Photoactive fluorescent compounds and their use for labeling proteins

Abstract

Provided herein are novel photoactive fluorescent compounds and their use in labeling proteins, e.g., tagged proteins, and for visualizing the location and dynamics of proteins in living cells.

Description

[Technical Field]

[0001] 1. Field This application claims the benefit of U.S. Provisional Application No. 63 / 481,961, filed January 27, 2023, the disclosure of which is incorporated by reference in its entirety.

[0002] Provided herein are novel photoactive fluorescent compounds and their use in labeling proteins, e.g., tagged proteins, and for visualizing the location and dynamics of proteins in living cells. [Background technology]

[0003] 2.Background Fluorescence microscopy is useful for visualizing the location and dynamics of biomolecules in living cells. This 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 were previously the gold standard for fluorescence imaging because they allow genetic specificity in labeling. Many efforts have been made to improve protein-associated dyes by improving their photostability and other properties. These efforts include enzyme-based self-labeling tags, e.g., HaloTag, which allow labeling of specific protein fusions with synthetic fluorophores, enabling a variety of imaging experiments inside living cells. See U.S. Pat. No. 10,161,932.

[0004] Rhodamine dyes have been and remain widely used, particularly due to their brightness and photostability. The photophysics of rhodamines is known, among other things, for their importance as biological probes. See Grimm et al., “Deuteration Improves Small-Molecule Fluorophores,” 2020, BioRxiv preprint (https: / / doi.org / 10.1101 / 2020.08.17.250027 (Grimm et al., 2020)). Methods for enhancing the brightness and photostability of fluorophores have been described. Such methods involve incorporating 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, the rhodamine dyes do not have the cell permeability to allow for optimized live cell labeling experiments, see U.S. Patent No. 10,161,932.

[0005] Azetidine-substituted fluorescent compounds have been developed to enhance cell permeability and brightness. These compounds include molecules that are azetidine-substituted derivatives of known fluorescent tags. These compounds can exhibit higher quantum yields compared to their parent compounds. See U.S. Patent No. 10,161,932. Subsequently, the fluorescence quantum yield of rhodamine and other dyes was improved by incorporating deuterium into their alkylamino substituents. However, deuteration was found to prevent or slow down undesirable properties such as photochemically induced spectral shifts and irreversible photobleaching. See Grimm et al., 2020.

[0006] Thus, there remains a need, and the present disclosure addresses, for photoactive fluorescent compounds with improved photostability, brightness, labeling specificity, concentration control, and other properties for use in labeling proteins. Summary of the Invention

[0007] 3. Overview In one aspect, provided herein is a compound of formula (A): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein L A is a linker, Z comprises a moiety that covalently binds to a tagged protein, n is an integer from 1 to 3, and other variables are as defined herein; and the moiety L A -Z is located at the 5th or 6th position.

[0008] In one aspect, provided herein is a compound of formula (A-1): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein L A is a linker, Z comprises a moiety that covalently binds to the tagged protein, n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3, and the other variables are as defined herein.

[0009] In one aspect, provided herein is a compound of formula (A-2): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein L A is a linker, Z comprises a moiety that covalently binds to the tagged protein, n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3, and the other variables are as defined herein.

[0010] In one aspect, provided herein is a compound of formula (VIII): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein: R 1 is H or -C(=O)N(C 1-3 alkyl)2, Each L 1 is, independently, -OC 1-6 -Alkylene-, -C(O)NH-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)(C 1-6 -alkylene)-, or -C 1-6 -Alkylene-OC 1-6 -alkylene-, Each L 2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] and R 1 If H and a is 1, then L 1 teeth, [ka] Instead, a wavy line [ka] represents the point of attachment to the rest of the compound.

[0011] Also provided herein is a compound of formula (IX): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein: L B is (i)-OC 1-6 -Alkylene-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)-, or -C 1-6 -alkylene-O-.

[0012] Also provided herein is a compound of formula (X): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof; In the formula, R 1 is H or -C(=O)N(C 1-3 alkyl)2.

[0013] In another aspect, provided herein are methods for making the compounds disclosed herein to obtain compounds that are substantially chemically pure and / or substantially free of chemical impurities.

[0014] In another aspect, provided herein is a method for labeling a protein, comprising contacting a sample containing a tagged protein with a compound disclosed herein to obtain a labeled protein. [Brief explanation of the drawings]

[0015] 4. Brief description of the drawings [Figure 1] A schematic representation 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 is provided.

[0016] [Figure 2] The preparation of photoactivatable fluorescently tagged (e.g., labeled) proteins is shown.

[0017] [Figure 3]Figure 1 shows the relative labeling specificity of PA-JF549 compared to the photoactivatable dye compound of Example 4. Labeling specificity was calculated using histone H2B, which is located in the nucleus of wild-type cells that do not express the HaloTag® protein.

[0018] [Figure 4] 1 shows the signal-to-noise ratio (SNR) of JF549, PA-JF549, and the photoactivatable dye compounds of Examples 1-4.

[0019] [Figure 5] An exemplary field of view of U2OS expressing the ER-HaloTag fusion co-stained with Potomac Red (CAS: 2127150-65-4, Grimm et al., 2017) is shown. The labeling specificity of the photoactivatable dye compound of Example 4 was measured and compared to PA-JF549.

[0020] [Figure 6] 1 shows the signal-to-noise ratio (SNR) of JF549, PA-JF549, and the photoactivatable dye compounds of Examples 1, 4, 7, and 10.

[0021] [Figure 7] The number of spots detected by JF549-HaloTag (left), photoactivatable JF549-HaloTag (center), and compound 39-10c (compound 4) (right) in HaloTag-negative U2OS cells is shown, demonstrating that compound 39-10c reduces nonspecific labeling compared to photoactivatable JF549-HaloTag.

[0022] [Figure 8] A time course over 4000 frames is shown showing the photoactivation event of compound 39-10c at 1 mW, 4 mW, and 8 mW, and the subsequent loss of signal due to photobleaching.

[0023] [Figure 9]16 is a graphical representation of the presence of compound 39-10c tagged to a β-catenin-HaloTag fusion protein, marked as slow (<0.1 μm / sec), intermediate (0.1-1 μm / sec), and fast (>1 μm / sec) diffusing populations across the diffusion coefficient.

[0024] [Figure 10A] A field of view containing four cells with the population shown in Figure 9 is shown. The boundaries of these cells can be superimposed on Figures 10B-E to show cell location in the fluorescence image. This figure therefore maps the location of the four cells in the image, as a reference for Figures 10B-E.

[0025] [Figure 10B] An overlay of all three populations shown in Figure 9. The cutout zooms in on two distinct slow and medium populations in the focal adhesion region between cell 1 and cell 3.

[0026] [Figure 10C] Separately shown channels clearly showing the location of each population and the cellular localization map. [Figure 10D] Separately shown channels clearly showing the location of each population and the cellular localization map. [Figure 10E] Separately shown channels clearly showing the location of each population and the cellular localization map. DETAILED DESCRIPTION OF THE INVENTION

[0027] 5. Detailed Description 5.1 Definition To facilitate understanding of the disclosure set forth herein, several 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 used in the art.

[0028] As used herein, the terms "comprising" and "including" may be used interchangeably. The terms "comprising" and "including" are intended to specify the presence of stated features or components, but do not exclude the presence or addition of one or more features or components, or groups thereof. Furthermore, the terms "comprising" and "including" are intended to include examples encompassed by the term "consisting of." Thus, the term "consisting of" may be used in place of the terms "comprising" and "including" to indicate more specific embodiments of the invention.

[0029] 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 the following: "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 inherently inconsistent with each other in some way.

[0030] As used herein, the term "about" or "approximately" refers to the tolerance of 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 certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means 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.

[0031] 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. Specific non-toxic acids include hydrochloric acid, hydrobromic acid, maleic acid, phosphoric acid, sulfuric acid, and methanesulfonic acid. Specific examples of salts include hydrochloride and mesylate salts. Others are known in the art.

[0032] As used herein, unless otherwise indicated, the terms "stereoisomer" or "stereoisomerically pure" refer to one stereoisomer of a compound disclosed herein that is substantially free of other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of that compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of that compound. A typical stereoisomerically pure compound will contain greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of that compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of that compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of that compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of that 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.

[0033] The use of such stereomerically pure forms of the compounds disclosed herein, as well as mixtures of these forms, are encompassed within the embodiments disclosed herein. For example, mixtures containing equal or unequal amounts of the 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, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, Ed., University of Notre Dame Press, Notre Dame, IN, 1972).

[0034] It should also be noted that the compounds disclosed herein can include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In certain embodiments, the compounds disclosed herein are isolated as either the E or Z isomer. In other embodiments, the compounds disclosed herein are a mixture of E and Z isomers.

[0035] As used herein, the terms "isotopic form" or "isotope" refer to compounds that, for example, contain tritium ( 3 H), iodine-125( 125 I), sulfur 35( 35 S), or carbon-14 ( 14 Even if it is radiolabeled with a radioisotope such as carbon-13( 13 C), or nitrogen-15( 15This means that the compound may be enriched in an isotope, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 120, 121, 130, 141, 142, 150, 151, 162, 170, 181, 191, 192, 193, 194, 195, 196, 197, 1 As used herein, "deuterated" means that at least one hydrogen (H) has been replaced with a deuterium (D or 2 H), meaning that the compound is enriched with deuterium at at least one position.

[0036] As used herein, unless otherwise specified, a compound that is "substantially chemically pure" is substantially free of other compounds (i.e., chemical impurities). In certain embodiments, a substantially chemically pure compound contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% by weight of one or more other compounds. Detection of other compounds can be achieved by any method apparent to one of skill in the art, including, but not limited to, methods of chemical analysis, such as mass spectrometry, spectroscopic analysis, thermal analysis, elemental combustion analysis, and / or chromatographic analysis.

[0037] As used herein, "alkyl" refers to a straight-chain or branched saturated hydrocarbon group containing 1 to 10 carbon atoms. In certain embodiments, an alkyl contains 1 carbon atom ("C alkyl"). In certain embodiments, an alkyl contains 1 to 2 carbon atoms ("C 1-2 In certain embodiments, alkyl contains 1 to 3 carbon atoms ("C 1-3 In certain embodiments, alkyl contains 1 to 4 carbon atoms ("C 1-4 In certain embodiments, alkyl contains 1 to 6 carbon atoms ("C 1-6 In certain embodiments, alkyl contains 1 to 10 carbon atoms ("C 1-10 In 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.

[0038] As used herein, "alkylene" refers to a linear or branched saturated divalent hydrocarbon group containing 1 to 10 carbon atoms, and in certain embodiments, 1 to 6 carbon atoms. In certain embodiments, alkylene contains 1 to 3 carbon atoms ("C 1-3 In certain embodiments, alkylene contains 1 to 4 carbon atoms ("C 1-4 In certain embodiments, alkylene contains 1 to 6 carbon atoms ("C 1-6 In certain embodiments, alkylene contains 1 to 10 carbon atoms ("C 1-10 alkylene).

[0039] As used herein, "alkenylene" refers to a group in which one hydrogen of a straight or branched chain alkenyl group is removed to make the group divalent. In certain embodiments, alkenylene contains 1 to 3 carbon atoms ("C1-3 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 1-10 Non-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, but- buta-1,2-ene-2,3-diyl, buta-3-ene-1,1-diyl, buta-3-ene-1,2-diyl, buta-3-ene-1,3-diyl, buta-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. The alkenylene group can be unsubstituted or substituted as described for alkyl (eg, optionally substituted alkenylene).

[0040] As used herein, "alkynylene" refers to a straight or branched chain divalent substituent containing one or two carbon-carbon triple bonds, and, if unsubstituted, containing only C and H. In certain embodiments, alkynylene contains 1 to 3 carbon atoms ("C 1-3In 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 1-10 alkynylene"). 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. The alkynylene group can be unsubstituted or substituted as described for an alkynyl group (e.g., optionally substituted alkynylene).

[0041] As used herein, "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic ring of 5 to 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, wherein the monocyclic ring is aromatic and at least one ring within the bicyclic or tricyclic ring is aromatic (although not necessarily the ring containing the heteroatom, e.g., tetrahydroquinolinyl, dihydroisoquinolinyl, dihydrobenzodioxinyl, 2,3-dihydrobenzo[b][1,4]dioxinyl, etc.). In certain embodiments, a heteroaryl is a monocyclic ring of 5 to 6 ring atoms. Unless otherwise specified, valency may be assigned to any atom of any ring of the heteroaryl group, as permitted by valency 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]pyridinyl, 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.

[0042] As used herein, unless otherwise specified, "halogen" is fluorine, chlorine, bromine, or iodine.

[0043] 5.2 Embodiment (a) compound In one aspect, provided herein are photoactive fluorescent compounds. In one embodiment, the photoactive fluorescent compounds are for use in labeling, e.g., labeling proteins. In one embodiment, the compounds disclosed herein are useful for visualizing the location and dynamics of proteins in living cells.

[0044] These compounds exhibit unexpectedly improved properties that are desirable for photoactivatable fluorescent compounds, such as improved photostability, brightness, labeling specificity, and concentration control. 549 ), commercially available photoactivatable dyes (PA-JF 549 ) and Figure 1, which provides a schematic of the specificity, brightness, and density control of an ideal dye. 549 shows high specificity and brightness, but the concentration control is insufficient. 549 The ideal dyes exhibit high specificity, brightness, and density control, properties that are not predictable based on structural features alone. For example, PA-JF 549 Modification of the core structure of PA-JF can not only change the photophysical properties of the resulting photoconversion dye, but also change the photoactivation propensity of the dye after exposure to blue light. 549 The compounds disclosed herein, having a core structure of the formula: exhibit unexpectedly improved properties.

[0045] In certain embodiments, the compound is of formula (A): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein L Ais a linker, Z comprises a moiety that covalently binds to a tagged protein, n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3, and the moiety L A -Z is located at the 5th or 6th position, and 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)-, and R is [ka] and the wavy line [ka] represents the point of attachment to the rest 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)-.

[0046] In certain embodiments, the compound is of formula (A-1): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein L A is a linker, Z comprises a moiety that covalently binds to a tagged protein, n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3, and 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)-, and R is [ka] and the wavy line [ka] represents the point of attachment to the rest 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)-.

[0047] In certain embodiments, the compound is of formula (A-2): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein L A is a linker, Z comprises a moiety that covalently binds to a tagged protein, n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3, and 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)-, and R is [ka] and the wavy line [ka] represents the point of attachment to the rest 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)-.

[0048] In certain embodiments, the compound is of formula (I): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein L A is a linker, Z comprises a moiety that covalently binds to the tagged protein, and the other variables are as defined herein.

[0049] In certain embodiments, L A is a linker of formula (IA): -(L 2 ) b -(L 1 ) a - (IA) During the ceremony, Each L 1 is, 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 independently, -C 1-6 -Alkylene-, -(OCH2) p , -(CHO) p -, -(OCH2CH2) p - or -(CH2CH2O) p - and p is an integer from 1 to 3, a and b are each independently an integer of 1 or 2.

[0050] In certain embodiments, L A is represented by -(L 2 ) b - is oriented so that it bonds to Z: [ka]

[0051] In certain embodiments, the compound of formula (IB) is [ka] and R 1 is H and X is H, then Z-(L 2) b -(L 1 ) a -teeth, [ka] It is not a wavy line. [ka] represents the point of attachment to the rest of the compound.

[0052] In certain embodiments of compounds of Formula (I) or (IB), G is —O—, —S—, —SO—, —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)-.

[0053] In certain embodiments of compounds of Formula (I) or (IB), R is [ka] is.

[0054] 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 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), [ka] is.

[0055] In certain embodiments of compounds of Formula (I) or (IB), R 2 is -O- or -N(C 1-3 alkyl)-.

[0056] In certain embodiments of compounds of Formula (I) or (IB), X is hydrogen or halogen.

[0057] In certain embodiments of compounds of Formula (I) or (IB), X is halogen.

[0058] In certain embodiments, the compound of formula (IB) is [ka] and R 1 is H and X is halo.

[0059] In certain embodiments of compounds of Formula (I) or (IB), Z is [ka] is.

[0060] In certain embodiments, the compound of formula (I): [ka] is in the formula: 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] and the wavy line [ka] represents the point of attachment to the rest of the compound; R 1 is H, -OH, -C(=O)OH, -C(=O)O(C1-3 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 the linker, Z, [ka] That is what it is.

[0061] In certain embodiments, the compound of formula (I) is a compound of formula (I) wherein L A is a linker of formula (IA) and R is [ka] and R 1 is H and X is H, then Z-(L 2 ) b -(L 1 ) a -teeth, [ka] It is not a wavy line. [ka] represents the point of attachment to the rest of the compound.

[0062] In certain embodiments, a compound of formula (IB): [ka] is in the formula: 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] and the wavy line [ka] represents the point of attachment to the rest 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 But 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(C1-6 -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 But independently, -C 1-6 -Alkylene-, -(OCH2) p , -(CHO) p -, -(OCH2CH2) p - or -(CH2CH2O) p - and p is an integer from 1 to 3, a and b are each independently an integer of 1 or 2; Z, [ka] That is what it is.

[0063] In some such embodiments of compounds of Formula (IB), Z is: [ka] is.

[0064] In some such embodiments of compounds of Formula (IB), Z is [ka] and X is hydrogen.

[0065] In some such embodiments of compounds of Formula (IB), Z is [ka] and X is fluorine.

[0066] In certain embodiments, compounds of formula (I) are those in which R is [ka] and R 1 is H and X is H, then Z-(L 2 ) b -(L 1 ) a -teeth, [ka] It is not a wavy line. [ka] represents the point of attachment to the rest of the compound.

[0067] In certain embodiments, the tagged protein comprises a HaloTag®, a SNAP-tag®, or a CLIP-tag®.

[0068] In certain embodiments, provided herein is a compound of formula (II): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein: The asterixis "**" and "*" represent the linker L B and other variables are as defined herein.

[0069] In certain embodiments of compounds of Formula (II), G is —O—, —S—, —SO—, —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)-.

[0070] In certain embodiments of compounds of Formula (II), R is [ka] is.

[0071] In certain embodiments of compounds 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] is.

[0072] In certain embodiments of compounds of Formula (II), X is hydrogen or halogen. In some such embodiments, X is hydrogen. In some such embodiments, X is halogen.

