Phenoxy-dioxetanes-based chemiluminescence probes and uses thereof

Abstract

The present invention provides phenoxy-dioxetane-based chemiluminescence probes having enhanced chemiexcitation and chemical stability, in which either an adamantyl derivative equipped with a hetero -functional group or a six-member carbocyclic or heterocyclic ring having an electron-withdrawing motif is spiro fused to the dioxetane. The chemiluminescence probes disclosed are useful for both diagnostics and in vivo imaging.

Description

PHENOXY-DIOXETANES-BASED CHEMILUMINESCENCE PROBES AND USES THEREOFTECHNICAL FIELD

[0001] The present invention provides phenoxy-dioxetane-based chemiluminescence probes having enhanced chemiexcitation and chemical stability, and compositions thereof for diagnostics and in vivo imaging.BACKGROUND ART

[0002] The discovery of adamantyl-phenoxy-l,2-dioxetane chemiluminescent luminophores by the Schaap group in 1987 enabled the design of modular turn-on probes by simply masking the phenol with a triggering group, which is used as a substrate for a specific analyte of interest (Figs. 1A-1B) (Schaap et al., 1987). However, the light emission generated by these dioxetanes is quenched in the presence of water, and detergent-like additives are required to enhance the light signal under physiological conditions (Schaap et al., 1989). Since these additives are highly toxic to cells, only a limited number of biocompatible applications were reported for these chemiluminescent luminophores. In 2017, our group discovered that incorporating an acrylate electron-withdrawing substituent at the ortho position of a phenoxy-adamantyl-l,2-dioxetane chemiluminescent luminophore prevents water-mediated quenching and amplifies the light-emission intensity of the luminophore by a factor of 3000-fold (Fig. IB) (Green et al., 2017). Noticeably, this development ended almost 30 years of dormancy in the field and enabled the use of chemiluminescent probes, for the first time, as a sole component in water with no required additives. Numerous research groups worldwide (Kagalwala et al. , 2022; Huang et al. , 2022; Huang et al., 2021; An et al., 2019; Cao et al., 2018), including ours, used the ortho- substituted phenoxy-adamantyl-l,2-dioxetane luminophore to develop new turn-on chemiluminescent probes for the detection and imaging of a variety of enzymes and analytes in living systems (Shelef et al., 2024; Shelef et al., 2022; Peukert et al., 2022; Scot et al., 2021; Gutkin et al., 2021; Gholap et al., 2021; Babin et al., 2021; Ye et al., 2020; Gutkin et al., 2020; Das etal., 2020; Son et al., 2019; Roth-Konforti etal., 2019; Hananya etal., 2019; Hananya et al., 2019a; Roth-Konforti et al., 2017; Hananya et al., 2017; Green et al., 2017a; Cheng, et al., 2019; Gutkin et al., 2023).

[0003] Despite the encouraging progress in the field, further advancements could be attained by gaining a deeper understanding of the chemiexcitation mechanism of dioxetanes. Indeed, the chemiluminescent light-emission pathway of phenoxy- 1,2-dioxetane luminophores has been capturing growing interest within the scientific community (Blau et al., 2023; Kagalwala et al., 2022a; Haris and Lippert, 2023; Hananya and Shabat, 2017). When the chemiexcitation rate of a given molecule is fast, light emission occurs in a flash mode. Contrariwise, if the chemiexcitation is slow, light emission takes place in a glow mode. Flash-type chemiluminescence assays generate more intense light emission signals in comparison to glow-type assays, primarily because they produce a higher number of photons within a given time interval.

[0004] The brightness of a fluorescence dye is determined by both the quantum yield and the extinction coefficient. In the case of a chemiluminescence luminophore, the brightness depends on the number of photons, emitted within a specific time interval. Consequently, the luminophore's brightness is directly affected by the rate of chemiexcitation. In order to obtain a bright chemiluminophore, the compound should have both a high quantum yield and a fast chemiexcitation rate (Gutkin et al., 2023). A year ago, we discovered a distinct molecular motif that enables efficient enhancement of the chemiexcitation rate of dioxetane luminophores (Tannous et al., 2024). The chemiexcitation rate of phenoxy- 1,2-dioxetanes was significantly accelerated through a spiro -strain-release effect, generated by a fused spiro-cyclobutane-dioxetane (Fig. 1C). Remarkably, chemiluminescent 1,2-dioxetane probes, equipped with a strained cyclobutyl substituent, exhibited a diagnostic signal with a significantly high signal-to-noise (S / N) ratio, resulting in unprecedented detection sensitivity toward the target enzyme.

[0005] In a structure-activity-relationship screen, we noticed that spiro-dioxetanes featuring strained four-member rings, either substituted with electron-withdrawing groups (EWGs) or with a hetero atom incorporated in the ring, exhibited higher chemiexcitation rates in comparison to their non- substituted cyclobutane counterparts (Tannous et al., 2024). However, the chemical stability of these dioxetanes was noticeably compromised due to the presence of electron-withdrawing functional groups. This observation implies that dioxetanes fused to non-strained rings, with hetero atoms or inductive EWGs, may exhibit both accelerated chemiexcitation rates and elevated chemical stability.SUMMARY OF INVENTION

[0006] In one aspect, disclosed herein is a dioxetane -based chemiluminescence probe, more specifically a compound of formula I:I whereinR1is selected from a linear or branched (C1-C18)alkyl, or (C3-C7)cycloalkyl;R2and R3together with the carbon atom to which they are attached form:(a) an adamantane analogue of formula II:whereinRB, RD, RF, RH, and R1, each independently is selected from -C(O)-, -C(O)- O-, -O-, and -C(X)2-, wherein X each independently is selected from H, (C1- C8)alkyl, preferably (C1-C3)alkyl, -COOH, -COO(C1-C8)alkyl, preferably - COO(C1-C3)alkyl, and -O-(C1-C8)alkyl, preferably -O-(C1-C3)alkyl; andRA, RC, RE, and RG, each independently is tertiary carbon optionally substituted with (C1-C8)alkyl, preferably (C1-C3)alkyl, -COOH, -COO(C1- C8)alkyl, preferably -COO(C1-C3)alkyl, or -O-(C1-C8)alkyl, preferably -O-(C1- C3)alkyl, tertiary nitrogen, or quaternary nitrogen wherein the nitrogen atom is further alkylated;provided that at least one of RB, RD, RF, RH, and R1is not -CH2-, -CH(C1- C8)alkyl-, or -C((C1-C8)alkyl)2-; and / or at least one of RA, Rc, RE, and RGis not tertiary carbon optionally substituted with (C1-C8)alkyl; or(b) a 6-membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electron withdrawing group such as halogen, -NO2, -CN, =0, -C00R9, -C(=O)R10, - 0-(C1-C8)alkylene-(C6-Cio)aryl, and -SO2R9, but excluding unsubstituted cyclohexyl, wherein two non-adjacent carbon atoms of said carbocyclic or heterocyclic ring optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; or two alkyl groups attached to the same carbon atom of said carbocyclic or heterocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring;R4, R5and R6each independently is selected from H, an electron acceptor group such as halogen, -NO2, -CN, -C00R9, -C(=O)R10, and -SO2R9, and a π* acceptor group of the formula -CH=CH-E, wherein E is selected from -CN, -C00H, -COO(C1-C18)alkyl optionally interrupted in the alkylene chain with one or more -O- groups, -C00(C2- C18)alkenyl such as -COO-allyl, 4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H- indolyl, l,3,3-trimethyl-3H-indol-l-ium-2-yl, 4-(dicyanomethylene)-4H-chromen-2-yl, 4H- chromen-2-yl-4-one, 9-(dicyanomethylene)-9H-xanthen-3-yl, 9H-xanthen-3-yl-9-one, 2- dicy anomethylenemethyl-thieno [3 ,2-b] thiophene-5 -yl, 3 -methylbenzo [d] thiazol-2-yl-3 - ium, tetrathiafulvalenyl, l,2,3,5,6,7-hexahydropyrido[3,2, l-zj]quinolin-9-yl, benzo [<7]oxazol-2-yl, benzo [<7]thiazol-2-yl, l / Z-benzo[z / ]imidazol-2-yl, 2,6-di-tert- butylpyrylium-4-yl, 1 -methylquinolin- 1 -ium-4-yl, 4-dicyanomethylene-2-methy 1-4 / 7 - pyran-6-yl, 5,5-dimethyl-3-cyano-2-dicyanomethylene-2,5-dihydrofuran-4-yl, and 5,5- dimethy 1-3 -cyano -2-oxo-2,5 -dihy drofuran-4 -y 1 ; or one of R5and R6, together with R7, forms an optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring, and the other one of R5and R6is H;R7is H, or together with one of R5and R6forms said optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring;R8is H, an analyte-responsive group, or a boron-containing group having the formula -B(Z)(Z') or -B(Z")3’ Kat+;R9each independently is H, (C1-C18)alkyl, or (C6-Ci4)aryl;R10each independently is H, (C1-C18)alkyl, (C6-Ci4)aryl, or halogen such as chlorine and bromine;Z and Z' each independently is -OR11or -O' Kat+;Z" is selected from F, Cl, Br, and I;R11is selected from H, (C1-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl, (C2- C4)heteroalkyl, (C2-C4)heteroalkenyl, (C2-C4)heteroalkynyl, (C6-Cio)aryl, and (C5- C6)heteroaryl, or two R11together with their intervening atoms form a 5- to 7-membered optionally substituted heterocyclic ring, preferably a saturated optionally substituted heterocyclic ring;Kat+each independently is an organic cation, or an inorganic cation such as an alkali metal cation;Y is absent or is -O-; andL is absent or is a linker of the formula LI, L2 or L3:optionally substituted at the aromatic ring with one or more substituents each independently selected from (C1-C18)alkyl and (C3-C7)cycloalkyl, wherein M is absent or is -O-, -NH-, - N(C1-C8)alkyl-, or -N+((C1-C8)alkyl)2-, and the asterisk represents the point of attachment to the group Y, provided that: (1) when R8is H, Y is -O-, and L is absent; (2) when R8is said analyte-responsive group, Y is -O-; and (3) when R8is said boron-containing group, Y is absent, and either L is absent or L is said linker wherein M is absent.

[0007] In another aspect, disclosed herein is a composition comprising a carrier, and a dioxetane-based chemiluminescence probe of the formula I as defined above.

[0008] In a further aspect, disclosed herein is a method for diagnostics or in vivo imaging comprising: applying a composition as defined above to a sample or administering such a composition to a subject, wherein R8in the compound of the formula I is a group cleavableby an analyte, wherein upon exposure to said analyte, R8is cleaved (fully or partially) from the compound of formula I, thereby generating an unstable phenolate-dioxetane compound, which in turn decomposes through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light; and imaging to detect the emission of light. The method disclosed may be used for either determining the presence, or measuring the level, of said analyte in said sample or said subject. When used for in vivo imaging, the chemiluminescence probe may be administered systemically or locally, e.g., to a particular organ of said subject, so as to determine the presence, or measure the level, of said analyte in general, or in particular said organ.BRIEF DESCRIPTION OF DRAWINGS

[0009] Figs. 1A-1C show (1A) general activation and chemi excitation pathway of 1,2- dioxetanes with or without ortho-acrylate substituent; (IB) general structure and characteristics of adamantyl substituted dioxetanes (1987, 2017); and (1C) general structure and characteristics of cyclobutyl substituted dioxetanes (2023).

[0010] Figs. 2A-2B show (2A) visual demonstration of the light emitted by Diox 1, Diox 9, and Diox 8 [500 pM] during selected times in the presence of TBAF [20 mM] in DMSO; and (2B) normalized total light emission kinetic profile (time is represented in logarithmic scale) of Diox 1, Diox 9, and Diox 8. The relative calculated chemiexcitation rates are taken from Table 6.

[0011] Figs. 3A-3B show (3A) the chemiexcitation mechanism of a general spirocycloalkyl phenoxy- 1,2-dioxetane; and (3B) comparison between the computed Gibbs free energy of the rate-determining step of chemiexcitation for five selected phenoxy- 1,2- dioxetane. Detailed density functional theory (DFT) calculations are not shown.

[0012] Figs. 4A-4E show (4A) chemiluminescent activation pathway of methyl-acrylate 1,2-dioxetanes bearing a β-galactosidase-responsive trigger; (4B) total light emission of methyl-acrylate 1,2-dioxetanes probes [ 10pM] in the presence ofβ-g alactosidase [2 U / mL]; (4C) chemiluminescence kinetic profiles and signal-to-noise of total light emission of MA- Diox-1, MA-Diox-2, MA-Diox-8, MA-Diox-9 [10μM] in the presence and absence of / 3- galactosidase [2 U / mL]; (4D) signal-to-noise of total light emission vs. time of MA-Diox-1, MA-Diox-2, MA-Diox-8, MA-Diox-9 [10μM] in the presence and absence of / 3- galactosidase [0.001 U / mL]; and (4E) signal-to-noise of total light emission of MA-Diox-1, MA-Diox-2, MA-Diox-8, MA-Diox-9 [10μM] in the presence and absence of / 3-galactosidase [0.001 U / mL] after 10 min measurements. All measurements were conducted in PBS, pH 7.4, with 10% ACN at 27°C.

[0013] Figs. 5A-5D show (5A) graphical demonstration of the activation pathway of methyl-acrylate dioxetanes probes in the presence ofβ-g alactosidase-expressing bacteria; (5B) stability of MA-Diox-8 and MA-Diox-9 [100 M] in LB medium, 10% ACN at room temperature; product distribution was determined using RP-HPLC (30-100% ACN in water with 0.1% TFA); (5C) signal-to-noise of total light emission vs. time and after 10 min measurements of MA-Diox-1, MA-Diox-2, MA-Diox-8, and MA-Diox-9 [10μM] in the presence of E. Coli ATCC 25922 [O.D.= 0.4] in LB medium with 1% ACN at 37°C; (5D) determination of the limit of detection values of MA-Diox-1 and MA-Diox-8 [ 10μM]. Measurements were taken with various concentrations of E. coli ATCC 25922 [1.95xl03to 3.20xl07cells), in LB medium with 1% ACN at 37°C.

[0014] Figs 6A-6G show (6A) chemiluminescent kinetic profiles of the methyl acrylate probes MA-Diox-1, MA-KetoneAD and MA-LactoneAD [10μM] in the presence of [>- galactosidase [2 U / mL]; (6B) chemiluminescent kinetic profiles of the acrylic acid probes AA-Diox-1, AA-KetoneAD and AA-LactoneAD [10μM] in the presence of β-g alactosidase [2 U / mL]; (6C) total light emission of said methyl acrylate- and acrylic acid probes [10μM] in the presence ofβ-g alactosidase [2 U / mL]; (6D) saturation kinetic profiles of said methyl acrylate probes [10μM] in the presence ofβ-g alactosidase [0.001 U / mL]; (6E) saturation kinetic profiles of said acrylic acid probes [10μM] in the presence ofβ-g alactosidase [0.001 U / mL]; (6F) S / N of total light emission over 20 min of said methyl acrylate- and acrylic acid probes 10 M] in the presence ofβ-g alactosidase [2 U / mL]; and (6G) S / N over time of said methyl acrylate- and acrylic acid probes [ 10μM] in the presence ofβ-g alactosidase [2 U / mL]. All measurements were conducted in PBS, pH 7.4, 10% ACN at 37°C.DETAILED DESCRIPTION

[0015] Dioxetane probes undergoing rapid flash-type chemiexcitation exhibit higher detection sensitivity than those with a slow glow-type chemiexcitation rate. As found in accordance with the present invention and shown in Study 1 herein, dioxetanes fused to nonstrained six-member rings with hetero atoms or inductive EWGs exhibit substantially higher chemiexcitation rates, when compared to their spiro-adamantyl-dioxetane counterpart; and elevated chemical stability compared to the spiro-dioxetanes featuring strained four-memberrings equipped with electronegative polar motifs, disclosed in Tannous et al. (2024). The observed acceleration effect was adequately backed up by density functional theory (DFT) computational simulations. For example, a spiro-dioxetane with a six-member sulfone ring exhibited a chemiexcitation rate that is 293-fold faster than that of spiro-adamantyl- dioxetane, and a turn-on dioxetane probe containing said six-member sulfone ring (identified herein as MA-Diox-8), for the detection of the enzyme β-g alactosidase, exhibited a S / N value of 108 in lysogeny broth (LB) cell growth medium. This probe demonstrated substantially increased detection sensitivity towards E. coli bacterial cells expressing f>- galactosidase, with a limit-of-detection (LOD) value that indicates a 44-fold increase in sensitivity compared to that obtained by the spiro-adamantyl-dioxetane counterpart. The acceleration of chemiexcitation and increased chemical stability exhibited by MA-Diox-8 make it an ideal candidate for designing efficient and innovative turn-on chemiluminescent probes with exceptionally high detection sensitivity.

[0016] Although the chemical stability of the compounds shown in Study 1 is remarkably elevated compared to that of the spiro-dioxetanes disclosed in Tannous et al., 2024, it is severely hampered compared to that of their spiro-adamantyl-dioxetane counterparts. It has therefore been decided to investigate whether a spiro-fused hetero-substituted adamantyl unit would exhibit a chemiexcitation rate that is higher than that of the parent spiro- adamantyl-dioxetane probe, while retaining the chemical stability of the latter.

[0017] As described in Study 2, specific such ortho-acrylate substituted phenoxy- 1,2- dioxeatne chemiluminescent probes, identified herein as MA-KetoneAD and MA- LactoneAD, were prepared, and their chemiluminescence behavior was measured compared to that of the spiro-adamantyl-dioxetane counterpart identified herein as MA-Diox-1. As shown, the probes bearing hetero-adamantyl derivatives reach a plateau signal faster compared to the control probes, wherein the S / N values measured for these probes were substantially higher than the S / N values of the unsubstituted adamantyl compounds. Moreover, the hetero-substituted probes reach peak S / N value significantly faster than the control probes while also retaining higher absolute values for the duration of the measurement. These results highlight the advantage of the hetero-substituted adamantyl probes, leading to overall higher sensitivity obtained in very short times, and present promising evidence that hetero -functional adamantyl units can enhance the chemiexcitation rate and sensitivity of phenoxy- 1,2-dioxetanes while maintaining their chemical stability.

[0018] In one aspect, the present invention thus provides a compound of the formula I:I whereinR2and R3together with the carbon atom to which they are attached form:(a) an adamantane analogue of formula II:whereinRB, RD, RF, RH, and R1, each independently is selected from -C(O)-, -C(O)- O-, -O-, and -C(X)2-, wherein X each independently is selected from H, (C1-excluding unsubstituted cyclohexyl, wherein two non-adjacent carbon atoms of said carbocyclic or heterocyclic ring optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; or two alkyl groups attached to the same carbon atom of said carbocyclic or heterocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring;R4, R5and R6each independently is selected from H, an electron acceptor groupindolyl, l,3,3-trimethyl-3H-indol-l-ium-2-yl, 4-(dicyanomethylene)-4H-chromen-2-yl, 4H- chromen-2-yl-4-one, 9-(dicyanomethylene)-9H-xanthen-3-yl, 9H-xanthen-3-yl-9-one, 2- dicy anomethylenemethyl-thieno [3 ,2-b] thiophene-5 -yl, 3 -methylbenzo [d] thiazol-2-yl-3 -pyran-6-yl, 5,5-dimethyl-3-cyano-2-dicyanomethylene-2,5-dihydrofuran-4-yl, and 5,5- dimethy 1-3 -cyano -2-oxo-2,5 -dihy drofuran-4 -y 1 ; or one of R5and R6, together with R7, forms an optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring, and the other one of R5and R6is H;R7is H, or together with one of R5and R6forms said optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring;R8is H, an analyte-responsive group, or a boron-containing group having the formula -B(Z)(Z') or -B(Z")3’ Kat+; or halogen such aschlorine and bromine;Z and Z' each independently is -OR11or -O' Kat+;Z" is selected from F, Cl, Br, and I;C6)heteroaryl, or two R11together with their intervening atoms form a 5- to 7-membered optionally substituted heterocyclic ring, preferably a saturated optionally substituted heterocyclic ring;Kat+each independently is an organic cation, or an inorganic cation such as an alkali metal cation;Y is absent or is -O-; andL is absent or is a linker of the formula LI, L2 or L3:optionally substituted at the aromatic ring with one or more substituents each independentlythe group Y, provided that: (1) when R8is H, Y is -O-, and L is absent; (2) when R8is said analyte-responsive group, Y is -O-; and (3) when R8is said boron-containing group, Y is absent, and either L is absent or L is said linker wherein M is absent.

[0019] In one particular such aspect, disclosed herein is a compound of the formula I as defined above, wherein R2and R3together with the carbon atom to which they are attached form an adamantane analogue of the formula II as defined above, provided that at least one

[0020] In another particular such aspect, disclosed herein is a compound of the formula I as defined above, wherein R2and R3together with the carbon atom to which they are attached form a 6-membered carbocyclic or heterocyclic ring as defined above.

[0021] The term "alkyl" typically means a linear or branched hydrocarbyl, i.e., a univalent group derived from a saturated linear or branched aliphatic chain by removal of hydrogen atom from any of the carbon atoms, having, e.g., 1-18 carbon atoms and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec -butyl, isobutyl, tert-butyl, n-pentyl, isoamyl, neopentyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n- dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, and the like. Preferred are (C1- C8)alkyls, more preferably (C1-C4)alkyls, most preferably methyl, ethyl, n-propyl, isopropyl, and tert-butyl.

[0022] The term "alkylene" as used herein refers to a hydrocarbylene, i.e., a divalent group of the formula Cntkn wherein n is an integer of at least 1, derived from a saturated linear or branched aliphatic chain having, e.g., 1-18 carbon atoms, by removal of two hydrogen atoms. The term “alkylene chain” refers to a linear alkylene group of the formula -(CH2)n- wherein n is an integer of at least 2, derived from a saturated linear aliphatic chain having at least 2 carbon atoms, after removal of a hydrogen atom from each one of the terminal carbon atoms, but may further refer to such a linear chain being a part of an alkyl group.

[0023] The terms "alkenyl" and "alkynyl" typically mean linear or branched hydrocarbyls having 2-12, e.g., 2-8, carbon atoms and at least one double or triple bond, respectively, and include ethenyl, propenyl, 3-buten-l-yl, 2-ethenylbutyl, 3-octen-l-yl, 3-nonenyl, 3 -decenyl, and the like, and propynyl, 2-butyn-l-yl, 3-pentyn-l-yl, 3-hexynyl, 3-octynyl, 4-decynyl, and the like Particular alkenyls and alkynyls are (C2-C6)alkenyls and (C2-C6)alkynyls, wherein (C2-C4)alkenyls and (C2-C4)alkynyls are preferred.

[0024] The terms "heteroalkyl", "heteroalkenyl" and "heteroalkynyl", as used herein, refer to the corresponding hydrocarbyl (i.e., alkyl, alkenyl, and alkynyl), which contains one or more heteroatoms each independently selected from O, N, and S within the backbone residue; thus, at least one carbon atom of a corresponding alkyl, alkenyl, or alkynyl is replaced by one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl, or heteroalkynyl group. Preferred are (C2-C4)heteroalkyls, (C2-C4)heteroalkenyls, and (C2- C4)heteroalkynyls.

[0025] The term “aliphatic ring” or “carbocyclic ring” used herein interchangeably refers to a mono-, bi-, or poly-cyclic non-aromatic hydrocarbon having, e.g., 3-12, but preferably3-8, carbon atoms. The carbocyclic ring may be saturated, such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, and the like; or unsaturated, i.e., having at least one double bond, such as cyclopropene, cyclobutene, cyclopentene, cyclohexene, and the like. The carbocyclic ring may be substituted as defined herein with at least one group each independently selected from alkyl optionally substituted with at least one aryl group, aryl, and an electron withdrawing group, and may also be oxidized at a carbon atom thereof. The term “cycloalkyl” means a univalent group derived from a saturated carbocyclic ring by removal of hydrogen atom from any of the carbon atoms. Examples of such groups include, without limiting, (C3-C10)cycloalkyls, preferably (C3-C8)cycloalkyls, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0026] The term "heterocyclic ring" as used herein refers to a mono-, bi-, or poly-cyclic non-aromatic ring having, e.g., 3-12, but preferably 3-8, atoms, and consisting of at least one carbon atom and at least one heteroatom selected from oxygen, nitrogen, and sulfur (optionally oxidized), which may be saturated or unsaturated, i.e., containing at least one unsaturated bond. The heterocyclic ring may be substituted as defined herein with at least one group each independently selected from alkyl optionally substituted with at least one aryl group, aryl, and an electron withdrawing group, and may also be oxidized at either a carbon atom or a heteroatom thereof (in cases said heteroatom is sulfur, it may also be dioxidized). Non-limiting examples of heterocyclic rings include oxetane, azetidine, thietane, 1-oxidothietan, 1,1-dioxidothietan, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxazolidine, thiazolidine, imidazolidine, oxazoline, thiazoline, imidazoline, dioxole, dioxolane, dihydrooxadiazole, pyran, dihydropyran, tetrahydropyran, thiopyran, dihydrothiopyran, tetrahydrothiopyran, 1-oxidotetrahydrothiopyran, 1,1- dioxidotetrahydrothiopyran, tetrahydrofuran, pyrazolidine, pyrazoline, tetrahydropyrimidine, dihydrotriazole, tetrahydrotriazole, azepane, dihydropyridine, tetrahydropyridine, and the like. The term "heterocyclyl" as used herein refers to a univalent group derived from a heterocyclic ring by removal of hydrogen atom from any of the ring atoms.

