Fast, high intensity chemiluminescent dioxetanes

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 926,985, filed October 28, 2019, which is incorporated herein by reference as if fully set forth herein. Background Technology

[0003] Chemiluminescent dioxane is a strained cyclic peroxide that can undergo rapid decomposition to produce an excited transient substance, which then decays to the ground state via photoemission.

[0004] Such compounds can be used as luminescent probes in a range of assays, including enzyme activity assays, immunoassays, and DNA detection assays. Chemiluminescence-based assays can offer excellent sensitivity because, unlike fluorescence and absorption-based assays, they do not require photoexcitation.

[0005] Dioxanes can be generated in situ upon use or prepared in a stable form beforehand and subsequently activated. When generated in situ via precursor olefin oxidation, chemiluminescent dioxanes can also be used as a detection or imaging method for reactive oxygen species (ROS). Some examples of stable chemiluminescent dioxanes are 4-methoxy-4-(3-phosphophenyl)spiro[1,2-dioxane-3,2'-adamantane]. This compound, also known as... PPD can be activated upon treatment with alkaline phosphatase (ALP). ALP is an enzyme that catalyzes the hydrolysis of phosphate groups. Once activated, the resulting compound then undergoes 1,2-diphosphate hydrolysis. The alkyl ring is fragmented and luminescent, thus serving as a luminescent probe in alkaline phosphatase labeling assays.

[0006] Dioxane compounds that exhibit sensitive and strong emission under non-aqueous conditions have been developed. However, such compounds emit weakly in aqueous media and require a long time to reach maximum luminescence after contact with the desired analyte. Surfactant-based luminescence enhancers have been added to dioxane probes to amplify the weak emission in aqueous environments, but the use of such enhancers is neither desirable nor suitable for a wide range of applications. Invention Overview

[0008] Dioxanes that provide a rapid response to the presence of analytes are required. Dioxanes with strong emission and suitability for aqueous environments are also needed, without the need for surfactant-based enhancers. Several compounds disclosed herein offer these characteristics.

[0009] This disclosure provides compounds of formula I and their salts.

[0010]

[0011] R 1 and R 2 each independently is C3-C 10 alkyl, or R 1 and R 2 together with the carbon to which they are attached provide a C5-C 10 cycloalkyl ring. R 3 is C1-C 10 alkyl, C6-C 10 aryl, or heteroaryl.

[0012] R 4 , R 5 , R 6 , and R 7 each independently is H, Q, X, hydroxy, halogen, amino, sulfanyl, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 trialkylammonium salt, C1-C 10 alkylsulfanyl, C2-C 10 acyl, C1-C 10 alkoxycarbonyl, C1-C 10 alkylaminocarbonyl, C1-C 10 alkylthiocarbonyl, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkylcarbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, C1-C 10 alkylsulfinyl, C1-C 10 alkylsulfonyl, arylsulfanyl, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylsulfanyl, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 alkylphosphonate, C1-C 10 alkylphosphinate, C1-C 10 trialkyl salt, C4-C 10 heterocycloamino, C6-C 10 aryl, or π-excessive heteroaryl, and R 4R 5 R 6 and R 7 At least one of them is Q.

[0013] Q is a π-conjugated electron-donating group.

[0014] X is -OH, -OG, -O - Salt or borate ester / salt group.

[0015] G is an alcohol protecting group.

[0016] This disclosure also provides aqueous compositions comprising one or more chemiluminescent dioxane compounds, which, after treatment with a pH 9.7 buffer at 37°C, exhibit a peak emission intensity greater than 1000 photons / second and a Tg of 3 minutes or less. 1 / 2 Furthermore, the composition is substantially free of surfactant-based luminescence enhancers.

[0017] This 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 luminescence of the sample.

[0018] Several advantages, some of which are unexpected, are achieved through various embodiments of this disclosure. The various compounds described herein can advantageously provide rapid, high-intensity luminescence signals in non-aqueous media, aqueous media, or both. A significant advantage is that determinations involving such compounds can be performed more quickly than those using compounds lacking the currently described characterization of the compounds. Furthermore, the compounds of this disclosure provide enhanced luminescence intensity, including in aqueous media. Another advantage of the compounds of the invention is that their aqueous compositions may be free of surfactant-based luminescence enhancers. Due to these advantageous properties, various embodiments of this disclosure can provide methods or kits that can detect 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 description of the attached diagram

[0020] Figure 1 The graph shows the time-varying chemiluminescence intensity of the compound from Example 1, using 20 μL of a 1 mg / mL compound sample in methanol, which was triggered at 37°C with 200 μL of an amine-based buffer.

[0021] Figure 2 The graph shows the time-varying chemiluminescence intensity of the compound in Example 2, using 10 μL of a 1 mg / mL compound sample in THF, which was triggered at 37°C with 200 μL of an amine-based buffer.

[0022] Figure 3is a graph showing the time profile of chemiluminescent intensity for the compound of Example 3, using 10 μL of a 1 mg / mL compound sample in THF, triggered with 200 μL of amine-based buffer at 37 °C.

[0023] Figure 4 is a graph showing the time profile of chemiluminescent intensity for the compound of Example 4, using 10 μL of a 1 mg / mL compound sample in THF, triggered with 200 μL of amine-based buffer at 37 °C.

[0024] Figure 5 is a graph showing the time profile of chemiluminescent intensity for the compound of Example 5, using 10 μL of a 0.1 mg / mL compound sample in dioxane, triggered with 200 μL of amine-based buffer at 37 °C.

[0025] Figure 6 is a graph showing the time profile of chemiluminescent intensity for the compound of Example 7, using 10 μL of a 1 mg / mL compound sample in dioxane, further diluted with 90 μL of water, triggered with 200 μL of amine-based buffer at 37 °C.

[0026] Figure 7 is a graph showing the time profile of chemiluminescent intensity for the compound of Example 8, using 10 μL of a 0.001 mg / mL compound sample in dioxane, further diluted with 90 μL of water, triggered with 200 μL of amine-based buffer at 37 °C.

[0027] Figure 8 is a graph showing the time profile of chemiluminescent intensity for the compound of Example 9, using 10 μL of a 0.01 mg / mL compound sample in dioxane, further diluted with 90 μL of water, triggered with 200 μL of amine-based buffer at 37 °C.

[0028] Figure 9 is a graph showing the time profile of chemiluminescent intensity for the compound of Example 10, using 10 μL of a 0.01 mg / mL compound sample in dioxane, further diluted with 90 μL of water, triggered with 200 μL of amine-based buffer at 37 °C.

[0029] Figure 10 is a graph showing the time profile of chemiluminescent intensity for the compound of Example 11, using 10 μL of a 0.01 mg / mL compound sample in dioxane, further diluted with 90 μL of water, triggered with 200 μL of amine-based buffer at 37 °C.

[0030] Figure 11is a graph showing the time profile of chemiluminescent intensity of the compound of Example 12, using 100 μL of a 1.25 mg / mL compound sample in 5 mg / mL TBE enhancer amine-based buffer and 10 μL alkaline phosphatase (AP8) at 37°C.

[0031] Figure 12 is a graph showing the time profile of chemiluminescent intensity of the compound of Example 13, using 100 μL of a 0.125 mg / mL compound sample in 2.5 mg / mL TBE enhancer amine-based buffer and 10 μL alkaline phosphatase (AP8) at 37°C.

[0032] Figure 13 is a graph showing the time profile of chemiluminescent intensity of the compound of Example 14, using 100 μL of a 0.25 mg / mL compound sample in 5 mg / mL TBE enhancer amine-based buffer and 10 μL alkaline phosphatase (AP8) at 37°C.

[0033] Figure 14 is a graph showing the time profile of chemiluminescent intensity of the compound of Example 15, using 100 μL of a 0.25 mg / mL compound sample in 5 mg / mL TBE enhancer amine-based buffer and 10 μL alkaline phosphatase (AP8) at 37°C.

[0034] Figure 15 is a graph showing the time profile of chemiluminescent intensity of the compound of Example 16, using 100 μL of a 0.25 mg / mL compound sample in 2.5 mg / mL TBE enhancer amine-based buffer and 10 μL alkaline phosphatase (AP8) at 37°C.

[0035] Figure 16 is a graph showing the time profile of chemiluminescent intensity of the compound of Example 16, using 100 μL of a 0.1 mg / mL compound sample in water and 10 μL alkaline phosphatase (AP4) and 300 μL of 5 mg / mL TBE enhancer amine-based buffer at 37°C.

[0036] Figure 17 is a graph showing the time profile of chemiluminescent intensity of the compound of Example 17, using 100 μL of a 0.2 mg / mL compound in amine-based buffer without poly(vinylbenzyl tri-n-butylphosphonium chloride (TBE) and 10 μL alkaline phosphatase (AP9) at 37°C.

[0037] Figure 18 is a graph showing the time profile of chemiluminescent intensity of the compound of Example 12, a plot of the time course of chemiluminescent intensity of PPD) at 37°C using 100 μL of 2 mg / mL compound in an amine-based buffer without TBE enhancer and 20 μL alkaline phosphatase (AP8). DETAILED DESCRIPTION

[0039] Reference will now be made in detail to some embodiments of the disclosed subject matter, examples of which are partially illustrated in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplary subject matter is not intended to limit the claims to the disclosed subject matter.

[0040] The compounds of the present disclosure can be used in chemiluminescent applications, such as assays and chemical probes.

[0041] The present disclosure provides a compound of Formula I or a salt thereof.

[0042]

[0043] R 1 and R 2 each independently is C3-C 10 alkyl, or R 1 and R 2 together with the carbon to which they are attached provide a C5-C 10 cycloalkyl ring, such as a monocyclic, bicyclic, or tricyclic ring. R 1 and R 2 may be substituted or unsubstituted. In various embodiments, R 1 and R 2 are connected such that they together with the carbon to which they are attached provide a spirocyclic bridged bicyclic or tricyclic group. For example, R 1 and R 2 together with the carbon to which they are attached can be spirocyclo adamantane, norbornane, or bornane.

[0044] R 3 is C1-C 10 alkyl, C6-C 10 aryl, or heteroaryl, each of which is optionally substituted. R 3 may be substituted or unsubstituted. For example, R 3 may be unsubstituted C1-C 10 alkyl or C1-C 10 alkyl substituted with one or more halogen, hydroxyl, amino, sulfido, alkoxy, alkylamino, alkylsulfido, sulfate, or carboxylate.

[0045] R 4 , R 5 , R 6 and R 7each independently H, Q, X, hydroxyl, halogen, amino, sulfanyl, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 trialkylammonium, C1-C 10 alkylthio, C2-C 10 acyl, C1-C 10 alkoxycarbonyl, C1-C 10 alkylaminocarbonyl, C1-C 10 alkylthiocarbonyl, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkylcarbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, C1-C 10 alkylsulfinyl, C1-C 10 alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroaryl- amino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 alkylphosphonate, C1-C 10 alkylphosphinate, C1-C 10 trialkyl salt, C4-C 10 heterocyclic amino, C6-C 10 aryl or a π-excessive heteroaryl, and R 4 , R 5 , R 6 and R 7 are each independently Q. R 4 , R 5 , R 6 and R 7 may be substituted or unsubstituted.