[0073] In certain embodiments of compounds of Formula (II), R 2 is -O- or -N(C 1-3 alkyl)-.

[0074] In certain embodiments of compounds of Formula (II), L B is (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-, -C1-6 -Alkylene-O-, -OC 1-6 -Alkylene-, -C(O)-C 1-6 -Alkylene- or -C 1-6 -alkylene-C(O)-.

[0075] In certain embodiments of compounds of Formula (II), Z is [ka] In some such embodiments of compounds of Formula (II), Z is: [ka] is.

[0076] In some such embodiments of compounds of Formula (II), Z is [ka] and X is hydrogen.

[0077] In some such embodiments of compounds of Formula (II), Z is [ka] and X is fluorine.

[0078] In certain embodiments, the compound of formula (II): [ka] is in the formula: 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] and 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] and n is an integer from 1 to 3. R 2 is -O- or -N(C 1-3 alkyl)-, X is hydrogen or halogen; L B However, (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 -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 -alkylene-C(O)-.

[0079] In certain embodiments of compounds of Formula (II), G is —O—, —S—, or —SO 2 —; and R is [ka] and 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 alkyl), and L B is —NHC(O)—, or —C(O)NH—, and X is hydrogen.

[0080] In certain embodiments of compounds of Formula (II), G is —O—, or —S—, and R is [ka] and 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 alkyl), and L B is —NHC(O)—, or —C(O)NH—, and X is hydrogen.

[0081] In certain embodiments, provided herein is a compound of formula (III): [ka]

[0082] In certain embodiments of a compound of Formula (III), G is —O—, —S—, —N(CH 3 )—, —Si(CH 3 ) 2 —, —C(CH 3 ) 2 —, or —SO 2 —.

[0083] In certain embodiments of compounds of Formula (III), L B teeth, [ka] and * and ** are linkers L BIt should be noted that it is not relevant to one skilled in the art whether the placement of the wavy bond is shown in the middle or at the end of the bond, e.g. [ka] It is understood that all structures in the above are equivalent to each other.

[0084] In certain embodiments of compounds of Formula (III), L B includes heteroaryl.

[0085] In certain embodiments of a compound of Formula (III), the heteroaryl is triazole or imidazole.

[0086] In certain embodiments of compounds of Formula (III), R 1 teeth, [ka] is.

[0087] In certain embodiments of compounds of Formula (III), R 1 is H and L B teeth [ka] is.

[0088] In certain embodiments, provided herein is a compound of formula (IV): [ka] During the ceremony, 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] and n is an integer of 1 to 3.

[0089] In certain embodiments, R 1 is -N(CH3)2.

[0090] In certain embodiments, R 1 is -OH.

[0091] In certain embodiments, R 1 teeth, [ka] is.

[0092] In certain embodiments, R 1 teeth, [ka] is.

[0093] In certain embodiments, R 1 teeth, [ka] is.

[0094] In certain embodiments, R 1 teeth, [ka] is.

[0095] In certain embodiments, R 1 teeth, [ka] is.

[0096] In certain embodiments, R 1 teeth, [ka] is.

[0097] In certain embodiments, R 1 teeth, [ka] is.

[0098] In certain embodiments, provided herein is a compound of formula (V): [ka] During the ceremony,

[0099] 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] and n is an integer of 1 to 3.

[0100] In certain embodiments, provided herein is a compound of formula (V): [ka] During the ceremony, 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] and n is an integer of 1 to 3.

[0101] In certain embodiments, provided herein is a compound of formula (V): [ka] During the ceremony,

[0102] 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] and n is an integer of 1 to 3.

[0103] In certain embodiments, R 1 is -N(CH3)2.

[0104] In certain embodiments, R 1 is -OH.

[0105] In certain embodiments, R 1 teeth, [ka] is.

[0106] In certain embodiments, R 1 teeth, [ka] is.

[0107] In certain embodiments, R1 teeth, [ka] is.

[0108] In certain embodiments, R 1 teeth, [ka] is.

[0109] In certain embodiments, R 1 teeth, [ka] is.

[0110] In certain embodiments, R 1 teeth, [ka] is.

[0111] In certain embodiments, R 1 teeth, [ka] is.

[0112] In certain embodiments, provided herein is a compound of formula (VI): [ka] During the ceremony, 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] and n is an integer of 1 to 3.

[0113] In certain embodiments, R 1 teeth, [ka] is.

[0114] In certain embodiments, R 1 teeth, [ka] is.

[0115] In certain embodiments, R 1 teeth, [ka] is.

[0116] In certain embodiments, provided herein is a compound of formula (VIa): [ka] During the ceremony, 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] and n is an integer of 1 to 3.

[0117] In certain embodiments, R 1 teeth, [ka] is.

[0118] In certain embodiments, R 1 teeth, [ka] is.

[0119] In certain embodiments, R 1 teeth, [ka] is.

[0120] In certain embodiments, the compound is [ka] is.

[0121] In certain embodiments, the compound is [ka] The file is TIFF2026503623000102.tif58165.

[0122] In certain embodiments, the compound is [ka] The file is TIFF2026503623000104.tif68165.

[0123] In certain embodiments, the compound is [ka] is.

[0124] In certain embodiments, the compound of formula (VIa) is [ka] is.

[0125] In certain embodiments, the compound of formula (VIa) is [ka] is.

[0126] In certain embodiments, provided herein is a compound of formula (VII): [ka] During the ceremony, R is [ka] and R 2 is -CH3, L B teeth, [ka] and * and ** are linkers L B 1 shows the orientation of the embodiment in FIG.

[0127] In certain embodiments of compounds of Formula (VII), R is [ka] isn't it.

[0128] In certain embodiments of compounds of Formula (VII), L B teeth, [ka] isn't it.

[0129] In certain embodiments of compounds of Formula (VII), R is [ka] and L B teeth [ka] isn't it.

[0130] In certain embodiments of compounds of Formula (VII), R is [ka] and L B teeth [ka] is.

[0131] In certain embodiments of compounds of Formula (VII), R is [ka] and L B teeth [ka] is.

[0132] In certain embodiments, the compound is of formula (VIII): [ka]

[0133] or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof; During the ceremony, R 1 is H or -C(=O)N(C 1-3 alkyl)2, Each L 1 is, independently, -OC 1-6 -Alkylene-, -C(O)NH-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)(C 1-6 -alkylene)-, or -C 1-6 -Alkylene-OC 1-6 -alkylene-, Each L2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] and R 1 If H and a is 1, then L 1 teeth, [ka] Instead, a wavy line [ka] represents the point of attachment to the rest of the compound.

[0134] In one embodiment of Formula (VIII), each L 1 is, independently, -OC 1-6 -Alkylene-, -C(O)NH-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)(C 1-6 -alkylene)-, or -C 1-6 -Alkylene-OC 1-6 -alkylene-, Each L 2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] and R 1 If H and a is 1, then L 1 teeth, [ka] Instead, a wavy line [ka] represents the point of attachment to the rest of the compound.

[0135] In certain embodiments of a compound of Formula (VIII), p is 1, a is 1, and b is an integer from 0-1.

[0136] In certain embodiments of Formula (VIII), R 1 is H, and each L 1 is, independently, -OC 1-6 -Alkylene-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)(C 1-6 -alkylene)-, or -C 1-6 -Alkylene-OC 1-6 -alkylene-, Each L 2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] is.

[0137] In some such embodiments of compounds of Formula (VIII), p is 1, a is 1, and b is 1.

[0138] In certain embodiments of compounds of Formula (VIII), R 1 is -C(=O)N(C 1-3 alkyl)2, Each L 1 is —C(O)NH—, Each L 2 is -(OCH2CH2) p- and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] is.

[0139] In certain embodiments of compounds of Formula (VIII), R 1 is -C(=O)N(C 1-3 alkyl)2, Each L 1 is —C(O)NH—, Each L 2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] is.

[0140] In some such embodiments of compounds of Formula (VIII), a is 1 and b is 0.

[0141] Also provided herein are compounds of formula (IX): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers, or isotopic form, wherein: L B is (i)-OC 1-6 -Alkylene-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)-, or -C 1-6 -alkylene-O-.

[0142] In certain embodiments of compounds of Formula (IX), L B teeth, A linker containing -OCH2-, -CH2N(CH3)-, or -CH2O-.

[0143] Also provided herein are compounds of formula (X-1): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers, or isotopic form, wherein: R 1 is H or -C(=O)N(C 1-3 alkyl)2.

[0144] In certain embodiments of compounds of Formula (X-1), R 1 is H.

[0145] In certain embodiments of compounds of Formula (X-1), R 1 is -C(=O)N(C 1-3 alkyl)2.

[0146] In certain embodiments of compounds of Formula (X-1), R 1 teeth, [ka] is.

[0147] Also provided herein is a compound of formula (X): [ka] or a salt thereof, single stereoisomer, mixture of stereoisomers, or isotopic form thereof;

[0148] In the formula, R 1 is H or -C(=O)N(C 1-3 alkyl)2.

[0149] In certain embodiments of compounds of Formula (X), R1 is H.

[0150] In certain embodiments of compounds of Formula (X), R 1 is -C(=O)N(C 1-3 alkyl)2.

[0151] In certain embodiments of compounds of Formula (X), R 1 teeth, [ka] is.

[0152] In certain embodiments, the compound is [ka] is.

[0153] In certain embodiments, the compound is [ka] is.

[0154] In certain embodiments, the compound is [ka] is.

[0155] In certain embodiments, the compound is [ka] is.

[0156] In certain embodiments, the compound is [ka] is.

[0157] In certain embodiments, 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).

[0158] 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).

[0159] 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.

[0160] 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).

[0161] In certain embodiments, 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.

[0162] In certain embodiments, 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.

[0163] 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).

[0164] In certain embodiments, 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).

[0165] 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.

[0166] In certain embodiments, 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).

[0167] In certain embodiments, 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).

[0168] 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).

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] In certain embodiments, the compound is 3',6'-di(azetidin-1-yl)-6-(3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one.

[0175] In certain embodiments, the compound is 3',6'-di(azetidin-1-yl)-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one.

[0176] In certain embodiments, the compound is 3',6'-di(azetidin-1-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-2-diazospiro[indene-1,9'-xanthen]-3(2H)-one.

[0177] In certain embodiments, the compound is 1,1'-(6-((4-(((2-aminopyrimidin-4-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).

[0178] In certain embodiments, the compound is 1,1'-(6-((4-(((4-aminopyrimidin-2-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).

[0179] It should be noted that if there is a discrepancy between a depicted structure and the name for that structure, the depicted structure is given weight.

[0180] The above paragraphs present several embodiments of the compounds provided herein. In each instance, the embodiment includes both the listed compound(s), as well as salts, single stereoisomers, mixtures of stereoisomers, or isotopic forms thereof.

[0181] (b) Tagged protein In another aspect, provided herein are tagged proteins.

[0182] In certain embodiments, the tagged protein is a kinase. In certain embodiments, the tagged protein is a transcription factor. In certain embodiments, the tagged protein is a chromatin modulator. In certain embodiments, the tagged protein is an adaptor. In certain embodiments, the tagged protein is a transporter. In certain embodiments, the tagged protein is a pathogenic aggregator.

[0183] In certain embodiments, the tagged protein is a histone, hi certain embodiments, the histone is an H2B-HaloTag protein.

[0184] In certain embodiments, the tag is 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 those skilled in the art know how to generate proteins with fused HaloTag®.

[0185] In certain embodiments, the HaloTag® is derived from a bacterial enzyme. In certain embodiments, the bacterial enzyme is a haloalkane dehalogenase. In certain embodiments, the HaloTag® is part of a protein fused to a HaloTag™. In certain embodiments, the HaloTag® is expressed using standard recombinant protein expression techniques. In certain embodiments, the coding region for the HaloTag® protein is inserted adjacent to a gene of interest. In certain embodiments, the HaloTag® is self-labeling. In certain embodiments, the HaloTag® specifically binds to a chloroalkane linker. In certain embodiments, the binding of the HaloTag® to the chloroalkane linker is irreversible under physiological conditions. In certain embodiments, the HaloTag® is used as a protein label in enzymatic assays. In certain embodiments, the HaloTag® is used as a protein label in cellular imaging. In certain embodiments, the HaloTag® is used as a protein tag in fluorescence microscopy. In certain embodiments, the HaloTag® is used as a protein tag in protein sequencing. In certain embodiments, the HaloTag® is used as a protein tag to identify the subcellular location of a protein.

[0186] In certain embodiments, the HaloTagged protein is a kinase, a transcription factor, a chromatin modulator, an adaptor, a transporter, or a pathogenic aggregator.

[0187] In certain embodiments, 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 those skilled in the art know how to make SNAP-tagged proteins.

[0188] In certain embodiments, the SNAP-tag® is engineered from the enzyme alanine dioxylate transaminase. In certain embodiments, the CLIP-tag® is self-labeling. In certain embodiments, the SNAP-tag® is O 6 In certain embodiments, the SNAP-tag® is encoded by the O-methylguanine-DNA methyltransferase (MGMT) gene. 6 It reacts covalently with benzylguanine derivatives. In certain embodiments, the SNAP-tag® is used as a protein label in enzymatic analysis. In certain embodiments, the SNAP-tag® is used as a protein label in cell imaging. In certain embodiments, the SNAP-tag® is used as a protein label in fluorescence microscopy. In certain embodiments, the SNAP-tag® is used as a protein label in protein sequencing. In certain embodiments, the SNAP-tag® is used as a protein label to identify the subcellular localization of proteins.

[0189] In certain embodiments, the SNAP-tagged protein is a kinase, a transcription factor, a chromatin modulator, an adaptor, a transporter, or a pathogenic aggregator.

[0190] In certain embodiments, 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 those skilled in the art know how to make CLIP-tagged proteins.

[0191] In certain embodiments, the CLIP-tag® is self-labeling. In certain embodiments, the CLIP-tag® is an orthogonal tag. In certain embodiments, the CLIP-tag® is an O 6 -methylguanine-DNA methyltransferase (MGMT) gene. In certain embodiments, the CLIP-tag® covalently reacts with benzylcytosine derivatives. In certain embodiments, the CLIP-tag® is used as a protein label in protein complementation assays. In certain embodiments, the CLIP-tag® is used as a protein label in protein-protein interaction studies. In certain embodiments, the CLIP-tag® is used as a protein label in enzyme analysis. In certain embodiments, the CLIP-tag® is used as a protein label in cell imaging. In certain embodiments, the CLIP-tag® is used as a protein label in fluorescence microscopy. In certain embodiments, the CLIP-tag® is used as a protein label in protein sequencing. In certain embodiments, the CLIP-tag® is used as a protein label to identify the subcellular localization of a protein.

[0192] In certain embodiments, the CLIP-tagged protein is a kinase, a transcription factor, a chromatin modulator, an adaptor, a transporter, or a pathogenic aggregator.

[0193] (c) Labeled Proteins and Methods In another aspect, provided herein is a method for producing labeled proteins for measuring individual protein translocation within a cellular environment.

[0194] In certain embodiments, the method includes contacting a sample containing a tagged protein with a compound described herein to obtain a labeled protein. Without being bound by any mechanism or theory, it is understood that the tagged protein generally has an engineered active site that can specifically bind to a reactive linker of a photoactive fluorescent dye compound, forming 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 tagged protein-compound covalent complex is referred to herein as a labeled protein. The covalent bond forms rapidly and essentially irreversibly under physiological conditions.

[0195] The labeled protein can then be exposed to light, for example, 405 nm light, which causes the covalently attached photoactive dye compound to fluoresce.

[0196] 2 shows a schematic diagram for preparing a photoactivated labeled protein. As shown in the diagram and as described above, a tagged protein described herein is contacted with a compound, such as a photoactivated fluorescent dye compound described herein, to form a covalent bond between the tagged protein and the compound, forming the labeled protein. The labeled protein is then treated with light, such as 405 nm light, which causes the covalently bound compound to fluoresce.

[0197] In certain embodiments, a tagged protein described herein is contacted with a compound, such as a photoactive fluorescent dye compound described herein, to form the labeled protein. In certain embodiments, the compound is a fluorophore. In certain embodiments, the compound comprises a moiety that binds to the tagged protein to form the labeled protein. In certain embodiments, the tagged protein is covalently bound to the compound to form the labeled protein. In certain embodiments, the compound is covalently bound to a lysine residue of the tagged protein to form the labeled protein. In certain embodiments, the compound is covalently bound to a cysteine ​​residue of the tagged protein to form the labeled protein. In certain embodiments, the compound is covalently bound to an aspartic acid residue of the tagged protein to form the labeled protein.

[0198] In certain embodiments, the compound portion of the labeled protein emits fluorescence when exposed to light. Without being bound by any mechanism or theory, the compound portion of the labeled protein may undergo Wolff rearrangement when exposed to light, and then the compound is usually decarboxylated. In certain embodiments, the labeled protein is illuminated with a 405 nm light source. In certain embodiments, the intensity of the 405 nm light source is about 365 mW. In certain embodiments, the labeled protein is illuminated for about 5 minutes.

[0199] In certain embodiments, the solution of labeled protein is filtered after incubation, hi certain embodiments, the solution is filtered through a desalting column.

[0200] In certain embodiments, the labeled protein fluoresces when exposed to light. In certain embodiments, the light is a laser. In certain embodiments, the wavelength (λ) of the light is about 405 nm. In certain embodiments, the intensity of the 405 nm light is less than 1 mW. In certain embodiments, the intensity of the 405 nm light is about 12 mW. In certain embodiments, the intensity of the 405 nm light is between about 0 mW and about 12 mW. In certain embodiments, the intensity of the 405 nm light is greater than 12 mW, e.g., 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 certain embodiments, the laser is pulsed. In certain embodiments, the laser intensity increases over time during pulsation. In certain embodiments, light at 561 nm (λ) is used to excite the fluorophore of the labeled protein. In certain embodiments, the wavelength (λ) of the light is about 561 nm. In certain embodiments, the intensity of the 561 nm light is about 500 mW. In certain embodiments, the sample comprises one or more living cells, and the protein is labeled in the one or more living cells. In certain embodiments, the protein is labeled in an intracellular compartment of the one or more living cells. In certain embodiments, the protein is labeled in the nucleus of the one or more living cells. In certain embodiments, the protein is labeled in the cytoplasm of the one or more living cells. In certain embodiments, the protein is labeled in the cell membrane of the one or more living cells. In certain embodiments, the protein is labeled in the mitochondria of the one or more living cells. In certain embodiments, the protein is labeled in the outer membrane of the mitochondria of the one or more living cells. In certain embodiments, the protein is labeled in the inner membrane of the mitochondria of the one or more living cells. In certain embodiments, the protein is labeled in the mitochondrial matrix of the one or more living cells.In certain embodiments, the protein is labeled in the Golgi apparatus of the one or more living cells.In certain embodiments, the protein is labeled in the lysosome of the one or more living cells.In certain embodiments, the protein is labeled in the endosome of the one or more living cells.In certain embodiments, the protein is labeled in the endoplasmic reticulum of the one or more living cells.In certain embodiments, the protein is labeled in the endoplasmic reticulum membrane of the one or more living cells.In certain embodiments, the protein is labeled in the rough endoplasmic reticulum of the one or more living cells.

