Photoactive fluorescent compounds and their use for labeling proteins

Abstract

Provided herein are novel photoactive fluorescent compounds and their use in labeling proteins, such as tagged proteins, and their use to visualize the position and dynamics of proteins in living cells.

Description

1. TECHNICAL FIELD

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

[0002] Provided herein are novel photoactive fluorescent compounds and their use in labeling proteins, such as tagged proteins, and their use for visualizing the location and dynamics of proteins in living cells. 2. BACKGROUND

[0003] Fluorescence microscopy can be used to visualize the location and dynamics of biomolecules within living cells. The process involves labeling biomolecules with bright, photostable fluorescent dyes that absorb photons and then emit them at a different wavelength. Green fluorescent protein (GFP) and other genetically encoded fluorophores have previously been the gold standard for fluorescence imaging because they allow for labeling with genetic specificity. Numerous efforts have been made to improve protein dyes by enhancing photostability and other properties; these efforts include enzyme-based self-labeling tags, such as HaloTag, which allows for labeling of specific protein fusions with synthetic fluorophores and enables a variety of imaging experiments within living cells. See U.S. Patent No. 10,161,932.

[0004] Rhodamine dyes have been widely used for their brightness and photostability properties, among others. The photophysics of rhodamines are well known for their importance as biological probes. See Grimm et al., “Deuteration Improves Small-Molecule Fluorophores,” 2020, BioRxiv preprint, available at https: / / doi.org / 10.1101 / 2020.08.17.250027 (Grimm et al., 2020). Methods have been described to increase the brightness and photostability of fluorophores. These methods include 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, rhodamine dyes do not possess cell penetration properties that would allow for optimization of live cell labeling experiments. See U.S. Patent No. 10,161,932.

[0005] To enhance cell permeability and improve brightness, azetidine-substituted fluorescent compounds were developed. Such 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 fluorescent quantum yields of rhodamines and other dyes were improved by introducing deuterium into the alkylamino substituents of these dyes. However, it was found that deuteration can prevent or slow down adverse properties such as photochemically induced spectral shifts and non-reparable photobleaching. See Grimm et al., 2020.

[0006] Accordingly, there remains a need for photoactive fluorescent compounds with improved photostability, brightness, labeling specificity, density control, and other properties for labeling proteins, and the present disclosure addresses this need. 3. SUMMARY

[0007] In one aspect, provided herein is a compound of Formula (A):

[0008] (A) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein L A is a linker, Z comprises a moiety that covalently bonds to a tagged protein, n is an integer from 1 to 3, and other variables are as defined herein; and wherein moiety L A -Z is located at position 5 or 6.

[0009] In one aspect, provided herein is a compound of Formula (A-1):

[0010] (A-1) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein L A is a linker, Z comprises a moiety that covalently bonds to a tagged protein, n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3, and other variables are as defined herein.

[0011] In one aspect, provided herein is a compound of Formula (A-2):

[0012] (A-2) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein L A is a linker, Z comprises a moiety that covalently bonds to a tagged protein, n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3, and other variables are as defined herein.

[0013] In one aspect, provided herein is a compound of Formula (VIII):

[0014] (VIII) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein: R 1 is H or –C(=O)N(C 1-3 alkyl)2, 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 –(OCH2CH2) p –; 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 or ; and wherein when R 1 is H and a is 1, then L 1 is not , wherein the wavy line represents the point of attachment to the rest of the compound.

[0015] Also provided herein is a compound of Formula (IX):

[0016] (IX) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form 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–.

[0017] Also provided herein is a compound of Formula (X):

[0018] (X) or salts, single stereoisomers, mixtures of stereoisomers, or isotopic forms thereof, wherein: R 1 is H or -C(=0)N(C 1-3 alkyl)2.

[0019] In another aspect, provided herein are methods of making a compound disclosed herein to produce a compound that is substantially chemically pure and / or substantially free of chemical impurities.

[0020] In another aspect, provided herein are methods of labeling a protein, comprising contacting a sample comprising a tagged protein with a compound disclosed herein, thereby producing a labeled protein. 4. Brief Description of the Drawings

[0021] Figure 1 Provided are the specificities, brightness, and density control of the commercial non- photoactivatable dye Janelia Fluor ® 549 (JF 549 ), the commercial photoactivatable dye (PA-JF 549 ), and a hypothetical ideal dye.

[0022] Figure 2 Shown is the preparation of photoactivated fluorescently tagged (e.g., labeled) proteins.

[0023] Figure 3 Shown is the relative labeling specificity of PA-JF 549 compared to the photoactivatable dye compounds of Example 4. Labeling specificity was calculated using histone H2B located in the nucleus of wild-type cells that do not express any HaloTag® protein.

[0024] Figure 4 Shown is the signal-to-noise ratio (SNR) of JF 549 , PA-JF 549 , and the photoactivatable dye compounds of Examples 1-4.

[0025] Figure 5 Shown are exemplary fields of view of U2OS expressing ER-HaloTag fusions co- stained with Potomac Red (CAS: 2127150-65-4; Grimm et al., 2017). Labeling specificity of the photoactivatable dye compound of Example 4 was measured and compared to PA-JF 549 .

[0026] Figure 6 Shown is the signal-to-noise ratio (SNR) of JF 549 , PA-JF549 Signal-to-noise ratio (SNR) of the photoactivatable dye compounds of Examples 1, 4, 7 and 10.

[0027] Figure 7 The number of spots detected in HaloTag negative U2OS cells using JF549-HaloTag (left), photoactivatable JF549-HaloTag (middle) and compound 39-10c (compound 4) (right) is shown, depicting that compound 39-10c can reduce non-specific labeling compared to photoactivatable JF549-HaloTag.

[0028] Figure 8 A time course of 4000 frames is shown, depicting photoactivation events of compound 39-10c at 1 mW, 4 mW and 8 mW and subsequent loss of signal due to photobleaching.

[0029] Figure 9 A graphical representation of the appearance of compound 39-10c labeled as a β-catenin-HaloTag fusion protein at different diffusion coefficients is shown, labeled as slow (< 0.1 pm / s), medium (0.1 - 1 pm / s) and fast (> 1 pm / s) diffusion populations. 2 2 2

[0030] Figure 10A A field of view containing four cells, whose populations are as described in Figure 9 , is shown. These cell boundaries can be overlaid on the Figure 10B -E to indicate the cell positions in the fluorescence image. Thus, this map maps the positions of the four cells in the image as a reference for Figure 10B -E.

[0031] Figure 10B :An overlay of all three cell populations depicted in Figure 9 is shown. The zoomed-in view of the shear graph shows two different slow and medium populations at the area of the focal adhesion between cell 1 and cell 3.

[0032] Figure 10C , Figure 10D , Figure 10E : The three channels depicted separately clearly indicate the position of each population and the cell localization map. 5. DETAILED DESCRIPTION

[0033] 5.1 DEFINITIONS ​​To facilitate understanding of the present disclosure described herein, some 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.

[0034] As used herein, the terms “comprising” and “including” can be used interchangeably. The terms “comprising” and “including” shall be construed as specifying the presence of stated features or components, but do not preclude the presence or addition of one or more other features, components, or groups thereof. Furthermore, the terms “comprising” and “including” are intended to include instances of the term “consisting of.” Thus, the terms “comprising” and “including” can be replaced by the term “consisting of” to provide still more specific embodiments of the application.

[0035] As used herein, the term “or” shall be construed in the inclusive sense, meaning either one or any combination of the listed items. 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.” The exception to this definition will occur when an element, function, step or act is in some way inherently mutually exclusive from another.

[0036] As used herein, the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which is dependent on how the value is being 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.

[0037] As used herein, the term "salt" 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, metal or ammonium salts of 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 acids and organic acids such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Particular non-toxic acids include hydrochloric, hydrobromic, maleic, phosphoric, sulfuric, and methanesulfonic acids. Thus, particular examples of salts include hydrochloride and methanesulfonate salts. Others are well-known in the art.

[0038] As used herein and unless otherwise indicated, the term "stereoisomer" or "stereoisomerically pure" means one stereoisomer of a compound disclosed herein that is substantially free of other stereoisomers of the compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. Typical stereoisomerically pure compounds include greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. The compounds disclosed herein can have chiral centers and can exist in the form of racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms of these compounds are included in the embodiments disclosed herein, including mixtures thereof.

[0039] The embodiments disclosed herein cover the use of the pure stereoisomers of such compounds as disclosed herein, as well as the use of mixtures of these forms. For example, mixtures comprising equal or unequal amounts of enantiomers of a particular compound can be used in the methods and compositions disclosed herein. These isomers can be synthesized asymmetrically 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, EL, Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions Page 268 (EL Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0040] It should also be noted that the compounds disclosed herein may include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In some embodiments, the compounds disclosed herein are isolated as either E or Z isomers. In other embodiments, the compounds disclosed herein are mixtures of E and Z isomers.

[0041] As used herein, the terms "isotopic form" or "isotope" mean, for example, that a compound can be radiolabeled with a radioactive isotope, such as, for example, tritium ( 3 H), Iodine-125 ( 125 I), sulfur-35( 35 S) or carbon-14 ( 14 C), or it could be isotopically enriched, such as with carbon-13 (C). 13 C) or nitrogen-15 ( 15N). As used herein, an “isotopologue” is an isotopically enriched compound. The term “isotopically enriched” refers to an atom having an isotopic composition other than the natural isotopic composition of the atom. “Isotopically enriched” can also refer to a compound containing at least one atom that has an isotopic composition other than the natural isotopic composition of that atom. The term “isotopic composition” refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer and inflammation therapeutic agents, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds disclosed herein are provided, e.g., isotopologues are carbon-13 or nitrogen-15 enriched compounds. As used herein, “deuterated” means a compound in which at least one hydrogen (H) has been replaced by deuterium (indicated by D or 2 H represents) i.e., the compound is enriched in deuterium at at least one position.

[0042] As used herein and unless otherwise indicated, a “substantially chemically pure” compound is substantially free of other chemical 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 chemical compounds. Detection of other chemical compounds can be accomplished by any method apparent to one of ordinary skill in the art, including but not limited to chemical analytical methods such as, for example, mass spectrometric analysis, spectroscopic analysis, thermal analysis, elemental combustion analysis, and / or chromatographic analysis.

[0043] As used herein, “alkyl” refers to a straight or branched chain saturated hydrocarbon group containing 1-10 carbon atoms. In certain embodiments, an alkyl group includes one carbon atom (“C1alkyl”). In certain embodiments, an alkyl group includes 1-2 carbon atoms (“C 1-2 alkyl”). In certain embodiments, an alkyl group includes 1-3 carbon atoms (“C 1-3 alkyl”). In certain embodiments, an alkyl group includes 1-4 carbon atoms (“C 1-4 alkyl”). In certain embodiments, an alkyl group includes 1-6 carbon atoms (“C 1-6 alkyl”). In certain embodiments, an alkyl group includes 1-10 carbon atoms (“C 1-10alkyl") groups. In certain embodiments, alkyl includes methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylhexyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.

[0044] As used herein, "alkylene" refers to a straight-chain or branched saturated divalent hydrocarbon radical containing 1-10 carbon atoms, and in certain embodiments includes 1-6 carbon atoms. In certain embodiments, alkylene includes 1-3 carbon atoms ("C 1-3 alkylene") groups. In certain embodiments, alkylene includes 1-4 carbon atoms ("C 1-4 alkylene") groups. In certain embodiments, alkylene includes 1-6 carbon atoms ("C 1-6 alkylene") groups. In certain embodiments, alkylene includes 1-10 carbon atoms ("C 1-10 alkylene") groups.

[0045] As used herein, "alkenylene" refers to a straight-chain or branched chain alkenyl group minus one hydrogen so that the group is divalent. In certain embodiments, alkenylene includes 1-3 carbon atoms ("C 1-3 alkenylene") groups. In certain embodiments, alkenylene includes 1-4 carbon atoms ("C 1-4 alkenylene") groups. In certain embodiments, alkenylene includes 1-6 carbon atoms ("C 1-6 alkenylene") groups. In certain embodiments, alkenylene includes 1-10 carbon atoms ("C 1-10ethen-1,2-diyl; ethen-1,3-diyl; ethen-1,4-diyl; ethen-2,3-diyl; prop-1 - en-1,1 -diyl; prop-1 -en-1,2-diyl; prop-1 -en-1,3-diyl; prop-2-en-1,1 -diyl; prop-2- en-1,2-diyl; prop-2-en-1,3-diyl; prop-2-en-2,3-diyl; but-1 -en-1,1 -diyl; but-1 -en- 1,2-diyl; but-1 -en-1,3-diyl; but-1 -en-1,4-diyl; but-2-en-1,1 -diyl; but-2-en-1,2-diyl; but-2-en-1,3-diyl; but-2-en-1,4-diyl; but-2-en-2,3-diyl; but-3-en-1,1 -diyl; but-3- en-1,2-diyl; but-3-en-1,3-diyl; but-3-en-2,3-diyl; but-1,2-dien-1,1 -diyl; but-1,2- dien-1,3-diyl; but-1,2-dien-1,4-diyl; but-1,3-dien-1,1 -diyl; but-1,3-dien-1,2-diyl; but-1,3-dien-1,3-diyl; but-1,3-dien-1,4-diyl; but-1,3-dien-2,3-diyl; but-2,3-dien-1,1 - diyl; and but-2,3-dien-1,2-diyl. Alkenylene groups can be unsubstituted or substituted (e.g., optionally substituted alkenylene), as described for alkyl groups.

[0046] As used herein, "alkynylene" refers to a straight or branched chain bivalent substituent that contains one or two carbon-carbon triple bonds and only contains C and H when unsubstituted. In certain embodiments, an alkynylene group includes 1-3 carbon atoms ("C 1-3 alkynylene"). In certain embodiments, an alkynylene group includes 1-4 carbon atoms ("C 1-4 alkynylene"). In certain embodiments, an alkynylene group includes 1-6 carbon atoms ("C 1-6 alkynylene"). In certain embodiments, an alkynylene group includes 1-10 carbon atoms ("C 1-10 alkynylene"). In certain embodiments, an alkynylene group includes 1-10 carbon atoms ("C ethen-1,2-diyl; ethen-1,3-diyl; ethen-1,4-diyl; ethen-2,3-diyl; prop-1 - en-1,1 -diyl; prop-1 -en-1,2-diyl; prop-1 -en-1,3-diyl; prop-2-en-1,1 -diyl; prop-2- en-1,2-diyl; prop-2-en-1,3-diyl; prop-2-en-2,3-diyl; but-1 -en-1,1 -diyl; but-1 -en- 1,2-diyl; but-1 -en-1,3-diyl; but-1 -en-1,4-diyl; but-2-en-1,1 -diyl; but-2-en-1,2-diyl; but-2-en-1,3-diyl; but-2-en-1,4-diyl; but-2-en-2,3-diyl; but-3-en-1,1 -diyl; but-3- en-1,2-diyl; but-3-en-1,3-diyl; but-3-en-2,3-diyl; but-1,2-dien-1,1 -diyl; but-1,2- dien-1,3-diyl; but-1,2-dien-1,4-diyl; but-1,3-dien-1,1 -diyl; but-1,3-dien-1,2-diyl; but-1,3-dien-1,3-diyl; but-1,3-dien-1,4-diyl; but-1,3-dien-2,3-diyl; but-2,3-dien-1,1 - diyl; and but-2,3-dien-1,2-diyl. Alkenylene groups can be unsubstituted or substituted (e.g., optionally substituted alkenylene), as described for alkyl groups.

[0047] As used herein, "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic ring having 5 to 14 ring atoms, which contains one or more ring heteroatoms independently selected from O-, S-, -N= (trivalent nitrogen), and N(H)-, and the remaining ring atoms are carbon atoms, wherein the monocyclic ring is aromatic, and wherein at least one ring in the bicyclic or tricyclic ring is aromatic (but not necessarily the ring containing the heteroatom, e.g., tetrahydroquinolinyl, dihydroisoquinolinyl, dihydrobenzodioxinyl, 2,3-dihydrobenzo[b][l,4]dioxinyl, and the like). In certain embodiments, the heteroaryl is a monocyclic ring having 5 to 6 ring atoms. Unless otherwise specified, valence can be on any atom of any ring of the heteroaryl, as valence rules permit. 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[l,2-b]pyridazinyl, imidazo[l,2-a]pyridinyl, benzo[d][l,3]dioxolyl, 2,3-dihydrobenzo[b][l,4]dioxinyl, furo[2,3-d]thiazolyl, thieno[2,3-d]oxazolyl, thieno[3,2-b]furyl, 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][l,4]dioxinyl.

[0048] As used herein and unless otherwise indicated, "halogen" is fluorine, chlorine, bromine, or iodine.

[0049] 5.2 Embodiments (a) Compounds In one aspect, provided herein are photoactivatable fluorescent compounds. In one embodiment, the photoactivatable fluorescent compounds are used for labeling, e.g., labeling proteins. In one embodiment, the compounds disclosed herein can be used to visualize the location and dynamics of proteins in live cells.

[0050] These compounds exhibit unexpected improved properties, such as improved photostability, brightness, labeling specificity, and density control, which are desirable properties for photoactivatable fluorescent compounds. See Figure 1 , which provides a commercial non-photoactivatable dye, Janelia Fluor ®549(JF 549 ), commercially available photoactivated dyes (PA-JF) 549 Schematic diagram of the specificity, brightness, and density control of an ideal dye. (JF) 549 It exhibits high specificity and brightness, but poor density control. On the other hand, PAJF 549 It exhibits high brightness and density control, but poor specificity. An ideal dye should exhibit high specificity, brightness, and density control—properties that cannot be predicted based solely on structural characteristics. For example, PA-JF... 549 Modification of the core structure can not only alter the photophysical properties of the resulting light-converting dye, but also change its tendency to be photoactivated upon exposure to blue light. As described above, the modified PA-JF disclosed herein... 549 Compounds with a core structure exhibited unexpectedly improved properties.

[0051] In some embodiments, the compound has formula (A):

[0052] (A) Or in the form of its salts, single stereoisomers, mixtures of stereoisomers, or isotopes, wherein L A It is a linker, Z contains the portion covalently bonded to the tagged protein; n is an integer from 1 to 3, X is hydrogen, halogen, or –OCH3; and part of L... A -Z is located at position 5 or 6; 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 , or Among them, the wavy line ( () indicates the connection point with the rest of the compound; where: R 1 It 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 group 2, –(CH2) n O(C 1-3 alkyl), , or ; and R 2 Is it –O– or –N(C)? 1-3alkyl)2—, —N(C

[0053] In certain embodiments, the compound is of Formula (A-1):

[0054] (A-1) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein L A is a linker, Z comprises a moiety covalently bonded to a tagged protein; n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3; 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 , or wherein the wavy line represents the point of attachment to the remainder of the compound; wherein: 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), , or ; and wherein R 2 is -O- or —N(C 1-3 alkyl)—.

[0055] In certain embodiments, the compound is of Formula (A-2):

[0056] (A-2) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein L A is a linker, Z comprises a moiety covalently bonded to a tagged protein; n is an integer from 1 to 3, X is hydrogen, halogen, or -OCH3; 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 1-3alkyl)– or –P(=O)(Ph)–; R is , or Among them, the wavy line ( () indicates the connection point with the rest of the compound; where: R 1 It 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 group 2, –(CH2) n O(C 1-3 alkyl), , or ; and R 2 Is it –O– or –N(C)? 1-3 alkyl)-.

[0057] In some embodiments, the compound has formula (I):

[0058] (I) Or in the form of its salts, single stereoisomers, mixtures of stereoisomers, or isotopes, wherein L A Z is the linker, containing the portion covalently bonded to the tagged protein, and other variables are as defined in this paper.

[0059] In some implementations, L A Type IA connector:

[0060] (IA) in: Each L 1 Independently: (i) –C 1-6 -alkylene–、–C 1-6 -Idenoyl–or–C 1-6 -Imyynyl–; (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-, -O-C 1-6 -alkylene-, -C(O)-C 1-6 -alkylene- or -C 1-6 -alkylene-C(O)-; each L 2 independently is -C 1-6- -alkylene-, -(OCH2) p , -(CH2O) p -, -(OCH2CH2) p - or -(CH2CH2O) p -; p is an integer from 1 to 3; and a and b are each independently an integer of 1 or 2.

[0061] In certain embodiments, L A is oriented such that -(L 2 ) b - is bonded to Z, as shown in the following formula (IB):

[0062] (IB).

[0063] In certain embodiments, the compound of formula (IB) is one in which: when R is , R 1 is H and X is H, then Z-(L 2 ) b -(L 1 ) a - is not where the wavy line represents the point of attachment to the remainder of the compound.

[0064] In certain embodiments of the compound of formula (I) or (IB), G is -O-, -S-, -SO2-, -C(C 1-3 alkyl)2-, -N(C 1-3 alkyl)-, -Si(C 1-3 alkyl)2-, -P(=O)(OH)-, -P(=O)(C 1-3 alkyl)-, or -P(=O)(Ph)-.

[0065] In certain embodiments of the compound of formula (I) or (IB), R is , or .

[0066] In certain embodiments of the compounds of Formula (I) or (IB), R 1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), , or .

