Rapid, high-intensity chemiluminescent dioxanes

Abstract

To provide dioxetanes that rapidly respond to the presence of an analyte.SOLUTION: Provided herein are 1,2-dioxetanes that are useful as chemiluminescent probes, diagnostic agents, and imaging agents, where the 1,2-dioxetanes are represented by the formula in the figure, for example. Also described herein are compositions containing such compounds and methods of using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Application No. 62 / 926,985, filed October 28, 2019. No. 60 / 699,999, filed on Oct. 1, 2003, which claims the benefit of the same, which is incorporated by reference as if fully set forth herein. can be. [Background technology]

[0002] Chemiluminescent dioxetanes undergo rapid decomposition to generate excited transient species, which It is a strained cyclic peroxide that can then decay to the ground state via photoemission. do.

[0003] Such compounds are useful in enzyme activity assays, immunoassays, and DNA detection assays. They are useful as luminescent probes in a variety of assays, including chemiluminescence-based assays. Unlike fluorescence and absorbance-based assays, no light excitation is required, making it an excellent choice. Sensitivity can be provided.

[0004] Dioxanes are either generated in situ at the time of their use or pre-prepared in a stable form. can be prepared in situ via oxidation of a precursor alkene and then subsequently activated. In this case, chemiluminescent dioxetanes are used to detect reactive oxygen species (ROS). Examples of stable chemiluminescent dioxetanes include 4 -Methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2 This compound, also known as LUMIGEN® PPD, is The compound was activated by treatment with alkaline phosphatase (ALP). ALP is an enzyme that catalyzes the hydrolysis of phosphate groups. When activated, The compound then undergoes fragmentation of the 1,2-dioxatane ring, releasing light and thus It functions as a luminescent probe in alkaline phosphatase-labeled assays.

[0005] Dioxane compounds that emit strongly and sensitively under non-aqueous conditions have been developed. Therefore, such compounds are weakly luminescent in aqueous media and exhibit maximum luminescence after contact with the desired analyte. The problem was that it took a long time for the light to reach the target. To achieve this, surfactant-based emission enhancers have been added to the dioxetane probes. The use of such enhancers is neither desirable nor suitable in a variety of applications. Summary of the Invention

[0006] There is a need for dioxetanes that respond rapidly to the presence of analytes. There is also a need for dioxetanes that are highly luminescent without the need for sensors and that are suitable for use in aqueous environments. Various compounds disclosed herein provide such characteristics.

[0007] The present disclosure provides compounds of Formula I and salts thereof: [ka]

[0008] R 1 and R 2 Each of these is independently C3 to C 10 alkyl or R 1 and R 2 teeth , together with the carbons to which they are attached, C5 to C 10 Provides a cycloalkyl ring. R 3 is C1~C 10 Alkyl, C6-C 10 It is aryl or heteroaryl.

[0009] R 4 , R 5 , R 6 and R 7 each independently represents H, Q, X, hydroxy, halogen, Amino, thio, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkini Lu, C1~C 10 Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Trial Quilammonium salts, C1-C 10 Alkylthio, C2-C 10 Acyl, C1-C 10 a Alkyloxycarbonyl, C1-C 10 Alkylaminocarbonyl, C1-C 10 Archi Ruthiocarbonyl, C2-C 10 Acyloxy, C2-C 10 Acylamino, C2-C1 0 Acylthio, C1-C 10 Alkyl carbonates, C1-C 10 Alkyl Carbamate , C1~C 10 Carbamide, aryloxy, C1-C 10 Alkylsulfinyl, C1 ~C 10 Alkyl sulfonyl, arylthio, arylamino, arylsulfinyl, Arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryl Oxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, Heterarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1~ C10 Alkylphosphonates, C1-C 10 Alkyl phosphinates, C1-C 10 Bird Alkylphosphonium salts, C4-C 10 Heterocycloamino, C6-C 10 Aryl, or is a π-excess heteroaryl, and R 4 , R 5 , R 6 and R 7 At least one of It's Q.

[0010] Q is a π-conjugated electron donating group.

[0011] X is an -OH, -OG, -O' salt, or boronate group.

[0012] G is an alcohol protecting group.

[0013] The present disclosure also provides a fluorescent compound having a peak emission intensity of over 1000 photons / second in a pH 9.7 buffer solution. Treated at 37°C for less than 3 minutes. 1 / 2 one or more chemiluminescent dioxetane compounds having and wherein the composition is substantially free of surfactant-based luminescence enhancers. .

[0014] The present disclosure also provides a method for determining the presence of an analyte in a sample, comprising: and monitoring the sample for emission of light.

[0015] Advantages, some of which are unexpected, are achieved by various embodiments of the present disclosure. The various compounds described herein exhibit rapid, high activity in non-aqueous media, aqueous media, or both. Advantageously, the assays containing such compounds can provide an intense luminescent signal. perform more quickly than assays with compounds lacking the characteristics of the compounds described herein; Furthermore, the compounds of the present disclosure exhibit high luminescence intensity, including in aqueous media. The aqueous composition may be free of surfactant-based luminescence enhancers. This is another advantage of the present compounds. Such advantageous properties allow various implementations of the present disclosure to be The form can be in aqueous or non-aqueous samples in less than 3 minutes, less than 1 minute, less than 30 seconds, or about 15 seconds or less. In one embodiment, a method or kit can be provided that can detect the analyte. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 1 using a 20 μL sample of 1 mg / mL of the compound in methanol triggered with 200 μL of an amine-based buffer at 37° C.

[0017] [Figure 2] 1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 2, using a 10 μL sample of 1 mg / mL of the compound in THF, triggered with 200 μL of an amine-based buffer at 37° C.

[0018] [Figure 3] 1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 3, using a 10 μL sample of 1 mg / mL of compound in THF, triggered with 200 μL of an amine-based buffer at 37° C.

[0019] [Figure 4] 1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 4, using a 10 μL sample of 1 mg / mL of compound in THF, triggered with 200 μL of an amine-based buffer at 37° C.

[0020] [Figure 5]1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 5, using a 10 μL sample of 0.1 mg / mL of the compound in dioxane, triggered with 200 μL of an amine-based buffer at 37° C.

[0021] [Figure 6] FIG. 1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 7, triggered with 200 μL of an amine-based buffer at 37° C. using a 10 μL sample of 1 mg / mL of the compound in dioxane, further diluted with 90 μL of water.

[0022] [Figure 7] FIG. 12 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 8, triggered with 200 μL of an amine-based buffer at 37° C. using a 10 μL sample of a 0.001 mg / mL sample of the compound in dioxane, further diluted with 90 μL of water.

[0023] [Figure 8] FIG. 1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 9, triggered with 200 μL of an amine-based buffer at 37° C. using a 10 μL sample of a 0.01 mg / mL sample of the compound in dioxane, further diluted with 90 μL of water.

[0024] [Figure 9] FIG. 1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 10, triggered with 200 μL of an amine-based buffer at 37° C. using a 10 μL sample of a 0.01 mg / mL sample of the compound in dioxane, further diluted with 90 μL of water.

[0025] [Figure 10] FIG. 12 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 11, triggered with 200 μL of an amine-based buffer at 37° C. using a 10 μL sample of a 0.01 mg / mL sample of the compound in dioxane, further diluted with 90 μL of water.

[0026] [Figure 11] FIG. 12 is a graph showing the time profile of chemiluminescence intensity for the compound of Example 12, using a 100 μL sample of 1.25 mg / mL of compound in 5 mg / mL TBE Enhancer Amine Buffer containing 10 μL of alkaline phosphatase (AP8) at 37° C.

[0027] [Figure 12] FIG. 12 is a graph showing the time profile of chemiluminescence intensity for the compound of Example 13, using a 100 μL sample of 0.125 mg / mL of compound in 2.5 mg / mL TBE Enhancer Amine Buffer containing 10 μL of alkaline phosphatase (AP8) at 37° C.

[0028] [Figure 13] FIG. 12 is a graph showing the time profile of chemiluminescence intensity for the compound of Example 14, using a 100 μL sample of 0.25 mg / mL of compound in 5 mg / mL TBE Enhancer Amine Buffer containing 10 μL of alkaline phosphatase (AP8) at 37° C.

[0029] [Figure 14] FIG. 12 is a graph showing the time profile of chemiluminescence intensity for the compound of Example 15, using a 100 μL sample of 0.25 mg / mL of compound in 5 mg / mL TBE Enhancer Amine Buffer containing 10 μL of alkaline phosphatase (AP8) at 37° C.

[0030] [Figure 15] FIG. 12 is a graph showing the time profile of chemiluminescence intensity for the compound of Example 16, using a 100 μL sample of 0.25 mg / mL of compound in 2.5 mg / mL TBE Enhancer Amine Buffer containing 10 μL of alkaline phosphatase (AP8) at 37° C.

[0031] [Figure 16]FIG. 12 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 16, using a 100 μL sample of 0.1 mg / mL of the compound in water containing 10 μL of alkaline phosphatase (AP4) and 300 μL of 5 mg / mL TBE Enhancer Amine Buffer at 37° C.

[0032] [Figure 17] FIG. 12 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 17, using a 100 μL sample of a 0.2 mg / mL sample of the compound in an amine-based buffer without poly(vinylbenzyltributylphosphonium chloride) (TBE) and 10 μL of alkaline phosphatase (AP9) at 37° C.

[0033] [Figure 18] FIG. 10 is a graph showing the chemiluminescence intensity time profile of the compound of Example 12 (Lumigen® PPO) using a 100 μL sample of 2 mg / mL of compound in an amine-based buffer without TBE enhancer and containing 20 μL of alkaline phosphatase (AP8) at 37° C. DETAILED DESCRIPTION OF THE INVENTION

[0034] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in the accompanying drawings. The disclosed subject matter is described in conjunction with the enumerated claims, but is not limited to the examples. The subject matter disclosed is not intended to limit the scope of the claims to the disclosed subject matter. It is understood that:

[0035] The compounds of the present disclosure are useful in chemiluminescent applications such as assays and chemical probes .

[0036] The present disclosure provides a compound of Formula I, or a salt thereof: [ka]

[0037] R 1 and R 2 Each of these is independently C3 to C 10 alkyl or R 1 and R 2 teeth , together with the carbons to which they are attached, C5 to C 10 Cycloalkyl rings, e.g., monocyclic R 1 and R 2 may be substituted or unsubstituted In various embodiments, R 1 and R 2 together with the carbon to which they are attached form a spiro are linked to provide a cyclic bridged bicyclo or tricyclo group. For example, R 1 and R 2 together with the carbon to which they are attached form spirocyclic adamantanes, norbornanes, Or it can be bornane.

[0038] R 3 is C1~C 10 Alkyl, C6-C 10 aryl or heteroaryl; Each of them is optionally substituted. 3 can be substituted or unsubstituted. Ba, R 3 is unsubstituted C1 to C 10 Alkyl, or one or more halogen, hydroxy, amino no, thio, alkoxy, alkylamino, alkylthio, sulfate, or carboxyle C1-C substituted with carboxylate 10 It can be alkyl.

[0039] R 4 , R 5 , R 6 and R 7 each independently represents H, Q, X, hydroxy, halogen, Amino, thio, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkini Lu, C1~C 10 Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Trial Quilammonium salts, C1-C 10 Alkylthio, C2-C 10 Acyl, C1-C 10 a Alkyloxycarbonyl, C1-C 10 Alkylaminocarbonyl, C1-C 10 Archi Ruthiocarbonyl, C2-C 10 Acyloxy, C2-C 10 Acylamino, C2-C1 0 Acylthio, C1-C 10 Alkyl carbonates, C1-C 10 Alkyl Carbamate , C1~C 10 Carbamide, aryloxy, C1-C 10 Alkylsulfinyl, C1 ~C 10 Alkyl sulfonyl, arylthio, arylamino, arylsulfinyl, Arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryl Oxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, Heterarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1~ C 10 Alkylphosphonates, C1-C 10 Alkyl phosphinates, C1-C 10 Bird Alkylphosphonium salts, C4-C 10 Heterocycloamino, C6-C 10 Aryl, or is a π-excess heteroaryl, and R 4 , R 5 , R 6and R 7 At least one of Q. R 4 , R 5 , R 6 and R 7 Each of may be substituted or unsubstituted.

[0040] In various embodiments, R 4 , R 5 , R 6 and R 7 are each independently H, Q, X, halo Gen, C1~C 10 Alkyl, hydroxy, C1-C 10 Alkyloxy, amino, C1 ~C 10 Alkylamino, thio, C1-C 10 Alkylthio, C2-C 10 Acyloxy , C2~C 10 Acylamino, C2-C 10 Acylthio, C1-C 10 Alkyl Carbonate C1~C 10 Alkyl carbamates, C1-C 10 Carbamide, aryloxy, Arylthio, arylamino, heteroaryloxy, heteroarylthio, and heteroaryloxy. R is selected from the group consisting of: 4 , R 5 , R 6 and R 7 At least one of The other one is Q.

[0041] In various further embodiments, R 4 , R 5 , R 6 and R 7 are attached to the aromatic rings to which they are attached. Provides a net electron-donating effect. For example, X, Q, R 4 , R 5 , R 6 and R 7 is combined Aromatic rings are Q and R4 , R 5 , R 6 and R 7 compared to an otherwise identical compound where It is rich in electrons.

[0042] In some embodiments, R 4 , R 5 , R 6 and R 7 Exactly one, two or 3 is X or R 4 , R 5 , R 6 and R 7 Exactly one, two, or three of , Q, or any combination thereof. In the form R is not X or Q 4 , R 5 , R 6 and R 7 The remainder of is H. For example, R 4 , R 5 , and R 6 can be H, but R 7 is Q.