[0027] The term “aromatic ring” as used herein refers to an aromatic carbocyclic ring having, e.g., 6-14 carbon atoms, and consisting of a single ring or multiple rings either condensed or linked by a covalent bond. Non-limiting examples of aromatic rings include benzene, naphthalene, anthracene, naphthacene, phenanthrene, pyrene, chrysene, tetracene,and triphenylene. The aromatic ring may optionally be substituted with one or more groups each independently selected from alkyl, e.g., (C1-C8)alkyl or (C1-C4)alkyl. The term "aryl" denotes a univalent aromatic carbocyclic group derived from an aromatic ring by removal of hydrogen atom from any of the ring atoms.

[0028] The term “heteroaromatic ring” as used herein refers to a mono-, bi-, or poly-cyclic aromatic ring having, e.g., 4-12 atoms, and consisting of at least one carbon atom and at least one heteroatom selected from oxygen, nitrogen, and sulfur (optionally oxidized). Nonlimiting examples of heteroaromatic rings include thiophene, imidazole, pyridine, furan, pymole, oxazole, thiazole, purine, indole, pyrrole, pyrazine, isoquinoline, pyrazole, isoxazole, thiazole, isothiazole, pyrazine, pyrimidine, pyridazine, carbazole. The heteroaromatic ring may optionally be substituted, at any one of the atoms thereof, with one or more groups each independently selected from alkyl, e.g., (C1-C8)alkyl or (C1-C4)alkyl. The term “heteroaryl” refers to a univalent group derived from a heteroaromatic ring by removal of hydrogen atom from any of the ring atoms.

[0029] The term "halogen" as used herein refers to a halogen and includes fluoro, chloro, bromo, and iodo, but it is preferably fluoro or chloro.

[0030] The term “electron withdrawing group”, as used herein, refers to a group reducing electron density in a molecule through the carbon atom to which it is bonded. Non-limiting examples of such groups include halogen, preferably fluoro and chloro, -NO2, -CN, =0, - C(=O)OR, -C(=O)R’, -C(=O)NR2, -0-(C1-C8)alkylene-(C6-Cio)aryl, and -SO2R, wherein R each independently may be, e.g., hydrogen, (C1-C18)alkyl, or (C6-C14)aryl; and R’ may be, e.g., hydrogen, (C1-C18)alkyl, (C6-Ci4)aryl, or halogen such as chlorine and bromine. Particular such electron acceptor groups include fluoro, chloro, -NO2, -CN, -C(=O)OR, - C(=O)R’, -C(=O)NR2, -O-CH2 -phenyl (benzyloxy), and -SO2R, wherein R each independently is H, (C1-C4)alkyl, or phenyl; and R’ is H, (C1-C4)alkyl, phenyl, or halogen.

[0031] The term "electron acceptor group", as used herein, refers to a group with a high electron affinity. Non-limiting examples of such groups include halogen, preferably fluoro and chloro, -NO2, -CN, -C(=O)OR, -C(=O)R’, C(=O)NR2, and -SO2R, wherein R each independently may be, e.g., hydrogen, (C1-C18)alkyl, or (C6-Ci4)aryl; and R’ may be, e.g., hydrogen, (C1-C18)alkyl, (C6-Ci4)aryl, or halogen such as chlorine and bromine. Particular such electron acceptor groups include halogen, -NO2, -CN, -C(=O)OR, -C(=O)R’, C(=O)NR2, and -SO2R, wherein R each independently is H, (C1-C4)alkyl, or phenyl; and R’ is H, (C1-C4)alkyl, phenyl, or halogen.

[0032] The term "K* acceptor group", as used herein, refers to any group containing a π* acceptor system capable of accepting electrons. Specific such groups are those of the formula -CH=CH-E, wherein E is -CN, -COOH, -COO(C1-C18)alkyl optionally interrupted in the alkylene chain with one or more -O- groups, -COO(C2-C18)alkenyl such as -COO-allyl, or any of the groups shown in Table 1.Table 1: Certain π* acceptor groups A of the formula -CH=CH-E (names refer to group E)Optionally substituted at one or more of the carbon atoms of the aromatic or heteroaromatic ring with a substituent each independently selected from halogen, -CN, -COOH, -COO(C1-C18)alkyl, and -C(O)(C1-Ci8)alkyl.

[0033] In certain embodiments, disclosed herein is a compound of the formula I, wherein R1is a linear or branched (C1-C8)alkyl. In particular such embodiments, R1is (C1-C4)alkyl, preferably methyl, ethyl, propyl, isopropyl, or tert-butyl.

[0034] In certain embodiments, disclosed herein is a compound of the formula I, wherein R2and R3together with the carbon atom to which they are attached form: (a) an adamantane analogue of the formula II, wherein at least one of RB, RD, RF, RH, and R1is selected from - C(O)-, -C(O)-O-, -O-, and -C(X)2- wherein at least one of the X groups is -COOH, -COO(C1- C3)alkyl, or -O-(C1-C3)alkyl, and the others of RB, RD, RF, RH, and R1each is -CH2-; or at least one of RA, Rc, RE, and RGis tertiary carbon substituted with -COOH, -COO(C1- C3)alkyl, or -O-(C1-C3)alkyl, tertiary nitrogen, or quaternary nitrogen, and the others of RA, Rc, RE, and RGeach is unsubstituted tertiary carbon; (b) cyclohexyl substituted with one or more groups each independently selected from (C1-C4) alkyl optionally substituted with one or two (C6-Cio)aryl groups, (Ce)aryl, halogen, =0, -COOR9, -C(=O)R10, and -O-(C1- C4)alkylene-(C6-Cio)aryl, wherein two non-adjacent carbon atoms of said cyclohexyl optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1- C18)alkyl; or two alkyl groups attached to the same carbon atom of said cyclohexyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; (c) oxanyl (tetrahydropyranyl) or dioxanyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1- C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (Ce)aryl, halogen, =0, -COOR9, -C(=O)R10, and -0-(C1-C4)alkylene-(C6-Cio)aryl; (d) tetrahydrothiopyranyl (thianyl), 1-oxido tetrahydro thiopyranyl or 1,1-dioxido tetrahydro thiopyranyl, optionallysubstituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, - COOR9, -C(=O)R10, and -SO2R9; or (e) piperidinyl or piperidinyl- 1 -oxide, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR9, -C(=O)R10, and -0-(C1-C4)alkylene-(C6-Cio)aryl, said piperidinyl being further optionally substituted at the nitrogen atom with one or two groups each independently selected from (C1-C4)alkyl, (C3-C7)cycloalkyl, and (C6-Cio)aryl, wherein two non-adjacent carbon atoms of said piperidinyl optionally form an additional 3- 4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; or two alkyl groups attached to the same carbon atom of said piperidinyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, wherein R9each independently is H, (C1-C4)alkyl, or (C6-Cio)aryl such as phenyl; and R10each independently is H, (C1- C4)alkyl, (C6-Cio)aryl, or halogen such as chlorine and bromine. In particular such embodiments, R2and R3together with the carbon atom to which they are attached form (a) an adamantane analogue of formula II selected from (lr,3r,5r,7r)-adamantan-2-one, 4- oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)- adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3 -diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium (Table 2); or (b) a 6-membered carbocyclic or heterocyclic ring selected from cyclohexanecarboxylic acid (wherein the -C00H group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), methyl cyclohexanecarboxylate (wherein the -COOCH3 group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), ethyl cyclohexanecarboxylate (wherein the - COOC2H5 group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), cyclohexanecarbonyl chloride, cyclohexanecarbonyl bromide, fluorocyclohexane, difluorocyclohexane (wherein the fluoro groups each is linked to a different carbon atom of the cyclohexane, or both are linked to the same carbon atom, e.g.,meta or para to the point of spiro attachment to the dioxetane), chlorocyclohexane, dichlorocyclohexane (wherein the chloro groups each is linked to a different carbon atom of the cyclohexane, or both are linked to the same carbon atom, e.g., meta or para to the point of spiro attachment to the dioxetane), cyclohexanecarbonitrile (wherein the -CN group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), nitrocyclohexane (wherein the -NO2 group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), tctrahydro-2 / 7-pyran, 1,3-dioxane, 2,2-dimethyl-l,3-dioxane, tctrahydro-2 / 7- thiopyran, tetrahydro -2H- thiopyran 1 -oxide, tctrahydro-2 / 7-thiopyran 1,1 -dioxide, and (lR,5S)-8-methyl-8-azabicyclo[3.2.1] octane (Table 3).Table 2: Specific adamantane analogues of formula II that may be formed by R2and R3(the asterisk represents the point of spiro attachment to the dioxetane)Table 3: Specific 6-membered carbocyclic or heterocyclic rings that may be formed by R2and R3(the asterisk represents the point of spiro attachment to the dioxetane)

[0035] In certain embodiments, disclosed herein is a compound of the formula I, wherein (i) R4, R5and R6each independently is selected from H, halogen, preferably chlorine, -CN, and a π* acceptor group of the formula -CH=CH-E as defined above; or (ii) R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6is H; and the other one ofR5and R6, together with R7, forms a 6-membered structure selected from Othe central aromatic ring, wherein R12is (C1-Ci2)alkyl, preferably (C1-C4)alkyl; and R13each independently is selected from -CN, -NO2, (C2-Ci2)alkenyl, preferably (C2-C4)alkenyl, aryl, -COOR14, -COO' Kat+, and -C(O)N(R14)2, wherein R14each independently is H or (C1- Ci2)alkyl, preferably (C1-C4)alkyl, or the two R14together with the nitrogen atom to which they are attached form a heterocyclic ring.

[0036] In certain particular such embodiments, one of R4, R5and R6is a π* acceptor group of the formula -CH=CH-E, and the other two of R4, R5and R6each independently is H or halogen, preferably chlorine, i.e., (i) R4and R5each independently is H or halogen; and R6is a π* acceptor group of the formula -CH=CH-E; (ii) R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E; or (iii) R5and R6each independently is H or halogen; and R4is a π* acceptor group of the formula -CH=CH-E. In preferred such embodiments, R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E, more preferably R4is halogen; R5is a π* acceptor group of the formula -CH=CH-E; and R6is H. Particular such embodiments, are those wherein E is -CN; -COOH; -COO(C1-C8)alkyl, preferably -COO(C1-C4)alkyl such as - COOCH3 and -COOC(CH3)3; or -COO(C2-C8)alkenyl, preferably -COO(C2-C4)alkenyl such as -COOCH=CH-CH3.

[0037] In other particular such embodiments, R4, R5and R6each independently is H or halogen, preferably chlorine, i.e., none of R4, R5and R6is a π* acceptor group. In preferred such embodiments, R4is halogen, and R5and R6each is H.

[0038] In further particular such embodiments, R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6, preferably R6, together with R7, forms a 6-memberedthose wherein R12is (C1-C4)alkyl; and R13is selected from -CN, -NO2, (C2-C4)alkenyl, phenyl, -COOR14, -COO' Kat+, and -C(O)N(R14)2, wherein R14each independently is H or CH3. In particular such embodiments, R4is H or halogen; R5is H; and R6together with R7forms said 6-membered structure. In preferred such embodiments, the 6-membered structure formed i

[0039] In certain embodiments, disclosed herein is a compound of the formula I, wherein L is absent or is a linker of the formula LI, L2 or L3, preferably LI, wherein M is absent or is selected from -O-, -NH-, and -N+(CH3)2-.

[0040] In certain embodiments, disclosed herein is a compound of the formula I, wherein (i) R1is a linear or branched (C1-C8)alkyl, preferably (C1-C4)alkyl; (ii) R2and R3together with the carbon atom to which they are attached form (a) an adamantane analogue of the formula II, wherein at least one of RB, RD, RF, RH, and R1is selected from -C(O)-, -C(O)-O- , -O-, and -C(X)2- wherein at least one of the X groups is -COOH, -COO(C1-C3)alkyl, or - O-(C1-C3)alkyl, and the others of RB, RD, RF, RH, and R1each is -CH2-; or at least one of RA, Rc, RE, and RGis tertiary carbon substituted with -COOH, -COO(C1-C3)alkyl, or -O-(C1- C3)alkyl, tertiary nitrogen, or quaternary nitrogen, and the others of RA, Rc, RE, and RGeach is unsubstituted tertiary carbon; (b) cyclohexyl substituted with one or more groups each independently selected from (C1-C4) alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR9, -C(=O)R10, and -0-(C1-C4)alkylene-(C6-Cio)aryl, wherein two non-adjacent carbon atoms of said cyclohexyl optionally form an additional 3- 4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; or two alkyl groups attached to the same carbon atom of said cyclohexyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring; (c) oxanyl or dioxanyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4) alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR9, -C(=O)R10, and -0-(C1-C4)alkylene-(C6-Cio)aryl; (d) tetrahydrothiopyranyl, 1-oxidotetrahydrothiopyranyl or 1,1- dioxidotetrahydrothiopyranyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4) alkyl optionally substitutedwith one or two (C6-Cio)aryl groups, -COOR9, -C(=O)R10, and -SO2R9; or (e) piperidinyl or piperidinyl- 1 -oxide, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4) alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR9, -C(=O)R10, and -O-(C1- C4)alkylene-(C6-Cio)aryl, said piperidinyl being further optionally substituted at the nitrogen atom with one or two groups each independently selected from (C1-C4)alkyl, (C3- C7)cycloalkyl, and (C6-Cio)aryl, wherein two non-adjacent carbon atoms of said piperidinyl optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1- C18)alkyl; or two alkyl groups attached to the same carbon atom of said piperidinyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, wherein R9each independently is H, (C1-C alkyl, or (C6-Cio)aryl; and R10each independently is H, (C1-C alkyl, (C6-Cio)aryl, or halogen such as chlorine and bromine; (iii) either (a) R4, R5and R6each independently is selected from H, halogen, preferably chlorine, -CN, and a π* acceptor group of the formula -CH=CH-E; or (b) R4is selected from H, halogen, preferably chlorine, and -CN; one of R5and R6is H; and the other one of R5and R6, together with R7, forms a 6-membered structure selected fromwherein R12is (C1-C 12) alkyl; and R13each independently is selected from -CN, -NO2, (C2-Ci2)alkenyl, aryl, -COOR14, - COO' Kat+, and -C(O)N(R14)2, wherein R14is H or (C1-Ci2)alkyl, or the two R14together with the nitrogen atom to which they are attached form a heterocyclic ring; and (iv) L is absent or is a linker of the formula LI, L2 or L3, preferably LI, wherein M is absent or is selected from -O-, -NH-, and -N+(CH3)2-. Particular such embodiments are those wherein R1is methyl; and R2and R3together with the carbon atom to which they are attached form (a)an adamantane analogue selected from (lr,3r,5r,7r)-adamantan-2-one, 4- oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)- adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3 -diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium; or (b) a 6-membered carbocyclic or heterocyclic ring selected from cyclohexanecarboxylic acid (wherein the -COOH group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), methyl cyclohexanecarboxylate (wherein the -COOCH3 group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), ethyl cyclohexanecarboxylate (wherein the - COOC2H5 group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), cyclohexanecarbonyl chloride, cyclohexanecarbonyl bromide, fluorocyclohexane, difluorocyclohexane (wherein the fluoro groups each is linked to a different carbon atom of the cyclohexane, or both are linked to the same carbon atom, e.g., meta or para to the point of spiro attachment to the dioxetane), chlorocyclohexane, dichlorocyclohexane (wherein the chloro groups each is linked to a different carbon atom of the cyclohexane, or both are linked to the same carbon atom, e.g., meta or para to the point of spiro attachment to the dioxetane), cyclohexanecarbonitrile (wherein the -CN group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), nitrocyclohexane (wherein the -NO2 group is linked, e.g., meta or para to the point of spiro attachment to the dioxetane), tctrahydro-2 / 7-pyran, 1,3-dioxane, 2,2-dimethyl-l,3-dioxane, tctrahydro-2 / 7- thiopyran, tetrahydro -2 / 7- thiopyran 1 -oxide, tctrahydro-2 / 7-thiopyran 1,1 -dioxide, and (lR,5S)-8 -methyl- 8 - azabicyclo [3.2.1 ] octane .

[0041] In certain particular such embodiments, (i) R4and R5each independently is H or halogen; and R6is a π* acceptor group of the formula -CH=CH-E; (ii) R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E; or (iii) R5and R6each independently is H or halogen; and R4is a π* acceptor group of the formula -CH=CH-E. In preferred such embodiments, R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E, more preferably R4is halogen; R5is a π* acceptor group of the formula -CH=CH-E; and R6is H. Particular suchembodiments are those wherein E is -CN; -COOH; -COO(C1-C8)alkyl, preferably -COO(C1-C4)alkyl such as -COOCH3 and -COOC(CH3)3; or -COO(C2-C8)alkenyl, preferably -COO(C2-C4)alkenyl such as -COOCH=CH-CH3.

[0042] In other particular such embodiments, R4, R5and R6each independently is H or halogen, i.e., none of R4, R5and R6is a π* acceptor group. In preferred such embodiments, R4is halogen, and R5and R6each is H.

[0043] In further particular such embodiments, R4is selected from H, halogen and -CN; one of R5and R6, preferably R6, together with R7, forms a 6-membered structure selectedone of R5and R6is H. Preferred such 6-membered structures are those wherein R12is (C1- C4)alkyl; and R13is selected from -CN, -NO2, (C2-C4)alkenyl, phenyl, -COOR14, -COO' Kat+, and -C(O)N(R14)2, wherein R14each independently is H or CH3. In particular such embodiments, R4is H or halogen; R5is H; and R6together with R7forms said 6-membered structure. In preferred such embodiments, the 6-membered structure formed i

[0044] In certain embodiments, disclosed herein is a compound of the formula I, according to any one of the embodiments above, wherein R8is H.

[0045] In other embodiments, disclosed herein is a compound of the formula I, according to any one of the embodiments above, wherein R8is an analyte -responsive group, i.e., a group that can be removed, i.e., cleaved (at least in part), or modified, by means of a specific analyte, wherein removal or modification of said group generates an unstable phenolate - dioxetane compound, which decomposes through a chemiexcitation process to yield an excited intermediate, which in turn decays to its ground-state through emission of light. In certain such embodiments, the analyte -responsive group is an enzyme-labile group, i.e., a group that can be removed (at least in part), or modified, by means of a specific enzyme.Examples of analyte-responsive groups include, without limiting, those listed in Table 4, wherein Pep (last group in the Table) is a group comprising a peptide moiety consisting of at least two amino acid residues and linked to the aniline group via a carbocyclic group of said peptide moiety.Table 4: Specific analyte-responsive groups suitable as cleavable groups (R8)

[0046] In further embodiments, disclosed herein is a compound of the formula I, according to any one of the embodiments above, wherein R8is a boron-containing group, which is in fact a particular analyte -responsive group. Examples of boron-containing groups include, without being limited to, -B(OH)2, 4,4,5,5-tetramethyl-l,3,2-dioxaborolanyl, and 4-[4, 4,5,5- tetramethy 1- 1 , 3 ,2-dioxaborolany 1] benzyl .

[0047] In particular embodiments, disclosed herein is a compound of the formula I as defined above, wherein: R1is methyl; R2and R3together with the carbon atom to which they are attached form an adamantane analogue selected from (lr,3r,5r,7r)-adamantan-2-one, 4- oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)- adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3-diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium; or a 6-membered carbocyclic or heterocyclic ring selected from methyl cyclohexanecarboxylate wherein the -COOCH3 group is linked para to the point of spiro attachment to the dioxetane, tetrahydro -2 / 7-pyran wherein the point of spiro attachment to the dioxetane is positioned para or meta to the oxygen atom, difluorocyclohexane wherein the fluoro groups both are linked to the carbon atom para to the point of spiro attachment to the dioxetane, 2,2-dimethyl-l,3-dioxane, or tetrahydro-2H- thiopyran 1,1-dioxide; R4is H or Cl; R5and R6each is H; or R5is -CH=CH-E, wherein E is-COOH, -COOCH3, or -CN, and R6is H; or R5is H, and R6together with R7form; Y is -O-; L is absent, or is a linker of the formula LI wherein M is -O-; and R8is an analyte- responsive group such as each one of those listed in Table 4, or a boron-containing group such as -B(0H)2, 4,4,5,5-tetramethyl-l,3,2-dioxaborolanyl, or 4-[4,4,5,5-tetramethyl-l,3,2- dioxaborolanyl] benzyl. For the purpose of clarity, it should be noted that each one of the combinations falling within the scope of these particular embodimens, i.e., any combination of the carbocyclic ring, heterocyclic ring or adamantane analogue formed by R2and R3, R4, R5, and L, regardless of the specific group represented by R8, is considered a separate embodiment.

[0048] In certain specific embodiments exemplified herein, disclosed herein is a compound of the formula I as defined above, wherein R1is methyl; R4, R5and R6each is H; Y is -O-; L is absent; R8is TBDMS; and R2and R3together with the carbon atom to which they are attached form an adamantane analogue selected from (lr,3r,5r,7r)-adamantan-2-one, 4-oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)- adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3-diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium; or a 6-membered carbocyclic or heterocyclic ring selected from methyl cyclohexanecarboxylate wherein the -COOCH3 group is linked para to the point of spiro attachment to the dioxetane, tetrahydro -2 / 7-pyran wherein the point of spiro attachment to the dioxetane is positioned para or meta to the oxygen atom, difluorocyclohexane wherein the fluoro groups both are linked to the carbon atom para to the point of spiro attachment to the dioxetane, 2,2-dimethyl-l,3-dioxane, or tetrahydro-2H- thiopyran 1,1 -dioxide, herein identified Diox 3, Diox 4, Diox 5, Diox 6, Diox 7, and Diox 8, respectively (Table 5).Table 5: The specific compounds identified herein as Diox 1 - Diox 9

[0049] In other specific embodiments exemplified herein, disclosed herein is a compound of the formula I as defined above, wherein R1is methyl; R2and R3together with the carbon atom to which they are attached form an adamantane analogue selected from (lr,3r,5r,7r)- adamantan-2-one, 4-oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2- oxoadamantane-l,3-dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl ( 1R,3S ,5r,7r)-adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3-diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium; or a 6-membered carbocyclic or heterocyclic ring selected from methyl cyclohexanecarboxylate wherein the -COOCH3 group is linked para to the point of spiro attachment to the dioxetane, tetrahydro -2 / 7-pyran wherein the point of spiro attachment to the dioxetane is positioned para or meta to the oxygen atom, difluorocyclohexane wherein the fluoro groups both are linked to the carbon atom para to the point of spiro attachment to the dioxetane, 2,2-dimethyl-l,3-dioxane, or tetrahydro-2H- thiopyran 1,1-dioxide; R4is Cl; R5is -CH=CH-E, wherein E is -COOH, -COOCH3, or -CN; R6is H; Y is -O-; L is a linker of the formula LI, wherein M is -O-; and R8is galactosyl. The specific compounds wherein R2and R3together with the carbon atom to which they are attached form a 6-membered carbocyclic or heterocyclic ring, and E is -COOH are herein identified AA-Diox-3, AA-Diox-4, AA-Diox-5, AA-Diox-6, AA-Diox-7, and AA-Diox-8, respectively; the corresponding compounds wherein E is -COOCH3 are herein identified MA-Diox-3, MA-Diox-4, MA-Diox-5, MA-Diox-6, MA-Diox-7, and MA-Diox-8, respectively; and the corresponding compounds wherein E is -CN are herein identified CN- Diox-3, CN-Diox-4, CN-Diox-5, CN-Diox-6, CN-Diox-7, and CN-Diox-8, respectively (Table 6). The specific compounds wherein R2and R3together with the carbon atom to which they are attached form selected from (lr,3r,5r,7r)-adamantan-2-one or 4- oxatricyclo[4.3.1.13,8]undecan-5-one, and E is -COOH are herein identified AA-KetoneAD and AA-LactoneAD, respectively; the corresponding compounds wherein E is -COOCH3 are herein identified MA-KetoneAD and MA-LactoneAD, respectively; and the corresponding compounds wherein E is -CN are herein identified CN-KetoneAD and CN- LactoneAD, respectively (Table 7)Table 6: The compounds identified herein as AA-Diox-1 - AA-Diox-9*Table 7: The compounds identified herein as AA-KetoneAD and AA-LactoneAD

[0050] In another aspect, the present invention provides a composition comprising a carrier, and a dioxetane-based chemiluminescence probe of the formula I according to any one of the embodiments above.

[0051] As described above, the compounds of the formula I have a cleavable caging group (R8), e.g., an analyte-responsive group or a boron-containing group, wherein removal of said cleavable group by an analyte of interest, upon exposure to said analyte, generates an unstable phenolate-dioxetane species that decomposes through a chemiexcitation process to yield an excited intermediate, which in turn decays to its ground-state through emission of photons. Such a composition may thus be used for diagnostics or in vivo imaging.

[0052] Dioxetane-based chemiluminescence probes according to the present invention, wherein one or more of R4, R5and R6is a particular π* acceptor group emitting near-infrared (NIR) light under physiological conditions with high efficiency, may thus be used for diagnostics and / or imaging both in vitro and in vivo. Such dioxetane-based chemiluminescence probes are those wherein, e.g., at least one of R4, R5and R6is 1,3,3- trimethyl-3H-indol- 1 -ium-2-yl, 4-(dicyanomethylene)-4H-chromen-2-yl, 4H-chromen-2-

[0053] In certain embodiments, the composition of the present invention is thus a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0054] Pharmaceutical compositions according to the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19thEd., 1995. The compositions can be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., the dioxetane-based chemiluminescence probe, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation. The compositions may be in liquid, solid or semisolid form and may further include pharmaceutically acceptable fillers, carriers, diluents or adjuvants, and other inert ingredients and excipients.

[0055] The pharmaceutical composition of the invention may be in the form of a sterile injectable aqueous or oleaginous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include, e.g., water, Ringer's solution and isotonic sodium chloride solution.