[0067] In certain embodiments of the compounds of Formula (I) or (IB), R 2 is -O- or -N(C 1-3 alkyl)-.

[0068] In certain embodiments of the compounds of Formula (I) or (IB), X is hydrogen or halogen.

[0069] In certain embodiments of the compounds of Formula (I) or (IB), X is halogen.

[0070] In certain embodiments, the compounds of Formula (IB) are those wherein: R is , R 1 is H and X is halo.

[0071] In certain embodiments of the compounds of Formula (I) or (IB), Z is , , or .

[0072] In certain embodiments, the compounds of Formula (I) are those wherein:

[0073] (I) are those wherein: G is -0-, -S-, -S02-, -C(C 1-3 alkyl)2-, -N(C 1-3 alkyl)-, -Si(C 1-3 alkyl)2-, -P(=0)(OH)-, -P(=0)(C 1-3 alkyl)-, or -P(=0)(Ph)-; R is , or , wherein the wavy line represents the point of attachment to the remainder of the compound. wherein: R 1 is H, -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), 1-3 -C(=0)N(C 1-3 alkyl)2, -N(C n alkyl)2, -(CH2) 1-3 O(C 2 alkyl), , or ; R 1-3 is -0- or -N(C A alkyl)-; X is hydrogen or halogen; L A is a linker; and Z is , , or .

[0074] In certain embodiments, the compound of Formula (I) is wherein: when L 1 is a linker of Formula (IA), R is , R 2 is H and X is H, then Z-(L b ) 1 -(L a ) wherein the wavy line represents the point of attachment to the remainder of the compound.

[0075] In certain embodiments, the compound of Formula (IB) is:

[0076] (IB) wherein: G is -0-, -S-, -S02-, -C(C 1-3 alkyl)2-, -N(C 1-3 alkyl)-, -Si(C 1-3 alkyl)2-, -P(=0)(OH)-, -P(=0)(C 1-3 alkyl)-, or -P(=0)(Ph)-; R is , or wherein the wavy line represents the point of attachment to the remainder of the compound; wherein: R 1 is H, -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), , or ; R 2 is -0- or -N(C 1-3 alkyl)-; X is hydrogen or halogen; 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-, -O-C 1-6 -alkylene-, -C(O)-C 1-6 -alkylene- or -C 1-6 -alkylene-C(O)-; each L 2 is independently -C 1-6 -alkylene-, -(OCH2) p -, -(CH2O) p -, -(OCH2CH2) p - or -(CH2CH2O) p -; p is an integer from 1 to 3; a and b are each independently an integer of 1 or 2; and Z is , , or .

[0077] In some such embodiments of the compound of formula (IB), Z is .

[0078] In some such embodiments of the compound of formula (IB), wherein Z is , X is hydrogen.

[0079] In some such embodiments of the compound of formula (IB), wherein Z is , X is fluorine.

[0080] In certain embodiments, the compound of formula (I) is wherein: when R is , R 1 is H and X is H, then Z–(L 2 ) b –(L 1 ) a – is not wherein the wavy line indicates the point of attachment to the remainder of the compound.

[0081] In certain embodiments, the tagged protein comprises a HaloTag®, a SNAP-tag® or a CLIP-tag®.

[0082] In certain embodiments, provided herein is a compound of formula (II):

[0083] (II) or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein: the asterisks "**" and "*" indicate the orientation of the linker L B ; and the other variables are as defined herein.

[0084] In certain embodiments of the compound of formula (II), 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)–.

[0085] In certain embodiments of the compound of formula (II), R is , or .

[0086] In certain embodiments of the compound of formula (II), R 1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), , or .

[0087] In certain embodiments of the compound of formula (II), X is hydrogen or halogen. In some such embodiments, X is hydrogen. In some such embodiments, X is halogen.

[0088] In certain embodiments of the compound of formula (II), R 2 is -0- or -N(C 1-3 alkyl)-

[0089] In certain embodiments of the compound 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)0-, -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-, -O-C 1-6 -alkylene-, -C(O)-C 1-6 -alkylene-, or -C 1-6 -alkylene-C(O)-.

[0090] In certain embodiments of the compound of formula (II), Z is , , or . In some such embodiments of the compound of formula (II), Z is .

[0091] In some such embodiments of the compound of formula (II), wherein Z is X is hydrogen.

[0092] In some such embodiments of the compound of formula (II), wherein Z is X is fluorine.

[0093] In certain embodiments, the compound of formula (II):

[0094] (II) is wherein: G is -0-, -S-, -S02-, -C(C 1-3 alkyl)2-, -N(C 1-3 alkyl)-, -Si(C 1-3 alkyl)2-, -P(=0)(OH)-, -P(=0)(C 1-3 alkyl)-, or -P(=0)(Ph)-; R is or wherein: R 1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n 0(C 1-3 alkyl), , or ; wherein n is an integer from 1 to 3; and R 2 is -0- or -N(C 1-3 alkyl)-; X is hydrogen or halogen; L B is (i) -C 1-6 -alkylene-, -C 1-6 -alkenylene-, or -C 1-6 -alkynylene-; (ii) heteroaryl; or (iii) -NHC(0)-, -C(0)NH-, -OC(0)-, -C(0)0-, -N(C 1-3 alkyl)-C(0)-, -C(0)-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(C1-6 -alkylene)-C 1-6 -alkylene-, -C 1-6 -alkylene-O-, -O-C 1-6 -alkylene-, -C(O)-C 1-6 -alkylene- or -C 1-6 -alkylene-C(O)-.

[0095] In certain embodiments of the compound of formula (II), G is -O-, -S-, or -SO2-; R is ; 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); L B is -NHC(O)- or -C(O)NH-, and X is hydrogen.

[0096] In certain embodiments of the compound of formula (II), G is -O- or -S-; R is ; R 1 is -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); L B is -NHC(O)- or -C(O)NH-, and X is hydrogen.

[0097] In certain embodiments, provided herein is a compound of formula (III):

[0098] (III).

[0099] In certain embodiments of the compound of formula (III), G is -O-, -S-, -N(CH3)-, -Si(CH3)2-, -C(CH3)2-, or -SO2-.

[0100] In certain embodiments of the compound of formula (III), L B is a linker, including , , , , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or wherein * and ** indicate linkers L B in the orientation of the above embodiments.

[0101] It should be noted that the position of the wavy line bond (whether depicted in the middle of the bond or at the end of the bond) is immaterial such that, for example, 、 、 and structures are all equivalent to one another.

[0102] In certain embodiments of the compound of Formula (III), L B comprises a heteroaryl group.

[0103] In certain embodiments of the compound of Formula (III), the heteroaryl group is a triazole or an imidazole.

[0104] In certain embodiments of the compound of Formula (III), R 1 is 、 、 、 、 、 、 、 or .

[0105] In certain embodiments of the compound of Formula (III), R 1 is H and L B is .

[0106] In certain embodiments, provided herein is a compound of Formula (IV):

[0107] (IV) wherein: R1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), , or ; wherein n is an integer from 1 to 3.

[0108] In certain embodiments, R 1 is -N(CH3)2.

[0109] In certain embodiments, R 1 is -OH.

[0110] In certain embodiments, R 1 is .

[0111] In certain embodiments, R 1 is .

[0112] In certain embodiments, R 1 is .

[0113] In certain embodiments, R 1 is .

[0114] In certain embodiments, R 1 is .

[0115] In certain embodiments, R 1 is .

[0116] In certain embodiments, R 1 is .

[0117] In certain embodiments, provided herein is a compound of Formula (V):

[0118] (V) wherein: R 1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2)n O(C 1-3 alkyl), , or ; wherein n is an integer from 1-3.

[0119] In certain embodiments, provided herein is a compound of Formula (V):

[0120] (V) wherein: R 1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)NH2, -C(=0)NH(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), , or ; wherein n is an integer from 1-3.

[0121] In certain embodiments, provided herein is a compound of Formula (V):

[0122] (V) wherein: R 1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)NH2, -C(=0)NH(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), , or ; wherein n is an integer from 1-3.

[0123] In certain embodiments, R 1 is -N(CH3)2.

[0124] In certain embodiments, R 1 is -OH.

[0125] In certain embodiments, R 1 is .

[0126] In certain embodiments, R 1 is .

[0127] In certain embodiments, R 1 is .

[0128] In certain embodiments, R 1 is .

[0129] In certain embodiments, R 1 is .

[0130] In certain embodiments, R 1 is .

[0131] In certain embodiments, R 1 is .

[0132] In certain embodiments, provided herein is a compound of Formula (VI):

[0133] (VI) wherein: R 1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), , or ; wherein n is an integer from 1-3.

[0134] In certain embodiments, R 1 is .

[0135] In certain embodiments, R 1 is .

[0136] In certain embodiments, R 1 is .

[0137] In certain embodiments, provided herein is a compound of Formula (Via):

[0138] (VIa) wherein: R 1 is -OH, -C(=0)OH, -C(=0)0(C 1-3 alkyl), -C(=0)N(C 1-3 alkyl)2, -N(C 1-3 alkyl)2, -(CH2) n O(C 1-3 alkyl), , or ; wherein n is an integer from 1 to 3.

[0139] In certain embodiments, R 1 is .

[0140] In certain embodiments, R 1 is .

[0141] In certain embodiments, R 1 is .

[0142] In certain embodiments, the compound is: .

[0143] In certain embodiments, the compound is: , , , or .

[0144] In certain embodiments, the compound is: , , , or .

[0145] In certain embodiments, the compound is: .

[0146] In certain embodiments, the compound of formula (Via) is: .

[0147] In certain embodiments, the compound of Formula (Via) is: .

[0148] In certain embodiments, provided herein is a compound of Formula (VII):

[0149] (VII) wherein: R is , or

[0150] wherein: R 2 is -CH3and L B is a linker comprising: , , or , wherein * and ** indicate the orientation of the linker L B in the above embodiments.

[0151] In certain embodiments of the compound of Formula (VII), R is not .

[0152] In certain embodiments of the compound of Formula (VII), L B is not .

[0153] In certain embodiments of the compound of Formula (VII), when R is , L B is not .

[0154] In certain embodiments of the compound of Formula (VII), when R is , L B is .

[0155] In certain embodiments of the compound of Formula (VII), when R is , L B is or .

[0156] In certain embodiments, the compound is of Formula (VIII):

[0157] (VIII) or salts, single stereoisomers, mixtures of stereoisomers, or isotopic forms thereof, wherein: R 1 is H or -C(=0)N(C 1-3 alkyl)2, 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 -(OCH2CH2) p -; p is an integer from 1 to 3; a is an integer from 1 or 2; b is an integer from 0 to 2; Z is or ; and wherein when R 1 is H and a is 1, then L 1 is not , wherein the wavy line represents the point of attachment to the remainder of the compound.

[0158] In one embodiment of Formula (VIII), 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 –(OCH2CH2) p –; p is an integer from 1 to 3; a is an integer from 1 or 2; b is an integer from 0 to 2; Z is or ; and wherein when R 1 is H and a is 1, then L 1 is not wherein the wavy line ( ) indicates the point of attachment to the remainder of the compound.

[0159] In certain embodiments of the compound of formula (VIII), p is 1, a is 1, and b is an integer from 0 to 1.

[0160] In certain embodiments of the compound of formula (VIII), R 1 is H, each L 1 is 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 is –(OCH2CH2) p –; p is an integer from 1 to 3; a is an integer from 1 or 2; b is an integer from 0 to 2; and Z is .

[0161] In some such embodiments of the compound of formula (VIII), p is 1, a is 1, and b is 1.

[0162] In certain embodiments of the compound 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 –; p is an integer from 1 to 3; a is an integer from 1 or 2; b is an integer from 0 to 2; and Z is .

[0163] In certain embodiments of the compound 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 –; p is an integer from 1 to 3; a is an integer from 1 or 2; b is an integer from 0 to 2; and Z is .

[0164] In some such embodiments of the compound of Formula (VIII), a is 1 and b is 0.

[0165] Also provided herein are compounds of Formula (IX):

[0166] (IX) or salts, single stereoisomers, mixtures 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–.

[0167] In certain embodiments of the compound of Formula (IX), L B is a linker, including: –OCH2–, –CH2N(CH3)–, or –CH2O–.

[0168] Also provided herein are compounds of Formula (X-1):

[0169] (X-1) or salts, single stereoisomers, mixtures of stereoisomers, or isotopic forms thereof, wherein: R 1 is H or –C(=O)N(C 1-3 alkyl)2.

[0170] In certain embodiments of the compound of Formula (X-1), R 1 is H.

[0171] In certain embodiments of the compound of Formula (X-1), R 1 is –C(=O)N(C 1-3 alkyl)2.

[0172] In certain embodiments of the compound of Formula (X-1), R 1 is .

[0173] Also provided herein are compounds of Formula (X):

[0174] (X) or salts, single stereoisomers, mixtures of stereoisomers, or isotopic forms thereof, wherein: R 1 is H or -C(=0)N(C 1-3 alkyl)2.

[0175] In certain embodiments of the compound of Formula (X), R 1 is H.

[0176] In certain embodiments of the compound of Formula (X), R 1 is -C(=0)N(C 1-3 alkyl)2.

[0177] In certain embodiments of the compound of Formula (X), R 1 is .

[0178] In certain embodiments, the compound is:

[0179] In certain embodiments, the compound is:

[0180] In certain embodiments, the compound is:

[0181] In certain embodiments, the compound is:

[0182] In certain embodiments, the compound is:

[0183] 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'- xanthen]-3',6'-diyl)bis(azetidine-3-carboxylic acid dimethyl ester).

[0184] 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'- xanthen]-3',6'-diyl)bis(azetidine-3-carboxylic acid).

[0185] In certain embodiments, the compound is N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)- 2-diazo-3',6'-bis((R)-2-(methoxymethyl)azetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'- xanthen]-6-formamide.

[0186] In certain embodiments, the compound is l,l'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthen]-3',6'- diyl)bis(N,N-dimethylazetidine-3-carboxamide).

[0187] 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]hept-6-yl)-3-oxo-3H-spiro[isobenzofuran-l,9'- xanthen]-6-formamide.

[0188] In certain embodiments, the compound is 3',6'-di(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-N- (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthen]-6- formamide.

[0189] In certain embodiments, the compound is l,l'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthen]-3',6'- diyl)bis(N-methylazetidine-3-carboxamide).

[0190] In certain embodiments, the compound is (2S,2')S)-l,l'-(6-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-l,9'- xanthen]-3',6'-diyl)bis(N,N-dimethylazetidine-2-carboxamide).

[0191] In certain embodiments, the compound is N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)- 2-diazo-3',6'-bis((R)-2-(hydroxymethyl)azetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'- xanthen]-6-formamide.

[0192] 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'- xanthen]-3',6'-diyl)bis(N,N-dimethylpyrrolidine-3-carboxamide).

[0193] 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] silinane-10,1 '-isochromene]-3,7-diyl)bis(N,N-dimethylazetidine-3- carboxamide).

[0194] 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'- xanthen]-3',6'-diyl)bis(azetidine-3-carboxamide).

[0195] In certain embodiments, the compound is 3',6'-bis(azetidin-1-yl)-6-((2-(2- ((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-3H-spiro[isochromene-1,9'-xanthen]-3-one.

[0196] In certain embodiments, the compound is 3-(2-((6-chlorohexyl)oxy)ethoxy)-N- (3',6'-bis(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthen]-6-yl)propanamide.

[0197] In certain embodiments, the compound is (E)-3',6'-bis(azetidin-1-yl)-6-(4-(2- ((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isochromene-1,9'-xanthen]-3-one.

[0198] In certain embodiments, the compound is (Z)-3',6'-bis(azetidin-1-yl)-6-(4-(2- ((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isochromene-1,9'-xanthen]-3-one.

[0199] In certain embodiments, the compound is 3',6'-bis(azetidin-l-yl)-6-(4-(2-((6- chlorohexyl)oxy)ethoxy)butyl)-2-diazo spiro[indene-l,9'-xanthene]-3(2H)-one.

[0200] In certain embodiments, the compound is 3',6'-bis(azetidin-l-yl)-6-(3-(2-((6- chlorohexyl)oxy)ethoxy)propoxy)-2-diazo spiro[indene-l,9'-xanthene]-3(2H)-one.

[0201] In certain embodiments, the compound is 3',6'-bis(azetidin-l-yl)-6-(3-(2-((6- chlorohexyl)oxy)ethoxy)propoxy)-2-diazo spiro[indene-l,9'-xanthene]-3(2H)-one.

[0202] In certain embodiments, the compound is 3',6'-bis(azetidin-l-yl)-6-(3-(2-((6- chlorohexyl)oxy)ethoxy)propoxy)-2-diazo spiro[indene-l,9'-xanthene]-3(2H)-one.

[0203] In certain embodiments, the compound is 3',6'-bis(azetidin-l-yl)-6-(3-(2-((6- chlorohexyl)oxy)ethoxy)propoxy)-2-diazo spiro[indene-l,9'-xanthene]-3(2H)-one. N , N - dimethylazetidine-3-carboxamide).

[0204] In certain embodiments, the compound is 3',6'-bis(azetidin-l-yl)-6-(3-(2-((6- chlorohexyl)oxy)ethoxy)propoxy)-2-diazo spiro[indene-l,9'-xanthene]-3(2H)-one.

[0205] It should be noted that if there is a discrepancy between a depicted structure and the name of the structure, the depicted structure controls.

[0206] The above paragraph presents multiple embodiments of the compounds provided herein. In each case, the embodiments include the recited compound and salts, single stereoisomers, mixtures of stereoisomers thereof, or isotopically-labeled forms thereof.

[0207] (b) Tagged protein In another aspect, provided herein is a tagged protein.

[0208] 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 adapter. In certain embodiments, the tagged protein is a transport protein. In certain embodiments, the tagged protein is a pathogenic aggregate.

[0209] In certain embodiments, the tagged protein is a histone. In certain embodiments, the histone is a H2B-HaloTag protein.

[0210] In certain embodiments, the tag is a HaloTag® (see, e.g., England et al., “HaloTag Technology: A Versatile Platform for Biomedical Applications,” Bioconjugate Chem. 2015, 26(6), 975-986 (England et al., 2015)). It should be noted that one of skill in the art knows how to make a protein with a fused HaloTag®.

[0211] 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 the HaloTag®. In certain embodiments, the HaloTag® is expressed using standard recombinant protein expression techniques. In certain embodiments, the HaloTag® protein coding region is inserted near the 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 label in fluorescence microscopy. In certain embodiments, the HaloTag® is used as a protein label in protein arrays. In certain embodiments, the HaloTag® is used as a protein label to determine the subcellular localization of a protein.

[0212] In certain embodiments, the protein tagged with Halo is a kinase, a transcription factor, a chromatin modulator, an adapter, a transport protein, or a pathogenic aggregate.

[0213] 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 one of skill in the art knows how to make a protein tagged with SNAP.

[0214] In certain embodiments, the SNAP-tag® is engineered from the enzyme alanine glyoxylate aminotransferase. In certain embodiments, the CLIP-tag® is self-labeling. In certain embodiments, the SNAP-tag® is orthogonal to O 6 - methylguanine-DNA methyltransferase (MGMT) gene. In certain embodiments, the SNAP-tag® covalently reacts with O 6 benzylguanine derivatives. In certain embodiments, the SNAP-tag® is used as a protein label in enzymatic assays. In certain embodiments, the SNAP-tag® is used as a protein label in cellular 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 arrays. In certain embodiments, the SNAP-tag® is used as a protein label to determine subcellular localization of proteins.

[0215] In certain embodiments, the protein tagged with SNAP is a kinase, a transcription factor, a chromatin modulator, an adaptor, a transport protein, or a pathogenic aggregate.

[0216] In certain embodiments, the tag is a CLIP-tag® (see, e.g., Corrêa et al., “Considerations and Protocols for the Synthesis of Custom Protein Labeling Probes,” Methods Mol Biol. 2015, 1266, 55-79 (Corrêa et al., 2015)). It should be noted that one of skill in the art knows how to make a protein tagged with CLIP.

[0217] In certain embodiments, the CLIP-tag® is self-labeling. In certain embodiments, the CLIP-tag® is orthogonal. In certain embodiments, the CLIP-tag® is engineered from the enzyme O 6- the methylguanine-DNA methyltransferase (MGMT) gene encodes. In certain embodiments, CLIP-tag® covalently reacts with a benzylcytosine derivative. In certain embodiments, CLIP-tag® is used as a protein label in protein complementation arrays. In certain embodiments, CLIP-tag® is used as a protein label in protein-protein interaction studies. In certain embodiments, CLIP-tag® is used as a protein label in enzymatic assays. In certain embodiments, CLIP-tag® is used as a protein label in cellular imaging. In certain embodiments, CLIP-tag® is used as a protein label in fluorescence microscopy. In certain embodiments, CLIP-tag® is used as a protein label in protein arrays. In certain embodiments, CLIP-tag® is used as a protein label to determine subcellular localization of proteins.

[0218] In certain embodiments, the protein tagged with CLIP is a kinase, a transcription factor, a chromatin regulator, an adaptor, a transport protein, or a pathogenic aggregate.

[0219] (c) labeled proteins and methods In another aspect, provided herein are methods of making a labeled protein to measure single protein motion within a cellular environment.

