptides, small molecules, cofactors, nucleic acids, lipids, carbohydrates, antibodies, etc. An interacting pair may consist 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 may be of the same type (e.g., polypeptides) or two different types (e.g., a polypeptide and a small molecule). In some embodiments where complex formation by interacting pairs is studied, the interacting pair may be referred to as the “target pair” or “target pair,” and the individual interacting elements may be referred to as the “target element” (e.g., “target peptide,” “target polypeptide,” etc.) or “target element” (e.g., “target peptide,” “target polypeptide,” etc.).

[0092] As used herein, the terms “fusion,” “fusion polypeptide,” and “fusion protein” refer to a chimeric protein containing a target protein or polypeptide (e.g., a target sequence) bound to a second different peptide, polypeptide, or protein (e.g., a detectable sequence, an isolable 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 classification 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 derivatives or variants, and their natural analogs, which include, in addition to those disclosed in WO2003 / 040100; U.S. Patent No. 12 / 056,073 (paragraph

[0086] ); and U.S. Patent No. 8,669,103 (these disclosures are incorporated herein by reference in their entirety), flimazine, 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.

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

[0095] The term “energy receptor” or “receptor molecule” refers to any small molecule (e.g., a chromophore), macromolecule (e.g., an autofluorescent protein, phycobiliprotein, nanoparticle, surface, etc.), or molecular complex that generates an readily detectable signal in response to energy absorption (e.g., resonance energy transfer). In a given embodiment, the energy receptor 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., proflavin, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porfin, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA Examples of fluorophores 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 dye (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, the energy acceptor includes, but is not limited to, small molecule fluorescent dyes such as NCTs, fluorescent particles such as quenchers and quantum dots, luminescent metal complexes, and any other known energy acceptors.

[0096] The terms “luminescent enzyme,” “bioluminescent enzyme,” or “luciferase,” as used interchangeably herein, refer to a classification of oxidative enzymes used in bioluminescence, in which an enzyme produces and emits light when given a substrate. A luciferase may be a naturally occurring luciferase, a recombinant luciferase, or a mutant luciferase that uses a luciferase substrate. The luciferase substrate may be luciferin, a luciferin derivative or analog, a pre-luciferin derivative or analog, coelenterazine, or a coelenterazine derivative or analog. If a luminescent enzyme is naturally occurring, it can be readily obtained from living organisms by those skilled in the art. If a luminescent enzyme is naturally occurring, or 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 readily obtained from cultures such as bacteria, yeast, mammalian cells, insect cells, or plant cells transformed to express the nucleic acid encoding the luminescent enzyme. Furthermore, recombinant or mutant luminescent enzymes can be induced from in vitro cell-free systems using nucleic acids encoding luciferase.Suitable bioluminescent enzymes include luciferases from bioluminescent decapod crustaceans, such as 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, chia luciferases, such as Gaussia luciferase (e.g., Gaussia princeps luciferase), Metridia luciferases, such as Metridia longa and Metridia pacifica luciferase, Vargula luciferases, such as Vargula hilgendorfii luciferase, and Pleuromamma luciferase. This includes xiphias luciferases and photoproteins, such as aequorin, as well as their variants, recombinants, and mutants.

[0097] A "luminescent reaction mixture" contains a substance that enables a luminescent enzyme to produce a light signal, i.e., luminescence. The mixture may also contain an enzyme, such as luciferase enzyme or luciferase. The substance required to produce the luminescence signal, as well as the specific concentration and / or amount, will vary depending on the luminescent enzyme used and the type of assay being performed. Often, other substances 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, and washing agents.

[0098] As used herein, the terms “Oplophorus luciferase” and “Oplophorus-derived luciferase” are interchangeable and refer to the luciferase secreted from the deep-sea shrimp Opophorus gracilirostris (e.g., Sequence ID No. 1) (including its wild type, variants, and mutants). For example, preferred Opophorus luciferase variants are described in U.S. Patents 8,557,970 and 8,669,103, each of which is incorporated herein by reference in whole.

[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, e.g., acidic (e.g., aspartate, glutamic acid), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and non-charged (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The "sequence identity rate" (or "sequence similarity rate") is calculated by (1) comparing two optimally aligned sequences across a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, or a specific window), (2) determining the number of positions containing identical (or similar) monomers (e.g., the same amino acid present in both sequences, or similar amino acids present in both sequences) to obtain the number of matching positions, (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, or a specific window), and (4) multiplying the result by 100 to obtain the sequence identity rate or sequence similarity rate. For example, if both peptides A and B are 20 amino acids long and have the same amino acid at all positions except one, then peptides A and B have 95% sequence identity. If the amino acids at the non-identical positions share the same biophysical characteristics (e.g., both are acidic), then peptides A and B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to those in peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity with respect to the optimal comparison window of peptide C.For the purpose of calculating the “sequence identity” (or “sequence similarity”) in this specification, any gaps in the aligned sequences are treated as mismatches at their respective locations.

[0100] As used herein, the term “reporter portion” may refer to a portion that, under appropriate conditions, directly or indirectly generates a detectable signal. Exemplary reporter portions include, but are not limited to, fluorophores, luminescent molecules, dyes, radiolabels, and substrates for enzymes such as luciferase. In some embodiments, a reporter portion may indirectly generate a detectable signal, for example, if the reporter portion is a substrate for an enzyme. The reaction between the enzyme and the substrate then generates a detectable signal, such as fluorescence or luminescence. As used herein, the term “bioluminescent reporter portion” may refer to a portion that is a substrate for luciferase. For example, a bioluminescent reporter portion may be luciferin, luciferin derivatives, such as pre-luciferin, aminoluciferin, chyonorylluciferin, naptylluciferin, fluoroluciferin, chloroluciferin, precursors of luciferin derivatives, coelenterazine, or coelenterazine derivatives or analogs, such as flimazine. The resulting luminescence signal may be detected using a luminometer. As used herein, the term “fluorescent reporter portion” may refer to the portion that emits fluorescence. For example, the fluorescent reporter portion may be a fluorophore such as coumarin, R110, fluoroceine, DDAO, resolphin, cresyl violet, silixanthene, or carbopyronine. Fluorescence may be detected using a fluorometer.

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

[0102] 2. Compound This specification provides compounds that can selectively inhibit bioluminescent complexes derived from Oplophorus luciferase, for example, compounds that can inhibit the luciferase activity of bioluminescent complexes. In one aspect, a compound of formula (I), or a salt thereof: TIFF2023178294000004.tif43170(wherein: R 1 is aryl, cycloalkyl, heteroaryl, heterocycle, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocycle thereof are optionally substituted with one or more R W ; each R W is independently C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-10 alkylene-OR A , -CO-R A , -C 1-10 alkylene-CO-R A , -CO-OR A , -C 1-10 alkylene-CO-OR A , -CO-NHR A , -C 1-10 alkylene-CO-NHR A , -NR B R C , -C 1-10 alkylene-NR B R C , -NH-CO-C 1-4 alkyl, -C 1-10 alkylene-NH-CO-C 1-4 alkyl, phenyl, and phenyl substituted with 1, 2, 3, or 4 R D groups; each R 2 is independently C 1-10 alkyl, C 1-10 haloalkyl, halogen, -CN, -OR A , -C 1-4 alkylene-OR A , -CO-RA , -C 1-4 Alkilen-CO-R A , -CO-OR A , -C 1-4 Alkilen-CO-OR A ,-CO-NHR A , -C 1-4 Alkilen-CO-NHR A , -NR B R C , -C 1-4 Alkilen-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 groups substituted with -C≡CR A , or -C≡CC 1-4 Alkilen-OR A and or two R 2 However, they are combined Together with the carbon atoms in the TIFF2023178294000005.tif1315 portion, they form a 5- or 6-membered condensed ring; Each R 3 Independently, C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, -OR A , -C 1-4 Alkilen-OR A ,-CO-R A , -CO-OR A , or -CO-NHR A and or two R 3 However, they are combined Together with the carbon atoms in the TIFF2023178294000006.tif1720 portion, they form a 5- or 6-membered condensed ring; R 4 is H or C 1-4 It is alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R in each appearanceA H and C are independent of each other. 1-4 Alkyl, or C 1-4 It is a haloalkyl; R in each appearance B and R C H or C 1-4 Alkyl, or R B and R C These, together with the N atoms to which they are bonded, form a 5- or 6-membered heterocycle; R in each appearance D Independently, C 1-4 Alkyl, -OC 1-4 (It is alkyl, -CN, or halogen.) It will be disclosed.

[0103] In some embodiments, in a compound of formula (I): R 1 is an aryl, cycloalkyl, heteroaryl, or heterocycle, and the aryl, cycloalkyl, heteroaryl, and heterocycle are one or more R W It is arbitrarily substituted, and each R W Independently, C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, -OR A , -C 1-10 Alkilen-OR A ,-CO-R A , -C 1-10 Alkilen-CO-R A , -CO-OR A , -C 1-10 Alkilen-CO-OR A ,-CO-NHR A , -C 1-10 Alkilen-CO-NHR A , -NR B R C , -C 1-10 Alkilen-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 Selected from the group consisting of phenyl compounds substituted with a group; Each R 2 Independently, C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, -OR A , -C 1-4 Alkilen-OR A ,-CO-R A , -C 1-4 Alkilen-CO-R A , -CO-OR A , -C 1-4 Alkilen-CO-OR A ,-CO-NHR A , -C 1-4 Alkilen-CO-NHR A , -NR B R C , -C 1-4 Alkilen-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 groups substituted with -C≡CR A , or -C≡CC 1-4 Alkilen-OR A and or two R 2 However, they are combined Together with the carbon atoms in the TIFF2023178294000007.tif1315 portion, they form a 5- or 6-membered condensed ring; Each R 3 Independently, C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, -OR A , -C 1-4 Alkilen-OR A ,-CO-R A , -CO-OR A , or -CO-NHR A and or two R 3 However, they are combined Together with the carbon atoms in the TIFF2023178294000008.tif1720 portion, they form a 5- or 6-membered condensed ring; R 4 is H or C 1-4 It is alkyl; p is 0, 1, 2, 3, or 4; q is 0, 1, 2, 3, or 4; R in each appearance A H and C are independent of each other. 1-4 Alkyl, or C 1-4 It is a haloalkyl; R in each appearance B and R C H or C 1-4 Alkyl, or R B and R C These, together with the N atoms to which they are bonded, form a 5- or 6-membered heterocycle; R in each appearance D Independently, C 1-4 Alkyl, -OC 1-4 It is alkyl, -CN, or halogen.

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

[0105] In some embodiments, R 1 These are unsubstituted aryl, unsubstituted cycloalkyl, unsubstituted heteroaryl, or unsubstituted heterocycles.

[0106] In some embodiments, R 1 is aryl(C 6-20 It is an aryl (such as an aryl) or a heteroaryl (such as a 5-12 member heteroaryl), and the aryl and heteroaryl are one or more R W It is optionally replaced with R. In some embodiments, 1 C 6-20 It is an aryl, and that aryl is one or more R WIt is optionally replaced with R. In some embodiments, 1 is one or more R W It is a 5- to 12-membered heteroaryl that is optionally substituted with R. In some embodiments, 1 is one or more R W C arbitrarily replaced by 5-10 It is cycloalkyl. In some embodiments, R 1 is one or more R W It is a 5- to 12-membered complex ring with arbitrary substitutions.

[0107] In some embodiments, R 1 teeth, The filename is TIFF2023178294000009.tif22170, and each contains one or more R values. W It is optionally replaced with R. In some embodiments, 1 teeth, TIFF2023178294000010.tif22170, and each of these is non-substitutable. In some embodiments, R 1 teeth, TIFF2023178294000011.tif22170, and each of these is C 1-4 Alkyl or C 1-4 It is substituted with a haloalkyl group.

[0108] In some embodiments, R 1 teeth, TIFF2023178294000012.tif22170, and each of these contains one or more R W It is optionally replaced with R. In some embodiments, 1 teeth, This is TIFF2023178294000013.tif22170. In some embodiments, each R W Independently, C 1-10 Alkyl, C 1-10 Selected from the group consisting of haloalkyl, halogen, or -CN. In some embodiments, R W is -OR A , -C 1-4Alkilen-OR A ,-CO-R A , -C 1-4 Alkilen-CO-R A , -CO-OR A , -C 1-4 Alkilen-CO-OR A ,-CO-NHR A , or -C 1-4 Alkilen-CO-NHR A In some embodiments, R W -NR B R C , -C 1-4 Alkilen-NR B R C , -NH-CO-C 1-4 Alkyl, or -C 1-4 Alkylene-NH-CO-C 1-4 It is alkyl. In some embodiments, R W is phenyl or 1, 2, 3, or 4 R D It is substituted with a group and is phenyl. In some embodiments, R W C 1-10 Alkyl, C 1-10 It is a haloalkyl, halogen, or -CN. In some embodiments, R W C is a compound such as methyl, ethyl, propyl, or butyl. 1-4 It is alkyl.