[0201] In certain embodiments, individual protein movement within the cellular environment of the one or more cells is measured. In certain embodiments, individual protein movement within an intracellular compartment of the one or more cells is measured. In certain embodiments, individual protein movement within the nucleus of the one or more cells is measured. In certain embodiments, individual protein movement within the cytoplasm of the one or more living cells is measured. In certain embodiments, individual protein movement within the plasma membrane of the one or more living cells is measured. In certain embodiments, individual protein movement within the mitochondria of the one or more living cells is measured. In certain embodiments, individual protein movement within the outer membrane of the mitochondria of the one or more living cells is measured. In certain embodiments, individual protein movement within the inner membrane of the mitochondria of the one or more living cells is measured. In certain embodiments, individual protein movement within the mitochondrial matrix of the one or more living cells is measured. In certain embodiments, individual protein movement within the Golgi apparatus of the one or more living cells is measured. In certain embodiments, individual protein movement within the lysosomes of the one or more living cells is measured. In certain embodiments, individual protein movement within the endosomes of the one or more living cells is measured. In certain embodiments, measure the movement of individual proteins in the endoplasmic reticulum of one or more living cells.In certain embodiments, measure the movement of individual proteins in the endoplasmic reticulum membrane of one or more living cells.In certain embodiments, measure the movement of individual proteins in the rough endoplasmic reticulum of one or more living cells.In certain embodiments, measure the movement of individual proteins in the rough endoplasmic reticulum of one or more living cells.In certain embodiments, measure in real time.

[0202] In certain embodiments, the one or more live cells are prepared for imaging by, for example, incubating at about 37°C. In certain embodiments, the one or more live cells are incubated, for example, in the presence of about 5% CO. In certain embodiments, the one or more live cells are incubated overnight or for about 8-10 hours. In certain embodiments, the one or more live cells are prepared for imaging by incubating with a compound, e.g., a photoactive fluorescent dye compound described herein, at a concentration of about 1 nM. In certain embodiments, the one or more live cells are prepared for imaging by incubating with a compound, e.g., a photoactive fluorescent dye compound described herein, at a concentration of about 200 nM. In certain embodiments, the one or more live cells are prepared for imaging by incubating with a compound, e.g., a photoactive fluorescent dye compound described herein, at a concentration of about 1 to about 200 nM. In certain embodiments, the one or more live cells are incubated with a compound, e.g., a photoactive fluorescent dye compound described herein, for about 45 minutes.

[0203] In certain embodiments, protein movement within the cellular environment is observed by confocal microscopy. In certain embodiments, protein movement within the cellular environment is observed by localization microscopy. In certain embodiments, protein movement within the cellular environment is observed by super-resolution microscopy. In certain embodiments, protein movement within the cellular environment is observed by single molecule localization microscopy ("SMLM"). In certain embodiments, protein movement within the cellular environment is observed by photoactivated localization microscopy ("PALM"). In certain embodiments, protein movement within the cellular environment is observed by stochastic optical reconstruction microscopy ("STORM").

[0204] In certain embodiments, protein trafficking within the cellular environment is analyzed via a maximum likelihood estimator model to detect single molecule fluorescence.

[0205] In certain embodiments, signal-to-noise ratio ("SNR") is used as a proxy for single-molecule brightness. In certain embodiments, SNR is analyzed via a log-likelihood ratio test. In certain embodiments, the compounds, e.g., photoactive fluorescent dye compounds described herein, exhibit an SNR similar to that of commercially available photoactivatable fluorescent dyes. In certain embodiments, the compounds exhibit an SNR similar to that of commercially available non-photoactivatable fluorescent dyes. In certain embodiments, labeling specificity is determined using a microscopy method, e.g., confocal microscopy, localization microscopy, super-resolution microscopy, SMLM, PALM, or STORM. In certain embodiments, labeling specificity is calculated by comparing the number of spots detected via a microscopy method, e.g., localization microscopy, super-resolution microscopy, SMLM, PALM, or STORM, with a control sample. In certain embodiments, the labeling specificity of the compounds described herein is higher than that of commercially available photoactivatable fluorescent dyes.

[0206] 6. Compound manufacturing method The compounds of formula (I), (IB), (II), (III), (IV), (V), (VI), and (VIa) can be prepared using conventional organic synthesis and commercially available starting materials. By way of example and not limitation, the compounds of formula (I), (IB), (II), (III), (IV), (V), (VI), and (VIa) can be prepared as outlined in Scheme 1a and Scheme 1b shown below, as well as in the Examples described herein. It should be noted that those skilled in the art will know how to modify the procedures described in the exemplary schemes to arrive at the desired products.

[0207] Scheme 1a [ka] As shown in Scheme 1a, R, G, and R 1Compounds of formula (I), (IB), (II), (III), (IV), (V), (VI), and (VIa), 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 reacted with a suitable catalyst, such as a bromine or triflate derivative. Intermediate (C), as described herein, can be prepared, for example, by converting 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid to the corresponding triflate derivative by conventional organic synthesis methods. Intermediate (C) may also be prepared according to 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, 13, 985-988 (Grimm et al., 2016). Alternatively, intermediate (C) can be prepared starting from 1,2,4-benzenetricarboxylic acid reacted 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 reacted with an appropriately substituted azetidine and a palladium catalyst, such as Pd(dba) in the presence of a ligand and a base, such as cesium carbonate, in a solvent, such as dioxane, and heated at a temperature ranging from about 25 to about 100° C. to provide intermediate (D).Intermediate D is then reacted with an acid, such as TFA, or alternatively, a base, such as lithium hydroxide or trimethyltin hydroxide, at a temperature of about 0 to about 25° C., followed by coupling with a suitable linker moiety under basic conditions to provide intermediate (E). Intermediate (E) is reacted with oxalyl chloride in a suitable solvent, such as dichloromethane, at a temperature of about 0 to about 25° C., followed by reaction with a solution of freshly prepared diazomethane in EtO (see, e.g., F. Arndt, “Diazomethane,” Org. Synth. 1935, 15, 3) at a temperature of about 0° C. to provide a compound of formula (IV).

[0208] Alternatively, intermediate (E) can be reacted with a solution of 1-chloro-N,N,2-trimethylprop-1-en-1-amine in a suitable solvent at room temperature in an aprotic solvent in the presence of 4 Å molecular sieves, followed by reaction with trimethylsilyldiazomethane to give a compound of formula (IV). Such synthetic methods may be used to optimize the synthesis of specific 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.

[0209] Scheme 1b [ka] As shown in Scheme 1b, R 1Compounds of formula (IV) and (V), where , are as defined herein, can be prepared from intermediate (D') via a cross-coupling reaction using an appropriately substituted azetidine and a palladium catalyst, such as Pd(dba) , in the presence of a ligand and a base, such as cesium carbonate, in a solvent, such as dioxane, at temperatures ranging from about 25 to about 100 °C. Subsequent Arndt-Eistert reaction (see, e.g., F. Arndt, "Diazomethane," Org. Synth. 1935, 15, 3) affords compounds of formula (IV) and (V). Intermediate D' can be prepared from intermediate B1 in the presence of a peptide coupling reagent, such as T3P (propanephosphonic anhydride), in a solvent, as described herein.

[0210] Scheme 2 [ka] As shown in Scheme 2, R' can be -C(=O)OCH3 or -L A Compounds of Formulas (A) and (I)-(X), wherein -Z, and all other variables are as defined herein, can be prepared using Ghosez reagent and a diazomethane reagent, such as TMSCHN2.

[0211] In one embodiment, provided herein is a method for preparing a compound of formula (VIII): [ka] During the ceremony, R 1 is H or -C(=O)N(C 1-3 alkyl)2, Each L 1 is, independently, -OC 1-6 -Alkylene-, -C(O)NH-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)(C 1-6 -alkylene)-, or -C 1-6 -Alkylene-OC1-6 -alkylene-, Each L 2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] and R 1 If H and a is 1, then L 1 teeth, [ka] Instead, a wavy line [ka] represents a point of attachment to another moiety of the compound, and the process comprises: A compound of formula (a): [ka] During the ceremony, R 1 is H or -C(=O)N(C 1-3 alkyl)2, R is -C(=O)OCH3 or -C 1-6 -Alkylene-OC 1-6 -alkylene-(OCH2CH2)-Z, Z is [ka] A compound The method includes converting the compound of formula (VIII) using Ghosez's reagent in the presence of a diazomethane agent and a solvent to prepare the compound of formula (VIII).

[0212] In one embodiment, provided herein is a method for preparing a compound of formula (VIII-a): [ka] During the ceremony, R' is [ka] and R 1 is H or -C(=O)N(C 1-3 alkyl)2, Each L 1 is, independently, -OC 1-6 -Alkylene-, -C(O)NH-, -C(O)NH(C 1-3 -alkylene)-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)(C 1-6 -alkylene)-, or -C 1-6- Alkylene-OC 1-6 -alkylene-, Each L 2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] and R 1 If H and a is 1, then L 1 teeth, [ka] Instead, a wavy line [ka] represents a point of attachment to another moiety of the compound, and the process comprises providing a compound of formula (a-1): [ka] During the ceremony, R'= [ka] R 1 is H or -C(=O)N(C 1-3 alkyl)2, R is -C(=O)OCH3 or -C(=O)NH(C 1-3 alkylene)-(OCH2CH2)-Z, Z is [ka] A compound The method includes converting the compound of formula (VIII-a) using Ghosez's reagent in the presence of a diazomethane agent and a solvent to prepare the compound of formula (VIII-a).

[0213] In one embodiment, provided herein is a method for preparing a compound of formula (VIII-a): [ka] During the ceremony, R' is [ka] and R 1 is H or -C(=O)N(C 1-3 alkyl)2, Each L 1 is, independently, -OC 1-6 -Alkylene-, -C(O)NH-, -C(O)NH(C 1-3 -alkylene)-, -C 1-6 -Alkylene-N(C 1-3 -alkyl)(C 1-6 -alkylene)-, or -C 1-6- Alkylene-OC 1-6 -alkylene-, Each L 2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] The process comprises the step of reacting a compound of formula (a-1) [ka] During the ceremony, R'= [ka] R 1 is H or -C(=O)N(C 1-3 alkyl)2, R is -C(=O)OCH3 or -C(=O)NH(C 1-3 alkylene)-(OCH2CH2)-Z, Z is [ka] A compound The method includes converting the compound of formula (VIII-a) using Ghosez's reagent in the presence of a diazomethane agent and a solvent to prepare the compound of formula (VIII-a).

[0214] In another embodiment, provided herein is a method for preparing a compound of formula (VIII-a): [ka] During the ceremony, R' is [ka] and R 1 is H or -C(=O)N(C 1-3 alkyl)2, Each L 1 teeth, -C(O)NH(C 1-3 -alkylene)-, Each L2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] and The process comprises reacting a compound of formula (a-1) [ka] During the ceremony, R'= [ka] R 1 is H or -C(=O)N(C 1-3 alkyl)2, R is -C(=O)OCH3 or -C(=O)NH(C 1-3 alkylene)-(OCH2CH2)-Z, Z is [ka] using Ghosez's reagent in the presence of a diazomethane agent and a solvent to prepare a compound of formula (VIII-a).

[0215] In one embodiment of the compounds of Formula (VIII) and Formula (VIII-a), p is 1, a is 1, and b is 1.

[0216] In one embodiment, provided herein is a method for preparing a compound of formula (XI): [ka] During the ceremony, R 1 is H or -C(=O)N(C 1-3 alkyl)2, Each L1 teeth, -C(O)NH(C 1-3 -alkylene)-, Each L 2 is -(OCH2CH2) p - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2, Z is [ka] The process comprises the step of: [ka] During the ceremony, R 1 is H or -C(=O)N(C 1-3 alkyl)2, R is -C(=O)OCH3 or -C(=O)NH(C 1-3 alkylene)-(OCH2CH2)-Z, Z is [ka] using Ghosez's reagent in the presence of a diazomethane agent and a solvent to prepare a compound of formula (XI).

[0217] In one embodiment of the compound of formula (XI), p is 1, a is 1, and b is 1.

[0218] In one embodiment, the diazomethane agent is trimethylsilyldiazomethane diethyl ether. In one such embodiment, the solvent comprises dichloromethane. In one such embodiment, the solvent is dichloromethane. In one such embodiment, the solvent is a mixture of dichloromethane and acetonitrile. In one such embodiment, the solvent is dichloromethane and acetonitrile (1:1).

[0219] In one embodiment, the method further comprises using a desiccant and a metal oxide. In one such embodiment, the desiccant is a molecular sieve and the metal oxide is calcium oxide. In one embodiment, the method further comprises using an activator. In one embodiment, the activator is KF, KBr, or KI. In one embodiment, the activator is KF.

[0220] In one embodiment, the method comprises using equal ratios of trimethylsilyldiazomethane diethyl ether and KF. In one such embodiment, the method comprises using trimethylsilyldiazomethane diethyl ether (8 equivalents) and KF (8 equivalents). In one embodiment, the method further comprises using 2.5 equivalents of molecular sieves and 3 equivalents of calcium oxide.

[0221] In one embodiment, the compounds of Formula (VIII), Formula (VIII-a), and Formula (XI) prepared by the methods described herein are substantially chemically pure. In one embodiment, the compounds of Formula (VIII), Formula (VIII-a), and Formula (XI) prepared by the methods described herein are substantially free of chemical impurities.

[0222] 7. How to prepare labeled proteins By way of example and not limitation, the labeled proteins described herein can be prepared as outlined in Scheme 3, shown below, and in the Examples described herein. It should be noted that one of skill in the art will know how to modify the procedures described in the exemplary scheme to arrive at a desired product.

[0223] Scheme 3 [ka] As shown in Scheme 3, R, G, X, and L ACompounds of formula (I), (IB), (II), (III), (IV), (V), (VI), and (VIa), where Z and Z are as defined herein, can be prepared by contacting a tagged protein described herein with a compound, e.g., a photoactive fluorescent dye compound described herein, resulting in the formation of a covalent bond between the tagged protein and the compound, forming the non-fluorescently labeled protein. The non-fluorescently labeled protein is then treated with light (e.g., 405 nm light), which causes the compound, and thus the labeled protein, to fluoresce. See also Figure 2. [Example]

[0224] 8. Working Example The following examples are offered by way of illustration and not by way of limitation, and one skilled in the art will be able to modify the procedures set forth in the illustrative examples to arrive at the desired product.

[0225] Abbreviations used [Table 1] TIFF2026503623000174.tif160165

[0226] Intermediates

[0227] Intermediate A

[0228] Synthesis of tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate. Intermediate A [ka]

[0229] Step 1: Preparation of 3',6'-diacetoxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid. Compound A1 [ka]

[0230] 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 at 110 °C for 3 h. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: 50-55% EtOAc in 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. 1 H NMR: (400 MHz, CDCl3): δ 8.35 (d, 1H), 1.13 (d, 1H), 7.87 (s, 1H), 7.12 (s, 2H), 6.78-6.84 (dd, 4H), 2.31 (s, 6H) ppm. m / z = 461.5 [M+H]+.

[0231] Step 2: Preparation of 6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl diacetate. Compound A2 [ka]

[0232] 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) as a brown solid, which was used in the next step without further purification. m / z = 517.5 [M+H]+.

[0233] Step 3: Preparation of tert-butyl 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate. Compound A3 [ka]

[0234] 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 (17.0 mL) and methanol (17 mL) was added sodium hydroxide (1 M, 10.2 mL, 10.2 mmol). The mixture was stirred at room temperature for 12 hours and then concentrated under reduced pressure. The residue was diluted with water (50 mL) and acidified with saturated aqueous citric acid. The aqueous layer was extracted with ethyl acetate (2 × 50 mL), and the combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 35% EtOAc in 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]+.

[0235] Step 4: Preparation of tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate. Compound A4 [ka]

[0236] 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 hours. The mixture was quenched with ice-water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were 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 in 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. 1 H NMR: (400 MHz, DMSO-d6): δ 8.25 (d, 2H), 7.98 (s, 1H), 7. 69 (s, 2H), 7.31-7.32 (d, 2H), 7.14-7.16 (d, 2H), 1.5 (s, 9H) ppm. m / z = 697.5[M+H]+.

[0237] Intermediate B

[0238] Synthesis of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate Intermediate B [ka]

[0239] Step 1: Preparation of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate pyridinium salt Compound B1 [ka]

[0240] 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 deep purple solution was poured into 200 mL of ice water, and the slurry was vigorously stirred. The green-yellow solid was collected by vacuum filtration and dried under suction. The solid was recrystallized from a mixture of 750 mL of acetic anhydride and 250 mL of pyridine to give a white solid. The white solid was recrystallized three times from 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]+.

[0241] Step 2: Preparation of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate. Compound B2 [ka]

[0242] 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 CHCl3 containing 10% IPA (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 in 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]+.

[0243] Intermediate C

[0244] Synthesis of tert-butyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate. Intermediate C [ka]

[0245] 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) was added 1,1-di-tert-butoxy-N,N-dimethylmethanamine (9.2 g, 47.2 mmol), and the mixture was stirred under reflux for 24 hours. 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 brown solid, which was used without further purification.

[0246] Intermediate D'

[0247] Synthesis of 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide. Intermediate D' [ka]

[0248] 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. T3P solution (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 hours. 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 brine (3x) at 0°C. The organic layers were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 0–50% EtOAc in 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.

