Coelenterazine analogues

Abstract

Coelenterazine analogs, methods for making the analogs, kits containing the analogs, and methods for using the compounds to detect luminescence in luciferase-based assays are described. For example, the compounds can be used for in vivo imaging.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 379,573, filed October 14, 2022, and U.S. Provisional Patent Application No. 63 / 457,624, filed April 6, 2023, each of which is incorporated by reference in its entirety.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates to coelenterazine analogs, methods for making coelenterazine analogs, and methods for using coelenterazine analogs in luciferase-based assays. [Background technology]

[0003] Bioluminescence assays are widely used to investigate cellular physiology, particularly processes related to gene expression. Luciferase reporter enzymes, in particular, are extremely valuable tools in this field, and to date, extensive protein engineering has been conducted to obtain small, environmentally insensitive luciferases that can be useful in bioluminescence assays. Efficient luciferase reporters exist that enable whole-cell biosensor measurements, drug discovery through high-throughput screening, and in vivo imaging. They also enable the study of protein-protein interactions, apoptosis, and cell viability in living cells. Luciferases using coelenterazine and coelenterazine analogs as substrates are one of the most widely used systems due to their brightness and acceptability in whole-cell applications. Summary of the Invention

[0004] Many known coelenterazine analogs have deficiencies that limit their effectiveness as luciferase substrates and their utility in luciferase-based assays. These deficiencies include cytotoxicity, light sensitivity, thermodynamic instability, low aqueous solubility, and low cell permeability. Therefore, there is a need for coelenterazine analogs with improved properties and methods for synthesizing the analogs.

[0005] In one aspect, disclosed herein is a compound of formula (I): [ka] or a tautomer thereof or a salt thereof, wherein R 1 teeth, [ka] where X is selected from O and S; R a is selected from hydrogen, fluoro, C1-C4 alkyl, and C1-C4 fluoroalkyl; R 2 is selected from H and F; R 3 is selected from H and F.

[0006] In some embodiments, R 1 teeth, [ka] In some embodiments, R 1 teeth, [ka] In some embodiments, R 1 teeth [ka] In some embodiments, R 1 teeth [ka] is.

[0007] In some embodiments, R 2 is H. In some embodiments, R 2 is F.

[0008] In some embodiments, R 3 is H. In some embodiments, R 3 is F.

[0009] In one aspect, disclosed herein is a method for treating a pulmonary arthritis, comprising: [ka] and tautomers thereof and salts thereof.

[0010] In one aspect, disclosed herein is a compound of the formula: [ka] The compound or a tautomer thereof or a salt thereof.

[0011] In one aspect, disclosed herein is a compound of the formula: [ka] or a tautomer or a salt thereof.

[0012] In one aspect, disclosed herein are kits that include a compound disclosed herein (e.g., a compound of Formula (I), or a specific compound disclosed herein, or a tautomer or salt thereof). In some embodiments, the kit further includes a luciferase. In some embodiments, the kit further includes a buffer reagent. In some embodiments, the kit further includes instructions for performing a luminescence assay. [Brief explanation of the drawings]

[0013] [Figure 1] A-E show the effect of furimazine analogs on the intensity of bioluminescence produced by purified NLuc-HaloTag fusions or chimeras (i.e., HT178-cpNLuc-179, generated by insertion of cpNLuc into HaloTag). A: Structures of furimazine analogs. B-C: Total bioluminescence and bioluminescence spectral scans of 6 nM NLuc-HaloTag fusions treated with 20 μM of either furimazine, fluorofurimazine, or compound 10. D-E: Total bioluminescence and bioluminescence spectral scans of 6 nM chimeras treated with 20 μM of furimazine, fluorofurimazine, or compound 10. [Figure 2] A-D show the effect of furimazine analogs on the intensity of bioluminescence produced by transiently expressed NLuc-HaloTag fusions or chimeras (i.e., HT178-cpNLuc-179, generated by insertion of cpNLuc into HaloTag). The structures of the furimazine analogs are the same as those shown in Figure 1A. A-B: Total bioluminescence and bioluminescence spectral scans of HeLa cells transiently expressing NLuc-HaloTag fusions and treated with 10 μM of either furimazine, fluorofurimazine, or compound 10. C-D: Total bioluminescence and bioluminescence spectral scans of HeLa cells transiently expressing chimeras and treated with 10 μM of furimazine, fluorofurimazine, or compound 10. [Figure 3] Figures A-F show additional data demonstrating the effect of furimazine analogs on the intensity of bioluminescence produced by transiently expressed NLuc-HaloTag fusions or chimeras (i.e., HT178-cpNLuc-179, generated by insertion of cpNLuc into HaloTag). The structures of the furimazine analogs are shown in the Examples. [Figure 4A] 1 shows data demonstrating the stability and purity of compound 6 in different reconstitution buffers. [Figure 4B] 1 shows data demonstrating the stability and purity of compound 6 in different reconstitution buffers. [Figure 4C] 1 shows data demonstrating the stability and purity of compound 6 in different reconstitution buffers. [Figure 5A]

[0033] Figure 1 shows additional data demonstrating the stability and purity of compound 6 in different reconstitution buffers. [Figure 5B]

[0033] Figure 1 shows additional data demonstrating the stability and purity of compound 6 in different reconstitution buffers. DETAILED DESCRIPTION OF THE INVENTION

[0014] Disclosed herein are coelenterazine analogs useful for luciferases and photoproteins found in various marine organisms, such as proteins that utilize coelenterazine (coelenterazine-utilizing enzymes) to generate light, including, but not limited to, cynomolgus luciferases (e.g., Renilla luciferase), jellyfish (e.g., aequorin from Aequorea jellyfish), and decapod luciferases (e.g., the luciferase complex of Oplophorus gracilirostris).

[0015] In some embodiments, the disclosed compounds exhibit improved aqueous solubility compared to furimazine. In some embodiments, the disclosed compounds exhibit improved bioluminescence signal kinetics compared to coelenterazine compounds with similar substitutions at the para-position of the 6-phenyl group. Thus, the compounds may be useful in in vivo luminescence imaging applications, as well as other applications that utilize bioluminescence.

[0016] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0017] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0018] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with measurement of the particular quantity). The modifier "about" should also be considered to disclose the range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses the range "2 to 4." The term "about" can refer to plus or minus 10% of the indicated number. For example, "about 10%" could indicate a range of 9% to 11%, and "about 1" could mean 0.9 to 1.1. Other meanings of "about" may be apparent from the context, such as rounding; for example, "about 1" could also mean 0.5 to 1.4.

