Coelenterazine analogs

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 379,573 filed on October 14, 2022 and U.S. Provisional Patent Application No. 63 / 457,624 filed on April 6, 2023, which applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to coelenterazine analogs, methods for preparing coelenterazine analogs, and methods of using coelenterazine analogs in luciferase-based assays. Background Art

[0004] Bioluminescence assays are widely used in the study of cell physiology, particularly processes associated with gene expression. Specifically, luciferase reporter enzymes are very valuable tools in this field, and to date, a large amount of protein engineering has been performed to obtain small and environmentally insensitive luciferases that can be used for bioluminescence assays. There are many efficient luciferase reporter genes that can achieve whole-cell biosensor measurements, drug discovery by high-throughput screening, and in vivo imaging, thereby also allowing the study of protein-protein interactions, apoptosis, and cell viability in living cells. The luciferase using coelenterazine and coelenterazine analogs as substrates has become one of the most widely used systems due to its brightness and acceptance in whole-cell applications. Summary of the invention

[0005] Many known coelenterazine analogs have defects that limit their effectiveness as luciferase substrates and their usability in luciferase-based assays. These defects include cytotoxicity, photosensitivity, thermodynamic instability, low water solubility, and low cell permeability. Therefore, there is a need for coelenterazine analogs with improved properties and methods for synthesizing the same.

[0006] In one aspect, disclosed herein is a compound of formula (I):

[0007]

[0008] or a tautomer or salt thereof, wherein:

[0009] R 1 Selected from: Wherein X is selected from O and S, and R a Selected from hydrogen, fluorine, C 1 -C 4 Alkyl and C 1 -C 4 Fluoroalkyl;

[0010] R2 is selected from H and F; and

[0011] R 3 Selected from H and F.

[0012] In some embodiments, R 1 Selected from In some embodiments, R 1 Selected from: In some embodiments, R 1 for In some embodiments, R 1 for

[0013] In some embodiments, R 2 is H. In some embodiments, R 2 For F.

[0014] In some embodiments, R 3 is H. In some embodiments, R 3 For F.

[0015] In one aspect, disclosed herein is a compound selected from the group consisting of:

[0016]

[0017]

[0018] and tautomers and salts thereof.

[0019] In one aspect, disclosed herein is a compound of the formula:

[0020]

[0021] or a tautomer or a salt thereof.

[0022] In one aspect, disclosed herein is a compound of the formula:

[0023]

[0024] or a tautomer or a salt thereof.

[0025] In one aspect, disclosed herein is a kit comprising 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 comprises a luciferase. In some embodiments, the kit further comprises a buffer reagent. In some embodiments, the kit further comprises instructions for performing a luminescence assay. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1A-1E Furimazine analogs are shown to be effective against purified NLuc-HaloTag fusions or chimeras (ie, HT 178 -cpNLuc- 179， The effect of the bioluminescence intensity produced by inserting cpNLuc into HaloTag). Figure 1A : Structures of formazan analogs. Figure 1B-1C : Total bioluminescence and bioluminescence spectral scans of 6 nM NLuc-HaloTag fusion treated with 20 μM Formazan, FluoroFurimazine or Compound 10. Figure 1D-1E : Total bioluminescence and bioluminescence spectral scans of 6 nM chimeras treated with 20 μM formazan, fluoroformazan or compound 10.

[0027] Figures 2A-2D The effects of formazan analogs on transiently expressed NLuc-HaloTag fusions or chimeras (i.e., HT 178 -cpNLuc- 179 , generated by inserting cpNLuc into HaloTag) produces an effect on the bioluminescence intensity. Figure 1A The structures are the same as those shown in . Figure 2A-2B : Total bioluminescence and bioluminescence spectra scans of HeLa cells transiently expressing NLuc-HaloTag fusion and treated with 10 μM formazan, fluoroformazan, or compound 10. Figure 2C-2D : Total bioluminescence and bioluminescence spectra scans of HeLa cells transiently expressing the chimera and treated with 10 μM formazan, fluoroformazan, or compound 10.

[0028] Figures 3A-3F The results show that the formazan analogs can be used to express transiently expressed NLuc-HaloTag fusions or chimeras (i.e., HT 178 -cpNLuc- 179 Additional data on the effect of bioluminescence intensity produced by inserting cpNLuc into HaloTag). The structures of formazan analogs are shown in the Examples.

[0029] Figures 4A-4C Shown are data demonstrating the stability and purity of Compound 6 in different reconstitution buffers.

[0030] Figure 5A -5C shows additional data demonstrating the stability and purity of Compound 6 in different reconstitution buffers. DETAILED DESCRIPTION

[0031] Disclosed herein are coelenterazine analogs that are useful substrates for proteins that utilize coelenterazine to produce luminescence ("coelenterazine-utilizing enzymes"), 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 Aequorea jellyfish), and decapod luciferases (e.g., the luciferase complex of Oplophorus gracilirostris).

[0032] In some embodiments, the disclosed compounds exhibit improved water solubility compared to formazan. In some embodiments, the disclosed compounds exhibit improved bioluminescent signal kinetics compared to coelenterazine compounds with similar substitutions at the para position of the 6-phenyl group. Thus, the compounds can be used in in vivo luminescent imaging applications and other applications utilizing bioluminescence.

[0033] 1. Definition

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of a conflict, this document (including definitions) shall prevail. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described below. 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.

[0035] As used herein, the terms "comprising," "including," "having," "has," "may," "containing," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates additional embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements set forth herein, whether or not explicitly stated.

[0036] The modifier "about" used in conjunction 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 the measurement of a particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the expression "from about 2 to about 4" also discloses a range of "from 2 to 4". The term "about" can refer to plus or minus 10% of the specified number. For example, "about 10%" can mean a range of 9% to 11%, and "about 1" can mean 0.9-1.1. Other meanings of "about" may be apparent from the context, such as rounding, so that, for example, "about 1" can also mean from 0.5 to 1.4.