[0056] In a further aspect, the present invention relates to a method for diagnostics or in vivo imaging comprising: applying a dioxetane-based chemiluminescence probe or a composition, each according to any one of the embodiments above, to a sample, or administering such a dioxetane-based chemiluminescence probe or composition to a subject, wherein R8in the compound of the formula I is a group cleavable by an analyte, wherein upon exposure to said analyte, R8is cleaved (fully or partially) from the compound of formula I, thereby generating an unstable phenolate-dioxetane compound, which in turn decomposes through a chemiexcitation process to produce an excited intermediate that decays to its ground-state through emission of light; and imaging to detect the emission of light. The method disclosed herein may be used for either determining the presence, or measuring the level, of said analyte in said sample or said subject. When used for in vivo imaging, the chemiluminescence probe may be administered systemically or locally, e.g., to a particular organ of said subject, so as to determine the presence, or measure the level, of said analyte in general, or in particular said organ.

[0057] The sample analyzed according to this method may be any sample, e.g., a biological sample. The term “biological sample” as used herein refers to a tissue biopsy sample; a bodily fluid such as an amniotic fluid, aqueous humour, vitreous humour, bile,blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), endolymph, perilymph, female ejaculate, gastric juice, mucus, peritoneal fluid, saliva, sebum (skin oil), semen, sweat, tears, vaginal secretion, vomit and urine; or a bodily fluid-based solution, i.e., an aqueous solution in which a bodily fluid is dissolved.

[0058] The term “subject” as used herein refers to a mammal, e.g., a human, non -human primate, horse, ferret, dog, cat, cow, and goat, but it preferably denotes a human, i.e., an individual.

[0059] The chemiluminescence emission of the probes of the present invention can be detected utilizing any technique or procedure known in the art.

[0060] Optical molecular imaging is a promising technique that provides a high degree of sensitivity and specificity in tumor margin detection. Furthermore, existing clinical applications have proven that optical molecular imaging is a powerful intraoperative tool for guiding surgeons performing precision procedures, thus enabling radical resection and improved survival rates. An example of a clinically approved instrument for minimally invasive surgical procedures under fluorescence guidance is the da Vinci Surgical System. This instrument is featured with a 3D HD vision system for a clear and magnified view inside a patient's body and allows surgeons to perform complex and routine procedures through a few small openings, similar to traditional laparoscopy. In addition, the following systems have already been applied in surgeries for breast cancer, liver metastases and bypassing graft surgery: The Hamamatsu's Photodynamic Eye (PDE™), Artemis™ and Novadaq SPY™ (Novadaq Technologies Inc., Toronto, Canada) (Chi et al., 2014). Several existing intraoperative NIR fluorescence molecular imaging systems were evaluated in clinical trials, including Fluobeam®, FLARE™ and GXMI Navigator. They have played an important role in operation convenience, improving image assessment and increasing detection depth.

[0061] In recent years, there has been great progress in the development of cameras and lasers for optical fluorescence imaging in the IR range. In parallel, there is a vast clinical use of low MW organic dyes such as ICG and methylene blue for determining cardiac output, hepatic function and liver blood flow, and for ophthalmic angiography. In 2015, the fluorescence imaging system, Xiralite®, gained FDA approval for visualization of microcirculation in the hands (for inflammation and perfusion-related disorders).

[0062] The invention will now be illustrated by the following non-limiting Examples.EXAMPLESAbbreviations

[0063] ACN, acetonitrile; CHCl3, chloroform; DCM, dichloromethane; DMBA, 1,3- dimethylbarbituric acid; DMSO, dimethylsulf oxide; DMF, N, N' -dimethylformamide; EtOAc, ethyl acetate; EtsN, triethylamine; Hex, hexane; LDA, lithium diisopropylamide, MB, methylene blue; MeOH, methanol; TBAF, tetra-n-butylammonium fluoride; TBS, terZ-butyldimethylsilyl; THF, tetrahydrofuran; TFA, trifluoroacetic acid.Study 1. Chemiexcitation acceleration of 1,2-dioxetanes by spiro-fused six-member rings with electron-withdrawing motifsExperimental

[0064] General methods. All reactions requiring anhydrous conditions were performed under an Argon atmosphere. All reactions were carried out at room temperature unless stated otherwise. Chemicals and solvents were either A.R. grade or purified by standard techniques. Thin-layer chromatography (TLC): silica gel plates Merck 60 F254: compounds were visualized by irradiation with UV light. Column chromatography (FC): silica gel Merck 60 (particle size 0.040-0.063 mm), eluent given in parentheses. Reverse-phase high-pressure liquid chromatography (RP-HPLC): C18 5u, 250x4.6mm, eluent given in parentheses. Preparative RP-HPLC: C18 5u, 250x21mm, eluent given in parentheses. ' H-NMR spectra were measured using Bruker Avance operated at 400MHz.13C-NMR spectra were measured using Bruker Avance operated at 101 MHz. Chemical shifts were reported in ppm on the 6 scale relative to a residual solvent (CDCI3: 6 = 7.26 for ' H-NMR and 77.16 for13C-NMR and MeOH-d4: 6 = 3.31 for ' H-NMR and 49.00 for13C-NMR). Mass spectra were measured on Waters Xevo TQD. Chemiluminescence was recorded on Molecular Devices Spectramax iD3. All general reagents, including salts and solvents, were purchased from Sigma- Aldrich. Light irradiation for photochemical reactions: LED PAR38 lamp (19W, 3000K).Synthesis and characterizationScheme 1: General synthetic procedures for the formation of TBS -masked Diox 1-9 Procedure

[0065] Diox 1, 2 and 9 were previously reported (Tannous et al., 2024). All compounds were synthesized according to general procedures A and B below.

[0066] Procedure A - Wittig-Horner reaction. Phosphonate I (Green et al., 2017) (1.0 equiv.) was dissolved in dry THF under argon atmosphere at -78°C. LDA (1.5 equiv.) was added dropwise, and the solution was stirred for 20 minutes. Cycloalkyl ketone (1 equiv.) was added, and then the reaction was warmed to room temperature. The reaction was monitored by TLC (Hex: EtOAc mixture). Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and brine. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification of the crude residues by column chromatography (Hex: EtOAc mixture) afforded the desired enolethers (compounds la-6a).

[0067] Procedure B - Oxidation reaction. Enol-ether (Compound la-6a) was dissolved in 10 mL of DCM. A catalytic amount of methylene blue was added, and the solution was cooled to 0°C. Then, oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC (gradient of 70-100% or 90-100% ACN in water, 0.1% TFA). Upon completion, the crude product was immediately passed through a silica gel column (DCM or Hex: EtOAc mixture) to filter out the methylene blue. The solvents were removed under reduced pressure while cooling the bath to 20°C. The compounds were further purified by preparative RP-HPLC (gradient of ACN in water, 0.1% TFA or 100% ACN) to obtain Diox 3-8.Synthesis and characterization of Diox 3-8

[0068] Compound la. As depicted in Scheme 2, compound la was synthesized according to Procedure A: Phosphonate I (Green et al., 2017) (406 mg, 1.13 mmol), dry THF (4 mL), LDA (2.0M in THF, 0.848 mL, 1.70 mmol), and methyl 4 -ketocyclohexanecarboxylate (158 mL, 1.13 mmol). The crude residue was purified by column chromatography (98.5: 1.5, Hex: EtOAc) to afford compound la as a yellow oil (291 mg, 66% yield).Scheme 2: Synthesis of compound la

[0069] ' H NMR (400 MHz, CDCl3) 6 7.20 (t, J = 7.7 Hz, 1H), 6.87 (dd, J = 7.5, 1.1 Hz, 1H), 6.81-6.75 (m, 2H), 3.67 (s, 3H), 3.27 (s, 3H), 3.05-2.98 (m, 1H), 2.52-2.37 (m, 2H), 2.08-1.99 (m, 1H), 1.95-1.83 (m, 3H), 1.66-1.45 (m, 2H), 0.98 (s, 9H), 0.19 (s, 6H).

[0070] 13C NMR (101 MHz, CDCl3) 6 176.23, 155.53, 147.51, 136.43, 129.13, 122.92,121.42, 120.66, 119.80, 57.48, 51.69, 43.27, 30.12, 29.85, 28.39, 25.81, -4.30.

[0071] Diox 3. As depicted in Scheme 3, compound la (22 mg, 0.056 mmol) was reacted according to Procedure B. Reaction was monitored by RP-HPLC (90-100% ACN in water, 0.1% TFA). Upon completion, the crude product was filtered through a short silica column (DCM) and then the solvent was evaporated under reduced pressure. The obtained product was purified by preparative RP-HPLC (100% ACN) to afford Diox 3 as an oil (12.3 mg, 52% yield).

[0072] 1H NMR (400 MHz, CDCl3) (diastereomeric mixture) 8 7.32-7.26 (m, 2H), 7.15- 6.80 (m, 6H), 3.63 (s, 3H), 3.61 (s, 3H), 3.17 (s, 3H), 3.14 (s, 3H), 2.82-2.77 (m, 1H), 2.52- 2.62 (m, 1H), 2.28-2.36 (m, 1H), 2.12-2.21 (m, 1H), 1.93-1.76 (m, 3H), 1.75-1.93 (m, 3H), 1.34-1.23 (m, 2H), 0.98 (s, 9H), 0.98 (s, 9H), 0.19 (s, 12H).

[0073] 13C NMR (101 MHz, CDCl3) (diastereomeric mixture) 8 175.35, 175.15, 156.10,136.43, 136.11, 129.74, 121.39, 121.25, 111.97, 91.54, 90.96, 51.76, 51.70, 50.22, 41.53, 40.12, 32.40, 31.51, 29.12, 28.97, 25.78, 24.51, 23.83, 23.41, 23.21, 18.33, -4.30.Scheme 3: Synthesis of Diox 3

[0074] Compound 2a. As depicted in Scheme 4, compound 2a was synthesized according to Procedure A: Phosphonate I (Green et al., 2017) (406 mg, 1.13 mmol), dry THF (4 mL), LDA (2.0M in THF, 0.848 mL, 1.70 mmol), and tetrahydro -4 / 7-pyran-4 -one (104 mL, 1.13 mmol). The crude residue was purified by column chromatography (95: 5, Hex: EtOAc) to afford compound 2a as a yellow oil (223 mg, 59% yield).

[0075] 1H NMR (400 MHz, CDCl3) 6 7.23-7.18 (m, 1H), 6.88 (dt, J = 7.6, 1.1 Hz, 1H), 6.79 (dt, J = 10.3, 1.8 Hz, 2H), 3.73 (t, J = 5.5 Hz, 2H), 3.60 (t, J = 5.4 Hz, 2H), 3.29 (s, 3H), 2.54 (t, J = 5.4 Hz, 2H), 2.22 (t, J = 5.6 Hz, 2H), 0.98 (s, 9H), 0.19 (s, 6H).

[0076] 13C NMR (101 MHz, CDCl3) 6 155.64, 148.17, 135.94, 129.24, 122.92, 121.39, 119.94, 117.59, 69.01, 68.92, 57.49, 30.50, 27.97, 25.80, 18.36, -4.28.Scheme 4: Synthesis of compound 2a

[0077] Diox 4. As depicted in Scheme 5, compound 2a (23 mg, 0.069 mmol) was reacted according to Procedure B. Reaction was monitored by RP-HPLC (90-100% ACN in water, 0.1% TFA). Upon completion, the crude product was filtered through a short silica column (DCM) and then the solvent was evaporated under reduced pressure. The obtained product was purified by preparative RP-HPLC (100% ACN) to afford Diox 4 as an oil (13.2 mg, 52% yield).

[0078] 1H NMR (400 MHz, CDCl3) 6 7.30 (t, J = 7.9 Hz, 1H), 7.07 (d, J = 5.8 Hz, 1H), 6.94 (s, 1H), 6.88 (ddd, J = 8.1, 2.4, 1.0 Hz, 1H), 3.78 (dt, J = 11.9, 4.5 Hz, 1H), 3.58-3.45 (m, 3H), 3.18 (s, 3H), 2.46-2.37 (m, 1H), 2.18-2.09 (m, 1H), 1.78 (ddd, J = 13.8, 5.3, 3.4 Hz, 1H), 1.53-1.41 (m, 1H), 0.99 (s, 9H), 0.20 (s, 6H).

[0079] 13C NMR (101 MHz, CDCl3) 6 156.26, 135.98, 129.91, 121.44, 120.53, 119.23, 111.90, 89.58, 63.19, 63.01, 50.31, 33.99, 31.42, 25.78, 18.35, -4.26.Scheme 5: Synthesis of Diox 4

[0080] Compound 3a. As depicted in Scheme 6, compound 3a was synthesized according to Procedure A: Phosphonate I (Green et al., 2017) (406 mg, 1.13 mmol), dry THF (4 mL), LDA (2.0M in THF, 0.848 mL, 1.70 mmol), and tetrahydro -4 / 7-pyran-3 -one (103 mL, 1.13 mmol). The crude residue was purified by column chromatography (95: 5, Hex: EtOAc) to afford compound 3a as a yellow oil (241 mg, 64% yield). Compound 3a was obtained as a diastereomeric mixture of trans and cis (E:Z) in 3:2 ratio, respectively.

[0081] 1H NMR (400 MHz, CDCl3) (major) 8 7.22 (dd, J = 15.7, 7.9 Hz, 1H), 6.84-6.91 (m, 1H), 6.83-6.72 (m, 2H), 4.06 (s, 2H), 3.77-3.71 (m, 2H), 3.31 (s, 3H), 2.59-2.55 (m, 2H), 1.69-1.61 (m, 2H), 0.98 (s, 9H), 0.19 (s, 6H).

[0082] 1H NMR (400 MHz, CDCl3) (minor) 8 7.22 (dd, J = 15.7, 7.9 Hz, 1H), 6.84-6.91 (m, 1H), 6.83-6.72 (m, 2H), 4.39 (s, 2H), 3.78-3.71 (m, 2H), 3.29 (s, 3H), 2.26-2.21 (m, 2H), 1.77-1.70 (m, 2H), 0.98 (s, 9H), 0.20 (s, 6H).

[0083] 13C NMR (101 MHz, CDCl3) (diastereomeric mixture) 8 155.64, 149.21, 148.63, 135.49, 135.30, 129.31, 129.27, 122.98, 122.79, 121.34, 120.28, 120.24, 117.47, 116.96,68.89, 68.75, 68.55, 66.31, 57.46, 57.32, 27.90, 27.60, 26.68, 25.80, 24.24, -4.29.Scheme 6: Synthesis of compound 3a

[0084] Diox 5. As depicted in Scheme 7, compound 3a (21 mg, 0.063 mmol) was reacted according to Procedure B. Reaction was monitored by RP-HPLC (90-100% ACN in water, 0.1% TFA). Upon completion, the crude product was filtered through a short silica column (DCM) and then the solvent was evaporated under reduced pressure. The obtained product was purified by preparative RP-HPLC (70-100% ACN in water, 0.1% TFA) to afford Diox 5 as an oil (8.2 mg, 36% yield).

[0085] NMR (400 MHz, CDCl3) (diastereomeric mixture) 8 7.68-7.58 (m, 2H), 7.50- 7.18 (m, 6H), 4.51 (dd, 7 = 70.2, 12.3 Hz, 2H), 4.03-3.86 (m, 4H), 3.78-3.65 (m, 4H), 3.52-3.45 (m, 6H), 2.62 (t, J = 6.2 Hz, 2H), 2.19-1.88 (m, 4H), 1.32 (s, 18H), 0.54 (d, J = 2.7 Hz, 12H).

[0086] 13C NMR (101 MHz, CDCl3) (diastereomeric mixture) 8 156.21, 156.13, 135.86, 135.40, 129.83, 129.49, 124.93, 122.71, 121.56, 121.49, 121.16, 120.49, 119.23, 87.59, 87.41, 71.10, 69.67, 67.25, 52.26, 50.34, 50.03, 30.66, 27.89, 25.79, 22.23, 21.78, 18.36, - 4.31.Scheme 7: Synthesis of Diox 5

[0087] Compound 4a. As depicted in Scheme 8, compound 4a was synthesized according to Procedure A: Phosphonate I (Green et al., 2017) (424 mg, 1.18 mmol), dry THF (4 mL), LDA (2.0M in THF, 0.885 mL, 1.77 mmol), and 4,4-difluorocyclohexanone (158 mg, 1.18 mmol). The crude residue was purified by column chromatography (99: 1, Hex: EtOAc) to afford compound 4a as a yellow oil (303 mg, 7x0% yield).

[0088] 1H NMR (400 MHz, CDCl3) 6 7.23 (t, J = 7.8 Hz, 1H), 6.89-6.86 (m, 1H), 6.84- 6.80 (m, 1H), 6.77-6.75 (m, 1H), 3.29 (s, 3H), 2.59 (t, J = 6.6, 2H), 2.26 (t, J = 6.6, 2H), 1.98(ddd, J = 20.3, 13.5, 6.6 Hz, 2H), 1.85 (ddd, J = 19.9, 13.4, 6.4 Hz, 2H), 0.99 (s, 9H), 0.20 (s, 6H).

[0089] 13C NMR (101 MHz, CDCl3) 6 155.67, 148.83, 135.87, 129.33, 122.85, 121.38, 120.18, 117.24, 57.45, 34.88 (t, J = 20.1 Hz), 34.44 (t, J = 20.0 Hz), 25.80, 25.51 (t, J = 5.0 Hz), 22.81 (t, J = 5.0 Hz), 18.36, -4.29.Scheme 8: Synthesis of compound 4a

[0090] Diox 6. As depicted in Scheme 9, compound 4a (28 mg, 0.076 mmol) was reacted according to Procedure B. Reaction was monitored by RP-HPLC (90-100% ACN in water, 0.1% TFA). Upon completion, the crude product was filtered through a short silica column (DCM) and then the solvent was evaporated under reduced pressure. The obtained productwas purified by preparative RP-HPLC (90-100% ACN in water, 0.1% TFA) to afford Diox 6 as an oil (12.9 mg, 42% yield).

[0091] 1H NMR (400 MHz. CDCL) 5 7.31 (t, J = 7.9 Hz, 1H), 7.14-6.86 (m, 3H), 3.17 (s, 3H), 2.69-2.60 (m, 1H), 2.16-1.69 (m, 7H), 1.53-1.42 (m, 1H), 0.99 (s, 9H), 0.21 (s, 6H).

[0092] 13C NMR (101 MHz, CDCL) 6 156.34, 135.98, 130.01, 122.33, 121.56, 120.26, 111.65, 90.30, 50.42, 29.88 (d, J = 7.5 Hz), 28.90 (t, J = 25.2 Hz), 28.86 (t, J = 25.4 Hz) ,26.76 (d, J = 6.7 Hz), 25.79, 18.38, -4.26.Scheme 9: Synthesis of Diox 6

[0093] Compound 5a. As depicted in Scheme 10, compound 5a was synthesized according to Procedure A: phosphonate I (Green etal., 2017) (428 mg, 1.19 mmol), dry THF(4 mL), LDA (2.0M in THF, 0.892 mL, 1.78 mmol), and 2,2-dimethyl-l,3-dioxan-5-one (142 mL, 1.19 mmol). The crude residue was purified by column chromatography (97.5: 2.5, Hex: EtOAc) to afford compound 5a as a yellow oil (285 mg, 66% yield).

[0094] 1H NMR (400 MHz, CDCl3) 6 7.22 (t, J = 7.8 Hz, 1H), 6.85-6.79 (m, 2H), 6.73- 6.70 (m, 1H), 4.57 (s, 2H), 4.26 (s, 2H), 3.34 (s, 3H), 1.42 (s, 6H), 0.98 (s, 9H), 0.19 (s, 6H).

[0095] 13C NMR (101 MHz, CDCL) 6 155.78, 147.77, 134.40, 129.45, 122.25, 120.62, 120.57, 116.34, 99.41, 59.83, 59.02, 57.32, 25.79, 24.11, 18.34, -4.27.Scheme 10: Synthesis of compound 5a

[0096] Diox 7. As depicted in Scheme 11, compound 5a (19 mg, 0.052 mmol) was reacted according to Procedure B. Reaction was monitored by RP-HPLC (90-100% ACN in water, 0.1% TFA). Upon completion, the crude product was filtered through a short silica column (DCM) and then the solvent was evaporated under reduced pressure. The obtained product was purified by preparative RP-HPLC (100% ACN) to afford Diox 7 as an oil (5.8 mg, 28% yield).

[0097] 7.32 (t, J = 7.9 Hz, 1H), 7.10 (d, J = 7.3 Hz, 1H), 6.98 (s, 1H), 6.94-6.87 (m, 1H), 4.58 (d, J = 13.4 Hz, 1H), 4.14 (d, J = 13.4 Hz, 1H), 3.77 (d, J = 13.0 Hz, 1H), 3.56 (d, J = 13.0 Hz, 1H), 3.16 (s, 3H), 1.32 (s, 3H), 1.28 (s, 3H), 0.99 (s, 9H), 0.21 (s, 6H).

[0098] 13C NMR (101 MHz, CDCl3) 6 156.37, 135.14, 130.11, 121.81, 120.25, 118.99, 110.80, 98.82, 86.74, 63.47, 61.61, 50.23, 25.78, 24.23, 22.47, 18.35, -4.28.Scheme 11: Synthesis of Diox 7

[0099] Compound 6a. As depicted in Scheme 12, compound 6a was synthesized according to Procedure A: phosphonate I (Green et al., 2017) (200 mg, 0.555 mmol), dry THF (2 mL), LDA (2.0M in THF, 0.416 mL, 0.832 mmol), and tetrahydrothiopyran-4-one 1,1-dioxide (82 mg, 0.555 mmol). The crude residue was purified by column chromatography (85: 15, Hex: EtOAc) to afford compound 6a as a solid (84 mg, 40% yield).

[0100] 1H NMR (400 MHz, CDCl3) 6 7.25 (t, J = 7.8 Hz, 1H), 6.87-6.81 (m, 2H), 6.73- 6.70 (m, 1H), 3.29 (s, 3H), 3.09-3.03 (m, 2H), 3.01-2.97 (m, 2H), 2.91-2.86 (m, 2H), 2.60 (dd, J = 7.3, 5.1 Hz, 2H), 0.98 (s, 9H), 0.19 (s, 6H).

[0101] 13C NMR (101 MHz, CDCl3) 6 155.95, 151.71, 134.61, 129.75, 122.71, 121.29, 120.87, 112.01, 57.35, 52.60, 52.40, 27.11, 25.77, 24.10, 18.37, -4.27.Scheme 12: Synthesis of compound 6a

[0102] Diox 8. As depicted in Scheme 13, compound 6a (26 mg, 0.068 mmol) was reacted according to Procedure B. Reaction was monitored by RP-HPLC (70-100% ACN in water, 0.1% TFA). Upon completion, the crude product was filtered through a short silica column (50:50, Hex:EtOAc) and then the solvent was evaporated under reduced pressure. The obtained product was purified by preparative RP-HPLC (70-100% ACN in water, 0.1% TFA) to afford Diox 8 as an oil (15.1 mg, 54% yield).

[0103] 1H NMR (400 MHz, CDCl3) 6 7.33 (t, J = 8.0 Hz, 1H), 7.21-6.74 (m, 3H), 3.18 (s, 3H), 3.15-2.96 (m, 4H), 2.79-2.71 (m, 1H), 2.53 (ddd, J = 15.7, 12.4, 3.5 Hz, 1H), 2.33 (dtd, J = 6.3, 5.5, 3.6 Hz, 1H), 2.01-1.92 (m, 1H), 0.99 (s, 9H), 0.21 (s, 6H).

[0104] 13C NMR (101 MHz, CDCl3) 6 156.59, 135.12, 130.37, 121.91, 111.26, 88.19, 50.70, 46.08, 46.04, 31.51, 28.52, 25.77, 18.37, -4.23.Scheme 13: Synthesis of Diox 8

[0105] TBS-SO2 ene-product. As depicted in Scheme 14, compound 6a (95 mg, 0.248 mmol) was dissolved in 10 mL of DCM, and a catalytic amount of methylene blue was added to the solution. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC (gradient of 70-100% ACN in water, 0.1% TFA). The obtained product was purified by preparative RP-HPLC (70-100% ACN in water, 0.1% TFA) to afford TBS-SO2 ene-product as a white solid (7 mg, 7% yield).

[0106] 1H NMR (400 MHz, CDCl3) 6 7.23 (t, J = 7.9 Hz, 1H), 6.98 (ddd, J = 7.8, 1.6, 1.0 Hz, 1H), 6.89-6.87 (m, 1H), 6.82 (ddd, J = 8.1, 2.5, 1.0 Hz, 1H), 6.23 (t, J = 4.3 Hz, 1H), 3.78-3.65 (m, 2H), 3.22 (s, 3H), 3.05-2.97 (m, 1H), 2.95-2.87 (m, 1H), 2.79-2.69 (m, 1H), 2.46 (dddd, J = 17.7, 8.2, 4.9, 1.6 Hz, 1H), 1.25 (s, 1H), 0.98 (s, 9H), 0.19 (s, 6H).