[0019] Definitions of certain functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. (endpaper), and specific functional groups are generally defined as described therein. In addition, for general principles of organic chemistry and specific functional moieties and reactivity, see Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March's Advanced Organic Chemistry, 5 th Edition, John Wiley&Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the contents of each of which are incorporated herein by reference in their entirety.

[0020] The term "alkyl," as used herein, means a straight or branched saturated hydrocarbon chain. The term "Ci_4 alkyl" refers to a straight or branched saturated hydrocarbon containing from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0021] As used herein, the term "fluoroalkyl" refers to an alkyl group, as defined herein, in which one or more hydrogen atoms have been replaced with fluorine. Representative examples of fluoroalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, and 2,2,2-trifluoroethyl.

[0022] As used herein, "animal" refers to any vertebrate, including, but not limited to, mammals, amphibians, birds, fish, insects, reptiles, etc. Mammals can include, but are not limited to, humans, non-human primates (e.g., gorillas, monkeys, baboons, and chimpanzees, etc.), dogs, cats, goats, horses, pigs, cows, sheep, etc., and laboratory animals (e.g., rats, guinea pigs, mice, gerbils, hamsters, etc.). In some embodiments, animals can be human or non-human. Suitable animals include both males and females and animals of any age, including embryos (e.g., in utero or in ovo), infants, juveniles, adolescents, adults, and geriatric animals.

[0023] As used herein, "fusion protein" and "fusion polypeptide" refer to a fusion comprising at least one bioluminescent protein in combination with a heterologous protein of interest, such as a fluorescent protein, as part of a single continuous chain of amino acids, which does not occur in nature.

[0024] As used herein, "operably linked" means that the expression of a gene is under the control of a spatially connected promoter. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As known to those skilled in the art, variations in this distance can be accommodated without loss of promoter function.

[0025] As used herein, "transgene" refers to a gene or genetic material containing a genetic sequence that has been isolated and / or manipulated from one organism and introduced into a different organism. A transgene may comprise a transgenic sequence or a native or wild-type DNA sequence. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism. For example, a transgene may encode a fusion protein, such as a fusion protein containing luciferase. A transgenic sequence may be partially or completely species-heterologous, i.e., the transgenic sequence or a portion thereof may originate from a different species than the cell into which it is introduced.

[0026] "Transgenic animal" refers to a genetically engineered animal or the offspring of a genetically engineered animal. Transgenic animals typically contain genetic material from at least one unrelated organism, such as a virus, plant, or other animal.

[0027] The terms "transformation," "transfect," and "transduction," used interchangeably herein, refer to the introduction of a heterologous nucleic acid molecule, such as genetic material, into a cell. Such introduction into a cell can be stable or transient. Thus, in some embodiments, a host cell or host organism is stably transformed with a heterologous nucleic acid molecule, such as genetic material. In other embodiments, a host cell or host organism is transiently transformed with a heterologous nucleic acid molecule, such as genetic material. "Transient transformation," in the context of a polynucleotide, means that the polynucleotide is introduced into a cell and is not integrated into the genome of the cell. "Stably introducing" or "stably introduced," with respect to a polynucleotide introduced into a cell, intends that the introduced polynucleotide is stably integrated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide. As used herein, "stable transformation" or "stably transformed" means that a nucleic acid molecule is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule can be inherited by its progeny, and more particularly, by multiple successive progeny. As used herein, "genome" also includes nuclear, plasmid, and plastid genomes, and thus includes, for example, integration of a nucleic acid construct into a chloroplast genome or a mitochondrial genome. As used herein, stable transformation can also refer to a transgene that is maintained extrachromosomally, for example, as a minichromosome or plasmid. In some embodiments, nucleotide sequences, constructs, expression cassettes can be transiently expressed and / or they can be stably integrated into the genome of a host organism.

[0028] In the compounds described herein, groups and substituents thereof may be selected according to the allowed valences of atoms and substituents so that the selection and substitution results in stable compounds that do not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like.

[0029] In the recitation of numerical ranges herein, each intervening number is expressly contemplated to the same precision. For example, in the range of 6 to 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are also expressly contemplated.

[0030] 2.Compound Disclosed herein are compounds of formula (I): [ka] or a tautomer thereof or a salt thereof, wherein: R 1 teeth, [ka] where X is selected from O and S; R a is selected from hydrogen, fluoro, C1-C4 alkyl, and C1-C4 fluoroalkyl; R 2 is selected from H and F; R 3 is selected from H and F.

[0031] In some embodiments, X is O. In some embodiments, X is S. In some embodiments, R a is hydrogen. In some embodiments, R a is C1-C4 alkyl (e.g., methyl). In some embodiments, R a is fluoro.

[0032] In some embodiments, R 1 teeth, [ka] In some embodiments, R 1 teeth [ka] In some embodiments, R 1 teeth [ka] In some embodiments, R 1 teeth [ka] In some embodiments, R 1 teeth [ka] is.

[0033] In some embodiments, R 2 is H. In some embodiments, R 2 is F.

[0034] In some embodiments, R 3 is H. In some embodiments, R 3 is F.

[0035] In some embodiments, R 2 is F and R 3 is H. In some embodiments, R 2 is H and R 3 is F. In some embodiments, R 2 is F and R 3 is F.

[0036] Disclosed herein is a method for producing a medicament for the treatment of a pulmonary arthritis. [ka] and tautomers thereof and salts thereof.

[0037] In some embodiments, the compound is [ka] or a tautomer thereof or a salt thereof.

[0038] In some embodiments, the compound is [ka] or a tautomer thereof or a salt thereof.

[0039] In some embodiments, the compound is [ka] or a tautomer thereof or a salt thereof.

[0040] Compound names are assigned using the Struct=Name naming algorithm as part of CHEMDRAW® ULTRA.

[0041] Compounds may exist as stereoisomers where asymmetric or chiral centers exist. Stereoisomers are "R" or "S" depending on the configuration of substituents around the chiral carbon atom. As used herein, the terms "R" and "S" refer to the configurations defined below: IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45:13-30. The present disclosure contemplates various stereoisomers and mixtures thereof, which are specifically included within the scope of the present invention. Stereoisomers include enantiomers and diastereomers, as well as mixtures of enantiomers or diastereomers. Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials containing asymmetric or chiral centers, or by preparing racemic mixtures followed by resolution methods known to those skilled in the art. These resolution methods are exemplified by: (1) coupling the enantiomeric mixture to a chiral auxiliary, separating the resulting diastereomeric mixture by recrystallization or chromatography, and, optionally, liberating the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, "Vogel's Textbook of Practical Organic Chemistry," 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England; or (2) directly separating the mixture of optical enantiomers on a chiral chromatographic column; or (3) fractional recrystallization.

