2-Diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene] derivatives and analogues as photoactivatable fluorescent compounds for labeling proteins

Abstract

2. The method of claim 1, wherein the nucleotide sequence of the formula (I) is a nucleotide sequence of the nucleotide sequence of the formula (I) for use in labeling proteins, e.g., tag fusion proteins, and for use in visualizing the location and dynamics of proteins in living cells. [Formula 1] Photoactivated fluorescent 2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene] derivatives and other similar compounds of TIFF2024528902000281.tif25170 are provided. Exemplary compounds include, for example, dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate). [chemical 2] TIFF2024528902000282.tif40170 (Example 1, Compound 1). Labeling assays and experimental data for example compounds are provided.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 226,141, filed July 27, 2021, and U.S. Application No. 63 / 339,707, filed May 9, 2022, the disclosures of each of which are incorporated by reference in their entirety herein.

[0002] (2. Field) Provided herein are novel photoactivatable fluorescent compounds and their use in labeling proteins, e.g., tagged fusion proteins, and for visualizing the location and dynamics of proteins within living cells. [Background technology]

[0003] (3.Background) Fluorescence microscopy is useful for visualizing the location and dynamics of biomolecules in living cells. The process involves labeling biomolecules with bright, photostable fluorescent dyes that absorb photons and then emit them at different wavelengths. Green fluorescent protein (GFP) and other genetically encoded fluorophores have been the known gold standard for fluorescence imaging because they allow for genetic specific labeling. Many efforts have been made to improve proteinaceous dyes by improving their photostability and other properties, including enzyme-based self-labeling tags such as the Halo tag, which allow for labeling of specific protein fusions with synthetic fluorophores, enabling a variety of imaging experiments in living cells. See U.S. Pat. No. 10,161,932.

[0004] Rhodamine dyes have been used extensively due to their brightness and photostability, among other properties. The photophysics of rhodamines is known, among other things, due to their importance as biological probes. See Grimm et al., "Deuteration Improves Small-Molecule Fluorophores," 2020, BioRxiv (preprint) (Grimm et al., 2020), available at https: / / doi.org / 10.1101 / 2020.08.17.250027. Methods to improve the brightness and photostability of fluorophores have been reported. These methods include the incorporation of deuterium into the alkylamino auxochromes of rhodamine and other dyes. See Grimm et al., "A General Method to Improve Fluorophores Using Deuterated Auxochromes," JACS Au 2021, 1(5), 690-696 (Grimm et al., 2021). However, rhodamine dyes do not have cell membrane permeability that would allow for optimization of live cell labeling experiments. See U.S. Patent No. 10,161,932.

[0005] In an effort to enhance cell membrane permeability and improve brightness, azetidine-substituted fluorescent compounds have been developed. Such compounds include molecules that are azetidine-substituted derivatives of known fluorescent tags. These compounds can exhibit greater quantum yields compared to their parent compounds. See U.S. Pat. No. 10,161,932. The fluorescence quantum yields of rhodamine dyes and other dyes were subsequently improved by incorporating deuterium into their alkylamino substituents. However, deuteration was found to prevent or delay undesirable properties such as photochemically induced spectral shifts and irreparable photobleaching. See Grimm et al., 2020.

[0006] Thus, there exists a need, and the present disclosure addresses, for photoactivatable fluorescent compounds with improved photostability, brightness, labeling specificity, density control, and other properties for use in protein labeling. Summary of the Invention

[0007] (4. Overview) In one aspect, provided herein is a compound of formula (I): [ka] where: G is -O-, -S-, -SO2-, -C(C 1-3 Alkyl)2-, -N(C 1-3 Alkyl)-, -Si(C 1-3 alkyl)2-, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, or -P(=O)(Ph)-; R is [ka] (In the formula, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] (wherein n is an integer of 1 to 3); R 2 is -O- or -N(C 1-3 alkyl)-; X is hydrogen or halogen; L A is a linker; and Z comprises a moiety that covalently binds to a tag fusion protein, or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof.

[0008] In one embodiment, the tagged fusion protein comprises a Halo tag, a SNAP tag, or a CLIP tag.

[0009] In one embodiment, L A is expressed by the following formula (IA): [ka] (In the formula, Each L 1 Independently: (I C 1-6 Alkylene-, -C 1-6 Alkenylene- or -C 1-6 Alkynylene-; (ii) heteroaryl; or (iii)-NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -N(C 1-3 Alkyl)-C(O)-, -C(O)-N(C 1-3 Alkyl)-, -C 1-6 Alkylene-NH-, -NH-C 1-6 Alkylene-, -C 1-6 Alkylene-O-, -OC 1-6 Alkylene-, -C(O)-C 1-6 Alkylene- or -C 1-6 alkylene-C(O)-; Each L 2 are independent, -C 1-6 Alkylene-, -(OCH2) p , -(CH2O) p -, -(OCH2CH2) p - or -(CH2CH2O) p - (wherein p is an integer from 1 to 3); a and b are each independently an integer of 1 or 2; and L 1 and L 2 But -(L 2 ) b - is oriented in the direction that bonds to Z in formula (I).

[0010] In one embodiment, Z is [ka] It is.

[0011] In another aspect, provided herein is a method of labeling a protein, comprising contacting a sample containing a tagged fusion protein with a compound of formula (I) to obtain a labeled protein. [Brief description of the drawings]

[0012] 5. BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows an overview of the specificity, brightness and concentration control of a commercially available non-photoactivatable dye, Janelia Fluor® 549 (JF549), a commercially available photoactivatable dye (PA-JF549), and a hypothetical ideal dye.

[0013] [Diagram 2] FIG. 2 shows the preparation of a photoactivated fluorescent tagged (eg, labeled) protein.

[0014] [Diagram 3] Figure 3 shows the relative labeling specificity of PA-JF549 compared to the photoactivatable dye compound of Example 4. Labeling specificity was calculated using histone H2B located in the nucleus of wild-type cells not expressing the Halo Tag® protein.

[0015] [Figure 4] FIG. 4 shows the signal-to-noise ratio (SNR) of JF549, PA-JF549, and the photoactivatable dye compounds of Examples 1-4.

[0016] [Diagram 5]Figure 5 shows an example field of view of U2OS expressing an ER-Halo tag fusion co-stained with Potomac Red (CAS:2127150-65-4; Grimm et al., 2017). The labeling specificity of the photoactivatable dye compound of Example 4 was measured and compared to PA-JF549.

[0017] [Figure 6] FIG. 6 shows the signal-to-noise ratio (SNR) of JF549, PA-JF549, and the photoactivatable dye compounds of Examples 1, 4, 7, and 10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] (6. Detailed Description) (6.1 Definition) To facilitate understanding of the disclosure set forth herein, certain terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well known and commonly employed in the art.

[0019] As used herein, the terms "comprising" and "including" may be used interchangeably. The terms "comprising" and "including" are intended to specify the presence of the stated features or components as stated, but do not exclude the presence or addition of one or more features, components, or groups thereof. Furthermore, the terms "comprising" and "including" are intended to include examples that are encompassed by the term "consisting of." As a result, the term "consisting of" may be used in place of the terms "comprising" and "including" to provide more specific embodiments of the present invention.

[0020] As used herein, the term "or" is to be interpreted as an inclusive "or" meaning any one or any combination. Thus, "A, B, or C" means any of "(A), (B), (C), (A and B), (A and C), (B and C), (A, B, and C)." Exceptions to this definition occur only when combinations of elements, features, steps, or acts are in some way inherently mutually exclusive.

[0021] As used herein, the term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In some embodiments, the term "about" or "approximately" refers to within 1, 2, 3, or 4 standard deviations. In some embodiments, the term "about" or "approximately" refers to within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0022] As used herein, the term "salt(s)" refers to salts prepared from non-toxic acids or bases, including inorganic acids and bases, and organic acids and bases. Suitable base addition salts of the compounds disclosed herein include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl-glucamine), and procaine. Suitable non-toxic acids include, but are not limited to, inorganic and organic acids such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Particular non-toxic acids include hydrochloric acid, hydrobromic acid, maleic acid, phosphoric acid, sulfuric acid, and methanesulfonic acid. Thus, examples of particular salts include hydrochloride and mesylate salts. Others are well known in the art.

[0023] As used herein, unless otherwise specified, the term "stereoisomer" or "stereoisomerically pure" refers to one stereoisomer of a compound disclosed herein that is substantially free of other stereoisomers of the compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound is greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of the other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound. The compounds disclosed herein may contain chiral centers and may exist as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms, including mixtures thereof, are included in the embodiments disclosed herein.

[0024] The use of stereoisomerically pure forms of such compounds disclosed herein, as well as the use of mixtures of those forms, are encompassed by the embodiments disclosed herein. For example, mixtures containing equal or unequal amounts of enantiomers of a particular compound may be used in the methods and compositions disclosed herein. These isomers may be asymmetrically synthesized or resolved using standard techniques, such as chiral columns or chiral resolving agents. See, e.g., Jacques et al., Enantiomers, Racemates and Resolutions, Wiley Interscience, New York, 1981; Wilen et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds, McGraw Hill, NY, 1962; and Wilen, Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0025] It should also be noted that the compounds disclosed herein may include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In some embodiments, the compounds disclosed herein are isolated as either E or Z isomers. In other embodiments, the compounds disclosed herein are mixtures of E and Z isomers.

[0026] As used herein, the term "isotopic form" or "isotope" refers to an isotope of a compound that is, for example, an isotope of tritium ( 3 H), iodine-125( 125 I), Sulfur-35( 35 S), or carbon-14 ( 14 C), or may be radiolabeled with a radioisotope such as carbon-13 ( 13C) or nitrogen-15( 15 N) may be isotopically enriched. As used herein, an "isotopologue" is an isotope-enriched compound. The term "isotopically enriched" refers to an atom having an isotopic composition that is not the natural isotopic composition of that atom. "Isotopically enriched" may also refer to a compound that includes at least one atom having an isotopic composition that is not the natural isotopic composition of that atom. The term "isotopic composition" refers to the amount of each respective isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, such as cancer and inflammation therapeutic agents, research reagents, such as binding assay reagents, and diagnostic agents, such as in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds disclosed herein are provided, for example, the isotopologues are carbon-13 or nitrogen-15 enriched compounds. As used herein, "deuterated" refers to an atom in which at least one hydrogen (H) is replaced with a deuterium (D or 2 H), that is, the compound is enriched with deuterium at at least one position.

[0027] As used herein, "alkyl" refers to a linear or branched saturated hydrocarbon group containing from 1 to 10 carbon atoms. In certain embodiments, an alkyl group contains 1 carbon atom ("C1 alkyl"). In certain embodiments, an alkyl group contains 1 to 2 carbon atoms ("C 1-2 In some embodiments, alkyl contains 1 to 3 carbon atoms ("C 1-3 In some embodiments, alkyl contains 1 to 4 carbon atoms ("C 1-4 In some embodiments, alkyl contains 1 to 6 carbon atoms ("C 1-6 In one embodiment, alkyl contains 1 to 10 carbon atoms ("C 1-10In certain embodiments, alkyl includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylhexyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.

[0028] As used herein, "alkylene" refers to a linear or branched saturated divalent hydrocarbon group containing from 1 to 10 carbon atoms, and in certain embodiments, from 1 to 6 carbon atoms. In certain embodiments, alkylene contains from 1 to 3 carbon atoms ("C 1-3 In some embodiments, alkylene contains 1 to 4 carbon atoms ("C 1-4 In some embodiments, alkylene contains 1 to 6 carbon atoms ("C 1-6 In some embodiments, alkylene contains 1 to 10 carbon atoms ("C 1-10 alkylene").

[0029] As used herein, "alkenylene" refers to a linear or branched alkenyl group from which one hydrogen has been removed, resulting in a divalent group. In certain embodiments, alkenylene contains 1 to 3 carbon atoms ("C 1-3 In certain embodiments, alkenylene contains 1 to 4 carbon atoms ("C 1-4 In certain embodiments, alkenylene contains 1 to 6 carbon atoms ("C 1-6 In certain embodiments, alkenylene contains 1 to 10 carbon atoms ("C 1-10Non-limiting examples of alkenylene groups include ethene-1,1-diyl; ethene-1,2-diyl; prop-1-ene-1,1-diyl, prop-2-ene-1,1-diyl; prop-1-ene-1,2-diyl, prop-1-ene-1,3-diyl; prop-2-ene-1,1-diyl; prop-2-ene-1,2-diyl; but-1-ene-1,1-diyl; but-1-ene-1,2-diyl; but-1-ene-1,3-diyl; but-1-ene-1,4-diyl; but-2-ene-1,1-diyl; but-2-ene-1,2-diyl; but-2-ene-1,3-diyl; but-2-ene-1,4-diyl; These include ut-2-ene-2,3-diyl; but-3-ene-1,1-diyl; but-3-ene-1,2-diyl; but-3-ene-1,3-diyl; but-3-ene-2,3-diyl; buta-1,2-diene-1,1-diyl; buta-1,2-diene-1,3-diyl; buta-1,2-diene-1,4-diyl; buta-1,3-diene-1,1-diyl; buta-1,3-diene-1,2-diyl; buta-1,3-diene-1,3-diyl; buta-1,3-diene-1,4-diyl; buta-1,3-diene-2,3-diyl; buta-2,3-diene-1,1-diyl; and buta-2,3-diene-1,2-diyl. Alkenylene groups can be unsubstituted or substituted (eg, optionally substituted alkenylene) as described for alkyl.

[0030] As used herein, "alkynylene" refers to a straight or branched chain divalent substituent that contains one or two carbon-carbon triple bonds and, if unsubstituted, contains only C and H. In certain embodiments, alkynylene contains 1 to 3 carbon atoms ("C 1-3 In certain embodiments, alkynylene contains 1 to 4 carbon atoms ("C 1-4 In certain embodiments, alkynylene contains 1 to 6 carbon atoms ("C 1-6 In certain embodiments, alkynylene contains 1 to 10 carbon atoms ("C 1-10alkynylene"). Non-limiting examples of alkynylene groups include ethyne-1,2-diyl; prop-1-yne-1,3-diyl; prop-2-yne-1,1-diyl; but-1-yne-1,3-diyl; but-1-yne-1,4-diyl; but-2-yne-1,1-diyl; but-2-yne-1,4-diyl; but-3-yne-1,1-diyl; but-3-yne-1,2-diyl; but-3-yne-2,2-diyl; and buta-1,3-diyne-1,4-diyl. Alkynylene groups can be unsubstituted or substituted (e.g., optionally substituted alkynylene), as described for alkynyl groups.

[0031] As used herein, "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic ring of 5-14 ring atoms containing one or more ring heteroatoms independently selected from O-, S-, -N=(trivalent nitrogen), and N(H)-, with the remaining ring atoms being carbon atoms, where the monocyclic ring is aromatic and where at least one ring of the bicyclic or tricyclic ring is aromatic (although the ring need not be a heteroatom-containing ring, e.g.; tetrahydroquinolinyl, dihydroisoquinolinyl, dihydrobenzodioxinyl, 2,3-dihydrobenzo[b][1,4]dioxinyl, etc.). In certain embodiments, a heteroaryl is a monocyclic ring of 5-6 ring atoms. Unless otherwise specified, chemical bonds may be present at any atom of any ring of a heteroaryl group, to the extent permitted by valence rules. In certain embodiments, heteroaryl includes, but is not limited to, triazolyl, tetrazolyl, pyrrolyl, imidazolyl, thienyl, furanyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, indolyl, indolinyl, isoindolinyl, indazolyl, benzimidazolyl, benzoxazolyl, benzofuranyl, benzothienyl, benzopyranyl, benzothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, dihydroisoquinolinyl, pyrrolo[3,2-c]pyridin ... aryl, pyrrolo[1,2-b]pyridazinyl, imidazo[1,2-a]pyridinyl, benzo[d][1,3]dioxolyl, 2,3-dihydrobenzo[b][1,4]dioxinyl, furo[2,3-d]thiazolyl, thieno[2,3-d]oxazolyl, thieno[3,2-b]furanyl, furo[2,3-d]pyrimidinyl, furo[3,2-b]pyridinyl, furo[3,2-c]pyridinyl, 6,7-dihydro-5H-cyclopenta[b]pyridinyl, 7,8-dihydro-6H-cyclopenta[g]quinoxalinyl, dihydrobenzodioxinyl, or 2,3-dihydrobenzo[b][1,4]dioxinyl.