[0046] In various embodiments, R 4 , R 5 , R 6 and R 7 are each independently selected from the group consisting of H, Q, X, halogen, C1-C 10 alkyl, hydroxyl, C1-C 10 alkoxy, amino, C1-C 10alkylamino, C1-C 10 alkylthio, C1-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkyl carbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, and heteroarylamino, wherein R 4 , R 5 , R 6 , and R 7 are Q.

[0047] In various additional 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, the aromatic ring to which X, Q, R 4 , R 5 , R 6 , and R 7 are attached is electron rich relative to otherwise identical compounds in which Q, R 4 , R 5 , R 6 , and R 7 are H.

[0048] In some embodiments, exactly one, two, or three of R 4 , R 5 , R 6 , and R 7 are X; exactly one, two, or three of R 4 , R 5 , R 6 , and R 7 are Q; or any combination thereof. In some additional such embodiments, the remainder of R 4 , R 5 , R 6 , and R 7 that are not X or Q are H. For example, R 4 , R 5 , and R 6 may each be H, while R 7 is Q.

[0049] 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-C10 heterocycloalkenyl, C6-C 10 aryl or heteroaryl. Q can be substituted or unsubstituted. In some embodiments, when Q is C2-C 10 alkenyl, it is substituted with one or more electron donating groups, it is free of electron withdrawing groups, or both. In various embodiments, when Q is C2-C 10 alkenyl, the vinyl position and the allyl position, if present, are unsubstituted. In other embodiments, for example, Q can be unsubstituted vinyl. In some embodiments, when Q is C6-C 10 aryl, it is substituted with one or more electron donating groups, it is free of 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 electron donating substituents. As another example, Q can be vinyl or phenyl substituted with substituents such that the net effect of the substituents is electron donating. In another example, Q is a π-excessive heteroaryl, such as thienyl, furanyl, pyrrolyl, benzothienyl, benzofuranyl, or indolyl. In certain embodiments, Q is substituted or unsubstituted thien-2-yl or thien-3-yl.

[0050] X is -OH, -O-G, -O - salt or boronate / salt group. In various embodiments, X is a group that generates an oxygen-containing anion upon chemical or enzymatic triggering. When X is a boronate group, it has the following structure:

[0051]

[0052] R 8 and R 9 are each independently H or C1-C 10 alkyl, or R 8 and R 9 together with the boronate to which they are attached are C2-C 10 cyclic boronate. For example, X can be 4,4,5,5-tetramethyl-1,3,2-dioxaborolan- yl or -B(OH)2.

[0053] In various embodiments, X is -O-G, where G is an alcohol protecting group, an analyte-responsive group, or both. For example, G can be a trialkylsilyl, alkylarylsilyl, arylsulfonyl, dioxobenzyl, trityl, alkyl carbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranosyluronic, furanoyl, acyl, benzoyl, or benzyl group. In some embodiments, G is a pyranosyl or pyranosyluronic group, such as a galactosyl, glucosyl, or glucuronyl group. In other such embodiments, G is a β-galactosyl, β-glucosyl, or β-glucuronyl group. G can also be a phosphorus-containing group, such as a phosphate, phosphonate, or the like. For example, G can be -PO3H2or a salt or ester thereof. In other embodiments, G is a 2,4-dinitrophenylsulfonyl, 3,4,6-trimethyl-2,5-dioxobenzyl, 4-azidobenzyloxy, t-butyldimethylsilyl, acetyl, pivaloyl, 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 with a pendant protecting group such that removal of the pendant protecting group triggers fragmentation of the linker and removal of the protecting group G. Thus, G can comprise a bivalent fragmentable linker, such as a 4-aminobenzyl, 4-(alkylamino)benzyl, 4-oxobenzyl, 4-(oxymethyl)benzyl, oxymethyl, aminomethyl, alkylaminomethyl, or the like, and a terminal protecting group such as a trialkylsilyl, alkylarylsilyl, arylphenylsulfonyl, dioxobenzyl, trityl, alkyl carbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranosyluronic, furanoyl, acyl, benzoyl, benzyl, or boronate ester group.

[0054] Some examples of X and -O-G include the following structures:

[0055]

[0056] The present disclosure also provides a compound of Formula II, or a salt thereof.

[0057]

[0058] R 10 and R 11 each independently is H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, C 6- C 10 aryl. In some embodiments, R 10 and R 11 independently is H or halogen.

[0059] The present disclosure also provides compounds of Formulae IIa and IIb, or salts thereof.

[0060]

[0061] R 10 and R 11 each independently is H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, C6-C 10 aryl. In some embodiments, R 10 and R 11 are independently H or halogen.

[0062] The present disclosure also provides compounds of Formula III, or salts thereof.

[0063]

[0064] R 12 and R 13 each independently is H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 trialkylammonium salt, C1-C 10 alkylthio, C2-C 10 acyl, C1-C 10 alkoxycarbonyl, C1-C 10 alkylaminocarbonyl, C1-C 10 alkylthiocarbonyl, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkylcarbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, C1-C 10 alkylsulfinyl, C1-C 10 alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylaminol heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 alkylphosphonate, C1-C 10 alkylphosphinate, C1-C 10 trialkyl salt, C4-C 10heteroaryloxy, heteroarylthio, or heteroarylamino, C4-C 10 aryl or π-excessive heteroaryl, or R 12 and R 13 together with the carbon to which they are attached provide a C5-C 10 cycloalkenyl, C2-C 10 heterocycloalkenyl, C6-C 10 aryl or π-excessive heteroaryl. In some embodiments, R 12 and R 13 are independently H, C1-C 10 alkyl, C2-C 10 alkenyl, C6-C 10 aryl or π-excessive heteroaryl, or R 12 and R 13 together with the carbon to which they are attached provide a C5-C 10 cycloalkenyl, C2-C 10 heterocycloalkenyl, C6-C 10 aryl or π-excessive heteroaryl, or R 12 and R 13 together with the carbon to which they are attached provide a C5-C 10 cycloalkenyl, C2-C 10 heterocycloalkenyl, C6-C 10 aryl or π-excessive heteroaryl. In other embodiments, R 12 and R 13 are independently H, C1-C 10 alkyl, C2-C 10 alkenyl, C6-C 10 aryl or π-excessive heteroaryl.

[0065] R 14 is H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 alkylthio, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkylcarbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, C4-C 10 heteroaryloxy, heteroarylthio, or heteroarylamino, C4-C 10 aryl or π-excessive heteroaryl. In some embodiments, R 14H, C1-C 10 alkyl, C2-C 10 alkenyl, C6-C 10 aryl or π-excessive heteroaryl.

[0066] 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 electron rich relative to the otherwise identical compound in which R 12 , R 13 and R 14 are H.

[0067] In various embodiments, at least one or two of R 12 , R 13 and R 14 are H.

[0068] The present disclosure also provides compounds of Formula IIIa and IIIb, or salts thereof.

[0069]

[0070] R 12 and R 13 are each independently H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 trialkylammonium salt, C1-C 10 alkylthio, C2-C 10 acyl, C1-C 10 alkoxycarbonyl, C1-C 10 alkylaminocarbonyl, C1-C 10 alkylthiocarbonyl, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkylcarbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, C1-C 10 alkylsulfinyl, C1-C 10alkylsulfonyl, arylsulfenyl, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylsulfenyl, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 alkylphosphonate, C1-C 10 alkylphosphinate, C1-C 10 trialkyl salt, C4-C 10 heterocyclic amino, C6-C 10 aryl or π-excessive heteroaryl, or R 12 and R 13 together with the carbon to which they are attached provide a C5-C 10 cycloalkenyl, C2-C 10 heterocycloalkenyl, C6-C 10 aryl, or π-excessive heteroaryl. In some embodiments, R 12 and R 13 are independently H, C1-C 10 alkyl, C2-C 10 alkenyl, C6-C 10 aryl or π-excessive heteroaryl, or R 12 and R 13 together with the carbon to which they are attached provide a C5-C 10 cycloalkenyl, C2-C 10 heterocycloalkenyl, C6-C 10 aryl or π-excessive heteroaryl, or R 12 and R 13 together with the carbon to which they are attached provide a C5-C 10 cycloalkenyl, C2-C 10 heterocycloalkenyl, C6-C 10 aryl, or π-excessive heteroaryl. In other embodiments, R 12 and R 13 are independently H, C1-C 10 alkyl, C2-C 10 alkenyl, C6-C 10 aryl or π-excessive heteroaryl.

[0071] R 14 is H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 alkylsulfenyl, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C10 acylthio, C1-C 10 alkyl carbonate, C1-C 10 alkyl carbamate, C1-C 10 ureido, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, C4-C 10 heterocyclic amino, C6-C 10 aryl or π-excessive heteroaryl. In some embodiments, R 14 is H, C1-C 10 alkyl, C2-C 10 alkenyl, C6-C 10 aryl or π-excessive heteroaryl.

[0072] 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 aryl ring to which X is attached is electron rich relative to the otherwise identical compound in which R 12 , R 13 , and R 14 are H.

[0073] In various embodiments, at least one or two of R 12 , R 13 , and R 14 are H.

[0074] The present disclosure also provides a compound according to one or more of the following formulae:

[0075]

[0076]

[0077] The present disclosure also provides a compound according to one or more of the following formulae:

[0078]

[0079]

[0080] Z, L, and J are each S, O, Se, NR 15 or (CR 16 R 17 )n, where each R 15 is independently H, alkyl, acyl, benzyl, alkoxycarbonyl, arylsulfonyl; and R 14 , R 16 , R 17 , R 18 , R 19 , R20 and R 21 , if present, are each independently H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 trialkylammonium salt, C1-C 10 alkylthio, C2-C 10 acyl, C1-C 10 alkoxycarbonyl, C1-C 10 alkylaminocarbonyl, C1-C 10 alkylthiocarbonyl, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkylcarbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, C1-C 10 alkylsulfinyl, C1-C 10 alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroaryl- amino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 alkylphosphonate, C1-C 10 alkylphosphinate, C1-C 10 trialkyl salt, C4-C 10 heterocycloamino, C6-C 10 aryl or π-excessive heteroaryl, or any two of R 14 , R 16 , R 17 , R 18 and R 20 together with the carbon to which they are attached provide a C5-C 10 cycloalkenyl, C2-C 10 heterocycloalkenyl, C6-C 10 aryl or π-excessive heteroaryl.

[0081] In some embodiments, R 14 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 , if present, are each independently H, halogen, C1-C10 alkyl, C2-C 10 alkenyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 alkylthio, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkylcarbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, C4-C 10 heterocyclicamino, C6-C 10 aryl or π-excessive heteroaryl, or R 14 , R 16 , R 17 , R 18 and R 20 any two of which, together with the carbon to which they are attached, provide a C5-C 10 cycloalkenyl, C2-C 10 heterocycloalkenyl, C6-C 10 aryl or π-excessive heteroaryl.

[0082] In various embodiments, at least one of R 14 , R 16 , R 17 , R 18 and R 20 is an electron donating group. In other embodiments, R 14 , R 16 , R 18 and R 20 , if present, are each hydrogen.