[0042] It should be understood that the compounds may have tautomeric and geometric isomeric forms and that these also form aspects of the present invention. The compounds of the present invention or tautomers or salts thereof include the compounds, salts of the compounds, tautomers of the compounds, and tautomers of salts of the compounds.

[0043] The present disclosure also includes isotopically labeled compounds that are identical to those recited in Formula (I) or to the specific compounds exemplified herein, except for the replacement of one or more atoms with an atom having an atomic mass or mass number different from that normally found in nature. Examples of isotopes suitable for incorporation into compounds of the present invention include, but are not limited to, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36 The elements are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as Cl. Deuterium, i.e., 2 Substitution with heavier isotopes, such as H, may result in certain therapeutic benefits, such as increased in vivo half-life or reduced required dosage, resulting from greater metabolic stability, and may therefore be preferred in some cases. Positron-emitting isotopes can be incorporated into compounds for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that can be incorporated into compounds include: 11 C. 13 N, 15 O, and 18 F. 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, substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent.

[0044] The compounds described herein can be in the form of salts. The selection of a salt that is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and that is commensurate with a reasonable benefit / risk ratio, is within the normal scope of sound medical judgment. Pharmaceutically acceptable salts are known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or 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, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, Examples of the salts include 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, and valerate.Base addition salts can be prepared during the final isolation and purification of the disclosed compounds by reaction of the carboxyl group with a suitable base (e.g., hydroxide, carbonate, or bicarbonate) of a metal cation (e.g., 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.

[0045] A. Compound Properties The disclosed compounds can be substrates for luciferase to generate luminescence. The compounds can have improved aqueous solubility, improved stability, improved cell permeability, increased biocompatibility with cells, reduced self-luminescence, and / or reduced toxicity.

[0046] "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. Light output can be measured as an instantaneous or near-instantaneous measurement of light output at the start of the luminescence reaction, which may begin upon addition of the coelenterazine substrate (sometimes referred to as "T=0" luminescence or "flash"). In various embodiments, the luminescence reaction is carried out in solution. In other embodiments, the luminescence reaction is carried out on a solid support. The liquid comprises, for example, a lysate from 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 the extracellular medium, obviating the need to produce a lysate. In some embodiments, the reaction is initiated by injecting appropriate materials, e.g., a coelenterazine analog, buffer, etc., into a reaction chamber (e.g., a well of a multiwell plate, such as a 96-well plate) containing the luminescent protein. In yet other embodiments, luciferase and / or a coelenterazine analog (e.g., a compound disclosed herein) are introduced into a host, and luminescence measurements are made in the host or a portion thereof, which may include a whole organism or a cell, tissue, explant, or extract thereof. The reaction chamber can be placed in a reader that can measure light output, for example, using a luminometer or photomultiplier tube. Light output or luminescence may be measured over time, for example, over a period of seconds, minutes, hours, etc., in the same reaction chamber. Light output or luminescence may be reported as an average over time, a signal decay half-life, a sum of the signal over a period of time, or a peak output. Luminescence can be measured as relative light units (RLU).

[0047] In some embodiments, the disclosed compounds may have an RLU of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more compared to coelenterazine or known coelenterazine analogs such as furimazine.

[0048] In some embodiments, the disclosed compounds can have a λ of 450-700 nanometers, 460-600 nanometers, 470-600 nanometers, 480-600 nanometers, 490-600 nanometers, 500-600 nanometers, 510-600 nanometers, 520-600 nanometers, 530-600 nanometers, 540-600 nanometers, 550-600 nanometers, 560-600 nanometers, 570-600 nanometers, 580-600 nanometers, 590-600 nanometers, 470-590 nanometers, 480-580 nanometers, 490-570 nanometers, 500-560 nanometers, or 510-550 nanometers. The compounds disclosed herein can have a λ of 450 nanometers or greater, 460 nanometers or greater, 470 nanometers or greater, 480 nanometers or greater, 490 nanometers or greater, 500 nanometers or greater, 510 nanometers or greater, 520 nanometers or greater, 530 nanometers or greater, 540 nanometers or greater, 550 nanometers or greater, 560 nanometers, 570 nanometers or greater, 580 nanometers or greater, 590 nanometers or greater, 600 nanometers or greater, 610 nanometers or greater, 620 nanometers or greater, 630 nanometers or greater, 640 nanometers or greater, 650 nanometers or greater, 660 nanometers or greater, 670 nanometers or greater, 680 nanometers or greater, 690 nanometers or greater, or 700 nanometers or greater.

[0049] "Biocompatibility" refers to the resistance of a cell (e.g., a prokaryotic or eukaryotic cell) to a coelenterazine analog (e.g., a compound disclosed herein). The biocompatibility of a coelenterazine analog is related to the stress it causes to the host cell.

[0050] The enhanced biocompatibility of a coelenterazine analog (e.g., a compound disclosed herein) can be determined by measuring cell viability and / or cell proliferation rate. For example, the enhanced biocompatibility of a coelenterazine analog can be determined by measuring cell viability in the absence of luciferase expression of cells exposed to the coelenterazine analog compared to native or known coelenterazine to determine how compatible and / or toxic the coelenterazine analog is to cells.

[0051] In particular, enhanced biocompatibility may be measured by cell viability assays (e.g., using the CELLTITER-GLO® Luminescent Cell Viability assay), apoptosis assays (e.g., using CASPASE-GLO® technology), or other methods known in the art. The effects of the disclosed compounds on cell viability or apoptosis can be compared to the effects of naturally occurring or known coelenterazine analogs on cell viability or apoptosis.

[0052] Enhanced biocompatibility can also be determined by measuring the effect of a coelenterazine analog (e.g., a compound disclosed herein) on cell proliferation or gene expression. For example, the enhanced biocompatibility of a compound disclosed herein can be determined by measuring cell number after a period of time, or by comparing the expression of stress response genes in a cell sample exposed to a compound disclosed herein with cells exposed to a natural or known coelenterazine, or to a compound without coelenterazine. The effect of a disclosed compound on cell proliferation or gene expression can be compared with that of a natural or known coelenterazine.

[0053] B. Synthesis method The compounds disclosed herein may be prepared by synthetic processes or by metabolic processes, including those that occur within the human or animal body (in vivo) or in vitro.