[0220] In certain embodiments, the method comprises contacting a sample comprising a tagged protein with a compound described herein, thereby producing a labeled protein. Without being bound by any mechanism or theory, it is understood that the tagged protein generally has a genetically modified active site that can specifically bind to the reactive linker of a photoactive fluorescent dye compound, thereby 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 Corrêa et al., 2015). The tagged protein-compound covalent complex is referred to herein as a labeled protein. The covalent bond forms rapidly and is essentially irreversible under physiological conditions.

[0221] The labeled protein can then be exposed to light, e.g., 405 nm light, causing the covalently bound photoactive dye compound to emit fluorescence.

[0222] Figure 2 A schematic showing the preparation of a photoactivated labeled protein is shown. As shown, and as described above, a tagged protein described herein is contacted 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, thereby forming a labeled protein. The labeled protein is then treated with light (e.g., light at 405 nm), resulting in the fluorescent emission of the covalently attached compound.

[0223] In certain embodiments, a tagged protein described herein is contacted with a compound (e.g., a photoactive fluorescent dye compound described herein) to form a labeled protein. In certain embodiments, the compound is a fluorophore. In certain embodiments, the compound contains a moiety that binds to the tagged protein to form the labeled protein. In certain embodiments, the tagged protein is covalently bonded to the compound to form the labeled protein. In certain embodiments, the compound is covalently bonded to a lysine residue on the tagged protein, thereby forming the labeled protein. In certain embodiments, the compound is covalently bonded to a cysteine residue on the tagged protein, thereby forming the labeled protein. In certain embodiments, the compound is covalently bonded to an aspartic acid residue on the tagged protein, thereby forming the labeled protein.

[0224] In certain embodiments, the compound portion of the labeled protein emits fluorescence when exposed to light. Without being bound by any mechanism or theory, it is understood that the compound portion of the labeled protein can undergo a Wolff rearrangement upon exposure to light, typically followed by decarboxylation of the compound. In certain embodiments, the labeled protein is irradiated 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 irradiated for about 5 minutes.

[0225] In certain embodiments, after incubation, the solution of labeled protein is filtered. In certain embodiments, the solution is filtered through a desalting column.

[0226] In certain embodiments, the labeled protein emits fluorescence when exposed to light. In certain embodiments, the light is a laser. In certain embodiments, the wavelength of light (λ) 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 the pulse. In certain embodiments, 561 nm (λ) light is used to stimulate the fluorophore of the labeled protein. In certain embodiments, the wavelength of light (λ) 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 a subcellular 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 plasma 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 membrane of the endoplasmic reticulum 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.

[0227] In certain embodiments, single protein movement within the cellular environment of one or more cells is measured. In certain embodiments, single protein movement within a cellular compartment of one or more cells is measured. In certain embodiments, single protein movement within the nucleus of one or more cells is measured. In certain embodiments, single protein movement within the cytoplasm of one or more live cells is measured. In certain embodiments, single protein movement within the plasma membrane of one or more live cells is measured. In certain embodiments, single protein movement within the mitochondria of one or more live cells is measured. In certain embodiments, single protein movement within the outer membrane of the mitochondria of one or more live cells is measured. In certain embodiments, single protein movement within the inner membrane of the mitochondria of one or more live cells is measured. In certain embodiments, single protein movement within the mitochondrial matrix of one or more live cells is measured. In certain embodiments, single protein movement within the Golgi apparatus of one or more live cells is measured. In certain embodiments, single protein movement within the lysosome of one or more live cells is measured. In certain embodiments, single protein movement within the endosome of one or more live cells is measured. In certain embodiments, single protein movement within the endoplasmic reticulum of one or more live cells is measured. In certain embodiments, single protein movement within the membrane of the endoplasmic reticulum of one or more live cells is measured. In certain embodiments, single protein movement within the rough endoplasmic reticulum of one or more live cells is measured. In certain embodiments, the measurement is real-time.

[0228] In certain embodiments, one or more live cells are prepared for imaging, for example, by incubation at about 37 °C. In certain embodiments, one or more live cells are incubated, for example, in the presence of about 5% CO2. In certain embodiments, one or more live cells are incubated overnight or for about 8-10 hours. In certain embodiments, one or more live cells are prepared for imaging by incubation with a compound (e.g., a photoactive fluorescent dye compound described herein) at a concentration of about 1 nM. In certain embodiments, one or more live cells are prepared for imaging by incubation with a compound (e.g., a photoactive fluorescent dye compound described herein) at a concentration of about 200 nM. In certain embodiments, one or more live cells are prepared for imaging by incubation with a compound (e.g., a photoactive fluorescent dye compound described herein) at a concentration of between about 1 nM and about 200 nM. In certain embodiments, one or more live cells are incubated with a compound (e.g., a photoactive fluorescent dye compound described herein) for about 45 minutes.

[0229] In certain embodiments, protein movement within a cellular environment is monitored by confocal microscopy. In certain embodiments, protein movement within a cellular environment is monitored by localization microscopy. In certain embodiments, protein movement within a cellular environment is monitored by super-resolution microscopy. In certain embodiments, protein movement within a cellular environment is monitored by single molecule localization microscopy (“SMLM”). In certain embodiments, protein movement within a cellular environment is monitored by photoactivated localization microscopy (“PALM”). In certain embodiments, protein movement within a cellular environment is monitored by stochastic optical reconstruction microscopy (“STORM”).

[0230] In certain embodiments, protein movement within a cellular environment is analyzed by maximum likelihood estimation models to detect single molecule fluorescence.

[0231] In certain embodiments, signal-to-noise ratio (“SNR”) is used as a proxy for single molecule brightness. In certain embodiments, SNR is analyzed by a log-likelihood ratio test. In certain embodiments, compounds (e.g., photoactive fluorescent dye compounds described herein) exhibit similar SNR to commercially available photoactivatable fluorescent dyes. In certain embodiments, compounds exhibit similar SNR to commercially available non-photoactivatable fluorescent dyes.

[0232] In certain embodiments, microscopy, e.g., confocal microscopy, localization microscopy, super-resolution microscopy, SMLM, PALM, or STORM, is used to determine labeling specificity. In certain embodiments, labeling specificity is calculated by comparing the number of spots detected by microscopy, e.g., localization microscopy, super-resolution microscopy, SMLM, PALM, or STORM, to a control sample. In certain embodiments, labeling specificity of compounds described herein is higher than labeling specificity of commercially available photoactivatable fluorescent dyes. 6. Methods of making compounds

[0233] Compounds of Formula (I), (IB), (II), (III), (IV), (V), (VI), and (Via) can be prepared using conventional organic synthesis and commercially available starting materials. For example, but not limited to, 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 and outlined in the examples described herein. It should be noted that one skilled in the art knows how to modify the procedures set forth in the illustrative schemes and obtain the desired products.

[0234] Scheme 1a

[0235] Compounds of formula (I), (IB), (II), (III), (IV), (V), (VI) and (Via) (wherein R, G and R 1 (VIa) (as defined herein) can be prepared starting from an appropriately derivatized intermediate, wherein Q represents a group capable of undergoing a cross-coupling reaction upon treatment with a suitable catalyst, such as a bromo or triflate derivative. Intermediate (C) can be prepared as described herein, for example, by converting 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid into its corresponding triflate derivative by conventional organic synthesis methods. Intermediate (C) can 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 . Methods2016, 13, 985-988 (Grimm et al. 2016). Alternatively, intermediate (C) can be prepared starting from 1,2,4-benzene tricarboxylic acid treated with 3-bromophenol under appropriate conditions. For example, in the preparation of compounds of formula (IV), intermediate (C) (where G is O) is treated with an appropriately substituted azetidine and a palladium catalyst such as Pd2(dba)3 in the presence of a ligand and a base such as cesium carbonate in a solvent such as dioxane and heated at a temperature of about 25 to about 100 °C to give intermediate (D). Intermediate D is then treated with an acid such as TFA or alternatively with a base such as lithium hydroxide or tin trimethyl hydroxide at a temperature of about 0 to about 25 °C and then coupled with an appropriate linker under basic conditions to give intermediate (E). Intermediate (E) is treated with oxalyl chloride in a suitable solvent such as dichloromethane at a temperature of about 0 to about 25 °C followed by treatment with freshly prepared diazomethane in Et2O (see for example, F. Arndt, “Diazomethane,” Org. Synth. 1935, 15, 3) to give compounds of formula (IV).

[0236] Alternatively, intermediate (E) is treated with 1-chloro-2,2-trimethylprop-1- ene-1-amine in a suitable solvent in the presence of 4 A molecular sieves at room temperature followed by treatment with trimethylsilyldiazomethane to give compounds of formula (IV) and this synthetic method can be used to optimize the synthesis of specific compounds and is necessary for the synthesis of other compounds. Examples of the use of this synthetic method are set out in the Examples section. N , N

[0237] Scheme 1b

[0238] As shown in Scheme 1b, compounds of formula (IV) and (V) (where R 1 is as defined herein) can be prepared starting from intermediate (D’) by cross-coupling reaction with an appropriately substituted azetidine and a palladium catalyst such as Pd2(dba)3 in the presence of a ligand and a base such as cesium carbonate in a solvent such as dioxane and heated at a temperature of about 25 to about 100 °C. The Arndt-Eistert reaction (see for example, F. Arndt, “Diazomethane,” Org. Synth. 1935, 15, 3) to give compounds of formula (IV) and (V). Intermediate D’ can be prepared from intermediate B1 as described herein in a solvent in the presence of a peptide coupling reagent such as T3P (propane phosphonic anhydride). ​

[0239] Scheme 2

[0240] As shown in Scheme 2, compounds of Formula (A) and (I) to (X) (where R' is -C(=0)OCH3or -L A -Z, and wherein all other variables are as defined herein), can be prepared by using Ghosez reagent and diazomethane reagent (such as TMSCHN2).

[0241] In one embodiment, provided herein is a method for preparing a compound of Formula (VIII):

[0242] (VIII) wherein: R 1 is H or -C(=0)N(C 1-3 alkyl)2, 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 -(OCH2CH2) p -; 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 or ; and wherein when R 1 is H and a is 1, then L 1 is not , wherein the wavy line represents the point of attachment to the remainder of the compound, the method comprising: converting a compound of formula (a):

[0243] (a) wherein: R 1 is H or -C(=0)N(C1-3 Alkyl)2, R is –C(=O)OCH3 or –C 1-6 -alkylene-OC 1-6 -alkylene–(OCH2CH2)–Z; and wherein Z is , Thus, compounds of formula (VIII) are prepared.

[0244] In one embodiment, this document provides a method for preparing compounds of formula (VIII-a):

[0245] (VIII-a) in: R' is , ; R 1 Is it H or –C(=O)N(C) 1-3 Alkyl)2, Each L 1 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 It is –(OCH2CH2) p –; p is an integer from 1 to 3; a is an integer of 1 or 2; b is an integer between 0 and 2; Z is or ;and in When R 1 When H is 1 and a is 1, then L 1 no Among them, the wavy line ( The symbol () represents the connection point with the remainder of the compound, and the method comprises: using a Ghosez reagent, in the presence of a diazomethane agent and a solvent, converting the compound of formula (a-1) into:

[0246] (a-1) wherein: R'= , ; R 1 is H or -C(=0)N(C 1-3 alkyl)2, R is -C(=0)0CH3or -C(=0)NH(C 1-3 alkylene)-(OCH2CH2)-Z; and wherein Z is , to thereby produce a compound of formula (VIII-a).

[0247] In one embodiment, provided herein is a method for producing a compound of formula (VIII-a):

[0248] (VIII-a) wherein: R' is , ; R 1 is H or -C(=0)N(C 1-3 alkyl)2, 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 -(OCH2CH2) p -; 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 or ; the method comprising: converting a compound of formula (a-1) below:

[0249] (a-1) wherein: R' 、 ; R 1 is H or -C(=0)N(C 1-3 alkyl)2, R is -C(=0)OCH3or -C(=0)NH(C 1-3 alkylene)-(OCH2CH2)-Z; and wherein Z is , to thereby produce a compound of formula (VIII-a).

[0250] In another embodiment, provided herein is a method for producing a compound of formula (VIII-a):

[0251] (VIII-a) wherein: R' is , ; R 1 is H or -C(=0)N(C 1-3 alkyl)2, each L 1 is: -C(0)NH(C 1-3 -alkylene)-; each L 2 is -(OCH2CH2) p -; 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 ; The method comprises: converting a compound of formula (a-1) below using Ghosez reagent in the presence of diazomethane agent and a solvent:

[0252] (a-1) wherein: R'= , ; R 1 is H or -C(=0)N(C 1-3 alkyl)2, R is -C(=0)OCH3or -C(=0)NH(C 1-3 alkylene)-(OCH2CH2)-Z; and wherein Z is , to thereby produce a compound of Formula (VIII-a).

[0253] In one embodiment of the compounds of Formula (VIII) and Formula (VIII-a), p is 1, a is 1, and b is 1.

[0254] In one embodiment, provided herein is a method for preparing a compound of Formula (XI):

[0255] (XI) wherein: R 1 is H or -C(=0)N(C 1-3 alkyl)2, each L 1 is: -C(O)NH(C 1-3 -alkylene)-; each L 2 is -(OCH2CH2) p -; p is an integer from 1 to 3; a is an integer from 1 or 2; b is an integer from 0 to 2; Z is ; and wherein the method comprises: converting a compound of Formula (a-2) below:

[0256] (a-2) wherein: R 1 is H or -C(=0)N(C 1-3 alkyl)2, R is -C(=0)OCH3or -C(=0)NH(C 1-3 alkylene)-(OCH2CH2)-Z; and wherein Z is , to thereby produce a compound of Formula (XI).

[0257] In one embodiment of the compounds of Formula (XI), p is 1, a is 1, and b is 1.

[0258] 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 embodiment, the solvent is a mixture of dichloromethane and acetonitrile. In one such embodiment, the solvent is dichloromethane and acetonitrile (1 : 1).

[0259] In one embodiment, the method further comprises using a drying agent and a metal oxide. In one such embodiment, the drying agent is molecular sieves and the metal oxide is calcium oxide. In one embodiment, the method further comprises using an activating agent. In one embodiment, the activating agent is KF, KBr, or KI. In one embodiment, the activating agent is KF.

[0260] In one embodiment, the method comprises using trimethylsilyldiazomethane diethyl ether and KF in the same ratio. 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.

[0261] In one embodiment, the compounds of Formula (VIII), Formula (VIII-a), and Formula (XI) prepared using the methods described herein are substantially chemically pure. In one embodiment, the compounds of Formula (VIII), Formula (VIII-a), and Formula (XI) prepared using the methods described herein are substantially free of chemical impurities. 7. A method of preparing a labeled protein

[0262] For example, but not by way of limitation, the labeled proteins described herein can be prepared as outlined in Scheme 3 shown below as well as the examples described herein. It should be noted that one skilled in the art knows how to modify the procedures set forth in the illustrative schemes and obtain the desired products.

[0263] Scheme 3

[0264] As shown in Scheme 3, compounds of Formula (I), (IB), (II), (III), (IV), (V), (VI), and (Via) (wherein R, G, X, L A 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, thereby forming a non-fluorescent labeled protein. The non-fluorescent labeled protein is then treated with light (e.g., light at 405 nm), resulting in the compound, and thus the labeled protein, becoming fluorescent. See also Figure 2 . 8. Examples

[0265] The following examples are presented by way of illustration and not limitation. One skilled in the art can modify the procedures set forth in the illustrative examples to produce the desired products.

[0266] Abbreviations used

[0267] Intermediate Intermediate A Synthesis of tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3H- spiro[isobenzofuran-l,9'-xanthene]-6-carboxylate.

[0268] Intermediate A

[0269] Step 1: Preparation of 3',6'-diacetoxy-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-6- carboxylic acid. H

[0270] Compound A1

[0271] A solution of 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-6- carboxylic acid (5.0 g, 13.3 mmol) in acetic anhydride (25 mL) was stirred under 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-l,9'-xanthene]-6-carboxylic acid (3.7 g, yield 61%) 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]+.

[0272] Step 2: ​Preparation of 6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-3',6'-dimethyldiacetate.

[0273] Compound A2

[0274] To 3',6'-diacetoxy-3-oxo-3 H 1,1-Di-tert-butoxy- N , N -Dimethylmethylamine (9.8 g, 48.2 mmol), and the mixture was stirred under 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'-oxazanthracene]-3',6'-dimethyldiacetate (3.7 g, crude) is a brown solid that can be used in the next step without further purification. m / z = 517.5 [M+H]+.

[0275] Step 3: 3',6'-Dihydroxy-3-oxo-3 H Preparation of tert-butyl spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylate.

[0276] Compound A3

[0277] Sodium hydroxide (1M, 10.2 mL, 10.2 mmol) was added to a stirred solution of 6-(tert-butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-3',6'-dimethyldiacetate (3.7 g, 7.2 mmol) in THF (17 mL) and methanol (17 mL). The mixture was stirred at room temperature for 12 h and then concentrated under reduced pressure. The residue was diluted with water (50 mL) and acidified with saturated aqueous citric acid solution. The aqueous layer was extracted with ethyl acetate (2 x 50 mL) and the combined organic layers were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (elution: 35% EtOAc in petroleum ether) to give tert-butyl 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylate (2.8 g) as a solid. m / z = 433.6 [M+H]+.

[0278] Step 4:3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3 H - spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid tert tert-butyl ester.

[0279] Compound A4

[0280] To a solution of tert-butyl 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'- xanthene]-6-carboxylate (2.8 g, 6.5 mmol) in DMF (28 mL) was added N , N diisopropylethylamine (4.5 mL, 25.9 mmol) followed by N phenyl-O-((trifluoromethyl)sulfonyl)- N (((trifluoromethyl)sulfonyl)oxy)hydroxylamine (5.5 g, 14.2 mmol) at 0 °C. The mixture was stirred at room temperature for 4 h. The mixture was quenched with ice cold water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layer was washed with ice cold brine, then the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 22-25% EtOAc in petroleum ether) to get tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3 H - spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid tert tert-butyl ester (2.0 g, yield 47%) 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]+.

[0281] Intermediate B 3',6'-dibromo-3-oxo-3 H synthesis of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6- carboxylate.

[0282] Intermediate B

[0283] Step 1:3',6'-dibromo-3-oxo-3 H - Preparation of spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid pyridinium salt.

[0284] Compound B1

[0285] To a stirred solution of 1,2,4-benzene tricarboxylic acid (50 g, 238 mmol) in methanesulfonic acid (250 mL) was added 3-bromophenol (86.4 g, 500 mmol). The mixture was stirred at 140 °C for 72 h. After cooling to room temperature, the dark purple solution was poured into 200 mL of ice water and the slurry was stirred vigorously. The greenish yellow solid was collected by vacuum filtration and suction dried. 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 from a 2:1 mixture of acetic anhydride and pyridine three more times to give 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid pyridinium salt (15.0 g, yield 12%) as a powder. m / z = 503.2 [M+H]+.

[0286] Step 2: 3',6'-dibromo-3-oxo-3 H - Preparation of spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid methyl ester.

[0287] Compound B2

[0288] To a stirred solution of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid 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 solution (50 mL) was added until the reaction was basic (pH > 7) and then the aqueous solution was extracted with 10% IPA in CHCI3(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 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid methyl ester (6.0 g, yield 58%) as a solid. H - Preparation of spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid methyl ester. m / z = 517.12 [M+H]+.

[0289] Intermediate C Synthesis of tert-butyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate.

[0290] Intermediate C

[0291] To a solution of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6- carboxylic acid pyridinium salt (4.0 g, 8.0 mmol, Compound B1) in toluene (16 mL) was added 1,1-ditert-butoxy-dimethylmethanamine (9.2 g, 47.2 mmol) and the mixture was stirred at reflux for 24 h. The mixture was concentrated under reduced pressure to give tert-butyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate as a brown solid which was used without further purification. N , N - dimethylmethanamine (9.2 g, 47.2 mmol) and the mixture was stirred at reflux for 24 h. The mixture was concentrated under reduced pressure to give tert-butyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate as a brown solid which was used without further purification.

[0292] Intermediate D' Synthesis of 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide.

[0293] Intermediate D'

[0294] At 0 °C, N,N'-diisopropylethylamine (0.52 mL, 3.0 mmol) was added to a solution of pyridinium salt of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-6-carboxylic acid (0.5 g, 1.0 mmol, compound B1) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl-1-amine (0.56 g, 2.5 mmol) in DMF (5.0 mL). T3P solution (50% in ethyl acetate; 0.8 g, 2.5 mmol) was added dropwise at 0 °C. The mixture was heated to room temperature and stirred for 16 h. Ice-cold water (50 mL) was added, and the mixture was extracted with ethyl acetate (3x). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution (2x) at 0 °C and then with 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 (elution: 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'-oxazanthracene]-6-carboxamide (0.3 g, yield 42%) as a grayish-white solid.