[0249] 8.1 Reference 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): compound 1 [ka]

[0250] 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). Compound 1.1 [ka]

[0251] 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 and then charged with Pd2(dba)3 (0.13 g, 0.14 mmol) and XPhos (0.14 g, 0.29 mmol). The mixture was stirred at 100 °C for 16 h. 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 × 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 in 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]+.

[0252] Step 2: Preparation of 3',6'-bis(3-(methoxycarbonyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid. Compound 1.2 [ka]

[0253] 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 at room temperature for 12 h 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].

[0254] 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). Compound 1.3 [ka]

[0255] 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 hour. 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 warmed to room temperature and stirred for 12 hours. The mixture was diluted with ice-cold water (20 mL) and extracted with ethyl acetate (2 x 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 in 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]+.

[0256] 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). Compound 1.4 [ka]

[0257] 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 warmed to room temperature, stirred for 30 min, and then concentrated under reduced pressure. The residue was dissolved in anhydrous CHCl (20 mL), and a freshly prepared solution of diazomethane in EtO (see, e.g., 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 in petroleum ether). The residue was further purified by chiral SFC (Chiralcel-OJ-3, mobile phase 30% MeOH in CO) 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. 1 H NMR (400 MHz, DMSO-d6): δ 8.69 (t, J = 5.6 Hz, 1H), 7.99-7.96 (m, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 6.72 (d, J = 8.4 Hz, 2H), 6.25 (d, J = 2.4 Hz, 2H), 6.19-6.16 (m, 2H), 4.07-4.03 (m, 4H), 3.94-3.89 (m, 4H), 3.67 (s, 6H), 3.65-3.57 (m, 4H), 3.48-3.40 (m, 10H), 1.67-1.64 (m, 2H), 1.43-1.39 (m, 2H), 1.33-1.31 (m, 2H), 1.25-1.23 (m, 2H) ppm. m / z =800.3 [M+H]+.

[0258] 8.2 Reference 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). compound 2 [ka]

[0259] 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 achiral SFC (YMC-PAK DIOL, mobile phase, 30% MeOH in CO) 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. 1H NMR (400 MHz, DMSO-d6): δ 8.69 (t, J = 8.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.48 (s, 1H), 6.66 (d, J = 8.8 Hz, 2H), 6.18-6.10 (m, 4H), 3.92-3.82 (m, 8H), 3.60 (t, J = 13.2 Hz, 2H), 3.46-3.39 (m, 12H), 1.65 (t, J = 14.8 Hz, 2H), 1.42-1.28 (m, 4H), 1.24-1.22 (m, 2H). m / z = 770.23 [M+H]+.

[0260] 8.3 Reference 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. compound 3 [ka]

[0261] Step 1: Preparation of methyl 3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate. Compound 3.1 [ka]

[0262] 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, and 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 at 110 °C for 2 h. The mixture was filtered through Celite, and the filtrate was diluted with water (50 mL) and extracted with EtOAc (2 × 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 in 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]+.

[0263] Step 2: Preparation of 3',6'-bis((R)-2-(methoxymethyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid. Compound 3.2 [ka]

[0264] 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, diluted with water (5 mL), and extracted with EtOAc (10 mL). The aqueous layer was acidified with 1 N HCl and extracted with 10% MeOH in CHCl (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]+.

[0265] 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. Compound 3.3 [ka]

[0266] 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 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 minutes, and 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 hours. 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 in 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].

[0267] 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. compound 3.4 [ka]

[0268] 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 warmed to room temperature and stirred at room temperature for 30 minutes. The mixture was concentrated under a stream of nitrogen gas. The residue was dissolved in dry CHCl (20 mL), and freshly prepared diazomethane (see, e.g., F. Arndt, “Diazomethane,” Org. Synth. 1935, 15, 3) in EtO (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 chromatography using neutral alumina (eluent: 0–10% acetone in petroleum ether). The residue was further purified by achiral SFC (YMC PAK-DIOL, mobile phase: 25% MeOH in CO) 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. 1 H NMR (400 MHz, DMSO-d6): δ 8.69 (t, J = 5.2 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 6.68 (d, J = 8.4 Hz, 2H), 6.38 (s, 2H), 6.29-6.27 (m, 2H), 4.17-4.16 (m, 2H), 3.84 (bs, 2H), 3.60-3.37 (m, 24H), 2.32-2.27 (m, 2H), 2.13-2.07 (m, 2H), 1.67-1.63 (m, 2H), 1.42-1.29 (m, 6H) ppm. m / z = 772.7 [M+H]+.

[0269] 8.4 Reference 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). compound 4 [ka]

[0270] Step 1: Preparation of methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate. Compound 4.1 [ka]

[0271] 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) in a microwave oven. The vial was degassed with nitrogen for 15 min, and then Pd(dba) (0.18 g, 0.19 mmol) and RuPhos Pd G (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 CHCl (100 mL). The filtrate was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: 0-20% MeOH in CHCl) 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].

[0272] Step 2: Preparation of 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid. Compound 4.2 [ka]

[0273] 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 hours. After adding water (2 mL), the mixture was cooled to 0°C and acidified to pH 5 with 1N HCl. 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]+.

[0274] 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). Compound 4.3 [ka]

[0275] 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 minutes, and then propylphosphonic anhydride solution (50% in ethyl acetate, 0.3 g, 0.96 mmol) was added dropwise. The mixture was warmed to room temperature and stirred for 16 hours. 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 in 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].

[0276] 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). Compound 4.4 [ka]

[0277] 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 warmed 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, e.g., F. Arndt, “Diazomethane,” Org. Synth. 1935, 15, 3) (approximately 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 using neutral alumina (eluent: 0-5% MeOH in CHCl). The residue was further purified by SFC (DCPAK P4VP, mobile phase, 45% CO with (0.2% DEA in ACN)) 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. 1H NMR (400 MHz, DMSO-d6): δ 8.70 (t, J = 5.6 Hz, 1H), 7.99-7.96 (m, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 6.70 (d, J = 8.4 Hz, 2H), 6.22 (d, J = 8.4 Hz, 2H), 6.17 (d, J = 8.4 Hz, 2H), 4.04-4.01 (m, 4H), 3.91-3.81 (m, 6H), 3.60 (t, J = 6.4 Hz, 2H), 3.46-3.39 (m, 8H), 3.32-3.28 (m, 2H), 2.88 (s, 6H), 2.83 (s, 6H), 1.67-1.63 (m, 2H), 1.42-1.28 (m, 4H), 1.24-1.22 (m, 2H) ppm. m / z = 826.63 [M+H]+.

[0278] 8.5 Reference 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). compound 5 [ka]

[0279] 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. Compound 5.1 [ka]

[0280] The title compound was prepared using a procedure similar to Steps 1-3 of Example 7, substituting N,N-dimethylazetidine-3-carboxamide hydrochloride for 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.

[0281] Alternatively, the title compound was prepared according to the procedure in Step 2 of Example 28.

[0282] 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). Compound 5.2 [ka]

[0283] 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: X-BRIDGE C8, mobile phase: 0-100% aqueous ACN) to give the title compound (0.008 g, 12%) as a light brown solid. 1H NMR (400 MHz, DMSO-d6): δ 9.21 (t, J = 12.0 & 6.0 Hz, 1H), 8.21 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.50 (s, 1H), 7.44 (d, J = 8 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 8.4 Hz, 4H), 6.21 (s, 2H), 6.16 (d, J = 8.4 Hz, 2H), 5.46 (s, 2H), 4.45-4.35 (m, 2H), 4.06-4.01 (m, 4H), 3.91-3.81 (m, 6H), 2.88 (s, 6H), 2.83 (s, 6H) ppm. m / z = 873.3 [M+H]+.

[0284] 8.6 Reference 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). compound 6 [ka]

[0285] The title compound was prepared using a procedure similar to steps 1-4 of Example 1, replacing methyl azetidine-3-carboxylate hydrochloride in step 1 with methyl (R)-azetidine-2-carboxylate hydrochloride, and replacing intermediate A in step 1 with intermediate C. The residue was purified by preparative HPLC (column: XBridge C18, mobile phase: 0-100% ACN in water) to give the title compound (0.0032 g, 16% yield) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.71-8.69 (m, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8 Hz, 1H), 7.44 (d, J = 4.8Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H), 6.28 (dd, J = 3.2 Hz, 2H), 6.24-6.21 (m, 2H), 4.68-4.63 (m, 2H), 3.89-3.81 (m, 2H), 3.71-3.67 (m, 8H), 3.61-3.57 (m, 2H), 3.48-3.40 (m, 6H), 3.31-3.29 (m, 4H), 2.53-2.48 (m, 4H), 1.67-1.64 (m, 2H), 1.41-1.20 (m, 6H) ppm. m / z = 800.6 [M+H]+.

[0286] 8.7 Reference 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. compound 7 [ka]

[0287] Step 1: Preparation of methyl 3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate. Compound 7.1 [ka]

[0288] 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 minutes, and 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 minutes and heated at 120 °C for 16 hours. 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 obtain methyl 3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.90 g, yield 47%) as a purple solid.

[0289] 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. Compound 7.2 [ka]

[0290] 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 oxalyl chloride (1 g, 8 mmol) at 0 °C. The mixture was warmed 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 EtO (ca. 0.5 M, 100 mmol) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 15 min and then warmed to room temperature. The mixture was stirred 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 in petroleum ether). The residue was further purified by SFC (column: YMC PACK DIOL-120, mobile phase: CO2 containing 20% ​​MeOH) 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.

[0291] 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. Compound 7.3 [ka]

[0292] 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.

[0293] 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. Compound 7.4 [ka]

[0294] 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 minutes, and 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 hours. The mixture was concentrated under reduced pressure and purified by preparative HPLC (column: XSELECT-C18, mobile phase: 0-100% aqueous ACN) 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. 1 H NMR (400 MHz, DMSO-d6): δ 8.68 (t, J = 5.6 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 6.68 (d, J = 8.8 Hz, 2H), 6.19 (s, 2H), 6.14 (d, J = 8.4 Hz, 2H), 5.60 (d, J = 6.8 Hz, 2H), 4.57-4.52 (m, 2H), 4.09-4.05 (m, 4H), 3.61-3.41 (m, 16H) 1.68-1.62 (m, 2H), 1.43-1.37 (m, 2H), 1.37-1.30 (m, 2H), 1.30-1.23 (m, 2H) ppm. m / z = 716.7[M+H]+.

[0295] 8.8 Reference 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. compound 8 [ka]

[0296] Step 1: Preparation of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate. Compound 8.1 [ka]

[0297] 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% solution in mineral oil, 29.2 mmol). The mixture was stirred at 0 °C for 30 min, and 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 in petroleum ether) to give tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (2.6 g, 33% yield) as a colorless oil.

[0298] Step 2: Preparation of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)carbamate. Compound 8.2 [ka]

[0299] 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) was added sodium hydride (60% dispersion in mineral oil, 0.93 g, 23.2 mmol) at 0 °C. The mixture was warmed 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 × 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 in 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.

[0300] Step 3: Preparation of 2-(2-((6-chlorohexyl)oxy)ethoxy)-N-methylethan-1-amine trifluoroacetate. Compound 8.3 [ka]

[0301] 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) was added trifluoroacetic acid (1.0 mL, 13.0 mmol) 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. The residue was triturated with EtO to give the title compound as a yellowish oil.

[0302] Step 4: Preparation of 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid. Compound 8.4 [ka]

[0303] The title compound was prepared using a procedure similar to that of Example 7, replacing azetidin-3-ol hydrochloride with azetidine hydrochloride in Step 1. The residue was purified by silica gel column chromatography (eluent: 0-8% MeOH in DCM). The residue was further purified by preparative HPLC (column: C18RP, mobile phase: 0-100% ACN in 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.

[0304] 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. Compound 8.5 [ka]

[0305] 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 minutes, and 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 hours. The mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: X-BRIDGE-C18, mobile phase: 0-100% water in 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. 1H NMR (400 MHz, DMSO-d6): δ 7.81-7.76 (m, 1H), 7.49 (d, J = 8.0 Hz, 1H), 6.99-6.94 (m, 1H), 6.71 (d, J = 8.4 Hz, 2H), 6.14-6.11 (m, 4H), 3.83 (t, J = 7.2 Hz, 8H), 3.60-3.41 (m, 6H), 3.26-3.12 (m, 6H), 2.87 (s, 3H), 2.32-2.25 (m, 4H), 1.67-1.62 (m, 2H), 1.43-1.29 (m, 6H) ppm. m / z = 698.7 [M+H]+.

[0306] 8.9 Reference 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. compound 9 [ka]

[0307] 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). Compound 9.1 [ka]

[0308] 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) was added a solution of lithium hydroxide monohydrate (0.021 g, 0.50 mmol) in water (0.35 mL) at 0° C. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated to give the desired product as a pink solid, which was used without further purification.

[0309] 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. Compound 9.2 [ka]

[0310] 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 hour, and 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 hours. The mixture was diluted with ice-cold water (20 mL) and extracted with ethyl acetate (2x). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: C18, mobile phase: 32% ACN in 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.

[0311] 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. Compound 9.3 [ka]

[0312] 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, 2 M solution in DCM) at 0 °C. The mixture was warmed 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 EtO (ca. 0.5 M, 50 mmol) was added under nitrogen at 0 °C. 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 in petroleum ether) and further purified by preparative HPLC (column: XBridge C18, mobile phase: 0-100% ACN in 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, yield 7.6%) as a solid. 1 H NMR (400 MHz, DMSO-d6): δ 8.68 (dd, J = 5.6 Hz, 1H), 7.98 (d, J = 1.6 Hz, 1H), 7.96 (d, J = 1.6 Hz, 1H),7.47 (s, 1H), 6.70 (d, J = 8.8 Hz, 2H), 6.22 (d, J = 2.0 Hz, 2H), 6.16 (dd, J = 2.4 Hz, 2H), 4.04-3.82 (m, 10H), 3.38-3.33 (m, 28H), 1.65 (m, 2H), 1.42-1.22 (m, 6H) ppm. m / z = 910.8[M+H]+.

[0313] 8.10 Reference 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. compound 10 [ka]

[0314] 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, T3P solution (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 hour. 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, and the residue was purified by preparative HPLC (column: Sunfire C18, mobile phase A: 0-100% aqueous ACN) 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. 1H NMR (400 MHz, DMSO-d6): δ 7.95 (dd, J = 1.2 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.43 (s 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.25-6.20 (m, 4H), 4.02-3.91 (m, 4H), 3.89-3.85 (m, 4H), 3.75-3.73 (m, 2H), 3.57-3.54 (m, 2H), 3.48-3.19 (m, 20H), 1.89-1.86 (m, 4H), 1.80-1.77 (m, 4H), 1.62-1.61 (m, 2H), 1.38-1.18 (m, 6H) ppm. m / z = 878.9 [M+H]+.

[0315] 8.11 Reference 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. compound 11 [ka]

[0316] 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 hour. 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, and the residue was purified by preparative HPLC (column: X-SELECT C18, mobile phase: 0-100% acetonitrile with 10 mM ammonium acetate in water) 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, yield 9.5%) as a solid. 1 H NMR (400 MHz, DMSO-d6): δ 8.69 (t, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 6.69 (d, J = 8.4 Hz, 2H), 6.22 (d, J = 2 Hz, 2H), 6.16 (d, J = 6.8 Hz, 2H), 4.06-4.01 (m, 4H), 3.92-3.79 (m, 6H), 3.42-3.29 (m, 20H), 2.35-2.20 (m, 8H), 2.17 (s, 6H), 1.69-1.63 (m, 2H), 1.42-1.20 (m, 6H) ppm. m / z = 936.7 [M+H]+.

[0317] 8.12 Reference 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). compound 12 [ka]

[0318] The title compound was prepared using the same procedure as in Example 11, replacing N-methylpiperazine with methylamine (2 M in THF). The residue was purified by preparative HPLC (column: X SELECT-C18, mobile phase: 0-100% ACN in water) to give the title compound. 1 H NMR (400 MHz, DMSO-d6): δ 8.70 (t, J = 5.2 Hz, 1H), 7.98-7.92 (m, 3H), 7.83 (d, J = 8.0 Hz, 1H), 7.48 (s 1H), 6.68 (d, J = 8.4 Hz,), 6.21 (d, J = 8.4 Hz, 2H), 6.14 (dd, J = 2.0 Hz and 2.4 Hz, 2H), 3.97-3.95 (m, 4H), 3.85-3.80 (m, 4H), 3.58 (t, J = 6.4 Hz, 2H), 3.47-3.28 (m, 12H), 2.60-2.49 (m, 6H), 1.67-1.63 (m, 2H), 1.42-1.23 (m, 6H) ppm. m / z = 798.8 [M+H]+.

[0319] 8.13 Reference 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. compound 13 [ka]

[0320] The title compound was prepared using a procedure similar to that in Example 7, replacing methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate, Intermediate B, and azetidin-3-ol hydrochloride in Step 1 with 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. The residue was purified by silica gel column chromatography (eluent: 50-100% ethyl acetate in 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. 1 H NMR (400 MHz, DMSO-d6): δ 8.68 (br s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H) 6.69 (d, J = 8.8 Hz, 2H), 6.19 (d, J = 2.4 Hz, 2H), 6.15 (d, J = 8.8 Hz, 2H), 3.90-3.87 (m, 4H), 3.61-3.57 (m, 6H), 3.47-3.39 (m, 10H), 3.18-3.15 (m, 2H), 2.09 (s, 12H), 1.67-1.62 (m, 2H), 1.42-1.39 (m, 2H), 1.32-1.30 (m, 2H), 1.26-1.23 (m, 2H) ppm. m / z = 770.7[M+H]+.

[0321] 8.14 Reference 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). compound 14 [ka]

[0322] The title compound was prepared using a procedure similar to that in Example 7, replacing methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate, Intermediate B, and azetidin-3-ol hydrochloride with 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 in Step 1. The residue was purified by silica gel column chromatography (eluent: 90% ethyl acetate in petroleum ether). The residue was further purified by preparative HPLC (column: X-BRIDGE C18, mobile phase: 0-100% aqueous ACN) 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. 1H NMR (400 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.98-795 (m, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.48-7.41 (m, 1H), 6.67 (dd, J = 3.2 Hz & 8.8 Hz, 2H), 6.14-6.10 (m, 4H), 4.81 (d, J = 8.0 Hz, 2H), 3.81-3.29 (m, 16H), 2.91-2.87 (m, 12H), 2.63-2.58 (m, 2H), 2.31-2.26 (m, 2H), 1.68-1.65 (m, 2H), 1.44-1.40 (m, 2H), 1.35-1.31 (m, 2H), 1.26-1.23 (m, 2H) ppm. m / z = 826.3 [M+H]+.