[0032] As used herein, and unless otherwise specified, "halogen" means fluorine, chlorine, bromine, or iodine.

[0033] (6.2 Implementation) (a) compound In one aspect, provided herein are photoactivatable fluorescent compounds. In one embodiment, the photoactivatable fluorescent compounds are for use in labeling, such as protein labeling. In one embodiment, the compounds disclosed herein are useful for visualizing the location and dynamics of proteins in living cells.

[0034] These compounds unexpectedly exhibit improved properties desirable in photoactivatable fluorescent compounds, such as photostability, brightness, labeling specificity, and concentration control. 549 ), commercially available photoactivatable dyes (PA-JF 549 ) and Figure 1, which provides an overview of the specificity, brightness, and density control of ideal dyes. 549 shows high specificity and brightness, but the concentration control is insufficient. 549 The ideal dye exhibits high specificity, brightness, and density control, but poor specificity. These properties cannot be predicted based on structural features alone. For example, PA-JF 549 Modification of the core structure of PA-JF can not only change the photoconvertible photophysical properties of the prepared dyes, but also the tendency of the dyes to become photoactivated after exposure to blue light. 549 The compounds disclosed herein in which the core structure has been modified exhibit unexpected and improved properties.

[0035] In one embodiment, the compound has formula (I): [ka] (In the formula, L A is a linker, Z comprises a moiety that covalently attaches to the tag fusion protein, and the other variables are defined herein), or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof.

[0036] In one embodiment, L A is expressed by the following formula (IA): [ka] (In the formula, Each L 1 Independently: (I C 1-6 Alkylene-, -C 1-6 Alkenylene- or -C 1-6 Alkynylene-; (ii) heteroaryl; or (iii)-NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -N(C 1-3 Alkyl)-C(O)-, -C(O)-N(C 1-3 Alkyl)-, -C 1-6 Alkylene-NH-, -NH-C 1-6 Alkylene-, -C 1-6 Alkylene-N(C 1-6 alkylene)-, -N(C 1-6 Alkylene)-C 1-6 Alkylene-, -C 1-6 Alkylene-O-, -OC 1-6 Alkylene-, -C(O)-C 1-6 Alkylene- or -C 1-6 alkylene-C(O)-; Each L 2 are independent, -C 1-6 Alkylene-, -(OCH2) p , -(CH2O) p -, -(OCH2CH2) p - or -(CH2CH2O) p - (wherein p is an integer from 1 to 3); and a and b are each independently an integer of 1 or 2.

[0037] In one embodiment, L A is expressed as follows (IB): [ka] As shown in the figure, -(L 2 ) b - is the direction in which it bonds to Z.

[0038] In one embodiment, the compound of formula (IB) is: R, [ka] and R 1 If is H and X is H, then Z-(L 2 ) b -(L 1 ) a -teeth, [ka] (Wherein, the wavy line [ka] represents the point of attachment to the remainder of the compound).

[0039] In certain embodiments of the compound of Formula (I) or (IB), G is -O-, -S-, -SO2-, -C(C 1-3 Alkyl)2-, -N(C 1-3 Alkyl)-, -Si(C 1-3 alkyl)2-, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, or -P(=O)(Ph)-.

[0040] In certain embodiments of compounds of Formula (I) or (IB), R is [ka] It is.

[0041] In certain embodiments of compounds of Formula (I) or (IB), R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] It is.

[0042] In certain embodiments of compounds of Formula (I) or (IB), R 2 is -O- or -N(C 1-3 alkyl)-.

[0043] In certain embodiments of the compounds of Formula (I) or (IB), X is hydrogen or halogen.

[0044] In certain embodiments of compounds of Formula (I) or (IB), X is halogen.

[0045] In one embodiment, the compound of formula (IB) is: R is [ka] and R 1 is H and X is halo.

[0046] In certain embodiments of compounds of Formula (I) or (IB), Z is [ka] It is.

[0047] In one embodiment, the compound of formula (I) is: [ka] Here, G is -O-, -S-, -SO2-, -C(C 1-3 Alkyl)2-, -N(C 1-3 Alkyl)-, -Si(C 1-3 alkyl)2-, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, or -P(=O)(Ph)-; R is [ka] (Wherein, the wavy line [ka] represents the point of attachment to the remainder of the compound; R 1 is H, -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] and; R 2 is -O- or -N(C 1-3 alkyl)-; X is hydrogen or halogen; L A is a linker; and Z is [ka] It is what it is.

[0048] In one embodiment, the compound of formula (I) is: L A is a linker of formula (IA), and R is [ka] and R 1 If is H and X is H, then Z-(L 2 ) b -(L 1 ) a -teeth, [ka] (Wherein, the wavy line [ka] represents a point of attachment to the remainder of the compound.

[0049] In one embodiment, the compound of formula (IB) is: [ka] Here, G is -O-, -S-, -SO2-, -C(C 1-3 Alkyl)2-, -N(C 1-3 Alkyl)-, -Si(C 1-3 alkyl)2-, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, or -P(=O)(Ph)-; R is [ka] (Wherein, the wavy line [ka] represents the point of attachment to the remainder of the compound, R 1 is H, -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] and; R 2 is -O- or -N(C 1-3 alkyl)-; X is hydrogen or halogen; Each L 1 Independently: (I C 1-6 Alkylene-, -C 1-6 Alkenylene- or -C 1-6 Alkynylene-; (ii) heteroaryl; or (iii)-NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -N(C 1-3 Alkyl)-C(O)-, -C(O)-N(C 1-3 Alkyl)-, -C 1-6 Alkylene-NH-, -NH-C 1-6 Alkylene-, -C 1-6 Alkylene-N(C 1-6 alkylene)-, -N(C 1-6 Alkylene)-C 1-6 Alkylene-, -C 1-6 Alkylene-O-, -OC 1-6 Alkylene-, -C(O)-C 1-6 Alkylene- or -C 1-6 alkylene-C(O)-; Each L 2 are independent, -C 1-6 Alkylene-, -(OCH2) p , -(CH2O) p -, -(OCH2CH2) p - or -(CH2CH2O) p - (wherein p is an integer from 1 to 3); a and b are each independently an integer of 1 or 2; and Z is [ka] It is what it is.

[0050] In such an embodiment of the compound of formula (IB), Z is [ka] It is.

[0051] In such an embodiment of the compound of formula (IB), Z is [ka] and X is hydrogen.

[0052] In such an embodiment of the compound of formula (IB), Z is [ka] and X is fluorine.

[0053] In one embodiment, the compound of formula (I) is: R, [ka] and R 1 If is H and X is H, then Z-(L 2 ) b -(L 1 ) a -teeth, [ka] (Wherein, the wavy line [ka] represents the point of attachment to the remainder of the compound).

[0054] In certain embodiments, the tagged fusion protein comprises a Halo tag®, a SNAP tag®, or a CLIP tag®.

[0055] In certain embodiments, provided herein is a compound of formula (II): [ka] (In the formula, the asterisks "**" and "*" represent the linker L B and other variables are defined herein), or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof.

[0056] In certain embodiments of the compound of Formula (II), G is -O-, -S-, -SO2-, -C(C 1-3Alkyl)2-, -N(C 1-3 Alkyl)-, -Si(C 1-3 alkyl)2-, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, or -P(=O)(Ph)-.

[0057] In certain embodiments of the compound of Formula (II), R is [ka] It is.

[0058] In certain embodiments of the compound of Formula (II), R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] It is.

[0059] In certain embodiments of compounds of formula (II), X is hydrogen or halogen. In such embodiments, X is hydrogen. In such embodiments, X is halogen.

[0060] In certain embodiments of the compound of Formula (II), R 2 is -O- or -N(C 1-3 alkyl)-.

[0061] In certain embodiments of compounds of Formula (II), L B (i)-C 1-6 Alkylene-, -C 1-6 Alkenylene- or -C 1-6 (ii) alkynylene-; (iii) heteroaryl; or (iv) -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -N(C 1-3 Alkyl)-C(O)-, -C(O)-N(C 1-3Alkyl)-, -C 1-6 Alkylene-NH-, -NH-C 1-6 Alkylene-, -C 1-6 Alkylene-N(C 1-6 alkylene)-, -N(C 1-6 Alkylene)-C 1-6 Alkylene-, -C 1-6 Alkylene-O-, -OC 1-6 Alkylene-, -C(O)-C 1-6 Alkylene- or -C 1-6 It is alkylene-C(O)-.

[0062] In certain embodiments of the compound of Formula (II), Z is [ka] In such an embodiment of the compound of formula (II), Z is [ka] It is.

[0063] In such an embodiment of the compound of Formula (II), Z is [ka] When it is, X is hydrogen.

[0064] In such an embodiment of the compound of Formula (II), Z is [ka] When it is, X is fluorine.

[0065] In one embodiment, the compound of formula (II) is: [ka] Here, G is -O-, -S-, -SO2-, -C(C 1-3 Alkyl)2-, -N(C 1-3 Alkyl)-, -Si(C 1-3alkyl)2-, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, or -P(=O)(Ph)-; R is [ka] (In the formula, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] (wherein n is an integer from 1 to 3); and R 2 is -O- or -N(C 1-3 alkyl)-; X is hydrogen or halogen; L B (i)-C 1-6 Alkylene-, -C 1-6 Alkenylene- or -C 1-6 (ii) alkynylene-; (iii) heteroaryl; or (iv) -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -N(C 1-3 Alkyl)-C(O)-, -C(O)-N(C 1-3 Alkyl)-, -C 1-6 Alkylene-NH-, -NH-C 1-6 Alkyl, -C 1-6 Alkylene-N(C 1-6 alkylene)-, -N(C 1-6 Alkylene)-C 1-6 Alkylene-, -C 1-6 Alkylene-O-, -OC 1-6 Alkylene-, -C(O)-C 1-6 Alkylene- or -C 1-6 It is alkylene-C(O)-.

[0066] In certain embodiments of the compound of Formula (II), G is -O-, -S-, or -SO2-; R is [ka] (In the formula, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, or -(CH2) n O(C 1-3 L B is -NHC(O)- or -C(O)NH- and X is hydrogen.

[0067] In certain embodiments of a compound of Formula (II), G is -O- or -S-; R is [ka] (In the formula, R 1 -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 alkyl)2, or -(CH2) n O(C 1-3 L B is -NHC(O)- or -C(O)NH- and X is hydrogen.

[0068] In one embodiment, provided herein is a compound having formula (III): [ka] It is a compound of the formula:

[0069] In certain embodiments of the compound of Formula (III), G is -O-, -S-, -N(CH3)-, -Si(CH3)2-, -C(CH3)2-, or -SO2-.

[0070] In certain embodiments of compounds of Formula (III), L B teeth, [ka] (In the formula, "**" and "*" represent linkers L B A person skilled in the art will recognize that the position of the wavy bond is irrelevant whether it is displayed in the middle of the bond or at the end of the bond, and can, for example, [ka] It should be noted that the above structures are all understood to be equivalent to one another.

[0071] In certain embodiments of compounds of Formula (III), L B includes heteroaryl.

[0072] In certain embodiments of a compound of Formula (III), heteroaryl is triazole or imidazole.

[0073] In certain embodiments of the compound of Formula (III), R 1 teeth, [ka] It is.

[0074] In certain embodiments of the compound of Formula (III), R 1 is H and L B teeth, [ka] It is.

[0075] In certain embodiments, provided herein is a compound of formula (IV): [ka] (In the formula, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] (wherein n is an integer of 1 to 3).

[0076] In one embodiment, R 1 is -N(CH3)2.

[0077] In one embodiment, R 1 is -OH.

[0078] In one embodiment, R 1 teeth, [ka] It is.

[0079] In one embodiment, R 1 teeth, [ka] It is.

[0080] In one embodiment, R 1 teeth, [ka] It is.

[0081] In one embodiment, R 1 teeth, [ka] It is.

[0082] In one embodiment, R 1 teeth, [ka] It is.

[0083] In one embodiment, R 1 teeth, [ka] It is.

[0084] In one embodiment, R 1 teeth, [ka] It is.

[0085] In certain embodiments, provided herein is a compound of Formula (V): [ka]

[0086] Here, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] (wherein n is an integer of 1 to 3).

[0087] In certain embodiments, provided herein is a compound of Formula (V): [ka] Here, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] (wherein n is an integer of 1 to 3).

[0088] In certain embodiments, provided herein is a compound of Formula (V): [ka]

[0089] Here, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)NH2, -C(=O)NH(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] (wherein n is an integer of 1 to 3).

[0090] In one embodiment, R 1 is -N(CH3)2.

[0091] In one embodiment, R 1 is -OH.

[0092] In one embodiment, R 1 teeth, [ka] It is.

[0093] In one embodiment, R 1 teeth, [ka] It is.

[0094] In one embodiment, R 1 teeth, [ka] It is.

[0095] In one embodiment, R 1 teeth, [ka] It is.

[0096] In one embodiment, R 1 teeth, [ka] It is.

[0097] In one embodiment, R 1 teeth, [ka] It is.

[0098] In one embodiment, R 1 teeth, [ka] It is.

[0099] In certain embodiments, provided herein is a compound of formula (VI): [ka] Here, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] (wherein n is an integer of 1 to 3).

[0100] In one embodiment, R 1 teeth, [ka] It is.

[0101] In one embodiment, R 1 teeth, [ka] It is.

[0102] In one embodiment, R 1 teeth, [ka] It is.

[0103] In certain embodiments, provided herein is a compound of Formula (VIa): [ka] Here, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 Alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] (wherein n is an integer of 1 to 3).

[0104] In one embodiment, R 1 teeth, [ka] It is.

[0105] In one embodiment, R 1 teeth, [ka] It is.

[0106] In one embodiment, R 1 teeth, [ka] It is.

[0107] In one embodiment, the compound is [ka] It is.

[0108] In one embodiment, the compound is [ka] It is.

[0109] In one embodiment, the compound is [ka] The file is TIFF2024528902000088.tif181170.

[0110] In one embodiment, the compound is [ka] It is.

[0111] In one embodiment, the compound of formula (VIa) is [ka] It is.

[0112] In one embodiment, the compound of formula (VIa) is [ka] It is.

[0113] In certain embodiments, provided herein is a compound of formula (VII): [ka] where R is [ka] (In the formula, R 2 is -CH3), and L B teeth, [ka] (In the formula, "*" and "**" represent linkers L B is a linker comprising:

[0114] In certain embodiments of the compound of Formula (VII), R is [ka] isn't it.

[0115] In certain embodiments of compounds of Formula (VII), L B teeth, [ka] isn't it.

[0116] In certain embodiments of the compound of Formula (VII), R is [ka] If L B teeth, [ka] isn't it.

[0117] In certain embodiments of the compound of Formula (VII), R is [ka] If L B teeth, [ka] It is.

[0118] In certain embodiments of the compound of Formula (VII), R is [ka] If L B teeth, [ka] It is.

[0119] In one embodiment, the compound is dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate).

[0120] In certain embodiments, the compound is 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid).

[0121] In certain embodiments, the compound is N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide.

[0122] In certain embodiments, the compound is 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide).

[0123] In one embodiment, the compound is N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide.

[0124] In one embodiment, the compound is 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide.

[0125] In certain embodiments, the compound is 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N-methylazetidine-3-carboxamide).

[0126] In one embodiment, the compound is (2S,2'S)-1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-2-carboxamide).

[0127] In certain embodiments, the compound is N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2-(hydroxymethyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide.

[0128] In one embodiment, the compound is (3S,3'S)-1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylpyrrolidine-3-carboxamide).

[0129] In one embodiment, the compound is 1,1'-(6'-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-3,7-diyl)bis(N,N-dimethylazetidine-3-carboxamide).

[0130] In certain embodiments, the compound is 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxamide).

[0131] In certain embodiments, the compound is 3',6'-di(azetidin-1-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one.

[0132] In certain embodiments, the compound is 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthen]-6-yl)propanamide.

[0133] In certain embodiments, the compound is (E)-3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one.

[0134] In certain embodiments, the compound is (Z)-3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one.

[0135] In certain embodiments, the compound is 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-2-diazospiro[indene-1,9'-xanthen]-3(2H)-one.