[0037] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the CAS version of the Periodic Table of the Elements, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry and specific functional moieties and reactivity are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th 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, 3rd edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0038] As used herein, the term "alkyl" refers to a straight or branched saturated hydrocarbon chain. 1-4 “Alkyl” refers to a straight or branched chain 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.

[0039] As used herein, the term "fluoroalkyl" refers to an alkyl group as defined herein in which one or more hydrogen atoms are replaced by fluorine. Representative examples of fluoroalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, and 2,2,2-trifluoroethyl.

[0040] As used herein, "animal" refers to any vertebrate, including but not limited to mammals, amphibians, birds, fish, insects, reptiles, etc. Mammals may include but are not limited to humans, non-human primates (e.g., gorillas, monkeys, baboons, and chimpanzees, etc.), dogs, cats, goats, horses, pigs, cattle, sheep, etc., and laboratory animals (e.g., rats, guinea pigs, mice, gerbils, hamsters, etc.). In some embodiments, the animal may be human or non-human. Suitable animals include male and female and animals of any age, including embryos (e.g., in utero or in eggs), infants, teenagers, youth, adults, and elderly animals.

[0041] 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 contiguous amino acid chain that does not occur in nature.

[0042] As used herein, "operably linked" means that the expression of a gene is under the control of a promoter to which it is spatially linked. A promoter may be located 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance may be tolerated without loss of promoter function.

[0043] As used herein, "transgenic" refers to a gene or genetic material that has been separated and / or manipulated from an organism and introduced into a different organism.Transgenic can contain a transgenic sequence or a natural or wild-type DNA sequence.This non-natural DNA segment can retain the ability to produce RNA or protein in a transgenic organism.For example, transgenic can encode a fusion protein, such as a fusion protein that comprises luciferase.The transgenic sequence can be partly or entirely of a species heterologous, that is, the transgenic sequence or its portion can come from a species different from the cell into which it is introduced.

[0044] A "transgenic animal" refers to a genetically engineered animal or the offspring of a genetically engineered animal. A transgenic animal typically contains genetic material from at least one unrelated organism, such as a virus, plant, or other animal.

[0045] The terms "transformation", "transfection" and "transduction" as used interchangeably herein refer to the introduction of a heterologous nucleic acid molecule (such as genetic material) into a cell. This introduction into a cell can be stable or transient. Therefore, in some embodiments, a host cell or host organism is stably transformed by a heterologous nucleic acid molecule (such as genetic material). In other embodiments, a host cell or host organism is transiently transformed by a heterologous nucleic acid molecule (such as genetic material). In the context of a polynucleotide, "transient transformation" refers to the introduction of a polynucleotide into a cell and not being integrated into the genome of the cell. In the context of the introduction of a polynucleotide into a cell, "stably introducing" or "stably introduced" is intended to indicate that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed by the polynucleotide. "Stable transformation" or "stably transformed" as used herein refers to the introduction of a nucleic acid molecule into a cell and being integrated into the genome of the cell. Therefore, the integrated nucleic acid molecule can be inherited by its progeny, more specifically, by successive generations of progeny. As used herein, "genome" also includes nuclear, plasmid and plastid genomes, and therefore includes the integration of nucleic acid constructs into, for example, chloroplast or mitochondrial genomes. Stable transformation as used herein can also refer to transgenes maintained extrachromosomally, for example as minichromosomes or plasmids. In some embodiments, the nucleotide sequence, construct, expression cassette can be transiently expressed and / or they can be stably incorporated into the genome of the host organism.

[0046] For the compounds described herein, radicals and substituents may be chosen with regard to the allowed valences of atoms and substituents such that selection and substitution results in stable compounds, eg, which do not spontaneously transform such as by rearrangement, cyclization, elimination, and the like.

[0047] For the recitation of numerical ranges herein, each intervening number with the same degree of precision is expressly 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 of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0048] 2. Compounds

[0049] Disclosed herein is a compound of formula (I):

[0050]

[0051] or a tautomer or salt thereof, wherein:

[0052] R 1 Selected from: Wherein X is selected from O and S, and R a Selected from hydrogen, fluorine, C 1 -C 4 Alkyl and C 1 -C 4 Fluoroalkyl;

[0053] R 2 is selected from H and F; and

[0054] R 3 Selected from H and F.

[0055] In some embodiments, X is O. In some embodiments, X is S. In some embodiments, R a In some embodiments, R a C 1 -C 4 In some embodiments, R a For fluorine.

[0056] In some embodiments, R 1 Selected from: In some embodiments, R 1 for In some embodiments, R 1 for In some embodiments, R 1 for In some embodiments, R 1 for

[0057] In some embodiments, R 2 is H. In some embodiments, R 2 For F.

[0058] In some embodiments, R 3 is H. In some embodiments, R 3 For F.

[0059] In some embodiments, R 2 is F and R 3 is H. In some embodiments, R 2 H and R 3 is F. In some embodiments, R 2 is F and R 3 For F.

[0060] Disclosed herein are compounds selected from the group consisting of:

[0061]

[0062]

[0063] and tautomers and salts thereof.

[0064] In some embodiments, the compound is:

[0065]

[0066] or a tautomer or a salt thereof.

[0067] In some embodiments, the compound is:

[0068]

[0069] or a tautomer or a salt thereof.

[0070] In some embodiments, the compound is:

[0071]

[0072] or a tautomer or a salt thereof.

[0073] Compound names were generated using the Struct=Name algorithm as in CHEMDRA ULTRA part to specify.

[0074] Compounds can exist as stereoisomers, wherein there is an asymmetric or chiral center. A stereoisomer is "R" or "S", depending on the configuration of the substituents around the chiral carbon atom. The terms "R" and "S" used herein are configurations as defined in 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, and these are specifically included within the scope of the present invention. Stereoisomers include enantiomers and diastereomers and mixtures of enantiomers or diastereomers. A single stereoisomer of a compound can be synthesized from commercially available starting materials containing an asymmetric or chiral center, or by preparing a racemic mixture and then using a resolution method well known to those of ordinary skill in the art. Examples of these resolution methods are: (1) attaching a mixture of enantiomers 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 methods.