[0323] 8.15 Reference 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. compound 15 [ka]

[0324] The title compound was prepared using a procedure similar to that in Example 7, substituting methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate, Intermediate B, and azetidin-3-ol hydrochloride with 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. The residue was purified by silica gel column chromatography (eluent: 90% ethyl acetate in petroleum ether). The residue was further purified by preparative HPLC (column: X-BRIDGE C18, mobile phase: 0-100% aqueous ACN) 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. 1 H NMR (400 MHz, DMSO-d6): δ 8.69 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.45 (s, 1H), 6.66 (d, J = 8.4 Hz, 2H), 6.45 (d, J = 6.0 Hz, 2H), 6.28-6.24 (m, 2H), 4.96 (d, J = 3.2 Hz, 2H), 4.0-4.02 (m, 2H), 3.88-3.80 (m, 2H), 3.60-3.45 (m, 18H), 2.10-2.08 (m, 2H), 2.10-2.07 (m, 2H), 1.67-1.64 (m, 2H), 1.43-1.39 (m, 2H), 1.38-1.20 (m, 4H) ppm. m / z = 744.8 [M+H]+.

[0325] 8.16 Reference 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). compound 16 [ka]

[0326] The title compound was prepared using a procedure similar to that in Example 7, substituting methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate, Intermediate B, and azetidin-3-ol hydrochloride with 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. The residue was purified by silica gel column chromatography (eluent: 50-100% ethyl acetate in petroleum ether). The residue was further purified by preparative HPLC (column: X-BRIDGE C18, mobile phase: 0-100% aqueous ACN) 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. 1H NMR (400 MHz, DMSO-d6): δ 8.67 (t, J = 5.6 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 6.67 (d, J = 8.0 Hz) 2H), 6.29-6.26 (m, 4H), 3.57 (t, J = 6.8 Hz, 2H), 3.51-3.39 (m, 10H), 3.31-3.25 (m, 10H), 3.06 (s, 6H), 2.84 (s, 6H),2.17-2.16 (m, 2H), 2.07-2.05 (m, 2H), 1.67-1.63 (m, 2H), 1.42-1.22 (m, 6H) ppm. m / z = 854.9[M+H]+.

[0327] 8.17 Reference 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. compound 17 [ka]

[0328] 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. Compound 17.1 [ka]

[0329] 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 x 2). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash chromatography using silica gel in petroleum ether containing 0-100% ethyl acetate. The desired product was eluted in petroleum ether containing 90-95% ethyl acetate 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 + ).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. Compound 17.2 [ka]

[0330] 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:HO (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 hours. 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 (pH approx. 2) using 1N HCl. The resulting 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]+.

[0331] 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. Compound 17.3 [ka]

[0332] 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% ethyl acetate solution, 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 it was purified by reverse-phase column purification using a C18 column in 0-100% acetonitrile in water, and the desired product eluted in 22% acetonitrile in 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.

[0333] 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. Compound 17.4 [ka]

[0334] 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. Then, 1-chloro-N,N,2-trimethylprop-1-en-1-amine (20.6 μL, 0.156 mmol) was added via syringe, and the mixture was stirred at room temperature for 45 minutes. N,N-Diisopropylethylamine (27 μL, 0.156 mmol) was added, followed by TMSCHN2 (2 M in hexane, 78 μL, 0.156 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 in water). The residue was purified again by reverse-phase flash chromatography (30–100% ACN in water). A third purification was performed using a pipette column packed with basic alumina, eluting with 50–100% EtOAc in toluene. The product-containing fractions were concentrated, redissolved in dioxane, and dried on a lyophilizer to give a solid (1.7 mg, 6%). 1H NMR (400 MHz, Pyr) δ 9.39 (t, J = 5.7 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.32 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 8.5 Hz, 2H), 6.52 (d, J = 2.2 Hz, 2H), 6.35 (dd, J = 8.5, 2.2 Hz, 2H), 4.37 (dd, J = 22.3, 10.6 Hz, 4H), 4.19 (d, J = 6.0 Hz, 4H), 3.70 - 3.60 (m, 8H), 3.55- 3.47 (m, 6H), 3.35 (t, J = 6.5 Hz, 2H), 2.65 - 2.60 (m, 2H), 1.82 (d, J = 8.0 Hz, 2H), 1.65 - 1.58 (m, 2H), 1.51 - 1.45 (m, 2H), 1.35 - 1.19 (m, 4H). m / z = 768.2 [M+H + ].

[0335] 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. Then, 1-chloro-N,N,2-trimethylprop-1-en-1-amine (20.6 μL, 0.156 mmol) was added via syringe, and the mixture was stirred at room temperature for 45 minutes. N,N-Diisopropylethylamine (27 μL, 0.156 mmol) was added, followed by TMSCHN2 (2 M in hexane, 78 μL, 0.156 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 to 100% ACN in water). The residue was further purified by reverse-phase flash chromatography (30 to 100% ACN in water). The residue was further purified by flash chromatography using basic alumina (eluent: 50 to 100% EtOAc in toluene) to give the title compound as a solid (1.7 mg, 6%). 1H NMR (400 MHz, pyridine-d5) δ 9.39 (t, J = 5.7 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.32 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 8.5 Hz, 2H), 6.52 (d, J = 2.2 Hz, 2H), 6.35 (dd, J = 8.5, 2.2 Hz, 2H), 4.37 (dd, J = 22.3, 10.6 Hz, 4H), 4.19 (d, J = 6.0 Hz, 4H), 3.70 - 3.60 (m, 8H), 3.55- 3.47 (m, 6H), 3.35 (t, J = 6.5 Hz, 2H), 2.65 - 2.60 (m, 2H), 1.82 (d, J = 8.0 Hz, 2H), 1.65 - 1.58 (m, 2H), 1.51 - 1.45 (m, 2H), 1.35 - 1.19 (m, 4H). m / z = 768.2 [M+H]+.

[0336] 8.18 Reference 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. compound 18 [ka]

[0337] 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. Compound 18.1 [ka]

[0338] 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, CsCO (3.156 g, 9.687 mmol) was added at room temperature, and the mixture was purged with argon gas for 20 minutes. Pd(dba) (0.089 g, 0.097 mmol) and RuPhos (0.135 g, 0.291 mmol) were then added at room temperature. The mixture was again purged with argon gas for 10 minutes and stirred at 110 °C for 16 hours. 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]+.

[0339] 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. Compound 18.2 [ka]

[0340] 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:HO (1:1) (2.0 mL) was added lithium hydroxide monohydrate (0.072 g, 1.728 mmol) in 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 material was diluted with water (2 mL), and the mixture was acidified to pH ∼2 using concentrated HCl. A solid precipitated, which was filtered and dried in vacuo. The residue was purified by C18 reverse-phase column chromatography (eluent: aqueous ACN (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 + ).

[0341] 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. Compound 18.3 [ka]

[0342] 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 it was purified by reverse-phase column purification using a C18 column in 0-100% acetonitrile in water, and the desired product eluted in 15% acetonitrile in 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. Compound 18.4 [ka]

[0343] Method A: To a solution of -(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. Then, 1-chloro-N,N,2-trimethylprop-1-en-1-amine (6.9 μL, 0.052 mmol) was added via syringe, and the mixture was stirred at room temperature for 45 minutes. N,N-Diisopropylethylamine (9 μL, 0.052 mmol) was added, followed by TMSCHN (2 M in hexane, 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 (10–100% ACN in water). The residue was again purified using a pipette column packed with basic alumina, eluting with 10% MeOH in CHCl. ​​The product-containing fractions were concentrated, redissolved in dioxane, and dried on a lyophilizer to give a solid (1.4 mg, 13%). 1H NMR (400 MHz, Pyr) δ 9.42 (t, J = 5.6 Hz, 1H), 8.35 (dd, J = 8.0, 1.5 Hz, 1H), 8.25 (d, J = 1.5 Hz, 1H), 8.10 (d, J = 1 = 8.9). 8.5 Hz, 2H), 6.47 (d, J = 2.2 Hz, 2H), 6.27 (dd, J = 8.5, 2.2 Hz, 2H), 4.28 - 4.11 (m, 4H), 3.75 - 3.67 (m, 3. 4H), - 3.35 (t, J = 6.5 Hz, 2H), 3.05 (t, J = 11.7 Hz, 4H), 2.71 - 2.64 (m, 4H), 2.43 (q, J = 6.3 Hz, 2H), 2.04 (s, J = 6H (d, 4), 1.65 - 1.57 (m, 2H), 1.51 - 1.45 (m, 2H), 1.45 - 1.14 (m, 4H). m / z = 794.2 [M+H + ].

[0344] 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. Then, 1-chloro-N,N,2-trimethylprop-1-en-1-amine (6.9 μL, 0.052 mmol) was added via syringe, and the mixture was stirred at room temperature for 45 minutes. N,N-Diisopropylethylamine (9 μL, 0.052 mmol) was added, followed by TMSCHN (2 M in hexane, 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 (10–100% ACN in water). The residue was further purified by flash chromatography using basic alumina (eluent: 10% MeOH in CHCl) to give the title compound (1.4 mg, 13%) as a solid. 1H NMR (400 MHz, pyridine-d5) δ 9.42 (t, J = 5.6 Hz, 1H), 8.35 (dd, J = 8.0, 1.5 Hz, 1H), 8.25 (d, J = 1.5 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 6.47 (d, J = 2.2 Hz, 2H), 6.27 (dd, J = 8.5, 2.2 Hz, 2H), 4.28 - 4.11 (m, 4H), 3.75 - 3.67 (m, 4H), 3.61 - 3.44 (m, 6H), 3.35 (t, J = 6.5 Hz, 2H), 3.05 (t, J = 11.7 Hz, 4H), 2.71 - 2.64 (m, 4H), 2.43 (q, J = 6.3 Hz, 2H), 2.04 (s, 6H), 2.02 (d, J = 7.4 Hz, 2H), 1.65 - 1.57 (m, 2H), 1.51 - 1.45 (m, 2H), 1.45 - 1.14 (m, 4H). m / z = 794.2 [M+H]+.

[0345] 8.19 Reference 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). compound 19 [ka]

[0346] 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. Compound 19.1 [ka]

[0347] A solution of 1,4-di-tert-butyl 2-bromobenzene-1,4-dicarboxylate (1.53 g, 4.29 mmol, WO2018046753A1) in 2:1 anhydrous THF / pentane (15 mL) was cooled to −100 °C in a diethyl ether / liquid nitrogen bath. After sparging the solution with Ar(g) for 10 min, n-BuLi (2.5 M in hexane, 1.75 mL, 1.72 mmol) was added dropwise down the side of the flask. The solution was stirred at −100 °C for 10 min, during which time it turned purple and then brown. A solution of 5,5-dimethyl-3,7-bis((triisopropylsilyl)oxy)dibenzo[b,e]silin-10(5H)-one (1.0 g, 1.72 mmol, WO2018046753A1) in THF (5 mL) was added dropwise down the side of the flask and stirred at −78 °C in a dry ice / acetone bath for 10 min. The mixture was then allowed to warm to room temperature with stirring for 2.5 h. The mixture was adsorbed onto Celite and purified by flash chromatography on a silica gel column (25–50% CHCl in hexane) to give the title compound (436 mg, 32%) as a crystalline solid. 1 H NMR (400 MHz, CDCl3) δ 7.92 (dd, J = 8.0, 1.3 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H), 6.98 (d, J = 2.7 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 6.56 (dd, J = 8.7, 2.7 Hz, 2H), 1.35 (s, 9H), 1.07 (sept, J = 6.9 Hz, 6H ), 0.90 (d, J = 7.3 Hz, 36H), 0.46 (s, 3H), 0.38 (s, 3H).

[0348] 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 Compound 19.2 [ka]

[0349] 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 (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 solution color changed to deep purple. After 30 min, ½ saturated NH₄Cl was added. The solution color changed to light orange. The mixture was extracted with ethyl acetate (3 × 50 mL), and the combined organic layers were preabsorbed onto silica gel and purified by flash chromatography (eluent: 0–10% ethyl acetate in DCM) to give the title compound (126 mg, 97%) as a translucent film. m / z = 475.1 [M+2H]+.

[0350] 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. Compound 19.3 [ka]

[0351] 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, absorbed onto Celite, and purified by silica gel flash chromatography (eluent: 0-8% MeOH in DCM) to give the title compound (341 mg, 83%) as a white foam. m / z = 739.1 [M+H]+.

[0352] 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 Compound 19.4 [ka]

[0353] An oven-dried 1-dram vial was charged with N,N-dimethylazetidine-3-carboxamide hydrochloride (61.9 mg, 0.308 mmol), CsCO, 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 minutes. Pddba (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, absorbed onto silica gel, and purified by flash chromatography (eluent: 2-20% MeOH in CH2Cl2) to give the title compound as an orange-brown solid (81 mg, 95%). m / z = 695.4 [M+H]+.

[0354] 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 Compound 19.5 [ka]

[0355] 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. Trifluoroacetic acid (0.750 mL) was then 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 stirred 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].

[0356] 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). Compound 19.6 [ka]

[0357] 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 color 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 stirred at room temperature overnight. The mixture was concentrated, and the 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 pale green solid (4 mg, 19%). m / z = 844.4 [M+H]+.

[0358] 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). Compound 19.7 [ka]

[0359] 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 colored solution turned deep blue. The mixture was stirred for 5 min, then TMSCHN (2 M in EtO, 16.6 μL, 33.2 μmol) and N,N-diisopropylethylamine (5.78 μL, 33.2 μmol) were added dropwise. The mixture was stirred at room temperature for 30 min. 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].

[0360] 8.20 Reference 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). compound 20 [ka]

[0361] 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% in ethyl acetate, 0.132 g, 0.207 mmol) at 0° C. The mixture was stirred at 0° C. for 1 hour, and then ammonium bicarbonate (0.041 g, 0.518 mmol) was added. The mixture was warmed to room temperature and stirred at room temperature for 12 hours. After that, 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% aqueous ACN) to give the title compound (0.0052 g, 13% yield) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6): δ 8.69 (t, J = 5.6 Hz, 1H), 7.97 (dd, J = 1.6 Hz and 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.46 (s, 2H), 6.99 (s, 2H), 6.68 (d, J = 8.8 Hz, 2H), 6.21 (d, J = 2.4 Hz, 2H), 6.14 (dd, J = 2.0 Hz and 8.4 Hz, 2H), 3.97-3.95 (m, 4H), 3.85-3.80 (m, 4H), 3.60-3.57 (m, 2H), 3.47-3.34 (m, 10H), 1.67-1.63 (m, 2H), 1.67-1.63 (m, 2H), 1.40-1.38 (m, 2H), 1.24-1.23 (m, 4H) ppm. m / z 770.72 [M+H] + .

[0362] 8.21 Reference example 21 Synthesis of 3',6'-di(azetidin-1-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one. compound 21 [ka]

[0363] Step 1: Preparation of 2-((6-chlorohexyl)oxy)ethan-1-ol). Compound 21.1 [ka]

[0364] To a solution of ethane-1,2-diol (5 g, 80.5 mmol) in DMF (50 mL) at 0 °C under nitrogen, NaH (60% solution in mineral oil) (3.86 g, 161.10 mmol) was added, 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 organic layers were combined, dried over Na SO and concentrated. The residue was purified by flash chromatography (eluent: 45% EtOAc in petroleum ether) to give the title compound as a colorless oil (2 g, 13%).

[0365] Step 2: tert-Butyl 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoate. Compound 21.2 [ka]

[0366] To a solution of 2-((6-chlorohexyl)oxy)ethan-1-ol (2.5 g, 13.9 mmol) in ACN (25 mL) was added N-benzyl-trimethylammonium hydroxide in HO (0.277 g, 4.16 mmol) at room temperature over 10 min. tert-Butyl acrylate (8.9 g, 69.4 mmol) was then 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 x 50 mL). The combined organic layers were dried over NaSO and concentrated. The residue was purified by flash chromatography (eluent: 45% EtOAc in petroleum ether) to give the title compound as a colorless oil (3 g, 70%).

[0367] Step 3: 3-(2-((6-chlorohexyl)oxy)ethoxy)propanal). Compound 21.3 [ka]

[0368] 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 stirred at −78° C. for 1 h. The resulting mixture was quenched with Rochelle's salt solution and extracted with EtOAc (2×30 mL). The organic fractions were combined, dried over Na2SO4, and concentrated. The residue was purified by flash chromatography (eluent: 45% EtOAc in petroleum ether) to give the title compound (0.2 g, 52%) as a colorless oil.

[0369] Step 4: Preparation of tert-butyl (3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)carbamate Compound 21.4 [ka]

[0370] 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 slowly warmed to room temperature and stirred for 16 h. The mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with NaHCO and brine, dried over NaSO, 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 in petroleum ether) to give the title compound as an off-white solid. m / z = 574.32 [M+H]+.

[0371] Step 5: Preparation of tert-butyl (3',6'-di(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)carbamate Compound 21.5 [ka]

[0372] 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. Pd2(dba)3 (2.80 g, 3.06 mmol) was then 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% aqueous ACN) to give the title compound (1.4 g, 43%) as a dark pink solid. m / z = 574.32 [M+H]+.

[0373] Step 6: Preparation of 6-amino-3',6'-di(azetidin-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one Compound 21.6 [ka]

[0374] 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 (40 mL), and extracted with 10% MeOH in CHCl (3 × 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 in water) to give the title compound (0.2 g, 25%) as a purple solid. m / z = 426.35 [M+H].