[0054] The compounds disclosed herein can be synthesized by a variety of methods, including those illustrated in the Examples. Optimal reaction conditions and reaction times for each individual step may vary depending on the specific reactants employed and the substituents present in the reactants. The reactions can be worked up in a conventional manner, for example, by removing the solvent from the residue, and further purified according to methodologies generally known in the art (e.g., but not limited to, crystallization, distillation, extraction, trituration, and chromatography). Unless otherwise noted, starting materials and reagents are commercially available or can be prepared by one of ordinary skill in the art from commercially available materials using methods described in the chemical literature. If the starting materials are not commercially available, they can be prepared by procedures selected from standard organic chemistry techniques, techniques analogous to the synthesis of known structurally similar compounds, or procedures analogous to those described in the Schemes or Synthetic Examples sections above.

[0055] Routine experimentation, including appropriate manipulation of reaction conditions, reagents, and sequence of synthetic routes, protection of chemical functional groups incompatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the method, is within the scope of the present invention. Suitable protecting groups and methods for protecting and deprotecting various substituents using such suitable protecting groups are known to those skilled in the art, and examples can be found in: PGM Wuts and TW Greene, in Greene's book titled "Protective Groups in Organic Synthesis" (4th ed.), John Wiley & Sons, NY (2006) (incorporated herein by reference in its entirety). Synthesis of the compounds of the present invention can be achieved by methods similar to those described in the above synthetic schemes and specific examples.

[0056] If an optically active form of a disclosed compound is required, it can be obtained by carrying out one of the procedures described herein using optically active starting materials (prepared, for example, by asymmetric induction in an appropriate reaction step), or by resolving a stereoisomeric mixture of the compound or intermediate using standard procedures (such as chromatographic separation, recrystallization, or enzymatic resolution).

[0057] Similarly, if a pure geometric isomer of a compound is required, it can be obtained by carrying out one of the procedures described above using the pure geometric isomer as the starting material, or by resolving a geometric isomeric mixture of the compound or intermediate using standard procedures such as chromatographic separation.

[0058] It can be understood that the synthetic schemes and specific examples described are illustrative and should not be construed as limiting the scope of the invention as defined by the appended claims. All alternatives, modifications, and equivalents of the synthetic methods and specific examples are intended to be included within the scope of the claims.

[0059] 3. Instructions for use and kit The compounds of the present disclosure can be used in any method in which luciferase substrates, such as coelenterazine analogs, are used. For example, they can be used in bioluminescence assays that use coelenterazine analogs to detect one or more molecules, such as enzymes, enzyme reaction cofactors, enzyme substrates, enzyme inhibitors, enzyme activators, or OH radicals, or one or more conditions, such as redox conditions, in a sample. Samples can include animals (e.g., vertebrates), plants, fungi, physiological fluids (e.g., blood, plasma, urine, mucus secretions), cells, cell lysates, cell supernatants, or purified fractions of cells (e.g., subcellular fractions). The presence, amount, spectral distribution, luminescence kinetics, or specific activity of such molecules can be detected or quantified. Molecules can be detected or quantified in solution, including multiphase solutions (e.g., emulsions or suspensions), or on solid supports (e.g., particles, capillaries, or assay vessels).

[0060] In certain embodiments, the compounds disclosed herein can be used to quantify molecules of interest. In some embodiments, coelenterazine analogs (e.g., natural or known coelenterazines or compounds disclosed herein) can be used as probes for specific biochemical activities, such as apoptosis or drug metabolism.

[0061] In certain embodiments, the compounds disclosed herein can be used with inhibitors of Oplophorus luciferase and / or Oplophorus-luciferase derived bioluminescence complexes. Exemplary inhibitors of Oplophorus luciferase and / or Oplophorus-luciferase derived bioluminescence complexes are described, for example, in International Patent Publication Nos. WO 2016 / 210294, WO 2018 / 125992, WO 2019 / 232384, and WO 2019 / 213119, each of which is incorporated by reference in its entirety.

[0062] In certain embodiments, the compounds disclosed herein can be used to detect luminescence in live cells, e.g., in vivo. In some embodiments, luciferase can be expressed in cells (as a reporter or otherwise), and the cells are treated with a coelenterazine analog (e.g., a compound disclosed herein), which permeates the cells in culture, reacts with luciferase, and emits light. In addition to being cell-permeable, the compounds disclosed herein exhibit biocompatibility comparable to native coelenterazine in terms of cell viability. In some embodiments, compounds disclosed herein are synthesized containing chemical modifications known to increase the stability of native coelenterazine in culture media and can be used in more robust live-cell luciferase-based reporter assays. In still other embodiments, samples (including cells, tissues, animals, etc.) containing luciferase and the compounds disclosed herein can be assayed using various microscopy and imaging techniques, e.g., in vivo imaging. In still other embodiments, secreted luciferase is expressed in cells as part of a live-cell reporter system.

[0063] In certain embodiments, the compounds disclosed herein may be provided as part of a kit. In some embodiments, the kit may include one or more luciferases (in the form of a polypeptide, polynucleotide, or both) and a coelenterazine analog disclosed herein, 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. In some embodiments, the kit may further include an inhibitor of the Oplophorus luciferase and / or Oplophorus-luciferase bioluminescence complex described above.

[0064] Buffers include citric acid or citrate buffer, MES, 1,4-piperazineditanesulfonic acid, or HEPES; inorganic phosphates (e.g., in the form of pyrophosphate or potassium phosphate); chelating agents such as EDTA, CDTA, or 1,2-diaminocyclohexanetetraacetic acid; salts such as sodium fluoride, magnesium sulfate; surfactants or detergents such as TERGITOL® (e.g., non-ionic nonylphenol ethoxylate), dodecyltrimethylammonium bromide (DTAB), or THESIT® (hydroxypolyethoxydodecane); defoamers such as INDUSTROL® DF204 (organic defoamer) or MAZU® DF (silicone defoamer); protein stabilizers such as gelatin, PRIONEX® 10% (gelatin, type A) or albumin (e.g., BSA, HSA), or glycerol; adenosine triphosphate (ATP) or adenosine monophosphate (AMP). Other components may include polyethylene glycol, polyvinylpyridine, crown ethers, or cyclodextrins.