[0075] It should be understood that compounds may have tautomeric forms as well as geometric isomers, and these also constitute an aspect of the present invention. The compounds of the present invention or their tautomers or salts include: the compound, the salt of the compound, the tautomer of the compound, and the tautomer of the salt of the compound.

[0076] The present disclosure also includes isotopically labeled compounds, which are identical to those described in formula (I) or specific compounds exemplified 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 present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as, but not limited to, respectively 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 31 P.32 P. 35 S. 18 F and 36 Cl. With heavier isotopes such as deuterium, 2 Substitution with H may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. The compounds may incorporate positron emitting isotopes for use in medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron emitting isotopes that may be incorporated into the compounds are 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 using an appropriate isotopically labeled reagent in place of a non-isotopically labeled reagent.

[0077] The compounds described herein may be in the form of salts. Suitable for contact with tissues of humans and lower animals without causing excessive toxicity, irritation, allergic reactions, etc. and the selection of salts commensurate with a reasonable benefit / risk ratio is within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. The 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 formed by amino groups 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) formed salts. 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, and the like. Base addition salts can be prepared during the final isolation and purification of the disclosed compounds by reaction of a carboxyl group with a suitable base such as a 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 ammonium salts 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-diphenylhydroxymethylamine and N,N′-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like can be prepared.

[0078] A. Properties of Compounds

[0079] The disclosed compounds can be substrates for luciferase to produce luminescence. The compounds can have improved water solubility, improved stability, improved cell permeability, increased cell biocompatibility, reduced autoluminescence and / or reduced toxicity.

[0080] "Luminescence" refers to the light output of luciferase under appropriate conditions, for example, in the presence of a suitable substrate (such as a coelenterazine analog). The light output can be measured as an instantaneous or quasi-instantaneous measurement of the light output at the start of a luminescent reaction (sometimes referred to as "T = 0" luminescence or "flash"), which can be triggered after the addition of a coelenterazine substrate. The luminescent reaction in various embodiments is carried out in solution. In other embodiments, the luminescent reaction is carried out on a solid support. The solution may contain a lysate, such as a lysate from a cell 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 matrix, so that in the latter case, there is no need to produce a lysate. In some embodiments, the reaction is initiated by injecting appropriate materials (e.g., coelenterazine analogs, buffers, etc.) into a reaction chamber (e.g., a well of a multi-well plate such as a 96-well plate) containing a luminescent protein. In other embodiments, luciferase and / or coelenterazine analogs (e.g., compounds disclosed herein) are introduced into a host, and luminescence is measured on the host or a portion thereof (which may include a whole organism or a cell, tissue, explant, or extract thereof). The reaction chamber may be located in a reading device that can measure light output, for example, using a photometer or a photomultiplier tube. Light output or luminescence can also be measured over time, for example, for a period of seconds, minutes, hours, etc. in the same reaction chamber. Light output or luminescence can be reported as an average value over time, a half-life of signal decay, a sum of signals over a period of time, or a peak output. Luminescence can be measured in relative light units (RLU).

[0081] In some embodiments, the disclosed compounds can have an RLU greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 10, greater than or equal to 20, greater than or equal to 30, greater than or equal to 40, greater than or equal to 50, or greater than or equal to 100 relative to coelenterazine or a known coelenterazine analog, such as formazine.

[0082] In some embodiments, the disclosed compounds can have a λmax of 450-700 nm, 460-600 nm, 470-600 nm, 480-600 nm, 490-600 nm, 500-600 nm, 510-600 nm, 520-600 nm, 530-600 nm, 540-600 nm, 550-600 nm, 560-600 nm, 570-600 nm, 580-600 nm, 590-600 nm, 470-590 nm, 480-580 nm, 490-570 nm, 500-560 nm, or 510-550 nm. The compounds disclosed herein can have a λmax of greater than or equal to 450 nm, greater than or equal to 460 nm, greater than or equal to 470 nm, greater than or equal to 480 nm, greater than or equal to 490 nm, greater than or equal to 500 nm, greater than or equal to 510 nm, greater than or equal to 520 nm, greater than or equal to 530 nm, greater than or equal to 540 nm, greater than or equal to 550 nm, greater than or equal to 560 nm, greater than or equal to 570 nm, greater than or equal to 580 nm, greater than or equal to 590 nm, greater than or equal to 600 nm, greater than or equal to 610 nm, greater than or equal to 620 nm, greater than or equal to 630 nm, greater than or equal to 640 nm, greater than or equal to 650 nm, greater than or equal to 660 nm, greater than or equal to 670 nm, greater than or equal to 680 nm, greater than or equal to 690 nm, or greater than or equal to 700 nm.

[0083] "Biocompatibility" refers to the tolerance of cells (e.g., prokaryotic or eukaryotic) to a coelenterazine analog (e.g., a compound disclosed herein). The biocompatibility of a coelenterazine analog is related to the stress it causes to host cells.

[0084] The enhanced biocompatibility of a coelenterazine analog (e.g., a compound disclosed herein) can be determined by measuring cell viability and / or the growth rate of the cells. For example, the enhanced biocompatibility of a coelenterazine analog can be determined by measuring the cell viability of cells exposed to a coelenterazine analog in the absence of luciferase expression compared to native or known coelenterazine to determine how compatible and / or toxic the coelenterazine analog is to the cells.

[0085] Specifically, cell viability assays (e.g., using luminescent cell viability assay), apoptosis assay (e.g., using Enhanced biocompatibility can be determined by PCR (eg, PCR technology) or other methods known in the art. The effects of the disclosed compounds on cell viability or apoptosis can be compared with the effects of natural or known coelenterazine analogs on cell viability or apoptosis.

