Rapid, high-intensity chemiluminescence in dioxane

Abstract

Described herein are 1,2-dioxetanes that are useful as chemiluminescent probes, diagnostic agents, and imaging agents. Also described herein are compositions containing such compounds and methods of using them.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 62 / 926,985, filed October 28, 2019, which is incorporated by reference as if fully set forth herein. [Background technology]

[0002] Chemiluminescent dioxetanes are strained cyclic peroxides that can undergo rapid decomposition to generate excited transient species that then decay to the ground state via the emission of light.

[0003] Such compounds are useful as luminescent probes in a variety of assays, including enzyme activity assays, immunoassays, and DNA detection assays. Chemiluminescence-based assays can offer superior sensitivity because, unlike fluorescence- and absorbance-based assays, no light excitation is required.

[0004] Dioxanes can be generated in situ at the time of their use or prepared in advance in a stable form and then activated later. When generated in situ via the oxidation of a precursor alkene, chemiluminescent dioxetanes can also function as a method for detecting or imaging reactive oxygen species (ROS). An example of a stable chemiluminescent dioxetane is 4-methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2'-adamantane]. This compound, also known as LUMIGEN® PPD, can be activated upon treatment with alkaline phosphatase (ALP). ALP is an enzyme that catalyzes the hydrolysis of phosphate groups. Upon activation, the resulting compound subsequently undergoes fragmentation of the 1,2-dioxatane ring, releasing light and thus functioning as a luminescent probe in alkaline phosphatase-labeled assays.

[0005] Dioxane compounds have been developed that exhibit high sensitivity and strong luminescence under non-aqueous conditions. However, these compounds exhibit weak luminescence in aqueous media and require a long time to reach maximum luminescence after contact with the desired analyte. To amplify the weak luminescence in aqueous environments, surfactant-based luminescence enhancers have been added to dioxetane probes, but the use of such enhancers is neither desirable nor suitable for various applications. Summary of the Invention

[0006] There is a need for dioxetanes that respond rapidly to the presence of an analyte. There is also a need for dioxetanes that are highly luminescent and suitable for use in aqueous environments without the need for surfactant-based enhancers. 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 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 Alkynyl, C1-C 10Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Trialkylammonium salts, C1-C 10 Alkylthio, C2-C 10 Acyl, C1-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 Alkylsulfinyl, C1-C 10 Alkyl sulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 Alkylphosphonates, C1-C 10 Alkyl phosphinates, C1-C 10 Trialkylphosphonium salts, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl; R 4 , R 5 , R 6 and R 7 At least one of them is 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 novel fluorophore having a peak emission intensity of more than 1000 photons / second and a T of less than 3 minutes at 37°C when treated with a pH 9.7 buffer. 1 / 2 Also provided is an aqueous composition comprising one or more chemiluminescent dioxetane compounds having the formula:

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

[0015] Advantages, some of which are unexpected, are achieved by various embodiments of the present disclosure. Various compounds described herein can advantageously provide rapid, high-intensity luminescent signals in non-aqueous media, aqueous media, or both. An important advantage is that assays containing such compounds can be performed more quickly than assays with compounds lacking the characteristics of the compounds described herein. Furthermore, compounds of the present disclosure provide increased intensity of luminescence, including in aqueous media. Another advantage of the present compounds is that their aqueous compositions do not require surfactant-based luminescence enhancers. Due to such advantageous properties, various embodiments of the present disclosure can provide methods or kits capable of detecting analytes 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. [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 part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the scope of the claims to the disclosed subject matter.

[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 together with the carbons to which they are attached, C5 to C 10 Cycloalkyl rings, for example, monocyclic, bicyclic, or tricyclic rings, are provided. R 1 and R 2 can be substituted or unsubstituted. In various embodiments, R 1 and R 2 are linked together with the carbons to which they are attached to provide a spirocyclic bridged bicyclo or tricyclo group. For example, R 1 and R 2 together with the carbon to which they are attached can be a spirocyclic adamantane, norbornane, or bornane.

[0038] R 3 is C1~C 10 Alkyl, C6-C 10 aryl, or heteroaryl, each of which is optionally substituted. R 3 can be substituted or unsubstituted. For example, R 3 is unsubstituted C1 to C 10 Alkyl or C1-C substituted with one or more halogen, hydroxy, amino, thio, alkoxy, alkylamino, alkylthio, sulfate, or carboxylate 10 It can be alkyl.

[0039] R 4 , R 5 , R6 and R 7 each independently represents H, Q, X, hydroxy, halogen, amino, thio, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C1-C 10 Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Trialkylammonium salts, C1-C 10 Alkylthio, C2-C 10 Acyl, C1-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 Alkylsulfinyl, C1-C 10 Alkyl sulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 Alkylphosphonates, C1-C 10 Alkyl phosphinates, C1-C 10 Trialkylphosphonium salts, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl, and R 4 , R 5 , R 6 and R 7 At least one of the R is Q. 4 , R 5 , R 6 and R 7Each 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, halogen, 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 carbonates, C1-C 10 Alkyl carbamates, C1-C 10 carbamido, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, and heteroarylamino; R 4 , R 5 , R 6 and R 7 At least one of them is Q.

[0041] In various further embodiments, R 4 , R 5 , R 6 and R 7 provide a net electron-donating effect to the aromatic ring to which they are attached. For example, X, Q, and R 4 , R 5 , R 6 and R 7 The aromatic rings to which Q and R are bonded are 4 , R 5 , R 6 and R 7 is electron-rich compared to the otherwise identical compound where

[0042] In some embodiments, R 4 , R 5 , R 6 and R 7 Exactly one, two, or three of the4 , R 5 , R 6 and R 7 In some further such embodiments, exactly one, two, or three of R are Q, or any combination thereof. 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 electron-donating group. In various embodiments, Q is a C2-C 10 Alkenyl, C2-C 10 Heterocycloalkenyl, C6-C 10 aryl, or heteroaryl. Q can be substituted or unsubstituted. In some embodiments, Q is C2-C 10 When Q is alkenyl, it is substituted with one or more electron donating groups, contains no electron withdrawing groups, or both. 10 When alkenyl, the vinyl and allylic positions, if present, are unsubstituted in some further embodiments. For example, Q can be an unsubstituted vinyl. In some embodiments, Q is a C-C 10When Q is aryl, it is substituted with one or more electron-donating groups, does not contain electron-withdrawing groups, or both. For example, Q can be unsubstituted phenyl, phenyl substituted with one or more electron-donating groups, or phenyl substituted with one or more substituents selected from the group consisting of electron-donating substituents. As another example, Q can be vinyl or phenyl substituted with a substituent such that the net effect of the substituent is electron-donating. In a further example, Q is a π-excess heteroaryl such as thiophenyl, furanyl, pyrrolyl, benzothiophenyl, benzofuranyl, or indolyl. In certain embodiments, Q is substituted or unsubstituted thiophen-2-yl or thiophen-3-yl.

[0044] X is an -OH, -OG, -O' salt, or boronate group. In various embodiments, X is a group that generates an oxyanion upon chemical or enzymatic trigger. When X is a boronate 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 alkyl or R 8 and R 9 together with the boronates to which they are bonded, C2 to C 10 It is a cyclic boronic ester. For example, X can be 4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl or -B(OH)2.

[0046] In various embodiments, X is -OG, and G is an alcohol protecting group, an analyte-responsive group, or both. For example, G can be trialkylsilyl, alkylarylsilyl, arylsulfonyl, dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranuronyl, furanosyl, acyl, benzoyl, or benzyl. In some embodiments, G is pyranosyl or pyranuronyl, such as galactosyl, glucosyl, or glucuronyl. In further such embodiments, G is β-galactosyl, β-glucosyl, or β-glucuronyl. G can also be a phosphorus-containing group, such as phosphate or phosphonate. For example, G can be -PO3H2 or a salt or ester thereof. In further embodiments, G is 2,4-dinitrobenzenesulfonyl, 3,4,6-trimethyl-2,5-dioxobenzyl, 4-azidobenzyloxy, tert-butyldimethylsilyl, acetyl, pivaloyl, or an enzyme-cleavable moiety. For example, G can be a phosphatase-cleavable moiety or a peptidase-cleavable moiety. G can also comprise a bivalent fragmentable linker having a pendant protecting group such that removal of the pendant protecting group causes fragmentation of the linker and removal of the protecting group G. Thus, G can contain a divalent fragmentable linker such as 4-aminobenzyl, 4-(alkylamino)benzyl, 4-oxybenzyl, 4-(oxymethyl)benzyl, oxymethyl, aminomethyl, alkylaminomethyl, and the like, along with a terminal protecting group such as a trialkylsilyl, alkylarylsilyl, arylbenzenesulfonyl, dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranuronyl, furanosyl, acyl, benzoyl, 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 10 Alkyl, 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 10Alkynyl, C1-C 10 Alkyloxy, C1-C 10 Alkylamino, C1-C 10 Trialkylammonium salts, C1-C 10 Alkylthio, C2-C 10 Acyl, C1-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 Alkylsulfinyl, C1-C 10 Alkyl sulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 Alkylphosphonates, C1-C 10 Alkyl phosphinates, C1-C 10 Trialkylphosphonium salts, C4-C 10 Heterocycloamino, 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 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, C6-C 10 In some embodiments, R 12 and R 13 are independently H, C1 to C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10aryl, 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 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 10 In some further embodiments, R 12 and R 13 are independently H, C1 to C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 It is an aryl or a π-excess heteroaryl.