[0107] 13C NMR (101 MHz, CDCl3) 6 156.01, 137.87, 137.60, 129.79, 120.83, 119.51, 118.51, 117.79, 106.34, 50.51, 50.38, 47.44, 25.81, 25.17, 18.39, -4.26.Scheme 14: Synthesis of TBS-SO2 ene-productSynthesis and characterization of MA-Diox 1-9

[0108] MA-Diox-1 and MA-Diox-9 were previously reported (Tannous et al., 2024;Green et al., 2017). All compounds were synthesized according to general procedures C-H (Scheme 15).Scheme 15: General synthetic procedures for the formation of P-gal masked MA-Diox 1-9

[0109] Procedure C - Wittig-Horner reaction and TBS deprotection. Phosphonate II (Hananya et al., 2016) (1.0 equiv.) was dissolved in dry THF under argon atmosphere and cooled to -78°C. LDA (2.0 M in THF, 1.5 equiv.) was added dropwise, and the solution was stirred for 20 minutes. Cycloalkyl ketone (1.0 equiv.) was added, and the reaction was warmed to room temperature. The reaction was monitored by TLC (Hex: EtOAc mixture). Upon completion, TBAF (1.0 M in THF, 1.1 eq) was added, and the reaction was monitored by TLC (Hex: EtOAc mixture). Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and brine. The organic layer was dried overNa2SO4 and concentrated under reduced pressure. Purification by column chromatography (Hex: EtOAc mixture) afforded compound lb -7b.

[0110] Procedure D - Ortho-formylation reaction. Compound lb-7b (1 equiv.) and EtaN (8 equiv.) were dissolved in dry THF in a pressure flask. MgCl2(4 equiv.) and paraformaldehyde (16 equiv.) were added simultaneously, and the pressure flask was inserted into an oil bath that was pre-heated to 80°C. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc mixture) to afford compound lc-7c.

[0111] Procedure E - Wittig reaction. Compound lc-7c (1 equiv.) was dissolved in DCM. Methyl (triphenylphosphoranylidene) acetate (1.5 equiv.) was added, and the reaction was stirred at room temperature. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with brine. The organic phase was dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc mixture) to afford compound ld-7d.

[0112] Procedure F - SN2 reaction. Compound ld-7d (1 equiv.) was dissolved in dry DMF. K2CO3 (2 equiv.) and compound III (Green et al., 2017) (1 equiv.) were added, and the reaction was stirred at room temperature. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and Na2S2O3. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc mixture) or preparative HPLC (50-100% ACN in water, 0.1% TFA) to afford compound le-7e.

[0113] Procedure G - Acetate deprotection. Compound le-7e (1 equiv.) was dissolved in MeOH, and K2CO3 (5-10 equiv.) was added, and the reaction was stirred at room temperature. The reaction was monitored by RP-HPLC (30-100% ACN in water, 0.1% TFA). Upon completion, the solvent was evaporated under reduced pressure, and the product was purified by preparative RP-HPLC (gradient of ACN in water, 0.1% TFA) or (21-70% ACN, ammonium carbonate buffer [30 mM]) to afford compound lf-7f.

[0114] Procedure H - Oxidation reaction. Compound lf-7f was dissolved in DCM / MeOH (10 mL) followed by the addition of a catalytic amount of methylene blue or rose bengal. The solution was cooled to 0°C (except for the oxidation of compound 7f), then oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC (30-100% ACN in water, 0.1% TFA). Upon completion, the solvent was evaporated under reduced pressure at 10°C. The crude product was purified by preparative RP-HPLC (gradient of ACN in water, 0.1% TFA) or (35-70% ACN, ammonium carbonate buffer [30 mM]) to afford MA-Diox 2-8.

[0115] Compound lb. As depicted in Scheme 16, compound lb was synthesized according to Procedure C: Phosphonate II (Hananya et al., 2016) (490 mg, 1.24 mmol), dry THF (5 mL), LDA (2.0M in THF, 0.93 mL, 1.86 mmol), cyclohexanone (128 mL, 1.24 mmol), and TBAF (1.0M in THF, 1.36 mL, 1.36 mmol). The crude residue was purified by column chromatography (95: 5, Hex: EtOAc) to afford compound lb as an oil (154 mg, 49% yield).

[0116] 1H NMR (400 MHz, CDCl3) 6 7.15 (t, J = 8.0, 1H), 6.99 (dd, J = 8.1, 1.5 Hz, 1H), 6.83 (dd, J = 7.5, 1.5 Hz, 1H), 3.29 (s, 3H), 2.40 (d, J = 5.9 Hz, 2H), 1.86-1.77 (m, 2H), 1.66-1.31 (m, 8H).

[0117] 13C NMR (101 MHz, CDCl3) 6 151.77, 142.88, 135.11, 127.45, 124.28, 124.04, 120.77, 115.62, 56.90, 29.96, 27.79, 27.69, 26.88, 26.85.

[0118] MS (ES-): m / z calc, for CI4HI7C1O2: 252.09; found: 251.3 [M-H]’.Scheme 16: Synthesis of compound lb

[0119] Compound 2b. As depicted in Scheme 17, compound 2b was synthesized according to Procedure C: Phosphonate II (Hananya et al., 2016) (550 mg, 1.39 mmol), dry THF (5.5 mL), LDA (2.0M in THF, 1.04 mL, 2.08 mmol), methyl 4- ketocyclohexanecarboxylate (196 mL, 1.39 mmol), and TBAF (1.0M in THF, 1.53 mL, 1.53 mmol). The crude residue was purified by column chromatography (92.5: 7.5, Hex: EtOAc) to afford compound 2b as an oil (197 mg, 46% yield).

[0120] 1H NMR (400 MHz, CDCl3) 6 7.17 (t, J = 7.5 Hz, 1H), 7.03-6.98 (m, J = 7.8 Hz, 1H), 6.87-6.77 (m, J = 9.6 Hz, 1H), 3.66 (s, 3H), 3.28 (s, 3H), 3.10-2.97 (m, 1H), 2.52-2.29 (m, 2H), 2.13-1.73 (m, 5H), 1.68-1.54 (m, 1H).

[0121] 13C NMR (101 MHz, CDCl3) 6 176.25, 151.87, 134.69, 127.62, 123.96, 123.92, 115.91, 115.86, 56.84, 51.74, 43.16, 39.85, 29.77, 29.63, 28.64.

[0122] MS (ES-): m / z calc, for C16H19CIO4: 310.10; found: 309.3 [M-H]’.Scheme 17: Synthesis of compound 2b

[0123] Compound 3b. As depicted in Scheme 18, compound 3b was synthesized according to Procedure C: Phosphonate II (Hananya et al., 2016) (408 mg, 1.03 mmol), dry THF (4 mL), LDA (2.0M in THF, 1.04 mL, 2.08 mmol), tetrahydro -4H-pyran-4-one (95.4 mL, 1.03 mmol), and TBAF (1.0M in THF, 1.13 mL, 1.13 mmol). The crude residue was purified by column chromatography (90: 10, Hex: EtOAc) to afford compound 3b as an oil (74 mg, 28% yield).

[0124] 1H NMR (400 MHz, CDCl3) 6 7.20-7.12 (m, 1H), 7.00 (dd, J = 8.2, 1.5 Hz, 1H), 6.83 (dd, J = 7.5, 1.5 Hz, 1H), 6.20 (s, 1H), 3.75 (d, J = 5.1 Hz, 2H), 3.61 (t, J = 5.4 Hz, 2H), 3.29 (s, 3H), 2.65-2.44 (m, 2H), 1.96 (t, J = 5.4 Hz, 2H).

[0125] 13C NMR (101 MHz, CDCl3) 6 152.05, 144.55, 134.19, 127.67, 123.83, 120.64, 117.99, 116.15, 68.81, 68.76, 56.74, 30.27, 27.48.

[0126] MS (ES-): m / z calc, for C13H15CIO3: 254.07; found: 253.2 [M-H]’.Scheme 18: Synthesis of compound 3b

[0127] Compound 4b. As depicted in Scheme 19, compound 4b was synthesized according to Procedure C: Phosphonate II (Hananya et al., 2016) (460 mg, 1.16 mmol), dry THF (4.5 mL), LDA (2.0M in THF, 0.87 mL, 1.74 mmol), tetrahydro -2H-pyran-3 -one (107mL, 1.16 mmol), and TBAF (1.0M in THF, 1.28 mL, 1.28 mmol). The crude residue was purified by column chromatography (80: 20, Hex: EtOAc) to afford compound 4b as an oil (99 mg, 33% yield). Compound 4b was obtained as a diastereomeric mixture of trans and cis (E:Z) in 3:2 ratio, respectively.

[0128] 1H NMR (400 MHz, CDCl3) (major) 8 7.21-7.12 (m, 1H), 7.02-6.97 (m, 1H), 6.83 (dd, J = 7.5, 1.5 Hz, 1H), 4.43 (d, J = 5.2 Hz, 1H), 3.84 (s, 1H), 3.78-3.70 (m, 2H), 3.27 (s, 3H), 2.70-2.38 (m, 1H), 1.99 (t, J = 6.2 Hz, 1H), 1.77-1.70 (m, 1H), 1.63 (s, 1H).

[0129] ‘H NMR (400 MHz, CDCl3) (minor) 8 7.21-7.12 (m, 1H), 7.02-6.97 (m, 1H), 6.83 (dd, J = 7.5, 1.5 Hz, 1H), 4.43 (d, J = 5.2 Hz, 1H), 3.84 (s, 1H), 3.78-3.70 (m, 2H), 3.29 (s, 3H), 2.70-2.38 (m, 1H), 1.99 (t, J = 6.2 Hz, 1H), 1.77-1.70 (m, 1H), 1.63 (s, 1H).

[0130] 13C NMR (101 MHz, CDCl3) (diastereomeric mixture) 8 152.09, 145.61, 145.06, 133.71, 133.35, 127.66, 123.84, 123.53, 117.34, 117.11, 116.53, 116.30, 68.70, 68.41, 65.69, 56.57, 56.44, 29.77, 27.32, 26.38, 23.69.

[0131] MS (ES-): m / z calc, for C13H15CIO3: 254.07; found: 253.2 [M-H]’.Scheme 19: Synthesis of compound 4b

[0132] Compound 5b. As depicted in Scheme 20, compound 5b was synthesized according to Procedure C: Phosphonate II (Hananya et al., 2016) (550 mg, 1.39 mmol), dry THF (5.5 mL), LDA (2.0M in THF, 1.04 mL, 2.08 mmol), 4,4-difluorocyclohexanone (187 mg, 1.39 mmol), and TBAF (1.0M in THF, 1.53 mL, 1.53 mmol). The crude residue was purified by column chromatography (95: 5, Hex: EtOAc) to afford compound 5b as an oil (222 mg, 55% yield).

[0133] 1H NMR (400 MHz, CDCl3) 6 7.23-7.14 (m, 1H), 7.03 (dd, J = 8.2, 1.5 Hz, 1H), 6.83 (dd, J = 7.5, 1.5 Hz, 1H), 5.38 (s, 1H), 3.29 (s, 3H), 2.60 (ddd, J = 53.0, 13.2, 6.5 Hz, 2H), 2.05-1.93 (m, 4H), 1.91-1.77 (m, J = 13.1, 6.6 Hz, 2H).

[0134] 13C NMR (101 MHz, CDCl3) 6 151.93, 145.01, 134.10, 127.83, 123.75, 120.56, 117.92, 116.30, 56.69, 34.91-33.88 (m), 25.52 (t, J = 5.1 Hz), 22.27 (t, J = 5.1 Hz).

[0135] MS (ES-): m / z calc, for C14H15CIF2O2: 288.07; found: 287.2 [M-H]’.Scheme 20: Synthesis of compound 5b

[0136] Compound 6b. As depicted in Scheme 21, compound 6b was synthesized according to Procedure C: Phosphonate II (Hananya et al., 2016) (550 mg, 1.39 mmol), dryTHF (5.5 mL), LDA (2.0M in THF, 1.04 mL, 2.08 mmol), 2,2-dimethyl-l,3-dioxan-5-one (171 mL, 1.39 mmol), and TBAF (1.0M in THF, 1.53 mL, 1.53 mmol). The crude residue was purified by column chromatography (80: 20, Hex: EtOAc) to afford compound 6b as an oil (247 mg, 62% yield).

[0137] 1H NMR (400 MHz, CDCl3) 6 7.16 (t, J = 7.8 Hz, 1H), 7.02 (dd, J = 8.2, 1.5 Hz, 1H), 6.83 (dd, J = 7.5, 1.5 Hz, 1H), 5.72 (s, 1H), 4.62 (s, 2H), 4.03 (s, 2H), 3.30 (s, 3H), 1.42 (s, 6H).

[0138] 13C NMR (101 MHz, CDCl3) 6 152.58, 144.71, 132.35, 127.91, 123.35, 120.50, 117.11, 114.60, 99.25, 59.80, 58.67, 56.25, 24.22.

[0139] MS (ES-): m / z calc, for CI4HI7C1O4: 284.08; found: 283.2 [M-H]’.Scheme 21: Synthesis of compound 6b

[0140] Compound 7b. As depicted in Scheme 22, compound 7b was synthesized according to Procedure C: Phosphonate II (Hananya et al., 2016) (860 mg, 2.18 mmol), dry THF (8.5 mL), LDA (2.0M in THF, 1.64 mL, 3. 1 mmol), tetrahydrothiopyran-4-one 1,1- dioxide (373 mg, 2.18 mmol), and TBAF (1.0M in THF, 2.40 mL, 2.78 mmol). The crude residue was purified by column chromatography (40: 60, Hex: EtOAc) to afford compound 7b as an oil (382 mg, 58% yield).

[0141] 1H NMR (400 MHz. CDCl3) 5 7.21 (dd. J = 8.1. 7.5 Hz. 1H), 7.06 (dd, J = 8.2, 1.5 Hz, 1H), 6.80 (dd, J = 7.5, 1.5 Hz, 1H), 3.29 (s, 3H), 3.17-3.05 (m, 3H), 2.99-2.78 (m, 3H),2.42-2.36 (m, 2H).

[0142] 13C NMR (101 MHz, CDCl3) 6 152.27, 147.83, 132.83, 128.08, 123.22, 120.21, 117.02, 112.10, 56.42, 52.15, 52.10, 27.03, 23.52.

[0143] MS (ES-): m / z calc, for C13H15CIO4S: 302.04; found: 301.2 [M-H]’.Scheme 22: Synthesis of compound 7b

[0144] Compound 1c. As depicted in Scheme 23, compound 1c was synthesized according to Procedure D: Compound lb (236 mg, 0.934 mmol), dry THF (7 mL), EtsN (1.04 mL, 7.47 mmol), paraformaldehyde (449 mg, 14.9 mmol), and MgCh (356 mg, 3.74 mmol). The crude residue was purified by column chromatography (90: 10, Hex: EtOAc) to afford compound 1c as an oil (163 mg, 62% yield).

[0145] 1H NMR (400 MHz, CDCl3) 6 9.90 (s, 1H), 7.49 (d, J = 7.9 Hz, 1H), 6.99 (d, J = 7.9 Hz, 1H), 3.31 (s, 3H), 2.41 (dd, J = 11.2, 5.1 Hz, 2H), 1.86-1.76 (m, 2H), 1.63-1.49 (m, 6H).

[0146] 13C NMR (101 MHz, CDCl3) 6 195.84, 157.64, 143.59, 142.00, 131.00, 125.96, 123.00, 120.48, 115.57, 57.35, 29.95, 27.58, 26.88, 26.74.

[0147] MS (ES-): m / z calc, for C15H17CIO3: 280.09; found: 279.3 [M-H]’.Scheme 23: Synthesis of compound 1c

[0148] Compound 2c. As depicted in Scheme 24, compound 2c was synthesized according to Procedure D: Compound 2b (117 mg, 0.376 mmol), dry THF (4 mL), EtsN (0.42 mL, 3.01 mmol), paraformaldehyde (181 mg, 6.02 mmol), and MgCh (143 mg, 1.51 mmol). The crude residue was purified by column chromatography (85: 15, Hex: EtOAc) to afford compound 2c as an oil (69 mg, 54% yield).

[0149] H NMR (400 MHz, CDCl3) 6 11.63 (s, 1H), 9.91 (s, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.01 (dd, J = 8.1, 1.4 Hz, 1H), 3.67 (s, 3H), 3.31 (s, 3H), 3.03 (t, J = 13.5 Hz, 1H), 2.52-2.40 (m, 1H), 2.10-1.83 (m, 5H), 1.70-1.52 (m, 2H).

[0150] 13C NMR (101 MHz, CDCl3) 6 195.89, 176.01, 157.64, 143.03, 131.15, 127.55, 123.84, 122.81, 120.63, 115.95, 57.24, 51.73, 43.06, 42.86, 29.79, 29.56, 25.18.

[0151] MS (ES-): m / z calc, for C17H19CIO5: 338.09; found: 337.3 [M-H]’.Scheme 24: Synthesis of compound 2c

[0152] Compound 3c. As depicted in Scheme 25, compound 3c was synthesized according to Procedure D: Compound 3b (210 mg, 0.824 mmol), dry THF (2 mL), EtsN (0.92 mL, 6.60 mmol), paraformaldehyde (396 mg, 13.2 mmol), and MgCh (314 mg, 3.30 mmol). The crude residue was purified by column chromatography (90: 10, Hex: EtOAc) to afford compound 3c as an oil (150 mg, 64% yield).

[0153] H NMR (400 MHz, CDCl3) 6 11.64 (s, 1H), 9.91 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H), 3.74 (dd, J = 9.7, 4.9 Hz, 2H), 3.66-3.53 (m, 2H), 3.32 (s, 3H), 2.63-2.41 (m, 2H), 2.00-1.90 (m, 2H).

[0154] 13C NMR (101 MHz, CDCl3) 6 195.83, 157.69, 143.46, 142.46, 131.18, 123.86, 122.76, 120.69, 120.04, 77.16, 68.61, 68.52, 57.19, 30.27, 27.54.

[0155] MS (ES-): m / z calc, for C14H15CIO4: 282.07; found: 281.2 [M-H]’.Scheme 25: Synthesis of compound 3c

[0156] Compound 4c. As depicted in Scheme 26, compound 4c was synthesized according to Procedure D: Compound 4b (200 mg, 0.785 mmol), dry THF (5 mL), EtsN (0.876 mL, 6.28 mmol), paraformaldehyde (377 mg, 12.6 mmol), and MgCh (299 mg, 3.14 mmol). The crude residue was purified by column chromatography (70: 30, Hex: EtOAc) to afford compound 4c as an oil (152 mg, 68% yield). Compound 4c was obtained as a diastereomeric mixture of trans and cis (E:Z) in 3:2 ratio, respectively.

[0157] H NMR (400 MHz, CDCl3) (major) 8 9.91 (d, J = 2.6 Hz, 1H), 7.54-7.49 (m, 1H), 7.04-6.97 (m, 1H), 4.41 (d, J = 10.1 Hz, 2H), 3.82 (s, 1H), 3.73 (t, J = 5.0 Hz, 2H), 3.31 (s, 3H), 2.04-1.94 (m, 2H), 1.79-1.71 (m, 1H).

[0158] 1H NMR (400 MHz, CDCl3) (minor) 8 9.91 (d, J = 2.6 Hz, 1H), 7.54-7.49 (m, 1H), 7.04-6.97 (m, 1H), 4.41 (d, J = 10.1 Hz, 2H), 3.82 (s, 1H), 3.73 (t, J = 5.0 Hz, 2H), 3.32 (s, 3H), 2.04-1.94 (m, 2H), 1.79-1.71 (m, 1H).

[0159] 13C NMR (101 MHz, CDCl3) (diastereomeric mixture) 8 195.87, 157.66, 143.84, 141.97, 131.23, 127.72, 123.13, 122.82, 122.56, 120.90, 120.79, 119.48, 119.13, 68.69, 68.44, 68.36, 65.52, 57.08, 56.95, 27.29, 27.15, 26.42, 23.81.

[0160] MS (ES-): m / z calc, for C14H15CIO4: 282.07; found: 281.3 [M-H]’.Scheme 26: Synthesis of compound 4c

[0161] Compound 5c. As depicted in Scheme 27, compound 5c was synthesized according to Procedure D: Compound 5b (240 mg, 0.831 mmol), dry THF (6 mL), EtsN (0.927 mL, 6.65 mmol), paraformaldehyde (399 mg, 13.3 mmol), and MgCh (317 mg, 3.33 mmol). The crude residue was purified by column chromatography (85: 15, Hex: EtOAc) to afford compound 5c as an oil (72 mg, 27% yield).

[0162] H NMR (400 MHz, CDCl3) 8 11.65 (s, 1H), 9.91 (s, 1H), 7.53 (d, J = 7.9 Hz, 1H), 6.98 (d, J = 7.9 Hz, 1H), 3.31 (s, 3H), 2.76-2.64 (m, 1H), 2.51 (dt, J = 13.7, 6.9 Hz, 1H), 2.07-1.77 (m, 6H).

[0163] 13C NMR (101 MHz, CDCl3) 8 195.91, 157.69, 144.06, 142.32, 131.34, 123.55, 122.62, 120.84, 119.42, 77.19, 57.09, 34.23 (t, 7 = 23.7 Hz), 34.18 (t, J = 23.6 Hz), 25.51 (t, 7 = 5.1 Hz), 22.36 (t, 7 = 5.1 Hz).

[0164] MS (ES-): m / z calc, for C15H15CIF2O3: 316.07; found: 315.3 [M-H]’.Scheme 27: Synthesis of compound 5c

[0165] Compound 6c. As depicted in Scheme 28, compound 6c was synthesized according to Procedure D: Compound 6b (200 mg, 0.702 mmol), dry THF (5 mL), EtsN (0.783 mL, 5.62 mmol), paraformaldehyde (337 mg, 11.2 mmol), and MgCh (268 mg, 2.81 mmol). The crude residue was purified by column chromatography (DCM) to afford compound 6c as an oil (97 mg, 44% yield).

[0166] 1H NMR (400 MHz, CDCl3) 6 9.90 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.01 (d, J = 7.6 Hz, 1H), 4.61 (s, 2H), 4.02 (s, 2H), 3.32 (s, 3H), 1.41 (s, 6H).

[0167] 13C NMR (101 MHz, CDCl3) 6 195.73, 157.80, 143.33, 140.15, 131.34, 122.08, 121.04, 116.73, 99.23, 59.41, 58.42, 56.60, 29.68.

[0168] MS (ES-): m / z calc, for C15H17CIO5: 312.08; found: 311.3 [M-H]’.Scheme 28: Synthesis of compound 6c

[0169] Compound 7c. As depicted in Scheme 29, compound 7c was synthesized according to Procedure D: Compound 7b (295 mg, 0.974 mmol), dry THF (3 mL), EtsN (1.09 mL, 7.79 mmol), paraformaldehyde (468 mg, 15.6 mmol), and MgCh (371 mg, 3.90 mmol). The crude residue was purified by column chromatography (60: 40, Hex: EtOAc) to afford compound 7c as an oil (65 mg, 20% yield).

[0170] H NMR (400 MHz, CDCl3) 6 11.67 (s, 1H), 9.94 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 6.98 (d, J = 7.9 Hz, 1H), 3.33 (s, 3H), 3.23-3.14 (m, 1H), 3.12-3.05 (m, 2H), 3.05-2.96 (m, 1H), 2.90-2.81 (m, 2H), 2.44-2.32 (m, 2H).

[0171] 13C NMR (101 MHz, CDCl3) 6 195.85, 157.78, 146.75, 140.90, 131.67, 122.28, 121.21, 113.80, 56.92, 52.02, 51.92, 27.12, 23.74.

[0172] MS (ES-): m / z calc, for C14H15CIO5S: 330.03; found: 329.2 [M-H]’.Scheme 29: Synthesis of compound 7c

[0173] Compound Id. As depicted in Scheme 30, compound Id was synthesized according to Procedure E: Compound 1c (143 mg, 0.509 mmol), DCM (1.5 mL), and methyl (triphenylphosphoranylidene)acetate (256 mg, 0.764 mmol). The crude residue was purified by column chromatography (85: 15, Hex: EtOAc) to afford compound Id as an oil (151 mg, 88% yield).

[0174] 1H NMR (400 MHz, CDCl3) 6 7.95 (d, J = 16.2 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.61 (d, J = 16.2 Hz, 1H), 6.37 (s, 1H), 3.81 (s, 3H), 3.28 (s, 3H), 2.40 (t, J = 5.6 Hz, 2H), 1.86-1.78 (m, 2H), 1.65-1.38 (m, 6H).

[0175] 13C NMR (101 MHz, CDCl3) 6 167.78, 150.67, 142.60, 139.29, 136.67, 126.82, 125.25, 123.77, 122.07, 121.70, 119.83, 57.14, 51.88, 30.00, 27.78, 27.67, 26.89, 26.82.

[0176] MS (ES-): m / z calc, for CI8H2IC1O4: 336.11; found: 335.3 [M-H]’.Scheme 30: Synthesis of compound Id

[0177] Compound 2d. As depicted in Scheme 31, compound 2d was synthesized according to Procedure E: Compound 2c (69 mg, 0.204 mmol), DCM (2 mL), and methyl (triphenylphosphorranylidene) acetate (102 mg, 0.306 mmol). The crude residue was purified by column chromatography (85: 15, Hex: EtOAc) to afford compound 2d as an oil (62 mg, 78% yield).

[0178] 1H NMR (400 MHz, CDCl3) 6 7.95 (d, J = 16.2 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 6.59 (d, J = 16.2 Hz, 1H), 3.79 (s, J = 8.9 Hz, 3H), 3.64 (s, 3H), 3.26 (s, 3H), 3.01 (s, 1H), 2.42 (s, 1H), 2.07-1.72 (m, 5H), 1.66-1.54 (m, 2H).

[0179] 13C NMR (101 MHz, CDCl3) 6 176.13, 167.82, 150.88, 139.30, 127.53, 126.86, 123.82, 123.55, 122.43, 121.73, 119.87, 115.98, 57.01, 51.91, 51.75, 43.06, 29.79, 29.73, 29.61, 25.27.