[0086] Enhanced biocompatibility can also be determined by measuring the effect of coelenterazine analogs (e.g., compounds disclosed herein) on cell growth or gene expression. For example, the enhanced biocompatibility of compounds disclosed herein can be determined by measuring the number of cells after a period of time in a cell sample exposed to compounds disclosed herein compared to cells exposed to natural or known coelenterazine or not exposed to coelenterazine, or by determining the expression of stress response genes therein. The effects of the disclosed compounds on cell growth or gene expression can be compared to natural or known coelenterazine.

[0087] B. Synthesis Method

[0088] The compounds disclosed herein can be prepared by synthetic processes or metabolic processes. Preparation of compounds by metabolic processes include those that occur within the human or animal body (in vivo) or processes that occur in vitro.

[0089] Compounds disclosed herein can be synthesized by a variety of methods, including those shown in the examples. The optimal reaction conditions and reaction times for each individual step can vary according to the specific reactants used and the substituents present in the reactants used. The reaction can be carried out in a conventional manner, for example, by removing the solvent from the residue, and further purified according to methods generally known in the art, such as but not limited to crystallization, distillation, extraction, wet grinding and chromatography. Unless otherwise described, starting materials and reagents are commercially available or can be prepared from commercially available materials by those skilled in the art using the methods described in the chemical literature. Starting materials, if not commercially available, can be prepared by procedures selected from standard organic chemistry techniques, techniques similar to the synthesis of known structurally similar compounds, or techniques similar to the procedures described in the above-mentioned schemes or synthetic examples.

[0090] Routine experiments, including appropriate manipulation of reaction conditions, reagents and synthetic route sequences, protection of any chemical functional groups that may be incompatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the method are all included within the scope of the present invention. Suitable protecting groups and methods of using such suitable protecting groups to protect different substituents and deprotection are well known to those skilled in the art; examples thereof can be found in PGM Wuts and TW Greene, in Greene's booktitled Protective Groups in Organic Synthesis (4th edition), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. The synthesis of the compounds of the present invention can be accomplished by methods similar to those described in the synthetic schemes and specific embodiments described above.

[0091] 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 optically active starting materials (e.g., by asymmetric induced preparation by appropriate reaction steps) or by resolving a mixture of stereoisomers of the compounds or intermediates using standard procedures (such as chromatographic separation, recrystallization or enzymatic resolution).

[0092] Similarly, when pure geometric isomers of compounds are required, they may be obtained by carrying out one of the above procedures using the pure geometric isomers as starting materials, or by resolving a mixture of geometric isomers of compounds or intermediates using standard procedures such as chromatographic separation.

[0093] It should 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 in the appended claims. All alternatives, modifications and equivalents of the synthetic methods and specific examples are included within the scope of the claims.

[0094] 3. Usage Method and Kit

[0095] The compounds of the present disclosure can be used in any manner that has used luciferase substrates (e.g., coelenterazine analogs). For example, they can be used in bioluminescent methods that use coelenterazine analogs to detect one or more molecules in a sample, such as an enzyme, a cofactor of an enzymatic reaction, an enzyme substrate, an enzyme inhibitor, an enzyme activator, or an OH radical, or one or more conditions, such as redox conditions. Samples may include animals (e.g., vertebrates), plants, fungi, physiological fluids (e.g., blood, plasma, urine, mucous secretions), cells, cell lysates, cell supernatants, or purified fractions of cells (e.g., subcellular fractions). The presence, amount, spectral distribution, emission kinetics, or specific activity of such molecules can be detected or quantified. The molecule can be detected or quantified in a solution, including a multiphase solution (e.g., an emulsion or suspension), or on a solid support (e.g., a particle, a capillary, or an assay container).

[0096] 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 coelenterazine or compounds disclosed herein) can be used as probes for specific biochemical activities (e.g., apoptosis or drug metabolism).

[0097] In certain embodiments, the compounds disclosed herein can be used with inhibitors of luciferase from Oplophorus and / or bioluminescent complexes derived from Oplophorus. Exemplary inhibitors of luciferase from Oplophorus and / or bioluminescent complexes derived from Oplophorus are described in, for example, International Patent Publication Nos. WO2016 / 210294, WO 2018 / 125992, WO 2019 / 232384, and WO 2019 / 213119, each of which is incorporated herein by reference in its entirety.

[0098] In certain embodiments, compounds disclosed herein can be used to detect luminescence in living cells (e.g., in vivo). In some embodiments, luciferase can be expressed in cells (as a reporter gene or otherwise), and the cells treated with coelenterazine analogs (e.g., compounds disclosed herein) that will penetrate the cultured cells react with luciferase and produce luminescence. In addition to being cell permeable, compounds disclosed herein also show biocompatibility comparable to natural coelenterazine in terms of cell viability. In some embodiments, chemically modified compounds containing known increases in the stability of natural coelenterazine in culture medium disclosed herein can be synthesized and used for more robust reporter gene assays based on living cell luciferase. In other embodiments, various microscopy and imaging techniques (e.g., in vivo imaging) can be used to measure samples (including cells, tissues, animals, etc.) containing luciferase and compounds disclosed herein. In other embodiments, secretable luciferase is expressed in cells as part of a living cell reporter system.

[0099] 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 polypeptides, polynucleotides or both) and coelenterazine analogs disclosed herein, as well as suitable reagents and instructions to enable users 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 also include an inhibitor of a luciferase derived from a spiky shrimp as described above and / or a spiky shrimp-luciferase derived bioluminescent complex.

[0100] Buffers include citric acid or citrate buffer, MES, 1,4-piperazine diethanesulfonic acid or HEPES; inorganic phosphates, for example in the form of pyrophosphate or potassium phosphate; chelating agents such as EDTA, CDTA or 1,2-diaminocyclohexane tetraacetic acid; salts such as sodium fluoride, magnesium sulfate; surfactants or detergents such as (e.g. nonionic nonylphenol ethoxylate), dodecyltrimethylammonium bromide (DTAB) or (hydroxypolyethoxydodecane); defoaming agents such as DF204 (organic defoamer) or DF (organic silicon defoamer); protein stabilizers, such as gelatin, 10% (gelatin, type A) or albumin (eg BSA, HSA) or glycerol; adenosine triphosphate (ATP) or adenosine monophosphate (AMP). Other components may include polyethylene glycol, polyvinyl pyridine, crown ethers or cyclodextrins.