[0295] 8.1 Refer to Example 1 Synthesis of 1,1'-(6-((2-(2-(((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[inden-1,9'-oxazanthene]-3',6'-diyl)bis(azircyclobutane-3-carboxylic acid dimethyl ester): Compound 1

[0296] Step 1: 1,1'-(6-( tert (-Butoxycarbonyl)-3-oxo-3 H Preparation of spiro[isobenzofuran-1,9'-oxazanthracene]-3',6'-diyl)bis(dimethyl aziridine-3-carboxylate).

[0297] Compound 1.1

[0298] 3-Oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3 H -spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylic acid tertButyl ester (1.0 g, 1.4 mmol, intermediate A) and methyl 3-oxane hydrochloride (0.55 g, 3.59 mmol) in dioxane (20 mL) were added to a solution of Cs₂CO₃ (1.4 g, 4.3 mmol). The mixture was purged with argon for 15–20 min, and then loaded with Pd₂(dba)₃ (0.13 g, 0.14 mmol) and XPos (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 diatomaceous earth (celite) pad. The filtrate was diluted with water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (elution: 85% EtOAc in petroleum ether) to give 1,1'-(6-( tert (butoxycarbonyl)-3-oxo-3 H -spiro[isobenzofuran-1,9'-oxazanthracene]-3',6'-diyl)bis(azircyclobutane-3-carboxylic acid dimethyl ester) (0.35 g, yield 39%), is a solid. m / z = 627.8[M+H]+.

[0299] Step 2: Preparation of 3',6'-bis(3-(methoxycarbonyl)azacyclobutane-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-6-carboxylic acid.

[0300] Compound 1.2

[0301] At 0℃, towards 1,1'-(6-( tert (-Butoxycarbonyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-3',6'-diyl)bis(azacyclobutane-3-carboxylic acid dimethyl ester) (0.25 g, 0.40 mmol) was added dropwise to a solution of TFA (0.2 mL) in CH2Cl2 (2 mL). The mixture was stirred at room temperature for 12 h, and then concentrated under reduced pressure. The residue was then... normal 3',6'-bis(3-(methoxycarbonyl)azacyclobutane-1-yl)-3-oxo-3- H Spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylic acid (180 mg, 79% yield) is a solid. m / z = 571.9 [M+H]+.

[0302] Step 3:1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3 H Preparation of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3

[0303] Compound 1.3

[0304] To a solution of 3',6'-bis(3-(methoxycarbonyl)azetidin-1-yl)-3-oxo-3 H To a solution of 3',6'-bis(3-(methoxycarbonyl)azetidin-1-yl)-3-oxo-3 N,N'- succinimidyl carbonate (0.18 g, 0.69 mmol) and DMAP (3.9 mg, 0.032 mmol) were added. The mixture was stirred at 0 °C for 1 h. Then, a solution of 2-(2-((6- chlorohexyl)oxy)ethoxy)ethan-1 -amine (0.17 g, 0.79 mmol) in DMF (0.8 mL) was added dropwise. The mixture was allowed to warm to room temperature and stirred for 12 h. The mixture was diluted with ice-cold water (20 mL) and extracted with ethyl acetate (2 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 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3 H - 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3 m / z = 777.0 [M+H]+.

[0305] Step 4: Preparation of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid dimethyl ester).

[0306] Compound 1.4

[0307] To a solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3 H To a solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3 Org . Synth . 1935, 15, 3) (ca. 0.5 M, 5 mmol) and stirred for 30 min. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column (eluent: 22% EtOAc in petroleum ether). The residue was further purified by chiral SFC (Chiralcel-OJ-3; mobile phase 30% MeOH in CO2) to give 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'- diyl)bis(dimethyl 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]+.

[0308] 8.2 Reference Example 2 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo- 2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid) was synthesized according to the following scheme:

[0309] Compound 2

[0310] To a solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2- diazo-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(azetidine-3- carboxylic acid dimethyl ester) (18 mg, 0.022 mmol, Example 1) in MeOH (0.2 mL) and THF (0.1 mL) at 0 °C was added a solution of LiOH (5 mg, 0.11 mmol) in water (0.1 mL). The mixture was stirred at room temperature for 24 h. The mixture was concentrated under reduced pressure, and the residue was purified using achiral SFC (YMC-PAK DIOL; mobile phase, 30% MeOH in CO2) to give 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo- 2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid) (11 mg, 63%) as a solid. 1 H NMR (400 MHz, DMSO- d 6): 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]+.

[0311] 8.3 Refer to Example 3 Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2-(methoxymethyl)azacyclobutane-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-oxazanthene]-6-carboxamide.

[0312] Compound 3

[0313] Step 1: 3',6'-double(( R )-2-(methoxymethyl)azacyclobutane-1-yl)-3-oxo-3 H Preparation of methyl spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylate.

[0314] Compound 3.1

[0315] In a microwave container, 3',6'-dibromo-3-oxo-3 H methyl spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylate (0.50 g, 0.96 mmol, intermediate B), ( R Cesium carbonate (0.95 g, 2.9 mmol) was added to a solution of 2-(methoxymethyl)azacyclobutane (0.24 g, 2.42 mmol) in 1,4-dioxane (10 mL). The mixture was purged with argon 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 diatomaceous earth, and the filtrate was diluted with water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (elution: 85% EtOAc in petroleum ether) to give 3',6'-bis((R)-2-(methoxymethyl)azacyclobutane-1-yl)-3-oxo-3 H methyl spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylate (0.17 g, yield 31%) is a solid. m / z = 557.52 [M+H]+.

[0316] Step 2: 3',6'-double(( R)-2-(methoxymethyl)azacyclobutane-1-yl)-3-oxo-3 H Preparation of spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylic acid.

[0317] Compound 3.2

[0318] At 0℃, towards 3',6'-double(( R Methyl 2-(methoxymethyl)azacyclobutane-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-6-carboxylate (0.17 g, 0.30 mmol) in MeOH (1.0 mL) and THF (0.5 mL) was mixed with a solution of LiOH (0.06 g, 1.53 mmol) in water (0.5 mL). 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 1N HCl and extracted with 10% MeOH in CH2Cl2 (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative HPLC (C18, mobile phase, 20% ACN / water (0.05% formic acid)) to give 3',6'-bis(( R )-2-(methoxymethyl)azacyclobutane-1-yl)-3-oxo-3 H -spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylic acid (0.12 g, yield 72%) is a solid. m / z = 543.45 [M+H]+.

[0319] Step 3: N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(( R )-2-(methoxymethyl)azacyclobutane-1-yl)-3-oxo-3 H Preparation of spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxamide.

[0320] Compound 3.3

[0321] 3',6'-bis(( R )-2-(methoxymethyl)azacyclobutane-1-yl)-3-oxo-3 H- To a stirred solution of spiro[isobenzo furan-1,9'-xanthene]-6-carboxylic acid (0.12 g, 0.22 mmol) in DMF (1.2 mL) was added 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1 -amine (0.099 g, 0.442 mmol) in DMF (0.1 mL) at 0 °C. 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). The mixture was stirred at 0 °C for 10 min, then propyl phosphonic anhydride (50% in ethyl acetate; 0.18 g, 0.550 mmol) was added dropwise. The mixture was warmed to room temperature and stirred for 16 h. The mixture was diluted with ice-cold water (20 mL) and extracted with ethyl acetate (2 x 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 CH2CI2) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis((R)-2- (methoxymethyl)azetidin-1 -yl)-3-oxo-3 H - spiro[isobenzo furan-1,9'-xanthene]-6-carboxamide (110 mg, 66% yield) as a pink solid. m / z = 749.14 [M+H]+.

[0322] Step 4: 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. R 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.

[0323] Compound 3.4

[0324] To a stirred solution of spiro[isobenzo furan-1,9'-xanthene]-6-carboxylic acid (0.12 g, 0.22 mmol) in DMF (1.2 mL) was added 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1 -amine (0.099 g, 0.442 mmol) in DMF (0.1 mL) at 0 °C. N N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis((R)-2- (methoxymethyl)azetidin-1 -yl)-3-oxo-3 R -2-(methoxymethyl)azetidin-1 -yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6- carboxamide. H- To a solution of spiro[isobenzofuran-l,9'-xanthene]-6-carboxamide (0.09 g, 0.12 mmol) in CH2Cl2(5 mL) was added oxalyl chloride (0.10 mL, 1.20 mmol). The mixture was warmed to room temperature and stirred at room temperature for 30 min. The mixture was concentrated with a stream of nitrogen. The residue was dissolved in dry CH2Cl2(20 mL) at 0 °C and diazomethane freshly prepared in Et2O (ca. 0.5 M, 3 mmol) was added (see for example, F. Arndt, “Diazomethane,” Org . Synth . 1935, 15, 3). The mixture was stirred at 0 °C for 30 min and then concentrated under reduced pressure. The residue was purified using neutral alumina by flash column purification (eluent: 0-10% acetone in petroleum ether). The residue was further purified by chiral SFC (YMC PAK-DIOL; mobile phase, 25% MeOH in CO2) to give N -(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis( R )-2-(methoxymethyl)azetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6- carboxamide (0.0032 g, 3%) as a solid. 1 H NMR (400 MHz, DMSO- d 6): 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]+.

[0325] 8.4Reference 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).

[0326] Compound 4

[0327] Step 1: 3',6'-Bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6- carboxylic acid. H Preparation of methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthen]-6-carboxylate.

[0328] Compound 4.1

[0329] A mixture of methyl 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-carboxylate (1.0 g, 1.94 mmol, Intermediate B), 3-(dimethylcarbamoyl)azetidine-3-carboxamide hydrochloride (0.62 g, 4.84 mmol) and cesium carbonate (1.90 g, 5.810 mmol) was suspended in anhydrous 1,4-dioxane (10.0 mL). The vial was degassed with nitrogen for 15 min before Pd2(dba)3 (0.18 g, 0.19 mmol) and RuPhos Pd G4 (0.27 g, 0.50 mmol) were added. The mixture was stirred at 110 °C for 2 h. The mixture was filtered through celite and washed with 10% MeOH in CH2Cl2(100 mL). The filtrate was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: 0-20% MeOH in CH2Cl2) to give methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-carboxylate (0.28 g, 23% yield) as a solid. N , N - dimethylazetidine-3-carboxamide hydrochloride (0.62 g, 4.84 mmol) and cesium carbonate (1.90 g, 5.810 mmol) was suspended in anhydrous 1,4-dioxane (10.0 mL). The vial was degassed with nitrogen for 15 min before Pd2(dba)3 (0.18 g, 0.19 mmol) and RuPhos Pd G4 (0.27 g, 0.50 mmol) were added. The mixture was stirred at 110 °C for 2 h. The mixture was filtered through celite and washed with 10% MeOH in CH2Cl2(100 mL). The filtrate was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: 0-20% MeOH in CH2Cl2) to give methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-carboxylate (0.28 g, 23% yield) as a solid. H - dimethylazetidine-3-carboxamide hydrochloride (0.62 g, 4.84 mmol) and cesium carbonate (1.90 g, 5.810 mmol) was suspended in anhydrous 1,4-dioxane (10.0 mL). The vial was degassed with nitrogen for 15 min before Pd2(dba)3 (0.18 g, 0.19 mmol) and RuPhos Pd G4 (0.27 g, 0.50 mmol) were added. The mixture was stirred at 110 °C for 2 h. The mixture was filtered through celite and washed with 10% MeOH in CH2Cl2(100 mL). The filtrate was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: 0-20% MeOH in CH2Cl2) to give methyl 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-carboxylate (0.28 g, 23% yield) as a solid. m / z = 612.0 [M+H]+.

[0330] Step 2: Preparation of 3',6'-bis(3-(dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-carboxylic acid.

[0331] Compound 4.2

[0332] To a solution of 3',6'-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-3-oxo-3 H - spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid methyl ester (0.28 g, 0.46 mmol) in THF (2.8 mL) was added trimethyltin hydroxide (0.5 g, 2.75 mmol). The mixture was warmed to room temperature and stirred for 2 h. Water (2 mL) was added, then the mixture was cooled to 0 °C and acidified to pH 5 with 1 N HC1. 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-l-yl)-3-oxo-3 H - spiro[isobenzofuran-l,9'-xanthene]-6-carboxylic acid methyl ester (0.28 g, 0.46 mmol) in THF (2.8 mL) was added trimethyltin hydroxide (0.5 g, 2.75 mmol). The mixture was warmed to room temperature and stirred for 2 h. Water (2 mL) was added, then the mixture was cooled to 0 °C and acidified to pH 5 with 1 N HC1. 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-l-yl)-3-oxo-3 m / z = 597.41 [M+H]+.

[0333] Step 3: 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3 H - spiro[isobenzofuran-l,9'-xanthene]-3',6'-diyl)bis( N , N - dimethylazetidine-3-carboxamide) was prepared.

[0334] Compound 4.3

[0335] To a solution of 3',6'-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-3-oxo-3 H- To a solution of spiro[isobenzo furan-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). The mixture was stirred at 0 °C for 10 min, then a solution of propyl phosphonic anhydride (50% in ethyl acetate; 0.3 g, 0.96 mmol) was added dropwise. The mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was diluted with ice-cold water (10 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with brine (50 mL) and ice-cold water (30 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: 0-8% MeOH in CH2CI2) to give 1,1 '-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3 H - spiro[isobenzo furan-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]+.

[0336] Step 4: 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) N , N Preparation of 1,1 '-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3

[0337] Compound 4.4

[0338] To a solution of 1,1 '-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3 H - spiro[isobenzo furan-1,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-3- carboxamide) N , N- dimethylazetidine-3-carboxamide) in CH2Cl2(4 mL) was added oxalyl chloride (0.07 mL, 0.872 mmol). The mixture was warmed to room temperature and stirred for 30 min. The mixture was concentrated under reduced pressure. The residue was dissolved in anhydrous CH2Cl2(20 mL) at 0 °C and freshly prepared diazomethane in Et2O (see, e.g., F. Arndt, “Diazomethane,” Org . Synth . 1935, 15, 3) (ca. 0.5 M, 2.2 mmol). 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 using neutral alumina (eluent: 0-5% MeOH in CH2Cl2). The residue was further purified by SFC (DCPAK P4VP; mobile phase, 45% CO2in 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(2-(dimethylazetidin-3-yl)carboxamide) (0.0052 g, 7% yield) as a solid. N , N - dimethylazetidine-3-carboxamide) in CH2Cl2(4 mL) was added oxalyl chloride (0.07 mL, 0.872 mmol). The mixture was warmed to room temperature and stirred for 30 min. The mixture was concentrated under reduced pressure. The residue was dissolved in anhydrous CH2Cl2(20 mL) at 0 °C and freshly prepared diazomethane in Et2O (see, e.g., F. Arndt, “Diazomethane,” 1 H NMR (400 MHz, DMSO- d 6): 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]+.

[0339] 8.5 Reference Example 5 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) was prepared according to the procedure of Example 28, Step 2.

[0340] Compound 5

[0341] 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.

[0342] Compound 5.1

[0343] The title compound was prepared using a procedure analogous to Example 7, Steps 1-3, 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.

[0344] Alternatively, the title compound was prepared according to the procedure of Example 28, Step 2.

[0345] Step 2: 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) was prepared according to the procedure of Example 28, Step 2.

[0346] Compound 5.2

[0347] To a stirred solution of 3',6'-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-3-oxo-3H- spiro[isobenzofuran-l,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, propyl phosphonic anhydride solution (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% ACN in water) to give the title compound (0.008 g, 12%) as a light brown solid. 1 H 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]+.

[0348] 8.6Reference Example 6 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3- dihydros piro[indene- 1,9'-xanthene] -3',6'-diyl)(2R,2'R)bis(azetidine-2-carboxylate) synthesis.

[0349] Compound 6

[0350] The title compound was prepared using a procedure analogous to Example 1, Steps 1-4, replacing (R)-azetidine-2-carboxylic acid methyl ester hydrochloride with azetidine-3-carboxylic acid methyl ester hydrochloride in Step 1, and intermediate C with intermediate A in Step 1. 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 light yellow solid. 1 H 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]+.

[0351] 8.7Reference 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.

[0352] Compound 7

[0353] Step 1: Preparation of methyl 3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran- 1,9'-xanthene]-6-carboxylate.

[0354] Compound 7.1

[0355] To a stirred solution of 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'- xanthene]-6-carboxylic acid methyl ester (Intermediate B, 2.0 g, 3.88 mmol) and azetidin-3-ol-hydrochloride (1.06 g, 9.69 mmol) in 1,4-dioxane (40 mL) was added cesium carbonate (6.31 g, 19.38 mmol) at room temperature. The mixture was purged with argon for 20 min, then Pd2(dba)3 (0.35 g, 0.38 mmol) and Xphos (0.55 g, 1.16 mmol) were added. The mixture was purged with argon for 10 min and heated to 120 °C for 16 h. The mixture was cooled to room temperature and filtered through celite. The celite was washed with MeOH and the solution was concentrated under reduced pressure. The residue was purified by reverse phase column chromatography (Column C-18 column; Eluent: 0-20% ACN in water with 0.1% formic acid) to give 3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'- xanthene]-6-carboxylic acid methyl ester (0.90 g, 47% yield) as a purple solid.

[0356] Step 2: Preparation of 2-diazo-3',6'-bis(3-hydroxyazetidin-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'- xanthene]-6-carboxylic acid methyl ester.

[0357] Compound 7.2

[0358] 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) at 0 °C was added dropwise oxalyl chloride (1 g, 8 mmol). The mixture was allowed to warm to room temperature and stirred at room temperature for 60 min. The mixture was concentrated under reduced pressure. The residue was dissolved in dry DCM (100 mL) and freshly prepared diazomethane in Et20 (ca. 0.5 M, 100 mmol) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 15 min, then allowed to warm 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: 20% MeOH in C02) 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.

[0359] 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.

[0360] Compound 7.3

[0361] 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) at 0 °C was added lithium hydroxide monohydrate (8 mg, 0.19 mmol). The mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the residue was triturated with Et20 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.

[0362] Step 4:Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3- hydroxyazetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-carboxamide.

[0363] Compound 7.4

[0364] To a stirred solution of 2-diazo-3',6'-bis(3-hydroxyazetidin-l-yl)-3-oxo-2,3- dihydrospiro[indene-l,9'-xanthene]-6-carboxylic acid (0.048 g, 0.094 mmol) and 2-(2- ((6-chlorohexyl)oxy)ethoxy)ethan-l -amine (0.053 g, 0.24 mmol) in DMF (1 mL) at 0 °C was added N , N diisopropylethylamine (0.05 g, 0.38 mmol). The mixture was stirred at 0 °C for 10 min, then propane phosphonic anhydride (50% in ethyl acetate; 0.18 g, 0.282 mmol) was added dropwise at 0 °C. The mixture was warmed to room temperature and stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure and purified by preparative HPLC (column: XSELECT-C18; mobile phase, 0-100% ACN in water) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3- hydroxyazetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-carboxamide (5 mg, yield 7%) 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]+.

[0365] 8.8 Reference Example 8 3',6'-Bis(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-formamide.

[0366] Compound 8

[0367] Step 1: Preparation of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate.

[0368] Compound 8.1

[0369] 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) was added NaH (1.17 g, 60% in mineral oil, 29.2 mmol) at 0 °C. The mixture was stirred at 0 °C for 30 min, then 6-chloro-1- iodohexane (8.4 g, 34.1 mmol) was added. The mixture was allowed to warm 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 Na2SO4and 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, yield 33%) as a colorless oil.

[0370] Step 2: Preparation of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)carbamate.

[0371] Compound 8.2

[0372] To a stirred solution of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (3.0 g, 9.3 mmol) and iodomethane (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 allowed to warm to room temperature and stirred at room temperature for 3 h. Saturated aqueous NH4Cl (50 mL) was added and the mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over Na2SO4and 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 light yellow oil.

[0373] Step 3: Preparation of 2-(2-((6-chlorohexyl)oxy)ethoxy)-N-methylethan-1-amine trifluoroacetate salt.

[0374] Compound 8.3

[0375] 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 Et2O to give the title compound as a light yellow oil.

[0376] Step 4: Preparation of 3',6'-bis(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid.

[0377] Compound 8.4

[0378] The title compound was prepared using a similar procedure to Example 7, replacing azetidine-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: C18 RP; mobile phase: 0-100% ACN in water) to give 3',6'-bis(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic acid as a solid.

[0379] Step 5: 3',6'-Bis(azetidin-l-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-N- methyl-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-carboxamide.

[0380] Compound 8.5

[0381] To a stirred solution of 3',6'-bis(azetidin-l-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-l -amine trifluoroacetate salt (0.088 g, 0.25 mmol) in DMF (1 mL) at 0 °C was added N,N diisopropylethylamine (0.1 mL, 0.62 mmol). The mixture was stirred at 0 °C for 10 min, then T3P (50% in ethyl acetate; 0.22 g, 0.38 mmol) was added dropwise at 0 °C. The mixture was warmed to room temperature and stirred at room temperature for 4 h. The mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: X-BRIDGE -C18; mobile phase: 0-100% water in ACN) to give 3',6'-bis(azetidin-l-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-N-methyl-3-oxo-2,3-dihydrospiro[indene-l,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]+.

[0382] 8.9Reference Example 9 Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis(3-(morpholine- 4-carbonyl)azetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-carboxamide.

[0383] Compound 9

[0384] Step 1: Preparation of l,l'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo- 3H-spiro[isobenzofuran-l,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid).