[0375] Step 7: 3',6'-Di(azetidin-1-yl)-6-((3-(2-((6-chlorohexyl)oxy)ethoxy)propyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one. Compound 21.7 [ka]

[0376] 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 hours. Sodium triacetoxyborohydride (0.199 g, 0.941 mmol) was then added portionwise over 20 minutes at 0 °C. The resulting mixture was stirred for 2 hours, then quenched with ice water and extracted with 10% MeOH in CHCl (2 x 25 mL). The organic layers were combined, dried over NaSO, and concentrated. The residue was purified by reverse-phase column chromatography (eluent: 0-100% aqueous ACN) to give the title compound (0.1 g, 32%) as a pink solid. m / z 646.50 [M+H] + .

[0377] Step 8: 3',6'-Di(azetidin-1-yl)-6-((3-(2-((6-chlorohexyl)oxy)ethoxy)propyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one. Compound 21.8 [ka]

[0378] To a solution of 3',6'-di(azetidin-1-yl)-6-((3-(2-((6-chlorohexyl)oxy)ethoxy)propyl)amino)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.1 g, 0.155 mmol) in CHCl (5 mL) was added a solution of freshly distilled thionyl chloride (0.058 mL, 0.774 mmol) in CHCl (0.025 mL) at 0 °C under a nitrogen atmosphere. The resulting solution was stirred 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 equiv.) 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% aqueous ACN) to afford the title compound (5 mg, 4.82%) as a brown gum. 1 H NMR (400 MHz, DMSO-d6): δ 7.45 (d, J = 8.8 Hz, 1H), 6.70-6.62 (m, 4H), 6.16-6.10 (m, 4H), 6.01 (s, 1H), 3.83-3.80 (m, 8H), 3.58 (t, M / z 668.37 [MH] - .

[0379] 8.22 Reference 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. compound 22 [ka]

[0380] Step 1: Synthesis of 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid. Compound 22.1 [ka]

[0381] 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 a 4 M solution of hydrochloric acid in dioxane (12 mL) at 0 °C under an Ar(g) atmosphere. The resulting mixture was allowed to warm to room temperature and stirred for 4 h. The mixture was then concentrated to give 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid (0.45 g, crude) as an off-white solid. m / z 284.1 [M+H] + .

[0382] 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. Compound 22.2 [ka]

[0383] 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) dropwise at 0 °C, followed by the addition of methanesulfonyl chloride (0.082 mL, 1.06 mmol). The mixture was allowed to warm to room temperature and stirred for 1 h. The mixture was then concentrated and purified by reverse-phase HPLC (eluent: 0-100% acetonitrile in 10 mM aqueous ammonium acetate) to afford the title compound (0.17 g, 36% yield) as a pink solid. m / z 660.31 [M+H] + .

[0384] 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. Compound 22.3 [ka]

[0385] 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 an argon atmosphere. The mixture was warmed to room temperature and stirred for 1 h. Freshly prepared diazomethane (approximately 30 mL, >25 equiv.) 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 in water) to give Example 22 (0.012 g, 8%) as a light brown solid. NMR (400 MHz, DMSO-d6): δ 10.2 (s, 1H), 7.70-7.63 (m, 2H), 7.37 (d, J = 1.2 Hz, 1H), 6.70 (d, J = 8.4 Hz, 2H), 6.15-6.11 (m, 4H), 3.82-3.80 (m, 8H), 3.62-3.56 (m, 4H), 3.45-3.38 (m, 4H), 3.31-3.27 (m, 2H), 2.47-2.45 (m, 2H), 2.33-2.27 (m, 4H), 1.66-1.63 (m, 2H), 1.41-1.30 (m, 4H), 1.25-1.20 (m, 2H) m / z 684.5 [M+H] + .

[0386] 8.23 Reference 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. compound 23 [ka]

[0387] Step 1: Preparation of 2-((6-chlorohexyl)oxy)ethan-1-ol). Compound 23.1 [ka]

[0388] 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 minutes. 1-Chloro-6-iodohexane (19.86 g, 80.55 mmol) was added to the mixture at −10°C, and the mixture was then warmed to room temperature and stirred at room temperature for 2 hours. Ice water (0.5 L) was then added to the mixture, and the mixture was extracted with ethyl acetate (2 × 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 in petroleum ether) to give the title compound (0.9 g, 6%) as a pale yellow liquid.

[0389] Step 2: Preparation of 2-((6-chlorohexyl)oxy)ethyl methanesulfonate. Compound 23.2 [ka]

[0390] 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 warmed to room temperature and stirred at room temperature for 1 hour. Ice water (50 mL) was then added and the mixture was extracted with dichloromethane (2×50 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to afford the title compound (1.5 g, 70%) as a pale yellow liquid, which was used in the next step without further purification.

[0391] Step 3: Preparation of 1-(2-(but-3-yn-1-yloxy)ethoxy)-6-chlorohexane. Compound 23.3 [ka]

[0392] 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 minutes. 2-((6-chlorohexyl)oxy)ethyl methanesulfonate (1.48 g, 5.71 mmol) was then added. The mixture was warmed to room temperature and stirred at room temperature for 16 hours. Ice water (80 mL) was then added, and the mixture was extracted with ethyl acetate (80 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and then concentrated. The residue was purified by silica gel column chromatography (eluent: 0-15% ethyl acetate in petroleum ether) to afford the title compound as a colorless liquid.

[0393] Step 4: Preparation of 3',6'-di(azetidin-1-yl)-6-iodo-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one Compound 23.4 [ka]

[0394] 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) was added CuI (0.67 g, 3.52 mmol) and tBuONO (0.57 mL, 4.70 mmol) at 0 °C. The mixture was stirred at 50 °C for 2 h. Aqueous sodium thiosulfate was then 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% aqueous ACN) to give the title compound (1.0 g, 31%) as a purple solid.

[0395] 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'-xanthene]-3-one. Compound 23.5 [ka]

[0396] 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'-xanthene]-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 (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 elution with 50-60% aqueous acetonitrile containing 10 mM ammonium bicarbonate to give the title compound (0.12 g, 24%) as a pink solid.

[0397] 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. Compound 23.6 [ka]

[0398] 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'-xanthen]-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 hydrogen for 12 hours. The mixture was then filtered through Celite and concentrated. The residue was purified by reverse-phase column (elution with 50-80% aqueous acetonitrile containing 10 mM ammonium bicarbonate) to give the title compound as the first eluting mixture (0.025 g, 24% yield).

[0399] 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. Compound 23.7 [ka]

[0400] 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) was added oxalyl chloride (0.03 mL, 0.31 mL) at 0°C under an argon atmosphere, and the mixture was allowed to warm to room temperature and stirred for 1 hour. The mixture was then concentrated and back-filled under argon. 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 hour. 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] + .

[0401] 8.24 Reference example 24 Synthesis of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one. compound 24 [ka]

[0402] Step 1: Preparation of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one. Compound 24.1 [ka]

[0403] Further elution of compound 23 on the reverse phase column in step 6 afforded the title compound (0.02 g, 20% yield) as a pink solid.

[0404] Step 2: Preparation of 3',6'-di(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one. Compound 24.2 [ka]

[0405] 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) was added oxalyl chloride (0.03 mL, 0.31 mL) at 0°C under an argon atmosphere. The mixture was warmed to room temperature and stirred at room temperature for 1 h, after which the mixture was concentrated under an argon atmosphere. A solution of freshly prepared 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, after which the mixture was concentrated. The residue was purified by preparative HPLC (C18, mobile phase: 0-100% aqueous ACN) to give the title compound (0.025 g, 12%) as a brown gum. 1 HNMR (400 MHz, DMSO-d6): δ 7.67 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 6.81 (s, 1H), 6.64 (d, J = 8.4 Hz, 2H), 6.14 (dd, J = 2.0 Hz, 2.0 Hz, 4H), 3.83 (t, J = 7.2 Hz, 8H), 3.59 (t, J = 6.8 Hz, 2H), 3.38-3.31 (m, 8H), 2.57-2.53 (m, 2H), 2.32-2.27 (m, 4H), 1.67-1.63 (m, 2H), 1.48-1.31 (m, 10H) ppm. m / z 669.5 [M+H] + .

[0406] 8.25 Example 25 Synthesis of 3',6'-di(azetidin-1-yl)-6-(3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one. compound 25 [ka]

[0407] Compound 25.1 [ka] Step 1: To a solution of 3-(benzyloxy)propan-1-ol (100 g, 601 mmol) in dichloromethane, triethylamine (91.3 g, 902 mmol) was added at 0° C. and stirred for 30 minutes. p-Toluenesulfonyl chloride (141 g, 721 mmol) was then added portionwise at 0° C., and the mixture was allowed to warm to room temperature and stirred for 16 hours. The solution was then diluted with water (1000 mL) and extracted with dichloromethane (3×200 mL). The organic layer was washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (100% petroleum ether) to give the title compound (71.0 g, 37%) as a colorless liquid.

[0408] Compound 25.2 [ka] Step 2: To a solution of ethane-1,2-diol (12.6 g, 203 mmol) in DMF (40 mL) was added sodium hydride (7.31 g, 60% oil dispersion, 305 mmol) at 0 °C and stirred for 30 min. 3-(Benzyloxy)propyl 4-methylbenzenesulfonate (65 g, 203 mmol) was added dropwise at 0 °C. The resulting solution was stirred at room temperature for 16 h. After completion of the reaction by TLC, the solution was quenched with cold water (400 mL) and extracted with ethyl acetate (400 mL). The organic layer was washed with brine solution (2 × 400 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by flash chromatography (0–10% ethyl acetate in petroleum ether) to give 2-(3-(benzyloxy)propoxy)ethan-1-ol (8 g, 18%) as a colorless liquid.

[0409] Compound 25.3 [ka] Step 3: To a solution of 2-(3-(benzyloxy)propoxy)ethan-1-ol (4.5 g, 21 mmol) in DMF (40 mL) was added sodium hydride (0.77 g, 60% oil dispersion, 32.11 mmol) at 0 °C and stirred for 30 min. 1-Chloro-6-iodohexane (12.6 g, 31.7 mmol) in DMF (10 mL) was added dropwise at 0 °C, and the resulting solution was stirred at room temperature for 16 h. After TLC showed the reaction was complete, the solution was quenched with cold ammonium chloride solution (200 mL) and extracted with ethyl acetate (2 × 100 mL). The organic layer was washed with brine solution, dried over sodium sulfate, and concentrated. The crude product was triturated with pentane to give ((3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)methyl)benzene (4.3 g, 21%) as a colorless liquid.

[0410] Compound 25.4 [ka] Step 4: To a stirred ethanolic solution of ((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)benzene (4.2 g, 12 mmol) in an autoclave, 10% palladium on carbon (50% wet) (4.2 g) was added, followed by 100 psi of hydrogen pressure and stirring at room temperature for 16 hours. After TLC showed the reaction was complete, the mixture was carefully filtered through a pad of Celite and washed with ethyl acetate. The filtrate was concentrated to give 3-(2-((6-chlorohexyl)oxy)ethoxy)propan-1-ol (3.0 g, 98%) as a colorless liquid.

[0411] Compound 25.5 [ka] Step 5: To a stirred solution of 3-(2-((6-chlorohexyl)oxy)ethoxy)propan-1-ol (1.0 g, 4.2 mmol) in DCM (20 mL) was added DIPEA (2.20 mL, 12.6 mmol) followed by mesyl chloride (0.4 mL, 5 mmol) at 0 °C. The resulting solution was stirred at room temperature for 1 h. After TLC showed the reaction was complete, the reaction mixture was diluted with dichloromethane (20 mL) and washed with cold water (2 x 20 mL) and brine solution (20 mL). The organic layer was dried over sodium sulfate and concentrated. The crude material was purified by silica gel flash chromatography (0-10% ethyl acetate in petroleum ether) to give 3-(2-((6-chlorohexyl)oxy)ethoxy)propyl methanesulfonate (1.0 g, 75%) as a light brown solid. m / z found 317.1 [M+H] + .

[0412] Compound 25.6 [ka] Step 6: To a stirred solution of 4-methoxyphthalic acid (50.0 g, 237 mmol) in methanesulfonic acid (250 mL) was added 3-bromophenol (86.4 g, 500 mmol). The reaction mixture was stirred at 140 °C for 48 h. The reaction was then cooled to room temperature and poured into ice water (1000 mL). This mixture was then washed with ethyl acetate (3 x 1 L), and the organic layer was dried over sodium sulfate and concentrated. The crude product was purified by reverse-phase chromatography (0–60% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give peak 1, 3',6'-dibromo-6-hydroxy-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (1.2 g, 1% yield) as an off-white solid, and peak 2, 3',6'-dibromo-5-hydroxy-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.12 g, 0.1% yield). m / z found 475.18 [M+H]. + .

[0413] Compound 25.7 [ka] Step 7: To a stirred solution of 3',6'-dibromo-6-hydroxy-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (1.0 g, 2.1 mmol) in DMF (10 mL) under an argon atmosphere at room temperature, cesium carbonate (2.0 g, 6.4 mmol) was added, followed by 3-(2-((6-chlorohexyl)oxy)ethoxy)propyl methanesulfonate (1.0 g, 3.3 mmol, Compound 25.5). The resulting mixture was warmed to 70 °C and stirred for 16 h. The reaction mixture was then cooled to room temperature, quenched with ice-water (100 mL), and extracted with ethyl acetate (100 mL). The organic layer was washed with brine solution (2 x 100 mL), dried over sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (0-20% ethyl acetate in petroleum ether) to give 3',6'-dibromo-6-(3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (0.52 g, 35% yield) as a colorless liquid. m / z 697.39 [M+H] + .

[0414] Compound 25.8 [ka] Step 8: To a stirred solution of 3',6'-dibromo-6-(3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.3 g, 0.4 mmol) in anhydrous 1,4-dioxane (10 mL) in a sealed tube was added davephos-Pd-G3 (0.033 g, 0.043 mmol) and cesium carbonate (0.56 g, 1.7 mmol) and degassed with argon for 15 minutes. Azetidine (0.10 g, 1.7 mmol) was then added and the mixture was warmed to 110 °C and stirred for 16 hours. The reaction mixture was cooled to room temperature, filtered through Celite, washed with ethyl acetate, and concentrated. The crude material was purified by preparative HPLC (0-67% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give 3',6'-di(azetidin-1-yl)-6-(3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (0.09 g, 16% yield). m / z 647.48 [M+H] + .

[0415] 1 H NMR (400 MHz, DMSO-d6): δ 7.86 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.4, 2.0 Hz, 1H), 6.64 (d, J = 1.6 Hz, 1H), 6.52 (d, J = 8.4 Hz, 2H), 6.19-6.15 (m, 4H), 4.02 (t, J = 6.0 Hz, 2H), 3.86 (t, J = 7.2 Hz, 8H), 3.59 (t, J = 6.4 Hz, 2H), 3.48 (t, J = 6.4 Hz, 2H), 3.40 (t, J = 3.6 Hz, 4H), 3.31 - 3.27 (m, 2H), 2.35 - 2.29 (m, 4H), 1.88 (t, J = 6.0 Hz, 2H), 1.68 - 1.61 (m, 2H), 1.43 - 1.30 (m, 6H).

[0416] [ka] Step 9: To a stirred solution of 3',6'-di(azetidin-1-yl)-6-(3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.025 g, 0.039 mmol) in dichloromethane (50 mL) at 0 °C under an argon atmosphere, oxalyl chloride (0.3 mL, 0.579 mmol, 2 M in DCM) was added dropwise. The resulting solution was warmed to room temperature and stirred for 1 h. The reaction mixture was concentrated under an argon atmosphere. The crude product was dissolved in dichloromethane (50 mL) and freshly prepared diazomethane (2.5 g, 1.0 equiv) in diethyl ether (20 mL) was added. The reaction was then warmed to room temperature and stirred for 1 h. The reaction mixture was concentrated and purified by reverse phase chromatography (10 mM acetic acid in water containing 0-25% acetonitrile) to give the title compound (2.5 mg, 10% yield). m / z 671.54 [M+H] + . 1 H NMR (400 MHz, DMSO-d6): δ 7.69 (d, J = 8.4 Hz, 1H), 7.08 (dd, J = 8.4, 2.0 Hz, 1H), 6.67 (d, J = 9.2 Hz, 2H), 6.40 (d, J = 2.0 Hz, 1H), 6.14-6.11 (m, 4H), 3.95 (t, J = 6.4 Hz, 2H), 3.83 (t, J = 7.2 Hz, 8H), 3.46-3.37 (m, 6H), 3.32-3.28 (m, 2H), 2.33 - 2.28 (m, 4H), 1.85 (t, J = 6.4 Hz, 2H), 1.67 - 1.63 (m, 2H), 1.42 - 1.23 (m, 8H).

[0417] 8.26 Example 26 Synthesis of 3',6'-di(azetidin-1-yl)-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one. compound 26 [ka]

[0418] Compound 26.1 [ka] Step 1: To a stirred solution of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (20 g, 40 mmol, prepared according to the procedure described in Tetrahedron, 2005, 61, 3097-3105) in THF (200 mL) was added 1,1'-carbonyldiimidazole (12.9 g, 79.6 mmol) in portions at 0 °C. The resulting solution was stirred at room temperature for 3 hours. Then, sodium borohydride (7.5 g, 200 mmol) in 400 mL of water was added dropwise at 0 °C and stirred at room temperature for 10 minutes. After completion of the reaction, the mixture was extracted with ethyl acetate (2 x 250 mL). The organic layer was dried over sodium sulfate and concentrated. The crude material was triturated with cold DCM to give 3',6'-dibromo-6-(hydroxymethyl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (5.8 g, 30%) as an off-white solid. m / z found 487.07 [M+H] + .

[0419] Compound 26.2 [ka] Step 2: To a stirred solution of 3',6'-dibromo-6-(hydroxymethyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (5.8 g, 12 mmol) in DCM (58 mL) was added TEA (8.4 g, 83 mmol) followed by mesyl chloride (6.8 g, 60 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The mixture was then diluted with water (100 mL), and the aqueous layer was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated. The crude material was purified by silica gel column (0–30% ethyl acetate in petroleum ether, 0.1% TEA) to give 3',6'-dibromo-6-(chloromethyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (4.2 g, 70%) as an off-white solid. m / z Measured value 505.20 [M+H] + .