[0043] Q is a π-conjugated group, an electron-donating group, or both. In various embodiments, Q is a π-conjugated group. In various embodiments, Q is a C2-C 10 Alkenyl, C2-C1 0 Heterocycloalkenyl, C6-C 10 Q is aryl or heteroaryl. It can be substituted or unsubstituted. In some embodiments, Q is C2 to C 10 With alkenyl If present, they may be substituted with one or more electron-donating groups, contain no electron-withdrawing groups, or both. In various embodiments, Q is C2 to C 10 When present, the alkyl group is alkenyl. In this case, the vinyl and allylic positions are unsubstituted in some further embodiments. For example, Q In some embodiments, Q is a C-C 10 With Aryl If present, they may be substituted with one or more electron-donating groups, contain no electron-withdrawing groups, or both. For example, Q can be an unsubstituted phenyl, a phenyl substituted with one or more electron-donating groups, or or a phenyl substituted with one or more substituents selected from the group consisting of electron donating substituents. As another example, Q may be a substituent such that the net effect of the substituent is an electron donating effect. In a further example, Q can be thiophenyl, π-peroxy groups such as furanyl, pyrrolyl, benzothiophenyl, benzofuranyl, or indolyl; In certain embodiments, Q is a substituted or unsubstituted thiophene-2 -yl or thiophen-3-yl.

[0044] X is an -OH, -OG, -O' salt, or boronate group. X is a group that generates an oxyanion upon chemical or enzymatic trigger. X is a boronate. When it is a group, it has the following structure: [ka]

[0045] R of the boronate group 8 and R 9 Each of the groups is independently H or C1 to C 10 With alkyl Or R 8 and R 9 together with the boronates to which they are bonded, C2 to C1 For example, X is 4,4,5,5-tetramethyl-1, It can be 3,2-dioxaborolanyl or -B(OH)2.

[0046] In various embodiments, X is -OG, and G is an alcohol protecting group, an analyte-responsive For example, G can be a trialkylsilyl, alkylarylsilyl, or both. , arylsulfonyl, dioxobenzyl, trityl, alkyl carbonate, phosphoryl dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl pyranosyl, pyranuronyl, furanosyl, acyl, benzoyl, or In some embodiments, G can be galactosyl, glucosyl, or is pyranosyl or pyranuronyl, such as glucuronyl. G is β-galactosyl, β-glucosyl, or β-glucuronyl. G is also G can be a phosphorus-containing group such as -POH, phosphate, phosphonate, etc. 2 or a salt or ester thereof. In a further embodiment, G is 2,4-dinitro Benzene sulfonyl, 3,4,6-trimethyl-2,5-dioxobenzyl, 4-azide benzyloxy, tert-butyldimethylsilyl, acetyl, pivaloyl, enzymatic cleavage For example, G is a phosphatase-cleavable moiety or a peptidase-cleavable moiety. G may also be a functional group that allows removal of the pendant protecting group to fragment and deprotect the linker. The fragmentable linker comprises a bivalent linker having a pendant protecting group to cause removal of the group G. Therefore, G can be 4-aminobenzyl, 4-(alkylamino)benzyl, 4 -oxybenzyl, 4-(oxymethyl)benzyl, oxymethyl, aminomethyl, alkane Divalent fragmentable linkers such as trialkylsilyl, alkylaminomethyl, Arylsilyl, arylbenzenesulfonyl, dioxobenzyl, trityl, alkyl Carbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranic acid tetrahydrofuranyl, pyranosyl, pyranuronyl, furanosyl, acyl, benzoyl It may contain a terminal protecting group such as phenyl, benzyl or boronate group.

[0047] Examples of X and -OG include the following structures: [ka]

[0048] The present disclosure also provides a compound of formula II, or a salt thereof: [ka]

[0049] R 10 and R 11 each independently represents H, halogen, C1-C 10 Alkyl, C2~ C 10 Alkenyl, C6-C 10 In some embodiments, R 10 and R 11 are independently H or halogen.

[0050] The present disclosure also provides compounds of formula IIa and IIb, or salts thereof. [ka]

[0051] R 10 and R 11 each independently represents H, halogen, C1-C 10Alkyl, C2~ C 10 Alkenyl, C6-C 10 In some embodiments, R 10 and R 11 are independently H or halogen.

[0052] The present disclosure further provides a compound of formula III, or a salt thereof: [ka]

[0053] R 12 and R 13 each independently represents H, halogen, C1-C 10 Alkyl, C2~ C 10 Alkenyl, C2-C 10 Alkynyl, C1-C 10 Alkyloxy, C1-C1 0 Alkylamino, C1-C 10 Trialkylammonium salts, C1-C 10 Alkylchi O, C2~C 10 Acyl, C1-C 10 Alkyloxycarbonyl, C1-C 10 Archi Aminocarbonyl, C1-C 10 Alkylthiocarbonyl, C2-C 10 Acyloxy , C2~C 10 Acylamino, C2-C 10 Acylthio, C1-C 10 Alkyl Carbonate C1~C 10 Alkyl carbamates, C1-C 10 Carbamide, aryloxy, C1~C 10 Alkylsulfinyl, C1-C 10 Alkyl sulfonyl, arylthio, Arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, Heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroaryl Amino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, thiazolinone Trifluoromethyl, phosphonate, C1-C 10 Alkylphosphonates, C1-C 10 a Alkylphosphinate, C1-C 10 Trialkylphosphonium salts, C4-C 10 Hetero Cycloamino, C6-C 10 aryl or π-excess heteroaryl; or R 12 and R 13 together with the carbons to which they are attached, C5 to C 10 Cycloalkane Nil, C2~C 10 Heterocycloalkenyl, C6-C 10 Aryl or π-excess In some embodiments, R 12 and R 13 is, independently, H, C1~C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 aryl, or π-excess heteroaryl, or R 12 and R 13 are identical to the carbons to which they are attached. Together, C5~C 10 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, C 6~C 10 aryl, or π-excess heteroaryl, or R 12 and R 13 together with the carbons to which they are attached, C5 to C 10 Cycloalkenyl, C2~ C 10 Heterocycloalkenyl, C6-C 10Aryl or π-excess heteroaryl In some further embodiments, R 12 and R 13 are independently H, C 1~C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 Aryl or π-excess helical It is a teraryl.

[0054] R 14 H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C1-C 10 Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Alkylthio, C2~ C 10 Acyloxy, C2-C 10 Acylamino, C2-C 10 Acylthio, C1-C1 0 Alkyl carbonate, C1-C 10 Alkyl carbamates, C1-C 10 Carbamide , aryloxy, arylthio, arylamino, heteroaryloxy, heteroaryl arylthio or heteroarylamino, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl. In some embodiments, R 14 is H , C1~C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 Aryl or π-excess is a heteroaryl of the formula:

[0055] R 12 , R 13 and R 14 together, the net electron-donating effect on the phenyl ring to which X is attached is For example, the aromatic ring to which X is attached has R 12 , R 13 and R 14 Other points where H is electron-rich compared to the same compound.

[0056] In various embodiments, R 12 , R 13 and R 14 At least one or two of It's H.

[0057] The present disclosure further provides compounds of formula IIIa and IIIb, or salts thereof: [ka]

[0058] R 12 and R 13 each independently represents H, halogen, C1-C 10 Alkyl, C2~ C 10 Alkenyl, C2-C 10 Alkynyl, C1-C 10 Alkyloxy, C1-C1 0 Alkylamino, C1-C 10 Trialkylammonium salts, C1-C 10 Alkylchi O, C2~C 10 Acyl, C1-C 10 Alkyloxycarbonyl, C1-C 10 Archi Aminocarbonyl, C1-C 10 Alkylthiocarbonyl, C2-C 10 Acyloxy , C2~C 10 Acylamino, C2-C 10 Acylthio, C1-C 10 Alkyl Carbonate C1~C 10 Alkyl carbamates, C1-C 10 Carbamide, aryloxy, C1~C 10Alkylsulfinyl, C1-C 10 Alkyl sulfonyl, arylthio, Arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, Heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroaryl Amino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, thiazolinone Trifluoromethyl, phosphonate, C1-C 10 Alkylphosphonates, C1-C 10 a Alkylphosphinate, C1-C 10 Trialkylphosphonium salts, C4-C 10 Hetero Cycloamino, C6-C 10 aryl or π-excess heteroaryl; or R 12 and R 13 together with the carbons to which they are attached, C5 to C 10 Cycloalkane Nil, C2~C 10 Heterocycloalkenyl, C6-C 10 Aryl or π-excess In some embodiments, R 12 and R 13 is, independently, H, C1~C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 aryl, or π-excess heteroaryl, or R 12 and R 13 are identical to the carbons to which they are attached. Together, C5~C 10 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, C 6~C 10 aryl, or π-excess heteroaryl, or R 12 and R 13together with the carbons to which they are attached, C5 to C 10 Cycloalkenyl, C2~ C 10 Heterocycloalkenyl, C6-C 10 Aryl or π-excess heteroaryl In some further embodiments, R 12 and R 13 are independently H, C 1~C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 Aryl or π-excess helical It is a teraryl.

[0059] R 14 H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C1-C 10 Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Alkylthio, C2~ C 10 Acyloxy, C2-C 10 Acylamino, C2-C 10 Acylthio, C1-C1 0 Alkyl carbonate, C1-C 10 Alkyl carbamates, C1-C 10 Carbamide , aryloxy, arylthio, arylamino, heteroaryloxy, heteroaryl arylthio or heteroarylamino, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl. In some embodiments, R 14 is H , C1~C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 Aryl or π-excess is a heteroaryl of the formula:

[0060] R 12 , R 13 and R 14 together, the net electron-donating effect on the phenyl ring to which X is attached is For example, the aromatic ring to which X is attached has R 12 , R 13 and R 14 Other points where H is electron-rich compared to the same compound.

[0061] In various embodiments, R 12 , R 13 and R 14 At least one or two of It's H.

[0062] The present disclosure provides compounds of the formula: [ka] Further provided is a compound according to one or more of the following, or a salt thereof:

[0063] The present disclosure provides compounds of the formula: [ka] Further provided is a compound according to one or more of the following, or a salt thereof:

[0064] Each of Z, L and J is S, O, Se, NR 15 , or (CR 16 R 17 ) n and Each R 15 are independently H, alkyl, acyl, benzyl, alkyloxycarbonyl, arylsulfonyl, and R 14 , R 16 , R 17 , R 18 , R 19 , R 20 and R 2 1each, if present, independently represents H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C1-C 10 Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Trialkylammonium salts, C1-C 10 Alkylthio, C2-C 10 Achill, C 1~C 10 Alkyloxycarbonyl, C1-C 10 Alkylaminocarbonyl, C1~ C 10 Alkylthiocarbonyl, C2-C 10 Acyloxy, C2-C 10 Acylamino , C2~C 10 Acylthio, C1-C 10 Alkyl carbonates, C1-C 10 Alkyl Carbamates, C1-C 10 Carbamide, aryloxy, C1-C 10 Alkyl sulfur Vinyl, C1~C 10 Alkyl sulfonyl, arylthio, arylamino, aryls sulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, Heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulf phenyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate C1~C 10 Alkylphosphonates, C1-C 10 Alkyl phosphinate, C1 ~C 10 Trialkylphosphonium salts, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl, or R 14 , R 16 , R17 , R 18 , and R 20 Any two of these, together with the carbons to which they are attached, form C5 to C1 0 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, C6-C 10 Aryl, young or a π-excess heteroaryl.

[0065] In some embodiments, R 14 , R 16 , R 17 , R 18 , R 19 , R 20 and R 2 1 each, if present, independently represents H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C1-C 10 Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Alkylthio, C2-C 10 Acyloxy, C2-C 10 Acylamino, C2-C1 0 Acylthio, C1-C 10 Alkyl carbonates, C1-C 10 Alkyl Carbamate , C1~C 10 Carbamide, aryloxy, arylthio, arylamino, heteroaromatic Aryloxy, heteroarylthio, or heteroarylamino, C4-C 10 Haitai Isocycloamino, C6-C 10 aryl or π-excess heteroaryl, or is R 14 , R 16 , R 17 , R 18 , and R 20 Any two of them are bonded to the carbon Together with the element, C5~C10 Cycloalkenyl, C2-C 10 Heterocycloalkeny Lu, C6~C 10 An aryl or a π-excess heteroaryl is provided.

[0066] In various embodiments, R 14 , R 16 , R 17 , R 18 , and R 20 At least one of Another is an electron donating group. 14 , R 16 , R 18 and R 20 of Each, when present, is hydrogen.

[0067] Compounds of formula I, II, IIa, III, IIIa, IIIb, and XII (below) In any of the foregoing compounds, including 4 or R 5 is C1~C 10 Alkyl (e.g., CH3) or halo (e.g., chloro). Further, compounds of formulas XII-XIV are included. In any of the foregoing compounds, including 12 is C1~C 10 Alkyl (e.g., CH3 ) can be.

[0068] The present disclosure further provides compounds according to formulas (IV)-(XV), or salts thereof. [ka] [ka]

[0069] The present disclosure provides a compound having the following structure: [ka] [ka] The present invention provides a compound or a salt thereof according to one or more of the following:

[0070] The present disclosure provides a method for producing one or more of the compounds described herein, their olefin precursors, or their The olefin precursor is a compound obtained by reacting an analyte, an oxidizing agent, an alkaline phosphatase, and a salt thereof with an olefin precursor. When treated with an oxidizing agent, such as a hydroxybenzoate, or photooxidizing conditions, any of the compounds described herein (e.g., compounds of Formula I, Compounds II, IIa, III, IIIa, IIIb and IV to XV are provided.

