Coelenterazine analogues

Abstract

To provide methods for preparing coelenterazine analogues.SOLUTION: The present invention provides a method for preparing a compound of formula (I), where R1 represents alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl or the like, and q represents 0-2. The method includes converting a compound (vi) into the compound of formula (I).SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 295,363, filed February 15, 2016, the entire contents of which are incorporated herein by reference.

[0002] Technical Field 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 in the study of cell physiology, especially in processes related to gene expression. In particular, luciferase reporter enzymes are extremely useful tools in this technical field, and intensive protein engineering has been carried out to obtain small and environmentally insensitive luciferases that can be useful in bioluminescence assays. There are many effective luciferase reporters that enable whole cell biosensor measurements, drug discovery by high throughput screening, and in vivo imaging, which also allow the study of protein-protein interactions, apoptosis, and cell viability in living cells. Luciferases using coelenterazine and coelenterazine analogs as substrates are the most widely used systems in whole cell applications due to their brightness and tolerance. Summary of the Invention

[0004] Many known coelenterazine analogues have deficiencies that limit their effectiveness as luciferase substrates and their usefulness in luciferase-based luminescence assays. These deficiencies include cytotoxicity, photosensitivity, thermodynamic instability, poor aqueous solubility, and poor cell permeability. Therefore, there is a need for coelenterazine analogues with improved properties and synthesis methods.

[0005] In one embodiment, the compound of formula (I): The present invention discloses a compound or tautomer of TIFF0007669430000001.tif58164, or a pharma- ceutically acceptable salt thereof, wherein R 1 is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate, and q is 0 to 2, wherein said alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate are independently substituted or unsubstituted at each occurrence.

[0006] Also disclosed are methods for making the compounds, kits containing the compounds, and methods for using the compounds as luciferase substrates in luciferase-based luminescence assays. [Brief description of the drawings]

[0007] [Figure 1] 1 is a graph depicting the cellular bioluminescence activity of representative compounds in HEK293 cells compared to coelenterazine. [Diagram 2] 1 is a graph showing the ranking of compounds of formula (I) according to their signal intensities relative to coelenterazine. [Diagram 3] 1 is a graph showing the results of a cell viability assay with representative compounds in HeLa cells. [Figure 4] 1 is a graph showing the results of a cell viability assay with representative compounds in HeLa cells. [Diagram 5] 1 is a graph showing the results of a cell viability assay with representative compounds in HEK293 cells. [Figure 6] 1 is a graph showing the results of a cell viability assay with representative compounds in HEK293 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description of the Invention Coelenterazine analogues are disclosed herein. The coelenterazine analogues may be compounds of formula (I) and may be useful substrates for proteins that utilize coelenterazine to emit light, including but not limited to luciferases and photoproteins found in various marine organisms, such as cnidaria (e.g. Renilla luciferase), jellyfish (e.g. Aequorin from Aequorea jellyfish) and decapod luciferases (e.g. Oplophorus gracilirostris luciferase complex). Compared to coelenterazine, the compounds of formula (I) may have at least one of improved aqueous solubility, improved stability, improved cell permeability, high biocompatibility with cells, reduced autobioluminescence, and low toxicity.

[0009] Additionally, methods for the preparation of coelenterazine analogs [compounds of formula (I)] are disclosed herein. Three robust and versatile approaches are disclosed for the preparation of coelenterazine analogs. These methods allow for the preparation of analogs that could not be prepared using existing synthetic methods. The methodology described involves the synthesis of R 1 The disclosed synthetic methodology provides access to a variety of substituents at the positions and can be carried out under mild conditions using a variety of readily available starting materials. The disclosed synthetic methodology unexpectedly provides a variety of novel applications and advances in bioluminescence methods based on coelenterazine analogues.

[0010] 1.Definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will prevail. Preferred methods and materials are described below, although 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.

[0011] As used herein, the terms "comprise," "include," "having," "has," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words and do not exclude the possibility of additional features or structures. The singular forms "a," "an," and "the" include plural meanings unless the context clearly dictates otherwise. The present disclosure further contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or components set forth herein, whether or not expressly stated.

[0012] The modifier "about" used with a quantity is inclusive of the recited value and has a meaning dictated by the context (e.g., it encompasses at least some degree of error for a particular numerical measurement). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the phrase "from about 2 to about 4" also discloses the range "from 2 to 4". The term "about" may refer to ±10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may represent 0.9 to 1.1. Alternative meanings of "about" may be apparent from the context, such as rounding, e.g., "about 1" may represent 0.5 to 1.4.

[0013] Definitions of specific 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 The general principles of organic chemistry and specific functional moieties and reactivities are generally defined in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito (1999), Smith and March March's Advanced Organic Chemistry, 5th Ed. (Cover), and specific functional groups are generally defined as described therein. 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 entire contents of which are incorporated herein by reference.

[0014] The term "alkoxy" as used herein refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert-butoxy.

[0015] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon chain containing 1 to 10 carbon atoms. The term "lower alkyl" or "C1-C6 alkyl" refers to a linear or branched hydrocarbon chain containing 1 to 6 carbon atoms. The term "C1-C3 alkyl" refers to a linear or branched hydrocarbon chain containing 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0016] As used herein, the term "alkenyl" refers to a hydrocarbon chain containing 2 to 10 carbon atoms and having at least one carbon-carbon double bond. An alkenyl group may be substituted or unsubstituted. For example, an alkenyl group may be substituted with an aryl group, such as phenyl.

[0017] As used herein, the term "alkynyl" refers to a hydrocarbon chain containing 2 to 10 carbon atoms and having at least one carbon-carbon triple bond. An alkynyl group may be substituted or unsubstituted. For example, an alkynyl group may be substituted with an aryl group, such as phenyl.

[0018] The term "alkoxyalkyl," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0019] As used herein, the term "alkylene" refers to a divalent group derived from a straight or branched hydrocarbon chain of 1 to 10 carbon atoms, for example, 2 to 5 carbon atoms. Representative examples of alkylene include, but are not limited to, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-.

[0020] As used herein, the term "aryl" refers to a phenyl group, or a bicyclic aryl fused ring system or a tricyclic aryl fused ring system. A bicyclic fused ring system is exemplified by a phenyl group attached to the parent molecular moiety and fused to a phenyl group. A tricyclic fused ring system is exemplified by a phenyl group attached to the parent molecular moiety and fused to two other phenyl groups. Representative examples of bicyclic aryls include, but are not limited to, naphthyl. Representative examples of tricyclic aryls include, but are not limited to, anthracenyl. Monocyclic aryls, bicyclic aryls, and tricyclic aryls are attached to the parent molecular moiety by any carbon atom in the ring and may be unsubstituted or substituted.

[0021] As used herein, the term "cycloalkyl" refers to a carbocyclic ring system containing 3 to 10 carbon atoms, 0 heteroatoms and 0 double bonds. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl.

[0022] As used herein, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having 5 to 10 carbon atoms per ring. Representative examples of monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0023] As used herein, the term "fluoroalkyl" refers to an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by fluorine. Representative examples of fluoroalkyl include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl, such as 3,3,3-trifluoropropyl.

[0024] The term "alkoxyfluoroalkyl," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through a fluoroalkyl group, as defined herein.

[0025] As used herein, the term "fluoroalkoxy" refers to at least one fluoroalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of fluoroalkyloxy include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0026] As used herein, the term "halogen" or "halo" means Cl, Br, I or F.

[0027] As used herein, the term "haloalkyl" means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by halogen.

[0028] The term "haloalkoxy," as used herein, means at least one haloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0029] As used herein, the term "heteroalkyl" refers to an alkyl group, as defined herein, in which one or more carbon atoms are replaced with a heteroatom selected from S, Si, O, P, and N. The heteroatoms may be optionally oxidized. Representative examples of heteroalkyl include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, amides, and alkyl sulfides.

[0030] As used herein, the term "heteroaryl" refers to an aromatic monocyclic ring system or an aromatic bicyclic ring system or an aromatic tricyclic ring system. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. A bicyclic heteroaryl group is exemplified by a monocyclic cycloalkyl group, as defined herein, a monocyclic aryl group, as defined herein, a monocyclic heteroaryl group, as defined herein, or a monocyclic heterocyclic ring fused to a monocyclic cycloalkyl group, as defined herein, a monocyclic aryl group, as defined herein, a monocyclic heteroaryl group, as defined herein, or a monocyclic heterocyclic ring, as defined herein, attached to a parent molecular moiety. Tricyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring attached to a parent molecular moiety and fused to two of a monocyclic cycloalkyl group, as defined herein, a monocyclic aryl group, as defined herein, a monocyclic heteroaryl group, as defined herein, or a monocyclic heterocycle, as defined herein. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-dihydropyridinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, chromenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranyl, indolyl, quinolinyl, imidazopyridine, benzoxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic, and tricyclic heteroaryls are attached to the parent molecular moiety by any carbon atom or any nitrogen atom contained in the ring, and may be unsubstituted or substituted.