[0065] In vivo imaging The compounds of the present disclosure can be used for imaging live cells, such as in vivo and ex vivo bioluminescence imaging. For example, the compounds of the present disclosure can be used with luciferase-based coelenterazine for bioluminescence imaging of tissue sections or cells in live animals. In vivo bioluminescence imaging is a versatile and sensitive tool based on the detection of emitted light from cells or tissues. Bioluminescence has been used to noninvasively track tumor cells, bacterial and viral infections, gene expression, and treatment responses. Bioluminescence imaging provides longitudinal monitoring of disease progression in the same animal, which is a desirable alternative to analyzing multiple animals at multiple time points during the disease course. In some embodiments, the compounds of the present disclosure can be used in vivo to observe biological processes such as cell movement, tumor progression, gene expression, and viral infection in various animal models. In some embodiments, the compounds of the present disclosure can be used for imaging in transgenic animals, such as transgenic mice. Transgenic animals containing cells or tissues can represent models of cellular function and disease in humans. These animals are therefore useful for studying the mechanisms behind cellular function and related events, for generating and testing products (e.g., antibodies, small molecules, etc.), and for treating and diagnosing related human diseases, including cancer and autoimmune conditions. In some embodiments, transgenic animals may further provide an indication of the safety of particular drugs for administration to humans. The effects of drugs can be studied by administering specific drugs and compounds of the present disclosure to specific cells or systemically and performing bioluminescence imaging to examine the specific effects. The animal- and cell-based models and compounds of the present disclosure can be used to identify drugs, pharmaceuticals, therapies, and interventions that may be effective in treating disease.

[0066] In some embodiments, compounds of the present disclosure can be used for bioluminescence imaging of cells or animals transformed to express a fusion protein, such as a fusion protein containing luciferase. In some embodiments, the transgenic animal or cell can express a fusion protein containing luciferase. In some embodiments, the luciferase can be a coelenterazine-utilizing luciferase, such as Oplophorus or Oplophorus-derived luciferase, Renilla luciferase, Gaussia luciferase, such as Gaussia princeps luciferase, Metridia luciferase, such as Metridia longa and Metridia pacifica luciferase, Vargula luciferase, Vargula hilgendorfii luciferase, Pleuromamma xiphias luciferase, and variants, recombinants, and mutants thereof. In some embodiments, the polynucleotide sequence encoding the fusion protein is operably linked to a promoter. In some embodiments, the promoter can be a constitutive promoter, an inducible promoter, a compact promoter, or a regulatable promoter. In some embodiments, the promoter may also be a tissue-specific promoter.

[0067] In some embodiments, fusion proteins of a bioluminescent protein and a heterologous protein of interest, such as a fluorescent protein, may be directly linked to each other by a peptide bond or separated by an intervening amino acid sequence. In some embodiments, the fusion polypeptide may also contain sequences exogenous to the heterologous protein of interest, such as the bioluminescent protein and the fluorescent protein. For example, the fusion protein may include a targeting or localization sequence, a tag sequence, the sequence of another fluorescent or bioluminescent protein, or another chromophore. In some embodiments, the targeting sequence may localize the fusion protein to a particular tissue, cell type (e.g., muscle, heart, or neuronal cell), cellular compartment (e.g., mitochondria or other organelles, nucleus, cytoplasm, or plasma membrane), or protein. Furthermore, fusions may include sequences from multiple fluorescent or bioluminescent proteins, or variants thereof, and / or other selected proteins. In some embodiments, luciferase is fused to a HALOTAG® protein or a fluorescent protein, such as green fluorescent protein (GFP), red fluorescent protein (RFP), or orange fluorescent protein.

[0068] Bioluminescence produced in cells, such as cells of transgenic animals, can be imaged or detected by various means known in the art. For example, fusion proteins and compounds of the present disclosure that have localized to their intended sites in transgenic animals can be imaged in several ways. A reasonable estimate of the time required to achieve localization can be made by those skilled in the art. Furthermore, the bioluminescence produced from the fusion proteins and compounds of the present disclosure can be imaged after localization as a function of time. Because imaging, or measuring photon emission from a subject, can last up to several tens of minutes, transgenic animals can be immobilized during the imaging process.

[0069] In vivo imaging can be performed using the naked eye or any type of camera (still or video). Imaging bioluminescence, for example, involves the use of a photodetector that can detect extremely low levels of light (generally single photon events) and integrate the photon emissions until an image can be constructed. Examples of such highly sensitive photodetectors include devices that enhance single photon events before they are detected by the camera, and cameras (e.g., cooled with liquid nitrogen) that can detect single photons above the background noise inherent in the detection system. The "photodetection device" used must be sensitive enough to enable imaging of low-level light from within a mammal within a reasonable time and to use the signal from such a device to construct an image.

[0070] Bioluminescent signals can be detected with a highly sensitive charge-coupled device (CCD) camera. In certain embodiments, an intensified CCD camera is used for imaging, which is sensitive enough to detect bioluminescent signals and has a wide enough dynamic range to also detect fluorescent signals. Suitable cameras are known in the art and include, but are not limited to, the Olympus LV200 Bioluminescent Imaging System, an integrated imaging system (IVIS™ Imaging System, Caliper Life Sciences) controlled using LivingImage™ software (Caliper Life Sciences), or a custom-built two-photon fluorescence lifetime imaging microscope (Yasuda Curr Opin Neurobiol. 2006;16:551-561). In some embodiments, the camera is mounted in a light-tight container that provides anesthesia, a platform for the animal, such as a mouse, and internal illumination.

[0071] In vivo imaging can be non-invasive whole animal imaging, as described herein (Contag, C., US Pat. No. 5,650,135, July 22, 1997; Contag, P., et al., Nature Medicine 4(2):245-247, 1998; Contag, C., et al., OSA TOPS on Biomedical Optical Spectroscopy and Diagnostics 3:220-224, 1996; Contag, CH, Photochemistry and Photobiology 66(4):523-531, 1997; Contag, CH, et al., Molecular Microbiology 18(4):593-603, 1995). The sensitivity of detecting light emitted from internal organs depends on several factors, including the expression level of luciferase, the depth of the labeled cells in the body (the distance photons must travel through the tissue), and the sensitivity of the detection system.

[0072] "Photon amplification devices" amplify photons before they enter the detection screen. This class includes CCD cameras equipped with intensifier tubes, such as microchannel intensifiers. Microchannel intensifiers typically contain a metal array of channels that are perpendicular to and coextensive with the camera's detection screen. The microchannel array is placed between the camera and the specimen, subject, or animal to be imaged. Photons entering the channels of the array mostly contact the sides of the channels before exiting. When a voltage is applied across the array, many electrons are released from each photon collision. Electrons from such collisions exit the channels in a "shotgun" pattern and are detected by the camera.

[0073] The image processor processes the signals generated by the photon-counting light-detecting device to construct an image that can be displayed on a monitor or printed on a video printer, for example. Such image processors are typically sold as part of a system that includes the sensitive photon-counting camera described above, and are therefore available from the same sources. The image processor is typically connected to a personal computer, such as an IBM-compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif.), and may or may not be included as part of a purchased image processing system. Once the image is in digital file format, it can be processed with various image processing programs (such as "ADOBE PHOTOSHOP," Adobe Systems, Mt. View, Calif.) and printed.