[0109] In some embodiments, R W C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, -OR A , -C 1-10 Alkilen-OR A , -CO-OR A , -C 1-10 Alkilen-CO-OR A Selected from the group consisting of , and phenyl. In some embodiments, each R A This is independently selected from hydrogen, methyl, and ethyl.

[0110] In some embodiments, R 1is one or more R W These are arylalkyls (such as benzyl) that have been arbitrarily substituted.

[0111] In some embodiments, R 1 is an aryl, cycloalkyl, heteroaryl, heterocyclic, or arylalkyl, and the aryl, cycloalkyl, heteroaryl, and heterocyclic are one R W It is replaced with R W C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, -OR A , -C 1-10 Alkilen-OR A , -CO-OR A , -C 1-10 Alkilen-CO-OR A Selected from the group consisting of , and phenyl, each R A This is independently selected from hydrogen, methyl, and ethyl.

[0112] In some embodiments, R 1 teeth, TIFF2023178294000014.tif43170, and each of these is non-substitutive, or one R W It is replaced with R W 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 (For example, -OCH3), -C 1-10 Alkilen-OR A (For example, -(CH2)4-OH or -(CH2)6-OH), -CO-OR A (For example, -COOH, -COOCH3, or -COOCH2CH3), -C 1-10 Alkilen-CO-OR ASelected from the group consisting of (for example, -CH2COOH, -CH2COOCH2CH3, -(CH2)5-COOH, or -(CH2)5-COOCH3) and phenyl.

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

[0114] In some embodiments, R 2 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 It is alkyl. In some embodiments, R 2 C 1-10 It is alkyl. In some embodiments, R 2 C is a compound such as methyl, ethyl, propyl, or butyl. 1-4 It is alkyl. In some embodiments, R 2 is methyl. In some embodiments, R 2 is halogen, C 1-4 Haloalkyl, -OH, C 1-4 Alkylene-OH,-OC 1-4 It is alkyl or -NH2.

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

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

[0117] In some embodiments, p is 2. In some embodiments, 2 R2 They are joined together Together with the carbon atoms in the TIFF2023178294000015.tif1315 portion, they form a 5- or 6-membered fused ring. For example, two R 2 They are joined together Together with the TIFF2023178294000016.tif1315 section It is possible to form TIFF2023178294000017.tif1326.

[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 C 1-10 Alkyl, C 1-10 Haloalkyl, halogen, -CN, and -OR A Selected from, R A is hydrogen and C 1-4 Selected from alkyl groups. In some embodiments, R 3 C 1-10 Alkyl (e.g., methyl, ethyl, isopropyl, n-propyl, or n-butyl C) 1-4 Alkyl), C 1-10 Haloalkyl (e.g., trifluoromethyl C) 1-4 Haloalkyl), halogen (e.g., fluoro, chloro, or bromo), -CN, and -OR A (For example, selected from -OH or -OCH3). In some embodiments, R 3 C 1-4 Alkyl, halogen, -CN, -OH, or -OC 1-4 It is alkyl. In some embodiments, R 3 is a halogen. In some embodiments, R 2 C 1-4 Alkyl (such as methyl), R 3 C 1-4 Alkyl, halogen, -CN, -OH, or -OC 1-4It is alkyl.

[0120] In some embodiments, q is 2. In some embodiments, 2 R 3 They are joined together Together with the carbon atoms in the TIFF2023178294000018.tif1720 portion, they form a 5- or 6-membered condensed ring. For example, two R 3 They are joined together Together with the TIFF2023178294000019.tif1720 section It is possible to form TIFF2023178294000020.tif2622.

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

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

[0123] R in each appearance A (R 1 , R 2 , or R 3(As substituents in) are independently H, C 1-4 Alkyl, or C 1-4 In some embodiments, R A H is H. In some embodiments, R A C 1-4 It is alkyl. In some embodiments, R A is methyl. In some embodiments, R A C such as -CF3 1-4 It is a haloalkyl group.

[0124] R in each appearance B and R C (R 1 or R 2 (As substituents in) independently, H or C 1-4 Alkyl, or R B and R C These, together with the N atoms to which they are bonded, form a 5- or 6-membered heterocycle. In some embodiments, R B and R C Both are H. In some embodiments, R B H is R C C 1-4 It is alkyl. In some embodiments, R B C 1-4 It is alkyl, R C C 1-4 It is alkyl. In some embodiments, R B and R C These, together with the N atoms to which they are bonded, form a 5- or 6-membered heterocycle.

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

[0126] In some embodiments, R 1 teeth, TIFF2023178294000021.tif22170, and each of these is C 1-4 Alkyl or C 1-4 Substituted with haloalkyl, or R 1 teeth TIFF2023178294000022.tif22170, R 2 C 1-4 Alkyl, halogen, C 1-4 Haloalkyl, -OH, C 1-4 Alkylene-OH,-OC 1-4 Alkyl or -NH2, R 3 C 1-4 Alkyl, halogen, -CN, -OH, or -OC 1-4 It is an alkyl group, p is 1, and q is 0 or 1.

[0127] In some embodiments, if p is 0, then R 1 is one or more R W (For example, one R W ) is replaced by. In some embodiments, p is 0 and R 1 If it is phenyl, then R 1 is one or more R W (For example, one R W ) is replaced by. In some embodiments, R 1 If is unsubstituted, then p is 1, 2, 3, or 4 (for example, p is 1 or 2, or p is 1). In some embodiments, R 1 If is an unsubstituted phenyl, then p is 1, 2, 3, or 4 (for example, p is 1 or 2, or p is 1). In some embodiments, the compound is not N-phenyl-2-(phenylsulfonamide)benzamide.

[0128] In some embodiments, the compound of formula (I) is the compound of formula (Ia), or a salt thereof. TIFF2023178294000023.tif37170 (in the formula, R 1 C 6-20 It is an aryl or a 5-12 member heteroaryl, and the aryl and heteroaryl are one or more R W It is arbitrarily replaced with; p is 0, 1, or 2; q is either 0 or 1; R 3 C 1-4 Alkyl, halogen, -CN or -OR A and; R A is H or C 1-4 It is alkyl; R 2 and R W (This is as defined herein.) That is the case.

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

[0130] In some embodiments, a compound of formula (Ia) or a salt thereof (wherein R 1 teeth, The filename is TIFF2023178294000024.tif22170, and each contains one or more R W A (which is optionally substituted by) is disclosed. In some embodiments, R of formula (Ia) is disclosed. 1 teeth, The filename is TIFF2023178294000025.tif22170.

[0131] In some embodiments, a compound of formula (Ia) or a salt thereof (wherein R 1 C 1-4 Alkyl or C 1-4 A haloalkyl is disclosed. For example, R of formula (Ia) 1 teeth, It could be TIFF2023178294000026.tif22170, and each is C 1-4 Alkyl or C 1-4 It is substituted with a haloalkyl group.

[0132] In some embodiments, a compound of formula (Ia) or a salt thereof (wherein R 1 teeth, TIFF2023178294000027.tif22170, and each of these contains one or more R W (Optionally substituted with) is disclosed. In some embodiments, R 1 teeth, The filename is TIFF2023178294000028.tif22170.

[0133] In some embodiments, a compound of formula (Ia) or a salt thereof (wherein R 1 teeth, TIFF2023178294000029.tif43170, and each of these is non-substitutive, or one R W It is replaced with R W 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 (For example, -OCH3), -C 1-10 Alkilen-OR A (For example, -(CH2)4-OH or -(CH2)6-OH), -CO-OR A (For example, -COOH, -COOCH3, or -COOCH2CH3), -C 1-10 Alkilen-CO-OR A (For example, selected from the group consisting of -CH2COOH, -CH2COOCH2CH3, -(CH2)5-COOH, or -(CH2)5-COOCH3) and phenyl) is disclosed.

[0134] In some embodiments, a compound of formula (Ia) or a salt thereof (wherein R 2 C 1-4 Alkyl (methyl, ethyl, propyl, or butyl, etc.), 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 They are joined together TIFF2023178294000030.tif1315 discloses a part of the carbon atom that, together with the carbon atoms, forms a 5 or 6-membered fused ring. For example, the two R in formula (Ia) 2 It is that it is joined together Together with the TIFF2023178294000031.tif1315 section TIFF2023178294000032.tif1326 can be formed. In some embodiments, a compound of formula (Ia) or a salt thereof (wherein R 2 C 1-4 Alkyl compounds (such as methyl compounds) are disclosed. In some embodiments, compounds of formula (Ia) or salts thereof (wherein R 2 is -OH, -C 1-4 Alkylene -OH (-CH2OH, etc.), -OC 1-4 Alkyl compounds (such as -OCH3) or -NH2 are disclosed. In some embodiments, compounds of formula (Ia) or salts thereof (wherein p is 1 and q is 0) are disclosed. In some embodiments, compounds of formula (Ia) or salts thereof (wherein R 2 C 1-4 Alkyl (methyl, etc.), -OH, -C 1-4 Alkylene -OH (-CH2OH, etc.), -OC 1-4Alkyl (-OCH3, etc.) or -NH2, where p is 1 and q is 0) is disclosed. In some embodiments, a compound of formula (Ia) or a salt thereof (wherein p is 1 and q is 1) is disclosed. In some embodiments, a compound of formula (Ia) or a salt thereof (wherein R 2 It is methyl, and R 3 A is disclosed in which is -CH3, a halogen (such as F), -OCH3, or -CN, where p is 1 and q is 1.

[0135] In some embodiments, a compound of formula (Ia), or a salt thereof (wherein p is 0, R 1 If it is phenyl, then R 1 is one or more R W Compounds of formula (Ia) or salts thereof (wherein R 1 If is an unsubstituted phenyl, then p is 1, 2, 3, or 4 (for example, p is 1 or 2, or p is 1)). In some embodiments, a compound of formula (Ia) or a salt thereof (wherein if p is 0, then R 1 is one or more R W (For example, one R W Compounds of formula (Ia) or salts thereof (wherein p is 0 and R) are disclosed. In some embodiments, compounds of formula (Ia) or salts thereof are disclosed. 1 If it is phenyl, then R 1 is one or more R W (For example, one R W Compounds of formula (Ia) or salts thereof (wherein R) are disclosed. In some embodiments, compounds of formula (Ia) or salts thereof are disclosed. 1 If is unsubstituted, then p is 1, 2, 3, or 4 (for example, p is 1 or 2, or p is 1)). In some embodiments, a compound of formula (Ia) or a salt thereof (wherein R 1If is an unsubstituted phenyl, then p is 1, 2, 3, or 4 (for example, p is 1 or 2, or p is 1) is disclosed. In some embodiments, compounds of formula (Ia) or salts thereof are disclosed, wherein the compound is not N-phenyl-2-(phenylsulfonamide)benzamide.

[0136] In some embodiments, the compound of formula (Ia) is the compound of formula (Ia-1), or a salt thereof. TIFF2023178294000033.tif43170(in the formula: R 1 teeth, The filenames are TIFF2023178294000034.tif22170, and each represents a halogen and a C. 1-4 Alkyl, C 1-4 Optionally substituted with haloalkyl or phenyl; or R 1 teeth, The filename is TIFF2023178294000035.tif20150; R 2 is halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, -OH, -C 1-4 Alkylene-OH,-OC 1-4 Alkyl or -NH2; R 3 (and q are as defined in equation (Ia)) That is the case.

[0137] In some embodiments, a compound of formula (Ia-1) or a salt thereof (wherein R) 1 teeth, A compound of formula (Ia-1) or a salt thereof (wherein R 1 teeth, TIFF2023178294000037.tif22170, and each of these is halogen, C 1-4 Alkyl, C 1-4Compounds of formula (Ia-1) or salts thereof (wherein R 1 teeth, The file TIFF2023178294000038.tif22170 is disclosed.

