Bioluminescence pro-substrates

Abstract

Disclosed herein are pro-substrates of furimazine and analogs thereof, methods of using the pro-substrates in live-cell bioluminescence applications, and kits comprising the pro-substrates.

Description

[0001] PRMG2-43218.601 BIOLUMINESCENCE PRO-SUBSTRATES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 678,633, filed on August 2, 2024, the disclosure of which is incorporated herein by reference in its entirety. FIELD Provided herein are pro-substrates of furimazine and derivatives thereof, methods of using the pro-substrates in bioluminescence applications, including live-cell bioluminescence assays. Also disclosed herein are kits comprising the pro-substrates. BACKGROUND Luminescence pro-substrates are chemically protected luminescence substrates which, upon introduction to assay systems, undergo chemical transformation to release the unprotected luminescence substrates for generating light signals in assays. Compared to their unprotected counterparts, pro-substrates can generate steadier signals that can last for several hours to several days. SUMMARY Disclosed herein are luminescence pro-substrates based on furimazine and derivatives thereof, which have improved brightness and sensitivity. The probe substrate compounds also release their corresponding luminescence substrates over longer periods of time, making them particularly useful for longer-term assays. Accordingly, in one aspect, disclosed herein is a compound of formula (I): or a salt thereof, wherein: R1is selected from hydrogen, C1-C4 alkyl, halo, and C1-C4 haloalkyl; m is 0, 1, 2, 3, 4, or 5; PRMG2-43218.601 each R2is independently selected from C1-C4alkyl, halo, C1-C4haloalkyl, -OR2a, - NR2bR2c, nitro, and cyano; n is 0, 1, 2, 3, 4, or 5; each R3is independently selected from C1-C4alkyl, halo, C1-C4haloalkyl, -OR3a, - NR3bR3c, nitro, and cyano; R2a, R2b, R2c, R3a, R3b, and R3care each independently selected from hydrogen, C1-C4 alkyl, and C1-C4haloalkyl; L is a linker; and M is a water soluble and cell impermeable moiety selected from a peptide, an alkyl or aryl sulfonate, an alkyl or aryl phosphonate, a monosaccharide, a disaccharide, and an oligosaccharide, wherein M is optionally substituted with a moiety of formula (A): In some embodiments, R1is hydrogen. In some embodiments, R1is methyl. In some embodiments, m is 0, 1, or 2. In some embodiments, m is 0. In some embodiments, m is 1 or 2. In some embodiments, each R2is independently selected from halo, -OR2a, and -NR2bR2c, wherein R2a, R2b, R2care independently selected from hydrogen and methyl. In some embodiments, each R2is independently selected from fluoro and -NH2. In some embodiments, n is 0, 1, or 2. In some embodiments, n is 0. In some embodiments, n is 1 or 2. In some embodiments, each R3is independently selected from halo and C1-C4 alkyl. In some embodiments, the compound is a compound of formula (Ia): PRMG2-43218.601 or a salt thereof, wherein: R1is selected from hydrogen and C1-C4 alkyl; R2xand R2yare independently selected from halo and -NR2bR2c; and R3is halo. In some embodiments, M is a peptide. In some embodiments, M is a peptide comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, M is a peptide comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the amino acids are independently selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, lysine, and NN%NN&:?A;F>I@@IE?B;' In some embodiments, M is a peptide having a formula selected from: PRMG2-43218.601 . In some embodiments, M is a moiety of formula: . In some embodiments, M is an alkyl or aryl sulfonate moiety. In some embodiments, M is a moiety of formula; PRMG2-43218.601 . In some embodiments, M is an alkyl or aryl phosphonate moiety. In some embodiments, M is a -CH2CH2-PO3H2group. In some embodiments, M is a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, M is selected from: In some embodiments, M is selected from: PRMG2-43218.601 PRMG2-43218.601 . In some embodiments, L is a linker comprising any combination of moieties selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -C(O)-, -NH-, -NR’-, -O-, and monocyclic heteroarylene, wherein R’ is an optionally substituted C1-C6 alkyl group, and wherein the linker comprises a total of about 5 to about 100 atoms. In some embodiments, L is a linker comprising any combination of moieties selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -C(O)-, -NH-, -NCH3-, -O-, and . In some embodiments, L is a linker comprising a group of formula – (OCH2CH2)q–, wherein q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L is a linker having a formula selected from: PRMG2-43218.601 5 In some embodiments, the compound is selected from: PRMG2-43218.601 PRMG2-43218.601