[0180] MS (ES-): m / z calc, for C20H23CIO6: 394.12; found: 393.4 [M-H]’.Scheme 31: Synthesis of compound 2d

[0181] Compound 3d. As depicted in Scheme 32, compound 3d was synthesized according to Procedure E: Compound 3c (150 mg, 0.531 mmol), DCM (2 mL), and methyl (triphenylphosphoranylidene)acetate (266 mg, 0.796 mmol). The crude residue was purified by column chromatography (85: 15, Hex: EtOAc) to afford compound 3d as an oil (115 mg, 64% yield).

[0182] 1H NMR (400 MHz, CDCl3) 8 7.96 (d, J = 16.2 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 16.2 Hz, 1H), 3.79 (s, 3H), 3.72 (t, J = 5.0 Hz, 2H), 3.58 (t, J = 5.4 Hz, 2H), 3.28 (s, 3H), 2.53 (d, J = 15.0 Hz, 2H), 1.95 (t, J = 5.4 Hz, 2H).

[0183] 13C NMR (101 MHz, CDCl3) 8 167.81, 151.08, 144.24, 139.28, 135.67, 126.88, 123.48, 122.73, 121.66, 119.91, 119.00, 68.72, 56.95, 51.92, 30.30, 27.52.

[0184] MS (ES-): m / z calc, for C17H19CIO5: 338.09; found: 337.3 [M-H]’.Scheme 32: Synthesis of compound 3d

[0185] Compound 4d. As depicted in Scheme 33, Compound 4d was synthesized according to Procedure E: Compound 4c (152 mg, 0.538 mmol), DCM (3 mL), and methyl (triphenylphosphoranylidene)acetate (270 mg, 0.806 mmol). The crude residue was purified by column chromatography (85: 15, Hex: EtOAc) to afford compound 4d as an oil (133 mg, 73% yield). Compound 4d was obtained as a diastereomeric mixture of trans and cis (E:Z) in 3:2 ratio, respectively.

[0186] 1H NMR (400 MHz, CDCl3) (major) 8 7.95 (d, J = 16.2 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.62 (d, J = 16.2 Hz, 1H), 6.50 (s, 1H), 4.41 (s, J = 11.8 Hz, 1H), 3.82 (s, 3H), 3.74 (t, J = 5.1 Hz, 2H), 3.29 (s, 3H), 2.64-2.49 (m, 1H), 2.00 (t, J = 6.2 Hz, 2H), 1.78-1.69 (m, 1H), 1.64 (s, 1H).

[0187] 1H NMR (400 MHz, CDCl3) (minor) 8 7.94 (d, J = 16.2 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.61 (d, J = 16.2 Hz, 1H), 6.50 (s, 1H), 4.41 (s, J = 11.8 Hz, 1H), 3.81 (s, 3H), 3.74 (t, J = 5.1 Hz, 2H), 3.30 (s, 3H), 2.64-2.49 (m, 1H), 2.00 (t, J = 6.2 Hz, 2H), 1.78-1.69 (m, 1H), 1.64 (s, 1H).

[0188] 13C NMR (101 MHz, CDCl3) (diastereomeric mixture) 8 167.70, 150.77, 144.53, 139.06, 135.13, 127.05, 123.65, 123.39, 122.90, 122.70, 121.58, 120.28, 120.22, 118.66, 118.34, 68.73, 68.61, 68.40, 65.64, 56.86, 56.73, 51.93, 31.75, 29.81, 27.33, 26.49, 23.84.

[0189] MS (ES-): m / z calc, for C17H19CIO5: 338.09; found: 337.3 [M-H]’.Scheme 33: Synthesis of compound 4d

[0190] Compound 5d. As depicted in Scheme 34, compound 5d was synthesized according to Procedure E: Compound 5c (72 mg, 0.227 mmol), DCM (2.5 mL), and methyl (triphenylphosphoranylidene)acetate (114 mg, 0.341 mmol). The crude residue was purified by column chromatography (85: 15, Hex: EtOAc) to afford compound 5d as an oil (60 mg, 71% yield).

[0191] 1H NMR (400 MHz, CDCl3) 6 7.98 (d, J = 16.2 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.62 (d, J = 16.2 Hz, 1H), 3.83 (s, 3H), 3.29 (s, 3H), 2.65 (s, 1H), 2.59-2.51 (m, 1H), 2.05-1.92 (m, 4H), 1.90-1.77 (m, 2H).

[0192] 13C NMR (101 MHz, CDCl3) 6 167.78, 150.86, 144.69, 139.11, 135.53, 127.01,123.59 (t, J = 240.8 Hz), 123.38, 122.76, 121.57, 120.13, 118.74, 56.88, 51.95, 34.88-33.70 (m), 25.52 (t, J = 4.4 Hz), 22.31 (t, J = 4.8 Hz).

[0193] MS (ES-): m / z calc, for C18H19CIF2O4: 372.09; found: 371.4 [M-H]’.Scheme 34: Synthesis of compound 5d

[0194] Compound 6d. As depicted in Scheme 35, compound 6d was synthesized according to Procedure E: Compound 6c (87 mg, 0.278 mmol), DCM (1 mL), and methyl (triphenylphosphoranylidene)acetate (140 mg, 0.417 mmol). The crude residue was purified by column chromatography (75: 25, Hex: EtOAc) to afford compound 6d as an oil (84 mg, 82% yield).

[0195] 1H NMR (400 MHz, CDCE) 6 7.95 (d, 7 = 16.2 Hz, 1H), 7.38 (d, 7 = 8.0 Hz, 1H), 6.82 (d, 7 = 8.0 Hz, 1H), 6.61 (d, 7 = 16.2 Hz, 1H), 5.17 (s, 1H), 4.60 (s, 2H), 4.03 (s, 2H), 3.80 (s, 3H), 3.30 (s, 3H), 1.41 (s, 6H).

[0196] 13C NMR (101 MHz, CDCE) 6 167.64, 150.94, 144.04, 138.91, 133.62, 127.93, 127.16, 123.26, 120.48, 116.89, 116.04, 99.33, 59.74, 59.62, 58.61, 56.49, 51.95, 24.14.

[0197] MS (ES-): m / z calc, for Ci8H2iC106: 368.10; found: 367.4 [M-H]’.Scheme 35: Synthesis of compound 6d

[0198] Compound 7d. As depicted in Scheme 36, compound 7d was synthesized according to Procedure E: Compound 7c (65 mg, 0.196 mmol), DCM (1 mL), and methyl (triphenylphosphoranylidene)acetate (99 mg, 0.295 mmol). The crude residue was purified by column chromatography (65: 35, Hex: EtOAc) to afford compound 7d as an oil (57 mg, 75% yield).

[0199] 1H NMR (400 MHz, CDCE) 6 7.95 (d, J = 16.2 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 6.84 (d, 7 = 7.9 Hz, 1H), 6.61 (d, J = 16.2 Hz, 1H), 3.81 (s, 3H), 3.30 (s, 3H), 3.13-3.02 (m, 3H), 2.96-2.81 (m, 3H), 2.40 (t, 7 = 6.2 Hz, 2H).

[0200] 13C NMR (101 MHz, CDCE) 6 167.65, 150.99, 147.47, 138.75, 134.15, 127.32, 123.51, 123.09, 120.64, 113.14, 99.47, 56.71, 52.09, 52.02, 29.76, 27.10, 23.66.

[0201] MS (ES-): m / z calc, for Ci7Hi9C106S: 386.06; found: 385.3 [M-H]’.Scheme 36: Synthesis of compound 7d

[0202] Compound le. As depicted in Scheme 37, compound le was synthesized according to Procedure F: Compound Id (56 mg, 0.166 mmol), dry DMF (4 mF), K2CO3 (46 mg, 0.332 mmol), and benzyl iodide III (Green et al., 2017) (94 mg, 0.166 mmol). The crude residue was purified by column chromatography (50: 50, Hex: EtOAc) to afford compound le as an oil (114 mg, 88% yield).

[0203] 1H NMR (400 MHz, CDCl3) 6 7.88 (d, J = 16.2 Hz, 1H), 7.44-7.36 (m, 3H), 7.04 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 8.6 Hz, 2H), 6.41 (d, J = 16.2 Hz, 1H), 5.49-5.41 (m, 2H), 5.10 (dd, J = 10.4, 3.5 Hz, 1H), 5.04 (d, J = 7.9 Hz, 1H), 4.91 (s, 2H), 4.22-4.11 (m, 2H), 4.07 (q, J = 7.1 Hz, 2H), 3.76 (s, 3H), 3.25 (s, 3H), 2.39-2.34 (m, 2H), 2.15 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.97 (s, 3H), 1.77 (d, J = 4.9 Hz, 2H), 1.61-1.44 (m, 5H).

[0204] 13C NMR (101 MHz, CDCl3) 6 170.45, 170.35, 170.19, 169.50, 167.15, 157.33,153.70, 142.58, 138.89, 138.28, 131.08, 130.51, 129.95, 129.77, 127.97, 125.20, 124.87, 120.02, 117.08, 99.74, 75.70, 71.16, 70.93, 68.76, 67.03, 61.49, 60.46, 57.03, 51.87, 30.00,27.70, 27.62, 26.85, 26.81, 20.80, 20.74, 20.67.

[0205] MS (ES+): m / z calc, for C39H45CIO14: 772.25; found: 795.7 [M+Na]+.Scheme 37: Synthesis of compound le

[0206] Compound 2e. As depicted in Scheme 38, compound 2e was synthesized according to Procedure F: Compound 2d (62 mg, 0.157 mmol), dry DMF (4 mb), K2CO3 (43 mg, 0.314 mmol), and benzyl iodide III (Green et al., 2017) (89 mg, 0.157 mmol). The crude residue was purified by column chromatography (80: 20, Hex: EtOAc) to afford compound 2e as an oil (97 mg, 75% yield).

[0207] 1H NMR (400 MHz, CDCl3) 6 7.88 (d, J = 16.2 Hz, 1H), 7.45-7.37 (m, 3H), 7.09- 7.00 (m, 1H), 6.99 (dd, J = 8.6, 1.4 Hz, 2H), 6.43 (d, J = 16.2 Hz, 1H), 5.49-5.42 (m, 2H), 5.13-5.01 (m, 2H), 4.91 (s, 2H), 4.17 (qd, J = 11.2, 6.6 Hz, 2H), 4.11-4.02 (m, 1H), 3.77 (s, 3H), 3.63 (s, 3H), 3.25 (s, 3H), 3.00 (t, J = 13.3 Hz, 1H), 2.48-2.38 (m, 1H), 2.15 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.97-1.68 (m, 5H), 1.65-1.52 (m, 2H).

[0208] 13C NMR (101 MHz, CDCl3) 6 175.93, 170.37, 170.27, 170.11, 169.42, 167.03, 157.26, 153.65, 138.68, 137.68, 137.57, 130.92, 130.41, 129.95, 127.73, 125.26, 122.28, 121.68, 120.14, 117.00, 99.64, 75.67, 71.07, 70.83, 68.67, 66.94, 61.40, 56.90, 56.80, 51.82, 51.61, 42.96, 42.88, 29.54, 25.21, 25.10, 20.72, 20.67, 20.59.

[0209] MS (ES+): m / z calc, for C41H47CIO16: 830.26; found: 853.8 [M+Na]+.Scheme 38: Synthesis of compound 2e

[0210] Compound 3e. As depicted in Scheme 39, compound 3e was synthesized according to Procedure F: Compound 3d (46 mg, 0.136 mmol), dry DMF (1 mL), K2CO3 (38 mg, 0.272 mmol), and benzyl iodide III (Green et al., 2017) (77 mg, 0.136 mmol). The crude residue was purified by column chromatography (50: 50, Hex: EtOAc) to afford compound 3e as an oil (88 mg, 84% yield).

[0211] 1H NMR (400 MHz, MeOD) 8 7.84 (d, J = 16.2 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.0 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 6.52 (d, J = 16.2 Hz, 1H), 5.46 (dd, J = 3.4, 0.9 Hz, 1H), 5.39-5.34 (m, 1H), 5.30-5.23 (m, 2H), 4.99 (s, 2H), 4.30 (td, J = 6.5, 0.9 Hz, 1H), 4.18 (d, J = 6.5 Hz, 2H), 3.79 (s, 3H), 3.71 (t, J = 5.5 Hz, 2H), 3.59 (dd, J = 10.1, 4.6 Hz, 2H), 3.28 (s, 3H), 2.52 (dt, J = 11.3, 5.3 Hz, 2H), 2.18 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.90 (t, J = 5.4 Hz, 2H).

[0212] 13C NMR (101 MHz, MeOD) 8 172.01, 171.97, 171.43, 171.29, 168.62, 158.70, 154.85, 145.61, 139.79, 138.32, 132.00, 131.86, 131.69, 130.82, 129.01, 126.67, 121.11, 119.31, 117.68, 100.00, 76.99, 72.25, 72.18, 70.18, 69.55, 68.74, 62.65, 57.06, 52.37, 31.82, 31.28, 30.77, 28.44, 20.65, 20.50.

[0213] MS (ES+): m / z calc, for C38H43CIO15: 774.23; found: 797.7 [M+Na]+.Scheme 39: Synthesis of compound 3e

[0214] Compound 4e. As depicted in Scheme 40, compound 4e was synthesized according to Procedure F: Compound 4d (20 mg, 0.059 mmol), dry DMF (1 mL), K2CO3 (16 mg, 0.118 mmol), and benzyl iodide III (Green et al., 2017) (33 mg, 0.059 mmol). Thecrude residue was purified by column chromatography (50: 50, Hex: EtOAc) to afford compound 4e as an oil (37 mg, 84% yield). Compound 4e was obtained as a diastereomeric mixture of trans and cis (E:Z) in 3:2 ratio, respectively.

[0215] 1H NMR (400 MHz, MeOD) (major) 8 7.83 (d, J = 16.2 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.40-7.31 (m, 2H), 7.13 (d, J = 8.1 Hz, 1H), 7.04-6.97 (m, 2H), 6.52 (d, J = 16.2 Hz, 1H), 5.50-5.43 (m, 1H), 5.39-5.32 (m, 1H), 5.32-5.22 (m, 2H), 5.00 (s, 2H), 4.39 (s, 1H), 4.31 (t, J = 6.5 Hz, 1H), 4.18 (d, J = 6.6 Hz, 2H), 3.79 (s, 4H), 3.73 (t, J = 5.2 Hz, 2H), 3.28 (s, 3H), 2.18 (s, 3H), 2.07 (s, 4H), 2.04 (s, 3H), 1.98 (s, 4H), 1.76-1.57 (m, 2H).

[0216] 1H NMR (400 MHz, MeOD) (minor) 8 7.82 (d, J = 16.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.40-7.31 (m, 2H), 7.12 (d, J = 8.1 Hz, 1H), 7.04-6.97 (m, 2H), 6.51 (d, J = 16.2 Hz, 1H), 5.50-5.43 (m, 1H), 5.39-5.32 (m, 1H), 5.32-5.22 (m, 2H), 5.00 (s, 2H), 4.39 (s, 1H), 4.31 (t, J = 6.5 Hz, 1H), 4.18 (d, J = 6.6 Hz, 2H), 3.79 (s, 4H), 3.73 (t, J = 5.2 Hz, 2H), 3.29 (s, 3H), 2.18 (s, 3H), 2.07 (s, 4H), 2.04 (s, 3H), 1.98 (s, 4H), 1.76-1.57 (m, 2H).

[0217] 13C NMR (101 MHz, MeOD) (diastereomeric mixture) 8 172.04, 171.97, 171.44, 171.31, 168.63, 158.77, 154.87, 146.04, 139.75, 137.77, 137.50, 131.99, 131.88, 131.14, 128.96, 128.78, 126.73, 126.64, 121.20, 118.84, 118.64, 117.73, 100.04, 77.00, 72.26, 72.19, 70.19, 69.54, 69.28, 68.75, 66.34, 62.63, 57.00, 56.82, 52.37, 30.74, 28.33, 27.38, 24.51, 20.64, 20.49.

[0218] MS (ES+): m / z calc, for C38H43CIO15: 774.23; found: 797.7 [M+Na]+.Scheme 40: Synthesis of compound 4e

[0219] Compound 5e. As depicted in Scheme 41, compound 5e was synthesized according to Procedure F: Compound 5d (24 mg, 0.064 mmol), dry DMF (1 mL), K2CO3(18 mg, 0.129 mmol), and benzyl iodide III (Green et al., 2017) (36 mg, 0.064 mmol). The crude residue was purified by column chromatography (60: 40, Hex: EtOAc) to afford compound 5e as an oil (41 mg, 79% yield).

[0220] 1H NMR (400 MHz, CDCl3) 6 7.89 (d, J = 16.2 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.39 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.6 Hz, 2H), 6.44 (d, J = 16.2 Hz, 1H), 5.49-5.42 (m, 2H), 5.10 (dd, J = 10.5, 3.4 Hz, 1H), 5.04 (d, J = 7.9 Hz, 1H), 4.92 (s, 2H), 4.26-4.03 (m, 4H), 3.78 (s, 3H), 3.27 (s, 3H), 2.65 (dd, J = 13.0, 6.5 Hz, 1H), 2.50 (dd, J = 13.4, 6.2 Hz, 1H), 2.16 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.97-1.92 (m, 3H), 1.86-1.75 (m, 2H).

[0221] 13C NMR (101 MHz, CDCl3) 6 170.39, 170.29, 170.14, 169.44, 166.97, 157.29, 153.67, 144.53, 138.53, 136.97, 130.76, 130.48, 130.35, 129.82, 127.55, 125.49, 123.55 (t, J = 240.8 Hz), 120.46, 118.44, 117.00, 99.61, 75.79, 71.09, 70.85, 68.67, 66.93, 61.40, 56.73, 51.87, 34.65-33.67 (m), 29.69, 25.49, 22.22, 20.73, 20.67, 20.60.

[0222] MS (ES+): m / z calc, for C39H43CIF2O14: 808.23; found: 831.8 [M+Na]+.Scheme 41: Synthesis of compound 5e

[0223] Compound 6e. As depicted in Scheme 42, compound 6e was synthesized according to Procedure F: Compound 6d (45 mg, 0.122 mmol), dry DMF (1 mL), K2CO3 (34 mg, 0.244 mmol), and benzyl iodide III (Green et al., 2017) (69 mg, 0.122 mmol). The crude residue was purified by column chromatography (60: 40, Hex: EtOAc) to afford compound 6e as an oil (63 mg, 64% yield).

[0224] 1H NMR (400 MHz, MeOD) 8 7.81 (d, J = 16.2 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.37 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.0 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 16.2 Hz, 1H), 5.47-5.42 (m, 1H), 5.39-5.32 (m, 1H), 5.28-5.21 (m, 2H), 4.96 (d, J = 1.5 Hz, 2H), 4.59 (s, 2H), 4.30-4.25 (m, 1H), 4.19-4.14 (m, 2H), 4.00 (s, 2H), 3.78 (s, 3H), 3.29 (s, 3H), 2.17 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.38 (s, 6H).

[0225] 13C NMR (101 MHz, MeOD) 8 171.96, 171.94, 171.40, 171.23, 168.44, 158.71, 154.94, 145.65, 139.48, 136.30, 132.41, 131.98, 131.82, 130.52, 128.68, 126.93, 121.52,117.77, 115.93, 100.31, 100.08, 77.01, 72.19, 70.14, 68.71, 62.64, 60.60, 59.25, 56.79,52.41, 32.15, 30.72, 30.08, 24.37, 20.70, 20.53.

[0226] MS (ES+): m / z calc, for C39H45CIO16: 804.24; found: 827.8 [M+Na]+.Scheme 42: Synthesis of compound 6e

[0227] Compound 7e. As depicted in Scheme 43, compound 7e was synthesized according to Procedure F: Compound 7d (54 mg, 0.140 mmol), dry DMF (1 mL), K2CO3 (39 mg, 0.279 mmol), and benzyl iodide III (Green et al., 2017) (79 mg, 0.140 mmol). The crude residue was purified by column chromatography (40: 60, Hex: EtOAc) to afford compound 7e as an oil (81 mg, 70% yield).

[0228] H NMR (400 MHz, MeOD) 8 7.85 (dd, J = 16.2, 2.3 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.35 (dd, J = 8.6, 2.8 Hz, 2H), 7.16 (dd, J = 8.0, 2.3 Hz, 1H), 6.99 (dd, J = 8.6, 1.6 Hz, 2H), 6.55 (dd, J = 16.2, 2.4 Hz, 1H), 5.45 (d, J = 2.8 Hz, 1H), 5.36 (dd, J = 10.2, 7.9 Hz, 1H), 5.31-5.23 (m, 2H), 5.08-4.95 (m, 2H), 4.30 (t, J = 6.3 Hz, 1H), 4.21-4.15 (m, 2H), 3.80 (s, 3H), 3.30 (s, 3H), 3.15-3.06 (m, 2H), 2.99-2.92 (m, 4H), 2.30 (dt, J = 12.5, 5.6 Hz, 2H), 2.18 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H).

[0229] 13C NMR (101 MHz, MeOD) 8 172.02, 171.44, 171.31, 168.55, 158.70, 154.82, 148.64, 139.60, 137.16, 132.34, 132.20, 132.13, 131.65, 128.74, 127.06, 121.55, 117.64, 117.58, 114.37, 99.96, 77.14, 72.24, 70.19, 68.74, 62.70, 56.96, 52.61, 52.41, 28.16, 24.59, 20.66, 20.51.

[0230] MS (ES+): m / z calc, for C38H43CIO16S: 822.20; found: 845.7 [M+Na]+.Scheme 43: Synthesis of compound 7e

[0231] Compound If. As depicted in Scheme 44, compound If was synthesized according to Procedure G: Compound le (60 mg, 0.078 mmol), MeOH (1 mL), and K2CO3 (54 mg, 0.388 mmol). The crude residue was purified by preparative RP-HPLC (50-100% ACN in water, 0.1% TFA) to afford compound If as a white solid (22 mg, 47% yield).

[0232] 1H NMR (400 MHz, MeOD) 8 7.86 (d, J = 16.2 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 6.5 Hz, 2H), 7.08 (d, J = 5.8 Hz, 1H), 6.51 (d, J = 16.2 Hz, 1H), 4.96 (s, 2H), 4.88 (s, 1H), 3.92 (d, J = 3.1 Hz, 1H), 3.84-3.75 (m, 6H), 3.73-3.66 (m, 1H), 3.59 (dd, J = 9.7, 3.4 Hz, 1H), 3.27 (s, 3H), 2.44-2.34 (m, 2H), 1.86-1.77 (m, 2H), 1.67-1.38 (m, 6H).

[0233] 13C NMR (101 MHz, MeOD) 8 168.77, 159.57, 154.88, 143.99, 140.03, 139.30, 131.73, 131.24, 131.12, 130.88, 129.05, 126.39, 125.36, 120.78, 117.74, 102.89, 76.94, 74.82, 72.24, 70.18, 62.38, 57.16, 52.38, 30.96, 28.69, 28.58, 27.79, 27.71.

[0234] MS (ES+): m / z calc, for C31H37CIO10: 604.21; found: 643.6 [M+K]+.Scheme 44: Synthesis of compound If

[0235] Compound 2f. As depicted in Scheme 45, compound 2f was synthesized according to Procedure G: Compound 2e (56 mg, 0.067 mmol), MeOH (1 mL), and K2CO3 (93 mg, 0.674 mmol). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound 2f as a white solid (24 mg, 53% yield).

[0236] H NMR (400 MHz, CDCl3) 6 7.85-7.74 (m, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.31 (d, J = 5.8 Hz, 2H), 7.09-6.95 (m, 3H), 6.35 (dd, J = 15.9, 9.5 Hz, 1H), 4.94-4.77 (m, 3H), 4.57 (s, 4H), 4.16-3.96 (m, 2H), 3.87-3.73 (m, 3H), 3.69 (s, 3H), 3.64 (s, 4H), 3.24 (s, 3H), 2.99 (d, J = 9.8 Hz, 1H), 2.43 (d, J = 9.7 Hz, 1H), 2.04-1.99 (m, 1H), 1.96-1.69 (m, 4H), 1.63- 1.35 (m, 2H).

[0237] 13C NMR (101 MHz, CDCl3) 6 176.11, 167.45, 157.56, 153.80, 143.63, 138.92, 137.76, 130.69, 130.27, 130.03, 129.85, 127.80, 125.24, 121.75, 119.97, 116.91, 101.31, 76.06, 74.55, 73.48, 71.13, 69.20, 61.55, 56.99, 52.08, 51.72, 42.99, 33.72, 29.64, 29.45, 25.21.

[0238] MS (ES+): m / z calc, for C33H39CIO12: 662.21; found: 701.6 [M+K]+.Scheme 45: Synthesis of compound 2f

[0239] Compound 3f. As depicted in Scheme 46, compound 3f was synthesized according to Procedure G: Compound 3e (25 mg, 0.032 mmol), MeOH (1 mL), and K2CO3 (45 mg, 0.322 mmol). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound 3f as a white solid (13 mg, 66% yield).

[0240] 1H NMR (400 MHz, MeOD) 8 7.87 (d, J = 16.2 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 8.5 Hz, 2H), 7.14-7.07 (m, 3H), 6.53 (d, J = 16.2 Hz, 1H), 4.99 (s, 2H), 3.91 (d, J= 3.3 Hz, 1H), 3.83-3.75 (m, 6H), 3.74-3.68 (m, 3H), 3.64-3.56 (m, 3H), 3.29 (s, 3H), 2.60- 2.43 (m, 2H), 1.93 (t, J = 5.4 Hz, 2H).

[0241] 13C NMR (101 MHz, MeOD) 8 168.73, 159.65, 154.95, 145.67, 139.91, 138.31, 131.82, 131.72, 131.04, 130.84, 128.97, 126.65, 121.07, 119.23, 117.74, 102.92, 77.00, 74.86, 72.25, 70.22, 69.57, 62.42, 57.04, 52.39, 31.30, 28.44.