[0031] As used herein, the term "heterocycle" or "heterocyclic" refers to a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The 7- and 8-membered rings contain 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, These include, but are not limited to, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiadiananyl, 1,3-thiadiananyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms of the ring are joined by an alkylene bridge of 1, 2, 3 or 4 carbon atoms or an alkenylene bridge of 2, 3 or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are joined by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 Monocyclic, bicyclic, and tricyclic heterocycles are attached to the parent molecular moiety by any carbon atom or any nitrogen atom in the ring, and can be unsubstituted or substituted.

[0032] As used herein, the term "hydroxyl" refers to an --OH group.

[0033] In some cases, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is indicated by the prefix "C x ~C y -" where x is the smallest number of carbon atoms in the substituent and y is the largest number of carbon atoms in the substituent. So, for example, "C1-C3-alkyl" refers to an alkyl substituent containing 1 to 3 carbon atoms.

[0034] The term "substituent" refers to an aryl, heteroaryl, phenyl, or pyridinyl group "substituted" at any atom of said group. Any atom may be substituted.

[0035] The term "substituted" refers to a group that may be further substituted with one or more non-hydrogen substituents. Substituents include, but are not limited to, halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0036] In the compounds described herein, the groups and substituents may be selected according to the allowed valences of atoms and substituents, such selection and substitution resulting in stable compounds that do not undergo spontaneous transformation, e.g., by rearrangement, cyclization, elimination, and the like.

[0037] In describing numerical ranges herein, each intervening number in the range is expressly contemplated with the same precision. For example, in the range from 6 to 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and in the range from 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.

[0038] 2.Compound Formula (I) The present invention discloses a compound or tautomer of TIFF0007669430000002.tif58164, or a pharma- ceutically acceptable salt thereof, wherein R 1 is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate, and q is 0 to 2, wherein said alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate are independently substituted or unsubstituted at each occurrence.

[0039] In certain embodiments, R 1is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate; q is 0-2; and wherein said alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate are each independently selected from halogen, =O, =S, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, hetero, and is independently substituted or unsubstituted with 1, 2, 3, 4, 5, 6, or 7 functional groups independently selected from the group consisting of aryl, heterocycle, heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyloxy, t-butyldimethylsilyloxy, alkylsulfanyl, sulfanyl, thiotriazolyl, and acyl.

[0040] In certain embodiments, R 1is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate, and q is 0 to 2, wherein said alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate are independently substituted or unsubstituted at each occurrence; Here, if q is 0, R 1 is C1-C5 alkyl or TIFF0007669430000003.tif1437, but in the formula, R a is NH2, halogen, OH, NHC(O)C1-C7-alkyl, or CO2C1-C7-alkyl, Here, if q is 1, R 1 is C1-C4 alkyl, TIFF0007669430000004.tif21164, but in the formula, R c is H, OH, OC(O)C1-C7-alkyl, OCH2OC(O)C1-C7-alkyl, X is S, O, NH, NCH3 or NCH2CH3 and Z is CH or N, and Here, if q is 2, R 1 is not C1-C3 alkyl, and wherein the compound of formula (I) is not 8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

[0041] In certain embodiments, R 1is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate; q is 0-2; and wherein alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate, at each occurrence, are independently selected from halogen, =O, =S, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaromatic, aryl, heterocyclic ... and is independently substituted or unsubstituted with 1, 2, 3, 4, 5, 6, or 7 functional groups independently selected from the group consisting of aryl, heterocycle, heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyloxy, t-butyldimethylsilyloxy, alkylsulfanyl, sulfanyl, thiotriazolyl, and acyl; Here, if q is 0, R 1 is C1-C5 alkyl or TIFF0007669430000005.tif1639, but in the formula, R a is NH2, halogen, OH, NHC(O)C1-C7-alkyl, or CO2C1-C7-alkyl, Here, if q is 1, R 1 is C1-C4 alkyl, TIFF0007669430000006.tif21164, but in the formula, R c is H, OH, OC(O)C1-C7-alkyl, OCH2OC(O)C1-C7-alkyl, X is S, O, NH, NCH3 or NCH2CH3 and Z is CH or N, and Here, if q is 2, R 1 is not C1-C3 alkyl, and wherein the compound of formula (I) is not 8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

[0042] In certain embodiments, R 1 is phenyl, pyridinyl, benzodioxolyl, benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl, pyrimidinyl, pyrazinyl, thienothienyl, thienyl, diethylphosphonate, isoxazolyl, imidazothiazolyl, 1,3-dimethylpyrimidin-2,4(1H,3H)-dionyl, furanyl, pyrazolyl, benzothienyl, C 10 ~C 12 alkyl, benzothiazolyl, cinnamyl, dibenzofuranyl, chromenyl or naphthalenyl, and q is 0 to 2, wherein the phenyl, pyridinyl, benzodioxolyl, benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl, pyrimidinyl, pyrazinyl, thienothienyl, thienyl, diethylphosphonate, isoxazolyl, imidazothiazolyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dionyl, furanyl, pyrazolyl, benzothienyl, C 10 ~C 12 alkyl, benzothiazolyl, cinnamyl, dibenzofuranyl, chromenyl and naphthalenyl are independently substituted or unsubstituted in each occurrence; Here, if q is 0, R 1 is C1-C5 alkyl or TIFF0007669430000007.tif1437, but in the formula, Ra is NH2, halogen, OH, NHC(O)C1-C7-alkyl, or CO2C1-C7-alkyl, Here, if q is 1, R 1 is C1-C4 alkyl, TIFF0007669430000008.tif21164, but in the formula, R c is H, OH, OC(O)C1-C7-alkyl, OCH2OC(O)C1-C7-alkyl, X is S, O, NH, NCH3 or NCH2CH3 and Z is CH or N, and Here, if q is 2, R 1 is not C1-C3 alkyl, and wherein the compound of formula (I) is not 8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

[0043] In certain embodiments, R 1 is phenyl, pyridinyl, benzodioxolyl, benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl, pyrimidinyl, pyrazinyl, thienothienyl, thienyl, diethylphosphonate, isoxazolyl, imidazothiazolyl, 1,3-dimethylpyrimidin-2,4(1H,3H)-dionyl, furanyl, pyrazolyl, benzothienyl, C 10 ~C 12 alkyl, benzothiazolyl, cinnamyl, dibenzofuranyl, chromenyl or naphthalenyl, and q is 0 to 2, wherein the phenyl, pyridinyl, benzodioxolyl, benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl, pyrimidinyl, pyrazinyl, thienothienyl, thienyl, diethylphosphonate, isoxazolyl, imidazothiazolyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dionyl, furanyl, pyrazolyl, benzothienyl, C 10 ~C 12Alkyl, benzothiazolyl, cinnamyl, dibenzofuranyl, chromenyl and naphthalenyl, at each occurrence, are selected from the group consisting of halogen, =O, =S, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl. , alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyloxy, t-butyldimethylsilyloxy, alkylsulfanyl, sulfanyl, thiotriazolyl, and acyl; Here, if q is 0, R 1 is C1-C5 alkyl or TIFF0007669430000009.tif1437, but in the formula, R a is NH2, halogen, OH, NHC(O)C1-C7-alkyl, or CO2C1-C7-alkyl, Here, if q is 1, R 1 is C1-C4 alkyl, TIFF0007669430000010.tif21164, but in the formula, R c is H, OH, OC(O)C1-C7-alkyl, OCH2OC(O)C1-C7-alkyl, X is S, O, NH, NCH3 or NCH2CH3 and Z is CH or N, and Here, if q is 2, R 1is not C1-C3 alkyl, and wherein the compound of formula (I) is not 8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one.