[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-C 10 Alkyl carbonates, C1-C 10 Alkyl carbamates, C1-C 10 Carbamide, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl. In some embodiments, R 14 H, C1~C 10 Alkyl, C2-C 10 Alkenyl, C6-C10 It is an aryl or a π-excess heteroaryl.

[0055] R 12 , R 13 and R 14 together have a net electron-donating effect on the phenyl ring to which X is attached. For example, the aromatic ring to which X is attached is R 12 , R 13 and R 14 is electron-rich compared to the otherwise identical compound where

[0056] In various embodiments, R 12 , R 13 and R 14 At least one or two of are 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-C 10 Alkylamino, C1-C 10 Trialkylammonium salts, C1-C 10 Alkylthio, C2-C 10 Acyl, C1-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-C10 Carbamide, aryloxy, C1-C 10 Alkylsulfinyl, C1-C 10 Alkyl sulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 Alkylphosphonates, C1-C 10 Alkyl phosphinates, C1-C 10 Trialkylphosphonium salts, C4-C 10 Heterocycloamino, 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 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, C6-C 10 In some embodiments, R 12 and R 13 are independently H, C1 to C 10 Alkyl, C2-C 10 Alkenyl, 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 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, 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 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, C6-C 10 In some further embodiments, R 12 and R13 are independently H, C1 to C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 It is an aryl or a π-excess heteroaryl.

[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-C 10 Alkyl carbonates, C1-C 10 Alkyl carbamates, C1-C 10 Carbamide, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl. In some embodiments, R 14 H, C1~C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 It is an aryl or a π-excess heteroaryl.

[0060] R 12 , R 13 and R 14 together have a net electron-donating effect on the phenyl ring to which X is attached. For example, the aromatic ring to which X is attached is R 12 , R 13 and R 14 is electron-rich compared to the otherwise identical compound where

[0061] In various embodiments, R 12 , R 13 and R 14 At least one or two of are 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, or arylsulfonyl; R 14 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 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 Trialkylammonium salts, C1-C 10 Alkylthio, C2-C 10 Acyl, C1-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 10Alkyl carbamates, C1-C 10 Carbamide, aryloxy, C1-C 10 Alkylsulfinyl, C1-C 10 Alkyl sulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 Alkylphosphonates, C1-C 10 Alkyl phosphinates, C1-C 10 Trialkylphosphonium salts, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl, or R 14 , R 16 , R 17 , R 18 , and R 20 Any two of these, together with the carbons to which they are attached, form C5 to C 10 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, C6-C 10 An aryl or a π-excess heteroaryl is provided.

[0065] In some embodiments, R 14 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 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-C 10 Acylthio, C1-C 10Alkyl carbonates, C1-C 10 Alkyl carbamates, C1-C 10 Carbamide, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, C4-C 10 Heterocycloamino, C6-C 10 aryl, or π-excess heteroaryl, or R 14 , R 16 , R 17 , R 18 , and R 20 Any two of these, together with the carbons to which they are attached, form C5 to C 10 Cycloalkenyl, C2-C 10 Heterocycloalkenyl, 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 R is an electron donating group. 14 , R 16 , R 18 and R 20 Each of, if present, is hydrogen.

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

[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 compositions comprising one or more of the compounds described herein, their olefin precursors, or salts thereof, which upon treatment with an analyte, an oxidizing agent, alkaline phosphatase, or photooxidative conditions provide any of the compounds described herein (e.g., compounds of Formulas I, II, IIa, III, IIIa, IIIb, and IV-XV).

[0071] The composition may be an aqueous composition or a non-aqueous composition. The composition may be a mixture of both aqueous and non-aqueous solvents. In various embodiments, the composition is substantially free of surfactant-based luminescence enhancers, surfactants, or both. For example, the composition may be substantially free of surfactants having a tail group that is an acyclic alkyl group (e.g., an acyclic group of at least 8 carbons) or an aromatic group (e.g., an aromatic group containing at least 6 carbons) and one or more of a quaternary ammonium salt, a pyridinium salt, a quaternary phosphonium surfactant salt, an ethylene glycol, or a fluorescein head group. As further examples, the compositions may be substantially free of cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'-tributylphosphonium-p-xylene chloride, poly(vinylbenzyltrioctylphosphonium chloride) (TBE), poly(vinylbenzyltrioctylphosphonium chloride), Triton X-100, Tween surfactants, surfactants with long alkyl chains having a polyethylene glycol head, poly(vinylbenzyltrioctylphosphonium chloride), Brij® surfactants, IGEPAL® surfactants, octylphenoxypolyethoxyethanol, and the like. The composition may be free of fluorescein-containing compounds such as N-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthan]-5-yl)tetradecanamide and other fluorescein-containing surfactants.

[0072] In various embodiments, the composition contains a buffer. The buffer can be, but is not necessarily, an alkaline or amine-based buffer. An exemplary amine-based buffer is 221 buffer, available from Sigma-Aldrich (St. Louis, MO). Similarly, the composition need not necessarily have a basic pH. For example, the composition can have a pH of about 4-12, about 5-12, about 6-12, about 7-12, about 8-12, about 9-12, about 10-12, about 4-11, about 4-10, about 4-9, about 4 to about 8, about 4-7, about 4-6, or about 4-5. The pH of the composition can be selected based on whether it will emit light immediately upon removal of the analyte trigger, as in the case of an alkaline pH value, or whether the pH of the composition will be acidic so that it will emit light upon treatment with a base.

[0073] In various embodiments, the composition has a peak luminescence intensity of greater than 1,000 photons / second and a T of 3 minutes or less at 37° C. when treated with a pH 9.7 buffer. 1 / 2 In various examples, the peak emission intensity can be greater than about 2,000 photons / second, greater than about 3,000 photons / second, greater than about 4,000 photons / second, greater than about 5,000 photons / second, greater than about 6,000 photons / second, greater than about 7,000 photons / second, greater than about 8,000 photons / second, greater than about 9,000 photons / second, or greater than about 10,000 photons / second, and T 1 / 2 The T may be about 3 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 10 seconds or less. For example, the present disclosure provides a T of about 3 minutes or less at 37° C. when treated with a pH 9.7 buffer solution, with a peak emission intensity of more than 1000 photons / second. 1 / 2 wherein the composition is substantially free of surfactant-based emission enhancers.

[0074] The present disclosure also provides methods for detecting an analyte in a sample, comprising contacting the sample with one or more of the compounds described herein, their olefin precursors, their salts, or compositions comprising them, and then monitoring the sample for luminescence. In various embodiments, the method involves measuring the intensity of the resulting luminescence and correlating the intensity to the presence of the analyte.

[0075] In some embodiments, the method further involves increasing the pH of the sample. For example, 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 detected in about 3 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 10 seconds or less. For example, the sample can be monitored for about 3 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 10 seconds or less.

[0077] The analyte can be any substance that generates any compound described herein (e.g., compounds of Formulas I, II, IIa, III, IIa, IIIb, and IV-XV) from the composition, where X is an oxyanion. For example, in various embodiments, the analyte can be alkaline phosphatase, peptidase, glucosidase, an oxidizing agent such as hydrogen peroxide or other reactive oxygen species, 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 herein, their olefin precursors, their salts, or compositions comprising them. The kit can include instructions according to the methods described herein.

[0079] The compounds and compositions described herein can be directly triggered by the analyte to produce a signal that identifies the presence of the analyte and probe, or can be triggered in a two-step process, one step involving contacting the analyte and another step involving increasing the pH.

[0080] The compounds of the present disclosure can be configured as probes for detecting a variety of different analytes by modifying the X or G groups. For example, the compounds described herein can be configured to detect β-galactosidase by providing a glucosyl group at G, hydrogen peroxide or other oxidizing agents by providing a boronic ester at X, alkaline phosphatase by providing a phosphate at X or a phosphoryl group at G, glutathione by providing a dinitrobenzenesulfonaminobenzyl group at G, fluorine by providing a trialkylsilyl group at G, and alkaline conditions when X is OH.

[0081] It is desirable for the compound to emit all possible light in the shortest possible time when induced by the analyte to provide the strongest possible signal. If the chemiluminescence is released gradually over a period of time, the light intensity (photons / second) may decrease, compromising detection sensitivity. An example of a 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 luminescence half-life (T 1 / 2 ) can be explained by either

[0083] The compounds described herein can be used as enzyme substrates. For example, various compounds in which G is a phosphorus-containing group can be used as substrates for alkaline phosphatase (ALP) enzymes, etc. Without being limited by theory, one exemplary mechanism involves the ALP enzyme hydrolyzing the phosphorus-containing group to provide a phenol that is immediately deprotonated due to the alkaline environment of the solution (e.g., pH 9.7 buffer). With the generation of an oxyanion, the 1,2-dioxetane decomposes into two compounds: 2-adamantanone and a phenyl ester in an excited state. The excited phenyl ester then immediately decays to the ground state by emitting light.

