A covalent fluorescent probe for cannabinoid receptor 2

Abstract

The present invention relates to a compound represented by general formula (I) TIFF2025532470000400.tif50161 (Wherein X, n, p and R 1 ~R 4 are as described herein) The present invention provides a covalent fluorescent probe for cannabinoid receptor 2 ("CB2") having the formula:

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to organic compounds useful as affinity-driven covalent probes for cannabinoid receptor 2 (CB2R), achieved through a novel convergent synthetic blueprint from a central reactive motif. [Background technology]

[0002] Background of the Invention Dysregulation of the endocannabinoid system, particularly the signaling pathways mediated by CB2R, has been implicated in a variety of diseases, including tissue injury, neurodegenerative conditions, and inflammation. Despite the clear importance of CB2R, no compounds have been introduced into the clinic. This is primarily due to a poor understanding of CB2R biology, particularly its expression and localization in healthy and disease states. Fluorescent imaging probes have recently emerged as high-resolution tools for investigating the localization, expression levels, and distribution of CB2R in live cells and in vivo (R.C. Sarott, et al., J. Am. Chem. Soc., 2020, 142, 16953-16964; T. Gazzi, et al., Chem. Sci. 2022, 13, 5539-5545). However, these reported probes are standard affinity fluorescent probes that bind the orthosteric site of CB2R, and their potent agonist ligands trigger the relevant signaling pathways, ultimately resulting in desensitization via receptor internalization. Even in the case of antagonist or inverse agonist CB2R probes (S. Singh, et al., ACS Med. Chem. Lett. 2019, 10, 209-214), which do not inherently trigger signaling, the probe alters cellular homeostasis by constantly occupying the orthosteric site of CB2R and thus directly altering cellular tone by preventing endogenous ligand binding. Therefore, there are currently no available probes for studying CB2R without simultaneously altering receptor signaling pathways, localization, expression, trafficking, and / or distribution. Summary of the Invention

[0003] Summary of the Invention The limitations outlined above are addressed by the present invention, which reports a CB2R affinity-driven probe with a cleavable motif that allows the covalent transfer of a reporter unit to study target proteins, accompanied by the simultaneous release of a targeting ligand (T. Tamura, I. Hamachi, J. Am. Chem. Soc. 2019, 141, 2782-2799). Such probes can be applied to cellular studies using flow cytometry fluorescence-activated cell sorting (FACS) experiments or confocal live-cell imaging, for example, to quantify the transport of low-abundance targets. Furthermore, such probes offer the possibility of generating equilibrium and kinetic binding data in a high-throughput manner without handling radioactive materials, for example, using time-resolved Förster resonance energy transfer (TR-FRET). Furthermore, these probes are particularly well suited for constructing FRET biosensors on and within live cells to study ligand-endogenous protein binding interactions in real time (K. Matsuo, et al., Angew. Chem. Int. Ed. 2018, 57, 659-662). Due to the covalent nature of the reporter transfer, such probes are ideally suited for targeting low-expressing targets, such as CB2R, in the healthy brain. These probes can also assist in the translation of preclinical pharmacological animal data to the clinic. In a first aspect, the present invention provides a compound of formula (I) [ka] or a pharmaceutically acceptable salt thereof, wherein X, n, p and R 1 ~R 4 is as defined herein.

[0004] In a further aspect, the present invention provides methods for producing fluorescent and bioorthogonal probes for proteins of interest comprising compounds of formula (I) described herein.

[0005] In a further aspect, the present invention provides a method that allows for the orthogonal formation of amides in the presence of labile, highly electrophilic functional groups.

[0006] In a further aspect, the present invention provides certain synthetic intermediates useful in the methods according to the invention.

[0007] In a further aspect, the present invention provides the use of a compound of formula (I) as described herein as a fluorescent probe or probe for secondary bioorthogonal conjugation for cannabinoid receptor 2 (CB2R). DETAILED DESCRIPTION OF THE INVENTION

[0008] Detailed Description of the Invention definition It should be understood that any feature, integer, characteristic, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, except where inconsistent therewith. All features disclosed herein (including any accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel one or any novel combination of features disclosed herein (including any accompanying claims, abstract, and drawings), or any novel one or any novel combination of steps of any method or process so disclosed.

[0009] The term "alkyl" refers to a monovalent or polyvalent, e.g., monovalent or divalent, straight-chain or branched saturated hydrocarbon group of 1 to 12 carbon atoms. In some preferred embodiments, the alkyl group contains 1 to 6 carbon atoms, e.g., 1, 2, 3, 4, 5, or 6 carbon atoms ("C1-C6-alkyl"). In other embodiments, the alkyl group contains 1 to 3 carbon atoms, e.g., 1, 2, or 3 carbon atoms. Some non-limiting examples of alkyl include methyl, ethyl, propyl, 2-propyl (isopropyl), n-butyl, iso-butyl, sec-butyl, tert-butyl, and 2,2-dimethylpropyl. A particularly preferred, but non-limiting, example of alkyl is methyl.

[0010] The term "cycloalkyl" as used herein refers to a saturated or partially unsaturated, monocyclic hydrocarbon group of 3 to 10 ring carbon atoms ("C 3~10 In some preferred embodiments, the cycloalkyl group is a saturated monocyclic hydrocarbon group of 3 to 8 ring carbon atoms. Preferably, the cycloalkyl group is a saturated monocyclic hydrocarbon group of 3 to 6 ring carbon atoms, particularly 3 to 5 ring carbon atoms, e.g., 3, 4, or 5 carbon atoms. Some non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. A preferred, but non-limiting, example of cycloalkyl is cyclopropyl.

[0011] The term "halogen" or "halo" refers to fluoro (F), chloro (Cl), bromo (Br), or iodo (I). Preferably, the term "halogen" or "halo" refers to fluoro (F), chloro (Cl), or bromo (Br). Particularly preferred, but non-limiting, examples of "halogen" or "halo" are fluoro (F) and chloro (Cl).

[0012] The term "cyano" refers to a -CN (nitrile) group.

[0013] The term "azido" refers to the group -N3.

[0014] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, without being biologically or otherwise undesirable. Salts are formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., especially hydrochloric acid, as well as organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, etc. Furthermore, these salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts, etc. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resins, etc. Particular pharmaceutically acceptable salts of compounds of formula (I) are the hydrochloride and trifluoroacetate salts.

[0015] The compounds of formula (I) may contain several asymmetric centers and may exist in the form of optically pure enantiomers, mixtures of enantiomers, e.g. racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereomeric racemates or mixtures of diastereomeric racemates.

[0016] According to the Cahn-Ingold-Prelog rules, the asymmetric carbon atom may be of the "R" or "S" configuration.

[0017] The abbreviation "CB2" refers to cannabinoid receptor 2.

[0018] Compounds of the Invention In a first aspect (A1), the present invention provides a compound of formula (I) [ka] or a pharmaceutically acceptable salt thereof, wherein: (i)R 1 teeth, [ka] ;or [ka] and X is selected from O, S and SO, or (ii)R 1 teeth, [ka] ;or [ka] and X is selected from O, S, SO and NHCO; R 2 and R 3 is selected from hydrogen and C1-C4-alkyl, or R 2 and R 3 together with the carbon atoms to which they are attached form a C3-C5-cycloalkyl, R 4 is selected from hydrogen, halogen, cyano and azide; R 5 teeth, [ka] ; [ka] ; [ka] ; and [ka] is selected from R 6 teeth, [ka] ; and [ka] is selected from R 7 teeth, [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; [ka] ;

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[0019] In one embodiment, the present invention provides (i)R 1 but, [ka] ;or [ka] and X is selected from O, S and SO, or (ii)R 1 but, [ka] ;or [ka] and X is NHCO; R 5 but, [ka] ; [ka] ; [ka] ; and [ka] is selected from R 6 but, [ka] ; [ka] is selected from R 7 but, [ka] ; and [ka] is selected from R 8 but, [ka] [ka] ; and [ka] is selected from p is selected from 3, 4 and 5; q is selected from 2, 3 and 5; r is selected from 1 and 2; s is selected from 1 and 4; u is selected from 0, 4 and 5; W is carbonyl or absent; Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Y is selected from Se and O.

[0020] In a preferred embodiment, the present invention comprises: (i)R 1 but, [ka] ;or [ka] and X is O, or (ii)R 1 but, [ka] ;or [ka] and X is NHCO; R 5が , [ka] ; [ka] ; [ka] ; and [ka] is selected from R 6 but, [ka] ; [ka] is selected from R 7 but, [ka] ; and [ka] is selected from R 8 but, [ka] [ka] ; and [ka] is selected from p is 3, q is selected from 2, 3 and 5; r is 1, s is 1, Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein u is 5.

[0021] In a particularly preferred embodiment, the present invention comprises: R 1 but, [ka] ;or [ka] and R 5 but, [ka] or [ka] and R 6 but, [ka] and R 7 but, [ka] and R 8 but, [ka] and X is NHCO; p is 3, q is 2 or 3; r is 1, Provided herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein u is 5.

[0022] In one embodiment, the present invention provides R 1 but, [ka] and Provided are compounds of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein X is selected from O, S and SO.

[0023] In one embodiment, the present invention provides R 1 but, [ka] and Provided are compounds of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein X is selected from O, S and SO.

[0024] In one embodiment, the present invention provides R 1 but, [ka] and X is NHCO; R 5 , R 6 and q is as defined herein, or a pharmaceutically acceptable salt thereof.

[0025] In one embodiment, the present invention provides R 1 but, [ka] and X is NHCO; R 8 and r is as defined herein, or a pharmaceutically acceptable salt thereof.

[0026] In a preferred embodiment, the present invention comprises: R 1 but, [ka] ;or [ka] and X is NHCO; R 5 , R 6 , R 8 The present invention provides compounds of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein q, q, and r are as defined herein.

[0027] In one embodiment, the present invention provides R 5 but, [ka] ; [ka] ; [ka] ; and [ka] (R 7 , W and u are as defined herein. or a pharmaceutically acceptable salt thereof.

[0028] In a preferred embodiment, the present invention comprises: R 5 but, [ka] or [ka] (R 7 and u is as defined herein. or a pharmaceutically acceptable salt thereof.

[0029] In one embodiment, the present invention provides R 6 but, [ka] or a pharmaceutically acceptable salt thereof.

[0030] In one embodiment, the present invention provides R 6 but, [ka] or a pharmaceutically acceptable salt thereof.

[0031] In one embodiment, the present invention provides R 7 but, [ka] ; and [ka] or a pharmaceutically acceptable salt thereof.

[0032] In a particularly preferred embodiment, the present invention comprises: R 7 but, [ka] or a pharmaceutically acceptable salt thereof.

[0033] In a preferred embodiment, the present invention comprises: R 7 but, [ka] or a pharmaceutically acceptable salt thereof.

[0034] In one embodiment, the present invention provides R 8 but, [ka] [ka] ; and [ka] wherein s is as defined herein. or a pharmaceutically acceptable salt thereof.

[0035] In a preferred embodiment, the present invention comprises: R 8 but, [ka] or a pharmaceutically acceptable salt thereof.

[0036] In a preferred embodiment, the present invention comprises: R 8 but, [ka] wherein s is as defined herein. or a pharmaceutically acceptable salt thereof.

[0037] In a particularly preferred embodiment, the present invention comprises: R 8 but, [ka] or a pharmaceutically acceptable salt thereof.

[0038] In one embodiment, the present invention provides Provided herein is a compound of formula (I) as described herein, wherein p is selected from 1, 3, 4 and 5, or a pharmaceutically acceptable salt thereof.

[0039] In a preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein p is 1.

[0040] In a particularly preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein p is 3.

[0041] In a preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein p is 4.

[0042] In a preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein p is 5.

[0043] In one embodiment, the present invention provides Provided herein is a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein q is selected from 2, 3 and 5.

[0044] In a preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein q is 2 or 3.

[0045] In one embodiment, the present invention provides Provided herein is a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein r is selected from 1 and 2.

[0046] In a preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein r is 1.

[0047] In one embodiment, the present invention provides Provided herein is a compound of formula (I) as described herein, wherein s is selected from 1 and 4, or a pharmaceutically acceptable salt thereof.

[0048] In one embodiment, the present invention provides Provided herein is a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein u is selected from 0, 4 and 5.

[0049] In a preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein u is 5.

[0050] In one embodiment, the present invention provides Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Y is selected from Se and O.

[0051] In a preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein s is 1.

[0052] In a preferred embodiment, the present invention comprises: Provided herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein u is 5.

[0053] In one embodiment, the present invention provides R 2 and R 3 is selected from hydrogen and C1-C4-alkyl, R 4 is hydrogen or azide, Provided herein is a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein n is 6.

[0054] In one embodiment, the present invention provides R 2 and R 3 are all C1-C4 alkyl, R 4 is an azide, Provided herein is a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein n is 6.

[0055] In one embodiment, the present invention provides R 2 and R 3 However, all of them are methyl. R 4 is an azide, Provided herein is a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein n is 6.

[0056] In one embodiment, the present invention provides R 2 and R 3 is selected from hydrogen and C1-C4-alkyl, or a pharmaceutically acceptable salt thereof.

[0057] In a preferred embodiment, the present invention comprises: R 2 and R3 are both C1-C4-alkyl, or a pharmaceutically acceptable salt thereof.

[0058] In one embodiment, the present invention provides R 2 and R 3 and R are each hydrogen, or a pharmaceutically acceptable salt thereof.

[0059] In a particularly preferred embodiment, the present invention comprises: R 2 and R 3 and R are each methyl, or a pharmaceutically acceptable salt thereof.

[0060] In one embodiment, the present invention provides R 4 is hydrogen or azido, or a pharmaceutically acceptable salt thereof.

[0061] In one embodiment, the present invention provides R 4 is hydrogen, or a pharmaceutically acceptable salt thereof.

[0062] In a preferred embodiment, the present invention comprises: R 4 is azide, or a pharmaceutically acceptable salt thereof.

[0063] In one embodiment, the present invention provides Provided herein is a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein n is 6.

[0064] In one embodiment, the present invention provides (i)R 1 but, [ka] ;or [ka] and X is selected from O, S and SO, or (ii)R 1 but, [ka] ;or [ka] and X is NHCO; R 2 and R 3 is selected from hydrogen and C1-C4-alkyl, R 4 is hydrogen or azide, R 5 but, [ka] ; [ka] ; [ka] ; and [ka] is selected from R 6 but, [ka] ; [ka] is selected from R 7 but, [ka] ; and [ka] is selected from R 8 but, [ka] ; [ka] and [ka] is selected from n is 6, p is selected from 1, 3, 4 and 5; q is selected from 2, 3 and 5; r is selected from 1 and 2; s is selected from 1 and 4; u is selected from 0, 4 and 5; W is carbonyl or absent; Provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Y is selected from Se and O.

[0065] In a preferred embodiment, the present invention comprises: (i)R 1 but, [ka] ;or [ka] and X is O, or (ii)R 1 but, [ka] ;or [ka] and X is NHCO; R 2 and R 3 are all C1-C4 alkyl, R 4 is an azide, R 5 but, [ka] ; [ka] ; [ka] ; and [ka] is selected from R 6 but, [ka] ; [ka] is selected from R 7 but, [ka] ; and [ka] is selected from R 8 but, [ka] ; [ka] and [ka] is selected from n is 6, p is 3, q is selected from 2, 3 and 5; r is 1, s is 1, Provided herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein u is 5.

[0066] In a particularly preferred embodiment, the present invention comprises: R 1 but, [ka] ;or [ka] and R 2 and R 3 However, all of them are methyl. R 4is an azide, R 5 but, [ka] or [ka] and R 6 but, [ka] and R 7 but, [ka] and R 8 but, [ka] and X is NHCO; n is 6, p is 3, q is 2 or 3; r is 1, Provided herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein u is 5.

[0067] In one embodiment, the present invention provides a compound of formula (I): N-(9-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-9-oxononyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(5-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-5-oxopentyl)-1-((3-((2-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(14-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; 1-(6-((2-(2-(4-((3-((5-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-((E)-2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide; 1-(6-((2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate; 3,7-Di(azetidin-1-yl)-N-(2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfinyl)octanamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]siline-10,1'-isobenzofuran]-6'-yl)oxy)heptanamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(16-oxo-20-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12-tetraoxa-15-azaicosyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide; and N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-6-oxo-1-((3-(((1-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamide or a pharmaceutically acceptable salt thereof.

[0068] In one embodiment, the present invention provides a compound of formula (I): N-(9-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-9-oxononyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(5-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-5-oxopentyl)-1-((3-((2-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(14-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; 1-(6-((2-(2-(4-((3-((5-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-((E)-2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide; 1-(6-((2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate; 3,7-Di(azetidin-1-yl)-N-(2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfinyl)octanamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamide; and N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-yl)oxy)heptanamide or a pharmaceutically acceptable salt thereof.

[0069] In a preferred embodiment, the present invention provides a compound of formula (I) 1-(6-((2-(2-(4-((3-((5-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-((E)-2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanamide; 1-(6-((2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate; 3,7-Di(azetidin-1-yl)-N-(2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamide; and N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-yl)oxy)heptanamide or a pharmaceutically acceptable salt thereof.

[0070] In a particularly preferred embodiment, the present invention relates to a compound of formula (I) comprising 1-(6-((2-(2-(4-((3-((5-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-((E)-2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate or a pharmaceutically acceptable salt thereof.

[0071] In a particularly preferred embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide.

[0072] In a particularly preferred embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide.

[0073] In a particularly preferred embodiment, the present invention relates to a compound of formula (I) comprising N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl)amino) Provided is a compound of formula (I) as described herein which is ethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide, or a pharmaceutically acceptable salt thereof.

[0074] In a particularly preferred embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanamide.

[0075] In a particularly preferred embodiment, the present invention relates to a compound of formula (I) comprising 1-(6-((2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyano Provided is a compound of formula (I) as described herein which is (methyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate, or a pharmaceutically acceptable salt thereof.

[0076] In a particularly preferred embodiment, the present invention relates to a compound of formula (I) comprising 3,7-di(azetidin-1-yl)-N-(2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamate. bamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide, or a pharmaceutically acceptable salt thereof.

[0077] In a particularly preferred embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamide.

[0078] In a particularly preferred embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamide.

[0079] In a particularly preferred embodiment, the present invention provides a compound of formula (I) as described herein, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) is N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]siline-10,1'-isobenzofuran]-6'-yl)oxy)heptanamide.

[0080] In certain embodiments, the present invention provides pharmaceutically acceptable salts of compounds according to formula (I) described herein. In more particular embodiments, the present invention provides compounds according to formula (I) described herein as the free base.

[0081] Manufacturing method The preparation of the compounds of formula (I) of the present invention can be carried out by sequential or convergent synthetic routes. The synthesis of the present invention is shown in the following general scheme. The skills required to carry out the reactions and purification of the resulting products are known to those skilled in the art. The substituents and indices used in the following method descriptions have the meanings indicated herein unless otherwise indicated.

[0082] If one of the starting materials, intermediates or compounds of formula (I) contains one or more functional groups that are not stable or reactive under the reaction conditions of one or more reaction steps, suitable protecting groups (e.g., as described in "Protective Groups in Organic Chemistry", T.W. Greene and P.G.M. Hutts, 5th Edition, 2014, John Wiley & Sons, NY) can be introduced prior to a critical step, applying methods well known in the art. Such protecting groups can be removed at a later stage of the synthesis using standard methods described in the literature.

[0083] When the starting material or intermediate contains a stereocenter, the compound of formula (I) may be obtained as a mixture of diastereomers or enantiomers, which can be separated by methods known in the art, such as chiral HPLC, chiral SFC, or chiral crystallization. Racemates can be separated into their antipodes, for example, via diastereomeric salts by crystallization with optically pure acids, or by separating the antipodes by specific chromatographic methods using either chiral adsorbents or chiral eluents. Starting materials and intermediates containing a stereocenter can also be separated to obtain diastereomerically / enantiomerically enriched starting materials and intermediates. The use of such diastereomerically / enantiomerically enriched starting materials and intermediates in the synthesis of the compound of formula (I) typically results in the respective diastereomerically / enantiomerically enriched compounds of formula (I).

[0084] Those skilled in the art will recognize that in the synthesis of compounds of formula (I), an "orthogonal protecting group strategy" may be applied (unless otherwise desired), allowing for the cleavage of several protecting groups one at a time, without affecting other protecting groups in the molecule. The principle of orthogonal protection is well known in the art and has been described in the literature (e.g., Barany and R.B. Merrifield, J. Am. Chem. Soc. 1977, 99, 7363; H. Waldmann et al., Angew. Chem. Int. Ed. Engl. 1996, 35, 2056).

[0085] Those skilled in the art will recognize that the reaction sequence may vary depending on the reactivity and nature of the intermediates.

[0086] More specifically, compounds of formula (I) can be prepared by the methods set forth below, in the Examples, or by analogous methods. Suitable reaction conditions for the individual reaction steps are known to those skilled in the art. For literature-described reaction conditions affecting the described reactions, see, for example, "Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 2nd Edition, Richard C. Larock, John Wiley & Sons, New York, NY, 1999." We have found it convenient to carry out the reactions in the presence or absence of a solvent. There are no particular limitations on the nature of the solvent used, so long as it does not adversely affect the reaction or the reagents involved and is capable of dissolving the reagents to at least some extent. The described reactions can occur over a wide range of temperatures, and the precise reaction temperature is not critical to the present invention. It is convenient to carry out the described reactions at temperatures ranging from -78°C to reflux. The reaction time required for the reaction can also vary widely, depending on many factors, particularly the reaction temperature and the nature of the reagents. However, a period of 0.5 hours to several days is usually sufficient to obtain the intermediates and compounds described. The reaction sequence is not limited to the sequence shown in the scheme, but the order of the reaction steps can be freely changed depending on the starting materials and their respective reactivities.

[0087] In cases where the starting materials or intermediates are not commercially available or their synthesis is not described in the literature, they can be prepared analogously to existing procedures for similar analogs or as outlined in the experimental section.

[0088] The following abbreviations are used herein:

[0089] AcOH = acetic acid, ACN = acetonitrile, Boc = tert-butyloxycarbonyl, CAS RN = Chemical Abstracts Registry Number, CH2Cl2 = dichloromethane, COMU = (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate, [Cu(MeCN)4]PF6 = tetrakis(acetonitrile)copper(I) hexafluorophosphate, DCC = N,N'-dicyclohexylcarbodiimide, DIC = N,N'-diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide, DMAP = 4-dimethylaminopyridine, DMA = N,N-dimethylacetamide, DMF = N,N-dimethylformamide, DMSO = dimethyl sulfoxide, i-Pr2NEt = N,N-diisopropylethylamine, EDCI = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, EDCI·HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, ESI = electrospray ionization, Et3N = triethylamine, Et2O = diethyl ether, EtOAc = ethyl acetate, EtOH = ethanol, h = hour, GABA = γ-aminobutyric acid, H2O = water, HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate, HCl = hydrochloric acid, HOAt = 1-hydroxy-7-azabenzotriazole, HOBt = 1-hydroxy-1H-benzotriazole; HPLC = high-performance liquid chromatography, KO t Bu = potassium tert-butoxide, K2CO3 = potassium carbonate, MeOH = methanol, MgSO4 = magnesium sulfate, min = minute, mL = milliliter, MS = mass spectrum, NaH = sodium hydride, NaHCO3 = sodium bicarbonate, NaOH = sodium hydroxide, Na2SO4 = sodium sulfate, NEt3 = triethylamine (TEA), NH4Cl = ammonium chloride, O tBu = tert-butoxy, PG = protecting group, PyAOP = (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, PyBOP = (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, R = any group, rt = room temperature, SiO = silicon dioxide, T3P = propylphosphonic anhydride, TBTU = O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, TFA = trifluoroacetic acid, THF = tetrahydrofuran, TLC = thin layer chromatography.

[0090] R 4 , R 7 Compounds of formula I, where p, r, s, t, W are as described herein, can be synthesized analogously to the literature and / or as shown, for example, in Scheme 1. [ka] Scheme 1.