[0385] Compound 9.1

[0386] To a solution of l,l'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo- 3H-spiro[isobenzofuran-l,9'-xanthene]-3',6'-diyl)bis(azetidine-3-carboxylic acid dimethyl ester) (0.35 g, 0.45 mmol, Compound 1.3) in MeOH (0.35 mL) and THF (0.7 mL) at 0 °C was added a solution of lithium hydroxide monohydrate (0.021 g, 0.50 mmol) in water (0.35 mL). The mixture was stirred at room temperature for 2 h. The mixture was concentrated to give the desired product as a pink solid which was used without further purification.

[0387] Step 2: Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-(morpholine-4- carbonyl)azetidin-l-yl)-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-6-carboxamide.

[0388] Compound 9.2

[0389] 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) at 0 °C was added N,N-diisopropylethylamine (0.6 mL, 3.3 mmol) followed by T3P (50% in ethyl acetate, 0.64 g, 1.0 mmol). The mixture was stirred at 0 °C for 1 h, then a solution of morpholine (0.145 g, 1.666 mmol) in DMF (1 mL) was added dropwise. The mixture was allowed to warm to room temperature and stirred at room temperature for 12 h. The mixture was diluted with ice-cold water (20 mL) and extracted with ethyl acetate (2x). The combined organic layers were dried over Na2SO4and 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.

[0390] 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.

[0391] Compound 9.3

[0392] At 0 °C, oxaloyl chloride (1.015 mL, 2.0 mmol, 2 M solution in DCM) was added to a solution of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-(morpholino-4-carbonyl)azacyclobutan-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-6-carboxamide (0.18 g, 0.20 mmol) in DCM (3.6 mL). The mixture was heated 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 diazomethane (approximately 0.5 M, 50 mmol) from freshly prepared Et₂O 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 (elution: 85% EtOAc in petroleum ether). The residue was 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-(morpholin-4-carbonyl)azacyclobutane-1-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-oxazanthracene]-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]+.

[0393] 8.10 Refer to Example 10 Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3-oxo-3',6'-bis(3-(pyrrolidine-1-carbonyl)azacyclobutane-1-yl)-2,3-dihydrospiro[indene-1,9'-oxazanthracene]-6-carboxamide.

[0394] Compound 10

[0395] To a stirred solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2- diazono-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(azetidine-3- carboxylate) (0.03 g, 0.039 mmol, Compound 2) in DMF (0.6 mL) at 0 °C was added N,N- diisopropylethylamine (0.07 mL, 0.39 mmol). 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 h. Then a solution of pyrrolidine (0.011 g, 0.16 mmol) in DMF (0.1 mL) was added. The mixture was allowed to warm to room temperature and stirred at room temperature for 12 h. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Sunfire C18 mobile phase A: 0-100% ACN in water) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazono-3-oxo-3',6'-bis(3-(pyrrolidine-l- carbonyl)azetidin-l-yl)-2,3-dihydrospiro[indene-l,9'-xanthene]-6-carboxamide (0.004 g, 11.72% yield) as a light yellow solid. 1 H 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]+.

[0396] 8.11Reference Example 11 Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazono-3',6'-bis(3-(4- methylpiperazine-l-carbonyl)azetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6- carboxamide.

[0397] Compound 11

[0398] To a stirred solution of 1,1'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2- diazene-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(azetidine-3- carboxylic acid) (0.045 g, 0.058 mmol, Compound 2) in DMF (0.9 mL) at 0 °C was added N,N-diisopropylethylamine (0.065 mL, 0.35 mmol) followed by T3P (50% in ethyl acetate, 0.074 g, 0.117 mmol). The mixture was stirred at 0 °C for 1 h. Then a solution of N-methylpiperazine (0.015 g, 0.146 mmol) in DMF (0.1 mL) was added dropwise. The mixture was allowed to warm to room temperature and stirred at room temperature for 12 h. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: X-SELECT C18; mobile phase: 0-100% acetonitrile in water and 10 mM ammonium acetate) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazen-3',6'-bis(3-(4- methylpiperazine-l-carbonyl)azetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6- formamide (5.2 mg, yield 9.5%) as a solid. 1 H NMR (400MHz, 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]+.

[0399] 8.12Reference Example 12 Synthesis of 1,1 '-((6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2- diazene-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-3',6'- diyl)bis(N,N-dimethylazetidine-3-carboxamide).

[0400] Compound 12

[0401] The title compound was prepared using a similar procedure to 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, 2H), 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]+.

[0402] 8.13 Reference Example 13 Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazen-3',6'-bis(3- (dimethylamino)azetidin-1 -yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6- carboxamide.

[0403] Compound 13

[0404] The title compound was prepared using a similar procedure to Example 7, in Step 1, substituting 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H- spiro[isobenzofuran-l,9'-xanthene]-6-carboxamide (Intermediate D') and N,N- dimethylazetidine-3-amine hydrochloride for 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran- 1,9'-xanthene]-6-carboxylic acid methyl ester (Intermediate B) and azetidin-3-ol- 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-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-carboxamide as a light 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]+.

[0405] 8.14 Reference Example 14 Synthesis of (2S,2'S)-l,l'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)- 2-diazo-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(N,N- dimethylazetidine-2-carboxamide).

[0406] Compound 14

[0407] The title compound was prepared using a similar procedure to Example 7, in Step 1, substituting 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H- spiro[isobenzofuran-l,9'-xanthene]-6-carboxamide (Intermediate D') and (S)-N,N- dimethylazetidine-2-carboxamide hydrochloride for 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran- 1,9'-xanthene]-6-carboxylic acid methyl ester (Intermediate B) and azetidin-3-ol- 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% ACN in water) to give (2S,2'S)-l,l'-(6-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-l,9'- xanthene]-3',6'-diyl)bis(N,N-dimethylazetidine-2-carboxamide) as an off-white solid. 1 H NMR (400MHz, 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]+.

[0408] 8.15Reference Example 15 Synthesis of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2- (hydroxymethyl)azetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-carboxamide.

[0409] Compound 15

[0410] The title compound was prepared using a similar procedure to Example 7, substituting 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H- spiro[isobenzofuran-l,9'-xanthene]-6-carboxamide (Intermediate D') and (R)-azetidin-2- ylmethanol hydrochloride for 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-6- carboxylic acid methyl ester (Intermediate B) and azetidin-3-ol-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% ACN in water) to give N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3',6'-bis((R)-2- (hydroxymethyl)azetidin-l-yl)-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-carboxamide as a light 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]+.

[0411] 8.16Reference Example 16 Synthesis of (3S,3'S)-l,l'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2- diazo-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(N,N- dimethylpyrrolidine-3-carboxamide).

[0412] Compound 16

[0413] The title compound was prepared using a similar procedure to Example 7, substituting 3',6'-dibromo-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H- spiro[isobenzofuran-l,9'-xanthene]-6-carboxamide (Intermediate D') and (S)-N,N- dimethylpyrrolidine-3-carboxamide for 3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-l,9'- xanthene]-6-carboxylic acid methyl ester (Intermediate B) and azetidin-3-ol hydrochloride. 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% ACN in water) to give (3S,3'S)-l,l'-(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-diazo-3-oxo- 2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(N,N-dimethylpyrrolidine-3- carboxamide) as a solid. 1 H 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]+.

[0414] 8.17Reference Example 17 Synthesis of 3',6'-bis(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-l,9'-xanthene]-6- carboxamide.

[0415] Compound 17

[0416] Step 1: Preparation of 3',6'-Bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid methyl ester.

[0417] Compound 17.1

[0418] 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-sulfonic acid ester (0.657 g, 2.422 mmol) in 1,4-dioxane (10 mL) in a dry sealed tube was added cesium carbonate (1.578 g, 4.844 mmol) at room temperature and the reaction mixture was purged with argon for 20 min. Then XPhos-Pd-G4 (0.083 g, 0.097 mmol) was added at room temperature. The reaction mixture was again purged with argon for 10 min and stirred at 100 °C for 12 h. 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), extracted with ethyl acetate (30 mL X 2) and the combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude was purified by flash chromatography using silica gel in 0-100% ethyl acetate in petroleum ether, the desired product was eluted in 90-95% ethyl acetate in petroleum ether to get methyl 3',6'-bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.35 g, yield 65%) as a pink solid. m / z 553.51 (M+H + ). Step 2 Preparation of 3',6'-Bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid.

[0419] Compound 17.2

[0420] To a stirred solution of 3',6'-bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid methyl ester (Compound 17.1) (0.35 g, 0.633 mmol) in THF (8.0 mL) and MeOH:H2O (1 :1) (8.0 mL) was added lithium hydroxide monohydrate (0.053 g, 1.266 mmol) in portions at 0 °C. The reaction mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was concentrated under reduced pressure and diluted with water (2 mL) and then acidified by using 1 N HCI (pH ~ 2). The precipitate formed was filtered and dried under high vacuum. The dried solid was washed with diethyl ether to get 3',6'-bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.25 g, yield 70%) as a pink solid. m / z 539.36 [M+H]+.

[0421] Step 3: 3',6'-bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid methyl ester (Compound 17.1) was prepared as described in Scheme 17. N -(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3 H - spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide.

[0422] Compound 17.3

[0423] At 0 °C, N,N'-diisopropylethylamine (0.4 mL, 2.321 mmol) was added to a stirred solution of 3',6'-bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-6-carboxylic acid (compound 17.2) (0.250 g, 0.464 mmol) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl-1-amine (0.259 g, 1.160 mmol) in DMF (5 mL). After 10 min, propanephosphonic anhydride (T3P) (50% in ethyl acetate; 0.886 g, 1.392 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC and LCMS. After the reaction was completed, the reaction mixture was directly evaporated to remove the DMF solvent, and then purified by reverse-phase column purification in 0-100% acetonitrile in water using a C18 column. The desired product was eluted in 22% acetonitrile in water to give 3',6'-bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxamide (0.097 g, yield 28%) as a pink solid.

[0424] Step 4: 3',6'-Di(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)- N Preparation of -(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-oxazanthene]-6-carboxamide.

[0425] Compound 17.4

[0426] Method A. Under argon atmosphere, 3',6'-bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)- N -(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3 H Spiro[isobenzofuran-1,9'-oxazanthracene]-6-carboxamide (compound 17.3) (29 mg, 0.039 mmol) was added to a solution of 4 Å powdered molecular sieve (15 mg) in anhydrous CH2Cl2 (1 mL). Then, 1-chloro- N , N,2-trimethylprop-l-en-l-amine (20.6 µL, 0.156 mmol) and the mixture was stirred at room temperature for 45 min. N,N-diisopropylethylamine (27 µL, 0.156 mmol) was added, followed by TMSCHN2 (2M in hexanes, 78 µL, 0.156 mmol). After stirring at room temperature for 2.5 hours, the mixture was filtered and concentrated. The residue was purified by reverse-phase flash chromatography (0-100% ACN in water). The residue was purified a second time by reverse-phase flash chromatography (30-100% ACN in water). A third purification was performed using a pipette column packed with basic alumina and eluted with 50-100% EtOAc in toluene. The fractions containing product were concentrated, re-dissolved in dioxane and dried on a lyophilizer to yield a solid (1.7 mg, 6%). 1 H 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 + ]。

[0427] Method B. To 3',6'-bis(3-oxa-6-azabicyclo[3.1.1]hept-6-yl)-2- methoxyphenol (0.100 g, 0.300 mmol) was added 2-bromo-2-methylpropionitrile (0.050 mL, 0.450 mmol) and the mixture was heated to 80 °C for 2 hours. The mixture was filtered and concentrated. The residue was purified by reverse-phase flash chromatography (0-100% ACN in water). The residue was purified a second time by reverse-phase flash chromatography (30-100% ACN in water). A third purification was performed using a pipette column packed with basic alumina and eluted with 50-100% EtOAc in toluene. The fractions containing product were concentrated, re-dissolved in dioxane and dried on a lyophilizer to yield a solid (0.050 g, 40%). N- (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-oxo-3 H A solution of spiro[isobenzofuran-l,9'-xanthene]-6-carboxamide (Compound 17.3) (29 mg, 0.039 mmol) in anhydrous CH2Cl2(1 mL) was charged with 4A powdered molecular sieves (15 mg). Then 1-chloro- N , N ,2-trimethylprop-l-en-l-amine (20.6 μL, 0.156 mmol) was added via syringe and the mixture was stirred at room temperature for 45 min. N,N-diisopropylethylamine (27 μL, 0.156 mmol) was added, followed by TMSCHN2(2M in hexanes, 78 μL, 0.156 mmol). After stirring 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 further purified by reverse-phase flash chromatography (30-100% ACN in water). The residue was further purified by flash chromatography using basic alumina (eluent: 50-100% EtOAc in toluene) to give the title compound as a solid (1.7 mg, 6%). 1 H NMR (400MHz, 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]+.

[0428] 8.18Reference 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]hept-6-yl)-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6- formamide.

[0429] Compound 18

[0430] Step 1: Preparation of methyl 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]hept-6-yl)-3-oxo- 3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate.

[0431] Compound 18.1

[0432] 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 was added Cs2CO3(3.156 g, 9.687 mmol) at room temperature and the mixture was purged with argon for 20 min. Then Pd2(dba)3(0.089 g, 0.097 mmol) and RuPhos (0.135 g, 0.291 mmol) were added at room temperature. The mixture was again purged with argon for 10 min and stirred at 110 °C for 16 h. The reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was filtered through celite, washed with methanol (100 mL) and concentrated. The residue was purified by silica gel chromatography eluting with (1% MeOH in DCM to 5% ammonia in DCM) to afford methyl 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]hept-6-yl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (0.20 g, yield 35.68%) as a pink solid. m / z579.58 [M+H]+.

[0433] Step 2: Preparation of 3',6'-Bis(3-methyl-3,6-diazabicyclo[3.1.1]oct-6-yl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid.

[0434] Compound 18.2

[0435] To a stirred solution of methyl 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]oct-6-yl)-3- oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (compound 18.1) (0.2 g, 0.346 mmol) in THF (2.0 mL) and MeOH:H2O (1:1) (2.0 mL) was added lithium hydroxide monohydrate (0.072 g, 1.728 mmol) in 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 solvents were removed under reduced pressure, the remaining crude material was diluted with water (2 mL) and the mixture was acidified to pH ~ 2 with concentrated HCl. The solid precipitated, which was then filtered and dried under vacuum. The residue was purified by C18 reverse phase column chromatography (eluent: ACN in water (0.05% formic acid)) to afford 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]oct-6-yl)-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (0.14 g, yield 72%) as a pink solid. m / z 565.43 (M+H + ).

[0436] Step 3: Preparation of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6- diazabicyclo[3.1.1]oct-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide.

[0437] Compound 18.3

[0438] To a stirred solution of 3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]oct-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% 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 DMF solvent and then purified using a C18 column in 0-100% acetonitrile in water by reverse phase column purification, the desired product was eluted in 15% acetonitrile in water to get N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]oct-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (0.110 g, 56% yield) as a pink solid.

[0439] Compound 18.4

[0440] Method A. To a solution of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3- methyl-3,6-diazabicyclo[3.1.1]oct-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6- carboxamide (Compound 18.3) (10 mg, 0.013 mmol) in anhydrous CH2Cl2(350 µL) was added 4 Å powdered molecular sieves (5 mg) under argon. Then 1-chloro-4-oxo- butane-2,3-dione (2.5 mg, 0.015 mmol) was added via syringe. The reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was directly evaporated to remove CH2Cl2solvent and then purified using a C18 column in 0-100% acetonitrile in water by reverse phase column purification, the desired product was eluted in 15% acetonitrile in water to get N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]oct-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (Compound 18.4) (5 mg, 48% yield) as a pink solid. N , N, 2-trimethylprop-l-en-l-amine (6.9 µL, 0.052 mmol) and the mixture was stirred at room temperature for 45 min. N,N-diisopropylethylamine (9 µL, 0.052 mmol) was added, followed by TMSCHN2 (2M in hexanes, 26 µL, 0.052 mmol). After stirring at room temperature for 2.5 hours, the mixture was filtered and concentrated. The residue was purified by reverse-phase flash chromatography (10-100% ACN in water). The residue was purified a second time using a pipette column packed with basic alumina and eluted with 10% MeOH in CH2Cl2. Fractions containing product were concentrated, re-dissolved in dioxane and dried on a lyophilizer to yield a solid (1.4 mg, 13%). 1 H 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 = 1.5Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 6.47 (d, J = 2.2Hz, 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 + ]。

[0441] Method B. Under argon atmosphere, 4 Å powdered molecular sieve (5 mg) was added to a solution of N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3',6'-bis(3-methyl-3,6-diazabicyclo[3.1.1]hept-6-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxamide (compound 18.3) (10 mg, 0.013 mmol) in anhydrous CH2Cl2 (350 µL). Then, 1-chloro- N , N 2-Trimethylprop-1-en-1-amine (6.9 µL, 0.052 mmol) was added, and the mixture was stirred at room temperature for 45 min. N,N-Diisopropylethylamine (9 µL, 0.052 mmol) was added, followed by TMSCHN2 (2M in hexane, 26 µL, 0.052 mmol). After stirring at room temperature for 2.5 hours, the mixture was filtered and concentrated. The residue was purified by reversed-phase rapid chromatography (10-100% ACN in water). The residue was further purified by rapid chromatography using basic alumina (elution: 10% MeOH in CH2Cl2) to give the title compound as a solid (1.4 mg, 13%). 1 H 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]+.

[0442] 8.19 Reference Example 19 1,1'-(6'-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-5,5-dimethyl-3'-oxo- 3'H,5H-spiro[di benz[b,e] silinane-10,1'-isochromene]-3,7-diyl)bis(N,N-dimethylazetidine- 3-carboxamide) synthesis.

[0443] Compound 19

[0444] Step 1: 5,5-dimethyl-3'-oxo-3,7-bis((triisopropylsilyl)oxy)-3'H,5'H-spiro[di benz[b,e] silinane- 10,1'-isochromene]-6'-carboxylic acid tert-butyl ester synthesis. H ,5 H -oxo-3,7-bis((triisopropylsilyl)oxy)-3'H,5'H-spiro[di benz[b,e] silinane-10,1'-isochromene]- 6'-carboxylic acid tert-butyl ester synthesis. b , e 5,5-dimethyl-3'-oxo-3,7-bis((triisopropylsilyl)oxy)-3'H,5'H-spiro[di benz[b,e] silinane- 10,1'-isochromene]-6'-carboxylic acid tert-butyl ester synthesis.

[0445] Compound 19.1

[0446] A solution of 2-bromobenzene-1,4-dicarboxylic acid 1,4-di-tert-butyl ester (1.53 g, 4.29 mmol, WO2018046753A1) in 2:1 anhydrous THF / pentane (15 ml) was cooled to -100°C in an ethyl ether / liquid nitrogen bath. The solution was purged with Ar (g) for 10 min, then n BuLi (2.5M in hexanes, 1.75 mL, 1.72 mmol) was added dropwise along the wall of the flask. The solution was stirred at -100°C for 10 minutes, during which time the solution turned purple, then brown. 5,5-dimethyl-3,7-bis((triisopropylsilyl)oxy)dibenzo[ b , e ]silinane-10(5 HA solution of 1.0 g (1.72 mmol, WO2018046753A1) in THF (5 mL) was prepared and stirred for 10 min in a dry ice / acetone bath at -78 °C. The mixture was then heated to room temperature and stirred for 2.5 h. The mixture was adsorbed onto diatomaceous earth and purified by rapid chromatography using a silica gel column (25-50% CH2Cl2 in hexane) to give the title compound as a crystalline solid (436 mg, 32%). 1 H NMR (400MHz, 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 = 7.3Hz, 36H), 0.46 (s, 3H), 0.38 (s, 3H).

[0447] Step 2: 3,7-Dihydroxy-5,5-dimethyl-3'-oxo-3' H 5 H -spiro[dibenzo[] b , e Preparation of tert-butyl silane-10,1'-isobenzofuran-6'-carboxylate.

[0448] Compound 19.2

[0449] 5,5-Dimethyl-3'-oxo-3,7-bis((triisopropylsilyl)oxy)-3' H 5 H -spiro[dibenzo[] b , eSilacyclohexane-10,1 '-isobenzofuran]-6'-carboxylic acid tert-butyl ester (216 mg, 0.274 mmol) was dissolved in THF (2.16 mL) and cooled to 0 °C. To this solution was added TBAF (1 M in THF, 1.1 mL, 0.274 mmol). The solution turned a deep purple colour. After 30 minutes, ½ saturated NH4CI was added. The solution turned a light orange colour. The mixture was extracted with ethyl acetate (3 x 50 mL), the organic layers were combined, pre-absorbed onto silica gel and purified by flash chromatography (eluent: 0-10% ethyl acetate in DCM) to give the title compound as a translucent film (126 mg, 97%). m / z = 475.1 [M+2H]+.

[0450] Step 3: 5,5-Dimethyl-3'-oxo-3',7-bis(((trifluoromethyl)sulfonyl)oxy)-3' H ,5 H -spiro[di benzofuran]-6'-carboxylic acid tert-butyl ester. b , e Preparation of tert-butyl silacyclohexane-10,1 '-isobenzofuran]-6'-carboxylate.