[0071] The composition may be an aqueous composition or a non-aqueous composition. In various embodiments, the composition may be a mixture of both surfactant-based luminescence enhancers and surfactant-based luminescence enhancers. For example, the composition may be substantially free of acyclic alcohols, surfactants, or both. alkyl groups (e.g., acyclic groups of at least 8 carbons) or aromatic groups (e.g., at least Aromatic groups containing 6 carbon atoms) and one or more quaternary ammonium salts, pyridinium salts, quaternary Phosphonium surfactant salts, ethylene glycol, or fluorescein head groups The surfactant may be substantially free of any of the following tail groups: The composition contains cetrimonium bromide (CTAB), cetylpyrifolic acid chloride, and Cetylpyridinium chloride (CPC), benzalkonium chloride chloride (BAC), benzethonium chloride (BZT), dimethicone Dioctadecyl ammonium chloride, Dioctadecyl dimethyl ammonium bromide (dioctadecyldimethylammonium bromide, DODAB), α'-tributylphosphonium p-xylene chloride, poly(vinylbenzyltrioctylphosphonium chloride) (TBE), poly(vinylbenzyltrioctylphosphonium chloride), Triton X-100, a Tween surfactant with a long alkyl group containing a polyethylene glycol head group surfactants with alkyl chains, poly(vinylbenzyltrioctylphosphonium chloride), Brij® surfactants, IGEPAL® surfactants, octyl phenol The composition may be substantially free of N-(3'-hydroxypropyl)ethanol, etc. ,6'-Dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxa fluorescein-containing surfactants, such as It may be free of fluorescein-containing compounds.

[0072] In various embodiments, the composition contains a buffer. The buffer does not necessarily have to be Although not required, it can be an alkaline or amine-based buffer. Exemplary amine-based buffers include Si 221 buffer available from gma-Aldrich (St. Louis, MO). Similarly, the composition does not necessarily have to have a basic pH, but this is not necessarily the case. For example, the composition may contain about 4 to 12, about 5 to 12, about 6 to 12, about 7 to 12, about 8 to 12, Approximately 9-12, approximately 10-12, approximately 4-11, approximately 4-10, approximately 4-9, approximately 4-approximately 8, approximately 4-7 The pH of the composition may be about 4 to 6, or about 4 to 5. As in the case of H values, light is emitted immediately upon removal of the analyte trigger, or upon treatment with base. The selection can be based on whether the pH of the composition is acidic so that it glows.

[0073] In various embodiments, the composition has a peak luminescence intensity of greater than 1,000 photons / second; When treated with pH 9.7 buffer, T 1 / 2 In various examples, The peak emission intensity is approximately over 2,000 photons / second, approximately over 3,000 photons / second, and approximately 4,000 photons / second. / second, approximately 5,000 photons / second, approximately 6,000 photons / second, approximately 7,000 photons / second, More than about 8,000 photons / second, more than about 9,000 photons / second, or more than about 10,000 photons / second Riteku, T 1 / 2 is approximately 3 minutes or less, approximately 2 minutes or less, approximately 1 minute or less, approximately 30 seconds or less, approximately 20 seconds or less For example, the present disclosure provides a method for detecting 1000 photons / second. and when treated with pH 9.7 buffer, the T 1 / The present invention provides an aqueous composition comprising one or more dioxetane compounds having a surfactant, The agent is substantially free of luminescence enhancers.

[0074] The present disclosure also provides a method for detecting an analyte in a sample, comprising: and contacting one or more of the compounds, an olefin precursor thereof, a salt thereof, or a composition containing them. and then monitoring the sample for luminescence. This involves measuring the intensity of the emitted light and correlating the intensity to the presence of the analyte.

[0075] In some embodiments, the method further involves increasing the pH of the sample. The pH can be adjusted to 7 or greater, 8 or greater, 9 or greater, 10 or greater, or 11 or greater.

[0076] In various embodiments, the analyte is analyzed in about 3 minutes or less, about 2 minutes or less, about 1 minute or less, or about 30 seconds or less. For example, the sample may be detected in about 3 seconds or less, about 20 seconds or less, about 15 seconds or less, or about 10 seconds or less. minutes or less, about 2 minutes or less, about 1 minute or less, about 30 seconds or less, about 20 seconds or less, about 15 seconds or less, or about It can be monitored for less than 10 seconds.

[0077] The analyte can be isolated from the composition by isolating any compound described herein (e.g., Formula I, II, IIa, III, IIa, IIIb, and IV-XV). , X is an oxyanion. For example, in various embodiments, the analyte is an alkaline phosphatase. Oxidative enzymes such as catalysis, peptidases, glucosidases, hydrogen peroxide or other reactive oxygen species The inhibitor may be a phosphate buffer, glutathione, fluoride, or a base under alkaline conditions.

[0078] The present disclosure further provides a kit for determining the presence of an analyte, the kit comprising: Any one or more of the compounds described in the document, their olefin precursors, their salts, or The kits may include instructions according to the methods described herein. This can be done.

[0079] The compounds and compositions described herein provide signals that identify the presence of analytes and probes. The method may be directly induced by the analyte to produce Two steps: one step involving increasing the pH, and another step involving increasing the pH. It can be induced in the process.

[0080] The compounds of the present disclosure detect a variety of different analytes by modifying the X or G groups. For example, the compounds described herein can be configured as probes for configured to detect β-galactosidase by providing a glucosyl group; X may be used to detect hydrogen peroxide or other oxidizing agents by providing a boronic ester. and by providing a phosphate group at X or a phosphoryl group at G, an alkali phosphate can be obtained. configured to detect phospholipase A, G, and dinitrobenzenesulfonaminobenzyl and G is a trialkylsilyl group. and X is an alkyl group, wherein X is OH. The device may be configured to detect a risk condition.

[0081] This compound is derivatized by the analyte to provide the strongest signal possible. When chemiluminescence occurs, it is desirable to emit all possible light in the shortest possible time. If the light is emitted slowly over time, the light intensity (photons / second) will decrease, compromising detection sensitivity. An example of the luminescence signal profile is shown in Figure 1.

[0082] The rate of increase in luminescence, or rise time, is the time to maximum luminescence (t max ) or half-emission period(T 1 / 2 ) can be explained by either

[0083] The compounds described herein can be used as enzyme substrates. For example, Various compounds containing the group act as substrates for enzymes such as alkaline phosphatase (ALP). Without being limited by theory, one exemplary mechanism is The ALP enzyme hydrolyzes the phosphorus-containing group to form the alkaline environment of the solution (e.g., pH 9.7). This involves providing a phenol that is readily deprotonated due to the presence of a buffer solution. Upon generation of the anion, 1,2-dioxetanes give two compounds: 2-adamantanone and The excited phenyl ester then reacts with the light to form a Upon emission, it immediately decays to the ground state.

[0084] The resulting light intensity is a linear function of the amount of enzyme. For example, a dioxetane may be used to detect a labeled enzyme used in an assay. The steps of the chemical process provided are the following steps: (i) X+S→X+S′ (ii) S'→P * and (iii) p * → This can be explained according to p+ light. Step (i) represents the catalytic turnover of the substrate, and X is an enzyme or other component that converts the substrate to its activated form. minutes, step (ii) represents the decomposition of the activated substrate into transient excited species, and step (iii) represents the decomposition of the activated substrate into transient excited species. iii) represents the decay of the excited species to the ground state and emission of light. The light intensity is determined by the and the catalytic turnover of the substrate in step (ii) and the resulting photogenerated compound P * Longevity Step (ii) is usually a linear function with a rate constant k, and its half-life is :T 1 / 2 = (In 2) / k. Step (iii) is , is very short compared to the other steps and generally does not have a significant effect on the reaction rate.

[0085] The chemiluminescence intensity / time profile shows an initial rise in luminescence intensity followed by a steady Includes a period of steady-state intensity. S' → P * The slow first-order reaction of Since it takes time to reach the point where the voltage is equal to the voltage S', the rise time is longer. * High-speed reaction The response corresponds to a shorter initial rise period, thus providing a rapid rise. In the case of enzymatic chemiluminescence reactions, the intensity typically plateaus at a high level, and the resulting signal The curve corresponds to the shape of the signal shown in Figure 5. The absence of a steady intensity indicates substrate depletion or The detection of enzyme-generated chemiluminescence was performed by measuring the light intensity at any time point. This provides flexibility in the measurement process, as the level can be related to the amount of enzyme, but The process can have drawbacks, for example, due to the size and "sticky" nature of the enzyme label. To maximize sensitivity, the maximum intensity (I max ) or It is best to measure in that vicinity.

[0086] The compounds described herein also act as complementary binding partners in immunoassays. The compounds described herein can be used as a direct label for one of the large organisms. As labels, they are small molecules, in contrast to luminescent molecules and other types of enzyme labels. be used advantageously.

[0087] Thus, the present disclosure also provides for the use of the compounds described herein as chemiluminescent probes. This relates to an assay.

[0088] In various embodiments, the assay is homogenous, where bound and unbound ligand do not need to be separated. The assay can be a quantitative (non-separation) assay, or the assay can be a quantitative (non-separation) assay in which the labeled binding pair complex is separated from the unbound target. The assay may be a heterogeneous assay separated from the recognition reaction. The assay can be configured to run in a test tube or can be automated and run robotically. The method can be performed in a tube, cuvette, microwell, or a combination thereof. In various embodiments, the test tube, cuvette, microwell, or the like in which the assay is performed. Other containers used may be at least partially opaque, completely opaque, black, white, or any combination thereof. It's a combination.

[0089] Assays were performed on immobilized proteins in Western blots, Southern blots, or non- Western blots. It can be performed on immobilized nucleic acids in a Lusan blot.

[0090] Imaging can be performed using a luminometer, X-ray film, or a charge-coupled device. The video can be recorded using a video camera system (Vice, CCD).

[0091] Chemiluminescence measurements have advantages over fluorescence and absorption spectroscopy. Absorption spectroscopy suffers from interference signals generated from either the incident light or background signals. It can be done.

[0092] The assays described herein were carried out by J.E. Wampler, Instrumentation The L ight and Measuring It,in Chemi-and Biolu minescence,JGBurr,ed.,Marcel Dekker,Ne w York, 1-44 (1985), AKCampbell, Detection and Quantification of Chemiluminescence ,in Chemiluminescence Principles and App lications in Biology and Medicine,Ellis Norwood,Chichester,68-126(1988),F.Bertho ld,Instrumentation for Chemiluminescence Immunoassays,in Luminescence Immunoassays y and Molecular Applications, K. Van Dyke and R. Van Dyke, eds., CRC Press, Boca Raton. ,11-25(1990),and T.Nieman,Chemiluminesce nce:Theory and Instrumentation,Overview, in Encyclopedia of Analytical Science,Ac Academic Press, Orlando, pp. 608-613 (1995). each of which is incorporated herein by reference in its entirety.

[0093] Several approaches are used to attach the compounds of the present disclosure to biological molecules. For example, if the compound is a carboxyl, carboxyl halide, sulfur N-succinyl halide, carboalkoxy, carboxamide, carboxim, or N-succinyl When a reactive group such as imidyl carboxy is contained, such a group may be, for example, carbodiimide. using a conjugation reagent such as 1,1-carbonyldiimidazole or 1,1-carbonyldiimidazole The N-maleimide group can be covalently bonded to hydroxyl or amino functional groups. It reacts directly with sulfhydryl residues in proteins. If the compound contains an aromatic amino group, When present, these are converted into diazonium salts, which are then reacted with tyrosine groups in proteins. Any phenolic group can react with a polycyclic aromatic moiety or other photogenerating group, or The compounds of the invention can be converted to biologically active compounds using either one of the reactive groups present in the leaving group. It can be attached to the child.

[0094] Compounds of the present disclosure can generally be prepared according to the synthetic procedures described, for example, in Scheme 1. This can be done. [ka]

[0095] 2-Adamantanone and trisubstituted benzoic acid esters are oxophilic titanium and reducing agents. can be bonded together by subjecting them to McMurry reaction conditions including The resulting olefin can be further functionalized, for example, by adding a protecting group G or a further functionalization position R on the ring. 4 , R 5 , R 6 , and R 7 The olefin can then be further modified by removing or substituting and subjecting the product to photooxygenation conditions to provide the 1,2-dioxetane product. R 3 , R 4 , R 5 , R 6 , R 7 , R 10 , R 11 , G and X are, for example, As described in any of the various embodiments of the present application. In some embodiments, R 4 , R 5 , R 6 , R 10and R 11 is H. Some In an embodiment of the present invention, R 3 is a substituted or unsubstituted alkyl and R 7 is an electron donating group.

[0096] As used herein, the terms light "intensity" or luminescence "intensity" refer to the emission of photons / second. Refers to the speed of light. Intensity can be measured by using a luminometer. A luminometer measures the intensity of ambient light. The photodetector is a photomultiplier tube and a photodiode in a housing that excludes the Any suitable luminometer may be used, including a luminometer.

[0097] The term "luminescence rate" refers to the rate of increase in luminescence, i.e., the change in light intensity over time. .

[0098] As used herein, the term "sensitivity" refers to the signal of an analyte or product being measured. refers to the lowest level at which a protein can be reproducibly detected.