[0002] PRMG2-43218.601

[0003] PRMG2-43218.601

[0004] PRMG2-43218.601

[0005] PRMG2-43218.601

[0006] PRMG2-43218.601

[0007] PRMG2-43218.601 and salts thereof. In another aspect, disclosed herein is a method for detecting luminescence in a sample, the method comprising: contacting a sample with a compound of formula (I); contacting the sample with a coelenterazine-utilizing luciferase, if no coelenterazine- utilizing luciferase is present in the sample; and detecting luminescence in the sample. In some embodiments, the sample comprises live cells. In some embodiments, the sample comprises a coelenterazine-utilizing luciferase or a fragment complementary luciferase. In some embodiments, the sample comprises live cells that express a coelenterazine-utilizing luciferase or a fragment complementary luciferase. In some embodiments, the method further comprises contacting the sample with a deprotection enzyme. In some embodiments, the deprotection enzyme comprises an esterase. In another aspect, disclosed herein is a method for detecting luminescence in a transgenic animal comprising administering a compound of formula (I) to a transgenic animal; and detecting luminescence; wherein the transgenic animal expresses a coelenterazine-utilizing luciferase. In another aspect, disclosed herein is a kit comprising a compound of formula (I). In some embodiments, the kit further comprises a luciferase or a fragment complementary luciferase. In some embodiments, the kit further comprises a deprotection enzyme. In some embodiments, the deprotection enzyme comprises an esterase. In some embodiments, the kit further comprises a buffer reagent. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 shows luminescence signals (RLU) over time (min.) for Compound 1 in a NanoLuc enzymatic system. PRMG2-43218.601 FIG.2 shows luminescence signals (RLU) over time (min.) for Compound 2 in a NanoLuc enzymatic system. DETAILED DESCRIPTION Provided herein are compounds that are luminescence pro-substrates of furimazine and furimazine derivatives. The compounds can be useful pro-substrates for proteins that utilize coelenterazine (“coelenterazine-utilizing enzymes”) to produce luminescence, including, but not limited to, luciferases and photoproteins found in various marine organisms such as cnidarians (e.g., Renilla luciferase), jellyfish (e.g., aequorin from the Aequorea jellyfish), and decapods luciferases (e.g., luciferase complex of Oplophorus gracilirostris), and variants thereof. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. As used herein, the term “and / or” includes any and all combinations of listed items, including any of the listed items individually. For example, “A, B, and / or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and / or C.” For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in PRMG2-43218.601 accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Sorrell, Organic Chemistry, 2ndedition, University Science Books, Sausalito, 2006; Smith, March’s Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7thEdition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rdEdition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference. As used herein, the term “alkyl” refers to a radical of a straight or branched saturated hydrocarbon chain. The alkyl chain can include, e.g., from 1 to 24 carbon atoms (C1-C24 alkyl), 1 to 16 carbon atoms (C1-C16alkyl), 1 to 14 carbon atoms (C1-C14alkyl), 1 to 12 carbon atoms (C1-C12alkyl), 1 to 10 carbon atoms (C1-C10alkyl), 1 to 8 carbon atoms (C1-C8alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 1 to 3 carbon atoms (C1-C3alkyl), or 1 to 2 carbon atoms (C1-C2alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso- butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. As used herein, the term “alkylene” refers to a divalent alkyl group. As used herein, the term “amino acid” refers to any and all amino acids, includingB7FGD7@@I C99GDD?B= 7A?BC 79?:E ";'='% M&7A?BC 79?:E#% GBB7FGD7@ 7A?BC 79?:E% AC:?<?;: 7A?BCacids, and non-natural amino acids. It includes both D- and L-amino acids. Naturally occurring amino acids include those found in nature, such as, e.g., the twenty-three amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These include the twenty “standard” amino acids and the additional three “non-standard” amino acids pyrrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N- formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. “Unnatural” or “non-natural” amino acids are non- proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. For the most part, the names of naturally occurring and non-naturally occurring amino acids used herein follow the naming PRMG2-43218.601 conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out inJ3CA;B9@7FGD; C< M&0A?BC 09?:E "5;9CAA;B:7F?CBE% ).-+#K Biochemistry, 14(2), (1975).Throughout the present specification, unless naturally-occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). As used herein, the term “aryl” refers to a radical of a monocyclic, bicyclic, orFD?9I9@?9 +B$* 7DCA7F?9 D?B= EIEF;A ";'='% >7H?B= ,% )(% CD )+ P ;@;9FDCBE E>7D;: ?B 79I9@?9array) having 6-14 ring carbon atoms and zero heteroatoms (“C6-C14aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6aryl,” i.e., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl,” e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14aryl,” e.g., anthracenyl and phenanthrenyl). As used herein, the term “arylene” refers to a divalent aryl radical. As used herein, the term “cycloalkyl” refers to a radical of a saturated carbocyclic ring system containing three to ten carbon atoms and zero heteroatoms. The cycloalkyl may be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl. As used herein, the term “cycloalkylene” refers to a divalent cycloalkyl radical. As used herein, the term “cyano” refers to a -CN group. As used herein, the term “halogen” or “halo” refers to F, Cl, Br, or I. As used herein, the term “haloalkyl” refers to an alkyl group, as defined herein, in which at least one hydrogen atom (e.g., one, two, three, four, five, six, seven or eight hydrogen atoms) is replaced with a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced with a halogen (“perhaloalkyl”). Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2- fluoroethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl. As used herein, the term “heteroaryl” refers to a radical of a 5-10 memberedACBC9I9@?9 CD 8?9I9@?9 +B$* 7DCA7F?9 D?B= EIEF;A ";'='% >7H?B= , CD )( P ;@;9FDCBE E>7D;: ?Ba cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In PRMG2-43218.601 heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6- bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. As used herein, the term “heteroarylene” refers to a divalent heteroaryl radical. As used herein, the term “heterocyclyl” refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, PRMG2-43218.601 bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl (e.g., 2,2,6,6-tetramethylpiperidinyl), tetrahydropyranyl, dihydropyridinyl, pyridinonyl (e.g., 1-methylpyridin-2-onyl), and thianyl. Exemplary 6- membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, pyridazinonyl (2-methylpyridazin-3-onyl), pyrimidinonyl (e.g., 1- methylpyrimidin-2-onyl, 3-methylpyrimidin-4-onyl), dithianyl, dioxanyl. Exemplary 6- membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as a 5,6-bicyclic heterocyclyl ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 5-membered heterocyclyl groups fused to a heterocyclyl ring (also referred to PRMG2-43218.601 herein as a 5,5-bicyclic heterocyclyl ring) include, without limitation, octahydropyrrolopyrrolyl (e.g., octahydropyrrolo[3,4-c]pyrrolyl), and the like. Exemplary 6- membered heterocyclyl groups fused to a heterocyclyl ring (also referred to as a 4,6- membered heterocyclyl ring) include, without limitation, diazaspirononanyl (e.g., 2,7- diazaspiro[3.5]nonanyl). Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclyl ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as a 6,7-bicyclic heterocyclyl ring) include, without limitation, azabicyclooctanyl (e.g., (1,5)-8- azabicyclo[3.2.1]octanyl). Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as a 6,8-bicyclic heterocyclyl ring) include, without limitation, azabicyclononanyl (e.g., 9-azabicyclo[3.3.1]nonanyl). As used herein, the term “heterocyclylene” refers to a divalent heterocyclyl radical. As used herein, the term “hydroxy” refers to an -OH group. As used herein, the term “nitro” refers to an -NO2 group. As used herein, the term “oligosaccharide” refers to a polymeric saccharide moiety having from 3 to about 12 monosaccharide moieties, linked to each other by glycosidic bonds. As used herein, the term “peptide” refers to a polymer compound of 2 to 25 amino acids joined through the main chain by peptide amide bonds (-C(O)NH-). As used herein, in chemical structures the indication: represents a point of attachment of one moiety to another moiety (e.g., a substituent group to the rest of the compound). For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. When substituent groups are specified by their conventional chemical formulae, written from left to right, such indication also encompass substituent groups resulting from writing the structure from right to left. For example, if a bivalent group is shown as -CH2O-, such indication also encompasses -OCH2-; similarly, -OC(O)NH- also encompasses - PRMG2-43218.601 NHC(O)O-. When linker moieties are shown, the linkers can be attached to other moieties of the compound in either direction. Compounds Disclosed herein are compounds of formula (I): and salts thereof, wherein: R1is selected from hydrogen, C1-C4 alkyl, halo, and C1-C4 haloalkyl; m is 0, 1, 2, 3, 4, or 5; each R2is independently selected from C1-C4alkyl, halo, C1-C4haloalkyl, -OR2a, - NR2bR2c, nitro, and cyano; n is 0, 1, 2, 3, 4, or 5; each R3is independently selected from C1-C4alkyl, halo, C1-C4haloalkyl, -OR3a, - NR3bR3c, nitro, and cyano; R2a, R2b, R2c, R3a, R3b, and R3care each independently selected from hydrogen, C1-C4 alkyl, and C1-C4haloalkyl; L is a linker; and M is a water soluble and cell impermeable moiety selected from a peptide, an alkyl or aryl sulfonate, an alkyl or aryl phosphonate, a monosaccharide, a disaccharide, and an oligosaccharide, wherein M is optionally substituted with a moiety of formula (A): PRMG2-43218.601 In some embodiments, R1is hydrogen. In some embodiments, R1is C1-C4alkyl. In some embodiments, R1is methyl. In some embodiments, R1is halo. In some embodiments, R1is fluoro. In some embodiments, R1is C1-C4 haloalkyl. In some embodiments, R1is trifluoromethyl. In some embodiments, R1is hydrogen or C1-C4alkyl. In some embodiments, R1is hydrogen or methyl. In some embodiments, m is 0, 1, or 2. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 1 or 2. In some embodiments, each R2is independently selected from halo, -OR2a, and - NR2bR2c. In some embodiments, each R2is independently selected from halo, -OR2a, and - NR2bR2c, wherein R2a, R2b, R2care independently selected from hydrogen and methyl. In some embodiments, m is 2, and each R2is independently selected from halo and -NR2bR2c. In some embodiments, m is 2, and each R2is independently selected from fluoro and -NH2. In some embodiments, m is 2, and each R2is fluoro. In some embodiments, m is 2, one R2is fluoro, and one R2is -NH2. In some embodiments, n is 0, 1, or 2. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 0 or 1. In some embodiments, n is 1 or 2. In some embodiments, each R3is independently selected from halo and C1-C4 alkyl. In some embodiments, each R3is independently selected from fluoro and methyl. In some embodiments, n is 1, and R3is halo. In some embodiments, n is 1, and R3is fluoro. In some embodiments, the compound of formula (I) is a compound of formula (Ia): or a salt thereof, wherein: R1is selected from hydrogen and C1-C4 alkyl; R2xand R2yare independently selected from halo and -NR2bR2c; and R3is halo. In some embodiments, R1is hydrogen. In some embodiments, In some embodiments, R1is C1-C4 alkyl. In some embodiments, R1is methyl. PRMG2-43218.601 In some embodiments, R2xis halo. In some embodiments, R2xis fluoro. In some embodiments, R2xis -NR2bR2c, and R2band R2care each hydrogen. In some embodiments, R3is halo. In some embodiments, R3is fluoro. In some embodiments, M is a peptide. For example, in some embodiments, M is a peptide comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, M is a peptide comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, M is a peptide comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids independently selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, lysine, and NN%NN&:?A;F>I@@IE?B;' In some embodiments, M is a peptide comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids independently selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, and lysine. In embodiments in which M is a peptide, it can be bound to the linker L by either the N-terminus or the C- terminus of the peptide, and the opposite end can be either a carboxylic acid or amino group, or such a group can be capped or protected with a suitable moiety. For example, when attached to the linker at the N-terminus of the peptide, the C-terminus can include an amide group. Similarly, when attached to the linker at the C-terminus of the peptide, the N-terminus can include a protecting group, such as an acetyl group or a tert-butyloxycarbonyl group. In some embodiments, M is a peptide having a formula selected from: PRMG2-43218.601 . In some embodiments, M is a triaspartate peptide of formula: PRMG2-43218.601 . In some embodiments, M is an alkyl or aryl sulfonate moiety, i.e., a group comprising a moiety of formula -SO3H substituted on an aryl group or an alkyl group (e.g., a -CH2- group). For example, in some embodiments, M is a moiety comprising one or more - CH2CH2-SO3H groups. In some embodiments, M is a moiety comprising one or more -CH2- SO3H groups. In some embodiments, M is a -CH2CH2-SO3H group. In some embodiments, M is a moiety comprising a group: . In some embodiments, M is selected . In some embodiments, M is an alkyl or aryl phosphonate moiety, i.e., a group comprising a moiety of formula -PO3H2substituted on an aryl group or an alkyl group (e.g., a -CH2- group). For example, in some embodiments, M is a moiety comprising one or more - CH2CH2-PO3H2 groups. In some embodiments, M is a moiety comprising one or more -CH2- PO3H2groups. In some embodiments, M is a -CH2CH2-PO3H2group. PRMG2-43218.601 In some embodiments, M is a monosaccharide, a disaccharide, or an oligosaccharide. For example, in some embodiments, M is a monosaccharide. In some embodiments, M is a disaccharide. In some embodiments, M is selected from: In some embodiments, M is selected from:

[0008] PRMG2-43218.601 PRMG2-43218.601 . In compounds of formula (I) (including compounds of formula (Ia)), L is a linker. The linker is a chain of atoms that separates the tri-aspartate moiety of formula (I) from the furimazine (or derivative thereof). In some embodiments, the linker separates the groups by about 5 Å, about 10 Å, about 20 Å, about 50 Å, about 100 Å, about 150 Å, about 200 Å, about 300 Å, about 400 Å, about 500 Å, about 600 Å, about 700 Å, about 800 Å, about 900 Å, about 1000 Å, or any suitable range therebetween (e.g., about 5-100 Å, about 50-500 Å, about 150-700 Å, etc.). In some embodiments, the linker separates the moiety of formula (I) from the polymer backbone by about 1-200 atoms (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 200, or any suitable ranges therebetween (e.g., about 2-20, about 10-50, etc.)). In some embodiments, the linker comprises one or more -CH2-, -CH(CH3)-, -C(CH3)2-% &12 / 12&% &1O1&% &4&% &1"4#&% &1"6#&% &32&% &3"123)-, -S-, arylene, heteroarylene,cycloalkylene, or heterocyclylene moieties, each of which may be optionally substituted, or any combination thereof. In some embodiments, the linker comprises one or more groups that represents a combination of one or more of the above moieties, e.g., -C(O)O-, -C(O)NH-, - NHC(O)O-, -OC(O)O-, -NHC(O)NH-, -C(O)S-, -C(S)NH-, -NHC(S)O-, -OC(S)O-, or - NHC(S)NH-. PRMG2-43218.601 In some embodiments, the linker comprises one or more alkylene groups (e.g., - (CH2)n-, wherein n is 1-12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any suitable range therebetween). In some embodiments, the linker comprises one or more branched alkylene groups. In some embodiments, the linker comprises a heteroarylene group. For example, in some embodiments, the linker comprises a monocyclic heteroarylene group having 1, 2, or 3 nitrogen atoms. In particular embodiments, the linker comprises a 1,2,3-triazole ring, i.e., a ring of formula , which one skilled in the art will recognize is a reaction product of an alkyne and an azide. For example, linkers may include such a ring when it is synthesized using a copper-catalyzed azide-alkyne cycloaddition reaction. In some embodiments, L is a linker comprising about 3 to about 100 atoms, comprising any combination of moieties selected from , -C(CH3)2-, -C(O)-, -NH-, -NCH3-, -O-, and monocyclic heteroarylene some embodiments, L is a linker comprising about 3 to about 50 atoms, comprising any combination of moieties selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -C(O)-, -NH-, -NCH3-, -O-, and monocyclic heteroarylene (e.g., In some embodiments, L is a linker comprising about 40 to about 50 atoms, comprising any combination of moieties selected from -CH2-, -C(CH3)2-, - In some embodiments, L is a linker having a formula selected from: wherein:R4, R5, R6x, and R6yare each independently alkyl, aryl, or heterocyclyl, each of which is independently substituted or unsubstituted; wherein R6xand R6yare optionally taken together with the carbon atom to which they are attached to form a 3- to 7 membered ring; and m, n, and o are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. PRMG2-43218.601 In some embodiments, L is a linker having a formula selected from: In some embodiments, the linker is: PRMG2-43218.601 . In some embodiments, the compound is selected from: PRMG2-43218.601