[0138] In some embodiments, a compound of formula (Ia-1) or a salt thereof (wherein R) 2 C 1-4 Alkyl (such as methyl), halogen (such as Br), or C 1-4 A haloalkyl compound (such as -CF3) is disclosed. In some embodiments, a compound of formula (Ia-1) or a salt thereof (wherein R 2 C 1-4 Alkyl compounds (such as methyl compounds) are disclosed. In some embodiments, compounds of formula (Ia-1) or salts thereof (wherein R 2 is -OH, -C 1-4 Alkylene -OH (-CH2OH, etc.), -OC 1-4 Alkyl compounds (such as -OCH3) or -NH2 compounds are disclosed. In some embodiments, compounds of formula (Ia-1) or salts thereof (wherein R 2 C 1-4 Alkyl (methyl, etc.), halogen (Br, etc.), -OH, C 1-4 Alkylene -OH (-CH2-OH, etc.), -OC 1-4 Alkyl (such as -OCH3) or -NH2, where q is 0) is disclosed. In some embodiments, a compound of formula (Ia-1) or a salt thereof (wherein R 2 C 1-4 Alkyl (such as methyl), where q is 0 is disclosed. In some embodiments, a compound of formula (Ia-1) or a salt thereof (wherein R is 0) is disclosed. 2 C 1-4 Alkyl (such as methyl), R 3 A is disclosed in which (where is -CH3, halogen (such as F), -OCH3, 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)sulfonamide)benzamide; N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(3-cyanothiophen-2-yl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(2-cyanophenyl)-2-((4-methylphenyl)sulfonamide)benzamide; 2-((4-methylphenyl)sulfonamide)-N-phenylbenzamide; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamide)benzamide; N-(benzo[b]thiophen-2-yl)-2-((4-formylphenyl)sulfonamide)benzamide; Methyl 3-(4-(N-(2-(benzo[b]thiophene-2-ylcarbamoyl)phenyl)sulfamoyl)phenyl)propanoate; N-(benzo[b]thiophen-2-yl)-2-((3-methylphenyl)sulfonamide)benzamide; N-(benzo[b]thiophen-2-yl)-2-((4-(3-hydroxypropyl)phenyl)sulfonamide)benzamide; 2-([1,1'-biphenyl]-3-sulfonamide)-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)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-aminophenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-(hydroxymethyl)phenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-(butylcarbamoyl)phenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-bromophenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-(butylamino)phenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-(hexy-1-in-1-yl)phenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-hexylphenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-(3-hydroxyprop-1-in-1-yl)phenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-(3-hydroxypropyl)phenyl)sulfonamide)-N-(p-tolyl)benzamide; N-(p-tolyl)-2-((4-(trifluoromethyl)phenyl)sulfonamide)benzamide; 2-((4-methoxyphenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((4-bromophenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-([1,1'-biphenyl]-4-sulfonamide)-N-(p-tolyl)benzamide; N-(p-tolyl)-2-((3-(trifluoromethyl)phenyl)sulfonamide)benzamide; N-(p-tolyl)-2-((3-(trifluoromethoxy)phenyl)sulfonamide)benzamide; N-(benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamide)benzamide; N-cyclohexyl-2-((4-methylphenyl)sulfonamide)benzamide; 2-((4-methylphenyl)sulfonamide)-N-(naphthalene-2-yl)benzamide; 2-((4-methylphenyl)sulfonamide)-N-(5,6,7,8-tetrahydronaphthalene-2-yl)benzamide; Methyltrans-4-(2-((4-methylphenyl)sulfonamide)benzamide)cyclohexane-1-carboxylate; trans-4-(2-((4-methylphenyl)sulfonamide)benzamide)cyclohexane-1-carboxylic acid; 2-((4-methylphenyl)sulfonamide)-N-(p-tolyl)benzamide; Ethyl 2-(4-(2-((4-methylphenyl)sulfonamide)benzamide)phenyl)acetate; N-(3-isopropylphenyl)-2-((4-methylphenyl)sulfonamide)benzamide; Ethyl 3-(2-((4-methylphenyl)sulfonamide)benzamide)benzoate; 2-(4-(2-((4-methylphenyl)sulfonamide)benzamide)phenyl)acetic acid; 3-(2-((4-methylphenyl)sulfonamide)benzamide)benzoic acid; 2-((4-methylphenyl)sulfonamide)-N-(m-tolyl)benzamide; N-(benzo[b]thiophen-2-yl)-3-((4-methylphenyl)sulfonamide)-2-naphthamide; 2-((4-methylphenyl)sulfonamide)-N-(2-propylphenyl)benzamide; N-(benzo[b]thiophen-2-yl)-5-methyl-2-((4-methylphenyl)sulfonamide)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(benzo[b]thiophen-2-yl)-5-cyano-2-((4-methylphenyl)sulfonamide)benzamide; N-(4-butylphenyl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(benzo[b]thiophen-2-yl)-2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamide)benzamide; N-(4-hexylphenyl)-2-((4-methylphenyl)sulfonamide)benzamide; 2-((4-methylphenyl)sulfonamide)-N-(4-octylphenyl)benzamide; Methyl 6-(4-(2-((4-methylphenyl)sulfonamide)benzamide)phenyl)hexanoate; 6-(4-(2-((4-methylphenyl)sulfonamide)benzamide)phenyl)hexanoic acid; N-(benzo[b]thiophen-2-yl)-2-((4-butylphenyl)sulfonamide)benzamide; N-(4-(6-hydroxyhexyl)phenyl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(benzo[b]thiophen-2-yl)-2-((4-pentylphenyl)sulfonamide)benzamide; N-(benzo[b]thiophen-2-yl)-5-butyl-2-((4-methylphenyl)sulfonamide)benzamide; N-(4-(4-hydroxybutyl)phenyl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(4-(4-bromobutyl)phenyl)-2-((4-methylphenyl)sulfonamide)benzamide; 5-Methoxy-2-((4-methylphenyl)sulfonamide)-N-(p-tolyl)benzamide; N-(benzo[b]thiophen-2-yl)-5-methoxy-2-((4-methylphenyl)sulfonamide)benzamide; 4-Methoxy-2-((4-methylphenyl)sulfonamide)-N-(p-tolyl)benzamide; N-(benzo[b]thiophen-2-yl)-4-methoxy-2-((4-methylphenyl)sulfonamide)benzamide; 2-((3-methoxyphenyl)sulfonamide)-N-(p-tolyl)benzamide; N-(benzo[b]thiophen-2-yl)-2-((3-methoxyphenyl)sulfonamide)benzamide; 5-Hydroxy-2-((4-methylphenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-hydroxyphenyl)sulfonamide)-N-(p-tolyl)benzamide; 2-((3-butoxyphenyl)sulfonamide)-N-(p-tolyl)benzamide; N-(2-bromophenyl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(3-bromophenyl)-2-((4-methylphenyl)sulfonamide)benzamide; N-([1,1'-biphenyl]-4-yl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(2-methoxyphenyl)-2-((4-methylphenyl)sulfonamide)benzamide; N-([1,1'-biphenyl]-3-yl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(3-methoxyphenyl)-2-((4-methylphenyl)sulfonamide)benzamide; N-(4-methoxyphenyl)-2-((4-methylphenyl)sulfonamide)benzamide; 2-((N-ethyl-4-methylphenyl)sulfonamide)-N-(4-methoxyphenyl)benzamide; N-(benzo[b]thiophen-2-yl)-4-fluoro-2-((4-methylphenyl)sulfonamide)benzamide; N-(benzo[b]thiophen-2-yl)-5-fluoro-2-((4-methylphenyl)sulfonamide)benzamide; N-benzyl-2-((4-methylphenyl)sulfonamide)benzamide; N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamide)benzamide; and N-(benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamide)-5-(trifluoromethyl)benzamide, or a salt thereof is included.

[0140] (1) Salt form The thienopyrrole compounds described herein may be in salt form. 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 in other respects, the salt is equivalent to the parent form of the compound for the purposes of this disclosure.

[0141] For example, when a compound is anionic or has a functional group that can become anionic (for example, -COOH is -COO - (This can happen), in which case the salt can be formed with a suitable cation. Examples of suitable inorganic cations include Na + and K + Alkali metal ions such as Ca 2+ and Mg 2+ This includes, but is not limited to, alkaline earth cations such as NH4, as well as other cations. Examples of suitable organic cations include ammonium ions (i.e., NH4) + ) and substituted ammonium ions (e.g., NH3R1 + NH2R2 + NHR3 + NR4 + Examples of 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, as well as those derived from amino acids such as lysine and arginine.

[0142] If a compound is cationic or has a functional group that can become cationic (for example, -NH2 is -NH3),+ (This may occur), in which case the salt can be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrite, 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-acetyloxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphor sulfonic 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, mucinic 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. Suitable polymeric organic anions include, but are not limited to, the following polymeric acids: tannic acid and those derived from carboxymethylcellulose.

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

[0145] (2) Isomers A given thienopyrrole compound may exist in one or more specific geometric, optical, enantiomer, diastereomer, epimer, atropic, stereoisomer, tautomer, 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 1 forms; (+) and (-) forms; keto, enol, and enolate forms; syn and anti forms; syncrinal and anticrinal forms; a and β forms; axial and equatorial forms; boat, chair, twist, envelope, and half-chair forms; and combinations thereof, which are collectively referred to below as “isomers” (or “isomer forms”).

[0146] In some embodiments, the compounds described herein may be enantiomerically enriched isomers of the stereoisomers described herein. For example, a compound may have an enantiomer 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%. As used herein, an enantiomer refers to either of a pair of chemical compounds whose molecular structures are mirror images of each other.

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

[0148] In some embodiments, the compositions described herein include preparations of the compounds disclosed herein that are concentrated with respect to a selected stereochemistry at a selected stereocenter, for example, one or more structures having R or S. Exemplary R / S configurations may be provided in the examples described herein.

[0149] When used herein, “concentrated preparation” refers to a preparation that is concentrated with respect to a selected stereochemistry of one, two, three or more selected stereocenters within the compound of interest. Exemplary selected stereocenters and their exemplary stereochemistrys may be selected herein from, for example, those provided in the examples described herein. Concentrated 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%. Concentrated refers to the level of the molecule(s) of interest and does not imply any limitations on the process unless otherwise specified.

[0150] The compound can be prepared in racemic form or as individual enantiomers or diastereomers by either stereospecific synthesis or resolution. The compound can be divided into enantiomers or diastereomers of its components by standard techniques, such as the formation of stereoisomer pairs by salt formation using optically active bases, followed by fractional crystallization and regeneration of free acids. The compound can also be divided by the formation of stereoisomer esters or amides, followed by chromatographic separation and removal of chiral auxiliaries. Alternatively, the compound can be divided using a chiral HPLC column. Enantiomers can also be obtained from the kinetic resolution of the racemic compound of the corresponding ester using lipase enzymes.

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

[0152] The above exclusions do not apply to tautomer forms, such as the following tautomer pairs: keto / enol, imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, N-nitroso / hydroxyazo, and nitro / acy-nitro, which are keto-, enol-, and enolate-forms.

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

[0154] 3. Bioluminescent complex derived from Oplophorus luciferase The disclosed compounds may be used to inhibit the bioluminescent complex derived from Oplophorus luciferase. The disclosed compounds may inhibit the luciferase activity of the bioluminescent complex derived from Oplophorus luciferase. Oplophorus luciferase may be wild-type Oplophorus luciferase or a variant of Oplophorus luciferase. Oplophorus luciferase may be a variant of the luciferase of Sequence ID No. 7. Variants of Oplophorus luciferase are described in U.S. Patents No. 8,557,970 and No. 8,669,103, each of which is incorporated herein by reference in whole.

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

[0156] 4. Coelenterazine substrates The compounds disclosed in this invention can be used to inhibit luciferase activity by competing with or interfering with coelenterazine or coelenterazine derivative substrates because they bind to luciferase. Coelenterazine substrates are a class of reporter molecules that emit light when acted upon by luciferase and other bioluminescent proteins. Examples of coelenterazine substrates include coelenterazine; disclosed in WO2003 / 040100, U.S. Patent Publication 2008 / 0248511, and U.S. Patent Publication US2012 / 0117667, as well as coelenterazine derivatives and / or analogs, e.g., 2-furanylmethyl-deoxy-coelenterazine (flimazine), coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine- Coelenterazine substrates include, but are not limited to, cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl-coelenterazine; pro-coelenterazine (i.e., compounds that are not substrates of non-luminescent enzymes that convert the compound into a substrate for luciferase), quinoline-masked coelenterazine, and others. Further examples of coelenterazine substrates are described, for example, in U.S. Publication 2012 / 0107849, U.S. Publication 2013 / 0130289, U.S. Patent Application 14 / 608,910, and U.S. Patent Application 14 / 609,372, each of which is incorporated herein by reference.