[0091] Thus, reaction of ω-aminoester linker type A with 6-hydroxynicotinic acid gives B (step a). This type of amide coupling can be achieved by using one of the well-known coupling reagents, such as DCC, HATU, EDCI, TBTU, T3P, and a base, such as i-Pr2NEt, Et3N, or DMAP, in a suitable solvent, such as DMF, DMA, CHCl2, or dioxane, preferably at 0 °C to room temperature. This is followed by the addition of a base, preferably KO, in a suitable solvent, such as THF, CHCl2, or dioxane. tReaction of B with an activated sulfonate, such as, but not limited to, a sulfonyl halide, in the presence of Bu, i-Pr2NEt, TEA, DMAP, among others, affords C (step b). Those skilled in the art will recognize that the order of addition of reagents and the choice of base and solvent can be important in this type of reaction due to the reactivity and stability of the two tautomers of the deprotonated 2-pyridone anion, which leads to the formation of N- or O-sulfonylpyridone structural isomers. Compounds of type C can be directly linked to ligands used in the covalent transfer of universal cargoes, such as alkynes, for further functionalization by secondary conjugation with, for example, azides in the presence of a metal catalyst. Alternatively, Cu I N-pyridone motif C can be coupled to functionalized N-pyridone scaffolds D (step c) via a [3 + 2]-azide-alkyne cycloaddition, optionally catalyzed by a metal catalyst such as . The use of protic, non-nucleophilic solvent mixtures (CHCl, AcOH), which allow the reaction to proceed in higher yields at ambient temperature, is preferred, but not essential, as for example, MeOH, CHCl at 40 °C also works well. The functionality is preferentially a fluorescent label, but can also be any cargo for secondary reactions in vitro or in vivo, preferentially bioorthogonal, such as strained alkenes, strained alkynes, tetrazenes, alkynes, azides, diazirines, aldehydes, boronic acids, etc. Compound type D is subsequently deprotected with an acid such as HCl or TFA in various solvents such as CHCl, EtO, or dioxane, and the acid is coupled to the desired target protein ligand in one step without further purification (step d) to give E. The ligand bears an amino function that is coupled with one of the well-known coupling reagents (DCC, EDCI, TBTU, T3P), preferentially HATU, PyAOP, or, generally, an activating reagent that generates an HOAt ester from a carboxylic acid. The reaction is carried out in a suitable solvent such as DMF, DMA, CHCl, or dioxane and an acid / base buffer system, e.g., i-PrNEt, TFA, preferably at 0°C to room temperature.

[0092] A crucial part of this invention is the synthesis of KO in THF. t It was discovered that using Bu, N-sulfonylpyridone C can be formed substantially exclusively rather than the O-pyridone structural isomer.

[0093] Another crucial part of this invention is the discovery and use of an acid / base buffer system, such as TFA / i-Pr2NEt, in the amide bond formation step in the presence of an N-sulfonylpyridone. Preferentially, the HATU-mediated reaction is applied with a TFA / i-Pr2NEt buffer system (step d). The HOAt active ester promotes rapid amide bond formation, but if left unbuffered, free HOAt reacts with the electrophilic N-sulfonylpyridone and mediates its decomposition. Selecting an appropriate acid / base buffer system prevents this critical side reaction, allowing final amide bond formation to proceed in the presence of highly electrophilic motifs. Those skilled in the art will recognize that similar reactivity is expected for other coupling reagents, such as TBTU, PyAOP, PyBOP, and COMU, in which the post-bond activation agent is sufficiently acidic and nucleophilic to cleave the electrophile.

[0094] Those skilled in the art will recognize that the order of functionalization of C is interchangeable and depends on the desired application and the availability of any functionalizing components.

[0095] R 4 , R 5 Compounds of formula I, where p, q are as described herein, can be synthesized analogously to the literature and / or as shown, for example, in Scheme 2. [ka] Scheme 2.

[0096] Thus, ω-aminoester linker type A is reacted with an activated 4-sulfobenzoic acid, such as an acyl halide or sulfonyl form, followed by the addition of amine B to give compounds of formula C (step a). Subsequently, N-alkylsulfonamide C is reacted with a functionalized acid to give N-acyl-N-alkylsulfonamide D (step b). This type of amide coupling can be achieved by using one of the well-known coupling reagents, such as DCC, HATU, EDCI, TBTU, or T3P, and a base, such as i-Pr2NEt, Et3N, or DMAP, in a suitable solvent, such as DMF, DMA, CHCl2, or dioxane, preferably at 0 °C to room temperature. Compound type D is then deprotected with an acid, such as HCl or TFA, in a variety of solvents, such as CHCl2, Et2O, or dioxane, and the acid is coupled to the desired protein ligand of interest in one step to give E (step c) without further purification required. The ligands have an amino function that is coupled with one of the well-known coupling reagents (DCC, EDCI, TBTU, T3P), preferentially HATU, PyAOP, or generally with an activating reagent that generates the HOAt ester from the carboxylic acid. The reaction is carried out in a suitable solvent such as DMF, DMA, CHCl or dioxane and an acid / base buffer system, e.g., i-PrNEt, TFA, preferably at 0°C to room temperature.

[0097] R 4 , R 5 Compounds of formula I, where p, q are as described herein, can be synthesized analogously to the literature and / or as shown, for example, in Scheme 3. [ka] Scheme 3.

[0098] Thus, reaction of ω-aminoester linker type A with 4-sulfamoylbenzoic acid affords B (Step a). This type of amide coupling can be achieved by using one of the well-known coupling reagents, such as DCC, HATU, EDCI, TBTU, or T3P, catalyzed preferably by HOBt, and a base, such as i-Pr2NEt, Et3N, or DMAP, in a suitable solvent, such as DMF, DMA, CHCl2, or dioxane, preferably at 0°C to RT. Subsequently, N-acylation of the sulfonamide of B with a carboxylic acid and one of the well-known coupling reagents, such as DCC, HATU, EDCI, TBTU, or T3P, and a base, such as i-Pr2NEt or Et3N, catalyzed preferably by DMAP, in a suitable solvent, such as DMF, DMA, CHCl2, or dioxane, preferably at 0°C to RT, affords C1 and C2 (Step b).

[0099] Following the route to Example 10, N-acylsulfonamide C1 is alkylated with iodoacetonitrile with the aid of a suitable base to give compound type D (step c). The tert-butyl ester of compound type D is subsequently deprotected with an acid such as HCl or TFA in a variety of solvents, such as CHCl, EtO, or dioxane, and the acid is coupled to the desired target protein ligand E in one step without further purification (step d). The ligand bears an amino function that is coupled with one of the well-known coupling reagents (DCC, EDCI, TBTU, T3P), preferentially HATU, PyAOP, or, in general, an activating reagent that generates a HOAt ester from a carboxylic acid. The reaction is carried out in a suitable solvent, such as DMF, DMA, CHCl, or dioxane, and an acid / base buffer system, e.g., i-PrNEt, TFA, preferably at temperatures between 0°C and room temperature.

[0100] Following the route towards Example 15, deprotection of C2 with an acid such as HCl or TFA and reaction with an activated carboxylic acid derivative such as an N-hydroxysuccinimide ester or acid anhydride affords N-acylsulfonamide F (step e). Compound type F is then coupled to the desired target protein ligand. The ligand bears a complementary functional group, such as an amine or alcohol, which is coupled with one of the well-known coupling reagents, such as DCC, HATU, EDCI, TBTU, or T3P, and a base, such as i-Pr2NEt, Et3N, or DMAP, in a suitable solvent, such as DMF, DMA, CHCl, or dioxane, preferably at 0 °C to room temperature. Finally, alkylation of the intermediate N-acylsulfonamide with the desired alkyl halide affords probe G (step f).

[0101] In step g of the synthesis for Examples 11-14, B is acylated with 4-pentynoic acid to give H under conditions similar to those in step b. In step h, the N-acylsulfonamide in H is alkylated under conditions similar to those in step c to give the activated N-acyl-N-alkylsulfonamide platform reagent I.

[0102] Following the route towards Example 14, I is deprotected with an acid such as HCl or TFA in a variety of solvents such as CHCl, EtO, dioxane, etc., and the acid is coupled to the desired protein ligand of interest in one step (step i) without further necessary purification.

[0103] In the synthesis of Examples 11-13, the alkyne of I is functionalized with an azide-bearing cargo, such as a fluorophore, in a manner similar to step c of Scheme 1 to give K (step j), which is then deprotected and coupled with the desired ligand under conditions similar to step d of Scheme 1 to give the final compound of type L (step k).

[0104] A crucial part of this invention is the discovery and use of an acid / base buffer system, such as TFA / i-Pr2NEt, in the amide bond formation step in the presence of N-acyl-N-alkylsulfonamides. Preferentially, HATU-mediated reactions are applied with a TFA / i-Pr2NEt buffer system. While the HOAt activated ester promotes rapid amide bond formation, if left unbuffered, free HOAt reacts with the electrophilic N-acyl-N-alkylsulfonamide and mediates its decomposition. Selecting an appropriate acid / base buffer system prevents this critical side reaction, allowing final amide bond formation to proceed in the presence of highly electrophilic motifs. Those skilled in the art will recognize that similar reactivity is expected for other coupling reagents, such as TBTU, PyAOP, PyBOP, and COMU, in which the post-bond activation agent is sufficiently acidic and nucleophilic to cleave the electrophile.

[0105] Those skilled in the art will recognize that amide bond formation depends on differences in the acidity of the nucleophilic catalytic activator (e.g., HOAt), the carboxylic acid, and the acid / base (e.g., TFA / i-PrNEt) buffer system. The same reaction outcome would be expected if different activators were applied with matching buffer systems. In the example above, the acidity of HOAt (pKa ≈ 3.3) is located between the carboxylic acid (pKa ≈ 5) and TFA (pKa ≈ 0.5). Therefore, during the reaction, the released nucleophilic HOAt anion is immediately protonated by TFA and therefore does not participate in the nucleophilic decomposition of the electrophilic motif. The resulting non-nucleophilic TFA anion does not cleave the electrophilic motif. Using the logic illustrated above, those skilled in the art can construct a system tailored to their preferred coupling reagents and buffer systems.

[0106] Those skilled in the art will recognize that the order of functionalization of I is interchangeable and depends on the desired application and the availability of any functionalizing components.

[0107] R 1 , R 4Compounds of formula I, where , p, X are as described herein, can be synthesized analogously to the literature and / or as shown, for example, in Scheme 4. [ka] Scheme 4.

[0108] Thus, ester type A, where X can be N, O, S, Se, or P, is reacted with a fluorophore bearing a leaving group (L) to give compound type B (step a). Subsequently, the ester of B is cleaved with acid using standard conditions to give a carboxylic acid, which can be coupled to the desired targeting ligand to give compound type C (step b), similar to the methods previously described herein. Alternatively, B can be oxidized to give compound type D (step c). This can be achieved with oxidizing agents such as hydrogen peroxide, meta-chloroperbenzoic acid, dimethyldioxirane, or oxone. After deprotection and amide bond formation with the desired ligand in a manner similar to C, compound type E is obtained (step d).

[0109] In one aspect, the present invention provides a fluorescent probe P1 or P2 for a protein of interest. [ka] [ka] (where LG is a ligand for the protein of interest, W, R 6 , R 7 , p, q, r and s are as defined herein) 1. A method for producing (a) Alkyne C or I [ka] [ka] (In the formula, R 6 , p, q and r are as defined herein) Azide-substituted fluorophore M [ka] (In the formula, W, R 7 , s and t are as defined herein, Compound D or K [ka] [ka] (In the formula, W, R 6 , R 7 , p, q, r and s are as defined herein) and forming Next, (b) removing the t-Bu protecting group from compound D or K using an acid, such as HCl or TFA, to give compound D1 or K1 [ka] [ka] (In the formula, W, R 6 , R 7 , p, q, r and s are as defined herein) and Next, (c) Binding compound D1 or K1 to an amino-substituted ligand N [ka] where LG is a ligand for the protein of interest. in the presence of a coupling reagent and an acid / base buffer system. obtaining the fluorescent probe P1 or P2 for a protein of interest; The present invention provides a method comprising:

[0110] In the process according to the invention described above, the reaction order may be reversed so that steps (b) and (c) are carried out before step (a).

[0111] Thus, in a further aspect, the present invention provides a fluorescent probe P1 or P2 for a protein of interest. [ka] [ka] (where LG is a ligand for the protein of interest, W, R 6 , R 7 , p, q, r and s are as defined herein) 1. A method for producing (a) removing the t-Bu protecting group from compound C or I using an acid, such as HCl or TFA; [ka] [ka] (In the formula, R 6 , p, q and r are as defined herein) Remove from Compound Ci or Ii [ka] [ka] (In the formula, R6 , p, q and r are as defined herein) and Next, (b) coupling said compound Ci or Ii to an amino-substituted ligand N [ka] where LG is a ligand for the protein of interest. in the presence of a coupling reagent and an acid / base buffer system. Compound Cii or Iii [ka] [ka] where LG is the ligand for the protein of interest and R 6 , p, q and r are as defined herein) and Next, (c) converting said compound Cii or Iii into an azide-substituted fluorophore M [ka] (In the formula, W, R 7 , s and t are as defined herein, obtaining the fluorescent probe P1 or P2 for a protein of interest; The present invention provides a method comprising:

[0112] In a further aspect, the present invention provides a fluorescent probe P1 or P2 for a protein of interest. [ka] [ka] (where LG is a ligand for the protein of interest, W, R 6 , R 7 , p, q, r and s are as defined herein) 1. A method for producing (a) Compound D1 or K1 [ka] [ka] (In the formula, W, R 6 , R 7 , p, q, r and s are as defined herein) , an amino-substituted ligand N to a protein of interest. [ka] where LG is a ligand for the protein of interest. in the presence of a coupling reagent and an acid / base buffer system. obtaining the fluorescent probe P1 or P2 for a protein of interest; The present invention provides a method comprising:

[0113] In a further aspect, the present invention provides a fluorescent probe P1 or P2 for a protein of interest. [ka] [ka] (where LG is a ligand for the protein of interest, W, R 6 , R 7 , p, q, r and s are as defined herein) 1. A method for producing (a) Compound Cii or Iii [ka] [ka] where LG is the ligand for the protein of interest and R 6 , p, q and r are as defined herein) , an azide-substituted fluorophore M [ka] (In the formula, W, R 7 , s and t are as defined herein, obtaining the fluorescent probe P1 or P2 for a protein of interest; The present invention provides a method comprising:

[0114] In one aspect, the present invention provides compounds D or K [ka] [ka] (In the formula, W, R 6 , R 7 , p, q, r and s are as defined herein) 1. A method for producing (a) Alkyne C or I [ka] [ka] (In the formula, R 6, p, q and r are as defined herein) Azide-substituted fluorophore M [ka] (In the formula, W, R 7 , s and t are as defined herein) React with forming said compound D or K; The present invention provides a method comprising:

[0115] In a further aspect, the present invention provides compounds Cii or Iii [ka] [ka] where LG is the ligand for the protein of interest and R 6 , p, q and r are as defined herein) 1. A method for producing (a) the compound Ci or Ii [ka] [ka] (In the formula, R 6 , p, q and r are as defined herein) , an amino-substituted ligand N to a protein of interest. [ka] where LG is a ligand for the protein of interest. in the presence of a coupling reagent and an acid / base buffer system. obtaining said compound Cii or Iii, The present invention provides a method comprising:

[0116] In a further aspect, the present invention provides an amide of general formula 1 [ka] (In the formula, R x and R y are chemical moieties attached to the parent amide functional group through a carbon atom, R x N-sulfonylpyridone (SP) motif i or N-acyl-N-alkylsulfonamide (NASA) motif ii [ka] [ka] (In the formula, R 6 is as defined herein) (including 1. A method for producing Carboxylic Acid 2 [ka] Amine 3 [ka] in the presence of a coupling reagent and an acid / base buffer system. obtaining said amide 1; The present invention provides a method comprising:

[0117] In one embodiment, the coupling reagent used in the amide coupling according to the present invention is selected from DCC, PyBOP, COMU, EDCI, TBTU and T3P.

[0118] In a preferred embodiment, the coupling reagent used in the amide coupling according to the present invention generates an HOAt ester from a carboxylic acid in the presence of a suitable catalyst. Examples of coupling reagents that generate an HOAt ester from a carboxylic acid in the presence of a suitable catalyst are well known in the art. Non-limiting examples include DCC and HOAt. Similarly, suitable catalysts for use in this type of coupling reaction are well known in the art. Non-limiting examples include EDCI and HOAt, and DIC and HOAt.

[0119] In a particularly preferred embodiment, the coupling reagent used in the amide coupling according to the present invention is selected from HATU and PyAOP.

[0120] In a further particularly preferred embodiment, the coupling reagent used in the amide coupling according to the present invention is HATU.

[0121] In one embodiment, 1 to 5 equivalents, for example 1, 2, 3, 4 or 5 equivalents of coupling reagent relative to each carboxylic acid are used in the amide coupling according to the present invention.

[0122] In a preferred embodiment, 1 to 2 equivalents of coupling reagent for each carboxylic acid are used in the amide coupling according to the present invention.

[0123] In a particularly preferred embodiment, 1.1 equivalents of coupling reagent for each carboxylic acid are used in the amide coupling according to the present invention.

[0124] In a further particularly preferred embodiment, the coupling reagent used in the amide coupling according to the present invention is PyAOP.

[0125] In one embodiment, 1 to 2 equivalents of coupling reagent for each carboxylic acid are used in the amide coupling according to the present invention.

[0126] In a preferred embodiment, 1 to 1.5 equivalents of coupling reagent for each carboxylic acid are used in the amide coupling according to the present invention.

[0127] In a particularly preferred embodiment, 1.1 equivalents of coupling reagent for each carboxylic acid are used in the amide coupling according to the present invention.

[0128] In one embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr2NEt.

[0129] In one embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr2NEt, with a ratio of TFA to i-Pr2NEt between 1 / 3 and 11 / 13, for example, 1 / 3, 3 / 5, 5 / 7, 7 / 9, 9 / 11 or 11 / 13.

[0130] In a preferred embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr2NEt, with a ratio of TFA to i-Pr2NEt of 3 / 5 to 3.5 / 5.5.

[0131] In a particularly preferred embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr2NEt, with a ratio of TFA to i-Pr2NEt of 3.3 / 5.3.

[0132] In a preferred embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr2NEt, with 3-3.5 equivalents of TFA and 5-5.5 equivalents of i-Pr2NEt used for each carboxylic acid.

[0133] In a particularly preferred embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr2NEt, with 3.3 equivalents of TFA and 5.3 equivalents of i-Pr2NEt used for each carboxylic acid.

[0134] In one embodiment, the acid / base buffer system used in the amide coupling according to the present invention is TFA / i-Pr2NEt, with 1-10 equivalents of TFA relative to the coupling reagent.

[0135] In a preferred embodiment, the coupling reagent used in the amide coupling according to the present invention is HATU and the acid / base buffer system is TFA / i-Pr2NEt.

[0136] In one embodiment, the amide coupling according to the present invention is carried out in a solvent selected from DMF, DMSO, DMA, CH2Cl2 and dioxane or mixtures thereof.

[0137] In a preferred embodiment, the amide coupling according to the present invention is carried out in a mixture of DMF and CH2Cl2.

[0138] In a preferred embodiment, the ligand for the protein of interest is a ligand for CB2, in particular the following ligand: [ka] (In the formula, R 2 , R 3 and R 4 is as defined herein, and the wavy line indicates the point of attachment of the ligand to the remainder of the compound of formula (I) as described herein. is.

[0139] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; [ka] ; and [ka] (In the formula, R 2 , R 3 , R 4 , R 6 , R 7 , p, q, r, s, t are as described herein, and LG is a ligand for the protein of interest). The present invention provides a compound selected from:

[0140] TR-FRET hCB2R binding assay Cell culture: Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) containing blasticidin (5 μg / mL; Invitrogen) and Zeocin (20 μg / mL; Invitrogen) in a humidified atmosphere at 37°C and 5% CO2. For inducible expression, SNAP-tagged human CB2 receptor cDNA in pcDNA4 / TO was introduced into HEK293TR cells (Invitrogen, which express the Tet repressor protein to enable inducible expression) using PEI. Mixed population stable lines were selected by resistance to blasticidin (TR vector, 5 μg / mL) and Zeocin (receptor plasmid, 20 μg / mL). For receptor-inducible expression, cells were transfected into t175cm. 2 Cells were seeded in flasks, grown to 70% confluence, and supplemented with DMEM containing 1 μg / ml tetracycline. After 24 hours, cells were labeled with SNAP-Lumi4-Tb (CisBio) and membranes were prepared as described in detail below.

[0141] Terbium labeling of SNAP-tagged CB2R HEK293-TR cells: Cell culture medium was removed from a 175 cm flask containing confluent, adherent CB2R HEK293-TR cells. The cells were washed once with PBS (GIBCO, Carlsbad, CA) and then once with Tag-lite labeling medium (LAMBED, CisBio) to remove excess cell culture medium. Ten milliliters of LAMBED containing 100 nM SNAP-Lumi4-Tb was then added to the flask and incubated at 37°C under 5% CO for 1 hour. The cells were washed once with PBS (GIBCO, Carlsbad, CA) to remove excess SNAP-Lumi4-Tb. They were then detached using 5 mL of GIBCO enzyme-free Hank's-based cell dissociation buffer (GIBCO, Carlsbad, CA) and collected in a vial containing 5 mL of DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum. Cells were pelleted by centrifugation (1500 rpm for 5 min) and the pellet was frozen at −80° C. To prepare membranes, a homogenization step was performed at 4° C (to avoid receptor degradation) as described in C.K. Herenbrink, et al., Nat. Commun., 2016, 7, 10842.

[0142] Fluorescent Ligand Binding Assay: All fluorescent ligand binding experiments were performed in white 384-well Optiplate plates in an assay binding buffer of either Hanks' Balanced Salt Solution (HBSS), 5 mM HEPES, 0.5% BSA, 0.02% Pluronic F-127 pH 7.4, and 100 μM GppNHp. Because the Motulsky-Mahan model is only appropriate for ligands that compete at a single site, GppNHp was included to eliminate G protein-coupled populations of receptors, which may result in two distinct populations of binding sites in membrane preparations. In all cases, nonspecific binding was determined by the presence of 1 μM SR144528. Data are shown as the mean ± SEM from a representative of 3–8 experiments.

[0143] Determination of fluorescent ligand binding kinetics and equilibrium affinity: To accurately determine the association rate (k) and dissociation rate (koff) values, at least five different concentrations of 8-SiR (see T. Gazzi et al. Chem. Sci., 2022, 13, 5539-5545 for a description of 8-SiR and the assay) were used to calculate the observed association rate (koff). An appropriate concentration of fluorescent ligand was incubated with human CB2R HEK293-TR cell membranes (4 μg / well) in assay binding buffer (final assay volume, 40 μL). The extent of fluorescent ligand bound to the receptor was assessed at multiple time points by HTRF detection, allowing the construction of an association rate curve. The resulting data were globally fitted to an association rate model (Equation 1, see the Signal Detection and Data Analysis section below) to derive single best-fit estimates for koff and koff, as described in Data Analysis. Saturation analysis was performed at equilibrium by simultaneously fitting total and nonspecific (NSB) binding data (Equation 2; see Signal Detection and Data Analysis section below), allowing for the determination of fluorescent ligand binding affinity.

[0144] Competitive Binding: To determine the affinity of CB2R-specific ligands, we used a simple competitive kinetic binding assay. This approach involves the simultaneous addition of both fluorescent ligand and competitor to a CB2R preparation. 62.5 nM 8-SiR, a concentration that avoids ligand depletion in the assay volume, was added to CB2R cell membranes (4 μg / well) simultaneously with increasing concentrations of unlabeled compound in 40 μL of assay buffer in a 384-well plate, incubated at room temperature with orbital mixing. The extent of fluorescent ligand bound to the receptor was assessed at equilibrium by HTRF detection. Nonspecific binding was determined as the amount of HTRF signal detected in the presence of SR144528 (1 μM) and subtracted from total binding to yield the IC 50 The specific binding was calculated to construct the curves.