[0083] In any of the above compounds (including any of the compounds of Formulae I, II, Ila, III, Ilia, Illb) and XII (below), R 4 or R 5 may be C1-C 10 alkyl (e.g., CH3) or halogen (e.g., chloro). Further, in any of the above compounds including the compounds of Formula XII to Formula XIV, R 12 may be C1-C 10 alkyl (e.g., CH3).

[0084] The present disclosure also provides compounds according to Formulae (IV) to (XV), or salts thereof.

[0085]

[0086]

[0087] The present disclosure provides a compound according to one or more of the following structures or a salt thereof:

[0088]

[0089]

[0090] The present disclosure provides a composition comprising one or more compounds described herein, an olefin precursor thereof, or a salt thereof. The olefin precursor provides any of the compounds described herein (e.g., compounds of Formulae I, II, Ila, III, Ilia, Illb, and IV-XV) upon treatment with an analyte, an oxidizing agent, an alkaline phosphatase, or photooxidation conditions.

[0091] The composition can be an aqueous composition or a non-aqueous composition. The composition can be a mixture of both aqueous and non-aqueous solvents. In embodiments, the composition is substantially free of a surfactant-based luminescence enhancer, a surfactant, or both. For example, the composition can be substantially free of a surfactant having a tail group that is a non-cyclic alkyl group (e.g., a non-cyclic group of at least eight carbons) or an aromatic group (e.g., an aromatic group comprising at least six carbons) and a head group that is one or more quaternary ammonium salts, pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl pyridinium salts, ethylene glycol, or fluorescein. As another example, the composition can be substantially free of cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'- tributyl

[0092] In various embodiments, the composition contains a buffer solution. The buffer solution can be, but is not necessarily, a basic buffer solution or an amine-based buffer solution. One example of an amine-based buffer is 221 buffer, which is available from Sigma-Aldrich (St. Louis, MO). Likewise, the composition can be, but is not necessarily, required to have a basic pH. For example, the pH of the composition can be about 4 to 12, about 5 to 12, about 6 to 12, about 7 to 12, about 8 to 12, about 9 to 12, about 10 to 12, about 4 to 11, about 4 to 10, about 4 to 9, about 4 to about 8, about 4 to 7, about 4 to 6, or about 4 to 5. The pH of the composition can be selected depending on whether the luminescence is intended to be triggered immediately after analyte-triggered removal, which is typically the case for basic pH values, or the pH value of the composition can be acidic so as to luminesce upon treatment with a base.

[0093] In various embodiments, the composition has a peak luminescence intensity of greater than 1,000 photons / second and a T 1 / 2 In various examples, the peak luminescence intensity can be greater than about 2,000 photons / second, about 3,000 photons / second, about 4,000 photons / second, about 5,000 photons / second, about 6,000 photons / second, about 7,000 photons / second, about 8,000 photons / second, about 9,000 photons / second, or greater than about 10,000 photons / second, and the T 1 / 2 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 an aqueous composition comprising one or more dioxetane compounds and having a peak luminescence intensity of greater than 1000 photons / second and a T 1 / 2 wherein the composition is substantially free of a surfactant-based luminescence enhancer.

[0094] The present disclosure also provides methods of detecting an analyte in a sample, comprising contacting the sample with one or more of the compounds described herein, olefin precursors thereof, salts thereof, or compositions comprising the same, and subsequently monitoring the sample for luminescence. In various embodiments, the method involves measuring the intensity of the resulting luminescence and correlating the intensity with the presence of the analyte.

[0095] In some embodiments, the method further comprises increasing the pH of the sample. For example, the pH can be adjusted to 7 or higher, 8 or higher, 9 or higher, 10 or higher, or 11 or higher.

[0096] 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.

[0097] The analyte can be any species derived from a composition of any of the compounds described herein (e.g., compounds of Formulae I, II, Ila, III, Ilia, Illb, and IV-XV), wherein X is an oxygen-containing anion. 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 basic conditions.

[0098] The present disclosure also provides kits for determining the presence of an analyte, the kits comprising one or more of any of the compounds described herein, olefin precursors thereof, salts thereof, or compositions comprising the same. The kits can contain instructions according to the methods described herein.

[0099] The compounds and compositions described herein can be triggered directly by the analyte in order to produce the signal identifying the probe and the presence of the analyte, or can be triggered in a two-step process, i.e., one step involving contact with the analyte, and another step involving an increase in pH.

[0100] The compounds of the present disclosure can be configured as probes to detect a variety of different analytes by modifying the group X or G. For example, the compounds described herein can be configured to detect beta-galactosidase by providing a glucosyl group at G, to detect hydrogen peroxide or other oxidizing agents by providing a borate at X, to detect alkaline phosphatase by providing a phosphate at X or a phosphoryl group at G, to detect glutathione by providing a dinitrophenylsulfenylaminobenzyl group at G, to detect fluoride by providing a trialkylsilyl group at G, and to detect a base when X is OH.

[0101] It is desirable that the compound emits all possible light in as short a time period as possible after being triggered by the analyte, thereby providing as strong a signal as possible. When chemiluminescence is emitted gradually over a period of time, the light intensity (photons / second) is attenuated and the detection sensitivity is compromised. Figure 1 One example of a luminescence signal time curve is shown.

[0102] The rate of luminescence enhancement, or rise time, can be measured as the time (t maxor emission half-life (T) 1 / 2 To describe it.

[0103] 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 theoretically limited, an exemplary mechanism involves the ALP enzyme hydrolyzing the phosphorus-containing group to provide phenol, which is immediately deprotonated due to the alkaline environment of the solution (e.g., a pH 9.7 buffer). The formation of the oxyanion triggers the decomposition of 1,2-dioxane into two compounds: 2-adamantanone and an excited-state phenyl ester. The excited phenyl ester then immediately decays to the ground state by emitting light.

[0104] The resulting light intensity is a linear function of the enzyme amount. Therefore, the compounds described herein can be used to detect labeled enzymes used in assays. For example, the steps of the chemical process in which dioxane provides light can be described as follows: (i) X + S → X + S', (ii) S' → P*, and (iii) P* → P + light. Step (i) represents the catalytic conversion of the substrate, where X is an enzyme or other component that converts the substrate into its activated form, step (ii) represents the degradation of the activated substrate into a transiently excited substance, and step (iii) represents the decay of the excited substance into its ground state and the emission of light. The light intensity is the product of the catalytic conversion of the substrate in step (i) and the lifetime of the light-generating compound P* in step (ii). Step (ii) is generally first-order, has a rate constant k, and can be characterized by its half-life: T 1 / 2 = (ln 2) / k. Step (iii) is very short compared to the other steps and generally has no meaningful effect on reaction kinetics.

[0105] Chemiluminescence intensity / time curves include the initial rise in emission intensity and the subsequent steady-state intensity period. The slow first-order reaction S'→P* corresponds to a prolonged rise time because it takes longer to reach the steady-state concentration of S'. The rapid reaction S'→P* corresponds to a shorter initial rise period and thus provides a rapid rise. For enzyme-catalyzed chemiluminescence reactions, the intensity typically remains high, and the resulting signal will generally correspond to... Figure 5 The signal shape is shown in the figure. The absence of a stable intensity indicates substrate depletion or subsequent enzyme inactivation. Detection of enzyme-generated chemiluminescence provides flexibility in the measurement process because the light intensity at any point in time can be related to the amount of enzyme; however, the enzyme-generating process can have drawbacks, for example, due to the size and "viscous" nature of the enzyme label. However, for maximum sensitivity, it is best to use the maximum intensity (I0.05) during the steady-state intensity period. max Measurements were taken at or near the location.

[0106] The compounds described herein can also be used as direct labels of one of the complementary binding partners in immunoassays. The compounds described herein are advantageously used as labels compared to large bioluminescent molecules and other types of enzyme labels because they are small molecules.

[0107] Accordingly, the present disclosure also relates to assays using the compounds described herein as chemiluminescent probes.

[0108] In various embodiments, the assay can be a homogeneous (non-separation) assay, in which bound and unbound ligand need not be separated, or it can be a heterogeneous assay, in which the labeled binding pair complex is separated from unbound labeled reactants. The assay can be configured to be performed manually, or it can also be performed automatically and by robots. The assay can be performed in test tubes, cuvettes, micro-wells, or combinations thereof. In various embodiments, the test tubes, cuvettes, micro-wells, or other containers in which the assay is performed are at least partially opaque, fully opaque, black, white, or combinations thereof.

[0109] The assay can be performed on immobilized proteins in western blots, immobilized nucleic acids in Southern or Northern blots.

[0110] Imaging can be recorded using a luminometer, X-ray film, or a charge coupling device (CCD) camera system.

[0111] Measuring chemiluminescence is advantageous over fluorescence and absorption spectroscopy. For example, fluorescence and absorption spectroscopy can be affected by interfering signals generated by incident light or background signals.

[0112] The assays described herein can be configured to measure chemiluminescence according to the non-limiting examples described in J. E. Wampler, Instrumentation: Seeing 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 Horwood, 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, Orlando, 608-613 (1995), each of which is incorporated herein by reference in its entirety.

[0113] A variety of methods can be used to attach the compounds of the disclosure to biomolecules. For example, when the compound contains a reactive group (e.g., carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxamide, carboxime, or N-succinimidyl carboxyl), such groups can be covalently conjugated to hydroxyl or amino functional groups using conjugation reagents (e.g., carbodiimides or 1,1-carbonyldiimidazole). N-maleimide groups react directly with sulfhydryl residues in proteins. If the compound contains aromatic amines, they can be converted to diazonium salts and reacted with phenolic groups (such as those found in the tyrosine groups of proteins). Reactive groups present in polycyclic aromatic moieties or other light generating groups, or reactive groups present in leaving groups, can be used to attach the compounds of the invention to biomolecules.

[0114] The compounds of the present disclosure can generally be prepared, for example, according to the synthetic methods described in Scheme 1.

[0115]

[0116] 2-adamantanone and 3-substituted benzoate can be coupled together by subjecting them to McMurry reaction conditions involving a titanium oxyphilic and a reducing agent. The resulting olefin can be further modified, for example, by removing or replacing protecting group G or further functionalizing the R 4 , R 5 , R 6 , and R 7 groups on the ring. Next, the olefin is subjected to photooxidation reaction 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 one 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 are H. In some embodiments, R 3 is a substituted or unsubstituted alkyl group and R 7 is an electron donating group.

[0117] The term light “intensity” or luminescence “intensity” as used herein refers to the rate of emission in photons / second. Intensity can be measured by using a luminometer. A luminometer is a photodetector in a housing that does not include ambient light. Any suitable luminometer can be used, including photomultiplier tubes and photodiodes.

[0118] The term “luminescence speed” refers to the rate at which luminescence increases, i.e., the change in light intensity over time.

[0119] The term “sensitivity” as used herein refers to the lowest level of signal of a measured analyte or product that can be reproducibly detected.