[0451] Compound 19.3

[0452] Will 3,7-Dihydroxy-5,5-dimethyl-3'-oxo-3' H ,5 H -spiro[di benzofuran]-6'-carboxylic acid tert-butyl ester. b , e Preparation of tert-butyl silacyclohexane-10,1 '-isobenzofuran]-6'-carboxylate. N -phenyl bistrifluoromethanesulfonyl (792 mg, 2.22 mmol) was suspended in THF (12 mL). To the mixture was added slowly N , N -diisopropylethylamine (579 μ L, 3.33 mmol). 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 as a white foam (341 mg, 83%). m / z = 739.1 [M+H]+.

[0453] Step 4:3,7-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-5,5-dimethyl-3'-oxo-3' H ,5 H - spiro [dibenzo[ b , e ] silinane-10, 1'-isochromene] -6'-carboxylic acid tert-butyl ester.

[0454] Compound 19.4

[0455] A dried one dram vial was charged with N , N - dimethylazetidine-3-carboxamide hydrochloride (61.9 mg, 0.308 mmol), Cs2CO3, and dioxane (1.23 mL). 5,5-dimethyl-3'-oxo-3,7-bis(((trifluoromethyl)sulfonyl)oxy)-3' H ,5 H - spiro [dibenzo[ b , e ] silinane-10, 1'-isochromene] -6'-carboxylic acid tert-butyl ester (91 mg, 0.123 mmol) and XPhos (17.6 mg, 0.037 mmol). The mixture was purged with Ar for 5 min. Then Pd2dba3(11.3 mg, 0.012 mmol) was added. The vial was sealed and heated to 100 °C overnight. The mixture was cooled to rt, diluted with MeOH, taken up in 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]+.

[0456] Step 5: 3,7-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-5,5-dimethyl-3'-oxo-3' H ,5 H - spiro [dibenzo[ b , e ] silinane-10, 1'-isochromene] -6'-carboxylic acid.

[0457] Compound 19.5

[0458] A dried one dram vial was charged with 3,7-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-5,5-dimethyl-3'-oxo-3' H ,5H - spiro [dibenzo[ b , e ] silinane-10, 1 '-isobenzofuran] -6'-carboxylic acid tert-butyl ester (70 mg, 0.101 mmol) in CH2CI2 (3.73 mL) was cooled to 0 °C. Then trifluoroacetic acid (0.750 mL) was added dropwise. The solution color changed from yellow to green, then 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]+.

[0459] Step 6: 1,1'-(6'-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-5,5-dimethyl-3'-oxo-3' H ,5 H - spiro [dibenzo[ b , e ] silinane-10, 1 '-isobenzofuran] -3,7-diyl)bis( N , N -dimethylazetidine-3-carboxamide) Preparation.

[0460] Compound 19.6

[0461] To a solution of 3,7-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-5,5-dimethyl-3'-oxo-3' H ,5 H - spiro [dibenzo[ b , e ] silinane-10, 1 '-isobenzofuran] -6'-carboxylic acid (16 mg, 0.025 mmol) in DMF (0.764 mL) was added TBTU (9.65 mg, 0.030 mmol), HOBT (5.75 mg, 0.030 mmol) and N , N -diisopropylethylamine (8.73 μL, 0.050 mmol). The solution changed from deep blue to light green, then to light brown. 1-[2-(2-aminoethoxy)ethoxy]-6-chlorohexane (7.82 mg, 0.030 mmol) was then added, and the mixture was stirred overnight at room temperature. The mixture was concentrated and the blue-green film was diluted with DMSO and filtered. The mixture was purified by reversed-phase preparative HPLC (10-100% ACN / water (0.05% formic acid)) to give the title compound as a light green solid (4 mg, 19%). m / z =844.4 [M+H]+.

[0462] 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]silazane-10,1'-indene]-3,7-diyl)bis(N,N-dimethylazacyclobutane-3-carboxamide).

[0463] Compound 19.7

[0464] 1,1'-(6'-((2-(2-(((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-5,5-dimethyl-3'-oxo-3' H 5 H -spiro[dibenzo[] b , e ]silanecyclohexane-10,1'-isobenzofuran]-3,7-diyl)bis( N , N -Dimethylazonylbutane-3-carboxamide (7 mg, 8.29 mg) μ Add 1-chloro- (mol) to a 1:1 CH2Cl2 / ACN (2 mL) solution. N , N 2-Trimethylprop-1-en-1-amine (4.39) μ L, 33.2 μ mol). The solution color turned dark blue. Stir the mixture for 5 minutes, then add TMSCHN2 (2M Et2O, 16.6 mol). μ L, 33.2 μ mol) and N , N -Diisopropylethylamine (5.78) μ L, 33.2 μmol). 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]+.

[0465] 8.20 Reference Example 20 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) synthesis.

[0466] Compound 20

[0467] 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) at 0 °C was added N,N- diisopropylethylamine (0.054 mL, 0.311 mmol) followed by T3P (50% in ethyl acetate, 0.132 g, 0.207 mmol). The mixture was stirred at 0 °C for 1 h, then ammonium bicarbonate (0.041 g, 0.518 mmol) was added. The mixture was allowed to warm to room temperature and stirred at room temperature for 12 h, then ice-cold water (10 mL) was added and the mixture was extracted with ethyl acetate (10 mL x 2). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative HPLC (C18; mobile phase, 0-100% ACN in water) to give the title compound (0.0052 g, 13% yield) as a light yellow solid. 1 H NMR (400 MHz, DMSO- d 6): δ8.69 (t, J = 5.6 Hz, 1H), 7.97 (dd, J = 1.6 Hz and 8.0 Hz, 1H), 7.83 (d, J = 8.0Hz, 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] + .

[0468] 8.21 Reference Example 21 3',6'-Diazolidin-1-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-3 H Synthesis of spiro[isobenzofuran-1,9'-xanthene]-3-one.

[0469] Compound 21

[0470] Step 1: Preparation of 2-((6-chlorohexyl)oxy)ethan-1-ol.

[0471] Compound 21.1

[0472] To a solution of ethane-1,2-diol (5 g, 80.5 mmol) in DMF (50 mL) was added NaH (60% in mineral oil) (3.86 g, 161.10 mmol) followed by 1-chloro-6- iodohexane (19.85 g, 80.55 mmol) at 0 °C under nitrogen. 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 x 100 mL). The organic layers were combined, dried over Na2S04and 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%).

[0473] Step 2: 3-(2-((6-Chlorohexyl)oxy)ethoxy)propan-1-ol.

[0474] Compound 21.2

[0475] To a solution of 2-((6-chlorohexyl)oxy)ethan-1-ol (2.5 g, 13.9 mmol) in ACN (25 mL) was added a solution of N-benzyl-trimethylammonium hydroxide in H2O (0.277 g, 4.16 mmol) at room temperature over 10 min. Then, acrylic acid butyl ester (8.9 g, 69.4 mmol) was added dropwise. The resulting solution was stirred at room temperature for 16 h, 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 Na2SO4and 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%). tert

[0476] Step 3: 3-(2-((6-chlorohexyl)oxy)ethoxy)propanal).

[0477] Compound 21.3

[0478] To a solution of 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid butyl ester (0.5 g, 1.623 mmol) in anhydrous THF (5 mL) at -78 °C was added DIBAL-H (1 M in THF, 3.2 mL, 3.247 mmol) 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 x 30 mL). The organic fractions were combined, dried over Na2SO4and concentrated. The residue was purified by flash chromatography (eluent: 45% EtOAc in petroleum ether) to give the title compound as a colorless oil (0.2 g, 52%). tert

[0479] Step 4: Preparation of tert-butyl (3',6'-dibromo-3-oxo-3 H -spi[isobenzofuran-1,9'-xanthene]-6-yl)carbamate.

[0480] Compound 21.4

[0481] To a solution of pyridinium 3',6'-dibromo-3-oxo-3 H ​​To a solution of spiro[isobenzofuran-l,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). The mixture was stirred at 0 °C for 30 min. The solution was allowed to warm 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 NaHC03 and brine, dried over Na2S04 and concentrated. The residue was suspended in t BuOH (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 NaHC03 solution and brine, then dried over Na2S04, 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]+.

[0482] Step 5: Preparation of tert-butyl (3',6'-diazolidin-1-yl-3-oxo-3H-spiro[isobenzofuran-l,9'- xanthene]-6-yl)carbamate.

[0483] Compound 21.5

[0484] To a solution of tert-butyl (3',6'-dibromo-3-oxo-3H-spiro[isobenzofuran-l,9'-xanthene]-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 purged with Ar for 15 min, then Pd2(dba)3 (2.80 g, 3.06 mmol) was added followed by XPhos (0.87 g, 1.83 mmol) and the mixture was heated to 110 °C for 16 h. The mixture was cooled to room temperature, filtered through a pad of celite and concentrated. The residue was purified by reverse phase C-18 chromatography (eluent: 0-100% ACN in water) to give the title compound (1.4 g, 43%) as a deep pink solid, m / z = 574.32 [M+H]+.

[0485] Step 6:Preparation of 6-amino-3',6'-bis(azetidin-l-yl)-3H-spiro[isobenzofuran-l,9'-xanthene]-3- one.

[0486] Compound 21.6

[0487] To a solution of tert-butyl (3',6'-bis(azetidin-l-yl)-3-oxo-3H-spiro[isobenzofuran-l,9'- xanthen]-6-yl)carbamate (1.0 g, 1.90 mmol) in CH2Cl2(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 NaHCO3(40 mL), and extracted with 10% MeOH in CH2Cl2(3 x 30 mL). The combined organic layers were dried over Na2SO4and 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]+.

[0488] Step 7: 3',6'-Bis(azetidin-l-yl)-6-((3-(2-((6-chlorohexyl)oxy)ethoxy)propyl)amino)-3H- spiro[isobenzofuran-l,9'-xanthene]-3-one.

[0489] Compound 21.7

[0490] To a solution of 6-amino-3',6'-bis(azetidin-l-yl)-3H-spiro[isobenzofuran-l,9'-xanthene]-3- one (0.2 g, 0.471 mmol) and 3-(2-((6-chlorohexyl)oxy)ethoxy)propanal (0.11 g, 0.471 mmol, compound 21.3) in dichloroethane (2 mL) was added 2-3 drops of acetic acid at room temperature. The resulting solution was stirred for 2 h. Then, sodium triacetoxyborohydride (0.199 g, 0.941 mmol) was added portionwise at 0 °C over 20 min. The resulting mixture was stirred at room temperature for 2 h, then quenched with ice water and extracted through 10% MeOH in CH2Cl2(2 x 25 mL). The organic layers were combined, dried over Na2SO4and concentrated. The residue was purified by reverse phase column chromatography (eluent: 0-100% ACN in water) to give the title compound (0.1 g, 32%) as a pink solid. m / z 646.50 [M+H]+ .

[0491] Step 8: 3',6'-Bis(azetidin-1-yl)-6-((3-(2-((6-chlorohexyl)oxy)ethoxy)propyl)amino)-3H- spiro[isobenzofuran-1,9'-xanthene]-3-one.

[0492] Compound 21.8

[0493] To a solution of 3',6'-bis(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 CH2Cl2(5 mL) under nitrogen atmosphere at 0 °C was added a freshly distilled solution of thionyl chloride (0.058 mL, 0.774 mmol) in CH2Cl2(0.025 mL). The resulting solution was allowed to warm to room temperature for 30 mn, then concentrated. The residue was diluted with anhydrous CH2Cl2(0.025 mL), then freshly prepared diazomethane in Et2O (10 mL, >25 eq) was added. The mixture was stirred at 0 °C for 30 min, the mixture was concentrated, and the residue was purified by preparative reverse-phase HPLC (eluent: 0-100% ACN in water) to give the title compound (5 mg, 4.82%) as a brown gum. 1 H NMR (400 MHz, DMSO- d 6): 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, J = 6.4 Hz, 2H), 3.39-3.38 (m, 8H),3.00-2.95 (m, 2H), 2.32-2.27 (m, 4H), 1.68-1.63 (m, 4H), 1.46-1.42 (m, 6H),ppm. m / z 668.37 [M-H] - .

[0494] 8.22Reference Example 22 Synthesis of 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-bis(azetidin-l-yl)-2-diazo-3- oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-yl)propanamide.

[0495] Compound 22

[0496] Step 1: Synthesis of 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid.

[0497] Compound 22.1

[0498] To a solution of 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid (0.45 g, 1.447 mmol) in CH2Cl2(10 mL) was added 3',6'-bis(azetidin-l-yl)-3-oxo-3H-spiro[isobenzofuran-l,9'- xanthene]-6-carboxylic acid (0.5 g, 1.447 mmol) and HATU (0.6 g, 1.6 mmol) at 0 °C under Ar (g) atmosphere. The resulting mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was then diluted with water and extracted with CH2Cl2(3 x 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (silica gel, 12 g, 60-120 mesh, 10% CH3OH in CH2Cl2) to give 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-bis(azetidin-l-yl)-3-oxo-3H- spiro[isobenzofuran-l,9'-xanthene]-6-yl)propanamide (0.5 g; 0.8 mmol) as a white solid. tert To a solution of 3-(2-((6-chlorohexyl)oxy)ethoxy)propanoic acid (0.45 g, 1.447 mmol) in CH2Cl2(10 mL) was added 3',6'-bis(azetidin-l-yl)-3-oxo-3H-spiro[isobenzofuran-l,9'- xanthene]-6-carboxylic acid (0.5 g, 1.447 mmol) and HATU (0.6 g, 1.6 mmol) at 0 °C under Ar (g) atmosphere. The resulting mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was then diluted with water and extracted with CH2Cl2(3 x 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (silica gel, 12 g, 60-120 mesh, 10% CH3OH in CH2Cl2) to give 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-bis(azetidin-l-yl)-3-oxo-3H- spiro[isobenzofuran-l,9'-xanthene]-6-yl)propanamide (0.5 g; 0.8 mmol) as a white solid. m / z 284.1 [M+H] + .

[0499] Step 2: Synthesis of 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-bis(azetidin-l-yl)-2-diazo-3- oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-6-yl)propanamide.

[0500] Compound 22.2

[0501] At 0 °C, n-methylimidazolium (0.28 mL, 3.53 mmol) was added dropwise to a solution of 6-amino-3',6'-bis(azacyclobutan-1-yl)-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-3-one (compound 21.6, 0.3 g, 0.705 mmol) and 3-(2-((6-chlorohexyl)oxy)ethoxy)propionic acid (0.2 g, 0.705 mmol) in CH2Cl2 (15 mL), followed by the addition of methanesulfonyl chloride (0.082 mL, 1.06 mmol). The mixture was heated to room temperature and stirred for 1 h. The mixture was then concentrated and purified by reversed-phase HPLC (elution: 0-100% acetonitrile in 10 mM ammonium acetate in water) to give the title compound (0.17 g; yield 36%) as a pink solid. m / z 660.31 [M+H] + .

[0502] Step 3: 3-(2-((6-chlorohexyl)oxy)ethoxy)-N-(3',6'-bis(azacyclobutane-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-oxazanthene]-6-yl)propionamide.

[0503] Compound 22.3

[0504] Oxaloyl chloride (0.2 mL, 2.28 mmol) was added dropwise to a solution of N-(3'-(azacyclobutan-1-yl)-6'-cyclobutyl-3-oxo-3H-spiro[isobenzofuran-1,9'-oxazanthracene]-6-yl)-3-(2-((6-chlorohexyl)oxy)ethoxy)propionamide (0.15 g, 0.228 mmol) in CH2Cl2 (30 mL) under an argon atmosphere at 0 °C. The mixture was heated to room temperature and stirred for 1 h. Then, freshly prepared diazomethane (approximately 30 mL, >25 equivalents) was added dropwise at 0 °C. The mixture was stirred for 1 h. The resulting solution was then concentrated and purified by reversed-phase preparative HPLC (elution: 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] - .

[0505] 8.23 Reference Example 23 Synthesis of (E)-3',6'-bis(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'-bis(azetidin-1-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-1-en-1-yl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one.

[0506] Compound 23

[0507] Step 1: Preparation of 2-((6-chlorohexyl)oxy)ethan-1-ol.

[0508] Compound 23.1

[0509] To a stirred solution of ethane-1,2-diol (5.0 g, 80.55 mmol) in DMF (50 mL) was added 60% sodium hydride (6.44 g, 161.11 mmol) at -10 °C and the mixture was stirred at -10 °C for 15 min. To the mixture was added 1-chloro-6-iodohexane (19.86 g, 80.55 mmol) at -10 °C, then the mixture was allowed to warm to room temperature and stirred at room temperature for 2 h. To the mixture was then added ice water (0.5 L) and the mixture was extracted with ethyl acetate (2 x 500 mL). The combined organic layers were washed with brine solution, dried over sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: 0-20% ethyl acetate in petroleum ether) to give the title compound (0.9 g, 6%) as a light yellow liquid.

[0510] Step 2:Preparation of 2-((6-chlorohexyl)oxy)ethyl methanesulfonate.

[0511] Compound 23.2

[0512] To a stirred solution of 2-((6-chlorohexyl)oxy)ethan-1-ol (1.5 g, 8.302 mmol) in dichloromethane (15.0 mL) was added triethylamine (1.4 mL, 9.963 mmol) followed by methanesulfonyl chloride (1.05 g, 9.133 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature for 1 h. Ice water (50 mL) was added and the mixture was extracted with dichloromethane (2 x 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to afford the title compound (1.5 g, 70%) as a light yellow liquid which was used for the next step without further purification.

[0513] Step 3: Preparation of 1-(2-(but-3-yn-1-yloxy)ethoxy)-6-chlorohexane.

[0514] Compound 23.3

[0515] 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 60% sodium hydride (0.457 g, 11.41 mmol) at 0 °C and the mixture was stirred at 0 °C for 30 min. Then 2-((6-chlorohexyl)oxy)ethyl methanesulfonate (1.48 g, 5.71 mmol) was added. The mixture was allowed to warm to room temperature and stirred at room temperature for 16 h. Then ice water (80 mL) was added and the mixture was extracted with ethyl acetate (80 mL). The combined organic layers were washed with brine, dried over sodium sulfate and 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.

[0516] Step 4: Preparation of 3',6'-di(azetidin-1-yl)-6-iodo-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one.

[0517] Compound 23.4

[0518] To a stirred solution of 6-amino-3',6'-bis(azetidin-l-yl)-3H-spiro[isobenzofuran-l,9'- xanthen]-3-one (2.5 g, 5.88 mmol, Compound 21.6) in ACN (10 mL) was added Cul (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. Then sodium thiosulfate solution in water was added and the mixture was extracted with 10% MeOH in DCM (3 x 80 mL). The combined organic layers were dried over Na2S04and concentrated. The residue was purified by preparative HPLC (mobile phase, 0-100% ACN in water) to give the title compound (1.0 g, 31%) as a purple solid.

[0519] Step 5: Preparation of 3',6'-bis(azetidin-l-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-l-yn-l-yl)- 3H-spiro[isobenzofuran-l,9'-xanthen]-3-one.

[0520] Compound 23.5

[0521] To a stirred solution of l-(2-(but-3-yn-l-yloxy)ethoxy)-6-chlorohexane (0.15 g, 0.644 mmol, Compound 23.3) and 3',6'-bis(azetidin-l-yl)-6-iodo-3H-spiro[isobenzofuran-l,9'-xanthen]-3-one (0.346 g, 0.644 mmol, Compound 23.4) in DMF (4.5 mL) was added triethylamine (4.5 mL), copper iodide (0.012 g, 0.064 mmol) and bis(triphenylphosphine)palladium(II) chloride (0.045 g, 0.064 mmol). The mixture was stirred at room temperature for 2 h. Then the mixture was concentrated under reduced pressure. The residue was purified by reverse phase column eluting with 50-60% acetonitrile in water with 10 mM ammonium bicarbonate to give the title compound (0.12 g; 24%) as a pink solid.

[0522] Step 6:Preparation of (E)-3',6'-bis(azetidin-l-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-l-en-l-yl)- 3H-spiro[isobenzofuran-l,9'-xanthene]-3-one and (Z)-3',6'-bis(azetidin-l-yl)-6-(4-(2-((6- chlorohexyl)oxy)ethoxy)but-l-en-l-yl)-3H-spiro[isobenzofuran-l,9'-xanthene]-3-one.

[0523] Compound 23.6

[0524] To a stirred solution of 3',6'-bis(azetidin-l-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-l-yn-l- yl)-3H-spiro[isobenzofuran-l,9'-xanthene]-3-one (0.1 g, 0.156 mmol) in ethanol (10.0 mL) was added 10% palladium on carbon (50% humidity) (0.1 g). The mixture was stirred under hydrogen at room temperature for 12 h. The mixture was then filtered through celite and concentrated. The residue was purified by reverse phase column (eluted with 50-80% acetonitrile in water with 10 mM ammonium bicarbonate) to give the title compound as the first eluted mixture (0.025 g; 24% yield).

[0525] Step 7: Preparation of (E)-3',6'-bis(azetidin-l-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-l-en-l-yl)- 3H-spiro[isobenzofuran-l,9'-xanthene]-3-one and (Z)-3',6'-bis(azetidin-l-yl)-6-(4-(2-((6- chlorohexyl)oxy)ethoxy)but-l-en-l-yl)-3H-spiro[isobenzofuran-l,9'-xanthene]-3-one.