[0101] A. In vivo imaging

[0102] The compounds of the present disclosure can be used for imaging of living cells, such as in vivo and ex vivo bioluminescent imaging. For example, the compounds of the present disclosure can be used together with coelenterazine using luciferase to perform bioluminescent imaging on cells in tissue sections or living animals. In vivo bioluminescent imaging is a versatile and sensitive tool based on the detection of light emitted from cells or tissues. Bioluminescence has been used to track tumor cells, bacterial and viral infections, gene expression, and therapeutic responses in a non-invasive manner. Bioluminescent imaging provides longitudinal monitoring of the disease process of the same animal, which is a desired alternative to analyzing multiple animals at multiple time points during the disease process. In some embodiments, the compounds of the present disclosure can be used in vivo to monitor biological processes in a variety of animal models, such as cell movement, tumor progression, gene expression, and viral infection. In some embodiments, the compounds of the present disclosure can be used for imaging in transgenic animals (such as transgenic mice). Transgenic animals, including cells or tissues, can represent models of human cell function and disease. Therefore, these animals can be used to study the mechanisms behind cell function and related events, generate and test products (e.g., antibodies, small molecules, etc.) and treat and diagnose related human diseases, including cancer and autoimmune conditions. In some embodiments, transgenic animals can further provide an indication of the safety of administering a particular agent to a human. The effects of a particular agent and the compounds of the present disclosure can be studied by administering the agent to specific cells or the whole body and performing bioluminescent imaging to look for specific effects. Animal and cell-based models and the compounds of the present disclosure can be used to identify drugs, medicines, therapies, and interventions that are effective in treating diseases.

[0103] In some embodiments, the compound of the present disclosure can be used for the bioluminescent imaging of the cell or animal transformed to express fusion protein (such as the fusion protein comprising luciferase).In some embodiments, transgenic animals or cells can express the fusion protein comprising luciferase.In some embodiments, luciferase can be the luciferase utilizing coelenterazine, such as luciferase in thorn shrimp or thorn shrimp source, sea renilla luciferase, Gaussia (Gaussia) luciferase such as Gaussia princeps (Gaussia princeps) luciferase, long abdominal water flea (Metridia) luciferase such as elongated long abdominal water flea (Metridia longa) and Taiping long abdominal water flea (Metridia pacifica) luciferase, curved throat sea firefly (Vargula) luciferase such as Xi's curved throat sea firefly (Vargula hilgendorfii) luciferase, sword milk point water flea (Pleuromamma xiphias) luciferase and its variant, recombinant and mutant.In some embodiments, the polynucleotide sequence encoding fusion protein is operably connected to a promoter. In some embodiments, the promoter can be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter. In some embodiments, the promoter can also be a tissue-specific promoter.

[0104] In some embodiments, the fusion protein of the bioluminescent protein and the heterologous protein of interest (such as a fluorescent protein) can be directly connected to each other by a peptide bond, or can be separated by an intermediate amino acid sequence. In some embodiments, the fusion polypeptide may also contain exogenous sequences of the bioluminescent protein and the heterologous protein of interest (such as a fluorescent protein). For example, the fusion protein may include a targeting or localization sequence, a tag sequence, a sequence of other fluorescent proteins or bioluminescent proteins or other chromophores. In some embodiments, the targeting sequence can guide the fusion protein to localize to a specific tissue, cell type (e.g., muscle, heart, or nerve cells), cell compartment (e.g., mitochondria or other organelles, nucleus, cytoplasm, or plasma membrane), or protein. In addition, the fusion may contain sequences from a variety of fluorescent or bioluminescent proteins or variants thereof and / or other selected proteins. In some embodiments, luciferase and The GFP, RFP or green fluorescent protein can be fused to the GFP or red fluorescent protein.

[0105] Bioluminescence generated in cells (such as cells of transgenic animals) can be imaged or detected by a variety of means well known in the art. For example, fusion proteins and compounds of the present disclosure that have been localized to their intended sites in transgenic animals can be imaged in a variety of ways. Those skilled in the art can make reasonable estimates of the time to achieve localization. In addition, the localization state that changes over time can be tracked by imaging the bioluminescence generated by the fusion proteins and compounds of the present disclosure. Since imaging or measuring photon emission from a subject can last for tens of minutes, the transgenic animal can be fixed during the imaging process.

[0106] In vivo imaging can be performed with the naked eye or with any type of camera (still or video). Imaging of bioluminescence involves the use of, for example, a photodetector capable of detecting extremely low levels of light (typically single photon events) and integrating the photon emissions until an image can be constructed. Examples of such 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 despite the background noise inherent in the detection system. The "photodetector device" used should have a sufficiently high sensitivity to be able to image the faint light from within a mammal in a reasonable amount of time, and to construct an image using the signal from such a device.

[0107] Bioluminescent signals can be detected using a highly sensitive enhanced charge coupled device (CCD) camera. In certain embodiments, imaging is performed using an enhanced CCD camera that is sensitive enough to detect bioluminescent signals and has a dynamic range wide enough to additionally detect fluorescent signals. Suitable cameras are known in the art and include, but are not limited to, the Olympus LV200 bioluminescent imaging system, the Living Image TM Integrated Imaging System (IVIS) controlled by software (Caliper Life Sciences) TM Imaging system, Caliper Life Sciences) or a custom 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 an animal (such as a mouse), and internal lighting.

[0108] In vivo imaging can be non-invasive whole animal imaging, which has been described (Contag, C., U.S. Pat. No. 5,650,135, July 22, 1997), which is incorporated herein by reference; 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 detection sensitivity of light emitted from the viscera depends on several factors, including the expression level of luciferase, the depth of the labeled cells in the body (the distance the photons must travel through the tissue), and the sensitivity of the detection system.