[0044] Representative compounds of formula (I) include, but are not limited to: 8-benzyl-2-(4-fluorobenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(2,4-difluorobenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-fluoro-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-fluoro-3-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-fluoro-4-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-fluoro-2-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, tert-butyl (3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)carbamate, 8-benzyl-2-((5-methoxythiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(3-aminobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-(tert-butyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(naphthalen-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-([1,1'-biphenyl]-4-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-((2H-chromen-3-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(dibenzo[b,d]furan-4-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-cinnamyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-((3-acetylbenzo[d]thiazol-2(3H)-ylidene)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, tert-butyl (4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)carbamate, 2-(4-aminobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(4-(allyloxy)-3-methoxybenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-hydroxy-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-((4-methyl-4H-1,2,4-triazol-3-yl)thio)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-((5-(pyridin-2-yl)thiophen-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one, 2-(benzo[b]thiophen-3-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-dodecyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(benzo[b]thiophen-2-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-cyclohexylthiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-([2,2'-bithiophene]-5-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-isobutylthiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-((5-(trifluoromethyl)furan-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one, 4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzoic acid, 8-benzyl-2-((5-(methoxymethyl)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((perfluorophenyl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((1-methyl-3-(thiophen-2-yl)-1H-pyrazol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3,5-bis(trifluoromethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-chloro-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-methoxyfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzoic acid, 8-benzyl-2-((5-(morpholinomethyl)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(3-(aminomethyl)benzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 5-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione, 8-benzyl-2-(imidazo[2,1-b]thiazol-6-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, N-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)methanesulfonamide, 8-benzyl-2-((3,5-dimethylisoxazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-(pyridin-3-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one, Diethyl (8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)phosphonate, 8-benzyl-2-(4-methoxy-3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-(dimethylamino)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-(methoxymethyl)thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-(hydroxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2-methoxypyrimidin-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((3,4-dimethylthieno[2,3-b]thiophen-2-yl)methyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-(pyrazin-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-((2-(propylthio)pyrimidin-5-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((4-methyl-4H-thieno[3,2-b]pyrrol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-((1,2,3-thiadiazol-5-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((1-methyl-1H-benzo[d][1,2,3]triazol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3,4-dimethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-(methylthio)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(benzo[d][1,3]dioxol-5-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-((dimethylamino)methyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-ethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-methylpyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-(dimethylamino)pyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-(2,4,6-trifluorobenzyl)imidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2,6-dimethoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-chloro-4-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-fluoro-2-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2-fluoro-6-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2,6-difluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-bromopyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-chloropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2,6-dichloropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-styryl-1,7-dihydroimidazo[1,2-a]pyrazin-3(2H)-one, 8-benzyl-2-(3-(2-methoxyethoxy)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzonitrile, and pharma-ceutically acceptable salts thereof.

[0045] Compound names were assigned using the Struct=Name naming algorithm as part of CHEMDRAW® ULTRA v.12.0.

[0046] Compounds may exist as stereoisomers containing asymmetric or chiral centers. Stereoisomers are "R" or "S" depending on the structure of the substituents around the asymmetric carbon atom. As used herein, the terms "R" and "S" refer to the structures 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, 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 may be synthetically prepared from commercially available starting materials, which contain asymmetric or chiral centers, or may be prepared by preparation of racemic mixtures followed by resolution methods well known to those skilled in the art. The resolution method is as follows: (1) Furniss, Hannaford, Smith and Tatchell, “Vogel's Textbook of Practical Organic Chemistry”, 5 th This is exemplified by (1) coupling the mixture of enantiomers to a chiral auxiliary and separating the resulting diastereomeric mixture by recrystallization or chromatography to selectively liberate the optically pure products from the auxiliary, as described in U.S. Pat. No. 6,339,341, issued Dec. 29, 1989, Longman Scientific & Technical, Essex CM20 2JE, England, or (2) directly separating the optically pure mixture of enantiomers on a chiral chromatographic column, or (3) by fractional recrystallization techniques.

[0047] It should be understood that compounds may have structural isomers as well as tautomeric forms and these also form a further aspect of the present invention.

[0048] The present disclosure further encompasses isotopically labeled compounds, which are identical to those set forth in formula (I) except for the fact that one or more atoms have been replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, e.g., 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36 Deuterium, i.e., but not limited to Cl. 2 Substitution with heavier isotopes such as H may confer certain therapeutic advantages due to greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some circumstances. The compounds may also 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 compounds of formula (I) are: 11 C. 13 N, 15 O. 18 F. Isotopically labeled compounds of formula (I) may 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 appropriate isotopically labeled reagents for non-isotopically labeled reagents.

[0049] A. Properties of the Compounds of Formula (I) The compound of formula (I) can be a substrate for luciferase to generate luminescence. The compound may have improved water solubility, improved stability, improved cell permeability, high biocompatibility with cells, reduced autobioluminescence, and low toxicity.

[0050] "Luminescence" refers to the light output of 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 (sometimes referred to as "T=0" luminescence or "flash"), which can be initiated upon addition of the coelenterazine substrate. In various embodiments, the luminescence reaction is performed in solution. In other embodiments, the luminescence reaction is performed on a solid support. The solution may contain a lysate, e.g., from cells in a prokaryotic or eukaryotic expression system. In another embodiment, expression occurs in a cell-free system or the luciferase protein is partitioned into the extracellular medium, in the latter case, no lysate needs to be generated. In some embodiments, the reaction is initiated by introducing appropriate materials, e.g., a coelenterazine analog, a buffer, etc., into the reaction chamber (e.g., a well of a multi-well plate, such as a 96-well plate) containing the luminescent protein. In yet another embodiment, luciferase and / or coelenterazine analogs (e.g., compounds of formula (I)) can be introduced into a host and luminescence measurements can be made in the host or portions thereof, including whole organisms or cells, tissues, explants, or extracts thereof. The reaction chamber can be placed in a reading device, which allows light output to be measured, for example, using a luminometer or photomultiplier tube. Light output or luminescence can also be measured over time, for example, over seconds, minutes, hours, etc., in the same reaction chamber. Light output or luminescence can be reported as an average over time, half-life of signal decay, sum of signal over time, or peak output. Luminescence can be measured in Relative Light Units (RLU).

[0051] The RLU of a compound of formula (I) may be 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 relative to coelenterazine or a known coelenterazine analogue.

[0052] "Biocompatibility" refers to the tolerance of a cell (e.g., a prokaryotic or eukaryotic organism) to a coelenterazine analog (e.g., a compound of formula (I)). The biocompatibility of a coelenterazine analog is related to the stresses that occur in the host cell.

[0053] The high biocompatibility of a coelenterazine analog (e.g., a compound of formula (I)) can be determined by measuring cell viability and / or cell growth rate. For example, the high biocompatibility of a coelenterazine analog can be determined by measuring cell viability in the absence of luciferase expression in 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.

[0054] Specifically, high biocompatibility can be determined using cell viability analysis (e.g., using CELLTITER-GLO® luminescent cell viability assay), apoptosis assay (e.g., using CASPASE-GLO® method), or other methods known in the art. The effect of the compound of formula (I) on cell viability or apoptosis can be compared with the effect of natural or known coelenterazine analogs on cell viability or apoptosis.

[0055] High biocompatibility can also be determined by measuring the effect of coelenterazine analogs (e.g., compounds of formula (I)) on cell growth or gene expression. For example, high biocompatibility of compounds of formula (I) can be determined by measuring cell number after a period of time in a cell sample exposed to compounds of formula (I) or by measuring the expression of stress response genes in comparison with cells exposed to natural or known coelenterazine or cells not exposed to coelenterazine. The effect of compounds of formula (I) on cell growth or gene expression can be compared with natural or known coelenterazine.

[0056] 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, such as a hydroxide, carbonate or bicarbonate of a metal cation, such as lithium, sodium, potassium, calcium, magnesium or aluminum, or a primary, secondary or tertiary organic amine. Quaternary amine salts can also 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.

[0057] B. Synthesis of Compounds of Formula (I) Current methods for producing coelenterazine analogs use harsh reaction conditions. The limitations of this synthetic protocol have slowed the search for new analogs. Furthermore, the incompatibility of the process conditions with various functional groups and the inability to synthesize ketoaldehyde / keto acid precursors have also limited the structural changes at the C-2 position of coelenterazine. TIFF0007669430000011.tif60164

[0058] Here, three robust and versatile methods are described to accomplish the preparation of compounds of formula (I). The methods are useful for the preparation of analogs that could not be produced using existing synthetic methods. The disclosed methodology allows access to a variety of substituents at the C-2 position and can be carried out under relatively mild conditions utilizing a variety of readily available aldehydes as starting materials.

[0059] The compounds of formula (I) may be prepared by synthetic processes or by metabolic processes, including those occurring in the human or animal body (in vivo) or in vitro.

[0060] Compounds of formula (I), in which, unless otherwise specified, R 1 Compounds in which the groups and q have the meanings described in the Summary of the Invention can be synthesized as shown in Schemes 1 to 3.

[0061] The following abbreviations are used in the description of the schemes below: Ac2O is acetic anhydride, CDI is carbonyldiimidazole, MeOH is methanol, TMG is 1,1,3,3-tetramethylguanidine, and TFA is trifluoroacetic acid.

[0062] Scheme 1. Synthesis of intermediates A–C As shown in Scheme 1, intermediates A-C can be prepared from aminopyrazine i. Treatment of i with diazocarbonyls ii-iv in the presence of Rh2(OAc)4 can form aminopyrazine acetophosphonates A-C. Intermediates A-C can be stable at room temperature and may provide starting materials for a variety of analogues.