[0157] 5. Method for inhibiting the activity of the bioluminescent complex derived from Oplophorus luciferase. The disclosed compounds can be used in methods to inhibit the Oplophorus luciferase-derived bioluminescent complex, for example, to inhibit the luciferase activity of the bioluminescent complex. The method may involve contacting the disclosed compounds with cells expressing or containing the Oplophorus luciferase-derived bioluminescent complex or with non-luminescent peptides and / or polypeptides of the Oplophorus luciferase-derived bioluminescent complex, thereby selectively inhibiting the Oplophorus luciferase-derived bioluminescent complex. The method may involve contacting the disclosed compounds with non-luminescent peptides and / or polypeptides of the Oplophorus luciferase-derived bioluminescent complex, thereby inhibiting the Oplophorus luciferase-derived bioluminescent complex when the complex assembles. The disclosed compounds can be used to selectively inhibit signals from the Oplophorus luciferase-derived bioluminescent complex in assays used to detect the presence or activity of enzymes utilizing the Oplophorus luciferase-derived bioluminescent complex. For example, the disclosed compounds may 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 target protein (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 redox conditions. While the coelenterazine substrate acts as a substrate for the Oplophorus luciferase-derived bioluminescent complex, the disclosed compounds may act to inhibit the luciferase-derived bioluminescent complex to selectively suppress the luminescence signal in embodiments, in which such suppression may be desired, for example, in applications involving the temporal multiplexing of multiple bioluminescent systems or in some plate-based luminescence assays. For example, the disclosed compounds may be used to inhibit the activity of intracellular and / or extracellular Oplophorus luciferase-derived bioluminescent complexes.

[0158] (1) Protein complementation assay Accordingly, the disclosed compounds may be used to inhibit such bioluminescent complexes when Oplophorus luciferase-derived bioluminescent complexes are used in other methods for detecting ligand-protein and protein-protein interactions or proximity or co-localization, such as protein complementation assays (PCA) or enzyme fragment complementation (EFC) assays. PCA and EFC assays provide means for detecting interactions between two interacting elements, e.g., biomolecules, polypeptides. PCA utilizes two fragments of the same protein, e.g., an enzyme, that are in close proximity to each other via binding interactions with their fusion partners, e.g., target polypeptides, and fused to a functionally active protein, e.g., an enzyme, which generates light only when reassembled into a target polypeptide. For example, NANOBIT® technology (Promega Corporation) may be used to detect molecular proximity due to the reassembly of a luminescent enzyme via binding interactions of enzyme units. The NanoBiT® system may comprise 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 may comprise NanoBiT® LgBiT non-luminescent polypeptide (NLpoly) and NanoBiT® SmBiT non-luminescent peptide (NLpep). In some embodiments, the two units may comprise NanoBiT® LgBiT non-luminescent polypeptide (NLpoly) and NanoBiT® HiBiT non-luminescent peptide (NLpep). Bioluminescent complexes derived from Oplophorus luciferase are described in U.S. Patents 9,797,889 and 9,797,890, each of which is incorporated herein by reference in whole.

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

[0160] (2) Molecular detection assay Accordingly, the disclosed compounds may be used to inhibit bioluminescent complexes derived from Oplophorus luciferase when such complexes are used in other methods for detecting the target molecule. For example, NANOBIT® technology (Promega Corporation) may be used to detect the target molecule. The NanoBiT® system may comprise two or more nonluminescent peptide and / or polypeptide units. One or more of the nonluminescent peptide and / or polypeptide units may be fused to the target molecule. The two units may comprise NanoBiT® LgBiT nonluminescent polypeptide (NLpoly) and NanoBiT® HiBiT nonluminescent peptide (NLpep). In some embodiments, NanoBiT® HiBiT NLpep may be fused to the target molecule. A sample containing NanoBiT® HiBiT NLpep fused to the target molecule may be contacted with 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, and inhibited with compounds disclosed herein. In some embodiments, NanoBiT® LgBiT NLpoly can be fused to a target molecule. A sample containing NanoBiT® LgBiT NLpoly fused to a target molecule 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 the compounds described herein modified to be cell-impermeable. In such embodiments, the disclosed compounds and methods may be used to construct initial luminescence in a high-throughput screening operation assay format and then selectively inhibit any Oplophorus luciferase-derived bioluminescent complex that may be expelled from cells, thereby selectively inhibiting luminescence that may occur outside the cell. Such methods may provide more selective intracellular signaling.

[0162] (4) Use of cell-permeable compounds In certain embodiments, the methods disclosed herein involve contacting a sample (e.g., cells) with 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 the Oplophorus luciferase-derived bioluminescent complex within them. Such methods may be advantageous in multiplexed assays involving the use of two or more luciferases, which may allow for the inhibition of luminescence from the Oplophorus luciferase-derived bioluminescent complex so that luminescence from another luciferase within the cell can be selectively observed.

[0163] (5) Use with a transfer reporter The disclosed compounds may be used in conjunction with a gene transcription reporter system. In a given embodiment, a method is provided for measuring the activity of a promoter in a sample, wherein the promoter is functionally linked to a gene encoding a non-luminescent unit of the Oplophorus luciferase-derived bioluminescent complex, e.g., a non-luminescent polypeptide. The method comprises (a) contacting a sample expressing a non-luminescent polypeptide of the Oplophorus luciferase-derived bioluminescent complex fused to a promoter with a coelenterazine substrate and a non-luminescent peptide of the Oplophorus luciferase-derived bioluminescent complex that can form a bioluminescent complex with the non-luminescent polypeptide of the Oplophorus luciferase-derived bioluminescent complex, and (b) determining the activity of the promoter by measuring the luminescence of the sample. The method may further include contacting the sample with the compounds described herein to selectively inhibit the luminescence produced from the bioluminescent complex. The promoter may be functionally linked to a gene via translation or transcriptional fusion. The target biological pathway can be investigated, for example, by treating cells containing a promoter functionally linked to a non-luminescent unit of the Oplophorus luciferase-derived bioluminescent complex, such as a gene encoding a non-luminescent polypeptide, with a pathway inducer. The activity of this promoter is then measured and monitored, and any correlations between promoter activity and the target pathway can be studied, and kinetic measurements related to gene expression (e.g., inducibility, repression, and activation) can be obtained. The compounds described herein can be used to selectively inhibit luminescence from the Oplophorus luciferase-derived bioluminescent complex.

[0164] (6) Multiplexing The disclosed compounds can be used to inhibit the Oplophorus luciferase-derived bioluminescent complex when applied to temporal multiplexing with other luciferases and assays. In some embodiments, the Oplophorus luciferase-derived bioluminescent complex can be multiplexed with another enzyme (e.g., luciferase) that emits light at different wavelengths, such as green firefly luciferase, e.g., Photinus pyralis (e.g., Luc2; Promega Corp.) or red click mushroom luciferase (CHROMA-LUC® luciferase; Promega Corp.). For example, when the Oplophorus luciferase-derived bioluminescent complex is used as a functional reporter, the green firefly luciferase or red CHROMA-LUC® luciferase can be used to control nonspecific effects on gene regulation or to normalize transfection efficiency. In some embodiments, the luminescence (approximately 460 nm) and red Chroma-LUC (approximately 610 nm) produced from the Oplophorus luciferase-derived bioluminescent complex can be readily degraded using a luminometer with a wavelength-discriminating filter that allows measurement of both signals from the same sample. In such embodiments, the compounds described herein may be used to selectively inhibit the Oplophorus luciferase-derived bioluminescent complex so that signals from other luciferases can be selectively observed.

[0165] In another example, the Oplophorus luciferase-derived bioluminescent complex may be used as a transcription reporter and paired with luciferases that emit light at different wavelengths, contained in an assay reagent. In yet another example, the Oplophorus luciferase-derived bioluminescent complex may be used with one or more additional luciferases, and the luminescence of each luciferase and bioluminescent complex may be measured separately by the use of selective enzyme inhibitors. For example, the luminescence of the Oplophorus luciferase-derived bioluminescent complex may be measured by the addition of a suitable substrate and buffer, followed by the measurement of the second luciferase by the subsequent addition of a suitable substrate and buffer, as well as 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 may comprise two or more non-luminescent peptides and / or polypeptide units. One or more of the non-luminescent peptides and / or polypeptide units can be fused to a target molecule. In some embodiments, NanoBiT® LgBiT non-luminescent polypeptides and / or NanoBiT® HiBiT non-luminescent peptides can be fused to a target molecule. For example, the NanoBiT® LgBiT non-luminescent polypeptide can be added to a detection reagent containing flimazine as a means for detecting and quantifying a target protein fused to the NanoBiT® HiBiT non-luminescent peptide. As another example, NanoBiT® HiBiT non-luminescent peptides may be added to a detection reagent containing flimazine as a means for detecting and quantifying a target protein fused to NanoBiT® LgBiT non-luminescent polypeptides. The disclosed compounds may 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 by which an Oplophorus luciferase-derived bioluminescent complex is used to detect ligand-protein and / or protein-protein interactions. In various embodiments, the Oplophorus luciferase-derived bioluminescent complex can be used to transfer energy to an energy receptor. One such method is bioluminescent resonance energy transfer (BRET). In BRET, energy transfer from a bioluminescent donor to a fluorescent receptor 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, the Oplophorus luciferase-derived bioluminescent complex used in BRET analysis may be used to determine whether two molecules can bind to each other or co-localize within a cell. For example, the Oplophorus luciferase-derived bioluminescent complex may be used as a bioluminescent donor molecule, in which case one of the non-luminescent units combines with the target molecule or protein to produce a first fusion protein. In some embodiments, a non-luminescent peptide may combine with the target molecule or protein to produce a first fusion protein. In other embodiments, a non-luminescent polypeptide may combine with the target molecule or protein to produce a first fusion protein. In various embodiments, a first fusion protein containing the non-luminescent unit of the Oplophorus luciferase-derived bioluminescent complex (e.g., a non-luminescent peptide or non-luminescent polypeptide) may be used in BRET analysis to detect protein / protein interactions in systems including, but not limited to, cell lysates, intact cells, and living animals. In various embodiments, HALOTAG may be used as a fluorescent receptor molecule. In some embodiments, HALOTAG can be fused to a complementary non-luminescent unit (e.g., a non-luminescent polypeptide or non-luminescent peptide) of a second target protein or bioluminescent complex. For example, a non-luminescent polypeptide from an Oplophorus luciferase-derived bioluminescent complex can be fused to HALOTAG, expressed in cells or animals, and labeled with a fluorescent HALOTAG® ligand such as HALOTAG® TMR ligand. The fusion can then be excited to fluoresce in the presence of a cell-permeable luminescent enzyme substrate. As another example, a non-luminescent peptide from an Oplophorus luciferase-derived bioluminescent complex can be fused to HaloTag, expressed in cells or animals, and labeled with a fluorescent HaloTag® ligand such as HaloTag® TMR ligand. The fusion can then be excited to fluoresce in the presence of a cell-permeable luminescent enzyme substrate.In some embodiments, HALOTAG may be fused to a second target protein and a complementary non-luminescent unit (e.g., a non-luminescent polypeptide or non-luminescent peptide) of the Oplophorus luciferase-derived bioluminescent complex added via a detection reagent. In some embodiments, BRET may be carried out using the Oplophorus luciferase-derived bioluminescent complex in combination with fluorescent proteins, including but not limited to, green fluorescent protein (GFP) or red fluorescent protein (RFP) or fluorescent labels containing fluorescein, rhodamine green, Oregon green, or Alexa488, to give some non-limiting examples.

[0169] In some embodiments, quenching the signal from the 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 may be used to inhibit intracellular BRET. In certain embodiments, cell-impermeable compounds may be used to inhibit extracellular BRET. In certain embodiments, cell-impermeable compounds may be used in a target engagement model.

[0171] 6. Sample The disclosed compounds may be used in conjunction with samples containing biological components. The samples may include cells. The samples may contain heterogeneous mixtures of components (including intact cells, cell extracts, cell lysates, bacteria, viruses, organelles, exosomes, and mixtures thereof) or single components or homogeneous groups of components (e.g., natural or synthetic amino acids, nucleic acids or carbohydrate polymers, or lipid membrane complexes). The disclosed compounds are generally non-toxic to viable cells and other biological components within the range of concentrations used.

[0172] Samples may include animals (e.g., vertebrates), plants, fungi, physiological fluids (e.g., blood, plasma, urine, mucus secretions, etc.), cells, cell lysates, cell supernatants, or purified cell fractions (e.g., intracellular fractions). In certain embodiments, the sample may be cells. In some embodiments, the sample may be viable cells. Cells may be eukaryotic cells, such as yeast, bird, plant, insect, or mammalian cells, including but not limited to human, ape, mouse, dog, cattle, horse, cat, sheep, goat, or pig cells, or prokaryotic cells, or cells from two or more different organisms, or their cell lysates or supernatants. Cells may be non-recombinant cells, or recombinant cells transiently transfected with recombinant DNA, and / or recombinant cells whose genomes are stably enlarged with recombinant DNA, or recombinant cells whose genomes are modified to disrupt genes, for example, by disrupting promoters, introns, or open reading frames, or by replacing one DNA fragment with another. Recombinant DNA or substituted DNA fragments may encode molecules detected by the method of the present invention, regions that alter the level or activity of the detected molecules, and / or gene products unrelated to the molecules or regions that alter the level or activity of the molecules. Cells may or may not express luciferase. Cells may be genetically modified by recombinant technology.