[0242] MS (ES+): m / z calc, for C30H35CIO11: 606.19; found: 629.6 [M+Na]+.Scheme 46: Synthesis of compound 3f

[0243] Compound 4f. As depicted in Scheme 47, compound 4f was synthesized according to Procedure G: Compound 4e (32 mg, 0.041 mmol), MeOH (1 mL), and K2CO3 (57 mg, 0.413 mmol). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound 4f as a white solid (15 mg, 60% yield).

[0244] H NMR (400 MHz, MeOD) (major) 8 7.86 (d, J = 16.2 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.13-7.06 (m, 3H), 6.53 (d, J = 16.2 Hz, 1H), 4.99 (d, J = 1.2 Hz, 2H), 4.39 (s, 1H), 3.91 (d, J = 3.0 Hz, 1H), 3.86-3.66 (m, 11H), 3.59 (dd, J = 9.7, 3.4 Hz, 1H), 3.28 (s, 3H), 2.59 (d, J = 59.7 Hz, 1H), 2.02-1.96 (m, 1H), 1.75-1.59 (m, 2H).

[0245] 1H NMR (400 MHz, MeOD) (minor) 8 7.85 (d, J = 16.2 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H), 7.13-7.06 (m, 3H), 6.52 (d, J = 16.2 Hz, 1H), 4.99 (d, J =1.2 Hz, 2H), 4.39 (s, 1H), 3.91 (d, J = 3.0 Hz, 1H), 3.86-3.66 (m, 11H), 3.59 (dd, J = 9.7, 3.4 Hz, 1H), 3.29 (s, 3H), 2.59 (d, J = 59.7 Hz, 1H), 2.02-1.96 (m, 1H), 1.75-1.59 (m, 2H).

[0246] 13C NMR (101 MHz, MeOD) (diastereomeric mixture) 8 168.69, 159.65, 154.93, 146.57, 146.05, 139.84, 137.73, 137.46, 132.06, 131.90, 131.82, 130.99, 130.83, 128.88, 128.69, 126.73, 126.64, 121.23, 121.19, 118.78, 118.58, 117.75, 102.90, 77.10, 76.97, 74.85, 72.24, 70.20, 69.54, 69.28, 66.34, 62.40, 57.00, 56.82, 52.40, 28.32, 28.26, 27.37, 24.50.

[0247] MS (ES+): m / z calc, for C30H35CIO11: 606.19; found: 629.6 [M+Na]+.Scheme 47: Synthesis of compound 4f

[0248] Compound 5f. As depicted in Scheme 48, compound 5f was synthesized according to Procedure G: Compound 5e (38 mg, 0.047 mmol), MeOH (1 mL), and K2CO3 (65 mg, 0.470 mmol). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound 5f as a white solid (18 mg, 60% yield).Scheme 48: Synthesis of compound 5f

[0249] 1H NMR (400 MHz, MeOD) 8 7.87 (d, J = 16.2 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 8.6 Hz, 2H), 7.16-7.05 (m, 3H), 6.53 (d, J = 16.2 Hz, 1H), 4.99 (s, 2H), 3.91 (d, J = 2.8 Hz, 1H), 3.83-3.74 (m, 6H), 3.74-3.67 (m, 1H), 3.59 (dd, J = 9.7, 3.4 Hz, 1H), 3.29 (s, 3H), 2.69-2.51 (m, 2H), 2.05-1.78 (m, 6H).

[0250] 13C NMR (101 MHz, MeOD) 8 168.70, 159.66, 154.95, 146.15, 139.86, 138.26, 131.80, 131.03, 130.83, 128.84, 126.72, 124.62, 121.16, 119.09, 117.76, 102.93, 77.10, 76.99, 74.86, 72.25, 70.21, 62.41, 57.00, 52.39, 35.64-34.75 (m), 26.55, 23.17.

[0251] MS (ES+): m / z calc, for C31H35CIF2O10: 640.19; found: 663.6 [M+Na]+.

[0252] Compound 6f. As depicted in Scheme 49, compound 6f was synthesized according to Procedure G: Compound 6e (35 mg, 0.044 mmol), MeOH (1 mL), and K2CO3 (60 mg, 0.435 mmol). The crude residue was purified by preparative RP-HPLC (21-70% ACN, ammonium carbonate buffer [30 mM]) to afford compound 6f as a white solid (18 mg, 60% yield).

[0253] 1H NMR (400 MHz, MeOD) 8 7.84 (d, J = 16.2 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.0 Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.53 (d, J = 16.2 Hz, 1H), 4.99 (s, 2H), 4.60 (s, 2H), 4.02 (s, 2H), 3.90 (d, 7= 3.1 Hz, 1H), 3.84-3.74 (m, 6H), 3.70 (dd, J = 13.0, 7.1 Hz, 1H), 3.58 (dd, 7 = 9.7, 3.4 Hz, 1H), 3.31 (s, 3H), 1.39 (s, 6H).

[0254] 13C NMR (101 MHz, MeOD) 8 168.63, 159.68, 155.07, 145.79, 139.68, 136.31, 132.51, 131.83, 130.96, 130.62, 128.62, 126.92, 121.48, 117.77, 115.77, 102.91, 100.36, 77.15, 76.99, 74.86, 72.25, 70.20, 62.41, 60.67, 59.29, 56.74, 52.41, 24.31.

[0255] MS (ES+): m / z calc, for C31H37CIO12: 636.20; found: 675.6 [M+K]+.Scheme 49: Synthesis of compound 6f

[0256] Compound 7f. As depicted in Scheme 50, compound 7f was synthesized according to Procedure G: Compound 7e (56 mg, 0.068 mmol), MeOH (1 mL), and K2CO3 (94 mg, 0.680 mmol). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound 7f as a white solid (38 mg, 84% yield).

[0257] 1H NMR (400 MHz, MeOD) 8 7.87 (d, J = 16.2 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.34 (d, J = 8.7 Hz, 2H), 7.16 (d, 7 = 7.9 Hz, 1H), 7.09 (d, J = 8.7 Hz, 2H), 6.55 (d, J = 16.2 Hz, 1H), 5.05-4.97 (m, 2H), 3.90 (d, 7 = 0.5 Hz, 1H), 3.84-3.74 (m, 6H), 3.71 (dd, 7 = 13.0, 6.8 Hz, 1H), 3.59 (dd, 7 = 9.7, 3.4 Hz, 1H), 3.31 (s, 3H), 3.12 (dd, 7 = 11.3, 5.9 Hz, 2H), 3.02-2.94 (m, 4H), 2.33 (t, 7 = 6.1 Hz, 2H).

[0258] 13C NMR (101 MHz, MeOD) 8 168.62, 159.68, 154.95, 148.72, 139.70, 137.14, 132.36, 131.94, 130.86, 130.76, 128.69, 127.03, 121.50, 117.71, 114.22, 102.91, 77.21, 77.01, 74.84, 72.24, 70.21, 62.43, 56.95, 52.63, 52.43, 28.18, 24.59.

[0259] MS (ES+): m / z calc, for C30H35CIO12S: 654.15; found: 693.5 [M+K]+.Scheme 50: Synthesis of compound 7f

[0260] MA-Diox-2. As depicted in Scheme 51, MA-Diox-2 was synthesized according to Procedure H: Compound If (22 mg, 0.036 mmol), a catalytic amount of methylene blue, and DCM (10 ml). The crude residue was purified by preparative RP-HPLC (50-100% ACN in water, 0.1% TFA) to afford compound MA Diox-2 as a white solid (15 mg, 65% yield).

[0261] 1H NMR (400 MHz, MeOD) 8 7.84 (d, J = 16.3 Hz, 1H), 7.71 (dd, J = 8.3, 0.4 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.36-7.30 (m, 2H), 7.10 (d, J = 8.5 Hz, 2H), 6.55 (dd, J = 16.2, 1.2 Hz, 1H), 4.96 (s, 2H), 4.87 (d, J = 0.9 Hz, 1H), 4.85 (d, J = 0.9 Hz, 1H), 3.91 (d, J = 3.2 Hz, 1H), 3.83-3.75 (m, 6H), 3.74-3.67 (m, 1H), 3.59 (dd, J = 9.7, 3.4 Hz, 1H), 3.04 (s, 3H), 2.75 (d, J = 13.5 Hz, 1H), 1.87-1.74 (m, 2H), 1.72-1.49 (m, 3H), 1.42-1.15 (m, 5H).

[0262] 13C NMR (101 MHz, MeOD) 8 168.57, 159.68, 155.35, 139.55, 137.87, 132.67, 131.85, 130.87, 127.50, 126.81, 126.68, 121.77, 117.77, 112.13, 102.90, 93.11, 77.24, 77.20, 76.98, 74.84, 72.24, 70.19, 62.40, 52.43, 50.50, 33.47, 31.37, 25.90, 21.98, 21.51.

[0263] MS (ES+): m / z calc, for C31H37CIO12: 636.20; found: 659.6 [M+Na]+.Scheme 51: Synthesis of MA-Diox-2

[0264] MA-Diox-3. As depicted in Scheme 52, MA-Diox-3 was synthesized according to Procedure H: Compound 2f (9 mg, 0.014 mmol), a catalytic amount of methylene blue, and DCM (10 ml). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound MA Diox-3 as a white solid (5 mg, 53% yield).

[0265] 1H NMR (400 MHz, CDC13) (diastereomeric mixture) 8 7.81-7.73 (m, 2H), 7.55- 7.44 (m, 4H), 7.29 (t, J = 7.9 Hz, 4H), 7.01 (t, J = 6.7 Hz, 4H), 6.44-6.34 (m, 2H), 4.95-4.83 (m, 6H), 4.12 (s, 2H), 4.01 (d, J = 6.0 Hz, 2H), 3.88 (s, 4H), 3.77 (s, 3H), 3.76 (s, 3H), 3.63 (s, 6H), 3.07 (s, 6H), 2.90 (d, J = 14.4 Hz, 2H), 2.64 (d, J = 13.8 Hz, 2H), 2.56 (s, 2H), 2.23 (s, 2H), 2.13-1.28 (m, 22H).

[0266] 13C NMR (101 MHz, CDCh) (diastereomeric mixture) 8 175.48, 175.01, 167.16, 157.43, 153.97, 153.85, 138.44, 136.20, 131.43, 130.85, 130.77, 129.94, 126.40, 126.26, 125.71, 125.62, 125.46, 120.86, 120.73, 116.72, 110.89, 110.64, 100.92, 91.79, 90.91, 76.12, 74.44, 73.39, 71.18, 69.33, 62.02, 52.05, 51.77, 50.28, 41.76, 37.85, 31.31, 29.72, 29.39, 29.00, 26.94, 23.26, 22.93, 21.96, 21.61.

[0267] MS (ES+): m / z calc, for C33H39CIO14: 694.20; found: 717.7 [M+Na]+.Scheme 52: Synthesis of MA-Diox-3

[0268] MA-Diox-4. As depicted in Scheme 53, MA-Diox-4 was synthesized according to Procedure H: Compound 3f (20 mg, 0.033 mmol), a catalytic amount of methylene blue, and DCM (10 ml). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound MA Diox-4 as a white solid (11 mg, 52% yield).Scheme 53: Synthesis of MA-Diox-4

[0269] 1H NMR (400 MHz, MeOD) 8 7.85 (dd, J = 16.2, 2.4 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.49 (dd, J = 8.2, 0.9 Hz, 1H), 7.35-7.29 (m, 2H), 7.11-7.05 (m, 2H), 6.57 (dd, J = 16.2, 2.4 Hz, 1H), 5.01-4.96 (m, 2H), 3.92-3.84 (m, 2H), 3.83-3.67 (m, 9H), 3.58 (ddd, J = 9.7, 3.4, 0.9 Hz, 1H), 3.48-3.32 (m, 2H), 3.07 (s, 3H), 2.58 (t, J = 12.6 Hz, 1H), 2.25-2.14 (m, 1H), 1.80-1.61 (m, 2H).

[0270] 13C NMR (101 MHz, MeOD) 8 168.53, 159.75, 155.36, 139.50, 137.01, 132.99, 131.99, 130.75, 127.02, 126.63, 121.98, 117.77, 117.71, 111.83, 102.90, 90.43, 77.33, 76.99, 74.84, 72.24, 70.23, 63.58, 63.41, 62.45, 52.45, 50.65, 34.09, 32.59, 31.29.

[0271] MS (ES+): m / z calc, for C30H35CIO13 : 638.18; found: 661.6 [M+Na]+.

[0272] MA-Diox-5. As depicted in Scheme 54, MA-Diox-5 was synthesized according to Procedure H: Compound 4f (8 mg, 0.013 mmol), a catalytic amount of methylene blue, and DCM (10 ml). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound MA Diox-5 as a white solid (2 mg, 24% yield).

[0273] 1H NMR (400 MHz, MeOD) (diastereomeric mixture) 8 7.89-7.81 (m, 2H), 7.78- 7.72 (m, 2H), 7.54-7.49 (m, 2H), 7.37-7.30 (m, 4H), 7.12-7.07 (m, 4H), 6.62-6.54 (m, 2H), 5.01-4.97 (m, 4H), 4.68 (d, J = 12.5 Hz, 2H), 3.94-3.87 (m, 4H), 3.83-3.67 (m, 18H), 3.59 (d, J = 3.4 Hz, 1H), 3.57 (d, J = 3.4 Hz, 1H), 3.08-3.00 (m, 6H), 2.72 (d, J = 13.5 Hz, 1H), 1.90 (d, J = 11.5 Hz, 1H), 1.81-1.44 (m, 6H), 1.34-1.27 (m, 2H).

[0274] 13C NMR (101 MHz, MeOD) (Diastereomeric mixture) 8 168.53, 159.74, 155.39, 139.46, 136.52, 133.15, 131.97, 130.76, 127.06, 126.72, 126.53, 122.05, 117.75, 111.66, 102.91, 89.07, 88.67, 77.33, 77.00, 74.86, 72.24, 71.72, 71.11, 70.21, 68.13, 62.42, 52.45, 50.60, 50.22, 30.49, 28.71, 22.15.

[0275] MS (ES+): m / z calc, for C30H35CIO13 : 638.18; found: 661.6 [M+Na]+.Scheme 54: Synthesis of MA-Diox-5

[0276] MA-Diox-6. As depicted in Scheme 55, MA-Diox-6 was synthesized according to Procedure H: Compound 5f (9 mg, 0.014 mmol), a catalytic amount of methylene blue, and DCM (10 ml). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound MA Diox-6 as a white solid (5.5 mg, 58% yield).

[0277] H NMR (400 MHz, MeOD) 8 7.85 (d, J = 16.1 Hz, 1H), 7.75 (dd, J = 8.3, 1.5 Hz, 1H), 7.49 (dd, J = 8.2, 2.7 Hz, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.10 (dd, J = 8.6, 1.6 Hz, 2H), 6.57 (d, J = 16.2 Hz, 1H), 4.98 (d, J = 2.6 Hz, 2H), 3.91 (d, J = 2.9 Hz, 1H), 3.83-3.75 (m, 6H), 3.74-3.67 (m, 1H), 3.58 (dd, J = 9.7, 3.4 Hz, 1H), 3.07 (s, 3H), 2.89 (d, J = 12.9 Hz, 1H), 2.16 (td, J = 14.3, 4.8 Hz, 1H), 2.11-1.90 (m, 4H), 1.90-1.66 (m, 3H).

[0278] 13C NMR (101 MHz, MeOD) 8 168.52, 159.72, 139.45, 137.13, 133.09, 131.90, 131.88, 130.81, 130.76, 127.07, 126.46, 122.02, 117.78, 102.91, 91.12, 77.31, 77.26, 77.00, 74.86, 72.24, 70.21, 62.41, 52.45, 50.74, 29.95 (d, J = 9.5 Hz), 29.47 (t, 7 = 20.1 Hz), 29.01 (t, J = 19.9 Hz), 27.93 (d, J = 9.5 Hz).

[0279] MS (ES+): m / z calc, for C31H35CIF2O12: 672.18; found: 695.7 [M+Na]+.Scheme 55: Synthesis of MA-Diox-6

[0280] MA-Diox-7. As depicted in Scheme 56, MA-Diox-7 was synthesized according to Procedure H: Compound 6f (8 mg, 0.013 mmol), a catalytic amount of methylene blue, and 10:1 DCM: MeOH (10 ml). The crude residue was purified by preparative RP-HPLC (21- 70% ACN, ammonium carbonate buffer [30 mM]) to afford compound MA Diox-7 as a white solid (1.5 mg, 18% yield).Scheme 56: Synthesis of MA-Diox-7

[0281] 1H NMR (400 MHz, MeOD) 8 7.85 (d, J= 16.2 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.33 (dd, J = 8.6, 2.2 Hz, 2H), 7.09 (d, J = 8.6 Hz, 2H), 6.58 (d, J = 16.2 Hz, 1H), 5.02-4.99 (m, 2H), 4.67 (dd, J = 13.6, 1.2 Hz, 1H), 4.29 (d, J = 13.5 Hz, 1H), 3.91 (d, J = 3.3 Hz, 1H), 3.86-3.67 (m, 10H), 3.58 (dd, J = 9.7, 3.4 Hz, 1H), 3.06 (s, 3H), 1.39 (s, 3H), 1.29 (s, 3H).

[0282] 13C NMR (101 MHz, MeOD) 8 168.48, 159.75, 155.43, 139.35, 135.41, 133.56, 131.95, 130.72, 127.28, 126.35, 122.26, 117.77, 110.24, 102.89, 100.13, 87.57, 77.35, 77.01, 74.86, 72.24, 70.20, 64.60, 63.21, 62.42, 52.46, 50.40, 26.65, 20.67.

[0283] MS (ES+): m / z calc, for C31H37CIO14: 668.19; found: 691.6 [M+Na]+.

[0284] MA-Diox-8. As depicted in Scheme 57, MA-Diox-8 was synthesized according to Procedure H: Compound 7f (9 mg, 0.014 mmol), a catalytic amount of rose bengal, and 10:1 DCM: MeOH (10 ml). The crude residue was purified by preparative RP-HPLC (30-100% ACN in water, 0.1% TFA) to afford compound MA Diox-8 as a white solid (6 mg, 64% yield).

[0285] 1H NMR (400 MHz, MeOD) (diastereomeric mixture) 6 7.88 (d, J = 4.2 Hz, 1H), 7.84 (d, J = 4.2 Hz, 1H), 7.76 (d, 7 = 2.8 Hz, 1H), 7.74 (d, J = 2.8 Hz, 1H), 7.45 (d, J = 7.1 Hz, 1H), 7.43 (d, J = 7.3 Hz, 1H), 7.33 (d, J = 6.1 Hz, 2H), 7.31 (d, J = 6.1 Hz, 2H), 7.09 (d, J = 6.4 Hz, 2H), 7.07 (d, J = 6.4 Hz, 2H), 6.62 (d, J = 4.5 Hz, 1H), 6.58 (d, J = 4.5 Hz, 1H), 5.07-4.99 (m, 4H), 3.91 (s, 1H), 3.90 (s, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.80-3.74 (m, 7H), 3.74-3.67 (m, 3H), 3.61-3.56 (m, 2H), 3.29-3.11 (m, 6H), 3.08 (m, 8H), 3.05-2.93 (m, 2H), 2.67-2.56 (m, 2H), 2.36-2.05 (m, 4H).

[0286] 13C NMR (101 MHz, MeOD) (diastereomeric mixture) 8 168.48, 159.77, 155.30, 139.43, 136.33, 133.41, 132.16, 132.03, 130.66, 130.49, 127.25, 126.24, 122.25, 117.78, 117.69, 111.26, 102.95, 102.81, 89.18, 77.35, 77.04, 74.85, 72.25, 70.21, 62.44, 52.48, 51.00, 46.55, 46.23, 31.68, 29.81.

[0287] MS (ES+): m / z calc, for C30H35CIO14S: 686.14; found: 709.6 [M+Na]+.Scheme 57: Synthesis of MA-Diox-8Experimental protocols

[0288] Determination of ene-product and 1,2-dioxetane ratio obtained during oxidation of compound 6a. Compound 6a (95 mg, 0.248 mmol) was dissolved in 10 mL of DCM, and a catalytic amount of methylene blue was added to the solution. Oxygen was bubbled through the solution while irradiating with yellow light. The product ratio was determinedby RP-HPLC (gradient of 70-100% ACN in water, 0.1% TFA). TBS-SO2 ene-product is fully characterized in the synthetic procedures.

[0289] Stability assays of Diox 1 - Diox 9. 500 pM solutions of Diox 1-Diox 9 in PBS, pH 7.4, with 15% ACN were prepared in small vials. The vials were vortexed for about 1 minute. Then, the vials were put into an incubator that was pre-heated to 37°C. RP HPLC analysis (gradient of 70-100% or 90-100% ACN in water, 0.1% TFA) was conducted for each solution at different time intervals: t= 0, 0.5, 4, 8, 22, 29 hours (for Diox 1 the time intervals that were measured were t= 0, 24, 48 hours). The percentage of decomposition at each time point was determined by calculating the ratio of the area under the peak of the dioxetane and the peak of the formed benzoate (decomposition product) at 270 nm.

[0290] Chemiluminescent kinetic measurements of Diox 1 - Diox 9. Chemiluminescent kinetic profiles were recorded using Spectramax iD3 with an injector cartridge. The injector settings were fixed on the following parameters: Integration time: 50 msec, injection volume: 10 mL, measuring interval time: 50 msec. The injector was pre-washed with water, EtOH (70%), water, air, and ACN. Then, the injector was primed with a solution of 100 nM of Diox 1 - Diox 9 in ACN before the measurement of each dioxetane. Measurements were conducted in a white 96-well Corning™ plate. Each well contained 89 mL DMSO or acetone and 1 mL of TBAF (IM in THF), with a final volume of lOOmL after the addition via injection of lOmL of Diox 1 - Diox 9 [100 nM], TBAF was added to each well at the beginning of a measurement.

[0291] Stability assays of MA-Diox-1 - MA-Diox-9. 100 pM solutions of MA-Diox-1- MA-Diox-9 in LB, with 10% ACN were prepared in small vials. The vials were vortexed for about 1 minute. The vials were kept at room temperature in the dark. RP HPLC analysis (gradient of 30-100% ACN in water, 0.1% TFA) was conducted for each solution at different time intervals. The percentage of decomposition at each time point was determined by calculating the ratio of the area under the peak of the dioxetane and the peak of the decomposition products formed at 280 nm.

[0292] Visual demonstration of Diox 1, Diox 8, Diox 9. Light emission was recorded using iPhone XS. To a vial containing 1 mL of DMSO was added 20mL of TBAF (IM in THF) and stirred for a few seconds, then 50mL of Diox 1 / Diox 8 / Diox 9 was added, and the light emission was recorded for 2 minutes in total.Bacterial experiments

[0293] Chemiluminescence measurements of MA-Diox-1 - MA-Diox-9 in PBS / LB. E. coli ATCC 25922 was cultured in LB at 37°C for 18 hours under aerobic conditions. Subsequently, the initial culture was subjected to a PBS or LB wash (centrifuged at 5000 rpm, 10 minutes), and the bacterial pellet obtained was reconstituted in 4 mL of PBS or LB, aiming for an OD600of 0.8. Following this, a 96-well plate was utilized, and each well was pre-loaded with 50 pL of the chemiluminescent probes MA-Diox-1 - MA-Diox-9 [20 pM, 0.2% ACN]. Next, 50 pL of bacterial aliquot was introduced into each well, bringing the final OD600to 0.4. The resultant chemiluminescence signal was monitored using a Molecular Devices Spectramax iD3 at 37°C.

[0294] Limit of detection measurements of MA-Diox-1 and MA-Diox-8 in LB media. E. coli (ATCC 25922) was cultured in LB at 37°C for 18 hours under aerobic conditions. The initial culture was diluted with LB to OD600of 0.8, to facilitate a 1:4 dilution experiment. For the subsequent procedure, a 96-well plate was utilized, with each well initially loaded with 50pL of the MA-Diox-1 or MA-Diox-8 [20pM, 0.2% ACN]. Subsequently, 50pL of bacterial aliquot was introduced into each well, marking the commencement of the 1:4 dilution experiment (which was initiated with an OD600of 0.8). The ensuing chemiluminescence signal was monitored over 10 minutes of incubation at 37°C using a Molecular Devices Spectramax i3x.Computational methods

[0295] All the calculations were carried out using Gaussian 16 program (Gaussian 16, Revision C.01). The geometries were optimized using coB97X-D functional with 6-31G(d) basis set with SMD solvent model (Marenich et al., 2009) to describe the DMSO environment. Intrinsic Reaction Coordination (IRC) was used to confirm all transitions located are connected to the correct minimum. Single point energies were calculated using CAM-B3LYP-D3(BJ) functional, 6-311++G(2d,2p) basis set and SMD solvent model (Marenich et al., 2009). The stability of the wavefunction of diradical species was checked to ensure we converged to the lowest energy wavefunction. Quasiharmonic and concentration corrections to enthalpy and entropy were made using Paton’s GoodVibes software. The rate constants reported are calculated via TST using Eyring’s equation (where the free energies of TS1 are used as the overall free energy barriers since it is the rate-determining step.Results and discussion

[0296] In order to evaluate the chemiexcitation effect of a spiro-fused inductive electronwithdrawing six-member rings, we synthesized several new phenoxy 1,2-dioxetanes, equipped with various spiro-fused six-member rings. Tert-butyldimethylsilyl (TBS) triggering group was used to mask the phenols. The chemiexcitation of the dioxetanes was triggered by the removal of the TBS groups through addition of fluoride (tetra-n- butylammonium fluoride). The molecular structures of nine different spiro-cycloalkyl- dioxetanes, their stability (PBS 7.4, 37°C), total light emission half-life value (ti / 2 in DMSO or acetone), and relative chemiexcitation rate, are presented in Table 8. Adamantyl and cyclobutyl dioxetanes, referred to herein as Diox 1 and Diox 9, respectively, served as reference compounds. In general, the chemiexcitation of dioxetanes is much faster in polar organic solvents like DMSO. Therefore, the ti / 2 values of total light emission for dioxetanes with a relatively slow chemiexcitation rate were determined in DMSO, and for dioxetanes with a fast chemiexcitation rate measurements were conducted in acetone.