[0074] It will be appreciated that the entire animal or subject does not necessarily need to be within the detection field of the light detection device. For example, if one is measuring a fusion protein targeted to a particular region of a subject, it is necessary to measure only the light from that region, and a sufficient surrounding "dark" zone, to obtain the desired information.

[0075] Once the photon emission image is generated, it is typically overlaid on a "normal" reflected light image of the object to provide a frame of reference for the source of the emitted photons (i.e., localize the fusion protein with respect to the object). The "composite" image formed by the overlay of the photon emission image and the reflected light image is then analyzed to determine the location and / or quantity of the target within the object.

[0076] Bioluminescence Resonance Energy Transfer (BRET) The disclosed compounds can be used in any method for detecting ligand-protein and / or protein-protein interactions. In some embodiments, the disclosed compounds can be used in in vivo or in vitro bioluminescence resonance energy transfer (BRET) systems. With BRET, energy transfer from a bioluminescent donor to a fluorescent acceptor results in a shift in the spectral distribution of light emission. This energy transfer can enable real-time monitoring of protein-protein or ligand-protein interactions in vitro or in vivo, including the interaction and dissociation of partners. Examples of BRET systems, such as the NanoBRET™ system, are described, for example, in U.S. Pat. No. 10,024,862, U.S. Patent Publication No. 2014 / 0194307, U.S. Patent No. 10,067,149, and U.S. Patent Publication No. 2014 / 0194325.

[0077] In some embodiments, the luciferase used in BRET analysis can be used to determine whether two molecules can bind to each other or colocalize within a cell. For example, the luciferase can be used as a bioluminescent donor molecule combined with a molecule or protein of interest to create a first fusion protein. In some embodiments, the luciferase can be conjugated to an antibody, protein, receptor, drug, drug carrier, peptide, sugar, fatty acid, nanoparticle, or other biomolecule. In various embodiments, the first fusion protein contains the luciferase and the protein of interest. In various embodiments, the luciferase-containing first fusion protein can be used in BRET analysis to detect protein / protein interactions in systems including, but not limited to, cell lysates, intact cells, and live animals. In some embodiments, the BRET analysis can also include an inhibitor of the Oplophorus luciferase and / or the Oplophorus-luciferase bioluminescent complex described above.

[0078] In some embodiments, the fluorescent acceptor can be a fluorophore, such as a fluorescent protein, a fluorescent molecule, a fluorescent label, or a fluorescent tracer. In some embodiments, the fluorescent tracer can be a small molecule tagged with a fluorophore. In some embodiments, the fluorescent acceptor can be a second fusion protein comprising the fluorescent acceptor conjugated to an antibody, protein, receptor, drug, drug carrier, peptide, sugar, fatty acid, nanoparticle, or other biomolecule.

[0079] In various embodiments, HALOTAG® can be used as a fluorescent acceptor molecule. In some embodiments, HALOTAG® can be fused to a second protein of interest or a luciferase. For example, a luciferase can be fused to HALOTAG®, expressed in a cell or animal, and labeled with a fluorescent HALOTAG® ligand, such as the HALOTAG® TMR ligand. In another example, a luciferase can be fused to a fluorescent protein and expressed in a cell or animal. In some embodiments, BRET can be performed using a luciferase in combination with a fluorescent protein, including, but not limited to, GFP, RFP, orange fluorescent protein, or a fluorescent label, including, as some non-limiting examples, fluorescein, rhodamine green, Oregon green, or Alexa 488.

[0080] In some embodiments, the disclosed compounds can be used in target binding assays, such as the NANOBRET™ Target Engagement (TE) Assay, to measure compound binding at a selected target protein, such as a drug:target interaction, in intact cells in real time. For example, a NANOBRET™ TE Assay can include four components: an expressed cellular target protein fused to the bright NANOLUC® luciferase; a cell-permeable fluorescent tracer that specifically binds to the target protein; one or more compounds of the present disclosure used as substrates for NANOLUC® luciferase; and a cell-impermeable inhibitor of NANOLUC® luciferase. The assay uses bioluminescence resonance energy transfer (BRET), which is achieved by transferring luminescent energy from NANOLUC® luciferase to a fluorescent tracer bound to the target protein-NANOLUC® fusion. This energy transfer allows for direct measurement of compound binding affinity and compound-target residence time.

[0081] In some embodiments, compounds are applied to cells that can specifically bind to the intracellular target protein-NANOLUC™ fusion, resulting in a decrease in BRET. In some embodiments, to ensure accurate assessment of intracellular target engagement, NANOLUC® inhibitors can be used to mitigate any extracellular NANOLUC® signal that may arise from cells compromised during manipulation, without adversely affecting NANOLUC® luciferase expressed in healthy, living cells.

[0082] The BRET system may further comprise a photodetector or imaging device for detecting light emitted from the bioluminescent fusion protein, such as, but not limited to, a light microscope, a digital microscope, a luminometer, a charge-coupled device (CCD) image sensor, a complementary metal-oxide semiconductor (CMOS) image sensor, or a digital camera.

[0083] Formulation and Administration For whole animal testing, the disclosed imaging probe is preferably formulated for parenteral administration.Parenteral formulation can be prepared as aqueous composition by using techniques known in the art.Generally, this composition is prepared as solution or suspension; solid form suitable for preparing solution or suspension when adding reconstitution medium; emulsion, such as water-in-oil (w / o) emulsion, oil-in-water (o / w) emulsion and their microemulsions, liposome or emulsome.

[0084] The term "parenteral," as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0085] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and mixtures thereof.

[0086] Solutions and dispersions of the active compounds as free acids or bases or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersion medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersing agents, emulsifying agents, pH-adjusting agents, and combinations thereof.

[0087] Suitable surfactants may be anionic, cationic, amphoteric, or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Anionic surfactants include sodium, potassium, and ammonium long-chain alkyl and alkylaryl sulfonates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-(ethylthiol)-sulfosuccinate); and alkyl sulfates, such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyldimethylbenzylammonium chloride, polyoxyethylene, and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate. , propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000® cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0088] The formulation may contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent the decomposition of the active agent(s).

[0089] Typically, the formulation, upon reconstitution, is buffered for parenteral administration to a pH of 3 to 8. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0090] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0091] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent or dispersion medium with one or more of the excipients listed above, and then optionally sterilizing by filtration.Generally, dispersions can be prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above.For the preparation of sterile injectable solutions, the preferred method for preparing sterile powders is vacuum drying and freeze-drying technology, which can obtain a powder containing the active ingredient and any desired additional ingredients from a solution that has been previously sterile-filtered.Powder can be prepared in a way that the particles are inherently porous, which can facilitate the dissolution of the particles.The method for producing porous particles is known in the art.