[0420] Compound 26.3 [ka] Step 3: To a stirred solution of 3',6'-dibromo-6-(chloromethyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (2.0 g, 3.5 mmol) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (1.2 g, 5.3 mmol) in DMF (40 mL) was added potassium carbonate (1.5 g, 11 mmol). The resulting solution was stirred at room temperature for 4 hours. The reaction mixture was then diluted with ice water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography (5% methanol in dichloromethane) to give 3',6'-dibromo-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (1.3 g, 52%) as a yellowish semi-solid. m / z 692.41 [M+H] + .

[0421] Compound 26.4 [ka] Step 4: A stirred solution of 3',6'-dibromo-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (1.3 g, 1.873 mmol) and formaldehyde (0.51 mL, 5.6 mmol, 37% aqueous solution) in DCE (26 mL) was cooled at 0 °C and acetic acid (catalytic) was added. The reaction was stirred at 0 °C and allowed to warm to room temperature over 2 h, after which NaCNBH (0.35 g, 5.6 mmol) was added portionwise to the solution and stirring was continued for 12 h. After completion of the reaction, the solution was quenched with water (50 mL) and extracted with DCM (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography (65% ethyl acetate in petroleum ether) to give 3',6'-dibromo-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (0.9 g, 68%) as a colorless semi-solid. m / z 705.36 [M+H] + .

[0422] Compound 26.5 [ka] Step 5: To a stirred solution of 3',6'-dibromo-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.25 g, 0.35 mmol) in anhydrous 1,4-dioxane (12.5 mL) was added tris(dibenzylideneacetone)dipalladium(0) (0.032 g, 0.035 mmol), dicyclohexyl[2',4',6'-tris(propan-2-yl)[1,1'-biphenyl]-2-yl]phosphane, XPhos (0.051 g, 0.11 mmol), and cesium carbonate (0.46 g, 1.4 mmol) in a sealed tube and degassed with argon for 15 minutes. Azetidine (0.081 g, 1.4 mmol) was added, and the reaction was then heated to 110° C. with stirring for 16 hours. The reaction mixture was cooled to room temperature, filtered through Celite, and washed with ethyl acetate. The crude product was purified by preparative HPLC (0-67% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give 3',6'-di(azetidin-1-yl)-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (0.03 g, 13% yield). 1 H NMR (400 MHz, DMSO-d6): δ 7.90 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.12 (s, 1H), 6.48 (d, J = 8.8 Hz, 2H), 6.20 (d, J = 2.4 Hz, 2H), 6.16 (dd, J = 14, 8.8 Hz, 2H), 6.38 (dd, J = 8.4 & 2.0 Hz, 2H), 3.86 (t, J = 7.2 Hz, 8H), 3.65-3.55 (m, 2H), 3.44 (t, J = 6.0 Hz, 2H), 3.37 - 3.27 (m, 6H), 2.49 - 2.44 (m, 2H), 2.34 - 2.37 (m, 4H), 2.09 (s, 3H), 1.67-1.62 (m, 2H), 1.43-1.22 (m, 6H). m / z 660.48 [M+H]+ .

[0423] compound 26 [ka] Step 6: To a stirred solution of 3',6'-di(azetidin-1-yl)-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.02 g, 0.030 mmol) in dichloromethane (50 mL) was added oxalyl chloride (0.23 mL, 0.45 mL) at 0 °C under an argon atmosphere. The reaction was then warmed to room temperature and stirred for 1 h. The reaction mixture was concentrated under an argon atmosphere. The crude product was taken up in dichloromethane (50 mL) and a solution of freshly prepared diazomethane in diethyl ether (1.0 equiv.) in diethyl ether (20 mL) was added dropwise. After completion of the reaction, the mixture was concentrated and the crude product was purified by preparative HPLC (10 mM aqueous ammonium bicarbonate solution containing 0-37% acetonitrile) to give the title compound (4 mg, 19% yield). 1 H NMR (400 MHz, DMSO-d6): δ 7.70 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.8, 1H), 6.95 (s, 1H), 6.65 (d, J = 8.4, 2H), 6.14 (dd, J = 6.4, 2.4 Hz, 2H), 6.10 (d, J = 2.4, 2H), 3.83 (t, J = 7.6 Hz, 8H), 3.60 (t, J = 6.4 Hz, 2H), 3.48 (s, 2H), 3.36 - 3.29 (m, 6H), 2.38 (t, J = 6.0 Hz, 2H), 2.33 - 2.27 (m, 4H), 2.05 (s, 3H), 1.68 - 1.64 (m, 2H), 1.45 - 1.41 (m, 2H), 1.36 - 1.23 (m, 6H). m / z 684.54 [M+H] + .

[0424] 8.27 Example 27 Synthesis of 3',6'-di(azetidin-1-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-2-diazospiro[indene-1,9'-xanthene]-3(2H)-one. [ka] compound 27 [ka]

[0425] Compound 27.1 [ka] Step 1: To a solution of 2-(2-(benzyloxy)ethoxy)ethan-1-ol (5 g, 25.5 mmol) in DMF (40 mL) was added sodium hydride (2 g, 60% oil dispersion, 51 mmol) at 0 °C and stirred for 30 min. Then, 1-chloro-6-iodohexane (12.6 g, 31.7 mmol) in DMF (10 mL) was added dropwise at 0 °C and stirred for 16 h. After completion of the reaction, the reaction mixture was quenched with cold ammonium chloride solution (50 mL) and extracted with ethyl acetate (2 × 100 mL). The organic layer was washed with brine solution, dried over sodium sulfate, and concentrated. The crude product was purified by silica gel column (0–15% ethyl acetate in petroleum ether) to give ((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)benzene (5 g, 62%) as a pale yellow liquid.

[0426] Compound 27.2 [ka] Step 2: To a stirred solution of ((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)benzene (5 g, 16 mmol) in THF (20 mL) was added 10% Pd / C (5 g, 50% wet basis) and stirred under hydrogen (1 atm) for 16 h. After completion of the reaction, the reaction mixture was carefully filtered through a pad of Celite. The pad was washed with ethyl acetate (3 x 50 mL). The filtrate was concentrated to give 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-ol (3.5 g, 99%) as a colorless liquid.

[0427] Compound 27.3 [ka] Step 3: To a solution of 3',6'-dibromo-6-(chloromethyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (4.2 g, 8.3 mmol, compound 26.2) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-ol (2.8 g, 12 mmol) in anhydrous DMSO (42 mL) was added potassium hydroxide (1.4 g, 25 mmol) at room temperature and stirred for 16 hours. The reaction mixture was then diluted with ethyl acetate (100 mL) and washed with water (50 mL). The organic layer was dried over sodium sulfate and concentrated. The crude product was purified by silica gel chromatography (0–30% ethyl acetate in petroleum ether, 0.1% TEA) to give 3′,6′-dibromo-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one (2 g, 35%) as a viscous liquid.

[0428] Compound 27.4 [ka] Step 4: A suspension of 3',6'-dibromo-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.1 g, 0.14 mmol)) and cesium carbonate (230 mg, 0.7 mmol) in 1,4-dioxane (1.5 mL) was purged with nitrogen for 10 minutes. Pd2(dba)3 (12.8 mg, 0.014 mmol) and XPhos (20 mg, 0.042 mmol) were then added, followed by purging with nitrogen for 5 minutes. Azetidine (24.6 mg, 0.42 mmol) was added, and the reaction was stirred at 100 °C for 16 hours. The reaction mixture was then filtered through Celite and washed with 10% methanol in dichloromethane (3 x 10 mL). The organic layer was concentrated and purified by preparative HPLC (0-70% acetonitrile in water, 0.1% formic acid) to give 3',6'-di(azetidin-1-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one (30 mg, 32%) as a purple sticky solid. m / z found 647.72 [M+H] + .

[0429] compound 27 [ka] Step 5 (Method C): To an oven-dried 20 mL vial was added 3',6'-di(azetidin-1-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (50 mg, 0.077 mmol). The solid was dissolved in 1:1 dichloromethane / acetonitrile (2 mL). Molecular sieves (82.3 mg) and CaO (13 mg, 0.23 mmol) were added to the solution, and the vial was capped and purged with argon for 5 minutes. Ghosez's reagent (81.8 μL, 0.618 mmol) was added, causing the solution to darken. Two minutes after the addition of Ghosez's reagent, trimethylsilyldiazomethane (0.31 mL, 0.62 mmol, 2 M in diethyl ether) was added, followed by potassium fluoride (35.9 mg, 0.618 mmol). The reaction mixture was concentrated directly onto Celite and purified by silica gel chromatography (0-20% EtOAc in toluene) to give the title compound. 1 H NMR (400 MHz, DMSO-d6): δ 7.72 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 6.94 (s, 1H), 6.66 (d, J = 8.4 Hz, 2H), 6.14-6.10 (m, 4H), 4.48 (s, 2H), 3.83 (t, J = 7.2 Hz, 8H), 3.59 (t, J = 6.8 Hz, 2H), 3.49 (br s, 4H), 3.42-3.32 (m, 6H), 2.32-2.27 (m, 4H), 1.68-1.63 (m, 2H), 1.48-1.31 (m, 6H). m / z Measured value 671.46 [M+H] + .

[0430] 8.28 Example 28 Synthesis of 1,1'-(6-((4-(((2-aminopyrimidin-4-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] compound 28 [ka]

[0431] Compound 28.1 [ka] Step 1 (Method C): To a stirred 1:1 DCM / acetonitrile (2 mL) mixture of methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (13 mg, 0.021 mmol, compound 4.1), 4 Å molecular sieves (80 mg), and CaO (3.6 mg, 0.063 mmol) was added dropwise at room temperature. After stirring at room temperature for 2 min, TMSCHN2 (0.085 mL, 0.17 mmol, 2 M solution in diethyl ether) was added immediately followed by KF (9.9 mg, 0.17 mmol). After 20 min, the mixture was concentrated. The crude product was purified by silica gel flash chromatography (0–100% ethyl acetate in toluene) to give methyl 2-diazo-3′,6′-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9′-xanthene]-6-carboxylate (3 mg, 22%). m / z found 316.4 [M+H] 2+ .

[0432] Compound 28.2 (also referred to herein as Compound 5.1) [ka] Step 2: To a stirred solution of methyl-2-diazo-3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate (0.055 g, 0.086 mmol) in THF (1.0 mL) and 1:1 MeOH / HO (1.0 mL) was added lithium hydroxide monohydrate (0.008 g, 0.173 mmol) in portions at 0° C. The reaction mixture was stirred at room temperature for 2 h. The reaction was concentrated and the crude product 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 (0.05 g, 93%) as a brown solid. m / z found 621.6 [M+H] + .

[0433] Compound 28.3 [ka] Step 3: To a stirred solution of 2-diazo-3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid (0.05 g, 0.081 mmol) and 2-((4-(aminomethyl)benzyl)oxy)pyrimidin-4-amine (0.037 g, 0.161 mmol) in DMF (1 mL) was added N,N'-ethyldiisopropylamine (0.06 g, 0.4 mmol) at 0°C. After stirring for 10 min, T3P (50% ethyl acetate solution, 0.22 g, 0.32 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, the reaction mixture was concentrated and the crude product was purified by preparative HPLC (10 mM aqueous ammonium bicarbonate solution containing 0-70% acetonitrile) to give the title compound (0.0156 g, 20%). 1H NMR (400 MHz, DMSO-d6): δ 9.17 (t, J = 6.0 Hz, 1H), 8.03-8.01 (dd, J = 1.2 Hz, 8.0 Hz, 1H), 7.94 (d, J = 5.6 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.50 (s, 1H), 7.34 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H). 6.70 (d, J = 8.8 Hz, 2H), 6.53 (s, 2H), 6.21 (d, J = 2.0 Hz, 2H), 6.17-6.14 (dd, J = 2.4 Hz, 8.4 Hz, 2H), 8.99 (d, J = 5.6 Hz, 1H), 5.23 (s, 2H), 4.38 (d, J = 5.6 Hz, 2H), 4.06-4.01 (m, 4H), 3.91 -3.79 (m, 6H), 2.88 (s, 6H), 2.83 (s, 6H). m / z actual value 833.52 [M+H] + .

[0434] 8.29 Reference Example 29: Kinetic solubility in PBS at 7.4 190 μL of buffer (PBS, pH 7.4) was added to the wells of a 96-well Millipore Solubility filter plate, followed by 10 μL of test compound in DMSO (10 mM) to give a final concentration of 500 μM.

[0435] The filter plate was shaken in the dark at room temperature for 1.5 hours, and the samples were filtered into a new 96-well plate via a vacuum system. Samples were diluted to 500 μM (highest concentration) with DMSO and further diluted (1:10) to generate a three-point calibration curve. HPLC / UV analysis was used to measure absorbance at 220 nm, 254 nm, and 280 nm. Data are reported as the average of three runs for each test compound and are shown in Table 1 below.

[0436] [Table 2]

[0437] 8.30 Reference Example 30: Passive Permeability MDCK-MDR1 cells were plated at 7,500 cells / 75 μL / well in a 96-well Millipore Millicell-96 plate and incubated at 37°C with 5% CO for 3 days. The cells were then washed with Hank's balanced salt solution (HBSS) containing 5 mM HEPES for 30 minutes. A DMSO (10 mM) solution of the test compound was added to HBSS buffer containing 10 μM GF-120918 to obtain a final DMSO concentration of 0.2% and a test compound concentration of 5 μM. The transport plate was incubated at 37°C for 1 hour in a humidified incubator with 5% CO. After 1 hour, samples were collected from the apical and basolateral compartments and analyzed by liquid chromatography with tandem mass spectrometry (LC / MS / MS, AB Sciex API4000 instrument, connected to a Shimadzu LC-20AD LC Pump system). Analytical samples were separated using a Waters Atlantis T3 dC18 reverse-phase HPLC column (20 mm x 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 100% acetonitrile containing 0.1% formic acid (solvent B).

[0438] Apparent permeability (Papp, A2B) values ​​were calculated using the following formula:

[0439] Papp=(dQ / dt) / A / C0 where dQ / dt is the initial rate of transport of the amount of test compound through the cell monolayer, A is the surface area of ​​the filter membrane, and C is the initial concentration of the test compound calculated for each direction using a four-point calibration curve by LC / MS / MS. Data are reported as the average of two runs and are shown in Table 2 below.

[0440] [Table 3]

[0441] 8.31 Reference Example 31: Protein Labeling with Photoactivatable Dyes 60 μM 6xHis-tagged HaloTag protein (expressed and purified from the pH6HTC His6HaloTag T7 vector from Promega) was incubated with 200 μM dye compound in 200 μL of 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 at 4°C for 18 h. The solution was illuminated for 5 min with the center of a 405 nm laser (365 mW) with a 6x beam expansion. 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 MgCl, 1 mM DTT, and 10% glycerol). The labeled protein conjugate in the supernatant was analyzed on an SDS-PAGE protein gel.

[0442] 8.32 Reference Example 32: Halo-Protein Specificity Wild-type (WT) U2OS cells or U2OS cells ectopically expressing a histone H2B-HaloTag fusion 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 and 5% CO2. The following day, cells were incubated with PA-JF549 or Examples 1-4 at concentrations ranging from 200 nM to 1 nM for 45 minutes. After incubation, cells were washed three times with PBS, and the medium was finally replaced with phenol-free imaging medium. Samples were imaged using a Nikon Ti2 microscope equipped with a fiber-optic-coupled illumination source. Fluorophore emission was stimulated using 561 nm light (approximately 500 mW at the coverslip). Images were collected every 10 ms for 5 seconds per field, and the dye molecules were photoactivated using pulses of increasing intensity 405 nm light (0-12 mW at the coverslip). Approximately 20 fields were collected for each cell line / dye compound / concentration set.

[0443] All frames from each field of view were analyzed using a maximum likelihood estimator model to detect single-molecule fluorescence emission within the image with subpixel precision. For each dye compound, linear regression was used to determine the relationship between the dye compound concentration and the number of fluorescent spots detected by the microscope. In WT cells, an ideal dye would have no correlation between compound concentration and the number of measured spots. The specificity coefficient was calculated using the PA-JF 549 The slope was calculated as the ratio of the slope of the compound in question to the slope calculated for

[0444] [Table 4]

[0445] 8.33 Reference example 33: Label specificity The labeling specificity of the compounds of Examples 1-4 was measured using the method described in Example 32 above, and PA-JF 549 U2OS cells expressing wild-type or H2B-HaloTag fusion were compared with PA-JF. 549 or Example 4 for 45 minutes. Figure 3 shows an exemplary field of view of H2B-HaloTag fusion-expressing U2OS cells, i.e., nuclei labeled with Hoechst 33342 dye. Labeling specificity was calculated by dividing the number of spots detected at a given concentration of dye in H2B-HaloTag-expressing U2OS cells by the number of spots detected at the same concentration in U2OS wild-type cells.

[0446] 8.34 Reference Example 34: Signal-to-Noise Ratio The SNR, a proxy for single-molecule brightness, was calculated for Examples 1-4 (see Figure 4). Each spot in the SPT experiment described in Example 32 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. This calculation uses a fixed-width 2D Gaussian PSF model with sigma / standard deviation equal to 0.183 μm. Due to the stringency of our spot detection filter, only spots with an SNR of 14 or greater were included in this calculation. The bars in Figure 4 are the average SNR across all biological replicates, and the error bars are the standard error of the average SNR across all biological replicates.

[0447] 8.35 Example 35: Reduction of Nonspecific Labeling Wild-type (WT) U2OS cells or U2OS cells ectopically expressing an estrogen receptor-HaloTag (ER-Halo) fusion were plated at 6000 cells / 50 μL / well in glass-bottom 384-well plates and incubated overnight at 37°C, 5% CO2, and 95% humidity. The next day, cells were treated with 100 nM (conventional JF 549Cells were incubated with JF549, PA-JF549, or Examples 1-28 at concentrations of 0.01 pM (20 pM for 0.01 pM) and 100 nM Potomac Red for 1 hour. After incubation, cells were washed three times with PBS, and the medium was finally replaced with phenol-free imaging medium. Samples were imaged on a Nikon Ti2e microscope equipped with a fiber-optic-coupled illumination source using a 60x 1.27NA objective and an sCMOS camera. Fluorophore emission was stimulated using 561 nm light (approximately 500 mW integrated intensity at the coverslip). Images were collected every 10 ms for 2 seconds per field. To test the sensitivity of each dye variant to 405 nm light, each well was imaged multiple times at increasing 405 nm light intensities (0-5 mW at the coverslip). Approximately 60 fields were collected for each cell line / dye compound set.