[0009] PRMG2-43218.601

[0010] PRMG2-43218.601

[0011] PRMG2-43218.601

[0012] PRMG2-43218.601

[0013] PRMG2-43218.601

[0014] PRMG2-43218.601

[0015] PRMG2-43218.601 , and salts thereof. Certain compounds described herein may have at least one asymmetric center. Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Compounds with asymmetric centers give rise to enantiomers (optical isomers), diastereomers (configurational isomers) or both, and it is intended that all of the possible enantiomers and diastereomers, in mixtures and as pure or partially purified compounds, are included within the scope of this disclosure. The independent syntheses of the enantiomerically or diastereomerically enriched compounds, or their chromatographic separations, may be achieved as known in the art by appropriate modification of the methodology disclosed herein. Their absolute stereochemistry may be determined by the x-ray crystallography of crystalline products or crystalline intermediates that are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration. If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is often the formation of salts using an enantiomerically pure acid or base. The diastereomeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods using chiral stationary phases, which methods are well known in the art. Alternatively, any enantiomer of a compound may be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art. PRMG2-43218.601 The compounds disclosed herein may also possess tautomeric forms, and all tautomers also constitute embodiments of the disclosure. The present disclosure also includes isotopically-labeled compounds, which are identical to those recited in formula (I) or the specific compounds illustrated herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to,2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F, and36Cl, respectively. Substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain advantages resulting from greater stability, for example increased in vivo half-life or reduced dosage requirements, and, hence, may be preferred in some circumstances. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron-emitting isotopes that can be incorporated in the compounds are11C,13N,15O, and18F. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically-labeled reagent in place of non-isotopically-labeled reagent. A compound described herein can be in the form of a salt. Acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. Examples of acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange. Other acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Basic addition salts may be prepared during the final isolation and purification of the PRMG2-43218.601 disclosed compounds by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine and N,N’-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. Compounds disclosed herein can exist in solvated as well as unsolvated forms with solvents such as water, ethanol, and the like, and it is intended that the disclosure encompass both solvated and unsolvated forms. In one embodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in a crystalline form. Compounds disclosed herein can be prepared by a variety of methods, including those illustrated in the Examples. Compounds and intermediates may be isolated and purified by methods well-known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds can include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperature with an optional pretreatment with activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described for instance in “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), by Furniss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM202JE, England. Reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Reactions can be worked up in a conventional manner, e.g., by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration, and chromatography. Unless otherwise described, the starting materials and reagents are either commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. PRMG2-43218.601 Standard experimentation, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the disclosure. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in PGM Wuts and TW Greene, in Greene's book titled Protective Groups in Organic Synthesis (4thed.), John Wiley & Sons, NY (2006). When an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using an optically active starting material (prepared, for example, by asymmetric induction of a suitable reaction step), or by resolution of a mixture of the stereoisomers of the compound or intermediates using a standard procedure (such as chromatographic separation, recrystallization, or enzymatic resolution). Similarly, when a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the procedures described herein using a pure geometric isomer as a starting material, or by resolution of a mixture of the geometric isomers of the compound or intermediates using a standard procedure such as chromatographic separation. The synthetic schemes and specific examples as described are illustrative and are not to be read as limiting the scope of the disclosure or the claims. Alternatives, modifications, and equivalents of the synthetic methods and specific examples are contemplated. Compounds of formula (I) are pro-substrates of luciferases, which produce luminescence upon chemical transformation to release the active luminescence substrates. The chemical transformation can occur via hydrolysis, or via action of a non-luciferase enzyme in the sample, to release the luciferase substrate (e.g., furimazine or a derivative thereof) over a certain time period. In some embodiments, the compound may react with a deprotection enzyme (such as an esterase) to release the luciferase substrate. The compounds may have improved stability, improved water solubility, reduced autoluminescence, and / or reduced toxicity. In some embodiments, the disclosed compounds may exhibit unexpectedly superior serum stability in comparison to other coelenterazine and furimazine ester analogues. In particular embodiments, the disclosed compounds are stable in a media containing a serum, which may allow for a stable signal in various live cell assays up to 24 hours and longer. PRMG2-43218.601 In general, “enhanced” or “improved” means that the particular property (such as luminescence, or signal stability) is increased relative to that of the reference luciferase plus the furimazine analog under consideration, where the increase is at least 1 %, at least 5%, at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, at least 500%, or at least 1000% greater than the reference luciferase plus furimazine or the furimazine analog under consideration. “Luminescence” refers to the light output of a luciferase under appropriate conditions, e.g., in the presence of a suitable substrate such as a coelenterazine analog. The light output may be measured as an instantaneous or near-instantaneous measure of light output (which is sometimes referred to as “T=0” luminescence or “flash”) at the start of the luminescence reaction, which may be initiated upon addition of the pro-coelenterazine substrate. The luminescence reaction in various embodiments is carried out in a solution. In other embodiments, the luminescence reaction is carried out on a solid support. In some embodiments, the solution may contain live cells in a prokaryotic or eukaryotic expression system. In other embodiments, expression occurs in a cell-free system, or the luciferase protein is secreted into an extracellular medium, such that, in the latter case, it is not necessary to produce a lysate. In some embodiments, the reaction is started by injecting appropriate materials, e.g., pro-coelenterazine analog, buffer, etc., into a reaction chamber (e.g., a well of a multiwell plate such as a 96-well plate) containing the ester deprotection enzyme and the luminescent protein. In still other embodiments, the luciferase and / or pro- coelenterazine analogs (e.g., compounds of formula (I)) are introduced into a host, and measurements of luminescence are made on the host or a portion thereof, which can include a whole organism or cells, tissues, explants, or extracts thereof. The reaction chamber may be situated in a reading device which can measure the light output, e.g., using a luminometer or photomultiplier. The light output or luminescence may also be measured over time, for example in the same reaction chamber for a period of seconds, minutes, hours, etc. The light output or luminescence may be reported as the average over time, the half-life of decay of signal, the sum of the signal over a period of time, or the peak output. Luminescence may be measured in Relative Light Units (RLUs). For a pro-substrate compound, “stability” may also refer to how stable the compound is in certain conditions such that it maintains the ability to release the furimazine substrate gradually over certain time period (for example, by the actions of live cell enzymes) when used for live cell assays by a luciferase. Stability for the disclosed pro-coelenterazine compounds may be demonstrated by the percentage of degradation for the particular PRMG2-43218.601 compound in a specific environment over time. The percentage of purity for a particular compound can be determined by a variety of techniques known to those skilled in the art. These techniques include, for example, nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC). “Serum stability” refers to the stability of a compound in media or culture that includes serum. The serum as used herein may include, but is not limited to, fetal bovine serum (FBS). Serum stability of a compound as used herein generally may be characterized by the extent of degradation of such compound in a serum over a period of time. For example, a compound may degrade by less than 25%, less than 20%, less than 15%, or even less than 10% in FBS over a period of 24 hours or longer. For live cell assays, serum (such as FBS) is a component to maintain cell health. The degree of degradation of the disclosed