[0145] Signal detection and data analysis: Signal detection was performed on a Pherastar FSX (BMG Labtech, Offenburg, Germany). The terbium donor was constantly excited with eight laser flashes at a wavelength of 337 nm. When using the red acceptor fluorescent ligand 8-SiR, the TR-FRET signal was collected at 665 (acceptor) and 620 nm (donor). The HTRF ratio was obtained by dividing the acceptor signal by the donor signal and multiplying this value by 10,000. All experiments were analyzed by nonlinear regression using Prism 8.0 (GraphPad Software, San Diego, USA).

[0146] Using GraphPad Prism 8.0, the fluorescent ligand association data were fitted to a global fitting model as follows to obtain k using the following equation: on and k off were calculated simultaneously. Y=Y max *(1-exp(-1*k ob *X)) (Formula 1) In the formula, k ob =[L]*k on +k off

[0147] k ob is equal to the observed rate of ligand association, and k on and k off are the association and dissociation rate constants of the fluorescent ligand, respectively. In this globally fitted model of tracer binding, the tracer concentration [L] is fixed and k on and k off is a shared parameter, but k obs can be varied, where Y is the level of tracer bound to the receptor and Y max is the level of tracer binding at equilibrium, X is expressed in units of time (e.g., minutes), and k obs is the rate at which the equilibrium state is approached (e.g., min -1 )

number

[0148] Saturation binding data were analyzed by nonlinear regression according to a one-site equation by globally fitting the total and NSB. The fluorescent ligand dissociation constant (K) was calculated using the following equation: d ) where L is the fluorescent ligand concentration:

[0149] By fitting the total and NSB datasets globally (simultaneously) and sharing the slope values, K d and B max A single best fit value is obtained for both

[0150] The competitive displacement binding data was fitted to a sigmoidal (variable slope) curve using the "four parameter logistic equation": Y = Lowest value + (Highest value - Lowest value) / (1 + 10 (logIC50-X).ヒル係数 ) (Formula 3)

[0151] The IC obtained from the inhibition curve using the method of Cheng and Prusoff (C. Yung-Chi, W.H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099-3108) 50 Value K i converted to a value. [Table 1]

[0152] Uses of the Compounds of the Invention The compounds of formula (I) are fluorescent or bioorthogonal probes with high affinity for CB2R. Therefore, they can be used as high-resolution tools to investigate the localization, expression levels and protein distribution in health and disease, structure, dynamics, and function of CB2R in live cells. They can also be applied to cellular trafficking studies using flow cytometry (fluorescence-activated cell sorting) experiments or confocal live-cell imaging. These probes, functionalized with a cleavable motif that allows ligand release after covalent labeling, provide an excellent tool for conducting these experiments without disrupting cellular homeostasis. Due to the covalent nature of the labeling, these probes are particularly well-suited for imaging low-abundance target proteins. These probes can be used to construct FRET sensors for membrane-bound and intracellular protein targets, which can then be used to investigate ligand-protein interactions in real time via TR-FRET.

[0153] In one aspect, the present invention provides a compound of formula (I) as described herein for use as a fluorescent or bioorthogonal probe for cannabinoid receptor 2 (CB2R).

[0154] In a further aspect, the present invention provides the use of a compound of formula (I) as described herein as a fluorescent or bioorthogonal probe for cannabinoid receptor 2 (CB2R).

[0155] In a further aspect, the present invention provides a method for imaging cannabinoid receptor 2 (CB2R), comprising contacting cannabinoid receptor 2 (CB2R) with a compound of formula (I) described herein. [Example]

[0156] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the claims to the examples.

[0157] Where preparations are obtained as mixtures of enantiomers, the pure enantiomers may be separated by the methods described herein or by methods known to those skilled in the art, such as chiral chromatography (e.g., chiral SFC) or crystallization.

[0158] Unless otherwise noted, all reactions and intermediates were prepared under an argon atmosphere.

[0159] Example 1 N-(5-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-5-oxopentyl)-1-((3-((2-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide [ka]

[0160] Step a) tert-butyl 5-(6-hydroxynicotinamido)pentanoate A solution of 6-hydroxynicotinic acid (155 mg, 1.11 mmol, 1.0 equiv., CAS RN5006-66-6), tert-butyl 5-aminopentanoate (193 mg, 1.11 mmol, 1.0 equiv., CAS RN63984-03-2), HOBt·HO (213 mg, 1.39 mmol, 1.25 equiv.), EDCI·HCl (267 mg, 1.39 mmol, 1.25 equiv.), and EtN (388 μL, 2.79 mmol, 2.5 equiv.) in DMF (2.60 mL) was stirred at ambient temperature for 16 h. The mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO, dry loading onto SiO, 5% MeOH in CHCl) to afford the title compound as a white solid (285 mg, 87%). 1 H NMR(500MHz,CD2Cl2)δ12.58(bs,1H), 8.10(d,J=2.5Hz,1H), 7.86(dd,J=9.5,2.6Hz,1H), 7.54(t,J=5.6Hz, 1H), 6.43(dd,J=9.5,0.6Hz,1H), 3.49-3.22(m,2H), 2.22(t,J=7.0Hz,2H), 1.69-1.52(m,4H), 1.40(s,9H). 13 C NMR (126MHz, CD2Cl2) δ173.6, 165.0, 164.9, 140.4, 137.5, 119.6, 115.4, 80.7, 40.1, 35.5, 29.4, 28.4, 22.9. IR(neat, ν max / cm -1 )3311, 3058, 2978, 2930, 1724, 1654, 1632, 1611, 1542, 1460, 1428, 1392, 1367, 1313, 1251, 1148, 1096. HRMS(ESI):m / z=317.1475[M+Na] + (C 15 H 22 Calculated m / z for N2NaO4 = 317.1472)

[0161] Step b) tert-butyl 5-(1-((3-(but-3-yn-1-ylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)pentanoate tert-Butyl 5-(6-hydroxynicotinamido)pentanoate (15.0 mg, 51 μmol, 1.0 equiv.) was dissolved in anhydrous THF (0.50 mL) at room temperature and cooled to −78° C. t Bu (1 M in THF, 102 μL, 102 μmol, 2.0 equiv.) was added dropwise at −78 °C, and the solution was stirred at −78 °C for 10 min. Subsequently, 3-(but-3-yn-1-ylcarbamoyl)benzenesulfonyl chloride (41.5 mg, 153 μmol, 3.0 equiv., CAS RN1344252-51-2) dissolved in anhydrous THF (0.30 mL) was added dropwise, and the mixture was stirred at −78 °C for 2 h before warming to room temperature. The solvent was removed in vacuo, and the residue was redissolved in CHCl and adsorbed onto silica. The residue was purified by flash column chromatography (SiO, dry loading onto SiO, 1–4% MeOH in CHCl) to afford the title compound as a pale yellow waxy solid (19.5 mg, 72%). 1 H NMR(500MHz,CD3OD)δ8.80(dd,J=2.5,0.7Hz,1H), 8.53(td,J=1.9,0.5Hz,1H), 8.29(ddd, J=8.0,2.0,1.1Hz,1H), 8.21(ddd,J=7.8,1.8,1.1Hz,1H), 7.89(dd,J=9.6,2.5Hz,1H), 7. 75(td,J=7.9,0.5Hz,1H), 6.45(dd,J=9.6,0.7Hz,1H), 3.53(t,J=7.1Hz,2H), 3.37(t,J=6 .6Hz,2H), 2.51(td,J=7.1,2.7Hz,2H), 2.33-2.27(m,3H), 1.71-1.59(m,4H), 1.45(s,9H). 13C NMR(126MHz,CD3OD)δ174.8, 167.7, 165.7, 161.4, 141.5, 138.2, 136.8, 135.2, 134.9, 133. 8, 130.6, 129.7, 122.9, 116.4, 82.1, 81.5, 71.0, 40.7, 40.3, 35.9, 29.8, 28.4, 23.6, 19.7. IR(neat, ν max / cm -1 )3299, 3075, 2931, 2483, 1725, 1693, 1635, 1536, 1454, 1368, 1312, 1255, 1191, 1171, 1153, 1085, 1020, 881, 594. HRMS(ESI):m / z=552.1776[M+Na] + (C 26 H 31 Calculated m / z for N3NaO7S = 552.1775

[0162] Step c) tert-butyl 5-(1-((3-((2-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)pentanoate To a vial containing N-(2-(2-azidoethoxy)ethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (6.6 mg, 22.7 μmol, 2.0 equiv., CAS RN2449214-44-0) and tert-butyl 5-(1-((3-(but-3-yn-1-ylcarbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)pentanoate (6.0 mg, 11.3 μmol, 1.0 equiv.) was added [Cu(MeCN)]PF (21.1 mg, 56.7 μmol, 5.0 equiv.) in a glove box. The mixture was dissolved in argon-degassed anhydrous MeOH (0.11 mL) and CHCl (0.11 mL), and the reaction mixture was capped, heated to 40 °C, and stirred under argon for 16 h. The reaction mixture was loaded directly onto a SiO plate, and the crude product was purified by preparative TLC (SiO, 6–10% MeOH in CHCl) and triturated with hexanes to give the title product as an orange solid (8.2 mg, 88%). 1 H NMR(500MHz,CD3OD)δ8.78(dd,J=2.5,0.7Hz,1H), 8.47(t,J=1.7Hz,1H), 8.44(d,J=8.9Hz,1H), 8.26(ddd,J=7.9,2.0,1. 1Hz,1H), 8.15(ddd,J=7.8,1.7,1.0Hz,1H), 7.85(dd,J=9.6,2.5Hz,1H), 7.78(s,1H), 7.72(td,J=7.9,0.5Hz,1H), 6.42(d d,J=9.7,0.7Hz,1H), 6.31(d,J=8.9Hz,1H), 4.54(t,J=5.2Hz,2H), 3.88(t,J=5.1Hz,2H), 3.72(t,J=5.2Hz,2H), 3.65(bs ,2H), 3.58(t,J=7.0Hz,2H), 3.38-3.33(m,2H), 2.89(t,J=6.9Hz,2H), 2.31-2.25(m,2H), 1.71-1.56(m,4H), 1.44(s,9H). 13C NMR(126MHz,CD3OD)δ174.8, 167.7, 165.7, 161.4, 146.1, 141.5, 138.0, 137.0, 135.2, 134.9, 133. 7, 130.6, 129.8, 124.5, 122.9, 116.3, 81.5, 70.3, 70.0, 51.3, 40.7, 35.9, 29.8, 28.4, 26.4, 23.6. IR(neat, ν max / cm -1 )3300, 3076, 2925, 2855, 1724, 1693, 1644, 1583, 1532, 1443, 1368, 1301, 1257, 1188, 1150. HRMS(ESI):m / z=845.2641[M+Na] + (C 36 H 42 N 10 NaO 11 Calculated m / z for S = 845.2647

[0163] Step d) N-(5-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-5-oxopentyl)-1-((3-((2-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide To a solution of tert-butyl 5-(1-((3-((2-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)pentanoate (3.8 mg, 4.6 μmol, 1.0 equiv) in anhydrous CHCl (200 μL) was added TFA (100 μL) and the reaction mixture was stirred at room temperature for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (50 μL). TFA (0.5 M solution in anhydrous DMF, 30.5 μL, 15.2 μmol, 3.3 equiv.) and i-Pr2NEt (0.5 M solution in anhydrous DMF, 49.0 μL, 24.4 μmol, 5.3 equiv.) were added at room temperature, and the resulting solution was cooled to 0 °C. HATU (0.1 M solution in DMF, 50.8 μL, 5.0 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0° C. for 15 minutes, and ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.1 M solution in anhydrous CHCl, 50.8 μL, 5.0 μmol, 1.1 equiv., Chem. Eur. J., 2020, 26, 1380-1387) was added. The reaction mixture was then warmed to ambient temperature and stirred for an additional 35 minutes. i-PrNEt (0.5 M solution in anhydrous DMF, 2.3 μL, 1.1 μmol, 0.25 equiv.) was added at room temperature, and the resulting solution was stirred for an additional 10 min. The residue was dissolved in CHCl (10 mL) and washed with saturated aqueous NHCl (5 mL), saturated aqueous NaHCO (5 mL), and brine (5 mL). The organic layer was dried over NaSO, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO, 4–5% MeOH in CHCl) to afford the product (5.1 mg, 92%) as an orange solid. 1H NMR(600MHz,CD2Cl2)δ8.67(d,J=2.4Hz,1H)、8.42(d,J=8.6Hz,1H)、8.37(s,1H)、8.19(d,J=8.0Hz,1H)、8.09(d,J=7.8Hz,1H)、7.79(dd,J=9.7,2.5Hz,1H)、7.64-7.56(m,3H)、7.53(bs,1H)、6.48(s,2H)、6.33(d,J=9.6Hz,1H)、6.21(d,J=5.4Hz,1H)、5.80(bs,1H)、5.59(dt,J=3.1,1.5Hz,1H)、4.51(t,J=4.9Hz,2H)、3.98-3.92(m,1H)、3.90-3.85(m,3H)、3.84-3.78(m,1H)、3.76-3.73(m,2H)、3.73-3.72(m,2H)、3.71(s,6H)、3.66(bs,2H)、3.41-3.32(m,2H)、3.21(t,J=7.0Hz,2H)、2.95(t,J=6.4Hz,2H)、2.27(t,J=7.0Hz,2H)、2.17-2.11(m,1H)、2.06(t,J=5.7Hz,1H)、2.03-1.97(m,1H)、1.76-1.49(m,8H)、1.32-1.20(m,6H)、1.26(s,3H)、1.26(s,6H)、1.16-1.07(m,2H)、0.92(s,3H)。 13 C NMR(151MHz,CD2Cl2)δ173.8、165.7、164.0、160.2、159.0、150.1、145.8、145.0、144.7、140.5、138.8、137.3、137.0、136.3、134.2、134.0、133.3、129.9、128.7、124.4、123.3、122.8、117.9、115.8、103.3、69.9、68.9、56.3、52.0、50.7、47.9、45.0、44.9、44.7、41.2、40.2、40.0、38.5、38.0、36.2、30.4、30.2、29.3、29.3、28.7、28.1、27.1、26.6、25.9、25.1、23.2、21.3。IR(neat、ν max / cm -1)3300, 3075, 2926, 2855, 2096, 1645, 1578, 1532, 1502, 1449, 1411, 1302, 1258, 1189, 1121, 1033. HRMS(ESI):m / z=624.2558[M+Na2] 2+ (C 59 H 74 N 14 NaO 12 Calculated value for S (m / z = 624.2558) [α] D =+26.400±0.321(c=0.23, CH2Cl2).

[0164] Example 2 N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide [ka]

[0165] Step a) tert-butyl 7-(6-hydroxynicotinamide)heptanoate A solution of 6-hydroxynicotinic acid (346 mg, 2.48 mmol, 1.0 equiv.), tert-butyl 7-aminoheptanoate (500 mg, 2.48 mmol, 1.0 equiv., CAS RN105974-64-9), HOBt·HO (476 mg, 3.11 mmol, 1.25 equiv.), EDCI·HCl (595 mg, 3.11 mmol, 1.25 equiv.), and EtN (865 μL, 6.21 mmol, 2.5 equiv.) in DMF (8.0 mL) was stirred at ambient temperature for 16 h. The mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO, dry loading onto SiO, 60% hexanes (EtOAc:EtOH, 3:1)) to afford the title compound as a white solid (540 mg, 67%). 1 H NMR(500MHz,CD2Cl2)δ8.04(dd,J=2.7,0.7Hz,1H), 7.80(dd,J=9.6,2.6Hz,1H), 6.56(t,J=5.6Hz,1H), 6.49(dd,J=9. 5,0.7Hz,1H), 3.34(td,J=7.2,5.7Hz,2H), 2.18(t,J=7.4Hz,2H), 1.62-1.51(m,4H), 1.42(s,9H), 1.40-1.29(m,4H). 13 C NMR (126MHz, CD2Cl2) δ173.6, 165.2, 164.6, 140.2, 137.4, 119.8, 115.5, 80.4, 40.5, 36.0, 29.9, 29.2, 28.4, 27.2, 25.5. IR(neat, ν max / cm -1 )3320, 3070, 2976, 2933, 2861, 1728, 1654, 1635, 1610, 1537, 1461, 1426, 1391, 1366, 1313, 1252, 1219, 1152, 1102. HRMS(ESI):m / z=345.1782[M+Na] + (C 17 H 26 Calculated m / z for N2NaO4 = 345.1785)

[0166] Step b) tert-butyl 7-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)heptanoate tert-Butyl 7-(6-hydroxynicotinamido)heptanoate (20.5 mg, 63.5 μmol, 1.0 equiv.) was dissolved in anhydrous THF (0.50 mL) at room temperature and cooled to -78°C. t Bu (1 M in THF, 127 μL, 127 μmol, 2.0 equiv.) was added dropwise at −78 °C, and the solution was stirred at −78 °C for 10 min. Subsequently, 3-(prop-2-yn-1-ylcarbamoyl)benzenesulfonyl chloride (49.2 mg, 191 μmol, 3.0 equiv., CAS RN 1016841-21-6) dissolved in anhydrous THF (0.30 mL) was added dropwise, and the mixture was stirred at −78 °C for 2 h before warming to room temperature. The solvent was removed in vacuo, and the residue was redissolved in CHCl and adsorbed onto silica. The residue was purified by flash column chromatography (SiO, dry loading onto SiO, 30% (EtOAc:EtOH, 3:1) in hexanes) to afford the title compound as a white waxy solid (23.5 mg, 68%). 1 H NMR(500MHz,CD3OD)δ8.80(dd,J=2.5,0.7Hz,1H), 8.55(td,J=1.9,0.5Hz,1H), 8.31(ddd,J=8 .0,2.0,1.1Hz,1H), 8.22(ddd,J=7.8,1.7,1.1Hz,1H), 7.89(dd,J=9.6,2.5Hz,1H), 7.76(td,J =7.9,0.5Hz,1H), 6.45(dd,J=9.6,0.7Hz,1H), 4.17(d,J=2.5Hz,2H), 3.36(t,J=7.2Hz,2H), 2. 63(t,J=2.6Hz,1H), 2.24(t,J=7.4Hz,2H), 1.68-1.54(m,4H), 1.44(s,9H), 1.42-1.34(m,4H). 13C NMR(126MHz,CD3OD)δ175.1, 167.2, 165.7, 161.4, 141.6, 138.2, 136.4, 135.2, 135.1, 134.0, 130.7, 129.8, 122.9, 116.4, 81.4, 80.4, 72.4, 41.1, 36.3, 30.2, 29.8, 28.4, 27.7, 26.1, 23.7. IR(neat, ν max / cm -1 )3299, 3073, 2926, 2855, 2471, 2068, 1724, 1691, 1639, 1535, 1453, 1367, 1313, 1259, 1219, 1172, 1151, 1085. HRMS(ESI):m / z=566.1933[M+Na] + (C 27 H 33 Calculated for N3NaO7S (m / z = 566.1931)

[0167] Step c) tert-butyl 7-(1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate A vial containing N-(2-(2-azidoethoxy)ethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (2.5 mg, 8.4 μmol, 1.15 equiv.) and tert-butyl 7-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)heptanoate (4.0 mg, 7.3 μmol, 1.0 equiv.) was treated with [Cu(MeCN)]PF in a glovebox. 6(To the resulting solution was added 13.7 mg, 36.7 μmol, 5.0 equiv. The mixture was dissolved in argon-degassed CHCl (0.30 mL) and AcOH (10 μL), capped, and stirred under argon for 16 h. The reaction mixture was loaded directly onto a SiO plate, and the crude product was purified by preparative TLC (SiO, 5% MeOH in CHCl) to give the title product as an orange solid (5.3 mg, 86%). 1 H NMR(500MHz,CD3OD)δ8.77(dd,J=2.5,0.7Hz,1H), 8.53(td,J=1.9,0.5Hz,1H), 8.42(d,J=8.8Hz,1H), 8.27(ddd,J =8.0,2.0,1.1Hz,1H), 8.19(ddd,J=7.8,1.7,1.1Hz,1H), 7.92(s,1H), 7.85(dd,J=9.6,2.5Hz,1H), 7.72(td,J=7.9 ,0.5Hz,1H), 6.40(dd,J=9.6,0.7Hz,1H), 6.31(d,J=8.9Hz,1H), 4.60-4.51(m,4H), 3.95-3.87(m,2H), 3.78-3.71( m,2H), 3.66(bs,2H), 3.38-3.32(m,2H), 2.22(t,J=7.4Hz,2H), 1.69-1.54(m,4H), 1.43(s,9H), 1.41-1.34(m,4H). 13 C NMR (126MHz, CD3OD) δ175.1, 167.5, 165.7, 161.4, 145.8, 141.5, 138.1, 136.5, 135.2, 135.0, 133.9, 130.6, 129.8, 125.3, 122.9, 116.4, 81.4, 70.3, 70.1, 51.4, 41.1, 36.3, 30.2, 29.8, 28.4, 27.7, 26.1. IR(neat, ν max / cm -1 )3300, 3075, 2926, 2855, 1725, 1646, 1583, 1532, 1443, 1367, 1302, 1258, 1189, 1149, 1036. HRMS(ESI):m / z=859.2808[M+Na] + (C 37 H 44 N 10 NaO 11Calculated m / z for S = 859.2804)

[0168] Step d) N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide To a solution of tert-butyl 7-(1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate (2.1 mg, 2.6 μmol, 1.0 equiv) in anhydrous CHCl (200 μL) was added TFA (100 μL) and the reaction mixture was stirred at room temperature for 30 minutes. The solvent was then removed in vacuo, and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (50 μL). TFA (0.5 M solution in anhydrous DMF, 17.8 μL, 8.8 μmol, 3.3 equiv.) and i-PrNEt (0.5 M solution in anhydrous DMF, 28.5 μL, 14.2 μmol, 5.3 equiv.) were added at room temperature, and the resulting solution was cooled to 0° C. HATU (0.1 M solution in DMF, 29.6 μL, 2.9 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0° C. for 15 minutes, and the amine ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.1 M solution in anhydrous CHCl, 29.6 μL, 2.9 μmol, 1.1 equiv.) was added. The reaction mixture was then warmed to ambient temperature and stirred for an additional 45 minutes. The reaction mixture was then concentrated in vacuo, and the residue was dissolved in CH2Cl2 and loaded directly onto a silica plate. Purification by preparative TLC (SiO2, 4% MeOH in CH2Cl2) afforded the product (2.6 mg, 79%) as an orange solid. 1H NMR(600MHz,CD2Cl2)δ8.62(d,J=2.4Hz,1H)、8.46(d,J=8.7Hz,1H)、8.43(s,1H)、8.26(d,J=8.1Hz,1H)、8.13(d,J=7.8Hz,1H)、7.74(dd,J=9.7,2.4Hz,1H)、7.72(s,1H)、7.63(t,J=7.9Hz,1H)、6.49(s,2H)、6.35(d,J=9.6Hz,1H)、6.22(d,J=8.8Hz,1H)、5.58(s,1H)、4.65(d,J=5.5Hz,2H)、4.53(t,J=4.9Hz,2H)、3.94(s,1H)、3.90(t,J=5.0Hz,2H)、3.86-3.77(m,2H)、3.76-3.73(m,2H)、3.72(s,6H)、3.66(s,2H)、3.42-3.34(m,2H)、3.22(t,J=7.0Hz,2H)、2.23-1.96(m,8H)、1.67(d,J=8.4Hz,1H)、1.64-1.05(m,16H)、1.26(s,9H)、0.93(s,3H)。 13 C NMR(151MHz,CD2Cl2)δ173.6、165.5、163.9、162.9、160.1、159.0、150.1、145.1、144.9、144.7、140.5、139.0、137.2、137.1、135.7、134.3、133.7、133.6、130.5、130.3、129.8、128.7、124.2、122.9、118.0、115.8、103.3、69.8、69.0、56.3、52.1、50.8、47.9、46.0、44.9、44.9、44.8、40.3、38.5、38.0、36.8、36.3、32.5、29.9、29.4、28.3、28.1、27.7、27.2、26.6、26.1、25.2、23.3、21.3。IR(neat,ν max / cm -1 ):2959、2924、2854、2096、1649、1583、1457、1411、1377、1302、1260、1186、1092、1018。HRMS(ESI):m / z=1217.5570[M+H] + (C 60 H 77 N 14 O 12Calculated m / z for S = 1217.5561)

[0169] Example 3 N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide [ka]