[0099] As used herein, the term "alkyl" refers to an alkyl group having 1 to 20 carbon atoms, ~20 carbon atoms, 12-18 carbon atoms, 6-approximately 10 carbon atoms, 1-10 Carbon atoms, 1-8 carbon atoms, 2-8 carbon atoms, 3-8 carbon atoms, 4-8 carbon atoms Carbon atoms, 5-8 carbon atoms, 1-6 carbon atoms, 2-6 carbon atoms, 3-6 carbon atoms Carbon atoms or substituted or unsubstituted straight-chain, branched-chain or cyclic saturated hydrocarbons having 1 to 3 carbon atoms It refers to a monovalent or divalent group. Examples of linear monovalent groups (C1-C 20 )-alkyl groups include methyl (i.e., CH3), ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl Examples include those having 1 to 8 carbon atoms such as n-cyl, n-heptyl, and n-octyl groups. Examples of branched monovalent (C1 to C 20 )-Alkyl groups include isopropyl and isobutyl , sec-butyl, t-butyl, neopentyl, and isopentyl. Valence examples (C1 to C 20 )-Alkyl groups include -CH2-, -CH2CH2-, -CH 2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2C Examples of branched divalent alkyl groups include those having 1 to 6 carbon atoms such as H2. Examples include -CH(CH3)CH2- and -CH2CH(CH3)CH2. Examples of alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cyclohexyl, cyclooctyl, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl Cycloalkyl includes cycloalkyl, bicyclo[2.2.1]heptyl, and adamantyl. The alkyl group includes, but is not limited to, norbornyl, adamantyl, bornyl, camphanyl, isopropyl, and the like. Substituted and unsubstituted polycyclic cycloalkyl groups such as cambenyl and carmyl groups, and decacyclic groups such as cyclohexyl and decacyclic groups. Further fused rings include, but are not limited to, cycloaliphatic alkyl groups such as, for example, cycloaliphatic alkyl groups. Alkyl may have one, two, three, four or more substituents, e.g., methylene. In some embodiments, the tertiary bridgehead positions of the bridge include adamantyl substituted therein. Alkyl includes combinations of substituted and unsubstituted alkyl. (C1) alkyl includes methyl and substituted methyl. Specific examples include (C1) alkyl By way of further example, alkyl includes methyl and substituted (C2-C8) alkyl. Alkyl can also include substituted methyl and unsubstituted (C2-C8) alkyl. In some embodiments, alkyl can include methyl and C2-C8 alkyl groups. In some embodiments, alkyl can be methyl and substituted (C C8) alkyl. The term methyl is understood to mean an unsubstituted -CH3. The term methylene is understood to be an unsubstituted -CH2. Therefore, the term (C1) alkyl refers to a substituted or unsubstituted -CH3 or substituted or unsubstituted -CH3. is understood to be unsubstituted -CH2-. Representative substituted alkyl groups are listed herein. any of the groups listed above, for example, cycloalkyl, heterocyclyl, aryl, amino, halo Alkyl, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen By way of further example, representative substituted alkyl groups may be substituted one or more times with a group. one or more of fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, Alkylamide, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, Arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloa alkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethyl Fluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, dialkyl alkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamino In some embodiments, representative substituted alkyl groups include amino, hydroxy, Contains hydroxy, cyano, carboxy, nitro, thio, and alkoxy groups, but does not contain halogen groups. Thus, in some embodiments, alkyl can be substituted with a non-halogenated alkyl group. For example, representative substituted alkyl groups include alkyl groups with halogens other than bromo. fluoro groups substituted with bromo groups substituted with halogens other than fluoro; In some embodiments, representative substituted alkyl groups include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 3 They may be substituted with one, two, three or more non-fluoro groups. For example, alkyl may be substituted with trifluoromethyl, difluoromethyl, or can be fluoromethyl, or alkyl can be trifluoromethyl, difluoromethyl The alkyl may be haloalkyl or substituted alkyl other than fluoromethyl. It may be alkyl, and may be substituted alkyl other than haloalkyl.

[0100] As used herein, the term "alkenyl" refers to an alkyl group consisting of at least one carbon-carbon Carbon atoms with double bonds and 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms 6 to about 10 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms Carbon atoms, 4-8 carbon atoms, 5-8 carbon atoms, 2-6 carbon atoms, 3-6 carbon atoms carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms It refers to a substituted or unsubstituted, linear, branched, or cyclic saturated monovalent or divalent group. The double bond may be terminal or internal. An alkenyl group may be , can be attached via a portion of the alkenyl group containing a double bond, e.g., vinyl , propen-1-yl and but-1-yl, or the alkenyl group may be The bond may be via a portion of the alkenyl group that does not contain, for example, penten-4-yl. If specified, the parent moiety is at the vinyl position of the double bond rather than the non-vinyl position. For example, when the aromatic ring is a π-conjugated alkenyl group, When substituted with a vinyl group, it is understood that the substitution is at the vinyl position, not the non-vinyl position. As a further example, an aromatic ring substituted with a π-conjugated propenyl group should be It is understood that the group is a propen-1-yl or prop-2-ene group rather than a propen-3-yl group. Examples of univalent (C2~C 20 The )-alkenyl group includes vinyl, propenyl, propen-1-yl, propan-2-yl, butenyl, butenyl-1-yl, buton-2-yl sec-buten-1-yl, sec-buten-3-yl, pentenyl, hexenyl, Examples include those having 1 to 8 carbon atoms, such as heptenyl and octenyl groups. Examples of branched monovalent (C2~C 20 )-alkenyl groups include isopropenyl and isobutenyl , sec-butenyl, t-butenyl, neopentenyl, and isopentenyl Examples of linear divalent alkyl chains (C2 to C 20 ) The alkenyl group is, for example, -CHCH-, -CHCHCH 2-, -CHCHCH2CH2-, and -CHCHCH2CH2CH2-, etc. Examples of branched divalent alkyl groups include those having carbon atoms of -C(CH3 Examples of cyclic alkenyl groups include: cyclopentenyl, cyclohexenyl, and cyclooctenyl. Alkenyl can be vinyl and substituted vinyl. For example, alkenyl can be vinyl and substituted (C3-C6) alkenyl. Alkenyl also includes substituted vinyl and unsubstituted (C3 Representative substituted alkenyl groups include C-C6 alkenyl. Any of the groups described above, for example, monoalkylamino, dialkylamino, cyano, acetyl one or more of the following groups: aryl, amido, carboxy, nitro, alkylthio, alkoxy, and halogen By way of further example, representative substituted alkenyl groups can be substituted with one or more alkyl groups. Fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkyl Amido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, aryl carbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, Hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethyl thiol, alkylthiol, arylthiol, alkylsulfonyl, dialkylamino sulfonyl, sulfonic, carboxylic, dialkylamino and dialkylamide In some embodiments, representative substituted alkenyl groups include monoalkylamino, dialkylamino, and Alkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio and a It can be substituted with a range of groups including alkoxy but not including halogen groups. In some embodiments, the alkenyl can be substituted with a non-halogen group. In the example, representative substituted alkenyl groups are substituted with halogens other than bromo, or fluoro. It may be substituted with a fluoro group substituted with a bromo group substituted with a halogen other than Alkenyl is 1-fluorovinyl, 2-fluorovinyl, 1,2-difluorovinyl, 1,2,2-trifluorovinyl, 2,2-difluorovinyl, trifluoropropene 2-yl, 3,3,3-trifluoropropenyl, 1-fluoropropenyl, 1-chloro Vinyl, 2-chlorovinyl, 1,2-dichlorovinyl, 1,2,2-trichlorovinyl, or 2,2-dichlorovinyl. In some embodiments, representative substituted alkenes include The aryl groups may be substituted with one, two, three or more fluoro groups, or they may be substituted with one, It may be substituted with two, three or more non-fluoro groups.

[0101] As used herein, the term "alkynyl" refers to an alkyl group having at least one triple bond. Substituted or unsubstituted straight and branched chain alkyl groups, except that Thus, an alkynyl group can be any group having 2 to 50 carbon atoms, 2 to 20 carbon atoms, 10-20 carbon atoms, 12-18 carbon atoms, 6 to about 10 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 3-8 carbon atoms, 4-8 carbon atoms, 5-8 carbon atoms, 2-6 carbon atoms, 3-6 carbon atoms, 4-6 carbon atoms, 2-4 or 2-3 carbon atoms. Examples include ethynyl, propynyl, Propyn-1-yl, propyn-2-yl, butynyl, butyn-1-yl, butyn-2-yl yl, butyn-3-yl, butyn-4-yl, pentynyl, pentyn-1-yl, hexyl Examples include, but are not limited to, -C≡CH, -C≡C( CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3).

[0102] The term "aryl" as used herein refers to an alkyl group having 6 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 20 carbon atoms, 6 to about 10 carbon atoms, or 6 to 8 carbon atoms by removing a hydrogen atom from an arene, a cyclic aromatic hydrocarbon with (C6-C 20 ) Examples of aryl groups include , phenyl, naphthalenyl, azulenyl, biphenyl, indacenyl, fluorenyl, fluorenyl phenanthrenyl, triphenylenyl, pyrenyl, naphthenyl, chrysenyl, anthracenyl Examples include substituted phenyl, substituted naphthalenyl, substituted azulenyl, and substituted phenyl groups. Substituted biphenyl, substituted indacenyl, substituted fluorenyl, substituted phenanthrenyl, substituted triphenyl Phenylenyl, substituted pyrenyl, substituted naphthenyl, substituted chrysenyl, and substituted anthracenyl Examples include unsubstituted phenyl, unsubstituted naphthalenyl, unsubstituted azulenyl, unsubstituted biphenyl, unsubstituted indacenyl, unsubstituted fluorenyl, unsubstituted phenanthrenyl unsubstituted triphenylenyl, unsubstituted pyrenyl, unsubstituted naphthacenyl, unsubstituted chrysenyl Aryl also includes phenyl and non-phenyl groups. These examples also include aryl groups (C6-C 20 The term aryl refers to a monocyclic and polycyclic (C6~C 20 ) aryl groups, including fused and non-fused polycyclic (C6-C2 0) It is clear that it contains an aryl group.

[0103] As used herein, the term "heterocyclyl" refers to a heterocyclic group having three or more atoms in the ring. refers to substituted aromatic, unsubstituted aromatic, substituted non-aromatic, and unsubstituted non-aromatic rings containing One or more of which are heteroatoms such as, but not limited to, N, O, and S. The term "heteroaryl" refers to a fully aromatic heterocyclyl and therefore to a heteroaryl. The term "heterocycloalkenyl" is a subset of the term "cycloalkenyl." Heterocyclic rings containing an olefin within a non-aromatic ring such that the olefin is the point of attachment to the parent moiety. A heterocyclyl group is therefore a heterocycloalkyl, ... It may be chloroalkenyl, or heteroaryl, or if polycyclic, any of these. In some embodiments, the heterocyclyl group may be any combination of 3 to about 20 ring members, while other such groups have from 3 to about 15 ring members. In the form, heterocyclyl groups are substituted with 3 to 8 carbon atoms (C3-C6), 3 to 6 carbon atoms (C6-C6), Heterocyclyl groups containing 6 to 8 carbon atoms (C3 to C6) or 6 to 8 carbon atoms (C6 to C6) A heterocyclyl group designated as a C2-heterocyclyl has two carbon atoms and three a 5-membered ring having 1 heteroatom, a 6-membered ring having 2 carbon atoms and 4 heteroatoms Similarly, a C4-heterocyclyl may have one heteroatom. It can be a five-membered ring with two heteroatoms, a six-membered ring with two heteroatoms, etc. The number of heteroatoms is equal to the total number of ring atoms. A heterocyclyl ring can also have one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The term "group" includes fused ring species, including those containing fused aromatic and non-aromatic groups. Suitable heterocyclyl groups include piperidinyl, pyrrolidinyl, piperazinyl, and morpho For example, heterocyclyl groups include, but are not limited to, linyl. [ka] In particular, the formula includes, but is not limited to, 1 is H, (C1~C 20 ) alkyl , (C6~C20 ) aryl or amine protecting groups (e.g., t-butyloxycarbonyl groups) ), where the heterocyclyl group can be substituted or unsubstituted. Representative Heteroaryl Groups Examples include furanyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophene, nyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, imidazolyl, triazinyl, Examples include tetrazolyl, benzoxazolinyl, and benzimidazolinyl groups. In some embodiments, the heteroaryl is a 5-membered heteroaryl. In embodiments, heteroaryl is pyridine, pyrimidine, pyridazine, pyrazine, or any of its derivatives. The π-excess heteroaryl can function as an electron donating group. The heteroaryl is electron-rich so that it can be Examples are furan, thiophene, indole, pyrrole, benzofuran, and benzothiophene. It is.

[0104] As used herein, the term "alkoxy" means any of the groups defined herein. refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group. Examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, pentyloxy, and hexyl. Examples of branched alkoxy include, but are not limited to, iso, oxy, etc. Propoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyl Examples of cyclic alkoxy include, but are not limited to, cycloalkoxy ... cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy Alkoxy groups include, but are not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, may contain from about 12 to 20 or from about 12 to 40 carbon atoms, and may further contain double or triple bonds. It may also contain heteroatoms. Thus, an alkyloxy may also be attached to an alkynyl group. It includes an alkylenyl group attached to an oxygen atom and an oxygen atom attached to an oxygen atom. For example, allyloxy The group is an alkoxy group within the meaning of the present specification. A methoxyethoxy group also has the structure is a methylenedioxy group in the situation where two adjacent atoms of Alkoxy groups within the meaning of this specification.

[0105] As used herein, the term "aryloxy" means an aryl group, as defined herein. , refers to the oxygen atom attached to the aryl group. The point of substitution into the parent moiety is the oxygen atom.

[0106] As used herein, the term "arylcarbonyl" means an aryl group, as defined herein. It refers to a carbonyl (CO) group attached to an aryl group, as in the case of aryl groups. is a carbonyl group.