[0173] 7. Kit A kit for determining the presence or activity of an Oplophorus luciferase-derived bioluminescent complex is disclosed. The kit may comprise one or more of the following: a compound or composition of the present invention capable of inhibiting an Oplophorus luciferase-derived bioluminescent complex; a coelenterazine or coelenterazine derivative substrate; and an Oplophorus luciferase-derived bioluminescent complex, e.g., a polynucleotide for the expression of non-luminescent peptides and / or polypeptides of the Oplophorus luciferase-derived bioluminescent complex; instructions for performing a luminescence assay; and a reaction buffer(s). The reaction buffer(s) may be present in individual formulations for non-luciferase enzyme reactions and luminescent enzyme reactions, or in a single formulation for a single-step assay. The reaction buffer(s) may contain non-luminescent units of the Oplophorus luciferase-derived bioluminescent complex, e.g., non-luminescent peptides. The kit may also comprise other inhibitors, activators and / or enhancers for the non-luciferase enzyme(s). The kit may also comprise positive and / or negative controls for the assay. [Examples]

[0174] 8. Examples Example 1 Compound Synthesis General procedure A: Formation of sulfonamide bonds.

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

[0176] General procedure B: Saponification.

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

[0178] General procedure C1: Formation of amide bonds via acid chlorides.

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

[0180] General procedure C2: Formation of amide bonds via carboxylates.

[0181] A solution of a carboxylic acid derivative (1 equivalent) in DMF was mixed with amine (1.2 equivalents), HBTU (2 equivalents), and diisopropylethylamine (3 equivalents). The reaction mixture was heated at 60-85°C for 2-18 hours. 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] A solution of a nitro derivative (1 equivalent) in ethyl acetate was mixed with tin chloride hydrate (3-5 equivalents). The mixture was heated under reflux for 24-48 hours. Saturated potassium carbonate was added, and the mixture was stirred at room temperature for 1 hour. The layers were separated, the organic layer was washed with water, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography.

[0184] The exemplary compounds can be prepared according to the typical synthetic methods of Scheme 1 or Scheme 2. Scheme 1. Representative synthesis of amide analogs via acid chlorides. TIFF2023178294000039.tif53128

[0185] Example 2 N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamide)benzamide (JRW-0998) TIFF2023178294000040.tif2659 Step 1. N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-nitrobenzamide (JRW-0994) TIFF2023178294000041.tif2744

[0186] Following the 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 obtain 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) TIFF2023178294000042.tif2543

[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 obtain 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)sulfonamide)benzamide (JRW-0998) TIFF2023178294000043.tif2661

[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 obtain 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)sulfonamide)benzamide (JRW-1004) TIFF2023178294000044.tif2661 Step 1. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-nitrobenzamide (JRW-1000) TIFF2023178294000045.tif2847

[0192] Following the 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 obtain 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) TIFF2023178294000046.tif2847

[0194] Ammonium chloride (474 ​​mg, 8.8 mmol) and iron scrap (100 mg, 1.8 mmol) were added 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 mL / 2 mL). The suspension was heated at 60 °C for 18 hours. The reaction product was filtered, the filtrate was added to Celite, concentrated, and purified by silica gel chromatography to obtain the desired product (57 mg, 21%) as a light 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)sulfonamide)benzamide (JRW-1004) TIFF2023178294000047.tif3268

[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 obtain 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)sulfonamide)benzamide (JRW-1006) TIFF2023178294000048.tif2654 Process 1. N-(3-cyanothiophene-2-yl)-2-nitrobenzamide (JRW-1001). TIFF2023178294000049.tif2134

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

[0199] Step 2.2-Amino-N-(3-cyanothiophene-2-yl)benzamide (JRW-1003). TIFF2023178294000050.tif2134

[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 obtain the desired product (90 mg, 53%) as a light brown solid. ESI MS m / z 244[M+H] + .

[0201] Step 3. N-(3-cyanothiophen-2-yl)-2-((4-methylphenyl)sulfonamide)benzamide (JRW-1006). TIFF2023178294000051.tif2654

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

[0203] Example 5 N-(2-cyanophenyl)-2-((4-methylphenyl)sulfonamide)benzamide (JRW-1008) TIFF2023178294000052.tif2656 Process 1. N-(2-cyanophenyl)-2-nitrobenzamide (JRW-1005). TIFF2023178294000053.tif2639

[0204] Following the general procedure C1, 2-nitrobenzoyl chloride (189 mg, 1.0 mmol) was reacted with 2-aminobenzonitrile (120 mg, 1.0 mmol) to obtain 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). TIFF2023178294000054.tif2540

[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 obtain 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)sulfonamide)benzamide (JRW-1008). TIFF2023178294000055.tif2656

[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 obtain the desired product (91 mg, 68%) as a white solid. ESI MS m / z 392[M+H]+.

[0209] Example 6 2-((4-methylphenyl)sulfonamide)-N-phenylbenzamide (JRW-1011) TIFF2023178294000056.tif2656 Process 1,2-Nitro-N-phenylbenzamide (JRW-1009) TIFF2023178294000057.tif2639

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

[0211] Step 2.2-Amino-N-phenylbenzamide (JRW-1010). TIFF2023178294000058.tif2538

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

[0213] Step 3.2 - ((4-methylphenyl)sulfonamide)-N-phenylbenzamide (JRW-1011). TIFF2023178294000059.tif3063

[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 obtain 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-(phenylsulfonamide)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 obtain the desired product (17 mg, 26%) as a light brown solid. ESI MS m / z 424[M+H] + .

[0216] Example 8 N-(benzo[b]thiophen-2-yl)-2-((4-formylphenyl)sulfonamide)benzamide (HL-0038) TIFF2023178294000061.tif2664 Step 1. N-(benzo[b]thiophen-2-yl)-2-nitrobenzamide (HL-0028). TIFF2023178294000062.tif2847

[0217] Following the general procedure C1, 2-nitrobenzoyl chloride (373 mg, 2.0 mmol) was reacted with benzo[b]thiophene-2-amine (300 mg, 2.0 mmol) to obtain 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). TIFF2023178294000063.tif2747

[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 obtain 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)sulfonamide)benzamide (HL-0038). TIFF2023178294000064.tif2662

[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 obtain 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]thiophene-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 obtain the desired product (12 mg, 63%) as a light brown solid. ESI MS m / z 495[M+H] + .

[0223] Example 10 N-(benzo[b]thiophen-2-yl)-2-((3-methylphenyl)sulfonamide)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 obtain 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)sulfonamide)benzamide (HL-0062) Lithium borohydride (4 mg, 0.19 mmol) was added to a solution of methyl 3-(4-(N-(2-(benzo[b]thiophene-2-ylcarbamoyl)phenyl)sulfamoyl)phenyl)propanoate (31 mg, 0.063 mmol) in DMF (2 mL) at 40°C. The reaction mixture was warmed to room temperature and stirred overnight. The mixture was diluted with ethyl acetate and water, and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain 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-sulfonamide)-N-(p-tolyl)benzamide (JRW-1248) TIFF2023178294000068.tif2969 Process 1,2-Nitro-N-(p-Tolyl)benzamide (JRW-1243). TIFF2023178294000069.tif2143

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

[0227] Step 2.2-amino-N-(p-tolyl)benzamide (JRW-1247). TIFF2023178294000070.tif2549

[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 obtain 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-sulfonamide)-N-(p-tolyl)benzamide (JRW-1248). TIFF2023178294000071.tif2869

[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 obtain 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 obtain 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 obtain the desired product (580 mg, 92%) as a white solid. ESI MS m / z 411[M+H] + .

[0233] Example 15 2-((3-acetamidophenyl)sulfonamide)-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 obtain the desired product (690 mg, 92%) as a white foam. ESI MS m / z 424[M+H] + .

[0234] Example 16 2-((3-aminophenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1255) TIFF2023178294000075.tif2970 A solution of 2-((3-acetamidophenyl)sulfonamide)-N-(p-tolyl)benzamide (690 mg, 1.6 mmol) in methanol (20 mL) was mixed with sodium hydroxide (5 mL, 2 M). The mixture was heated at 85 °C for 5 hours. The reaction mixture 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 obtain the desired product (62 mg, 10%) as a white solid. ESI MS m / z 382[M+H]+.

[0235] Example 17 2-((3-(hydroxymethyl)phenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1261) TIFF2023178294000076.tif2669 A mixture of 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoic acid (54 mg, 0.13 mmol) in DCM (5 mL) was mixed with HOBt (20 mg, 0.13 mmol) and EDC (25 mg, 0.13 mmol). The mixture was stirred at room temperature for 30 minutes. The solution was concentrated to form a white foam, and the solid was then 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 mixture was stirred at room temperature for 18 hours, quenched with HCl, and diluted with ethyl acetate and water. The organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain the desired product (29 mg, 55%) as a white solid. ESI MS m / z 397[M+H] + .

[0236] Example 18 2-((3-(butylcarbamoyl)phenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1263) Following general procedure C2 (TIFF2023178294000077.tif3092), 3-(N-(2-(p-tolylcarbamoyl)phenyl)sulfamoyl)benzoic acid (50 mg, 0.12 mmol) was reacted with butylamine (17 mg, 0.21 mmol) to obtain the desired product (43 mg, 76%) as a white solid. ESI MS m / z 466[M+H] + .

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

[0238] Example 20 2-((3-(butylamino)phenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1269) Butyraldehyde (14 mg, 0.20 mmol) was added to a solution of 2-((3-aminophenyl)sulfonamide)-N-(p-tolyl)benzamide (50 mg, 0.13 mmol) in THF (5 mL). The mixture was stirred at room temperature for 30 minutes, then sodium triacetoxyborohydride (55 mg, 0.26 mmol) was added. The reaction mixture 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 obtain the desired product (27 mg, 47%) as a white solid. ESI MS m / z 438[M+H] + .

[0239] Example 21 2-((3-(hexy-1-in-1-yl)phenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1270) To a solution of 2-((3-bromophenyl)sulfonamide)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in TIFF2023178294000080.tif2687DMF (5 mL), 1-hexine (36 mg, 0.45 mmol), triethylamine (68 mg, 0.67 mmol), triphenylphosphine (6 mg, 0.022 mmol), and PdCl2(PPh3)2 (8 mg, 0.011 mmol) were added. The suspension was purged with nitrogen. Copper iodide (4 mg, 0.022 mmol) was added, and the reaction mixture was stirred at 60°C for 18 hours. The reaction mixture was diluted with ethyl acetate and water, extracted with ethyl acetate, the organic layers were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain the desired product (50 mg, 50%) as an orange oil. ESI MS m / z 447[M+H] + .

[0240] Example 22 2-((3-hexylphenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1271) TIFF2023178294000081.tif2990 A solution of 2-((3-(hexy-1-in-1-yl)phenyl)sulfonamide)-N-(p-tolyl)benzamide (40 mg, 0.090 mmol) in ethanol (10 mL) was mixed with palladium-supported carbon (5 mg). The reaction mixture was stirred with hydrogen at 40 psi at room temperature for 2 hours. The mixture was filtered through Celite, concentrated, and purified by silica gel chromatography to obtain 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-in-1-yl)phenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1272) To a solution of 2-((3-bromophenyl)sulfonamide)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in TIFF2023178294000082.tif2981DMF (5 mL), propargyl alcohol (25 mg, 0.45 mmol), triethylamine (68 mg, 0.67 mmol), triphenylphosphine (6 mg, 0.022 mmol), and PdCl2(PPh3)2 (8 mg, 0.011 mmol) were added. The suspension was purged with nitrogen. Copper iodide (4 mg, 0.022 mmol) was added, and the reaction mixture was stirred at 85°C for 48 hours. The reaction mixture was diluted with ethyl acetate and water, extracted with ethyl acetate, combined with the organic layers, dried over sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain the desired product (30 mg, 31%) as a pale yellow gum-like substance. ESI MS m / z 421[M+H] + .

[0242] Example 24 2-((3-(3-hydroxypropyl)phenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1275) TIFF2023178294000083.tif2981 A solution of 2-((3-(3-hydroxyprop-1-in-1-yl)phenyl)sulfonamide)-N-(p-tolyl)benzamide (23 mg, 0.055 mmol) in ethanol (10 mL) was mixed with palladium-supported carbon (5 mg). The reaction mixture was stirred with hydrogen at 40 psi at room temperature for 1 hour. The mixture was filtered through Celite, concentrated, and purified by silica gel chromatography to obtain 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)sulfonamide)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 obtain the desired product (86 mg, 89%) as a white solid. ESI MS m / z 434[M+H] + .