[0084] The resulting light intensity is a linear function of the amount of enzyme. Thus, the compounds described herein can be used to detect labeled enzymes used in assays. For example, the steps in the chemical process by which a dioxetane provides light are the following: (i) X + S → X + S' (ii) S' → P * and (iii) p * →p + light. Step (i) represents the catalytic turnover of the substrate, X is an enzyme or other component that converts the substrate to its activated form, step (ii) represents the decomposition of the activated substrate into transient excited species, and step (iii) represents the decay of the excited species to the ground state and emission of light. The light intensity is a function of the catalytic turnover of the substrate in step (i) and the resulting photogenerated compound P in step (ii). * Step (ii) is usually a linear function with a rate constant k and its half-life: T 1 / 2 = (In 2) / k. Step (iii) 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 includes an initial rise period of luminescence intensity followed by a period of steady-state intensity. S' → P * The slow first-order reaction of S' corresponds to a longer rise time because it takes longer for the concentration of S' to reach a steady state. * A fast reaction corresponds to a shorter initial rise period and therefore provides a rapid rise. In the case of an enzymatic chemiluminescent reaction, the intensity typically plateaus at a high level, and the resulting signal corresponds to the signal shape shown in Figure 5. The absence of a steady-state intensity indicates either substrate depletion or subsequent inactivation of the enzyme. While detection of enzyme-generated chemiluminescence offers flexibility in the measurement process, as the light intensity at any time can be related to the amount of enzyme, enzyme-generated processes can have drawbacks due, for example, to the size and "sticky" nature of the enzyme label. However, to maximize sensitivity, it is desirable to maintain a maximum intensity (I) during the period of steady-state intensity. max ) or nearby.

[0086] The compounds described herein can also be used as direct labels for one of the complementary binding partners in immunoassays. The compounds described herein are advantageously used as labels because they are small molecules, as opposed to large bioluminescent molecules and other types of enzyme labels.

[0087] Thus, the present disclosure also relates to assays that utilize the compounds described herein as chemiluminescent probes.

[0088] In various embodiments, the assay can be a homogeneous (non-separation) assay, in which bound and unbound ligands do not need to be separated, or the assay can be a heterogeneous assay, in which the labeled binding pair complex is separated from the unbound labeled reactants. The assay can be configured to be performed manually or can be automated and performed robotically. The assay can be performed in a test tube, cuvette, microwell, or combination thereof. In various embodiments, the test tube, cuvette, microwell, or other container in which the assay is performed is at least partially opaque, completely opaque, black, white, or a combination thereof.

[0089] Assays can be performed on immobilized proteins in Western blots, or on immobilized nucleic acids in Southern or Northern blots.

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

[0091] Chemiluminescence measurements have advantages over fluorescence and absorption spectroscopy, for example, which can suffer from interfering signals generated from either the incident light or background signals.

[0092] The assays described herein can be configured to measure chemiluminescence according to non-limiting examples set forth in J.E. Wampler, "Instrumentation of the Light and Measuring It," in Chemi- and Bioluminescence, J.G. Burr, ed., Marcel Dekker, New York, 1-44 (1985), A.K. Campbell, "Detection and Quantification of Chemiluminescence," in "Chemiluminescence Principles and Applications in Biology and Medicine," Ellis Norwood, Chichester, 68-126 (1988), F. Berthold, "Instrumentation for Chemiluminescence Immunoassays," in "Luminescence Immunoassay and Molecular Applications," K. Van Dyke and R. Van Dyke, eds., CRC Press, Boca Raton, 11-25 (1990), and T. Nieman, "Chemiluminescence: Theory and Instrumentation, Overview," in "Encyclopedia of Analytical Science," Academic Press, 1990. Press, Orlando, 608-613 (1995), each of which is incorporated herein by reference in its entirety.

[0093] Several approaches can be used to attach the compounds of the present disclosure to biological molecules. For example, if the compound contains a reactive group, such as carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxamide, carboxim, or N-succinimidyl carboxy, such a group can be covalently attached to a hydroxyl or amino functional group using a conjugation reagent, such as carbodiimide or 1,1-carbonyldiimidazole. N-maleimide groups react directly with sulfhydryl residues in proteins. If the compound contains aromatic amino groups, they can be converted to diazonium salts and reacted with phenolic groups, such as those found in tyrosine groups in proteins. The compounds of the present invention can be attached to biological molecules using either a polycyclic aromatic moiety or other photogenerating group, or a reactive group present in a leaving group.

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

[0095] 2-Adamantanone and trisubstituted benzoates can be coupled together by subjecting them to McMurry reaction conditions involving oxophilic titanium and a reducing agent. 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 may then be subjected to photooxygenation conditions to provide the 1,2-dioxetane product. In various embodiments, R 3 , R 4 , R 5 , R 6 , R 7 , R 10 , R 11, G, and X are as described in any of the various embodiments of the present application, for example, as described according to Example 1. In some embodiments, R 4 , R 5 , R 6 , R 10 and R 11 is H. In some embodiments, 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 rate of light emission in photons / second. Intensity can be measured through the use of a luminometer. A luminometer is a light detector in a housing that filters out ambient light. Any suitable luminometer can be used, including photomultiplier tubes and photodiodes.

[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 lowest level at which the signal of the analyte or product being measured can be reproducibly detected.

[0099] As used herein, the term "alkyl" refers to a substituted or unsubstituted straight-chain, branched-chain, or cyclic, saturated monovalent or divalent group having 1 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to about 10 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 1 to 6 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 1 to 3 carbon atoms. Examples of straight-chain monovalent groups include (C1-C 20 Examples of branched monovalent (C1-C3) alkyl groups include those having 1 to 8 carbon atoms, such as methyl (i.e., CH3), ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. 20)-alkyl groups include isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, and isopentyl. Examples of straight chain divalent (C1-C 20)-Alkyl groups include those having 1 to 6 carbon atoms, such as -CH-, -CHCH-, -CHCHCH-, -CHCHCHCH-, -CHCHCHCHCH-, and -CHCHCHCHCHCH. Examples of branched divalent alkyl groups include -CH(CH)CH- and -CHCH(CH)CH. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, and adamantyl. Cycloalkyl groups further include fused rings, including, but not limited to, substituted and unsubstituted polycyclic cycloalkyl groups such as norbornyl, adamantyl, bornyl, camphanyl, isocambenyl, and carnyl groups, as well as decalinyl. For example, cycloalkyl includes one, two, three, four, or more substituents, such as adamantyl, substituted at the tertiary bridgehead positions of the methylene bridge. In some embodiments, alkyl includes a combination of substituted and unsubstituted alkyl. As an example, alkyl, also referred to as (C1)alkyl, includes methyl and substituted methyl. As a specific example, (C1)alkyl includes benzyl. As a further example, alkyl can include methyl and substituted (C2-C8)alkyl. Alkyl can also include substituted methyl and unsubstituted (C2-C8)alkyl. In some embodiments, alkyl can be methyl and C2-C8 straight chain alkyl. In some embodiments, alkyl can be methyl and substituted (C2-C8)alkyl. The term methyl is understood to mean unsubstituted -CH3. The term methylene is understood to mean unsubstituted -CH2. For comparison, the term (C1)alkyl is understood to mean substituted or unsubstituted -CH3 or substituted or unsubstituted -CH2-. Representative substituted alkyl groups may be substituted one or more times with any of the groups enumerated herein, such as cycloalkyl, heterocyclyl, aryl, amino, haloalkyl, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.As a further example, representative substituted alkyl groups may be substituted with one or more of fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. In some embodiments, representative substituted alkyl groups may be substituted with a group including amino, hydroxy, cyano, carboxy, nitro, thio, and alkoxy, but not including halogen groups. Thus, in some embodiments, alkyl may be substituted with a non-halogen group. For example, representative substituted alkyl groups may be substituted with a fluoro group substituted with a halogen other than bromo, or with a bromo group substituted with a halogen other than fluoro. In some embodiments, representative substituted alkyl groups can be substituted with one, two, three or more fluoro groups, or they can be substituted with one, two, three or more non-fluoro groups. For example, alkyl can be trifluoromethyl, difluoromethyl, or fluoromethyl, or alkyl can be substituted with alkyl other than trifluoromethyl, difluoromethyl, or fluoromethyl. Alkyl can be haloalkyl or alkyl, and can be substituted alkyl other than haloalkyl.