compounds or the other pro-coelenterazine analogs in DMSO control, or DMEM media with / without FBS, can be monitored by HPLC using specific elution solvent / buffer (e.g., 0.1% TFA and acetonitrile) under the same condition at certain time of points. The percentage of purity can be calculated by the peak area of the tested compound divided by the total peak areas including tested compound and degradation compounds at their corresponding retention time at certain absorbance wavelength (e.g., 260 nm) at certain time point in media with / without FBS. The changes of purity for a given compound over time indicate the degree of instability of the compound in media / culture with / without serum. In some embodiments, compounds of formula (I) have improved water-solubility compared to the corresponding furimazine compound (i.e., furimazine or a derivative thereof). In particular, the tri-aspartic acid group is a polar moiety that imparts significantly higher water-solubility to the compounds. The improved water solubility allows more pro- substrate to be added to an assay system (e.g., in a method described herein), which can lead to an increase in overall signal level (due to more substrate being available for light generation), as well as longer assay time periods (as it takes longer to deplete the substrate). In some embodiments, the compounds have decreased cell permeability, such that only the released furimazine compound (i.e., furimazine or a derivative thereof) can enter the cell and produce a luminescence signal. This may provide advantages in assays (e.g., in methods described herein), for example, if the pro-substrate acts as an inhibitor of the luciferase enzyme. The decreased cell permeability of the pro-substrate would prevent its entry into the cell and thus prevent this potential inhibition. PRMG2-43218.601 Methods of Use The present compounds may be used in any way that luciferase substrates, e.g., coelenterazine and furimazine compounds, have been used. In particular, the present compounds may be used in live cell assays, including reporter assays, NanoBiT® assays and NanoBRET® assays, Annexin assays, and the like. When a stable pro-furimazine compound is used, the length of time for live-cell, non-lytic assays can be extended, e.g., from hours to days. Accordingly, compounds disclosed herein, including compounds of formula (I), may be used in a method for detecting luminescence in a sample, the method comprising: contacting a sample with a compound disclosed herein; contacting the sample with a coelenterazine-utilizing luciferase, if no coelenterazine-utilizing luciferase is present in the sample; and detecting luminescence. In some embodiments, the method further comprises a step of contacting the sample with a deprotection enzyme, if no deprotection enzyme is present in the sample or if levels are insufficiently low. Compounds of formula (I) can undergo simple hydrolysis to release the corresponding furimazine compound or derivative thereof, or may be cleaved by native enzymes already present in the sample, such as esterases. In other embodiments, addition of an exogenous deprotection enzyme may accelerate the process. A deprotection enzyme, as used herein, refers to an enzyme that is capable of converting the compound of formula (I) to furimazine (or a derivative thereof). Various deprotection enzymes may be used, for example, esterases. In certain embodiments, the sample comprises live cells. The live cells may include those from an animal (e.g., a vertebrate), a plant, a fungus, physiological fluid (e.g., blood, plasma, urine, mucus secretions), or cell culture. In certain embodiments, the sample comprises a coelenterazine-utilizing luciferase or a fragment complementary luciferase. In certain embodiments, compounds of formula (I) may be used as assay reagents. Assays using luciferases are well known in the art. Such assays are particularly useful for analyzing biological mechanisms or processes, such as gene expression and regulation in live cells. In some embodiments, cells are transfected with a nucleic acid encoding a luciferase, and the presence of luciferase is determined by the addition of reagents with cells, including the pro-furimazine analog. In some embodiments, a compound of formula (I) is used in a live cell assay comprising expression of a luciferase or a luciferase fusion protein (e.g., NanoLuc or other PRMG2-43218.601 coelenterazine-utilizing enzyme), where esterases present in living cells or the extracellular medium catalyze deprotection of the protected substrate. In some embodiments, a compound of formula (I) is used in live cell assays comprising protein-NanoLuc® fragment complementary assays for reporter gene expression and regulation in live cells. In some embodiments, a compound of formula (I) is used in a live cell assay comprising HiBiT fused to a protein of interest in cells expressing Large BiT (LgBiT) to continuously monitor changes in expression of the HiBiT-tagged protein in real time. In some embodiments, a compound of formula (I) is used in live cell assays comprising proteins of interest fused with NanoLuc® fragments for NanoLuc® complementary assays to investigate protein-protein interaction. In some embodiments, a compound of formula (I) is used in a live cell assay proteins of interest fused to Large BiT (LgBiT) and Small BiT (SmBiT) to investigate protein-protein interactions in the living cells. In some embodiments, a compound of formula (I) is used in a real time assay comprising Annexin-complementary NanoLuc® fragments (e.g., LgBiT and SmBiT fused to Annexin) for a real time apoptosis assay. Annexins are a family of calcium-dependent phospholipid-binding proteins. In healthy cells, phosphatidylserine is predominantly located along the cytosolic side of the plasma membrane. Phosphatidyl serine actively translocates to the extracellular membrane as a result of the induction of apoptosis. This early biomarker of the apoptotic cascade can be measured by NanoLuc® complementary fragment-fused Annexin V proteins (e.g., LgBiT and SmBiT fused to Annexin V) which bind to phosphatidylserine and can react with furimazine or coelenterazine to detect apoptotic cells by luminescence. In some embodiments, a compound of formula (I) is used in live cell bioluminescence resonance energy transfer (BRET) assay. BRET can be determined if two molecules are capable of binding each other or are co-localized in a cell. BRET involves the use of bioluminescent donor molecule and a fluorescent acceptor molecule. The molecules are chosen such that the emission wavelength of the donor is within the excitation spectra of the acceptor. Furthermore, the excitation spectrum of the donor and the emission spectrum of the acceptor should overlap minimally if at all. When the molecules are in close proximity, excitation of the donor leads to energy transfer to the acceptor, with subsequent emission from the acceptor at a longer wavelength (high acceptor:donor BRET ratio).. When the molecules are separated from each other, no energy transfer occurs (low acceptor:donor BRET ratio). By linking the donor to a first protein and the acceptor to a second protein or small molecule, molecular interactions can be determined by BRET. In preferred PRMG2-43218.601 embodiments, the donor is NanoLuc® luciferase and the acceptor is a fluorescent chloroalkane dye (for use with a HaloTag fusion protein) or a fluorescent dye fused to a small molecule to be used as a tracer for NanoBRET® Target Engagement (NanoBRET TE) applications. In some embodiments, a compound of formula (I) is used in an assay wherein a deprotection enzyme can be added as exogenous enzyme along with the addition of the assay reagents. Such assays may be useful in methods of releasing the active luminophore (e.g., furimazine or a derivative thereof) rapidly in a sample for certain live cell assays that require reaching the maximal brightness over a short time period but still require maintaining a reasonably stable signal over a certain time period. In certain embodiments, a compound of formula (I) is used for in vivo studies. Applications of the compounds to in vivo luminescent analysis will be readily apparent to those skilled in the art. In the embodiments involving live cell assays, fetal bovine serum (FBS) might be necessary component to maintain cell health in media or culture. Compounds of formula (I), by virtue of their increased stability in media containing FBS, may be used for more robust, live cell, luciferase-based assays. In still other embodiments, a sample (including cells, tissues, animals, etc.) containing a luciferase or fragment complimentary luciferase and a compound of formula (I) may be assayed using various microscopy and imaging techniques, e.g., in vivo imaging. In still other embodiments, a secretable luciferase is expressed in cells as part of a live-cell assay system. In certain embodiments, the present compounds can be used in a method for detecting luminescence in a transgenic animal. The method comprises administering a compound of formula (I) to a transgenic animal expressing a coelenterazine-utilizing luciferase, and detecting luminescence. Kits In certain embodiments, the compounds of formula (I) disclosed herein may be provided as part of a kit. The kit may include one or more luciferases (in the form of a polypeptide, a polynucleotide, or both) and a pro-furimazine compound of formula (I) along with suitable reagents and instructions to enable a user to perform assays such as those disclosed herein. The kit may also include one or more buffers such as those disclosed herein. PRMG2-43218.601 Examples In the Examples below, the following abbreviations are used: DCM is dichloromethane; DIPEA is N,N-diisopropylethylamine; DMF is N,N-dimethylformamide; EtOAc is ethyl acetate; HPLC is high pressure liquid chromatography; LRMS is low- resolution mass spectrometry; MeCN is acetonitrile; MPLC is medium pressure liquid chromatography; THF is tetrahydrofuran; and substrates D and E are as illustrated below. . Example 1 Compound Syntheses Synthesis of Compound 1 Substrate