[0136] It should be noted that if there is a discrepancy between a depicted structure and the name of that structure, the depicted structure is given more weight.

[0137] The above paragraphs present numerous embodiments of the compounds provided herein. The embodiments in each example include both the recited compound(s) as well as salts, single stereoisomers, mixtures of stereoisomers, or isotopic forms thereof.

[0138] (b) Tagged protein In another aspect, provided herein are tagged fusion proteins.

[0139] In some embodiments, the tagged fusion protein is a kinase. In some embodiments, the tagged fusion protein is a transcription factor. In some embodiments, the tagged fusion protein is a chromatin regulator. In some embodiments, the tagged fusion protein is an adaptor. In some embodiments, the tagged fusion protein is a transporter. In some embodiments, the tagged fusion protein is a pathogenic aggregation factor.

[0140] In one embodiment, the tagged fusion protein is a histone, hi one embodiment, the histone is an H2B-Halo tagged protein.

[0141] In one embodiment, the tag is a HaloTag® (see, e.g., England et al., "HaloTag Technology: A Versatile Platform for Biomedical Applications," Bioconjugate Chem. 2015, 26(6), 975-986 (England et al., 2015)). It should be noted that one of skill in the art would know how to make proteins fused to a HaloTag®.

[0142] In some embodiments, the Halo Tag® is derived from a bacterial enzyme. In some embodiments, the bacterial enzyme is a haloalkane dehalogenase. In some embodiments, the Halo Tag® is part of a protein that is fused to the Halo Tag®. In some embodiments, the Halo Tag® is expressed using standard recombinant protein expression techniques. In some embodiments, the Halo Tag® protein coding region is inserted near a gene of interest. In some embodiments, the Halo Tag® is self-labeling. In some embodiments, the Halo Tag® specifically binds to a chloroalkane linker. In some embodiments, the binding of the Halo Tag® to the chloroalkane linker is irreversible under physiological conditions. In some embodiments, the Halo Tag® is used as a protein label in an enzyme assay. In some embodiments, the Halo Tag® is used as a protein label in cell imaging. In some embodiments, the Halo Tag® is used as a protein label in fluorescence microscopy. In some embodiments, Halo Tags® are used as protein labels in protein arrays. In some embodiments, Halo Tags® are used as protein labels to determine the subcellular localization of proteins.

[0143] In some embodiments, the Halo tag fusion protein is a kinase, a transcription factor, a chromatin regulator, an adaptor, a transporter, or a pathogenic aggregation factor.

[0144] In one embodiment, the tag is a SNAP-tag® (see, e.g., Kolberg et al., "SNAP-Tag Technology: A General Introduction," Current Pharmaceutical Design, 2013, 19(30), 5406-5413 (Kolberg et al., 2013)). It should be noted that one of skill in the art would know how to make a SNAP-tag fusion protein.

[0145] In one embodiment, the SNAP Tag® is genetically engineered from the enzyme alanine glyoxylate transaminase. In one embodiment, the CLIP Tag® is self-labeling. In one embodiment, the SNAP Tag® is O 6 In one embodiment, the SNAP tag is encoded by the O-methylguanine-DNA methyltransferase (MGMT) gene. 6 Covalently reacts with benzylguanine derivatives. In some embodiments, SNAP-tags® are used as protein labels in enzyme assays. In some embodiments, SNAP-tags® are used as protein labels in cell imaging. In some embodiments, SNAP-tags® are used as protein labels in fluorescence microscopy. In some embodiments, SNAP-tags® are used as protein labels in protein arrays. In some embodiments, SNAP-tags® are used as protein labels to determine the subcellular localization of proteins.

[0146] In some embodiments, the SNAP tagged fusion protein is a kinase, a transcription factor, a chromatin regulator, an adaptor, a transporter, or a pathogenic aggregation factor.

[0147] In one embodiment, the tag is a CLIP-tag® (see, e.g., Correa et al., "Considerations and Protocols for the Synthesis of Custom Protein Labeling Probes," Methods Mol Biol. 2015, 1266, 55-79 (Correa et al., 2015)). It should be noted that one of skill in the art would know how to make a CLIP-tagged fusion protein.

[0148] In some embodiments, the CLIP tag is self-labeling. In some embodiments, the CLIP tag is an orthogonal tag. In some embodiments, the CLIP tag is an orthogonal tag. 6 -methylguanine-DNA methyltransferase (MGMT) gene. In some embodiments, CLIP-tags® covalently react with benzylcytosine derivatives. In some embodiments, CLIP-tags® are used as protein labels in protein complementation assays. In some embodiments, CLIP-tags® are used as protein labels in protein-protein interaction studies. In some embodiments, CLIP-tags® are used as protein labels in enzyme assays. In some embodiments, CLIP-tags® are used as protein labels in cell imaging. In some embodiments, CLIP-tags® are used as protein labels in fluorescence microscopy. In some embodiments, CLIP-tags® are used as protein labels in protein arrays. In some embodiments, CLIP-tags® are used as protein labels for determining the subcellular localization of proteins.

[0149] In certain embodiments, the CLIP tagged fusion protein is a kinase, a transcription factor, a chromatin regulator, an adaptor, a transporter, or a pathogenic aggregation factor.

[0150] (c) Labeling Proteins and Methods In another aspect, provided herein is a method for producing labeled proteins for measuring the movement of individual proteins within a cellular environment.

[0151] In some embodiments, the method includes contacting a sample containing the tag fusion protein with a compound described herein to obtain a labeled protein. Without being bound by any mechanism or theory, it will be understood that the tag fusion protein generally has a genetically engineered active site that can specifically bind to a reactive linker of a photoactivatable fluorochrome compound to form a covalent bond between the tag and the linker (see, e.g., Jradi et al., "Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy," ACS Chem. Bio. 2019, 14(6), 1077-1090 (Jradi et al., 2019); see also, e.g., England et al., 2015; Kolberg et al., 2013; and Correa et al., 2015). The tag fusion protein-compound covalent complex is referred to herein as the labeled protein. This covalent bond forms rapidly and essentially irreversibly under physiological conditions.

[0152] The labeled protein is then exposed to light, for example 405 nm light, causing the covalently attached photoactivatable dye compound to become fluorescent.

[0153] Figure 2 shows a schematic diagram for preparing a photoactivated labeled protein. As shown in this diagram and described above, a tagged fusion protein described herein is contacted with a compound described herein, such as a photoactivated fluorescent dye compound, to form a covalent bond between the tagged fusion protein and the compound to form a labeled protein. The labeled protein is then treated with light, such as 405 nm light, causing the covalently attached compound to become fluorescent.

[0154] In some embodiments, a tagged fusion protein as described herein is contacted with a compound as described herein, e.g., a photoactivatable fluorochrome compound, to form a labeled protein. In some embodiments, the compound is a fluorophore. In some embodiments, the compound includes a moiety that binds to the tagged fusion protein to form the labeled protein. In some embodiments, the tagged fusion protein is covalently bound to a compound to form the labeled protein. In some embodiments, the compound is covalently bound to a lysine residue on the tagged fusion protein to form the labeled protein. In some embodiments, the compound is covalently bound to a cysteine ​​residue on the tagged fusion protein to form the labeled protein. In some embodiments, the compound is covalently bound to an aspartic acid residue on the tagged fusion protein to form the labeled protein.

[0155] In one embodiment, the compound moiety of the labeled protein fluoresces when exposed to light. Without being bound by any mechanism or theory, it is understood that the compound moiety of the labeled protein undergoes Wolff rearrangement when exposed to light, usually followed by decarboxylation of the compound. In one embodiment, the labeled protein is illuminated with a 405 nm light source. In one embodiment, the intensity of the 405 nm light source is about 365 mW. In one embodiment, the labeled protein is illuminated for about 5 minutes.

[0156] In some embodiments, the labeled protein solution is filtered after incubation, hi some embodiments, the solution is filtered through a desalting column.

[0157] In some embodiments, the labeled protein fluoresces when exposed to light. In some embodiments, the light is a laser. In some embodiments, the wavelength (λ) of the light is about 405 nm. In some embodiments, the intensity of the 405 nm light is less than 1 mW. In some embodiments, the intensity of the 405 nm light is about 12 mW. In some embodiments, the intensity of the 405 nm light is about 0 mW to about 12 mW. In some embodiments, the intensity of the 405 nm light is greater than 12 mW, e.g., greater than about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 200, about 300, or about 400 mW. In some embodiments, the laser is pulsed. In some embodiments, the laser intensity increases over time during pulsing. In some embodiments, 561 nm (λ) light is used to excite the fluorophores of the labeled proteins. In some embodiments, the wavelength (λ) of the light is about 561 nm. In some embodiments, the intensity of the 561 nm light is about 500 mW. In some embodiments, the sample comprises one or more living cells and the proteins are labeled in the one or more living cells. In some embodiments, the proteins are labeled in intracellular compartments of the one or more living cells. In some embodiments, the proteins are labeled in the nuclei of the one or more living cells. In some embodiments, the proteins are labeled in the cytoplasm of the one or more living cells. In some embodiments, the proteins are labeled in the plasma membrane of the one or more living cells. In some embodiments, the proteins are labeled in the mitochondria of the one or more living cells. In some embodiments, the proteins are labeled in the outer membrane of the mitochondria of the one or more living cells. In some embodiments, the proteins are labeled in the inner membrane of the mitochondria of the one or more living cells. In some embodiments, the proteins are labeled in the mitochondrial matrix of the one or more living cells. In some embodiments, the protein is labeled in the Golgi apparatus of one or more live cells. In some embodiments, the protein is labeled in the lysosomes of one or more live cells. In some embodiments, the protein is labeled in the endosomes of one or more live cells. In some embodiments, the protein is labeled in the endoplasmic reticulum of one or more live cells.In some embodiments, the proteins are labeled within the endoplasmic reticulum membrane of one or more living cells. In some embodiments, the proteins are labeled within the rough endoplasmic reticulum of one or more living cells.

[0158] In some embodiments, the movement of individual proteins within the cellular environment of one or more cells is measured. In some embodiments, the movement of individual proteins within an intracellular compartment of one or more cells is measured. In some embodiments, the movement of individual proteins within the nucleus of one or more cells is measured. In some embodiments, the movement of individual proteins within the cytoplasm of one or more living cells is measured. In some embodiments, the movement of individual proteins within the plasma membrane of one or more living cells is measured. In some embodiments, the movement of individual proteins within the mitochondria of one or more living cells is measured. In some embodiments, the movement of individual proteins within the outer mitochondrial membrane of one or more living cells is measured. In some embodiments, the movement of individual proteins within the inner mitochondrial membrane of one or more living cells is measured. In some embodiments, the movement of individual proteins within the mitochondrial matrix of one or more living cells is measured. In some embodiments, the movement of individual proteins within the Golgi apparatus of one or more living cells is measured. In some embodiments, the movement of individual proteins within lysosomes of one or more living cells is measured. In some embodiments, the movement of individual proteins within endosomes of one or more living cells is measured. In some embodiments, the movement of individual proteins within the endoplasmic reticulum of one or more living cells is measured. In some embodiments, the movement of individual proteins within the endoplasmic reticulum membrane of one or more living cells is measured. In some embodiments, the movement of individual proteins within the rough endoplasmic reticulum of one or more living cells is measured, hi some embodiments, the measurements are made in real time.

[0159] In some embodiments, one or more live cells are prepared for imaging by incubating at, for example, about 37° C. In some embodiments, one or more live cells are incubated in the presence of, for example, about 5% CO2. In some embodiments, one or more live cells are incubated overnight or for about 8-10 hours. In some embodiments, one or more live cells are prepared for imaging by incubating with a compound described herein, for example, a photoactivatable fluorescent dye compound, at a concentration of about 1 nM. In some embodiments, one or more live cells are prepared for imaging by incubating with a compound described herein, for example, a photoactivatable fluorescent dye compound, at a concentration of about 200 nM. In some embodiments, one or more live cells are prepared for imaging by incubating with a compound described herein, for example, a photoactivatable fluorescent dye compound, at a concentration of about 1 to about 200 nM. In some embodiments, one or more live cells are incubated with a compound described herein, for example, a photoactivatable fluorescent dye compound, for about 45 minutes.

[0160] In some embodiments, the movement of proteins within the cellular environment is monitored by confocal microscopy. In some embodiments, the movement of proteins within the cellular environment is monitored by localization microscopy. In some embodiments, the movement of proteins within the cellular environment is monitored by super-resolution microscopy. In some embodiments, the movement of proteins within the cellular environment is monitored by single molecule localization microscopy ("SMLM"). In some embodiments, the movement of proteins within the cellular environment is monitored by photoactivated localization microscopy ("PALM"). In some embodiments, the movement of proteins within the cellular environment is monitored by stochastic optical reconstruction microscopy ("STORM").

[0161] In one embodiment, the movement of proteins within a cellular environment is analyzed by maximum likelihood models and single molecule fluorescence is detected.

[0162] In some embodiments, signal-to-noise ratio ("SNR") is used as a surrogate for single molecule brightness. In some embodiments, SNR is analyzed by a log-likelihood ratio test. In some embodiments, the compounds described herein, such as photoactivatable fluorochrome compounds, exhibit similar SNRs to commercially available photoactivatable fluorochromes. In some embodiments, the compounds exhibit similar SNRs to commercially available non-photoactivatable fluorochromes.

[0163] In some embodiments, the labeling specificity is determined by microscopy, such as confocal microscopy, localization microscopy, super-resolution microscopy, SMLM, PALM, or STORM. In some embodiments, the labeling specificity is calculated by comparing the number of spots detected by microscopy, such as localization microscopy, super-resolution microscopy, SMLM, PALM, or STORM, with a control sample. In some embodiments, the labeling specificity of the compounds described herein is higher than that of commercially available photoactivatable fluorescent dyes.

[0164] 7. Method for preparing compounds The compounds of formula (I), (IB), (II), (III), (IV), (V), (VI), and (VIa) can be prepared using conventional organic synthesis methods and commercially available starting materials. As a non-limiting example, the compounds of formula (I), (IB), (II), (III), (IV), (V), (VI), and (VIa) can be prepared as outlined in Scheme 1 and Scheme 1a below, and in the Examples described herein. It should be noted that a person skilled in the art would know how to modify the procedures described in the exemplary schemes to arrive at the desired product.

[0165] [ka] As shown in Scheme 1, compounds of formula (I), (IB), (II), (III), (IV), (V), (VI), and (VIa), where R, G, and R 1(wherein Q is as defined herein) can be prepared starting from an appropriately derivatized intermediate, where Q represents a group capable of undergoing a cross-coupling reaction when treated with a suitable catalyst, such as a bromine or triflate derivative. Intermediate (C) can be prepared, for example, by converting 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid to its corresponding triflate derivative by conventional organic synthesis methods, as described herein. Intermediate (C) may also be synthesized as described in Grimm et al., "A general method to improve fluorophores for live-cell and single-molecule microscopy," Nat. Methods 2015, 12, 244-250 (Grimm et al., 2015); Woodroofe et al., "Synthesis of isomerically pure carboxylate-and sulfonate-substituted xanthene fluorophores," Tetrahedron 2005, 61(12), 3097-3105 (Woodroofe et al., 2005); and Grimm et al., "Bright photoactivatable fluorophores for single-molecule imaging," Nat. Methods 2016, 137-141 (Grimm et al., 2015). 13, 985-988 (Grimm et al., 2016). Alternatively, intermediate (C) can be prepared starting from 1,2,4-benzenetricarboxylic acid by treatment with 3-bromophenol under appropriate conditions.For example, in preparing a compound of formula (IV), an appropriately substituted intermediate (C) (where G is O) is treated with an appropriately substituted azetidine and a palladium catalyst such as Pd2(dba)3, a ligand and a base such as cesium carbonate in a solvent such as dioxane and heated at a temperature ranging from about 25° C. to about 100° C. to provide intermediate (D). Intermediate D is then treated with an acid such as TFA or a base such as lithium hydroxide or trimethyltin hydroxide at a temperature ranging from about 0 to about 25° C., and then coupled with an appropriate linker moiety under basic conditions to provide intermediate (E). Treatment of intermediate (E) with oxalyl chloride in a suitable solvent such as dichloromethane at a temperature of about 0 to about 25 °C, followed by treatment with freshly prepared diazomethane in EtO (see, for example, F. Arndt, "Diazomethane," Org. Synth. 1935, 15, 3) at a temperature of about 0 °C provides compound of formula (IV).