[0100] As used herein, the term "alkenyl" refers to a substituted or unsubstituted, linear, branched, or cyclic, saturated monovalent or divalent group having at least one carbon-carbon double bond 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, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. The double bond may be in the trans or cis orientation. The double bond may be terminal or internal. The alkenyl group may be bonded through a portion of the alkenyl group that contains a double bond, such as vinyl, propen-1-yl, and but-1-yl, or the alkenyl group may be bonded through a portion of the alkenyl group that does not contain a double bond, such as penten-4-yl. Where specified, it should be understood that the parent moiety is attached to the alkenyl group at the vinylic position of the double bond, rather than at the non-vinylic position. For example, if an aromatic ring is substituted with a π-conjugated alkenyl group, it should be understood to be substituted at the vinylic position, rather than at the non-vinylic position. As a further example, an aromatic ring substituted with a π-conjugated propenyl group would be understood to be a propen-1-yl or prop-2-ene group, rather than a propen-3-yl group. Monovalent examples (C2-C 20 Examples of branched monovalent (C2-C3)-alkenyl groups include those having 1 to 8 carbon atoms, such as vinyl, propenyl, propen-1-yl, prop-2-yl, butenyl, butenyl-1-yl, buton-2-yl, sec-buten-1-yl, sec-buten-3-yl, pentenyl, hexenyl, heptenyl, and octenyl groups. 20 )-alkenyl groups include isopropenyl, iso-butenyl, sec-butenyl, t-butenyl, neopentenyl, and isopentenyl. Examples of straight chain divalent (C-C 20Alkenyl groups include those having 2 to 6 carbon atoms, such as -CHCH-, -CHCHCH2-, -CHCHCH2CH2-, and -CHCHCH2CH2CH2-. Examples of branched divalent alkyl groups include -C(CH3)CH- and -CHC(CH3)CH2. Examples of cyclic alkenyl groups include cyclopentenyl, cyclohexenyl, and cyclooctenyl. For example, alkenyl can be vinyl and substituted vinyl. For example, alkenyl can be vinyl and substituted (C3-C6) alkenyl. Alkenyl can also include substituted vinyl and unsubstituted (C3-C6) alkenyl. Representative substituted alkenyl groups can be substituted one or more times with any of the groups enumerated herein, such as monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio, alkoxy, and halogen groups. As a further example, representative substituted alkenyl groups may be substituted with one or more of fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. In some embodiments, representative substituted alkenyl groups may be substituted with a group that includes monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio, and alkoxy, but does not include halogen groups. Thus, in some embodiments, alkenyl may be substituted with a non-halogen group. In some embodiments, representative substituted alkenyl groups may be substituted with a fluoro group substituted with a halogen other than bromo, or with a bromo group substituted with a halogen other than fluoro.For example, alkenyl can be 1-fluorovinyl, 2-fluorovinyl, 1,2-difluorovinyl, 1,2,2-trifluorovinyl, 2,2-difluorovinyl, trifluoropropen-2-yl, 3,3,3-trifluoropropenyl, 1-fluoropropenyl, 1-chlorovinyl, 2-chlorovinyl, 1,2-dichlorovinyl, 1,2,2-trichlorovinyl, or 2,2-dichlorovinyl. In some embodiments, representative substituted alkenyl groups can be substituted with one, two, three, or more fluoro groups, or they can be substituted with one, two, three, or more non-fluoro groups.

[0101] As used herein, the term "alkynyl" refers to substituted or unsubstituted straight- and branched-chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have 2 to 50 carbon atoms, 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, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples include, but are not limited to, ethynyl, propynyl, propyn-1-yl, propyn-2-yl, butynyl, butyn-1-yl, butyn-2-yl, butyn-3-yl, butyn-4-yl, pentynyl, pentyn-1-yl, and hexynyl. Examples include, but are not limited to, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3), among others.

[0102] The term "aryl," as used herein, refers to a substituted or unsubstituted monovalent group derived by removing a hydrogen atom from an arene, which is a cyclic aromatic hydrocarbon 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. (C6-C 20Examples of aryl groups include phenyl, naphthalenyl, azulenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthenyl, chrysenyl, and anthracenyl groups. Examples include substituted phenyl, substituted naphthalenyl, substituted azulenyl, substituted biphenyl, substituted indacenyl, substituted fluorenyl, substituted phenanthrenyl, substituted triphenylenyl, substituted pyrenyl, substituted naphthenyl, substituted chrysenyl, and substituted anthracenyl groups. Examples also include unsubstituted phenyl, unsubstituted naphthalenyl, unsubstituted azulenyl, unsubstituted biphenyl, unsubstituted indacenyl, unsubstituted fluorenyl, unsubstituted phenanthrenyl, unsubstituted triphenylenyl, unsubstituted pyrenyl, unsubstituted naphthenyl, unsubstituted chrysenyl, and unsubstituted anthracenyl groups. Aryl also includes phenyl and non-phenylaryl groups. From these examples, (C6-C 20 The term aryl includes monocyclic and polycyclic (C6-C 20 ) aryl groups, including fused and non-fused polycyclic (C6-C 20 ) aryl groups.

[0103] As used herein, the term "heterocyclyl" refers to substituted aromatic, unsubstituted aromatic, substituted nonaromatic, and unsubstituted nonaromatic rings containing three or more atoms in the ring, 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 is therefore a subset of the term heterocyclyl. The term "heterocycloalkenyl" refers to a heterocyclyl group that contains an olefin in the non-aromatic ring such that the olefin is the point of attachment to the parent moiety. Thus, a heterocyclyl group can be a heterocycloalkyl, heterocycloalkenyl, or heteroaryl, or, if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups contain from 3 to about 20 ring members, while other such groups have from 3 to about 15 ring members. In some embodiments, heterocyclyl groups include heterocyclyl groups containing 3 to 8 carbon atoms (C3-C6), 3 to 6 carbon atoms (C3-C6), or 6 to 8 carbon atoms (C6-C6). A heterocyclyl group designated as a C2-heterocyclyl can be a 5-membered ring having 2 carbon atoms and 3 heteroatoms, a 6-membered ring having 2 carbon atoms and 4 heteroatoms, etc. Similarly, a C4-heterocyclyl can be a 5-membered ring having 1 heteroatom, a 6-membered ring having 2 heteroatoms, etc. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also contain one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The term heterocyclyl group includes fused ring species, including those containing fused aromatic and non-aromatic groups. Representative heterocyclyl groups include, but are not limited to, piperidinyl, pyrrolidinyl, piperazinyl, and morpholinyl. For example, the heterocyclyl group is [ka] In particular, the formula includes, but is not limited to, 1 is H, (C1~C 20 ) Alkyl, (C6-C 20) represents an aryl or amine protecting group (e.g., a t-butyloxycarbonyl group), and the heterocyclyl group can be substituted or unsubstituted. Representative heteroaryl groups include furanyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, imidazolyl, triazinyl, tetrazolyl, benzoxazolinyl, and benzimidazolinyl groups. In some embodiments, the heteroaryl is a 5-membered heteroaryl. In some embodiments, the heteroaryl is other than pyridine, pyrimidine, pyridazine, pyrazine, or a fused derivative thereof. A π-excess heteroaryl is a heteroaryl that is electron-rich so that it can function as an electron-donating group. Examples of π-excess heteroaryls are furan, thiophene, indole, pyrrole, benzofuran, and benzothiophene.

[0104] As used herein, the term "alkoxy" refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as defined herein. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can contain from 1 to about 12 to 20 or about 12 to 40 carbon atoms bonded to the oxygen atom, and can further contain double or triple bonds and heteroatoms. Thus, alkoxy also includes an oxygen atom bonded to an alkylenyl group and an oxygen atom connected to an alkynyl group. For example, an allyloxy group is an alkoxy group within the meaning of this specification. A methoxyethoxy group is also an alkoxy group within the meaning of the present specification, which is a methylenedioxy group in the situation where two adjacent atoms of the structure are substituted therewith.

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

[0106] The term "arylcarbonyl," as used herein, refers to a carbonyl (CO) group attached to an aryl group, as defined herein. The point of substitution onto the parent moiety is the carbonyl group.

[0107] The term "heteroarylcarbonyl," as used herein, refers to a carbonyl (CO) group attached to a heteroaryl group, as defined herein. The point of substitution onto the parent moiety is the carbonyl group.

[0108] As used herein, the term "arylalkyl" refers to an alkyl group, as defined herein, in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to an aryl group, as defined herein. Representative aralkyl groups include benzyl, biphenylmethyl, and phenylethyl groups, and fused (cycloalkylaryl) alkyl groups such as 4-ethyl-indanyl. An alkenyl group is an alkenyl group, as defined herein, in which a hydrogen or carbon bond of the alkenyl group is replaced with a bond to an aryl group, as defined herein. The point of substitution on the parent moiety is the alkyl group.

[0109] The terms "halo," "halogen," or "halide" group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0110] As used herein, the term "amino" refers to -NH2, -NHR, -NR2, -NR3, where each R is independently selected. + Substituents of the form -NR3, and -NR3 which cannot be protonated +" refers to the protonated form of each, excluding: ". Thus, any compound substituted with an amino group can be considered an amine. An "amino group" within the meaning of this specification can be a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group includes monoalkylamino, dialkylamino, and trialkylamino groups.