[0120] As used herein, the term "alkyl" refers to a substituted or unsubstituted straight chain, branched chain, or cyclic, saturated, monovalent or divalent radical having from 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 (Ci-C 20 Some examples of straight chain monovalent (Ci-C 20 Some examples of branched chain monovalent (Ci-C 20Some examples of alkyl groups include those having 1 to 6 carbon atoms, such as CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-. Some examples of branched divalent alkyl groups include -CH(CH3)CH2- and -CH2CH(CH3)CH2-. Some examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, bicyclo[l. l. l]pentyl, bicyclo[2. l. l]hexyl, bicyclo[2.2. l]heptyl, and adamantyl. Cyclic alkyl groups also include substituted and unsubstituted polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, camphanyl, camphene, isocamphene, and caryophyllane groups, as well as fused rings such as, but not limited to, decahydronaphthyl, and the like. For example, a cycloalkyl group includes adamantyl groups substituted with one, two, three, four, or more substituents, such as at the tertiary bridgehead positions of the methylene bridges. In some embodiments, alkyl groups include combinations of substituted and unsubstituted alkyl groups. As an example, alkyl groups, and (Ci)alkyl groups, include methyl and substituted methyl groups. As a specific example, (Ci)alkyl groups include benzyl groups. As another example, alkyl groups can include methyl and substituted (C2-C8)alkyl groups. Alkyl groups can also include substituted methyl and unsubstituted (C2-C8)alkyl groups. In some embodiments, alkyl groups can be methyl and C2-C8straight chain alkyl groups. In some embodiments, alkyl groups can be methyl and C2-C8branched chain alkyl groups. The term methyl is understood to be -CH3, which is unsubstituted. The term methylene is understood to be -CH2-, which is unsubstituted. For comparison, the term (Ci)alkyl is understood to be -CH3or -CH2- which are substituted or unsubstituted. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, such as cycloalkyl, heterocyclyl, aryl, amino, haloalkyl, hydroxyl, cyano, carboxyl, nitro, thio, alkoxy, and halogen groups. As another example, representative substituted alkyl groups can be substituted with one or more of fluorine, chlorine, bromine, iodine, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxyl, haloalkyl, hydroxyl, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido groups. In some embodiments, representative substituted alkyl groups can be substituted from a group of groups that includes amino, hydroxyl, cyano, carboxyl, nitro, thio, and alkoxy, but not halogen groups. Thus, in some embodiments, alkyl groups can be substituted with non-halogen groups.For example, a representative substituted alkyl can be substituted with a fluoro group, substituted with a bromo group, substituted with a halogen other than bromo, or substituted with a halogen other than fluoro. In some embodiments, a representative substituted alkyl 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, an alkyl can be trifluoromethyl, difluoromethyl, or fluoromethyl, or an alkyl can be a substituted alkyl other than trifluoromethyl, difluoromethyl, or fluoromethyl. An alkyl can be a haloalkyl, or an alkyl can be a substituted alkyl other than a haloalkyl.

[0121] The term "alkenyl" as used herein refers to a straight, branched, or cyclic, saturated, monovalent or divalent radical, 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 can be in the trans or cis orientation. The double bond can be terminal or internal. The alkenyl group can be attached through an alkenyl moiety containing the double bond (e.g., ethenyl, propen-1-yl, and buten-1-yl), or the alkenyl group can be attached through an alkenyl moiety not containing the double bond (e.g., penten-4-yl). Where specified, the parent moiety is to be understood as being attached to the alkenyl group at the vinylic position of the double bond and not the non-vinylic position. For example, when an aromatic ring is substituted with a π-conjugated alkenyl group, it is to be understood as being substituted at the vinylic position and not the non-vinylic position. As another example, an aromatic ring substituted with a π-conjugated propenyl group is to be understood as a propen-1-yl or propen-2-yl group and not a propen-3-yl group. Some examples of monovalent (C2-C 20 Some examples of monovalent (C2-C 20 Some examples of monovalent (C2-C 20Some examples of alkenyl groups include those having 2 to 6 carbon atoms, such as -CHCH-, -CHCHCH2-, -CHCHCH2CH2-, and -CHCHCH2CH2CH2-. Some examples of branched divalent alkyl groups include -C(CH3)CH- and -CHC(CH3)CH2-. Some 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-C8)alkenyl. Alkenyl can also include substituted vinyl and unsubstituted (C3-C8)alkenyl. Representative substituted alkenyl groups can be substituted one or more times with any of the groups listed herein, such as monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxyl, nitro, alkylthio, alkoxy, and halogen groups. As another example, representative substituted alkenyl groups can be substituted with one or more fluorine, chlorine, bromine, iodine, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxyl, haloalkyl, hydroxyl, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, sulfido, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. In some embodiments, representative substituted alkenyl groups can be substituted from the group consisting of monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxyl, nitro, alkylthio, and alkoxy, but not halogen groups. Thus, in some embodiments, alkenyl can be substituted with non-halogen groups. In some embodiments, representative substituted alkenyl groups can be substituted with fluorine groups, substituted with bromine groups, substituted with halogens other than bromine, or substituted with halogens other than fluorine. 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 fluorine groups, or they can be substituted with one, two, three, or more non-fluorine groups.

[0122] 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-l-yl, propyn-2-yl, butynyl, butyn-l-yl, butyn-2-yl, butyn-3-yl, butyn-4-yl, pentynyl, pentyn-l-yl, hexynyl, examples include, but are not limited to, -CºCH, -CºC(CH3), -CºC(CH2CH3), -CH2CºCH, -CH2CºC(CH3), and -CH2CºC(CH2CH3), and the like.

[0123] As used herein, the term "aryl" refers to substituted or unsubstituted monovalent radicals derived from an aromatic hydrocarbon, which is a cyclic hydrocarbon having from 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 20 Some examples of (C6-C 20 )aryl groups include phenyl, naphthyl, azulenyl, biphenyl, indacenyl, fluorenyl, phenanthryl, triphenylenyl, pyrenyl, tetracenyl, benzophenanthryl, anthryl groups. Examples include substituted phenyl, substituted naphthyl, substituted azulenyl, substituted biphenyl, substituted indanyl, substituted fluorenyl, substituted phenanthryl, substituted triphenylenyl, substituted pyrenyl, substituted tetracenyl, substituted benzophenanthryl, and substituted anthryl groups. Examples also include unsubstituted phenyl, unsubstituted naphthyl, unsubstituted azulenyl, unsubstituted biphenyl, unsubstituted indanyl, unsubstituted fluorenyl, unsubstituted phenanthryl, unsubstituted triphenylenyl, unsubstituted pyrenyl, unsubstituted tetracenyl, unsubstituted benzophenanthryl, and unsubstituted anthryl groups. Aryl includes phenyl as well as non-phenyl aryl groups. As is clear from these examples, the term (C6-C 20 )aryl encompasses both monocyclic and polycyclic (C6-C 20 )aryl groups, including fused and non-fused polycyclic (C6-C

[0124] As used herein, the term "heterocyclic group" refers to a substituted aromatic, unsubstituted aromatic, substituted non-aromatic, and unsubstituted non-aromatic ring containing three or more atoms, one or more of which are heteroatoms, such as, but not limited to, N, O, and S. The term "heteroaryl" is a fully aromatic heterocyclic group and is therefore a subset of the term "heterocyclic group." The term "heterocyclic alkenyl" refers to a heterocyclic group containing an alkene within a non-aromatic ring, such that the alkene is the linking point to the parent moiety. Thus, a heterocyclic group can be a heterocyclic alkyl, heterocyclic alkenyl, or heteroaryl, or, if polycyclic, any combination thereof. In some embodiments, the heterocyclic group comprises 3 to about 20 ring members, while other such groups have 3 to about 15 ring members. In some embodiments, the heterocyclic group comprises a heterocyclic group containing 3 to 8 carbon atoms (C3-C8), 3 to 6 carbon atoms (C3-C6), or 6 to 8 carbon atoms (C6-C8). A heterocyclic group, referred to as a C2-heterocyclic group, can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms, etc. Similarly, a C4-heterocyclic group can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, etc. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. The heterocyclic ring can also contain one or more double bonds. A heteroaryl ring is one embodiment of a heterocyclic group. The term heterocyclic group includes fused-ring substances, including those containing fused aromatic and non-aromatic groups. Representative heterocyclic groups include, but are not limited to, piperidinyl, pyrrolidinyl, piperazine, and morpholinyl. For example, heterocyclic groups include, but are not limited to:

[0125]

[0126] Where X 1 Represents H, (C1-C 20 )alkyl, (C6-C 20 The heterocyclic group may contain an aryl or amine protecting group (e.g., a tert-butyloxycarbonyl group), and may be substituted or unsubstituted. Representative heterocyclic groups include furanyl, pyridyl, pyrazinyl, pyrimidinyl, triazineyl, thiopheneyl, tetrahydrofuranyl, pyrroleyl, etc. Azolyl, imidazole, triazolyl, tetrazolyl, benzo[] Azoline and benzimidazolinyl groups. In some embodiments, the heteroaryl group is a 5-membered heteroaryl group. In some embodiments, the heteroaryl group is not pyridine, pyrimidine, pyridazine, pyrazine, or a fused derivative thereof. π-Excess heteroaryl groups are electron-rich heteroaryl groups, allowing them to be used as electron-donating groups. Some examples of π-excess heteroaryl groups are furan, thiophene, indole, pyrrole, benzofuran, and benzothiophene.

[0127] The term "alkoxy" as used herein refers to an oxygen atom linked to an alkyl group (including cycloalkyl groups) as defined herein. Some examples of straight chain alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Some examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, t-butoxy, isopentoxy, isohexoxy, and the like. Some examples of cyclic alkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy, and the like. Alkoxy groups can comprise from 1 to about 12 to 20 or about 12 to 40 carbon atoms bonded to the oxygen atom, and can also comprise double or triple bonds, and can also comprise heteroatoms. Thus, alkoxy also includes oxygen atoms linked to alkynyl groups and oxygen atoms linked to alkynyl groups. For example, allyloxy is an alkoxy group within the meaning herein. Methoxyethoxy is also an alkoxy group within the meaning herein, as is methylenedioxy in the context of two adjacent atoms of a structure being substituted thereby.

[0128] The term "aryloxy" as used herein refers to an oxygen atom linked to an aryl group as defined herein. The point of substitution of the parent moiety is at the oxygen atom.

[0129] The term "arylcarbonyl" as used herein refers to a carbonyl (CO) group linked to an aryl group as defined herein. The point of substitution of the parent moiety is at the carbonyl group.

[0130] The term "heteroarylcarbonyl" as used herein refers to a carbonyl (CO) group linked to a heteroaryl group as defined herein. The point of substitution of the parent moiety is at the carbonyl group.

[0131] The term "aralkyl" as used herein 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, as well as fused (cycloalkylaryl)alkyl groups, such as 4-ethyl-indan. An aralkenyl 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 of the parent moiety is at the alkyl group.

[0132] The term "halo," "halogen," or "halide" group as used herein by itself or as part of another substituent means a fluorine, chlorine, bromine, or iodine atom, unless otherwise specified.

[0133] The term "amino" as used herein refers to -NH2, -NHR, -NR2, -NR3 + substituents of the form where each R is independently selected, and each of the protonated forms thereof (except -NR3 +), which cannot be protonated. Thus, any compound substituted with an amino group can be considered an amine. An "amino group" within the meaning herein can be a primary amino, secondary amino, tertiary amino, or quaternary amino group. "Alkylamino" groups include monoalkylamino, dialkylamino, and trialkylamino groups.

[0134] The term "acyl" as used herein refers to a group containing a carbonyl moiety, wherein the group is bonded through the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl group, and the like.