[0107] As used herein, the term "heteroarylcarbonyl" means any heteroaryl group as defined herein. This refers to a carbonyl (CO) group attached to a heteroaryl group, as in the case of a heteroaryl group. The dots are carbonyl groups.

[0108] As used herein, the terms "arylalkyl" and "arylalkyl" refer to a hydrogen or wherein the carbon bond is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and biphenyl. Methyl and phenylethyl groups, and fused (cycloalkyl) groups such as 4-ethyl-indanyl An alkenyl group includes an alkenyl (aryl) alkyl group as defined herein. The hydrogen or carbon bond of the alkenyl group is an aryl group as defined herein. The point of substitution to the parent moiety is the alkyl group.

[0109] The terms "halo," "halogen," or "halide" groups are used herein. When present, by itself or as part of another substituent, fluorine, chlorine, It means a bromine or iodine atom.

[0110] As used herein, the term "amino" refers to an amino group where each R is independently selected from -NH, -N HR, -NR2, -NR3 + Substituents of the form -NR3, and -NR3 which cannot be protonated + Excluding , respectively, refer to their protonated forms. Therefore, any compound substituted with an amino group The compound can be considered an amine. An "amino group" within the meaning of this specification is a primary An "alkylamino" group can be a monoalkylamino group. These include amino, dialkylamino, and trialkylamino groups.

[0111] As used herein, the term "acyl" refers to a group containing a carbonyl moiety. The carbonyl carbon atom can also be used as a carbon atom bonded to another and the carbon atom of the alkyl group is bonded to the carbon atom of the alkyl group, which is a substituted or unsubstituted alkyl, alkenyl, alkyl It can be part of a group such as aryl, alkoxy, aryl, cycloalkyl, heterocyclyl, etc. do.

[0112] As used herein, the term "formyl" refers to a group containing an aldehyde moiety. The point of substitution onto the parent moiety is the carbonyl group.

[0113] As used herein, the term "alkoxycarbonyl" refers to an alkoxy group containing a carbonyl moiety. The carbonyl carbon atom is attached to the group. It is also bonded to an oxygen atom which is further bonded to an alkyl group. The carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkylenyl group. Alkoxycarbonyl also includes groups having an oxygen atom further bonded to an alkynyl group. As defined herein, an alkoxy group includes a group having a carbonyl carbon atom attached to the alkyl group. Further groups included within the definition of aryloxycarbonyl and included within the term "aryloxycarbonyl" are In some cases, the carbonyl carbon atom is bonded to an oxygen atom attached to an aryl group instead of an alkyl group. is connected to the child.

[0114] As used herein, the term "alkylamide" refers to a group containing a carbonyl moiety. The carbonyl carbon atom is also and is bonded to a nitrogen group which is bonded to one or more alkyl groups, as defined herein. In further cases, such as alkylamides, the carbonyl carbon atom is Alternatively or in addition to an alkyl group, a nitrogen atom bonded to one or more aryl groups may be In a further case, which is also an alkylamide as defined herein, The carbonyl carbon atom may be in place of or in addition to one or more alkyl and / or aryl groups. In addition, the nitrogen atom is bonded to one or more alkenyl groups. In a further case, which is also an alkylamide as defined, the carbonyl carbon atom is Instead of or in addition to the above alkyl, alkenyl and / or aryl groups, It is bonded to a nitrogen atom which is bonded to one or more alkynyl groups.

[0115] As used herein, the term "carboxy" refers to a group containing a carbonyl moiety. The carbonyl carbon atom is also a carbonyl carbon atom. , which binds to a hydroxy group or an oxygen anion to give a carboxylic acid or carboxylate. Carboxy also includes both the protonated and salt forms of the carboxylic acid. For example, Carboxy can be understood as COOH or CO2H.

[0116] The term "alkylthio" as used herein means an alkyl group as defined herein. The sulfur atom attached to the alkyl, alkenyl, or alkynyl group. is a sulfur atom.

[0117] The term "arylthio" as used herein means an aryl group, as defined herein. refers to the sulfur atom attached to the aryl group. The point of substitution into the parent moiety is the sulfur atom.

[0118] As used herein, the term "alkylsulfonyl" means any group defined herein. It refers to a sulfonyl group attached to an alkyl, alkenyl, or alkynyl group, such as The point of substitution onto the moiety is the sulfonyl group.

[0119] As used herein, the term "alkylsulfinyl" refers to a group as defined herein. It refers to a sulfinyl group attached to an alkyl, alkenyl, or alkynyl group such as The point of substitution into the moiety is the sulfinyl group.

[0120] As used herein, the term "dialkylaminosulfonyl" means any of the groups defined herein. a sulfonyl group attached to a nitrogen atom which is further connected to two alkyl groups, as defined by which may optionally be joined together to form a ring with the nitrogen. Also included are groups in which the nitrogen is further connected to one or two alkenyl groups instead of an alkyl group. The point of substitution onto the parent moiety is the sulfonyl group.

[0121] As used herein, the term "dialkylamino" means any of the groups defined herein. refers to an amino group connected to two alkyl groups, optionally joined together, to form a nitrogen The term also refers to a ring in which one or two nitrogen atoms are substituted for the alkyl group. It also includes groups that are further attached to two alkenyl groups. The point of substitution onto the parent moiety is the nitrogen atom.

[0122] As used herein, the term "dialkylamide" means a dialkylamide, as defined herein. refers to an amide group connected to two alkyl groups, optionally bonded together, and The term also refers to a ring in which one or two nitrogen atoms are substituted for the alkyl group. The point of substitution onto the parent moiety is an amide group.

[0123] Each of the various substituents described herein can be substituted or unsubstituted. When used, the term "substituted" refers to the following groups: deuterium (D), halogen (e.g., F, Cl, Br and I), R, OR, OC(O)N(R)2, CN, NO, NO2, ON O2, azide, CF3, OCF3, methylenedioxy, ethylenedioxy, (C3-C2 0) Heteroaryl, N(R)2, Si(R)3, SR, SOR, SO2R, SO2N( R)2, SO3R, P(O)(OR)2, OP(O)(OR)2, C(O)R, C(O) C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C (O)N(R)2, C(O)N(R)OH, OC(O)N(R)2, C(S)N(R)2 , (CH2) 0-2 N(R)C(O)R, (CH2) 0-2 N(R)N(R)2, N(R )N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R) 2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C (O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R )2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR )R, or C(=NOR)R (wherein R is hydrogen, (C1-C 20 ) alkyl or (C6 ~C 20 ) aryl). Substitutions also include, but are not limited to, the following groups: fluoro, chloro, bromo, and methyl. Bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, Alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryl Oxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, Nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkyl Ruthio, arylthiol, alkylsulfonyl, dialkylaminosulfonyl, sulfonyl including, but not limited to, carboxylic acids, dialkylaminos and dialkylamides. When two or more adjacent substituents are present, the substituents are The groups can be linked to form a carbocyclic or heterocyclic ring. The groups can be adjacent or have an atomic relationship, or can be, for example, ortho. The substituents may be adjacent on the ring in an arrangement. Each instance of substitution is understood to be independent. For example, The substituted aryl can be substituted with bromo, and the substituted heterocycle on the same compound can be substituted with alkyl. It is contemplated that the substituents may be substituted with one or more non-fluoro groups. As another example, the substituent may be substituted with one or more non-cyano groups. As yet another example, the substituent may be tert- As yet another example, the substituents may be trifluoro. As a further example, the substituents may be substituted with one or more groups other than methyl. , other than methyl, other than methoxymethyl, other than dialkylaminosulfonyl, other than bromo, Other than chloro, other than amido, other than halo, other than benzodioxepinyl, other than polycyclic heterocyclyl Other than polycyclic substituted aryl, other than methoxycarbonyl, other than alkoxycarbonyl, thio One or more groups other than phenyl or other than nitrophenyl, or combinations of such descriptions Further, substitutions may be made with groups that satisfy the match, such as fluoro, cyano, haloalkyl, t ert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkyl Aminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic hetero Cyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophene In various embodiments, the substituents are also understood to include aryl, phenyl, and nitrophenyl groups. a carbonyl-containing group, other than a nitro, cyano, sulfinyl, sulfonyl, or halogen-containing group In various embodiments, the substituents may be substituted with groups other than electron-withdrawing groups. Some substituents in certain embodiments are substituted only with one or more electron-donating groups. obtain.

[0124] As used herein, the term "boronate group" refers to the following structure: R 8 and R 9 Each of the groups is independently H or C1 to C 10 alkyl or R 8 and R 9 teeth , together with the boronates to which they are bonded, C2 to C 10 Cyclic boronic esters provide. [ka]

[0125] As used herein, the term "π-conjugated" group refers to a group to which a π-conjugate is attached. hybridized P orbitals that overlap or align with non-hybridized P orbitals in the parent moiety refers to a substituent with an unshrunk P orbital, so that electrons are Exemplary parent moieties are X, R 4 , R 5 , R 8 , and BiR 7 The substituent that is a π-conjugated group is also a phenyl group to which the substituent and the Examples of π-conjugated groups are: As for substituted or unsubstituted C2 to C 10 Alken-1-yl, C2-C 10 Alkene-2- Il, C2~C10 Alken-1-yl, C2-C 10 Heterocycloalken-1-yl , C2~C 10 Heterocycloalken-2-yl, C6-C 10 Aryl or heteroaryl Further examples of π-conjugated groups include C2-C 10 alkene-1-yl, C2~C 10 Alken-2-yl, C2-C 10 Alken-1-yl, C2-C 10 Haitai Cycloalken-1-yl, C2-C 10 Heterocycloalken-2-yl, C6-C 10 substituted or unsubstituted vinyl, ethynyl, which are further substituted with aryl or heteroaryl; C6~C 10 Further examples include aryl, aryl, and heteroaryl. , substituted or unsubstituted biaryl, biheterol aryl vinyl, heteroaryl vinyl , and C2~C 10 Alken-1-ylaryl, phenylheteroaryl, and heteroaryl Examples of the alkyl groups include aryl and aryl.

[0126] As used herein, the term "electron donating group" refers to a group that has a net electron donating group relative to hydrogen. Electron-donating groups are well known in the art. For example, J erry March, Michael B. Smith, March's Advan ced Organic Chemistry 6th edition,2007,W iley Interscience and J. McMurry,Organic Chemistry,5th Ed.(Brooks / Cole,Pacific Gr. ove, 2000), each of which is incorporated herein by reference in its entirety. Electron-donating groups, sometimes abbreviated as EDG, are also known as sigma values ​​(o values). It can be defined according to Hammett's substituent constants. The donor group has a sigma value of 0.3 or less, 0.2 or less, 0.1 or less, or a negative sigma value. In further embodiments, the electron donating group has a Cd of 0.3 or less, 0.2 or less, 0.1 or less, or a negative ...1 or less. The position of the substituent is substantially equal to the sigma value. If so, the sigma value should be determined relative to the position of the X group. , σ meta The value is R 6 can be provided to determine the sigma value of the substituent at σ para The value is R 5 This can be used to determine the sigma value of the substituent at The Λ value can be obtained according to published tables or experimentally. For example, JELe ffler and E. Grunwald,Rates and Equilibri a of Organic Reactions,Wiley,1963(Dover reprint), which is incorporated herein by reference in its entirety. Various examples of donor groups include oxyanion, hydroxyl, amino, thio, alkyl, etc. Amino, dialkylamino, alkoxy, alkylthio, acylamino, acyloxy, These include alkyl, alkenyl, vinyl, aryl, and electron-rich heteroaryl. do.

[0127] A further method for determining whether a particular substituent on a given structure is electron donating is to The pKa of the phenol group in the substituted structure (i.e., X=OH) is compared with that of the unsubstituted but otherwise For example, by comparing the pKa of a phenol with the same structure. 4 , R 5 , and R 6 is H and R 7 is vinyl and R 4 , R 5 , R 6 , R 7 is H The phenolic pKa values ​​of certain compounds can be compared.

[0128] In various embodiments, R 4 , R 5 , R 6 , and R 7 is the pKa of the phenol group at X If it is 9.0 or higher, 9.5 or higher, 10.0 or higher, 10.5 or higher, or 11.0 or higher, It can be seen that this provides a net benefit. 4 , R 5 , R 6 , and R 7 is the pKa of the phenol group at X, R 4 , R 5 , R 6 , and R 7 is H When the amount of the hydroxyl group is greater than that of the hydroxyl group, it provides a net electron-donating effect.

[0129] In further embodiments, X is OH or an oxyanion and is 9.0 or greater, 9.5 or greater , 10.0 or greater, 10.5 or greater, or 11.0 or greater.

[0130] "Electron-withdrawing groups," sometimes abbreviated as EWG, have a net electron-withdrawing effect on hydrogen. Electron-withdrawing groups are well known in the art. For example, Jerry March,Michael B.Smith,March's Advanced O rganic chemistry 6th edition,2007,Wiley Interscience and J.McMurry,Organic Chemistry stry,5th Ed.(Brooks / Cole,Pacific Grove,2 000), each of which is incorporated herein by reference in its entirety. The presence of WG is believed to slow the rate of formation of reactive luminescent intermediates, Some embodiments include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 38, 39, For example, in some embodiments, R 4 , R 5 , R 6 , and BiR 7 may contain one or more electron withdrawing groups (EWG), provided that R 4 , R 5 , R 6 , and R 7 have an overall net electron donating effect on the aryl ring to which they are attached. Examples of electron-withdrawing groups include acrylates (e.g., CH3C(O)CH=CH- ) and cyanoacrylate (NCCH=CH-) groups.