[0111] As used herein, the term "acyl" refers to a group containing a carbonyl moiety, wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is in turn bonded to another carbon atom, which may be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl, group, etc.

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

[0113] The term "alkoxycarbonyl" as used herein refers to a group containing a carbonyl moiety, wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkyl group. Alkoxycarbonyl also includes groups in which the carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkylenyl group. Alkoxycarbonyl also includes groups in which the carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkynyl group. In further cases included in the definition of alkoxycarbonyl and included in the term "aryloxycarbonyl," as defined herein, the carbonyl carbon atom is bonded to an oxygen atom which is bonded to an aryl group instead of an alkyl group.

[0114] The term "alkylamide" as used herein refers to a group containing a carbonyl moiety, which is bonded via a carbonyl carbon atom. The carbonyl carbon atom is also bonded to a nitrogen group bonded to one or more alkyl groups. In a further case that is also an alkylamide as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom bonded to one or more aryl groups instead of, or in addition to, one or more alkyl groups. In a further case that is also an alkylamide as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom bonded to one or more alkenyl groups instead of, or in addition to, one or more alkyl and / or aryl groups. In a further case that is also an alkylamide as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom bonded to one or more alkynyl groups instead of, or in addition to, one or more alkyl, alkenyl, and / or aryl groups.

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

[0116] The term "alkylthio," as used herein, refers to a sulfur atom attached to an alkyl, alkenyl, or alkynyl group, as defined herein. The point of substitution onto the parent moiety is the sulfur atom.

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

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

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

[0120] As used herein, the term "dialkylaminosulfonyl" refers to a sulfonyl group, as defined herein, attached to a nitrogen atom which is further attached to two alkyl groups, optionally joined together to form a ring with the nitrogen atom. This term also includes groups in which the nitrogen atom is further attached to one or two alkenyl groups instead of an alkyl group. The point of substitution on the parent moiety is the sulfonyl group.

[0121] The term "dialkylamino," as used herein, refers to an amino group connected to two alkyl groups, as defined herein, which may optionally be joined together to form a ring with the nitrogen. This term also includes groups in which the nitrogen is further connected to one or two alkenyl groups instead of an alkyl group. The point of substitution into the parent moiety is the nitrogen atom.

[0122] The term "dialkylamide," as used herein, refers to an amide group connected to two alkyl groups, as defined herein, optionally joined together to form a ring with the nitrogen. This term also includes 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 amide group.

[0123] Each of the various substituents described herein can be substituted or unsubstituted. As used herein, the term "substituted" refers to the following groups: deuterium (D), halogen (e.g., F, Cl, Br, and I), R, OR, OC(O)N(R), CN, NO, NO, ONO, azido, CF, OCF, methylenedioxy, ethylenedioxy, (C-C 20 )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(which may be aryl). Substitution also includes groups substituted with one or more groups, including, but not limited to, the following groups: fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. When two or more adjacent substituents are present, the substituents can be linked to form a carbocyclic or heterocyclic ring. Such adjacent groups can have a vicinal or atomic relationship, or can be adjacent on the ring, for example, in an ortho configuration. Each instance of substitution is understood to be independent. For example, a substituted aryl can be substituted with bromo, and a substituted heterocycle on the same compound can be substituted with alkyl. It is contemplated that a substituent can be substituted with one or more non-fluoro groups. As another example, a substituent can be substituted with one or more non-cyano groups. As another example, a substituent can be substituted with one or more groups other than haloalkyl. As yet another example, a substituent can be substituted with one or more groups other than tert-butyl. As yet another example, a substituent can be substituted with one or more groups other than trifluoromethyl. As a still further example, a substituent can be substituted with one or more groups other than nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophenyl, or nitrophenyl, or a combination of such descriptions.Substitution is further understood to include fluoro, cyano, haloalkyl, tert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophenyl, and nitrophenyl groups. In various embodiments, substituents can be substituted with groups other than carbonyl-containing groups, nitro, cyano, sulfinyl, sulfonyl, or halogen-containing groups. In various embodiments, substituents can be substituted with groups other than electron-withdrawing groups. Some substituents in certain embodiments can be substituted only with one or more electron-donating groups.

[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 together with the boronates to which they are bonded, C2 to C 10 A cyclic boronic acid ester is provided. [ka]

[0125] As used herein, the term "π-conjugated" group refers to a substituent having an unhybridized P orbital that overlaps or aligns with an unhybridized P orbital in the parent moiety to which the π-conjugate is attached, such that electrons can be shared between the two P orbitals and a lower energy state. Exemplary parent moieties include X, R 4 , R 5 , R 8 , and R 7 is a phenyl group to which is attached. Substituents that are π-conjugated groups can also have the π-bonding electrons delocalized through both the substituent and the parent moiety to which it is attached. Examples of π-conjugated groups include substituted or unsubstituted C2-C 10 Alken-1-yl, C2-C 10Alken-2-yl, C2-C 10 Alken-1-yl, C2-C 10 Heterocycloalken-1-yl, C2-C 10 Heterocycloalken-2-yl, C6-C 10 Further examples of π-conjugated groups include C2-C 10 Alken-1-yl, C2-C 10 Alken-2-yl, C2-C 10 Alken-1-yl, C2-C 10 Heterocycloalken-1-yl, C2-C 10 Heterocycloalken-2-yl, C6-C 10 Substituted or unsubstituted vinyl, ethynyl, C6-C further substituted with aryl or heteroaryl 10 Further examples include substituted or unsubstituted biaryl, biheterol aryl vinyl, heteroaryl vinyl, and C2-C 10 Included are alkene-1-ylaryl, phenylheteroaryl, and heteroarylaryl.

[0126] As used herein, the term "electron-donating group" refers to a group that has a net electron-donating effect relative to hydrogen. Electron-donating groups are well known in the art. See, for example, Jerry March, Michael B. Smith, March's Advanced Organic Chemistry 6th edition, 2007, Wiley Interscience, and J. McMurry, Organic Chemistry, 5th Ed. (Brooks / Cole, Pacific Grove, 2000), each of which is incorporated herein by reference in its entirety. Electron-donating groups, sometimes abbreviated as EDG, can be defined according to the Hammett substituent constant, also known as the sigma value (o value). In various embodiments, the electron-donating 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 is a non-halogen group having a sigma value of 0.3 or less, 0.2 or less, 0.1 or less, or a negative sigma value. If the position of a substituent substantially affects its sigma value, then the sigma value should be determined relative to the position of the X group. For example, 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 The sigma values ​​of the substituents at the sigma values ​​can be determined according to published tables or experimentally. See, for example, J. E. Feffler and E. Grunwald, Rates and Equilibria of Organic Reactions, Wiley, 1963 (Dover reprint), which is incorporated herein by reference in its entirety. Various examples of electron-donating groups include oxyanion, hydroxyl, amino, thio, alkylamino, dialkylamino, alkoxy, alkylthio, acylamino, acyloxy, alkyl, alkenyl, vinyl, aryl, and electron-rich heteroaryl.

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

[0128] In various embodiments, R 4 , R 5 , R 6 , and R 7 It can be seen that R provides a net donating effect when the pKa of the phenol group at X is 9.0 or greater, 9.5 or greater, 10.0 or greater, 10.5 or greater, or 11.0 or greater. 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 greater than that of the compound where H provides a net electron donating effect.

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

[0130] "Electron-withdrawing group," sometimes abbreviated as EWG, refers to a group that has a net electron-withdrawing effect on hydrogen. Electron-withdrawing groups are well known in the art. See, for example, Jerry March, Michael B. Smith, March's Advanced Organic Chemistry 6th edition, 2007, Wiley Interscience, and J. McMurry, Organic Chemistry, 5th Ed. (Brooks / Cole, Pacific Grove, 2000), each of which is incorporated herein by reference in its entirety. While the presence of an EWG is believed to slow the rate of formation of a reactive emissive intermediate, some embodiments of the present disclosure may contain one or more EWGs, provided that the net overall effect of the substituents is an electron-donating effect. For example, in some embodiments, R 4 , R 5 , R 6 , and R 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 acrylate (e.g., CHC(O)CH=CH-) and cyanoacrylate (NCCH=CH-) groups.

[0131] As used herein, the term alcohol protecting group refers to a substituent on an oxy group that renders the alcohol inert to oxygen against various conditions with which it typically reacts, but is easily removed when subjected to certain conditions. The alcohol protecting groups described herein typically improve the stability of the dioxetane moiety and, upon their removal, promote decomposition of the dioxetane. Thus, alcohol protecting groups include phosphates such as PO3Na2, PO3CI2, and PO3H2, glycosyl groups, dinitrobenzenesulfonaminobenzyl groups, and other groups that can be enzymatically hydrolyzed to provide the unprotected alcohol. Some alcohol protecting groups are described in Theodora W. Greene, Peter G.M.Wuts (1999). Protecting Groups in Organic Synthesis (3rd ed.). J. Wiley, which is incorporated herein in its entirety. Alcohol protecting groups include acetyl, benzoyl, benzyl, methoxyethoxymethyl, dimethyltrityl, methoxymethyl, methylthiomethyl, pivaloyl, tetrahydropyranyl, tetrahydrofuranyl, trityl, trialkylsilyl, trialkylsiloxymethyl, dialkylarylsilyl, glycosyl, pyranyl, galactosyl, and ethoxyethyl groups. Alcohol protecting groups also include groups in which the alcohol is substituted with a fragmentable linker further substituted with a protecting group, and deprotecting such a protecting group deprotects the linker fragment and eliminates it from the alcohol. The following compounds are further examples of alcohols substituted with alcohol protecting groups: [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 an analyte of interest in the presence of an enzyme capable of cleaving the enzyme-cleavable group provides an unstable phenolate-dioxetane species that subsequently decomposes and emits light. For example, G can be a peptide moiety consisting of two or more amino acid residues that is cleavable by a specific enzyme.