[0135] The term "formyl" as used herein refers to a group containing an aldehyde moiety. The point of substitution of the parent moiety is at the carbonyl group.

[0136] The term "alkoxycarbonyl" as used herein refers to a group containing a carbonyl moiety, wherein the group is bonded through 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 alkenyl 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 another instance, which is included in the definition of the term alkoxycarbonyl as defined herein, and also in the term "aryloxycarbonyl", the carbonyl carbon atom is bonded to an oxygen atom, which is bonded to an aryl group instead of an alkyl group.

[0137] The term "alkylcarbonylamido" as used herein refers to a group containing a carbonyl moiety, wherein the group is bonded through the carbonyl carbon atom. The carbonyl carbon atom is also bonded to a nitrogen group, which is bonded to one or more alkyl groups. In another instance of the term alkylcarbonylamido as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom, which is bonded to one or more aryl groups instead of, or in addition to, one or more alkyl groups. In another instance of the term alkylcarbonylamido as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom, which is bonded to one or more alkenyl groups instead of, or in addition to, one or more alkyl and / or aryl groups. In another instance of the term alkylcarbonylamido as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom, which is bonded to one or more alkynyl groups instead of, or in addition to, one or more alkyl, alkenyl, and / or aryl groups.

[0138] The term "carboxyl," as used herein, refers to a group that contains a carbonyl moiety in which the group is bonded through the carbonyl carbon atom. The carbonyl carbon atom is also bonded to a hydroxyl group or an oxygen-containing anion to produce a carboxylic acid or carboxylate salt. Carboxyl includes both the protonated form of the carboxylic acid and the salt form. For example, carboxyl can be understood as COOH or CO2H.

[0139] The term "alkylthio," as used herein, refers to a sulfur atom linked to an alkyl, alkenyl, or alkynyl group, as defined herein. The point of attachment of the parent moiety is at the sulfur atom.

[0140] The term "arylthio," as used herein, refers to a sulfur atom linked to an aryl group, as defined herein. The point of attachment of the parent moiety is at the sulfur atom.

[0141] The term "alkylsulfonyl," as used herein, refers to a sulfonyl group linked to an alkyl, alkenyl, or alkynyl group, as defined herein. The point of attachment of the parent moiety is at the sulfonyl group.

[0142] The term "alkylsulfinyl," as used herein, refers to a sulfinyl group linked to an alkyl, alkenyl, or alkynyl group, as defined herein. The point of attachment of the parent moiety is at the sulfinyl group.

[0143] The term "dialkylaminosulfonyl," as used herein, refers to a sulfonyl group linked to a nitrogen, which is further linked to two alkyl groups, as defined herein, and which can optionally be linked together to form a ring with the nitrogen. The term also includes groups in which the nitrogen is further linked to one or two alkenyl groups instead of alkyl groups. The point of attachment of the parent moiety is at the sulfonyl group.

[0144] The term "dialkylamino," as used herein, refers to an amino group linked to two alkyl groups, as defined herein, and which can optionally be linked together to form a ring with the nitrogen. The term also includes groups in which the nitrogen is further linked to one or two alkenyl groups instead of alkyl groups. The point of attachment of the parent moiety is at the nitrogen atom.

[0145] The term "dialkylamido," as used herein, refers to an amido group linked to two alkyl groups, as defined herein, and which can optionally be linked together to form a ring with the nitrogen. The term also includes groups in which the nitrogen is further linked to one or two alkenyl groups instead of alkyl groups. The point of attachment of the parent moiety is at the amido group.

[0146] Each of the plurality of substituents described herein can be substituted or unsubstituted. As used herein, the term "substituted" means substituted with one or more moieties (substituents) including, but not limited to, the following: deuterium (D), halogen (e.g., F, Cl, Br, and I), R, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, methylenedioxy, ethylenedioxy, (C3-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, where R can be hydrogen, (C1-C 20 )alkyl, or (C6-C 20) aryl. Substituted also includes groups substituted with one or more groups including, but not limited to, the following: 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, sulfenyl, alkylsulfenyl, arylthiol, alkylsulfonyl, alkylsulfenyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. When there are two or more adjacent substituents, the substituents can be joined to form a carbocyclic or heterocyclic ring. Such adjacent groups can have a vicinal or germinal relationship, or they can be adjacent on a ring, for example, in an ortho arrangement. Each instance of substituted is understood to be independent. For example, a substituted aryl can be substituted with a bromo, and a substituted heterocycle on the same compound can be substituted with an alkyl. It is contemplated that a substituted group can be substituted with one or more non-fluoro groups. As another example, a substituted group can be substituted with one or more non-cyano groups. As another example, a substituted group can be substituted with one or more groups other than haloalkyl. As yet another example, a substituted group can be substituted with one or more groups other than t-butyl. As yet another example, a substituted group can be substituted with one or more groups other than trifluoromethyl. As further examples, a substituted group can be substituted with one or more groups other than nitro, other than methyl, other than methoxymethyl, other than dialkylaminosulfonyl, other than bromo, other than chloro, other than amido, other than halo, other than benzodioxepinyl, other than polycyclic heterocyclyl, other than polycyclic substituted aryl, other than methoxycarbonyl, other than alkoxycarbonyl, other than thienyl, or other than nitrophenyl, or with groups that satisfy combinations of such descriptions. Further, substituted is also understood to include fluoro, cyano, haloalkyl, t-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thienyl, and nitrophenyl. In various embodiments, a substituted group can be substituted with groups other than carbonyl-containing groups, nitro, cyano, sulfinyl, sulfonyl, or halogen-containing groups. In various embodiments, a substituted group can be substituted with groups other than electron-withdrawing groups. Some of the substituted groups in certain embodiments can be substituted with only one or more electron-donating groups.

[0147] The term "boronate group" as used herein refers to the following structure, wherein R 8 and R 9 are each independently H or C1-C 10 alkyl, or R 8 and R9 with the boronic ester to which they are attached provide C2-C 10 cyclic boronic ester.

[0148]

[0149] The term "π-conjugated" group as used herein 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-conjugated group is attached, such that electrons can be shared between the two p-orbitals and a lower energy state is achieved. One exemplary parent moiety is X, R 4 , R 5 , R 6 , and R 7 to which it is attached. Substituents that are π-conjugated groups can also have π-bonded electrons that are delocalized through the substituent and the parent moiety to which it is attached. Some examples of π-conjugated groups include substituted or unsubstituted C2-C 10 alken-1-yl, C2-C 10 alken-2-yl, C2-C 10 alkenyn-1-yl, C2-C 10 heterocycloalken-1-yl, C2-C 10 heterocycloalken-2-yl, C6-C 10 aryl, or heteroaryl. Other examples of π-conjugated groups include substituted or unsubstituted vinyl, acetylenyl, C6-C 10 aryl, or heteroaryl further substituted with 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 aryl, or heteroaryl. Other examples include, for example, substituted or unsubstituted biaryl, diheteroaryl aryl vinyl, heteroaryl vinyl, and C2-C 10 alken-1-yl aryl, phenyl heteroaryl, and heteroaryl aryl.

[0150] 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, e.g., 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 in terms of their Hammett Substituent Constant (also known as sigma value (σ value)). In various embodiments, the σ value of the electron donating group is 0.3 or lower, 0.2 or lower, 0.1 or lower, or a negative σ value. In other embodiments, the electron donating group is a non-halogen group with a σ value of 0.3 or lower, 0.2 or lower, 0.1 or lower, or a negative σ value. In cases where the position of the substituent significantly affects its σ value, the σ value should be determined relative to the position of the group X. For example, a σ meta value can be provided to determine the σ value of the substituent at R 6 . A σ para value can be provided to determine the σ value of the substituent at R 5 . The σ value can be obtained according to published tables or experimentally. See, e.g., J. E. Leffler and E. Grunwald, Rates and Equilibria of Organic Reactions, Wiley, 1963 (Dover reprint), which is incorporated herein by reference in its entirety. Examples of electron donating groups include oxygen-containing anions, hydroxyl, amino, sulfido, alkylamino, dialkylamino, alkoxy, alkylthio, acylamino, acyloxy, alkyl, alkenyl, vinyl, aryl, electron-rich heteroaryl.

[0151] Another way to determine whether a particular substituent on a given structure is electron donating is by comparing the pKa of the phenol group (i.e., X = OH) of the substituted structure to the pKa of the phenol group of an otherwise identical structure that is not substituted. For example, the phenol pKa of a compound in which R 4 , R 5 , and R 6 are H and R 7 is vinyl can be compared to the phenol pKa of a compound in which R 4 , R 5 , R 6 , and R 7 are H.

[0152] In various embodiments, if the pKa of the phenolic 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, then R 4 , R 5 , R 6 , and R 7 can be understood to provide a net donating effect. In various embodiments, if the pKa of the phenolic group at X is greater than the pKa of a compound in which R 4 , R 5 , R 6 , and R 7 are H, then R 4 , R 5 , R 6 , and R 7 provide a net electron donating effect.

[0153] In other embodiments, X is OH or an oxygen-containing anion and the pKa is 9.0 or greater, 9.5 or greater, 10.0 or greater, 10.5 or greater, or 11.0 or greater.

[0154] An “electron-withdrawing group,” sometimes abbreviated as EWG, refers to a group that has a net electron-withdrawing effect relative to hydrogen. Electron-withdrawing groups are well known in the art. See, e.g., 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 reactive luminescent intermediates, some embodiments of the present disclosure can contain one or more EWGs, provided that the net overall effect of the substituents is electron-donating. For example, in some embodiments, R 4 , R 5 , R 6 , and R 7 may include one or more electron-withdrawing groups (EWGs), 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. Some examples of electron-withdrawing groups include acrylate groups, such as alkyl acrylate (e.g., CH3C(O)CH=CH-) and cyano acrylate (NCCH=CH-) groups.

[0155] The term alcohol protecting group as used herein refers to a substituent on an oxygen group that renders the oxygen inert to the various conditions under which alcohols typically react, but which is readily removed when subjected to certain conditions. Alcohol protecting groups as described herein will generally increase the stability of the dioxetane moiety and will facilitate the decomposition of the dioxetane after their removal. Thus, alcohol protecting groups include phosphates such as PO3Na2, PO3Cl2, and PO3H2, glycosyl groups, dinitrobenzenesulfonyl aminobenzyl 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 (3 ed.). J. Wiley, the entirety of which is incorporated herein. Alcohol protecting groups include acetyl, benzoyl, benzyl, methoxyethoxymethyl, dimethyltrityl, methoxymethyl, methylthiomethyl, pivaloyl, tetrahydropyranyl, tetrahydrofuranyl, trityl, trialkylsilyl, trialkylsilyloxymethyl, dialkylarylsilyl, glycosyl, pyranyl, galactosyl, and ethoxyethyl. Alcohol protecting groups also include groups in which the alcohol is further substituted with a protecting group that can fragment a linker, where upon deprotection of such a protecting group, the linker fragments and is eliminated from the alcohol. The following compounds are additional examples of alcohols substituted with alcohol protecting groups:

[0156]

[0157] In various embodiments, the protecting group G can be an enzyme cleavable group, where the cleavable group is removed by the analyte of interest, for example in the presence of an enzyme capable of cleaving the enzyme cleavable group, providing 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 can be cleaved by a particular enzyme.