[0063] Scheme 2. Synthesis of compounds of formula (I) Scheme 2 depicts the conversion of intermediates A and B to compounds of formula (I), where n is 1. Intermediates A and B can be treated with 1,1,3,3-tetramethylguanidine to undergo a Horner-Wadsworth-Emmons olefination reaction with aldehyde v (where R 1 is as defined in the Summary of the Invention) to produce intermediate vi, which can be reduced to give compounds of formula (I).

[0064] Scheme 3. Synthesis of compounds of formula (I) Scheme 3 depicts the preparation of compounds of formula (I) by two routes from intermediate C. Intermediate C can be treated with 1,1,3,3-tetramethylguanidine to afford aldehyde v in a Horner-Wadsworth-Emmons olefination reaction (R 1 is as defined in the Summary of the Invention) to provide intermediate vii. Intermediate vii can be hydrogenated to provide intermediate viii, which can then be converted to compounds of formula (I) by treatment with TFA followed by cyclization promoted by the addition of carbonyldiimidazole. Alternatively, intermediate vii can be treated with TFA to hydrolyze the t-butyl ester to form intermediate ix. Subsequent addition of acetic anhydride followed by sodium borohydride can provide compounds of formula (I).

[0065] The optimum reaction conditions and reaction times for each step may vary depending on the specific reactants and the substituents contained in the reactants used. Specific procedures are developed in the Examples section. The reactions may be worked up in a conventional manner, for example, by removing the solvent from the residue and further purification according to methodologies generally known in the art, such as, but not limited to, crystallization, distillation, extraction, trituration and chromatography. Unless otherwise specified, the starting materials and reagents are commercially available or may be prepared by those skilled in the art from commercially available materials using methods described in the chemical literature. If the starting materials are not commercially available, they may be prepared by procedures selected from standard organic chemistry techniques, techniques analogous to the synthesis of known structurally similar compounds, or techniques analogous to the schemes described above, or procedures described in the Synthesis Examples section.

[0066] Routine experimentation, including appropriate control of reaction conditions, reagents and the sequence of synthetic routes, protection of any chemical functional groups that are incompatible with the reaction conditions, and deprotection at suitable positions within the reaction sequence of the method, is within the scope of the invention. Suitable protecting groups, and methods for protecting and deprotecting other substituents with said suitable protecting groups, are well 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 (4 th ed.), John Wiley & Sons, NY (2006), which is incorporated by reference herein in its entirety. Synthesis of the compounds of the invention can be accomplished by methods analogous to those described in the synthetic schemes and specific examples above.

[0067] If an optically active form of a disclosed compound is required, it may be obtained by carrying out one of the procedures described herein using optically active starting materials (prepared, for example, by asymmetric induction of a suitable reaction step) or by resolving a stereoisomeric mixture of the compound or intermediate using standard procedures (such as chromatographic separation, recrystallization or enzymatic resolution).

[0068] Similarly, if a pure stereoisomer of a compound is required, it may be obtained by carrying out one of the procedures described above using a pure stereoisomer as a starting material, or by resolving a mixture of stereoisomers of the compound or intermediates using standard procedures (such as chromatographic separation).

[0069] It will be understood that the synthetic schemes and specific examples as described are illustrative and should not be construed as limiting the scope of the invention as defined in the appended claims. All variations, modifications and equivalents of the synthetic methods and specific examples are intended to be encompassed within the scope of the claims.

[0070] 3. Instructions for use and kit The disclosed compounds and proteins can be used in any method where luciferase substrates, such as coelenterazine analogs, have been used. For example, coelenterazine analogs can be used in bioluminescence assays to detect one or more molecules, such as enzymes, cofactors for enzyme reactions, enzyme substrates, enzyme inhibitors, enzyme activators, or OH radicals, or one or more conditions, such as redox conditions, in a sample. The sample can include animals (e.g., vertebrates), plants, fungi, physiological fluids (e.g., blood, plasma, urine, mucosal secretions), cells, cell lysates, cell supernatants, or purified cell fractions (e.g., subcellular fractions). The presence, amount, spectral distribution, emission kinetics, or specific activity of the 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).

[0071] In certain embodiments, the compound of formula (I) can be used to quantify coelenterazine. In some embodiments, coelenterazine (e.g., natural or known coelenterazine or a compound of formula (I)) can also be used as a probe of specific biochemical activity, such as apoptosis or drug metabolism. In some embodiments, coelenterazine concentration is linked to a specific enzyme activity by a "pro-coelenterazine" or "pro-substrate" that can be acted upon by a specific enzyme of interest. In some embodiments, pro-coelenterazine is a molecule that cannot directly support luminescence when combined with luciferase, but can be converted to coelenterazine via a catalytic process by a specific enzyme of interest. In some embodiments, the approach can be used in the case of enzymes used in drug metabolism, such as cytochrome P450 enzymes, monoamine oxidase, and glutathione S-transferase, as well as enzymes used in apoptosis, such as caspases. For example, coelenterazine (e.g., natural or known coelenterazine, or a compound of formula (I)) can be modified to contain a cleavable group, such as 6'-O-methyl. In some embodiments, when incubated with a specific cytochrome P450 enzyme, the 6'O-methyl is cleaved and pro-coelenterazine is converted to coelenterazine, which can be detected by luciferase. In some embodiments, pro-coelenterazine can be combined with another component that needs to support light emission, for example, a light-emitting protein such as luciferase, to provide a single reagent and homogeneous assay. For example, when the reagent is added to a sample, light is generated when pro-coelenterazine is converted to coelenterazine. In various embodiments, similar assays can be developed for other enzymes, small cells, or other cellular processes that may be involved in the generation of coelenterazine from pro-coelenterazine.

[0072] In certain embodiments, compounds of formula (I) may be used to detect luminescence in live cells. In some embodiments, luciferase is expressed in cells (as a reporter or otherwise) and cells treated with coelenterazine (e.g., compounds of formula (I)) to permeate the cells in culture and react with luciferase to produce luminescence. In addition to cell permeability, compounds of formula (I) exhibit biocompatibility comparable to native coelenterazine in terms of cell viability. In some embodiments, compounds of formula (I) containing chemical modifications known to increase the stability of native coelenterazine in culture medium may be synthesized and used in more robust live cell luciferase-based reporter assays. In yet another embodiment, samples (including cells, tissues, animals, etc.) containing luciferase and compounds of formula (I) may be examined using a variety of microscopy and imaging techniques. In yet another embodiment, secreted luciferase is expressed in cells as part of a live cell reporter system.

[0073] In certain embodiments, the compounds of formula (I) disclosed herein may be provided as part of a kit. In some embodiments, the kit may also include one or more luciferases (in polypeptide or polynucleotide form, or both) and coelenterazine, along with suitable reagents and instructions that allow a user to carry out an assay as disclosed herein. The coelenterazine may be naturally occurring, known, or a compound of formula (I) disclosed herein. The kit may further include one or more buffers as disclosed herein. EXAMPLES

[0074] 4. Working Example Example 1. Synthesis of common intermediates (A, B, C) Scheme 4. Synthesis of intermediates A-C General synthetic procedure for TIFF0007669430000015.tif49168A-C: A 5 mL round bottom flask was charged with aminopyrazine (1, 500 mg, 1.9 mmol), diazo compounds (2-4, 3.83 mmol, 2 equiv.), Rh2(OAc)4 (84.57 mg, 10 mol%) and 3 mL of chlorobenzene. The reaction mixture was heated at 100 °C for 24 h. The progress of the reaction was monitored by LCMS. After 24 h, the reaction reached 100% conversion. The mixture was adsorbed onto Celite and purified on a silica column using 40% EtOAc in heptane as eluent, and the desired products were isolated pure as brown solids in 78-90% yields. The required diazophosphonoacetates were synthesized according to Wang et al. Synlett, 2007, 11, 1683-1686.

[0075] Methyl 2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate (A): Yield 90%, 1 H NMR(300 MHz, CDCl3) δ=8.40(s,1H),7.95-7.91(m,2H),7.49-7.40(m,2H), 7.40-7.19 (m, 6H), 5.33-5.19 (m, 2H),4.25(s,2H),4.18-3.85(m,4H),3.73(s, 3H),1.25 (t, J=6, 3H), 1.20 (t, J=6, 3H), 13 C NMR (75 MHz, CDCl3) δ = 168.34, 168.32, 149.76, 149.64, 142.24, 141.72, 137.14, 136.66, 136.40, 128.85, 128.77, 128.76 128.06, 126.96, 125.78, 63.72, 63.65, 63.63, 63.56, 53.55, 52.96, 51.61, 40.84, 16.33, 16.29, 16.25, 16.21. ESI-MS (m / z) [M+H] (C24H29N3O5P) found 470.