A was dissolved in dry THF, followed by adding bis(pentafluorophenyl)dicarbonate (1.1 equiv) and triethylamine (5 equiv) at room temperature. The mixture was stirred at room temperature for 3 min., followed by adding N- Methyl-3-butyn-1-amine (2 equiv). The resulting mixture was stirred at room temperature for 1 h, and the reaction mixture was concentrated in vacuo. The residue was purified by MPLC PRMG2-43218.601 (EtOAc in heptane) to afford the intermediate B (50 %). The product was characterized by LRMS (calculated [m + H]+= 559.2, found 559.1). Intermediate B was dissolved in DMF / H2O (4:1 by volume), followed by adding Asp- Asp-Asp-N3(2.0 equiv), sodium ascorbate (2.0 equiv), and CuSO4(20 mol%) at room temperature. The resulting mixture was stirred at room temperature for 2 h. The crude reaction mixture was purified by preparatory HPLC (C18 column, 10% formic acid (aq) in MeCN) to afford the desired product (46%) as a white powder. The product was characterized by LRMS (calculated [m + H]+= 1018.3, found 1018.4) Synthesis of Compound 2 The alkyl chloride linker was dissolved in dry DMF, followed by adding KI (3.0 equiv) and K2CO3 (3.0 equiv) at room temperature. The mixture was stirred at room temperature for3 h. To the reaction mixture, Substrate A was added at room temperature, and the reaction wasstirred at room temperature for 2 h. Upon completion, the reaction was concentrated in vacuo. The residue was purified by MPLC (EtOAc in heptane) to afford the intermediate C (83%). The product was characterized by LRMS (calculated [m + H]+= 659.3, found 659.1). The click chemistry protocol was the same as one for Compound 1. Compound 2 was afforded as a white powder (24%), and it was characterized by LRMS (calculated [m + H]+= 1118.3, found 1118.8). PRMG2-43218.601 Synthesis of Compound 3 Compound 3 was synthesized in the same method of Compound 2, except using substrate D as the substrate and triethylamine (3 equiv) in the alkylation step. The final product was isolated as a white powder (3.5%), and it was characterized by LRMS (calculated [m + H]+= 1101.4, found 1101.2) Synthesis of Compound 4 Compound 4 was synthesized in the same method of Compound 1, except using substrate D as the substrate. The final product was isolated as a white powder (57%), and it was characterized by LRMS (calculated [m + H]+= 1001.3, found 1001.3) Synthesis of Compound 5 Compound 5 was synthesized in the same method of Compound 2, except using substrate E as the substrate. The final product was isolated as a white powder (5.1%), and it was characterized by LRMS (calculated [m + H]+= 1050.4, found 1050.7) Synthesis of Compound 6 Compound 6 was synthesized in the same method of Compound 1, except using substrate E as the substrate. The final product was isolated as a white powder (22%), and it was characterized by LRMS (calculated [m + H]+= 950.3, found 950.6) Synthesis of Compound 7 Compound 7 was synthesized in the same method of Compound 1, except using N- methyl-N-(2-((5-morpholinopentyl)amino)ethyl)pent-4-ynamide as the coupling partner. The final product was isolated as a white powder (3.6%), and it was characterized by LRMS (calculated [m + H]+= 1243.5, found 1243.6) Synthesis of Compound 8 Compound 8 was synthesized in the same method of Compound 1, except using N- methyl-N-(2-((4-morpholinobutyl)amino)ethyl)pent-4-ynamide as the coupling partner. The final product was isolated as a white powder (15%), and it was characterized by LRMS (calculated [m + H]+= 1230.5, found 1230.4) PRMG2-43218.601 Synthesis of Compound 9 Compound 9 was synthesized in the same method of Compound 1, except using N- methyl-N-(2-((3-morpholinopropyl)amino)ethyl)pent-4-ynamide as the coupling partner. The final product was isolated as a white powder (9.9%), and it was characterized by LRMS (calculated [m - H]- = 1214.5, found 1214.9) Synthesis of Compound 10 Compound 10 was synthesized in the same method of Compound 2, except using Boc(Me)2Gly-Gly-Ala-N3 as the click chemistry partner. The final product was isolated as a white powder (5.1%), and it was characterized by LRMS (calculated [m + H]+= 1222.1, found 1200.0) Compound 11 was synthesized in the same method of Compound 1, except using chloromethyl 4-((tert-butoxycarbonyl)amino)-2,2-dimethylbutanoate as the alkylation partner, and triethylamine (3 equiv) as the base to afford intermediate F. In addition, the alkylation PRMG2-43218.601 product was deprotected by stirring the isolated product in TFA / DCM (1:1 by volume) at room temperature for 1 h, and the reaction mixture was concentrated in vacuo to afford the crude substrate, which was used in the following step without purification. The deprotected substrate in dry DMF was treated with commercially available AF594-SE (0.2 equiv) and DIPEA (5 equiv) at room temperature for 24 h, and the crude reaction mixture was purified by preparatory HPLC (C18 column, 10% formic acid (aq) in MeCN) to afford the desired product (3.3%) as a dark purple powder. The product was characterized by LRMS (calculated [m - H]- = 1295.4, found 1294.5) Compound 12 was synthesized in a similar manner to the one for synthesizing Compound 11, except Ac-Gly-Ph-SE was used in the second step. The crude product was purified by preparatory HPLC (C18 column, 10% formic acid (aq) in MeCN) to afford the desired product (10.8%) as a white powder. The product was characterized by LRMS (calculated [m + H]+= 839.3,found 839.6). Synthesis of Compound 13 Compound 13 was synthesized in the same method of Compound 2, except using N3- QPHNE as the click chemistry partner. The final product was isolated as a white powder (4.7%), and it was characterized by LRMS (calculated [m - H]+ = 1377.2, found 1377.6). PRMG2-43218.601 Synthesis of Compound 14 Compound 14 was synthesized in the same method of Compound 11, except using Trip9-SE as the coupling partner. The final product was isolated as a white powder (3.2%), and it was characterized by LRMS (calculated [m + 3H] / 3+ = 887.8, found 887.4; calculated [m + 4H] / 4+ = 666.1, found 887.4; calculated [m + 5H] / 5+ = 533.1, found 533.3; calculated [m + 6H] / 6+ = 444.4, found 444.1). Synthesis of Compound 15 Compound 15 was synthesized in the same method of Compound 2, except using SO3-PEG4-N3as the click chemistry partner. The final product was isolated as a white powder (16.6%), and it was characterized by LRMS (calculated [m + H]+ = 986.4, found 986.4). Synthesis of Compound 16 Compound 16 was synthesized in a similar manner to the one for synthesizing Compound 12. Compound G-2 was obtained from G by deprotecting with TFA and the crude product was concentrated in vacuo and then used directly without purification. Compound G- 2 (2.3 equiv) was dissolved in DMSO, followed by adding H (1 equiv) and Et3N (5 equiv). The crude product was purified by preparatory HPLC (C18 column, 10% TFA (aq) in MeCN) to afford the desired product (6.5%) as a white powder. The product was characterized by LRMS (calculated [m]- = 1431.54,found 1431.2). PRMG2-43218.601 Synthesis of Compound 17 Compound 17 was synthesized in the same method of Compound 2, except using (PO3H2)-PEG3-N3 as the click chemistry partner. The final product was isolated as a white powder (15.5%), and it was characterized by LRMS (calculated [m + H]+ = 942.3, found 942.7). Synthesis of Compound 18 Compound 18 was synthesized in the same method of Compound 2, except using beta-D-glocose-PEG4-N3 as the click chemistry partner. The final product was isolated as a white powder (12.6%), and it was characterized by LRMS (calculated [m + H]+ = 1040.4, found 1040.9). Synthesis of Compound 19 Compound 19 was synthesized in the same method of Compound 2, except using trehalose -N3 as the click chemistry partner. The final product was isolated as a white powder (4.8%), and it was characterized by LRMS (calculated [m + H]+ = 1026.4, found 1026.1). Synthesis of Compound 20 Compound 20 was synthesized in the same method of Compound 1, except using Substrate F as the substrate. The final product was isolated as a white powder (42.2%), and it was characterized by LRMS (calculated [m + H]+ = 1104.4, found 1104.5). Synthesis of Compound 21 Compound 21 was synthesized in the same method of Compound 2, except using Substrate F as the substrate. The final product was isolated as a white powder (57.0%), and it was characterized by LRMS (calculated [m + H]+ = 1004.3, found 1004.4). PRMG2-43218.601 Example 2 Assays To examine the efficiency of the above compounds, in vivo experiments were conducted in which NanoLuc is over expressed, and new pro-substrates Compound 1 and Compound 2 were used to generate bioluminescence signals over ca.5000 minutes. The commercially available pro-substrates Endurazine and Vivazine (Promega Corporation, Madison, WI) were also tested under the same conditions. The experimental results are shown in FIGS.1 and 2. As shown in FIG.1, when Compound 1 was used as the pro-substrate, and stable RLUs were observed over 5000 minutes as suggested by the flight curvature of the signal curve. In addition, compared with that of Vivazine, the signal becomes significantly higher after ca.1000 minutes. The data in FIG.1 also indicate that Compound 1 could potentially continue to generate signal while maintaining brightness for much longer time than Endurazine and Vivazine. As shown in FIG.2, when Compound 2 was used as the pro-substrate, and stable RLUs were observed over 5000 minutes as suggested by the flight curvature of the signal curve. In addition, compared with that of Vivazine, the signal becomes significantly higher after ca.1000 minutes. When compared with Endurazine, the Compound 2 signals are still higher on average over the course of the experiment. The data in FIG.2 also indicates Compound 2 could potentially continue to generate signal while maintaining brightness for much longer time than Endurazine and Vivazine. For both Compounds 1 and 2, there was no sign of a precipitate in the samples due to the much-improved solubility. Accordingly, even more prosubstrate could be introduced and thus further increase signal intensity and longevity.