[0297] In agreement with our expectations, all of the phenoxy 1,2-dioxetanes with spiro- fused inductive electron-withdrawing six-member rings, exhibited a significantly enhanced chemiexcitation rate in comparison to that of the spiro-adamantyl and cyclohexyl dioxetanes (Diox 1 and Diox 2). Remarkably, spiro-dioxetanes fused to 1,3-dioxane and sulfone electron-withdrawing motifs (Diox 7 and Diox 8) presented a chemiexcitation rate, which is 103 -fold and 293 -fold faster than that of the adamantyl counterpart, respectively. Intriguingly, the chemiexcitation rates of these two spiro-dioxetanes were even faster than that of the cyclobutyl-dioxetane, Diox 9 (2-fold and 6-fold respectively).

[0298] The chemical stabilities of the spiro -six-member ring dioxetanes were lower than that of the parent adamantyl derivative. Moreover, the stability of dioxetanes bearing EWG or a hetero atom (Diox 3 -Diox 8) was lower compared to the non-substituted cyclohexyl Diox-2. This phenomenon is caused by the steric hindrance decrease in the vicinity of the dioxetane unit and the electron-withdrawing effect of the hetero atoms or polar groups presented in the six-member ring. However, all dioxetanes with spiro-fused inductive EWG rings exhibited better chemical stabilities than the cyclobutyl derivative (Diox 9). Thesulfone derivative Diox 8 exhibited both relatively high stability (ti / 2=42.8h) and a fast chemiexcitation rate (293 -fold greater than that of Diox 1).

[0299] The chemiexcitation acceleration effect obtained by the cyclic -sulfone dioxetane Diox 8 can be clearly visualized in comparison to the chemiexcitation of the adamantyl and the cyclobutyl-dioxetanes. Fig. 2 shows images of vials taken at selected time intervals for a period of 30 sec, for the chemiexcitation of the three dioxetanes in DMSO. The adamantyl- dioxetane emitted light with a relatively slow chemiexcitation rate that lasted much beyond 30 sec. The cyclobutyl-dioxetane emitted light with a significantly higher chemiexcitation rate that lasted for almost 2 sec. The 6-member cyclic-sulfone dioxetane exhibited an ultrafast chemiexcitation rate that lasted for less than a second. The relative chemiexcitation rates of the three dioxetanes were calculated by measuring their total light emission ti / 2 values according to the plots presented in Fig. 2 (right). Data for processing the plots were taken from measurements obtained in Table 8.Table 8: Molecular structures and chemiluminescent properties of different spiro-cycloalkyl phenoxy- 1 ,2-dioxetanes*The stability of Diox 1-Diox 9 [500 mM] was measured in PBS, pH 7.4, 15% ACN at 37°C; product distribution was determined using RP-HPLC (70-100% or 90-100% ACN in water with 0.1% TFA). Chemiexcitation properties of Diox 1-Diox 9 [10 nM] were measured in DMSO or acetone, with TBAF [10 mM], with 10% ACN. Half-life value (ti / 2) is defined as the time point by which half of the total light emission was observed. Relative chemiexcitation rate is defined as the ratio between the ti / 2 values of Diox 1-Diox 9. The ti / 2 of Diox 1 in DMSO was used as a reference. All measurements were conducted using SpectraMax iD3, with injector settings fixed on an integration time of 50 msec.

[0300] To better understand the chemiexcitation acceleration effect obtained by the spiro - fused inductive electron-withdrawing motifs, we conducted quantum mechanical studies with reliable DFT methods (CAM-B3LYP-D3(BJ) / 6-311++G(2d,2p),SMD / (DB97XD / 6- 31G(d),SMD) (Yeh et al., 2023). It was previously shown that the CIEEL mechanism of dioxetane ring opening involves charge transfer from phenoxide to the dioxetane fragment during the 0-0 cleavage transition state, followed by C-C cleavage that leads in part to the luminescent excited state of the aryl ester (Fig. 3A) (Tannous et al., 2024). The CIEEL process has been investigated extensively, and the back electron transfer (BET) step, inwhich the excited benzoate is formed, is yet not fully understood. The rate-determining step of the phenoxy- 1,2-dioxetane chemiexcitation involves the 0-0 cleavage of the dioxetane, which is accompanied by a single electron transfer (SET) from the phenolate to the dioxetane to generate the diradical intermediate Inti, which can then undergo a C-C cleavage transition state TS2_CC to form the excited product via passing through a conical intersection (CI) point that nears TS2_CC. The computational results of the rate-determining step (0-0 cleavage transition state) for five phenoxy- 1,2-dioxetanes are presented in Fig. 3B.

[0301] The adamantyl phenoxy- 1,2-dioxetane (Phenolate-Diox 1) exhibits a comparatively slow chemiexcitation rate, predicted by the relatively high barrier of 18.2 kcal / mol, for a transition state with 0-0 cleavage and electron transfer. Subsequently, a slightly lower barrier of 16.0 kcal / mol is predicted for the cyclohexyl phenoxy- 1,2- dioxetanes (Phenolate-Diox 2), which corresponds with a 4-fold increase in relative chemiexcitation rate observed experimentally. The introduction of electron -withdrawing substituents to the cyclohexyl ring further lowers the barrier to 14.7 kcal / mol for a single oxygen atom, 13.3 kcal / mol for two oxygens, and 11.0 kcal / mol for sulfone. This effect can be attributed to the electronegativity of the oxygen (in Phenolate-Diox 3 and 4) and the sulfone EWG (in Phenolate-Diox 5), which stabilizes the anion intermediate and facilitates the electron transfer process.

[0302] The ultrafast chemiexcitation acceleration observed for phenoxy- 1,2-dioxetanes with spiro-fused six-member rings (especially for Diox 8) suggests that turn-ON probes composed of such luminophores are expected to exhibit higher detection sensitivity. To evaluate this postulate, we synthesized several new ort / zo-acrylate substituted phenoxy- 1,2- dioxetanes chemiluminescent probes for the detection of alactosidaβ-sge (P-gal) activity (Fig. 4A). The probes were equipped with alactoβ-sge as a triggering substrate. The known adamantyl and cyclobutyl-phenoxy- 1,2-dioxetanes were used as control probes. The relative chemiluminescence quantum yields of the probes were determined by measuring the total light emission produced upon activation with a high concentration of P-gal [2U / mL] in PBS 7.4 (Fig. 4B). Interestingly, the quantum yields of all dioxetanes with fused six-member rings were up to almost 4-fold higher than that of the parent adamantyl dioxetane. The quantum yield of the cyclobutyl-dioxetane MA-Diox-9 was also 3.3-fold higher compared to the adamantyl dioxetane.

[0303] The full light emission profiles of probes MA-Diox-1, MA-Diox-2, MA-Diox-8 and MA-Diox-9 are presented in Fig. 4C. Predictably, probes MA-Diox-8 and MA-Diox-9generate a fast light emission response with high intensity that completely decayed after 25 min. On the other hand, the light emission profile of probes MA-Diox-8 and MA-Diox-9 was significantly less intense and lasted over more than 100 min. The light emission signals of the four selected probes were then measured under saturation kinetics conditions (low enzyme concentration). Under such conditions, the generated signal is gradually increased to a plateau level. The S / N values measured for the six-member ring sulfone and the cyclobutyl probes, MA-Diox-8 and MA-Diox-9, after 10 min (230 and 262 respectively), were substantially higher than the S / N values measured for the adamantyl and the cyclohexyl probes MA-Diox-1 and MA-Diox-2 (Fig. 4E). The obtained S / N values of MA-Diox-8 and MA-Diox-9 highlight their kinetic advantage by exhibiting higher detection sensitivity within a short measurement time.

[0304] The superior chemical stability of sulfone Diox 8, in comparison to cyclobutyl Diox 9, indicates that probe MA-Diox-8 is a more suitable candidate for assessment of P-gal activity in bacterial cell assays conducted in cell-growth media (Fig. 5A). We next sought to evaluate the ability of the four selected probes, MA-Diox-1, MA-Diox-2, MA-Diox-8, and MA-Diox-9 to detect P-gal activity with P-gal-expressing bacterial strain E. coli. The stability of four probes was first measured in LB microbial growth medium. Probes MA- Diox-1 and MA-Diox-2 exhibited high stability, with half-lives with ti / 2of >150h and 23.5h respectively. Probe MA-Diox-8 demonstrated moderate stability, with ti / 2of 3.6 hours, while the highly strained cyclobutyl probe MA-Diox-9 exhibited extremely low chemical stability in the growth medium, undergoing rapid decomposition (Fig. 5B). The lower stability of probe MA-Diox-9 in LB medium, compared to that of MA-Diox-8, is attributed to the high reactivity of the strained cyclobutyl-dioxetane toward the reducing agent’ s presence in the bacterial medium. Although the stability of MA-Diox-8 in LB medium is lower compared to that of MA-Diox-1 and MA-Diox-2, it is more than sufficient for applications that require short measurement times (up to Ih).

[0305] The four probes were then incubated in the presence of Escherichia coli ATCC 25922 in LB growth medium, and the light emission signal was monitored over 10 min. Noticeably, the S / N plot over time produced by probe MA-Diox-8 was significantly more intense than those produced by the other three probes. The S / N value obtained after 10 min with the sulfone probe MA-Diox-8 was 90-fold higher than that achieved with the adamantyl probe MA-Diox-1. As expected, the signal of the cyclobutyl probe, MA-Diox-9, owing to its high instability, resulted in a low S / N value (Fig. 5B). Next, we sought to harness theenhanced detection sensitivity of probe MA-Diox-8 towards P-gal and its elevated chemical and thermal stability, to determine the LOD for E. coli bacterial cells. Remarkably, the LOD value (3.9xl04cells) obtained by the sulfone probe MA-Diox-8 was significantly lower, indicating a 44-fold increase in sensitivity compared to that obtained by the previously known adamantyl analogue, probe MA-Diox-1 (Fig. 5C). Notably, due to the moderate but sufficient chemical stability of probe MA-Diox-8, the bacterial assay could be performed directly in LB growth medium with no need for prewashing with buffer. These data clearly highlight the superior ability of probe MA-Diox-8 to detect P-gal activity in bacteria, in comparison to the other tested probes in the selected panel.

[0306] As explained above, the brightness of a chemiluminescence luminophore depends on the number of photons emitted within a specific time interval. Accordingly, the luminophore's brightness increases when the rate of chemiexcitation is faster. Cyclohexyl dioxetane MA-Diox-2 has a two-fold higher chemiluminescence quantum yield compared to that of sulfone MA-Diox-8. However, its rate of chemiexcitation is substantially slower. Given these circumstances and the higher background signal observed for MA-Diox-2 vs. that of MA-Diox-8, the S / N value achieved by probe MA-Diox-8 is significantly higher than that resulting by probe MA-Diox-2 and its adamantyl analogue probe MA-Diox-1 (77-fold and 90-fold respectively, Fig. 5B).

[0307] The synthesis of the spiro-fused dioxetanes was readily achieved as described above. The last step of the synthesis involves oxidation by singlet oxygen of an enolether precursor to form a dioxetane. A side ene -product of this oxidation can be obtained through the elimination of a proton positioned at the allylic position of the enolether. For a cyclobutyl-enolether, the formation of the side product is unfavored because the elimination reaction leads to the generation of a highly constrained cyclic alkene. We have previously reported that oxidation of cyclopentyl, and cycloheptyl-enolethers resulted in a full formation of the undesired ene -product (Tannous et al., 2024). The corresponding cyclohexyl derivative gave about 1:1 ratio of ene- and dioxetane products. Interestingly, oxidation of enolether attached to six-member rings with inductive electron-withdrawing properties resulted in a relatively high yield of the dioxetane product. Particularly, oxidation of the six-member ring sulfone enolether has resulted in a 91% yield of the corresponding dioxetane and only 9% of the ene -product.

[0308] The introduction of the spirofused-cyclobutyl-dioxetane unit as a chemiluminophore resulted in a substantial chemiexcitation acceleration effect due to therelease of angular strain. Indeed, this flash chemiluminescence led to a notable enhancement in the detection sensitivity achieved by such dioxetanes, as compared to non-strained dioxetanes. Unfortunately, the chemical stability of the spiro -cyclobutyl-dioxetane was significantly compromised, and bacterial detection assays could only be performed after removing the cell growth media through prewashing with a buffer solution. Stability measurements of a spiro-cyclobutyl-dioxetane in cell-growth media resulted in the rapid decomposition of the strained dioxetane, most likely due to a reduction reaction of the peroxide bond. In this study, we observed that the inclusion of spiro-fused six-member rings with inductive electron-withdrawing properties induces a chemiexcitation acceleration effect of dioxetanes, which is comparable to the strain effect generated by the cyclobutyl units. The chemical stability of these spiro-fused six-member ring dioxetanes was notably higher and enabled their use in LB cell growth medium. The flash chemiexcitation and elevated chemical stability presented by sulfone MA-Diox-8 have resulted in a chemiluminescent probe with exceptionally high detection sensitivity and extended shelf life.Study 2. Phenoxy- 1,2-dioxeatne probes chemiluminescent equipped with adamantyl hetero-functional unitsExperimentalSynthesis and characterization of lactone -dioxetane (AA-LactoneAD)

[0309] Lactone-dioxetane (AA-LactoneAD) was synthesized according to the procedure depicted in Scheme 58.

[0310] Compound I. A flame-dried round bottom flask equipped with a stir bar was charged with adamantane-2, 6-dione (500 mg, 3.045 mmol, 1 equiv.) and CH2CI2 (0.2 M), and then cooled to 0°C with an ice-water bath. Next, meta-chloroperoxybenzoic acid (mCPBA, 578 mg, 3.35 mmol, 1.1 equiv.) was added in 3 equal portions at 5-minute intervals. After the final addition of mCPBA, the resulting solution was stirred for an additional 10 minutes at 0°C before the ice-bath was removed and the solution allowed to warm to room temperature. The solution was stirred at room temperature until complete consumption of the ketone starting material as determined by TLC. Upon complete consumption of the ketone, excess mCPBA was quenched with ^2826)3 (sat. aq.) followed by NaHCCh (sat. aq.). The layers were then separated and the aqueous phase extracted with additional CH2CI2 (x3). The combined organic extracts were washed with brine, dried overNa2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (30 to 50% EtOAc / hexanes, 252 mg, 46% yield).

[0311] Compound la. Phosphonate (521 mg, 1.32 mmol, 1.1 equiv.) was dissolved in dry THF under argon atmosphere, and the solution was cooled to -78°C. LDA (2.0 M in THF, 0.9 mF, 1.5 equiv.) was added dropwise, and the solution was stirred for 20 minutes. Cycloalkyl ketone I (218 mg, 1.2 mmol, 1 equiv.) was added, and the reaction was allowed to warm to room temperature. The reaction was monitored by TLC (Hex: EtOAc, 70:30). Upon completion, TBAF (1.0 M in THF, 1.4 mL 1.1 eq.) was added, and the deprotection reaction was monitored by TLC (Hex: EtOAc, 60: 40). Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The product was purified by column chromatography (Hex: EtOAc mixture) afforded compound la (170 mg, 42% yield).

[0312] ' H NMR (400 MHz, Chloroform-^ / ) 8 7.12 (td, J = 7.9, 2.6 Hz, 1H), 6.90 (dd, J = 8.1, 1.5 Hz, 1H), 6.82 (td, J = 7.9, 1.6 Hz, 1H), 4.49 (tt, J = 4.5, 2.3 Hz, 1H), 3.45 (dq, J = 1.0, 3.2 Hz, 1H), 3.28 (s, 3H), 3.08 (td, J = 6.0, 3.9 Hz, 1H), 2.23-2.02 (m, 5H), 1.99-1.80 (m, 4H).

[0313] 13C NMR (101 MHz, CDCI3) 6 152.05, 142.25, 133.72, 127.83, 123.43, 123.29, 116.40, 73.48, 73.35, 70.99, 56.64, 44.70, 41.28, 38.68, 37.71, 37.39, 37.30, 37.10, 33.76, 32.80, 32.47, 32.38, 32.21, 31.02, 29.70, 27.61.

[0314] Compound lb. Compound la (170 mg, 0.51 mmol, 1 equiv.) and EtaN (206 mg, 2.03 mmol, 4 equiv.) were dissolved in dry THF in a pressure flask. MgCh (97 mg, 1.02 mmol, 2 equiv.) and paraformaldehyde (123 mg, 4.06 mmol, 8 equiv.) were added simultaneously, and the pressure flask was inserted into an oil bath that was pre-heated to 80°C. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and brine. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc, 70:30) to afford compound lb (104 mg, 56% yield).

[0315] ' H NMR (400 MHz, Chloroform-^ / ) 8 9.92 (s, 1H), 7.54 (dd, J = 7.9, 2.8 Hz, 1H), 6.96 (t, J = 8.0 Hz, 1H), 4.52 (dh, J = 7.0, 2.3 Hz, 1H), 3.49 (s, 1H), 3.33 (s, 3H), 3.16-3.03 (m, 1H), 2.39-2.08 (m, 5H), 2.02-1.85 (m, 3H), 1.85-1.61 (m, 1H).Scheme 58: Synthetic procedure for the formation of AA-LactoneAD

[0316] Compound 1c. Compound lb (104 mg, 0.287 mmol, 1 equiv.) was dissolved in DCM. Allyl (triphenylphosphoranylidene) acetate (124 mg, 0.344 mmol, 1.2 equiv.) was added, and the reaction was stirred at room temperature. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with brine. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc, 50: 50) to afford compound 1c (109 mg, 86% yield).

[0317] ' H NMR (400 MHz, Chloroform- 8 7.96 (d, J = 16.2 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 6.64 (d, J = 16.1 Hz, 1H), 5.98 (ddt, J = 17.2, 10.4, 5.7 Hz, 1H), 5.36 (dq, J = 17.3, 1.5 Hz, 1H), 5.26 (dq, J = 10.5, 1.3 Hz, 1H), 4.71 (dt, J = 5.6, 1.5 Hz, 2H), 4.52 (tt, J = 4.5, 2.3 Hz, 1H), 3.47 (dd, J = 6.4, 3.4 Hz, 1H), 3.31 (s, 3H), 3.12 (dd, J = 8.5, 3.9 Hz, 1H), 2.32-2.05 (m, 5H), 2.01-1.83 (m, 4H).

[0318] 13C NMR (101 MHz, CDCE) 6 178.76, 166.74, 150.91, 142.43, 141.90, 139.05, 135.10, 132.24, 127.12, 125.82, 125.64, 120.31, 118.89, 118.34, 73.28, 65.34, 56.85, 52.95, 41.21, 37.74, 37.39, 37.29, 37.10, 32.83, 32.46, 32.36, 32.21, 31.07, 29.68, 27.67.

[0319] Compound Id. Compound 1c (50 mg, 0.112 mmol, 1 equiv.) was dissolved in dry DMF. K2CO3 (46 mg, 0.336 mmol, 3 equiv.) and Iodide (63 mg, 0.112mmol, 1 equiv.) were added, and the reaction was stirred at room temperature. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and ^2826)3. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc, 70:30) to afford compound Id (70 mg, 71% yield).

[0320] Compound le. Compound Id (70 mg, 0.079 mmol, 1.0 equiv.) dissolved in DCM (2 ml), followed by the addition of DMBA (25 mg, 0.159 mmol, 2.0 equiv.) and tetrakis(triphenylphosphine)palladium(9 mg, 0.008 mmol, 0.1 equiv.). The reaction was stirred at room temperature and allyl deprotection was monitored by TLC. Upon completion, the solvent was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography (Hex: EtOAc, 40:60) to afford compound le (35 mg, 52% yield).

[0321] Compound 1g (AA-LactoneAD) . Compound le (35 mg, 0.042 mmol, 1 equiv.) was dissolved in MeOH, K2CO3 (29 mg, 0.21 mmol, 5 equiv.) was added, and the reaction was stirred at room temperature. The reaction was monitored by RP-HPLC (30-100% ACN in water, 0.1% TFA). Upon completion, the reaction mixture was filtered to remove potassium carbonate particles and diluted with DCM (solution need add 10μL AcOH to acidify), followed by the addition of a catalytic amount of methylene blue. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC (30-100% ACN in water, 0.1% TFA). Upon completion, the solvent was evaporated under reduced pressure at 30°C. The crude product was purified by preparative RP-HPLC (gradient of ACN in water, 0.1% TFA) to afford 1g (15 mg, 50% yield).

[0322] MS (ES+): m / z calc, for C34H37CIO14: 704.2; found: 705.7 [M-H]+.Synthesis and characterization of ketone -acid-dioxetane (AA-KetoneAD)

[0323] Ketone-acid-dioxetane (AA-KetoneAD) was synthesized according to the procedure depicted in Scheme 59.

[0324] Compound 2a. Phosphonate (300 mg, 0.759 mmol, 1.1 equiv.) was dissolved in dry THF under argon atmosphere, and the solution was cooled to -78°. LDA (2.0 M in THF, 0.46 mL, 1.2 equiv.) was added dropwise, and the solution was stirred for 20 minutes. adamantane-2, 6-dione (174 mg, 1.063 mmol, .5 equiv.) was added, and the reaction was allowed to warm to room temperature. The reaction was monitored by TLC (Hex: EtOAc, 70:30). Upon completion, TBAF (1.0 M in THF, 1.4 mL 1.1 eq.) was added, and the deprotection reaction was monitored by TLC (Hex: EtOAc, 60: 40). Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The product was purified by column chromatography (Hex: EtOAc mixture) afforded compound 2a (175 mg, 72% yield).

[0325] ' H NMR (400 MHz, Chloroform-^) 8 7.14 (t, J = 7.8 Hz, 1H), 6.90 (td, J = 7.9, 7.4, 1.6 Hz, 2H), 3.45-3.39 (m, 1H), 3.34 (s, 3H), 2.75-2.45 (m, 2H), 2.23-2.09 (m, 6H), 2.02-1.88 (m, 3H).

[0326] Compound 2b. Compound 2a (175 mg, 0.549 mmol, 1 equiv.) and EtaN (222 mg, 2.2 mmol, 4 equiv.) were dissolved in dry THF in a pressure flask. MgCh (105 mg, 1.09 mmol, 2 equiv.) and paraformaldehyde (123 mg, 4.4 mmol, 8 equiv.) were added simultaneously, and the pressure flask was inserted into an oil bath that was pre-heated to 80°C. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and brine. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc, 70:30) to afford compound 2b (129 mg, 68% yield).

[0327] ' H NMR (400 MHz, Chloroform-^) 8 11.66 (s, 1H), 9.92 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 3.48-3.40 (m, 1H), 3.37 (s, 3H), 2.73-2.65 (m, 9H), 2.65- 2.58 (m, 2H).

[0328] 13C NMR (101 MHz, CDCI3) 8 213.45, 195.74, 131.24, 122.57, 57.23, 46.71, 45.35, 40.51, 40.19, 39.99, 39.78, 31.84, 28.57.

[0329] Compound 2c. Methyl-(triphenylphosphoranylidene) acetate (48 mg, 0.143 mmol, 1.5 equiv.) was added to DCM solution of compound 2b (33 mg, 0.095 mmol, 1 equiv.), and the reaction was stirred at room temperature. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with brine. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc, 70:30) to afford compound 2c (32 mg, 83% yield).

[0330] ' H NMR (400 MHz, Chloroform-7) 8 7.92 (d, J = 16.2 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 6.87 (d, 7= 7.9 Hz, 1H), 6.59 (d, J = 16.2 Hz, 1H), 3.79 (s, 2H), 3.42-3.37 (m, 1H), 3.32 (s, 3H), 2.68-2.64 (m, 9H), 2.62-2.56 (m, 2H).

[0331] 13C NMR (101 MHz, CDCl3) 6 217.35. 213.49, 167.51, 150.84, 141.23, 138.90, 135.47, 127.63, 126.97, 123.34, 122.66, 120.11, 57.00, 51.79, 46.71, 45.32, 39.76, 31.76, 29.66, 28.52.

[0332] Compound 2d. Compound 2c (25 mg, 0.062 mmol, 1 equiv.) was dissolved in dry DMF. K2CO3 (25 mg, 0.18 mmol, 3 equiv.) and Iodide (35 mg, 0.062 mmol, 1 equiv.) were added, and the reaction was stirred at room temperature. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH4CI and Na2S2O3. The organic phase was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography (Hex: EtOAc, 50:50) to afford compound 2e (45 mg, 86% yield).

[0333] Compound 2e. Compound 2d (30 mg, 0.035 mmol, 1.0 equiv.) and NaOH (14 mg, 0.35mmol, 10 equiv.) were dissolved in a mixture of THF: H2O (4:1). The reaction mixture was stirred at 60°C and monitored by RP-HPLC. Upon completion, the reaction mixture was diluted with EtOAc and washed with 1 M HC1. The organic layer was separated, washed twice with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was directly used for next step without further purified.