[0158] The term "surfactant-based luminescence enhancer" as used herein refers to a class of compounds that are typically used to improve dioxetane intensity in aqueous solutions. Emerald™ and Emerald-ITM enhancers are some examples of surfactant-based dyes commercially available from Thermo Fisher Scientific (Waltham, MA). Additional examples of surfactant-based luminescence enhancers are described in Schaap, A. P.; Akhavan, H.; Romano, L. J. Clin. Chem. 1989, 35 (9), 1863, which is incorporated by reference in its entirety. In various embodiments, the surfactant-based luminescence enhancer contains a tail portion that is a non-cyclic alkyl of at least 8 carbons and a head portion that is one or more quaternary ammonium salts, pyridinium salts, 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 cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'-tributyl quaternary ammonium salts, pyridinium salts, 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 cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a'-tributyl -para-dichlorobenzene, poly(vinylbenzyltributylammonium chloride )(TBE), poly(vinylbenzyltrioctylammonium chloride )(TBE), poly(vinylbenzyltrioctylammonium chloride ), and the like. In various other embodiments, the surfactant-based luminescence enhancer is a non-ionic Triton X-100, a Tween surfactant, a surfactant with long alkyl chains with a polyethylene glycol head, surfactant, surfactant, surfactant, octylphenoxy polyethoxyethanol, and the like. The surfactant-based luminescence enhancer can also include surfactants with a fluorescein head group, such as N-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-5-yl)tetradecanamide (fluorescein surfactant), and the like.

[0159] In some cases, the compounds described herein (e.g., compounds of Formula (I)-(X) can contain chiral centers. All diastereomers of the compounds described herein, as well as racemates, are contemplated herein.

[0160] The terms "salt" and "pharmaceutically acceptable salt" as used herein refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of salts include alkali and alkaline earth metal salts of the ionized form of the disclosed compounds. For example, lithium, sodium, potassium, calcium, or magnesium salts. The disclosed compounds can be salts comprising a cationic metal and an anionic organic compound, for example, a compound with an oxygen-containing anion and a sodium cation. 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. The pharmaceutically acceptable salts include the conventional nontoxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.

[0161] Pharmaceutically acceptable salts can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. In some cases, such salts can be prepared by reacting the free acid or free base forms of these compounds with a stoichiometric (or greater) amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of both; typically, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference.

[0162] The term "solvate" means a compound or salt thereof, which further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.

[0163] Values ​​expressed in range form should be interpreted flexibly to include not only the numerical value explicitly stated as the limit of the range, but also all individual numerical values ​​or subranges covered within that range, as if each numerical value and subrange were explicitly stated. 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%, but also individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges within the indicated range (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). Unless otherwise stated, the statement “about X to Y” has the same meaning as “about X to about Y”. Similarly, unless otherwise stated, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z”.

[0164] In this document, unless the context clearly specifies otherwise, nouns without quantifiers are used to include one or more than one. Unless otherwise stated, the term "or" is used to indicate a non-exclusive "or". Furthermore, it should be understood that the wording or terms used herein without further definition are for descriptive purposes only and not restrictive. Any use of section headings is intended to aid reading the document and should not be construed as limiting. Additionally, information relating to section headings may appear within or outside that particular section. Moreover, all publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety as if individually incorporated by reference. If there is any inconsistency between the use of this document and those documents incorporated by reference, the usage in the incorporated references shall be considered supplementary to the usage in this document; in the case of contradictory inconsistencies, the usage in this document shall prevail.

[0165] In the methods described herein, steps may be performed in any order without departing from the principles of the invention, except where the timing or sequence of operations is explicitly stated. Furthermore, unless the explicit language of the claims specifies that a particular step may be performed individually, specific steps may be performed simultaneously. For example, claimed step X and claimed step Y may be performed simultaneously in a single operation, and the resulting process will fall within the scope of the claimed method.

[0166] The term “about” as used in this document may allow for a certain degree of variability in the value or range, for example, within 10%, 5%, or 1% of the specified value or range limit.

[0167] It is contemplated that each of the above embodiments can be applicable to each combination with the other embodiments described herein. For example, embodiments corresponding to Formula (I) are likewise contemplated to be applicable to Formulas (II) through (X). As another example, embodiments corresponding to Formula (II) are likewise contemplated to be applicable to each of Formulas (I) and (III) through (X).

[0168] The term "about" as used herein can allow for a degree of variability in a value or range, for example within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

[0169] The term "substantially" as used herein refers to a majority or a substantial part of, such as 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.

[0170] The term "substantially free of" or "substantially free from" as used herein means less than about 1%, 0.5%, 0.1%, 0.05%, 0.001%, or less than about 0.0005% or less, about 0%, below the limit of quantitation, below the limit of detection, or 0%.

[0171] Those skilled in the art will appreciate that many modifications can be made to the embodiments described herein without departing from the spirit and scope of the disclosure. Therefore, it is intended that the description not serve as a limitation of the presented examples but rather be accorded the full scope provided by the appended claims and their equivalents. Additionally, some features of the disclosure can be utilized without the corresponding use of other features. Accordingly, the purpose of the above description and exemplified embodiments is to illustrate the principles of the present disclosure and not to limit it, and modifications and arrangements can be included therein. Examples

[0172] The present invention can be better understood by reference to the following examples, which are offered by way of illustration. The present invention is not limited to the examples given herein.

[0173] General Methods

[0174] The various compounds of the present disclosure can be synthesized according to a variety of methods, including but not limited to the synthetic methods described in PCT International Application WO 1996 / 015122 Al, U.S. Patent No. 4,962,192, or U.S. Patent No. 5,004,565, each of which is incorporated herein by reference in its entirety.

[0175] Chemiluminescence (emission) intensity can be measured using a Turner Designs (Sunnyvale, CA) TD-20e photometer, a BMG Labtech light-emitting plate reader, a charge-coupled device (CCD) camera photometer, or any other suitable light intensity measurement device. In the examples listed below, solutions containing different concentrations of alkaline phosphatase (e.g., AP4, AP6, AP8, and AP9) were used, where each number represents a sequential dilution of 10 from the initial stock solution. 4 10 6 10 8 and 10 9 The compounds and enhancers (if used) were tested at their near-optimal concentrations.

[0176] Nuclear magnetic resonance (NMR) spectra were obtained in D2O and CDCl3 solutions using a 400 MHz spectrometer.

[0177] Amine-based buffers “221” or “σ-221” are available from Sigma-Aldrich (St. Louis, MO).

[0178] Example 1

[0179] Dioxane compounds with the following structures were obtained:

[0180]

[0181] 1 H NMR (400MHz, CDCl3) δppm 7.30-7.05 (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).

[0182] An initial solution of the compound was prepared in dioxane (1 mg of compound per 1 mL of dioxane), then mixed with water (20 μL dioxane solution in 180 μL of water) and subsequently treated with 200 μL of amine-based 221 buffer at 37 °C. Chemiluminescence intensity was measured over time after treatment with the compound in an alkaline buffer. Figure 1 The diagram provides a graph showing the luminescence intensity over time, illustrating the drawout over long periods. The compound of Example 1 shows a chemiluminescent half-life of 3.79 minutes and a ∑ value of 1.13E+5.

[0183] Example 2

[0184] A dioxetane compound having the following structure was obtained:

[0185]

[0186] 1 H NMR (400 MHz, CDC13) δ ppm 7.63 (m, 1 H), 7.31 (m, 1 H), 7.15 (m, 1 H), 6.0 (br, 1 H), 3.22 (s, 3 H), 30 (s, 1 H), 224 (s, 1 H), 21-14 (m, 12 H)

[0187] Example 2 was tested in a similar manner to Example 1 using 10 μL of a 1 mg / mL sample of the test compound in THF. The compound of Example 2 showed a chemiluminescent half-life of 4.91 minutes. The addition of an electron withdrawing chlorine on the ortho phenyl ring resulted in a slower increase in emission rate and a longer luminescent half-life.

[0188] Example 3

[0189] A dioxetane compound having the following structure was obtained:

[0190]

[0191] 1 H NMR (400 MHz, CDC13) δ ppm 7.05 (m, 2 H), 3.44 (s, 3 H), 3.24 (s, 1 H), 2.54 (s, 1 H), 2.1-1.6 (m, 12 H).

[0192] Example 3 was tested in a similar manner to Example 2. The compound of Example 3 showed a chemiluminescent half-life of 9.22 minutes. The addition of two electron withdrawing chlorine groups resulted in an even slower increase in emission rate and a longer luminescent half-life.

[0193] Example 4

[0194] A dioxetane compound having the following structure was obtained:

[0195]

[0196] 1 H NMR (400 MHz, CDC13) δ ppm 7.70 (m, 1 H), 745-665 (m, 2 H), 559 (br, 1 H), 3.20 (s, 3 H), 3.00 (s, 1 H), 2.16 (s, 1 H), 1.90-1.40 (m, 10 H), 1.27 (m, 1 H), 1.04 (m, 1).

[0197] Example 4 was tested in a similar manner to Example 2. The compound of Example 4 showed a chemiluminescence half-life of 2.10 minutes. Compared to Examples 1-3, the slight addition of an electron donating iodine atom resulted in a slightly increased emission rate and a slightly shorter half-life.

[0198] Example 5

[0199] A dioxetane compound having the following structure was obtained:

[0200]

[0201] 1 H NMR (400 MHz, CDC13) δ ppm 744 (m, 1H), 7.40-690 (m, 3H), 582 (d, J= 16 Hz, 1H), 5.65 (br, 1H), 5.41 (d, J= 16 Hz, 1H), 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).

[0202] Example 5 was tested in a similar manner 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 1 mL of dioxane), which was then mixed with water (10 μL of the dioxane solution in 100 μL of water) and subsequently treated with 200 μL of the amine-based 221 buffer at 37°C.

[0203] A plot showing the luminescence intensity over time is provided in Figure 5 Example 5 showed a sharp signal and a chemiluminescence half-life of 23 seconds and a ∑ value of 1.28E+5. Compared to Examples 1-4, the addition of an electron donating vinyl group on the benzene ring of the PPD resulted in a higher emission intensity, a faster emission rate increase, and a significantly shorter luminescence half-life.

[0204] Example 6

[0205] A dioxetane compound having the following structure was obtained:

[0206]

[0207] 1H NMR (400 MHz, CDC13) δ ppm 7.51 (m, 1 H), 7.48 (m, 1 H), 7.41 (m, 1 H), 7.35 (m, 1 H), 7.40-701 (m, 2 H), 5.52 (br, 1 H), 326 (s, 3 H), 3.04 (s, 1 H), 226 (s, 1 H), 190-1.46 (m, 10 H), 1.28 (m, 1 H), 1.13 (m, 1 H).

[0208] Example 6 was tested in a similar manner to Example 1. The compound of Example 6 showed a chemiluminescence half-life of 12.7 seconds. The addition of even more electron donating 3-thienyl groups resulted in higher emission intensity, faster emission rate increase, and shorter luminescence half-life compared to Examples 1-5.