Claims

PRMG2-43218.601 CLAIMS 1. A compound of formula (I):or a salt thereof, wherein: R1is selected from hydrogen, C1-C4alkyl, halo, and C1-C4haloalkyl; m is 0, 1, 2, 3, 4, or 5; each R2is independently selected from C1-C4 alkyl, halo, C1-C4 haloalkyl, -OR2a, - NR2bR2c, nitro, and cyano; n is 0, 1, 2, 3, 4, or 5; each R3is independently selected from C1-C4 alkyl, halo, C1-C4 haloalkyl, -OR3a, - NR3bR3c, nitro, and cyano; R2a, R2b, R2c, R3a, R3b, and R3care each independently selected from hydrogen, C1-C4alkyl, and C1-C4 haloalkyl; L is a linker; and M is a water soluble and cell impermeable moiety selected from a peptide, an alkyl or aryl sulfonate, an alkyl or aryl phosphonate, a monosaccharide, a disaccharide, and an oligosaccharide, wherein M is optionally substituted with a moiety of formula (A):

2. The compound of claim 1, or a salt thereof, wherein R1is hydrogen.PRMG2-43218.601 3. The compound of claim 1, or a salt thereof, wherein R1is methyl.

4. The compound of any one of claims 1-3, or a salt thereof, wherein m is 0, 1, or 2.

5. The compound of claim 4, or a salt thereof, wherein m is 0.

6. The compound of claim 4, or a salt thereof, wherein m is 1 or 2.

7. The compound of claim 6, or a salt thereof, wherein each R2is independently selected from halo, -OR2a, and -NR2bR2c, wherein R2a, R2b, R2care independently selected from hydrogen and methyl.

8. The compound of claim 7, or a salt thereof, wherein each R2is independently selected from fluoro and -NH2.

9. The compound of any one of claims 1-8, or a salt thereof, wherein n is 0, 1, or 2.

10. The compound of claim 9, or a salt thereof, wherein n is 0.

11. The compound of claim 9, or a salt thereof, wherein n is 1 or 2.

12. The compound of claim 11, or a salt thereof, wherein each R3is independently selected from halo and C1-C4alkyl.

13. The compound of claim 1, or a salt thereof, wherein the compound is a compound of formula (Ia):or a salt thereof, wherein:PRMG2-43218.601 R1is selected from hydrogen and C1-C4alkyl; R2xand R2yare independently selected from halo and -NR2bR2c; and R3is halo.

14. The compound of any one of claims 1-13, or a salt thereof, wherein M is a peptide.

15. The compound of claim 14, or a salt thereof, wherein M is a peptide comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

16. The compound of claim 14, or a salt thereof, wherein M is a peptide comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

17. The compound of claim 15 or claim 16, or a salt thereof, wherein the amino acids are independently selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, serine, cysteine, threonine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, histidine, lysine, and NN%NN&:?A;F>I@@IE?B;' 18. The compound of any one of claims 1-17, or a salt thereof, wherein M is a peptide having a formula selected from:PRMG2-43218.

601.

19. The compound of any one of claims 1-15, or a salt thereof, wherein M is a moiety of formula:PRMG2-43218.

601.

20. The compound of any one of claims 1-13, or a salt thereof, wherein M is an alkyl or aryl sulfonate moiety.

21. The compound of claim 19, wherein M is a moiety of formula:.

22. The compound of any one of claims 1-13, or a salt thereof, wherein M is an alkyl or aryl phosphonate moiety.

23. The compound of claim 22, or a salt thereof, wherein M is a -CH2CH2-PO3H2 group.

24. The compound of any one of claims 1-13, or a salt thereof, wherein M is a monosaccharide, a disaccharide, or a trisaccharide.

25. The compound of claim 24, or a salt thereof, wherein M is selected from:PRMG2-43218.60126. The compound of any one of claims 1-13, or a salt thereof, wherein M is selected from:PRMG2-43218.601PRMG2-43218.

601.

27. The compound of any one of claims 1-26, or a salt thereof, wherein L is a linker comprising any combination of moieties selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -C(O)-, -NH-, -NR’-, -O-, and monocyclic heteroarylene, wherein R’ is an optionally substituted C1- C6alkyl group, and wherein the linker comprises a total of about 5 to about 100 atoms.

28. The compound of claim 27, or a salt thereof, wherein L is a linker comprising any combination of moieties selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -C(O)-, -NH-, -NCH3-,29. The compound of any one of claims 1-26, or a salt thereof, wherein L is a linker comprising a group of formula –(OCH2CH2)q–, wherein q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

30. The compound of any one of claims 1-26, or a salt thereof wherein L is a linker having a formula selected from:PRMG2-43218.601 O31. The compound of claim 1, wherein the compound is selected from:PRMG2-43218.601PRMG2-43218.601PRMG2-43218.601PRMG2-43218.601PRMG2-43218.601PRMG2-43218.601PRMG2-43218.601PRMG2-43218.601PRMG2-43218.601and salts thereof.

32. A method for detecting luminescence in a sample, the method comprising: contacting a sample with a compound of any one of claims 1-31; contacting the sample with a coelenterazine-utilizing luciferase, if no coelenterazine- utilizing luciferase is present in the sample; and detecting luminescence in the sample.

33. The method of claim 32, wherein the sample comprises live cells.

34. The method of claim 32 or claim 33, wherein the sample comprises a coelenterazine- utilizing luciferase or a fragment complementary luciferase.

35. The method of claim 32 or claim 33, wherein the sample comprises live cells that express a coelenterazine-utilizing luciferase or a fragment complementary luciferase.

36. The method of any one of claims 32-35, further comprising contacting the sample with a deprotection enzyme.

37. The method of claim 36, wherein the deprotection enzyme comprises an esterase.

38. A method for detecting luminescence in a transgenic animal comprising administering a compound of any one of claims 1-31 to a transgenic animal; and detecting luminescence; wherein the transgenic animal expresses a coelenterazine-utilizing luciferase.

39. A kit comprising a compound of any one of claims 1-31.PRMG2-43218.601 40. The kit of claim 39, further comprising a luciferase or a fragment complementary luciferase.

41. The kit of claim 39 or claim 40, further comprising a deprotection enzyme.

42. The kit of claim 41, wherein the deprotection enzyme comprises an esterase.

43. The kit of any one of claims 39-42, further comprising a buffer reagent.