[0244] Example 26 2-((4-methoxyphenyl)sulfonamide)-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 obtain the desired product (82 mg, 94%) as a white solid. ESI MS m / z 397[M+H] + .

[0245] Example 27 2-((4-bromophenyl)sulfonamide)-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 obtain 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-sulfonamide)-N-(p-tolyl)benzamide (JRW-1293) TIFF2023178294000087.tif2669 A solution of 2-((4-bromophenyl)sulfonamide)-N-(p-tolyl)benzamide (100 mg, 0.22 mmol) in dioxane (5 mL) was mixed with 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 mixture was heated at 80°C for 2 hours. 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 obtain 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)sulfonamide)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 obtain 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)sulfonamide)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 obtain 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. TIFF2023178294000090.tif60153 Example 31 N-(benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamide)benzamide (HL-0005) TIFF2023178294000091.tif2661 Step 1. Methyl 2-((4-methylphenyl)sulfonamide)benzoate (HL-0001). TIFF2023178294000092.tif2651

[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 obtain the desired product (1.25 g, 62%) as a white solid. ESI MS m / z 306[M+H] + .

[0251] Step 2.2-((4-methylphenyl)sulfonamide)benzoic acid (HL-0003). TIFF2023178294000093.tif2646

[0252] Following general procedure B, methyl 2-((4-methylphenyl)sulfonamide)benzoate (1.2 g, 4.1 mmol) was reacted with LiOH (294 mg, 12.3 mmol) to obtain 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)sulfonamide)benzamide (HL-0005). TIFF2023178294000094.tif2661

[0254] Following general procedure C2, 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with benzo[b]thiophene-2-amine (31 mg, 0.21 mmol) to obtain 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)sulfonamide)benzamide (HL-0006) Following the general procedure C2 (TIFF2023178294000095.tif2656), 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with cyclohexylamine (20 mg, 0.21 mmol) to obtain the desired product (14 mg, 22%) as a pale yellow solid. ESI MS m / z 373[M+H] + .

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

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

[0258] Example 35 Methyltrans-4-(2-((4-methylphenyl)sulfonamide)benzamide)cyclohexane-1-carboxylate (HL-0009) Following general procedure C2 (TIFF2023178294000098.tif2672), 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with methyl trans-4-aminocyclohexane-1-carboxylate HCl (40 mg, 0.21 mmol) to obtain the desired product (41 mg, 55%) as a solid. ESI MS m / z 431[M+H] + .

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

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

[0261] Example 38 Ethyl 2-(4-(2-((4-methylphenyl)sulfonamide)benzamide)phenyl)acetate (HL-0023) Following general procedure C2, 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with ethyl 2-(4-aminophenyl)acetate (37 mg, 0.21 mmol) to obtain 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)sulfonamide)benzamide (HL-0025) Following general procedure C2, 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with 3-isopropylaniline (28 mg, 0.21 mmol) to obtain 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)sulfonamide)benzamide)benzoate (HL-0026) Following general procedure C2, 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with ethyl 3-aminobenzoate (34 mg, 0.21 mmol) to obtain the desired product (50 mg, 67%) as a solid. ESI MS m / z 439[M+H] + .

[0264] Example 41 2-(4-(2-((4-methylphenyl)sulfonamide)benzamide)phenyl)acetic acid (HL-0030) Following general procedure B (TIFF2023178294000104.tif2672), ethyl 2-(4-(2-((4-methylphenyl)sulfonamide)benzamide)phenyl)acetate (23 mg, 0.052 mmol) was reacted with LiOH (3 mg, 0.11 mmol) to obtain the desired product (17 mg, 79%) as a brown solid. ESI MS m / z 425[M+H] + .

[0265] Example 42 3-(2-((4-methylphenyl)sulfonamide)benzamide)benzoic acid (HL-0031) Following general procedure B (TIFF2023178294000105.tif2974), ethyl 3-(2-((4-methylphenyl)sulfonamide)benzamide)benzoate (23 mg, 0.054 mmol) was reacted with LiOH (3 mg, 0.11 mmol) to obtain the desired product (12 mg, 54%) as a pale yellow solid. ESI MS m / z 411[M+H] + .

[0266] Example 43 2-((4-methylphenyl)sulfonamide)-N-(m-tolyl)benzamide (HL-0044) Following general procedure C2 (TIFF2023178294000106.tif2968), 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with m-toluidine (22 mg, 0.21 mmol) to obtain 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)sulfonamide)-2-naphthamide (HL-0057) TIFF2023178294000107.tif3765 Step 1. Methyl 3-((4-methylphenyl)sulfonamide)-2-naphthoate (HL-0049). TIFF2023178294000108.tif3657

[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 obtain the desired product (340 mg, 64%) as a solid. ESI MS m / z 356[M+H]+.

[0269] Step 2.3 - ((4-methylphenyl)sulfonamide)-2-naphthoic acid (HL-0052). TIFF2023178294000109.tif3346

[0270] Following general procedure B, methyl 3-((4-methylphenyl)sulfonamide)-2-naphthoate (340 mg, 0.96 mmol) was reacted with LiOH (69 mg, 2.9 mmol) to obtain 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)sulfonamide)-2-naphthamide (HL-0057). TIFF2023178294000110.tif3766

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

[0273] Example 45 2-((4-methylphenyl)sulfonamide)-N-(2-propylphenyl)benzamide (HL-0059) Following general procedure C2 (TIFF2023178294000111.tif3864), 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with 2-propylaniline (23 mg, 0.17 mmol) to obtain 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)sulfonamide)benzamide (HL-0061) TIFF2023178294000112.tif3867 Step 1. Ethyl 5-methyl-2-((4-methylphenyl)sulfonamide)benzoate (HL-0053). TIFF2023178294000113.tif3761

[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 obtain the desired product (404 mg, 72%) as a solid. ESI MS m / z 334[M+H] + .

[0276] Process 2.5-Methyl-2-((4-methylphenyl)sulfonamide)benzoic acid (HL-0058). TIFF2023178294000114.tif3446

[0277] Following general procedure B, ethyl 5-methyl-2-((4-methylphenyl)sulfonamide)benzoate (404 mg, 1.2 mmol) was reacted with LiOH (87 mg, 3.6 mmol) to obtain 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)sulfonamide)benzamide (HL-0061). TIFF2023178294000115.tif3461

[0279] Following general procedure C2, 5-methyl-2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.16 mmol) was reacted with benzo[b]thiophene-2-amine (24 mg, 0.16 mmol) to obtain 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)sulfonamide)benzamide (HL-0070) Following general procedure C2 (TIFF2023178294000116.tif2984), 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with 3-butylaniline (25 mg, 0.17 mmol) to obtain 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)sulfonamide)benzamide (HL-0071) TIFF2023178294000117.tif3866 Step 1. Methyl 5-cyano-2-((4-methylphenyl)sulfonamide)benzoate (HL-0056). TIFF2023178294000118.tif3351

[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 obtain the desired product (360 mg, 64%) as a solid. ESI MS m / z 331[M+H] + .

[0283] Step 2.5-Cyano-2-((4-methylphenyl)sulfonamide)benzoic acid (HL-0060). TIFF2023178294000119.tif3751

[0284] Following general procedure B, methyl 5-cyano-2-((4-methylphenyl)sulfonamide)benzoate (360 mg, 1.1 mmol) was reacted with LiOH (78 mg, 3.3 mmol) to obtain 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)sulfonamide)benzamide (HL-0071). TIFF2023178294000120.tif3361

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

[0287] Example 49 N-(4-butylphenyl)-2-((4-methylphenyl)sulfonamide)benzamide (JRW-1076) Following general procedure C2, 2-((4-methylphenyl)sulfonamide)benzoic acid (55 mg, 0.19 mmol) was reacted with 4-butylaniline (42 mg, 0.28 mmol) to obtain the desired product (68 mg, 86%) as a light brown oil. ESI MS m / z 423[M+H] + .

[0288] Example 50 N-(benzo[b]thiophen-2-yl)-2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamide)benzamide (JRW-1077) TIFF2023178294000122.tif2661 Step 1. Methyl 2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamide)benzoate (HL-0068). TIFF2023178294000123.tif2649

[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 obtain 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-sulfonamide)benzoic acid (HL-0072) TIFF2023178294000124.tif2646

[0291] Following general procedure B, methyl 2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamide)benzoate (289 mg, 0.84 mmol) was reacted with LiOH (60 mg, 2.5 mmol) to obtain 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-sulfonamide)benzamide (JRW-1077). TIFF2023178294000125.tif2661

[0293] Following general procedure C2, 2-((5,6,7,8-tetrahydronaphthalene)-2-sulfonamide)benzoic acid (50 mg, 0.15 mmol) was reacted with benzo[b]thiophene-2-amine (27 mg, 0.18 mmol) to obtain 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)sulfonamide)benzamide (JRW-1090) Following general procedure C2 (TIFF2023178294000126.tif3092), 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with 4-hexylaniline (36 mg, 0.21 mmol) to obtain the desired product (61 mg, 79%) as a white solid. ESI MS m / z 451[M+H] + .

[0295] Example 52 2-((4-methylphenyl)sulfonamide)-N-(4-octylphenyl)benzamide (JRW-1091) Following general procedure C2 (TIFF2023178294000127.tif2694), 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with 4-octylaniline (42 mg, 0.21 mmol) to obtain 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)sulfonamide)benzamide)phenyl)hexanoate (JRW-1107) Following general procedure C2, 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with methyl 6-(4-aminophenyl)hexanoate (45 mg, 0.21 mmol) to obtain 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)sulfonamide)benzamide)phenyl)hexanoic acid (JRW-1110) Following general procedure B, methyl 6-(4-(2-((4-methylphenyl)sulfonamide)benzamide)phenyl)hexanoate (55 mg, 0.11 mmol) was reacted with LiOH (8 mg, 0.33 mmol) to obtain the desired product (50 mg, 94%) as a light brown solid. ESI MS m / z 481[M+H] + .

[0298] Example 55 N-(benzo[b]thiophen-2-yl)-2-((4-butylphenyl)sulfonamide)benzamide (JRW-1114) TIFF2023178294000130.tif3083 Step 1. Methyl 2-((4-butylphenyl)sulfonamide)benzoate (JRW-1111) TIFF2023178294000131.tif3068

[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 obtain a crude product as a light brown oil. ESI MS m / z 348[M+H] + .

[0300] Step 2.2-((4-butylphenyl)sulfonamide)benzoic acid (JRW-1112) TIFF2023178294000132.tif2864

[0301] Step 3. N-(benzo[b]thiophen-2-yl)-2-((4-butylphenyl)sulfonamide)benzamide (JRW-1114) TIFF2023178294000133.tif2881

[0302] Following general procedure C2, 2-((4-butylphenyl)sulfonamide)benzoic acid (120 mg, 0.36 mmol) was reacted with benzo[b]thiophene-2-amine (54 mg, 0.36 mmol) to obtain 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)sulfonamide)benzamide (JRW-1120) Following general procedure C2, 2-((4-methylphenyl)sulfonamide)benzoic acid (150 mg, 0.51 mmol) was reacted with 6-(4-aminophenyl)hexane-1-ol (100 mg, 0.51 mmol) to obtain 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)sulfonamide)benzamide (JRW-1121) TIFF2023178294000135.tif2679 Step 1. Methyl 2-((4-pentylphenyl)sulfonamide)benzoate (JRW-1115) TIFF2023178294000136.tif2667

[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 obtain a crude product (2.3 g) as an orange oil. ESI MS m / z 362[M+H] + .

[0306] Step 2.2 - ((4-pentylphenyl)sulfonamide)benzoic acid (JRW-1116) TIFF2023178294000137.tif2664

[0307] Following general procedure B, methyl 2-((4-pentylphenyl)sulfonamide)benzoate (2.3 g, 6.4 mmol) was reacted with NaOH (6.4 mL, 2 M, 12.7 mmol) to obtain a 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)sulfonamide)benzamide (JRW-1121) TIFF2023178294000138.tif2679

[0309] Following general procedure C2, 2-((4-pentylphenyl)sulfonamide)benzoic acid (100 mg, 0.29 mmol) was reacted with benzo[b]thiophene-2-amine (34 mg, 0.23 mmol) to obtain 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)sulfonamide)benzamide (JRW-1146) TIFF2023178294000139.tif5070 Process 1.5-Butyl-2-((4-methylphenyl)sulfonamide)benzoic acid (JRW-1142) TIFF2023178294000140.tif4652

[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 obtain 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)sulfonamide)benzamide (JRW-1146) TIFF2023178294000141.tif4667

[0313] Following general procedure C2, 5-butyl-2-((4-methylphenyl)sulfonamide)benzoic acid (275 mg, 0.79 mmol) was reacted with benzo[b]thiophene-2-amine (118 mg, 0.79 mmol) to obtain 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)sulfonamide)benzamide (JRW-1150) Following general procedure C2 (TIFF2023178294000142.tif2682), 2-((4-methylphenyl)sulfonamide)benzoic acid (250 mg, 0.86 mmol) was reacted with 4-(4-aminophenyl)butan-1-ol (170 mg, 1.0 mmol) to obtain the desired product (140 mg, 37%) as oil. ESI MS m / z 439[M+H] + .