[0109] "Photon amplification devices" amplify photons before they hit a detection screen. This category includes CCD cameras with intensifiers such as microchannel intensifiers. Microchannel intensifiers typically contain an array of metal channels that are perpendicular and coextensive with the camera's detection screen. The microchannel array is placed between the sample, subject, or animal to be imaged and the camera. Most photons that enter the array's channels contact one side of the channel before exiting. A voltage applied to the array causes many electrons to be released for each photon collision. The electrons produced by this collision leave their original channels in a "shotgun" pattern and are detected by the camera.

[0110] The image processor processes the signals generated by the photodetector device that counts the photons in order to construct an image that can be displayed, for example, on a monitor or printed on a video printer. Such image processors are typically sold as part of a system that includes the sensitive photon counting cameras 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 Apple Macintosh (Apple Computer, Cupertino, Calif.), which may or may not be included as part of the purchased imaging system. Once the image is in the form of a digital file, it can be manipulated and printed by a variety of image processing programs (such as "ADOBE PHOTOSHOP", Adobe Systems, Adobe Systems, Mt. View, Calif.).

[0111] It should be understood that the entire animal or subject does not necessarily need to be within the detection field of the light detection device. For example, if a fusion protein targeted to a specific area of ​​the subject is being measured, it is only necessary to measure the light from that area and sufficient surrounding "dark" areas to obtain the desired information.

[0112] Once the photon emission image is generated, it is typically superimposed on a "normal" reflected light image of the subject to provide a frame of reference for the source of the emitted photons (i.e., to locate the fusion protein relative to the subject). The "composite" image formed by the superposition of the photon emission image on the reflected light image is then analyzed to determine the location and / or amount of the target within the subject.

[0113] B. Bioluminescence Resonance Energy Transfer (BRET)

[0114] 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 an in vivo or in vitro bioluminescence resonance energy transfer (BRET) system. With respect to 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 enables real-time monitoring of protein-protein or ligand-protein interactions in vitro or in vivo, such as interaction and dissociation of partners. BRET systems (such as NanoBRET TM Examples of systems) are described in, for example, U.S. Patent 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.

[0115] In some embodiments, the luminescent enzyme used in the BRET analysis can be used to determine whether two molecules can bind to each other or co-localize in a cell. For example, a luminescent enzyme can be used as a bioluminescent donor molecule, which is combined with a molecule or protein of interest to produce a first fusion protein. In some embodiments, the luminescent enzyme can be conjugated to an antibody, a protein, a receptor, a drug, a drug carrier, a peptide, a sugar, a fatty acid, a nanoparticle, or other biological molecules. In various embodiments, the first fusion protein contains a luminescent enzyme and a protein of interest. In various embodiments, the first fusion protein containing a luminescent enzyme can be used in a BRET analysis to detect protein / protein interactions in systems including but not limited to cell lysates, intact cells, and living animals. In some embodiments, the BRET analysis can also include inhibitors of luciferases derived from spiky shrimp and / or spiky shrimp-luciferase-derived bioluminescent complexes as described above.

[0116] In some embodiments, the fluorescent receptor can be a fluorophore, such as a fluorescent protein, a fluorescent molecule, a fluorescent marker, or a fluorescent tracer. In some embodiments, the fluorescent tracer can be a small molecule labeled with a fluorophore. In some embodiments, the fluorescent receptor can be a second fusion protein, which includes a fluorescent receptor conjugated to an antibody, protein, receptor, drug, drug carrier, peptide, sugar, fatty acid, nanoparticle or other biomolecule.

[0117] In various embodiments, Can be used as a fluorescent receptor molecule. In some embodiments, It can be fused to a second protein of interest or to a luminescent enzyme. For example, a luminescent enzyme can be fused to Fusion, expression in cells or animals, and fluorescence Ligands (such as In some embodiments, BRET can be performed using a combination of a luminescent enzyme and a fluorescent protein (including but not limited to GFP, RFP, orange-red fluorescent protein) or a fluorescent marker (including fluorescein, rhodamine green, Oregon green, or Alexa 488, to name a few non-limiting examples).

[0118] In some embodiments, the disclosed compounds can be used in target engagement assays such as NANOBRET TM Target engagement (TE) assays to measure the binding of compounds to selected target proteins in intact cells in real time, such as drug:target interactions. For example, NANOBRET TM TE assays can include four components: luciferase fused to an expressed cellular target protein; a cell-permeable fluorescent tracer that specifically binds to the target protein; used as one or more disclosed compounds that are substrates for luciferase; and The assay uses bioluminescence resonance energy transfer (BRET) by combining The luminescence energy of luciferase is transferred to the target protein This energy transfer allows direct measurement of compound binding affinity as well as compound-target residence time.

[0119] In some embodiments, the compound applied to the cell can specifically bind to a target protein within the cell. In some embodiments, to ensure accurate assessment of target engagement within cells, the fusion may be used. inhibitors to mitigate any extracellular signal, while not expressing luciferase.

[0120] The BRET system may also include a light detector or imaging device for detecting light emitted from the bioluminescent fusion protein, such as, but not limited to, an optical microscope, a digital microscope, a photometer, a charge coupled device (CCD) image sensor, a complementary metal-oxide-semiconductor (CMOS) image sensor, or a digital camera.

[0121] C. Formulation and Administration

[0122] For whole animal studies, the disclosed imaging probes are preferably formulated for parenteral administration. Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions are prepared as solutions or suspensions; solid forms suitable for preparing solutions or suspensions after adding a reconstitution medium; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions and microemulsions thereof, liposomes or emulsomes.

[0123] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion.

[0124] The carrier can be a solvent and dispersion medium containing, for example, water, ethanol, one or more polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol), oils such as vegetable oils (eg, peanut oil, corn oil, sesame oil, etc.), and combinations thereof.

[0125] Solutions and dispersions of the active compound as a free acid or base or a pharmacologically acceptable salt 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, dispersants, emulsifiers, pH adjusters, and combinations thereof.