[0131] As used herein, the term alcohol protecting group refers to a group in which an alcohol is typically reacted with It is oxygen inert to various conditions but easily removed when subjected to specific conditions. The alcohol protecting groups described herein are typically These compounds improve the stability of the dioxetane moieties and promote the decomposition of the dioxetanes upon their removal. Therefore, the alcohol protecting groups are PO3Na2, PO3CI2, and P Phosphates such as O3H2, glycosyl groups, dinitrobenzenesulfonaminobenzyl groups, as well as other groups that can be enzymatically hydrolyzed to provide unprotected alcohols. Some alcohol protecting groups are described in Theodora W. Greene, Pete r GMWuts(1999).Protecting Groups in Or ganic Synthesis (3rd ed.). J. Wiley, and the whole The alcohol protecting groups include acetyl, benzoyl, benzyl, , methoxyethoxymethyl, dimethyltrityl, methoxymethyl, methylthiomethyl, Valoyl, tetrahydropyranyl, tetrahydrofuranyl, trityl, trialkylsilyl aryl, trialkylsiloxymethyl, dialkylarylsilyl, glycosyl, pyranyl, Alcohol protecting groups also include galactosyl and ethoxyethyl groups. the linker comprises a group substituted with a fragmentable linker which is further substituted with a protecting group, Upon deprotection of the protective group, the linker fragment is deprotected and eliminated from the alcohol. The compound is a further example of an alcohol substituted with an alcohol protecting group. [ka]

[0132] [ka]

[0133] In various embodiments, the protecting group G can be an enzyme-cleavable group, e.g., Removal of the cleavable group by the analyte of interest in the presence of an enzyme capable of cleaving the cleavable group. The removal provides an unstable phenolate-dioxetane species that subsequently decomposes and emits light. For example, G is a peptide moiety consisting of two or more amino acid residues that can be cleaved by a specific enzyme. It could be.

[0134] As used herein, the term "surfactant-based luminescence enhancer" refers to a surfactant-based luminescence enhancer that is an aqueous solution. Refers to a class of compounds typically used to increase the strength of dioxetanes in Emerald™ and Emerald-ll™ enhancers are available from Thermo Scientific Commercially available from Fisher Scientific (Waltham, MA) Further examples of surfactant-based luminescence enhancers are given by Schaap et al. ,AP,Akhavan,H.,Romano,LJClin.Chem.19 89,35(9),1863, which is incorporated by reference in its entirety. In embodiments, the surfactant-based luminescence enhancer comprises an acyclic alkyl group of at least 8 carbons. The tail portion is a group, and one or more of a quaternary ammonium salt, a pyridinium salt, a quaternary phosphonium salt, a head moiety that is an ammonium surfactant salt, an ethylene glycol chain, or a fluorescein moiety; In various embodiments, the surfactant-based luminescence enhancer comprises cetrimonium bromide. Cetylpyridinium chloride (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC) , benzethonium chloride (BZT), dimethyldioctadecane ammonium chloride, di octadecyldimethylammonium (DODAB), a'-tributylphosphonium-p- Xylene chloride, poly(vinylbenzyltributylphosphonium chloride) (TBE) , cationic surfactants such as poly(vinylbenzyltrioctylphosphonium chloride) In various further embodiments, surfactant-based luminescence enhancers are used. The detergents used are non-ionic Triton X-100, Tween surfactant, polyethylene glycol Coalhead, Brij® surfactants, IGEPAL® surfactants , octylphenoxypolyethoxyethanol, etc. The surfactant-based luminescence enhancer also includes N-(3',6'-dihydroxybenzoyl) -3-oxo-3H-spiro[isobenzofuran-1,9'-xanthan]-5-yl) Containing a fluorescein head group, such as tetradecaneamide (fluorescein surfactant) It is possible.

[0135] In some cases, the compounds described herein (e.g., compounds of Formulas (l)-(X)) may be All diastereomers of the compounds described herein may be racemic. as well as are contemplated herein.

[0136] As used herein, the terms "salt" and "pharmaceutically acceptable salt" refer to parenteral compounds. Derivatives of the disclosed compounds, wherein the compound is modified by making an acid or base salt thereof. Examples of salts include alkali salts and alkaline earth salts of the disclosed compounds in ionized form. For example, lithium salt, sodium salt, potassium salt, calcium salt, The disclosed compounds are cationic metal and anionic organic compounds, e.g., magnesium salts. For example, it may be a salt containing a compound having an oxyanion and a sodium cation. Examples of physiologically acceptable salts include mineral or organic acid salts of basic groups such as amines, and carbohydrates. Examples of suitable pharmaceutical compounds include, but are not limited to, alkali or organic salts of acidic groups such as carboxylic acids. Suitable salts include, for example, the salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Examples include conventional non-toxic salts or quaternary ammonium salts. Salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid. and those derived from acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, Lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxy Maleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanyl tri-, 2- -Acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid Included are salts prepared from organic acids such as sulfonic acid, oxalic acid, and isethionic acid.

[0137] Pharmaceutically acceptable salts are prepared by conventional chemical methods from parent compounds containing a basic or acidic moiety. In some cases, such salts can be synthesized from the compounds The free acid or base form can be stoichiometrically ( can be prepared by reacting with an appropriate amount (or greater) of a base or acid. Generally, ether, ethyl acetate, ethanol, isopropanol, or acetonitrile Non-aqueous solutions such as HCl, ... aceutical Sciences,17th ed.,Mack Publish ing Company, Easton, Pa., 1985, the disclosure of which is incorporated herein by reference. is incorporated herein by reference.

[0138] The term "solvate" refers to a stoichiometric or non-stoichiometric solvate bound by non-covalent intermolecular forces. It means a compound or a salt thereof further comprising a stoichiometric amount of a solvent. The solvates are hydrates.

[0139] Values ​​expressed in range format include each number, not just the numbers explicitly listed as the limits of the range. All individual values ​​and subranges included within the range are treated as if the values ​​and subranges were explicitly recited. The terms "value" and "values" should be interpreted in a flexible manner to include any number or subrange. For example, the range "about 0.1% to about 5%" or "about 0.1% to 5%" means approximately within the indicated range. Not just 0.1% to about 5%, but individual values ​​(e.g., 1%, 2%, 3%, and 4%) and Subranges (e.g., 0.1%-0.5%, 1.1%-2.2%, 3.3%-4.4%) are also possible. A statement such as "about X to Y" should be interpreted as including "about X to about Y" unless otherwise indicated. Similarly, a statement such as "about X, Y, or about Z" has the same meaning as "about X, Y, or about Z" unless otherwise indicated. and has the same meaning as "about X, about Y, or about Z."

[0140] As used herein, the terms "a," "an," or "the" may be used unless the context clearly indicates otherwise. Unless otherwise indicated, the term "or" is used to include one or more. Unless otherwise specified, the term "or" is used to refer to a non-exclusive "or." or terminology is for descriptive purposes only and not for purposes of limitation. The section headings are intended to aid in the reading of this specification and should not be construed as limiting. Furthermore, information relating to a section heading may not be used within or on the basis of that particular section. Furthermore, all publications, patents, and patent documents referred to herein are the property of their respective owners. No. 6,239,793, filed Dec. 1, 2003, which is incorporated by reference in its entirety as if individually incorporated by reference. In the event of inconsistent usage between this specification and the specification incorporated by reference, the The usage in the incorporated references should be considered complementary to that of this specification. In case of conflict, the usage set forth herein will govern.

[0141] In the methods described herein, the steps are not explicitly recited in terms of temporal or operational sequence. Except as otherwise indicated, the steps may be performed in any order without departing from the principles of the present invention. Furthermore, unless express claim language indicates otherwise, a particular The steps may be performed simultaneously. For example, a claimed step of performing X and The claimed steps of performing Y may be performed simultaneously in a single operation, resulting in The resulting process falls within the literal scope of the claimed process.

[0142] As used herein, the term "about" refers to the extent to which a value or range, e.g., the reference to a stated value or range, is exceeded. Some variation within 10%, 5%, or 1% of the limit may be tolerated. can.

[0143] Each of the above embodiments may be applied in combination with other embodiments described herein. For example, an embodiment corresponding to formula (I) may be represented by formulas (II) to (X). As another example, an embodiment corresponding to formula (II) is It is equally contemplated that this is applicable to each of formulas (I) and (III)-(X).

[0144] As used herein, the term "about" refers to the extent to which a value or range, e.g., the reference to a stated value or range, is exceeded. Some variation within 10%, 5%, or 1% of the limit may be tolerated. can.

[0145] As used herein, the term "substantially" means at least about 50%, 60% ,70%,80%,90%,95%,96%,97%,98%,99%,99.5%,9 This refers to a majority of 9.9%, 99.99%, or at least about 99.999% or more.

[0146] As used herein, "substantially free" or "substantially free" means The term "substantially free of" means less than about 1%, less than 0.5%, less than 0.1% Less than, less than 0.05%, less than 0.001%, or less than about 0.0005%, about 0% or less, It refers to below the limit of quantity, below the detectable limit, or 0%.

[0147] Those skilled in the art will readily appreciate that the embodiments described herein may be practiced without departing from the spirit and scope of the present disclosure. It will be appreciated that many modifications to the state are possible, and therefore the description is not intended. The present invention is not limited to the embodiments given and should not be construed as being limited to the embodiments set forth in the accompanying patent claims. should be granted the full breadth of protection afforded by the scope of the claim and its equivalents. Furthermore, the use of some of the features of this disclosure without the corresponding use of other features may be Therefore, the foregoing description and exemplary embodiments illustrate the principles of the present disclosure. are provided for illustrative purposes only, including, but not limited to, modifications and permutations thereof. This can be done. [Example]

[0148] The invention will be better understood by reference to the following examples, which are provided by way of illustration. The present invention is not limited to the examples given herein. General method

[0149] Various compounds of the present disclosure may be prepared using methods described in WO 1996 / 015122(A1), U.S. Pat. No. 4,962,192 or the synthetic approach described in U.S. Pat. No. 5,004,565. They can be synthesized according to a variety of methods, including, but not limited to, each of which is incorporated herein by reference in its entirety.

[0150] Chemiluminescence (luminescence) intensity was measured using a chemiluminescence (CT) assay kit from Turner Designs (Sunnyvale, CA). ) Model TD-20e Luminometer with BMG Labtech Luminescence Plates a reader, or a charge-coupled device (CCD) camera luminometer, or any other suitable The light intensity can be measured using a light intensity measuring device. In the examples listed below, different densities are measured. Solutions containing alkaline phosphatase (e.g., AP4, AP6, AP8, and AP 9) for example, 10 from the initial stock 4 , 10 6 , 10 8 and 10 9 Present serial dilutions At each value, compounds and enhancers (if used) were used at their near optimum. Various concentrations were tested.

[0151] Nuclear magnetic resonance (NMR) spectrum Spectra were obtained using a 400 MHz spectrometer in a solution of D2O and CDCI3.

[0152] Amine buffer solution "221" or "Sigma-221" is available from Sigma-Aldric. h (St. Louis, MO). Example 1

[0153] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.30-7.05(m,3H ),6.90(m,1H),6.28(br,1H),3.22(s,3H),3.04 (s,1H),2.21(s,1H),1.94-1.6(m,10H),1.24(m ,1H),1.04(m,1H).

[0154] Prepare an initial solution of the compound in dioxane (1 mg of compound per mL of dioxane) and then mixed with water (20 μL of dioxane in 180 μL of water), followed by heating at 37° C. The compound was treated with 200 μL of amine-based 221 buffer. The intensity of chemiluminescence was measured over time. A graph showing the luminescence intensity over time is provided in Figure 1. The compound of Example 1 was luminescent for 3.79 minutes, demonstrating that luminescence can be elicited over a long period of time. and a Σ value of 1.13E+5. Example 2

[0155] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.63(m,1H),7.3 1(m,1H),7.15(m,1H),6.0(br,1H),3.22(s,3H) ,3.0(s,1H),2.24(s,1H),2.1-1.4(m,12H).

[0156] Example 2 was tested in the same manner as Example 1, with a 1 mg / mL test compound in THF. 10 μL of sample was used. The compound of Example 2 exhibits a chemiluminescence half-life of 4.91 minutes. The addition of electron-withdrawing chlorine on the phenyl ring at the 2-position slowed down the rate of increase in emission, and the The half-life becomes longer. Example 3

[0157] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.05(m,2H),3.4 4(s,3H),3.24(s,1H),2.54(s,1H),2.1-1.6(m, 12H).

[0158] Example 3 was tested in the same manner as Example 2. The compound of Example 3 was tested in a 9.22 minute chemical reaction. The luminescence half-life is shown. The addition of two electron-withdrawing chlorine groups further slows down the rate of luminescence. This increases the half-life of the luminescence. Example 4

[0159] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.70(m,1H),7.4 5-6.65(m,2H),5.59(br,1H),3.20(s,3H),3.00 (s,1H),2.16(s,1H),1.90-1.40(m,10H),1.27( m,1H),1.04(m,1).

[0160] Example 4 was tested in the same manner as Example 2. The compound of Example 4 was tested in a 2.10 minute chemical The luminescence half-life is shown. The addition of a slightly electron-donating iodine atom significantly increases the luminescence rate. The half-life is slightly increased compared to Examples 1-3. Example 5

[0161] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.44(m,1H),7.4 0-6.90(m,3H),5.82(d,J=16Hz,1H),5.65(br,1 H),5.41(d,J=16Hz,1H),3.23(s,3H),3.02(s,1 H),2.18(s,1H),1.90-1.40(m,10H),1.25(m,1H) ),1.09(m,1H).