[0170] Step d) N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide To a solution of tert-butyl 7-(1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate (2.5 mg, 3.2 μmol, 1.0 equiv) in anhydrous CHCl (200 μL) was added TFA (100 μL) and the reaction mixture was stirred at room temperature for 30 minutes. The solvent was then removed in vacuo and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (50 μL). TFA (0.5 M solution in anhydrous DMF, 21.1 μL, 10.5 μmol, 3.3 equiv.) and i-Pr2NEt (0.5 M solution in anhydrous DMF, 33.9 μL, 16.9 μmol, 5.3 equiv.) were added at room temperature and the resulting solution was cooled to 0 °C. HATU (0.1 M solution in DMF, 35.2 μL, 3.5 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0 °C for 15 min, and the amine ((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.1 M solution in anhydrous CHCl, 35.2 μL, 3.5 μmol, 1.1 equiv., Chem. Eur. J., 2020, 26, 1380-1387) was added. The reaction mixture was then warmed to ambient temperature and stirred for an additional 45 min. The residue was dissolved in CHCl (10 mL) and washed with saturated aqueous NHCl (5 mL), saturated aqueous NaHCO (5 mL), and brine (5 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. Purification by preparative TLC (SiO2, 4% MeOH in CH2Cl2) afforded the product (2.7 mg, 72%) as an orange solid. 1H NMR(500MHz,CD2Cl2)δ8.61(dd,J=2.5,0.7Hz,1H)、8.46(d,J=8.7Hz,1H)、8.41(t,J=1.7Hz,1H)、8.25(ddd,J=8.0,1.9,1.1Hz,1H)、8.12(ddd,J=7.8,1.7,1.1Hz,1H)、7.74(dd,J=9.7,2.5Hz,1H)、7.70(s,1H)、7.63(td,J=7.9,0.5Hz,1H)、7.44(s,1H)、6.71(t,J=5.7Hz,1H)、6.49(s,2H)、6.35(dd,J=9.6,0.7Hz,1H)、6.22(d,J=8.7Hz,1H)、5.58(dt,J=2.9,1.5Hz,1H)、5.53-5.48(m,1H)、4.65(d,J=5.7Hz,2H)、4.57-4.49(m,2H)、3.94(t,J=2.4Hz,1H)、3.90(dd,J=5.4,4.5Hz,2H)、3.88-3.77(m,2H)、3.77-3.72(m,2H)、3.72(s,6H)、3.66(s,2H)、3.38(q,J=6.6Hz,2H)、2.24-1.94(m,8H)、1.67(d,J=8.4Hz,1H)、1.64-1.05(m,16H)、1.26(s,9H)、0.93(s,3H)、0.85(t,J=7.7Hz,3H)。 13 C NMR(126MHz,CD2Cl2)δ173.3、165.5、163.8、159.0、150.2、140.4、139.1、137.2、135.7、134.3、133.5、129.9、128.7、124.2、122.9、117.9、115.8、103.3、69.8、69.0、56.3、50.8、48.0、44.9、44.9、44.8、41.2、40.3、38.5、38.0、37.0、36.1、32.3、30.6、29.9、29.5、29.3、28.8、28.1、26.6、26.6、26.0、25.2、23.2、21.3、14.4。IR(neat,ν max / cm -1):3311, 3074, 2957, 2924, 2853, 1736, 1645, 1575, 1532, 1458, 1411, 1377, 1301, 1260, 1189, 1116, 1022. HRMS(ESI):m / z=1198.5360[M+Na] + (C 60 H 77 N 11 NaO 12 Calculated m / z for S = 1198.5366)

[0171] Example 4 N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(14-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide [ka]

[0172] Step c) tert-butyl 7-(1-((3-(((1-(14-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate To a heart-shaped flask charged with tert-butyl 7-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)heptanoate (5.8 mg, 10.7 μmol, 1.3 equiv.), N-(14-azido-3,6,9,12-tetraoxatetradecyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (3.5 mg, 8.2 μmol, 1.0 equiv.), and Cu[MeCN]PF (15.3 mg, 41.1 μmol, 5.0 equiv.) was added anhydrous CHCl (0.3 mL, pre-degassed with argon) and 20 μL of AcOH. The flask was then flushed with argon, stoppered with a glass stopper, and the resulting deep orange solution was stirred for 16 h. The reaction mixture was then loaded directly onto a silica plate and purified by preparative TLC (SiO 2 , 5% MeOH in CH 2 Cl 2 ) to give the product (7.2 mg, 90%) as an orange solid. 1 H NMR(500MHz,CD3OD)δ=8.76(dd,J=2.5,0.7Hz,1H), 8.56(d,J=1.8Hz,1H), 8.47(d,J=8.9Hz,1H), 8.27(ddd,J=8.1,1.9,1.0Hz,1H), 8.21(d,J=7.8Hz,1 H), 8.01(s,1H), 7.86(dd,J=9.6,2.5Hz,1H), 7.73(t,J=7.9Hz,1H), 6.42(d d,J=9.6,0.7Hz,1H), 6.39(d,J=8.9Hz,1H), 4.64(t,J=5.0Hz,2H), 4.57-4. 50(m,2H), 3.84(t,J=4.6Hz,2H), 3.79(t,J=5.5Hz,2H), 3.71(bs,1H), 3.65 -3.61(m,1H), 3.60-3.57(m,2H), 3.56-3.52(m,2H), 3.52-3.49(m,4H), 3.4 8-3.43(m,2H), 3.38-3.33(m,2H), 3.32-3.28(2H,HSQC),2.23(t,J=7.4Hz, 2H), 1.61(td,J=10.0,7.0Hz,4H), 1.44(s,9H), 1.39(tt,J=5.2,2.3Hz,4H). 13C NMR(126MHz,CD3OD)δ=175.08, 167.38, 165.63, 161.36, 141.49, 138.12, 135.19, 135.09, 133.90, 130.64, 129.80, 122. 94, 116.35, 81.37, 71.61, 71.56, 71.54, 71.44, 71.42, 70.31, 51.49, 41.09, 36.34, 30.24, 29.82, 28.36, 27.74, 26.11. IR(neat,ν max / cm -1 )3300, 3073, 2924, 2857, 1724, 1691, 1651, 1621, 1583, 1531, 1499, 1443, 1367, 1299, 1258, 1188, 1144, 1094, 1031. HRMS(ESI):m / z=969.3757[M+H] + (C 43 H 57 N 10 O 14 Calculated m / z for S = 969.3771)

[0173] Step d) N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(14-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide To a solution of tert-butyl 7-(1-((3-(((1-(14-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate (7.2 mg, 7.4 μmol, 1.0 equiv) in anhydrous CHCl (200 μL) was added TFA (100 μL) and the resulting orange reaction mixture was stirred for 30 min. The solvent was then concentrated in vacuo and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (0.1 mL). TFA (0.5 M solution in anhydrous DMF, 49.0 μL, 24.5 μmol, 3.3 equiv.) and i-PrNEt (0.5 M solution in anhydrous DMF, 78.8 μL, 39.4 μmol, 5.3 equiv.) were added, and the resulting pale orange solution was cooled to 0° C. HATU (0.1 M solution in DMF, 81.7 μL, 8.2 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0° C. for 15 minutes, and ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.1 M solution in anhydrous CHCl, 81.7 μL, 8.2 μmol, 1.1 equiv.) was added. The reaction mixture was then warmed to ambient temperature and stirred for an additional 30 minutes. LCMS reaction control indicated incomplete conversion, and more i-PrNEt (0.5 M solution in anhydrous DMF, 7.4 μL, 3.7 μmol, 0.5 equiv.) was added. The reaction mixture was then concentrated in vacuo, and the residue was dissolved in CHCl and loaded directly onto a silica plate. Purification by preparative TLC (SiO, 7% MeOH in CHCl) afforded the product (5.7 mg, 57%) as an orange solid. 1H NMR(500MHz,CD2Cl2)δ=8.63(d,J=2.4Hz,1H)、8.57(s,1H)、8.45(d,J=8.7Hz,1H)、8.34(d,J=7.9Hz,1H)、8.29(s,1H)、8.21(s,1H)、8.19(d,J=7.9Hz,1H)、7.75(dd,J=9.6,2.4Hz,1H)、7.64(t,J=7.9Hz,1H)、7.36(s,1H)、7.23(s,1H)、6.49(s,2H)、6.36(d,J=9.5Hz,1H)、6.21(d,J=8.7Hz,1H)、5.97(s,1H)、5.64-5.59(m,1H)、4.80(d,J=5.2Hz,2H)、4.59(t,J=4.5Hz,2H)、3.95(s,1H)、3.86(dt,J=12.1,5.1Hz,4H)、3.84-3.80(m,2H)、3.72(s,6H)、3.70-3.65(m,4H)、3.65-3.61(m,2H)、3.60-3.56(m,4H)、3.54-3.49(m,4H)、3.37(q,J=6.4Hz,2H)、3.22(t,J=7.0Hz,2H)、2.29(t,J=7.6Hz,2H)、2.22-2.13(m,1H)、2.07(t,J=5.5Hz,1H)、2.02(d,J=5.8Hz,1H)、1.67(d,J=8.4Hz,1H)、1.64-1.50(m,8H)、1.40-1.20(m,8H)、1.27(s,3H)、1.26(s,6H)、1.15-1.07(m,2H)、0.94(s,3H)。 13C NMR(126MHz,CD2Cl2)δ=173.55, 166.05, 165.20, 164.06, 160.22, 159.00, 150.05, 145.03, 140.82, 139.01, 137.28 , 137.12, 135.48, 134.74, 133.87, 133.68, 129.67, 128.49, 124.22, 122.82, 117.94, 115.96, 103.29, 71.12, 71.04 , 70.91, 70.81, 69.43, 56.26, 52.06, 51.78, 47.95, 44.94, 44.88, 44.77, 41.23, 40.26, 38.49, 38.03, 37.02, 35.23 , 32.50, 30.39, 30.26, 29.51, 29.36, 29.28, 28.95, 28.11, 27.74, 27.15, 26.76, 26.57, 26.11, 25.15, 23.26, 21.30. IR(neat,ν max / cm -1 )3289, 3074, 2932, 2863, 2096, 1648, 1579, 1531, 1448, 1410, 1380, 1349, 1302, 1257, 1189, 1121, 1036. HRMS(ESI):m / z=1349.6341[M+H] + (C 66 H 89 N 14 O 15 Calculated m / z for S = 1349.6347

[0174] Example 5 1-(6-((2-(2-(4-((3-((5-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate [ka]

[0175] Step c) 1-(6-((2-(2-(4-((3-((5-((7-(tert-butoxy)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate In a glove box, [Cu(MeCN)]PF (8.9 mg, 23.9 μmol, 5.0 equiv) was added to a flask charged with tert-butyl 7-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)heptanoate (3.9 mg, 7.1 μmol, 1.5 equiv) and 1-(6-((2-(2-azidoethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate (3.0 mg, 4.7 μmol, 1.0 equiv). The solid was dissolved in anhydrous CHCl (0.30 mL) and AcOH (10 μL) that had been degassed with argon. The flask was then re-flushed with argon, stoppered, and stirred at room temperature for 16 h. The reaction mixture was then loaded directly onto a SiO plate and purified by preparative TLC (SiO, 15% MeOH in CHCl) to give the product as a red solid (4.6 mg, 82%). 1H NMR(400MHz,CD3OD,E / Z dielectric coating and solvent)δ=9.20(d,J=1 .8Hz,1H,Z),9.06(d,J=1.9Hz,1H,E),8.76(dd,J=2.5,0.7Hz,1H )、8.57(dd,J=8.3,1.8Hz,1H,E),8.55(s,1H)、8.51(dd,J=8.3,1.8Hz,1H,Z),8.42(d,J=8.7Hz,1H,E),8.29-8.22(m,1H)、8.19( dt,J=7.9,1.3Hz,1H), 8.12(s,1H,E), 8.09(d,J=15.4Hz,1H,E), 7.96(s,1H), 7.91(s,1H,Z), 7.89(d,J=8.4Hz,1H,Z), 7.85(dd, J=9.7,2.4Hz,1H), 7.79(d,J=15.3Hz,1H,E),7.71(t,J=7.9Hz,1H),7.48(d,J=9.1Hz,1H,E),7.36(d,J=9.0Hz,1H,Z),7.09(dd, J=12.3,0.9Hz,1H,Z), 6.81(dd,J=9.0,2.5Hz,1H,E),6.79(dd,J=12.3,0.9Hz,1H,Z),6.72(dd,J=9.0,2.5Hz,1H,Z),6.52(d,J= 2.4Hz,1H,E),6.44(d,J=2.4Hz,1H,Z),6.40(dd,J=9.6,0.8Hz,1H),4.69(t,J=7.8Hz,2H),4.61(s,2H),4.55(t,J=4.9Hz,2H). .86-3.78(m,2H) 3.55(t,J=7.2Hz,2H) 3.52-3.45(m,4H) 3.3 6-3.26(m,4H) 2.22(t,J=7.3Hz,2H) 2.20-2.15(m,2H) 2.10-1 .92(m,4H) 1.73-1.53(m,6H) 1.51-1.46(m,2H) 1.43(s,9H) 1.40-1.35(m,2H) 1.24(t,J=7.1Hz,3H) 1.20(t,J=7.1Hz,3H) 13C NMR(101MHz,CD3OD)δ=168.9, 161.3, 158.3, 155.4, 154.5, 149.2, 145.9, 143.8, 142.8, 141.5, 135.1, 132.3, 125.4, 116.4, 114.7, 97.6, 81.4, 70.5, 70.1, 51.5, 46.1, 41.1, 40.2, 36.3, 30.8, 30.2, 29.8, 28.4, 27.8, 26.7, 26.1, 12.8. IR(neat,ν max / cm -1 )3311, 3064, 2956, 2924, 2854, 1718, 1657, 1618, 1579, 1556, 1504, 1457, 1419, 1377, 1356, 1259, 1191, 1170, 1134, 1081, 1040, 1016. HRMS(ESI):m / z=1170.4638[M+H] + (C 57 H 72 N9O 14 Calculated m / z for S2 = 1170.4635)

[0176] Step d) 1-(6-((2-(2-(4-((3-((5-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate To a solution of the ester 1-(6-((2-(2-(4-((3-((5-((7-(tert-butoxy)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate (4.4 mg, 3.7 μmol, 1.0 equiv.) in anhydrous CHCl (200 μL) was added TFA (100 μL) and the resulting deep red reaction mixture was stirred at room temperature for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (75 μL). TFA (0.5 M solution in anhydrous DMF, 24.8 μL, 12.4 μmol, 3.3 equiv.) and i-PrNEt (0.5 M solution in anhydrous DMF, 39.9 μL, 19.9 μmol, 5.3 equiv.) were added at room temperature, and the resulting pale red solution was cooled to 0° C. HATU (0.1 M solution in DMF, 41.4 μL, 4.1 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0 °C for 15 min, and the amine ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.1 M solution in anhydrous CHCl, 41.4 μL, 4.1 μmol, 1.1 equiv.) was added. The reaction mixture was then warmed to ambient temperature and stirred for an additional 30 min. Reaction control by LCMS indicated incomplete conversion, so more i-PrNEt (0.5 M solution in anhydrous DMF, 3.8 μL, 1.9 μmol, 0.5 equiv.) was added, and the mixture was stirred for an additional 5 min. The reaction mixture was then concentrated in vacuo and the residue was dissolved in CH2Cl2 and loaded directly onto a silica plate. Purification by preparative TLC (SiO2, 10% MeOH in CH2Cl2) afforded the product as a red solid (4.4 mg, 75%). 11H NMR (600 MHz, CD2Cl2, reported with E / Z signal sets as complete signals) δ = 9.64 (s, 1H, Z), 9.59 (s, 1H, E), 9.01 (bs, 1H, E), 8.96 (bs, 1H, Z), 8.78 - 8.75 (m, 1H), 8.71 (t, J = 1.7 Hz, 1H), 8.69 (d, J = 9.2 Hz, 1H, E), 8.60 (dd, J = 8.3, 1.7 Hz, 1H, Z), 8.51 - 8.44 (m, 1H), 8.36 (ddd, J = 6.2, 3.1, 1.6 Hz, 1H), 8.26 (d, J = 8.5 Hz, 1H, E), 8.13 (d, J = 15.3 Hz, 1H, E), 8.09 (bs, 1H, E), 8.02 (bs, 1H, Z), 7.93 (s, 1H), 7.87 (dt, J = 9.7, 2.7 Hz, 1H), 7.81 (s, 1H, Z), 7.80 (s, 1H, E), 7.71 (d, J = 8.3 Hz, 1H, Z), 7.69 - 7.66 (m, 1H), 7.63 (d, J = 15.3 Hz, 1H, E), 7.40 (d, J = 9.0 Hz, 1H, E), 7.26 (d, J = 9.0 Hz, 1H, E), 7.01 (dd, J = 12.3, 0.9 Hz, 1H, Z), 6.68 (dd, J = 9.0, 2.4 Hz, 1H, E), 6.62 (dd, J = 9.0, 2.5 Hz, 1H, Z), 6.53 (d, J = 2.8 Hz, 1H, E), 6.52 (d, J = 7.1 Hz, 1H, Z), 6.48 (s, 2H, Z), 6.48 (s, 2H, E), 6.43 (d, J = 2.4 Hz, 1H, Z), 6.35 (dd, J = 9.9, 2.8 Hz, 1H), 6.32 (bt, J = 6.3 Hz, 1H, Z), 6.25 (t, J = 5.4 Hz, 1H, E), 5.59 - 5.56 (m, 1H, E), 5.56 - 5.53 (m, 1H, Z), 4.78 (t, J = 7.7 Hz, 2H, E), 4.72 (t, J = 7.8 Hz, 2H, Z), 4.64 (dd, J = 5.7, 3.0Hz,2H)、2.21-2.16(m,2H)、2.13(dt,J=8.4,5.5Hz,1H)、2.06(t,J=6.6Hz,2H)、2.03-1.94(m,3H)、1.91(td,J=7.2,3.0Hz,1H)、1.69-1.48(m,10H)、1.42-1.38(m,4H)、1.33-1.30(m,6H)、1.29-1.27(m,2H)、1.26(s,9H)、1.25-1.23(m,3H,E),1.20(t,J=7.1Hz,3H,Z),1.14-1.02(m,2H)、0.92(s,3H,E),0.92(s,3H,Z)。. 13C NMR(151MHz,CD2Cl2)δ175.6、173.4、173.3、173.2、173.2、165.2、165.1、164.1、164.1、160.6、160.4、159.0、157.7、157.6、154.6、153.8、153.6、153.0、150.0、150.0、148.3、146.5、145.8、145.8、145.8、144.4、143.6、143.3、142.2、142.0、141.9、141.9、139.3、139.1、137.2、137.1、136.7、135.6、135.5、135.4、135.3、135.1、134.9、133.2、133.2、131.7、131.3、130.7、130.5、130.3、129.4、128.5、128.3、127.4、127.3、125.0、124.9、124.8、124.1、123.9、122.5、118.1、118.0、117.7、116.0、115.9、114.4、114.0、110.8、110.4、109.5、108.7、103.3、97.4、97.3、69.7、69.6、69.6、69.4、66.0、59.1、58.7、56.3、52.1、50.8、50.8、48.0、45.8、45.6、44.9、44.9、44.8、44.8、44.7、41.2、40.5、40.4、40.1、38.5、38.0、37.6、37.3、37.1、36.3、36.1、36.1、36.0、32.5、31.7、31.2、30.6、30.3、30.3、30.1、29.9、29.9、29.8、29.8、29.7、29.5、29.4、29.3、28.1、27.7、27.7、27.3、27.2、27.1、26.6、26.4、26.2、26.1、26.0、25.1、25.0、24.9、23.3、14.4、12.8、12.8。IR(neat,ν max / cm -1 )3358、2956、2923、2853、2096、1632、1578、1555、1504、1459、1420、1378、1378、1355、1321、1259、1238、1191、1134、1121、1080、1041。HRMS(ESI):m / z=775.8653[M+H] 2+ (C 80 H105 N 13 O 15 Calculated m / z for S2 = 775.8642)

[0177] Example 6 N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide [ka] 3,7-Di(azetidin-1-yl)-N-(2-(2-azidoethoxy)ethyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide To a solution of tert-butyl 3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxylate (9.5 mg, 17.2 μmol, 1.0 equiv., prepared as described in Nat. Methods, 2020, 17, 815-821) in anhydrous CHCl (200 μL), TFA (100 μL) was added, and the resulting dark blue solution was stirred for 1 h. The solvent was then evaporated in vacuo, and the residue was redissolved in CHCl and co-evaporated (×3). To the crude product was then added i-PrNEt (0.5% solution in DMF, 172 μL, 86.4 μmol, 5.0 equiv.), and the resulting pale blue solution was cooled to 0 °C. HATU (0.1 M solution in DMF, 258 μL, 25.8 μmol, 1.5 equiv.) was then added, the reaction was stirred at 0 °C for 15 min, and 2-(2-azidoethoxy)ethanamine (2.4 mg, 5.3 μmol, 1.0 equiv., CAS RN464190-91-8) in CHCl (0.2 mL) was added at 0 °C, the mixture was warmed to ambient temperature, and stirred for an additional 30 min. The reaction mixture was then diluted with saturated aqueous NaHCO (5 mL) and CHCl (10 mL), and the phases were separated. The aqueous phase was extracted with CHCl (2 × 10 mL), dried over NaSO, and concentrated in vacuo. Purification by preparative TLC (SiO, 3% MeOH in CHCl) afforded the product (6.0 mg, 57%) as a blue solid. 1 H NMR(500MHz,CD2Cl2)δ=7.96(dd,J=8.0,0.8Hz,1H), 7.89(dd,J=8.0,1.4Hz,1H), 7.60 (dd,J=1.4,0.8Hz,1H), 6.73(d,J=8.7Hz,2H), 6.68(d,J=2.6Hz,2H), 6.56(s,1H), 6.28 (dd,J=8.7,2.6Hz,2H), 3.89(t,J=7.3Hz,8H), 3.69-3.63(m,2H), 3.61(dtd,J=4.8,3.6 ,1.3Hz,4H), 3.37-3.32(m,2H), 2.36(tt,J=7.8,6.8Hz,4H), 0.62(s,3H), 0.54(s,3H). 13C NMR(126MHz,CD2Cl2)δ=170.16, 166.50, 155.83, 151.77, 140.60, 136.85, 132.39, 129.16, 128.37 , 127.96, 126.30, 123.57, 116.08, 112.92, 70.68, 70.10, 52.85, 51.20, 40.46, 17.43, 0.41, -0.97. IR(neat,ν max / cm -1 )3332, 2924, 2854, 2103, 1755, 1662, 1594, 1547, 1478, 1405, 1352, 1306, 1267, 1246, 1163, 1124, 1097, 1039. HRMS(ESI):m / z=609.2641[M+H] + (C 33 H 37 Calculated m / z for N6O4Si = 609.2640