[0126] Suitable surfactants can be anionic, cationic, amphoteric or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long-chain alkyl sulfonates and alkyl aryl sulfonates, such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinates, such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinates, such as sodium bis-(2-ethylthioxy) 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, stearyl dimethyl benzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of the nonionic surfactant 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, 401, stearyl monoisopropanolamide and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoylamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0127] The preparation 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 preparation may also contain an antioxidant to prevent degradation of the active agent.

[0128] The formulation is typically buffered to a pH of 3-8 for parenteral administration after reconstitution. Suitable buffers include, but are not limited to, phosphate buffer, acetate buffer, and citrate buffer.

[0129] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinyl pyrrolidone, dextran, carboxymethyl cellulose, and polyethylene glycol.

[0130] The required amount of active compound can be mixed with one or more of the excipients listed above in a suitable solvent or dispersion medium, and then filtered and sterilized as needed to prepare a sterile injectable solution. Generally, dispersions are prepared by mixing various sterilized active ingredients into a sterile vehicle containing an alkaline dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying techniques and freeze drying techniques, which produce active ingredients plus powders of any additional desired components from their previous sterile filtered solutions. Powders can be prepared in such a way that particles are porous in nature, which can increase the dissolution of particles. Methods for preparing porous particles are well known in the art.

[0131] 4. Examples

[0132] Example 1. Compound synthesis and characterization

[0133] The compounds were prepared using the synthetic route shown in Scheme 1, which has been described previously (Su et al. Nat Methods 17, 852-860 (2020); Shakhmin et al. Chemistry 22, 10369-10375 (2016)). Abbreviations in Scheme 1 include the following: ACN is acetonitrile; CDI is carbonyldiimidazole; DMA is dimethylacetamide; DCM is dichloromethane; eq is equivalent; h is hour; MeOH is methanol; min is minute; rt is room temperature; TFA is trifluoroacetic acid; and THF is tetrahydrofuran.

[0134] Solution 1.

[0135]

[0136] 8-Benzyl-6-(3-fluorophenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 2)

[0137]

[0138] 1 H NMR (400 MHz, methanol-d 4 )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 FN 3 O 2HRMS (ESI+) calcd. [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).

[0139] 8-Benzyl-6-(2,3-difluorophenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 3)

[0140]

[0141] 1 H NMR (400 MHz, methanol-d 4 C 24 H 17 F 2 N 3 O 2 HRMS (ESI+) calcd. [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).

[0142] 8-(2-Fluorobenzyl)-6-(3-fluorophenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 4)

[0143]

[0144] 1 H NMR (400 MHz, methanol-d 4 )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),7.3 8–7.22(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);C 24 H17 F 2 N 3 O 2 HRMS (ESI+) calcd. [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).

[0145] 8-(2-Fluorobenzyl)-6-(2-fluorophenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 5)

[0146]

[0147] 1 H NMR (400 MHz, methanol-d 4 )δ7.98(s,1H),7.75(s,1H),7.51–7.45(m,1H),7.40(t,J=2.8Hz,1H),7.35–7.20(m,4H) ,7.18–7.04(m,2H),6.36–6.28(m,1H),6.12(d,J=3.2Hz,1H),4.50(s,2H),4.20(s,2H);C 24 H 17 F 2 N 3 O 2 HRMS (ESI+) calculated value [M+H]+ m / z 418.1368, found value 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).

[0148] 6-(2,3-difluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 6)

[0149]

[0150] 1 H NMR (400 MHz, methanol-d 4)δ8.12(s,1H),7.57(t,J=7.3Hz,1H),7.42–7.18(m,5H),7.20–7.10(m,2H),6 .33(dd,J=3.2,1.9Hz,1H),6.11(d,J=3.2Hz,1H),4.50(s,2H),4.20(s,2H);C 24 H 16 F 3 N 3 O 2 HRMS (ESI+) calculated value [M+H]+ m / z 436.1274, found value 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).

[0151] 8-Benzyl-6-(2,3-difluorophenyl)-2-((5-methylfuran-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 7)

[0152]

[0153] 1 H NMR (400 MHz, methanol-d 4 )δ7.89(s,1H),7.53(s,1H),7.46–7.20(m,8H),5.97(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 F 2 N 3 O 2 HRMS (ESI+) calcd. [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).

[0154] 6-(2,3-difluorophenyl)-8-(2-fluorobenzyl)-2-(thien-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 8)

[0155]

[0156] 1 H NMR (400 MHz, methanol-d4 )δ8.07(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 F 3 N 3 HRMS (ESI+) calcd. [M+H]+ m / z 452.1045, found 452.1008 for OS; HPLC 88.2% (AUC at 254 nm) 3.25 min (Accucore C8, 50x2.1 mm, 2.6 μm, water / ACN 0.1% TFA).

[0157] 6-(2,3-difluorophenyl)-8-(2-fluorobenzyl)-2-(4-fluorobenzyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 9)

[0158]

[0159] 1 H NMR (400 MHz, methanol-d 4 )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 F 4 N 3 HRMS (ESI+) calcd. [M+H]+ m / z 464.1387 for O, 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).

[0160] 6-(2,3-difluorophenyl)-8-(2-fluorobenzyl)-2-((5-methylfuran-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 10)

[0161]

[0162] 1 H NMR (400 MHz, methanol-d 4)δ8.11(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,2 H),5.96(d,J=3.0Hz,1H),5.88(d,J=3.0Hz,1H),4.50(s,2H),4.14(s,2H),2.22(s,3H);C 25 H 18 F 3 N 3 O 2 HRMS (ESI+) calcd. [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).

[0163] 8-Benzyl-6-(2,3-difluorophenyl)-2-(4-fluorobenzyl)imidazo[1,2-a]pyrazin-3(7H)-one (Compound 11)

[0164]

[0165] Yield: 34 mg.