[0526] Compound 23.7

[0527] To a stirred solution of 3',6'-bis(azetidin-l-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)but-l-en-l-yl)-3H-spiro[isobenzofuran-l,9'-xanthene]-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 mmol) at 0 °C under argon, then the mixture was warmed to room temperature and stirred for 1 h. The mixture was then concentrated and backfilled with argon. A freshly prepared solution of diazomethane in ether (4.0 mL) was added to the residue at 0 °C and the mixture was stirred for 1 h. The mixture was then concentrated and the residue was purified by preparative HPLC (C18; mobile phase: 0-100% ACN / water) to give the title compound (0.0022 g, 10%) as a brown gum. m / z 667.5 [M+H] + .

[0528] 8.24 Reference Example 24 Synthesis of 3',6'-bis(azetidin-l-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-2-diazo- spiro[indene-l,9'-xanthene]-3(2H)-one.

[0529] Compound 24

[0530] Step 1: Preparation of 3',6'-bis(azetidin-l-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-3H- spiro[isobenzofuran-l,9'-xanthene]-3-one.

[0531] Compound 24.1

[0532] Further elution of the reverse phase column of Step 6 of Compound 23 gave the title compound (0.02 g; yield 20%) as a pink solid.

[0533] Step 2: Preparation of 3',6'-bis(azetidin-l-yl)-6-(4-(2-((6-chlorohexyl)oxy)ethoxy)butyl)-2-diazo- spiro[indene-l,9'-xanthene]-3(2H)-one.

[0534] Compound 24.2

[0535] To a stirred solution of 3',6'-bis(azetidin-l-yl)-6-(4-(2-((6- chlorohexyl)oxy)ethoxy)butyl)-3H-spiro[isobenzofuran-l,9'-xanthene]-3-one (0.02 g, 0.031 mmol) in dichloromethane (4.0 mL) was added oxalyl chloride (0.03 mL, 0.31 mmol) at 0 °C under argon atmosphere. The mixture was allowed to warm to room temperature and stirred at room temperature for 1 h, then the mixture was concentrated under argon atmosphere. Freshly prepared diazomethane solution in ether (4.0 mL) was added to the residue at 0 °C and the mixture was stirred at 0 °C for 1 h, then the mixture was concentrated. The residue was purified by preparative HPLC (C18; mobile phase, 0-100% ACN in water) 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] + .

[0536] 8.25Example 25 Synthesis of 3',6'-bis(azetidin-l-yl)-6-(3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)-2- diazospiro[indene-l,9'-xanthene]-3(2 H )-one.

[0537] Compound 25

[0538] Compound 25.1

[0539] Step 1 To a solution of 3-(benzyloxy)propan-1 -ol (100 g, 601 mmol) in dichloromethane at 0 °C was added triethylamine (91.3 g, 902 mmol) and stirred for 30 min. Then p-toluenesulfonyl chloride (141 g, 721 mmol) was added portion wise at 0 °C and allowed to warm to room temperature, then allowed to stir for 16 h. The solution was then diluted with water (1000 mL) and extracted with dichloromethane (3 x 200 mL). The organic layer was washed with brine, then 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 colourless liquid.

[0540] Compound 25.2

[0541] Step 2 To a solution of ethane-1,2-diol (12.6 g, 203 mmol) in DMF (40 mL) at 0 °C was added sodium hydride (7.31 g, 60% oil dispersion, 305 mmol) 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. TLC confirmed the completion of the reaction, the solution was quenched with cold water (400 mL) then extracted with ethyl acetate (400 ml). The organic layer was washed with brine solution (2 x 400 mL) and dried over anhydrous sodium sulfate, then 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 colourless liquid.

[0542] Compound 25.3

[0543] Step 3To a stirred solution of 2-(3-(benzyloxy)propoxy)ethan-1-ol (4.5 g, 21 mmol) in DMF (40 mL) was added sodium cyanide (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. TLC confirmed completion of the reaction, the solution was quenched with cold ammonium chloride solution (200 mL) and then extracted with ethyl acetate (2 x 100 mL). The organic layer was washed with brine solution, dried over sodium sulfate and concentrated. The crude was triturated with pentane to get ((3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)methyl)benzene (4.3 g, 21%) as a colorless liquid.

[0544] Compound 25.4

[0545] Step 4 In a pressure tube, to a stirred solution of ((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)benzene (4.2 g, 12 mmol) in ethanol was added 10% palladium on carbon (50% moisture) (4.2 g) followed by hydrogen pressure of 100 psi and stirred at room temperature for 16 h. After TLC indicated completion of the reaction, the mixture was carefully filtered through a pad of celite and washed with ethyl acetate. The filtrate was concentrated to get 3-(2-((6-chlorohexyl)oxy)ethoxy)propan-1-ol (3.0 g, 98%) as a colorless liquid.

[0546] Compound 25.5

[0547] 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 methanesulfonyl chloride (0.4 mL, 5 mmol) at 0 °C. The resulting solution was stirred at room temperature for 1 h. After TLC indicated completion of the reaction, 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 get 3-(2-((6-chlorohexyl)oxy)ethoxy)propanesulfonic acid methyl ester (1.0 g, 75%) as a light brown solid. m / zFound 317.1 [M+H] + .

[0548] Compound 25.6

[0549] 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). The mixture was then washed with ethyl acetate (3 x 1 L), 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 as 3',6'-dibromo-6-hydroxy-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (1.2 g, yield 1%) as off-white solid; and peak-2 as 3',6'-dibromo-5-hydroxy-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.12 g, yield 0.1%). m / z Found 475.18 [M+H] + .

[0550] Compound 25.7

[0551] Step 7 To a stirred solution of 3',6'-dibromo-6-hydroxy-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (1.0 g, 2.1 mmol) in DMF (10 mL) was added cesium carbonate (2.0 g, 6.4 mmol) followed by 3-(2-((6-chlorohexyl)oxy)ethoxy)propane sulfonic acid propyl ester (1.0 g, 3.3 mmol, compound 25.5) at room temperature under argon atmosphere. 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 into ethyl acetate (100 mL). The organic layer was washed with brine solution (2 x 100 mL), the organic layer was 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'-xanthene]-3-one (0.52 g, yield 35%) as colorless liquid. m / z 697.39 [M+H] + .

[0552] Compound 25.8

[0553] Step 8 : In a sealed tube, to a stirred solution of 3',6'-dibromo-6-(3-(2-((6- chlorohexyl)oxy)ethoxy)propoxy)-3H-spiro[isobenzofuran-l,9'-xanthene]-3-one (0.3 g, 0.4 mmol) in anhydrous 1,4-dioxane (10 mL) 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 min. Then azetidine (0.10 g, 1.7 mmol) was added, the mixture was warmed to 110 °C and stirred for 16 h. 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'-bis(azetidin-l-yl)-6-(3-(2-((6-chlorohexyl)oxy)ethoxy)propoxy)-3H- spiro[isobenzofuran-l,9'-xanthene]-3-one (0.09 g, 16% yield). m / z 647.48 [M+H] + .

[0554] 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).

[0555]

[0556] Compound 25 Step 9 To a stirred solution of 3',6'-bis(azetidin-l-yl)-6-(3-(2-((6- chlorohexyl)oxy)ethoxy)propoxy)-3H-spiro[isobenzofuran-l,9'-xanthene]-3-one (0.025 g, 0.039 mmol) in dichloromethane (50 mL) was added dropwise oxalyl chloride (0.3 mL, 0.579 mmol, 2M in DCM) at 0 °C under argon atmosphere. The resulting mixture was then allowed to warm to room temperature and stirred for 1 h. The reaction mixture was concentrated under argon atmosphere. The crude was dissolved in dichloromethane (50 mL) and diazomethane (2.5 g; 1.0 eq) in freshly prepared diethyl ether (20 mL) was added. The reaction was then allowed to warm to room temperature and stirred for 1 h. The reaction mixture was concentrated and purified by reverse phase chromatography (0-25% acetonitrile in 10 mM aqueous acetic acid) to afford the title compound (2.5 mg; yield 10%). m / z 671.54 [M+H] + .

[0557] 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).

[0558] 8.26 Example 26 3',6'-Di(azacyclobutane-1-yl)-6-(((2-(2-(((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-2-diazospiro[indene-1,9'-oxazanthene]-3(2 H Synthesis of )-ketones.

[0559] Compound 26

[0560] Compound 26.1

[0561] Step 1 At 0°C, 3',6'-dibromo-3-oxo-3 H -spiro[isobenzofuran-1,9'-oxanthracene]-6-carboxylic acid (20 g, 40 mmol, according to Tetrahedron The reaction was prepared according to the procedure described in 2005, 61, 3097-3105. 1,1'-carbonyldiimidazole (12.9 g, 79.6 mmol) was added dropwise to a stirred solution in THF (200 mL). The resulting solution was stirred at room temperature for 3 h. Then, sodium borohydride (7.5 g, 200 mmol) in 400 mL of water was added dropwise at 0 °C, and the mixture was stirred at room temperature for 10 min. After the reaction was complete, the mixture was extracted with ethyl acetate (2 × 250 mL). The organic layer was dried over sodium sulfate and concentrated. The crude material was ground with cold DCM to give 3',6'-dibromo-6-(hydroxymethyl)-3 H 3-spiro[isobenzofuran-1,9'-oxanthracene]-3-one (5.8 g, 30%) is a grayish-white solid. m / z Measured value: 487.07 [M+H] + .

[0562] Compound 26.2

[0563] Step 2 At 0°C, 3',6'-dibromo-6-(hydroxymethyl)-3 H- To a stirred solution of spiro[isobenzo furan-1,9'-xanthene]-3-one (5.8 g, 12 mmol) in DCM (58 mL) was added TEA (8.4 g, 83 mmol) followed by methanesulfonyl chloride (6.8 g, 60 mmol). 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 x 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 containing 0.1 % TEA) to afford 3',6'-dibromo-6-(chloromethyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (5.8 g, 70%) as off-white solid. H - spiro[isobenzo furan-1,9'-xanthene]-3-one (4.2 g, 70%) as off-white solid. m / z Found 505.20 [M+H] + .

[0564] Compound 26.3

[0565] Step 3 : To a stirred solution of 3',6'-dibromo-6-(chloromethyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (1.3 g, 3.3 mmol) in DCM (30 mL) was added TEA (1.8 g, 17 mmol) followed by 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1 -amine (1.2 g, 5.0 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (50 mL) and the aqueous layer was extracted with DCM (3 x 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 chromatography (5% methanol in dichloromethane) to afford 3',6'-dibromo-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (1.3 g, 70%) as yellow semi solid. H - To a stirred solution of spiro[isobenzo furan-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 h. The reaction mixture was then diluted with ice cold 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 was purified by silica gel column chromatography (5% methanol in dichloromethane) to afford 3',6'-dibromo-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (1.3 g, 52%) as yellowish semi solid. m / z 692.41 [M+H] + .

[0566] Compound 26.4

[0567] Step 4A 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 to 0 °C and acetic acid (saturated) was added. The reaction was stirred at 0 °C and allowed to warm to room temperature over 2 h, then NaCNBH3 (0.35 g, 5.6 mmol) was added portionwise to the solution and stirring was continued for 12 h. Upon 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 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'-xanthene]-3-one (0.9 g, 68%) as a colorless semi-solid. m / z 705.36 [M+H] + .

[0568] Compound 26.5

[0569] Step 5To 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]phosphine; 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 min. Azetidine (0.081 g, 1.4 mmol) was added and the reaction was heated to 110 °C with stirring for 16 h. The reaction mixture was cooled to room temperature, filtered through celite and washed with ethyl acetate. The crude was purified by preparative HPLC (0-67% acetonitrile in 10 mM aqueous ammonium bicarbonate) to give 3',6'-bis(azetidin-1-yl)-6-(((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (0.03 g, 13% yield). 1 HNMR (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]+ .

[0570] Compound 26

[0571] Step 6 To a stirred solution of 3',6'-bis(azetidin-l-yl)-6-(((2-(2-((6- chlorohexyl)oxy)ethoxy)ethyl)(methyl)amino)methyl)-3H-spiro[isobenzofuran-l,9'- xanthen]-3-one (0.02 g, 0.030 mmol) in dichloromethane (50 mL) was added oxalyl chloride (0.23 mL, 0.45 mmol) at 0 °C under argon atmosphere. The reaction was then allowed to warm to room temperature and stirred for 1 h. The reaction mixture was concentrated under argon atmosphere. The crude was dissolved in dichloromethane (50 mL) and a freshly prepared solution of diazomethane in diethyl ether (1.0 equivalent) was added dropwise. After completion of the reaction, the mixture was concentrated and the crude was purified by preparative HPLC (0-37% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford 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] + .

[0572] 8.27 Example 27 3',6'-Bis(azetidin-l-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-2- diazospiro[indene-l,9'-xanthene]-3'(2 H )-one.

[0573]

[0574] Compound 27

[0575] Compound 27.1

[0576] Step 1: To a solution of 2-(2-(benzyloxy)ethoxy)ethan-l-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 x 100 mL). The organic layer was washed with brine solution, dried over sodium sulfate and concentrated. The crude was purified by silica gel column (0-15% ethyl acetate in petroleum ether) to afford ((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)benzene (5 g, 62%) as a light yellow liquid.

[0577] Compound 27.2

[0578] 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 base) and stirred under hydrogen gas (1 atm) for 16 h. After completion of the reaction, the reaction mixture was carefully filtered under a pad of celite. The pad was washed with ethyl acetate (3 x 50 mL). The filtrate was concentrated to obtain 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-l-ol (3.5 g, 99%) as a colorless liquid.

[0579] Compound 27.3

[0580] Step 3: To a solution of 3',6'-dibromo-6-(chloromethyl)-3 H - A solution of spiro[isobenzofuran-l,9'-xanthene]-3-one (4.2 g, 8.3 mmol, compound 26.2) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-l-ol (2.8 g, 12 mmol) in anhydrous DMSO (42 mL) was added potassium hydroxide (1.4 g, 25 mmol) and stirred for 16 h. 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 containing 0.1% TEA) to give 3',6'-dibromo-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-3 H - spiro[isobenzofuran-l,9'-xanthene]-3-one (2 g, 35%) as a viscous liquid.

[0581] Compound 27.4

[0582] Step 4 : To a solution of 3',6'-dibromo-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-3 H - A suspension of spiro[isobenzofuran-l,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 min. Pd2(dba)3 (12.8 mg, 0.014 mmol) and XPhos (20 mg, 0.042 mmol) were then added, followed by a 5 min purge with nitrogen. Azetidine (24.6 mg, 0.42 mmol) was added and the reaction stirred at 100 °C for 16 h. 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 containing 0.1% formic acid) to give 3',6'-bis(azetidin-l-yl)-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)methyl)-3 H - spiro[isobenzofuran-l,9'-xanthene]-3-one (30 mg, 32%) as a purple viscous solid. m / z Found 647.72 [M+H] + .

[0583] Compound 27

[0584] Step 5 (Method C) To a dried 20 mL vial was added 3',6'-bis(azetidin-l-yl)-6-((2-(2-((6- chlorohexyl)oxy)ethoxy)ethoxy)methyl)-3 H - spiro[isobenzofuran-l,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, the vial was capped, and purged with argon for 5 minutes. Ghosez reagent (81.8 μL, 0.618 mmol) was added and the solution turned dark. Two minutes after the addition of Ghosez reagent, trimethylsilyldiazomethane (0.31 mL, 0.62 mmol, 2M in ether) was added, followed by potassium fluoride (35.9 mg, 0.618 mmol). The reaction mixture was concentrated directly onto the silica gel and purified by silica gel chromatography (0-20% EtOAc in toluene) to give the title compound. 1 HNMR (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 Found 671.46 [M+H] + .

[0585] 8.28Example 28 1,1'-(6-((4-(((2-aminopyrimidin-4-yl)oxy)methyl)benzyl)carbamoyl)-2- diazo-3-oxo-2,3-dihydrospiro[indene-l,9'-xanthene]-3',6'-diyl)bis(azetidin-3- yl) (Compound 28) N , NSynthesis of (3-(dimethylcarbamoyl)azetidin-3-yl)methyl 3',6'-bis(3- (dimethylcarbamoyl)azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6- carboxylate.

[0586]

[0587] Compound 28

[0588] Compound 28.1

[0589] Step 1 (Method C) To a stirred 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 A molecular sieves (80 mg), and CaO (3.6 mg, 0.063 mmol) in 1:1 DCM / acetonitrile (2 mL) was added Ghosez reagent (0.022 mL, 0.17 mmol) dropwise at room temperature. After stirring at room temperature for 2 min, TMSCHN2 (0.085 mL, 0.17 mmol, 2 M in ether) was added, followed immediately 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+ .

[0590] Compound 28.2 (also referred to herein as Compound 5.1)

[0591] Step 2:To a stirred solution of 2-diazo-3',6'-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-3-oxo-2,3- dihydrospiro[indene-l,9'-xanthene]-6-carboxylic acid methyl ester (0.055 g, 0.086 mmol) in THF (1.0 mL) and 1:1 MeOH / H20 (1.0 mL) was added lithium hydroxide monohydrate (0.008 g, 0.173 mmol) portion wise at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction was concentrated and the crude was purified by trituration with diethyl ether to afford 2-diazo-3',6'-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-3-oxo-2,3- dihydrospiro[indene-l,9'-xanthene]-6-carboxylic acid (0.05 g, 93%) as a brown solid. m / z Found 621.6 [M+H] + .

[0592] Compound 28.3

[0593] Step 3 To a stirred solution of 2-diazo-3',6'-bis(3-(dimethylcarbamoyl)azetidin-l-yl)-3-oxo-2,3- dihydrospiro[indene-l,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% in ethyl acetate; 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 was purified by preparative HPLC (0-70% acetonitrile in 10 mM aqueous ammonium bicarbonate) to afford the title compound (0.0156 g, 20%). 1 H 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 = 2.4Hz, 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 Obs. 833.52 [M+H] + .

[0594] 8.29Reference Example 29: Kinetic solubility in 7.4 PBS 190 μL of buffer solution (PBS, pH 7.4) was added to the wells on a 96-well Millipore solubility filter plate. Then 10 μL of test compound in DMSO (10 mM) was added to give a final concentration of 500 μM.

[0595] The filter plate was shaken in the dark at room temperature for 1.5 hours, then the samples were filtered into a new 96-well plate by a vacuum system. The samples were diluted in DMSO to 500 μM (top concentration) and further diluted (1 :10) to provide a three-point calibration curve. The absorbance at 220 nm, 254 nm and 280 nm was measured using HPLC / UV analysis. Data is reported as the average of three runs for each test compound and is shown in Table 1 below.

[0596] Table 1: Kinetic solubility values

[0597] 8.30Reference Example 30: Passive permeation rate MDCK-MDR1 cells were seeded at 7,500 cells / 75 μL / well into 96-well Millipore Millicell-96 plates and incubated at 37°C, 5% CO2for three days. Cells were then washed with Hank's Balanced Salt Solution (HBSS) containing 5 mM HEPES for 30 minutes. Solutions of test compounds in DMSO (10 mM) were added to HBSS buffer containing 10 μM GF-120918 to a final DMSO concentration of 0.2% and test compound concentration of 5 μM. Transport plates were incubated at 37°C in a humidified incubator containing 5% CO2for one hour. Samples were collected from the apical and basolateral compartments after one hour and analyzed by liquid chromatography with tandem mass spectrometry (LC / MS / MS, AB Sciex API 4000 instrument coupled with 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 0.1% formic acid in 100% acetonitrile (solvent B).

[0598] The apparent permeability (Papp, A2B) values were calculated using the following equation: Papp = (dQ / dt) / A / C0 where dQ / dt is the initial rate of the amount of test compound transported across the cell monolayer, A is the surface area of the filter, and Co is the initial concentration of the test compound, calculated for each direction by LC / MS / MS using a 4-point calibration curve. Data is reported as the average of two runs and is shown in Table 2 below.

[0599] Table 2: Passive permeability values

[0600] 8.31 Reference Example 31: Protein labeling with photoactivatable dyes HaloTag protein tagged with W60 µM 6xHis (expressed and purified from pH6HTC His6HaloTag T7 vector from Promega) was incubated with 200 µM dye compound in 200 µL solution (50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% NP40 substitute, 0.5 mM EDTA, 1 mM DTT, and 2% DMSO) for 30 min at room temperature, then incubated at 4°C for 18 h. The solution was irradiated with a 6x beam expanding 405 nm laser (365 mW) for 5 min. The solution was filtered twice through 2 mL 7K MWCO Zeba spin desalting columns (Thermo Scientific) pre-equilibrated with 25 mM HEPES pH 7.6, 100 mM KCl, 0.1 mM EDTA, 12.5 mM MgCl2, 1 mM DTT, and 10% glycerol. The supernatant was analyzed for labeled protein conjugate using SDS-PAGE protein gels.