[0076] Benzyl 2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate (B): Yield 78%, 1 H NMR(300 MHz, CDCl3) δ=8.35(s,1H),7.94(d, J=7.2, 2H), 7.50-7.42 (m, 2H), 7.40 - 7.18 (m, 11H), 5.43 - 5.06 (m, 4H), 4.25 (s, 2H), 4.17 - 3.76 (m, 4H), 1.19 (t, J=7.1, 3H), 1.16 (t, J=7.0, 3H); 13 C NMR (75 MHz, CDCl3) δ = 167.70, 167.68, 149.82, 149.70, 142.27, 141.73, 137.18, 136.63, 136.39, 135.20, 128.85, 128.79, 128.77, 128.43, 128.30, 128.29, 128.07, 126.94, 125.80, 67.57, 63.72, 63.64, 63.60, 63.51, 53.96, 52.04, 40.84, 16.29, 16.24, 16.21, 16.16;ESI-MS (m / z) [M+H] (C30H32N3O5P) Found 546.

[0077] tert-Butyl 2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate (A): Yield 83%, 1 H NMR(300 MHz, CDCl3) δ=8.41(s,1H),7.95 (d, J=5.2, 2H), 7.52 - 7.16 (m, 8H), 5.30 - 5.06 (m, 2H), 4.31 - 4.17 (m, 2H), 4.15 - 3.83 (m, 4H), 1.44 (s, 9H), 1.25 (t, J=9, 3H), 1.19 (t, J=9 3H); 13C NMR (75 MHz, CDCl3) δ = 166.54, 166.52, 149.89, 149.79, 141.94, 141.60, 137.25, 136.58, 136.49, 128.80, 128.75, 127.98, 126.84, 125.73, 82.86, 63.46, 63.38, 63.29, 63.20, 54.43, 52.50, 40.75, 27.84, 16.34, 16.26;ESI-MS (m / z) [M+H] (C27H35N3O5P) Actual value 512.

[0078] Example 2. Method for the synthesis of compounds of formula (I) Scheme 5 Method I TIFF0007669430000016.tif78158The development of the methodology for the synthesis of compounds of formula (I) began with studying the reaction conditions for the Horner-Wadsworth-Emmons (HWE) olefination reaction shown in the scheme above. Various bases were screened for the HWE olefination reaction of p-fluorobenzaldehyde with compound A in THF. These initial experiments showed that the reaction did not proceed when the base used had a pKa below 11 (K2CO3 or DIPEA). Meanwhile, strong nitrogen or alkali metal bases with a pKa above 12 produced a complex mixture of decomposition products. Surprisingly, the use of 1,1,3,3-tetramethylguanidine (TMG) in THF (pKa ≈13.6) resulted in a reduced yield of the olefination product (see entry 2 in Table 1).

[0079] Most aldehydes, when used in HWE reactions with compound A, did not exclusively afford the unsaturated ester x. Instead, x tended to cyclize to give vi (see Scheme 5). However, these compounds were generally unstable, especially in aprotic solvents and at high pH. Further experiments with solvent / base combinations showed that a variety of protic solvents, preferably MeOH, stabilized the cyclized product vi (see Scheme and Table 1 below), while pretreatment of A with TMG in methanol afforded the cyclized diethylphosphonate [diethyl (8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)phosphonate, compound 1].

[0080] In a model system using p-fluorobenzaldehyde, several nitrogen bases afforded the desired cyclization product E in the reaction of A with p-fluorobenzaldehyde. In general, there was no advantage to using bases other than TMG in MeOH (Table 1). TIFF0007669430000017.tif39163 [Table 1] The acronyms of the bases and solvents used in screening the reaction conditions: TMG: 1,1,3,3-tetramethylguanidine, DBU: 1,8-diazabicycloundec-7-ene, t-BuTMG: 2-tert-butyl-1,1,3,3-tetramethylguanidine, TBD: 1,5,7-triazabicyclo[4.4.0]dec-5-ene, MeTBD: 7-methyl-1,5,7-triazabicyclo[4.4.40]dec-5-ene. * The yields of products D and E were obtained by integration of the LCMS traces of the corresponding reaction mixtures. Conditions: room temperature, A 10 mg, solvent (4 mL), TMG (3 eq), 4-fluorobenzaldehyde aldehyde 3 eq.

[0081] In the reaction of A with p-fluorobenzaldehyde, the product ratio of D to E was determined by the equilibrium established in the TMG-methanol solution, and it was speculated that the instability of E under said conditions led to the complete destruction of the reactants within 24 hours. Re-exposure of individually isolated D and E to the reaction conditions led to similar ratios of D and E. Furthermore, when the reaction was carried out in n-butanol, similar ratios of D and E were produced, but only a small proportion of the n-Bu-ester (R 5 = n-Bu) was also formed. Thus, for the widest range of substrates, methanol as a solvent was accessible to E. Typically, the synthesis of E in methanol required less than 4 hours and proceeded in good yields if the reaction was stopped before significant decomposition of E was observed.

[0082] General procedure for Method I: A 20 mL vial was charged with methyl 2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate (A) (100 mg, 0.21 mmol, 1 equiv.), aldehyde (0.23 mmol, 1.1 equiv.) and 12 mL of methanol. To the solution was added 1,1,3,3-tetramethylguanidine (74 mg, 0.64 mmol, 3 equiv.). The reaction mixture was stirred at room temperature until maximum conversion was reached (2-6 h). The progress of the reaction was monitored by LCMS. The mixture was poured into water, extracted with ethyl acetate and dried over MgSO4. The drying agent was filtered off and the solvent was concentrated under reduced pressure. The residue was flash chromatographed on silica gel with dichloromethane as eluent. Dehydrocoelenterazine, corresponding to the general structure vi, was isolated as a red solid and used in the next step without further purification. Dehydrocoelenterazine vi was dissolved in 25 mL of dichloromethane and 10 mL of methanol and cooled to 0° C. To this solution, NaBH4 (24.2 mg, 0.64 mmol, 3 equiv.) was added and the reaction mixture was stirred at 0° C. for 30 min. The reaction mixture was quenched with 50 mL of 0.1 M HCL, extracted with dichloromethane, and dried by MgSO4. The drying agent was filtered off, the solvent was concentrated under reduced pressure, and the residue was purified on silica gel using dichloromethane / methanol as eluent. The desired coelenterazine analog was isolated pure as a yellow solid and dried under high vacuum.

[0083] Using common intermediate A and the general procedure of Method I above, the following compounds were prepared. Yields are calculated for the two-step procedure starting from intermediate A. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0084] Example 3. Modifications of Method I for the Synthesis of Compounds of Formula (I) Scheme 6. Modified Method I TIFF0007669430000024.tif89148 Further investigation of method I showed that the use of intermediate B is useful when the equilibration between the cyclized and uncyclized olefin intermediates takes time. Immediately after the completion of the olefination reaction, vi was formed, and the uncyclized intermediate was not detected in the reaction mixture. On the other hand, vi also underwent ring-opening by methoxy attack to give x. To prevent the formation of x, the reaction was stopped after 100% conversion of B (10-20 min). Nevertheless, the compound of formula (I) was smoothly obtained by reducing vi. This two-step procedure could be completed in 10-20 min. Furthermore, the yield of the target substrate could be increased.

[0085] Throughout this study, R 1 It was observed that electron-withdrawing substituents at the R position tend to favor the formation of the closed ring form vi. Taking advantage of this, a modified version of Method I was used to obtain 1 Compounds of formula (I) containing a pyridinyl group at the aryl position were prepared.

[0086] The following compounds were prepared in an analogous manner using common intermediate B and the general procedure of Method I of Example 2. Yields were calculated for the two-step procedure starting from intermediate B. [Table 3]

[0087] Example 4. Methods II and III for synthesizing compounds of formula (I) Method I is a convenient and rapid method with short reaction times that allows the use of a wide variety of compounds of formula (I). However, it is difficult to combine electron-deficient aromatic rings or reactive heterocycles with R 1Many compounds of formula (I) containing the aryl group at the aryl position cannot be prepared by Method I. In some cases, the olefination and subsequent cyclization were time consuming and significant decomposition of the product was observed. On the other hand, electron-rich aldehydes tended to be converted slowly in the HWE olefination reaction and also led to the formation of significant amounts of the non-productive intermediate 1 (see Example 2).