[0166] Alternatively, intermediate (E) can be treated with 1-chloro-N,N,2-trimethylprop-1-en-1-amine in a suitable solvent in the presence of 4 Å molecular sieves in an aprotic solvent at room temperature, followed by treatment with trimethylsilyldiazomethane to give compounds of formula (IV), such synthetic methods may be used for the optimization of the synthesis of particular compounds, or may be required for the synthesis of other compounds. Examples of the use of such synthetic methods are described in the Examples section.

[0167] [ka] As shown in Scheme 1a, compounds of formula (IV) and (V), 1(wherein is as defined herein) can be prepared starting from intermediate (D') via a cross-coupling reaction in the presence of an appropriately substituted azetidine and a palladium catalyst such as Pd2(dba)3, a ligand and a base such as cesium carbonate in a solvent such as dioxane and heating at a temperature ranging from about 25 to about 100° C. Intermediate D' can be prepared from intermediate B1 as described herein in the presence of a peptide coupling reagent such as T3P (propanephosphonic anhydride) in a solvent.

[0168] (8. Method for preparing labeled proteins) As a non-limiting example, the labeled proteins described herein can be prepared as outlined in Scheme 2 below, as well as in the Examples described herein. It should be noted that one of ordinary skill in the art would know how to modify the procedures described in the exemplary schemes to arrive at a desired product.

[0169] [ka] As shown in Scheme 2, compounds of formula (I), (IB), (II), (III), (IV), (V), (VI), and (VIa) (wherein R, G, X, L A (wherein Z is as defined herein) can be prepared by contacting a tagged fusion protein as described herein with a compound as described herein, e.g., a photoactivatable fluorescent dye compound, to form a covalent bond between the tagged fusion protein and the compound, resulting in the formation of a non-fluorescent labeled protein. This non-fluorescent labeled protein is then treated with light (e.g., 405 nm light) which causes the compound to become fluorescent, thereby rendering the labeled protein fluorescent. See also FIG. 2. EXAMPLES

[0170] 9. Working Examples The following examples are given by way of illustration and not by way of limitation. Those skilled in the art can modify the procedures described in the illustrative examples to arrive at the desired products.

[0171] (Abbreviations used) [Table 1] TIFF2024528902000107.tif171170

[0172] (Intermediate)

[0173] (Intermediate A)

[0174] (Synthesis of tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate) [ka]

[0175] Step 1: Preparation of 3',6'-diacetoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid [ka]

[0176] A solution of 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (5.0 g, 13.3 mmol) in acetic anhydride (25 mL) was stirred at reflux for 3 h at 110 °C. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: 50-55% EtOAc / petroleum ether) to give 3',6'-diacetoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (3.7 g, 61% yield) as a solid. [ka]

[0177] (Step 2: Preparation of 6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl diacetate) [ka]

[0178] To a stirred solution of 3',6'-diacetoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (3.7 g, 8.0 mmol) in toluene (14 mL) was added 1,1-di-tert-butoxy-N,N-dimethylmethanamine (9.8 g, 48.2 mmol) and the mixture was stirred at reflux for 1 h. The mixture was cooled to room temperature and concentrated under reduced pressure to give 6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl diacetate (3.7 g, crude product) as a brownish solid, which was used in the next step without further purification. m / z=517.5[M+H] + .

[0179] Step 3: Preparation of tert-butyl 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate [ka]

[0180] To a stirred solution of 6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl diacetate (3.7 g, 7.2 mmol) in THF (17 mL) and methanol (17 mL) was added sodium hydroxide (1 M, 10.2 mL, 10.2 mmol). The mixture was stirred for 12 hours at room temperature and then concentrated under reduced pressure. The residue was diluted with water (50 mL) and acidified with aqueous citric acid (saturated). The aqueous layer was extracted with ethyl acetate (2 x 50 mL) and the combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 35% EtOAc / petroleum ether) to give tert-butyl 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (2.8 g) as a solid. m / z=433.6[M+H] + .

[0181] Step 4: Preparation of tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate [ka]

[0182] To a solution of tert-butyl 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (2.8 g, 6.5 mmol) in DMF (28 mL) was added N,N-diisopropylethylamine (4.5 mL, 25.9 mmol) followed by N-phenyl-O-((trifluoromethyl)sulfonyl)-N-(((trifluoromethyl)sulfonyl)oxy)hydroxylamine (5.5 g, 14.2 mmol) at 0 °C. The mixture was stirred at room temperature for 4 h. The mixture was quenched with ice water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layer was washed with ice-cold brine, then the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 22-25% EtOAc / petroleum ether) to give tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (2.0 g, 47% yield) as a solid. [ka]

[0183] (Intermediate B)

[0184] (Synthesis of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate) [ka]

[0185] (Step 1: Preparation of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate pyridinium salt) [ka]

[0186] To a stirred solution of 1,2,4-benzenetricarboxylic acid (50 g, 238 mmol) in methanesulfonic acid (250 mL) was added 3-bromophenol (86.4 g, 500 mmol). The mixture was stirred at 140 °C for 72 h. After cooling to room temperature, the dark purple solution was poured into 200 mL of ice water and the slurry was stirred vigorously. A greenish yellow solid was obtained by vacuum filtration and suction drying. The solid was recrystallized in a mixture of 750 mL of acetic anhydride and 250 mL of pyridine to give a white solid. The white solid was recrystallized three more times in a 2:1 mixture of acetic anhydride and pyridine to give 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid pyridine salt (15.0 g, 12% yield) as a powder. m / z=503.2 [M+H] + .

[0187] Step 2: Preparation of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate [ka]

[0188] To a solution of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate pyridinium salt (10.0 g, 19.9 mmol) in MeOH (330 mL) was added H2SO4 (98%, 2.1 mL, 39.8 mmol) dropwise at room temperature. The mixture was stirred at 80 °C for 72 h. The mixture was cooled to room temperature and concentrated under reduced pressure. Saturated aqueous sodium bicarbonate (50 mL) was added until the reaction was basic (pH > 7), and the aqueous solution was then extracted with 10% IPA in CHCl3 (2 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 10% EtOAc / petroleum ether) to give methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (6.0 g, 58% yield) as a solid. m / z=517.12 [M+H] + .

[0189] (Intermediate C)

[0190] (Synthesis of tert-butyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate) [ka]

[0191] To a solution of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid pyridine salt (4.0 g, 8.0 mmol, compound B1) in toluene (16 mL), 1,1-di-tert-butoxy-N,N-dimethylmethanamine (9.2 g, 47.2 mmol) was added and the mixture was stirred at reflux for 24 h. The mixture was concentrated under reduced pressure to give tert-butyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate as a brownish solid, which was used without further purification.

[0192] (Intermediate D')

[0193] (Synthesis of 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide) [ka]

[0194] To a solution of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid pyridine salt (0.5 g, 1.0 mmol, compound B1) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (0.56 g, 2.5 mmol) in DMF (5.0 mL) was added N,N'-diisopropylethylamine (0.52 mL, 3.0 mmol) at 0 °C. A solution of T3P (50% in ethyl acetate; 0.8 g, 2.5 mmol) was added dropwise at 0 °C. The mixture was allowed to warm to room temperature and stirred for 16 h. Ice-cold water (50 mL) was added and the mixture was extracted with ethyl acetate (3x). The combined organic layers were washed with saturated aqueous sodium bicarbonate (2x) at 0 °C and with brine (3x) at 0 °C. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 0-50% EtOAc / petroleum ether) to give 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (0.3 g, 42% yield) as an off-white solid.

[0195] 9.1 Example 1 (Synthesis of dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate)) [ka]

[0196] (Step 1: Preparation of dimethyl 1,1'-(6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate)) [ka]

[0197] To a solution of tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (1.0 g, 1.4 mmol, intermediate A) and methyl azetidine-3-carboxylate hydrochloride (0.55 g, 3.59 mmol) in dioxane (20 mL) was added Cs2CO3 (1.4 g, 4.3 mmol). The mixture was purged with argon gas for 15-20 min, then Pd2(dba)3 (0.13 g, 0.14 mmol) and XPhos (0.14 g, 0.29 mmol) were charged. The mixture was stirred for 16 h at 100 °C. The mixture was cooled to room temperature and filtered through a pad of Celite. The filtrate was diluted with water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 85% EtOAc / petroleum ether) to give dimethyl 1,1'-(6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate) (0.35 g, 39% yield) as a solid. m / z=627.8[M+H] + .

[0198] Step 2: Preparation of 3',6'-bis(3-(methoxycarbonyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid [ka]

[0199] To a solution of dimethyl 1,1'-(6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate) (0.25 g, 0.40 mmol) in CHCl (2 mL) was added TFA (0.2 mL) dropwise at 0 °C. The mixture was stirred for 12 h at room temperature and then concentrated under reduced pressure. The residue was triturated with n-pentane and then diethyl ether to give 3',6'-bis(3-(methoxycarbonyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (180 mg, 79% yield) as a solid. m / z=571.9[M+H] + .

[0200] (Step 3: Preparation of dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate)) [ka]

[0201] To a solution of 3',6'-bis(3-(methoxycarbonyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.18 g, 0.35 mmol) in DMF (3.6 mL) was added triethylamine (0.27 mL, 1.9 mmol) followed by N,N'-disuccinimidyl carbonate (0.18 g, 0.69 mmol) and DMAP (3.9 mg, 0.032 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h. Then, a solution of 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (0.17 g, 0.79 mmol) in DMF (0.8 mL) was added dropwise. The mixture was allowed to warm to room temperature and stirred for 12 h. The mixture was diluted with ice-cold water (20 mL) and extracted with ethyl acetate (2×20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 87% EtOAc / petroleum ether) to give dimethyl 1,1′-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-3′,6′-diyl)bis(azetidine-3-carboxylate) (0.18 g, 66% yield) as a solid. m / z=777.0[M+H] + .

[0202] (Step 4: Preparation of dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate)) [ka]

[0203] To a solution of dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate) (0.15 g, 0.20 mmol) in CHCl (6 mL) was added oxalyl chloride (2 M in CHCl, 0.18 mL, 1.94 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred for 30 min, then concentrated under reduced pressure. The residue was dissolved in dry CHCl (20 mL) and a freshly prepared solution of diazomethane in EtO (see, for example, F. Arndt, "Diazomethane," Org. Synth. 1935, 15, 3) (ca. 0.5 M, 5 mmol) was added at 0° C. and stirred for 30 min. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column purification (eluent: 22% EtOAc / petroleum ether). The residue was further purified by chiral SFC (Chiralcel-OJ-3; mobile phase, 30% MeOH / CO2) to give dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate) (0.03 g, 19% yield) as a solid. [ka]

[0204] 9.2 Example 2 (Synthesis of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid)) [ka]

[0205] To a solution of dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate) (18 mg, 0.022 mmol, Example 1) in MeOH (0.2 mL) and THF (0.1 mL) was added a solution of LiOH (5 mg, 0.11 mmol) in water (0.1 mL) at 0° C. The mixture was stirred at room temperature for 24 hours. The mixture was concentrated under reduced pressure and the residue was then purified using chiral SFC (YMC-PAK DIOL; mobile phase, 30% MeOH / CO2) to give 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid) (11 mg, 63%) as a solid. [ka]

[0206] 9.3 Example 3 (Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0207] Step 1: Preparation of methyl 3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate [ka]

[0208] To a solution of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.50 g, 0.96 mmol, intermediate B), (R)-2-(methoxymethyl)azetidine (0.24 g, 2.42 mmol) in 1,4-dioxane (10 mL) was added cesium carbonate (0.95 g, 2.9 mmol). The mixture was purged with argon gas for 15-20 min, then Pd2(dba)3 (0.09 g, 0.097 mmol) and RuPhos Pd G4 (0.14 g, 0.29 mmol) were added. The mixture was stirred for 2 h at 110 °C. The mixture was filtered through Celite, then the filtrate was diluted with water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 85% EtOAc / petroleum ether) to give methyl 3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.17 g, 31% yield) as a solid. m / z=557.52[M+H] + .

[0209] Step 2: Preparation of 3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid [ka]

[0210] To a solution of methyl 3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.17 g, 0.30 mmol) in MeOH (1.0 mL) and THF (0.5 mL) was added a solution of LiOH (0.06 g, 1.53 mmol) in water (0.5 mL) at 0 °C. The mixture was stirred at room temperature for 24 h. The mixture was concentrated to half its volume under reduced pressure, then diluted with water (5 mL) and extracted with EtOAc (10 mL). The aqueous layer was acidified with 1N HCl and extracted with 10% MeOH in CH2Cl2 (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative HPLC (C18; mobile phase, 20% ACN / water (0.05% formic acid)) to give 3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.12 g, 72% yield) as a solid. m / z=543.45[M+H] + .

[0211] Step 3: Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide [ka]

[0212] To a stirred solution of 3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.12 g, 0.22 mmol) in DMF (1.2 mL) at 0 °C was added a solution of N,N'-diisopropylethylamine (0.12 mL, 0.66 mmol) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (0.099 g, 0.442 mmol) in DMF (0.1 mL) at 0 °C. The mixture was stirred at 0 °C for 10 min, then propylphosphonic anhydride (50% solution in ethyl acetate; 0.18 g, 0.550 mmol) was added dropwise. The mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was diluted with ice-cold water (20 mL) and extracted with ethyl acetate (2×20 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 6% MeOH / CHCl) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (110 mg, 66% yield) as a pink solid. m / z=749.14[M+H] + .

[0213] (Step 4: Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0214] To a solution of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (0.09 g, 0.12 mmol) in CHCl (5 mL) was added oxalyl chloride (0.10 mL, 1.20 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 30 min. The mixture was concentrated with a stream of nitrogen gas. The residue was dissolved in dry CHCl (20 mL) and a freshly prepared solution of diazomethane in EtO (see, for example, F. Arndt, "Diazomethane," Org. Synth. 1935, 15, 3) (ca. 0.5 M, 3 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 30 min and then concentrated under reduced pressure. The residue was purified by flash column purification using neutral alumina (eluent: 0-10% acetone / petroleum ether). The residue was further purified by chiral SFC (YMC PAK-DIOL; mobile phase, 25% MeOH / CO2) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide (0.0032 g, 3%) as a solid. [ka]

[0215] 9.4 Example 4 (Synthesis of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide)) [ka]

[0216] Step 1: Preparation of methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate [ka]

[0217] In a microwave vessel, a mixture of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (1.0 g, 1.94 mmol, intermediate B), N,N-dimethylazetidine-3-carboxamide hydrochloride (0.62 g, 4.84 mmol), and cesium carbonate (1.90 g, 5.810 mmol) was suspended in anhydrous 1,4-dioxane (10.0 mL). The vial was degassed with nitrogen for 15 min, and then Pd2(dba)3 (0.18 g, 0.19 mmol) and RuPhos Pd G4 (0.27 g, 0.50 mmol) were added. The mixture was stirred at 110 °C for 2 h. The mixture was filtered through Celite and washed with 10% MeOH in CHCl2 (100 mL). The filtrate was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: 0-20% MeOH / CH2Cl2) to give methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.28 g, 23% yield) as a solid. m / z=612.0[M+H] + .

[0218] Step 2: Preparation of 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid [ka]

[0219] To a solution of methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.28 g, 0.46 mmol) in THF (2.8 mL) was added trimethyltin hydroxide (0.5 g, 2.75 mmol) at 0 °C. The mixture was warmed to room temperature and stirred for 2 h. Water (2 mL) was added, then the mixture was cooled to 0 °C and acidified with 1N HCl to pH 5. The aqueous layer was extracted with 10% isopropanol in chloroform (3 x 30 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative HPLC (C18; mobile phase, 0–100% ACN / water (0.05% formic acid) to give 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.23 g, 84% yield) as a solid. m / z=597.41 [M+H] + .