[0092] 4. Working Example Example 1. Compound synthesis and characterization The compounds were prepared using the synthetic route shown in Scheme 1, previously described (Su et al. Nat Methods 17, 852-860 (2020); Shakhmin et al. Chemistry 22, 10369-10375 (2016)). Abbreviations in Scheme 1 include: ACN is acetonitrile, CDI is carbonyldiimidazole, DMA is dimethylacetamide, DCM is dichloromethane, eq is equivalents, h is hour(s), MeOH is methanol, min is minutes, rt is room temperature, TFA is trifluoroacetic acid, and THF is tetrahydrofuran.

[0093] Scheme 1. [ka]

[0094] 8-benzyl-6-(3-fluorophenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (compound 2) [ka] 1 H NMR (400MHz, methanol-d4) δ7.91(s,1H),7.65-7.30(m,6H),7.39-7.24(m,3H),7.27-7. 13(m,2H),6.33(d, J=3.2,Hz,1H),6.12(d,J=3.2Hz,1H),4.44(s,2H),4.20(s,2H),C 24 H 18 HRMS (ESI+) calculated for FN3O2 [M+H]+ m / z 400.1462, found 400.1429; HPLC 92.4% (AUC at 254 nm) 2.93 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0095] 8-Benzyl-6-(2,3-fluorophenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 3) [ka] 1 H NMR (400 MHz, methanol-d4) δ 7.89 (s, 1H), 7.54 (s, 1H), 7.46-7.37 (m, 4H), 7.41-7.21 (m, 5H), 6.37-6.31 (m, 1H), 6.12 (d, J = 3.2 Hz, 1H), 4.43 (s, 2H), 4.22 (s, 2H); C 24 H 17 HRMS (ESI+) calculated for F2N3O2 [M+H]+ m / z 418.1368, found 418.1327; HPLC 99.0% (AUC at 254 nm) 2.94 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0096] 8-(2-Fluorobenzyl)-6-(3-fluorophenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 4) [ka] 1 H NMR (400MHz, methanol-d4) δ8.17(s,1H),7.61(d,J=7.8Hz,1H),7.54(d,J=10.4Hz,1H),7.45(td,J=8.0,5.9Hz,1H),7.40(s,1H) ),7.38-7.22(m,2H),7.20-7.05(m,3H),6.33(d,J=3.2Hz,1H),6.12(d,J=3.2Hz,1H),4.51(s,2H),4.19(s,2H),HRMS(ESI+)C 24 H 17 Calculated for F2N3O2 [M+H]+ m / z 418.1368, found 418.1327, HPLC 95.7% (AUC at 254 nm) 3.08 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0097] 8-(2-Fluorobenzyl)-6-(2-fluorophenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 5) [ka] 1 H NMR (400 MHz, methanol-d4) δ 7.98 (s, 1H), 7.75 (s, 1H), 7.51-7.45 (m, 1H), 7.40 (t, J = 2.8 Hz, 1H), 7.35-7.20 (m, 4H), 7.18-7.04 (m, 2H), 6.36-6.28 (m, 1H), 6.12 (d, J = 3.2 Hz, 1H), 4.50 (s, 2H), 4.20 (s, 2H); HRMS (ESI+) C 24 H 17 Calculated for F2N3O2 [M+H]+ m / z 418.1368, found 418.1327, HPLC 92.1% (AUC at 254 nm) 2.88 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0098] 6-(2,3-Difluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 6) [ka] 1 H NMR(400MHz, methanol-d4)δ8.12(s,1H),7.57(t,J=7.3Hz,1H),7.42-7.18(m,5H),7.20-7.10(m, HRMS(ESI+)C 24 H 16 Calculated for F3N3O2 [M+H]+ m / z 436.1274, found 436.1233, HPLC 96.9% (AUC at 254 nm) 3.08 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0099] 8-Benzyl-6-(2,3-fluorophenyl)-2((5-methylfuran-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 7) [ka] 1 H NMR (400MHz, methanol-d4) δ7.89(s,1H),7.53(s,1H),7.46-7.20(m,8H),5.97(d,J=3.0Hz,1 H),5.90(d,J=3.0Hz,1H),5.90(d,J=3.0Hz,1H),4.42(s,2H),4.15(s,2H),2.23(s,3H);C 25 H 19 HRMS (ESI+) calculated for F2N3O2 [M+H]+ m / z 432.1524, found 432.1490; HPLC 98.3% (AUC at 254 nm) 3.11 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0100] 6-(2,3-Difluorophenyl)-8-(2-fluorobenzyl)-2-(thiophen-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 8) [ka] 1 H NMR (400 MHz, methanol-d4) δ 8.07 (s, 1H), 7.56 (s, 1H), 7.41-7.17 (m, 4H), 7.17-7.04 (m, 2H), 6.98-6.86 (m, 2H), 4.50 (s, 2H), 4.36 (s, 2H); C 24 H 16 HRMS (ESI+) calculated for F3N3OS [M+H]+ m / z 452.1045, found 452.1008; HPLC 88.2% (AUC at 254 nm) 3.25 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0101] 6-(2,3-difluorophenyl)-8-(2-fluorobenzyl)-2-(4-fluorobenzyl)imidazo[1,2-a]pyrazin-3(7H)-one (compound 9) [ka] 1 H NMR (400MHz, methanol-d4) δ8.07(s,1H),7.56(s,1H),7.42-7.20(m,6H),7.18-7.08(m,2H),7.02(t,J=8.8Hz,2H),4.49(s,2H),4.17(s,2H),C 26 H 17 HRMS (ESI+) calculated for F4N3O [M+H]+ m / z 464.1387, found 464.1356; HPLC 99.1% (AUC at 254 nm) 3.40 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0102] 6-(2,3-difluorophenyl)-8-(2-fluorobenzyl)-2-((5-methylfuran-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 10) [ka] 1 H NMR (400 MHz, methanol-d4) δ 8.11 (s, 1H), 7.60-7.49 (m, 1H), 7.38-7.23 (m, 3H), 7.27-7.17 (m, 1H), 7.15-7.05 (m, 2H), 5.96 (d, J = 3.0 Hz, 1H), 5.88 (d, J = 3.0 Hz, 1H), 4.50 (s, 2H), 4.14 (s, 2H), 2.22 (s, 3H); HRMS (ESI+) C 25 H 18 Calculated for F3N3O2 [M+H]+ m / z 450.1430, found 450.1381, HPLC 98.8% (AUC at 254 nm) 3.29 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0103] 8-Benzyl-6-(2,3-difluorophenyl)-2-(4-fluorobenzyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 11) [ka] Yield: 34 mg.