[0178] Step c) tert-butyl 7-(1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate tert-Butyl 7-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)heptanoate (2.0 mg, 3.7 μmol, 1.5 equiv.), 3,7-di(azetidin-1-yl)-N-(2-(2-azidoethoxy)ethyl)-5,5-dimethyl-3'-oxo-3 To a heart-shaped flask charged with 'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide (1.5 mg, 2.5 μmol, 1.0 equiv.) and Cu[MeCN]PF (4.6 mg, 12.3 μmol, 5.0 equiv.) was added anhydrous CHCl (0.3 mL, pre-degassed with argon) and 10 μL of AcOH. The flask was then flushed with argon, stoppered with a glass stopper, and the resulting deep blue solution was stirred for 16 h. The reaction mixture was then loaded directly onto a silica plate and purified by preparative TLC (SiO, 6% MeOH in CHCl) to afford the product (1.4 mg, 49%) as a blue solid. 11H NMR (500 MHz, CD3OD) δ = 8.77 (dd, J = 2.5, 0.7 Hz, 1H), 8.53 (td, J = 1.9, 0.5 Hz, 1H), 8.26 (ddd, J = 8.0, 2.0, 1.1 Hz, 1H), 8.15 (ddd, J = 7.9, 1.7, 1.1 Hz, 1H), 8.01 - 7.92 (m, 2H), 7.88 (s, 1H), 7.82 (dd, J = 9.7, 2.5 Hz, 1H), 7.71 - 7.64 (m, 2H), 6.71 (dd, J = 2.7, 0.4 Hz, 2H), 6.69 (dd, J = 8.7, 0.4 Hz, 2H), 6.37 (dd, J = 9.6, 0.7 Hz, 1H), 6.29 (dd, J = 8.7, 2.7 Hz, 2H), 4.52 (t, J = 4.8 Hz, 2H), 4.37 (s, 2H), 3.85 (t, J = 7.2 Hz, 8H), 3.82 (t, J = 4.3 Hz, 2H), 3.56 (t, J = 5.4 Hz, 3H), 3.48 (t, J = 5.3 Hz, 3H), 3.34 - 3.32 (m, 2H), 2.34 (p, J = 7.3 Hz, 4H), 2.22 (t, J = 7.4 Hz, 2H), 1.66 - 1.53 (m, 4H), 1.43 (s, 9H), 1.40 - 1.35 (m, 4H), 0.59 (s, 3H), 0.51 (s, 3H). 13 13C NMR (126 MHz, CD3OD) δ = 196.21 (HMBC), 161.31 (HSQC), 152.79, 141.44, 138.10, 136.51, 135.18, 133.91, 133.28, 129.01, 126.75, 125.43, 124.67, 122.91, 116.89, 113.86, 70.15, 53.36, 51.42, 36.33, 30.23, 29.81, 28.37, 27.74, 26.11, 24.08, 17.77, 17.28, 10.35, 0.26, -1.31. IR (neat, ν max / cm -1 ) 3310, 2928, 2856, 1756, 1654, 1594, 1545, 1454, 1367, 1306, 1247, 1189, 1151, 1058. HRMS (ESI): m / z = 1152.4678 [M + H] + (C 60 H 70 N9O 11 SSi calculated m / z = 1152.4679)

[0179] Step d) N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide To a solution of tert-butyl 7-(1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)heptanoate (4.0 mg, 3.5 μmol, 1.0 equiv) in anhydrous CHCl (200 μL) was added TFA (100 μL) and the resulting dark blue reaction mixture was stirred for 1 h. The solvent was then evaporated in vacuo and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (0.1 mL). TFA (0.3 M solution in anhydrous DMF, 38.2 μL, 11.5 μmol, 3.3 equiv.) and i-PrNEt (0.5 M solution in anhydrous DMF, 43.7 μL, 21.9 μmol, 6.3 equiv.) were added, and the resulting pale green solution was cooled to 0° C. HATU (0.1 M solution in DMF, 38.2 μL, 3.8 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0 °C for 15 min, and ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine was added (0.05 M solution in anhydrous CHCl, 72.8 μL, 3.6 μmol, 1.05 equiv.). The reaction mixture was then warmed to ambient temperature and stirred for an additional 30 min. The reaction mixture was then concentrated in vacuo, and the residue was dissolved in CHCl and loaded directly onto a silica plate. Purification by preparative TLC (SiO, 10% MeOH in CHCl) afforded the product (2.2 mg, 41%) as a blue solid. 11H NMR (600 MHz, CDCl3) δ = 8.66 (s, 1H), 8.50 (s, 1H), 8.37 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 7.7 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.74 (s, 2H), 7.67 (s, 1H), 7.62 (t, J = 7.9 Hz, 1H), 6.78 (d, J = 8.7 Hz, 2H), 6.64 (d, J = 2.6 Hz, 2H), 6.47 (s, 2H), 6.35 (d, J = 9.6 Hz, 1H), 6.24 (dd, J = 8.7, 2.7 Hz, 2H), 5.61 (s, 1H), 4.56 - 4.35 (m, 4H), 3.96 (s, 1H), 3.93 - 3.87 (m, 8H), 3.85 - 3.83 (m, 2H), 3.83 - 3.77 (m, 2H), 3.73 (s, 6H), 3.58 (dd, J = 9.2, 4.9 Hz, 2H), 3.32 - 3.27 (m, 1H), 3.22 (t, J = 7.0 Hz, 1H), 2.36 (p, J = 15.8, 7.5 Hz, 4H), 2.22 (t, J = 9.8 Hz, 2H), 2.19 - 2.14 (m, 1H), 2.07 - 2.04 (m, 1H), 2.01 (q, J = 6.3 Hz, 2H), 1.96 (s, 1H), 1.77 - 1.46 (m, 9H), 1.38 - 1.16 (m, 17H), 1.​​​​​​​​​​​​​​​​​​ Example 7 N-(9-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-9-oxononyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide [ka]

[0181] Step a) tert-butyl 9-(6-hydroxynicotinamide)nonanoate A solution of 6-hydroxynicotinic acid (91.0 mg, 654 μmol, 1.0 equiv.), tert-butyl 9-aminononanoate (150 mg, 654 μmol, 1.0 equiv., CAS RN134857-22-0), HOBt·HO (125 mg, 818 μmol, 1.25 equiv.), EDCI·HCl (157 mg, 818 μmol, 1.25 equiv.), and EtN (228 μL, 1.64 mmol, 2.5 equiv.) in DMF (1.5 mL) was stirred at ambient temperature for 16 h. The mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO2, dry loading onto SiO2, 50% hexanes (EtOAc:EtOH, 3:1)) to afford the title compound as a white solid (162 mg, 71%). 1 H NMR(400MHz,CD2Cl2)δ12.64(bs,1H), 8.09(d,J=2.6Hz,1H), 7.84(dd,J=9.5,2.5Hz,1H), 7.11(t,J=5.6Hz,1H), 6 .45(d,J=9.5Hz,1H), 3.39-3.26(m,2H), 2.16(t,J=7.5Hz,2H), 1.65-1.46(m,4H), 1.41(s,9H), 1.37-1.23(m,8H). 13C NMR (101MHz, CD2Cl2) δ173.7, 165.2, 164.7, 140.3, 137.4, 119.7, 115.5, 80.3, 40.6, 36.1, 30.1, 29.8, 29.7, 29.6, 28.4, 27.5, 25.6. IR(neat,ν max / cm -1 )3327, 2930, 2853, 2700, 1727, 1690, 1625, 1608, 1531, 1474, 1432, 1420, 1390, 1365, 1312, 1254, 1154, 1102. HRMS(ESI):m / z=373.2098[M+Na] + (C 19 H 30 Calculated m / z for N2NaO4 = 373.2098)

[0182] Step b) tert-butyl 9-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)nonanoate tert-Butyl 9-(6-hydroxynicotinamide)nonanoate (20.0 mg, 57.0 μmol, 1.0 equiv.) was dissolved in anhydrous THF (0.50 mL) at room temperature using sonication and cooled to -78°C. t Bu (1 M in THF, 114 μL, 114 μmol, 2.0 equiv.) was added dropwise at −78 °C, and the solution was stirred at −78 °C for 10 min. Subsequently, 3-(prop-2-yn-1-ylcarbamoyl)benzenesulfonyl chloride (44.1 mg, 171 μmol, 3.0 equiv., CAS RN1016841-21-6) dissolved in anhydrous THF (0.30 mL) was added dropwise, and the mixture was stirred at −78 °C for 2 h before warming to room temperature. The solvent was removed in vacuo, and the residue was redissolved in CHCl and adsorbed onto silica. The residue was purified by flash column chromatography (SiO, dry loading onto SiO, 30% (EtOAc:EtOH, 3:1) in hexanes) to afford the title compound as a white waxy solid (23 mg, 70%). 1H NMR(400MHz,CD3OD)δ8.80(dd,J=2.5,0.7Hz,1H), 8.55(td,J=1.9,0.6Hz,1H), 8.31(ddd,J=8 .0,2.0,1.1Hz,1H), 8.22(ddd,J=7.9,1.8,1.1Hz,1H), 7.89(dd,J=9.7,2.5Hz,1H), 7.76(td,J =7.9,0.5Hz,1H), 6.45(dd,J=9.6,0.7Hz,1H), 4.17(d,J=2.6Hz,2H), 3.35(t,J=7.2Hz,2H), 2. 64(t,J=2.6Hz,1H), 2.21(t,J=7.3Hz,2H), 1.69-1.51(m,4H), 1.44(s,9H), 1.42-1.30(m,8H). 13 C NMR(101MHz,CD3OD)δ175.1, 167.2, 165.7, 161.4, 141.6, 138.2, 136.4, 135.2, 135.1, 134.0, 130.7 , 129.8, 122.9, 116.4, 81.3, 80.4, 72.4, 41.1, 36.4, 30.4, 30.3, 30.3, 30.1, 30.1, 28.4, 28.0, 26.2. IR(neat,ν max / cm -1 )3298, 3073, 2928, 2856, 1726, 1692, 1638, 1534, 1460, 1368, 1300, 1252, 1187, 1153, 1096. HRMS(ESI):m / z=594.2245[M+Na] + (C 29 H 37 Calculated m / z for N3NaO7S = 594.2244

[0183] Step c) tert-butyl 9-(1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide)nonanoate To a vial containing N-(2-(2-azidoethoxy)ethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (7.0 mg, 23.7 μmol, 2.0 equiv.) and tert-butyl 9-(6-oxo-1-((3-(prop-2-yn-1-ylcarbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamido)nonanoate (6.8 mg, 11.8 μmol, 1.0 equiv.) in a glovebox, [Cu(MeCN)4]PF6 (22.2 mg, 59.4 μmol, 5.0 equiv.) was added. The mixture was dissolved in argon-degassed MeOH (0.12 mL) and CHCl2 (0.12 mL). The reaction mixture was heated to 40 °C and stirred under argon for 16 h. The reaction mixture was loaded directly onto a SiO2 plate and the crude product was purified by preparative TLC (SiO2, 2-6% MeOH in CH2Cl2) and triturated with hexanes to give the title product as an orange solid (7.5 mg, 73%). 1 H NMR(400MHz,CD3OD)δ8.77(dd,J=2.5,0.7Hz,1H), 8.53(t,J=1.8Hz,1H), 8.43(d,J=8.9Hz,1H), 8.27(ddd,J=8.0, 2.0,1.1Hz,1H), 8.19(ddd,J=7.9,1.7,1.1Hz,1H), 7.92(s,1H), 7.85(dd,J=9.7,2.5Hz,1H), 7.72(td,J=7.9,0.5 Hz,1H), 6.40(dd,J=9.7,0.7Hz,1H), 6.31(d,J=8.9Hz,1H), 4.64-4.49(m,4H), 3.94-3.86(m,2H), 3.79-3.71(m,2 H), 3.69(bs,2H), 3.34(d,J=7.2Hz,2H), 2.21(t,J=7.3Hz,2H), 1.70-1.52(m,4H), 1.43(s,9H), 1.40-1.23(m,8H). 13C NMR(101MHz,CD3OD)δ175.2, 167.5, 165.6, 161.4, 145.8, 141.5, 138.1, 136.5, 135.2, 135.0, 133.9, 130.6 , 129.8, 125.3, 122.9, 116.4, 81.3, 70.3, 70.1, 51.4, 41.2, 36.4, 36.3, 30.3, 30.3, 30.1, 28.4, 28.0, 26.2. IR(neat,ν max / cm -1 )3300, 3075, 2925, 2854, 1727, 1691, 1643, 1583, 1532, 1456, 1368, 1301, 1257, 1189, 1148. HRMS(ESI):m / z=865.3298[M+H] + (C 39 H 49 N 10 O 11 Calculated m / z for S = 865.3296)

[0184] Step d) N-(9-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-9-oxononyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide To a solution of tert-butyl 9-(1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamido)nonanoate (3.0 mg, 3.4 μmol, 1.0 equiv) in anhydrous CHCl (200 μL) was added TFA (100 μL) and the reaction mixture was stirred at room temperature for 30 min. The solvent was then removed in vacuo, and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (50 μL). TFA (0.5 M solution in anhydrous DMF, 22.9 μL, 11.4 μmol, 3.3 equiv.) and i-PrNEt (0.5 M solution in anhydrous DMF, 36.8 μL, 18.3 μmol, 5.3 equiv.) were added at room temperature, and the resulting solution was cooled to 0° C. HATU (0.1 M solution in DMF, 38.2 μL, 3.8 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0° C. for 15 minutes, and ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.1 M solution in anhydrous CHCl, 38.2 μL, 3.8 μmol, 1.1 equiv.) was added. The reaction mixture was then warmed to ambient temperature and stirred for an additional 45 minutes. The residue was dissolved in CHCl (10 mL) and washed with saturated aqueous NHCl (5 mL), saturated aqueous NaHCO (5 mL), and brine (5 mL). The organic layer was dried over NaSO, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO, 4% MeOH in CHCl) to afford the product (3.5 mg, 81%) as an orange solid. 1H NMR(500MHz,CD2Cl2)δ8.59(dd,J=2.5,0.7Hz,1H)、8.46(d,J=8.7Hz,1H)、8.40(t,J=1.7Hz,1H)、8.25(ddd,J=8.0,1.9,1.1Hz,1H)、8.11(d,J=7.8Hz,1H)、7.72(dd,J=9.6,2.5Hz,1H)、7.70(s,1H)、7.63(t,J=7.9Hz,1H)、7.43(bs,1H)、6.57(t,J=5.9Hz,1H)、6.49(s,2H)、6.36(dd,J=9.7,0.6Hz,1H)、6.23(d,J=8.7Hz,1H)、5.58(dt,J=3.0,1.5Hz,1H)、5.48(t,J=5.7Hz,1H)、4.64(d,J=5.6Hz,2H)、4.53(t,J=4.9Hz,2H)、3.96-3.93(m,1H)、3.90(t,J=5.0Hz,2H)、3.87-3.77(m,2H)、3.77-3.73(m,2H)、3.72(s,6H)、3.66(bs,2H)、3.40-3.33(m,2H)、3.22(t,J=7.0Hz,2H)、2.21-2.13(m,4H)、2.11-1.98(m,3H)、1.67(d,J=8.4Hz,1H)、1.63-1.50(m,6H)、1.36-1.21(m,12H)、1.27(s,3H)、1.26(s,6H)、1.15-1.06(m,2H)、0.94(s,3H)。 13 C NMR(126MHz,CD2Cl2)δ173.3、165.5、163.7、160.0、159.0、150.0、140.4、139.2、137.2、137.0、135.8、134.2、133.7、133.4、129.8、128.7、124.6、124.0、123.0、118.0、115.8、103.3、69.8、69.1、56.2、52.0、50.8、48.0、44.9、44.8、41.2、40.7、38.5、38.0、37.3、36.1、32.5、30.4、30.2、29.6、29.5、29.4、29.3、28.1、27.7、27.2、27.1、26.6、26.2、25.1、23.3、21.3。IR(neat,ν max / cm -1)3322, 2924, 2853, 2096, 1651, 1534, 1463, 1411, 1377, 1302, 1189, 1123. HRMS(ESI):m / z=645.2793[M+Na2] 2+ (C 62 H 80 N 14 NaO 12 Calculated m / z for S = 645.2793

[0185] Example 8 N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide [ka]

[0186] Step a) tert-butyl 7-(4-(N-(pyridin-2-ylmethyl)sulfamoyl)benzamido)heptanoate A solution of 4-chlorosulfonylbenzoyl chloride (855 mg, 3.56 mmol, 3.75 equiv., CAS RN 7516-60-1) in anhydrous CHCl (30 mL) was cooled to 0 °C, and EtN (1.04 mL, 5.96 mmol, 6.25 equiv.) was added dropwise, followed by tert-butyl 7-aminoheptanoate (240 g, 954 μmol, 1.00 equiv., CAS RN 105974-64-9). The solution was stirred at 0 °C for 1 h, concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO, 10% EtOAc in CHCl). The sulfonyl chloride was used immediately in the next step. tert-Butyl 7-(4-(chlorosulfonyl)benzamido)heptanoate (260 mg, 644 μmol, 1.0 equiv.) was dissolved in anhydrous dioxane (1.5 mL). Pyridin-2-ylmethanamine (73 μL, 708 μmol, 1.1 equiv.) and i-PrNEt (170 μL, 966 μmol, 1.5 equiv.) were added, and the solution was stirred at room temperature overnight. The mixture was diluted with EtOAc (50 mL) and saturated aqueous NHCl (50 mL). The layers were separated, and the aqueous phase was extracted with EtOAc (2 × 50 mL). The combined organic phases were washed with brine (30 mL), dried over MgSO, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO, 70% EtOAc in hexanes) to afford the title compound as an amorphous solid (232 mg, 76%). 1 H NMR(500MHz,CD3Cl)δ8.44(ddd,J=4.9,1.8,1.0Hz,1H), 7.94-7.86(m,2H), 7.83-7.78(m,2H), 7.60(td,J=7.7,1.8Hz,1H), 7.19-7.12(m,2H), 6.27( bs,1H), 6.16(bs,1H), 4.26(d,J=5.1Hz,2H), 3.44(td,J=7.2,5.8Hz,2H), 2.21(t,J=7.4Hz,2H), 1.71-1.52(m,4H), 1.43(s,9H), 1.41-1.30(m,4H). 13 C NMR(126MHz,CD3Cl)δ173.34, 166.16, 154.37, 149.14, 142.28, 138.76, 137.04, 127.74, 127.56, 122.93, 122.05, 80.26, 47.36, 40.27, 35.53, 29.45, 28.71, 28.26, 26.67, 25.00. IR(neat,ν max / cm -1 ):3289, 2931, 2859, 1726, 1645, 1597, 1543, 1439, 1393, 1367, 1334, 1152, 1094. HRMS(ESI):m / z=498.2034[M+Na] + (C 24 H 33Calculated m / z for N3NaO5S = 498.2033

[0187] Step b) tert-butyl 7-(4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamido)heptanoate 6-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoic acid (14.5 mg, 44.4 μmol, 1.25 equiv., CAS RN88235-25-0), EDCI HCl (14.0 mg, 73.0 μmol, 2.00 equiv.), i-PrNEt (18.6 μL, 106 μmol, 3.00 equiv.), and DMAP (5.4 mg, 44.4 μmol, 1.25 equiv.) were dissolved in anhydrous DMF (200 μL), and the mixture was stirred at room temperature for 5 min. tert-Butyl 7-(4-(N-(pyridin-2-ylmethyl)sulfamoyl)benzamido)heptanoate (16.9 mg, 35.5 μmol, 1.00 equiv.) was added to anhydrous DMF (100 μL), and the reaction mixture was stirred at room temperature overnight. The mixture was partitioned between CH2Cl2 (20 mL) and saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous phase was extracted with CH2Cl2 (2 x 20 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO2, 70% EtOAc in hexanes) to afford the title compound as an orange solid (6.1 mg, 23%). 11H NMR (500 MHz, CD2Cl2) δ 8.49 - 8.44 (m, 2H), 7.93 - 7.87 (m, 2H), 7.84 - 7.80 (m, 2H), 7.73 (td, J = 7.7, 1.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.23 (ddd, J = 7.6, 4.9, 1.1 Hz, 1H), 6.50 (bs, 1H), 6.29 (t, J = 5.7 Hz, 1H), 6.16 (d, J = 8.7 Hz, 1H), 5.19 (s, 2H), 3.50 - 3.37 (m, 4H), 2.61 (t, J = 7.1 Hz, 2H), 2.19 (t, J = 7.4 Hz, 2H), 1.75 - 1.65 (m, 2H), 1.64 - 1.52 (m, 6H), 1.42 (s, 9H), 1.40 - 1.26 (m, 6H). 13 13C NMR (126 MHz, CD2Cl2) δ 173.54, 173.34, 166.00, 156.01, 149.72, 144.86, 144.50, 142.08, 140.15, 137.36, 137.10, 129.16, 127.56, 123.21, 122.06, 99.07, 80.15, 51.10, 44.04, 40.52, 35.88, 35.75, 29.71, 29.01, 28.41, 28.23, 26.94, 26.35, 25.32, 23.99. IR (neat, ν max / cm -1 ): 3335, 2926, 2856, 1708, 1649, 1622, 1585, 1531, 1490, 1445, 1365, 1298, 1261, 1169, 1123, 1088. HRMS (ESI): m / z = 774.2904 [M + Na] + (C 36 H 45 N7NaO9S calculated m / z = 774.2892)

[0188] Step c) N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide tert-Butyl 7-(4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamido)heptanoate (6.4 mg, 8.4 μmol, 1.1 equiv) was dissolved in a mixture of CHCl (200 μL) and TFA (100 μL), and the solution was stirred at room temperature for 2 h. The volatiles were removed in vacuo, and the residue was dissolved in anhydrous DMF (0.1 mL) and cooled to 0 °C. Stock solutions of TFA (0.5 M in DMF, 69 μL, 34.6 μmol, 4.5 equiv.), i-Pr2NEt (0.5 M in DMF, 123 μL, 61.6 μmol, 8.0 equiv.), and HATU (0.1 M in DMF, 115 μL, 11.5 μmol, 1.5 equiv.) were then added, and the mixture was stirred for 15 min at 0 °C. ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (3.5 mg, 7.6 μmol, 1.0 equiv.) dissolved in anhydrous CHCl2 (50 μL) was added, and the reaction mixture was warmed to room temperature and stirred for 45 min. The crude mixture was concentrated in vacuo and purified by preparative TLC (SiO 2 , 95% EtOAc in hexanes) and triturated with hexanes to afford the title compound as an orange solid (6.2 mg, 71%). 1H NMR1H NMR(500MHz,CD2Cl2)δ8.52-8.48(m,1H)、8.47(d,J=8.7Hz,1H)、7.93(d,J=8.5Hz,2H)、7.90-7.80(m,3H)、7.47(d,J=7.8Hz,1H)、7.34(bs,1H)、6.59(bs,1H)、6.48(s,2H)、6.16(d,J=8.7Hz,1H)、5.58(dt,J=2.9,1.4Hz,1H)、5.45(t,J=5.7Hz,1H)、5.26(s,2H)、3.98-3.92(m,1H)、3.88-3.75(m,2H)、3.72(s,6H)、3.48-3.37(m,4H)、3.21(t,J=7.0Hz,2H)、2.64(t,J=7.0Hz,2H)、2.22-2.12(m,3H)、2.07(td,J=5.7,1.4Hz,1H)、2.03-1.98(m,1H)、1.76-1.48(m,13H)、1.44-1.21(m,10H)、1.27(s,3H)、1.26(s,6H)、1.16-1.05(m,2H)、0.94(s,3H)。 13 C NMR(126MHz,CD2Cl2)δ173.48、172.89、165.92、158.91、155.65、149.92、140.37、139.09、137.09、129.05、127.86、123.91、117.92、103.22、56.13、51.93、47.87、44.85、44.64、44.09、41.11、40.40、38.36、37.92、36.94、36.02、32.36、30.25、30.12、29.58、29.23、29.14、28.98、28.44、27.98、27.02、26.79、26.46、26.40、25.98、25.02、24.08、21.17。IR(neat、ν max / cm -1 ):3315、2925、2855、2095、1707、1647、1622、1573、1531、1448、1410、1353、1297、1261、1239、1169、1120、1088、1032。HRMS(ESI):m / z=1154.5472[M+H] + (C 59 H 77 N 11 NaO 10Calculated m / z for S = 1154.5468

[0189] Example 9 N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide [ka]