[0220] (Step 3: Preparation of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide)) [ka]

[0221] To a solution of 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.23 g, 0.39 mmol) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (0.17 g, 0.77 mmol) in DMF (2.3 mL) was added diisopropylethylamine (0.2 mL, 1.15 mmol) at 0 °C. The mixture was stirred at 0 °C for 10 min, then a solution of propylphosphonic anhydride (50% in ethyl acetate; 0.3 g, 0.96 mmol) was added dropwise. The mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was diluted with ice-cold water (10 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with brine (50 mL) and ice-cold water (30 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 0-8% MeOH / CHCl) to give 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide) (0.20 g, 64% yield) as a solid. m / z=802.73[M+H] + .

[0222] (Step 4: Preparation of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide)) [ka]

[0223] To a solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide) (0.07 g, 0.087 mmol) in CHCl (4 mL) was added oxalyl chloride (0.07 mL, 0.872 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred for 30 min. The mixture was concentrated under reduced pressure. The residue was dissolved in dry CHCl (20 mL) and a freshly prepared solution of diazomethane in EtO (see, for example, F. Arndt, "Diazomethane," Org. Synth. 1935, 15, 3) (ca. 0.5 M, 2.2 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 30 min. The mixture was concentrated under reduced pressure and the residue was purified by flash column purification over neutral alumina (eluent: 0-5% MeOH / CHCl). The residue was further purified by SFC (DCPAK P4VP; mobile phase, 45% (0.2% DEA in ACN) / CO2) to give 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide) (0.0052 g, 7% yield) as a solid. [ka]

[0224] 9.5 Example 5 (Synthesis of 1,1'-(6-((4-(((2-amino-7H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide)) [ka]

[0225] Step 1: Preparation of 2-diazo-3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid [ka]

[0226] The title compound was prepared similarly to steps 1-3 of Example 7, using N,N-dimethylazetidine-3-carboxamide hydrochloride instead of azetidin-3-ol hydrochloride in step 1. The residue was purified by trituration with diethyl ether to give 2-diazo-3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid as a brown solid.

[0227] (Step 2: Preparation of 1,1'-(6-((4-(((2-amino-7H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3-carboxamide)) [ka]

[0228] To a stirred solution of 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.05 g, 0.081 mmol, compound 4.2) and 6-((4-(aminomethyl)benzyl)oxy)-7H-purin-2-amine (0.05 g, 0.161 mmol) in DMF (1 mL) was added diisopropylethylamine (0.06 g, 0.403 mmol) at 0 °C. After 10 min, a solution of propylphosphonic anhydride (50% in ethyl acetate; 0.22 g, 0.32 mmol) was added dropwise at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 3 h. The mixture was concentrated and the residue was purified by preparative HPLC (column: XBridge C8; mobile phase, 0-100% ACN / water) to afford the title compound (0.008 g, 12%) as a light brown solid. [ka]

[0229] 9.6 Example 6 (Synthesis of dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)(2R,2'R)-bis(azetidine-2-carboxylate)) [ka]

[0230] The title compound was prepared by a procedure similar to steps 1-4 of Example 1, except that methyl (R)-azetidine-2-carboxylate hydrochloride was used instead of methyl azetidine-3-carboxylate hydrochloride in step 1, and intermediate C was used instead of intermediate A in step 1. The residue was purified by preparative HPLC (column: XBridge C18; mobile phase: 0-100% ACN / water) to give the title compound (0.0032 g, 16% yield) as a pale yellow solid. [ka]

[0231] 9.7 Example 7 (Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0232] Step 1: Preparation of methyl 3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate [ka]

[0233] To a stirred solution of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (Intermediate B, 2.0 g, 3.88 mmol) and azetidin-3-ol hydrochloride (1.06 g, 9.69 mmol) in 1,4-dioxane (40 mL) was added cesium carbonate (6.31 g, 19.38 mmol) at room temperature. The mixture was purged with argon for 20 min, then Pd2(dba)3 (0.35 g, 0.38 mmol) and Xphos (0.55 g, 1.16 mmol) were added. The mixture was purged with argon for 10 min and heated to 120 °C for 16 h. The mixture was cooled to room temperature and filtered through Celite. The Celite was washed with MeOH and the solution was concentrated under reduced pressure. The residue was purified by reverse phase column chromatography (column C-18; eluent: 0-20% ACN / water (0.1% formic acid)) to give methyl 3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.90 g, 47% yield)) as a violet solid.

[0234] (Step 2: Preparation of methyl 2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate) [ka]

[0235] To a stirred solution of methyl 3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.2 g, 0.4 mmol) in DCM (20 mL) was added dropwise at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 60 min. The mixture was concentrated under reduced pressure. The residue was dissolved in dry DCM (100 mL) and a freshly prepared solution of diazomethane in Et2O (ca. 0.5 M, 100 mmol) was added dropwise at 0 °C. The mixture was allowed to stir at 0 °C for 15 min and then allowed to warm to room temperature. The mixture was allowed to stir at room temperature for 15 min. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 50-100% ethyl acetate / petroleum ether). The residue was further purified by SFC (column: YMC PACK DIOL-120; mobile phase, 20% MeOH / CO2) to give methyl 2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate (0.055 g, 7% yield) as a light brown solid.

[0236] Step 3: Preparation of 2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid [ka]

[0237] To a stirred solution of methyl 2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate (0.05 g, 0.095 mmol) in THF (1.0 mL), MeOH (0.5 mL) and water (0.5 mL) was added lithium hydroxide monohydrate (8 mg, 0.19 mmol) at 0 °C. The mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the residue was triturated with EtO to give 2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid (0.048 g, 98% yield) as a brown solid.

[0238] (Step 4: Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0239] To a stirred solution of 2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid (0.048 g, 0.094 mmol) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (0.053 g, 0.24 mmol) in DMF (1 mL) was added N,N'-diisopropylethylamine (0.05 g, 0.38 mmol) at 0° C. The mixture was stirred at 0° C. for 10 min, then propanephosphonic anhydride (50% solution in ethyl acetate; 0.18 g, 0.282 mmol) was added dropwise at 0° C. The mixture was allowed to warm to room temperature and stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure and purified by preparative HPLC (column: X-SELECT-C18; mobile phase, 0–100% ACN / water) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3′,6′-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9′-xanthene]-6-carboxamide (5 mg, 7% yield) as a light brown solid. [ka]

[0240] 9.8 Example 8 (Synthesis of 3',6'-di(azetidin-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-N-methyl-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0241] Step 1: Preparation of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate [ka]

[0242] To a stirred solution of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (5.0 g, 24.4 mmol) in THF (35 mL) and DMF (18 mL) at 0 °C was added NaH (1.17 g, 60% in mineral oil, 29.2 mmol). The mixture was stirred at 0 °C for 30 min, then 6-chloro-1-iodohexane (8.4 g, 34.1 mmol) was added. The mixture was warmed to room temperature and stirred at room temperature for 30 min. Saturated NH4Cl solution was added and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water and brine. The mixture was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 30% ethyl acetate / petroleum ether) to give tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (2.6 g, 33% yield) as a colorless oil.

[0243] Step 2: Preparation of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)carbamate [ka]

[0244] To a stirred solution of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (3.0 g, 9.3 mmol) and methyl iodide (2.3 mL, 46.3 mmol) in DMF (50 mL) at 0 °C was added sodium hydride (60% dispersion in mineral oil; 0.93 g, 23.2 mmol). The mixture was allowed to warm to room temperature and stirred at room temperature for 3 h. Saturated aqueous NH4Cl (50 mL) was added and the mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 5-10% ethyl acetate / petroleum ether) to give tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)carbamate (2.3 g, 45% yield) as a pale yellow oil.

[0245] Step 3: Preparation of 2-(2-((6-chlorohexyl)oxy)ethoxy)-N-methylethan-1-amine trifluoroacetate [ka]

[0246] To a stirred solution of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)carbamate (2.2 g, 6.5 mmol) in DCM (22 mL) at 0 °C was added trifluoroacetic acid (1.0 mL, 13.0 mmol). The mixture was allowed to warm to room temperature and stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure. The residue was triturated with EtO to give the title compound as a slightly yellow oil.

[0247] Step 4: Preparation of 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid [ka]

[0248] The title compound was prepared similarly to Example 7, using azetidine hydrochloride instead of azetidin-3-ol hydrochloride in step 1. The residue was purified by silica gel column chromatography (eluent: 0-8% MeOH / DCM). The residue was further purified by preparative HPLC (column: C18 RP; mobile phase, 0-100% ACN / water) to give 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid as a solid.

[0249] Step 5: Preparation of 3',6'-di(azetidin-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-N-methyl-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide [ka]

[0250] To a stirred solution of 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid (0.06 g, 0.12 mmol) and 2-(2-((6-chlorohexyl)oxy)ethoxy)-N-methylethan-1-amine trifluoroacetate (0.088 g, 0.25 mmol) in DMF (1 mL) was added N,N'-diisopropylethylamine (0.1 mL, 0.62 mmol) at 0° C. The mixture was stirred at 0° C. for 10 min, then propanephosphonic anhydride (T3P) (50% solution in ethyl acetate; 0.22 g, 0.38 mmol) was added dropwise at 0° C. The mixture was allowed to warm to room temperature and stirred at room temperature for 4 h. The mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC (column: XBridge C18; mobile phase, 0-100% water / ACN) to give 3',6'-di(azetidin-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-N-methyl-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide (0.02 g, 22% yield) as a light brown solid. [ka]

[0251] 9.9 Example 9 (Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-(morpholine-4-carbonyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0252] (Step 1: Preparation of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid)) [ka]

[0253] To a solution of dimethyl 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylate) (0.35 g, 0.45 mmol, compound 1.3) in MeOH (0.35 mL) and THF (0.7 mL) at 0 °C was added a solution of lithium hydroxide monohydrate (0.021 g, 0.50 mmol) in water (0.35 mL). The mixture was stirred at room temperature for 2 h. The mixture was concentrated to give the desired product as a pink solid, which was used without further purification.

[0254] Step 2: Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-(morpholine-4-carbonyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide [ka]

[0255] To a solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid) (0.25 g, 0.33 mmol) in DMF (5 mL) was added N,N-diisopropylethylamine (0.6 mL, 3.3 mmol) followed by T3P (50% in ethyl acetate, 0.64 g, 1.0 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h, then a solution of morpholine (0.145 g, 1.666 mmol) in DMF (1 mL) was added dropwise. The mixture was allowed to warm to room temperature and stirred at room temperature for 12 h. The mixture was diluted with ice-cold water (20 mL) and extracted with ethyl acetate (2x). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: C18; mobile phase, 32% ACN / water) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-(morpholine-4-carbonyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (0.18 g, 61% yield) as a solid.

[0256] (Step 3: Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-(morpholine-4-carbonyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0257] To a solution of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-(morpholine-4-carbonyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (0.18 g, 0.20 mmol) in DCM (3.6 mL) was added oxalyl chloride (1.015 mL, 2.0 mmol, 2M in DCM) at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 1 h. The mixture was then concentrated under reduced pressure. The residue was dissolved in DCM (20 mL) and a freshly prepared solution of diazomethane in Et2O (ca. 0.5 M, 50 mmol) was added at 0 °C under nitrogen. The mixture was stirred at 0 °C for 30 min and then concentrated. The residue was purified by silica gel column chromatography (eluent: 85% EtOAc / petroleum ether). The residue was further purified by preparative HPLC (column: XBridge C18; mobile phase, 0-100% ACN / water) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-(morpholine-4-carbonyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide (0.014 g, 7.6% yield) as a solid. [ka]

[0258] 9.10 Example 10 (Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3-oxo-3',6'-bis(3-(pyrrolidine-1-carbonyl)azetidin-1-yl)-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0259] To a stirred solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid) (0.03 g, 0.039 mmol, compound 2) in DMF (0.6 mL) was added N,N-diisopropylethylamine (0.07 mL, 0.39 mmol) at 0 °C. Then, a solution of T3P (50% in ethyl acetate, 0.058 g, 0.117 mmol) was added at 0 °C, and the mixture was stirred at 0 °C for 1 h. Then, a solution of pyrrolidine (0.011 g, 0.16 mmol) in DMF (0.1 mL) was added. The mixture was allowed to warm to room temperature and stirred at room temperature for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Sunfire C18; mobile phase A, 0-100% ACN / water) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3-oxo-3',6'-bis(3-(pyrrolidine-1-carbonyl)azetidin-1-yl)-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide (0.004 g, 11.72% yield) as a pale yellow solid. [ka]

[0260] (9.11 Example 11) (Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-(4-methylpiperazine-1-carbonyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0261] To a solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid) (0.045 g, 0.058 mmol, compound 2) in DMF (0.9 mL) was added N,N-diisopropylethylamine (0.065 mL, 0.35 mmol) followed by T3P (50% solution in ethyl acetate, 0.074 g, 0.117 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h. Then, a solution of N-methylpiperazine (0.015 g, 0.146 mmol) in DMF (0.1 mL) was added dropwise. The mixture was allowed to warm to room temperature and stirred at room temperature for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: X-SELECT C18; mobile phase, 0-100% acetonitrile / 10 mM aqueous ammonium acetate) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-(4-methylpiperazine-1-carbonyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide (5.2 mg, 9.5% yield) as a solid. [ka]

[0262] 9.12 Example 12 (Synthesis of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N-methylazetidine-3-carboxamide)) [ka]

[0263] The title compound was prepared by a procedure similar to that of Example 11, using methylamine (2 M in THF) instead of N-methylpiperazine. The residue was purified by preparative HPLC (column: X-SELECT-C18; mobile phase, 0-100% ACN / water) to give the title compound. [ka]

[0264] 9.13 Example 13 (Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-(dimethylamino)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0265] The title compound was prepared in a similar manner to Example 7, using 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide, intermediate D', and N,N-dimethylazetidin-3-amine hydrochloride instead of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate, intermediate B, and azetidin-3-ol hydrochloride in step 1. The residue was purified by silica gel column chromatography (eluent: 50-100% ethyl acetate / petroleum ether). The residue was further purified by SFC to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-(dimethylamino)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide as a pale yellow solid. [ka]

[0266] 9.14 Example 14 (Synthesis of (2S,2'S)-1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-2-carboxamide)) [ka]

[0267] The title compound was prepared in a similar manner to Example 7, using 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide, intermediate D', and (S)-N,N-dimethylazetidine-2-carboxamide hydrochloride instead of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate, intermediate B, and azetidin-3-ol hydrochloride in step 1. The residue was purified by silica gel column chromatography (eluent: 90% ethyl acetate / petroleum ether). The residue was further purified by preparative HPLC (column: XBridge C18; mobile phase, 0-100% ACN / water) to give (2S,2'S)-1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-2-carboxamide) as an off-white solid. [ka]

[0268] 9.15 Example 15 (Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2-(hydroxymethyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0269] The title compound was prepared in a similar manner to Example 7 using 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide, intermediate D', and (R)-azetidin-2-ylmethanol hydrochloride instead of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate, intermediate B, and azetidin-3-ol hydrochloride. The residue was purified by silica gel column chromatography (eluent: 90% ethyl acetate / petroleum ether). The residue was further purified by preparative HPLC (column: XBridge C18; mobile phase, 0-100% ACN / water) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2-(hydroxymethyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide as a pale yellow solid. [ka]

[0270] 9.16 Example 16 (Synthesis of (3S,3'S)-1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylpyrrolidine-3-carboxamide)) [ka]

[0271] The title compound was prepared in a similar manner to Example 7 using 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide, intermediate D', and (S)-N,N-dimethylpyrrolidine-3-carboxamide instead of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate, intermediate B, and azetidin-3-ol hydrochloride. The residue was purified by silica gel column chromatography (eluent: 50-100% ethyl acetate / petroleum ether). The residue was further purified by preparative HPLC (column: XBridge C18; mobile phase, 0-100% ACN / water) to give (3S,3'S)-1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylpyrrolidine-3-carboxamide) as a solid. [ka]

[0272] (9.17 Example 17) (Synthesis of 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0273] Step 1: Preparation of methyl 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate [ka]

[0274] To a stirred solution of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (Intermediate B) (0.5 g, 0.969 mmol) and 3-oxa-6-azabicyclo[3.1.1]heptane 4-methylbenzene-1-sulfonate (0.657 g, 2.422 mmol) in 1,4-dioxane (10 mL) in a dry sealed tube, cesium carbonate (1.578 g, 4.844 mmol) was added at room temperature and the reaction mixture was purged with argon gas for 20 minutes. XPhos-Pd-G4 (0.083 g, 0.097 mmol) was then added at room temperature. The reaction mixture was again purged with argon gas for 10 minutes and stirred at 100 °C for 12 hours. The reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was filtered through Celite and washed with ethyl acetate. The filtrate was diluted with water (30 mL) and extracted with ethyl acetate (30 mL×2), the combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel using 0-100% ethyl acetate / petroleum ether and the desired product was eluted with 90-95% ethyl acetate / petroleum ether to give methyl 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.35 g, 65% yield) as a pink solid. m / z=553.51 (M+H + ).