[0209] Example 7

[0210] A dioxetane compound having the following structure was obtained:

[0211]

[0212] 1 H NMR (400 MHz, CDC13) δ ppm 7.51 (m, 1 H), 7.42 (m, 1 H), 7.45-7.01 (m, 4 H), 5.80 (br, 1 H), 3.26 (s, 3 H), 3.04 (s, 1 H), 2.08 (s, 1 H), 2.1-1.5 (m, 10 H), 1.25 (m, 1 H), 1.10 (m, 1 H).

[0213] Example 7 was tested in a similar manner to Example 1 using 10 μΐ^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 1 mL of dioxane), which was then mixed with water (10 μΐ^of the dioxane solution in 90 μΐ^of water), and subsequently treated with 200 μΐ^of the amine-based 221 buffer at 37 °C.

[0214] A plot showing luminescence intensity over time is provided in Figure 6 Example 7. The compound of Example 7 showed a sharp signal and a chemiluminescence half-life of 11.8 seconds and a ∑ value of 1.32E+05. Here, the 2-thienyl group places the electron-rich sulfur atom closer to the benzene ring and thus provides a stronger electron donating effect. The compound of Example 7 resulted in higher emission intensity, faster emission rate increase, and shorter luminescence half-life compared to Examples 1-6.

[0215] Example 8

[0216] A dioxetane compound having the following structure is obtained:

[0217]

[0218] 1 H NMR (400 MHz, CDC13) δ ppm 7.68 (d, J = 2 Hz, IH), 7.62 (d, J = 1.2 Hz, IH), 7.56 (s, IH), 7.50-7.10 (br, 2H), 6.80 (br, IH), 3.27 (s, 3H), 3.05 (s, IH), 2.23 (s, IH), 1.88-1.45 (m, 10H), 1.31-1.26 (m, IH), 1.12-1.08 (m, IH).

[0219] Example 8 is tested in a similar manner to Example 1 using 10 μL of a 0.001 mg / mL sample of the test compound in dioxane. Specifically, an initial solution of the compound is prepared in dioxane (0.001 mg of compound per 1 mL of dioxane), which is then mixed with water (10 μL of the dioxane solution in 90 μL of water) and subsequently treated with 100 μL of the amine-based 221 buffer at 37°C.

[0220] A graph showing the luminescence intensity over time is provided in Figure 7 Example 8 shows a sharp signal and a chemiluminescence half-life of 23 seconds and a ∑ value of 9.76E+04. While not wishing to be bound by any particular theory, it is believed that the compound of Example 8 results in a higher emission intensity than Example 7 due to the extended π conjugation system. The half-life of sample 8 is greater than sample 7 due to the 4-CN electron withdrawing effect.

[0221] Example 9

[0222] A dioxetane compound having the following structure is obtained:

[0223]

[0224] 1 H NMR (400 MHz, CDC13) δ ppm 7.68 (d, J = 2 Hz, IH), 7.62 (d, J = 1.2 Hz, IH), 7.56 (s, IH), 7.50-7.10 (br, 2H), 6.80 (br, IH), 3.27 (s, 3H), 3.05 (s, IH), 2.23 (s, IH), 1.88-1.45 (m, 10H), 1.31-1.26 (m, IH), 1.12-1.08 (m, IH).

[0225] Example 9 was tested in a similar manner 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 1 mL of dioxane), which was then mixed with water (10 μL of the dioxane solution in 90 μL of water), and subsequently treated with 100 μL of amine-based Buffer 221 at 37 °C. In Figure 8 A plot showing luminescence intensity over time is provided in

[0226] Example 10

[0227] A dioxetane compound having the following structure was obtained:

[0228]

[0229] 1 H NMR (400 MHz, CDC13) δ ppm 7.72 (d, J = 7.2 Hz, 2H), 7.68 (m, 2H), 7.61 (d, J = 8 Hz, IH), 7.52 (d, J = 8 Hz, IH), 7.45 (d, J = 4 Hz, IH), 7.44-77.05 (m, 2H), 5.81 (br, IH), 3.27 (s, 3H), 3.04 (s, IH), 2.25 (s, IH), 1.90-1.45 (m, 10H), 1.31-1.26 (m, IH), 1.15-1.08 (m, IH).

[0230] Example 10 was tested in a similar manner 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 1 mL of dioxane), which was then mixed with water (10 μL of the dioxane solution in 90 μL of water), and subsequently treated with 100 μL of amine-based Buffer 221 at 37 °C. In Figure 9 A plot showing luminescence intensity over time is provided in

[0231] Example 11

[0232] A dioxetane compound having the following structure was obtained:

[0233]

[0234] 1H NMR (400 MHz, CDC13) δ ppm 7.56 (m, 2 H), 7.42 (m, 1 H), 7.36 (m, 2 H), 7.22 (d, J=6 Hz, 2 H), 6.93 (d, J=8 Hz, 2 H), 5.91 (s, 1 H), 3.85 (s, 3 H), 3.27 (s, 3 H), 3.04 (s, 1 H), 2.27 (s, 1 H), 1.90-1.45 (m, 10 H), 1.31-1.26 (m, 1 H), 1.15-1.08 (m, 1 H).

[0235] Example 11 was tested in a similar manner 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 1 mL of dioxane), which was then mixed with water (10 μL of the dioxane solution in 90 μL of water) and subsequently treated with 100 μL of the amine-based 221 buffer at 37 °C. The chemiluminescent intensity was measured over time. Figure 10 A plot showing the luminescent intensity over time is provided in FIG. 2.

[0236] Example 12

[0237] A dioxetane compound having the following structure was obtained PPD]:

[0238]

[0239] 1 H NMR (400 MHz, D20) δ ppm 7.40-7.15 (m, 4 H), 3.24 (s, 3 H), 289 (s, 1 H), 2.28 (s, 1 H), 1.90-1.50 (m, 10 H), 1.28 (d, J=13.2 Hz, 1 H), 0.99 (d, J=10 Hz, 1 H).

[0240] An initial solution of the compound was prepared in the 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. The chemiluminescent intensity was measured over time after the compound was combined with the alkaline phosphatase solution. In Figure 11 A plot showing the luminescent intensity over time is provided in FIG. 2. The compound showed a slow and gradual increase in emission intensity that did not reach a maximum light intensity at a steady state plateau over 15 minutes and provided a ∑ value of 2.27E+05.

[0241] Example 13

[0242] A dioxetane compound having the following structure was obtained:

[0243]

[0244] An initial solution of the 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 a solution of Alkaline Phosphatase (AP8) at 37°C. Chemiluminescent intensity was measured over time after the compound was combined with the alkaline phosphatase solution. In Figure 12 A graph showing luminescent intensity over time is provided in FIG. 6. The compound showed a slow and gradual increase in emission intensity, which did not reach a maximum light intensity at a steady state plateau over 15 minutes, and provided a ∑ value of 1.91E+05.

[0245] Example 14

[0246] A dioxetane compound was prepared according to the following structure:

[0247]

[0248] 1 H NMR (400 MHz, D20) δ ppm 7.86 (br, IH), 7.81 (d, J=8.4 Hz, IH), 7.74 (S, IH), 7.51 (d, J=5.2 Hz, IH), 7.21 (br, IH), 7.18 (d, J=7.2 Hz, IH), 3.28 (s, 3H), 2.90 (s, IH), 2.33 (s, IH), 1.85-1.55 (m, 10H), 1.28 (d, J=8 Hz, IH), 1.10 (d, J=12 Hz, IH).

[0249] An initial solution of the compound was prepared in 221 buffer (Compound: 0.25 mg / mL, TBE Enhancer: 5 mg / mL). Next, a 100 μL aliquot of the initial solution was combined with a solution of Alkaline Phosphatase (AP8) at 37°C. Chemiluminescent intensity was measured over time after the compound was combined with the alkaline phosphatase solution. In Figure 13 A graph showing luminescent intensity over time is provided in FIG. 6. The compound showed a slow and gradual increase in emission intensity, which did not reach a maximum light intensity at a steady state plateau over 15 minutes, and provided a ∑ value of 1.91E+05.

[0250] Example 15

[0251] A dioxetane compound was prepared according to the following structure:

[0252]

[0253] 1 H NMR (400 MHz, D20) δ ppm 7.95 (m, 1 H), 7.85 (br, 1 H), 7.65-7.55 (m, 2 H), 7.52 (m, 1 H), 7.25 (br, 1 H), 3.28 (s, 3 H), 2.90 (s, 1 H), 2.34 (s, 1 H), 1.90-1.55 (m, 10 H), 1.38 (m, 1 H), 1.08 (m, 1 H).

[0254] An initial solution of the compound was prepared in 221 buffer (compound: 0.25 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. Chemiluminescent intensity was measured over time after the compound was combined with the alkaline phosphatase solution. Figure 14 A graph showing luminescent intensity over time is provided in FIG. 16. The compound showed a rapid increase in intensity, reaching a maximum steady state intensity at 2 minutes, and provided a sum value of 1.01E+06. The compound of Example 15 provided luminescence with higher intensity and a faster response compared to the compounds of Examples 12-13.

[0255] Example 16

[0256] A dioxetane compound was prepared according to the following structure:

[0257]

[0258] 1H NMR (400 MHz, D20) δ ppm 7.67 (d, J=8.4 Hz, 1 H), 7.66 (br, 1 H), 7.26 (br, 1 H), 7.16-6.06 (m, 1 H), 5.88 (d, J=16 Hz, 1 H), 5.37 (d, J=12.4 Hz, 1 H), 3.27 (s, 3 H), 2.88 (s, 1 H), 2.30 (s, 1 H), 1.84-1.56 (m, 10 H), 1.28 (d, J=9.2 Hz, 1 H), 1.05 (d, J=12.8 Hz, 1 H).

[0259] 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. Chemiluminescent intensity was measured over time after the compound was combined with the alkaline phosphatase solution. Figure 15A plot showing luminescence intensity over time is provided in FIG. 16. The compound shows a rapid increase in intensity, reaching a maximum steady state intensity in less than 3 minutes, and provides a ∑ value of 1.59E+06. The compound of Example 16 provides luminescence with higher intensity and faster response compared to the compounds of Examples 12-15.

[0260] In another experiment, an initial solution of the compound was prepared in water (compound: 0.1 mg / mL). Next, 100 μL of the initial solution was combined with 10 μL of an alkaline phosphatase (AP4) solution and 300 uL of a TEB 221 buffer at 5 mg / mL at 37 °C. The chemiluminescence intensity was measured over time after the compound was combined with the alkaline phosphatase solution. The results are shown in FIG. 17. Figure 16 A plot showing luminescence intensity over time is provided in FIG. 16. The compound shows a rapid increase in intensity, reaching a maximum steady state intensity in less than 3 minutes, and provides a ∑ value of 1.59E+06. The compound of Example 16 provides luminescence with higher intensity and faster response compared to the compounds of Examples 12-15.

[0261] Example 17

[0262] A dioxetane compound was prepared according to the following structure:

[0263]

[0264] 1 H NMR (400 MHz, D20) δ ppm 7.88 (m, 2H), 7.75 (m, 1H), 7.52 (m, 1H), 7.30 (br, 1H), 3.26 (s, 3H), 288 (s, 1H), 229 (s, 1H), 1.90-1.55 (m, 10H), 124 (m, 1H), 1.04 (m, 1H).