[0134] As used herein, the term "surfactant-based luminescence enhancer" refers to a class of compounds typically used to increase the intensity of dioxetanes in aqueous solution. Emerald™ and Emerald-11™ enhancers are examples of surfactant-based dyes commercially available from Thermo Fisher Scientific (Waltham, MA). Further examples of surfactant-based luminescence enhancers are described in Schaap, AP, Akhavan, H., Romano, LJ, Clin. Chem. 1989, 35(9), 1863, incorporated by reference in its entirety. In various embodiments, the surfactant-based luminescence enhancer contains a tail portion that is an acyclic alkyl group of at least 8 carbons and a head portion that is one or more quaternary ammonium salts, pyridinium salts, quaternary phosphonium surfactant salts, ethylene glycol chains, or fluorescein moieties. In various embodiments, the surfactant-based luminescence enhancer is a cationic surfactant-based luminescence enhancer, such as cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecane ammonium chloride, dioctadecyldimethylammonium (DODAB), α'-tributylphosphonium-p-xylene chloride, poly(vinylbenzyltributylphosphonium chloride) (TBE), poly(vinylbenzyltrioctylphosphonium chloride), etc. In various further embodiments, the surfactant-based luminescence enhancer is a surfactant having a long alkyl chain with a polyethylene glycol head, such as non-ionic Triton X-100, Tween surfactants, Brij® surfactants, IGEPAL® surfactants, octylphenoxypolyethoxyethanol, etc. Surfactant-based emission enhancers can also include a fluorescein head group, such as N-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthan]-5-yl)tetradecanamide (fluorescein surfactant).

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

[0136] As used herein, the terms "salt" and "pharmaceutically acceptable salt" refer to derivatives of the disclosed compounds in which the parent compound is modified by making acid or base salts thereof. Examples of salts include alkali salts and alkaline earth salts of ionized forms of the disclosed compounds, such as lithium, sodium, potassium, calcium, or magnesium salts. The disclosed compounds may also be salts containing cationic metals and anionic organic compounds, such as compounds having oxyanions and sodium cations. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines, and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanyltrimethyl, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, and isethionic acid.

[0137] Pharmaceutically acceptable salts can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. In some cases, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric (or greater) amount of an appropriate base or acid in water or an organic solvent, or a mixture thereof; generally, non-aqueous solutions such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is incorporated herein by reference.

[0138] The term "solvate" means a compound or a salt thereof that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0139] Values ​​expressed in range format should be interpreted in an open manner to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5% within the stated range, but also individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). A statement of "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, a statement of "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise indicated.

[0140] As used herein, the terms "a," "an," or "the" are used to include one or more unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. Furthermore, it should be understood that any phraseology or terminology used herein is for purposes of description only and not of limitation. Section headings are intended to aid in browsing the specification and are not to be construed as limiting. Furthermore, information associated with a section heading may occur within or outside that particular section. Furthermore, all publications, patents, and patent documents mentioned herein are incorporated by reference in their entirety, just as if individually incorporated by reference. In the event of any inconsistent usage between this specification and the specification incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this specification. In the event of a conflict, the usage set forth herein shall govern.

[0141] In the methods described herein, steps may be performed in any order without departing from the principles of the invention, unless a temporal or operational sequence is explicitly recited. Furthermore, certain steps may be performed simultaneously unless express claim language dictates that they be performed separately. For example, a claimed step of performing X and a claimed step of performing Y may be performed simultaneously in a single operation, with the resulting process falling within the literal scope of the claimed process.

[0142] As used herein, the term "about" may allow for some variation in a value or range, for example, within 10%, within 5%, or within 1% of the stated limits of the stated value or range.

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

[0144] As used herein, the term "about" may allow for some variation in a value or range, for example, within 10%, within 5%, or within 1% of the stated limits of the stated value or range.

[0145] As used herein, the term "substantially" refers to at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a majority.

[0146] As used herein, the terms "substantially free" or "substantially free of" refer to less than about 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.001%, or less than about 0.0005%, about 0% or less, below the limit of quantitation, below the limit of detectability, or 0%.

[0147] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Accordingly, the description is not intended and should not be construed as being limited to the examples given, but should be granted the full breadth of protection afforded by the appended claims and their equivalents. Furthermore, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Thus, the foregoing description or exemplary embodiments are provided for the purpose of illustrating the principles of the present disclosure and are not intended to be limiting thereof, and may include modifications and permutations thereof. [Example]

[0148] The present invention may be better understood by reference to the following examples, which are provided by way of illustration and not by way of limitation. General method

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

[0150] Chemiluminescence (light emission) intensity can be measured using a Turner Designs (Sunnyvale, CA) Model TD-20e luminometer, a BMG Labtech luminescence plate reader, or a charge-coupled device (CCD) camera luminometer, or any other suitable light intensity measuring device. In the examples listed below, solutions containing alkaline phosphatase at different concentrations (e.g., AP4, AP6, AP8, and AP9) were diluted, e.g., from an initial stock to a 10 4 , 10 6 , 10 8 and 10 9 Serial dilutions were used at each value presented. Compounds and enhancers (if used) were tested at their near optimal concentrations.

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

[0152] Amine-based buffer "221" or "Sigma-221" is available from Sigma-Aldrich (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] An initial solution of the compound was prepared in dioxane (1 mg of compound per mL of dioxane) and then mixed with water (20 μL of dioxane solution in 180 μL of water), followed by treatment with 200 μL of amine-based 221 buffer at 37° C. Upon treatment of the compound with the alkaline buffer, the intensity of chemiluminescence was measured over time. A graph showing luminescence intensity over time is provided in FIG. 1, demonstrating that luminescence is elicited over an extended period of time. The compound of Example 1 exhibits a chemiluminescence half-life of 3.79 minutes 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),7.31(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 a manner similar to Example 1, using a 10 μL sample of 1 mg / mL of the test compound in THF. The compound of Example 2 exhibits a chemiluminescence half-life of 4.91 minutes. The addition of an electron-withdrawing chlorine on the ortho phenyl ring slows the rate of increase in light emission and increases the half-life of light emission. Example 3

[0157] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR (400MHz, CDCI3) δ ppm 7.05 (m, 2H), 3.44 (s, 3H), 3.24 (s, 1H), 2.54 (s, 1H), 2.1-1.6 (m, 12H).

[0158] Example 3 was tested in a similar manner to Example 2. The compound of Example 3 exhibits a chemiluminescence half-life of 9.22 minutes. The addition of two electron-withdrawing chlorine groups further slows the rate of increase in luminescence and increases the luminescence half-life. Example 4

[0159] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.70(m,1H),7.45-6.65(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 a similar manner to Example 2. The compound of Example 4 exhibits a chemiluminescence half-life of 2.10 minutes. The addition of the slightly electron-donating iodine atom results in a slightly increased rate of luminescence and a slightly shorter half-life compared to Examples 1-3. Example 5

[0161] A dioxetane compound having the following structure was obtained: [ka] 1H NMR(400MHz,CDCI3)δ ppm7.44(m,1H),7.40-6.90(m,3H),5.82(d,J=16Hz,1H),5.65(br,1H),5.41(d,J=16Hz,1 H),3.23(s,3H),3.02(s,1H),2.18(s,1H),1.90-1.40(m,10H),1.25(m,1H),1.09(m,1H).

[0162] Example 5 was tested in a manner similar to Example 1, using 10 μL of a 0.1 mg / mL sample of the test compound in dioxane. Specifically, an initial solution of the compound was prepared in dioxane (0.1 mg of compound per mL of dioxane) and then mixed with water (10 μL of dioxane solution in 100 μL of water), followed by treatment with 200 μL of Amine-Based 221 buffer at 37° C.

[0163] A graph showing the luminescence intensity over time is provided in Figure 5. The compound of Example 5 exhibited a chemiluminescence half-life of 23 seconds and a Σ value of 1.28E+5. Addition of an electron-donating vinyl group onto the phenyl ring of PPD resulted in a higher luminescence intensity, a more rapid increase in the luminescence rate, and a dramatically shorter luminescence half-life compared to Examples 1-4. Example 6

[0164] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.51(m,1H),7.48(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 chemiluminescence half-life of 12.7 seconds. Addition of more electron donating 3-thienyl groups resulted in higher luminescence intensity, a more rapid increase in luminescence rate, and a shorter luminescence half-life compared to Examples 1-5. Example 7

[0166] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.51(m,1H),7.42(m,1H),7.45-7.01(m,4H),5.80(br,1H),3.26(s,3 H),3.04(s,1H),2.08(s,1H),2.1-1.5(m,10H),1.25(m,1H),1.10(m,1H).