[0162] Example 5 was tested in the same manner as Example 1, with 0.1 mg of the test compound in dioxane 10 μL of 1 mL / mL sample was used. Specifically, the initial solution of the compound was dissolved in dioxane (1 mL of 0.1 mg of compound per dioxane) and then in water (10 in 100 μL of water). 200 μL of dioxane solution) followed by 200 μL of amine-based 221 buffer at 37 °C. Processed.

[0163] A graph showing the luminescence intensity over time is provided in Figure 5. The compound of Example 5 exhibited a 23 second conversion. It showed a chemiluminescence half-life and a Σ value of 1.28E+5. The addition of the aryl group resulted in a higher luminescence intensity and a more rapid increase in the luminescence rate, compared to Examples 1 to 4. The half-life of the luminescence was dramatically shortened. Example 6

[0164] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.51(m,1H),7.4 8(m,1H),7.41(m,1H),7.35(m,1H),7.40-7.01( m,2H),5.52(br,1H),3.26(s,3H),3.04(s,1H), 2.26(s,1H),1.90-1.46(m,10H),1.28(m,1H),1 .13(m,1H).

[0165] Example 6 was tested in the same manner as Example 1. The compound of Example 6 exhibited a chemical reaction time of 12.7 seconds. The emission half-life was shown. As a result, the luminescence intensity was higher and the luminescence rate increased more rapidly, and the luminescence was The half-life of has shortened. Example 7

[0166] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.51(m,1H),7.4 2(m,1H),7.45-7.01(m,4H),5.80(br,1H),3.26 (s,3H),3.04(s,1H),2.08(s,1H),2.1-1.5(m,1 0H), 1.25(m,1H), 1.10(m,1H).

[0167] Example 7 was tested in the same manner as Example 1, with 1 mg / m of the test compound in dioxane. A 10 μL sample was used. Specifically, the initial solution of the compound was dissolved in dioxane (1 mL of dioxane). Prepare the solution in water (1 mg of compound per xanthate) and then in water (10 μL of distilled water in 90 μL of water). The mixture was mixed with 200 μL of amine-based 221 buffer at 37°C.

[0168] A graph showing the luminescence intensity over time is provided in Figure 6. The compound of Example 7 was 11.8 seconds and a Σ value of 1.32E+05. Moving the electron-rich sulfur atom closer to the nyl ring and thus providing a stronger electron-donating effect The compound of Example 7 has a higher luminescence intensity and a faster emission rate than those of Examples 1 to 6. The emission increased more rapidly and the half-life of the luminescence decreased. Example 8

[0169] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.68(d,J=2Hz,1 H),7.62(d,J=1.2Hz,1H),7.56(s,1H),7.50-7. 10(br,2H),6.80(br,1H),3.27(s,3H),3.05(s, 1H), 2.23(s, 1H), 1.88-1.45(m, 10H), 1.31-1.2 6(m,1H),1.12-1.08(m,1H).

[0170] Example 8 was tested in the same manner as Example 1, with 0.001% of the test compound in dioxane. 10 μL of mg / mL sample was used. Specifically, the initial solution of the compound was dissolved in dioxane (1 m Prepare a solution in 0.001 mg of compound per L of dioxane and then in water (90 μL of water) 10 μL of dioxane solution), followed by 100 μL of amine-based 221 buffer at 37 °C. Treated with liquid.

[0171] A graph showing the luminescence intensity over time is provided in Figure 7. The compound of Example 8 exhibited a 23 second conversion. A chemiluminescence half-life and Σ value of 9.76E+04 were shown. Without being bound by any particular theory However, due to the extended π-conjugated system, the compound of Example 8 has a higher luminescence than Example 7. The half-life of sample 8 is thought to be longer than that of sample 7 due to the 4-CN electron-withdrawing effect. It's also bigger. Example 9

[0172] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.72(d,J=7.2Hz ,2H),7.67(d,J=8Hz,2H),7.60(d,J=8Hz,1H),7 .52(d,J=4Hz,1H),7.45(d,J=4Hz,1H),7.44-7. 10(m,3H,5.75(br,1H),3.27(s,3H),3.04(s,1H) ),2.23(s,1H),1.90-1.45(m,10H),1.31-1.26( m,1H),1.15-1.08(m,1H).

[0173] Example 9 was tested in the same manner as Example 1, with 0.01 ml of the test compound in dioxane. 10 μL of 10 μg / mL sample was used. Specifically, the initial solution of the compound was dissolved in dioxane (1 mL dioxane) and then water (10 in 90 μL of water). 100 μL of dioxane solution) followed by 100 μL of amine-based 221 buffer at 37 °C. A graph showing luminescence intensity over time is provided in FIG. Example 10

[0174] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.72(d,J=7.2Hz ,2H),7.68(m,2H),7.61(d,J=8Hz,1H),7.52(d, J=8Hz,1H),7.45(d,J=4Hz,1H),7.44-7.7.05(m ,2H),5.81(br,1H),3.27(s,3H),3.04(s,1H),2 .25(s,1H),1.90-1.45(m,10H),1.31-1.26(m,1 H), 1.15-1.08(m,1H).

[0175] Example 10 was tested in the same manner as Example 1, with 0.01% of the test compound in dioxane. 10 μL of mg / mL sample was used. Specifically, the initial solution of the compound was dissolved in dioxane (1 m Prepare a solution in water (0.01 mg of compound per L of dioxane) and then in water (1 in 90 μL of water). 100 μL of dioxane solution), followed by 100 μL of amine-based 221 buffer at 37 °C. A graph showing luminescence intensity over time is provided in FIG. Example 11

[0176] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.56(m,2H),7.4 2(m,1H),7.44-7.30(m,2H),7.22(d,J=6Hz,2H) ,6.93(d,J=8.8Hz,2H),5.91(s,1H),3.85(s,3H ),3.27(s,3H),3.04(s,1H),2.27(s,1H),1.90- 1.45(m,10H),1.31-1.26(m,1H),1.15-1.08(m, 1H).

[0177] Example 11 was tested in the same manner as Example 1, with 0.1 ml of the test compound in dioxane. 10 μL of 10 μg / mL sample was used. Specifically, the initial solution of the compound was dissolved in dioxane (1 mL dioxane) and then water (10 in 90 μL of water). 100 μL of dioxane solution) followed by 100 μL of amine-based 221 buffer at 37 °C. A graph showing luminescence intensity over time is provided in FIG. Example 12

[0178] A dioxetane compound (Lumigen® PPD) was obtained having the following structure: . [ka] 1 H NMR(400MHz,D2O)δ ppm7.40-7.15(m,4H), 3.24(s,3H),2.89(s,1H),2.28(s,1H),1.90-1. 50(m,10H),1.28(d,J=13.2Hz,1H),0.99(d,J=1 0Hz,1H).

[0179] Prepare the initial solution of the compound in 221 buffer (compound: 1.25 mg / ml, TBE enhancer Next, 100 μL of the initial solution was diluted with 10 μL of alkali at 37°C. The compounds were combined with a solution of alkaline phosphatase (AP8). When combined with the solution, the intensity of chemiluminescence was measured over time. The results are provided in Figure 11. The compounds reached a steady-state plateau maximum light intensity over 15 minutes. The luminescence intensity showed a slow and gradual increase of 2.27E+05 provided the Σ value of Example 13

[0180] A dioxetane compound having the following structure was obtained: [ka]

[0181] The initial solution of the compound was prepared in 221 buffer (compound: 0.125 mg / mL, TBE enhancer: A 100 μL aliquot was added to 10 μL of alkali solution at 37°C. The compounds were combined with a solution of alkaline phosphatase (AP8). When combined with the solution, the intensity of chemiluminescence was measured over time. The results are provided in Figure 12. The compounds reached a steady-state plateau maximum light intensity over 15 minutes. The luminescence intensity showed a slow and gradual increase of 1.91E+05 provided the Σ value of Example 14

[0182] Dioxetane compounds were prepared according to the following structures: [ka]

[0183] 1 H NMR(400MHz,D2O)δ ppm7.86(br,1H),7.81 (d,J=8.4Hz,1H),7.74(S,1H),7.51(d,J=5.2Hz ,1H),7.21(br,1H),7.18(d,J=7.2Hz,1H),3.28 (s,3H),2.90(s,1H),2.33(s,1H),1.85-1.55(m ,10H),1.28(d,J=8Hz,1H),1.10(d,J=12Hz,1H) .

[0184] Prepare the initial solution of the compound in 221 buffer (compound: 0.25 mg / mL, TBE enhancer Next, 100 μL of the initial solution was diluted with 10 μL of alkali at 37°C. The compounds were combined with a solution of alkaline phosphatase (AP8). When combined with the solution, the intensity of chemiluminescence was measured over time. The results are provided in Figure 13. The compound exhibited a rapid increase in intensity, reaching a maximum steady-state intensity. The compound of Example 14 reached a Σ value of 9.97E+05 over 1 minute. Compared to the compounds of Examples 12 and 13, the compounds exhibited higher intensity and faster response. Provided. Example 15

[0185] Dioxetane compounds were prepared according to the following structures: [ka] 1H NMR(400MHz,D2O)δ ppm7.95(m,1H),7.85( br,1H),7.65-7.55(m,2H),7.52(m,1H),7.25(b r,1H),3.28(s,3H),2.90(s,1H),2.34(s,1H),1 .90-1.55(m,10H),1.38(m,1H),1.08(m,1H).

[0186] Prepare the initial solution of the compound in 221 buffer (compound: 0.25 mg / mL, TBE enhancer Next, 100 μL of the initial solution was diluted with 10 μL of alkali at 37°C. The graph showing the luminescence intensity over time is shown in Figure 1. 14. The compound exhibited a rapid increase in strength, reaching a maximum steady-state strength. The compound of Example 15 provided a Σ value of 7.38E+05 over a 2 minute period. Compared with compounds 12 and 13, the compounds provided luminescence with higher intensity and faster response. Ta. Example 16

[0187] Dioxetane compounds were prepared according to the following structures: [ka]

[0188] 1 H NMR(400MHz,D2O)δ ppm7.67(d,J=8.4Hz,1 H),7.66(br,1H),7.26(br,1H),7.16-6.06(m,1 H),5.88(d,J=16Hz,1H),5.37(d,J=12.4Hz,1H) ,3.27(s,3H),2.88(s,1H),2.30(s,1H),1.84-1 .56(m,10H),1.28(d,J=9.2Hz,1H),1.05(d,J=1 2.8Hz,1H).

[0189] Prepare the initial solution of the compound in 221 buffer (compound: 0.25 mg / mL, TBE enhancer Next, 100 μL of the initial solution was diluted with 10 μL of acetone at 37°C. The compound was combined with a solution of alkaline phosphatase (AP8). When combined with the enzyme solution, the chemiluminescence intensity was measured over time. A graph is provided in Figure 15. The compound exhibited a rapid increase in potency, reaching a maximum steady-state potency. The compound of Example 16 reached a temperature of 1.59E+06 over a period of 3 minutes. The compounds of Examples 12 to 15 emit light with higher intensity and faster response. was provided.

[0190] In further experiments, initial solutions of the compounds were prepared in water (compound: 0.1 mg / mL) and then 100 μL of the initial solution was added to 10 μL of alkaline phosphatase (AP4) at 37 °C. The compound was combined with 300 uL of 5 mg / mL TEB221 buffer. When combined with potassium phosphatase solution, the intensity of chemiluminescence was measured over time. A graph showing the luminescence intensity is provided in FIG. Example 17

[0191] Dioxetane compounds were prepared according to the following structures: [ka] 1 H NMR(400MHz,D2O)δ ppm7.88(m,2H),7.75( m,1H),7.52(m,1H),7.30(br,1H),3.26(s,3H), 2.88(s,1H),2.29(s,1H),1.90-1.55(m,10H),1 .24(m,1H),1.04(m,1H).

[0192] The initial solution of the compound was prepared in 221 buffer (compound: 0.2 mg / mL, TBE enhancer: Next, 100 μL of the initial solution was diluted with 10 μL of alkaline phosphatase at 37°C. The mixture was combined with a solution of phospholipase A (AP9, [1.24E-20 mol / μL]). A graph showing the typical luminescence intensity is provided in Figure 17. The compound showed a rapid increase in intensity, Maximum steady-state intensity was reached over 2 minutes, giving a Σ value of 2.77E+05.

[0193] Figure 18 shows the luminescence intensity over time for Lumigen® PPD under similar conditions. show. [ka]

[0194] The results demonstrate the direct addition of π-conjugated and electron-donating groups to the aryl ring attached to the dioxetane. This can result in an increase in the rate and intensity of light emission upon dioxetane fragmentation (see Examples 5- 11 and Figures 5 to 10). Such substitutions include, for example, π-conjugated groups and electron-donating groups. , R of Formula I 4 , R 5 , R 6 , or R 7 or corresponds to being placed at Q in formula III.

[0195] For example, vinyl substituents produce emission with a rapidity at least 20 times greater than the corresponding unsubstituted compounds. This resulted in a dioxetane that provided a fast and intense burst (see Example 5, Figure 5 and Figure 1). Substitution with a thiophene group provided another example of such an advantage (see Examples 6 and (See Figures 6 and 7, compare Figures 6 and 1). The examples also show the degree of electron donating properties of π-conjugated electron donating groups. Adjusting the rate can result in a more intense emission, a more rapidly increasing emission rate, and a shorter half-life of emission. (See Examples 7 and 8.) Specifically, π-conjugated electron donors The strength can be improved by reducing the electron-donating effect of the group by directly replacing it with an electron-withdrawing group. The emission rate can be increased and the half-life of the luminescence can be increased (compare Figure 7 and Figure 6). In addition, the effect of direct substitution with thiophene, a type of π-conjugated electron-donating group, is significantly greater than that of the corresponding non-substituted group. These compounds offer significant advantages over their replacement compounds (compare Figure 7 and Figure 1). A remote placement of a pull-out or donor group (e.g., cyano or methoxy) is attached to the dioxetane. The advantages of having π-conjugated electron donating groups located directly on the central aryl ring are not outweighed by the (See Examples 9, 10, and 11; compare Figures 8, 9, and 10 with each other; see Figure 10.) 1). Therefore, π-conjugated electron donating groups, such as vinyl, aryl, or hydroxyl groups, The heteroaryls can be further substituted and modified without destroying the improved luminescent properties.