[0315] Example 60 N-(4-(4-bromobutyl)phenyl)-2-((4-methylphenyl)sulfonamide)benzamide (JRW-1152) To a solution of N-(4-(4-hydroxybutyl)phenyl)-2-((4-methylphenyl)sulfonamide)benzamide (40 mg, 0.091 mmol) in TIFF2023178294000143.tif2679DCM (5 mL), carbon tetrabromide (60 mg, 0.18 mmol) and triphenylphosphine (47 mg, 0.18 mmol) were added. The reaction mixture 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 obtain the desired product (18 mg, 40%) as a clear oil. ESI MS m / z 502[M+H] + .

[0316] Example 61 5-Methoxy-2-((4-methylphenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1166) TIFF2023178294000144.tif3769 Step 1. Methyl 5-methoxy-2-((4-methylphenyl)sulfonamide)benzoate (JRW-1159) TIFF2023178294000145.tif3860

[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 obtain the crude product as a purple solid. ESI MS m / z 336[M+H] + .

[0318] Step 2.5-Methoxy-2-((4-methylphenyl)sulfonamide)benzoic acid (JRW-1161) TIFF2023178294000146.tif3852

[0319] Following general procedure B, methyl 5-methoxy-2-((4-methylphenyl)sulfonamide)benzoate (27.6 mmol) was reacted with NaOH (27.6 mL, 2 M, 55.2 mmol) to obtain 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)sulfonamide)-N-(p-tolyl)benzamide (JRW-1166) TIFF2023178294000147.tif3768

[0321] Following general procedure C2, 5-methoxy-2-((4-methylphenyl)sulfonamide)benzoic acid (500 mg, 1.6 mmol) was reacted with p-toluidine (200 mg, 1.9 mmol) to obtain 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)sulfonamide)benzamide (JRW-1167) Following general procedure C2 (TIFF2023178294000148.tif3866), 5-methoxy-2-((4-methylphenyl)sulfonamide)benzoic acid (100 mg, 0.31 mmol) was reacted with benzo[b]thiophene-2-amine (51 mg, 0.34 mmol) to obtain the desired product (34 mg, 24%) as a light brown solid. ESI MS m / z 453[M+H] + .

[0323] Example 63 4-Methoxy-2-((4-methylphenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1202) TIFF2023178294000149.tif3267 Step 1. Methyl 4-methoxy-2-((4-methylphenyl)sulfonamide)benzoate (JRW-1195) TIFF2023178294000150.tif3356

[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 obtain a crude product (1.9 g) as a white foam. ESI MS m / z 336[M+H] + .

[0325] Step 2. 4-Methoxy-2-((4-methylphenyl)sulfonamide)benzoic acid (JRW-1198) TIFF2023178294000151.tif3351

[0326] Following general procedure B, methyl 4-methoxy-2-((4-methylphenyl)sulfonamide)benzoate (5.5 mmol) was reacted with NaOH (5.6 mL, 2 M, 11.3 mmol) to obtain 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)sulfonamide)-N-(p-tolyl)benzamide (JRW-1202) TIFF2023178294000152.tif3370

[0328] Following general procedure C2, 4-methoxy-2-((4-methylphenyl)sulfonamide)benzoic acid (510 mg, 1.6 mmol) was reacted with p-toluidine (200 mg, 1.9 mmol) to obtain 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)sulfonamide)benzamide (JRW-1203) Following general procedure C2, 4-methoxy-2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.16 mmol) was reacted with benzo[b]thiophene-2-amine (27 mg, 1.8 mmol) to obtain the desired product (30 mg, 42%) as an orange solid. ESI MS m / z 453[M+H] + .

[0330] Example 65 2-((3-methoxyphenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1205) TIFF2023178294000154.tif2973 Step 1. Methyl 2-((3-methoxyphenyl)sulfonamide)benzoate (JRW-1196) TIFF2023178294000155.tif2960

[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 obtain the desired product (1.3 g, 87%) as a white solid. ESI MS m / z 322[M+H] + .

[0332] Step 2.2-((3-Methoxyphenyl)sulfonamide)benzoic acid (JRW-1199) TIFF2023178294000156.tif2953

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

[0334] Step 3.2-((3-methoxyphenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1205) TIFF2023178294000157.tif2667

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

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

[0337] Example 67 5-Hydroxy-2-((4-methylphenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1232) TIFF2023178294000159.tif4069 A solution of 5-methoxy-2-((4-methylphenyl)sulfonamide)-N-(p-tolyl)benzamide (260 mg, 0.63 mmol) in DCM (10 mL) was mixed with boron tribromide (1.6 mL, 1.0 M, 1.6 mmol) at 0°C. The reaction mixture was warmed to room temperature, stirred for 18 hours, and quenched with a saturated solution of NaHCO3. 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 obtain the desired product (29 mg, 11%) as a brown solid. ESI MS m / z 397[M+H]+.

[0338] Example 68 2-((3-hydroxyphenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1236) A solution of 2-((3-methoxyphenyl)sulfonamide)-N-(p-tolyl)benzamide (190 mg, 0.48 mmol) in 10 mL of DCM was mixed with boron tribromide (0.96 mL, 1.0 M, 0.96 mmol) at 0°C. The reaction mixture was warmed to room temperature, stirred for 18 hours, and quenched with a saturated solution of NaHCO3. 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 obtain the desired product (140 mg, 76%) as a light brown solid. ESI MS m / z 383[M+H] + .

[0339] Example 69 2-((3-butoxyphenyl)sulfonamide)-N-(p-tolyl)benzamide (JRW-1266) A solution of 2-((3-hydroxyphenyl)sulfonamide)-N-(p-tolyl)benzamide (40 mg, 0.10 mmol) in TIFF2023178294000161.tif2988THF (3 mL) was mixed with n-butanol (15 mg, 0.20 mmol), triphenylphosphine (30 mg, 0.12 mmol), and DIAD (46 mg, 0.23 mmol). The mixture was stirred at room temperature for 18 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 obtain 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)sulfonamide)benzamide (JRW-1282) Following general procedure C2 (TIFF2023178294000162.tif2961), 2-((4-methylphenyl)sulfonamide)benzoic acid (200 mg, 0.68 mmol) was reacted with 2-bromoaniline (141 mg, 0.82 mmol) to obtain 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)sulfonamide)benzamide (JRW-1283) Following general procedure C2 (TIFF2023178294000163.tif2971), 2-((4-methylphenyl)sulfonamide)benzoic acid (200 mg, 0.68 mmol) was reacted with 3-bromoaniline (141 mg, 0.82 mmol) to obtain 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)sulfonamide)benzamide (JRW-1288) Following general procedure C2, 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with [1,1'-biphenyl]-4-amine (34 mg, 0.20 mmol) to obtain 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)sulfonamide)benzamide (JRW-1292) Following general procedure C2 (TIFF2023178294000165.tif2961), 2-((4-methylphenyl)sulfonamide)benzoic acid (190 mg, 0.65 mmol) was reacted with 2-methoxyaniline (96 mg, 0.78 mmol) to obtain the desired product (155 mg, 60%) as a light brown solid. ESI MS m / z 397[M+H] + .

[0344] Example 74 N-([1,1'-biphenyl]-3-yl)-2-((4-methylphenyl)sulfonamide)benzamide (JRW-1297) TIFF2023178294000166.tif2974 A solution of N-(3-bromophenyl)-2-((4-methylphenyl)sulfonamide)benzamide (110 mg, 0.24 mmol) in dioxane (5 mL) was mixed with phenylboronic acid (36 mg, 0.29 mmol) and Pd(dppf)Cl2 (20 mg, 0.024 mmol). The mixture was purged with nitrogen, and then aqueous Cs2CO3 (0.74 mL, 1 M) was added. The reaction mixture was heated at 80 °C for 2 hours. 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 obtain 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)sulfonamide)benzamide (JRW-1299) Following general procedure C2 (TIFF2023178294000167.tif3072), 2-((4-methylphenyl)sulfonamide)benzoic acid (190 mg, 0.65 mmol) was reacted with 3-methoxyaniline (96 mg, 0.78 mmol) to obtain 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)sulfonamide)benzamide (JRW-1300) Following general procedure C2 (TIFF2023178294000168.tif2971), 2-((4-methylphenyl)sulfonamide)benzoic acid (190 mg, 0.65 mmol) was reacted with 4-methoxyaniline (96 mg, 0.78 mmol) to obtain the desired product (156 mg, 60%) as a light brown solid. ESI MS m / z 397[M+H] + .

[0347] Example 77 2-((N-ethyl-4-methylphenyl)sulfonamide)-N-(4-methoxyphenyl)benzamide (JRW-1325) To a solution of N-(4-methoxyphenyl)-2-((4-methylphenyl)sulfonamide)benzamide (50 mg, 0.12 mmol) in TIFF2023178294000169.tif2871DMF (3 mL), diisopropylethylamine (49 mg, 0.38 mmol) and ethyl iodide (0.5 mL) were added. The solution was stirred at 60°C for 18 hours. 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 obtain 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)sulfonamide)benzamide (JRW-1346) TIFF2023178294000170.tif2961 Step 1. Methyl 4-fluoro-2-((4-methylphenyl)sulfonamide)benzoate (JRW-1342). TIFF2023178294000171.tif3357

[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 obtain 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)sulfonamide)benzoic acid (JRW-1344). TIFF2023178294000172.tif3151

[0351] Following general procedure B, methyl 4-fluoro-2-((4-methylphenyl)sulfonamide)benzoate (1.5 g, 4.6 mmol) was reacted with NaOH (7 mL, 2 M, 14 mmol) to obtain a 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)sulfonamide)benzamide (JRW-1346). TIFF2023178294000173.tif3266

[0353] Following general procedure C2, 4-fluoro-2-((4-methylphenyl)sulfonamide)benzoic acid (100 mg, 0.32 mmol) was reacted with benzo[b]thiophene-2-amine (58 mg, 0.39 mmol) to obtain 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)sulfonamide)benzamide (JRW-1347) TIFF2023178294000174.tif3766 Step 1. Methyl 5-fluoro-2-((4-methylphenyl)sulfonamide)benzoate (JRW-1343). TIFF2023178294000175.tif3351

[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 obtain 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)sulfonamide)benzoic acid (JRW-1345). TIFF2023178294000176.tif3752

[0357] Following general procedure B, methyl 5-fluoro-2-((4-methylphenyl)sulfonamide)benzoate (1.9 g, 5.9 mmol) was reacted with NaOH (9 mL, 2 M, 18 mmol) to obtain a 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)sulfonamide)benzamide (JRW-1347). TIFF2023178294000177.tif3868

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

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

[0361] Example 81 N-(4-Methoxybenzyl)-2-((4-methylphenyl)sulfonamide)benzamide (JRW-1384) Following general procedure C2 (TIFF2023178294000179.tif2872), 2-((4-methylphenyl)sulfonamide)benzoic acid (50 mg, 0.17 mmol) was reacted with 4-methoxybenzylamine (31 mg, 0.21 mmol) to obtain the desired product (25 mg, 35%) as a light brown solid. ESI MS m / z 411[M+H] + .

[0362] Example 82 N-(benzo[b]thiophen-2-yl)-2-((4-methylphenyl)sulfonamide)-5-(trifluoromethyl)benzamide (JRW-1388) TIFF2023178294000180.tif3362 Step 1. Methyl 2-((4-methylphenyl)sulfonamide)-5-(trifluoromethyl)benzoate (JRW-1382) TIFF2023178294000181.tif3350

[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 obtain the desired product (1.4 g, 81%) as a white solid. ESI MS m / z 374[M+H] + .