[0601] 8.32Reference Example 32: Halo-protein specificity Wild type (WT) U2OS cells or U2OS cells ectopically expressing histone H2B-HaloTag fusion under the control of the CMV promoter (H2B) were seeded at 6000 cells / 50 µL / well in a glass bottom 384 well plate and incubated at 37°C and 5% CO2 overnight. The next day, cells were incubated with PA-JF549 or Examples 1-4 at a concentration range between 200 nM and 1 nM for 45 min. After incubation, cells were washed three times with PBS and finally the media was replaced with phenol-free media for imaging. Samples were imaged on a Nikon Ti2 microscope with a fiber-coupled illumination source. 561 nm light (approximately 500 mW at the coverslip) was used to stimulate emission of the fluorophores. Images were collected every 10 milliseconds for 5 seconds per field of view and dye molecules were photoactivated using an intensity increasing 405 nm light pulse (between 0 and 12 mW at the coverslip). Approximately 20 fields of view were collected per cell line / dye compound / concentration group.

[0602] All frames of each field of view were analyzed using a maximum likelihood estimation model to detect single-molecule fluorescence emission within the image with sub-pixel accuracy. For each dye compound, linear regression was used to determine the relationship between the concentration of the dye compound and the number of fluorescent spots detected on the microscope, where an ideal dye has no correlation between compound concentration and measured spot number in WT cells. The specificity factor was calculated as the slope of the compound in question relative to PA-JF549, and is shown in Table 3 below. 549 The ratio of the calculated slopes was calculated, and is shown in Table 3 below.

[0603] Table 3: Specificity factors

[0604] 8.33 Reference Example 33: Label specificity The label specificity of the compounds of Examples 1-4 was measured using the method described in Example 32 above and compared to PA-JF 549 . U2OS cells either wild type or expressing H2B-HaloTag fusion were incubated with PA-JF 549 or Example 4 for 45 min. Figure 3 An example of a field of view of U2OS expressing H2B-HaloTag fusion is shown, nuclei are labeled with Hoechst 33342 dye. The calculation method for label specificity is the number of spots detected in U2OS cells expressing H2B-HaloTag at a given dye concentration divided by the number of spots detected in U2OS wild type cells at the same concentration.

[0605] 8.34 Reference Example 34: Signal to noise ratio The SNR, which is a representation of the single molecule brightness, was calculated for Examples 1-4 (see Figure 4 ). The log-likelihood ratio test was used to calculate whether each spot in the SPT experiment described in Example 32 was a 2D Gaussian spot. See Sergé et al., “Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes,” Nat . Methods 2008, 5(8), 687-694 (Sergé et al., 2008). The SNR of each spot was reported as its peak log-likelihood ratio. This calculation used a 2D Gaussian PSF model with a fixed width, whose sigma / standard deviation was equal to 0.183 pm. Due to the stringency of our spot detection filter, only spots with SNR >= 14 were included in this calculation. Figure 4 The bar graphs in Figure 8 are the mean SNR across biological replicates, while the error bars are the standard error of the mean SNR across biological replicates.

[0606] 8.35 Example 35: Reduction of non-specific labeling Wild type (WT) U20S cells or U20S cells ectopically expressing an estrogen receptor-HaloTag (ER-Halo) fusion were seeded at 6000 cells / 50 pL / well in glass bottom 384 well plates and incubated overnight at 37 °C and 5% C02 and 95% humidity. The next day, cells were incubated with JF549, PA-JF549 or Examples 1-28 at a concentration of 100 nM (20 pM in the case of regular JF 549 PA-JF549 or Examples 1-28, and 100 nM Potomac Red. After incubation, cells were washed three times with PBS and finally the medium was replaced with phenol-free medium for imaging. Samples were imaged on a Nikon Ti2e microscope with a fiber-coupled illumination source using a 60x 1.27 NA objective and sCMOS camera. Emission of the fluorophores was stimulated using 561 nm light with an integrated intensity of approximately 500 mW at the coverslip. Images were collected every 10 milliseconds for 2 seconds per field of view. To test the sensitivity of each dye variant to 405 nm light, each well was imaged multiple times with increasing 405 nm light intensity (between 0 and 5 mW at the coverslip). Approximately 60 fields of view were collected for each cell line / dye compound set.

[0607] For the measurement of Reference Example 19, where the fluorophore is a Si-containing far-red emitting dye, 642 nm light (approximately 500 mW at the coverslip) was used to stimulate emission of the fluorophore instead of using a 561 nm light source, and cells were stained with 100 nM Potomac Yellow instead of Potomac Red.

[0608] After acquiring images for each compound, the images were individually inspected to qualitatively assess the performance of each test compound. It will be appreciated by one skilled in the art that modifications to the PA-JF 549 Modifications to the core structure not only change dye properties, but also change photoactivation under 405 nm light; therefore, the performance of each dye variant was assessed as follows, and these performances are not predictable from structural features alone. Compounds that are obviously unsuitable for single molecule imaging (such as those that cause intracellular dye aggregation) were flagged and removed from subsequent analysis. The remaining dyes that reliably produced spots composed of a single fluorophore and were therefore quantifiable using single molecule detection algorithms were further processed to generate individual tracks. All frames of each field of view were analyzed using a maximum likelihood estimation model in order to detect single molecule fluorescence emission within the image with sub-pixel accuracy. Subsequently, individual detections were linked into tracks across consecutive camera frames. Statistical data generated from detections (e.g. signal-to-noise ratio) and tracks (e.g. number of tracks) were used to compare dye variants.

[0609] The number of nuclear tracks per test compound in the ER-Halo cell line was determined at each 405 nm light activation level. To set a common baseline between different test compounds, the number of nuclear tracks was selected for each test compound at the light activation level where the most tracks were obtained. This level was chosen as the reference level for each test compound. The reference level is indicated in the tables below as "ref". 549 The best matching light activation condition was determined, and this condition was used for all subsequent comparisons.

[0610] Table 4a: Specificity factor (all values)

[0611] For the definitions of -; +; ++; +++ see Example 36.

[0612] Table 4b: Specificity factor (all values)

[0613] For the definitions of -; +; ++; +++ see Example 36.

[0614] 8.36 Example 36: Label specificity improvement The label specificity of the compounds of Examples 1-28 was measured using the method described in Tables 4a and 4b above and compared to PA-JF 549 or PA-JF 549 or Examples 1-28 was incubated for 1 h. Figure 5 Exemplary fields of U2OS expressing the ER-HaloTag fusion co-stained with Portmouth Red (CAS: 2127150-65-4; Grimm et al., 2017) are shown. The label specificity, as measured by the fold reduction of non-specific labeling by WT cells, was calculated by comparing the number of tracks measured in WT cells for Examples 1-28 to the number of tracks in WT cells for PA-JF 549 or PA-JF 549 as a ratio of the molecule.

[0615] The sensitivity of photoconversion under 405 nm illumination was qualitatively determined according to the amount of 405 nm light required to achieve a comparable number of tracks. Compounds that exhibited high levels of activation at 0.25 mW 405 nm laser input (the lowest intensity tested) received a “+++”, while compounds that were unable to reach high levels of activation at 5 mW 405 nm intensity received a “-”. For compounds that required more than 1 and 2.5 mW of 405 nm laser power at the target, “+” and “++” were assigned, respectively.

[0616] The SNR was calculated for Examples 1, 4, 7, and 10, which is a representation of the single molecule brightness (see Example 37). The SNR was calculated as the ratio of the mean intensity of the brightest 10% of the tracks to the standard deviation of the mean intensity of the brightest 10% of the tracks.Figure 6 ). Log-likelihood ratio test was used to calculate whether each point in the SPT experiment described in Example 34 was present or not. See Sergé et al., “Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes,” Nat . Methods 2008, 5(8), 687-694 (Sergé et al., 2008). The SNR of each point was reported as its peak log-likelihood ratio. This calculation used a 2D Gaussian PSF model with a fixed width, whose sigma / standard deviation was equal to 0.183 µm. Due to the stringency of the blob detection filter, only blobs with SNR ≥ 14 were included in this calculation. Figure 6 in Figure 8.36 are the average SNR of all detection calculations for the dye variant across biological replicates, while the error bars are the standard error of the mean SNR across biological replicates. The above examples show that the compounds disclosed herein show surprisingly improved labeling specificity when used to label proteins, while maintaining control over brightness and density.

[0617] 8.37 Example 37: Method C and Comparative Data Method C: Rhodamine (1 eq) was charged into a 20 mL oven-dried vial with a crimp cap with septum and dissolved in a 1:1 solution of dichloromethane and acetonitrile (2 mL / eq). To the resulting solution was added crushed 4 Å molecular sieves (8 eq) and CaO (3 eq) before sealing the vial. The mixture was purged with argon for 5 minutes before the addition of Ghosez reagent (4 eq), which caused the solution to darken. An aliquot was removed from the reaction and quenched with 2 M methylamine in THF to monitor the formation of the acyl chloride by LC-MS. After complete conversion to the acyl chloride (5 min), TMSCHN2 (8 eq, 2 M in diethyl ether) was added, followed immediately by KF (8 eq). After complete conversion to the diazoketone, the reaction mixture was directly concentrated onto celite and purified by silica gel column chromatography. Comparative data was generated using reference compounds and compounds with sample numbers 27 and 28.1, as follows:

[0618]

[0619] For reference compound JF 549, using methods B and C. Reference a, b, and c are sample reference compounds used to generate data comparisons. nd = not detected. Yields were calculated as isolated yield of title compound after purification. Product ratios were determined using peak integration in LC-MS equipped with a 254 nm detector. Ratios include the relative amount of desired product (1) and chlorinated byproducts (2 and 3) compared.

[0620] Traditional Arndt-Eistert conditions using oxalyl chloride, TEA, TMSCHN2, DCM (Method A), i.e. traditional diazoketone synthesis, resulted in azetidine ring opening to produce 3-chloropropylamine. Surprisingly, when using Ghosez reagent, CaO, 4A molecular sieves, TMSCHN2, and KF (Method C), these conditions provided the desired diazoketone without azetidine ring opening occurring on a set of substrates. Specifically, as shown in the table above, compound JF 549 , Reference a, Reference b, Reference c, Compound 27, and Compound 28.1, resulted in an azetidine product (1) that was substantially chemically pure with no detectable amount of other undesired 3-chloropropylamines (2 and 3) (product ratio of 100:nd:nd).

[0621] Method C described above can be used to synthesize compounds described herein.

[0622] 8.38 Example 38: Synthesis Investigation

[0623] The synthesis of acyl chloride (38-1e) and conversion to diazoketone (38-1b) was performed following the procedure of Example 37 (Method C). Table 5 shows the impact of differences between standard conditions (Method C) by screening using various reagents. Product distribution is expressed as the percentage observed by LC-MS analysis of the reaction mixture. Reactions using Method C gave consistent yields on a scale ranging from 0.1 mmol (50 mg) to 2.25 mmol (1.0 g) on the test substrate (38-1a).

[0624] Table 5:

[0625] 8.39 Example 39: Synthesis of Photoactivatable Rhodamines by Method C

[0626]

[0627] In this pathway, compound 39-10b is obtained by saponifying and coupling 2-((4-(aminomethyl)benzyl)oxy)pyrimidine-4-amine to 39-9b, rather than directly from the corresponding rhodamine lactone.

[0628] 8.40 Example 40: Label Specificity like Figure 7 As shown, the addition of dimethylamide (compounds 39-10c, also referred to herein as compound 4) at the 2-position of aziridine increases solubility and passive permeability across the cell membrane, resulting in a significant reduction in nonspecific labeling compared to the photoactivated JF549-HaloTag.

[0629] To evaluate the behavior of compound 39-10c, U2OS cells with endogenous knock-in of a HaloTag fusion with β-catenin were labeled. β-catenin is a protein with multiple cellular functions, including activation of the Wnt response gene pathway via transcriptional activation. β-catenin plays a role in diseases such as colorectal cancer. Cells were labeled with 50 nM compound 39-10c and then imaged under conditions where the location of individual molecules could be identified with subpixel precision and traced in live cells.

[0630] Results: α-diazotone was sensitive to 405 nm laser uncaging, and the number of fluorescent emitters increased with increasing laser intensity before the final depletion of the labeled protein aggregate. Figure 8 Following localization and tracing, the dynamic states present in the sample were characterized. At least four distinct dynamic states were identified, ranging from very slow diffusion (<0.1 µm). 2 From 0.5 µs to very fast diffusion (>5 µm) 2 / s), which is consistent with the multiple functional roles of β-catenin in cells ( Figure 9 (SG Pai et al., Journal of Hematology & Oncology (2017, 10, 101). Due to the photoactivatable nature of compound 39-10c, thousands of protein trajectories in each cell could be observed, and individual super-resolution images of β-catenin could be generated for each observed dynamic state. Different dynamic states were found to correspond to different spatial regions of the cell: the slowest populations were all associated with the cell membrane, but due to their slow diffusion, it could be inferred that spatially distinct subsets were involved in intercellular gap junctions (Fig. 10). In contrast, the fastest populations were primarily in the cytoplasm and nucleus, consistent with the role of β-catenin in activating Wnt response genes in the nucleus.

[0631] 8.41 Example 41: Preparation of Single-Molecular Tracer Samples U2OS cells (HBT-96, ATCC) or U2OS cells with endogenous HaloTag knock-in were seeded at 6000 cells per well on tissue culture treated 384-well glass bottom plates. The seeded cells were then incubated overnight at 37 °C and 5% CO2 to allow them to adhere. For all SMT experiments, cells were incubated with 50 nM of compound 38-1b or PA-JF 549 or 50 pMJF 549 (GA1110, Promega) in complete media for one hour. Cells were then washed three times in DPBS and twice in imaging media, which is fluoroBrite DMEM media (A1896701, Thermo Fisher) supplemented with GlutaMAX (35050079, Thermo Fisher) and the same serum and antibiotics as the growth media.

[0632] 8.42 Example 42: Single molecule tracking image acquisition Image acquisition using SMT was performed on a custom HILO microscope based on a Nikon Ti2, motorized stage, stage-top environmental chamber (OKO Labs), four-band filter cube (Chroma), custom laser launchers for 405 nm and 561 nm wavelengths described in McSwiggen et al. (“A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms,” (2023) eLife 12:RP93183), delivering 0 - 10 mW and >150 mW of power to the back focal plane of the objective, respectively. Fluorescence emission was passed through a high-speed filter wheel (Finger Lakes Instruments) and collected with a back-illuminated CMOS camera (Prime 95b, Teledyne). Images were acquired using a 60X 1.27 NA water immersion objective (Nikon). The environmental chamber was set to 37 degrees Celsius, 95% humidity, and 5% CO2. 5000 frame images were collected, and the 405 illumination intensity was increased stepwise to account for depletion of the unimaged molecule population over time.

[0633] 8.43 Example 43: Single molecule tracking image analysis Tracking is performed as described by McSwiggen et al. (“A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms,” (2023) eLife12:RP93183), but only briefly here. A single SMT image is processed in three consecutive steps using a combination of existing methods: detection, subpixel localization, and concatenation. Briefly, a generalized log-likelihood ratio detector is used to detect blobes. After detection, a 2D Gaussian blob model, fitted and integrated with Levenberg-Marquardt, refines the estimated location of each emitter to subpixel resolution, starting from an initial guess provided by a radially symmetric method. A custom modification of a hill-climbing algorithm is used to concatenate the detected points into trajectories. The result is a table of point coordinates and their diffusion coefficient estimates. To visualize the dynamic state as a function of its position in the image, blobs falling within a defined diffusion coefficient range are incorporated into an image reconstruction with the same pixel size as the original image.

[0634] To recover dynamic information from the trajectory, a state array (Heckert), a Bayesian inference method, is used, along with an RBME likelihood function and 125 values ​​ranging from 0.003 to 30 µm. 2 s -1 The diffusion coefficient and seven grids with positioning error amplitudes ranging from 0.02 to 0.08 µm were used. Through inference, the positioning error was marginalized, resulting in a one-dimensional distribution of the diffusion coefficient for each field of view.

[0635] 8.44 Example 44: Solubility and Permeability Data Table 6: Figure 7 Solubility and permeability data of the compounds characterized in the study were collected. The data indicate that compound 39-10c exhibits superior properties compared to the photoactivated JF549-HaloTag, and is more similar to JF549-HaloTag. Data were collected to demonstrate that 39-10c reduces non-specific labeling compared to the photoactivated JF549-HaloTag.

[0636] Table 6:

[0637] Numerous references have been cited, and the public content of each reference is incorporated into this paper in its entirety by reference for all purposes.

Claims

1. A compound of Formula (VIII): or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, (VIII) wherein: p is an integer from 1 to 3; R 1 is H or -C(=0)N(C 1-3 alkyl)2, Each L 1 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 is - (OCH2CH2) - ; and 2 is - (OCH2CH2) p - ; a is an integer of 1 or 2; b is an integer from 0 to 2; and wherein Z is or ; and 2. The compound of claim 1, wherein: when R 1 is H and a is 1, then L 1 is not where the wavy line represents the point of attachment to the rest of the compound. p is 1, a is 1, and b is an integer from 0 to 1.

3. The compound of claim 1 or claim 2, wherein: p is an integer from 1 to 3; R 1 is H, each L 1 is 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 is - (OCH2CH2) 2 is - (OCH2CH2) p -; a is an integer of 1 or 2; b is an integer from 0 to 2; and 4. The compound of claim 1 or claim 2, wherein: Z is . p is an integer from 1 to 3; R 1 is -C(=O)N(C 1-3 alkyl)2; Each L 1 It is –C(O)NH– each L is - (OCH2CH2) - ; and 2 is - (OCH2CH2) p - ; a is an integer of 1 or 2; b is an integer from 0 to 2; and 5. A compound of Formula (IX): or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, wherein Z is . –OCH2–, –CH2N(CH3)–, or –CH2O–. (IX) 7. A compound of Formula (X): or a salt, single stereoisomer, mixture of stereoisomers, or isotopic form thereof, L B is (i) -O-C 1-6 -alkylene-, -C 1-6 -alkylene-N(C 1-3 -alkyl)- or -C 1-6 -alkylene-O-.

6. The compound of claim 5, wherein L B is a linker comprising: wherein:

11. The compound of claim 5, having the structure: (X) 12. The compound of claim 5, having the structure:

13. The compound of claim 5, having the structure: R 1 is H or -C(=0)N(C 1-3 alkyl)2.

8. The compound of claim 7, wherein R 1 is H.​ 9. The compound of claim 7, wherein R 1 is -C(=0)N(C 1-3 alkyl)2.

10. The compound of claim 7, wherein R 1 is .

14. The compound of claim 7, having the structure: 。 15. A method of labeling a protein, comprising contacting a sample comprising a tagged protein with a compound of any one of claims 1-14, thereby producing a labeled protein. 。 16. The method of claim 15, wherein the tagged protein is a kinase, a transcription factor, a chromatin regulator, an adaptor, a transport protein, or a pathogenic aggregate. 。 17. The method of claim 15, wherein the tagged protein is a histone. 。 18. The method of claim 17, wherein the histone is H2B.

19. The method of any one of claims 15-18, wherein the labeled protein emits fluorescence upon exposure to light.

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

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

22. A method for preparing a compound of Formula (VIII-a): wherein: p is an integer from 1 to 3; a is an integer of 1 or 2; b is an integer from 0 to 2; (VIII) using a Ghosez reagent, in the presence of a diazomethane agent and a solvent, a compound of Formula (a-1) below: R' is , ; R 1 is H or -C(=0)N(C 1-3 alkyl)2, Each L 1 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 is - (OCH2CH2) -; and 2 is - (OCH2CH2) p -; wherein: and wherein to thereby prepare a compound of Formula (VIII-a). Z is or ; and wherein when R 1 is H and a is 1, then L 1 is not wherein the wavy line represents the point of attachment to the remainder of the compound, the method comprising:

23. A method for preparing a compound of Formula (XI): wherein: (a-1) p is an integer from 1 to 3; R’= 、 ； R 1 is H or -C(=0)N(C 1-3 alkyl)2, R is -C(=0)OCH3or -C(=0)NH(C 1-3 alkylene)-(OCH2CH2)-Z; a is an integer of 1 or 2; Z is , b is an integer from 0 to 2; using a Ghosez reagent, in the presence of a diazomethane agent and a solvent, a compound of Formula (a-2) below: wherein: R 1 is H or -C(=0)N(C 1-3 alkyl)2, Each L 1 yes: –C(O)NH(C 1-3 -alkylene)–; each L is - (OCH2CH2) - ; and 2 is - (OCH2CH2) p - ; to thereby prepare a compound of Formula (XI).

24. The method of claim 22 or 23, wherein the diazomethane agent is trimethylsilyldiazomethane ethyl ether. ​ Z is ; the method comprising: ​ (a-2) ​ R 1 is H or -C(=0)N(C 1-3 alkyl)2, R is -C(=0)OCH3or -C(=0)NH(C 1-3 alkylene)-(OCH2CH2)-Z; and wherein Z is , ​ ​ 25. The method of claim 22 or 23, wherein the solvent comprises dichloromethane.

26. The method of any one of claims 22-25, further comprising the use of a drying agent and a metal oxide.

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

28. A compound of formula (VIII-a) prepared by the method of claim 22.

29. A compound of formula (XI) prepared by the method of claim 23.

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

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

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

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

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