[0196] In comparison, compounds substituted with groups that are not π-conjugated or electron-donating are less likely to produce emissive groups. The light is slow and the half-life of the luminescence is long (see Examples 2 to 4 and Figures 2 to 4). The results showed that adding a second chlorine atom resulted in an additive (compare Figures 2 and 3). Iodine, which neither strongly donates nor strongly withdraws, did not significantly improve luminescence. had a significantly smaller effect (see Example 4).

[0197] The 3-phosphate phenyl derivative is activated with alkaline phosphatase (ALP). Further preparations and tests were carried out under aqueous conditions by using the method described above (Examples 12 to 16 and Figures 11 to 16). 6). Two examples are commercially available dioxetanes, namely Lumigen (registered trademark). PPD (Example 12) and Tropix CDP-Star (Example 13) Example 13), both of which lack an electron-donating group on the central aromatic ring attached to the dioxetane. Both of the dioxetanes sold reached a maximum light intensity or a steady-state intensity plateau over 15 min. In comparison, the dioxetanes with π-conjugated electron-donating groups showed slow luminescence. The active composition was a commercially available dioxetane (see Examples 14-16, and Figures 13-16 in Figures 11 and 12). 12) showed greatly improved speed and intensity of light emission.

[0198] Without intending to be limited by any theory, it is surprising that aromatic rings The increase in electron density on the π-conjugated substituents generates transient excited state species that undergo chemical decay. Based on these results, it appears that the rate-limiting step of Dioxetanes can be obtained by using substituents that are ductive, electron donating, or both. It can increase the speed and intensity of chemiluminescence and provide surprisingly high-intensity luminescence. The examples were tested in aqueous conditions without the surfactant-based luminescence enhancer. The compounds and compositions of the present disclosure represent a significant improvement over commercially available dioxetanes. Example 18

[0199] Dioxane compounds were prepared according to the following structure, consistent with the synthetic methods described herein: did. [ka]

[0200] (D2Oppm): 7.51 - 7.41 (m, 2H), 7.29 - 7.20 (m, 1H ), 5.66 (d, J = 17.2 Hz, 1H), 5.19 (d, J = 11.2 Hz, 1H ), 3.07 (s, 3H), 2.72 (s, 1H), 2.36 (s, 3H), 1.99( s, 1H), 1.95 - 1.38 (m, 10H), 1.19 - 1.15 (m, 1H), 0 .92 - 0.88 (m, 1H).

Chem.

[0201] (D2O) 7.56 - 7.49 (m, 2H), 7.26 - 7.19 (m, 1H), 5. 71 (d, J = 16.4 Hz, 1H), 5.26 (d, J = 12.4 Hz, 1H), 3. 10 (s, 3H), 2.66 (s, 1H), 2.01 (s, 1H), 1.67 - 1.43 (m, 10H), 1.39 - 1.18 (m, 1H), 1.10 - 1.06 (m, 1H).

Chem.

[0202] (CD3OD) 7.53 - 7.50 (m, 1H), 7.40 - 6.85 (m, 3H), 5.74 (d, J = 16.5 Hz, 1H), 5.29 - 5.24 (m, 1H), 4.96 - 4.91 (m, 1H), 4.20 (s, 1H), 4.12 - 4.02 (m, 1H), 3 .85 - 3.75 (m, 5H), 3.17 (s, 3H), 2.98 (s, 1H), 2.1 0 (s, 1H), 1.85 - 1.44 (m, 10H), 1.24 - 1.19 (m, 1H) , 1.0 - 0.85 (m.1H). [Chemical formula]

[0203] (CDCI3) 7.50 - 7.40 (m, 2H), 7.35 - 7.30 (s, 1H), 6.91 (s, 1H), 5.50 (d, J = 16Hz, 1H), 5.20 (d, J = 16 Hz, 1H), 3.17 (s, 3H), 3.01 (s, 1H), 2.02 (s, 1H), 1.95 - 1.58 (m, 10H), 1.34 - 1.26 (m, 1H), 1.20 - 1. 10 (m, 1H). [Chemical formula] <00​​​​​​​​​​​​​​​​​​​​​​​​​​​.30 - 1.26 (m, 1H), 1.24 - 1.21 (m, 1H).

Chem.

[0206] (CDCI3) 7.40 - 6.80 (m, 3H), 5.80 (s, 1H), 5.44 ( s, 1H), 5.20 (s, 1H), 3.23 (s, 3H), 3.02 (s, 1H), 2 .24 (s, 1H), 2.13 (s, 3H), 1.96 - 1.44 (m, 10H), 1. 32 - 1.24 (m, 1H), 1.10 - 0.96 (m, 1H).

Claims

1. Formula III 【Chemistry 1】 (In the formula, R ３ However, C １ ~C １０ Alkyl, C ６ ~C １０ It is an aryl or heteroaryl, R ４ 、 R ５ 、 R ６ and R ７ each independently is H, Q, X, hydroxy, halogen, amino, thio, C １ to C １０ alkyl, C ２ to C １０ alkenyl, C<000..... １０ ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Alkylphosphonate, C １ ~C １０ Alkyl phosphinate, C １ ~C １０ Trialkylphosphonium salt, C ４ ~C １０ Heterocycloamino, C ６ ~C １０ It is an aryl or π-rich heteroaryl, Q is a π-conjugated electron-donating group, X is a -OH, -O-G, -O' salt, or a boron group having the following structure: 【Chemistry 2】 R ８ and R ９ Each of these independently is H or C １ ~C １０ Alkyl or R ８ and R ９ Together with the boronate to which they bond, C ２ ~C １０ We provide cyclic boronic acid esters, R １０ and R １１ Each of these is independently H or halogen, R １２ and R １３ each independently is H, halogen, C １ ~C １０ alkyl, C ２ ~C １０ alkenyl, C ２ ~C １０ alkynyl, C １ ~C １０ alkyloxy, C １ ~C １０ alkylamino, C １ ~C １０ trialkylammonium salt, C １ ~C １０ alkylthio, C ２ ~C １０ acyl, C １ ~C １０ alkyloxycarbonyl, C １ ~C １０ alkylaminocarbonyl, C １ ~C １０ alkylthiocarbonyl, C ２ ~C １０ acyloxy, C ２ ~C １０ acylamino, C ２ ~C １０ acylthio, C １ ~C １０ alkyl carbonate, C １ ~C １０ alkyl carbamate, C １ ~C １０ carbamide, aryloxy, C １ ~C １０ alkylsulfinyl, C １ ~C １０ alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C １ ~C １０ alkyl phosphonate, C １ ~C １０ alkyl phosphinate, C １ ~C １０ Trialkylphosphonium salt, C ４ ~C １０ Heterocycloamino, C ６ ~C １０ It is either an aryl or a π-excess heteroaryl, or R １２ and R １３ However, together with the carbon atoms to which they bond, C ５ ~C １０ Cycloalkenyl, C ２ ~C １０ Heterocycloalkenyl, C ６ ~C １０ We provide aryl or π-rich heteroaryls. R １４ However, H, halogen, C １ ~C １０ Alkyl, C ２ ~C １０ Alkenil, C １ ~C １０ Alkyloxy, C １ ~C １０ Alkylamino, C １ ~C １０ Alkylthio, C ２ ~C １０ Acyloxy, C ２ ~C １０ Acylamino, C ２ ~C １０ Acyrchio, C １ ~C １０ Alkyl carbonate, C １ ~C １０ Alkylcarbamate, C １ ~C １０ Carbamide, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, C ４ ~C １０ Heterocycloamino, C ６ ~C １０ It is an aryl or π-rich heteroaryl, G is the following protecting group: a) A divalent, fragmentable linker, and optionally substituted trialkylsilyl, alkylarylsilyl, arylsulfonyl, dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, furanosyl, acyl, benzoyl, benzyl, or boronate group; b) Trialkylsilyl, alkylarylsilyl, arylsulfonyl, dioxobenzyl, trityl, alkyl carbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranulonyl, furanosyl, acyl, benzoyl, or benzyl, which may have substituents; or c) - PO ３ H ２ A compound having either a salt or ester thereof.

2. R １２ and R １３ However, together with the carbon that they bond to, C ５ ~C １０ Cycloalkenyl, C ２ ~C １０ Heterocycloalkenyl, C ６ ~C １０ The compound according to claim 1, which provides an aryl or π-excess heteroaryl.

3. The following formula: 【Transformation 3】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 (In the formula, R ３ However, C １ ~C １０ It is alkyl, R １０ and R １１ Each of these is independently H or halogen, Z, L, and J are each S, O, Se, NR １５ , or (CR １６ R １７ ) ｎ And, Each R １５ However, independently, these are H, alkyl, acyl, benzyl, alkyloxycarbonyl, and arylsulfonyl. R １４ , R １６ , R １７ , R １８ , R １９ , R ２０ and R ２１ If each of these exists, independently, H, halogen, C １ ~C １０ Alkyl, C ２ ~C １０ Alkenil, C １ ~C １０ Alkyloxy, C １ ~C １０ Alkylamino, C １ ~C １０ Trialkylammonium salt, C １ ~C １０ Alkylthio, C ２ ~C １０ Ashiru, C １ ~C １０ Alkyloxycarbonyl, C １ ~C １０ Alkylaminocarbonyl, C １ ~C １０ Alkylthiocarbonyl, C ２ ~C １０ Acyloxy, C ２ ~C １０ Acylamino, C ２ ~C １０ Acyrchio, C １ ~C １０ Alkyl carbonate, C １ ~C １０ Alkylcarbamate, C １ ~C １０ Carbamide, aryloxy, C １ ~C １０ Alkyl sulfinyl, C １ ~C １０ Alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C １ ~C １０ Alkylphosphonate, C １ ~C １０ Alkyl phosphinate, C １ ~C １０ Trialkylphosphonium salt, C ４ ~C １０ Heterocycloamino, C ６ ~C １０ It is either an aryl or a π-excess heteroaryl, or R １４ , R １６ , R １７ , R １８ , and R ２０ Any two of them, together with the carbon to which they are bonded, C ５ ~C １０ Cycloalkenyl, C ２ ~C １０ Heterocycloalkenyl, C ６ ~C １０ The compound according to claim 1 or 2, having one of the following (providing an aryl or a π-excess heteroaryl).

4. The above formula is, 【Chemistry 4】 【change】 The compound according to claim 3.

5. R ４ , R ５ , and R ６ The compound according to any one of claims 1 to 4, wherein each of them is H.

6. R １０ The compound according to any one of claims 1 to 5, wherein each of them is H.

7. R １４ , R １６ , R １７ , R １８ , R １９ , R ２０ and R ２１ If each of these exists, independently, H, halogen, C １ ~C １０ Alkyl, C ２ ~C １０ Alkenil, C １ ~C １０ Alkyloxy, C １ ~C １０ Alkylamino, C １ ~C １０ Alkylthio, C ２ ~C １０ Acyloxy, C ２ ~C １０ Acylamino, C ２ ~C １０ Acyrchio, C １ ~C １０ Alkyl carbonate, C １ ~C １０ Alkylcarbamate, C １ ~C １０ Carbamide, aryloxy, arylthio, cyano, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, C ４ ~C １０ Heterocycloamino, C ６ ~C １０ The compound according to any one of claims 1 to 6, which is an aryl or π-excess heteroaryl.

8. The compound according to any one of claims 1 to 7, wherein each of Z and J is S if present.

9. If present, R １４ , R １６ , R １７ and R ２０ Each of them is hydrogen, and R １８ However, C １ ~C １０ Alkyloxy, halogen, cyano, or C １ ~C １０ C may be substituted with alkyloxy, halogen, or cyano. ６ ~C １０ The compound according to any one of claims 1 to 8, wherein it is an aryl compound.

10. The compound according to any one of claims 1 to 9, wherein G is β-galactosyl, β-glucosyl, β-glucuronyl, 2,4-dinitrobenzenesulfonyl, 3,4,6-trimethyl-2,5-dioxobenzyl, 4-azidobenzyloxy, tert-butyldimethylsilyl, acetyl, pivaloyl, peptidase-cleavable group, 4-aminobenzyl, 4-(alkylamino)benzyl, 4-oxybenzyl, 4-(oxymethyl)benzyl, oxymethyl, aminomethyl, or alkylaminomethyl.

11. A composition comprising a compound according to any one of claims 1 to 10, wherein the composition is aqueous.

12. The composition according to claim 11, wherein the composition substantially does not contain a surfactant-based luminescence enhancer or a surfactant, and exhibits a peak luminescence intensity of more than 1,000 photons / second at 37°C when treated with a buffer solution at pH 9.

7.

13. A method for detecting an analyte in a sample, comprising contacting the sample with a compound according to any one of claims 1 to 10, and monitoring the sample for luminescence.

14. The method according to claim 13, further comprising measuring the intensity of the luminescence obtained and correlating the intensity with the presence of the analyte.

15. The method according to claim 14, wherein the analyte is alkaline phosphatase, hydrogen peroxide, glutathione, fluoride, or a base.

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