[0104] Example 2. Luminescence properties During the development of the embodiments herein, purified NLuc-HaloTag fusions or purified chimeras (i.e., HT generated by insertion of cpNLuc into HaloTag) were obtained. 178 -cpNLuc- 179 Experiments were performed to evaluate the effect of furimazine analogs on the intensity of bioluminescence generated by either NLuc-HaloTag or 10. Briefly, purified NLuc-HaloTag fusions or chimeras were diluted to a final concentration of 6 nM in TBS + 0.1% BSA and treated with substrate at a final concentration of 20 μM. After 1 min of incubation, total bioluminescence was measured using a GloMax® Discover plate reader (Promega, n = 3) or bioluminescence intensity over a 400-600 nm range using an Infinite M1000 plate reader (Tecan, n = 1). The results in Figures 1A-1E show that both NLuc-HaloTag fusions and chimeras generate significantly brighter signals using compound 10 as a substrate, especially when compared to furimazine.

[0105] During development of the embodiments herein, transiently expressed NLuc-HaloTag fusions or chimeras (i.e., HT generated by insertion of cpNLuc into HaloTag) were generated. 178 -cpNLuc- 179 Additional experiments were performed to evaluate the effect of furimazine analogs on the intensity of bioluminescence produced by either 2.2 × 10 5HeLa cells diluted to 1000 cells / mL were transfected with DNA encoding either the NLuc-HaloTag fusion or chimera, plated in 96-well plates at 90 μL / well, and incubated overnight at 37°C + 5% CO2. The following day, cells were treated with substrate at a final concentration of 10 μM and incubated for 1 minute. Total bioluminescence was measured using a GloMax® Discover plate reader (Promega, n=3) or bioluminescence intensity over a 400-600 nm range using an Infinite M1000 plate reader (Tecan, n=1). The results in Figures 2A-2D show that both the NLuc-HaloTag fusion and chimera produced significantly brighter signals using compound 10 as a substrate, especially when compared to furimazine.

[0106] Additional results are shown in Figures 3A-3F.

[0107] Example 3. Formulation of Compound 6 During the development of embodiments herein, experiments were conducted to evaluate the stability of compound 6 in various reconstitution buffer formulations. Briefly, 12 mg of Poloxamer 407 (P-407) was dissolved at 70-75°C, and 4.2 μmol of compound 6 was dissolved in 1 mL of EtOH. The resulting solution was then transferred to the dissolved P-407. The resulting mixture was rotated at 65-75°C to obtain a homogeneous solution. The resulting solution was concentrated under vacuum and resuspended in 6 mL of MQ water. Aliquots (1 mL) of the resuspended compound 6 were then dispensed into sample vials, frozen, and lyophilized to obtain formulation cakes. The sample formulation cakes were then reconstituted in DPBS, HEPES, or Tris (pH 6.5, 7.5, or 8.0) buffer. Stability and purity were determined by LC-MS.

[0108] The data shown in Figures 4A-4C indicate that compound 6 in Tris buffer (Figure 4C) provided the highest stability, maintaining nearly 90% purity after 3 hours. Reconstitution with DPBS (Figure 4A) demonstrated the lowest stability and purity, and was shown to have many degradation products. On the other hand, reconstitution with HEPES (Figure 4B) demonstrated improved stability compared to DPBS, but compound 6 degraded very rapidly, maintaining only approximately 50% purity after 3 hours.

[0109] Example 4. Systematic Study for Reconstitution of Formulated Compound 6 In addition to the stability experiments in Example 3, additional systematic, controlled, and comprehensive stability studies were conducted to evaluate the stability of formulated Compound 6 in various buffer formulations. Briefly, 4.2 μmol of Compound 6 was reconstituted in 1 mL of each buffer at the concentrations and pHs outlined in Table 1. Concentration and purity were determined by LC-MS. Briefly, 1 μL of sample was injected onto an Eclipse RRHD C8 (50 × 2; 1 mm; 1.8 microns; λ = 254 nm) with an aqueous mobile phase of 0.1% TFA in Nanopure water and an organic mobile phase of acetonitrile. [Table 1]

[0110] The data are shown in Figures 5A-5B. Specifically, the data shown in Figure 5A demonstrate that compound 6 reconstituted using saline, water, Tris (pH = 8), glycerol, citrate, and acetate buffers was able to maintain greater than 90% purity over a 6-hour period. The data shown in Figure 5B demonstrate that compound 6 reconstituted using bicarbonate, DPBS, Tris (pH = 6.5), and glycerol exhibited visible precipitation, indicating reduced solubility. Based on the purity and concentration data, it was found that the samples could be successfully stored in water, saline, and Tris (pH = 8) buffers.

[0111] It should be understood that the above detailed description and accompanying examples are illustrative only and should not be construed as limitations on the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0112] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention, including but not limited to, its chemical structures, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use.

Claims

1. Compounds of formula (I): 【Chemistry 1】 or a tautomer thereof or a salt thereof, wherein R 1 teeth, 【Chemistry 2】 where X is selected from O and S; a is hydrogen, fluoro, C 1 ~C 4 Alkyl, and C 1 ~C 4 fluoroalkyl; R 2 is selected from H and F; R 3 is selected from H and F, or a tautomer or salt thereof.

2. R 1 but, 【Transformation 3】 2. The compound of claim 1, or a tautomer or salt thereof, selected from:

3. R 1 but, 【Chemistry 4】 2. The compound of claim 1, or a tautomer or salt thereof, selected from:

4. R 1 but, 【Transformation 5】 2. The compound of claim 1, wherein:

5. R 1 but, 【Transformation 6】 2. The compound of claim 1, wherein:

6. R 2 The compound according to any one of claims 1 to 5, wherein is H, or a tautomer or salt thereof.

7. R 2 The compound according to any one of claims 1 to 5, or a tautomer or salt thereof, wherein is F.

8. R 3 The compound according to any one of claims 1 to 7, or a tautomer or salt thereof, wherein is H.

9. R 3 The compound according to any one of claims 1 to 7, or a tautomer or salt thereof, wherein is F.

10. A compound comprising: 【Transformation 7】 and tautomers and salts thereof.

11. formula: 【Transformation 8】 or a tautomer or a salt thereof.

12. formula: 【Chemistry 9】 or a tautomer or a salt thereof.

13. A kit comprising the compound according to any one of claims 1 to 12, or a tautomer or salt thereof.

14. The kit of claim 13 further comprising luciferase.

15. 15. The kit of claim 13 or 14, further comprising a buffer reagent.

16. The kit of any one of claims 13 to 15, further comprising instructions for performing a luminescent assay.

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