[0334] Compound 2g (AA-KetoneAD) . Compound 2e (16 mg, 0.024 mmol) was dissolved in MeOH, K2CO3 (5 equiv.) was added, and the reaction was stirred at room temperature. The reaction was monitored by RP-HPLC (30-100% ACN in water, 0.1% TFA). Upon completion, the reaction mixture was filtered to remove potassium carbonate particles and diluted with DCM (with 10 pL AcOH to acidify), followed by the addition of a catalytic amount of methylene blue. Oxygen was bubbled through the solution while irradiating withyellow light. The reaction was monitored by RP-HPLC (30-100% ACN in water, 0.1% TFA). Upon completion, the solvent was evaporated under reduced pressure at 30°C. The crude product was purified by preparative RP-HPLC (gradient of ACN in water, 0.1% TFA) to afford 2g (8 mg, 45 % yield).

[0335] MS (ES+): m / z calc, for C34H37CIO13: 689.1; found: 689.8 [M-H]+.Scheme 59: Synthetic procedure for the formation of AA-KetoneADResults and discussion

[0336] The light emission from adamantyl-phenoxy- 1,2-dioxetanes can glow from minutes to hours, depending on the solvent and the specific substituent on the dioxetane molecule. To improve the light emission properties (S / N ratio), the rate of chemiexcitation should be significantly enhanced to a flash mode. Tannous et al., 2024 shows that spiro- dioxetanes featuring strained four-member rings equipped with electronegative polar motifs, either substituted with an EWG or having a hetero atom incorporated in the ring, undergo accelerated chemiexcitation compared to their adamantyl counterparts. Study 1 herein shows that six-membered rings with inductive electron- withdrawing units enhance the chemiexcitation rate of phenoxy- 1,2-dioxetanes compared to their adamantyl counterparts as well. Yet, although the chemical stability of the compounds disclosed in Study 1 is significantly elevated compared to that of the spiro-dioxetanes disclosed in Tannous et al., 2024, it is still severely hampered compared to that of the adamantyl-phenoxy- 1,2- dioxetanes disclosed in Green et al., 2017.

[0337] It was therefore postulated that a hetero-substituted adamantyl unit would similarly accelerate the chemiexcitation rate while retaining the high chemical stability of the parent adamantyl probe. Representative examples of adamantanones substituted with hetero atoms (adamantyl-like ketones bearing EWGs) include (lr,3r,5r,7r)-adamantan-2-one; 4- oxatricyclo[4.3.1.13,8]undecan-5-one; diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate; (lR,3S,5r,7r)-adamantane-2-carboxylic acid; methyl (lR,3S,5r,7r)- adamantane-2-carboxylate; (lr,3r,5r,7r)-2-oxaadamantane (Tercel, Moana. "New routes to spiro-oxindoles for the synthesis of gelsemine", Thesis, University of Cambridge, 1989); (ls,5s,7s)-2,4,10-trioxaadamantane; (lR,3r,5S,6R,7r,8S)-6,8-dimethoxy-2,4,10- trioxaadamantane (a similar compound, having two -O-CH2(Ph) groups instead of -OCH3 groups is disclosed in Lee and Kishi, J. Org. Chem., 1985, 50(22), 4402-4404); (3s, 5s, 7s)- 1 -azaadamantane (Komarov et al., J Am Chem Soc., 2015, 137(2), 926-930); (lr,3r,5r,7r)- 1,3 -diazaadamantane (Gonikberg and le Noble, The Journal of Organic Chemistry, 1995, 60(24), 7751-7755); (lr,3r,5r,7r)-2,2,5,7-tetramethyl-l,3-diazaadamantane (Dalinger et al., Russ Chem Bull., 2021, 70, 1002-1005); (3s,5s,7s)-l-methyl-l-azaadamantan-l-ium; (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium; ( lr,3r,5r,7r)- 1 ,3-dimethyl- 1,3- diazaadamantane- 1 ,3-diium; and ( lr,3r,5r,7r)- 1 ,2,2,3 ,5,7 -hexamethyl- 1,3- diazaadamantane-l,3-diium (Table 2).

[0338] In the present Study, the effect of several adamantyl derivatives equipped with hetero-functional groups on the chemiexcitation of its corresponding phenoxy- 1,2-dioxetane was studied. Thus, the commercially available adamantane-2, 6-dione was selectively oxidized through Baeyer-Villiger oxidation to form adamantane-lactone. The latter was used to synthesize the ort / zo-substituted methyl-acrylate and acrylic acid P-gal dioxetanes. Additionally, adamantane-2, 6-dione was used to synthesize the corresponding P-gal dioxetanes (Schemes 58-59).

[0339] The chemiluminescence behavior of those probes was measured in comparison to that of the previously reported adamantyl-phenoxy- 1,2-dioxetane controls referred to herein as AA-Diox-1 and MA-Diox-1 (Table 6). The light emission profile of the probes was initially measured in relatively high P-gal concertation (2 U / ml) in PBS 7.4 (Figs. 6A-6B). Under such conditions, the probes bearing hetero-adamantyl derivatives exhibited a faster chemiexcitation rate than the probes AA-Diox- 1 and MA-Diox- 1. Similarly to the previously reported cyclohexyl derivatives with EWGs, the hetero-adamantyl dioxetanes exhibited slightly lower relative quantum yield (Fig. 6C). The light emission signals of the probes were then measured under saturation kinetic conditions (low enzyme concentration, 0.001 U / mL). Under such conditions, the signal generated gradually increases to a plateau level (Figs. 6D-6E).

[0340] As shown, the hetero-adamantyl derivatives reach a plateau signal faster compared to the control probes, both the methyl acrylate (MA-Diox-1) and the acrylic acid (AA-Diox- 1). The S / N values measured for the probes MA-KetoneAD, MA-LactoneAD and AA- KetoneAD were substantially higher than the S / N values of the unsubstituted adamantyl compounds (Fig. 6F). Moreover, all of the hetero-substituted probes reach peak S / N value significantly faster than the control probes while also retaining higher absolute values for the duration of the measurement (Fig. 6G). These results highlight the advantage of the heterosubstituted adamantyl probes, which exhibit faster chemiexcitation kinetics and higher S / N values, leading to overall higher sensitivity obtained in very short times. 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Claims

CLAIMS1. A compound of formula I:whereinR1is selected from a linear or branched (C1-C18)alkyl, or (C3-C7)cycloalkyl;R2and R3together with the carbon atom to which they are attached form:(a) an adamantane analogue of formula II:whereinRB, RD, RF, RH, and R1each independently is selected from -C(O)-, -C(O)-O-, -O-, and -C(X)2-, wherein X each independently is selected from H, (C1-C8)alkyl, - COOH, -COO(C1-C8)alkyl, and -O-(C1-C8)alkyl; andRA, RC, RE, and RGeach independently is tertiary carbon optionally substituted with (C1-C8)alkyl, -COOH, -COO(C1-C8)alkyl, or -O-(C1-C8)alkyl, tertiary nitrogen, or quaternary nitrogen wherein the nitrogen atom is further alkylated; provided that at least one of RB, RD, RF, RH, and R1is not -CH2-, -CH(C1- C8)alkyl-, or -C((C1-C8)alkyl)2-; and / or at least one of RA, Rc, RE, and RGis not tertiary carbon optionally substituted with (C1-C8)alkyl; or(b) a 6-membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from (C1-C8)alkyl optionally substituted with at least one (C6-Cio)aryl group, (C6-Cio)aryl, or an electronwithdrawing group such as halogen, -NO2, -CN, =0, -C00R9, -C(=O)R10, -O- (C1-C8)alkylene-(C6-Cio)aryl, and -SO2R9, but excluding unsubstituted cyclohexyl, wherein two non-adjacent carbon atoms of said carbocyclic or heterocyclic ring optionally from an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (Ci -C18) alkyl; or two alkyl groups attached to the same carbon atom of said carbocyclic or heterocyclic ring, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring;or one of R5and R6, together with R7, forms an optionally substituted cyclic or heterocyclic structure that extends the pi-system of the central aromatic ring, and the other one of R5and R6is H;R7is H, or together with one of R5and R6forms said optionally substituted cyclic or heterocyclic structure;R8is H, an analyte-responsive group, or a boron-containing group having the formula -B(Z)(Z') or -B(Z")3’ Kat+;R9each independently is H, (C1-C18)alkyl, or (C6-Ci4)aryl;R10each independently is H, (C1-C18)alkyl, (C6-Ci4)aryl, or halogen;Z and Z' each independently is -OR11or -O' Kat+;Z" is selected from F, Cl, Br, and I;R11is selected from H, (C1-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl, (C2- C4)heteroalkyl, (C2-C4)heteroalkenyl, (C2-C4)heteroalkynyl, (C6-Cio)aryl, and (C5- C6)heteroaryl, or two R11together with their intervening atoms form a 5- to 7-membered optionally substituted heterocyclic ring;Kat+each independently is an organic cation, or an inorganic cation such as an alkali metal cation;Y is absent or is -O-; andL is absent or is a linker of the formula LI, L2 or L3:optionally substituted at the aromatic ring with one or more substituents each independently selected from (C1-C18)alkyl and (C3-C7)cycloalkyl, wherein M is absent or is -O-, -NH-, - N(C1-C8)alkyl-, or -N+((C1-C8)alkyl)2-, and the asterisk represents the point of attachment to the group Y, provided that: when R8is H, Y is -O-, and L is absent; when R8is said analyte-responsive group, Y is -O-; and when R8is said boron-containing group, Y is absent, and either L is absent or L is said linker wherein M is absent.

2. The compound of claim 1, wherein R1is a linear or branched (C1-C8)alkyl.

3. The compound of claim 2, wherein R1is methyl.

4. The compound of claim 1, wherein R2and R3together with the carbon atom to which they are attached form:(a) an adamantane analogue of the formula II, wherein at least one of RB, RD, RF, RH, and R1is selected from -C(O)-, -C(O)-O-, -O-, and -C(X)2- wherein at least one of the X groups is -COOH, -COO(C1-C3)alkyl, or -O-(C1-C3)alkyl, and the others of RB, RD, RF, RH, and R1each is -CH2-; or at least one of RA, Rc, RE, and RGis tertiary carbon substituted with -COOH, -COO(C1-C3)alkyl, or -O-(C1-C3)alkyl, tertiary nitrogen, or quaternary nitrogen, and the others of RA, Rc, RE, and RGeach is unsubstituted tertiary carbon;(b) cyclohexyl substituted with one or more groups each independently selected from (C1-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -C00R9, -C(=O)R10, and -O-(C1-C4)alkylene-(C6- Cio)aryl, wherein two non-adjacent carbon atoms of said cyclohexyl optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; or two alkyl groups attached to the same carbon atom of said cyclohexyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring;(c) tetrahydropyranyl (oxanyl) or dioxanyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -C00R9, -C(=O)R10, and -O-(C1-C4)alkylene-(C6- Cio)aryl;(d) tetrahydro thiopyranyl (thianyl), 1-oxido tetrahydro thiopyranyl or 1,1- dioxido tetrahydro thiopyranyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1- C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, -C00R9, - C(=O)R10, and -SO2R9; or(e) piperidinyl or piperidinyl-1 -oxide, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1- C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -C00R9, -C(=O)R10, and -0-(C1-C4)alkylene-(C6-Cio)aryl, said piperidinyl being further optionally substituted at the nitrogen atom with one or two groups each independently selected from (C1-C4)alkyl, (C3-C7)cycloalkyl, and (C6-Cio)aryl, wherein two non-adjacent carbon atoms of said piperidinyl optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; or two alkyl groups attached to the same carbon atom of said piperidinyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring,whereinR9each independently is H, (C1-C alkyl, or (C6-Cio)aryl; andR10each independently is H, (C1-C alkyl, (C6-Cio)aryl, or halogen.

5. The compound of claim 4, wherein R2and R3together with the carbon atom to which they are attached form (a) an adamantane analogue selected from (lr,3r,5r,7r)-adamantan- 2-one, 4-oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)- adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3 -diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium; or (b) a 6-membered carbocyclic or heterocyclic ring selected from cyclohexanecarboxylic acid, methyl cyclohexanecarboxylate, ethyl cyclohexanecarboxylate, cyclohexanecarbonyl chloride, cyclohexanecarbonyl bromide, fluorocyclohexane, difluorocyclohexane, chlorocyclohexane, dichlorocyclohexane, cyclohexanecarbonitrile, nitrocyclohexane, tctrahydro-2 / 7-pyran, 1,3-dioxane, 2,2-dimethyl-l,3-dioxane, tetrahydro-2 / Z-thiopyran, tctrahydro-2 / 7-thiopyran 1-oxide, tctrahydro-2 / 7-thiopyran 1,1-dioxide, and (lR,5S)-8- methyl-8-azabicyclo[3.2.1]octane.

6. The compound of claim 1, wherein:(i) R4, R5and R6each independently is selected from H, halogen, -CN, and a π* acceptor group of the formula -CH=CH-E; or(ii) R4is selected from H, halogen, and -CN; one of R5and R6is H; and the other one of R5and R6, together with R7, forms a 6-membered structure selected fromwherein R12is (C1-C 12) alkyl; and R13each independently is selected from -CN, -NO2, (C2-C 12) alkenyl, aryl, -COOR14, -COO' Kat+, and -C(O)N(R14)2, wherein R14is H or (Ci -Ci 2) alkyl, or the two R14together with the nitrogen atom to which they are attached form a heterocyclic ring.

7. The compound of claim 6, wherein:(i) R4and R5each independently is H or halogen; and R6is a π* acceptor group of the formula -CH=CH-E;(ii) R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E; or(iii) R5and R6each independently is H or halogen; and R4is a π* acceptor group of the formula -CH=CH-E.

8. The compound of claim 7, wherein R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E.

9. The compound of claim 8, wherein R4is halogen; R6is H; and R5is a π* acceptor group of the formula -CH=CH-E.

10. The compound of any one of claims 6-9, wherein E is -CN, -COOH, -COO(C1- C8)alkyl, or -COO(C2-C8)alkenyl.

11. The compound of claim 10, wherein E is -CN, -COOH, -COO(C1-C4)alkyl such as - COOCH3 and -COOC(CH3)3, or -COO(C2-C4)alkenyl such as -COOCH=CH-CH3.

12. The compound of claim 6, wherein R4, R5and R6each independently is H or halogen.

13. The compound of claim 12, wherein R4is halogen, and R5and R6each is H.

14. The compound of claim 6, wherein R4is selected from H, halogen and -CN; R5is H;and R6together with R7forms a 6-membered structure selected from O15. The compound of claim 14, wherein R4is H or halogen; R5is H; and R6together with R7forms said 6-membered structure.

16. the compound of claim 1, wherein L is absent or is a linker of the formula LI, L2 or L3, wherein M is absent or is -O-, -NH-, or -N+(CH3)2-.

17. The compound of claim 1, wherein:R1is a linear or branched (C1-C8)alkyl;R2and R3together with the carbon atom to which they are attached form (a) an adamantane analogue of the formula II, wherein at least one of RB, RD, RF, RH, and R1is selected from -C(O)-, -C(O)-O-, -O-, and -C(X)2- wherein at least one of the X groups is - COOH, -COO(C1-C3)alkyl, or -O-(C1-C3)alkyl, and the others of RB, RD, RF, RH, and R1each is -CH2-; or at least one of RA, Rc, RE, and RGis tertiary carbon substituted with -COOH, - COO(C1-C3)alkyl, or -O-(C1-C3)alkyl, tertiary nitrogen, or quaternary nitrogen, and the others of RA, Rc, RE, and RGeach is unsubstituted tertiary carbon; (b) cyclohexyl substituted with one or more groups each independently selected from (C1-C4) alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -C00R9, -C(=O)R10, and -0-(C1-C4)alkylene-(C6-Cio)aryl, wherein two non-adjacent carbon atoms of said cyclohexyl optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1-C18)alkyl; or two alkyl groups attached to the same carbon atom of said cyclohexyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic orheterocyclic ring; (c) oxanyl or dioxanyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4) alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR9, -C(=O)R10, and -0-(C1-C4)alkylene-(C6-Cio)aryl; (d) thianyl, 1-oxidotetrahydrothiopyranyl or 1,1- dioxidotetrahydrothiopyranyl, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4) alkyl optionally substituted with one or two (C6-Cio)aryl groups, -COOR9, -C(=O)R10, and -SO2R9; or (e) piperidinyl or piperidinyl-1 -oxide, optionally substituted at one or more of the carbon atoms with one or more groups each independently selected from (C1-C4)alkyl optionally substituted with one or two (C6-Cio)aryl groups, (C6)aryl, halogen, =0, -COOR9, -C(=O)R10, and -O-(C1- C4)alkylene-(C6-Cio)aryl, said piperidinyl being further optionally substituted at the nitrogen atom with one or two groups each independently selected from (C1-C4)alkyl, (C3- C7)cycloalkyl, and (C6-Cio)aryl, wherein two non-adjacent carbon atoms of said piperidinyl optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, optionally substituted with at least one group each independently selected from halogen and (C1- C18)alkyl; or two alkyl groups attached to the same carbon atom of said piperidinyl, together with said carbon atom, optionally form an additional 3-4 membered carbocyclic or heterocyclic ring, wherein R9each independently is H, (C1-C alkyl, or (C6-Cio)aryl; and R10each independently is H, (C1-C alkyl, (C6-Cio)aryl, or halogen; either(i) R4, R5and R6each independently is selected from H, halogen, -CN, and a π* acceptor group of the formula -CH=CH-E; or(ii) R4is selected from H, halogen, and -CN; one of R5and R6is H; and the other one of R5and R6, together with R7, forms a 6-membered structure selected fromwherein R12is (C1-C 12) alkyl; and R13each independently is selected from -CN, -NO2, (C1-Ci2)alkenyl, aryl, -COOR14, -COO' Kat+, and -C(O)N(R14)2, whereinR14is H or (Ci -Ci 2) alkyl, or the two R14together with the nitrogen atom to which they are attached form a heterocyclic ring; andL is absent or is a linker of the formula LI, L2 or L3, wherein M is absent or is -O-, -NH-, or -N+(CH3)2-.

18. The compound of claim 17, wherein R1is methyl; and R2and R3together with the carbon atom to which they are attached form (a) an adamantane analogue selected from (lr,3r,5r,7r)-adamantan-2-one, 4-oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (Is, 3s, 5s, 7 s)-2-oxoadamantane- 1 ,3 -dicarboxylate, ( lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)-adamantane-2-carboxylate, (lr,3r,5r,7r)-2-oxaadamantane, (ls,5s,7s)-2,4,10-trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8-dimethoxy-2,4,10- trioxaadamantane, (3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3-diazaadamantane,( lr,3r,5r,7r)-2,2,5,7 -tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 - azaadamantan- 1-ium, ( lr,3r,5r,7r)- 1 ,3-dimethyl- 1 ,3-diazaadamantane- 1 ,3-diium, and ( lr,3r,5r,7r)- 1 ,2,2,3 ,5,7 -hexamethyl- 1 ,3-diazaadamantane- 1 ,3-diium; or (b) cyclohexanecarboxylic acid, methyl cyclohexanecarboxylate, ethyl cyclohexanecarboxylate, cyclohexanecarbonyl chloride, cyclohexanecarbonyl bromide, fluorocyclohexane, difluorocyclohexane, chlorocyclohexane, dichlorocyclohexane, cyclohexanecarbonitrile, nitrocyclohexane, tctrahydro-2 / 7-pyran, 1,3-dioxane, 2,2- dimethyl-l,3-dioxane, tetrahydro-2 / Z-thiopyran, tctrahydro-2 / 7-thiopyran 1-oxide, tctrahydro-2 / 7-thiopyran 1,1-dioxide, and (lR,5S)-8-methyl-8-azabicyclo[3.2.1]octane.

19. The compound of claim 17 or 18, wherein:(i) R4and R5each independently is H or halogen; and R6is a π* acceptor group of the formula -CH=CH-E;(ii) R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E; or(iii) R5and R6each independently is H or halogen; and R4is a π* acceptor group of the formula -CH=CH-E.

20. The compound of claim 19, wherein R4and R6each independently is H or halogen; and R5is a π* acceptor group of the formula -CH=CH-E.

21. The compound of claim 20, wherein R4is halogen; R6is H; and R5is a π* acceptor group of the formula -CH=CH-E.

22. The compound of any one of claims 17-21, wherein E is -CN, -COOH, -COO(C1- C8)alkyl, or -COO(C2-C8)alkenyl.

23. The compound of claim 22, wherein E is -CN, -COOH, -COO(C1-C4)alkyl such as - COOCH3 and -COOC(CH3)3, or -COO(C2-C4)alkenyl such as -COOCH=CH-CH3.

24. The compound of claim 17 or 18, wherein R4, R5and R6each independently is H or halogen.

25. The compound of claim 24, wherein R4is halogen, and R5and R6each is H.

26. The compound of claim 17 or 18, wherein R4is selected from H, halogen and -CN;27. The compound of claim 26, wherein R4is H or halogen; R5is H; and R6together with R7forms said 6-membered structure.

28. The compound of any one of claims 1-27, wherein:(i) said analyte-responsive group is selected from:wherein Pep is a group comprising a peptide moiety consisting of at least two amino acid residues and linked to the aniline group via a carbocyclic group thereof; or(ii) said boron-containing group is selected from -B(OH)2, 4,4,5,5-tetramethyl- 1,3,2-dioxaborolanyl, and 4-[4,4,5,5-tetramethyl-l,3,2-dioxaborolanyl]benzyl.

29. The compound of claim 28, wherein:R1is methyl;R2and R3together with the carbon atom to which they are attached form an adamantane analogue selected from (lr,3r,5r,7r)-adamantan-2-one, 4- oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)- adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3 -diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium; or a 6-membered carbocyclic or heterocyclicring selected from methyl cyclohexanecarboxylate wherein the -COOCH3 group is linked para to the point of spiro attachment to the dioxetane, tctrahydro-2 / 7-pyran wherein the point of spiro attachment to the dioxetane is positioned para or meta to the oxygen atom, difluorocyclohexane wherein the fluoro groups both are linked to the carbon atom para to the point of spiro attachment to the dioxetane, 2,2-dimethyl-l,3-dioxane, or tetrahydro-2H- thiopyran 1,1 -dioxide;R4is H or Cl;R5and R6each is H; orR5is -CH=CH-E, wherein E is -COOH, -COOCH3, or -CN, and R6is H; orR5is H; and R6together with R7formY is -O-;L is absent, or is a linker of the formula LI wherein M is -O-; and R8is said analyte-responsive group or said boron-containing group.

30. The compound of claim 29, wherein:R1is methyl;R2and R3together with the carbon atom to which they are attached form an adamantane analogue selected from (lr,3r,5r,7r)-adamantan-2-one, 4- oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)- adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3 -diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium; or a 6-membered carbocyclic or heterocyclic ring selected from methyl cyclohexanecarboxylate wherein the -COOCH3 group is linked para to the point of spiro attachment to the dioxetane, tctrahydro-2 / 7-pyran wherein the point of spiro attachment to the dioxetane is positioned para or meta to the oxygen atom, difluorocyclohexane wherein the fluoro groups both are linked to the carbon atom para tothe point of spiro attachment to the dioxetane, 2,2-dimethyl-l,3-dioxane, or tetrahydro-2H- thiopyran 1,1 -dioxide;R4, R5and R6each is H;Y is -O-;L is absent; andR8is TBDMS.

31. The compound of claim 29, wherein:R1is methyl;R2and R3together with the carbon atom to which they are attached form an adamantane analogue selected from (lr,3r,5r,7r)-adamantan-2-one, 4- oxatricyclo[4.3.1.13,8]undecan-5-one, diethyl (ls,3s,5s,7s)-2-oxoadamantane-l,3- dicarboxylate, (lR,3S,5r,7r)-adamantane-2-carboxylic acid, methyl (lR,3S,5r,7r)- adamantane-2-carboxylate, ( lr,3r,5r,7r)-2-oxaadamantane, ( 1 s,5s,7s)-2,4, 10- trioxaadamantane, (lR,3r,5S,6R,7r,8S)-6,8 -dimethoxy-2 ,4 , 10-trioxaadamantane,(3 s, 5 s, 7 s)- 1 -azaadamantane, ( lr,3r,5r,7r)- 1 ,3 -diazaadamantane, ( lr,3r,5r,7r)-2,2,5,7 - tetramethyl- 1 ,3-diazaadamantane, (3 s, 5 s, 7 s)- 1 -methyl- 1 -azaadamantan- 1 -ium,(lr,3r,5r,7r)-l,3-dimethyl-l,3-diazaadamantane-l,3-diium, and (lr,3r,5r,7r)-l,2,2,3,5,7- hexamethyl-l,3-diazaadamantane-l,3-diium; or a 6-membered carbocyclic or heterocyclic ring selected from methyl cyclohexanecarboxylate wherein the -COOCH3 group is linked para to the point of spiro attachment to the dioxetane, tetrahydro -2 / 7-pyran wherein the point of spiro attachment to the dioxetane is positioned para or meta to the oxygen atom, difluorocyclohexane wherein the fluoro groups both are linked to the carbon atom para to the point of spiro attachment to the dioxetane, 2,2-dimethyl-l,3-dioxane, or tetrahydro-2H- thiopyran 1,1 -dioxide;R4is Cl;R5is -CH=CH-E, wherein E is -COOH, -COOCH3, or -CN;R6is H;Y is -O-;L is a linker of the formula LI, wherein M is -O-; andR8is galactosyl.

32. A composition comprising a compound according to any one of claims 1 to 31, and a carrier.

33. The composition of claim 32, for use in diagnostics or in vivo imaging.

34. A method for diagnostics or in vivo imaging comprising: applying a composition according to claim 32 to a sample, e.g., a biological sample, or administering a composition according to claim 32 to a subject, wherein R8in the compound of the formula I is a group cleavable by an analyte, wherein upon exposure to said analyte, R8is cleaved from the compound of formula I, thereby generating an unstable phenolate-dioxetane compound, which in turn decomposes through a chemiexcitation process to yield an excited intermediate that decays to its ground-state through emission of light; and imaging to detect the emission of light.

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