[0275] Step 2: Preparation of 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid [ka] To a stirred solution of methyl 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (compound 17.1) (0.35 g, 0.633 mmol) in THF (8.0 mL) and MeOH:H2O (1:1) (8.0 mL) was added lithium hydroxide monohydrate (0.053 g, 1.266 mmol) in portions at 0 °C. The reaction mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was concentrated under reduced pressure, diluted with water (2 mL), and then acidified using 1N HCl (pH approx. 2). The formed precipitate was filtered and dried under high vacuum. The dried solid was washed with diethyl ether to give 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.25 g, 70% yield) as a pink solid. m / z=539.36[M+H] + .

[0276] Step 3: Preparation of 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide [ka]

[0277] To a stirred solution of 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (compound 17.2) (0.250 g, 0.464 mmol) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (0.259 g, 1.160 mmol) in DMF (5 mL) was added N,N'-diisopropylethylamine (0.4 mL, 2.321 mmol) at 0 °C. After 10 min, propanephosphonic anhydride (T3P) (50% solution in ethyl acetate; 0.886 g, 1.392 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was directly evaporated to remove the DMF solvent, and then purified by reverse phase column purification using a C18 column, 0-100% acetonitrile / water, and the desired product was eluted with 22% acetonitrile / water to give 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (0.097 g, 28% yield) as a pink solid.

[0278] (Step 4: Preparation of 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0279] Method A. To a solution of 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (compound 17.3) (29 mg, 0.039 mmol) in dry CHCl (1 mL) was added 4 Å powdered molecular sieves (15 mg) under argon. 1-Chloro-N,N,2-trimethylprop-1-en-1-amine (20.6 μL, 0.156 mmol) was then added via syringe and the mixture was stirred at room temperature for 45 min. N,N-Diisopropylethylamine (27 μL, 0.156 mmol) was added, followed by TMSCHN2 (2 M in hexanes, 78 μL, 0.156 mmol). After stirring for 2.5 h at room temperature, the mixture was filtered and concentrated. The residue was purified by reversed-phase flash chromatography (0–100% ACN / water). The residue was purified a second time by reversed-phase flash chromatography (30–100% ACN / water). A third purification was performed using a pipette column packed with basic alumina and eluted with 50–100% EtOAc / toluene. The product-containing fractions were concentrated, redissolved in dioxane, and dried on a lyophilizer to give a solid (1.7 mg, 6%). [ka]

[0280] Method B. To a solution of 3',6'-di(3-oxa-6-azabicyclo[3.1.1]heptan-6-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (compound 17.3) (29 mg, 0.039 mmol) in dry CHCl (1 mL) was added 4 Å powdered molecular sieves (15 mg) under argon. 1-Chloro-N,N,2-trimethylprop-1-en-1-amine (20.6 μL, 0.156 mmol) was then added via syringe and the mixture was stirred at room temperature for 45 min. N,N-Diisopropylethylamine (27 μL, 0.156 mmol) was added followed by TMSCHN2 (2M in hexanes, 78 μL, 0.156 mmol). After stirring for 2.5 h at room temperature, the mixture was filtered and concentrated. The residue was purified by reverse phase flash chromatography (0-100% ACN / water). The residue was further purified by reverse phase flash chromatography (30-100% ACN / water). The residue was further purified by flash chromatography using basic alumina (eluent: 50-100% EtOAc / toluene) to give the title compound as a solid (1.7 mg, 6%). [ka]

[0281] 9.18 Example 18 (Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide) [ka]

[0282] Step 1: Preparation of methyl 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate [ka]

[0283] To a stirred solution of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (Intermediate B) (0.5 g, 0.969 mmol) and 3-methyl-3,6-diaza-bicyclo[3.1.1]heptane dihydrochloride (0.447 g, 2.422 mmol) in 1,4-dioxane (10 mL) in a dry sealed tube, Cs2CO3 (3.156 g, 9.687 mmol) was added at room temperature and the mixture was purged with argon gas for 20 min. Then, Pd2(dba)3 (0.089 g, 0.097 mmol) and RuPhos (0.135 g, 0.291 mmol) were added at room temperature. The mixture was again purged with argon gas for 10 min and stirred at 110 °C for 16 h. The reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was filtered through Celite, washed with methanol (100 mL) and concentrated. The residue was purified by silica gel chromatography eluting with (1% MeOH in DCM to 5% ammonia in DCM) to give methyl 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.20 g, 35.68% yield) as a pink solid. m / z=579.58[M+H] + .

[0284] (Step 2: Preparation of 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid) [ka]

[0285] To a stirred solution of methyl 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (compound 18.1) (0.2 g, 0.346 mmol) in THF (2.0 mL) and MeOH:H2O (1:1) (2.0 mL) was added lithium hydroxide monohydrate (0.072 g, 1.728 mmol) in small portions at 0 °C. The mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC and LCMS. After completion of the reaction, the organic solvent was removed under reduced pressure and the remaining crude product material was diluted with water (2 mL) and the mixture was acidified to pH approx. 2 using concentrated hydrochloric acid. The precipitated solid was then filtered and dried under vacuum. The residue was purified by C18 reverse phase column chromatography (eluent: ACN / water (0.05% formic acid)) to give 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.14 g, 72% yield) as a pink solid. m / z=565.43 (M+H + ).

[0286] (Step 3: Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide) [ka]

[0287] To a stirred solution of 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (compound 18.2) (0.14 g, 0.248 mmol) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (0.139 g, 0.620 mmol) in DMF (2 mL) was added N,N'-diisopropylethylamine (0.216 mL, 1.240 mmol) at 0 °C. After 10 min, propanephosphonic anhydride (T3P) (50% solution in ethyl acetate; 0.237 g, 0.744 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was directly evaporated to remove the DMF solvent, and then purified by reverse phase column purification using a C18 column, 0-100% acetonitrile / water, and the desired product was eluted with 15% acetonitrile / water to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (0.110 g, 56% yield) as a pink solid.

[0288] [ka] Method A. To a solution of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (compound 18.3) (10 mg, 0.013 mmol) in dry CHCl (350 μL) was added 4 Å powdered molecular sieves (5 mg) under argon. 1-Chloro-N,N,2-trimethylprop-1-en-1-amine (6.9 μL, 0.052 mmol) was then added via syringe and the mixture was stirred at room temperature for 45 min. N,N-Diisopropylethylamine (9 μL, 0.052 mmol) was added followed by TMSCHN2 (2M in hexanes, 26 μL, 0.052 mmol). After stirring at room temperature for 2.5 h, the mixture was filtered and concentrated. The residue was purified by reverse-phase flash chromatography (0-100% ACN / water). The residue was purified a second time using a pipette column packed with basic alumina and eluted with 10% MeOH / CH2Cl2. The product-containing fractions were concentrated, redissolved in dioxane, and dried on a lyophilizer to give a solid (1.4 mg, 13%). [ka]

[0289] Method B. To a solution of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]heptan-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (compound 18.3) (10 mg, 0.013 mmol) in dry CHCl (350 μL) was added 4 Å powdered molecular sieves (5 mg) under argon. 1-Chloro-N,N,2-trimethylprop-1-en-1-amine (6.9 μL, 0.052 mmol) was then added via syringe and the mixture was stirred at room temperature for 45 min. N,N-Diisopropylethylamine (9 μL, 0.052 mmol) was added followed by TMSCHN2 (2M in hexanes, 26 μL, 0.052 mmol). After stirring for 2.5 h at room temperature, the mixture was filtered and concentrated. The residue was purified by reverse phase flash chromatography (0-100% ACN / water). The residue was further purified by flash chromatography using basic alumina (eluent: 10% MeOH / CH2Cl2) to give the title compound as a solid (1.4 mg, 13%). [ka]

[0290] 9.19 Example 19 (Synthesis of 1,1'-(6'-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-3,7-diyl)bis(N,N-dimethylazetidine-3-carboxamide)) [ka]

[0291] (Step 1: Preparation of tert-butyl 5,5-dimethyl-3'-oxo-3,7-bis((triisopropylsilyl)oxy)-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylate) [ka]

[0292] A solution of 1,4-di-tert-butyl 2-bromobenzene-1,4-dicarboxylate (1.53 g, 4.29 mmol, WO2018046753 A1) in 2:1 anhydrous THF / pentane (15 mL) was cooled to -100 °C in a diethyl ether / liquid nitrogen bath. The solution was sparged with Ar (gas) for 10 min, after which n-BuLi (2.5 M in hexanes, 1.75 mL, 1.72 mmol) was added dropwise down the flask wall. The solution was allowed to stir at -100 °C for 10 min, at which point the solution turned purple and then brown. A solution of 5,5-dimethyl-3,7-bis((triisopropylsilyl)oxy)dibenzo[b,e]silylin-10(5H)-one (1.0 g, 1.72 mmol, WO2018046753 A1) in THF (5 mL) was added dropwise down the flask wall and stirred for 10 min at -78 °C in a dry ice / acetone bath. The mixture was then warmed to room temperature and stirred for 2.5 h. The mixture was adsorbed onto Celite and purified by flash chromatography (25-50% CH2Cl2 / hexanes) using a silica gel column to give the title compound as a crystalline solid (436 mg, 32%). [ka]

[0293] (Step 2: Preparation of tert-butyl 3,7-dihydroxy-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylate) [ka]

[0294] tert-Butyl 5,5-dimethyl-3'-oxo-3,7-bis((triisopropylsilyl)oxy)-3'H,5H-spiro[dibenzo[b,e]silyline-10,1'-isobenzofuran]-6'-carboxylate (216 mg, 0.274 mmol) was dissolved in THF (2.16 mL) and cooled to 0 °C. To the solution was added TBAF (1 M in THF, 1.1 mL, 0.274 mmol). The color of the solution changed to dark purple. After 30 min, 1 / 2 saturated NH4Cl was added. The color of the solution became light orange. The mixture was extracted with ethyl acetate (3 x 50 mL) and the combined organic layers were pre-adsorbed onto silica gel and purified by flash chromatography (eluent: 0-10% ethyl acetate / DCM) to give the title compound as a translucent film (126 mg, 97%). m / z=475.1[M+2H] + .

[0295] (Step 3: Preparation of tert-butyl 5,5-dimethyl-3'-oxo-3,7-bis(((trifluoromethyl)sulfonyl)oxy)-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylate) [ka]

[0296] tert-Butyl 3,7-dihydroxy-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylate (263 mg, 0.554 mmol) and N-phenylbistriflamide (792 mg, 2.22 mmol) were suspended in THF (12 mL). N,N-diisopropylethylamine (579 μL, 3.33 mmol) was slowly added to the mixture. The resulting mixture was sealed and heated to 60 °C overnight. The mixture was cooled to room temperature, adsorbed onto Celite, and purified by silica gel flash chromatography (eluent: 0-8% MeOH / DCM) to give the title compound as a white foam (341 mg, 83%). m / z=739.1 [M+H] + .

[0297] (Step 4: Preparation of tert-butyl 3,7-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylate) [ka]

[0298] An oven-dried one-dram vial was charged with N,N-dimethylazetidine-3-carboxamide hydrochloride (61.9 mg, 0.308 mmol), Cs2CO3, and dioxane (1.23 mL). Tert-butyl 5,5-dimethyl-3'-oxo-3,7-bis(((trifluoromethyl)sulfonyl)oxy)-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylate (91 mg, 0.123 mmol) and XPhos (17.6 mg, 0.037 mmol) were added. The mixture was sparged with Ar for 5 min. Pd2dba3 (11.3 mg, 0.012 mmol) was then added. The vial was sealed and heated to 100 °C overnight. The mixture was cooled to room temperature, diluted with MeOH, adsorbed onto silica gel, and purified by flash chromatography (eluent: 2-20% MeOH / CH2Cl2) to give the title compound as an orange-brown solid (81 mg, 95%). m / z=695.4 [M+H] + .

[0299] (Step 5: Preparation of 3,7-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylic acid) [ka]

[0300] A solution of tert-butyl 3,7-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylate (70 mg, 0.101 mmol) in CHCl (3.73 mL) was cooled to 0° C. Then, trifluoroacetic acid (0.750 mL) was added dropwise. The color of the solution changed from yellow to green to dark red. The mixture was allowed to warm to room temperature and continued to stir overnight. The dark green mixture was then concentrated to give the title compound, which was used directly in the next step without further purification. m / z=639.3 [M+H] + .

[0301] (Step 6: Preparation of 1,1'-(6'-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-3,7-diyl)bis(N,N-dimethylazetidine-3-carboxamide)) [ka]

[0302] To a solution of 3,7-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylic acid (16 mg, 0.025 mmol) in DMF (0.764 mL) was added TBTU (9.65 mg, 0.030 mmol), HOBT (5.75 mg, 0.030 mmol), and N,N-diisopropylethylamine (8.73 μL, 0.050 mmol). The solution changed from dark blue to light green to light brown. 1-[2-(2-aminoethoxy)ethoxy]-6-chlorohexane (7.82 mg, 0.030 mmol) was then added and the mixture was left stirring at room temperature overnight. The mixture was concentrated and the resulting blue-green film was diluted with DMSO and filtered. The mixture was purified by reverse phase preparative HPLC (10-100% ACN / water (0.05% formic acid)) to give the title compound as a light green solid (4 mg, 19%). m / z=844.4[M+H] + .

[0303] (Step 7: Preparation of 1,1'-(6'-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2'-diazo-5,5-dimethyl-3'-oxo-2',3'-dihydro-5H-spiro[dibenzo[b,e]silyne-10,1'-indene]-3,7-diyl)bis(N,N-dimethylazetidine-3-carboxamide)) [ka]

[0304] To a solution of 1,1'-(6'-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-3,7-diyl)bis(N,N-dimethylazetidine-3-carboxamide) (7 mg, 8.29 μmol) in 1:1 CHCl / ACN (2 mL) in an oven-dried vial was added 1-chloro-N,N,2-trimethylprop-1-en-1-amine (4.39 μL, 33.2 μmol). The color of the solution changed to deep blue. The mixture was stirred for 5 min, then TMSCHN2 (2M in Et2O, 16.6 μL, 33.2 μmol) and N,N-diisopropylethylamine (5.78 μL, 33.2 μmol) were added dropwise. The mixture was stirred for 30 min at room temperature. The mixture was concentrated. The residue was purified by reverse phase preparative HPLC (10-100% can / water (0.1% formic acid)) to give the title compound as a solid (0.5 mg, 7%). m / z=868.4[M+H] + .

[0305] 9.20 Example 20 Synthesis of (1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxamide) [ka]

[0306] To a stirred solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid) (0.040 g, 0.052 mmol, Example 2) in DMF (1 mL) was added N,N'-diisopropylethylamine (0.054 mL, 0.311 mmol) followed by T3P (50% solution in ethyl acetate, 0.132 g, 0.207 mmol) at 0° C. The mixture was stirred at 0° C. for 1 h and then ammonium bicarbonate (0.041 g, 0.518 mmol) was added. The mixture was allowed to warm to room temperature and stirred at room temperature for 12 h, then ice-cold water (10 mL) was added and the mixture was extracted with ethyl acetate (10 mL×2). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative HPLC (C18; mobile phase, 0-100% ACN / water) to give the title compound (0.0052 g, 13% yield) as a pale yellow solid. [ka]

[0307] 9.21 Example 21 (Synthesis of 3',6'-di(azetidin-1-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) [ka]

[0308] (Step 1: Preparation of 2-((6-chlorohexyl)oxy)ethan-1-ol) [ka]

[0309] To a solution of ethane-1,2-diol (5 g, 80.5 mmol) in DMF (50 mL) under nitrogen at 0° C. was added NaH (60% in mineral oil) (3.86 g, 161.10 mmol) followed by 1-chloro-6-iodohexane (19.85 g, 80.55 mmol). The mixture was stirred at room temperature for 2 h. The mixture was then poured into ice water (500 mL) and extracted with EtOAc (2×100 mL). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (eluent: 45% EtOAc / petroleum ether) to give the title compound as a colorless oil (2 g, 13%).