[0190] Step c) N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide tert-Butyl 7-(4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamido)heptanoate (3.0 mg, 3.9 μmol, 1.0 equiv) was dissolved in a mixture of CHCl (200 μL) and TFA (100 μL), and the solution was stirred at room temperature for 2 h. The volatiles were removed in vacuo, and the residue was dissolved in anhydrous DMF (0.1 mL) and cooled to 0 °C. Stock solutions of TFA (0.5 M in DMF, 35.9 μL, 17.9 μmol, 4.5 equiv.), i-Pr2NEt (0.5 M in DMF, 60 μL, 29.9 μmol, 7.5 equiv.), and HATU (0.1 M in DMF, 60 μL, 5.9 μmol, 1.5 equiv.) were then added, and the mixture was stirred for 15 min at 0 °C. ((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (1.7 mg, 4.1 μmol, 1.05 equiv.) dissolved in anhydrous CHCl2 (50 μL) was added, and the reaction mixture was allowed to warm to room temperature and stirred for 45 min. Additional i-PrNEt (0.5 M in DMF, 10 μL, 5.0 μmol, 1.25 equiv.) was added, and the reaction mixture was stirred at room temperature for 1.5 h. The crude mixture was concentrated in vacuo and purified by preparative TLC (SiO, 95% EtOAc in hexanes) to afford the title compound as an orange solid (4.2 mg, 96%) after trituration with hexanes. 1H NMR1H NMR(500MHz,CD2Cl2)δ8.50-8.42(m,2H)、7.93-7.88(m,2H)、7.87-7.80(m,2H)、7.72(td,J=7.7,1.8Hz,1H)、7.36(dt,J=7.8,1.0Hz,1H)、7.23(ddd,J=7.6,4.8,1.1Hz,1H)、6.52(s,1H)、6.49(s,2H)、6.43(t,J=5.8Hz,1H)、6.16(d,J=8.7Hz,1H)、5.59(dt,J=2.9,1.4Hz,1H)、5.47-5.41(m,1H)、5.19(s,2H)、3.95(t,J=2.3Hz,1H)、3.89-3.74(m,2H)、3.72(s,6H)、3.48-3.37(m,4H)、2.61(t,J=7.0Hz,2H)、2.22-2.13(m,3H)、2.08(td,J=5.7,1.4Hz,1H)、2.05-1.97(m,1H)、1.74-1.54(m,13H)、1.45-1.19(m,10H)、1.28(s,3H)、1.26(s,6H)、1.15-1.05(m,2H)、0.95(s,3H)、0.84(t,J=4.4Hz,3H)。 13 C NMR(126MHz,CD2Cl2)δ173.51、172.87、165.96、158.83、156.04、150.03、149.74、142.07、140.14、139.01、137.34、137.10、129.12、127.62、123.90、123.19、122.05、117.77、103.17、56.09、51.11、47.81、44.78、44.71、44.61、44.04、41.07、40.35、38.34、37.87、36.92、35.88、32.18、30.43、30.10、29.55、29.16、28.94、28.42、27.94、26.75、26.42、26.36、25.95、25.08、24.00、23.07、21.14、14.25。IR(neat,ν max / cm -1 ):3321、2926、2855、1706、1648、1572、1531、1448、1410、1353、1297、1260、1170、1122、1019。HRMS(ESI):m / z=1113.5443[M+Na]+ (C 59 H 78 N8NaO 10 Calculated m / z for S = 1113.5454)

[0191] Example 10 N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamide [ka]

[0192] Step a) tert-Butyl 7-(4-sulfamoylbenzamido)heptanoate To a solution of 4-sulfamoylbenzoic acid (550 mg, 2.73 mmol, 1.0 equiv., CAS RN 138-41-0) in anhydrous DMF (11.0 mL) was added tert-butyl 7-aminoheptanoate (688 mg, 2.73 mmol, 1.0 equiv., CAS RN 105974-64-9), HOBt·HO (628 mg, 4.10 mmol, 1.5 equiv.), EDCI·HCl (786 mg, 4.10 mmol, 1.5 equiv.), and i-Pr·NEt (1.43 mL, 8.20 mmol, 3.0 equiv.). The solution was stirred at room temperature for 16 h. The mixture was diluted with EtOAc (100 mL) and saturated aqueous NaHCO (50 mL). The layers were separated, and the aqueous phase was extracted with EtOAc (2 × 100 mL). The combined organic phase was washed with brine (30 mL), dried over MgSO, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO, 4-6% MeOH in CHCl) to afford the title compound as a white solid (700 mg, 67%). 1H NMR (400MHz, CD3OD) δ8.00-7.92(m,4H), 3.39(t,J=7.2Hz,2H), 2.23(t,J=7.3Hz,2H), 1.71-1.54(m,4H), 1.44(s,9H), 1.42-1.34(m,4H). 13 C NMR (101MHz, CD3OD) δ175.1, 168.7, 147.6, 139.2, 128.9, 127.3, 81.4, 41.1, 36.3, 30.2, 29.8, 28.3, 27.7, 26.1. IR(neat,ν max / cm -1 ):3321, 2926, 2854, 1730, 1629, 1559, 1456, 1367, 1342, 1166, 1151, 1097. HRMS(ESI):m / z=407.1605[M+Na] + (C 18 H 28 Calculated m / z for N2NaO5S = 407.1611)

[0193] Step b) tert-butyl 7-(4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamido)heptanoate 6-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoic acid (112 mg, 343 μmol, 1.1 equiv., CAS RN88235-25-0), EDCI HCl (90.0 mg, 468 μmol, 1.5 equiv.), i-PrNEt (272 μL, 1.56 mmol, 5.0 equiv.), and DMAP (11.4 mg, 93.6 μmol, 0.3 equiv.) were dissolved in anhydrous DMF (1.2 mL), and the mixture was stirred at room temperature for 5 min. tert-Butyl 7-(4-sulfamoylbenzamido)heptanoate (120 mg, 312 μmol, 1.0 equiv.) was added, and the reaction mixture was stirred at room temperature overnight. The mixture was concentrated in vacuo. The crude product was purified by flash column chromatography (1% AcOH, 1-4% MeOH in CH2Cl2) to give the title compound as an orange amorphous solid, which was recrystallized in CHCl3 (59 mg, 29%). Unreacted sulfonamide starting material was also recovered (62 mg). 1 H NMR(400MHz,CD3OD)δ8.60(t,J=5.7Hz,1H), 8.42(d,J=8.8Hz,1H), 8.09-8.02(m,2H), 7.98-7.92(m,2H), 6.23(d,J=8.9Hz ,1H), 3.49-3.33(m,4H), 2.27(t,J=7.2Hz,2H), 2.20(t,J=7.4Hz,2H), 1.75-1.52(m,8H), 1.42(s,9H), 1.39-1.30(m,6H). 13 C NMR(101MHz,CD3OD)δ175.05, 173.63, 168.34, 146.50, 145.74, 145.43, 143.20, 140.67, 138.49, 129.34, 12 8.81, 122.86, 99.61, 81.34, 44.48, 41.13, 36.69, 36.30, 30.17, 29.77, 28.34, 27.71, 27.19, 26.07, 25.09. IR(neat,ν max / cm -1 ):3288, 2932, 2859, 1721, 1641, 1622, 1584, 1531, 1495, 1447, 1366, 1297, 1170, 1151, 1088. HRMS(ESI):m / z=683.2466[M+Na] + (C 30 H 40 Calculated m / z for N6NaO9S = 683.2470

[0194] Step c) tert-butyl 7-(4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamido)heptanoate To a stirred solution of tert-butyl 7-(4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamido)heptanoate (7.0 mg, 10.6 μmol, 1.0 equiv) in anhydrous DMF (0.1 mL) was added iodoacetonitrile (8 μL, 106 μmol, 10 equiv) and i-PrNEt (0.5 M in DMF, 106 μL, 53.0 μmol, 5.0 equiv). The mixture was stirred at room temperature overnight. The mixture was concentrated in vacuo, and the crude product was purified by preparative TLC (SiO, 70% EtOAc in hexanes) and triturated with hexanes to give the title compound as an orange solid (7.3 mg, 98%). 1 H NMR(500MHz,CD2Cl2)δ8.48(d,J=8.6Hz,1H), 8.06-8.01(m,2H), 8.01-7.96(m,2H), 6.54(bs,1H), 6.35(bs,1H), 6.19(d,J=8.7Hz,1 H), 4.76(s,2H), 3.51-3.39(m,4H), 2.70(t,J=7.1Hz,2H), 2.19(t,J=7.4Hz,2H), 1.80-1.52(m,8H), 1.42(s,9H), 1.40-1.27(m,6H). 13 C NMR(126MHz,CD2Cl2)δ173.36, 172.08, 165.64, 144.89, 144.50, 141.44, 140.69, 137.09, 128.78, 128.39, 115. 16, 99.11, 80.18, 44.03, 40.62, 36.24, 35.73, 33.74, 29.64, 28.97, 28.50, 28.22, 26.92, 26.43, 25.29, 24.27. IR(neat, ν max / cm -1 ):3338, 2926, 2856, 1716, 1653, 1621, 1585, 1532, 1491, 1447, 1401, 1367, 1298, 1169, 1126, 1089. HRMS(ESI):m / z=722.2574[M+Na] + (C 32 H 41 Calculated m / z for N7NaO9S = 722.2579

[0195] Step d) N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamide tert-Butyl 7-(4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamido)heptanoate (3.6 mg, 5.1 μmol, 1.0 equiv) was dissolved in a mixture of CHCl (200 μL) and TFA (100 μL), and the solution was stirred at room temperature for 2 h. The volatiles were removed in vacuo, and the residue was dissolved in anhydrous DMF (0.1 mL) and cooled to 0 °C. Stock solutions of TFA (0.5 M in DMF, 46 μL, 23.2 μmol, 4.5 equiv.), i-Pr2NEt (0.5 M in DMF, 82 μL, 41.2 μmol, 8.0 equiv.), and HATU (0.1 M in DMF, 77 μL, 77.2 μmol, 1.5 equiv.) were then added, and the mixture was stirred for 15 min at 0 °C. ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (2.4 mg, 5.1 μmol, 1.0 equiv.) dissolved in anhydrous CHCl2 (50 μL) was added, and the reaction mixture was warmed to room temperature and stirred for 45 min. The crude mixture was concentrated in vacuo and purified by preparative TLC (SiO 2 , 95% EtOAc in hexanes) and triturated with hexanes to afford the title compound as an orange solid (3.2 mg, 58%). 11H NMR (500 MHz, CD2Cl2) δ 8.48 (d, J = 8.7 Hz, 1H), 8.06 - 7.99 (m, 4H), 6.61 (bs, 1H), 6.56 (bt, J = 5.7 Hz, 1H), 6.49 (s, 2H), 6.18 (d, J = 8.7 Hz, 1H), 5.58 (dt, J = 2.8, 1.5 Hz, 1H), 5.46 (bt, J = 5.7 Hz, 1H), 4.76 (s, 2H), 3.98 - 3.92 (m, 1H), 3.89 - 3.74 (m, 2H), 3.72 (s, 6H), 3.53 - 3.38 (m, 4H), 3.22 (t, J = 7.0 Hz, 2H), 2.70 (t, J = 7.1 Hz, 2H), 2.23 - 2.12 (m, 3H), 2.10 - 2.04 (m, 1H), 2.04 - 1.95 (m, 1H), 1.76 - 1.47 (m, 13H), 1.43 - 1.29 (m, 10H), 1.27 (s, 3H), 1.26 (s, 6H), 1.16 - 1.06 (m, 2H), 0.94 (s, 3H). 13 13C NMR (126 MHz, CD2Cl2) δ 173.03, 172.08, 165.64, 158.84, 149.90, 141.40, 140.67, 138.96, 137.11, 128.86, 128.34, 123.94, 117.81, 115.17, 103.16, 56.11, 51.90, 47.80, 44.79, 44.64, 44.61, 41.07, 40.36, 38.33, 37.87, 36.84, 36.23, 33.72, 30.22, 30.09, 29.35, 29.20, 29.11, 28.75, 28.48, 27.94, 26.99, 26.58, 26.41, 25.83, 24.99, 24.27, 21.13. IR (neat, ν max / cm -1 ): 3363, 2925, 2855, 2096, 1715, 1653, 1583, 1532, 1456, 1410, 1365, 1297, 1261, 1170, 1122, 1031. HRMS (ESI): m / z = 1080.5312 [M + H] + (C 55 H 74 N 11 O 10 S calculated value for m / z = 1080.5335)

[0196] Example 11 N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide [ka]

[0197] Step g) tert-butyl 7-(4-(N-(pent-4-ynoyl)sulfamoyl)benzamido)heptanoate Pent-4-ynoic acid (60.3 mg, 614 μmol, 1.05 equiv., CAS RN6089-09-4), EDCI HCl (168 mg, 878 μmol, 1.5 equiv.), i-PrNEt (510 μL, 2.93 mmol, 5.0 equiv.), and DMAP (21.6 mg, 176 μmol, 0.30 equiv.) were dissolved in anhydrous DMF (1.3 mL), and the mixture was stirred at room temperature for 15 min. Then, tert-butyl 7-(4-sulfamoylbenzamido)heptanoate (225 mg, 585 μmol, 1.0 equiv.) was added to anhydrous DMF (1.0 mL), and the reaction mixture was stirred at room temperature overnight. The mixture was partitioned between CHCl (60 mL) and saturated aqueous NHCl (20 mL). The layers were separated, and the aqueous phase was extracted with CHCl (2 × 60 mL). The combined organic layers were dried over MgSO, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO, 0.5% AcOH in CHCl, 0-4% MeOH) to afford the title compound as a white solid (190 mg, 70%), with unreacted sulfonamide starting material also recovered (52 mg). 1H NMR(400MHz,CD3OD)δ8.68(bt,J=5.8Hz,1H), 8.12-8.05(m,2H), 8.02-7.93(m,2H), 3.47-3.34(m,2H), 2.48-2.43(m,2) H), 2.39-2.33(m,2H), 2.23(t,J=7.3Hz,2H), 2.19(t,J=2.6Hz,1H), 1.71-1.53(m,4H), 1.44(s,9H), 1.42-1.33(m,4H). 13 C NMR(126MHz,CD3OD)δ175.07, 171.88, 168.53, 143.24, 140.72, 129.40, 128.82, 82 .81, 81.36, 70.46, 41.12, 36.31, 35.99, 30.20, 29.80, 28.34, 27.72, 26.10, 14.36. IR(neat,ν max / cm -1 ):3254, 2928, 2856, 2510, 1712, 1635, 1570, 1480, 1464, 1434, 1415, 1351, 1172. HRMS(ESI):m / z=487.1862[M+Na] + (C 23 H 32 Calculated m / z for N2NaO6S = 487.1873

[0198] Step h) tert-butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamido)heptanoate To a solution of tert-butyl 7-(4-(N-(pent-4-ynoyl)sulfamoyl)benzamido)heptanoate (75.0 mg, 161 μmol, 1.0 equiv.) in anhydrous DMF (0.25 mL) was added i-PrNEt (141 μL, 807 μmol, 5.0 equiv.) and iodoacetonitrile (117 μL, 1.61 mmol, 10 equiv.). The clear reaction mixture turned brown after 10 min and was stirred at room temperature for an additional 16 h. The reaction mixture was then concentrated in vacuo, redissolved in CHCl, and adsorbed onto silica gel. Purification by flash column chromatography (SiO, 30% EtOAc in hexanes) afforded the product (63.5 mg, 78%) as a yellowish wax. 1 H NMR(500MHz,CD2Cl2)δ=8.06-8.02(m,2H), 8.01-7.97(m,2H), 6.34(s,1H), 4.74(s,2H), 3.43(td,J=7.2,5.8Hz,2H), 2.97(t,J=7.1H) z,2H), 2.49(td,J=7.1,2.7Hz,2H), 2.19(t,J=7.4Hz,2H), 1.98(t,J=2.7Hz,1H), 1.69-1.50(m,4H), 1.42(s,9H), 1.41-1.31(m,4H). 13 C NMR(126MHz,CD2Cl2)δ173.5, 170.8, 165.6, 141.6, 140.5, 129.0, 128.5, 115.1 , 82.4, 80.3, 69.7, 40.7, 36.1, 35.9, 33.9, 29.8, 29.1, 28.4, 27.1, 25.5, 14.4. IR(neat,ν max / cm -1 )3300, 2934, 2861, 1718, 1649, 1542, 1487, 1367, 1293, 1169, 1154, 1088, 1070, 1001. HRMS(ESI):m / z=526.1978[M+Na] + (C 25 H 33 Calculated m / z for N3NaO6S = 526.1982

[0199] Step j) tert-butyl 7-(4-(N-(cyanomethyl)-N-(3-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamido)heptanoate To a heart-shaped flask charged with tert-butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamido)heptanoate (5.7 mg, 11.3 μmol, 1.0 equiv.), N-(2-(2-azidoethoxy)ethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (5.0 mg, 16.9 μmol, 1.5 equiv., CAS RN2449214-44-0), and Cu[MeCN]PF (21.1 mg, 56.6 μmol, 5.0 equiv.) was added anhydrous CHCl (0.3 mL, pre-degassed with argon) and 10 μL of AcOH. The flask was then flushed with argon, stoppered with a glass stopper, and the orange solution was stirred for 16 h. The reaction mixture was then loaded directly onto a silica plate and purified by preparative TLC (SiO 2 , 8% MeOH in CH 2 Cl 2 ) to give the product (3.3 mg, 37%) as an orange solid. 1 H NMR(500MHz,CD2Cl2)δ=8.48(d,J=8.4Hz,1H), 8.05-7.85(m,4H), 7.34(s,1H), 6.73( s,1H), 6.49(s,1H), 6.23(d,J=8.6Hz,1H), 4.74(s,2H), 4.53-4.41(m,2H), 3.88(s,2 H), 3.74(s,2H), 3.65(d,J=11.5Hz,2H), 3.43(t,J=6.4Hz,2H), 3.17-3.08(m,2H), 3. 00-2.92(m,2H), 2.19(t,J=7.4Hz,2H), 1.67-1.52(m,4H), 1.42(s,9H), 1.38(s,4H). 13 C NMR(126MHz,CD2Cl2)δ=173.50, 172.16, 171.97, 165.94, 145.06, 141.59, 140.56, 137.17, 128.89, 128.49, 123.54, 122.95 , 115.33, 98.84, 80.30, 69.94, 68.93, 50.60, 44.04, 40.64, 36.02, 35.92, 34.05, 29.84, 29.19, 28.39, 27.14, 25.48, 20.99. IR(neat,ν max / cm -1)3325, 3080, 2932, 2863, 1718, 1648, 1622, 1583, 1532, 1493, 1443, 1399, 1366, 1301, 1191, 1168, 1152, 1037. HRMS(ESI):m / z=819.2860[M+Na] + (C 35 H 44 N 10 NaO 10 Calculated m / z for S = 819.2855

[0200] Step k) N-(3-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-3-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide To a solution of tert-butyl 7-(4-(N-(cyanomethyl)-N-(3-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamido)heptanoate (3.3 mg, 4.1 μmol, 1.0 equiv.) in anhydrous CHCl (200 μL) was added TFA (100 μL), and the resulting yellow reaction mixture was stirred for 1 h. The solvent was then evaporated in vacuo, and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (0.1 mL). TFA (0.5 M solution in anhydrous DMF, 27.3 μL, 13.7 μmol, 3.3 equiv.) and i-PrNEt (0.5 M solution in anhydrous DMF, 43.9 μL, 30.0 μmol, 5.3 equiv.) were added, and the resulting pale yellow solution was cooled to 0° C. HATU (0.1 M solution in DMF, 45.6 μL, 4.5 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0° C. for 15 min, and ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.1 M solution in anhydrous CHCl, 45.6 μL, 4.6 μmol, 1.1 equiv.) was added. The reaction mixture was then warmed to ambient temperature and stirred for an additional 30 minutes. The reaction mixture was then concentrated in vacuo, and the residue was dissolved in CH2Cl2 and directly loaded onto a silica plate. Purification by preparative TLC (SiO2, 7% MeOH in CH2Cl2) afforded the product (1.7 mg, 35%) as an orange solid. In the absence of residual TFA, the compound readily undergoes methanolysis. 11H NMR (500 MHz, CD2Cl2) δ = 8.46 (d, J = 8.5 Hz, 1H), 8.04 - 7.87 (m, 4H), 7.71 (s, 1H), 6.83 (s, 1H), 6.49 (s, 2H), 6.23 (d, J = 8.7 Hz, 1H), 5.91 (s, 1H), 5.62 (s, 1H), 4.73 (bs, 2H), 4.58 (bs, 2H), 3.96 (bs, 1H), 3.92 (bs, 2H), 3.85 (d, J = 14.2 Hz, 2H), 3.79 (s, 2H), 3.72 (s, 6H), 3.70 - 3.66 (m, 2H), 3.43 (d, J = 6.4 Hz, 2H), 3.22 (t, J = 6.9 Hz, 2H), 3.14 (td, J = 7.5, 4.0 Hz, 2H), 3.06 (s, 2H), 2.27 - 2.22 (m, 2H), 2.20 - 2.15 (m, 1H), 2.10 - 2.05 (m, 1H), 2.02 (t, J = 4.6 Hz, 1H), 1.68 (d, J = 8.4 Hz, 1H), 13 13C NMR (126 MHz, CD2Cl2) δ = 178.48 (HMBC), 158.98, 147.61, 120.83, 120.62, 116.18, 112.43, 103.29, 102.47, 83.82, 56.26, 55.93, 52.06, 44.96, 44.76, 41.27, 40.60, 38.50, 38.05, 30.38, 29.88, 29.27, 27.15, 26.56, 25.14, 21.31. IR (neat, ν max / cm -1 ) 3325, 2929, 2864, 2097, 1717, 1635, 1574, + 1541, 58 1449, 76 1410, 14 1364, 11 1302, 1172, 1122, 1030. HRMS (ESI): m / z = 1,199.5422 [M + Na]

[0201] Example 12 1-(6-((2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate [ka]

[0202] Step j) 1-(6-((2-(2-(4-(3-((4-((7-(tert-butoxy)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate In a glovebox, [Cu(MeCN)4]PF6 (5.9 mg, 16.0 μmol, 5.0 equiv.) was added to a heart-shaped flask charged with tert-butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamido)heptanoate (2.4 mg, 4.7 μmol, 1.5 equiv.) and 1-(6-((2-(2-azidoethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate (2.0 mg, 3.1 μmol, 1.0 equiv.). The solid was dissolved in anhydrous, argon-degassed CHCl2 (300 μL) and AcOH (10 μL). The flask was then flushed with argon, stoppered, and the plum-colored solution was stirred at room temperature for 16 h. The reaction mixture was then loaded directly onto a silica plate and purified by preparative TLC (SiO, 15% MeOH in CHCl) to afford the product (3.4 mg, 94%) as a red solid. 1H NMR (500MHz, CD3OD, E / Z シグナルセットをcomplete なシグナルとして report) δ=9.17(dd ,J=1.8,0.6Hz,1H,Z), 9.02(dd,J=1.9,0.5Hz,1H,E), 8.58( dd,J=8.7,1.9Hz,1H,E), 8.51(dd,J=8.3,1.8Hz,1H,Z), 8.44(d,J=8.6Hz,1H,E), 8.15(s,1H,E), 8.11(d,J=15.3Hz,1H, E), 8.07(d,J=8.8Hz,2H), 8.02-7.98(m,2H), 7.94(s,1H,Z), 7.89(d,J=8.4Hz,1H,Z), 7.81(d,J=15.5Hz,1H,E), 7.65(s ,1H), 7.51(d,J=9.1Hz,1H,E), 7.40(d,J=9.1Hz,1H,Z), 7.10(dd,J=12.3,0.9Hz,1H,Z), 6.83(dd,J=9.0,2.5Hz,1H,E), 6.79(d,J=12.3Hz,1H,Z), 6.75(dd,J=9.0,2.5Hz,1H,Z), 6.57(dd,J=2.4,0.6Hz,1H,E), 6.47(d,J=2.4Hz,1H,Z), 4.74- 4.63(m,2H), 4.48(dd,J=5.5,4.4Hz,2H), 3.79(dd,J=5.6,4.5Hz,2H), 3.58-3.52(m,4H), 3.51-3.47(m,4H), 3.42-3.35 (m,2H), 3.35-3.30(m,2H), 3.16-3.10(m,2H), 2.99-2.92(m,2H), 2.28-2.18(m,4H), 2.11-1.97(m,2H), 1.74-1.67(m,2 H), 1.65-1.57(m,4H), 1.54-1.46(m,2H), 1.43(s,9H), 1.41-1.36(m,4H), 1.25(t,J=7.1Hz,6H,E), 1.22-1.18(m,6H,Z). 13C NMR(126MHz,CD3OD)δ175.8, 175.0, 172.8, 162.1, 158.4, 155.4, 154.5, 153. 8, 149.3, 146.9, 143.6, 142.8, 142.0, 141.6, 132.3, 129.6, 129.2, 128.4, 126 .1, 124.4, 116.8, 116.7, 114.6, 111.7, 110.4, 97.6, 81.4, 70.5, 70.0, 51.3, 46.1, 40.2, 36.7, 36.3, 34.6, 30.2, 29.8, 28.4, 27.8, 26.8, 26.1, 21.4, 12.8. IR(neat,ν max / cm -1 )2924, 2854, 1710, 1620, 1579, 1556, 1504, 1459, 1423, 1357, 1278, 1198, 1171, 1136, 1053. HRMS(ESI):m / z=1152.4512[M+Na] + (C 55 H 71 N9NaO 13 Calculated m / z for S2 = 1152.4505)