[0167] Example 7 was tested in a manner similar to Example 1, using 10 μL of a 1 mg / mL sample of the test compound in dioxane. Specifically, an initial solution of the compound was prepared in dioxane (1 mg of compound per mL of dioxane) and then mixed with water (10 μL of dioxane solution in 90 μL of water), followed by treatment 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 exhibited a chemiluminescence half-life of 11.8 seconds and a Σ value of 1.32E+05. Here, the 2-thienyl transfers the electron-rich sulfur atom closer to the phenyl ring, thus providing a stronger electron-donating effect. Compared with Examples 1 to 6, the compound of Example 7 exhibited higher luminescence intensity, a more rapid increase in emission rate, and a shorter luminescence half-life. Example 8

[0169] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.68(d,J=2Hz,1H),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),1.31-1.26(m,1H),1.12-1.08(m,1H).

[0170] Example 8 was tested in a manner similar to Example 1, using a 10 μL sample of a 0.001 mg / mL test compound in dioxane. Specifically, an initial solution of the compound was prepared in dioxane (0.001 mg compound per mL of dioxane) and then mixed with water (10 μL of dioxane solution in 90 μL of water), followed by treatment with 100 μL of Amine-Based 221 buffer at 37° C.

[0171] A graph showing the luminescence intensity over time is provided in Figure 7. The compound of Example 8 exhibited a chemiluminescence half-life of 23 seconds and a Σ value of 9.76E+04. Without wishing to be bound by any particular theory, it is believed that the compound of Example 8 provides a higher luminescence intensity than Example 7 due to the extended π-conjugated system. The half-life of Sample 8 is greater than that of Sample 7 due to the 4-CN electron-withdrawing effect. Example 9

[0172] A dioxetane compound having the following structure was obtained: [ka] 1H 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 a manner similar to Example 1, using 10 μL of a 0.01 mg / mL sample of the test compound in dioxane. Specifically, an initial solution of the compound was prepared in dioxane (0.01 mg of compound per mL of dioxane) and then mixed with water (10 μL of dioxane solution in 90 μL of water), followed by treatment with 100 μL of Amine-Based 221 buffer at 37° C. A graph showing the luminescence intensity over time is provided in FIG. 8. 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, 1H), 1.15-1.08 (m, 1H).

[0175] Example 10 was tested in a manner similar to Example 1, using 10 μL of a 0.01 mg / mL sample of the test compound in dioxane. Specifically, an initial solution of the compound was prepared in dioxane (0.01 mg of compound per mL of dioxane) and then mixed with water (10 μL of dioxane solution in 90 μL of water), followed by treatment with 100 μL of Amine-Based 221 buffer at 37° C. A graph showing the luminescence intensity over time is provided in FIG. 9. Example 11

[0176] A dioxetane compound having the following structure was obtained: [ka] 1 H NMR(400MHz,CDCI3)δ ppm7.56(m,2H),7.42(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 a manner similar to Example 1, using 10 μL of a 0.1 mg / mL sample of the test compound in dioxane. Specifically, an initial solution of the compound was prepared in dioxane (0.01 mg of compound per mL of dioxane) and then mixed with water (10 μL of dioxane solution in 90 μL of water), followed by treatment with 100 μL of Amine-Based 221 buffer at 37° C. A graph showing the luminescence intensity over time is provided in FIG. 10. Example 12

[0178] A dioxetane compound (Lumigen® PPD) having the following structure was obtained: [ka] 1H 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=10Hz,1H).

[0179] An initial solution of the compound was prepared in 221 buffer (compound: 1.25 mg / mL, TBE enhancer: 5 mg / mL). Next, 100 μL of the initial solution was combined with 10 μL of a solution of alkaline phosphatase (AP8) at 37°C. Upon combining the compound with the alkaline phosphatase solution, the intensity of chemiluminescence was measured over time. A graph showing the luminescence intensity over time is provided in Figure 11. The compound exhibited a slow, gradual increase in luminescence intensity that did not reach a steady-state plateau maximum light intensity beyond 15 minutes, providing a Σ value of 2.27E+05. Example 13

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

[0181] An initial solution of compound was prepared in 221 buffer (compound: 0.125 mg / mL, TBE enhancer: 2.5 mg / mL), and a 100 μL aliquot was combined with 10 μL of a solution of alkaline phosphatase (AP8) at 37°C. Upon combining the compound with the alkaline phosphatase solution, the intensity of chemiluminescence was measured over time. A graph showing luminescence intensity over time is provided in Figure 12. The compound exhibited a slow, gradual increase in luminescence intensity that did not reach a steady-state plateau maximum light intensity beyond 15 minutes, providing a Σ value of 1.91E+05. 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] An initial solution of the compound was prepared in 221 buffer (compound: 0.25 mg / mL, TBE enhancer 5 mg / mL). 100 μL of the initial solution was then combined with 10 μL of a solution of alkaline phosphatase (AP8) at 37°C. Upon combining the compound with the alkaline phosphatase solution, the intensity of the chemiluminescence was measured over time. A graph showing the luminescence intensity over time is provided in FIG. 13. The compound exhibited a rapid increase in intensity, reaching a maximum steady-state intensity and providing a Σ value of 9.97E+05 over 1 minute. The compound of Example 14 provided luminescence with higher intensity and a faster response than the compounds of Examples 12 and 13. Example 15

[0185] Dioxetane compounds were prepared according to the following structures: [ka] 1 H NMR(400MHz,D2O)δ ppm7.95(m,1H),7.85(br,1H),7.65-7.55(m,2H),7.52(m,1H),7.25(br,1H),3.2 8(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] An initial solution of the compound was prepared in 221 buffer (compound: 0.25 mg / mL, TBE enhancer 5 mg / mL). 100 μL of the initial solution was then combined with 10 μL of a solution of alkaline phosphatase (AP8) at 37° C. A graph showing luminescence intensity over time is provided in FIG. 14. The compound exhibited a rapid increase in intensity, reaching a maximum steady-state intensity over 2 minutes, providing a Σ value of 7.38E+05. The compound of Example 15 provided luminescence with higher intensity and a faster response compared to the compounds of Examples 12 and 13. 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,1H),7.66(br,1H),7.26(br,1H),7.16-6.06(m,1H),5.88(d,J=16Hz,1H),5.37(d,J=12.4H) z,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=12.8Hz,1H).

[0189] An initial solution of the compound was prepared in 221 buffer (compound: 0.25 mg / mL, TBE enhancer 2.5 mg / mL). Next, 100 μL of the initial solution was combined with 10 μL of a solution of alkaline phosphatase (AP8) at 37°C. Upon combining the compound with the alkaline phosphatase solution, the intensity of the chemiluminescence was measured over time. A graph showing the luminescence intensity over time is provided in Figure 15. The compound exhibited a rapid increase in intensity, reaching a maximum steady-state intensity over 3 minutes, providing a Σ value of 1.59E+06. The compound of Example 16 provided luminescence with higher intensity and a faster response compared to the compounds of Examples 12-15.

[0190] In further experiments, an initial solution of compound was prepared in water (compound: 0.1 mg / mL), and then 100 μL of the initial solution was combined with 10 μL of a solution of alkaline phosphatase (AP4) and 300 μL of 5 mg / mL TEB221 buffer at 37° C. Upon combining the compound with the alkaline phosphatase solution, the intensity of chemiluminescence was measured over time. A graph showing the luminescence intensity over time is provided in FIG. 16. 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] An initial solution of the compound was prepared in 221 buffer (compound: 0.2 mg / mL, without TBE enhancer). 100 μL of the initial solution was then combined with 10 μL of a solution of alkaline phosphatase (AP9, [1.24E-20 mol / μL]) at 37°C. A graph showing luminescence intensity over time is provided in Figure 17. The compound exhibited a rapid increase in intensity, reaching a maximum steady-state intensity over 2 minutes, providing a Σ value of 2.77E+05.