[0166] Example 2. Luminescence properties

[0167] During the development of the embodiments herein, experiments were performed to evaluate the effects of formazan analogs on purified NLuc-HaloTag fusions or purified chimeras (i.e., HT 178 -cpNLuc- 179 , generated by inserting cpNLuc into HaloTag). Briefly, purified NLuc-HaloTag fusions or chimeras were diluted to a final concentration of 6 nM in TBS + 0.1% BSA, treated with substrate at a final concentration of 20 μM and incubated for 1 min, then Total bioluminescence was measured on a Promega Discover plate reader (Promega; n=3), or bioluminescence intensity in the range of 400-600 nm was measured on an Infinite M1000 plate reader (Tecan; n=1). Figures 1A-1E The results in show that both NLuc-HaloTag fusions and chimeras produced significantly brighter signals with compound 10 as substrate, especially when compared with formazine.

[0168] Additional experiments were performed during the development of the embodiments herein to evaluate the effects of formazan analogs on the expression of transiently expressed NLuc-HaloTag fusions or chimeras (i.e., HT 178 -cpNLuc- 179 , generated by inserting cpNLuc into HaloTag). Briefly, cells were diluted to 2.2 x 10 5 HeLa cells at 100 cells / mL were transfected with DNA encoding NLuc-HaloTag fusions or chimeras, plated at 90 μL / well in 96-well plates, and cultured at 37°C + 5% CO. 2 The next day, cells were treated with substrate at a final concentration of 10 μM and incubated for 1 min, then Total bioluminescence was measured on a Discover plate reader (Promega; n=3), or bioluminescence intensity in the range of 400-600 nm was measured on an Infinite M1000 plate reader (Tecan; n=1). Figures 2A-2D The results in show that both NLuc-HaloTag fusions and chimeras produced significantly brighter signals with compound 10 as substrate, especially when compared with formazine.

[0169] Other results in Figures 3A-3F Shown in.

[0170] Example 3. Preparation of Compound 6

[0171] Experiments were conducted during the development of the embodiments herein to evaluate the stability of compound 6 in various reconstitution buffer formulations. Briefly, 12 mg poloxamer 407 (P-407) was melted at 70-75 ° C, and 4.2 μmol of compound 6 was dissolved in 1 mL of EtOH, and the resulting solution was then transferred to the molten P-407. The resulting mixture was rotated at 65-75 ° C to obtain a homogeneous solution. The resulting solution was vacuum concentrated and resuspended in 6 ml of MQ water. Then 1 mL of aliquots of resuspended compound 6 were dispensed into sample vials, frozen and lyophilized to obtain a prepared cake. The cake prepared by the sample was then reconstructed in DPBS, HEPES or Tris (pH 6.5, 7.5 or 8.0) buffer. Stability and purity were determined by LC-MS.

[0172] Figures 4A-4C The data shown in Figure 2 show that compound 6 ( Figure 4C ) provided the highest stability and maintained nearly 90% purity after 3 h. Reconstitution in DPBS ( Figure 4A) showed the lowest stability and purity and had many decomposition products, while reconstitution in HEPES ( Figure 4B ) showed improved stability compared to DPBS, but compound 6 decomposed very quickly and only retained about 50% purity after 3 h.

[0173] Example 4. Systematic study of the reconstitution of formulated compound 6

[0174] In addition to the stability experiments in Example 3, additional systematic, controlled and comprehensive stability studies were performed 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 with concentrations and pH summarized in Table 1. Concentration and purity were determined by LC-MS. Briefly, 1 μL of sample was injected into an Eclipse RRHD C8 (50x2; 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.

[0175] Table 1.

[0176] Buffer pH concentration Sodium acetate 5.2 3M Bicarbonate 9.5 0.1M / 0.2M Citrate 6.0 10x brine not applicable 0.9％ DP8S 7.0 1x HEPES 7.8 1M Tris 6,5 1M Tris 7.5 1M Tris 8.0 2M glycerin not applicable 2％ water not applicable not applicable Solutol HS15 not applicable 10％

[0177] The data is shown in Figure 5A-5B middle. Specifically, Figure 5A The data presented indicate that Compound 6 reconstituted using saline, water, Tris (pH=8), glycerol, citrate, and acetate buffers can maintain greater than 90% purity over a 6 hour period. Figure 5B The data presented show that compound 6 reconstituted with bicarbonate, DPBS, Tris (pH=6.5) and glycerol showed visible precipitation, indicating low solubility. Based on the purity and concentration data, the samples were found to be well preserved in water, saline and Tris (pH=8) buffer.

[0178] It should be understood that the foregoing detailed description and the accompanying examples are merely illustrative and should not be taken as limiting the scope of the present invention, which is defined solely by the appended claims and their equivalents.

[0179] 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 those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the invention.

Claims

1. A compound of formula (I): or a tautomer or a salt thereof, in: R 1 Selected from: Wherein X is selected from O and S, and R a Selected from hydrogen, fluorine, C 1 -C 4 Alkyl and C 1 -C 4 Fluoroalkyl; R 2 is selected from H and F; and R 3 Selected from H and F.

2. The compound according to claim 1 or its tautomer or salt, wherein R 1 Selected from 3. The compound as claimed in claim 1 or its tautomer or salt, wherein R 1 Selected from:

4. The compound according to claim 1 or its tautomer or salt, wherein R 1 for 5. The compound according to claim 1 or its tautomer or salt, wherein R 1 for 6. A compound or a tautomer or salt thereof as claimed in any one of claims 1 to 5, wherein R 2 For H.

7. A compound or a tautomer or salt thereof as claimed in any one of claims 1 to 5, wherein R 2 For F.

8. A compound or a tautomer or salt thereof as described in any one of claims 1 to 7, wherein R 3 For H.

9. The compound or tautomer or salt thereof as claimed in any one of claims 1 to 7, wherein R 3 For F.

10. A compound selected from the group consisting of: and tautomers and salts thereof.

11. A compound of the formula: or a tautomer or a salt thereof.

12. A compound of the formula: or a tautomer or a salt thereof.

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

14. The kit of claim 13, further comprising luciferase.

15. The kit according to claim 13 or 14, further comprising a buffering agent.

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

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