[0364] Step 2.2-((4-methylphenyl)sulfonamide)-5-(trifluoromethyl)benzoic acid (JRW-1386) TIFF2023178294000182.tif3346

[0365] Following general procedure B, methyl 2-((4-methylphenyl)sulfonamide)-5-(trifluoromethyl)benzoate (1.4 g, 3.7 mmol) was reacted with NaOH (3.7 mL, 2 M, 7.4 mmol) to obtain a 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)sulfonamide)-5-(trifluoromethyl)benzamide (JRW-1388) TIFF2023178294000183.tif3362

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

[0368] Example 83 Selective inhibition of bioluminescent complexes The following examples provide the use of disclosed inhibitors to selectively inhibit various bioluminescent complexes, such as the Oplophorus luciferase-derived bioluminescent complex, 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 diluted in PBS + 0.01% BSA (final concentrations of 0.1 nM and 100 nM, respectively) and incubated at room temperature for 2 hours in the presence of RPMI medium (final concentration of 2.5% FBS) with sequentially diluted concentrations of the indicated compounds. Samples were analyzed using a GloMax®-Multi+ plate reader after the addition of flimazine (final concentration of 10 μM). Each sample was normalized to a “no inhibitor” control. IC50 was then calculated. 50 Values ​​were determined using GraphPad Prism (log[inhibitor] vs. normalized response). Figure 1B shows the inhibition of NanoBiT® SmBiT / LgBiT bioluminescent complexes by exemplary compounds of the present invention. NanoBiT® SmBiT-annexin fusions and NanoBiT® LgBiT-annexin fusions were diluted in PBS / 0.01% BSA to final concentrations of 60 nM and 30 nM, respectively, and incubated at room temperature for 2 hours with sequentially diluted concentrations of the indicated compounds in the presence of K562 cell lysates diluted in RPMI medium (2.5% FBS final). Samples were analyzed using a GloMax®-Multi+ plate reader after the addition of flimazine (final concentration of 10 μM). Each sample was normalized to a “no inhibitor” control. IC20 was then calculated. 50 The values ​​were determined using GraphPad Prism (log[inhibitor] vs. normalized reaction). Figure 1C shows the calculated IC25 between NanoBiT® HiBiT / LgBiT and SmBiT / LgBiT complexes of the exemplary compounds shown in Figures 1A and 1B. 50 This is a bar graph that compares 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. The inhibitors were diluted in CO2-independent medium with 10% FBS. Nluc was diluted to 2 ng / ml in NanoGlo® buffer with 100 μM flimazine. A series of inhibitor dilutions were added to NanoLuc / flimazine / NanoGlo® solution, and the samples were immediately analyzed using a GloMax®-Multi+ plate reader. As shown in Figure 2, JRW-1004, HL-0005, and HL-0010 showed no sensible inhibition of NanoLuc, demonstrating the selectivity of the compounds for the bioluminescent complex derived from Oplophorus luciferase.

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

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

[0372] Example 85 IC of inhibitors 50 decision The following examples illustrate ICs of compounds disclosed herein. 50 The values ​​are provided. The results are shown in Table 1. Detection reagents were prepared by diluting NanoBiT® HiBiT non-luminescent peptide and LgBiT non-luminescent non-polypeptide to 0.1 nM and 1 nM, respectively, in TBS buffer containing 0.01% BSA. Then, a 3-fold dilution series of each inhibitor was prepared in the detection reagent. A "no inhibitor" control was also prepared for each sample. 50 μl dilutions of each inhibitor were mixed with 6 μM (final) flimazine, and luminescence was measured. Each sample was normalized to the "no inhibitor" control. Then, IC50 was performed. 50 The values ​​were determined using GraphPad Prism (log[inhibitor] vs. normalized response). Table 1 TIFF2023178294000184.tif203143 TIFF2023178294000185.tif203143 TIFF2023178294000186.tif62131 NA - No activity NT-test not performed.

[0373] It is understood that the above-mentioned detailed description and accompanying embodiments are illustrative only and should not be construed as limitations on the scope of the invention as 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 those relating to the chemical structure, substituents, derivatives, intermediates, synthesis, composition, formulation, or method of use of the present invention, may be made without departing from the spirit and scope thereof. Appendix

[0375] Sequence ID 1 - Amino acid sequence of naturally grown Oplophorus luciferase FTLDF

[0376] Sequence ID 2 - Wild-type NLPep MGVTGWRLCERILA

[0377] Sequence ID 3 - Wild-type NLpoly MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINV

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

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

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

[0381] Sequence ID 7 - NanoLuc MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLFRVTINGVTGWRLCERILA

Claims

1. 1. A method of inhibiting a bioluminescent complex derived from Oplophorus luciferase, said method comprising: 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide 20. A method of excising a medical procedure on a human comprising contacting the human with a compound selected from the group consisting of:

2. The method of claim 1, 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.

3. The method of claim 2, 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.

4. The bioluminescent complex comprises: a) a peptide comprising an amino acid sequence having less than 100% and more than 40% identity to SEQ ID NO:2; and b) a polypeptide comprising an amino acid sequence having less than 100% and more than 40% identity to SEQ ID NO:3; Including, The method of claim 1 , wherein the bioluminescent conjugate exhibits a detectable luminescence in the presence of a coelenterazine substrate.

5. The method of claim 4, 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.

6. 1. A method for modulating the luminescence of an Oplophorus luciferase-derived bioluminescent complex in a sample, the method comprising: (a) subjecting the sample to a coelenterazine substrate, and 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide and contacting the compound selected from the group consisting of: (b) detecting luminescence in the sample; and Including, The compound causes a decrease in the luminescence from the bioluminescent complex, the method comprising the step of: eliminating medical treatment for a human;

7. 1. A method for detecting an interaction or co-localization between a first molecule and a second molecule in a sample, the method comprising: (a) The sample was treated with a coelenterazine substrate, and 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide wherein the sample is contacted with a compound selected from the group consisting of: (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, 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; and Including, The method of claim 1, wherein detection of said luminescence indicates an interaction or co-localization between said first protein and said second protein.

8. 1. A method for detecting an interaction or co-localization between a first molecule and a second molecule in a sample, the method comprising: (a) The sample was treated with a coelenterazine substrate, and 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide wherein the sample is contacted with a compound selected from the group consisting of: (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; and Including, The method of claim 1, wherein detection of said emission indicates an interaction or co-localization between said first molecule and said second molecule.

9. 1. A method for detecting an interaction or co-localization of a first molecule and a second molecule in a sample, the method comprising: (a) The sample was prepared by mixing a non-luminescent polypeptide of Oplophorus luciferase, a coelenterazine substrate, and 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide wherein the sample is contacted with a compound selected from the group consisting of: (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 receptor 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; Methods other than medical procedures on humans, including

10. 1. A method for detecting an interaction or co-localization of a first molecule and a second molecule in a sample, the method comprising: (a) The sample was prepared by mixing a non-luminescent peptide of Oplophorus luciferase, a coelenterazine substrate, and 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide wherein the sample is contacted with a compound selected from the group consisting of: (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 receptor 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; Methods other than medical procedures on humans, including

11. 1. A method for detecting molecular interactions or co-localization in a sample, the method comprising: (a) The sample was treated with a coelenterazine substrate, and 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide wherein the sample is contacted with a compound selected from the group consisting of: (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, the non-luminescent peptide being capable of forming a bioluminescent complex with the non-luminescent polypeptide; and (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; and Methods other than medical procedures on humans, including

12. 1. 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) a coelenterazine substrate; (ii) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide A compound selected from the group consisting of: (iii) a non-luminescent polypeptide of Oplophorus luciferase, said non-luminescent polypeptide being capable of forming a bioluminescent complex with said non-luminescent peptide; and contacting the (b) detecting luminescence in the sample; and Including, The method of exempting medical treatment of a human, wherein detection of luminescence indicates formation of a bioluminescent complex between said non-luminescent peptide and said non-luminescent polypeptide.

13. 1. A method for detecting a molecule of interest in a sample, the method comprising: (a) subjecting a sample containing the molecule of interest fused to a non-luminescent polypeptide of Oplophorus luciferase; (i) a coelenterazine substrate; (ii) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide A compound selected from the group consisting of: (iii) a non-luminescent peptide of Oplophorus luciferase, said non-luminescent peptide being capable of forming a bioluminescent complex with said non-luminescent polypeptide; and contacting the (b) detecting luminescence in the sample; and Including, The method of exempting medical treatment of a human, wherein detection of luminescence indicates formation of a bioluminescent complex between said non-luminescent peptide and said non-luminescent polypeptide.

14. 1. 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) a coelenterazine substrate; (ii) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide A compound selected from the group consisting of: (iii) a fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a fluorescent moiety, said non-luminescent polypeptide being capable of forming a bioluminescent complex with said non-luminescent peptide; and contacting the (b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of detection of the molecule; and Methods other than medical procedures on humans, including

15. 1. A method for detecting a molecule of interest in a sample, the method comprising: (a) subjecting a sample containing the molecule of interest fused to a non-luminescent polypeptide of Oplophorus luciferase; (i) a coelenterazine substrate; (ii) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide A compound selected from the group consisting of: (iii) a fusion comprising a non-luminescent peptide of Oplophorus luciferase and a fluorescent moiety, said non-luminescent peptide being capable of forming a bioluminescent complex with said non-luminescent polypeptide; and contacting the (b) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of detection of the molecule; and Methods other than medical procedures on humans, including

16. The method of any one of claims 6 to 15, comprising contacting the sample with the coelenterazine substrate prior to contacting the sample with the compound.

17. The method according to any one of claims 6 to 11, 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.

18. The method of any one of claims 6 to 17, wherein the sample comprises a cell.

19. The method of any one of claims 6 to 18, wherein the coelenterazine substrate is coelenterazine, a coelenterazine derivative, a coelenterazine analogue, pro-coelenterazine, or a quinone-masked coelenterazine.

20. 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; The method according to any one of claims 6 to 19, wherein b) the non-luminescent polypeptide comprises an amino acid sequence having a sequence identity of less than 100% and more than 40% with SEQ ID NO:

3.

21. 1. A bioluminescence resonance energy transfer (BRET) system comprising: (a) a first fusion comprising a non-light-emitting peptide of Oplophorus luciferase and a first molecule; (b) a second fusion comprising a non-luminescent polypeptide of Oplophorus luciferase and a fluorescent moiety, the non-luminescent polypeptide being capable of forming a bioluminescent complex with the non-luminescent peptide; and (c) a coelenterazine substrate; (d) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide and a compound selected from the group consisting of The bioluminescence resonance energy transfer (BRET) system comprises:

22. 1. 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 of Oplophorus luciferase and a fluorescent moiety, the non-luminescent peptide being capable of forming a bioluminescent complex with the non-luminescent polypeptide; and (c) a coelenterazine substrate; (d) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide and a compound selected from the group consisting of The bioluminescence resonance energy transfer (BRET) system comprises:

23. 1. 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; and (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) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide and a compound selected from the group consisting of The bioluminescence resonance energy transfer (BRET) system comprises:

24. 1. A bioluminescence resonance energy transfer (BRET) system comprising: (a) a first fusion comprising a first molecule and a non-luminescent polypeptide of Oplophorus derived luciferase; (b) a second fusion comprising a second molecule and a fluorescent receptor molecule; and (c) a non-light emitting peptide of Oplophorus luciferase capable of forming a bioluminescent complex with said non-light emitting polypeptide of Oplophorus luciferase; and (d) a coelenterazine substrate; (e) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide and a compound selected from the group consisting of The bioluminescence resonance energy transfer (BRET) system comprises:

25. 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; A bioluminescent resonance energy transfer system according to any one of claims 21 to 24.

26. (a) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide and a compound selected from the group consisting of (b) a bioluminescent complex derived from Oplophorus luciferase; Including the kit.

27. 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; 27. The kit of claim 26, comprising:

28. (a) Below 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; N-(3-cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-2-(phenylsulfonamido)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; 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; 2-((4-methylphenyl)sulfonamido)-N-(2-propylphenyl)benzamide; N-(3-butylphenyl)-2-((4-methylphenyl)sulfonamido)benzamide; N-(4-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-(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; and N-(4-methoxybenzyl)-2-((4-methylphenyl)sulfonamido)benzamide and a compound selected from the group consisting of (b) a first polynucleotide encoding a non-light-emitting peptide of Oplophorus luciferase; (c) a second polynucleotide encoding a non-luminescent polypeptide of Oplophorus luciferase, the non-luminescent polypeptide being capable of forming a bioluminescent complex with the non-luminescent peptide; and Including the kit.

29. The kit of claim 28, wherein the non-luminescent peptide comprises an amino acid sequence having less than 100% and more than 40% sequence identity with SEQ ID NO:2, and the non-luminescent polypeptide comprises an amino acid sequence having less than 100% and more than 40% sequence identity with SEQ ID NO:

3.

30. The kit according to any one of claims 26 to 29, further comprising a coelenterazine substrate.

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