[0203] Step k) 1-(6-((2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate To a solution of 1-(6-((2-(2-(4-(3-((4-((7-(tert-butoxy)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate (2.5 mg, 2.2 μmol, 1.0 equiv) in anhydrous CHCl (200 μL) was added TFA (100 μL) and the resulting plum-colored reaction mixture was stirred for 30 min. The solvent was then removed in vacuo and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (50 μL). TFA (14.6 μL, 7.2 μmol, 3.3 equiv. of a 0.5 M solution in anhydrous DMF) and i-Pr2NEt (23.4 μL, 11.7 μmol, 5.3 equiv. of a 0.5 M solution in anhydrous DMF) were added, and the resulting colorless solution was cooled to 0° C. HATU (24.3 μL, 2.4 μmol, 1.1 equiv. of a 0.1 M solution in DMF) was then added, and the reaction was stirred at 0° C. for 15 minutes. The amine ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.1 M solution in anhydrous CHCl, 24.3 μL, 2.4 μmol, 1.1 equiv.) was then added. The reaction mixture was allowed to warm to ambient temperature and stirred for an additional 30 min. The reaction mixture was then concentrated in vacuo, and the residue was dissolved in CHCl and loaded directly onto a silica plate. Purification by preparative TLC (SiO, 7% MeOH in CHCl) afforded the product (2.2 mg, 66%) as a red solid. HRMS(ESI):m / z=755.8670[M+2H] 2+ (C 78 H 105 N 13 O 14 Calculated m / z for S2 = 755.8667)

[0204] Example 13 3,7-Di(azetidin-1-yl)-N-(2-(2-(4-(3-((4-((7-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide [ka]

[0205] Step j) tert-butyl 7-(4-(N-(cyanomethyl)-N-(3-(1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyl-10,1'-isobenzofuran]-6'-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamido)heptanoate To a heart-shaped flask charged with tert-butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamido)heptanoate (3.1 mg, 6.2 μmol, 1.5 equiv.), 3,7-di(azetidin-1-yl)-N-(2-(2-azidoethoxy)ethyl)-5,5-dimethyl-3′-oxo-3′H,5H-spiro[dibenzo[b,e]silyne-10,1′-isobenzofuran]-6′-carboxamide (2.5 mg, 4.1 μmol, 1.0 equiv.), and Cu[MeCN]PF (7.7 mg, 20.5 μmol, 5.0 equiv.) was added anhydrous CHCl (0.3 mL, pre-degassed with argon) and 10 μL of AcOH. The flask was then flushed with argon, stoppered with a glass stopper, and the dark blue solution was stirred for 16 h. The reaction mixture was then loaded directly onto a silica plate and purified by preparative TLC (SiO, 3% MeOH in CHCl) to afford the product (3.1 mg, 68%) as a blue solid. 1 H NMR(500MHz,CD2Cl2)δ=8.09-7.85(m,6H), 7.74(d,J=4.9Hz,1H), 7.29(s,1H), 6.83-6.68(m,4H), 6.37 -6.28(m,2H), 4.70(s,2H), 4.43-4.37(m,2H), 3.96(s,8H), 3.81-3.76(m,2H), 3.59-3.50(m,4H), 3.33 (td,J=6.6,2.2Hz,2H), 3.05(t,J=13.4Hz,2H), 2.87-2.81(m,2H), 2.44-2.35(m,4H), 2.17(t,J=10.3H) z,2H), 1.68-1.48(m,4H), 1.41(s,9H), 1.36-1.30(m,4H), 0.61(d,J=1.7Hz,3H), 0.56(d,J=2.4Hz,3H). 13C NMR(126MHz,CD2Cl2)δ=171.90, 145.87, 140.47, 129.06, 128.31, 122.83(HMBC), 122.37(HMBC), 86.61, 80.50, 80.27, 69.88, 69.51, 69.46, 50.5 1, 43.32, 40.73, 40.44, 40.31, 36.08, 35.93, 33.93, 30.26, 29.79, 29.2 1, 28.40, 27.17, 25.50, 20.96, 17.29(HSQC), 14.44(HSQC), 9.07, -1.07. IR(neat,ν max / cm -1 )3344, 2926, 2855, 1755, 1722, 1654, 1594, 1546, 1480, 1365, 1291, 1235, 1159, 1091. HRMS(ESI):m / z=1134.4562[M+Na] + (C 58 H 69 N9NaO 10 Calculated m / z for SSi = 1134.4550

[0206] Step k) 3,7-di(azetidin-1-yl)-N-(2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide To a solution of tert-butyl 7-(4-(N-(cyanomethyl)-N-(3-(1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamido)heptanoate (3.1 mg, 2.8 μmol, 1.0 equiv) in anhydrous CHCl (200 μL) was added TFA (100 μL), and the resulting dark blue reaction mixture was stirred for 1 h. The solvent was then concentrated in vacuo, and the residue was redissolved in CHCl and co-evaporated (×3). The residue was then dissolved in anhydrous DMF (0.1 mL). TFA (0.5 M solution in anhydrous DMF, 18.4 μL, 9.2 μmol, 3.3 equiv.) and i-PrNEt (0.5 M solution in anhydrous DMF, 35.1 μL, 17.6 μmol, 6.3 equiv.) were added, and the resulting colorless solution was cooled to 0° C. HATU (0.1 M solution in DMF, 30.7 μL, 3.1 μmol, 1.1 equiv.) was then added, the reaction was stirred at 0° C. for 15 min, and ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (0.05 M solution in anhydrous CHCl, 61.3 μL, 3.1 μmol, 1.1 equiv.) was added. Monitoring the reaction by LCMS indicated incomplete conversion, and additional i-PrNEt (0.5 M solution in anhydrous DMF, 11.1 μL, 5.6 μmol, 2.0 equiv.) was added until complete conversion was achieved. The reaction mixture was then warmed to ambient temperature and stirred for an additional 30 min. The reaction mixture was then concentrated in vacuo, and the residue was dissolved in CHCl and loaded directly onto a silica plate. Purification by preparative TLC (SiO, 0.5% AcOH, 7% MeOH in CHCl) afforded the product (2.1 mg, 38%). HRMS (ESI): m / z = 1492.7302 [M+H] + (C 81 H 102 N 13 O 11Calculated m / z for SSi = 1492.7306)

[0207] Example 14 N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamide [ka]

[0208] Step i) N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamide tert-Butyl 7-(4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamido)heptanoate (2.4 mg, 4.8 μmol, 1.1 equiv.) was dissolved in a mixture of CHCl (200 μL) and TFA (100 μL), and the solution was stirred at room temperature for 1 h. The volatiles were removed in vacuo, and the residue was dissolved in anhydrous DMF (50 μL) and cooled to 0 °C. Then, stock solutions of TFA (0.5 M in DMF, 29 μL, 14.5 μmol, 3.3 equiv.), i-PrNEt (0.5 M in DMF, 47 μL, 23.3 μmol, 5.3 equiv.), and HATU (0.1 M in DMF, 48 μL, 4.8 μmol, 1.1 equiv.) were added, and the mixture was stirred at 0 °C for 15 min. ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (2.0 mg, 4.3 μmol, 1.0 equiv) dissolved in anhydrous CHCl (50 μL) was added, and the reaction mixture was warmed to room temperature and stirred for 10 min. The crude mixture was concentrated in vacuo and purified by preparative TLC (SiO, 85% EtOAc in hexanes) to afford the title compound as a colorless solid (2.0 mg, 51%). 1 H NMR1H NMR(500MHz,CD2Cl2)δ8.07-8.00(m,4H), 6.49(s,2H), 6.47(d,J=6.0Hz,1H), 5.61-5.56(m,1H), 5.42(t,J=5.8Hz,1H), 4.7 4(s,2H), 3.98-3.93(m,1H), 3.88-3.74(m,2H), 3.72(s,6H), 3.48-3.39(m,2H), 3.22(t,J=7.0Hz,2H), 2.97(dd,J=7.4,6.8H z,2H), 2.49(ddd,J=7.5,6.8,2.6Hz,2H), 2.22-2.13(m,3H), 2.08(td,J=5.7,1.4Hz,1H), 2.05-1.98(m,1H), 1.98(t,J=2.7 Hz,1H), 1.72-1.48(m,7H), 1.47-1.36(m,4H), 1.35-1.22(m,6H), 1.28(s,3H), 1.26(s,6H), 1.17-1.06(m,2H), 0.95(s,3H).13 C NMR(126MHz,CD2Cl2)δ173.04(HMBC), 170.81(HMBC), 165.51, 159.01, 150.04, 140.50 , 139.17, 129.07, 128.51, 124.03, 117.98, 115.08, 103.30, 82.37, 69.71, 56.26, 52.06 , 47.97, 44.95, 44.77, 41.23, 40.53, 38.49, 38.04, 37.02, 36.07, 33.93, 32.50, 30.39 , 30.26, 29.62, 29.36, 29.28, 28.98, 28.11, 27.16, 26.81, 26.58, 26.06, 25.15, 21.30. IR(neat,ν max / cm -1 ):3309, 2960, 2920, 2851, 2094, 1715, 1648, 1571, 1464. HRMS(ESI):m / z=884.4736[M+H] + (C 48 H 66 Calculated m / z for N7O7S = 884.4739

[0209] Example 15 N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamide [ka]

[0210] Step b) tert-butyl 7-(4-(N-(4-((tert-butoxycarbonyl)amino)butanoyl)sulfamoyl)benzamido)heptanoate To a solution of tert-butyl 7-(4-sulfamoylbenzamido)heptanoate (115 mg, 299 μmol, 1.0 equiv.) in anhydrous DMF (1.3 mL) was added Boc-GABA-OH (76 mg, 374 μmol, 1.25 equiv., CAS RN57294-38-9), EDCI·HCl (86 mg, 449 μmol, 1.5 equiv.), i-Pr₂NEt (260 μL, 1.50 mmol, 5.0 equiv.), and DMAP (11 mg, 90.1 μmol, 0.3 equiv.), and the solution was stirred at room temperature overnight. The mixture was diluted with saturated aqueous NH₄Cl (50 mL) and EtOAc (50 mL), and the phases were separated. The aqueous layer was extracted with EtOAc (2 × 50 mL), and the combined organic layers were washed with brine (25 mL), dried over MgSO₄, filtered, and concentrated in vacuo. The crude material was purified by flash column chromatography (SiO2, 1% AcOH, 2-5% MeOH in CH2Cl2) to give the product as a white solid (140 mg, 82%). 1 H NMR(400MHz,CD3OD)δ8.66(t,J=5.7Hz,1H), 8.13-8.03(m,2H), 8.01-7.90(m,2H), 3.44-3.35(m ,2H), 2.96(t,J=6.9Hz,2H), 2.24(dt,J=9.3,7.4Hz,4H), 1.71-1.55(m,6H), 1.45-1.37(m,4H). 1.44(s,9H), 1.41(s,9H). 13 C NMR(101MHz,CD3OD)δ171.98, 167.14, 157.11, 141.96, 139.29, 127.93, 127.43, 79.96 , 78.60, 39.72, 39.00, 34.92, 32.77, 28.81, 28.40, 27.35, 26.95, 26.32, 24.71, 24.40. IR(neat,ν max / cm -1 ):3366, 3110, 2978, 2934, 2866, 1729, 1213, 1678, 1635, 1515, 1444, 1357. HRMS(ESI):m / z=592.2661[M+Na] + (C 27 H 43 Calculated m / z for N3NaO8S = 592.2663

[0211] Step e) 7-(4-(N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamido)heptanoic acid To a solution of tert-butyl 7-(4-(N-(4-((tert-butoxycarbonyl)amino)butanoyl)sulfamoyl)benzamido)heptanoate (38.0 mg, 66.7 μmol, 1.0 equiv.) in CHCl (200 μL) was added TFA (200 μL) and the reaction was stirred at room temperature for 1 h. The volatiles were removed in vacuo and residual TFA was azeotropically removed with toluene (3 × 3 mL). The crude material was dissolved in anhydrous DMF (0.2 mL) and i-PrNEt (58.1 μL, 334 μmol, 5.0 equiv.), followed by the addition of 2,5-dioxopyrrolidin-1-yl-2-methylcycloprop-2-ene-1-carboxylate (15.6 mg, 80.0 μmol, 1.2 equiv.), and the mixture was stirred at room temperature overnight. The mixture was diluted with 1 M aqueous HCl (30 mL) and EtOAc (30 mL), and the phases were separated. The aqueous layer was extracted with EtOAc (2 × 30 mL), and the combined organic layers were dried over MgSO, filtered, and concentrated in vacuo. The crude material was purified by flash column chromatography (1% AcOH, 5–10% MeOH in CHCl) to afford the product as a white solid (31 mg, 85%). 1 H NMR(500MHz,MeOD)δ8.11-8.04(m,2H), 7.99-7.93(m,2H), 6.66-6.34(m,1H), 3.39(t,J=7.2Hz,2H), 3.11(td,J=6.9,1.6Hz,2H), 2 .30(t,J=7.4Hz,2H), 2.24(t,J=7.3Hz,2H), 2.12(d,J=1.3Hz,3H), 1.96(d,J=1.6Hz,1H), 1.73-1.58(m,6H), 1.41(h,J=2.6Hz,4H). 13C NMR(126MHz,MeOD)δ179.39, 177.70, 173.64, 168.57, 143.49, 140.65, 129.30, 128.81, 113 .99, 96.45, 41.12, 39.58, 34.89, 34.38, 30.22, 29.90, 27.75, 26.01, 25.53, 22.72, 10.40. IR(neat,ν max / cm -1 ):3096, 2935, 2861, 2504, 1711, 1632, 1571, 1459, 1346, 1178. HRMS(ESI):m / z=494.1951[M+H] + (C 23 H 32 Calculated m / z for N3O7S = 494.1955

[0212] Step f) N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamide To a solution of 7-(4-(N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamido)heptanoic acid (2.7 mg, 5.4 μmol, 1.25 equiv.) in anhydrous DMF (50 μL) was added HATU (2.1 mg, 5.4 μmol, 1.25 equiv.) and i-PrNEt (3.1 μL, 17 μmol, 4.0 equiv.). The reaction mixture was stirred at room temperature for 5 min, after which ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (2.0 mg, 4.3 μmol, 1.0 equiv.) in anhydrous CHCl (25 μL) was added. The reaction mixture was stirred at room temperature for 1 h and concentrated in vacuo. The crude product was purified by preparative TLC (SiO2, 1% AcOH, 6% MeOH in CHCl2) to give the intermediate as a colorless, waxy solid, which was immediately redissolved in DMF (50 μL). 2-Iodoacetonitrile (2.0 μL, 27 μmol, 6.3 equiv.) and i-Pr2NEt (2.3 μL, 13 μmol, 3.0 equiv.) were added, and the yellowish mixture was stirred at room temperature overnight. The volatiles were removed in vacuo, and the crude product was purified by preparative TLC (SiO2, 5–7% MeOH in CHCl2) to give the final compound as a white amorphous solid (2.4 mg, 56%). 11H NMR (600 MHz, CD2Cl2) δ 8.06 - 7.99 (m, 2H), 7.99 - 7.91 (m, 2H), 7.12 (t, J = 5.2 Hz, 1H), 6.49 (s, 2H), 6.46 - 6.39 (m, 1H), 5.61 - 5.55 (m, 1H), 5.48 - 5.40 (m, 2H), 4.77 (s, 2H), 3.95 (t, J = 2.4 Hz, 1H), 3.87 - 3.74 (m, 2H), 3.72 (s, 6H), 3.43 - 3.38 (m, 2H), 3.22 (t, J = 7.0 Hz, 2H), 3.05 (q, J = 6.6 Hz, 2H), 2.65 (td, J = 7.1, 0.9 Hz, 2H), 2.21 - 2.14 (m, 3H), 也 2.13 (d, J = 1.3 Hz, 3H), 2.10 - 2.05 (m, 1H), 2.02 (tt, J = 6.0, 1.8 Hz, 1H), 1.90 (d, J = 1.6 Hz, 1H), 1.71 - 1.48 (m, 9H), 1.45 - 1.35 (m, 4H), 1.34 - 1.22 (m, 6H), 1.28 (s, 3H), 也 1.26 (s, 6H), 1.13 - 1.07 (m, 2H), 0.95 (s, 3H). 13 13C NMR (151 MHz, CD2Cl2) δ 值 176.72, 173.04, 172.43, 166.39, 159.02, 150.04, 142.17, 140.40, 139.21, 129.21, 128.64, 128.40, 128.02, 124.01, 118.01, 115.45, 114.59, 103.31, 96.58, 56.26, 52.06, 47.98, 44.95, 44.77, 40.55, 38.85, 38.49, 38.04, 37.09, 34.09, 33.77, 30.39, 也 30.22, 29.69, 29.37, 29.28, 29.19, 28.11, 27.16, 27.02, 26.59, 26.16, 25.29, 25.15, 22.86, 21.31, 14.45, 10.95. IR (neat, ν max ​​​​​​​​​

[0213] Example 16 N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanamide [ka]

[0214] Step a) tert-butyl 7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanoate Sodium hydride (60% suspension in oil, 4.2 mg, 104 μmol, 1.2 equiv.) was suspended in anhydrous THF (0.35 mL) at 0 °C. tert-Butyl 7-hydroxyheptanoate (17.5 mg, 86.5 μmol, 1.0 equiv., CAS RN 86013-78-7) was added, and the solution was stirred for 15 minutes. 4-Fluoro-7-nitrobenzo[c][1,2,5]selenadiazole (32.0 mg, 130 μmol, 1.5 equiv., CAS RN 2351940-09-3) was then added, and the solution was allowed to warm to room temperature and stirred overnight. The reaction mixture was quenched by the addition of saturated aqueous NH4Cl (15 mL) and extracted with CHCl (3 × 30 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. The crude oil was purified by flash column chromatography (SiO2, 2% MeOH in CH2Cl2) to give the product as a yellow solid (11.5 mg, 31%). 1 H NMR(500MHz,CDCl3)δ8.65(d,J=8.5Hz,1H), 6.71(d,J=8.6Hz,1H), 4.33(t,J=6.6Hz,2H), 2 .23(t,J=7.4Hz,2H), 2.09-1.97(m,2H), 1.69-1.54(m,4H), 1.47-1.41(m,2H), 1.43(s,9H). 13C NMR (126MHz, CDCl3) δ173.18, 157.15, 154.09, 152.71, 134.87, 132.20, 102.88, 80.23, 70.48, 35.54, 28.85, 28.68, 28.26, 25.85, 25.02. IR(neat,ν max / cm -1 ):2932, 2859, 1727, 1610, 1532, 1511, 1463, 1406, 1392, 1320, 1249. HRMS(ESI):m / z=452.0698[M+Na] + (C 17 H 23 Calculated m / z for N3NaO5Se = 452.0695

[0215] Step b) N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanamide To a solution of tert-butyl 7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanoate (2.9 mg, 6.8 μmol, 1.0 equiv.) in anhydrous CHCl (200 μL) was added TFA (100 μL), and the resulting deep red reaction mixture was stirred at room temperature for 30 min. The solvent was then removed in vacuo, and the residue was redissolved in CHCl and co-evaporated (×3). To the residue dissolved in anhydrous DMF (100 μL) were added anhydrous i-PrNEt (2.9 μL, 16.5 μmol, 3.0 equiv.), HATU (2.6 mg, 6.8 μmol, 1.25 equiv.), and the reaction mixture was stirred at room temperature for 15 min. ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (2.5 mg, 5.4 μmol, 1.0 equiv) was added to the mixture in anhydrous CHCl (100 μL), and the reaction mixture was stirred at room temperature for 45 min, followed by quenching with saturated aqueous NaHCO (5 mL). The aqueous layer was extracted with CHCl (3 × 15 mL), and the combined organic extracts were washed with brine (10 mL). The organic layer was dried over NaSO, filtered, and concentrated in vacuo. The crude product was purified by preparative TLC (SiO, 2% MeOH in CHCl) to afford the title product as a bright yellow solid (3.0 mg, 67%). 1 H NMR(500MHz,CD2Cl2)δ8.59(d,J=8.4Hz,1H), 6.69(d,J=8.6Hz,1H), 6.49(s,2H), 5.58(dt,J=2.9,1.5Hz ,1H), 5.40(t,J=5.4Hz,1H), 4.30(t,J=6.6Hz,2H), 3.95(t,J=2.3Hz,1H), 3.89-3.75(m,2H), 3.72(s,6H) ), 3.21(t,J=7.0Hz,2H), 2.21-2.13(m,3H), 2.08(td,J=5.6,1.4Hz,1H), 2.04-1.93(m,3H), 1.74-1.63( m,3H), 1.62-1.42(m,8H), 1.37-1.21(m,4H), 1.27(s,3H), 1.26(s,6H), 1.17-1.06(m,2H), 0.94(s,3H).13 C NMR(126MHz,CD2Cl2)δ172.58, 158.86, 157.51, 149.88, 139.10, 132.34, 123.76, 117.84, 103.16, 103.13, 70.67, 56.09, 51.89, 47.81, 44.78, 44.61, 44.57, 41.07, 38.32, 37.87, 37.01, 30.23, 29.24, 29.20, 29.11, 28.94, 27.95, 26.99, 26.42, 26.10, 26.03, 24.99, 21.14. IR(neat,ν max / cm -1 )3309, 2927, 2856, 2094, 1649, 1607, 1571, 1523, 1463, 1410, 1324, 1294, 1239. HRMS(ESI):m / z=832.3280[M+Na] + (C 40 H 55 Calculated m / z for NNaOSe = 832.3275

[0216] Example 17 N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanamide [ka]

[0217] Step a) 8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanoic acid To 8-sulfanylheptanoic acid (150 mg, 851 μmol, 1.0 equiv., CAS RN74328-61-3) in a mixture of EtOH (1.5 mL) and water (4.5 mL) was added NBD-Cl (255 mg, 1.28 mmol, 1.5 equiv., CAS RN10199-89-0), followed by pyridine (343 μL, 4.26 mmol, 5.0 equiv.). The mixture gradually turned black and was stirred vigorously at room temperature for 16 h. The mixture was partitioned between EtOAc (150 mL) and 1 M aqueous HCl (150 mL), the layers were separated, and the aqueous layer was extracted with EtOAc (2 × 150 mL). The combined organic fractions were dried over MgSO4, filtered, and concentrated in vacuo. The crude product was adsorbed onto silica and purified by flash column chromatography (SiO2, 30-50% EtOAc, 1% AcOH in hexanes) to give the product as a cloudy brown solid (182 mg, 63%). 1 H NMR(400MHz,DMSO)δ11.99(s,1H), 8.56(d,J=8.0Hz,1H), 7.50(d,J=8.1Hz,1H), 3.41-3.3 0(m,2H), 2.20(t,J=7.3Hz,2H), 1.84-1.70(m,2H), 1.57-1.41(m,4H), 1.39-1.23(m,4H). 13 C NMR (101MHz, DMSO) δ174.52, 149.21, 142.72, 140.04, 132.38, 132.12, 122.17, 33.61, 30.58, 28.39, 28.19, 28.08, 27.39, 24.41. IR(neat,ν max / cm -1 ):3031, 2936, 2853, 1697, 1615, 1513, 1433, 1364, 1334, 1303. HRMS(ESI):m / z=340.0969[M+H] + (C 14 H 18 Calculated m / z for N3O5S = 340.0962