[0310] (Step 2: tert-Butyl 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoate) [ka]

[0311] To a solution of 2-((6-chlorohexyl)oxy)ethan-1-ol (2.5 g, 13.9 mmol) in ACN (25 mL) was added an aqueous solution of N-benzyl-trimethylammonium hydroxide (0.277 g, 4.16 mmol) at room temperature over 10 min. Then, tert-butyl acrylate (8.9 g, 69.4 mmol) was added dropwise. The resulting solution was stirred at room temperature for 16 h and then concentrated. The resulting mixture was poured into ice water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (eluent: 45% EtOAc / petroleum ether) to give the title compound as a colorless oil (3 g, 70%).

[0312] (Step 3: 3-(2-((6-chlorohexyl)oxy)ethoxy)propanal) [ka]

[0313] To a solution of tert-butyl 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoate (0.5 g, 1.623 mmol) in anhydrous THF (5 mL) at -78 °C, DIBAL-H (1 M in THF, 3.2 mL, 3.247 mmol) was added over 20 min. The resulting solution was allowed to stir at -78 °C for 1 h. The resulting mixture was quenched with Rochelle's salt solution and extracted with EtOAc (2 x 30 mL). The organic fractions were combined, dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (eluent: 45 % EtOAc / petroleum ether) to give the title compound as a colorless oil (0.2 g, 52 %).

[0314] Step 4: Preparation of tert-butyl (3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)carbamate [ka]

[0315] To a solution of pyridinium 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (25 g, 49.8 mmol, compound B1) in THF (250 mL) was added triethylamine (23 mL, 174.3 mmol) and diphenylphosphoryl azide (14.8 mL, 64.72 mmol) at 0 °C. The mixture was stirred at 0 °C for 30 min. The solution was gradually warmed to room temperature and continued to stir for 16 h. The mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with NaHCO3 and brine, dried over Na2SO4 and concentrated. The residue was suspended in tBuOH (500 mL) and stirred at 95 °C for 12 h. The mixture was cooled to room temperature, diluted with water (250 mL) and extracted with ethyl acetate (2 x 1 L). The combined organic layers were washed with NaHCO3 solution and brine, then dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (eluent: 2-20% EtOAc / petroleum ether) to give the title compound as an off-white solid. m / z=574.32 [M+H]+ .

[0316] Step 5: Preparation of tert-butyl (3',6'-di(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)carbamate [ka]

[0317] To a solution of tert-butyl (3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)carbamate (3.5 g, 6.13 mmol) and azetidine (1.4 g, 24.52 mmol) in 1,4-dioxane (70 mL) was added cesium carbonate (9.9 g, 30.65 mmol). The mixture was sparged with Ar for 15 min. Then, Pd2(dba)3 (2.80 g, 3.06 mmol) was added followed by XPhos (0.87 g, 1.83 mmol) and the mixture was heated to 110 °C for 16 h. The mixture was cooled to room temperature, filtered through a celite pad and concentrated. The residue was purified by reverse phase C-18 chromatography (eluent: 0-100% ACN / water) to give the title compound (1.4 g, 43%) as a dark pink solid. m / z=574.32 [M+H] + .

[0318] Step 6: Preparation of 6-amino-3',6'-di(azetidin-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one [ka]

[0319] To a solution of tert-butyl (3',6'-di(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)carbamate (1.0 g, 1.90 mmol) in CHCl (10 mL) was added TFA (5 mL) at 0 °C and stirred at room temperature for 6 h. The resulting solution was concentrated, quenched with saturated NaHCO solution (40 mL) and extracted with 10% MeOH in CHCl (3 x 30 mL). The combined organic layers were dried over NaSO and concentrated. The residue was purified by reverse phase C-18 column chromatography (eluent: 0-100% ACN / water) to give the title compound (0.2 g, 25%) as a purple solid. m / z=426.35[M+H] + .

[0320] (Step 7: 3',6'-Di(azetidin-1-yl)-6-((3-(2-((6-chlorohexyl)oxy)ethoxy)propyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) [ka]

[0321] To a solution of 6-amino-3',6'-di(azetidin-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (0.2 g, 0.471 mmol) and 3-(2-((6-chlorohexyl)oxy)ethoxy)propanal (0.11 g, 0.471 mmol, compound 21.3) in dichloroethane (2 mL) was added 2-3 drops of acetic acid at room temperature. The resulting solution was stirred for 2 h. Sodium triacetoxyborohydride (0.199 g, 0.941 mmol) was then added in small portions over 20 min at 0 °C. The resulting mixture was stirred at room temperature for 2 h, then quenched with ice water and extracted with 10% MeOH in CH2Cl2 (2 x 25 mL). The organic layers were combined, dried over Na2SO4 and concentrated. The residue was purified by reverse phase column chromatography (eluent: 0-100% ACN / water) to give the title compound (0.1 g, 32%) as a pink solid. m / z=646.50 [M+H] + .

[0322] (Step 8: 3',6'-Di(azetidin-1-yl)-6-((3-(2-((6-chlorohexyl)oxy)ethoxy)propyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) [ka]

[0323] To a solution of 3',6'-di(azetidin-1-yl)-6-((3-(2-((6-chlorohexyl)oxy)ethoxy)propyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (0.1 g, 0.155 mmol) in CHCl (5 mL) was added freshly distilled thionyl chloride (0.058 mL, 0.774 mmol) in CHCl (0.025 mL) at 0 °C under nitrogen. The resulting solution was allowed to warm to room temperature for 30 min and then concentrated. The residue was diluted with anhydrous CHCl (0.025 mL) and freshly prepared diazomethane in EtO (10 mL, >25 eq) was added. The mixture was stirred at 0 °C for 30 min. The mixture was concentrated and the residue was purified by preparative reverse-phase HPLC (eluent: 0-100% ACN / water) to afford the title compound (5 mg, 4.82%) as a brown gum. [ka]

[0324] 9.22 Example 22 (Synthesis of 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthen]-6-yl)propanamide) [ka]

[0325] (Step 1: Synthesis of 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid) [ka]

[0326] To a solution of tert-butyl 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoate (compound 21.2, 0.6 g, 1.947 mmol) in CHCl (12.0 mL) was added dropwise 4M hydrochloric acid in dioxane (12 mL) under Ar (gas) at 0°C. The resulting mixture was allowed to come to room temperature and continued to stir for 4 h. The mixture was then concentrated to give 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid (0.45 g, crude product) as an off-white solid. m / z=284.1[M+H] + .

[0327] (Step 2: Synthesis of 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-di(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)propanamide) [ka]

[0328] To a solution of 6-amino-3',6'-di(azetidin-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (compound 21.6, 0.3 g, 0.705 mmol) and 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid (0.2 g, 0.705 mmol) in CHCl (15 mL) was added n-methylimidazole (0.28 mL, 3.53 mmol) followed by methanesulfonyl chloride (0.082 mL, 1.06 mmol) dropwise at 0 °C. The mixture was allowed to come to room temperature and continued to stir for 1 h. The mixture was then concentrated and purified by reverse phase HPLC (eluent: 0-100% acetonitrile / 10 mM aqueous ammonium acetate) to give the title compound (0.17 g; 36% yield) as a pink solid. m / z=660.31[M+H] + .

[0329] (Step 3: 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthen]-6-yl)propanamide) [ka]

[0330] To a solution of N-(3'-(azetidin-1-yl)-6'-cyclobutyl-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)-3-(2-((6-chlorohexyl)oxy)ethoxy)propanamide (0.15 g, 0.228 mmol) in CHCl (30 mL) was added dropwise at 0° C. under argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 1 h. Freshly prepared diazomethane (approximately 30 mL, >25 eq) was then added dropwise at 0° C. The mixture was stirred for 1 h. The resulting solution was then concentrated and purified by reverse phase preparative HPLC (eluent: 0-100% ACN / water) to give Example 22 (0.012 g, 8%) as a light brown solid. [ka]

[0331] 9.23 Example 23 (Synthesis of (E)-3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one and (Z)-3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one) [ka]

[0332] Step 1: Preparation of 2-((6-chlorohexyl)oxy)ethan-1-ol [ka]

[0333] To a stirred solution of ethane-1,2-diol (5.0 g, 80.55 mmol) in DMF (50 mL) was added sodium hydride 60% (6.44 g, 161.11 mmol) at -10 °C and the mixture was stirred at -10 °C for 15 min. To the mixture was added 1-chloro-6-iodohexane (19.86 g, 80.55 mmol) at -10 °C and then the mixture was warmed to room temperature and stirred at room temperature for 2 h. Then ice water (0.5 L) was added to the mixture and the mixture was extracted with ethyl acetate (2 x 500 mL). The combined organic layers were washed with brine solution, dried over sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: 0-20% ethyl acetate / petroleum ether) to give the title compound (0.9 g, 6%) as a pale yellow liquid.

[0334] Step 2: Preparation of 2-((6-chlorohexyl)oxy)ethyl methanesulfonate [ka]

[0335] To a stirred solution of 2-((6-chlorohexyl)oxy)ethan-1-ol (1.5 g, 8.302 mmol) in dichloromethane (15.0 mL) at 0 °C was added triethylamine (1.4 mL, 9.963 mmol) followed by methanesulfonyl chloride (1.05 g, 9.133 mmol). The mixture was allowed to warm to room temperature and stirred at room temperature for 1 h. Then, ice water (50 mL) was added and the mixture was extracted with dichloromethane (2 x 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to give the title compound (1.5 g, 70%) as a pale yellow liquid which was used in the next step without further purification.

[0336] Step 3: Preparation of 1-(2-(but-3-yn-1-yloxy)ethoxy)-6-chlorohexane [ka]

[0337] To a stirred solution of but-3-yn-1-ol (0.8 g, 11.41 mmol, compound 23.2) in DMF (8.0 mL) was added sodium hydride 60% (0.457 g, 11.41 mmol) at 0 °C, and the mixture was stirred at 0 °C for 30 min. Then, 2-((6-chlorohexyl)oxy)ethyl methanesulfonate (1.48 g, 5.71 mmol) was added. The mixture was warmed to room temperature and stirred at room temperature for 16 h. Then, ice water (80 mL) was added, and the mixture was extracted with ethyl acetate (80 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: 0-15% ethyl acetate / petroleum ether) to give the title compound as a colorless liquid.

[0338] Step 4: Preparation of 3',6'-di(azetidin-1-yl)-6-iodo-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one [ka]

[0339] To a stirred solution of 6-amino-3',6'-di(azetidin-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (2.5 g, 5.88 mmol, compound 21.6) in ACN (10 mL) at 0 °C, CuI (0.67 g, 3.52 mmol) and tBuONO (0.57 mL, 4.70 mmol) were added. The mixture was stirred at 50 °C for 2 h. Then, aqueous sodium thiosulfate was added and the mixture was extracted with 10% MeOH in DCM (3 x 80 mL). The combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC (mobile phase 0-100% ACN / water) to give the title compound (1.0 g, 31%) as a purple solid.

[0340] Step 5: Preparation of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-yn-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one [ka]

[0341] To a stirred solution of 1-(2-(but-3-yn-1-yloxy)ethoxy)-6-chlorohexane (0.15 g, 0.644 mmol, compound 23.3) and 3',6'-di(azetidin-1-yl)-6-iodo-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (0.346 g, 0.644 mmol, compound 23.4) in DMF (4.5 mL) was added triethylamine (4.5 mL), copper iodide (0.012 g, 0.064 mmol), and bis(triphenylphosphine)palladium(II) chloride (chloride / hydrochloride) (0.045 g, 0.064 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure. The residue was purified by reverse phase column (eluted with 50-60% acetonitrile / 10 mM aqueous ammonium bicarbonate) to give the title compound (0.12 g; 24%) as a pink solid.

[0342] (Step 6: Preparation of (E)-3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one and (Z)-3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one) [ka]

[0343] To a stirred solution of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-yn-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.1 g, 0.156 mmol) in ethanol (10.0 mL) was added 10% palladium on carbon (50% wet) (0.1 g). The mixture was stirred at room temperature under a hydrogen atmosphere for 12 hours. The mixture was then filtered through Celite and concentrated. The residue was purified by reverse phase column (eluted with 50-80% acetonitrile / 10 mM aqueous ammonium bicarbonate) to give the title compound as the first eluted mixture (0.025 g; 24% yield).

[0344] (Step 7: Preparation of (E)-3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one and (Z)-3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one) [ka]

[0345] To a stirred solution of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (E:Z mixture) (0.02 g, 0.031 mmol, compound 23.6) in dichloromethane (4.0 mL) at 0 °C under an argon atmosphere was added oxalyl chloride (0.03 mL, 0.31 mmol), and the mixture was then allowed to warm to room temperature and stirred for 1 h. The mixture was then concentrated and backfilled under an argon atmosphere. A freshly prepared solution of diazomethane in diethyl ether (4.0 mL) was added to the residue at 0 °C, and the mixture was stirred for 1 h. The mixture was then concentrated and the residue was purified by preparative HPLC (C18; mobile phase: 0-100% ACN / water) to give the title compound (0.0022 g, 10%) as a brown gum. m / z=667.5[M+H] + .

[0346] (9.24 Example 24) (Synthesis of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-2-diazospiro[indene-1,9'-xanthen]-3(2H)-one) [ka]

[0347] Step 1: Preparation of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one [ka]

[0348] Further elution of compound 23 on a reverse phase column of step 6 afforded the title compound (0.02 g; 20% yield) as a pink solid.

[0349] Step 2: Preparation of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-2-diazospiro[indene-1,9'-xanthen]-3(2H)-one [ka]

[0350] To a stirred solution of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (0.02 g, 0.031 mmol) in dichloromethane (4.0 mL) at 0 °C under an argon atmosphere was added oxalyl chloride (0.03 mL, 0.31 mmol). The mixture was allowed to warm to room temperature and stirred at room temperature for 1 h, then the mixture was concentrated under an argon atmosphere. A freshly prepared solution of diazomethane in diethyl ether (4.0 mL) was added to the residue at 0 °C and the mixture was stirred at 0 °C for 1 h, then the mixture was concentrated. The residue was purified by preparative HPLC (C18; mobile phase, 0-100% ACN / water) to give the title compound (0.025 g, 12%) as a brown gum. [ka]

[0351] 9.25 Example 25: Kinetic solubility in PBS at 7.4 190 μL of buffer (PBS, pH 7.4) was poured into wells on a 96-well Millipore solubility filter plate, and then 10 μL of a 10 mM DMSO solution of the test compound was added to a final concentration of 500 μM.

[0352] The filter plate was shaken for 1.5 hours at room temperature in the dark and the samples were filtered into a new 96-well plate using a vacuum system. Samples were diluted in DMSO to a 500 μM (maximum concentration) solution and further diluted (1:10) to prepare a three-point calibration curve. Absorbance was measured at 220 nm, 254 nm, and 280 nm using HPLC / UV analysis. Data is reported as the average of triplicates for each test compound and is shown in Table 1 below.

[0353] (Table 1: Kinetic solubility values) [Table 2]

[0354] 9.26 Example 26: Passive Membrane Permeability MDCK-MDR1 cells were plated at 7,500 cells / 75 μL / well in 96-well Millipore Millicell-96 plates and incubated at 37 °C and 5 % CO2 for 3 days. Afterwards, cells were washed with Hank's Balanced Salt Solution (HBSS) containing 5 mM HEPES for 30 min. Test compounds were added in DMSO (10 mM) to HBSS buffer containing 10 μM GF-120918 to give a final DMSO concentration of 0.2% and test compound concentration of 5 μM. The transport plate was incubated at 37 °C for 1 h in a humidified incubator with 5 % CO2. After 1 h, samples were taken from the apical and basolateral compartments and analyzed by liquid chromatography with tandem mass spectrometry (LC / MS / MS, AB Sciex API 4000 instrument, coupled with Shimadzu LC-20AD LC pump system). Analytical samples were separated using a Waters Atlantis T3 dC18 reversed-phase HPLC column (20 mm × 2.1 mm) at a flow rate of 0.5 mL / min. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in 100% acetonitrile (solvent B).