[0088] To circumvent this challenge, two alternative routes were developed. Utilizing C suppressed cyclization to the imidazolinone ring during the HWE reaction, providing only the unsaturated ester vii. Subsequent treatment with TFA produced the unsaturated acid ix, which was converted to compounds of formula (I) via a two-step cyclization and reduction sequence (Method II). In this method, vii was typically isolated in excellent yield. After the hydrolysis procedure, Method II avoids exposure of sensitive intermediates to high pH conditions. Thus, provided that the product ix was stable to TFA treatment, Method II provided compounds of formula (I) in higher yield than Method I, although the procedure was more time consuming.

[0089] Scheme 7. Method II TIFF0007669430000026.tif75153 General procedure for Method II: A 20 mL vial was charged with tert-butyl 2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate (C) (200 mg, 0.39 mmol, 1 eq.), aldehyde (0.43 mmol, 1.1 eq.) and 15 mL of methanol. To the solution was added 1,1,3,3-tetramethylguanidine (135 mg, 1.2 mmol, 3 eq.) and the reaction mixture was stirred at room temperature for 2 h. The progress of the reaction was monitored by LCMS. The reaction mixture was poured into water, extracted with ethyl acetate and dried by MgSO4. The drying agent was filtered off, the solvent was concentrated under reduced pressure and the product having the general structure vii was used in the next step without further purification.

[0090] To a solution of vii in dichloromethane (5 mL) was added trifluoroacetic acid (2 mL). The reaction mixture was stirred at room temperature for 4 hours. The progress of the reaction was monitored by LCMS. After completion of the reaction, all volatiles were removed under reduced pressure and the residue was dried under high vacuum. The resulting unsaturated acid was dissolved in THF (10 mL) and to this solution was added acetic anhydride (398 mg, 3.9 mmol, 10 equiv), triethylamine (395 mg, 3.9 mmol, 10 equiv) and DMAP (4.8 mg, 0.039 mmol, 0.1 equiv). The reaction mixture was stirred at room temperature for 30 minutes and poured into water, then extracted with dichloromethane and dried by MgSO4. The drying agent was filtered off and the solvent was concentrated under reduced pressure. The residue was flash chromatographed on silica gel with dichloromethane as eluent. Dehydrocoelenterazine corresponding to the general structure vi was isolated as a red solid and used in the next step without further purification.

[0091] Dehydrocoelenterazine vi was dissolved in 25 mL of dichloromethane and 10 mL of methanol and cooled to 0° C. To this solution, NaBH4 (44.26 mg, 1.2 mmol, 3 equiv.) was added and the reaction mixture was stirred at 0° C. for 30 min. The reaction mixture was quenched with 100 mL of 0.1 M HCL, extracted with dichloromethane, and dried by MgSO4. The drying agent was filtered off, the solvent was concentrated under reduced pressure, and the residue was purified on silica gel with dichloromethane / methanol as eluent. The desired coelenterazine analog of formula (I) was isolated pure as a yellow solid and dried under high vacuum.

[0092] Method III is a second alternative that avoids potentially unstable synthetic intermediates. Hydrogenation of unsaturated ester vii is accomplished under high pressure hydrogen in the presence of Wilkinson's catalyst, typically affording intermediate viii, which is easy to isolate and purify. Treatment of viii with TFA, followed by intramolecular cyclization of the corresponding acid with carbonyldiimidazole (CDI), afforded compounds of formula (I). Method III uses mild reaction conditions to avoid oxygen- and moisture-sensitive reaction intermediates, but is suitable for both scale-up and preparation of coelenterazine analogs containing sensitive functional groups. However, high pressure and long reaction times are required.

[0093] Scheme 8. Method III TIFF0007669430000027.tif71154 General procedure for Method III: A 20 mL vial was charged with tert-butyl 2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-2-(diethoxyphosphoryl)acetate (C) (200 mg, 0.39 mmol, 1 equiv), aldehyde (0.43 mmol, 1.1 equiv) and 15 mL of methanol. To the solution, 1,1,3,3-tetramethylguanidine (135 mg, 1.2 mmol, 3 equiv) was added and the reaction mixture was stirred at room temperature for 1-2 h. The progress of the reaction was monitored by LCMS. After completion of the reaction, the mixture was poured into water, extracted with ethyl acetate and dried over MgSO4. The drying agent was filtered off and the solvent was concentrated under reduced pressure. The residue was purified using flash chromatography on silica gel with heptane / ethyl acetate as eluent, and the target compound vii was isolated as a colorless oil.

[0094] A Parr-shaker reaction flask was charged with tert-butyl 2-(3-benzyl-5-phenylpyrazin-2-ylamino)-acrylate derivative vii (0.3 mmol, 1 equiv.), Rh(PPh3)3Cl (0.03 mmol, 0.1 equiv.) and 50 mL of ethanol. The reaction vessel was flushed with H2 (50 psi) and shaken at room temperature for 20 h. The progress of the reaction was monitored by LCMS. All volatiles were then removed under reduced pressure and the residue was flash chromatographed on silica gel using heptane / ethyl acetate as eluent. The reduced aminopyrazine derivative vii was isolated pure as a colorless oil.

[0095] Compound viii (0.25 mmol, 1 equiv.) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (2 mL) was added to the solution. The reaction mixture was stirred at room temperature for 12 h. The progress of the reaction was monitored by LCMS. After completion of the reaction, all volatiles were removed under reduced pressure and the residue was coevaporated with 10 mL of toluene three times to remove all TFA to give compound xi. After drying under high vacuum, compound xi was dissolved in dichloromethane (15 mL) and carbodiimidazole (CDI) (122 mg, 0.75 mmol, 3 equiv.) was added to the solution. The reaction mixture was stirred at room temperature for 20 min, then poured into 0.01 M HCl (50 mL), extracted with dichloromethane, and dried by MgSO4. The drying agent was filtered off and the solvent was concentrated under reduced pressure. The residue was flash chromatographed on silica gel eluting with dichloromethane / methanol. The coelenterazine corresponding to formula (I) was isolated pure as a yellow solid.

[0096] The following representative intermediates were isolated and characterized using Method III: tert-Butyl (E)-2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-3-(3-cyanophenyl)acrylate TIFF0007669430000028.tif5750 1H NMR (300 MHz, CDCl3) δ = 10.03 (s, 1H), 8.31 (s, 1H), 8.15 (td, J=0.6, 1.6, 1H), 8.12 - 8.08 (m, 1H), 7.96 - 7.87 (m, 2H), 7.68 (t, J=7.7, 1H), 7.51 - 7.35 (m, 4H), 7.33 - 7.23 (m, 3H), 7.21 - 7.07 (m, 1H), 6.98 (s, 1H), 6.34 (s, 1H), 4.30 (s, 2H), 1.46 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ = 189.91, 164.29, 148.16, 143.74, 142.89, 137.18, 136.85, 136.79, 136.76, 136.62, 136.13, 133.27, 133.12, 132.29, 132.23, 131.08, 130.23, 130.09, 129.20, 129.12, 128.83, 128.54, 128.49, 127.41, 126.02, 121.99, 118.39, 117.54, 113.68, 112.55, 104.99, 82.43, 41.15, 27.89;ESI-MS (m / z) [M+H] (C31H29N4O2) found 489. tert-Butyl 2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-3-(3-cyanophenyl)propanoate TIFF0007669430000029.tif5851 1 H NMR (300 MHz, CDCl3) δ = 8.40 (s, 1H), 7.98 - 7.92 (m, 2H), 7.50 - 7.43 (m, 3H), 7.38 - 7.23 (m, 7H), 7.22 - 7.18 (m, 1H), 7.11 (dt, J=1.5, 7.8, 1H), 5.02 (d, J=6.8, 1H), 4.93 - 4.79 (m, 1H), 4.15 (q, J=15.3, 3H), 3.10 (ddd, J=5.8, 13.9, 34.0, 2H), 1.38 (s, 9H);13 C NMR (75 MHz, CDCl3) δ = 170.81, 150.01, 141.21, 141.14, 138.34, 137.35, 136.79, 136.45, 133.91, 132.94, 130.36, 128.93, 128.91, 128.77, 128.58, 127.88, 127.03, 125.66, 118.72, 112.15, 82.50, 55.09, 40.94, 37.46, 27.96 (C31H31N4O2) Found value 491. tert-Butyl (E)-2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-3-(1,2,3-thiadiazol-5-yl)acrylate TIFF0007669430000030.tif5551 1 H NMR (300 MHz, CDCl3) δ = 9.45 (s, 1H), 8.51 (s, 1H), 8.27 (s, 1H), 8.03 - 7.96 (m, 2H), 7.52 - 7.45 (m, 4H), 7.43 - 7.31 (m, 3H), 7.30 - 7.22 (m, 1H), 6.70 (s, 1H), 4.43 (s, 2H), 1.48 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ = 164.35, 159.03, 147.85, 144.44, 143.61, 136.90, 136.87, 136.42, 135.80, 133.05, 129.07, 128.85, 128.74, 128.54, 126.81, 126.09, 104.18, 82.10, 40.34, 27.87;ESI-MS (m / z) [M+H] (C26H26N5O2S) Actual value 472 tert-Butyl 2-((3-benzyl-5-phenylpyrazin-2-yl)amino)-3-(1,2,3-thiadiazol-5-yl)propanoate TIFF0007669430000031.tif5552 1H NMR (300 MHz, CDCl3) δ = 8.42 (s, 1H), 7.99 - 7.93 (m, 2H), 7.51 - 7.42 (m, 3H), 7.41 - 7.23 (m, 6H), 5.41 (d, J=6.6, 1H), 5.00 (dt, J=5.0, 6.5, 1H), 4.19 (dd, J=15.2, 58.4, 2H), 3.82 - 3.62 (m, 2H), 1.36 (s, 9H); 13 C NMR (75 MHz, CDCl3) δ = 170.52, 158.41, 149.87, 141.26, 141.12, 137.38, 136.91, 136.87, 132.95, 129.02, 128.81, 128.78, 127.88, 127.01, 125.65, 82.60, 53.92, 40.98, 30.06, 27.83;ESI-MS (m / z) [M+H] (C26H28N5O2S) Actual value 474.