[0448] To measure Reference Example 19, where the fluorophore was 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.

[0449] After acquiring images for each compound, the images were individually inspected to qualitatively assess the performance of each test compound. 549It is understood that modifications to the core structure of dyes can alter not only dye properties but also photoactivation with 405 nm light. Therefore, the performance of each dye variant, which was not predictable based on structural features alone, was evaluated as follows. Compounds that were clearly unsuitable for single-molecule imaging (e.g., those that caused dye aggregation inside cells) were flagged and excluded from subsequent analysis. The remaining dyes, which reliably yielded spots consisting of a single fluorophore and were therefore quantifiable using a single-molecule detection algorithm, were further processed to generate individual trajectories. All frames from each field of view were analyzed using a maximum likelihood estimator model to detect single-molecule fluorescence emission within the image with subpixel accuracy. Individual detections were then connected to form trajectories across consecutive camera frames. Statistics generated from detections (e.g., signal-to-noise) and tracks (e.g., track counts) were used to compare dye variants.

[0450] The number of nuclear tracks in the ER-Halo cell line was determined for each test compound at each level of 405 nm light activation. To establish a common baseline between different test compounds, the number of nuclear tracks was compared with the JF 549 The photoactivation conditions that most closely matched those of the control were selected for each test compound and these conditions were used for all subsequent comparisons.

[0451] [Table 5]

[0452] [Table 6]

[0453] 8.36 Example 36: Improved labeling specificity The labeling specificity of the compounds of Examples 1 to 28 was measured using the methods described in Tables 4a and 4b above, and the PA-JF 549 U2OS cells expressing wild-type or ER-HaloTag fusions were compared with PA-JF. 549or Examples 1-28 for 1 hour. Figure 5 shows an exemplary field of U2OS expressing the ER-HaloTag fusion co-stained with Potomac Red (CAS: 2127150-65-4, Grimm et al., 2017). Labeling specificity, as measured by the fold reduction in nonspecific labeling of WT cells, was measured using PA-JF. 549 The numerator is PA-JF, calculated by the number of tracks measured in WT cells for Examples 1-28 compared to the number of tracks in WT cells for Examples 1-28. 549 It is expressed as a ratio of.

[0454] The sensitivity of phototransduction under 405 nm illumination was determined qualitatively based on the amount of 405 nm light required to achieve a comparable number of tracks. Compounds that showed high levels of activation at 0.25 mW of 405 nm laser input (the lowest intensity tested) were given a "+++" and compounds that failed to achieve high levels of activation at 5 mW of 405 nm intensity were given a "-". "+" and "++" were assigned to compounds that required greater than 1 mW and 2.5 mW of 405 nm laser power at the objective, respectively.

[0455] The SNR, a proxy for single-molecule brightness, was calculated for Examples 1, 4, 7, and 10 (see Figure 6). Each spot in the SPT experiment described in Example 34 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. This calculation uses a fixed-width 2D Gaussian PSF model with sigma / standard deviation equal to 0.183 μm. Due to the stringency of this spot detection filter, only spots with an SNR of 14 or greater were included in this calculation. The bars in Figure 6 are the average SNR calculated for all detections from the dye variants summed across biological replicates, and the error bars are the standard error of the average SNR across biological replicates. The above examples demonstrate that the compounds disclosed herein, when used to label proteins, surprisingly exhibit improved labeling specificity while maintaining brightness and concentration control.

[0456] 8.37 Example 37: Method C and Comparative Data Method C: Rhodamine (1 equiv.) was charged to an oven-dried 20 mL vial equipped with a septum-equipped screw cap and dissolved in a 1:1 solution of dichloromethane and acetonitrile (2 mL / eq.). Crushed 4 Å molecular sieves (8 equiv.) and CaO (3 equiv.) were added to the resulting solution, and the vial was sealed. The mixture was sparged with argon for 5 minutes, after which Ghosez reagent (4 equiv.) was added, causing the solution to darken. An aliquot was taken from the reaction and quenched with 2 M methylamine in THF, and the formation of the acyl chloride was monitored by LC-MS. After complete conversion to the acyl chloride (5 min), TMSCHN2 (8 equiv., 2 M in diethyl ether) was added, followed immediately by KF (8 equiv.). After complete conversion to the diazoketone, the reaction mixture was concentrated directly onto Celite and purified by silica gel column chromatography. Comparative data was generated using the reference compounds and sample numbers of compounds 27 and 28.1 as shown below.

[0457] [ka] [Table 7]

[0458] Conventional Arndt-Eistad conditions, i.e., conventional diazoketone synthesis using oxalyl chloride, TEA, TMSCHN2, and DCM (Method A), result in the ring-opening of azetidine to produce 3-chloropropanamine. Surprisingly, when Ghosez reagent, CaO, 4 Å molecular sieves, TMSCHN2, and KF (Method C) were used, these conditions afforded the desired diazoketone without ring-opening of azetidine across a panel of substrates. Specifically, as shown in the table above, compound JF 549 , Reference a, Reference b, Reference c, Using Method C to make Compound 27 and Compound 28.1 produced a substantially chemically pure azetidine product (1), which contained undetectable amounts of other undesired 3-chloropropanamine (2 and 3) (product ratio was 100:nd:nd).

[0459] Method C above may be used to synthesize the compounds described herein.

[0460] 8.38 Example 38: Synthesis Testing [ka] The synthesis of acid chloride 38-1e and its conversion to diazoketone 38-1b was carried out according to the procedure in Example 37 (Method C). Table 5 shows the effect of variations from the standard conditions (Method C) upon screening with various reagents. Product distribution is expressed as a percentage as observed by LC-MS analysis of the reaction mixture. The reaction gave consistent yields on a scale ranging from 0.1 mmol (50 mg) to 2.25 mmol (1.0 g) of test substrate 38-1a when Method C was applied. [Table 8]

[0461] 8.39 Example 39: Synthesis of Photoactivatable Rhodamine by Method C [ka] TIFF2026503623000309.tif135165 In this route, compound 39-10b was obtained via saponification and coupling of 2-((4-(aminomethyl)benzyl)oxy)pyrimidin-4-amine to 39-9b rather than directly from the corresponding rhodamine lactone.

[0462] 8.40 Example 40: Labeling Specificity Introduction of a dimethylcarboxamide at the 2-position of azetidine (compound 39-10c, also referred to herein as compound 4) increased solubility and passive permeability through cell membranes, resulting in a dramatic reduction in nonspecific labeling compared to photoactivatable JF549-HaloTag, as shown in Figure 7.

[0463] To evaluate the behavior of compound 39-10c, we labeled U2OS cells with endogenous knock-in of a HaloTag fusion to β-catenin, a protein with multiple cellular roles, including activation of Wnt-responsive gene pathways via transcriptional activation. β-catenin plays a role in diseases such as colorectal cancer. Cells were labeled with 50 nM of compound 39-10c and then imaged under conditions that allowed the location of individual molecules to be identified with subpixel precision and tracked within live cells.

[0464] Results: α-diazoketones were sensitive to 405 nm laser uncaging, and increasing the laser intensity resulted in an increase in the number of corresponding fluorophores, ultimately depleting the labeled protein pool (Figure 8). After localization and tracking, a profile of the kinetics present in the sample was constructed. Very slow diffusion (<0.1 μm) 2 / sec) to very fast diffusion (>5 μm 2 At least four distinct kinetics were observed, ranging from 100 s to 100 s (100 s / s), consistent with the many functional roles of β-catenin in cells (Figure 9) (SGPai, et al., Journal of Hematology & Oncology, 2017, 10, 101). The photoactivatable nature of compounds 39-10c allowed us to observe thousands of protein trajectories per cell, generating individual super-resolution images of β-catenin for each observed kinetic. The distinct kinetics were also found to correspond to spatially distinct regions of the cell. While the slowest populations were both associated with the cell's plasma membrane, spatially distinct subsets can be presumed to be involved in intercellular gap junctions due to their slow diffusion (Figure 10). In contrast, the fastest populations were primarily cytoplasmic and nuclear, consistent with β-catenin's role in activating Wnt-responsive genes in the nucleus.

[0465] 8.41 Example 41: Preparation of Single Molecule Tracking Samples U2OS cells (HBT-96, ATCC) or U2OS cells with endogenous HaloTag knock-in were seeded at 6000 cells per well in tissue culture-treated 384-well glass-bottom plates. The seeded cells were then incubated overnight at 37°C and 5% CO2 to allow attachment. For all SMT experiments, cells were incubated with 50 nM of compound 38-1b or PA-JF in complete medium. 549 , or 50 pM JF 549 (Cat. No. GA1110, Promega) for 1 hour. Cells were then washed three times with DPBS and twice with imaging medium: GlutaMAX (Cat. No. 35050079, Thermo Fisher Scientific) and fluoroBrite DMEM medium (Cat. No. A1896701, Thermo Fisher Scientific) supplemented with the same serum and antibiotics as the growth medium.

[0466] 8.42 Example 42: Acquisition of Single Molecule Tracking Images Image acquisition using SMT was performed on a custom-built HILO microscope, as described by McSwiggen et al. ("A high-throughput platform for single-molecule tracking identifies drug interactions and cellular mechanisms," (2023) eLife 12:RP93183), based on a Nikon Ti2 microscope, a motorized stage, a stage-top environmental chamber (OKO labs), a quad-band filter cube (Chroma), custom-built laser launches at wavelengths of 405 nm and 561 nm, and powers of 0–10 mW and >150 mW delivered to the back focal plane and objective, respectively. Fluorescence emission was passed through a high-speed filter wheel (Finger Lakes Instruments) and collected with a backlit CMOS camera (Prime 95b, Teledyne). Images were acquired with a 60x 1.27NA water-immersion lens (Nikon). The environmental chamber was set at 37°C, 95% humidity, and 5% CO2. To account for depletion of the unimaged pool of molecules over time, 5000 frames of images were collected at stepwise increases in 405 illumination intensity.

[0467] 8.43 Example 43: Analysis of Single Molecule Tracking Images Tracking proceeded as described by McSwiggen et al. (“A high-throughput platform for single-molecule tracking identifies drug interactions and cellular mechanisms,” (2023) eLife 12:RP93183) and will be briefly described here. Individual SMT videos were processed in three sequential steps using a combination of existing methods: detection, subpixel localization, and connection. Briefly, spots were detected using a generalized log-likelihood ratio detector. After detection, the estimated location of each emitter was refined to subpixel resolution using Levenberg-Marquardt fitting with an integrated 2D Gaussian spot model starting from an initial estimate provided by a radial symmetry method. Detected spots were connected into trajectories using a custom modification of the hill-climbing algorithm. The result is a table of spot coordinates and estimates of their diffusion coefficients. To visualize kinetics as a function of their position in the image, spots falling within a defined range of diffusion coefficients were reconstructed with the same pixel dimensions as the original image.

[0468] To recover dynamic information from the trajectories, we use a Bayesian estimation approach, State Array (Heckert), with a "RBME" likelihood function and a time scale of 0.003 to 30 μm. 2 s -1 A grid of 125 diffusion coefficients and seven localization error magnitudes ranging from 0.02 to 0.08 μm was used. After estimation, localization errors were ignored to obtain a one-dimensional distribution over the diffusion coefficients for each field of view.

[0469] 8.44 Example 44: Solubility and Permeability Data Table 6: Solubility and permeability data for compounds featured in Figure 7. The data show that compound 39-10c has improved properties over photoactivatable JF549-HaloTag, similar to JF549-HaloTag. Data was obtained to demonstrate reduced nonspecific labeling with 39-10c over photoactivatable JF549-HaloTag. [Table 9]

[0470] A number of references have been cited, the disclosures of each of which are incorporated herein by reference in their entirety.

Claims

1. Compound of formula (VIII): 【Chemistry 1】 or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, During the ceremony, R １ is H or -C(=O)N(C １－３ alkyl) ２ and Each L １ is, independently, -O-C １－６ -Alkylene-, -C(O)NH-, -C １－６ -Alkylene-N(C １－３ -alkyl)(C １－６ -alkylene)-, or -C １－６ -Alkylene-O-C １－６ -alkylene-, Each L ２ is -(OCH ２ CH ２ ) ｐ - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2; Z is 【Chemistry 2】 and R １ is H and a is 1, then L １ teeth, 【Transformation 3】 Instead, a wavy line 【Chemistry 4】 represents a point of attachment to another moiety of said compound, or a salt, single stereoisomer, mixture of stereoisomers or isotopic form thereof.

2. 2. The compound of claim 1 , The compound wherein p is 1, a is 1, and b is an integer of 0 to 1.

3. A compound according to claim 1 or claim 2, R １ is H, and each L １ But independently, -O-C １－６ -Alkylene-, -C １－６ -Alkylene-N(C １－３ -alkyl)(C １－６ -alkylene)-, or -C １－６ -Alkylene-O-C １－６ -alkylene-, Each L ２ But -(OCH ２ CH ２ ) ｐ - and p is an integer from 1 to 3, a is an integer of 1 or 2; b is an integer from 0 to 2; Z is, 【Transformation 5】 The compound,

4. A compound according to claim 1 or claim 2, R １ But -C(=O)N(C １－３ alkyl) ２ and Each L １ is —C(O)NH—, Each L ２ But -(OCH ２ CH ２ ) ｐ - and p is an integer from 1 to 3; a is an integer of 1 or 2, b is an integer from 0 to 2; Z is, 【Transformation 6】 The compound,

5. Compound of formula (IX): 【Transformation 7】 or a salt thereof, single stereoisomer, mixture of stereoisomers or isotopic forms thereof, wherein: L Ｂ is (i) -O-C １－６ -Alkylene-, -C １－６ -Alkylene-N(C １－３ -alkyl)-, or -C １－６ -alkylene-O-, or a salt, single stereoisomer, mixture of stereoisomers or isotopic forms thereof.

6. 6. The compound of claim 5, wherein L Ｂ but, -OCH ２ -, -CH ２ N (CH ３ ) - or -CH ２ The compound, wherein the linker comprises O—.

7. Compound of formula (X): 【Transformation 8】 or a salt thereof, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, During the ceremony, R １ is H or -C(=O)N(C １－３ alkyl) ２ or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein

8. R １ The compound of claim 7, wherein is H.

9. R １ -C(=O)N(C １－３ alkyl) ２ 8. The compound of claim 7, wherein:

10. R １ but 【Chemistry 9】 8. The compound of claim 7, wherein:

11. The compound of claim 5 having the following structure: 【Chemistry 10】

12. The compound of claim 5 having the following structure: 【Chemistry 11】

13. The compound of claim 5 having the following structure: 【Chemistry 12】

14. 8. The compound of claim 7 having the following structure: 【Chemistry 13】

15. A method for labeling a protein, comprising contacting a sample containing a tagged protein with a compound according to any one of claims 1 to 14 to obtain a labeled protein.

16. 16. The method of claim 15, wherein the tagged protein is a kinase, a transcription factor, a chromatin modulator, an adaptor, a transporter, or a pathogenic aggregator.

17. 16. The method of claim 15, wherein the tagged protein is a histone.

18. 18. The method of claim 17, wherein the histone is H2B.

19. The method of any one of claims 15 to 18, wherein the labeled protein fluoresces when exposed to light.

20. 20. The method of claim 19, wherein the light is a laser.

21. 21. The method of claim 20, wherein the wavelength (λ) of the light is about 405 nm.

22. A process for preparing a compound of formula (VIII-a) comprising: 【Chemistry 14】 During the ceremony, R' is 【Chemistry 15】 and R １ is H or -C(=O)N(C １－３ alkyl) ２ and Each L １ is, independently, -O-C １－６ -Alkylene-, -C(O)NH-, -C(O)NH(C １－３ -alkylene)-, -C １－６ -Alkylene-N(C １－３ -alkyl)(C １－６ -alkylene)-, or -C １－６－ Alkylene -O-C １－６ -alkylene-, Each L ２ is -(OCH ２ CH ２ ) ｐ - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2; Z is 【Chemistry 16】 and R １ is H and a is 1, then L １ teeth, 【Chemistry 17】 Instead, a wavy line [Chemistry 18] represents a point of attachment to another moiety of said compound, and said process comprises: A compound of the following formula (a-1): 【Chemistry 19】 During the ceremony, R'= 【Chemistry 20】 R １ is H or -C(=O)N(C １－３ alkyl) ２ and R is -C(=O)OCH ３ or -C(=O)NH(C １－３ alkylene)-(OCH ２ CH ２ )-Z, Z is 【Chemistry 21】 The compound The method comprises converting using Ghosez's reagent in the presence of a diazomethane agent and a solvent to prepare a compound of formula (VIII-a).

23. A process for preparing a compound of formula (XI) comprising: During the ceremony, R １ is H or -C(=O)N(C １－３ alkyl) ２ and Each L １ teeth, -C(O)NH(C １－３ -alkylene)-, Each L ２ is -(OCH ２ CH ２ ) ｐ - and p is an integer from 1 to 3, a is an integer of 1 or 2, b is an integer from 0 to 2; Z is 【Chemistry 22】 and the process comprises: A compound of the following formula (a-2): 【Chemistry 23】 During the ceremony, R １ is H or -C(=O)N(C １－３ alkyl) ２ and R is -C(=O)OCH ３ or -C(=O)NH(C １－３ alkylene)-(OCH ２ CH ２ )-Z, Z is 【Chemistry 24】 The compound The process comprises converting using Ghosez's reagent in the presence of a diazomethane agent and a solvent to prepare a compound of formula (XI).

24. 24. The method of claim 22 or 23, wherein the diazomethane agent is trimethylsilyldiazomethane diethyl ether.

25. 24. The method of claim 22 or 23, wherein the solvent comprises dichloromethane.

26. The method of any one of claims 22 to 25, further comprising using a desiccant and a metal oxide.

27. 27. The method of claim 26, wherein the desiccant is a molecular sieve and the metal oxide is calcium oxide.

28. 23. A compound of formula (VIII-a) produced by the process of claim 22.

29. 24. A compound of formula (XI) produced by the process of claim 23.

30. 30. The compound of claim 28 or 29, which is substantially chemically pure.

31. 30. The compound of claim 28 or 29, which is substantially free of chemical impurities.

32. 10. The compound of claim 1, which is substantially chemically pure.

33. 10. The compound of claim 1, substantially free of chemical impurities.

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