[0265] An initial solution of the compound was prepared in 221 buffer (compound: 0.2 mg / mL, without TBE enhancer). Next, 100 μL of the initial solution was combined with 10 μL of an alkaline phosphatase (AP9, [1.24E-20 moles / μL]) solution at 37 °C. The chemiluminescence intensity was measured over time after the compound was combined with the alkaline phosphatase solution. The results are shown in FIG. 18. Figure 17 A plot showing luminescence intensity over time is provided in FIG. 16. The compound shows a rapid increase in intensity, reaching a maximum steady state intensity in less than 3 minutes, and provides a ∑ value of 1.59E+06. The compound of Example 16 provides luminescence with higher intensity and faster response compared to the compounds of Examples 12-15.

[0266] Figure 18 A plot showing luminescence intensity over time is provided in FIG. 16. The compound shows a rapid increase in intensity, reaching a maximum steady state intensity in less than 3 minutes, and provides a ∑ value of 1.59E+06. The compound of Example 16 provides luminescence with higher intensity and faster response compared to the compounds of Examples 12-15. Luminescence intensity of PPD over time under similar conditions:

[0267]

[0268] The results indicate that the addition of a π-conjugated group and an electron donating group directly to the aromatic ring attached to the dioxetane results in an increase in the speed and intensity of luminescence upon fragmentation of the dioxetane (see Examples 5-11 and 16-18). Figures 5 to 10). Such substitutions correspond, for example, to placing π-conjugated and electron-donating groups at R 4 , R 5 , R 6 or R 7 or Q in Formula III.

[0269] For example, the vinyl substituent produces dioxetanes that provide a rapid, intense burst of luminescence that is at least 20 times stronger than the corresponding unsubstituted compound (see Examples 5, comparing Figure 5 and Figure 1 ). Substitution with a thiophene group provides another example of such advantages. (See Examples 6 and 7, comparing Figure 6 and Figure 1 ). The examples also show that adjusting the electron-donating extent of the π-conjugated electron-donating group can affect the emission intensity, the faster increased emission rate, and the shorter luminescent half-life (see Examples 7 and 8). Specifically, reducing the electron-donating effect by directly substituting the π-conjugated electron-donating group with an electron-withdrawing group results in a lower intensity, a slower increased emission rate, and a longer luminescent half-life (compare Figure 7 and Figure 6 ). Nonetheless, the effect of directly substituting with thiophene, a type of π-conjugated electron-donating group, provides significant advantages over the corresponding unsubstituted compound (compare Figure 7 and Figure 1 ). Moreover, the examples show that placing electron- withdrawing or electron-donating groups (e.g., cyano or methoxy) remotely does not outperform the advantages of directly placing a π-conjugated electron-donating group on the central aryl ring that binds the dioxetane (see Examples 9, 10, and 11; compare Figure 8 , 9 and 10 with each other and with Figure 1 ). Thus, the π-conjugated electron-donating group (e.g., vinyl, aryl, or heteroaryl) can be further substituted and modified without destroying the improved luminescent properties.

[0270] In contrast, compounds substituted with non-π conjugated groups or non-electron-donating groups result in slower luminescence and longer luminescent half-lives (see Examples 2 through 4 and Figures 2 to 4 ). In the case of chloro, the effect is additive upon addition of a second chloro group (compare Figure 2 and Figure 3 ). Iodo (not a π-conjugated group, and neither a strong electron-donor nor a strong electron-acceptor) results in a relatively small effect, with no significant increase in luminescence (see Example 4).

[0271] 3-Phosphonophenyl derivatives were also prepared and tested under aqueous conditions by activation with alkaline phosphatase (ALP) (see Examples 12 through 16 and Figures 11 to 16). Two of the examples correspond to commercially available dioxetanes, namely PPD (Example 12) and Tropix (Example 13), both lack electron donating groups on the central aromatic ring to which the dioxetane is bound. Both commercially available dioxetanes exhibit slow luminescence, which does not reach a maximum light intensity or plateau of steady state intensity for more than 15 minutes. In contrast, aqueous compositions of dioxetanes with π-conjugated electron donating groups show significantly increased luminescence speed and luminescence intensity compared to the commercially available dioxetanes (see Examples 14 to 16, which compare Figures 13 to 16 to Figure 11 and Figure 12 ).

[0272] Without wishing to be bound by any theory, it was surprisingly shown that increased electron density on the aromatic ring and the π-conjugated substituent accelerates the rate limiting step that generates the transient excited-state species that undergoes chemiluminescent decay. Based on these results, the speed and intensity of dioxetane chemiluminescence can be increased by using π-conjugated, electron donating or both substituents. Furthermore, one example was tested under aqueous conditions without surfactant-based luminescence enhancer, which provided surprisingly high intensity luminescence. Thus, the compounds and compositions of the present disclosure represent a significant improvement over commercially available dioxetanes.

[0273] Example 18

[0274] The dioxetane compounds were prepared according to the following structure, consistent with the synthetic methods described herein:

[0275]

[0276] (D2O ppm) : 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).

[0277]

[0278] (D2O)7.56-7.49(m,2H),7.26-7.19(m,1H),5.71(d,J=16.4Hz,1H),5.26(d,J=12.4Hz,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).

[0279]

[0280] (CD3OD)7.53-7.50(m,1H),7.40-6.85(m,3H),5.74(d,J=16.5Hz,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).

[0281]

[0282] (CDCl3)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).

[0283]

[0284] (CDCl3)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).

[0285]

[0286] (CDCl3) 7.50 (d, J = 4 Hz, IH), 7.30-7.18 (m, 2H), 6.95-6.85 (m, 2H), 5.48 (br, IH), 3.28 (s, 3H), 2.86 (s, IH), 2.19 (s, IH), 1.90-1.46 (m, 10H), 1.30-1.26 (m, IH), 1.24-1.21 (m, IH).

[0287]

[0288] (CDCl3) 7.40-6.80 (m, 3H), 5.80 (s, IH), 5.44 (s, IH), 5.20 (s, IH), 3.23 (s, 3H), 3.02 (s, IH), 2.24 (s, IH), 2.13 (s, 3H), 1.96-1.44 (m, 10H), 1.32-1.24 (m, IH), 1.10-0.96 (m, IH).

Claims

1. A compound of Formula I ###0001### wherein: G is an alcohol protecting group.

2. The compound of claim 1, wherein Q is unsubstituted vinyl. R 1 and R 2 together with the carbon to which they are attached provide a spirocyclophane, norbornane or camphane; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 4 , R 5 , R 6 , and R 7 are each independently H, Q, X, hydroxyl, halogen, amino, sulfanyl, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, C1-C 10 alkoxy, C1-C 10 alkylamino, C1-C 10 trialkylammonium salt, C1-C 10 alkylsulfanyl, C2-C 10 acyl, C1-C 10 alkoxycarbonyl, C1-C 10 alkylaminocarbonyl, C1-C 10 alkylthiocarbonyl, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylsulfanyl, C1-C 10 alkylcarbonate, C1-C 10 alkylcarbamate, C1-C 10 ureido, aryloxy, C1-C 10 alkylsulfinyl, C1-C 10 alkylsulfonyl, arylsulfanyl, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylsulfanyl, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C 10 alkylphosphonate, C1-C 10 alkylphosphinate, C1-C 10 trialkyl phosphonium salt, C4-C 10 heterocycloamino, C6-C 10 aryl or π-excessive heteroaryl, and at least one of R 4 , R 5 , R 6 , and R 7 is Q; Q is selected from unsubstituted vinyl groups, C2-C substituted with one or more electron-donating groups. 10 π-conjugated electron-donating groups of alkenyl, unsubstituted thiophene-2-yl, substituted thiophene-2-yl, unsubstituted thiophene-3-yl and substituted thiophene-3-yl; X is -OH, -O-G, -O - a salt or boron group having the following structure: R 8 and R 9 each independently is H or C1-C 10 alkyl, or R 8 and R 9 together with the boronic acid ester to which they are attached provide a C2-C 10 cyclic boronic ester; 3. The compound of claim 1, wherein the vinyl position and allyl position of Q, if present, are unsubstituted.

5. The compound of claim 1, wherein G is trialkylsilyl, alkylarylsilyl, arylsulfonyl, dioxobenzyl, trityl, alkyl carbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranosyl uronic, furanosyl, acyl, benzoyl, or benzyl.

6. The compound of claim 1, wherein G is -PO3H2 or a salt or ester thereof.

4. The compound of claim 1, wherein X, Q, R 4 , R 5 , R 6 , and R 7 are attached to the aromatic ring in the ortho position relative to where Q, R 4 , R 5 , R 6 , and R 7 are H. The other aspects of the compound are otherwise identical.

7. The compound of claim 1, wherein G comprises a bivalent releasable linker and a terminal trialkylsilyl, alkylarylsilyl, arylphenylsulfonyl, dioxobenzyl, trityl, alkyl carbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranosyl uronic, furanosyl, acyl, benzoyl, benzyl, or boronate ester group.

12. The compound of claim 1 having Formula II ###0002### 13. The compound of claim 1 having the following structure: ###0003### or a salt thereof.

8. The compound of claim 1, wherein R 3 is unsubstituted C1-C 10 alkyl or C1-C 10 alkyl substituted with one or more halogen, hydroxyl, amino, sulfido, alkoxy, alkylamino, alkylsulfido, sulfate, or carboxylate.

9. The compound of claim 1, wherein R 4 , R 5 , R 6 , and R 7 are each independently H, Q, halo, C1-C 10 alkyl, hydroxy, C1-C 10 alkoxy, amino, C1-C 10 alkylamino, thio, C1-C 10 alkylthio, C2-C 10 acyloxy, C2-C 10 acylamino, C2-C 10 acylthio, C1-C 10 alkyl carbonate, C1-C 10 alkyl carbamate, C1-C 10 ureido, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino, and exactly one of R 4 , R 5 , R 6 , and R 7 is Q.

10. The compound of claim 1, wherein one of R 4 , R 5 , R 6 , and R 7 is Q, and the rest are H.

11. The compound of claim 1, wherein R 7 is Q.

14. The compound of claim 1 having the following structure: ###0004### or a salt thereof. wherein R 10 and R 11 each independently is H or halo.

15. A composition comprising the compound of claim 1, wherein the composition is substantially free of a surfactant.

18. Use of the compound of any one of claims 1 to 14 in the manufacture of a kit for detecting an analyte in a sample, wherein the kit is configured to contact the sample with the compound and monitor luminescence of the sample. ​ 16. The composition of claim 15, wherein the composition is substantially free of hexadecyltrimethylammonium bromide (CTAB) and hexadecylpyridine chloride. (CPC), benzalkonium chloride (BAC), benzyl chloride (BZT), dimethyl dioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide (DODAB), α'-tributyl - p-Dichloroxylene, poly(vinylbenzyltributyl chloride) (TBE) and poly(vinylbenzyltrioctyl chloride) ).

17. An aqueous chemiluminescent composition comprising the compound of any one of claims 1 to 14, and which has a peak luminescence intensity of greater than 1000 photons / second and a T 1 / 2 and wherein the composition is substantially free of a surfactant-based luminescence enhancer. ​

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