[0097] The following compounds were prepared by Methods II and III above. Yields were calculated for the four step procedure starting from Intermediate C. [Table 4] a: Compound was prepared by Method II except that the final reduction step with NaBH4 was eliminated.

[0098] Therefore, several synthesis methodologies have been established, but the scope and utility of these methods are unclear. 1 This has been demonstrated by synthesizing compounds of formula (I) bearing a variety of substituents at the aryl groups. As shown above, aromatic groups bearing electron-withdrawing substituents, aromatic groups bearing electron-donating substituents, sterically demanding groups, and polar heterocyclic motifs are all amenable to synthesis using one of these new methods.

[0099] Example 5. Luminescence properties Luminescence assay procedure: Each compound to be screened was dissolved in DMSO (5 mM) and then further diluted to 100 uM in NANO-GLO® Luciferase Assay Buffer. Each diluted substrate was then mixed with an equal volume of purified NanoLuc® Luciferase diluted in CO2-independent medium + 10% FBS. The initial light output of each substrate was measured in a GloMax® Multi+ Luminometer 3 minutes after addition of the substrate and at 5 minute intervals thereafter as a method for determining signal half-life.

[0100] Some of the synthesized coelenterazine analogs (compounds of formula (I)) were evaluated for their suitability as luciferase substrates. NANOLUC® luciferase was used in the screen because it is a small (19 kDa), stable, and particularly bright enzyme. Table 2 demonstrates that the relative light units (RLU) and half-life data (relative to native coelenterazine) indicate that moderate electron donating and electron withdrawing functionalities are well tolerated. Various pyridinyl groups were also well tolerated, with a significant number demonstrating superiority over coelenterazine. Overall, a significant number of the analogs were superior to coelenterazine in producing light. [Table 5]

[0101] The compounds of formula (I) were also evaluated for their potential as luciferase substrates in an additional assay, in which the analogs were compared to a known coelenterazine analog, 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one (C-1), which is known to be a better luciferase substrate than coelenterazine. Table 3 demonstrates that a significant number of the analogs are well tolerated and perform as well as or better than 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one. [Table 6] TIFF0007669430000035.tif237168 TIFF0007669430000036.tif27168

[0102] Example 6. Cell permeability and bioluminescence activity Cell Culture: HeLa and HEK293 cells were maintained in DMEM containing 0.3 mg / ml glutamine, 100 IU / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum at 37° C. in 5% CO2. Dulbecco's modified Eagle's medium (DMEM), Opti-MEM, penicillin / streptomycin, and trypsin-EDTA were obtained from Life Technologies (Carlsbad). Fetal bovine serum (FBS) was obtained from HyClone (GE Healthcare). Microtiter plates were obtained from Corning.

[0103] Cell-based luciferase assay: HEK293 cells stably expressing NANOLUC® luciferase under the control of the CMV promoter were plated at a density of 10,000 cells per well in 100 μl of growth medium (DMEM supplemented with 10% FBS) in the wells of white TC-treated 96-well plates and cultured for 24 hours. The growth medium was then replaced with 100 μl of OptiMEM containing 12.5 μM of the substrate. Immediately after addition of the substrate, the luminescence signal was analyzed using a GLOMAX® Discove multimode detection plate reader (Promega).

[0104] The cell permeability and bioluminescence activity of selected compounds (64-77) were compared to coelenterazine using HEK293 cells stably expressing NANOLUC® luciferase (Figure 1). Most of the tested compounds showed similar or stronger luminescence signals than coelenterazine, and at least six showed a significant increase (>5-fold) over coelenterazine. Furthermore, the ranking of the compounds by signal intensity comparison with coelenterazine showed a strong correlation between the biochemical assay and the cell-based luciferase assay (Figure 2). Together, the results suggest that the tested analogs can freely permeate biological membranes and show good bioluminescence activity in living cells.

[0105] Example 7. Cell viability Cell viability assay: HEK293 or HeLa cells were seeded in 100 μl of growth medium (DMEM supplemented with 10% FBS) in the wells of a white TC-treated 96-well plate at a density of 10,000 cells per well and cultured for 24 hours. The growth medium was then replaced with 100 μl of Opti-MEM medium containing serial dilutions of the compounds. The change in cell viability was then measured after 24 hours of culture using the CELLTITER® Green cell viability assay (Promega) according to the manufacturer's instructions. All luminescence measurements were performed on a GLOMAX® Discover multimode plate reader (Promega).

[0106] Evaluation of compound-induced cytotoxicity in HeLa cells (Figures 3 and 4) and HEK293 cells (Figures 5 and 6) revealed no fundamental differences in substrate toxicity patterns across the two different cell lines. Interestingly, fluorinated compounds 65-68 generally demonstrated lower toxicity in both cell lines compared to coelenterazine. Moreover, compound 76 further demonstrated particularly lower toxicity in both cell lines and allowed the use of higher substrate concentrations in cell-based assays.

[0107] It is understood that the foregoing detailed description of the invention and the accompanying examples are merely illustrative and should not be construed as limiting the scope of the invention, which is defined solely by the appended claims and equivalents thereof.