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

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

[0195] For example, vinyl substitution resulted in dioxetanes that provided rapid, intense bursts of emission at least 20 times stronger than the corresponding unsubstituted compounds (see Example 5, compare Figures 5 and 1). Substitution with a thiophene group provided another example of such advantages (see Examples 6 and 7, compare Figures 6 and 1). The examples also show that adjusting the degree of electron-donating property of the π-conjugated electron-donating group can result in the intensity of the emission, a more rapidly increasing emission rate, and a shorter half-life of the emission (see Examples 7 and 8). Specifically, reducing the electron-donating effect by directly replacing the π-conjugated electron-donating group with an electron-withdrawing group can reduce the intensity, increase the emission rate, and prolong the half-life of the emission (compare Figures 7 and 6). Furthermore, the effect of direct substitution with thiophene, a type of π-conjugated electron-donating group, provides significant advantages over the corresponding unsubstituted compounds (compare Figures 7 and 1). Furthermore, the examples show that remote placement of the abstracting or donating group (e.g., cyano or methoxy) does not outweigh the benefits of having the π-conjugated electron-donating group located directly on the central aryl ring attached to the dioxetane (see Examples 9, 10, and 11; compare Figures 8, 9, and 10 with each other and to Figure 1). Thus, the π-conjugated electron-donating groups, e.g., vinyl, aryl, or heteroaryl, 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 exhibited slower emission and longer half-lives of emission (see Examples 2-4 and Figures 2-4). In the case of chlorine, the effect was additive when a second chlorine was added (compare Figures 2 and 3). Iodine, which is not a π-conjugated group and is neither strongly donating nor strongly withdrawing, had a relatively small effect that did not significantly improve emission (see Example 4).

[0197] Further 3-phosphate phenyl derivatives were prepared and tested under aqueous conditions by activation with alkaline phosphatase (ALP) (see Examples 12-16 and Figures 11-16). Two of the examples correspond to commercially available dioxetanes, namely, Lumigen® PPD (Example 12) and Tropix CDP-Star® (Example 13), both of which lack electron-donating groups on the central aromatic ring attached to the dioxetane. Both commercially available dioxetanes exhibited slow emission that did not reach maximum light intensity or a steady-state intensity plateau beyond 15 minutes. In comparison, aqueous compositions of dioxetanes bearing π-conjugated electron-donating groups exhibited greatly improved speed and intensity of emission compared to commercially available dioxetanes (see Examples 14-16; compare Figures 13-16 with Figures 11 and 12).

[0198] While not intending to be limited by any theory, it appears surprising that increasing electron density on aromatic rings and π-conjugated substituents accelerates the rate-limiting step that generates transient excited-state species that undergo chemical decay. Based on these results, the rate and intensity of dioxetane chemiluminescence can be increased by using substituents that are π-conjugated, electron-donating, or both. Furthermore, examples were tested in aqueous conditions without surfactant-based luminescence enhancers, which provided surprisingly high-intensity luminescence. Thus, 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 structures, consistent with the synthetic methods described herein. [Chemical]

[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). [Chemical]

[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). [Chemical]

[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), 2.10 (s, 1H), 1.85 - 1.44 (m, 10H), 1.24 - 1.19 (m, 1H), 1.0 - 0.85 (m.1H). [Chemical]

[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 = 16Hz, 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).

Chem.

[0204] (CDCI3) 7.80 (s, 1H), 7.46 (s, 1H), 7.35 - 7.25 (m, 1H), 7.10 - 6.90 (m, 1H), 5.87 (d, J = 18Hz, 1H), 5.54 (d, J = 20Hz, 1H), 5.41 (d, J = 11.2Hz, 1H), 5.21 (d, J = 11.2Hz, 1H), 3.18 (s, 3H), 3.01 (s, 1H), 2.15 (s, 1H), 2.05 - 1.46 (m, 10H), 1.26 - 1.24 (m, 1H), 1.20 - 1.16 (m, 1H).

Chem.

[0205] (CDCI3) 7.50 (d, J = 4Hz, 1H), 7.30 - 7.18 (m, 2H), 6.95 - 6.85 (m, 2H), 5.48 (br, 1H), 3.28 (s, 3H), 2.86 (s, 1H), 2.19 (s, 1H), 1.90 - 1.46 (m, 10H), 1.30 - 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). The present invention also includes the following aspects. <1> Formula I

Chem.

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Claims

1. Formula I 【Chemistry 1】 (In the formula, R 1 and R 2 together with the carbon to which they are attached, C 5 ~C 10 providing a cycloalkyl ring, R 3 But C 1 ~C 10 is alkyl, R 4 , R 5 and R 6 are hydrogen atoms; R 7 is a vinyl group or a substituted or unsubstituted thiophenyl; X is —OH or —OP(O)(ONa) 2 .

2. a) R 1 and R 2 together with the carbons to which they are attached provide a spirocyclic bridged bicyclo or tricyclo group selected from spirocyclic adamantane, norbornane, or bornane.

3. Formula I 【Chemistry 2】 (In the formula, R 1 and R 2 together with the carbons to which they are attached provide a C 5 -C 10 cycloalkyl ring; R 3 is C 1 -C 10 alkyl; R 4 , R 5 , R 6 and R 7 are each independently H, Q, X, hydroxy, halogen, amino, thio, C 1 to C 10 alkyl, C 2 to C 10 alkenyl, C 2 to C 10 alkynyl, C 1 to C 10 alkyloxy, C 1 to C 10 alkylamino, C 1 to C 10 trialkylammonium salt, C 1 to C 10 alkylthio, C 2 to C 10 acyl, C 1 to C 10 alkyloxycarbonyl, C 1 to C 10 alkylaminocarbonyl, C 1 to C 10 alkylthiocarbonyl, C 2 to C 10 acyloxy, C 2 to C 10 acylamino, C 2 to C 10 acylthio, C 1 to C 10 alkylcarbonate, C 1 to C 10 alkyl carbamate, C 1 -C 10 carbamido, 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 alkylphosphonate, C 1 -C 10 alkylphosphinate, C 1 -C 10 trialkylphosphonium salt, C 4 -C 10 heterocycloamino, C 6 -C 10 aryl, or a π-excess heteroaryl selected from thiophenyl, furanyl, pyrrolyl, benzothiophenyl, benzofuranyl, or indolyl; and at least one of R 4 , R 5 , R 6 , and R 7 is Q; Q is a π-conjugated electron donating group that is an unsubstituted vinyl; X is —OH, —O—G, —O − salt, or a boron group having the structure: 【Transformation 3】 each of R 8 and R 9 is independently H or C 1 -C 10 alkyl, or R 8 and R 9 together with the boronate to which they are attached provide a C 2 -C 10 cyclic boronic acid ester; G, a) trialkylsilyl, alkylarylsilyl, arylsulfonyl, dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, furanosyl, acyl, benzoyl, or benzyl; b) is galactosyl, glucosyl, or glucuronyl; c) β-galactosyl, β-glucosyl, or β-glucuronyl; d) —PO 3 H 2 or a salt or ester thereof; e) 2,4-dinitrobenzenesulfonyl, 3,4,6-trimethyl-2,5-dioxobenzyl, 4-azidobenzyloxy, tert-butyldimethylsilyl, acetyl, or pivaloyl, or f) comprising (i) a divalent releasable linker selected from 4-aminobenzyl, 4-(alkylamino)benzyl, 4-oxybenzyl, 4-(oxymethyl)benzyl, oxymethyl, aminomethyl, or alkylaminomethyl, and (ii) a terminal protecting group selected from a trialkylsilyl, alkylarylsilyl, arylbenzenesulfonyl, dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, furanosyl, acyl, benzoyl, benzyl, or boronate group; is an alcohol protecting group selected from wherein the alkyl, alkenyl, alkynyl, aryl, and heterocyclo are optionally substituted, and the compound is A) A structure comprising formula IV 【Chemistry 4】 a compound having B) The following structure: 【Transformation 5】 or a salt thereof, C) a molecule having the following structure: 【Transformation 6】 or a salt thereof, or D) A structure comprising Formula IIa or IIb: 【Transformation 7】 (In the formula, R 10 and R 11 wherein each of is independently H or halogen.

4. A composition for detecting an analyte in a sample, comprising a compound according to any one of claims 1 to 3.

5. The composition comprises: a) does not contain surfactants; b) does not contain surfactants having acyclic alkyl or aromatic groups and one or more of quaternary ammonium salts, pyridinium salts, quaternary phosphonium surfactant salts, ethylene glycol; c) does not contain cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'-tributylphosphonium-p-xylene chloride, poly(vinylbenzyltrioctylphosphonium chloride) (TBE), and poly(vinylbenzyltrioctylphosphonium chloride); d) further comprising an amine buffer; e) in the form of an aqueous composition, or f) having a peak emission intensity of more than 1000 photons / sec and, when treated with a pH 9.7 buffer, a T of 30 seconds or less at 37°C; 1 / 2 wherein the composition is free of surfactants. The composition of claim 4.

6. 10. A method for detecting an analyte in a sample, the method comprising contacting the sample with a compound according to any one of claims 1 to 3 and monitoring the sample for emission of light.

7. a) the method further comprises measuring the intensity of the resulting luminescence and correlating said intensity to the presence of said analyte; b) the contacted sample is monitored for less than one minute; or c) the analyte is alkaline phosphatase; The method of claim 6.

8. 8. The method of claim 6 or 7, wherein the analyte is an oxidizing agent selected from hydrogen peroxide, glutathione, fluoride or a base, and the sample is an aqueous sample having a pH of 4 to 12 or 8 to 12.

9. A kit for determining the presence of an analyte, said kit comprising a compound according to any one of claims 1 to 3 or a composition according to claim 4 or 5.

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