[0218] Step b) N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanamide To a solution of 8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanoic acid (2.7 mg, 7.9 μmol, 1.2 equiv.) in anhydrous DMF (50 μL) at 0 °C was added HATU (3.0 mg, 7.9 μmol, 1.2 equiv.) and i-PrNEt (3.5 μL, 20 μmol, 3.0 equiv.). The reaction mixture was stirred at 0 °C for 10 min and then added to ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (3.0 mg, 6.5 μmol, 1.0 equiv.) in anhydrous CHCl (50 μL). The ice bath was removed and the reaction mixture was stirred at room temperature for 1 h, concentrated in vacuo and purified by preparative TLC (SiO 2 , 70% EtOAc in hexanes) to give the final product as an orange solid (1.9 mg, 37%). 1 H NMR(500MHz,CD2Cl2)δ8.41(d,J=7.9Hz,1H), 7.15(d,J=7.9Hz,1H), 6.49(s,2H), 5.66-5.52(m,1H), 5.37(t,J=5.7Hz,1H), 3.99-3.91(m,1H), 3.89-3.76(m,2H), 3.72(s,6H), 3.24(t,J=7.4Hz,2H), 3.22 (t,J=7.0Hz,2H), 2.19-2.13(m,3H), 2.08(td,J=5.7,1.4Hz,1H), 2.04-1.99(m,1H), 1.89-1.78(m,2 H), 1.69-1.49(m,7H), 1.44-1.21(m,10H), 1.28(s,3H), 1.26(s,6H), 1.17-1.05(m,2H), 0.95(s,3H). 13C NMR(126MHz,CD2Cl2)δ172.25, 158.44, 149.47, 138.66, 131.04, 123.35, 120.44, 117.42, 102.71, 55.66, 51.48, 47.40, 44.37, 44.1 5, 40.65, 37.91, 37.45, 36.66, 31.70, 29.81, 29.68, 28.79, 28.77, 28.70, 28.64, 27.75, 27.51, 26.58, 26.00, 25.64, 24.57, 20.71. IR(neat,ν max / cm -1 )3316, 2929, 2857, 2094, 1725, 1648, 1571, 1522, 1508, 1411, 1362, 1328. HRMS(ESI):m / z=798.3969[M+Na] + (C 41 H 57 Calculated m / z for N7NaO6S = 798.3983

[0219] Example 18 N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfinyl)octanamide [ka]

[0220] Step c) 8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfinyl)octanoic acid A solution of mCPBA in anhydrous CHCl (0.15 M, 1.08 mL, 161 μmol, 1.5 equiv.) was added dropwise to a solution of 8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanoic acid (35 mg, 108 μmol, 1.0 equiv.) in anhydrous CHCl (0.5 mL) at 0 °C. The resulting mixture was allowed to reach room temperature and stirred for 2 h. The mixture was directly purified by flash column chromatography (30–40% EtOAc, 1% AcOH in hexanes) to afford the product as a cloudy green solid (22.8 mg, 62%). 1 H NMR(400MHz,CD2Cl2)δ8.64(d,J=7.5Hz,1H), 8.13(d,J=7.5Hz,1H), 3.38(ddd,J=13.5,9.6,6.0Hz,1 H), 3.12(ddd,J=13.5,10.0,4.6Hz,1H), 2.31(t,J=7.5Hz,2H), 2.04-1.88(m,1H), 1.66-1.24(m,9H). 13 C NMR(101MHz,CD2Cl2)δ=178.69, 146.08, 143.38, 142.97, 137.92, 129.67, 128.25, 54.12, 33.62, 28.65, 28.59, 28.18, 24.43, 21.63. IR(neat,ν max / cm -1 ):3096, 2930, 2859, 1706, 1548, 1534, 1464, 1407, 1368, 1340. HRMS(ESI):m / z=378.0730[M+Na] + (C 14 H 17 Calculated m / z for N3NaO6S = 378.0730

[0221] Step b) N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfinyl)octanamide To a solution of 8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfinyl)octanoic acid (2.4 mg, 6.8 μmol, 1.25 equiv.) in anhydrous DMF (50 μL) at 0 °C was added HATU (2.6 mg, 6.8 μmol, 1.25 equiv.) and i-PrNEt (2.9 μL, 17 μmol, 3.0 equiv.). The reaction mixture was stirred at 0 °C for 10 min and then added to ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine (2.5 mg, 5.5 μmol, 1.0 equiv.) in anhydrous CHCl (50 μL). The ice bath was removed and the reaction mixture was stirred at room temperature for 30 min, concentrated in vacuo and purified by preparative TLC (SiO 2 , 80% EtOAc in hexanes) to give the final product as a brown solid (1.5 mg, 35%). 1 H NMR(500MHz,CD2Cl2)8.62(d,J=7.5Hz,1H),8.11(d,J=7.5Hz,1H),6.49(s,2H),5.60-5.55(m,1H) , 3.97-3.91(m,1H), 3.87-3.75(m,2H), 3.72(s,6H), 3.41-3.31(m,1H), 3.22(t,J=7.0Hz,2H), 3.15 -3.04(m,1H), 2.19-2.10(m,3H), 2.09-2.05(m,1H), 2.03-1.99(m,1H), 1.67(dd,J=8.5,1.5Hz,1H) , 1.65-1.44(m,10H), 1.37-1.22(m,8H), 1.27(s,3H), 1.26(s,6H), 1.16-1.05(m,2H), 0.94(s,3H). 13 C NMR(126MHz,CD2Cl2)δ158.44(HMBC), 129.62, 123.37(HSQC), 117.62(HMBC), 102.71, 55.67, 54.22, 51.48, 47.40, 44.35, 44.14, 40.65, 37.91, 37.45, 36.62, 29.82, 29.68, 26.58, 26.00, 25.57, 24.57, 21.66, 20.71. IR(neat,ν max / cm -1)2929, 2856, 2095, 1725, 1649, 1604, 1571, 1543, 1365, 1336, 1121. HRMS(ESI):m / z=792.4101[M+H] + (C 41 H 58 Calculated m / z for N7O7S = 792.4113

[0222] Example 19 N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-yl)oxy)heptanamide [ka]

[0223] Step a) tert-butyl 7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-yl)oxy)heptanoate To a solution of tert-butyl 7-hydroxyheptanoate (1.7 mg, 8.3 μmol, 1.1 equiv., CAS RN86013-78-7) in THF (0.1 mL) at 0 °C, NaH (60% dispersion in mineral oil, 0.4 mg, 8.3 μmol, 1.1 equiv.) was added to give a gray slurry. 3,7-Di(azetidin-1-yl)-4',5',6',7'-tetrafluoro-5,5-dimethyl-3'H,5H-spiro[dibenzo[b,e]silylin-10,1'-isobenzofuran]-3'-one (4.0 mg, 7.6 μmol, 1.0 equiv., prepared as described in Nat. Methods, 2020, 17, 815-821) was then added in THF (0.1 mL), and the reaction mixture was stirred at 0 °C for 1 h. The mixture was then diluted with water (5 mL) and CHCl (5 mL), the phases were separated, and the aqueous phase was extracted with CHCl (3 × 5 mL). The combined organic layers were dried over NaSO and concentrated in vacuo. Purification by preparative TLC (SiO, 7% MeOH in CHCl) afforded the product (3.9 mg, 5.5 μmol, 72% yield) as a pale blue solid. 1 H NMR(500MHz,CD2Cl2)δ=6.74(dd,J=8.6,1.2Hz,2H), 6.67(dd,J=2.7,0.5Hz,2 H), 6.34(dd,J=8.7,2.6Hz,2H), 4.40(td,J=6.6,1.0Hz,2H), 3.93(t,J=7.3Hz ,8H), 2.42-2.34(m,4H), 2.20(t,J=7.2Hz,2H), 1.84(dt,J=14.6,6.7Hz,2H), 1.64-1.48(m,4H), 1.42(s,9H), 1.41-1.36(m,2H), 0.56(s,3H), 0.51(s,3H). 13 C NMR(126MHz,CD2Cl2)δ=173.43(HMBC), 143.53(HMBC), 130.48(HMBC), 127.86, 116.39, 11 2.94, 80.20, 76.99, 52.92, 35.99, 30.42, 29.30, 28.39, 25.83, 25.56, 17.39, 1.33, 0.60. 19F NMR (471MHz, CD2Cl2) δ = -143.02 (dd, J = 21.9, 2.8 Hz), -147.22 (t, J = 19.5 Hz), -147.76 (d, J = 17.6 Hz). (decoupled)IR(neat,ν max / cm -1 )2930, 2854, 1768, 1728, 1595, 1484, 1405, 1365, 1306, 1265, 1164, 1103, 1005. HRMS(ESI):m / z=707.3123[M+H] + (C 39 H 46 Calculated m / z for F3N2O5Si = 707.3123

[0224] Step b) N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-yl)oxy)heptanamide To a solution of tert-butyl 7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-yl)oxy)heptanoate (2.0 mg, 2.8 μmol, 1.0 equiv.) in anhydrous CHCl (200 μL) was added TFA (100 μL), and the resulting dark blue solution was stirred for 1 h. The solvent was then concentrated in vacuo, and the residue was redissolved in CHCH and co-evaporated (×3). To the crude product was then added i-PrNEt (0.5 M solution in DMF, 34.0 μL, 16.9 μmol, 6.0 equiv.), and the resulting pale blue solution was cooled to 0 °C. HATU (0.1 M solution in DMF, 34.0 μL, 3.4 μmol, 1.2 equiv.) was then added, the reaction was stirred at 0 °C for 15 min, and ((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methanamine was added (0.05 M solution in anhydrous CHCl, 59.4 μL, 3.0 μmol, 1.05 equiv.). The mixture was warmed to ambient temperature and stirred for an additional 30 min. The reaction mixture was then diluted with saturated aqueous NaHCO (5 mL) and CHCl (5 mL). The phases were separated, and the aqueous phase was extracted with CHCl (3 × 5 mL), dried over NaSO, and concentrated in vacuo. Purification by preparative TLC (SiO2, 2% MeOH in CH2Cl2) afforded the product (0.9 mg, 29%) as a bluish solid. HRMS (ESI): m / z = 1087.5695 [M+H] + (C 62 H 78 Calculated m / z for F3N6O6Si = 1087.5699)

Claims

1. Compounds of formula (I) 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein: (i) R １ teeth, 【Chemistry 2】 ； or 【Chemistry 3】 and X is selected from O, S and SO; or (ii) R １ teeth, 【Chemistry 4】 ； or 【Chemistry 5】 and X is selected from O, S, SO, and NHCO; R ２ and R ３ is hydrogen and C １ ~C ４ - alkyl, or R ２ and R ３ together with the carbon atoms to which they are attached, form C ３ ~C ５ - forms a cycloalkyl, R ４ is selected from hydrogen, halogen, cyano and azide; R ５ teeth, 【Chemistry 6】 ； 【Chemistry 7】 ； 【Chemistry 8】 ； and 【Chemistry 9】 is selected from R ６ teeth, 【Chemistry 10】 and 【Chemistry 11】 is selected from R ７ teeth, 【Chemistry 12】 ； 【Chemistry 13】 ； 【Chemistry 14】 ； 【Chemistry 15】 ； 【Chemistry 16】 ； 【Chemistry 17】 ； 【Chemistry 18】 ； 【Chemistry 19】 ； 【Chemistry 20】 ； 【Chemical 21】 ； 【Chemical 22】 ； 【Chemical 23】 ； 【Chemistry 24】 ； 【Chemistry 25】 ； 【Chemical 26】 ； 【Chemical 27】 ； 【Chemical Formula 28】 ； 【Chemical 29】 ； 【Chemistry 30】 ； 【Chemical 31】 ； 【Chemical 32】 ； 【Chemical 33】 ； 【Chemical Formula 34】 ； 【Chemical 35】 ； and 【Chemical 36】 is selected from R ａ , R ｂ and R ｃ are each independently selected from hydrogen and halogen; n is selected from 1, 2, 3, 4, 5 and 6; p is selected from 1, 2, 3, 4, 5, 6 and 7; q is selected from 1, 2, 3, 4 and 5; r is selected from 1 and 2; s is selected from 1, 2, 3 and 4; t is selected from 0, 1, 2, 3, 4 and 5; u is selected from 0, 1, 2, 3, 4 and 5; W is carbonyl or absent; A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein Y is selected from Se, S and O.

2. (i) R １ but, 【Chemical 37】 ； or 【Chemical Formula 38】 and X is selected from O, S and SO, or (ii) R １ but, 【Chemical Formula 39】 ； or 【Chemistry 40】 and X is selected from O, S, SO, and NHCO; R ２ and R ３ is hydrogen and C １ ~C ４ - alkyl, or R ２ and R ３ together with the carbon atoms to which they are attached, C ３ ~C ５ - forms a cycloalkyl, R ４ is selected from hydrogen, halogen, cyano and azide; R ５ but, 【Chemistry 41】 ； 【Chemistry 42】 ； 【Chemistry 43】 ； and 【Chemical 44】 is selected from R ６ but, 【Chemistry 45】 ； 【Chemistry 46】 is selected from R ７ but, 【Chemistry 47】 ； 【Chemistry 48】 and 【Chemistry 49】 is selected from R ８ but, 【Chemistry 50】 ； 【Chemistry 51】 ； 【Chemistry 52】 ； and 【Chemistry 53】 is selected from R ａ , R ｂ and R ｃ are each independently selected from hydrogen and halogen; n is selected from 1, 2, 3, 4, 5 and 6; p is selected from 1, 2, 3, 4, 5, 6 and 7; q is selected from 1, 2, 3, 4 and 5; r is selected from 1 and 2; s is selected from 1, 2, 3 and 4; u is selected from 0, 1, 2, 3, 4 and 5; v is selected from 1, 2, 3 and 4; W is carbonyl or absent; 2. The compound of formula (I) according to claim 1, wherein Y is selected from Se, S and O, or a pharmaceutically acceptable salt thereof.

3. (i) R １ but, 【Chemical 54】 ； or 【Chemistry 55】 and X is selected from O, S and SO, or (ii) R １ but, 【Chemical 56】 ； or 【Chemical 57】 and X is NHCO; R ５ but, 【Chemistry 58】 ； 【Chemical Formula 59】 ； 【Chemistry 60】 ； and 【Hua 61】 is selected from R ６ but, 【Hua 62】 ； 【Chemistry 63】 is selected from R ７ but, 【Hua 64】 ； and 【Chemistry 65】 is selected from R ８ but, 【Hua 66】 ； 【Hua 67】 and 【Chemistry 68】 is selected from p is selected from 1, 3, 4 and 5; q is selected from 2, 3 and 5; r is selected from 1 and 2; s is selected from 1 and 4; u is selected from 0, 4 and 5; W is carbonyl or absent; 3. A compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein Y is selected from Se and O.

4. (i) R １ but, 【Chemical Formula 69】 ； or 【Chemistry 70】 and X is O, or (ii) R １ but, [Chemical Formula 71] ； or 【Chemical 72】 and X is NHCO; R ５ but, 【Chemical 73】 ； 【Chemical 74】 ； 【Chemistry 75】 ； and 【Chemical 76】 is selected from R ６ but, 【Chemical 77】 ； 【Chemical 78】 is selected from R ７ but, 【Chemical 79】 ； and 【Chemistry 80】 is selected from R ８ but, 【Chemistry 81】 ； 【Chemistry 82】 and 【Chemistry 83】 is selected from p is 3, q is selected from 2, 3 and 5; r is 1, s is 1, 4. The compound of formula (I) according to claim 3, or a pharmaceutically acceptable salt thereof, wherein u is 5.

5. R １ but, 【Chemistry 84】 ;or 【Chemistry 85】 and R ５ but, 【Chemistry 86】 or 【Hua 87】 and R ６ but, 【Hua 88】 and R ７ but, 【Chemistry 89】 and R ８ but, 【Chemistry 90】 and X is NHCO; p is 3, q is 2 or 3; r is 1, 5. The compound of formula (I) according to claim 4, or a pharmaceutically acceptable salt thereof, wherein u is 5.

6. R ２ and R ３ is hydrogen and C １ ~C ４ - alkyl, R ４ is hydrogen or azide, 6. The compound of formula (I) according to any one of claims 1 to 5, wherein n is 6, or a pharmaceutically acceptable salt thereof.

7. R ２ and R ３ However, both are C １ ~C ４ - alkyl, R ４ is an azide, 7. The compound of formula (I) according to claim 6, wherein n is 6, or a pharmaceutically acceptable salt thereof.

8. R ２ and R ３ However, all of them are methyl. R ４ is an azide, 8. The compound of formula (I) according to claim 7, or a pharmaceutically acceptable salt thereof, wherein n is 6.

9. (i) R １ but, 【Chemistry 91】 ； or 【Chemistry 92】 and X is selected from O, S and SO, or (ii) R １ but, 【Chemistry 93】 ;or 【Chemistry 94】 and X is NHCO; R ２ and R ３ is hydrogen and C １ ~C ４ - alkyl, R ４ is hydrogen or azide, R ５ but, 【Chemistry 95】 ； 【Chemistry 96】 ； 【Chemistry 97】 ； and 【Chemistry 98】 is selected from R ６ but, 【Hua99】 ； 【Chemistry 100】 is selected from R ７ but, 【Chemistry 101】 ； and 【Chemistry 102】 is selected from R ８ but, 【Chemistry 103】 ； 【Chemistry 104】 and 【Chemistry 105】 is selected from n is 6, p is selected from 1, 3, 4 and 5; q is selected from 2, 3 and 5; r is selected from 1 and 2; s is selected from 1 and 4; u is selected from 0, 4 and 5; W is carbonyl or absent; 2. The compound of formula (I) according to claim 1, wherein Y is selected from Se and O, or a pharmaceutically acceptable salt thereof.

10. (i) R １ but, 【Chemistry 106】 ； or 【Chemistry 107】 and X is O, or (ii) R １ but, 【Chemistry 108】 ； or 【Chemistry 109】 and X is NHCO; R ２ and R ３ However, both are C １ ~C ４ - alkyl, R ４ is an azide, R ５ but, 【Chemistry 110】 ； 【Chemistry 111】 ； 【Chemistry 112】 ； and 【Chemistry 113】 is selected from R ６ but, 【Chemistry 114】 ； 【Chemistry 115】 is selected from R ７ but, 【Chemistry 116】 ； and 【Chemistry 117】 is selected from R ８ but, 【Chemistry 118】 ； 【Chemistry 119】 and 【Chemistry 120】 is selected from n is 6, p is 3, q is selected from 2, 3 and 5; r is 1, s is 1, 10. The compound of formula (I) according to claim 9, or a pharmaceutically acceptable salt thereof, wherein u is 5.

11. R １ but, 【Chemistry 121】 ;or 【Chemistry 122】 and R ２ and R ３ However, all of them are methyl. R ４ is an azide, R ５ but, 【Chemical 123】 or 【Chemical 124】 and R ６ but, 【Chemistry 125】 and R ７ but, 【126】 and R ８ but, 【Chemistry 127】 and X is NHCO; n is 6, p is 3, q is 2 or 3; r is 1, 11. The compound of formula (I) according to claim 10, or a pharmaceutically acceptable salt thereof, wherein u is 5.

12. wherein said compound of formula (I) N-(9-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-9-oxononyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(5-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-5-oxopentyl)-1-((3-((2-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(14-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; 1-(6-((2-(2-(4-((3-((5-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-2-oxopyridin-1(2H)-yl)sulfonyl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-((E)-2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)-N-(pyridin-2-ylmethyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-1-((3-(((1-(2-(2-(3,7-di(azetidin-1-yl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-6-oxo-1,6-dihydropyridine-3-carboxamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((7-nitrobenzo[c][1,2,5]selenadiazol-4-yl)oxy)heptanamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide; 1-(6-((2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-6-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridin-1-ium-3-sulfonate; 3,7-di(azetidin-1-yl)-N-(2-(2-(4-(3-((4-((7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)carbamoyl)-N-(cyanomethyl)phenyl)sulfonamido)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-carboxamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(pent-4-ynoyl)sulfamoyl)benzamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)octanamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-8-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfinyl)octanamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(4-(2-methylcycloprop-2-ene-1-carboxamido)butanoyl)sulfamoyl)benzamide; N-(((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)-7-((3,7-di(azetidin-1-yl)-4',5',7'-trifluoro-5,5-dimethyl-3'-oxo-3'H,5H-spiro[dibenzo[b,e]silyne-10,1'-isobenzofuran]-6'-yl)oxy)heptanamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide; N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-4-(N-(cyanomethyl)-N-(3-(1-(16-oxo-20-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12-tetraoxa-15-azaicosyl)-1H-1,2,3-triazol-4-yl)propanoyl)sulfamoyl)benzamide; and N-(7-((((1S,4S,5S)-4-(4-(8-azido-2-methyloctan-2-yl)-2,6-dimethoxyphenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)amino)-7-oxoheptyl)-6-oxo-1-((3-(((1-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)sulfonyl)-1,6-dihydropyridine-3-carboxamide 2. The compound of formula (I) according to claim 1, selected from: or a pharmaceutically acceptable salt thereof.

13. A process for preparing a compound of formula (I) according to claim 1, comprising: (a) Alkyne C or I 【128】 【129】 (In the formula, R ６ , p, q and r are as defined in claim 1. azido-substituted fluorophore M 【Chemistry 130】 (In the formula, W, R ７ , s and t are as defined in claim 1 Compound D or K 【Chemistry 131】 【132】 (In the formula, W, R ６ , R ７ , p, q, r and s are as defined in claim 1. and forming Next, (b) removing the t-Bu protecting group from compound D or K using an acid, such as HCl or TFA, to give compound D1 or K1 【Chemistry 133】 【134】 (In the formula, W, R ６ , R ７ , p, q, r and s are as defined in claim 1. and Next, (c) Reacting Compound D1 or K1 with Amine N1 【Chemistry 135】 (In the formula, R ２ , R ３ and R ４ is as defined in claim 1) in the presence of a coupling reagent and an acid / base buffer system, obtaining said compound of formula (I); A method comprising:

14.

136. ； 【Chemistry 137】 ； 【Chemistry 138】 ； 【Chemistry 139】 ； 【Chemistry 140】 ； 【Chemistry 141】 ； 【142】 ； 【Chemistry 143】 ； 【Chemical 144】 ； 【Chemistry 145】 ； 【Chemistry 146】 ； and 【147】 (In the formula, R ２ , R ３ , R ４ , R ６ , R ７ , p, q, r, s, t are as defined in claim 1, and LG is 【148】 is) A compound selected from:

15. Amides of general formula 1 【149】 (In the formula, R ｘ and R ｙ are chemical moieties attached to a parent amide functional group through a carbon atom, R ｘ is an N-sulfonylpyridone (SP) motif i or an N-acyl-N-alkylsulfonamide (NASA) motif ii 【Chemistry 150】 【Chemistry 151】 (In the formula, R ６ is as defined in claim 1) (including 1. A method for producing Carboxylic Acid 2 【Chemistry 152】 to amine 3 【Chemistry 153】 in the presence of a coupling reagent and an acid / base buffer system. obtaining said amide 1.

16. Fluorescent probe P1 or P2 for the protein of interest 【Chemistry 154】 【Chemistry 155】 where LG is a ligand for the protein of interest, W, R ６ , R ７ , p, q, r and s are as defined herein) 1. A method for producing (a) Alkyne C or I 【Chemistry 156】 【Chemistry 157】 (In the formula, R ６ , p, q and r are as defined in claim 1. azido-substituted fluorophore M 【158】 (In the formula, W, R ７ , s and t are as defined in claim 1 Compound D or K 【Chemistry 159】 【Chemistry 160】 (In the formula, W, R ６ , R ７ , p, q, r and s are as defined in claim 1. and forming Next, (b) removing the t-Bu protecting group from compound D or K using an acid, such as HCl or TFA, to give compound D1 or K1 【Chemistry 161】 【Chemistry 162】 (In the formula, W, R ６ , R ７ , p, q, r and s are as defined in claim 1. and Next, (c) Binding Compound D1 or K1 to an amino-substituted ligand N 【Chemistry 163】 where LG is a ligand for the protein of interest. in the presence of a coupling reagent and an acid / base buffer system. obtaining the fluorescent probe P1 or P2 for a protein of interest; A method comprising:

17. Cannabinoid receptor 2 (CB ２ A compound of formula (I) according to any one of claims 1 to 12 for use as a fluorescent probe for R).

18. Cannabinoid receptor 2 (CB ２ Use of a compound of formula (I) according to any one of claims 1 to 12 as a fluorescent probe for R).

19. Cannabinoid receptor 2 (CB ２ A method for imaging cannabinoid receptor 2 (CB R), comprising: ２ R) with a compound of formula (I) according to any one of claims 1 to 12.

20. 10. An invention as hereinbefore described.

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