[0108] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, including but not limited to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of using the invention, which can be made without departing from the spirit and scope thereof. Yet another aspect of the present invention may be as follows. [1] Compound of formula (I) TIFF0007669430000037.tif52164 or a tautomer or a pharma- ceutically acceptable salt thereof, wherein: R 1 is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate; q is 0 to 2; said alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, and phosphonate are independently substituted or unsubstituted at each occurrence; However, if q is 0, R 1 is C 1 ~C 5 Alkyl or TIFF0007669430000038.tif1333 Instead, here R a NH 2 , halogen, OH, NHC(O)C 1 ~C 7 Alkyl, or CO 2 C 1 ~C 7 is alkyl, If q is 1, R 1 is C 1 ~C 4 Alkyl, TIFF0007669430000039.tif21164 Instead, here R c are H, OH, OC(O)C 1 ~C 7 Alkyl, OCH 2 O.C.(O)C 1 ~C 7 alkyl, and X is S, O, NH, NCH 3 or NCH 2 CH 3 and Z is CH or N; If q is 2, R 1 is C 1 ~C 3 Not alkyl, and The compound of formula (I) is not 8-benzyl-2-((1-methyl-1H-imidazol-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one; The compound, or a tautomer or a pharma- ceutically acceptable salt thereof. 〔2〕R 1 However, phenyl, pyridinyl, benzodioxolyl, benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl, pyrimidinyl, pyrazinyl, thienothienyl, thienyl, diethylphosphonate, isoxazolyl, imidazothiazolyl, 1,3-dimethylpyrimidin-2,4(1H,3H)-dionyl, furanyl, pyrazolyl, benzothienyl, C 10 ~C 12 The compound according to the above item [1], which is alkyl, benzothiazolyl, cinnamyl, dibenzofuranyl, chromenyl or naphthalenyl. [3] 8-benzyl-2-(4-fluorobenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(2,4-difluorobenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-fluoro-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-fluoro-3-hydroxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-fluoro-4-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-fluoro-2-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, tert-butyl (3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)carbamate, 8-benzyl-2-((5-methoxythiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(3-aminobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-(tert-butyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(naphthalen-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-([1,1'-biphenyl]-4-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-((2H-chromen-3-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(dibenzo[b,d]furan-4-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-cinnamyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-((3-acetylbenzo[d]thiazol-2(3H)-ylidene)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, tert-butyl (4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)carbamate, 2-(4-aminobenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(4-(allyloxy)-3-methoxybenzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(4-hydroxy-3-methoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-((4-methyl-4H-1,2,4-triazol-3-yl)thio)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-((5-(pyridin-2-yl)thiophen-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one, 2-(benzo[b]thiophen-3-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-dodecyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(benzo[b]thiophen-2-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-cyclohexylthiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-([2,2'-bithiophene]-5-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-isobutylthiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-((5-(trifluoromethyl)furan-2-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one, 4-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzoic acid, 8-benzyl-2-((5-(methoxymethyl)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((perfluorophenyl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((1-methyl-3-(thiophen-2-yl)-1H-pyrazol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3,5-bis(trifluoromethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-chloro-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzoic acid, 8-benzyl-2-((5-(morpholinomethyl)furan-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(3-(aminomethyl)benzyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 5-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione, 8-benzyl-2-(imidazo[2,1-b]thiazol-6-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, N-(3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)phenyl)methanesulfonamide, 8-benzyl-2-((3,5-dimethylisoxazol-4-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-(pyridin-3-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one, Diethyl (8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)phosphonate, 8-benzyl-2-(4-methoxy-3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-(dimethylamino)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-(methoxymethyl)thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-(methoxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-(hydroxymethyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2-methoxypyrimidin-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((3,4-dimethylthieno[2,3-b]thiophen-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-(pyrazin-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-((2-(propylthio)pyrimidin-5-yl)methyl)imidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((4-methyl-4H-thieno[3,2-b]pyrrol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-((1,2,3-thiadiazol-5-yl)methyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((1-methyl-1H-benzo[d][1,2,3]triazol-5-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3,4-dimethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-(methylthio)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 2-(benzo[d][1,3]dioxol-5-ylmethyl)-8-benzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-((dimethylamino)methyl)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-(3-ethoxybenzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-methylpyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-(dimethylamino)pyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-(2,4,6-trifluorobenzyl)imidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2,6-dimethoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-chloro-4-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-fluoro-2-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2-fluoro-6-methoxypyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((5-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2,6-difluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-bromopyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((6-chloropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2-fluoropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-2-((2,6-dichloropyridin-3-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, 8-benzyl-6-phenyl-2-styryl-1,7-dihydroimidazo[1,2-a]pyrazin-3(2H)-one, 8-benzyl-2-(3-(2-methoxyethoxy)benzyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one, and 3-((8-benzyl-3-oxo-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-2-yl)methyl)benzonitrile, or a pharma- ceutically acceptable salt thereof The compound according to [1] above, selected from the group consisting of: [4] A kit comprising the compound according to [1] above. [5] The kit according to [4] above, further comprising luciferase. [6] The kit according to [4] above, further comprising a buffer reagent. [7] A method for detecting luminescence in a sample, comprising: Contacting a sample with the compound described in [1] above; contacting the sample with a coelenterazine-utilizing luciferase if not present in the sample; and detecting the emission of light; The method comprising: [8] The method according to [7], wherein the sample contains living cells. [9] The method according to [7], wherein the sample contains a luciferase that utilizes coelenterazine.

[10] A method for detecting luminescence in a transgenic animal, comprising: Administering the compound according to [1] to a transgenic animal; and detecting the emission of light; wherein the transgenic animal expresses a coelenterazine-utilizing luciferase.

[11] Compound of formula (I) TIFF0007669430000040.tif52164 wherein R 1 is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate; q is 0 to 2; The method further comprises the step of: TIFF0007669430000041.tif37164 to a compound of formula (I).

[12] The method according to

[11] above, wherein converting compound (vi) to a compound of formula (I) comprises reduction with a hydride reducing agent.

[13] The method according to [2] above, wherein the hydride reducing agent is sodium borohydride.

[14] Compound (A) TIFF0007669430000042.tif42164 in the presence of a solvent and a base R 1 The method according to claim 11, further comprising reacting with -C(O)H to produce compound (vi).

[15] The method according to

[14] above, wherein the base is 1,1,3,3-tetramethylguanidine.

[16] The method according to

[14] above, wherein the solvent is methanol.

[17] Compound (B) TIFF0007669430000043.tif42164 in the presence of a solvent and a base R 1 The method according to claim 11, further comprising reacting with -C(O)H to produce compound (vi).

[18] The method according to

[17] above, wherein the base is 1,1,3,3-tetramethylguanidine.

[19] The method according to

[17] , wherein the solvent is methanol.

[20] Compound (ix) TIFF0007669430000044.tif42164 The method according to claim 11, further comprising reacting the compound (vi) with acetic anhydride and a base.

[21] Compound (vii) TIFF0007669430000045.tif44150 The method according to claim 20, further comprising reacting with trifluoroacetic acid to produce compound (ix).

[22] Compound (C) TIFF0007669430000046.tif42164 in the presence of a solvent and a base R 1 The method according to claim 21, further comprising reacting with -C(O)H to produce compound (vii).

[23] The method according to

[22] above, wherein the base is 1,1,3,3-tetramethylguanidine.

[24] The method according to

[22] above, wherein the solvent is methanol.

[25] Compound of formula (I) TIFF0007669430000047.tif52164 wherein R 1 is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, heteroarylcarbonyl, or phosphonate; q is 0 to 2; The process further comprises the step of: TIFF0007669430000048.tif42164 to a compound of formula (I).

[26] The method according to

[25] above, wherein converting compound (xi) to a compound of formula (I) comprises reacting compound (xi) with carbonyldiimidazole.

[27] Compound (viii) TIFF0007669430000049.tif44150 The method according to

[25] above, further comprising reacting with trifluoroacetic acid to produce compound (xi).

[28] Compound (vii) TIFF0007669430000050.tif44150 The method according to claim 27, further comprising hydrogenating the compound (viii) in the presence of a metal catalyst.

[29] The method according to

[28] , wherein the metal catalyst comprises rhodium.

[30] Compound (C) TIFF0007669430000051.tif42164 in the presence of a solvent and a base R 1 The method according to claim 28, further comprising reacting with -C(O)H to produce compound (vii).

[31] The method according to

[30] above, wherein the base is 1,1,3,3-tetramethylguanidine.

[32] The method according to

[30] above, wherein the solvent is methanol. 〔33〕R 1 However, phenyl, pyridinyl, benzodioxolyl, benzotriazolyl, thiazolyl, thiadiazolyl, thienopyrrolyl, pyrimidinyl, pyrazinyl, thienothienyl, thienyl, diethylphosphonate, isoxazolyl, imidazothiazolyl, 1,3-dimethylpyrimidin-2,4(1H,3H)-dionyl, furanyl, pyrazolyl, benzothienyl, C 10 ~C 12 The method according to any one of claims 11 to 25, wherein the aryl group is alkyl, benzothiazolyl, cinnamyl, dibenzofuranyl, chromenyl or naphthalenyl.

Claims

1. Compounds of formula (I) wherein R 1 is alkyl, alkenyl, alkynyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, or cycloalkyl; q is 1; The method further comprising: (I) Compound (vi) with compound (A), compound (B), or compound (C). from, by reacting compound (A) with a base and R 1 —C(O)H in the presence of a solvent to produce compound (vi); by reacting compound (B) with a base and R 1 --C(O)H in the presence of a solvent to produce compound (vi); or Compound (C) is reacted with a base and R 1 —C(O)H in the presence of a solvent to give compound (vii). and reacting compound (vii) with trifluoroacetic acid to give compound (ix). and reacting compound (ix) with acetic anhydride and a base to produce compound (vi). preparing, and (II) converting compound (vi) to a compound of formula (I) by reduction with a hydride reducing agent; The method comprising:

2. 2. The method of claim 1, wherein the hydride reducing agent is sodium borohydride.

3. Compound (A) in the presence of a solvent. 1 2. The method of claim 1, comprising preparing compound (vi) by reacting with -C(O)H to produce compound (vi).

4. The method of claim 3, wherein the base is 1,1,3,3-tetramethylguanidine.

5. The method of claim 3 wherein the solvent is methanol.

6. Compound (B) in the presence of a solvent. 1 2. The method of claim 1, comprising preparing compound (vi) by reacting with -C(O)H to produce compound (vi).

7. The method of claim 6, wherein the base is 1,1,3,3-tetramethylguanidine.

8. The method of claim 6 , wherein the solvent is methanol.

9. Compound (C) with a base and R 1 —C(O)H in the presence of a solvent to obtain compound (vii). and reacting compound (vii) with trifluoroacetic acid to give compound (ix). and reacting compound (ix) with acetic anhydride and a base to produce compound (vi).

10. The method of claim 9, wherein the base is 1,1,3,3-tetramethylguanidine.

11. The method of claim 9, wherein the solvent is methanol.

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