[0355] Apparent permeability (Papp, A2B) values ​​were calculated using the following formula: Papp = (dQ / dt) / A / C0 (where dQ / dt is the initial rate of transport of the amount of test compound across the cell monolayer, A is the surface area of ​​the filter membrane, and C0 is the initial concentration of test compound, calculated for each direction using a four-point calibration curve by LC / MS / MS.) Data are reported as the average of duplicate runs and are shown in Table 2 below.

[0356] (Table 2: Passive membrane permeability values) [Table 3]

[0357] 9.27 Example 27: Protein labeling with photoactivatable dyes 60 μM of 6xHis tag fused Halo tag protein (expressed and purified from Promega's pH6HTC His6Halo tag T7 vector) was incubated in 200 μL of 200 μM dye compound solution (50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% NP40 substitute, 0.5 mM EDTA, 1 mM DTT, and 2% DMSO) at room temperature for 30 min and then incubated at 4 °C for 18 h. The solution was irradiated with a 405 nm laser (365 mW) at the center of a 6x expansion beam for 5 min. The solution was filtered twice through a 2 mL 7K MWCO Zeba spin desalting column (Thermo Scientific) (pre-equilibrated with 25 mM HEPES, pH 7.6, 100 mM KCl, 0.1 mM EDTA, 12.5 mM MgCl2, 1 mM DTT, and 10% glycerol). The labeled protein conjugates in the supernatant were analyzed on SDS-PAGE protein gels.

[0358] 9.28 Example 28: Halo-Protein Specificity Wild-type (WT) U2OS cells or U2OS cells ectopically expressing histone H2B-Halo tag fusions under the control of the CMV promoter (H2B) were plated at 6000 cells / 50 μL / well in glass-bottom 384-well plates and incubated overnight at 37 °C with 5% CO2. The next day, cells were incubated with PA-JF at concentrations ranging from 200 nM to 1 nM. 549 or Examples 1-4, and incubated for 45 min. After incubation, cells were washed three times with PBS and finally the medium was replaced with phenol-free medium for imaging. Samples were imaged on a Nikon Ti2 microscope coupled to an illumination source and fiber optics. 561 nm light (approximately 500 mW at the coverslip) was used to stimulate fluorophore emission. Images were taken 10 ms per 5 s in one field of view, and pulsing of 405 nm light (0-12 mW at the coverslip) with increasing intensity was used to photoactivate the dye molecules. Approximately 20 fields of view were imaged per set of cell line / dye compound / concentration.

[0359] All frames from each field of view were analyzed using a maximum likelihood estimator model to detect the fluorescence emission of single molecules in the images with subpixel precision. For each dye compound, linear regression was used to determine the relationship between dye compound concentration and the number of fluorescent spots detected by the microscope, assuming that an ideal dye would have no correlation between compound concentration and the number of measured slots in WT cells. The specificity factor was calculated using the PA-JF 549 The slope was calculated as the ratio of the slope of the compound being analyzed to the slope calculated for

[0360] (Table 3: Specificity factors) [Table 4]

[0361] 9.29 Example 29: Labeling Specificity The labeling specificity of the compounds of Examples 1-4 was measured using the method described in Example 8 above, and the PA-JF 549Wild-type U2OS cells or U2OS cells expressing the H2B-Halo tag fusion were cultured in PA-JF 549 or Example 4 for 45 min. Figure 3 shows an example of a field of view of U2OS expressing an H2B-Halo tag fusion with nuclei labeled with Hoechst 33342 dye. Labeling specificity was calculated by dividing the number of spots detected at a given dye concentration in U2OS cells expressing the H2B-Halo tag by the number of spots detected at the same concentration in U2OS wild type cells.

[0362] 9.30 Example 30: Signal to Noise Ratio The SNR, a proxy for single molecule brightness, was calculated for Examples 1-4 (see FIG. 4). Each spot in the SPT experiment described in Example 8 was calculated using a log-likelihood ratio test for the presence of a 2D Gaussian spot. See Serge et al., Dynamic multipl-target tracing to probe spatiotemporal cartography of cell membranes, Nat. Methods 2008, 5(8), 687-694 (Serge et al., 2008). The SNR of each spot was reported as its peak log-likelihood ratio. The calculation uses a 2D Gaussian PSF model with a fixed width including sigma / standard deviation equal to 0.183 μm. Only spots with SNR ≥ 14 were included in the calculation by tightening the spot detection filter. The bar graph in FIG. 4 is the SNR average across biological replicates, and the error bars are the standard error of the SNR average across biological replicates.

[0363] 9.31 Example 31: Reduction of non-specific labeling Wild-type (WT) U2OS cells or U2OS cells ectopically expressing an estrogen receptor-Halo tag (ER-Halo) fusion were plated at 6000 cells / 50 μL / well in glass-bottom 384-well plates and incubated overnight at 37 °C with 5% CO2 and 95% humidity. The next day, cells were incubated with 100 nM (conventional JF 549 20 pM for JF 549 , P.A.-J.F. 549 , or Examples 1-24, and 100 nM Potomac Red for 1 hour. After incubation, cells were washed three times with PBS, and finally the medium was replaced with phenol-free medium for imaging. Samples were imaged on a Nikon Ti2e microscope with an optical fiber coupled illumination source, using a 60x 1.27 NA objective and an sCMOS camera. Fluorophore emission was stimulated with 561 nm light with an integrated intensity of approximately 500 mW at the coverslip. Images were taken for 10 ms every 2 seconds in one field of view. To test the degree of sensitivity of each dye variant to 405 nm light, each well was imaged multiple times with increasing intensities of 405 nm light (between 0 and 5 mW at the coverslip). Approximately 60 fields of view were imaged per cell line / dye compound set.

[0364] To measure Example 19, where the fluorophore is a Si-containing far-red emitting dye, 642 nm light (approximately 500 mW at the coverslip) rather than a 561 nm light source was used to stimulate fluorophore emission, and cells were stained with 100 nM Potomac Yellow rather than Potomac Red.

[0365] After acquiring images for each compound, the images were individually inspected and the performance of each test compound was qualitatively evaluated. 549Those skilled in the art will appreciate that modifications to the core structure of the dye may alter not only the dye properties but also its photoactivation with 405 nm light. Thus, the performance of each dye variant, which could not be predicted based on structural features alone, was evaluated as follows. Compounds that were clearly unsuitable for single molecule imaging (e.g., those that cause dye aggregation in cells) were flagged and excluded from further analysis. The remaining dyes, which could be quantified using single molecule detection algorithms because spots consisting of a single fluorophore were reliably obtained, were further processed to generate individual trajectories. All frames of each field of view were analyzed using a maximum likelihood estimator model to detect single molecule fluorescence emission in the image with subpixel accuracy. The individual detection values ​​were then concatenated into trajectories of sequential camera frames. Statistics generated from detection values ​​(e.g., signal to noise) and tracks (e.g., track number) were used to compare between dye variants.

[0366] The number of nuclear tracks in the ER-Halo cell line was determined for each test compound at each level of activation with 405 nm light. To establish a common baseline between the different test compounds, the number of nuclear tracks was calculated using the JF 549 The photoactivation conditions that most closely matched those of were selected for each test compound and these conditions were used in all subsequent comparisons.

[0367] Table 4a: Specificity factors (full values) [Table 5] For definitions of "-", "+", "++", and "+++", see Example 32.

[0368] Table 4b: Specificity factors (full values) [Table 6] For definitions of "-", "+", "++", and "+++", see Example 32.

[0369] 9.32 Example 32: Improving labeling specificity The labeling specificity of the compounds of Examples 1 to 24 was measured using the methods described in Tables 4a and 4b above, and the PA-JF 549 Wild-type U2OS cells or U2OS cells expressing the ER-Halo tag fusion were cultured in PA-JF 549 or Examples 1-24 were incubated for 1 hour. Figure 5 shows examples of real fields of U2OS expressing ER-Halo tag fusions co-stained with Potomac Red (CAS:2127150-65-4; Grimm et al., 2017). Labeling specificity, as measured by the fold reduction relative to non-specific labeling of WT cells, was measured by comparing the track counts of Examples 1-24 measured in WT cells with the track counts of PA-JF in WT cells. 549 Calculated by comparing with the number of tracks in PA-JF 549 It is expressed as the numerator in terms of the ratio of

[0370] Sensitivity to photoconversion by 405 nm illumination was determined qualitatively based on the amount of 405 nm light required to achieve a comparable number of tracks. Compounds that demonstrated high levels of activation at 0.25 mW of 405 nm laser input (minimum tested intensity) were given a "+++", whereas compounds that failed to achieve high levels of activation at 5 mW intensity at 405 nm were given a "-". "+" and "++" were assigned to compounds that required >1 mW and >2.5 mW of 405 nm laser power at the objective, respectively.

[0371] The SNR, a proxy for single molecule brightness, was calculated for Examples 1-4 (see FIG. 6). Each spot in the SPT experiment described in Example 31 was calculated using a log-likelihood ratio test for the presence of a 2D Gaussian spot. See Serge et al., Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes, Nat. Methods 2008, 5(8), 687-694 (Serge et al., 2008). The SNR of each spot was reported as its peak log-likelihood ratio. The calculation uses a 2D Gaussian PSF model with a fixed width including sigma / standard deviation equal to 0.183 μm. Only spots with SNR≧14 were included in the calculation by tightening the spot detection filter. The bar graph in Figure 6 is the SNR average calculated from the total detection values ​​of the dye variants across biological replicates, and the error bars are the standard error of the SNR average across biological replicates. The above examples show that the compounds disclosed herein surprisingly exhibit improved labeling specificity when used to label proteins while maintaining control over brightness and concentration.

[0372] A number of references are cited throughout this specification, the disclosures of each of which are incorporated herein by reference in their entirety for all intents and purposes.

Claims

1. Compounds of formula (I): 【Chemical 1】 where: G is -O-, -S-, -SO 2 -, -C(C 1-3 alkyl) 2 -, -N(C 1-3 alkyl)-, -Si(C 1-3 alkyl) 2 -, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, or -P(=O)(Ph)-; R is 【Chemistry 2】 (In the formula, R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 alkyl) 2 , -N(C 1-3 alkyl) 2 , -(CH 2 ) n O(C 1-3 alkyl), 【Chemistry 3】 wherein n is an integer from 1 to 3; R 2 is -O- or -N(C 1-3 alkyl)-; X is hydrogen or halogen; L A is a linker; and Z is the compound, or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, comprising a moiety that covalently binds to a tag fusion protein.

2. The compound of claim 1 , wherein the tag fusion protein comprises a Halo tag, a SNAP tag, or a CLIP tag.

3. L A is represented by the following formula (IA): 【Chemistry 4】 (In the formula, Each L 1 Independently: (I C 1-6 Alkylene-, -C 1-6 Alkenylene- or -C 1-6 Alkynylene-; (ii) heteroaryl; or (iii)-NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -N(C 1-3 alkyl)-C(O)-, -C(O)-N(C 1-3 alkyl)-, -C 1-6 Alkylene-NH-, -NH-C 1-6 Alkylene-, -C 1-6 Alkylene-O-, -OC 1-6 Alkylene-, -C(O)-C 1-6 Alkylene- or -C 1-6 alkylene-C(O)-; Each L 2 are independent, -C 1-6 Alkylene-, -(OCH 2 ) p , -(CH 2 O) p -, -(OCH 2 CH 2 ) p - or -(CH 2 CH 2 O) p - (wherein p is an integer from 1 to 3); a and b are each independently an integer of 1 or 2; and L 1 and L 2 But, -(L 2 ) b The compound of claim 1, wherein - is a linker of formula (I) in the orientation that binds to Z.

4. Z is 【Chemistry 5】 The compound according to any one of claims 1 to 3, wherein

5. Compound of formula (II): 【Chemistry 6】 (wherein the asterisks "**" and "*" represent linkers L B is a symbol to indicate the orientation of; G is -O-, -S-, -SO 2 -, -C(C 1-3 alkyl) 2 -, -N(C 1-3 alkyl)-, -Si(C 1-3 alkyl) 2 -, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, -P(=O)(Ph)-; R is 【Chemistry 7】 (where R 1 is -OH, -C(=O)OH, -C(=O)O(C 1-3 alkyl), -C(=O)N(C 1-3 alkyl) 2 , -N(C 1-3 alkyl) 2 , -(CH 2 ) n O(C 1-3 alkyl), 【Chemistry 8】 where n is an integer from 1 to 3; R 2 is -O- or -N(C 1-3 alkyl)-; X is hydrogen or halogen; and L B is (i)-C 1-6 Alkylene-, -C 1-6 Alkenylene- or -C 1-6 (ii) alkynylene-; (iii) heteroaryl; or (iv) —NHC(O)—, —C(O)NH—, —OC(O)—, —C(O)O—, —N(C 1-3 alkyl)-C(O)-, -C(O)-N(C 1-3 alkyl)-, -C 1-6 Alkylene-NH-, -NH-C 1-6 Alkylene-, -C 1-6 Alkylene-N(C 1-6 alkylene)-, -N(C 1-6 Alkylene)-C 1-6 Alkylene-, -C 1-6 Alkylene-O-, -OC 1-6 Alkylene-, -C(O)-C 1-6 Alkylene- or -C 1-6 alkylene-C(O)-) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof.

6. Structure of formula (III): 【Chemistry 9】 6. The compound of claim 5, having the formula:

7. G is -O-, -S-, -N(CH 3 )-, -Si(CH 3 ) 2 -, -C(CH 3 ) 2 - or -SO 2 7. The compound according to claim 5 or 6, wherein:

8. L B teeth, 【Chemistry 10】 (In the formula, "*" and "**" represent the linker L B 7. The compound according to claim 5 or 6, wherein the linker comprises:

9. L B 7. The compound of claim 5 or 6, wherein is a linker comprising a heteroaryl.

10. 10. The compound of claim 9, wherein the heteroaryl is triazole or imidazole.

11. R 1 teeth, 【Chemistry 11】 7. The compound according to claim 5 or 6, wherein:

12. Structure of formula (IV): 【Chemistry 12】 6. The compound of claim 5, having the formula:

13. R 1 is -N(CH 3 ) 2 , -OH, 【Chemistry 13】 13. The compound of claim 12, wherein:

14. Structure of formula (V): 【Chemistry 14】 6. The compound of claim 5, having the formula:

15. R 1 is -N(CH 3 ) 2 , -OH, 【Chemistry 15】 15. The compound of claim 14, wherein:

16. Structure of formula (VI): 【Chemistry 16】 6. The compound of claim 5, having the formula:

17. R 1 teeth, 【Chemistry 17】 17. The compound of claim 16, wherein:

18. The compound is 【Chemistry 18】 2. The compound of claim 1, wherein:

19. The compound is 【Chemistry 19】 【change】 【change】 【change】 6. The compound of claim 1 or 5, wherein:

20. The following structure: 【Chemistry 20】 or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof.

21. The following structure: 【Chemical 21】 or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof.

22. The following structure: 【Chemical 22】 or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof.

23. Compound of formula (VII): 【Chemical 23】 (wherein the asterisks "**" and "*" represent linkers L B is a symbol to indicate the orientation of; R is 【Chemistry 24】 (where R 2 Ha-CH 3 ) and L B teeth, 【Chemistry 25】 is a linker comprising: Here, R is 【Chemical 26】 If L B teeth, 【Chemical 27】 ) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof.

24. 10. An ex vivo method for labeling a protein, comprising contacting a sample containing a tagged fusion protein with a compound of claim 1 to obtain a labeled protein.

25. 25. The ex vivo method of claim 24, wherein the tagged protein is a kinase, a transcription factor, a chromatin regulator, an adaptor, a transporter, or a pathogenic aggregation factor.

26. 25. The ex vivo method of claim 24, wherein the tagged protein is a histone.

27. 27. The ex vivo method of claim 26, wherein the histone is H2B.

28. 25. The ex vivo method of claim 24, wherein the labeled protein fluoresces when exposed to light.

29. 30. The ex vivo method of claim 28, wherein the light beam is a laser.

30. 29. The ex vivo method of claim 28, wherein the wavelength (λ) of the light is about 405 nm.

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